Root knot nematode, Meloidogyne incognita, is an obligate sedentary endoparasite that infects a large number of crop species and causes substantial yield losses. Non-chemical based control strategies for these nematodes are gaining importance. In the present study, we have demonstrated the significance of two FMRFamide like peptide genes (flp-14 and flp-18) for infection and development of resistance to M. incognita through host-derived RNAi. The study demonstrated both in vitro and in planta validation of RNAi-induced silencing of the two genes cloned from J2 stage of M. incognita. In vitro silencing of both the genes interfered with nematode migration towards the host roots and subsequent invasion into the roots. Transgenic tobacco lines were developed with RNAi constructs of flp-14 and flp-18 and evaluated against M. incognita. The transformed plants did not show any visible phenotypic variations suggesting the absence of any off-target effects. Bioefficacy studies with deliberate challenging of M. incognita resulted in 50-80% reduction in infection and multiplication confirming the silencing effect. We have provided evidence for in vitro and in planta silencing of the genes by expression analysis using qRT-PCR. Thus the identified genes and the strategy can be used as a potential tool for the control of M. incognita. This is the first ever report that has revealed the utility of host delivered RNAi of flps to control M. incognita. The strategy can also be extended to other crops and nematodes.

BackgroundAnalysis of gene expression patterns leads to functional understanding of biological processes. Quantitative real-time PCR has become the most commonly used technique for in-depth studies of gene expression. To quantify variation in specific gene expression, accurate and reliable normalization across different samples and tissues is necessary. This can be achieved by selecting one or more suitable reference genes to compare the target mRNA transcript levels. In the present work, we illustrate the first evaluation of potential internal control or reference genes across different developmental stages of eggplant for reliable quantification of transcripts by real-time PCR.ResultsWe have evaluated the stability in expression of six candidate reference genes (18s rRNA, APRT, GAPDH, Cyclophilin, Actin, and RuBP) in a set of tissues representing six developmental stages of eggplant. The candidate genes were cloned from cDNA and analysed by real-time PCR. The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant. This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant.Conclusion18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant.

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Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80–100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25–80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.

Highly tolerant herbicide-resistant transgenic rice was developed by expressing codon-modified synthetic CP4--EPSPS. The transformants could tolerate up to 1% commercial glyphosate and has the potential to be used for DSR (direct-seeded rice). Weed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T0, T1 and T2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1% of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace.

Burgeoning population growth, industrial demand, and the predicted global climate change resulting in erratic monsoon rains are expected to severely limit fresh water availability for agriculture both in irrigated and rainfed ecosystems. In order to remain food and nutrient secure, agriculture research needs to focus on devising strategies to save water in irrigated conditions and to develop superior cultivars with improved water productivity to sustain yield under rainfed conditions. Recent opinions accruing in the scientific literature strongly favor the adoption of a “trait based” crop improvement approach for increasing water productivity. Traits associated with maintenance of positive tissue turgor and maintenance of increased carbon assimilation are regarded as most relevant to improve crop growth rates under water limiting conditions and to enhance water productivity. The advent of several water saving agronomic practices notwithstanding, a genetic enhancement strategy of introgressing distinct physiological, morphological, and cellular mechanisms on to a single elite genetic background is essential for achieving a comprehensive improvement in drought adaptation in crop plants. The significant progress made in genomics, though would provide the necessary impetus, a clear understanding of the “traits” to be introgressed is the most essential need of the hour. Water uptake by a better root architecture, water conservation by preventing unproductive transpiration are crucial for maintaining positive tissue water relations. Improved carbon assimilation associated with carboxylation capacity and mesophyll conductance is important in sustaining crop growth rates under water limited conditions. Besides these major traits, we summarize the available information in literature on classifying various drought adaptive traits. We provide evidences that Water-Use Efficiency when introgressed with moderately higher transpiration, would significantly enhance growth rates and water productivity in rice through an improved photosynthetic capacity.

The complex parasitic strategy of Meloidogyne incognita appears to involve simultaneous expression of its pharyngeal gland-specific effector genes in order to colonize the host plants. Research reports related to effector crosstalk in phytonematodes for successful parasitism of the host tissue is yet underexplored. In view of this, we have used in planta effector screening approach to understand the possible interaction of pioneer genes (msp-18 and msp-20, putatively involved in late and early stage of M. incognita parasitism, respectively) with other unrelated effectors such as cell-wall modifying enzymes (CWMEs) in M. incognita. Host-induced gene silencing (HIGS) strategy was used to generate the transgenic eggplants expressing msp-18 and msp-20, independently. Putative transformants were characterized via qRT-PCR and Southern hybridization assay. SiRNAs specific to msp-18 and msp-20 were also detected in the transformants via Northern hybridization assay. Transgenic expression of the RNAi constructs of msp-18 and msp-20 genes resulted in 43.64–69.68% and 41.74–67.30% reduction in M. incognita multiplication encompassing 6 and 10 events, respectively. Additionally, transcriptional oscillation of CWMEs documented in the penetrating and developing nematodes suggested the possible interaction among CWMEs and pioneer genes. The rapid assimilation of plant-derived carbon by invading nematodes was also demonstrated using 14C isotope probing approach. Our data suggests that HIGS of msp-18 and msp-20, improves nematode resistance in eggplant by affecting the steady-state transcription level of CWME genes in invading nematodes, and safeguard the plant against nematode invasion at very early stage because nematodes may become the recipient of bioactive RNA species during the process of penetration into the plant root.

Peanut, a major edible oil seed crop globally is predominantly grown under rainfed conditions and suffers yield losses due to drought. Development of drought-tolerant varieties through transgenic technology is a valid approach. Besides superior water relation traits like water mining, intrinsic cellular level tolerance mechanisms are important to sustain the growth under stress. To achieve this objective, the focus of this study was to pyramid drought adaptive traits by overexpressing a stress responsive helicase, PDH45 in the background of a genotype with superior water relations. PCR, Southern, and RT-PCR analyses confirmed stable integration and expression of the PDH45 gene in peanut transgenics. At the end of T₃ generation, eight transgenic events were identified as promising based on stress tolerance and improvement in productivity. Several transgenic lines showed stay-green phenotype and increased chlorophyll stability under stress and reduced chlorophyll retardation under etherel-induced simulated stress conditions. Stress-induced root growth was also substantially higher in the case of transformants. This was reflected in increased WUE (low Δ¹³C) and improved growth rates and productivity. The transgenics showed 17.2 and 26.75 % increase in yield under non-stress and stress conditions over wild type ascertaining the feasibility of trait pyramiding strategy for the development of drought-tolerant peanut.