Androgenesis is the production of haploid plants through the culture of male gametophytes or microspores. There are two main methods - anther culture and isolated pollen/microspore culture. Anther culture involves excising anthers from flower buds and culturing them on nutrient media, while microspore culture isolates microspores from anthers. Several factors influence androgenesis success, including genotype, anther wall components, culture medium, growth regulators, and physical conditions. Androgenic haploids can develop directly from microspores or indirectly through a callus phase, following various developmental pathways. Androgenesis allows for the efficient production of haploid plants for breeding programs.

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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