Morpho- histological observations on somatic embryogenesis in mature embryo derived callus of Oryza sativa L. cv. Sakha 101

         Cellular totipotency is one of the fundamental principles of plant biotechnology. The mature embryo is increasingly recorded as a valuable explant for somatic embryogenesis in rice biotechnology. In present study, rice cells dedifferentiation, proliferation and re-differentiation were investigated by culturing mature rice (Oryza sativa L. cv. Sakha 101) embryos in modified MS media fortified with different growth regulators alone and in combination. Mature embryo tissues competent for tissue culture and the chronological changes of cells morphology and histology were observed. The results showed that callus was induced only from mature rice seed (explant) cultured on MS media supplemented with         2, 4-D (2 or 2.5 mg/l) alone while the rest treatments showed negative response. Callus was initiated after 5 days of culture in MS media fortified with the lower 2, 4-D dose as clusters of undifferentiated cell masses while callus initiation was delayed 4 days more by increasing the applied dose. At morphological level, Pale yellowish and friable calli was noted in both tested doses. Calli texture exhibited different appearance, while was slightly nodular in the lower dose it was soft in the higher one. High callus induction frequency (70%) was estimated for 2mg/l 2, 4-D application while decreased frequency (40%) was concomitant with dose increment. Histological analysis for somatic embryogenesis revealed that within two weeks of culturing explants on callus induction medium (CIM), somatic embryos development began as clusters of embryonic cells at the peripheral parts of the proliferated calli while non-embryonic cells were observed at the inner regions of the induced callus. Embryogenic cells at the outer cell layer were observed as small and isodiametric with dense cytoplasm and clear nucleus located in the center of the cells, whereas the non embryogenic cells were large, vacuolated and had a very small nucleus located near the cell wall. Embryogenic cells undergo series of ordered divisions and protodermis observed surrounding globular embryo was recorded at the end of culturing in CIM fortified with lower 2, 4-D dose.
On the other hand, culture on MS fortified with higher dose delayed rice cells differentiation and globular stage was recorded two days after subculture embryonic callus into free hormone MS medium. After 2 days of subculture into free hormone MS medium, heart shaped embryo was observed in low dose of 2, 4-D.        
This study assists to draw attention to the use of a histological approach as a helpful tool to follow the chronological series of embryo development in vitro.