information of androgenisis

1. Anderogenesis
Laraib Zafar Iqbal (L1F17BSBC0010)
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2. Contents
• Introduction
• Methods for production
• Stages and pathways of development
• Factors affecting androgenesis
• Importance of androgenesis
• Conclusion
• References
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3. Introduction
• The dominant life form of higher plants is free living from sporophytes.
• Sporophytes fertilization of male and female gamates.
• Genomic constituent is 2n.
• Gametophyte carry half of the sporophyte set.
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4. • Several strategies and methods have been worked
for the production of haploid plants.
• Two methods:
• Androgenic methods
• Gynogenic methods
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5. • Androgenic method:
Haploid plant production.
Uses anther or microscopic culture.
Referred as androgenesis.
• Gynogenic method:
 it is also the production of haploid plants
through the ovary or ovule culture.
Referred as gynogenesis.
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6. Androgenesis
• Formation of sporophyte from the
male gametophyte.
• Artificial medium is uses.
• Found in family solanaceae and
poaceae.
• Immature pollen grains are induced.
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7. Methods for production:
• There are two methods for in vitro production of androgenic haploids
• They are :
 Anther culture
Isolated Pollen [microspore] culture.
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8. Anther culture
• Stamen: Male reproductive part of
flower.
• It is composed of log tube known as
filament.
• The oval-shaped structure is called
the anther.
• It produces the male gametophyte
known as pollen.
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9. • Anther culture is the process of using anthers to culture haploid plantlets.
• 1964 by Guha and Maheshwari.
• This technique can be used in over 200 species including Solanaceae, cruciferae,
gramineae/Poaceae are most common.
• It is an artificial technique by which the developing anthers at a precise and critical
stage are excised aseptically from unopened flower bud.
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10. Sources of anther culture:
• Success of androgenesis depend upon:
Variety used
Growth condition
Quality of donor
• The normal flower condition is the best environment for the anther production.
• Age of donor plant should must be noted.
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11. • Usually anther from the flower buds will give better response during
androgenesis.
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12. Pretreatment of anther
• Once the donor plant is selected,
it requires the specific
pretreatment conditions.
• It can be done at different levels of explants.
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13. Cont…
• Pretreatment plays a key role for anther callus induction.
• The main pretreatments applied to anther culture are
 cold treatment
hot treatment
• With regard to different explants, the type, levels, and duration of
pretreatments are different.
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14. Cold treatment
• 3-6 degree C
• 3- 15 days gives good
response.
• As a result, weak or non-viable
anther and microspores are
killed.
• It will also retards aging of the
anther wall.
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15. Hot treatment
• Plant in some species when subjected to 30 degree C for 24h or 40
degree for 1h stimulates embryogenesis.
• Dissolution of microtubules.
• Dislodging of the spindle which causes abnormal division.
• Sterilization of flower buds was carried out in the Laminar Air Flow
Cabinet.
• Young flower buds are surface sterilized.
• Calyx from the flower buds will be removed by flamed forceps.
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16. Media and growth regulator
• Media:
 Vary with species
 MS media for anther culture.
 The basal medium and combinations of growth regulators are also an
important factor.
• Solidified with Agar
• Agar contain compounds inhibitory
to some species.
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17. Cont…
• The use of liquid medium has
been advocated by some
researchers.
Anthers may be placed on the
surface of the medium.
• Microspores isolated in liquid
medium.
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18. • Growth regulator:
Complete nutrient medium.
2-3 % sucrose is added.
 One or more hormone has been found necessary for an androgenic
response.
Activated charcoal to agar medium is advocated.
In some species it is not necessary in anther culture.
A low concentration of some form of auxin.
Cytokinin is sometimes used in combination with auxin.
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19. Stages and pathways of development
• After inoculation haploid plants develop from anther culture either
directly or indirectly through a callus phase.
 Direct androgenesis
Indirect androgenesis
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20. Direct androgenesis:
• It is also called pollen derived embryogenesis.
• Pollen grains directly acts as a zygote.
• Similar to zygotic embryogenesis.
• When the pollen grains has reached globular stage of embryo, the
wall of the pollen is broken and embryo is released.
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21. • The released embryo develop
cotyledons, which ultimately give
rise to plantlets.
• Eg: Datura , Brassica campestris
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22. Indirect androgenesis
• In indirect androgenesis the pollen
grains divide erratically to develop
callus.
• Callus tissue which is finally
redifferentiates and forms haploid
plantlets.
• Eg: rice , wheat, tomato
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23. Pathways for development
• Depending on the composition of the medium, development pollen
may leads to the formation of callus.
• Based on the few initial divisions in the pollen grains or responds of
pollen grains.
• 4 pathways have been identified in in vitro androgenesis.
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24. Pathway I
• 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
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25. Pathway II
• The uninucleate pollen divides
unequally.
• Formation of Vegetative and
generative cells.
• Further division in the vegetative
cell.
• While the generative cell does not
divide.
• Examples: Nicotiana tabacum ,
Hordeum vulgare , Triticum
aestivum
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26. Pathway III
• Uninucleate pollen undergoes a
normal division.
• pollen embryos are predominantly
formed from generative cell alone.
• The vegetative cell does not divide.
• Examples: Hyoscyamus niger
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27. PATHWAY IV
• The uninucleate pollen grains divide
unequally.
• Producing generative and vegetative
cell.
• these cells divide repeatedly to
contribute to the development of
sporophyte .
• Examples: Datura metal
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29. Microspore culture
• Anther are collected from sterilized flower buds.
• The microspore are then squeezed out of the anthers by pressing them
against the side of beaker with a glass rod.
• Anther tissue debris is removed by filtering the suspension through a
nylon sieve.
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30. • This pollen suspension is then centrifuged.
• The supernatant containing fine debris is discarded.
• Resuspended in fresh media.
• Washed at least twice.
• Then pipetted in to small petri dishes.
• Incubated at 28 degree C.
• 14 days of culture.
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31. • After 14 days ,the culture are transferred to suitable media.
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32. Factors affecting androgenesis
• Genotype of donor:
important for determining the success or failure of androgenesis.
• Anther wall factor:
Growth inhibiting substances leaking out of the anther wall in contact with
nutrient medium.
• Culture medium:
The culture medium also play a vital role in the correct amount and
proportion of inorganic nutrients.
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33. • Growth regulators:
Kinetin or cytokinins are essential for induction of pollen embryos. Sucrose plays
an important role in induction of pollen haploid plants.
• Activated charcoal:
It removes the inhibitors from the medium and helps in the adsorption of 5-
hydroxymethylfurfural.
• Physical factor:
Temperature and light are two physical factors which plays an important role in the
culture of anthers.
Chilling of anthers before inoculation , increases the number of pollen embryoids.
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34. • Other factor:
organic supplements added to culture medium.
 products of proteins such as casein (found in milk),nucleic acids.
 Coconut milk.
Amino acids like glutamine, proline, serine, etc. enhance the frequency of responsive
anthers.
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35. Conclusion
• Androgenesis involves the control and reprogramming of
developmental switches, it provides opportunities to investigate
key elements in developmental control. Moreover, via
androgenesis, fertile homozygous progeny from a heterozygous
parent can be obtained in a single generation,
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36. Reference:
• https://www.slideshare.net/pillaiaswathy/androgenesis-by-
aswathy-iswanath
• Dubey R.C,A textbook of biotechnology, (2004),published by
S.Chand and company LTD.
• Bajaj, Y.P.S. 1983. In vitroproduction of haploids. In: Handbook of
Plant Cell Culture, Vol. 1: Techniques for Propagation and Breeding
. Ed. D.A. Evans et al. Macmillan, New York. 228–287
• http://www.plantphysiology.org/content/124/2/523
• http://www.hos.ufl.edu/mooreweb/TissueCulture/tccla ss.html
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