| In regard to harvested area and total yield, China is the world’s leading producer of potato, but the average yield of it is less than that of the whole world. Potato is the sixth crop in China after maize, rice, wheat, rape seed and soybean in terms of harvested area, and it’s also the fourth most important food crop in China.Potato production is threatened by various diseases as mosaic, leafroll, black leg, early blight, black scurf and late blight etc. in China, the pathogens include 5 viruses,2 bacteria,2 fungi and loomycetes. With the ever-increasing scale of potato cultivation, potato diseases, which represented by late blight, has become a problem with which the development of potato industry is confronted.Among integrated pest managements (IPM) of potato diseases, the development of new cultivars that are more resistant to economically important diseases is therefore one of the top priorities for potato breeding programs worldwide. In order to be successful, potato breeding strategy has changed from making crosses between varieties originated from S. tuberosum to introgressing disease resistance genes from their wild relatives. During the past 30 years, the development of molecular biology technology for species other than potato could offer new possibilities in potato breeding, such as disease resistance management. In addition, with the available of technologies on regeneration and genetic transformation of potato, and with the property of colonal propagation, plant genetic engineering has become one of the potent tools for improving disease resistance of potato.It is the first time in China to report that transferred ribosome inactivating protein gene (RIPs) cloned from maize (Zea mays L.) to cultivated potato variety which is susceptible to both Late Blight and Rhizoctonia canker(Black scurf) by method of plat genetic engineering. The expression of the transgene in transgenic potato plants was identified by means of molecular biology techniques, and assays was carried out to evaluate the resistance to both fungus Rhizoctonia solani and oomycetes Phytophthora infestans pathogen of transgenic clones expressing the RIPs gene. The main results as follows:1. Thirty-six kanamycin resistant plants of 5 lines of putative trans formants generated from kanamycin resistant calli were obtained by Agrobacterium-mediated transformation using leaf disc as explants, the percentage of transgenic transformation for the protocol used is 6.25%.2. Thirty-four plants from 3 lines developed in selective medium were positive for the corresponding gene using the PCR technique. Southern blot analysis demonstrated that the tested 12 transgenic plants of 3 lines integrated 2 or 4 copies of RIPs gene into their genome. RT-PCR and RT-qPCR analysis showed that RIPs gene can be transcribed correctly, and the transcription level of the transgene was higher in roots than in both stems and leaves for all transgenic plants tested. The translation of the RIPs gene was confirmed in the crude protein extracts of leaves from the transgenic clones by analysis of depurination activity to ribosome.3. Resistance evaluation of 3 transgenic plants from 2 lines in greenhouse conditions showed that disease severity of only 1 plant were reduced for R. solani AG2-1, in planta assays showed that the resistance to stem canker of 3 transgenic lines had not been improved significantly.4. Resistance to P. infestans were also evaluated using detached leaves, both 4 plants of 1 transgenic line and 3 plants of 3 transgenic lines expressing the transgene RIPs showed significant improved resistance to P. infestans than untransformed control. |
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