History of orchid propagation: a mirror of the history of biotechnology

Part I Orchid seeds are nearly microscopic in size. Because of that, many fanciful theories were proposed for the origin of orchids. Almost 400 years separate the time when orchid seeds were seen for the first time and the development of a practical asymbiotic method for their germination. The seeds were first observed and drawn during the sixteenth century. Seedlings were first described and illustrated in 1804. The association between orchid and fungi was observed as early as 1824, while the requirement for mycorrhiza for seed germination was established in 1899. An asymbiotic method for orchid seed germination was developed in 1921. After Knudson’s media B and C were formulated, orchids growing and hybridization became widespread. Hybrids which early growers may not have even imagined became possible. Part II A commonly held view is that Prof. Georges Morel is the sole discoverer of orchid micropropagation and that he was the first to culture an orchid shoot tip in 1960. In fact, the first in vitro orchid propagation was carried out by Dr. Gavino Rotor in 1949. Hans Thomale was the first to culture an orchid shoot tip in 1956. The methods used by Morel to culture his shoot tips were developed by others many years before he adapted them to orchids. This review also traces the history of several techniques, additives, and peculiarities (agitated liquid cultures, coconut water, banana pulp, a patent and what appears to be an empty claim) which are associated with orchid micropropagation. A summary of plant hormone history is also outlined because micropropagation could not have been developed without phytohormones.

In vitro culture of Chloraea gavilu Lindl., an endemic terrestrial orchid from Chile

Orchids are the second most diverse plant family, recognized for their importance as ornamental species; this has driven research development in propagation. One of the most common culture methodologies is in vitro asymbiotic germination, in which the nutritional conditions that provide orchids with a fungal partner are emulated. Although Chile possesses more than 60 terrestrial orchid species, in vitro cultivation protocols have only been developed for Chloraea crispa. In Southern Chile, Chloraea gavilu stands out due to its floral characteristics. We evaluate different explants and cultivation conditions for C. gavilu. The best germination and development results were achieved in the MM medium +0.1% yeast extract +1% sucrose +0.454 µmol l^?1 TDZ, using immature seeds of 24–30 days after pollination, which we cultivated into seedlings in order to be acclimatized and mycorrhized. In addition, induction of protocorm-like bodies was achieved from germinated seeds, using the same culture media as in the germination and development of immature C. gavilu seeds. This resulted in the successful asymbiotic germination of immature seeds, along with the micropropagation of a terrestrial, temperate orchid. We hope to use our protocol in the commercial production of Chilean orchid species as well as to propagate threatened species.     

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

Summary In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO₂ concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO₂ concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO₂ concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.     

Conservation of rare and endangered plants using in vitro methods

Summary Many botanic gardens now have tissue culture laboratories for the micropropagation of plants that are difficult to propagate by conventional horticultural techniques. In many cases the work centers on rare and endangered species. Examples of the use of different techniques including micropropagation, in vitro seed germination, dual culture with symbiotic fungi, and regeneration from callus are discussed with reference to their application to plant germplasm conservation. Presented in the Session-in-Depth Cell Culture of Endangered Species at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Micropropagation of Cypripedium macranthos var. rebunense through Protocorm-like Bodies Derived from Mature Seeds

Cypripedium macranthos var. rebunense is the most famous terrestrial orchid in Japan, since the variety has large beautiful yellowish-white flowers and is threatened with extinction. Establishment of an efficient method for micropropagation is urgently needed. When imbibed mature seeds of the orchid, that had been pre-chilled at 4°C for 3 months, were sown onto Hyponex-peptone medium that contained both α-naphthaleneacetic acid (NAA) and cytokinin, protocorm-like bodies (PLBs) were formed from germinated seeds. Although the growth of PLBs was very slow, plantlets were easily regenerated from the PLBs on hormone-free medium. The PLBs were subcultured eight times along 2 years without loss of ability to regenerate plantlets, and one aggregate of PLBs (ca. 5 mm in diameter) produced ca. 10 plants within a year. A reduction of commercial value through a large-scale micropropagation by this method will be able to prevent illegal collection from the wild populations.     

Recent advancement in research on photoautotrophic micropropagation using large culture vessels with forced ventilation

Summary Successful fundamental or basic research, while being stimulated by applied studies, provides the development of new technologies for the benefit of mankind. Photoautotrophic micropropagation or micropropagation using sugar-free medium is no exception from this generalization. The concept of photoautotrophic micropropagation is derived from research that revealed the relatively high photosynthetic abilities of chlorophyllous cultures such as leafy explants and plantlets in vitro. To meet the ever-increasing demand for quality transplants, the scaling-up of photoautotrophic micropropagation systems, for commercialization, has become necessary. This article reviews the recent advancement in the development and utilization of large culture vessels for photoautotrophic micropropagation with special emphasis on the feasibility of the system for the commercial-scale propagation. The review also includes choices for supporting material, ventilation type, planting density, vessel volume, and vessel sterilization procedure, and problems and solutions to achieve uniform growth in a large culture vessel. A case study of the commercial application of a photoautotrophic micropropagation system using large culture vessles, which recently has been established in Kunming, China, is also presented in this article.     

Micropropagation of oregano (<Emphasis Type="Italic">Origanum vulgare×applii</Emphasis>) from meristem tips

Summary ‘Mendocino’ oregano (Origanum vulgare × applii) cultivated in Argentina has been subject to progressive yield loss as a consequence of continuous vegetative propagation. A method of reducing damage to the shoot tip during sterilization procedures for micropropagation is proposed. Single shoot cuttings are less sensitive to disinfecting substances than green tips. Meristems taken from young plantlets that grew in vitro produced less oxidation during the culture than those dissected immediately after disinfecting from plants grown in the field. This work describes the effects of the growth regulators benzyladenine (BA) and naphthaleneacetic acid (NAA) at various concentrations and combinations on micropropagation of O. vulgare × applii. Treatment with 0.28 μM BA and 0.53 μM NAA gave greatest effiency (mean: 22.2 nodes per plantlet). After 60 d 100% of rooted plantlets could be formed per explant under optimum conditions.     

Establishment of a novel tissue culture method, stem-disc culture, and its practical application to micropropagation of garlic (Allium sativum L.)

A restricted part of the undeveloped stem of the garlic clove, called the “stem disc”, which is just under the basement of the immature foliage leaves, proved to be a very potent explant for the micropropagation of garlic. Twenty to thirty tissue-cultured shoots consistently were differentiated from a single clove during 1 month of culture on phytohormone-free Linsmaier and Skoog medium. In addition, more than 90% of the shoots formed bulblets in vitro during an additional 1 month of culture. Pretreatment of the garlic bulbs at 4 °C for approximately 8 weeks before preparing the stem discs enhanced both shoot development and bulblet formation. This novel method for culturing garlic was designated the stem-disc culture method. Shoot development in this type of in vitro culture apparently is divided into four stages: expansion of tissue zones surrounded by the basal parts of the immature foliage leaves, formation of dome-shaped structures, bud differentiation directly from each dome, and development into shoots and bulblets. The dome-shaped structures appeared within 5 days of the onset of culture and had developed independently into shoots approximately 1 cm high 3 weeks later. Histological observations showed that both the internal cell organization and formation process of the dome-shaped structures were similar to those in the meristem. In addition, events leading to the formation of these dome-shaped structures appeared to be initiated by vigorous cell division in the epidermis of concentric tissue zones surrounded by the basements of immature foliage leaves. The results of several field trials showed that the stem-disc culture method is useful for the production of garlic seed plants, including virus-free plantlets. Furthermore, it is a novel field cultivation system for garlic in that the seedlings produced by in vitro-induced bulblets are used as seed instead of the usual cloves. Received: 25 November 1997 / Revision received: 4 February 1998 / Accepted: 21 February 1998     

In what situations isin vitro culture appropriate to plant conservations?

Variousin vitro techniques are available for plant propagation, including seed germination, micropropagation, meristem culture and callus culture. The role of these techniques in the conservation of endangered plants is discussed, using examples drawn from the work of the Micropropagation Unit at Kew.     

Ploidy mosaics: does endopolyploidy in explants affect the cytogenetic stability of orchids regenerated from PLBs?

The induction and regeneration of protocorm-like bodies (PLBs) is a morphogenetic pathway widely used for orchid micropropagation. As endopolyploidy, i.e., the coexistence of cells with different ploidy levels, is a common feature in orchid tissues, a natural question arises when using somatic tissues as explants for orchid micropropagation: does endopolyploidy in explants affect the cytogenetic stability of regenerated plantlets? To answer this question, Epidendrum fulgens was used as a model plant, and flow cytometry was used to analyze endopolyploidy in pollinia, petals, labella, leaf bases, leaf tips, root tips, and protocorm bases and apices, which were subsequently used as explants for PLB induction and plant regeneration. Ploidy screenings showed contrasting ploidy patterns in samples, endopolyploidy being detected in all tissues, with C-values ranging from 1 to 16C. Protocorm bases and root tips presented the highest proportion of endopolyploidy, while petals and protocorm apices showed the lowest proportion. Flower parts exhibited high oxidation for PLB induction and pollinia failed to produce PLB or callus. The highest induction rate occurred at 10 µM TDZ, with 92%, 22%, and 0.92% for protocorm bases, leaves, and root tips, respectively. Plantlets were more easily regenerated from PLBs induced from protocorm bases than from leaves and roots. Doubled ploidy levels were registered in a proportion of 11% and 33% for PLB-regenerated plantlets obtained from protocorm bases and leaf bases, respectively, which was not directly associated with the proportion of endopolyploid cells or cycle value of explants.

     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.     

Improved in vitro rooting in liquid culture using a two piece scaffold system

Plant tissue culture techniques have been used to propagate horticultural crops at a commercial scale for more than three decades. However, due to the high cost it is generally only used for high value crops. To increase production efficiency and make micropropagation viable for a wider range of species, new approaches to address key steps of the process with high labor inputs need to be evaluated. For this study, a two‐piece scaffold system was designed, prototyped using 3D printing, and tested to physically hold plants upright thereby facilitating liquid based rooting. This system was evaluated with Malus domestica, Betula lenta, and Musa sp. using static liquid culture as well as rocker based temporary immersion system and compared to rooting in semi‐solid based medium as is commonly practiced. Significantly, earlier rooting was observed in all three species in liquid when compared to semi‐solid culture system, and plants cultured in liquid on the rocker generally performed better than those in static liquid. In addition to quicker, more uniform rooting, reducing labor requirements, and preventing root damage. This newly designed system is simple, easy to use, will help to improve efficiency, and reduce the cost of micropropagation.     

Application of a novel disposable film culture system to photoautotrophic micropropagation of <Emphasis Type="Italic">Eucalyptus uro-grandis (Urophylia x grandis)</Emphasis>

Summary To overcome various disadvantages of conventional culture vessls for plant micropropagation, we previously developed the photoautotrophic micropropagation technique, with special mention for the first practical film culture system, the ‘Miracle Pack’ (MP), which was made of fluorocarbon polymer film (Neoflo^? PFA film) and supported by a polycarbonate frame. While the PFA film has superior thermal stability, high light transmittance and high gas permeability, making the MP system (MP-PFA) superior to conventional culture vessels for the micropropagation of various plant species, its high cost is a disadvantage. In this study, a possible alternative of lower-cost OTP^? film made of TPX (4-methyl-1-pentane polymer) and CPP (a polypropylene), which possesses similar characteristics to PFA film, is evaluated to develop a novel disposable film culture vesel, termed ‘Vitron’, for culturing Eucalyptus (urophylla x grandis), plantlets. The three film culture systems, MP-PFA, MP-OTP (MP with OTP film), and Vitron, were placed under CO₂ enrichment, low photosynthetic photon flux density (PPFD; 45 μmol m⁻² s⁻¹), and sugar-free medium, using phenol resin foam (Oasis^?) as a substrate. In vitro and ex vitro growth and development of Eucalyptus shoots from the four-leaf stage to the rooting stage were compared for all three culture systems. The effects of the duration and concentration of CO₂ enrichments on in vitro growth of Eucalyptus cultured in the Vitron film system were also examined. The best growth and quality of Eucalyptus plantlets was obtained for the Vitron vessel placed in 3000 ppm CO₂ enrichment for 24 hours per day at low PPFD with sugar-free liquid medium and Oasis as substate. Results of this study suggest that the novel Vitron culture system is suitable for the photoautotrophic micropropagation of Eucalyptus. These authors contributed equally to the research results.     

兰花组织培养和分子生物学研究进展

兰科植物是开花植物中最大的家族之一,其科研和经济价值越来越受到全世界的重视。兰花的组织培养近年来发展迅速,对兰花组织培养中原球茎的诱导和培养基选择的国内外研究进行了综述;并对近年来应用分子标记、转基因等分子生物学技术研究兰花的遗传多样性、系统分类和基因功能进行综述。     

Micropropagation ofDendrobium fimbriatum Hook. by green pod culture

Present studies have demonstrated that immature seeds obtained from green pod ofDendrobium fimbriatum Hook., an endangered epiphytic forest orchid having horticultural importance, can be germinated asymbioticallyin vitro for rapid micropropagation. Vacin and Went medium containing 0.1 mg L^-1 NAA and 15% coconut water was found most effective for high percentage (80-90%) seed germination and seedling development. This method can be exploited for their rapid micropropagation and conservation.     

The role of activated charcoal in plant tissue culture

Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.     

Control of microbial contamination for large-scale photoautotrophic micropropagation

Summary Photoautotrophic (sugar-free) micropropagation has been examined for many different plant species. One of the advantages of photoautotrophic micropropagation is the low risk of contamination, which facilitates the use of large culture vessels, and thus contributes to the reduction in production cost. In this article, pathogenic and nonpathogenic contaminations are discussed, and guidelines for development of large-scale, pathogen-free, photautotrophic micropropagation systems are introduced. A preliminary experiment was conducted for producing pathogen-free plantlets by large-scale photoautotrophic micropropagation. Addition of AgNO₃ in the medium was shown to suppress growth of nonpathogenic contaminants without reducing fresh and dry weight, and number of leaves of the tomato plantlets.     

Elimination of cymbidium mosaic virus and odontoglossum ringspot virus from orchids by meristem culture and thin section culture with chemotherapy

Most commercially cultivated orchid plants are generally infected with cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV). Two methods were used in order to generate virus-free plants: meristem culture and thin section culture with chemotherapy. Meristems (0.10 mm to 1.00 mm) were excised from infected axillary shoots of an infected monopodial orchid hybrid (Mokara Char Kuan ‘Pink’) and cultured in modified Vacin and Went medium. Only larger meristem explants survived and the regenerated plantlets remained virus-infected. In contrast, high percentages of virus-free plantlets were obtained from thin section cultures of infected plantlets and protocorm-like bodies with ribavirin treatment. Interestingly, regenerants from thin section cultures without ribavirin treatment were also found to be free from CyMV and ORSV. All plantlets were tested by enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR).     

In vitro culture of Aloe barbadensis Mill.: Morphogenetic ability and nuclear DNA content

In vitro culture of Aloe barbadensis is very difficult for both callus Induction and plant regeneration. By contrast, micropropagation from shoot meristems is easily achieved. A DNA microdensitometric study was performed on different organs of A. barbadensis and during in vitro culture of different explants. It was shown that morphogenetic ability is correlated to nuclear DNA content in the explant and/or in the callus. In particular, plant regeneration (or micropropagation) is obtained only from calli (or from explants) in which the amount of DNA per nucleus falls strictly within the diploid limits (2C and 4C) of the species.