当前位置:首页 >> 农学 >>

Anthocyanins in Catharanthus roseus in vivo and in vitro


Phytochem Rev (2007) 6:235–242 DOI 10.1007/s11101-006-9052-y

Anthocyanins in Catharanthus roseus in vivo and in vitro: a review
Anna Piovan ? Raffaella Filippini

Received: 13 July 2005 / Accepted: 30 November 2006 / Published online: 6 March 2007 ? Springer Science+Business Media B.V. 2007

Abstract The production of anthocyanin in Catharanthus roseus ?owers from both ?eldgrown and regenerated by somatic embryogenesis plants and cell cultures was described. The anthocyanins were identi?ed as the 3-O-glucosides, and the 3-O-(6-O-p-coumaroyl) glucosides of hirsutidin, malvidin and petunidin, respectively both in vivo and in vitro. The in?uence of environmental conditions on in vitro anthocyanin accumulation is described. The relationship between in vivo and in vitro anthocyanin production is discussed. Keywords Anthocyanins ? Catharanthus roseus ? Flavonol O-methyltransferase ? Plant cell cultures ? Somatic embryogenesis

Introduction Catharanthus roseus (L.) G. Don (Apocynaceae) is one of the most important medicinal plant, being a valuable source of the antitumour agents

A. Piovan ? R. Filippini (&) Department of Biology, University of Padua, Via U. Bassi, 58/B, Padua 35131, Italy e-mail: raffaella.?lippini@unipd.it

vinblastine and vincristine, used in chemotherapy of leukemia and treatment of Hodgkin’s disease. C. roseus is also a popular ornamental plant with pink or white ?owers. In the past few decades a large number of publications have covered the improving knowledge on the antitumour alkaloids of Catharanthus. Many reports in the literature detail the effects of modi?cations to culture conditions upon the yield of valuable secondary metabolites in cultured cells (van der Heijden et al. 2004). On the contrary, the anthocyanic pigments production in vivo and in vitro of this species have attracted little attention. The term anthocyanin, initially coined to designate the substance responsible for the color of the corn?ower (from the greek anthos, ?ower and kuanos, blue) applies to a group of water soluble pigments responsible for the red, pink, mauve, purple, blue, or violet color of most ?owers and fruits. These pigments occur as glycosides (anthocyanins), and their aglycones (anthocyanidins) are derived from the 2-phenylbenzopyrylium cation, more commonly referred to as the ?avylium cation, a name that emphasizes the fact that these molecules belong to the vast group of ?avonoids in the broad sense of the term (Bruneton 1999). This review gives an overview about the production of anthocyanins in the ?owers both of C. roseus ?eld-grown and regenerated by

123

236

Phytochem Rev (2007) 6:235–242

somatic embryogenesis plants, and C. roseus in vitro cell cultures.

Anthocyanins in ?eld-grown plants Three basic corolla color patterns are known in C. roseus, white, pink and red-eyed (Flory 1944; Morton 1977). The partial identi?cation of the pigments extracted from the pink ?owers revealed that hirsutidin was the major anthocyanin and that malvidin and petunidin were also present in small amounts (Forsyth and Simmonds 1957), and the sugar residues revealed only the presence of glucose. The exact position(s) of attachment of the glucose of the anthocyanins was not determined (Carew and Krueger 1976). Flory (1944) studied at ?rst the genetic of C. roseus ?ower color and proposed a two-gene model. In a successive study Milo et al. (1985) studied the inheritance of corolla color and the pigments involved in three unrelated pure lines with different ?ower colors, and elaborated a more comprehensive genetic model. Chemical analysis of the ?ower pigments revealed the presence of the three anthocyanidins previously identi?ed, petunidin, malvidin and hirsutidin. Each was present in two different glycosides, and their amounts varied with the corolla color patterns. The total and relative quantities of the pigments varied in the different ?owers types (Table 1). In the red-eyed line, three different anthocyanins were obtained: A1, B1 and C1. These pigments were found also in all the phenotypes with a pink center in the corolla. Three additional anthocyanins, A2, B2 and C2, were recovered in the pink materials. No ?owers were found with A2, B2 and C2 pigments only. There were no phenotypic differences in the corolla color between the
Table 1 The distribution of six anthocyanins and their relative total amount in the parental lines and their F1 hybrids (Milo et al. 1985) Lines

reciprocal hybrids. The phenotypes of the three parents and their hybrids were very easily distinguished visually and by their pigment patterns. The results suggest that corolla color patterns in C. roseus are controlled by three interacting genes, while in Flory’s work (1944) a two gene model was described. The third was found in this study, because additional phenotypes were considered. In 1998 we identi?ed by ESI–MS/MS the pigments of C. roseus pink ?owers as the 3-Oglucoside, and the three 3-O-(6-O-p-coumaroyl) glucosides of hirsutidin, malvidin, and petunidin respectively (Piovan et al. 1998). Vimala and Jain (2001) con?rmed by HPLC– ESI–MS the presence in intact mature ?ower petals of hirsutidin, petunidin and malvidin. They detected several m/z peaks corresponding to anthocyanidins in trihydrate and pentahydrate forms and a molecular ion peak indicating malvidin with a possible pentose glycone moiety. Anthocyanins in plants regenerated by somatic embryogenesis We have previously described the achievement of somatic embryogenesis from C. roseus cell line V17 (Piovan et al. 2000). The analysis of the regenerated plant ?owers showed the same anthocyanins identi?ed in pink ?owers of C. roseus ?eldgrown plants (1–6) (Fig. 1) (Piovan et al. 1998).

Anthocyanins in plant cell cultures Plant cell and tissue are used as experimental tools for studying plant cell metabolism and selecting a superior cell line to produce valuable metabolites. However, also non-selected cells
HPLC peak, %a A1 A2 B1 B2 C1 C2 Relative pigment amount 100 – 4 51 12 59

Corolla color

a

A1, A2 = petunidin; B1, B2 = malvidin; C1, C2 = hirsutidin

Parents Pink White Red-eyed Hybrids Pink · white Red-eyed · white Pink · red-eyed

Pink White White with a eye red Light pink Pale, pink center Light pink

0.3 – 7.6 0.3 7.4 0.3

9.0 – – 8.7 0.3 7.5

0.3 – 22.1 0.6 21.3 1.9

11.0 – – 8.3 6.0 7.3

0.9 – 70.3 2.4 52.3 4.8

78.5 – – 79.7 12.7 78.2

123

Phytochem Rev (2007) 6:235–242
R OH R'' + O R' O OH OH OH O OH OR'''

237

R 1 2 3 4 5 6 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3

R’ OH OCH3 OCH3 OH OCH3 OCH3

R’’ OH OH OCH3 OH OH OCH3

R’’’ H H H p-coumaroyl p-coumaroyl p-coumaroyl

Fig. 1 Anthocyanins found in Catharanthus roseus

have showed unpredicted and interesting biosynthetic capability: anthocyanins in C. roseus cell cultures. The ?rst work on anthocyanins in callus cultures of C. roseus reports that the line Wc13S, which had been continuously subcultured in the dark since 1961, in 1972 was placed under 2150 lx continuous cool ray ?uorescent light and the cells became increasingly red. Peak pigment production occurred at approximately 21 days after inoculation after which the pigments were seen to degrade. The rate of pigment accumulation increased with light intensity and by addition of either phenylalanine or trans-cinnamic acid into the medium. Pigment biosynthesis was inhibited in the dark and by medium sucrose concentration exceeding 2%. The anthocyanidins isolated from the callus tissue were identi?ed as petunidin, malvidin and hirsutidin; the analysis of the sugar residues revealed only the presence of glucose (Carew and Krueger 1976). In a study on the in?uence of environmental conditions on the regulation of secondary metabolism in C. roseus cell culture, Knobloch et al. (1982) observed that after transfer of cell suspension cultures of C. roseus, grown in Murashige and Skoog (MS) medium containing 2 · 10–6 M of 2,4-dichlorophenoxyacetic acid (2,4-D), into a

10-fold volume of an 8% aqueous sucrose solution and upon continuous irradiation with ?uorescent lamps, the cultures turned red due to the formation of anthocyanins. The authors had not previously found these pigments in dark-grown cultures. The formation of anthocyanins during illumination was easily observed to occur 4–7 days after cell transfer. The anthocyanins were accumulated within the vacuolar space of the cells, whereas no signi?cant amounts were found in the medium; the culture was highly heterogeneous with regard to the accumulation of pigments. After irradiation, only few cells (ca. 5%) showed a high content of anthocyanins, whereas the majority had only a slight or no red color. The mean value for the intracellular concentration of anthocyanins was 0.6 mM, whereas the concentration of a single, high producing cell was estimated 2.8 mM. Highest accumulation of anthocyanins was observed in the absence of phosphate and mineral nitrogen. The addition of phosphate or KNO3 and NH4NO3 inhibited the anthocyanin accumulation by 90%. In this experiment it was found that raising the sucrose concentration from 2 to 11% there was a parallel increase of anthocyanins by more than 300%. The chemical analysis of the cell cultures showed the presence of the same anthocyanidins (petunidin, malvidin and hirsutidin) identi?ed previously in callus tissue (Carew and Krueger 1976) and in intact ?owers (Forsyth and Simmonds 1957). The relative amounts, however, differed in ?owers and cell suspensions: hirsutidin was the most prominent pigment in petals, whereas the extracts from cell suspensions contained mainly the less methylated compounds petunidin and malvidin. These results indicate that in C. roseus cell cultures the accumulation of anthocyanins are dependent upon the environmental conditions (phosphate, mineral nitrogen, phenylalanine, trans-cinnamic acid, sucrose) and that light is an essential stimulus. Signi?cantly, however, resulted that even in the continuous presence of light, the accumulation of anthocyanins by cell cultures was distinctly discontinuous, and to different extent, if at all, by cells within a plant tissue culture. Hall and Yeoman (1986a) performed a study directly on the variation present in tissue cultures

123

238

Phytochem Rev (2007) 6:235–242

of C. roseus in relation to the accumulation of anthocyanin pigments. A detailed analysis of the growth and accumulation of anthocyanins throughout one complete culture cycle in cell cultures of C. roseus was performed. The results revealed a distinct pattern of temporal heterogeneity with respect to the accumulation of these pigments. The cell cultures were exposed to light immediately following initiation from a stock culture, which had previously been grown in darkness. The accumulation of anthocyanins was not detectable until the tenth day of culture, after which levels increased steadily. In these cultures the proportion of visibly pigmented cells was observed to reach low plateau level (10%) on day 16. The same experiment was repeated using cultures, which had previously been grown in the light for over 3 months and thus already pigmented. The results indicated that once again anthocyanin accumulations was delayed for a period coincident with the lag and cell division phases of the culture growth cycle. During this time both the percentage and total number of pigmented cells decreased, reaching minimum values on day 8 by which time the cultures had become almost pure white in color. Thereafter anthocyanins accumulated rapidly, the cultures once again became visibly pigmented and by day 16 maximum levels had been obtained. It was observed that the productive cell population constituted less than 10% of the total (microscopic and microdensitometric examinations). The pigmented cell population consisted primarily of cells with magenta vacuoles but also included a small number which had vacuoles which were more deeply pigmented and appeared

dark purple in color. A similar analysis performed on pigmented cells from mature whole plant tissues taken from both vegetative and reproductive organs revealed that these cells all had closely qualitative characteristics (kmax values), irrespective of the tissue of origin and all were magenta in color. Further analysis (TLC) indicated that despite the visible differences between cells, no qualitative difference in the anthocyanin content was apparent between any of the tissue isolated. Three glycosides of hirsutidin, petunidin and malvidin were detected in all cases. The quantitative analysis revealed considerable differences between the mean intracellular anthocyanin content (Table 2). The content was four times greater at the mouth of the perianth tube in comparison to the rest of the petal lamina. The equivalent value for the somatic tissue was greater still but it was, to a considerable extent, due to the much larger mean cell size for this tissue. The mean cellular anthocyanin concentration values indicated that cells of the petal located at the top of the perianth tube contained the greatest concentration of pigment. Comparison of the data obtained from the intact plant with those from cell cultures revealed that both the mean intracellular anthocyanin content and concentration values for the cultured cells fell within the limits of those observed for whole plant tissues. However, much in contrast to the data for the in vitro system, the range of values observed within speci?c whole plant tissues was considerably restricted and was, in the case of intracellular anthocyanin content (IOD) values, more than one order of magnitude less in all cases.

Table 2 Microdensitometric analysis of individual cells of C. roseus obtained from whole-plant tissues and illuminated, stationary phase suspension cultures (Hall and Yeoman 1986a) Tissue source IOD Range Volume Range IOD/volume Range Somatic tissue 508.1 ± 45.6 272–1107 (4·) 1718 ± 152 342–2582 (8·) 0.333 ± 0.021 0.191–0.686 (4·) Petal lamina 66.6 ± 3.7 47.6–110 (2·) 304.2 ± 28.4 148–463 (3·) 0.227 ± 0.015 0.157–0.322 (2·) Perianth tube 265.4 ± 32.3 164.4–576 (3.5·) 599.1 ± 60.5 227–1130 (5·) 0.440 ± 0.035 0.235–0.677 (3·) Cell culture (14-day-old) 182.6 ± 12.72 14.3–615 (43·) 798.0 ± 53 103–2184 (21·) 0.261 ± 0.018 0.112–0.899 (8·)

Mean values (±SE) for the intracellular anthocyanin content (IOD) and concentration (IOD/volume) are presented with their corresponding minimum and maximum values. One hundred cells were examined for each tissue. All values in machine units

123

Phytochem Rev (2007) 6:235–242

239

Comparison of the concentration data produced a similar result although the in vivo/in vitro differences were reduced. The range of concentration values for the cultured cells appeared to be extended, in comparison with the overall equivalent for plant tissues, not only at the lower end of the scale but also at the upper end. Two important points emerge from this study: in the cultures used only a very small proportion of cell population accumulated signi?cant amounts of the secondary metabolites and within this productive population extensive variation in the intracellular anthocyanin content occurred. It is interesting to note that the properties of the cell cultures studied were very much in contrast to those of whole plant. This indicates the precise degree of intracellular control of anthocyanin accumulation which must operate within the whole plant. The level at which the control operates is variable and apparently tissue-speci?c. In a successive work Hall and Yeoman (1986b) studied the in?uence of chemical and physical factors on anthocyanin accumulation in these cultures. High levels of 2,4-D (20 lM) proved to reduce anthocyanin accumulation in comparison to the control (2 lM 2,4-D). This was more or less entirely due to a decrease in the proportion of anthocyanin-accumulating cells as the estimated concentration of anthocyanin in the productive cell population remained relatively constant. On medium depleted of iP and iN sources together a doubling in fresh weight was observed which was entirely due to a doubling of the mean cell volume. In comparison to the original inoculum an approx. 4-fold increasing in total anthocyanin yield was evident. This was found to have resulted from the 2-fold increase in cell size and an approx. 2-fold increase in the proportion of visibly pigmented cells. The estimated mean anthocyanin concentration within the pigmented cells of the treated culture was not signi?cantly different from either the original inoculum or control values. In an experiment in which callus cultures were grown at one of two different light levels, it was determined that increased irradiance brought about an approximate doubling in the proportion of pigmented cells in association with an 80%

increase in the mean intracellular anthocyanin content. However, due to an increase in mean cell size only an approx. 30% increase in the mean intracellular anthocyanin concentration occurred. The same authors (Hall and Yeoman 1987) in order to test the stability of the cell lines re-examined 26 cell lines of C. roseus over 6 months after the ?rst analysis. In the cell lines, the difference observed in secondary metabolite yield were, once again, primarily the result of differences in the proportion of the pigmented cells. The levels of anthocyanin accumulated per culture in these cell lines varied by more than 30-fold. Determination of the proportion of pigmented cells in these cultures also revealed approx. 30-fold difference in response. Despite these variations only an approx. 2-fold variation in the mean intracellular anthocyanin concentration was found. No differences in culture morphology were apparent between any of the 26 cell lines and each produced the same three anthocyanins as the stock culture and in similar proportions. This study shows that even though the stock culture consistently contained both non-productive and productive cell populations (9:1 ratio), all of the cell lines were capable of anthocyanin synthesis, albeit with reproducibility different capacities for accumulation. This suggests that the stock culture did not contain two distinctly different cell types (producing and not producing anthocyanins) characterized to be unstable and easily interconvertible. This work con?rms again the critical importance of the size of the productive cell population in determining the anthocyanin yield in the culture, being yield changes exclusively the result of an increase/reduction in the size of the productive cell population. Berglund et al. (1993a, b) studied the in?uence of nicotinamide (NIC) exposure on anthocyanin production in an anthocyanin accumulating C. roseus tissue culture. NIC exerted a positive effect on the glutathione (GSH) accumulation. GSH is known to have several functions in plants, a major possible involvement being protection against oxidative biotic and abiotic stress (irradiation) by inducing the activity of defence-related genes coding for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). PAL and CHS are involved in ?avonoid and iso?avonoid

123

240

Phytochem Rev (2007) 6:235–242

phytoalexin synthesis. Plant anthocyanins serve as signals to pollinators and plant feeding animals. It has been suggested that anthocyanin accumulation can be induced by stress and that anthocyanins show UV-B protecting effects. NIC causes a pronounced increase in the GSH level of the anthocyanin accumulating tissue culture. This effect of NIC was concentration dependent with a maximum at a NIC concentration of 8.2 mM. The glutathione content increased with time after NIC addition. The increases were observed in GSHtot, GSH and GSSG (oxidized form of glutathione) levels. After NIC treatment of the culture on day 9, anthocyanin content increased in a manner similar to that of glutathione and reached the maximum (180% of their respective controls NIC absent) at day 19. The glutathione biosynthesis inhibitor buthioninesulfoximine (BSO) counteracted the stimulatory effect of NIC on the anthocyanin accumulation. This indicates that the effect of NIC on anthocyanin accumulation may depend on glutathione metabolism. ? ndez and Loyola-Vargas (1997) Godoy-Herna examined the effect of acetylsalicylic acid (ASA) on secondary metabolism in a C. roseus tumor cell line cultivated in vitro in the absence of growth regulators and with corner starch as a carbon source under continuous light. At 20 mM ASA an increase of 1476% total anthocyanins was detected. The addition of a Aspergillus fumigatus homogenates increased the anthocyanins more than 3-fold with respect to the control. However, when it was added in combination with ASA the effect diminished by almost 50%. The addition of trans-cinnamic acid also increased the anthocyanin content and did not affect the increase produced by ASA. Results suggest that ASA may act as modulator of phenylpropanoid metabolism. The role of ASA on anthocyanin production in C. roseus transformed cell suspension cultures is unknown, it was hypothesized that ASA could be activating the key enzyme (CHS) in the anthocyanin biosynthetic pathway. We have previously characterized by ESI–MS/ MS the anthocyanins of V31R cell suspension culture accumulating anthocyanins. The extract investigated showed the same qualitative anthocyanic pro?le of ?owers from both ?eld-grown

and in vitro regenerated plants (Piovan at al. 1998). Ohlsson and Berglund (2001) studied the in?uence of gibberellic acid (GA3) on glutathione metabolism in anthocyanin accumulating cell culture of C. roseus (CR19 PINK). While treating this culture with GA3 they observed an increase in the pigmentation. The anthocyanin content and PAL activity in the culture were determined. GA3 caused a dose-dependent decrease in the level of oxidized glutathione (GSSG) in CR19 PINK cell line. Glutathione reductase (GR) activity was enhanced by GA3 addition, to a maximal activity of 135% of the control in CR19 PINK and the content of anthocyanins was increased about 137% of the control. PAL activity was considerably higher in the cultures treated with GA3 compared to untreated cultures: GA3 increased the PAL activity to almost 500% of the control. GA3 caused a decrease in the GSSG content along with an increased GR activity and consequently a decreased GSSG/GSH ratio in CR19 PINK. The strong increase in PAL activity observed in this study probably contributes to the increase in anthocyanin accumulation observed. The increased accumulation of anthocyanins observed after GA3 treatment may depend on increased synthesis, re?ected in increased PAL activity and improved transport to the vacuole by the GSH pump, as the glutathione pool becomes more reduced. In a successive study (Filippini et al. 2003) we obtained a V32R cell strain, derived from calli V17, the same from which derived the embryogenic cell line, having high and stable anthocyanin production. This cell line was obtained by subculturing alternatively in MS hormone-containing and MS hormone-free medium and by continuous pigmented cell-aggregate selection. Callus capability to produce anthocyanic pigments was maintained only when subcultured alternatively in the above mentioned media. The suspension cultures, established in the same conditions from calli V32R, maintained the anthocyanin productivity after 15 months of subculture. We studied the growth, anthocyanin production and percentage of visibly pigmented cells of V32R cell suspension. After a 2-day lag phase following transfer to fresh medium, the cells grew rapidly and attained

123

Phytochem Rev (2007) 6:235–242

241

the stationary phase after 1 week. Anthocyanin production was associated with the second half of log phase and reached a maximum in the stationary phase, after 10 days from transfer. The proportion of pigmented cells was approximately 30% and the percentage was rather constant during the growth cell cycle. We observed color differences in the pigmented cells, indicating that within this productive population extensive variation in the biosynthetic expression occurred, as observed by Hall and Yeoman (1986b). Delaying medium replacement, in the last stationary phase, a cell browning occurred; this probably resulted from an increased vacuolar sap pH, perhaps as a consequence of the cell senescence, causing a rapid decrease both in the anthocyanin content and percentage of pigmented cells. V32R supension cultures were maintained by several subcultures and in order to test the cell line stability, 10 months after the ?rst analysis the cell suspension was re-examined. The results were superimposable on those described above and indicated that no changes in the ability of the cells to accumulate anthocyanin occurred. Furthermore we analyzed the extracts of the stable cell line V32R and compared with those of ?owers both from regenerated and ?eld-grown plants, in order to evaluate the total and the relative contents of anthocyanins. In Table 3 the total anthocyanin contents and the relative abundance of the molecular ions of the six glucosides detected in the ESI mass

Table 3 Anthocyanin contents and the relative abundance of the molecular ions of the six glucosides (standard deviations less than 3%) (Filippini et al. 2003) Anthocyanin Pigmented Flowers of Flowers of ?eldregenerated grown plants cells (M+ rel. ab. %) plants (M+ (M+ rel. ab. %) rel. ab. %) 1 2 3 4 5 6 Totala
a

1.2 3.2 4.3 6.5 18.7 66.1 213

0.8 2.1 12 2.7 12.4 70 324

3.5 1.5 28 11.5 6.2 49.3 340

Total anthocyanin concentration is in lg per g fr. wt

spectrum of the anthocyanin extracts, are quoted. The anthocyanin contents in the ?owers from both plant types were comparable and about 1.5 times higher than that obtained from cell suspensions. In all the analyzed samples, hirsutidin 3-O-(6-O-p-coumaroyl)glucoside (6) was the major pigment; the 3-O-(6-O-p-coumaroyl)glucosides (4,5,6) were more abundant than the simple glucosides (1,2,3). The extracts from both embryogenic plant ?owers and cell cultures contained the malvidin derivatives (2,5) in higher amount than the petunidin ones (1,4). On the other hand, in the ?owers from ?eld-grown plants anthocyanins of the less methylated aglycone petunidin were more abundant. It is to note that although petunidin and malvidin acylated with p-coumaroyl glucoside are present in nature, the corresponding hirsutidin derivative has been detected only in C. roseus. Our results con?rm that the C. roseus anthocyanin biosynthesis enzymes are expressed both in vivo and in vitro. Cell line selection yields a cell suspension which exhibits an increased proportion of productive cells (30% pigmented cells) compared to the results of Hall and Yeoman (1987) (10% pigmented cells). However, the differentiated petal cells showed a higher capacity for the metabolite accumulation than the undifferentiated suspension cells. In this case, the results indicate that the changes in the ability of cells to accumulate anthocyanin involve no change in the genetic information of the cells but perhaps modi?cations on the control mechanism determining intracellular accumulation. Interestingly, Cacace et al. (2003) evidentiated a ?avonol O-methyltransferase that was expressed in C. roseus dark-grown cell suspensions. Its presence was unexpected because these suspension cultures do not synthesize ?avonoids in the dark (Knobloch et al. 1982), and it is also known that the expression of the key enzyme in the precursor formation, CHS, is strictly lightdependent (Kaltenbach et al. 1999). The enzyme represented a new type of O-methyltransferase that performs two sequential methylations at the 3?- and 5?-positions of the B-ring in myricetin and dihydromyricetin. The resulting methylation pattern is characteristic for C. roseus anthocyanins,

123

242

Phytochem Rev (2007) 6:235–242 ? ndez G, Loyola-Vargas VM (1997) Effect of Godoy-Herna acetylsalicylic acid on secondary metabolism of Catharanthus roseus tumor suspension cultures. Plant Cell Rep 16:287–290 Hall RD, Yeoman MM (1986a) Temporal and spatial heterogeneity in the accumulation of anthocyanins in cell cultures of Catharanthus roseus (L.) G. Don. J Exp Bot 37(174):48–60 Hall RD, Yeoman MM (1986b) Factors determining anthocyanin yield in cell cultures of Catharanthus roseus (L.) G. Don. New Phytol 103:33–43 Hall RD, Yeoman MM (1987) Intercellular and intercultural heterogeneity in secondary metabolite accumulation in cultures of Catharanthus roseus following cell line selection. J Exp Bot 38(193):1391–1398 ¨ der G, Schmelzer E, Lutz V, Schro ¨Kaltenbach M, Schro der J (1999) Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties, and cell-type speci?c expression in plants. Plant J 19:183–193 Knobloch KH, Bast G, Berlin J (1982) Medium- and lightinduced formation of serpentine and anthocyanins in cell suspension cultures of Catharanthus roseus. Phytochemistry 21(3):591–594 Milo J, Levy A, Akavia N, Ashri A, Palevitch D (1985) Inheritance of corolla color and anthocyanin pigments in periwinkle (Catharanthus roseus [L.] G. Don). Z ¨ chtg 95:352–360 P?anzenzu Morton JF (1977) Periwinkle. In: Thomas CC (ed) Major medicinal plants—botany, culture and uses. Spring?eld, USA, pp 237–241 Ohlsson AB, Berglund T (2001) Gibberellic acid-induced changes in glutathione metabolism and anthocyanin content in plant tissue. Plant Cell Tiss Org Cult 64:77–80 Piovan A, Filippini R, Favretto D (1998) Characterization of the anthocyanins of Catharanthus roseus (L.) G. Don in vivo and in vitro by electrospray ionisation ion trap mass spectrometry. Rapid Commun Mass Spectrom 12:361–367 Piovan A, Filippini R, Caniato R, Dalla Vecchia F, Innocenti G, Cappelletti EM, Puricelli L (2000) Somatic embryogenesis and indole alkaloid production in Catharanthus roseus. Plant Biosyst 134(2):179– 184 van der Heijden R, Jacobs DI, Snoeijer W, Hallared D, Verpoorte R (2004) The Catharanthus alkaloids: pharmacognosy and biotechnology. Current Med Chem 11(5):607–628 Vimala Y, Jain R (2001) A new ?avone in mature Catharanthus roseus petals. Indian J Plant Physiol 6(2):187–189

petunidin and malvidin. The enzyme for the methylation in position 7 of the A-ring in hirsutidin has not yet been identi?ed.

Conclusion Catharanthus roseus ?owers and cell cultures have been shown to accumulate anthocyanins in the same chemical types. The differentiated petal cells showed a higher capacity for the metabolite accumulation than the undifferentiated cell suspension cells. Many factors contribute to the in vitro anthocyanin production. A temporal and spatial heterogeneity in the accumulation of antocyanins were evidentiated in cell cultures.
Acknowledgment The authors gratefully acknowledge Professor Verpoorte for his consideration.

References
¨ m J (1993a) NicotinBerglund T, Ohlsson AB, Rydstro amide increases glutathione and anthocyanin in tissue culture of Catharanthus roseus. J Plant Physiol 141:596–600 ¨ m J, Ohlsson Berglund T, Strid A, Naaranlahti T, Rydstro AB (1993b) Nicotinamide induces defence-related and/or secondary metabolism in plant tissue cultures of Catharanthus roseus and Pisum sativum. Planta Med 59(suppl):A660–A661 Bruneton J (1999) Anthocyanins. In: Pharmacognosy—phytochemistry medicinal plants, 2nd ed. Intercepts, London, p 355 ¨ der G, Wehinger E, Strack D, Schmidt J, Cacace S, Schro ¨ der J (2003) A ?avonol O-methyltransferase Schro from Catharanthus roseus performing two sequential methylations. Phytochemistry 62:127–137 Carew DP, Krueger RJ (1976) Anthocyanidins of Catharanthus roseus callus cultures. Phytochemistry 15:442 Filippini R, Caniato R, Piovan A, Cappelletti EM (2003) Production of anthocyanins by Catharanthus roseus. Fitoterapia 74:62–67 Flory WS Jr (1944) Inheritance studies of ?ower color in periwinkle. Proc Am Soc Hort Sci 44:525–526 Forsyth WGC, Simmonds NW (1957) Anthocyanidins of Lochnera rosea. Nature 180:247

123


赞助商链接
相关文章:
更多相关标签: