Establishment Of Ac/Ds Transgenic Mutants And Transposition Analysis In Upland Rice

Rice drought resistance is of great significance for crop sustainable development. We plan to get series of drought resistant or sensitive mutants from AclDs transposable elements so as to give an insight into molecular mechanisms of upland rice drought resistance. This study focuses on transformation of Ds to upland rice and transposition analysis of AclDs. The main results and conclusions of this study are as follows:1. We set up different concentration of sorbitol in callus induction, subculture and regeneration media, respectively, and found the10-20-40treatment got the highest callus regeneration rate for the two cultivars HD297(Handao297) and9311with45.1%and32.6%increase compared to the control (0-0-0). On the basis of different concentration of sorbitol in induction and subculture stage, we supplemented sufficient sorbitol in regeneration medium and reduced sucrose concentration from30g/L to5.5g/L and0g/L, respectively. Amazingly, no matter whether we added sorbitol in induction or subculture medium we found the combination of sucrose concentration at5.5g/L significantly improved callus regeneration rate, increased to from82.7%to95.2%for the two cultivars. Those calli that were compact and fresh yellow produced much more green buds. More over, callus browning of9311was suppressed significantly.2. Through Agrobacterium-mediated approach, we transferred Ds construct into upland rice HD297. Finally, we got24transgenic Ds lines of HD297and verified Ds integration at molecular level. Interestingly, in the process of transformation, we found that more resistant calli were obtained when transformed calli were screened once and then transferred immediately to regeneration media which obviously shortened transformation period. Further more, we observed that addition of TDZ (Thidiazuron) in regeneration media showed better effect on green spots regeneration.3. To screen transgenic lines, we germinated Ac and Ds transgenic seeds on G418-and Hygromycin-containing solution, respectively. From126T2lines by generational screening to T5generation, we eventually got64homozygous and23heterozygous Ac transgenic materials, totally87independent transgenic events with94different lines. In parallel,13homozygous ones from16indepentant Ds transgenic events of T3generation were obtained.4. To activate Ds transposition, we made12AclDs combinations from5Ds lines by crosses and got1289F2plants. By PCR assay, we found that the proportion of Ds transposition in this population was29.6%. Among the5Ds lines,3lines harbor single copy of Ds and2lines harbor double copies. There were more than50%Ds transposed plants in single-copy Ds hybrid combinations, most of which were germinal transposition, while there were few Ds transposed plants in double-copy Ds hybrid combinations, most of which were somatic transposition. Also we found that90%of Ds transposed plants were Ac positive in the F2generation.In this part, we treated leaf samples of AclDs hybrid F2generation with Basta, G418and Hygromycin solution to find consistency between existence of the corresponding gene and the resistance of antibiotics, the resistance consistency of Basta and the two antibiotics was high. In Hygromycin screening of single-copy Ds hybrid combinations, Ds screening consistency was higher than81%, but it was very low for double-copy Ds hybrid combinations as a result of the Hygromycin resistance gene silencing. In G418screening of the combinations, most of the Ac PCR positive plants were sensitive to G418. The G418resistance gene silencing also happened.5. Variation characters of11agronomic traits related to rice yield were analyzed by ANOVA(Analysis of Variance), correlation analysis, PCA(Principal Component Analysis) and hierarchical cluster analysis. Results are as follows:Compared to wild type HD297, most agronomic traits of lines performed a wide variation; There was complex correlation among different traits; In Ac lines, first six principle components revealed most information of11traits, among which plant height, seed set and spikelets contributed most to them. On this basis, Ac lines were divided to six types by cluster analysis. Type4and Type1had better yield potential; Ds lines were divided to four types. Type3and Type4had better yield potential. Above all, this study can provide theoretical and practical references of germplasm innovation for rice transgenic breeding.Mutants for drought resistance or sensitive need to be further established and screened.