Biotechnological approaches for cultivation and enhancement of secondary metabolites in Arnica montana L.

Arnica montana L. (Asteraceae) is a valuable medicinal plant with anti-inflammatory and cicatrizing properties attributed to the sesquiterpene lactones, flavonoids and essential oil produced in the flower heads. In many European countries, the populations of A. montana are close to extinction in their natural habitats because of uncontrolled eradication and indiscriminate collection of the plants. Various approaches for in vitro propagation of the species and also, biosynthesis of secondary metabolites in tissue and cell cultures are assessed in the current review. Special attention is paid to the biological activity and chemical composition of compounds produced by the species as well as the opportunities of in vitro methods to isolate high-productive clones. The influence of different factors on the micropropagation, callusogenesis, genetic transformation and identification of certain biologically active substances is discussed in detail. By the reference to the available issues we concluded that biotechnology applied to A. montana cultivation may improved the plant preservation and increased the production of sesquiterpene lactones and other secondary metabolites.     

<Emphasis Type="Italic">Rhodiola rosea</Emphasis> L.: from golden root to green cell factories

Rhodiola rosea L. is a worldwide popular plant with adaptogenic activities that have been and currently are exploited in the traditional medicine of many countries, as well as, examined in a number of clinical trials. More than 140 chemical structures have been identified which belong to several natural product classes, including phenylpropanoid glycosides, phenylethanoids, flavonoids and essential oils, and are mainly stored in the rhizomes and the roots of the plant. A number of mechanisms contribute to the adaptogenic activities of R. rosea preparations and its phytochemical constituents. Among them, the intrinsic inducible mammalian stress responses and their effector proteins, such as heat shock protein 70 (Hsp70), are the most prominent. Due to its popular medicinal use, which has led to depletion of its natural habitats, R. rosea is now considered as endangered in most parts of the world. Conservation, cultivation and micropropagation are all implemented as potential preservation strategies. A number of in vitro systems of R. rosea are being developed as sources of pharmaceutically valuable secondary metabolites. These are greatly facilitated by advances in elucidation of the biosynthetic pathways and the enzymes, which catalyse the production of these secondary metabolites in the plant. In addition, biotechnological approaches show promise towards achieving sustainable production of R. rosea secondary metabolites.     

RETRACTED ARTICLE: The AtCCX1 transporter mediates salinity tolerance in both <Emphasis Type="Italic">Arabidopsis</Emphasis> and yeast

The only species in the genus Passiflora (Passifloraceae) known to produce resin glands is P. foetida. These glands are secretory trichomes mainly present on the floral bracts and leaf stipules. The secretion produced by these glands has received attention recently due to the presence of substances with pharmacological properties. Attempts to apply in vitro cell culture methods for the large scale production of highly valuable metabolites has been rather limited due to the fact that these compounds are produced by highly differentiated secretory cells in trichomes which are seldom obtained or because differentiation is inhibited by in vitro conditions. Here we describe the in vitro plant regeneration of P. foetida obtained via organogenesis, using mature zygotic embryos as explants. Differentiated plantlets and, more important, the de novo differentiation of secretory trichomes in vitro could be observed in less than 30 days. There was a clear effect of the concentration of 2,4-dichlorophenoxyacetic acid in the culture media on the regeneration of plants and on the differentiation of glandular trichomes. Our results should be useful for the micropropagation of P. foetida, as well as for studies of the process of secretory trichome differentiation and the implemention of biotechnological methodologies for in vitro mass production of passifloricin and/or other substances present in the P. foetida resin.     

Evaluation of genetic stability using FRAPD markers as novel method along with antioxidant and anti-diabetic properties of micropropagated <Emphasis Type="Italic">Salacia chinensis</Emphasis> L.

Salacia chinensis L., a perennial medicinal plant, is well-known for its well-documented anti-diabetic properties. The daily growing demand in pharmaceutical industry is stimulating the conservation and wide-ranging production of the plant using plant tissue culture techniques (micropropagation). In the present study, the plants generated by direct micropropagation from nodal explants were assessed using fluorescently labeled RAPD (FRAPD) primers. Although standard RAPD primer bands in agarose gel showed genetic stability, using FRAPD analysis in genetic DNA sequencer as a novel strategy showed more accurate and reliable method has indicated by the evidence in 5% genetic variation. Antioxidant and anti-diabetic activities of micropropagated plants versus mother plant were examined using DPPH, FRAP, α-amylase, and α-glucosidase assays. The results showed that the micropropagated plants, which are able to produce higher amount of secondary metabolites than the mother plant, possess higher in vitro antioxidant and anti-diabetic properties.     

Micropropagation of apple — A review

Micropropagation of apple has played an important role in the production of healthy, disease-free plants and in the rapid multiplication of scions and rootstocks with desirable traits. During the last few decades, in apple, many reliable methods have been developed for both rootstocks and scions from a practical, commercial point of view. Successful micropropagation of apple using pre-existing meristems (culture of apical buds or nodal segments) is influenced by several internal and external factors including ex vitro (e.g. genotype and physiological state) and in vitro conditions (e.g., media constituents and light). Specific requirements during stages of micropropagation, such as the establishment of in vitro cultures, shoot multiplication, rooting of microshoots and acclimatization are summarized in this review. New approaches for increasing shoot multiplication and rooting for apple and current use of micropropagated plantlets as tools in basic and applied research are also discussed.     

Picrorhiza kurrooa: current status and tissue culture mediated biotechnological interventions

Picrorhiza kurrooa, one of the important plant species among the various medicinal plants, is endemic to Himalaya. As the plant is useful in the treatment of various diseases, e.g., hepatic disorders, gastric troubles, anemia, asthma, etc., illegal collection from the wild is increasing and now this plant is banned for export in any form and listed as ‘endangered’. Ecological studies carried out on this species in last few decades suggested that the availability of this species in its specific habitats is comparatively lower than other associate species. Possible factors responsible for this depletion are increasing demand in the pharmaceutical industries, habitat specificity, heavy exploitation from the wild, unorganized cultivation practices etc. Biotechnology is playing a crucial role to conserve this important plant species. The past 23 years have witnessed a progressive biotechnological advances made in P. kurrooa. People have published various reports on establishments of in vitro culture techniques including micropropagation, synthetic seed production, plant regeneration via callus-mediated shoot organogenesis, adventitious shoot regeneration, genetic transformation through Agrobacterium rhizogenes, secondary metabolite analysis etc. This review attempts to focus on present ecological status and provide a comprehensive account on the tissue culture-mediated biotechnological interventions made in P. kurrooa for improvement and conservation of this medicinally important plant.     

Tissue culture-mediated biotechnological intervention in pomegranate: a review

The past 30 years have witnessed a series of systematic biotechnological advances made in pomegranate. These encompass optimization and establishment of in vitro culture techniques including micropropagation, somatic embryogenesis, synthetic seed production, plant regeneration via callus-mediated shoot organogenesis, adventitious shoot regeneration, anther culture, tetraploid induction and genetic transformation. This review attempts to provide a comprehensive account on the tissue culture-mediated biotechnological interventions made in pomegranate aimed at complementing conventional programmes for improvement of this nutraceutically important fruit crop.     

Shoot proliferation and rooting treatments influence secondary metabolite production and antioxidant activity in tissue culture-derived Aloe arborescens grown ex vitro

Aloe species are valuable plants with great ornamental and medicinal value. Although micropropagation protocols have been developed to meet the increasing global demand, the effects of the series of events during micropropagation on the phytochemical and pharmacological efficacy of ex-vitro plants remains poorly understood. Thus, we evaluated the effects of cytokinin and rooting compounds used during the shoot regeneration and rooting phases respectively, on secondary metabolite production in greenhouse-grown in vitro-derived Aloe arborescens. Shoots derived from meta-methoxytopolin (MemT)-containing medium and rooted with either smoke–water (SW) or indole butyric acid (IBA) had higher levels of total phenolics and flavonoids than those rooted on plant growth regulator (PGR)-free medium. Iridoid content was significantly reduced in cytokinin-regenerated shoots rooted with IBA in comparison to PGR-free regenerated shoots rooted with IBA. Conversely, the use of SW for rooting in cytokinin-regenerated shoots significantly increased iridoid content when compared to PGR-free regenerated shoots rooted with SW. These findings suggest an antagonistic interaction between cytokinins used in this study and IBA as well as a possible synergistic or additive interaction of the cytokinins with SW on iridoid production. Significantly higher antioxidant activity was recorded in shoots regenerated from meta-topolin riboside (mTR) and MemT and rooted with IBA or SW when compared to those rooted without PGR. Overall, the type of cytokinin and rooting treatments individually and interactively had a significant carry-over effect on secondary metabolite production and antioxidant potential of tissue culture-derived A. arborescens. Therefore, when micropropagating plants for medicinal uses, it is prudent to select the right cytokinin and rooting compound for optimal production of secondary metabolites and ultimately the pharmacological efficacy of acclimatized plants.     

In vitro propagation,clonal fidelity and phytochemical analysis of <Emphasis Type="Italic">Rhodiola imbricata</Emphasis> Edgew: a rare trans-Himalayan medicinal plant

The present study focuses on development of a micropropagation protocol for true to type plants of Rhodiola imbricata, an endangered medicinal plant found in trans-Himalayan Leh-Ladakh region of India. It also aims at analyzing the pharmaceutically important secondary metabolites and antioxidant potential of in vitro and in vivo plants. Various cytokinins and auxins were tested for shoot proliferation and in vitro rooting of the microshoots, respectively. Random primers were used for checking genetic uniformity at different stages of micropropagation. Pharmaceutically important secondary metabolites of R. imbricata such as Rosavin, total polyphenols and free radical scavenging activity were analyzed by HPLC. Among different cytokinins used, BAP (5 µM) and TDZ (1 µM) were found to perform better in terms of shoot proliferation, shoot length and number of leaves as compared to other concentrations. For rooting of microshoots, a lower concentration of NAA (0.5 µM) yielded more efficient rooting of micro shoots (17.33 roots per micro shoot). In vitro rooted microshoots were hardened and showed 60% survival rate. The content of gallic acid, chlorogenic acid and 4-hydroxybenzoic acid was higher in the in vivo plant. The amount of ferulic acid was higher in the in vitro raised plant when compared to field grown plant. Furthermore, caffeic acid and p-coumaric acid were higher in the in vitro raised plants as compared to field grown plants. This work will facilitate in conservation of this endangered herb and provide necessary plant materials for various biotechnological and pharmaceutical applications.     

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.     

Gerbera micropropagation

Gerbera jamesonii (gerbera) is an important cut-flower in the global floricultural industry. Micropropagation is the main system used to clonally propagate gerbera in vitro resulting in the production of millions of plantlets each year. Numerous types of explants and protocols for micropropagation have been established and used for gerbera. Shoot tips are the commonly used explant while adventitious shoot induction from the capitulum is also a popular method. Most papers in the literature have focused on testing the influence of different types and combinations of plant growth regulators with the aim of improving the regeneration and multiplication stage of one or few cultivars. Genotype is one of the most influential factors on the response of gerbera in vitro. Despite this, no successful universal protocol has yet been developed for multiple cultivars, limiting the usefulness of current protocols for commercial biotechnology labs. Slow-growing endogenous bacteria are one of the most important problems in gerbera micropropagation but require more studies on control and prevention. Individual shoots are normally easy to root, usually in excess of 90% of plantlets, but the acclimatization stage requires improvements and new technologies to increase the survival of plants. Epigenetic variations in micropropagated gerbera are frequently observed only with high concentrations of cytokinins in the culture medium but somaclonal variation is rare.     

Direct plant regeneration from different explants through micropropagation and determination of secondary metabolites in the critically endangered endemic Rhaponticoides mykalea

Direct plant regeneration from different explants, micropropagation and determination of secondary metabolites were studied in the critically endangered endemic Rhaponticoides mykalea (Hub.-Mor.) M.V. Agab & Greuter. Seed germination was achieved by damaging the seed coat and cultivating the embryos on Woody Plant Medium (WPM), of which 40% germinated. The epicotyls and cotyledonary petioles of seedlings were used as initial explants and direct shoot regeneration was obtained on WPM containing 2.22 μM 6-benzyladenine (BA). WPM medium supplemented with 2.22 μM BA and 4.92 μM indole-3-butyric acid (IBA) significantly improved the production of multiple shoots, resulting in an average of 5.6 shoots per explants. The highest rooting of shoots (35.6%) was observed with WPM medium containing 19.68 μM IBA with 990 μM putrescine. Plantlets with well-developed roots were transferred to soil and acclimatised within a plant growth chamber. Acclimatised plants showed 100% survival rate and remained healthy. As a part of our study, the content of secondary metabolites in three tissue culture regenerated lines were determined by HPLC analysis. Chlorogenic acid, Quercetin and scutellarin were confirmed secondary metabolites of R. mykalea.     

Application of cell technologies for production of plant-derived bioactive substances of plant origin

Bioactive substances (BAS) of plant origin are known to play a very important role in modern medicine. Their use, however, is often limited by availability of plant resources and may jeopardize rare species of medicinal plants. Plant cell cultures can serve as a renewable source of valuable secondary metabolites. To the date, however, only few examples of their commercial use are known. The main reasons for such a situation are the insufficient production of secondary metabolites and high cultivation costs. It is possible to increase the performance of plant cell cultures by one or two orders of magnitude using traditional methods, such as selection of highly productive strains, optimization of the medium composition, elicitation, and addition of precursors of secondary metabolite biosynthesis. The progress in molecular biology methods brought about the advent of new means for increasing of the productivity of cell cultures based on the methods of metabolic engineering. Thus, overexpression of genes encoding the enzymes involved in the synthesis of the target product or, by contrast, repression of these genes significantly influences the cell biosynthetic capacity in vitro. Nevertheless, the attempts of the production of many secondary metabolites in plant cell culture were unsuccessful so far, probably due to the peculiarities of the cell culture as an artificial population of plant somatic cells. The use of plant organ culture or transformed roots (hairy root) could turn to be a considerably more efficient solution for this problem. The production of plant-derived secondary metabolites in yeast or bacteria transformed with plant genes is being studied currently. Although the attempts to use metabolic engineering methods were not particularly successful so far, new insights in biochemistry and physiology of secondary metabolism, particularly in regulation and compartmentation of secondary metabolite synthesis as well as mechanisms of their transport and storage make these approaches promising.     

Influence of abiotic stress signals on secondary metabolites in plants

Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors, and industrially important biochemicals. Accumulation of such metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. Secondary metabolites play a major role in the adaptation of plants to the environment and in overcoming stress conditions. Environmental factors viz. temperature, humidity, light intensity, the supply of water, minerals, and CO₂ influence the growth of a plant and secondary metabolite production. Drought, high salinity, and freezing temperatures are environmental conditions that cause adverse effects on the growth of plants and the productivity of crops. Plant cell culture technologies have been effective tools for both studying and producing plant secondary metabolites under in vitro conditions and for plant improvement. This brief review summarizes the influence of different abiotic factors include salt, drought, light, heavy metals, frost etc. on secondary metabolites in plants. The focus of the present review is the influence of abiotic factors on secondary metabolite production and some of important plant pharmaceuticals. Also, we describe the results of in vitro cultures and production of some important secondary metabolites obtained in our laboratory.     

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.     

Sage in vitro cultures: a promising tool for the production of bioactive terpenes and phenolic substances

Extracts of Salvia species are used in traditional medicine to treat various diseases. The economic importance of this genus has increased in recent years due to evidence that some of its secondary metabolites have valuable pharmaceutical and nutraceutical properties.The bioactivity of sage extracts is mainly due to their content of terpenes and polyphenols. The increasing demand for sage products combined with environmental, ecological and climatic limitations on the production of sage metabolites from field-grown plants have led to extensive investigations into biotechnological approaches for the production of Salvia phytochemicals. The purpose of this review is to evaluate recent progress in investigations of sage in vitro systems as tools for producing important terpenoids and polyphenols and in development of methods for manipulating regulatory processes to enhance secondary metabolite production in such systems.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

In vitro culture: an epigenetic challenge for plants

In vitro plant cell and tissue culture techniques are the basis of many micropropagation and breeding programs for scientific research. Plant tissue culture (PTC) involves organogenesis and embryogenesis, and the outcome depends on the different conditions to which the tissue is exposed. PTC is a stressful environment—high relative humidity, low ventilation rate, high concentrations of plant growth regulators, and low light availability—for plants that need to rapidly change their molecular regulation in order to respond fast and efficiently during cell division and growth. For instance, somatic embryogenesis (SE), which plays an important role in plant multiplication, requires complex cellular, biochemical and molecular processes for embryo formation and development. New data has come out about a connection between plant morphogenesis and epigenetics. Epigenetics is a very sensitive regulatory mechanism, which in most of cases is affected by the environment. Although it is known that, under plant morphogenesis, the genome has little or no change, DNA methylation and histone modifications are very susceptible to those in vitro environmental conditions. In the present review, we highlight the most used in vitro systems such as organogenesis and SE in plants and discuss how epigenetics plays a pivotal role in the phenotype outcome. Furthermore, we discuss the big role that the small RNAs have during cell division and propagation and propose different challenges and opportunities to study epigenetics in plant cell tissue and organ cultures.     

Biotechnological advances in jojoba [Simmondsia chinensis (Link) Schneider]: recent developments and prospects for further research

Jojoba (Simmondsia chinensis), is a medicinal and oil-yielding, multi-purpose species of the family Simmondsiaceae. The most valuable product of jojoba seed is the liquid wax or jojoba oil which is used extensively in the cosmetic and bio-fuel industry. Propagation of jojoba is possible using conventional methods, but it is time consuming and cumbersome owing to long rotation periods, male-biased population, and long flowering and seed set time. The development of an efficient regeneration system is a prerequisite for a number of biotechnological interventions for the improvement of jojoba, such as genetic transformation, production of useful metabolites in vitro, etc. During the past decade, therefore, several attempts have been made for in vitro propagation of jojoba. Organogenesis has been achieved in this species from mature as well as juvenile explants. Present communication reports an overview of the in vitro regeneration of jojoba via organogenesis and somatic embryogenesis. Factors affecting organogenesis as well as production of synthetic seeds using shoot tips and axillary buds have also been discussed; however, efforts need to be made to develop an efficient genetic transformation system in jojoba. The purpose of this review is to focus upon the current information on in vitro propagation and biotechnological advances made in jojoba.