ORGAN CULTURE,

ANTHER AND POLLEN CULTURE

UNIT 5: Plant tissue culture

Presented by
Santhiya.K
II M.Sc biotechnology
18PBT014
• Plant tissue culture (PTC)- aseptic culture of plant parts
in vitro in a suitable nutrient medium at optimized
environmental conditions like pH, temperature and
appropriate photoperiod.
• Why PTC?
• Production of secondary metabolites from cultured organs.
• Agricultural land insufficiency.
• Transgenic plants with desirable traits.
• Production of haploids
• Many more…
 Types of culture
(Explant base)
Embryo culture Seed culture Meristem culture

Cell culture Plant tissue culture Protoplast culture

(suspension culture)

Organ culture
Bud culture
Callus culture
 Organ culture

• In vitro culture and maintenance of an isolated organ

primordia or whole/part of an organ in a way that allows
differentiation and preservation of the architecture and
function.
• Types of organ culture: vegetative organs and reproductive
organs.
• Ex: Meristem or shoot tip culture, root culture, nucellus
culture, endosperm culture, ovary and ovule culture, anther
and pollen culture.
• Anther and pollen culture- production of androgenic haploids.
• Ovary and ovule culture- gynogenic haploids.
• Culture of plant organs occur in three modes
• Organized: whole plant (embryo, seeds) or organ is cultured
and the organized structure is maintained identical to that of
the original explant
• Non organized: cells or tissue isolated- dedifferentiated-
cultured as non organized callus tissue- “suspension cultures”-
low genetic stability.
• Non organized/organized: intermediate of above two; cells
isolated- dedifferentiate to form callus - redifferentiate to
form organs or embryos; progeny not completely identical to
original plant material.
 Importance of organ culture

• Excellent experimental system to study nutrients and growth

factors promoting plant growth.
• Study of morphogenesis.
• Study of secondary metabolites and its biosynthesis.
• Can lead to new developments in agriculture and horticulture
 Root culture
• The aseptic culture of excised radical tips obtained from
aseptically germinated seeds in the liquid or solid medium
where they are induced to grow independently under
controlled condition.
• Subculture possible.
• Growth of excised roots in culture- fresh and dry weight,
increase in the length of main axis, number of emergent
laterals, total length of laterals per culture
• Mostly carried out in an agitated liquid medium with
appropriate auxin concentration.
• Kotte and Robbins (1922) first successfully cultured wheat root tips.
• In 1934 White first successfully cultured tomatoroots.
Root and important parts
 Media composition

• Iron source: Fe(SO4)3 canbereplacedwithchelated iron like

NaFeEDTA(sodium-ferric-ethyl-diaminetetracetate).
• Sugar concentration of 1.5—2% is sufficient.
• Vitamin requirement varies according to theplant.
• Thiamine is needed by all species.
• Cereal roots require more auxin than dicotroots.
• A medium supplemented with sugar, IAA,
sometimes cytokinin and meso- inositol helps
cambial development in excisedroots.
 protocol

• Initiation of Isolated RootCulture:

• Seeds-surfaced sterilized – germinated in White’s basal medium at
25°Cin the dark .
• seedling roots of 20 to 40 mm length- excised 10 mm radical tipwith a
scalpel-transferred to 40 ml of liquid medium contained in
100 ml wide- neckedErlenmeyer flask
• Initiation of Clones: genetically identical
• Roots maintained for 10 days- main axis bearing 4 or 5 laterals
isolated and subcultured in fresh media- after some days –
growing sector of 4 to 5 laterals subcultured again.
Root organ culture
APPLICATIONS
Study of Nodulation of Leguminous Root inCulture
• Atechnique has been developed for culturing legume roots with
Rhizobium.
• Apical parts of the roots are dipped in a petridish
containing an inorganic medium inoculated with Rhizobium.
• In vitro nodulation helps to understand the relationship between
symbiotic NIFbacteria and higher plants.
• Roots of Glycine maxand Phaseolusvulgaris
Regeneration of Shoots onRoots

• Culture of isolated roots can be maintained continuously for manyyears

• Shoots can be induced to regenerate from culturedroots

• The shoot primordia can be derived from callus at the cut ends of the
roots
E.g. Atropa, Convolvulus arvensis
 Study of Synthesis of SecondaryMetabolites

• Used to increase the synthesis of such compounds in cultured root by some

nutritional manipulations.

• Hairy root culture, also called transformed root culture, is a type of plant
tissue culture that is used to study plant metabolic processes or to produce
valuable secondary metabolites or recombinantproteins.
 secondary metabolites produced in root culture
 L-DOPA: a precursor of catecholamines, an important neurotransmitter used in the treatment
of Parkinson’s disease

 Shikonin: used as an anti-bacterial and anti-ulcer agent

 Anthraquinone: used for dyes and medicinal purpose

 Opiate alkaloids: particularly codeine and morphine for medical purposes

 Berberine: an alkaloid with medicinal uses for cholera and bacterial dysentery

 Valepotriates: used as a sedative

 Ginsenosides: for medicinal purposes

 Rosmarinic acid: for antiviral, suppression of endotoxin shock and other medicinal purposes

 Quinine: for malaria

 Cardenolides or Cardioactive glycosides: for treatment of heart disease

10 3/30/2011
 NUCELLUS CULTURE

• Formation of embryo from cells of the nucellus or integument.

• Observed in citrus and mango.
• Nucellus proliferated into callus mass- differentiated into embryo
like structures called pseudobulbils- gave rise to plants
• medium: sucrose concentration of 5%, casein hydrolysate, malt or
yeast extract.
• Immature citrus fruits of 2.5 to 3 cm dia harvested after 100 days of
pollination- ovules removed- viewed in microscope- nucellus
removed from micropylar end- placed in media and incubated for
50 days in dark- embryo develop- placed in fresh media- secondary
embryo structures from cotyledons are formed.
 Applications

• Embryos- genetically uniform; inherit maternal

characters only; no variations due to gametic fusion.
• Disease free clones obtained from polyembryonic
citrus species
• Desirable plant vigor, fruiting characteristics
associated with juvenility seen in trees developed
from nucellar seedlings.
• Seedless citrus varieties without risk of viral diseases
• in vitro development of the immature or isolated mature
endosperm from seed at proper stage on a suitable culture
medium to obtain triploid plantlet.
• formed by Double fertilization
• triploid endosperm (fusion product of 2 polarnuclei and one
male gamete)
• function as reserve food for developing embryo.
• Explant: Endosperm- excisedat proper period of development
• cereals -12 days after pollination
• maize :8-11 days after pollination
• 4-7 days for rice
• 8 days after pollination for wheat.
• In some families, entire seed are used asexplant.
Inoculation of explant: in vitro culture of mature endosperm-seeds with
massive endosperm-decoated , surface sterilized -2 to 3 washings with sterile
distilled water - planted on the nutrient medium .
• in vitro culture of an immature endosperm -the entire seed or kernel is
surface sterilized -endosperm tissue excised under aseptic conditions.
• White’s or MS medium- induce callus from immature endosperm.
• Medium is also supplemented with tomato juice ,grapejuice.
• Sucrose is carbon source .
3. Incubation of culture: initiation of callus culture occurs at darkness.
Differentiation of callus at bright light (2000 lux- 4000lux) and 25°C

4. Embryogenesis:
Embryo differentiation occurs when proliferated tissue is transferred
to basal medium with or withoutgibberellins – leads to development
of plantlet.

5. Plantlets are transferred to soil:

Plantlets are hardened in green house by transferring to vermiculture media
and maintaining proper humidity.
APPLICATIONS:

• Production of triploid plant

• Triploid plant – sterile and seedless.
• Increases the edibility of fruits - grape ,watermelon.
• Timber and fuel yielding plants -triploids show better performance
over relative diploids or tetraploids.
• Used as nurse tissue- for hybrid embryos – rise hybrid plants.
• Ex: 9 to 12 day hybrid embryos of Hordeum*Triticum cultured using
endosperm nurse tissue of hordeum.
• Banana, apple, beet, tea, mulberry- triploids produced by endosperm
culture.
 Meristem and shoot tip culture

• Culture of the apical portion of shoot containing

meristem (0.1 mm dia; 0.25 mm length) along with
one to three young leaf primordia (1 to 0.5 mm).
• V shaped cut- 0.3 to 0.5mm below the tip of the
dome- remove explant along with procambial tissue-
place in MS media. 2cm length, 0.2 to 0.5mm
meristem tips produce virus free plants.
• Actively growing shoot tips used- strong growth
potential, low virus concentration.
 new leaf
tunica
apical
meristem
corpus
leaf trace

axillary
meristem
procambium

cortex pith
 3 stages of culture

• Stage 1: culture establishment stage- explants develop into either

single or multiple shoots.
• Media: cytokinin (BA, Kinetin, 2iP), Low conc of weak auxin like
NAA, IBA, antioxidants like ascorbic acid, citric acid etc to prevent
blackening or browning of explant tissue on exposure to polyphenol
oxidases.
• Stage 2: high levels (4.5 to 25µM) of cytokinin prevent apical
dominance of shoots. BA commonly used followed by kinetin and
2iP.
• STAGE 3: de novo regeneration of adventitious roots from the
shoots of stage 2 or 1; low cytokinin to high auxin ratio; NAA, IBA,
IAA, 2,4-D.
• Optimal temperature: 20 to 28°C; Light intensity 1 to 10 Klux;
duration: 16h day Vs 8h night.
 Advantages

 Production of virus free germplasm.

 Mass production of desirable genotypes.

 Cryopreservation (cold storage) or in vitro conservation of germplasm .

 Bud culture

• 2 types of culture
• Single node culture: bud from axil of the leaves – placed in
nutrient media- develop shoots- transferred to rooting media
and then to soil.
• Shoot proliferation requires no cytokinin.
• Axillary bud culture: from shoot tip- axillary bud - allowed to
develop under the influence of high cytokinin concentration-
stops apical dominance. Ex: strawberry and gerbera.
 ORGANOGENESIS

• Two types
• Organogenesis via callus formation: initiation of basal callus- shoot
bud differentiation- form plant organs like roots, shoots, bud,
flowers, stem etc.
• Good Explants: meristem, shoot tips, axillary buds, immature leaf,
embryos.
• Explants from mature or immature plants; mitotically active cells for
callus initiation.
• MS, B5,White’s medium.
• Cultured on solid or liquid media; suspension cell culture- free cells
of 2-100 number
• AUXIN – 2,4-D at moderate to high concentration – initiate callus
• On formation of new organ cells- transfer to regeneration medium
and subculture continuously
• Direct adventitious organ formation: development of organs
(roots, shoots, buds) or embryos (-like structure) from unusual
point of origin of an organized explant.
• Somatic tissues of higher plants- adventitious plants
regenerate without intervening callus phase.
• Formation of adventitious organs – reactivation of genes
concerned with the embryonic phase of development.
• Auxins, cytokinins- different concentrations- based on explant
taken, age of plant, growth conditions.
 In vitro production of haploids

• Haploid : plants with single set of chromosome in their

sporophytes.
• Spontaneous production : through apomixis or
parthenogenesis.
• 1964- Guha and Maheshwari- culture of microspores from
Datura innoxia- direct development of embryos and plantlets.
• two groups: monoploids (half the number of chromosome
from diploid species); polyhaploids ( half the number of
chromosome from polyploid species)
1. Androgenesis:

Haploid production occurs through anther or pollen culture, and they are
referred to as androgenic haploids.

2. Gynogenesis:

Ovary or ovule culture that results in the production of

haploids, known as gynogenic haploids.
 GYNOGENESIS

• formation of sporophyte from the female gametophyte

on anartificial medium.

• Explant removal: mature embryo sac

• Used as a alternative technique in species where anther or

pollen culture is unsuccessful .

• Two methods for production of gynogenic haploids:

1. ovaryculture
2. ovule culture
• Culture of ovaries isolated either from pollinated or
un-pollinated flowers on a suitable nutrient medium.

• Also refers asgynogenesis.

• First studied by Sannoeum in1976

In barley.
• Pollinated flower is cultured on a simple nutrient medium where as un-
pollinated flower cultured on special medium of synthetic auxin 2,4-D

• common medium used are white’s medium ,MS medium ,N6 medium .

• liquid medium - the ovaries are placed on a filter paper or allowed to

float over the medium with pedicel inserted through filter paper.

• Success rate varies with eachspecies

• In sunflower, cold pretreatment 24-48 hr at 4°C before ovary culture

enhancesgynogenesis.
 ADVANTAGES
• Study of early stages of embryo development.
• Study of fruitdevelopment and its physiology including
maturation .
• Effects of phytohormones on parthenocarpic fruit can be
studied .
• Induce polyembryony and in the study ofmutation.
• Helps to understand the hybridization
process of crossing interspecific and intergenic
species.
• hybrids of diploid Brassicachinensis and
autotetraploid B.pekinensis are obtained.
LIMITATIONS
• Frequency of responding ovaries are low ,1-5%.

• Number of plantlets per ovary are quite low1-2.

• Requires high technical skills and management.

• Besides a haploid plant ,some different traits

may develop.

• Employed in limited species like wheat ,rice

maize , sugarbeet.

• Successful only in less than two dozen species so

far.
• ovules are aseptically isolated from the ovary and are
grown on chemically defined nutrient medium under
controlled conditions.

PRINCIPLE

• Ovule is a mega sporangium covered with integument.

• Ovules attached to placenta inside ovary by funicle.
• It contain eggcell.
• After fertilization forms zygote and lead to mature
embryo possessing shoot and root primordial.
• Ovule can be isolated and cultured in nutrient medium.
 PROTOCOL
1) unfertilized ovules or fertilized ovules – collected from open flower

(2) Remove sepals, petals, androecium etc. from the ovaries containing either
fertilized or unfertilizedovules.

(3) Soak the ovaries in 6% NaClO solution.

4) Rinse the ovaries 3-4 times with sterile distilledwater.

(5) Using sterile techniques, ovules are gently prodded with the help of spoon
shaped spatula by breaking the funicles at its junction withplacental tissue.
6) The spatula with ovules is gently lowered into sterile solid or liquid
medium as the culture vial is slanted about45°.

(7)Damaged or undersized ovules are rejected during transfer.

(8)Incubate the ovule culture in either dark or light (16 hrs. 3,000 lux) at 25°C

(9)After two weeks ,haploid plantlets grow either through embryogenesis

or through plant regeneration from callus.
 APPLICATIONS
1.Test tube pollination and fertilization :
• Possible to germinate pollen in the same culture as the excised ovule and to
induce in vitro fertilization.

• Excised unfertilized ovules of Argemone mexicana, Papaver somniferum have

been cultured along with their respective pollengrains.
 Production of HaploidCallus
• Uchimiya et al. (1971) attempted culturing unfertilized ovules of
Solanum melongena and obtained vigorous callus formation on a
medium supplemented with IAA and kinetin.

Ovule Culture and Angiospermic Parasites

• Studies on ovule culture of Orobanche aegyptica and Cistanche tubulosa
have demonstrated that the formation of shoots in vitro can be
induced in anyabsenceof anystimulus from thehost.

• Generally believed that in

obligate root parasites such as
Striga or Orobanke the
formation of seedlings is
dependent on some stimulus
from the hostroot.
Induction of Poly-embryo

• It has been observed that the nucellus of

mono-embryonic ovuleof citrus can be induced
to form adventive embryos in culture.

Virus Irradiation through Ovule Culture

• In the varieties of Citrus which are impossible to free

of virus by other means, the ovule culture has proved
decisively advantageous to make them virus free.
ANTHER AND POLLEN
CULTURE
 ANTHER: A part of stamen
contaning pollen.
POLLEN: A fertilising powder
discharged from anther.
ANTHER AND MICROSPORE(POLLEN)
CULTURE: It is the process of formation of
haploid plants from microspores (pollen)
cultured individually or anthers.
Anther culture for production of
haploids reported in about 250 species.
Solanaceae, cruciferae, gramineae are most common.
Anther/pollen culture is referred as
ANDROGENESIS : occurs when
pollen/microspore shift from a gametophytic to
sporophytic pathway of embryo formation.
shift occur prior to premitotic & postmitotic :
either vegetative or generative divide to
undergo androgenesis.
 In experiments using Datura innoxia,
induction frequencies of almost 100% and
a yield of more than one thousand
plantlets or calluses have occurred under
optimal conditions from one anther.
Success can be determined within 24
hours as cells begin to divide.
 figure : Anther culture and
haploid plants regeneration.

• (a) Anther at the onset of the

culture. (b) Anther after 6 days
in culture. (c, d) Embryos
emerging from the anthers
after 30 days in culture,
showing roots
• (c) and shoots (d). (e–g)
Plantlets with cotyledons (e)
and with leaves (f, g)
subcultured in growing medium.
(h) 80- day-old regenerated
haploid plant from anther
culture (left-hand side).
1. Pathway I
2. Pathway II
3. Pathway III
4. Pathway IV
5. Pathway V
The microspores divide by an equal
division and two identical daughter cells
developed.
Vegetative and generative cells are not
distinctly formed in the pathway.
Example: Datura innoxia
The division of uninucleate microspores is
unusual , resulting in the formation of
vegetative and generative cell.
The sporophyte arises through further
division in the vegetative cell and
generative cell does not divide.
Examples: Nicotiana tabacum , Hordeum
vulgare , Triticum aestivum , Triticale.
 The uninucleate microspore undergoes a
normal division but pollen embryos are formed
from generative cell alone.
The vegetative cell does not divide.
Examples: Hyoscyamus niger
 Both generative and vegetative cell
divide further to the development of
sporophyte.
Examples: Datura metal, Atropa
belladona, Datura innoxia(occasionally).

PATHWAY V
 In Brassica napus , 1st division is
symmetric and the pollen embryos
develop the vegetative cell.
Cold Treatment (3 to 5 C) Enhances Symmetric Division of
Microspores or Division of Vegetative Nuclei

3 to 5°C

Vegetative
Microspore
Similar nuclei

Generative

3 to 5°C

Embryo
 Cold Pretreatment of Anthers Enhances the
Embryogenic Response
Cold treatment imposed prior to the first pollen
mitosis increases the frequency of symmetric
divisions of the microspore leading to embryo
formation.
100 3C
Producing Embryos

80
5C C
% Anthers

60

40
C
20

0
Tobacco Datura
1)Selection of explants(eg. Flower bud)
2) preparation of explant
3) disinfection of bud
4) selected buds are pretreated
5) surface sterlization
6) inoculation
7) transfer to culture room
8)transplanted to small pots in
greenhouse
 simple.
 less time consuming.
 responsive.
 Requires skill to remove anthers without
causing damage.
 Not much successful in case of cereal
crop.
 Risk of chimera and callus formation from
anther wall.
1. Often fail to grow in-vitro.
2. Tissue or callus comprises a chimera of
diploid, tetraploid and haploid cells.
3. Formation of albinos especially with cereals
and effect the loss of plants due to albinism.
4. It is not economically viable for haploid
production.
5. Callus in a medium supplemented with growth
regulators is usually detrimental for haploid
production.
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