Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid

This review provides an informative and broad overview of orchid biotechnology, addressing several important aspects such as molecular systematics, modern breeding, in vitro morphogenesis, protoplast culture, flowering control, flower color, somaclonal variation, orchid mycorrhiza, pathogen resistance, virus diagnosis and production of virus-free plants, functional genomics, genetic transformation, conservation biotechnology and pharmaceutical biotechnology. This resource will provide valuable insight to researchers who are involved in orchid biology and floriculture, using biotechnology to advance research objectives. Producing an improved orchid through biotechnology for industrial purposes or to serve as a model plant for pure and applied sciences is well within reach and many of the current techniques and systems are already employed at the commercial production level.

The Application of Biotechnology to Orchids

The present review illustrates the implementation of synthetic seed technology for mass propagation and short-term storage of several medicinal plants, popularly grown throughout the world. Biotechnology-based research with special reference to in vitro plant cell and tissue culture intervention created a new outlook in terms of mass propagation, germplasm storage and cryoconservation, production of secondary metabolites as well as genetic transformation. Synthetic seed technology involving alginate encapsulation of in vitro or in vivo generated explants proved to be a competent system to deal with multiplication, storage and exchange of seedless medicinal plants having traits of choice that are intricate to propagate via conventional approach. Nevertheless, optimization of production, storage and exchange of synthetic seeds are influenced by several factors. Manipulation of those factors such as explant selection, encapsulating agent and matrix determined the success of synthetic seed technology in medicinal plants. The present review elucidates an outline of past progress, present status and future prospects of synthetic seed technology intervention in medicinal plants with special emphasis on the factors which determine the success of this technology.

Synthetic seed production of medicinal plants: a review on influence of explants, encapsulation agent and matrix

Cryopreservation as a tool used in long-term storage of ornamental species - A review

High frequency regeneration protocol for Dendrobium nobile: A model tissue culture approach for propagation of medicinally important orchid species

An efficient protocol for cryopreservation of protocorm like bodies (PLBs) of Dendrobium nobile, based on encapsulation–dehydration (ED) and encapsulation–vitrification (EV), was established. In both cryogenic procedures, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix [comprising 3% (w/v) sodium alginate and 0.1 M CaCl2]. Out of the two methods, EV resulted in higher survival (78.1%) and regrowth (75.9%) than ED (53.3 and 50.2% respectively). Incorporation of 0.4 M sucrose and 2 M glycerol in the encapsulation matrix resulted in higher survival percentage after cryopreservation. In both the cases (ED and EV), shoots regenerated from cryopreserved PLBs with an intermediary PLB formation. Regenerated shoots were successfully rooted in the medium containing 1.5 mg/l Indole-3 butyric acid. Successful acclimatization of plantlets was obtained in the compost containing brick pieces and charcoal chunks (1:1) + a top layer of moss with a maximum survivability (82%). EV method proved to be most appropriate way to cryopreserve the PLBs of D. nobile. Regenerated plantlets showed normal morphology as that of control plants.

http://dspace.nehu.ac.in/bitstream/1/8669/1/fulltext3.pdf

High-efficiency cryopreservation of the medicinal orchid Dendrobium nobile Lindl.

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), a-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 mM BAP and 2.5 mM NAA. The most effective auxin source promoting root production (7.46+ 0.09 per shoot) was 10.0 mM 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 mM BAP and 2.5 mM NAA. In vitro raised plantlets acclimatized in a greenhouse were closely similar to the mother plants in morphology.

A simple and efficient protocol for the mass propagation of Cymbidium mastersii: an ornamental orchid of Northeast India

Synthetic seed technology is an exciting and rapidly growing area of research as deals with conservation and storage of rare, endangered and desirable genotypes along with its easy handling and transportation. As propagation of many ornamental and medicinally important plant species is labour intensive, application of different growth retardants and osmotica in simple artificial seed system would dramatically reduce labour requirement by storing the germplasm in vitro. Moreover, the primary aim of developing in vitro storage methods is to reduce the frequent demands of subculturing and preserving the unique genetic constituent of the germplasm. Dendrobium nobile is a pharmaceutically important orchid mostly used in the Chinese herbal drug industry for its medicinal property. Commercial exploitation of this species has considerably depleted their population in wild. Hence, for conserving this valuable germplasm, short term in vitro storage of Protocorm-Like Bodies (PLBs) of D. nobile was carried out using different osmotica (sucrose and mannitol). It was observed that incorporation of low sucrose and mannitol (3 and 5 %) in the encapsulating matrix showed almost similar results with that of control. In all these cases, more than half of PLBs burst out from the matrix thus making these concentrations of sucrose and mannitol along with control not suitable for storage studies. However, with the increase in concentration to 7.5 and 12.5 % in the encapsulating matrix, no outburst of encapsulated PLBs was recorded till 60 days of storage; hence it can be concluded that these concentrations play an important role in minimizing the growth of PLBs during storage condition.

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Short-term storage of alginate-encapsulated protocorm-like bodies of Dendrobium nobile Lindl.: an endangered medicinal orchid from North-east India.

Our present study constitutes the successful and efficient protocol for cryopreservation of Dendrobium chrysanthum. D. chrysanthum Wall. ex Lindl. is a pharmaceutically valuable, ornamental epiphytic orchid of temperate and subtropical regions. On account of excellent herbal medicinal value and horticultural importance, D. chrysanthum is becoming rare due to over exploitation. For long-term conservation of this orchid, protocorm-like bodies of D. chrysanthum were excised and used for cryopreservation by encapsulation–vitrification. In this cryogenic procedure, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix (comprising of 3 % (w/v) sodium alginate and 0.1 M CaCl2). Encapsulated protocorm-like bodies (PLBs) were then precultured on MS liquid medium supplemented with different concentrations of sucrose (0.06, 0.3, 0.5, 0.7 M), and loaded in a loading solution (comprised of 2 M glycerol and 0.4 M sucrose) for different duration to make the precultured PLBs tolerant to plant vitrification solution 2 (PVS2). Subsequently, the PLBs were subjected to PVS2 (Sakai et al. 1990) treatment at different time of exposure (minutes) and temperatures (0 °C and 25 °C). Encapsulated–vitrified PLBs were plunged directly into liquid nitrogen and stored for 1 h. Optimum result (survival 63.2 % and regrowth 59.9 %) was obtained when the beads treated with loading solution for 80 min followed by PVS2 treatment for 100 min. Regenerated plants showed normal morphology as that of control plants.

Cryopreservation of pharmaceutically important orchid Dendrobium chrysanthum Wall. ex Lindl. using vitrification based method

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.

Padmaja Mohanty

Papers

High-efficiency cryopreservation of the medicinal orchid Dendrobium nobile Lindl.

A simple and efficient protocol for the mass propagation of Cymbidium mastersii: an ornamental orchid of Northeast India

Short-term storage of alginate-encapsulated protocorm-like bodies of Dendrobium nobile Lindl.: an endangered medicinal orchid from North-east India.

Cryopreservation of pharmaceutically important orchid Dendrobium chrysanthum Wall. ex Lindl. using vitrification based method

Cymbidium mastersii, an ornamental orchid of Northeast India