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With the rapid development of industry in our country lawn, turfgrass breeding work more and more people concerned about biotechnology in turfgrass genetic improvement has great potential. Drought and high salt is to limit the growth of turfgrass main environmental factors, especially the lack of China's current drought tolerant turf grass varieties. Use of plant genetic engineering techniques, drought-tolerant gene was cloned and transformed to express the lawn grass, you can significantly speed up the breeding of new varieties of drought-tolerant, and has broad application prospects. Arabidopsis vacuolar proton pyrophosphatase Arabidopsis Vacuolar H ~-Pyrophosphatase (AVP1) by adjusting the vacuolar proton gradient across the membrane to control auxin transport, with the attendant impact on a variety of developmental events dependent auxin, the plant at high salt and drought stress generated adaptability. Arabidopsis rd29A (Responsive to Dehydration, rd29A) promoter is a stress inducible promoter, in a drought, low temperature and high salt conditions drive the downstream gene expression. In this paper, Agrobacterium AVP1 cloned from Arabidopsis gene into creeping bentgrass, rd29A in inducible promoter-driven expression under, and to obtain transgenic creeping bentgrass for physiological drought tolerant determination obtained enhanced drought tolerant lawn grass. The main results are as follows: 1, the establishment of turfgrass callus genetic transformation receptor system. In the cold season turfgrass and creeping bentgrass tall fescue seed as explants, callus induction rate was 53.0 ± 8.7% and 60.7 ± 7.9%, could induce callus, embryogenic callus growth and stability, growth rate month after subculture up to 206.0 ± 43.7% and 159.3 ± 34.4%. The warm season turfgrasses bermudagrass seed as explants to callus induction rate was 14.7 ± 4.0%, and only produce non-embryogenic callus, the callus growth rate was 78.4 ± 23.2%; to bermudagrass sections as explants can induce callus, but after several subculture to non-embryogenic callus. 2, by Agrobacterium-mediated method rd29A AVP1 gene promoter-driven expression vector into pCambia1302 creeping bentgrass. Respectively, at a concentration of 50,75,100 mg / L hygromycin transformed callus, three concentrations of the filter was 64.1 ± 5.0%, 81.5 ± 1.5% and 33.3 ± 3.8%. After one and a half screening, 50mg / L hygromycin transformed callus after hpt-PCR positive plants ratio was only 0.9%, 75mg / L concentration differentiation plants hpt-PCR-positive rate as high as 96.7%. Thus, determining 75mg / L of creeping bentgrass transformed callus best screening concentration. Three transgenic creeping bentgrass plants by PCR. Reborn creeping bentgrass plants 90, wherein hpt-PCR positive strains 87, the positive rate was 96.7%; AVP1-PCR positive strains 13, the positive ratio of 14/4%. 4, genetically modified creeping bentgrass callus AVP1 gene expression by RT-PCR. The results showed that in normal culture conditions transgenic callus AVP1 gene micro expression, whereas in the dry (drying 48h), low temperature (4 ℃, 24h) or high salt (200mM NaCl) stress AVP1 gene expression were significantly enhanced; untransformed The wild-type materials, whether or not detected processing AVP1 gene expression. This result indicates that the non-stress conditions rd29A promoter activity is low, and in the dry, low temperature or high salt treatment conditions significantly enhanced activity, which initiates the expression of the downstream gene AVP1. Thus verified drought, low temperature and high salt conditions can promote genetically modified creeping bentgrass AVP1 target gene expression levels. 5, genetically modified creeping bentgrass callus resistance physiological analysis. Dried for 2 days and 3 days, the recovery of transgenic callus growth rate was 645% and 543% of wild-type and 135% to 423%, showing that genetically modified creeping bentgrass has more than wild-type drought tolerance ; the high salt stress, with different concentrations of 0-200 mM NaCl for transgenic callus, and its growth rate is higher than the same in case of wild-type callus. Especially in the NaCl concentration of 50mmol / L, 200mmol / L when the difference between the two larger, respectively, 170% and 148% difference. Showed that gene transfer AVP1 creeping bentgrass high salt tolerance increased. 6 transgenic creeping bentgrass plants resistant physiological analysis. After nine days of drought stress, the transgenic bentgrass seedling leaves bright green, while the wild-type grass blade is obviously wilting yellow. Seedlings to drought relative water content before and after treatment were measured before drought transgenic and wild-type seedlings bentgrass no significant difference between the moisture content and drought stress of transgenic bentgrass after a water content of 82.6%, significantly higher than the wild-type 77.5%, indicating that genetically modified creeping bentgrass than wild-type drought. Transgenic root biomass measurement results show that transgenic plants root dry weight averaged 0.17 ± 0.02g, while the wild-type plants averaged only 0.07 ± 0.01g, significant differences between the two. These results indicate that transgenic plants with well-developed root system, is conducive to plant in the soil absorb water and nutrients, enhance drought tolerance.
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