P. Ananda Kumar

The insecticidal proteins of Bacillus thuringiensis.

Publisher Summary The chapter focuses on the classification and mode of action of the insecticidal proteins of Bacillus thuringiensis (Bt) or Bt toxin. Bt is a gram-positive, aerobic, endospore-forming bacterium. Bt toxin gene is classified into four major classes: cry I, cry II, cry III, and cry IV. Cry is a Bt gene which codes a parasporal inclusion protein that exhibits pesticide activity. Bt toxin genes are usually plasmid borne but also chromosomally located. The chapter discusses on various aspects of the bacterium such as Bt fermentation, biology and genetics, molecular biology, and mechanism of action. The chapter also reviews application of Bt toxin as biopesticide. Bt is the most popular biological control agent. The most effective delivery system for Bt toxins is the transgenic plant. The importance and features of transgenic plants are summarized. Strategies to screen new Bt strains/genes, expression of the toxin protein in transgenic microorganisms and plants and various resistance management strategies in agricultural systems are examined. The review puts emphasis on agricultural application of Bt.

Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance

We evaluated the insecticidal toxicity of Cry1Aa, Cry1Ab and Cry1Ac toxins against neonate larvae of sugarcane shoot borer Chilo infuscatellus Snellen (Lepidoptera: Crambidae) in vitro on diet surface. With the lowest LC50 value, Cry1Ab emerged as the most effective among the three toxins. Sugarcane cultivars Co 86032 and CoJ 64 were transformed with cry1Ab gene driven by maize ubiquitin promoter through particle bombardment and Agrobacterium-mediated transformation systems. Gene pyramiding was also attempted by retransforming sugarcane plants carrying bovine pancreatic trypsin inhibitor (aprotinin) gene, with cry1Ab. Southern analysis confirmed multiple integration of the transgene in case of particle bombardment and single site integration in Agrobacterium-mediated transformants. The expression of cry1Ab was demonstrated through Western analysis and the toxin was quantified using ELISA. The amount of Cry1Ab protein in different events varied from 0.007 to 1.73% of the total soluble leaf protein; the events transformed by Agrobacterium method showed significantly higher values. In in vivo bioassay with neonate larvae of shoot borer, transgenics produced considerably lower percentage of deadhearts despite suffering feeding damage by the borer compared with the untransformed control plants. Expressed Cry1Ab content was negatively related to deadheart damage. Aprotinin-expressing sugarcane pyramided with cry1Ab also showed reduction in damage. The potential of producing sugarcane transgenics with cry1Ab and aprotinin genes resistant to early shoot borer was discussed in the light of the results obtained.

Transgenic tomato plants resistant to fruit borer (Helicoverpa armigera Hubner)

Abstract A synthetic cry 1 Ac gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis ( Bt ) was transferred to tomato by cocultivating cotyledonary explants with Agrobacterium tumefaciens . Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and gene copy number in the transgenic plants. Double-antibody sandwich ELISA analysis revealed high levels of Bt ICP expression in the leaves of transgenic plants. The expression resulted in a high level of protection of transgenic plant leaves and fruits against the larvae of tomato fruit borer ( Helicoverpa armigera ). Limited field trial of the transgenic plants (T 1 generation) confirmed the high levels of insect protection.

Photosynthesis and Nitrogen-Use Efficiency

In C3 crop plants about 60–80% of leaf nitrogen (N) is invested in the photosynthetic apparatus, and N nutrition plays a crucial role in determining photosynthetic capacity. The proportion of leaf N invested in photosynthetic components is fairly constant. By contrast, both N per unit leaf area and the allocation of N between the component photosynthetic processes depend on environmental factors such as N availability, irradiance and CO2 concentration. Light-harvesting and electron transport components often show a co-ordinated and equivalent response to N nutrition. In contrast, most studies have shown disproportionately large changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in response to N supply, demonstrating the importance of this protein in leaf N economy. At low light, for a given N availability, more protein is allocated towards light harvesting components in order to maximize light capture and, expressed per unit Chl, electron transport and carboxylation capacities are relatively small. High irradiance tends to alter the partitioning of N away from thylakoid protein to soluble proteins, particularly Rubisco. Growth at elevated CO2 often leads to decreases in the amounts of Rubisco and other photosynthetic components on a leaf area basis. This is explicable in terms of greater N sinks elsewhere in the plant as a result of increased carbohydrate availability and acclimatory changes. Models predict that in order to arrive at optimal N use efficiency (NUE) at likely future ambient CO2 concentrations, leaves will need to achieve a redistribution of N so that the ratio between the capacities for regeneration of ribulose-1,5-bisphosphate and carboxylation increases by 30–40%. Human intervention to improve the NUE of crops would have economic and environmental benefits, reducing pollution of water supply by nitrates. The NUE of photosynthesis could be increased either through manipulation of Rubisco amounts or properties, or by decreasing photorespiration. While decreasing Rubisco content could enhance NUE by only about 5%, eliminating photorespiration could produce a change of more than 50%.

Plant regeneration via somatic embryogenesis in pigeonpea (Cajanus cajan L. Millsp)

Abstract Efficient plant regeneration via somatic embryogenesis has been developed in pigeonpea. Cotyledon and leaf explants from 10-day-old seedlings produced embryogenic callus and somatic embryos when cultured on Murashige and Skoog (MS) medium supplemented with 10 µm thidiazuron (TDZ). Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal medium. The rooted plantlets were transferred and acclimatized on vermiculite where they showed normal morphological characters.

Plant RNA Interference Pathways: Diversity in Function, Similarity in Action

Small non-coding RNA-mediated gene-silencing pathways, collectively called RNA interference (RNAi), are involved in regulation of endogenous gene expression and plant defence. It is manifested through two broad classes of small non-coding regulatory RNAs, small interfering RNA (siRNA) and microRNA (miRNA). siRNAs, generated from cleavage of long hairpin RNA by RNase III-class endonuclease, Dicer-like, mediate transcriptional or post-transcriptional gene silencing. At transcriptional level, 24-nucleotide (nt)-long-siRNAs guide an effector complex for DNA methylation, which leads to heterochromatinisation of target loci and consequently transcriptional silencing. At post-transcriptional level, a different size class of 21-nt-long siRNAs guides a silencing complex, called RNA-induced silencing complex, for cleavage of target mRNA. cis-acting siRNAs are involved in plant defence against viruses and transposons, and trans-acting siRNAs regulate endogenous genes involved in plant growth. miRNAs are generated from processing of imperfect stem-loop RNA precursors by Dicer-like. They regulate plant growth and adaptive stress responses by either degradation or translational repression of target mRNAs.

Insect Pest Resistant Transgenic Crops

Insect pests are the major scourge of agriculture down the ages. It is estimated that 14% of crop productivity is lost to insect pests on a global scale (Krattiger, 1997). Agronomically important crops and their high-yielding genotypes are highly susceptible to insect pests. Introduction of chemical pesticides has brought about a significant change in the pest management practices but, unfortunately, resulted in adverse effects on human health, other biological organisms and environment. Figure 1 depicts the amount of money spent annually on pesticides on the global scale. Although complete elimination of pesticides is neither feasible nor advisable, it is imperative to reduce drastically the consumption of pesticides in agriculture and environment for practising safe and sustainable farming. Effective alternatives are now available in the form of genetically engineered crops resistant to insect pests that can be integrated in agricultural ecosystems (Schuler et al., 1998). Many insecticidal proteins are available in nature which are highly specific to agronomically important insect pests but at the same time harmless to man, mammals and other organisms including beneficial insects. These proteins can be expressed in plant systems in sufficient quantities so as to confer insect resistance.

Potential for nitrate reduction in wheat (triticum aestivum L.)

Abstract Following the prevalent agronomic practice of applying N fertilizer in two splits at optimum levels recommended for maximum yield viz. 120 kg N ha, to two wheat (Triticum aestlvum L.) cultivars, which differ in in vivo nitrate reductase (NR) activity, it was observed that the activity is high in the first formed leaf blades and declines in the successively formed ones. Enhancement in the activity subsequent to incubation of excised leaf blades in NO3 ‐ suggests that the substrate (NO‐ 3) is limiting and that the leaf blades, particularly the upper ones, have the potential to reduce additional amounts of NO3 ‐. High NR cultivar has greater potential than the low NR cultivar. The studies suggest that it may be possible to increase the NO3 ‐ moles reduced and thus enhance the reduced N content in case the nitrogen is available at later stages of growth.

Bioinsecticidal activity of Murraya koenigii miraculin‐like protein against Helicoverpa armigera and Spodoptera litura

Miraculin-like proteins, belonging to the Kunitz superfamily, are natural plant defense agents against pests and predators, and therefore are potential biopesticides for incorporation into pest-resistant crops. Here, a miraculin-like protein from Murraya koenigii was assessed for its in vitro and in vivo effects against two polyphagous lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura. M. koenigii miraculin-like protein (MKMLP) inhibited the trypsin-like activity and total protease activity of H. armigera gut proteinases (HGP) by 78.5 and 40%, respectively, and S.litura gut proteinases (SGP) by 81 and 48%, respectively. The inhibitor was stable and actively inhibited the proteolysis of both HGP and SGP enzymes for up to 72 h. Incorporation of MKMLP into artificial diet adversely affected the growth and development of pests in a dose-dependent manner. After 10 days of feeding on diets containing 200 µM MKMLP, larval weight was reduced to 69 and 44.8% and larval mortality was increased to 40 and 43.3% for H. armigera and S litura, respectively. The LC(50) of MKMLP was 0.34 and 0.22% of the diet for H.armigera and S. litura, respectively. These results demonstrate the efficacy of MKMLP as a potential plant defense agent against H. armigera and S. litura.

Tissue specific response of Agrobacterium tumefaciens attachment to Sorghum bicolor (L) Moench

Agrobacterium mediated genetic transformation of plants have advantages over other methods, especially for making single copy transgenic plants with reduced chances of gene silencing and instability. However, monocotyledonous plant species could not utilize the full potential of this system because of possible limitations in Agrobacterium interaction with monocot plant cells. Agrobacterium attachment as a factor in genetic transformation was studied in the leaf, shoot apex, and leaf derived callus of sorghum (Sorghum bicolor (L) Moench). Pre-induction of Agrobacterium with acetosyringone was found necessary for Agrobacterium attachment to sorghum tissues. All the explants responded positively, with preferential Agrobacterium attachment and colonization around the tissues having actively dividing cells. Callus proved to be the best explant for Agrobacterium attachment as observed in scanning electron microscopy and transient GUS expression. Loss of Agrobacterium attachment was observed with an increase in the degree of tissue differentiation.