50. Transgene integration in rice transformants derived from independent transformation events via particle bombardment
      X.L. WANG’, SM. ZHOU2, D.N. HUANG’, R. XuE’, Z.H. HUA’ and Z.Y. GAo’
      1) Biotechnology Department, China National Rice Research Institute, Hangzhou, 311006 China
      2) Biology of Department, Hangzhou University, Hangzhou, 310028 China
 
     Transformation via particle bombardment, which introduces foreign genes into rice varieties is an efficient approach for rice genetic improvement. Recently, a number of useful genes have been introduced into rice via this method. However, the copy number and insertion sites of the transgene in the chromosome complement are random. The transgene expression is related to site of insertion and the copy number which are different in each transformation event. Hence, it is necessary to analyze transgene integration in different transformants, then asses the fate and function of transgenes in host and its progeny.
     Using callus derived from immature embryos and suspension culture system as target tissue, plasmid pCB 1 (Fig. 1) containing cecropin B gene (that is an anti-bacterial polypeptide of insects) and bar gene (that confers resistance to the herbicide, bialaphos) was introduced into rice varieties Jia 59, Chun Jiangzhao 4, and Bing 93-63 via particle bombardment using procedures described earlier (Huang et a!. 1996). Fourteen transgenic plants were regenerated from Jia59, twenty from chunJiangzhao 4 and twenty-four from Bing 93-63.
     Eight transgenic plants (RO) of Bing 93-63 derived from five independent transformation events were analyzed by southern blot to determine the integration pattern of target gene (cecropin B gene) and selectable gene (bar gene) (Fig. 2).
     The DNA samples were subjected to southern blot analysis using cecropin B or bar gene as probes. Undigested DNA of all 8 transgenic plants showed a smear of hybridization signal in the high molecular weight region, indicating multiple, full length and rearranged fragments and three distinct hybridization patterns were identified.
     Two things were evident from Fig. 1. First, target gene was similar to selected gene in the integration pattern. The complexity of the southern patterns was comparable from one gene probe to another, suggesting that transformation plasmid generally integrated as a complete unit. Second, transformation events were different: (i) in different independent transformation events, there were five or more insertion sites of cecropin B gene and seven or more sites for Bar gene in Bing 2, Bing 4 and Bing 5, while up to seven and nine or more in Bing 9, (ii) different transformants derived from same transformation events had different southern patterns. The insertion sites in Bing 1 (lane-2) were up to five, while that in Bing 1 (lane 3) only one.
     These findings showed that transgene integration was complicated and diverse. We are in process of investigating transgene expression in their progeny to understand the relationship between gene integration and gene expression.


 


 
 

Reference

Huang, 
D.N. et a!., 1996. Introduction of cecropin B gene into rice (Oryza sativa L.) by particle bombardment 
and analysis of transgenic plants. Science in China (Series C) 39 (6): 652-661.