This article throws light upon the five techniques used in conventional breeding programme.
The five techniques are: (1) Embryo Culture (2) Ovary and Ovule Culture (3) Nucellus Culture (4) Control of Fertilization and (5) Endosperm Culture.
Technique # i. Embryo Culture:
Inter-specific crosses may fail because of several reasons, but when the development of embryo is arrested owing to the degeneration of the endosperm, or when the embryo aborts at an early stage of development, embryo culture is the only technique to recover hybrid plants.
Incidentally, embryo has been the first plant part successfully cultured in isolation. It was in 1904 that Hanning could rear seedlings from cruciferous embryo excised from immature pods.
Laibach (1925) for the first time, employed embryo culture for obtaining hybrids from a cross between Linum perenne and L. austriacum. More recently, a number of hybrids have been successfully raised through embryo culture: Hordeum vulgare X Secale cereale, H. vulgare X Agropyron repens, H. vulgare X Triticum sp. inter-specific hybrids in Abelmoschus and Secale. Embryo culture has helped in overcoming self-sterility of seeds, especially of crop plants propagated vegetatively, when the seeds do not germinate in nature. In a wild relative of commercial banana, Musa balbisiana for example, the seeds do not germinate under natural conditions. However, if the embryos are excised and grown on a simple culture medium of mineral salts, seedlings are readily obtained. Likewise, tuber crop Colocasia esculenta propagates only vegetatively and the seeds do not germinate in nature. This natural sterility barrier could be overcome by resorting to culture of embryos.
In vitro zygotic embryo culture continues as a means of rescuing hybrid embryos during interspecific gene transfer. Parker and Michaels (1986) employed this technology to recover hybrids of Phaseolus vulgaris and P. acutifolius. P. acutifolius has improved tolerance to common bean bacterial blight (Xanthomonas compestris) than P. vulgaris (common field bean) but hybrid and early backcross embryos from crosses between these species frequently abort. Embryos rescue assisted the transfer of improved bacterial blight tolerance to economically important field beans.
In another case, embryo rescue technique has been utilized in the transfer of cytoplasmically inherited herbicide tolerance from Brassica napus to B. oleracea. The orchid industry owes a great deal to the technique of embryo culture. The culture of orchid embryos was initiated at the Singapore Botanic Gardens in 1928. To date, they have succeeded in producing 1,431 hybrids, and 141 selfed (plants raised through selfpollination) species. Callus induction from embryos of orchids can be very useful in obtaining more hybrids.
Technique # ii. Ovary and Ovule Culture:
Embryos at very early stages of development have not been amenable to culture, primarily because of the problems involved in excision and the complex nutrient requirements. Where such a problem exists, it is logical to culture either the entire ovary or the ovule. This facilitates the availability of maternal tissue and eliminates the cumbersome problem of dissecting very young embryos and selecting a very exacting medium. The culture of pollinated ovaries and ovule in vitro has been successful in a number of plants (Fig. 27.1).
The credit for developing and firmly establishing the technique of ovary culture goes to J.P. Nitsch (1951), who experimented with the ovaries of Phaseolus vulgaris, Cucumis anguria, Fragaria chilensis, F. virginiana, and Lycopersicon esculentum. Ovaries excised several days after pollination showed satisfactory growth on a simple medium. However, the size of the fruit development in vitro was smaller than that of field grown fruits. Later on ovaries of tomato, wheat, Pisum and Phlox were grown in culture.
Beasley (1971) observed good growth of fibres of cotton (Gossypium hirsutum) ovules, excised 2 days post-anthesis, and grown on liquid medium. The unfertilized ovules were found to shrivel and turn brown on the basal medium. However, the addition of GA3 or IAA (0.5 to 50 µM) resulted in larger ovules and the production of fibres. Cotton breeders have long been interested in producing hybrids between Gossypium arboreum and Ghirsutum in order to combine several desirable characters.
The crosses between these species are often unsuccessful because of the abortion of hybrid endosperm and embryo at early stages of development. The technique of culturing in vitro offers an alternative to overcome such handicaps in making the crosses successful. Ovules and embryos of the hybrids can be cultured on suitable media to obtain viable plants.
Technique # iii. Nucellus Culture:
The culture of nucellar tissue has also yielded encouraging results and is now being employed in the improvement of Citrus crop. Rangaswamy (1958) evolved a novel technique of studying nucellar polyembryony. He excised the nucellus from pollinated ovules of Citrus japonica (C. microcarpa) and cultured on different media. The micropylar half of the nucellus proliferated on white’s medium containing casein hydrolysate and callus differentiated into pseudo bulbils, which eventually developed into plantlets.
During recent years nucellus culture has been attempted in many vegetable and fruit crops towards clonal propagation of selected materials, e.g., Aegle marmelos, Cucumis melo, Luffa cylindrica, Trichosanthes anguina, Vitis vinifera. Nucellar culture also helps in the elimination of viruses, e.g., in Citrus species.
Technique # iv. Control of Fertilization:
In his attempts to evolve a desirable variety, the plant breeder is often confronted with several barriers.
The common ones are:
(a) Disharmony in the flowering periods of the parents,
(b) Short life span of pollen,
(c) Failure of pollen germination,
(d) Slow growth of pollen tube,
(e) Inhibition or bursting of pollen tube in style, and
(f) Failure of male gametes to cause fertilization.
Using the techniques of tissue culture, embryologists have successfully evolved methods for overcoming these barriers to cross-ability. Test-tube pollination and fertilization, first employed at the University of Delhi, have now been shown to have wider implications. With the success of intra-ovarian pollination by Kanta (1960), investigations were extended to demonstrate the feasibility of the technique in pollinating and fertilizing excised pistils and ovules in vitro.
For successful pollination and fertilization in vitro, the following considerations are important:
(a) Culture of pollen grains and ovules at the right stage of development,
(b) A suitable nutrient medium, and
(c) Appropriate cultural conditions. Success has been achieved in Nicotiana tabacum, where the entire pistil is cultured and stigma pollinated artificially.
Consequent to such test-tube pollination, pollen germination, growth of pollen tubes, entry of tubes into ovules, fertilization, development of embryo and endosperm, viable seed-setting and seed germination take place in vitro. This technique has been used for cross pollination in N. rustica and Petunia violacea. In another method of ovule from un-pollinated ovaries are aseptically excised and cultured on a suitable nutrient medium.
Pollen from freshly dehisced anthers is then dusted the next day on cultured ovules. The pollen grains germinate, and growth of pollen tube and fertilization are brought about followed by normal development of endosperm, embryo and seed. This has already been successfully achieved in Argemone mexicana, Eschscholzia californica and Papaver somniferum (Fig. 27.1).
Technique # v. Endosperm Culture:
Endosperm, usually a triploid tissue in angiosperms, can be suitable explant for raising triploids of cereals. As early as 1949, LaRue reported the proliferation of immature endosperm of maize. In Oryza induction of callus was observed at the cellular stage of endosperm containing starch grains. Endosperm culture to obtain triploid plants has been used in Putranjiva roxburghii, Nigella damascena, Petroselinum crispum, Citrus, apple and Vitis vinifera. The successful culture of endosperm tissue has opened up new avenues for plant breeders who can apply this technique to economically important crop plants, especially where conventional breeding methods prove futile.