Production of Transgenic Rice Homozygous Lines with Enhanced Resistance to the Rice Brown Planthopper

Mature seed‐derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was cotransformed with two plasmids, pWRG1515 and pRSSGNA1,containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β‐glucuronidase gene (gusA) and the snow‐drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin‐containing medium, resistant callus was transferred to hygromycin‐containing regeneration medium for plant regeneration. Twenty‐six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy‐three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parentplants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes.Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding.This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis‐based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.     

An efficient particle bombardment system for the genetic transformation of asparagus (Asparagus officinalis L.)

The microprojectile bombardment method was used to transfer DNA into embryogenic callus of asparagus (Asparagus officcinalis L.) and to produce stably transformed asparagus plants. Embryogenic callus, derived from UC 157 and UC72 asparagus cultivars, was bombarded with tungsten particles coated with plasmid DNA that contained genes encoding hygromycin phosphotransferase, phosphinothricin acetyl transferase and -glucuronidase. Putatively transformed calli were identified from the bombarded tissue after 4 months selection on 25 mg/L hygromycin B plus 4 mg/L phosphinothricin (PPT). By selecting embryogenic callus on hygromycin plus PPT the overall transformation and selection efficiencies were substantially improved over selection with hygromycin or PPT alone, where no transgenic clones were recovered. The transgenic nature of the selected material was demonstrated by GUS histochemical assays and Southern blot hybridization analysis. Transgenic asparagus plants were found to withstand the prescribed levels of the PPT-based herbicide BASTATM for weed control.Abbreviations GUS -glucuronidase - HPT hygromycin phosphotransferase - bar phosphinothricin acetyl transferase gene - PPT phosphophinothricin - NAA naphthalene acetic acid - 2iP 2-isopentenyl adenine     

Genetic transformation and regeneration of rubber tree (Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress superoxide dismutase gene

Agrobacterium tumefaciens-mediated genetic transformation and the regeneration of transgenic plants was achieved in Hevea brasiliensis. Immature anther-derived calli were used to develop transgenic plants. These calli were co-cultured with A. tumefaciens harboring a plasmid vector containing the H. brasiliensis superoxide dismutase gene (HbSOD) under the control of the CaMV 35S promoter. The -glucuronidase gene (uidA) was used for screening and the neomycin phosphotransferase gene (nptII) was used for selection of the transformed calli. Factors such as co-cultivation time, co-cultivation media and kanamycin concentration were assessed to establish optimal conditions for the selection of transformed callus lines. Transformed calli surviving on medium containing 300 mg l^-1 kanamycin showed a strong GUS-positive reaction. Somatic embryos were then regenerated from these transgenic calli on MS2 medium containing 2.0 mg l^-1 spermine and 0.1 mg l^-1 abscisic acid. Mature embryos were germinated and developed into plantlets on MS4 medium supplemented with 0.2 mg l^-1 gibberellic acid, 0.2 mg l^-1 kinetin (KIN) and 0.1 mg l^-1 indole-3-acetic acid. A transformation frequency of 4% was achieved. The morphology of the transgenic plants was similar to that of untransformed plants. Histochemical GUS assay revealed the expression of the uidA gene in embryos as well as leaves of transgenic plants. The presence of the uidA, nptII and HbSOD genes in the Hevea genome was confirmed by polymerase chain reaction amplification and genomic Southern blot hybridization analyses.Communicated by L. Peña     

Oryzacystatin Exogenously Introduced into Protoplasts and Regeneration of Transgenic Rice

Oryzacystatin (OC) is a proteinaceous cysteine proteinase inhibitor involved in the biodefense of rice seeds. To create transgenic rice plants with increased OC activity, we introduced an OC expressing vector into rice protoplasts and obtained transformed calli. The expression vector contained a bacterial inaA DNA fragment in the 3′-noncoding region as a tag to distinguish the introduced DNA from the intrinsic OC gene. The OC vector and a selection marker gene conferring hygromycin resistance were used together to transfect into rice protoplasts. A number of hygromycin-resistant calli were obtained and studied by polymerase chain reaction and genomic Southern blotting to find if the exogenous OC gene had been integrated. The calli were studied by northern blotting as well to examine mRNA expression. The results showed that integration and expression of the introduced OC gene occurred in 51% and 27%, respectively, of 156 subcultures from 15 hygromycin-resistant calli. As a final step, transgenic rice plants were regenerated from the calli expressing OC. Leaves and seeds from the plants had higher OC activities than those from nontransgenic plants.     

An improved rice transformation system using the biolistic method

Immature embryos and embryogenic calli of rice, both japonica and indica subspecies, were bombarded with tungsten particles coated with plasmid DNA that contained a gene encoding hygromycin phosphotransferase (HPH, conferring hygromycin resistance) driven by the CaMV 35S promoter or Agrobactenum tumefaciens NOS promoter. Putatively transformed cell clusters were identified from the bombarded tissues 2 weeks after selection on hygromycin B. By separating these cell clusters from each other, and by stringent selection not only at the callus growth stage but also during regeneration and plantlet growth, the overall transformation and selection efficiencies were substantially improved over those previously reported. From the most responsive cultivar used in these studies, an average of one transgenic plant was produced from 1.3 immature embryos or from 5 pieces of embryogenic calli bombarded. Integration of the introduced gene into the plant genome, and inheritance to the offspring were demonstrated. By using this procedure, we have produced several hundred transgenic plants. The procedure described here provides a simple method for improving transformation and selection efficiencies in rice and may be applicable to other monocots.Abbreviations bp base pairs - CaMV cauliflower mosaic virus - GUS -glucuronidase - HPH hygromycin phosphotransferase - hyg B hygromycin B - hyg^r hygromycin resistance - NOS Agrobactenum tumefaciens nopaline synthase - PCR polymerase chain reaction - X-Gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide     

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.     

Effects of antibiotics on the elimination of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) zygotic embryo explants and on transgenic plant regeneration

Three antibiotics were evaluated for their effects on the elimination of Agrobacterium tumefaciens during the genetic transformation of loblolly pine ( Pinus taeda L.) using mature zygotic embryos as targets. Agrobacterium tumefaciens strains, EHA105, GV3101, and LBA 4404, all harbouring the plasmid pCAMBIA1301, which carries the selectable marker gene, hygromycin phosphotransferase ( hpt) controlled by the cauliflower mosaic virus 35S promoter and terminator, and the uidA reporter gene (GUS) driven by the cauliflower mosaic virus 35S promoter and the terminator of nopaline synthase gene, were used in this study. Exposure to 350 mg l^-1 carbenicillin, claforan, and timentin respectively for up to 6 weeks did not eliminate the Agrobacterium, while antibiotics at 500 mg l^-1 eradicated them from the co-cultivated zygotic embryos. All three antibiotics increased callus growth and shoot regeneration at 350 and 500 mg l^-1 each, but reduced callus growth and shoot regeneration at 650 mg l^-1 when compared with controls. Putative transgenic calli were selected for continued proliferation and differentiation on 4.5 mg l^-1 hygromycin-containing medium. Transformed calli and transgenic plants produced on a selection medium containing 4.5 mg l^-1 hygromycin were confirmed by GUS histochemical assays, by polymerase chain reaction (PCR), and by Southern blot analysis. These results are useful for future studies on optimizing genetic transformation procedures in loblolly pine.     

Introduction of <Emphasis Type="Italic">OsglyII</Emphasis> gene into <Emphasis Type="Italic">Oryza sativa</Emphasis> for increasing salinity tolerance

Mature seed-derived embryogenic calli of indica rice (Oryza sativa L. cv. PAU201) were induced on semisolid Murashige and Skoog medium supplemented with 2.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 0.5 mg dm⁻³ kinetin + 560 mg dm⁻³ proline + 30 g dm⁻³ sucrose + 8 g dm⁻³ agar. Using OsglyII gene, out of 3180 calli bombarded, 32 plants were regenerated on medium containing hygromycin (30 mg dm⁻³). Histochemical GUS assay of the hygromycin selected calli revealed GUS expression in 50 % calli. Among the regenerants, 46.87 % were GUS positive. PCR analysis confirmed the presence of the transgene of 1 kb in 60 % of independent plants. Further, these plants have been grown to maturity in glasshouse. In vitro screening for salt tolerance showed increase in fresh mass of OsglyII putative transgenic calli (185.4 mg) as compared to control calli (84.2 mg) on 90 mM NaCl after 15 d. When exposed to 150 mM NaCl, OsglyII putative transgenic plantlets showed normal growth while the non-transgenic control plantlets turned yellow and finally did not survive.     

Effect of selectable gene to reporter gene ratio on the frequency of co-transformation and co-expression of uidA and hpt transgenes in protoplast-derived plants of tall fescue

Forty-six independent transformed plants were regenerated under hygromycin selection from cell-suspension derived protoplasts of Festuca arundinacea (Schreb.) after PEG-mediated transformation. Protoplasts were co-transformed with varying molar gene ratios (0.7:1–6:1) of a marker -glucuronidase (uidA) gene and a selective hygromycin (hpt) resistance gene. Logistic regression analysis indicated that, as expected, the proportion of co-transformed plants tended to increase as the proportion of the marker gene was increased. However, although the proportion of plants co-expressing both genes tended to increase up to a molar ratio of 4:1, it appeared to fall at a molar ratio of 6:1. No statistically significant differences were found in the average copy number of the integrated uidA or hpt transgenes, either in GUS expressing, or in non-GUS expressing plants at the different molar ratios. When using naked-DNA gene transformation methods most authors use a molar ratio of 1:1; our data suggest that adding non-selected and selected transgenes at a higher Molar Gene Ratio would probably improve the proportion of plants regenerated which express both transgenes.     

Luciferase as a reporter gene for transformation studies in rice (Oryza sativa L.)

Transformed rice plants of var `TN1' were regenerated from immature embryos following particle bombardment with a construct containing the firefly luciferase gene as a reporter gene and the hygromycin resistance gene as a selectable marker. Expression of the luciferase gene in the presence of the substrate luciferin was visualised in the calli derived from bombarded immature embryos and in the leaves and roots of the regenerated transformed plants using a low light imaging system (luminograph). Embryogenic callus proliferation and plant regeneration were unaffected by luciferin treatment and luminograph screening. The quantitative Luc assay using samples of leaf tissue from the segregating generations gave early information about the homozygous and hemizygous state of the luc transgene. Received: 25 August 1998 / Revision received: 2 November 1998 / Accepted: 13 November 1998     

农杆菌介导的苏云金杆菌抗虫基因cryIA(b)和cryIA(c)在水稻中的遗传转化及蛋白表达

通过农杆菌介导法用含有抗潮霉素和 Ｇ Ｕ Ｓ 基因的双元载体将杀虫结晶蛋白基因ｃｒｙ Ｉ Ａ（ ｂ） 和ｃｒｙ Ｉ Ａ（ｃ） 导入到籼、粳稻幼穗愈伤组织中,然后经过在含有不同浓度潮霉素的培养基上进行数次筛选,获得一批 Ｂｔ 转基因株。经 Ｐ Ｃ Ｒ、 Ｓｏｕｔｈｅｒｎ 杂交及 Ｗｅｓｔｅｒｎ 印迹分析证实此二基因已整合进水稻中,饲虫试验结果表明,转基因株具有１００ ％ 杀虫率。     

Selection of co-transformed Dendrobium Sonia 17 using hygromycin and green fluorescent protein

Dendrobium Sonia 17 calluses were used for co-transformation study using particle bombardment. The bombarded transformed callus tissues were selected using half-strength MS medium containing 25 mg dm⁻³ hygromycin. Expression of green fluorescent protein (GFP) was observed in the callus and protocorm-like body (PLBs) tissues survived on the selection medium. The presence of green fluorescence protein (sgfp), hygromycin-B-phosphotransferase (hptII) and β-glucuronidase (uidA) genes in the transformed tissues were verified using PCR, Southern blot and dot blot analyses. Based on the results from PCR and expression of sgfp and uidA genes in the calluses and PLBs survived from hygromycin selection, we reported the co-transformation of sgfp, hptII and uidA genes into Dendrobium Sonia 17. GFP-expressing tissues were also observed in the regenerated transformed plantlets.     

Fertile,transgenicTriticale ( ×Triticosecale Wittmack)

Fertile transgenicTriticale ( ×Triticosecale Wittmack) plants expressing the-glucuronidase (uidA) and phosphinothricin acetyltransferase (bar) genes were obtained after microprojectile bombardment of scutellar tissue with the plasmid pDB1 containing theuidA gene under the control of the actin-1 promoter (Act1) from rice and the selectable marker genebar under the control of the CaMV 35S promoter. From 465 bombarded scutella about 4000 plantlets were regenerated; 300 plants survived the selection. These regenerants were screened for enzyme activity by the histological GUS assay and by spraying the plants with a herbicide (Basta). Twenty-five regenerants showed GUS activity and survived repeated Basta spraying. Southern blot analysis showed the presence of both marker genes introduced into the genome of analysed plants.All transgenic plants were fertile. They were grown to maturity and set seed. Pollen and progeny analyses provided evidence for inheritance of the introduced genes to the next generation.     

Timing of Transposition of Ac Mobile Element in Potato

The timing of excision of maize transposable element Ac was studied using visual histochemical assay based on Ac excision restoring activity of -glucuronidase (GUS). The Solanum tuberosum L. cv. Bintje was used for Agrobacterium-mediated transformation with pTT230 plasmid harbouring Ac-interrupted gus A gene and npt II gene as a selectable marker gene. Twenty-eight out of 72 kanamycin resistant calli did not express any GUS activity, 31 calli showed partial GUS expression and 13 out of assayed calli revealed strong expression of gus A gene. Plants were regenerated from calli without and/or with partial expression of gus A gene. The regenerated transformants which did not express GUS during the callus phase often contained many small GUS expressing spots on leaves. A phenotypic selection assay for excision of Ac has been also used. This non-detectable excision of Ac in callus tissue could be followed by a "late" timing excision during leaf development. After transformation with pTT224 plasmid harbouring Ac-interrupted hpt II gene and npt II gene transgenic calli containing Ac within the hygromycin resistance gene were derived and hygromycin sensitive plants were regenerated from them. Protoplasts isolated from leaves of transgenic regenerated plants were selected on hygromycin. Hygromycin resistant minicalli showed to harbour multiple copies of Ac and mark out low uniqueness of integration sites.     

Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method

A system for the production of transgenic papaya (Carica papaya L.) plants using zygotic embryos and embryogenic callus as target cells for particle bombardment is described. Phosphinothricin (bar ) and kanamycin (npt II) resistance genes were used as selectable markers, and the gus gene (uidA) as a reporter gene. Selection with 100 mg/l kanamycin and 4 mg/l phosphinothricin (PPT) yielded a total of over 90 resistant embryogenic colonies from three independent experiments using embryogenic callus as a target tissue. This represents an efficiency of 60 transgenic clones per gram of fresh weight callus bombarded. The efficiency of genetic transformation using zygotic embryos was lower, as only 8 independent resistant clones were recovered out of 645 bombarded zygotic embryos, giving a efficiency of 1.24%. Subsequent subculture of transgenic somatic embryos both from zygotic embryos and embryogenic callus led to the development of plants with apparently normal morphology. Histological, fluorimetric assay for GUS, NPT II assay and DNA analysis (Southern hybridization) showed that kanamycin /PPT resistant plants carried and expressed the transgenes.Abbreviations Gus -glucuronidase - NPTII neomycin phophotransferase II - bar phophinothricin acetyl transferase gene - Pat phosphinothricin acetyl transferase - PPT phosphinothricin - Km kanamycin - 2,4-D 2,4-dichlorophenoxyacetic acid - K kinetin - BAP benzylaminopurine - IBA indolbutyric acid     

Efficient selection and regeneration of creeping bentgrass transformants following particle bombardment

We have established an efficient genetic transformation system for creeping bentgrass (Agrostis palustris Huds.) using particle bombardment. The transformation was performed using the plasmid pZO1052 which contains the reporter β-glucuronidase (uidA) gene and the selectable marker hygromycin phosphotransferase (hph) gene. Transformed calli and plants were obtained via particle bombardment followed by selection of transformants on medium containing 200 mg/l of hygromycin. An average of 4.6 resistant colonies per bombardment were obtained. Southern analysis confirmed the integration of foreign genes in 19 of 21 putative transformants, indicating that selection by hygromycin was highly effective. Received: 6 February 1997 / Revision received: 16 April 1997 / Accepted: 9 May 1997     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of meadow fescue (<Emphasis Type="Italic">Festuca pratensis</Emphasis> Huds.)

Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene. C. Gao and J. Liu contributed equally to the work.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

药用野生稻TAC克隆转化籼稻的体系初探

采用已构建的药用野生稻TAC文库,比较研究4个籼稻品种在不同培养基的诱导率及愈伤组织对潮霉素抗性。通过2种不同诱导方法产生的愈伤组织,将携带药用野生稻大片段DNA的TAC克隆转化到籼稻品种中的结果表明,除了‘粤香占’以外,其它3个品种在心诱导培养基中添加微量B,后,其愈伤诱导率最高。不同品种的潮霉素筛选适合浓度存在差异,其中‘华粳籼74’的适合筛选浓度为50mg．L-1,‘粤香占’为40mg．L-1。用预培养5d的愈伤组织进行遗传转化,在潮霉素筛选之前先以头孢拉定抑菌处理的,容易获得转基因植株。4个品种中以‘粤香占’的抗性愈伤组织筛选率和分化率最高,分别为22．58％和14．86％。分子检测11株转基因水稻的结果表明,其中8株含有抗性标记基因。据此认为,药用野生稻TAC文库在水稻创新育种中可能有一定的利用前景。     

Stable wheat transformation obtained without selectable markers

Transgenic wheat plants without the selectable marker gene were obtained either in the presence or in the absence of selective pressure during the transformation protocol. When using hygromycin as selective agent in a co-transformation experiment involving a mixture of plasmids pGL2, containing the hpt gene, and pAI₁Gus, containing the uidA gene, 3 out of 19 transgenic wheat plants had the uidA gene alone as shown by Southern blots. The gene was transmitted to the progeny following Mendelian rules. Segregation and loss of the selectable marker gene was also found in three out of six events from other experiments where high-molecular-weight glutenin genes were expressed or over-expressed. On the other hand, in 7 experiments where no selective pressure was applied and that involved 1016 bombarded explants, 23 transgenic wheat plants were obtained. The uidA gene was stably integrated as suggested by its transmission to the progeny.these authors contributed equally to the work