双丙氨磷在橡胶树遗传转化中的应用

本研究以GV3101::pMP90RK为工程菌株,bar基因作为筛选标记基因,研究了双丙氨磷在橡胶树根癌农杆菌介导法转基因体系中对转基因愈伤组织的筛选效果,并利用该体系将橡胶树转录因子HbWRKY5转入橡胶树无性系热研8-79中。实验结果表明,3mg/L双丙氨磷可作为橡胶树转基因体系中抗性愈伤组织筛选的最佳浓度。本研究中共获得了3个抗性愈伤组织系,抗性愈伤经过体细胞胚发生共获得234个体细胞胚,经植株再生培养后共获得22株再生植株。对再生植株进行的PCR鉴定结果表明,所获得的再生植株均为转基因植株。因此,bar基因结合双丙氨磷的筛选体系可以作为橡胶树转化体系中的有效筛选体系。     

转基因油菜研究进展

随着分子生物技术研究的快速发展,油菜的遗传转化研究日趋成熟。2003年,转基因油菜在全球种植面积为352万公顷。油菜基因工程研究的重点主要放在转化的目的基因、筛选标记、再生体系的建立和遗传转化方法方面。目前油菜已建立了子叶、下胚轴、茎段、原生质体培养、小孢子培养的再生体系;用于油菜基因转化的方法有根癌农杆菌介导法、基因枪法、PEG法、激光微束穿刺法、显微注射法和花粉介导法等。在油菜转基因中所转化的目的基因趋于多样化,如:品质改良、抗病、抗逆、不育基因等;较常用的筛选标记基因是新霉素磷酸转移酶基因(NPTII);本文主要从油菜转基因的再生体系建立、筛选标记及转化方法方面进行综述,并对油菜转基因上存在的问题及前景进行了探讨和展望。     

牡丹再生及遗传转化体系构建的研究进展

组织培养和转基因技术是当今植物研究的热点。近年来,关于牡丹再生体系的建立仍存在再生体系不完善、再生效率低、生根难、褐化率高等问题。而转基因研究是以成熟的再生体系的建立为基础,因此,关于牡丹的转基因研究更为匮乏。为了建立高效的再生体系和遗传转化体系,本研究综述了国内外近年来关于牡丹组织培养的方法,分析了不同方法的诱导效率,同时提出了防止褐化,提高生根率的对策。     

雄性不育基因对棉花的遗传转化

利用TA29、A6、A9三启动子功能区与barnase基因融合构建的不育基因以农杆菌介导法对棉花下胚轴进行了遗传转化,通过胚状体途径获得了转基因再生植株.利用150 mg·L-1高浓度卡那霉素(Km)对转化初期筛选出的再生苗进行再次筛选,提高了转化株的选出率.通过PCR检测和Southern dot blot分析从转基因胚状体再生植株中获得了带有barnase不育基因的120株转基因植株.进行转基因植株生物学性状检测和观察表明,所获得的转基因植株对溴苯腈表现出了明显的抗性,并从不育基因转化植株中筛选出了具有明显不育特征的雄性不育株.     

科技大世界

转基因芥菜能消除土壤硒污染美国科学家试验证实,三种转基因印度芥菜吸收土壤硒污染的能力很强。他们认为,转基因植物清除土壤污染可能是一种高效廉价的办法。由美国加利福尼亚大学伯克利分校和美国农业部农业研究中心科学家在新一期《环境科学和技术》上发表论文说,野外试验地中生长的转基因印度芥菜,清除土壤硒污染的效果比在温室试验还要好,表明转基因印度芥菜有很强的适应能力。天然的印度芥菜吸收土壤中硒的能力就很强。美国科学家培育出了三种转基因印度芥菜,一种能增强三磷酸腺苷酶的生成,有助于将硒酸盐分解成对生物无害的物质。另…     

甘薯细胞工程和分子育种的研究现状

概述了甘薯细胞工程和分子育种的研究现状,主要包括细胞培养与植株再生体系的建立、体细胞杂交与有益种间杂种的生产、细胞诱变与同质突变体的筛选、基因工程与转基因植株的获得以及分子标记辅助育种……     

基因枪轰击转化棉花茎尖分生组织影响因素的探讨

以农大KB18的茎尖分生组织为受体材料,利用基因枪轰击法将植酸酶基因(PhyA)导入棉花茎尖分生组织,经过再生、筛选、分子检测等过程获得若干转基因植株。通过研究碳源、外源激素、活性炭、轰击距离和轰击次数以及茎尖的恢复培养与抗性筛选等,建立了较为完善的棉花基因枪茎尖转化体系。结果表明,植株再生率与再生培养基的成分,如激素的有无、活性炭的含量和碳源等有关,轰击距离以及筛选程序中卡那霉素的起始筛选时间、筛选浓度和浓度梯度等因素对转化周期和转化率有直接影响。     

农杆菌介导的马铃薯茎段遗传转化体系优化研究

以甘肃主栽品种的茎段为外植体,研究了不同转化条件(菌液浓度、感染时间、预培养及共培养时间)对转化效率的影响,以及选择压和培养基对抗性出芽的影响,初步建立了马铃薯品种“陇薯3号”和“台湾红皮”的遗传转化体系并获得了转化植株。卡那霉素生根筛选和PCR鉴定结果表明,再生的转化植株假阳性率比较高,要获得预期的转基因植株群体需加大遗传转化工作中转基因植株的数量。     

高粱转基因再生体系建立初探

为建立高粱转基因再生体系,本研究以XL52、‘三尺三’、M81-E、07-27、BJ-285幼穗和幼胚的愈伤组织及幼胚为材料,利用农杆菌介导法进行高粱转Bar基因转基因体系研究。结果表明:以XL52幼胚、XL52和M81-E幼胚愈伤组织、‘三尺三’幼穗愈伤组织为外植体转化都获得了抗性愈伤组织,通过分化培养只有XL52幼胚转化所获得的愈伤组织获得转基因苗。本研究成功获得了抗性愈伤组织,为以后高粱转基因再生体系建立提供借鉴。     

粳稻恢复系C418成熟胚转基因再生体系的建立

为了建立粳稻恢复系C418转基因再生体系,本研究以不同培养基、不同2,4-D浓度、不同KT浓度及不同6-BA浓度的不同组合培养基来诱导C418成熟胚愈伤组织,以获得最适C418诱导的培养基,然后以获得的愈伤组织利用农杆菌介导法进行转基因再生体系的建立。结果表明:NBD培养基为6种培养基中最适培养基;2,4-D浓度为2.0 mg/L时,诱导率最高;随着6-BA的浓度增加诱导率降低;KT对C418成熟胚愈伤组织的诱导没有影响。本研究成功建立了C418转基因再生体系。     

Linamarin content and genetic stability of cassava plants derived by somatic embryogenesis

A protocol was established for high frequency cyclic somatic embryogenesis for different varieties of cassava. An efficient plant regeneration system was developed for the high cyanogenic variety PRC60a. Linamarin content and linamarase activity were determined in various tissues of secondary somatic embryos and regenerated plants of PRC 60a. Both linamarin and linamarase activity were not detected in embryogenic callus, roots induced from callus and somatic embryo tissues. The stems and leaves of regenerated plants (in vitro) and storage roots and leaves of mature plants (in vivo), however, contained variable amounts of linamarin and linamarase activity whereas in the non storage root tissues (in vitro) only linamarin was detected. The present study suggested that the linamarin biosynthetic pathway may be absent or not switched on in the embryogenic callus and somatic embryos. The ploidy level and somatic chromosome number of the regenerated plants were found to be same as the source plants. The availability of this regeneration system would be useful not only for investigating cyanogenesis but also for genetic manipulation in cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plant regeneration from protoplasts isolated from primary calli using mature embryos of two Brazilian rice cultivars

A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regeneration of Fertile Plants from Cell-suspension-derived Small-cell Colonies of Lolium perenne (L.)

A defined method for the rapid establishment of cell suspensions releasing small-cell colonies and their regeneration to fertile plants is described. Cell suspensions were obtained from 10 mature, lengthwise-divided embryos. Five of the 10 suspensions regenerated green plants. Filtration of suspensions through a 150 μm mesh followed by regeneration was carried out 28 days after initiation of the suspensions, to demonstrate a rapid release of regenerable small cell colonies from cell suspensions using the protocol described. Two of the 10 filtered suspensions regenerated green plants from small cell colonies.     

Selection of regenerative genotypes from highly productive cultivars of alfalfa

Summary Seedlings from Canadian alfalfa cultivars Algonquin and Apica were selected on the basis of ability to regenerate plants from hypocotyl callus. A total of eleven genotypes were selected, resulting in regeneration frequencies of 4.5 and 11.7% for Algonquin and Apica, respectively. Regeneration capacity within these cultivars was also compared with cultivars and breeding lines for which regeneration frequencies have previously been documented. Two genotypes (A1 93 and Ap 20) exhibited prolific embryogenesis from hypocotyl and petiole callus and also regenerated plants from suspension culture. Flowering and seed set have been observed for regenerated plants.     

甘薯（Ipomoea batatas (L.) Lam.）及其近缘野生种原生质体的植株再生

对甘薯品种高系14号及其近缘野生种I.triloba L、和I.lacunosa L,进行原生质体植株再生研究。从离体培养植株的叶柄分离出原生质体,将其培养在含有0.05mg/L 2,4-D和0.5mg/L激动素(KT)的MS培养基中,从原生质体获得了高频率的愈伤组织。培养8-12周后,将直径达2—3mm的小愈伤组织转移到添加0.05mg/L 2,4-D的MS培养基上。转移3-6周后,将愈伤组织进一步转移到添加吲哚乙酸(IAA)和6-苄基嘌呤(BAP)的MS培养基上,一些愈伤组织再生出植株。未再生植株的愈伤组织进一步在MS基本培养基上培养,它们也再生出植株。本研究从I.triloba原生质体获得高频率的植株再生;首次从I.lacunosa原生质体再生出植株;从高系14号原生质体也再生出完整植株。     

高频率生菜植株再生及转化体系的建立

通过对两种生菜及其不同外植体在培养基上不定芽发生能力的筛选，得到“大湖３６６”叶切片不定芽发生率高达８７％的培养体系，并且丛生状不定芽可陆续发生。将叶切片与农杆菌共培养，对经ｋｍ筛选后所得再生株进行Ｘ－Ｇｌｕ染色，兰色反应阳性率为５０％。     

木薯淀粉分支酶SBEⅠ反义基因遗传转化木薯的研究

旨在获得转淀粉分支酶反义SBEⅠ基因的‘华南木薯8号’转基因植株,为利用转基因技术改良木薯淀粉品质打下基础。在建立了木薯从胚状体子叶到完整植株的再生体系的基础上,用块根特异表达启动子Sporamin驱动的木薯淀粉分支酶SBEⅠ反义基因,通过农杆菌介导法对‘华南木薯8号’进行遗传转化。共接种‘华南木薯8号’子叶517块,获得7株生长良好的转化再生植株,转化再生频率达到1.35%。经PCR检测,其中5株转化再生植株扩增出目的条带,初步证实木薯淀粉分支酶SBEⅠ反义基因已整合进了‘华南木薯8号’基因组中。通过农杆菌介导法可以将淀粉分支酶SBEⅠ反义基因导入到‘华南木薯8号’基因组中,获得了5株转基因植株。     

矮丛蓝莓茎段再生植株研究

以矮丛蓝莓‘Blomidon’品种的试管苗茎段为外植体,研究了不同的培养基、激素、pH值、暗培养时间、接种茎段部位对植株再生的影响以及不同封口材料对再生植株茎段繁殖的影响。结果表明：适宜的培养基为WPM;ZT 1.0 mg?L- 诱导茎段后植株直接再生效果最佳,ZT 5.0 mg?L-1+IBA 1.0 mg?L-1诱导茎段后植株经由愈伤组织再生效果最佳,再生频率分别为100%和91.67%;适宜的茎段再生pH值为5.0±0.2;适宜的暗培养时间为0 d或10 d;接种的最佳部位为茎段上部;适宜的封口材料为玻璃纸。     

An assessment of morphogenic and transformation efficiency in a range of varieties of potato (Solanum tuberosum L.)

Summary To provide a truly genotype-independent transformation system, it is necessary to be able to transform a wide range of potato genotypes. The ability to regenerate shoots in vitro was determined for 34 potato varieties using tuber disc explants. Following a culture regime used extensively in previous studies with the variety Desiree, half of the varieties could be regenerated from tuber discs and half could not. From a sample of varieties that could be regenerated from tuber discs, all but one variety gave transgenic plants. Twelve varieties were evaluated for the capacity to regenerate shoots from leaf and internode explants excised from in vitro grown plants. All of the varieties tested regenerated adventitious shoots. Leaf and internode explants from 5 varieties were subsequently used for transformation, and transgenic plants were produced from two potato varieties that did not give transgenic plants from tuber disc explants. Some varieties could not be transformed by either method, and will require modification of the in vitro regeneration and transformation system to be successful.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid     

Optimization of culture conditions for improved plant regeneration efficiency from wheat microspore culture

The production of haploid plants through microspore culture is a very important tool for plant breeding. However, progress in microspore culture for many species has been hampered by a number of factors that have resulted in low recovery of regenerated green plants. In this study, a series of experiments were conducted to increase the regeneration of haploid green plants from isolated wheat microspores. The use of different basal media and variations in media components resulted in the increased recovery (approximately double) of regenerated haploid wheat plants. Our findings demonstrate that CHB medium, in combination with 2,4-d, was a better medium for embryoids induction and plant regeneration than medium MC17 with either 2,4-d or PAA growth hormones. Wheat microspores cultured without ovary co-cultivation did not respond. Furthermore, high efficiency of microspore derived embryoids (up to 296 MDEs per 100 anthers) and green plant regeneration (up to 71 green plants per 100 anthers) were achieved by the use of gelrite instead of agarose as a gelling agent, and by the addition of media additives such as spent medium or MET.