ORGAN CULTURE, Anther & Pollen Culture
ORGAN CULTURE, Anther & Pollen Culture
ORGAN CULTURE, Anther & Pollen 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
Organ culture
Bud culture
Callus culture
Organ culture
• 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
• 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
Berberine: an alkaloid with medicinal uses for cholera and bacterial dysentery
Rosmarinic acid: for antiviral, suppression of endotoxin shock and other medicinal purposes
4. Embryogenesis:
Embryo differentiation occurs when proliferated tissue is transferred
to basal medium with or withoutgibberellins – leads to development
of plantlet.
axillary
meristem
procambium
cortex pith
3 stages of 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 production occurs through anther or pollen culture, and they are
referred to as androgenic haploids.
2. Gynogenesis:
• common medium used are white’s medium ,MS medium ,N6 medium .
PRINCIPLE
(2) Remove sepals, petals, androecium etc. from the ovaries containing either
fertilized or unfertilizedovules.
(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°.
(8)Incubate the ovule culture in either dark or light (16 hrs. 3,000 lux) at 25°C
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.
THANK YOU