Characterization and manipulation of embryogenic response from in-vitro-cultured immature inflorescences of rice (Oryza sativa L.)

Characterization and optimization of the embryogenic response from in-vitro-cultured immature inflorescences of rice (Oryza sativa L. sub-species indica and japonica) are described. Histological and morphological analyses revealed that the parenchymatous ground tissue present in the region between the second whorl of sterile bracts and the base of the fertile bracts, the embryogenically competent region (ECR), was involved in the embryogenic response. Initial cell divisions within the ECR occurred in the vicinity of the pro-vascular regions of the spikelet. Continued cell divisions resulted in groups of proliferating units and each single proliferating unit was the product of a coordinated behavior of neighboring cells functioning as a morphogenic group. Further proliferation of this embryogenic tissue was due to the development of cambium-like tissue(s) often forming an embryogenic stratum which under optimal culture conditions produced plants at a high frequency. The morphogenic pathways governing plant regeneration from spikelets of the immature rice inflorescence were dependent upon the growth-regulator composition of the culture medium. Three different modes of plant regeneration were observed: (i) direct plant regeneration, (ii) plant regeneration with an intervening callus phase (prolific non-embryogenic growth associated with unorganized, loose and mucilaginous tissue), and (iii) plant regeneration without an intervening callus phase (compact embryogenie tissue with highly organized growth). The efficiency of plant regeneration, via somatic embryogenesis without an intervening callus phase, was increased by optimizing the culture conditions. In a two-step procedure, immature inflorescences of rice were first cultured on a conditioning medium supplemented with 2.0 mg · 1^–1 2,4-dichlorophenoxyacetic acid + 1.5 mg · 1^–1 kinetin + 0.75 mg · 1^–1 -naphthaleneacetic acid for a period of two weeks. The conditioning medium, with the appropriate culture conditions, allowed redirection of partially differentiated cells of the ECR into embryogenically competent pro-embryogenic groups. Maturation of these pro-embryogenic groups was achieved by transferring them to an embryo proliferation medium, and plants could then be regenerated at a high frequency upon their transfer to the regeneration medium.Abbreviations CM conditioning medium - 2,4-D 2,4-dichlorophenoxyacetic acid - ECR embryogenically competent region - NAA -naphthaleneacetic acid - SEM scanning electron micrograph Thanks are extended to Stephanie Lara, Barbara Bricks, Kay Robbinson-Beers, James Haudenshield, Dave Bayer and Gene Nester, for their invaluable help during the course of this research. The research was partly supported by the Rockefeller Foundation through the Rice Biotechnology program as a grant to W.J.L. and a Postdoctoral Fellowship to J.R.R.