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1.  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
2.  A simple and efficient protocol for the mass propagation of Cymbidium mastersii: an ornamental orchid of Northeast India 
AoB Plants  2012;2012:pls023.
The present investigation was undertaken to mass propagate Cymbidium mastersii, an ornamental orchid of Northeast India by in vitro propagation method. This approach could also help for the conservation as well as commercialization of C. mastersii and other threatened and ornamental orchids.
Background and aims
Cymbidium mastersii is an epiphytic orchid distributed mainly in Northeast India. Owing to its high commercial value in the floricultural industry, natural populations are under threat from over-exploitation. Mass propagation provides an alternative means of satisfying the demand. Unfortunately, conventional propagation is slow and difficult, suggesting in vitro methods for mass multiplication may be more appropriate. The objective of this study was to develop an efficient protocol.
Methodology and principal results
Four nutrient media were evaluated for seed germination and early protocorm development: Murashige and Skoog (MS), half-strength MS, Knudson ‘C’ (KC), and Vacin and Went (VW). In addition, the effects of plant growth regulators 6-benzylaminopurine (BAP), kinetin (KN), α-naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) were studied alone and in combination. The maximum percentage seed germination (93.58 ± 0.56) was obtained in MS basal medium after 8–9 weeks of culture. Secondary protocorms (protocorm-like bodies) were developed from primary protocorms on MS medium fortified with different concentrations and combinations of cytokinins (BAP and KN) and auxins (NAA and IBA). The highest numbers of secondary protocorms (20.55 ± 0.62)/primary protocorms were obtained in MS medium supplemented with 5.0 µM BAP and 2.5 µM NAA. The most effective auxin source promoting root production (7.46 ± 0.09 per shoot) was 10.0 µM IBA. The plants were acclimatized effectively (survival percentage 88 %) in a greenhouse using a rooting medium of crushed sterile brick and charcoal (1 : 1 v/v) and vermicompost (leaf litter + cow dung, 1 : 1 v/v).
Conclusions
An efficient protocol was established for in vitro propagation of C. mastersii using seed as the starting material. The percentage seed germination varied with the composition of the nutrient media and was highest in full-strength MS basal medium. The number of secondary protocorms that developed from primary protocorms was increased by the addition of 5.0 µM BAP and 2.5 µM NAA. In vitro raised plantlets acclimatized in a greenhouse were closely similar to the mother plants in morphology.
doi:10.1093/aobpla/pls023
PMCID: PMC3447538  PMID: 22997547
3.  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
4.  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

Results 1-4 (4)