The phenomenon of somatic embryogenesis in maize has been studied by Green (Proc. Fifth Int. Cong. Plant Tissue Cell Culture, Japan, pp. 107-108, 1982) and Lu et al. (Theor. Appl. Genet. 62:109-112, 1982) using immature embryos as the explant. We have reported callus induction and plantlet regeneration from immature glume explants (MGNL 57:51-52, 1983). These friable cultures, upon closer examination, revealed the presence of somatic embryos. The present study deals with the characterization of glume cultures exhibiting somatic embryogenesis. Glumes of sweet corn and seed corn were cultured on MS medium with 2 mg/l 2,4-D. After callus induction, callus was transferred and maintained on MS medium with 1.0 mg/l 2,4-D for subsequent studies.
Factors affecting somatic embryogenesis (such as sucrose, auxins, 2,4-D analogues and nitrates in MS medium) were tested. In order to find the optimal sucrose concentration for callus and embryoid production, five concentrations of sucrose (2, 3, 6, 9 and 12%) were used; 2-3% sucrose was found to be optimal, whereas 6 and 9% decreased the embryogenic potential and higher concentrations (9 and 12%) inhibited callus growth. Among the auxins, 2,4-D was tested in concentrations ranging from 0.5-4.0 mg/l, of which 2 mg/I was effective. Of the two analogues of 2,4-D used, 2,4,5 trichlorophenoxy propionic acid and 2,4,5 trichloro phenoxy acetic acid, 2,4-D was superior both in callus induction and embryogenesis. Effect of nitrates was also tested; increased levels of NH4NO3 (25-75%), compared to the normal levels in MS medium, yielded a high frequency of embryogenic calli and embryoids.
Anatomical observations of isolated embryoids revealed typical scutellum, shoot meristem and coleoptile structures, similar to zygotic embryos. Different developmental stages, from the early globular stage to clearly differentiated somatic embryos, were observed.
Preliminary studies on certain isozymes, peroxidase, esterase, malate dehydrogenase (MDH), polyphenol oxidase (PPO) and IAA oxidase, indicate clear differences in their activity and/or pattern between embryogenic and non-embryogenic calli. Quantitative studies of peroxidase, PPO and IAA oxidase exhibited more activity in embryogenic calli than in non-embryogenic calli under similar nutrient and hormonal conditions. Qualitative studies of peroxidase, esterase and MDH isozymes exhibited specific banding patterns. Embryogenic calli showed two additional slow migrating peroxidase isozymes at Rf 0.11 and 0.14, which were absent in non-embryogenic calli. Esterase isozymes showed two specific isozymes at Rf 0.51 and 0.96 in embryogenic calli and at Rf 0.34 and 0.43 in non-embryogenic calli, while MDH isozymes at Rf 0.68 were present only in non-embyogenic calli.
K.V. Rao, P. Suprasanna and G.M. Reddy
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