Secondary metabolite content in rhizomes, callus cultures and in vitro regenerated plantlets of Solidago chilensis

An in vitro culture system leading to the formation of callus and plant regeneration, starting from nodal sections and shoot tips, was developed for Solidago chilensis (Asteraceae). The content of the gastroprotective diterpene solidagenone as well as the phenolics chlorogenic acid (CA) and rutin was determined either in rhizomes from wild growing plants and in callus and in in vitro regenerated plantlets by analytical HPLC. Additionally, total phenolic and flavonoid content was assessed in plant samples, callus and cell suspensions. In terms of dry starting material, the percentual solidagenone content in nine S. chilensis samples ranged from 0.5-3.5% for rhizomes from wild growing plants, 0.1-0.3% for callus and 0.3% for an in vitro regenerated plantlet, respectively. The highest solidagenone contents were found in the wild plant during the late summer in the months of March and April (3.5-2.2%) while highest values for chlorogenic acid (0.5%) and rutin (0.4%) were detected in May, before senescence. The callus tissue and cell suspensions contained some 1.8-2.0 and 1.2% of total phenolics, respectively. CA was the main phenolic in the cell suspension while only traces were found in the callus. Rutin was not detected in the callus nor cell culture.     

Improved growth and quality of <Emphasis Type="Italic">Siraitia grosvenorii</Emphasis> plantlets using a temporary immersion system

The effects of temporary immersion system (TIS) culture on the growth and quality of Siraitia grosvenorii plantlets were investigated. The TIS promoted the growth and quality of S. grosvenorii plantlets. Proliferation rate, shoot length, fresh weight (FW) and dry weight (DW) of shoots, and total biomass production were significantly (P ≤ 0.05) higher in the TIS than in gelled and liquid medium, respectively. The TIS also decreased callus formation at the base of shoots. Callus diameter was significantly (P ≤ 0.05) lower in the TIS (3.30 mm) than in gelled medium (6.31 mm) and liquid medium (6.77 mm), respectively. FW (50.83 mg) and DW (7.08 mg) of callus in the TIS were also significantly (P ≤ 0.05) lower than those in gelled medium (80.00 and 10.56 mg, respectively) and liquid medium (218.75 and 23.75 mg, respectively). During rhizogenesis, minimal callus was evident at the base of shoots in the TIS, with a well-developed root system. However, the plantlets in gelled medium just produced thick, brown and easily broken roots with obvious callus and fewer secondary roots. The natural-like plantlets of S. grosvenorii obtained in the TIS would probably have positive effects on ex vitro rooting and transplanting in large-scale commercial production.     

Production of bakuchiol by in vitro systems of <Emphasis Type="Italic">Psoralea drupacea</Emphasis> Bge

In vitro production of the meroterpene bakuchiol by Psoralea drupacea Bge (Fabaceae) has been studied using aseptically-grown plants, callus cultures of different origin, cell suspensions and transgenic hairy root cultures. The effect of phytohormones and methyl jasmonate on bakuchiol production was also investigated. Bakuchiol was not detected in cell suspensions or hairy root preparations of P. drupacea. In contrast, aerial parts of P. drupacea grown in vitro were found to accumulate up to 11% dry weight of bakuchiol and can therefore be regarded as a potentially useful source of this antimicrobial compound.     

Micropropagation of <Emphasis Type="Italic">Arnebia hispidissima</Emphasis> (Lehm). DC. and production of alkannin from callus and cell suspension culture

Alkannin, a red-purple dye and bioactive compound found in the roots of Arnebia hispidissima has antibiotic and anti-inflammatory properties and is also used in cosmetic and textile industries at a large-scale. In the present communication, we demonstrate the establishment of callus and cell suspension culture of A. hispidissima with the aim of optimizing the production of alkannin. Highest alkannin content was recorded in cell suspension and callus culture established on M-9 medium. Production of alkannin was influenced by the different culture medium. Evaluation of alkannin content of roots of field-grown plants and in vitro grown cell, tissue and organ showed that alkannin production was higher in all in vitro grown culture systems (cell suspension, callus and roots) than the roots of field-grown plants. The present investigation may be applicable in designing systems for the large-scale cultivation of A. hispidissima cell suspensions for the production of alkannin.     

Accumulation of coumarins in Arabidopsis thaliana

The biosynthesis of coumarins in plants is not well understood, although these metabolic pathways are often found in the plant kingdom. We report here the occurrence of coumarins in Arabidopsis thaliana ecotype Columbia. Considerably high levels of scopoletin and its beta-d-glucopyranoside, scopolin, were found in the wild-type roots. The scopolin level in the roots was approximately 1200nmol/gFW, which was approximately 180-fold of that in the aerial parts. Calli accumulated scopolin at a level of 70nmol/gFW. Scopoletin and scopolin formation were induced in shoots after treatment with either 2,4-dichlorophenoxyacetic acid (at 100microM) or a bud-cell suspension of Fusarium oxysporum. In order to gain insight into the biosynthetic pathway of coumarins in A. thaliana, we analyzed coumarins in the mutants obtained from the SALK Institute collection that carried a T-DNA insertion within the gene encoding the cytochrome P450, CYP98A3, which catalyzes 3'-hydroxylation of p-coumarate units in the phenylpropanoid pathway. The content of scopoletin and scopolin in the mutant roots greatly decreased to approximately 3% of that in the wild-type roots. This observation suggests that scopoletin and scopolin biosynthesis in A. thaliana are strongly dependent on the 3'-hydroxylation of p-coumarate units catalyzed by CYP98A3. We also found that the level of skimmin, a beta-d-glucopyranoside of umbelliferone, was slightly increased in the mutant roots.     

Saporin production from in vitro cultures of the soapwort Saponaria officinalis L.

We report here the successful establishment of callus, cell and root cultures from explants of in-vitro-grown plantlets of the soapwort Saponaria officinalis L. The production of saporin in the different tissue systems was evaluated by determining the capability of crude extracts to inactivate protein synthesis and by Western blotting analysis. Protein synthesis inhibition varied in callus and derived cell suspensions and in cultured roots, the latter, in particular, showing the lowest specific activity. The ribosome-inactivating principle from root cultures was purified to homogeneity by cation exchange chromatography. Received: 2 October 1996 / Revision received: 24 January 1997 / Accepted: 15 February 1997     

Growth and development of carnation ‘Dreambyul’ plantlets in a temporary immersion system and comparisons with conventional solid culture methods

The aim of the current study was to compare the effects of the culture method—conventional solid medium culture and temporary immersion system (TIS)—on the growth and development of carnation ‘Dreambyul’ plantlets. At the same time, different immersion intervals and immersion durations of TIS culture were also tested to find the optimal setting for mass production of high-quality carnation plantlets in vitro. In the first experiment, the results showed that the shoot length, root length, and number of nodes of plantlets cultured in the TIS were highest when the immersion interval was 8 h. Compared with that of plantlets cultured in the conventional solid medium culture, the fresh weight of plantlets cultured in the TIS was at least 3 times greater. The greatest total chlorophyll content, stomata with normal shapes was observed for plantlets grown in the TIS with an 8-h immersion interval. The lowest H₂O₂ level was recorded in plantlets cultured with the 8-h immersion interval. In the second study, growth traits such as the shoot length, root length, and stem diameter, as well as the number of shoots and roots tended to increase with immersion durations, and reached their peaks when the immersion duration was 90 s. Excessive water accumulation in tissues and a higher incidence of hyperhydricity were observed in plantlets where the immersion duration was 120 and 150 s. These findings suggest that an immersion interval of 8 h, combined with an immersion duration of 90 s, could be the optimal setting for growth and development of carnation ‘Dreambyul’ plantlets cultured in the TIS.

     

Comparison of somatic embryogenesis-derived coffee (Coffea arabica L.) plantlets regenerated in vitro or ex vitro: morphological,mineral and water characteristics

Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi-solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro-regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0.86 cm2) had the highest rates of plant conversion ex vitro (63%), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi-solid media, those produced in soil were branched, fine (30-50% had a diameter of less than 0-5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm-2). Moreover, they were more turgid, as indicated by higher pressure potential (psiP) (0.91 s. 0.30 MPa) and relative water content values (97 vs. 93%). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.     

Plant regeneration from embryogenic cell suspensions derived from anther cultures of barley (Hordeum vulgare L.)

Summary We have established embryogenic cell suspension cultures of barley (Hordeum vulgare L. cultivars Igri, Gimpel, Princesse, and Baronesse) from anther-derived embryogenic callus. Suspension cultures of cultivars Igri and Gimpel were regenerable. The most successful cultivar was Igri, from which a number of independent cell lines producing plantlets were established. Plants could be transferred to soil; up to now, 50% of more than 200 regenerated plants were morphologically normal and fertile. The relative frequency of sterile plants increased as suspensions aged. Suspensions older than 1 year produced embryogenic callus but only albino plantlets could be regenerated.     

In vitro shoot and root organogenesis, plant regeneration and production of tropane alkaloids in some species of Schizanthus

A rapid in vitro propagation system leading to formation of shoots from callus, roots, and plantlets was developed for Schizanthus hookeri Gill. (Solanaceae), an endemic Chilean plant. The genus Schizanthus is of particular interest due to the presence of several tropane alkaloids. So far, in vitro propagation of species of this genus has not been reported. Propagation of S. hookeri consisted of two phases, the first one for callus initiation and shoot formation and the second for rhizogenesis and plantlet regeneration. From a single callus that rapidly increased in cell biomass (from approximately 50 mg to approximately 460 mg/culture tube [25 x 130 mm] in 60 days) in the presence of 2.69 microM NAA and 2.22 microM BA, more than 10 shoots/callus explant were formed. From the latter, approx. twenty plantlets formed after 90-110 days shoot subculture in medium devoid of growth regulators that favored root formation. Ten alkaloids ranging from simple pyrrolidine derivatives to tropane esters derived from angelic, tiglic, senecioic or methylmesaconic acids were obtained from in vitro regenerated plantlets. One of them, 3alpha-methylmesaconyloxytropane, was not previously described. The same growth conditions, as well as other growth regulator levels tested, were required to induce callus and root formation in S. grahamii Gill. Root organogenesis occurred despite a high level of BA vs. NAA used, (i.e., 4.44 microM BA and 0.54 microM NAA); however no shoot formation was achieved. In the case of S. tricolor Grau et Gronbach, only callus formation was obtained in the presence of various growth regulators.     

杯山药零余子愈伤组织诱导及植株再生的研究

对怀山药（Ｄｉｏｓｃｏｒｅａ ｏｐｐｏｓｉｔａ）零余子愈伤组织的诱导、分化、再生苗的生根和移栽进行了研究。结果表明：⑴在不同激素组合的培养基上怀山药零余子均能产生愈作组织,而且具有一次成苗的能力。ＢＡ２ｍｇ／Ｌ＋ＮＡＡ２ｍｇ／Ｌ的培养基对诱导愈伤组织最有利,其出愈率达１００％;⑵在愈伤组织的分化中,ＢＡ１ｍｇ／Ｌ＋ＮＡＡ１ｍｇ／Ｌ的激素组合是最佳的,其分化率为６３．６％,且多形成丛生芽;⑶再生植株     

Untersuchungen über Beziehungen zwischen Zellteilung und Morphogenese bei Gewebekulturen von Daucus carota

Relationship between cell division and morphogenesis of tissue cultures of Daucus carota. I. Observations on rhizogenesis and formation of whole plants.—By using a chemically defined medium the conditions for formation of roots and embryos in callus of various tap root parts of domestic Daucus carota forms were investigated and compared. Whole carrot plants capable of generative reproduction were produced from callus as well as from cell and tissue suspensions. The influence of various phytohormones on differentiation and cell division activity of explants was studied. A close relationship between cell division activity and differential stains was established and explained as a result of the phytohormone action on the cell division rate.     

Stem Apex Culture and Quality Improvement of Tubercles of Pinellia ternata

Formation of Plantlets was achieved when stem apex of Pinellia ternata Brier. Cultured in vitro on MS medium with KT 0. 5 mg/L + NAA 0.2 mg/L (MSI). With petioles of the plantlet as explants callus could be induced after cultured for a week on MS medium with 2, 4-D 2.0 mg/L + KT 0.5 mg/L (MSII). Calli were subcultured once in every month. After 3--4 months a kind of friable calli could be selected, from which the tubercles could be differentiate and the plantlets formed when transfered onto MSI. But before callus differentiation, a lot of roots were formed on callus. The plantlets could be produced directly from the petiole segment. It was found that the stem growing tip was always covered by the leaf primordium and the former leaf primordium was covered by the latter leaf primordium during the differentiation of the apical bud of tubercle. The frenquency of plantlet differentiation from callus and petioles was over 70%. The rate of regeneration of plantlet on liquid static culture was twice as much as that on solid culture. All plantlets grew well after being transfered into the plot. The fresh weight of tuber-plant was 103 % higher than that of control (cultivated plant come from tubers). The alkaloid content of tubers come from tuberplant was 0. 344%, that of control was 0. 203% and 0. 264% for the wild tuber.     

Regeneration of somatic embryos in Theobroma cacao L. in temporary immersion bioreactor and analyses of free amino acids in different tissues

The present study aimed at developing temporary immersion bioreactor techniques for multiplication of cacao somatic embryos. Temporary Immersion System (TIS), i.e. flooding of plant tissue at regular time intervals provides an efficient way to propagate plants. Somatic embryos were regenerated in twin flask bioreactors. The TIS proved to be suitable for mass regeneration of somatic embryos and for their subsequent direct sowing. The number of embryos after 3 months of culture was significantly higher in TIS cultures than in the solid medium variant. TIS also improved embryo development regarding the conversion to torpedo shaped forms. Matured embryos derived from TIS and pre-treated with 6% sucrose were converted into plants after direct sowing. Additionally to the influence of culture conditions on the development of somatic embryogenesis the content and composition of free amino acids were analysed. The content of free amino acids in somatic embryos rose as immersion frequency increased. The endogenous free GABA content in embryogenic callus was significantly higher than in non-embryogenic callus.     

Effect of differentiation on the regulation of indole alkaloid production in Catharanthus roseus hairy roots

In vitro cultures of hairy root derived from Catharanthus roseus accumulate higher levels of indole alkaloids than cell suspension cultures. Hairy roots were interconverted to undifferentiated cells by manipulation of the culture medium. When the concentration of micronutrients in the culture medium was five times that of Phillips and Collins (1979) medium, cell suspensions formed from the hairy roots. The alkaloid content was five times lower in the cell suspensions than in the control, but upon regeneration of the roots the alkaloid content regained its original level. The formation of cell suspensions from hairy roots was also accompanied by a reduction in tryptophan decarboxylase and the strictosidine synthase activity to less than 5% and 30%, respectively. 3-Hydroxymethylglutaryl coenzyme A reductase activity was the same in the cell suspension and in the regenerated line. Received: 12 February 1998 / Revision received: 21 May 1998 / Accepted: 5 June 1998     

An efficient plant regeneration system with <Emphasis Type="Italic">in vitro</Emphasis> flavonoid accumulation for <Emphasis Type="Italic">Hylotelephium tatarinowii</Emphasis> (Maxim.) H. Ohba

An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid was as high as 2.0 mg l⁻¹ in combination with 0.5 mg l⁻¹ 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed to 1.0 mg l⁻¹ 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile of callus and regenerated plants was similar to that of wild plants.     

Establishment of oil palm cell suspensions and plant regeneration

Primary globular callus from immature zygotic embryos and friable embryogenic tissue derived from mature zygotic embryos were used to establish suspension cultures. Callus cultures were established either on modified Y3 or MS medium containing 475–500 M 2,4-D or 250 M picloram and 0.3% (w/v) activated charcoal. Suspension cultures of both cell lines were established in modified Y3 medium containing 10 M 2,4-D. The establishment of cell suspensions from friable embryogenic tissue took only 2 months, in contrast with suspensions from primary globular callus which took 3–5 months to establish. Embryo differentiation was observed only in cell suspensions derived from the friable embryogenic tissue after plating aliquots on regeneration medium. Germinated embryos were recovered and plantlets were successfully established under greenhouse conditions.Abbreviations CET compact embryogenic tissue - FET friable embryogenic tissue - CIM callus induction medium - PGC primary globular callus - 2,3-D 2,4-dichlorphenoxyacetic acid Y3-Eeuwens' medium - MS Murashige & Skoog medium - PVP-40 polyvinylpyrrolidone - KM Kao & Michayluk vitamins - ABA abscisic acid     

The relationship between polyploidy and organogenetic potential in embryo- and root-derived tissue cultures of Vicia faba L.

The cell cycle was examined in embryo and root explants of Vicia faba in culture to test whether or not polyploidy and aneuploidy affected organogenetic potential. Nuclear DNA contents and the mitotic index were measured in the 0–1 mm apical segment of primary roots of 5-day old seedlings and at various times following transfer to modified MS in darkness or Chu's N6 medium in an 8 h light/16h dark cycle (N6-MS programme) at 20°C. Mature embryos were dissected and cut longitudinally. Each half was cultured on the N6-MS programme. Root explants grown on MS in darkness developed into callus but there was no subsequent organogenesis. Only on the N6-MS programme were new roots initiated from root-derived callus. Using the N6-MS programme, embryo-derived callus became green and after 3 to 4 months, produced roots and shoots. Approximately 40% of these cultures regenerated plantlets. Polyploidy occurred within 24 h of culture irrespective of both tissue source and culture protocol. Variations in chromosome number from 2n=2x=12 were also routinely observed. Thus, calluses had the ability to initiate roots and shoots regardless of persistent polyploidy and aneuploidy. Compared with the baseline of cell cycle data for roots in vivo, the proportions of cells in the different cell cycle phases remained constant. Thus, in V. faba induction of organogenesis seems more related to culture protocols than to specific changes to the cell cycle. The mitotic index was significantly lower in vitro compared with meristems of intact roots.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of antioxidants from <Emphasis Type="Italic">Hemidesmus indicus</Emphasis> R. Br. cultures

Summary Shoot cultures and callus cultures from roots and leaves of Hemidesmus indicus R. Br (Asclepiadaceae) were established on Murashige and Skoog medium with various hormonal combinations. The production of antioxidants (lupeol, vanillin, and rutin) in shoot cultures, callus cultures derived from leaf cells and root cells, was compared with root and aerial portions of the parent plant. Shoot cultures and leaf callus cultures produced more antioxidants than root callus cultures. In vitro culture of this species might ofter an alternative method for production of these important pharmaccuticals, which would reduce the collection pressure on this rare plant.