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1.  Multiple shoot induction from axillary bud cultures of the medicinal orchid, Dendrobium longicornu 
AoB Plants  2012;2012:pls032.
The present investigation was undertaken to propagate D. longicornu, a medicinally important orchid using axillary bud segments. This approach could also help in conserving other threatened orchids as well.
Background and aims
Dendrobium longicornu, commonly known as the ‘Long-horned Dendrobium’, is an endangered and medicinally important epiphytic orchid. Over-exploitation and habitat destruction seriously threaten this orchid in Northeast India. Our objective was to develop an efficient protocol for the mass propagation of D. longicornu using axillary bud segments.
Methodology and principal results
Axillary buds cultured in Murashige and Skoog semi-solid medium supplemented with α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) readily developed into plantlets. These formed either directly from shoot buds or from intermediary protocorm-like bodies (PLBs). The maximum explant response (86.6 %) was obtained in medium supplemented with NAA at 30 µM, while the maximum number of shoots (4.42) and maximum bud-forming capacity (3.51) were observed in medium containing 15 µM BAP and 5 µM NAA in combination. Protocorm-like bodies were obtained when the medium contained 2,4-D. The maximum number of explants forming PLBs (41.48 %) was obtained in medium containing 15 µM BAP and 15 µM 2,4-D. Well-developed plantlets obtained after 20–25 weeks of culture were acclimatized and eventually transferred to the greenhouse. Over 60 % of these survived to form plants ∼3–4 cm tall after 90 days in glasshouse conditions using a substrate of crushed brick and charcoal, shredded bark and moss.
Conclusions
The method described can readily be used for the rapid and large-scale regeneration of D. longicornu. Its commercial adoption would reduce the collection of this medicinally important and increasingly rare orchid from the wild.
doi:10.1093/aobpla/pls032
PMCID: PMC3491754  PMID: 23136638
2.  Ex situ conservation of Cymbidium eburneum Lindl.: a threatened and vulnerable orchid, by asymbiotic seed germination 
3 Biotech  2012;2(4):337-343.
The population of many splendid orchids is reducing from their natural habitats at an alarming rate and their conservation is becoming a matter of global concern. Asymbiotic seed germination has been applied for ex situ conservation of rare, endangered and threatened orchid taxa and could provide rapid means their multiplication. In the present study reported here, seeds of an epiphytic and rare orchid, Cymbidium eburneum were germinated asymbiotically in different basal media viz., Murashige and Skoog (MS), Knudson C, Mitra et al. (Mitra), Gamborg et al. (B5) and Nitsch. The highest germination rate was observed in Mitra medium, whereas the development of the protocorms was found to be best in MS medium. Effects of growth regulators viz., indole-3 acetic acid (IAA), α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-d), thidiazuron (TDZ), 6-benzyl aminopurine (BAP) and kinetin (Kn) both singly and in combination incorporated in the MS medium were studied on growth and development of seedlings. It was observed that MS medium nourished with 15 μM each of BAP and NAA in combination was found to enhance shoot number and length, and root number and length in the seedlings. The rooted seedlings were successfully acclimatized.
doi:10.1007/s13205-012-0062-8
PMCID: PMC3482442
Ex situ conservation; Endangered; Asymbiotic seed germination; Protocorms; Cymbidium eburneum
3.  Asymbiotic seed germination and in vitro conservation of Coelogyne nervosa A. Rich. an endemic orchid to Western Ghats 
Coelogyne nervosa is an epiphytic orchid endemic to Western Ghats, South India. The mature seeds of C. nervosa were cultured on ½ MS (Murashige and Skoog), MS, Kn (Knudson) and VW (Vacin and Went) media to evaluate the seed germination response. Of the four basal media used, MS medium supported maximum seed germination. Further experiments to enhance seed germination were done on MS medium supplemented with various concentrations (10, 20, 30 and 40 %) of coconut water (CW). Thirty percent CW gave the highest response in terms of percent seed germination (96), fresh weight (7.2 mg/seedling) and protocorm length (15.2 mm). Since CW containing medium did not support further seedling growth, each seedling was isolated and cultured on MS medium supplemented with either BA (6-benzylaminopurine) or Kin (kinetin) alone (1.0–4.0 mg/l each) or in combination with NAA (1-naphthaleneacetic acid; 0.2–1.0 mg/l). Maximum growth was observed on MS medium supplemented with BA (3.0 mg/l) and NAA (0.5 mg/l). On this medium, the seedlings reached an average length of 3.6 cm with 2.8 well expanded green leaves per seedling. Similarly optimum, healthy, white root induction (3.3 roots/seedlings) was also observed on the same medium. The rooted seedlings were successfully transplanted to pots with 91 % success. The 2-year-old tissue culture derived plants produced normal flowers and fruits.
doi:10.1007/s12298-012-0118-6
PMCID: PMC3550512  PMID: 23814439
Coelogyne nervosa; Epiphytic orchid; Micropropagation; Seedlings; Tissue culture
4.  An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast India 
AoB Plants  2011;2012:plr032.
Submergence inhibits photosynthesis by terrestrial wetland plants, but less so in species that possess leaf gas films when submerged. Floodwaters are often supersaturated with dissolved CO2 enabling photosynthesis by submerged terrestrial plants, although rates remain well-below those in air. This important adaptation that enhances survival in submerged conditions is reviewed.
Background and aims
Dendrobium hookerianum is a rare and threatened epiphytic orchid of northeast India. Prospects for conservation would be strengthened by developing an in vitro method for mass propagation. Seeds are minute and difficult to use directly in the field for this purpose, being non-endospermous with a low nutrient content and dependent on a specific fungus for germination and early seedling development. Although produced in large numbers (2–3 million per capsule), <5 % germinate naturally in the wild. Our objective was to develop a rapid and successful method for in vitro propagation based on an initial in vitro asymbiotic seed germination step that achieved high percentages.
Methodology
Effects of four different media, i.e. (i) Murashige and Skoog (MS), (ii) Mitra et al., (iii) Knudson (KC) and (iv) Gamborg et al. (B5), were evaluated for large-scale multiplication by asymbiotic seed germination. Seedling leaf number, shoot number, shoot length, root number and root length were scored. After 7–8 months, large numbers of well-rooted plantlets were transferred to a glasshouse in thermocol pots containing compost. Six different composts based on broken brick and charcoal were compared for their ability to support further development over 90 days of hardening.
Principal results
The fastest and highest percentage seed germination was achieved using MS medium. Seeds on MS medium germinated in 3–4 weeks compared with 7–8 weeks on B5 medium. Seedling development was also superior on MS medium. The inclusion of plant growth regulators was unnecessary. Compost comprising broken brick and charcoal with an upper layer of moss was found to be the most suitable for the survival of transferred plantlets. Ninety per cent survival of plantlets was achieved 90 days after transfer to a glasshouse.
Conclusions
The use of MS culture medium is well suited for the mass multiplication of D. hookerianum plants intended for re-introducing this threatened orchid into the wild.
doi:10.1093/aobpla/plr032
PMCID: PMC3260561
5.  Effects of phytohormones on thermal denaturation profiles of Cymbidium DNA: indication of differential DNA replication. 
Nucleic Acids Research  1976;3(8):2033-2039.
Protocorm pieces of the orchid Cymbidium were aseptically cultured either without phytohormones, or with one of the growth promoting substances, auxin cytokinin, and gibberellin. The derivative melting profiles of the extracted DNA's differ from each other with respect to the size of various AT- and GC- rich fractions. Evidence has been obtained for the increase of the more AT--rich fractions in auxin-treated cultures, while gibberellin stimulated the expansion of the GC-rich fractions. These results are consistent with earlier cytological and cytochemical findings and indicate the involvement of hormone-controlled differential DNA replication in the development of Cymbidium protocorms in vitro.
PMCID: PMC343059  PMID: 967687
6.  Micropropagation of Ilex khasiana, a critically endangered and endemic holly of Northeast India 
AoB Plants  2011;2011:plr012.
The paper describes in vitro techniques for mass propagation of IIex khasiana, a rare and critically endangered holly endemic to Khasi Hills Hills of Meghalaya, India. The approach will help conserve I. khasiana and other endangered species.
Background and aims
Ilex khasiana is a rare and critically endangered holly endemic to the Khasi Hills of Meghalaya, India, and confined to a small number of pocket areas. In addition to conventional methods of propagation, endemic and threatened plants such as this could be more effectively multiplied and conserved using in vitro methods. Such techniques have the additional advantage of having a low impact on wild populations because they require a minimum of starting material. Our objective was to develop methodologies for the successful in vitro mass propagation of I. khasiana.
Methodology
Seedlings were germinated in vitro under sterile conditions and nodal explants from these were transferred to Murashige and Skoog (MS) medium supplemented with 8.88 µM 6-benzyladenine and 4.64 µM kinetin.
Principal results
This generated ∼10 shoots per explant. In a second approach, callus was obtained from seedling-derived leaf discs cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Approximately 12 adventitious shoots per callus were regenerated from 83.33 % of the calli after transfer to MS medium supplemented with 6.63 µM 6-benzyladenine. The most effective treatment for inducing root formation on the shoots was transfer of shoots to half-strength MS medium with 9.84 µM indole-3-butyric acid. Regenerated plantlets with well-developed shoots and roots were hardened and transferred to open soil with 70 % survival after 4 weeks.
Conclusions
Both the methods described here are well suited for the mass multiplication of this critically endangered tree species.
doi:10.1093/aobpla/plr012
PMCID: PMC3129536  PMID: 22476482
7.  Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage 
BMC Plant Biology  2010;10:82.
Background
Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus.
Results
For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l-1) and TDZ (0.5 mg l-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l-1) and TDZ (0.3 mg l-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation.
Conclusion
The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin.
doi:10.1186/1471-2229-10-82
PMCID: PMC3095354  PMID: 20444255
8.  Perspectives on MADS-box expression during orchid flower evolution and development 
The diverse morphology of orchid flowers and their complex, often deceptive strategies to become pollinated have fascinated researchers for a long time. However, it was not until the 20th century that the ontogeny of orchid flowers, the genetic basis of their morphology and the complex phylogeny of Orchidaceae were investigated. In parallel, the improvement of techniques for in vitro seed germination and tissue culture, together with studies on biochemistry, physiology, and cytology supported the progress of what is now a highly productive industry of orchid breeding and propagation. In the present century both basic research in orchid flower evo-devo and the interest for generating novel horticultural varieties have driven the characterization of many members of the MADS-box family encoding key regulators of flower development. This perspective summarizes the picture emerging from these studies and discusses the advantages and limitations of the comparative strategy employed so far. I address the growing role of natural and horticultural mutants in these studies and the emergence of several model species in orchid evo-devo and genomics. In this context, I make a plea for an increasingly integrative approach.
doi:10.3389/fpls.2013.00377
PMCID: PMC3779858  PMID: 24065980
Orchidaceae; evo-devo; MADS-box gene; peloric mutant; gene family; transcriptome; model species
9.  Comparative karyomorphological study of some Indian Cymbidium Swartz, 1799 (Cymbidieae, Orchidaceae) 
Comparative Cytogenetics  2012;6(4):453-465.
Understanding the genetic resources and diversity is very important for the breeding programs and improvement of several economically important orchids like Cymbidium. Karyomorphological studies have been carried out on seven Cymbidium species, Cymbidium aloifolium (Linnaeus, 1753), Cymbidium devonianum Paxton,1843, Cymbidium elegans Lindley, 1828, Cymbidium iridioides D. Don, 1825, Cymbidium lowianum Rchb. f.,1877, Cymbidium tigrinum Parish ex Hook. f., 1864, and Cymbidium tracyanum L. Castle,1890, most of them endangered/threatened in their natural habitat. As reported earlier, the somatic chromosome number (2n = 40) has been observed in all the seven species. Distinct inter-specific variation was recorded in the arm ratio of few homologous pairs in the complements. Symmetrical or almost symmetrical karyotypes were prevalent; however significant asymmetry was reported in Cymbidium iridioides and Cymbidium tracyanum. The significance of karyotypic variation in speciation of the genus Cymbidium has been discussed. This study provides useful chromosome landmarks and evidence about genome evolution, heteromorphic chromosomes based heterozygosity, basic chromosome number and ploidy level in the genus Cymbidium.
doi:10.3897/CompCytogen.v6i4.3461
PMCID: PMC3834576  PMID: 24260684
Orchidaceae; mitosis; karyotype; heteromorphism; symmetry
10.  In vitro propagation of Homalomena aromatica Schott., an endangered aromatic medicinal herb of Northeast India 
A successful report on the in vitro propagation of Homalomena aromatica via rhizome axillary bud multiplication is presented. Rhizome bud explants were cultured on Murashige and Skoog medium supplemented with various concentrations of cytokinins to induce multiple shoot formation for micropropagation. The highest number of shoots was achieved in MS medium supplemented with 2.0 mg l−1 6-benzylaminopurine. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 0.5 mg l−1 α-naphthalene acetic acid. The regenerated plantlets showed no morphological differences from the parent plant. This protocol takes approximately 6 months to reach the acclimatization stage from the initiation stage and facilitates commercial and rapid propagation of H. aromatica.
doi:10.1007/s12298-013-0168-4
PMCID: PMC3656179  PMID: 24431499
Cytokinins; Homalomena aromatica; In vitro propagation; Multiple shoots; Rhizome axillary bud
11.  In vitro clonal propagation and genetic fidelity of the regenerants of Spilanthes calva DC. using RAPD and ISSR marker 
An efficient in vitro protocol has been established for clonal propagation of elite plant of Spilanthes calva DC., an important source of spilanthol, an antimalarial larvicidal compound. Nodal explants excised from field grown plant of S. calva DC. when reared on Murashige and Skoog’s medium augmented with different cytokinins, viz. N6-Benzyladenine (BA), N6-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn), differentiated multiple shoots from the axils. BA at 10 μM proved optimum for elicitation of multiple shoots in 91.6 % cultures with an average of 7.12 shoots per explant within 6 weeks. The excised shoots rooted on half strength Murashige and Skoog’s medium supplemented with 0.1 μM IBA. Micropropagated plants were hardened and transferred to field for acclimatization, where 95 % plants survived and were phenotypically similar to the donor plant. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to evaluate the genetic fidelity amongst the regenerants. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 71 scorable bands, ranging in size from 100 bp to 1,100 bp were generated by a combination of the two markers in the aforesaid plants. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant. The similarity values amongst the aforesaid plants varied from 0.967 to 1.000. The dendrogram generated through UPGMA (Unweighted Pair Group Method with arithmetic mean) analysis revealed 98 % similarity amongst them, thus confirming the genetic fidelity of the in vitro clones.
doi:10.1007/s12298-012-0152-4
PMCID: PMC3656186  PMID: 24431493
Cytokinin; Fidelity; ISSR; Micropropagation; RAPD; Spilanthes calva DC.
12.  Embryo Development in the Lady's Slipper Orchid, Paphiopedilum delenatii, with Emphasis on the Ultrastructure of the Suspensor 
Annals of Botany  2006;98(6):1311-1319.
• Background and Aims Owing to large-scale collecting, the lady's slipper orchid, Paphiopedilum delenatii, is under threat of extinction. Asymbiotic germination provides a useful way to re-establish plants in the wild and for commercial propagation. A detailed study of embryo development would provide information on subsequent germination events and aid in the propagation of the species.
• Methods Developing capsules were collected for histochemical and ultrastructural studies by using both light and transmission electron microscopy.
• Key Results The suspensor of this species consists of three vacuolated cells. During the early globular stage of embryo development, structural differentiation occurs, revealing an abundance of smooth endoplasmic reticulum cisternae and wall ingrowths within the suspensor cells. These features are not present in cells of the embryo proper. Furthermore, the results of Nile red staining demonstrate that a cuticular layer is present only in the embryo proper, but absent from the suspensor. Cuticular material is also present in the inner walls of the seed coat, and persists through seed maturation.
• Conclusions The morphological features of the transfer cell and the absence of cuticular material in the suspensor cell wall corroborate the hypothesis that the suspensor is the major nutrient uptake site for the developing embryo in the lady's slipper orchid. The absence of an endosperm and presence of cuticular material in the inner walls of the seed coat enclosing the embryo proper further support the notion that nutrient uptake by the embryo is confined to the micropylar end of the seed through the suspensor.
doi:10.1093/aob/mcl222
PMCID: PMC2803589  PMID: 17056612
Cuticular material; embryology; lady's slipper orchid; Paphiopedilum; suspensor
13.  Mycorrhizal associations and reproductive isolation in three closely related Orchis species 
Annals of Botany  2010;107(3):347-356.
Background and Aims
The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid–mycorrhizal associations may be involved in acting as a post-mating barrier.
Methods
We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.
Results
Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.
Conclusions
Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.
doi:10.1093/aob/mcq248
PMCID: PMC3043927  PMID: 21186239
DNA array; gene flow; hybrid zones; mycorrhizal associations; reproductive barriers; seed germination
14.  Mycorrhizal preference promotes habitat invasion by a native Australian orchid: Microtis media 
Annals of Botany  2012;111(3):409-418.
Background and Aims
Mycorrhizal specialization has been shown to limit recruitment capacity in orchids, but an increasing number of orchids are being documented as invasive or weed-like. The reasons for this proliferation were examined by investigating mycorrhizal fungi and edaphic correlates of Microtis media, an Australian terrestrial orchid that is an aggressive ecosystem and horticultural weed.
Methods
Molecular identification of fungi cultivated from M. media pelotons, symbiotic in vitro M. media seed germination assays, ex situ fungal baiting of M. media and co-occurring orchid taxa (Caladenia arenicola, Pterostylis sanguinea and Diuris magnifica) and soil physical and chemical analyses were undertaken.
Key Results
It was found that: (1) M. media associates with a broad taxonomic spectrum of mycobionts including Piriformospora indica, Sebacina vermifera, Tulasnella calospora and Ceratobasidium sp.; (2) germination efficacy of mycorrhizal isolates was greater for fungi isolated from plants in disturbed than in natural habitats; (3) a higher percentage of M. media seeds germinate than D. magnifica, P. sanguinea or C. arenicola seeds when incubated with soil from M. media roots; and (4) M. media–mycorrhizal fungal associations show an unusual breadth of habitat tolerance, especially for soil phosphorus (P) fertility.
Conclusions
The findings in M. media support the idea that invasive terrestrial orchids may associate with a diversity of fungi that are widespread and common, enhance seed germination in the host plant but not co-occurring orchid species and tolerate a range of habitats. These traits may provide the weedy orchid with a competitive advantage over co-occurring orchid species. If so, invasive orchids are likely to become more broadly distributed and increasingly colonize novel habitats.
doi:10.1093/aob/mcs294
PMCID: PMC3579446  PMID: 23275632
Terrestrial orchid; mycorrhizal fungi; disturbed habitats; south-western Australia; invasive species; Microtis media
15.  Collection and trade of wild-harvested orchids in Nepal 
Background
Wild orchids are illegally harvested and traded in Nepal for use in local traditional medicine, horticulture, and international trade. This study aims to: 1) identify the diversity of species of wild orchids in trade in Nepal; 2) study the chain of commercialization from collector to client and/or export; 3) map traditional knowledge and medicinal use of orchids; and 4) integrate the collected data to propose a more sustainable approach to orchid conservation in Nepal.
Methods
Trade, species diversity, and traditional use of wild-harvested orchids were documented during field surveys of markets and through interviews. Trade volumes and approximate income were estimated based on surveys and current market prices. Orchid material samples were identified to species level using a combination of morphology and DNA barcoding.
Results
Orchid trade is a long tradition, and illegal export to China, India and Hong Kong is rife. Estimates show that 9.4 tons of wild orchids were illegally traded from the study sites during 2008/2009. A total of 60 species of wild orchids were reported to be used in traditional medicinal practices to cure at least 38 different ailments, including energizers, aphrodisiacs and treatments of burnt skin, fractured or dislocated bones, headaches, fever and wounds. DNA barcoding successfully identified orchid material to species level that remained sterile after culturing.
Conclusions
Collection of wild orchids was found to be widespread in Nepal, but illegal trade is threatening many species in the wild. Establishment of small-scale sustainable orchid breeding enterprises could be a valuable alternative for the production of medicinal orchids for local communities. Critically endangered species should be placed on CITES Appendix I to provide extra protection to those species. DNA barcoding is an effective method for species identification and monitoring of illegal cross-border trade.
doi:10.1186/1746-4269-9-64
PMCID: PMC3846542  PMID: 24004516
Commercialization; DNA barcoding; Orchids; CITES; Traditional medicine
16.  In vitro plantlet regeneration from nodal segments and shoot tips of Capsicum chinense Jacq. cv. Naga King Chili 
3 Biotech  2011;2(1):31-35.
An in vitro regeneration protocol was developed for Capsicum chinense Jacq. cv. Naga King Chili, a very pungent chili cultivar and an important horticultural crop of Nagaland (Northeast India). Maximum number of shoot (13 ± 0.70) was induced with bud-forming capacity (BFC) index of 10.8, by culturing nodal segments in Murashige and Skoog (MS) medium supplemented with 18.16 μM Thidiazuron (TDZ) followed by 35.52 μM 6-benzylaminopurine (BAP). Using shoot tips as explants, multiple shoot (10 ± 0.37) (BFC 8.3) was also induced in MS medium fortified with either 18.16 μM TDZ or 35.52 μM BAP. Elongated shoots were best rooted in MS medium containing 5.70 μM indole-3-acetic acid (IAA). Rooted plantlets thus developed were hardened in 2–3 weeks time in plastic cups containing potting mixture of a 1:1 mix of soil and cow dung manure and then subsequently transferred to earthen pots. The regenerated plants did not show any variation in the morphology and growth as compared to the parent plant.
doi:10.1007/s13205-011-0025-5
PMCID: PMC3339594  PMID: 22582155
Capsicum chinense Jacq.; Nodal segments; Plant regeneration; Shoot tips; Chemistry; Bioinformatics; Agriculture; Stem Cells; Biomaterials; Biotechnology; Cancer Research
17.  In vitro plantlet regeneration from nodal segments and shoot tips of Capsicum chinense Jacq. cv. Naga King Chili 
3 Biotech  2011;2(1):31-35.
An in vitro regeneration protocol was developed for Capsicum chinense Jacq. cv. Naga King Chili, a very pungent chili cultivar and an important horticultural crop of Nagaland (Northeast India). Maximum number of shoot (13 ± 0.70) was induced with bud-forming capacity (BFC) index of 10.8, by culturing nodal segments in Murashige and Skoog (MS) medium supplemented with 18.16 μM Thidiazuron (TDZ) followed by 35.52 μM 6-benzylaminopurine (BAP). Using shoot tips as explants, multiple shoot (10 ± 0.37) (BFC 8.3) was also induced in MS medium fortified with either 18.16 μM TDZ or 35.52 μM BAP. Elongated shoots were best rooted in MS medium containing 5.70 μM indole-3-acetic acid (IAA). Rooted plantlets thus developed were hardened in 2–3 weeks time in plastic cups containing potting mixture of a 1:1 mix of soil and cow dung manure and then subsequently transferred to earthen pots. The regenerated plants did not show any variation in the morphology and growth as compared to the parent plant.
doi:10.1007/s13205-011-0025-5
PMCID: PMC3339594  PMID: 22582155
Capsicum chinense Jacq.; Nodal segments; Plant regeneration; Shoot tips
18.  Shoot Organogenesis and Plant Regeneration from Leaf Explants of Lysionotus serratus D. Don 
The Scientific World Journal  2013;2013:280384.
The gesneriaceous perennial plant, Lysionotus serratus, has been used in traditional Chinese medicine. It also has a great development potential as an ornamental plant with its attractive foliage and beautiful flowers. An efficient propagation and regeneration system via direct shoot organogenesis from leaf explant was established in this study. High active cytokinin (6-benzyladenine (BA) or thidiazuron (TDZ)) was effective for direct organogenesis of initial induction. Murashige and Skoog (MS) growth media containing 0.5 mg L−1 BA alone or with combination of 0.1 mg L−1  α-Naphthaleneacetic acid (NAA) were the most effective for shoot proliferation. High BA concentration (1.0 mg L−1) in the media caused high percentage of vitrified shoots though they introduced high shoot proliferation rate. Histological observation indicated that adventitious shoot regeneration on the medium containing 0.5 mg L−1 BA alone occurred directly from leaf epidermal cells without callus formation. Regenerated shoots rooted well on medium containing half-strength MS medium with 0.5 mg L−1 indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA), and the plantlets successfully acclimatized and grew vigorously in the greenhouse with a 94.2% and 92.1% survival rate.
doi:10.1155/2013/280384
PMCID: PMC3747412  PMID: 23983626
19.  In vitro propagation of spine gourd (Momordica dioica Roxb.) and assessment of genetic fidelity of micropropagated plants using RAPD analysis 
An efficient protocol for rapid in vitro clonal propagation of spine gourd (Momordica dioica Roxb.) genotype RSR/DR15 (female) and DR/NKB-28 (male) was developed through enhanced axillary shoot proliferation from nodal segments. Maximum shoot proliferation of 6.2 shoots per explant with 100 % shoot regeneration frequency was obtained from the female genotype on Murashige and Skoog’s (1962) medium supplemented with 0.9 μM N6-benzyladenine (BA) and 200 mg l-1 casein hydrolysate (CH). While from the male genotype the optimum shoot regeneration frequency (86.6 %) and 6.4 shoots per explant was obtained on MS medium supplemented with 2.2 μM BA. CH induced vigorous shoots, promoted callus formation, and proved inhibitory for shoot differentiation and shoot length, especially in explants from male genotype. Rooting was optimum on half-strength MS medium (male 92.8 %, female 74.6 %) containing 4.9 μM indole-3-butyric acid (IBA). Plantlets were transferred to plastic cups containing a mixture of cocopit and perlite (1:1 ratio) and then to soil after 2–3 weeks. 84 % female and 81 % male regenerated plantlets survived and grew vigorously in the field. Genetic stability of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD). The amplification products were monomorphic in the in vitro propagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of in vitro propagated plants. This micropropagation procedure could be useful for raising genetically uniform planting material of known sex for commercial cultivation or build-up of plant material of a specific sex-type.
doi:10.1007/s12298-012-0109-7
PMCID: PMC3550508  PMID: 23814442
Momordica dioica; Dioecious; Axillary shoot proliferation; Micropropagation; Nodal explants; Genetic fidelity
20.  In vitro Asymbiotic Germination of Immature Seed and Formation of Protocorm by Cephalanthera falcata (Orchidaceae) 
Annals of Botany  2006;98(6):1197-1206.
• Background and Aims Many Orchidaceous species are threatened globally by development and over-collection from their natural habitats for horticultural purposes. Artificial propagation from seeds is difficult in most terrestrial orchids native to temperate regions. Seed production is another limiting factor in the artificial propagation for these species because of the lessened probability of pollination and the destruction of fruit by insect larvae. Members of the genus Cephalanthera are distributed across Europe, Asia and North America. C. falcata is a temperate species of East Asia and an endangered species in Japan. As successful propagation from seeds of this species has never been reported, a reproducible method is described here for seed production in situ and propagation using immature seeds in asymbiotic culture in vitro.
• Methods Effects of hand-pollination and bagging treatment of ovaries were examined. Young capsules were collected every 10 d from 50 d after pollination until 120 d after pollination. Immature seeds obtained from these capsules were cultured asymbiotically on modified Kano medium and ND medium. Seed viability was examined within TTC (2,3,5-triphenyl tetrazolium chloride) test solution and histological observations were made on viable seeds by paraffin embedding at each collection stage.
• Key Results and Conclusions Hand-pollination followed by bagging treatment of ovaries with aluminium foil was effective for insect control during fruit development, and successfully yielded capsules. Of the capsules, 74·5 % survived to full maturity. The highest frequency (39·8 %) of seed germination was obtained with seeds harvested 70 d after pollination. The frequency declined with progress of seed maturity on the mother plant. Minimal germination was observed with seeds harvested 100 d or later after pollination. Histological observation suggests that accumulation of such substances as lignin in the inner integument surrounding the embryo during seed maturation plays an important role in induction of dormancy.
doi:10.1093/aob/mcl223
PMCID: PMC3292276  PMID: 17071633
Orchidaceae; Cephalanthera falcata; seed dormancy; seed germination; seed production; immature seed; inner integument
21.  In vitro zygotic embryo germination and propagation of an endangered Boswellia serrata Roxb., a source of boswellic acid 
This study aims to establish an efficient protocol for development of seedlings of an endangered medicinally important forest tree Boswellia serrata Roxb., for mass plantation and consistent supply of salai guggul. The green mature fruits served as source of seeds. The excised green zygotic embryos were cultured on Gamborg (B5), McCown and Loyd (WPM) and Schenk and Hildebrandt (SH) media fortified with different concentration of sucrose and on Murashige and Skoog (MS) medium containing 3 % sucrose, polyvinylpyrrolidone (PVP) (0–300 mg l−l), Gibberellic acid (GA3), Indoleacetic acid (IAA), Naphthaleneacetic acid (NAA), Indole-3-Butyric acid (IBA) or 2,4-dichlorophenoxyacetic acid (2,4 D) and 6-benzylaminopurine (BA) or kinetin (Kin) individually. The highest frequency of embryo germination (96 %) and conversion into seedling was obtained on MS medium containing 3 % sucrose together with 200 mg l−l PVP; other media were either inferior or induced abnormalities in the seedlings including callus formation from the zygotic embryos. Fully developed seedlings could be successfully established in soil with about 94 % survival. The embryos from mature dry seeds did not respond for germination in any of the experiments. In conclusion, selection of zygotic embryo from green mature seeds and their in vitro germination is important for propagation of B. serrata.
doi:10.1007/s12298-010-0017-7
PMCID: PMC3550602  PMID: 23572965
Endangered; frankincense; green mature embryo; mass plantation; seed viability
22.  Micropropagation of Pithecellobium dulce (Roxb.) Benth—a multipurpose leguminous tree and assessment of genetic fidelity of micropropagated plants using molecular markers 
An efficient and reproducible protocol has been developed for in vitro propagation of Pithecellobium dulce (Roxb.) Benth (a multipurpose leguminous tree) from field grown nodal segments (axillary bud). Shoot bud induction occurred from nodal explants of 15-years-old tree on Murashige and Skoog (MS) basal medium supplemented with 4.4 μM 6-benzyladenine (BA) and multiplication was achieved on MS medium supplemented with 4.4 μM BA + 0.73 μM phenylacetic acid (PAA) i.e. up to 7 shoot buds in the period of 5–6 weeks. Addition of adenine sulphate (AdS) to this medium further enhanced the number of shoot buds up to 10. Proliferating shoot cultures were established by repeatedly subculturing primary culture on fresh medium (MS + 4.4 μM BA + 0.73 μM PAA) after every 25 days. In vitro rooting was achieved on MS medium supplemented with 2.46 μM Indole-3-butyric acid (IBA) + 41.63 μM activated charcoal (AC). The micropropagated shoots with well developed roots were acclimatized in green house in pots containing sand, soil and manure (1:1:1). Genetic stability of micropropagated clones was evaluated using Random amplified polymorphic DNA (RAPD) and Inter simple sequence repeat (ISSR) markers. The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic uniformity of micropropagated plants. This is the first report of an efficient protocol for regeneration of P. dulce through organogenesis, which can be used for further genetic transformation and pharmaceutical purposes.
doi:10.1007/s12298-012-0112-z
PMCID: PMC3550498  PMID: 23573054
Legume tree; Nodal segments; Activated charcoal; Adenine sulphate; RAPD, ISSR marker
23.  Mycorrhizal Acquisition of Inorganic Phosphorus by the Green-leaved Terrestrial Orchid Goodyera repens 
Annals of Botany  2007;99(5):831-834.
Background and Aims
Mycorrhizal fungi play a vital role in providing a carbon subsidy to support the germination and establishment of orchids from tiny seeds, but their roles in adult orchids have not been adequately characterized. Recent evidence that carbon is supplied by Goodyera repens to its fungal partner in return for nitrogen has established the mutualistic nature of the symbiosis in this orchid. In this paper the role of the fungus in the capture and transfer of inorganic phosphorus (P) to the orchid is unequivocally demonstrated for the first time.
Methods
Mycorrhiza-mediated uptake of phosphorus in G. repens was investigated using spatially separated, two-dimensional agar-based microcosms.
Results
External mycelium growing from this green orchid is shown to be effective in assimilating and transporting the radiotracer 33P orthophosphate into the plant. After 7 d of exposure, over 10 % of the P supplied was transported over a diffusion barrier by the fungus and to the plants, more than half of this to the shoots.
Conclusions Goodyera repens
can obtain significant amounts of P from its mycorrhizal partner. These results provide further support for the view that mycorrhizal associations in some adult green orchids are mutualistic.
doi:10.1093/aob/mcm018
PMCID: PMC2802910  PMID: 17339276
33P; phosphate; Goodyera repens; myco-heterotrophy; mineral nutrition; orchid; mycorrhizal networks
24.  Micropropagation and cytogenetic assessment of Zingiber species of Northeast India 
3 Biotech  2012;3(6):471-479.
An improved micropropagation protocol was developed for Zingiber moran and Z. zerumbet, two wild species of the genus Zingiber, found in Northeast India. The effects of growth regulators, sugar concentrations, and nutrients were tested on the rate of shoot initiation and multiplication. An increase in proliferation and multiplication occurred in modified Murashige and Skoog (MS) medium supplemented with benzyladenine and kinetin. About 2 % sucrose and 0.7 % agar were found to be the optimum for shoot multiplication and regeneration. Naphthalene acetic acid at 0.5 mg/L produced the best rooting response for both the species. Regenerated plantlets were acclimatized successfully and cytogenetic stability was confirmed by RAPD profiling and ploidy checks.
doi:10.1007/s13205-012-0108-y
PMCID: PMC3824790
Axillary bud; Micropropagation; Northeast India; RAPD; Zingiber moran
25.  Asymbiotic Germination Response to Photoperiod and Nutritional Media in Six Populations of Calopogon tuberosus var. tuberosus (Orchidaceae): Evidence for Ecotypic Differentiation 
Annals of Botany  2008;102(5):783-793.
Background and Aims
Ecotypic differentiation has been explored in numerous plant species, but has been largely ignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliable due to inherent physiological or genetic differences in localized populations. It is crucial to determine whether ecotypic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, a widespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germination requirements, as this species occupies diverse habitats spanning a wide geographical range.
Methods
Mature seeds were collected from south Florida, north central Florida, three locations in South Carolina, and the upper Michigan peninsula. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) were examined on asymbiotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development was monitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additional experiment, asymbiotic seed germination and development was monitored for 8 weeks on six culture media (BM-1 terrestrial orchid medium, Knudson C, Malmgrem, half-strength MS, P723, and Vacin and Went). A tetrazolium test for embryo viability was performed.
Key Results
Short days promoted the highest germination among Florida populations, but few differences among photoperiods in other seed sources existed. Different media had little effect on the germination of Michigan and Florida populations, but germination of South Carolina seeds was higher on media with higher calcium and magnesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability was higher in Florida seeds. Seed germination and corm formation was rapid in Michigan seeds across all treatments. Michigan seedlings allocated more biomass to corms compared with other seed sources.
Conclusions
Rapid germination and corm formation may be a survival mechanism in response to a compressed growing season in northern populations. Ecotypic differentiation may be occurring based on seed germination and corm formation data.
doi:10.1093/aob/mcn163
PMCID: PMC2712386  PMID: 18757881
Asymbiotic germination; corm development; Calopogon tuberosus; ecotypic differentiation; native orchid; orchid seed germination; seedling development

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