Novel phenylanthraquinones,isofuranonaphthoquinones, homoisoflavonoids,and biflavonoids from African plants in the genera Bulbine,Scilla, Ledebouria,and Rhus

The sale of non-cultivated plants poses a real threat to the survival of a large number of plant species. On the other hand, the fact that a given plant has become a traded item is an important lead in the effort to investigate plants for the purpose of discovering novel secondary metabolites that may have marketable properties. This report is largely based on the investigations of plants collected from vendors of medicinal plants. The identification is based on botanical specimens derived from the purchased plant parts either by growing them in a garden or through micropropagation using tissue culture techniques. Bulbine species yielded novel phenyl anthraquinones and isofuranonaphthoquinones, which were found to exhibit antiparasitic and antioxidant properties. Scilla nervosa subsp. rigidifolia and Ledebouria graminifolia yielded twenty homoisoflavonoids and two xanthones. Bulbs derived from tissue culture based specimens of L. graminifolia contained all the compounds isolated from the marketed samples. Some of the homoisoflavonoids were found to possess activity against colon and breast cancer cell lines. Rhus pyroides furnished six novel bichalcones some of which exhibited insect antifeedant, cytotoxic and antiproliferative activities. Structures of the compounds described in this report were elucidated by spectroscopic means including HRMS and 1D-, and 2D-NMR experiments. Interconversion of some of the phenyl anthraquinones was accomplished as part of their structure proof. The total synthesis of three of the biflavonoids was achieved using a synthetic route which involves a microwave irradiation promoted Ulmann synthesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of Ranunculus Asiaticus: A Review and Perspectives

Ranunculus asiaticus is an important ornamental species mainly cultivated in the countries surrounding the Mediterranean sea. So far the multiplication of this plant has been mainly carried out by seed and rhizome division; however, these systems present many drawbacks. Tissue culture is an attractive alternative for accelerated propagation of selected and indexed genotypes. In this paper we review the work carried out on in vitro culture of R. asiaticus and present a flow chart for its commercial production, using axillary budding and embryogenesis. Although the price of micropropagated plants is higher compared to traditional material (seedlings and rhizomes from seed populations), it should be considered that tissue cultured plants have a better rhizome yield per plant; moreover, the tissue culture approach allows to offer clonal material of selected lines.     

Comparison of carbonic anhydrase activity among various species of plantlets

During plant tissue culture, the culture container is small and sealed; the concentration of CO₂ in the microenvironment is relatively low. The plantlet growth is restrained for the shortage of CO₂ in the culture container. Carbonic anhydrase is a zinc-containing metalloenzyme that catalyzes the reversible conversion of bicarbonate to CO₂. The determination of carbonic anhydrase of leaves from Atractylodes lancea (thunb.) DC, Orychophragmus violaceus (L.) O.E. Schulz, Brassica juncea (L.) Czern.et Coss. cv. Luzhousileng, Brassica campestris L. cv. Chuanyou No.8, Brassica napus L cv. Oro, Brassica carinata Braun, Raphanus sativa L. var. raphanistroides Makino and their plantlets indicates that the carbonic anhydrase activity of leaves from both plantlets and fields varies from plant species to plant species, the carbonic anhydrase activity of leaves of Atractylodes lancea (thunb.) DC is the lowest among those plants, and the leaves of all plantlets are lower in carbonic anhydrase activity than the same species of plants from fields. The comparison of the growth rates of those plantlets shows that their relative growth rates are significantly different, plantlets of Atractylodes lancea have the slowest relative growth rate among those plants, and plantlets of Brassica juncea have the greatest relative growth rate. The relationship between RGR of plantlets and their CA activities is a significant linear function. It seems that there was certain correlation between carbonic anhydrase activities of plants and their growth rates. It suggests that in vitro, the greater the carbonic anhydrase activity of plantlet is, the higher its net photosynthetic rate, and the faster its growth rate. Those results offer a foundation to a rational medium choice in plant tissue culture.     

<Emphasis Type="Italic">Echinacea</Emphasis> biotechnology: Challenges and opportunities

Echinacea, better known as purple coneflower, has received a global attention because of its increasing medicinal value. There is enormous potential for the discovery of new medicinal compounds in this species and an immediate need for techniques to facilitate the production of high quality, chemically consistent plant material for drug development and clinical trials. In vitro tissue culture of Echinacea can play a vital role in the development of novel germplasm, rapid multiplication, and genetic modifications for an enhanced phytochemical production. Recent establishment of liquid culture techniques, large-scale bioreactors, and Agrobacterium-mediated transformation are changing the production parameters of the Echinacea species. This review provides an overview of the recent developments in in vitro technologies and challenges that remain in the Echinacea biotechnology.     

Adventitious bud development and regeneration in Tillandsia eizii

Summary The bromeliad Tillandsia eizii is a stricking species with large, colorful, and persistent inflorescences that can reach 1 m in length. The value of this plant as an ornamental and its importance in cultural and religious activities has led to its overcollection in the wild. Clonal propagation via tissue culture may be a means to repopulate native stands while meeting the demands for this species as an ornamental and ceremonial plant. Adventitious bud proliferation was induced from axenically germinated scedling material. Parameters evaluated were the age of explant material at the time of transfer onto bud-induction medium, the concentration of plant growth regulators, and the period of exposure to induction medium. Light and seanning electron microscopy (SEM) established the origin and development of buds. Twelve-week-old seedling explants rapidly initiated adventitious buds after a 30-d induction period on shoot-initiation medium. Adventitious buds were induced in 40% of the explants placed on media with 2 mg l⁻¹ 6-benzylaminopurine (BA) (8.88 μM) plus 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA) (0.54 μM) with some cultures becoming highly prolific after repeated subeulture. Shoots elongated in proliferating cultures, and plants were successfully acclimatized and planted into the greenhouse. The results indicate that tissue culture may be used as a means to propagate this epiphytic bromeliad species, which is being seriously affected by deforestation and habitat destruction. In addition, adventitious bud proliferation can provide a means to propagate superior genotypes.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Conservation of medicinal plants of Western Ghats,India and its sustainable utilization through <Emphasis Type="Italic">in vitro</Emphasis> technology

Climate change, alien species, and use of land for intensive farming and development are causing severe threat to the plant genetic diversity worldwide. Hence, conservation of biodiversity is considered fundamental and also provides the livelihoods to millions of people worldwide. Medicinal plants play a key role in the treatment of a number of diseases, and they are only the source of medicine for majority of people in the developing world. The tropical regions of the world supply the bulk of current global demand for “natural medicine,” albeit with increasing threat to populations in the world and its genetic diversity. India is a major center of origin and diversity of crop and medicinal plants. India poses out 20,000 species of higher plants, one third of it being endemic and 500 species are categorized to have medicinal value. The Western Ghats is one of the major repositories of medicinal plants. It harbors around 4,000 species of higher plants of which 450 species are threatened. Currently, the number of species added to the red list category in this region is increasing, and the valuable genetic resources are being lost at a rapid rate. Demand for medicinal plants is increasing, and this leads to unscrupulous collection from the wild and adulteration of supplies. Providing high-quality planting material for sustainable use and thereby saving the genetic diversity of plants in the wild is important. During the last 25 years of intensive research, Tropical Botanic Garden and Research Institute has developed in vitro protocol for rapid regeneration and establishment of about 40 medicinally important rare and threatened plants of Western Ghats. In situ conservation alone would not be effective in safeguarding these important species. Thus, utilizing the biotechnoligical approach to complement ex situ conservation program is becoming vital. Propagating biotechnology tools in plant conservation program is a prerequisite to succeed in sustainable use and to complement the existing ex situ measures. In addition to propagation, storage of these valuable genetic resources is equally important. In vitro slow growth of 35 species and cryopreservation using embryo/meristem/seed in 20 different species of rare medicinal plants of this region is accomplished. Plants developed in vitro of ten medicinal plants, which have restricted distribution, were reintroduced in the natural habitat as well.     

Goldenseal (<Emphasis Type="Italic">Hydrastis canadensis</Emphasis> L.): <Emphasis Type="Italic">In vitro</Emphasis> regeneration for germplasm conservation and elimination of heavy metal contamination

Summary Hydrastis canadensis L. (Goldenseal) is an endangered medicinal plant used in the treatment of many ailments, such as gastrointestinal disturbances, urinary disorders, hemorrhage, skin, mouth and eye infections, and inflammation. Commercial preparations of wild-harvested goldenseal were found to contain heavy metal contaminants including aluminum (848 μgg⁻¹), cadmium (0.4μgg⁻¹), lead (18.7μgg⁻¹), and mercury (0.1 μgg⁻¹). As well, goldenseal is an endangered species listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix II. Therefore, the practice of wild-harvest is actually decimating natural populations of goldenseal and endangering its genetic diversity. In vitro propagation protocol by tissue culture was developed for producing high-quality tissues of goldenseal. Significantly more de novo regeneration was induced on stem explants of 3-mo.-old plants cultured on a medium containing 10 μM 6-benzylaminopurine (BA) (22 regenerants per explant) than any other treatment. Subculture of the regenerants on a medium devoid of growth regulators resulted in the development of complete plants that were acclimatized and thrived in standard greenhouse conditions. The plants regenerated in vitro contained the lowest levels of heavy metals. The findings of this study provide the first evidence that heavy metal contaminants bioaccumulate in goldenseal tissues and also provide a method for germplasm conservation, mass multiplication, and production of goldenseal tissues free from abiotic contamination.     

五加科植物发状根诱导研究进展

五加科植物多为重要的中药材,利用发根农杆菌诱导五加科药用植物产生发状根,并从中获取有用的次生代谢产物,是保护五加科珍稀药用植物资源和实现有效次生代谢物质工业化生产的有效途径。该文在概述发根农杆菌转化药用植物研究历程和转化机理研究的基础上,对近年来在发根农杆菌诱导五加科植物的种类及诱导率、影响发根农杆菌诱导五加科植物的各种因素和利用发根农杆菌诱导五加科植物获得再生植株等方面研究进行了重点分析,并对今后亟需研究的几个重点方向进行了展望,以期为五加科药用植物的良性开发和合理利用提供参考。     

A leaf-based regeneration and transformation system for maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic and applied research. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants.     

Cytotoxicity of Fusarium species mycotoxins and culture filtrates of Fusarium species isolated from the medicinal plant Tribulus terrestris to mammalian cells

Ayurvedic medicine, which uses decoctions made of medicinal plants, is used to cure diseases in many Asian countries including Sri Lanka. Although proper storage facilities for medicinal plants are unavailable in Sri Lanka, neither the potential for growth of toxigenic fungi nor their ability to produce mycotoxins in stored medicinal plants has been investigated. We isolated three Fusarium species, F. culmorum, F. acuminatum and F. graminearum from the medicinal plant Tribulus terrestris. Culture extracts of the 3 Fusarium spp. were cytotoxic to mammalian cell lines BHK-21 and HEP-2. Three toxic metabolites produced by Fusarium spp; T-2 toxin, zearalenone, and diacetoxyscirpenol were also cytotoxic to the same mammalian cell lines. The 3 Fusarium spp. grown on rice media produced zearalenone. Plant material destined for medicinal use should be stored under suitable conditions to prevent growth of naturally occurring toxigenic fungi prior to its use.     

Doubled haploid production via anther culture in annual,winter type of caraway (<Emphasis Type="Italic">Carum carvi</Emphasis> L.)

Caraway (Carum carvi L.) is a traditional medicinal and spice cross-pollinated plant species. Although in vitro techniques are recently extensively applied in plant breeding programmes, these are not commonly utilized in caraway. Therefore, based on the protocol for anther culture in carrot (Daucus carota L., a closely related species of caraway in Daucaceae family), in vitro androgenesis in caraway has been studied with the aim to produce completely homozygous inbred lines. Various induction conditions, such as temperature pretreatments, carbon sources and combination of growth regulators in a culture medium as well as the effect of genotype on in vitro androgenesis were examined. Ten breeding lines of winter caraway representing third generation of forced (artificial) self-pollination were used as donor plant material. Cultured anthers produced embryogenic calli, and subsequently two types of regenerated plants were obtained, namely haploids with evident microspore origin, and diploids which may represent somatic (anther wall) regenerants or spontaneous doubled haploids. The ploidy status of regenerated plants was determined by flow cytometry. This is the first report on androgenic doubled haploid production in caraway.     

<Emphasis Type="Italic">In vitro</Emphasis> induction and identification of autotetraploids of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

Dioscorea zingiberensis is an important medicinal plant and a source of diosgenin in China. We report research on the induction, characteristics, and chemical assays of polyploid plants of D. zingiberensis. Immersing calli in 0.3% colchicine solution for 16 h prior to culture induced a high number of autotetraploid plants. The induction rate reached as high as 36.7% of treated calli. More than 50 lines of autotetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Six elite lines have been selected for further selection and breeding into new varieties for commercial production.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

Conservation In vitro of threatened plants—Progress in the past decade

Summary In vitro techniques have found increasing use in the conservation of threatened plants in recent years and this trend is likely to continue as more species face risk of extinction. The Micropropagation Unit at Royal Botanic Gardens, Kew, UK (RBG Kew) has an extensive collection of in vitro plants including many threatened species from throughout the world. The long history of the unit and the range of plants cultured have enabled considerable expertise to be amassed in identifying the problems and developing experimental strategies for propagation and conservation of threatened plants. While a large body of knowledge is available on the in vitro culture of plants, there are limited publications relating to threatened plant conservation. This review highlights the progress in in vitro culture and conservation of threatened plants in the past decade (1995–2005) and suggests future research directions. Works on non-threatened plants are also included wherever methods have applications in rare plant conservation. Recalcitrant plant materials collected from the wild or ex situ collections are difficult to grow in culture. Different methods of sterilization and other treatments to establish clean material for culture initiation are reviewed. Application of different culture methods for multiplication, and use of unconventional materials for rooting and transplantation are reviewed. As the available plant material for culture initiation is scarce and in many cases associated with inherent problems such as low viability and endogenous contamination, reliable protocols on multiplication, rooting, and storage methods are very important. In this context, photoautotrophic micropropagation has the potential for development as a routine method for the in vitro conservation of endangered plants. Long-term storage of material in culture is challenging and the potential applications of cryopreservation are significant in this area. Future conservation biotechnology research and its applications must be aimed at conserving highly threatened, mainly endemic, plants from conservation hotspots.     

Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production

Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as “Indian Ginseng”, is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and the secondary metabolites produced by this plant can be exploited further for the benefit of human health in a sustainable way.     

Somatic embryogenesis in Jatropha curcas Linn., an important biofuel plant

Jatropha curcas L. is one potential source of non-edible biofuel-producing energy crop. Its importance also lies in its medicinal properties. The species is primarily propagated through heterozygous seeds, and thus the seed oil content varies from 4 to 40%. Moreover, due to its perennial nature, seed setting requires 2 to 3 years time. The seed viability and rate of germination are low, and quality seed screening is another laborious task; thus, seed propagation alone cannot provide quality planting material for sustainable use. Somatic embryogenesis, a powerful tool of plant biotechnology for faster and quality plant production has been successfully applied to regenerate plants in Jatropha curcas for the first time. Embryogenic calli were obtained from leaf explants on MS basal medium supplemented with only 9.3 μM Kn. Induction of globular somatic embryos from 58% of the cultures was achieved on MS medium with different concentrations of 2.3–4.6 μM Kn and 0.5–4.9 μM IBA; 2.3 μM Kn and 1.0 μM IBA proved to be the most effective combination for somatic embryo induction in Jatropha curcas. Addition of 13.6 μM adenine sulphate stimulated the process of development of somatic embryos. Mature somatic embryos were converted to plantlets on half strength MS basal medium with 90% survival rate in the field condition. The whole process required 12–16 weeks of culture for completion of all steps of plant regeneration. This protocol of somatic embryogenesis in Jatropha curcas may be an ideal system for future transgenic research.     

Micropropagation systems for the mexican redbud (Cercis canadensis var. Mexicana L.) and other woody plants of the chihuahuan desert

Summary There are many Chihuahuan desert species that have potential as landscape plants for the arid communities of the southwestern United States [agarita, Berberis trifoliata Moric.; Mexican buckeye, Ungnadia speciosa Endl.; Texas madrone, Arbutus xalapensis var. texana (Buckl.). A. Gray]. Within these plant populations, there are superior genotypes that offer even greater interest for the landscape. However, it is difficult to clonally propagate many of these species with conventional techniques, and the seed-derived populations often do not breed true. Therefore, selection of superior genotypes in wild populations coupled with clonal propagation through tissue culture may offer an attractive option. It is relatively easy to achieve disinfestation of explants from desert plants due to a general lack of natural surface contamination by fungi and bacteria, even though interference from numerous trichomes can impede good contact with disinfesting agents. However, there is only a narrow window of time that is ideal for explant collection, because of the brief, periodic flushes of growth that characterize this unusual plant group. There may be years when, due to the harsh environment, the amount of suitable explant material is severely limited. Phenolics and exudates are also problematic in this group of plants, and acclimatization of ex vitro plantlets to the harsh desert environment is a particular challenge. For these reasons, specific adaptations and modifications were necessary to achieve success with micropropagation of desert plant species such as Mexican redbud (Cercis canadensis var. mexicana L.).     

A Panax-centric view of invasive species

Plant-centric sampling provides a novel approach to quantifying the potential impact of invasive species on native plant species. The aim of this study was to determine the level of exposure of individuals and populations of Panax quinquefolius to invasive plant species using this approach in thirty natural ginseng populations. A high level of invasion was found with 63–70% of ginseng populations containing at least one invasive species. Approximately one-third of all individuals were found in close proximity to invasive plants. The most prevalent invasive species were Rosa multiflora and Berberis thunbergii. The exposure to invasives of plants in different size classes varied among populations. Invasive species presence increased with greater ginseng population sizes and presence of harvest. The abundance of invasives plants within forest interiors near this valuable medicinal herb suggests that the economic and ecological costs of competitive interactions with native species could be high.