Somatic embryogenesis from mature <Emphasis Type="Italic">Bambusa balcooa</Emphasis> Roxburgh as basis for mass production of elite forestry bamboos

A reliable protocol for mass propagation via somatic embryogenesis in mature bamboos has been established using pseudospikelets of Bambusa balcooa. Fourty percent of the explants gave rise to multiple regenerants within 4 months. This conversion rate is sufficiently high to use the process in commercial mass production. Further, shoot apical meristems can also be used as primary explants without lost of efficiency. Regenerated plants were uniform and identical to the mother plant and to plants obtained by axillary branching with respect to growth characteristics and morphology. Furthermore, epigenetic changes could not be detected by Methylation Sensitive AFLP (MSAP). During the complete process no changes in ploidy level could be observed. The process allows for a cost reduction for this tropical bamboo for forestry of up to 57% compared to micropropagation via axillary branching. For the first time, a reliable process based on somatic embryogenesis has been developed that is well suited for commercial micropropagation of elite mature bamboos.     

Conservation and Propagation of High Altitude Medicinal and Aromatic Plant: Hedychium spicatum

The micropropagation of H.spicatum, a medicinal and aromatic plant was investigated as an option for conservation and propagation, as wild populations are fast depleting. The source of raw material is rhizomes of plants that are collected from the wild. There is no planned cultivation of the plant. Multiple shoot cultures were established on MS medium supplemented with BAP and IAA from the pre-existing buds on the rhizome. Prolonged cultivation on the same medium or transfer to hormone free medium induced roots/rhizome formation; liquid medium proved more suitable. Greenhouse hardened plants were transferred to field. A successful protocol with 99% root formation and 80–85.5% field survival has been formulated.     

Regeneration and assessment of genetic fidelity of the endangered tree Moringa peregrina (Forsk.) Fiori using Inter Simple Sequence Repeat (ISSR)

Moringa peregrinais an endangered species of Moringaceae.M. peregrinais a multipurpose tree with a wide variety of potential uses including its medicinal activity. In our study, a rapid and efficient micropropagation protocol for M. peregrina has been established. In vitro germinated seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different levels of either 6-benzyladenine (BA) or kinetin (Kin). The maximum shoot proliferation of 6.5 shoots per explant with 100 % shoot proliferation rate was observed on MS medium supplemented with 1.0 mg/l BA. On the other hand, MS medium supplemented with 1 mg/l indole-3-butyric acid (IBA) resulted in the maximum number of roots. Micropropagated plants were successfully acclimatized. Genetic stability of micropropagated plants was assessed using Inter-Simple Sequence Repeat (ISSR). The amplification products were monomorphic in all in vitro grown plants. No polymorphism was detected indicating the genetic integrity of in vitro propagated plants. This micropropagation protocol could be useful for raising genetically uniform plants for plant propagation and commercial cultivation.     

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.     

Biochemical and molecular profiling of micropropagated and conventionally grown Kaempferia galanga

Kaempferia galanga L., family Zingiberaceae, is used extensively in the preparation of both traditional and modern medicines. Buds of rhizomes of K. galanga were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/l benzyladenine and 0.5 mg/l indole-3-acetic acid (IAA) to induce shoot proliferation. Micropropagated plantlets subjected to random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker-based molecular profiling revealed uniform banding patterns similar to those of the mother plants. After 2 years of culture in vitro, plantlets were transplanted to the field, evaluated for different agronomic traits, and their rhizomes were subjected to biochemical profiling using quantitative and qualitative assays of essential oils. Gas chromatography and mass spectroscopy analysis of rhizome oil of micropropagated plants showed the presence of 10 major components which were similar to those detected in the mother plants, and accounted for 95.5% of the total compounds. The compound ethyl-p-methoxy cinnamate accounted for 59.5% of the total compounds detected, followed by ethyl cinnamate, 3-carene, pentadecane, borneol, bornyl acetate, delta-selinene, camphor, alpha-piene and immidazole, 5-carbonylvinyl-4-nitro. Biochemical and molecular profiling of micropropagated clones revealed that an in vitro micropropagation protocol could be effectively used for commercial propagation of true-to-type K. galanga for a stable supply of the medicinal compounds present in this plant species.     

Tissue culture of Hypericum brasiliense Choisy: Shoot multiplication and callus induction

Hypericum brasiliense, a non-domesticated plant has been shown to have useful medicinal properties. This plant has not been cultivated so a protocol for mass propagation based on selection of superior clones was developed and a protocol established for the culture of callus cells that could be used for in vitro metabolite production. A micropropagation method based on amplification of nodal buds was developed, by selection, from ten seedling clones that were examined for growth rate, multiplication rate and rooting. The effect of various basal media, growth regulator types and concentrations were examined for optimal callus induction. Optimal callus induction occured on either Murashige and Skoog's or Gamborg's media supplemented with 1 to 2 mg l^–1 of 2,4-dichlorophenoxyacetic acid.Abbreviations B5 Gamborg's medium - 2,4-Dscd 2,4-dichlorophenoxyacetic acid - IAA indolacetic acid - MS Murashige & Skoog's medium - NAA naphtaleneacetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>, a critically endangered ethnomedicinal plant

Summary This study reports a protocol for successful micropropagation of Decalepis arayalpathra (Joseph and Chandras) Venter. (Janakia arayalpathra Joseph and Chandrasekhran; Periplocaceae), a critically endangered and endemic ethnomedicinal plant in the southern forests of the Western Ghats which is overexploited for its tuberous medicinal roots by the local Kani tribes. Natural regeneration is rare and conventional propagation is difficult. Conservation of the species through micropropagation was attempted. The nodal explants of greenhouse-raised plants, were more desirable than cotyledonary nodal explants of aseptic seedlings. The basal nodes (73%) of 12–16-wk-old greenhouse-grown plants cultured in Murashige and Skoog (MS) medium containing 12.96 μM 6-benzyladenine (BA), 2.48 μM 2-isopentenyladenine (2-ip) and 2.68 μM α-naphthaleneacetic acid (NAA) formed 16–17 cm long unbranched robust solitary shoots in 8 wk. Cotyledonary nodal explants cultured in the same medium showed multiple shoot formation and axillary branching. But the shoots were thin, fragile and not suitable for mass propagation. Single nodes of a solitary shoot subcultured on MS medium containing 2.22 μM BA and 0.24 μM 2-ip together produced 9.8±0.3 nodes from 18.0±0.6 cm long shoots within 5–6 wk. The basal nodes of the shoots so formed were repeatedly subcultured to increase the stock of propagules while the 2.5–3.0 cm terminal cuttings were used for rooting. The best root induction (68%) and survival (86%) was achieved on half-strength MS medium supplemented with 1.07 μM NAA. Field-established plants showed uniform growth and phenotypic similarity to parental stock.     

In vitro propagation of medicinal and aromatic plants: the case of selected Greek species with conservation priority

Worldwide, many medicinal and aromatic plants (MAPs) are still collected from the wild and only a small fraction of them are exclusively sourced from cultivation. This practice when performed non-sustainably threatens species and populations. Micropropagation of MAPs is a powerful tool to conserve rare, threatened, and valuable MAPs, and to massively produce high-value plant material for cultivation without seasonal constraints. In this study, the in vitro propagation protocols of 22 Greek native MAPs assigned with conservation priority were assessed (herbaceous perennials, bulbous, subshrubs, and trees), including 17 range-restricted plants and 5 taxa of Orchidaceae. For the latter, current micropropagation efforts include seed germination, callus induction, and protocorm formation for successful plantlet development; however, these propagation protocols are still fragmentary. For the rest (n = 17), a five-stage detailed procedure is outlined (plant material, establishment, proliferation, rooting, and acclimatization), while materials, treatments, and data per stage are shown comparatively and discussed. Emphasis is given on the selection and preparation of plant material obtained from nature for research, sustainable use, and ex situ conservation actions, and on their effectiveness for conservation purposes or mass production needs. The protocol effectiveness was calculated using a specific equation to estimate the potential number of acclimatized plants raised from a single explant within a year. All protocols can facilitate conservation, and almost half of them could be used for commercialization with high cost (five cases), intermediate cost (eight), or low cost (four), which enables their possible sustainable use.

     

Micropropagation and β-ecdysone content of the Brazilian ginsengs <Emphasis Type="Italic">Pfaffia glomerata</Emphasis> and <Emphasis Type="Italic">Pfaffia tuberosa</Emphasis>

The aim of this study was to investigate both a mass in vitro propagation system and the β-ecdysone content in roots and aerial parts of Pfaffia glomerata and Pfaffia tuberosa. Nodal segments of two genotypes (BRA and JB-UFSM) of P. glomerata, originated from aseptically grown plants, were cultivated on hormone-free Murashige and Skoog medium. For the proliferation of P. tuberosa shoots, nodal segments, originated from aseptically grown plants, were either cultivated on hormone-free Murashige and Skoog (MS) medium or were supplemented with 1.0 μM thidiazuron (TDZ); the elongation and rooting of these plants were carried out on MS medium without TDZ. Plantlets of both species were acclimatized and transferred to field conditions. The β-ecdysone content in the plants was determined by high performance liquid chromatography. The BRA genotype showed a greater in vitro proliferation rate and β-ecdysone content than that of the JB-UFSM genotype. The culture of nodal segments of P. tuberosa on medium with 1.0 μM TDZ with subsequent subcultivation of shoots on hormone-free medium was shown to be a suitable method for micropropagation due to the high multiplication rate and good plant development. Both species showed good adaptation to ex vitro conditions. The β-ecdysone content in micropropagated P. tuberosa was similar to that found in field-grown plants. For both species, the aerial parts accumulated higher β-ecdysone content than roots. These results reveal that micropropagation is a successful, alternative method for rapid plant multiplication of both species of Brazilian ginseng. Furthermore, this study demonstrates that these two species have a potential for cultivation that is associated with high β-ecdysone production.     

In vitro regeneration of Echinacea purpurea L.: Direct somatic embryogenesis and indirect shoot organogenesis in petiole culture

Summary An in vitro propagation system was developed for Echinacea purpurea L. (purple coneflower), a medicinal plant commonly used in the treatment of colds, flu and related ailments. Echinacea seeds were found to be contaminated with systemic fungi and therefore an optimized minimal concentration of Plant Preservation Mixture (PPM) was incorporated in the seed germination medium to recover sterile seedlings. Regeneration was induced on petiole explants from 2-month-old sterile seedlings cultured on medium supplemented with benzylaminopurine (BAP) or thidiazuron (TDZ) in combination with indoleacetic acid (IAA). Two distinct forms of regeneration were identified in cultured petiole explants with histological and morphological observations, viz. the direct formation of somatic embryos on the epidermis and the de novo development of shoots from callus tissues formed in subepidermal cell layers. the results of this study have established a micropropagation system for E. purpurea that will provide sterile plant material for further investigations into medicinally active biochemicals and may facilitate mass production of high-quality E. purpurea plants for the commercial market.     

Plant Thin Cell Layers: A 40-Year Celebration

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.     

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.     

Conservation strategies for threatened medicinal plant – Barleria prionitis L. – using in vitro and ex vitro propagation techniques

Over-utilisation and continuous depletion of medicinal plants have affected their supply and loss of genetic diversity. Hence the current study is based on conservation strategies for threatened medicinal plants with special reference to Barleria prionitis L. using in vitro and ex vitro propagation techniques. We have developed here a protocol for plant regeneration of Barleria prionitis L. We have also developed an efficient system of vegetative propagation of Barleria prionitis L. through stem cuttings using revive rooting hormones. These studies can be useful for conservation strategies of this important medicinal plant.     

In vitro manipulation and propagation of medicinal plants

Well developed techniques are currently available to help growers meet the demand of the pharmaceutical industry in the next century. These protocols are designed to provide optimal levels of carbohydrates, organic compounds (vitamins), mineral nutrients, environmental factors (e.g. light, gaseous environment, temperature, and humidity) and growth regulators required to obtain high regeneration rates of many plant species in vitro and thereby facilitate commercially viable micropropagation. Well-defined cell culture methods have also been developed for the production of several important secondary products. An overview of the regeneration of medicinal plants by direct and indirect organogenesis and by somatic embryogenesis from various types of explants is presented, and the use of these techniques combined with other biotechnological approaches to improve medicinal plants through somaclonal variation and genetic transformation is reviewed.     

Thidiazuron-induced regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Micropropagation in solid and liquid culture systems

The goals of this study were to investigate thidiazuron (TDZ)-induced morphogenesis of Echinacea purpurea L. and to assess the possibility of developing a liquid-based protocol for rapid micropropagation. Callus development and root organogenesis were observed on leaf explants cultured on media containing 2,4-dicholorophenoxyacetic acid or dicamba, but no plantlets were regenerated. Addition of TDZ to the culture medium as the sole growth regulator resulted in the production of regenerable callus cultures. The highest rate of regeneration was observed for explants cultured on medium with TDZ at 2.5 μM or higher. Tissue derived from 1.0 μM TDZ treatments was used to initiate liquid cultures. All liquid treatments produced a similar number of regenerants but significantly more healthy plants were obtained from cultures grown in the presence of 0.1 and 1.0 μM TDZ. This TDZ-based micropropagation system is the first liquid, large-scale propagation protocol developed for the mass production of E. purpurea plants.     

Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production

Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as “Indian Ginseng”, is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and the secondary metabolites produced by this plant can be exploited further for the benefit of human health in a sustainable way.     

Metabolic profiling of in vitro micropropagated and conventionally greenhouse grown ginger (Zingiber officinale)

Ginger is an important medicinal and culinary herb, known worldwide for its health promoting properties. Because ginger does not reproduce by seed, but is clonally propagated via rhizome division and replanting, it is susceptible to accumulation and transmittance of pathogens from generation to generation. In addition, such propagation techniques lead to slow multiplication of particularly useful stocks. We have developed an in vitro propagation method to alleviate these problems. Metabolic profiling, using GC/MS and LC-ESI-MS, was used to determine if chemical differences existed between greenhouse grown or in vitro micropropagation derived plants. Three different ginger lines were analyzed. The constituent gingerols and gingerol-related compounds, other diarylheptanoids, and methyl ether derivatives of these compounds, as well as major mono- and sesquiterpenoids were identified. Principal component analysis and hierarchical cluster analysis revealed chemical differences between lines (yellow ginger vs. white ginger and blue ring ginger) and tissues (rhizome, root, leaf and shoot). However, this analysis indicated that no significant differences existed between growth treatments (conventional greenhouse grown vs. in vitro propagation derived plants). Further statistical analyses (ANOVA) confirmed these results. These findings suggest that the biochemical mechanisms used to produce the large array of compounds found in ginger are not affected by in vitro propagation.     

Micropropagation of Gagnepainia godefroyi K Schum and Gagnepainia thoreliana (Baill) K Schum — Rare Medicinal Plants of Thailand

A micropropagation protocol for Gagnepainia godefroyi K Schum and G thoreliana (Baill) K Schum, two rare medicinal plants of Thailand, has been developed using terminal buds. After a total of twenty weeks of culture (4 weeks on MS added with 36.33 μM TDZ and 16 weeks on PGR-free MS), 28.77 and 13.50 shoots/explant were obtained, respectively. Rooting was spontaneous and regenerated plants were successfully transplanted to soil.     

Regeneration of Echinacea purpurea: Induction of root organogenesis from hypocotyl and cotyledon explants

An in vitro propagation system was developed for Echinacea purpureaL. (purple coneflower), a medicinal plant commonly used in the treatment of colds, flu and related ailments. Root organogenesis from Echinacea purpurea hypocotyl explants was effectively induced by indolebutyric acid. Indoleacetic acid was found to be less effective than indolebutyric acid while treatments with naphthaleneacetic acid were ineffective for induction of root organogenesis. The results of this study have established a micropropagation system for Echinacea purpurea that will provide axenic plant material for further investigations into medicinally active biochemicals and the mass production of high-quality Echinacea purpurea root tissues for the commercial market. This revised version was published online in June 2006 with corrections to the Cover Date.     

Density and potential utilisation of medicinal grassland plants from Abe Bailey Nature Reserve, South Africa

The Abe Bailey Nature Reserve, South Africa has been identified as a potential community resource reserve compatible with biodiversity conservation. As part of the planning and public participation phase this study investigated the potential for sustainably harvesting medicinal plants. Medicinal plant use data on local species, parts used and harvesting techniques, in collaboration with neighbouring traditional healers were collected. A census of these plants was conducted in the reserve and frequency, density and abundance were calculated. A medicinal plant rating system for assessing conservation priorities was used to combine both social-qualitative and ecological-quantitative data. Five medicinal species suitable for high impact harvesting were identified. Thirty-four species have potential to be set up as harvesting trials to determine sustainable quotas. Thirty-one medicinal species including Boophane disticha, Dicoma anomala, Eucomis autumnalis, Hypoxis hemerocallidea, Pentanisia prunelloides and Indigofera daleoides are considered to be site-specific sensitive species and should not be harvested from the reserve. Following this research an on site nursery has been started at the reserve. A multi-pronged approach involving harvesting trials, propagation and plant rescue from developments to cope with escalating demand and dwindling natural populations needs to be implemented.