Transformation Of Low Phorsphorus Tolerance GmPAP4and AtPHRl Into Soybean And Development Of Elite Transgenic Germplasms

Soybean requires a lot of phosphorus, but organic phosphorus (Po) is the main formof phosphorus in soil, and phosphorus availability is a major limiting factor forimproving the yield and quality of crops. In this study, the influence factors onefficiency of Agrobacterium-mediated soybean cotyledonary-node transformationwere analyzed firstly, and then acid phosphatase GmPAP4and the transcription factorAtPHR1were introduced into soybean varieties by Agrobacterium-mediatedcotyledonary-node and pollen tube pathway methods to obtain transgenic germplasms.The characteristics of transgenic materials were also studied under low phosphorusstress treatment. The main results were as follows:1. Agrobacterium-mediated soybean cotyledonary-node transformation was optimiz-ed. A much higher induction rate of resistance shoots was obtained when theseedling age was5d from germination with1d preculture, strain EHA105,density (OD600) at0.7, infectious concentration at the level of0.5or0.7and thekanamycin concentration at100mg/L. The soybean variety with the highesttransformation efficiency was WX2and its transformation efficiency was up to2%.2. The transgenic plants with GmPAP4and AtPHR1were obtained via Agroba-ctetium-mediated method. We got positive materials with GmPAP4, includingJD12T5generation (47plants), JD12T4(19plants), SD4T2(5plants), M9793-1T1(23plants), LGD1T1(6plants) and WX2T1(3plants); The plants with AtPHR1were also acquired, including JD17T3generation (1plant), WX2T2(14plants),JD12T1(7plants), LGD1T1(6plants) and JD7T1(4plants). By mixing straisco-transformation method, GmPAP4and AtPHR1were introduced into soybeanvarieties together at the same time, and three T1positive plants with both GmPAP4and AtPHR1were obtained.3. The transgenic soybean plants with GmPAP4and AtPHR1were obtained viapollen tube pathway transformation method, and the soybean varieties ZH41,SN14, ZH15had higher podding rates. PCR analysis results showed that positive materials with GmPAP4, including29plants (9lines) of SN14T2generation,11plants (one line) of ZH15and four plants (two lines) of JD17were acquired;Seventeen T0positive plants of JD12with a vector backbone-free and selectablemarker-free linear GmPAP4cassette were also obtained. We also gained six T2positive plants (3lines) of ZH41with PX6-AtPHR1.4. The characteristics of tolerance to low phosphorus in soybean germplasms withGmPAP4were analyzed. The results showed that the transgenic plants grew betterthan the wild-type, and the secreted phytase activities of roots of three transgeniclines were significantly increased by11%and22%, respectively, compared withthe wild type when phytate was used as the sole phosphorus source.