Development Of Transgenic Bt+Sck Insect-resistant Cotton And Its Genetic Stability

Since 1996, the application and dissemination of transgenic cotton in USA, Australiaand China have brought about great economic profits to the cotton production and reducedenvironmental pollution from synthetic insecticides. Whereas, along with thecommercialization on a large scale in the world of transgenic Bt cotton, the insect resistanceto Bt cotton has become a serious problem in cotton genetic engineering at present.Moreover, the phenomena that 'the efficiency of Bt transgenic cotton in the early stages washigh, and then it would decrease thereafter' is prevalence in the current commercializedtransgenic insect resistant cotton, which has a direct effect on cotton yield. At present,scientists are engaged in engineering plants with two or more resistant genes in order todelay and prevent adaptation in pest species, prolong the usage of life span of transgenicplants and broaden resistant spectrum. The transgenic cotton harboring double genes(Bt+CpTI), SGK321, was developed and released commercially in China in 1999, whichcould greatly delay the resistance development than that of transgenic Bt cotton. In thisstudy, the Bt and Sck genes were transformed into the cotton genome (Sumian16) bypollen-tube pathway mediated transformation, which would broaden resistant spectrum,and be valuable to develop new transgenic cotton varieties with high efficiency againstcotton bollworm. Transformation with the pollen-tube pathway was first reported by Zhouet al. (1983). Up to the present, lots of Bt transgenic cotton lines were developed by thepollen-tube pathway-mediated transformation, in which some cultivars or hybrids werealready released commercially. However the study on the mechanism of this transformationis relatively few, thus the research on the integration pattern and copy number of transgeniccotton developed by pollen-tube pathway-mediated transformation is necessary, which isimportant to further use the transgenic cotton. TAIL-PCR, Thermal Asymmetric InterlacePCR, was originated by Liu et al in 1995, and was applied to amplify the flankingsequences of T-DNA insertion in Arabidopsis thaliana plants. In this study TAIL-PCR wasused for obtain the flanking sequence of T-DNA insertion in transgenic cotton withBt+Sck, which developed by pollen-tube pathway-mediated transformation, the results wasa good help to study on the mechanism of this transformation method. The Bt and Sck resistance genes were introduced to elite cotton (Gossypium hirsutum L.)cultivar Sumian 16 by pollen-tube pathway-mediated transformation and five fertiletransgenic Bt+Sck plants were confirmed by molecular biology techniques and insectbioassays in present paper. Laboratory bioassays showed that the mortality of bollwormlarvae (Helicoverpa armigera) accounted for more than 90ï¼…during peak flowing and bolldeveloping stages after 5d fed with new expanded leaves on main stem nodes of the fivetransgenic plants 301-5, 305-5, 312-5, 314-4 and 332-2, which are high toxic to neonatelarvae. The resistance of these five transgenic cotton plants to bollworm was controlled byone pair of dominant genes and inherited in a classical Mendelian manner in firstgeneration progeny plants from the self-fertilized transformed parent tested by Kanamycinresistance as an indirect identification marker and bollworm bioassays, and the conclusionwas further confirmed by using F₂ population crossed two homologous transgenic lines(312-5T₂ and 332-2T₂) with original parent Sumian16, respectively, tested by the samemethods. Additionally, the resistant and susceptible plants fit 15:1 segregation ratiorespectively for (312-5T₂×SGK321) F₂ and (332-2T₂×SGK321) F₂. Independentinheritance or different chromosome insertion of Bt genes for the two transgenichomologous lines and SGK321 strains was concluded. Segregation of resistant andsusceptible plants fits 15:1 ratio in F₂ populations crossed among 312-5T₂ and R19, too,indicating independent inheritance or different chromosome insertion of Bt genes for312-5T₂ and R19. In the population of (332-2T₂×R19) F₂, all the plants had highefficiency to bollworm, implying linkage inheritance or same chromosome insertion of Btgenes for 332-2T₂ and R19. The high level of resistance to bollworm was the same as thatof their parents in F₁ hybrids crossed among all those insect-resistant transgenic cottonmentioned above and there doesn't happen co-suppression between Bt genes. The Nptâ…¡, Btand Sck genes were always co-inherited, exhibited transgene activities and stablytransmitted over successive generations monitored by PCR analysis, resistanceidentification of Kanamycin and insect bioassays. The leaf bioassays showed nosignificantly difference in the progenies of transformed plants till T₄, indicating theresistance to bollworm was stably inherited over successive generations.All the Bt+Sck transgenic lines showed high and stable resistance to bollworm in everygrowing stage(seedling, peak flowering and boll developing stages). Especially in the laststage of cotton development, the transgenic lines showed higher efficiency to bollwormthan bivalent transgenic cotton SGK321. The content of Bt toxoprotein, which measured by ELISA, also showed apparently spatio-temporal fluctuation(seedling＞peak flowering＞bolldeveloping stage), and the spatio-temporal tendency was similar with their resistance of therespective transgenic cotton.The morphological characters, important yield traits, insect resistance and fiber quality offive transgenic homologous lines were compared with that of Sumian 16, showing theeffects of exogenous Bt+Sck genes on agronomic traits of cotton. The effects were thatbolls per plant and resistance to bollworm increased, leaf changed smaller, leaf colordeepened, boll size and lint percentage decreased, the lint percentage of 305-5T₂ and314-4T₂ was significantly lower than Sumian 16. The plant height and the fiber quality hadno significant difference among transgenic homologous lines and Sumian 16. The lint yieldof all the transgenic lines except for 305-5T₂ were the same as Sumianl6 plants.TAIL-PCR is a useful method for obtaining the flanking sequence of T-DNA insertionsite in the transgenic cotton plants genome.Our results showed that the flanking sequencescontained a section of vector sequence and are rich in 'A+T' base pairs which bind tonuclear matrices.This results also indicated the left border or the right border of T-DNA wasabsent during the T-DNA insertion process.