An efficient field screening procedure for identifying transposants for constructing an Ac/Ds-based insertional-mutant library of rice.

An efficient system was developed, and several variables tested, for generating a large-scale insertional-mutagenesis population of rice. The most important feature in this improved Ac/Ds tagging system is that one can conveniently carry out large-scale screening in the field and select transposants at the seedling stage. Rice was transformed with a plasmid that includes a Basta-resistance gene (bar). After the Ds element is excised during transposition, bar becomes adjacent to the ubiquitin promoter, and the rice plant becomes resistant to the herbicide Basta. In principle, one can plant up to one million plants in the field and select those plants that survive after spraying with Basta. To test the utility of this system, 4 Ds starter lines were crossed with 14 different Ac plants, and many transposants were successfully identified after planting 134,285 F2 plants in the field. Over 2,800 of these transposants were randomly chosen for PCR analysis, and the results fully confirmed the reliability of the field screening procedure.

Isolation and physiological characteristics of a premature senescence mutant in rice (Oryza sativa L.).

A rice pse(t) (premature senescence, tentatively) mutant line, was isolated from 4,500 independent T-DNA inserted transgenic lines. The symptoms of premature senescence appeared more severely than those of the control plants (Zhonghua 11, japonica) at the last development stage. To characterize the mutant and provide basic information on the candidate genes by mapping to a physical region of 220-kb, experiments were carried out in two phytotrons under controlled temperature of 24 degrees C and 28 degrees C, respectively. The content of chlorophyll, soluble protein and MDA (malondialdehyde), net photosynthesis, the antioxidant enzyme activities of SOD (superoxide dismuase) (EC 1.15.1.1) and POD (peroxidase) (EC 1.11.1.7) and the peptidase activities of leaves were measured from top to bottom according to the leaf positions at the flowering stage. Compared with the control plant, the mutant showed the following characteristics: (1) Higher net photosynthesis rate (P(n)) appeared in the 1st and 2nd leaves, contents of chlorophyll and soluble protein were also higher in the 1st leaf; (2) The activities of SOD, POD and peptidase were higher according to the leaf position from top to bottom; (3) The symptom of premature senescence was accelerated in the mutant at 28 degrees C treatment. The MDA content and the SOD and POD activities between the 24 degrees C and 28 degrees C treatment mutants were not significantly different. Content of chlorophyll and soluble protein of leaves mutant decreased rapidly at 28 degrees C treatment. The results show that pse(t) is sensitive to high temperature. The probable function of PSE(T) is discussed.

[Comparison between two methods of establishing DH population for mapping lox-3 gene in rice (Oryza sativa L.)].

Crosses were produced by using a storable rice variety (Daw Dam) and 2070, Ming Hui 63, as well as Dulur/Xie Qing Zao B as parents, among these materials there are abundant polymorphisms of DNA molecular markers. Their F1's were applied to generate DH lines by an improved method of direct induction of pollen plant. The method of the direct induction of pollen plant was used as check treatment to evaluate the improved method. The results showed that the improved method increased the re-differentiation percentage of pollen callus, the percentage of regeneration plantlets, and the percentage of green regeneration plantlets and produced more vigorous seedlings. The improved method was a competent method of establishing DH population for mapping lox-3 gene. It was also showed that the efficiency of the new method was different among genotypes and out of three experimental crosses Daw Dam/2070 and Daw Dam/Minghui 63 showed more efficient. More than 60 DH lines have been obtained in this experiment.