影响农杆菌介导的木薯基因转化因素的研究

本文研究了影响农杆菌介导的木薯基因转化的因素。结果表明,供试的4个菌种中,LBA4404(pBin9GusInt)及LBA4404(pTOK)瞬时表达效果较好。对农杆菌的诱导处理能增强瞬时表达效果,外植体的预处理对瞬时表达无影响,而外植体的预培养显著降低瞬时表达。所有供试的木薯品种都能被农杆菌侵染,但外植体的类型及生理状况对农杆菌的侵染力影响很大,成熟胚状体的子叶(萌发15d)及试管苗完全展开的叶片对农杆菌亲和性最高。四种筛选剂(kanamycin、hygromycin、phosphinothricin及geneticin)均表现出剂量效应且能同步抑制芽器官发生、愈伤生长及芽切段生根。     

影响根癌农杆菌介导的木薯遗传转化因素分析

采用根癌农杆菌介导的叶盘转化法,以我国南方地区主栽木薯品种—华南8号的胚状体子叶为受体,对影响木薯遗传转化效率的主要因素进行了分析。研究结果表明,在木薯的遗传转化中,选用GV3101作为浸染外植体的农杆菌菌株,将感染时间和共培养时间分别控制在30~45 min和3~4 d、菌液浓度（OD₆₀₀）采用0.45、并添加200 μmol·L^-1的乙酰丁香酮（AS）均可明显提高其转化效率,但若对外植体进行预培养反而会降低其转化效率。利用该体系从453块外植体中共转化获得10株抗性再生植株,经PCR和Southern杂交检测,有8株木薯的基因组中已整合进了外源基因glgC336,转化率为1.77%。     

影响农杆菌介导植物基因转化的因素问题

简要综述了农杆菌介导的植物基因转化的机理及其特点,着重分析了影响农杆菌介导植物基因转化的各种因素。     

影响农杆菌介导的柑桔基因转化因素研究

以柑桔（ Ｃｉｔｒｕｓ） 中的枳壳、甜橙、柠檬、四季桔上胚轴为对象, 进行了农杆菌介导的柑桔衰退病病毒外壳蛋白基因转化影响因素研究, 最终将衰退病病毒外壳蛋白基因转入枳壳。研究结果表明： 以卡那霉素为选择试剂, 且外植体水平放置时, 枳壳的选择浓度为５０μｇ／ｍＬ, 其它品种为２０ ～３０μｇ／ｍＬ。外植体与农杆菌的共培养时间３ ｄ 最好, ７０ ～１００μｍｏｌ／Ｌ的酚类化合物（Ａｓ） 的添加对外植体Ｇｕｓ 瞬时表达阳性率有明显促进作用。转化所用外植体年龄以２０ ｄ 最好, 品种之间抗性芽产生率与Ｇｕｓ 阳性芽产生率明显不同。以质粒ＤＮＡＮｃｏⅠ酶切产物为探针, 进行了Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 分析, 结果表明外源基因已稳定整合到了枳壳植株的核基因组中。     

农杆菌介导的木本植物遗传转化

木本植物由于本身的特殊性使其遗传转化较为困难,但根据植物类型及其生长特点,选取不同的受体系统,采用农杆菌介导进行转化,已在多种木本植物上获得成功。本文综述了农杆菌介导的木本植物在转化方法、受体系统和转化机理方面的研究,对Ｖｉｒ 基因活化、外植体预培养、共培养方法等影响转化的一些因素及存在的问题进行了探讨。     

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。     

农杆菌介导的天绿香遗传转化影响因素的研究

通过农杆菌介导的方法将新疆准噶尔小胸鳖甲抗冻蛋白基因Mpafp149导入天绿香中,对影响农杆菌转化频率的各种因素进行了研究。结果表明农杆菌介导天绿香遗传转化的潮霉素最佳筛选浓度为8 mg·L^-1,预培养时间2 d,共培养时间2 d,菌液浓度OD₆₀₀为0.2～0.25,浸染时间为8 min时适宜天绿香不定芽的诱导,有利于天绿香转基因植株的获得。通过PCR检测,初步证明外源基因Mpafp149已整合到天绿香基因组中。     

农杆菌介导Bt基因遗传转化高粱

高粱是全球仅次于小麦、水稻、玉米、大豆和马铃薯等的重要作物之一。以高粱幼穗愈伤组织为转化受体,通过农杆菌介导法和含有抗潮霉素和gus基因的双元载体将杀虫晶体蛋白基因cry1Ab导入高粱品种115、ICS21B和5-27,经Hyg筛选共获得21个独立的转基因株系,52株转基因植株,平均转化率为1.9%。经PCR、Southern杂交和RT-PCR分析表明cry1Ab基因已整合入高粱基因组中并得到正确转录。Bt蛋白Westernblotting分析和ELISA定量测定显示,cry1Ab基因在转基因高粱植株中表达,但不同转基因植株表达量有差异。饲虫试验表明,转基因高粱对大螟(Sesamiainferens)具有一定抗性。     

农杆菌介导的玉米遗传转化

Several maize inbreds were transformed with Agrobacterium tumefaciens EHA101 (pGIH). Transgenic maize plants were obtained. Frequency of transformation of maize inbred Suyu No. 1 can reach 8.1%. Results of PCR and Southern blot analysis proved that T-DNA was stably integrated into the genome of maize. Staining with X-gluc confirmed the expression of GUS gene in maize cells. The band amplified by inverse PCR showed that the copy number of transgene in three transformants was single. After long term of subculture, some hygromycin resistant calli lost their regeneration ability. Although Southern blot probed the integration of gusA gene in their genome, GUS activity cannot be detected in those calli. Southern blot analysis of HpaII digest DNA showed that transgenic gusA gene was highly methylated.     

农杆菌介导籼稻优良恢复系bar基因的遗传转化研究

应用农杆菌介导转化体系,成功地将含有CaMv35s启动子启动的bar基因导入籼稻幼胚来源的愈伤组织,获得籼稻优良恢复系T461、R402和752三个品种（系）共47个抗除草剂Basta的转基因株系,Southem分析结果表明,转基因植株基因组中检测到bar基因的整合,转基因植株自交后代Basta除草剂抗性鉴定表现出分离,且大多数为1-2个整合位点的孟德尔方式遗传。结果表明,根癌农杆菌介导法可以有效且可靠地转化籼稻。     

Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration

Various aspects of transformation and regeneration processes were examined in efforts to improve the efficiency of production of transgenic cotton (Gossypium hirsutum L.). Green fluorescent protein (GFP) proved to be a valuable tool in elucidating the timing and localization of transient gene expression and in visualizing conversion of transient events to stable transformation events. By day 4 after infection, there was maximal transient activity in the cells at the cut edge of Agrobacterium-infected cotyledon disks. We were able to visualize conversion of some of these events to stable transformation by day 8. The effects of Agrobacterium strains, acetosyringone, and temperature on stable transformation were also evaluated. Strain LBA4404 proved to be significantly better than EHA105. Acetosyringone increased significantly the stable transformation efficiency in cotton. Cocultivation at 21°C, compared to 25°C, consistently resulted in higher transformation frequencies. GFP expression in stably transformed callus was useful in studying the efficiency of selection during early stages of culture. We found that the survival of individual callus lines on selection medium was influenced by their original size and initial transgene expression status. Larger-size calluses and calluses expressing the transgene (GFP) had a higher rate of survival. Survival could be improved by an additional two-week culture on medium high in cytokinin and low in auxin before transfer to a medium to induce embryogenesis. However, this treatment delayed embryogenesis. Various other important aspects of the regeneration process are described and an overall scheme for producing transgenic cotton is presented.     

木薯的组织培养和基因转化

木薯是重要的热带作物之一, 其块根富含淀粉, 是热带、亚热带地区近５ 亿人粮食的主要来源。简单介绍近年来运用生物技术改良木薯的研究进展。     

Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.)

Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.     

An experimental assessment of the factors influencing Agrobacterium-mediated transformation in tomato

Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pTOK233, which contained the GUS reporter gene and a kanamycin-resistance gene nptII, was employed for optimizing the transformation efficiency evaluated by a GUS gene transient expression level. Eight factors including explant types, explant size and source, the concentration of cytokinin, inoculation time, pH of inoculation and cocultivation media, bacterial concentration, acetosyringone concentration, and cocultivation duration were investigated in detail. This optimized protocol was then adopted to obtain transgenic tomato plants resistant to cucumber mosaic virus (CMV) mediated by Agrobacterium tumefaciens, strain LBA4404, carrying a binary vector pR-ΔGDD containing the kanamy cin-resistance gene and CMV replicase gene with GDD deletion. The presence of the CMV-RNA2 gene was confirmed by genomic DNA Southern blot analysis in all transformants analyzed. Field spray test showed that the transgenic tomato plants were resistant to 100 mg/l kanamycin. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 280–284. The text was submitted by the authors in English.     

Factors influencing T-DNA transfer in Agrobacterium-mediated transformation of sugarbeet

Agrobacterium-mediated transformation of sugarbeet (Beta vulgaris L.) was investigated for T-DNA transfer efficiency, using an intron containing -glucuronidase gene. Preculture and coculture of hypocotyl and cotyledon explants with acetosyringone upon infection was studied. Seven seed lots which included several hundred genotypes, were screened, and were all susceptible to T-DNA transfer but with variable frequencies. Cotyledon explants were more readily transformed than those from hypocotyls. Transformation frequency of hypocotyl explants increased with acetosyringone. Both preculture treatment and acetosyringone improved transformation in cotyledon explants. Callus assayed with fluorometric procedures confirmed that the GUS gene had been transferred into sugarbeet.Abbreviations BAP N6-benzylaminopurine - TIBA 2,3,5 triiodobenzoic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - AS acetosyringone - GUS gene -glucuronidase gene - MS Murashige and Skoog medium - NPTII Neomycin PhosphoTransferase II - MU 4-Methyl-Umbelliferone - UV UltraViolet light     

Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration

The effects of cocultivation with Agrobacterium tumefaciens, regeneration and selection conditions on the transformation efficiency of citrange (Citrus sinensis L. Osbeck×Poncirus trifoliata L. Raf.) have been investigated. Factors such as cocultivation period, preculture of explants, use of acetosyringone or feeder plates during cocultivation, cocultivation on a medium rich in auxins, postcultivation in darkness, and different kanamycin concentrations for selection were assessed. A 3-day cocultivation on a medium rich in auxins improved transformation frequencies, since it increased the number of dividing cells competent for transformation, at the cut ends of the explants. Exposure of explants to darkness for 4 weeks on selection medium resulted in further callus development and increased the regeneration frequency of transgenic shoots. Furthermore, this treatment drastically reduced the number of regenerated escape shoots. A transformation efficiency of 41.3% was achieved using the optimized transformation procedure. Received: 4 November 1997 / Revision received: 7 January 1998 / Accepted: 13 February 1998     

影响农杆菌介导白细胞介素-2基因转化番茄的因素研究

利用农杆菌介导法将白细胞介素-2基因（il-2）导入番茄中,对影响其转化的因素进行了分析。结果表明：农杆菌菌种（EHA105和C58C1）、外植体类型（子叶和下胚轴）、带有不同筛选标记（Kan^r、PPT^r、Hyg^r）的载体质粒几个因素对芽诱导分化及转化均有影响。实验共接种转化2018个子叶和下胚轴外植体,获得了47株抗性再生株,对其进行il-2的PCR扩增检测,有44株呈阳性。PCR-Southern杂交证实PCR结果可靠,显示il-2基因已导入到番茄中。     

Evaluation of factors influencing Agrobacterium-mediated spinach transformation and transformant selection by EGFP fluorescence under low-selective pressure

We developed an efficient Agrobacterium-mediated transformation protocol for spinach (Spinacia oleracea L.) that uses root-derived callus. Evaluation of this protocol was based on the systematic evaluation of factors that influence transformation efficiency. Four of the five factors that were tested significantly affected the transformation efficiency, including spinach cultivar, Agrobacterium tumefaciens strain and density, and the duration of co-cultivation. Transgenic spinach plants were generated based on optimized conditions, consisting of callus explants of the cultivar Gyeowoonae, A. tumefaciens strain EHA105 with OD₆₀₀ of 0.2, a co-cultivation period of 4 d, and 100 μM acetosyringone supplemented in the inoculation and co-cultivation media. After co-cultivation with A. tumefaciens, explants were cultured in low-selective and then non-selective conditions to enhance the growth of putative transgenic explants. Visualization of the fluorescent marker, enhanced green fluorescent protein (EGFP), was used to select transgenic explants at several stages, including callus, somatic cotyledonary embryo, and plantlet. The best results for fluorescence visualization screening were obtained at the somatic cotyledonary embryo stage. On average, 24.96?±?6.05% of the initial calli regenerated shoots that exhibited EGFP fluorescence. The putative transgenic plants were subjected to β-glucuronidase (GUS)-staining assay, phosphinothricin acetyltransferase (PAT) strip test, and molecular analyses to assess the transgene incorporation into plant genome and its expression. All EGFP-positive plants tested were confirmed to be transgenic by GUS-staining assay, PAT strip test, and molecular analyses. The transformation system described in this study could be a practical and powerful technique for functional genetic analysis and genetic modification of spinach.     

根癌农杆菌介导的芦荟遗传转化条件的研究

以美国库拉索芦荟(Aloe.arborescens)的横切薄层切片(transverse thin cell layer, tTCL)作为转化受体, 通过受体材料对抗生素的敏感性实验和Gus 基因瞬时表达率的研究, 找出了较适合的外植体转化条件。研究表明:芦荟对头雹霉素(cefotaxime) 和羧苄霉素(carbenicillin)不敏感, 而对卡那霉素(kanamycin)和潮霉素(hygromycin)敏感;用靠近顶芽的材料得到的横切薄层切片芽再生率高, 有较高的Gus 基因瞬时表达率;乙酰丁香酮(acetosyringone)在芦荟转化是不可缺少的, 对其转化有明显的促进作用。     

Agrobacterium－mediated transformation and assessment of factors influencing transgene expression in loblolly pine（Pinus taeda L.）

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.