A Cytological Observation On The Pollen Abortion In Male Populus Tomentosa And Study On Transformation With Two Genes Of Insect-resistant

It has been a main aspect to cultivate new variety of poplar that is insect-resistant and sureness to enviroment. In order to avoid the pollution that caused by the seed of female poplar and the pollen drifting of male poplar, the research selected new variety of male populus tomentosa as material to be contransformed. On the base of the study of the morphology and histology of pollen abortion , the modified Bacillus thuringiensis (Bt) -endotoxin gene and arrowead proteinase inhibitor (API) gene were contransformed to the male poplar by Agrobacterium tumefaciens. The transgenic plants were selected on the medium containing kan. The resistant plants were tested by molecular biology measures and raising worms experiment. The main results as follow:1) The anther modality and the process of pollen growth of the male poplar were observed for two continual years. The result shows that it is no pollen during the process of anther growth. The reason of pollen abortion probably is tapetum cell delayed disintegration .The sporule can't growth normally. So the male populus tomentosa was ascertained to be male sterility.2) The transformation regeneration system of the male populus tomentosa was established. The bud differentiation media is MS+TDZ0.1mg/L +IAA0.1mg/L. The bud multiplication media is MS+6-BA0.3mg/L +IBA0.1mg/L .The rooting media is 1/2 MS+IBA0.5mg/L.3) By Agrobacterium tumefaciens, 258 leaf-explant were contransformed with two genes of insect-resistantance. The number of differentiation is 81. The differentiation rate is 31.39%. 108 resistant buds were obtained. The transformed buds were selected on the medium containing kan and obtained 21 resistant plants. The PCR analysis and southern hybridization proved that Bt gene and protease inhibitor have already combined in genome DNA of male poplar.4) Feeding Clostera anachoreta(Fabricius) and Lymantria dispar(Linnaeus) with the young tender leaves of transgenic male white poplar. The result showed: Toxicity of transgenic poplar is significantly higher than contrast. According to the total death rate of Clostera anachoreta(Fabricius) , the transformed plants were devided into 4 grades: high-insect resistance, middling insect-resistance, low insect-resistance and none insect-resistance. Four high insect-resistance clones (D-84 D-87, D-94 D-95) and three middling insect-resistance clones (D-74 D-85 D-88 ) were sieved. In addition, the insect's growth and development were tested. The result indicated: the transformed clones inhibited the insects' growth and development significantly. The auxeticduration that eating transformed clones was obviously delayed contrasting with control. The growth rate of larva's weight that fetched the transformed clones was obviously slower than CK. That means Bt gene and Api gene can express effectively in the plant's cell and give off strong toxicity. Meanwhile, it proved these 2 genes have different insecticidal mechanism which conspire to poison function. So the insect-resistance of transgenic male poplar was improved.