Expression of an endochitinase gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis> confers enhanced tolerance to <Emphasis Type="Italic">Alternaria</Emphasis> blight in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) czern and coss lines

An endochitinase gene ‘ech42’ from the biocontrol fungus ‘Trichoderma virens’ was introduced to Brassica juncea (L). Czern and Coss via Agrobaterium tumefaciens mediated genetic transformation method. Integration and expression of the ‘ech42’ gene in transgenic lines were confirmed by PCR, RT-PCR and Southern hybridization. Transgenic lines (T₁) showed expected 3:1 Mendelian segregation ratio when segregation analysis for inheritance of transgene ‘hpt’ was carried out. Fluorimetric analysis of transgenic lines (T₀ and T₁) showed 7 fold higher endochitinase activity than the non-transformed plant. Fluorimetric zymogram showed presence of endochitinase (42 kDa) in crude protein extract of transgenic lines. In detached leaf bioassay with fungi Alternaria brassicae and Alternaria brassicicola, transgenic lines (T₀ and T₁) showed delayed onset of lesions as well as 30–73 % reduction in infected leaf area compared to non-transformed plant.     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.     

Improving tobacco freezing tolerance by co-transfer of stress-inducible <Emphasis Type="Italic">CbCBF</Emphasis> and <Emphasis Type="Italic">CbICE53</Emphasis> genes

Cold stress is one of the major limitations to crop productivity worldwide. We investigated the effects of multiple gene expression from cold tolerant Capsella bursa-pastoris in transgenic tobacco (Nicotiana tabaccum) plants. We combined CblCE53 and CbCBF into a reconstruct vector by isocaudomers. Plant overexpression of CbICE53 under the stress inducible CbCOR15b promoter and CbCBF under a constitutive promoter showed increased tolerance to both chilling and freezing temperatures in comparison to wild-type plants, according to the electrolyte leakage and relative water content. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a and NtERD10b) were obviously upregulated under normal and low temperature conditions. These results suggest that the CbICE53 + CbCBF transgenic plants showed a much greater cold tolerance as well as no dwarfism and delayed flowering. Thus they can be considered as a potential candidate for transgenic engineering for cold tolerant tobacco.     

Over-expression of <Emphasis Type="Italic">JrsHSP17.3</Emphasis> gene from <Emphasis Type="Italic">Juglans regia</Emphasis> confer the tolerance to abnormal temperature and NaCl stresses

Small heat shock proteins (sHSPs) are an HSP subgroup and involved in environmental stress response. In the current study, to understand the role of sHSP protein in a widely distribution nut woody tree, a sHSP gene was cloned from Juglans regia (JrsHSP17.3, GeneBank No.: KT277704). Compared with control condition, the expression of JrsHSP17.3 was induced to 58.1-fold (6 h) in the roots, 86.8-fold in the stems (9 h), 50.9-fold in the leaves (6 h) under 10°C; and was up-regulated to 2.9- ～ 79.9-fold response to 40°C for 3～9 h; meanwhile, it was transcribed to 5.9 - ～39.7-fold under 9 h NaCl treatment, suggesting the potential role of JrsHSP17.3 to cold, heat and NaCl stimulus. Further, JrsHSP17.3 transgenic yeasts showed improved tolerance to freezing, heat and salt stresses compared with control yeast. JrsHSP17.3 was transient over-expressed in J. regia leaves. The leaves non-transgenic (NT) and vector prokII transgenic (empty, PT) were used as control. The expression of JrsHSP17.3 was 81.6-, 125.4-, and 54.2-fold of the control lines under normal conditions, indicating the success over-expression of JrsHSP17.3. Cell damage staining and physiological index determination showed that JrsHSP17.3 transformed lines, NT and PT displayed no obvious difference under control conditions, however, after treated with 16°C, 40°C and NaCl, JrsHSP17.3 transformed lines displayed weaker cell damage, lower level of electrolyte leakages (EL) rate, malondialdehyde (MDA) and H₂O₂ content, and higher activities of catalase (CAT), glutathione transferase (GST), superoxide dismutase (SOD) and peroxidase (POD) as well as more accumulation of proline than NT and PT. Meanwhile, NT and PT were similar and showed no significant difference under all conditions. All of these results indicated that JrsHSP17.3 can improve plant tolerance to abnormal temperatures and NaCl stresses, it represents a potential candidate gene for molecular breeding to enhance stress tolerance in plants.     

Establishment of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of seashore paspalum (<Emphasis Type="Italic">Paspalum vaginatum</Emphasis> O. Swartz)

Seashore paspalum (Paspalum vaginatum O. Swartz) is an important warm-season turfgrass with great salinity tolerance. Based on establishment of embryogenic callus induction and regeneration from different mature seeds of ‘Sea Spray’, an Agrobacterium tumefaciens-mediated transformation was established and optimized in this study. Three clones of callus were selected for examining transformation conditions using Agrobacterium tumefaciens strain AGL1 carrying the binary vector pCAMBIA1305.2, containing β-glucuronidase (GUS) as a reporter gene and hygromycin phosphotransferase (HPT) as a selective marker gene. The results showed that a high transient transformation efficiency was observed by using Agrobacterium concentration of OD₆₀₀?=?0.6, 5 min of sonication treatment during Agrobacterium infection, and 2 d of co-cultivation. By using the optimized transformation conditions, transgenic seashore paspalum plants were obtained. PCR and Southern blot analysis showed that T-DNA was integrated into the genomes of seashore paspalum. GUS staining experiments showed that the GUS gene was expressed in transgenic plants. Our results suggested that the transformation protocol will provide an effective tool for breeding of seashore paspalum in the future.     

Genotype independent and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of the medicinal plant <Emphasis Type="Italic">Withania somnifera</Emphasis> Dunal

Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD₆₀₀ 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36?±?13.2 to 277.6?±?22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T₀ transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.     

An ATP-Binding Cassette Transporter Gene from <Emphasis Type="Italic">Cucumis sativus</Emphasis> L., <Emphasis Type="Italic">CsABC19</Emphasis>, Is Involved in Propamocarb Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A propamocarb-responsive gene named CsABC19 was isolated from a cucumber cultivar ‘D0351’ using a homologous cloning strategy. The full-length cDNA of CsABC19 was 921 bp with a complete ORF encoding 306 amino acids. Quantitative real-time PCR analysis revealed that CsABC19 was induced in the root, stem, leaf, and fruit by propamocarb and the expression levels of CsABC19 seemed to be different in different tissues. Further functional analysis showed that CsABC19 transgenic Arabidopsis plants appeared better growth performance under propamocarb stress and lower propamocarb residues. Our findings suggest that CsABC19 plays a crucial role in plant responses to propamocarb stress and also provide new clues for the mechanism regulation of the responses to propamocarb stress in cucumber.     

A <Emphasis Type="Italic">Vitis vinifera</Emphasis> xanthine dehydrogenase gene, <Emphasis Type="Italic">VvXDH</Emphasis>, enhances salinity tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.     

Over-expressing a UDP-glucosyltransferase gene (<Emphasis Type="Italic">Ta-UGT</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript>) enhances <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight resistance of wheat

Wheat Fusarium Head Blight (FHB), mainly caused by Fusarium graminearum (F.g), is a destructive fungal disease worldwide. FHB can not only cause considerable reduction in yield, but more seriously, can contaminate grain by trichothecene toxins released by the fungus. Here, we report new insights into the function and underlying mechanisms of a UDP-glycosyltransferase gene, Ta-UGT ₃ , that is involved in FHB resistance in wheat. In our previous study, Ta-UGT ₃ was found to enhance host tolerance against deoxynivalenol (DON) in Arabidopsis. In this study, four transgenic lines over-expressing Ta-UGT ₃ in a FHB highly susceptible wheat variety, Alondra’s, were obtained and characterized. 3 years of assays using single floret inoculation with F.g indicated that all four transgenic lines exhibited significantly enhanced type II resistance to FHB and less DON accumulation in the grains compared to the untransformed control. Histological observation using GFP labelled F.g was in agreement with the above test results since over-expression of Ta-UGT ₃ dramatically inhibited expansion of F.g. To explore the putative mechanism of resistance mediated by Ta-UGT ₃ , microarray analysis, qRT-PCR and hormone measurements were performed. Microarray analysis showed that DON up-regulated genes, such as TaNPR1, in the susceptible control, and down-regulated genes in F.g inoculated transgenic lines, while qRT-PCR showed that some defence related genes were up-regulated in F.g inoculated transgenic lines. Ta-UGT ₃ over-expression also changed the contents of the endogenous hormones SA and JA in the spikes. These data suggest that Ta-UGT ₃ positively regulates the defence responses to F.g, perhaps by regulating defence-related and DON-induced downstream genes.     

Molecular cloning and characterization of the <Emphasis Type="Italic">MsHSP17.7</Emphasis> gene from <Emphasis Type="Italic">Medicago sativa</Emphasis> L.

Heat shock proteins (HSPs) are ubiquitous protective proteins that play crucial roles in plant development and adaptation to stress, and the aim of this study is to characterize the HSP gene in alfalfa. Here we isolated a small heat shock protein gene (MsHSP17.7) from alfalfa by homology-based cloning. MsHSP17.7 contains a 477-bp open reading frame and encodes a protein of 17.70-kDa. The amino acid sequence shares high identity with MtHSP (93.98 %), PsHSP17.1 (83.13 %), GmHSP17.9 (74.10 %) and SlHSP17.6 (79.25 %). Phylogenetic analysis revealed that MsHSP17.7 belongs to the group of cytosolic class II small heat shock proteins (sHSP), and likely localizes to the cytoplasm. Quantitative RT-PCR indicated that MsHSP17.7 was induced by heat shock, high salinity, peroxide and drought stress. Prokaryotic expression indicated that the salt and peroxide tolerance of Escherichia coli was remarkably enhanced. Transgenic Arabidopsis plants overexpressing MsHSP17.7 exhibited increased root length of transgenic Arabidopsis lines under salt stress compared to the wild-type line. The malondialdehyde (MDA) levels in the transgenic lines were significantly lower than in wild-type, although proline levels were similar between transgenic and wild-type lines. MsHSP17.7 was induced by heat shock, high salinity, oxidative stress and drought stress. Overexpression analysis suggests that MsHSP17.7 might play a key role in response to high salinity stress.     

Pistil abortion in Japanese apricot (<Emphasis Type="Italic">Prunus mume</Emphasis> Sieb. et Zucc.): isolation and functional analysis of <Emphasis Type="Italic">PmCCoAOMT</Emphasis> gene

The abnormal pistils widely occur in Japanese apricot (Prunus mume Sieb. et Zucc) and seriously affect the fruit production. In this study, a CCoAOMT homologue, PmCCoAOMT, was cloned in Japanese apricot and the bioinformatics software analyzed the structural characteristics. The PmCCoAOMT protein was detected to be located in the cell cytoplasm by onion transient expression experiment. Analysis of the real-time PCR data showed that PmCCoAOMT gene expressed in the prophase development of pistil and the expression level in ‘Daqiandi’ was higher than ‘Longyan.’ The expression level in ‘Longyan’ was higher than ‘Daqiandi’ in the late period development of pistil, and the expression level of perfect flower (perfect pistil) was higher than imperfect flower (pistil deformity and no pistil). Compared with the control, the over-expression of PmCCoAOMT transgenic tobacco lines showed bigger flowers, darker petals. The lignin monomer composition in transgenic tobacco lines was also measured, and the results showed that transgenic tobacco lines had a higher S (Syringyl)/G (Guaiacyl) ratio (22.3 %) than control lines (11.8 %). Also, the perfect flower buds contained more S/G ratio (92.62 %) than imperfect flower buds (83.55 %) in ‘Daqiandi.’ Our results indicated that the PmCCoAOMT gene might have function in lignin accumulation, which contributed to pistil development in Japanese apricot.