In vitro pollination

In vitro pollination technique

1. In-Vitro Pollination and Fertilization Presented by: Yashika Shoolini University, Solan

2. INTRODUCTION Cultivation of plant tissue or other organs on artificial media in a test tube or conical flask is called in vitro technique. Under controlled conditions aseptic transfer of pollen grains on stigma to produce hybrid embryos among plants that can’t cross by conventional method of plant breeding is called as in vitro pollination and fertilization.

3. HISTORY German Botanist Harberlandt (1902) develops the concept of in vitro culture. This in vitro pollination technique was developed at university of Delhi to produce hybrid among species of P avaceraceae and Solanaceae . [Ref: Maheshwari and kanta , 1964]

4. Barriers during pollination and fertilization of in vitro technique Pollination and fertilization under in vitro condition offer an opportunity for producing hybrid embryos among plants that can’t be crossed by conventional method of plant breeding. In hybridization programs, transferring viable pollen from one parent to another does not always lead to seed setting.

5. Some of the barriers to fertilizations are P re-fertilization/pre zygotic Barriers: I nability of pollen to germinate on foreign stigma. Failure of the pollen tube to reach the ovule due to excessive length of the style or slow growth of the pollen tube, which fails to reach the base of the style before the ovary abscises. Bursting of the pollen tube in the style. Post Zygotic Barriers: Fertilization may occur normally, but the hybrid embryo fails to attain maturing due to embryo-endosperm incompatibility, or poor development of endosperm. It is called post zygotic barriers.

6. Overcome the Barriers: From time to time various techniques have been developed to overcome the pre zygotic Barriers to fertility. Some of these are: Bud pollination Stub pollination Heat treatment of the style Mixed pollination Irradication The possibility of effecting fertilization by introducing pollen grains directly into the ovary (intra-ovarian pollination) is yet another approach to bypass the pre-fertilization barriers. A still more promising and proven technique developed by Maheshwari and his students to overcome the pre-zygotic barriers to fertility is what they have described as ‘test tube fertilization’. Post fertilization barrier may overcome by the culture of ovaries by ovule culture after pollination

7. Why in vitro pollination is needed? For the production of homozygous plant. For the conservation of extinct plant species. Hybrid production Reducing the breeding cycle. Overcome the dormant period. Production of hybrid species by distant hybridization. Production of haploid plant. Conservation of germplasm .

8. In vitro fertilization using isolated single gamete A landmark technique developed recently in the field of plant biotechnology has been the successful fusion of male and female gametes isolated from higher plants in vitro and subsequent regeneration of fusion product into embryo and finally a plant.[ Kraz and Lorz , 1993]. This process is known as in vitro fertilization requires isolation of male gametes sperms from germinating pollen grainer tube and of the female gamete (egg) from embryo sac . Purpose of in vitro pollination Intergeneric hybridization Intraspecific hybridization Interspecific hybridization Intra-familiar crossing/

9. Types of In Vitro Pollination Ovular pollination: Application of pollen to excised ovule. Ovarian pollination: Application of pollen to excised ovary. Placental pollination: Application of pollen to ovules attached to the placenta. Stigmatic pollination: Application of pollen to stigma.

10. I n vitro P ollination Stigmatic pollination Ovarian pollination Placental pollination

11. Technique Conditions required for successful IVF Ovaries large in size and contain many ovules are the best experimental material for in vitro pollination. Isolation of ovules without damage as possible. Pollen which should be viable and able to germinate. There must be abundant growth of pollen tubes all over the ovules and placenta in culture. 1% CaCl 2 solution that favour the growth of pollen tube. Other requirements: Before start, the information on Time of anthesis Time of dehiscence Time of germination of pollen tubes into ovules. Viability of ovules.

12. Disinfection of materials A reasonable disinfection of ovule and pollen is the principle requirement for in vitro pollination. Flower buds are emasculated before anthesis and bagged in order to prevent pollination. The buds are brought to the laboratory for aseptic culture. The whole pistil are sterilized by 70% alcohol surface sterilized with a suitable agent and finally washed with distil water. To collect pollen under aseptic condition, anthers removed from the flower are kept in sterile petri plates containing a filter paper until their dehiscence. The pollen is then aseptically deposited on the cultured ovules, placental or stigma depending on the nature of the experiment.

13. Culture of ovules, ovary and stigma Ovules: The growth of pollen tube attached to bare ovules is inhibited by the presence of water on the surface of the ovules. This film of water should be dried with filter paper and later the dried ovules covered by the pollen grain. In Nicotiana tabacum , Allium cepa , Gynandropsis gynandra seeds are raised from ovules which contain globular or older embryo. 6 days after in vitro pollination ovules contain a single celled zygote which requires more complex growth condition.

14. For the development of subsequent embryonic stages, ovules which have been self-pollinated are usually kept on the placenta until seed formation while cross pollinated ovules regain placenta only during the initial 6-8 days of culture. Afterwards, they can be transferred to fresh medium without placenta. Use: Ovule culture has proved to be very useful technique for raising inter specific hybrids within genus Gossypium herbacium and Trifalium .

15. 2. Ovary: The technique of ovary culture was developed by Nitsch in 1951 by the ovaries of Cucumis and Lycopersicum excised from pollinated flower in vitro to develop into mature fruits The addition of vitamin B to the medium resulted in the development of fruits of normal size with viable seeds Further enrichment of medium with IAA or coconut milk induced even larger fruits than the fruits formed in in vivo conditions. Successful culture excised ovaries from a number of species such as Linaria macroccana , Hyoscymus niger on a medium containing mineral salts and sucrose.

16. Floret envelops: The floret envelops play an important role in the development of the fruit and the embryo of monocots. Ovary excised soon after pollination only when the floret envelop remain intact. E.g . Triticum aestivum and Triticum spelta . Hull factor: This requirement of the floret envelops associating with excised monocot ovules in vitro is known as ‘hull factor’. In the elongation of barely embryo cells can take place but cell division doesn’t occur. Use of ovary culture: Several interspecific and intergeneric hybrids can be produced between sexually incompatible parents in the family Cruciferae with the aid of ovary culture.

17. 3. Stigma The entire placenta or part of it bearing the ovules is used in placenta pollination. To perform in vitro stigmatic pollination the excised pistils are carefully surface sterilized without wetting the stigma with the sterilant solution. Sometimes the entire pistil in which the placental bearing ovules have been exposed are cultured to study the effect of placental and stigmatic pollination in the same pistil. In stigmatic pollination presence of perianth is important factor in dicot.

18. Factor affecting seed set Physiological state of the explant The physiological state of the pistil at the time of excising the ovules or ovary influences the seed set after in vitro pollination. Wetting surface of the ovules or stigma may lead to poor pollen germination or bursting of the pollen tubes and poor seed set. Pollen germination on the stigma and growth of the pollen tube influences the synthesis of proteins which may sometimes inhibit the entry of the pollen tube into the ovary. To improve the chances of success of in vitro pollination the level of incompatibility should be reduced. The time of excising the ovules from pistil has a definite influences on seed set after in vitro pollination. Ovules excised 1-2 days after anthesis show a higher seed set.

19. Culture medium: The nutrient medium plays an important role in supporting the normal development of ovary and ovules in culture until seed formation. Nutrient medium on successful culture of ovules include Nitsch’s mineral salts, white’s vitamins and 5% sucrose. Several orchid ovules isolated from pollinated ovaries can grow successful on simple 10% sucrose solution and Zephyranthes require coconut milk. Source of reduced N 2 as a complete amino acid mixture is require for optimal kernel development and growth. Kinetin promotes the initial growth of the embryo. IAA or kinetin improves the no. of seeds per ovule. Nitsch’s medium is appropriate for in vitro culture of pollinated ovules of most species. Osmolarity of the culture medium also affects the development of excised ovule.

20. Storage condition Usually the first step of this process occurs at room temperature and without special lighting. [ Zenk teller-1980] The ovary cultures are maintained at 22-26 C and other suitable conditions favouring embryogenesis. Genotype: The response of in vitro ovaries in relation to the seed set depends on the species. Pollen grains of crucifers are difficult to germinate in culture. Brassica oleracea ovules in 1% solution of CaCl 2 with 10% gelatin and then pollinated with pollen transferred to Nitsch’s medium until seed set.

21. Application of in vitro pollination In plant breeding programs, the technique of in vitro pollination has potential application in different areas- Overcoming self-incompatibility: Petunia axilaris and Petunia hybrida are self-incompatible species. Germination of pollen is good on self- pollinated pistils but a barrier exists in the zone of the ovary as a result, the pollen tube cannot fertilize the ovule. The barrier of these taxa can be overcome by in vitro pollination. Overcoming cross-incompatibility: Successful culture of in vitro pollinated ovules has raised the possibility of producing hybrids which are unknown because of pre-fertilization incompatibility barriers. Production of haploid plant: Another application of in vitro pollination reported, is the production of haploids of Mimulus luteus CV. Tigrinus grandiflorus by pollinating its exposed ovules with Torenia fournieri . The haploids of Mimulus luteus developed parthenogenetically , which otherwise could not be obtained through anther culture.

22. Some other examples that produces hybrid parthenogenitically through in vitro pollination are Nicotiana tabacum , Hordeum vulgare , Triticum aestivum . Production of stress-tolerant plant: Maize plants tolerant to beat stress have been produced through in vitro pollination at high temperature. Additionally these plants exhibited increased vigour and grain yield. Development of young hybrid embryo: Development of young hybrid embryos can be achieved in extremely widely crosses through in vitro pollination. The efficiency of this technique needs much improvement.

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