Preliminary Evaluation On Transformation Efficiency Of Improved Lines From Kasalath In Rice

Rice production is one of the most important economic activities. China is the largest consumer and producer of rice in the world. With increasing of biotic and abiotic stress, rice production is facing tremendous pressure. Breeding of new varieties by genetic engineering is a suitable way to increase the unit yield and total yield of rice in the world. Since most of the indica rice varieties have poor characteristic of tissue culture and transformation, the application of transgenic technology is severely restricted in indica rice. The indica rice varieties with high transformation efficiency and elite agronomic traits are urgently needed. This study evaluates the transformation efficiency of improved lines from indica rice Kasalath via Agrobacterium mediated transformation method using the Bar gene as a selectable marker, hopes for selecting lines with high transformation efficiency, as well as obtaining elite herbicide resistance transformants. The results are as follows:(1) The derived lines from HD9802S/Kasalath,9K08-3,9K18-7,9K21-3,9K28-5, 9K29-9 and 9K30-0, were transformed by agrobacterium mediated method, followed by parent Kasalath and HD9802S. Eighteen regenerat plantlets were obtained, and sixteen approved to be true transformants by PCR, including nine from Kasalath, two from 9K18-7, one from 9K28-5, three from 9K29-9, and one from 9K30-0.(2) The integration and expression of exogenous gene in the positive regenerated plantlets were verified. Southern blotting of sixteen PCR positive regenerated plantlets showed that the foreign gene Bar was integrated into rice genome. Fifteen independent transformants were obtained, in which eight plantlets were of single copy and eight plantlets were of multiple copies (two plantlets shared a same transformation event). The stably inheritance and expression of Bar gene in the transformants were verified by herbicide resistance test, PCR detection and analysis of PAT protein in two consecutive generations.(3) The relative contents of PAT protein in T1 and T2 generation plants were detected by ELISA. The relative contents of PAT protein in eight transformants, i.e. BK-1, BK-3, BK-5, BK-8, BK-9, B9K-1, B9K-6 and B9K-8, are higher than those of others. The relative contents of PAT protein of them are as high as more than 25 ?g/g.(4) The transformation efficiencies of several improved lines from Kasalath (including 9K08-3,9K18-7,9K21-3,9K28-5,9K29-9, and 9K30-0) were preliminary evaluated by using parent Kasalath and HD9802S as check and herbicide resistant gene Bar as selective marker. Fifteen independent transformants were obtained in this study, and the transformation efficiencies of line 9K08-3,9K18-7,9K21-3,9K28-5,9K29-9, and 9K30-0 were 0,0.65%,0,0.23%,0.40% and 0.22%, respectively. It primary indicates that the correlation between the transformation efficiency and the tissue culture characteristics of improved lines from Kasalath was not significant and line 9K18-7 has higher transformation efficiency and better tissue culture characteristics comparatively.(5) The single copy transformants, BK-1, B9K-1, B9K-6 and B9K-8, were selected by preferable agronomic characters. Then the insertion sites of exogenous gene in these transformants were identified.