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’
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.
1) Biotechnology Department, China National Rice Research Institute, Hangzhou, 311006 China 2) Biology of Department, Hangzhou University, Hangzhou, 310028 China 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
and analysis of transgenic plants. Science in China (Series
C) 39 (6): 652-661.
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