Introduction Of Transgenic Plants Via Non-Tissue-Culture Methods

Agrobacterium-mediated method and particle bombardment method are the most commonly methods in the research of transgenic crops and they have achieved remarkable achievement. But these approaches rely on tissue culture which complex operation and expensive instruments are needed. Still, it is not convenient to integrate these methods into conventional breeding programs, therefore, the research on non-tissue-culture transformation method has important significance.In this study the antiportor protein gene OsSIK1 was introduced into three maize inbreds via pollen-mediated transformation method. At the same time we also studied the variation of transformation efficiency among various inbreds. Besides, in this research the vector p3301ubiAc was introduced into soybean via co-cultivation of germinating seeds with Agrobacterium tumefaciens, In order to establish an effective soybean transformation system, we also studied factors affecting the transformation efficiency of germinating seed approach in soybean. The results were as follows.1. Introduction of OsSIK1 gene into maize via pollen-mediated method:(1) OsSIK1 gene was introduced into maize inbreds, Zheng58,98-2-19 and Chang7-2 via pollen mediation, by which To seeds were obtained in the same season. Seed setting were 272 (setting rate of 63.4%) for Zheng58,27 seeds (14.1%) for 98-2-19 and 167 for Chang7-2 (31.2%).(2) T1 generation plants were detected by Kanamycin screening and PCR amplification. The results indicated that the target gene had been introduced into the maize genome. we totally obtained 59 transgenic lines, including 35 lines (transformation efficiency 28.5%) for Zheng58,4 lines (20.0%) for 98-2-19, and 20 lines (37.1%) for Chang7-2. (3) The inheritance of exotic gene among the 3 maize inbreds in T2 generation were:Zheng58 34.3%,98-2-19 12.5% and Chang7-2 55.0%. By comparing the seed setting rate, transformation efficiency and inheritance of exotic gene among the 3 maize inbreds, it is suggested that the inbreds Chang 7-2 and Zheng 58 were more suitable for pollen-mediated transformation.2. Introduction of cry1Ac gene into soybean plants via co-cultivation of germinating seeds with Agrobacterium tumefaciens:(1) The vector with the bar gene and an insect-resistant gene, cry 1 Ac was introduced into soybean. The germinating seeds were taken as the receptor of exogenous gene. T1 plants were detected by screening for phosphinothricin resistance and PCR amplification. The results indicated that the average transformation efficiency was 6.9%.(2) We established a soybean transformation system, including the concentration of Agrobactium OD6oo were 0.5 to 0.6, concentration of acetosyringone was 100μmol/L and power of ultrasonication was 800W.(3) Concentration of 1.5mg/L phosphinothricin was used to selected T1 plants, which could avoid many following work.