何首乌毛状根的诱导和培养(简报)

发根农杆菌（Agrobacteriumrhizogenes）侵染后,通过其Ri质粒的T－DNA片段在植物细胞基因组中整合,诱导形成毛状根（hairyroot）l’l。利用生长迅速、生产效能高而稳定的毛状根培养物生产药用植物的次生物质,已有不少报道「一习。但未见发根农杆菌对何首乌遗传转化的报道。何首乌（Polygonu。。ltij7orumThunb．）为常用中药,其块根为乌须发、悦颜色、益精养血和抗衰老的要药。本文报道发根农杆菌对何首乌遗传转化的实验结果,为今后用毛状根培养物生产何首乌的次生物质打下基础。l材料和方法细菌菌株及培养发根农杆菌（Agrobacteri…     

黄芪毛状根的大量培养

毛状根（hairyroots）是发根农杆菌（他如加加洲南栩前议）感染双子叶植物后,其R质粒上的TDNA片断整合进植物细胞核基因组中诱导产生的一种特殊表现型,近10年来已发展成一种新的培养系统［‘j。与植物细胞培养相比,毛状根培养具有生长速度快、激素自养、分化程度较高以及遗传性状相对稳定等优点,因此通过毛状根大规模培养,从中提取有价值的植物次生代谢产物具有应用前景。由于近三分之一传统药材的药用部位是根,所以这一培养系统在传统药材生产中具有更重要的意义。自1997年Ache＿。nn［fi首先成功地用发根农杆菌转化高等植物以来…     

甘草毛状根培养系统的建立及化学成分分析

利用发根农杆菌转化甘草（Glycyrrhiza uralensis Fisch.)外植体获得毛状根,经PCR法检测,表明已转化成功。应用均匀设计法与比较法,建立了适合甘草毛状根的培养系统。化学成分分析结果表明,甘草毛状根含半胱亚磺酸,不含胱氨酸,商品甘草却含胱氨酸而不含半胱亚磺酸;甘草毛状根能合成多种黄酮成分,其中甘草查尔酮A的含量高达干生的0.18％。     

毛喉鞘蕊花毛状根培养及二萜化合物的形成

发根农杆菌ｐＲｉ１５８３４菌株感染毛喉鞘蕊花的根、茎、叶及小苗,均诱导出毛状根。通过纸电泳,在毛状根中检测到甘露碱。该毛状根不仅具有典型的形态特征,并能合成二萜化合物,对其中３个主要成分ｆｏｒｓｋｏｌｉｎ,１,９－ｄｉｄｅｏｘｙｆｏｒｓｋｏｌｉｎ和ｃｏｌｅｏｌ分别进行了定量测定。ＭＳ培养基有利于ｆｏｒｓｋｏｌｉｎ和ｃｏｌｅｏｌ的合成,而Ｂ５培养基促进１,９－ｄｉｄｅｏｘｙｆｏｒｓｋｏｌｉｎ的合成。     

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。     

地不容毛状根的培养

以Ｒ１０００,１５８３４,１８５５,８１９６等４种发根农杆菌菌株,在不同条件下,感染地不溶,结果在光照条件下,Ｒ１０００诱导植物幼嫩组织产生毛状根。毛状根在ＭＳ液体培养基中生长４２ｄ后,趋于稳定;异喹吉林生物碱含量在第２８ｄ时达到峰值１１０４μｇ／ｇＦＷ。６种培养基中,毛状根生长及异喹啉生物碱含量的差异很大,在在Ｂ５２基中毛状根生长最快,Ｎｉｔｓｃｈ培养基中毛状根异喹啉生物碱含量最讥。Ｂ５培养基中     

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%。     

植物毛状根的培养及其化学进展：1.植物毛状根的诱导形成？…

本文综述了发根农杆菌诱导植物毛状根的方法及其分子机制。对毛状根的鉴定技术进行了介绍。诱导出毛状根的植物主要集中在双子叶植物,对单子叶植物毛状根诱导的可能性进行了讨论。由于毛状根的激素自主性、稳定性和高产性,这一技术为植物有用成分的大量生产提供了新的途径。     

大豆毛状根-VA菌根真菌双重培养体系的建立

以大豆毛状根为宿主,接种VA菌根真菌珠状巨孢囊霉（Gigaspora margarita）,经过3.5个月的双重培养,观察到VA菌根真菌珠状巨孢囊霉对大豆毛状根的侵染,辅助细胞形成,并获得VA菌根真菌成熟孢子,在无菌条件下建立了大豆毛状根-VA菌根真菌双重培养体系,为研究菌根真菌侵染大豆根部形成共生体系及相关分子机制提供了一种有效的研究方法。     

膜荚黄芪毛状根培养系统的建立和外界因子对其生长的影响

建立了由发根农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓ）感染膜荚黄芪（Ａｓｔｒａｇａｌｕｓｍｅｍｂｒａｎａｃｅｕｓ（Ｆｉｓｃｈ．）Ｂｇｅ．）叶片外植体形成的毛状根培养系统并研究了外界因子对其生长的影响。ＭＳ基本培养基（无激素）适合于黄芪毛状根的生长,但是由于ＮＨ４ＮＯ３具有抑制作用而被省略。３％的蔗糖和培养基ｐＨ６适宜毛状根生长。光抑制毛状根生长。每天１２ｈ的１５℃处理对黄芪毛状根生长的影响不明显。与黄芪药用根生物活性成分比较表明,毛状根组织含有较多的粗皂甙和可溶性多糖。讨论了毛状根培养技术在中药材生产中的应用和潜力。     

Phenylethanoid and lignan glycosides from Verbascum thapsus.

Verbascum thapsus afforded, in addition to three known phenylethanoid glycosides and four lignan ones, five new phenylethanoid glycosides and one new lignan glycoside. Structures of the compounds were elucidated by spectroscopic methods and chemical evidence.     

Fitness consequences of branching in Verbascum thapsus (Scrophulariaceae)

The reserve meristem hypothesis proposes that strong apical dominance suppresses lateral meristems and branches to escape from predictable damage (herbivory). This hypothesis was tested for Verbascum thapsus and its seed predator the weevil Gynmnetron tetrum by two mensurative experiments. The following predictions were made under this hypothesis: the proportion of individuals branched within a population will increase with increased damage, the main stalk of branched plants will be more damaged, and branching increases net seed production. Fifty populations of V. thapsus were extensively surveyed, and one pair of similar-sized individuals (branched vs. unbranched) were selected from each population to determine damage patterns and measure seed production. Two of the predictions of the reserve meristem hypothesis were clearly supported. The proportion of fruits damaged on the main stalk of branched plants was significantly greater than unbranched plants, and branched plants produced significantly more seeds. Hence, the reserve meristem hypothesis is supported as an adaptive interpretation of apical dominance in this species. This study is a potential example of overcompensation following granivory in the field.     

Temperature relations of photosynthetic response in populations of Verbascum thapsus L.

Summary Seedlings representative of Verbascum thapsus L. populations from thermally diverse habitats were grown under uniform, controlled conditions. The plants were used to obtain temperature response curves for net photosynthesis over a range of 15–40°C. In general, all experimental plants exhibited similar rates of net photosynthesis at 20, 25, 30, and 35°C. Plants representative of cool habitat populations (high-latidude and high-altitude) had greatest rates of net photosynthesis at the lower temperatures and much lower rates at 40°C. Plants representative of warm habitat populations (low-latitude and low-altitude) exhibited rates of net photosynthesis at 40°C which were nearly twice those of plants representative of cool habitat populations. Carbon dioxide transfer resistances are discussed with reference to plant control of photosynthesis at different temperatures. Patterns of photosynthesis and resistance response among plants representative of different habitats suggest ecotypic variation has occurred only to a very limited extent. Therefore, the patterns exhibited by experimental plants suggest that Verbascum thapsus' success in a number of diverse sites is related to the ability of all members of the species to photosynthesize over a broad range of temperatures.     

生物反应器与药用植物毛状根的大规模培养

利用药用植物毛状根培养生产次生代谢产物具有极大的生产潜力,而开发适合毛状根培养的反应器,又是毛状根生产天然产物工业化的关键。本文系统地介绍了各种用于毛状根培养的生物反应器,对通气搅拌式、气升式、超声雾化式等生物反应器各自的特点及优势进行了详细阐述,进一步讨论了各种反应器对于毛状根生长和次生代谢物积累的影响,并提出药用植物毛状根大规模培养的生物反应器在今后的发展方向。     

In vitro culture of common mullein (Verbascum thapsus L.)

Summary Verbascum thapsus L. is a medicinal herb and has been used to treat inflammatory disease, asthma, spasmodic coughs and migraine headaches. Studies were initiated to establish an in vitro culture protocol for V. thapsus. Explants (leaf dises, petioles and roots) were cultured on Murashing and Skoog minimal organics (MSMO) medium with benzyladenine (BA) or kinetin. Best shoot proliferation was obtained from leaf dise and petiole explants at 13.32 μM BA. Leaf dises were cultured on MSMO medium with 13.32 μM BA in combination with naphthalene acetic acid (NAA) or 2,4-dichlorphenoxyacetic acid (2,4-D). More shoot development was obtained with 13.32 μM BA and 5.37 μM NAA. Shoots were transferred to rooting media containing different levels of NAA and 2,4-D. Most of the shoots formed roots on media with 5.37 μM NAA. Plants were transferred to vermiculite and subsequently to potting media and maintained in the greenhouse.     

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

ABSTRACT

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

外源激素对何首乌毛状根生长及蒽醌类成分生物合成的影响

研究了外源激素对液体培养何首乌毛状根生长及其蒽醌类化合物生物合成的影响。结果表明外源激素2,4-D、NAA和6-BA对何首乌毛状根的生长及蒽醌生物合成有较大的影响。在MS培养基中添加2,4-D对何首乌毛状根的生长和蒽醌类化合物的生物合成有很强的抑制作用,而添加适量浓度NAA和6-BA则可促进何首乌毛状根的生长以及蒽醌类物质的生物合成。     

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%。     

A test of the reserve meristem hypothesis using Verbascum thapsus (Scrophulariaceae)

The reserve meristem hypothesis predicts that latent meristems may act as a bet-hedging strategy given high-cost, predictable herbivory. Under this hypothesis, damage to a plant should elicit greater branching. This prediction was tested in Verbascum thapsus with three experiments manipulating the intensity and type of damage to reproductive tissue. In the first experiment, seed set was prevented in the treatment group by stigma excision and lanolin application to 80% of the flowers of each plant. In the second experiment, a minimum of two mating pairs of weevils were added to treated plants prior to the onset of flowering. In the third experiment, all fruits were sliced lengthwise twice. All three treatments significantly reduced seed set. In the first two experiments, treated plants significantly increased degree of branching (branch number and total branch length). This supports the reserve meristem hypothesis as an explanation for greater branching in larger plants of V. thapsus. Interestingly, the fruit destruction experiment failed to elicit a branching response, which suggests that the timing of damage is important.