Expression of a synthetic cholera toxin B subunit in tobacco using ubiquitin promoter and bar gene as a selectable marker

A protocol has been developed to produce a cholera toxin B subunit (CTB) in tobacco tolerant to the herbicide phosphinothricin (PPT) by means of in vitro selection. The synthetic CTB subunit gene was altered to modify the codon usage to that of tobacco plant genes. The gene was then cloned into a plant expression vector and was under the control of the ubiquitin promoter and transformed into tobacco plants by Agrobacterium-mediated transformation. Transgenic plantlets were selected in a medium supplemented with 5 mg/L PPT. Polymerase chain reaction analysis confirmed stable integration of the synthetic CTB gene into a chromosomal DNA. A high level of CTB (1.8% of total soluble protein) was expressed in transgenic plants, which was 18-fold higher than that under the control of the expressed CaMV 35S promoter with native gene. The transgenic plants when transferred to a greenhouse proved to be resistant to 2% PPT.     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols

Transgenic plants of strawberry cultivar Totem were developed by Agrobacterium-mediated transformation using a plasmid vector containing gus and nptII genes. Parallel experiments were carried out with and without repeated subculturing (iterative cultures) for generation of transgenic shoots on selection medium. The selection levels in the non-iterative pathway were kept constant, while in the iterative protocol, stepwise increase of selection pressure was applied at different stages of tissue growth. Rooted transgenic plants obtained via both protocols were outplanted in soil. Random leaf samples of greenhouse-grown transgenics were analysed for the presence of gus gene sequences by Southern hybridization as well as gus expression on leaf and petiole tissues by X-Gluc histological assay. Random leaf samples analysed from individual transgenic events developed under iterative culture were positive for the gus insert as verified by Southern analysis confirming the presence of transgenes and lack of chimaeras. Leaf samples of the transgenic events from the non-iterative protocol were either positive or negative on Southern analysis indicating the chimaeric nature of the transgenic plants. The absence of gus sequences in the transgenic plants grown under the non-iterative protocol reinforced the necessity of iterative cultures along with stepwise increase in selection levels for generating non-chimaeric transgenics in strawberry. The gus expression was highly variable, irrespective of the iterative or non-iterative protocol used for transformation. We conclude that strawberry is highly prone to develop chimaeric transgenics if derived from primary regenerants and that the iterative culture technique effectively converts chimaeras to pure line transgenic plants     

Transformation of peanut with a soybean vspB promoter‐uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut ( Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi‐quantitation of transient expression. One hundred and sixty hygromycin‐resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R₁ seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.     

Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified delta-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud

A synthetic version of the CRY1Ac gene of Bacillus thuringiensis has been used for the transformation of loblolly pine (Pinus taeda L.) using particle bombardment. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Expression vector pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences, and the neomycin phosphotransferase II (NPTII) gene controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected on media with kanamycin. Shoot regeneration was induced from the kanamycin-resistant calli, and transgenic plantlets were then produced. More than 60 transformed plants from independent transformation events were obtained for each loblolly pine genotype tested. The integration and expression of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern hybridization, by Northern blot analysis, and by Western blot analysis. Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects. The transgenic plants recovered could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform.     

Agrobacterium-mediated transformation and plant regeneration of Brassica oleracea var. capitata

An efficient protocol for Agrobacterium tumefaciens-mediated transformation of four commercial cultivars of Brassica oleracea var. capitata is described. A strain of A. tumefaciens LBA4404 with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette were used for co-cultivation. Preliminary selection of regenerated transgenic plants was performed on kanamycin-containing medium. The frequency of transgenic plants was calculated on the basis of GUS (β-glucuronidase) activity detected by the histochemical X-gluc test. Tissue-specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. The transformation rates of the commercial cultivars of B. oleracea was higher than in previous reports. Southern blot analysis revealed that integration of marker genes occurred in single and multiple loci in the genome. All transgenic plants grew normally after a brief vernalization period and showed stable inheritance of the marker gene. The present study demonstrates that morphologically normal, fertile transgenic plants of B. oleracea can be obtained. Received: 24 August 1999 / Revision received: 23 November 1999 / Accepted: 3 December     

Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.     

Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum)

We have established an efficient particle-bombardment transformation protocol for the diploid non-apomictic genotype of the warm season forage crop Paspalum notatum (bahiagrass). A vector containing a herbicide resistance gene (bar) together with the GUS reporter gene was used in transformation experiments. The bar gene confers resistance to the herbicide bialaphos. An improved culture system, highly regenerative callus, dense in compact polyembryogenic clusters, was produced on medium with a high CuSO4 content at elevated temperature. Target tissue (360 calli) produced under these conditions yielded 52 rooted plants on herbicide-containing medium, and 22 of these plants were PCR-positive. DNA gel blot analysis revealed a copy number of 1-5 for the GUS gene in different independent transformants. There was no correlation between copy number and GUS activity. While conventional cultures yielded exclusively albino plants on herbicide-containing medium, improved culture conditions for the target tissue resulted in the recovery of 100% green transgenic plants. All green herbicide-resistant regenerants were morphological normal and fertile.     

Transgene expression in strawberries driven by a heterologous phloem-specific promoter

Strawberry is susceptible to diseases caused by phytoplasmas, mycoplasma-like prokaryotes restricted to sieve elements in the phloem tissue of infected plants. One strategy to improve strawberry resistance to phytoplasmas involves transgenic expression of anti-microbial peptide genes in phloem. For targeted phloem-specific expression, we constructed a binary vector with an expression cassette bearing the -glucuronidase (GUS) reporter gene (uidA) under control of the Arabidopsis sucrose-H⁺ symporter gene (AtSUC2) promoter. Transgenic strawberry lines were generated with high efficiencies by a modified transformation protocol, which combines the adoption of a 3-day pre-selection period following transformation, and the addition of 10-M thidiazuron to the regeneration medium. Histological GUS activity indicated that the reporter gene was expressed specifically in phloem of leaves, petioles, and roots of transgenic plants. The results suggest that the transformation protocol and the AtSUC2 promoter may be useful for engineering phytoplasma-resistant transgenic strawberries.     

Efficient genetic transformation of Withania coagulans (Stocks) Dunal mediated by Agrobacterium tumefaciens from leaf explants of in vitro multiple shoot culture

An efficient and reproducible Agrobacterium-mediated genetic transformation of Withania coagulans was achieved using leaf explants of in vitro multiple shoot culture. The Agrobacterium strain LBA4404 harboring the binary vector pIG121Hm containing β-glucuronidase gene (gusA) under the control of CaMV35S promoter was used in the development of transformation protocol. The optimal conditions for the Agrobacterium-mediated transformation of W. coagulans were found to be the co-cultivation of leaf explants for 20 min to agrobacterial inoculum (O.D. 0.4) followed by 3 days of co-cultivation on medium supplemented with 100 μM acetosyringone. Shoot bud induction as well as differentiation occurred on Murashige and Skoog medium supplemented with 10.0 μM 6-benzylaminopurine, 8.0 μM indole 3-acetic acid, and 50.0 mgl^?1 kanamycin after three consecutive cycles of selection. Elongated shoots were rooted using a two-step procedure involving root induction in a medium containing 2.5 μM indole 3-butyric acid for 1 week and then transferred to hormone free one-half MS basal for 2 weeks. We were successful in achieving 100 % frequency of transient GUS expression with 5 % stable transformation efficiency using optimized conditions. PCR analysis of T₀ transgenic plants showed the presence of gusA and nptII genes confirming the transgenic event. Histochemical GUS expression was observed in the putative transgenic W. coagulans plants. Thin layer chromatography showed the presence of similar type of withanolides in the transgenic and non-transgenic regenerated plants. A. tumefaciens mediated transformation system via leaf explants developed in this study will be useful for pathway manipulation using metabolic engineering for bioactive withanolides in W. coagulans, an important medicinal plant.     

Highly efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cell suspensions of <Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Mas (AA) via a liquid co-cultivation system

A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0–490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.     

Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation

A new method has been developed for guayule tissue culture and transformation. Guayule leaf explants have a poor survival rate when placed on normal MS medium and under normal culture room light conditions. Low light and low ammonium treatment greatly improved shoot organogenesis and transformation from leaf tissues. Using this method, a 35S promoter driven BAR gene and an ubiquitin-3 promoter driven GUS gene (with intron) have been successfully introduced into guayule. These transgenic guayule plants were resistant to the herbicide ammonium-glufosinate and were positive to GUS staining. Molecular analysis showed the expected band and signal in all GUS positive transformants. The transformation efficiency with glufosinate selection ranged from 3 to 6%. Transformation with a pBIN19-based plasmid containing a NPTII gene and then selection with kanamycin also works well using this method. The ratio of kanamycin-resistant calli to total starting explants reached 50% in some experiments.     

AtNHX1基因对草木樨状黄芪的转化和耐盐性表达研究

应用RT-PCR技术从100mmol/LNaCl胁迫处理的拟南芥幼中克隆得到编码液泡膜Na /H 逆向转运蛋白的AtNHX1基因cDNA 编码ORF.并在该ORF上游分别插入CaMV 35启动子和TMV RNA5'UTR的Ω片段,而在下游插入NOS polyA构建真核表达盒,进而将该表达盒插入双元植物表达栽体pNT质粒的T-DNA区构建了携带AtNHX1 基因的植物表达载体质粒pNT-AtNHX1.将pNT-AtNHX1 导入农杆菌LBA4404,用农杆菌介导法将AtNHX1 基因导入豆科牧草草木樨状黄芪中,共获得103株Kan抗性再生植株.通过对农杆菌茵液浓度、侵染时间和乙酰丁香酮浓度等影响转化效率的因素进行优化,初步建立了稳定的草木樨状黄芪农杆菌转化体系.经过PCR检测、Southern杂交和RT-PCR检测表明,AtNHX1 基因已被成功整合到草木樨状黄芪基因组中,并且能够正常转录.野生型和转基因株系诱发的愈伤组织进行耐盐生长实验,结果显示相同盐胁迫条件下,转基因愈伤组织的相对生长率显著高于野生型愈伤组织.施加梯度NaCl胁迫后,植株叶片K ,Na 含量和叶片相对电导率测定结果显示,转基因植物叶片比野生型积累更多的Na 和K ,维持较高的K /Na ;转基因株系叶片相对电导率显著低于野生型.上述结果表明,AtNHX1 基因的导入和表达在提高草木樨状黄芪耐盐性的同时减轻了盐胁迫对植物细胞膜的伤害.关键词: AtNHX1 草木樨状黄芪农杆菌遗传转化耐盐性.     

Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane

A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8–4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and β-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1–3 transgene insertion events that were randomly integrated in the majority of the plants produced.     

Improving freezing tolerance of transgenic tobacco expressing sucrose: sucrose 1-fructosyltransferase gene from Lactuca sativa

Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The 1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants.     

Enhanced resistance against bacterial wilt in transgenic tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) lines expressing the <Emphasis Type="Italic">Xa21</Emphasis> gene

To enhance bacterial wilt resistance in tomato plants and simplify the protocol of Agrobacterium tumefaciens mediated gene transfer, parameters affecting transformation efficiency in tomato have been optimized. A. tumefaciens strain EHA101, harboring a recombinant binary expression vector pTCL5 containing the Xa21 gene under the control of the CaMV 35S promoter was used for transformation. Five cultivars of tomato (Rio Grande, Roma, Pusa Ruby Pant Bahr and Avinash) were tested for transformation. Transformation efficiency was highly dependent on preculture of the explants with acetosyringone, acetosyringone in co-cultivation media, shoot regeneration medium and pre-selection after co-cultivation without selective agent. One week of pre-selection following selection along with 400 μM acetosyringone resulted in 92.3% transient GUS expression efficiency in Rio Grande followed by 90.3% in Avinash. The presence and integration of the Xa21 gene in putative transgenic plants was confirmed by polymerase chain reaction (PCR) and Southern blot analyses with 4.5–42.12% PCR-positive shoots were obtained for Xa21 and hygromycin genes, respectively. Transgenic plants of the all lines showed resistance to bacterial wilt. T₁ plants (resulting from self-pollination of transgenic plants) tested against Pseudomonas solanacearum inoculation in glasshouse, showed Mendelian segregation.     

Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.