Sarvjeet Kaur

Insect-resistant transgenic brinjal plants

A synthetic cry1Ab gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis (Bt) was transferred to brinjal (eggplant) by cocultivating cotyledonary explants with Agrobacterium tumefaciens. Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and mRNA expression. Double-antibody sandwich ELISA analysis revealed Bt toxin protein expression in the transgenic plants. The expression resulted in a significant insecticidal activity of transgenic brinjal fruits against the larvae of fruit borer (Leucinodes orbonalis). The results also demonstrated that a synthetic gene based on monocot codon usage can be expressed in dicotyledonous plants for insect control.

Molecular approaches towards development of novel Bacillus thuringiensis biopesticides.

Bacillus thuringiensis (Bt) has been used for control of lepidopteran, dipteran and coleopteran insects for over three decades. Novel Bt strains harbouring new types of insecticidal genes are being discovered worldwide. Recombinant strains with enhanced toxicity and broadened insecticidal spectrum have been constructed. To increase the field persistence of insecticidal crystal proteins (ICPs), alternative modes of their delivery in Pseudomonas sp. and endophytes have been developed. ICPs have been modified by site-directed mutagenesis to improve their insecticidal efficacy. Higher yields of ICPs have been achieved by use of strong expression promoters and other regulatory elements. Gene-disabling of the sporulation-specific protease has led to yield enhancement of ICPs. Interestingly, Bt toxins have been found to act synergistically with some other pesticidal agents. Optimization of fermentation conditions is an essential requirement for cost-effective commercial production of Bt biopesticides. The environmental impact of deployment of genetically engineered biopesticides has been assessed. Recombinant Bt strains that do not carry any non-Bt DNA, endophytes, encapsulation in killed bacteria (such as Pseudomonas) and asporogenous Bt strains are ecologically safe approaches. Efficient resistance management strategies require judicious use of Bt transgenic plants in conjunction with refugia and Bt biopesticides in an Integrated Pest Management (IPM) program.

Natural occurrence of Bacillus thuringiensis in leguminous phylloplanes in the New Delhi region of India

Bacillus thuringiensis (Bt) isolates were present on the phylloplanes of chickpea (Cicer arietinum), pigeon pea (Cajanus cajan), pea (Pisum sativum) and mung bean (Vigna radiata). Bt index (ratio of the number of Bt colonies to the total number of spore-forming colonies per g of leaves) differed significantly among these plants, with the highest (0.20) in the chickpea phylloplane, followed by pigeon pea (0.17). Bt population of the chickpea phylloplane varied with plant age, being maximal in 45-day-old plants. Diversity was observed among Bt isolates for growth (up to 10-fold difference), antibiotic resistance, PCR product profile and toxicity to Helicoverpa armigera. Two isolates with high activity towards H. armigera were found.

Phytotoxicity of solanapyrones produced by the fungus Ascochyta rabiei and their possible role in blight of chickpea (Cicer arietinum)

Solanapyrones A, B and C have been extracted from the culture filtrates and spore germination fluids of Ascochyta rabiei and purified by reverse phase HPLC. Maximum production of solanapyrones was observed in the chickpea seed extract medium at the onset of sporulation. Supplementation of the culture medium with the extract of the host plant stimulated solanapyrone production. Production of solanapyrones by different isolates of Ascochyta rabiei was well correlated with the degree of pathogenicity of these isolates on chickpea. Phytotoxicity of solanapyrones to different chickpea varieties in a host-selective and concentration-dependent manner suggests their role as a factor in the development of chickpea blight.

A membrane-associated auxin-binding site from chickpea (Cicer arietinum L.) epicotyls☆

Abstract A membrane-associated indoleacetic acid (IAA)-binding site with high affinity, high specificity and finite capacity has been found in etiolated chickpea epicotyls. Dissociation constant ( K d ) and binding site concentration are 3 × 10 −7 M and 0.25 pmol/mg protein, respectively. IAA binding is optimum at pH 5.5 and 0°C. The sensitivity of specific binding to pretreatment with proteases including trypsin, sodium dodecylsulfate (SDS) and thiol-reactive agents such as parachloromercuribenzoate (PCMB) and mercuric chloride (HgCl 2 ) indicates that the binding moiety is a protein containing a sulfhydryl group.

Risk Assessment of Bt Transgenic Crops

The global demand, likely to escalate for at least another 40 years, requires a multifaceted strategy to ensure food security. Augmentation of crop yields in a sustainable manner is essential. Pests destroy on an average 14–25% of the total global agricultural production. Biopesticides are an important component of integrated pest management (IPM) strategies employed for minimization of insect pests-incurred crop yield losses as also for reduction of use of environmentally harmful chemical pesticides. Bacillus thuringiensis (Bt) is an aerobic, gram-positive, spore-forming bacterium producing crystal proteins (Cry), which are selectively toxic to target insects. Cry proteins act by insertion into the microvillar brush-border membranes in the midgut of susceptible insects, leading to disruption of osmotic balance, lysis of epithelial cells and eventually death of insect. Some Bt strains additionally secrete vegetative insecticidal proteins (Vips), which cause toxicity in susceptible insects by midgut epithelial cell lysis and gut paralysis. Bt has been used as a microbial biopesticide for the past five decades because of the advantages of specific toxicity against target insects, lack of polluting residues and safety to non-target organisms, and accounts for 95% of the 1% market share of biopesticides in the total pesticide market. However, the use of Bt microbial biopesticide formulations has been rather limited due to the problems of narrow host range, low persistence on plants and inability of foliar application to reach the insects feeding inside the plants, notwithstanding several biotechnological approaches for the development of improved Bt biopesticides. Bt transgenic crops have been developed to overcome the problems of Bt biopesticides and for more effective insect control. Risk assessment in relation to certain concerns raised about environmental and food safety of Bt transgenic crops has been addressed in this chapter.