Optimization Of Barley Tissue Culture System And Genetic Transformation Of MiR528

Barley is an important grain crop in the world.Because of its wide range of uses,it is called a �Ternary crop� or �crop of multiple values�.Barley is a diploid self-pollination crop,with a small number of chromosomes(2n=14)and a small genome.It is relatively easy to develop molecular biology research such as gene cloning and functional analysis in wheat crops.Currently,what has been completed is sequencing of the whole genome and illustration of partial gene function.The mature genetic transformation system is one of the main technical supports for gene cloning and functional analysis.With the continuous discovery of barley functional genes and improvement of tissue culture techniques,the transgenic technology in barley has developed a lot,and many positive achievements have been made in the study of gene function,one of which is the functional analysis of MicroRNAs.Many studies have shown that in many crops,MicroRNAs are involved in their growth,disease resistance,stress resistance and so on.MiR528 is one of the microRNAs discovered in recent years.It plays an important role in the regulation of plant growth and development and tolerance to saline and low nitrogen soil.The effective use and functional analysis of miR528 using transgenic technology will provide important information for crop genetic improvement.With the immature and mature embryo callus of ten barley varieties as materials,and the hygromycin hptII gene as the screening marker,this study makes use of Agrobacterium tumefaciens-mediated genetic transformation technology,introduces the double 35 S osa-miR528 gene vector into barley,and obtains transgenic positive plants of different barley varieties containing the target gene osa-miR528.The main results are as follows:(1)A total of 60 resistant plants were obtained by Agrobacterium tumefaciens-mediated genetic transformation of osa-miR528 into immature embryo callus of barley.Among them,transformation of Huadamai5 was most efficient,which had obtained 30 resistant plants.According to the results of PCR,there were 3 positive plants containing hygromycin hptII gene,including 2 positive plants from Huadamai5 and 1positive plant from Meili Gold.(2)A total of 55 resistant plants were obtained by Agrobacterium tumefaciens-mediated genetic transformation of osa-miR528 into mature embryo callus of barley.Also,transformation of Huadamai5 was most efficient,which had obtained 38 resistant plants.It shows that,of the ten varieties tested,Huadamai5 is a relatively good material as genetic transformation receptor.According to the results of PCR,there were 2positive plants containing hygromycin hptII gene,the receptor material of which were both from Huadamai5.(3)If seedlings were cultivated through hydroponics,and then transplanted into the soil,the survival rate of transplanted seedlings can be increased.In the genetic transformation experiments of barley callus,the regenerated seedlings that were stripped from the rooting medium were first hydroponically cultivated for a month,and then transplanted into the soil.The length of the part of seedlings above the ground,the length of roots below the ground,and the number of tillers were significantly increased than those cultivated in soil(i.e.transplanted into the soil).It shows that,if seedlings were cultivated through hydroponics,and then transplanted into the soil,the survival rate of transplanted seedlings can be increased.(4)Salt-tolerance experiments of transgenic positive plants showed that,after cultivation with 15 g/L NaCl hydroponic solution,stress phenomenon of T1 generation positive seedlings of Huadamai5 was relatively lighter than that of the corresponding wild Huadamai5.It concluded that,over-expression of osa-miR528 in barley may improve its tolerance to salt.