Transgenic expression of insecticidal toxins under different promoters for the control of insect pests.

Abstract

Insect pests are the major reasons for low yield in agricultural crops. Crop losses due to insect pests can be as high as 25 %, depends upon the climatic conditions and other factors; several strategies have been adopted for the control of insect pests. Recently developed biotechnological applications, several transgenic plants have been developed for insect resistance. Bacillus thuringiensis is one of the most successful examples of insecticidal toxins being used for insect control (especially bollworms). Sucking pests are also major cause of reduced yield in agricultural crops. Sucking insects suck the cell sap from the phloem tissues of plants and also act as a vector for virus transmission. Recent trends in agriculture towards reducing pesticide use and bringing ecological sustainability have led to increased interest in spiders as potential biological control agents. Spider venoms are complex cocktails of toxins that have evolved specifically to kill insects. Spider toxin (Hvt) gene from the Australian funnel web spider which is a calcium channel antagonist has been expressed in tobacco plants to develop resistance against some major insect pests (Bollworms).

In the present PhD research work, the main focus was to develop resistance against major insect pests; American bollworm (Heliothis armigera) and mealybug (Phenococcus solenopsis). Different gene constructs were prepared and transformed in tobacco plants (Nicotiana tabacum) through Agrobacterium mediated plant transformation. Spider toxin (Hvt) gene was cloned under phloem specific RSs1 and RolC promoters and lectin gene from onion Allium cepa L agglutinin (ACA) and kallar grass Leptochloa fusca (LfL) was cloned under 2X35S promoter. PCR, Southern hybridization and real time qPCR analysis showed successful transformation and expression of insecticidal toxin genes in N. tabacum.

Insect bioassays of transgenic plants were carried out in the laboratory and glasshouse conditions. Results showed 93.75 and 100 % mortality of H. armigera larvae when fed on detached leaves from transgenic tobacco plants expressing spider toxin (Hvt) gene under RolC and RSs1 promoters within 72 hour respectively. H. armigera larvae released on the detached leaves of transgenic plants expressing lectin gene from kallar grass and onion under 2X35S promoter showed, as 75 and 81.25 % mortality within 72 hours respectively. No mortality of H. armigera larvae was observed on the detached leaves of non-transformed tobacco plants up to 72 hours.

Transgenic plants expressing spider toxin under RSs1 and RolC promoters gave good resistance against P. solenopsis on detached leaves; 68.75 and 75 % and on live transgenic tobacco plants 70 and 62.5 %, up to 10 days respectively. Transgenic plants expressing lectin gene from onion and kallar grass under 2X35S promoter showed high level resistance against nymphs of P. solenopsis on detached leaves; 87.5 and 81.25 % and on live transgenic plants; 90 and 87.5 % mortality of P. solenopsis within 10 days respectively. No mortality of P. solenopsis was recorded on non-transformed (control) tobacco plants up to 10 days.

This study described expression of Hvt gene under RSs1 and RolC promoters and lectin gene under 2X35S promoter. Both genes are helpful for controlling chewing insects like H. armigera larvae and sap-sucking insect like P. solenopsis. This study has a potential for developing insect resistant transgenic crops. These crops can reduce the use of harmful pesticides, fuel use, input cost and yield losses, while increase profitability and enhance biodiversity and save the environment.