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Treatment with medicinal plants consists of the ancient tradition of India and which has been continued for years. Our ancestors collected rare plants from forests and mountains for treatment purpose. At the same time, they give deep concern for the conservation of the species on the earth. Their knowledge and services are not profitable but valuable. The study is focused on the phytochemical and antimicrobial activity of the medicinal plant Rhynchostylis retusa of the family Orchidaceae. The plant is growing in sacred groves and local gardens of Payyanur of Kannur district, Kerala, India and used for traditional healing practices. The Orchidaceae family includes many plants having medicinal properties, but most of them were not studied or exploited yet. Qualitative analysis of primary metabolites and secondary metabolites indicates the presence of phytochemicals in Rhynchostylis retusa. The total alkaloid, carbohydrate, protein, phenolic compounds and flavonoids were seen in significant amount in this plant. In vitro antibacterial (agar well diffusion) activity was done by gram-negative Pseudomonas aeruginosa (ATCC 27853), gram-positive Streptococcus mutans (MTCC 890), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and antifungal activity by Aspergillus niger (ATCC 16404). The plant extracts exhibited potent antimicrobial activity against these strains. The studies with the medicinal plant R.retusa make clear that the plant is rich in active components and phytochemicals. The plant contains a favourable amount of phytochemicals and this makes them cure various diseases. The plant also shows antioxidant and antibacterial activities.

Isolation and identification of mycorrhizal fungi from the roots of Rhynchostylis retusa indicated that the cultural and microscopic features, namely colony appearance, colony colour, diameter of vegetative hyphae, presence of monilioid cells, right angle branching pattern of the fungal endophyte corroborated the identity of the fungus Rhizoctonia like anamorphs of Ceratobasidium species. Fungal identity was further confirmed through sequencing and analysis of internal transcribed spacer (ITS) sequences of the nuclear ribosomal DNA (nrDNA). ITS sequence of the isolate RhY10A6 (accession no. MN120903.1) showed > 99% similarity with several isolates of the teleomorphic fungus Ceratobasidium sp. in NCBI mega blast search. The phylogenetic analysis based on maximum parsimony method, this orchid mycorrhizal fungus clustered with several isolates of Ceratobasidium sp. or Rhizoctonia like fungi. It showed near distant relation with Ceratobasidium ramicola (GeneBank accession no. NR_138368.1) which is an orchid mycorrhizal fungus. Therefore, molecular characterization validated the morphological data. The techniques established in this orchid will facilitate to isolate and accurate identification of mycorrhizal fungus.

The present study on in vitro seed germination of Rhynchostylis retusa obtained on nutrient medium made clear pathway for the in vitro propagation of orchids by seed culture. The presence of coconut water in the medium enhanced seed germination and growth of seedlings. For enhancing the growth of seedlings further the individual seedlings were cultured on MS medium supplemented with 3 mg/l BAP and 1g/l activated charcoal. These seedlings were sub cultured on rooting medium (IBA) supplemented with activated charcoal. The plantlets were transferred to soil with 90% success. Hence the present investigation made possible for the large scale multiplication of R.retusa and thus to conserve this endemic orchid.

This investigation portrayed phytochemical screening, antioxidant, anti-inflammatory, antimicrobial, cytotoxic and analgesic activities of three medicinally important epiphytic orchids of Bangladesh, namely Rhynchostylis retusa (L.) Bl., Luisia zeylanica Lindl. and Papilionanthe teres (Roxb.) Schltr. The methanolic extracts of the leaves and roots of R. retusa and the leaves and stems of L. zeylanica and P. teres were used in the present investigation. Quantitative phytochemical screening revealed the presence of a number of bioactive phytochemicals, such as alkaloids, carbohydrates, flavonoids, glycosides, tannins, phenols, xanthoproteics, proteins, saponins, cardiac glycosides, coumarins, quinines, flavonols, steroids, terpenoids, phytosterols, phlobatannins etc. Antioxidant activity of the selected orchids showed significant DPPH free radical scavenging activity compared with that of ascorbic acid at 100 μg/ml. Excellent anti-inflammatory activity was also showed in these orchids, and the highest anti-inflammatory activity was recorded in P. teres (88.22%). Total phenol content was measured by using Folin-Ciocalteu reagent gallic acid was used as a reference standard. The maximum total phenol content was 205.33 mg/g GAE in L. zeylanica, and the minimum was 36.67 mg/g GAE in the roots of R. retusa. Antibacterial activity of methanolic extracts was tested against Bacillus subtilis, Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa and Escherichia coli. The maximum antibacterial activity was observed against Bacillus subtilis. The antifungal activity was also studied against Aspergillus niger and Fusarium oxysporum, and the highest antifungal activity was recorded against Aspergillus niger (70%) by P. teres. In brine shrimp lethality bioassay, encouraging cytotoxicity was observed after 24 h, and the maximum mortality of nauplii was recorded in L. zeylanica (LC50 value 77.175 μg/ml) as compared with anticancer drugs ‘vincristine sulphate’ (LC50 value 0.25 μg/ml). In analgesic assay, remarkable inhibitory effect was observed in acetic-acid-induced writhing test on mice by the extract of P. teres (86.19%). The percentage of paw licking inhibition was high in the late phase than in the early phase. The findings of the present investigation will facilitate the designing of new drugs to comate challenging diseases.

Rhynchostylis retusa (L.) is an epiphytic orchid having both medicinal and ornamental value. The present study aimed to develop a protocol for mass propagation of R. retusa and evaluate the genetic stability of in vitro regenerants. The immature seeds obtained from capsule of mother plants (wild type) were cultured on different strength of MS medium viz. full (FMS), half (HMS), quarter (QMS) supplemented with BAP (0.5 mg/L), NAA (0.5 mg/L), and 5% or 10 % coconut water (CW). The earliest germination and protocorm development were observed on HMS and QMS media. Mature protocorms produced higher number of shoots (12.8) and longest shoots (5.3 cm) on FMS supplemented with 10 % CW. The maximum number of roots (7.3) and root length (5.0 cm) were observed on FMS when supplemented with fungal elicitor CVS4 (extracted from the stem of Vanda cristata). Ten RAPD primers were used to analyze genetic stability among six samples (five in vitro and one mother plant) produced a total of 23 fragments ranging from 275bp to 1100bp. The polymorphic information content (PIC) values ranged from 0.28 to 0.50. The amplified bands of all the samples of in vitro plants were similar to bands of mother plant. The result demonstrated the genetic stability of the micropropagated plants of R. retusa. Research reported here is indicating the applicability of tissue culture for true-to-type plant production and conservation of R. retusa.

International audience; The present study aims to identify the diverse endophytic fungi residing in the roots of Dendrobium moniliforme and their role in plant growth and development. Nine endophytic fungi were isolated from the root sections and characterized by molecular technique. Quantification of the indole acetic acid (IAA) compound by these endophytes was done. Further, Chemical profiling of R11 and R13 fungi was done by Gas Chromatography-Mass Spectroscopy (GC-MS). Asymbiotic seed derived protocorms of Rhynchostylis retusa was used for the plant growth assay to investigate the growth promoting activities of the fungal elicitor prepared from the isolated fungi from D. moniliforme. Among the isolated fungi, the relative dominant fungus was Fusarium sp. The R13 and R6 fungi were identified only at the genus level which concludes the fungi are of new species or strain. The indole acetic acid production was relatively higher in R10. Bioactive compound diversity was observed in the organic extract of R11 and R6. The presence of phenolic compound and essential oil suggest their contribution for the antimicrobial and antioxidant properties to their host plant, D. moniliforme. The plant growth assay result concluded, the fungal elicitor prepared from R10, Colletotrichum alatae was the best among all other for the plant growth activities.

Orchid is one ofthe flowering plants with a very high level of diversity, including the diversity of morphological characters it has. This study aims to determine the diversity of orchids (orchidaceae) seen morphologically in the Ngalian sub-district forest in Semarang city. The method used in this study is cruise method and characterization techniques. Analysis of research data was carried out descriptively and displayed in the form of tables and photos. Orchids in the teak forest cover 940 ha, spread in 4 villages, namely Wonosari village, Gondoriyo village, Podorejo village and Wates village. The results showed that there were 3 types of orchid plants (orchidaceae). The types of orchids include Rhynchostylis retusa (L.) Bi., Rhynchostylis gigantea (Lindl.) Rindl. and Dendrobium crumenatum (L.) Sw. The diversity of morphological characters in each species of orchid such as diversity in leaf color, leafshape, leaftip shape, stem color, root type etc.

The aimed of this research is to increase Rhynchostylis retusa callus formation through immersing explants in ascorbic acid and planting it on activated charcoal contained medium. It's an experimental research that consist of three factors; i.e. immersion explants in ascorbic acid (immersed and not immersed), kind of in-vitro culture medium (not-contained (as control), pro-analysis activated charcoal contained and commercial activated charcoal contained), and the type of expants (leaf and root). The source of explants used are 18 months after planting (map) Rhynchostylis retusa orchids plants in in-vitro cultures. The explants condition (the level of browning) and callus formation (the percentage of callus forming explants, the time of callus appearance, and callus diameters) were observed at8 weeks after planting (wap).The result show that callus formation can be induced by medium that contain activated charcoal of both kind (pro-analysis or commercial).Combination of the treatment with explants immersionin ascorbic acid before planting can increase the callus formation. The root explants form higher callus diameters (0,563 mm) than leaf (0,298 mm).

Two fungal endophytes isolated from the roots of epiphytic orchids Rhynchostylis retusa and Aerides multiflorum were employed for biological hardening of asymbiotically raised seedlings of Cymbidium aloifolium and C. giganteum. These fungal isolates showed resemblance to Rhizoctonia-like fungi in their cultural characteristics, microscopic features and ITS region sequences. The ITS region sequences of Ceratobasidium sp. strain RR showed 100% identity with Rhizoctonia sp. M2ao1 and Ceratobasidium sp. FPUB 168, whereas Ceratobasidium sp. strain AM displayed 89% identity with Ceratobasidium sp. JTO 031. The fungal isolates promoted growth and survival of seedlings of both species of Cymbidium, with greater promotory effects of Ceratobasidium sp. strain RR. The higher survival and growth increment in inoculated seedlings of C. aloifolium emphasized the importance of biological hardening of asymbiotically grown orchid seedlings.

For high frequency regeneration of Rhyncnostylis retusa (Lin.) Blume apical nodal segments were used. Half strength MS + 2% sucrose + 1.5 mg/l BA + 0.5 mg/l NAA + 2 g/l peptone + 10% (v/v) coconut water (CW) + 0.5 g/l activated charcoal (AC) was the best nutrient medium, on which 89% cultures induced 8 microshoots per culture. Subculture of microshoots for further 8 weeks on the same nutrient medium enhanced the number of microshoots up to 95. For further proliferation of microshoots, their development into shoots as well as formation of secondary microshoots from the base of the old ones, the best medium was half strength of MS + 2% sucrose + 2 g/l peptone + 10% (v/v) CW + 0.5 g/l AC + 150 mg/l L-glutamine. Plantlets with roots were obtained in half strength of MS + 2% sucrose + 2 g/l peptone + 10% (v/v) CW + 0.5 g/l AC + 5.0 g/l banana powder, on which cent per cent shoots rooted within eight weeks. The pH of all the categories of cultures were maintained at 5.6 before adding 2.2 g/l gelrite and autoclaving, and the cultures were incubated at 2000 - 3000 lux for 16/8 hrs light/dark at 24 ± 2ºC. Regeneration of plantlets continued due to repeated subculture of microshoots and regenerants were acclimatized and established in the nursery. Plant Tissue Cult. & Biotech. 22(1): 1-11, 2012 (June) DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11242

We studied distribution pattern of the epiphytic orchid Rhynchostylis retusa (L.) Blume with respect to (i) site characteristics and host conditions, and (ii) the type and intensity of land use in Kathmandu Valley, central Nepal. We established a 1.5 km grid net and analyzed epiphytic orchids at each point, searching for 10 trees as close as possible to the grid point. There we analyzed bark water-holding capacity, bark pH, bark roughness and light intensity. We assessed the probability of the occurrence of R. retusa in different land use patterns. Our results indicated that R. retusa was not a host-specific orchid species. It was found on different host tree species. However, Ficus religiosa was the most common host species. The correlation between R. retusa occurrence and microclimate condition was weak. R. retusa, to a certain degree, preferred light intensity of 40-80% of full sun light, rough bark with pH around 6.5 and bark with a wide range of water holding capacity. The distribution pattern of R. retusa was influenced by certain types of land use. The probability to find R. retusa was highest in forest patches and parks and lowest in agricultural and dense populated area. The study reveals that to improve the population size of R. retusa, trees (mainly Alnus nepalensis, Ficus religiosa and Schima wallichii) should be planted in areas where the orchid species is recently missing.doi: http://dx.doi.org/10.3126/botor.v8i0.5956 Botanica Orientalis – Journal of Plant Science (2011) 8: 90-99

This brief chapter discusses the botanical and medicinal usage of Renanthera and Rhynchostylis, two very popular tropical orchids in cultivation, and Robequetia. Rhynchostylis retusa has a prominent place among Indian medicinal plants described by van Rheede in 1703 in his Hortus Indicus Malabaricus, the first European publication in which medicinal Asian orchid species were described.

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L−1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.

SummaryThe effects of two gelling agents (agar or phytagel) and two light sources (cool fluorescent light or photosynthetically active radiation) on micropropagation of Rosa damascena Mill. and Rhynchostylis retusa (L.) Bl. were studied. Enhanced growth and better shoot proliferation was observed in cultures grown on medium gelled with phytagel (0.22%, w/v) and supplied with photosynthetically active radiation (PAR) as compared with cultures grown on medium gelled with agar (0.8%, w/v) and maintained under cool fluorescent lights (CFL). Direct protocorm-like bodies (PLBs) differentiated from the roots of regenerated plantlets of R. retusa on MS medium supplemented with 2.5 μM BA when the cultures were kept under PAR. While PLB differentiation was observed from the leaves of similar cultures incubated under CFL, better response was observed in cultures grown on medium containing phytagel. Differences were also recorded in respect of some physiological parameters, e.g. the relative water content, osmotic po...

Physical seed characteristics of 32 threatened Western Himalayan orchids belonging to 23 genera of 3 subfamilies were studied using light and scanning electron microscopy. The species showed remarkable seed shape variations (fusiform, spathulate, ovoid, filiform) irrespective of their taxonomic position. Epiphytes produced relatively small-sized seeds compared to their terrestrial, lithophytic, or mycoheterotrophic counterparts. Seeds were truncated (length/width < 6.0) in 27 species, and were mostly elongated (length/width = 19.957 ± 1.459) in the orchidoid species Goodyera biflora (Lindl.) Hook.f. The number or size of testa cells was found fairly consistent at the genus level, and their walls were straight, sinuous, or undulate. Periclinal walls possessed vertical or oblique ornamentation in 6 species, which is of taxonomic implication. Embryos were rather tiny and most of the seed space (even >90% in 7 species) was occupied by air. The highest seed to embryo volume ratio was recorded in terrestrial Liparis odorata (Willd.) Lindl. and the lowest in epiphytic Rhynchostylis retusa (L.) Blume, both of which are members of the advanced Epidendroideae. Seed characteristics are important in elucidating taxonomic, phylogenetic, and phytogeographic relationships among different orchid taxa.

The possibility of using anthers for initiating in vitro cultures of Rhynchostylis retusa (Orchidaceae), has been positively tested. The study was designed to study the effects of physiological status, stage of microspore development and pre-cold treatment on androgenic response in R.retusa. The anthers from open flowers (2 days of anthesis) in tetrad stage failed to respond despite variations in the chemical regime; whereas those from unopened buds (1.25-1.35cm long) with an intact operculum & pre-cold treatment with microspores in early & late-uninucleate decussate regenerated provided their nutritional complexities were satisfied through an exogeneous supply of Peptone (2mg/l) in the BAP (10mg/l) and NAA enriched Sharma medium. The best response was obtained when a 24hrs cold treatment was employed at 4°C in darkness. The complete plantlet (2-3 leaves &1-2 roots) was formed in 24wks.The plantlet was accilimatized & the survival rate is 70%.

An experiment was carried out during 2013–14 and 2014–2015. The experiment was laid out in factorial CRD with two factors, mineral nutrients viz., M1 (RDF+Zn 250 ppm), M2 (RDF+Zn 500 ppm), M3 (RDF+Zn 750 ppm), M4 (RDF+Mg 250 ppm), M5 (RDF+Mg 500 ppm), M6 (RDF+Mg 1000 ppm), M7 (RDF+Ca 250 ppm), M8 (RDF+Ca 500 ppm), M9 (RDF+Ca 1000 ppm) and M10 (control) and growing supports viz., G1 (side perforated earthen pot), G2(bamboo basket), G3(coco husk block) and G4 (wooden log). M8 was found superior in respect of growth characters viz., plant height (41.14 cm), number of leaves per plant (8.33) and M3 in respect of flower characters viz., minimum days to full bloom (84.83), highest spike length (41.65 cm), number of florets per spike (88.79), number of spikes per plant (3.58), self life (16.50 days) and vase life (11.17 days). However, the highest plant height (38.72 cm) and number of leaves per plant (7.13) were recorded in G1 as well as minimum days for full bloom (87.38), the highest spike length (36.97 cm), number of florets per spike (79.23), number of spikes per plant (2.70), self life (13.33 days) and vase life (9.25 days) were recorded in G2. M8G1 and M3G2 considered as the suitable combinations for growth and flowering parameters, respectively.

Epidendrum retusum Linnaeus, Species Plantarum 2: 953. 1753. "Habitat in India." RCN: 6897. Lectotype (Majumdar & Bakshi in Taxon 28: 354. 1979): [icon] " Ansjeli-maravara " in Rheede, Hort. Malab. 12: 1, t. 1. 1693. Current name: Rhynchostylis retusa (L.) Blume (Orchidaceae)., Published as part of Jarvis, Charlie, 2007, Chapter 7: Linnaean Plant Names and their Types (part E), pp. 490-515 in Order out of Chaos. Linnaean Plant Types and their Types, London :Linnaean Society of London in association with the Natural History Museum on page 495, DOI: 10.5281/zenodo.291971

Meru Betiri National Park is located in southern part of East Java Province. Inventory of orchid species was conducted to study orchid diversity in Meru Betiri National Park, especially in Bandealit coastal area. Observation of orchid within host trees was also done to study the preference host trees for orchid growth. It was recorded that there were 25 orchid species belonging to 20 genera. Twenty species of which are epiphyte and 5 species are terrestrial. The most common epiphyte orchids were Pomatocalpa latifolia, Pomatocalpa spicata, Rhynchostylis retusa, Micropera pallida and Grosourdya appendiculata. While terrestrial orchid was only found in a small number, with common terrestrial orchids were Corymborkis veratrifolia and Goodyera rubicunda. The most preference host trees for epiphyte orchid were Tectona grandis (Teak), Clausena indica, Lagerstroemia speciosa, Mangifera indica (Mango), but there is no specific relationshipbetween host trees and epiphyte orchid© 2007 Jurusan Biologi FMIPA UNS SurakartaKey words: orchid, host trees, Meru Betiri National Park

Young leaf segments from plants growing both in vivo and in vitro were cultured on Mitra et al medium (BM) supplemented variously with auxins (IAA, NAA, 2,4-D), kinetin (KN), GA3, and organic supplements (peptone, urea, casein hydrolysate, and yeast extract). The explants from mature leaves did not show any growth and turned necrotic, while those obtained from juvenile leaves of 6-mo-old plantlets growing in vitro developed protocorm-like bodies (plbs) at their cut surfaces within 4-7 wk, depending on the growth medium. Histological studies revealed their epidermal origin. Formation of plbs subsequently spread all along the explant and suggests a meristematic/generative potential of the epidermal cells in immature leaves. Upon subculture in basal medium, the plbs differentiated into plantlets within 4-5 wk. Although regeneration of plbs was achieved in BM and the auxins or GA3, KN in combination with IAA/NAA in the presence of peptone accelerated their formation.