Strain GV3101 Research Articles

Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

Overexpression of Arabidopsis BBX 21 Gene in Bg 250 Rice Enhances Its Architecture and Productivity

Arabidopsis BBX21 protein was identified as a transcription activator which can activate the light dependent transcription and positively regulate light mediated development of plants. The productivity of crop plants can be enhanced by over-expressing central regulators of light signaling pathway. The present study was conducted to enhance the productivity of Bg 250 Sri Lankan rice variety by overexpressing Arabidopsis BBX21 gene. The BBX21 gene was cloned into pPZP 200 binary vector and the cloned vector gene construct was transformed into Agrobarcteium tumefaciens GV3101 strain by freeze thaw method. The selected recombinant colonies of A. tumefaciens strain GV3101 harboring BBX 21 gene were transformed into three weeks old Bg 250 rice callus. Selection of the transformed callus was conducted on N6B5 selection medium containing 50 mg/L gentamycin and 500mg/L cefotaxime. A total of putative 83 plants which showed resistant to gentamycin were subjected to PCR analysis and the results showed 69 of the regenerated plants are transgenic. The T1 seeds collected from putative transgenic plants were characterize to check its integration and inheritance. The transgenic plants which showed 3:1 ratio was selected and further grown to obtain its homozygous lines. The expression of BBX21 gene was demonstrated by northern blot analysis and the results showed 4 fold higher expression of BBX21 gene in the transgenic plants compared with its non-transgenic wild type plants. The phenotypic analysis of homozygous BBX21 transgenic plants showed improved plant height, leaf length, leaf area, number of panicles and bushy appearance compared with its non-transgenic plants. Taken together, these results demonstrate an involvement of BBX21 gene in rice modifying architecture and productivity of Bg 250 transgenic plants. Int J Appl Sci Biotechnol, Vol 4(2): 240-246

Artificial Agrobacterium tumefaciens strains exhibit diverse mechanisms to repress Xanthomonas oryzae pv. oryzae-induced hypersensitive response and non-host resistance in Nicotiana benthamiana.

Xanthomonas oryzae pv. oryzae (Xoo) rapidly triggers a hypersensitive response (HR) and non-host resistance in its non-host plant Nicotiana benthamiana. Here, we report that Agrobacterium tumefaciens strain GV3101 blocks Xoo-induced HR in N.benthamiana when pre-infiltrated or co-infiltrated, but not when post-infiltrated at 4h after Xoo inoculation. This suppression by A.tumefaciens is local and highly efficient to Xoo. The HR-inhibiting efficiency of A.tumefaciens is strain dependent. Strain C58C1 has almost no effect on Xoo-induced HR, whereas strains GV3101, EHA105 and LBA4404 nearly completely block HR formation. Intriguingly, these three HR-inhibiting strains employ different strategies to repress HR. Strain GV3101 displays strong antibiotic activity and thus suppresses Xoo growth. Comparison of the genotype and Xoo antibiosis activity of wild-type A.tumefaciens strain C58 and a set of C58-derived strains reveals that this Xoo antibiosis activity of A.tumefaciens is negatively, but not solely, regulated by the transferred-DNA (T-DNA) of the Ti plasmid pTiC58. Unlike GV3101, strains LBA4404 and EHA105 exhibit no significant antibiotic effect on Xoo, but rather abolish hydrogen peroxide accumulation. In addition, expression assays indicate that strains LBA4404 and EHA105 may inhibit Xoo-induced HR by suppression of the expression of Xoo type III secretion system (T3SS) effector genes hpa1 and hrpD6. Collectively, our results unveil the multiple levels of effects of A.tumefaciens on Xoo in N.benthamiana and provide insights into the molecular mechanisms underlying the bacterial antibiosis of A.tumefaciens and the non-host resistance induced by Xoo.

Towards better insect management strategy: restriction of insecticidal gene expression to biting sites in transgenic cotton

Most of the commercialized Bt crops express cry genes under 35S promoter that induces strong gene expression in all plant parts. However, targeted foreign gene expression in plants is esteemed more important as public may be likely to accept ‘less intrusive’ expression of transgene. We developed plant expression constructs harboring cry1Ac gene under control of wound-inducible promoter (AoPR1) to confine Bt gene expression in insect wounding parts of the plants in comparison with cry1Ac gene under the control of 35S promoter. The constructs were used to transform four Turkish cotton cultivars (GSN-12, STN-468, Ozbek-100 and Ayhan-107) through Agrobacterium tumefaciens strains GV2260 containing binary vectors p35SAcBAR.101 and AoPR1AcBAR.101 harboring cry1Ac gene under control of 35S and AoPR1, respectively. Phosphinothricin (PPT) was used at concentration of 5 mg L−1 for selection of primary transformants. The primary transformants were analyzed for transgene presence and expression standard molecular techniques. The transformants exhibited appreciable mortality rates against larvae of Spodoptera exigua and S. littoralis. It was found that mechanical wounding of T 1 transgenic plants was effective in inducing expression of cry1Ac protein as accumulated levels of cry1Ac protein increased during post-wounding period. We conclude that use of wound-inducible promoter to drive insecticidal gene(s) can be regarded as a valuable insect-resistant management strategy since the promoter activity is limited to insect biting sites of plant. There is no Bt toxin accumulation in unwounded plant organs, seed and crop residues, cotton products and by-products, thus minimizing food and environmental concerns.

RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.

Establishment of an in vitro propagation and transformation system of Balanites aegyptiaca

Balanites aegyptiaca (Balanitaceae) is a drought-tolerant but salt-sensitive tree species distributed in the tropical and arid lands in Africa and Asia. The tree contains many secondary metabolites and a high percentage of oil in the kernels that can be used for biodiesel production. This study aimed to establish an in vitro propagation system of two B. aegyptiaca provenances (El-Kharga and Wadi El-Alaqi) from nodal and cotyledon explants. The explants were placed on Murashige and Skoog medium supplemented with different concentrations of 6-benzyladenine (BA) and thidiazuron (TDZ) for shoot induction. BA was significantly more effective in shoot induction from nodal explants and treatment with BA also resulted in higher regeneration rates of about 40–60 % of adventitious shoots on cotyledon explants, whereas on TDZ-containing medium slightly higher shoot numbers per explant but a negative effect on shoot length were recorded. Rooting was achieved in 40–60 % of the shoots on medium containing between 1.2 and 4.8 µM indole-3-butyric acid. Three different Agrobacterium tumefaciens strains (EHA105, GV3101, and LBA4404) harboring the plasmid pCAMBIA2301 containing the nptII marker and gus reporter genes were used to establish a transformation system in B. aegyptiaca. Strain GV3101 resulted in the highest survival rates and highest number of explants positive in the GUS assay. This selected A. tumefaciens strain was used to introduce pBinAR containing the sequence encoding ERD10 (early responsive to dehydration 10) to produce salt-tolerant B. aegyptiaca plants. The presence of the ERD10 and the nptII gene were detected by PCR in transformed B. aegyptiaca plants.

Novel antibiotics regeneration and genetic transformation with RsMYB1 gene of recalcitrant chrysanthemum cv. Shinma

This research was conducted to develop genetic transformation of the recalcitrant chrysanthemum cv. Shinma by application of appropriate antibiotics and selective agents. Clavamox had the least inhibitory effect on shoot regeneration compared to timentin, carbenicillin, and cefotaxime. Clavamox, at a concentration of 125 mg L−1, was found to be the most suitable for shoot regeneration and production of quality shoots, suppressing the growth of Agrobacterium in explants infected with strains GV3101 or C58C1 for 3 and 4 weeks, respectively. The concentration of phosphinothricin (PPT) was found to be 1.0 mg L−1 for screening of putative transgenic shoots. Moreover, transgenic chrysanthemums were obtained by culturing explants co-cultivated with A. tumefaciens strain GV3101 harboring an anthocyanin regulatory gene RsMYB1 isolated from radish (Raphanus sativus), which was placed under the control of cauliflower mosaic virus promoter (CaMV) 35S and petal-specific promoter InMYB1 isolated from the morning glory (Ipomoea nil), on shoot regeneration medium supplemented with recommended concentration of antibiotic and selective agent. Flow cytometry analysis revealed that there was no variation in ploidy level between transgenic plants and donor plants (non-transformants). To our knowledge, this is the first report of the use of Clavamox and MYB transcription factor for genetic transformation of this chrysanthemum.

Efficient In Vitro Regeneration, Analysis of Molecular Fidelity and Agrobacterium tumifaciens - Mediated Genetic Transformation of Grewia asiatica L.

Grewia asiatica is a dietotheraphtically important fruit bearing shrub, indigenous to India. It is a rich resource of triterpinoids and flavonoids and possesses many putative health benefits. Two of the drawbacks which include short shelf life of its fruits and larger seed volume impedes its full exploitation. Seed abortion for developing seedless cultivars through biotechnological interventions is a viable option. One of the prerequisites for such strategy is to develop an efficient plant regeneration and transformation protocols in G. asiatica. Against this backdrop multiple shoot induction was achieved from nodal explants with axillary buds, on culturing in Woody Plant medium (WM) fortified with 3% (w/v) sucrose, 2 × 10-5M Kinetin (Kn) and 1 × 10-5M indole-3-butyric acid (IBA) giving rise to an average of 4.25 ± 0.71 microshoots per explant. More than 90% of the explants formed micro-shoots with mean shoot length of 10.5 ± 1.96 cm leading to whole plant regeneration. Healthy regenerated shoots showed prolific rooting of more than 95% on WM supplemented with 4.8 × 10-6M indole-3-butyric acid (IBA). Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with about 92% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD). Additionally, Agrobacterium-mediated genetic transformation protocol was developed using A. tumefaciens strain GV2260 harboring binary vector p35SGUSINT containing hygromycin phosphotransferase gene (hpt). Transformation was verified by GUS assay and detection of the hygromycin phosphotransferase (hpt) by polymerase chain reaction. In vitro regeneration and ensuing molecular fidelity of regenerated plants and transformation studies are hitherto unreported for G. asiatica.

Genotype independent and efficient Agrobacterium-mediated genetic transformation of the medicinal plant Withania somnifera 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.

A Novel Phenolic Compound, Chloroxynil, Improves Agrobacterium-Mediated Transient Transformation in Lotus japonicus.

Agrobacterium-mediated transformation is a commonly used method for plant genetic engineering. However, the limitations of Agrobacterium host-plant interactions and the complexity of plant tissue culture often make the production of transgenic plants difficult. Transformation efficiency in many legume species, including soybean and the common bean, has been reported to be quite low. To improve the transformation procedure in legumes, we screened for chemicals that increase the transformation efficiency of Lotus japonicus, a model legume species. A Chemical library was screened and chemicals that increase in transient transformation efficiency of L. japonicus accession, Miyakojima MG-20 were identified. The transient transformation efficiency was quantified by reporter activity in which an intron-containing reporter gene produces the GUS protein only when the T-DNA is expressed in the plant nuclei. We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105. Characterization of the mode of chloroxynil action indicated that it enhanced Agrobacterium-mediated transformation through the activation of the Agrobacterium vir gene expression, similar to acetosyringone, a phenolic compound known to improve Agrobacterium-mediated transformation efficiency. Transient transformation efficiency of L. japonicus with 5 μM chloroxynil was 60- and 6- fold higher than that of the control and acetosyringone treatment, respectively. In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation. Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops.

Evaluation of Agrobacterium-mediated Transformation of Chlamydomonas reinhardtii using a Synthetic amorpha-4, 11-diene Synthase Gene

Amorpha-4,11-diene synthase (ADS) is a key enzyme in biochemical pathway of the antimalarial agent artemisinin. An Agrobacterium-mediated transformation was carried out to express a synthetic ADS gene in green microalga Chlamydomonas reinhardtii strain 125C with bacterial strains GV3101 and LBA4404. The foreign gene was optimized based on codon usage bias of the microalga. Integration of the ADS in nuclear genome of C. reinhardtii was confirmed by polymerase chain reaction assay. The transgenic colonies cultured on selective medium turned yellow after three days and gradually died. Transformation procedure, growth habit of the transgenic microalgae together with probable causes of transformants loss is discussed. The present study is the first investigation for production of ADS enzyme in a microalgal system.

MOLECULAR BREEDING OF PETUNIA HYBRID 'FAMOUS LILAC DARK VEIN' FOR COMPACT GROWTH INDUCTION BY MKS1 AND GA2OX CONSTITUTIVE OVEREXPRESSION

One of the top targets of recent breeding programs in ornamental plants is focused on developing an alternative way to the chemical growth retardants application in production of plants with compact growth habit. We describe work on Petunia hybrid ‘Famous Lilac Dark Vein’ plants with overexpressed MAP kinase 4 nuclear substrate 1 (MKS1) and gibberellin 2-oxidase (GA2ox) genes. Petunia plants were transformed with pCAMBIA modified vectors in separate experiments, containing constructs either with the MKS1 or the GA2ox gene under the control of Ca35S promoter. The transformation was performed with the use of Agrobacterium tumefaciens strain GV3101. The integration and expression of the transgene was investigated using Southern blot and RT-PCR analysis. The mean internode lengths of the MKS1-transgenic lines were 2.5-fold shorter than those of the wild-type plants. The flowering delay in MKS1-transgenic lines was observed with a range of 6 to 11 days in comparison to the wild-type plants. The mean internode lengths of the GA2ox-transgenic lines were 6.5-fold shorter than those of the wild-type plants. The flowering delay in GA2ox-transgenic lines was observed with a range of 7 to 12 days in comparison to the wild-type plants. The flower morphology either in MKS1- or GA2ox-transgenic lines did not differ from wild-type plants. Constitutive overexpression of both MKS1 and GA2ox genes results in compact phenotype but also lead to delayed flowering.

Overexpression of the genes coding ascorbate peroxidase from Brassica campestris enhances heat tolerance in transgenic Arabidopsis thaliana

Previously, the ascorbate peroxidase (APX1) activity and gene expression in Chinese cabbage (Brassica campestris, Bc) heat-tolerant cv. ASVEG2 were found to be significantly higher than in heat-sensitive cv. RN720 under a heat stress. Furthermore, BcAPX2 and BcAPX3, isoforms of BcAPX1, were cloned in this study. Our objective was to transfer BcAPX cDNA under the control of the ubiquitin promoter to Arabidopsis via Agrobacterium tumefaciens strain GV3101. We found that BcAPX genes were overexpressed in transgenic Arabidopsis, and the expression of APX, and the APX activity in transgenic lines were higher than in non-transgenic (NT) plants under high temperatures. The chlorophyll content and the germination rate were significantly higher, and the malondialdehyde content was lower in BcAPX1-3, 2-1, and 3–5 lines subjected to the heat-stress treatment than those in the NT plants. Compared to the NT plants, a lower heat-induced H2O2 accumulation was detected by diaminobenzidine staining in leaves of the transgenic lines with a high APX activity indicating that the overexpression of BcAPX in Arabidopsis could enhance heat tolerance by eliminating H2O2.

Co-expression of hepatitis C virus polytope–HBsAg and p19-silencing suppressor protein in tobacco leaves

Context: Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition.Objective: We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine.Materials and methods: A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc–HBsAg was also evaluated by co-infiltration of a p19 expression vector.Results: Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc–HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants.Discussion and conclusion: The results indicated the possibility of expression of HCVpc–HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

Effects of squirting cucumber (Ecballium elaterium) fruit juice onAgrobacterium tumefaciens-mediated transformation of plants

Different concentrations of squirting cucumber (Ecballium elaterium (L.) A.Rich.) fruit juice were added to Agrobacterium tumefaciens growth, leaf disc inoculation, and cocultivation media, to investigate its effect on the transformation frequency of tobacco and potato. A. tumefaciens strain GV2260 harboring p35S GUS-INT and pAoPR1-GUS-INT plasmids were used separately in the transformation experiments. Neomycin phosphotransferase (NPT-II) gene was used as a plant selectable marker at a concentration of 100 mg L -1 . The addition of 5-10 mg L -1 squirting cucumber fruit juice to bacterial nutrient medium increased A. tumefaciens growth significantly by 6 h. Moreover, the use of high concentrations (2.5-20 mL L -1 ) of fruit juice resulted in excessive bacterial growth on cocultivation and selection media around the explants, which was difficult to eliminate by subculture or higher levels of antibiotics. On the other hand, lower concentrations (0.2-1.6 mL L -1 ) of squirting cucumber fruit juice significantly increased the transformation frequency in both tobacco and potato. Kanamycin-resistant tobacco shoots, rooted in a medium containing 100 mg L -1 kanamycin, were transferred to pots containing organic soil and perlite in growth cabinets for acclimatization. Transgenic plants grew normally and set seeds. The presence of T-DNA in these transformants was confirmed by PCR and GUS analysis.

Transformation of Campanula carpatica to alter flower colour and ethylene sensitivity

SUMMARYThis study is the first report of gene manipulation to alter flower colour in the genus Campanula. The experiment was designed using Campanula carpatica ‘Improved Blue Uniform’ (IBU) with two main purposes: to generate a red flower colour by down-regulation of delphinidin production through RNAi silencing of the flavonoid 3′, 5′ hydroxylase (F3′5′H) gene, and to improve vase-life by inducing insensitivity to ethylene using the ethylene-resistant 1–1 (etr1–1) gene. Three independent transgenic lines were obtained after genetic transformation via Agrobacterium tumefaciens strain GV3101. Southern blot analysis was performed to determine the integrated T-DNA copy number, and reverse transcription-polymerase chain reactions (RT-PCR) were performed to evaluate expression of the F3′5′H and flavonoid 3′ hydroxylase (F3′H) genes. An ethylene sensitivity test was conducted to evaluate the tolerance of the transformed plants to exposure to ethylene. Southern blot analysis revealed the integration of two or three T-DNA copies in each of the three transformed lines. The flower colour of the transformed lines was not visually altered, and expression of the F3′5′H and F3′H genes in all three transformed and in non-transformed lines was confirmed by RTPCR. Although all flowers on non-transformed Campanula plants were senescent 6 d after exposure to ethylene, 63.3% of the flowers on transgenic line 11–213 and 86.4% of the flowers on transgenic line 21–1 survived. The ethylene sensitivity test showed that transformed plants exposed to ethylene showed a significant delay in senescence compared to non-transformed plants.

Improving fruit quality in tomato (Lycopersicum esculentum Mill) under heat stress by silencing the vis 1 gene using small interfering RNA technology

Constantly elevated temperatures cause an array of physio-biochemical changes in tomato (Lycopersicum esculentum Mill.) which make the fruit ripen quickly and up to 50 % yield loss. The development of tomato cultivars, using genetic engineering approaches which delay ripening, offers a new way to keep tomatoes healthy under heat stress. Over-expression of small heat shock protein gene, viscosity 1 (vis 1) plays a role in increasing juice viscosity, early ripening and tissue soften which emphasizes the importance of this gene in premature ripening. The aim of this work was therefore to develop a useful system for silencing the vis1 gene using small interfering RNA strategy. Agrobacterium strain GV3101 harbouring the binary vector pICBV19 containing the gus and bar genes was used to adapt the transformation process in this study. The primers were designed to amplified the first exon of the vis 1 gene and the amplified fragment was used for cloning into the pFGC5941 at XhoI/NcoI site at sense orientation then additional fragment was subsequently cloned at BamHI/XbaI to form sense/antisense cloned fragment interrupted by the CHSA-intron Agrobacterium strain LBA4404 with the binary vector pFGC5941 which harbors vis1 gene under the control of the 35S promoter containing bar gene under the control of a mannopine synthase 2′(Mas2′) as selectable marker, was used to reduce the expression of vis1 gene in fruit. Polymerase chain reaction (PCR), RT–PCR and northern blotting analysis were applied to detect putative transgenic plants. Significantly, silencing of vis 1 gene was potently occur and new transgenic tomato cultivars were produced with enhanced ripening qualities for recommendation for growing under heat stress.

Agroinfiltration by cytokinin-producing Agrobacterium sp. strain GV3101 primes defense responses in Nicotiana tabacum.

Transient infiltrations in tobacco are commonly used in plant studies, but the host response to different disarmed Agrobacterium strains is not fully understood. The present study shows that pretreatment with disarmed Agrobacterium tumefaciens GV3101 primes the defense response to subsequent infection by Pseudomonas syringae in Nicotiana tabacum. The presence of a trans-zeatin synthase (tzs) gene in strain GV3101 may be partly responsible for the priming response, as the tzs-deficient Agrobacterium sp. strain LBA4404 only weakly imparts such responses. Besides inducing the expression of defense-related genes like PR-1 and NHL10, GV3101 pretreatment increased the expression of tobacco mitogen-activated protein kinase (MAPK) pathway genes like MEK2, WIPK (wound-induced protein kinase), and SIPK (salicylic acid-induced protein kinase). Furthermore, the GV3101 strain showed a stronger effect than the LBA4404 strain in activating phosphorylation of the tobacco MAPK, WIPK and SIPK, which presumably prime the plant immune machinery. Lower doses of exogenously applied cytokinins increased the activation of MAPK, while higher doses decreased the activation, suggesting a balanced level of cytokinins is required to generate defense response in planta. The current study serves as a cautionary warning for plant researchers over the choice of Agrobacterium strains and their possible consequences on subsequent pathogen-related studies.

Transient GUS gene expression in cassava (Manihot esculenta Crantz) using Agrobacterium tumefaciens leaf infiltration

ABSTRACTObjective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz) leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS). A. tumefaciens infiltration (agroinfiltration) was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan) and CM6438-14 (Vergara), respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.

First Report of Ageratum enation virus, Betasatellite and Alphasatellite Causing Leaf Curl and Enation Disease of Amaranthus hypochondriacus in India.

During a survey in February 2011, severe symptoms of upward leaf curling, vein enation on lower side of the leaves, and shortening of internodes were observed on 20 out of 117 Amaranthus hypochondriacus plants (17% disease incidence) examined at breeding plots of CSIR-NBRI, Lucknow. These symptoms are typical of begomovirus infection. PCR with begomovirus-specific primers (3) produced the expected ~1.1-kb product from DNA extracts of 20 symptomatic plants but not from a non-symptomatic plant, suggesting the association of a begomovirus. The full-length begomoviral genome from a representative sample was amplified by rolling circle amplification using Ø-29 DNA polymerase and digested by BamHI, which resulted in a ~2.7 kb product when electrophoresed in 1.0% agarose gel. The product obtained was cloned, sequenced, and sequence data of 2,753 nucleotides was deposited in GenBank (Accession No. JF682242). BLASTn analysis revealed 97 to 98% nucleotide identity and forms a distinct clade with Ageratum enation virus (AEV) isolates. This shows the virus in A. hypochondriacus to be an isolate of AEV. The separate PCRs were also performed with betasatellite and alphasatellite specific primers (1,2) that resulted in ~1.3-kb amplicons from all samples, suggesting their association. The amplification products were cloned and sequenced. An analysis of betasatellite (JX512904) revealed highest 98% nucleotide identity and close phylogenetic relationship with Ageratum leaf curl betasatellite (ALCB, JQ710745). The alphasatellite (JX512905) showed highest 95% identity and close relationship with Hibiscus leaf curl alphasatellite (HLCA, FN794199). This shows the betasatellite and alphasatellite in A. hypochondriacus to be isolates of ALCB and HLCA, respectively. The partial direct repeat clones of the begomovirus (pCAM-AEV), betasatellite (pCAM-ALCB), alphasatellite (pCAM-HLCA) were generated and mobilized into Agrobacterium tumefaciens strain GV3101 and infiltrated in A. hypochondriacus seedlings. The plants inoculated with pCAM-AEV, pCAM-ALCB, and pCAM-HLCA; pCAM-AEV and pCAM-ALCB developed severe leaf curl and enation symptoms on 5/5 plant at 35 days post inoculation, which were similar to those of naturally infected plants, satisfying Koch's postulates. On the other hand, plants inoculated with pCAM-AEV alone or in combination with pCAM-HLCA developed mild symptoms. Plants inoculated with pCAM-ALCB and pCAM-HLCA did not develop symptoms. The results here show that leaf curl and enation disease of A. hypochondriacus in India is caused by AEV and ALCB and that an alphasatellite may be associated with symptomatic plants.