Ovule and seed culture by Prerna Jain

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1. O vule and Seed culture T echnique and Applications Prerna jain 11615509 1628 MSC botany (H) A06

2. Contents Ovule culture Principle Protocol Historical perspective of ovule culture Applications and advantages with examples In vitro pollination and fertilisation Ovule culture in hybridization Production of haploid callus

3. Contents Ovule culture of parasitic plants Ovule culture of orchids plants Induction of poly- embryony by ovule culture Virus irradiation through ovule culture Seed culture Protocol

4. Contents Applications and advantages of seed culture Seed culture of plants with reduced embryos Seed culture of parasitic plants Seed culture of orchid plants Examples of seed culture In –vitro technologies - a challenge

5. Ovule culture is an elegant experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions. Ovule culture

6. Principle An ovule is a mega sporangium covered by integument. Ovules are attached with placenta inside the ovary by means of its funiculus . An ovule contains a megaspore or an egg cell. After fertilization, a single cell zygote is formed which ultimately leads to form a mature embryo possessing shoot and root primordia . Ovules can be isolated and cultured in nu­trient medium. In vitro ovule culture helps to understand the factors that regulate the devel­opment of a zygote through organised stages to a mature embryo. Alternatively, it may be possible to germi­nate pollen in the same culture as the excised ovule and to induce in vitro fertilisation and sub­sequently embryo production.

7. Protocol (1) Collect the open flower (unfertilized ovules). If fertilized ovules are desired, collect the open flower where the anthers are dehisced and pollination has taken place. To ensure the fertilization, collect the flower after 48 hrs. of anther dehiscence. (2) Remove sepals, petals, androecium etc. from the ovaries containing either fertilized or unfertilized ovules. (3) Soak the ovaries in 6% NaOCl solution. (4) Rinse the ovaries 3-4 times with sterile distilled water.

8. Protocol (5) Using sterile techniques, ovules are gently prodded with the help of spoon shaped sptula by breaking the funicles at its junction with placental tissue. (6) The spatula with ovules is gently lowered into the sterile solid or liquid medium as the culture vial is slanted about 45°. (7) Damaged or undersized ovules are rejected when possible, during transfer. (8) Incubate the ovule culture in either dark or light (16 hrs. 3,000 lux ) at 25°.C

9. Ovule culture in laminar air flow

10. Historical perspective of ovule culture First attempt to isolate ovules and culture them under aseptic conditions was made by white (1932) in Antirrhinum majus . But perfected by Maheshwari (1958). Maheshwari (1958) and Lal (1961) raise viable seeds of Papaver somniferum starting with ovules excised 6 days after pollination when they contained zygote or 2-celled proembryo and a few endosperm nuclei.

11. Historical perspective of ovule culture Paddubnaya - arnoldi (1959,1960) grew ovules from pollinated ovaries of several orchids on simply 10% sucrose solution. Nakajima (1969) cultured ovules at the zygote or 2-celled proembryo stage in the medium supplemented with cucumber and watermelon juices.

12. Historical perspective of ovule culture Joshi and Johri (1972) made extensive studies on the nutritional and hormonal factors concerned in ovule and fibre development after the unsuccessful attempts at the culture of Gossypium hirsutum due to interference of environmental conditions. Pundir (1967) successfully produced hybrids by using a cross between Gossypium arboreum and G. hirsutum .

13. Historical perspective of ovule culture Ramming (1971) reported the various in vitro conditions for culture of ovules of immature embryo of peach. Ramming discovered when medium supplemented with activated charcoal produced larger embryos. Doi et al, demonstrated that the unfertilized ovule culture techniques of gentians is a powerful tool for obtaining haploids and double haploids because of its reproducible and reliable nature and application to a wide range of genotypes.

14. Image of ovule culture

15. Applications and advantages of ovule culture

16. Test Tube Pollination and Fertilization An important achievement of research on ovule culture has been the development of the technique of test tube pollination and fertilization . In this technique, the ovules are planted on a suitable culture medium which supports growth of ovules as well as germination of pollen grain. Pollen grains are dusted around the ovules. T he pollen germinate and affect fertilization.

17. Examples Normal viable seeds develop within a few days example Argemone mexicana , Nicotiana developed by P. M aheshwari in 1962. Using the same method, it has been possible to fertilize the ovules of Melandrium album with pollen grains from other species of caryophyllaceae and subsequently even with pollen of Datura stramonium . Employing ovule culture technique, the incompatibility barrier in Petunia axillaris has been overcome.

18. Advantage In-vitro pollination via ovule culture has been used to circumvent various problems associated with hybridization such as failure of pollen germination on stigmas, insufficient growth of pollen tube, or precocious abscission of flowers and self incompatibility or sterility.

19. Application of Ovule Culture in Hybridization ovule culture has been successfully employed to obtain hybrid seedlings. It has been observed that in several inter specific crosses; the hybrid embryo of Abelmoschus fails to develop beyond the heart or torpedo-shaped embryo. By ovule culture, viable hybrids have been obtained in three out of five interspecific crosses attempted, namely, A esculentus x A ficuneus Aesculentus x A moschatus and A tuberculatus x A moschatus . Similarly, a true hybrid be­tween Brassica chinensis and B. pekinensis has been obtained by culturing the fertilized ovule in vitro. A hybrid between L oluim perenne and F estuca rubra has also been obtained successfully by means of ovule culture . Pundir successfully produced hybrids by using a cross between Gossypium hirsutum and G.arboreum .

20. Advantage In a normal process these hybrids fail to develop due to early embryo abortion and premature abscission of fruits. T hus ovule culture can be used to rescue them. This technique omits excision of the embryo by culturing the entire ovule. Thus greatly facilitating the time and effort involved. Also, ovule culture of cotton offers an unique method for the studies on the effect of phytohormones on fibre and seed development.

21. Production of Haploid Callus through Ovule Culture Uchimiya et al. (1971) attempted culturing unfertilized ovules of Solanum melongena and obtained vigorous callus formation on a medium supplemented with IAA and kinetin. A cytological assay revealed it to be haploid in nature . So it is an important attempt to obtain a haploid cell line or plant from an alternative source rather than anther or pollen culture.

22. Advantage For haploid production , one can go for anther culture but anther cultures of some plant such as Mimulus luteus did not form haploids. So it is an important attempt to obtain a haploid cell line or plant from an alternative source rather than anther or pollen culture.

23. Ovule Culture and Angiospermic Parasites It is generally believed that in obligate root parasites such as Striga or Orobanke the formation of seedlings is dependent on some stimulus from the host root. Studies on ovule culture of Orobanche aegyptica and Cistanche tubulosa have demonstrated that the formation of shoots in vitro can be induced in any absence of any stimulus from the host.

24. Advantage In ovule cultures of these parasites, it is possible to substitute the host stimulus with some known chemical like medium enriched with cytokinin , GA, scopoletin or strigol . Thus one can able to study hormonal requirement or nutritional requirement of ovules of parasitic plants.

25. Ovule Culture of Orchid Plants Poddubnaya-Arnoldi (1959, ’60) successfully grew the fertilized ovule of orchids in vitro. T hey grew the fertilized ovule of Calanthe veitchn , Cypripedium insigne, Dendrobium and Phalaenopsis schilleriana .

26. Phaleonopsis -Orchid flower resembling bee

27. Advantage In nature, the seeds of orchid germinate only in association with a proper fungus. As a result numerous seeds are lost due to unavailability of proper fungus. Secondly , the seed capsule of many orchid takes a long time to mature. thirdly ,minute size of seeds. To overcome such problems, several attempts have been made to culture the fertilized ovule of orchid in vitro.( asymbiotic germination).

28. Induction of Poly-embryo by Ovule Culture Ovule culture induces polyembryony in various parts of the plant. It has been observed that the nucellus of mono-embryonic ovule of citrus can be induced to form adventive embryos in culture.

29. A dvantage In horticultural practices, the artificial induction of polyembryo holds a great potential. Therefore, such achievement is very significant. Polyembryony associated with polyploids helps them to counteract the disadvantage of being sexually sterile and thus help them in their perpetuation generation after generation.

30. Virus Irradiation through Ovule Culture In the varieties of Citrus which are impossible to free of virus by other means, the ovule culture has proved decisively advantageous to make them virus free.

31. Thus , due to all these advantages and applications, ovule culture is a boon for the plant breeders in obtaining seedlings from crosses which are normally unsuccessful because of abortive embryos.

32. Seed culture Germination of seeds in vitro conditions involves the proper inoculation of seed in the medium. It is better to inoculate the seeds without seed coats as it reduce the germination time and increases the germination potential.

33. Surface sterilize the seeds with teepol and then 0.1% Hgcl2 for about 5 min. Wash 3-4 times thoroughly with deionized water and then inoculate the seeds in basal MS media under laminar air flow. Protocol

34. Applications and advantages of seed culture

35. Seed culture of plants with reduced embryos Plants such as Eranthis ( ranunculaceae ) characterized by the presence of embryos that lack differentiation into various embryonic organs, namely radicle , plumule and cotyledons. Thus in such a case the culture of seeds to raise sterile seedlings is the best method.

36. Eranthis -Plant with reduced embryo

37. Seed culture of parasitic plants In obligate root parasites such as O robanche and S triga , the seeds germinate close to host roots ,and it is only after the parasite establishes contact with the host –roots that it develops shoot . Thus ,through seed cultures of these parasites it is possible to substitute the host stimulus with some known chemicals.

38. Example of seed culture of parasitic plants The seeds of Orobanche germinate in vitro in the absence of a natural host if the nutrient medium is enriched with a cytokinin ,GA, scopoletin or strigol The seeds of Scurrula pulverulenta a stem parasite can also germinate on plain white’s basal medium also seedlings grow better when casein hydrolysate is added to the medium.

39. Seed culture of orchid plants There are several plant species in which seed sizes are minute and do not germinate well. orchid is one of them. Thus , to meet requirements and to conserve natural resources in-vitro seed germination has been utilized to produce large quantities of uniform seedlings. In vitro seed culturing of such plants is dependent on the growth medium supplemented and not required fungus/orchid symbiotic relationship dependent germination and thus known as asymbiotic germination.

40. Seed culture of orchids plants

41. Examples of seed culture Seed culture is widely used for large, rapid propagation and large-scale seedling production. For producing large-scale seedlings of Capparis spinosa sterilized the seeds with 0.1% HgCl2 (12 min) and utilized optimum MS medium supplemented with activated carbon. Several examples of seed cultures are given in following table-

42. Examples of seed culture Plant description method Effective media Cephalanthera falcata Asymbiotic germination Kano medium and ND medium Malaxis khasiana Rapid in-vitro propagation Ms medium 2 % sucrose + casein hydrolysate +BAP Dendrobium hookerianum Asymbiotic seed germination MS medium Coelogyne nervosa Asymbiotic seed germination Ms medium + 30% coconut water Cymbidium mastersii Mass propagation Ms medium + BAP +NAA

43. In –vitro technology –a challenge In-vitro culture technologies are still a challenge because of the slow growth of plantlets, low multiplication rate, poor rooting, and somaclonal variation. In this regard, micropropagation through protocorm -like bodies obtained from germinating embryos and somatic tissues is an important strategy in obtaining genetically stable plants and the improvement of quality.

44. Thank you