Cross-protection and selectable marker genes in plant transformation

Summary Selectable marker genes play an important role in plant transformation. The level of selection pressure is generally established by generating a kill curve for the selectable marker. In most cases, the lowest concentration which kills all explants is used. This study examined two selectable marker genes, phosphinothricin acetyl transferase (PAT) and hygromycin phosphotransferase (HPT), in transformation of tobacco leaf disks. Experiments to determine the lethal level of the herbicide, glufosinate-ammonium (phosphinothricin) (PPT) using a leaf-disk regeneration assay established that no shoots regenerated at 2 to 4 mg PPT per 1. Likewise with the antibiotic, hygromycin (HYG), no plants regenerated at 50 mg hygromycin per 1. In contrast, after cocultivation of the leaf disks withAgrobacterium tumefaciens containing either the PAT or HPT gene in combination with a Bt gene for insect resistance, plants were successfully regenerated from leaf disks at 2 to 4 mg PPT per 1 and 50 mg hygromycin per 1. However, most plants regenerated at 2 and 3 mg PPT per 1 were found to be nontransformed (95–100% escapes) by i) Southern-blot analysis, ii) herbicide application test, and iii) insect feeding bioassay. On the other hand, plants that regenerated on 50 mg hygromycin per 1 and 4 mg PPT per 1 were transgenic as determined by Southern analysis, leaf assay for PPT or HYG resistance, and death of tobacco budworms feeding on these leaves. This study showed a significant level of cross-protection and/or transient expression of the PAT selectable marker gene allowing escapes (95–100%) at selection levels of 2 and 3 mg PPT per 1 which completely kill controls. On the other hand, the HPT gene at 50 mg is efficient in selecting for T-DNA integration.     

Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system.

After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.     

Thansgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system

INTRODUCTIONArachiS hypogaea L., Peanut or groundnut, isan importal commercial crop worldwide. It provides an excellellt source of protein and other nutrients. Its production and quality can be severelyimpacted under stressful growing conditions such ascdriate factors, pests and diseajses. Genetic engineering provides a prospective way to reduce certainproblems by transferring individual genes for pestresistance or other traits into elite germplasm of acultiVated species. Thansgenic pea…     

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Festuca arundinacea</Emphasis> (Schreb.) and <Emphasis Type="Italic">Lolium multiflorum</Emphasis> (Lam.)

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24-68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.     

Agrobacterium-mediated transformation of the wetland monocot Typha latifolia L. (Broadleaf cattail)

An Agrobacterium-mediated model transformation system was standardized for the wetland monocot Typha latifolia L. to achieve the long-term objective of introducing candidate genes for phytoremediation. Two binary plasmid vectors, pCAMBIA1301/EHA105 and pTOK233/LBA4404, both containing the gus (beta-glucuronidase) and hptII (hygromycin phosphotransferase II) genes, were used for transformation. Fifty-day-old 5 mg/l picloram-derived calli were cocultivated and selected on medium containing 20 mg/l or 40 mg/l hygromycin. Resistant calli were regenerated on medium supplemented with 5 mg/l 6-benzylaminopurine, with or without 20 mg/l or 40 mg/l hygromycin and with or without charcoal (10 g/l). Transient GUS activity in explants ranged between 28% and 36%. Hygromycin-resistant calli, selected after 3 months, showed stable GUS expression. A total of 46 plants were regenerated and established in the greenhouse; 13 showed stable GUS expression. Cocultivation of dark culture-derived calli, directly selected on regeneration medium containing 20 mg/l hygromycin and rooted on medium with 20 mg/l hygromycin was the best protocol. The addition of charcoal did not have any effect on regeneration. PCR and Southern analyses of transgenic calli and transgenic plants confirmed the presence of the introduced genes. In conclusion, T. latifolia could be genetically transformed by Agrobacterium tumefaciens.     

Development of transgenic rice pure lines by particle bombardment‐mediated transformation and genetic analysis‐based selection

Mature seed‐derived callus from an elite Chinese japonica rice cv. Eyl 105 was transformed with a plasmid containing the selectable marker hygromycin phosphotransferase (hpt) and the reporter β‐glucuronidase (gusA) genes via particle bombardment. After two rounds of selection on hygromycin (30 mg/l)‐containing medium, resistant callus was transferred to hygromycin (30 mg/l)‐containing regeneration medium for plant regeneration. Twenty‐three independent transgenic rice plants were regenerated from 127 bombarded callus with a transformation frequency of 18.1%. All the transgenic plants contained both gusA and hpt genes, revealed by PCR/Southern blot analysis. GUS assay revealed 18 out of 23 plants (78.3%) proliferated on hygromycin‐containing medium had GUS expression at various levels. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation, we identified three independent homozygous transgenic rice lines. The homozygous lines were phenotypically normal and fertile compared to the control plants. We demonstrate that homozygous transgenic rice lines can be obtained via particle bombardment‐mediated transformation and through genetic analysis‐based selection.     

Efficient Production of Transgenic Cassava Using Negative and Positive Selection

In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene. Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays. In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants. A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system.     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants

We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol. Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days. After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively. Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome. Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration. Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse. Two Taipei 309 and 35 Nipponbare plants set viable seeds. Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed.     

Transgenic Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>) plants obtained by biolistic transformation of embryogenic suspension cells

Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates. We are interested in developing biotechnological methods to improve this monocot forage species. Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric -glucuronidase (gusA) gene was co-transformed with hph. Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin. Plants were regenerated from 45% of the hygromycin resistant calli. Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed. Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues. Fertile transgenic plants were obtained after two winters of vernalization under field conditions. This is the first report on the generation of transgenic plants in Russian wildrye.     

Expression of a fungal cyanamide hydratase in transgenic soybean detoxifies cyanamide in tissue culture and in planta to provide cyanamide resistance

Embryogenic tissue cultures of soybean were transformed by particle bombardment with a vector pCHZ-II that carries the coding sequence for cyanamide hydratase (Cah), an enzyme that converts toxic cyanamide to urea, from the soil fungus Myrothecium verrucaria. The Cah gene was driven by the constitutive Arabidopsis thaliana actin-2 promoter and terminated with its cognate terminator. This vector also carries the hygromycin phosphotransferase gene (hpt) driven by the potato (Solanum tuberosum) ubiquitin-3 promoter. Twelve individual lines of transgenic plants that were obtained under hygromycin selection expressed Cah mRNA and exhibited resistance to hygromycin in leaf tissue culture, while the untransformed tissues were sensitive. Cah enzyme activity was present in extracts of transformed leaves and embryogenic tissue cultures when measured by a colorimetric assay and the presence of the Cah protein was confirmed by enzyme-linked immunosorbent assay (ELISA). Cah expression detoxified cyanamide in leaf callus and embryogenic cultures as well as in whole plants as shown by cyanamide resistance. The Cah-expressing plants grew and set seeds normally indicating that the Cah enzyme activity did not affect soybean plant metabolism. We also describe a test whereby callus was formed on cultured leaf tissue in the presence of hygromycin or cyanamide only if the hpt or Cah gene was expressed, respectively. This test is a convenient and cost-effective way to follow the marker gene in the primary regenerated plants and subsequent generations, which is particularly reliable for the hpt gene expression using hygromycin.     

Over Expression of Rice chitinase Gene in Transgenic Peanut (Arachis hypogaea L.) Improves Resistance Against Leaf Spot

A Rice chitinase-3 under enhance version of CaMV 35S was introduced into peanut (Arachis hypogaea L.) through Agrobacterium mediation. Agrobacterium tumefaciens strain LB4404 was used harboring the binary vector (pB1333-EN4-RCG3) containing the chitinase (chit) and hygromycin resistance (hpt) gene as selectable marker. Putative transgenic shoots were regenerated and grown on MS medium supplemented with 5 mg/l BAP, 1 mg/l kinetin, and 30 mg/l hygromycin. Elongated shoots were examined for the presence of the integrated rice chitinase gene along with hygromycin gene as selectable. The integration pattern of transgene in the nuclear genome of the putative transformed plants (T₀) was confirmed through Southern hybridization analysis of the genomic DNA. Survival rate of the in vitro regenerated plantlets was over 60% while healthy putatively transgenic (T₀) plants with over 42% transformation frequency were produced through Agrobacterium mediated gene transfer of the rice chitinase gene and all the plants flowered and set seed normally. T1 plants were tested for resistance against Cercospora arachidicola by infection with the microspores. Transgenic strains exhibited a higher resistance than the control (non-transgenic plants). chitinase gene expression in highly resistant transgenic strains was compared to that of a susceptible control. A good correlation was observed between chitinase activity and fungal pathogen resistance.     

Molecular analysis of genomic DNA-mediated transformation in Zea mays

Total genomic DNA isolated from maize hygromycin B resistant cell line(hygr-G204) was used to transform the maize hygromycin B sensitive cell line(hygs-G204) to the hygr-phenotype using polyethyleneglycol treatment and the transformed calli were selected using hygromycin B. The primary transformant maize plants were regenerated and analysed at the molecular level using DNA hybridization, transgenome rescue and histochemical β-glucuronidase assay. The results indicated that genomic DNA-mediated transformation can lead to transfer, expression and stable integration of a DNA fragment into the host genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

拟南芥HyPRP蛋白AZI1在转基因烟草中的亚细胞定位及其对灰葡萄孢的抗性

通过农杆菌转化法得到了整合有拟南芥AZII基因的烟草植株,进一步利用转基因烟草分析了AZI1蛋白的亚细胞定位及其对真菌病原体的抗性特征。在上下游引物5’端分别引入NcoI和SpeI酶切位点,采用高保真耐热DNA聚合酶彤Pfu从拟南芥Co1-0生态型基因组DNA扩增AZII基因的编码序列,用NcoI和Spel对扩增片段和pCAMBIA1302质粒载体进行双酶切,通过T4DNA连接酶构建产生AZII-GFP融合表达载体。用包含融合表达载体的农杆菌细胞转化烟草叶片,经潮霉素选择获得了完整的再生植株,并收取了T。代种子。激光共聚焦显微观察发现,AZI1蛋白主要定位于细胞表面。病原体侵染结果显示,AZI1基因能够明显提高烟草对灰葡萄孢的抗性。说明AZI1蛋白通过分泌途径被定位到细胞表面后,能够抑制真菌病原体对植物组织的侵染过程。     

Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T₁ progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation using embryogenic suspension cultures in sweetpotato, <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.

Efficient Agrobacterium tumefaciens-mediated transformation was achieved using embryogenic suspension cultures of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lizixiang. Cell aggregates from embryogenic suspension cultures were cocultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hygromycin phosphotransferase II gene (hpt II) genes. Selection culture was conducted using 25 mg l⁻¹ hygromycin. A total of 2,218 plants were regenerated from the inoculated 1,776 cell aggregates via somatic embryogenesis. β-glucuronidase (GUS) assay and PCR, dot blot and Southern blot analyses of the regenerated plants randomly sampled showed that 90.37% of the regenerated plants were transgenic plants. The number of integrated T-DNA copies varied from 1 to 4. Transgenic plants, when transferred to soil in a greenhouse and a field, showed 100% survival. No morphological variations were observed in the ex vitro transgenic plants. These results exceed all transformation experiments reported so far in the literature in quantity of independent events per transformation experiment in sweetpotato.