2. Factors controlling callus proliferation and plant regeneration
2.1. Endosperm at culture
Usually culture of endosperm needs the selection of seeds at proper stage of development. This is usually calculated as days after pollination (DAP) and it varies from plant to plant as 9-10 days after pollination (DAP) in Lolium perenne (Norstog 1956), 8-11 DAP in Zea mays (Tamaoki and Ullstrup 1958), 8 DAP in Triticum aestivum and Hordeum vulgare (Sehgal 1974) and 4-7 DAP in Oryza sativa (Nakano et al. 1975). Usually free nuclear endosperm did not produce any callus and the intensity of response depends on the level of organization of endosperm cells.
2.2. Plant growth regulators and other supplements
Selection of a suitable basal medium and the addition of proper growth regulators and other adjuvants are the decisive factors that determine the success of triploid plant development. The culture of immature endosperm requires yeast extract (YE), casein hydrolysate (CH), coconut milk (CM), corn extract (CE), potato extract (PE), grape juice (GJ), cow's milk (CWM) or tomato juice (TJ) despite a suitable medium and growth regulators. Murashige and Skoog (1962) basal medium was mostly used to initiate and improve the response in in vitro endosperm cultures. White (1963) basal medium (WM) was also employed in some cases. La Rue (1949) used various organic supplements like CE, PE, TJ, GJ, YE or CWM to raise endosperm callus cultures. Of these, TJ was found to be superior over other additives due to cytokinin-like activities.
Later it was found that the TJ could be replaced by YE. YE induced callus proliferation was reported in Zea mays, Croton, Jatropha panduraefolia, Lolium, Ricinus communis, Oryza sativa, Coffea arabica, and Juglans regia. Other additives like CH in Zea mays, Exocarpus cupressiformis, Dendrophthoe falcata, Nuytsia floribunda, Putrangiva roxburghii, Hordeum vulgare, Achras sapota, Citrus grandis, Prunus persica, Actinidia chinensis, Actinidia urguta, A. deliciosa and CM in Acacia nilotica, and Codiaeum variegatum were also employed by different workers.
Most of the immature endosperm needs the presence of one or more growth regulators for plant regeneration except in few , where MS basal medium is sufficient for endosperm embryogenesis. In majority of reports, an auxin, preferably 2,4-D is necessary for callus induction from immature endosperms. In case of mature endosperm, the optimum callus growth was observed either on a cytokinin or a cytokinin in combination with an auxin and for autotrophic taxa, cytokinin, auxin, CH or YE is necessary. In most of the cases the time required to initiate proliferation varies from 10 days to 20 days, but pre-soaking of endosperms with GA3 have reduced the time period from 10 days to 7 days.
2.3. Physical factors
This includes effect of temperature, light and pH on endosperm proliferation. Straus and La Rue (1954) observed that corn endosperms develop better in dark than light conditions. But in Ricinus reverse is the case where a continuous light period was found optimum for endosperm proliferation (Srivastava 1971). In some cases, the endosperms were cultured along with the embryo and kept in the diffuse light with 16 h photoperiod. Light conditions facilitate the early germination of embryo and the embryos can be removed easily due to their characteristic green colour (Thomas et. al. 2000). In coffee, the endosperm callus grows better under 12 h light/dark conditions (Keller et al. 1972). In Lolium the light doesn't have any significant role on endosperm proliferation (Norstog 1956).
Not much research has been carried out till date with regard to the effect of temperature and pH on endosperm proliferation. In available literature the optimum temperature for endosperm growth was reported to be 25°C (Johri and Srivastava 1973). The pH varies from 4.0 for Asimina to 5.0 for Ricinus , 5.6 for Jatropa and Putranjiva and 6.1 for Zea mays . In general, 5.5 to 5.8 pH seem to support the best growth of endosperm tissues in cultures.