Tomatoes are grown in 159 countries for marketing purpose. In Egypt, there was an intense loss of tomato crop due to the damage caused by the American bollworm called Helicoverpa armigera, cotton leafworm called Spodoptera littoralis and potato tuber moth called Phthorimaea operculella. The growth of transgenic plants expressing delta-endotoxin could effectively tackle pest management issues. Transgenic plants have diminished the use of chemical pesticides which are the cause of ecological damage. As per 2009 statistics, Bacillus thuringiensis (Bt) represents 15 percent of the entire area of the crops that are genetically modified. Agrobacterium mediated transfer of genes has become a popular method for transforming di-cotyledons. Many research studies have concluded that incorporation of Cry genes of Bt into tomato plant will offer protection to the plant against different insect pests. This study concentrates on the generation of transgenic tomato plants expressing Bt gene-Cry2Ab using Agrobacterium mediated transformation. This study also assesses the resistance developed by the transgenic plants against insects belonging to Lepidoptera causing endemic in Egypt.
Plant Transformation Methodology
The tomato seeds are made to germinate upon and the early seedlings are agitated in the bacterial suspension. Then they are allowed to co-cultivate along with bacteria for 2 days in the laboratory. Later, the tomato explants are washed thoroughly to eliminate the bacterial extra growth. Hygromycin resistant shoots of transgenic tomato seedlings were grown in a selective medium. The infected explants could proliferate in the medium with hygromycinB. A part of the proliferated tissue of the explant is taken and grown in the medium stimulating the root system. The tomato plants that are derived by tissue culture having well developed roots and those with the height of 11cm to 14cm were allowed to adjust to ex vitro environment.
Histochemical GUS Assay
The stable expression of GUS gene was observed with the histochemical GUS assay. The integration and expression of GUS gene was identified in plantlets that turned clearly into blue color. The screening of putative transformants was performed using PCR technique. The PCR primers that were complimentary to Cry2Ab gene were used for molecular analyses using PCR. The presence of Cry2Ab sequence was confirmed to be present in 30 plants out of 34 that were investigated for the purpose.
The tomato seedlings that were transformed with Agrobacterium mediated approach were able to proliferate the shoots on the selective medium compared to the plants that were not transformed. The vigor of the transformed plants was checked with the bioassays. The toxic nature of the genes introduced into the tomato plants against the Lepidopteran insects mentioned above was investigated. The larvae of these insects were fed with the leaves of transgenic tomato. The larvae fed with the transgenic tomato leaves died in 1 to 7 days period. The death of various larval instars compared to the insect pests was evaluated among 50 larvae of the various insect species in a bioassay.
Results of the Study
In the insect Phthorimaea operculella, the first and second larval stages were found to be affected very early compared to the third and fourth stage instars. However, the mortality was observed in two to three days period in the case of first two stages. The mortality rate of the second stage larvae was 84 percent on the second day of feeding with the tested plant clones. While on the third day, the rate of mortality reached 100 percent. The mortality rate in the case of third and fourth instar larvae fed with tested plant clones was 100 percent after 4 to 5 days period.
In the insect Helicoverpa armigera, the early instars were more prone to death compared to the later stages. The first instar showed 91.7 percent mortality after feeding the tested clones for two days while the death rate was 80.4 percent in the case of second instar larvae. The younger instars are not so tolerant compared to the older larvae. The mortality was 50 percent in the case of third instar fed for 2 days and 75 percent when it is fed for 3 days. The mortality was 75 percent in the case of fourth instar fed for 3 days and 95 percent when it is fed for 4 days. The mortality rate of fifth instar after 4 days was evaluated as 93 percent. The sixth instar showed 88 percent of mortality after 4 days of feeding while the same stage larvae showed mortality rate of 7.7 percent in 5 days, 3.5 percent in 6 days and 0.4 percent in 7 days.
In the insect Spodoptera littoralis, the first and second instar larvae showed mortality rate of 47.3 percent and 36.3 percent respectively after 7 days of feeding with transgenic leaves. The mortality rates of third and fourth instars were 44 percent and 32 percent respectively, after they are fed with transgenic leaves for 7 days.
Though the transformation becomes successful only if the gene properly integrates and expresses, there are many benefits of the transgenic plants in reducing their reliance on chemical pesticides.
M.M.Saker, H.S.Salama, M.Salama, A.El-Banna, N.M.Abdel Ghany. Production of transgenic tomato plants expressing Cry2Ab gene for the control of some lepidopterous insects endemic in Egypt. Journal of Genetic engineering and Biotechnology (2011) 9, 149-155.
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