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.

     

<Emphasis Type="Italic">In Vitro</Emphasis> Preservation of Spanish Biodiversity

Spanish territories contain many of the hot spots of plant biodiversity among European countries. Most of the Spanish territory is found in the Mediterranean basin and in the Canary Islands, a region of great floral singularity and diversity (Macaronesian bioregion). Therefore, an important effort must be made to contribute to its conservation. Several strategies can be considered, but seed conservation under standard conditions is the most resource-efficient method. However, the application of this methodology is not always possible for recalcitrant seeds or species for which vegetative propagation is necessary or convenient under some circumstances. Micropropagation is one of the measures suggested for preserving endangered species. During the 1990s, several in vitro culture protocols for Spanish endemics were established. The main purpose of this strategy was to obtain a considerable number of individuals to reduce the loss of natural populations. Likewise, diverse slow growth protocols were developed for this material. However, these efforts usually did not lead to the establishment of in vitro collections. The advantages and disadvantages of the in vitro conservation strategy will be reviewed for some cases. The establishment of the in vitro protocols together with the development of cryopreservation techniques created the ideal conditions to generate cryogenic collections. In this paper, we review the knowledge and experience accumulated during the last decades in micropropagation, slow growth, and cryopreservation for Spanish plant wild species. Their application in the development ex situ collections and their contribution toward an integrated system to conserve threatened species will be discussed.     

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.     

In vitro culture of the endangered plant Eryngium viviparum as dual strategy for its ex situ conservation and source of bioactive compounds

Different Eryngium species have been used with ornamental, agricultural and medicinal purposes, as a consequence of their chemical constituents. In the southwest Europe the endemic Eryngium viviparum, presents a high risk of extinction and ex situ strategies are high recommended for efficient conservation and re-introduction program. The objective of this study was to satisfy a dual objective: (i) to develop an ex situ conservation strategy through micropropagation and (ii) taking advantage of the extraordinary potential of plant tissue culture, produce a considerable amount of plant material to carry out a preliminary phytochemical study, based on the accumulation of phenolic compounds and their associated antioxidant activity. First a factorial design was conducted in order to study the effect of two cytokinins (6- benzylaminopurine, BAP, and kinetin, KIN), at three levels (0, 1 and 2 mg L^?1), on shoot multiplication. Later another factorial design was applied, by using three levels of MS medium salt strength (full, half and quarter- strength) and four sucrose levels (0, 1, 2, and 3%) for improving shoot elongation and rooting. In parallel, a preliminary quantification of total phenolic and flavonoid contents from E. viviparum aerial parts was determined. The simple micropropagation protocol designed allowed obtaining a high rates of shoot multiplication (5.1–5.8 new shoots), rooting (100%) with healthy long roots (3.1–3.5 cm) and plantlet acclimatization (96%). Moderate antioxidant activity was recorded in hydromethanolic extracts from E. viviparum aerial parts. High correlation between total phenolic content and BAP levels in the culture media was found. In conclusion, the micropropagation procedure described here for the endangered E. viviparum can be used as new and very efficient ex situ conservation strategy, and as potential source of antioxidants, conferring an added-value to this plant.

     

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 ornamental <Emphasis Type="Italic">Prunus</Emphasis> spp. and GF305 peach,a <Emphasis Type="Italic">Prunus</Emphasis> viral indicator

A micropropagation approach was developed for nine ornamental Prunus species, P. americana, P. cistena, P. glandulosa, P. serrulata ‘Kwanzan’, P. laurocerasus, P. sargentii, P. tomentosa, P. triloba, P. virginiana ‘Schubert’, commercially important in North America, and GF305 peach, commonly used for Prunus virus indexing. The micropropagation cycle based on proliferation of vegetative tissues includes establishment of tissue culture through introduction of shoot meristems in vitro, shoot proliferation, root induction and plant acclimatization steps and can be completed in 5 months. A meristem sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation in ornamental Prunus was obtained through the use of 1 mg l⁻¹ 6-benzyladenine. For GF305 peach, alteration in the sugar composition, fructose instead of sucrose, and addition of 1 mg l⁻¹ ferulic acid had a significant impact on the shoot proliferation rate and maintenance of long-term in vitro culture. Rooting and plant acclimatization conditions were improved using a two-step protocol with a 4-day root induction in indole-3-butiric acid (IBA)-containing media with consequent 3-week root elongation in IBA-free media. One-month incubation of rooted shoots in a vermiculite-based medium resulted in additional shoot and root growth and provided better acclimatization and plant recovery. The micropropagation approach can be used for maintenance of the clonal properties for Prunus spp. as well as a protocol to support meristem therapy against viral infection.     

Micropropagation and in vitro conservation of wild <Emphasis Type="Italic">Arachis</Emphasis> species

Wild species of Arachis constitute potential sources of novel genes for groundnut improvement programs and some of them are also considered as new agricultural usage. The majority of these species occur in regions under intensive human activity and their areas of distribution are being drastically reduced, thus requiring effective conservation measures. Conservation of Arachis germplasm is usually carried out in seed banks or as live plants. However, seed renewal can be impaired by loss of germinative potential, and plant multiplication under field conditions can be limited by specific soil and environmental requirements or by low seed yield. Therefore, complementary in vitro methodologies represent an important tool for ex situ conservation of Arachis germplasm. In this work, we analyse the state of art of micropropagation and the use of in vitro conservation methodologies for wild relatives of the groundnut.     

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions. Received: 12 January 1998 / Revision received: 10 October 1998 / Accepted: 28 October 1998     

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.     

In vitro propagation of four saponin producing <Emphasis Type="Italic">Maesa</Emphasis> species

A successful micropropagation system was developed for four different medicinal Maesa species. Multiple shoots were induced through both axillary bud formation and adventitious shoot regeneration from leaf explants. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), thidiazuron (TDZ) and/or α-naphthalene acetic acid (NAA). The success of regeneration varied for different species and depended on the type and concentration of plant growth regulators. Regenerated shoots spontaneously developed roots within 6 weeks on MS hormone-free medium. The rooted shoots were transferred to the greenhouse with a 100% success rate. Furthermore, flow cytometry analysis indicated that there were no changes in ploidy level of those regenerated shoots as compared with wild type adult plants. Thin layer chromatography (TLC) analysis revealed that common and distinguishing spot of saponins were similarly observed in regenerated shoots compared to the control plants. Therefore, the protocol also provides an effective means for the in vitro conservation of Maesa spp. that produce pharmaceutically interesting saponins.     

Biodiversity conservation and conservation biotechnology tools

This special issue is dedicated to the in vitro tools and methods used to conserve the genetic diversity of rare and threatened plant species from around the world. Species that are on the brink of extinction because of the rapid loss of genetic diversity and habitat come mainly from resource-poor areas of the world and from global biodiversity hotspots and island countries. These species are unique because they are endemic, and only a few small populations or sometimes only a few individuals remain in the wild. Therefore, the challenges to support conservation by in vitro measures are many and varied. The editors of this invited issue solicited papers from experts from Asia, Africa, Europe, Australia, and North and South America. This compilation of articles describes the efforts in these diverse regions toward saving plants from extinction, and details the direct application of in vitro and cryopreservation methods. In addition, these contributions provide guidance on propagation of rare plants, including techniques for large-scale propagation, storage, and reintroduction. The in vitro techniques for conserving plant biodiversity include shoot apical or axillary-meristem-based micropropagation, somatic embryogenesis, cell culture technologies and embryo rescue techniques, as well as a range of in vitro cold storage and cryopreservation protocols, and they are discussed in depth in this issue.     

Effect of different media composition on the micropropagation of Erica andevalensis, a metal accumulator species growing in mining areas (SW Spain)

We describe a micropropagation and acclimatization protocol of Erica andevalensis Cabezudo & Rivera, using stem pieces collected from wild plants. This species always grows in soils enriched with metals such as Fe, Pb, Zn and in acid pH (2.5–4). Among the different media and hormone concentration tested, MS medium with ammonium nitrate concentration reduced to a quarter, 2 mg l⁻¹ indole-acetic acid (IAA) and 0.5 mg l⁻¹ kinetin was found to yield the best growth response in the plants. This medium produced an average of 3.6 ± 1.2 shoots and 16.9 ± 8.3 verticils per explant after 30 days of the onset of the culture. MS media achieved rooting without phytohormones. Rooted plantlets were successfully transferred to plastic containers with autoclaved perlite, watered with MS half-strength, and then to pots with commercial substrate after 2 weeks. New plants were produced in about 100 days. As far as we know, this is the first time that a replicable and complete micropropagation protocol for E. andevalensis has been developed. The plants looked healthy with no visual detectable phenotypic variations. The protocol provides a successful technique that could be used for the conservation of the species, as well as new researches on phenolics and triterpenoids compounds found in plant extracts.     

Biotechnology for saving rare and threatened flora in a biodiversity hotspot

The Southwest Australian Floristic Region (SWAFR) is a plant biodiversity hotspot with a geographically isolated and predominantly endemic flora. Known threatening processes (i.e. excessive clearing of native vegetation, soil salinity, soil erosion and chronic weed infestation) combined with uncertain but potentially deleterious environmental (climate) changes pose great challenges for conservation and restoration efforts. With a paucity of nature reserves, in situ protection of species can be problematic. For many species, ex situ conservation becomes the only viable strategy for saving species from extinction via seed banking or living collections established through vegetative propagation, or tissue (in vitro) culture methods. Development of specific in vitro protocols is necessary to successfully initiate culture lines, with considerable development of nutrient media, plant growth regulator regimes and incubation conditions required to optimise shoot regeneration and multiplication, especially with woody species of the SWAFR. In addition, integration of root induction and acclimatization stages has allowed significant improvements in speed and success of plant production of both endangered and difficult-to-propagate woody species. We contend that there is also considerable potential for expansion of alternative in vitro technologies such as somatic embryogenesis for difficult taxa to complement existing ex situ conservation and restoration strategies in biodiversity hotspots such as SWAFR.     

Development and application of photoautotrophic micropropagation plant system

Research has revealed that most chlorophyllous explants/plants in vitro have the ability to grow photoautotrophically (without sugar in the culture medium), and that the low or negative net photosynthetic rate of plants in vitro is not due to poor photosynthetic ability, but to the low CO₂ concentration in the air-tight culture vessel during the photoperiod. Moreover, numerous studies have been conducted on improving the in vitro environment and investigating its effects on growth and development of cultures/plantlets on nearly 50 species since the concept of photoautotrophic micropropagation was developed more than two decades ago. These studies indicate that the photoautotrophic growth in vitro of many plant species can be significantly promoted by increasing the CO₂ concentration and light intensity in the vessel, by decreasing the relative humidity in the vessel, and by using a fibrous or porous supporting material with high air porosity instead of gelling agents such as agar. This paper reviews the development and characteristics of photoautotrophic micropropagation systems and the effects of environmental conditions on the growth and development of the plantlets. The commercial applications and the perspective of photoautotrophic micropropagation systems are discussed.     

Morphogenesis and in vitro production of caffeoylquinic and caffeic acids in Baccharis conferta Kunth

We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and formed roots on MS medium without PGRs. The micropropagated plantlets and the organogenic callus showed similar chemical profiles in HPLC-mass spectrometry analyses. The main compounds present in the cultures were caffeoylquinic acids. Only plantlets contained small amounts of triterpenes (erythrodiol and ursolic acid). These findings will be useful for the micropropagation of this important native resource, and for further studies on its biology.

     

Evaluation of phytomedicinal yield potential and molecular profiling of micropropagated and conventionally grown turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.)

Drug yielding potential of turmeric (Curcuma longa L.) is due to the presence of important phytoconstituents such as curcumin, oleoresin and essential oil. Slow multiplication rate, high susceptibility to rhizome rot and leaf spot disease and restricted availability of elite genotype necessitated application of tissue culture technique to alleviate the problems. A protocol has been developed for in vitro micropropagation of an elite genotype (cv. suroma) using latent axillary bud explants from unsprouted rhizome, available throughout the year. MS media containing 3 mg/l 6-Benzyladenine (BA) and 1 mg/l Indole Acetic acid (IAA) was found optimum for regeneration, multiplication and in vitro conservation of plantlets. After 3 years of in vitro conservation regenerants were transplanted to field and assessed for drug yielding potential through evaluation of curcumin, oleoresin and essential oil contents of rhizomes and leaves. One year of field grown tissue culture derived turmeric were found uniform in all the characteristics examined, when compared with those grown conventionally. Micropropagated turmeric showing stable drug yielding potential also proved to have genetic basis of stability as revealed by RAPD based molecular profiling.     

Micropropagation of Populus trichocarpa ‘Nisqually-1’: the genotype deriving the Populus reference genome

Populus serves as a model tree for biotechnology and molecular biology research due to the availability of the reference genome sequence of Populus trichocarpa (Torr. & Gray) genotype ‘Nisqually-1’. However, ‘Nisqually-1’ has been shown to be very recalcitrant to micropropagation, regeneration and transformation. In this study, a highly efficient micropropagation protocol from greenhouse-grown shoot tips of ‘Nisqually-1’ was established. The optimal micropropagation protocol involves growing in vitro shoots in plant growth regulator-free Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 0.3% Gelrite^? and 5–10 g L⁻¹ of activated charcoal. Plants grown on this medium were significantly longer, and contained significantly higher concentrations of chlorophyll. This highly effective protocol provides a consistent supply of quality leaf and stem materials throughout the year for transformation experiments and other in vitro manipulations, therefore eliminating inconsistency due to seasonal and greenhouse environmental variations and the need for repetitive tissue sterilization.     

Conservation Strategy for African Medicinal Species: <i>In Vitro</i> Biotechnological Approach

The use of medicinal plants for different therapeutic values is well documented in African continent. African diverse biodiversity hotspots provide a wide range of endemic species, which ensures a potential medicinal value. The feasible conservation approach and sustainable harvesting for the medicinal species remains a huge challenge. However, conservation approach through different biotechnological tools such as micropropagation, somatic embryogenesis, synthetic seed production, hairy root culture, molecular markers based study and cryopreservation of endemic African medicinal species is much crucial. In this review, an attempt has been made to provide different in vitro biotechnological approaches for the conservation of African medicinal species. The present review will be helpful in further technology development and deciding the priorities at decision-making levels for in vitro conservation and sustainable use of African medicinal species.     

A simple,economical, and high efficient protocol to produce in vitro miniature rose

Due to its high commercial value, many studies on rose (Rosa hybrida L.) micropropagation have been published. However, there are a limited number of studies on rose in vitro flowering. These studies only focused on the roles of plant growth regulators in the formation and morphogenesis of flowers. In this protocol, cytokinin was confirmed to positively function in the induction of in vitro rose flowers. In fact, more than 40% of in vitro shoots were induced to flower when they were grown on a medium supplemented with benzylaminopurine (BA) (2 mg L⁻¹) and IAA (0.1 mg L⁻¹). In addition, this study showed that the growth medium supplemented with only coconut water (15 or 20% v/v) was very efficient to induce flowering of in vitro miniature rose plants (> 70%) after 60 d of subculture. In addition, the in vitro flowers were normal and almost similar to ex vitro flowers in terms of flower shape and color. Based on these results, a detailed procedure for in vitro miniature rose flower production is provided.

     

Enhanced growth and quality of St. John's wort (Hypericum perforatum l.) under photoautotrophic in vitro conditions

Summary Photomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many rescarchers for transplant production of St. John's wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21d of culture, Pa conditions enhanced the growth and quality of St. John's wort plantlets in vitro, and these plantlets showed faster growth after transplantaing ex vitro compared with those cultured under Pm conditions.