Production of caffeic,chlorogenic and rosmarinic acids in plants and suspension cultures of Glechoma hederacea

Glechoma hederacea L. (Lamiaceae) is a perennial plant, which is distributed widely in Europe, Asia and America. Important anti-oxidant compounds are caffeic acid esters like rosmarinic acid (RA) and chlorogenic acid (CA). Phenylalanine ammonia-lyase (PAL) and rosmarinic acid synthase (RAS, 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) contribute to the formation of RA. Our aim in this study was to follow the accumulation of RA, CA and caffeic acid in a suspension culture of G. hederacea. Growth, medium and secondary metabolism parameters were determined during a culture period of 14 days. The maximal PAL activity was observed on day 5 and the maximal RAS activity on day 8. The RA content was exceedingly high and reached 25.9% of the dry mass on day 7. Caffeic acid and CA contents remained rather low. Furthermore, the presence of RA, CA and caffeic acid and the expression patterns of RAS and hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HST), an important enzyme of monolignol formation, in leaves, flowers, stems and roots of naturally grown G. hederacea were assessed. The expression of RAS and HST genes was detectable in all organs except roots. Flowers accumulated 12.5% RA in their dry mass, leaves, stems and roots about 1%. CA was highest in leaves (2.0%), while it was at 1.6% in flowers, 1.3% in stems and almost undetectable in roots. The caffeic acid content remained at or below 0.4% of the dry weight in all organs.     

Establishment of Salvia officinalis L. hairy root cultures for the production of rosmarinic acid

Shoots of Salvia officinalis, a medicinally important plant, were infected with Agrobacterium rhizogenes strains ATCC 15834 and A4 which led to the induction of hairy roots in 57% and 37% of the explants, respectively. Seven lines of hairy roots were established in WP liquid medium under light and dark conditions. The transformed nature of the root lines was confirmed by polymerase chain reaction using rolB and rolC specific primers. Transformed root cultures of Salvia officinalis showed variations in biomass and rosmarinic acid production depending on the bacterial strain used for transformation and the root line analyzed. Both parameters (growth and rosmarinic acid content) of ATCC 15834-induced lines were significantly higher than the A4-induced lines. The maximum accumulation of rosmarinic acid (about 45 mg g(-1) of dry weight) was achieved by hairy root line 1 (HR-1) at the end of the culture period (45-50 days). The level was significantly higher than that found in untransformed root culture (19 mg g(-10 of dry wt).     

腐殖酸对丹参生长的促进作用及其机理的研究

本文旨在研究腐殖酸对丹参生长的促进作用及其机理以提高丹以的产量和品质。水培试验研究表明：20ppm－80ppm腐殖酸促进了丹参根干物质的积累,80ppm时达到了最大,丹参根重比对照增加了49％,其作用机理是：通过提高根系活力,增加根系吸收能力,促进谷氨酸合成酶的活性、提高氮同化的效率,促进丹参根系分泌酸性磷酸酯酶以及改变体内酸性和中性磷酸酯酶的活性,以充分利用内的磷而适应低磷环境。     

Establishment of Withania somnifera hairy root cultures for the production of withanolide A

Withanla sominifera (Indian ginseng) was transformed by Agrobacterlum rhizogenes.Explants from seedling roots,stems,hypocotyls,cotyledonary nodal segments,cotyledons and young leaves were inoculated with A.rhizogenes strain R1601.Hairy (transformed) roots were induced from cotyledons and leaf explants.The transgenic status of hairy roots was confirmed by polymerase chain reaction using nptll and roIB specific primers and,subsequently,by Southern analysis for the presence of nptll and roIB genes in the genomes of transformed roots.Four clones of hairy roots were established;these differed in their morphology.The doubling time of faster growing cultures was 8-14 d with a fivefold increase in biomass after 28 d compared with cultured,non-transformed seedling roots.MS-based liquid medium was superior for the growth of transformed roots compared with other culture media evaluated (SH,LS and N6),with MS-based medium supplemented with 40 g/L sucrose being optimal for biomass production.Cultured hairy roots synthesized withanolide A,a steroidal lactone of medicinal and therapeutic value.The concentration of withanolide A in transformed roots (157.4 μg/g dry weight) was 2.7-fold more than in non-transformed cultured roots (57.9 μg/g dry weight).     

Artemisinin production by transformed roots of Artemisia annua

Summary Transformed root cultures of several strains of Artemisia annua were obtained by infection with Agrobacterium rhizogenes ATCC 15834. Production of artemisinin, measured by HPLC, ranged from 0–0.42 % of dry weight (DW) in 10 different clones. Artemistene, artemisinic acid, and arteannuin B were also measured. Comparisons to literature reports suggest that the commercial production of artemisinic compounds using transformed roots is feasible.     

Diterpenoid production in hairy root culture of Salvia sclarea L

Growth and diterpenoid accumulation (salvipisone, ferruginol, aethiopinone and 1-oxoaethiopinone) during the growth cycle of a Salvia sclarea hairy root culture are described. The roots transformed by Agrobacterium rhizogenes (LBA 9402) were cultured in half-strength B5 liquid medium supplemented with 30 g L(-1) sucrose under light (16 h/8 h light/dark). A culture period of 30 days was optimal for both biomass and diterpenoid production. The total content of four diterpenoids in the hairy roots [(27.3 +/- 0.6) mg g(-1) dry weight] was higher than that of roots of field-grown S. sclarea plants [(3.15 +/- 0.15) mg g(-1) dry weight]. In transformed roots, aethiopinone was the main diterpenoid, whereas the principal diterpenoid of natural roots was salvipisone.     

The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza

Hairy root cultures of Salvia miltiorrhiza transformed with Agrobacterium rhizogenes ATCC 15834 produced a tiny amount of tanshinones and a constituent level of phenolic acids under normal growth conditions. Upon elicitation with yeast elicitor, the production of both phenolic acids and tanshinones was enhanced. For example, the contents of two phenolic acids, rosmarinic acid and lithospermic acid B were elevated from 1.24% and 2.59% to 2.89% and 2.98% of dry wt, respectively while the intracellular content of cryptotanshinone increased from 0.001% to as much as 0.096% of dry wt. Yeast elicitor also improved the growth of hairy roots (from 3.9 g/l to 7.3 g/l on a dry wt basis). Liquid chromatography-mass spectrometry (LC-MS) was developed for simultaneous detection and identification of phenolic acids and tanshinones in the extracts of S. miltiorrhiza. Rosmarinic acid, lithospermic acid B, cryptotanshinone, tanshinone I, tanshinone IIA and tanshinone IIB were identified by comparison with standards available. Dihydrotanshinone I and methylenetanshiquinone were tentatively identified by the molecular weights and the elution comparable with the literature. An unknown compound with a molecular weight of 280 was found in yeast-elicitor treated hairy root cultures, which was one of the major tanshinones induced.     

Synergistic effect of auxins and polyamines in hairy roots of <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. during growth,coumarin production and morphogenesis

Hairy roots of Cichorium intybus obtained by infecting with different Agrobacterium rhizogenes strains (LMG-150 and A20/83) were studied for total endogenous indole-3-acetic acid (IAA) levels and indole-3-acetic acid oxidase (IAAO) activity. The roots initiated by LMG- 150 showed higher endogenous IAA levels as well as IAAO activity as compared to the roots from A20/83. Coumarin production in roots obtained by both of these strains strictly correlated with growth, with higher content in the roots obtained by LMG- 150. Moreover roots from LMG-150 showed increased growth index, length of primary roots and number of secondary and tertiary roots. The roots derived from LMG-150 were studied for total endogenous IAA and IAAO activity under the exogenous administration of polyamines and fungal elicitors. The treatment with putrescine (Put) at 1.5 mM level showed maximum endogenous IAA levels and IAAO activity as compared to the control and other polyamine administration, it also supported faster growth in terms of biomass accumulation, and total coumarin production. Of the various treatments, mycelial extract (ME) and culture media filtrate (CMF) of Pythium aphanidermatum and Phytopthora parasitica var. nicotiana, the treatment with 1 % CMF of P. parasitica var. nicotiana, resulted in maximum IAA levels and IAAO activity, which was supported by maximum biomass, coumarin production as compared to the control and other elicitor treatments. Two different regenerants of chicory obtained through A. rhizogenes LMG-150 designated as T-I and T-II, were studied for total endogenous IAA levels and IAAO activity. T-II showed higher titers of IAA with higher activity of IAAO as compared to T-I. Endogenous titer of IAA and IAAO activity was found to be maximum in transformed roots as compared to T-I, T-II, normal roots and normal plants. Our work showed a variation in endogenous auxin levels in these transformed plants. There exists a synergistic effect of endogenous IAA titers and polyamines in regulating root morphogenesis. Fungal elicitors influenced growth and coumarin production and an elicitor preparation of 1 % CMF of P. parasitica var. nicotiana gave spontaneous regeneration of shoots. The implications of these results are discussed.     

松嫩平原不同株型玉米品种根系分布特征比较研究

采用土柱模拟栽培法与大田试验相结合的方法,对松嫩平原不同株型玉米的根系分布特征进行了比较。结果表明,平展型玉米和紧凑型玉米根干重最大值出现的时期不同,二者根干重分别在抽丝后15d和抽丝后30d时达到最大值,成熟时紧凑型玉米根干重比平展型高12．2％,二者根系垂直分布有明显的差异,在20cm以下的根干重比率,平展型玉米在19％以下,而紧凑型玉米高于23％,紧凑型玉米的深层根量较多,在深40～100cm土层内根干重比率比平展型高42．3％,二者的根系水平分布也不同,紧凑型玉米根系水平分布较集中,在距植株0～10cm水平范围内,根系分布比率比平展型玉米高9．6％,紧凑型玉米深层根量较多,水平分布集中,耐密植,是易获得高产的重要原因之一。     

间作对植株生长及养分吸收和根际环境的影响

通过盆栽实验研究了线辣椒和玉米间作对其植株生长、矿质养分吸收、根际环境以及铁载体分泌的影响,以探索间作促进铁、磷等养分吸收利用的可能生理机制.结果表明:(1)与单作相比,间作线辣椒地上部干重降低23.0%,根系干重增加44.2%,玉米地上部和根系的干重分别增加8.7%和22.9%;间作线辣椒根冠比和根系活力分别显著提高86.4%和29.8%;间作线辣椒、玉米叶绿素含量分别显著提高12.6%和7.8%.(2)与单作相比,间作线辣椒的铁、锌、锰含量分别增加1.50倍、1.39倍和1.34%,而间作玉米则无显著变化;间作线辣椒和玉米的钙含量都显著低于相应单作,氮含量没有显著变化,但磷、钾含量显著增加.(3)间作线辣椒和玉米的根际土、非根际土的酸性磷酸酶活性及根系酸性磷酸酶活性都显著高于相应单作,而其根际土和非根际土的pH值无显著变化;间作玉米根系的铁载体分泌比单作减少32.8%,间作线辣椒根系的铁还原酶活性是单作的1.10倍.研究发现,线辣椒/玉米间作能通过影响根际生物学特征和化学过程提高植株的铁、锌、磷和钾养分水平,缓解养分胁迫,是一种很有推广价值的种植模式.     

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of <Emphasis Type="Italic">Withania somnifera</Emphasis>

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the T_L T-DNA, and 90% of them carried the T_R T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the T_L and the T_R-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the T_R T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the T_L T-DNA genes, while all the callusing lines induced by strain A4 contained the T_L DNA. Four of these callus lines produced both withaferin A (0.15–0.21% dry weight) and withanolide D (0.08–0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.     

Inhibition of glutathione synthesis reduces chilling tolerance in maize

 The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine (γEC) synthetase, the first enzyme of GSH synthesis. At 25 °C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 °C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or γEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity. Received: 9 November 1999 / Accepted: 17 February 2000     

Influence of exogenous hormones on the growth and secondary metabolite formation in transformed root cultures

Transformed organ cultures formed following transformation of plant tissues with Agrobacterium species owe their phenotypes to alterations in hormone metabolism. Exogenously supplied hormones have been used to probe the relationship between the growth and morphology of transformed root cultures of a number of species and their ability to accumulate secondary products. Auxins in the presence of low levels of kinetin induce the rapid disorganisation of transformed roots of Nicotiana rustica ultimately toform suspension cultures of transformed cells and this process is associated with a decrease in nicotine content of the cells. This is related to cells in the culture losing competence in alkaloid biosynthesis. In contrast, exogenously supplied GA₃ enhanced branching in two transformed root clones of the tropane-alkaloid producing species, Brugmansia candida and so enhanced their typical hairy root phenotype. This growth substance had the effect of reducing the overall alkaloid accumulation but in one case significantly altered the relative concentrations of different tropine esters.In transformed roots of Cucumis sativus, the phenotype of the roots is influenced by the expression of auxin synthesis genes on T_R-DNA resulting in roots with two distinct morphologies. The pattern of expression of the enzyme ascorbate oxidase in populations of control roots of different morphologies is described. The significance of these phenotypic variations on the utility of transformed root cultures for the study of secondary metabolic pathways will be discussed.Abbreviations AO ascorbate oxidase - DW dry weight - FW fresh weight - GA₃ gibberellic acid     

Nucleic Acid Metabolism of Vicia faba during Germination and Growth

During the growth of Vicia faba seedlings in the absence of an external nitrogen supply, the cotyledons decreased rapidly in dry weight and nucleic acid content. In the developing shoot the dry weight increased rapidly for four weeks and then very slowly over the next two weeks growth; the nucleic acid content of the shoot increased to a maximum after 4 weeks growth and decreased in amount during the next 2 weeks. On the other hand the roots increased in both dry weight and nucleic acid content throughout the growth period, although they only accounted for a small proportion of the total dry weight and nucleic acid content of the plant. These changes during germination and growth are discussed in relation to those occurring during these developmental stages in other plants.     

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.     

Localization of newly synthesized indoIe-3-methylglucosinolate (= glucobrassicin) in vacuoles from horseradish (Armoracia rusticana)

The presence and concentration of indole-3-methylglucosinolate [= glucobrassicin; 0.49 μmol (g dry weight)-1] and its 1-methoxy derivative [0.38 umol (g dry weight)⁻¹] in Armoracia rusticana P. Gärtner, B. Meyer and Scherb, storage roots were measured. The storage tissue contains L-tryptophan [1.63 μmol (g dry weight)^-1], which is the common precursor amino acid of the indoleglucosinolates. Tissue cylinders convert [¹⁴C]- l -tryptophan efficiently to the indoleglucosinolates (25%) in vivo. The conversion of [¹⁴C]- l -tryptophan to indole-3-methylgluco-sinolate shows biphasic kinetics.
A fraction rich in vacuoles was prepared from tissue sections to which [¹⁴C]- l -tryp-tophan had been fed and allowed to be metabolized. The predominantly vacuolar location of both L-tryptophan and the indoleglucosinolates is demonstrated by correlation with the vacuolar marker acid phosphatase.
The significance for the regulation of the indoleglucosinolate biosynthesis and the role of indole-3-methylglucosinolate as a potential auxin precursor are discussed.     

Composition of essential oil in genetically transformed roots of <Emphasis Type="Italic">Ruta graveolens</Emphasis>

The composition of essential oil obtained by hydrodistillation from genetically transformed roots of common rue (Ruta graveolens L.) was analyzed. Using gas chromatography and complex gas chromatography-mass spectrometry, it was established that the major component of rue essential oil was a root-specific sesquiterpene geijerene comprising 67% of total amount of volatile compounds. In essential oil of cultured rue roots, furocoumarins characteristic of intact plant roots were found, viz. osthole, halepensin, and rutacultin. The content of essential oil in genetically transformed rue roots was 0.23% of root dry weight, which is comparable with that in the roots of intact plants. The long-term maintenance in the in vitro cultured rue roots of a capability for the synthesis of essential oil major components characteristic of intact plants allows their usage for studying the physiological activity of these volatile compounds and their putative role in the plant root interaction in biocenoses.     

新疆雪莲毛状根的诱导及其植株再生体系的建立

利用发根农杆菌R1601、R1000、LBA9402感染新疆雪莲的叶片、叶柄和根段外植体,诱导产生毛状根。毛状根接种量为2.8 g/L(FW)时,20d生长量可达66.7 g/L,黄酮含量达到干重的10.23%。冠瘿碱的检测和rolB基因的PCR分析表明,Ri质粒中的T_DNA片段已经整合到毛状根细胞的基因组中。预培养时间、外植体类型以及发根农杆菌的菌株属性对毛状根诱导有着重要的影响。其中预培养2 d的新疆雪莲根段外植体,经过R1601感染后,毛状根的诱导率可达100%。诱导产生的毛状根在附加生长素的液体培养基中,有少量愈伤组织产生。由毛状根再生的植株与雪莲外植体再生的植株在形态上无明显区别,但前者的黄酮含量仅为后者的53%。     

Enhanced production of phytoestrogenic isoflavones from hairy root cultures of Psoralea corylifolia L. Using elicitation and precursor feeding

The effect of biotic elicitors (yeast extract, chitosan), signaling molecule (salicylic acid), and polyamines (putrescine and spermidine) was studied with respect to isoflavones accumulation in hairy root cultures of Psoralea corylifolia L. Untreated hairy roots (control) accumulated 1.55% dry wt of daidzein and 0.19% dry wt of genistein. In precursor feeding experiment, phenylalanine at 2 mM concentration led to 1.3 fold higher production of daidzein (1.91% dry wt) and genistein (0.27% dry wt). In biotic elicitors, chitosan (2 mg/L) was found to be the most efficient elicitor to induce daidzein (2.78% dry wt) and genistein (0.279% dry wt) levels in hairy roots. Salicylic acid at 1 mM concentration stimulated the maximum accumulation of daidzein (2.2% dry wt) and genistein (0.228% dry wt) 2 days after elicitation. In case of polyamines, putrescine (50 mM) resulted in highest accumulation of daidzein (3.01% dry wt) and genistein (0.227% dry wt) after 5 days of addition. Present results indicated the effectiveness of elicitation and precursor feeding on isoflavones accumulation in hairy roots of P. corylifolia. This is the first report of elicitation on isoflavones production by hairy roots of P. corylifolia.     

Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogenes

Hairy root culture of Ginseng (Panax ginseng) was established after roots were induced on callus following infection with Agrobacterium rhizogenes. The transformed cultures of ginseng could be subcultured as an axenic root culture in the absence of phytohormones, and grew with extensive lateral branches more rapidly than the ordinary cultured roots induced by hormonal control from ginseng callus. The hairy roots synthesized the same saponins, ginsenosides, as those of the native root, up to about 2.4 times in the quantity, and up to about 2 times in comparison with that of the ordinary cultured roots, on dry weight basis.Abbreviations Ms medium Murashige and Skoog's medium - 2,4-D 2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - K kinetin This paper is Part 52 in the series "Studies on Plant Tissue Culture". For Part 51, see Ayabe S, Udagawa A, Furuya T (1987).