Ansellia africana (Leopard orchid): A medicinal orchid

South African Journal of Botany 106 (2016) 181–185

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Ansellia africana (Leopard orchid): A medicinal orchid species with untapped reserves of important biomolecules—A mini review Paromik Bhattacharyya, Johannes Van Staden ⁎ Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa

a r t i c l e

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Article history: Received 29 March 2016 Accepted 28 June 2016 Available online 25 July 2016 Edited by AK Jäger Keywords: South African medicinal orchids Alzheimer's disease Drug discovery Phylogeography Phytochemicals

a b s t r a c t Ansellia africana Lindl., the “Leopard orchid” is a species endemic to Africa. Its ethnobotanical usage has been documented in various traditional African pharmacopeias. It has activity on the Central Nervous System (CNS) and has shown potentiality in the treatment of Alzheimer's disease. However, due to over-exploitation and habitat destruction, the plant is facing the risk of extinction and it has been categorized in the red list of plants as “vulnerable” by IUCN. To protect the remaining natural populations of A. africana, a sustainable conservation strategy coupled with systematic scientific exploration of its medicinal potential is of utmost importance. Coupled with this, the advances made in the field of plant metabolomics and transcriptome data mining of putative plant genes will throw more light in understanding the secondary metabolite biosynthesis in this medicinal plant. This article briefly reviews the botany, pharmacology, biochemistry and scope of future research of this important medicinal orchid species. As there is very little literature available on the scientific documentation of the African, especially South African medicinal orchids, we are attempting to compile and document information on different aspects of A. africana and highlight the need for research and development on the ethno-pharmacology of this medicinal orchid species. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Ansellia africana—the plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Medicinal uses of A. africana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. The concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Future research prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Phylogeography and DNA barcoding studies . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Conservation through plant tissue culture and maintenance of genetic diversity . . . . . . . . . . 2.3. Drug discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Transcriptome analysis and mining of putative genes controlling secondary metabolite bio-synthesis 3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The Orchidaceae is one of the world's largest angiosperm families of flowering plants. Coupled with their tremendous horticultural importance, orchids have been used in various traditional pharmacopeias for ⁎ Corresponding author. Tel.: +27 33 260 5130. E-mail address: [email protected] (J. Van Staden).

http://dx.doi.org/10.1016/j.sajb.2016.06.010 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

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centuries (Hossain, 2011). Advances in research of medicinal plants and especially orchids have resulted in the discovery of various active metabolites which have shown potentiality in the treatment of chronic disorders. The phyto-constituents obtained from orchids have shown potential as anti-rheumatic, anti-inflammatory, anti-carcinogenic, anticonvulsive, diuretic, neuroprotective, relaxation, anti-aging, wound healing, hypoglycemic, anti-tumor, anti-cancer, anti-microbial and antiviral activities (Miyazawa et al., 1997; Bulpitt, 2005; Gutiérrez, 2010;

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P. Bhattacharyya, J. Van Staden / South African Journal of Botany 106 (2016) 181–185

Hossain, 2011; Pant, 2013). Research in recent years have led to the isolation of various active principles from orchids such as anthocyanins, orchinol, hircinol, cypripedin, bibenzyl derivatives, phenanthrenes, jibantine, nidemin and loroglossin which are present in leaves, pseudobulbs, roots and flowers or in the whole plant (Okamoto et al., 1966; Ye et al., 2002; Bulpitt et al., 2007; Pant, 2013). The above findings largely emphasize that knowledge of different ethno-pharmacological reports on traditional medicinal systems, especially orchids, helps to provide a more pragmatic research approach. This makes the chances of drug discovery much more comprehensive than with random collection. Like in other traditional pharmacopoeia of the world, orchids also form an integral part of the African traditional medicinal system. However, there are no authentic reports of the exact time when the Africans started to use orchids as medicine (Chinsamy et al., 2011; Hossain, 2011). In South African traditional pharmacopeias, approximately 49 orchid species are being used (Hutchings et al., 1996; Germishuizen and Meyer, 2003). Amongst the various orchid species used, Ansellia africana (Fig. 1A) deserves special mention for its broad-spectrum of medicinal properties especially for its activity on the Central Nervous System (CNS). Coupled with an existent lacunae on systematic studies of medicinal orchids of southern Africa, there is also very little research on the identification and diversity amongst the various natural populations of A. africana and its related species. This severe deficiency of documented scientific reports in the related research domain raises the need for a systematic study of the species, concentrating on the aspects of their conservation and mining down its ethno-pharmacological attributes for potential medicinal activities.

1.1. Ansellia africana—the plant A. africana is an epiphytic orchid, growing in spectacular clumps on trees in the subtropical areas of southern Africa (Fig. 1A). The roots, which anchor the plant to the tree, are specially adapted to absorb water and nutrients very quickly. Unlike other epiphytic orchids, the most characteristic feature of A. africana is the needle-like roots pointing upwards which forms a dense mass around the pseudobulbs and which collects senescing leaves and detritus upon which the plant feeds (Fig. 1B). The plant flowers during the dry winter months producing a mass of yellow or greenish yellow blooms, which can be lightly or heavily marked with brown spots. It is found in tropical Africa and into Namibia, Botswana, Swaziland, and in South Africa in the Northern Province, the Lowveld and KwaZulu-Natal, mainly in hot, dry river valleys (Vasudevan and Van Staden, 2011).

1.2. Medicinal uses of A. africana Traditionally, stem infusions are used by the Zulu as antidotes to bad dreams. Smoke from burning roots is inhaled for the same purpose (Hutchings et al., 1996) whereas the leaves and stems are used to make an infusion for treating madness in the Mpika district of Zambia (Gelfand, 1985). It is also used for various protective charm purposes and as an aphrodisiac in Zimbabwe (Gelfand, 1985). Recent studies have shown that A. africana has potent anti-acetylcholinesterase activity and can be used as an important source of various biomolecules for the treatment of Alzheimer's disease (Table 1) (Chinsamy et al., 2014).

Fig. 1. (A) Photograph of A. africana (B) growing A. africana plants with needle-like roots pointing upwards (C) shoot originating from PLB (bar = 2 cm) (D) greenhouse acclimatized plants of A. africana.


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Table 1 Biological activities attributed to A. africana. Sl.

Name of the biological activity

Plant parts used

Salient findings

Reference

1.

COX-1 and COX-2 assay

Leaves, stems, roots

Chinsamy et al. (2014)

2.

Acetylcholinesterase inhibitory activity


3. 4.

β- carotene bleaching activity Mutagenic activity

Leaves, stems, roots Leaves, stems, roots

5.

Antioxidant power


1. Roots of A. africana showed high COX-1 and COX-2 inhibition activity. 2. The dichloromethane (DCM) root extract of A. africana exhibited the highest EC50 activity amongst seven South African medicinal orchid species tested i.e. (0.25 ± 0.10 mg/ml). Highest acetylcholinesterase inhibitory activity exhibited amongst the seven most potential medicinal orchid species tested. The most potent extract was the ethanolic root extract. Exhibited moderate β-carotene bleaching activity. DCM root extract and ethanolic leaf, stem and root extract exhibited mutagenic effects. High antioxidant levels determined by FRAP assay.

1.3. The concerns Due to indiscriminate collection from the wild and heavy deforestation, the natural populations of A. africana are severely threatened and presently they are categorized as “vulnerable” in the IUCN Red Data List (http://www.iucnredlist.org/details/44392142/0). Being an epiphytic orchid, A. africana seeds require fungal association to germinate in nature coupled with favorable environmental conditions. Coupled with this requirement and various other anthropogenic pressures and over-collection, its natural populations have been severely threatened. However, recent findings obtained by Papenfus et al. (2015) reported that the use of smoke extracts can overcome the need for fungi to a great extent and may solve the issue of germination failures in nature, which resolves the aspect of reduced germination rate of A. africana in nature augmented with the previous findings of Vasudevan and Van Staden (2010). Novel techniques of in vitro propagation can further help in conservation and production of large numbers of disease free, true-to-type plants. Vasudevan and Van Staden (2011) reported that a very large number of A. africana plantlets can be regenerated using topolins, a next generation group of plant growth regulators helping in the sustainable utilization and commercial exploitation of plants (Fig. 1C–D). Considering the range of different niches occupied by the plant, there is the possibility that many ecotypes and/or chemotypes of A. africana exist. It would be interesting to study the morphological, molecular and biochemical variations amongst different natural populations of A. africana. These studies become more significant as this is the only species in the genus Ansellia (Papenfus et al., 2015). Some of the A. africana populations may be particularly significant in terms of the degree of diversity they possess (Rao and Hodgkin, 2002). Populations with maximum diversity can be identified and isolated for conservation without any duplication within the conserved germplasm. This argument is strongly supported by the finding of Bhattacharyya et al. (2015) with Dendrobium nobile.

2. Future research prospects 2.1. Phylogeography and DNA barcoding studies In the designing of conservation strategies of Rare Endangered and Threatened (RET) plants scientific analysis of the phytogeography plays an important role. Phylogeographic studies are largely influenced by the phenomenon of concerted evolution. Concerted evolution is a biological phenomenon in which hundreds to thousands of tandemly repeated copies of DNA such as ribosomal DNA (nrDNA), do not evolve independently but in a concerted way as a consequence of which, the copies of these related genes are more similar within species than between species. The primary factor influencing the phenomenon of concerted evolution is the homogenization of these multiple copy genes through unequal crossing over and high frequency of gene conversion.

However, under certain circumstances, the integral nature of the tandem repeats of such genes gets disturbed. In such cases, incomplete intra-genomic variation appears. Incomplete concerted evolution with respect to nr-DNA occurs in many plant species including orchids. South Africa in particular houses a wide array of representatives from the family Orchidaceae. In orchid species such as A. africana which is heavily threatened due to heavy rates of deforestation and habitat destruction, studies on phylogeography and existing genetic variations using molecular markers is of significance. Studies on the genetic diversity of various orchid species depicted an uneven distribution of genetic diversity amongst various geographic areas. Similar studies in related plant groups have revealed the existence of ribotypes which in turn might be nonfunctional copies or pseudogenes. Furthermore, being an endangered plant taxa, DNA barcoding of A. africana and other related South African orchid species is of relevance. At present, rbcL, matK, psbA-trnH, rpoC1, and ITS2 are popularly used worldwide as DNA barcodes in plants. In recent years, some researches employed the psbA-trnH barcode to identify species of medicinal pteridophytes and within the genus Dendrobium. Chen et al. (2010) have shown that ITS2 is a universal barcode in the identification of plants and allied species, as ITS2 correctly identified 92.7% cases of over 6600 samples in seven phyla (Angiosperms, Gymnosperms, Ferns, Mosses, Liverworts, Algae, and Fungi). Since then, the ITS2 region has been shown to be useful in discrimination amongst a wide range of plants including the orchidaceae. Thus, using these potential DNA barcodes, an important orchid species such as A. africana can be molecularly authenticated. 2.2. Conservation through plant tissue culture and maintenance of genetic diversity In vitro propagation to produce plants for commercial production and to repopulate decimated populations is an alternative that might help in decreasing pressure on natural populations. In vitro culture is a useful method to propagate endemic or endangered plant species for conservation purposes (Arditti and Krikorian, 1996). Many orchids are propagated through seeds, which require specific mycorrhizal associations (Rubluo et al., 1993; Buyun et al., 2004; Hernández et al., 2005). Propagation of orchids by in vitro germination allows for the maintenance of higher genetic variability than other methods. Establishment of protocols for in vitro culture of orchid seeds is species-specific and depends on several factors such as capsule maturity, components of culture media, light, and temperature conditions (Arditti, 1984). The International Union for Conservation of Nature and Natural Resources (IUCN) designates genetic diversity as one of three levels of biodiversity requiring conservation besides ecosystem and species diversity (McNeely et al., 1990). Genetic diversity means the extent of genetic variation amongst individuals and/or populations within a species or across a group of species (Frankham et al., 2002). As such it is the raw basis for adaptation (Geffen et al., 2006). As genetic variation is a precondition for the ability of the species to respond to continuously changing environmental

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conditions, it is a key factor for long-term survival in the wild (Frankham et al., 2002). However, in the short-term, genetic diversity is important for the maintenance of reproductive fitness at the population level (Leimu et al., 2006). Threatened species tend to have small and/or declining populations. With decreasing population size, the probability of biparental inbreeding or selfing increases, making small populations more susceptible to inbreeding depression (Mustajärvi et al., 2001). Inbreeding has long been known to have deleterious consequences for reproduction and survival in naturally outbreeding species (Charlesworth and Willis, 2009). There is now strong evidence that the loss of genetic diversity and inbreeding contribute to the extinction risk of populations in the wild (Newman and Pilson, 1997; Saccheri et al., 1998). Hence, the protection and restoration of genetic diversity is of major importance for the longterm survival or the sustainable management of (endangered) species such as A. africana. Ignoring genetic factors may lead to inappropriate conservation strategies (Frankham, 2005). The existing genetic diversity amongst the natural populations of A. africana can be assessed using PCR-based markers like inter simple sequence repeats (ISSR), start codon targeted polymorphism (SCoT) and direct amplification of minisatellite DNA (DAMD). Furthermore, a positive correlation exists between genetic variability and variation in biochemical constituents (Hong et al., 2005; Kuroda et al., 2007; Han et al., 2008; Tori et al., 2008). Quantitative and qualitative status of active constituents along with genetic diversity in a medicinal plant could help to design conservation strategies and selection of the best plant genetic material (Ali et al., 2013). Although a few studies have been done with medicinal plants, reports dealing with orchids are very limited (Ali et al., 2013; Jugran et al., 2013; Bhattacharyya et al., 2015). The studies on the relation of molecular markers with the biochemical markers in a medicinal plant species will provide vital information about rare diagnostic marker(s) which will help in identification of genes responsible for high antioxidant activity, which can be further utilized in clonal propagation and also in marker-assisted selection (MAS) of the species so as to commercially cultivate the novel genotypes along with defining genetic relationships amongst the genotypes. 2.3. Drug discovery Current research in drug discovery from medicinal plants involves a multidirectional approach constituting of botanical, biological, phytochemical and molecular biological techniques. Mining of biologically important biomolecules within medicinal plants continues to provide new and important leads against catastrophic disorders which are threatening the existence of mankind such as HIV/AIDS, Alzheimer's disease, and malaria (Hossain, 2011, Chinsamy et al., 2014). Several natural product drugs of herbal origin like arteether, galantamine, nitisinone, and tiotropium have made a major impact in the domain of drug discovery and ethno-pharmacology (Balunas and Kinghorn, 2005). Our survey on the orchids of South Africa, with special focus on A. africana, revealed that it is an untapped reserve for various biologically active compounds that requires thorough, systematic evaluation. The traditional Zulu healers use this plant in the treatment of madness which indicates that it contains biological entities which may prove to be significant in the treatment of CNS disorders (Chinsamy et al., 2014). 2.4. Transcriptome analysis and mining of putative genes controlling secondary metabolite bio-synthesis The emergence of next generation sequencing (NGS) has paved the way for large scale sequencing of several non-model plants which can be valuable in investigating the basis of medicinal properties. Different NGS technologies and their potential applications in plant biology including transcriptome investigations have been reviewed (Egan et al., 2012). Strategies and tools which can be employed in transcriptome studies of non-model plants using second generation sequencing have been discussed (Bräutigam and Gowik, 2010). The understanding of the therapeutic potential of A. africana will help to establish the basic

understanding about the genes involved in the regulation and channelization of the secondary metabolites like phenyl propanoids and terpenoids. Cribb et al. (2003) have made mention of the “gaps” in our knowledge of orchids, particularly in their sustainable utilization and conservation. Further research on metabolomics of A. africana will be a significant advance in terms of genomic resources in the domain of comparative studies of closely related medicinal plants with special reference to Alzeimer's disease. The medicinal bio-entities isolated from orchid species are in general polysaccharides (Ng et al., 2012). The NGS approach provides the researcher with a new tool for the better understanding of sucrose metabolism and polysaccharide enrichment which will ultimately help in the generation of more efficient biomolecules with higher efficacy (Yan et al., 2014). First-hand knowledge of the alkaloid biosynthetic pathway helped to extend to the generation of 16-epivellosomine with the existence of the polyneuridine-aldehyde esterase enzyme (Yan et al., 2014). Thus, the NGS sequencing of A. africana could be useful in deciphering the complex genome for a better understanding of its metabolome. Researches on related orchid species of medicinal and horticultural importance have shown that the genome data obtained by NGS helps to understand important biological features like drought resistance, symbiosis with fungi, completeness of the floral gene sets in orchids, and the biosynthesis of medicinal components. We anticipate that an improved understanding of the biology of A. africana would ultimately facilitate in the modernization of the traditional South African pharmacopeia. 3. Conclusions The ethnobotanical uses of orchids in South Africa have not been exploited pharmacologically. Being an important medicinal member of the traditional “Zulu” pharmacopeia, a wide scope of exploring different aspects of A. africana still exist. Furthermore, medicinal plants especially orchids hold an unexplored reserve of various biomolecules which might provide answers to incurable disorders like HIV/AIDS and Alzheimer's. Apart from two reports of seed germination (Vasudevan and Van Staden, 2010; Papenfus et al., 2015) and one micropropagation protocol from Vasudevan and Van Staden (2011), there is no other studies on the conservation strategies of A. africana which is presently categorized as “vulnerable” by IUCN. The present era of research have put forward a huge untapped potential hidden within medicinal orchid species such as A. africana and others. A comprehensive strategic approach constituting of biochemical, molecular techniques at metabolomics level will enable the research community to realize the tremendous medicinal potential within the existing germplasms of A. africana and hence will assist in their sustainable utilization. Acknowledgements P. Bhattacharyya thanks the University of KwaZulu-Natal, South Africa for support in the form of a postdoctoral fellowship. References Ali, Z., Ganie, S.H., Narula, A., Sharma, M.P., Srivastava, P.S., 2013. Intra-specific genetic diversity and chemical profiling of different accessions of Clitoria ternatea L. Industrial Crops and Products 43, 768–773. Arditti, J., 1984. Physiology of germinating orchid seeds. Orchid Biology Reviews and Perspectives 3, 177–222. Arditti, J., Krikorian, A.D., 1996. Orchid micropropagation: the path from laboratory to commercialization and an account of several unappreciated investigators. Botanical Journal of the Linnean Society 122, 183–241. Balunas, M.J., Kinghorn, A.D., 2005. Drug discovery from medicinal plants. Life Sciences 78, 431–441. Bhattacharyya, P., Kumaria, S., Tandon, P., 2015. Applicability of ISSR and DAMD markers for phyto-molecular characterization and association with some important biochemical traits of Dendrobium nobile, an endangered medicinal orchid. Phytochemistry 117, 306–316. Bräutigam, A., Gowik, U., 2010. What can next generation sequencing do for you? Next generation sequencing as a valuable tool in plant research. Plant Biology 12, 831–841.

P. Bhattacharyya, J. Van Staden / South African Journal of Botany 106 (2016) 181–185 Bulpitt, C.J., 2005. The uses and misuses of orchids in medicine. QJ. - Monthly Journal of the Association of Physicians 98, 625–631. Bulpitt, C.J., Li, Y., Bulpitt, P.F., Wang, J., 2007. The use of orchids in Chinese medicine. Journal of the Royal Society of Medicine 100, 558–563. Buyun, L., Lavrentyeva, A., Kovalska, L., Ivannikov, R., 2004. In vitro germination of seeds of some rare tropical orchids. Acta Universitatis Latviensis, Biology 159–162. Charlesworth, D., Willis, J.H., 2009. The genetics of inbreeding depression. Nature Reviews Genetics 10, 783–796. Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., Zhu, Y., Ma, X., Gao, T., Pang, X., 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PloS One http://dx.doi.org/10.1371/journal.pone.0008613. Chinsamy, M., Finnie, J.F., Van Staden, J., 2011. The ethnobotany of South African medicinal orchids. South African Journal of Botany 77, 2–9. Chinsamy, M., Finnie, J.F., Van Staden, J., 2014. Anti-inflammatory, antioxidant, anticholinesterase activity and mutagenicity of South African medicinal orchids. South African Journal of Botany 91, 88–98. Cribb, P.J., Kell, S.P., Dixon, K.W., Barrett, R.L., 2003. Orchid conservation: a global perspective. Orchid Conservation. Natural History Publications, Kota Kinabalu, pp. 1–24. Egan, A.N., Schlueter, J., Spooner, D.M., 2012. Applications of next-generation sequencing in plant biology. American Journal of Botany 99, 175–185. Frankham, R., 2005. Genetics and extinction. Biological Conservation 126, 131–140. Frankham, R., Briscoe, D.A., Ballou, J.D., 2002. Introduction to Conservation Genetics. Cambridge University Press. Geffen, E., Luikart, G., Waples, R.S., 2006. Impacts of modern molecular genetic techniques on Conservation Biology. Key Topics in Conservation Biology, pp. 46–63. Gelfand, M., 1985. The Traditional Medical Practitioner in Zimbabwe: his Principles of Practice and Pharmacopoeia. Mambo Press. Germishuizen, G., Meyer, N.L., 2003. Plants of southern Africa: an Annotated Checklist. National Botanical Institute Pretoria. Gutiérrez, R.M.P., 2010. Orchids: a review of uses in traditional medicine, its phytochemistry and pharmacology. Journal of Medicinal Plants Research 4, 592–638. Han, T., Hu, Y., Zhou, S., Li, H., Zhang, Q., Zhang, H., Huang, B., Rahman, K., Zheng, H., Qin, L., 2008. Correlation between the genetic diversity and variation of total phenolic acids contents in Fructus xanthii from different populations in China. Biomedical Chromatography 22, 478–486. Hernández, L., Martínez-García, M., Campos, J.E., Aguirre-León, E., 2005. In vitro propagation of Laelia albida (Orchidaceae) for conservation and ornamental purposes in Mexico. Hortscience 40, 439–442. Hong, D.Y.Q., Lau, A.J., Yeo, C.L., Liu, X.K., Yang, C.R., Koh, H.L., Hong, Y., 2005. Genetic diversity and variation of saponin contents in Panax notoginseng roots from a single farm. Journal of Agricultural and Food Chemistry 53, 8460–8467. Hossain, M.M., 2011. Therapeutic orchids: traditional uses and recent advances—an overview. Fitoterapia 82, 102–140. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A.B., 1996. Zulu Medicinal Plants: an Inventory. University of Natal Press. Jugran, A., Rawat, S., Dauthal, P., Mondal, S., Bhatt, I.D., Rawal, R.S., 2013. Association of ISSR markers with some biochemical traits of Valeriana jatamansi Jones. Industrial Crops and Products 44, 671–676.

185

Kuroda, C., Kiuchi, K., Torihata, A., Takeshita, K., Gong, X., Shen, Y., Hirota, H., Onuki, H., Hanai, R., 2007. Chemical and genetic diversity of Ligularia latihastata and Ligularia villosa in Yunnan Province of China. Chemistry and Biodiversity 4, 2210–2217. Leimu, R., Mutikainen, P.I.A., Koricheva, J., Fischer, M., 2006. How general are positive relationships between plant population size, fitness and genetic variation? Journal of Ecology. 94, 942–952. McNeely, J.A., Miller, K.R., Reid, W.V., Mittermeier, R.A., Werner, T.B., 1990. Conserving the world's Biological Diversity. IUCN Gland. Miyazawa, M., Shimamura, H., Nakamura, S., Kameoka, H., 1997. Antimutagenic activity of gigantol from Dendrobium nobile. Journal of Agriculture and Food Chemistry 45, 2849–2853. Mustajärvi, K., Siikamäki, P., Rytkönen, S., Lammi, A., 2001. Consequences of plant population size and density for plant–pollinator interactions and plant performance. Journal of Ecology 89, 80–87. Newman, D., Pilson, D., 1997. Increased probability of extinction due to decreased genetic effective population size: experimental populations of Clarkia pulchella. Evolution (N. Y) 354–362. Ng, T.B., Liu, J., Wong, J.H., Ye, X., Sze, S.C.W., Tong, Y., Zhang, K.Y., 2012. Review of research on Dendrobium, a prized folk medicine. Applied Microbiology and Biotechnology 93, 1795–1803. Okamoto, T., Natsume, M., Onaka, T., Uchimaru, F., Shimizu, M., 1966. The structure of dendroxine. The third alkaloid from Dendrobium nobile. Chemcal and Pharmaceutical Bulluletin. (Tokyo) 14, 672. Pant, B., 2013. Medicinal orchids and their uses: tissue culture a potential alternative for conservation. African Journal of Plant Science 7, 448–467. Papenfus, H.B., Naidoo, D., Pošta, M., Finnie, J.F., Van Staden, J., 2015. The effects of smoke derivatives on in vitro seed germination and development of the leopard orchid Ansellia africana. Plant Biology http://dx.doi.org/10.1111/plb.12374. Rao, V.R., Hodgkin, T., 2002. Genetic diversity and conservation and utilization of plant genetic resources. Plant Cell Tissue Organ Culture 68, 1–19. Rubluo, A., Chávez, V., Martínez, A.P., Martínez-Vázquez, O., 1993. Strategies for the recovery of endangered orchids and cacti through in-vitro culture. Biological Conservation 63, 163–169. Saccheri, I., Kuussaari, M., Kankare, M., Vikman, P., Fortelius, W., Hanski, I., 1998. Inbreeding and extinction in a butterfly metapopulation. Nature 392, 491–494. Tori, M., Nakamizo, H., Mihara, K., Sato, M., Okamoto, Y., Nakashima, K., Tanaka, M., Saito, Y., Sono, M., Gong, X., 2008. Chemical and genetic diversity of Ligularia vellerea in Yunnan, China. Phytochemistry 69, 1158–1165. Vasudevan, R., Van Staden, J., 2010. In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl. Scientia Horticulturae (Amsterdam) 123, 496–504. Vasudevan, R., Van Staden, J., 2011. Cytokinin and explant types influence in vitro plant regeneration of Leopard orchid (Ansellia africana Lindl.). Plant Cell, Tissue Organ Culture 107, 123–129. Yan, L., Wang, X., Liu, H., Tian, Y., Lian, J., Yang, R., Hao, S., Wang, X., Yang, S., Li, Q., 2014. The genome of Dendrobium officinale illuminates the biology of the important traditional chinese orchid herb. Molecular Plant 6, 922–934. Ye, Q., Qin, G., Zhao, W., 2002. Immunomodulatory sesquiterpene glycosides from Dendrobium nobile. Phytochemistry 61, 885–890.