Investigations of copy number of transgene, fertility and expression level of an introduced GUS gene in transgenic NERICA produced by Agrobacterium-mediated methods

In the use of genetic transformation in breeding, there are several possible problems including multiple copy insertion of transgene, sterility caused by somaclonal variation and gene silencing. In this study, we characterized transgenic New Rice for Africa (NERICA) produced by Agrobacterium-mediated methods with respect to copy number of transgene, fertility, and expression level of an introduced GUS gene. Southern blot analysis of primary transformants demonstrated that about half of the events carried a single copy of the transgene regardless of the cell density of Agrobacerium for inoculation. We examined ten procedures, consisting of different time periods and times of subculture for callus formation and the starting times of hygromycin-based selection of transformed cells, for transformation of NERICA cultivars to produce transformants within a short culture period at high frequency. A new culture method developed in this study required only about 1.5 mo from the beginning of tissue culture to transformants, whereas a standard protocol we developed previously needed about 2 mo of culture; however, it did not significantly reduce percentages of sterile plants. Fertile T-0 plants produced fertile T-1 plants at higher frequency. However, fertility was not inherited in a simple fashion: both fertile and partially sterile T-0 plants produced fertile, partially sterile and sterile T-1 plants. Expression assay of an introduced GUS gene revealed position effects in seven independent homozygous transformed lines carrying one copy of the transgene. Points to pay attention to in the use of genetic transformation in breeding are discussed.