不同理化因子对发根农杆菌Ri质粒转化骆驼刺的影响

骆驼刺苗茎切段有很强的离体培养再生能力,用野生型发根农杆菌Ａ,菌株转化骆驼刺度轩茎段,并建立了发根转化体的培养体系。高压纸电泳显示转化组织可合成冠瘿。试验不同试验经条件及理化因子对提高转化率和加快发根生长的影响。结果表明,经过３天预培养及０．３ｍｏｌ／Ｌ甘露醇１２ｈ高渗处理后培养于ＭＳ培养基中可有效提高转化率。而２％～３％蔗糖,３ｍｇ／Ｌ,ＧＡ３,１０μｍｏｌ／ＬＣｕ＾２＋,１０ｍｇ／ＬＡｇ＾＋,     

青蒿发根生长及青蒿素生物合成动态的研究

从747条发根农杆菌ATCC15834转化的青蒿株系025发根中,筛选出7个生长较快的发根系,这7个系在生长速度和青蒿素含量上均有显著差异,其中发根系HR9青蒿素产率最高,达到每月3325mg/L。青蒿发根的生长量和青蒿素含量极显著高于未转化根和愈伤组织。青蒿发根在分批培养中没有明显的迟滞期,接种后第7天进入指数生长期,第11天生长最快,第20天进入稳定期。青蒿发根中青蒿素含量呈明显的“与生长相关”特性,在指数生长期,青蒿素含量缓慢下降,生长速度减缓后,青蒿素含量上升,发根生长停止后,继续延长培养时间,青蒿素含量也不再提高。在分批培养中,青蒿发根适宜的培养时间为21d。     

乙酰丁香酮对发根农杆菌遗传转化黄瓜的影响

乙酰丁香酮对发根农杆菌遗传转化黄瓜的影响施和平李玲潘瑞炽（华南师范大学生物系,广州５１０６３１）ＥＦＦＥＣＴＯＦＡＣＥＴＯＳＹＲＩＮＧＯＮＥＯＮＴＲＡＮＳＦＯＲＭＡＴＩＯＮＯＦＣＵＣＵＭＩＳＳＡＴＩＵＳＢＹＡＧＲＯＢＡＣＴＥＲＩＵＭＲＨＩＺＯＧＥＮＥ...     

发根农杆菌研究进展

ReviewofStudiesonObtainingTransgenicPlantsbyAgrobacteriumrhizogenesMediatedGeneTransferSystemZhouYanqingZhanggenfaYuanBaojun(DepartmentofBiology,HenanNormalUniversity,Xinxiang453002)②苑保军现在河南省周口地区农业科学技术研究所工作．在植物基因工程中,农杆菌质粒介导的基因转移系统[37]是比较完善与有效的基因转移方法。目前,在根癌农杆菌Ti质粒的结构、功能及其被改造为载体系统与应用等方面均已取得很大进展的情况下,与之同属于根瘤菌科的发根农杆菌及其所携带的Ri质粒开始被广泛研究。本文就发根农杆菌Ri…     

发根农杆菌转化大豆的研究

本文利用发根农杆菌感染大豆不同外植体,在成熟胚靠近子叶节部位诱导产生毛状根。经冠瘿碱检测表明,毛状根及由此产生的愈伤组织均有甘露碱存在,说明Ri质粒的T-DNA已整合到大豆的转化根及愈伤组织中。转化根再生实验表明,在含NAA和IAA8mg/L的MS培养基上得到不定根的分化,MS和B_3培养基及6％蔗糖对转化丛生芽的诱导有利。转化的丛生芽在MS基本培养基上进一步长成小植株。     

发根农杆菌转化龙胆再生植株的研究

本文利用具Ri质粒的发根农杆菌(Agrobacterium rhizogenes)通过叶盘法对药用植物龙胆(Gentiana manshurica Kitagawa)进行了转化实验,发根农杆菌15834感染龙胆,诱发产生毛状根,并得到再生的龙胆植株,冠瘿碱检测实验表明,再生植株显示甘露碱带(mannopine),说明发根农杆菌Ri质粒的T-DNA部分已转移到龙胆植物细胞中。再生的龙胆丛生苗可以在不含激素的简化培养基上快速无限繁殖,转化的龙胆植株明显具有发达的根系,且根部龙胆苦甙含量比对照高,从而为东北龙胆栽培事业的发展以及龙胆有效成份的工业化生产提供了新的途径。     

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1 ^pro-1 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1 ^pro-1 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture. Received: 25 November 1997 / Revision received: 26 May 1998 / Accepted: 15 June 1998     

Artemisia tilesii Ledeb hairy roots establishment using Agrobacterium rhizogenes-mediated transformation

An efficient and rapid protocol for the establishment of Artemisia tilesii “hairy” root culture is reported. Leaf explants of aseptically growing plants were cocultured with Agrobacterium rhizogenes A4 wild strain or A. rhizogenes carrying the plasmids with nptII and ifn-α2b genes. Root formation on the explants started in 5–6 days after their cocultivation with bacterial suspension. Prolongation of explant cultivation time on the medium without cefotaxime led to stimulation of root growth. The effects of sucrose concentration as well as of the levels of synthetic indole-3-butyric acid (IBA) and native growth regulator Emistim on the stimulation of A. tilesii “hairy” root growth were studied. Maximum stimulating effect both for the control and for transgenic roots was observed in case of root cultivation on the media supplemented with IBA—up to 7.95- and 9.1-fold biomass increase, respectively. Cultivation on the medium with 10 μl/L Emistime has also led to the control roots growth stimulation (up to 2.75-fold). Emistime at 5 μl/L concentration led to 5.46-fold mass increase in only one “hairy” root line. Higher sucrose content (40 g/L) stimulated growth of two hairy root lines but had no effect on growth of the control roots.     

Sesame hairy root cultures for extra-cellular production of a recombinant fungal phytase

Sesame (Sesamum indicum L.) hairy roots were transformed with a fungal (Aspergillus) phytase and their culture conditions were surveyed for the extra-cellular production of the recombinant phytase protein in shake flasks. Kanamycin resistance of sesame hairy roots was observed at 50 μg ml⁻¹ kanamycin sulfate and southern hybridization analysis confirmed the existence of the phytase gene in the hairy root genomic DNA. The continuous dark condition was more effective for both the root growth and phytase production than light. Slightly higher root growth was determined at 30 °C than 26 °C in Murashige & Skoog (MS) medium supplemented with 3% sucrose, while the final phytase production was greatest in MS medium with 5 or 3% sucrose at both temperatures of 26 and at 30 °C. Among the culture media used, full-strength MS medium was exclusively efficient for production of the recombinant phytase. Most rapid increase rates in both the root growth and phytase production were detected at the 4th week of the culture periods and thereafter their rates began to decrease. Our results indicated that 5–6-week culture periods may be necessary for the maximal phytase production. Western analysis revealed that even though the phytase proteins expressed were measured with greater activities in the liquid medium than in the root tissues, they were still retained in the tissues.     

发根农杆菌介导的药用植物遗传转化研究

利用发根农杆菌(Agrobacterium rhizogenes)诱导药用植物产生的毛状根具有生长迅速,合成能力强和遗传性稳定等优点,已成为一种新的培养系统。就影响发根农杆菌介导的药用植物遗传转化的因素作一概述。     

Genetic transformation of Rhamnus fallax and hairy roots as a source of anthraquinones

Hairy roots of Rhamnus fallax Boiss. were induced using Agrobacterium rhizogenes strain A4M70GUS. The culture established on Woody plant media (WPM) showed a typical hairy root phenotype: rapid growth, reduced apical dominance and root plagiotropism. Seven clones of R. fallax were selected on the basis of their differences in colour and the root branching. The growth of hairy root culture, measured through gain in fresh mass, was done under 16-h photoperiod or in the dark. An increase in anthraquinone (AQ) content was obtained in clones with yellow and less branched roots, like clone 1 [16.43 mg g⁻¹(d.m.)] and clone 7 [14.21 mg g⁻¹(d.m.)], compared with other analysed transformed and non-transformed tissue. This study presents the first report of successful transformation of any species from family Rhamnaceae by A. rhizogenes and analysis of AQ production in transformed tissue.     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

Optimized genetic transformation of Zanthoxylum zanthoxyloides by Agrobacterium rhizogenes and the production of chelerythrine and skimmiamine in hairy root cultures

Zanthoxylum zanthoxyloides is an endangered African tree producing numerous bioactive substances including antileukemic and antisickling agents. Here, the potential of Z. zanthoxyloides hairy root cultures was tested for the production of bioactive substances with limited natural resources. The efficiency of Agrobacterium rhizogenes LBA9402‐mediated transformation of leaf material was evaluated using different techniques. An optimal transformation frequency of 77% was obtained after 11 days by inoculating A. rhizogenes directly onto the central vein of 14‐week‐old leaves followed by a co‐cultivation period of 3 days. Different treatments in immersion mode (manual wounding, acetosyringone, CaCl₂, ultrasonication) never exceeded these results. A maximum growth rate of 0.37 cm/day was determined during the exponential phase. Liquid chromatography‐diode array detection analysis showed the presence of skimmiamine, sesamine, chelerythrine, and chelerythrine derivatives in Z. zanthoxyloides hairy root lines. The maximum production of skimmiamine and chelerythrine in 28‐day‐old hairy root cultures was 45 ± 2 and 107 ± 4 mg/100 g dry weight, respectively. The present results highlight the potential of Z. zanthoxyloides hairy root cultures for the sustainable production of skimmiamine and chelerythrine.     

除虫菊发状根的诱导及培养条件优化

以除虫菊（Pyrethrum cinerariifolium Trey.）无菌苗为外植体,研究除虫菊发状根的诱导、培养条件优化,并对发状根中的除虫菊素进行检测和生物活性测定。结果显示,乙酰丁香酮能促进除虫菊下胚轴和子叶发状根的诱导,当乙酰丁香酮浓度为150 μmol/L时除虫菊下胚轴和子叶的诱导率为对照的2.29倍和2.66倍,预培养6 d时,下胚轴发状根诱导率为对照的2.25倍,发根农杆菌A4的诱导率均高于ATCC15834,愈伤组织较适合发状根的诱导,愈伤组织侵染后适合在无激素的MS培养基上进行发状根诱导,250 mL三角瓶中添加50 mL MS培养基较适合发状根的生长。对除虫菊发状根进行PCR检测发现,发根农杆菌含有的Ri T-DNA的rolB基因已整合进入发状根基因组中。通过GCMS检测发现,愈伤组织中除虫菊素的6种成分均未检测到,而发状根中检测到瓜菊素Ⅰ、茉酮菊素Ⅰ和茉酮菊素Ⅱ 3种成分,发状根对粘虫的拒食作用明显优于愈伤组织。本研究为通过组织培养方式生产除虫菊素奠定了基础。     

Regeneration of plants from callus tissues of hairy roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi

INTRoDUCTIONThe hairy root disease is a patholOgical syndromeof dicotyledonous plants fOllowing wounding and in-fection with Agrobacterum rhjZOgenesI1]. The rhi-zogenicity is conferred to p1ant cel1s by a fragmentof DNA (Ri T-DNA), which is transferred from thelarge root--inducting (Ri) plasmid, haJrboured by thebacterium, to the genome, where it is stably inte-grated and expressed. Illtegration of a DNA segment(T-DNA) of pRi into the host genome 1eads to ac-tive proliferation of…     

Hairy roots induced by Agrobacterium rhizogenes and production of regenerative plants in hairy root cultures in maize

Hairy roots of maize were induced by infecting 15-d calli with Agrobacterium rhizogenes. The hairy roots cultured in hormone-free media showed the vigorous growth and typical hairy root features. The regenerated plants were produced from hairy roots in MS media supplemented with 1.6 mg/L ZT and 0.4 mg/L NAA. The PCR-Southern hybridization demonstrated that T-DNA had been integrated into the chromosome of regenerated plants. These authors contributed equally to this work.