GFP基因转化藿香的研究

以藿香无菌苗幼嫩茎段为受体,利用根癌农杆菌介导法进行了绿色荧光蛋白基因(GFP)的遗传转化研究。经农杆菌侵染,通过共培养、选择培养后获得其抗性愈伤组织,对抗性愈伤组织的诱导过程进行了GFP荧光检测。结果表明,GFP基因能在抗性愈伤组织中强烈表达,证明GFP基因能够在藿香遗传转化中得到应用。对抗性愈伤组织的PCR检测初步证实外源GFP基因已整合到藿香愈伤组织的基因组中。     

GFP基因转化香樟胚性愈伤组织的研究

以香樟胚性愈伤组织作为受体,利用根癌农杆菌介导法进行了绿色荧光蛋白基因(GFP)的遗传转化研究。经农杆菌侵染后的胚性愈伤组织通过共培养、选择培养后获得抗性愈伤组织和体胚,对抗性愈伤组织及体胚的诱导过程进行了GFP荧光检测。结果表明,GFP基因能在抗性愈伤组织和体胚中强烈表达,证明GFP基因能够在香樟遗传转化中得到应用。对抗性愈伤组织的PCR检测初步证实外源GFP基因已整合到香樟胚性愈伤组织的基因组中。     

伪狂犬病毒gD基因转化马铃薯的初步研究

以可直接饲用的马铃薯作为伪狂犬病病毒糖蛋白基因gD的表达植物,探索用马铃薯茎段作为外植体的再生体系和遗传转化体系.结果表明:添加2 mg/L ZT和0.1 mg/L NAA的MS培养基,可使马铃薯茎段愈伤组织诱导率达70%,出芽率达38%;添加0.5 mg/L的硝酸银可明显减少愈伤组织在继代过程中的褐化程度,并且促进其分化.在农杆菌的菌液OD_(600)值为0.1～0.2,侵染茎段1 h,共培养3 d的条件下转化效果最好,抗性愈伤率达到52.3%.实验共获得21株转化植株,挑选7株进行RT-PCR检测,结果证明gD基因已被整合到马铃薯基因组中.     

诺丽茎段愈伤组织诱导优化及细胞悬浮系的建立

为获取诺丽茎段中的次生代谢物并为建立遗传转化体系奠定基础,以诺丽茎段(无腋芽)为外植体诱导愈伤组织,并建立细胞悬浮系,对影响愈伤组织的诱导及细胞悬浮系的因子进行了研究。结果表明:愈伤组织诱导的最优培养基是MS+1.0mg/L6-BA+0.1mg/L2,4-D;悬浮培养的最佳培养基为MS+1.0mg/L6-BA+0.1mg/L2,4-D+3%蔗糖,pH为5.85,当初始接种量为37.5g/L、摇床转速为110r/min且(25±2)℃暗培养时,悬浮细胞生长良好,生长速率最大;诺丽茎段悬浮细胞生长曲线呈"S"型,最适继代周期为12–20 d;培养过程中,培养基的pH呈先下降后缓慢升高的变化趋势,诺丽茎段愈伤组织悬浮细胞培养的最适pH在4.5–5.0之间。文中成功建立了以诺丽茎段为外植体的稳定的细胞悬浮系。     

PSAG12-ipt嵌合基因转化马铃薯体系的研究

以根癌农杆菌介导法将PSAG12-ipt嵌合基因导入马铃薯栽培品种,对影响马铃薯遗传转化的多种因素进行系统研究.结果表明:马铃薯茎段分化效率高于叶片,马铃薯愈伤诱导和芽分化最适培养基为MS+6-BA 0.25mg/L+NAA 0.25mg/L+2,4-D 0.25mg/L,添加1%Na2SO3能有效防止褐化;茎段愈伤诱导和分化苗生根最适的Kan浓度分别为50mg/L和75mg/L;外植体预培养2d,OD600为0.2～0.5的农杆菌浓度侵染8min、共培养3d后进行选择培养能有效地提高植株再生能力.用PSAG12和ipt双重PCR检测再生植株,阳性转化率为65.8%.Southern blotting结果表明,转基因植株多以单拷贝形式整合进马铃薯基因组中.     

环境激素DBP对拟南芥试管形态发生的影响

报道了酞酸酯类化合物DBP对拟南芥试管形态发生的影响。利用不同浓度的DBF处理拟南芥茎段外植体,切片观察拟南芥茎段试管形态发生过程中脱分化、分化及再分化的变化情况。结果发现：拟南芥茎段外植体在对照组培养基上生长良好,首先是愈伤组织产生,而后愈伤组织块增大,颜色呈淡绿色,生长旺盛,并有分化出芽现象;添加0．01mg／L、0．1mg／L和1．0mg／LDBP,茎段愈伤组织诱导率低,且高浓度时愈伤组织出现褐化、活力差的现象;培养12h、24h、2d、3d、7d、14d、21d和28d茎段显微切片结果显示,DBP具有抑制拟南芥试管形态发生的作用,随着DBP浓度的增加呈现剂量抑制效应。     

绿色荧光蛋白基因导入胡萝卜愈伤组织并获得表达

目的:将绿色荧光蛋白基因(green fluorescent protein,GFP)重组到胡萝卜愈伤组织细胞中,使其获得表达,为今后利用GFP基因作为植物报告基因提供条件。方法:通过冻融法将含有GFP基因的重组表达载体PBI1121转入到根癌农杆菌EHA105中,再利用根癌农杆菌介导的方法将GFP基因导入到胡萝卜愈伤组织细胞中,经过除菌和抗性筛选后观测转化结果。结果:荧光显微镜观测到被转化的愈伤组织在受蓝光激发后发出绿色荧光,利用PCR法扩增出约740bp的目的基因片断。结论:GFP基因在胡萝卜愈伤组织细胞中获得了表达。     

农杆菌介导的胀果甘草愈伤组织遗传转化

目的:旨在建立一个农杆菌介导的甘草愈伤组织遗传转化的可行方案,并对转化条件进行优化。方法:选择EHA105和LBA4404两种根癌农杆菌菌株,热激法转入含有绿色荧光蛋白GFP基因的植物表达载体pBI121-gfp,挑选转化的农杆菌用于侵染胀果甘草愈伤组织。设置不同培养时间的愈伤组织作为受体材料和农杆菌不同侵染时间两组条件,经共培养后的甘草愈伤组织进行荧光检测。结果:愈伤组织在含有100mg/l卡那霉素的继代培养基上进行筛选培养,得到了具有卡那霉素抗性的转化甘草愈伤组织,转化愈伤组织经继代培养基后在紫外光下仍可见绿色荧光,PCR检测转化愈伤组织基因组中含有gfp基因。结论:试验建立了农杆菌介导的甘草愈伤组织的遗传转化体系,为目的基因导入甘草细胞利用基因工程手段调控甘草次生代谢产物生物合成的研究奠定了基础。     

农杆菌介导小麦成熟胚愈伤组织转化的主要因子优化

目的：提高小麦成熟胚愈伤组织的转化效率,为建立快速高效的小麦遗传转化体系奠定基础。方法：以普通小麦HB341、SN2618成熟胚愈伤组织为受体,通过农杆菌GV3101/pBI121介导,进行选择剂选择压、外植体预培养时间、接种菌液浓度及侵染时间等遗传转化主要因子的优化研究。结果：最适宜的转化条件是：胚性愈伤组织在附加200μmol/l乙酰丁香酮的分化培养基上预培养3d,在OD600=0.6的接种菌液中侵染30min,然后在附加40～60mg/l卡那霉素的分化培养基上筛选培养30d,即可获得假阳性率很低的转基因小麦抗性芽。结论：该研究为快速高效小麦遗传转化体系的建立提供了依据。     

毛白杨高效转化系统的研究(英文)

毛白杨是一种具有多种优良特性、用途广泛并有重要经济价值的杨属树种,又是农杆菌的天然寄主植物。因此,毛白杨一直是进行木本植物组培及遗传转化研究的重要对象。本研究通过比较培养基组成(Table 1)、外植体类型(Table 2)、农杆菌侵染浓度以及预培养(Table 3&4, Fig.1)和共培养条件(Table 5)等影响农杆菌转化及植株再生效率的诸多重要条件后,建立了毛白杨的农杆菌高效转化系统。实验选用毛白杨叶片为外植体,在MS + 4.0 mg/L 2,4-D + 1.0 mg/L 6-BA培养基上预培养2 d后制成叶盘,并做戳伤处理,再与5×108 Cell/mL浓度的农杆菌新鲜菌液共培养2 d,然后以MS + 4.0 mg/L 2,4-D + 1.0 mg/L 6-BA附加50 mg/L Km和1 000 mg/L AP为筛选培养基(Table 6, Fig.4) ,获得最佳转化效果。2－3周后,在叶盘周围长出许多Km抗性愈伤组织,转入分化培养基分化后,再转入生根培养基3－4周,获得完整植株(Fig.3)。经PCR扩增实验鉴定,转化愈伤组织的阳性率达到83.8％(Fig.2)。多次重复实验结果表明,该转化系统与以往的文献报道相比,其遗传转化率大大提高。抗性愈伤组织的平均转化率高达56.2%,最终得到的再生转化植株的比率达19.9%。其中,毛白杨叶盘经戳伤处理,其转化率可提高至20~40% (Table 7)。利用此转化系统     

Green fluorescent protein as a reporter system in the transformation of barley cultivars

A cereal transformation vector, pN1473, containing the strong constitutive rice actin promoter Act-1 , a multiple cloning site, and the nos terminator, was constructed. Fusion of a plant-optimized gfp gene to Act-1 in pN1473 resulted in the vector pN1473GFP. To assess the suitability of pN1473, and GFP as a reporter system in barley transformation, two barley cultivars (Baronesse and Golden Promise) were transformed by microprojectile bombardment. Transient gfp expression in transformed embryogenic callus material was detectable by fluorescence microscopy less than 12 h after transformation. The presence of the gfp gene in callus and regenerated plantlets was confirmed by PCR amplification and DNA gel-blot analysis.     

Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea)

Agrobacterium tumefaciens-mediated transformation of callus culture, combined with a visual selection of GFP-tagged fimbrin actin binding domain (FABD₂) expression is described for parasitic species (Cuscuta europaea). The conditions for callus induction from 1 cm-long explants from the basal part of 7-day-old dodder seedlings were defined. We obtained light-green calli, which were transformed with A. tumefaciens bacterial strain GV3101 carrying plasmid pCB302 (35S::ABD₂:gfp) with neomycin phosphotransferase (nptII) gene. The limitations of selection procedures based on antibiotics were avoided using green fluorescent protein (GFP) detection, as a visual selection marker subcellularly targeted to the actin cytoskeleton. Fluorescence microscopy analyses demonstrated a network of nucleus-associated actin arrays and dense cortical actin arrangements in stably transformed Cuscuta callus cells. RT-PCR analyses confirmed gfp expression in transformed calli 7, 14 and 21 days after transformation. Although the GFP fluorescence associated with the actin cytoskeleton has retained for at least six months without silencing, no shoot regeneration was observed. It can be concluded that, C. europaea callus cells are competent for transformation, but under given conditions, these cells failed to realize their morphogenic and regeneration potentials.     

Detection of vector- and selectable marker-free transgenic maize with a linear GFP cassette transformation via the pollen-tube pathway

The pollen-tube pathway is feasible to transform vector- and selectable marker-free linear gene cassettes into plants to address the biosafety issues. However, its transformation frequency is low and the screening of selectable marker-free transformants by PCR analysis is time-consuming and expensive. In this study, a linear GFP cassette (Ubi-GFP-nos) flanked by 25bp T-DNA borders was transformed into maize via the pollen-tube pathway. The forepart of each maize ear was divided into five segments (segments I-V) at an interval of two rows of kernels. The segments that were most likely to contain transgenic kernels were identified by monitoring GFP expression in the immature embryos. A total of 21 ears were transformed with the linear GFP cassette. Seven out of 19 ears exhibited positive GFP expression in the immature embryos. Transgenic kernels were primarily identified in segments III and IV. A total of 121 plants derived from kernels located within segments III and IV of the remaining two ears were screened by PCR analysis. Six plants (4.96%) showed the presence of the GFP cassette. Southern blot analysis showed that the transgenic plants had simple integration patterns. The identification of transgenic kernels would facilitate PCR screening for marker-free transgenic plants.     

Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper

In order to establish a reliable and highly efficient method for genetic transformation of pepper, a monitoring system featuring GFP (green fluorescent protein) as a report marker was applied to Agrobacterium-mediated transformation. A callus-induced transformation (CIT) system was used to transform the GFP gene. GFP expression was observed in all tissues of T₀, T₁ and T₂ peppers, constituting the first instance in which the whole pepper plant has exhibited GFP fluorescence. A total of 38 T₀ peppers were obtained from 4,200 explants. The transformation rate ranged from 0.47 to 1.83% depending on the genotype, which was higher than that obtained by CIT without the GFP monitoring system. This technique could enhance selection power by monitoring GFP expression at the early stage of callus in vitro. The detection of GFP expression in the callus led to successful identification of the shoot that contained the transgene. Thus, this technique saved lots of time and money for conducting the genetic transformation process of pepper. In addition, a co-transformation technique was applied to the target transgene, CaCS (encoding capsaicinoid synthetase of Capsicum) along with GFP. Paprika varieties were transformed by the CaCS::GFP construct, and GFP expression in callus tissues of paprika was monitored to select the right transformant.     

Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes

Experiments were conducted to produce transgenic barley plants following infection of immature embryos with Agrobacterium tumefaciens. Transformed callus was obtained using hygromycin resistance as a selectable marker and either green fluorescent protein (GFP) or -glucuronidase (GUS) as a reporter. Significantly reduced plant transformation frequencies were obtained with the GFP gene compared to GUS. However, GFP proved to be an excellent reporter of early transformation events and was used to compare four barley cultivars for efficiency in two phases of transformation: the generation of stably transformed barley callus and the regeneration of plantlets from transformed callus. Transformed callus was generated at a high frequency (47–76%) in all four cultivars. Regeneration of transformed plantlets was also achieved for all four cultivars although the frequency was much higher for Golden Promise than for the other three genotypes, reiterating that genotype is an important determinant in the regenerative ability of barley. This study has demonstrated for the first time that Agrobacterium-mediated transformation can be used to transform the Australian cultivars Sloop and Chebec.Communicated by W. Harwood     

Transformation of sweet potato tissues with green-fluorescent protein gene

Summary The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1×10⁶ protoplasts ml⁻¹ was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm⁻¹. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

发根农杆菌介导丹参牻牛儿基牻牛儿基焦磷酸合酶1基因RNA干扰载体的转化

目的：利用发根农杆菌ACCC10060介导丹参牻牛儿基牻牛儿基焦磷酸合酶1基因（SmGGPS1）RNA干扰（RNAi）载体转化丹参叶片,生成SmGGPS1的RNAi转基因毛状根。方法：根据已克隆到的SmGGPS1特异区域设计并合成2段RNAi序列,分别插入RNAi双元载体pK7GWIWG2D（Ⅱ）中,构建2个含卡那霉素（Kan）和绿色荧光蛋白（GFP）双筛选标记的植物表达载体pK7GWIWG2D（Ⅱ）-SmGGPS1-RNAi2和pK7GWIWG2D（Ⅱ）-SmGGPS1-RNAi3;利用带有上述2个RNAi载体的发根农杆菌ACCC10060侵染丹参叶片,诱导生成转基因毛状根;通过Kan抗性筛选和GFP绿色荧光观察统计转化率。结果：分别得到SmGGPS1-RNAi2和SmGGPS1-RNAi3转基因毛状根301根和399根,平均转化率为60.34%。结论：首次建立了发根农杆菌介导的外源基因转化丹参的体系。     

板栗愈伤组织瞬时转化体系的优化与应用

愈伤组织瞬时转化体系可初步快速地验证基因功能和鉴定相关表型.为提高板栗愈伤组织瞬时转化体系的转化效率和稳定性,并鉴定板栗淀粉合成酶基因的功能,该研究以板栗'燕山红栗'幼胚胚芽诱导形成的愈伤组织为材料,通过不同状态的愈伤组织和表达载体优化板栗愈伤组织瞬时转化体系;并构建板栗淀粉合成关键酶基因CmSS Ⅰ沉默载体,验证Cm...     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.