Flavonoid Production in Transformed Scutellaria baicalensis Roots and Ways of Its Regulation

A root culture of skullcap (Scutellaria baicalensisGeorgi) transformed with pRi T-DNA was initiated by the inoculation of sterile seedlings with Agrobacterium rhizogenes(wild-type strain A-4). The flavonoid concentration in cultured roots comprised 5% of the root dry weight and was maintained essentially constant during a subculture. For four weeks of culturing, the weight of the roots increased by 20–30 times; when the roots were cultured for a longer time and with periodic enrichment of the nutrient medium, their weight increased 50-fold. Skullcap roots were shown to synthesize flavones characteristic of intact roots (wogonin, baicalein, and baicalin). The addition of 0.01–1 mM L-phenylalanine (a precursor of flavonoids) to the nutrient medium affected neither root growth, nor their flavonoid concentration. Root elicitation with 100 M methyl jasmonate for 72 h increased the flavonoid content per flask and per root dry weight by 1.8 and 2.3 times, respectively.     

Identification and production of flavonoids in a cell suspension culture of Scutellaria baicalensis G.

A high yielding cell line of Scutellaria baicalensis G. has successfully been developed to produce flavonoids. Major components of the flavonoids were identified as baicalin and wogonin-7-O-glucuronic acid by a series of instrumental analyses using UV, IR, MASS, and NMR. After 12 days in suspension culture, the cell growth reached 14 g ^DW/l, and baicalin and wogonin-7-O-glucuronic acid were obtained in concentrations of 2.9 g/l and 1.07 g/l, respectively. The culture temperature was found to be an important parameter for improving production yield of the flavonoids. The yield of baicalin was observed to increase to 4.2 g/l by shifting the temperature from 30 °C to 25 °C after 72 h of suspension culture.Abbreviations DW cell dry weight - FW cell fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - PSH medium phytohormone added Schenk and Hildebrandt medium - FPM a modified Schenk and Hildebrandt medium for flavonoid production     

Induced accumulation of triterpenoids in Scutellaria baicalensis suspension cultures using a yeast elicitor

When growth-phase cell suspension cultures of Scutellaria baicalensis were treated with 50 g of yeast elicitor preparation ml^–1, both oleanolic acid and ursolic acid transiently increased in the culture medium rather than in the cells. The maximal triterpenoid concentration was 13.7 mg l^–1 media approx. 35 h after treatment, whereas the maximum concentration was 2.1 mg l^–1 media after about 20 h following treatment with methyl jasmonate. Elicitor treatment also doubled phospholipase A₂ activity (25 pmol mg^–1 min) and the simultaneous treatment of aristolochic acid, a phospholipase A₂ inhibitor, inhibited triterpenoids accumulation as well as phospholipase A₂ activity.     

酶解法提取黄芩中黄芩素的工艺研究

本文对黄芩药材中黄芩苷的酶解条件和黄芩素的提取工艺进行研究,以样品中黄芩素的含量为指标,采用正交实验对影响酶解的温度、时间和药材粒度进行考察;以黄芩素的提取率为指标,经正交实验优选乙醇浓度、用量、回流次数和时间。得出如下较理想的酶解条件和提取工艺:黄芩药材粉碎至10~20目,加水6倍,于60℃保温10 h;酶解后的黄芩药材加8倍量70%乙醇回流提取2次,每次1 h。黄芩素的提取率大于70%,工艺稳定可行。     

Flavones in genetically transformed <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> roots and induction of their synthesis by elicitation with methyl jasmonate

The composition and content of flavones were estimated in pRi T-DNA-transformed skullcap (Scutellaria baicalensis Georgi) roots obtained by the inoculation of axenically grown seedlings with a wild A4 strain of the soil bacterium Agrobacterium rhizogenes. It was elucidated by analytical and preparative HPLC of phenolic compounds in the extracts from the pRi T-DNA-transformed roots and also by ultraviolet spectroscopy and ¹H and ¹³C NMR that cultured skullcap roots contained similar basic flavones as intact roots of this plant species, i.e., baicalein and wogonin and corresponding glucuronides, baicalin and wogonoside. The content of these flavones in cultured roots was threefold lower than in the roots of intact five-year-old plants. When skullcap roots were cultured on B5 or Murashige and Skoog medium, the ratios between major flavones changed but their total content remained unchanged. The treatment of three-week-old cultured roots with methyl ether of jasmonic acid (MeJa) doubled the total concentration of major flavones in roots; the content of aglycons, baicalein and wogonin, increased to a greater degree, e.g., by 2.3 and 3.3 times, respectively. The induction of flavone production by elicitors indicates that flavones behave as phytoanticipins because major flavones of skullcap manifest a distinct antimicrobial activity. The results of the short-term treatment of skullcap roots with MeJa show that stress biotic factors can considerably increase the content of physiologically active flavones.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 90–96.Original Russian Text Copyright © 2005 by Kuzovkina, Guseva, Kovács, Szöke, Vdovitchenko.     

Flavone-catalyzed apoptosis in Scutellaria baicalensis

In response to mechanical damage, roots of Scutellaria baicalensis undergo cell death within 24 h. The flavone baicalein was identified as the factor regulating apoptosis in the damaged roots of S. baicalensis. Plant apoptosis is known to be triggered by oxidative damage of DNA through oxidative bursts, whereas baicalein causes apoptosis in Scutellaria cells by a copper-dependent oxidation of nuclear DNA without inducing an oxidative burst. S. baicalensis possesses an interesting system for quickly producing this apoptosis-inducing flavone in its cells. Intact Scutellaria cells contain little baicalein but store a large amount of baicalin (baicalein 7-O-β-D-glucuronide). Stress treatment of Scutellaria cells immediately initiates hydrolysis of baicalin by endogenous β-glucuronidase, and the resulting baicalein is immediately translocated to the nucleus, leading to apoptosis. Thus, S. baicalensis possesses a unique apoptosis-inducing system that is linked with metabolism of baicalin.     

Relation between electrokinetic potentials and growth in callus cultures ofTrigonella foenum-graecum

Callus cultures ofTrigonella foenum-graecum were initiated from radicle or cotyledon portions of seedlings and young leaves and maintained on modified 1-B5 medium. The callus mass was disaggregated by mechanical agitation and the discrete cells thus obtained were used to measure their electrokinetic potential. Studies pertaining to the effects of ageing on electrokinetic potential and growth index revealed a relationship between these two parameters. Thus, the rate of change of electrokinotie potential with age could be employed as a parameter to study the growth kinetics of cells in callus cultures.     

The in vitro production of an anthocyanin from callus cultures of Oxalis linearis

Callus growth and the production of anthocyanins were sustained on the salts and vitamins of Murashige and Skoog. Callus growth was stimulated at a concentration of 8–32 M -naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d). Benzyladenine (BA) and zeatin at 8 M inhibited callus growth whereas isopentenyladenine (iP) stimulated callus growth. NAA repressed anthocyanin production with an increase in NAA from 8–32 M. Anthocyanin synthesis was promoted by an increase in 2,4-d from 0.5 to 2 M and decreased thereafter up to a concentration 32 M 2,4-d. A concentration of 8 M BA, thidiazuron and zeatin, respectively stimulated pigment production. Sucrose stimulated callus growth at 60 mM and pigment production at 120–360 mM.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - iP isopentenyladenine - TZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-yl-urea - Bu-HCl Butanol-2N HCl - BAW Butanol-acetic acid-water     

Effects of incubation temperature on microspore callus production and plant regeneration in wheat anther cultures

Anthers of wheat cultivars Orofen and Pitic 62 were incubated for 8 days at 15, 20, 25, 30, 35 and 40°C before transfer to 25°C. Compared with anthers cultured at 25°C constantly, anthers treated at 30°C produced 40% more microspore callus and green plants in both cultivars whereas those treated at 35°C produced 2–3 fold more green plants. Treatment at 40°C was deleterious. Possible modes of action of high temperature on callus production and albinism were discussed.     

Secondary metabolites produced by callus cultures of various Ruta species

Callus cultures were established from hypocotyl explants of R. bracteosa, R. chalepensis and R. macrophylla. Calli were maintained for more than three years on MS-medium supplemented with 1 mg l^-1 of each 2,4-D and kinetin. Acridone and furoquinoline alkaloids and coumarins have been isolated from four week old calli grown on a hormone containing and hormone-free medium. A new chlorinated acridone alkaloid has been detected.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog [6]     

Induction of pollen callus in anther cultures of Feijoa sellowiana Berg. (Myrtaceae)

Summary Anthers of Feijoa sellowiana Berg. (feijoa) produced pollen callus when cultured in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine or in nurse cultures. Somatic callus was also formed in large amounts from the connective and from the cut end of the filament. Anthers containing microspores at the stage immediately prior to the first pollen mitosis cultured in the presence of 3% sucrose, presented the highest frequencies of induction. Androgenetic divisions were initiated by the formation of two morphologically equal cells, the so-called B-pathway. Attempts to regenerate pollen plants were unsuccessful but leaf-like structures could be obtained in regeneration media containing combinations of gibberellic acid and benzyladenine.Abbreviations 2,4-D 2,4-diclorophenoxyacetic acid - BA benzyladenine - FDA fluorescein diacetate - GA₃ gibberellic acid - Kn kinetin - MS Murashige and Skoog (1962) medium     

In vitro anti-microbial activity of extracts from the callus cultures of some Nigella species

Crude methanol extracts from callus cultures of Nigella arvensis, N. damascena, N. hispanica, N. integrifolia, and N. sativa were investigated for their anti-microbial activity. Growth inhibition was determined in Gram-positive and Gram-negative bacterial strains as well as in yeast by using a broth-microdilution method. The results showed that the extracts of all calli tested exhibited significant anti-microbial activity, especially against Bacillus cereus, Staphylococcus aureus and Staphylococcus epidermidis. Compared with other Nigella species, a callus culture of N. hispanica was the most effective against the microorganisms used in this study.     

Effect of sucrose on polyploidization in early callus cultures of Solanum tuberosum

Leaf segments of a monohaploid, dihaploid and tetraploid genotype of the potato (Solanum tuberosum; x = 12) were cultured on callus-inducing medium with 10, 20, 30 or 40 gl^–1 sucrose. After 5 and 7 days of culture, metaphases contained the somatic or polyploidized number of mono- or diplochromosomes. The percentages of polyploidized metaphases were inversely correlated with the number of chromosome sets of the genotypes. In monohaploid leaf segments the percentages of polyploidized metaphases and of metaphases with diplochromosomes increased when the sucrose concentration was raised from 10 or 20 to 30 gl^–1 and remained constant or decreased from 30 to 40 gl^–1. Higher concentrations of sucrose but not higher osmolalities of the medium due to mannitol induced endoreduplication in more cells. The frequency of polyploidized metaphases and metaphases with diplochromosomes in dihaploid and tetraploid leaf segments remained constant through increases in sucrose concentrations.     

Attempts to produce solasodine in callus and suspension cultures of Solanum mauritianum Scop.

Callus cultures of Solanum mauritianum Scop. were initiated from green berry explants on a hormone-free Murashige and Skoog (1962) medium excluding glycine, and containing 0.1 g L^–1 myo-inositol and 3% sucrose. Such cultures contained 10.08±0.59 g g^–1 DW of solasodine, which is equivalent to that in the leaves of mature S. mauritianum plants, but far less than that extracted from the green berries (185 g g^–1 DW). In vitro solasodine productivity could be increased by reducing the strength of the medium by half, substituting 3% glucose for 3% sucrose as carbon source, or by the addition of certain combinations of BA and NAA. Phosphate limitation and alterations in the carbon: nitrogen ratio were not able to increase solasodine productivity. Suspension cultures of S. mauritianum were initiated and maintained in a Murashige and Skoog (1962) medium with the RT vitamins of Khanna and Staba (1968), 0.1 g L^–1 myo-inositol, 3% sucrose and 1 mg L^–1 2,4-D. No solasodine was detectable in these cultures, or slight modifications thereof.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

Retention of phytoalexin regulation in legume callus cultures

Legume callus cultures were examined to assess whether regulation of phytoalexin biosynthetic pathways is retained in cultured tissues. Callus tissue cultures ofCanavalia ensiformis (jackbean),Medicago sativa (alfalfa), and nine species ofTrifolium (clover) were established (six clover species for the first time) and maintained on modified Gamborg's B5 medium. Phytoalexins educed in cultures incubated for 48 h with an abiotic elicitor (3.15 mM HgCl₂) were detected by their antifungal activity and were purified by column chromatography and high-performance liquid chromatography. Following crystallization, phytoalexins were identified by ultraviolet and proton nuclear magnetic resonance spectroscopy. None of the treated cultures yielded the same complement of phytoalexins reported for fungal-inoculated leaves of the corresponding plants. Callus from all species exceptT. pratense yielded medicarpin, the only phytoalexin reported in treated leaves of all the corresponding plants. A second phytoalexin, maackiain, was found in treatedT. pratense andT. medium calli; maackiain has been reported in fungal-inoculated leaves of those plant species as well asT. hybridum. The phytoalexins sativan and vestitol were not found in treated callus tissues even though they were reported to be present in fungal-inoculated leaves of the same species. These results suggest that (a) the pathway for medicarpin biosynthesis is of central importance for this group of legumes, (b) some phytoalexin anabolic pathways contain metabolic blocks in cells of cultured tissue, and (c) the mechanism for regulating phytoalexin accumulation in tissues is not lost in culture. Contribution no 8113 of the US Regional Pasture Research Laboratory, USDA-ARS, University Park, PA, USA     

赤霉素GA3调节黄芩组织培养中芽和根的形成

应用组织培养技术对黄芩进行外源激素调控研究。在培养不同时间进行的不同培养基之间的转移培养研究表明,组织培养条件下,在培养基中添加赤霉素,可显著刺激黄芩外植体芽的形成,同时抑制根的生长。在加有GA3的IAA培养基上,GA3显著影响黄芩组织培养物中的黄酮含量。在黑暗条件下,开始在2.5μmol/LIAA培养基中培养6d,随后转移到5μmol/LGA3培养基上培养,黄芩外植体中黄岑苷、黄岑素和汉黄芩苷的含量最高,分别为14.90,2.70和0.54μgmg-1(干重)。     

Berberine synthesis by callus and cell suspension cultures of Coscinium fenestratum

Callus and cell suspension cultures of Coscinium fenestratum were established from sterile petiole segments on Murashige & Skoog (MS) medium, supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyl amino purine (BAP). The cells in the culture produced berberine as the major compound. NAA stimulated the product synthesis over 2,4-D. Presence of light inhibited the growth and enhanced the berberine synthesis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - TLC thin layer chromatography     

Molecular characterization of Mutator systems in maize embryogenic callus cultures indicates Mu element activity in vitro

Summary Active Mutator lines of maize (Zea mays L.) are characterized by their ability to generate new mutants at a high rate and by somatic instability at Mutator-induced mutant alleles. Mutagenically active lines with fewer than ten Mu elements per diploid genome have not been observed. Alteration of Mutator activity has been shown to correlate with the state of modification of Hinfl restiction sites that lie within inverted terminal repeats of Mu elements. To determine whether active Mutator systems can be established and maintained in culture, copy number and modification state of Mu elements were investigated in embryogenic callus lines derived from F₁S of crosses of active Mutator stock with the inbred lines A188 and H99. All callus lines studied maintain high Mu-element copy numbers, and more than half show a continued lack of modification at the Mu element Hinfl sites; thus, parameters associated with mutagenic activity in planta are present in some, but not all, callus lines. Mutator activity was then tested directly by restriction fragment analysis of subclonal populations from A188/Mu ² and H99/Mu ² embryonic cultures. Novel Mu-homologous restriction fragments occurred in 38% of the subpopulations which contained unmodified Mu elements, but not in control cultures containing modified, genetically inactive Mu elements. We conclude that Mu elements from active Mutator parents can remain transpositionally active in embryogenic cell culture. Active Mutator cell lines may be useful for the production of mutations in vitro.     

Effect of bialaphos and phosphinothricin on plant regeneration from long- and short-term callus cultures of Gladiolus

Summary Callus was initiated from in vitro-grown plants of Gladiolus cultivars ‘Jenny Lee’ and ‘Florida Flame.’ The age of callus used for regeneration of plants was either 9 mo. old or 8 yr old from ‘Jenny Lee,’ and 4 mo. old from ‘Florida Flame.’ Regeneration medium consisted of Murashige and Skoog’s basal salts medium supplemented with 2.0 mg/l (9.3 μM) kinetin. This medium was supplemented with various concentrations of either bialaphos (Meiji Seika, Tokyo, Japan) or phosphinothricin (Hoechst-Roussell, Frankfurt, Germany). Bialaphos was more effective than phosphinothricin at stimulating plant regeneration. Plants regenerated from 8-yr-old callus of ‘Jenny Lee’ only when the regeneration medium was supplemented with 0.10 mg/l bialaphos. A bialaphos concentration of 0.01 mg/l stimulated regeneration from 9-mo.-old callus of cultivar ‘Jenny Lee’ and 4-mo.-old callus of ‘Florida Flame.’     

In vitro isoflavonoid production in callus from different organs of Pueraria lobata (Wild.) Ohwi

Pueraria lobata (Wild.) Ohwi is a medicinal plant producing large amounts of isoflavonoid glycosides. Here, the ability of in vitro callus cultures to synthesize isoflavonoids was tested. Callus cultures have been initiated from different explants of in vitro germinated plants using modified MS medium. Roots, leaves and stem segments were the best sources of callus tissue. The isoflavonoid profile and content was determined by means of chromatographic methods. Callus from all organs contained isoflavonoid aglycones: genistein and daidzein and daidzein glycosides: daidzin, puerarin and 3'-methoxypuerarin. The differences between each kind of explant were observed in both the total amount of isoflavonoids and in the proportion of individual compounds. The highest content was in root callus, followed by leaf- and stem callus.