Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.     

银杏毛状根褐化机制的初步研究

该实验依据现有发根农杆菌侵染法,以银杏无菌苗叶片为外植体,对农杆菌诱导银杏毛状根的实验操作进行优化,并观察记录银杏毛状根继代生长情况,测定继代不同天数的毛状根组织内多酚含量、多酚合成相关酶基因和POD基因相对表达量,以探讨银杏毛状根褐化的生理机制。结果显示:(1)银杏农杆菌侵染最适预培养时间为3d、菌种为R1601、共培养基为White、侵染时间为30min,优化侵染操作后的银杏毛状根诱导率可达72.36%;银杏毛状根继代生长的最适IBA浓度为0.5mg/L。(2)银杏毛状根初始发根时颜色为乳白色,多在伤口处簇生,伸长生长时无向地性,不分叶正背面都能在伤口处诱导出根,且在除菌培养20d时毛状根长可达0.4～0.8cm;继代培养25d时毛状根为白色,继代35d时部分毛状根从根部至中部开始转黄,继代45d时大部分毛状根由白转黄,根部至中部开始有褐化出现。(3)继代35d时毛状根的PAL、C4 H、4CL和POD基因表达量分别为25d的68%、44%、51%、52%;继代45d时PAL、C4 H、4CL、POD基因表达量分别为35d的19%、75%、86%、105%。(4)继代35d、45d的银杏毛状根总酚类含量分别为17.14和13.78mg/g。研究发现,PAL基因表达量变化与其下游的两个关键酶基因(C4 H、4CL)表达量变化不一致,且PAL基因表达量的大幅降低并未对下游C4 H、4CL基因表达有明显影响,说明PAL基因表达量变化对其下游合成速率影响不大,可能不是银杏毛状根苯丙氨酸代谢途径的限速酶;而C4 H与4CL基因表达量变化趋势高度一致,说明C4H应是该途径中的限速酶之一;推测继代前期可能是由PPO作为关键酶参与毛状根褐化反应,导致后期毛状根生长受抑制,POD基因表达水平随之上升,POD参与褐化反应。     

Ri质粒介导的毛状根体系建立及其在植物次生代谢产物合成中的研究进展

发根农杆菌Ri质粒可诱导植物产生毛状根体系,该体系具有遗传性状稳定且增殖速度快的特点,可用于药用植物次生代谢产物的生产研究,为利用生物反应器技术进行药用植物有效成分工业化水平的发酵培养开辟了新途径。本文主要综述了发根农杆菌Ri质粒介导的植物毛状根体系遗传转化机理,并对毛状根体系在药用植物次生代谢产物生产中的研究现状进行了深入分析,为从基因水平上调控植物次生代谢产物的合成提供新思路。     

Infection of turnip and radish storage roots with Agrobacterium rhizogenes

Within about 10 days after inoculation with Agrobacterium rhizogenes, the vascular bundles of storage root disks of turnip or radish developed small outgrowths with numerous root hairs. Thereafter, adventitious roots (hairy roots) emerged extensively from these outgrowths. The hairy roots which emerged fully supported the growth of host plants, though they lacked geotropism. An excised hairy root could be subcultured as an axenic root culture in the absence of phytohormones. Hairy root cultures with extensive lateral branches grew much more rapidly than those with few lateral branches or ordinary roots. Calli were induced from hairy root cultures in the presence of 2,4-D, and root proliferation from these calli occurred in the absence of 2,4-D. Both the primary hairy roots and the roots which grew from them synthesized agropine and mannopine.     

3种农杆菌对茶树发状根诱导的影响

以茶树‘福云6号’和‘铁观音’成熟种子下胚轴、未成熟种子下胚轴和愈伤组织为材料,以发状根诱导率为指标,探究菌液浓度、农杆菌菌株、外植体类型和预培养时间对发状根诱导的影响。结果表明:(1)菌液浓度OD600在0.4~1.2范围内,‘福云6号’成熟种子下胚轴发状根诱导率先升高后降低,ATCC15834在OD600为0.6时发状根诱导率最高为23.96%,A4和K599在OD600为0.8时,发状根诱导率最高,分别为10.51%和13.11%。(2)3种发根农杆菌致根能力不同,ATCC15834侵染力最强,致根能力大小依次为ATCC15834>K599>A4。(3)发状根诱导率与外植体有关,茶树成熟种子下胚轴可被诱导产生发状根,诱导率‘福云6号’大于‘铁观音’,未成熟种子下胚轴和愈伤组织难以产生发状根。(4)不经预培养和预培养时间为1~2 d的愈伤组织易褐变,无法产生发状根;预培养3 d的愈伤组织经侵染可产生发状根,诱导率为1.85%(‘福云6号’)和0.59%(‘铁观音’)。(5)PCR琼脂糖凝胶电泳检测和GUS组织化学染色证实GUS基因已被整合进‘福云6号’和‘铁观音’愈伤组织和成熟种子下胚轴发状根基因组中并表达。研究发现,发根农杆菌ATCC15834在OD600为0.6时,对茶树‘福云6号’成熟种子下胚轴发状根诱导率最高。该研究结果对改进发根农杆菌介导的茶树遗传转化系统提供理论依据,对茶树高效遗传转化体系的优化和新型受体的开发具有重要意义。     

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。     

Selection of high ginsenoside producing ginseng hairy root lines using targeted metabolic analysis

To develop an experimental system for studying ginsenoside biosynthesis, we generated thousands of ginseng (Panax ginseng C.A. Meyer) hairy roots, genetically transformed roots induced by Agrobacterium rhizogenes, and analyzed the ginsenosides in the samples. 27 putative ginsenosides were detected in ginseng hairy roots. Quantitative and qualitative variations in the seven major ginsenosides were profiled in 993 ginseng hairy root lines using LC/MS and HPLC-UV. Cluster analysis of metabolic profiling data enabled us to select hairy root lines, which varied significantly in ginsenoside production. We selected hairy root lines producing total ginsenoside contents 4-5 times higher than that of a common hairy root population, as well as lines that varied in the ratio of the protopanaxadiol to protopanaxatriol type ginsenoside. Some of the hairy root lines produce only a single ginsenoside in relatively high amounts. These metabolites represent the end product of gene expression, thus metabolic profiling can give a broad view of the biochemical status or biochemical phenotype of a hairy root line that can be directly linked to gene function.     

Transgenic hairy roots. recent trends and applications

Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic roots produced by A. rhizogenes infection is characterized by high growth rate and genetic stability. These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants. Hairy root cultures offer promise for production of valuable secondary metabolites in many plants. The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel. Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots. Development of bioreactor models for hairy root cultures is still a recent phenomenon. It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium. Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants. In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest. In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement. Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest. This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A. rhizogenes.     

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.     

Root hairiness: effect on fluid flow and oxygen transfer in hairy root cultures

The effect of root hairiness on fluid flow and oxygen transfer in hairy root cultures was investigated using wild-type, transgenic and root-hair mutants of Arabidopsis thaliana. The root hair morphologies of the A. thaliana lines were hairless, short hairs, moderately hairy (wild-type) and excessively hairy, and these morphologies were maintained after transformation of seedlings with Agrobacterium rhizogenes. Filtration experiments were used to determine the permeability of packed beds of roots; permeability declined significantly with increasing root hairiness as well as with increasing biomass density. Hairy roots of wild-type A. thaliana grew fastest with a doubling time of 6.9 days, but the hairless roots exhibited the highest specific oxygen uptake rate. In experiments using a gradientless packed bed reactor with medium recirculation, the liquid velocity required to eliminate external mass transfer boundary layer effects increased with increasing root hairiness, reflecting the greater tendency towards liquid stagnation near the surface of roots covered with hairs. External critical oxygen tensions also increased with increasing root hairiness, ranging from 50% air saturation for hairless roots to ca. 150% air saturation for roots with excessive root hairs. These results are consistent with root hairs providing a significant additional resistance to oxygen transfer to the roots, indicating that very hairy roots are more likely than hairless roots to become oxygen-limited in culture. This investigation demonstrates that root hairiness is an important biological parameter affecting the performance of root cultures and suggests that control over root hair formation, either by use of genetically modified plant lines or manipulation of culture conditions, is desirable in large-scale hairy root systems.     

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。     

甘薯、胡萝卜发根单寄主培养体系繁殖马铃薯腐烂线虫的研究

利用发根农杆菌A4转化甘薯品种徐薯18和胡萝卜品种天红2号的发根,建立甘薯茎线虫病病原线虫(马铃薯腐烂线虫)的单寄主培养体系。通过该体系对马铃薯腐烂线虫的行为进行观察以及繁殖情况进行调查。结果表明:(1)马铃薯腐烂线虫在甘薯和胡萝卜发根上都能正常发育和繁殖,完成其生活周期;(2)培养4周和8周后,在甘薯发根上线虫繁殖倍数分别为2.6和50.6倍;在胡萝卜发根上线虫繁殖倍数分别为1.7和9.9倍;相同培养时间内,线虫在甘薯发根上的繁殖数极显著高于在胡萝卜发根上的繁殖数。(3)利用发根系统繁殖马铃薯腐烂线虫,便于研究其行为,在显微镜下可以直接观察到线虫在发根上活动情况,这对研究发根和线虫相互关系十分有利。基于上述结果,初步证实构建甘薯发根单寄主培养体系繁殖马铃薯腐烂线虫是可行的,且优于胡萝卜发根繁殖马铃薯腐烂线虫体系。     

Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation

Valerenic acid (VA) is a pharmacologically-active sesquiterpene found in valerian (Valeriana officinalis L., Valerianaceae) roots and rhizomes. The plant produces only small amounts of this metabolite naturally. So, induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, Wild-type strain ‘A13’ of Agrobacterium rhizogenes was used to induce hairy roots in valerian. The influence of three different elicitors including Fusarium graminearum extract (FE), methyl jasmonate (MJ) and salicylic acid (SA) on VA production in the selected hairy root line ‘LeVa-C4’ was also investigated. The 23-day-old cultures were treated with different concentrations of the elicitors at exposure time of 3 and 7 days. FE (1%) and MJ (100 µM L^?1) highly promoted VA production at 7 days after elicitation, to a level of 12.31- and 6-fold higher than that of non-elicited controls, respectively, and FE did not exert any negative effects on biomass yield of hairy root. SA did not significantly increase the production of VA. This is the first time study to assess the elicitation of hairy root cultures to promote VA biosynthesis in valerian and the resulting experiments demonstrated that F. graminearum extract and MJ were indeed a potent inducer of VA biosynthesis.     

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%。     

滇黄芩毛状根的诱导及其黄芩苷含量测定

本文利用发根农杆菌A grobacterizum rhizogenes1.2556感染滇黄芩再生苗的茎段和叶片,建立了毛状根培养及其植株再生体系。毛状根可直接从受伤的茎、叶外植体表面产生,在无外源激素的MS固体和液体培养基上自主生长,表现出典型的发根特征。毛状根茎段的诱导率较叶片高,最高可达到14.44%;经rolB基因PCR分析和甘露碱纸电泳检测,证明Ri质粒T-DNA已整合到滇黄芩基因组中并表达;毛状根在附加6-BA2mg/L和NAA0.2mg/L的MS固体培养基上直接诱导不定芽,并在MS培养基上生根,形成再生植株。获得的毛状根系经MS液体培养基培养30d后通过HPLC都能检测到黄芩苷,其中1个转化系黄芩苷含量为2.59%,是药材黄芩的0.20倍,而从3年单位时间黄芩苷生成量计算,毛状根是药材黄芩的7.18倍。本研究建立的毛状根培养体系,将对滇黄芩转基因技术的完善和利用毛状根生产黄芩苷的生物转化提供了实验基础。     

Viability of plant hairy roots is sustained without propagation in low sugar medium kept at ambient temperature

The effect of sucrose concentration in the medium on the growth and resumption ability to form lateral roots was investigated using the hairy roots of pak-bung and tobacco. It was found that the growth evaluated by root tip elongation of pak-bung and tobacco hairy roots was suppressed in the medium having an initial sucrose concentration of <2.5kg/m(3), and that the resumption abilities of both the hairy roots could be preserved when the hairy roots were kept at an initial sucrose concentration of 2.5kg/m(3) under ambient temperature conditions. The values of maintenance energy for pak-bung and tobacco hairy roots were determined to be 0.11 and 0.12 per day, respectively, from the total sugar consumption rates. Under the oligotrophic condition of the sucrose concentration of 2.5kg/m(3), the hairy roots were considered to exist as resting cells with maintenance metabolism, and the minimum demand for the energy source to ensure survival of the cells was met because the cells hardly multiplied and sugar consumption was not significant. In addition, long-term storage of pak-bung hairy roots in the liquid medium with 2.5kg/m(3) sucrose was performed at 25 degrees C. It was demonstrated that the hairy roots could maintain their resumption abilities without a serious loss of viability over 600 days and that the number of budding lateral roots per unit length of the main roots remained a value of 72 roots/m after the 600-day storage.     

Production and secretion of recombinant thaumatin in tobacco hairy root cultures

Production of recombinant proteins in plant cell or organ cultures and their secretion into the plant cell culture medium simplify the purification procedure and increase protein yield. In this study, the sweet-tasting protein thaumatin I was expressed and successfully secreted from tobacco hairy root cultures. The presence of an ER signal peptide appears to be crucial for the secretion of thaumatin: without an ER signal peptide, no thaumatin was detectable in the spent medium, whereas inclusion of the ER signal peptide calreticulin fused to the N terminus of thaumatin led to the secretion of thaumatin into the spent medium of hairy root cultures at concentrations of up to 0.21 mg/L. Extracellular thaumatin levels reached a maximum after 30 days (stationary phase) and the subsequent decline was linked to the rapid increase of proteases in the medium. Significant amounts of thaumatin were trapped in the apoplastic space of the root cells. The addition of polyvinylpyrrolidone and sodium chloride into the culture medium led to an increase of extracellular thaumatin amounts up to 1.4 and 2.63 mg/L, respectively. Thaumatin production compares well with yields from other transgenic plants, so that tobacco hairy roots can be considered an alternative production platform of thaumatin.     

Antioxidative enzymes activities and accumulation of steroids in hairy roots of Trigonella

Steroidal sapogenins and phytosterols are a group of secondary metabolites which are very considerable in the pharmaceutical industry. Fenugreek (Trigonella foenum-graecum L.) is the good source of these compounds. In recent decades, there is a great interest to production of these compounds by cultivation of transformed roots. In present study, hairy roots induction in two Trigonella species (T. foenum-graeceum, T. monantha) with three strains of Agrobacterium rhizogenes (15,834, A4 and wt) was investigated. Transgenic status of roots was confirmed by PCR using rolB specific primers. Virulence of these strains was examined on explants of leaf, leaf cotyledone and hypocotyle in both species. The best strain was wt for hairy root induction in hypocotyle and leaf explants of T. foenum-graeceum and T. monantha. Significant quantitative differences were showed between shoot, root and hairy roots in both species. Protein content in root and hairy root of both species was significantly lower in comparison with shoot. Superoxide dismutase (SOD) and peroxidase (POX) activities in hairy roots of both species were higher as compared to other organs. The hairy roots of both species showed an ability to synthesize steroidal sapogenins. These results presented that hairy roots could be a suitable procedure for producing sapogenins compounds that have medicinal value in Trigonella.