Lignans in plant cell and organ cultures: An overview

Lignans are found in a wide variety of plant species. The lignan podophyllotoxin is of special interest, since its derivatives like e.g. etopophos® are used in anticancer therapy. As chemical synthesis of podophyllotoxin is not yet economic, it still has to be isolated from wild growing Podophyllum species, some of which are considered to be endangered species. Therefore plant in vitro cultures may serve as alternative sources for podophyllotoxin and for other types of lignans as well. This review describes the establishment of plant cell and tissue cultures for lignan production and the experiments to improve product yields by changing the cultivation parameters, addition of elicitors and feeding of precursors. It also summarizes the use of plant cell and organ cultures to study the biosynthesis of lignans on enzymological level. Abbreviations: DOP – deoxypodophyllotoxin; LARI – lariciresinol; MATAI – matairesinol; 6MPTOX – 6-methoxypodophyllotoxin; PINO – pinoresinol; PTOX – podophyllotoxin; SECO – secoisolariciresinol     

Plant cell factories as a source for anti-cancer lignans

The review places podophyllotoxin, a powerful anti-cancer material used in clinical treatment of small cell cancers, in focus. The economical synthesis of podophyllotoxin is not feasible and demand for this material outstrips supply. At present, Podophyllum hexandrum (Indian May apple) is the commercial source but it grows in an inhospitable region (the Himalayas) where it is collected from wild stands. Furthermore, the plant is now an endangered species. Alternative sources of podophyllotoxin are considered, e.g., the supply of podophyllotoxin and related lignans by establishing plant cell cultures that can be grown in fermentation vessels. Increase of product yields, by variation of medium and culture conditions or by varying the channelling of precursors into side-branches of the biosynthetic pathway by molecular approaches, are discussed.     

The production of cytotoxic lignans by plant cell cultures

Cytotoxic lignans derived from podophyllotoxin are currently used in cancer chemotherapy. Podophyllotoxin for semi-synthetic derivatization is isolated from the rhizomes of Podophyllum plants growing wild, some of which are counted as endangered species. An alternative source for podophyllotoxin or related lignans may in future be cell cultures derived from different plant species, such as Podophyllum spp or Linum spp. These cell cultures were shown to accumulate considerable amounts of podophyllotoxin or 5-methoxypodophyllotoxin. Optimization of the cell cultivation regime might lead to a renewable source of cytotoxic lignans for medicinal uses. This Mini-Review summarizes the attempts to establish plant cell cultures for the production of podophyllotoxin and related lignans and their optimization towards high levels of these target compounds. It also summarizes the results of studies on the biosynthesis of podophyllotoxin and 5-methoxypodophyllotoxin.     

Podophyllotoxin content in <Emphasis Type="Italic">Podophyllum hexandrum</Emphasis> Royle plants of known age of seed origin and grown at a lower altitude

Podophyllum hexandrum Royle, an important alpine herb, and a source of the highly valued aryltetralin-type lignan, podophyllotoxin, has been subjected to heavy collection from the wild due to ever increasing demand. The present study deals with an attempt to bring this plant under cultivation at a relatively lower altitude and to evaluate (1) various growth parameters including above and below ground biomass accumulation, net assimilation rate and relative growth rate, etc., and (2) podophyllotoxin content from the resulting above and below ground biomass in seed raised plants of a known age series of 1–5 years. The podophyllotoxin content was estimated on the basis of HPLC analyses. The levels were found to increase with the plant age and the maximum amount was found in 5-year old plants. This study demonstrates that (1) seeds can be conveniently used for raising healthy propagules in easily approachable locations at a relatively lower altitude, and that the plants can be maintained in such sites over long periods, and (2) this approach of “conservation through cultivation” can be suggested as an effective tool for the management of this “critically endangered status” species.     

Tissue culture and metabolome investigation of a wild endangered medicinal plant using high definition mass spectrometry

An increasing effort is dedicated to investigate the potential of native plants used in traditional medicine as a source of bioactive compounds for numerous industries. The bioprospection of the metabolome of medicinal and/or endangered plants has two important merits: confirming or revealing the biotechnological potential of that species, and assisting in its conservation. In addition, biotechnological techniques, such as tissue culture, are key strategies in conservation and multiplication of medicinal plants. This is the first in vitro development and non-targeted metabolome study by UPLC–QTOF–MS^E of extracts from C. menthoides, an endangered medicinal plant. In vitro development investigation with a wide range of plant growth regulators resulted in maximum survival rate (81%) and the highest growth rate (1.74 cm?±?0.36) for plantlets cultured on Murashige and Skoog medium, supplemented with 1 µM gibberellic acid. Maximum rooting occurred on medium supplemented with 4.4 µM 6-benzyladenine, which also resulted in more leaves per plantlet (10.16?±?1.7). We developed a protocol that can be used for the clonal propagation and ex situ conservation of this species. In terms of metabolome analysis, a total of 107 metabolites from several classes were detected and identified in its hydrophilic extract (HE), including organic acids and derivatives, glucosinolates, terpenes, phenolic compounds as well as other polar metabolites. The metabolites in HE with the greatest signal intensity included the isoquinoline alkaloid magnoflorine; the coumaric acid rosmarinic acid; the steroid-cardanolide convallatoxin; two anthraquinones including the poorly investigated ventinone A. Several molecules identified here carry potential pharmacological benefits such as anti-inflammatory and anticancer applications.     

Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs

Podophyllum hexandrum Royle of family Berberidaceae is an endangered medicinal plant. Rhizome ofP.hexandrum contains several lignans which posses antitumor activity. Podphyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. Availabilityof podophyllotoxin from plants has its limitations because of its intense collection from nature and lack of organized cultivation. The chemical synthesis of podophyllotoxin is considered to be very complicated as yet. The use of biotechnological approaches for the production of podophyllotoxin using cell cultures, organ cultures, and biotransformation route or by manipulating biosynthetic pathway proves to be an attractive alternative for production of podophyllotoxin. The present paper discusses the current status of research, limitations and future prospects for theproduction of podophyllotoxinin vitro.     

Evaluating costs for the <Emphasis Type="Italic">in vitro</Emphasis> propagation and preservation of endangered plants

In vitro methods provide opportunities for propagating and preserving endangered plant species when seed-based methods are not adequate. Such species include those that produce few or no seeds, as well as species with recalcitrant seeds. Tissue culture propagation methods can be used to produce such plants for reintroduction, research, education, display, and commerce. They can also be the basis for tissue banking as a way to preserve genetic diversity when seeds cannot be banked. With some recalcitrant species, embryo banking, a method which also utilizes in vitro culture for recovery germination, is possible. The number of endangered species that will require in vitro methods is estimated to be at least 5,000 worldwide. Further information is needed to identify these species, and the ongoing collection of information into databases on endangered species and recalcitrant species will help provide this. The costs of these methods are higher than for traditional propagation and preservation, but they may be necessary for species under higher threat. The multiplication rate of a culture, as well as the rates of rooting and acclimatization, has a major effect on the number of transfers needed for producing plants or tissue for banking, and improvements that will increase the efficiency of these steps can help lower costs. Further research into factors affecting the growth of tissues in vitro, as well as coordination of efforts among institutions with infrastructure for in vitro work, should facilitate the application of in vitro methods to the endangered species that cannot be propagated or preserved using seeds.     

Low temperature enhanced the podophyllotoxin accumulation vis-a-vis its biosynthetic pathway gene(s) expression in Dysosma versipellis (Hance) M. Cheng – A pharmaceutically important medicinal plant

Dysosma versipellis (Hance) M. Chang is an endemic and endangered Chinese medicinal herb. Podophyllotoxin, a lead compound for the semi-synthesis of clinically useful anticancer compounds, is the main active aryltetralin lignan accumulated in D. versipellis. In this study, morphology and physiology traits in response to podophyllotoxin accumulation and its biosynthetic pathway genes expression were investigated in D. versipellis plants grown at low temperature (4∼6 °C) and greenhouse (25∼30 °C) temperature. Our study found that low temperature significantly increased plant biomass (2.7-fold), chlorophyll, and carotenoid content compared to greenhouse temperature. The mRNA expression analysis of podophyllotoxin pathway genes secoisolariciresinol dehydrogenase and pinoresionol lariciresinol reductase expression levels were also up-regulated by low temperature. In addition, plants grown at low temperature markedly increased podophyllotoxin accumulation to 3.49-fold in rhizome of D. versipellis than the greenhouse temperature. These results suggest that plants at low temperature enhance the accumulation of podophyllotoxin in rhizome of D. versipellis by over-expressing podophyllotoxin pathway genes. This study finding could be useful in the producing pharmaceutically important podophyllotoxin without overexploiting this endangered species.     

Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva

Podophyllotoxin, a well-known naturally occurring aryltetralin lignan occurs in few plant species that is used as a precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophose phosphate. The availiability of this lignan is becoming increasingly limited because of the scarce occurance of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. This paper reports first time the production of podophyllotoxin by an endophytic fungus Fusarium oxysporum isolated from the medicinal plant Juniperus recurva. Further confirmation and quantification of podophyllotoxin was performed by HPLC, LC-MS, and LC-MS/MS.     

An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Boiss.

An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid (IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3 weeks of culture. Optimal regeneration was achieved on medium containing 3 mg/l kinetin and 0.5 mg/l IAA. This particular medium composition significantly improved the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators. Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation and mass propagation of this species.     

西藏米林县珍稀濒危药用植物优先保护评价

通过设置样方和路线踏查,对米林县的野生药用植物资源进行调查,并利用多个指标定量计算各濒危植物的优先保护值,确定其珍稀濒危物种优先保护序列,对珍稀濒危药用植物的种类、药效和入药部位多样性进行了分析。结果表明:米林县共有药用保护植物37科72属97种,分别占药用植物总科数、总属数、总种数的48.05%、32.00%、30.70%。其中,被子植物有35科69属95种,裸子植物有2科2属2种。97种珍稀濒危药用植物中,处于Ⅰ级保护的药用植物有9科11属11种,处于Ⅱ级保护的药用植物有18科25属27种,处于Ⅲ级保护的药用植物有28科46属59种。从珍稀濒危药用植物的药效来看,清热功效和补虚功效的药用植物较多,分别有32种和20种,各占濒危药用植物总种数的32.99%、20.62%;从药用部位统计,根与根茎类和全草类的药用植物分别有34种和26种,占珍稀濒危药用植物总数比例较大,分别为35.05%、26.80%。该区评估的优先保护等级和濒危等级与保护植物名录有一定差异,桃儿七在本区的保护等级由Ⅲ级升为Ⅰ级,另外有7种植物的濒危程度在本区加强。基于调查和研究结果分析了米林县珍稀濒危药用植物的致濒原因,并就此提出了一些具体的保护建议和措施。     

Conservation of important plants from the Ionian Islands at the Balkan Botanic Garden of Kroussia,N Greece: using GIS to link the in situ collection data with plant propagation and <Emphasis Type="Italic">ex situ</Emphasis> cultivation

The Balkan Botanic Garden of Kroussia (BBGK) is dedicated to the ex situ conservation of native plants of Greece and the Balkans. The BBGK has formulated a conservation strategy for the collection of wild plant material for propagation, prioritizing mainly the endemic, rare, endangered, threatened and vulnerable plants of Europe found in different regions of Greece. Its aim is to contribute to the implementation of Target 8 of the Global and European Strategies for Plant Conservation at local, regional and international scales. In order to (i) define the ecological profile of the in situ requirements preferred and/or tolerated by each selected species, (ii) develop rapid and effective species-specific propagation protocols, and (iii) improve the cultivation of species of conservation concern in BBGK’s nurseries and ex situ conservation sections, geographical coordinates and in situ collection data obtained for each taxon were imported into a Geographic Information System environment (GIS). This information was then linked with several digital GIS thematic layers, including topographic, geological, edaphic, climatic, precipitation and temperature data derived from digital databases. Based on this approach, sexual and asexual propagation of plants from the Ionian Islands were conducted and rapid and effective baseline protocols were developed for 29 taxa (species and subspecies); four are presented here in detail and species-specific ex situ propagation and cultivation guidelines are given. Most of the taxa originating from the Ionian Islands were propagated by cuttings (55.2%) or seeds (34.5%), while the rest were propagated by root division at a rate from 1.7 to 2. The first round of propagation achieved a success rate ranging from 15 to 50% for 3 taxa, from 60 to 80% for 8 taxa and from more than 80 to 100% for 16 taxa, while the ex situ cultivation of the wild and propagated plant material has, so far, been successful. The application of GIS exemplified here presents a sensible and invaluable tool with a broad-scale potential in enhancing the prospects of the ex situ conservation of priority species collected from diverse environmental conditions in man-made habitats such as botanic gardens.     

江西九岭山脉种子植物区系研究

九岭山脉植物区系发生历史悠久,对研究我国早期种子植物演化、动植物迁徙、物种分布格局均具有重要作用。为探讨九岭山脉种子植物区系成因和濒危保护植物的保护利用,该文通过野外调查、标本采集和鉴定,结合经典植物地理学方法,对该山脉种子植物区系进行了研究。结果表明:(1)九岭山脉共有野生种子植物211科956属2928种,占江西种子植物1064属4028种的比例分别为89.8%和72.69%。(2)该植物区系的热带成分科占该地区种子植物总科数(不含世界分布科)的81.35%,但缺乏典型热带成分科,区系中的热带性属和温带性属数量接近,显示该植物区系由热带性质向温带性质过渡,这与九岭山脉地处重要的气候和生态交错地带有一定关联。(3)该地区共分布中国特有植物6科33属41种,特有属主要为古特有属,体现了该植物区系起源的古老性,推断该山脉在第四纪冰期环境较为稳定,为众多植物提供了“避难所”。(4)该地区的种子植物中有30科63属109种为国家重点保护植物,其中,国家I级保护植物15种,极危植物3种、濒危植物11种、易危植物20种。该山脉中的九岭山、官山和大围山已有相应的自然保护区发挥生态保护作用,而其余的山地则未建立有效保护,应对相关濒危植物所在区域加以监管,可采取就地保护或迁地保护等措施,建立相关的种质资源库,确保濒危保护植物的可持续利用。     

珍稀药用植物桃儿七细胞工程及内生菌研究进展

桃儿七的主要活性成分是鬼臼毒素,在医药领域具有重要的应用价值。目前商用桃儿七主要来自野生资源,过度采挖已造成桃儿七在世界范围内濒临灭绝。综述了桃儿七的植物学特性,以及近年来通过细胞工程(组织培养、人工快繁、大规模细胞培养、毛状根培养)和内生菌发酵等生物技术手段生产鬼臼毒素的研究进展及存在的问题,旨在为该领域今后的相关研究提供背景资料。     

Interest of lignans in prevention and treatment of cancers

Lignans are diphenolic compounds widely distributed in the plant kingdom. They are mainly localised in lignified tissues, seeds and roots. These molecules are involved in plant defence mechanisms, but are also interesting for human health. Flax lignans belonging to the phytoestrogens are metabolised after ingestion into enterolignans that may offer a protection against the onset and development of hormono-dependant cancers. In vitro studies based on mammalian cellular models tend to confirm their beneficial effects observed during epidemiological studies and give us insights about their mechanisms of action. The most studied lignan, podophyllotoxin, and its semi-synthetic derivatives (etoposide, teniposide, etoposide phosphate), are particularly interesting at a curative level due to their cytotoxic properties. These semi-synthetic derivatives are used in chemotherapy of lung cancer for example. However, the extensive use of these anticancer drugs will lead to the problem of podophyllotoxin supply. This molecule is currently extracted from the rhizomes and roots of an Indian species Podophyllum hexandrum which has subsequently become endangered. Strategies are investigated to obtain economically viable alternative sources of Podophyllotoxin from plants and in vitro cultures of several species. Among them, north american Podophyllum peltatum, Linum wild species, Hyptis, Anthriscus, Juniperus or Dysosma species which accumulate Podophyllotoxin or closely related derivatives, are good candidates. double dagger.     

Importance of <Emphasis Type="Italic">in vitro</Emphasis> technology to future conservation programmes worldwide

The latest IUCN statistics show that of over 12,000 plant species, 70% are threatened, 19% are critically endangered and 28 species are extinct in the wild. Target 8 of the Global Strategy for Plant Conservation (GSPC) highlights the importance of ex situ conservation of critically endangered plants. Long-term germplasm storage for species with recalcitrant seeds needs alternative measures. In vitro methods complement seed banking and other ex situ measures and are vital for long-term conservation. Conservation Biotechnology at RBG Kew is currently working on a number of rare and threatened recalcitrant species from biodiversity-rich areas of the world to develop good quality in vitro propagules for cryopreservation, recovery and restoration projects. The importance of successful in vitro propagation methods, transplantation technologies, cryopreservation and international networking for the integrated conservation of these species are discussed in detail.     

The Proportion of Three Foundation Plant Species and Their Genotypes Influence an Arthropod Community: Restoration Implications for the Endangered Southwestern Willow Flycatcher

As part of a restoration project, multiple genotypes of two tree species, Fremont cottonwood (Populus fremontii) and Goodding's willow (Salix gooddingii), and one shrub species, Coyote willow (S. exigua), were experimentally planted in different proportions at the Palo Verde Ecological Reserve near Blythe, California, U.S.A. These common woody plant species are important to the endangered southwestern willow flycatcher, providing perch, nesting, and foraging habitat. We conducted this study to evaluate plant species proportion and plant genotype effects on the arthropod community, the prey base for the endangered southwestern willow flycatcher. Three patterns emerged. First, plant species proportions were important; the arthropod community had the greatest richness and diversity (H′) when Goodding's willow proportion was high and Fremont cottonwood proportion was lower; that is, fewer Fremont cottonwoods are required to positively affect overall arthropod diversity. Second, we found significant genotypic effects, for all three plant species, on arthropod species accumulation. Third, while both planting proportion and genotype effects were significant, we found that the effect of planting proportion on arthropod richness was about twice as large as the effect of plant genotype. This shows that both plant species proportions and genotype should be utilized in restoration projects to maximize habitat heterogeneity and arthropod richness. Similar studies can determine which planting proportion and specific genotypes may result in a more favorable arthropod prey base for the southwestern willow flycatcher and other species of concern. Greater attention to planting design and genotype can result in significant gains in diversity at little or no additional project cost.     

Propagation and Seedling Cultivation of the Endemic Species Nothofagus alessandrii Espinosa in Central Chile

Nothofagus alessandrii is an endemic and endangered species from the Maule Region of Central Chile. The forests it once dominated have been severely degraded and fragmented by human activities, and it is estimated that only 350 ha remain. Yet, available information for propagation and nursery plant cultivation of this species is conspicuously lacking. Future efforts to restore this ecosystem type will rely, in large part, on advancing the capacity to propagate and cultivate this highly threatened species. To this end, we studied the response (germination process and nursery growth) of viable seeds of N. alessandrii to different pre‐germination treatments, sowing times, and slow‐release fertilizer. The highest germination percentages were obtained with cold stratification for periods of 30 and 45 days (84.9 and 82.6%, respectively). Sowing time was also relevant, with seeds obtaining highest germination rates (53%) when sowing during spring season (September) as well as the best growth rates. Both cold stratification and GA₃ treatments can provide adequate germination percentage and similar seedling quality. After one nursery season, no clear effect of slow‐release fertilizer on plant growth was observed. The results of this study provide important baseline information for propagation and nursery techniques for restoration programs of N. alessandrii.     

Use of biotechnologies for the conservation of plant biodiversity

In vitro techniques are very useful for conserving plant biodiversity, including (a) genetic resources of recalcitrant seed and vegetatively propagated species, (b) rare and endangered plant species and (c) biotechnology products such as elite genotypes and genetically engineered material. Explants from recalcitrant seed and vegetatively propagated species can be efficiently collected under field conditions using in vitro techniques. In vitro culture techniques ensure the production and rapid multiplication of disease-free material. Medium-term conservation is achieved by reducing growth of plant material, thus increasing intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, −196°C) allows storing plant material without modification or alteration for extended periods, protected from contaminations and with limited maintenance. Slow growth storage protocols are routinely employed for a large number of species, including numerous endangered plants, from temperate and tropical origin. Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases. Research is much less advanced for recalcitrant species due to their seed characteristics, viz., very high sensitivity to desiccation, structural complexity and heterogeneity in terms of developmental stage and water content at maturity. However, various technical approaches should be explored to develop cryopreservation techniques for a larger number of recalcitrant seed species. A range of analytical techniques are available, which allow understanding physical and biological processes taking place in explants during cryopreservation. These techniques are extremely useful to assist in the development of cryopreservation protocols. In comparison with crop species, only limited research has been performed on cryopreservation of rare and endangered species. Even though routine use of cryopreservation is still limited, an increasing number of examples where cryopreservation is used on a large scale can be found both in genebanks for crops and in botanical gardens for endangered species.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of some important chinese medicinal plants and their sustainable usage

Summary Medicinal plants are valuable sources of medicinal and many other pharmaceutical products. The conventional propagation method is the principal means of propagation and takes a long time for multiplication because of a low rate of fruit set, and/or poor germination and also sometimes clonal uniformity is not maintained through seeds. The plants used in the phyto-pharmaceutical preparations are obtained mainly from the natural growing areas. With the increase in the demand for the erude drugs, the plants are being overexploited, threatening the survival of many rate species. Also, many medicinal plant species are disappearing at an alarming rate due to rapid agricultural and urban development, uncontrolled deforestation, and indiscriminate collection. Advanced biotechnological methods of culturing plant cells and tissues should provide new means for conserving and rapidly propagating valuable, rare, and endangered medicinal plants. The purpose of the present review is to focus the application of tissue culture technology for in vitro propagation via somatic embryogenesis and organogenesis and the cell suspension culture with suitable examples reported earlier. An overview of tissue culture studies on important Chinese medicinal plants and related species is presented.