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Gene Stacking Through Agrobacterium Mediated Co-Transformation in Tomato to Engineer Resistance Against Root-Knot Nematode, Meloidogyne incognita and Tomato Leaf Curl Virus Koulagi Ramanna*, Sirohi Anil Division of Nematology, ICAR-Indian Agricultural Research Institute, New Delhi-110012, India *Corresponding author, E-mail: koulagiramu@gmail.com
Online published on 21 June, 2016. Abstract The aim of the present study was to develop a protocol for stacking multiple genes through Agrobacterium mediated cotransformation in tomato. Agrobacterium strains GV-3101 and LB4404 carrying Meloidogyne incognita specific dsRNA of Integrase and ToLCV specific dsRNA of AC4 constructs respectively were used for co-transformation in order to provide dual resistance against both the pathogens. Binary vectors pBC-142 and pCAMBIA-2301 were used for cloning Integrase and AC4 hairpin RNA respectively. Zeatin riboside (1mg/L) and IAA (0.5mg/L) were used as plant hormones in the pre-culture, shoot induction and shoot elongation medium while only IAA (0.5mg/L) was used for rooting media. Transformed plants were molecularly confirmed for the presence of nptII (Kanamycin resistance gene), Integrase and AC4 genes by PCR. A transformation frequency of 62.50% was obtained for the co-transformed dual constructs based on PCR positive transgenic plants as compared to 82.60% for the single construct. The results strongly verify multiple genes stacking in tomato through co-transformation which is reported for the first time in tomato. This protocol also paves way for developing resistance against two different pathogens by host generated RNAi through co-transformation. Top Keywords Tomato, Co-transformation, Agrobacterium, Meloidogyne, tomato leaf curl virus. Top | |
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