Indo American Hybrid Seeds, Bangalore, 56004 India

The work is under review

Summary:

This work was carried out in IAHS, Bangalore while I was working as Principal Scientist in the Plant Genetic Engineering Lab.

In India tomato crop is as important as potato for it is extensively consumed as fruit and for cooking and culinary product. Cultivation of this crop is not seasonal, but cultivated through out the year. Most the farmers involved in this farming are small holders. Very often this crop is devastated by two viruses; Tomato mosaic virus (ToMv) and Tomato leaf curl virus (ToLCv). The disease is so wide spread and loss to the crop can range 50-70%.

To find remedy for this problem, I have collected the infected plants from different areas of Karnataka and also from north India. On observation I found the infection is often combined with ToLCv and ToMv. The mixed infection was found to be very severe, where the plants show leathery leaves and little color tinged and all show drooped nature.

From the samples collected, leaves were used to extract viruses from differential centrifugation method, using sucrose-buffered solution as step gradient columns. Each of the fractions are collected and looked for the viruses. From the leaves with single infection I was able isolate pure forms of ToMv and ToLCv. The identity was confirmed by viewing under TEM. Once the isolates were confirmed as pure forms, they were used for molecular studies.

Genomic RNA (ToMv) and genomic ss-circular DNA (TolCv) were isolated from the purified viruses. The RNA was reverse transcribed to generate first cDNA using Reverse transcriptase, then the second strand was produced by DNA pol I. The ds cDNAs were treated with enzymes for to generate blunt ends and the same was eluted from the agarose gel. The ds cDNA was cloned into pUC plasmid which was cut and blunt ended. The cDNAs cloned were not of the same length and showed different sizes. Two of the largest cDNAs were cloned into Ti plasmid PGA 100 (developed from pGA170 from Galvin).

The same were characterized for the insert and selected for transformation of Agrobacterium strain lb 0044. The transformed bacteria were grown under favorable conditions. When the growth reached exponential stage, they are used for co-cultivation of bacteria and sliced tomato leaves. After overnight incubation the leaves were taken out washed in tissue culture buffer and transferred to regeneration medium with carbamicllin. This process is repeated till all the bacteria are killed and regenerated plant lets were propagated. The regenerated plant lets were used for detecting the insert by PCR and found that the cloned DNA is found in plant tissues. Propagated plants were subjected acclimatization in the green house and the plants were then transferred to fields.

We expected to generate transgenic plants to develop capsid mediated resistance, but it turned out to be RNAi mediated resistance; we are surprised. The plants were found to resistant to ToMv viruses. The clonal DNA inserted, some how produced dsRNA, which has lead to the production of RNAi s, that took care of resistance to viral infections.

Similarly the ss-circular DNA from Gemini viruses were used for generating ds strand by using designed primers. Many of the products were found to be linear and the same were made blunt ended. After characterization they were used for cloning into Ti plasmid pGA100. After screening for the insert, the same was used for transformation of Agrobacterium and selected for the insert and the same was used for transfection of tomato plants. The regenerated plants were screened for the insert by PCR. Once the transgenic plants were obtained they are propagated and acclimatized and transferred to fields. And they are found to be resistant to the said viruses. After grown for some period of time transgenic plants for ToMv and ToLCv were crossed and to obtain a hybrid for developing transgenics resistant to both viruses.

Materials and Methods given else where.