小麦抗白粉病相关基因的转化

利用玉米花青素苷合成调节基因C1-Lc作为报告基因, 通过瞬间表达后愈伤组织表面红色斑点的统计分析, 优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是2个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的2个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中, 使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株, 进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株, 转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明, 外源基因的导入不同程度上增强了植株的白粉病抗性, 表现为延缓了白粉菌的发育。利用玉米花青素苷合成调节基因C1-Lc作为报告基因,通过瞬间表达后愈伤组织表面红色斑点的统计分析,优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是两个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的两个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中,使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株,进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株,转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明,外源基因的导入不同程度上增强了植株的白粉病抗性,表现为延缓了白粉菌的发育。     

Genetic transformation of the commercial barley (Hordeum vulgare L.) cultivar Conlon by particle bombardment of callus

Commercial barley cultivars are difficult to transform because of the lack of an efficient regeneration system. By modifying certain components in the standard culture medium, we have developed a reproducible and more efficient regeneration system. Herbicide-resistant transgenic plants from barley (Hordeum vulgare L. cv. Conlon) were obtained using this medium. Embryo-derived callus was bombarded with pAHC25, which contains the screenable marker gus (#-glucuronidase) and the selectable marker bar (bialaphos resistance gene), both driven by the maize ubiquitin promoter (Ubi1) and followed by the nos terminator. Following bombardment, callus was transferred to callus-induction medium supplemented with 5 mg/l bialaphos for selection. Resistant calli were subsequently transferred to maintenance medium containing 5 mg/l bialaphos for further selection and finally transferred to regeneration medium with 5 mg/l bialaphos. Green shoots that developed on the regeneration medium were transferred to rooting medium containing 3 mg/l bialaphos. Eighty-five transgenic plants were obtained from 13 independent transformation events. Progeny tests showed Mendelian inheritance for the transgenes. This is the first report of the production of large numbers of transgenic plants from a commercial cultivar adapted to Midwestern US barley production.     

An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration

 A method for producing large numbers of transgenic wheat plants has been developed. With this approach, an average of 9.7% of immature embryo explants were transformed and generated multiple self-fertile, independently transformed plants. No untransformed plants, or escapes, were regenerated. This transformation procedure uses morphogenic calli derived from scutellum tissue of immature embryos of Triticum aestivum cv. Bobwhite co-bombarded with separate plasmids carrying a selectable marker gene (bar) and a gene of interest, respectively. Transformed wheat calli with a vigorous growth phenotype were obtained by extended culture on media containing 5.0 mg/l bialaphos. These calli retained morphogenic potential and were competent for plant regeneration for as long as 11 months. The bar gene and the gene of interest were co-expressed in T0 progeny plants. This wheat transformation protocol may facilitate quantitative production of multiple transgenic plants and significantly reduce the cost and labor otherwise required for screening out untransformed escapes. Received: 15 June 1998 / Revision received: 6 April 1999 / Accepted: 26 April 1999     

Microprojectile bombardment-mediated transformation of Lilium longiflorum

We have obtained transgenic lily (Lilium longiflorum) plants after microprojectile bombardment, using the Biolistics PDS 1000/He system, of morphogenic calli derived from bulblet scales, followed by bialaphos selection. Parameters which gave the highest transient uidA expression were used: a bombardment pressure of 1100 psi, a target distance of 6 cm and a 48-h preculture on medium with 3% sucrose. A total of 1800 morphogenic calli were co-bombarded with plasmids containing either the uidA reporter or PAT selectable marker genes. After bombardment, the calli were exposed to 2 mg/l bialaphos. Only 72 of the shoot-forming calli (4%) survived. The 72 shoot clusters produced 342 shoots on elongation medium containing 0.5 mg/l bialaphos. Only 55 plantlets survived subsequent exposure to 2.0 mg/l bialaphos. PCR analysis indicated that 19 of these plantlets contained the PAT transgene. Southern analysis of 3 of the plants indicated that all contained the PAT gene. Received: 21 March 1997 / Revision received: 8 July 1997 / Accepted: 7 August 1997     

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.     

提高小麦基因枪法转化频率的研究

用基因枪法将带Ｂａｒ－ＧＵＳ双标记基因的质粒较入普通春小麦品种中－６０６３４的幼胚盾片,并获基因植株。在轰击的经预培养３－４天的３４２块幼胚片再经筛选再生的植株中,经ＰＣＲ和Ｓｏｕｔｈｅｒｎ分析表明,     

Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

Genetic transformation of two species of orchid by biolistic bombardment

We report here the transformation of two species of orchid, Dendrobium phalaenopsis and D. nobile,by biolistic bombardment. Calli or protocorm-like bodies (PLBs) were used as target explants. Gold particles (1.0 microm) coated with plasmid DNA (pCAMBIA1301) encoding an intron-containing beta-glucuronidase gene (gus-int) and a hygromycin phosphotransferase (hpt) gene were introduced into the PLBs or calli using the Bio-Rad PDS-1000/He Biolistic Particle Delivery System. Calli and PLBs were then chopped up and pre-cultured in 1/2-strength MS medium supplemented with 0.4 M mannitol for a 1-h osmoticum treatment before bombardment. Immediately after bombardment, the calli and PLBs were transferred to 1/2-strength MS medium without mannitol for recovery. Putatively transformed plantlets were obtained by selection and regeneration on medium supplemented with 30 mg/l hygromycin. The highest efficiency of transformation was obtained when selection was conducted at 2 days post-bombardment. For D. phalaenopsis and D. nobile, respectively, about 12% and 2% of the bombarded calli or PLBs produced independent transgenic plants. Integration and expression of the transgenes were confirmed by Southern hybridization and Northern hybridization. No nontransformed plants were regenerated, indicating a tight selection scheme. However, separate incorporation of the gus gene and the hpt gene was observed, and in one transgenic line the gus gene was integrated into the genome of the transgenic plant, but not expressed.     

Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event

Elite white maize lines W506 and M37W were transformed with a selectable marker gene (bar) and a reporter gene (uidA) or the polygalacturonase-inhibiting protein (pgip) gene after bombardment of cultured immature zygotic embryos using the particle inflow gun. Successful transformation with this device did not require a narrow range of parameters, since transformants were obtained from a wide range of treatments, namely pre-culture of the embryos for 4-6 days, bombardment at helium pressures of 700-900 kPa, selection-free culture for 2-4 days after bombardment and selection on medium containing bialaphos at 0.5-2 mg l^-1. However, bombardments with helium pressures below 700 kPa yielded no transformants. The culture of immature zygotic embryos of selected elite white maize lines on medium containing 2 mg l^-1 2,4-dichlorophenoxyacetic acid and 20 mM L-proline proved to be most successful for the production of regenerable embryogenic calli and for the selection of putative transgenic calli on bialaphos-containing medium after transformation. Transgenic plants were obtained from four independent transformation events as confirmed by Southern blot analysis. Transmission of the bar and uidA genes to the T₄ progeny of one of these transformation events was demonstrated by Southern blot analysis and by transgene expression. In this event, the transgenes bar and uidA were inserted in tandem.     

Comparative analysis of transgenic tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) plants obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and particle bombardment

Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants.     

Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker

A selectable marker system for plant transformation that does not require the use of antibiotics or herbicides was developed. The selectable marker consists of the manA gene from Escherichia coli under the control of a plant promoter that encodes for phosphomannose isomerase, pmi. Only transgenic plants were able to metabolize the selection agent, mannose, into a usable source of carbon, fructose. Transgenic plants were produced efficiently after delivery by Biolistics™ of the pmi gene into maize and wheat tissues, with mean transformation frequencies of 45% for maize and 20% for wheat. Adjustment of the sucrose and mannose levels in the selection medium essentially eliminated escapes. Transgenic events can be identified as early as 2 months for wheat and 4 months for maize. A simple test, a modified chlorophenol red assay, was used for early identification of transgenic events expressing the pmi gene. Transformation frequencies for both crops exceeded those obtained with the bar and pat genes with selection on either Basta^® or bialaphos.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.) using root explants

An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes.     

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     

绿色荧光蛋白基因作为报告基因在水稻基因转化中的应用研究

本研究中 ,构建了含有编码绿色荧光蛋白的改进型基因质粒pJPM5。用基因枪法分别把pJPM5和另一带有绿色荧光蛋白基因的质粒pSBG70 0转入水稻TNG6 7愈伤组织。用South ern杂交法证实了转基因的存在 ,而且表明多数转基因植株含有 1到 8个拷贝的转基因。取 2个月的转基因植株上的叶片用于分析绿色荧光蛋白基因表达。用SLM - 80 0 0荧光分析仪定量测定绿色荧光蛋白。多数转基因植株具有很高的绿色荧光蛋白信号。虽然水稻植株有少量自发荧光 ,但是绿色荧光蛋白基因表达出的绿色荧光蛋白信号比植株的自发荧光强得多 ,其测定不会受自发荧光的太大影响。在荧光显微镜下观察到了绿色荧光蛋白基因的表达。借助观察分析绿色荧光蛋白基因的瞬时表达 ,本研究还发现基因枪法转化中 ,如果两枪的气压为90 0psi& 135 0psi,比两枪的气压都为 90 0psi或者 135 0psi更好 ,因其能使质粒进入更多的细胞。研究结果表明 ,绿色荧光蛋白基因可以作为水稻 (甚至小麦、玉米 )转基因研究中的报告基因。研究还显示 ,MAR序列能明显增强绿色荧光蛋白基因的表达能力 (这一结果在另文讨论 ) .     

Developing phosphinothricin-resistant transgenic sunflower (Helianthus?annuus L.) plants

Most investigations on genetic transformations of sunflower have used the neomycin transferase (nptII) gene as the selectable marker. We previously reported a PPT-based selection system for sunflower transformation that uses the bialaphos resistance (bar) gene as the selectable marker and 20 mg/l of phosphinothricin (PPT) as the selective agent. Sunflower (Helianthus annuus L.) variety Skorospeliy 87 was genetically transformed via Agrobacterium tumefaciens strain EHA 105 harbouring the binary plasmid vector pBAR. Two-day-old explants from mature embryos competent for direct shooting were used. Southern blot and ELISA experiments confirmed the stability of expression in two generations of transgenic plants. Transformed plants transferred to soil in the greenhouse exhibited resistance to the herbicide Basta^? at 3 l/ha.     

Stably transformed callus of wheat by electroporation-induced direct gene transfer

Fertile plants of wheat have been regenerated from protoplasts in several laboratories. The objective of this study was to develop a transformation system using protoplasts as target cells. Protoplasts were isolated from cell suspensions initiated from an anther-derived callus. The protoplasts were transformed by electroporation using pBARGUS or pBAS, both carrying the Basta resistance (BAR) gene. A total of 2,761 calli were produced from electroporation transformed protoplasts in 3 independent experiments. Six calli survived selective culture on 10 mg/l phosphinothricin (PPT), a concentration that completely inhibited the growth of non-transformed wheat callus. Five PPT resistant calli showed phosphinothricin acetyltransferase (PAT) activity, whereas the sixth probably was a mutant. The transformed wheat calli could tolerate PPT concentrations up to 2,560 mg/l. Southern blot analyses confirmed the integration of the BAR gene in wheat genomes. The integrated DNA sequence may have partially methylated and tandemly repeated at least once. These results demonstrate the production of stably transformed wheat calli by electroporation-mediated direct gene transfer into protoplasts.     

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.     

Agrobacterium-mediated transformation of Bangladeshi Indica rices

Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233. Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation. Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis. Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number. Mendelian segregation of transgenes occurred in T1 seed progeny.     

A high-throughput <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for the grass model species <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> L.

In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA((R)), while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T(0) plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.