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


Affiliations
1 Division of Crop Protection, ICAR-Indian Institute of Sugarcane Research, Biological Control Centre, Pravaranagar 413 712, India
2 Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
 

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 XhoI to release nptII 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 HindIII. 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.

Keywords

Agroinfiltration, Bud Necrosis Disease, Orthotospovirus, Watermelon.
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  • Development and Validation of Marker-Free Constructs Based on Nucleocapsid Protein Gene of Watermelon Bud Necrosis Orthotospovirus in Watermelon

Abstract Views: 336  |  PDF Views: 130

Authors

S. K. Holkar
Division of Crop Protection, ICAR-Indian Institute of Sugarcane Research, Biological Control Centre, Pravaranagar 413 712, India
Bikash Mandal
Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
R. K. Jain
Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India

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 XhoI to release nptII 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 HindIII. 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.

Keywords


Agroinfiltration, Bud Necrosis Disease, Orthotospovirus, Watermelon.

References





DOI: https://doi.org/10.18520/cs%2Fv114%2Fi08%2F1742-1747