**2. Insect resistance**

One of the most difficult issues in plant crop cultivation is dealing with insect pests. Insect pests are primarily managed using insecticides, but the rising occurrence of insect resistance genes in many organisms could be harnessed and introduced to crop plants through the effective use of transgenic technology. The cloning of genes codes for insecticidal δ-endotoxins dates back to the early 1980s [1]. Transgenic tomato and tobacco produce modified toxin genes which provide insect resistance res the first examples of genetically modified plants [2–4]. As insect-resistance genes transferred into plants predominantly act on the digestive system of insects, researchers are currently identifying genes with distinct modes of action to combat the development of resistance in the target insects, and to enhance potency. Few noteworthy insect control proteins (ICPs) such as protease inhibitors, different enzymes, ribosomal inactivating proteins, and lectins derived from various genus and domains that have an antimetabolic or toxic effect on insect digestion are being viewed as an alternative to control insect infestation or confer resistance to plants.

#### **2.1 Source of transgenes**

#### *2.1.1 Resistance gene from microorganisms*

#### *2.1.1.1 Cry gene from Bacillus thuringiensis*

The most important and successful example of a transgene derived from the bacterium *B. thuringiensis* is Bt gene toxin [5]. It is a significant soil borne sporeforming bacteria that produces insecticidal crystal (Cry) proteins encoded by *cry*

#### *Genetically Modified Crops and Their Impact on New Era of Agriculture DOI: http://dx.doi.org/10.5772/intechopen.105937*

genes (*cry*-represents gene; Cry-represents protein) during sporulation [5, 6]. Proteinases in the insect gut proteolytically cleave the inactive protoxins to produce the active 60–65Kd toxin made up of high homology regions interspersed with (hyper)variable regions. Sequence analysis [7, 8] and X-ray crystallography were used to infer the structure and functional roles of the toxin's three domains [9] that bind to glycoprotein receptors in the brush border membrane of the midgut epithelium of susceptible insects. The nature of the receptors that explicitly play a significant role in establishing susceptibility/resistance to a specific Bt toxin, is under intensive investigation, with a number of midgut integral membrane glycoproteins, including aminopeptidase and a cadherin-like protein, being identified [10–13]. Following binding, the toxin rapidly and irreversibly inserts into the cell membrane resulting in the formation of a pore supposedly by Cry proteins leading to disruption of the electrical, K+ and pH gradients eventually causing irreversible damage to the midgut wall which gives rise to epithelial cell lysis [6, 14] paving way to gut paralysis, cessation of feeding and finally (typically after 1–3 days) death from starvation and/or septicaemia. These toxins are mainly targeted enzymes and lectins of digestive systems of Lepidopteron and coleopteran pests.

#### *2.1.1.1.1 Codon optimization for higher expression*

Specificity, efficiency and insecticidal activity of toxins are more in codon optimised proteins than native form of Bt toxin and hence the former was introduced into various plants for increased level of expression [14]. Plants harbouring modified codon with plant preference rather than bacterial preference in which G:C rich codons are preferred over A:T rich codons, and undesirable mRNA secondary structure and polyadenylation signals eliminated, produced 100X higher than plants were transformed with unmodified (native) Bt gens [15, 16]. Genes are frequently inserted with constitutive promoters such as maize ubiquitin, CaMV35S, or rice actin 1, promoted protein expression at all times and in all parts of the plant allowing broader-spectrum ICPs to be targeted at different components of the pest complex. The use of tissue specific (e.g., RsS1 promoter for phloem-specific expression or PHA-L promoter for seed-specific expression) or inducible (e.g., potato pin2 wound-induced promoter) promoters is recommended to conserve the space and time of expression of toxic proteins and thus avoid unfavourable interactions with the beneficial insect's ecosystem [17].

The highest risk of resistance development would most likely arise from prolonged exposure to ineffective levels of the transgene, a situation that farmers would not tolerate and would almost certainly necessitate additional (different) control measures (deployment of which would in fact reduce the risk of resistance development). CaMV35S has a notoriously low/no expression in pollen. Temporal and spatial promoters are used to target the pest at its most vulnerable stage and time of infestation.

#### *2.1.1.1.2 Cry protein classification*

Cry protein classification is based on the degree of homology of Cry proteins. Primary Cry protein group: Cry proteins with less than 45 percent amino acid similarity fall into this category. Cry 1 to Cry 78, for example (in 2018). Secondary Cry protein group: Cry proteins with less than 78 percent amino acid similarity fall into this category. Cry 1A and Cry 1B are two examples. Tertiary Cry protein group: Cry proteins with less than 95% amino acid similarity fall into this category. Cry 1Aa, Cry 1Ac, etc. Cry proteins that share more than 95% of their amino acid sequences are


#### **Table 1.**

*Classification of cry proteins based on their amino acids classification.*

classified as part of the Quandary Cry protein group. Cry 1Aa1, Cry 1Aa2 (Cry 1Aa25 was recently discovered in 2019). Each Cry protein of the Bt bacterium has a distinct host range cry protein (**Table 1**) [18].

Even within the Cry protein subfamily, the toxic spectrum varies depending on the host. Cry1A and Cry1C proteins, for example, are toxic to larvae of lepidopteran pests such as the codling moth (*Cydia pomonella*), the European corn borer (*Ostrinia nubilalis*) [19], and heliothine bollworms, respectively. The Cry3A protein, on the other hand, is toxic to coleopteran pests such as the Colorado potato beetle (*Leptinotarsa decemlineata*) [20]. So far, 26 plant species have been genetically engineered and expressed for the Bt toxin [18].
