Effects of non-frozen cold storage on the growth,organogenesis and secondary metabolism of callus cultures

Callus tissues derived from chilling-tolerant herbaceous plant, Atractylodes lancea, Atropa belladonna, Bupleurum falcatum, Dioscorea tokoro, Lithospermum erythrorhizon and Phytolacca americana could be cold-stored at 4°C for three months or more, whereas those from chilling-sensitive herbaceous plants such as Datura innoxia and Perilla frutescens var. crispa and a deciduous tree, Mallotus japonicus, could not survive after cold storage for two to three months. Tobacco callus cultures could be stored at 4°C for two or four months depending on a callus strain. The effect of cold storage on secondary metabolite production varied. Nicotine and betalain production suffered from cold storage of tobacco and Phytolacca americana callus cultures, respectively. However, production of anthocyanin in cultures of Mallotus japonicus and Bupleurum falcatum and shikonin derivatives in Lithospermum erythrorhizon callus was affected very little. Root-forming ability was retained for more than one year in cold-stored callus tissues of Bupleurum falcatum, while the control callus tissues maintained at 25°C completely lost the organogenetic ability six months after the first subculture.     

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).     

Combined effects of phytohormone, indole-3-butyric acid, and methyl jasmonate on root growth and ginsenoside production in adventitious root cultures of Panax ginseng C.A. Meyer

Indole-3-butyric acid at 25 μM with methyl jasmonate (MJ) at 100 μM in Panax ginseng synergistically stimulated both root growth and ginsenoside accumulation compared with 100 μM MJ alone. Productivity of ginsenoside was 10 mg l⁻¹ d⁻¹ compared to 7.3 mg l⁻¹ d⁻¹ with MJ elicitation alone.     

Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures

The differentiation of sieve and tracheary elements was studied in callus culture of Daucus carota L., Syringa vulgaris L., Glycine max (L.) Merr., Helianthus annuus L., Hibiscus cannabinus L. and Pisum sativum L. By the lacmoid clearing technique it was found that development of the phloem commenced before that of the xylem. In not one of the calluses was differentiation of tracheary elements observed in the absence of sieve elements. The influence of indole-3-acetic acid (IAA) and sucrose was evaluated quantitatively in callus of Syringa, Daucus and Glycine. Low IAA levels resulted in the differentiation of sieve elements with no tracheary cells. High levels resulted in that of both phloem and xylem. IAA thus controlled the number of sieve and tracheary elements, increase in auxin concentration boosting the number of both cell types. Changes in sucrose concentration, while the IAA concentration was kept constant, did not have a specific effect on either sieve element differentiation, or on the ratio between phloem and xylem. Sucrose did, however, affect the quantity of callose deposited on the sieve plates, because increase in the sucrose concentration resulted in an increase in the amount of callose. It is proposed that phloem is formed in response to auxin, while xylem is formed in response to auxin together with some added factor which reaches it from the phloem.     

Spectral composition of irradiation regulates the cell growth and flavonoids biosynthesis in callus cultures of Saussurea medusa Maxim

Cell growth, flavonoids biosynthesis and L-phenylalanine ammonia-lyase (PAL) activity were studied in callus cultures of Saussurea medusa Maxim. under different types of spectral radiance. After 21 days, red light significantly improved the callus growth, but inhibited the biosynthesis of flavonoids in callus cultures. However, blue light was found to enhance the biosynthesis of flavonoids, although callus growth under this spectrum was comparable with that under white and other coloured spectra, such as green and yellow. The accumulation of flavonoids in callus cultures was related to the PAL activity, which was found to be stimulated by the spectral composition of irradiation.     

Production of tropan alkaloids in the in vitro and callus cultures of Hyoscyamus aureus and their genetic stability assessment using ISSR markers

Green wild plants (dirctly before flowering) and seeds of Hyoscyamus aureus were collected from natural habitat at Al Qalamon region in Syria. Seeds were surface sterilized and cultured in vitro, after 21 days from germination stem-derived callus was induced on two different nutrient media. Tropane alkaloids were extracted from wild plants and 30 days old in vitro plants and callus, and then analyzed using GC-MS. Genetic variation was also studied between the wild and in vitro plants and the callus culture lines using twenty ISSR markers. The results showed that there were significant variations in tropane alkaloids contents between the wild plants, the in vitro plants and the callus culture lines. The highest content of hyoscyamine was in callus on line A medium, but the highest content of scopolamine was in the wild plants. However, the lowest content of tropane alkaloids was in callus on line B medium. Also the ISSR analyses showed that there was genetic variation between the wild and in vitro plants and the callus culture lines.     

The effects of methyl jasmonate and salicylic acid on the production of secondary metabolites in organ culture of Ziziphora persica

Plant Cell, Tissue and Organ Culture (PCTOC) - Ziziphora persica (Lamiaceae) has been used as infusions and decoctions in traditional medicine for various purposes such as sedative, carminative,...     

24-Epibrassinolide and methyl jasmonate can encourage cell growth and the production of secondary metabolites in immobilized cells of Hyoscyamus niger

In Vitro Cellular & Developmental Biology - Plant - This study was carried out to determine the effects of 24-epibrassinolide (24-eBL) and methyl jasmonate (MeJA) on cell growth and...     

Optimized genetic transformation of Zanthoxylum zanthoxyloides by Agrobacterium rhizogenes and the production of chelerythrine and skimmiamine in hairy root cultures

Zanthoxylum zanthoxyloides is an endangered African tree producing numerous bioactive substances including antileukemic and antisickling agents. Here, the potential of Z. zanthoxyloides hairy root cultures was tested for the production of bioactive substances with limited natural resources. The efficiency of Agrobacterium rhizogenes LBA9402‐mediated transformation of leaf material was evaluated using different techniques. An optimal transformation frequency of 77% was obtained after 11 days by inoculating A. rhizogenes directly onto the central vein of 14‐week‐old leaves followed by a co‐cultivation period of 3 days. Different treatments in immersion mode (manual wounding, acetosyringone, CaCl₂, ultrasonication) never exceeded these results. A maximum growth rate of 0.37 cm/day was determined during the exponential phase. Liquid chromatography‐diode array detection analysis showed the presence of skimmiamine, sesamine, chelerythrine, and chelerythrine derivatives in Z. zanthoxyloides hairy root lines. The maximum production of skimmiamine and chelerythrine in 28‐day‐old hairy root cultures was 45 ± 2 and 107 ± 4 mg/100 g dry weight, respectively. The present results highlight the potential of Z. zanthoxyloides hairy root cultures for the sustainable production of skimmiamine and chelerythrine.     

Two NAD-dependent alcohol dehydrogenases (E.C. 1.1.1.1) in callus cultures of wheat,rye and triticale

Summary Two NAD-dependent alcohol dehydrogenases ADH-1 and ADH-2, under independent genetic control of genes designated as Adh-1 and Adh-2 located on chromosomes 4A, 4B and 4D, have been reported in aestivum wheat (Hart 1980). Only ADH-1 is expressed in developing seeds, dry seeds, pollen and germinating seedlings. ADH-2 can be induced in seedling roots or shoots under conditions of partial anaerobiosis or by certain chemicals. Expression of ADH-1 and ADH-2 isoenzymes was investigated in undifferentiated calli from aestivum and durum wheats, rye, triticale and also in in vitro regenerated roots and leaves from aestivum cultures. Wheat callus cultures originating from seed, mature and immature embryos, mesocotyl and root, as well as cultures grown on media containing different supplements did not show any variation in the overall expression of ADH-1 or ADH-2, although differences in the band intensities were observed. The callus isoenzyme pattern was similar to that observed in roots under anaerobic conditions. Both ADH-1 and ADH-2 were expressed in in vitro regenerated roots but were absent in regenerated leaves. Expression of ADH-1 and ADH-2 in wheat calli seems to be related to the type of differentiation.     

Enhancement of lignan biosynthesis in suspension cultures of <Emphasis Type="Italic">Linum nodiflorum</Emphasis> by coronalon,indanoyl-isoleucine and methyl jasmonate

The effect of the two synthetic elicitors coronalon and indanoyl-isoleucine and of methyl jasmonate (MeJA) on the accumulation and biosynthesis of lignans by cell suspension cultures of Linum nodiflorum (Linaceae) was investigated. The production of 6-methoxypodophyllotoxin (MPTOX) could be increased more than tenfold, the maximal content reaching up to over 2.5% of the cell dry weight. The highest yield was achieved by administering 50 μM of the synthetic elicitors on the fourth day and extracting the products on the tenth day of the culture period. An additional lignan accumulated in elicitor-treated cultures. Its structure was elucidated by extensive 1D and 2D NMR measurements, revealing its identity as 5′-demethoxy-MPTOX (5′-dMPTOX). Its average content amounted up to over 5% of the cell dry weight. Growth was only slightly affected by the addition of the elicitors. Methyl jasmonate exerted a moderate stimulating effect on the L. nodiflorum cells with MPTOX and 5′-dMPTOX contents going up to 1.4 and 2.1% of the cell dry weight, respectively. The activities of deoxypodophyllotoxin 6-hydroxylase and β-peltatin 6-O-methyltransferase, two enzymes involved in MPTOX biosynthesis, were increased up to 21.9-fold and 14.6-fold, respectively, in the treated cultures.     

Comparison of photomixotrophic and heterotrophic callus and suspension cultures of Pinus elliottii. 1. Photosynthetic properties and ultrastructural evidence for coexistence of starch granules and secondary metabolites

Photomixotrophic callus and suspension cultures of salsh pine (Pinus elliottii var. elliottii Engelm.) have been established. Callus tissues contained up to 2.76 g chlorophyll mg^-1 dry wt and suspensions 2.98 g chlorophyll mg^-1 dry wt. Maximum photosynthetic oxygen evolution was 25–32 mol O₂ h^-1 mg^-1 chlorophyll for callus and 35–39 mol O₂h^-1 mg^-1 chlorophyll for suspension, respectively. Photomixotrophic callus was friable with a high moisture content during early and exponential growth, but evolved into a compact and dense tissue during the latter stage of growth. Compact photomixotrophic callus accumulated and deposited secondary metabolites in the central vacuole and developed large starch granules in the chloroplasts. Secondary metabolites were not observed in photomixotrophic suspensions or in heterotrophic calli and suspensions. Photomixotrophic callus contained numerous mitochondria closely associated with well-developed chloroplasts containing 2–6 thylakoids per granum. Heterotrophic callus was characterized by a poorly developed cytoplasm and cup-shaped mitochondria.     

Establishment of callus and cell suspension culture of Scrophularia striata Boiss.: an in vitro approach for acteoside production

In the present study, a protocol was optimized for establishment of callus and cell suspension culture of Scrophularia striata Boiss. as a strategy to obtain an in vitro acteoside producing cell line for the first time. The effects of growth regulators were analyzed to optimize the biomass growth and acteoside production. The stem explant of S. striata was optimum for callus induction. Modified Murashige and Skoog medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine was the most favorable medium for callus formation with the highest induction rate (100 %), the best callus growth and the highest acteoside content (1.6 μg/g fresh weight). Incompact and rapid growing suspension cells were established in the liquid medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine. The optimum time of subculture was found to 17–20 days. Acteoside content in the cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The maximum content of acteoside (about 14.25 μg/g cell fresh weight) was observed on the 17th day of the cultivation cycle. This study provided an efficient way to further regulation of phenylethanoid glycoside biosynthesis and production of valuable acteoside, a phenylethanoid glycoside, on scale-up in S. striata cell suspension culture.     

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.     

Effects of methyl jasmonate and salicylic acid on the production of bilobalide and ginkgolides in cell cultures of Ginkgo biloba

Summary In an attempt to increase productivity, the effects of the elicitors methyl jasmonate (MJ) and salicylic acid (SA) on the production of bilobalide (B), ginkgolide A (GA), and ginkgolide B (GB) were studied in cell suspension cultures of Ginkgo biloba. MJ treatments increased the amounts of B, GA, and GB, concomitant with a slight decrease in cell growth. After treatment of 0.01 mM MJ, levels of GA and GB increased 4.3-and 8.2-fold over controls by 12 h and declined after 24h. The 1.0mM MJ treatment produced a maximal release of B after 12h of exposure and increased the concentration of B in the culture medium up to 6.25-fold compared with the controls. Treatment with 1.0mM SA transiently enhanced GA and GB production up to 3.1-and 6.1-fold, respectively, compared with the control. However, treatment 1.0 mM SA did not have a significant effect on B production. When treated with 0.01 mM SA, the level of B in the cells was increased 5.4-fold over controls by 12h and declined after 24h. The concentrations and exposure times of both MJ and SA were factors that strongly affected the production of B, GA, and GB. The results from this study suggest that MJ and SA directly or indirectly increased the production of B, GA, and GB in cells, and stimulated the release of these metabolites into the culture medium.     

Arbuscular mycorrhizal fungi alter the content and composition of secondary metabolites in Bituminaria bituminosa L.

• Secondary metabolites may be affected by arbuscular mycorrhizal fungi (AMF), which are beneficial symbionts associated with the roots of most plant species. Bituminaria bituminosa (L.) C.H.Stirt is known as a source of several phytochemicals and therefore used in folk medicine as a vulnerary, cicatrising, disinfectant agent. Characteristic metabolites found in B. bituminosa are furanocoumarins and pterocarpans, which are used in cosmetics and as chemotherapeutic agents. Here we address the question whether AMF inoculation might affect positively the synthesis of these phytochemicals.
• B. bituminosa plants were inoculated with different AMF and several metabolites were assessed during full vegetative stage and flowering phase. Pigments (chlorophylls and carotenoids), polyphenols and flavonoids were spectrophotometrically determined; specific isoflavones (genistein), furanocoumarins (psoralene and angelicin), pterocarpans (bitucarpin A and erybraedin C) and plicatin B were assessed with HPLC; leaf volatile organic compounds were analysed using SPME and identified by GC‐MS.
• During the vegetative stage, the inoculated plants had a high amount of furanocoumarins (angelicin and psoralen) and pterocarpans (erybraedin C and bitucarpin A). The analysis of volatile organic compounds of inoculated plants showed different chemical composition compared with non‐mycorrhizal plants.
• Given the important potential role played by furanocoumarins and pterocarpans in the pharmaceutical industry, AMF inoculation of B. bituminosa plants may represent a suitable biotechnological tool to obtain higher amounts of such metabolites for pharmaceutical and medicinal purposes.

     

花椒资源与开发利用现状调查

本文介绍了我国重点产区的花椒资源与开发利用现状。     

Glutamate dehydrogenase regulation in callus cultures of Nicotiana plumbaginifolia: effect of glucose feeding and carbon source starvation on the isoenzymatic pattern

Abstract. In callus cultures of Nicotiana plumbaginifolia , the activity of glutamate dehydrogenase was repressed by glucose, whereas, on the contrary, carbon and energy source deprivation induced a remarkable increase in specific activity. Definition of these two opposite types of response was made possible by the use of glycerol as a non-repressing carbon source: in this condition, glutamate dehydrogenase activity reached an intermediate level, which was similar to the derepressed values of activity obtainable when cultures were allowed to exhaust the glucose supply in the medium. Isoelectric focusing analysis revealed the existence of three different isoenzymatic patterns which could be correlated to the three different levels of specific activity: repressed (glucose), induced (carbon starvation) and intermediate (glycerol). Repression affected mainly the four more cathodic bands which were predominant in non-repressed conditions. The possible catabolic role of these isoenzymes is discussed.     

Indoleamines and calcium channels influence morphogenesis in in vitro cultures of Mimosa pudica L.

The present article reports the interplay of indoleamine neurohormones viz. serotonin, melatonin and calcium channels on shoot organogenesis in Mimosa pudica L. In vitro grown nodal segments were cultured on MS medium with B5 vitamins containing Serotonin (SER) and Melatonin (MEL) at 100 µM and indoleamine inhibitors viz. serotonin to melatonin conversion inhibitor p-chlorophenylalanine (p-CPA) at 40 µM, serotonin reuptake inhibitor (Prozac) 20 µM. In another set of experiment, calcium at 5 mM, calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) 100 µM, and calcium channel blocker varapamil hydrochloride (1 mM) a calcium chelator EGTA (100 µM) were administered to the culture medium. The percentage of shoot multiplication, endogenous MEL and SER were monitored during shoot organogenesis. At 100 µM SER and MEL treatment 60% and 70% explants responded for shoot multiplication respectively. Medium supplemented with either SER or MEL along with calcium (5 mM) 75%–80% explants responded for organogenesis. SER or MEL along with calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) at 100 µM 70% explants responded for shoot multiplication. p-CPA, prozac, verapamil and EGTA, shoot multiplication was reduced and endogenous pools of SER, MEL decreased by 40–70%. The results clearly demonstrated that indoleamines and calcium channels positively influenced shoot organogenesis in M. pudica L.