Establishment of synchrony by starvation and readdition of auxin in suspension cultures of Catharanthus roseus cells

A system of synchronous cell division was established by starvation of auxin and its readdition to suspension cultures of cells of Catharanthus roseus L. cv. Little-Pinky. When cells in the stationary phase were transferred to fresh medium free of 2,4-dichlorophenoxyacetic acid (2,4-D), cells were arrested preferentially at the G₁ phase. After cells had been cultured for 2 days in medium without 2,4-D, readdition of 2,4-D induced the synchronous division of cells. In this system, 70–80% of cells divided synchronously within 3 to 4h, and the mitotic index increased sharply in parallel with the increase in cell number. Active synthesis of DNA was demonstrated by measurements of incorporation of [³H]-thymidine into the DNA fraction. The induction of cell division by the addition of 2,4-D was inhibited by treating cells with analogues of auxin, such as 2,4,6-trichlorophenoxyacetic acid and p-chlorophenoxyisobutyric acid.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - IAA indole-3-acetic acid - MS Murashige & Skoog - NAA -naphthalenacetic acid - PCIB p-chlorophenoxyisobutyric acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid     

Growth and activities of enzymes of primary metabolism in batch cultures of Catharanthus roseus cell suspension under different pCO2 conditions

In vitro enzyme activities of glycolysis, pentose-phosphate pathway and dark CO₂ fixation were assayed in batch cultures of heterotrophic Catharanthus roseus cells under various gassing rates and partial pressures of carbon dioxide. Detrimental effects of low pCO₂ culture conditions on the growth characteristics could be linked to marked changes in levels of enzymes of primary metabolism during growth. The enzyme levels observed during the early stages of growth were found to be more stable when a constant pCO₂ (20 mbar) was maintained and enabled exponential growth to be reached more rapidly.The importance of carbon dioxide as a conditioning factor of the culture medium is discussed.     

Large scale fermentation and alkaloid production of cell suspension cultures of Catharanthus roseus

Cell cultures of Catharanthus roseus were scaled up to volumes of 50001 using conventional reactors equipped with flat-blade impellers. The behavior of the fermenter grown cells was compared with corresponding shake flask experiments with respect to growth and indole alkaloid inducibility and production. The limits and problems of transferring shake flask experiments of culture systems such as Catharanthus, in which alkaloid production depends greatly upon the physiological state of the cells, to large scale multistage processes is discussed.     

The cryopreservation of shoot tips of Rosa multiflora

Shoot tips of in vitro grown plantlets of Rosa multiflora were cryopreserved using an encapsulation/dehydration procedure. The influence of sucrose and silica gel pretreatments on pre- and post-freeze shoot growth were examined. Shoot tips recovered from liquid nitrogen only grew after 24h pretreatment in medium containing 0.5 M sucrose, followed by 2 h drying with silica gel and rapid freezing.Abbreviations RSC1 modified Murashige and Skoog medium for Rosa multiflora shoot culture     

The use of TTC reduction assay for assessment of Gentiana spp. cell suspension viability after cryopreservation

This study was aimed at improving the 2,3,5-triphenyl-tetrazoliumchloride (TTC) reduction test for initial assessment of cell survival after cryopreservation. Experiments were carried out on three embryogenic cell suspensions of different ages: 9-year-old Gentiana tibetica (King ex Hook. F.), 2-year-old G. kurroo (Royle), and 1-year-old G. cruciata (L.). The suspensions were maintained in MS medium supplemented with 1.0 mg 1⁻¹ 3,6-dichloro-o-anisic acid, 0.1 mg 1⁻¹ naphthaleneacetic acid, 2.0 mg l⁻¹ 6-benzylaminopurine, 80.0 mg 1⁻¹ adenine sulphate and 0.09 M sucrose. Four weeks before freezing, part of the tissue was subcultured to the same medium with sucrose concentrations elevated from 0.09 M (3%sMS) to 0.175 M (6%sMS) or 0.26 M (9%sMS). In freezing treatments without cryoprotection, tissue was plunged directly into liquid nitrogen (LN) or cooled gradually. In freezing treatments with cryoprotection, the cells were pretreated with 1 M sucrose, or with 0.4 M sorbitol + 0.25 M proline or + 0.08 M DMSO, or with vitrification solution (PVS2). Encapsulation was another variant. TTC reduction activity was spectrophotometrically assessed immediately, 1, 3, 5, 24 and 48 h after thawing. Cells without cryoprotection were lethally damaged, but TTC reduction activity in those cells ranged from 6.5% (tissue from 3%sMS) to 73 % (tissue from 9%sMS) directly after thawing. Formazan production was reduced to zero after 24 h. The TTC test showed 50% formazan content immediately after thawing of DMSO-protected G. tibetica tissue, but only 22.47% after 24 h and 2.9% after 48 h. Ultrastructural analysis of those cells showed lethal damage in many of them. For the PVS2 treatment, the formazan content was similar in samples analyzed directly after thawing and 24 h later. Cells treated with PVS2 did not show structural disturbances. Encapsulated cell aggregates of G. cruciata treated with concentrations of sucrose increasing up to 1 M produced 2.6 times more formazan. When applied at least 48 h after thawing, the TTC test can reflect cell viability and can be used to compare the effectiveness of cryoprotectant performance and freezing protocols, but it must be carefully evaluated, with appropriate controls.     

Effect of acetylsalicylic acid on secondary metabolism ofCatharanthus roseus tumor suspension cultures

Summary Addition of various concentrations (0.5–20 mM) of acetylsalicylic acid (ASA) to tumor lines ofCatharanthus roseus cultivatedin vitro and requiring corn starch as carbon source, produced remarkable effects on secondary metabolite production. An increase of 505% total alkaloids per culture (cells plus liquid medium), 1587% total phenolics (liquid medium), 612% total furanocoumarins (liquid medium) and 1476% total anthocyanins (liquid medium) was detected. 1 mM ASA in combination with other elicitors, such as homogenates ofAspergillus fumigatus or trans-cinnamic acid, did not further increase the metabolite content substantially. The results suggest that ASA could act as a new biotic elicitor of metabolite production inC. roseus cell suspension culture.     

花烛胚性悬浮细胞玻璃化超低温保存条件研究

为建立适宜的花烛(Anthurium andraeanum Lind. )胚性悬浮细胞玻璃化超低温保存技术,采用单因素实验方法对影响玻璃化超低温保存后细胞相对存活率的主要因素进行了研究.结果表明,经玻璃化超低温保存后花烛悬浮细胞的相对存活率与悬浮细胞的继代培养时间、渗透调节剂的种类和浓度及预培养时间、装载液种类和预处理时间、PVS2脱水时间以及超低温保存后的化冻温度均有一定的关系.继代培养3和5 d,细胞的相对存活率较高(约20%);分别以0.3、0.5、0.7 mol·L-1山梨醇和60、80、100、120 g·L-1蔗糖为渗透调节剂预培养0～4 d,以0.5 mol·L-1山梨醇预培养2 d的效果最好,细胞的相对存活率为26.2%;用体积分数25%PVS2预处理15 min,细胞的相对存活率最高(29.0%);分别用体积分数100%PVS2脱水0、5、10、15、20、25和30 min,其中脱水10 min的悬浮细胞相对存活率最高(32.1%);分别在10 ℃、20 ℃、30 ℃、40 ℃、50 ℃和60 ℃水浴条件下进行化冻处理,其中用40 ℃水浴化冻的悬浮细胞相对存活率最高(32.1%).花烛胚性悬浮细胞玻璃化超低温保存和化冻的适宜流程为:将继代培养3～5 d的胚性悬浮细胞团(直径2 mm)在含0.5 mol·L-1山梨醇的1/2MS液体培养基中预培养2 d后,于4 ℃条件下处理24 h,然后先用体积分数25%PVS2室温预处理15 min,再用体积分数100%PVS2 在0 ℃条件下脱水10 min,最后迅速投入液氮中冷冻保存;将经过冷冻保存的细胞置于40 ℃水浴中化冻3 min,用含1.2 mol·L-1蔗糖的1/2MS液体培养基洗涤3次(每次10 min),之后即可进行恢复培养.     

The exposure to trans-cinnamic acid of osmotically stressed Catharanthus roseus cells cultured in a 14-l bioreactor increases alkaloid accumulation

A Catharanthus roseus cell line was cultured in a 14-l bioreactor. Total alkaloid production decreased more than 80% while scaling up this cell line from 250 ml batch cultures to the bioreactor. However, the subsequent application of an osmotic stress and 1 mM trans-cinnamic acid, which inhibits the synthesis of phenolic compounds, restored the original alkaloid amounts.     

Alkaloid accumulation in Catharanthus roseus cell suspension cultures fed with stemmadenine

Feeding stemmadenine to Catharanthus roseus cell suspension culture resulted in the accumulation of catharanthine, tabersonine and condylocarpine. Condylocarpine is not an intermediate in the pathway to catharanthine or tabersonine when it is fed to the cultures. The results support the hypothesis that stemmadenine is an intermediate in the pathway to catharanthine and tabersonine.     

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

An interchangeable system of hairy root and cell suspension cultures ofCatharanthus roseus for indole alkaloid production

Hairy roots ofCatharanthus roseus obtained by co-cultivation of hypocotyl segments withAgrobacterium rhizogenes, and cultured in SH (Schenk and Hildebrandt) basal medium, formed two types of calli when subcultured in SH medium with 1 mg/1 -naphthaleneacetic acid and 0.1 mg/l kinetin. One of them, a compact callus, when re-subcultured in SH basal medium gave rise to hairy roots again. A rhizogenic cell suspension culture was established from this type of callus. When cultured in SH medium with growth regulators, the rhizogenic callus produced catharanthine at a level of 41% of the level in the initial hairy roots. Upon transfer to SH basal medium, regenerated hairy roots produced this alkaloid at the original level of 1.5 mg/g dry wt. Using this cell/hairy root interchange system a new management system for hairy root culture in bioreactors has been devised and examined involving production of biomass in the form of a cell suspension in medium supplemented with growth regulators, and catharanthine production by hairy roots regenerated from these cells in medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - SH Schenk and Hildebrandt - SHNK SH medium + 1 mg 1^–1 NAA + 0.1 mg 1^–1 kinetin     

Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus

Catharanthus roseus cell suspension cultures converted exogenously supplied curcumin to a series of glucosides, none of which has been found in nature so far. The efficiency of glucosylation was dependent on culture stage of the cells and medium sucrose concentration. Methyl jasmonate and salicylic acid enhanced the glucoside formation only when they were added to the cultures prior to the addition of curcumin. The glucoside yield was 2.5 micromol/g fresh weight of the cells at an optimal culture condition. The water solubility of curcumin-4',4"-O-beta-D-digentiobioside was 0.65 mmol/ml, which was 20 million-fold higher than that of curcumin.     

Tryptophan decarboxylase from Catharanthus roseus cell suspension cultures: purification,molecular and kinetic data of the homogenous protein

The purification of tryptophan decarboxylase from Catharanthus roseus (TDC, E.C.:4.1.1.27), to apparent homogeneity, is described. The enzyme represents a soluble protein with a molecular weight of 115 000±3 000, consisting of 2 identical subunits of 54 000±1 000. The pI was estimated to be 5.9 and the K_m for L-tryptophan was found to be 7.5×10^-5 M. Phenylalanine, tyrosine and DOPA were not decarboxylated by tryptophan decarboxylase from Catharanthus cells. Similar to the aromatic amino acid decarboxylase from hog kidney the enzyme does not appear to be obligatorily dependent on exogenously supplied pyridoxal phosphate, as it seems to contain a certain amount of this cofactor. The average percentage of TDC in the cells was found to be 0.002% in the growth medium while the level increased up to 0.03% when indole alkaloid biosynthesis was induced. The role of the protein as a bottleneck enzyme of indole alkaloid biosynthesis is discussed.     

Improved alkaloid production in Catharanthus roseus suspension cell cultures by various chemicals

Fourteen chemicals were used to treat Catharanthus roseussuspension cell cultures to improve ajmalicine, catharanthine or serpentine biosynthesis. Ajmalicine production was increased by betaine (to 55 mg l^–1), n-propyl gallate (to 27 mg l^–1), succinic acid (to 31 mg l^–1), malic acid (to 60 mg l^–1) and tetramethyl ammonium bromide (to 64 mg l^–1). Ajmalicine and catharanthine yields were about 5–6 fold higher than the control. A large portion (up to 50–85%) of total indole alkaloids was released into the medium. For maximal catharanthine production, the optimal doses of malic acid and tetramethyl ammonium bromide were 50 mg l^–1and 120 mg l^–1, respectively. The mechanisms which may be responsible for these treatment effects are discussed.     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Ultrastructure ofCatharanthus roseus cells culturedin vitro and exposed to conditions for alkaloid accumulation

Summary Periwinkle (Catharanthus roseus) cells cultured in 1-B 5 medium display the ultrastructure of parenchyma cells. The parenchyma character remained unchanged when cells were exposed to any one of three different conditions effecting alkaloid accumulation. Transfer of cells to alkaloid production medium for 2 weeks (condition 1) accorded two special features,i.e., unusually big lipid droplets in the cytoplasm and, upon fixation, one or several electron-dense droplets of spongy precipitate in vacuoles. Among hormone-autotrophic cultures (condition 2) some cells showed a fine electron-dense vacuolar precipitate. Addition ofPhythium homogenate (fungal elicitor) to cells cultured in 1-B 5-medium for 10 days (condition 3), cells showed a frequent appearance of singular big lipid droplets in the cytoplasm, whereas vacuoles remained devoid of precipitate. The appearance of big lipid droplets and of vacuolar precipitate is interpreted as progressing cytodifferentiation, but is coincidental with alkaloid accumulation.NRCC no. 24524.     

High frequency plant regeneration from anther-derived cell suspension cultures via somatic embryogenesis in Catharanthus roseus

Summary A system for high frequency plant regeneration from cell suspension cultures in Catharanthus roseus is described. Calli were obtained from anthers cultured on Murashige and Skoog's medium supplemented with 1 mgl^-1 -naphthaleneacetic acid and 0.1 mgl^-1 kinetin. After the second subculture on solid medium, embryogenic callus was identified and transferred to liquid medium to initiate suspension cultures. Cells dispersed finely in the medium were subcultured at 14-day intervals. Upon plating onto the basal medium, yellowish compact colonies proliferated from the cells and more than 80% of them gave rise to somatic embryos. Subsequently, plantlets developed from the embryos. Both the plantlets and the source plants showed the normal somatic chromosome number of 2n=2x=16.Abbreviations MS Murashige and Skoog - MSNK MS medium + 1 mgl^-1 NAA + 0.1 mgl^-1 kinetin - NAA -naphthaleneacetic acid     

Impact of hypoxia on the growth and alkaloid accumulation in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension

The Madagascar periwinkle (Catharanthus roseus) produces numerous indole alkaloids, several of which have an important pharmaceutical uses such as ajmalicine, vinblastine and vincristine. The relationship between hypoxia and ajmalicine production in a cell suspension culture of C. roseus were investigated during the cycle of cell culture, in correlation with the effects on growth. The results show that the lack of oxygenation in C20D cells provokes a very strong inhibition in accumulation of the alkaloids and of other possible substances. Moreover, the addition of loganin, a metabolic intermediate of the biosynthetic pathway, in the culture medium of cells subjected to hypoxia restored the alkaloid production. Also, the results showed that the addition of benzyladenine (BA) to the culture medium increased the ajmalicine production and that the inhibitory effect of hypoxia was almost absent in these conditions. Therefore, it could be suggested that BA can without doubt decrease the effects of the hypoxia and increase the ajmalicine production in periwinkle cell suspensions.     

Plant regeneration from rice anthers cryopreserved by an encapsulation/dehydration technique

Summary Anther-derived rice (Oryza sativa L. ssp. japonica variety Yerua P.A.) plants were obtained after cryopreservation by an encapsulation/dehydration technique. Immature anthers, excised from spikelets pretreated at 8°C for 8d, were encapsulated in calcium alginate beads. The beads were cultured on N6 medium with 11.5 μM naphthalenaecetic acid (NAA) and 2.3 μM 6-furfurylaminopurine (KIN). Fifteen percent of the encapsulated anthers formed calluses when pretreated with sucrose for 3 d in liquid medium, desiccated on silica gel, slowly cooled to −30°C, immersed in liquid nitrogen (LN), thawed, and recultured. The cryopreserved encapsulated anthers produced 1.67 shoots/callus, in contrast to the control (non-cooled encapsulated anthers), which produced 6 shoots/callus. Eighty percent of the plantlets developed into normal plants after being transferred to greenhouse conditions. Histological observations showed that the origin of the plants was not modified by the cryopreservation process.     

Phenolic compounds in Catharanthus roseus

Besides alkaloids Catharanthus roseus produces a wide spectrum of phenolic compounds, this includes C6C1 compounds such as 2,3-dihydoxybenzoic acid, as well as phenylpropanoids such as cinnamic acid derivatives, flavonoids and anthocyanins. The occurrence of these compounds in C. roseus is reviewed as well as their biosynthesis and the regulation of the pathways. Both types of compounds compete with the indole alkaloid biosynthesis for chorismate, an important intermediate in plant metabolism. The biosynthesis C6C1 compounds is induced by biotic elicitors.