Browsing by Author "Major Singh"

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Assessment of factors on shoot proliferation potential of nodal explants of Phyllanthus fraternus and assessment of genetic fidelity of micropropagated plants using RAPD marker
(Versita, 2014) Richa Upadhyay; Sarvesh Pratap Kashyap; Chandrashekhar Singh; Kavindra Nath Tiwari; Karuna Singh; Major Singh
Phyllanthus fraternus is an important medicinal plant, popularly known for its hepatoprotective and antiviral activities since ancient times. Various physiological factors like carbon sources, concentration of agar, pH of the media and effect of season of explants collection were optimized for high frequency regeneration of P. fraternus. The frequency of regeneration, average number and length of shoots were highly influenced by the type and concentration of carbon sources (monosaccharides and disaccharides, 1 to 4%), agar concentration (0.2 to 1%) and pH (4.5 to 6.8) of the media. Media containing 3% sucrose, 0.6% agar at pH 5.8 was best for regeneration. Seasonal variation of explants collection significantly affected the axillary shoots proliferation from explants and best proliferation was observed from explants collected during April to June. Genetic fidelity of regenerated plants was assessed by random amplified polymorphic DNA markers. No polymorphism was detected in micropropagated plants and the mother plant, revealing the genetic homogeneity of the in vitro raised plantlets. This is the first report regarding establishment of genetic fidelity of micropropagated P. fraternus plants, which could be successfully applied for the mass multiplication, germplasm conservation and further genetic transformation assays to meet the ever increasing demand of this medicinally potent plant for industrial and pharmaceutical uses. © 2014, Versita Warsaw and Springer-Verlag Wien.
Assessment of molecular diversity in chickpea (Cicer arietinum L.) rhizobia and structural analysis of 16S rDNA sequences from Mesorhizobium ciceri
(Polish Society of Microbiologists, 2013) Akhilesh Yadav; Asha Lata Singh; Govind Kumar Rai; Major Singh
Molecular diversity studies of 19 rhizobia isolates from chickpea were conducted using simple sequence repeats (SSR) and 16S rDNA-RFLP markers. Phenotypic characterization with special reference to salinity and pH tolerance was performed. These isolates were identified as different strains of Mesorhizobium, Rhizobium, Bradyrhizobium, and Agrobacterium. Twenty SSR loci of Mesorhizobium ciceri, distributed across the other rhizobial genome, clearly differentiated 19 rhizobial isolates. Analogous clustering supported the results of 16S rDNA sequence-based phylogeny. Analysis of the 16S rDNA sequences from M. ciceri strains revealed that nucleotide variables (signature sites) were located at 20 different positions; most of them were present in the first 820 bp region from 5' terminal. Interestingly, 14 signature sites were located in two main regions, the variable region V1 (nt 527-584), and variable region V2 (nt 754-813). The secondary structure and minimal free energy were determined in these two regions. These results will be useful in characterizing the micro-evolutionary mechanisms of species formation and increase understanding of the symbiotic relationship.
Bacterial Community Structure in the Rhizosphere of a Cry1Ac Bt-Brinjal Crop and Comparison to Its Non-transgenic Counterpart in the Tropical Soil
(2013) Amit Kishore Singh; Govind Kumar Rai; Major Singh; Suresh Kumar Dubey
To elucidate whether the transgenic crop alters the rhizospheric bacterial community structure, a 2-year study was performed with Cry1Ac gene-inserted brinjal crop (Bt) and their near isogenic non-transformed trait (non-Bt). The event of Bt crop (VRBT-8) was screened using an insect bioassay and enzyme-linked immunosorbent assay. Soil moisture, NH4 +-N, NO3 --N, and PO4 --P level had non-significant variation. Quantitative polymerase chain reaction revealed that abundance of bacterial 16S rRNA gene copies were lower in soils associated with Bt brinjal. Microbial biomass carbon (MBC) showed slight reduction in Bt brinjal soils. Higher MBC values in the non-Bt crop soil may be attributed to increased root activity and availability of readily metabolizable carbon compounds. The restriction fragment length polymorphism of PCR-amplified rRNA gene fragments detected 13 different bacterial groups with the exclusive presence of β-Proteobacteria, Chloroflexus, Planctomycetes, and Fusobacteria in non-Bt, and Cyanobacteria and Bacteroidetes in Bt soils, respectively, reflecting minor changes in the community structure. Despite the detection of Cry1Ac protein in the rhizospheric soil, the overall impact of Cry1Ac expressing Bt brinjal was less compared to that due to seasonal changes. © 2013 Springer Science+Business Media New York.
Bioactive compounds of tomato fruits from transgenic plants tolerant to drought
(Academic Press, 2015) Kavita Shah; Major Singh; Avinash Chandra Rai
Advances in agricultural biotechnology bringforth the need for experimental evidence for benefits and risks of engineered crops and the quality of fruit products obtained from them. Tomato fruits from non-transgenic (NT) and BcZAT12-transformed tomato lines ZT1-ZT6 (cv. H-86, var. Kashi vishesh) tolerant to drought, were assessed for nutritional quality, changes in physico-chemical characteristics and health-related bioactive compounds. Fruits from transgenics were evaluated for size, pH, total-soluble solids, total sugars, phenolics, flavonoids, vitamin C, lycopene and β-carotene. An early ripening of tomatoes from transgenics with more red but smaller fruits, high sugar levels, elevated phenolics, flavonoids, lycopene and β-carotene with an unaltered vitamin C levels as compared to tomatoes from non-transgenics were noted. Results suggest that although differences between tomatoes from transgenics and non-transgenics do exist, yet tomato fruits from transgenic plants have relatively improved antioxidant capacity than those from non-transgenics and therefore may be products safe for consumption. © 2015 Elsevier Ltd.
Biotechnological Interventions in Tomato (Solanum lycopersicum) for Drought Stress Tolerance: Achievements and Future Prospects
(MDPI, 2022) Ram Krishna; Waquar Akhter Ansari; P.S. Soumia; Akhilesh Yadav; Durgesh Kumar Jaiswal; Sudhir Kumar; Achuit Kumar Singh; Major Singh; Jay Prakash Verma
Tomato production is severely affected by abiotic stresses (drought, flood, heat, and salt) and causes approximately 70% loss in yield depending on severity and duration of the stress. Drought is the most destructive abiotic stress and tomato is very sensitive to the drought stress, as cultivated tomato lack novel gene(s) for drought stress tolerance. Only 20% of agricultural land worldwide is irrigated, and only 14.51% of that is well-irrigated, while the rest is rain fed. This scenario makes drought very frequent, which restricts the genetically predetermined yield. Primarily, drought disturbs tomato plant physiology by altering plant–water relation and reactive oxygen species (ROS) generation. Many wild tomato species have drought tolerance gene(s); however, their exploitation is very difficult because of high genetic distance and pre- and post-transcriptional barriers for embryo development. To overcome these issues, biotechnological methods, including transgenic technology and CRISPR-Cas, are used to enhance drought tolerance in tomato. Transgenic technology permitted the exploitation of non-host gene/s. On the other hand, CRISPR-Cas9 technology facilitated the editing of host tomato gene(s) for drought stress tolerance. The present review provides updated information on biotechnological intervention in tomato for drought stress management and sustainable agriculture. © 2022 by the authors.
Changes in Actinomycetes community structure under the influence of Bt transgenic brinjal crop in a tropical agroecosystem
(2013) Amit Kishore Singh; Major Singh; Suresh Kumar Dubey
Background: The global area under brinjal cultivation is expected to be 1.85 million hectare with total fruit production about 32 million metric tons (MTs). Brinjal cultivars are susceptible to a variety of stresses that significantly limit productivity. The most important biotic stress is caused by the Brinjal fruit and shoot Borer (FSB) forcing farmers to deploy high doses of insecticides; a matter of serious health concern. Therefore, to control the adverse effect of insecticides on the environment including the soil, transgenic technology has emerged as the effective alternative. However, the reports, regarding the nature of interaction of transgenic crops with the native microbial community are inconsistent. The effect of a Bt transgenic brinjal expressing the bio-insecticidal protein (Cry1Ac) on the rhizospheric community of actinomycetes has been assessed and compared with its non-transgenic counterpart. Results: Significant variation in the organic carbon observed between the crops (non-Bt and Bt brinjal) may be due to changes in root exudates quality and composition mediated by genetic attributes of Bt transgenic brinjal. Real time quantitative PCR indicated significant differences in the actinomycetes- specific 16S rRNA gene copy numbers between the non-Bt (5.62-27.86) × 1011 g-1 dws and Bt brinjal planted soil (5.62-24.04) × 1011 g-1 dws. Phylogenetic analysis indicated 14 and 11, actinomycetes related groups in soil with non-Bt and Bt brinjal crop, respectively. Micrococaceaea and Nocardiodaceae were the dominant groups in pre-vegetation, branching, flowering, maturation and post-harvest stage. However, Promicromonosporaceae, Streptosporangiaceae, Mycobacteriaceae, Geodermatophilaceae, Frankiaceae, Kineosporaceae, Actisymmetaceae and Streptomycetaceae were exclusively detected in a few stages in non-Bt brinjal rhizosphere soil while Nakamurellaceae, Corynebactericeae, Thermomonosporaceae and Pseudonocardiaceae in Bt brinjal counterpart. Conclusion: Field trails envisage that cultivation of Bt transgenic brinjal had negative effect on organic carbon which might be attributed to genetic modifications in the plant. Changes in the organic carbon also affect the actinomycetes population size and diversity associated with rhizospheric soils of both the crops. Further long-term study is required by taking account the natural cultivar apart from the Bt brinjal and its near-isogenic non-Bt brinjal with particular reference to the effects induced by the Bt transgenic brinjal across different plant growth stages. © 2013 Singh et al.; licensee BioMed Central Ltd.
Characterization of Allium germplasms for conservation and sustainable management using SSR markers
(National Institute of Science Communication and Information Resources (NISCAIR), 2019) Kuldip Jayaswall; Abhishek Bhandawat; Himanshu Sharma; Vinod Kumar Yadav; Vijay Mahajan; Major Singh
Allium species are very important due to their medicinal values. Quercetin and allicin are medicinally important compound of onion and garlic, respectively which are proved useful to treat various diseases. However, highly heterozygous nature, self-incompatibility and long gestation period limits genetic improvement of Allium species. Further, the existing germplasms in Indian subcontinents are largely cultivated ones with poor genetic characterization, which limits the germplasm conservation and future management. A total of thirty polymorphic Simple Sequence Repeats (SSRs) were utilized for characterisation of popular onion germplasms and their cross-transferability revealed relatedness with fifteen garlic and wild relatives. Average number of alleles per SSR locus, PIC and heterozygosity was found to be 3.9, 0.51 and 0.57,respectively.Overall genetic diversity recorded was higher in wild relative compared to cultivated A. cepa, possibly because most of the A. cepa variety is derived by domestication but wild relatives are open pollinated and undergoes extensive gene pool shuffling leading to higher heterogeneity. In this study, SSR markers were successfully utilized to assess genetic variations in popular Indian A. cepa, A. sativum and establish genetic relationships with wild Allium species. These markers can be harnessed for molecular breeding, varietal identification and planning germplasm conservation strategies in future. © 2019, National Institute of Science Communication and Information Resources (NISCAIR). All rights reserved.
Chloroplast derived SSRs reveals genetic relationships in domesticated alliums and wild relatives
(Springer Science and Business Media B.V., 2022) Kuldip Jayaswall; Himanshu Sharma; Abhishek Bhandawat; R. Sagar; Deepanshu Jayaswal; Akash Kumar; Pratibha Chaturvedi; Vijay Mahajan; Sanjay Kumar; Major Singh
Alliums comprise of popular spices and used for various culinary purposes and nutraceuticals. Poor genetic characterization and scarce information regarding domestication, evolution of important alliums like onion, garlic and wild relatives are the major bottleneck to alliums improvement programs. In present study, chloroplast simple sequence repeat (cpSSR) markers were developed from Allium paradoxum plastome to examine the genetic relationships among onion, garlic and wild germplasms. A total of 15 cpSSR markers were used to establish genetic relationships in 18 individuals of alliums, producing 40 alleles ranging from 1 to 6 alleles per SSR locus and polymorphism potential of 100%.Average PIC and heterozygosities was found to be 0.392 and 0.330, respectively. The developed cpSSR significantly assisted the phylogenetic study and evolutionary relationships among various Allium species. In future, these cpSSR markers will be useful for varietal identification, purity testing and genetic improvement of alliums. © 2021, The Author(s), under exclusive licence to Springer Nature B.V.
Co-overexpression of AtDREB1A and BcZAT12 increases drought tolerance and fruit production in double transgenic tomato (Solanum lycopersicum) plants
(Elsevier B.V., 2021) Ram Krishna; Waquar Akhter Ansari; Durgesh Kumar Jaiswal; Achuit Kumar Singh; Jay Prakash Verma; Major Singh
Drought is the major problem in agricultural production due to loss of moisture content in soil as well as climate variations. Our main aim is to enhance drought tolerance and yield potential in the present study pyramided Arabidopsis thaliana Dehydration Responsive Element Binding1A (AtDREB1A) and Brasica caranata Zinc finger proteins (BcZAT12) transcription factor genes driven by ectopic promoter rd29 A of Arabidopsis thaliana and Brassica carinata lea1, respectively. Co-overexpression of both the genes provides tolerance to multiple abiotic stresses but the AtDREB1A overexpression has been reported to cause retarded growth and dwarf phenotype; however BcZAT12 overexpressing transgenic plants does not show retarded growth and dwarf phenotype. Co-overexpressing of AtDREB1A and BcZAT12 in five (DZ1-DZ5) double transgenic (DT) tomato lines has been observed under 0, 07, 14 and 21 Days of Water Deficit (DWD). The DT plants showed enhanced drought tolerance and yield potential than single transgenic (ST) and non transgenic (NT) plants. Furthermore, AtDREB1A and BcZAT12 co-overexpressed plants showed reduced level of electrolyte leakage (EL), hydrogen peroxide and membrane lipid peroxidation and elevated level of relative water content (RWC), proline, chlorophyll color index (CCI) and photosynthetic efficiency as compared to ST and NT. The plant growth and yield attributes were improved by the co-overexpression of AtDREB1A and BcZAT12 in DT plants. The transcript analysis showed the increased level of DREB1A, ZAT12 and P5CS genes expression which were higher in DT tomato plants, and indicate that both the genes induce together in the DT plants. The present study which is first report of co-overexpressing AtDREB1A and BcZAT12 in tomato will provide a base for genetic engineering in plants through the multigenic transgenic approach to cope against various biotic and abiotic stresses. © 2021 Elsevier B.V.
Comparative in vitro shoot organogenesis and plantlet regeneration in tomato genotypes
(Indian Academy of Horticultural Sciences, 2010) Anita Singh; Major Singh; B.D. Singh
A study was conducted for achieving high frequency regeneration in tomato genotypes as a pre-requisite for genetic transformation. Regeneration efficiencies were compared in five tomato genotypes using hypocotyl and cotyledon segments as explant source. Two cytokinins, namely BAP (0.0 to 3.0 mg l-1) in combination with or without kinetin (0.5 and 1.0 mg l -1) were used in Murashige and Skoog's medium. Irrespective of genotypes, 2.0 mg l-1 BAP supplementation gave the best response for both explants. The highest frequency shoot regeneration (96.6 and 92.2%) as well as the maximum number of shoots per explant (10.2 and 8.4) was obtained from the hypocotyl and cotyledon explants, respectively in genotype H-86. The cultures on this medium were green and showed good shoot bud organogenesis. The individual shootlets were separated and inoculated on growth regulator-free MS medium. After two weeks of root induction, the individual plantlets were transferred to glass jars filled with autoclavable polyproplylene (PP) caps filled with sterile peat: vermiculite (2:1). This hardening strategy lead to over 90.0% plant survival at greenhouse stage. The results suggested that of the two explants tested, hypocotyl segment was more responsive compared to cotyledon segment. The order of genotype response was H-86> H-24> DVRT-1> Sel-7> DVRT-2 with regards to shoot organogenesis and multiplication frequency.
De novo assembly of bitter gourd transcriptomes: Gene expression and sequence variations in gynoecious and monoecious lines
(Public Library of Science, 2015) Anjali Shukla; V.K. Singh; D.R. Bharadwaj; Rajesh Kumar; Ashutosh Rai; A.K. Rai; Raja Mugasimangalam; Sriram Parameswaran; Major Singh; P.S. Naik
Bitter gourd (Momordica charantia L.) is a nutritious vegetable crop of Asian origin, used as a medicinal herb in Indian and Chinese traditional medicine. Molecular breeding in bitter gourd is in its infancy, due to limited molecular resources, particularly on functional markers for traits such as gynoecy. We performed de novo transcriptome sequencing of bitter gourd using Illumina next-generation sequencer, from root, flower buds, stem and leaf samples of gynoecious line (Gy323) and a monoecious line (DRAR1). A total of 65,540 transcripts for Gy323 and 61,490 for DRAR1 were obtained. Comparisons revealed SNP and SSR variations between these lines and, identification of gene classes. Based on available transcripts we identified 80 WRKY transcription factors, several reported in responses to biotic and abiotic stresses; 56 ARF genes which play a pivotal role in auxin-regulated gene expression and development. The data presented will be useful in both functions studies and breeding programs in bitter gourd. © 2015 Shukla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
De novo assembly, differential gene expression and pathway analyses for anthracnose resistance in chilli (Capsicum annuum L.)
(Springer, 2022) Rajesh Kumar; Ashutosh Rai; Avinash Chandra Rai; Vinay Kumar Singh; Major Singh; Prabhakar Mohan Singh; Jagdish Singh
Chilli (Capsicum annuum L.) is one of the most important vegetable as well as spice crops grown worldwide for its wide usage. Production of chilli is profoundly affected by anthracnose disease which is one of the most serious and destructive fungal diseases of chilli, especially in tropical and subtropical regions causing around 60–80% yield loss under severe infection. In order to understand the pattern of gene expression and development of molecular markers in chilli for management of anthracnose disease, transcriptome analysis of a resistant (IIVRC-452) and susceptible (Pusa Jwala) genotypes was carried out. Transcriptome data yielded a total of 53,921,012 and 50,079,890 reads with HQ bases 5.18 GB and 4.78 GB of FASTAq sequences and were used for de novo assembly of transcriptome for IIVRC-452 and Pusa Jwala, respectively. More than 50 thousand unigenes were identified in each genotype and around 40,000 of them could be annotated with 30% cut off identity. Digital gene expression analysis revealed a total of 3124 transcripts differentially expressed in resistant and susceptible lines. A total of 871 differential transcripts were annotated and 814 differential genes were present in both resistant and susceptible lines. Furthermore, five key candidate genes (CaLOX, CaLAG-1, CaPG, CaCYP76A2, and CaSAP-13) in pathogen-responsive pathway were identified by quantitative real-time PCR (qRT-PCR). Transcriptome data generated in the present study is a valuable resource for focused investigation on plant–pathogen-interaction, to study the pathways involved in resistance mechanism and to identify markers for use in resistance breeding programmes. © 2021, Society for Plant Biochemistry and Biotechnology.
Determination of proper gamma radiation dose for creating variation in indian garlic varieties
(National Institute of Science Communication and Information Resources (NISCAIR), 2019) Ashwini P Benke; Somnath Dukare; Kuldip Jayaswall; Vinod Kumar Yadav; Major Singh
Garlic (Allium sativum L.) an important neutraceutical crop. It is used as traditional medicine to aid digestion, respiration, parasitic infestation and to treat leprosy and various other diseases. But biologically garlic is sterile in nature hence for creating variation; traditional breeding methods are not applicable. Further clonal propagation method leads garlic more prone for various seed and soil borne diseases including virus infection. And natural genetic variation in morphological as well as biochemical traits is limited in garlic. Considering this is in view, among various methods of creating variation, treating bulbs with radiation is simple and easy technique. Main activity of mutation breeding using gamma radiation is to determine optimum dose (LD-50). Hence in present experiment, two garlic varieties were subjected to gamma radiation treatments (0, 1, 5, 10, 25, 50, 75 and 100 gray). Aim of this experiment is to identify LD-50 dose and then generate variation in garlic like increase in bulb size, bulb shape, high neutraceutical compounds like allicin etc. In results, both treated varieties recorded 50 percent germination at dose 10 grays as LD-50. Further on basis of survival of plantlets and final matured plants count, optimum gamma radiation dose of 5 gray for Bhima Omkar and in between 5 to 10 gray for Bhima Purple is identified as LD-50 respectively. In case of Bhima Purple, there is further need to screen the population for exact dose value of LD-50. All other morphological traits in both varieties recorded decreasing trend with increase in radiation doses. However no significant alterations observed visually in any treatment. This optimized radiation dose will be helpful for subjecting other genotypes for getting mutants and generating diversity in garlic which may have high neutracutical compounds and would suitable for processing and export. © 2019, National Institute of Science Communication and Information Resources (NISCAIR). All right reserved.
Development of Allium cepa potential intron polymorphism markers for molecular breeding of Alliums
(Elsevier B.V., 2024) Kuldip Jayaswall; R. Sagar; Deepanshu Jayaswal; Akash Kumar; Surendra Pratap Singh; Romit Seth; Himanshu Sharma; Deepesh Kumar; Vijay Mahajan; Sanjay Kumar; Major Singh
Allium cepa is a widely grown crop for its spice and culinary properties. For molecular breeding of Allium cepa, mining and utilization of various sequence-based markers have been widely reported. Unfortunately, none has reported its molecular breeding using potential intron polymorphism (PIP) markers. Due to advantage of intron positioning prediction and practical utility, the PIP markers used for screening the possible polymorphism and cross-transferability in the Alliums. Screening results showed that among 500 Allium cepa potential intron polymorphism (AcPIP) markers, 275 are cross-transferable and polymorphic. Among the 275 AcPIP, 111 polymorphic markers were physically mapped on to 1st to 8th chromosomes of the Allium cepa. Out of the 275 AcPIP containing functionally significant markers, 118 were mapped in onion with Arabidopsis proteome. A set of 10 Alliums were utilized to interpret the polymorphic possibilities of the 500 AcPIP markers. In totality, 55 % AcPIP markers were polymorphic and cross-transferable among the Alliums. The polymorphic information content (PIC) of AcPIP markers ranged from 0.03 to 0.47 and heterozygosity index (H) varied between 0.16 and 0.80. The constructed phylogenetic tree based on the AcPIP markers of 10 Alliums revealed different clusters due to differences in their taxonomic positions. Out of 275 AcPIP markers, 10 AcPIP markers genotyping based Jaccard dissimilarity-based NJ tree of 96 individuals of Allium cepa showed two distinct groups (high total soluble solid; HTSS and low total soluble solid; LTSS). NJ tree and dissimilarity matrix reveal that group I genotypes are distinct, and dissimilar from group II and III genotypes hence it seems that group II and III genotype evolve from group I genotypes. Further we identified nine diverse Allium cepa subsample among which, genotype number 74 has HTSS and could be used for identification and introgression of HTSS coding genes in elite cultivars. Further DNA fingerprint of Alliums with 20 AcPIP markers suggested geographical reach of the released onion varieties. Hence results suggest that 275 AcPIP markers may be useful for accelerating the breeding programme of the Alliums and other species. © 2023 SAAB
Development of an embryo germination protocol for shy-seeded grape (Vitis vinifera L.)
(Cambridge University Press, 2021) Ashwini P. Benke; Ram Krishna; Roshni R. Samarth; Shweta S. Dhumal; Waquar A. Ansari; Poonam V. Shelke; Somnath S. Dukare; Major Singh
Acquisition and germination of seeds are the most desired targets for the improvement of vegetatively propagated crops. In the present study, we developed a potential embryo germination protocol for the Red Globe grape cultivar having a low seed germination rate. Three grape berries at different developmental stages, viz. 50, 60 and 70 days after flowering (DAF), were selected for in-vitro embryo germination. Three growth media, namely Emershad and Ramming (ER), Nitsch and Nitsch (NN) and Murashige and Skoog (MS), and plant growth regulators (benzyl amino purine (BA), 0.5, 0.7 and 0.9 mg/l; indole butyric acid (IBA), 1.0, 1.5 and 2.0 mg/l; and gibberellic acid (GA), 0.1, 0.3 and 0.9 mg/l) were screened individually in different combinations with three amino acids, namely cysteine, glutamine and proline (2.0 μmol/l each). The maximum embryos germination percentage recorded at 70 DAF was 63.33, 47.78 and 45.56% in ER, NN and MS media, respectively, supplemented with 0.9 mg/l BA, 2.0 mg/l IBA, 0.9 mg/l GA and 2.0 μmol glutamine. Glutamine was found to have the most significant impact, and it improved the rescued embryos germination. The present study provides a potential recipe for a medium that can facilitate efficient germination of grape embryos. © The Author(s), 2021. Published by Cambridge University Press on behalf of NIAB.
Development of chloroplast derived SSR markers for genus Allium and their characterization in the allies for genetic improvement of Alliums
(Elsevier B.V., 2023) Kuldip Jayaswall; Himanshu Sharma; Deepanshu Jayaswal; R. Sagar; Abhishek Bhandawat; Akash Kumar; Isha Sharma; Pratibha Chaturvedi; Dalasanuru Chandregowda Manjunathagowda; Deepesh Kumar; Vijay Mahajan; Sanjay Kumar; Major Singh
Alliums are the most popular for their culinary usage and nutraceutical benefits. Their production is greatly affected by the multiple biotic and abiotic stresses. Poor characterizations of genetic resources are the major bottleneck in the genetic improvement of Alliums. Chloroplast-derived simple sequence repeat has recently gained much popularity due to their maternal inheritance and low recombination along with their hypervariable nature. In this study, 22 of the 28 chloroplast-derived markers from chloroplast genomes of A. cepa and A. sativum were reported as polymorphic and cross-transferable. Tetra nucleotide repeats were more prevalent (52 %) in these 22 cpSSR markers as compared to other repeat motifs. Further polymorphic information content of these 22cpSSR markers ranged from 0.007 to 0.427. Based on the genotyping of 22cpSSR markers, 25 Alliums were clustered into two groups (groups I, IIA, and IIB). This showed that cultivated Allium cepa and Allium sativum belong to group II rather than the majority of wild Alliums, which belong to group I. Hence This proved the widespread use of these AccpSSR and AscpSSR markers. Additionally, it was clear from 79 Alliums morphological and genotyping-based clustering that these 79 wild Alliums exhibit strong clustering consistency in genotyping and phenotyping-based dendrograms, hence confirming these cpSSR's discrimination potential. Allium tuberosum Rottler ex. Spreng is quite distinct from Allium fistulosum L, A. schoenoprasumas as well as Allium cepa var. aggregatum G.Don. Further Allium fistulosum L, A. schoenoprasum and Allium cepa var. aggregatum are closely related, according to genotyping-based dendrograms of 79 wild Alliums. Hence Allium fistulosum L, A. schoenoprasum, and Allium cepa provide a brilliant opportunity for introgression of the high total soluble content, high quercetin content, and allicin content of cultivated Allium into wild Allium as well as the biotic and abiotic stress tolerance of wild Alliums into cultivated Alliums. Subsequent clustering of 79 Alliums further allowed us to identify diverse Alliums for constructing a core collection. Hence present study will be useful for molecular breeding and genomic selection-based crop improvement. © 2023 SAAB
Development of intron length polymorphic (ILP) markers in onion (Allium cepa L.), and their cross-species transferability in garlic (A. sativum L.) and wild relatives
(Springer Netherlands, 2019) Kuldip Jayaswall; Himanshu Sharma; Abhishek Bhandawat; Ram Sagar; Vinod Kumar Yadav; Vikas Sharma; Vijay Mahajan; Joy Roy; Major Singh
Onion (Allium cepa L.) is a popular spice and a plant of high medicinal value. Conventional breeding and genetic improvement efforts were largely limited due to self-incompatibility and heterozygosity. Recently, marker assisted breeding has significantly reduced time and labour in developing elite varieties. But very limited polymorphic and cross-transferable markers are available in onion. There is an urgent need to develop polymorphic markers in Allium to expedite and introgress desirable traits from wild relatives (which are rich bioresource of various biotic and abiotic resistance genes) to A. cepa. Considering limited availability of reliable molecular markers in Allium and wild relatives, in current study, 20,204 ESTs (3750 contigs and 8364 singletons), of A. cepa were successfully utilized for identification of over 2689 intron length polymorphic (ILP) markers. A set of 30 markers was tested for polymorphism in onion and cross-transferability in garlic and related wild species. Among these, eighteen markers amplified at least one of the accessions of A. cepa. Transferability of these ILP markers was ranged from 21.7 to 95.7% in Allium spp. Low level of polymorphism in A. cepa compared to wild Allium species is reported. Based on the Jaccard dissimilarity matrix, a neighbour-joining tree was constructed, which clustered all the 23 varieties/accessions under three groups. All the varieties of A. cepa were clearly clustered separately under group I. However, there was intermixing of varieties/accessions of A. sativum L. and wild relatives, which may possibly be due to less number of markers validated for cross-transferability. In future, larger set of markers will be used to resolve the genetic variations among wild varieties and A. sativum These 18 polymorphic ILP markers could be utilised for diversity characterization of Allium spp., varietal identification, mapping of genes and introgression of desirable traits from wild relatives. © 2019, Springer Nature B.V.
Effect of heat-shock induced oxidative stress is suppressed in BcZAT12 expressing drought tolerant tomato
(Elsevier Ltd, 2013) Kavita Shah; Major Singh; Avinash Chandra Rai
The transcription factor ZAT12 is a member of stress-responsive C 2H2 type zinc finger protein (ZFP) reported to control the expression of stress-activated genes mediated via ROS in plants. BcZAT12-transformed tomato cv. H-86, var. Kashi vishesh (lines ZT1-ZT6) over-expressing the gene product is demonstrated herein to be tolerant to heat-shock (HS)-induced oxidative stress. Results reveal that the relative expression of ZAT12 as well as heat induced Hsp17.4 and Hsp21 gene transcripts increased in transgenic upon exposure to HS. The transformed tomato lines ZT1 and ZT5 had significantly lowered free radical formation, improved electrolyte leakage, relative water content and chlorophyll levels with an enhanced activities of antioxidant enzymes viz. superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase when exposed to HS. HS-induced oxidative stress by over-expression of the BcZAT12 gene transcripts in tomato as well as by largely enhancing the ROS-scavenging capacity and up regulation of Hsp transcripts. This enables the transgenic tomato plants to acquire a greater ability to counteract HSinduced oxidative stress, being endowed with more reduced antioxidant pools. The use of these HS-tolerant tomato lines could possibly be used for tomato cultivation in the areas affected by sudden temperature changes. © 2013 Elsevier Ltd. All rights reserved.
Effect of water withdrawal on formation of free radical, proline accumulation and activities of antioxidant enzymes in ZAT12-transformed transgenic tomato plants
(2012) Avinash Chandra Rai; Major Singh; Kavita Shah
Water stress often leads to the accumulation of reactive oxygen species (ROS) and their excessive production alters the activities of enzymes involved in their removal. ZAT12 is a member of stress-responsive C2H2 type Zinc Finger Protein (ZFP) reported to control the expression of several stress-activated genes in plants through ROS signaling. The ZAT12-transformed tomato lines (cv. H-86 variety Kashi Vishesh) when subjected to water withdrawal for 7, 14 and 21 days revealed significant and consistent changes in activities of enzymes SOD, CAT, APX, GR and POD paralleled with an increased proline levels. Unlike that in wild-type tomato, the leaf superoxide anion and hydrogen peroxide concentrations in the transformed tomato plants did not alter much, suggesting a well regulated formation of free radicals suppressing oxidative stress in the latter. Results suggest BcZAT12-transformed tomato lines ZT1, ZT2 and ZT6 to be better adapted to drought stress tolerance by accumulation of osmolyte proline and increased antioxidant response triggered by the ZAT12 gene. Therefore, the ZAT12-transformed tomato cv. H-86 lines will prove useful for higher yield of tomato crop in regions affected with severe drought stress. © 2012 Elsevier Masson SAS.
Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium-mediated transformation of tomato
(2012) Govind Kumar Rai; Neha Prakash Rai; Sanjeev Kumar; Akhilesh Yadav; Sushma Rathaur; Major Singh
An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog's (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l -1 thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l -1 in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T 1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars. © 2012 The Society for In Vitro Biology.