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Agricultural and Biological Sciences » "Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives", book edited by Arun K. Shanker and Chitra Shanker, ISBN 978-953-51-2250-0, Published: February 17, 2016 under CC BY 3.0 license. © The Author(s).

Chapter 14

Molecules and Methods for the Control of Biotic Stress Especially the Insect Pests — Present Scenario and Future Perspective

By Santosh Kumar Upadhyay and Sudhir P. Singh
DOI: 10.5772/62034

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Molecules and Methods for the Control of Biotic Stress Especially the Insect Pests — Present Scenario and Future Perspective

Santosh Kumar Upadhyay1 and Sudhir P. Singh2

1. Introduction

World population is projected to increase over 1,000 million in the next four decades. An immediate priority for agriculture industry is to achieve maximum production in an environmentally sustainable and cost-effective manner. Food security is on high agenda at the political and social level [1]. Our progeny can face a severe shortage of food supply due to the over demand of continuously increasing population. Jaques Diouf, the Director General FAO, stated (2011) “The silent hunger crisis, affecting one sixth of all humanity, poses a serious risk for world peace and security”. The current challenge is to increase primary crop production in agricultural sustainability manner. In order to achieve these goals, it is important to identify and address the major limitations of productivity. Crop damage caused by insect pests is one of the major confinements, which is estimated to be around 35–100%, globally [2]. Synthetic insecticides have made significant contributions in food production, but they are also responsible for environmental and health hazards.

Transgenic crops with enhanced biotic or abiotic stress tolerance have shown promising contribution in achieving greater food security. A milestone was established about 25 years ago with the development of genetically engineered tobacco expressing the entomotoxic Cry protein from the bacterium Bacillus thuringiensis (Bt) [3, 4]. Presently, a number of Bt-Cry protein containing products are in the market of the United States ( and some other countries. In March 2002, the Government of India permitted the release of transgenic cotton (Bollgard) expressing Bt toxins-Cry1Ac for commercial cultivation, which conferred resistance against bollworms [5]. Subsequently, BollgardII was released, which expresses Cry2Ab toxin along with Cry1Ac. Bt-Cry proteins have increased the productivity of crops substantially by controlling the major insects of order Lepidoptera and Coleoptera [6]. However, a concomitant increase in the population of minor pests (like whiteflies, aphids, leafhoppers and others) has threatened the success of Bt-transgenic crops [79]. An alternative strategy is to take advantage of the plant’s own defense mechanisms, either by maneuvering the expression of the endogenous defense proteins or by introduction of an insect toxic gene (like lectins) derived from another plant. Several insecticidal proteins encoding genes have now been isolated from different sources and introduced into crop genomes to combat the issue of various groups of insect pests [10, 11]. Simultaneously, a new approach based on RNA interference is also reported for the control of crop insects [1214]. Present chapter briefs about the insecticidal proteins and transgenic strategies for the control of crop insects.

2. Insecticidal proteins

2.1. Cry toxins of Bacillus thuringiensis

Introduction of Bt-Cry toxins revolutionized the area of insect control through transgenic technology. These are toxic to the insects of orders Lepidoptera, Diptera, Hymenoptera, Coleoptera and also to nematodes. These are produced as parasporal crystalline inclusions in B. thuringiensis. More than 500 Cry proteins/genes have been discovered till date, which are classified into 67 groups (Cry1–Cry67) on the basis of the primary structure [15, 16]. The genes are further divided into four phylogenetically unrelated protein families with different modes of action. These are: (1) three domain Cry toxins (3D) family, (2) mosquitocidal Cry toxins (Mtx) family, (3) binary-like (Bin) family and (4) the Cyt family of toxins [17]. Some Bt strains produce an additional insecticidal toxin called as VIP (vegetative insecticidal proteins) during the vegetative growth phase. Three VIP toxins: VIP1/VIP2, a binary toxin, and VIP3 have been characterized till date [18, 19].

Several insect-resistant transgenic crops have been developed by expressing Bt-Cry proteins, among which corn, cotton, soybean and canola are the most important crops. These transgenic crops are mostly expressing the Cry1Ac and Cry2Ab to control the chewing pests like H. armigera, H. zea and Pectinophora gossypiella, Heliothis virescens and Ostrinia nubilalis [20]. Some other cry toxin based products are also commercialized, which express Cry1A, Cry1F, Cry1EC, Cry34Ab/Cry35Ab binary toxin, Cry1Ab and Cry3Bb for the control of lepidopteran insect Spodoptera frugiperda, S. litura and coleopteran insect Diabrotica virgifera [6, 20, 21]. Further detail about the commercialized insecticidal crops are given in a later section.

2.2. Lectins

Lectins are carbohydrate-binding proteins, which possess at least one non-catalytic domain for specific and reversible binding to mono- or oligosaccharides [22, 23]. A typical lectin is multivalent in nature, therefore agglutinate cells. Lectins are extensively distributed in nature from prokaryotes to eukaryotes. The specific interaction with glycoconjugates makes them valuable in biomedical sciences and biotechnology [24]. Carbohydrates present in viruses, microorganisms, fungi, nematodes or phytophagous insects interact with plant lectins [25, 26]. In the past decades, many plant lectins are reported to be toxic to several economically important insect pests of various orders [2729]. To analyze the insecticidal properties under natural conditions, many transgenic plants expressing lectins have been developed. The toxic effects of different lectins have been demonstrated on several insect species; these effects range from a severe delay in development to high mortality in insects [11].

2.2.1. GNA-related lectins

Galanthus nivalis agglutinin (GNA) purified from snowdrop bulbs is the best studied plant lectin for insecticidal properties. The snowdrop lectin specifically binds to terminal mannose residues in high-mannose-N-glycans, which occur very frequently on insect glycoproteins [30]. Toxicity of GNA has been shown for a wide range of insects; but homopteran insects are highly sensitive to GNA. Several GNA-related lectins have been isolated from different Allium species which have shown the potential for insect control [11, 29]. Further, accumulation of some lectins like Allium porrum agglutinin in the phloem sap in natural situations support the defensive role of lectins against sap-sucking insects [31].

GNA and related lectins have been successfully expressed for resistance against insect pests into a variety of crops [32]. Transgenic rice expressing ASAL caused significant mortality in nymph of hemipteran insect pests [33]. Onion (Allium cepa) lectin has shown more potential against mustard aphid (Lipaphis erysimi) in comparison to GNA and ASAL (Allium sativum leaf agglutinin) [34]. Transgenic rice expressing ASAL exhibited protection against tungro disease also, after infestation with the N. virescens [35]. Vajhala et al. [36] recently demonstrated significant protection in ASAL expressing transgenic cotton against jassid and whitefly. ASAL is also reported to be toxic to chewing insects like Helicoverpa armigera and Spodoptera litura [27] and several other sucking insects like Nephotettix virescens and Nilaparvata lugens [37]. Studies related to the mechanism of toxicity showed that ASAL shares the similar receptors with Bt-Cry toxin [28], but both the proteins interact at different positions without steric hindrance and increased the toxicity of each other [29]. Therefore, they can be pyramided together for broad-range insect resistance.

2.2.2. Legume lectins

Legume lectins are purified from seeds and bind to carbohydrate structures like Thomsen-nouveau (Tn) antigen or complex N-glycan with terminal galactose and sialic acid residues. Pea lectin (Pisum sativum agglutinin, PSA) expressed in transgenic oilseed rape (Brassica napus) shows growth retardation of the pollen beetle larvae (Meligethes aeneus) [38] and no effect on the adult beetles [39]. A legume lectin known as Gleheda purified from ground ivy (Glechoma hederacea) exhibits high insecticidal activity against the Colorado potato beetle larvae (Leptinotarsa decem-lineata) [40]. GS-II lectin isolated from the seed of Griffonia simplicifolia shows toxicity to Cowpea weevil (Callosobruchus maculates) [41]. A mannose-binding legume lectin concanavalin A (ConA) from jackbean has shown toxicity to the hemipteran pea aphid (Acyrthosiphon pisum) [42, 43] and tara plant hopper (Tarophagous proserpina) [44].

2.2.3. Hevein-related lectins

Hevein-related plant lectins exhibit specificity for chitin (chitin forms endo- and exo-arthropods, nematodes and fungi). These are also studied for insecticidal properties [45]. Due to the absence of chitin in mammals, hevein-related lectins are considered safe for the usage in genetically modified crops. Wheat germ agglutinin (WGA) has shown a negative effect on the development of the cowpea weevil (Callosobruchus maculatus) larvae and southern corn root worm (Diabrotica undecimpunctata) [46, 47]. WGA is active against lepidopteran insect larvae also [47, 48].

2.2.4. Other insecticidal lectins

Several other plant lectins have shown insecticidal property. Transgenic tobacco plants expressing tobacco leaf lectin (NICTABA) is detrimental to the cotton leafworm (S. littoralis) and the tobacco hornworm (M. sexta) [49]. Another protein, phloem protein 2 (PP2) belonging to the NICTABA family, also possesses insecticidal activity [50, 51]. The amaranthins and the jacalin-related lectins have also shown the potential for insect control, especially against sap-sucking insect pests. Transgenic cotton expressing Amaranthus caudatus agglutinin (ACA) under the control of a phloem-specific promoter shows a strong resistance against nymphs of the cotton aphid (Aphis gossypii) [52]. Transgenic tobacco expressing Heltuba, a jacalin-related lectin from the Helianthus tuberosus, showed reduced development and fecundity of the peach–potato aphid (M. persicae) [53]. Another promising jacalin-related lectin HFR1 is produced in resistant varieties of wheat (T. aestivum) during infestation by the Hessian fly larvae. Although HFR1 has not shown any toxicity against Hessian fly, it shows the strong insecticidal activity to the larvae of fruit fly (D. melanogaster) [54].

2.3. Proteinase inhibitors

Proteinase inhibitors (PIs) are small molecular weight proteins which affect several metabolic pathways. They are the major components in seeds and storage organs of crops. Mickel and Standish [55] demonstrated the role of PIs in plant defense for the first time and noticed the abnormality in the development of larvae of certain insects fed on soybean products. The feature was attributed to trypsin inhibitors, and it was found to be toxic to the larvae of flour beetle (Tribolium confusum) [56].

PIs inhibit the digestion of proteins in midgut and cause mortality of insects due to nutritional imbalance [57, 58]. PIs also interfere with several metabolic processes (like moulting) by blocking the proteolytic activation of enzymes [59]. They affect growth and development, multiplication rate and insect life span [6062]. PIs have been expressed in several transgenic plants for resistance against insect pests of several classes [6365]. Pea and soybean trypsin–chymotrypsin inhibitors (PsTI-2, SbBBI) belonging to the Bowman–Birk family [66] and mustard-type trypsin–chymotrypsin variant Chy8 [67] cause significant mortality of pea aphid A. pisum. Plant-derived PIs have been used for the development of insect-resistant transgenic plants and projected as an alternative to Bt-Cry proteins [68, 69].

The majority of plant PIs originate from three main families, namely Solanaceae, Leguminosae and Gramineae [70]. Plant PIs can be grouped into four classes: serine, thiol, metallo and aspartyl. Most plant PIs are inhibitors of microbial and animal serine proteases, such as chymotrypsin, trypsin, elastase and subtilisin [71]. Specificity of protease inhibitor families is mainly based on the amino acid residues present in the active site [72].

2.3.1. Serine (Serpin) protease inhibitors

It is found in almost all kingdoms of organisms [7376]. Several serine PIs have been purified from plants and characterized [77, 78]. Plant serine PIs show inconsistent and varied specificities towards plant proteases [79]. Hordeum vulgare serine PI inhibits trypsin, chymotrypsin [80], thrombin, plasma kallikrein, Factor VIIa and Factor Xa [81]. Triticum aestivum serine PI inhibits chymotrypsin and cathepsin G [82]. Serine protease inhibitors have been used most commonly for the development of transgenic plants for the control of insect pests [8385].

2.3.2. Cysteine protease inhibitors

An inhibitor of cysteine proteinases was first described in egg white by Sen and Whitaker [86] and was later named cystatin [87]. Cysteine proteinases inhibitors are widely distributed in plants, animals and microorganisms [88]. Their role in defense has been explored by in vitro analysis on inhibition of digestive proteinases from insect pests and nematodes [8991]. First plant cystatin was isolated from rice seeds and as of now, more than 80 members of different plant species have been characterized [92, 93]. Barley cystatin in artificial diets hampered the life cycle of two aphid species and also in transgenic Arabidopsis [94]. Expression of such inhibitors in maize enhanced the resistance against phytophagous mites [95]. Inhibition of these proteases provides a promising control on insects and therefore PIs can be employed as a potential source of defense in plants against insect pests.

2.3.3. Aspartyl protease inhibitors

It is relatively less studied class, due to the rare occurrence [91]. Potato tubers possess cathepsin D, an aspartic proteinase inhibitor which showed substantial amino acid sequence similarity with the soybean trypsin inhibitor [96]. Aspartic proteases have been found in coleoptera species, such as Callosobruchus maculatus [97] and H. hampei [98], in which the acidic pH in midgut provides a favourable condition for these proteases [58].

2.3.4. Metallo-proteases inhibitors

The metallo carboxypeptidase inhibitors (MCPIs) have been identified in solanaceaous plants tomato and potato [99]. The MCPIs are 38–39 amino acid residues long polypeptide [100, 101]. Plants have evolved at least two families of metalloproteinase inhibitors, the metallo-carboxypeptidase inhibitor family in potato and tomato [102] and a cathepsin D inhibitor family in potato [103]. The inhibitor is produced in potato tubers and accumulates with potato inhibitor I and II families (serine proteinase inhibitors) during tuber development. The inhibitor also accumulates in potato leaf with inhibitor I and II in response to wounding and have the potential to inhibit all the major digestive enzymes (like trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B) of higher animals and many insects [104].

2.4. α-Amylase inhibitors

α-Amylases (α-1,4-glucan-4-glucanohydrolases) are hydrolytic enzymes, which catalyze the hydrolysis of α-1,4-glycosydic bonds in polysaccharides. They are present in microorganisms, animals and plants [105107]. They are the most important digestive enzymes of many insects which feed exclusively on seed products. Inhibition of α-amylase impairs the digestion in an organism and causes shortage of free sugar for energy. α-Amylase inhibitors (α-AI) are found in many plants as a part of the defense system and abundant in cereals and legumes [108111].

α-AI of Phaseolus vulgaris is the most studied amylase inhibitor and have shown toxic effects to several insect pests [110, 111]. Like lectins, they possess carbohydrate-binding property. There are at least four types of Phaseolus amylase inhibitors on the basis of α-AIs: AI-1, AI-2, AI-3 and the null type [112]. AI-1 is present in the most cultivated common bean varieties and inhibits mammalian α-amylases. It also inhibits α-amylases in insects like C. chinensis, C. maculatus and B. pisorum [106]. AI-2 is 78% homologous to AI-1 and found in few wild accessions. It inhibits the Z. subfasciatus larval α-amylase and pea bruchid α-amylase [106, 111, 113]. This inhibitor is a good example of co-evolution of insect digestive enzymes and plant defense proteins.

They are potential molecules for the development of insect-resistant transgenic plants [114, 115]. Seeds of transgenic pea and azuki, expressing α-AI-1 inhibitor of P. vulgaris, shows resistance against pea weevil (Bruchus pisorum), cowpea weevil (C. maculatus) and azuki bean weevil (Callosobruchus chinensis) [110, 113, 116].

2.5. Chitinase

Chitinases are being employed in plant defense in many ways. It has been used in controlling the growth of fungi and insects. Expression of poplar chitinase in tomato leads to growth inhibition in Colorado potato beetle [117]. Secretome analysis of tobacco cell suspension represents chitinase as the major defense protein [118]. A chitinase-like domain containing 56-kDa defense protein (MLX56) provides strong resistance against cabbage armyworm, Mamestra brassicae, and Eri silkworm, Samia ricini [119]. Two chitinase like proteins LA-a and LA-b (latex abundant) from Mulberry (Morus sp.) latex are found to be toxic against Drosophila melanogaster [120].

Chitinases have also been isolated from insects and found to be equally promising in plant defense. Transgenic tobacco plants expressing chitinase of tobacco hornworm (Manduca sexta) shows resistance to tobacco budworm Heliothis virescens [121]. Hornworm chitinase expressing transgenic plants are also resistant against fungal infection [122]. Further, a recombinant baculovirus expressing chitinase of hard tick (Haemaphysalias longicornis) has been shown as bio-acaricide for tick control [123].

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 day-by-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 environmental 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 [133135]. 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.

3.2.3. Multiple insecticidal proteins containing transgenic crop

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 (Cnaphalocrocis 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 accumulation 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 insect-specific 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 methylesterase 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.

4. Conclusions and perspectives

Transgenic technology (especially Bt crops) has contributed significantly in increasing the crop production worldwide. The crops are protected from being damaged by insect pests. Certainly, this methodology provides an environmentally safe alternative for the synthetic pesticides. Further, it has also been proven to be useful in enhancing nutritional values of crops, improvement of stress tolerance and production of pharmaceutical proteins. Introduction of Bt cotton varieties in India has tremendously increased the yields of cotton and thereby profits to the farmers. Bt proteins are able to control the damage caused by Lepidopteran and Coleopteran insects, but not effective against sap-sucking Homopteran pests [8, 9]. Therefore, an unusual increase in the population of homopteran pests like whiteflies, aphids and leafhoppers on transgenic cotton has been reported [7]. Further, development of resistance in insects against toxins is also going to be a major point of concern, which might ultimately challenge the future of Bt crops. Some defense-related proteins like plant lectins, PIs and chitinases are reported to be toxic to various homopteran insect pests. However, several safety and societal concerns are raised from time to time. Further, there is non-availability of an effective and safe protein against several important and emerging insects, which need an ab initio approach to resolve this issue. A promising and biosafe strategy to defeat the above problem can be: (a) exploration of the plant’s own defense mechanisms and manipulation of their expressions or (b) by introducing a gene for insect control derived from other plants, especially derived from non-host plants, and (c) pyramiding of insecticidal proteins for the control of multiple insect pests. Exploration of RNAi mediated insect control by targeting high expressing and/or important vital genes can also be an effective approach (12-14, 163, 164). Besides this, in our country, we need a dedicated forum to popularize the use of genetically modified crops and convince the government as well as citizens at ethical issues.


SKU acknowledges Department of Science and Technology-INSPIRE faculty fellowship.


1 - Anon (2010) Food 2030, London UK: Department for Food and Rural Affairs.
2 - Ferry N, Gatehouse AMR (2010) Transgenic crop plants for resistance to biotic stress, In Transgenic crop plants: utilization and biosafety 2 (eds C Kole, CH Michler, AG Abbott & TC Hall) pp. 1–66, Germany: Springer-Verlag.
3 - Andrews RE, Faust RM, Wabiko H, Raymond KC, Bulla LA (1987) The biotechnology of Bacillus thuringiensis, Crit Rev Biotech 6 163–232.
4 - Vaeck M, Reynaerts A, Hofte H, Jansens S, de Beuckleer M, Dean C, Zabeau M, Van Montagu M, Leemans J (1987) Transgenic plants protected from insect attack, Nature 328 33–37.
5 - Xie R, Zhuang M, Ross LS, Gomez I, Oltean DI, Bravo A, Soberon M, Gill S (2005) Single amino acid mutation in the cadherin recepter from Heliothis virescens affect its toxin binding ability to cry1A toxins, J Biol Chem 280 8416–8425.
6 - Sanahuja G, Banakar R, Twyman RM, Capell T, Christou P (2011) Bacillus thuringiensis: a century of research development and commercial applications, Plant Biotechnol J 9 283–300.
7 - Abro GH, Syed TS, Tunio GM, and Khuhro MA (2004) Performance of transgenic Bt cotton against insect pest infestation, Biotechnology 3 75–81.
8 - Dutt U (2007) Mealy bug infestation in Punjab: Bt cotton falls flat, Environment News Service 21 August (
9 - Virla EG, Casuso M, Frias E A (2010) A preliminary study on the effects of a transgenic corn event on the non targrt pest Dalbulus Maiid (Hemitera: Cicadeliidae), Crop protection 29 635–638.
10 - Carlini CR, Grossi-de-Sa MF (2002) Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides, Toxicon 40 1515–1539.
11 - Vandenborre G, Smagghe G, and VanDamme EJM (2011) Plant lectins as defense proteins against phytophagous insects, Phytochemistry 72 1538-50.
12 - Upadhyay SK, Chandrashekar K, Thakur N, Verma PC, Singh PK, Tuli R (2011) RNA interference (RNAi) for the control of whitefly (Bemisia tobaci), Journal of Biosciences 36 153–161.
13 - Upadhyay SK, Dixit S, Sharma S, Singh H, Kumar J, Verma PC, Chandrashekar K (2013) siRNA machinery in whitefly (Bemisia tabaci), Plos ONE 8(12) e83692 doi: 10.1371/journal.pone.0083692.
14 - Thakur N, Upadhyay SK, Verma PC, Chandrashekar K, Tuli R, Singh PK (2014) Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene, PLoS ONE 9 e87235.
15 - Crickmore N, Zeigler DR, Schnepf E, Van Rie J, Lereclus D, Baum J, Bravo A, Dean DH (2010) Bacillus thuringiensis toxin nomenclature. Crickmore/Bt/index.html.
16 - Upadhyay SK, Singh PK (2011) Role of alkaline phosphatase in insecticidal action of Cry1Ac against Helicoverpa armigera larvae, Biotechnology lettters 33 2027-2036.
17 - Bravo A, Gill SS, Soberón M (2005) Bacillus thuringiensis mechanisms and use, In: Gilbert LI, Iatrou K, Gill SS, (Eds) Comprehensive Molecluar Insect Science, Elsevier BV ISBN 0-4 4-451516-X 175–206.
18 - Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG (1996) Vip3A a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects, Proc Natl Acad Sci USA 93 5389–5394.
19 - Warren G (1997) Vegetative insecticidal proteins: novel proteins for control of corn pests, In: Carozzi N, Koziel M, (Eds) Advances in Insect Control: The Role of Transgenic Plants, Taylor & Francis Ltd p. 109.
20 - Christou P, Capell T, Kohli A, Gatehouse JA, Gatehouse AM (2006) Recent developments and future prospects in insect pest control in transgenic crops, Trends Plant Sci 11 302–308.
21 - Singh PK, Kumar M, Chaturvedi CP, Yadav D, Tuli R (2004) Development of hybrid delta endotoxin and its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura, Transgenic Res 13 397–410.
22 - Vijayan M, Chandra N (1999) Lectins, Curr Opin Struct Biol 9 707–714.
23 - Upadhyay SK, Saurabh S, Singh R, Rai P, Dubey NK, Chandrashekar K, Negi KS, Tuli R, Singh PK, (2011) Purification and Characterization of a Lectin with High Hemagglutination Property Isolated from Allium altaicum, Protein J 30 374–83.
24 - VanDriessche E, Fischer J, Beeckmans S, Bog-Hanse TC (1996) Lectins Biology Biochemistry Clinical Biochemistry Textop Hellerup Denmark 11 215–219.
25 - Upadhyay SK, Singh S, Chandrashekar K, Singh PK, Tuli R (2012) Compatibility of garlic (Allium sativum L.) leaf agglutinin and Cry1Ac δ-endotoxin for gene pyramiding, Applied microbiology and Biotechnology 93 2365–2375.
26 - Ripoll C, Favery B, Lecompte P, Van Damme EJM, Peumans WJ, Abad P, Jouanin L (2003) Evaluation of the ability of lectin from snowdrop (Galanthus nivalis) to protect plants against root-knot nematodes, Plant Sci 164 517–523.
27 - Upadhyay SK, Saurabh S, Rai P, Singh R, Chandrashekar K, Verma PC, Singh PK, Tuli R (2010a) SUMO fusion facilitates expression and purification of garlic leaf lectin but modifies some of its properties, J Biotechnol 146 1–8.
28 - Upadhyay SK, Mishra M, Singh H, Ranjan A, Chandrashekar K, Verma PC, Singh PK, Tuli R (2010b) Interaction of Allium sativum leaf agglutinin (ASAL) with midgut brush border membrane vesicle proteins and its stability in Helicoverpa armigera, Proteomics 10 4431–4440.
29 - Upadhyay SK, Singh PK (2012) Receptors of garlic (Allium sativum) lectins and their role in insecticidal action, Protein J 31(6) 439–446.
30 - Schachter H (2009) Paucimannose N-glycans in Caenorhabditis elegans and Drosophila melanogaster, Carbohydr Res 344 1391–1396.
31 - Peumans WJ, Smeets K, VanNerum K, VanLeuven F, VanDamme EJM (1997) Lectin and alliinase are the predominant proteins in nectar from leek (Allium porrum L,) flowers, Planta 201 298–302.
32 - Wang Z, Zhang K, Sun X, Tang K, Zhang J (2005) Enhancement of resistance to aphids by introducing the snowdrop lectin gene gna into maize plants, J Biosci 30 627–638.
33 - Yarasi B, Sadumpati V, Immanni CP, Vudem DR, Khareedu VR (2008) Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests, BMC Plant Biology 8 102.
34 - Hossain MA, Maiti MK, Basu A, Sen S, Ghosh AK, Sen SK (2006) Transgenic expression of onion leaf lectin gene in Indian mustard offers protection against aphid colonization, Crop Sci 46 2022–2032.
35 - Saha P, Dasgupta I, Das S (2006) A novel approach for developing resistance in rice against phloem limited viruses by antagonizing the phloem feeding hemipteran vectors, Plant Mol Biol 62 735–752.
36 - Vajhala CSK, Sadumpati VK, Nunna HR, Puligundla SK, Vudem DR, et al. (2013) Development of transgenic cotton lines expressing Allium sativum agglutinin (ASAL) for enhanced resistance against major sap-sucking pests. PLoS ONE 8 e72542.
37 - Bala A, Roy A, Behura N, Hess D, Das S (2013) Insight to the mode of action of Allium sativum leaf agglutinin (ASAL) expressing in T3 rice lines on brown plant hopper, Am J Plant Sci 4 400–407.
38 - Melander M, Ahman I, Kamnert I, and Strömdahl AC (2003) Pea lectin expressed transgenically in oilseed rape reduces growth rate of pollen beetle larvae, Transgenic Res 12 555–567.
39 - Lehrman A, Ahman I, Ekbom B (2007) Influence of pea lectin expressed transgenically in oilseed rape (Brassica napus) on adult pollen beetle (Meligethes aeneus), J Appl Entomol 131 319–325.
40 - Wang W, Hause B, Peumans WJ, Smagghe G, Mackie A, Fraser R, VanDamme EJM (2003) The Tn antigen-specific lectin from ground ivy is an insecticidal protein with an unusual physiology, Plant Physiol 132 1322–1334.
41 - Zhu K, Huesing JE, Shade RE, Bressan RA, Hasegawa PM, Murdock LL (1996) An insecticidal N-acetylglucosamine-specific lectin gene from Griffonia simplicifolia (Leguminosae), Plant Physiol 110 195–202.
42 - Sauvion N, Charles H, Febvay G, Rahbé Y (2004a) Effects of jackbean lectin (ConA) on the feeding behavior and kinetics of intoxication of the pea aphid Acyrthosiphon pisum, Entomol Exp Appl 10 34–44.
43 - Sauvion N, Nerdon C, Febvay G, Gatehouse AMR, Rahbé Y (2004b) Binding of the insecticidal lectin Concanavalin A in pea aphid Acyrthosiphon pisum (Harris) and induced effects on the structure of midgut epithelial cells, J Insect Physiol 50 1137–1150.
44 - Powell KS (2001) Antimetabolic effects of plant lectins towards nymphal stages of the planthoppers Tarophagous proserpina and Nilaparvata lugens, Entomol Exp Appl 99 71–78.
45 - Merzendorfer H (2006) Insect chitin synthases: a review, J Comp Physiol 176 1–15.
46 - Huesing JE, Murdock LL, Shade RE (1991) Effect of wheat germ isolectins on the development of the cowpea weevil, Phytochemistry 30 785–788.
47 - Czapla TH, Lang BA (1990) Effect of plant lectins on the larval development of the European corn borer (Lepidoptera: Pyralidae) and the Southern corn rootworm (Coleoptera: Chrysomelidae), J Econ Entomol 83 2480–2485.
48 - Hopkins TL, Harper MS (2001) Lepidopteran peritrophic membranes and the effect of dietary wheat germ agglutinin on their formation and structure, Archiv Insect Biochem Physiol 47 100–109.
49 - Vandenborre G, Groten K, Smagghe G, Lannoo N, Baldwin IT, VanDamme EJM (2010a) Nicotiana tabacum agglutinin is active against Lepidopteran pest insects, J Exp Bot 61 1003–1014.
50 - Beneteau J, Renard D, Marché L, Douville E, Lavenant L, Rahbé Y, Dupont D, Vilaine F, Dinant S, (2010) Binding properties of the N -acetylglucosamine and high-mannose N-glycan PP2–A1 phloem lectin in Arabidopsis, Plant Physiol 153 1345–1361.
51 - Dinant S, Clark AM, Zhu Y, Vilaine F, Palauqui JC, Kusiak C, Thompson GA (2003) Diversity of the superfamily of phloem lectins (phloem protein 2) in angiosperms, Plant Physiol 131 114–128.
52 - Wu J, Luo X, Guo H, Xiao J, Tian Y (2006) Transgenic cotton expressing Amaranthus caudatus agglutinin confers enhanced resistance to aphids, Plant Breeding 125 390–394.
53 - Chang T, Chen L, Chen S, Cai H, Liu X, Xiao G, Zhu Z (2003) Transformation of tobacco with genes encoding Helianthus tuberosus agglutinin (HTA) confers resistance to peach-potato aphid (Myzus persicae), Transgenic Res 12 607–614.
54 - Subramanyam S, Smith DF, Clemens JC, Webb MA, Sardesai N, Williams CE (2008) Functional characterization of HFR-1 a high-mannoseN-glycan-specific wheat lectin induced by Hessian fly larvae, Plant Physiol 147 1412–1426.
55 - Mickel CE, Standish J (1947) Susceptibility of processed soy flour and soy grits in storage to attack by Tribolium castaneum, University of Minnesota, Agricultural Experimental Station, Technical Bulletin, 178 1–20,
56 - Lipke H, Fraenkel GS, Liener I (1954) Effect of soybean inhibitors on growth of Tribolium confusum, J Sci Food Agri 2 410–415.
57 - Broadway R, Duffey S (1986) Plant proteinase inhibitors: mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua, J Insect Physiol 32 827–833.
58 - Ryan CA (1990) Proteinase inhibitors in plants: genes for improving defenses against insects and pathogens, Annu Rev Phytopathol 28 425–449.
59 - Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D (1987) A novel mechanism of insect resistance engineered into tobacco, Nature 330 160–163.
60 - Gatehouse AM, Norton E, Davison GM, Babbé SM, Newell CA, Gatehouse JA (1999) Digestive proteolytic activity in larvae of tomato moth Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo, J Insect Physiol 45 545–558.
61 - Annadana S, Peters J, Gruden K, Schipper A, Outchkourov NS, Beekwilder MJ, Udayakumar M, Jongsma MA (2002) Effects of cysteine protease inhibitors on oviposition rate of the western flower thrips Frankliniella occidentalis, J Insect Physiol 48 701–706.
62 - Oppert B, Morgan TD, Hartzer K, Lenarcic B, Galesa K, Brzin J, Turk V, Yoza K, Ohtsubo K, Kramer KJ (2003) Effects of proteinase inhibitors on digestive proteinases and growth of the red flour beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), Comp Biochem Physiol Toxicol Pharmacol 134 481–90.
63 - De Leo F, Ceci LR, Jouanin L, Gallerani R (2001) Analysis of mustard trypsin inhibitor-2 gene expression in response to developmental or environmental induction, Planta 212 710–717.
64 - Upadhyay SK, Chandrashekar K (2012) Interaction of salivary and midgut proteins of Helicoverpa armigera with soybean trypsin inhibitor. Protein J 31 259–64.
65 - Alfonso-Rubi J, Ortego F, Castanera P, Carbonero P, Diaz I (2003) Transgenic expression of trypsin inhibitor Cme from barley in indica and japonica rice confers resistance to the rice weevil Sitophilus oryzae, Transgenic Res 12 23–31.
66 - Rahbe Y, Ferrasson E, Rabesona H, Quillien L (2003a) Toxicity to the pea aphid Acyrthosiphon pisum of anti-chymotrypsin isoforms and fragments of Bowman-Birk protease inhibitors from pea seeds, Insect Biochem Molecul Biol 33 299–306.
67 - Ceci LR, Volpicella M, Rahbe Y, Gallerani R, Beekwilder J, Jongsma MA (2003) Selection by phage display of a variant mustard trypsin inhibitor toxic against aphids, The Plant J 33 557–566.
68 - Abdeen A, Virgós A, Olivella E, Villanueva J, Avilés X, Gabarra R, Prat S (2005) Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors, Plant Mol Biol 57 189–202.
69 - Ferry N, Edwards M, Gatehouse JA, Capell T, Christou P, Gatehouse AMR (2006) Transgenic plants for insect pest control: a forward looking scientific perspective, Transgen Res 15 13–19.
70 - Richardson MJ (1991) Seed storage proteins: The enzyme inhibitors, In Methods in Plant Biochemistry, Edited by: Richardson MJ. New York: Academic Press; pp. 259-305.
71 - Michaud D (2000) Recombinant Protease Inhibitors in Plants. Eurekah Georgetown.
72 - Koiwa H, Bressan RA, Hasegawa PM (1997) Regulation of protease inhibitors and plant defense, Trends in Plant Sci 2 379–384.
73 - Gettins PG (2002) Serpin structure mechanism and function, Chem Rev 102 4751–4804.
74 - Christeller J, Liang W (2005) Plant serine protease inhibitors, Protein Peptide Lett 12 439–447.
75 - Law R, Zhang Q, McGowan S, Buckle AM, Silverman GA, Wong W, Rosado CJ, Langendorf CG, Bird PI, Whisstock JC (2006) An overview of the serpin superfamily, Genome Biol 7 216.
76 - Irving JA, Pike RN, Lesk AM, Whisstock JC (2000) Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function, Genome Res 10 1845–1864.
77 - Yoo BC, Aoki K, Xiang Y, Campbell LR, Hull RJ, Xoconostle-Cazares B, Monzer J, Lee JY, Ullman DE, Lucas WJ (2000) Characterization of Cucurbita maxima phloem serpin-1 (CmPS-1), A developmentally regulated elastase inhibitor, J Biol Chem 275 35122–35128.
78 - Tsybina T, Dunaevsky Y, Musolyamov A, Egorov T, Larionova N, Popykina N, Belozersky M (2004) New protease inhibitors from buckwheat seeds: properties partial amino acid sequences and possible biological role, Biol Chem 385 429–434.
79 - Al-Khunaizi M, Luke CJ, Cataltepe S, Miller D, Mills DR, Tsu C, Brömme D, Irving JA, Whisstock JC, Silverman GA (2002) The serpin SON-5 is a dual mechanistic class inhibitor of serine and cysteine proteinases, Biochemistry 41 3189–3199.
80 - Dahl SW, Rasmussen SK, Hejgaard J (1996a) Heterologous expression of three plant serpins with distinct inhibitory specificities, J Biol Chem 271 25083–25088.
81 - Dahl SW, Rasmussen SK, Petersen LC, Hejgaard J (1996b) Inhibition of coagulation factors by recombinant barley serpin BSZX, FEBS Lett 394 165–168.
82 - Roberts TH, Marttila S, Rasmussen SK, Hejgaard J (2003) Differentia gene expression for suicide-substrate serine proteinase inhibitor (serpins) in vegetative and grain tissues of barley, J Exp Bot 54 2251–2263.
83 - Savić JM, Smigocki AC (2012) Beta vulgaris L. serine proteinase inhibitor gene expression in insect resistant sugar beet, Euphytica 188 187–198.
84 - Schlüter U, Benchabane M, Munger A, Kiggundu A, Vorster J, Goulet MC, Cloutier C, Michaud D (2010) Recombinant protease inhibitors for herbivore pest control: a multitrophic perspective, J Exp Bot 61 4169–4183.
85 - Smigocki AC, Ivic-Haymes S, Li H, Savić J (2013) Pest protection conferred by a beta vulgaris serine proteinase inhibitor gene, PLoS ONE 8 e57303.
86 - Sen LC, Whitaker JR (1973) Some properties of a ficin-papain inhibitor from avian egg white, Arch Biochem Biophys 158 623–632.
87 - Anastasi A, Brown A, Kembhavi AA, Nicklin MJH, Sayers CA, Sunter DC, Barrett AJ (1983) Cystatin a protein inhibitor of cysteine proteinases, Improved purification from egg white characterization and detection in chicken serum, Biochem J 211 129–138.
88 - Oliveira AS, Filho JX, Sales MP (2003) Cysteine proteinases cystatins, Braz Arch Biol Technol 46 91–104.
89 - Pernas M, Sanchez M,R, Gomez L, Salcedo G (1998) A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests, Plant Mol Biol 38 1235–1242.
90 - Siqueira-Junior CL, Fernandes KVS, Machado OLT, Cunha M, Gomes VM, Moura D, Jacinto T (2002) 87 kDa tomato cystatin exhibits properties of a defence protein and forms crystals in prosystemin over-expressing transgenic plants, Plant Physiol Biochem 40 247–254.
91 - Haq SK, Atif SM, Khan RH (2004) Protein proteinase inhibitor genes in combat against insects pests and pathogens: natural and engineered phytoprotection, Archiv Biochem Biophys 431 145–159.
92 - Abe K, Emori Y, Kondo H, Suzuki K, Arai S (1987a) Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin): homology with animal cystatins and transient expression in the ripening process of rice seeds, J Biol Chem 262 16793–16797.
93 - Abe K, Kondo H, Arai S (1987b) Purification and characterization of a rice cysteine proteinase inhibitor, Agric Biol Chem 51 2763–2768.
94 - Carrillo L, Martinez M, Alvarez-Alfageme F, Castañera P, Smagghe G, Diaz I, Ortego F (2011a) A barley cysteine proteinase inhibitor reduces the performance of two aphid species in artificial diets and transgenic Arabidopsis plants, Transgenic Res 20 305–319.
95 - Carrillo L, Martinez M, Ramessar K, Cambra I, Castañera P, Ortego F, Díaz I (2011b) Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases, Plant Cell Rep 30 101–112.
96 - Mares M, Meloun B, Pavlik M, Kostka V, Baudys M (1989) Primary structure of Cathepsin-D inhibitor from potatoes and its structural relationship to trypsin inhibitor family, FEBS Lett 251 94–98.
97 - Silva CP, Xavier-Filho J (1991) Comparison between the levels of aspartic and cysteine proteinases of the larval midguts of Callosobruchus maculatus (F.) and Zabrotes subfasciatus (Boh.) (Coleoptera: Bruchidae), Comp Biochem Physiol 99B 529–533.
98 - Preciado DPR, Valencia AJ (2000) Partial characterization of digestive proteinases from coffee berry borer adults (Hypothenemus hampei), Insect physiology neurosciences immunity and cell biology symposium and Poster Session abstract book II – XXI-International Congress of Entomology Brazil August 20–26.
99 - Habib H, Fazili KM (2007) Plant protease inhibitors: a defense strategy in plants, Biotechnol Molecul Biol Rev 2 068–085.
100 - Hass GM, Nau H, Biemann K, Grahn DT, Ericsson LH, Neurath H (1975) The amino acid sequence of a carboxypeptidase inhibitor from potatoes, Biochem 14 1334–1342.
101 - Hass GM, Hermodson MA (1981) Amino acid sequence of a carboxy-peptidase inhibitor from tomato fruit, Biochemistry 20 2256–2260.
102 - Rancour JM, Ryan CA (1968) Isolation of a carboxypeptidase B inhibitor from potatoes, Arch Biochem Biophys 125 380–382.
103 - Keilova H, Tomasek V (1976) Isolation and properties of cathepsin D inhibitor from potatoes, Collect Czech Chem Commun 41 489–497.
104 - Hollander-Czytko H, Andersen JL, Graham JS, Ryan CA (1997) Accumulation of metallocarboxy-peptidase inhibitor in leaves of wounded potato plants, Biochem Biophys Res Commun 101 1164–1170.
105 - Qian MX, Haser R, Payan F (1993) Structure and molecular-model refinement of pig pancreatic alpha-amylase at 2,1 angstrom resolution, J Mol Biol 231 785–799.
106 - Grossi-de-Sa MF, Chrispeels MJ (1997) Molecular cloning of bruchid (Zabrotes subfasciatus) alpha-amylase cDNA and interactions of the expressed enzyme with bean amylase inhibitors, Insect Biochem Mol Biol 27 271–281.
107 - Strobl S, Maskos K, Betz M, Wiegand G, Huber R, Gomis-Ru°th FX, Glockshuber R (1998) Crystal structure of yellow meal worm a-amylase at 1,64 A˚ resolution, J Mol Biol 278 617–628.
108 - Franco OL, Rigden DJ, Melo FR, Bloch C Jr, Silva CP, Grossi-de-Sa´ MF (2000) Activity of wheat α–amylase inhibitors towards bruchid α-amylases and structural explanation of observed specificities, Eur J Biochem 267 2166–2173.
109 - Iulek J, Franco OL, Silva M, Slivinski CT, Bloch C Jr, Rigden DJ, Grossi-de-Sa´ MF (2000) Purification biochemi-cal characterisation and partial primary structure of a new a -amylase inhibitor from Secale cereale (Rye), Int J Biochem Cell Physiol 32 1195–1204.
110 - Ishimoto M, Chrispeels MJ (1996) Protective mechanism of the Mexican bean weevil against high levels of α-amylase inhibitor in the common bean, Plant Physiol 111 393–401.
111 - Grossi-de-Sa MF, Mirkov TE, Ishimoto M, Colucci G, Bateman KS, Chrispeels MJ (1997) Molecular characterization of a bean α-amylase inhibitor that inhibits the α-amylase of the Mexican bean weevil Zabrotes subfasciatus, Planta 203 295–303.
112 - Suzuki K, Ishimoto M (1999) Characterization of the third alpha-amylase inhibitor alpha AI-3 in the common bean (Phaseolus vulgaris L.), Breed Sci 49 275–280.
113 - Morton RL, Schroeder HE, Bateman KS, Chrispeels MJ, Armstrong E, Higgins TJV (2000) Bean α-amylase inhibitor-I in transgenic peas (Pisum sativum) provides complete protection from pea weevil (Bruchus pisorum) under field conditions, Proc Natl Acad Sci USA 97 3820–3825.
114 - Gatehouse AMR, Gatehouse JA (1998) Identifying proteins with insecticidal activity: use of encoding genes to produce insect-resistant transgenic crops, Pest Sci 52 165–175.
115 - Valencia A, Bustillo AE, Ossa GE, Chrispeels MJ (2000) α-Amylases of the coffee berry borer (Hypothenemus hampei) and their inhibition by two plant amylase inhibitors, Insect Biochem Mol Biol 30 207–213.
116 - Chrispeels MJ (1996) Transfer of bruchid resistance from the common bean to other starchy grain legumes by genetic engineering with the a-amylase inhibitor gene, In: Carozzi N, Koziel M, (Eds,) Transgenic Plants for Control of Insect Pests, Taylor & Francis London pp. 1–10.
117 - Lawrence SD, Novak NG (2006) Expression of poplar chitinase in tomato leads to inhibition of development in colorado potato beetle, Biotechnol Lett 28 593–599.
118 - Lipmann R, Kaspar S, Rutten T, Melzer M, Kumlehn J, Matros A, Mock HP (2009) Protein and metabolite analysis reveals permanent induction of stress defense and cell regeneration processes in a tobacco cell suspension culture, Int J Mol Sci 10 3012–3032.
119 - Wasano N, Konno K, Nakamura M, Hirayama C, Hattori M, Tateishi K (2009) A unique latex protein MLX56 defends mulberry trees from insects, Phytochemistry 70 880–888.
120 - Kitajima S, Kamei K, Taketani S, Yamaguchi M, Kawai F, Komatsu A, Inukai Y (2010) Two chitinase-like proteins abundantly accumulated in latex of mulberry show insecticidal activity, BMC Biochemistry 11 6.
121 - Ding X, Gopalakrishnan B, Johnson LB, White FF, Wang X, Morgan TD, Kramer KJ, Muthukrishnan S (1998) Insect resistance of transgenic tobacco expressing an insect chitinase gene, Transgenic Res 7 77–84.
122 - Kramer KJ, Muthukrishnan S (1997) Insect chitinases: Molecular biology and potential use as biopesticides, Insect Biochem Mol Bio 27 887–900.
123 - Assenga SP, You M, Shy CH, Yamagishi J, Sakaguchi T, Zhou J, Kibe MK, Xuan X, Fujisaki K (2006) The use of a recombinant baculovirus expressing a chitinase from the hard tick Haemaphysalias longicornis and its potential application as a bioacaricide for tick control, Parasitol Res 98 111–118.
124 - James C (2005) Preview: Global Status of Commercialized Biotech/ GM Crops: 2005. ISAAA Briefs No 34, ISAAA ( pdf).
125 - Sankula S, Marmon G, Blumenthal E (2005). Biotechnology-derived crops planted in 2004 – impacts on US agriculture. National Center for Food and Agricultural Policy (
126 - High SM, Cohen MB, Shu QY, Altosaar I (2004) Achieving successful deployment of Bt rice, Trends Plant Sci 9 286–292.
127 - Qaim M, Zilberman D (2003) Yield effects of genetically modified crops in developing countries, Science 299 900–902.
128 - Huang J, Rozelle S, Pray C, and Wang Q (2002) Plant biotechnology in China, Science 295 674–677.
129 - Eizaguirre M, Albajes R, López C, Eras J, Lumbierres B, Pons X (2006) Six years after the commercial introduction of Bt maize in Spain: field evaluation impact and future prospects, Transgenic Res 15 1–12.
130 - De Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world, Trends Genet 17 193–199.
131 - Karlova R, Weemen WMJ, Naimov S, Ceron J, Dukiandjiev S, Maagd RA de (2005) Bacillus thuringiensis delta endotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some but not all Cry1-Cry1Ac hybrids, J Invertebr Pathol 88 169–172.
132 - Naimov S, Dukiandjiev S, De Maagd RA (2003) A hybrid Bacillus thuringiensis delta endotoxin gives resistance against a coleopteran and a lepidopteran pest in transgenic potato, Plant Biotechnol J 1 51–57.
133 - Fitches E, Audsley N, Gatehouse JA, Edwards JP (2002) Fusion proteins containing neuropeptides as novel insect control agent s: snowdrop lectin delivers fused allatostatin to insect haemolymph following oral ingestion, Insect Biochem Mol Biol 32 1653–1661.
134 - Fitches E, Edwards MG, Mee C, Grishin E, Gatehouse AM, Edwards JP, Gatehouse JA (2004) Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion, J Insect Physiol 50 61–71.
135 - Fitches E, Wiles D, Douglas EA, Hinchliffe G, Audsley N, and Gatehouse JA (2008) The insecticidal activity of recombinant garlic lectins towards aphids, Insect Biochem Mol Biol 38 905–915.
136 - Dutta I, Saha P, Majumder P, Sarkar A, Chakraborti D, Banerjee S, Das S (2005a) The efficacy of a novel insecticidal protein Allium sativum leaf lectin (ASAL) against homopteran insects monitored in transgenic tobacco, Plant Biotech J 3 601–611.
137 - Mehlo L, Gahakwa D, Nghia PT, Loc NT, Capell T, Gatehouse A, Gatehouse AMR, Christou P (2005) An alternative stra tegy for sustainable pest resistance in genetically enhanced crops, Proc Natl Acad Sci USA 102 7812–7816.
138 - Gruden K, Strukelj B, Popovic T, Lenarcic B, Bevec T, Brzin J, Kregar I, Herzog-Velikonja J, Stiekema WJ, Bosch D, Jongsma MA (1998) The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts which is insensitive to potato protease inhibitors is inhibited by thyroglobulin type-1 domain inhibitors, Insect Biochem M ol Biol 28 549–560.
139 - Martínez M, Abraham Z, Carbonero P, and Díaz I (2005a) Comparative phylogenetic analysis of cystatin gene families from Arabidopsis rice and barley, Mol Genet Genomics 273 423–432.
140 - Martínez M, Rubio-Somoza I, Fuentes R, Lara P, Carbonero P, Díaz I, (2005b) The barley cystatin gene (Icy) is regulated by DOF transcription factors in aleur one cells upon germination, J Exp Bot 56 547–556.
141 - Outchkourov NS, de Kogel WJ, Wiegers GL, Abrahamson M, Jongsma MA (2004) Engineered multidomain cysteine protease inhibitors yield resistance against western flower thrips (Franklinielia occidentalis) in greenhouse trials, Plant Biotechnol J 2 449–458.
142 - Zhu-Salzman K, Ahn JE, Salzman R, A, Koiwa H, Shade RE, Balfe S (2003) Fusion of a soybean cysteine protease inhibitor and a legume lectin enhances anti-insect activity synergistically, Agric Amd Forest Entomol 5 317–323.
143 - Gahan LJ, Ma YT, Cobble MLM, Gould F, Moar WJ, Heckel DG (2005) Genetic basis of resistance to Cry1Ac and Cry2Aa in Heliothis virescens (Lepidoptera:Noctuidae), J Econ Entomol 98 1357–1368.
144 - Jackson RE, Bradley Jr JR, VanDuyn JW (2004) Performance of feral and Cry1Ac-selected Helicoverpa zea (Lepidoptera: Noctuidae) strains on transgenic cottons expressing one or two Bacillus thuringiensis kurstaki proteins under greenhouse conditions, J Entomol Sci 39 46–55.
145 - Bano-Maqbool S, Riazuddin S, Loc NT, Gatehouse AMR, Gatehouse JA, Christou P (2001) Expression of multiple insecticidal genes confers broad resistance against arange of different rice pests, Mol Breed 7 85–93.
146 - Cao J, Zhao JZ, Tang JD, Shelton AM, Earle ED (2002) Broccoli plants with pyramided Cry1Ac and Cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins Theor Appl Genet, 105 258–264.
147 - Zhao JZ, Cao J, Li Y, Collins HL, Roush R, Earle ED, Shelton AM (2003) Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution, Nat Biotechnol 21 1493–1497.
148 - Zhao JZ, Fan YL, Fan XL, Shi XP, Lu MG (1999) Evaluation of transgenic tobacco expressing two insecticidal genes to delay resistance development of Helicoverpa armigera, Chin Sci Bull 44 1871–1874.
149 - Bharathi Y, VijayaKumar S, Pasalu I,C, Balachandran S,M, Reddy VD, Rao KV (2011) Pyramided rice lines harbouring Allium sativum (asal) and Galanthus nivalis (gna) lectin genes impart enhanced resistance against major sap-sucking pests, J Biotechnol 152 63–71.
150 - Hooker TS, Millar AA, Kunst L (2002) Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis, Plant Physiol 129 1568–1580.
151 - Guo HN, Chen X, Zhang H, Fang R, Yuan Z, Zhang Z, Tian Y (2004) Characterization and activity enhancement of the phloem-specific pumpkin PP2 gene promoter, Transgenic Res 13 559–566.
152 - Nagadhara D, Ramesh S, Pasalu IC, Rao YK, Sarma NP, Reddy VD, Rao KV (2004) Transgenic rice plants expressing the snow drop lectin gene (gna) exhibit high-level resistance to the white backed plant hopper (Sogatella furcifera), Theor Appl Genet 109 1399– 1405.
153 - Okumoto S, Koch W, Tegeder M, Fischer WN, Biehl A, Leister D, Stierhof YD, and Frommer WB (2004) Root phloem-specific expression of the plasma membrane amino acid proton cotransporter AAP3, J Exp Bot 55 2155–2168.
154 - Sadeghi A, Broeders S, De Greve H, Hernalsteens JP, Peumans WJ, VanDamme EJ, Smagghe G (2007) Expression of garlic leaf lectin under the control of the phloem-specific promoter Asus1 from Arabidopsis thaliana protects tobacco plants against the tobacco aphid (Myzus nicotianae), Pest Manag Sci 63 1215–1223.
155 - Chakraborti D, Sarkar A, Mondal HA, Das S (2009) Tissue specific expression of potent insecticidal Allium sativum leaf agglutinin (ASAL) in important pulse crop chickpea (Cicer arietinum L.) to resist the phloem feeding Aphis craccivora, Transgenic Res 18 529–544.
156 - Dubey N (2012) Identification and characterization of phloem specific promoter induced by sap sucking pests. Ph. D. Thesis, Banaras Hindu University, India.
157 - Dixit S, Upadhyay SK, Singh H, Sidhu OP, Verma PC, Chandrashekar K (2013) Enhanced methanol production in plants provides broad spectrum insect resistance, PLoS ONE 8 e79664.
158 - Mishra P, Pandey A, Tiwari M, Chandrashekar K, Sidhu OP, Asif MH, Chakrabarty D, Singh PK, Trivedi PK, Nath P, Tuli R (2010) Modulation of transcriptome and metabolome of tobacco by Arabidopsis transcription factor, AtMYB12, leads to insect resistance, Plant Physiol 152 2258–2268.
159 - Pandey A, Misra P, Chandrashekar K, Trivedi PK (2012) Development of AtMYB12-expressing transgenic tobacco callus culture for production of rutin with biopesticidal potential, Plant Cell Rep 31 1867–1876.
160 - Pandey V, Niranjan A, Atri N, Chandrashekhar K, Mishra MK, Trivedi PK, Misra P (2014) WsSGTL1 gene from Withania somnifera, modulates glycosylation profile, antioxidant system and confers biotic and salt stress tolerance in transgenic tobacco, Planta doi 10.1007/s00425-014-2046-x.
161 - Kim YS, Uefuji H, Ogita S, Sano H (2006) Transgenic tobacco plants producing caffeine: a potential new strategy for insect pest control, Transgenic Res 15 667–672.
162 - Dixit S, Yadav S, Upadhyay SK, Verma PC, Chandrashekar K (2013) A method to produce insect resistance in plant by altering amino acid content in sap, Int J Biotech Res 3 13-20.
163 - Upadhyay SK, Sharma S, Singh H, Dixit S, Kumar J, Verma PC, Chandrashekar K (2015) Whitefly genome expression reveals host-symbiont interaction in amino acid biosynthesis. Plos ONE 10(5) e0126751 DOI: 10.1371/journal.pone.0126751.
164 - Thakur N, Mundey JK, SK Upadhyay (2015) RNAi: Implications in Entomological Research and Pest Control. ISBN 979-953-307-112-0: InTech - Open Access Publisher.