Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean)

Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker. Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium. Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants. Transgenic character of tissues was confirmed by GUS assay and Southern analysis. Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium.     

抗氧化剂对农杆菌介导的大豆下胚轴GUS基因瞬时表达的影响

大豆(Glycine max)下胚轴作为大豆遗传转化的外植体材料,能快速高频再生不定芽。然而,在遗传转化过程中褐化影响基因转化效率。在该研究中,我们用含有GUS染色基因和hpt II(Hygromycin phosphotransferase II)筛选基因的农杆菌(Agrobacterium tumefaciens) LBA4404侵染大豆下胚轴,并用组织化学定位法测定了GUS基因的瞬时表达,以确定大豆的优化基因转化条件。结果显示,在共培养基中加入硫代硫酸钠、L_半胱氨酸以及二硫苏糖醇等抗氧化剂,可以有效地抑制大豆下胚轴在组培过程中褐化的发生,并大幅度提高农杆菌在下胚轴的瞬时表达率。这些结果说明抗氧化剂可以降低这种影响并有效提高基因转化效率。     

转基因培育抗除草剂水稻

以ｐＡＨＣ２０（含Ｂａｒ基因）和ｐＷＲＧ１５１５（含ＧＵＳ基因和潮霉素抗性基因）以及含Ｂａｒ基因和雪莲凝集素（ＧＮＡ）基因的ｐＣＡＭＢＩＡ３３００ ＲＧ为供体ＤＮＡ,选用水稻品系８７２０３、上农香糯及鄂宜１０５的成熟胚诱导出的愈伤组织及微不定芽为受体材料,分别采用基因枪和根癌农杆菌（ＬＢＡ４４０４,含ｐＡＬ４４０４）导入法进行基因转化;经抗性筛选、ＧＵＳ检测和ＰＣＲ分析。结果表明,外源基因已通过基     

草莓叶片再生芽及遗传转化系统的建立

本文从五个草莓品种中筛选了再生频率高的品种M14,并进一步提高其再生频率至100%,单叶盘再生芽数至10.0个;石蜡切片法观察其芽多起源于愈伤组织,少数由叶细胞脱分化后直接形成; 该再生体系用于根癌农杆菌菌株EHA105介导的基因转化,并以14.5%频率得到抗卡那霉素的抗性芽,初步鉴定是转化芽。上述结果表明高频率稳定的草莓品种M14 叶片再生芽及遗传转化系统已经形成,并可以用于转基因研究。     

植物激素和抗生素调节的转基因火炬松愈伤组织的生长和分化

包含质粒载体pBI121的农杆菌菌株LBA4404被用于转化火炬松的成熟合子胚。质粒载体pBI121含有胭脂碱合成酶基因的启动子驱动的新霉素磷酸转移酶基因（npt Ⅱ)和花椰菜花叶病毒的35S启动子驱动的GUS基因。器官发生的转基因愈伤组织和转基因的再生植株已经获得,并经GUS组织化学染色、聚合酶链式反应和Southern杂交分析证实。植物激素（BA/IBA）和抗生素对转基因愈伤组织的生长和分化的影响被研究。500mg/L羧苄青霉素和2mg/LBA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加54.2%,分化增加45.7%。500mg/L Claforan和2mg/L BA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加40.8%,分化增加38.7%。高浓度的植物激素和抗生素降低了转基因愈伤组织的生长和分化。实验结果有助于建立一个高效的农杆菌介导的火炬松遗传转化系统,也有助于未来针叶树的遗传转化研究。     

Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 g/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 g/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.NRCC No. 31491During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94).     

Expression of 10 kD Sulfur-Rice Prolamin Gene of Rice in Potato

Using 10 kD sulfur-rich prolamin gene of rice (PLG) as target gene, the authers constructed the expression vectors pBinLG and pBinLGP, which contained CaMV 35S promoter/PLG/NOS terminator, and Patafin Class Ⅰ promoter/PLG/NOS terminator respectively. They were transformed into Agrobacterium tumefaciens strain LBA4404 (pAL4404) by direct transformation method with incubating the leaf and tuber explants of potato (Solanum tuberosum L. ) with LBA4404 (pAL4404) and selecting in the medium containing 100 mg/L kanamycin, regenerated resistant plants were obtained. The NPT Ⅱ enzyme activity analysis, polymerase chain reaction (PCR), Southern blotting, Northern dot blotting and Western blotting demonstrated that the target gene was integrated into the genome of potato ceils and well expressed in the plant.     

Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.     

农杆菌介导GUS基因对多年生黑麦草转化的研究

通过检测愈伤组织中GUS基因的瞬间表达,研究农杆菌LBA4404/pCAMBIA1301介导多年生黑麦草的转化体系。通过对多年生黑麦草瞬间表达率的比较,确立了其遗传转化的最佳优化条件。研究发现,多年生黑麦草不同品种的转化率在25%~45%之间变化。多年生黑麦草遗传转化最佳优化条件是预培养10d的胚性愈伤组织、浓度为0.5~0.8OD的农杆菌菌液以及2d共培养时间。在共培养基中添加100μmol/L乙酰丁香酮能有效地提高植物瞬间表达率。两种侵染处理方法比较结果为滤纸滴加法比浸泡法更优。转化后对愈伤组织的干燥处理能抑制农杆菌过度繁殖,能改善愈伤状态,有利于提高转化率。     

A robust genetic transformation protocol to obtain transgenic shoots of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’

The genetic transformation of plants is an important biotechnological tool used for crop improvement for many decades. The present study was focussed to investigate various factors affecting genetic transformation of potato cultivar ‘Kufri Chipsona 1’. It was observed that explants pre-cultured for 2 days on MS2 medium (MS medium containing 10 µM silver nitrate, 10 µM BA, 15 µM GA3), injured with a surgical blade and co-cultivated with Agrobacterium tumefaciens strain EHA105 [O.D600 (0.6)] for 2 days results in maximum transient β-glucuronidase (GUS) expression. The addition of 100 µM acetosyringone in MS2 medium also increased rate of transient GUS expression in both the explants. Clumps of putative transgenic shoots were regenerated using the optimised culture conditions from leaf and internodal explants. The stable integration of T-DNA was established using histochemical staining for GUS and amplification of DNA fragment specific to nptII and uidA genes. Within the clumps, around 67.85% of shoots showed uniform GUS expression in all the tissues and about 32.15% shoots show intermittent GUS expression establishing chimeric nature. Uniform GUS staining of the tissue was used as initial marker of non-chimeric transgenic shoots. Quantitative expression of nptII transgene was found to be directly proportional to uniformity of GUS staining in transgenic shoots. The present investigation indicated that manipulation of culture conditions and the medium composition may help to get transgenic shoots with uniform expression of transgene in all the tissues of potato cultivar ‘Kufri Chipsona 1’.     

过量表达大叶补血草LgNHX1基因对拟南芥耐盐性的影响

利用植物表达载体pCAMBIA1301和农杆菌GV3101将LgNHX1(全长1 656 bp)基因在拟南芥中过量表达.在含30 mg/L潮霉素的培养基上筛选获得LgNHX1的纯合转化子,并对其进行了分子鉴定和耐盐性分析.结果显示,经PCR和RT-PCR鉴定,野生型植株(对照)没有出现扩增条带,而转基因株系有相应的扩增条带,表明LgNHX1的确已经整合到拟南芥的基因组中,并已正常转录.在不同盐浓度处理下,转基因株系生长情况好于野生型对照;转基因植株地上部分和根的干重、鲜重相对高于野生型对照,但差异没有达到显著水平;当盐浓度达到150-200 mmol/L时,两个特基因株系的Na+含量显著高于野生型,K+含量极显著高于野生型.以上结果表明,过量表达LgNHX1基因可能增强了拟南芥将Na+区隔化至液泡的能力,提高了转基因拟南芥的耐盐能力.     

Transformation and regeneration of transgenic aspen plants via shoot formation from stem explants

An Agrobacterium -mediated transformation procedure for aspen ( Populus tremula L.), involving the direct regeneration of shoot-buds from stem explants, is described. Disarmed Agrobacterium tumefaciens strain EHA101 harboring the binary plasmid pKIW1105 (which carries the uidA and nptII genes, coding for β-glucuronidase [GUS] and neomycin phosphotransferase II, respectively) was used for the transformation of stem explants. An incubation period of 48 to 72 h was found to be most effective in terms of transient GUS expression on the cut surface of the stem explants. Adventitious shoots regenerated after 2–3 weeks of culture in a woody plant medium (WPM) supplemented with TDZ (1-phenyl-3-[1,2,3-thiadiazol-5-yl]-urea, Thidiazuron) and carbenicillin. Three different kanamycin-based selection schemes were evaluated for optimization of transformation efficiency: (1) Kanamycin was added only to the rooting medium (5 to 6 weeks post-inoculation), or (2) to the regeneration medium 10–14 days after inoculation, or (3) after 2 days of co-cultivation. The third selection scheme was found to be optimal for adventitious shoots with regard to both the time required and the transformation efficiency, the latter being much higher than with the other schemes. Leaf samples from kanamycin-resistant shoots and plantlets were tested for GUS expression, and subjected to polymerase chain reaction (PCR) analysis of uidA and nptII genes. A Southern blot of the corresponding PCR-amplified fragments confirmed their authenticity and Southern blots of total plant DNA confirmed integration of the nptII gene into the plant genome.     

Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.)

We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.     

Seed-transmissible expression of mammalian metallothionein in transgenic tobacco

A binary plasmid was constructed to contain the mouse metallothionein c-DNA, the constitutive 35S promoter from cauliflower mosaic virus, the polyadenylation signal from the pea rbcS-E9 gene and several selectable markers. The plasmid was transferred to Agrobacterium tumefaciens and the leaf disc method was used to transform tobacco. Callus and shoots were regenerated in the presence of kanamycin and transformed plants were obtained. Southern, Northern and Western blot analysis demonstrated integration and expression of the metallothionein gene in transformed callus and transgenic plants. The gene is transmitted to and expressed in seed derived progeny as a dominant Mendelian trait.     

金鱼草基因转化和转基因植株再生

本实验采用根癌农杆菌ＬＢＡ４４０４（ｐ３５ＳＧＵＳＩＮＴ与金鱼草下胚轴切段共培养,将ＧＵＳ基因导入金鱼草细胞,通过不定芽发生途径获得抗Ｇ４１８再生植株．经ＤＮＡ／ＤＮＡ斑点杂交及ＧＵＳ活性原位组织检测初步证实外源基因ＧＵＳ已整合进金鱼草基因组并得到表达．     

Agrobacterium -mediated Transformation of Rice with Help of Bombardment

Oryza sativa L. ssp. japonica cv. Zhonghua 8, which is recalcitrant to infection of Agrobacterium tumefaciens (Smith et Townsend) Conn strain EHA105 with ordinary binary vector pCambia 1301, was transformed through Agrobacterium mediated transformation with help of bombardment. The transformation efficiency can be raised greatly. Single copy of gene insertion in the genome of transgenic rice plants was proved by Southern analysis and the expression of GUS gene was observed. GUS gene and hygromycin-resistant gene show 3∶1 segregation in progenies of the transgenic rice plants.     

Class I patatin基因5‘侧翼区驱动的GUS基因在马铃薯块茎中专一性表达

Binary vectors pPATIs (with partial signal sequence) and pPATI (without signal sequence) were constructed by fusing 1.4 kb 5' flanking regions of Class I patatin gene with GUS. Transient GUS expression was observed in in vitro tuber slices bombarded with pPATI. These constructs were then introduced into potato (cv. Desiree) via Agrobacterium tumefaciens transformation. Transgenic potato plants were confirmed by X-Gluc staining and PCR. Using in vitro tuberization system, GUS activities were assayed by fluorescence. It was shown that, in plants transformed with PATI-GUS, GUS specific activities were about 10-20 fold higher in tubers than in stems. Increased sucrose concentration could not induce PATI-GUS expression, but light enhanced PATI-GUS expression in cultured shoots.     

Agrobacterium-mediated transformation of rough lemon (citrus jambhiri lush) with yeast HAL2 gene

ABSTRACT: BACKGROUND: Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants. RESULTS: Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100 mg L-1along with 250 mg L-1 each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 muM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 mg L-1of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis. CONCLUSION: Rough lemon has been successfully transformed via grobacterium tumefaciens with beta-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated.     

Additional virulence genes and sonication enhance <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated loblolly pine transformation

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.