Studies On The Heterosis Of The Polymerzation Of Transgenes And The Transgenic Male Sterility Of Cotton

Ten hybrid combinations were made from 8 different transgenic lines with the same genetic background of a receptor variety in the transformation experiment, which were one transformed with rolB gene, four transformed with different constructs of mannosyltransferase gene, two transformed with monooxygenase gene in generating male sterile line (ms2P-mono) and its restorer (antimono) , and one with bar gene. All the hybrid combinations together with 7 parental lines, 3 check test varieties, including transformation receptor 4105(Huakangmian No.1) and 2 popularized varieties (Emian No18 and a hybrid Eza No.9) in total 20 materials were put in trial for the evaluation of heterosis induced by transgene polymerization and the effects of transgene expression on the development of cotton traits. The trial was arranged in accordance to the design of randomly grouped plots and repeated three times with single line in each group. Meanwhile, a cytological investigation of transgenic male sterile line generated by the construct of ms2P-mono was carried out for making clear of abortive stage in the process of meiosis.The results as following:(1) The event of transgene polymerization could increase the cotton yield and improve the fiber quality significantly, but the polymerizations of different transgenes generated different impacts on the cotton yield and contributed unequal value to the hybrid vigor. All the combinations had the heterosis over mean of parents in both of lint yield and unginned cotton yield. The heterosis over better of parents in the lint yield was found in five combinations, and that in the unginned cotton yield was found in six combinations. All the combinations in the unginned cotton yield, and eight of them in the lint yield had competitive hybrid vigor. The combinations located at the three top places were the lines, which were transformed with bar gene driven by enhancer quadruplet of 35S promoter and mannosyltransferase gene driven by 35S promoter. The competitive heterosis of combination No. 1 was made up to 31.75%.(2)Polymerization of the transgenes, ms2P-mant and bar, resulted not only in the enhancement of plant height, but also in the increment of cotton yield. Compared to the receptor 4105, the lint yield of the transgenic line increased by 63.18%, the lint percentage increased by 7.85%, both of which reached a statistically extreme significant level.(3) The lint percentages of transgenic lines anti-mono and anti-mant, two kinds of antisense RNA constructs, increased by 10.12% and 5.44% separately, the differences reached significant level. Yet the fiber length of the transgenic line anti-mono was significantly reduced by 8% compared with the receptor variety.(4) Different effects of the other three mant transgenic lines on the development of cotton characteristics were observed, among which the plant height of No.15 had significant difference from that of other two lines, the differences of the fiber lengths and lint percentages between No.13 and No.14 statistically reached significant level. However, the agronomic and yield traits of all the three lines had no significant difference while compared with transgenic receptor variety. (5) The lint percentage of rolB transgenic line No.16 was higher 6.10% than that of the receptor, and the difference reached significant level; its lint yield was even higher by 5% than that of the hybrid Eza No.9, but the fiber quality of this line declined comprehensively.(6) The yield trait of the bar transgenic line had no difference from that of the receptor 4105, however its fiber quality was significantly improved, such as the increment of the fiber length and strength. But the Micronaire value also increased, this exerted negative effects on the fiber quality in some extent.(7) The hybrid produced by transgenic male sterile line crossed with the restorer could set seeds normally, demonstrating that the expression of the antisense RNA construct was able to suppress the expression of the monooxygenase gene and restored the fertility of the male sterile line. But the hybrid did not show heterosis; the yields of ginned and unginned cotton reduced significantly, the fiber quality basically kept the same as that of the receptor variety.(8) The morphology of transgenic male sterile line obviously changed and appeared in the forms of lower plant height, smaller boll size, shortened internodes in the fruit branch of the plants. The pollen grains released from the shriveled anthers were abortive, while stained with 1% acetyl carmine. The percentage of sterile pollen grains was as high as 91.95%. Microscope observation on the process of pollen mother cell meiosis showed that lagging chromosomes, asymmetric division, asynchronous division, multipolar division, and micronuclei were the main cause to result in the abortion of pollen grains. Abnormal microspore formation was observed at tetrad stage, such as triads, degenerated tetrads, pentads, hexads, heptads, octads, and other forms of multipartites. The percentage of the abnormal tetrads was as high as 49.81%.(9) Through the observation of chromosome behaviors in the meiosis process of pollen mother cell, a preliminary conclusion was made that the abortion of pollen grains took place at the stage of metaphaseâ… , and that the inheritance of the male sterility was incomplete dominant and was controlled by a pair of alleles based on the characteristics of plant shape that was linked with male sterility, the pollen-shedding type of the male sterile plants and the identification results of the abortive pollen grains under microscope.