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     

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.     

商陆毛状根的诱导、培养及其扼甙的产生

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%。     

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%。     

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。     

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。     

发根农杆菌诱导桑树毛状根体系的建立

应用发根农杆菌ACCC10060,以直接接种和共培养2种方法侵染桑树10 d龄子叶,并将2种处理的外植体分别接种于MS+AS(乙酰丁香酮,100 μmol/L)的平板,暗培养2 d后转接至MS+CS(头孢霉素,200 mg/L)平板培养3周,每3 d转接1次以除去其中所含的发根农杆菌菌体,结果2种侵染方法均成功诱导桑树产生毛状根,诱导效率分别为14%和17%.在无激素MS培养基上离体培养除菌后的毛状根,呈现旺盛的生长态势和典型的发状根结构特点.CTAB法提取毛状根基因组并进行PCR检测,结果扩增出了423 bp的rolB基因片段,表明Ri质粒的T-DNA已经成功整合到桑树的基因组中.     

新疆紫草毛状根的诱导及培养

将处于对数生长期(A600为0.5)的发根农杆菌MSU440、A4、R1000、15834、1025和R1601与新疆紫草子叶外植体共培养.结果表明:(1)发根农杆菌不同菌种对转化率有显著影响,供试6个菌种中只有MSU440菌株获得转化株.PCR及序列分析表明发根农杆菌Ri质粒的rolC基因已在新疆紫草毛状根基因组中整合并得到表达,转化率达4.5%.(2)子叶较真叶的不定根发生率高,且生根持续时间长.(3)在B5无铵无激素固体培养基上,毛状根分支多且根较长,达2~3 cm,毛状根鲜重月平均增殖达7~9倍,是固体培养毛状根的适宜培养基.(4)毛状根在MS无铵无激素液体培养基中培养12 d时,毛状根鲜重平均增殖达12倍,MS无铵液体培养基有利于毛状根的扩大生产.首次获得了激素自主、快速伸长生长、多分支、多根毛的新疆紫草毛状根株系,初步建立了新疆紫草毛状根诱导体系,为大规模培养、生产紫草素奠定了基础.     

木豆毛状根的诱导及其培养条件的研究

利用发根农杆菌LBA9402对木豆叶片直接进行诱导产生毛状根。本实验研究出诱导木豆毛状根的最佳条件是,以木豆叶片为外植体,于1/2MS固体培养基上预培养2~4 d,菌液浓度OD₆₀₀=0.6~0.8,浸染20 min,共培养3 d,诱导率为60.00%。在分子水平用PCR检测表明,发根农杆菌9402Ri质粒上的T-DNA成功整合进木豆毛状根的基因组中。     

不同理化因子对黄芩毛状根诱导的影响

目的:利用发根农杆菌1.2556诱导黄芩,得到毛状根.方法:采用共培养法诱导黄芩毛状根,研究不同外植体,不同预培养时间,不同菌液浓度,不同感染时间,乙酰丁香酮,抗生素浓度等条件对转化率的影响.结果:利用预培养2d后的茎段为转化材料,当发根农杆菌浓度在OD600值为0.5时感染10min,转化率最高.在菌液中或培养基中添加100umol/L,乙酰丁香酮可以提高黄芩毛状根的转化效率.培养基中加入250mg/L抗生素Cef能较好地抑制发根农杆菌生长.结论:用共培养法诱导出黄芩毛状根,并确定了最佳诱导条件,以提高黄芩外植体的诱导率.     

光对商陆发状根生长和PAP基因表达的影响

利用发根农杆菌菌株Ar1334与美洲商陆(Phytolacca americana)叶片外植体共培养转化体系,共获得58个发状根无性系(SL-1~58).以发根农杆菌Ri质粒TL-DNA上的rol C基因设计特异引物,对发状根进行PCR检测,得到了预期的560 bp目的片段,表明Ri质粒T-DNA整合到发状根基因组中.将筛选出的株系SL-7接种在MS培养基上分别置于光、暗条件下进行培养.结果发现:SL-7在暗培养条件下呈乳白色,具有多分枝、多根毛、无向地性等典型的发状根特性;在光培养条件下,发根呈粉红色,少分支且生长缓慢;以商陆抗病毒蛋白(pokeweed antiviral protein,PAP)cDNA片段为探针,分别对光、暗条件下的发状根进行Northern blot检测,发现光对PAP基因的转录具有一定抑制作用;将发状根粗蛋白提取液与TMV病毒液混合后,摩擦接种于心叶烟(Nicotiana glutinosa)离体叶片,发现暗培养的发状根粗蛋白提取液对TMV抗性明显提高.表明商陆发状根的生长及PAP基因的表达都受到光的负向调控.该结果为商陆发状根的规模化培养和PAP蛋白的离体合成优化体系的建立奠定了基础.     

The novel paclitaxel-producing system: establishment of Corylus avellana L. hairy root culture

Hazelnut (Corylus avellana L.), contains a valuable medicinal substance known as Paclitaxel®, which is one of the most effective anticancer drugs. The original plants produce negligible amount of paclitaxel; therefore, tissue culture techniques, especially hairy root culture, could be one of the most practical methods to enhance the amount of paclitaxel. The main goal of this study was to assess the induction of hairy roots in C. avellana. The effects of different strains of Agrobacterium rhizogenes including c58c1pRiA₄, K599, and 15834, and six culture media, MS (Murashige and Skoog), half-strength MS, quarter-strength MS, WPM (woody plant media), half-strength WPM, and quarter-strength WPM, were evaluated. The results showed that the maximum amounts of the rooted explants were obtained with c58c1pRiA4 strain in quarter-strength WPM medium. The investigations of explant type (leafstalk, petiole, lamina, and stem) and different propagation media (quarter-strength WPM, half-strength MS, and half-strength SH ((Schenk and Hildebrandt) medium) showed that the leafstalk was the most optimal explant for hairy root induction, and half-strength SH was the best culture medium for growth of the hairy roots in liquid medium. HPLC analyses confirmed the presence of paclitaxel (3.2 μg g⁻¹ (DW)) in hairy root extracts.

     

Induction of hairy roots and plant regeneration from the medicinal plant <Emphasis Type="Italic">Pogostemon Cablin</Emphasis>

An efficient transformation system for the medicinal and aromatic plant, Pogostemon cablin Benth was developed by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots formed directly from the cut edges of leaf explants or via callus stage 8 days after inoculation with the bacterium. The highest frequency of leaf explant transformation by Agrobacterium rhizogenes ATCC15834 was about 80% after infection for 25 days. Hairy roots grew rapidly on plant growth regulators (PGRs)-free Murashige and Skoog (MS) or 6,7-V medium and had characteristics of transformed roots such as fast growth and high lateral branching. The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. The hairy root line, PL6, grew very slowly in the first 8 days, then grew very quickly between day 8 and day 24. The optimum medium for callus induction of hairy roots consisted of 2.0 mg l⁻¹ benzyladenine (BA) and 0.1 mg/l α-naphthaleneacetic acid (NAA); while optimum medium for adventitious shoot regeneration from these cultures consisted of 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Adventitious shoots could be rooted on 1/2MS. Southern blot analysis confirmed that rol genes of TL-DNA of Ri plasmid was integrated with at least three copies into the genome of hairy roots- regenerated P. cablin plants. The results presented provide a solid foundation for production of patchouli essential oil from hairy roots or its regenerated plants and also provide possibilities for utilization of artifical polyploidization or chemical mutation of hairy roots for improving germplasm and breeding of a new cultivar of P. cablin.     

Optimization of induction and culture conditions and tropane alkaloid production in hairy roots of <Emphasis Type="Italic">Anisodus acutangulus</Emphasis>

In this study, an efficient transformation system for the medicinal plant Anisodus acutangulus was successfully developed and optimized using Agrobacterium rhizogenes. Three bacterial strains, A4, R1601, and modified C58C1 and three explant types, leaf blade, petiole, and stem, were examined. The highest transformation efficiency of 94.44% was achieved using strain C58C1 with stem explants. Over 20 independent hairy root lines were successfully established with strain C58C1 using stem explants, all of which contained the ro/B and ro/C genes as confirmed by polymerase chain reaction (PCR). Out of four media compositions, the liquid 1/2 MS medium was found the most suitable for hairy root growth. The maximum biomass of one hairy root line increased up to 80 times in liquid 1/2 MS medium after a 30 day culture period. Different hairy root lines displayed a varied capacity for tropane alkaloid production and the best hairy root line (T4) from the C58C1-stem combination produced up to 10.21 mg/g (dw) of hyoscyamine, which was about 1.5-fold higher than in the wild type plants. To our knowledge, this is the first report to demonstrate the production of tropane alkaloids in hairy roots of A. acutangulus.     

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％。     

发根农杆菌介导的长春花高效转基因体系的建立

以长春花幼叶为外植体建立了发根农杆菌介导的长春花高效遗传转化体系,主要技术环节为：用携带有基因表达载体的发根农杆菌R1000侵染幼嫩叶片,侵染的叶片外植体与发根农杆菌共培养2d,外植体移至除菌培养基除菌培养2～3周,切取外植体上诱导长出的毛状根置于筛选培养基上培养1-2周,最后对筛选出的阳性毛状根无性系进行扩繁。筛选出的阳性毛状根经GUS染色和PCR分子鉴定表明,该方法的发根诱导率和阳性转化率分别为82％±2．49％和100％。该转化方法所获得的毛状根系数量大、质量高、遗传稳定且所需时间短,明显优于现有的长春花遗传转化技术,是长春花遗传转化的高效便捷体系。     

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%。     

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.