Abhay Kumar

Heterologous expression of the AtDREB1A gene in transgenic peanut-conferred tolerance to drought and salinity stresses.

Research on genetic transformation in various crop plants using the DREB1A transcription factor has shown better abiotic stress tolerance in transgenic crops. The AtDREB1A transgenic peanut (Arachis hypogaea L. cv. GG 20), which was previously developed, was characterized in terms of its physio-biochemical, molecular and growth parameters. The tolerance of this transgenic peanut to drought and salinity stresses was evaluated at the seedling (18 days old) and maturity stages. Transgenic peanut lines showed improved tolerance to both stresses over wild-type, as observed by delayed and less severe wilting of leaves and by improved growth parameters that were correlated with physio-biochemical parameters such as proline content, total chlorophyll content, osmotic potential, electrolytic leakage and relative water content. The expression pattern of the AtDREB1A gene evaluated using qPCR at different time points demonstrated that transgene expression was induced within two hours of stress imposition. The better performance of transgenic AtDREB1A peanut at the seedling stage and the improved growth parameters were due to the expression of the transgene, which is a transcription factor, and the possible up-regulation of various stress-inducible, downstream genes in the signal transduction pathway under abiotic stress.

Coat protein-mediated transgenic resistance of peanut (Arachis hypogaea L.) to peanut stem necrosis disease through Agrobacterium-mediated genetic transformation

The absence of resistance genes against biotic stresses like Tobacco streak virus (TSV) within compatible peanut germplasm necessitates the deployment of genetic engineering strategy to develop transgenic resistance. Transgenic resistance in peanut (Arachis hypogaea L.) to peanut stem necrosis disease caused by TSV was obtained by transferring coat protein (CP) gene of TSV through Agrobacterium-mediated transformation of de-embryonated cotyledons and immature leaves of peanut cultivars Kadiri 6 (K6) and Kadiri 134 (K134). Integration of the transgene in T1, T2 and T3 generations were confirmed by PCR with gene-specific primers. On the basis of segregation analysis of the PCR amplicons, homozygosity was confirmed in progeny from five transgenic lines. Six transgenic plants from three different single copy transgenic lines homozygous for the transgene were selected for challenge inoculation in T3 generations. The transgenic lines remained symptomless throughout and showed traces or no systemic accumulation of virus indicating the tolerance/resistance to the TSV infection. CP gene expression was observed in transgenic lines by RT-PCR, real-time PCR and ELISA. The findings provide an effective strategy for developing peanut with resistance to peanut stem necrosis disease.

Overexpression of Bacterial mtlD Gene in Peanut Improves Drought Tolerance through Accumulation of Mannitol

In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L.) crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated.

Stress Inducible Expression of AtDREB1A Transcription Factor in Transgenic Peanut (Arachis hypogaea L.) Conferred Tolerance to Soil-Moisture Deficit Stress

Peanut, an important oilseed crop, is gaining priority for the development of drought tolerant genotypes in recent times, since the area under drought is constantly on the rise. To achieve this, one of the important strategies is to genetically engineer the ruling peanut varieties using transcription factor regulating the expression of several downstream, abiotic-stress responsive gene(s). In this study, eight independent transgenic peanut (cv. GG20) lines were developed using AtDREB1A gene, encoding for a transcription factor, through Agrobacterium-mediated genetic transformation. The transgene insertion was confirmed in (T0) using PCR and Dot-blot analysis, while copy-number(s) was ascertained using Southern-blot analysis. The inheritance of AtDREB1A gene in individual transgenic plants (T1 and T2) was confirmed using PCR. In homozygous transgenic plants (T2), under soil-moisture deficit stress, elevated level of AtDREB1A transgene expression was observed by RT-PCR assay. The transgenic plants at 45-d or reproductive growth stage showed tolerance to severe soil-moisture deficit stress. Physio-biochemical parameters such as proline content, osmotic potential, relative water content, electrolytic leakage, and total-chlorophyll content were found positively correlated with growth-related traits without any morphological abnormality, when compared to wild-type. qPCR analysis revealed consistent increase in expression of AtDREB1A gene under progressive soil-moisture deficit stress in two homozygous transgenic plants. The transgene expression showed significant correlation with improved physio-biochemical traits. The improvement of drought-stress tolerance in combination with improved growth-related traits is very essential criterion for a premium peanut cultivar like GG20, so that marginal farmers of India can incur the economic benefits during seasonal drought and water scarcity.

Insights into the Indian Peanut Genotypes for ahFAD2 Gene Polymorphism Regulating Its Oleic and Linoleic Acid Fluxes

In peanut (Arachis hypogaea L.), the customization of fatty acid profile is an evolving area to fulfill the nutritional needs in the modern market. A total of 174 peanut genotypes, including 167 Indian cultivars, 6 advanced breeding lines and “SunOleic95R”—a double mutant line, were investigated using AS-PCRs, CAPS and gene sequencing for the ahFAD2 allele polymorphism, along with its fatty acid compositions. Of these, 80 genotypes were found having substitution (448G>A) mutation only in ahFAD2A gene, while none recorded 1-bp insertion (441_442insA) mutation in ahFAD2B gene. Moreover, 22 wild peanut accessions found lacking both the mutations. Among botanical types, the ahFAD2A mutation was more frequent in ssp. hypogaea (89%) than in ssp. fastigiata (17%). This single allele mutation, found affecting not only oleic to linoleic acid fluxes, but also the composition of other fatty acids in the genotypes studied. Repeated use of a few selected genotypes in the Indian varietal development programs were also eminently reflected in its ahFAD2 allele polymorphism. Absence of known mutations in the wild-relatives indicated the possible origin of these mutations, after the allotetraploidization of cultivated peanut. The SNP analysis of both ahFAD2A and ahFAD2B genes, revealed haplotype diversity of 1.05% and 0.95%, while Ka/Ks ratio of 0.36 and 0.39, respectively, indicating strong purifying selection pressure on these genes. Cluster analysis, using ahFAD2 gene SNPs, showed presence of both mutant and non-mutant genotypes in the same cluster, which might be due the presence of ahFAD2 gene families. This investigation provided insights into the large number of Indian peanut genotypes, covering various aspects related to O/L flux regulation and ahFAD2 gene polymorphism.

Transgenic Peanut (Arachis hypogaea L.) Overexpressing mtlD Gene Showed Improved Photosynthetic, Physio-Biochemical, and Yield-Parameters under Soil-Moisture Deficit Stress in Lysimeter System

Peanut, an important oilseed crop, frequently encounters drought stress (DS) during its life cycle. In this study, four previously developed mtlD transgenic (T) peanut lines were used for detailed characterization under DS, at the reproductive stage using lysimeter system under controlled greenhouse conditions. In dry-down experiments, T lines maintained better photosynthetic machinery, such as, photosynthesis rate, stomatal conductance, transpiration rate, and SPAD (Soil-Plant Analyses Development) values, and had lower oxidative damage, including lipid membrane peroxidation and hydrogen peroxide and superoxide radical accumulation than WT, when exposed to 24 days of DS. WT plants had a more negative water potential (WP; up to −3.22 MPa) than T lines did (−2.56 to −2.71 MPa) at day 24 of DS treatment. During recovery, T lines recovered easily whereas 67% of WT plants failed to recover. In T lines, the rate of photosynthesis strongly and positively correlated with the transpiration rate (r = 0.92), RWC (r = 0.90), WP (r = 0.86), and total chlorophyll content (r = 0.75), suggesting its strong correlation with water retention-related parameters. Furthermore, yield parameters such as, pod weight and harvest index of T lines were up to 2.19 and 1.38 times more than those of WT plants, respectively. Thus, the significantly better performance of mtlD T peanut lines than of WT plants under DS could be attributed to the accumulation of mannitol, which in turn helped in maintaining the osmoregulation and ROS scavenging activity of mannitol and ultimately conferred water-economizing capacity and higher yield in T lines than in WT plants.

Major Virus Diseases of Groundnut in India and Their Management

Several virus diseases of groundnut have been reported in India based on symptoms, host range, and biological properties. Among those, peanut bud necrosis virus (PBNV), tobacco streak virus (TSV), peanut mottle virus (PeMoV), and Indian peanut clump virus (IPCV) are the economically important viruses of groundnut in India. Peanut bud necrosis virus belongs to the genus Tospovirus, transmitted effectively by Thrips palmi. PBND alone may cause 30–90 % yield losses. Necrosis of the terminal buds occurs which is a characteristic symptom of PBNV. Extensive field screening of the several genotypes, released varieties, and wild species at the hot spots has revealed the field tolerance of some of those genotypes. Peanut stem necrosis disease (PSND) is caused by the TSV of the genus Ilarvirus of the family Bromoviridae. Necrotic lesions on terminal leaflets, complete stem necrosis, and often total necrosis of entire plant are the characteristic symptoms of this disease. The PSND spreads mainly through the weed of crop species. A desired level of resistance of TSV has not yet been found in cultivated varieties of groundnut. The peanut clump disease of groundnut in India is caused by the IPCV of the genus Pecluvirus, family Virgaviridae. Symptoms are severe stunting of the plant appeared first on newly emerged leaves of two- to three-week-old seedlings. The host range of IPCV includes many monocot and dicot crop plants and weed species tested. IPCV was reported to be transmitted by the obligate fungal parasite (Polymyxa graminis) which is soilborne. Germplasm accessions, viz., NCAc 17099, NCAc 17133 (RF), and NCAc 17536, have been reported resistant to IPCV. Peanut mottle virus disease has been reported to occur on rabi/summer groundnut mainly in Andhra Pradesh, Maharashtra, and Gujarat. Newly formed leaves show mild mottling and vein clearing, whereas older leaves show upward curling and interveinal depression with dark green islands. The peanut mottle virus (PeMoV) occurs in nature on several important legume crops. Aphids are efficient vectors of PeMoV. Several lines of Arachis species like A. glabrata are reported to be resistant to this disease. Peanut stripe virus (PStV) is of quarantine significance to India and is almost eradicated from India. Since in most of the viral diseases sources with desired levels of genetic resistance could not be identified so far, transgenic approaches to engineer resistance to viruses by expressing the glycoproteins of tospoviruses in transgenic plants to block virus acquisition by thrips, by expressing truncated or modified forms of movement protein(s) of heterologous viruses, or by expressing virus-specific antibody genes may be adopted to tackle the viral diseases in groundnut.