Recovery of transgenic plants by pollen-mediated transformation in <Emphasis Type="Italic">Brassica juncea</Emphasis>

The aroA-M1 encoding the mutant of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) was introduced into the Brassica juncea genome by sonication-assisted, pollen-mediated transformation. The plasmid DNA and collected pollen grains were mixed in 0.3 mol/L sucrose solution and treated with mild ultrasonication. The treated pollen was then pollinated onto the oilseed stigmas after the stamens were removed artificially. Putative transgenic plants were obtained by screening germinating seeds on a medium containing glyphosate. Southern blot analysis of glyphosate-resistant plants indicated that the aroA-M1 gene had been integrated into the oilseed genome. Western blot analysis further confirmed that the EPSPS coded by aroA-M1 gene was expressed in transgenic plants. The transgenic plants exhibited increased resistance to glyphosate compared to untransformed plants. Some of those transgenic plants had considerably high resistance to glyphosate. The genetic analysis of T₁ progeny further confirmed that the inheritance of the introduced genes followed the Mendelian rules. The results indicated that foreign genes can be transferred by pollen-mediated transformation combined with mild ultrasonication.     

Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker

 Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T₀) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected 35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T₀ and T₁ transgenic plants. Mendelian segregation of herbicide resistance was observed in some T₁ progenies. Interestingly, a majority of the T₁ progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated in T₁ transgenic wheat plants. Received: 12 May 1998 / Accepted: 15 May 1998     

<Emphasis Type="Italic">Agrobacterium</Emphasis> -mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) using a heterologous bean chitinase gene

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T₁ plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T₀ transformants and progeny plants (T₁) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.     

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.     

转大肠杆菌过氧化氢酶基因棉花的抗旱生理研究

以导入大肠杆菌过氧化氢酶基因KatE的T3代转基因棉花为供试材料,经卡那霉素检测和PCR鉴定,将筛选出的阳性转基因植株与对照棉花进行整个生育期的持续水分胁迫处理直至收获,比较材料间的生理生化指标的差异,鉴定转基因植株的耐旱能力。结果显示:(1)干旱胁迫持续至初蕾期时,转基因棉花与对照植株间各项抗旱生理指标差异均未达到显著水平。(2)水分胁迫持续至盛蕾和盛花期时,转基因棉花叶片相对含水量、光系统Ⅱ最大光化学效率(Fv/Fm)、CAT活性,以及叶片的净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)均显著或极显著高于对照植株,叶绿素含量也都明显高于对照植株。干旱胁迫持续至吐絮期时,转基因棉花的株高、果枝数和铃数均显著或极显著高于对照植株,且转基因棉花和对照的籽棉产量分别比正常灌溉处理降低57.5%和60.1%,全生育期的水分胁迫严重影响了棉花籽棉产量,但转基因棉花的籽棉产量仍显著高于对照。研究表明,在新疆石河子当地自然降水(干旱胁迫)条件下,转KatE基因棉花表现出了较好的生理和生长优势,KatE基因有助于提高棉花的抗旱性。     

转OvDREB2B基因陆地棉鉴定及其对水分渗透胁迫的分析

通过花粉管通道技术,以该实验室自育陆地棉品系TH₁和TH₂为材料,将诸葛菜(Orychophragmus vidaceus)抗逆转录因子OvDREB2B基因构建到植物表达载体后,导入棉花基因组,经卡那霉素筛选和分子鉴定表明目的基因已整合到棉花基因组中并表达。将T₁代转基因植株和受体对照在温室中栽培,待植株生长至四叶一心时,用不同渗透势的PEG-6000水溶液进行渗透胁迫处理,分析探讨转基因植株的抗旱效果及其抗旱机理。结果显示:当渗透势为0和0.5 MPa处理时,转基因植株和对照无明显差异;当渗透势为0.8 MPa和1.1 MPa处理时转基因植株较对照抗旱性明显提高。当渗透势为1.1 MPa处理96 h时,对照植株F_v/F_m降至0.2左右,而转基因植株仍正常生长,F_v/F_m值约为0.51,而且初始荧光（F₀）值、净光合速率（P_n）、胞间CO₂浓度（C_i）、蒸腾速率（T_r）等一系列参数转基因植株都明显优于对照,表明DREB2B基因能够提高棉花对水分胁迫的耐受性。     

Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance

 Transformation of barley and wheat via particle bombardment with a gene derived from Vitis vinifera L. (Vst1 gene) resulted in the expression of the foreign phytoalexin, resveratrol, in the transformed plants. Transgenic barley plants were regenerated from microspores and transgenic wheat plants from immature embryos were both selected on Basta. Stable integration of the gene in the genomes of transgenic barley and wheat plants, as well as their progeny, was analysed by Southern-blot analysis. The induction of the stilbene synthase promoter and the transient expression of stilbene synthase-specific mRNA after induction by wounding and infection were proofed in T₁ and T₂ progeny plants. An enhanced expression of the Vst1 gene under control of the stilbene synthase promoter was observed with enhancer sequences from the cauliflower mosaic virus 35s (CaMV 35s) promoter. The enzyme activity of the stilbene synthase was analysed in T₁ progeny plants. The first pathological results indicated an increased resistance of transgenic barley plants to Botrytis cinerea used as a model experimental system. Received: 5 November 1997 / Accepted: 11 November 1997     

Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease

 A 1.1-kb DNA fragment containing the coding region of a thaumatin-like protein (TLP-D34), a member of the PR-5 group, was cloned into the rice transformation vector pGL2, under the control of the CaMV 35S promoter. The Indica rice cultivars, ‘Chinsurah Boro II’, ‘IR72’, and ‘IR51500’ were transformed with the tlp gene construct by PEG-mediated direct gene transfer to protoplasts and by biolistic transformation using immature embryos. The presence of the chimeric gene in T₀, T₁, and T₂ transgenic plants was detected by Southern blot analysis. The presence of the expected 23-kDa TLP in transgenic plants was confirmed by Western blot analysis and by staining with Coomassie Brilliant Blue. Bioassays of transgenic plants challenged with the sheath blight pathogen, Rhizoctonia solani, indicated that over-expression of TLP resulted in enhanced resistance compared to control plants. Received: 11 August 1998 / Accepted: 26 August 1998     

Expression of a bacterial <Emphasis Type="Italic">aroA</Emphasis> mutant, <Emphasis Type="Italic">aroA-M1</Emphasis>, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. We have previously reported a strategy for engineering glyphosate-resistant class I EPSPS based on staggered-PCR technology. Selected mutant enzymes exhibited high K_i[glyphosate] and low K_m[PEP] values compared to the parental enzymes from Escherichia coli (EcaroA) and Salmonella typhimurium (StaroA). One mutant, aroA-M1, was further engineered with a tobacco chloroplast leader sequence, and then placed in the binary vector pCAMBIA1300 for Agrobacterium-mediated gene transfer to tobacco (Nicotiana tabacum cv. Xanthi). Transgenic plants with increased resistance to glyphosate were generated.     

Generation of marker‐free transgenic hexaploid wheat via an Agrobacterium‐mediated co‐transformation strategy in commercial Chinese wheat varieties

Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker‐free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium‐mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker‐free transgenic wheat plants from various commercial Chinese varieties and their F₁ hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T‐DNA regions. The average co‐integration frequency of the gus and the bar genes located on the two independent T‐DNA regions was 49.0% in T₀ plants. We further found that the efficiency of generating marker‐free plants was related to the number of bar gene copies integrated in the genome. Marker‐free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T₁ positive plants, but the gus gene was never found to be silenced in T₁ plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants.     

Inheritance of a co-transferred foreign gene in the progenies of transgenic rice plants

The integration pattern and the inheritance of exogenous DNA in transgenic rice plants were analysed. Plasmid pCH (4.8 kb), that contains chimaeric cauliflower mosaic virus 35S promoter-hygromycin phosphotransferase structural gene, and plasmid pGP400 (7.2 kb), possessing oat phytochrome promoter and structural gene of bacterial -glucuronidase, were co-transferred into protoplasts of rice (Oryza sativa L.) plants via electroporation. Primary transformants (T₀ generation) and their progenies (T₁, T₂ and T₃) were selected by hygromycin B. Southern blot analysis of inserted genes in transgenic rice plants suggests the integration of an intact hygromycin phosphotransferase gene and non-functional DNA fragments into host genome. Co-inheritance of the hygromycin phosphotransferase gene and -glucuronidase gene was also observed. There were no significant differences in terms of the morphology and size of seeds between untransformed and transgenic plants (T₃ generation).     

根癌农杆菌介导的转aroAM12基因棉花植株的草甘膦抗性

以中棉35无菌苗下胚轴为外植体,采用农杆菌介导法将含有通过基因优化技术获得的草甘膦抗性突变基因aroAM12导入棉花中.以aroAM12为选择标记,利用草甘膦直接筛选获得65棵再生植株.PCR和Southerablot分析表明,经过草甘膦筛选出的To代植株均整合有aroAM12基因.Western blot分析表明整合进的aroAM12基因得到了有效表达,且不同植株之间的表达不尽相同.大棚喷洒的实验结果表明To代转化植株具有很高的草甘膦抗性.对T1代棉花的草甘膦抗性遗传分析表明,aroAM12基因以孟德尔方式遗传.     

Rapid production of fertile transgenic barley (Hordeum vulgare L.) by direct gene transfer to primary callus-derived protoplasts

Protoplasts were isolated from primary calli of barley (Hordeum vulgare L.), and an antibiotic (G418) resistance gene was introduced into these protoplasts using a polyethylene glycol (PEG) DNA uptake method. Sixty-four G418 resistant calli were obtained in nine experiments, and two plants were regenerated from these calli. NPTII ELISA and Southern analysis indicated that the G418 resistance gene was introduced and expressed in two T₀ plants. These plants set seed and the introduced gene was transmitted to T₁ plants. These results suggest that our transformation system using primary callus-derived protoplasts is a useful method for the generation of transgenic barley. Received: 14 November 1997 / Revision received: 12 March 1998 / Accepted: 24 April 1998     

Resistance to Alternaria brassicicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene

Transgenic broccoli plants expressing a Trichoderma harzianum endochitinase gene were obtained by Agrobacterium tumefaciens-mediated transformation. PCR and Southern blot analysis confirmed the presence of the gene in plants initially selected via resistance to kanamycin. Primary transformants (T₀) and selfed progeny (T₁) were examined for expression of the endochitinase gene using a fluorometric assay and for their resistance to the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum. All transgenic plants with elevated endochitinase activity had the expected 42 kDa endochitinase band in western blot analysis, whereas no such band was detected in the non-transgenic control. Leaves of most mature T₀ plants had 14–37 times higher endochitinase activity than controls; mature T₁ plants had higher endochitinase activity (100–200 times that in controls), in part because of lower control values. T₀ plantlets in vitro or young plants in soil had higher absolute and relative endochitinase activity. When detached leaves of T₀ plants were inoculated with A. brassicicola, lesion size showed a significant negative correlation with endochitinase levels. After inoculation of two-month old T₀ plants with A. brassicicola, all 15 transgenic lines tested showed significantly less severe disease symptoms than controls. In contrast, lesion size on petioles of T₀ and T₁ plants inoculated with S. sclerotiorum was not statistically different from controls.     

Expression of the cry1EC gene in castor (Ricinus communis L.) confers field resistance to tobacco caterpillar (Spodoptera litura Fabr) and castor semilooper (Achoea janata L.)

Castor (cv. DCS-9) has been transformed through Agrobacterium-mediated and particle gun bombardment methods using appropriate vectors containing the Bt chimeric gene cry1EC driven by enhanced 35S promoter. About 81 and 12 putative transformants were regenerated following selection on hygromycin and kanamycin, respectively. Southern analysis of DNA extracted from T₀ plants confirmed integration of the introduced gene in castor genome. The integration and inheritance of the introduced genes was demonstrated up to T₄ generation by PCR and Southern analysis. Southern analysis of two events having single and two copies showed the same pattern of integration in the subsequent generations. Insect feeding experiments conducted in the laboratory by releasing neonate larvae of castor semilooper and S. litura on leaf tissues excised from transgenic and control plants showed varying degrees of larval mortality and slow growth in larvae fed on transgenic leaf tissue. Field bioassays against Spodoptera litura and castor semilooper conducted for eight events in T₁–T₄ generations under net confinement were more informative and events conferring resistance to the two major defoliators were identified.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Over-expression of the Gr5 aroA gene from glyphosate-contaminated soil confers high tolerance to glyphosate in tobacco

Herbicide resistance is the most widely used transgenic crop trait for broad-spectrum control of weeds. Here we report a novel 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (Gr5 _aroA ) isolated from glyphosate-contaminated soil. The full Gr5 _aroA gene was 1,819 bp and contained a 1,341-bp open reading frame encoding a 47-kDa protein. Phylogenetic analysis showed that Gr5_aroA is a class I EPSPS even though most such enzymes are naturally sensitive to glyphosate. Interestingly, Gr5_aroA protein contained highly conserved PEP and S3P binding residues (Glu-351) and several motifs insensitive to glyphosate. Transgenic Gr5 _aroA plants (T ₀) grew normally and produced seeds which we treated with a high-glyphosate solution (4× recommended spray). Analysis of the T ₁ progenies showed that Gr5 _aroA was inherited at a Mendelian 3:1 segregation ratios and that glyphosate tolerance in T ₁ plants was unchanged. Our results show the Gr5 _aroA gene to be a promising candidate for the development of commercial transgenic crops with high glyphosate tolerance.     

Development of cotton transgenics with antisense <Emphasis Type="Italic">AV2</Emphasis> gene for resistance against cotton leaf curl virus (CLCuD) via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Cotton transgenics for resistance against cotton leaf curl disease using antisense movement protein gene (AV2) were developed in an Indian variety (F846) via Agrobacterium-mediated transformation using the protocol developed previously. A binary vector pPZP carrying the antisense AV2 (350 bp) gene along with the nptII gene was used. Transgenic nature of the putative transgenics was confirmed by molecular analysis. Shoots were induced on selection medium and subcultured on rooting medium containing IBA and 75 mg l^–1 kanamycin. Transgenic plants were recovered in 12–16 weeks from the time of gene transfer to establishment in pots. Preliminary analysis of the field-established plantlets was conducted by PCR. T₁ plants were obtained from T₀ seeds, the presence of the AV2 and nptIIgenes in the transgenic plants was verified by PCR and integration of T-DNA with AV2 into the plant genome of putative transgenics was further confirmed by Southern blot analysis. Several T₁ lines were maintained in the greenhouse. Progeny analysis of these plants by PCR analysis showed a classical Mendelian pattern of inheritance.     

Mutation by DNA shuffling of 5‐enolpyruvylshikimate‐3‐phosphate synthase from Malus domestica for improved glyphosate resistance

A new 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate‐tolerant plants. However, wild‐type MdEPSPS is not suitable for the development of transgenic glyphosate‐tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate‐resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site‐directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single‐site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate‐resistant crops.