Enhancement of Vindoline and Catharanthine Accumulation, Antioxidant Enzymes Activities, and Gene Expression Levels in Catharanthus roseus Leaves by Chitooligosaccharides Elicitation.

Catharanthus roseus (L.) G. Don is a plant belonging to the genus Catharanthus of the Apocynaceae family. It contains more than one hundred alkaloids, of which some exhibit significant pharmacological activities. Chitooligosaccharides are the only basic aminooligosaccharides with positively charged cations in nature, which can regulate plant growth and antioxidant properties. In this study, the leaves of Catharanthus roseus were sprayed with chitooligosaccharides of different molecular weights (1 kDa, 2 kDa, 3 kDa) and different concentrations (0.01 µg/mL, 0.1 µg/mL, 1 µg/mL and 10 µg/mL). The fresh weights of its root, stem and leaf were all improved after chitooligosaccharides treatments. More importantly, the chitooligosaccharides elicitor strongly stimulated the accumulation of vindoline and catharanthine in the leaves, especially with the treatment of 0.1 µg/mL 3 kDa chitooligosaccharides, the contents of them were increased by 60.68% and 141.54%, respectively. Furthermore, as the defensive responses, antioxidant enzymes activities (catalase, glutathione reductase, ascorbate peroxidase, peroxidase and superoxide dismutase) were enhanced under chitooligosaccharides treatments. To further elucidate the underlying mechanism, qRT-PCR was used to investigate the genes expression levels of secologanin synthase (SLS), strictosidine synthase (STR), strictosidine glucosidase (SGD), tabersonine 16-hydroxylase (T16H), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT), peroxidase 1 (PRX1) and octadecanoid-responsive Catharanthus AP2-domain protein 3 (ORCA3). All the genes were significantly up-regulated after chitooligosaccharides treatments, and the transcription abundance of ORCA3, SLS, STR, DAT and PRX1 reached a maximal level with 0.1 µg/mL 3 kDa chitooligosaccharides treatment. All these results suggest that spraying Catharanthus roseus leaves with chitooligosaccharides, especially 0.1 µg/mL of 3 kDa chitooligosaccharides, may effectively improve the pharmaceutical value of Catharanthus roseus.

Induced genetic and chemical changes in medicinally important plant Catharanthus roseus (L.) G. Don: cold plasma and phytohormones.

BACKGROUND: Catharanthus roseus (L.) G. Donis a medicinal plant species belonging to the Apocynaceae family, which produces vinblastine and vincristine along with 100 other monoterpenoid indole alkaloids. The process of biosynthesis of C. roseus alkaloids is complex, in which many genes, enzymes, and regulators are involved. Induced mutations may be considered as a potential source for producing a higher amount of vinblastine and vincristine in this plant species. Therefore, the objective of the present study was to examine the effects of different treatments utilized on the induced genetic changes in C. roseus plants and enzyme activities. METHODS AND RESULTS: Spermine, jasmonic acid, methyjasmonate, putrescine, and cold plasma treatments were used for seed treatments. Different molecular markers, namely inter simple sequence repeat, inter retrotransposon amplified polymorphism, and retrotransposon microsatellite amplified polymorphism were employed to reveal the induced genetic changes. Antioxidant enzyme activities were also studied. The treated plants showed genetic variability and a significant increase in antioxidant enzyme activity compared to the control plants. The putrescine treatment resulted in the highest level of activity in superoxidase. A significant positive correlation occurred between the molecular markers data and antioxidant enzyme activities in treated plants. CONCLUSION: Our data revealed that the different phytohormones and cold plasma treatments could induce both genetic and chemical content changes in C. roseus plants.

Versatile Roles of the Receptor-Like Kinase Feronia in Plant Growth, Development and Host-Pathogen Interaction.

As a member of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein kinase subfamily, FERONIA (FER) has emerged as a versatile player regulating multifaceted functions in growth and development, as well as responses to environmental factors and pathogens. With the concerted efforts of researchers, the molecular mechanism underlying FER-dependent signaling has been gradually elucidated. A number of cellular processes regulated by FER-ligand interactions have been extensively reported, implying cell type-specific mechanisms for FER. Here, we provide a review on the roles of FER in male-female gametophyte recognition, cell elongation, hormonal signaling, stress responses, responses to fungi and bacteria, and present a brief outlook for future efforts.

Developmental Methylome of the Medicinal Plant Catharanthus roseus Unravels the Tissue-Specific Control of the Monoterpene Indole Alkaloid Pathway by DNA Methylation.

Catharanthus roseus produces a wide spectrum of monoterpene indole alkaloids (MIAs). MIA biosynthesis requires a tightly coordinated pathway involving more than 30 enzymatic steps that are spatio-temporally and environmentally regulated so that some MIAs specifically accumulate in restricted plant parts. The first regulatory layer involves a complex network of transcription factors from the basic Helix Loop Helix (bHLH) or AP2 families. In the present manuscript, we investigated whether an additional epigenetic layer could control the organ-, developmental- and environmental-specificity of MIA accumulation. We used Whole-Genome Bisulfite Sequencing (WGBS) together with RNA-seq to identify differentially methylated and expressed genes among nine samples reflecting different plant organs and experimental conditions. Tissue specific gene expression was associated with specific methylation signatures depending on cytosine contexts and gene parts. Some genes encoding key enzymatic steps from the MIA pathway were found to be simultaneously differentially expressed and methylated in agreement with the corresponding MIA accumulation. In addition, we found that transcription factors were strikingly concerned by DNA methylation variations. Altogether, our integrative analysis supports an epigenetic regulation of specialized metabolisms in plants and more likely targeting transcription factors which in turn may control the expression of enzyme-encoding genes.

Catharanthus roseus potential for phyto-stabilizing metals in sewage sludge.

This work evaluated the potential of Catharanthus roseus in phytoremediation of As, Ba, Cd, Cu, Cr, Ni, Pb, Se and Zn in sewage sludge-based substrates. C. roseus was cultivated for 108 days in a treatment containing sewage sludge:vermiculite (70:30%) and in the control with 100% commercial substrate. The plants cultivated in sludge showed approximately four times greater height, number of leaves and stem diameter, as well as 89% higher fresh mass than those of the control. The highest concentrations of the metals were obtained in the roots of plants grown in the sludge, and ranged from 2.04 (Cd) to 1121 mg kg-1 (Zn). Cu, Cd and Zn had a higher bioconcentration factor than 1 in both treatments. On the other hand, the translocation factor value in the control was greater than 1 for Ba, Cd, Cu, Ni, Se and Zn. The results showed that C. roseus was efficient in the phytoremediation of the evaluated metals, in which the translocation process was progressive in the treatment that presented higher metal content.

Improvement of Commercially Valuable Traits of Industrial Crops by Application of Carbon-based Nanomaterials.

Carbon-based nanomaterials (CBNs) have great potential as a powerful tool to improve plant productivity. Here, we investigated the biological effects of graphene and carbon nanotubes (CNTs) on fiber-producing species (cotton, Gossypium hirsutum) and ornamental species (vinca, Catharanthus roseus). The exposure of seeds to CNTs or graphene led to the activation of early seed germination in Catharanthus and overall higher germination in cotton and Catharanthus seeds. The application of CBNs resulted in higher root and shoot growth of young seedlings of both tested species. Cultivation of Catharanthus plants in soil supplemented with CBNs resulted in the stimulation of plant reproductive system by inducing early flower development along with higher flower production. Catharanthus plants cultivated in CNTs or graphene supplemented soil accelerated total flower production by 37 and 58%, respectively. Additionally, CBNs reduced the toxic effects caused by NaCl. Long-term application of CBNs to crops cultivated under salt stress conditions improved the desired phenotypical traits of Catharanthus (higher flower number and leaf number) and cotton (increased fiber biomass) compared to untreated plants of both species cultivated at the same stress condition. The drought stress experiments revealed that introduction of CBNs to matured Catharanthus plant increased the plant survival with no symptoms of leaf wilting as compared to untreated Catharanthus growing in water deficit conditions.

Carbon-based nanomaterials as stimulators of production of pharmaceutically active alkaloids in cell culture of Catharanthus roseus.

Carbon-based nanomaterials (CBNs) were previously described as regulators of plant cell division. Here, we demonstrated the ability of multi-walled carbon nanotubes (MWCNT) and graphene to enhance biomass production in callus culture of the medicinal plant Catharanthus roseus cultivated in dark conditions. Furthermore, both tested CBNs were able to stimulate biosynthesis of total produced alkaloids in CBN-exposed callus culture of Catharanthus. In one case, total alkaloids in CBN-exposed Catharanthus were double that of unexposed Catharanthus. Analysis of metabolites by HPLC revealed that production of the pharmaceutically active alkaloids vinblastine and vincristine was dramatically enhanced in callus exposed to MWCNT or graphene in both dark and light conditions of callus cultivation. In vitro assays (MTT, flow cytometry) demonstrated that total alkaloid extracts derived from Catharanthus callus treated with CBNs significantly reduced cell proliferation of breast cancer (MCF-7) and lung cancer (A549) cell lines compared to the application of extracts derived from untreated Catharanthus callus.

Melatonin-stimulated biosynthesis of anti-microbial ZnONPs by enhancing bio-reductive prospective in callus cultures of Catharanthus roseus var. Alba.

Melatonin as plant growth regulator induces differential effects on metabolites that are responsible for reduction, capping and stabilization of zinc oxide nanoparticles. Phytochemical analysis of callus cultures was performed and results were compared with callus cultures supplemented with other plant growth regulators (α-napthalene acetic acid, 2,4-dichlorophenoxy acetic acid and thidiazuron). Highest total phenolic and flavonoid content [42.23 mg of gallic acid equivalent (GAE) g-1 DW and 36.4 mg of (quercetin equivalent) g-1 DW, respectively] were recorded at melatonin (1.0 µM) + NAA (13.5 µM). ZnONPs were synthesized from NAA (13.5 µM) and melatonin (1.0 µM) + NAA (13.5 µM)-induced calli extracts separately and characterized via X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). FTIR analysis confirmed the presence of phenolics and flavonoids that were mainly found responsible for reduction and capping of ZnONPs. SEM analysis showed triangular shaped ZnONPs synthesized from melatonin + NAA callus extract and these NPs were more dispersed as compared to the spherical-agglomerates of ZnONPs synthesized from NAA-mediated callus extract. Melatonin + NAA callus extract-mediated ZnONPs (having smaller size) were more potent against multiple drug resistant bacterial strains, e.g. Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa by producing zone of inhibitions 17 ± 0.76 mm,10 ± 0.57 mm and 13 ± 0.54 mm, respectively.

A rich source of potential bioactive compounds with anticancer activities by Catharanthus roseus cambium meristematic stem cell cultures.

ETHNOPHARMACOLOGICAL IMPORTANCE: Catharanthus roseus (L.) G. Don. is an important medicinal plant with rich sources of remarkable health benefits consisting more than 100 alkaloids and significant amounts of bioactive compounds, which have been widely used as a folk medicine for treatment of several pathologies. THE AIM OF THE STUDY: In the present study, we isolated and cultured innately undifferentiated cambium meristematic cells (CMCs), which were observed stable cell growth, enhancement of bioactive compounds from C.roseus. MATERIALS AND METHODS: We attempted to determine the effect of association between time-course growth rates, bioactive compounds and terpenoids indole alkaloid (TIA) contents as well as antioxidant and anticancer efficacies of C. roseus CMC suspension culture treated by UV-C. RESULTS: The bioactive compounds, vincristine contents, and antioxidant power were noticed significantly higher in 60 min exposure at 5 cm distances and with the directly collected sample (T7). A similar trend has also been noticed from the anticancer activity. Demonstration of TIA accumulation was found higher at 5 min exposure, at 20 cm distances and 48 h of incubation (T21) and the result of TIA contents had the highest correlation effects of anticancer activities. CONCLUSION: In the current study, we demonstrated that UV-C light could enhance the production of the essential compounds and bioactivities in the CMCs of C. roseus, and thus, C. roseus CMCs have the potential to serve as an industrial platform for the production of bioactive alkaloids and antioxidant, anticancer activity. Moreover, additional efforts should be made to irradiate CMC suspension cultures from C. roseus with UV-C to achieve better pharmacological profiles.

Effect of Pseudomonas fluorescens RB4 and Bacillus subtilis 189 on the phytoremediation potential of Catharanthus roseus (L.) in Cu and Pb-contaminated soils.

The remediation of heavy metal-contaminated soils has become a critical issue due to toxic effects of these metals on living organisms. The current research was conducted to study the effect of Pseudomonas fluorescens RB4 and Bacillus subtilis 189 on the growth and phytoremediation potential of Catharanthus roseus in Cu- and Pb-contaminated soils. The bacterial strains exhibited significantly higher level of water-extractable Pb and Cu in Pb, Cu, and Cu+Pb-contaminated. The P. fluorescens RB4 inoculated plants, produced 102%, 48%, and 45% higher fresh weight (FW) in soils contaminated with Cu, Pb, and both elements, respectively, as compared to un-inoculated control plants. Similarly, B. subtilis 189 inoculated plants produced 108%, 43%, and 114% more FW in the presence of Cu, Pb, and both elements. The plants co-cultivated with both bacteria exhibited 121%, 102%, and 177% higher FW, in Cu, Pb, and both elements contaminated soils, as compared to respective un-inoculated control. Co-cultivation of P. fluorescens RB4, B. subtilis 189, and P. fluorescens RB4 + B. subtilis 189 resulted in higher accumulation of Cu and Pb in shoots of the C. roseus grown in contaminated soils as compared to un-inoculated control. Bacterial treatments also improved the translocation and metal bioconcentration factors. The growth and phytoextraction capability of C. roseus was improved by inoculation of P. fluorescens RB4 and B. subtilis 189.

Screening and kinetic studies of catharanthine and ajmalicine accumulation and their correlation with growth biomass in Catharanthus roseus hairy roots.

Context Catharanthus roseus (L.) G. Don (Apocynaceae) is still one of the most important sources of terpene indole alkaloids including anticancer and hypertensive drugs as vincristine and vinblastine. These final compounds have complex pathway and many enzymes are involved in their biosynthesis. Indeed, ajmalicine and catharanthine are important precursors their increase can lead to enhance levels of molecules of interest. Objective This study aims at selecting the highest yield of hairy root line(s) and at identifying best times for further treatments. We study kinetics growth and alkaloids (ajmalicine and catharanthine) accumulation of three selected hairy root lines during the culture cycle in order to determine the relationship between biomass production and alkaloids accumulation. Materials and methods Comparative analysis has been carried out on three selected lines of Catharanthus roseus hairy roots (LP10, LP21 and L54) for their kinetics of growth and the accumulation of ajamalicine and catharanthine, throughout a 35-day culture cycle. The methanolic extract for each line in different times during culture cycle is analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results Maximum accumulation of the alkaloids is recorded for LP10 line in which the peak of ajmalicine and catharanthine accumulation reached to 3.8 and 4.3 mg/g dry weight (DW), respectively. This increase coincides with an exponential growth phase. Discussion and conclusion Our results suggest that the evolution of accumulation of ajmalicine and catharanthine are positively correlated with the development of the biomass growth. Significantly, for LP10 line the most promising line to continue optimizing the production of TIAs. Additionally, the end of exponential phase remains the best period for elicitor stimuli.

Understanding CrRLK1L Function: Cell Walls and Growth Control.

To develop successfully in an ever-changing environment, it is essential for plants to monitor and control their growth. Therefore, cell expansion is carefully regulated to establish correct cell shape and size. In this review, we explore the role of the Catharanthus roseus receptor-like kinase (CrRLK1L) subfamily as regulators of cell expansion. Recently, the downstream signalling events of individual CrRLK1L pathways were discovered, implicating known modulators of cell expansion, such as reactive oxygen species (ROS) production, Ca(2+) dynamics, and exocytosis of cell wall material. Based on these intriguing new insights, we propose a model for a common pathway of CrRLK1L signalling that enables spatial and temporal control of cell wall extensibility throughout the plant.

A novel terpenoid indole alkaloid derived from catharanthine via biotransformation by suspension-cultured cells of Catharanthus roseus.

OBJECTIVE: Although catharanthine (1) is well known as a biosynthetic precursor of the anticancer alkaloid, vinblastine, its alternative metabolic pathways are unclear. RESULTS: Biotransformation of 1 by suspension-cultured cells of Catharanthus roseus gave a new oxidative-cleavage product (2). The structure of 2 was determined as 3-hydroxy-4-imino-catharanthine by spectroscopic methods. Maximum conversion (9.75 %) of 2 was observed after 120 h adding 6 mg of 1/100 ml to 12-day-old suspension-cultured cells of C. roseus. Furthermore, qRT-PCR experiment was performed to reveal the effect of 1 on the expression of the genes in the biosynthetic pathway of TIA 1 up-regulated the transcript level of D4H whilst down-regulating the transcript levels of G10H, LAMT, GES, and IRS. CONCLUSION: A new metabolite of catharanthine, 3-hydroxy-4-imino-catharanthine, is reported.

Effects of ambient and elevated CO2 on growth, chlorophyll fluorescence, photosynthetic pigments, antioxidants, and secondary metabolites of Catharanthus roseus (L.) G Don. grown under three different soil N levels.

Catharanthus roseus L. plants were grown under ambient (375 ± 30 ppm) and elevated (560 ± 25 ppm) concentrations of atmospheric CO2 at different rates of N supply (without supplemental N, 0 kg N ha(-1); recommended N, 50 kg N ha(-1); and double recommended N, 100 kg N ha(-1)) in open top chambers under field condition. Elevated CO2 significantly increased photosynthetic pigments, photosynthetic efficiency, and organic carbon content in leaves at recommended (RN) and double recommended N (DRN), while significantly decreased total nitrogen content in without supplemental N (WSN). Activities of superoxide dismutase, catalase, and ascorbate peroxidase were declined, while glutathione reductase, peroxidase, and phenylalanine-ammonia lyase were stimulated under elevated CO2. However, the responses of the above enzymes were modified with different rates of N supply. Elevated CO2 significantly reduced superoxide production rate, hydrogen peroxide, and malondialdehyde contents in RN and DRN. Compared with ambient, total alkaloids content increased maximally at recommended level of N, while total phenolics in WSN under elevated CO2. Elevated CO2 stimulated growth of plants by increasing plant height and numbers of branches and leaves, and the magnitude of increment were maximum in DRN. The study suggests that elevated CO2 has positively affected plants by increasing growth and alkaloids production and reducing the level of oxidative stress. However, the positive effects of elevated CO2 were comparatively lesser in plants grown under limited N availability than in moderate and higher N availability. Furthermore, the excess N supply in DRN has stimulated the growth but not the alkaloids production under elevated CO2.

Salicylic acid alters antioxidant and phenolics metabolism in Catharanthus roseus grown under salinity stress.

BACKGROUND: Salicylic acid (SA) acts as a potential non-enzymatic antioxidant and a plant growth regulator, which plays a major role in regulating various plant physiological mechanisms. The effects of salicylic acid (SA; 0.05 mM) on physiological parameters, antioxidative capacity and phenolic metabolism, lignin, alkaloid accumulation in salt stressed Catharanthus roseus were investigated. MATERIALS AND METHODS: Catharanthus roseus seeds were grown for two months in a glass house at 27-30°C in sunlight, and then divided into four different groups and transplanted with each group with the following solutions for one month: group I (non-saline control), group II, 100 mM NaCl, group III, 0.05 mM SA, group IV, 100 mM NaCl+0.05 mM SA and to determine the physiological parameters (DW, FW, WC), chlorophyll contents, carotenoid contents, lipid peroxidation, phenolics, lignin, alkaloid and enzymatic assays in each leaf pairs and roots. RESULTS: SA exhibited growth-promoting property, which correlated with the increase of dry weight, water content, photosynthetic pigments and soluble proteins. SA has additive effect on the significant increase in phenylalanine ammonia-lyase (PAL) activity, which is followed by an increase in total soluble phenolics and lignin contents in all leaf pairs and root of C. roseus. SA enhances malondialdehyde content in all leaf pairs and root. The antioxidant enzymes (catalase, glutathione reductase, glutathione-S-tranferase, superoxide dismutase, peroxidase) as well as alkaloid accumulation increased in all treatments over that of non-saline control but the magnitude of increase was found more in root. Further, the magnitude of increase of alkaloid accumulation was significantly higher in 100 mM NaCl, but highly significant was found in presence of 0.05 mM SA and intermediate in presence of both 0.05 mM SA+100 mM NaCl. CONCLUSION: We concluded that applied SA to salt stress, antioxidant and phenolic metabolism, and alkaloid accumulation were significantly altered and the extent of alteration varied between the SA and salt stress.

High-throughput transcriptome analysis of the leafy flower transition of Catharanthus roseus induced by peanut witches'-broom phytoplasma infection.

Peanut witches'-broom (PnWB) phytoplasma are obligate bacteria that cause leafy flower symptoms in Catharanthus roseus. The PnWB-mediated leafy flower transitions were studied to understand the mechanisms underlying the pathogen-host interaction; however, our understanding is limited because of the lack of information on the C. roseus genome. In this study, the whole-transcriptome profiles from healthy flowers (HFs) and stage 4 (S4) PnWB-infected leafy flowers of C. roseus were investigated using next-generation sequencing (NGS). More than 60,000 contigs were generated using a de novo assembly approach, and 34.2% of the contigs (20,711 genes) were annotated as putative genes through name-calling, open reading frame determination and gene ontology analyses. Furthermore, a customized microarray based on this sequence information was designed and used to analyze samples further at various stages of PnWB infection. In the NGS profile, 87.8% of the genes showed expression levels that were consistent with those in the microarray profiles, suggesting that accurate gene expression levels can be detected using NGS. The data revealed that defense-related and flowering gene expression levels were altered in S4 PnWB-infected leafy flowers, indicating that the immunity and reproductive stages of C. roseus were compromised. The network analysis suggested that the expression levels of >1,000 candidate genes were highly associated with CrSVP1/2 and CrFT expression, which might be crucial in the leafy flower transition. In conclusion, this study provides a new perspective for understanding plant pathology and the mechanisms underlying the leafy flowering transition caused by host-pathogen interactions through analyzing bioinformatics data obtained using a powerful, rapid high-throughput technique.

Pleiotropic phenotypes of the salt-tolerant and cytosine hypomethylated leafless inflorescence, evergreen dwarf and irregular leaf lamina mutants of Catharanthus roseus possessing Mendelian inheritance.

In Catharanthus roseus, three morphological cum salt-tolerant chemically induced mutants of Mendelian inheritance and their wild-type parent cv Nirmal were characterized for overall cytosine methylation at DNA repeats, expression of 119 protein coding and seven miRNA-coding genes and 50 quantitative traits. The mutants, named after their principal morphological feature(s), were leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill). The Southern-blot analysis of MspI digested DNAs of mutants probed with centromeric and 5S and 18S rDNA probes indicated that, in comparison to wild type, the mutants were extensively demethylated at cytosine sites. Among the 126 genes investigated for transcriptional expression, 85 were upregulated and 41 were downregulated in mutants. All of the five genes known to be stress responsive had increased expression in mutants. Several miRNA genes showed either increased or decreased expression in mutants. The C. roseus counterparts of CMT3, DRM2 and RDR2 were downregulated in mutants. Among the cell, organ and plant size, photosynthesis and metabolism related traits studied, 28 traits were similarly affected in mutants as compared to wild type. Each of the mutants also expressed some traits distinctively. The egd mutant possessed superior photosynthesis and water retention abilities. Biomass was hyperaccumulated in roots, stems, leaves and seeds of the lli mutant. The ill mutant was richest in the pharmaceutical alkaloids catharanthine, vindoline, vincristine and vinblastine. The nature of mutations, origins of mutant phenotypes and evolutionary importance of these mutants are discussed.

Salicylic acid restrains nickel toxicity, improves antioxidant defence system and enhances the production of anticancer alkaloids in Catharanthus roseus (L.).

Salicylic acid (SA) has been reported to ameliorate various stresses in plants. In order to explore the role of SA under nickel (Ni) stress, thirty-days old plants of periwinkle (Catharanthus roseus L.) were supplied with eight treatments comprising basal application of Ni (0, 50, 100 and 150 mg kg(-1)) and foliar application of SA (0 and 10(-5)M) under net house conditions. Ni application significantly reduced the growth attributes including plant height, leaf-area index and fresh and dry weights of shoot and root. Increasing Ni concentration led to a gradual decrease in photosynthetic parameters and activities of nitrate reductase and carbonic anhydrase. The plants, undergoing Ni stress, exhibited a significant increase in the activity of superoxide dismutase, catalase and peroxidase together with an increase in electrolyte leakage and proline content. Total alkaloid content was also declined in Ni-treated plants. Foliar application of SA (10(-5)M) reduced the deleterious effects of Ni on plant growth, accelerating the restoration of growth processes. SA also improved the total alkaloid content under normal as well as adverse conditions. Foliar spray of SA significantly improved the content of anticancer alkaloids vincristine (by 22.2%) and vinblastine (by 50.0%) in plants treated with 150 mg kg(-1) of Ni.

In vitro plant propagation of Catharanthus roseus and assessment of genetic fidelity of micropropagated plants by RAPD marker assay.

An investigation was carried out to develop an efficient micropropagation protocol for Catharanthus roseus. Experiments were conducted to optimize suitable media for in vitro shoot multiplication and root induction. Out of the different media compared for in vitro shoot multiplication, Murashige and Skoog (MS) medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.2 mg/l α-naphthaleneacetic acid showed better response in terms of the emergence of shoots from axillary buds as well as proliferation and multiplication of shoots. The shoots when placed on half strength of MS medium having 1 mg/l indole 3-butyric acid and 0.25 % charcoal showed cent percent root induction with maximum number of roots per shoot (4.2) as well as maximum root length (1.72 cm). Further, clonal fidelity of the in vitro-raised plants was carried out using randomly amplified polymorphic DNA marker and results indicated that all the tissue culture-derived plants are true-to-type and there were no somaclonal variations among these plants.

Techniques for the maintenance and propagation of phytoplasmas in glasshouse collections of Catharanthus roseus.

Phytoplasma collections are a vital resource for researchers and diagnosticians studying phytoplasma diseases. They provide material as a point of reference and a research tool to increase our understanding of phytoplasmas and the diseases they cause. This chapter describes the techniques required to create and maintain collections of phytoplasma-infected Catharanthus roseus (Madagascar periwinkle).