The genus Dendrobium is one of the largest genera of the Orchidaceae Juss. family, although some of its members are the most threatened today. The reason why many species face a vulnerable or endangered status is primarily because of anthropogenic interference in natural habitats and commercial overexploitation. The development and application of modern techniques and strategies directed towards in vitro propagation of orchids not only increases their number but also provides a viable means to conserve plants in an artificial environment, both in vitro and ex vitro, thus providing material for reintroduction. Dendrobium seed germination and propagation are challenging processes in vivo and in vitro, especially when the extreme specialization of these plants is considered: (1) their biotic relationships with pollinators and mycorrhizae; (2) adaptation to epiphytic or lithophytic life-styles; (3) fine-scale requirements for an optimal combination of nutrients, light, temperature, and pH. This review also aims to summarize the available data on symbiotic in vitro Dendrobium seed germination. The influence of abiotic factors as well as composition and amounts of different exogenous nutrient substances is examined. With a view to better understanding how to optimize and control in vitro symbiotic associations, a part of the review describes the strong biotic relations of Dendrobium with different associative microorganisms that form microbial communities with adult plants, and also influence symbiotic seed germination. The beneficial role of plant growth-promoting bacteria is also discussed.

The ability to germinate orchids from seeds in vitro presents a useful and viable method for the propagation of valuable germplasm, maintaining the genetic heterogeneity inherent in seeds. Given the ornamental and medicinal importance of many species within the genus Dendrobium, this review explores in vitro techniques for their asymbiotic seed germination. The influence of abiotic factors (such as temperature and light), methods of sterilization, composition of basal media, and supplementation with organic additives and plant growth regulators are discussed in context to achieve successful seed germination, protocorm formation, and further seedling growth and development. This review provides both a basis for the selection of optimal conditions, and a platform for the discovery of better ones, that would allow the development of new protocols and the exploration of new hypotheses for germination and conservation of Dendrobium seeds and seedlings.

An efficient in vitro plant regeneration protocol from protocorms of Dendrobium aqueum was developed. The uniformly developed protocorms (in vitro origin) having shoot initials were cultured on half macro strength MS medium (1/2 MS) supplemented with cytokinins (BA, 2iP, KIN and TDZ) at 1, 3, 5, 7, 10 mg l À1 , natural additives (BP and CW) at 1%, 3%, 5%, 7%, 10% and auxins (IBA, NAA, 2,4-D) at 1, 3, 5, 7, 10 mg l À1 to study their efficacy on complete plant development. A maximum of 9.4 shoots per explant were generated on 3 mg l À1 of NAA followed by 3% of BP (7.0 shoots). Shoot elongation (1.52 cm) was achieved on 1/2 MS medium fortified with NAA 7 mg l À1 followed by TDZ 7 mg l À1 (1.37 cm). Shoots cultured on 1/2 MS medium supplemented with IBA 5 mg l À1 produced an average of 8.75 roots per shoot, however the lengthiest roots (1.48 cm) were noted in NAA 7 mg l À1 . Healthy rooted plantlets successfully acclimatized in ex vitro condition. The role of complete plantlet production by natural additives could be useful for conservation and cost effective commercial production of orchids.ª 2015 Production and hosting by Elsevier B.V. on behalf

An efficient in vitro regeneration protocol through somatic embryogenesis was established from stem transverse thin cell layers (tTCLs) of Dendrobium aqueum Lindley, an imperiled orchid. This study outlines the induction and successive maturation stages of D. aqueum somatic embryos (SEs). The tTCLs (~ 0.5 mm thick) cultured on halfstrength Murashige and Skoog (MS) medium containing cytokinins and auxins, either individually or in combination, produced embryogenic callus (EC). Treatment with 0.5 mg dm -3 zeatin induced EC in 41.42 % of tTCLs. As many as 42.66 globular SEs per tTCL were formed in the presence of 1.5 mg dm -3 N 6 -(2-isopentyl) adenine (2iP) but only on 10.33 % of explants. The combined treatment of 2iP (1.5 mg dm -3 ) and 0.5 mg dm -3 6-benzyladenine resulted in 34 globular SEs on 14.7 % of tTCLs whereas the combination of 2iP and 1.0 mg dm -3 indole-3-butyric acid (IBA) induced 7.4 globular SEs on 52.33 % of tTCLs. Supplementation of activated charcoal, amino acids, and antioxidants alleviated browning at all the concentrations tested, but the EC response declined. The addition of 0.5 mg dm -3 polyvinylpyrrolidone to 1.5 mg dm -3 2iP and 1.0 mg dm -3 IBA produced 24 SEs on 19.89 % of tTCLs suggesting that the EC and SEs can be effectively induced by individual cytokinins whereas the synergistic treatments with other compounds can only enhance the induction of EC. Histological observations of EC showed the formation of globular SEs from sub-epidermal regions. Successive developmental stages of globular SEs and the intermediate stage of protocorm like bodies until the formation of plantlets were observed. The plantlets obtained through SEs showed no morphological variations, and inter simple sequence repeat profiles also confirmed the genetic fidelity of in vitroderived progeny with high monomorphism (97.78 %). In conclusion, the use of stem tTCLs is an effective method to produce SEs through indirect somatic embryogenesis in D. aqueum.

Dendrobium aqueum Lindley is an epiphytic orchid decreasing in nature owing to environmental disturbance and inherent lower germination rates of mature seeds. To conserve this species, in vitro asymbiotic media and photoperiod screening were experimented. Twenty asymbiotic orchid seed germination media were examined to get the optimal medium for germination and seedling development under 16/8 hrs photoperiod. Highest seed germination occurred in 1 / 2 MS medium (93.41%) followed by mMS (91.04%) and m ½ MS (90.23%) in 6 to 7 weeks. The biggest protocorms were observed in MS medium (788.8 µm) followed by ½ MS (615.60 µm). The yellowish embryos during germination exhibit varied colours from yellow to pale green, dark green, yellow, brown, black and white. MS, ½ MS, mMS, ½ mMS, and ¼ MS media only promote fully developed seedlings within 126 to 143 days. The influence of five different photoperiods with four media was also examined separately. 24/0 hrs (L/D) photoperiod provide highest germination (97.75%) on MS followed by ½ MS (96.01%). Dark Photoperiod 0/24 h (L/D) supports up to protocorm stage but with poor seedling growth. The well rooted seedlings were hardened and successfully reintroduced in its natural habitat with 56% survival rate.

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