2.4. The embryo factor
There is an absolute necessity of the so called “embryo factor” for the proliferation of endosperm (Bhojwani 1968; Srivastava 1971a, b). Some factors contributed by the germinating embryo is required for the stimulation of mature and dried endosperms of few plant species. In general, it has been found that mature endosperm requires the initial association of embryo to form callus but immature endosperm proliferates independent of the embryo. However, in neem the association of the embryo proved essential to induce callusing of immature endosperm; the best explant was immature seeds (Chaturvedi et al. 2003). Similar observation for mulberry was reported by Thomas et al. (2000). However, the embryo factor can be overcome by the use of GA3 as was observed in Croton bonplandianum and Putranjiva roxburghii (Srivastava 1973). It is reported that during germination, the embryo releases certain gibberellin like substances, which may promote the endosperm proliferation (Ogawa 1964; Ingale and Hageman 1965). However the mature endosperms of Achras sapota, Santalum album, Emblica officinalis and Juglans regia (Cheema and Mehra 1982; Tulecke et. al. 1988) could proliferate without the association of embryo or pre-soaking of them in GA3.
3. Shoot regeneration
Organogenesis from endosperm tissue was first reported in Exocarpus cupressiformis (a member of the family Santalaceae) by Johri and Bhojwani (1965). The pathway of plant regeneration includes shoot-bud differentiation or embryogenesis directly from the explants or indirectly from proliferating callus (Figure 10.1 A-C). In almost all the parasitic angiosperms, the endosperm shows a tendency of direct differentiation of organs without prior callusing, whereas in the autotrophic taxa the endosperm usually forms callus tissue followed by the differentiation of shoot buds, roots or embryos. Direct shoot regeneration from the cultured endosperm was observed in a number of semiparasitic angiosperms including Exocarpus, Taxillus, Leptomeria, Scurrula and Dendrophthoe .
Figure 10.1: Shoot regeneration from endosperm callus of Azadirachta indica : A. An immature seed in culture has split open after 2 weeks and releasing the green embryo and callused endosperm , B. White fluffy endosperm callus can be seen from the fully opened seed after three weeks. Embryo is lying at one end of the explant, C. 6 -week-old subculture of endosperm callus showing the differentiation of distinct shoots and nodules as well.