Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

Hormones in the grains and roots in relation to post-anthesis development of inferior and superior spikelets in japonica/indica hybrid rice

Grain filling is usually not adequate in later-flowering inferior spikelets in japonica/indica (J/I) hybrid rice (Oryza sativa) although it shows stronger hybrid vigor than indica/indica (I/I) hybrid. This study investigated the potential causes by examining changes in zeatin (Z) + zeatin riboside (ZR), indole-3-acetic acid (IAA), gibberellins (GAs, GA₁ + GA₄), and abscisic acid (ABA) in spikelets and roots during the grain filling period. The inferior spikelets of J/I hybrid exhibited low rate of endosperm cell division and slow grain filling. During the early grain filing period, they had less Z + ZR, IAA, and ABA, but more GAs, than the earlier-flowering superior spikelets. If compared to the inferior spikelets of the I/I hybrid, the J/I inferior spikelets also had less Z + ZR, IAA, and ABA. Rates of endosperm cell division and grain filling were positively and significantly correlated with Z + ZR and ABA contents in both grains and roots or IAA in grains, whereas not significantly correlated with GAs either in grains or roots or IAA in roots. Applications of kinetin, IAA, or ABA to spikelets, or kinetin and ABA to roots, enhanced cell division and grain filling in the inferior spikelets. Results suggest that low contents of cytokinins and ABA in both grains and roots and low contents of IAA in grains may result in the poor filling of inferior spikelets in the J/I hybrid.     

Gibberellic acid mediated induction of salt tolerance in wheat plants: Growth,ionic partitioning,photosynthesis, yield and hormonal homeostasis

In order to elucidate the GA₃-priming-induced physiochemical changes responsible for induction of salt tolerance in wheat, the primed and non-primed seeds of two spring wheat (Triticum aestivum L.) cultivars, namely, MH-97 (salt intolerant) and Inqlab-91 (salt tolerant) were sown in a field treated with 15 dS m⁻¹ NaCl salinity. Although all the three concentrations (100, 150 and 200 mg L⁻¹) of GA₃ were effective in improving grain yield in both cultivars, the effect of 150 mg L⁻¹ GA₃ was much pronounced particularly in the salt intolerant cultivar when under salt stress. Seed priming with GA₃ altered the pattern of accumulation of different ions between shoots and roots in the adult plants of wheat under saline conditions. Treatment with GA₃ (150 mg L⁻¹) decreased Na⁺ concentrations both in the shoots and roots and increased Ca²⁺ and K⁺ concentrations in the roots of both wheat cultivars. GA₃-priming did not show consistent effect on gaseous exchange characteristics and the concentrations of auxins in the salt stressed plants of both wheat cultivars. However, all concentrations of GA₃ reduced leaf free ABA levels in the salt intolerant, while reverse was true in the salt tolerant cultivar under saline conditions. Priming with GA₃ (150 mg L⁻¹) was very effective in enhancing salicylic acid (SA) concentration in both wheat cultivars when under salt stress. Treatment with GA₃ (100–150 mg L⁻¹) lowered leaf free putrescine (Put) and spermidine (Spd) concentrations in the plants of both wheat cultivars. The decrease in polyamines (Put and Spd) and ABA concentrations in the salt stressed plants of the salt intolerant cultivar treated with GA₃ suggested that these plants might have faced less stress compared with control. Thus, physiologically, GA₃-priming-induced increase in grain yield was attributed to the GA₃-priming-induced modulation of ions uptake and partitioning (within shoots and roots) and hormones homeostasis under saline conditions.     

The combined effects of gibberellic acid and salinity on some antioxidant enzyme activities,plant growth parameters and nutritional status in maize plants

The combined effects of salt stress and gibberellic acid (GA₃) on plant growth and nutritional status of maize (Zea mays L. cv., DK 647 F1) were studied in a pot experiment. Treatments were (1) control (C): nutrient solution alone, (2) salt stress (S): 100 mM NaCl, (3) S + GA1: 100 mM NaCl and 50 ppm GA₃ and (4) S + GA2: 100 mM NaCl and 100 ppm GA₃. Salt stress (S) was found to reduce the total dry matter, chlorophyll content, relative water content (RWC), but to increase proline accumulation, superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; 1.10.3.1) enzyme activities and electrolyte leakage. GA₃ treatments overcame to variable extents the adverse effects of NaCl stress on the above physiological parameters. GA₃ treatments reduced the activities of enzyme in the salt-stressed plants. Salt stress reduced some macro and micronutrient concentrations but exogenous application of GA₃ increased these to levels of control treatment. Foliar application of GA₃ counteracted some of the adverse effects of NaCl salinity with the accumulation of proline which maintained membrane permeability and increased macro and micronutrient levels.     

Formation of catharanthine,akuammicine and vindoline in Catharanthus roseus suspension cells

Catharanthine and akuammicine, together with ajmalicine and strictosidine, were isolated from a culture strain of Catharanthus roseus suspension cells. The biosynthetic capability of the cultured cells to produce akuammicine, catharanthine and vindoline was confirmed by feeding experiments with dl-tryptophan-[3-¹⁴C] to yield the radioactive alkaloids.     

Effects of water-deficit stress and paclobutrazol on growth,relative water content,electrolyte leakage,proline content and some antioxidant changes in Curcuma alismatifolia Gagnep. cv. Chiang Mai Pink

Effects of water-deficit stress and paclobutrazol (PBZ) on the physiological and biochemical changes in Curcuma alismatifolia Gagnep. cv. Chiang Mai Pink (Zingiberaceae) were investigated. One hundred rhizomes were grown for 30–35 days and then divided into the following 4 treatments: (1) well-watered, (2) not watered, (3) well-watered and treated with 1500 ppm PBZ being applied once to the soil, and (4) not watered but treated with 1500 ppm PBZ. After 50 days of growth, watering was withheld for 30 days. After water stress was initiated, plant height, plant fresh weight, soil water content, relative water content (RWC), electrolyte leakage (EL), proline content, vitamin C and E content, as well as the activities of catalase (CAT) and superoxide dismutase (SOD) in the leaves were determined every 10 days. The results showed that water-deficit stress decreased plant height and plant fresh weight, whereas this stress and PBZ did not result in a decrease in these parameters. Water stress reduced RWC, but induced EL and proline content in the leaves. However, the leaves showed opposite results when PBZ was added to the treatments. Some antioxidants such as vitamin C, vitamin E, and the activities of CAT and SOD were induced in the leaves by PBZ. Moreover, the content of vitamin C, vitamin E and CAT activity were higher in relation to water-deficit stress and PBZ treatments. This indicates that PBZ induced a number of some physiological and biochemical adaptations (maintaining growth and RWC, decreasing EL and proline content, increasing the vitamin C and vitamin E levels, and CAT and SOD activities) that enable the Curcuma plant to tolerate drought.     

Effect of nitrogen load on growth and photosynthesis of seedlings of the hybrid larch F1 (Larix gmelinii var. japonica × L. kaempferi) grown on serpentine soil

We studied the growth and photosynthesis of the hybrid larch F₁ (Larix gmelinii var. japonica × L. kaempferi) grown on serpentine soil and the effects of soil N load, to determine the performance of this species as reforestation material in serpentine regions. We prepared 16 experimental plots (2 m × 4 m each), eight on serpentine and eight on brown forest soil, and planted one-year-old cutting seedlings of the hybrid larch F₁ in each plot, in May 2007. Ammonium sulfate was supplied to half of the plots of each soil type in 2008 and 2009, at a load of 47 kg N ha⁻¹ year⁻¹. Although the growth and photosynthetic capacity of hybrid larch F₁ seedlings in the serpentine soil were limited, the rate of growth in serpentine soil was greater than that of Sakhalin spruce (Picea glehnii) that is dominant species in serpentine regions. There was significant interaction between soil type and N load for the growth and photosynthetic parameters. The N load adversely affected growth and photosynthetic parameters in the serpentine soil, while improved them in brown forest soil. Although the growth rate of hybrid larch F₁ without N loading showed high potential as an afforestation species in serpentine region, increasing deposition of N might be a threat to the growth and photosynthesis of the hybrid larch F₁ in serpentine soil.     

Ameliorative symbiosis of endophyte (Penicillium funiculosum LHL06) under salt stress elevated plant growth of Glycine max L.

Experiments were conducted to investigate the role of a newly isolated endophytic fungus GMC-2A on physiology of host plant (Glycine max. L cv. Hwangkeum-kong) growing under salinity stress. GMC-2A was identified as a new strain of Penicillium funiculosum on the basis of sequence homology and phylogenetic analysis of D1/D2 regions of 28S rDNA. Preliminary screening experiment showed that the culture filtrate (CF) of GMC-2A promoted the growth of Waito-C, a dwarf gibberellin (GA) biosynthesis mutant rice cultivar. Analysis of fungal CF revealed the presence of GAs (GA₁ 1.53 ng/ml; GA₄ 9.34 ng/ml; GA₈ 1.21 ng/ml; GA₉ 37.87 ng/ml) and indole acetic acid (14.85 μg/ml). GMC-2A also showed high phosphate solubilization of tricalcium phosphate. Besides that, GMC-2A application enhanced soybean seed germination as compared to control. Under salinity stress (70 and 140 mM), GMC-2A significantly promoted the soybean growth attributes (shoot length, shoot fresh/dry biomass, chlorophyll content, photosynthesis rate and leaf area) in comparison to control treatments. We also observed low endogenous abscisic acid and elevated jasmonic acid contents in GMC-2A treated plants under salt stress. GMC-2A treatment significantly enhanced levels of isoflavones (34.22% and 75.37%) under salinity stress as compared to control. In conclusion, P. funiculosum LHL06 has significantly ameliorated the adverse effects of salinity induced abiotic stress, and re-programmed soybean to higher growth and isoflavone biosynthesis.     

A differential response to chemical elicitors in Catharanthus roseus in vitro cultures

The effects of methyl jasmonate, salicylic acid and ethylene on alkaloid accumulation in in vitro cell suspension, hairy roots and rootless shoot cultures of Catharanthus roseus were analyzed. Ajmalicine, but not catharanthine, accumulation was promoted by jasmonate and ethylene treatments in cell suspensions. In hairy roots, jasmonate induced the accumulation of both alkaloids, whereas ethylene only induced catharanthine accumulation. In shoot cultures, positive effects of jasmonate and ethylene were recorded only in vindoline accumulation. Ethylene diminished catharanthine accumulation in these cultures. No effect of salicylic acid was observed in any of the studied in vitro culture systems. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of Alkaloid Uptake by Catharanthus roseus (L.) G. Don Protoplasts

The accumulation of alkaloids by protoplasts of Catharanthus roseus (L.) G. Don var. Little Bright Eye was studied to determine the specificity of uptake and the role of ion trapping in the storage of alkaloids. Accumulation of the indole alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation. The concentration and pH dependence of the initial burst of uptake for vindoline suggested that uptake occurred by simple diffusion. Uptake of nicotine was monophasic, with a half life of 5.2 minutes. The accumulation ratio (Ci/Ce) for nicotine at steady state and for the initial burst of uptake for vindoline and ajmalicine suggested that accumulation was driven by the pH gradient between the vacuole and the external assay medium. The second, sustained phase of uptake of vindoline was sensitive to inhibition by either 20 millimolar NaN₃ or 0.5 millimolar Cu²⁺. In azide-treated protoplasts, the uptake for vindoline conformed to the kinetics of simple diffusion, with a half life of 4 minutes. The second phase of uptake for ajmalicine, although sensitive to inhibition by Cu²⁺, was insensitive to inhibition by NaN₃. The biphasic uptake of the indole alkaloids was not due to any significant metabolism. It is concluded that accumulation and storage of the indole alkaloids is due only partly to ion trapping of the alkaloids by the low pH of the vacuole lumen. In the case of vindoline, there appears to be a specific energy-requiring uptake that is not seen with nicotine (which is not endogenous to Catharanthus). Accumulation of ajmalicine appears to involve both ion trapping and an azide-insensitive component, which may be due to complexation with organic counterions and phenolics.     

Effects of testosterone and 2-hydroxyflutamide on progesterone receptor expression in porcine ovarian follicles in vitro

The purpose of the study was to test the possible role of the androgen receptor (AR) agonist (testosterone; T), an AR antagonist (2-hydroxyflutamide; 2-Hf) or combination of both (T + 2-Hf) on progesterone receptor (PGR) expression in cultured porcine granulosa cells (GCs) or whole follicles. GCs isolated from mature pig follicles (6–8 mm in diameter) were cultured for 48 h. Experimental cultures were carried out with the addition of T (10^?7 M), 2-Hf (1.7 × 10^?4 M) or both T and 2-Hf for the last 24 h of culture. To better imitate in vivo conditions, isolated whole porcine follicles (6–8 mm in diameter) were cultured for 24 h in an organ culture system, with the addition of the same factors. The cells or sections obtained from cultured follicles were processed for PGR immunocytochemical or immuno-histochemical staining. In addition, expression of PGR protein was determined by Western blot and progesterone (P₄) concentrations in the culture media were measured by a radioimmunoassay. We found that isoform A of PGR is expressed in both granulosal and follicular cultures. The 2-Hf in the presence of T increased PGR protein expression in porcine GCs and whole follicles. In both granulosal and follicular cultures, 2-Hf or T alone inhibited P₄ secretion, but simultaneous addition of 2-Hf and T increased P₄ secretion. Our results indicate that androgens may be involved in the control of PGR expression in porcine GCs in vitro. Moreover, we suggest a potential auto/paracrine regulation of the follicular function by androgen-dependent signaling pathway.     

Effect of plant growth regulators on the biosynthesis of vinblastine,vindoline and catharanthine in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine.     

Photosynthetic and physiological responses of Kandelia candel L. Druce seedlings to duration of tidal immersion in artificial seawater

The present study demonstrates the influence of the duration of periodical waterlogging with artificial seawater on the photosynthetic and physiological responses of Kandelia candel L. Druce seedlings, the pre-dominant species of subtropical mangroves in China. Artificial tidal fluctuations applied here closely mimicked the twice daily tidal inundation which mangroves experience in the field. All the seedlings were immersed in artificial seawater during 70-day cultivation. Similar trends with increasing duration of immersion occurred in photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO₂ concentration, where significant decreases occurred only in long time treatments of 10 or 12 h. Water used efficiency and chlorophyll contents showed lower in medium periods and higher in long periods of immersion. This indicates that the increase in pigment contents of leaves was ineffective in promoting P_n under long time immersion. Light saturation points under short time waterlogging (0–4 h) occurred at light intensities of 800–1000 μmol/m²/s, and at around 400 μmol/m²/s in long time treatments (8–12 h). Long periods of tidal immersion therefore significantly inhibited photosynthesis of mature leaves. Alcohol dehydrogenase and oxidase activity in roots both increased under longer immersion periods, suggesting that roots are sensitive to anaerobiosis under long term waterlogging. The activities of peroxidase and superoxide dismutase in mature leaves increased in 8 h and 10 h treatments, respectively. The content of malondialdehyde in mature leaves increased under long time treatments. Abscisic acid accumulation in mature leaves also had a sharp increase from 8 h to 12 h inundation. Even though the anti-oxidative enzymes were induced by waterlogging, this was not sufficient to protect the seedlings from senescence. The results suggested that K. candel seedlings completely tolerated tidal immersion by seawater up to about 8 h in each cycle, which matches the natural distribution of K. candel in inter-tidal zones of China.     

Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering

Catharanthus roseus plants produce many pharmaceutically important indole alkaloids, of which the bisindole alkaloids vinblastine and vincristine are antineoplastic medicines and the monoindole alkaloids ajmalicine and serpentine are antihypertension drugs. C. roseus cell cultures have been studied for producing these medicines or precursors catharanthine and vindoline for almost four decades but so far without a commercially successful process due to biological and technological limitations. The research thus focused on the one hand on engineering the bioreactor process on the other engineering the cell factory itself. This review mainly summarizes the progress made on biochemical engineering aspects of C. roseus cell cultures in bioreactors in the past decades and metabolic engineering of indole alkaloid production in recent years. The paper also attempts to highlight new strategies and technologies to improve alkaloid production and bioreactor performance. Perspectives of metabolic engineering to create new cell lines for large-scale production of indole alkaloids in bioreactors and effective combination of these up- and down-stream processing are presented.     

Changes in root system structure,leaf water potential and gas exchange of maize and triticale seedlings affected by soil compaction

The physiological reasons associated with differential sensitivity of C₃ and C₄ plant species to soil compaction stress are not well explained and understood. The responses of growth characteristics, changes in leaf water potential and gas exchange in maize and triticale to a different soil compaction were investigated. In the present study seedlings of triticale and maize, representative of C₃ and C₄ plants were subjected to low (L – 1.10 g cm⁻³), moderate (M – 1.34 g cm⁻³) and severe (S – 1.58 g cm⁻³) soil compaction level. Distinct differences in distribution of roots in the soil profile were observed. Plants of treatments M or S in comparison to treatment L, showed a decrease in leaf number, dry mass of stem, leaves and roots, and an increase in the shoot to root ratio. A drastic decrease in root biomass in M and S treatments in the soil profile on depth from 15 to 40 cm was observed. Any level of soil compaction did not influence the number of seminal and seminal-adventitious roots but decreased their length. The number and total length of nodal roots decreased with compaction. Changes of growth traits in M and S treatments in comparison to the L were greater for maize than for triticale and were accompanied by daily changes in water potential (ψ) and gas exchange parameters (P_N, E, g_s). Differences between M and S treatments in daily changes in ψ for maize were in most cases statistically insignificant, whereas for triticale, they were statistically significant. Differences in the responses of maize and triticale to soil compaction were found in P_N, E and g_s in particular for the measurements taken at 12:00 and 16:00. The highest correlation coefficients were obtained for the relationship between leaf water potential and stomatal conductance, both for maize and triticale, which indicates the close association between stomata behavior and changes in leaf water status.     

Extraction of indole alkaloids fromCatharanthus roseus by using supercritical carbon dioxide

Summary Indole alkaloids, particularly vindoline and catharanthine, were extracted from the leaves ofCatharanthus roseus by supercritical extraction with CO₂. The contents of vindoline and catharanthine in the extracts were determined by HPLC and identified by LC/MS. About 52 %(w/w) of the initial vindoline content, 1.5 mg vindoline/g dry wt leaves, was recovered after extracting this material for 10 h with the CO₂ flow rate of 400 ml/min at 40°C and 150 bar. Vindoline concentration in the extract was 67 %(w/w).     

Gas composition strategies for the successful scale-up of Catharanthus roseus cell cultures for the production of ajmalicine

Ajmalicine, serpentine, catharanthine, and vindoline are monoterpenoid indole alkaloids (MIAs) of commercial interest which are produced by the Catharanthus roseus plant. Cultures of C. roseus have been investigated as a potential source of these pharmaceutically important compounds since the early 1960s. In addition, their production from C. roseus cultures has served as a model system for investigating secondary metabolism and for evaluating production-enhancing strategies. Initially, this review will survey (1) the MIAs of interest for large-scale production from plant cell cultures and (2) the volumetric productivities of a specific MIA, ajmalicine, achieved and projected using plant cell cultures. To meet the need for these valuable compounds, the production of these MIAs from plant cell cultures must be successfully reproduced in large-scale aerated and agitated reactors. While the large-scale cultivation of plant cell cultures is currently feasible, initial attempts at scale-up may yield results that differ from that optimized in flasks. To bridge the jump between production in flasks and production in large-scale bioreactors, changes introduced with scale-up such as gas composition must be identified and rationally manipulated to reproduce or even improve growth and secondary metabolite production. Hence, this review will (1) identify the effects of gas composition (i.e., O₂, CO₂, ethylene, or other endogenous volatile compounds) on growth and secondary metabolism and (2) draw operating strategies for optimizing the gas composition for growth of C. roseus cultures and the production of ajmalicine.     

Ornamental characters,ion accumulation and water status in Arbutus unedo seedlings irrigated with saline water and subsequent relief and transplanting

Arbutus unedo seedlings were grown in a greenhouse and submitted to three irrigation treatments (salinity period) using solutions with an EC of 0.85 dS m^?1 (control treatment), 5.45 dS m^?1 (S1) and 9.45 dS m^?1 (S2). After 16 weeks, growth and ornamental characters, leaf water potentials, gas exchange and ion concentrations were determined. After the salinity period, plants were exposed to a relief period for 1 month, whereby half of the plants were transplanted to field conditions and the other half into 24 cm diameter plastic pots. Salinity induced a significant decrease in shoot biomass and leaf area but root/shoot ratio was increased. Plant height was significantly inhibited by salinity. The ornamental characters were affected in the treated plants, with symptoms of salt injury, such as burning of leaf margin. Leaf water potentials decreased with increasing salinity, more significantly at predawn than at midday. The relationship between net photosynthesis (P_n) and leaf conductance (g_l) was linear for all treatments and the same values of P_n are associated with lower values of g_l for the saline treatments than for control treatment. The concentration of Cl^? in leaves increased with increasing salinity and was higher than the corresponding concentration of Na⁺. Na⁺ and Cl^? contents were higher in the leaves than in the roots in both saline treatments. The K⁺ and Ca²⁺ levels were lower in the treated plants than in control plants and applied salinity reduced the K⁺/Na⁺ ratio in leaves, stems and roots, the decrease being much greater for leaves than for roots. The Ca²⁺/Na⁺ ratio fell with salinity in all parts of the plants. At the end of the relief period leaf water potentials were recovered mainly in field conditions. S2 treatment showed lower values of P_n and g_l than control and S1 treatments in pot conditions and in field conditions S1 showed the lowest values for P_n and g_l.