商陆毛状根的诱导、培养及其皂甙的产生

发根农杆菌（Agrobacterium rhizogenes）R1601 感染商陆叶片外植体1周后,在其切口处产生毛状根,20d后产生毛状根的外植体比例达70%;毛状根可直接从叶片外植体叶脉处或从叶脉处产生的愈伤组织上产生。毛状根能在无激素的 MS培养基上自主生长,其呼吸速率比对照根提高85.6%。冠瘿碱检测和PCR扩增结果表明,发根农杆菌RiTDNA的冠瘿碱合成酶基因及其Ri质粒的rol 基因均已在商陆毛状根基因组中得到表达。毛状根中总皂甙含量约为自然根的154倍,但其多糖含量则仅为非转化根的70%。     

三裂叶野葛毛状根的诱导及其固体培养和液体培养

发根农杆菌（Agrobacterium rhizogenes）ATCC15834感染三裂叶野葛(Pueraria phaseoloides)叶片外植体20 d后产生毛状根,毛状根可直接从叶片外植体叶脉处或从叶脉处产生的愈伤组织上产生。感染35d后,约85%的叶片外植体产生毛状根。毛状根能在无外源生长调节剂的 MS固体和液体培养基上自主生长。PCR扩增结果表明,发根农杆菌Ri质粒的rolB和rolC基因已在三裂叶野葛毛状根基因组中整合并得到表达。与固体培养的毛状根相比,在液体培养基中培养的毛状根不仅生长迅速,也不会形成愈伤组织。在无外源生长调节剂的液体MS培养基中培养15d的三裂叶野葛毛状根的鲜重、干重、可溶性总糖含量及细胞内活性氧(ROS)含量分别为固体培养毛状根的1.59倍、1.18倍、5.25倍和1.16倍。     

新疆雪莲毛状根的诱导及其植株再生体系的建立

利用发根农杆菌R1601、R1000、LBA9402感染新疆雪莲的叶片、叶柄和根段外植体,诱导产生毛状根。毛状根接种量为2.8 g/L(FW)时,20d生长量可达66.7 g/L,黄酮含量达到干重的10.23%。冠瘿碱的检测和rolB基因的PCR分析表明,Ri质粒中的T_DNA片段已经整合到毛状根细胞的基因组中。预培养时间、外植体类型以及发根农杆菌的菌株属性对毛状根诱导有着重要的影响。其中预培养2 d的新疆雪莲根段外植体,经过R1601感染后,毛状根的诱导率可达100%。诱导产生的毛状根在附加生长素的液体培养基中,有少量愈伤组织产生。由毛状根再生的植株与雪莲外植体再生的植株在形态上无明显区别,但前者的黄酮含量仅为后者的53%。     

外植体龄和蔗糖浓度对黄瓜子叶产生毛状根的影响

研究了外植体龄和蔗糖浓度对发根农杆菌 R160 1介导黄瓜子叶产生毛状根的影响。结果表明 :以 10 d龄子叶外植体产生毛状根的能力最强 ,外植体的毛状根诱导率为 88.89% ;2 0 d龄子叶外植体的毛状根诱导率比 10 d龄子叶外植体降低 52 .86% ;30 d龄子叶外植体感染发根农杆菌R160 1后不产生毛状根。感染发根农杆菌 R160 1的黄瓜子叶外植体在不加或加 1%蔗糖的 MS培养基上的毛状根诱导率极低 ,子叶外植体逐渐变黄 ,腐烂 ;而培养基中添加 2 % ,3%或 4 %的蔗糖可显著提高子叶外植体的毛状根诱导率。黄瓜毛状根能在无外源植物激素的 MS液体培养基中自主生长。冠瘿碱的高压纸电泳检测表明毛状根已被 Ri T- DNA转化     

三裂叶野葛毛状根的培养及其葛根素的产生

发根农杆菌ATCC15834感染三裂叶野葛叶片外植体20天后,从其切口叶脉处产生的愈伤组织上产生毛状根。感染35天后约85%的叶片外植体产生毛状根。毛状根能在无外源生长调节剂的MS固体和液体培养基上自主生长,但在液体培养基中培养的毛状根生长更迅速,也不会形成愈伤组织。毛状根线粒体膜电势的荧光染色结果表明,液体培养的毛状根细胞线粒体的膜电势比固体培养的毛状根高11.8倍。PCR结果证实,发根农杆菌Ri质粒的rolB和rolC基因已在三裂叶野葛毛状根基因组中整合并得到表达。HPLC测定结果表明,三裂叶野葛毛状根中的葛根素含量约为对照根(种子萌发产生的幼苗根)的2.5倍,达1.190 mg/g.dry.wt;并比多年生葛根生药片的葛根素含量高6.7%。     

香石竹毛状根诱导、离体培养及其植株再生

为了探讨利用发根农杆菌遗传转化所产生的毛状根来创新香石竹种质的可能性,本文采用叶盘法,建立了发根农杆菌Agrobacterium rhizogenes对香石竹Dianthus caryophyllus L.叶片外植体的遗传转化及其植株再生体系。结果表明,发根农杆菌ATCC15834感染香石竹幼嫩叶片外植体12 d后,从叶片外植体切口中脉处产生白色毛状根,21 d后约90%的叶片外植体产生毛状根。所获得的无菌毛状根能在无外源激素的MS固体和液体培养基中快速自主生长。PCR扩增和硅胶薄层层析结果显示发根农杆菌Ri质粒的rol B和rol C基因以及冠瘿碱合成酶基因已在香石竹毛状根基因组中整合并得到表达。将毛状根置于MS+6-BA 1.0-3.0 mg/L+NAA 0.1-0.2 mg/L中培养15 d后产生淡黄绿色的疏松愈伤组织。愈伤组织不定芽分化的最适培养基为MS+6-BA 2.0 mg/L+NAA 0.02 mg/L,培养6周后不定芽分化率为100%;平均每个愈伤组织产生30-40个不定芽;将不定芽转至1/2 MS或1/2 MS+0.5 mg/L NAA的培养基中10 d后产生不定根,发育成再生植株。再生植株移植于栽培基质中20 d后,成活率达95%以上。     

美洲商陆毛状根诱导及其离体培养的影响因素

为了探讨利用美洲商陆毛状根生产其药用成分的可能性,研究了美洲商陆毛状根诱导及其离体培养的影响因素。结果表明,美洲商陆叶片外植体被发根农杆菌ATCC 15834感染约18 d后,从其叶片外植体形态学下端叶脉切口处产生毛状根,其中以预培养1 d,农杆菌感染20 min,共培养4 d时的毛状根诱导率最高,达到70%。PCR扩增和硅胶薄层层析结果显示,发根农杆菌Ri质粒的rol C基因以及冠瘿碱合成酶基因已在美洲商陆毛状根基因组中整合和表达。所获得的美洲商陆毛状根系都能在无外源激素的MS固体培养基上快速自主生长;其中以毛状根根系2的生长速度最快、分生侧根能力最强和根表面的根毛密度最高;毛状根根表面呈紫红色或呈白色。在供试的MS、1/2MS、B5和6,7-V液体培养基中,以无外源激素的MS培养基最适合美洲商陆毛状根根系生长。与无外源激素的MS培养基相比,6,7-V培养基更有利于毛状根中商陆皂苷甲的合成与积累。本文所建立的美洲商陆毛状根诱导及其离体培养的适宜条件为今后利用其毛状根株系的规模培养来生产其药用有效成分商陆皂苷甲奠定了实验和技术基础。     

三裂叶野葛毛状根的培养及其葛根素的产生

发根农杆菌ATCC15834感染三裂叶野葛叶片外植体20天后,从其切口叶脉处产生的愈伤组织上产生毛状根。感染35天后约85％的叶片外植体产生毛状根。毛状根能在无外源生长调节剂的MS固体和液体培养基上自主生长,但在液体培养基中培养的毛状根生长更迅速,也不会形成愈伤组织。毛状根线粒体膜电势的荧光染色结果表明,液体培养的毛状根细胞线粒体的膜电势比固体培养的毛状根高11．8倍。PCR结果证实,发根农杆菌Ri质粒的rolB和rolC基因已在三裂叶野葛毛状根基因组中整合并得到表达。HPLC测定结果表明,三裂叶野葛毛状根中的葛根素含量约为对照根(种子萌发产生的幼苗根)的2．5倍,达1．190mg/g．dry．wt;并比多年生葛根生药片的葛根素含量高6．7％。     

烟草毛状根多倍体诱导及其植株再生

为了提高烟草的烟碱含量,采用发根农杆菌遗传转化和人工染色体加倍技术,进行了烟草毛状根及其多倍体诱导、植株再生及其烟碱含量测定。结果表明,发根农杆菌ATCC15834感染烟草叶片外植体8 d后产生白色毛状根,15 d后所有叶片外植体产生毛状根。毛状根能在无外源激素的MS固体和液体培养基上自主生长。PCR扩增结果显示发根农杆菌Ri质粒的rol B和rol C基因以及冠瘿碱合成酶基因已在烟草毛状根基因组中整合并得到表达。烟草毛状根多倍体诱导的最适条件为0.1%的秋水仙素溶液处理36 h,其多倍体诱导率为64.71%。经秋水仙素加倍的烟草毛状根多倍体植株再生的最适宜培养基为MS+6-BA 2.0 mg/L+NAA0.2 mg/L。与对照(二倍体非转化植株)相比,烟草二倍体毛状根再生植株的顶端优势减弱,腋芽增多,叶片变窄;而烟草毛状根多倍体再生植株茎更粗,节间变短,叶色更深,叶片的宽度和厚度均较对照明显增大。根尖细胞染色体压片观察证实,所获得的烟草毛状根多倍体再生植株为四倍体,其根尖细胞染色体数约为4n=96。盆栽实验表明,烟草二倍体毛状根植株和多倍体毛状根再生植株比对照植株延迟约21 d开花。GC-MS检测表明,烟草毛状根多倍体再生植株的烟碱含量比对照及二倍体毛状根再生植株显著提高,分别约为对照及二倍体毛状根再生植株的6.90倍和4.57倍。     

黄瓜毛状根的诱导及细胞分裂素6-BA对其生长和形态的影响

含农杆碱型Ri质粒pRiA4b的发根农杆菌(Agrobacteriumrhizogenes)ATCC15834感染黄瓜子叶外植体5d后产生毛状根,毛状根可直接从叶片外植体叶脉处产生。感染10d后,约90%的子叶外植体产生毛状根。毛状根能在无外源生长调节剂的MS固体和液体培养基上自主生长。PCR扩增结果证实,发根农杆菌Ri质粒的rolB和rolC基因已在黄瓜毛状根基因组中整合并得到表达。挑选一无菌黄瓜毛状根系置于含不同浓度6-BA的MS培养基中悬浮培养来探讨细胞分裂素6-BA对黄瓜毛状根生长及其形态变化的影响。结果表明,细胞分裂素6-BA能促进黄瓜毛状根的生长及改变其生长形态。随着培养基中6-BA浓度的升高,黄瓜毛状根变得越来越短而粗,更少分枝。与对照相比,培养基中添加0·1~3·0mg/L6-BA能推迟毛状根最大生长峰的出现,降低其可溶性蛋白含量、SOD和POD酶活性;而黄瓜毛状根的乙烯释放高峰比其最大生长峰和SOD和POD酶活性高峰提前5天,但6-BA处理能降低毛状根的内源乙烯释放量。该结果表明细胞分裂素6-BA可能通过对乙烯生成和释放的调节来影响黄瓜毛状根的生长和形态并延缓毛状根的衰老。     

Transformation of Gynostermma pentaphyllum by Agrobacterium rhizogenes and Saponin Production in Hairy Root Cultures

Authors report here the establishment of an efficient transformation system for Gynosternrna pentaphyllum (Thunb.) Makino using Agrobacteriurn rhizogenes R1600. Hairy roots appeared on leaf explants 10 days after inoculation with the bacteria . Frequency of the explants transformed by R1600 was up to 94%. Transformation was confirmed by Southern analysis. Biomass of hairy root cultures suspended in hormone-free MS medium increased 9 times after 20 days of incubation. There was no callus formation on the hairy roots during suspension culture. Saponin content in the hairy root cultures was about 2 times as much as in the natural roots, saponins of the hairy root cultures were also released into growth medium as well.     

野葛毛状根离体培养与异黄酮生产

用发根农杆菌（Agrobacterium rhizogenes）R1601菌株感染野葛（Pueraria lobata(Willd.)Ohwi）叶片外植体后获得的毛状根,经连续4次继代培养,鲜重增加到最初的6.2倍。PH为5.5的1/2SH液体培养基较适合野葛毛状根的生长。与自然根相比,毛状根在无激素的1/2SH液体培养基中离体培养20d后,鲜重增加22.6倍,释放入培养液中的异黄酮量增加10.6倍。毛状根生产异黄酮的量略高于自然根,且根中含量明显高于愈伤组织及茎中异黄酮的含量。培养液体积影响毛状根的离体生长及异黄酮的释放。     

北玄参毛状根诱导及其植株再生

利用发根农杆菌A4和R1601感染北玄参(Scrophularia buergeriana)叶片外植体,诱导产生毛状根,产生的毛状根可在无激素的液体和固体MS培养基上快速生长。rol B基因的PCR检测表明,Ri质粒中的T-DNA片段整合到了北玄参毛状根的基因组中。毛状根在附加0.5 mg·L–1 6-BA及0.1 mg·L–1 NAA的MS固体培养基上形成绿色愈伤组织,之后形成不定芽,并获得再生植株;毛状根在附加0.5 mg·L–1 6-BA及0.02 mg·L–1 NAA的MS固体培养基上培养约15–20天可直接形成不定芽,且不定芽的诱导率达85%。     

Genetic transformation<Emphasis Type="Italic"> of Pueraria phaseoloides </Emphasis>with<Emphasis Type="Italic"> Agrobacterium rhizogenes</Emphasis> and puerarin production in hairy roots

An efficient transformation system for the medicinal plant Pueraria phaseoloides was established by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots could be obtained directly from the cut edges of petioles of leaf explants or via callus 10 days after inoculation with the bacteria. The highest frequency of explant transformation by A. rhizogenes ATCC15834 was about 70% after infection for 30 days. Hairy roots could grow rapidly on solid, growth regulator-free Murashige and Skoog medium and had characteristics of transformed roots such as fast growth and high lateral branching. Paper electrophoresis revealed that bacteria-free hairy roots of P. phaseoloides could synthesize agropine and mannopine. The polymerase chain reaction amplification of rooting locus genes showed that left-hand transferred DNA of the root inducing plasmid of A. rhizogenes was inserted into the genome of transformed P. phaseoloides hairy roots. The content of puerarin in hairy roots reached a level of 1.190 mg/g dry weight and was 1.067 times the content in the roots of untransformed plants.     

Genetic Transformation of Cucumis sativus by Agrobacterium rhizogenes

Hairy roots were obtained in vitro 10 days after inoculation of cucumber ( Cucumis sativus L. ) cotyledon explants with the strains of Agrobacterium rhiwgenes R1000 and R1601. The frequency of the cotyledon explants transformed by R1000 and R1601 was up to 87.5% and 88.9%, respectively. All hairy roots induced by the strains of R1000 and R1601 grew rapidly on solid hormone-free MS medium. The roots incited by A. rhizogenes R1000 could be divided into three phenotypes. The roots of phenotype Ⅰ were similar to the normal ones, but had more numerous lateral roots. Roots of phenotype m were much stouter and shorter, they elongated very slowly and were more highly branched than roots of phenotype Ⅰ . Roots of phenotype Ⅱ were of intermediate in appearance. However, the roots incited by A. rhizogenes R1601 appeared similar to phenotype Ⅰ roots incited by A. rhizogenes R1000. Transformation was confirmed by opine detection.     

褐脉少花龙葵毛状根的诱导、培养及其澳洲茄胺的产生

利用发根农杆茵的遗传转化和液体培养技术,研究了褐脉少花龙葵(solanum nigrum L.Var.Dauciforum)毛状根的诱导和离体培养及其澳洲茄胺的产生以及液体培养过程中培养基中N源和钙的消耗变化.结果表明.发根农杆菌ATCC15834感染褐脉少花龙葵叶片外植体5 d后产生毛状根.感染25d后,约90%的叶片外植体产生毛状根.毛状根能在无外源生长调节剂的MS固体和体培养基上自主生长.PCR扩增结果显示发根农杆菌Ri质粒的rilB和rolC基因已在少花龙葵毛状根基因组中整合并得到表达.所产生的毛状根能产生药用次生物质澳洲茄胺,其含量约为非转化植株根的1.3倍,达到582.05μg/g干重.少花龙葵毛状根液体培养0-5 d内处于生长迟滞期、5-15 d为快速生长期、15d后进入生长平台期.培养基的硝态氮和铵态氮在毛状根液体培养过程中被逐渐吸收和消耗,至培养15 d时铵态氮被消耗殆尽.而硝态氮仍剩余44.7%;培养基中钙的浓度在培养过程中虽逐渐降低,但在培养25d时仍未被完全消耗,其浓度约为起始浓度的43.5%.该结果为今后设计合适的培养基来规模培养褐脉少花龙葵毛状根生产药用次生物质澳洲茄胺提供了可能性.     

The Genetic Transformation of Pueraria as Medicinal Plant by Agrobacterium rhizogenes

Pueraria radix (the dried root of Pueraria plant) is known as a traditional Chinese drug. Hairy roots of Pueraria lobata (Willd.) Ohwi, P.lobata var. montana and P. phaseoloides (Roxb.) Benth. transformed by Agrobacterium rhizogenes (Riker et al .) Conn R1601 were developed directly from the surface of sterile leaves in vitro . The transformation frequency was 16.6%, 16.2% and 26.6%, respectively. All hairy roots in the three species displayed the typical phenotypes of rapid growth, highly branched and plagiotropism, and also exhibited hormone autotrophy and resistance to kanamycin.The genetic transformations were confirmed by opine paper electrophoretic analysis, rol gene PCR amplification and molecular hybridization.