‘光叶蔷薇’器官间接再生体系的建立及GUS基因转化的初步研究

以‘光叶蔷薇’（Rosa wichuriana ‘Basye’s thornless’）无菌苗的顶生幼嫩小叶为外植体,探讨了其愈伤组织诱导及植株再生的方法。结果表明,高浓度的生长素NAA能诱导外植体产生愈伤组织;由NAA诱导的愈伤组织在附加TDZ的MS培养基上,先暗培养再进行光照培养可直接分化出不定芽。诱导愈伤组织的最佳NAA浓度是7.0 mg/L、暗培养时间为10 d,而最佳分化培养基是MS + 5.0 mg/L TDZ + 30 g/L葡萄糖 + 2.5 g/L GEL,分化率达18.34%。以诱导产生的愈伤组织为侵染受体,初步建立了‘光叶蔷薇’GUS基因转化体系。农杆菌菌液浓度OD₆₀₀值为0.5、侵染30 min、共培养2 d、乙酰丁香酮的浓度为50 μmol/L是'光叶蔷薇’愈伤组织转基因的最优条件。     

农杆菌介导的抗菌肽A基因转入沙田柚影响因素研究

以沙田柚上胚轴为对象,进行了农杆菌介导的抗菌肽shivaA基因转入沙田柚影响因素研究。研究结果表明转化中抑菌抗菌素以羧苄青霉素为首选,最佳转化条件为菌液稀释至OD6000.5,浸泡时间10min,共培养3d,卡那霉素起始选择浓度50μg/mL。农杆菌再悬浮液与共培养基中加入150μmol/L阿魏酸可提高转化频率。PCR与Southern杂交证明,外源基因已转入沙田柚基因组中。     

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.     

根癌农杆菌介导的高效大豆遗传转化体系的建立

利用根癌农杆菌对来自大豆成熟种子的胚尖进行遗传转化,研究了影响农杆菌介导大豆转化的各种因素,建立了一套优化的大豆遗传转化体系。研究结果表明:菌株KYRT1比EHA105和LBA4404具有更强的侵染能力;较酸的共培养基(pH5.4)、较低的培养温度(22℃)均有利于提高转化效率;恢复培养和分步抗性筛选方式有利于提高抗性组织的存活率和分化率。同时应用这种优化的遗传转化体系,获得了7个大豆品系的转基因植株,转化频率为4.29%-18%。经过PCR和Southern分析证明外源的双价抗虫基因cryIA(c)和pta已经整合到大豆的基因组中。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of shoot apices of Kentucky bluegrass (<Emphasis Type="Italic">Poa pratensis</Emphasis> L.) and production of transgenic plants carrying a <Emphasis Type="Italic">betA</Emphasis> gene

Apical meristems of multiple shoots produced from axenic seedlings of Kentucky bluegrass (Poa pratensis L.) were used for Agrobacterium tumefaciens-mediated transformation. Transformation parameters were optimized for concentration of bacterial cells, duration of infection, and vacuum infiltration. The highest transformation frequency (1.42%) was obtained by infection with Agrobacterium suspension of OD₆₀₀ = 0.6 for 5 min, under a negative pressure of 0.5 × 10⁵ Pa. After co-cultivation, the herbicide-resistant plants were rooted and transplanted into flowerpots. Transgenic plants were confirmed by polymerase chain reaction (PCR) assay and Southern blot analysis. Using this transformation system, the betA gene encoding choline dehydrogenase and mutant als gene encoding the enzyme acetolactate synthase were introduced into three Kentucky bluegrass cultivars.     

‘光叶蔷薇’器官间接再生体系的建立及GUS基因转化的初步研究

以‘光叶蔷薇’(Rosa wichuriana‘Basye's thornless’)无菌苗的顶生幼嫩小叶为外植体,探讨了其愈伤组织诱导及植株再生的方法。结果表明,高浓度的生长素NAA能诱导外植体产生愈伤组织;由NAA诱导的愈伤组织在附加TDZ的MS培养基上,先暗培养再进行光照培养可直接分化出不定芽。诱导愈伤组织的最佳NAA浓度是7.0 mg/L、暗培养时间为10 d,而最佳分化培养基是MS+5.0 mg/L TDZ+30 g/L葡萄糖+2.5 g/L GEL,分化率达18.34%。以诱导产生的愈伤组织为侵染受体,初步建立了‘光叶蔷薇’GUS基因转化体系。农杆菌菌液浓度OD600值为0.5、侵染30 min、共培养2 d、乙酰丁香酮的浓度为50μmol/L是‘光叶蔷薇’愈伤组织转基因的最优条件。     

Factors affecting Agrobacterium tumefaciens-mediated transformation of peppermint

 Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic evaluation of co-cultivation parameters established that deletion of coconut water from the co-cultivation medium resulted in substantially increased transient β-Glucuronidase (GUS) activity, in both the frequency of explants expressing gusA and the number of GUS foci per explant (>700 explants). Co-cultivation on a tobacco cell feeder layer also enhanced A. tumefaciens infection. Enhanced transformation efficiencies were further facilitated by increased selection pressure mediated by higher concentrations of kanamycin in the medium during shoot induction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/regeneration medium and from 15 to 30 mg/l in rooting medium. Raising the concentration of kanamycin in media substantially lowered the number of "escapes" without significant reduction in plant regeneration. These modifications to the protocol yielded an average transformation frequency of about 20% (>2000 explants) based on expression of GUS activity or the tobacco antifungal protein, osmotin, in transgenic plants. Genetic transformation of peppermint has been enhanced to the extent that biotechnology is a viable alternative to plant breeding and clonal selection for improvement of this crop. Received: 7 December 1998 / Revision received: 27 April 1999 / Accepted: 14 May 1999     

A simple and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated procedure for transformation of tomato

We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 10⁸ cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and β-glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone.     

Factors affecting genetic transformation and shoot organogenesis of Bacopa monnieri (L.) Wettst

Efficient Agrobacterium tumefaciens mediated T-DNA delivery and subsequent shoot organogenesis has been achieved from Bacopa monnieri. Various factors influenced T-DNA delivery as evident from transient GUS assay. The transient GUS expression was significantly higher (97.7 %) in explants that were pre-cultured before bacterial infection on medium supplemented with 100 μM acetosyringone. Incorporation of acetosyringone into the co-cultivation medium also enhanced transient GUS activity. Explant injury with carborundum paper, co-cultivation period of 2 days and a bacterial density of 0.4 OD₆₀₀ showed higher transient GUS expression. Following co-cultivation, shoot organogenesis was achieved from leaf segments on basal Murashige and Skoog medium containing 58 mM sucrose. Supplementation of antibiotics (cefotaxime or carbenicillin) at > 250 μg/ml into the medium significantly promoted shoot organogenesis from leaf explants (71.5 % in control and > 83.0 % on medium containing 500 μg/ml of carbenicillin or cefotaxime). Stable transformation of regenerated shoots was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to reporter gene (uidA) and selection marker gene (nptII). The expression level of nptII gene in independent transgenic lines was studied using quantitative real time-PCR. Stable transformed shoots after rooting were successfully established in the pots.     

水稻悬浮细胞再生及根癌农杆菌介导转化的影响因素

已经成功报道的农杆菌介导的水稻遗传转化多以活力较高的胚性愈伤为材料,很少以水稻悬浮细胞作为受体．另外,利用农杆菌转化多数都是通过浸泡的方式进行侵染．本实验利用滴加浸染法进行农杆菌介导转化水稻悬浮细胞,探讨影响 DNA 转化效率的因素．研究显示,在转化前,将水稻悬浮细胞在愈伤诱导培养基上培养1～2周,诱导产生直径为2～3 mm的微小愈伤组织对转化非常重要.微小愈伤组织大小不应小于 2 mm;对悬浮细胞短时间培养不但会缩短植株再生时间,而且会提高转化效率．此外,侵染农杆菌的浓度、侵染时间和不同侵染方法也影响 T-DNA 插入基因组的效率．用 1 ml Ａ600值为 0.5 浓度的农杆菌悬液滴加在水稻悬浮细胞诱导的愈伤,培养3 d或直到可见农杆菌菌落,此方法可以得到较高转化效率．将再生的潮霉素抗性的转化植株在含有 50 mg/L 潮霉素的分化和生根培养基中筛选得到,并对转化植株 gus 基因的表达进行 PCR 检测.结果显示,用 Ａ600值为 0.5 浓度的农杆菌浸泡侵染 20 min和滴加浸染法,分别得到PCR阳性植株率为 70% 和92%.     

金山绣线菊遗传转化体系的建立

以金山绣线菊愈伤组织为受体材料,采用组织培养的方法,在附加不同浓度TDZ的1／2MS培养基上诱导培养,获得再生植株。在附加0．03mg·L^-1 TDZ的培养基上获得了92．5％不定芽的再生率,且再生芽发育良好。选择抗生素筛选试验的结果表明：金山绣线菊愈伤组织对潮霉素较为敏感,在培养基中添加浓度为5～35mg·L^-1的潮霉素均对愈伤组织分化影响较大,潮霉素浓度为5mg·L^-1时,4N时间可使外植体全部褐化死亡;在培养基中添加0～100mg·L^-1的卡那霉素,不同浓度卡那霉素均对愈伤组织分化产生一定程度的影响,当卡那霉素浓度为80mg·L^-1时,愈伤组织基本不发生分化。由此确定卡那霉素为绣线菊遗传转化中适用的选择抗生素,最适选择压为80mg·L^-1。抑菌抗生素的筛选试验结果表明：200mg·L^-1的头孢霉素和200mg·L^-1。的羧苄霉素都能有效抑制农杆菌菌株LBA4404的生长,却对金山绣线菊愈伤组织的芽分化影响不大,可确定为适宜的抑菌抗生素。利用农杆菌介导法对金山绣线菊愈伤组织进行遗传转化,得到卡那霉素抗性植株164株,并初步确定预培养1d、菌液稀释10倍、侵染4min、共培养2d为金山绣线菊最优遗传转化体系,为金山绣线菊的基因工程育种奠定基础。     

Influence of Agrobacterium rhizogenes strains on hairy root induction and ‘bacoside A’ production from Bacopa monnieri (L.) Wettst.

Stable lines of hairy roots were established from leaf explants of Bacopa monnieri using different strains (A4, R1000, SA79, MTCC 532 and MTCC 2364) of Agrobacterium rhizogenes. The efficiency of hairy roots induction of these strains varied significantly and the maximum transformation frequency (75 %) was observed in case of strain SA79 using leaf explants followed by internode (55 %) in the presence of acetosyringone. Different parameters such as cell density of Agrobacterium suspension, co-cultivation period and infection time influenced the root induction frequency. Maximum frequency of root induction was obtained with bacterial density of 0.6 OD₆₀₀, 2 days of co-cultivation period and 10 min of infection time. Integration of T-DNA in the genome of hairy roots was confirmed by PCR amplification of rolB gene. Elimination of Agrobacterium from the established root cultures was ascertained by amplifying the DNA fragment specific to 16S rDNA and virD gene. All lines of hairy roots except strain A4 induced showed higher growth rate and accumulated higher levels of ‘bacoside A’ than the untransformed roots. Maximum biomass accumulation (6.8 g l^?1) and ‘bacoside A’ content (10.02 mg g^?1 DW) were recorded in case of the hairy root line induced by strain MTCC 2364.     

Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.     

水稻双元细菌人工染色体载体系统转化体系的建立

普通双元载体己被广泛碰用于农杆菌介导的植物转化,但这类载体通常只能转移5～20kb的外源DNA片段;而双元细菌人工染色体（BIBAC）载休可以弥补普通双元裁体的不足,通过它已在烟草、番茄等双子叶植物中实现了大片段DNA（150kb）的转移。BIBAC载体在单子叶植物转化中的应用尚未见报道。面于单、双子叶植物间以及大、小片段转化间的转化体系存在明显差异,常规的农杆菌介导的水稻转化体系不能适应BIBAC系统转化的要求。因此,建立适于BIBAC系统的水稻转化体系是十分必要的。通过比较不同的受体材料,不同的预培养、其培养条件,不同的去除农杆菌及选择阳性愈伤的方式等对转化效率的影响,建矿了适合水稻BIBAC系统的转化体系。该体系的技术要点包括：以水稻品种H1493为转化受体：以含毒性辅助质粒pCH32的LBA4404菌株（HP4404）为侵染菌株;预培养的培养拱pH5．6：以N6A代替AAM悬浮农杆菌：侵染菌液浓度为OD600=1．0;共培养温度为24℃;采用过渡（Resting）培养除去农杆菌;采用二步法进行选择等。基于PCR检测、Southern印迹分析的结果表明,BIBAC载体所携带的插入片段及标记基因已整合到转化植株的基因组中。这个体系的建立为在水稻中利用BIBAC系统进行大片段DNA转化奠定基础。     

Enhancing the frequency of somatic embryogenesis following <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of immature cotyledons of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill.]

Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl⁻¹ hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl⁻¹ hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols for transformation of soybean should be established under both Agrobacterium and selection conditions.     

根癌农杆菌介导转化川草二号老芒麦胚性愈伤组织

以川草二号老芒麦成熟种子为外植体,经过对培养基的筛选和培养条件优化,建立了愈伤组织再生系统。转化载体为pCAMBIA1304质粒,其T—DNA上携有潮霉素抗性基因（hptII）和类产碱假单胞菌杀虫蛋白基因（ppIP）,经根癌农杆菌EHA105介导转化结构致窑、颗粒状、黄白色的胚性愈伤组织。通过潮霉素筛选和对抗性植株进行分子检测,获得了转基因植株。同时优化了农杆菌遗传基因转化的参数,建立了农杆菌介导的川草二号老芒麦程序化转基因方案。     

Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmeria nivea (L.) Gaud]

In this study, leaf midribs, the elite explants, were used for the first time to develop an efficient regeneration and transformation protocol for ramie [Boehmeria nivea (L.) Gaud.] via Agrobacterium-mediated genetic transformation. Sensitivity of leaf midribs regeneration to kanamycin was evaluated, which showed that 40 mg l^?1 was the optimal concentration needed to create the necessary selection pressure. Factors affecting the ramie transformation efficiency were evaluated, including leaf age, Agrobacterium concentration, length of infection time for the Agrobacterium solution, acetosyringone concentration in the co-cultivation medium, and the co-cultivation period. The midrib explants from 40-day-old in vitro shoots, an Agrobacterium concentration at OD₆₀₀ of 0.6, 10-min immersion in the bacteria solution, an acetosyringone concentration of 50 mg l^?1 in the co-cultivation medium and a 3-day co-cultivation period produced the highest efficiencies of regeneration and transformation. In this study, the average transformation rate was 23.25 %. Polymerase chain reactions using GUS and NPTII gene-specific primers, Southern blot and histochemical GUS staining analyses further confirmed that the transgene was integrated into the ramie genome and expressed in the transgenic ramie. The establishment of this system of Agrobacterium-mediated genetic transformation and regeneration of transgenic plants will be used not only to introduce genes of interest into the ramie genome for the purpose of trait improvement, but also as a common means of testing gene function by enhancing or inhibiting the expression of target genes.     

Highly efficient Agrobacterium-based transformation system for callus cells of the C3 halophyte Suaeda salsa

Genetic manipulation technologies have been limited in the halophyte Suaeda salsa L. due to the lack of an efficient transformation system. Here, we examined factors affecting transformation and developed an efficient transformation system at the cell level using S. salsa hypocotyl as starting material. S. salsa hypocotyl explants from 10-day-old seedlings were precultured for 2 days on a hygromycin (hyg)-free callus induction medium (CIM) and then inoculated with Agrobacterium tumefaciens suspension at a concentration of 0.5 at OD₆₀₀ for 5–10 min. After cocultivation with A. tumefaciens for 4 days in the dark, followed by selection on carbenicillin (carb) for 3 days, explants were placed on CIM containing 10 mg l⁻¹ hyg and 500 mg l⁻¹ carb with three to four consecutive subcultures for up to 45 days. β-Glucuronidase assays showed an average transformation frequency of 62.89%. Gene integration was confirmed by polymerase chain reaction analysis and Northern blot analysis. To our knowledge, this is the first study to show Agrobacterium-mediated transformation in the C₃ halophyte S. salsa.     

根癌农杆菌介导小麦幼胚遗传转化的影响因素

利用携带pC3301质粒(含bar和gus基因)的超毒根癌农杆菌菌株EHA105对普通小麦(Triticum aestivum L.)扬麦158进行了遗传转化,对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、幼胚预培养时间、乙酰丁香酮(AS)浓度、洗涤用液及筛选方式等影响转化的几个重要因素进行了讨论.对从294个小麦幼胚外植体中转化得到的5株成活植株进行了PCR和Southern blot分析,结果表明其中2株小麦基因组中整合了外源DNA,转化频率为0.68%.