The advancements made in tissue culture techniques has made it possible to regenerate various horticultural species in vitro as micropropagation protocols for commercial scale multiplication are available for a wide range of crops. Clonal propagation and preservation of elite genotypes, selected for their superior characteristics, require high degree of genetic uniformity amongst the regenerated plants. However, plant tissue culture may generate genetic variability, i.e., somaclonal variations as a result of gene mutation or changes in epigenetic marks. The occurrence of subtle somaclonal variation is a drawback for both in vitro cloning as well as germplasm preservation. Therefore, it is of immense significance to assure the genetic uniformity of in vitro raised plants at an early stage. Several strategies have been followed to ascertain the genetic fidelity of the in vitro raised progenies comprising morpho-physiological, biochemical, cytological and DNA-based molecular markers approaches. Somaclonal variation can pose a serious problem in any micropropagation program, where it is highly desirable to produce true-to-type plant material. On the other hand, somaclonal variation has provided a new and alternative tool to the breeders for obtaining genetic variability relatively rapidly and without sophisticated technology in horticultural crops, which are either difficult to breed or have narrow genetic base. In the present paper, sources of variations induced during tissue culture cycle and strategies to ascertain and confirm genetic fidelity in a variety of in vitro raised plantlets and potential application of variants in horticultural crop improvement are reviewed.

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Somaclonal variations and their applications in horticultural crops improvement

Cypripedioideae (Orchidaceae) have been the subject of numerous taxonomic treatments with conflicting interpretations of relationships among the five genera and the 150–170 species. We have produced nuclear ribosomal ITS nucleotide sequences for nearly 100 slipper orchid species and used parsimony analysis to investigate their relationships. Our results demonstrate that each genus, as currently circumscribed, is monophyletic (Mexipedium andSelenipedium being represented by a single taxon). LikerbcL data, ITS sequences placeMexipedium sister toPhragmipedium. Relationships at the sectional level inPaphiopedilum are largely as described byCribb. However, the division ofPaphiopedilum into subgg.Brachypetalum andPaphiopedilum is not supported; subg.Brachypetalum is paraphyletic to subg.Paphiopedilum. Phragmipedium species are divided into the same three major clades as in the taxonomic scheme ofMcCook. The plicate-leaved genera,Cypripedium andSelenipedium, are successive sister groups to the rest of the subfamily, confirming generally held opinions that they display plesiomorphic characters compared to the conduplicate-leaved genera. A survey of karyotypes in the context of the ITS tree reveals a general trend toward increased chromosome number, probably brought about by centric fission. These data also accord with a previously suggested biogeographic hypothesis of a widespread Northern Hemisphere distribution, followed by range fragmentation due to Miocene cooling.

Phylogenetics of the slipper orchids (Cypripedioideae, Orchidaceae): Nuclear rDNA ITS sequences

This perspective draws attention to the functional organization of orchid seed and protocorm during the course of development. The orchid embryos have a well-organized developmental plan generating a blue-print of a protocorm as they mature. The different phases of embryo development in orchids, i.e. histodifferentiation, storage product synthesis and accumulation, and maturation are essentially similar to other flowering plants. The protocorm is considered as a unique structure designed to establish symbiotic association with mycorrhizal fungi and with the primary goal to form a shoot apical meristem. This perspective brings forth arguments that the processes of embryo and protocorm development are highly programmed events, enhancing survival of orchid seeds and plantlets in their natural habitats. Furthermore, the ability of protocorm cells to divide, makes them ideal explants for micropropagation and transformation studies. Through seed germination and micropropagation using protocorms as explants, orchid conservation efforts are greatly enhanced.

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A perspective on orchid seed and protocorm development.

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.

Symbiotic in vitro seed propagation of Dendrobium: fungal and bacterial partners and their influence on plant growth and development

The concept of a thin cell layer (TCL) was initially presented by Tran Thanh Van in two key papers exactly 40 years ago. At that time, Nicotiana tabacum was the model plant used to establish three main pathways for de novo organogenesis by developing a flower, vegetative bud, and root “programme” from pedicel tissue. Over the last 40 years, a wealth of research in plant tissue culture based on TCLs has emerged, fortifying the importance of this very simple technique, and highlighting its fundamental importance as a key tool in plant cell and tissue differentiation as well as organ development. This review not only highlights the achievements made using TCLs in the plant kingdom over these 40 years, it also reports on the success of this technique in ornamentals, fruit and forestry species, vegetables, and medicinal plants. There is overwhelming evidence of the importance of this technique for plant biotechnology, and it provides one solution for the mass clonal propagation of plants, use in bioreactors, genetic transformation, or micropropagation.

Plant Thin Cell Layers: A 40-Year Celebration

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.

P T Van

Papers

The Application of Biotechnology to Orchids