Exploration And Establishment Of A Simple,Quick And Efficient Transformation Protocol For Rice Using Agrobacterium Tumefaciens

Human population,resources and environment have been the main problems which obstruct human being's survival and development.In recent years,many factors,such as population growth,climatic change,financial crisis,and so on,have made the world facing with food shortage and the price of the main agricultural products increasing rapidly,and also caused food crisis and people's panic in the world wide.As rice is one of the most important crops in the world,it's very urgent to improve its output.However,some facts,such as declining of arable land and fresh water,pollution by large-scale use of pesticides and fertilizers,etc,have increased the agricultural production cost and peasants' burden,so it's hard to depend upon traditional way to breed new varieties with great breakthrough in the output potential.While in the last two decades,Genetically-Modified Organism(GMO)have played an important role in crop breeding,because transgenic technology can improve the yield and quality of the crops in a sustainable pattern,it has a favorable development potentiality and prospect.So GMO would ease the food security burden and instill new power to the world's agriculture development.In this study,we developed a new simple and efficient transformation method for rice.Compared to the traditional agrobacterium-mediated transformation method,it does not need to induce callus and a series of tissue culture,so it can save a large number of labor and costs.Furthermore,the transformation rate of this new method is much higher than the traditional one,not only for japonica rice,but also for indica rice.So it is simple,quick,efficient,general and safe.The main results as follows:1.Plasmid of pBI121-GUS which had been transformed into Agrobacterium EHA105 infected the apical meristem of Nanjing45 seeds directly by inoculating needle.Among 90 rice seeds,48 of which survived,the survival rate was 53.3%;16 seedlings were gus positive plants by PCR assay and GUS histochemical staining,the transgenic frequency was 33.3%.After harvesting T0 seeds,choosing 8 populations from T0 positive plants randomly to reproduce T1 plants.PCR results showed that the gus genes in 4 populations were almost all expressed,the number of the 4 populations were 15,32,37 and 46,while,the other 4 populations showed segregated.The number of these 4 populations were 24,52,64 and 66,the segregated ratio of the positive plants vs the negative plants were 1:3,1:2.4,1:1.5 and 1:10.Then,we sampled 16 leaves from the eight populations,2 leaves on 2 plants on each population to carry out GUS histochemical staining and it showed that all the selected leaves expressed the gus gene.The present results implied that this transformation method is effective.2.Plasmid of pCDMARUBA-Hyg which contained two insect resistant genes(sbk and sck)had been transformed into Agrobacterium EHA105,and infected the apical meristem of Nanjing45,9311(indica rice),110183 directly with the same manipulation.There were 10 plants for 9311,5 plants for Nanjing45,and 1 plant for 110183 survived.PCR results showed that 9 plants for 9311,3 plants for Nanjing45 and one plant for 110183 were positive.3.Plasmid of pCDMARUBA-Hyg which contained two insect resistant genes(sbk and sck)had been transformed into Agrobacterium EHA105,and infected the naked apical meristem after being wipped off the seminal root and bud of Nanjing45.9 plants were survived.PCR results showed that all the 9 plants didn't contain hpt gene,but 4 plants of them contained divalent insect-resistant genes and 4 plants contained 1 insect-resistant gene(2 plants for sbk gene and 2 plants for sck gene).