激素等外源物质对马铃薯愈伤组织花色苷积累的影响

从来源于马铃薯(Solanum tuberosum cv.Chieftain)茎的愈伤组织中分离到白色和红色2种愈伤组织,用鲜重法和分光光度法分别测量愈伤组织的生长量和花色苷的含量,并对激素、抗菌素和糖对马铃薯愈伤组织生长和花色苷积累的影响进行研究。结果表明:低浓度的2,4-D有利于红色愈伤组织的花色苷积累,高浓度的2,4-D促进其生长而不利于花色苷的积累;高浓度的6-BA能促进红色愈伤组织中花色苷的积累并诱导白色愈伤组织花色苷的合成,但抑制其生长;卡那霉素能使白色愈伤组织变红并积累花色苷,高浓度的卡那霉素严重抑制愈伤组织的生长并最终变褐死亡;提高蔗糖浓度能促进愈伤组织花色苷的产生和积累,但超过70g/L时抑制生长。实验结果为今后花色苷生物合成机理研究和花色苷的工厂化生产奠定了基础。     

Influence of salicylic acid and methyl jasmonate elicitation on anthocyanin production in callus cultures of Rosa hybrida L.

This study was undertaken to investigate the effects of salicylic acid (SA) and methyl jasmonate (MeJA) on anthocyanin induction, biomass accumulation, and color value (CV) indices for both pigment content (PC) and pigment production (PP) in callus cultures of Rosa hybrida cv. Pusa Ajay. A concentration-dependent response was exhibited by cultures on SA and MeJA at different concentrations individually or in combinations to Euphorbia millii medium supplemented with 204.5 mM sucrose, 2.45 μM indole butyric acid and 2.33 μM kinetin. There was positive influence on both callus biomass and anthocyanin accumulation. Treatment with 0.5 μM MeJA was most effective in inducing anthocyanin biosynthesis in callus cultures. Anthocyanin accumulation in callus cultures was enhanced with the addition of SA and MeJA, but these did not differ significantly from control for the number of days required for pigment initiation and for color intensification. Moreover, the addition of 0.5 μM MeJA alone resulted in a higher frequency of color response (97.25 %), PC (3.48 ± 0.07 CV g^?1 FW), and PP (1.56 ± 0.03 CV test tube^?1) over control. In contrast, the presence of higher levels of SA (400 μM) and MeJA (5.0 μM) reduced frequency of color response, as well as levels of PC and PP. MeJA did not increase biomass accumulation but promoted frequency of color response, PC and PP. Hence, it was suggested that 0.5 μM MeJA promoted anthocyanin production in rose callus cultures. Significant correlation was found between frequency of response and each of the PC (r = 0.988) and PP (r = 0.990). Furthermore, PC and PP were also highly correlated (r = 0.998).     

Cell cultures of Centaurea cyanus produce malonated anthocyanin in UV light

Callus cultures which produce anthocyanin under continuous irradiation of UV and white light were derived from the stem of blue-floweredCentaurea cyanus. From the callus a suspension culture, in which anthocyanin synthesis can be induced by UV light, was obtained. The pigment in the cell cultures was identified as cyanidin 3-(6″-malonylglucoside) which occurs in the leaf, but not the flowers, of the parent plant.     

The role of calcium channels in anthocyanin production in callus cultures of Daucus carota

The involvement of Ca²⁺ ATPases in anthocyanin accumulation in callus cultures of Daucus carota was investigated under the influence of calcium and calcium channel modulators. Ionophore (I) treatment enhanced callus growth and anthocyanin accumulation. Increasing the amount of calcium applied to cultures enhanced the anthocyanin level. Ionophore treatment influenced the enhancement of Ca²⁺ATPase and endogenous titres of PAs. Addition of the calcium channel blocker verapamil or the calmodulin antagonist chlorpromazine to the A23187 (ionophore) treated cells caused a reduction in anthocyanin levels. Channel blockers reduced Ca²⁺ATPase activity, which was restored by ionophore treatment, showing the importance of calcium in anthocyanin production. Higher ethylene levels were also found in treatment with ionophore or 2X calcium. Thus the influence of ionophore in anthocyanin production and its inhibition by calcium channel modulators suggests that calcium plays an important role in the production of anthocyanin by carrot callus cultures.     

Isolation and Identification of an Anthocyanin from the Cell Suspension Culture of Poplar

During the course of the induction of many species of plant callus, it was found that callus of poplar, induced on the agar medium containing 0.5 mg per liter of 2,4-dichlorophenoxy acetic acid (2,4-D), upon exposure to white light produces an anthocyanin in its cell. The callus was inoculated into submerged cultures under light on a reciprocal shaker, and the pigment produced was isolated as pure crystals.

By the use of IR spectrum, UV spectrum and other physical and chemical methods, the anthocyanin was identified as cyanidin-3-monoglucoside (chrysanthemin).     

Genetically controlled anthocyanin synthesis in cell cultures of Matthiola incana

Callus cultures were derived from different parts of 8 anthocyanin producing and 2 white flowering lines of the crucifer Matthiola incana. The tissue cultures of the cyanic lines were shown to produce genotype specific anthocyanin patterns, whereas in the calli of the acyanic lines no anthocyanin synthesis occured.Abbreviations IAA indoleacetic acid - 2,4-D dichlorophenoxyacetic acid - MeOH methanol - Et₂O ether - ETOAc ethylacetate     

Elicitation of anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> and the involvement of methyl jasmonate and salicylic acid

The effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the anthocyanin accumulation, endogenous titres of polyamines and ethylene production in callus cultures of Daucus carota were studied. The interaction of these signaling molecules with elicitors from Aspergillus niger was investigated and the involvement of MeJA was elucidated through the use of the jasmonic acid (JA) biosynthetic inhibitor ibuprofen. MeJA and SA were both found to stimulate the anthocyanin production in the callus cultures. The highest levels of anthocyanin was observed in the cultures treated with 200 μM SA 0.36 % and 0.01 μM MeJA 0.37 %. The MeJA and SA treatments were also found to result in higher activity of Ca²⁺ ATPase suggesting that the enhancement of anthocyanin by SA and MeJA could be mediated through the involvement of the calcium channel. The treatment of the callus cultures with SA was found to result in marginally higher titres of endogenous polyamines (PAs) whereas MeJA resulted in lower levels of PAs as compared to the control. The SA treatment was found to result in lower ethylene production and the treatment with MeJA stimulated the ethylene production. These results suggest that the stimulation of anthocyanin production by MeJA and SA in callus cultures of D. carota is not related to the ethylene production.     

Axillary shoot proliferation of blue honeysuckle

Callus cultures of Ajuga reptans flowers produced a complex mixture of cyanidin- and delphinidin-based pigments, of which more than 90% were acylated. The anthocyanin composition varied little during one growth period. During a time span of 5 years no new anthocyanin classes appeared. Quantitative differences in anthocyanin composition between the callus lines and during a 5 year time span were more pronounced. In general, the accumulation of delphinidin-based anthocyanins decreased. The percentage of acylated anthocyanins was stable in time. The accumulation of metabolically evolved anthocyanins (5′-substituted and acylated) decreased during passage from solid culture to liquid culture. The accumulation of acylated anthocyanins was influenced by the type of aeration in liquid cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Accumulation of anthocyanin in callus cultures of red-pod okra [<Emphasis Type="Italic">Abelmoschus esculentus</Emphasis> (L.) Hongjiao] in response to light and nitrogen levels

Nitrogen and light are critical determinants of biomass accumulation and secondary metabolite production under in vitro culture conditions. In this study, we analyzed the effects of varied concentrations of total nitrogen in Murashige and Skoog (MS) medium and light intensity on the production of biomass, anthocyanin pigments, and bioactive antioxidants in callus cultures of Abelmoschus esculentus cv. ‘Hongjiao’. Maximum callus biomass accumulation (3 g FW) was achieved when calluses were cultured on MS medium containing 60 mM nitrogen under 40 μmol m^??2 s^??1 light intensity. In contrast, maximum values of total anthocyanin accumulation (TA; 7.3 CV/g FW), total phenolic content (TP; 12.07 mg/100 g FW), total flavonoid content (TF; 2.47?±?0.15 mg/100 g FW), and total antioxidant activity (TAA; 56.10 μmol Trolox/g FW) were observed when calluses were cultured on MS medium containing 40 mM total nitrogen under 80 μmol m^??2 s^??1 light intensity. In addition, callus grown under same culture condition exhibited high flavonoid content along with increased phenolic content and antioxidant activity. High performance liquid chromatography (HPLC) was performed for qualitative and quantity analysis of callus cultures. Most of the pigments from the callus extracts were identical with pod anthocyanins, and appeared on the ODS-column HPLC with lower concentration than the main pigments of the pod tissues. These findings indicate that callus cultures of red-pod okra represent a potential source of bioactive compounds with antioxidant properties for industrial applications.     

Effect of light quality and tissue origin on phenolic compound accumulation and antioxidant activity in Camellia japonica calli

In Camellia japonica callus culture, the effects of light quality on the accumulation of phenolic compounds were examined. To this end, the calli were cultured under three monochrome [white (W), red (R), and blue (B)] and two mixed [red/blue (RB) and red/green/blue (RGB)] light sources. After 4 weeks of culture, fresh and dry callus weights were determined, and phenolic content was analyzed using high-performance liquid chromatography. We also examined the effects of RGB light on calli derived from four origins (leaf: CL; root: CR; petal: CP; and ovary: CO). Total phenolic and flavonoid content and DPPH radical scavenging activity were highest in calli exposed to RB and RGB. Catechin was detected only in calli exposed to mixed light (RB and RGB). Interestingly, red pigmentation development in CL and CR calli after 2 weeks of culture and organogenic calli in CR and CP samples was observed. Furthermore, expression of the phenylalanine ammonia lyase and chalcone synthase genes was found to be higher in the CR line than in other lines, and total phenolic and flavonoid content was also higher in the CR and CP lines. The findings of the present study revealed that the accumulation of phenolics and flavonoids in callus tissue varies depending on light quality, which stimulates cell division, and may thus affect the proliferation and organogenesis of C. japonica callus. We also established that the effects of light on red pigment expression and the accumulation of bioactive compounds are dependent on the tissue origin of callus material.

     

Effects of surfactants on cell growth and pigment production in suspension cultures ofPerilla frutescens

The effects of surfactants, adecanol LG-294 and silicone A, on anthocyanin accumulation and the growth ofPerilla frutescens cells in suspension cultures were studied. Production of the red pigment was remarkably reduced from about 1.9 g/l to 0.4 g/l by adecanol LG-294 at 0.06 ml/l but not by silicone A up to 0.4 ml/l. Several repeated shake-flask cultures also demonstrated no adverse effects of silicone A on the metabolite accumulation by the suspended cells. Furthermore, the addition of silicone A to a culture in a stirred bioreactor produced a three-fold higher growth rate and a seven-fold increase in anthocyanin compared with surfactant-free cultures. The improvement was due to the substantial reduction or prevention of foaming and of cell adhesion to the bioreactor wall.     

Optimization of growth and jaceosidin production in callus and cell suspension cultures of Saussurea medusa

Calli cultures derived from the leaves of Saussurea medusa were selected on the basis of colour into three callus, A, B and C, which suggested different levels of metabolite accumulation. An improved reversed phase high performance liquid chromatographic method provided selective determination of the jaceosidin content of these samples. The jaceosidin concentration in callus B was higher than that of the callus A and C. By using 12-day old culture and 9-day old inoculum, jaceosidin yield of 72.91 mg l^–1was obtained from cell line B in cell suspension cultures. The influence of some factors affecting jaceosidin formation, i.e. temperature, light, inoculum size, type of media, phytohormones, nitrogen and carbon source etc. were also examined. Light irradiation and combination of 3% (w/v) sucrose with 1% glucose brought about a marked increase of jaceosidin production. The effect of blue light on jaceosidin was markedly superior to other kinds of monochromatic light (red and far-red) or white light. Analysis of growth and jaceosidin content of callus cultures and cell suspension cultures demonstrated that the production of jaceosidin was growth-dependent in both cell solid culture and cell suspension culture.     

Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception

Transgenic tomato [Lycopersicon esculentum (=Solanum lycopersicum)] lines overexpressing tomato PHYA, PHYB1, or PHYB2, under control of the constitutive double-35S promoter from cauliflower mosaic virus (CaMV) have been generated to test the level of saturation in individual phytochrome-signalling pathways in tomato. Western blot analysis confirmed the elevated phytochrome protein levels in dark-grown seedlings of the respective PHY overexpressing (PHYOE) lines. Exposure to 4 h of red light resulted in a decrease in phytochrome A protein level in the PHYAOE lines, indicating that the chromophore availability is not limiting for assembly into holoprotein and that the excess of phytochrome A protein is also targeted for light-regulated destruction. The elongation and anthocyanin accumulation responses of plants grown under white light, red light, far-red light, and end-of-day far-red light were used for characterization of selected PHYOE lines. In addition, the anthocyanin accumulation response to different fluence rates of red light of 4-d-old dark-grown seedlings was studied. The elevated levels of phyA in the PHYAOE lines had little effect on seedling and adult plant phenotype. Both PHYAOE in the phyA mutant background and PHYB2OE in the double-mutant background rescued the mutant phenotype, proving that expression of the transgene results in biologically active phytochrome. The PHYB1OE lines showed mild effects on the inhibition of stem elongation and anthocyanin accumulation and little or no effect on the red light high irradiance response. By contrast, the PHYB2OE lines showed a strong inhibition of elongation, enhancement of anthocyanin accumulation, and a strong amplification of the red light high irradiance response.     

Influence of thidiazuron on callus induction and crocin production in corm and style explants of <Emphasis Type="Italic">Crocus sativus</Emphasis> L.

Crocus sativus L., mostly famous as saffron, has gained more attention due to its crocin (crocetin ester) pigment responsible for its extensive pharmaceutical properties. In this study, we established two different callus cultures from corm and style explants of saffron to find out the best explant as a suitable source for crocin production. Comparative analyses of total phenolic, flavonoid, carotenoid and anthocyanin contents were also performed in the two callus cultures. For callus induction, different combinations of MS medium with name thidiazuron (TDZ), benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination were tested. Of the used media, all the combinations containing TDZ and NAA gave 100% callus induction. HPLC-DAD and HPLC–ESI-MS analysis were used for identification of crocin esters in established callus cultures. The highest amount of 0.35 mg g^?1 DW crocin was detected in style originated calli grown on the medium containing 3 mg L^?1 NAA?+?1 mg L^?1 TDZ while the corm calli showed the most abundant total carotenoid (0.73 mg g^?1 DW), phenolic (15.04 mg gallic acid equivalent g^?1 DW) and flavonoid (0.76 mg rutin equivalent g^?1 DW) contents. In general, style-derived calli showed longer time survival with a fine texture and good quality compared to corm-derived calli.     

Effect of the genes B and Pl on anthocyanin synthesis in maize endosperm culture

B and Pl are two genes involved in anthocyanin biosynthesis in maize (Zea mays) plant tissues. In this work the effect of B and Pl on pigment accumulation is analyzed in endosperm tissues, either cultured in vitro or scraped off from the seed. The results obtained indicate that the two genes play a different role in callus pigmentation: B exerts a qualitative change in pigment composition, while Pl controls the rate of pigment accumulation in the callus. Anthocyanin synthesis in all strains analyzed appears to be light independent. Two cases of instability in pigment production arisen in the endosperm cultures are described and discussed in relation to epigenetic variation in secondary metabolite content in plant tissue culture.Abbreviations BEAF Benzene/ethyl acetate/formic acid (40:10:5) - 2-4D 2,4-dichlorophenoxyacetic acid - Wi initial weight - Wt total weight     

The effect of hormones on anthocyanin accumulation in cell cultures of Haplopappus gracilis

Summary Suspension cultures of Haplopappus gracilis accumulated anthocyanin when grown in defined media with 4.5×10^-6M 2,4-D. Transfer of cells to media with 10^-5M kinetin or benzyladenine and no auxin or 10^-7M NAA for 6 days resulted in increased anthocyanin concentration of the cells but the total amount of pigment was unaffected due to differences in growth rates. The cultures yielded up to 35 mg pigment per gram dry weight.Cells grown in batch culture in media with 10^-5M kinetin and with 10^-7 M NAA or 5×10^-5M NAA sampled and analyzed daily grew at the same rate. The concentration of anthocyanin differed, being lower in cells at 5×10^-5M NAA. After 6 days there was a rapid increase in pigment formation, and by 14 days the concentration of anthocyanin in cells in the two media were the same.When the cells were cultured in 3.5-1 phytostats and 600 ml culture was replaced daily with 600 ml medium, anthocyanins accumulated when the NAA concentration was 10^-7M but not at 10^-6M. At 10^-7M NAA the cultures remained pigmented and anthocyanin accumulation could be restored after a temporary loss of pigmentation due to an earlier, higher auxin concentration. The changes in concentration of phenylalanine ammonia-lyase did not correspond to changes in the rate of anthocyanin accumulation. The enzyme showed a maximum 4–8 h after inoculation of cells to fresh media. Cells grown on agar plates and rich in anthocyanin were observed to divide without loss of pigmentation, demonstrating that cells differentiated with respect to anthocyanin production undergo mitosis.Issued as NRCC No. 11388.Abbreviations used: 2,4-D=2,4-dichlorophenoxyacetic acid, NAA + -naphthaleneacetic acid.     

Influence of putrescine on anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> mediated through calcium ATPase

The influence of Putrescine (Put) on the growth and elicitation of anthocyanin in callus cultures of Daucus carota var. Nantes scarlet was investigated through the use of α-DL-difluoromethylarginine (DFMA), the polyamine (PA) biosynthetic inhibitor. It was observed that the addition of Put (0.05 mM) resulted in enhancement of growth and anthocyanin content. The anthocyanin content was found to be enhanced by 1.68 fold on the 21^st day as compared to the untreated controls. The PA inhibitor was found to result in lowering of the growth and the anthocyanin accumulation, which could be partially restored by the addition of Put in combination with this inhibitor. The levels of Ca²⁺ ATPase were also found to be elevated in treatment with Put suggesting the involvement of calcium in the elicitation of anthocyanin. The endogenous titres of PAs and the ethylene production under these treatments were also studied. The treatment with DFMA resulted in lower levels of endogenous PAs and higher levels of ethylene. Lowering of ethylene by putrescine treatment shows that PA treatment also inhibited ethylene formation, which would also imply that endogenous ethylene does not influence anthocyanin production in carrot callus cultures.     

Responses of anthocyanin-producing and non-producing cells of Glehnia littoralis to radical generators.

The responses of anthocyanin-producing (violet) and non-producing (white) cells of Glehnia littoralis to radical generators were compared. Cell growth, anthocyanin content, phenylalanine ammonia-lyase (PAL) activity and furanocoumarin production were determined after treatment with H(2)O(2), 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), X-ray and yeast extract, independently. AAPH and H(2)O(2) repressed the growth of both violet and white cells, but violet cells grew better than white cells. On the other hand, the anthocyanin content in violet cells decreased. Neither X-ray nor yeast extract affected cell growth or pigment production. Treatment with H(2)O(2), yeast extract, and X-ray, but not AAPH, induced PAL activity and furanocoumarin production in white cell cultures, whereas violet cell cultures did not produce furanocoumarin following any of the treatment employed.     

<Emphasis Type="Italic">Bridelia stipularis</Emphasis>: a new source for anthocyanin production in vitro

The present study prospects Bridelia stipularis (L.) Blume as a new source of anthocyanins through leaf and internode explants-derived callus cultures. Murashige and Skoog (MS) medium fortified with 21.48 μM α-naphthaleneacetic acid was superior for callus growth. Of the different regimes, the anthocyanin production relied on synergic effects of plant growth regulators, pH, light, and carbon source. The calluses incubated in light on MS medium with 4% glucose containing 2.22 μM N⁶-benzyladenine (BA) and 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) at pH 3.5 yielded the highest amount (a mean of 0.42 mg g⁻¹ callus) of anthocyanins. Subsequent cultures of the calluses on the above medium yielded a stable production of anthocyanins. Medium containing glucose was superior to that with sucrose for anthocyanin formation. Kinetin was inhibitory to anthocyanin accumulation. Suspension cultures of MS medium containing 2.26 μM 2,4-D and 2.22 μM BA at pH 5.0 started excretion of anthocyanins into the medium on reaching to pH 4.4–4.6.