Adventitious shoot regeneration of Platanus acerifolia Willd. facilitated by Timentin,an antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation

The effects of Timentin and cefotaxime (Cef) on shoot regeneration of the London plane tree (Platanus acerifolia Willd.) and their use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were compared. Shoot regeneration was significantly reduced on the media with Cef at concentrations from 100 to 500 mg·L–1. Timentin showed negative effect on plant regeneration at concentrations of 100 and 500 mg·L–1; however, 300 mg·L–1 Timentin was shown to facilitate shoot regeneration significantly and the regeneration frequency increased from 64% (control) to 88%. Effective suppression of A. tumefaciens could be obtained with 500 mg·L–1 Cef, but plant regeneration was completely inhibited at this level. The A. tumefaciens on infected P. acerifolia leaf tissues was visually undetectable after three subcultures on a medium with 300 mg·L–1 Timentin. Con-sidering the effect of Cef and Timentin on plant regeneration and suppression of Agrobacteria, Timentin at 300 mg·L–1 is the pre-ferred application in A. tumefaciens-mediated transformation of P. acerifolia.

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.

Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low.The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.In this study,we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors,with 93.50%regeneration rate on average.Based on this,a simple and efficient Agrobacteriummediated genetic transformation methodwas established,without pre-culture procedure and concentration adjustment of hormone and AgNO_(3) in co-cultivation and selection media.Using this system,transformants could be obtained within 3.5–4.0 months.Average transformation frequency is up to 10.83%.The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.

Agrobacterium tumefaciens-mediated transformation of modern rose(Rosa hybrida)using leaf-derived embryogenic callus

Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.

Agrobacterium tumefaciens-mediated transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, CryⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma har- zianum with an efficiency of 60—180 transformants per 106 spores by using Agrobacterium tumefaciens-mediated trans- formation. Putative transformants were analyzed to test the presence of CryⅠA(b) gene by Southern blot. Most trans- formants contained a single T-DNA copy. RT-PCR analysis showed that the CryⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, sim- plicity of T-DNA integration, and genetic stability of transformants.

Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid

A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies (PLBs) has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics (including ampicillin, carbenicillin, cefotaxime, and cefoperazone), meropenem showed the highest activity in suppressing all tested A. tumefaciens strains (minimum inhibitory concentration [MIC] < 0.5 mg L-1, which is equal to minimum bactericidal concentration [MBC]). Meropenem, at all tested concentrations, except for 10 mg L-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector pIG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L-1 meropenem and 25 mg L-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.

Production of Transgenic Tall Fescue Plants with Enhanced Stress Tolerances by Agrobacterium tumefaciens-Mediated Transformation

In order to improve stress tolerances of turf-type tall fescue (Festuca arundinacea Schreb.), Agrobacterium tumefaciensstrain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidopsis thaliana wasused to transform mature seeds-derived embryogenic calli of four cultivars. A total of 112 transgenic plants were regeneratedfrom 32 independent lines and verified by histochemical detection of GUS activity, PCR assay and Southern hybridizationanalysis. The transformation frequency ranged from 0.92 to 2.87% with apparent differences among the cultivars. Stresstolerances of transgenic plants were enhanced, which was shown by the facts that transgenic plants had distinct growthsuperiority and significantly higher survival rate than non-transformed ones under high salinity and high osmosis stresses,and that relative electronic conductivity of in vitro leaves treated with low and high temperatures, dehydration and highsalinity stresses was 25-30% lower in transgenic plants than in control plants. In addition, it was observed that growth oftransgenic plants was inhibited due to constitutive overexpression of CBF1 gene under normal environmental conditions.

根癌农杆菌(Agrobacterium tumefaciens)介导培育富含叶黄素的基因工程小球藻(Chlorella vulgaris)的研究

采用基因克隆技术构建双元载体pCAMBIA2301-idi,通过电转转入农杆菌LBA4404中。利用根癌农杆菌介导的转化方法,将pCAMBIA2301-idi质粒的T-DNA区转入小球藻,以G418抗性基因(NPTⅡ)作为筛选标记,筛选出阳性转化子。通过PCR扩增表明idi基因和NPTⅡ基因已经整合到小球藻基因组中。测定转化子的生物量,结果表明大部分转化子的生物量与野生型相似。测定转化子小球藻干粉的叶黄素含量,发现转化子叶黄素含量最高达到0.84mg/g,与野生型相比提高了30.95%。进一步分析藻液中叶黄素的产量,发现转化子的叶黄素产量最高达到1.98mg/L,比野生型提高了36.77%。

Preliminary Studies on Establishment of Genetic Transformation System in Loblolly Pine

A transformation system was established for loblolly pine (Pimus taeda L ) mature zygotic embryos using Agrobacterium tumefaciens.The gene coding for the glucuronidase (GUS) gene was introduced into loblolly pine tissues andits transient expression was detected with histochemical staining. The influences of different genotypes. Agrobacterium concentrations. and cocultivation time on GUS expression and Kanamycin resistant callus and shoot regeheration were investigated. The results showed that the highest `GUS expression frequency (1 6.3%) and shoot regencration frequency(7.8%) wereobtained from genotype 9-1003 with, Agrobactemm concentration decreased 9 times and cocultivation time of 56 hours.respectively GUS expression was, obtained in all genotypes tested The successtul expression of the GUS gene in differentgenotypes suggested that it will be a useful transformation system for loblolly

哈茨木霉(Trichoderma harzianum)多种胞壁降解酶基因表达载体的构建及转化水稻

为提高水稻的抗病性,利用哈茨木霉(Trichodermahazianum)P1菌株的3个胞壁降解酶基因ech42、nag70与gluc78构建了7个植物表达载体,每个基因受独立的Act1启动子调控.构建的7个载体不仅包含3个外源基因的所有组合(A,B,C,A+B,A+C,B+C,A+B+C),而且具有双元载体本身携带的HPT基因与Gus基因,为研究不同T-DNA长度、不同基因组合与不同基因排列方向对植物遗传转化效率以及外源基因在转基因植株中表达的影响提供了一套比较完整的材料.利用本实验室的农杆菌高效转化体系,将所有组合的7个载体分别转入粳稻品种石狩白毛(OryzasativaLssp.Japonicacv.Ishikari-shiroge)中,共获得再生植株1800余株.对部分再生植株进行了PCR检测,证明96%的植株至少携带有外源基因中的一个,80%以上的植株整合有完整的外源基因片断.

Transformation CodA Gene to Lily Plants by Agrobacterium tumefaciences Mediated

Glycinebetaine(GB),an osmotic substance,could improve some stress tolerance in plants.CodA gene,originating from bacteria,could translate choline oxidase which stimulates the synthesis of GB in plants.To create lily lines resistant to heat,Belladonna lily and Yelloween lily had been transferred CodA gene through Agrobacterium tumefaciens.The bacteria harbored a binary vector carrying the hygromycin phosphotransferase,choline oxidase(CodA)and intron-containingβ-glucuronidase(Gus)genes were co-cultivated with lily bulb scales slides.The result showed that most the bulb scales had developed into bulblets in a regulator-free growth medium,while some expressed the hygromycin-resistance,heat tolerance and Gus gene expression.Among them,one line demonstrated primarily the transcription level expression through reverse transcription polymerase chain reaction(RT-PCR).Moreover,they were tested with the accumulation of GB which was evident that the transferred line had four times of GB volumes higher than that of wild type.The original evidence could open a right approach to enhance stress tolerance in lily plants.

耐盐芦苇高频遗传转化体系的建立

以耐盐芦苇(Phragmites australis)种子愈伤组织为材料,以根瘤农杆菌EHA105为媒介建立高效的芦苇遗传转化体系。结果表明:在潮霉素浓度为50 mg/L、6-BA浓度为0.8 mg/L时不定芽分化率达到最高。将沉默载体pFGC1008-GRPi通过根瘤农杆菌EHA105在芦苇愈伤组织中进行遗传转化,共获得8个抗性株系,转化率为3.9%。实时荧光定量PCR检测显示6个株系实现了基因沉默,表明该转化体系高效可用。

利用绿色荧光蛋白优化辣椒遗传转化体系

【目的】农杆菌介导的辣椒遗传转化较为困难,至今仍未建立高效转化体系。绿色荧光蛋白(GFP)基因是植物遗传转化中常用的报告基因,以GFP为报告基因,优化农杆菌介导的辣椒遗传转化体系。【方法】利用GFP表达系统,统计3个辣椒品种(‘HP’‘8214’和‘L55’)中3种不同外植体(子叶、下胚轴和Flamingo-bill外植体)的不定芽分化率、不定根分化率与荧光阳性率,探究农杆菌侵染浓度、侵染时间、预培养时间和共培养时间等因素对不定芽、不定根分化率及荧光阳性率的影响。【结果】3个辣椒品种的Flamingo-bill外植体不定芽分化率均显著高于下胚轴与子叶外植体,其中‘L55’Flamingo-bill外植体的不定芽分化率最高、达77.59%,故选用‘L55’Flamingo-bill外植体进行后续研究。4种农杆菌不同侵染浓度和时间组合下,‘L55’Flamingo-bill外植体均可产生不定芽、不定根及表达GFP的愈伤组织,当农杆菌侵染浓度为OD_(600)=0.05,侵染时间为30 min时,不定芽分化率和荧光阳性率最高,分别达48.39%、4.84%。在6种不同预培养与共培养时间组合处理下,Flamingo-bill外植体也均产生不定芽和不定根,预培养1 d、共培养1~2 d处理下的不定芽分化率和荧光阳性率最高,分别达48.44%、12.50%。最后对表达GFP荧光的不定根与愈伤组织进行PCR检测,结果显示GFP、Kan和Cas9基因在荧光阳性的组织中均被检测到,表明农杆菌介导的T-DNA插入成功且转化稳定。【结论】辣椒的外植体类型、农杆菌侵染浓度、侵染时间、预培养和共培养时间均对辣椒遗传转化效率有影响。以GFP为报告基因,筛选合适的转化条件可提高农杆菌介导的辣椒遗传转化效率。

Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein (GFP) in Carrot (Daucus carota L.) Callus

Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis (Southern and Western) of callus tissue (non-photosynthetic tissue) to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.

农杆菌介导的籼稻9311和华占遗传转化体系的研究

【目的】水稻转基因技术是推动水稻分子生物学研究和精准育种的重要工具。农杆菌介导法是水稻遗传转化的主流方法。然而受基因型限制,典型籼稻的转化效率仍然偏低,需提高其转化效率。【方法】以顽拗型籼稻品种9311和华占为受体材料,研究了不同大量元素、微量元素、有机物和碳源的组合,植物激素浓度,侵染培养基,乙酰丁香酮(AS)浓度和光周期对遗传转化效率的影响。【结果】在成熟种子组织培养试验中,MS大量元素、B5微量元素、N6有机物和麦芽糖为9311最优组合,华占最优组合为MS大量元素、MS微量元素、MS有机物和麦芽糖。诱导培养基中添加0.5mg/L的BAP或1.5mg/L的KT可显著提高9311和华占的组织培养再生率,可达70.0%以上。在侵染过程中,添加100μmol/LAS的转化效率高于200μmol/LAS。光周期实验显示9311和华占宜采用不同的光周期策略,9311在全黑暗条件下进行愈伤诱导和筛选,在全光照条件下进行分化,转化效率最高,达6.0%~6.4%,而华占在12h光照/12h黑暗条件下进行诱导、筛选和分化,获得的转化效率最高(5.0%~7.5%)。【结论】本研究优化了顽拗型籼稻9311和华占的组织培养和遗传转化体系,该体系也可以应用于其他相关籼稻基因型的遗传转化。

农杆菌介导水稻遗传转化的影响因素及应用研究进展

在水稻遗传转化过程中,农杆菌介导转化法与其他方法相比优势较多,比如转入的外源DNA结构完整及表达比较稳定、操作简便、转化率高等,已被广泛应用于转基因技术中。本文对根癌农杆菌介导水稻遗传转化的原理、影响遗传转化的因素进行了综述,并探讨了农杆菌介导转化法的应用前景。

农杆菌介导的葡萄风信子愈伤组织遗传转化体系的优化

葡萄风信子(Muscari spp.)为百合科葡萄风信子属植物的总称,该属植物多具有稀少的正蓝色花色,是研究单子叶植物花青苷合成机理的模式材料。为开发一种高效的葡萄风信子遗传转化体系,本试验以葡萄风信子常见种亚美尼亚(M.armeniacum)、栽培品种‘黑眼睛’(M.aucheri‘Dark Eyes’)为材料,用叶片和花蕾分别诱导获得愈伤组织,对愈伤组织的潮霉素敏感性进行测试,对根癌农杆菌介导的愈伤组织遗传转化体系进行优化。试验结果表明:葡萄风信子亚美尼亚和‘黑眼睛’愈伤组织的潮霉素临界浓度分别为150 mg·L^(-1)和120 mg·L^(-1)。GUS染色结果显示不同基因型、不同农杆菌菌株及不同功率超声处理的愈伤组织瞬时转化效率差异不显著。而液体培养的花源愈伤组织能获得更高的瞬时转化效率,为葡萄风信子遗传转化的最优受体。其最高瞬时转化效率为98.73%。试验共获得了209块抗性愈伤组织,经PCR和RT-PCR检测,阳性率可达90%。本研究为在葡萄风信子中快速有效的开展基因功能研究奠定了技术基础。

农杆菌介导将雪莲凝集素(GNA)基因转入籼稻单倍体微芽的初步研究

以含有双元载体（携带GNA基因，npt－Ⅱ基因）的根癌农杆菌菌系LBA4404转化籼稻单倍体无性系微芽Hu18，经共培养后在含G418的保存培养基中连续筛选G418抗性芽，抗性芽经生根培养基中壮苗获得单倍体转化植株。PCR分析证明GNA基因已进入到Hu18细胞中，抗虫鉴定表明转基因植株具有白背飞虱抗性。并探明了G418对单倍体微芽的致死浓度和时间，共培养时间对G418抗性芽产生的影响。

影响根癌农杆菌介导的木霉菌遗传转化因素分析

采用根癌农杆菌介导的转化方法,以木霉菌分生孢子为受体材料,对影响转化效率的主要因素进行了分析。结果表明,农杆菌菌株类型、初始菌液量、分生孢子浓度、共培养时间以及乙酰丁香酮的诱导等因素对转化效率都具有重要的影响。通过对这些因素的分析,基本得出了根癌农杆菌转化系统对木霉菌遗传转化的特点和规律,为将该转化系统用于其它丝状真菌的遗传转化提供了重要参考。

根癌农杆菌介导的哈茨木霉菌遗传转化的研究

利用根癌农杆菌EHA105进行了哈茨木霉Th-33的转化,在优化的转化条件下,EHA105对木霉菌的转化效率约45-100个转化子/106个孢子。利用该方法已建立了含4000多个转化子的木霉T-DNA插入突变体库。随机挑选24个转化子进行遗传稳定性分析,结果显示转化子经过5代无选择压力连续转接后都能在选择平板上正常生长;潮霉素抗性基因的PCR扩增和Southern blot分析表明,木霉转化子含有该基因,Southern blot进一步表明转化子多为单拷贝随机插入。该转化体系的改进将有利于木霉菌生防功能基因的克隆和作用机制的研究。