In vitro regeneration and agrobacterium mediated transformation in tomato (Lycopersicon esculentum mill.) in relation to disease resistance against groundnut bud necrosis virus
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Date
2006
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Department of Plant Biotechnology, College of Agriculture, Vellayani
Abstract
A study on in vitro regeneration and Agrobacterium mediated transformation in tomato (Lycopersicon esculentum Mill.) in relation to disease resistance against groundnut bud necrosis virus (GBNV) carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2004 to 2006.
Irrespective of explants of tomato 0.08 per cent mercuric chloride for seven minutes and one per cent sodium hypochlorite for 15 minutes were found to be the most effective treatment for surface sterilization.
Highest callus induction was obtained from cotyledonary leaves (78.00%) and leaves (68.00%) in MS medium supplemented with BA 2 mg l-1 and IAA 1 mg l-1 and regeneration from cotyledonary leaves (79.42 %) and leaves (78.00%) was highest in medium containing 2.5 mg l-1 BA and 1 mg l-1 IAA. The MS medium with 2.5 mg l-1 BA and 1 mg l-1 IAA was the best regeneration (74.62%) medium for the nodal segments.
Rooting was obtained on MS medium with 1 mg l-1 IAA. Among the rooted plants 66.8 per cent of plants were successfully established ex vitro.
Experiments were conducted to evaluate the tomato calli to different doses of antibiotics viz. kanamycin and cefotaxime. It was observed that kanamycin 75 mg l-1 induced bleaching of callus. At concentration of 100 mg l-1 and above there was complete browning of callus. The callus was not seriously affected by cefotaxime up to 150 mg l-1.
The Agrobacterium strain LBA 4404 containing plasmid pCAMBIA 2301 was used for the experiment.
Effect of kanamycin and cefotaxime on bacterial growth was studied. It was observed that no colonies were produced after 350 mg l-1 kanamycin and 75 mg l-1 Cefotaxime.
The transformation efficiency was high with the infection process for 15 minutes using the bacterial suspension. Highest transformants was recovered only with the wounded callus in dark for three days. The transformation efficiency was very high with the addition of 200 µM acetosyringone. The bacteria after transformation were killed with 75 mg l-1 Cefotaxime.
The selection of transformants was done by using 100 mg l-1 kanamycin. Out of the total kanamycin resistant tissues 95 per cent showed GUS activity. The distribution of GUS expression (blue spots and blue patches) on the surface of the callus was even. The transformation efficiency obtained was 96.6 per cent based on the GUS staining.
DAC-ELISA was done for the immunological detection of GBNV in tomato. A polyclonal antibody for tospo virus was used for detecting the GBNV virus in tomato. The results of the experiment revealed that the antibody gave high reactivity towards the virus isolates.
The molecular characterization of the coat protein gene of the GBNV was done by RT-PCR, with the primer pair derived from the N gene sequence of GBNV. The amplicon (800 bp) was visualized and documented using gel-documentation system.
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