Rice Transformation as a Means to Study Gene Expression

Abstract

An exceptionally effective transformation procedure has been established by using class I embryo-derived rice callus. Every treated callus clump yielded multiple independently transformed plants (average 40 plantlets). Analysis of genomic DNA blots and pollen expressing green fluorescent protein (GFP) from T0 plants revealed that 64% bore a single locus T-DNA insertion in which half had one T-DNA copy. Additive transgene expression was observed fromT0 plants with GFP driven by mUbi1 promoter. Transgenic plants could be rapidly characterized by analyzing GFP pollen from T0 plants without the need for further generations or genomic DNA blot analysis. Agrobacterium tumefaciens-mediated transformation of microspore-derived callus for generating large numbers of T-DNA haploid and doubled haploid(DH) plants has also been investigated. The established transformation procedure resulted in 100% transformation frequency for class I microspore-derived rice callus. Each callus typically yields multiple independent transgenic plants. Genomic DNA blot analysis suggested 98% of the transgenic plants are independent events. About half of the transgenic plants were identified as haploid plants, whereas half are DH hemizygous or homozygous transgenic plants. DH homozygous transgenic plants were obtained from T0plants and confirmed by pollen GFP expression and genomic blot analysis in T0transgenic DH plants. In this study, about 60% ofT0transgenic DH plants had a single locus T-DNA insertion of which 45% bore one T-DNA copy. Furthermore, in a population of over 2,000 haploid and doubled haploid T-DNA plants , about 25% showed phenotypic differences from non-transformed haploid plants. Approximately 5% were seriously phenotypically abnormal including lethal or semi-lethal mutants. This highly efficient transformation procedure using microspore-derived callus could be valuable in speeding up plant breeding and in new gene discovery. Diversification of the mUbi1 promoter led to a minimal promoter that has a similar function as the original mUbi1. Transient and stable transformation as measured from gene expression driven by the minimal promoter suggested that it has a similar function as the original wild type promoter.