Alok Das

Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature.

Rice blast resistant gene, Pi54 cloned from rice line, Tetep, is effective against diverse isolates of Magnaporthe oryzae. In this study, we prospected the allelic variants of the dominant blast resistance gene from a set of 92 rice lines to determine the nucleotide diversity, pattern of its molecular evolution, phylogenetic relationships and evolutionary dynamics, and to develop allele specific markers. High quality sequences were generated for homologs of Pi54 gene. Using comparative sequence analysis, InDels of variable sizes in all the alleles were observed. Profiling of the selected sites of SNP (Single Nucleotide Polymorphism) and amino acids (N sites ≥ 10) exhibited constant frequency distribution of mutational and substitutional sites between the resistance and susceptible rice lines, respectively. A total of 50 new haplotypes based on the nucleotide polymorphism was also identified. A unique haplotype (H_3) was found to be linked to all the resistant alleles isolated from indica rice lines. Unique leucine zipper and tyrosine sulfation sites were identified in the predicted Pi54 proteins. Selection signals were observed in entire coding sequence of resistance alleles, as compared to LRR domains for susceptible alleles. This is a maiden report of extensive variability of Pi54 alleles in different landraces and cultivated varieties, possibly, attributing broad-spectrum resistance to Magnaporthe oryzae. The sequence variation in two consensus region: 163 and 144 bp were used for the development of allele specific DNA markers. Validated markers can be used for the selection and identification of better allele(s) and their introgression in commercial rice cultivars employing marker assisted selection.

Advances in biotechnological applications in three important food legumes

Legumes are the third largest family of flowering plants, known for their unique capacity of symbiotic nitrogen fixation. The draft genome sequences of three important food legumes [soybean (Glycine max), pigeonpea (Cajanus cajan) and chickpea (Cicer arietinum)] have been completed. Coupled with a deluge of information on transcriptomics, proteomics and metabolomics, they present a huge amount of genomic resources for the genetic improvement of legume crops. Developed molecular markers, structurally and functionally annotated genes/quantitative trait loci/alleles and regulatory sequences can be utilized in improvement breeding programmes. Further, the genetic transformation of two valuable pulses (chickpea and pigeonpea) has now taken centre stage, realizing the potential of genetically modified soybean, for enhanced prospects of food production. Together, the advances in biotechnological tools and the research community’s capacity to develop imaginative strategies will help in framing a legume development programme for ensuring the nutritional security of the world.

Expression of chimeric Bt gene, Cry1Aabc in transgenic pigeonpea (cv. Asha) confers resistance to gram pod borer (Helicoverpa armigera Hubner.)

The gram pod borer (Helicoverpa armigera Hubner) is the most serious insect pest of pigeonpea. It is highly susceptible to the insecticidal proteins of Bacillus thuringiensis (Bt). A codon-optimized chimeric Cry1Aabc gene of Bt driven by a constitutive promoter was introduced in pigeonpea (cv. Asha) to confer resistance against the insect. A total of eight transgenic plants could be established with transformation frequency of 0.06%. Two transgenic events were selected for advancement based on high insect mortality, single locus integration, protein expression and fertility status. Quantitative ELISA indicated high protein expression in different plant parts viz., leaves (pre and post flowering), flowers, pod walls and immature seeds. Analysis for the stable integration, expression and insect mortality (detached leaf and pod bioassay) led to identification of lines with high efficacy. These events were further advanced for the identification of a viable event by selfing to create homozygosity. The chimeric Cry1Aabc expressed in pigeonpea is effective against gram pod borer and can be utilized in transgenic variety development programme.

Expression of a Chimeric Gene Encoding Insecticidal Crystal Protein Cry1Aabc of Bacillus thuringiensis in Chickpea (Cicer arietinum L.) Confers Resistance to Gram Pod Borer (Helicoverpa armigera Hubner.)

Domain swapping and generation of chimeric insecticidal crystal protein is an emerging area of insect pest management. The lepidopteran insect pest, gram pod borer (Helicoverpa armigera H.) wreaks havoc to chickpea crop affecting production. Lepidopteran insects were reported to be controlled by Bt (cryI) genes. We designed a plant codon optimized chimeric Bt gene (cry1Aabc) using three domains from three different cry1A genes (domains I, II, and III from cry1Aa, cry1Ab, and cry1Ac, respectively) and expressed it under the control of a constitutive promoter in chickpea (cv. DCP92-3) to assess its effect on gram pod borer. A total of six transgenic chickpea shoots were established by grafting into mature fertile plants. The in vitro regenerated (organogenetic) shoots were selected based on antibiotic kanamycin monosulfate (100 mg/L) with transformation efficiency of 0.076%. Three transgenic events were extensively studied based on gene expression pattern and insect mortality across generations. Protein expression in pod walls, immature seeds and leaves (pre- and post-flowering) were estimated and expression in pre-flowering stage was found higher than that of post-flowering. Analysis for the stable integration, expression and insect mortality (detached leaf and whole plant bioassay) led to identification of efficacious transgenic chickpea lines. The chimeric cry1Aabc expressed in chickpea is effective against gram pod borer and generated events can be utilized in transgenic breeding program.

Using AFLP-RGA markers to assess genetic diversity among pigeon pea (Cajanus cajan) genotypes in relation to major diseases

Resistance gene analog (RGA)-anchored amplified fragment length polymorphism (AFLP-RGA) marker system was used in order to evaluate genetic relationships among 22 pigeon pea genotypes with varied responses to Fusarium wilt and sterility mosaic disease. Five AFLP-RGA primer combinations (E-CAG/wlrk-S, M-GTG/wlrk-S, M-GTG/wlrk-AS, E-CAT/S1-INV and E-CAG/wlrk-AS) produced 173 scorable fragments, of which 157 (90.7%) were polymorphic, with an average of 31.4 fragments per primer combination. The polymorphism rates obtained with the five primers were 83.3%, 92.0%, 92.3%, 93.0% and 93.1%, respectively. Mean polymorphic information content (PIC) values ranged from 0.24 (with E-CAT/S1-INV) to 0.30 (with E-CAG/wlrk-AS), whereas resolving power (RP) values varied from 11.06 (with M-GTG/wlrk-S) to 25.51 (with E-CAG/wlrk-AS) and marker index (MI) values ranged from 5.98 (with M-GTG/wlrk-S) to 12.30 (with E-CAG/wlrk-AS). We identified a positive correlation between MI and RP (r2=0.98, p<0.05), stronger that that observed for the comparison between PIC and RP (r2=0.88, p<0.05). That implies that either MI or RP is the best parameter for selecting more informative AFLP-RGA primer combinations. The Jaccard coefficient ranged from 0.07 to 0.72, suggesting a broad genetic base in the genotypes studied. A neighbor-joining tree, based on the unweighted pair group method with arithmetic mean, distinguished cultivated species from wild species. The grouping of resistant genotypes in different clusters would help in the selection of suitable donors for resistance breeding in pigeon pea.

Insect Smart Pulses for Sustainable Agriculture

The development of high-yielding insect-tolerant cultivars using conventional methods has been slow due to a number of reasons. With the advent of recombinant tools and genetic transformation systems, it has been possible to harness gene pool(s) by crossing the species barrier and utilize them for desired trait. Insect pest resistance has largely been introgressed in many crops, including pulses, by using the cry genes from Bacillus thuringiensis. However, many plant genes like lectins, protein inhibitors, etc. are also available that impart tolerance to insect pests and can be used for developing insect-tolerant plants. In comparison with other crops, relatively less work is available in this context in pulses because of their recalcitrant nature and biosafety issues related to candidate gene(s). In the regime of climate change, plant-pest dynamics has also witnessed change, and the need to develop transgenic tolerant to both pest and diseases is desirable. In context of sustainable pulse production, it is essential to develop and use insect-tolerant transgenics that have been developed by following the biosafety regulations, are high yielding, fit into popular cropping systems, and are expected to be remunerative to the stakeholders.

Identification, Characterization, and Phylogenetic analysis of Pigeon pea (Cajanus cajan L. Mill sp.) Resistance Gene Analogs using PCR cloning and in silico methods

Pigeon Pea (Cajanus cajan), an important grain legume, is susceptible to Fusarium wilt (FW), sterility mosaic disease (SMD), and Phytophthora blight. Identification of resistance gene analogs (RGAs) is important for development of resistant varieties. In this study, degenerate primers targeting nucleotide binding sites (NBS) of known resistance (R) genes were used to amplify RGAs from two Pigeon Pea genotypes with differing disease resistance profiles. The translated cloned RGAs had high amino acid identity (68–71%) with putative disease resistance proteins in Glycine Max. Five RGA open reading frames were found in the whole Pigeon Pea genome after BLASTN analysis with the cloned sequences. Translated RGA proteins contained several characteristic features such as the NB-ARC domain (characteristic of death-related disease resistance genes) and four NBS motifs. A tryptophan residue at the kinase-2 motif was indicative of the non-TIR-NBS class of proteins. Phylogenetic analysis revealed two major clusters. The seven Pigeon Pea RGAs were in a non-TIR group alongside wilt resistance proteins from tomato. Specific primers were designed against the RGAs identified by BLASTN, and these successfully amplified sequences from all eight Pigeon Pea genotypes. The 40 resultant sequences were combined according to genotype and subjected to phylogenetic analysis. Genotypes clustered according to breeding pedigree. Multiple alignments of the 40 sequences revealed several single nucleotide polymorphisms (SNPs) that are useful in identifying candidate resistance genes associated with FW and SMD.

Transgenic Strategies Towards Nutritional Enrichment of Crops

All the essential nutrients in the human diet are ultimately derived from plants. However, most of the major food crops lack certain essential vitamins and minerals. Although a diversified diet regime provides adequate nutrition, majority of the population in developing countries relies on staple crops, such as rice, wheat, maize or cassava, which lacks full complement of essential nutrients. Malnutrition, thus, is a significant humanitarian issue in most of the developing world. A pertinent way to address this challenge is through biofortification of crops to increase their essential nutrient content. Transgenic approaches offer the most rapid and precise way to develop high-nutrient crops, thus complementing mineral fertilization and conventional breeding towards ameliorating the scourge.

Genetic transformation of pigeonpea (Cajanus cajan L.) and screening transgenic progenies based on lateral root inhibition

Production of transgenic pigeonpea is becoming increasingly important, but the methods currently employed in production and subsequent screening still requires improvement. Here, we describe Agrobacterium-mediated genetic transformation of pigeonpea with reporter uidA (gus) gene and selectable marker, neomycin phospho-transferase (nptII) gene. Histochemical assay demonstrate localization of gus activity in cells and transformed plants. Overall, a transformation frequency of 0.33% was achieved using the protocol. Grafting of in vitro-regenerated healthy shoots indicates higher survival percent (72.6%), when stock and scion are of the same variety. Seeds harvested from primary transgenic plants can be screened based on lateral root inhibition strategy. Approximately 87% of the screened T1 plants were found to be PCR positive. In conclusion, in vitro grafting of transgenic pigeonpea shoots leads to better plant establishment and screening based on lateral root inhibition leads to quick identification of positive segregants.

Ontogeny of in vitro shoot organogenesis from axillary meristem explants in chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is a major food legume crops for the tropics and sub-tropical regions and is the main dietary protein source for vegetarians in developing countries. Besides several abiotic factors, its production is constrained by insect pests, as well as many fungal diseases. The success of any attempt to produce resistant varieties through genetic engineering to a large extent depends on the availability of efficient and reproducible regeneration and transformation procedures. Further, the transformation techniques can be more successfully applied if the ontogeny of shoot development is well understood. Ontogeny of shoot development from axillary meristem explants (AME) in chickpea has been studied with the optimization of conditions for high-frequency multiple shoot induction. Preculture of seeds in TDZ significantly enhances the frequency of multiple shoot induction from the explants. Ontogeny and early events of multiple shoot induction revealed direct adventitious origin of the shoots. The understanding of the regeneration process could be further utilized in the designing of efficient transformation methods.