Anther and pollen culture. Androgenesis Principle and pathway

1. Topic:Anthercultureandpollenculture

2. Anther
culture and
Pollen
culture

3. History:
• W. TULECKE(1953) First observed the mature pollen
grain of zinkgo biloba (a gymnosperm) can be induced
to proliferate in culture to form haploid callus.
• S.Guha and S. C Maheshwari (1964 ) first reported the
direct development of embryos from microspores of
Datura innoxia by the anther culture.
• J. P. BOURGIN AND J. P. NITSCH(1967) Obtained
complete haploid plantlets from anther culture of
nicotiana tabacum

4. Anther culture :
Culturing of anthers obtained from
unopened flower bud in nutrient
medium under aseptic condition.
Callus tissue or embryoids from
anther, that give rise to haploid
plantlets either though organogenesis
or embryogenesis.

5. Pollen
Anther
Pollen culture :
Pollen or microspore culture is an in vitro
technique by which the pollen grains
preferably at the uni-nucleated stage, are
squeezed out aseptically from the intact
anther and then cultured on nutrient medium.
The microspore develop into haploid
embryoids or callus that give rise to haploid
plantlets by embryogenesis or organogenesis.

6. Androgenesis :
Androgenesis is the in vitro development
of haploid plants originating from
totipotent pollen grains through a series
of cell division and differentiation.
It is of two types :
1) Direct androgenesis
2) Indirect androgenesis

7. Direct
androgenesis :
Indirect
androgenesis :
The microspores behaves like a
zygote and undergoes change to
form embryoid which ultimately
give rise to a plantlet.
The microspores divide
repeatedly to form a callus tissue
which differentiates into plantlets.

8. Principle of anther and pollen culture :
 The production of haploid plants is to exploit
the totipotency of microspore.
 In the process the normal development and
function of the pollen cell to become a male
gamete is stopped and is diverted forcely to a new
metabolic pathway for vegetative cell division

9. Pathways of pollen development :
Pathway I
The microspore 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

10. Pathway II
 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.

11. Pathway III
 The uninucleate microspore undergoes
pollen embryos are formed from generative
cell alone.
 The vegetative cell does not divide .
 Examples : Hyoscyamus niger

12. Pathway IV
 Both generative and vegetative cell
divide further to the development of
sporophyte.
 Examples: Datura metal, Atropa
belladona, Datura innoxia.
Pathway V
In Brassica napus 1st division is symmetric and
the pollen embryos develop the vegetative cell.

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14. Procedure of Anther Culture
First, select the unopened buds of size about 17-22mm in
length.
Buds are washed under running tap water to remove the dirt.
Then, transfer the selected buds to the laminar airflow
chamber.
After that surface sterilization of buds is carried out in the
chamber to maintain the aseptic conditions. Firstly, the buds are
surface sterilized by 70% ethanol for 10 seconds and then 20% of
sodium hypochlorite for 10 minutes.
Then, wash the buds three times by the distilled water.

15. Continue……..
After washing, transfer the buds to the sterilized Petri plate.
Then, separate the stamen from the bud through a scalpel.
Remove the filaments from an anther.
After that, transfer an anther onto the solid or liquid
nutrient medium and incubate it for 3-4 weeks at 24-28
degree C in the dark.
Then either by embryogenesis and organogenesis, haploid
plantlets would appear from the anther culture within 3-4
weeks. During this stage, incubate the culture at 24-28 ֯C for
12-18 hours in light and 6-12 hours in the dark.

16. continue,…..
At last, about 50mm tall plantlets will appear
that are then transferred into the pot containing
bio compost followed by washing.

18. Factors Influencing Anther Culture
Anther wall: It provides nourishment during the developmental stages of the anther.
Stage of microspore : Anthers are more productive when cultured at uninucleate stage.
Bud size: Its preferable size is up to the length of 17-22 mm.
Age of plant: The anther culture prefers the buds from the younger plants.
Culture medium: In the culture medium sucrose, iron, vitamins, coconut milk,
hormones and growth regulators (auxins or cytokinins) play an essential role in the
induction of haploid plant.
Temperature: It varies with the different plant species. Example: In Datura stramonium,
the optimum temperature is 20˚C for the formation of embryoids. In Nicotiana tabacum,
the optimum temperature is 25˚C for the formation of embryoids. In Brassica campestris,
the optimum temperature is much higher, i.e. 35˚C for the formation of embryoids.

19. Advantages
• Simple
• Less time consuming
• Responsive
Disadvantages
• 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.

20. Applications:
• For mutation study .
• For plant breeding and crop improvement.
• Haploids are used in molecular biology and
genetic engineering. ( Haploids tissue of
Arabidopsis and Lycopersicon have been used
for transfer and expression of three genes
from Escherchia coli

21. •
Thankyou