Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia.

Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.

Protocol for in vitro propagation of Excoecaria agallocha L., a medicinally important mangrove species.

An in vitro propagation protocol has been developed for Excoecaria agallocha L. (Euphorbiaceae), a mangrove species. Nodal segments were used for axillary shoot proliferation. One shoot from each node of binodal explants was observed 3 weeks after inoculation. The best axillary sprouting was seen on a newly formulated medium containing BA, Zeatin and IBA in concentrations of 13.3 µM, 4.65 µM and 1.23 µM, respectively. The new medium, first used in this study, has a specific composition of major nutrients, MS micronutrients and iron compounds. Nodal segments from rooted cuttings and seedlings responded better than those of mature tree explants. Multiple shoot induction was complemented with efficient shoot elongation, and repeated subculture of binodal segments from axillary shoots resulted in 10-12 shoots per explant in 3 months. Rooting was achieved by growing shoots in the new medium with 0.23 µM IBA. Regenerated plants were successfully acclimatized to the natural environment, and about 85% of plantlets survived under ex vitro conditions. This is the first report of micropropagation in the genus Excoecaria and also in mangrove tree species.