The Gentianaceae:

characterization, conservation, propagation, genetic manipulation and application

 

more general  title

The Gentianaceae family at present

 

editors: Jan J. Rybczyński, Michael R. Davey*, Anna Mikuła (Poland/*England)

 

I. General introduction to the family

I. 1. Characteristic of family Gentianaceae        

Pringle J. (Canada)

 

The Gentianaceae include monocarpic and perennial herbs, shrubs, trees, and rarely lianas, terrestrial or rarely epiphytic. Most are autotrophic; a few are achlorophyllous and mycoheterotrophic. The stems are erect, decumbent, or rarely twining. The leaves are opposite, whorled, or rarely alternate, simple, usually entire, sessile or petiolate; stipules are usually absent, present as ocreae in one subtribe. The flowers are solitary or in cymes, thyrses, or verticillasters, The flowers are usually bisexual and monomorphic, rarely heterostylous or some unisexual, actinomorphic or somewhat zygomorphic, 4-, 5-, or in a few genera 6-16-merous, rarely 3-merous. The calyx is non-petaloid, usually persistent, of united or rarely separate sepals. The corolla is petaloid, often marcescent, of united petals. The stamens are in one cycle, epipetalous, isomerous and alternate with petals, rarely some with the anthers sterile or absent; filaments are generally present, separate or occasionally fused or connected by a corona, straight or declinate. The anthers are dithecal, dehiscent longitudinally or rarely with terminal pores. The pistil is solitary, of two united carpels; the ovary is superior, usually unilocular with parietal placentae, sometimes bilocular with axile placentation; the style is solitary, undivided or bifid, straight, declinate, or deflexed to one side, persistent or deciduous, or none; the stigmas are one or two, diverse in form. The fruit is usually a septicidal capsule containing few to many seeds, rarely irregularly dehiscent or indehiscent. Nectaries are often well developed, diverse in form and position.

 

I. 2.  Classification and evolution of the Gentianaceae family

Struwe L. (USA)

 

This chapter will describe the changes in classification in the family since the worldwide treatment of Struwe & Albert in 2002, ten years ago, based on new knowledge and research results.  Largely the classification still stands, but there has been some important changes based on new evolutionary work. This includes reclassifications of poly- or paraphyletic genera (e.g., Canscora, Centaurium, Sebaea) that have led to the description of new or resurrected genera such as Duplipetala, Exochaenium, Gyrandra, Lagenias, Klackenbergia, Phyllocyclus, Schenkia, and Zeltnera. New genera have been discovered in South America, e.g., Aripuana, Roraimaea, and Yanomamua. In some cases, some genera had to be incorporation into others to preserve monophyly (Wurdackanthus).  In at least one case, a genus that was tentatively placed in 2002, has now been moved to a different tribe (e.g., Bisgoeppertia).  Unsolved generic delimitation problems remain in Gentianeae-Swertiinae (mostly regarding Swertia and Gentianella) and Helieae (the Symbolanthus-clade, especially Calolisianthus, Chelonanthus and Irlbachia), and will be highlighted and discussed. The placement of the enigmatic mycotroph Voyria, classified as incertae sedis in 2002, is still uncertain. Biogeographic studies during the last decade addressing large-scale distribution patterns and vicariance events of gentians and the significance of these new results will also be reviewed.

 

I. 3.  Biochemical and morphological determination of the flowers colour in Gentianaceae

Młodzińska E. (Poland)

 

Since Charles Darwin wrote his excellent book about flower-colored polymorphism within the same species (Darwin, 1877) studies about plant dyes have become one of the most popular research subjects of general botany. After nearly a two hundred years of investigation into the biochemistry and physiological factors, involved in flower color determination, our knowledge of the pathway leading to the accumulation of pigments in petals is deeply understood. Flavonoids are the major pigments that confer flower color and their biosynthesis is well characterized at the genetic and enzymatic level in many of decorative plants, including Eustoma, Carnation, Petunia and Snapdragon. This chapter summarizes the current status of all the key biosynthesis steps and secondary modification of the pigments and co-pigments which are responsible for marvelous diversity of color patterns observed in Gentian species. To help with understanding of the molecular basis of color patterning in flowers a brief overview is also given about the cellular and subcellular localization of plant pigments. More specifically it has been described anthocyanin vacuolar inclusion structures (AVIs) that have been shown to affect the final color by accumulating high concentrations of anthocyanins. Finally, it has been also described about the shape and morphology of petal epidermal cells that themselves may play important roles in determining of flower color in Gentianaceae.

 

 

I. 4. Karyology of European species of genus  Gentiana L.  

Kunakh V.A. Mel'nyk V.M , Drobyk N.M., Andreev I.O.,. Spiridonova K.V. , Twardovska M.O.,. Konvalyuk I.I^. , Adonin ¹ V.I. (Ukraine)  

 

The work summarizes the literature data on chromosome numbers of Gentiana European species and our own results concerning their cytogenetics from various localities in Ukraine. Based on the comparison of results of our own studies with those of literature ones there were confirmed previously obtained chromosome numbers in G. lutea, G. punctata, G acaulis, G. verna, G. cruciata and G. pneumonanthe species, as well as those of one G. asclepiadea race. Karyological details for some sections and individual genus members in context of their propagation were considered. The necessity for further karyological investigations to elucidate Gentiana species evolution and classify its individual taxons was discussed. To date we studied exclusively the gentian from Ukrainian flora (G. lutea, G. punctata, G. acaulis, G. asclepiadea, G. cruciata, G. pneumonanthe, G. verna).

 

I. 5. Distribution of Gentianaceae in Ukraine

Shiyan  N. M. (Ukraine)

 

The chapter will be devoted to species chorology of Gentianaceae family, which grow in natural habitats on the Ukraine territory. The analysis of 38 herbarium collections of the world (KW, LW, LWS, MSUD, YALT, LE, KRA, KRAM, CAI at al), literature data and field studies 1992-2010 were presented. The text will contain a general description of localities, citation sites by herbarium specimens for each species and maps of their distribution in Ukraine.

 

1. 6.  21^st century centauries - a review of Centaurium Hill and allies (Gentianaceae)

Mansion G. (Germany)

 

Centauries have a long tradition in human medicine and botanical accounts. Early indications of centauries as medicinal herbs refer to famous works of Theophraste (4^th Century BC), Pline and Dioscoridis (1^st Century), while first - somehow idealistic - representations date back to the 14^th Century. Since Linné's typification of Gentiana centaurium (1753) and Hill's first description of Centaurium (1756), centauries have been diversely interpreted by renowned botanists until the end of the 20^th Century. This chapter presents an overview of the current and accepted delimitations of centauries, as well as insights into their taxonomic and evolutionary history.

 

 

II. Gentianaceae in  wild habitat

 

II. 1. Genes expressed in the overwinter buds of Gentiana triflora: application to phylogenetic, pedigree and phenotypic markers

Tsutsumi  K. , Hikage T. (Japan)

 

Proteins enriched in the overwinter buds and their genes will be described. Then, we focus on the W14/15 gene, which encodes novel esterases belonging to the alpha/beta hydrolase superfamily. This gene is highly polymorphic, so that it provides useful strategy for phylogenetics and pedigree analysis. Its function will also be discussed in relation to some characters of the overwinter buds.

 

II. 2. Exacum bicolor  Roxb.  an exquisite, under exploited wild ornamental

            Sreelatha Unniampurath Baburaj, T.S ., C. Narayanan Kutty (India)

 

Exacum bicolor an elegant flowering plant in the wild is distributed in peninsular India. Natural habitats of Exacum bicolor are grasslands of both high and low altitudes as well as scrub savannah. The unique feature of the plant is its restricted occurrence in small pockets. The natural habitats are converted for various purposes like: tourism, construction of highways and sites for agriculture and industry. Ex situ conservation is the best strategy in the context of habitat destruction.

The plant is an excellent ornamental suitable for regions of high humidity and heavy rain fall. Horticultural aspects like sturdy stem, long flowering periods, more flowers in a bunch, large flower size and extended field life of flowers attribute good ornamental value as potted plants and for flower beds. Natural seed germination is very low i.e. below 5%. Analysis of soil in regions where plant occurs show presence of mycorrhize. Pollination is insect assisted and no fruit set is obtained in case of selfing.

Seedling progeny show wide variation for vegetative and floral characters and hence there exists high potential for selection and improvement. Protocol for in vitro culture from seed as well as leaf is standardized for mass multiplication.

There are reports in many books regarding the febrifuge, stomachic and tonic properties of Exacum bicolor. Traditional healers in many regions of India like: Kerala, Chhattisgarh and Karnataka used to prescribe the plant for treatment of fever, malaria and blood purification. Identification tests for various compounds like phenols, alkaloids, flavanoids, tannins and saponins have shown positive results. Thin layer chromatography conducted for qualitative analysis showed responses for the metabolites mentioned above.

 

 

III. 3. Indian Swertia from Eastern Himalaya: Strategies of conservation and biotechnological improvements

Rituparna Kundu Chaudhuri¹, Priyanka Mukherjee² , Timir baran Jha³* (India)

Eastern part of Indian Himalaya is highly acclaimed for its rich bio-resources and traditional repository of medicinal plants. This region is considered natural habitat of many species and populations of Swertia, an important and diverse genus of Gentianaceae. The genus Swertia, popularly known as chirata is one of the most important indigenous medicinal plants found in India. The plants are well known for their bitter taste and possess many known and unknown bioactive compounds. Habitat destruction, other human-caused stresses and impacts of climate change has caused enormous loss of genetic diversity and necessitate reassessment, documentation, molecular and biochemical screening and well formulated biotechnological strategies for sustainable use and improvements of these high value medicinal crops. Swertia chirata is considered the prized Indian species because of its some well characterized bioactive molecules known as iridoid and secoiridoid glycosides, xanthones and xanthone derivatives, effective against many conventional and unconventional ailments.  Due to increasing demand of chirata in national and international market, paucity of agricultural practices and proper exomorphological and molecular level screening has resulted  intentional and unintentional adulteration. The prized species Swertia chirata, has been tagged as critically endangered  and  most of the other important species whose IUCN status are not known  may face  similar threats  in near future. The present review work will primarily focus on the present status of Swertia species in Eastern Himalayas and discuss all possible biotechnological methods applied so far for  their conservation and future improvements.

 

 

III. Cell manipulations for plant genetic improvement

 

III.1. Interspecies sexual hybridization.

Morgan E. (New Zealand)

 

Interspecific hybridization is an important force in evolution of flowering plants. Natural hybrids have been reported where species have overlapping habitats. There is a range of hybrids that have been developed for horticultural uses from non-sympatric species. In recent years there has been increased interest in Gentiana as an ornamental crop with techniques such as in-ovule embryo culture employed to generate the desired hybrids. Outcomes from this work have included the expected hybrids and additionally haploid plants derived, presumably, from unfertilized ovules. Other in vitro work has resulted in polyploid plants, both to restore fertility in sterile hybrids and to investigate polyploidy as a tool for creating new variation for horticultural uses.

Proposed section headings

Introduction, Hybridisation and Natural hybrids,"Artificial" hybrids and overcoming hybridization barriers, In vitro breeding tools, Verification of hybrids, Haploids, Polyploidy, Future opportunities. Conclusions

 

III. 2. Protoplast fusion and somatic cell hybridization of gentians

.           Tomiczak K.,   Mikuła A.,  Rybczyński J.J. (Poland)

 

Somatic hybridization as a tool enabling mixing of both nuclear and cytoplasmic genomes from two distantly related, to closely related "naked" plant cells (protoplasts) through their fusion has opened up several possibilities for the parasexual manipulation of plants. In Gentianaceae somatic hybrids representing different nucleocytoplasmic combinations would be very welcome for utilizing as new ornamental varieties and valuable sources of secondary metabolites.

Despite the fact, that in Gentiana genus the potentiality of obtaining novel genotypes by sexual crossings is moderate due to limited genetic variation (especially in case of such ornamental aspects as flower color, there is only one report concerning protoplast fusion trials in Gentianaceae. Using ECM200 (BTX) electrical equipment mesophyll protoplasts of Eustoma grandiflorum and G. scabra were fused with cell suspension-derived ones of G. lutea, but no further communications of heterokaryons culture and somatic hybrids regeneration are available. Among the reasons, lack of efficient regeneration system from mesophyll protoplasts, adequate for wide range of Gentiana species, seems to be the most feasible.

However, abundant somatic embryogenesis in Gentiana cell suspension-derived protoplast culture was shown. Using this great morphogenic potential of cell suspension protoplasts of G. kurroo and G. cruciata on the one hand, and, on the other, limited embryogenic capacity of leaf mesophyll protoplasts of G. cruciata and G. tibetica, we succeed in creation of over 160 new hybrid individuals. The hybridity of G. kurroo (+) G. cruciata and G. cruciata (+) G. tibetica plants and calluses was confirmed by AFLP markers, chloroplast inheritance was analyzed by PCR-RFLP and their genome size and genetic stability were verified with the help of cytometric and cytogenetic methods.

 

 

III. 3. Haploid and doubled haploid production in gentian (Gentiana spp.) Takahata Y. (Japan)

 

Several gentian species have been utilized as economically important ornamental plants as well as medicinal plants. Gentiana triflora and G. scabra have been cultivated commercially as F1 hybrid cultivars for more than 50 years in Japan. Since the F1 hybrid cultivar is usually produced from a cross between two inbred lines, the establishment of parental inbred line is important. However, in F1 hybrid breeding of gentian, a major problem is that the production of inbred lines for parents of F1 varieties is difficult, because gentians exhibit intense inbreeding depression. Therefore, parental lines for F1 varieties have been produced and maintained by sib-mating or tissue culture methods. Since some heterozygosity of parental lines remains, the genotype of the parental lines gradually changed during their maintenance, resulting in the breakdown of the F1 variety. Production of haploids and doubled haploids (DHs) from male and female gametophytic cells by in vitro culture has been used for breeding and genetic research in many plants. In this section, we mention recent success of haploid and DH production via androgenesis and gynogenesis. Factors influencing haploid and DH production, ploidy level of regenerants and utilization of molecular genetic marker for identification of DH are discussed. Especially, unfertilized ovule culture is more efficient methods to produce haploids and DHs than anther culture in gentian.

 

 

III. 4 Genetic transformation and its application to gentian breeding
Nishihara M., Mishiba K., Imamura T., Takahashi H., Nakatsuka T.,
 Nishihara M. (Japan)

 

We will give an introduction to the genetic transformation of Japanese gentians. We focus on modification of flower color, flowering time and plant height so on. Gene silencing by promoter methylation should also be addressed as a critical program to apply genetic engineering to gentians.

 

 

IV. Biotechnology for improvement of Gentian family

 

IV. 1.Tissue culture, biotechnology of Taiwan native species belonging to Gentianaceae family

 Tsay H-S. (Taiwan)

 

The chapter describes tissue culture, biotechnology of Taiwan native species belonging to Gentianaceae family. This chapter will include In vitro propagation of Gentiana sp., factors effecting the establishment of suspension culture and quantitative determination of secoiridoid glucosides

 

IV. 2. In vitro manipulation and propagation of Gentiana L. species from Ukrainian flora

Drobyk N.M. ¹,. Hrytsak L.R ¹,. Kravets N.B. ¹, Mel'nyk V.M. ²,. Kunakh V.A. ² (Ukraine)

 

Conditions for microclonal propagation, callus induction and proliferation, plant regeneration from tissue culture, direct organogenesis in vitro, generation and long-term maintenance of fast-growing normal root culture of Gentiana species from Ukrainian flora were developed. The basic growth parameters of obtained cultures in vitro were evaluated. In most cultures highly intensive growth and considerable biomass yield was achieved. The ability to form the tissue and organ cultures was shown to depend on the original genotype, phytohormone and mineral composition of the nutrient medium, kind of explants. The efficiency of regeneration from tissue culture was found to decline with the duration of callus maintenance.

1. Microclonal propagation, 2. Callus induction and proliferation, 3. Plant regeneration from tissue culture, 4. Direct organogenesis of Gentiana lutea L., 5. Fast-growing normal root culture

 

 

IV. 3 Protoplast culture and plant regeneration of gentians

Rybczyński J.J. Tomiczak K. *Davey M. R., Mikuła A. (Poland/ *England)

 

In case of gentians various plant regeneration protocols were already published but the most sophisticated is the one basing on single cell culture - protoplast culture  resulted in plant regeneration via somatic embryogenesis.

The chapter reviews obtained results concerning the plant regeneration system from protoplast culture. Protoplast cultures constitute the unique system of somatic value exploration of plant genome. For gentian two systems: green leaf mesophyll protoplast and embryogenic cell suspension protoplasts were developed. In already published papers protoplasts were obtained from green leaf mesophyll protoplasts of G. sacra, G. triflora, G. caulis, G. triflora x G.scasbra, G. lutea. In our experiments morphogenic potential of six Gentiana species (i.e. G. cruciata (L.), G. decumbens (L.f.), G. kurroo (Royle), G. lutea (L.), G. septemfida (Pall.) and G. tibetica (King)) was tested in leaf mesophyll protoplast cultures. On modified MS media supplemented with NAA and BAP or TDZ protoplasts of all species underwent cell wall regeneration and first cell divisions. In case of G. decumbens, G. kurroo and G. tibetica sustained divisions led to callus tissue formation. Somatic embryogenesis occurred in cultures of all three species, with the highest frequency (about 0,42 embryo per 100 µl of medium) achieved for G. decumbens.

Embryogenic cell suspension appeared to be very efficient source of protoplasts showing high level of regeneration abilities. Possible exploration of protoplast culture in somatic cell genetic manipulation will be discussed.  

 

 

IV. 4. Genetic stability of regenerants

Rajinder Kaur (India)

 

Plant tissue culture is recognized as one of the key areas of plant biotechnology because of its potential use to regenerate and conserve elite and valuable plant genetic resources. However, in commercial industry where micropropagation is fully developed, the most crucial concern is to retain genetic integrity of micropropagated plants vis-à-vis explant source. It is well known fact that in vitro culture techniques could induce genetic variability, commonly known as somaclonal variations. Several techniques have been developed for detecting genetic variations, such as flow cytometery, morphological descriptions, physiological supervisions, cytological studies, isozymes, field assessment and genomic variations(using DNA markers) . In recent years, with the advent of recombinant DNA technology and PCR, DNA markers are being used for a variety of studies. More recently, DNA markers have also been used for testing the genetic fidelity during micropropagation /ex vitro conservation on one hand and for characterization of plant genetic resources on the other.

DNA marker techniques such as RAPD, RFLP, ISSR and SSR at present are powerful and valuable tools for assessing genetic or genomic variations in regenerants irrespective of the explant and method of in vitro culturing, employed for obtaining the same. So far cytological, and various DNA analysis methods have been successfully employed in a number of medicinal plants for assessing genetic fidelity which have been regenerated and/or ex situ conserved.

 

IV. 5. The role of arabinogalactan proteins in Centaurium erythraea Rafn. morphogenesis in vitro

Subotić A., Trifunović M.,  Petrić M., Tadić V. , Jevremović S. (Serbia)

 

Arabinogalactan proteins (AGPs) are highly glycosylated cell wall proteins involved in plant growth and development which can be specifically recognized and bind easily with synthetic dye, phenylglycoside β-D-glucosyl Yariv reagent (βGlcY). The presence, concentration and immmunolocalization of specific epitops of AGP were analyzed during direct somatic embryogenesis and organogenesis in root culture of Centaurium erythraea Rafn. The effect of β-GlcY supplemented into the culture medium on induction of morphogenesis in vitro was also investigated..  The induction of somatic embryogenesis and organogenesis was notably inhibited in concetration-depented manner with maximum (40.3%) at 75 µMl^-1 β-GlcY.  The concentration of AGPs in different tissues of C. erythraea Rafn. was determinated with radial diffusion method.  Light microscopy observations revealed that βGlcY specifically bind AGPs in cells of root epidermis, as well as, developed somatic embryos and adventitious buds. The precise distribution of several AGPs with specific epitops was analyzed with the immunolocalization with several monoclonal antibodies: LM2, JIM15, JIM16, MAC207. Specific monoclonal antibody-binding AGPs are located in the epidermal cells and vascular tissue of roots of primary explants, as a well as regenerated globular somatic embryos and in de novo formed meristematic centers in root cortex. The results showed that AGPs are clearly involved in induction of morphogenesis in vitro, development of somatic embryos and adventitious shoots in root cultures of C.erythraea Rafn.

 

IV. 6. Biotechnlogy of Gentianella species

Vinterhalter D., Krstić D., Vinterhalter B. and Janković T. (Serbia)

 

The genus Gentianella Moench (Gentianeceae) comprises some 250 species growing worldwide in temperate or mountain habitats. Although numerous, species of this genus  have been neglected and are stil considered as poorly investigated. Their name suggest small Gentians but apart from morphological similarities they have a quite different complement of secondary metabolites with xanthones as dominant compound.

In the mountain ranges of Serbia and neigbouring areas there are six distict Gentianella species (G. ciliata, G. crispata, G. praecox, G. axillaris, G. austriaca and G. bulgarica) which we recently started to investigate. Main lines of investigation include:

- biotechnology, in vitro culture and clonal propagation and

- phytochemical analyses, including HPLC identification and quantification of secondary metabolites.

Chapter will provide detailed data on two species G. austriaca  and G. bulgarica  which we considered as more or less finished and fragmentary data on other species. Also chapter will present our recent resuts on phytochemical composition of four Gentianella species not studied previously: G. albanica, G. bulgarica, G. austriaca and G. crispata.

Data will be compared and discussed in comparison  to previously studies high mountain Gentians

 

V. Various aspects of tissue, plant and flower maintenance

 

V. 1. Cryopreservation of Gentianaceae: trends and applications

Mikuła A., Tomiczak K., Rybczyński J.J. (Poland)

Over the last twenty years, considerable progress has been made towards improving our understanding of the mechanisms involved in tolerance to liquid nitrogen treatment and protection of plant tissue. New cryopreservation techniques, which are based on vitrification of internal solutes, have been successfully employed to storage of seven various species

belonging to Gentiana and one species of Centaurium genus. Currently two separate trends of cryopreservation exploitation have been developed for gentian species. The trends are strictly connected with the type of in vitro plant regeneration. The first one strives for optimization of preculture and cryotreatment methods for long-term and safe storage of differentiated explants, i.e. axillary buds and shoot apices. These Gentiana accessions collected in Japan institutes as a potentially superior ornamental genotypes could be successfully preserved with LN using, now. Their regeneration rates varying  between 16.7 and 93.3% depending on the cultivar/line and the cryopreservation protocol used. The second trend concerns necessity to maintain the embryogenic capacity of cell suspension cultures which are the excellent system for somatic cell genetic manipulation. Recent studies showed that the cryopreservation of this undifferentiated plant material can be routinely performed with average survival from 45.8% to 100%. What's more, it facilitate simple and quick recovery of embryogenic suspension cultures, increase the somatic embryo productivity and protect the genetic uniformity of regenerated plantlets (irrespective of time cryostorage).

 

 

V. 2. The role of arbuscular mycorrhiza in the growth and development of plants from Gentianaceae family

Sykorova Z. (Czech)

Arbuscular mycorrhiza is an ancient symbiosis between land plants and fungi from the phylum Glomeromycota. It is estimated to occur in about 80% of plant species, including plants from the family Gentianaceae. Arbuscular mycorrhizal fungi (AMF) colonize plant roots and contribute to the mineral nutrient uptake of the hosts in exchange for carbohydrates, they also benefit the plants by e.g. increasing water absorption, tolerance to pathogens, drought, high soil temperature, toxic heavy metals, extremes in pH and transplant shock.

AMF were also reported to have a decisive influence on plant diversity and community productivity. Because more than one plant can be colonized by the same fungus and each plant may host several fungal species, a large plant-fungal network may be established. Non-photosynthetic plants known as mycoheterotrophs may parasitize their green neighboring plants via taking up the carbohydrates from the shared fungal network.

It has been shown that AMF may play an important role in the life cycle of gentians. For example, several lineages of achlorophyllous mycoheterotrophic gentians showed exceptionally high host specificity known so far in arbuscular mycorrhiza. The role of AMF in the growth and development of Gentianaceae will be discussed in detail in the chapter.

 

V.3. Postharvest cut flower physiology of Eustoma

Celikel F.G. (Turkey)

Eustoma grandiflorum (Raf.) Shinn. (Syn. Lisianthus russellianus), a member of Gentianaceae family, known as Texas bluebells or prairie gentian, has been an important commercial cut flower because of its attractive flowers with relatively long vase life. Lisianthus has a long stem bearing many flowers with different colors, patterns and forms depending on cultivars. The limited number of studies on postharvest physiology and handling of cut Eustoma flowers have showed an increase recently. Eustoma flowers usually are harvested commercially when one or two flowers on the stem are opened. The longevity of the inflorescence is determined by the life of the open flowers while many buds fail to open in vase water. Carbohydrate supply is of importance in bud opening and preventing early wilting in Eustoma inflorescence. In addition to sucrose and glucose treatment, germicides in vase solutions, the effect of preharvest growing conditions and postharvest storage, harvest maturity, ethylene sensitivity, effects of ethylene inhibitors and other plant growth regulators, the changes during development and senescence of gentian flowers will be discussed in the review.

 

VI Secondary products

 

VI. 1. Gentiana radix

Buchwald W., Mikołajczak P. Ł. (Poland)

Gentian root (Gentianae radix) consists of the dried rhizome and roots of Gentiana lutea L. (Gentianaceae), a perennial herb indigenous to Central and South Europe. The raw material is collected in the autumn and dried. When fresh they are yellowish-white internally, but gradually become darker by slow drying, during which a characteristic odour is developed. Gentian root occurs as single or branched subcylindrical pieces of various lengths and usually 10-40 mm thick. The smoothed, transversely cut surface shows a bark, occupying about one-third of the radius, separated by the well-marked cambium from an indistinctly radiate and parenchymatous xylem. Powdered Gentian roots show the following diagnostic characters: fragments of the subero-phellodermic layer, cortical and ligneous parenchymatous cells, and lignified vessels. The raw material contains gentiopicroside (also known as gentiopicrin), swertiamarin and sweroside and a very small amount of amarogentin, which causes the bitter taste. Also present are: xanthones (gentisin, isogentisin, gentioside), phytosterols, phenolic acid, trisaccharides (gentianose), polysaccharides (pectin), and essential oil. Pharmacopoeial grade Gentian root must have a bitterness value of not less than 10000 and must also contain not less than 33% water soluble extractive. The bitterness of raw material stimulates secretions in the gastrointestinal tract, especially of gastric juice. Traditionally Gentian root is

used to increase the appetite in recovery from acute atonic dyspepsia. From many experiments its antihepatotoxic, adaptogenic, and anti-inflammatory activities are also postulated. Recently, it has been investigated for Gentian root possible effects on the central nervous system, such as antidepressant, anticonvulsant, and analgesic activities in mice. Moreover, Gentiana lutea extract showed radioprotective activity probably due to its antioxidant activity.

 

 

VI. 2. Tissue and organ cultures of gentians as potential sources for xanthones and flavonoids

Drobyk N.M. , Mel'nyk V.M., Twardovska M.O., Konvalyuk I.I., Kunakh V.A. (Ukraine)

 

Comparative investigation into total flavonoid and xanthone contents in tissue cultures, cultures of isolated roots and wild plants of Gentiana species from Ukrainian flora has been carried out.

Capacity for synthesizing these biologically active substances in most obtained cultures was ascertained. These contents varied both in calli and isolated roots, derived from plants of various gentian species as well as in tissue and organ cultures of the same species, generated from plants grown in different habitats. Morphogenic and nonmorphogenic gentian cultures showed much lower flavanoid and xanthone contents than shoots of the intact plants but higher or close to that of the natural roots. Culture of isolated roots in most cases displayed higher amount of these biologically active substances than those of calli.

High yield of biomass from gentian culture in vitro, especially from isolated root culture, and their ability to synthesize and accumulate flavonoids and xanthones allow considering them as a promising raw material source for flavanoid and xanthone production.

1. Total xanthone contents in tissue cultures, 2. Total flavonoid contents in tissue cultures

3. Xanthone and flavonoid contents in the isolated root cultures

 

 

VI. 3. Bioactive secondary metabolites in several genus of Gentianaceae family: plant species of the Central Balkan Peninsula.

Menkovic N. (Serbia)

Plants that belong to the family Gentianaceae are cosmopolitan in distribution and comprise about 1600 species classified into 87 genera. The largest genera are Gentiana (360 species), Gentianella (250 species) and Swertia (135 species). In Europe, Gentianaceae species are arranged into 9 genera. In the flora of the Central Balkan Peninsula (Serbia, Montenegro, Bosnia and Herzegovina) 5 genera with 31 species were found- Gentiana, Gentianella, Centaurium, Swertia and Blackstonia, which consist about 50% of the flora of Europe. Comprehensive phytochemical investigations of the Gentianaceae species are directed to the main secondary metabolites, namely flavonoids, xanthones and secoiridoids. Xanthones need special attention as their distribution is not universal in the family which makes them useful systematic markers. Along with their taxonomic importance, xanthones display various biological effects which put them among perspective therapeutic agents. In this chapter, distribution, chemical classification and biological activity of xanthones in Gentianaceae species of Central Balkan will be discussed.

 

VI. 4. Phytochemical analysis and molecular genetic characterization (identification/authentication) together with ecotype studies of Gentiana lutea

            Franz Ch. (Austria)

 

 

VII. Conclusions

 

VII. 1 European social life in the "shadow" of gentians

Jolles C. (Switzerland)