Genotypic Variation And Associated Mechanism For Regeneration Frequency During Tissue Culture In Rice And Sorghum

Rice is one of the most important crops in the world, providing the staple food for almost half of the world's population. The first regenerated rice from tissue culture was reported in 1968, laying a fundamental base for the development of transformation. Although a highly efficient Agrobacterium-mediated transformation system has been established and greatly facilitated the wide spread application of transformation, it happened frequently only in a few rice varieties such as Nipponbare (Japonica) and Kasalath (Indica), but not in many elite varieties used for rice production, such as 93-11(Indica). Low regeneration ability from mature seed culture system observed in these varieties has becoming a serious obstacle to efficient production of transgenic plants. In order to find possible mechanism caused for different regeneration frequencies between japonica variety Nipponbare and indica variety 93-11, phenotypic observations and biochemical analyses were conducted by comparing the differences of physiological metabolism in callus, which obtained at different stages of callus induction and differentiation. The results obtained from this experiment were summarized as follows.1) Frequency of callus induced from mature embryo differed greatly between two genotypes. Average reduced rate of Nipponbare is 78.48%, nearly 2.5 times than that of 93-11. It was found that much more and heavier callus are induced from Nipponbare than that from 93-11 after 2 weeks of induction.2) Growth rates of callus during the subculture period of two weeks are different in two genotypes. Growth rate of callus, the ratio of two weights of the callus induced after two weeks'subculture and the callus started for subculture, is 5.98 for 93-11, whereas 8.98 for Nipponbare. 3) Total nitrogen, ammoniacal nitrogen and nitrate nitrogen in callus induced were different between the two genotypes. In different culture days of callus induction and differentiation, total nitrogen and ammoniacal nitrogen in Nipponbare callus is higher than that of 93-11. Nitrate nitrogen in Nipponbare is also higher than that in 93-11 before 25 days of callus differentiation.4) At the level of cells metabolism, soluble protein in Nipponbare is higher than that in 93-11 from callus induction to 15 days after differentiation.Sorghum [Sorghum bicolor (L.)], one of the most important cereal crops, is popular as a grain and forage crop in the semi-arid regions of Asia and Africa. Compared with rice and maize, progress of sorghum transformation is slow because of the low frequency of regeneration from tissue culture. In order to find possible causes for genotypic variation of regeneration, phenotypic observation and biochemical analyses were conducted by comparing the differences of callus obtained at different stages of callus induction and differentiation from immature embryo of grain sorghum variety 654 and sweet sorghum variety LTR108. Agrobacterium-mediated transformation of sorghum is also attempted. The results from this study are summarized as follows.1) Frequencies of callus induced from immature embryo are very different between two genotypes. Average induced rate of grain sorghum immature embryo is 78.7%, nearly 2.5 times than that of sweet sorghum; and the highest reduced rate is 82.62%, more than 3 times than the lowest reduced rate of sweet sorghum.2) Different treatments were conducted to optimize the tissue culture condition. Cold treatment at 4℃for 24 hours on immature seeds in addition of PVP and vitamin C were significantly reduced the production of black pigments in grain sorghum 654, whereas, no obvious effects were found in sweet sorghum LTR108.3) High differentiation was occurred in grain sorghum 654,5-6 sprouts could be differentiated from one callus in some cases. However, the differentiation rate is very low in the sweet sorghum LTR108 and much more black pigments were produced in differentiation medium.4) Agrobacterium-mediated transformation of sorghum was attempted but failed to get transgenic plants.