*3.2.3. Multiple insecticidal proteins containing transgenic crop*

varieties have been performed. The benefit of Bt-rice has been acknowledged to the level of small and poor farmers, it is due to the lesser crop damage by the insects and therefore higher crop yields and less use of pesticides. An improved health has also been observed in Bt-rice cultivating farmers compared to non-Bt rice cultivating farmers [126]. Government of India approved the cultivation of Bt-cotton in 2003, which resulted in a 70% reduction in insecticide applications. This saves up to US\$ 30 per ha in insecticide costs and results 80–87% increase in cotton yield [127]. A spectacular decrease in pesticide usage in Bt cotton fields has also been reported from China. The pesticide poisoning to the farmers reduced from 22% to 4.7% [128].

To assess probable hazards of Bt toxins on non-target insects, field evaluation was performed in Spain [129]. Bt-maize did not show negative impact on non-target pests. Similar numbers of cutworms and wireworms were present in Bt versus non-Bt fields. Surprisingly, higher

Most of the Cry toxins share common three-domain structure in activated form [130]. Domain I gets inserted into the target membrane and forms pore; domain II is associated with receptor binding and thus determines specificity, and domain III is also involved in receptor-binding specificity. It has been demonstrated in a couple of studies that hybrid Cry toxins exhibit higher toxicity. Domain III of Cry1Ac increased the efficacy of various other Cry1 proteins in Cry1– Cry1Ac hybrid [131]. Similarly, Singh et al. (2004) developed a hybrid toxin against *Spodoptera litura*. They replaced a region in domain III of Cry1Ea toxin by 70 amino acid homologous region of Cry1Ca. Transgenic tobacco and cotton expressing hybrid gene are highly effective/ toxic to all stages of larvae of *S. litura*. Another hybrid *Bt* gene was developed by replacing part of domain II of Cry1Ba with that of Cry2a [132]. The transgenic potato expressing the hybrid toxin showed resistance against Colorado potato beetle, potato tuber moth and European corn borer. The strategy provides new opportunities for resistance management as the target receptor recognition of hybrid toxins is expected to be different from currently used

Detail about lectins and protease inhibitors have been discussed in earlier section. Some other insecticidal roles are summarized here. Besides insecticidal potential, GNA and ASAL also serve as a carrier protein for other insecticidal peptides and proteins to the haemolymph of lepidopteran larvae. It has been demonstrated by feeding GNA-allatostatin and GNA-SFI1 fusions to the tomato moth *Lacanobia oleracea* [133–135]. SFI1 is a neurotoxin isolated from the spider *Segestria florentina*. The individual toxin did not cause toxicity through oral delivery;

Lectins are reported to be insecticidal towards sap-sucking insects, where Bt-toxins are not effective. Transgenic tobacco expressing garlic (*Allium sativum*) leaf lectin showed substantial

numbers of aphids and leafhoppers were observed in Bt field.

**3.2. Strategies for next-generation insect resistance**

346 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

*3.2.2. Plant-derived insecticidal lectins and protease inhibitors*

however, the fusion proteins with GNA were toxic.

*3.2.1. Engineering of Cry toxin by domains swapping*

Cry toxins.

Second-generation Bt transgenic cotton [Bollgard II (Cry1Ac + Cry 2Ab) and Widestrike (Cry1Ac + Cry1F)] are developed to increase the level of resistance against cotton bollworm [143, 144]. It has also been demonstrated that the expression of three insecticidal proteins (Cry1Ac, Cry2A and GNA) into Indica rice control three major pests, rice leaf folder (*Cnapha‐ locrocis medinalis*), yellow stem borer (*Scirpophaga incertulas*) and the brown plant hopper (*Nilaparvata lugens*) [145]. Cry proteins target the leaf folder and the stem borer, and GNA targets the plant hopper. Comparison of three different Bt-cotton lines (either single Cry1Ac or Cry2Ab, or both genes) for insect damage showed that the lines containing two Bt genes performed better [144]. Broccoli expressing both Cry1Ac and Cry1C exhibited increased resistance to diamondback moths and delayed the resistance development [146, 147]. Similarly, transgenic tobacco expressing Cry1Ac and cowpea trypsin inhibitor (CpT 1) delayed resistance development in *H. armigera* [148]. Recently, Bharathi et al. [149] pyramided two lectin genes ASAL and GNA and showed increased resistance against brown plant hopper, green leaf hopper and white backed plant hopper, as compared to their parental lines expressing single lectin. The performance of transgenic plant pyramided with genes has shown that the insecticidal functions of most of the toxins are non-overlapping and non-competitive.

### *3.2.4. Tissue-specific or regulated expression*

Insecticidal proteins are usually expressed under constitutive promoter for higher accumula‐ tion of the proteins. Although the constitutive expression has some advantages, tissue-specific or inducible expression is desirable under certain circumstances. Insect attacks epidermal cells first and therefore the expression of insecticidal proteins under epidermal cell-specific promoters can be a useful strategy. For example, CER6 is an epidermal cell-specific promoter responsible for the expression of an enzyme for cuticular wax production [150]. Similarly, phloem-feeding insects can be targeted by using phloem-specific promoter like PP2 promoter of pumpkin [151], rice sucrose synthase Rss promoter [152] and root phloem-specific promoter AAP3 [153]. Tissue-specific expression of several insecticidal proteins has demonstrated as a good potential for insect control in several studies. Phloem-specific expression of ASAL under promoter Asus1 protects tobacco against aphid, *Myzus nicotianae* [154]. Transgenic chickpea expressing ASAL under rolC promoter showed effective control over *A. craccivora* [155] and transgenic Indian mustard (*Brassica juncea*) expressing ASAL under Rss I promoter showed resistance against aphid *Lipaphis erysimi* [136]. Researchers are also working on sap-sucking pest inducible phloem-specific promoters, which are not only insect-inducible but also insectspecific in nature [156]. Another strategy is temporal expression of insecticidal proteins as some insects infest a crop in a particular phase only. For example, pink bollworm (*Pectinophora gossypiella*) attacks and feeds on the cotton bolls only. At this stage, cotton plants are mature; the expression of Cry toxins goes down and becomes insufficient for effective control.

#### *3.2.5. Strategies to over express secondary metabolites*

Secondary metabolites synthesized by the plants participate in a number of physiological and biochemical processes. Our group demonstrated that the over-expression of pectin methyles‐ terase of *Arabidopsis thaliana* and *Aspergillus niger* in transgenic tobacco plants enhances methanol production, which in turn provided resistance against sap-sucking as well as chewing insect pests [157]. Similarly, transgenic tobacco expressing AtMYB-12 gene showed enhanced production of rutin in leaves and callus, which confers resistance against *H. armigera* and *S. litura* larvae [158, 159]. WsSGTL1, a sterol glycosyltransferases isolated from *Withania somnifera*, was expressed and functionally characterized in transgenic tobacco plants, which showed significant resistance towards *S. litura* [160]. Tobacco plants were transformed by a multigene transfer vector containing three coffee N-methyltransferases genes CaMXT1, CaMXMT1 and CaDXMT1 responsible for producing caffeine in transgenic plants which showed tolerance to *S. litura* [161]. Dixit et al [162] demonstrated the insect resistance by altering the amino acid composition in sap.
