Expression of OsDREB2A in Transgenic Tomato Improves Drought Tolerance

Dehydration responsive element binding (DREB) are important regulatory molecules which have a crucial role in abiotic stress tolerance. The productivity of tomato, as a drought-sensitive crop, is highly restricted by drought stress. The current study aimed at introducing the OsDERB2A gene into two tomato genotypes via Agrobacterium-mediated transformation system. Cotyledonary explants were pre-cultured for two days with Agrobacterium strain LBA4404 harboring pCAMBIA1301 with OsDREB2A driven by the constitutive promoter CaMV35S for transformation. Shoots were directly regenerated on MS medium containing 1 mg l-1 zeatin and 1 mg l-1 BAP, and in presence of 30 mg l-1 hygromycin as selective agent. Only eight weeks were needed to regenerate transgenic tomato using this protocol. An OD600 of 0.4 resulted in 64.3-76.9% transformation efficiency. Stable integration and expression of the OsDREB2A gene were confirmed in transgenic tomato using PCR and RT-PCR analyses, and drought tolerance of T0 transgenic lines was confirmed by leaf disc assay in 300 mM mannitol. The superior biomass, photosynthetic pigments, free soluble sugars and proline accumulation of OsDREB2A transgenic lines over wild type in response to mannitol-stress revealed their enhanced drought tolerance and indicated that the constitutive expression of OsDREB2A might modulate the expression of other drought responsive genes.

Complete genomic sequence and phylogenomics analysis of Agrobacterium strain AB2/73: a new Rhizobium species with a unique mega-Ti plasmid

Background The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors. Results We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7012 genes (6948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin. Conclusions The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium.

Complete Genomic Sequence and Phylogenomics Analysis of Agrobacterium Strain AB2/73: A New Rhizobium Species with a Unique mega-Ti Plasmid

Background: The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors. Results: We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7,012 genes (6,948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin. Conclusions: The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium.

Agrobacterium-Mediated Capsicum annuum Gene Editing in Two Cultivars, Hot Pepper CM334 and Bell Pepper Dempsey

Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains—AGL1, EHA101, and GV3101—to investigate which Agrobacterium strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuumMLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.

Genotypic Variability in Soybean [Glycine max (L.) Merrill] through Agrobacterium-Mediated Transformation

Embryonic tip explants of 92 Indian soyabean and 7 advanced breeding lines derived from soaked mature seeds were inoculated and co-cultivated for 5-day with Agrobacterium strain EHA105 carrying the binary vector pCambia1305.1 containing a hygromycin and kanamycin resistance gene as plant and bacterial selectable markers, respectively. Transient expression of transgene was monitored by histochemical localization of β-glucouronidase (GUSPlus) reporter activity in transformed ET tissues. A high genetic variability for Agrobacterium-infection ranging from 3.8 to 100% was observed in the form of transient GUS expression. Five highly efficient genotypes, namely DS-228, JS 335, JS 72-44, KHSb2, and JS 72-280 with transient GUS expression of 100, 98.1, 96.5, 96 and 92%, respectively were identified. In addition, various infectivity patterns in these genotypes were observed. Genotypes with very high transient GUS expression identified in this study may improve success rate of development of transgenic soybean. Plant Tissue Cult. & Biotech. 30(2): 231-242, 2020 (December)

Degradation of 2,3,7,8-TCDD by a consortium of bacterial strains isolated from heavil herbicide/dioxin contaminated soil in Bienhoa airbase

From two different soil sources in Bienhoa airbase (heavy herbicide/dioxin contaminated West-South region and bioremediated cell), five microbial strains were isolated and their 2,3,7,8-TCDD biodegrability in consortium was investigated. Based on the colony and cell morphological characteristics as well as 16S rRNA gene sequences, these strains were classified into 5 genera, including Methylobacterium (strain BHBi1), Hydrocarboniphaga (strain BHBi4), Agrobacterium (strain BHBi5), Bosea (strain BHBi7) and Microbacterium (strain BH09). Two strains BHBi7 and BHBi4 were the first representatives of the genera Bosea and Hydrocarboniphaga that were isolated from heavyly herbicide/dioxin contaminated soil. All five strains were able to grow well in mineral salt medium (MSM) supplemented with soil extract (SE) containing 2,3,7,8-TCDD (this congener is the main soil total compound toxicity) and other congeners, including PCDDs, PCDFs, 2,4,5-T, 2,4-D, PAHs and their intermediates. This microbial consortium degraded 2,537.34 ngTEQ/kg of 2,3,7,8-TCDD congener in soil, equivalent to 59.1% lost of total toxicity in comparison to the control without bacterial seeding (4,294.12 ng TEQ/kg). Such a high ratio of dioxin degradation by a bacterial consortium was reported here for the first time, contributing more evidences for convincing the successful dioxin bioremediation of “Active Landfill” technology at large scale in Z1 area at Bienhoa airbase, Dongnai, Vietnam.

In Planta Transformation in Plants: A Review

In planta transformation has been established as an innovative and simple technique involving direct transformation of plant parts without involving the tedious tissue culture step. Methodologies of in planta transformation involve different plant parts and strategies. Agrobacterium strain carrying the gene of interest is targeted to the specific plant part either directly or in the induction medium. Vacuum infiltration is sometimes used to facilitate foreign gene integration. Initial studies of this novel technique involved treatment of whole plants with the inoculum, and later shifted to treatments of shoot tips and floral parts and finally the female reproductive parts have been targeted. Zygotes, embryos and seeds have recently been used extensively yielding successful transformation. The method is simple, convenient and overcomes the problem of tissue culture induced genetic variability in the transformants. The review traces the origin and development in the in planta methodologies used over the past and the various parameters considered by the workers for increased effectiveness viz. developmental stages, Agrobacterium stain, surfactant, induction medium etc., in the various crops compiled. Based on the review it may be inferred that there is immense potential in planta transformation, and the ease of regeneration and selection of transformants in the methods described, can be utilized for crop improvement.

Agrobacterium-mediated Genetic Transformation of Lentil (Lens culinaris Medik.) with Chitinase Gene followed by In vitro Flower and Pod Formation

To investigate the integration of chitinase gene in lentil (Lens culinaris Medik.) namely, BARI masur-4 (BM-4), BARI masur-5 (BM-5) and BARI masur-6 (BM-6) through Agrobacterium-mediated genetic transformation was performed using Agrobacterium strain EHA 105 harboring bar (resistant to phosphinotrycin) and chitinase (gene of interest) gene. Selection of transformed shoots was carried out by gradually increasing the concentration of phosphinotrycin (PPT) up to 2.0 mg/l. Transgenic lentil shoots were produced with an overall frequency of 0.36 in case of BM-4 and BM-6 and 0.34 in case of BM-5, respectively. Most of the selected shoots developed in vitro flowers and pods following their sub-culture on half strength of MS supplemented with 20 mg/l IBA, 0.5 mg/l NAA with 50 mg/l ticarcillin. Seedlings germinated from the seeds were successfully transferred to soil for the development of further progeny. Stable integration of target gene was confirmed through PCR analysis. Plant Tissue Cult. & Biotech. 29(1): 99-109, 2019 (June)

Reconstruction and validation of three different binary vectors suitable for generation of genetically engineered Helicoverpa protected crops

Availability of a suitable plant transformation binary vector is necessary for the generation of transgenic crops with an adequate expression of transgenic proteins. Therefore, three binary vectors were constructed viz., pBK204, pBK205, and pBK206 harboring either a truncated or a full-length version of a Cry1Ac gene for the generation of Helicoverpa protected crops. Two different promoters viz., Arabidopsis Rubisco small subunit (AtSSU) gene promoter or CaMV35S promoters were used to regulate the various versions of Cry1Ac gene. The binary vectors were reconstructed either by the Gibson assembly method and others by ligating the restriction enzyme digested fragments. The reconstructed binary vectors were mobilized into Agrobacterium strain AGL1 and validated by Agrobacterium infiltration assays of Nicotiana benthamiana. The amount of Cry1Ac protein accumulated in the Agroinfiltrated tobacco leaves was quantified using the quantitative ELISA assay. The expression of the Cry1Ac protein in the tobacco leaves ranged from 0.25 to 0.26 µg /g fresh weight (FW) when transformed with these three constructs. Thus, the vectors constructed in this study appeared to be suitable for generation of Helicoverpa resistant transgenic crops by Agrobacterium-mediated genetic transformation method.

Regeneration and Agrobacterium-Mediated Transformation of Japonica Rice Varieties Developed for a Cold Region

So far, a large number of transformation systems have been established for japonica rice, but only a few have been reported for cold-region varieties. In our study, we established highly efficient tissue culture systems for two cold-region rice cultivars, Dongnong 427 and Longdao 14. Plant growth regulator (PGR) levels were optimized by an orthogonal experimental design. The culture ability, constituted by induction and differentiation rate, served as the detection index of orthogonal experiments. The optimal combinations of PGRs for callus induction and regeneration of Dongnong 427 and Longdao 14 were 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) + 2 mg/l 6-benzyladenine (BA) + 4 mg/l kinetin (KIN) + 0.2 mg/l α-naphthaleneacetic acid (NAA) and 1 mg/l 2,4-D + 4 mg/l 6-BA + 4 mg/l KIN + 0.5 mg/l NAA, respectively. Agrobacterium strain EHA 105 containing the plasmid pCAMBIA1301 was used for transformation. The frequency of transient transformation was expressed as the ratio between the number of calli showing GUS expression and the total number of calli kept for staining. The highest transformation efficiency in Dongnong 427 was obtained when calli were immersed in 0.272 OD₆₀₀ (optical density determined at 600 nm) for 10 min. While it was best for Longdao 14 calli to be infected with 0.592 OD₆₀₀ for 20 min. Infected calli of the two varieties were co-cultivated on two pieces of sterile filter paper moistened with 1 ml liquid co-cultivation medium for three days. The expression of the GUS gene was confirmed by PCR analysis of plants of both varieties.