Studies of Transgenic Hybrid Poplar 741 Carrying Two Insect-resistant Genes

Partially modified Bt Cry1Ac gene and the arrowhead proteinase inhibitor (API) gene were used to construct a plant transformation vector pBtiA and this construct was transferred into the genome of the hybrid poplar 741 [ Populus alba L.×( P. davidiana Dode+ P. simonii Carr.)× P. tomentosa Carr.] by Agrobacterium _ mediated transformation. Ten kanamycin resistant plants have been regenerated. Upon insect bioassay using Clostera anachoreta (Fabricius), three of the examined plants were demonstrated to be highly resistant to the testing insects. The mortality of insect larvae on one plant was higher than 90% in 6 days after infestation and the growth of the survival larvae were seriously inhibited. Results of PCR and Southern blot analysis indicated that both Bt Cry1Ac gene and API gene were integrated as a single copy into the genomes of these three plants when Cry1Ac gene fragment was used as the probe. Protein dot blot immunoassay and ELISA analysis revealed that at least the Cry1Ac protein was produced in these three transgenic plants and the expression levels were estimated to be approximately 0.015% of the leaf total soluble protein. This is the first report on insect resistant transgenic hybrid poplar 741 that expresses two insecticidal protein genes.

Construction of Plant Expression Vector Carrying Two Insecticidal Genes and Obtain Insect resistant Transgenic Tobacco Plants

A plant expression vector carrying both pea lectin gene and Soybean trypsin inhibitor gene has been constructed and transferred into tobacco via Agrobacterium mediated transformation. Transgenic plants are further confirmed by ELISA, PCR and PCR Southern assays. Results of bioassays show that transgenic plants display notably inhibitory effects to larvae development and survival of Heliothis armigera Hubner.

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.

Advances in Insect-resistant Transgenic Rice and Its Developing Tendency

Focusing on several commonly used insect resistance genes,we reviewed the advances in insect-resistant transgenic rice,and analyzed the problems and developing tendency in transgenic rice research in this paper.

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.