Opportunities for Biofortification of
Cassava for Sub-Saharan Africa:
The BioCassava Plus Program
M. fregene, r. Sayre, C. fauquet, P. anderson and n. taylor
Donald Danforth Plant Science Center
St. Louis, Missouri
e. Cahoon
D. Siritunga
M. Manary
University of Nebraska
Lincoln, Nebraska
University of Puerto Rico
Mayaguez, Puerto Rico
Washington University
Children’s Hospital
St. Louis, Missouri
mfregene@danforthcenter.org
C
assava is an important staple crop in sub-Saharan africa. Between 1970
and 2007, its production and acreage tripled across africa and quadrupled in
nigeria, the continent’s and the world’s largest producer (fao, 00). SubSaharan africa produced over million tons of fresh roots of cassava in 00, of which
no less than 95% was consumed as food; the starch provides >5% of dietary energy for
an estimated 00 million africans (Dorosh, 00). frequent consumers of cassava are at
greater risk for malnutrition—especially deficiencies in vitamin-a, iron, and zinc—than
consumers of other diets, particularly those that are cereal-based (Gegios et al., 00). a
nutrition survey in cassava-consuming areas of nigeria and Kenya revealed inadequate
intake of vitamin a in 3% and % and inadequate iron intake in 3% and % of
pre-school-aged children, respectively (Gegios et al., 00). vitamin-a deficiency causes
a loss of 9,000 disability-adjusted life years (DaLYs1) in nigeria and ,000 DaLYs
in Kenya annually; iron deficiency causes loss of 59,000 DaLYs in nigeria and 03,000
DaLYs in Kenya (fielder, 009).
1a measure of overall disease burden, expressed as the number of years lost due to ill-health, disability or early
death.
Current efforts to combat micronutrient deficiencies in africa include supplementation,
through distribution of micronutrients to high-risk populations, addition to processed
food and biofortification, i.e. the genetic improvement of nutrient content of crops via
field-based breeding or genetic engineering. Supplementation requires tremendous effort
to exceed 90% coverage and must be sustained for many years (Berti and rowley 00);
few countries in africa are able to run effective supplementation programs. Biofortification,
on the other hand, can achieve 00% penetration and, although it requires a substantial
initial investment in research and dissemination, it is self-sustaining. Cost per DaLY saved
for biofortification is 0% less compared to supplementation (nestel et al., 00).
BioCassava Plus (BC+) is a cassava-biofortification project at the Donald Danforth
Center in St. Louis, Mo, funded by the Bill and Melinda Gates foundation. BC+
scientists are engineering cassava for increased accumulation of β-carotene, iron, and
protein to provide minimum daily allowances of these essential nutrients as a means of
ameliorating the burden of malnutrition that accompanies consumption of cassava as a
staple food. Proof of concept for the enrichment of these nutrients has been demonstrated
in the model cassava cultivar 0, which, in greenhouse and confined field trials in
Puerto rico, contains up to 0 µg/g dry weight (Dw) of β-carotene (provitamin a), 0
µg/g dry weight of iron, 0% protein storage roots, and reduced levels of anti-nutritional
cyanogens.
Biofortification of Cassava
β-carotene
β-carotene enrichment of storage roots in cassava is conferred by two transgenes: the
Erwinia crtB phytoene-synthase gene, and the Arabidopsis -deoxyxylulose-5-phosphate
synthase (DXS) gene. The crtB transgene includes a 0.-kb sequence for the plastid
transit peptide for the Δ-palmitoyl-acyl carrier protein desaturase from coriander. The
phytoene synthase encoded by the crtB gene catalyzes the committed step in β-carotene
synthesis using geranylgeranyl-diphosphate (GGDP) from the plastid isoprenoid pathway
as its substrate. DXS catalyzes the first step in the plastid isoprenoid pathway. Increased
expression of this enzyme is intended to enhance concentrations of GGDP for β-carotene
synthesis and also to ensure sufficient amounts of GGDP to maintain vitamin-e production at or above wild-type levels. The crtB and DXS genes are each under the control of
the Solanum tuberosum (potato) patatin promoter and flanked on their 3’ ends by the
3’ untranslated region (Utr) of the nopaline synthase (nos) gene from Agrobacterium
tumefaciens. The selectable marker is the neomycin phosphotransferase II (nptII) from E.
coli under control of the cauliflower mosaic virus 35S (CaMv 35S) promoter (with x
enhancer). The nptII gene is flanked on its 3’ end by the CaMv 35S 3’ Utr.
Plants expressing the crtB and DXS genes were initially evaluated in four-inch pots
under greenhouse conditions. amounts of total carotenoids ranged from 30 to 0 µg/g
Dw in storage roots of the top lines. By comparison, amounts of total carotenoids in
storage roots of control 0 plants in these and subsequent greenhouse and field studies
ranged from .5 to .5 µg/g Dw. Subsequent evaluations were conducted of storage roots
of plants growing in soil beds in the greenhouse and in confined field trials in Puerto rico.
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Concentrations of total carotenoids in roots from greenhouse beds typically ranged from
30 to 5 µg/g Dw. Concentrations of carotenoids from the confined field studies ranged
from 30 to 0 µg/g Dw. Concentrations of vitamin e were not significantly different
between storage roots from the top β-carotene lines and non-transformed controls. In
the transformed lines, the relative amounts of all-trans-β-carotene, the most nutritionally efficacious form of carotenoid provitamin a, were 5 to 90% of the total carotenoid
content. relative amounts of trans-β-carotene in non-transformed controls were only 50
to 0% of the total carotenoid content. In collaboration with partners in africa, BC+ has
confined field trials of the β-carotene-enriched GM events (i.e. transformed plant lines)
ongoing in nigeria and another trial is planned for fall 00 in Kenya.
In addition to the consumer benefit of improved nutrient levels, BC+ β-carotenerich GM events also have producer benefits in terms of extended shelf-life. five of the
events with the highest amounts of total carotenoids could be stored for up to days
after harvest, whereas the wild-type recorded up to 0% spoilage after days. reduced
shelf-life of cassava roots, a result of post-harvest physiological deterioration (PPD), is a
major limitation to marketing of fresh roots. PPD begins hours after harvest and can
render the roots unpalatable and unmarketable within hours. Short shelf-life affects
cassava value-added chains because it increases losses during processing and limits access
to markets distant from production sites. Longer shelf-life was correlated with total carotenoid content (r = 0.0) in the GM events, which is consistent with previous studies
indicating that high levels of carotenoids in the roots (> ppm fresh weight basis) delay
PPD (Sanchez et al., 005).
nutrient retention during food preparation was also evaluated in three transgenic lines
expressing higher levels of β-carotene. In three common nigerian food preparations—gari,
fufu and boiled—retention of provitamin a ranged from % to 3%, equal to, or better than, that seen in the wild-type 0 variety. values for in vitro bioavailability of
provitamin a, as measured by uptake into micelles of Caco human intestinal cells, were
similar for the transgenic and wild-type sources, i.e. % and 3%, respectively.
Iron
Increased iron content was achieved by the expression of the FEA1 gene, from Chlamydomonas reinhardtii, in cassava storage roots. The fea protein is an iron-specific metal
transporter. It transfers iron efficiently at very low concentrations or at high pHs at which
iron is largely insoluble. It does not transport toxic heavy metals unlike all other known
metal transporters in plants. as with the β-carotene trait, FEA1 expression was driven by
the patatin promoter and nos terminator. Seven GM events were tested in the greenhouse
and in confined field trials in Puerto rico; iron content in the GM events ranged from
30 to 0 µg/g Dw in storage roots compared to 0 µg/g dry weight in the wild type.
real-time PCr analyses strongly suggest that the additional iron is stored as a ferritin
complex in the transgenic plants.
The morphology, including branching and flowering, of transgenic plants was identical
to that of wild-type plants in Puerto rico.
fregene, Sayre, fauquet, anderson, taylor, Cahoon, Siritunga, and Manary 3
Protein
Increased protein content in cassava was achieved by the expression of sporazein, a storage fusion protein consisting of a 0-bp fragment of β-zein from maize, the sporamin
gene from sweet potato and a 50-bp fragment of β-zein. Sporazein is a nutritionally
balanced protein of 9. kD in which the zein components drive accumulation of the
product to form protein bodies within the endoplasmic reticulum. as with the other two
traits, the patatin promoter and nos terminator drive sporazein accumulation in cassava
storage roots.
Seven GM events expressing sporazein were tested in the greenhouse, in soil beds, and
in confined field trials in Puerto rico. total protein content of storage roots harvested
ranged from 9 to % Dw across all seven transgenic lines studied. The morphology
and growth habit of these transgenic plants have shown no significant differences to
non-transgenic controls in all three locations tested to date. no changes in expression
of trait accumulation have been observed over the months during which these plants
have been tested in the greenhouse and field. as protein bodies are water-insoluble, water
soaking overnight at room temperature resulted in 9% retention of the total protein
content of the cassava roots. Boiling for 30 min resulted in 95% retention of the total
protein content.
It is well known from other plant systems that the direction of reduced nitrogen to
the synthesis of new proteins may come at the expense of nitrogen allocation to essential
proteins required for metabolism. Thus, elevating expression of storage proteins in cassava
may impair the growth or biochemical properties of roots. to address this concern, we have
over-expressed a vacuolar targeted linamarase, an enzyme that breaks down linamarine,
a cyanogenic glucoside found in cassava leaves, stems, and roots. This has been shown to
increase the pool sizes of free amino acids in cassava.
Product Development of nutrient-enriched Cassava for africa
BC+ has achieved nutrient enrichment of cassava such that if a 5-year-old child consumes 00 g/day of roots from the β-carotene, iron, or protein-rich GM events, (s)he
will obtain 00% of the minimum daily allowance (MDa) of these nutrients. BC+ has,
therefore, embarked on the expression of genes for the aforementioned nutritional traits
in farmer-preferred cassava varieties from nigeria and Kenya, its two target countries.
Genetic transformations at the Donald Danforth Plant Science Center (DDPSC) have
successfully generated transgenic lines of oko-Iyawo, the most popular nigerian variety
of cassava currently grown on to % of total acreage (about million hectares) in
that country, and the Kenyan cultivar Serere.
Based on ex-ante impact studies for nutrient deficiency in Kenya and nigeria, a
β-carotene- and iron-enriched oko-Iyawo for nigeria, and a β-carotene-, iron- and
protein-enriched, and virus-resistant, Serere for Kenya were selected as first products.
oko-Iyawo is resistant to cassava mosaic disease (CMD), which is of viral origin and is
the principal production constraint of cassava in africa, whereas Serere is susceptible to
CMD and needs to be engineered for resistance. Product development is divided into
several stages, namely:
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• generation of transgenic events,
• greenhouse characterization and testing,
• field testing,
• selection of a lead event,
• food and environmental safety assessments in a regulatory field trial,
• biosafety approval for commercial release,
• on-farm trials, and finally
• variety release and dissemination.
Lead Event Selection
a commercial quality construct that is codon-optimized without extraneous genetic
elements, will be created for the crtB, DXS, FEAI genes (β-carotene and iron traits)
and transformed into friable embryogenic callus derived from oko-Iyawo. Six hundred
transgenic plants will be regenerated and screened for events that possess a single copy
of the construct, no vector backbone sequences and good rna expression. events, an
estimated 5, that meet these criteria will be planted in the greenhouse and roots evaluated for β-carotene and iron contents. events that meet the set targets of 0 µg/g Dw
for the two traits will be transferred to confined trials in Puerto rico and nigeria for
trait assessment under field conditions. This will be followed by two cycles of replicated
confined field trials at three locations to select a lead event and a back-up for regulatory
trials. a similar procedure will be followed for the Kenyan product, with the exception
that the gene construct will contain sporazein and rnai for CMD and cassava brown
streak disease (CBSD), a viral disease of cassava that is specific to east africa.
Biosafety Regulatory Activities
Cultivation and consumption of cassava expressing these genes will require the granting
of approval from regulatory bodies in nigeria, based upon food and environmental safety
assessments of each novel gene/protein that confers the targets traits. a safety assessment
is required for each event intended for commercial release. DXS, crtB, FEA1, sporazein,
and npt II proteins will be purified, characterized, and assessed for potential toxicity in
acute (single dose) oral gavage studies with mice. allergenicity of the DXS, crtB, and FEA1
proteins will be assessed in accordance with international guidelines. This is a ‘weight of
evidence’ assessment that includes the source of the gene, documented dietary exposure to
the protein, any amino-acid sequence homology to known allergens, and protein stability
upon incubation in simulated gastric fluid.
The purified proteins will also be used to develop western blot and enzyme-linked immunosorbent assays for detection of the proteins in plant material. as the DXS and crtB
proteins are enzymes and will be present at higher levels than what are typical in cassava,
regulatory authorities will likely require information on substrate specificity. evidence
from the peer-reviewed literature on known substrate specificity for these enzymes will
be sought.
fregene, Sayre, fauquet, anderson, taylor, Cahoon, Siritunga, and Manary 5
a key consideration of regulators is assessment of any unintended effects that result
from elevated biosynthesis of metabolites in the carotenoid biosynthetic pathway. Unintended effects are traditionally addressed in the safety assessment by performing extensive
nutrient-composition and agronomic performance analyses. Compositional analyses of
starch quantity and quality, fatty acids, total and free amino acids, minerals, vitamins,
cyanogenic glucosides and phytates will be conducted. Protocols for the compositional
and agronomic regulatory field trials—a final field trial conducted on a lead event to
generate agronomic performance, food and environmental safety information—will be
in accordance with international guidelines.
Reaching End-Users
agricultural development in africa is replete with examples of well intended scientific
advances that have had limited impact because they were not sustainably adopted by
producers and consumers. fortunately, there are also excellent examples of widespread
successful adoption of new varieties. The strategy to reach end-users should avoid the
mistakes of the former and build on the successes of the latter, while taking into account
the special issues associated with transgenic varieties. Many failures have resulted from
a narrow focus on preferred production characteristics. a critical aspect important for
reaching end-users is seed production and distribution. In africa, the vast majority of
cassava stakes for planting is generated from current plants by the farmer or obtained from
neighbors; the private sector has little interest in this crop. However, non-government
organizations (nGos) involved in development activities and some national agricultural
systems now have good experience with dissemination of new varieties. BC+ will partner
with organizations that have the best linkages to large numbers of farmers. tissue culture
and an inexpensive two-node multiplication system for rapid and massive propagation
will be used to bulk up foundation seed for distribution to large nGos and government
agencies.
Cost-effective strategies for farmer adoption and consumer acceptance of β-carotene
and iron-rich cassava entail a marketing and promotion plan. for example, gari is the
most important food staple of the rural and urban poor and the gari market chains have
extensive coverage; over 0% of cassava grown in nigeria is used for gari production. a
great advantage is that the deep yellow color of high-β-carotene and -iron cassava is similar
to a yellow gari product, made by addition of palm oil, already accepted by consumers.
although gari processing tends to lead to a depletion of β-carotene and iron, levels of
nutrient enrichment achieved in BC+ ensure that sufficient amounts of these nutrients
remain to meet minimum daily allowances based on the average quantity consumed and
bioavailability.
anti-GMo nGos may be expected to mount campaigns in opposition to dissemination and adoption of β-carotene- and iron-enriched cassava. opposition can be overcome
through demonstration of the benefits to consumers (improved nutritional quality) and
to producers/processors (extended shelf life of storage roots). other elements for countering such opposition include: adhering strictly to bio-safety protocols and regulatory
requirements; being transparent by engaging key stakeholders in constant dialogue to
communicate progress and building confidence that the process is being properly and
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