Establishment Of High Efficient Rice Transformation System By Marker-Free Salt Tolerant Genes VIA Agrobacterium

Saline land area accounts for 20% of the total arable land in the world. Because of the drought, unreasonable irrigation and fertilizer overuse, aggravation pollution of industry, the area of soil salinization is unceasingly enlarging, available area for farming land is decreasing. Rice is about one-third of the world population's stable, the yield of the rice has been in the shortage of supply, therefore, increase the rice yield, improve the rice quality relate to human survival and development. High salt tolerance of transgenic rice and extensive adaptability of rice varieties through transgenic technologies are new points of utilization salinity-alkalinity desert and increasing rice yield. Currently one of the most widely applied rice transformation technologies is Agrobacterium mediated transformation. Establishment of high efficient Agrobacterium mediated salt tolerance gene transformation of rice has important theoretical and practical guiding significance. According to salt tolerant character, establishment of high efficient Agrobacterium mediated marker-free salt tolerance gene transformation of rice is of great significance to apply commercialization, and avoid the potential hazards of the resistance marker genes that may exist on food and the ecological environment.The shortcomings of transformation efficience of Agrobacterium mediated transformation are the long cycle and transformation receptor state. This paper establishes high efficient Agrobacterium mediated transformation rice system starting from the two facts. According to salt tolerant character, NaCl used as a screening agent is explored, but traditional determine NaCl screening concentration needs to draw the growth curve between NaCl concentration gradient and callus weight increase ratio. This process is tedious, easy to pollute, and prone to errors. The feasibility of determine NaCl screening concentration through NaCl inhibition effect to rice seed germination is explored, meanwhile screening effect of NaCl is compared with hygohycine. Finally, with high efficient Agrobacterium mediated marker-free various types of salt resistance gene transformation of rice, this system is proved completely feasible and effective. The experimental results are as follows:(1)Establish a high efficient Agrobacterium mediated transformation rice systemIt is found after 32℃dark culture 7d, the callus induction rate of Nipponbare, Xiushui 11 and Chunjiang 06 are 60%above,92.87%,83.62%,64.83% respectively; after 28℃light culture 7d, the callus induction rate of Nipponbare, Xiushui 11 and Chunjiang 06 are 70%above,95.5%,93.07%,74.87%respectively. Callus surface protuberant, pale yellow, vigor that fully meet the transformation requirements, the time for callus induction shorten from 15d to 7d.(2) Establish high efficient Agrobacterium-mediated transformation of riceZhonghua 11, Xiushui 11 and Chunjiang 06 transformation efficiency improve rate are 58.50%,21.96%,27.85%with heat shock treatment; Zhonghua 11, Xiushui 11 and Chunjiang 06 transformation efficiency improve rate are 60.57%,23.54%,25.17%with liquid co-culture infection, efficient Agrobacterium-mediated transformation rice is established.(3)Establish a Simple selection method of effective screening concentration of NaClAccording to the growth curve between NaCl concentration gradient and callus weight increase ratio, it is showed when the weight gain inhibition rate reaches 80%, NaCl concentration appropriate for screening, the same as NaCl concentration when seed germination inhibition rate by 50%or more. Therefore, NaCl concentrations when seed germination inhibition rate by 50%as an important reference for screening in salt resistance genes transformation.(4) NaCl screening in salt-tolerant gene transformation is effective and feasibleTransformation effect of NaCl as screening agent is compared with hygohycine. For Zhonghua 11, the transformation efficient of NaCl screening is 11.98%, and hygohycine is 14.43%; For Xiushui 11, the transformation efficient of NaCl screening is 32.06%, and hygohycine is 42.64%; For Chunjiang 06, the transformation efficient of NaCl screening is 32.03%, and hygohycine is 41.39%, which prove NaCl screening is feasible and effective.(5)Establish safe and high effective Agrobacterium mediated transformation of riceAdopted NaCl screening and high efficient agrobacterium-mediated transformation rice combination, it is found Xiushui 11's transformation efficiency improving rate is 29.9%, safe and high effective Agrobacterium mediated transformation of rice is established.(6) Safe and high effective Agrobacterium mediated salt-tolerant gene transformation of Nipponbare is feasible and effectiveChoosing salt resistance genes of different salt resistance mechanisms as the exogenous gene using Agrobacterium mediated marker-free salt resistance gene transformation of Nipponbare, the results show that the transformation efficiency of BADH, CMO, PKCC are all above 40%, respectively 44.03%,46.29%,44.68%, which proof Agrobacterium-mediated marker-free salt resistant gene transformation is feasible and effective.All the results suggest that:safe, fast and efficient Agrobacterium mediated salt resistance gene transformation rice system has been established:callus after 32℃light training for 7d,43℃water-bath 30min, ice bath beforehand 1min, moved to three-layer filter paper infiltrated liquid co-culture medium on dark for 2d, turn to salt screening medium, and plantlet regeneration.