Androgenesis

information of androgenisis

1. Anderogenesis Laraib Zafar Iqbal (L1F17BSBC0010 ) 1

2. Contents Introduction Methods for production Stages and pathways of development Factors affecting androgenesis Importance of androgenesis Conclusion References 2

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. 3

4.  Several strategies and methods have been worked for the production of haploid plants . Two methods: Androgenic methods Gynogenic methods 4

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. 5

6. Androgenesis  Formation of sporophyte from the male gametophyte. Artificial medium is uses.  Found in family solanaceae and poaceae.  Immature pollen grains are induced. 6

7. Methods for production: There are two methods for in vitro production of androgenic haploids They are : Anther culture I solated Pollen [microspore] culture. 7

8. Anther culture Stamen: M ale reproductive part of flower. It is composed of log tube known as filament. The oval-shaped structure is called the  anther. I t produces the male gametophyte known as pollen . 8

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. 9

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. 10

11.  Usually anther from the flower buds will give better response during androgenesis . 11

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. 12

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. 13

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 . 14

15. Hot treatment P lant in some species when subjected to 30 degree C for 24h or 40 degree for 1h stimulates embryogenesis . D issolution of microtubules.   Dislodging of the spindle which causes abnormal division. S terilization 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. 15

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. 16

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 . 17

18. Growth regulator: Complete nutrient medium. 2-3 % sucrose is added.   One or more hormone has been found necessary for an androgenic response . A ctivated 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. 18

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 19

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. 20

21. The released embryo develop cotyledons, which ultimately give rise to plantlets. Eg : Datura , Brassica campestris 21

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 22

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 . 23

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 24

25. P athway II T he uninucleate pollen divides unequally.   Formation of Vegetative and generative cells. F urther division in the vegetative cell. W hile the generative cell does not divide . Examples: Nicotiana tabacum , Hordeum vulgare , Triticum aestivum 25

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 26

27. PATHWAY IV T he uninucleate pollen grains divide unequally. P roducing generative and vegetative cell. these cells divide repeatedly to contribute to the development of sporophyte . Examples: Datura metal 27

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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. 29

30. This pollen suspension is then centrifuged.  The supernatant containing fine debris is discarded.  R esuspended in fresh media.   Washed at least twice.   Then pipetted in to small petri dishes.   Incubated at 28 degree C. 14 days of culture. 30

31. After 14 days ,the culture are transferred to suitable media. 31

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. 32

33. Growth regulators:  Kinetin or cytokinins are essential for induction of pollen embryos.  S ucrose 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. C hilling of anthers before inoculation , increases the number of pollen embryoids . 33

34. Other factor: organic supplements added to culture medium.  products of proteins such as casein (found in milk),nucleic acids.  Coconut milk. A mino acids like glutamine, proline, serine, etc. enhance the frequency of responsive anthers. 34

35. Conclusion A ndrogenesis 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, 35

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 36

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