Selection of Homozygous Cotton Lines Transformed with Two Insect-Resistant Genes

A plant expression vector containing a chimeric Bt29K gene coding for the activated Cry1Ac protein and the arrowhead proteinase inhibitior gene API B were introduced into the cotton cultivar Jihe321 mediated by Agrobactertium tumefaciens. Based on the results of kanamycin resistant testing, PCR detection for both foreign genes and insect bioassay using Heliethis armigera , nine transgenic homozygous cotton lines with insect resistance of more than 90% and better agronomic traits were bred through six generations from the original transgenic plants. Results from insect bioassay and sequence analysis of the PCR products of plants from some homozygous lines indicated that the chimeric Bt29K gene was stably inherited in these transgenic cotton lines. The main agronomic characters of these homozygous cotton lines, such as boll productivity and fibre strength, were better than that of the original cotton cv. Jihe321.

Development of lepidopteran pest-resistant transgenic japonica rice harboring a synthetic cry2A* gene

A synthetic cry2A^＊ gene enco ding Bacillus thuringiensis（Bt） δ-endotoxi n that resi st ance to lepidopteran pest was transformed into japonica rice variety Jijing 88, which is the most widely cultivated variety in Jilin Province, Northeast China, by Agrobacterium-mediated transformation. A total of 106 independent transformants overexpressing cry2A^＊ gene driven by ubiquitin（Ubi） promoter was produced. Three single-copy homozygous transgenic lines were finally selected based on the results of PCR analysis, se gregation ratio of Bast a resistance, and Southern hybridiza tion analyse s. RT-PCR and enzyme linke dimmune sorbent assay（ELISA） revealed that cry2A^＊ transcripts and protein were highly expressed in these lines. The high level of Cry2A^＊ protein expression resulted in high resistance to rice striped stem borer as evidence d by insect feeding bioassays. Our results demonst rate that cry2A^＊ transgenic japonica rice confers resistance to the rice striped stem borer in the laboratory conditions.

Developing transgenic maize(Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

Application of the New Gene Gm6 Against Rice GallMidge in Resistance Breeding Through PCR-BasedMarker Aided Selection

The research results of marker aided selection(MAS)for resistant varieties and lines against rice gall midge Orseolia oryzae Wood-Mason successfully in 1999 - 2002 were reported in the present paper. The molecular markers linked to the gene Gm6 against rice gall midge were used to select and breed the resistant varieties and lines. The RAPD marker OPM06 was used to verify the existence actually of gene Gm6 in ten developed varieties resistant to gall midge such as Duokang1, Duokang2, Kangwen2, Kangwen3, Kang-wen5, Duokangzaozhan, Kangwenqinzhan, which were derived from Daqiuqi. For resistance breeding through PCRbased marker aided selection(MAS), the polymorphisms in the resistant and susceptible parents were i-dentified by RG476/Alu I and RG476/Sca I respectively. The RAPD marker OPM06(1.4 kb)was used to i-dentify 15 new resistance lines from F3 lines of Fengyinzhan1/Daqiuqi in 1999. 21 and 7 resistance lines were selected from F4 and F6 lines of KWQZ/Gui99(restored line of hybrid rice)using RG476/Alu I in 2000-2001 respectively. The Gm6 gene was transferred into the restored line of hybrid rice. In 2001 - 2002, RG214/ Hha I and G214/Sca I were used for selecting 11 and 5 resistance lines from F3 lines of KWQZ/IR56 and AXZ/KWQZ successfully. The application of the resistance gene through PCR-based marker aided selection is a new and effective approach in resistance breeding.

Efficient Regeneration and Agrobacterium-mediated Transformation of Brassica napus Cultivar Qingza No.5

Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB ＋ 5 mg/L thidiazuron （TDZ） ＋7.5 mg/L AgNO3 ＋ 0.1 mg/L NAA ＋ 2 mg/L proline （L-pro） ＋ 250 mg/L casein acid hydrolysate （CH） ＋ 3% sucrose; the appropriate growth medium was 1/2 MSB ＋ 1 mg/L IBA ＋ 2 mg/L L-pro ＋ 250 mg/L CH ＋ 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB ＋ 0.2 mg/L IAA ＋ 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.