The Cre/ lox site-specific recombination controls the excision of a target DNA segment by recombination between two loxsites flanking it, mediated by the Cre recombinase. We have studied the functional expression of the Cre/ lox system to excise a transgene from the rice genome. We developed transgenic plants carrying the target gene, hygromycin phosphotransferase ( hpt) flanked by two lox sites and transgenic plants harboring the Cre gene. Each lox plant was crossed with each Cre plant reciprocally. In the Cre /lox hybrid plants, the Cre recombinase mediates recombination between two lox sites, resulting in excision of the hpt gene. The recombination event could be detected because it places the CaMV 35S promoter of the hpt gene adjacent to a promoterless gusA gene; as a result the gusA gene is activated and its expression could be visualized. In 73 Cre /lox hybrid plants from various crosses of T0 transgenic plants, 19 expressed GUS, and in 132 Cre /lox hybrid plants from crosses of T2 transgenic plants, 77 showed GUS expression. Molecular data proved the excision event occurred in all the GUS(+) plants. Recombination occurred with high efficiency at the early germinal stage, or randomly during somatic development stages.

Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.

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Sixty one double haploid (DH) lines were evaluated for yield components and yield and compared with the three corresponding hybrids from which they were derived through anther culture. Analysis of a 6×6 diallel experiment led to the selection of these hybrids based on their high heterosis and revealed the nature of gene action of the characters under evaluation. The DH lines along with the hybrids and parents were planted following a simple lattice design with two replications. The results show that in DH lines the values of the characters expressing predominantly additive genetic effects could reach the heterotic level of the hybrids. For characters including yield showing predominantly dominance effects, values in the DH lines were significantly lower than those of the corresponding hybrids. The promising DH lines, however, possess a higher yield potential than the better parents.