Obtaining Amylose-Free And Virus Resistant Potato Lines Using RNAi Technology

Potato(Solanum tuberosum L.) starch has been widely used in the food and industrial applications. Amylopectin has two properties such as gelatinization temperature and faintish retrogradation, which has been used in paper, textile and adhesives industry. Amylose has been used in adipose substitute. Three major potato viruses:Potato virus X (PVX), Potato virus Y (PVY) and Potato leaf roll virus (PLRV) cause significant yield losses each year. It is possible to use gene technology that silences the appropriate virus genes to improve potato resistance to viruses and manipulate the key enzyme genes to change the properties of starch. In this study, RNA interference technology was used to silence the potato endogenous granule-bound starch synthase gene (GBSS) and virus coat protein exogenous genes to obtain new varieties which are amylose-free and resistant to virus.The first part of this study was to generate new varieties which had improved virus resistance and reduced contents of amylose using two RNAi expression vectors. One vector harbors a35S driven hairpin structure (RNAi) consisting of the5’ region of GBSS sequences and part of coat protein gene of PVY. Another vector consists of the same GBSS sequence and three viruses (PVY, PVX and PLRV) sequences. These constructs were introduced into an elite potato cultivar (Desiree) by Agrobac- terium mediated transformation. PCR and real-time PCR were performed on all the transgenic potato lines to screen the putative transgenic potato plants and examine the expression level of endogenous GBSS. The transgenic lines derived from the two constructs showed various degree of reduction of GBSS expression, with a few lines showing the reduction up to98.21%. When mini tubers from these highly silenced lines were stained with iodine solution, the starch grains were red brown, while the starch grains of the negative or lowly silenced plants were blue, suggesting that the amylose contents were reduced to minimum level in those highly silenced GBSS lines. PVY was inoculated to lines with various levels of GBSS knock down. The infection of virus in each line was monitored by real-time PCR. Our results showed that the knock down level of GBSS gene of each line is correlated to its resistance to virus Y. Those highly silenced GBSS lines of the two constructs showed no symptoms after virus infection, suggesting that a single RNAi artificial gene carrying multiple sequences targeting different genes could be used to improve potato traits simultaneously.The second part of this study was to excise a selection-marker gene (Hg) from two GBSS RNAi vectors and obtain the marker-free transgenic lines. Two marker-free vectors were constructed, one of which contained GBSS antisense driven by GBSS promoter, another hosted the same RNAi but driven by CaMV35S promoter. These two constructed vectors were introduced into potato plants (Atlantic) by Agrobacterium-mediated transformation. PCR detection was performed on all the455putative transgenic plants, and one was positive. The expression level of GBSS was also examined by Real-time PCR, and the results showed that the efficiency of the GBSS expression reduction was20.38%. Hence, marker-free transgenic potato plant with partial inhibition of GBSS expression was obtained. In future, more transgenic plants are required to obtain highly silenced GBSS lines.