Biotechnology and Biodiversity
Biotechnology and Biodiversity
Biotechnology and Biodiversity
Volume 4
Series Editor
Kishan Gopal Ramawat
M.L. Sukhadia University, Botany Department
Udaipur, Rajasthan, India
This book series provides complete, comprehensive and broad subject based
reviews about existing biodiversity of different habitats and conservation strate-
gies in the framework of different technologies, ecosystem diversity, and genetic
diversity. The ways by which these resources are used with sustainable management
and replenishment are also dealt with. The topics of interest include but are not
restricted only to sustainable development of various ecosystems and conservation
of hotspots, traditional methods and role of local people, threatened and endangered
species, global climate change and effect on biodiversity, invasive species, impact
of various activities on biodiversity, biodiversity conservation in sustaining liveli-
hoods and reducing poverty, and technologies available and required. The books in
this series will be useful to botanists, environmentalists, marine biologists, policy
makers, conservationists, and NGOs working for environment protection.
Biotechnology
and Biodiversity
2123
Editors
M.R. Ahuja K.G. Ramawat
Formerly Forestry Consultant Botany Department
Zobel Forestry Associates M.L. Sukhadia University
New Paltz Udaipur
New York Rajasthan
USA India
v
vi Preface
We believe that biotechnology can affectively solve the problems related to bio-
diversity management, protection and conservation. The field in plant biotechnol-
ogy has been kept wide and general to accommodate a wide ranging topics. This
book provides complete, comprehensive and broad subject-based reviews useful
for students, teachers, researchers, policy makers, conservationists and NGOs for
environmental protection, and others interested in the field of biotechnology and
biodiversity.
Prof. M.R. Ahuja
Prof. K.G. Ramawat
Contents
vii
viii Contents
Index 327
Contributors
M.R. Ahuja Formerly Forestry Consultant, Zobel Forestry Associates, New Paltz,
NY, USA
Jaya AroraLaboratory of Bio-Molecular Technology, Department of Botany,
M. L. Sukhadia University, Udaipur, India
Georgina D. Arthur Mangosuthu University of Technology, Durban, KwaZulu-
Natal, South Africa
M. Asif Department of Agricultural, Food and Nutritional Science, University of
Alberta, Edmonton, AB, Canada
Achille E. Assogbadjo Laboratory of Applied Ecology, University of Abomey
Calavi, Abomey Calavi, Benin
S. K. BasuDepartment of Biological Sciences, University of Lethbridge,
Lethbridge, AB, Canada
W. Cetzal-IxHerbarium CICY, Centro de Investigacin Cientfica de Yucatn,
A. C. (CICY), Mrida, YUC, Mxico
Carlos A. Cruz-Cruz Facultad de Ciencias Qumicas, Universidad Veraccruzana,
Orizaba, Veracruz, Mexico
Manjul Dhiman Department of Botany, Kanahiya Lal DAV PG College, Roorkee,
Uttarakhand, India
Chandrakanth EmaniDepartment of Biology, Western Kentucky University-
Owensboro, Owensboro, KY, USA
Florent Engelmann IRD, UMR DIADE, Montpellier cedex 05, France
Duan Gmry Faculty of Forestry, Technical University Zvolen, Zvolen, Slovakia
Maria Teresa Gonzalez-ArnaoFacultad de Ciencias Qumicas, Universidad
Veraccruzana, Orizaba, Veracruz, Mexico
Shaily Goyal Erie, PA, USA
Hely Hggman Department of Biology, University of Oulu, Oulu, Finland
ix
x Contributors
Chandrakanth Emani
Abstract The rapidly expanding field of commercial transgenic cultivation has its
greatest concern related to environmental well being as transgenic crops are seen
as a threat to the biodiversity in the agricultural fields. Since transgenic technology
is continuing to witness a rapid growth in terms of developing novel varieties, it
is imperative to examine whether the developed varieties contribute to preserving
biodiversity. Further, it is also necessary to focus future research towards develop-
ing transgenic varieties to contribute to preserving and enhancing the biodiversity.
The present review aims to present an overview of the current status of transgenic
technology in contributing to biodiversity and suggest future research strategies
enabling the preservation of biodiversity.
Keywords Crop biodiversity Transgenic crops Speciation Non-target species
1.1Introduction
C.Emani()
Department of Biology, Western Kentucky University-Owensboro,
4821 New Hartford Road, Owensboro, KY 42303, USA
e-mail: chandrakanth.emani@wku.edu
in different taxonomic groups of which 1.8 million species have been described to
date) and ecosystem diversity (representing the abiotic components determined by
soil parent material and climate) (Groombridge and Jenkins 2002).
Human intervention has been responsible for the continual degradation of biodi-
versity and this led to numerous economic, environmental, and social consequences.
The failure to preserve our natural biological resources especially the diverse plant
life on which we depend for food, clothing, pharmaceuticals, and more recently en-
ergy in the form of biofuels is indicative of a fact that we may be losing potentially
beneficial compounds and materials that have not yet been discovered from natural
resources (OECD 2005). More recently, research has shown that climate change is
having a significant effect on the world agricultural output and thus directly influ-
ences world food security (White etal. 2004). For decades, the development of
novel crops by both conventional breeding as well as biotechnological crop im-
provement strategies had a direct influence on world food security (Kropiwnicka
2005). Plant biotechnological strategies that focused on the development of im-
proved high-yielding and disease resistant crop varieties were a result of collabora-
tive efforts between conventional and molecular breeders (Van Buerren etal. 2010).
A logical continuation of such research collaborations can now culminate towards
focused research studies resulting in preserving and enhancing plant biodiversity.
Plant biotechnological research was witness to many path breaking and applica-
tion-oriented technologies (Kumar etal. 2009) and it sometimes is a challenge for
current researchers to identify contextual research strategies without getting lost in
the diverse array of biotechnological strategies that unfold every year. In order to
develop novel strategies to preserve plant biodiversity (Pijut etal. 2011), research-
ers will be best advised to focus on certain crucial aspects of plant molecular biol-
ogy to better utilize the tools of biotechnology that are available.
1.2Transgenic Crops
and christening it as a gene revolution has to overcome the purported empty rhetoric
to a concerted identification of strategies similar to the success of green revolution
related to the increased yields and the resulting economic benefits to farmers. Fur-
ther, the major limitation attributed to green revolution in terms of increased mono-
cultures that allegedly led to a decrease in biodiversity may also be a debatable
issue. Transgenic research is also seen largely as a private enterprise with varieties
available to farmers only on market terms (Pingali and Raney 2005) and this may
jeopardize its cause to contribute to increasing biodiversity.
Transgenic crops (or the popular term attribute to them being genetically modified
crops or GMOs) are increasingly becoming a common feature of cultivated land-
scapes with the total plantings seeing a significant increase from 3 million hectares
in 1996 to 67.5 million hectares in 2003 (James 2003). Transgenic technology has
also been showcased as having a positive impact on commercial farming (Carpenter
2010) and as an effective research alternative to meet to the global needs of food
security (Schiler and Pinstrup-Andersen 2009). The pioneering efforts as exem-
plified by the herbicide-tolerant soybean (Carpenter and Gianessi 1999), insect
tolerant Bt-corn (Hilbeck 2001) and the more recent successes of the genetically
engineered-vitamin A fortified golden rice (Beyer 2010) has led to the approval
of a new generation of transgenic crops that produce health benefitting vitamins
and vaccines, and economically important enzymes and industrial products (IUCN
2007). The rapidity with which over 16 million farmers of the global agricultural
community has taken up the transgenic technology has surpassed all innovative ag-
ricultural practices of the past 80 centuries (James 2011; Lawson etal. 2009). Since
the first commercial transgenic crops of China of 1992, the countries that rapidly
adapted the transgenic crop cultivation were the USA followed by Argentina, Bra-
zil, Canada and India (GM Compass 2009) with the total transgenic crop cultiva-
tion area registering an increase from 134 million hectares in 2009 to 160 million
hectares by the end of 2011(James 2011). The four decade old global biotechnol-
ogy industry has the United States leading the world in the rapidity of transgenic
technology acceptance with proportions of major transgenic crops as high as 73%
in maize, 87% in cotton and 91% for soybean (USDA 2007/2010). In contrast, the
opposition to transgenic technology has been so severe in the European Union with
only 114, 500ha that accounts for less than 0.01% of European agriculture area
mostly in Spain cultivates Bt-maize (James 2011).
In the present agricultural scenario, the most direct negative impact on biodiversity
is the conversion of natural ecosystems into agricultural land that has a tremendous
environmental impact in terms of a significant loss of natural habitats. In overcom-
ing this environmental hazard, transgenic crops have had a significant role to play
in the past decade with their potential to increase crop yields, decreasing insecticide
use, promoting the use of environmentally friendly herbicides and the facilitation
of conserved tillage practices (Carpenter 2010, 2011). The efficient utilization of
transgenic crops has a beneficial trade-off in terms of the requirements for lesser
land to produce high yielding pest and herbicide-resistant varieties as compared
to traditional crop practices that are low yielding extensive agricultural systems
requiring more land and pesticide usage. This will also free more land that would
otherwise be forcibly be converted to agricultural land, thus minimizing the nega-
tive impacts of biodiversity on non-arable lands. Many recent reviews have focused
on both well-researched as well as hypothetical scenarios of transgenic crop cultiva-
tion related to their impact on biodiversity (Garcia and Altieri 2005; Raven 2010;
Carpenter 2011; Jacobsen etal. 2013). The present review attempts to focus on an
overview of the most important factors of transgenic crop effect on biodiversity and
suggest some application-oriented research strategies.
One of the biggest concerns expressed against transgenic crops is their potential to
reduce species abundance or the levels of genetic diversity within cultivated vari-
eties that include traditional land races as the focus will be on a small number of
high value cultivars (Ammann 2005). Initial studies conducted on transgenic cotton
related to field genetic uniformity (Bowman etal. 2003) showed a significant reduc-
tion in uniformity as compared to a study conducted on conventionally bred glypho-
sate tolerant cotton (Sneller 2003) that showed no little impact on diversity. How-
ever, the scope of examining the consequences of transgenic varieties on diversity
needs to be expanded to consider the impacts at three levels, namely, the crop, farm
and landscape levels (Carpenter 2011) to accommodate all the levels of agricultural
biodiversity from the gens to the ecosystems. When examined under this umbrella,
it is seen that transgenic crops increase the crop diversity by enhancing underuti-
lized alternative crops and making them widely domesticated (Gressel 2008) as
seen in orphan crops such as sweet potato (Bhattacharjee 2009). In case of impact-
ing farm-scale diversity, transgenic crops had no significant effect on non-target soil
organisms and weed communities (Carpenter 2011). At the landscape level, intro-
duction of transgenic corn, soybean and canola resulted in reduction in encroach-
ing into non-arable lands, and also helped in environmental friendly expansions
of arable lands (Bindraban etal. 2009; Brookes etal. 2010; Trigo and Cap 2006).
8 C. Emani
Further, an area-wide suppression of target pests by Bt corn and cotton led to pest
management benefits in other cultivars such as soybean and vegetables (Carpenter
2011). Research over the past decade has shown that the commercial cultivation
of transgenic crops had a positive impact on biodiversity through increased yields
that resulted in an alleviation of pressure to encroach on non-arable land, increased
use of environmentally friendly herbicides, reduction of insecticide use adoption
of conserved tillage and a general increase in agricultural sustainability (Carpenter
2011). An enhanced knowledge on the part of farmers who are now better educated
in agricultural practices also takes care of the fact that the purity of traditional va-
rieties will be maintained as most of the farming community grow both traditional
and improved types in the same field (Bellon and Berthaud 2004). In fact, the situ-
ation faced due to transgenic crop commercialization is no different from the times
when agriculture was exposed to conventionally bred commercial crops (Ellstrand
2001). There is a mechanism in place to study the impact of transgenes in the form
of an environmental impact statement requests by the USDA (Aphis-USDA 2007)
that precedes the release of a new transgenic plant.
The potential scenario of a transgenic herbicide tolerant trait introgressing into a na-
tive wild relative gave rise to the concept of a hypothetical superweed that can take
over entire ecosystems and be completely resistant to existing herbicides (Chap-
man and Burke 2006). This challenge, though not based on fact, was not limited to
transgenic crops but was first observed as a gene flow from conventional domesti-
cated herbicide tolerant crops to its wild relatives (Ellstrand etal. 1999; Itoh 2000).
This never led to the exaggerated scenarios of environmental disasters, but only
limited the effectiveness of existing weed control strategies and hampered weed
management options. Conventional breeders did manage the situation efficiently
with strategies such as revolving dose sprays to effectively delay the evolution of
both quantitative and major monogene resistance traits acquired within field popu-
lations (Gressel etal. 1996; Gardner etal. 1998). Evidence for herbicide tolerance
trait tolerance from transgenic crops resulting in resistant weeds was seen in well
documented studies (Watrud etal. 2004; Nandula etal. 2005; Warwick etal. 2008;
Lemaux 2009). As in conventional breeding, this could be still be attributed to a
single herbicide overuse (Lemaux 2009), and this was effectively overcome with
developing transgenic crops that have herbicide tolerance with alternate mode of
action that can be used in crop rotations to slow the resistance in weeds (Behrens
etal. 2007).
Transgenic crops could potentially impact unintended target species such as ben-
eficial pollinators, soil organisms and endangered species and such indirect effects
were seen as threats mainly from the Bt crops (Marvier etal. 2007; Duan etal.
2008; Wolfenbarger etal. 2008). However, this concern could be equally applicable
to both transgenic crop fields as well as fields sprayed with conventional pesticides
(Whitehouse etal. 2005; Cattaneo etal. 2006). As with most challenges in a trans-
genic field, the potential for the negative impact on non-target species is assessed
closely monitored by regulatory agencies before commercial approval as can be il-
lustrated with Bt cotton trials in India (Bambawale etal. 2004) and China (Pray etal.
2002; Huang etal. 2002, 2005) where a tiered approach focused on a comprehensive
risk assessment related to the introduced transgenes and direct exposure to their
expressed products and the indirect exposure through feeding patterns and accu-
mulation of expressed gene products in the release environment. No significant dif-
ferences were observed between conventional and transgenic fields and the s tudies
indicated that Bt cotton cultivation had an overall beneficial effect on biodiversity
as compared to regular applications of insecticides (Pray et al. 2002; Gepts 2004).
10 C. Emani
The identification of model plant species to study the genetic, biochemical and mo-
lecular basis of plant biodiversity is still not fully realized. Plant molecular biol-
ogy has christened Arabidopsis thaliana as a model system due to its complete
genomic sequence in the public domain, easy transformation protocols, short gen-
eration times, availability of expressed sequence tags (EST), microarray and pro-
teomics data, and a large set of well-characterized mutants as exemplified by the
Arabidopsis information resource (TAIR) database (http://www.arabidopsis.org).
Efforts geared towards identifying model plant systems by utilizing the molecular
and genetic data available on TAIR through bioinformatic analyses can be a good
starting point for researchers. The successful completion of complete genomes of
rice (Yu etal. 2002; Goff etal. 2002) and sorghum (Paterson etal. 2009) and more
recently banana (DHont etal. 2012) opens the doors for analyses of specific genes
as illustrated by studies made in Arabidopsis (Denby and Gehring 2005). A com-
prehensive database for model experimental plants among edible crops, forest spe-
cies, pharmaceutically important plants and biofuel crops aimed at data related to
transformation protocols, ESTs, microarray, experimental mutants, transcriptome
and proteome data in line with the TAIR database will be an effective resource for
breeders aiming to develop novel plants to preserve biodiversity.
genes with crucial molecular functions related to preserving and enhancing plant
biodiversity can then be evaluated for their biotechnological potential by geneti-
cally engineering them into popular cultivars and forest species.
1.5Conclusion
Charles Darwin in his monumental work The Origin of Species, focused on bio-
diversity to unravel the biological mechanics behind the rich variety of life forms
on our planet (Darwin 1859). Darwin attributed the evolution of diverse species
on earth to the ability of plants and animals adapted to their environment to breed
and pass on their characteristics to their offspring. In the revolutionary conclusion
to his classic theory, Darwin reflected on the crucial principle of the importance of
relationships between species and contemplated an entangled bank where various
life forms live in unison. This unraveled the fact that no species including our own
Homo sapiens can exist in isolation from other living things. Every species on earth
is dependent on natural processes for its own survival and in doing so contributes to
the natural balance of the environment that translates into the very survival of our
planet. We as human beings can thus be the agents of change to the preservation as
well as degradation of the rich biodiversity on our planet. With the ever growing
field of plant biotechnology that has a diverse array of technological applications
to choose from, a planned contextual strategy to best utilize the available state of
art techniques will richly benefit future researchers and their studies. The planned
research strategies will help fulfill our existence as a human race in being the very
agents of change that are responsibly preserving and enhancing the rich plant biodi-
versity to benefit planet earth, while systematically exploiting its resources to better
our lives.
12 C. Emani
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1 Transgenic Crops to Preserve Biodiversity 15
Abstract Hunger and malnutrition are flammable pertinent issues that hinder prog-
ress of a nation and become an increasing risk. Biotechnology and food security
have very good relationship both in the present and the future, concurrently embrac-
ing technology that offer new opportunities with increase crop and animal produc-
tion. Additionally, they offer capacity building, collaboration, research and ensure
sustenance. There is the need to engage and address exploration of new techniques
and encourage various scientific and community debates with the support of respec-
tive governments. The way forward is to review biotechnology tools including bio-
safety processes, policies and proper implementation to sustain biodiversity.
KeywordsBiotechnology Environmental safety Africa Food security
Biodiversity Genetically modified organisms Biosafety
2.1Introduction
West Africa, East Africa Central Africa and Southern Africa form the Sub Saha-
ran region of the African continent. The majority of Agricultural practices in this
region are characteristic of typical developing region where agriculture is still an
economic backbone. The agriculture economy employs about 60% of the work-
force and contributes an average of 30% of gross domestic product (USAID 2003).
Agricultural growth rates for SSA declined in the 2000s and food insecurity is still
a concern, as the prevalence of malnourishment has only dropped from 3430%
G.D.Arthur()
Mangosuthu University of Technology, Jacobs,
P. O. Box 12363, 4026 Durban, KwaZulu-Natal, South Africa
e-mail: Georgina@mut.ac.za
K.S.Yobo
Discipline of Plant Pathology, University of KwaZulu-Natal,
Private Bag X01, Scottsville, 3209 Pietermaritzburg, South Africa
Biotechnology is not new to mankind and has been around for thousands of years in
which mankind has been cross breeding and manipulating living organisms to meet
his own dietary and industrial needs. Food fermentation for example is evidence
2 Genetically Modified Crops in Africa 19
of one of the oldest known uses of biotechnology (Campbell-Platt 1994). The bak-
ing of yeast-leavened and sour dough breads also represents one of the oldest bio-
technical processes, together with the brewing of beer, wine, and the production
of yoghurt and cheese (Fleet 2007). These forms of biotechnology are labelled as
traditional.
Current process of biotechnology in its widest sense makes use of the improve-
ment of cereal grains and starter cultures by recombinant DNA technology, through
the use of enzymes as processing aids, to application of the most advanced batch
and continuous fermentation technologies (Linko etal. 1997). Modern methods of
biotechnology for the purpose of altering or modifying the genes of organisms in-
clude; Red biotechnology, which involves the medical processes, white Biotechnol-
ogy which is also known as Gray Biotechnology, which is used for the industrial
processes, Green Biotechnology which involves the processes and development of
pest-resistant crops and disease resistant animals, and finally, Blue Biotechnology
which is used for marine and aquatic processes (DaSilva 2004). Biotechnology also
includes the application of a wide variety of biological, biochemical, bioengineer-
ing, genetic, microbiological and control techniques. Undeniable evidence in the
form of vast bodies of scientifically proven literature demonstrates that biotech-
nological tools such as tissue culture, genetic engineering and molecular breeding
(marker-assisted selection) continue to provide promising opportunities for achiev-
ing greater food security while improving the quality of life (ISAAA 2009). Crop
genetic engineering process shown in Fig.2.1
The use of genetically modified (GM) crop technology in tackling food security
problems and poverty reduction in Africa (south of Sahel) has been debated upon
countless occasions. Although policy makers from developing countries have in-
creasingly considered GM crops as a potential tool for increasing agricultural pro-
ductivity, contentious debates over both the benefits and concerns of implementing
GM crops have hindered its implementation. There are currently 29 countries in
the world that are cultivating GM crops. Out of these 29 countries 19 are develop-
ing countries (James 2011). Out of the 19 developing countries, only three come
from the entire African continent. In Sub Saharan Africa only two countries have
approved commercial cultivation of GM crops namely South Africa and Burkina
Faso (Racovita et al. 2013). The development of GM crop varieties in Africa has
raised a wide range of new legal, ethical and economic questions in agriculture
(Azadi and Ho 2010). GM crops are promoted as the solution to the prevalent is-
sues of food security and low agricultural productivity in sub Saharan Africa and
other parts of the developing world. The promotion is however not restricted to the
developing countries but also the first world. On the one hand, Sub Saharan African
farmers are encouraged to accept and implement GM crops because of their higher
productivity, while organic farming is encouraged because of socio-economic and
20 G. D. Arthur and K. S. Yobo
Fig. 2.1 Crop genetic engineering includes: 1 DNA isolation, 2 gene cloning, 3 gene design, 4 trans-
formation, and 5 plant breeding. (Source: http://oregonstate.edu/orb/terms/genetic-engineering)
environmental considerations (Azadi and Ho 2010). The types of concerns that ac-
company GM crops are from the time they are planted right through to the time that
they are consumed. Concerns of GM crops are present in the divisions pertaining
to both environmental health and human health. They have been labelled countless
times as a potential risk to animal and human health because of their potential toxic-
ity and allergenicity (Racovita etal. 2013).
The following according to Malarkey (2003) are four concerns within food and
feed safety issues that GM crop cultivation bring about:
1. The inherent toxicity of the novel genes and their products.
2. The potential to express novel antigenic proteins or alter levels of existing protein
allergens.
3. The potential for unintended effects resulting from alterations of host metabolic
pathways or over expression of inherently toxic or pharmacologically active
substances.
4. The potential for nutrient composition in the new food differing significantly
from a conventional counterpart.
Other reasons opposing GM include public attitudes, Socio-economic factors and
intellectual property rights have also been raised (Racovita etal. 2013). There have
been instances where traditional beliefs and ethical concerns have played a role in
making the implementation of GM crops abominable. Coe (2014) noted that beliefs,
habits and rituals are attached to religion and culture and are so deeply rooted that
there is instant approval or disapproval of agribiotic products. Since the functioning
and the future of biotechnology rest on network of a setup, awareness and under-
standing of how biotechnology relates to these affiliations are imperative.
2 Genetically Modified Crops in Africa 21
Although the risks of genetically modified crops have sometimes been exaggerated
or misrepresented, they do have the potential to cause a variety of health problems
and environmental impacts (UCSUSA 2012). For instance, they may produce new
allergens and toxins, spread harmful traits to weeds and non-GE crops, or harm
animals that consume them (UCSUSA 2012). At least one major environmental
impact of genetic engineering has already reached critical proportions: overuse of
herbicide-tolerant GE crops has spurred an increase in herbicide use and an epi-
demic of herbicide-resistant superweeds, (UCSUSA 2012).
With the evidence of escalating crop pests that suppress staple crop yields both
on a subsistence and commercial scales, Sub Saharan Africa need crops that are
disease-resistant, can fend off insect predators, and can withstand severe environ-
mental conditions to produce larger crop yields (Pinstrup-Andersen and Schiler
2001). It must be acknowledged that the implementation of GM crops alone will not
solve the worlds food problem, but they may be a useful element towards the fight
against hunger. The contentious debates surrounding GM crops are a bottleneck to
the implementation and probably the main reason that SSA is still lagging behind in
accruing the benefits of this technology. Generally, people in developing countries
should have ready access to information about both the benefits and the risks of the
implementation of GM crops (Pinstrup-Andersen and Schiler 2001). There have
been many debates raising the concern as to whether the implementations of GM
crops are feasible from both environmental and health perspective. Anti-GM activ-
ists argued that, due to monopoly power, GM crops would result in input costs and
decrease diversity of seed choice, thereby forcing poorer farmers out and allowing
a form of uniform, corporate-capitalist agriculture to dominate. These risks would
be compounded, by potential threats to biodiversity from the spread of GM genetic
material, and consumers could be at risk from potentially unsafe foods. Pro-GM ad-
vocates argued, by contrast, that GM seeds would reduce costs for farmers in a way,
allowing rich and poor alike to benefit. By removing farmers from the burden of
purchasing pesticides, for example, both health and economic benefits would result.
No known health or environmental risks existed, they claimed, and, if governed
by a streamlined regulatory system, all would be well, and the benefits of a gene
revolution would be realized.
Some commentators have dismissed anti-GM mobilizations as merely copycat
responses by elite activists, using links with farmers organizations as a way of rais-
ing funds (Paarlberg 2001).
2.3.2Biosafety
advancement while preserving public health and the environment. Developing and/
or emerging countries often face major barriers to access biotechnologies and
biotechnology derived products as they frequently lack the institutional capaci-
ties and professional competence in exercising regulatory oversight. To address
this need, intensive biosafety capacity building is required. Different training ap-
proaches can be used to train individuals in biosafety ranging from long-term
leading to a postgraduate certificate or a Masters degree, to short term courses.
The UNIDO e-Biosafety program annually organized at the Marche Polytechnic
University (MPU) in Italy and Ghent University (UGhent) in Belgium since 2006
has identified that proper institutional capacities need to be in place for countries
to deal with the complex issues related to the adoption of GM-technology. It is
therefore important to continuously bring to the attention of governments, devel-
opmental agencies and international organizations, the value of biosafety capac-
ity development including training through formal degrees to encourage them to
mobilize resources for these projects. From October 2006 to 2012, 100 students
from 37 different countries participated in the course at the UGent and MPU
network nodes. More than half of the students came from Africa (58%), followed
by Europeans (23%). Only a minority came from Asia, Russia and Middle-East
(10%), Central and South America (7%) and North-America (2%). East African
countries have been well represented and more than one fifth of the participants
were Kenyans (Pertry etal. 2014).
Biosafety capacity building is a complex task and requires a multidisciplinary
approach, the main components being human resource development, institutional
and policy development for regulatory bodies and relevant research institutions,
to enable them efficiently and effectively use biotechnology products particularly
GM crops, microbes and/or their processed products. In the last decade, various
developmental agencies and donors, notably the Food and Agricultural Organiza-
tion of the United Nations (FAO), the Global Environment Facility (GEF) and
the United Nations Environment Programme (UNEP), have been supporting the
biosafety capacity building needs of developing/emerging countries through their
technical assistance programs (FAO 2009; Hull etal. 2010). The range of activi-
ties include: (i) the development of national policies and formulation of regula-
tions; (ii) GMO detection and monitoring including equipping of laboratories
and harmonizing protocols among countries; (iii) facilitating effective communi-
cation and public awareness and (iv) human resource development in biosafety
(Pertry etal. 2014). Figure2.2 shows the various biosafety is stages in African
countries.
In October 2002 relief effort took an unexpected twist, as the governments of Mala-
wi, Mozambique, Zambia and Zimbabwe rejected US food aid because of concerns
2 Genetically Modified Crops in Africa 23
Fig. 2.2 Biosafety and confined field trials (CFTs) for GM crops at various stages in Africa
over the inclusion of genetically modified maize. Zerbe (2004) argues that geneti-
cally modified maize transported to Southern Africa in the form of aid, is not an
initiative to end hunger in the region, but rather it was an initiative to expand market
access and control of transnational corporations. In South Africa herbicide tolerant
maize has been grown commercially since 2003, and in 2011, about 1millionha
out of total plantings of 2.71millionha used this trait (Brookes and Barfoot 2012).
From an economic perspective Sub-Saharan African farmers opposition to the im-
plementation of the cultivation of GM crops on their farm lands is understandable.
Implementing the cultivation of GM crops would also disqualify their participation
in certain European markets, or restrict them to providing only animal feed. Another
problem with the adoption of GM crops seeds is that the farmers ability to tap into
the potential benefits of GM seeds can be limited by institutional issues (Falck-
Zepeda etal. 2013).
24 G. D. Arthur and K. S. Yobo
Fig. 2.3 Factors influencing the adoption and development of agbiotech in sub-Saharan Africa
(Ezezika etal. 2012)
In brief Ezezika etal. (2012) spelt out the factors influencing agbiotech adoption
and development in sub-Saharan Africa as in Fig.2.3.
Table 2.1 Status and trends in plant biotechnology in Africa (Brink etal. 1998)
Region Country Area of research
North Africa Egypt Genetic engineering of potatoes, maize and tomatoes
Morocco Micropropagation of forest trees, date palms
Development of disease-free and stress tolerant plants
Molecular biology of date palms and cereals
Molecular markers
Field tests for transgenic tomato
Tunisia Abiotic stress tolerance and disease resistance
Genetic engineering of potatoes
Tissue culture of date palms, Prunus rootstocks and citrus
DNA markers for disease resistance
West Africa Burkina Faso Biological nitrogen fixation, production of legume inocu-
lants, fermented foods, medicinal plants
Cameroon Plant tissue culture of Theobroma cacao (cocoa tree), Hevea
brasiliensis (rubber tree), Coffea arabica (coffee tree),
Dioscorea spp (yam) and Xanthosoma mafutta (cocoyam)
Use of in vitro culture for propagation of banana, oil-palm,
pineapple, cotton and tea
Cote dIvoire In vitro production of coconut palm (Cocos nucifera) and
yam
Virus-free micropropagation of egg-plant (Solanum spp)
Production of rhizobial-based biofertilizers
Gabon Large-scale production of virus-free banana, plantain and
cassava plantlets
Ghana Micropropagation of cassava, banana/plantain, yam, pine-
apple and cocoa
Polymerase Chain Reaction (PCR) facility for virus
diagnostics
Nigeria Micropropagation cassava, yam and banana, ginger
Long term conservation of cassava, yam and banana, and
medicinal plants
Embryo rescue for yam
Transformation and regeneration of cowpea, yam, cassava
and Banana
Genetic engineering of cowpea for virus and insect resistance
Marker assisted selection of maize and cassava
DNA fingerprinting of cassava, yams, banana, pests, and
microbial pathogens
Genome linkage maps for cowpeas, cassava, yams and
banana
Human resource development through group training, degree
related training, fellowships and networking
2 Genetically Modified Crops in Africa 27
would be better served if the genetic modification debates were less polarized and
were focused on the potential for complementarity of GM technologies within a
diversified farming system framework (Bennett etal. 2013).
quality of life for millions of people in Sub Saharan Africa, in Africa as a whole,
and also in developing countries collectively. In Africa preschool children that are
affected by vitamin A deficiency is estimated to be 250million. More than 1million
deaths in children under the age of 5 years old can be prevented through vitamin A
rich crops such as Pro-vitamin-A- enriched Golden Rice (Whitty etal. 2013). Fur-
thermore genetic engineering can help to increase the yields of staple crops in Sub
Saharan Africa. Cow pea is one of those crops and there are 200million consumers
of cowpea on the African continent. The Maroca Pod Bearer Resistant Cowpea has
the potential of increasing yields by 70% while the use of insecticide spray will
experience a 67% reduction use. Another Staple diet of Sub Saharan dwellers is
maize which 300million Africans depend on. They suffer yield loss ranging be-
tween 1025% due to drought. Genetically modified Water efficient maize has
capabilities to increase maize yields by 2030% (Whitty etal. 2013).
Another staple crop for millions in Sub-Saharan Africa is the cassava crop. Cas-
sava (Manihot esculenta Crantz) is an important source of calories for more than
a billion people in developing countries, and its potential industrial use for starch
and bioethanol in the tropics is increasingly being recognized (Chetty etal. 2013).
Global production of cassava is about 256milliont, out of which 146milliont are
produced in Africa (FAOSTAT 2012) Despite the importance of cassava there are
traits that need improvement, such as pest and disease susceptibilities, accumulation
of cyanogens, and post-harvest physiological deterioration (Ceballos etal. 2004;
Sayre etal. 2011). Genetic transformation of cassava offers great potential for cas-
sava improvement because it could help to address two foremost problematic viral
diseases that are known to attack cassava crops, namely the cassava mosaic disease
and the brown streak disease (Liu etal. 2011; Whitty etal. 2013). The Cassava mo-
saic disease (CMD), caused by several circular ss DNA begomoviruses (belonging
to the Family: Geminiviridae), is the most important disease affecting cassava pro-
duction on the African continent (Chetty etal. 2013). The cassava mosaic disease
stunts the growth of these crops while the brown streak disease attacks the roots of
the cassava causing it to rot. These diseases are known to affect the cassava crops
especially in the East African region of the continent (Whitty etal. 2013). In Ugan-
da and Kenya, an international team of researchers are currently investigating the
possibilities of addressing this issues using GM technology. The importance of food
composition in safety assessments of GM food is described for cassava that natu-
rally contains significantly high levels of cyanogenic glycoside toxicants in roots
and leaves. The assessment of the safety of GM cassava would logically require
comparison with a non-GM crop with a proven history of safe use. Although acute
and chronic toxicity benchmark values for human have been determined, intake
data are scarce. In considering the nutritional values for cassava cyanogen glyco-
sides residues in food should be a priority topic for research. Successful application
of transgenic technologies in cassava will depend not only on technical advances,
but also on successful transfer of knowledge, tools and expertise to the countries
in which cassava has an important socioeconomic role (Nyaboga etal. 2013). In
Mozambique and Uganda orange sweet potatoes being rich in vitamin A has been
introduced into some sectors of the populations (Whitty etal. 2013). Apart from the
32 G. D. Arthur and K. S. Yobo
2.7Conclusion
Africa is a continent that is plagued with sad cases of hunger and poverty. The
severity of threat to environmental and food security is becoming more apparent.
These forms of security are closely intertwined since food production is highly sen-
sitive to environmental conditions and conversion of natural land for agriculture is
a major cause of the deterioration of earths life support systems. Some developing
countries are already benefiting and should continue to benefit significantly from
advances in plant biotechnology. Insect-protected cotton containing a natural insec-
ticide protein from Bacillus thuringiensis (Bt cotton) is providing millions of farm-
ers with increased yields, reduced insecticide costs and fewer health risks. Other
orphan crops such as rice, tropical maize, wheat, sorghum, millet, banana, potato,
34 G. D. Arthur and K. S. Yobo
sweet potato and oil seed can also benefit from GM technology in developing coun-
tries (Adendle 2011). Genetic engineering has made it possible to produce crops
that are improved in terms of their yield traits and their qualities (Uzogara 2000).
The African continent, more than any other, urgently needs agricultural biotechnol-
ogy, including transgenic crops, to improve food production (Wambugu 1999). This
is because the priority of Africa is to feed her people with safe foods and to sustain
agricultural production and the environment. SSA through the formalisation of the
cultivation of GM crops can make up for the green revolution it lost out on; the
same green revolution that helped in making Asia and Latin America self-sufficient
in food production. The nature of the GM debate with a large opposition from SSA
population indicates that there is a need to engage the community with researchers,
policy makers and local communities in this discourse. In April 2007, biosafety and
biotechnology scientists, regulators, educators, and communicators from Kenya,
Tanzania, and Uganda, met to examine the status and needs of biosafety training
and educational programs in East Africa. Workshop participants emphasized the
importance of developing biosafety capacity within their countries and regions. Key
recommendations included identification of key biosafety curricular components for
university students; collaboration among institutions and countries; development of
informational materials for non-academic stakeholders and media; and organization
of study tours for decision makers. It was emphasized that biosafety knowledge is
important for all aspects of environmental health, food safety, human and animal
hygiene. Thus, development of biosafety expertise, policies and procedures can be
a stepping stone to facilitate improved biosafety for all aspects of society and the
environment. Basic biotechnology principles integrated into secondary and primary
school curricula with the latter involving cartoons. If children could be made to ap-
preciate astronautical events when they have not been to space, then biotechnology
of the crops they handle and eat daily will not be a problem to appreciate. Frequent
workshops on biotechnology and biodiversity career options involving profession-
als in the field should be organised. Database of Biotechnology professionals and
Network list of professional societies and organizations should be made available.
Environmentalists and stakeholders from anti-GM crops groups express their
concerns about the commercialization of GMOs, stating that the introduction of
agbiotech will indeed pose a threat to the survival of indigenous crops and affect
biodiversity (Ezezika etal. 2012). Food insecurity exacerbated by high and unaf-
fordable food prices is a formidable challenge to which biotech crops can contribute
but are not a panacea (James 2013) Despite public concerns, it is regarded that the
main increase in agricultural productivity will be achieved through the direct use of
genetic improvement and biotechnology (Villalabos 1995). If biosafety measures
are rigorously adhered to and trans-boundary movements of GE products are moni-
tored, Africa will reduce hunger and boost the health of its people.
Acknowledgements We would like to acknowledge the immense valuable input from Mr Kofi
Gyan Quartey and very grateful for the contributions made by Mrs Lister Dube and Miss Awo
Quartey.
2 Genetically Modified Crops in Africa 35
References
3.1Introduction
E.M. Badea()
Institute of Biochemistry of the Romanian Academy, Splaiul Independenei 296, Sector 6,
Bucharest 060031, Romania
e-mail: elenamarcelabadea@gmail.com
I.P. Otiman
Institute of Agricultural Economics, Romanian Academy,
Calea 13 Septembrie 13, Casa Academiei, Sector 5, Bucharest 050711, Romania
e-mail: otiman@acad.ro
micro-organisms; their deliberate release into the environment for purposes other
than being launched on the market; their placement on the market either as GMOs
or in products derived there from; and imports/exports of GMOs as or in products.
Among the first biotech crops approved for commercial cultivation under the
biotech GO 49/2000 were glyphosate-resistant soybeans (GRS) and the Superior
New Leaf Potato (Badea and Pamfil 2009).
To date, the Romanian biotech legislation is harmonized with the EU legislation.
Romania has transposed Directive 2001/18 into its regulatory framework by adopt-
ing Law No. 247/2009 regulating activities involving the deliberate release and
placement on the market of GMOs. The Cartagena Protocol on Biosafety came into
force on September 28, 2003.
Romania is one of the few European countries with favourable conditions for
soybean production. GRS were commercially-grown in this country beginning with
1999, accounting for 68% of all the soybean area planted in 2006. Farmers using
GRS indicated that the plant was the most profitable arable crop grown in Romania,
with gains derived from higher yields and improved quality of seeds, combined
with lower production costs (Brookes 2005). In 2006, Romania was among the
eight countries that grew the crop worldwide. In 2007, as a Member State of the EU,
it banned the cultivation of the crop, despite the fact that growing HT soybeans had
generated a substantially higher net farm income per hectare in this country than in
any other country using the technology (Brookes 2005). As a result, in only 2 years,
the area planted to soybeans shrunk by 70% and Romania became a net importer
of plant protein, just like the European Union (Dinu etal. 2011; Balaj etal. 2012a).
According to the Romanian biotech law GO No. 49/2000, post-marketing moni-
toring aims to confirm the conclusions of the environmental risk assessment and to
identify unpredictable adverse effects. In Romania, monitoring of GRS was carried
out by universities and research institutes, in close cooperation with the private
sectors having received licenses in that respect. In order to confirm some conclu-
sions on the environmental risk assessments submitted by the applicants, field ex-
periments (case-specific monitoring) were undertaken to evaluate the impact of RR
versus conventional technology on soil microorganisms, the arthropod fauna, and
weed populations. Case-specific monitoring activities were carried out in 2004 on a
monoculture (20022004) soybean experimental field at Moara Domneasc Didac-
tic Experimental Station, focusing on the structure and make-up of the weed popu-
lation, the invertebrate fauna population, and the heterotrophic bacteria and micro-
scopic fungi in the RR and conventional plant rhizospheres (Badea etal. 2006).
An overall monitoring took place in 20022004, where farmers cultivating RR
soybeans were asked about the plant behaviour in the new agro-ecosystems. Re-
sponses were regarded as indicators for soybean behaviour in terms of the plants
invasiveness, persistence, rate and/or way of reproduction, dissemination, surviv-
ability, etc. (Badea etal. 2004).
The present paper presents soybean data in general as well as data regarding the
agronomic and environmental impact of glyphosate-tolerant soybean cultivation in
3 Agriculture and Environmental Impacts of Glyphosate-Tolerant 41
Table 3.1 The 20 highest soybean-producing countries in 2012. (Source: FAOSTAT 2012)
Country Area harvested (ha) Production (tons)
1. United States of America 30,798,530 82,054,800
2. Brazil 24,937,814 65,700,605
3. Argentina 19,350,000 51,500,000
4. India 10,800,000 11,500,000
5. China 6,750,080 12,800,145
6. Paraguay 3,000,000 8,350,000
7. Canada 1,668,400 4,870,160
8. Ukraine 1,412,400 2,410,200
9. Russian Federation 1,375,200 1,806,203
10. Uruguay 1,130,000 3,000,000
11. Bolivia 1,090,000 2,400,000
12. Indonesia 567,871 851,647
13. South Africa 500,000 850,000
14. Nigeria 440,000 450,000
15. Democratic Peoples Republic of Korea 300,000 350,000
16. Serbia 162,714 280,638
17. Italy 153,000 422,100
18. Vietnam 120,751 175,295
19. Thailand 100,000 180,000
20. Romania 77,927 104,330
Romania. We also aim to evaluate the impact that the potential shift from conven-
tional to GRS crops would bring to farming systems, through a comparison between
the environmental impacts of herbicide treatments used in GM and non-GM crops,
respectively. For a study case, we have looked at the technology used for soybeans
on one of the largest farms in Europe, located on the Great Brila Island (Buzdugan
2011; Balaj etal. 2012b).
Soybeans are one of the worlds most important and fastest expanding crops, which
contribute considerably to worldwide human nutrition. The main world soybean
producers are: the U.S., Brazil, Argentina, India, and China, countries where the
harvested area exceeded 10millionha in 2012 (Table3.1).
Since 1996the first year of global marketing for the biotech cropsGRS has been
the most grown engineered crop. In 2012, global GRS area stood at 80.7millionha,
representing 81% of the world soybean area (James 2012). The crop has been com-
mercially grown in the U.S., Argentina, Brazil, Paraguay, Canada, Uruguay, Boliv-
42 E. M. Badea and I. P. Otiman
Table 3.2 Countries in which commercial cultivation of GRS is/was approved. (Source: James
2012)
Country 1996 1997, 1999, 2001, 2003, 2005, 2007, 20092012
1998 2000 2002 2004 2006 2008
USA x x x x x x x x
Canada x x x x x x x
Brazil x x x x
Argentina x x x x x x x x
Mexico x x x x x x
South Africa x x x x x
Romania x x x x
Paraguay x x x
Uruguay x x x x x
Costa Rica x
Chile x
Bolivia x
ia, South Africa, Mexico, Chile, and Costa Rica (Table3.2). With glyphosate as the
primary herbicide for their soybean crops, growers achieved greater flexibility in
timing herbicide applications, as well as simplicity, with less confusion of herbicide
mixes and rates, an effective control of perennial, and other problem, weeds, excel-
lent crop safety, and economical weed control. For these reasons, GRS has been
adopted faster than any other new technology in the history of agriculture (Sankula
etal. 2005). Since 2011, new soybean events have been approved for cultivation
(Table3.4).
The data in Table3.2 indicate that Romania is the only country where cultivation
of GM soybeans has been prohibited. The event took place after several years of
steady increase in its adoption rate (Otiman etal. 2008).
3.2.2Europe
In Europe, the main soybean producers are Ukraine and the Russian Federation
(Table3.3). In Eastern Europe, soybeans are grown in northern Serbia (Vojvodina)
and various regions of Romania: west (the Banat), south (the Danube Plain), and
northeast (FAOSTAT 2012).
In the EU, soybean production is fairly limited, mainly because of the less fa-
vourable climate. In 2006 and 2012, the major EU soybean growers were Romania,
Italy, and Serbia, and Serbia and Italy, respectively (Table3.3).
With a large protein deficit, Europe is highly dependent on soybean imports. In
2012, the EU imported an overall 32millionMT of soybeans and soybean meal
($16.5billion), of which soybean meal represented 61%. Europes main suppli-
ers are the large biotech-producing countries: Brazil (15millionMT), Argentina
(8.6millionMT), and the United States (2.8millionMT)(Eurostat 2012).
3 Agriculture and Environmental Impacts of Glyphosate-Tolerant 43
Roundup Ready soybeans (the 40-3-2 event) are approved for marketing in the
EU (Commission Decision 96/281/EC of April 3, 1996, amended in 2012). The
decision allows for the imports of seed into the EU for industrial processing into
non-viable products, including animal feeds, food, and any other products using
soybean fractions. In the EU, new soybean events have been approved for food and
feed for direct use, or processing (Table3.4).
Except for RR soybeanswhich Romania grew between 1999 and 2006no
other herbicide-tolerant plant (sugar beet, maize, cotton, rapeseed) has been includ-
ed among the European commercial crops. This is why most of the studies done so
far have focused on potential changes in herbicide applications and the economic
and environmental impacts of the possible adoption of herbicide-tolerant plants
(Kleter etal. 2008; Devos etal. 2008; Dewar 2009).
3.2.3Romania
Table 3.4 Herbicide-tolerant soybean events and types of approvals. (Source: EU approval
database)
Event Event code/ GM Traits Type of approval
trade name EU Other countries
1. GTS 40-3-2 MON-432-6 Tolerance to Food and feed: Cultivation
(40-3-2) Roundup glyphosate direct use or (Table3.2)
Ready processing
(2005, amended
in 2012)
2. MON87705 MON-8775-6 Tolerance to Cultivation
Vistive Gold glyphosate; U.S., Canada
modified oil/ (2011)
fatty acid
3. MON87701 x MON-8771-2 Tolerance to Food and feed: Cultivation
MON89788 x MON- glyphosate; direct use or Brazil (2010);
89788-1 resistance to processing Argentina, Uru-
Intacta Lepidopteran (2012) guay (2012);
Roundup insects Paraguay
Ready 2 Pro (2013)
4. MON87708 MON-8778-9 Tolerance to Cultivation:
Genuity glyphosate and Canada (2012)
Roundup Dicamba
Ready 2
Xtend
5. MON89788 MON-89788-1 Tolerance to Food and feed: Cultivation
Genuity glyphosate direct use or Canada, USA
Roundup Ready processing (2007); Costa
2 Yield (2008) Rica (2008)
6. A2704-12 ACS- Tolerance to Food and feed: USA 1996,
GM5-3 glyphosate direct use or Canada 1999,
Liberty Link processing Brazil 2010,
(2008) Argentina 2011,
Uruguay 2012
7. A5547-127 ACS- Tolerance to Food and feed:
GM6-4 glyphosate direct use or
Liberty Link processing
(2012)
8. DP356043 DP-35643-5 Tolerance to Food and feed:
Optimum glyphosate; direct use or
GAT tolerance to processing
Sulfonylurea (2012)
GRS Cultivation in Romania Romania is one of the few European countries that
has favourable conditions for soybean production. Commercial cultivation of GR
soybeans was approved in 1999. In 2006, Romania represented one of the nine
countries in the world growing this GM crop (James 2007).
Beginning with 2000, the countrys RR area expanded constantly, reaching a
peak in 2006 (the eighth year of using the technology) of 137,000ha (Table3.6)
(Otiman etal. 2008). The same year witnessed large GR soybean areas (Fig.3.1),
3 Agriculture and Environmental Impacts of Glyphosate-Tolerant 45
particularly in regions most favourable to the crop: the Danube Plain (the counties
of Clrai, Ialomia, Brila, and Galai), Dobrogea (Constana county), and Banat
(Timi and Arad counties).
Fourteen varieties were registered in the Romanian Official Catalogue (of which
three by Pioneer and eleven by Monsanto). Six varieties were marketed in 2006
(one by Pioneer and five by Monsanto). Romanias variable soil and climate condi-
tionsgoing from average temperate temperatures and heavy rainfalls inside the
Carpathian arch to low rainfalls in the southalong with the countrys specific
seasonal pattern, with significant differences between the seasons meant growing
different maturity group varieties. Low temperatures in winter destroy potential
46 E. M. Badea and I. P. Otiman
volunteer plants. The most favourable climate conditions for soybeans are in the
Danube Plain.
Based on the herbicide use data for 20002003, Brookes and Barfoot (2005) calcu-
lated that Romanias adoption of RR soybeans resulted in a small net increase in the
amount of active ingredients applied, but a net reduction in the EIQ/ha. However,
the authors do not deem the results conclusive, because the rates of herbicides ap-
plied to conventional crops during the same interval were below optimal levels.
More specifically: during the analysed interval, recommended herbicide programs
were often applied in part, if not completely ignored. Not least, information sources
were not always reliable.
The results of a survey conducted at the end of 2006 on a sample of 160 soybean
growers (running commercial farms equipped with an adequate input and technol-
ogy mix) in 14 key counties indicated that conventional soybeans received on aver-
age 2.3 herbicide treatments/crop year, with about 10% of growers applying four
treatments. GRS received on average 1.63 treatments/no more than two (Otiman
etal. 2008).
The most efficient and used weed management scheme for GRS was the two-
step treatment of 2L of Roundup per hectare each time, after emergence, depending
on the stage of development of the first weed generation and the evolution of the
second weed generation (Buzdugan 2011).
Weed resistance is probably the highest risk related to growing GRS. GR weeds
have already made their appearance in various U.S. states (totalling 13 weeds in
mid-2011), as well as in more than a dozen countries throughout the world (totalling
21 weeds in mid-2011; Bonny 2011).
In Brazil, five weed species: hairy fleabane (Conyza bonariensis), Canadian
fleabane (Conyza canadensis), Italian ryegrass (Lolium multiflorum), wild poinset-
tia (Euphorbia heterophylla), and sourgrass (Digitaria insularis) have developed
resistance to glyphosate in GRS and are potentially major problems. Glyphosate-
resistant biotypes of Johnson grass (Sorghum halepense) L. and Italian ryegrass
(Lolium multiflorum) have developed in GRS crops in Argentina, as well as one of
sourgrass (Digitaris insularis), in Paraguay (Via-Aiub etal. 2008).
Currently, some of the above weed species can be found in Europe, all with
widespread distribution (bindweed/Convolvulus arvensis, ryegrass/Lolium spp.,
amaranth/Amaranthus spp., Canadian fleabane/Conyza canadensis, Italian
ryegrass/Lolium multiflorum, morning glory/Ipomoea spp., and fat hen/Chenopodium
album). The North American weed species Conyza canadensis was introduced to
Europe in the seventeenth century. Almost nothing exists in the literature about
naturally glyphosate-resistant European weed biotypes or about European weed
biotypes that can potentially develop glyphosate resistance (Sandermann 2006).
No GR weeds were reported in Romania during the 8 years in which the country
used the RR technology.
48 E. M. Badea and I. P. Otiman
Most Romanian farmers indicated that their economic efficiency was mainly due
to their having adopted the GR technology (Badea and Otiman 2006; Brookes and
Barfoot 2005, 2010).
In an attempt to intensify the pace of bringing Romanias biotech regulatory
capacity in line with the acquis communautaire, the Romanian authorities were, as
of January 2006, already highly committed to discourage biotech plantings. There-
fore, they went on to announce a subsidy program for conventional soybeans for
the year. Despite the announcement, the hectarage of transgenic soybeans went up
to 137,000 (of a total 199,000). Thus for a second year in a row, with a production
of almost 350,000 t, Romania started shipping its exportable surplus of soybeans,
while its imports of soybean meal went down substantially (Dinu etal. 2011).
With no more access to RR technology, soybean area started to decline in 2007,
dropping to 109,000ha, and further down to no more than 46,000 in 2008 (FAO-
STAT 2012). This was the equivalent of a 70% reduction over the recent years.
In 2006, the national level of revenues obtained by farmers who grew glypho-
sate-tolerant soybeans totalled $7.6million. During 19992006, nominal farm in-
come rose by $44.6million. In 2006, GM soybean production growth equalled a
9% increase at the national level. During the 8 years in which the technology was
used, producers recorded an annual average production increase of 10.1%. In 2006,
the combined impact of the higher crops, improved quality, and lower production
costs upon farm incomes meant a production increase of 9.3% (33,230t).
As against 2006, in 2007, Romania had to make additional hard currency ef-
fortsamounting to 60.5millionin order to compensate its deficit of soy beans
(worth over 30million) and meals (almost 20million), as well as for the soybean
oil it never exported (Dinu etal. 2011).
In 2008, the countrys trade balance value deficit for the three commodi-
ties increased. The difference between the 2008 and 2006 trade balances reached
117.353million, of which 58.084million were due to the countrys addi-
tional imports of soybean meal, 39.322million to the imports of soybeans, and
19.947million to the soybean oil (Dinu etal. 2011).
In real terms, the higher trade deficit meant an indirect loss to soybean farmers,
particularly to those who had to renounce GM soybeans. As a result of the ban,
most Romanian farmers gave up growing soybeans in general, viewing the newly-
established subsidy program not remunerative enough to compensate for the lack of
competitiveness of conventional varieties.
Dinu etal. (2011) appreciate that the ban on GM soybean cultivation had the
following effects:
A dramatic fall in the soybean areas and implicitly of soybean production, lead-
ing to problems in the raw material supply chain to processors;
A significant increase in the countrys soybean imports, as Romania re-became
a net importer of soybeans, having undergone an additional hard currency effort
of 60.5million in 2007 and of 117.353million in 2008;
3 Agriculture and Environmental Impacts of Glyphosate-Tolerant 49
The risk analysis of GRS cultivation in Romania is based on the following main
assertions:
Soybeans are not sexually-compatible with any native or introduced wild plant
species present in Europe (OECD 2001);
Soybeans are a self-pollinated species, which is commercially propagated by
seed, cross-pollination incidence representing less than 1%, as a rule (OECD
2001);
50 E. M. Badea and I. P. Otiman
Soybeans cannot survive without human assistance and are unable to survive as
a weed;
Soybeans have few weedy tendencies, since they share few of the traits that are
specific to genuine weeds (Baker 1974);
GM soybeans will be commercially grown in pre-existing agro-ecological envi-
ronments and the direct/indirect environmental effects of RR technology would
likely be largely similar to those resulting from conventional chemical spraying;
In Romania, Glycine max is not found outside cultivation and so far, no hy-
bridization between soybeans and other spontaneous/cultivated pulses has been
known to occur;
The biology of soybeans has been studied and it is well known in Romania (Ci-
ocrlan 1990; Popescu and Sanda 1998).
Wild soybean species are native to China, Korea, Japan, Taiwan, and former USSR.
In Romania, there are no wild relatives of soybeans, nor any other plants that are
sexually-compatible with soybeans. Therefore, the potential of gene flow from cul-
tivated soybeans to other plants is virtually impossible.
Soybeans are almost entirely self-pollinated, out-crossing levels averaging 1%
(OECD 2001). As result, implementing field coexistence measures can be relatively
easy. According to Yoshimura etal. (2006), the greatest distances between the re-
ceptors (conventional soybeansGlycine max (L.) Merr.) and an adjacent pollen
source (namely a GM glyphosate-tolerant soybean crop) at which out-crossing was
observed were 7m in 2001, 2.8m in 2002, and 3.5m in 2004. Results regarding
airborne pollen density measured during the flowering period indicated that the
possibility of out-crossing by wind was minimal. In field conditions in Brazil, out-
crossing of conventional crops by adjacent GM soybean fields is minimal if the
fields are situated more than 10m apart (Bindraban etal 2009).
The soybean plant is not weedy in character. Soybeans possess few of the char-
acteristics of plants that are weeds (Baker 1974). Soybeans are not an overwinter-
ing crop: they are not frost-tolerant and do not survive freezing winter conditions
(OECD 2001). In Romania, Glycine max is not found outside cultivation (Ciocrlan
1990; Popescu and Sanda 1998). Monitoring activities undertaken in Romania since
1999 during the first years of cultivation confirmed the conclusion of Environmen-
tal Risk Assessment submitted by the producer: genetic modification did not en-
hance the weediness and invasiveness of herbicide-tolerant soybeans (Badea etal.
2004, 2006).
3.4.2Monitoring Results
Table 3.8 Environmental impact of some active ingredients used in weed management in soybean
crops
Active ingredient Total EIQ (kg Farm worker EI Consumer EI Environmental
a.i./ha) EI
Bentazon 18.7 16 9 31
Fluazihop-P butyl 28.7 10.6 3.3 72.1
Glyphosate 15.3 8 3 30
Imazamox 19.5 8 8 42.5
Metribuzin 28.4 8 8 69.1
Metsulfuron-metyl 16.7 8 8 33
Quizalofop-p-tefuril 13.3 18 10 12
Quizalofop-p-etyl 22.1 10.6 3.3 52.4
Trifluralin 18.8 9 5.5 42
Tifensulphuron metyl 7.3 6 4 12
Table 3.9 Herbicide active ingredients and the amounts used in weed management in GM or
conventional soybean crops. (Source: Buzdugan 2011)
Product name Active ingredient Dosage g/kg or a.i./ha Total a.i. (kg/ha)
(a.i.) g/L L/ha; kg/ha
Conventional soybeans (2006)
Roundup Glyphosate 360g/L 4.5 1.620
Basagran F Bentazon 480g/L 2.5 1.200
Treflan 24 CE Trifluralin 240g/L 2.0 0.480
Surdone Metribuzin 700g/kg 0.7kg/ha 0.490
Galaxy Bentazon 360g/L 2.0 0.720+0.160
Acifluorfen 80g/L
Fusilade Super Fluazihop-P butyl 150g/L 2.0 0.300
Total 4.970
RR soybean (2006)
Roundup Glyphosate 360g/L 4.5L/ha 1.620
Total 1.620
In the herbicide treatment programs used for GM soybeans, not only the amount
of active ingredients is lower, but also the impact upon health and the environment
(Table3.11). The values of the three impact components and of the total impact are
considerably lower than in the herbicide treatment programs used for conventional
54 E. M. Badea and I. P. Otiman
soybeans. For instance, the impact upon farm workers is 77% lower, whereas the
impact upon consumers is 84% lower when RR technology is used, compared to
conventional weed control methods.
If conventional soybean crops were extended on 500,000ha and they were treat-
ed with the herbicides mentioned in Table3.10, the environment would receive
1,797,775kg of active ingredients. If the same area were planted to GM cultivars
and the associated herbicide treatments were applied, the environment would re-
ceive only 737,568kg of active ingredients, that is 59% less than for conventional
crops, while the environmental impact coefficient would be 67% lower. Regardless
of the size of the GM soybean area, the associated herbicide treatment program has
a significantly lower impact upon human health compared to the herbicide program
used in conventional soybeans (Table3.11).
According to Brookes and Barfoot (2005), who used the same methodology to
calculate the amounts of herbicides used in conventional as well as GM soybean
crops in 19992006, RR technology led to a small increase in the amount of active
ingredients used and a net reduction in the EIQ compared to the data recorded and
calculated in exclusively conventional crops.
Kleter etal. (2007) calculated the EIQ using data collected by the NCFAP (Na-
tional Centre for Food and Agricultural Policy) regarding the use of herbicides in
GM and conventional soybean crops in 2000, 2003, and 2004, in the United States.
In 2004, collected data and calculation results revealed a 25% reduction in the active
herbicide ingredients used as well as a lower impact thereof on the environment
down by 59% for GM soybeans, compared to their conventional counterparts. The
3 Agriculture and Environmental Impacts of Glyphosate-Tolerant 55
herbicide program applied on GM soybeans had a smaller impact upon farm work-
ers (down by 68%), consumers (down by 59%), and ecosystems (down by 55%)
compared to the herbicide program used for conventional soybeans (Table3.12).
The environmental impact of herbicide treatments used as a result of GM crop
adoption becomes even more evident if one considers the behaviour of the her-
bicides in the environment. For instance, in Argentina, once soybean RR technol-
ogy was adopted, the use of Toxicity Classes II and III herbicides was reduced by
83100%, while the use of less toxic herbicides in Class IV rose by 248%. In North
Carolina, U.S., GM cotton crops use herbicides whose potential of leakage is 25%
lower than herbicides applied on conventional cotton (Kleter etal. 2007).
Stewart etal. (2011) studied the efficiency, environmental impact, and profit-
ability of various herbicide treatments used on glyphosate-resistant soybeans, for
3 years, in three different locations in the United States. The study also determined
the herbicide treatment with the lowest selection pressure in terms of glyphosate-re-
sistant weed species. In order to reduce the selection pressure related to the glypho-
sate-resistant weed biotypes, to reduce the environmental impact, and increase prof-
itability, the authors recommend using a mix of herbicide agents with two modes of
action. The results indicated that the best alternative method to the two-step glypho-
sate treatment was a combination between glyphosate and Imazethapyr.
Dill etal. (2008) recommend herbicide programs that include a preemergent her-
bicideeither combined with a glyphosate-base product, or followed by a treatment
with glyphosate. Using agents with different modes of action is the proper strategy
of preventing resistance to herbicides in weed populations. The risk of weeds de-
veloping glyphosate resistance is much smaller in Romania than in other countries
growing RR soybeans. Taking into account the agronomic practices, the social de-
mand, and the need to ensure maximum efficiency, the best rotation is 4 years, using
six crops: wheat/rapeseed+barley/corn+sunflower/soybeans (Buzdugan 2011).
Conclusion: both the amount and the impact of herbicide agents applied to GM
plants would go down, compared to the alternative programs used for conventional
crops.
Soil Management and Conservation TillageBiotech herbicide-tolerant soy-
beans have enabled the adoption of conservation tillage in most countries that have
56 E. M. Badea and I. P. Otiman
adopted the technology (Cerdeira and Duke 2010). In Romania, farmers have not
adopted the low-till or no-till systems.
Worldwide, several studies have shown that both previous and potential effects
of glyphosatein terms of soil, water, and air, contaminationare minimal, com-
pared to the impact of herbicides that would be otherwise used and which glyphosate
herbicides replace when GRS are adopted. In the U.S. and Argentina, the advent of
glyphosate-resistant soybeans has led to a significant shift to low-, and no-, tillage
practices, thereby significantly reducing environmental degradation by agriculture
(Cerdeira etal. 2010; Cerdeira and Duke 2010). According to Cerdeira and Duke
(2010), both glyphosate and aminomethylphosphonate (AMPA)its degradation
productare considered to be much more toxicologically and environmentally be-
nign than most herbicides replaced by glyphosate.
3.5Conclusion
Romania is one of the few European countries with favourable conditions for soy-
bean production. GRS were grown commercially in this country beginning with
1999 and accounted for 68% (or 137,000ha) of the entire soybean area planted in
2006. In 2006, Romania stood among the eight countries that cultivated the crop
worldwide. Since 2007, after its accession to the European Unionwhich had ap-
proved the imports, but not the cultivation of RR soybeans on its territorythe Ro-
manian farmers access to the technology was banned, at a time where it had started
being used on increasing areas, because of its efficiency in controlling weeds at low
costs. Glyphosate-tolerant soybeans have been regarded by farmers as their most
profitable crop, since it enabled them to obtain higher yields, at lower costs. As a
result, in only 2 years, the area planted to soybeans shrunk by 70%, while Romania
became a net importer of plant protein, just like the European Union.
The results of a field case-specific monitoring conducted in order to evaluate the
impact of GR versus conventional technology on the arthropod fauna showed no
significant differences among the epigeal and beneficial insects typical of soybeans
(in terms of population size and/or composition). No obvious adverse effects on
the soil biota (heterotrophic bacteria and microscopic fungi) were identified upon
growing RR soybeans. No GR weeds were reported during the 8 years in which the
RR technology was used in Romania in two-steps treatments of 2L/ha each, after
emergence.
Romania was the only country in Europe that grew a GM, herbicide-tolerant
plantglyphosate-resistant soybeansfor commercial purposes. In 2006 alone,
the amount of herbicide agents applied per hectare for conventional soybeans
was considerably higher than what was used for GM soybeans. In 2006 alone, the
137,300ha planted to glyphosate-tolerant soybeans received 176,388kg of herbi-
cides less than on the 53,500ha planted to conventional soybeans. The environmen-
tal impact coefficient was about 70% lowerboth per hectare and for the entire
GM cultivar area (which represented 72% of the total soybean area that year).
3 Agriculture and Environmental Impacts of Glyphosate-Tolerant 57
If soybeans were planted on 500,000haalmost half of the area that lends it-
self well to this highly-important economic crop in Romaniathe total amounts
of herbicide ingredients applied would be 2,100,100kg in case only conventional
varieties were grown and 765,000kg in case only RR varieties were grown. Which
means that in case glyphosate-resistant, GM, soybeans were grown, the environ-
ment would be spared 1,335,000kg of herbicide active ingredients, and the herbi-
cide impact coefficient would be 67% lower than for conventional crops.
A legal framework is a necessary, but not sufficient condition to make the right
decisions in a certain field and at a certain time. Equally important is the enforce-
menton a scientifically sound basis and in good willof existing laws, enabling
their use by a certain social group and, at the end of the day, by the whole society. At
the same time, an excessive legal framework, enforced without responsibility, may
trigger dramatic socioeconomic consequences.
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weed resistance, and some economic issues. Case USA Sustain 3:13021322
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West, Canterbury. http://www.pgeconomics.co.uk/pdf/
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resistant soybean cultivation in South America. J Agric Food Chem 59(11):57995807
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Chapter 4
The Effects of Transgenic Crops on Non-target
Organisms
Chandrakanth Emani
4.1Introduction
Transgenic crops are the fruits of biotechnological research that enable plant ge-
netic engineers to ensure the stable integration, desired level of expression, and
predictable inheritance of numerous agriculturally important genes. The present
agricultural revolution sometimes referred to as gene revolution, which continues
the green revolution of the 1960s resulted in the cultivation of transgenic crops
expressing herbicide tolerant and insect resistant genes. Herbicide resistant trans-
genics, especially glyphosate-resistant soybean, cotton and corn have contributed
to effective weed management strategies in the respective crops (Green and
C.Emani()
Department of Biology, Western Kentucky University-Owensboro,
4821 New Hartford Road, Owensboro, KY 42303, USA
e-mail: chandrakanth.emani@wku.edu
The much discussed and dissected study that started a serious discussion on the ef-
fect of transgenic crops on non-target insect populations was the Losey etal. (1999)
report that showed the Bacillus thuringenesis (Bt) corn plants pollen on monarch
butterflies. In a laboratory assay, it was demonstrated that larvae of monarch butter-
fly, Danaus plexippus, reared on milkweed leaves, dusted with pollen from Bt corn,
ate less, had slow growth and high mortality compared to those reared on normal
corn pollen (Losey etal. 1999). It was argued that the dispersal of the corn pollen by
wind to almost 60m and its deposition on other plants might affect non-target insect
populations. Subsequent studies examined various flaws in the experimental set up
(Hodgson 1999) such as non-relatable field conditions in terms of pollen availabil-
ity and existence of milk weed plants in the real world, inappropriate experimental
controls and the absence of stringent quantification of the amount of pollen used in
the experiments. However, environmental groups and media seized the opportunity
to sensationalize the issue of transgenic crops being a threat to non-target insect
populations.
4 The Effects of Transgenic Crops on Non-target Organisms 61
Since transgenic plants, just like the regular crop species depend on pollinators for
their optimal reproduction, it is imperative to consider the effects of the expressed
transgenic products on the various pollinator insect species that are non-target
population. Recent studies showed no deleterious effects of transgenic herbicide-
tolerant or insect-tolerant on pollinators (Malone and Burgess 2009). The one Bt
toxin that was shown to have a potent effect on Hymenopteran insects was Cry5
(Garcia-Robles etal. 2001), but no Cry5-exprssing plants have been approved for
commercial cultivation. The more popular Cry1 toxin expressing plants have no
effect on pollinators such as honeybees (Ramirez-Romero etal. 2005; Rose etal.
62 C. Emani
The study of Losey etal. (1999) combined with the charismatic and iconic christen-
ing by over enthusiastic environmentalists and media of the lepidopteran monarch
butterfly triggered numerous studies on the effect of transgenic corn on monarch
butterfly populations. Later field studies showed that the risk to monarch butterfly in
terms of toxic levels of transgenic pollen is minimal simply due to the limited spatial
distribution of pollen (Pleasants etal. 2001) and the insignificant exposure of larva
during the pollen shed (Oberhauser etal. 2001). Studies in USA transgenic Bt corn
fields specific to effects on monarch butterfly larvae continuously exposed to the
transgenic crop during anthesis showed insignificant effects on mortality (Dively
etal. 2004), though laboratory studies continued to show reduction in feeding and
weight gain (Anderson etal. 2004; Prasifka etal. 2007). The differences in field re-
sults was later attributed to the fact that early larval instars are less exposed to Bt pol-
len drift as they feed on the upper third of milkweed plants that have lesser densities
of anthers and the larva tending to move on the underside of leaves avoiding contact
with anthers (Pleasants etal. 2001; Anderson etal. 2004; Jesse and Obrycki 2003).
4.8Conclusion
After the rich dividends reaped by the agricultural community through green revo-
lution, the commercial cultivation of transgenic crops is being seen as its successor
and this important scientific event is being christened as the gene revolution (Birch
and Wheatley 2005). Though the adoption of transgenic technology was one of the
fastest across the world (James 2009), the backlash in Europe as compared to USA
can be attributed to societal and political differences (Marshall 2009). An important
factor when examining issues such as the subject of this review puts the onus on
the scientific community to properly educate the general public and the political
decision makers, and when disseminating their findings take into consideration the
larger perspective of the agro-ecosystem instead of jumping to unwarranted conclu-
sions based on individual laboratory studies.
64 C. Emani
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4 The Effects of Transgenic Crops on Non-target Organisms 65
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Chapter 5
Agricultural Biotechnology for Health
and the Environment
5.1Introduction
S.O.Hansson()
Royal Institute of Technology and Swedish University of Agricultural Sciences,
Uppsala, Sweden
e-mail: soh@kth.se
that as yet remain largely unrealized. (I will not discuss the potential negative ef-
fects or detrimental uses.)
Biotechnology is a broad and somewhat vague term (IAASTD 2008). The Ox-
ford English Dictionary defines it as [t]he application of science and technology to
the utilization and improvement of living organisms for industrial and agricultural
production and (in later use) other biomedical applications. In what follows, the
focus will be what is usually called genetic engineering, i.e. the introduction of
exogenous genes in an organism.
If the beneficial potentials of biotechnology had been fully unfolded then there
would have been no need for a text like this. Due to the social circumstances sur-
rounding this technology, some of its major positive capacities have not yet been
realized. Many governments have withdrawn from plant breeding, leaving the field
to breeding companies who cannot be expected to have the same breeding goals as
publicly funded research. In most of the world GM crops are subject to regulations
that on the one hand make the introduction of new crops very expensive but on
the other hand provide innovators with strong intellectual property rights to their
products.
The current legal structure surrounding plant breeding is counterproductive in
the sense of thwarting the aims that it was set up to achieve. Due to the high en-
trance costs for most of the more innovative new cultivars, breeding activities are
focused on very few crops that have a large market in the industrialized economies,
and much too little work is being done on crops that are suitable for subsistence
farming in the third world. The same mechanism has led to a focus of breeding on
too few cultivars for each crop. This may lead to a loss of traditional cultivars that
could (if improved for example with resistance properties) have contributed sig-
nificantly to biodiversity. Agribusiness companies have strong incentives to make
it necessary for farmers to buy new seed every year. Current incentives also lead to
a one-sided focus on yield at the expense of other breeding goals that would help
solving environmental problems or developing more healthy food products.
It should be acknowledged that the current regulatory system was constructed to
deal with legitimate worries that the new technology might have unforeseen nega-
tive effects. However, in the four decades that have passed since the short voluntary
moratorium on recombinant DNA research, knowledge about the effects of genetic
modification has increased dramatically (Berg etal. 1974; Berg and Singer 1995).
Furthermore, since the first field trials with transgenic plants in 1986 (James and
Krattiger 1996) knowledge about the potential risks of crop biotechnology has risen
to a new and much higher level (Magaa-Gmez and Caldern de la Barca 2009).
The original worries concerning health and the environment were rather vague and
often referred to the possibility of unknown effects (Price 1978). They can now
be replaced by much more specified issues concerning potential hazards that can
be assessed and regulated. But current regulations are still based on an outdated
view of the uncertainties in plant breeding technologies. Today, with a dramatically
increased scientific understanding of genetics, and with GM crops being grown on
about 11% of the soil used for agriculture (James 2011) it is no longer tenable to
treat biotechnology as a step into the completely unknown or as a gamble that can
5 Agricultural Biotechnology for Health and the Environment 69
lead to the construction of a monster that will destroy us. (But that is how the tech-
nology is still often portrayed, see for instance Smith 2007.) We need a risk policy
that deals efficiently with todays uncertainties, rather than with those of the past.
Instead we have counterproductive policies that prevent the realization of some of
the major positive potentials of agricultural biotechnology to which I will now turn.
Diet is one of the major environmental factors that have influence on health. This
can be seen for instance from the Global Burden of Disease Study 2010 that sum-
marizes the health effects of a large number of risk factors on a global scale in terms
of the calculated global number of excess deaths that they give rise to (Lim etal.
2012).
Obesity or overweight: 3.37million deaths/year. Obesity is a diet-related disease
that is associated with substantially increased risk of a large number of diseases,
including diabetes, ischaemic heart disease, and several types of cancer. Obesity
is a growing problem not only in rich countries but also in countries such as In-
dia where at the same time malnutrition and food shortage is still a problem for
significant parts of the population (Sarkar etal. 2012).
Childhood underweight: 0.86million deaths/year. The number of children dying
from starvation has decreased substantially in the last two decades but is still
high in many countries, in particular in Subsaharan Africa.
Too much processed meat: 0.84million deaths/year. By processed meat is meant
smoked, cured and salted meat and meat with added chemical preservatives.
Diet high in red meat: 0.04million deaths/year.
Diet high in sugar-sweetened beverages: 0.30million deaths/year.
Diet high in trans fatty acids: 0.52million deaths/year. Trans fat is primarily
found in fast food and bakery products.
Diet high in salt: 3.10million deaths/year.
Insufficient breastfeeding: 0.55million deaths/year.
Maternal and infant iron deficiency: 0.12million deaths/year.
Vitamin A deficiency in children: 0.12million deaths/year. Lack of vitamin A is
also a major cause of blindness.
Zinc deficiency: 0.10million deaths/year.
Diet low in fruits: 4.90million deaths/year.
Diet low in vegetables: 1.80million deaths/year.
Diet low in whole grains: 1.73million deaths/year.
Diet low in fibre: 0.74million deaths/year.
Diet low in nuts and seeds: 2.47million deaths/year.
Diet low in milk: 0.10million deaths/year.
Diet low in calcium: 0.13million deaths/year.
Diet low in seafood omega-3 fatty acids: 1.39million deaths/year.
Diet low in polyunsaturated fatty acids: 0.53million deaths/year.
70 S. O. Hansson
Toxic Substances Some staple foods contain components that are toxic or cause food
intolerance or allergy. Biotechnology can often eliminate or reduce the contents of
the harmful substances. The content in potatoes of solanine has been substantially
reduced in this way (Chassy 2004), and so have allergenic components in wheat,
rice and peanuts (Lemaux 2008). The toxic cyanogenic glucosides in cassava
(a staple food for 250million sub-Saharan Africans) are another important target of
genetic change (Powell 2007).
Energy Density Reduction in total energy intake is essential to reduce the incidence
of obesity, type 2 diabetes, and concomitant diseases. Food intake is regulated by
satiety, and foodstuffs with low energy density produce satiety at a lower level of
energy intake than foodstuffs with high energy density. This is one of the reasons
why intake of food with high contents of water and fibres, such as vegetables and
fruit, are important components of diets for the treatment and prevention of over-
weight and obesity (Ello-Martin etal. 2007; Ledikwe etal. 2006; Rolls etal. 2004).
It is a plausible hypothesis that reducing the energy density of traditional major
foodstuffs with high energy density might potentially contribute to the prevention
of obesity and related diseases.
Not much would be gained by producing healthy food that few consumers actu-
ally eat. Therefore the contributions of agricultural biotechnology to improved diets
have to be parts of a larger strategy that leads to changes in what people actually eat.
Such a strategy will have to take agricultural and agroeconomical considerations as
well as consumer behaviour into account. New, healthier foodstuffs can either be
introduced universally or optionally. Universal introduction is in practice only pos-
sible if there is no consumer demand for the previous, less healthy alternative. There
does not seem to be any demand for bakery products with trans fats, and presumably
there would not be much demand for poisonous cassava if a non-poisonous alterna-
tive became available.
Optional introduction, i.e. introduction as an alternative alongside the older vari-
ant, can have significant health effects if there is a consumer demand for the health-
ier product. One example might be low-energy but highly satiating foodstuffs that
can be chosen by consumers wishing to prevent or reduce overweight.
In the period from 1700 to 1990, the global area of cropland is estimated to have
increased 5.5-fold, and the area of pasture land 6.6-fold. In 1990, 29% of the
worlds forest areas and 49% of its grasslands, steppe and savannas had been
transformed into agricultural land (Goldewijk 2001). Currently, agriculture is
responsible for about 80% of the worlds deforestation (UNFCCC 2007, p.81)
This dramatic reduction in wildland has a large part in the ongoing loss in species
that threatens to substantially decrease the planets biodiversity (Pimm etal. 1995;
Butchart etal. 2010). In addition, agriculture gives rise to a number of well-known
72 S. O. Hansson
5.4Conclusion
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Chapter 6
Next Generation Plant Biotechnology
M.R. Ahuja
Abstract Modern plant biotechnology began with the transfer of foreign chimeric
genes into plants. Initially recombinant genes were derived from bacteria, animals
and plants for gene transfer. Gene transfer was accomplished by Agrobacterium-
mediated or biolistic methods into the plant genome. The first wave of transgenic
plants that were monitored for transgene integration and expression, the second
wave transgenic plants carried economically important genes for herbicide toler-
ance, pest resistance, drought and salt tolerance, growth traits, and flowering con-
trol. Subsequently, a number of genetically modified crops with several useful
traits have been commercialized. Although relatively stable transgene expression
has been observed in a number of plant species, there were also unintended unsta-
ble events in transgenic plants. This is due to the fact that transgene integration
achieved by the two traditional methods (Agrobacterium or biolistic) of gene trans-
fer in the plant genome is random, and one to several copies of the transgenes may
be integrated at one or several locations in the genome. In order to overcome the
problem of randomness of transgene integration, site-specific transgene integration
strategies have been experimentally tested in plants, and offer prospects of stable
gene integration and expression in transgenic plants. In order to broaden the scope
of transgenic plants, biotechnologists started looking for other useful avenues for
their utility. With finite reserves of fossil fuels and climate change, and growing
demands for fuels, plastics, and pharmaceuticals, transgenic plants have been also
explored as production platforms for these commodities. This paper is an overview
of next generation transgenic plants that can serve as bioreactors or biofactories
for the cost-effective production of biofuels, biopharmaceuticals, bioplastics, and
as a resource for nutritional supplements to meet human demands in the future.
New developments in nanobiotechnology offer prospects for improved production
of crop plants.
M.R.Ahuja()
Formerly Forestry Consultant, Zobel Forestry Associates, 60 Shivertown Road,
New Paltz, NY 12561, USA
e-mail: mrahuja@hotmail.com
6.1Introduction
The world population has been increasing at an accelerated rate during the last cen-
tury. The world population was around 3.25billion in the 1960s. By the year 2000
the world population reached the 6billion mark. During the 40 years (19602000)
it almost doubled. By the year 2010 it increased by another 1billion to reach 7bil-
lion. It is expected to reach the 9billion mark by the year 2050 (http://www.fao.
org/index-en.htm 2013). That is an increase of 2billion from the current world
population of 7billion over the next 40 years. The bulk of the population increase
has occurred in the developing countries; by the year 2050 the world population
would be around 8billion in the developing countries, and only 1billion in the
developed countries. How to feed the world by the year 2050 remains a daunting
challenge for the food security. In order to feed this larger world population food
production would necessarily have to increase by 70% (Godfray etal. 2010; Bog-
danski 2012). This explosion in world population requires an enormous increase
in food production, improvement of nutritional quality of the staple food (bio-
fortification), production of safe and natural pharmaceutical proteins (molecular
farming), and increase in energy and plastics by alternatives routes (biofuels and
bioplastics) in the future to meet the human demands. In addition to conventional
breeding, plant biotechnology can play a significant role for the improvement of
human resources in the future.
Modern plant biotechnology began with the transfer of chimeric genes, consti-
tuted by the DNA recombinant technology, into the plant genome in the1980s, and
the first transgenic plants were produced by Agrobacterium-mediated gene transfer
(Bevan etal. 1983; Herrera-Estrella etal. 1983; Fraley etal. 1983). Subsequently,
transgenic plants from a number of different plant species, including agricultural
crops and tress, were produced with novel genotypes by Agrobacterium-mediated
and particle bombardment gene transfers (Pea and Sguin 2001; Sharma etal.
2002; Boerjan 2005; Jauhar 2006; Herdt 2006; Dunwell 2010). The first wave of
transgenic crops carried transgenes for resistance to fruit rotting, herbicide toler-
ance, and pest resistance.
Genetically engineered FlavrSavr tomatoes, developed by Calgene (Kramer and
Radenbaugh 1994), carrying an antisense polygalacturonase (PG) transgene which
makes tomatoes resistant to rotting, was released in the market in 1994. Under nor-
mal conditions, the enzyme polygalacturonase which degrades the pectin in the
cells wall results in softening of the fruit and consequently makes it susceptible to
fungal infection. FlavrSavr tomatoes, on the other hand, have a relatively longer
shelf-life, and also can be allowed to ripen on the tomato vine. However, the Fla-
vrSavr tomatoes turned out to be a disappointment in that the antisense PG gene,
6 Next Generation Plant Biotechnology 79
Table 6.1 Global area of biotech crops in 2013. (Data from James (2013))
Rank Country Area (millions of hectares) GM crops
1. USA 70.1 Maize, soybean, cotton, canola,
sugar beet, alfalfa, papaya, squash
2. Brazil 40.3 Soybean, maize, cotton
3. Argentina 24.4 Soybean, maize, cotton
4. India 11.0 Cotton
5. Canada 10.8 Canola, maize, soybean, sugar beet
6. China 4.2 Cotton, papaya, poplar, tomato,
sweet pepper
7. Paraguay 3.6 Soybean, maize, cotton
8. South Africa 2.9 Maize, soybean, cotton
9. Pakistan 2.8 Cotton
10. Uruguay 1.5 Soybean, maize
11. Bolivia 1.0 Soybean
12. Philippines 0.8 Maize
13. Australia 0.6 Cotton, canola
14. Burkina Faso 0.5 Cotton
15. Myanmar 0.3 Cotton
16. Spain 0.1 Maize
17. Mexico 0.1 Cotton, soybean
18. Columbia 0.1 Cotton, maize
19. Sudan 0.1 Cotton
20. Chile <0.1 Maize, soybean, canola
21. Honduras <0.1 Maize
22. Portugal <0.1 Maize
23. Cuba <0.1 Maize
24. Czech <0.1 Maize
Republic
25. Costa Rica <0.1 Cotton, soybean
26. Romania <0.1 Maize
27. Slovakia <0.1 Maize
Total area 175.2
1 ha=2.47 acres
which had a positive effect on the shelf-life, resulted in a negative effect on fruit
firmness, and also had a very bland taste. Consequently, Calgene halted the produc-
tion of FlavrSavr tomatoes in 1997.
In the next decade a number of genetically modified crops that were resistant to
herbicide tolerance and insect and disease resistance were commercially released
in the marketplace in many countries (Table6.1). These included soybean, maize,
cotton, canola, sugar beet, papaya, squash, tomato, poplar, and sweet pepper. Ini-
tially, transgenic crops were engineered for either herbicide or pest resistance. How-
ever, later on herbicide and insect resistant transgenes were stacked in some of the
transgenic crops (James 2013). In spite of the commercially profitable transgenic
crops, there are questions regarding the genetic stability of and mode of inheritance
of transgenes in the subsequent generations, transgene containment to effectively
80 M.R. Ahuja
prevent escape of transgenic pollen and seed, and impact of genetically modified
crops on the ecosystem and human nutrition. In order to address some of these con-
cerns and, at the same time, improve human nutrition and other utilities of biotech
plants, new areas of biotechnology are being explored for the next generation of
genetically modified plants. These include: (a) gene targeting and genome editing;
(b) nanobiotechnology; (c) biofortification; (d) molecular farming; (e) biofuels; and
(f) bioplastics.
The established methods of gene transfer have so far led to unpredictable inser-
tion and integration of transgenes in plants, including trees (Finnegan and McEl-
roy 1994; Buteye etal. 2005; Filipecki and Malepszy 2006; Ahuja 1997, 2009,
2011). Gene transfer has been accomplished by Agrobacterium-mediated and par-
ticle bombardment methods in plants. Transgene integration in the plant genome
is a complex process. Generally, Agrobacterium-mediated genetic transformation
produces transgenic lines with a relatively low (13) transgene copy number (Kohli
etal. 2003; Olhoft etal. 2004; Oltamanns etal. 2010; Fladung etal. 2013). Particle
bombardment transformation method, on the other hand, typically integrates on av-
erage higher (110 or even up to 100) transgene copy number and complex integra-
tion in the genome (Svitashev and Somers 2001; Makarevitch etal. 2003; Kohli
etal. 2003). A large number of diverse recombinant genes have been transferred in
the genomes of agricultural crops and trees. Transgene integration occurs in plants
by illegitimate recombination (Gheysen etal. 1991; Mayerhofer etal. 1991) be-
tween T-DNA and host genome. Integration of transgene is a random process, and
transgenes may be integrated at one location or dispersed on different chromosomes
in plants (Gelvin and Kim 2007; Kohli etal. 2010). One to a number of copies of
a transgene may be generally integrated at one or several sites in the host genome.
Transgene integration can occur throughout the plant genome (Alonso etal. 2003).
Depending on the site of integration of a transgene in the genome, transgene ex-
pression may be fairly stable, or there may be variation in transgene expression, or
instability/silencing of the transgene.
In order to overcome possible problems of variable transgene expression arising
from randomness and multicopy insertions of a transgene in the plant genome, gene
targeting systems have been developed in the past decades for directing a single
copy of a transgene, or its multiple copies thereof, in a predefined site in the host
genome (Pazskowski etal. 1998; Lyznik etal. 2003; Kumar et al. 2006; Poczai
etal. 2013; Puchta and Fauser 2013; Ahuja and Fladung 2014). Site-specific re-
combination systems developed from viruses, bacteria and yeast has been proposed
as tools for gene targeting (Liu etal. 2000; Kumar and Fladung 2001; Srivastava
and Ow 2004). Two components are needed for site-specific recombination: (1) a
site-specific recombinase, and (2) its recognition site (that is a defined sequence).
The recombinase systems include, the Cre-lox system of bacteriophage P1 (Sauer
6 Next Generation Plant Biotechnology 81
and Henderson 1990), the FLP-FRT (Golic and Lindquist 1989), the R-RS (Onouchi
etal. 1991) system of yeast, and the Gin/gix system of the bacteriophage Mu (Odell
and Russell 1994). Site-specific recombination takes place at a recognition site or a
specific DNA sequence and involves cleavage, and reunion leading to integration of
a recombinant gene, or deletion or inversion of a DNA fragment (Wang etal. 2011).
Site-specific recombination systems, experimentally used for in vivo excision of
donor DNA sequence, have been suggested as strategy to remove the antibiotic
marker genes or even the whole transgene cassette from the genome of transgenic
plants (De Buck etal. 2007; Luo etal. 2007; Gidoni etal. 2008; Wang etal. 2011).
However, the same system, but in the reverse reaction, can be used for targeted
integration of DNA (Kumar and Fladung 2001; Lyznik etal. 2003; Tzfira and White
2005). As a prerequisite, one copy of the recognition site must be present in the
targeted region, and a second one is located in the DNA to be inserted (Fladung and
Becker 2010). If the respective site-specific recombinase is temporally expressed,
the desired DNA fragment can exactly be inserted in the targeted region.
A gene targeting approach routinely requires two rounds of transformation: in
round one, the target site (e.g. lox or FRT), is randomly introduced into the plant
genome, and in the second round, a lox- (or FRT) containing recombinant gene is in-
serted into the previously targeted genomic site (De Buck etal. 2007; Li etal. 2009).
Cre-mediated site-specific gene integration has been demonstrated in rice (Srivas-
tava etal. 2004; Srivastava 2013), Arabidopsis (Vergunst etal. 1998; Louwerse etal.
2007; De Buck etal. 2007), and hybrid aspen (Fladung and Becker 2010); while
FLP-mediated site-specific gene insertion has been shown in soybean (Li etal. 2009),
and hybrid aspen (Fladung etal. 2010). In addition, stacking multiple transgenes
via repeated recombinase-mediated transformation, at selected genomic sites is ex-
perimentally feasible in plants (Li etal. 2010; Ow 2011). The advantage in stacking
transgenes at the same site on a chromosome is that the linked-transgenes, following
crossings, will most likely be transmitted as a single locus to the progeny. However, it
still has to be demonstrated whether site-specific recombination is practically feasible
for targeted transfer of numerous stacked transgenes to one genomic position.
A second gene targeting system utilizes synthetic recombinases or nucleases for
site-specific insertion of transgenes in the plant genome (Carroll 2011; Curtin etal.
2012; Tzfira etal. 2012; Puchta and Fauser 2013). These nucleases are engineered
proteins that are designed to break the double stranded DNA at a specific site, and
then exploit homologous recombination to insert a gene at a predetermined location
in the host genome. Three sequence-specific nuclease systems have been devel-
oped for site-specific integration of genes and mutagenesis in plants. These include:
zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and
LAGLIDADG homing nucleases, also known as Meganucleases (Carroll 2011;
Bogdanove and Voytas 2011; Curtin etal. 2012). Targeted integration of herbicide
tolerance genes by site-directed homologous recombination using ZFNs has been
reported in maize (Shukla etal. 2009) and tobacco (Cai etal. 2009), and by TALENs
in tobacco (Townsend etal. 2009). Both recombinase- and nuclease-mediated gene
insertions also require transformation systems that include either Agrobacterium or
particle bombardment.
82 M.R. Ahuja
Although recombinases and nucleases are promising avenues for gene targeting,
alternative methods for plant genome editing are being developed because of the
complicated designs and laborious assembly of specific binding proteins for each
specific target site (Belhaj etal. 2013). Recently, new methods of gene editing/ge-
nome engineering have emerged that involve clustered regulatory interspaced short
palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases (Cong
etal. 2013; Mali etal. 2013; Belhaj etal. 2013; Gaj etal. 2013). The CRISPR/Cas
system based targeted cleavage of genomic DNA is guided by a small customized
non-coding RNA in gene targeting by both non-homologous and homology-direct-
ed repair mechanism.
The real power of the engineered nucleases, recombinase, and CRISPR/Cas sys-
tems lies in their ability to precisely engineer not only foreign genes, but also native
plant genes for the production of transgenic plants. Another utility of synthetic gene
editing systems lies in their potential for activating/editing native plant genes for
herbicide tolerance and disease resistance, drought resistance and other qualitative
and quantitative traits, rather than engineering exogenous genes for these traits.
Such innovations will pave the way for next generation biotech crops to be less
regulated or not regulated by federal oversights, as these novel genotypes will be,
more or less, substantially equivalent to genetically unmodified plants. Although,
site-specific gene integration by recombinases and nucleases, and CRISPR/Cas sys-
tems is a promising avenue for stable integration of transgenes in plants, it is still
in experimental stages and further research is necessary for their application to next
generation crop plants and trees.
6.3Nanobiotechnology
6.4Biofortification
Recent research in biotechnology has also been focussed on the nutritional en-
hancement of micronutrients and vitamins in genetically modified crops. Genetic
engineering has been employed for fortification of minerals, amino acids, anti-oxi-
dants, vitamins for improving the nutritional quality of the staple crops. Biofortified
crops can alleviate essential micronutrient malnutrition in the human population,
particularly in the developing countries (Mayer etal. 2008; Hirschi 2009; Beyer
2010; Bashir etal. 2013; Murgia etal. 2013; Prez-Masscot etal. 2013; Saltzman
etal. 2013; Zhu etal. 2007, 2013). More than 50% of the human population world-
wide has little or no access to healthy staple fresh foods (Christou and Twyman
2004). Malnutrition of humans, particularly children, is rampant in underdeveloped
countries. Strategies to develop genetically modified plants as a resource for nutri-
tionally enhanced crop plants for food security have been developed by plant bio-
technologists in the past decades. Of course, some of the transgenic crops for food
security are still in experimental stages, while others have moved to field trials and
may become available in the market place in the future. In this direction, the next
generation biofortified transgenic crops include:
Rice that produces -carotene (provitamin A) in the endosperm (Golden Rice)
(Ye etal. 2000; Beyer 2010), and has increased amounts of folate, and mineral
(iron and zinc) in the seed (Beyer 2010; Lee etal. 2009, 2012; Yang etal. 2013);
Wheat grain with enhanced levels of iron and zinc (Borg etal. 2012; Sui etal.
2012; Borrill etal. 2014);
Potatoes that are protein-rich (Chakraborthy etal. 2010), have better aroma and
less browning (Llorente etal. 2010), and exhibit reduced cold-induced sweeten-
ing and increased carotenoid content (Giuliano etal. 2006; Chen etal. 2008;
Bhaskar etal. 2010; Barrell etal. 2013);
Tomatoes with increased lycopene and -carotene (Guo etal. 2012; Liu etal. 2014);
Bananas that are resistant to fungal wilt (Panama wilt) and black leaf streak
diseases, and exhibit increased -carotene and iron (Aravanityonnis etal. 2008;
Kovcs etal. 2013; Cressey 2013);
Corn with enhanced levels of multivitamins, including -carotene (provitamin
A), ascorbate (vitamin C), and folate (vitamin B9) (Naqvi etal. 2009);
84 M.R. Ahuja
Soybean with lower levels of saturated fat and higher levels of unsaturated oleic
acid, and higher levels of omega-3-fatty acids (Herschi 2009);
Citrus with enhanced levels of -carotene (provitamin A) (Cao etal. 2012), and
antioxidants in the fruit (Pons etal. 2014);
Cassava with improved nutritional quality, including starch (Ihemere etal. 2006;
Zeeman etal. 2010), provitamin A, and other micronutrients (Montagnac etal.
2009; Welsch etal. 2010; Sayre etal. 2011; Adenle etal. 2012);
Apple resistant to apple scab, fire blight, and improved growth (Malnoy etal.
2008; Borejsza-Wysocka etal. 2010; Joshi etal. 2011; Xu 2013; Schfer etal.
2012; Krens etal. 2012); early flowering to reduce generation time for breeding
to create new cultivars, and earlier yield (Flachowsky etal. 2011; Yamagishi
etal. 2014);
Crop plants with improved nutrition (McGloughlin 2010; Winkler 2011; Murgia
etal. 2013; Prez-Masscot etal. 2013; Saltzman etal. 2013), fortified micronu-
trients (Mayer etal. 2008; White and Broadley 2009), antioxidants (Zhu etal.
2013), vitamin A (Giuliano etal. 2008), vitamin B1 (Pourcel etal. 2013),vitamin
C (Locato etal. 2013), vitamin E (Yabuta etal. 2013), amino acid lysine (Galili
and Amir 2013), polyunsaturated fatty acids (omega-3-fatty acid) (Rogalski and
Carrer 2011; Petrie etal. 2012; Haslam etal. 2013;), and multivitamins (Hirschi
2009; Fitzpatrick etal. 2012);
Microalgae as a resource for fatty acids, such as omega-3-fatty acid (Adame-
Vega etal. 2012; Vaezi etal. 2013; Martins etal. 2013).
6.5Molecular Farming
Plants have been used for medicinal purposes for thousands of years by mankind.
Molecular farming, or biopharming, is a recent development using transgenic
plants, including algae, for the production of high-value pharmaceuticals, including
recombinant proteins (vaccines, cytokines, growth hormones) and other second-
ary metabolites (Daniell etal. 2001; Fischer etal. 2004, 2009; Karg and Kallio
2009; Obeme etal. 2011). Plants offer great potential as production platforms for
important pharmaceuticals for safe and effective use by consuming edible plant
tissues and seed (Hefferon 2013). Although biopharmaceuticals are predominantly
produced in transgenic animal and microbial bioreactor systems, transgenic plants
also offer alternatives to large scale biopharmaceutical production in plant tissues
and plant cell bioreactors. Plants cells are capable of full post-translational modi-
fication of recombinant proteins to fold properly and maintain their structural and
functional integrity, with simple growth factor requirement, minerals and light, and
essentially unlimited biosynthetic capacity and scalability of biopharmaceuticals
in leaves, stems, tubers, seeds, or whole plant, whether grown in the field or in
bioreactors. Besides, plants do not harbour human or animal pathogens, including
prions, human viruses and oncogenes, making them as safe hosts for the production
of biopharmaceuticals (Ma etal. 2003; Davies 2010). Although in earlier works,
6 Next Generation Plant Biotechnology 85
transgenic plants or their cell cultures were used for the expression of recombinant
proteins, more recently transient expression systems, involving the agroinfiltration
methods (Kapila etal. 1997; Pouge etal. 2010; Chen and Lai. 2013), the virus infec-
tion method (Porta and Lomonossoff 2002; Varsani etal. 2006; McCormick etal.
2008), and the magnifection technology (Gleba etal. 2005), have been developed
in plants for the production of biopharmaceuticals. Transient expression platforms,
perhaps the most convenient and efficient platforms, allows the cultivation of plants
under stringent controlled conditions, without stable genetic transformation, for the
rapid production of high grade pharmaceutical proteins on a large competitively
commercial scale (Rybicki 2010; Komarova etal. 2010; Tremblay etal. 2010; Cir-
celli etal. 2010).
The first recombinant proteins (human growth hormone) and with therapeutic po-
tential was successfully expressed in transgenic plants (Barta etal. 1986). A few years
later, the use of transgenic plants producing edible vaccines was reported (Mason
etal. 1992). It was not until 1997 that a recombinant protein, avidin (an egg protein)
was produced for commercial purposes in transgenic maize (Hood etal. 1997). Sub-
sequently, it was shown that transgenic plants have the ability and capacity for the
expression of a number of functional mammalian proteins with therapeutic value,
such as human serum proteins, growth hormones, antibodies, vaccines, cytokines,
and enzymes (Daniell etal. 2009; Karg and Kallio 2009; Obeme etal. 2011; Franconi
etal. 2010; Penney etal. 2011; Sirko etal. 2011; Kumar etal. 2013; Rigano etal.
2013; Da Cunha etal. 2014; Specth and Mayfield 2014). Recombinant biopharma-
ceutical production is moving at a very fast pace since 2007 when it captured about
10% of the pharmaceutical market (Lowe and Jones 2007). By the year 2010, there
were more than 200 bio-drugs approved biopharmaceuticals on the global market,
generating more than $100billion in the global pharmaceutical market (Walsh 2010).
It is expected that the biopharmaceutical market will continue to expand in the future
and the bio-drug sales may reach up to $240billion by 2015 (Stewart 2010). Some
of the biotech plants, including microalgae, used for the production platforms of a
number of biopharmaceuticals are listed in Table6.2. For more detailed listings of
plant-based biopharmaceuticals see reviews by Daniell etal. (2009), Walsh (2010),
Obeme etal. (2011), Da Cunha etal. (2014), and Specht and Mayfield (2014).
86 M.R. Ahuja
6.6Biofuels
Poplar with down regulation of CCR gene to reduce lignin content that resulted
in increased saccharification and high ethanol yield (Van Acker etal. 2014); or
suppression of other genes (CAD; 4CL. C3H,COMT) involved in the biosynthe-
sis of lignin to enhance biofuel production (Coleman etal. 2008; Mansfield etal.
2012; Voelker etal. 2011; Ye etal. 2011)
Microalgae as a resource for production ofbiodiesel and bioethanol (Schenk
etal. 2008; Beer etal. 2009; Brennan and Owende 2010; Gouveia and Oliveira
2009; Mata etal. 2009; Bajhaiya etal. 2010; Dragone etal. 2010; Huang etal.
2010; Khola and Ghazala 2012; Wu etal. 20123; Harun etal. 2014);
Industrial waste agricultural residue biomass as a potential source of biofuel
(Mwithiga 2013);
Forest trees (especially poplars, eucalypts, and salix) as a resource of lignocel-
lulosic feedstock, and lignin modification for improved production of biofuels
(Rockwood etal. 2008; Simmons etal. 2010; Seguin 2011; Mizrachi etal. 2012;
Nieminen etal. 2012; Pilate etal. 2012).
6.7Bioplastics
In addition to current focus on using plants for biofuels, plants also produce a large
number of useful chemicals and biopolymers. Plants naturally produce a large num-
ber of biodegradable polymers, which include starch, cellulose, proteins and rub-
ber (Kulkarni etal. 2012). Starch and cellulose play a major role in food and fi-
bre production for mankind. However, plants do not produce bioplastics, but many
bacteria including, Ralstonia, Pseudomonas, Azobacter, and Rhizobium do (Dalton
etal. 2011). Bacteria (e.g. Ralstonia) accumulate the polyester polyhydroxyalkano-
ate (PHA) as a bioplastic for carbon and energy reserve in response to nutritional
stress (Anderson and Dawes 1990). Polyhydroxybutyrate (PHB), a short side-chain
polymer of PHA, in produced in bacteria from acetyl-coA via a three enzymes bio-
synthesis pathway. These enzymes are encoded by three genes, phaA, phaB, and
phaC respectively (Slater etal. 1988). Currently, the PHA bioplastics are being com-
mercially produced in bacterial fermentation systems, with renewable resources as
sucrose, glucose, fatty acids, or plant oils, or waste effluents (molasses, whey), and
glycerol as carbon substrates (Chee etal. 2010; Chen 2009, 2010; Du etal. 2012).
Wild type bacterial strain of Ralstonia eutropha (formerly known as Alcaligenes
eutropha) has been most commonly used for industrial production of bioplastic
(Chen 2009). However, the bacterially produced biodegradable bioplastic polymers
are not cost-competitive with non-biodegradable petroleum-based plastic polymers.
Alterative platforms to bacterial fermentation are being explored for production of
bioplastics using transgenic plants that might be more cost-effective than bacterial
fermentation and petroleum-based plastics (van Beilen and Poirier 2008; Mooney
2009; Somleva etal. 2013; Petrasovits etal. 2013).
A publication entitled In search of plastic potato by Pool (1989) generated great
expectation in the scientific community that bioplastic PBA could be produced by
88 M.R. Ahuja
genetic engineering on plants. A few years later, PHB (0.01% fresh weight; FW)
was first produced in the model plant Arabidopsis thaliana transgenics (Poirier etal.
1992), which initiated a continuing wave of research to optimize the production of
PHB in transgenic plants (Somleva etal. 2013). Some improvement in the PHB
yield (14% dry weight; DW) was reported in transgenic Arabidopsis, with no obvi-
ous effect on the growth and fertility of the transgenic plants (Nawrath etal. 1994).
Subsequently, a later study on transgenic Arabidopsis showed efficient production
of PHB (40% DW) in the chloroplasts of the leaves, but that was accompanied by
severe growth reduction of the transgenic plants (Bohmert etal. 2000). Since then
a large number of studies with many plant species have been conducted that show
variable production levels of the bioplastic PHB (0.00540% DW) in transgenic
plants (van Beilen and Poirier 2008; Somleva etal. 2013). Different tissues, includ-
ing whole plant, shoot, stem, leaves, and cell suspension were employed for PHB
production, which mostly accumulated in the plastids, but also in the cytoplasm
(Van Beilen and Poirier 2008; Somleva etal. 2013). In addition, transgenic micro-
algae, such as green algae Chlamydomonas reinhardtii, and diatomsPhaeodactylum
tricornutum engineered with PHB pathway genes from Ralstonia eutropha have
also been explored as bioreactors for bioplastic production (Chaogang etal. 2010;
Hempel etal. 2011). A large number of transgenic plants, both crops and non-crops,
have been employed, and show different amounts of biodegradable bioplastics (van
Beilen and Poirier 2008; Someleva etal. 2013). Some of transgenic plants for bio-
plastic production are listed in Table6.3.
6.8Future Prospects
A lot of progress has been made in different areas plant biotechnology in the last
two decades. Initially crop plants were engineered with foreign genes derived
mostly from bacteria for herbicide and pest resistance to improve crop yields.
Later on, other transgenes for lignin modification, early flowering, male sterility,
and abiotic stresses were experimentally tested in crop plants. Earlier studies in
plants used alien genes from bacteria, animals and plants for genetic engineering.
Recent trend has been towards development of cisgenic and intragenic transgenic
crop plants (Holme etal. 2013; Espinoza etal. 2013). Cisgenic plants are derived
6 Next Generation Plant Biotechnology 89
from transformation with identical copy of a gene from sexually compatible pool,
including promoter, intron and terminator regions that are derived from the do-
nor plant. On the other hand, intragenic plants are derived by transformation with
combinations of different genes from the same or sexually compatible species.
While both cisgenic and intragenics plants are guided by their own genes, they
both require genetic transformation by Agrobacterium or biolistic methods. Recent
research in gene targeting and directed genome engineering promises site-specific
integration of transgenes in predetermines regions of the host genome, or tinkering
of the endogenous genes of economic importance for their stable transgene expres-
sion and inheritance in the next generation crop plants. Next generation genome
sequencing is already providing useful information regarding gene discovery and
molecular markers associated with a number of diverse economic traits (Edwards
and Batley 2010; Hamilton and Buell 2012). DNA sequencing is providing insight
information genes that would be useful for plant improvement through plant bio-
technology.
While these promising investigations are progressing at a rapid pace for the
commercialization transgenic/biotech crops, plant biotechnology has also harvest-
ing other useful plant products. These include next generation transgenic plants as
future production platforms for biopharmaceuticals, biofuels and bioplastics, and
nutritional supplements. In a sense, plants are becoming biofactories/bioreactors
for useful bioproducts for mankind. Nevertheless, transgenic plants are subject to
regulatory oversights. Containment of transgenes must be in place to effectively
prevent escape of transgenic pollen seed, and vegetative propagules from the trans-
genic plants. In addition, these new areas in plant biotechnology must take into
account risk assessment and biosafety considerations (Shama and Peterson 2008;
Breyer etal. 2009; Sparrow and Twyman 2009; Domingo and Bardonaba 2011;
Snell etal. 2012; Buiatti etal. 2013; Jouzani and Tohidar 2013), including the im-
pact of these new developments on human health (new allergies), ecosystem, plant
biodiversity, and sustainable agriculture. New developments in nanobiotechnology
offer prospects for precise delivery of genetic material and enhanced production of
agricultural crops. It would be necessary to maintain genetic biodiversity in the next
generation biotech plants (Sharma and Sharma 2013) used for the bioproduction
platforms as well as for crop improvement.
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Part II
Section B: Biotechnology and conservation
of Biodiversity
Chapter 7
Conservation of Forest Genetic Resources
M.ijai-Nikoli() M.Noni
University of Belgrade, Faculty of Forestry, Kneza Vieslava 1, Belgrade 11030, Serbia
M.Noni
e-mail: marina.nonic@sfb.bg.ac.rs
J.Milovanovi
Singidunum University Faculty of Applied Ecology Futura, Poeka 83a,
Belgrade 11030, Serbia
Modern man, by its various activities, continually destroys and changes the nature
which leads to irreversible loss of biodiversity through the disappearance of a large
number of organic species or reducing their natural population to critical limits.
The destruction of species does not occur as planned and targeted human activ-
ity, but usually indirectly, by destroying habitats where species live. The causes of
biodiversity loss are numerous, mutually interdependent and usually anthropogenic
character (ijai-Nikoli and Milovanovi 2007).
7.1.1Biodiversity
material, the literature, the activities on informing public, cooperation with other
regional programs, eg. Central Asia, Africa (Geburek and Turok 2005).
The Third Ministerial Conference on the Protection of Forests in EuropeThe
Third Ministerial Conference on the Protection of Forests in Europe was held on
June 24, 1998 in Lisbon, Portugal. Responsible for the European forestry adopted
two resolutions, L1People, Forests and Forestryimproving the socio-economic
aspects of sustainable forest management and L2Pan-European criteria, indica-
tors and operational guidelines for sustainable forest management. As a result of
this cooperation, a Pan-European project called The Work Programme for the con-
servation and enhancement of biological and landscape diversity in forest ecosys-
tems, 19972000 was launched and successfully implemented.
COP-4 of the CBD During COP-4, held in 1998, forest biological diversity has
been identified as one of the five thematic areas of the Convention on Biological
Diversity. At the same meeting, the working program for forest biological diversity
focused mainly on the development of research cooperation and technology was
approved, and the Ad Hoc Group of Experts tasked with further development of the
Work Programme was established. Special emphasis was placed on in situ conser-
vation. In addition to in situ conservation, it is necessary to measure the existence of
complementary ex situ conservation. In all aspects of the Convention, it is necessary
to ensure the participation of the public, especially when it comes to the assessment
of the environmental impact of those that pose a threat to biodiversity.
The Fourth Ministerial Conference on the Protection of Forests in EuropeThe
Fourth Ministerial Conference on the Protection of Forests in Europe under the
name Living Forest Summit was held on April 2830, 2003 in Vienna, Austria.
Resolution 4 recognizes biological diversity as a key element in sustainable for-
est management. Annexes to the Resolution are specifically defined framework for
cooperation and coordination between the MCPFE and The World Conservation
Union, IUCN.
The Seventh United Nations Forum for Forests United Nations Forum for For-
ests at its seventh meeting, held in 2007, adopted the so-called Non-legally bind-
ing instrument on all types of forests, which, once again, expressed concern about
deforestation, forest degradation, low rates of afforestation, as well as feedback
from these factors on the survival of biodiversity and genetic resources.
The Fifth Ministerial Conference on the Protection of Forests in EuropeThe
Fifth MCPFE entitled Forests for Quality of Life was held on November 57,
2007 in Warsaw, Poland. Within the Warsaw Declaration, the Parties committed
themselves to insisting on sustainable forest management, which contributes sig-
nificantly to the ecological, economic, social and cultural dimension of sustainable
development, in particular the achievement of adopted international goals, includ-
ing the goals of the Convention on Biological Diversity.
COP-9 of the CBD At the Ninth Meeting of the Parties of the Convention on Bio-
logical Diversity, held in 2008 in Germany, the Decision 4/7 on Forest Biological
110 M. ijai-Nikoli et al.
nature conservation and natural resources, as well as the regulation of trade and
market issues of forest reproductive material;
Recognition of the concept and the problem of conservation of forest genetic re-
sources in the strategic documents, which are intended to indicate the desired di-
rection of development in the field of sustainable forest management, indicating
the existence of intentions and desires of society for the prevention of adverse
effects, and the lack of commitment to this category of natural value and capital.
Raising awareness of forest owners and users about the importance of conserving
genetic diversity can significantly contribute to the improvement and implemen-
tation of strategic priorities. Forest owners can be very useful in the design and
implementation of conservation programs, but they are not sufficiently aware of the
biological value of their forests, or existing sources of financial support for conser-
vation activities.
Genetic variability, which is the result of different genetic processes: mutation, re-
combination, gene flow, natural selection and genetic drift, presents the basis for
conservation of forest genetic resources. The principles of conservation of genetic
variability can be regarded as identical for all living beings. However, the meth-
ods which are applied vary depending on the specificity of the conservation goals,
distribution and biological nature of the material that is the object of conservation
(F 1989).
From the aspect of preserving genetic variability, there are different methods of
conservation. The term method is used in the context of a certain concept of con-
servation of genetic resources: in situ or ex situ, dynamic or static, while the species,
ecosystem, population, individual or part of an individual, present objects of con-
servation (ijai-Nikoli and Milovanovi 2009; ijai-Nikoli and Milovanovi
2010). Every process of conservation should start by clearly defining of its objec-
tives. When the process of conservation allows adaptation and changes in gene
frequency, in accordance with local selective influence, it is dynamic (evolutionary)
conservation. If the process of conservation is planned with the aim of preserving
the current gene frequencies of the original population, wherein lack the effects of
the genetic process, we are talking about a static conservation (Guldager 1975).
Forest genetic resources conservation model is presented in Fig.7.1.
In situ (at the site) conservation means the conservation of forest genetic resources in:
natural populations (seed stands, groups of trees or individual trees),
112
Fig. 7.1 Forest genetic resources conservation model (ijai-Nikoli and Milovanovi 2007)
M. ijai-Nikoli et al.
7 Conservation of Forest Genetic Resources 113
Botanical Garden represents a collection of living plants, which is mainly used for
improvement and dissemination of botanical knowledge (Potoi 1980). As forerun-
ner of todays botanical gardens can be considered private gardens where wealthy
people, in being and nature lovers, collected and cultivated different plants. The first
public botanical garden was founded by the Venetian Republic in 1545, in Padua
as a scientific institution of the university of that time. Modeled on the botanical
gardens in Padua, Pisa and Bologna, botanical gardens were founded throughout
Europe. Nowdays, there are being kept unique specimens of various plant species,
some of which a long time ago disappeared from the natural habitat. An arboretum
(lat. arbor-tree) is a separate space or part of the botanical gardens, in which are
grown trees and shrubs in the scientific, ornamental and breeding purposes (Potoi
1980). The oldest such collection of trees and shrubs was mentioned in botanical
garden in Tokyo, which is reportedly over 800 years old.
7.3.2.2Seed Orchads
orchards is to establish populations that will maintain the original genetic variability
to the maximum extent and allow long-term adaptation to local conditions, where
planting was done. In addition to preserving the original genetic variability, seed
orchards are used as sources of reproductive material for commercial forestry.
Seed orchard means specialized, artificial culture for long-lasting production of
genetic quality seeds of economically significant tree species (Tucovi 1990). Seed
orchards are established from phenotypic and genotypic best individuals of one, or
various, species, at area which is supposed to be spatially isolated from physically
mature trees of the same species, in order to prevent uncontrolled pollination of in-
dividuals that come out of the seed orchard. According to the character of planting
material, plantations can be vegetative, formed of clones or generative, formed of
plants originating from the half-sib or full-sib lines of selected genotypes.
7.3.2.3Progeny Tests
Progeny tests represent an opportunity for exploring the genetic potential of certain
species, provenances, populations or the genotype. The basic principle of the es-
tablishment of such experiments is to create uniform conditions for the cultivation
of plants that are being tested, so that mutually appeared differences are reflection
of various genotypes, not various environmental conditions. Progeny tests may be
established of full-sib lines, which represent offspring of controlled hybridization
(both mother and father are known), or half-sib lines, where the mother is known,
and the father is unknown (free pollination). Tests can be performed in the phyto-
trons, greenhouses, nursery or field conditions. According to duration of experi-
ment, there are early tests, short-term and long-term tests. A particular form of ge-
netic potential assessment, which are constantly gaining in importance are early
tests. Research conducted in the earliest stages of ontogenesis in Serbian spruce
have pointed to the importance of the same for exploring the variability of species,
its taxonomy, genetic potential, cultivation, breeding and conservation direction
(ijai-Nikoli and Milovanovi 2010). The assessment of adaptive and produc-
tion potentials of different lines of half-sibs in progeny tests is used for the selection
of elite trees, which represent a basis for seed collection and seedling production,
aimed at spreading the population of some species and preservation of genetic vari-
ability (Noni etal. 2012).
The age variability is the result of natural selection, which is expressed at each
stage of the life cycle. Therefore, the variability of plants in the juvenile develop-
ment phase can be considered as products of the interaction of hereditary basis
and selection. Genetic variability of the mature population is significantly reduced,
compared to the variability of baseline levels seedlings, 1 year or 2 years old plants,
considering that selection was the highest in the germination stage, after which it
was reduced, but continuous. Wherein the larger dimensions of the cotyledons, hy-
pocotyls and epicotyls characteristics, good root system characteristics of, and simi-
lar, indicate the potential superiority of adult individuals in comparison to the aver-
age. Every geneticist, breeder or nurseryman, who knows spontaneous v ariability,
116 M. ijai-Nikoli et al.
already after the formation of cotyledons, can observe possible deviations from the
usual form, in particular in the haploids, polyploids or polysomics, which provides
high speciation (Tucovi 1990). The results of the early tests should be considered
approximate they need to be checked in the following years of research.
7.3.2.4Provenance Trials
Provenance trials, within gene pool conservation, are applied as a method of as-
sessing the degree of diversity and potential, both autochthonous and allochthonous
trees species. Also, provenance trials may contribute to determining the potential
and the degree of divergence of isolated populations, in terms of higher productivity
and adaptability, respectively, can be used to determine the differences in genetic
variability between and within different provenances.
7.3.2.5Preservation
The gene banks are formed to preserve the total genetic variability on a scientific
basis. The task of gene banks was to collect, determine, document, reproduce and
preserve genetic variability at the long term, and made it available for use. A fully
and easily accessible information are the primary requirements for the use of ge-
netic variability that is stored in gene banks (Peni etal. 1997). Plant gene banks
are formed from the parts of plants that contain germplasmgenetic basis of spe-
cies. The most commonly applied method is cryopreservation, which allows long-
term storage of plant parts (Withers and Engelmann 1998). This term refers to seed
storage at extremely low temperatures, typically with the use of liquid nitrogen
(196). Applied together with the in vitro technique, cryopreservation is often
the only reliable, safe and economically viable method of storage of some species.
In cases where the seed is not suitable for cryopreservation, the extract of embryo
or the core of the embryo should be applied, at the appropriate stage of develop-
ment. Establishment of seed banks, where the seed is stored in refrigerators or under
other appropriate conditions, is another form of static ex situ conservation. Seed
banks can be used only for species whose seed storage is possible. Most species
have seeds with high germination rates sustainable a few years, which is extremely
short, compared to the long life span of trees, and therefore the seed supplies must
be renewed at regular intervals. It involves germination, seedling production, tree
growing to the beginning of fruiting, collection and storage of new seeds.
This rejuvenilization leads to the appearance of new genetic recombination
and new selection pressures during the propagation and growth. For most species,
seed banks have to be a short-term form of conservation. Seeds of endangered pop-
ulations can be collected and stored in a bank for a fixed period, until the most suit-
able moment for the seeding and seedlings development for the establishment of ex
situ conservation habitats.
7 Conservation of Forest Genetic Resources 117
One of the main developments in forestry practice over the last three decades has
been its evolution from a practical discipline with a primary, or even exclusive,
focus on management of forests for timber, to a more holistic approach recogniz-
ing that forests provide a wide range of environmental and social services and that
provision of these should form an objective of management. The development of
concepts such as forest ecosystem management and multi-purpose forestry are
symptomatic of this process.
The importance of forests to people has been increasingly recognized, as illus-
trated by the widespread implementation of forest management approaches explic-
itly aimed at or involving local communities, such as community forestry and social
forestry. The importance of actively involving local communities and other stake-
holders is consistently an element of approaches to sustainable forest management
(Newton 2007).
Participation in forest conservation is often associated with the concept of com-
munity forestry. Community forestry basically means that a forest is managed or
co-managed by people who live close to it (Wily etal. 2000). Local participation is
important in almost all forest conservation, but there are situations where it is ab-
solutely necessary, for instance in areas characterized by high population pressure
and conflicts of resource use; in areas under communal ownership; and in smaller
protected areas because of the vulnerability to surrounding human activities (FAO,
FLD, IPGRI 2004).
The impact of any form of property on conservation depends on a variety of
external and internal factors, such as the economic status of the owners and the
structure of ownership. Small properties can be good for conservation, since what-
ever management decision a particular owner makes, it has a limited impact. Thus
these holdings form a mosaic of habitats. Many small forest owners follow in fact
the traditional silvicultural systems. Although looked down upon by forestry profes-
sionals, private forests are a reservoir of unwanted genotypesirregular forms,
forked or twisted trunks. Often old trees, of no commercial value, are left to live
their natural life span. Woody debris is removed only if used as fuel. Larger private
properties, that are regularly managed, are influenced by traditions of even-age sil-
viculture. This, as in state forests, leads to simplification of ecosystems and loss of
biodiversity. Management mistakes in large properties have greater impact and are
more difficult to rectify.
The concept of conservation is poorly understood both in society at large, and
within the group of forest owners. Appreciation of the role of elements of the eco-
system is missingmany owners do not see that forest is not just trees. Forest own-
ers often understand a need to protect game, birds or some rare flowers. However,
they do not understand very well importance of conservation of genetic variability
(IUCN 2004).
In an effort to save genetic diversity, a number of approaches to conservation
have been suggested. Some approaches focus on species habitats, ecosystems, or
7 Conservation of Forest Genetic Resources 119
(Thompson etal. 2005). Participation has been described as both a means and an
end, a vehicle and a goal itself (Jennings 2000).
Participatory tools reflect the dual nature of participation. A practitioner might
use participatory activities purely to elicit local knowledge and perspectives. Local
peoples input is limited to providing information, while the information that the
tool generates is used by decision makers elsewhere.
On the other hand, involving local people in decision making might be the objective
for using a participatory tool. This participatory approach is called collaborative
management. There are several participatory tools that are particularly strong in
collaborative management while selection of appropriate tool depends on the objec-
tive that researchers want to reach (Table7.1).
Collaborative management actually brings the community into the decision mak-
ing process, involving local people in discussion, negotiation and planning. This
7 Conservation of Forest Genetic Resources 121
Table 7.1 Some of the participatory tools which can be used in collaborative forest genetic
resources conservation management
Participatory tool Objectives
Participatory mapping/resource mapping Elicit knowledge about local resources
Spidergrams Stakeholder identification, quantitative values, elicit
values about resources and social interactions
Venn diagrams Identify stakeholders, stakeholder relationships, elicit
values about resources
H diagram Elicit knowledge and opinions about local resources
and improvement of current situation
Brainstorming Elicit knowledge and opinions about local resources
and improvement of current situation
approach is the most appropriate for planning of forest genetic resources conserva-
tion, having in mind that people live with forests and vice-versa (Evans etal. 2006).
There is no real alternative to public involvement and collaboration among all
stakeholder groups in the development and implementation of policies for the con-
servation of forest genetic resources. This is the only way of generating policies that
meet the final and absolute test: to be sustainable, policies must be socially accept-
able (Lindenmayer and Franklin 2002).
factors, and their interactions. Threats to genetic diversity are further complicated
by interactions with deforestation, habitat loss and poor management (St. Clair and
Howe 2011). It is essential that the genetic diversity in plant genetic resources be
properly understood and efficiently conserved and used. If the level of genetic di-
versity in a species is greater, the chances for its survival are better (Ledig 1988;
Ramanatha Rao and Hodgkin 2002; Koskela etal. 2007; Ahuja 2011). Climate
change will have long-term and widespread consequences for many species, but it
is important to give greater priority to those species and populations at greatest risk.
Considering the longevity and the long regeneration cycle of forest tree species,
climate changes must be one of the key factors in creating conservation strategies at
the individual and population levels. Forest trees define the essential characteristics
of forests, and threats to forest genetic diversity include threats to species, popula-
tions, and genetic variation within populations (St. Clair and Howe 2007, 2011).
Trees can respond in different ways in the face of global climate change. They may
have high phenotypic plasticity and tolerance, which will play an increasing role
in the adaptation of forest stands to changing environmental conditions (Mtys
2006; Savolainen etal. 2007). The ability of forest tree species to respond to climate
change is limited by their long life spans, long juvenile phases and long generation
intervals. However, forest trees maintain high levels of gene flow and genetic varia-
tion, which should facilitate their ability to evolve in response to rapid changing
climate (Hamrick etal. 1992; Erickson etal. 2012).
According to Loo etal. (2011), survival will depend on the capacity to quickly
adapt genetically to new conditions at existing sites; high degree of phenotypic
plasticity and/or migration to newly evolving environments that match basic physi-
ological requirements (Loo etal. 2011).
In regions where climate change is expected to be rapid and extensive, many
forest tree species will be exposed to stress. Changing climates will result in new
species invasions, new pests and diseases, flooding, temperature extremes, altered
patterns of gene flow and the hybridization of species and populations, competitive
pressures, and wildfires are expected to be more frequent. Therefore, high mortality
due to extreme climatic change, in combination with regeneration failure, will result
in the loss of forest genetic resources and the local population extinction (Woods
etal. 2005; Carroll etal. 2006; Westerling etal. 2006; Koskela etal. 2007; Loo
etal. 2011; St. Clair and Howe 2011; Erickson etal. 2012). The specific effects of
climate change will vary depending on the degree of sensitivity, exposure and the
adaptive capacity of individual species and populations (Parry etal. 2007; Chimura
etal. 2011; Erickson etal. 2012).
According to Mtys (2006), there are various mechanisms (genetic and non-
genetic) balancing changes in environmental conditions on different levels: in-
dividual, population, species and ecosystem level. On individual genotype level,
environmentally induced phenotypic plasticity and carryover effects (Jablonka
etal. 1995) provide the ability to survive in a wider range of environments without
classic genetic change. On the level of populations, natural selection adjusts the
adaptability of the population to changing conditions through genetic adaptation,
and sufficiently large genetic diversity is precondition for fast and effective genetic
7 Conservation of Forest Genetic Resources 123
7.6Conclusions
The increasing demand for wood, as a raw material for various purposes, as well
as general useful forest functions, have made the protection (conservation) and di-
rected utilization of forest genetic resources, become a priority task of forestry sci-
ence and profession.
124 M. ijai-Nikoli et al.
the forest genetic resources conservation. Conservation priorities set by the commu-
nity represent the best strategic approach for forest genetic resources management
especially when we have in mind various threats such as climate changes.
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Chapter 8
Advances in Cryogenic Techniques for the
Long-Term Preservation of Plant Biodiversity
Abstract This chapter presents different technical aspects related to the develop-
ment and large-scale application of cryopreservation techniques, as a biotechno-
logical approach for the long-term storage of plant biodiversity. The main cryogenic
procedures and the key steps for their successful adaptation to diverse forms of
germplasm are described. Some representative examples of cryopreservation of
different plant species are presented to illustrate the significant progress achieved
in the practical utilization of cryopreservation as a complementary alternative for
germplasm conservation. In addition, other potential uses of this technology to sup-
port genetic breeding programs, and its recent utilization to eliminate systemic plant
pathogens through cryotherapy are discussed.
KeywordsBiodiversity Cryopreservation Storage Breeding Cryotherapy
8.1Introduction
F.Engelmann()
IRD, UMR DIADE, 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 05, France
e-mail: florent.engelmann@ird.fr
M.T.Gonzalez-Arnao C.A.Cruz-Cruz
Facultad de Ciencias Qumicas, Universidad Veraccruzana, Prol. Ote 6, No. 1009,
CP 94340 Orizaba, Veracruz, Mexico
e-mail: teregonzalez@uv.mx
C.A.Cruz-Cruz
e-mail: calcruz@uv.mx
M.E.Martinez-Montero
Centro de Bioplantas, Laboratorio de Mejoramiento de plantas, Universidad de Ciego de vila,
Car. a Moron km 9, CP 69450 Ciego de Avila, Cuba
e-mail: marcosem@bioplantas.cu
species that have asexual propagation and of species that are impossible to keep as
seeds or in field gene banks. Nowadays, biotechnology is also necessary to solve
important problems of management, breeding and storage of plant genetic resourc-
es. Biotechnology provides new tools to select genotypes with desirable traits, to
identify and incorporate important genes that induce disease resistance and toler-
ance to biotic and abiotic stress. It also offers diverse in vitro strategies to multiply
and preserve plant genetic biodiversity outside its natural habitat. In a general con-
text, biotechnological methods have been developed and used to conserve endan-
gered, rare, crop, ornamental, medicinal, and forest species for short, medium and
long-term (Cruz-Cruz etal. 2013). In addition, in vitro techniques offer a safe mean
for the international exchange of germplasm, allow the establishment of extensive
collections with minimal space requirements. They allow a valuable supply of ma-
terials for wild population recovery, they guarantee the storage of pathogen-free
material and elite plants, and facilitate the performance of molecular investigations
and ecological studies (Tandon and Kumaria 2005).
Until recently, most conservation efforts, apart from work on forest genetic re-
sources, have focused on ex situ conservation, and particularly focused on seed
genebanks. In the 1950s1960s, the major advances in plant breeding due to the
green revolution resulted in the wide-scale adoption of high-yielding varieties and
genetically uniform cultivars of staple crops, particularly wheat and rice. Conse-
quently, global concern about the loss of genetic diversity in these crops increased,
because farmers abandoned their locally adapted landraces and traditional varieties,
to replace them with improved, genetically uniform modern ones. In response to
this concern, the international organizations related to agricultural research started
to assemble germplasm collections of the major crop species, and coordinated a
global effort to systematically collect and conserve the worlds threatened plant
genetic diversity (Engelmann and Engels 2002). As a result of this great effort,
there are over 1750 genebanks worldwide today, about 130 of which hold more than
10,000 accessions each, and conserve around 7.4million accessions (FAO 2009).
There are also substantial ex situ collections in botanical gardens, of which there
are over 2500 around the world, containing around 4million accessions from over
80,000 species. In that way, botanical gardens and agricultural genebanks also rep-
resent important complementary strategies for ex situ conservation of plant biodi-
versity (Engels and Engelmann 1998). The establishment of field genebanks allows
conserving species for which seed conservation is not appropriated or is impossible,
but conservation in the field presents important drawbacks, either of biotic or abi-
otic character, and these limitations, seriously affect its efficacy and constitute a
permanent threat to the safety of the germplasm conserved under these conditions.
Therefore, the development of other storage technologies was a new common need
for the international community (Engelmann and Engels 2002).
During the last 40 years, in vitro culture techniques have been extensively estab-
lished and applied to more than 1000 different plant species (George 1996). Tissue
culture techniques are of great interest for collecting, multiplication and storage
of plant germplasm (Engelmann 1991). Tissue culture systems allow propagating
plant material with high multiplication rates in an aseptic environment. In addi-
8 Advances in Cryogenic Techniques for the Long-Term 131
In the case of cell suspensions, the exponential growth stage is the most suitable
for cells to successfully withstand liquid nitrogen exposure (Withers 1985). In that
phase, cells are young, small, and contain only few vacuoles, which implies that
they have low amounts of intracellular water, and are more tolerant. However, it is
also important to take in mind as a general rule, that the long-term application of
tissue culture techniques before cryopreservation may also significantly reduce the
ability of biological material to survive after cryopreservation (Harding etal. 1991).
Another important parameter is the size of explants when dealing with organized
structures as shoot-tips. Large explants usually maybe less tolerant to cryopreser-
vation because they are difficult to get sufficiently or homogeneously dehydrated
before their exposure to low temperatures, but at the same time, the dissection of too
small meristems (about 0.2mm) has to face the technical difficulties of excising,
in addition to the main problem that such small explants do not always survive and
regenerate new plants, especially when dealing with tropical species. Therefore, an
important prerequisite is to define the smaller size of explant suitable to ensure vi-
ability of in vitro cultures.
Contemporary cryopreservation research has made a good use of the fact that
sugars are natural plant cryoprotectants. Although the nature of the sugar employed
during a preculture treatment can have a strong impact on recovery of cryopre-
served explants, up to now, sucrose has played a major role in the acquisition of
desiccation and cold tolerance in plants that are cryopreserved (Engelmann 1997).
To date, there are many examples of its application at different stages for most
cryopreservation protocols. In general, sucrose has been either successfully used
alone or in combination with other cryoprotectants. Sucrose is also an important
component for the formulation of loading, unloading and plant vitrification solu-
tions (Kim etal. 2009a, b). Moreover, its addition to semisolid culture medium is
often used for preconditioning of explants or of donor plants (Gonzalez-Arnao and
Engelmann 2006). A 1-day-preculture of shoot-tips after dissection on semisolid
medium supplemented with 0.3M sucrose is the most commonly applied step of
any cryopreservation procedure for organized tissues. In general, the action mode
of sugars appears to be osmotic, but there are some evidences that they also have a
crucial role in the stabilization of glasses and that their biochemical properties af-
ford cell protection (Jitsuyama etal. 2002).
On the other hand, the unusual molecular characteristics of liquid water deter-
mine how it behaves throughout the cooling-warming cycle. Therefore, to provide
an effective cryoprotection during a cryogenic process is also essential controlling
the water phase transitions (Mazur 2004). In this sense, water removal and its inter-
play with the cooling and warming rates play a central role in preventing freezing
injury.
Different methods exist to reduce the temperature depending on the cooling rate:
ultra-rapid, rapid or slow cooling. In the latter case, a programmable freezing appa-
ratus is usually used in order to obtain precise and reproducible cooling conditions.
This characterizes the conventional methods for cryopreservation. Conventional
protocols involve the pretreatment of samples with cryoprotective solutions com-
posed by a single or a mixture of colligative chemical substances, followed by the
134 M. T. Gonzalez-Arnao et al.
Table 8.2 Examples of plant species cryopreserved using cell suspensions and callus cultures
Abies cephalonica Aronen etal. (1999)
Asparagus officianalis Nishizawa etal. (1993); Uragami etal. (1989); Jitsuyama etal. (2002)
Betula pendula Ryynnen etal. (2002)
Brassica campestris Langis etal. (1989)
Bromus inermis Ishikawa etal. (1996)
Carica papaya Tsai etal. (2009)
Castanea sativa Corredoira etal. (2007)
Catharanthus roseus Van Iren etal. (1995); Bachiri etal. (1995)
Citrus deliciosa Perez etal. (1999); Aguilar etal. (1993); Engelmann etal. (1994)
Citrus sinensis Sakai etal. (1990); Engelmann etal. (1994); Hao etal. (2003)
Citrus spp. Perez etal. (1997)
Cyclamen persicum Winkelmann etal. (2004)
Elaeis guineensis Chabrillange etal. (2000); Dumet etal. (2000)
Festuca spp. Wang etal. (1994)
Fragaria spp. Wu etal. (1997)
Grape Engelmann etal. (1994); Dussert etal. (1992)
Hevea brasiliensis Engelmann and Etienne (2000)
Hordeum vulgare Fretz and Lrz (1995)
8 Advances in Cryogenic Techniques for the Long-Term 141
8.6Conclusion
In this chapter we have attempted to illustrate how cryostorage has advanced and
become an important biotechnological tool with the realistic target of supporting
the management and the long-term conservation of plant biodiversity. Since the first
report on plant cryopreservation, the most important progress has been related to
the successful application of cryogenic techniques to different germplasm forms of
species which previously could not tolerate cooling in liquid nitrogen. In addition,
cryopreservation currently provides complementary solutions for the elimination
of systemic pathogens, and potentially ensures plant germplasm security useful to
breeding programs.
As reviewed in this chapter, various criteria should be considered to select the
most appropriate cryopreservation procedure depending on the biological material.
In fact, the formulation of any cryopreservation protocol always requires the input
of theoretical and practical expertise from diverse disciplines, in order to define the
most suitable and adaptable cryogenic (cryoprotectants and cooling rate) and non-
cryogenic (pre- and post-storage culture) factors.
Although there are general guidelines to follow, there is no universal proto-
col that can be used for all groups of plants, and this is due to the specific physi-
ological and biochemical characteristics of each species, which demand optimizing
protocols according to each individual behavior. However, the standard procedures
already developed are a very helpful starting point, because they can often be adapt-
ed to other groups of species with only minor modifications. In this context, it is
important to continue advancing our understanding of different protective mecha-
nisms, such as the conflicting effect of protection and toxicity resulting from the
use of highly concentrated cryoprotective solutions and their role in enhancing a
glassy state to avoid intracellular crystallization of water and lethal injuries. The
better understanding of the stress conditions involved in cryopreservation protocols
will allow improving the tolerance of explants to dehydration and cryopreservation,
thereby improving the efficiency of cryostorage for present and future use.
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Chapter 9
Biotechnology in Biodiversity Conservation:
Overview of its Application for Conservation
of Endangered African Tree Species
9.1Background
threatened species or those at risk in one part and extinction of many species (animals
and plants) in other part is becoming more perceptible. Extinction rate was estimated
100200 times higher than historical natural level (Dudley and Parish 2006). Accord-
ing to Engelmann (2010), the number of plants recorded as critically endangered on
IUCN Red List has increased by 60%during the period 19962004. However, for-
est species are among the one taxon that is mostly at risk. In Africa, over 1000 tree
species have been reported to be threatened with various levels of threat (WCWM
1998). Regarding the human population growth trend, biodiversity declining will be
more and more exacerbated in Africa in future. Indeed, in 1900, the population of
Africa was about one-quarter that of Europe one; by 2000, the two regions had about
the same demographic weight, and by 2050, Africa population will be three times
larger than Europe (Hirschman 2005). Thus, conservation of biodiversity in general
and particularly threatened African tree species, is an important task concerning the
human scientist population worldwide and mainly the ones in Africa. In this frame,
plant biodiversity is receiving great importance. In fact, plant biodiversity is a natural
source of medicinal and food products. It provides raw materials for several indus-
tries and is able to supply genetic information required for developing sustainable
use, management and conservation of most crops (Rao 2004; Cruz-Cruz etal. 2013).
Moreover, biotechnology is become a rapid developing field that has received
considerable attention since discover of DNA. Thus, their activities have been in-
tensively focused on plant resources in these last decades. It is applying on major
plant crop species as well as on tree species. Its use is allowing breeding programs
for improving crop species productivity and their resistance against biological and
environmental stress. Although biotechnology activities on plants resources have
recently increased rapidly (Dawson and Jaenicke 2006), tree species received little
attention on this matter. Biotechnology has been developed on few tree species and
those concerned were mostly from high income countries. However, African tree
species have been seldom emphasized by biotechnologies activities. This situation
is more exacerbated in case of endangered African tree species.
Additionally threatened tree species and particularly those endangered are re-
ceiving priorities in biodiversity conservation. Thus, conservation of endangered
African tree species by biotechnology should require a critical analysis for efficient
using of this tool in frame of biodiversity conservation. What is the actual situation
concerning biotechnology and endangered African tree species? What are problems
that prevent using of biotechnology in conservation of endangered African tree spe-
cies? How can we do in perspective to help biotechnology to conserve endangered
African tree species?
tree species are missing sufficient data for managing and designing their sustain-
able use or conservation. Indeed, although some African tree species were reported
as threatened with various levels of threats on international Red List of UICN, it
remains also many others ones without sufficient data and therefore their status is
misunderstood. In the tropics, in general and in Africa mainly, the flora was least
described (Maxted etal. 1997). Thus, many African tree species that are endangered
were not always mentioned in a report due to the lack of information on their dis-
tribution, biology, ecology etc. Considering endangered African tree species that
have been listed by international or local reports, knowledge related to conservation
ecology has been mostly highlighted on these tree species. For instance, in spite
of the fact that several African studies (Sinsin etal. 2004; GllKaka etal. 2009;
Adjonou etal. 2010; Houehanou etal. 2011, 2013), focused on endangered African
tree species, those studies addressed mostly ecological researches questions. More-
over, few applications were delighted from these ecological researches to secure
conservation or sustainable uses of these tree species. This is a general problem of
researches in low-income countries. Indeed, researches in developing countries are
mostly funding by high-income or developed countries and therefore, applications
are not always easy to be done.
Over the world, biotechnology activities as conservation strategies are advanc-
ing. Regarding taxa coverage by biotechnologies activities, more than 140 genera
received biotechnologies activities in the entire world but most of them took place
in high-income regions (Dawson and Jaenicke 2006). FAO (2004) recorded bio-
technologies activities in 76 countries with 71 and 3% undertaken respectively in
high-income countries and in Africa. Most of biotechnologies activities of Africa
were concentrated in South Africa (FAO 2004). Few biotechnological studies were
undertaken in others parts of Africa on endangered African tree species. Those
existent studies, concerned characterization of genetic structure of some endan-
gered tree species such as Adansonia digitata (Kyndt etal. 2009), Milicia excelsa
(Bizoux etal. 2009) and Vitis vinifera ssp. Sylvestris (Zoghlami etal. 2013). This
state of knowledge is showing that African taxa are very few covered at present
by biotechnologies activities. Consequently, from state of knowledge it can be
concluded that endangered African tree species have not taken advantage of bio-
technologies strategies yet, although their conservation is a priority in biodiversity
conservation frame.
9.3What is Biotechnology?
Biotechnology could be split up in two simply words such as biology and technol-
ogy. It is then any technological application that uses, living organisms or deriva-
tives, to make or modify products or processes for specific use. Thus, it concerns a
range of scientific tools that, provides powerful methods for the sustainable devel-
opment of agriculture, fisheries and forestry (Dawson and Jaenicke 2006).
174 T. D. Houehanou et al.
Genetic modification
Genetic modification is the use of recombinant DNA and asexual gene trans-
fer methods that alter the structure or expression of specific genes and traits
(Dawson and Jaenicke 2006). A genetic modified organism or transgenic, is one
that has been obtained by the insertion of one or more genes from another organ-
ism. Active research in this area has been ongoing since the 1980s.
There are two approaches (in situ and ex situ) for conservation, which are globally
used to conserve plant biodiversity in the entire world. In situ conservation involves
maintaining plant biodiversity in their natural areas while ex situ conservation on
the other hand, involves conservation outside the natural area (Rao 2004). Accord-
ing to this last author ex situ conservation approaches is generally suitable to con-
serve in danger species population. Therefore, ex situ conservation strategies should
be used mostly on endangered tree species. However, as the global strategy of plant
conservation states that at least 60% of threatened plant species should be within
protected areas (Vellak etal. 2009) there is increasing concern about the extent
to which in situ conservation strategies contribute to conserve mostly endangered
plant species. Thus, in situ and ex situ conservation approaches should be combined
to design conservation strategies and secure conservation of endangered tree spe-
cies particularly the African ones.
By past, most biotechnologies activities for conservation approaches focused
on crop species and agroforestry ones. This is revealing that wild tree species were
neglected in conservation strategies. Although, ex situ conservation approaches is
seen sometime as the only option for conserving some highly endangered and rare
species (Ramsay etal. 2000), the traditional conservation approach of in situ is used
generally to conserve wild tree species. Then, in the case of wild tree species, con-
servation approaches of ex and insitu could be combined for their conservation. In
this context, botanic gardens that play important role in ex situ conservation of plant
biodiversity (Engelmann 2010), are able tool that combine sometimes ex and in situ
conservation approaches. Indeed, in the botanic garden, some plants that are present
can be in their natural areas while others ones can be outside of their natural area.
Highlighting importance of botanic gardens in plant biodiversity conservation, it
has been estimated that botanic gardens conserve more than one third of the worlds
flowering plants which among more than 15,000 threatened species have been iden-
tified (UNEP 1995; Engelmann 2010; http://www.bgci.org/ourwork/1977/). This is
involving that Botanic gardens over the world are guarantying conservation of most
endangered African tree species.
Apart of Botanic gardens, value of agroforestry ecosystems for conserving
plant biodiversity through tree species diversity, has become more widely rec-
ognized by several researches (Kindt 2002; Kirschenmann 2007; Dawson etal.
2009). This approach named circa situ conservation permitted conservation of lots
tree species among which some endangered African tree species, in agricultural
landscape such as Adansonia digitata, Milicia excelsa etc. Thus, conservation of
diversity of endangered African tree species by agroforestry ecosystems is pro-
moting world widely.
9 Biotechnology in Biodiversity Conservation 177
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180 T. D. Houehanou et al.
Anca Manole-Paunescu
10.1Introduction
Concerns about biodiversity loss have made necessary complex studies and ap-
proaches. As a result, in March 2005, the Millennium Ecosystem Assessment (MA)
was released. This is a comprehensive report drawn up by 1300 researchers from
95 nations over 4 years, and funded by the Global Environment Facility, the United
A.Manole-Paunescu()
Department of Vegetal and Animal Cytobiology, Institute of Biology, Bucharest
297 Splaiul Independentei street, 060031 Bucharest, Romania
e-mail: anca.manole@ibiol.ro
Nations Foundation, the World Bank and others. One of the key conclusions of this
report is that Everyone in the world depends on nature and ecosystem services to
provide the conditions for a decent, healthy, and secure life. In the same report is
also highlighted that substantially and largely irreversible loss in the diversity of
life on Earth, occurred in the last century. The Millennium Ecosystem Assessment
analyzed for the first time the conditions and the trends in the world ecosystems
and services, they provide and underline two distinct focal points of biodiversity
and ecosystem functioning (BEF) and biodiversity and ecosystem services (BES)
research. These researches reveals how species and functional diversity of organism
control basic ecological processes and also how major habitat modifications influ-
enced provisioning and regulating services of ecosystems (Cardinale etal. 2012).
According to the latest version of the Red List of Threatened Plants issued by
International Union for Conservation of Nature (IUCN), from the 18,292 listed spe-
cies, 10,065 are threatened (IUCN 2013). These figures indicate that approximately
4% of described plant species are threatened.
Causes of plant endangerment are numerous and include:
habitat alteration, fragmentation or destruction
introduction of exotic species, intentionally or accidentally, with competitive ad-
vantages over native species
over-collecting, overexploiting and unsuitable use
air, soil, and water pollution
urbanisation
unsuitable agriculture, farming and forestry practices
global warming, severe droughts, salinization
natural causes (overspecialisation, loss of genetic diversity, catastrophic events)
(Pitman and Jorgensen 2002).
The most vulnerable plant species originate mainly from resource-poor areas of the
world, from global biodiversity hotspots and island countries. In the past, 15.7% of
the earths land surface was covered by the 34 global biodiversity hotspots which
host more than 50% of the worlds endemic plant species. Presently, global bio-
diversity hotspots area covers only 2.3% of the earths land surface. Each hotspot
faces extreme threats and has already lost at least 70% of its original natural veg-
etation (Reed etal. 2011). A broad international initiative for an integrate plant
conservation was taken under The Convention on Biological Diversity by adopting
the recommendations of the Gran Canaria Declaration for a Global Strategy for
Plant Conservation, which presently is signed by 220 countries. Under the Target
8, proposed for period 20112020, is stipulated that At least 75% of threatened
plant species in ex situ collections, preferably in the country of origin, and at least
20% available for recovery and restoration programmes (GSPC 2011). As a con-
sequence several conservation strategies were developed mainly in the terms of in
situ and ex situ conservation.
In situ conservation refers to on-site conservation of species and populations on
their natural habitats. Protection of natural areas is the main way to preserve natural
10 Biotechnology for Endangered Plant Conservation 183
biological resources. Protected areas remain one of the cornerstones for promoting
biodiversity, ecosystem services and human well-being. By 2012, the 177,547 na-
tionally designated protected areas around the world, listed in the World Database
on Protected Areas cover 12.7% of the worlds terrestrial area and 1.6% of the
global ocean area. They store 15% of the global terrestrial carbon stock, assist in
reducing deforestation, habitat and species loss, and support the livelihoods of over
one billion people (Bertzky etal. 2012).
In situ biodiversity preservation through the establishment of protected areas of-
fers distinct advantages over ex situ methods in terms of coverage, viability of the
resource, and the economic sustainability of the methods. Establishing a conserva-
tion area does not mean that its biodiversity is under complete protection and with-
out risk. Some risks, both natural and anthropogenic, remain. Habitat degradation
or elimination to make way for human settlement and associated development ac-
tivities is the most important risk factor contributing to the biodiversity loss. These
hazards can only be met with a full array of conservation programs, including those
that use ex-situ methods.
Ex situ (off-site) conservation involves preservation and maintenance of samples
of living organisms outside their natural habitat, in the form of whole plants, seed,
pollen, vegetative propagules, tissue or cell cultures. Ex situ techniques are gener-
ally used to complement in situ methods but in some cases are the only possible
techniques to conserve certain species (Ramsay etal. 2000). Among ex situ conser-
vation methods, the most common are cultivation in botanic gardens, seed storage,
and in vitro cultivation.
The worlds 2500 botanic gardens cultivate more than 100,000 species mean-
ing almost one third of the known vascular plant species of the world. Although
cultivation in botanic gardens is an efficient way to conserve endangered species
ex situ, it is limited in time and space and it has to overcome acclimatisation and
accommodation problems.
Among the various ex situ conservation methods, seed storage seems to be one
of the most convenient for long-term conservation. This involves desiccation and
storage at low temperatures. Many plant species produce so-called orthodox seeds
which support desiccation and can be stored for long periods. For example, an
experiment by the Arava Research Institute (Israel), an ancient seed of Phoenix
dactylifera (naturally desiccated in Israels warm and dry environment) has been
germinated and a date palm seedling produced. The seeds were recovered from
a jar during the excavations of Masada in the 1970s and were then kept in store.
Radiocarbon dating has confirmed that the seeds are around 2000 years old, mak-
ing this the oldest seed to have been successfully germinated (Sallon etal. 2008).
Currently, all over the world are established seed collections onto so-called seed
banks. For example, almost 24% of the endangered Spanish flora is stored in the
Plant Germplasm Bank of the Universidad Politecnica de Madrid as seed acces-
sions (Gonzales-Benito and Martin 2011) as well as more than 200 of endangered
Australian plant species which are accessioned in the Threatened Flora Seed Centre
and Western Australian Seed Technology Centre (Kaczmarczyk etal. 2011). The
184 A. Manole-Paunescu
most extensive projects for seed collections are Millennium Seed Bank Partnership
and The Svalbard Global Seed Vault. The Millennium Seed Bank Partnership is
considered the largest ex situ wild plant conservation project in the world. With a
network of partners across 80 countries, within this project over 11% of the worlds
wild plant species seeds were already banked. Also, aims to save 25% of those plant
species with bankable seeds until 2020 (about 75,000 species). The Svalbard Global
Seed Vaults mission is to preserve genetic diversity of the worlds food crops and
to provide a safety net against accidental loss of diversity in traditional gene banks.
Presently, more than 770,000 different seed samples are deposited in the vault for
long-term storage.
However, there are a large number of threatened species, which produce im-
mature, sterile or recalcitrant seeds that quickly lose viability and do not survive
desiccation. Predominantly tropical and sub-tropical species have recalcitrant or in-
termediate seeds, which do not support desiccation and low temperature treatments,
hence conventional seed storage strategies are not suitable (Engelmann and Engels
2002). There are estimated to be 5000 or more endangered species for which con-
ventional conservation methods are not adequate (Pence 2013). For these species,
biotechnological tools offer a valuable alternative for conservation. The concept of
biotechnology encompasses a wide range of procedures, but when refers to plants
(green biotechnology) is associated mainly with recombinant DNA technology and
with the designing of transgenic plants. Plant biotechnologies include advanced
tools like designing synthetic promoters, tunable transcription factors, genome-
editing tools and site-specific recombinases, as well as assembly and synthesis of
large DNA molecules, plant transformation with linked multigenes and design plant
artificial chromosomes in order to produce new products in plants and to generate
plants with new functions. When refers to conservation, biotechnologies include
mainly in vitro techniques like cell and tissue culture, micropropagation as well as
cold storage and cryopreservation. Several in vitro techniques have been developed,
mostly for vegetatively propagated and recalcitrant seed producing species, with
recent establishment of extensive germplasm collections (Engelmann and Engels
2002; Sarasan etal. 2006; Paunescu 2009b). This chapter aims to summarise the
biotechnological tools implemented worldwide for plant conservation with specific
examples of some successful accomplishments.
The term in vitro culture covers a wide range of techniques involving the growth
under sterile conditions of plant tissues (especially shoot tips, axillary meristems,
somatic embryos or embryogenic callus) on artificial semi-solid culture media. Al-
though each species require specific protocols, there are some common steps in es-
tablishing an in vitro collection: culture initiation, maintenance and multiplication,
followed by long-term storage.
10 Biotechnology for Endangered Plant Conservation 185
10.2.1Culture Initiation
The potential explant (the starting tissue originated from the donor plant) consist
mostly of shoot meristem (apical or axillary), leaf tissues (lamina segments with
ribs, petiole), flower pieces, immature embryos, hypocotyl fragments or cotyledons.
Generally, younger, more rapidly growing tissue or tissue in early developmental
stage are the most effective. Therefore, the initial quality of the explants will de-
termine the success of the conservation procedure. The criteria for a good quality
explants are: normal, true to type donor plant, vigorous and disease free (Fay 1992).
Plant fragments are initiated into axenic culture from various sterilization proce-
dures depending of the tissue used. As a common rule, fragile tissues (meristems,
immature embryos, cotyledons, hypocotyls) requires less exposure to sterilizing
agents than seeds or lignified organs. A successful sterilization is achieved when
the explant is fully decontaminated and remains viable. An alternative for obtain-
ing uncontaminated explants is to obtain explants from seedlings, which are asep-
tically grown from surface-sterilized seeds. Another approach, known as in vitro
collecting, is to introduce the explant in vitro under aseptic conditions directly in
the field (Withers 1995, 2002). This method is a valuable alternative for rare and
endangered species since this material is limited in supply and seed collection is re-
stricted (Cruz-Cruz etal. 2013). Also, in vitro collecting allows germplasm collec-
tion of species without seeds, or storage organs, or when buds would quickly loose
viability or is highly contaminated (Engelmann and Engels 2002). Disinfestation
protocols vary considerably from single step to more complex procedures involv-
ing a variety of sterilizing agents including chemicals (liquids or gases) or physical
agents. Most procedures consists in pre-treatments with 7095% ethanol, followed
by immersion in broad-spectrum biocide solution like hypochlorite solutions (so-
dium or calcium), sodium dichloroisocyanurate, mercuric chloride, silver nitrate,
bromine water, hydrogen peroxide, silver nitrate, etc. (Sarasan etal. 2006; Pau-
nescu 2009b). More elaborated protocols could include gas sterilization, ultrasonic
cleaner and ethanol dip and flame. Such protocols were reported to be successful
in establishment of embryo or ovule in vitro cultures of some endangered Hawaiian
taxa (Sugii 2011).
After sterilization step, the explant is placed on a sterile culture media containing
mineral nutrients, vitamins, carbohydrates, and plant growth regulators (Murashige
and Skoog 1962). Plant growth regulators are the critical media components in de-
termining the developmental pathway of the plant cells. A balance between auxin
and cytokinin growth regulator is most often required for initiation of the morpho-
genetic events. The interactions found are often complex and, more than one com-
bination of substances is likely to produce the desired results. Seven major types of
growth regulators are generally recognised:
auxins, usually involved in cell enlargement and differentiation
cytokinins associated with cell division
gibberellins stimulate cell elongation, seed germination
abscisic acid involved in dormancy by inhibiting cell growth
ethylene often associated with senescence
186 A. Manole-Paunescu
One major step in establishing in vitro germplasm collections is induction and mul-
tiplication of shoots. This step could be often critical for some species, particularly
woody plants. The media composition, especially the growth regulators, mineral
salts and supplement factors are of paramount importance to successfully obtain
viable tissue culture. In the last 20 years, there were many reports in developing
micropropagation protocols for endangered plant species, all over the world (Ta-
ble10.1). According to Sarasan (Sarasan etal. 2006), at Conservation Biotechnol-
ogy Unit (previously known as Micropropagation Unit) from Royal Botanic Gar-
dens, Kew, are micropropagated and maintained more than 3000 endangered plant
taxa, from all over the world. Under a national initiative several research Spanish
groups have developed micropropagation protocols for more than 60 endemic and
endangered species, most of them belonging to Plumbaginaceae and Asteraceae
families (Gonzales-Benito and Martin 2011). Since 1998 the Hawaiian Rare Plant
Program (Lyon Arboretum, Honolulu, Hawaii) has been successful in establishing
in vitro cultures of 135 endangered plant taxa (Sugii 2011). Some successful multi-
plication strategies were developed for a number of endangered plants in Romanian
flora (Paunescu 2009b). A recent overview of in vitro conservation technologies for
Eastern Australian endangered plant taxa was published by Ashmore etal. (2011)
including Alloxylon flammeum, Citrus spp., Davidsonia spp., Diploglotis campbel-
lii, Macadamia tetraphylla, Pimelea spicata, Stackhousia tryonii, Syzygium spp.,
Tectaria devexa, Wollemia nobilis and ten selected orchid taxa. A significant num-
ber of other rare native species from Australian flora are also preserved in vitro
collections in Kings Park and Botanic Garden, Australia (Kaczmarczyk etal. 2011).
During the last 25 years of research within Tropical Botanic Garden and Research
Institute, Palode (India) were developed in vitro protocol for rapid regeneration and
establishment of more than 40 medicinal rare and threatened plants from Western
Ghats (Krishnan etal. 2011). As well, some examples of successful micropropa-
gated endangered plants from South Africa, was recently reviewed, including
Haworthia limofolia and Siphonochilus aethiopicus (Berjak etal. 2011). Most of
the reviewed culture protocols were developed onto Murashige and Skoog basal
10 Biotechnology for Endangered Plant Conservation 187
Table 10.1 Selected endangered species for which micropropagation protocols has been reported
within last two decades
Species Explant type Culture media Author
Dianthus bohemicus Nodal segments MS Kovc (1995)
BAP1mgl1
Globularia ascanii Apical buds MS Cabrera-Prez (1995)
BAP1mgl1
NAA0.1mgl1
Trillium persistens Dormand buds 1/2MS Pence and Soukup
BAP1mgl1 (1995)
NAA0.1mgl1
Vella lucentina Apical buds MS Lled etal. (1995)
BAP0.52mgl1
Leontopodium alpinum Young inflorescences MS Zapartan (1996)
BAP2mgl1
IAA2mgl1
Gentiana cerina Axillary buds MS Morgan etal. (1997)
G. corymbifera BAP0.050.5mgl1
Saussurea lappa Shoot apex MS Johnson etal. (1997)
TDZ0.1mgl1
Hypericum foliosum Axillary buds CM Moura (1998)
BAP1mgl1
NAA0.5mgl1
Holostemma annulare Shoot apex, nodal MS Sudha etal. (1998)
segments BAP1mgl1
NAA0.1mgl1
Dianthus superbus nodal segments MS Mikulik (1999)
BAP1mgl1
Astragalus pterfii Nodal segments, foliar MS Suteu etal. (1999)
and flower cuttings BAP0.5mgl1
NAA0.3mgl1
Heracleum candicans Axillary buds MS Wakhlu et al. (1999)
BAP0.5mgl1
NAA0.1mgl1
Salvia blancoana Shoot apex MS Cuenca and Amo-
Nodal segments 2iP1mgl1 Marco (2000)
Salvia valentina Shoot apex MS Cuenca and Amo-
Nodal segments Kin12mgl1 Marco (2000)
Primula scotica Seedlings cuttings MS Benson etal. (2000)
BAP1mgl1
IAA0.1mgl1
Piper barberi Nodal segments MS Anand and Rao (2000)
BAP1mgl1
Kin0.5mgl1
Ochreinauclea Nodal segments MS Dalal and Rai (2001)
missionis BAP2mgl1
Ensete ventricosum Nodal segments MS Negash etal. (2001)
BAP24mgl1
Allium wallichii Shoot cuttings MS Wawrosch etal. (2001)
Ze4mgl1
Aerides maculosum Leaf cuttigs MS Murthy and Pyati
BAP2mgl1 (2001)
188 A. Manole-Paunescu
10.2.3Storage of Collections
In vitro storage techniques include the medium-term storage (for a determined pe-
rioda few months up to a few years) using slow growth strategy or artificial seed
production, and long-term storage (tentatively for an indeterminate period of time)
using cryopreservation. In slow growth, cultures are kept under the level of optimal
growth conditions. Generally, there are three recognised methods for reducing in
vitro growth rates, including physical (reduced temperature and light conditions),
chemical (using growth retardants), and a combination of the two (Engelmann and
Engels 2002). Temperature will vary upon the origin of stored species; temper-
ate species may be stored at 4C, whereas the tropical plants are requiring tem-
peratures in the range of 1520C. Light conditions may be darkness or a 1216h
photoperiod, the light intensity varying upon the light requirement of the species
stored. The humidity should be between 4050%. Some effective approaches for
slow growth include reduction of the oxygen level available achieved by cover-
ing explants with a layer of liquid medium, paraffin or mineral oil, or by placing
them in controlled atmosphere (Engelmann and Engels 2002). By optimization of
all parameters, the subculture time is greatly enhanced, the quality of the stored ma-
terial is maintained and the recovery of the shoot proliferative potential is assured.
The subculture periods could be extended from 12 month up to 4 years for many
plant species (Ashmore 1997). Although slow growth techniques are available for a
wide range of plant species, there are used in practice only for a limited number of
endangered species. This is mainly because the need for regular subculture raises
the risk of contamination and occurrence of somaclonal variation. For example,
this technique is applied only for a very few Malaysian species like Nephelium
lappaceum (shoot cultures at 18C with subcultures at 12 weeks), and Lansium
domesticum seeds onto full-strength Murashige and Skoog medium (Murashige and
Skoog 1962) at 25C with two subcultures in 15 months (Normah etal. 2011).
Slow growth storage strategy is also used to preserve some endangered phytotaxa
in the Institute of Biology (Bucharest) in vitro collections. The endangered species
conserved by medium-term tissue culture are Artemisia tschernieviana, Astragalus
pseudopurpureus, Cerastium transsilvanicum, Dianthus callizonus, D. spiculifo-
lius, D.tenuifolius, Erigeron nanus, Hieracium pojoritense, and Marsilea quadrifo-
lia (Paunescu 2009b). Minimum growth techniques have been also used for some
Spanish endemics (Centaurium rigualii, Coronopus navasii, Lavatera oblongifolia,
Limonium calaminare, L. catalaunicum, L. dichotomum, L. dufourii, L. estevei, and
L. gibertii) with relative success, reduction of temperature being the most effective
192 A. Manole-Paunescu
way of decreasing growth (Gonzales-Benito and Martin 2011). Slow growth condi-
tions were employed for medium term conservation of 16 threatened species and
subspecies of the genus Turbinicarpus (Cactaceae), all native to the Chihuahuan
Desert in Mexico, using osmotic agents (sorbitol and mannitol) and temperatures of
4C (Prez-Molphe-Balch etal. 2012).
Artificial seeds (synthetic seeds, manufactured seeds) were first introduced in
the 1970s as a novel analogue to the plant seeds, useful for propagation and medium
term storage (Redenbaugh etal. 1988). Artificial seeds are produced by encapsulat-
ing a plant propagule in a matrix, which will allow it to grow into a plant. A typical
synthetic seed has following parts:
plant propagule
matrix (synthetic gametophyte)
artificial coat (membrane)
Plant propagules may consist of shoot buds, shoot tip, somatic embryos, or any other
competent aggregate cells, able to regenerate the whole plant. The most used plant
regenerative units in artificial seed production are somatic embryos in post-heart
or early cotyledonary stage. They are enclosed in gel agents like: alginate, agarose,
polyoxyethylene, urethane polymers, guar gum, sodium pectate, carrageenan, poly-
acrylamide, etc. Among these, alginate encapsulation was found to be more suitable
and practicable. Alginate hydrogel is frequently selected as a matrix for synthetic
seed because of its moderate viscosity and low spinnability of solution, low toxic-
ity for propagules and quick gellation, low cost and bio-compatibility character-
istics. The chosen propagules are mixed with sodium alginate and the suspension
is dropped into the calcium salts solution. The principle involved is when sodium
alginate dropped into the calcium salt solutions it from round firm beads due to the
ion exchange between Na+ in sodium alginate and Ca2+ in calcium slat solutions
and sodium alginate form calcium alginate. The rigidity of capsular membranes de-
pends on concentration of the two solutions. Generally, a 3% (w/v) sodium alginate
solution combined with a 75mM calcium salt, generates a firm and efficient mem-
brane. The matrix of encapsulation should be enriched with nutrients and growth
regulator, which will serves as an artificial endosperm. This will increase the ef-
ficiency of germination and of seed viability. Superfluoro chemical oils are used as
oxygen carrier and are often mixed with gel. It increases oxygen supply and helps in
keeping the seed viable for longer time. Other materials like fungicides, pesticides,
herbicides, insecticides, antibiotics and mycorrhizal fungi or bacteria can also be
incorporated. Generally, there are recognized various types artificial seeds which
include: (i) uncoated non-quiescent somatic embryos, (ii) non-quiescent somatic
embryos in a hydrated encapsulation and, (iii) dehydrated quiescent somatic em-
bryos (Ravi and Anand 2012).
When cultured, the seed coat softness allowing the propagule to resume growth,
enlarging and emerging from the encapsulation. The advantages of propagules en-
capsulation include multiplication of recalcitrant or non-seed producing species,
easier manipulation of fragile tissues, and direct protection during dehydration and
thawing during the cryopreservation trial (Saiprasad 2001). Synthetic seed technol-
10 Biotechnology for Endangered Plant Conservation 193
ogy was applied for conservation purposes to various plant species. Particularly,
the method was successfully applied for a number of endangered orchid species. It
involves protocorms-like bodies (orchid somatic embryos, Lee etal. 2013) encap-
sulation and shows high efficiency, for example 70% viability after more than 6
months storage at 4C (Devi etal. 1998).
Despite of their availability the in vitro medium term storage techniques are
used routinely in a few botanical gardens, research institutes, universities, or other
worldwide conservation centres. This is because such techniques require high tech
equipment, trained staff and large storage spaces. Also, culturing with periodic re-
freshment of medium is laborious and subsequent culturing increases the risk of mi-
crobial contamination and somaclonal variation. Another disadvantage is that under
medium term storage the material could be preserved only for a limited period (up
to a few years).
To avoid the genetic alterations that may occur in long cultures storage, experi-
mental protocols have been developed to preserve germplasm at very low tem-
peratures known as cryopreservation (Martin etal. 1998). The temperature used are
those of liquid nitrogen (196C) or its vapour phase (150C). At these tempera-
tures, all metabolic activity is suppressed minimizing the risk of genetic alterations
and eliminating the requirement for refreshing the culture medium. Furthermore,
cryopreserved material is stored in a small volume for a theoretically indefinite
time. Also, it requires minimal space and maintenance, such as replenishment the
storage container with liquid nitrogen (Kaczmarczyk etal. 2011). The technology
is effective for a broad range of phytotaxa including unicellular to flowering higher
plants (Reed 2008). Cryopreservation provides a safe and cost-effective method for
the long-term storage of genetic resources. Apart from the use in plant long-term
conservation, exposure to extreme low temperature proved to be very effective in
elimination of systemic plant pathogens (viruses, phytoplasmas and bacteria), pro-
cedure known as cryotherapy (Wang and Valkonen 2009). Cryopreservation as a
conservation tool has been underlined by a number of authors (Engelmann 2004;
Stacey etal. 1999) and presently is recognised as the most effective technique for
long-term storage of plant germplasm. Under low temperatures tissues proved to re-
main viable for very long periods of time. Recently, was published a report concern-
ing whole plant regeneration using in vitro tissue culture of a species originate from
Arctic tundra (Silene stenophylla Ledeb., Caryophyllaceae) from maternal, fruit
tissue, of Late Pleistocene age. The fruits were isolated from permafrost deposits
of about 30,000-year-old. The regenerated plants successful developed flowers and
fertile seeds (Yashina etal. 2012). Presently, a number of studies are dedicated to
the effect of low temperatures upon living tissues and its applicability in plant long-
term conservation. Pioneer researches about successful cryopreservation of plant
cell suspension (Quatrano 1968) and regeneration of somatic embryos from cryo-
preserved cells (Nag and Street 1973) have led to numerous studies on cryopreser-
vation of plant system (Frinkle etal. 1985; Kartha 1985; Prithcard and Predergast
1986). Cryopreservation technique is based on the removal of all freezable water
from tissues by physical or osmotic dehydration, followed by ultra-rapid freezing,
resulting in vitrification of intracellular solutes. Vitrification refers to transition of
194 A. Manole-Paunescu
water directly from the liquid phase into an amorphous phase, whilst avoiding the
formation of crystalline ice (Fahy etal. 1984). Vitrification-based procedures of-
fers some advantages being more appropriate for complex structures (shoot tips,
buds, embryos) which contain a variety of cell types, each with unique require-
ments under condition of freeze-induced dehydration (Engelmann 2011). The main
advantages are simplicity and the applicability to a wide range of genotypes. Cur-
rently, are identified seven different vitrification-based procedures: (1) encapsula-
tion-dehydration, (2) vitrification, (3) encapsulation-vitrification, (4) dehydration,
(5) pregrowth, (6) pregrowth-dehydration, and (7) droplet-vitrification (Engelmann
2004, 2011). The encapsulation-dehydration procedure is based on the technology
developed for the production of artificial seeds. The method is suitable for shoot
apices, cell suspensions and somatic embryos (Engelmann 2011). This technique
was employed also for nodal explants of some Spanish endemic species (Gonzales-
Benito and Martin 2011). Vitrification consists of placing explants in the presence
of highly concentrated cryoprotective solution followed by rapid freezing. Encap-
sulationvitrification is a combination of the first two, where explants are encap-
sulated in alginate beads and treated with vitrification solutions before freezing.
Vitrification methods were successfully used for cryopreservation of more than 30
species of Western Australian endangered plants (Kaczmarczyk etal. 2011) as well
as for shoot tips of Centaurea ultreiae a critically endangered species from Spain
(Gonzales-Benito and Martin 2011).
Dehydration is the simplest procedure and consists of dehydrating explants and
freezing them rapidly by direct immersion in liquid nitrogen and it may be used
for freezing zygotic embryos or embryonic axes extracted from seeds (Engelmann
2004; Dumet etal. 1997). It was also successfully used for freezing the seeds of
68 endangered Western Australian species (Touchell and Dixon 1993), several
endangered species originate from Eastern Australia including indigenous Citrus
(Ashmore etal. 2011) as well as for the seed of some rare temperate orchid species
(Nikishina etal. 2007). Pregrowth involves in vitro culture of explants in the pres-
ence of cryoprotectants followed by rapid freezing by immersion in liquid nitrogen.
Pregrowth-dehydration follows the same steps like pregrowth method with the dif-
ference that explants are dehydrated under laminar airflow or with silica gel prior
the immersion in liquid nitrogen. Droplet-vitrification is the newest developed and
consist in pre-treatment of the explants with vitrification reagents then placed in
minute droplets of vitrification and finally frozen in liquid nitrogen (Engelmann
2011). This technique has been used for shoot apices of Thymus moroderi, an en-
demic of Southeast Spain (Marco-Medina etal. 2010), shoot tips of wild potatoes
(Yoon etal. 2007) and for two Diospyros species (Niu etal. 2009).
A critical step in developing a reliable cryopreservation protocol is the regenera-
tion of plants after a determined period (as long as possible) of exposure to extreme
low temperatures. Plant regeneration is dependent on genotype, age and physiologi-
cal state of the culture, cryoprotectant treatment, rate of freezing, method of thaw-
ing, etc. (Tsukara and Hirosawa 1992). The pregrowth phase of plant cells is consid-
ered a critical one because various changes may occur at the cellular level, including
a decrease in cell and vacuole size, changes in the flexibility and thickness of cell
walls and alteration of metabolic activities (Withers 1978). Somatic embryogen-
10 Biotechnology for Endangered Plant Conservation 195
10.4Conclusions
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202 A. Manole-Paunescu
AbstractAravalli hills are hot spot of subtropical plant biodiversity. The tribal
people of the region partially or fully depend upon herbal drugs for primary health-
care. Overexploitation of these plants has made several of them as endangered spe-
cies. The present paper aimed to document the biotechnological approaches being
used to conserve ethnomedicinal plants of Aravalli Hills, the bioactive molecules
present in them and their traditional uses and the modern scientific validation/assay
of biological activities. Plants of Aravalli hills are showing various promising bio-
logical activities and bioactive molecules. Though various biotechnological meth-
ods are attempted for enhanced production of these bioactive molecules and for
their micropropagation, yet the approaches are insufficient at mass scale level as
some of the endangered species may have unusual growth requirements and thus
may require modified procedures for in vitro culture. The review will be supportive
in deciding the priorities at various decision-making levels and further technology
development for sustainable use and conservation of these plants.
KeywordsBiotechnology Biodiversity Conservation Bacopa Centella
Commiphora Celastrus Curculigo Chlorophytum, Withania Pueraria
11.1Introduction
The demand for medicinal plants is continuously increasing not only in developing
countries but also in developed countries as drug, food supplement (nutraceuticals)
and cosmetics (Ramawat etal. 2004). Tyler defines herbal medicines as crude drug
K.G.Ramawat()
Botany Department, M. L. Sukhadia University,
221, Landmark Treasure Town, Badgaon, Udaipur, Rajasthan 313011, India
e-mail: kg_ramawat@yahoo.com
S.Goyal
5360 Rome Drive, Erie, PA, USA
J.Arora
Laboratory of Bio-Molecular Technology, Department of Botany,
M. L. Sukhadia University, Udaipur 313001, India
of vegetable origin utilized for the treatment of diseased state often of a chronic na-
ture or to attain or maintain a condition of improved health (Tyler 1994). If we look
at socio-economical scenario of the Asian and African countries, modern medicine
is neither affordable nor in reach of many villagers and tribes inhabiting remote ar-
eas and deep forests (Katewa and Jain 2006). Two of the largest users of medicinal
plants are China and India. Traditional Chinese medicine utilizes over 5000 plant
species while the major classical systems of medicine in Indian sub continent like
Ayurveda, Siddha, Unani, altogether use about 1200 plant species to treat human
ailments, but the tribals of India are using over more than 7500 plant species (Push-
pangadan 1994). However, Indias share in the world market is US$1billion as
compared to Chinas share of 6billion. Indigenous medicinal herbs provide about
75% of need for medicines of the third world countries (Rajshekharan 2002).
India is one of the worlds 12 hot spots having the largest plant biodiversity; it
has almost 45,000 plant species, out of which 15,00020,000 are used for medicinal
value (Ramawat and Goyal 2008). Aravalli ranges have about 8% of the flora of
India, consisting of 1378 species belonging to 126 families (Tiagi and Aery 2007).
Reserve sanctuaries of the region (Sitamata, Sajjangarh, Phulwari-ki-naal) harbor
rich biodiversity. Aravalli ranges dissect the state of Rajasthan into two parts: (1)
North-western desert, and (2) South-eastern hilly semi-arid forest. These ranges
lie between 25N to 7330E running approximately 800km from Delhi to Gujarat
states having highest peakGuru Shikhar in Mt Abu at 1722m. These geographical
conditions provide variable habitat for a wide range of flora including bryophytes,
pteridophytes, a lone gymnospermEphedra foliata, and subtropical angiospermic
flora (Arora etal. 2010a).
Historically, herbal drugs were used as tinctures, poultices, powders and teas
followed by formulations, and lastly as pure compounds. Medicinal plants or their
extracts are used by humans since time immemorial for different ailments and pro-
vide valuable drugs such as analgesic (morphine), antitussive (codeine), antihy-
pertensive (reserpine), cardiotonic (digoxin), antineoplastic (vinblastine and taxol)
and antimalarial (quinine and artemisinin). Some of the plants which continues to
be used from Mesopotamian civilization till today are Cedrus species, Cupressus
sempervirens, Glycyrrhiza glabra, Commiphora wightii, and Papaver somniferum
(Ramawat and Goyal 2008; Ramawat etal. 2009; Gurib-Fakim 2006). During
20002005, about two dozen new drugs derived from natural sources have been
approved by Food and Drug Administration, USA and put in market, which include
drugs for cancer, neurological, cardiovascular, metabolic and immunological dis-
eases, and genetic disorders. Seven plant derived drugs currently used clinically for
various types of cancers are taxol from Taxus species, vinblastine and vincristine
from Catharanthus roseus, topotecan and irinotecan from Camptotheca accumi-
nata, and etoposide and teniposide from Podophyllum peltatum. It is estimated that
market potential for herbal drugs in the world is forecast to reach $107billion
by the year 2017, spurred by growing aging population and increasing consumer
awareness about general health and well being, according to a new report from
Global Industry Analysts.
11 Biotechnological Approaches to Medicinal Plants of Aravalli Hills 205
11.2.1Micropropagation
C. trifolia (Roat etal. 2008), their production as influenced by plant growth regula-
tors (Roat and Ramawat 2009a) and biotic and abiotic elicitors in cell cultures (Roat
and Ramawat 2009b; Arora etal. 2010b) and in root cultures (Arora etal. 2009)
was reported. Use of an angiospermic elicitor was a novel finding in enhancing the
stilbene content of the cell cultures (Arora etal. 2010b).
Unlike C. trifolia, Centella asiatica (Apiaceae), popularly known as Mandu-
kaparni have different micropropagation protocols reported. The plant is a pros-
trate, stoloniferous herb grown in marshy areas. In India, it is found at the altitude
of 600m. The wild stock of this species has been markedly depleted due to its large
scale and unrestricted exploitation, and as a result, it is listed as threatened species
by International Union for Conservation of Nature and National resources (IUCN)
(Pandey etal. 1993) and an endangered species (Singh 1989). Hence, there is an ur-
gent need to conserve this valuable germplasm. Micropropagation work of C. asiat-
ica includes multiple shoot regeneration from nodal and shoots tip explants on MS
medium containing either BAP alone or in combination with Indole-3-acetic acid
(IAA), NAA and Kn. Maximum multiple shoots were found on MS supplemented
with 1.0mg/l BAP and 0.4mg/l NAA. Profuse healthy rooting was obtained on MS
medium containing 0.2mg/l Indole butyric acid (IBA). The well rooted plantlets
were successfully transplanted to the garden soil and their survival rate under natu-
ral conditions was 9095% (Jaheduzzaman etal. 2012). Multiple shoot induction
and in vitro flowering in C. asiatica was also reported (Gaddaguti etal. 2013).
Celastrus paniculatus (Celastraceae), commonly known as Malkangni is a
woody climber, traditionally used to stimulate intellect and to sharpen the memory.
C. paniculatus is valued for its seeds and seed oil is used commercially. Indiscrimi-
nate collection of this plant has threatened the species and ex-situ conservation is
the urge of the present scenario. To develop micropropagation methods, adventi-
tious root formation in semi hardwood cuttings with a 57% response was obtained
on the MS medium containing 3.0g/l IAA, rooted cuttings exhibited 100% survival
in the experimental field (Raju and Prasad 2010). In a highly efficient protocol,
maximum percentage of shoot multiplication (83.4%) with 8.2 shoots/explants
was achieved on MS basal medium supplemented with 0.5mg/l BAP and 0.1mg/l
NAA. After rooting and acclimatization, 91% of these plantlets survived at the
natural conditions. Random amplified polymorphic DNA (RAPD) and inter simple
sequence repeat (ISSR) marker study confirmed genetic stability of in vitro raised
plantlets by showing 100% monomorphism (Senapati etal. 2013). Similarly, shoot
multiplication was also optimized up to 47 shoots while confirming the genetic
stability of the plants (Phulwaria etal. 2013). Thus, these efforts help in process of
conservation of this plant.
Clitoria trenatea (Fabaceae), commonly known as Shankpushpi, is a peren-
nial climbing vine found in India, China, Philippines and Madagascar. The primary
method of propagation is by seed, but this method result in considerable variability
in phenology and reproductive traits among accessions. A reproducible protocol
for rapid mass propagation using cotyledonary nodal explants was developed. The
highest frequency (100%) and the maximum number of shoots (10.1) were induced
on MS medium supplemented with 1.5mg/l BAP. The highest rooting (100%) and
208 S. Goyal et al.
maximum number of roots (5.3) per shoot was obtained when shoots were dipped
in IBA solution (500mg/l) for 5min and further subcultured on half-strength MS
medium. Acclimatized plants grew normally in the field without showing any
morphological variation (Singh and Tiwari 2010). In vitro clonal propagation was
achieved by employing decapitated embryonic axes (DEAs) explants (Singh and
Tiwari 2012). Further work with different explants and thidiazuron on micropro-
propagation of C. ternatea resulted in establishing a protocol, which gave about
88% survival rate of in vitro developed plants in the fields (Mukhtar etal. 2012).
Chlorophytum borivilianum (Liliaceae) is a popular herb in traditional Indian
medicine commonly known as Safed Musli and constitutes a group of herbs used
as Rasayan or adaptogen. Due to poor seed set and viability, the plant is propa-
gated by root tubers containing a part of stem. This is a slow, tedious, labor oriented
method producing low yield of the root tubers (Arora etal. 2004). Therefore, many
in-vitro methods have been developed to conserve this beneficial plant including
somatic embryogenesis (Arora etal. 1999) and organogenesis (Dave etal. 2003,
2004). Through this protocol 15,000 plantlets were produced with 90% survival in
field conditions and the method was found cost effective (Dave etal. 2003, 2004).
Further, method of encapsulation and analysis of fidelity of the regenerants was
done using RAPD (Mathur etal. 2008; Arora etal 2006; Lattoo etal. 2006). Factors
like propagule size, subculture strategy, gelling agents, liquid pulse treatment of
BAP and vessel type were optimized for in vitro shoot multiplication in C. borivil-
ianum. These plants showed comparable or better growth and 90% survival than the
greenhouse hardened plants (Joshi and Purohit 2012). RAPD and ISSR analysis of
regenerated plants showed genetic similarity to mother plant (Kumar etal. 2010a).
In vitro tuberization was also reported in C. borivilianum using solid and liquid
culture systems (Farshad etal. 2013). These methods enable the cultivation of the
plants at very large acreage.
Commiphora wightii (Burseraceae), commonly known as Guggul, is a slow
growing tree of arid regions of western India. It is listed in IUCNs Red Data List
of threatened plants and now it is becoming endangered. The plant exhibits poor
regeneration and its population is fast depleting in its natural habitat, primarily due
to over-exploitation, unsustainable and destructive methods of gum-extraction (Jain
and Nadgauda 2013). Research on C. wightii has been supported by the Ministry of
Science and Technology (India) for the past 30 years. Many Biotechnological at-
tempts have been made in order to conserve this important medicinal plant (Kulhari
etal. 2012). Organogenesis has been induced through axillary shoot proliferation
from nodal segments, seedling explants, shoot tips, internodes and leaves, by differ-
ent group of workers (Soni 2010; Kant etal. 2010; Singh etal. 2010). Less number
of plantlet formation was major constraint in all these studies with low rate of es-
tablishment in the field (Kulhari etal. 2012). But in recent years few studies showed
some progressive propagation and field survival percentage of in vitro raised plants
from nodal segments of mature plants (Parmar and Kant 2012). With such not so
competent micropropagation protocol, alternative studies have been done which
indirectly assist in plant conservation by providing the important secondary me-
tabolites (guggulsterones) for which the plant is being destroyed.
11 Biotechnological Approaches to Medicinal Plants of Aravalli Hills 209
medium (Goyal and Ramawat 2008b). These cultures were scaled up-to 2L stirred
tank bioreactor (Sharma etal. 2009). Shoots developed from explants callus were
grown in growtek bioreactor with different aeration volume and maximum puera-
rin content was recorded with 20% v/v aeration, which was ~2.3 fold higher than
puerarin content recorded in control cultures (cultures grown in growtek without
aeration) (Sharma etal. 2011a).
Withania somnifera (Solanaceae), commonly known as Ashwagandha, (means
smell like a horse) is an annual plant and its tuberous roots are used for medicinal
purposes. According to a report multiple shoot cultures were developed from nodal
explants of field-grown plants on MS medium supplemented with BAP and IAA
with the addition of polyamine, spermidine. A total of 46.4 shoots were obtained
from nodal explants. After rooting these rooted plants were successfully hardened
and acclimatized with a survival rate of 100% (Sivanandhan etal. 2011). In another
study, cotyledonary nodes derived from axenic seedlings were used for micropropa-
gation. MS medium supplemented with BA was found to be optimum for produc-
tion of multiple shoots (100% shoot proliferation frequency and 16.93 shoots per
explant). Regenerated shoots were best rooted (95.2%, 38.7 roots per shoot) in half-
strength MS medium supplemented with IAA. The plantlets were successfully ac-
climated and established in soil. RAPD and ISSR analysis revealed a homogeneous
amplification profile for all micropropagated plants (Nayak etal. 2013). Besides
direct regeneration from explants, callus mediated regeneration was also studied in
W. somnifera. Among different types of calli, best shoot regeneration was observed
on green, compact calli produced on MS medium with a combination of BA and
IBA. MS medium supplemented with BAP (2.0mg/l) showed highest frequency
(98%) of shoot bud regeneration in the cultures. The micro-shoots were efficiently
rooted and rooted plants were transferred to soil-vermi-compost (1:3; w/w) medium
in greenhouse for acclimatization (Chakraborty et al. 2013a). Hairy root cultures
of the plant were also reported for the production of useful metabolites (Praveen
and Murthy 2013; Kumar etal. 2005). These efforts clearly demonstrate the feasi-
bility of micropropagation technique in conserving these medicinal plants. Novel
approaches like use of morphactin as one of the plant growth regulator and biotic
elicitor like plant gum and Cuscuta are highlights of these works. Nove combina-
tions may result in enhanced production of secondary metabolites.
11.2.2Somatic Embryogenesis
development of artificial seeds. These artificial seeds can be used for germplasm
conservation and large scale clonal propagation, breeding of plants producing non-
orthodox seeds or non-seed producing plants and facilitate the storage and transpor-
tation of samples making handling and direct planting easier.
Although standard in vitro propagation methods are, in general, accessible, en-
dangered species may have unusual growth requirements and, thus, may need modi-
fied procedures for in vitro culture. In addition, the limited amount of plant material
available from rare and endangered species poses major challenges in the applica-
tion of in vitro techniques. Plant regeneration via somatic embryogenesis has been
demonstrated in many medicinal plant species. Effective in vitro regeneration of
Bacopa has been reported via young leaf derived somatic embryo cultures. Leaf
explants cultured in 2,4-D and BAP medium, initiated high frequency somatic em-
bryogenesis. Regenerated plantlets were successfully transferred to soil with 100%
survival rate (Chakravarthy etal. 2013). Similarly, in Clitoria an efficient plant
regeneration protocol has been developed from embryogenic callus derived from
cotyledonary explants. Optimum embryogenic callus (75%) was induced on MS
medium supplemented with 2, 4-D. On subculturing the callus on MS medium sup-
plemented with BA, NAA, and ABA the highest embryogenic response, frequency
of 83% and mean number of 37 embryos per gram callus, was observed. Synthetic
seeds were produced by encapsulating embryos in calcium alginate gel and were
germinated with 92% frequency. The synthetic seeds were stored at 4C and lab
conditions (252C) up to 5months. The synthetic seeds kept at 4C showed 86%
viability even after 5months of storage. Both somatic embryos and synthetic seeds
germinated and were transferred to soil successfully (Kumar and Thomas 2012).
In Chlorophytum, moderate to good callus induction was observed on MS medium
containing kinetin and 2,4-D using in vitro grown seedlings. Regular subculturing
of callus on kinetin and 2,4-D supplemented medium induced somatic embryogene-
sis. Modified solid embryogenic medium and liquid embryogenic medium support-
ed better somatic embryo production and maturation. Highest germination (57.5%)
was observed at inoculum density of 0.4g/40ml of liquid medium. RAPD analysis
of C. borivilianum plants regenerated through somatic embryogenesis revealed that
they were genetically similar to the mother plant (Rizvi etal. 2012). Another plant
with a hard to grow in nature, Commiphora, has also produced somatic embryo-
genesis (Kumar etal. 2006b). Development of resin canal (Kumar etal. 2004) and
guggulsterone synthesis during somatic embryogenesis has been observed in these
cultures (Kumar etal. 2006a). Similarly, in Curculigo embryogenic callus mediated
somatic embryogenesis has been reported with a frequency of 90% embryos being
developed into complete plantlets and with 6570% field survival rate (Nagesh
etal. 2010). Genetic fidelity of somatic embryogenesis derived regenerant using
RAPD was also assessed (Patel etal. 2011).
It is evident from the above account that complete protocols have been developed
for A. marmelos, B. monniera, C. asiatica, C. borivilianum, C. orchioides and W.
somnifera, and these protocols are used for the production of plants while more
work is required for other plants. Production of bioactive molecules is being carried
out in our laboratory and more inputs are being tested to make the technology viable.
212 S. Goyal et al.
Plants have been used for prevention and cure of many ailments since time imme-
morial. And according to their usage as medicines many of them have been catego-
rized as medicinal plants. Most of our present day knowledge of medicinal plants
comes from there folk uses and there uses by our ancestors. In the current scenario
when the natural products are gaining importance due to their less or negligible side
effects more and more research is being done on the scientific validation of these
traditional plants. Many of the research have proved that these traditional usages of
plants hold true when tested on scientific parameters. Now to find the herbal based
formulations in the pharmacy stores is quite common. Below we have summarized
the scientific validations of few important medicinal plants of Aravallis.
Fruits of A. marmelos are highly valued for treatment for chronic dysentery, diar-
rhoea, and are considered as gastric stimulant. Charak Samhita (Vedic medicinal
text) has classified the fruits as curative of piles, haemorrhoids, oedema and swell-
ing. This is one of the ingredients of Dasmoolarisht (ten roots, a standard Ayurvedic
medicine for loss of appetite and inflammations of uterus). Fresh leaf juice is effec-
tive in diabetes, asthama and fever, and body malodour.
It contains diverse chemicals such as essential oils, coumarins (marmain, aurap-
ten, umbelliferone, marmenol), furoquinoline, alkaloids, triterpenoids, tannins and
sterols etc. (Dev 2006; Samarasekera etal. 2004). A new molecule, 24-epibrassino-
lide has also been reported from A. marmelos (Sondhi etal. 2008).
The plant shows many biological activities. Its antidiarrhoeal activity has been
reported in rat model (Mazumder etal. 2006) and antimicrobial activity against
diarrhoea causing microbes (Brijesh etal. 2009). The plants unripe fruit extract
shows anti-inflammatory, antioxidant, and mast cell stabilizing effects thus demon-
strating protective effect in inflammatory bowel disease (Behera etal. 2012). Other
biological activities of the plant extract include cardioprotective (Krushna etal.
2012) analgesic, antipyretic, sedative, anticonvulsant (Dev 2006), contraceptive
Table 11.1 Selected plant species of Aravalli Hills with their in vitro response on different media. MS, Murashige and Skoog medium; WPM, Woody plant
medium; BAP, 6-benzylaminopurinne; NAA, -napthaleneacetic acid; Kn, kinetin; IBA, indole-3-butyric acid; IAA, 3 indole-3-acetic acid
Plant species Plant part used Medium Regeneration Reference
Abrus Node MS+BAP (5.0 mg/l)+NAA (0.5 mg/l) Callus Biswas etal. (2007)
precatorius Callus MS+BAP (3.0 mg/l)+Kn (0.5 mg/l)+NAA Shoot regeneration -do-
(0.5mg/l) Rooting, the rooted plantlets were trans- -do-
In- vitro shoots MS+IBA (1.0mg/l) ferred to soil after proper acclimatization
Acacia nilotica Node MS+NAA (0.6 mg/L)+Kn (1.0 mg/L) Shoot proliferation Dhabhai etal. (2010)
Excised shoots MS+IBA (0.5mg/L) Rooting, micropropagated plantlets were -do-
(23cm) successfully transferred to natural condi-
tions with 75% survival rate
Achyranthes Leaf MS+2,4-D (1.02.0 mg/l)+NAA (0.5 mg/l) Callus Kayani etal. (2008)
aspera
Asparagus Node MS+2-isopentyl adenine (3.69M) Shoot proliferation Bopana and Saxena
racemosus In-vitro shoots MS+NAA (1.61 M)+Kn (0.46 M)+adenine 85% rooting, tissue-cultured plants were (2008)
sulfate (98.91M)+500mg/l malt extract+phlo- transferred to the field with a 100%
roglucinol (198.25M) survival rate
Balanites Axillary bud MS+BAP (2.5 mg/l)+NAA (0.1 mg/l) Shoot regeneration Ndoye etal. (2003)
aegyptiaca In-vitro shoots MS+IBA (20 mg/l) Rooting, rooted shoots acclimated and -do-
were successfully transferred into soil,
with 48% of the plantlets surviving
Axillary meristems MS+BAP (0.45 M) Shoot regeneration Rathore etal. (2004)
11 Biotechnological Approaches to Medicinal Plants of Aravalli Hills
Boerhavia Shoot tip and Node MS+BAP (1.5 mg/l)+NAA (0.5 mg/l) Multiple shoot regeneration Roy (2008)
diffusa In-vitro shoots MS+IBA (1.0 mg/l)+IAA(1.0 mg/l) Rooting, about 80% rooted plantlets -do-
survived under field conditions
Callus MS+NAA (1.0 mg/l)+BAP (1.0 mg/l) Shoot regeneration Gupta etal. (2004)
In-vitro shoots MS+NAA(0.25 mg/l)+I BA (0.25 mg/l) Rooting, Regenerated shoots were mor- -do-
phologically similar to the shoots of field
grown plants
213
214
It is a small prostrate herb that grows wild in marshy places throughout India. The
plant contains a complex mixture of dammarane type of triterpenoidal saponins
with jujubogenin or pseudojujubogenin moiety as aglycone. The saponins dif-
fer in the sugar moieties. Important saponins include bacoside A1, A2, A3, baco-
pasaponins A-G, bacopaside I-VIII, bacopaside N1,N2,X and jujubogenin. Other
chemical constituents of the plant are hersaponin, betulic acid, alkaloids- brah-
mine and herpestine, flavonoids- luteolin-7-glucoside, glucuronyl-7-apigenin
and glucuronyl-7-luteolin, luteolin-7-O--glucopyranoside, a triterpene bacosine
(lup-20(29)-ene-3-ol-27-oic acid) and several common phytosterols (Rajani 2008;
Zhao etal. 2007; Bhandari etal. 2006). High Performance Liquid Chromatogra-
phy (HPLC) was used to quantify several bacopa saponins (Murthy etal. 2006). A
method of enrichment of bacopa saponins from the plant has been patented for its
use as memory enhancer (Kahol etal. 2004).
Various investigations have attempted to substantiate and identify a scientific
basis for the reputed effects of memory enhancing (Howes and Hughton 2009). A
number of in-vivo studies have shown B. monnieri extracts to improve cognitive
function. The mode of action to explain these effects has yet to be fully elucidated.
Some studies suggest that the antioxidant effects of B. monnieri may protect the
Central Nervous System (CNS) from oxidative damage. In a study, the rat experi-
mental model of neonatal hypoglycaemia, Bacopa extract improved alterations in
Dopamine D1, D2 receptor expression, cAMP signaling and cell death resulting
from oxidative stress (Thomas etal. 2013). It has shown beneficial effect in hy-
poxia and epilepsy management by down regulating glutamate receptor gene ex-
pression in rat model (Paulose etal. 2008). In another study, alcoholic extract of B.
monnieri treatment ameliorated olfactory bulbectomized (OBX) induced cognition
dysfunction in mice via a mechanism involving enhancement of synaptic plasticity-
related signaling and brain-derived neurotrophic factor (BDNF) transcription and
protection of cholinergic systems from OBX-induced neuronal damage (Le etal.
2013). Although the majority of relevant studies, which have investigated the re-
puted cognitive-enhancing effects, have focused on extracts rather than isolated
216 S. Goyal et al.
c onstituents, it is the triterpenoid saponins that have been associated with the activ-
ity. Triterpenoid saponins, a mixture known as bacoside A which includes bacoside
A3, have been shown to protect rat brains from smoking-induced apoptosis and
from structural and functional impairment of mitochondria (Anbarasi etal. 2005).
Ten years of research at Swinburne University at Melbourne, with the extract
of B. monnieri, showed it to be a safe and efficacious cognitive enhancer. Studies
using this extract indicate that it has several modes of action on the human brain.
Promising indications for use in humans include improving cognition in the elderly
and in patients with neurodegenerative disorders (Stough etal. 2013). Some studies
also suggest B. monnieri to be an efficient antidepressant which is comparable to
well accepted antidepressant drug Fluoxetine hydrochloride (Hazra etal. 2013) and
also showed a potential as a possible anti-Parkinsonian agent (Jadiya etal. 2011).
It is therefore possible that B. monnieri may exert multiple beneficial effects on the
CNS and brain ageing (Aguiar and Borowski 2013; Singh etal. 2008; Howes and
Hughton 2009).
It is traditionally used for boils, for curing bone fracture, as an asrtringent and for
the treatment of leukorrhea (Choudhary etal. 2008). Phytoalexins from the Vitaceae
constitute a rather restricted group of polyphenolic secondary metabolites belong-
ing to the stilbenes family (piceid, resveratrol, viniferin). Stilbenes production is
mainly studied in cell cultures of Vitis vinifera (Waffo-Teguo etal. 2008).
In plants, stilbenes play significant role in constitutive and inducible defense
mechanisms including antibacterial and antifungal activities (Jeandent etal. 2002;
Kostecki etal. 2004). Stilbenes possess a broad spectrum of pharmacological and
therapeutic effects such as anti-epileptic effect (Wu etal. 2009), antioxidative, anti-
cancer, anti-atherosclerosis activities, as well as having cardioprotective, hepato-
protective, and neuroprotective effects (Nassiri-Asl and Hosseinzadeh 2009; Kumar
et al. 2011; Waffo-Teugo etal. 2008; Baur and Sinclair 2006; Delmas etal. 2006).
Some studies also showed that C. trifolia extract possesses antiulcerogenic as well
as ulcer healing properties, which might be due to its antisecretory activity (Gupta
etal. 2012). In another study water extract of C. trifolia leaf promised as a cost
effective and potent larvicidal agent against Culex quinquefasciatus (Chakraborty
etal. 2013b). The role of stilbenes in management of cognitive impairment through
increasing the activity of choline acetyl transferase and antioxidative mechanism
has been identified (Ruan etal. 2009). Antimicrobial activity of stilbenes against
oral pathogens has also been explored (Yim etal. 2009). Low incidence of athero-
sclerosis in red wine drinking society is often co-related with significant level of
stilbenes in red wine (Renaud etal. 2004; Waffo-Teguo etal. 2008).
11 Biotechnological Approaches to Medicinal Plants of Aravalli Hills 217
It is reputed to restore youth, memory, longevity and several minor ailments. Several
pentacyclic triterpenoids of ursane subtype (viz., asiatic acid, madasiatic acid, brah-
mic acid, isothankunic acid and their glycosides) are present in C. asiatica. The herb
contains an essential oil rich in sesquiterpenes (b-caryophyllene, trans-b-farnesene
etc.), flavonoids, steroid and an alkaloid (Dev 2006; Yu etal. 2006). Methods of
quantification of some of these compounds have been established (Rafamantanana
etal. 2009; Zhang etal. 2008).
The pharmacological basis to explain the reputed anti-amnesic effects of C.
asiatica has been explored in a number of studies (Howes and Houghton 2009).
Based on its traditional use as memory and intellect promoting plant, several works
showed its prominent effect in improving cognitive functions and protection in
Alzheimers disease. It also has anti-epileptic, anabolic, antiviral and antitumor
activity, and is mild sedative and has beneficial effects in psoriasis and ulcer (Dev
2006). Modern studies proved its antipyretic and anti-inflammatory effects (Wan
etal. 2013). It has been shown that asiatic acid plays an important role in cancer
apoptosis by inducing mitochondrial death apoptosis cascade (Tang etal. 2009).
Similarly, the other results indicate that derivatives of asiatic acid induces inhibi-
tion of cell proliferation via down regulation of the Ras/Raf/MEK/ERK pathway
and cell cycle arrest at G1/S and G2/M (Wang etal. 2013). Madecassoside, a triter-
penoid isolated from the herb, exhibits prominent antioxidant activities in collagen
induced arthritic conditions in mice model (Li etal. 2009). The plants activity
was explored in epilepsy, stroke and other degenerative conditions (Krishnamurthy
etal. 2009). An anxiolytic effect of C. asiatica extract was demonstrated in non-
stressed mice subjected to acute stress in all behavioral tests employed. These ef-
fects could be mainly accounted by madecassoside and asiaticoside, thereby sug-
gesting a possible use of this extract for the treatment of both acute and chronic
anxiety in the pathological state (Wanasuntronwong etal. 2012). C. asiatica was
also found to attenuate the neurobehavioral, neurochemical and histological chang-
es in transient focal middle cerebral artery occlusion rats (Tabassum etal. 2013).
The plant extract render radioprotection to DNA and membranes both in in-vitro
and in-vivo conditions (Joy and Nair 2009). Moreover, C. asiatica has also shown
to protect against UVB-induced HaCaT keratinocyte damage through microRNA
expression changes (An etal. 2012). In a study, asiaticoside enhanced the initial
skin cell adhesion, induced an increase in the number of normal human dermal
fibroblasts thereby promoting skin cell behaviors involved in wound healing (Lee
etal. 2012). C. asiatica extract capsule can be used for wound healing promotion
and also suppress the scar in diabetic wound patients. These capsules can shorten
the course of diabetic wound and can be prescribed to the diabetic patients safely
(Paocharoen 2010).
218 S. Goyal et al.
The plants seeds and seed oil are considered analgesic, sedative, anti-inflammato-
ry, antirheumatic, diuretic, alterative, nervine tonic and aphrodisiac, and beneficial
in gout, and paralysis (Dev 2006). Characteristic secondary metabolites of the plant
are a range of esterified bicarbocyclic sesquiterpene polyols occurring in the seeds
and seed oil. These polyols e.g., malkanguniol, are esterified with one or more of
the following acids: acetic acid, benzoic acid and cinnamic acid (e.g., celapanine)
(Dev 2006). A new sesquiterpene polyol ester has also been characterized from the
seeds (Borbone etal. 2007).
In a study, seed oil of C. paniculatus has shown to improve memory (Kumar
and Gupta 2002). Methanol extract of flowers from C. paniculatus has shown to
be anti-inflammatory (Ahmad etal. 1994), which may also have some relevance in
the management of neurodegenerative disorders. A poly-herbal formula (Abana)
containing C. paniculatus as a component amongst other herbs is used in Ayurvedic
medicine, and dose-dependently improved memory in both young and aged rodents
and reversed scopolamine- and diazepam-induced amnesia (Parle and Vasudevan
2007). The contribution of each of the component herbs of this formula to the ob-
served effects, or if any synergistic effect occurred, is unknown. In other studies, C.
paniculatus extract protected neuronal cells by virtue of their free radical scaveng-
ing properties, reducing lipid per oxidation, and also by their ability to induce the
antioxidant enzyme catalase (da Rocha etal. 2011). In addition, aqueous extracts
of its seed have dose-dependent cholinergic activity, thereby improving memory
performance. This enhanced cognition may be due to increased acetylcholine level
in rat brain (Bhanumathy etal. 2010). Besides, well known neurological benefits
C. paniculatus also exhibits different pharmacological activities like analgesic
(Debnath etal. 2012), hypolipidemic (Patil etal. 2010) and antiproliferative (Weng
etal. 2013). Seed extract has also shown potent relaxant effect in isolated rat and
human ileum (Borrelli etal. 2009) that could explain the traditional use of this herb
in the treatment of intestinal spasms.
11.3.6Clitoria ternatea L.
and for use as a vascular smooth muscle relaxant properties (Mukherjee etal. 2008).
Its antipyretic property was established by yeast induced pyrexia in albino rats
(Parimaladevi etal. 2004). The methanolic extract was found to possess nootropic,
anxiolytic, antidepressant, anticonvulsions, antistress (Jain etal. 2003), hepatopro-
tective (Nithianantham etal. 2013) and larvicidal activity (Mathew etal. 2009)
and the leaves fresh juice showed anthelmintic activity (Nahar etal. 2010). An-
other group studied the chemosensitizing activities of cyclotides from C. ternatea
in paclitaxel-resistant lung cancer cells (Sen etal. 2013). Oral administration of the
hydroalcoholic extract of the roots and seeds of the plant resulted in a significant
(p<0.05) reduction of serum total cholesterol, triglycerides, very low-density lipo-
protein cholesterol, and low-density lipoprotein cholesterol levels. This antihyper-
lipidemic activity might be attributed to increased biliary excretion and decreased
absorption of dietary cholesterol (Solanki and Jain 2010a). Its leaf and flower ex-
tracts exhibit antihyperglycaemic effect in rats with alloxan-induced diabetes melli-
tus (Daisy and Rajathi 2009). The root extract showed antiasthmatic property (Taur
and Patil 2011) and profound immunosupressive activity in male albino rat model.
The antioxidant and anti-inflammatory activities of plant may be playing major
role in immunoinhibition (Solanki and Jain 2010b). Bioassay-guided fractionation
of effective extracts may result in identification of useful molecules responsible
for these activities. The roots of the plant have a reputation for promoting intel-
lect. Memory enhancing property of root extract of plant was shown in neonatal
rat pups (7 days old) by improved retention and spatial learning performance (Rai
etal. 2001). This reputed effect may be related to effects on cholinergic activity in
the CNS (Howes and Houghton 2009). Further studies are necessary to establish the
mechanism of action to explain the observed effects of the root extract on the CNS,
and also to identify the compounds responsible for activity.
Chlorophytum species are reported as diploid, triploid, tetraploid and polyploid with
basic chromosome number 7 or 8 (Arora etal. 2004). The tuberous roots (mostly
powdered) are widely used in Indian system of medicine for the following proper-
ties: as a non-hormonal restorative tonic, in fatigue, general debility, weakness and
as a general purpose tonic, in impotency and sterility and to enhance male potency,
as cardiac and brain tonic, as curative agent in various diseases like piles, diabetes,
albuminorrhoea- leucorrhoea, menorrhoea, and as anti-pyretic, sialogogue, galac-
togue, diuretic, hemostatic (Arora etal. 2004). These properties have been inferred
on the basis of its use in folk medicine, traditional medicine and experiments on
man volunteers (Jain 1991; Arora etal. 2004). An Ayurvedic medicine known as
Musli power extra has become a house hold energetic medicine for all age groups.
Tuberous roots of C. arundinaceum, contain steroidal saponins (neohecogenin,
neotigogenin, stigmasterol, tokorogenin), a bibenzyl xyloside (2,4,4-trihydroxy-
2-xylopyranosyl bibenzyl), a disubstituted tetrahydrofuran (4-hydroxy-8,11-
220 S. Goyal et al.
elimination (Urizar etal. 2002; Capello etal. 2008). Besides this, guggulsterone
also exhibits potent cancer chemopreventive activities (Almazari and Surh 2013).
Guggulsterone inhibits smokeless tobacco and nicotine-induced NF-B and STAT3
pathways in head and neck cancer cells (Macha etal. 2011). It suppresses the
pro-inflammatory transcription factor, NF-B and NF-B-regulated gene products
involved in anti-apoptosis (IAP1, xIAP, Bfl-1/A1, Bcl-2, cFLIP, and survivin), pro-
liferation (cyclin D1 and c-Myc), and metastasis (MMP-9, COX-2, and VEGF) of
tumour cells (Shishodia etal. 2008; Lv etal. 2008; Lee etal. 2008). In colon cancer
cells, guggulsterone significantly increased apoptosis by activating caspases-3 and
-8 (An etal. 2009). Guggulsterone mediates gene expression through regulation of
various transcription factors including NF-B, STAT-3 and C/EBPa and various
steroid receptors such as androgen receptor and glucocorticoid receptors (Ahn etal.
2008; Kim etal. 2008; Leeman-Neill etal. 2009). In addition to these activities,
guggulsterone has also found to exert a melanogenic inhibitory effect through the
downregulation of tyrosinase expression (Koo etal. 2012).
Tuberous roots are widely used as tonic for health, vigour and vitality because of
the presence of flavanone glycosides and other steroidal saponins. Several bioactive
compounds isolated from the plant include flavones, glycosides, steroids, saponins
and triterpenoids (Tandon and Shukla 1995). New curculigosides from in-vitro tu-
bers of C. orchioides (Valls etal. 2006), and orchioside D and curculigoside E from
roots were isolated (DallAcqua etal. 2009).
The medicinal property of the herb is mainly attributed to curculigosides and
Curculigo saponins (Xu etal. 1992), which is used along with Withania somnifera,
C. borivilianum and Asparagus racemosus in several herbal formulations. Increased
activity in terms of reduction of mount latency, increase in mount frequency, in-
creased penile erection index and enhanced attraction towards female was recorded
from ethanolic extract of the plant (Thakur etal. 2009; Chauhan etal. 2007). Simi-
larly, the aqueous extract of the herb improved sexual performance in streptozoto-
cin induced hyperglycemic and subsequent sexual dysfunctional male rats (Thakur
etal. 2010). Curculigoside can improve cognitive function in aged animals, pos-
sibly by decreasing the activity of acetylcholinesterase in the cerebra and inhibiting
the expression of BACE1 in the hippocampus (Wu etal. 2012). Curculigoside also
improved osteogenesis and inhibited osteoclastogenesis of human amniotic fluid-
derived stem cells (hAFSC), suggesting its potential use to regulate hAFSC osteo-
genic differentiation for treating bone disorders (Liu etal. 2014). The methanolic
extract of C. orchioides has shown to enhance the antioxidant defense against reac-
tive oxygen species produced under hyperglycemic conditions, hence protecting
the liver, pancreatic and kidney tissue injuries (Anandakirouchenane etal. 2013).
Ethanolic extract of the rhizome exhibited potent activities such assignificant hy-
poglycemic activity in normal and streptozotocin-induced diabetic rats (Jain etal.
222 S. Goyal et al.
2010), antihypertensive (Joshi etal. 2012), antiosteoporosis (Jiao etal. 2009; Cao
etal. 2008), immunostimulant and anti-inflammatory activity (Venkatesh etal.
2009), and estrogenic activity (Vijayanarayana etal. 2007; Nie etal. 2013).
Its tuber contains isoflavonoids (puerarin, genistin, daidzein, and genistein etc.) and
is highly valued in Ayurveda since the time of Samhitaas (Dev 2006). Besides this,
kudzu root (Pueraria lobata) is a well-known Chinese herbal medicine, which is
being extensively investigated. Traditionally it is used as contraceptive, cardiotonic,
rejuvenating and in rheumatism.
These roots are the important crude drugs in the pharmaceutical industries
(Dev 2006; Keung 2002). P. tuberosa extract have significant anxiolytic and anti-
stress properties (Pramanik etal. 2010). Recent findings reveal that puerarin exerts
the hypoglycemic and hypolipidemic roles and is potential anti-diabetic which is as-
sociated with elevating insulin expression and maintaining metabolic homoeostasis
(Wu etal. 2013). Anti-osteoprotic action of puerarin is independent of the estrogen
receptor mediated pathway (Michihara etal. 2012). Besides these activities, puera-
rin is also beneficial against neurological disorders by preventing the dysfunction of
the neuronal cholinergic system and ameliorates the increase of -amyloid caused
by estrogen deficiency (Zhang etal. 2013). Puerarin has anti-Parkinsons (Zhu etal.
2014) and cardioprotective activity (Chung etal. 2008). Puerarin successfully re-
verses hepatotoxicity in CCl4-induced HF rats via the underlying mechanisms of
regulating serum enzymes and attenuating TNF-/NF-B pathway for anti-inflam-
mation response, as well as improving metabolic function in liver tissues (Li etal.
2013; Xu etal. 2013; Peng etal. 2013). The other compound of interest is the
genistein, which is a promising anticancer agent that inhibits platelet aggregation
and induces apoptosis (Lin etal. 2009). Genistein and daidzein both show phytoes-
trogenic activities (Dixon and Ferreira 2002) and exhibit effective antioxidant and
antiosteoporotic properties (Dai etal. 2008). Besides these molecules, tuberosin
also have efficient antioxidant properties (Pandey and Tripathi 2010).
The plant is cultivated in Rajasthan and Madhya Pradesh states of India. It is highly
valued in Ayurveda as an alternative, restorative and as an anabolic agent. It has
diuretic, antidepressant and cardioprotective activities. Characteristic compounds
present in the plant are steroids with ergostane skeleton, which have been named
withanoloides. More than 45 withanololides such as withanolide-A, withaferin-A,
sitoinodoside-IX, sitoinodoside-X, somniferine, somniferinine have been isolated
from the leaves, fruits and roots of W. somnifera (Dev 2006; Mirjalili etal. 2009).
This plant has several geno- and chemo-types and hence, the nature and percentage
11 Biotechnological Approaches to Medicinal Plants of Aravalli Hills 223
11.4Future Prospects
Plants of Aravalli hills are showing various promising biological activities such as
anticancerous, antitumour, anti-inflammatory, antimicrobial, hypolipidemic, hypo-
cholesteremic, etc. An array of prototype bioactive molecules of different classes
Table 11.2 Selected plant species of the Aravalli Hills, their bioactive molecules, traditional usage and scientific validation
224
Plant species Local name Bioactive molecules Traditional uses Biological activities References
(family) (part/s used)
Abrus precatorius Chirmi Abrine, abricin, arbidin, Abortifacient, antifer- Mitochondrial apoptosis induced by the Bhutia etal. (2009a)
(Fabaceae) (Leaves, root) precatorine, choline tility, aphrodisiac, in peptide fraction of abrin Ramnath etal. (2009)
leucoderma Lectins-immunostimulatory Bhutia etal. (2009b)
Seed oil-antimicrobial activity Adelowotan etal.
(2008); Zore etal. (2007)
Acacia nilotica Babul Kaempferol (AN-5), In asthma, cholera, Antioxidant Kalaivani and Mathew
(Mimosaceae) (Leaves, Stem D-pinitol, a sex hormone viz diabetes, diarrhoea, Immunosuppressive (2009)
bark, gum, 3 -acetoxy-17-hydroxy- liver complication, Chemopreventive potential Aderbauer etal. (2008)
flower) androst-5-ene, acanilol A,B, leprosy Anti-inflammatory Singh etal. (2009a)
triterpene lupenone, gallic Antiplasmodial Chaubal etal. (2006)
acid, ellagic acid, epicat- Antidiarrhoeal Kirira etal. (2006)
echin, rutin Antimicrobial activity against multi- Agunu etal. (2005)
drug resistant bacterial and fungal Khan etal. (2009)
strains Hussein etal. (2000)
Inhibitory effect on hepatitis C virus Gilani etal. (1999)
(HCV) protease
Antihypertensive and antispasmodic
activities
Achyranthes aspera Andhijara, Ecdysterone, Betaine Stimulant, in ulcer, Antiparasitic activity of leaf ethyl Zahir etal. (2009)
(Amaranthaceae) undhokanto piles, snake antidote, acetate extract Goyal etal. (2007)
(Root, leaves) hypoglycaemic, Treatment of leprosy, fistula-in-ano, Vasudeva and Sharma
diuretic bronchial asthma (2006)
Post coital antifertility activity Chakrabarti and
Immunity enhancement Vasudeva (2006)
Anti-inflammatory Vetrichelvan and
Antiarthritic Jegadeesan (2003)
Cancer chemopreventive Gokhale etal. (2002)
Prothyroidic, antiperoxidative Chakraborty etal. (2002)
Tahiliani and Kar (2000)
S. Goyal et al.
Table 11.2 (continued)
Plant species Local name Bioactive molecules Traditional uses Biological activities References
(family) (part/s used)
Asparagus racemo- Satawari Steroidal saponins like Antiageing, intellect Potent antioxidant activity Visavadiya etal. (2009)
sus (Liliaceae) (Roots) shatavaroside A (1),B(2), promoting, aphro- Aphrodisiac activity Thakur etal. (2009)
shatavarins VI-X, and disiac, improves Immunomodulatory Gautam etal. (2009)
saponin like filiasparoside digestion, abaptogen, Elimination of excess cholesterol Visavadiya and Narasim-
C, Racemoside A,B,C, rac- treatment of diarrhoea and elevation of hepatic antioxidant hacharya (2009)
emofuran (3) asparagamine and dysentery status in hypercholesteremic conditions Agrawal etal. (2008)
A (1), racemosol (2) Chemopreventive Singh etal. (2009b)
Antidepressant effect Dutta etal. (2007)
Racemoside A is a potent anti-leish- Hannan etal. (2007)
manial agent Bhatnagar and Sisodia
Root extracts have wide-ranging (2006)
stimulatory effects on physiological Venkatesan etal. (2005)
insulinotropic pathways Mandal etal. (2000)
Antiulcerogenic agent
Antidiarrhoeal potential
Antibacterial efficacy
Balanites aegypti- Hingota Balanitin-6 and-7: diosgenyl Snake antidote, in Antitumor activity Gnoula etal. (2008)
aca (Balanitaceae) (Seed kernel, saponins skin disorders, urine Larvicidal Chapagain etal. (2008)
fruit, root, complications, insect Anti-inflammatory, antinociceptive, Speroni etal. (2005)
bark) bite, pneumonia antioxidant Koko etal. (2000)
Fasciolicidal
11 Biotechnological Approaches to Medicinal Plants of Aravalli Hills
Boerhavia diffusa Punarnavaa Nonprenylated rotenoids viz Diuretic,analgesic, Antiproliferative and antiestrogenic Sreeja and Sreeja (2009)
(Nyctaginaceae) (Whole plant) boeravinones G(1), H(2), laxative, anti-inflam- properties Manu and Kuttan (2008)
I(10), J(11), punarnavoside, matory, curative for Cell mediated immune response Manu etal. (2007)
liriodendrin bronchitis, jaundice, Radioprotective Borrelli etal. (2006)
gonorrhoea Spasmolytic effects Manu and Kuttan (2009)
Immunomodulatory activity Agrawal etal. (2004)
Antifungal activity Satheesh and Pari (2004)
Antidiabetic activity with improvement Bharali etal. (2003)
225
in antioxidant status
Cancer chemopreventive
Table 11.2 (continued)
226
Plant species Local name Bioactive molecules Traditional uses Biological activities References
(family) (part/s used)
Boswellia serrata Salar (Resin) Incensole acetate, boswellic Frankincense prepara- Anti-depressive, immunomodulatory , Moussaieff and
(Burseraceae) acids tions used to cure neuroprotective Mechoulam (2009)
inflammatory diseases Apoptotic effects Liu and Duan (2009)
Tausch etal. (2009)
Cathepsin G as functional target in anti-
inflammatory activity Pang etal. (2009);
Anticancerous Bhushan etal. (2009);
Prevent hyperlipidemia and Kunnumakkara etal.
atherosclerosis (2009)
Antiosteoarthritis Tripathi (2009)
Sengupta etal. (2008a)
Butea monosperma Dhak Butrin, isobutrin, butea- Antihelmenthic Antidiabetic and antioxidant potential Sharma and Garg (2009)
(Fabaceae) (Leaves, spermin A,B buteasper- appetizer, aphrodisiac, Antimycobacterial activity Chokchaisiri etal. (2009)
flowers, bark, manol monospermoside, laxative Osteogenic activity Maurya etal. (2009)
seed , gum) monospermin, stigmasterol Thyroid inhibitory, antiperoxidative Panda etal. (2009)
dihydromonospermoside Anti-inflammatory activity Shahavi and Desai
Chemopreventive hepatic (2008)
carcinogenesis Sehrawat and Sultana
Dermal wound healing (2006)
Sumitra etal. (2005)
Cocculus hirsutus Jal-jammi Isoquinoline alkaloid In eczema, dysentery Diuretic Badole etal. (2009)
(Menispermaceae) (Leaves) d-trilobine, dl-coclaurine, and in urinary prob- larvicidal activity against Anopheles Elango etal. (2009)
cohirsinine, jamtinine, lems, eye diseases subpictus and Culex tritaeniorhynchus. Sangameswaran and
cohirsutine Antidiabetic and spermatogenic Jayakar (2007)
Ficus bengalensis Bar (Aerial Flavonoids, viz. leucopelar- In urinary prob- Antidiabetic Singh etal. (2009c)
(Moraceae) roots, bark, gonin, leucocyanin deriva- lems, ulcers, Antiallergic and antistress in asthma Taur etal. (2007)
latex, fruits) tive, quercetin sores, antipyretic, Antiatherogenic Daniel etal. (2003)
anti-inflammatory Antioxidant and hypolipidaemic Shukla etal. (2004)
S. Goyal et al.
Table 11.2 (continued)
Plant species Local name Bioactive molecules Traditional uses Biological activities References
(family) (part/s used)
Helicteres isora Marorphali Saponins and sapogenin, Antidiabetic, anti- Hypoglycaemic effect Kumar etal. (2009);
(Sterculiaceae) (Bark, fruit, flavonoid glucuronides viz spasmodic activity, Improving hyperlipidaemia and Bhavsar etal. (2009)
root) isoscutellarein 4-methyl in anaemia, asthma, hyperglycaemia by increasing the Kumar and Murugesan
ether 8-O-beta-D-glucuro- gastrointestinal gene expression of adipsin, Glut4 and (2008)
nide etc. complications PPARgamma Venkatesh etal. (2007)
Hypolipidaemic activity Kumar etal. (2006a)
Antinociceptive activity
Hepatoprotective activity
Phyllanthus Amla (Fruit, Emblicanin-A, B, gallic Memory and intellect Induce specifically programmed Piva etal. (2009); Peno-
emblica syn leaves) acid, ellagic acid, pyrogallol, enhancer, nervine cell death of mature osteoclasts lazzi etal. (2008)
Emblica officinalis apigenin 7-0-(6 butyryl-- tonic, antifatigue, without altering the process of Wang etal. (2009)
(Euphorbeaceae) glucopyranoside), quercetin, diuretic, promoter of osteoclastogenesis Poltanov etal. (2009)
putranjivain A, phyllaem- hair growth, and good Potential therapeutic agent for viral Sumantran etal. (2008)
blicin-A,B,E,F, 4-hydroxy- eyesight myocarditis Talwar etal. (2008);
phyllaemblicin B (1) Potent antioxidant Srikumar etal. (2007)
Chondroprotective activity in Reddy etal. (2009);
osteoarthritis Pinmai etal. (2008);
Antimicrobial, virucidal action against Arulkumaran etal.
HIV-1NL4.3 and HPV infections (2007); Deep etal.
Hepatoprotective (2005)
11 Biotechnological Approaches to Medicinal Plants of Aravalli Hills
Plant species Local name Bioactive molecules Traditional uses Biological activities References
(family) (part/s used)
Phyllanthus Bhumi amla E,E-2,4-octadienamide, In jaundice, cough, Hepatoprotective Sailaja and Setty (2006);
fraternus (Whole plant) E,Z-2,4-decadienamide, laboured breathing, Antinociceptive Khatoon etal. (2006)
(Euphorbeaceae) niruriside, phyllanthin malaria, inflammation Antiplasmodial Catapan etal. (2000)
Sittie etal. (1998)
Salvadora persica Miswak Four benzylamides of which Chewing sticks, in Antifungal properties against oral Noumi etal. (2009)
(Salvadoraceae) (Stem) N-benzyl-2-phenylacet- toothache, mouth Candida strains Sofrata etal. (2008);
amide is pharmacologically ulcer, antimalarial Carries prevention Darmani etal. (2006)
important Antiplasmodial Ali etal. (2002)
Anticonvulsant and sedative effects Monforte etal. (2002)
Antiulcer Sanogo etal. (1999)
Hypolipidaemic Galati etal. (1999)
Tinospora Guduchi, (1,4)-alpha-D-glucan (alpha- Improve immune In diabetes type 1 Patel etal. (2009)
cordifolia giloy (leaf, DG), tinosporine, tinospo- system and protect Polysaccharide from plant act as an Raghu etal. (2009)
(Menispermaceae) stem) ride, cordifolide, diterpenoid against infections, immunomodulator and adjuvant Koppada etal. (2009)
furanolactone, saponarin, antipyretic Activates human lymphocytes Sengupta etal. (2008b)
octacosanol Saponarin-hypoglycaemic activity Dhanasekaran etal.
Chemopreventive-hepatocellular (2009)
carcinoma Chaudhary etal. (2008)
Anti-tumour in skin carcinogenesis Thippeswamy etal.
Antiangiogenic activity (2008)
Antiosteoporotic agent Kapur etal. (2008)
Immunomodulation for ulcer healing Purandare and Supe
(2007)
S. Goyal et al.
11 Biotechnological Approaches to Medicinal Plants of Aravalli Hills 229
has been obtained from these plants, some of which led to important drugs that are
available on the market today. In last three decades, much of the ethnomedicinal
data have been documented and now priorities can be determined as per the usage
of these plants. Though drug discovery from natural products is long process, use
of modern tools like high-throughput screening techniques and NMR spectroscopy
can help in the rapid identification of novel molecules and leads. Adequate and
continuous supplies of plant-derived drugs are essential to meet the market demand.
Therefore, sustainable use of these plants associated with domestication and culti-
vation practices can meet the demand. More inputs in biotechnological methods
can also help in the enhanced production of these bioactive molecules. Use of cell
culture technology in fermenters will be helpful in the production of bioactive mol-
ecules where synthesis is not available. Further refinement of existing technology
for large-scale micropropagation and establishment of industry will be helpful in
continuous supply of plant material. In traditional therapy, where combination of
various herbs is given, should be encouraged and the synergism of various bioac-
tive molecules along with bioavailability enhancement should also be carried out
for its beneficial health effects. Along with domestication, biological problems like
reproductive biology and germplasm conservation need attention.
Acknowledgements This work was supported by financial assistance from UGC-DRS under spe-
cial assistance programme for medicinal plant research to KGR. JA and SG thanks CSIR, New
Delhi for financial assistance in the form of JRF and RA, respectively.
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Chapter 12
Biotechnological Approaches Towards
Micropropagation and Conservation
of Cycads and Ephedrales
Abstract Cycads are woody plants, usually trees, which on casual observation
resemble palms since many have a stock cylindrical stem bearing a crown of very
large, coarse, palm-like leaves. The cycads also attract a great deal of scientific
attention because they have retained the ciliated sperms. Cycads, the only sur-
viving representative of the class Cycadophyta, are facing extinction from their
natural habitats. In order to conserve these living fossils, immediate steps are
needed to propagate them. Ephedra is the only genus belonging to the family
Ephedraceae. It comprises nearly 68 species widely distributed in the arid regions
of New World and Old World. The major active ingredients of Ephedra are alka-
loids and are referred to as ephedrine type alkaloids. Ephedra has been used for
more than 5000 years in China and India to treat various ailments. It has also been
an ingredient in many dietary supplements and used for weight loss, increased
energy, and enhanced athletic performance. Its excess use as medicine has inten-
sified the pressure of landscapes bearing these species. This has necessitated
bioprospection and active planning to ensure safe conservation of the existing
gene pool and sustainable utilization of this land resource. Ex-situ conservation of
germplasm can be achieved using biotechnological approaches such as tissue cul-
ture techniques. Hence it is important that biotechnological tools be employed for
micropropagation of selected plants and for establishment of germplasm banks.
Present review describes various biotechnological aspects related to conservation
of Cycads and Ephedra.
KeywordsCycads Cycas Zamia Ephedrales Ephedra In vitro culture
Micropropagation
M.Dhiman() I.Rautela
Department of Botany, Kanahiya Lal DAV PG College, 247667 Roorkee, Uttarakhand, India
e-mail: manjul.dhiman@rediffmail.com
12.1Introduction
12.2Cycads
Cycads, the only surviving representative of the class Cycadophyta, are facing
extinction from their natural habitats. Of the 11 genera and about 182 species (Ste-
venson and Osborne 1993) that are found today, more than half are categorized
as endangered or rare by international Union for Conservation of Nature and
Natural Resources (Gilbert 1984).The cycads also attract a great deal of scientif-
ic attention because they have retained the ciliate sperms. This feature, which is
a characteristic of lower plants, together with the restricted distribution of some
12 Biotechnological Approaches Towards Micropropagation 249
cycads species, points towards the antiquity of the group. Cycads which were once
common in the mid Mesozoic, are now present only as relics of the past and are
aptly referred to as the Dinosaurs of the Plant Kingdom.
The cycads are present both in Western and Eastern Hemispheres. Five, namely
Ceratozamia, Chigua, Dioon, Microcycas and Zamia belong to the New World and
the remaining six genera viz. Bowenia, Cycas, Encephalartos, Lepidozamia, Mac-
rozamia and Stangeria belong to the Old World. In India, only Cycas is recorded
and four species grow wild, namely C. beddomei, C. circinalis, C. pentinata and C.
rumphii. Besides these, C. revoluta and C. siamensis are commonly cultivated in the
gardens (see Bhatnagar and Moitra 1996).
In many parts of the world, cycads such as Cycas, Zamia and Macrozamia are
used as a source of starch either from seed kernels or from stem pith. Young unfold-
ed leaves of some Cycas species (C. circinalis, C. pectinata and C.siamensis) are
cooked and eaten. The seed of many cycads are liked by rodents, baboons and other
wild animals including elephants. Some Cycas species have medicinal value also.
The stem of C. circinalis is used as remedy for general debility and rheumatism and
its leaf is used as cure for flatulence. The male cone of C. beddomei forms a major
ingredient of some rejuvenating tonics (Ahmedullah and Nayar 1986). Many cycad
species are excellent decorative specimens and have great ornamental value.
Cycads propagate either through seeds or asexually by means of adventitious
shoots. Nearly all cycads are slow growing and require a long period of growth
before they reach the stage of reproduction. The plants are strictly dioecious and, in
Nature, a particular stand may be dominated by plants of either sex. The distance
between male and female plants is important as this influences pollination. Some-
time the pollinating agents themselves may be missing and seed formation is thus
low. Another disadvantage faced by cycads is the long time gap between pollination
and fertilization, followed by a prolonged period (many months) of embryo devel-
opment. Even the few seeds that do mature may be eaten by the animals or, if they
fail to find favorable conditions, they lose their viability soon.
Population pressures, reduction in forest area, slash and burn agriculture, over-
exploitation for horticultural purposes and, at times, environmental conditions have
led to a drastic reduction in natural stands of cycads all over the world. In order
to conserve these living fossils, immediate steps are needed to propagate them.
These include not only awareness strategies, but also sustained efforts of re-estab-
lishment of saplings at the impoverished sites. This obviously requires large-scale
multiplication which is not very feasible by the conventional methods. There is an
urgent need to conserve and protect these beautiful plants of the bygone era (Webb
and Osborne 1989; Pant 1996).
Regeneration from somatic tissue of gymnosperms has been a major obstacle. In
cycads, availability of readily usable meristematic tissue is highly restricted. Since
most of the cycads have usually unbranched stems, they have only one shoot apex
and no axillary buds; therefore, non-meristematic tissues from mature plants serve
as the source material for in vitro culture studies and micropropagation of these
plants. In this context, leaf tissues offer the most promising prospect. Following is
a resume of published literature related to in vitro culture studies in cycads using
different explants (see Tables12.1, 12.2 and 12.3).
250 M. Dhiman and I. Rautela
12.2.1
In Vitro Culture Studies Using Embryo as Explant
In vitro germination and growth of Zamia pumila embryos in Knops solution were
studied by Brown (1966). He observed a faster development of Zamia embryos in
vitro than in vivo. Embryoids were produced from cultured mature embryos of Z.
pumila on Whites Medium having glutamine and alanine (Norstog 1965). Later,
Norstog and Rhamstine (1967) cultured proembryos of Z. integrifolia on differ-
ent culture media containing a relatively low concentration of auxin and kinetin.
Proembryos formed callus, which on transfer to modified Whites medium showed
pseudobulbils and adventive embryos. However, plantlets were not formed.
Webb and his co-workers studied in vitro culture responses of embryos of dif-
ferent cycads. Webb (1982a) stated that both megagametophyte and cotyledons are
important for primary and secondary root production of Z. floridana embryos in
Whites culture medium. Root elongation and nodulation were observed in embryos
12 Biotechnological Approaches Towards Micropropagation 251
Table 12.3 Resume of in vitro studies on cycads using somatic tissue as explants
Species Medium Adjuvant Response Reference
Cycas revoluta MS 2,4-D,Kn,GA3 Callus Brown and Teas
(1966)
Encephalartos sp. MS NAA, Kn Callus Koelman and
Small (1982)
Stangeria eriopus SH 2,4-D, Kn Callus, leaf Osborne and van
Staden (1987)
Ceratozamia MS+B5 2,4-D, Kn Callus, somatic Chavez etal.
mexicana embryo (1992a)
Ceratozamia MS+B5 2,4-D, Kn Callus, somatic Litz etal. (1995a)
hildae embryo
Cycas revoluta MS 2,4-D,BAP, Kn Callus, somatic Dhiman etal.
C. rumphii embryo (1998a)
Zamia furfuracea
252 M. Dhiman and I. Rautela
12.2.2
In Vitro Culture Studies Using
Megagametophyte as Explant
12.2.3
In Vitro Culture Studies Using Seeding Explants
Webb and his associates have extensively studied the root elongation and nodu-
lation in seeding of cycads under culture conditions (Webb 1981a, b, 1982b, c;
Webb etal. 1984). When germinating seeds of different cycads were cultured on
modified Whites basal medium in dark, a typical tap root system developed. Ex-
posure to light induction nodulation and apogeotropism in the regeneration roots
(Webb 1981a, b, 1982b; Webb etal. 1984). However, seeding of Dioon edule failed
to nodulate in vitro and light induced callus formation in primary and secondary
roots (Webb 1982b, 1984). Seedling cultures of Macrozamia grown in dark formed
apogeotropic nodules at the junction of the primary root and shoot. In light, later-
als developed along the primary tap root and were converted into coralloid roots
(Webb 1983).
12 Biotechnological Approaches Towards Micropropagation 255
12.2.4
In Vitro Culture Studies Using Leaf as Explants
12.2.5
In Vitro Culture Studies Using Root as Explants
Callus formation from root explants of nine species of Encephalartos was reported
by Koeleman and Small (1982). Callus initiation was slow and took 46 months.
Subsequent organization development was not possible. Successful regeneration
from primary roots of Stangeria eriopus was reported by Osborne and van Staden
in 1987. They cultured root segments on SH medium having 2,4-D and Kn. Callus
was produced which developed into small green meristematic zones followed by
emergence and expansion of a typical circinnate leaf.
12.2.6Somatic Embryogenesis
long time. Therefore, time needed for somatic embryos and their subsequent de-
velopment is biologically determined.
The number of cotyledons in cycads is variable even within a species. Cham-
berlain (1935) reports that usually there are two cotyledons, but number may vary
between 16. In Ceratozamia, De Luca etal. (1979) indicated that the somatic em-
bryo seems to have only a single cotyledon. Chavez etal. (1992c) also showed that
C. mexicana had only one cotyledon. They also reported that somatic proembryo
of C. hildae underwent successive cleavage divisions resulting in somatic embryo
having many cotyledons (Chavez etal. 1992b). They stated that single cotyledony
may result from either fused cotyledons or cotyledons may be surrounded by a co-
leoptiles like sheath, thus appearing to be monocotyledonous. In Zamia furfuracea
also, (Dhiman etal. 1998b) number of cotyledons varied between one to as many
as eight, single cotyledon being predominant. Saxton (1910) observed that in vivo
developed embryo of the cycads Encephalartos could sometimes be branched and
that each branch could bear an equally developed embryo. Thus, polycotyledonous
somatic embryo may be explained in term of the fact that branching may occur in
early development of a proembryo, resulting in many cotyledons.
Colour of the somatic embryo is also a unique feature in the cycads. Light pink
colouration is characteristic feature of certain cycads embryos. It is creamy white
to light pink in colour (Litz etal. 1995a). In Zamia furfuracea also, somatic em-
bryo were initially pinkish in colour; however some were green also (Dhiman etal.
1998b).
12.3Ephedrales
There are about 68 species of Ephedra (Sharma and Dhiman 2010) spreading
worldwide, in Europe, temperate Asia, South America and Afghanistan to Bhutan
(24005000m) adapted to semiarid and desert environment. These are widely dis-
tributed in both Eastern as well as Western Hemisphere.
In India, Ephedra is represented by nine species (Sahni 1990) namely E. foliata,
E. gerardiana, E. intermedia, E. nebrodensis, E. regeliana, E. saxatilis, E. pachyclada
and E. przewalskii. Recently, Sharma and Uniyal (2008) and Sharma etal. (2010),
discovered few new species viz. E. sumlingensis, E. kardangensis and E. khurikensis
from Sumling (district Spiti), Kardang (district Lahul) and Khurik (district Spiti) re-
spectively in Himachal Pradesh. Medicinally important species of Ephedra includes
E. gerardiana, E. nebrodensis, E. saxatilis, E. sinica and E. monosperma.
Earlier reports indicate that the ancient Aryans discovered Ephedra or Soma
plant as an energizer-cum-euphoriant. Use of Ephedra juice for longevity was a
part of ancient Indian Aryans custom mentioned in the Rigveda (the oldest of sa-
cred Sanskrit Vedas) and followed even by ancient Romans (Mahdihassan 1981;
Mahdihassan and Mehdi 1989). In China, the Ephedra species have been dispensed
in Traditional Chinese Medicines (TCM) for at least 5000 years (Morton 1977)
and is popularly known as Ma Huang. In TCM, dried stems of Ephedra species are
12 Biotechnological Approaches Towards Micropropagation 257
Table 12.6 In vitro studies in Ephedra foliata using female gametophyte as explant
Medium Adjuvants Response Reference
WM CM, 2, 4-D Kn Callus Sankhla etal. (1967a)
MS CM, 2, 4-D Kn Callus, root, shoot Konar and Singh
(1979)
MS CM, 2, 4-D, Kn NAA, Callus, root, shoot, Singh and Konar
BAP plantlet (1981)
MS CM, 2, 4-D, Kn NAA, Callus, root, shoot, Singh etal. (1981)
BAP plantlet
MS CM, 2, 4-D, Kn NAA, Callus, root, shoot, Bhatnagar and Singh
BAP plantlet (1984)
12.3.1
In Vitro Culture Studies Using Stem Segment as Explant
Initial study on callus culture in Ephedra goes dates back to 1963 in which Straus
and Gerding explained IAA oxidase activity in isolated callus culture from stem of
an unknown species of Ephedra. They observed that Ephedra tissue produces IAA
oxidase in cultures which destroy IAA but not 2, 4-D or NAA. Work on tissue cul-
ture of Ephedra stem segment mainly deals with studies on alkaloid content of the
callus. Khanna and Uddin (1976) extracted a compound from E. foliata stem callus
and identified it as ephedrine. Uddin (1977) studied the presence of different amino
acids in E. foliata suspension cultures. Callus was obtained from stem pieces in MS
medium supplemented with 2, 4-D. He stated that total amino acid contents increase
with the age of the culture. They reported a higher concentration of glutamic acid
and arginine as compared to leucine and serine.
ODowd etal. (1993) observed the effect of various plant growth regulators on
callus production from stem explant in many species of Ephedra viz. E. andina,
E. distachya, E. equisetina, E. fragilis, E. gerardiana, E. intermedia, E. major, E.
minima and E. saxatilis. All species produced callus on MS medium supplemented
with Kn and 2, 4-D or NAA. Neither IAA nor IBA induced any significant callus
formation. Suspension cultures of callus were also established. Trace quantities of
l-ephedrine and d-pseudoephedrine were produced in suspension cultures of all the
species except E. distachya, E. fragilis and E. saxatilis.
ODowd and Richardson (1993a) cultured internodal portions of many species
of Ephedra on MS medium supplemented with 2, 4-D and BAP. E. fragilis pro-
duced green callus and adventitious buds. However, E. andina, E. distachya, E.
gerardiana, E. gerardiana var. sikkimensis and E. saxatilis formed green nodular
structures with roots.
In vitro micropropagation of 11 species of Ephedra was carried out by ODowd
and Richardson (1993b). E. fragilis nodal explants were cultured on MS medium
supplemented with 0.05M IBA and 0.05M Kn, Zeatin or BAP. At high concen-
trations (3.755M) of cytokinin, multiple shoots were produced. Substituting IBA
with 2,4-D caused callus formation and distorted shoot growth. Shoots from nodal
explants of ten other species were also obtained on 0.05M IBA and 0.05M Kn.
Rooting of shoots was observed on medium containing 15M IBA. Plantlets, thus
obtained, were successfully transplanted to pots.
260 M. Dhiman and I. Rautela
Fig. 12.2 a Four-week-old culture on BM+5M 2,4-D+5M Kn showing compact green callus
with initiation of somatic embryos and roots. b Four-week-old culture on BM+5M 2,4-D+8M
Kn showing callus with initiation of somatic embryos. c, d 30 and 45 days old cultures, respec-
tively on BM+8 M 2,4-D+8 M Kn showing initiation and further development of somatic
embryos. Roots are also visible. (From Dhiman and Sharma 2010)
containing 2, 4-D and Kinetin. The somatic embryo showed germination in basal
medium and plants thus regenerated were transferred to pots. Histological studies
confirmed the somatic embryogenesis.
Garla etal. in 2011 cultured nodal segment of E. gerardiana and reported callus
formation followed by shoot bud production however, root induction or plantlet
production was not achieved. Hegazi and El-Lamey (2011) studied ephedrine con-
tent in callus obtained from stem segment culture of E. alata. They reported excess
ephedrine content in 2, 4-D and Kn derived callus as compared to stem of both wild
& cultured plants.
Sharma etal. (2012) cultured nodal and internodal segments of E. gerardiana
onto MS medium containing Thidiazuron. The internodal segments showed callus-
ing followed by somatic embryogenesis onto lower concentration of TDZ. Howev-
er, nodal segment exhibited direct and indirect shoot bud production onto different
TDZ supplemented medium (Fig.12.3ai).
262 M. Dhiman and I. Rautela
Fig. 12.3 Somatic embryogenesis, shoot bud formation and plant regeneration in Ephedra gerard-
iana. a Internodal explant showing callus initiation on to MS+1M TDZ after 2 weeks of culture.
b, c Embryoid formation from callus obtained on to MS+0.5M TDZ after 4 weeks. d Somatic
embryos obtained from same as c after 2 weeks of transfer onto basal medium. e Induction of shoot
buds from nodal segment cultured on to MS+0.5M TDZ after 1 week of culture. f Same as e,
after 6 weeks. g Elongation of shoot on to MS after 3 weeks of transfer. h Rooting of shoots on
to one fourth MS+20M IBA after 2 weeks. i Transplanted plants in plastic pots. (From Sharma
etal. 2012)
12.3.2
In Vitro Culture Studies Using Embryonal/
Seedling Explants
There are various reports of in vitro work on Ephedra using seedling explant. Cal-
lus induction from hypocotyls segments of E. gerardiana and subsequent differen-
tiation of tissues was reported by Ramawat and Arya (1976). Green to yellowish
brown callus was formed on MS medium containing Kn and NAA. Embryoids were
produced on medium supplemented with Kn and NAA but they did not differenti-
ated into plantlets.
Ramawat and Arya (1977, 1979d) studied the effect of various sugars and nitro-
gen sources on callus growth of E. gerardiana and E. foliata. Sucrose supported
best callus growth in both the species followed by glucose, maltose and fructose.
Various nitrogen sources such as ammonium nitrate, potassium nitrate, ammonium
sulphate, calcium nitrate, ammonium citrate and urea were added to the medium.
On a single source of nitrogen, callus failed to grow while on a mixture of nitrate
and ammonium nitrogen, the callus growth in both the species was satisfactory.
Protein content of E. gerardiana callus was nearly four times higher than that of E.
foliata, irrespective of nitrogen source.
Ramawat and Arya (1979a, b, c) also studied the alkaloid contents and ephedrine
production in callus culture of Ephedra. They found that the callus derived from E.
foliata was devoid of alkaloid. However, 8 week-old callus of E. gerardiana yielded
0.17% alkaloid on MS+Kn+NAA. Moreover, there was an increase in ephedrine
content (0.3%) in callus subculture on MS+Kn and IBA. They noticed a decline
in ephedrine level (0.13%) when medium was supplemented with 2,4-D. They also
observed the effect of some precursor amino acids (phenylalanine, methionine and
glycine) on ephedrine production in E. gerardiana callus cultures and found that
these amino acids increase the alkaloid contents as compared to control. Maximum
yield (0.6%) was obtained from callus grown in a medium supplemented with 4M
IBA and 0.1g/l phenylalanine.
ODowd and Richardson (1993a) obtained adventitious shoot bud primordial
formation from germinating seeds of E. fragilis onto MS medium supplemented
with 2, 4-D and BAP. When these shoot bud primordia were transferred to medium
containing 0.05M IBA and 0.05M Kn, only 5% grew into shoots. However,
they could not obtain plantlets.
Dhiman etal. (2010) reported somatic embryogenesis (Fig.12.4ac) from half
embryonal segments of E. foliata grown onto MS medium containing 2, 4-D and
Kn/BAP. The somatic embryos germinated on the basal medium to form emblings.
The plants thus produced were transferred to pots containing sterilized mixture of
coarse sand and garden soil (1:1). During the process of gradual hardening, nearly
70% plants survived (Fig.12.5ac).
264 M. Dhiman and I. Rautela
Fig. 12.4 a Induction of somatic embryos from half embryo explants of Ephedra foliata cultured
onto BM+2 M 2, 4-D+2 M Kn after 5 weeks of culture. Roots and callus are also visible
(2.4). b Somatic embryos onto BM+5M 2, 4-D+5M BAP after 45 days of culture (3.4). c
Somatic embryos produced onto BM+5M 2, 4-D+8M BAP after 45 days of culture (3.3).
(c-callus, se-somatic embryos) (From Dhiman etal. 2010)
Fig. 12.5 a, b Different stages of germination of somatic embryos after 1 and 3 weeks of transfer,
respectively onto BM (2A2.7, 2B2.0). C. Regenerated plant after 6 weeks of transplantation
(0.69). (From Dhiman etal. 2010)
12.3.3
In Vitro Culture Studies Using Female
Gametophyte as Explant
Sankhla etal. (1967a) reported callus formation from the female gametophyte of
E. foliata on Whites basal medium containing 2,4-D and Kn. Callus could not be
maintained and it did not undergo morphogenesis. A considerable amount of work
has been done onto the regeneration potentialities of female gametophyte of E. fo-
liata (Konar and Singh 1979; Singh etal. 1981; Singh and Konar 1981; Bhatnagar
and Singh 1984). They obtained haploid callus, roots, shoot buds and plantlets from
mature and immature female gametophytes cultured on MS medium with 2% su-
crose and 10% CM. It was found that the age of the explant, culture conditions
12 Biotechnological Approaches Towards Micropropagation 265
and a subtle balance of auxins (2, 4-D and NAA) and cytokinins (Kn and BAP)
could make the female gametophyte of E. foliata a plastic system for morphogenic
potential in general and induction of haploid roots and shoots in particular. Female
gametophytes at archegonial stage were more regenerative than at mature embryo
stage in terms of percentage of root and shoot bud regeneration as well as maximum
shoot production per explant. When 2, 4-D (9M) and Kn (9.3M) were added
to the medium, the explant showed maximum percentage of shoot bud regenera-
tion (75%). The regeneration of roots was dependent upon the presence of NAA
(0.2721.48M), while BAP (0.222.22M) enhanced the root promotion effect
of NAA. However, at higher concentrations of BAP (4.426.8M), both roots and
shoots were formed (Bhatnagar and Singh 1984).
12.3.4Somatic Embryogenesis
12.4Conclusion
The cycads are trees resembling palms and have manoxylic wood which is of no
commercial value. The antiquity of cycads is indicated by the presence of ciliate
sperms and their restricted distribution. These plants usually propagate through
seeds or asexually by adventitious shoots or bulbils. Nearly all cycads are slow
growing and require a long period of growth before reaching the stage of repro-
duction vegetative by or sexual means. Aptly referred as Living fossils and
Dinosaurs of plant kingdom, the cycads have survived up to the present age.
The presence of the poisonous glycosides, cycasin and macrozamin (Tadera etal.
1985a; Yagi and Tadera 1987) in cycad tissues may be a potential deterrent to the
266 M. Dhiman and I. Rautela
consumption of these plants and helped in the long term survival of this plant
group (Fosberg 1964). Cycasin (Methylazoxymethanol glycoside) is known as a
carcinogenic agent, neutroxic and some studies also report its interference with
germination and growth of seeds (Kobayashi etal. 1980; Tadera etal. 1982). Cy-
casin is also reported to alter the in vitro growth and development of some molds
as well as insect larvae (Kobayashi etal. 1980; Tadera etal. 1985b, 1987). Biosyn-
thesis of cycasin in callus culture of Cycas revoluta has been reported by Tadera
etal. (1995).
If cycasin is effective against microbes and insect, it could be screened as a po-
tential tool for biological control. To obtain the chemical, either destruction of the
plants would have to be carried out or the other option is it could be produced by
mass scale callus cultures. The latter is dependent on techniques of tissue culture.
Cycad tissue grown in vitro are extremely slow growing and mimic the slow growth
in Nature.
Ephedra is one of the source of the alkaloids mainly l-ephedrine and d-pseu-
doephedrine. Callus culture can be possibly used for ephedrine biosynthesis in re-
actors. It may also be feasible to screen Ephedra populations and then clonally
propagate the elite plants.
There are, successive losses in population number and changes in distribution
ranges over a period of time, this has led to the listing of Ephedra gerardiana, a
high alkaloid containing species as an endangered species. The plant is categorized
as IV ranked plant of cold desert zone based upon knowledge, cultivation prospect
and marketing. Therefore, micropropagation of E. gerardiana & re-introduction of
elite germplasm into its natural habitat can be an aid for conservation of endangered
medicinal germplasm.
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Chapter 13
Genetic Resources and Biodiversity
Conservation in Nigeria Through
Biotechnology Approaches
Justin U. Ogbu
Abstract The chapter presented a treatise on plant genetic resources (PGR) and
biodiversity conservation in Nigeria vis-a-vis the relevance of biotechnological
approaches. It showed synopsis of plant resources base of the world at global and
Tropical African perspectives, and the shrinking diversity of present day agroeco-
systems. Attempts were made to review floristic and economic crops diversity of
the country as well as the concerns for the current spiral depletion and loss of vital
national plant genetic resources. Biotechnology has been recognized as a versatile
tool for biodiversity conservation, management and use. It offers range of applica-
tions to improve the understanding and management of genetic resources for food
and agriculture. It has been proven that modern biotechnologies can help to coun-
teract trends of genetic erosion in all food and agriculture sectors. Biotechnology
procedures in conservation and management of PGR were clearly discussed, while
institutional framework for conservation of national PGR was highlighted.
KeywordsConservation biotechnology Extinction Genetic resources
Nigeria Plant diversity
13.1Introduction
J.U.Ogbu()
Department of Horticulture and Landscape Technology,
Federal College of Agriculture (FCA), Ishiagu 491105, Nigeria
e-mail: ogbujugo@gmail.com
development. The categories of PGR range from landraces and farmers varieties,
absolute cultivars, modern cultivars, breeding lines and genetic stocks, wild rela-
tive, weedy races and potential domesticate species, exotic and indigenous species
(Engels and Visser 2006; Ruane and Sonnino 2006; Sharma 2007; FAO 2011a).
Biotechnology has been described (according to Convention on Biological Di-
versityCBD) as any technological application that uses biological systems, liv-
ing organisms, or derivatives thereof, to make or modify products or processes for
specific use. Following FAO statement on biotechnology, a narrower sense of inter-
pretation has also been added which described biotechnology to include other range
of different molecular technologies such as gene manipulation and gene transfer,
DNA typing and cloning of plants and animals (FAO/SDRR 2006; Speedy 2007).
Biotechnology has been recognized as a veritable and vital tool for biodiversity
conservation, management and use. It offers range of applications to improve the
understanding and management of genetic resources for food and agriculture. It has
been proven that modern biotechnologies can help to counteract trends of genetic
erosion in all food and agriculture sectors (Ruane and Sonnino 2006).
Nigeria is one of the largest countries in West Africa, and has a land area of approxi-
mately 91.07millionha. Nigerias territorial area spanned from latitude 414N to
1348N and from longitude 242E to 1440E. The country is a physically, cli-
matically and biologically diverse one. Essentially the country encompasses three
major ecological regions, viz: a humid tropical forest region, a sub-humid region
with highland and a semi-arid region; with annual rainfall ranging from 250mm
in the Sahelian north to over 3000mm in the southern coastal areas. The countrys
climate is largely tropical humid, characterised by high humidity in south, high tem-
peratures and intense heat in the north. In some areas north of the country (Kano,
Kaduna, Bauchi, Plateau and other similar states), the harmattan wind from Saharan
desert results in a mild cold dry winter, and thus permits the growth of winter crops
such as wheat and cold loving vegetables crops during the cool harmattan period
between December and February. The natural vegetation varies from rainforest to
savanna. Nigeria is also endowed with substantial biological resources. These in-
clude 68millionha of arable land (but barely 32millionha are annually cultivated),
and fresh water resources covering 12millionha. Land use patterns in the country
shows that cropland takes 34% of total land area, pasture takes 23%, forest 16%,
rivers/lakes/reservoirs 13% and others 14% (Shaib etal. 1997; Akoroda 2010).
Reports by Federal Environmental Protection Agency (FEPA) (Adejuwon 2000),
showed that the floristic diversity in Nigeria comprised of 4903 species of angio-
sperms, 32 species gymnosperms, 155 pteridophytes, 80 species bryophytes, 784
species algae, 3423 species fungi and more than 500 species virus. According to
the reports also, 20 species of plants had become extinct since 1950, 431 species
are endangered, 45 species are classified as rare, 20 species are vulnerable, while
305 species are endemic. All these are of PGR conservation concern to the country
13 Genetic Resources and Biodiversity Conservation in Nigeria 275
Over the several millennia of human existence on earth, Plant Genetic Resources
(PGR) had constituted the basis of development and sustainability of agricultural
production systems. After 10,000 years of sedentary agriculture and the discovery
of about 75,000 varieties of edible plants, close to 7000 identified species have been
used in agriculture for food and fodder (Dhillon and Saxena 2003). However, today,
less than 2% of these are recognized as economically relevant at regional, national
or global levels (FAO 1996). Currently, only 30 cultivated plant species provide
90% of all the human food obtained from plants, while 12 plant and 14 animal
species together provide 70% of the world human diet (Spore 2010). Three crops
namely rice, wheat and maize, make up the basic food for two third of the world
population (Jaramillo etal. 2011; Bioversity International 2012).
Extinction of genetic resources and PGR in particular, has been a naturally oc-
curring phenomenon over millions of years, without any human involvement. How-
ever, due to unprecedented human activities in the past few scores of years and
their effect on the environment, species and ecosystems have become increasingly
threatened in an alarming way, thereby undermining the basis required for sustain-
able development. According to a recent CTA report on agrobiodiversity, 75% of all
276 J. U. Ogbu
known crops have disappeared in the past century (Spore 2010). On the other hand,
the United Nations FAO has projected that unless the spiral loss of genetic diversity
is controlled, about 60,000 plant species (quarter of the world plant capital) might
be lost by 2025 (WCMC 2002).
Table 13.1 Some National Agricultural Research Institutes with plant based mandates and similar
institutions relevant to PGR conservation
NIFORNigerian Institute for Oil Palm Research, Benin City
NIHORTNigerian Institute of Horticultural Research, Ibadan
NRCRINational Root Crops Research Institute, Umudike
NCCRINational Cereal Crops Research Institute, Badegi
CRINCocoa Research Institute of Nigeria, Ibadan
FRINForestry Research Institute of Nigeria, Ibadan
IARInstitute of Agricultural Research, Samarru Zaria
IARTInstitute of Agricultural Research and Training, Ibadan
RRINRubber Research Institute of Nigeria, Benin City
NACGRABNational Centre for Genetic Resources and Biotechnology, Ibadan
NABDANational Biotechnology Development Agency, Abuja
NPQSNigerian Plant Quarantine Services, Moor Plantation Ibadan
NISLTNigerian Institute of Science Laboratory Technology, Ibadan
13 Genetic Resources and Biodiversity Conservation in Nigeria 277
yam, potato, sweet potato, ginger, turmeric], Nigeria Institute for Oil Palm Re-
searchNIFOR [coconut, oil palm, date, ornamental palms], National Centre for
Genetic Resources and Biotechnology (NACGRAB), Forestry Research Institute
of Nigeria (FRIN) [for inputs on conservation/domestication of certain important
indigenous fruit trees, aromatic and medicinal plants, as well as the forest trees] and
other organization that share similar interest in PGR and biodiversity management.
With reference to organisations in the Nigeria that have plant biotechnology
activities (including conservation biotechnology of plant genetic resources), there
are several of them ranging from government owned, CGIAR owned and privately
owned institutions (Table13.2).
In vitro
conservaon
Plant
Micropropagation cryopreservaon
Molecular
marker DNA banking
technology
13.7.1
In Vitro Conservation and Cryopreservation Techniques
and natural disasters. It is commonly use for vegetatively propagated species, non-
orthodox seeded species and wild species which produce little or no seeds.
While cryopreservation at ultra-low temperature, usually that of liquid nitrogen
(196C), is the only option currently available for the long-term conservation of
these PGR avoiding exogenous contamination, requiring small space and minimum
maintenance. At this very low temperature, all metabolic activities of cell cease,
and theoretically the cell or tissue can be stored for an indefinitely period. Both in
vitro conservation and cryopreservation techniques use tissue culture principles for
conservation (Roca etal. 1989; Reed 1993; Mandal 2003).
The realization of the potential of in vitro conservation came about in the early
1970s, at a time when the storage of microbial cultures was a routine procedure.
Since then, tissue culture techniques have been applied to more than 1000 plant
species. Subsequently, the technique has progressed from mere speculation to de-
velopment, and today it is routinely being used for conservation of vegetatively
propagated crops and perennial species (Tyagi and Yusuf 2003). The art and science
of plant tissue culture is based on devising media for each genotype/species that
would elicit the optimal response in terms of growth rate of the explants. However,
when tissue techniques are employed for conservation, the aim is to devise a me-
dium that would decrease the growth rate of explants to the minimum, thereby in-
creasing the subculture intervals. Slow growth techniques have been developed for
medium-term conservation of crop species (Engelmann and Drew 1998; Sarkar and
Naik 1999). The various methods used to achieve this include the following: use of
growth retardants, use of minimal growth media, use of osmotic regulators, reduc-
tion in oxygen concentration, size and type of culture vessels, type of enclosures,
maintenance under reduced temperature and for reduced light intensity and com-
bination of more than one treatment. Explants used for in vitro conservation must
be of right type as well as physiological stage. The apical and auxiliary meristems
of very small size are the preferred explants for in vitro storage. In fact, organized
explants have proved better than unorganized tissues, in terms of genetic stability of
the germplasm (Mandal 2003; Reed etal. 2004; Chaudhury and Vasil 1993).
13.7.2Cryopreservation
to test stored materials frequently, thus making storage cost-effective. There have
been several reviews on the use of cryopreservation for storage of plant materials
(Kartha and Engelmann 1994; Engelmann and Takagi 2000; Towill and Bajaj 2002;
Chaudhury 2002; Mandal 2003; Reed etal. 2004). The choice of material for cryo-
genic storage will depend on the plant species as well as the objectives of storage.
For conservation of PGR, the explants can include shoot apices, auxiliary buds,
dormant buds, somatic embryos, seeds, zygotic embryos, embryonic axes or pol-
lens. Cryopreserved explants (but pollen) should eventually regenerate whole plants
to be used and therefore, regeneration protocols need to be clearly defined prior to
embarking on cryopreservation. Regenerated plants should also maintain genetic
integrity of the starting material. The various techniques currently under investiga-
tion or in use include: the classical freezing method, encapsulation-dehydration,
vitrification, encapsulationvitrification, desiccation, pre-growth droplet freezing,
and pre-growth desiccation (Mandal 2003; Panis 2007).
13.7.3DNA Bank/Library
DNA storage is a relatively new technique that is rapidly gaining recognition. Sev-
eral DNA libraries are being established which provide an easy access for scientists.
DNA from the nucleus, mitochondria and chloroplasts are now routinely extracted
and immobilized into nitrocellulose sheets where the DNA can be probed with nu-
merous cloned genes. With the development of PCR (polymerase chain reaction),
one can now routinely amplify specific genes or oligonucleolides from the entire
mixture of genomic DNA. This approach, according to Engelmann etal. (2003),
is relatively easy and of low cost. It is particularly useful for the conservation of
specific genes and it allows easy access to specific material. The exchange of germ-
plasm through DNA sequences is safe since infestations with pathogens can be sim-
ply avoided. Note however, that entire plant cannot be regenerated from conserved
DNA (Reed etal. 2004; Guimaraes etal. 2007).
13.7.4Micropropagation
of selected plant species line, elimination of pathogens from seedlings, lesser re-
quirement of space, time and labour, season-independent, ease of transportation,
ease of exchange of plant germplasm and less quarantine restrictions. Various ap-
proaches used in this biotechnology include but not limited to these only: in vi-
tro micrografting, artificial seed production, somatic embryogenesis, adventitious
regeneration, single node culture, meristems culture, anther culture, and axillary
branching (Speedy 2007; Sonnino etal. 2009).
13.7.5Molecular Markers
Conservation and management of PGR for food and agricultural production via bio-
technology procedure in developing countries including, Nigeria, at present prove
to be a knotty issue among the policy makers and ruling class as the immediate
economic gain for such venture is not always readily felt. Often times the hype and
hot debate about genetically modified (GM) crops/food have tend to becloud the
13 Genetic Resources and Biodiversity Conservation in Nigeria 283
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Chapter 14
Biotechnology Tools for Conservation
of the Biodiversity of European
and Mediterranean Abies Species
J.Krajkov()
Department of Agriculture and Environmental Science, University of Udine,
Via delle Science 91, 33100 Udine, Italy
e-mail: jana.krajnakova@uniud.it
D.Gmry
Faculty of Forestry, Technical University Zvolen, T.G. Masaryka 24, 960 53 Zvolen, Slovakia
J.Krajkov H.Hggman
Department of Biology, University of Oulu, PO Box 3000, 90014 Oulu, Finland
14.1Introduction
According to the United Nations Food and Agriculture Organization (FAO 2013),
the world forest area is slightly more than 4billionha and its importance as a car-
bon sink is enormous. In Europe, forests represent almost half of the land surface
(102millionha, which amount to 25% of the world total), of which 65% are co-
nifers. Over the last 20 years, the forest area has expanded in all European regions
and has gained 0.8millionha in each year (Forest Europe 2011). European forests
sequester increasing amounts of carbon in tree biomass, between 2005 and 2010,
about 870milliont of CO2 have been removed annually from the atmosphere by
photosynthesis and tree biomass growth in European countries. This corresponds
to about 10% of the greenhouse gas emissions in 2008 of these countries (For-
est Europe 2011). Moreover, increasing population numbers in combination with
accelerated climate change including weather extremes (Nellemann etal. 2009)
are predicted to increase the need for more wood production. Wood is the worlds
only large scale renewable, sustainable and environmentally friendly raw material
and more systematic use of its potential needs to be made at the global level, if
the aim of achieving true sustainability for the world is to be met (Sutton 2013).
In forested landscapes, trees play also essential roles in ecosystem structure
and functioning. They mediate energy and material flows and are associated with
processes such as water and nutrient cycling, biomass production, soil formation
etc. Genetic diversity, which is closely associated with adaptability and population
stability, is an inevitable prerequisite for fulfilling these functions (Pimm 1984;
Johnson etal. 1996; Lefvre etal. 2013). In spite of positive data about the increas-
ing forest area in Europe, about a fifth of all trees are damaged or dead and 11mil-
lionha (or 1%) of Europes forests are affected by forest damage, most frequently
caused by insects and diseases, followed by wildlife and grazing (Forest Europe
2011).
Currently, the IUCN Red list includes 6277 tree species that are threatened
with extinction in the wild (http://www.iucnredlist.org). Of these, 1002 tree spe-
cies are recorded as Critically Endangered, the most threatened category for spe-
cies based on the risk of extinction (Oldfield 2009) indicating an urgent need for
germplasm conservation. Fulfilling the commitments adopted within the Global
Strategy for Plant Conservation (Convention on Biological Diversity 2010), es-
pecially preservation and sustainable use of genetic resources, requires elabora-
tion and application of a wide spectrum of tools for in situ and ex situ conserva-
tion. Biotechnological approaches can substantially contribute to the success of
such efforts.
14 Biotechnology Tools for Conservation of the Biodiversity 289
Euro-Mediterranean firs (the genus Abies Mill.) belong to ecologically and com-
mercially most important tree genera in Europe (Table14.1). Fir forests represent a
major component of Central European, Alpine and Mediterranean mountain forests.
Their distribution ranges from 6W to 44E in longitude, from 35N to 52N in lati-
tude and from 135 to 2900m in altitude (Alizoti etal. 2011) (Fig.14.1 Abies alba,
Fig.14.2 Mediterranean fir species).
Like in the other tree species in Europe, the history of firs has been turbulent
and left profound traces in their species diversity and genetic structures. Glacial/
interglacial climatic cycles during the Pleistocene provoked large retreats and ex-
pansions of species ranges. Mediterranean Sea bordering Europe from the south
largely prevented southward migration; this obstacle drove several tree genera to
local extinction (e.g., Pseudotsuga, Cryptomeria, Sequoia, Taxodium; Martinetto
2001; Svenning 2003). At the species level, the consequences are manifested in
reduced species diversity. Only four fir species have survived in Europe until recent
times (A. alba, A. cephalonica, A. pinsapo, and A. nebrodensis).
Greek fir (Abies cephalonica Loudon) is endemic to Greece, where it grows be-
tween 400 and 1800 (2000)m a.s.l. on a variety of parent rocks such as limestones,
dolomites, serpentines, sandstones, and schist with soil pH ranging from 5 to 8
(Panetsos 1975). At present, the population of Greek fir is considered stable. On
the other hand, the remaining two fir species are truly rare. Spanish fir (A. pinsapo
Boiss.) range covers only 1200ha in southwestern Spain (Arista 1995), on dolo-
mitic and serpentine soils at elevations between 1000 and 1600m. Its population
decreases. Climate change associated with increasing incidence of wildfires, pests
and diseases might under circumstances drive the species to extinction. The single
existing natural population of the Sicilian fir (A. nebrodensis Mattei) is extremely
small, consisting of 29 adult trees only (Alizoti etal. 2011), and grows on a single
limestone site in Sicily at elevations around 1500m. Although population size is
stable and genetic diversity is surprisingly high, the species is logically considered
critically endangered.
Abies alba Mill., silver fir, is the only widespread and abundant species of the
genus Abies in Europe. Longitudinally, the range spans between the Central Mas-
sive in France and the Eastern Carpathians in Romania. Isolated occurrences can
be found even more westwards, in the Pyrenees and Normandy. Latitudinally, silver
fir is distributed between the Dinaric Mountains and central Poland. Again, isolated
290 J. Krajkov et al.
Table 14.1 List of the European and Mediterranean Abies species, threats to genetic diversity and
information about in situ and ex situ conservation
Scientific name Common Category accord- In situ Ex situ conservation
name ing to IUCN Red conservation Stands/seed Tissue cul-
List of threatened standsa orchard ture system
species
Section Abies
A. alba Mill. Silver fir Least concern 36.315hab Conservation Yes, SE
stands 307ha
A. nebrodensis Sicilian fir Critically One seed No
(Lojac) Mattei endangered orchard
A. cephalonica Greek fir Least concern 1.210hab Conservation Yes, SE
Loudon stands 6ha
A. borisii-regis Bulgarian fir Least concern 456hab No
Mattf.
A. nordmanni- Nordmann Least concern unknown Yes, SE
ana (Steven) fir, Cauca-
Spach. sian fir
A. bornmuelle- Bithynian fir Endangered 213hab No
riana Mattf. (A.
nordmanniana
ssp. bornmuel-
leriana)
A. equi-trojani Turkish fir, Endangered 293hab No
Coode and Kazdaghi fir 24.374hac
Cullen (A. nor-
dmanniana ssp.
equi-trojani)
Section Piceaster
A. pinsapo Spanish fir Endangered 100hab No
Boiss.
A. marocana Moroccan fir Critically Seven ex situ No
Trabut (A. endangered stands
pinsapo ssp.
marocana)
A. cilicica (Ant. Taurus fir, Near threatened 69hab Yes, SE
and Kotschy) Cilicia fir
Carrire
A. numidica Algerian Fir Critically Yes, SE
de Lannoy ex endangered
Carrire
a
Specific conservation measures beyond nature conservationb Dynamic gene conservation units
fulfilling the minimum criteria of Euforgen (http://portal.eufgis.org)c Multispecies Gene Manage-
ment Zones (Ozturk etal. 2010)
populations are scattered along the northeastern range limit (Poland, Ukraine) and
the southern part of the range (Apennine and Balkan peninsulas) is highly frag-
mented (Wolf 2003).
Silver fir forms pure stands, but more frequently it can be found in mixed stands
with European beech and Norway spruce, in the south with pines and oaks. It toler-
14 Biotechnology Tools for Conservation of the Biodiversity 291
Fig. 14.1 Distribution map of silver fir (Abies alba). EUFORGEN 2009. (http://www.euforgen.
org)
ates a wide range of soil conditions. Consequently, it can be found over a variety of
parent rocks, covered by soils with varying textures, nutrient levels and pH, avoid-
ing both waterlogged and dry soils. Nevertheless, the best growth and competition
ability of silver fir can be expected on deep, nutrient-rich, fine- to medium-textured
292 J. Krajkov et al.
and well-drained soils. Climatic niche of silver fir is also broad. The species is cold-
hardy, but sensitive to winter desiccation, late and early frosts, and water deficit
during shoot elongation (Hansen and Larsen 2004). Silver fir is very shade tolerant,
especially in young age. Although it is generally considered a typical climax spe-
cies, silver fir is able to colonize pioneer pine forests and even open lands.
In addition to Europe, other fir species occur around the Mediterranean. A. nor-
dmanniana Spach is distributed in eastern Turkey and the Caucasus. In spite of a
fragmented range its population is stable and not endangered. Two subspecies, A.
equi trojani Coode and Cullen and A. bornmuelleriana Mattf. (sometimes consid-
ered separate species or, alternatively, hybrids A. nordmanniana A. cephalonica),
grow in western and northern Turkey, respectively, the former having a very limited
area of occupancy of 164km2. A. cilicica de Lannoy occurs in the Turkish Taurus
Mts., Syria and Lebanon on an area of almost 3400km2. Although its range is not
small, population size decreases and especially Syrian and Lebanonian local popu-
lations are threatened. Both African fir species, Abies numidica Carrire (Kabylian
Mts. in Algeria), and A. marocana Trabut (sometimes considered a subspecies of
A. pinsapo; Rif Mts. in Morocco) have extremely small areas of occupancy (1 and
28km2, respectively), and are critically endangered.
2001; Kobliha etal. 2013). They have thus a potential also for forestry, but their
primary field of use is greenery and Christmas tree production.
Genetic structures of the extant fir populations in Europe have largely been deter-
mined by historical factors. As mentioned above, Pleistocene climatic fluctuations
severely reduced population sizes of all temperate species. Refugial population
of rare fir species (A. pinsapo, A. nebrodensis) did not expand; either due to de-
creased vitality caused by inbreeding and lowered genetic variation, or because
they remained trapped in islands of favorable environments surrounded by dry
highlands or by sea. Almost nothing is known about the population development
of fir species South and East of the Mediterranean Sea in the postglacial period;
nevertheless, these regions have been less influenced by the glaciation, so that lo-
cal fir populations may have persisted since the Tertiary. Holocene warming may,
however, have contributed to the contraction of ranges of A. numidica, A. cilicica
or A. marocana and fragmentation of A. nordmanniana. For A. cephalonica, Fady
and Conkle (1993) concluded that the divergence between A. alba and this species
occurred quite recently, at the beginning of the last glaciation. The reconstruction
of the Holocene history of A. cephalonica is difficult because the pollen of differ-
ent Abies species cannot be distinguished in the fossil pollen record (Terhrne-
Berson etal. 2004). Nevertheless, as the range of A. cephalonica is located in
southern Balkans, which served as an important refugial area during the Holocene,
population sizes, distribution and genetic structures of this species probably have
not changed substantially.
The history of A. alba is more complicated, as this species recurrently succeed-
ed to colonize Europe during the warm phases of the Pleistocene, and during the
Eemian interglacial it even covered larger area than the current range (Terhrne-
Berson etal. 2004). Pollen and macrofossils (mainly charcoal) documented that
cryptic Pleniglacial refugia of silver fir were localized as far north as in Hungary
or Moravia (Willis etal. 2000; Terhrne-Berson etal. 2004). Nevertheless, main
refugial areas were situated more in the south. The analysis of maternally inherited
mitochondrial DNA revealed two genetic lineages of silver fir, one distributed in
western and central Europe, the other in southern Balkans and Eastern Carpathians
(Liepelt etal. 2002). A synthesis of paleobotanical and genetic data by Liepelt
etal. (2009) suggested that the effective refugia for the western lineage could
have been localized in northern Apennines and possibly Maritime Alps, those for
the eastern lineage in southeastern Balkans. Nevertheless, some regional silver fir
populations have originated from local minor refugia, e.g. those in the Pyrenees or
southern Italy.
Not much information is available about the past of Abies species in Asia Minor
and Africa. Genetic diversity of conifers in the Mediterranean is relatively high
compared with other regions of the world (Fady-Welterlen 2005). The rear-edge
294 J. Krajkov et al.
populations are frequently highly differentiated and contain many private alleles
(Petit etal. 2005; Awad etal. 2014). Most rear-edge populations did not substantially
contribute to postglacial recolonization, but rather reacted to climate fluctuations by
altitudinal range shifts (Hampe and Petit 2005). Traces of such local extinction/ex-
pansion cycles can still be recognized in gene pools of A. cilicica (Awad etal. 2014).
During postglacial recolonization, genetic lineages met and formed broad hybrid
zones on both sides of the Danube plain (Gmry etal. 2012). However, natural
hybridization of firs is not limited to the intraspecific level. Mediterranean firs (at
least those within the section Abies) intercross easily. Fir in northern Greece, dis-
tinguished by growth vigour and capable of massive colonization of open areas,
shows intermediate traits between A. alba and A. cephalonica and was classified as
a separate taxon A. borisii-regis Mattf. Phylogeny of this taxon is still unclear, but
genetic analyses generally support the hypothesis of its hybridogenous origin (Fady
etal. 1992; Scaltsoyiannes etal. 1999). Two further taxa, A. equi-trojani Asch. and
A. bornmuelleriana Mattf. occurring in Turkey, are also suspected to be hybrids, in
this case between A. nordmanniana and A. cephalonica.
effects of selective pressures on silver fir gene pools have also been demonstrated
in association with climate (Bergmann and Gregorius 1993) or pollution (Longauer
etal. 2001). This underlines the significance of genetic variation for adaptive prop-
erties of fir populations.
Climatic niche offers much broader distribution of silver fir than the realized
spatial range (Tinner etal. 2013), which, in addition to interspecific competition,
is an indication of strong direct or indirect human pressures. First of all, the area
of forests as such has steadily decreased since the Neolithic, as they were convert-
ed into agricultural land (mainly pastures and meadows in the case of fir forests).
Moreover, since the eighteenth century, natural mixed forests have largely been
being replaced by commercial conifer monocultures in many European countries.
Improper silvicultural systems associated with clear cutting or shelterwood cutting
with rapid canopy opening were also unfavourable for fir (Mayer 1984). Among
indirect influences, game browsing is one of the most important limiting factors
for silver fir regeneration. Current game management practices in many parts of
Europe often support high stocks of red deer, which heavily damages fir juveniles.
Last but not least, fir is susceptible to industrial pollution. The composition of pol-
lutants changes, sulphur dioxide, which was a serious problem in Central Europe in
1970s and 1980s, was replaced by tropospheric ozone, but as a whole, air pollution
remains a serious threat at least locally.
It is difficult to predict the future of firs under the ongoing climate change. Ar-
guing by the extent of fundamental climatic niche based on the comparison of past
climates and past distribution of fir during the Holocene and the Eemian, Tinner
etal. (2013) suggested that silver fir may profit from changing climate almost all
over the range. On the other hand, their study does not take into account potential
genetic differentiation in the past and the complexity of the phenomenon of climate
change, which is not necessarily limited to altering overall levels of temperatures
and precipitations. Drought stress and increased incidence of wildfires are gener-
ally considered the cardinal problem linked to climate change, as most climate sce-
narios predict increasing temperatures and prolonged drought periods, resulting in
increased continentality in much of Europe. However, the effects of climate change
are not restricted to drought. Elevated-temperature events during winter may induce
winter desiccation associated with xylem cavitation and needle loss, which may
decrease productivity of fir forests. Heritable features of tree architecture such as
crown shape or branching form result from evolutionary adaptation to snow pres-
sure and occurrence of hoarfrost and ice (Geburek etal. 2008). Changed winter
precipitation patterns in terms of a shift of wet and heavy snow towards higher alti-
tudes may bring excessive damage. Vegetative phenology (budburst, shoot growth
cessation, frost hardening etc.) results from evolutionary tradeoffs between the
length of the growing season and the risk of frost damage. A part of circum-annual
ontogenetic rhythms is internally regulated and proceed almost regardless of exter-
nal signals, however, climate-associated environmental signals (chilling, thermal
accumulation) play essential role in the timing of growth and reproduction (Konnert
etal. 2014). Changed temperature distribution over the year may confuse the tem-
poral course of life processes and lead to important economical losses.
296 J. Krajkov et al.
In spite of the protection in national parks and reserves, overharvesting and graz-
ing remain the main threats for rare fir species in southern Europe, Asia Minor and
North Africa. Unfavorable consequences of climate change, such as drought and
wildfires are expected to be even more pronounced and thus more risky for the
persistence of fir populations in this area than in central or northern Europe (Alizoti
etal. 2011).
silver fir. The area of in situ gene conservation units meeting the newly defined pan-
European minimum requirements for dynamic gene conservation units (Koskela
etal. 2013) is over 38,000ha for firs (cf. http://portal.eufgis.org).
The rate of the environmental change may exceed the capacity of genetic sys-
tems of population to adapt through natural selection and gene flow or to disperse
into more favourable habitats. Assisted migration or ex situ conservation aimed at
safeguarding populations which are in danger of physical destruction or genetic de-
terioration become viable options under such conditions (Konnert etal. 2014). Con-
servation measures include establishing conservation stands, seed orchards, clonal
archives or storing genetic material in gene banks (Skrppa 2005). At present, there
are 307ha of ex situ conservation stands for A. alba and 6ha for A. cephalonica
(cf. http://portal.eufgis.org). In addition, all Mediterranean species are represented
on numerous experimental sites such as provenance or progeny tests, and are also
conserved in many botanical gardens throughout Europe.
As firs have orthodox seeds, they can be stored over longer period (5 years)
after decreasing water content to 510% with only a minor loss of viability (Bon-
ner 2008) and seeds as stored in the national seed banks. On the other hand, the
cryostorage of A. alba seeds was also successfully tested nearly 30 years ago (Ahuja
1986), but till now, this method has not been vigorously involved in seed storage
banks, as the seed preparation and cooling procedures are complicated (Chmie-
larz 2008). Therefore, practical application is limited to few seed banks (e.g., the
Kostrzyca Forest Gene Bank in Poland; http://www.lbg.jgora.pl).
Ex situ conservation may also be driven by the effort of preserving specific geno-
types, including products of breeding. However, not all measures mentioned above
are applicable in the case of firs. In such cases, non-conventional biotechnological
solutions including cryopreservation and tissue culture techniques may become the
primary method of choice (Blakesley etal. 1996; Li and Pritchard 2009).
In vitro conservation and cryopreservation are the most specialized form of ex situ
conservation of genetic resources and the detailed gene bank standards for in vitro
culture, slow growth storage, and cryopreservation were published by FAO (2013)
recently. Engelmann (2011) recognizes three possibilities of biotechnological ap-
plications for ex situ conservation: (i) in vitro cultures, (ii) slow growth storage and
(iii) cryopreservation.
The recent biorepositories or banks are mostly established by using in vitro pro-
duced plant material and they are depended on the success of in vitro propagation
techniques which have been used for particular species (Pence 2014). In some spe-
cific cases, like isolated embryos or dormant buds, the in vitro methods may only be
applied at the recovery stage.
298 J. Krajkov et al.
Fig 14.3 Somatic embryogenesis of Abies cephalonica. a Elite tree of A. cephalonica. b Devel-
oping green cone shortly after meiosis. c Initiation of somatic embryogenesis using immature
embryos and proliferation of embryogenic cell mass. d Proliferating embryogenic cell mass and
detail of proembryogenic cell masses after staining with acetocarmine and Evans blue. e Option
for cryopreservation of the germplasm. f Maturation of somatic embryos. g Conversion of somatic
embryo plants. h Experimental field trail
300 J. Krajkov et al.
14.3.2.2Cryopreservation
protocol for the embryonic cultures of conifers is the classical slow-cooling and
fast-thawing one (as reviewed by Hggman etal. 2000; Lambardi etal. 2008). Suc-
cessful cryopreservation relies on the removal of freezable water in order to avoid
damage from ice crystallization and on the stabilization of membranes and molecu-
lar structure of the cells to avoid damage from the loss of water (Benson 2008).
Preculturing embryogenic cell masses, somatic embryos or in vitro shoot tissues
with treatments such as cold, increased sugars, or ABA can also work to increase
survival through cryopreservation, presumably by triggering natural desiccation-
adaptive physiology (Kushnarenko etal. 2009). However, even with preculturing,
most plant tissues require the application of further cryoprotective procedures to
remove water and stabilize tissues to maintain viability through LN exposure.
The slow-cooling method requires the use of a controlled-freezing apparatus
to lower the temperature in a constant and controlled way, at rates of 0.11.0C
per min. When temperatures reach 35C or 40C, the samples are plunged into
LN. During the slow freezing, as intercellular water freezes, water moves out of the
cells into the intercellular spaces, slowly dehydrating the cells. Limitations of the
slow-cooling method include the expense of the equipment and the amount of LN
needed. Mr. Frosty and similar products provide a less expensive alternative for
slow cooling (Pence 2014). Cryovials containing samples in a bath of isopropanol
are kept in the freezer at 80C (cooling rate of the samples being 1C per min).
Thereafter the samples are transferred to LN (196C). For thawing and regrowth
of embryogenic cell masses, the cryovials are rapidly thawed in water bath at 37C
for 12min. Cryoprotectants are removed from the thawed embryogenic cellular
masses by gradual elution. The regrowth of culture is obtained and followed on
semi-solid proliferation medium for 46 weeks depending on species and cell line.
In order to overcome some of the limitations of the slow cooling method, Sakai
etal. (1990) reported a different approach, known as vitrification, which combined
rapid freezing with cryoprotectants to cause the formation of glass, rather than crys-
tals, within the tissues. For vitrification, tissues are cryoprotected using more con-
centrated cryoprotectant solutions, the most widely used being PVS2, a mixture of
30% glycerol, 15% ethylene glycol, 15% DMSO, and 0.4M sucrose. Till now,
there are only a few reports where embryogenic cultures of Picea mariana (Mill.)
B.S.P. and Picea sitchensis (Bong.) Carr. have been cryopreserved successfully by
vitrification (Touchell etal. 2002; Gale etal. 2008). Recently, vitrification method
based on a pregrowth-dehydration method was successfully applied to cryopreser-
vation of Picea omorica (Pani) Purk. and Picea abies embryogenic cell lines (Ha-
zubska-Przybyl etal. 2010, 2013) without using cryoprotectants. Other approaches
of elimination of toxic cryoprotectants, such as DMSO, have used the desiccation
tolerance of somatic embryos in preparation for cryostorage and have also been suc-
cessful (Bomal and Tremblay 2000; Kong and von Aderkas 2011).
For the species belonging to the genus Abies, the classical, slow cooling cryo-
preservation procedure has been described only for three Abies species: for A. alba
(Krajkov et al. 2013) A. cephalonica (Aronen etal. 1999), A. nordmanniana
(Nrgaard etal. 1993; Misson etal. 2006), and some fir hybrids (Salaj etal. 2010)
(Table 14.2). As preculture treatment, the culturing of embryogenic cell masses
Table 14.2 Cryopreservation protocols used for ex situ conservation and based on existing SE protocols of European and Mediterranean Abies species
302
Species Preculture Cryoprotectant Time in Cryo-method used Recovery and Genetic fidelity References
cryostorage regeneration tested by genetic
markers
Abies alba
12 embryogenic Cold hardening for 14 5% PGD (polyeth- 6 years Programmable con- 4 out of 12 cryo- No Krajkov
cell lines days, 5C, dark ylene glycol 6000, trolled-temperature preserved cell etal. (2013)
0.2M sucrose/24h glucose, DMSO) chamber lines recovered
0.4M sucrose/24h Maturation
5C, dark experiment
Abies cephalonica
Eight cell lines Cold hardening for 14 5% PGD (polyeth- 7 days Programmable con- All cell lines Yes Aronen etal.
days, 5C, dark ylene glycol 6000, trolled-temperature recovered (1999)
0.2M sucrose/ 24h glucose, DMSO) chamber
0.4M sucrose/24h
5C, dark
Two cell lines detto detto 6 years detto All cell lines Yes Krajkov
recovered etal. (2011a)
Maturation
experiments
Two cell lines 0.2M sucrose/24h detto 7 days Nalgene, Mr. Occurrence of No Krajkov
0.4M sucrose/24h Frosty oxidative stress etal. (2011b)
5C, dark monitored
Biochemical
parameters used
A. nordmanniana
Five cell lines 0.2M sorbitol/24h 5% DMSO 2h Programmable freezer All cell lines No Nrgaard
0.4 sorbitol/24h recovered etal. (1993)
Samples placed on a
120rpm rotary shaker
24C, dark
J. Krajkov et al.
Table 14.2 (continued)
Species Preculture Cryoprotectant Time in Cryo-method used Recovery and Genetic fidelity References
cryostorage regeneration tested by genetic
markers
15 cell lines 0.52M sucrose 7.5% DMSO 1h Isopropanol container All cell lines No Misson etal.
Samples placed on a recovered; recov- (2006)
100rpm rotary shaker ery rate depended
22C for 24h/dark from treatment
Abies hybrids
A. alba A. 0.4M or 0.8M sorbitol 5% of DMSO 1h Nalgene Mr. Frosty Cell viability Yes Salaj etal.
cephalonica, three applied for 24, 48 or container Maturation (2010)
cell lines 72h experiment
A. alba A. 241C, dark performed
numidica
one cell line
14 Biotechnology Tools for Conservation of the Biodiversity
303
304 J. Krajkov et al.
was done on solid or liquid media with increased concentration of sucrose (0.2 and
0.4M) or sorbitol (0.2 and 0.4M) applied for subsequent 24h. The most common
cryoprotectants which were used are 5% PGD (polyethylene glycol 6000, glucose,
DMSO) and DMSO reaching the final concentration 7.5% and 5%, respectively.
The duration of storage in LN2 varied from 1h (Misson etal. 2006; Salaj etal. 2010)
till 6 years (Krajkov etal. 2011a, 2013).
The first reports have evaluated only the recovery after cryopreservation moni-
tored as increase in proliferation rate or as vital staining of embryogenic cell masses
(Nrgaard etal. 1993; Aronen etal. 1999). The most recent studies compared also
occurrence of oxidative stress (histological localization of H2O2) and the biochemi-
cal parameters (cellular levels of ATP and glucose-6-phosphate) during each step of
cryo-procedure and thawing (Krajkov etal. 2011b). The evaluation of matura-
tion abilities after cryopreservation was done by Salaj etal. (2010) for fir hybrids
and by Krajkov etal. (2011a, 2013) for A. cephalonica and A. alba.
However, despite more than 20 years of experience in conifer cryopreservation,
including Abies species, there are only a limited number of reports on long-term
storage. The present scenarios for global forest management and conservation, the
need to conserve breeding material during clonal field testing and the consequences
of climate change, not only underline the importance of cryopreservation as a safe
storage against external threats but also emphasize the significance of the genetic
fidelity of cryopreserved material. Long-term cryopreservation of an Abies species
has only been reported for A. cephalonica (Krajkov etal. 2011a) and A. alba
(Krajkov etal. 2013).
The experience and reports on the effects of prolonged storage in liquid nitrogen
are still limited, and the genetic fidelity at DNA level of the cryopreserved mate-
rial has rarely been considered (Aronen etal. 1999; Salaj etal. 2010; Krajkov
etal. 2011a). However, cryopreservation as a cost-effective, low labor- and space-
demanding alternative will have an important role for conservation of coniferous
tree species, including European and Mediterranean fir species in the near future.
14.4Concluding Remarks
Despite the fact that five European and Mediterranean fir species and some hybrids
were regenerated using somatic embryogenesis technique and the successful cryo-
preservation protocols were applied to three species, there is still need for further
studies. First, the critically endangered and endangered fir species were not subject-
ed to above mentioned studies. Second, the current protocols for regeneration have
some limitations and have been applied only to a few embryogenic cell lines. Due to
the fact, that in vitro cultures are clonally propagated lines, it is important to remem-
ber that multiple genotypes of these tissues need to be banked in order to achieve a
high level of genetic diversity in the collection. This can dramatically increase the
labour and resources needed initially to establish the lines and cryopreserve the tis-
sues, but once the lines are banked, maintenance costs are similar to those of other
14 Biotechnology Tools for Conservation of the Biodiversity 305
cryopreserved materials, such as seeds (Li and Pritchard 2009). Thus, biotechno-
logical approaches have their place in the toolbox of conservation methods of firs.
Biotechnology means for ex situ conservation are of specific value in the context
of rare endangered species with small local populations like A. nebrodensis or A.
numidica, where populations are small and virtually all trees are worth of being
conserved. They also can be useful in the case of small local populations, mainly
fragmentary demes on the edges of the distribution range, potentially containing
specific alleles. Transfer of the biotechnology experience gained in widespread spe-
cies and development of reliable procedures for somatic embryogenesis and cryo-
preservation for the endemics remain, however, the tasks for the future.
Acknowledgment Authors thank the EUFORGEN as the source of information for downloading
the distribution maps from http://www.euforgen.org/distribution_maps.html.
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IPGRI, Rome
Chapter 15
Conservation of Global Wheat Biodiversity:
Factors, Concerns and Approaches
Abstract Wheat is an important food crop in the world. It is also one of the top
three global food crops produced after rice and maize that constitutes an immensely
significant role with respect to global food security. Due to finite land resources
that can be dedicated to agriculture global wheat production has been consistently
dependent on genetic improvement of wheat germplasm across the world. Tradi-
tional plant breeding has been an important tool in increasing global food produc-
tion by producing disease and stress resistant, high yielding and early maturing
wheat varieties. However, it is necessary to have a stable and divergent pool of
wheat genotypes grown under different environmental conditions and different land
races of wheat as genetic feedstock for enhanced genetic improvements. Due to
increased global human population, extensive anthropogenic pollution and dam-
ages to the vulnerable local ecosystems, existing genotypes and land races of wheat
are under constant threat of becoming extinct. Hence it is absolutely necessary to
conserve the global wheat biodiversity for securing the future of our food security.
W.Cetzal-Ix()
Herbarium CICY, Centro de Investigacin Cientfica de Yucatn, A. C. (CICY),
Calle 43. No. 130. Col. Chuburn de Hidalgo, 97200 Mrida, YUC, Mxico
e-mail: rolito22@hotmail.com
M.Asif
Department of Agricultural, Food and Nutritional Science, University of Alberta,
Edmonton, AB T6G 2P5, Canada
A.H.Hirani
Department of Plant Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
S.K.Basu
Department of Biological Sciences, University of Lethbridge, Lethbridge, AB TIK 3M4, Canada
E.Noguera-Savelli
Francisco de Montejo, 97203 Mrida, YUC, Mxico
P.Zandi
Department of Agronomy, Takestan Branch, Islamic Azad University,
Takestan 34819-49479, Iran
R.Sengupta
Department of Zoology, WB State University, Barasat, West Bengal 700126, India
Abbreviations
AFLP Amplified fragment length polymorphism
CAAS Chinese Academy of Agricultural Sciences
CIMMYT International Maize and Wheat Improvement Center
DArT Diversity arrays technology
FAO Food and Agriculture Organization
ICARDA International Center for Agricultural Research in the Dry Areas
ICGR Institute of Crop Germplasm Resources
NBPGR National Bureau of Plant Genetic Resources
NIAS National Institute of Agrobiological Sciences
NSGC National Small Grains Germplasm Research Facility
RAPD Random amplified polymorphic DNA
RFLP Restriction fragment length polymorphism
SNP Single nucleotide polymorphism
SSR Simple sequence repeats
15.1Introduction
and enhancement of biodiversity especially for major crop species. Cardinale etal.
(2012) reviewed results of research experiments of last two decades and revealed
impact of loss of biodiversity on functional ecosystems and goods and services sup-
ply. Long term research on grassland revealed diverse plant communities tolerate
more and recover fully from major biotic and abiotic stresses (Naeem etal. 1994;
Tilman and Downing 1994; Zavaleta etal. 2010). Dismantling of eco-systems cause
loss of biodiversity and this is a primary concern around the globe. Loss of biodi-
versity is mainly due to habitat fragmentation and destruction, overexploitation,
climate change, deterioration and extinction cascades, invasion by alien species and
many other factors (Brook etal. 2008; Dunn etal. 2009; Thomas etal. 2004; Tilman
etal. 2001).
Wheat is one of most cereal produced in the world followed by rice and maize.
It is a primary source of calories for 1.2billion people around the globe and thus
constitutes a main platform for the global food security. Traditional plant breeding
has been an important tool in increasing global food production by producing high
yielding, disease resistant cultivars with better agronomic practices. However, ge-
notypic variation is one of prerequisites to improve any trait including grain yield.
Therefore, scientific community often rely on land races or wild progenitors when
the genetic diversity/variation is not present in the immediate gene pool. Unfortu-
nately many existing genotypes and land races are now being threatened with threat
of extinction due to several natural and anthropogenic factors. In this chapter, we
have tried to investigate different factors and concerns jeopardizing global wheat
biodiversity and pinpoints some potential approaches for its successful conserva-
tion.
The knowledge of the ancestral lineages of wheat and their distribution are the key
to the conservation of the current taxa. The wheat is widely distributed from the
Arctic Circle to the equator (Curtis 2002). Wheat is divided into two broad cat-
egories i.e., spring and winter based on its growth habit. Wheat is also divided into
various classes depending on the grain color and texture (Jing-Song etal. 2012). It
has been reported that several cultivars are more successful between the latitudes of
3060N and 2740S (Nuttonson 1955).
The human being in their quest to identify their natural environment has assigned
names to wheat plants in their distribution range. First classification of wheat was
proposed by Linnaeus in 1753 that was based on phenological characters: Triticum
aestivum L. for spring wheat and T. hybernum L. (nom. rejic.) for winter wheat; in
addition he described T. spelta and T. polonicum based on its morphology. Triticum
aestivum L., commonly known as wheat, is a species belonging to grass family Poa-
ceae, in the Pooideae subfamily and Triticeae tribe. Later on, different classifica-
tions based on morphological characters were proposed. Dumortier (1823) divided
the wheat based on the consistency of the spike (fragile vs. not fragile). Bowden
314 M. Asif et al.
(1959) integrated the two genera: Triticum L. and Aegilops L. into a single genus
(representing 40 Triticum species) which was later supported by Morris and Sears
(1967). During post second world war period, the older classifications have been
strongly opposed based on later studies focusing on cytogenetics (Bowden 1959;
Morris and Sears 1967), genetics (MacKey 1966, 2005) and molecular biological
evidences (Goncharov 2002; Goncharov etal. 2009). Other important proposals in-
clude MacKeys classification of the intrageneric taxonomy of Triticum followed by
Mansfelds Encyclopedia of Agricultural and Horticultural Crops based on smaller
number of genes regulating morphometric variations among different wheat species
As a result, this particular classification system is quite simple comprising of only
10 Triticum species and 20 infra-specific taxa (Goncharov 2011).
One of the most recent classifications are the Goncharov (2002) and Goncharov
etal. (2009) after the traditional Krnicke-Flaksberger-Dorofeev system compris-
ing of 29 different species represented by five separate sections. However, the taxo-
nomic treatments carried out in wheat usually lack robust criteria to distinguish nat-
ural hybrids from artificial hybrids and this has caused confusion in wheat research.
The criteria commonly used by plant taxonomists are morphological, anatomical,
ecological, geographical, karyological, biochemical, cytogenetic, and molecular
genetic methods have also been recently provided. There are many artificial hy-
brids without assigned names or invalid names which complicates the systematics
of Triticum. Based on the actual phylogenetic studies, there is evidence that the long
evolutionary process of wheat was parallel to that of humans. It is estimated that ap-
proximately 3million years ago, a common ancestor of wheat suffered a divergence
and raised ancestral diploid genomes, called A, B, and D (Gill etal. 2004).
According to Golovnina etal. (2007), inter-generic hybridization of genomes
(TriticumAegilops) has been fundamental to the process of speciation of wheat.
Furthermore, Feldman and Levy (2005) also suggested wheat genome evolution
through two major approaches: allopolyploidization with rapid and sporadic ge-
nomic modifications. The evolutionary process of wheat was also coupled with
domestication. According to Harlan (1992), 1500 years ago humans domesticated
wheat, cultivating it over large areas in present-day Iraq, parts of middle-east Asia
including Iran, Turkey and Syria.
All the modern polyploid wheat species have originated from the tetraploid wild
ancestor (T. dicoccoides (Krn. ex Aschers. & Graebn.) Schweinf.)). Nesbitt and
Samuel (1996) and Tanno and Willcox (2006) indicated that the wild emmer wheat
(T. dicoccoides) was possibly the first cereal species domesticated by our human
ancestors. However, the exact period of domestication is still highly debated by
different scholars. It was subsequently identified into four subspecies for T. dicoc-
con Schrank ex Schbl.: (1) ssp. maroccanum Flaksb., (2) ssp. abyssinicum Vav.,
(3) ssp. europaeum Vav.=ssp. dicoccon, and (4) ssp. asiaticum Vav. (Gkgl 1955;
Dorofeev etal. 1979; Szab and Hammer 1996; Teklu etal. 2007). The process of
domestication holds the link to the evolution of bread wheat (Ozkan etal. 2010).
According to phylogenetic studies of Golovnina etal. (2007), the genus Triticum
represents three groups, each including wild and cultivated species: (I) Diploids
15 Conservation of Global Wheat Biodiversity: Factors, Concerns and Approaches 315
Major grain crops such as wheat, rice, maize, soybean, canola etc. have been losing
diversity due to long term monoculture of a few high yielding cultivars. High level
of selection pressure for favourable traits is the primary reason for narrow genetic
bases for numerous other characters. Single assemblage limits the multi-functional-
ity in a crop eco-system. Uniformity in genetic make-up of varieties in same genetic
pool causes reduction of overall performance and stability, at the same time bring in
risk of vulnerability to biotic and abiotic stresses (Pecetti etal. 1992; Porceddu etal.
1988). For instant, stress tolerance and high yield can be negatively related and very
difficult to maximize simultaneously, either one can be penalized in join functions
(Diaz etal. 2004; Grime 1974).
a. Loss of Wheat Biodiversity Biodiversity of Triticum species for physiological
performance, quickly adapting to biotic and abiotic stresses such as evolutionary
adaptation is reduced in elite germplasm compared to landraces and wild relatives.
It could be seriously threatened in the crop improvement by future epidemic, global
warming and high level of regional droughts. Sustainable performance such as
durable diseases and pest resistance, drought and salinity tolerance is highly respon-
sible traits on wider biodiversity. Uniqueness of individuals within population for
physiological, biochemical, metabolomic processes govern high biodiversity within
species. Biodiversity of crop plants could be directly impacted by the existence of
316 M. Asif et al.
genetic diversity. It has been known that wheat was domesticated about 10,000year
ago, since then courteous selection and breeding efforts has been significantly
eroded genetic diversity (Chatzav etal. 2010; Tanksley and McCouch 1997).
b. Genetic Erosion/Gene Pollution Genetic erosion term referred as the loss of
variability of crop production in the areas of domestication and secondary diversi-
fication i.e. centre of origin (Tsegaye and Berg 2007). Genetic variability of a crop
population is altered in ways that make negative genetic gain over a period. Genetic
erosion is one of the most important factors contributing to global wheat biodiver-
sity. de Carvalho etal. (2013) defined genetic erosion as the Steady reduction of
combination of alleles over time in a defined areas or lasting reduction in richness
of common alleles. Variability is coined to heterogeneity of alleles and genotypes
that reflect morphotypes and phenotypes composition. The numbers of crops grown
are declining steadily and crop with commercial importance enhancing production
areas with highly similar genetic constitution. As a result wild and weedy relatives
lose their own genetic make-up over long period of time. The primary cause of
genetic erosion is wide distribution of modern cultivars from crop breeding pro-
grams (Brush 1995).
Initially domesticated landraces were replaced by the cultivars selected based on
conventional breeding programs and recently those cultivars were replaced by mod-
ern high-quality homogenous new varieties or hybrid selected by molecular marker
assisted selection. Concentrated focus on breeding for crop yield and related traits
is highly responsible factors for genetic erosion of major crop species. In genetic
diversity analysis, about 20% genetic erosion of local gene pool of Russian origin
ancestors observed in 78 spring durum wheat genotypes introduced in Russia be-
tween 19292004 (Martynov etal. 2005). Gradually reduction in genetic diversity
reported in spring bread wheat from early domestication to traditional landrace cul-
tivars to modern breeding varieties and collected germplasm for long term breeding
program through 90 SSR markers distributed across the wheat genome (Reif etal.
2005). Similarly, decay in genetic diversity reported in 242 accession of common
wheat released in China since 1940s. The study revealed lower genetic diversity
found in cultivars released in 1990s compared to 1940s (Tian etal. 2005). In addi-
tion to that other gene pools also found similar pattern of decayed in the genetic di-
versity of wheat cultivars over times (Huang etal. 2007; Smale etal. 2002; Tsegaye
and Berg 2007). A study reported a survey based on in situ conservation of local
varieties and landraces protected by farmers in Ethiopia, results suggested that lo-
calized landraces of species Triticum polonicum and T. turgidum have great genetic
erosion. The primary causes of the genetic erosion of landraces of several species of
genus Triticum in Ethiopia is displacement of landraces by other high yield modern
wheat cultivars (Teklu and Hammer 2006).
15 Conservation of Global Wheat Biodiversity: Factors, Concerns and Approaches 317
Plant breeding continuously selects the favourable allelic combinations in gene pool
to improve per se performance of elite lines that eventually to be used as parents of
new variety development.
a. Changing Genetic Architect of Wheat Populations in Genetic Pools Long term
breeding operations change original genetic architect of plants as a result genetic
shift observed in improved gene pools in wheat, barley and maize (Donini etal.
2000; Fu 2006; Koebner etal. 2003). Improved gene pools could have desirable
allelic composition for traits like yield, quality and agronomy but that may not have
durable resistance capacity to diseases and pest, long term sustainability to chang-
ing climate and high performance stability.
b. Narrowing Genetic Base for Biotic and Abiotic Stresses Robust and rapid maker
assisted selection system for selection of foreground and background genome of
elite lines causes elimination of large genomic variations resulting genetic bases
of elite germplasm would be narrow for quantitative control of biotic and abiotic
stresses in the newly developed cultivars. Most recent concern of this genetic bottle-
neck has been taken into consideration and breeder perform wide inter-specific or
inter-generic crosses to enhance genetic variation within gene pool for sustainable
durable resistance to biotic and abiotic stresses in wheat.
c. Vanishing Original Genetic Composition of Wild and Weedy Relatives In the
current agriculture system, farmers prefer to grow genetically uniform crop variet-
ies and majority farmers select high yield varieties of same crop because of high
revenue. Cultivation of large area with highly genetically uniform varieties creates
pressure on original genetic composition of wild and weedy relatives which exist
in the surrounding areas. Selection pressure over many generations in wild popula-
tions due to pollen drift from cultivated areas causes loss in the genetic diversity in
wild and weedy relatives.
d. Insufficient Resources for Germplasm Conservation and UtilizationIn addi-
tion to above mention factors, inadequate resources for germplasm conservation
and long term maintenance highly impact on the biodiversity of important crop
like wheat. Collection, conservation, research and utilization of wheat germplasm
resources play an important role in wheat breeding program to improve production
and productivity in the world. More resources need to be allocated for the preser-
vation of germplasm that can be used in current and future breeding program to
maintain biodiversity and overall performance of wheat crop. Current collections in
the gene banks have limited or no contribution to the modern cultivar development
programs for most essential agricultural traits. Crop improvement, is still focused
on poor genetic base for all the major crop species including wheat (Tanksley and
McCouch 1997).
318 M. Asif et al.
Wheat is one of the most important cereal crops in term of its production and con-
sumption in the world. Various sub species of genus Triticum are cultivated in dif-
ferent regions of the world; therefore genetic diversity is one of the most crucial
factors for wheat crop improvement. It has been widely debated that the genetic
diversity of major self-pollinating crops such as wheat has suffered with reduction
over time due to pure line breeding and selection (Donini etal. 2000; Hoisington
etal. 1999). Advance in molecular techniques have been used in molecular marker
development for marker assisted selection of foreground and background of ge-
nome in elite lines. Numerous types of molecular markers are currently being used
for the study of genetic diversity such as Simple Sequence Repeats (SSR), Diversity
Arrays Technology (DArT), Amplified Fragment Length Polymorphism (AFLP),
Restriction Fragment Length polymorphism (RFLP) and Random Amplified Poly-
morphic DNA (RAPD) in different crop species to know the status of diversity in
different breeding materials and gene pools. Huang etal. (2007) reported existence
of genetic diversity among the widely adopted 511 European winter wheat varieties
developed by modern plant breeding and suggested that there was no quantitative
loss of genetic diversity in different decadal groups of varieties. However within
decadal groups very small percentage of variation observed by 42 microsatellite
markers. Interestingly, the grain yield related traits such as grain size and shape has
progressively increased indicating that instrumental favourable genetic variation
in these traits observed however it reduced phenotypic variation over generations
(Gegas etal. 2010).
Currently, spring bread wheat area in the developing world has about 77% re-
lated to International Maize and Wheat Improvement Center (CIMMYT) wheat
(Smale etal. 2002). However, genetic uniformity or diversity does not imply with-
out evidences of molecular analysis. Soleimani etal. (2002) reported significant
amount of genetic variation between and within 13 registered modern Canadian du-
rum wheat cultivar by polymorphism of AFLP molecular markers. In modern culti-
vars, in spite of rigorous genetic selection for pure breeder seeds, genetic variation
observed between individuals of same cultivars (Soleimani etal. 2002). Similarly,
modern cultivars and landraces developed in different gene pools displayed genetic
variation that revealed by polymorphic detection of different molecular markers
(Autrique etal. 1996; Bebiakin etal. 1976; Carvalho etal. 2009a; Carvalho etal.
2009b; Carver etal. 1989; Shoaib and Arabi 2006). Genetic diversity evaluated for
the 150 accessions of durum wheat collected from worldwide using Single Nucleo-
tide polymorphism (SNP) molecular markers. The SNP diversity analysis study in-
dicated significant loss of gene diversity in terms of landraces as well as older and
later released cultivars during initial stages of green revolution, however genetic
diversity increased during post green revolution (Ren etal. 2013). In comparative
analysis of genetic diversity in geographical regions Middle East showed moderate
compared to the North and South Americas and the European regions (Ren etal.
2013).
15 Conservation of Global Wheat Biodiversity: Factors, Concerns and Approaches 319
Several studies reported about the current status of genetic diversity of wheat in
different geographical regions of the world based on the molecular marker analysis
on old landraces and modern cultivars. Studies based on various molecular mark-
er systems suggested existence of genetic diversity at certain degree. There is no
evidence however reported for the existence or loss of physiological, agronomical
and overall fitness diversity status since plants undergo numerous biochemical and
physiological and metabolomic mechanisms during growth and development. Mo-
lecular markers revealed genetic however modification after transcriptional level,
protein level and metabolomic level are poorly understood and so their impact on
biodiversity and fitness performance of plants.
Plant germplasms including modern cultivated varieties, landraces, closely and dis-
tance wild and weedy relative can be conserved by ex situ and in situ methods.
These methods facilitate management, maintenance and databank that can be avail-
able for future use in the breeding programs for crop improvement (Benz 2012).
The ex situ germplasm conservation method is most traditional approach that has
been utilized for many crop species especially for the maintenance of landraces
and wild relatives. Earlier ex situ conservation was assumed to have limited usage,
however, germplasm collected in ex situ serve primary source for trait improvement
in most of the elite breeding materials in breeding programs. Ex situ conservation
is continuous requirement to preserve neglected landraces, disappearing wild and
weedy relative or distinct relative and cultivated wheat species. Such germplasm
collected in ex situ can be indispensable resources to restore cropping system after
major disease epidemic or other disasters. Ex situ conservation has played important
role in distribution of productive crop varieties and breeding lines to those countries
or regions where crop has challenges for producing enough to sustain agriculture
production (Benz 2012).
Bettencourt and Konopka (1990) reported that 83,377 out of 529,577 wheat
accessions were found to be landraces representing ~15.7% of total germplasm
collections maintained in 102 collection centres in 47 countries. Later, Knpffer
(2009) reported 732,262 wheat germplasm accessions stored in 223 germplasm col-
lection centers across the globe. Most recently, Food and Agriculture Organiza-
tion (FAO) reported a total 856,168 wheat accessions in 229 institutes representing
24% landraces (de Carvalho etal. 2013). Together with conservation of cultivated
320 M. Asif et al.
Tissue culture based techniques for plant regeneration via organogenesis or em-
bryogenesis is most common in almost all the crop species. However, it is important
to identify suitable culture systems to minimize unwanted genetic variations during
preservation and subsequent regeneration. Most likely, regeneration of plantlet from
callus culture is associated with somaclonal variation or chromosomal rearrange-
ments that restrict the tissue culture techniques to be used for germplasm conserva-
tion. Using explants such as apical meristems have been suggested as one of the
most successful approach for avoiding wide genetic variability during conservation
process. In addition, preserving both somatic as well as zygotic embryos are also
regarded as viable alternatives for successful conservation (Villalobos etal. 1991).
Collection and storage of viable seeds is most common and feasible approach for
germplasm conservation. Germplasm of cereal crops including wheat can be stored
in two types of collections (i) working collections, and (ii) preservation. Seeds can
be storage at near freezing and low humidity through this way viability can be
maintained for 10 years or longer (Sachs 2009). Long term storage of wheat seed
needs ambient temperature gradient between 10 to 20C. Seeds can be efficient-
ly stored for long term purposes after carefully drying the seed and then storing in
specialized sealed vessels. Another approach suggested is to vacuum pack the seeds
in specially designed aluminum foil envelopes or in storage cans. Seeds can also
be stored by using cryogenic methods like application of liquid nitrogen (Walters
etal. 2005).
322 M. Asif et al.
15.7Conclusion
Wheat represents an important food crop species that is closely related to global food
security. It is therefore important to conserve all existing landraces, genotypes and
germplasm of different types of wheat (including both wild and cultivated species)
to protect the wide global genotypic diversity of this crop. Such genetic variability
will be useful for future breeding programs and biotechnological improvements for
developing high yielding, disease and stress resistant varieties locally suitable for
different agro-climatic regions of the world. Rapid loss of genetic diversity of wheat
has been reported from different corners of the world and hence it is important to
initiate an efficient and effective integrated global wheat conservation program to
conserve diverse species including landraces and germplasm of this valuable food
crop. We have explored the current status of wheat biodiversity and identified sev-
eral factors hampering such diversity, globally. We have also suggested potential
measures for wheat conservation from a multi-disciplinary perspective with special
emphasis on modern biotechnological approaches.
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Index
Forest genetic resources, 105, 106, 108, 110, Gerding, R.K., 259
111, 113, 119, 121, 122, 124 Germplasm, 4, 117, 174, 193, 195, 211, 248,
conservation methods, 111 280, 281, 296, 316, 317
conservation of, 105 Germplasm accessions, 174, 319
in international initiatives, 106 Gheysen, G., 80
Fornale, S., 86 Ghormade, V., 82
Fosberg, F.R., 266 Gianessi, L., 5
Foster, S., 257 Gibson, L.G., 86
Fourre, J.L., 117 Gidoni, D., 81
Fowke, L.C., 265 Gilbert, S., 248
Foyer, C.H., 72 Gill, B.S., 314
Fraleigh, B., 272 Giorgi, C., 85
Fraley, R.T., 78 Giridhar, P., 188, 189
Francis, S.V., 189 Giuliano, G., 83, 84
Franconi, R., 85 Gleba, Y., 85
Franke, A.C., 51, 52 GllKaka, R., 173
Franklin, J., 121 Glyphosate, 7, 47, 55
Fu, G., 86 Glyphosate-resistant weeds, 47, 55
Fukai, S., 196 GM Compass, 5
Fu, Y.B., 317 Godfray, H.C., 78
Fu, Y.-Q., 83 Goff, S.A., 10
Gkgl, M., 314
G Goldewijk, K.K., 71
Gaddaguti, V., 207 Golic, K.G., 81
Gajdoov, A., 298 Golovnina, K.A., 314
Gaj, T., 82 Gmry, D., 294
Gale, S., 301 Goncharov, N.P., 314, 315
Galili, G., 84 Gonzales-Benito, E., 183, 186, 192, 194
Gao, Y., 85 Goyal, S., 204, 209, 210
Garcia, M.A., 6, 7 Greek fir, 289
Garcia-Robles, I., 61 Green, J.M., 60
Gardner, S.N., 9 Gregorius, H.R., 295
Garla, M., 261 Gressel, J., 7, 9, 86
Geburek, T., 109, 295, 296 Griffiths, B.S., 62
Gegas, V.C., 318 Grime, J.P., 315
Gehring, C., 10 Groombridge, B., 3, 4
Gelvin, S.B., 80 Grossnickle, S.C., 298
Gene banks, 114, 116, 177, 184, 317 Grubben, G.J.H., 273
Gene pool conservation, 116 Gruissem, W., 70
Gene targeting systems, 80 Guggulsterone production, 209
Genetically modified crops, 5, 79, 83, 320 Guimaraes, E.P., 281, 282
Genetically-modified organisms (GMOs), 39 Guldager, P., 111
Genetic diversity, 3, 7, 104, 105, 108, 110, Guo, B., 189
111, 114, 122, 124, 175, 177, 178, Guo, F., 83
184, 271, 276, 288, 289, 296 Gupta, A., 223
neutral, 121 Gupta, J., 216
Genetic engineering, 4, 68, 83, 88, 205 Gupta, P.K., 265, 300
Genetic resources, 105, 109, 110, 113, 273, Gupta, V.V.S.R., 62
276, 279, 297 Gurib-Fakim, A., 204
Genetic variability, 105, 111, 115, 116, 124, Gurr, G.M., 6
209, 316, 322 Guru Kumar, D., 216
George, S.K., 215 Guys, K.J., 88
Gepts, P., 9 Gymnosperms, 248, 249, 265
332 Index
H I
Hadar, Y., 86 Icoz, I., 60, 63
Hafez, H.F., 223 Ihemere, U., 84
Hggman, H., 296, 301 Immature embryos, 185, 252
Hamilton, J.P., 89 In situ conservation, 109, 176, 177, 179, 182,
Hammer, K., 314, 316 205, 296, 316, 320, 321
Hamrick, J.L., 122 methods, 113
Hancock, J.F., 8 strategy, 196
Hansen, J.K., 292 In vitro conservation, 117, 186, 195, 223, 280,
Hansen, O.K., 294 297
Hansson, S.O., 72 of plant germplasm, 184, 279
Harding, K., 195 In vitro techniques for plant conservation, 181
Harlan, J.R., 314 Iriondo, J.M., 196
Harris, P.J., 86 Isajev, V., 105
Hasna, A.S., 82 Itoh, K., 9
Haughton, A.J., 61, 62 Izge, A.U., 72
Hayashi, Y., 82
Hazra, S., 216 J
Hazubska-Przybyl, T., 301 Jablonka, E., 122
Heathcote, A.J., 73 Jadiya, P., 216
Hefferon, K.L., 84 Jaenicke, H., 172174, 178
Hegazi, G.A., 261 Jager, A.K., 252
Hempel, F., 88 Jagetia, G.C., 215
Henderson, N., 81 Jain, A., 204
Herbicide, 5, 6, 8, 9, 46, 51 Jain, A.K., 222
Herdt, R.W., 78 Jain, H.K., 4
Herren, H.R., 6 Jain, N., 206, 208, 219
Herrera-Estrella, L., 78 Jain, S.K., 219
He, W.T., 189 Jain, S.M., 219, 265
Hilbeck, A., 5 James, C., 5, 41, 44, 63, 68, 79
Hinesley, L.E., 298 Jang, G., 188
Hirosawa, T., 194 Jaramillo, E.H., 275
Hirschi, K.D., 70, 83, 84 Jauhar, P.P., 78
Hirschman, C., 172 Jeandent, P., 216
Hisano, H., 86 Jenkins, M.D., 3, 4
Hodgson, J., 60 Jennings, R., 120
Hoisington, D., 318 Jesse, L.C.H., 63
Hokanson, K., 8 Jha, S., 223
Holeksa, J., 292 Jiao, L., 222
Holme, I.B., 88 Jing-Song, S., 313
Hood, E.E., 85 Jin, S., 190
Hooper, L., 70 Joelsson, K., 72
Hosseinzadeh, H., 216 Johnson, T., 190
Hss, S., 62 Johnson, T.S., 187
Houghton, P.J., 215217, 219 Jones, C.S., 85
Howe, G.T., 121, 122 Jorgensen, U., 86
Howes, M.J.R., 215217, 219 Josefson, D., 257
Hristoforoglu, K., 298 Joshi, N., 208
Huang, G.H., 87 Joshi, P., 189
Huang, J., 9 Joshi, S.G., 84
Huang, X.-Q., 316, 318 Joshi, U.H., 222
Hydrogel, 192 Jouzani, G.S., 89
Index 333
T U
Tabassum, R., 217 Uddin, A., 259
Tacket, C.O., 85 Uniyal, P.L., 256
Tadera, K., 252, 265, 266 Urizar, N.L., 221
Takagi, H., 281
Index 339
Z Zhao, J-Z., 10
Zamia, 249, 250, 255 Zhao, Y., 215
Zanwar, A.A., 223 Zhu, C., 83, 84
Zapartan, M., 187 Zhu, G., 222
Zavaleta, E.S., 313 Zilberman, D., 73
Zhang, F.L., 217 Zinc deficiency, 70
Zhang, L., 11 Zinc finger nucleases (ZFNs), 81
Zhang, Y., 222 Zoghlami, N., 173
Zhang, Y.-H.P., 86 Zwalhen, C., 62