Optimization Of Agrobacterium-mediated Genetic Transformation And Verification Of StNTP Gene Function

Since 1983,the first time to get transgenic plant,Agrobacterium-mediated transformation has been improved and various protocols with different explants of a range of plant species estabolished,but there are still lots of problems,such as the transformation showed genotype-dependent feature and the transformation efficiency is low and unstable in some plant species.The genetic transformation is expected valuable to improve plant traits,such as disease resistance,among them many efforts have made to control potato late blight(Phtytophthora infestans),one of the most serious diseases of potato.StNTP is a NAC transcription factor which is supposed to be involved in potato late blight resistance and was employed in the present research for its function dissection with optimized transformation protocol.Three potato late blight-susceptible(Désirée,304413.19 and 304413.89)and two resistant(301071.3 and 304413.40)genotypes were used in the present research.The optimization of Agrobacterium-mediated transformation was based on the mictuber-system for which potato microtubers formed in vitro were used as the receptor.Therefore,tuber formation of these potato genotypes,effects of the medium components on transformation were investigated,followed by the late blight resistance evaluation of the St NAC gene in the obtained transformed potato lines.The main results are listed below.1.The microtuber formation of potato genotypes 304413.19,304413.40 and Désirée,the first three genotypes available for tissue culture,were investigated.The results showed that the first day microtuber-formed in SD for 304413.19 was significant earlier than Désirée and 304413.40.It's suitable to take microtubers of 304413.19 cultured 55 d and microtubers of 304413.40 and Désirée cultured 60d(the microtuber age is 4 weeks)as expalnts to optimize the protocol.2.Based on the phenotype of in vitro tuberization,photoperiod treatment has been designed for two genotypes(301071.3 and 304413.89)and sucrose treantment for 301071.3.The results showed that the microtuber formed in advance in the dark,while the number of tuber formed per planlet and mean tuber weight is higher in short-day.60g/L sucrose was beneficial for the microtuber formation of 301071.3.3.Only three genotypes 304413.19,304413.40 and Désirée were investigated for optimizing the Kan concentration and medium components.The results showed that the most suitable Kan concentration for Désirée,304413.19 and 304413.40 was 60mg/L,50mg/L,50mg/L,respectively.Among these genotypes,Désirée was the only one to succeed in abtaining transgenic lines on different medium.The best combination was MS+0.1mg/L IAA+2mg/L ZT+60mg/L Kan+400mg/L Cef and transformation efficiency for regeneration rate of plants and explant shooting rate was 52.27%,11.36%,respectively.4.Overexpression StNTP1 and StNTP2 vectors were constructed,5 and 3 positive transgenic plants were obtained,respectively,via the optimized protocol with Désirée.All transgenic lines were further selected to identify the late blight resistance.The data of lesion area showed that,compared to the control Désirée,the overexpression plants were more resistance to P.infestans.The result indicated that StNTP contributed to potato late blight resistance.