A modified method for routine Agrobacterium-mediated transformation of in vitro grown potato microtubers

Summary In vitro-grown potato (Solanum tuberosum L.) microtubers were used as an explant source in the production of transgenic plants by Agrobacterium-mediated gene transfer. In this study we tested four diverse potato cultivars, Lemhi Russet, Russet Burbank, Wauseon, and Yankee Chipper on various levels of zeatin riboside and 3-indoleacetyl-DL-aspartic acid for their ability to regenerate transgenic plants after infection with Agrobacterium tumefaciens. Culturing microtuber blocks from the medullary area separately from cortex and epidermal tissue containing the eyes resulted in fewer transgenic plants, with transgenic shoots arising only from the tissue with the eyes. Lemhi and Russet Burbank microtuber discs were also transformed with a chimeric gene, CLaSP, designed to increase resistance to blackspot bruise in the tuber. This method resulted in transformed plants in every experiment, with an efficiency that appeared to be genotype dependent.Abbreviations GUS -glucuronida (uidA) - IAA-AA 3-indoleacetyl-DL-aspartic acid - LB Luria-Bertani - LSP larval serum storage protein - nos nopaline synthase - npt II neomycin phosphotransferase - MS Murashige and Skoog - PHA phytohemaglutinin - ZR zeatin riboside     

Resistance to Potato Virus X Infection in Transgenic Tobacco Plants with Coat Protein Gene of Virus

A major commercial cultivar of tobacco was transformed via Agrobacterium mediated procedure. Tobacco leaves started to form shoots on shoot inducing medium containing kanamycin after infected by Agrobacterium containing the plasmid with PVX CP gene. Regenerated plants were obtained in two weeks on hormone-free MS medium containing kanamycin. The transgenic tobacco plants were identified with nopaline detection,enzyme-linked immunosorbent assay and western blot analysis, symptom appearance was significantly delayed and virus accumulation was either absent or reduced in PVX CP gene transformed plants. Progenies of transgenic tobacco plants also gained resistance to PVX infection to a certain degree. These experiments demonstrate that CP protection is effective against PVX.     

Transformation of carrot tissues derived from proembryogenic suspension cells: A useful model system for gene expression studies in plants

A method is described for the high frequency transformation of carrot proembryogenic suspension culture cells by a non-oncogenic Ti-plasmid vector (pGV3850::1103) which carried a chimaeric kanamycin resistance gene (nos-NPT-II). Plants were regenerated efficiently from transformed material by somatic embryogenesis in the presence of kanamycin. Transformed tissues expressed readily detectable levels of both NPT-II and nopaline. NPT-II could be detected in total protein extracts by Western blotting. This analysis indicated that NPT-II was produced as a single, full length polypeptide. The T-DNA copy number in individually selected transformants was analysed by Southern blotting and ranged from 1–8 per diploid genome. The copy number and organization of the T-DNA was retained in plants regenerated from these transformants by somatic embryogenesis. These data suggested a clonal origin for the selected kanamycin resistant colonies. NPT-II expression levels appeared to be directly related to gene dosage.     

高效烟草遗传转化体系的建立及甜蛋白基因的导入

以烟草无菌茁叶片为外植体,通过根癌农杆菌LBA4404介导法,将Thamnatin基因导人烟草中,经梯度卡那霉素(Kana-mycin,Km)筛选,获得可在含75mg／L、100mg／L Km选择生根培养基上再生的抗性植株,其中部分Km抗性植株经PCR检测为阳性,转化率为31．3％,初步鉴定已成功地建立了烟草遗传转化系统,为进一步探讨甜蛋白在植物中的转化和表达情况奠定基础。     

Co-expression and inheritance of foreign genes in transformants obtained by direct DNA transformation of tobacco protoplasts

Summary A plasmid containing two marker genes for expression in plants was constructed. This 16 kb vector, pCT1T3, contains an intact nopaline synthase gene and a chimaeric gene consisting of the promoter and terminator regions from cauliflower mosaic virus gene VI and a structural gene, aminoglycoside phosphotransferase (APH(3′)II), from the bacterial transposon Tn5. After transformation of tobacco mesophyll protoplasts with this plasmid, several kanamycin-resistant transformants were obtained. Intensive studies on the drug tolerance of growth and differentiation of the transformants showed that the chimaeric gene was stably expressed. Of 17 independent transformants, 3 (about 18%) expressed the two marker genes, regardless of the state of differentiation, as did individual plants regenerated from the same callus. Multiple copies of the inserted DNA were found in some transformants. Viable seeds were produced by 12 out of 15 independent transformants. Seeds obtained by self-pollination were germinated on medium containing kanamycin sulphate. With the exception of one clone, resistant seedlings with green leaves and sensitive seedlings with white leaves were found to segregate in a 3:1 ratio. This suggests that the inheritance of the inserted gene is Mendelian. A reciprocal cross between the transformants and wild-type tobacco also showed nuclear transmission of the APH(3′)II gene. This was consistently maintained in a subclone of the same transformant derived from the same callus line. Stable inheritance of the single dominant character was also seen among seeds formed in several different flower pods of the same individual plants. Two clones were also found to synthesize nopaline in addition to expressing APH(3′)II. Analysis of the progeny obtained by self-crosses of such transformants revealed the simultaneous expression of these two enzymes, indicating that the two marker genes are linked on the same chromosome.     

Genetic analysis of T-DNA insertions into the tobacco genome

A genetic test was performed on seeds from 283 transgenic tobacco plants obtained by T-DNA transformation. Seeds from self-fertilized transgenic plants were germinated on kanamycin-containing medium, and the percentage of seeds which germinated, as well as the ratio of kanamycin-resistant to kanamycin-sensitive seedlings were scored. Nine categories of transformants could be distinguished according to the number of loci into which T-DNA had inserted, and according to the effects of T-DNA integration on seed or seedling development. In most of the plants, T-DNA was inserted into a single site; others contained multiple independent copies of T-DNA. The number of T-DNA integration sites was found to be independent of whether a binary vector system or a cointegrate Ti plasmid had been used to obtain the transgenic plant. Loss of marker genes or marker gene expression from generation to generation appeared to be a quite frequent event. Plants which appeared to be insertional recessive embryo-lethal mutants did not exhibit this trait in the next generation.Abbreviations Kan^R kanamycin resistant - Kan^S kanamycin sensitive - NOP nopaline - NOS nopaline synthase - NPT II neomycin phosphotransferase II     

Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

Transformation of potato (Solanum tuberosum) using particle bombardment

Internodes, leaves and tuber slices from potato (Solanum tuberosum), genotype 1024-2, were subjected to particle bombardment. Transient expression was optimized using the uidA and the luc reporter genes that encode #-glucuronidase (GUS) and luciferase, respectively. Stable transformation was achieved using the neomycin phosphotransferase (nptII) gene, which confers resistance to the antibiotic kanamycin. The influence of biological parameters (tissue type, growth period before bombardment, pre- and post-bombardment osmoticum treatment) and physical parameters (helium pressure, tissue distance) that are known to possibly affect stable transformation were investigated. Putative transgenic plants, which rooted in media containing kanamycin, were obtained from all of the tissues tested although there were large differences in the efficiency: internodes (0.77 plants per bombarded explant), microtuber slices (0.10 plants per bombarded explant) and leaves (0.02 plants per bombarded explant). Southern blot analysis of putative transgenic plants confirmed the integration of the transgenes into plant DNA. The results indicate that an efficient particle bombardment protocol is now available for both transient and stable transformation of potato internodal segments, thus contributing to an enhanced flexibility in the delivery of transgenes to this important food crop.     

Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens

Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens harbouring pGV 38501103 neo (dimer) containing chimaeric kanamycin resistance reporter genes. Transformed protoplasts were regenerated to fertile and phenotypically normal plants. Transformation was confirmed by kanamycin resistance, nopaline production, neomycinphosphotransferase II activity, and Southern blot hybridization. Seed progeny from self-pollinated transformants expressed the introduced kanamycin resistance as a Mendelian trait.Abbreviations BAP 6-benzylaminopurine - Cf Claforan^R - 2.4D 2,4-dichlorophenoxy acetic acid - Km kanamycin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - NPT II neomycinphosphotransferase - npt II neomycinphosphotransferase II gene - NOS nopaline synthase - nos nopaline synthase gene - ocs octopine synthase gene - IAA indole-3-acetic acid     

Transformation of potato (Solanum tuberosum) cv. Mantiqueira using Agrobacterium tumefaciens and evaluation of herbicide resistance

Summary In search of establishing a system for genetic transformation of Brazilian potato cultivars, Agrobacterium tumefaciens carrying the plasmid pGV1040, was used to transform leaf discs of three cultivars of local importance, i.e., Aracy, Baronesa and Mantiqueira. This plasmid contains marker genes for resistance to kanamycin and phosphinothricin plus the gene for the enzyme -glucuronidase. A two step regeneration/selection procedure produced shoots of potato cultivar Mantiqueira with in vitro resistance to kanamycin and to phosphinothricin. After transfer to the greenhouse, the potentially transgenic plants, sprayed with the herbicide Finale^® (20% a.i.; Hoechst^®) remained green as compared to control clones that died immediately afterwards. Southern blot analysis and histochemical and fluorimetric assay for -glucuronidase indicated that the gene coding for the enzyme was integrated in the potato genome and could be expressed in potato tissues. No success was obtained for transformation of cultivars Aracy and Baronesa using this procedure.Abbreviations NAA Naphthalene Acetic Acid - BAP Benzyl-aminopurine - GA₃ Gibberellic Acid - PPT Phosphinothricin - PAT Phosphinothricin Acetyl Transferase     

Genetic transformation of willows (Salix spp.) byAgrobacterium tumefaciens

Anin vitro transformation method has been developed for stem explants of fast-growing willow clones (Salix spp.) usingAgrobacterium tumefaciens as a vector. Transformants obtained with the strains C58 and GV3101 (pGV3851::pLD1) were selected on hormone-free medium and on medium containing kanamycin, respectively. Transformation was confirmed by Southern blot analysis and nopaline assay. Inoculation of green-house grown plants with nopaline and octopine wildtype strains and shoot or root inducing mutant strains caused undifferentiated tumors at a frequency of 0 to 80%, depending on theSalix genotype and the bacterial strain used.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Km kanamycin - NPT neomycin phosphotransferase     

The use of in vitro-grown microtuber discs inAgrobacterium-mediated transformation of Russet Burbank and Lemhi Russet potatoes

We have usedin vitro-grown microtuber discs in the transformation of Russet Burbank and Lemhi Russet potato (Solanum tuberosum L.) cultivars byAgrobacterium-mediated gene transfer. Transformed plants were selected by their resistance to kanamycin and identified by -glucuronidase activity. Northern blot analysis confirmed the presence of the corresponding messenger RNA. The ability to transform these two cultivars promises significant improvements to agronomically important varieties.Abbreviations MS Murashige and Skoog - LB Luria-Bertani - KS high kanamycin selection medium - NAA naphthaleneacetic acid - BA benzyladenine - GUS -glucuronidase - EDTA ethylenediaminetetraacetic acid - MUG 4-methylumbelliferyl glucuronide     

Spread of recombinant DNA by roots and pollen of transgenic potato plants,identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII'; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII' and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.     

Inactivation of the nopaline synthase gene by double transformation: reactivation by segregation of the induced DNA

Tobacco plants were transformed with derivatives of a binary vector pMON505 and two kanamycin resistant lines that were nopaline positive were selected for second transformation. The plasmids used for the second transformation were derivatives of pMON850 which carries the nopaline synthase gene in addition to a gene for gentamicin resistance. Insertion of each transgene was confirmed by Southern hybridization. Surprisingly, we found that more than 50% of the doubly transformed tobacco plants were nopaline negative. Tobacco plants that were transformed only by the second vector exhibited nopaline accumulation. DNA methylation patterns at the HpaII site in the promoter region of the nopaline synthase gene did not correlate with the nopaline phenotype. In some plant lines, seedlings of the R1 generation which segregated out the second T-DNA insertion recovered the nop⁺ phenotype. These results indicate that nopaline accumulation was inhibited by the presence of the second T-DNA.Abbreviations T-DNA transferred DNA - NPTII neomycin phosphotransferase II - uidA -glucuronidase - Km kanamycin - Gm gentamicin - nop⁺ nopaline positive - nop^– nopaline negative - MS medium, Murashige-Skoog medium     

小球藻NADP-谷氨酸脱氢酶的cDNA克隆及转基因烟草分析

用RT-PCR方法从小球藻(Chlorella sorokiniana)中克隆了铵诱导表达的以辅酶Ⅱ为辅基的谷氨酸脱氢酶(NADP-GDH)基因的cDNA片段,DNA测序分析表明与已报道的该基因c DNA序列同源性为94%.将NADP-GDH基因先插入到SPDK621质粒的2CaMV35S启动子和Ω增强序列之后,然后将2CaMV35S-Ω-GDH-NOS表达单元构建到RokⅡ质粒的HindⅢ与Eco RⅠ之间,从而获得高效植物表达载体.将RokⅡ-GDH质粒转移到根癌土壤杆菌(Agro bacterium tumefaciens (Smith et Townsend) Conn) EHA105中,对烟草(Nico tiana tabacum L.)进行转化并得到阳性转化后代.对转基因烟草分析表明,在低氮培养基或在低氮蛭石中其生长速度和叶片数明显高于对照;铵毒性实验表明,无论在低铵或高铵条件下,接种在MS固化培养基上的转基因绿叶圆片存活时间长,叶绿素含量高.这些结果说明外源NADP-GDH增强了植物对氮素的吸收和利用.另外,转化后代还表现了对除草剂膦化麦黄酮(PPT)具有较强的抗性;培养在含有不同浓度PPT的MS固化培养基上的转基因绿叶圆片,其愈伤化程度明显高于对照;在MS培养基中用0.5 μg/mL 的PPT可以代替卡那霉素对转化后代进行筛选,这暗示 NADP-GDH基因可以作为一种新的选择标记用于植物基因工程的研究.