In planta transformation in wheat: an improved protocol to develop wheat transformants.

BACKGROUND: Lack of efficient transformation protocol continues to be a major bottleneck for successful genome editing or transgenic development in wheat. An in planta transformation method was developed in Indian bread wheat in earlier study (Vasil et al. in Nat Biotechnol 10:667-674, 1992) which was labour-intensive and time-consuming. In the present study, in planta transformation method was improved to make it simple, efficient, less labour-intensive and time-saving. METHODS AND RESULTS: PCR-based screening for generated transformants at T0 stage was introduced in this method. Shoot apical meristem of two days old wheat seedling was inoculated with the routine active culture of Agrobacterium tumefaciens harboring plasmid pCAMBIA1300-Ubi-GFP having gene GFP under the control of Zea mays ubiquitin promoter. PCR analysis at T0 stage confirmed 27 plants to be transgene positive. These 27 plants were only taken to the next generation (T1) and the rest were discarded. At T1 generation 6 plants were analyzed to be PCR positive. Out of them, 4 plants were confirmed to have stable integration of transgene (GFP). Fluorescent microscopy at T1 stage confirmed the 4 Southern hybridization positive plants to be expressing reporter gene GFP. CONCLUSIONS: Screening at T0 stage, reduced the load of plants to be taken to T1 generation and their screening thereof at T1 with no overall loss in transformation efficiency. We successfully transformed wheat genotype HD2894 with 3.33% transformation efficiency using a simple, effective method which was less labour-intensive and less time-consuming. This method may be utilized to develop wheat transgenic as well as genome edited lines for desirable traits.

Agro-morphological and nutritional assessment of chenopod and quinoa germplasm-Highly adaptable potential crops.

Quinoa belongs to the family Chenopodiaceae, a pseudo-grain having high nutritional value and is considered an underexploited vegetable crop with the potential to improve the nutritional security of millions. Therefore, assessing genetic diversity in Chenopodium germplasm to untap nutritional and site-specific adaptation potential would be of prime importance for breeders/researchers. The present study used 10 accessions of two Chenopodium species, that is, C. quinoa and C. album. Quantitative and qualitative phenotypic traits, proximate composition, minerals, and amino acids profiles were studied to compare the differences in nutritional value and extent of genetic diversity between these two species. Our results showed significant variation existed in yield attributing agro-morphological traits. All the traits were considered for hierarchical clustering and principal components analysis. Large genetic variability was observed in traits of Chenopodium accessions. The protein, dietary fiber, oil, and sugar content ranged from 16.6% to 19.7%, 16.8% to 26%, 3.54% to 8.46%, and 3.74% to 5.64%, respectively. The results showed that C. album and C. quinoa seeds had good nutritional value and health-promoting benefits. The C. quinoa was slightly ahead of than C. album in terms of nutritional value, but C. album accession IC415477 was at par for higher test weight, seed yield (117.02 g/plant), and other nutritional parameters with C. quinoa accessions. IC415477 and other potential accessions observed in this study may be taken up by breeders/researchers in the near future to dissect nutritional value of Chenopodium and related species for dietary diversity, which is imperative for the nutritional security of the ever-growing world's population.

Indexing Resilience to Heat and Drought Stress in the Wild Relatives of Rapeseed-Mustard.

Wild species are weedy relatives and progenitors of cultivated crops, usually maintained in their centres of origin. They are rich sources of diversity as they possess many agriculturally important traits. In this study, we analysed 25 wild species and 5 U triangle species of Brassica for their potential tolerance against heat and drought stress during germination and in order to examine the early seedling stage. We identified the germplasms based on the mean membership function value (MFV), which was calculated from the tolerance index of shoot length, root length, and biochemical analysis. The study revealed that B. napus (GSC-6) could withstand high temperatures and drought. Other genotypes that were tolerant to the impact of heat stress were B. tournefortii (RBT 2002), D. gomez-campoi, B. tournefortii (Rawa), L. sativum, and B. carinata (PC-6). C. sativa resisted drought but did not perform well when subjected to high temperatures. Tolerance to drought was observed in B. fruticulosa (Spain), B. tournefortii (RBT 2003), C. bursa-pastoris (late), D. muralis, C. abyssinica (EC694145), C. abyssinica (EC400058) and B. juncea (Pusa Jaikisan). This investigation contributes to germplasm characterization and the identification of the potential source of abiotic stress tolerance in the Brassica breeding programme. These identified genotypes can be potential sources for transferring the gene(s)/genomic regions that determine tolerance to the elite cultivars.

Mining legume germplasm for genetic gains: An Indian perspective.

Legumes play a significant role in food and nutritional security and contribute to environmental sustainability. Although legumes are highly beneficial crops, it has not yet been possible to enhance their yield and production to a satisfactory level. Amid a rising population and low yield levels, per capita average legume consumption in India has fallen by 71% over the last 50 years, and this has led to protein-related malnutrition in a large segment of the Indian population, especially women and children. Several factors have hindered attempts to achieve yield enhancement in grain legumes, including biotic and abiotic pressures, a lack of good ideotypes, less amenability to mechanization, poorer responsiveness to fertilizer input, and a poor genetic base. Therefore, there is a need to mine the approximately 0.4 million ex situ collections of legumes that are being conserved in gene banks globally for identification of ideal donors for various traits. The Indian National Gene Bank conserves over 63,000 accessions of legumes belonging to 61 species. Recent initiatives have been undertaken in consortia mode with the aim of unlocking the genetic potential of ex situ collections and conducting large-scale germplasm characterization and evaluation analyses. We assume that large-scale phenotyping integrated with omics-based science will aid the identification of target traits and their use to enhance genetic gains. Additionally, in cases where the genetic base of major legumes is narrow, wild relatives have been evaluated, and these are being exploited through pre-breeding. Thus far, >200 accessions of various legumes have been registered as unique donors for various traits of interest.

Expression of a Pennisetum glaucum gene DREB2A confers enhanced heat, drought and salinity tolerance in transgenic Arabidopsis.

BACKGROUND: Pearl millet (Pennisetum glaucum) is an essential cereal crop, whose growth and yield are not impacted by abiotic stresses, such as drought, heat, and cold. The DREB transcription factors (TF) are some of the largest groups of TFs in plants and play varied roles in plant stress response and signal transduction. METHODS AND RESULTS: In the present study, PgDREB2A gene encoding a DREB transcription factor in pearl millet was functionally characterized in Arabidopsis. DREB2A proteins contain a conserved domain that binds toethylene responsive element binding factors. Three different T1 transgenic lines overexpressing PgDREB2A gene were identified by Southern blot. Quantitative real-time polymerase chain reaction exhibited that PgDREB2A could be induced under drought conditions. As compared with the control, PgDREB2A overexpressing transgenic Arabidopsis showed increased rate of seed germination and root growth in transgenic lines under higher concentrations of mannitol, NaCl, ABA, heat and cold stress. Additionally, PgDREB2A transgenic lines showed enhanced durability after rehydration and tolerance to drought and salt stress was augmented with increased proline and reduced MDA build-up and diminishing water loss. CONCLUSIONS: Results from this study suggested that PgDREB2A as a transcription factor may improve endurance to various abiotic stresses and can be employed for developing crops tolerant to abiotic stresses.

Current Research Trends and Prospects for Yield and Quality Improvement in Sesame, an Important Oilseed Crop.

Climate change is shifting agricultural production, which could impact the economic and cultural contexts of the oilseed industry, including sesame. Environmental threats (biotic and abiotic stresses) affect sesame production and thus yield (especially oil content). However, few studies have investigated the genetic enhancement, quality improvement, or the underlying mechanisms of stress tolerance in sesame. This study reveals the challenges faced by farmers/researchers growing sesame crops and the potential genetic and genomic resources for addressing the threats, including: (1) developing sesame varieties that tolerate phyllody, root rot disease, and waterlogging; (2) investigating beneficial agro-morphological traits, such as determinate growth, prostrate habit, and delayed response to seed shattering; (3) using wild relatives of sesame for wide hybridization; and (4) advancing existing strategies to maintain sesame production under changing climatic conditions. Future research programs need to add technologies and develop the best research strategies for economic and sustainable development.

Novel ASR isolated from drought stress responsive SSH library in pearl millet confers multiple abiotic stress tolerance in PgASR3 transgenic Arabidopsis.

A genomic resource of drought stress responsive genes/ESTs was generated using Suppression Subtractive Hybridization (SSH) approach in a drought stress tolerant Pennisetum glaucum genotype 841B. Fifty five days old plants were subjected to drought stress after withholding water for different time intervals (10 days, 15 days, 20 days and 25 days). A forward subtractive cDNA library was prepared from isolated RNA of leaf tissue. Differential gene expression under drought stress was validated for selected nine contigs by RT-qPCR. A transcript homologous to Setaria italica ASR3 upregulated under drought stress was isolated from genotype 841B and characterized. Heterologous expression of PgASR3 was validated in Arabidopsis and confirmed under multiple abiotic stress conditions. A total of four independent transgenic lines overexpressing gene PgASR3 were analyzed by Southern blot at T1 stage. For drought stress tolerance, three independent lines (T2 stage) were analyzed by biochemical and physiological assays at seedling stage. The growth rate (shoot and root length) of transgenic seedlings improved as compared to WT seedling under differenct abiotic stress conditions. The three transgenic lines were also validated for drought stress tolerance and RT-qPCR analysis, at maturity stage. Under drought stress conditions, the mature transgenic lines showed higher levels of RWC, chlorophyll and proline but lower levels of MDA as compared to WT plants. PgASR3 gene isolated and validated in this study can be utilized for developing abiotic stress tolerant crops.

Heat stress inducible cytoplasmic isoform of ClpB1 from Z. nummularia exhibits enhanced thermotolerance in transgenic tobacco.

Previously, we isolated CDS of Ziziphus nummularia isoform ZnJClpB1-C from heat stress-tolerant genotype Jaisalmer. To further functionally validate ZnJClpB1-C assumed function in tobacco and to generate novel germplasm for heat stress tolerance, this gene was transformed in the Nicotiana tabacum. ClpB proteins are the major key player required for basal and induced heat stress tolerance in plant cells under heat stress. In Ziziphus nummularia ClpB1-C transcript from genotype Jaisalmer was highly upregulated under heat stress conditions, as reported earlier. Nine transgenic lines (T1) from three transgenic tobacco events with single-copy integration (T0 stage) were taken for heat stress analysis at seedling stage. Mature tobacco transgenic plants did not show any deformity as compared to wild plants when grown under normal conditions. Overexpression of ZnJClpB1-C in tobacco significantly increased the tolerance to heat stress. Under heat stress conditions (42 °C), T1 transgenic tobacco seedlings showed higher photosynthetic rate, relative water content, membrane stability index and lower levels of MDA, compared to the wild type untransformed plants. The qRT-PCR analysis revealed different level of transgene expression (1.08 to 3.89 folds) in 9 T1 transgenic lines. In vitro roles of ZnJClpB1-C regulating thermotolerance is not reported so far. These results demonstrated the positive roles of ZnJClpB1-C in enhancing thermotolerance and its use as a genomic resource in the near future for developing heat stress-tolerant germplasm.

A quick, easy and cost-effective in planta method to develop direct transformants in wheat.

Agrobacterium mediated in planta method was used to transform Indian elite wheat genotype HD2894 with herbicide-tolerant CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene. The apical meristems of germinated seeds were targeted for introgression of transgene. The obtained T1 plants were screened by spraying 1% glyphosate and only positive transformants survived. The presence of transgene was also confirmed by PCR and Southern hybridization. Using this method, 3.07% transformation rate was observed. To identify transgenic lines carrying stably integrated CP4-EPSPS gene, the transgenic populations were screened in T3 generation using 1% glyphosate and lines with 100% survival were considered as homozygous. No significant morpho-physiological variations were observed within the transgenic lines as compared to non-transgenic plants. The present study resulted in herbicide-tolerant transgenic wheat and provides a valuable tool for development of wheat genetic transformation.

Heat stress transcripts, differential expression, and profiling of heat stress tolerant gene TaHsp90 in Indian wheat (Triticum aestivum L.) cv C306.

To generate a genetic resource of heat stress responsive genes/ESTs, suppression subtractive hybridization (SSH) library was constructed in a heat and drought stress tolerant Indian bread wheat cultivar C306. Ninety three days old plants during grain filling stage were subjected to heat stress at an elevated temperature of 37°C and 42°C for different time intervals (30 min, 1h, 2h, 4h, and 6h). Two subtractive cDNA libraries were prepared with RNA isolated from leaf samples at 37°C and 42°C heat stress. The ESTs obtained were reconfirmed by reverse northern dot blot hybridization. A total of 175 contigs and 403 singlets were obtained from 1728 ESTs by gene ontology analysis. Differential expression under heat stress was validated for a few selected genes (10) by qRT-PCR. A transcript showing homology to Hsp90 was observed to be upregulated (7.6 fold) under heat stress in cv. C306. CDS of TaHsp90 (Accession no. MF383197) was isolated from cv. C306 and characterized. Heterologous expression of TaHsp90 was validated in E. coli BL21 and confirmed by protein gel blot and MALDI-TOF analysis. Computational based analysis was carried out to understand the molecular functioning of TaHsp90. The heat stress responsive SSH library developed led to identification of a number of heat responsive genes/ESTs, which can be utilized for unravelling the heat tolerance mechanism in wheat. Gene TaHsp90 isolated and characterized in the present study can be utilized for developing heat tolerant transgenic crops.

Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

BACKGROUND: Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42 °C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage was cloned, characterized and its role was validated under heat stress by proteomics and in-silico studies. In the present study we report the characterization at molecular and in-silico level of peroxisomal TapAPX gene isolated from heat tolerant wheat cultivar of India. RESULTS: qPCR studies of TapAPX gene displayed up to 203 fold level of expression at 42 °C heat stress exposure. A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling. TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting. CONCLUSIONS: TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

The Zebrafish GenomeWiki: a crowdsourcing approach to connect the long tail for zebrafish gene annotation.

A large repertoire of gene-centric data has been generated in the field of zebrafish biology. Although the bulk of these data are available in the public domain, most of them are not readily accessible or available in nonstandard formats. One major challenge is to unify and integrate these widely scattered data sources. We tested the hypothesis that active community participation could be a viable option to address this challenge. We present here our approach to create standards for assimilation and sharing of information and a system of open standards for database intercommunication. We have attempted to address this challenge by creating a community-centric solution for zebrafish gene annotation. The Zebrafish GenomeWiki is a 'wiki'-based resource, which aims to provide an altruistic shared environment for collective annotation of the zebrafish genes. The Zebrafish GenomeWiki has features that enable users to comment, annotate, edit and rate this gene-centric information. The credits for contributions can be tracked through a transparent microattribution system. In contrast to other wikis, the Zebrafish GenomeWiki is a 'structured wiki' or rather a 'semantic wiki'. The Zebrafish GenomeWiki implements a semantically linked data structure, which in the future would be amenable to semantic search. Database URL: http://genome.igib.res.in/twiki.