乌拉尔甘草毛状根的诱导及离体培养

为获得甘草的药用成分和保护甘草野生资源,利用发根农杆菌ATCC15834、A4 侵染乌拉尔甘草幼苗、子叶、下胚轴,获得毛状根,建立了乌拉尔甘草毛状根培养系统,经PCR和毛细管凝胶电泳检测,在248 bp 有rolA 基因,在490 bp 有rolC 基因,证实发根农杆菌基因片段在甘草毛状根的基因组中整合并得到表达,其中无菌实生苗毛状根诱导率达79.5%,毛状根分枝较多,生长速度快,在不添加激素的1/2MS液体培养基中生长明显,培养25 天后,鲜重平均增殖达19.13 倍,乌拉尔甘草毛状根培养体系的建立为进一步利用生物反应器规模化培养甘草药用活性成分提供了参考。     

栽培型与野生型青蒿愈伤组织及毛状根的诱导

为了比较栽培型与野生型引种青蒿愈伤组织及毛状根的诱导差异,以2种类型青蒿种子为材料,在不加激素的MS培养基中诱导青蒿苗,并利用青蒿的根、茎和叶片在MS+6-BA 0.5 mg/L+IBA 0.5 mg/L诱导愈伤组织,发根农杆菌C58C1、ATCC15834和Accc10600诱导毛状根。结果表明青蒿愈伤组织诱导效率以根最佳,其次为茎和叶片。农杆菌C58C1和ATCC15834分别以青蒿的根和叶片的诱导率可达100%和70%;Accc10600菌种不能诱导青蒿叶片毛状根发生,但是可诱导根生出毛根的芽点。该研究为青蒿素的生物反应器规模化生产奠定了基础。     

刺五加毛状根诱导条件的研究

为研究影响刺五加毛状根产生的因素,以农杆菌R15834、R1601、C58C1为试验菌株,叶片、叶柄、幼茎为外植体,探讨发根农杆菌种类、菌液浓度、共培养时间和乙酰丁香酮(As)等因素对刺五加毛状根转化率的影响。结果表明菌株R15834以浓度OD600=0.4左右对刺五加毛状根的诱导能力较好;叶片诱导毛状根能力较好;共培养时间以4天左右为最佳;As浓度以400 μmol/L左右有利于促进刺五加毛状根的产生。试验对刺五加毛状根的诱导体系进行了比较系统的研究,为产业化生产毛状根工作提供参考。     

桑树毛状根生长动力学研究

为建立桑树毛状根的高效培养体系,采用液体摇瓶培养法,以培养30天的毛状根湿重为指标,对桑树毛状根的培养基及培养条件进行研究。结果表明,桑树毛状根的最佳培养基是pH 6.0的MS培养基,最优培养条件为26℃,130 r/min,暗培养。桑树毛状根的生长表现为“慢—快—慢”的趋势,呈“S”型曲线,生长21天左右进入稳定期。     

中国甘草的组织培养研究进展

概述了国内在甘草植株再生、快速繁殖和毛状根培养方面的研究进展情况。甘草组织培养的基本培养基为MS,诱导培养基主要附加激素为2,4-D、6-BA和NAA,其中6-BA(0.2￣1.0mg/L)必不可少;分化培养基以MS无机元素和B5的有机成分为基本培养基,并附加ZT、BA、KT,三者可单独使用或配合NAA使用;生根培养基主要是添加NAA(0.05￣0.01mg/L)。能分化成苗的外植体仅有下胚轴,而且分化率极低,仅为3%￣6%。快速繁殖方法有腋芽丛状茎生根成苗和带叶茎段直接生根成苗两种,其中带2叶茎段成苗繁殖系数最高,达到98%。发根农杆菌ATCC15834感染甘草下胚轴诱导产生毛状根效果最好,诱导率高达30%。毛状根的最佳培养条件是:50mg/LKNO3,100mg/LCaCl2·2H2O,0￣225mg/LKH2PO4,640mg/LMgSO4·7H2O,微量元素及有机物质采用B5水平,32.5g/L蔗糖,转速100r/min,pH6.2左右,通过毛状根的培养可获得原植物中含有的或者不含有的化合物。     

发根农杆菌介导的向日葵遗传转化

本研究首次报道了发根农杆菌介导的向日葵遗传转化和毛状根形成的优化条件。由发根农杆菌诱导向日葵子叶和下胚轴形成的根在含卡那霉素（200 mg/L，kanamycin）的选择培养基(1/2 MS)上选择，12 d后存活的根转到无激素培养基上能快速生长，经分子检测证实其为毛状根。同时着重研究了不同诱导条件对向日葵毛状根形成的影响。结果发现适用于毛状根形成的最优条件为菌株R1205，菌液浓度（OD₆₀₀）1.0，15 min感染，共培养3 d，培养基pH 6.5，30 μmol/L乙酰丁香酮（acetosyingone）。在此条件下，子叶和下胚轴的毛状根形成率分别达到23.7％和81.6％。     

罗汉果遗传转化受体再生体系的建立及发根农杆菌转化初探

以罗汉果品种“青皮果”试管苗的茎段和叶片为外植体,研究适合于遗传转化的受体再生系统,并进行了发根农杆菌转化的初步研究。试验结果表明茎段的再生能力强于叶片,且对农杆菌敏感,是进行遗传转化合适的受体材料。在MS基本培养基附加BA 1mg/L、KT 1mg./L、NAA 0.2mg./L、蔗糖30 mg/L培养基中,20d龄的茎段不定芽诱导率为100%,平均每个外植体诱导的不定芽数达到9.7个,移苗成活率90%以上;外植体培养前的割伤处理有利于提高出芽数;发根农杆菌转化茎段诱导出毛状根。罗汉果毛状根的诱导和培养将为罗汉果药用成分的研究和开发提供新的途径。     

发根农杆菌转化银杏等药用植物研究进展

利用发根农杆菌转化药用植物,并对毛状根进行离体培养,大量提取重要成分,是药用资源植物可持续发展的有效途径之一。讨论了发根农杆菌影响发根诱导的因素即转化组合的研究、转化条件的研究及影响发根生长量和有效物质含量的研究进展,并对其发展前景和今后的研究方向及重点进行了展望     

谷子毛状根诱导方法的建立与优化

为建立一种快速鉴定谷子基因功能的技术体系,本研究通过比较谷子外植体、品种、乙酰丁香酮、菌液浓度和共培养时间对发根农杆菌K599介导的毛状根诱导效率的影响,发现发根农杆菌浓度在OD600为0.5、以谷子芽尖为外植体、在含有100μmol L–1乙酰丁香酮的毛状根诱导培养基上共培养3 d时,可使毛状根诱导率高达80.24%。将GFP基因进行毛状根遗传转化,通过GFP基因的PCR扩增和GFP荧光观察结果分析,发现谷子毛状根的转基因效率大于70%。利用该体系对谷子SiDVL1和SiDVL3的亚细胞定位和SiNHX2、SiCBL4和SiCBL7基因功能进行了分析和鉴定。结果表明SiDVL1和SiDVL3在谷子毛状根中的亚细胞定位与在烟草叶片中的一致;SiNHX2、SiCBL4和SiCBL7转基因谷子的存活率显著高于空载体转化谷子。说明本研究建立了一种高效快速鉴定谷子基因定位和功能的方法。     

四倍体菘蓝毛状根的诱导及其植株再生

利用发根农杆菌A4和R1000感染四倍体菘蓝子叶,毛状根诱导频率和得根率分别达72.5%和27%以上.A4诱导四倍体菘蓝毛状根优于R1000,光照对毛状根的诱导起着重要作用,外源激素IAA能明显地促进毛状根的诱导作用.rolB、rolC基因的PCR分析表明,Ri质粒中的T-DNA片段已整合到毛状根细胞基因组中.毛状根能在不含激素的MS固体培养基上分化出不定芽,6-BA能促进毛状根不定芽的诱导;NAA促进不定芽生根,再生完整植株.     

Induction of Shikonin production in hairy root cultures of <Emphasis Type="Italic">Arnebia hispidissima</Emphasis> via <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated genetic transformation

Data presented herein provides a rapid and efficient method for Agrobacterium rhizogenes-mediated genetic transformation of Arnebia hispidissima for hairy root cultures as well as for enhancing Shikonin production. Etiolated explants viz. shoot tip, nodal, leaf and internodal segments were co-cultivated with Agrobacterium rhizogenes for induction of hairy root. Among the various explants employed, leaf explant showed maximum 70.7% response followed by shoot tip 48.3%, nodal segment 38.7% and internodal segment 9.3%. Integration of Ri plasmid rolB gene in the transformed hairy root cultures was confirmed by PCR analysis using forward (FrolB) and reverse (RrolB) primers of rolB gene resulting in the amplification of 0 ∼ 0.8 kb fragments. Medium compositions have been optimized for in vitro induction of Shikonin in hairy root cultures of Arnebia hispidissima. Hairy roots on hormonefree MS medium showed red spots in the older part of the tissues which turned white after a second subculture. Whereas hairy roots cultured on RC medium showed faster growth and produced large amount of Shikonin. The Shikonin content in transformed hairy root culture was estimated by recording absorbance at 620 nm and quantified against authentic sample of Shikonin. Shikonin content was estimated to be 0.85 mg g⁻¹ fresh weight of tissue at the end of the 50 days of culture. The results presented herein will help to design strategies for bridging the gap between ever increasing demand and supply of raw products necessary for obtaining Shikonin for cosmetic, dyeing, food, medicinal, and pharmaceutical industries.     

Production of phenolic compounds in hairy root culture of tartary buckwheat (Fagopyrum tataricum Gaertn)

Fagopyrum tataricum Gaertn (tartary buckwheat) is an excellent medicinal and nutrient-rich crop. It has a high content of rutin and other phenolic compounds. An experiment was conducted to investigate in vitro production of phenolic compounds from hairy root culture of tartary buckwheat. Hairy root growth was promoted by increasing culture time in MS medium. The highest hairy root growth reached up to 11.2 g/l dry weight at 18 d after placement. Transformation was confirmed by PCR using rol genes, rol A (304 bp), B (797 bp), C (550 bp), and D (1035 bp) genes which is transferred into hairy roots from the Ri-plasmid in Agrobacterium rhizogenes and is responsible for the induction of hairy root from plant species. Rutin, quercetin, (−) epicatechin, (−) catechin hydrate, gallic acid, ferulic acid, chlorogenic acid, and caffeic acid were identified both in hairy and wild type roots of tartary buckwheat. The main compound found in the both types of root was epicatechin followed by rutin. The concentration of phenolic compounds in the hairy roots of tartary buckwheat was several-fold higher compared with wild type roots of same species. Our results indicate that hairy root culture of F. tataricum is a valuable alternative approach for the production of phenolic compounds.     

农杆菌介导谷子成熟胚遗传转化体系的建立与优化

为建立一个稳定的谷子遗传转化体系,对216份谷子种质资源进行筛选,确定材料178成熟胚为受体材料,从植物激素浓度、防褐化剂、农杆菌菌液浓度、侵染时间、乙酰丁香酮（AS）浓度等方面对谷子再生及转化体系进行优化。结果表明,在MS培养基中加入生长素2,4-D 9μmol/L、细胞分裂素Kinetin 4μmol/L、硝酸银8mg/L,愈伤组织诱导率最高且能有效防止褐化。在侵染液和共培养基中加入100μmol/L AS,gus表达效果最好。农杆菌溶液OD₆₀₀为0.3~0.5,侵染15min,共培养3d时,抗性愈伤组织率最高。获得的转基因植株,经PCR检测及gus染色分析,确定外源bar基因已成功整合到谷子基因组中。初步建立了一套谷子遗传转化体系,为今后谷子遗传转化提供一些参考。