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With the growing concern of the people of the living environment, the turf industry as an emerging industry has been quietly rising. Lawn and green environment, clean air, soil and water conservation, reduce noise pollution, and has a very important link in the human civilization, has become an indispensable part of people's daily lives. Plant genetic engineering technology began in the 1970s has been widely used in the study of improved varieties of plants including turfgrass, is one of the most effective breeding methods. Import the genes of other species to be able to expand the range of genetic improvement, and help solve some of the conventional breeding is difficult to solve special problems. Functional genes purpose of import turfgrass embryogenic callus improved transgenic plants can be obtained after regeneration, an effective way to improve turfgrass stress resistance, has become the focus of current research. The land salinization is to limit the growth of crops, one of the most serious abiotic stress factors in the development and production, cultivate salt-tolerant crop varieties is an important way to overcome such factors. Domestic and foreign scholars have salt damage to plants, plant salt tolerance mechanism of long-term studies, cloned Tolerance Related Gene. Transfer these salt tolerance gene through genetic engineering techniques, has received a batch of salt stress tolerance of transgenic plants to improve. Creeping bentgrass (Agrostis stolonifera L.) is a perennial grass bentgrass species, native to Eurasia, is one of the most cold hardiness cold season turfgrass. It has good resistance to flooded, and the emergence of even finer blades etc., have been widely used in countries around the world planting high-end ornamental turf and sports turf, such as City Center Mall, golf courses and bowling alleys. However, due to differences in ecological conditions, some introduced species in many parts of our country showed some unpleasant aspects, it is necessary to improved resistance to creeping bentgrass varieties. 1. Regeneration system and genetic transformation of embryogenic callus is genetic engineering the best, one of the most common receptor, a strong ability to regenerate. Many turfgrass different explants in vitro regeneration, but about the creeping bentgrass python (Viper), Panna 4 (Penn-A-4) and Pood (Putter) these three varieties vitro Regeneration System still no reports. This work tries to two aspects of python (Viper), Panna on the 4th (Penn-A-4) and Pood (Putter) Ying new varieties of creeping bentgrass embryogenic embryogenic callus induction and plant regeneration callus induction, somatic embryogenesis and plant regeneration research, intended to provide an experimental basis for the further use of modern genetic engineering techniques modified creeping bentgrass quality, breeding new varieties. In this study, the use of mature seeds as explants, 2,4-D Ying-embryogenic callus induction and plant regeneration of creeping bentgrass, and somatic embryogenesis process was observed. Experimental results show that the additional 2.0mg / L 2,4-D, 0.1 mg / L 6-BA embryogenic callus induction frequency of the highest. With the increase in the concentration of 2,4-D, callus induction and differentiation capacity decreased significantly. 1.0mg / L 6-BA to achieve a relatively good results in the regeneration process, the regeneration frequency of callus in most experiments more than 90%. Also found that an appropriate increase in inositol concentration seedlings grow more stout. The process of creeping bentgrass somatic embryos in the experiment were observed, found creeping bentgrass somatic embryogenesis go through globular, heart-shaped embryos, torpedo embryo and cotyledon stage. 0.6mol / L and 0.4mol / L mannitol, hypertonic treatment 5 hours and 12 hours respectively creeping bentgrass embryogenic callus as the target material, the use of PDS1000/He gene gun and Agrobacterium tumefaciens (Agrobacterium tumefaciens ) LBA4404 strain genetic transformation, the target gene for Na ~ / H ~ antiporter protein gene of AtNHX1. The with plasmid pHZX1 by pBI121 transformation from cloned Arabidopsis Na ~ / H ~ antiporter gene AtNHX1 of and selection marker gene NPT II upstream of the CaMV35S promoter, downstream of the NOS terminator. Will be transformed embryonic callus Go to the additional 100mg / L sulfuric acid kanamycin neomycin medium for differentiation, respectively 6 (Biolistic) and 3 (the Agrobacterium method) Green seedlings. Green seedlings obtained by PCR, were a (Biolistic) and two (Agrobacterium law) green seedlings can be amplified bands, initially identified as transgenic plants. 2. Na ~ / H ~ the antiporter gene sequence cloning and analysis of Na ~~ / H ~~ antiporter and plant salt tolerance are closely related. Higher Plants, the tonoplast Na ~~ / H to antiporter is mainly responsible for the Na ~ compartmentalization to vacuoles. Plant Na ~ / H ~ antiporter genes were highly conserved tonoplast Na has been reported ~~ / H ~~ antiporter genes conserved sequence design degenerate primers can be separated from the other species in the same gene. This work was first extracted the Bentgrass total RNA and anti-transcribed to cDNA first strand, and then using degenerate primers by gradient PCR technique amplified creeping bentgrass Ying tonoplast Na ~~ / H ~~ antiporter cDNA from the middle sequence , about the size of 300bp. Intermediate sequence by cloning, sequencing and Blast homology than right, known tonoplast Na ~~ / H ~~ antiporter protein gene sequence of high homology, the consistency of the amino acid sequence of up to 88% of the initial determine the sequence of the creeping bentgrass tonoplast Na ~ / H ~ antiporter the middle of the gene fragment. Further intends to obtain full length cDNA using 3'-RACE and 5'-RACE sequence specific primers were designed according to the obtained, and its expression analysis and functional verification.
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