Development and Validation of Marker-Free Constructs Based on Nucleocapsid Protein Gene of Watermelon Bud Necrosis Orthotospovirus in Watermelon

Abstract

Marker-free (MF) constructs were developed using 825 bp and 426 bp N genes of watermelon bud necrosis orthotospovirus (WBNV). N genes were amplified and cloned in pDrive (825 bp) and/or pGEMT (426 bp) vectors and recombinants were confirmed. pCAMBIA 2300 was restricted with Xho I to release npt II gene and re-circularized; it is called MF-pCAMBIA 2300. GUS cassette with promoter and terminator from pBI 121 was cloned into the MCS region of MF-pCAMBIA 2300 with EcoRI and Hind III. Eluted PCR products of 825 and 426 bp were cloned in already developed MF-pCAMBIA 2300. These N gene constructs were then mobilized in Agrobacterium tumefaciens strain EHA 105. Agroinfiltration of A. tumefaciens harbouring MF-pCAMBIA 2300 N gene constructs was practised by syringe method in 20-day-old watermelon seedlings. PCR amplification showed 825 and 426 bp amplicons which confirmed their presence in agroinfiltrated seedlings. Further, three days of post agroinfiltration seedlings were challenge-inoculated with WBNV and kept under artificial light for 3–6 days. Agroinfiltrated and inoculated seedlings were tested by ELISA and resulted positive with OD ranging from 0.31 to 0.46 and 0.35 to 0.43 in full and partial N genes respectively. Six days post-inoculation, necrosis and complete drying of the leaves were observed. The systemic upper young leaves were subjected to DAC-ELISA and results revealed negative reaction with OD ranging from 0.11 to 0.19. The N gene constructs conferred resistance against WBNV in watermelon.