Dactylorhiza Hatagirea Research Articles

De novo transcriptome provides insights into the growth behaviour and resveratrol and trans-stilbenes biosynthesis in Dactylorhiza hatagirea - An endangered alpine terrestrial orchid of western Himalaya

This is the first report on de novo transcriptome of Dactylorhiza hatagirea, a critically-endangered, terrestrial orchid of alpine Himalayas. The plant is acclaimed for medicinal properties but little is known about its secondary-metabolites profile or cues regulating their biosynthesis. De novo transcriptome analysis was therefore, undertaken to gain basic understanding on these aspects, while circumventing the acute limitation of plant material availability. 65,384 transcripts and finally, 37,371 unigenes were assembled de novo from a total of 236 million reads obtained from shoot, tuber and leaves of the plant. Dominance of differentially-expressing-genes (DEGs) related to cold-stress-response and plant-hormone-signal-transduction; and those involved in photosynthesis, sugar-metabolism and secondary-metabolite-synthesis provided insights into carbohydrate-partitioning in the plant during its preparation for freezing winter at natural habitat. DEGs of glucomannan, ascorbic acid, carotenoids, phylloquinone/naphthoquinones, indole alkaloids, resveratrol and stilbene biosynthesis revealed the secondary-metabolite profile of D. hatagirea. UHPLC results confirmed appreciable amounts of resveratrol and trans-stilbene in D. hatagirea tubers, for the first time. Expression analysis of 15 selected genes including those of phenylpropanoid pathway confirmed the validity of RNA-seq data. Opportunistic growth, temperature- and tissue-specific-differential-expression of secondary metabolite biosynthesis and stress tolerant genes were confirmed using clonal plants growing at 8, 15 and 25 °C.

Antipyretic Activity of Hydroalcoholic Extract of Dactylorhiza Hatagirea Roots & Lavandula Stoechas Flowers on Brewer’s Yeast Induced Pyrexia in Wistar Rats

Medicinal plants are the part and parcel of human society to combat against different diseases from the dawn of human civilization. According to World Health Organization, approximately 85% population of the developing countries are facing difficulties to afford synthetic drugs and are relying on traditional medicines mainly of plant origin in order to maintain their primary health care needs. Plants are being used in various disorders. The present pharmacological investigation was undertaken to study the anti-pyretic activity of hydroalcoholic extract of D. hatagirea roots (HEDH) & L. stoechas flowers (HELS) in albino rats against yeast induced pyrexia. Hydroalcoholic extract of D. hatagirea roots & L. stoechas flowers (5, 50, 300 and 2000 mg/ kg) orally to observe acute toxicity, and observed for 14 days. Eight groups of six animals were used for the experiment. The yeast induced pyrexia method was standardized first by injecting 15% yeast suspension (s.c) followed by recording the rectal temperature at regular intervals. Then the evaluation of anti-pyretic activity of hydroalcoholic extract of D. hatagirea roots & L. stoechas flowers (100, 200mg/kg & 300mg/kg) was carried out by using this standard procedure. The extract of D. hatagirea & L. stoechas plant showed a significant (P<0.01) dose dependent antipyretic effect in yeast induced elevation of body temperature in experimental rats. The data generated during study shows that D. hatagirea roots & L. stoechas flowers having significant anti-pyretic activity. Keywords: Dactylorhiza Hatagirea, Lavandula Stoechas, Anti-pyretic activity, Yeast induced pyrexia

Effect of Hydroalcoholic Extract of Dactylorhiza Hatagirea Roots & Lavandula Stoechas Flower on Thiopental Sodium Induced Hypnosis in Mice

Dactylorhiza hatagirea (D.Don) Soo belongs to the family orchidaceae. The plant is native and near endemic to Indian Himalayan region. Its distribution extends to Pakistan, Afghanistan, Nepal, Tibet and Bhutan. In India, it is reported from Jammu and Kashmir, Sikkim, Arunachal Pradesh, Uttarakhand and Himachal Pradesh. The Juice extracted from tuber is used as tonic and also used for the treatment of pyorrhea (inflammation of the gum & teeth). Root paste is externally applied as poultice on cuts and wounds and extract is given in intestinal disorders. Lavandula stoechas, the Spanish lavender or topped lavender or French lavender is a species of flowering plant in the family lamiaceae, ccurring naturally in several Mediterranean countries, including France, Spain, Portugal, Italy and Greece. It is used commercially in air fresheners and insecticides. Flower spikes have been used internally for headaches, irritability, feverish colds and nausea and externally for wounds, rheumatic pain, antiseptic, digestive, antispasmodic, healing, insect repellent and antibacterial. The neuropharmacological activities were examined by thiopental sodium induced sleeping time in mice at the doses of 100, 200 and 300 mg/kg p.o body weight. All the extracts exhibited significant reduction of onset and duration of sleep in thiopental sodium induced sleeping time test. Altogether, these results suggest that experimental hydroalcoholic extracts of D. hatagirea roots & L. stoechas flower possesses potent hypnotic properties, which support its use in traditional medicine.
 Keywords: Dactylorhiza hata, Lavandula stoechas, Sleeping time test, Thiopental sodium

Anti-diabetic activities of Dactylorhiza hatagirea leaf extract in 3T3-L1 cell line model

Background: Diabetes is an endocrine disorder that results in altered carbohydrate, protein, and lipid metabolism. Several synthetic drugs used to treat diabetes have adverse effects on prolonged usage. This has given the impetus to the search for alternative medicines with no or less side effects. The plants of Orchidaceae family have displayed antimicrobial, anti-inflammatory, antioxidant, anticancer, and antidiabetic activity. However, their antidiabetic properties are yet to be explored. Materials and Methods: The in vitro antidiabetic properties of Dactylorhiza hatagirea leaf extract were studied by biochemical assays such as α-amylase and α-glucosidase inhibition assays and in vitro cellular assays such as glucose uptake assay and glucose transporter type 4 (GLUT4) expression studies in 3T3-L1 cell line. Results: The methanolic extract of D. hatagirea, at varying concentrations (25 μg-400 μg/mL), did not exhibit cytotoxicity against 3T3-L1 cell line after 24 h of incubation. Methanolic extract of D. hatagirea leaves showed significant inhibition of α-Amylase and α-Glucosidase enzymes. After 24 h of exposure of 3T3-L1 cells to 100 μg/mL of D. hatagirea leaf extract and 100 μM of metformin, the relative expression rates of GLUT4 receptor were elevated when compared with untreated cells. The results also revealed that the amount of 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-Deoxyglucose taken up by 3T3-L1 cells treated with D. hatagirea leaf extract and metformin is higher than that of untreated cells. Conclusion: Our results suggest that methanolic extract of D. hatagirea leaves has potential antidiabetic activity and could be a plausible resource for antidiabetic agents.

Enhanced reproductive success revealed key strategy for persistence of devastated populations in Himalayan food‐deceptive orchid, Dactylorhiza hatagirea

AbstractAnthropogenic disturbances adversely affect populations of rare and endemic plants, resulting in reduction of their population size and performance. Among different plant groups, deceptive terrestrial orchids are vulnerable and possess greater extinction risks because of rarity in occurrence. To understand the response of food‐deceptive terrestrial orchids to disturbances, we selected Dactylorhiza hatagirea as our representative species, which is endemic to Himalaya, and studied its natural populations. This species is rare for being habitat specific, pollination limited and threatened in its natural habitats. We tested the hypothesis that disturbances lead to reduction in population size and plant performance of food‐deceptive terrestrial orchids. For assessing the impact of disturbance, two contrasting groups, heavily devastated (HD) and lightly devastated (LD), were identified on the basis of frequency and intensity of disturbance (harvesting of plant for tubers) by interviewing local people, medicinal plant extractors and shepherds. HD sites, in comparison to LD sites, were found to have smaller population sizes, but showed an increase in plant growth traits (plant height, specific leaf area, leaf N and specific shoot length). Similarly, plants at HD sites were found to have invested less in inflorescence (inflorescence size, inflorescence length, inflorescence length fraction and flowers per length), but despite that showed higher reproductive success. This was a clear indication of enhanced performance of its populations driven by disturbances. Our findings suggested that food‐deceptive species in small populations tend to reduce the probability of population extinction and have the capability to recover rapidly if conserved in time.

Inventory and Characterization of New Populations through Ecological Niche Modelling Improve Threat Assessment

Categorization of species under different threat classes is a pre-requisite for planning, management and monitoring of any species conservation programme. However, data availability, particularly at the population level, has been a major bottleneck in the correct categorization of threatened species. Till date, threat assessments have been mostly based on expert opinion and/or herbarium records. The availability of primary data on distribution of species and their p opulation attributes is limited in India because of inadequate field survey, which has been ascribed to resource constraints and inaccessibility. In this study, we demonstrate that ecological niche modelling (ENM) can be an economical and effective tool to guide surveys overcoming the above two constraints leading to the discovery of new populations of threatened species. Such data lead to improved threat assessment and more accurate categorization. We selected 14 threatened plants comprising 5 trees ( Acer hookeri Miq., Bhesa robusta (Roxb.) Ding Hou, Gynocardia odorata Roxb., Ilex venulosa Hook. f. and Lagerstroemia minuticarpa Debb. ex P.C. Kanjilal), 8 herbs ( Angelica glauca Edgew., Aquilegia nivalis Falc. ex Jackson, Artemisia amygdalina DC., Begonia satrapis C.B. Clarke, Corydalis cashmeriana Royle, Dactylorhiza hatagirea (D. Don) Soo, Podophyllum hexandrum Royle, and Rheum australe D. Don), and 1 pteridophyte ( Angiopteris evecta (Forst.) Hoffm.) having distribution range in North East India, Eastern and Western Himalaya, and Jammu and Kashmir. The study was carried out between 2012 and 2016. ENM-based survey led to the discovery and characterization of 348 new populations. The data so obtained helped in assigning conservation status to 10 species, which earlier were never classified due to data deficiency. Using the new population and distribution data of the remaining four species, only one was confirmed regarding its existing status and two species were classified as ‘Critically endangered’ instead of the present classification as ‘Endangered’. The fourth species was classified as ‘Critically endangered’ against the earlier category of ‘Least concerned’.

Medicinal plants conservation and development areas (MPCDAs)-An initiative towards conservation of medicinal plants

India is one of the hot spots of world's biodiversity having wealth of medicinal plants. Forests are the main habitat of Medicinal Plants and a large number of species are also endemic that gives India a unique position in the world. Medicinal plants form the major resource base of our traditional systems of medicine or AYUSH systems of medicines and folklore medicines. Even allopathic medicines also used plant based raw material for the preparation of medicine. However, due to various anthropogenic activities and unsustainable harvesting from wild, many medicinal plants species in India have become endangered and have been included in Red Data Book of threatened species. Thus, there is an urgent need to have measures taken to protect/conserve the natural habitats of these medicinal plants wealth of the Country. Some important medicinal Plants species of conservation concern in India are Aconitum heterophyllum (Atis), Aconitum atrox (Mitha), Bergenia ciliata (Pashan bhed), Commiphora wightii (Guglu), Dactylorhiza hatagirea (Salampanja), Nardostachys grandiflora (Jatamansi), Paris polyphylla (Satva), Picrorhiza kurrooa (Kutki), Podophyllum hexandrum (Bankakri), Rauvolfia serpentina (Sarpagandha), Rheum australe (Revand chini), Saraca asoaca (Ashok) etc. The National Medicinal Plants Board (NMPB), Government of India, in this regard, has taken initiatives to support in-situ conservation of medicinal plants through establishment of Medicinal Plants Conservation and Development Areas (MPCDAs) throughout the country. For in-situ conservation, the State Forest Departments are playing a vital role in establishing the MPCDAs, ensuring conservation of the Red Listed Medicinal Plants species along with associated flora. Considering the richness in diversity of medicinal plants in India, there is still a good scope for conservation of these resources in their natural habitats through establishment of MPCDAs throughout the country. In addition, considering the diversity of medicinal flora, the state forest department in collaboration with research institutions should come forward to carryout research and development studies on conservation as part of germplasm of endemic medicinal plants species and augment the medicinal plants biodiversity base in the country.

Ecological status of high altitude medicinal plants and their sustainability: Lingshi, Bhutan.

BackgroundHuman beings use plants for a multitude of purposes of which a prominent one across the globe is for their medicinal values. Medicinal plants serve as one of the major sources of income for high altitude inhabitants in the Himalaya, particularly in countries like Nepal, and Bhutan. People here harvest huge volumes of medicinal plants indiscriminately, risking their sustainability. This paper attempts to identify some of the priority medicinal plant species harvested in the wild and assess their ecological status for their judicious utilization, and to help provide policy guidance for possible domestication and support strategic conservation frameworks.ResultsOut of the 16 priority species identified by the expert group, collectors’ perception on ecological status of the priority species differed from survey findings. Chrysosplenium nudicaule (clumps) ranked as most threatened species followed by Corydalis dubia, and Meconopsis simplicifolia. Onosma hookeri, Corydalis crispa and Delphinium glaciale were some of the species ranked as threatened species followed by Halenia elliptica (not in priority list). Percent relative abundance showed irregular pattern of species distribution. High species evenness was recorded among Nardostachys grandiflora, Chrysosplenium nudicaule, Saussurea gossypiphora and Aconitum orochryseum with average species density of 8 plant m−2. Rhodiola crenulata, and Dactylorhiza hatagirea followed by Meconopsis horridula and Meconopsis simplicifolia were ranked as most threatened species with average species density of 0.4, 0.4, 5.6 and 6.0 plant m−2, respectively. The most abundant (common) species was Onosma hookeri (plant m−2). Species composition and density also differed with vegetation, altitude, slope and its aspects.ConclusionPriority species identified by expert group were found vulnerable and patchy in distribution. Survey results and collectors’ perceptions tally to an extent. Some of the species (Dactylorhiza hatagirea, Rhodiola crenulata, Meconopsis simplicifolia and Meconopsis horridula) were critically low in plant density with less than a plant per m2 while Delphinium glaciale, Fritillaria delavayi and Aconitum orochryseum were confined to narrow altitude range. Collectors were aware that most species identified in priority list are threatened and existing harvesting plan are hardly implemented as it is not pragmatic. Moreover, major chunk of medicinal plants harvested remain unaccounted as illegal harvest and marketing seemed to occur across the borders. Policing and monitoring would continue to be a challenge given the rugged terrain and harsh climate. In-depth study and further monitoring of low density species is suggested to ensure its sustainability through long term strategy development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12898-016-0100-1) contains supplementary material, which is available to authorized users.

Ecological status and diversity indices of Panchaule (Dactylorhiza hatagirea) and its associates in Lete village of Mustang district, Nepal

This paper focuses on the ecological status and diversity indices: Simson’s Index (C), Simson’s Index of Dominance (D) and Shannon-Weaver Index (H) of Dactylorhiza hatagirea and its associates- Rheum australe and Rumex nepalensis in Lete village of Mustang District within the Annapurna Conservation Area. The study was conducted during the monsoon season (June/July) of 2013 in the Lete VDC of Mustang District. The study site possessed an area of 4.5 ha. Altogether, 100 circular plots, each with 25 m2 area, were laid out purposively within the study area; the sampling intensity being 5.55%. The relative frequency, the relative density, the abundance, the relative coverage and the Important Value Index of the species were found to be 61.11, 53.91, 1,061.54, 72.2 and 187.24 respectively. Similarly, the Simson’s Index (C), the Simson’s Index of Dominance (D) and the Shannon-Weaver Index of the species were found to be 0.41, 0.59 and 3.27 respectively, indicating relatively even and relatively diverse community. The study showed relatively higher values of all the parameters of D. hatagirea as compared to its associates indicating good ecological value. However, threats remain due to the illegal harvesting of this valuable orchid and overgrazing in the study site.Banko JanakariA Journal of Forestry Information for NepalVol. 26, No. 1, Page: 45-52, 2016

Peoples’ Perception and Conservation of <i>Dactylorhiza hatagirea</i> (D. Don) Soó in Manaslu Conservation Area, Central Nepal

The present study analyzes the information and perception of the local community of Samagaun VDC, Manaslu Conservation Area Project (MCAP) regarding Dactylorhiza hatagirea (D. Don) Soó (Orchidaceae). We assessed the local peoples’ perception on its population status, its availability, factors causing its decline and management practices of this terrestrial orchids. A pre-designed questionnaire was used to gather information targeting the age group between 25 and 60 years (n = 75, 45 male and 30 female). Most of the informants (76%) believe that the abundance of this orchid is declining. Over grazing of domestic animals, over harvesting and lack of awareness among the local community were determined to be the major causes of decline of D. hatagirea in the study area. Protection measures as prescribed by the informants were control grazing, raising awareness among the individuals and sustainable harvestings for the long-term conservation of the species. Systematic management plans that incorporate the participation of local individuals and prioritization of their views will be applicable for the proper conservation of the species.

Microscopic Studies ofin vitroSeed Germination and Protocorm development inDactylorhiza hatagirea(D. Don) Soo: A critically Endangered Medicinal Orchid of Northwestern Himalaya

Dactylorhiza hatagirea (D. Don.) Soo is a critically endangered medicinal orchid of North-western Himalayan region. Dactylorhiza hatagi-rea is one of the very few orchids known for its therapeutic and medicinal significance. In nature, D. hatagirea plant regeneration takes place through seeds but the seeds are minute and without endosperm, therefore, mycorrhizal asso-ciation is needed for germination; as a result they do not germinate readily. In the present study, seed germination and protocorm development was standardized under in vitro conditions. Com-bination of 2-iP (0.5 mgl−1) and with BA (1.0 mgl−1) showed better performance in terms of per cent survival, multiplication and growth of proto-corms. Formation of protocorm and initiation of shoot meristem was observed in 70±5 days. Light and Scanning electron microscopic studies were also performed to describe the stages of germination, development of shoot apex and leaf primordia in a 225±3 days old protocorm.

Integrated analysis of the trees and associated under-canopy species in a subalpine forest of western Himalaya, Uttarakhand, India

Subalpine forests are known as outstanding habitats due to co-existence of both temperate and alpine vegetation and are classic example of ecotonal zones. Limited but diverse physiognomy of trees inhabiting in subalpine forest results in variability within under-canopy habitat conditions. Studies were undertaken to assess population status, habitat preference and interferences to the trees and associated under-canopy herbs in a subalpine forest of western Himalaya. A total of 10 woody and 23 under-canopy herbs were recorded in the selected subalpine forest area. At each stand, the number of tree species and under-canopy herbs ranged from 2 to 4 and 8 to 10 respectively. Abies spectabilis, Acer caesium, Quercus floribunda, Q. semecarpifolia and Rhododendron arboreum were key tree species in this area. The density of main woody species was 280 to 1190 individuals ha-1 at different stands. Herbaceous plants with rosette and clump growth habits were observed to have higher values for total basal cover and importance value index. Presence of some under-canopy herbs like; Dactylorhiza hatagirea, Malaxis muscifera, Picrorhiza kurrooa, Polygonatum cirrhifolium and Skimmia laureola in the specific habitats also showed that they are habitat specific. However, the presence of Frageria nubicola and Viola sp. was common in the selected stands. Felling of trees for timber, construction of temporary huts, fuel wood and lopping for fodder were main interferences for trees. On the other hand, trampling driven damage due to grazing, habitats degradation and overexploitation were observed key threats for under-canopy herbs. Integrated analysis including population studies, habitats preference and interferences to the trees and under-canopy herbs in this sensitive and important ecosystem will be useful for determining the conservation plans and ecosystem management.

Characterization of novel polymorphic microsatellite markers in Dactylorhiza hatagirea: a critically endangered orchid species from western Himalayas

Dactylorhiza hatagirea (D. Don) Soo, family Orchidaceae is an endangered medicinal herb inhabiting higher altitudes of western Himalayas. Due to reckless exploitation for its roots, it is of high conservation concern. In the present study fifteen microsatellites were developed and characterized across twenty collections of D. hatagirea. The total numbers of alleles amplified by these microsatellites were 64 with an average of 4.2 alleles per marker. Average observed and expected heterozygosity values for polymorphic loci were 0.623 and 0.631, respectively. Mean polymorphism information content value of the polymorphic markers was 0.532. Of the fourteen polymorphic microsatellites, 7 deviated from Hardy–Weinberg equilibrium. Microsatellites reported here can be utilized to address questions related to genetic characteristics in this species.

Status, Distribution, Conservation and Use Value of Medicinal and Aromatic Plants (MAPs) in Sagarmatha National Park, Nepal

The study attempts to assess the status, distribution, conservation and use value of Medicinal and Aromatic Plant (MAPs) in the Sagarmatha National Park. Altogether 62 species of MAPs belonging to 47 genera and 33 families have been recorded in the study area. 10 species, belonging to 9 families are categorized as the potential species. Out of the these species, most potential in local but threatened species are Allium hypsistum Stearn, Cordyceps sinensis Sacc, Dactylorhiza hatagirea Soo, Nardostachys grandiflora DC, Aconitum orochryseum, Ephedra gerardiana Wall. Ex. Stapf, Swertia multicaulis D. Don, Picrorhiza scrophulariflora Penne, Rheum australe. D. Don, Malva verticillataL and Swertia pedicallata Benerji. By analysis of data using Simpson’s diversity index (SI) and Shannon weaver function (H’), there was high diversity (more heterogeneous) MAPs species composition in the Manjo Gate to Large Dobhan. (0.98349). Less diversity (less heterogeneous) MAPs species composition was in Tyanboche to Pangoche (0.90419). Similarly, the Shannon weaver function shows that in plots laying out in Mongla to Phorche are evenly distributed than others However, higher MAPs species (i.e., 31) was found in the way of Manjo Gate to Larja Dobhan than others.

Altitudinal Gradients Affecting Floristic Diversity in Shingan Valley of Rakchham-Chitkul Wildlife Sanctuary District Kinnaur, Himachal Pradesh

A study to understand and assess the impact of altitudinal gradients on the floristic diversity in Shingan valley falling in Rakchham-Chitkul Wildlife Sanctuary district Kinnaur, Himachal Pradesh was carried out during the year 2008. The altitudinal gradient in the present case varied from 2800m to 4300m above msl. Total number of plant species as recorded was 118 belonging to 46 families and 87 genera. The dominant families registered were Rosaceae, Asteraceae, Lamiaceae, Ranunculaceae and Ploygonaceae. The number of tree species was 11 and 4 with the dominance of Pinus wallichiana and Betula utilis at 2800-3300m and 3300-3800m elevation respectively. The number of shrub species was 15, 13 and 6 with the dominance of Salix fragilis, Juniperus indica and Rhododendron anthopogon at the elevation of 2800-3300m, 3300-3800m and 3800-4300m. The number of herbs species was 63, 55 and 40 at 2800-3300m, 3300-3800m and 3800-4300m elevation respectively. Caltha palustris, Anaphalis triplinervis and Thymus linearis were the dominant shrub at these elevations respectively. The distribution pattern of species was mostly contiguous in all the altitude ranges. Index of diversity for herb species in different elevations ranges was 3.75, 3.66 and 3.44 for 2800-3300m 3300-3800m and 3800-4300m elevation respectively. Out of the 58 medicinal plant species as recorded from the valley, 14 species like Dactylorhiza hatagirea, Angelica glauca, Betula utilis, Podophyllum hexandrum, Picrorhiza kurrooa, Rheum australe, Taxus wallichiana, Heracleum lanatum, Polygonatum verticillatum, Rheum webbianum , Rhodiola heterodonta, Rhododendron anthopogon, Rhododendron campanulatum, Rhododendron lepidotum etc. fall in the category of threatened plants as per IUCN categorization.

Morphobiochemical Variability and Selection Strategies for the Germplasm ofDactylorhiza hatagirea(D. Don) Soo: An Endangered Medicinal Orchid

Dactylorhiza hatagirea(D. Don) Soo (Orchidaceae) is an important endangered medicinal herb, distributed in subalpine to alpine regions of the Himalayas. Its tubers are important constituents of many medicines and health tonics. Overexploitation for medicinal uses has decreased availability in natural habitats and this species has been enlisted as endangered, making conservation and cultivation studies necessary. Variability studies may serve as an important tool for effective conservation and for a crop improvement program. Therefore, natural populations ofD. hatagireawere analyzed for variability on the basis of morphological, biochemical, and isoenzyme patterns. The studied populations were grouped into two clusters. Existing variability among different populations opens up new areas for conservation and perspectives for a genetic improvement program forD. hatagirea.

Ecology and Conservation Status of Threatened Orchid <i>Dactylorhiza hatagirea</i> (D. Don) Soo in Manaslu Conservation Area, Central Nepal

Dactylorhiza hatagirea (D. Don) Soo, commonly known as “Panchaunle”, is an important medicinal plant species distributed throughout the Himalaya region. The present work was conducted in order to assess its availability in natural habitats because of its medicinal properties, cultural significance and declining population density. The work focused primarily on its ecological features and conservation status. The density of D. hatagirea was found to be 2.18 individuals·m-2 with frequency and abundance being 81.81% and 2.67 individuals·m-2, respectively. We considered grazing, trampling, number of cattle and distance to settlement as anthropogenic factors. While grazing and trampling were shown to have a significant, negative impact on population density, we observed no significant change in the number of the species with respect to number of cattle and distance to settlement. We conclude that the species is threatened and that anthropogenic factors have a significant effect on its habitation in the study area.

In vitro protocorm development and mass multiplication of an endangered orchid, Dactylorhiza hatagirea

Immature seeds were cultured on 10 different media for germination. Maximum germination was achieved on Lindeman orchid medium (37.12%) within 17 days of culturing. Protocorms with leaf primordia were cultured on BM-2 and 7 different modifications of Murashige and Skoog (MS) medium with various hormone combinations [0-3 mg/L indole butyric acid (IBA) and 0-3 mg/L kinetin (Kin)] for plantlet regeneration and mass multiplication. Maximum number of shoots (18.12 ± 0.3), highest shoot length (17.80 cm ± 2.16), maximum root number (8.25 ± 0.69), and highest root length (8.02 cm ± 1.45) were found on MS medium with 3 mg/L IBA and 1 mg/L Kin. Plantlets with 2-3 shoots were transferred to different potting mixtures for acclimatization to field conditions and further multiplication. One hundred percent survival was obtained in C-8 potting mixture consisting of cocopeat + vermiculite + perlite (1:1:1), which produced 75 shoots (25 plantlets) after 1 month of transplantation in the greenhouse. The current study describes for the first time a rapid in vitro protocorm development and mass multiplication protocol for Dactylorhiza hatagirea (D.Don) Soo that holds robust potential for large-scale propagation and metabolite production.

Population genetic structure and conservation of small fragmented locations of Dactylorhiza hatagirea in Ladakh region of India

Analysis of molecular variance (AMOVA) of ISSR analysis revealed a moderate level of genetic differentiation among populations (60% of the total variance components, P<0.001), with gene differentiation coefficients (Gst=0.258, Fst=0.135) and moderate gene flow (Nm=1.432). Both Principal coordinate's analysis (PCoA) and Neighbor-Joining (NJ) cluster analysis supported the grouping of all 96 sample sizes of nine locations into two collection groups. The present level and pattern of genetic diversity and structure of Dactylorhiza hatagirea were assumed to result largely from its habitat fragmentation, its unique biological traits and evolutionary history. The genetic structure could be attributed to an earlier period of more pronounced gene flow when the species had a more continuous distribution. A Mantel test revealed no significant positive correlation between genetic distances and geographic distances (R2=0.0165, P=0.290).

Development and characterization of polymorphic microsatellite marker for Dactylorhiza hatagirea (D. Don) Soo

Dactylorhiza hatagirea (D. Don) Soo is a critically endangered orchid distributed in eastern China and central Asia. In order to investigate its genetic diversity within and between populations and design effective conservation strategies, we developed microsatellite marker for this species in the present study. A total of 14 polymorphic markers were isolated and characterized using 68 individuals from three natural populations. The number of alleles per locus for a single population ranged from 2 to 15, and the polymorphism information content ranged from 0.043 to 0.908. No genotypic linkage disequilibrium within populations was detected. These microsatellite markers are the first characterized for D. hatagirea and they are expected to be useful for population level studies in this species.