Studies On Transforming P_SAG12-IPT Gene To Wheat Via Pollen-Tube Pathway

The transformation of wheat via pollen tube pathway doesn′t need special equipment, The methods are easy and low-cost, Receptors transformed can produce seeds and plant regeneration without tissue culture. So it is very suitable for applying in developing countries widely. Many have been accepted by scientists and applied into breeding, and obtained encouraging results. Currently, the different systems transformation with pollen tube pathway have been used. The study on the acting factors of transformation with pollen tube pathway has been rarely reported. Therefore, it is significant to research the possible factors which affect the efficiency of wheat pollen tube channel conversion, establish and improve the system of the transformation of the wheat. The plasmid pCMLA35-1 in the leaf senescence-inhibition Gene PSAG12-IPT is introduced by pollen tube pathway in the wheat Zhengmai 9023 and Yumai 18, as well as the impact of DNA concentration, DNA solvent, conversion time, pollination methods on genetic transformation and the selection by kanamycin into later generations are researched, a better wheat transgenic technology system is initially set up. The main findings are as follows :1. Research the reaction of the wheat seed of different varies to Kanamycin and do screening based on different concentrations of Kanamycin for Xinong 1376, Zhengmai 9023,Yumai18, Xiaoyan 22.The seed germination rate and the potentiality in the wheat seed treated by Kanamycin can be significantly reduced. The albino happenes to wheat seedlings. The concentration of 150mg/L Kanamycin can remarkably inhibit the germination rate and 250 mg/L can more remarkably inhibit the germination rate. It is also found that kanamycin at 150 mg/L can remarkably decrease germination potentiality, and the reactions on germination potentiality of different varieties for kanamycin show a significant difference. The germination potentiality of Xinong 1376 for kanamycin is the most sensitive and albino rate was markedly changed in 4d, and the albino rate hasn't any difference among varieties in 10d. The reaction speed of seeds for kanamycin is different among varieties, but eventually the tolerance is broadly similar.2. Research the reaction of the wheat seedling of different varies to Kanamycin and do screening based on different concentrations of Kanamycin for Xinong 1376,Zhengmai 9023,Yumai18,Xiaoyan 22. In artificial climate conditions, the optimum concentration handling wheat seedlings by kanamycin should be above 12,000 mg/L. The resistance of leaves for kanamycin is different for the different varieties. Besides, there are obvious differences in genotypes, the highest albino rate for Xiaoyan22, the lowest albino rate for Yumai 18. Thus, when getting substantial transformation seeds, the seedlings are further screened according to different species characteristics. which can be treated preliminarily with low concentration of kanamycin.3.Research the reaction of the wheat seedlings of different varies to Kanamycin and do screening based on different concentrations of Kanamycin for the different growing time of Xinong1376. Also under field,we screen the wheat the seedlings of Xinong 1376 transgened by Gene PSAG12-IPT and do PCR to identify. The resistance of leaves of Xinong 1376 for kanamycin markedly improves with the development of the reproductive phase. The resistance of the three-leave stage was significantly higher than that before that stage . Therefore, screening using kanamycin should be done before the three-leave period. Under artificial climatic conditions, the appropriate combination of leaf and concentration is the combination of the two-leaf stage +16000mg/L. In Field conditions, After the seedlings of Xinong 1376 transgened by Gene PSAG12-IPT are screened based on kanamyc initially, PCR results show that the percentage of the positive plants accounting for kanamycin-resistant strains is 16.6% .It indicates that kanamycin the initial screening is effective and that its appropriate concentration and time are one of two leaf - clover bent plus 20000mg/L kanamycin.4. Compare the different DNA concentration for the impact of fecundity of wheat by pollen-tube pathway The surface active agents significantly low the seed set propagation coefficient of the wheat ,inhibit development of seed, reduce germination rate, and it is not suitable as a transformation of exogenous DNA solvent.5. Research the exposed time of the different stigma for the impact of seed set propagation coefficient of the stigma. Stigma of vitality affect seed set propagation coefficient significantly, the seed set propagation coefficient of the stigma exposed for more than 8 days is significantly lower than that before 8 days, So the time for Stigma exposing should not exceed 8 days.6. Compare the diffierent DNA concentration and transformation-time for the impact of fecundity of wheat by pollen-tube pathway. Transformation-time affects seed set propagation coefficient significantly and the seed set propagation coefficient transforming for 30 minutes was significantly lower than that for the 60 minutes, 90 minutes, so transformation-time should not be less than 60 minutes. The difference of DNA concentration impacting on setting percentage may exist between the different varieties. DNA concentration does not affect setting rate of Yumai 18 significantly, but affects setting rate of Zhengmai 9023 significantly. The setting rate for concentration of 700 ng/ul is significantly higher than the 300 ng/ul, 500 ng/ul andit is not different from 0 ng/ul control significantly.7. It is the first time to insert the delay leaf senescence PSAG12-IPT gene into wheat Zhengmai 9023 and Yumai 18 by pollen tube pathway. There are One Zhengmai 9023, three Yumai 18, total transformation efficiency of 0.5‰. All positive plants are from artificial pollination, transforming for 60 minutes.DNA concentration is 500-700ng/L ,using TE or water as a solvent DNA.