Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker

A mannose selection system was adapted for use in the Agrobacterium-mediated transformation of Chinese cabbage. This system makes use of the pmi gene that encodes phosphomannose isomerase, which converts mannose-6-phosphate to fructose-6-phosphate. Hypocotyl explants from 4–5-day-old seedlings of Chinese cabbage inbred lines were pre-cultured for 2–3 days and then infected with Agrobacterium. Two genes (l-guluno-γ-lactone oxidase, GLOase, and jasmonic methyl transferase, JMT) were transformed into Chinese cabbage using the transformation procedure developed in this study. We found that supplementing the media with 7 g l⁻¹ mannose and 2% sucrose provides the necessary conditions for the selection of transformed plants from nontransformed plants. The transformation rates were 1.4% for GLOase and 3.0% for JMT, respectively. The Southern blot analysis revealed that several independent transformants (T ₀) were obtained from each transgene. Three different inbred lines were transformed, and most of the T ₁ plants had normal phenotypes. The transformation method presented here for Chinese cabbage using mannose selection is efficient and reproducible, and it can be useful to introduce a desirable gene(s) into commercially useful inbred lines of Chinese cabbage.     

Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system.

We developed a marker rescue system for study of competence development and genetic transformation in Streptococcus mutans. The system involved the recombinational rescue of a tetracycline resistance (Tcr) determinant by a homologous, inactive locus (Tcs because of a small deletion). Streptococcal cells harboring this in vitro-prepared Tcs construct (pVA1208) were restored to Tcr when plasmid (pVA981) DNA was used as donor material. pVA981 contained the intact streptococcal Tcr locus and was unable to autonomously replicate in streptococci. Marker rescue with this system followed first-order kinetics and occurred at a frequency 8- or 160-fold higher than did transformation with homologous chromosomal or plasmid DNA, respectively. By using the rescue system, we were able to confirm that competence of S. mutans appeared to be inducible. This was indicated by a sequential increase and then decrease in Tcr transformation frequencies during growth in complex medium. Also, donor DNA binding was not sequence specific, since the recovery of Tcr transformants was reduced by increasing the concentrations of heterologous DNA. We investigated the fate of donor DNA and the kinetics of plasmid establishment in the transformation of S. mutans with plasmid DNA. Monomeric plasmid molecules transformed S. mutans as a second-order process, whereas multimeric plasmid DNA and chromosomal markers were recovered as a first-order process. Approximately 50% of the initially bound donor plasmid DNA was found to remain in a trichloroacetic acid-insoluble form. Our results suggested that molecular cloning in S. mutans would be conducted most efficiently by using helper plasmid systems or shuttle vectors and that gene transfer by transformation of S. mutans occurred in a manner similar to that observed in Streptococcus sanguis.     

Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker

Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.     

Efficient transformation of wheat by using a mutated rice acetolactate synthase gene as a selectable marker

Acetolactate synthase (ALS) is a target enzyme for many herbicides, including sulfonylurea and imidazolinone. We investigated the usefulness of a mutated ALS gene of rice, which had double point mutations and encoded an herbicide-resistant form of the enzyme, as a selectable marker for wheat transformation. After the genomic DNA fragment from rice containing the mutated ALS gene was introduced into immature embryos by means of particle bombardment, transgenic plants were efficiently selected with the herbicide bispyribac sodium (BS). Southern blot analysis confirmed that transgenic plants had one to more than ten copies of the transgene in their chromosomes. Adjustment of the BS concentration combined with repeated selection effectively prevented nontransgenic plants from escaping herbicide selection. Measurement of ALS activity indicated that transgenic plants produced an herbicide-resistant form of ALS and therefore had acquired the resistance to BS. This report is the first to describe a selection system for wheat transformation that uses a selectable marker gene of plant origin.     

Green fluorescent protein as a visual marker for wheat transformation

 Wheat (Triticum aestivum L.) transformation via particle bombardment is now established in many laboratories, but transformation efficiencies are still largely low and the highest efficiencies can only be obtained with certain genotypes. For rapid optimization and improvement of wheat transformation protocols, a non-destructive marker which permits early detection of transformed cells is needed. We have assessed the ability of a modified version of the Aequorea victoria green fluorescent protein (GFP) to act as a marker for detecting transformed cells and tissues of wheat. Multicellular clusters emitting green fluorescence were observed 14 days after particle bombardment with a sGFPS65T gene construct, and gfp-expressing shoots (often with expressing roots) could be observed as early as 21 days after bombardment. These shoots can be removed from the callus and grown further until they are ready to transfer to soil. Transgenic wheat plants could be selected on the basis of gfp expression alone although the inclusion of antibiotic resistance as a selectable marker could improve the efficiency. Using sgfpS65T as a marker gene in an experiment comparing bombardment parameters allowed the rapid identification of variables that could be targeted for optimization. Received: 29 March 2000 / Accepted: 29 March 2000     

高丹草EST-SSR标记的开发及其遗传多样性

对NCBI数据库中210 878条高粱EST序列进行处理, 得到57 498条无冗余EST序列, 经SSR搜索, 发现3 338个SSR分布于3 116条EST序列中, 分布频率为1/11.28 kb, 包括215种基元重复类型。其中三核苷酸重复最高, 占68.33%, 二核苷酸重复占17.97%。3 338条SSR序列中有1 694条序列能够设计出引物, 所占比例为50.75%。选取14对引物进行合成, 对50份高丹草、7份高粱和3份苏丹草材料进行了EST-SSR扩增, 共检测到72个等位变异, 平均每对引物检测出5.14个基因位点。每对引物多态性指数范围为0.54~0.93, 遗传距离的变化范围0.1646~0.6398。结果显示：供试材料具有较丰富的遗传多样性, 根据EST-SSR数据的聚类分析, 将供试材料按亲缘关系远近分为5大类, 来源相同的品种大致聚在一类, 呈现出一定的地域性分布规律。同时发现4个特异分子标记, 其中引物D1763只对314A和白壳苏丹草杂交后代GB-4-2高丹草审定品种产生特异性, 此标记已作为该材料的特异性标记用于种质资源的鉴定中, 同时表明, EST-SSR标记是高丹草遗传多样性及特异性研究的一种有效方法。     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Efficient transformation of mammalian cells with constructs containing a puromycin-resistance marker

Recombinant plasmids have been obtained that lead to the accumulation of five- to ten-fold more puromycin-N-acetyl-transferase (PAC) mRNA and two- to three-fold more PAC activity than the already described plasmid pSV2pac [Vara et al., Nucl. Acids Res. 14 (1986) 4117-4124]. When these optimized recombinants were used for stable transformation to puromycin resistance, efficiencies up to 1 x 10(-2) were obtained, indicating that these pac-containing recombinants may be very useful dominant selectable markers for gene transfer in mammalian cells.     

Green fluorescent protein as a visual selection marker for coffee transformation

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.     

Efficient genetic transformation of red raspberry,Rubus ideaus L.

We have developed an efficient transformation system for red raspberry (Rubus ideaus L.) using Agrobacterium mediated gene transfer. Using this system we have successfully introduced a gene that encodes an enzyme, S-adenosylmethionine hydrolase (SAMase), in raspberry cultivars Meeker (MK), Chilliwack (CH) and Canby (CY). Leaf and petiole expiants were inoculated with disarmed Agrobacterium tumefaciens strain EHA 105 carrying either of two binary vectors, pAG1452 or pAG1552, encoding gene sequences for SAMase under the control of the wound and fruit specific tomato E4 promoter. Primary shoot regenerants on selection medium were chimeral containing both transformed and non-transformed cells. Non-chimeral transgenic clones were developed by iterative culture of petiole, node and leaf explants, on selection medium, from successive generations of shoots derived from the primary regenerants. Percent recovery of transformants was higher with the selection marker gene hygromycin phosphotransferase (hpt), than with neomycin phosphotransferase (nptII). Transformation frequencies of 49.6%, 0.9% and 8.1% were obtained in cultivars Meeker, Chilliwack and Canby respectively from petiole expiants using hygromycin selection. Genomic integration of transgenes was confirmed by Southern hybridization. Transgenic plants from a total of 218 independent transformation events (161 MK, 4 CH, 53 CY) have been successfully established in soil.Abbreviations ACCO amincocyclopropane-1-carboxylic acid oxidase - AS acetosyringone - BA 6-benzylaminopurine - CH cultivar Chilliwack - CY cultivar Canby - cv cultivar - hpt hygromycin phosphotransferase - IBA indolebutyric acid - MK cultivar Meeker - npt II neomycin phosphotransferase - SAMase S-adenosylmethionine hydrolase - TDZ Thidiazuron (N-phenyl-N'-l,2,3-thidiazol-5-ylurea)     

运用萎锈灵抗性基因构建香菇聚乙二醇介导的遗传转化方法

【背景】萎锈灵抗性基因作为筛选标记在植物和真菌中得到广泛应用。【目的】构建可以使用萎锈灵作为筛选标记的香菇遗传转化技术。【方法】利用溶壁酶消化培养4 d的香菇菌株411-4的菌丝体获得原生质体,加入适量pL-cbx质粒和聚乙二醇溶液,混合物涂布于再生筛选培养基上,培养后挑选菌落进行验证试验。【结果】在原生质体数目108个和添加4μg质粒DNA的情况下,得到了40个抗性转化子。利用PCR实验和转代实验对转化子进行验证,结果显示38个转化子的抗性可稳定遗传,表明萎锈灵抗性基因整合进入了供试菌株的基因组中。【结论】利用香菇411-4菌株建立了一套运用萎锈灵抗性基因作为分子标记的遗传转化技术体系。     

Efficient genotype-independent cotton genetic transformation and genome editing

Cotton(Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However,genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation. Here, we developed a shoot apical meristem(SAM) cell-mediated transformation system(SAMT) that allowed the transformation of r...     

Efficient shoots regeneration and genetic transformation of Bacopa monniera

Bacopa monniera is an important source of metabolites with pharmaceutical value. It has been regarded as a valuable medicinal plant and its entire commercial requirement is met from wild natural population. Recently, metabolic engineering has emerged as an important solution for sustained supply of assured and quality raw material for the production of active principles. Present report describes efficient in vitro multiplication and transformation method for genetic manipulation of this species. MS medium supplemented with 2 mgl⁻¹ BA and 0.2 mgl⁻¹ IAA was found optimum for maximum shoot regeneration (98.33 %) from in vitro leaves with 2–3 longitudinal cuts. Agrobacterium tumefaciens-mediated transformation method was used for generating transgenic B. monniera plants. Putative transformants were confirmed by GUS assay and PCR based confirmation of hptII gene. DNA blot analysis showed single copy insertion of transgene cassette. An average of 87.5 % of the regenerated shoots were found PCR positive for hptII gene and GUS activity was detected in leaves of transgenic shoots at a frequency of 82.5 % The efficient multiple shoots regeneration system described herein may help in mass production of B. monniera plant. Also, the high frequency transformation protocol described here can be used for genetic engineering of B. monniera for enhancement of its pharmaceutically important metabolites.     

Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation

Summary Development of new selectable markers is needed to increase the efficiency and flexibility of plant transformation, and to overcome drawbacks sometimes associated with use of existing markers. A useful alternative to chemical-based selection systems would be a system using visual screening to obtain transgenic lines. Investigations were carried out to determine if the green fluorescent protein (gfp) gene could be utilized alone as a visual screenable marker to produce stably transformed, fertile oat plants. Twelve experiments were conducted in which gfp-based selection was utilized to obtain routinely stable transgenic lines in oat. A synthetic gfp gene under the control of the maize ubiquitin promoter was delivered into embryogenic oat callus via microprojectile bombardment. Cell clusters (1–3 mm) expressing gfp were visually identified using epifluorescence microscopy and physically isolated approximately 3 wk post-bombardment. Fertile, gfp-expressing T0 plants were regenerated from 78% of the glowing cell sectors placed on regeneration medium. T0 plants from 55% of the events produced gfp-expressing progeny in a 3∶1 Mendelian ratio. Southern blot and PCR analysis confirmed transgene integration and transmission to progeny. Expression of gfp did not reduce plant growth or fertility. Transgene copy number and integration patterns were similar to those in transgenic plants derived from chemical-based selection systems. The mean transformation frequency, based on fertile events obtained per bombarded plate, was 1.8%. Over 180 independent transgenic oat lines were produced, to date, using only visual screening for expression of gfp, demonstrating efficiency and repeatability of the selection system. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the University of Wisconsin or the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Efficient Agrobacterium-mediated genetic transformation method using hypocotyl explants of radish (Raphanus sativus L.)

To investigate the gene function of radish (Raphanus sativus L.), several attempts have been made to generate genetically transformed radish. However, no efficient and relatively simple method for the genetic transformation of radish has been developed to date. In this study, we established an Agrobacterium-mediated genetic transformation method using the hypocotyl-derived explants of radish cultivar “Pirabikku”. Primarily based on the Brassica transformation procedure, we optimized it for radish transformation. Using this system, the transformation efficiency of radish hypocotyl explants by Agrobacterium tumefaciens strain GV3101 harboring pIG121-Hm was 13.3%. The copy number of transfer DNA integrated into the genome was either one or two in the four independent transgenic plants. Two of the four plants exhibited male sterility and did not produce self-pollinated seeds. Examination of the expression of the β-glucuronidase (GUS) gene in T₁ plants from fertile T₀ plants showed that the GUS genes were inherited. The improvement in the genetic transformation in this study might pave the way for accelerated molecular breeding and genetic analysis of radish.     

Maize transformation using xylose isomerase gene as a selection marker]

The xylA gene, encoding xylose isomerase, was cloned as a 1342-bp BamHI/SacI fragment from the E. coli. As a selection marker, the xylA gene was fused between the enhanced CaMV 35S promoter (E35S) and terminator (35St) in pBAC413 (Fig.2). pBAC413 was constructed to prevent the expression of sbeIIb in maize. PDS1000/He was used to bombard maize calli, which were induced to form by the elite inbred lines. The selection was carried out on the media containing concentrations of xylose from 0 to 100%. The results showed that the media containing 50% to 100% D-xylose were better, but differed with the genotype of maize (Tables 1 and 2). Successful integration of xylA gene into the maize genome was confirmed by DNA dot blotting, PCR and PCR-Southern hybridization (Figs.4 to 6). A method was established in which transformed maize cells were successively screened on a medium containing xylose instead of antibiotic and herbicide for bio-safety.