Articles
Agrobacterium-mediated transformation of Nicotiana benthamiana with Plum pox virus T genes
Article number
1163_2
Pages
7 – 12
Language
English
Abstract
Different strains of Plum pox virus (PPV), including M, D and T, are widely present in Turkey; the economic importance of stone fruits such as apricot trees highlights the need for PPV resistance programs.
It is well known that transgenic constructs with self-complementary sequences separated by an intron produce hairpin structures that trigger silencing of virus genes, thereby producing resistance in the host plant.
In this study, we have used a 771-bp fragment from the P1/HCPro gene region of PPV-T to induce gene silencing in Nicotiana benthamiana (NB). A gateway cloning system was used to achieve virus gene cloning.
An amplicon product of the P1/HCPro gene was introduced first into an entry vector (pDONR221) and subsequently into the destination vector (pHELLSGATE12). Finally, the destination vector was cloned in Agrobacterium tumefaciens, which was used to introduce the construct to NB leaf discs taken from in-vitro plants.
Eight transgenic plants were regenerated onto rifampicin and kanamycin selective medium.
Transgenic plants were confirmed with PCR amplification of the neomycin phosphotransferase II (nptII) gene and amplification with PPV P1/HCPro gene-specific primers.
Seeds of transgenic plants were obtained, and T1 lines are currently being evaluated against PPV-M, -D and -T strains.
It is well known that transgenic constructs with self-complementary sequences separated by an intron produce hairpin structures that trigger silencing of virus genes, thereby producing resistance in the host plant.
In this study, we have used a 771-bp fragment from the P1/HCPro gene region of PPV-T to induce gene silencing in Nicotiana benthamiana (NB). A gateway cloning system was used to achieve virus gene cloning.
An amplicon product of the P1/HCPro gene was introduced first into an entry vector (pDONR221) and subsequently into the destination vector (pHELLSGATE12). Finally, the destination vector was cloned in Agrobacterium tumefaciens, which was used to introduce the construct to NB leaf discs taken from in-vitro plants.
Eight transgenic plants were regenerated onto rifampicin and kanamycin selective medium.
Transgenic plants were confirmed with PCR amplification of the neomycin phosphotransferase II (nptII) gene and amplification with PPV P1/HCPro gene-specific primers.
Seeds of transgenic plants were obtained, and T1 lines are currently being evaluated against PPV-M, -D and -T strains.
Publication
Authors
M. Ghaderi, M. Akbulut, K. Gürcan
Keywords
sharka, RNA silencing, virus gene, transgenic, resistance
Online Articles (23)