**3. Insect-resistant transgenic crops**

Development of many transgenic crops has been reported for insect resistance. Both private and public sector organizations are involved in the process and they used δ-endotoxins of *Bacillus thuringiensis* to achieve resistance against insects. Among transgenic plants, cotton and maize were the most successful and released for commercial cultivation. These crops are being adopted annually at very high rates. In other words, area under Bt-crops are increasing dayby-day. Successful deployment of these crops has decreased the pesticide usage. However, the sustainability and durability of pest resistance are still a matter of discussion. It is also important to focus on next-generation insect-resistant transgenic crops.

#### **3.1. First-generation insect-resistant transgenic crops**

Insect-resistant transgenic crops have not only increased the economy but also the environ‐ mental and health benefits [69, 124]. Six transgenic crops (canola, corn, cotton, papaya, squash and soybean) were planted in 2003 in the USA alone. These crops increased farm income by US\$ 1.9 billion by producing an additional 2.4 million tonnes of food and fiber and reduced the use of pesticides by 21,000 tonnes.

In 2009, China government approved the cultivation of Bt-rice (the country has been growing Bt-cotton since 1997). Farm surveys of randomly selected households cultivating Bt-rice 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 numbers of aphids and leafhoppers were observed in Bt field.

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

#### *3.2.1. Engineering of Cry toxin by domains swapping*

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 Cry toxins.

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

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; however, the fusion proteins with GNA were toxic.

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 control over peach potato aphids [136]. Fusion of galactose-binding domain of the non-toxic ricin B-chain with Cry1Ac provides additional binding domains, which increases interactions with the gut receptors in target insects. Transgenic rice and maize expressing the fusion protein show high toxicity in comparison to the Bt-toxin alone [137].

Protease inhibitors (PIs) expressing transgenic plants are not as effective as Bt and insecticidal lectin expressing plants. This is due to the adaptation in gut proteases in phytophagous insects. High genetic diversity in gut proteases and low potency of protease inhibitors is responsible for such adaptation. The combination of inhibitors (potato PI–II and carboxypeptidase) is not enough to avoid the compensatory adaptation [68]. However, inhibitors like barley trypsin inhibitor [65], equistatin from sea anemone [138], other cystatins [139, 140] or use of multiple inhibitors [141] or combination of inhibitors and lectins [142] might also be useful to provide resistance against insects in transgenic plants.
