Anther culture

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Anther culture

  1. 1. SEMINAR PRESENTATION ON ANTHER CULTURE PRESENTED TO: by: Dr.Harsh kumar sir & Biotech Dr. Kundan sir presented DEEPTI (msc.Ag. 1st sem)
  2. 2. ANTHER: A part of stamen contaning pollen. POLLEN: A fertilising powder discharged from flowers anther.
  3. 3. ANTHER AND MICROSPORE(POLLEN) CULTURE: It is the process of formation of haploid plants from microspores (pollen) cultured individually or anthers.  Anther culture for production of haploids reported in about 250 species. Solanaceae, cruciferae, gramineae are most common.  Anther/pollen culture is referred as ANDROGENESIS : occurs when pollen/microspore shift from a gametophytic to sporophytic pathway of embryo formation.  shift occur prior to premitotic & postmitotic : either vegetative or generative divide to undergo androgenesis. 
  4. 4.  In experiments using Datura innoxia, induction frequencies of almost 100% and a yield of more than one thousand plantlets or calluses have occurred under optimal conditions from one anther. Success can be determined within 24 hours as cells begin to divide.
  5. 5. figure : Anther culture and haploid plants regeneration. (a) Anther at the onset of the culture. (b) Anther after 6 days in culture. (c, d) Embryos emerging from the anthers after 30 days in culture, showing roots (c) and shoots (d). (e–g) Plantlets with cotyledons (e) and with leaves (f, g) subcultured in growing medium. (h) 80day-old regenerated haploid plant from anther culture (left-hand side).
  6. 6. – 1964, 1966 Datura innoxia (Guha and Maheshwari), the Indian scientists. – 1967 ( Bourgin and Nitsch ) : 1st haploid plants from isolated anthers of Nicotiana. – during last decade : anther culture of rice, wheat , maize, brassica, pepper and crop sp.
  7. 7. 1. 2. 3. 4. 5. Pathway I Pathway II Pathway III Pathway IV Pathway V
  8. 8. 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 
  9. 9. 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 , Triticale. 
  10. 10. The uninucleate microspore undergoes a normal division but pollen embryos are formed from generative cell alone.  The vegetative cell does not divide.  Examples: Hyoscyamus niger 
  11. 11. Both generative and vegetative cell divide further to the development of sporophyte.  Examples: Datura metal, Atropa belladona, Datura innoxia(occasionally).   PATHWAY V In Brassica napus , 1st division is symmetric and the pollen embryos develop the vegetative cell.
  12. 12. Pathways to pollen development Normal pollen development
  13. 13. 1) Genotype of donor plant : determine the frequency of pollen plant production. _ eg. In Hordeum each genotype differs with respect to androgenic response in anther culture. _ high responsive anthers should be taken.
  14. 14. 2) Anther wall factor : act as conditioning factors and promote culture growth. _ report: glutamine alone or in combination with serine and myoinositol could replace the anther wall factor. 3) Stage of pollen : stage of pollen varies with species. _ before/after 1st pollen mitosis- Datura,
  15. 15. 4) Physiological status of donor plant : a) grown under best environmental conditions with good anthers. b) flowers obtained at the beginning of flowering season are highly responsive.
  16. 16. 5) Pretreatment of anthers : appropriate treatment required for good success of haploid production(depend on donor plant species). # Temperature influence: a) induction of androgenesis is better if stored at low temperature prior to culture.e.g. maize , rye. b) pretreatment of anthers at higher temperature stimulates androgenesis
  17. 17.  response is not known but interference with starch accumulation in pollen and degradation of tapetum cellular matrix,etc.may be involved.
  18. 18. 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
  19. 19. 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. % Anthers Producing Embryos 100 3C 80 5C 60 C 40 C 20 0 Tobacco Datura
  20. 20. PLANT SPECIES ANDROGENIC RESPONSE Brassica napus Heat treatment(320 c for 8 hrs ), gamma- rays, colchicine Hordeum vulgare Stress by mannitol, calcium and ABA Nicotiana tabacum(tobacco) Glutamine and sugar starvation (transfer to high glucose medium) Cold treatment( 4 - 100 c for 3- Triticum aestivum
  21. 21. 6) Effect of light : pretreatment of anthers at elevated temperatures( 3 0 c) stimulate androgenesis in some Brassica and Capsicum. 7) Culture medium : - it vary with the genotype and age of the anther. - culture maintained on an auxin medium for longer period develop a friable callus. - a compound related to auxin namely 2,3,5triiodobenzoic acid(TIBA) gives +ve result at low concentration. - incorporation of activated charcoal/2chloroethyl-phosphate stimulates androgenesis
  22. 22. DIFFERENT TYPES RELATED CULTURE OF PLANT MEDIUM SPECIES Nicotiana tabaccum and Datura innoxia Simple agar plates containing only sucrose Solanaceae species Complete nutrient medium Non- solanaceae plants Medium fortified with growth adjuvents (yeast extract, casein hydrosylate, coconut milk) Maize anthers N6 and Yu-Pei Wheat anthers Potato – 2 medium(potato-1 + six low salt concentration)
  23. 23. 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
  24. 24.    simple. less time consuming. responsive.
  25. 25.    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.
  26. 26. 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.
  27. 27. THANKS thank you

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