Containment evaluation, cold tolerance and toxicity analysis in Osmotin transgenic tomato (Solanum lycopersicum L. cv. Pusa Ruby).

The present study reports engineered cold tolerance and toxicity analysis in genetically modified tomato (Solanum lycopersicum L. cv. Pusa Ruby) developed through constitutive over expression of Nicotiana tabacum Osmotin gene. Rate of seed germination, seedling establishment and growth remained unaffected in the transgenic tomato in response to a low temperature (15 °C) treatment, but were significantly (P ≤ 0.05) reduced in the wild type. At reproductive stage, the wild type plants failed to recover at the low temperature (4.0 °C) treatment for 10 days but the transgenic plants survived successfully without any leaf senescence or other visible chilling injury symptoms. The quantitative transcript expression analysis confirmed up regulation of the transgene by 55% in the transgenic plants on cold treatment for 2 h whereas, the transcripts were not detected in the wild type. Containment evaluation under normal environmental conditions revealed similar morphology in both the transgenic and wild type tomato plants however an average fruit yield was higher in the transgenic plants (725.91 ± 39.27 g) than the wild type (679.84 ± 28.80 g). The composition of mature fruits in terms of element content was at par in both the transgenic and wild type except significantly higher Ca and Mg contents in the transgenic fruits than that of the wild type. Further, acute and sub-acute toxicity tests conducted in the adult female Wister rats revealed no mortality or significant changes in general and psychological behaviour, at par food intake and body weight and, normal biochemical, and hematological parameters for animals fed with the wild type or transgenic tomato fruits as compared to the control group, confirming its safety for animal consumption.

Overexpression of NAC gene from Lepidium latifolium L. enhances biomass, shortens life cycle and induces cold stress tolerance in tobacco: potential for engineering fourth generation biofuel crops.

We report elevated biomass and altered growth characteristics of tobacco plants up on transformation with a NAC (NAM, ATAF1/2,CUC2) gene (GenBank Accession FJ754254) isolated from Lepidium latifolium L. (LlaNAC). Transgenic plants showed significant differences in fresh weight, midrib length of longest leaf, leaf area, height of the plant, root and shoot weights, etc. during vegetative phase. On 100th day after sowing (DAS), plants of transgenic lines were 2-3 times taller than the wild type plants, though no significant difference was recorded in moisture contents of any of the plant tissues. Over-expression of NAC gene up to 2,000 fold was recorded in leaves of transgenic plants on 100th DAS. Interestingly, transgenic plants showed significantly shortened (P(t) = 0.02-0.04) life cycle, as they showed a completely altered growth behaviour. Transgenic plants entered reproductive phase earlier by 60 days, with lines NC2 and NC7b entering first, followed by line NC10. However, the time period spent in the reproductive phase by the plant was nearly twice in case of transgenic lines NC2, NC7b and NC10, as compared to the wild type plants. Despite that, these lines completed their life cycle in 45-60 days lesser than the time taken by wild-type tobacco plants. No difference was recorded in fruit and seed yield of transgenic or wild type plants. To the best of our knowledge, this is the first report on over-expression of NAC gene causing altered growth and biomass patterns. We expect this study to become an important reference towards future engineering of plants for fuel and fodder purposes.

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

Cloning and characterization of GPAT gene from Lepidium latifolium L.: a step towards translational research in agri-genomics for food and fuel.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes first and the rate limiting step in glycerolipid synthesis pathway, which in turn contribute to stabilization of plasma membrane structure and oil lipid synthesis in plant cells. Here, we report cloning and characterization of GPAT gene from Lepidium latifolium (LlaGPAT). The cDNA sequence (1,615 bp) of LlaGPAT gene consisted of 1,113 bp ORF encoding a protein of 370 aa residues, with deduced mass of 41.2 kDa and four acyltransferase (AT) motifs having role in catalysis and in glycerol-3-phosphate binding. Southern blot analysis suggested presence of a single copy of the gene in the genome. Tissue specific expression of the gene was seen more abundantly in aerial parts, compared to the roots. Quantitative real-time PCR indicated down-regulation of the gene by cold (4 °C), drought (PEG6000), salt (300 mM NaCl) and ABA (100 µM) treatments. Considering the vitality of the function of encoded enzyme, LlaGPAT can be considered a potential candidate gene for genetic engineering of oil yields and abiotic stress management in food as well as fuel crops.

Cold tolerance in Osmotin transgenic tomato (Solanum lycopersicum L.) is associated with modulation in transcript abundance of stress responsive genes.

Our containment trials have established cold tolerance in Nicotiana tabacum osmotin (Nt Osm) transgenic tomato (Solanum lycopersicum L. cv. Pusa Ruby). Though, the stress tolerance mechanisms have been studied at physio-biochemical levels, molecular mechanisms underlying the tolerant response are still not well studied. Therefore, quantitative transcript expression of Osmotin and other stress responsive genes (CBF1, P5CS and APX) was studied in response to cold (4°C; 2 and 24 h) treatment in the transgenic and wild type tomato plants. The expression analysis revealed differential transcript regulation in the transgenic and wild type plants on the cold exposure. In general, the genes were either earlier induced or the extent of fold change in transcript expression over the respective untreated controls was higher in transgenic than in the wild type plants on cold exposure. The transcript expression data also supported the metabolite analysis on free Proline and ascorbate content. The results thus suggest that constitutive over expression of the Osmotin modulate transcript abundance and functional expression products of the other stress responsive genes thereby, imparting cold tolerance in the transgenic tomato plants.

Cold tolerance in thiourea primed capsicum seedlings is associated with transcript regulation of stress responsive genes.

Benefits of seed priming in seedling establishment and tolerance to subsequent stress exposure are well reported. However, the molecular mechanisms underlying the priming mediated benefits are not much discovered. Results of our earlier experiments established that thiourea (TU) seed priming imparts cold tolerance to capsicum seedlings. Therefore, to understand molecular mechanisms underlying priming mediated cold stress tolerance, quantitative transcript expression of stress responsive genes involved in transcript regulation (CaCBF1A, CaCBF1B, Zinc Finger protein, CaWRKY30), osmotic adjustment (PROX1, P5CS, Osmotin), antioxidant defence (CAT2, APX, GST, GR1, Cu/Zn SOD, Mn SOD, Fe SOD), signaling (Annexin), movement of solutes and water (CaPIP1), and metabolite biosynthesis through phenylpropanoid pathway (CAH) was studied in response to cold (4 °C; 4 and 24 h) stress in seedlings grown from the TU primed, hydroprimed and unsoaked seeds. The transcript expression of CaWRKY30, PROX1, Osmotin, Cu/Zn SOD and CAH genes was either higher or induced earlier on cold exposure in thiourea priming than that of hydroprimed and unsoaked over the respective unstressed controls. The results thus suggest that the TU priming modulate expression of these genes thereby imparting cold tolerance in capsicum seedlings.

Halopriming mediated salt and iso-osmotic PEG stress tolerance and, gene expression profiling in sugarcane (Saccharum officinarum L.).

Seed priming is a well known pre-germination strategy that improves seed performance. However, biochemical and molecular mechanisms underlying priming mediated stress tolerance are little understood. Here, we report results of the study on growth, physiological characteristics and expression of stress responsive genes in salt primed sugarcane cv. Co 86032 plants in response to salt (NaCl, 150 mM) or iso-osmotic (-0.7 MPa) polyethylene glycol-PEG 8000 (20 % w/v) stress exposure for 15 days. Variable growth, osmolyte accumulation and antioxidant capacity was revealed among the primed and non-primed plants. The primed plants showed better tolerance to the salt or PEG stress, as revealed by better growth and lower membrane damage, through better antioxidant capacity as compared to the respective non-primed controls. Further, steady state transcript expression analysis revealed up regulation of sodium proton antiporter (NHX) while, down regulation of sucrose transporter (SUT1), delta ( 1 )-pyrolline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (PDH) in primed plants on exposure to the stress as compared to the non-primed plants. Transcript abundance of catalase (CAT2) decreased by about 25 % in leaves of non-primed stressed plants, however, the expression was maintained in leaves of the stressed primed plants to that of non-stressed controls. Thus, the results indicated priming mediated salt and PEG stress tolerance through altered gene expression leading to improved antioxidant capacity in sugarcane.

Transcript expression profiling of stress responsive genes in response to short-term salt or PEG stress in sugarcane leaves.

Semi-quantitative RT-PCR based transcript expression of stress responsive genes was studied in leaves of sugarcane plants exposed to short-term (up to 24 h) salt (NaCl, 200 mM) or polyethylene glycol-PEG 8000 (20% w/v) stress. Transient increase in expression of NHX (sodium proton antiporter), SUT1 (sucrose transporter1), PDH (proline dhydrogenase) and CAT2 (catalase2) was observed in response to 2-4 h PEG stress. However, salt stress imposed repression of NHX, PDH and CAT2 at these time points. The transcript level of the delta (1)-pyrolline-5-carboxylate synthetase (P5CS) increased slightly in salt treatment while in response to the PEG stress, the gene expression increased at 4 h treatment but then decreased considerably by 80% at 24 h. The results thus indicated differential regulation of these stress responsive genes in response to salt or PEG stress in sugarcane. Further, the transcript expression data was compared with that available for the Arabidopsis homologs at Arabidopsis eFP Browser and Genevestigator V3 tools. Understanding transcript gene expression patterns of the stress responsive genes may provide insights into complex regulatory network of stress tolerance.

Expression analysis of sugarcane shaggy-like kinase (SuSK) gene identified through cDNA subtractive hybridization in sugarcane (Saccharum officinarum L.).

Identification of genes whose expression enables plants to adapt to any kind of stresses is integral to developing stress tolerance in crop plants. In this study, PCR-based cDNA suppression subtractive hybridization technique was used to construct sugarcane salt (NaCl) stress specific forward and reverse subtracted cDNA library. For this, mRNAs were pooled from the shoot and root tissues stressed with NaCl (200 mM) for various time intervals (0.5 to 18 h). Sequencing the clones from the forward subtracted cDNA library, we identified shaggy-like protein kinase (hereafter referred as sugarcane shaggy-like protein kinase, SuSK; NCBI GenBank EST database Acc: FG804674). The sequence analysis of the SuSK revealed homology to Arabidopsis thaliana shaggy-related protein kinase delta (E value, 1e(-108)), dzeta and iota. Alignment of the catalytic domain sequence of GSK-3/shaggy-like kinase with partial sequence of SuSK performed using ClustalW tool indicated kinase active-site signature sequence. Spatial and temporal transcript expression profiling of the SuSK gene based on Real-Time PCR revealed significant induction of transcript expression in response to short-term salt (NaCl 200 mM) or polyethylene glycol-8,000 (PEG; 20% w/v) induced osmotic stress in leaves and shoots of sugarcane plants. The transcript expression increased progressively under salt stress and reached to 1.5-fold of the control up to 8 h treatment. In response to PEG stress, the transcript expression increased by 1.5-fold over the control in 2-h treatment in leaf, whereas in shoots, the expression remained unchanged in response to the various treatments. Differences in growth parameters, relative water content, and membrane damage rate were statistically insignificant in the short-term salt or PEG-stressed plants as compared to the control, non-stressed plants. Expression analysis revealed the differential and temporal regulation of this gene under salt and PEG stress and that its early induction may indicate involvement in stress signaling.