Plant Cell Tissue Culture Research Articles

Influence of metal and metal oxide nanoparticles on growth and total phenolic content accumulation of Anoectochilus roxburghii cultured in vitro

The application of metal nanoparticles in agriculture and related fields, especially in plant cell tissue culture has increased interest in recent years due to its potential benefits. This study used gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), copper nanoparticles (CuNPs) and magnetite nanoparticles (Fe3O4NPs) to evaluate their effect on the growth and total phenolic content (TPC) of Anoectochilus roxburghii. The results showed that Fe3O4 NPs were the most positively influenced metal nanoparticles in increasing biomass and TPC of A. roxburghii among metal nanoparticles tested. After 8 weeks of culture, the dry weight (DW) and TPC of the plants cultured on the medium containing 5 ppm Fe3O4NPs were 39.07 mg and 13.0 mg gallic acid respectively per g of dry weight (mg GAE/g DW). Meanwhile, on the medium without Fe3O4NPs, they were 30.47 mg and 6.71 mg GAE/g DW respectively. This study proposed an effective approach to improve the growth and accumulation of TPC in A. roxburghii. Moreover, it suggests the potential application of metal nanoparticles in plant tissue culture and the production of bioactive compounds.

Use of Statistics in Plant Cell Tissue Culture.

Statistics and experimental design are important tools for plant cell and tissue culture researchers and should be used when planning and conducting experiments as well as during the analysis and interpretation of experimental results. The chapter provides basic concepts important to the statistical analysis of data obtained from plant tissue culture experiments and illustrates the application of common statistical procedures to analyze binomial, count, and continuous data for experiments with different treatment factors as well as identifying trends of dosage treatment factors.

Propagation of a U16 hybid Eucalyptus line by plant cell tissue culture

Eucalyptus is one of the important plant species for paper pulp, fuel wood, and timber. In this study, a hybrid Eucalyptus, U16 line, was investigated for rapid in vitro propagation. The apical and adventitious shoots served as initial explants. These were free-disease symptoms that were collected for surface sterilization. Different disinfectants including mercuric and Javel solutions at different concentrations and treated times were performed. The results showed that explants were cleaned under taping water with soap, rinsed in 70oEtOH for 1 minute, and then immersed in 10% Javen solution for 15 minutes and 0.1% HgCl2 for 10 minutes gave the highest disinfection rate that was 46.5%. The modified MS medium included basal MS, 30 g/L sucrose, and 7 g/L agar supplemented with 20 ml/L of coconut water (MS medium*) was the most suitable for the rapid multiplication of Eucalyptus lines U16, the shoot multiplication coefficient reached 1.56. In vitro multiplication of eucalyptus U16 was carried out by culturing disinfected shoot segments on the MS* medium supplemented with BAP 1.5 mg/L and kinetin 1.0 mg/L which gave the highest shoot multiplication coefficient (3.24 shoot per explant). The in vitro rooting medium of the U16 line was basal MS supplemented with 1.0 mg NAA/L, the rooting rate was 90.49%, the number of roots was 8.3, and the roots expressed strong growth

Plant Growth Regulators and Activated Charcoal Selectively Affect Phenylethanoid and Flavone Glycoside Accumulation in Sideritis scardica Griseb. Tissue Culture.

Sideritis scardica Griseb. is a Balkan endemic species traditionally used for the treatment of pulmonary emphysema and angina pectoris. Recent research has also shown its phytotherapeutic potential as an anticancer and neuroprotective agent. These findings, as well as the endangered status of the species in its wild habitats, have motivated the present research on application of plant cell tissue and organ culture for the purposes of both valuable germplasm conservation and secondary metabolites production. Shoot cultures of the plant were initiated from sterile germinated seeds and the effects of activated charcoal (AC), as well benzyl adenine and 1-naphthaleneacetic acid treatments, were experimented. The phenolic profile analysis was performed by HPLC/DAD/MSn. Comparison with samples collected from wild plants in their natural habitat was performed. It was established that in vitro multiplication induced by plant growth regulators (PGRs) was accompanied by a higher impairment of leaf morphology and trichome formation, as well as by the occurrence of plantlet hyperhydricity and callus formation, as compared with the AC treatments. Shoot culture-derived plant material was shown to produce two phenylethanoids and five flavone glycosides, not detected in the wild collected plant material. In addition, the two types of in vitro culture treatments led to the stimulation of either flavone glycosides or phenylethanoids in the in vitro cultivated plants. Thus, AC stimulated, to a higher extent, flavone glycosides' accumulation, leading to an elevated flavone/phenylethanoid ratio, as compared with PGR treatments.

Producción de antraquinonas por cultivo in vitro de suspensiones celulares y raíces transformadas de Rubia Tinctorum: un modelo para la enseñanza en el estudio de bioprocesos por cultivo de tejidos de plantas

Plant cell culture bioprocesses constitute an excellent tool for the formation of undergraduate, magister, and Ph.D. students. In fact, students can acquire experience in the field of plant biochemistry and molecular biology; plant primary and secondary metabolism; and biochemical engineering. In the present manuscript, the production of anthraquinones in different plant cell tissue cultures is showed as easy and versatile model to introduce magister students in the field of plant secondary metabolite production. AQs are extracted with ethanol 80% and spectrophotometrically quantified, which also makes it environmentally friendly and easy to manipulate in the laboratory. The students were able to analyse biomass and product formation kinetics in batch suspension and hairy root cultures and evaluate the effect of MeJa elicitation an extended used strategy to trigger secondary metabolite production in plant cell cultures. Moreover, the performance of in situ product removal, which has been extensively used in biocatalyst and microbial fermentations, was evaluated. In summary, an inexpensive, accessible, and robust laboratory work is presented that can be adapted to undergraduate, magister, and/or Ph.D. students' courses in different plant biology, chemistry, and biochemical engineering programs.

Narrative review of production of antioxidants and anticancer compounds from Bryophyllum spp. (Kalanchoe) using plant cell tissue culture

: For centuries, plants have been widely used in traditional medicine worldwide for the treatment of many diseases. The subgenus Bryophyllum (genus Kalanchoe) have been used in ethnobotanic medicine across America, Africa and Asia. Traditionally, some formulations derived from leaves and roots of Bryophyllum spp. have been applied for the treatment of common illness such as coughs, fever, infections, insect bites, wounds and burns. Phenolic compounds and bufadienolides are the two major families of secondary metabolites identified in several species of the subgenus Bryophyllum. These compounds have gained much attention due to their associated antioxidant and cytotoxic activity, but they are synthetized by plants in fairly limited amounts. In this sense, plant tissue culture (PTC) technology provides a powerful methodology, able to overcome the limitations of low yields associated with conventional open field cultivation of medicinal plants. Several types of PTC methods are routinely employed in plant in vitro propagation, although micropropagation and cell culture are the most common. While micropropagation provides a reliable multiplication procedure, enabling a continuous in vitro production of great amounts of whole medicinal plantlets or just their organs producing the bioactive metabolites, such as their leaves and/or roots; the cell suspension culture procedure allows for the massive production of secondary metabolites using huge bioreactors. In both cases, the addition of biotic and abiotic elicitors and metabolic precursors trigger the bioaccumulation of secondary metabolites through the induction of plant defense mechanisms.

Enrichment of alliin in different in vitro grown tissues of Allium sativum through CdCl2 elicitation as revealed by high performance thin layer chromatography (HPTLC)

Plant cell tissue and organ culture techniques provide an efficient way for the biosynthesis of high-value secondary metabolites in which elicitation has been recognized to be a potent approach in enriching the bioactive compounds. In this study, an effort was made to assess the influence of Cadmium chloride (CdCl2) on alliin yield of different in vitro grown tissues such as callus, somatic embryos, leaves and roots of Allium sativum. The tissues were cultured in MS amended with various levels of CdCl2 [control (0 mM), T1 (0.05 mM), T2 (0.1 mM), T3 (0.15 mM) and T4 (0.2 mM)] and the yield of alliin was quantified by using high performance thin layer chromatography (HPTLC). Biochemical analyses revealed that sample tissues under increasing CdCl2 conditions accumulated higher levels of sugar, protein and proline with maximum content of sugar (19.61 mg g−1 FW), protein (6.01 mg g−1 FW) and proline (12.19 mg g−1 FW) in leaves at T3 after 4 days of elicitor treatment. Changes within the activities of varied antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) was assayed as the addition of CdCl2 in the medium causes stress. We noted that with an elevated level of CdCl2, antioxidant enzyme activity increased linearly with the highest activity observed in leaves at T4 of 6 days of elicitation (4.97 EU min−1 mg−1 protein CAT, 6.30 EU min−1 mg−1 protein SOD and 1.125 EU min−1 mg−1 protein APX). Increased activities of enzymes are involved in negating the cellular stress triggered due to the overproduction of ROS. The yield of alliin in response to varying levels of CdCl2 was estimated by using a simple, rapid, and sensitive HPTLC method. The best separation was achieved using butanol:propanol:acetic acid:water (3:1:1:1) as mobile phase. For analysis of alliin, the plates were scanned at 550 nm after spraying with ninhydrin and a good resolution was achieved with Rf value of 0.66. In our study, the alliin yield enhanced in all tested cultures with the elicitor level up to T3. In leaves, maximum accumulation of alliin (3.37 μg g−1 dry wt.) was noted on 0.15 mM (T3) CdCl2 added medium. Our data indicated the increase of alliin in CdCl2 added cultures, which suggests that the use of elicitors as a potential strategy for augmenting secondary metabolite synthesis.

Chemical elicitors versus secondary metabolite production in vitro using plant cell, tissue and organ cultures: recent trends and a sky eye view appraisal

Plant secondary biosynthetic pathways are exceedingly inducible by elicitors and facilitate enhanced metabolite production using plant cell tissue and organ cultures. Elicitors can regulate large number of control points and trigger the expression of key genes with increased cellular activities at biochemical and molecular level involving signal compounds. A large number of chemical elicitors viz: jasmonic acid (JA), methyl jasmonate (MeJA), salicylic acid (SA), acetyl salicylic acid (ASA), ethylene (ET) and ethrel (Ethe), heavy metals (HM), many types of chemical compounds (natural or synthetic) and their combinations are used for elicitation studies. Cell suspensions and hairy roots are commonly used culture systems followed by adventitious roots and multiple shoots for elicitation experiments. Amongst the elicitors and concentrations used 100 µM MeJA was found optimum for secondary metabolite enhancement in majority of experiments compared to SA and JA. Elicitor treatments promoted yield enhancements starting from 1.0 to maximum of 2230-fold across plant species studied. Elicitors singly with media additives, combination of elicitors and elicitors other than signal compounds along with hormones were found beneficial for enhanced secondary metabolite production. Further, a combination of target gene over expression and elicitor treatment also supported higher secondary product yield. The present communication presents information exclusively about the use of chemical elicitors for secondary metabolites production in vitro covering approximately more than a decade of research at one place in one review. Further, this extensive appraisal will be useful for the understanding and manipulation of secondary metabolites for enhanced production in vitro.

Propagación in vitro y aclimatización de Euchile mariae (Ames) Withner (Orchidaceae)

Orchid micropropagation of species Euchile mariae was achieved from the in vitro culture of protocorms sections, obtained from the germination of seeds. The top and bottom protocorms sections, used as explants, were cultivated in modified MS culture medium, added with different concentrations of α-naph- thaleneacetic acid (NAA) and 6-benzylaminopurine (BA). The morphogenetic response obtained of both types of explants, was the formation of protocorm like bodies (PLB’s), that gave place to the formation of new plantlets. Of two tested explants the biggest formation of PLB’s was obtained from the bottom proto- corms sections. The acclimatization of the plantlets was successful, with a 100% percent survival. Plant cell tissue culture represents a useful alternative for the study, conservation and massive propagation of this and other endangered species.

In vitro culture: an epigenetic challenge for plants

In vitro plant cell and tissue culture techniques are the basis of many micropropagation and breeding programs for scientific research. Plant tissue culture (PTC) involves organogenesis and embryogenesis, and the outcome depends on the different conditions to which the tissue is exposed. PTC is a stressful environment—high relative humidity, low ventilation rate, high concentrations of plant growth regulators, and low light availability—for plants that need to rapidly change their molecular regulation in order to respond fast and efficiently during cell division and growth. For instance, somatic embryogenesis (SE), which plays an important role in plant multiplication, requires complex cellular, biochemical and molecular processes for embryo formation and development. New data has come out about a connection between plant morphogenesis and epigenetics. Epigenetics is a very sensitive regulatory mechanism, which in most of cases is affected by the environment. Although it is known that, under plant morphogenesis, the genome has little or no change, DNA methylation and histone modifications are very susceptible to those in vitro environmental conditions. In the present review, we highlight the most used in vitro systems such as organogenesis and SE in plants and discuss how epigenetics plays a pivotal role in the phenotype outcome. Furthermore, we discuss the big role that the small RNAs have during cell division and propagation and propose different challenges and opportunities to study epigenetics in plant cell tissue and organ cultures.

Molecular Farming of Pharmaceutical Proteins Using Plant Suspension Cell and Tissue Cultures

Plants have been used for more than 20 years to produce recombinant proteins but only recently has the focus shifted away from proof-of-principle studies (i.e. is my protein expressed and is it functional?) to a serious consideration of the requirements for sustainable productivity and the regulatory approval of pharmaceutical products (i.e. is my protein safe, is it efficacious, and does the product and process comply with regulatory guidelines?). In this context, plant tissue and cell suspension cultures are ideal production platforms whose potential has been demonstrated using diverse pharmaceutical proteins. Typically, cell/tissue cultures are grown in containment under defined conditions, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. Recombinant proteins can also be secreted to the culture medium, facilitating recovery and subsequent purification because cells contain most of the contaminating proteins and can be removed from the culture broth. Downstream processing costs are therefore lower compared to whole plant systems, balancing the higher costs of the fermentation equipment. In this article, we compare different approaches for the production of valuable proteins in plant cell suspension and tissue cultures, describing the advantages and disadvantages as well as challenges that must be overcome to make this platform commercially viable. We also present novel strategies for system and process optimization, helping to increase yields and scalability.

Plant Cell Tissue and Organ Culture Biotechnology and Its Application in Medicinal and Aromatic Plants

MAPs are increasing worldwide and continue to attract growing interest for farmers, traders, economists, teachers, professionals, health officials and various industries. The MAPs are natural biological resources that have a great potential to synthesize a huge variety of important secondary metabolites, also referred to as natural products, far more than animal and even microorganisms. Are defined as secondary metabolites all specific compounds (characteristic of a species) which are not synthesized by metabolic pathways of primary metabolism that they are common to large groups of plants. Secondary metabolites are rather scarce, their structure is often very complex and their synthesis requires multistep enzymatic reactions. Their levels in plants are low, typically less than 0.1 to 5% of the biomass, and vary depending on numerous factors such as tissue type, plant developmental stage, environmental conditions, etc. These natural compounds are used as pharmaceuticals, agrochemicals, and cosmeceuticals. Recently, MAPs also are used as functional herbal food ingredients, nutraceuticals and health products [3]. The supply of the source plants however, is often limited due to diseases, changes in climate, and changes in the development in the growing regions.

Biotechnologically Produced Secondary Plant Metabolites for Cancer Treatment and Prevention

Secondary metabolites of higher plants exert numerous effects on tumorigenesis, on tumor cells in vitro, tumors in experimental animals in vivo, interact with anti-cancer drugs, thus affecting positively or negatively their efficacy, and protect normal tissues of the host organism against adverse effects of anti-cancer therapies. The industrial development of pharmaceutical and nutraceutical products based on secondary plant metabolites is limited due to the following: (i) limited availability of their natural sources, (ii) concern about rare extinguishing plants, (iii) unavoidable contamination of plant extracts with environmental pollutants, (iv) seasonal variations in plant harvesting, (v) poor standardization of the final product due to variable conditions for plant growth, and (vi) difficulties of secondary metabolite extraction from the parts of grown plant. There is now steadily growing interest in the biotechnological approach to produce secondary metabolites using plant cell or plant tissue cultures. In the present review, biosynthesis of secondary metabolites and their role(s) in plant physiology will be briefly discussed; the biotechnological approach to active substances production in the plant cell and plant tissue cultures will be described; examples and mechanisms of cancer preventive and anti-cancer action of some biotechnologically produced plant metabolites will be provided; and future perspectives for biotechnologically produced plant-derived substances in the combined protocols for cancer treatment will be suggested.

PRODUCTION OF ROSMARINIC ACID IN ECHIUM AMOENUM FISCH. AND C.A. MEY. CELL CULTURES

Echium amoenum Fisch. and C.A. Mey. (Boraginaceae) is a very popular medicinal plant which is used as a tonic, tranquillizer, diaphoretic, cough remedy, sore throat and pneumonia in Iran’s traditional medicine. Callus culture of medicinal plants is one of the ways for production of secondary metabolites. In this study, callus culture of E. amoenum and its major secondary metabolite were investigated. The callus culture of E. amoenum was initiated and established from seeds in MS media with three different ratios of plant growth regulatories: kinetin, 2,4-D and NAA. Methanolic extracts of freeze-dried calluses were compared by TLC and HPLC. The major secondary metabolite was separated by preparative HPLC and the structure of this pure compound was elucidated by UV, IR, one and two dimensional 1 H and 13 C-NMR and Mass spectroscopy. Rosmarinic acid was identified by various spectroscopic methods from callus culture of E. amoenum. Rosmarinic acid is widespread within the plant cell tissue culture of the Lamiaceae and Boraginaceae families, although in insignificant quantities. Rosmarinic acid has an antimicrobial, antiviral, and anti-inflammatory effect, which makes it a valuable product for the pharmaceutical and cosmetic industries.

Lotus hairy roots expressing inducible arginine decarboxylase activity.

Biotechnological uses of plant cell-tissue culture usually rely on constitutive transgene expression. However, such expression of transgenes may not always be desirable. In those cases, the use of an inducible promoter could be an alternative approach. To test this hypothesis, we developed two binary vectors harboring a stress-inducible promoter from Arabidopsis thaliana, driving the beta-glucuronidase reporter gene and the oat arginine decarboxylase. Transgenic hairy roots of Lotus corniculatus were obtained with osmotic- and cold-inducible beta-glucuronidase and arginine decarboxylase activities. The increase in the activity of the latter was accompanied by a significant rise in total free polyamines level. Through an organogenesis process, we obtained L. corniculatus transgenic plants avoiding deleterious phenotypes frequently associated with the constitutive over-expression of arginine decarboxylation and putrescine accumulation.

“Panasorb+ge” - new adsorbent from plant cell tissue culture: [formula omitted], L.I.Slepyan, S.A.Minina The State Academy of Chemistry and Pharmacy, 14, Prof. Popov, 197376, St.-Petersburg, Russia

“Panasorb+ge” - new adsorbent from plant cell tissue culture: [formula omitted], L.I.Slepyan, S.A.Minina The State Academy of Chemistry and Pharmacy, 14, Prof. Popov, 197376, St.-Petersburg, Russia

5409828 Method for stimulating cell multiplication, differentiation, embroyogenesis and respiration in plant cell tissue culture by the addition of a glycoprotein extensin to culture medium : Frenkel Chaim; Chin Chee-Kok; Havkin-Frenkel Daphn North Brunswick, NJ, United States Assigned to Rutgers The State University of New Jersey

5409828 Method for stimulating cell multiplication, differentiation, embroyogenesis and respiration in plant cell tissue culture by the addition of a glycoprotein extensin to culture medium : Frenkel Chaim; Chin Chee-Kok; Havkin-Frenkel Daphn North Brunswick, NJ, United States Assigned to Rutgers The State University of New Jersey

Bioprocess engineering: Basic concepts

Preface to the Second Edition. Preface to the First Edition. I. INTRODUCTION. 1. What is a Bioprocess Engineer? Introductory Remarks. Biotechnology and Bioprocess Engineering. Biologists and Engineers Differ in Their Approach to Research. The Story of Penicillin: How Biologists and Engineers Work Together. Bioprocesses: Regulatory Constraints. Suggestions for Further Reading. Problems. II. THE BASICS OF BIOLOGY: AN ENGINEER'S PERSPECTIVE. 2. An Overview of Biological Basics. Are All Cells the Same? Cell Construction. Cell Nutrients. Summary. Suggestions for Further Reading. Problems. 3. Enzymes. Introduction. How Enzymes Work. Enzyme Kinetics. Immobilized Enzyme Systems. Large-scale Production of Enzymes. Medical and Industrial Utilization of Enzymes. Summary. Suggestions for Further Reading. Problems. 4. How Cells Work. Introduction. The Central Dogma. DNA Replication: Preserving and Propagating the Cellular Message. Transcription: Sending the Message. Translation: Message to Product. Metabolic Regulation. How the Cell Senses Its Extracellular Environment. Summary. Appendix: Examples of Regulation of Complex Pathways. Suggestions for Further Reading. Problems. 5. Major Metabolic Pathways. Introduction. Bioenergetics. Glucose Metabolism: Glycolysis and the TCA Cycle. Respiration. Control Sites in Aerobic Glucose Metabolism. Metabolism of Nitrogenous Compounds. Nitrogen Fixation. Metabolism of Hydrocarbons. Overview of Biosynthesis. Overview of Anaerobic Metabolism. Overview of Autotrophic Metabolism. Summary. Suggestions for Further Reading. Problems. 6. How Cells Grow. Introduction. Batch Growth. Quantifying Growth Kinetics. How Cells Grow in Continuous Culture. Summary. Suggestions for Further Reading. Problems. 7. Stoichiometry of Microbial Growth and Product Formation. Introduction. Some Other Definitions. Stoichiometric Calculations. Theoretical Predictions of Yield Coefficients. Summary. Suggestions for Further Reading. Problems. 8. How Cellular Information is Altered. Introduction. Evolving Desirable Biochemical Activities through Mutation and Selection. Natural Mechanisms for Gene Transfer and Rearrangement. Genetically Engineering Cells. Genomics. Summary. Suggestions for Further Reading. Problems. III. ENGINEERING PRINCIPLES FOR BIOPROCESSES. 9. Operating Considerations for Bioreactors for Suspension and Immobilized Cultures. Introduction. Choosing the Cultivation Method. Modifying Batch and Continuous Reactors. Immobolized Cell Systems. Solid-state Fermentations. Summary. Suggestions for Further Reading. Problems. 10. Selection, Scale-Up, Operation, and Control of Bioreactors. Introduction. Scale-up and Its Difficulties. Bioreactor Instrumentation and Control. Sterilization of Process Fluids. Summary. Suggestions for Further Reading. Problems. 11. Recovery and Purification of Products. Strategies to Recover and Purify Products. Separation of Insoluble Products. Cell Disruption. Separation of Soluble Products. Finishing Steps for Purification. Integration of Reaction and Separation. Summary. Suggestions for Further Reading. Problems. IV. APPLICATIONS TO NONCONVENTIONAL BIOLOGICAL SYSTEMS. 12. Bioprocess Considerations in Using Animal Cell Cultures. Structure and Biochemistry of Animal Cells. Methods Used for the Cultivation of Animal Cells. Bioreactor Considerations for Animal Cell Culture. Products of Animal Cell Cultures. Summary. Suggestions for Further Reading. Problems. 13. Bioprocess Considerations in Using Plant Cell Cultures. Why Plant Cell Cultures? Plant Cells in Culture Compared to Microbes. Bioreactor Considerations. Economics of Plant Cell Tissue Cultures. Summary. Suggestions for Further Reading. Problems. 14. Utilizing Genetically Engineered Organisms. Introduction. How the Product Influences Process Decisions. Guidelines for Choosing Host-Vector Systems. Process Constraints: Genetic Instability. Considerations in Plasmid Design to Avoid Process Problems. Predicting HostDVector Interactions and Genetic Instability. Regulatory Constraints on Genetic Processes. Metabolic Engineering. Protein Engineering. Summary. Suggestions for Further Reading. Problems. 15. Medical Applications of Bioprocess Engineering. Introduction. Tissue Engineering. Gene Therapy Using Viral Vectors. Bioreactors. Summary. Suggestions for Further Reading. Problems. 16. Mixed Cultures. Introduction. Major Classes of Interactions in Mixed Cultures. Simple Models Describing Mixed-culture Interactions. Mixed Cultures in Nature. Industrial Utilization of Mixed Cultures. Biological Waste Treatment: An Example of the Industrial Utilization of Mixed Cultures. Summary. Suggestions for Further Reading. Problems. 17. Epilogue. Appendix: Traditional Industrial Bioprocesses. Anaerobic Bioprocesses. Aerobic Processes. Suggestions for Further Reading. Index.

High-performance liquid chromatographic—mass spectrometric assay of high-value compounds for pharmaceutical use from plant cell tissue culture: Cinchona alkaloids

The use of high-performance liquid chromatography (HPLC) interfaced with thermospray (TSP) mass spectrometry is described for the separation and identification of various alkaloids from Cinchona ledgeriana extracts. The use of water—acetonitrile—acetic acid (71:25:4) with 0.01 M ammonium acetate (pH 3.0) as the mobile phase gave good HPLC separation and good TSP sensitivity. The specificity obtained by single-ion monitoring allowed the analysis of commercially important alkaloids such as quinine and quinidine in plant material, transformed roots and in cells from tissue culture, with relatively simple extraction and work-up procedures. TSP gave protonated species with few fragment ions but collision-induced dissociation offers the promise of increased analytically specificity from the fragment ion data. This work has important implications for the biotechnological production of pharmaceuticals normally obtained from plant sources.

Peroxidase activity in calluses and cell suspension cultures of radishRaphanus sativus var. Cherry Bell

Calluses ofRaphanus sativus var. Cherry Bell were induced in a medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine. The biomass and peroxidase activities were determined, both in agar cultures and cell suspension cultures. Different growth-regulator concentrations induced different responses measured as peroxidase activity in callus. The suspended-cell cultures showed the importance of selecting the cell line, in order to obtain an optimal response in extracellular peroxidase activity. The commercial production of this enzyme by utilizing plant cell tissue cultures is discussed.