Agrobacterium-mediated Transformation Of Potato With Drought Resistance Gene BDN1

In this study, Kexin No.18and DongNong303min-tuber are taken as the researchobjects.BDN1gene was transferred into Kexin No.18and Dongnong303that are main localcultivars of microtuber discs, with Agrobacterium-mediated method.The transgenic plants weretested by PCR、Southern blotting to confirm BDN1gene has been integrated in the genome ofpotato.And the gene can be transcribed nomally by semi-quantitative RT-PCR.Test of theefficiency of potato regeneration and genetic transformation system is established, in which theconcentration ratio of influencing factors of genetic transformation-hormone, the concentration ofoff inocula type, the time of co-culture and the size of the selection pressure have also beenstudied.Through the determination of the relative water content of confrontational plants, Prolinecontent, MDA content, Chlorophyll content, as well as the activity of SOD and POD, it ispreliminarily proved that the validation transgenic potato plants have more significant resistance towater stress compared with the control plants.The major findings are as follows:1Identified that the budding rate of Kexin No.18is82.61%, and that of Dongnong303is86.36%in tubers regeneration medium: MS+ZT4mg/L+IAA1mg/L.2Identified that the Agrobacterium bacterial concentration is OD600=0.7, infection time is5min.The budding rate of Kexin No.18is up to83.33%, and contamination rate is12.5%; thebudding rate of Dongnong303is90.91%, and contamination rate is9.09%.The fact demonstratesthat the optimal transcription could be achieved, if the co-culture time of Kexin No.18with BDN1gene was3days, in which the budding rate was80%.The optimal co-culture time of Dongnong303with BDN1gene was2days, and the rate of tuber disc with was85%.The rate of pollution inboth was10%.3In the comparative experiment of two bactericides of different concentrations, the effectiveconcentration of Carbenicillin treated in BDN1gene was300mg/L.the effective concentration ofimported cefotaxime sodium treated in BDN1gene was150mg/L.4Kan was used as a selective agent in this assay.The manner of selection was7d delayscreening.The concentration was100mg/L for the budding at the stage of shoot, and125mg/L forthe root formation. 5The assay of target gene BDN1obtains50resistant plants of Kexin No.18, among which26plants pass the rooting screening,13were PCR positive reaction and4detectshybridization signalin Southern blot hybridization assay; And90resistant plants from Dongnong303, among which35pass rooting screening,27were PCR positive reaction and4detect the hybridization signal inSouthern blot hybridization assay, indicating the gene has been integrated into the potatogenome.The results of further semi-quantitative RT-PCR test show that the BDN1gene expressionin potato resistant plants can be transcribed.6The biological detection was carried out among the positive transgenic plants inhybridization, and the transgenic plants and non-transgenic gene controls are carried water stresstreatment for15d, after which the the changes of physiological indicators in transferred geneplants and the control have been analyzed, the results showed that: the comparison of the sixphysiological indicators of the8transgenic plants is, to some extent, different with the control,indicating after the treatment of drought stress, the exogenous BDN1gene has all expressed intransgenic lines, improving the drought tolerance of transgenic potato plants.