In vitro chili pepper biotechnology

Summary Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation, have lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation aimed at genetic improvement against pests and diseases. Some approaches, however, have made possible the regeneration of chili pepper plants from in vitro-cultured cells, tissues, and organs through organogenesis or embryogenesis. Anther culture has been successfully applied to obtain haploid and doubledhaploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation. Application of both tissue culture and genetic transformation techniques have led to the development of chili pepper plants more resistant to at least one type of virus. Cell and tissue cultures have been applied successfully to the selection of variant cells exhibiting increased resistance to abiotic stresses, but no plants exhibiting the selected traits have been regenerated. Production of capsaicinoids, the hot principle of chili pepper fruits, by cells and callus tissues has been another area of intense research. The advances, limitations, and applications of chili pepper biotechnology are discussed.     

Influence of cytokinin levels on <Emphasis Type="Italic">in vitro</Emphasis> propagation of shy suckering chrysanthemum “Arka Swarna” and activation of endophytic bacteria

In an effort to develop a sustainable protocol for the micropropagation of a shy suckering elite chrysanthemum cv. Arka Swarna (yellow pompon type), in vitro cultures were established using surface-sterilized nodal microcuttings (1–1.5 cm) from polyhouse-grown plants on MS medium containing 3% sucrose, 0.25% phytagel, and 5 μM benzyl adenine (BA) or kinetin. Microbial contamination in the range of 6–24% was encountered during the first in vitro passage. Apparently clean cultures after one passage on MS basal medium were transferred to medium with BA or kinetin (0, 1, 5, 10, or 20 μM) in culture bottles, and were monitored for eight in vitro passages (1 mo. each) for growth and microbial contamination. Plant growth regulator (PGR)-free medium was the best for sustainable micropropagation over successive in vitro passages yielding a single shoot from cultured microcuttings. Higher cytokinin levels inhibited rooting and induced one or more shorter shoots with close nodes resulting in low propagation rates. All apparently clean stocks revealed covert endophytic bacteria during tissue-indexing using bacteriological media. Three distinct bacterial morphotypes were isolated from such stocks, identified based on 16S rRNA gene sequence analysis as different morphotypes of Curtobacterium citreum. The endophytes tended to show obvious growth on chrysanthemum culture medium with increase in cytokinin levels (5–20 μM), but such growth was not noticed in inoculations on MS medium without plants. Sustainable micropropagation of cv. Arka Swarna for more than 2 yr with the resident endophytic bacteria in covert form was realized on PGR-free MS medium giving a net propagation rate of three to four times over a subculture cycle of 2–3 wk.     

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.     

Alterations in growth and crassulacean acid metabolism (CAM) activity of in vitro cultured cactus

Unlike C-3 plants, cacti possess a crassulacean acid metabolism (CAM) physiology that can alter the pattern of carbon uptake and affect plant growth under artificial environmental conditions, especially in tissue culture. In vitro-derived plantlets of Coryphantha minima grew 7-fold larger than plants cultured under similar ex vitro conditions. Growth regulators incorporated into the culture media during shoot proliferation stage of micropropagation had a strong influence on this increased growth. Other important factors that contributed to increased growth under in vitro conditions were high relative humidity and sugar in the culture medium. An analysis of gas exchange and daily fluctuations of malic acid levels revealed an increase in net photosynthetic rate, in terms of carbon assimilation, by in vitro plants compared with that of ex vitro plants. This stimulated photosynthesis in the presence of an external carbon source was unexpected but apparently true for cacti exhibiting CAM physiology. Unlike CAM plants grown in ex vitro conditions, net CO₂ uptake by in vitro-cultured cacti occurred continuously in the light as well as the dark. Once regenerated, cacti were transferred to ex vitro conditions where the normal CAM pathway resumed with a concomitant reduction in growth and CO₂ uptake. These results showed that growth of cacti can be considerably accelerated by in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro regeneration and micro-propagation of enset from Southwestern Ethiopia

Three clones of enset (Ensete ventricosum Welw. Cheesman) from southwestern Ethiopia (Keffa-Shaka zone) were investigated for their potential for micropropagation and regeneration in tissue culture. Corm and leaf tissue of greenhouse-grown plants as well as in vitro germinated zygotic embryos were used as starting material for micro-propagation and regeneration studies. Embryos were excised from disinfected seeds and cultured in vitro. Multiple shoots from both corm- and embryo-explants were obtained using regeneration medium supplemented with 10 μM or 20 μM BAP. Rooting of shoots was achieved using medium with 5 μM IBA, 1 μM BAP and 1 g l⁻¹ activated charcoal. Plantlets obtained by this process were transferred to soil under greenhouse conditions. Optimal conditions, which were determined for clonal propagation of three different genotypes of enset, allowing both in vitro micropropagation and regeneration, are described. This protocol makes for conservation of enset clones, rapid propagation of selected disease-free germplasm and more efficient breeding procedures. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro micropropagation of Primula scotica: a rare Scottish plant

A simple micropropagation method is reported for Primula scotica, a rare plant, endemic to the North of Scotland. The technique involves the clonal proliferation of seed-derived plantlets on either hormone free tissue culture medium or on medium containing benzyl amino purine and indole acetic acid. Average multiplication rates of 4–6 were obtained for plants grown on plant growth regulator supplemented medium. The micropropagation method was applied to four different clones of P. scotica and clonal differences were observed in relation to media type. Some plants from certain clones did display hyperhydricity, however, this was circumvented by using frequent sub-culturing intervals and transferring the plants to hormone-free medium. Plantlets rooted on both media types and displayed normal, true-to-type rosette morphology. The cultures did not callus and development proceeded via shoot and root production only. The in vitro-grown plants could be transferred to ex vitro conditions and a range of growth substrates were assessed for their efficacy in supporting ex vitro growth, with a view to developing longer-term strategies for the transfer and reintroduction of micropropagated P. scotica plants into natural habitats. The simple method described in this paper may offer the potential of being applied to other endangered Primula spp.     

Molecular Analysis of In Vitro Shoot Organogenesis

Shoot organogenesis is one of the in vitro plant regeneration pathways. It has been widely employed in plant biotechnology for in vitro micropropagation and genetic transformation, as well as in study of plant development. Morphological and physiological aspects of in vitro shoot organogenesis have already been extensively studied in plant tissue culture for more than 50 years. Within the last ten years, given the research progress in plant genetics and molecular biology, our understanding of in vivo plant shoot meristem development, plant cell cycle, and cytokinin signal transduction has advanced significantly. These research advances have provided useful molecular tools and resources for the recent studies on the genetic and molecular aspects of in vitro shoot organogenesis. A few key molecular markers, genes, and probable pathways have been identified from these studies that are shown to be critically involved in in vitro shoot organogenesis. Furthermore, these studies have also indicated that in vitro shoot organogenesis, just as in in vivo shoot development, is a complex, well-coordinated developmental process, and induction of a single molecular event may not be sufficient to induce the occurrence of the entire process. Further study is needed to identify the early molecular event(s) that triggers dedifferentiation of somatic cells and serves as the developmental switch for de novo shoot development.     

Conservation In vitro of threatened plants—Progress in the past decade

Summary In vitro techniques have found increasing use in the conservation of threatened plants in recent years and this trend is likely to continue as more species face risk of extinction. The Micropropagation Unit at Royal Botanic Gardens, Kew, UK (RBG Kew) has an extensive collection of in vitro plants including many threatened species from throughout the world. The long history of the unit and the range of plants cultured have enabled considerable expertise to be amassed in identifying the problems and developing experimental strategies for propagation and conservation of threatened plants. While a large body of knowledge is available on the in vitro culture of plants, there are limited publications relating to threatened plant conservation. This review highlights the progress in in vitro culture and conservation of threatened plants in the past decade (1995–2005) and suggests future research directions. Works on non-threatened plants are also included wherever methods have applications in rare plant conservation. Recalcitrant plant materials collected from the wild or ex situ collections are difficult to grow in culture. Different methods of sterilization and other treatments to establish clean material for culture initiation are reviewed. Application of different culture methods for multiplication, and use of unconventional materials for rooting and transplantation are reviewed. As the available plant material for culture initiation is scarce and in many cases associated with inherent problems such as low viability and endogenous contamination, reliable protocols on multiplication, rooting, and storage methods are very important. In this context, photoautotrophic micropropagation has the potential for development as a routine method for the in vitro conservation of endangered plants. Long-term storage of material in culture is challenging and the potential applications of cryopreservation are significant in this area. Future conservation biotechnology research and its applications must be aimed at conserving highly threatened, mainly endemic, plants from conservation hotspots.     

Nuclear rDNA instability in in vitro‐generated plants is amplified after sexual reproduction with conspecific wild individuals

Using micropropagation through tissue culture has become the most used approach worldwide for mass production for the conservation of endangered species. However, the screening of somaclonal variations generated using in vitro culture is usually restricted to the first generation of micropropagated plants, when they have not yet been released in the field. Accordingly, the fate of genetically modified regenerants after sexual reproduction is usually not assessed and changes in the genetic structures of species are unknown. In this work, we assess the cytogenetic stability of two rDNA gene families in the offspring of experimental crosses between accessions generated after in vitro culture and wild individuals of Cistus heterophyllus (Cistaceae). The cytogenetic rDNA profiles (45S rDNA, 5S rDNA) of 118 accessions including wild and in vitro micropropagated individuals and bi‐directional artificial crosses between wild and in vitro‐generated plants were assessed by fluorescence in situ hybridization (FISH) and Ag‐NOR staining. Plants regenerated by micropropagation showed a lower size of the FISH signals in a 45S rDNA site, but this condition was not present in the wild accessions. Three new cytogenetic and cytological variants were present in 36% of the experimental progeny, involving the amplification of one additional 45S rDNA site and the presence of heteromorphic nucleoli. rDNA‐based genomic instability was present after sexual reproduction between wild and in vitro‐generated plants. The results of this study discourage the use of micropropagated materials for plant conservation unless comprehensive surveys of the genetic integrity and stability of the regenerants are performed after crossing between wild and micropropagated plants.     

Bushiness and cytokinin sensitivity in micropropagated Zantedeschia

Bushiness, the development of short, multiple stems, has been reported in some commercial hybrids of Zantedeschia, particularly the yellow-flowered cultivar Florex Gold derived from tissue culture. This cultivar exhibited a greater sensitivity to 6-benzylaminopurine when compared to four other cultivars using an in vitro root length bioassay, suggesting it may be pre-disposed to bushiness. The carry-over effect of cytokinin from micropropagation on subsequent tuber formation and flower development, in three selections of the cultivar Florex Gold, was also investigated. Cytokinin levels used in commercial Zantedeschia micropropagation protocols did not alter tuber or flower development. However, excessively high levels did affect development, also indicating that cytokinin may influence bushiness. Multi-eyed tubers used to initiate in vitro cultures were shown to have a significant effect on first year tuber formation and subsequent flower weight, compared to few-eyed tubers, although these plants did not necessarily develop bushy symptoms. Plant location within the greenhouse also influenced flowering although these plants did not display multiple stemmed bushy symptoms.     

In vitro studies on Eucalyptus globulus rooting ability

Summary Micropropagation has the potential to quickly introduce selected genotypes of adult Eucalyptus globulus clones and it is now widely used in Portugal as a part of genetic improvement programs. Several clones have been established and multiplied in vitro. The different clones have individual requirements for successful rooting. Rejuvenation was achieved at different periods after culture initiation for the different clones. Subculturing preceding rooting in multiplication medium supplemented with riboflavin and cholene chloride allowed the increase of rooting ability for several clones tested. Removal of boron from the rooting medium increased rooting by 10%. Indolebutyric acid (IBA) dipping before transfer to the rooting medium resulted in a rooting percentage of 80–95% for the best clones tested. Acclimatization was performed without difficulties (90–95% success) and the rooted plants were either planted directly or used as mother plants for further cutting production, depending on the needs. The results described in this paper increase the commercial feasibility of the micropropagation system for E. globulus.     

Virus infections reduce in vitro multiplication of ‘Malling Landmark’ raspberry

Summary Virus-infected plants are often symptomless and may be inadvertently used as explant sources in tissue culture research. Our objective was to determine the effect of virus infection on micropropagation. We studied the effects of single and multiple infections of three common raspberry viruses on the in vitro culture of ‘Malling Landmark’ red raspberry (Rubus idaeus L.). Virus-infected reaspberry plants were produced by leaf-graft inoculation from known-infected plants onto virus-free ‘Malling Landmark’. Single-virus source plants were infected with either tobacco streak ilarvirus (TSV), tomato ringspot nepovirus (TomRSV), or raspberry bushy dwarf idaeovirus (RBDV) and were free of other viruses as determined by enzyme-linked immunosorbent assay (ELISA) and bioassay. Virus-free, single, and multiple virus-infected ‘malling Landmark’ explants were initiated into culture and multiplied on Anderson's medium with 8.9 μM N⁶-benzyladenine (BA). At the end of the multiplication tests, ELISA reconfirmed virus infections. In vitro multiplication of ‘Malling Landmark’ was significantly reduced by multiple infections, and multiplication of plants infected with all three viruses (RBDV+TomRSV+TSV) was less than half that of virus free cultures. Shoot height and morphology of in vitro cultures were not influenced by virus infection. The greenhouse stock plant with the three-virus infection was stunted and yellow compared to the control and the other infected plants. Part of a thesis submitted by C.-W.V.T. in partial fulfilment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.     

Progress in medicinal plant <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis>: Metabolite profiling,tissue culture,growth and its regulation,and functional genomics

As an important medicinal plant, Rehmannia glutinosa Libosch. is widely spread in East Asian countries, and its root, possessing multiple pharmacological values, is used as traditional Chinese medicine in clinics. Recently, much progress in R. glutinosa has been made. Tissue culture and micropropagation have been applied to generate virus-free germs or homogeneous plants. In vitro culture and generation of transgenic R. glutinosa plants has been recently setup, which is helpful to develop more genetically-modified germplasms. Multiple environmental factors (e.g. CO₂ concentration, humidity, transpiration, drought, and viral diseases) play an important role in growth and development of R. glutinosa plants. Gene cloning, genetic transformation and metabolite profiling are becoming attractive. The review aims to summarize advances on metabolomics, tissue culture and regeneration, growth and its regulation, and functional genomics of R. glutinosa, which is in favor of the cultivation, processing, in addition to the study of metabolic engineering and metabolite profiling in R. glutinosa.     

Plant regeneration from stem nodal segments of <Emphasis Type="Italic">Orthosiphon stamineus</Emphasis> benth., a medicinal plant with diuretic activity

Summary A micropropagation method for Orthosiphon stamineus, using stem nodal segments, has been developed. The highest number of regenerated shoots was obtained on Murashige and Skoog (MS) medium supplemented with 6.7 μM benzyladenine with the formation of an average of 6.1 shoots per explant over a period of 4 wk. The number of shoots increased with longer culture duration on proliferation medium. Multiple shoots which were maintained on the proliferation medium for 6 wk had the highest proliferation rate. Separation of multiple shoots and culturing in larger flasks significantly promoted the growth and formation of plantlets. All the in vitro plantlets survived when transferred to the field and showed no significant morphological differences from the mother plants.     

Efficient micropropagation of Robinia ambigua var. idahoensis (Idaho Locust) and detection of genomic variation by ISSR markers

Robinia ambigua var. idahoensis, presumably originated from interspecific hybridization of R. pseudoacacia L. and R. hispida L., is a multipurpose tree. Several reports have showed that in vitro micropropagation is a feasible method to produce large quantities of ‘clonal’ plants from R. pseudoacacia, however, no information is available on micropropagation of R. ambigua or the other assumed parental species, R. hispida. Here, we report on a tissue culture system for efficient micropropagation of R. ambigua plants by enhanced branching of axillary buds taken from a single branch of a donor tree. The culture system consists of sequential use of three media, namely, the bud-induction medium (MS medium supplemented with 0.8–1.4 mg l⁻¹ 6-BA, 0.05–0.08 mg l⁻¹ NAA and 0.07–0.1 mg l⁻¹ GA), elongation medium (MS medium added with 0.35–0.5 mg l⁻¹ 6-BA, 0.05–0.08 mg l⁻¹ NAA and 0.07–0.1 mg l⁻¹ GA) and root-induction medium (1/4 MS medium fortified with 1.7–2.5 mg l⁻¹ IAA and 0.1–0.5 mg l⁻¹ IBA). In addition, we investigated the genetic stability (relative to the donor plant) of a sample of 41 morphologically normal plants randomly taken from ca. 13,000 micropropagated plants, by using the inter-simple sequence repeat (ISSR) marker with 32 selected primers. We found that of the 226 reproducible bands scored, 24 were polymorphic (10.62%), thus pointing to the occurrence, though at a relatively low level compared with an earlier study on R. pseudoacacia, of genomic variation in these micropropagated plants. Further sequencing on seven loci underlying the variations showed that two had significant homology to known or predicted plant genes.     

Agrobacterium-mediated genetic transformation of the desiccation tolerant resurrection plant Ramonda myconi (L.) Rchb.

In this paper we describe the first procedure for Agrobacterium tumefaciens-mediated genetic transformation of the desiccation tolerant plant Ramonda myconi (L.) Rchb. Previously, we reported the establishment of a reliable and effective tissue culture system based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. This efficient plant regeneration via callus phase provided a basis for the optimisation of the genetic transformation in R. myconi. For gene delivery, both a standard (method A) and a modified protocol (method B) have been applied. Since the latter has previously resulted in successful transformation of another resurrection plant, Craterostigma plantagineum, an identical protocol was utilized in transformation of R. myconi, as this method may prove general for dicotyledonous resurrection plants. On this basis, physical and biochemical key variables in transformation were evaluated such as mechanical microwounding of plant explants and in vitro preinduction of vir genes. While the physical enhancement of bacterial penetration was proved to be essential for successful genetic transformation of R. myconi, an additional two-fold increase in the transformation frequency was obtained when the above physical and biochemical treatments were applied in combination. All R ₀ and R ₁ transgenic plants were fertile, and no morphological abnormalities were observed on the whole-plant level. Collaborator via a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agriculture Systems     

Micropropagation and slow growth conservation of cardamom (<Emphasis Type="Italic">Elettaria cardamomum</Emphasis> Maton)

Cardamom (Elettaria cardamomum Maton) has great commercial value as a spice crop in India. A one-step protocol for direct regeneration of plants and in vitro conservation by slow growth method has been developed. A maximum of 6.5 shoots/culture were obtained in 2 mo or 15.1 shoots/culture in 4 mo on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) + 5 μM benzylaminopurine gelled with 0.7% agar (micropropagation medium). Rooting also occurred simultaneously on the same medium. Using one shoot tip or nodal explant, about 30,375 plants can be regenerated in a year on the micropropagation medium. In vitro conservation by slow growth method was achieved on 1/2 MS (major salts) + 5 μM BAP + 0.7% agar (conservation medium); about 70% of the cultures survived up to 18 mo at 25 ± 2°C. Successful regrowth of plants on micropropagation medium was obtained by culturing nodal explants excised from 18-mo-old conserved plants. Some 96% of the plants survived the hardening treatment and grew normally in a greenhouse. If 24 cultures are conserved on the conservation medium, it is possible to regenerate at least 750 plants by using explants derived from 70% of the surviving shoots and culturing the same in micropropagation medium for 4 mo. These plants may be used for planting or as a source of explants for the next conservation cycle. On the basis of 20 random amplified polymorphic DNA and 13 inter-simple sequence repeat primers analyses, no significant reproducible variation was detected among the in vitro-conserved plants compared with the mother plants.     

Chrysanthemum: advances in tissue culture, cryopreservation, postharvest technology, genetics and transgenic biotechnology

Members of the Chrysanthemum-complex include important floricultural (cut-flower) and ornamental (pot and garden) crops, as well as plants of culinary, medicinal and (ethno)pharmacological interest. The last 35 years have seen a tremendous emphasis on their in vitro tissue culture and micropropagation, while the latter 10–15 years has seen a surge in transformation experiments, all aimed at ameliorating aesthetic and growth characteristics of the plants. This review highlights all available literature that exists on ornamental Chrysanthemum in vitro cell, tissue and organ culture, micropropagation and transformation.     

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.     

In vitro shoot proliferation of 5-leaf aralia explants from field grown plants and forced dormant stems

A. Sieboldianus (5-leaf aralia) is recalcitrant for micropropagation, but has very good landscaping potential. This research was conducted with the following objectives: (1) to study effects of BA, TDZ, CPPU, 2iP, kinetin and zeatin in woody plant medium on the performance of softwood shoot nodal explants produced by field grown 5-leaf aralia plants; (2) to investigate influences of BA or TDZ in the forcing solution on subsequentin vitro shoot initiation of nodal explants taken from forced softwood growth. Shoot initiation of softwood nodal explants from field-grown plants was promoted by adding BA, TDZ or CPPU to the culture medium. Kinetin, zeatin and 2iP were ineffective for micropropagation ofA. Sieboldianus. The forced softwood growth for use as explants was “primed” by forcing dormant stems in solution containing 200 mg 8-HQC per liter plus 2% sucrose, 44.4, 222, or 444 μM BA, or 45.4, 227, or 454 μM TDZ. BA and TDZ in the forcing solution enhanced subsequentin vitro axillary shoot initiation of nodal explants taken from forced stems by doubling the number of shoots produced per explant to 3.3 from 1.65 shoots per explant taken from field grown plants. This forcing solution technique also reduced the time needed from culture initiation to potted plants to half of the time needed for the conventional micropropagation method (12 to 14 vs. 25 to 27 weeks), thus expediting the micropropagation ofA. Sieboldianus.