Development of marker-free transgenic resistance against thrips-borne orthotospoviruses in tobacco plants

• Main Authors: Kitty Tsang, 曾淑媛
• Other Authors: Shyi-Dong Yeh
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/62905830441669630285

碩士 === 國立中興大學 === 植物病理學系所 === 105 === Cultivated Nicotiana tabacum plants are the common tobacco for cigarettes and cigars. However, thrips-borne orthotospoviruses including pepper chlorotic spot virus (PCSV), a member of watermelon silver mottle virus (WSMoV) serogroup, cause severe damages on tobacco crop in Yunnan Province, southern China. So far, there are no effective measures to control these viruses due to lack of resistant tobacco varieties. Transgenic resistance based on post transcriptional gene silencing (PTGS) is an effective approach to control viral diseases; however, the maker genes for regeneration selection are concerns for ecology and food safety. Hence, we previously developed a marker-free two-T-DNA-binary vector, which contains a selection marker (nptII) and a target gene separately constructed in two individual sets of T-DNA borders to generate maker-free transgenic plant. Transgenic Nicotiana benthamiana lines carrying the hairpin construct of pBI2T-HpL/NSs/N, which contains highly conserved tospoviral replicase (L) gene region, NSs coding sequence (NSs) and an antisense fragment of N coding sequence of WSMoV, have been proved conferring broad-spectrum resistance to orthotospoviruses. In this study, we attempted to use the same construct to develop marker-free transgenic resistance in the real crop of cultivated tobacco variety Yunyan-87. By Agrobacterium tumefaciens-mediated transformation, a total of 43 kanamycin positive tobacco lines were obtained and among them 28 lines were transgene positive, as detected by polymerase chain reaction. After challenged with PCSV, enzyme-linked immunosorbent assay (ELISA) was conducted at 14 days post inoculation (dpi) and 25 dpi to check the presence of the virus. A total of 11 lines showed delay in symptom development for 7-11 days, 3 highly resistant lines showed recovered symptom at 25 dpi, and 3 immune lines were symptomless and ELISA negative up to 25 dpi. The selected 6 lines with high levels of resistance to PCSV were challenged with tomato spotted wilt virus (TSWV) to validate the broad-spectrum resistance. Our results indicated that lines 11, 34, 43, 44 and 49 confer high levels of resistance to PCSV and TSWV. Southern hybridization analysis showed that line 44 has a single insert of the transgene and 43 and 49 has 2 and 3 transgene inserts, all can be used for further breeding. As PCSV and TSWV belong to two different serogroups of orthotospoviruses, the borad-spectrum resistance of our transgenic lines may solve the problem of orthotospoviruses infection in Yunan. Following by self-fertilization of selected transgenic lines, marker-free F2 tobacco progenies with broad-spectrum resistance to different species of orthotospoviruses are being screened.