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Study On The Transfer Of Insect-resistant Genes To Chinese Cabbage Via Vacuum Infiltration And On Its Mechanism

Posted on:2005-12-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:H J XuFull Text:PDF
GTID:1103360125460463Subject:Vegetable science
Abstract/Summary:PDF Full Text Request
Brassica consists of more than 100 species, many of which are important crops, such asoil crops, vegetables and grazing. In the last decades, the great progress has been made on thebreeding of Brassica crops, especially on the increase of output, improvement of quality andinnovation of the germplasm resources. It did contribute more to the plant science andagriculture. But the conventional methods of plant breeding were confronted with manydifficulties, which restricted the further development in genes utilization. The lack of somegenes within a species and the difficulties in wide cross are common restrictive factors. Therising of the biotechnology has made a new prospect. The gene resources could be collocatedreasonably by the gene engineering without the limitation of species. It has succeeded inimproving germplasm via gene engineering To date, one major gene engineering method used on the Brassica plants, is theAgrobacterium Tumefaciens-mediated transformation method. This protocol is based on theregeneration of the explants in vitro. This method has made a slow progress on the Brassicaplants, and a great success on the other plants. due to the difficulties of regeneration ofexplants in vitro,there are also many problems, such as abnormality and developmentalobstacles derived from somaclonal variation, reduced fertility, and genotype-dependence. The 4山东农业大学博士学位论文regeneration was further more difficult in the Brassica campestris plants. The regenerationratio was poor and not stable, and more dependent on the gene type. The explants of theBrassica campestris were too sensitive to the A. tumefaciens. Although some laboratoriesissued their successful results, routine transformational method was not established. Using anew method without regeneration in vitro can avoid these problems, and would promote thegenetic modification of Brassica campestris plants. The In Planta transformation successfullyused on Arabidopsis Thaliana gave a good alternative. In Planta transformations consist ofseed co-incubation, floral dip, wound inoculation, pollen-tube pathway, plant male germ linetransformation and vacuum infiltration transformation. These methods have contributedmore to the study on Arabidopsis Thaliana. But it was lack of important progress on the othermajor crops. This paper reports the study on Chinese cabbage via vacuum infiltrationtransformation.1. Obtaining transgenic Pakchoi plants with anti-pest genes. The Pakchoi variation 49caixinand Agrobacterium C58 harbouring plasmids pBBB-pinⅡ-sip or pBBB-sip were used in thetests. The plasmid contains potato trypsin inhibitorⅡ(pinⅡ) and spider insecticidal peptide(sip) which are poisonous to insects, and bar gene which mediate resistant to the herbicidePPT. Transgenic plants were obtained by the selection of basta. The molecular assays of PCRand Southern hybridization confirmed the integration of the foreign genes. The detection ofPINⅡ protein indicated the successful expression of the pinⅡgene. The soluble proteinextraction from the transgenic plant made significant inhibition to the activity of the bovinetrypsin by the statistic analysis. The test of feeding cole moth (Plutella xylostella L.) with theleaves of the transgenic plant resulted in good effects, and the statistic analysis gave asignificant difference between transgenic plants and non-transgenic plants. The leaves fromtransgenic plants impaired the growth of cole moth on the pupal time, the weight of pupae, theratio of pupation, and ratio of eclosion. The different transgenic lines with same copies offoreign genes gave different effects on the insect. The transgenic plants with goodcomprehensive performance were selected out.2. The inheritance and expression of the foreign genes. The results indicated a low transformational ratio in Pakchoi, which was stable on 0.01%.The most T2 plants gave 3:1 segregation. There were also 15:1, 63:1, 2:1, 1:1, and...
Keywords/Search Tags:Chinese cabbage, the vacuum infiltration transformation, pinⅡ gene, sip gene, mechanism, Plutella xylostella L.
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