Cell Fresh Weight Research Articles

Utilization of High-Salinity Crude Glycerol Byproduct from Biodiesel Production for Biosynthesis of γ-Aminobutyric Acid in Engineered Halomonas elongata

As the development of renewable energy has become imperative, biodiesel fuel (BDF) has been used as renewable biofuel with the advantage of having lower levels of greenhouse gas emissions. However, the transesterification reaction that produces BDF generates a high-salinity crude glycerol (CG) byproduct, which is difficult to recycle. Halomonas elongata is a moderately halophilic bacterium being used as a cell factory due to its ability to assimilate varieties of substrates for growth in high-salinity conditions. Previously, we engineered a recombinant H. elongata GOP-Gad to biosynthesize and accumulate γ-aminobutyric acid (GABA) as a high-value product. Here, we tested the ability of H. elongata GOP-Gad to use CG as a substrate for cell growth and GABA production. The CG byproduct was obtained from a BDF facility in Unzen City, Nagasaki, Japan, where geothermal energy catalyzed BDF production from waste cooking oil and biomethanol. Prior to use as the sole carbon (C) source in culture media, the CG byproduct was partially purified to remove soap substances and other impurities. Finally, we showed that H. elongata GOP-Gad could grow and accumulate GABA up to 28 μmol/g cell fresh weight in a minimal M63 medium containing 7% w/v NaCl with 4% w/v glycerol from the partially purified CG as a C source and 15 mM (NH4)2SO4 as a nitrogen (N) source. This result demonstrates a new circulatory bioprocess of C and N, in which H. elongata GOP-Gad can use partially purified CG and (NH4)2SO4 as the sole C and N sources for growth and GABA production.

Metabolic pathway engineering of high-salinity-induced overproduction of L-proline improves high-salinity stress tolerance of an ectoine-deficient Halomonas elongata.

Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. Halomonas elongata OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, H. elongata OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of H. elongata OUT30018's Ect biosynthetic operon with the artificial self-cloned proBm1AC gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GKD118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the putA gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate H. elongata HN6. While the Ect-deficient H. elongata KA1 could not grow in minimal media containing more than 4% NaCl, H. elongata HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in H. elongata OUT30018 in response to salt stress. With its genetic background, H. elongata HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.IMPORTANCEWe report here the evidence for de novo biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient Halomonas elongata. Remarkably, the concentration of Pro accumulated in H. elongata HN6 (∆ectABC::mCherry-proBm1AC ∆putA) is comparable to that of ectoine accumulated in H. elongata OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GKD118N/D119N and γ-GKD154A/E155A) designed to resemble the two known Escherichia coli feedback-inhibition insensitive γ-GKD107N and γ-GKE143A, the γ-GKD118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in H. elongata. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered H. elongata HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.

Long-term cell suspension culture of Thymus persicus (Lamiaceae): A novel approach for the production of anti-cancer triterpenic acids

Triterpenic acids (TAs) such as betulinic acid (BA), oleanolic acid (OA), and ursolic acid (UA) are well-known natural compounds found in many plant taxa possessive a wide spectrum of biological activities and potential medicinal uses. Thymus persicus has been already reported as a rich source of TAs. Nowadays, long-term culture is an efficient technique in increasing biosynthesis and production of specialized metabolites in the plant cell suspension cultures with the possibility of stimulation, metabolic regulation, permeabilization, and up-scaling to bioreactors. In the present study, both batch and long-term cell suspension culture (CSC) of the plant were developed for the production of these compounds. A maximum specific cell growth rate of 5.7 g day−1, a doubling time of 42 h, and the highest cell viability (94.7%) throughout the growth period were measured. The highest cell fresh weight in CSC and its long-term culture was 100.63 ± 4.2 and 171.6 ± 4.1 g L−1 at day 21 and 35 of culture, respectively. A positive correlation between cell growth and the content of TAs in long-term culture was observed. The plant cells produced the highest levels of TAs in the mid-exponential phase and held their production stability during the long-term culture period. The highest content (mg 100 g−1 DW) of BA (163.11 ± 4.3) was measured in the second week of culture, while the maximum level of OA (10.22 ± 0.1) and UA (11.01 ± 0.4) was recorded in the third week of culture period. The present findings can be interestingly considered for further commercial production of valuable TAs at large-scale cultures in bioreactors.

Simultaneous accumulation of astaxanthin and β-carotene in Chlamydomonas reinhardtii by the introduction of foreign β-carotene hydroxylase gene in response to high light stress.

Carotenoids are important photosynthetic pigments with many physiological functions, nutritional properties and high commercial value. β-carotene hydroxylase is one of the key enzymes in the carotenoid synthesis pathway of Chlamydomonas reinhardtii for the conversion of β-carotene to astaxanthin. The vector p64DZ containing the β-carotene hydroxylase gene crtZ from Haematococcus pluvialis was transformed into C. reinhardtii CC-503. The transformants were selected by alternate culture in solid-liquid medium containing spectinomycin (100µgmL-1). PCR results indicated that the gene crtZ and aadA were integrated into the genome of C. reinhardtii. RT-PCR analysis showed that the gene crtZ was transcribed in Chlamydomonas transformants. HPLC analysis showed that the content of astaxanthin and β-carotene in cells of C. reinhardtii were simultaneously increased. Under medium light intensity cultivation (60µmolm-2s-1), transgenic C. reinhardtii had an 85.8% increase in β-carotene content compared with the wild type. The content of astaxanthin and β-carotene reached 1.97 ± 0.13mgg-1 fresh cell weight (FCW) and 105.94 ± 5.84µgg-1 FCW, which were increased 18% and 42.4% than the wild type after 6h of high light treatment (200µmolm-2s-1), respectively. Our results indicate the regulatory effect on pigments in C. reinhardtii by β-carotene hydroxylase gene of H. pluvialis, and demonstrate the positive effect of high light stress on pigment accumulation in transgenic C. reinhardtii.

The Effects of 2,4-Dichlorophenoxyacetic Acid and α-Naphthaleneacetic Acid on Biomass Increment, Rhizogenesis and Somatic Embryogenesis of Suspension-cultured Dinh Lang Cells [Polyscias fruticosa (L.) Harms

Background: Dinh Lang [Polyscias fruticosa (L.) Harms] is a medicinal plant widely grown in Vietnam, with proven note-worthy health benefits. However, Dinh Lang’s amounts of triterpenoid saponins could not meet the need of the pharmaceutical industry. Thus, this study’s purpose is to figure out the optimal condition for raising Dinh Lang’s cell biomass, rhizogenesis and somatic embryogenesis to provide materials for bioactive compound productions. Methods: Different 2,4-dichlorophenoxyacetic acid and α-naphthaleneacetic acid concentrations (0.5, 1.0, 1.5 and 2.0 mg/L) were examined to determine the best amount of each plant growth regulator for raising cells’ biomass, rhizogenesis and somatic embryogenesis. In each treatment, two grams of eight-week-old calli were cultured in 50 mL of liquid MS medium. Result: It is demonstrated by the results that liquid MS medium containing 1.5 mg/L α-naphthaleneacetic acid has the capacity of producing the highest numbers of somatic embryos (489 embryos per flask) and rooted cells (259.5 cells per flask), while the fresh weight of cells cultured in the medium given 1.5 mg/L 2,4-dichlorophenoxyacetic acid reached its peak of 5.7 g.

Sodium nitroprusside enhances biomass and gymnemic acids production in cell suspension of Gymnema sylvestre (Retz.) R.Br. ex. Sm.

Gymnemic acids (a group of triterpenoid saponins) found in Gymnema sylvestre (Retz.) R.Br. ex Sm. works as the main hypoglycaemic active components. These can be the potential active pharmaceutical ingredient (API) to be used by pharmaceutical industries in modern medicines against diabetes. The present study aims to investigate the effectiveness of sodium nitroprusside (SNP) treatment for enhancement of cell suspension culture biomass and to quantify the production of deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII contents. Callus was obtained from in vitro derived leaves of G. sylvestre on MS medium fortified with 3.0 mg/L 2, 4-d (2, 4-dichlorophenoxyacetic acid) and 1.0 mg/L Kn (Kinetin), and the same were used further to produce cell suspension cultures. Cell suspensions were exposed to different concentrations of SNP (5, 10, 20 and 40 μM) and data were collected at 20, 30 and 40 days. Out of the tested concentrations, 20 μM SNP had the highest level of cell culture growth (398.94 ± 8.32 g/L Fresh cell weight (FCW) and 40.00 ± 0.75 g/L Dry cell weight (DCW) on 40-day as compared to control (MS with 3.0 mg/L 2, 4-d + 1.0 mg/L Kn). High-performance liquid chromatography analysis showed that maximum accumulation of deacylgymnemic acid (5.51 mg/g DCW), gymnemagenin (2.80 mg/g DCW) and gymnemic acid XVII (2.08 mg/g DCW) in 20 μM SNP treatment which is (13.43, 13.86 and17.33 folds) higher than the respective control at 40 days exposure. This research suggests that G. sylvestre cell suspension culture with optimal SNP elicitation treatment could be used as a good strategy for the large-scale production of these secondary metabolites at the industrial level. This research suggests that Gymnema sylvestre cell suspension culture with SNP elicitation treatment could be used as a good strategy for the large-scale production of these secondary metabolites at the industrial level.

Growth Dynamics and Cell Viability in Tomato Suspension Cultures Derived from Different Types of Calli

To establish a dynamic and fine suspension culture, four different methods of tomato cell suspension culture were compared. Hypocotyl explants of the tomato cultivar Jina were used for callus induction on Murashige and Skoog (MS) Medium supplemented with three different phytohormone combinations. Then, one gram of each type of calli was transferred to 50 mL of liquid MS medium with four combinations of auxins and cytokinins to produce cell suspensions. The growth rate, judged by cell turbidity, cell fresh weight, and cell viability was evaluated. The best suspension culture was obtained by using friable calli formed on MS medium containing 1 mg L-1 NAA and 0.1 mg L-1 kinetin, transferred to the liquid MS supplemented with 2 mg L-1 NAA, 0.2 mg L-1 2, 4-D and 0.2 mg L-1 zeatin.

Application of nanoparticles (ZnO, TiO2 and CuO), a new opportunity for the stimulation of cell growth and azadirachtin production in cell suspension culture of Azadirachta indica

Nanoparticles have unique physicochemical properties and provide great opportunities in plant science studies. In this study, we investigated the impact of ZnO, TiO2 and CuO nanoparticles (0, 20, 40, 60, and 80 mg/L) and sampling times (2, 4, and 6 days) on cell suspension growth and azadirachtin accumulation and production. Factorial experiments based on a completely randomized design with three replications were used. Results demonstrated that different nanoparticles had a different effect on the studied characters. When ZnO nanoparticles were used, the highest fresh (540.73 g/L), dry cell weight (15.93 g/L), azadirachtin accumulation (5.15 mg/g DW) and production (68.27 mg/L) were obtained at control condition, 80 and 40 mg/L ZnO nanoparticles, and control condition after 6 days, respectively. The highest amount of fresh (526.95 g/L) and dry (17.05 g/L) cell weight and azadirachtin production (82.21 mg/L) and accumulation (5.93 mg/g DW) were observed in 20 mg/L TiO2 nanoparticles, 40 mg/L of TiO2 nanoparticles after 2 days, 20 mg/L TiO2 nanoparticles in 4 days and 60 mg/L of TiO2 nanoparticles, respectively. With applying CuO nanoparticles, the highest fresh cell weight and azadirachtin accumulation were 422.59 g/L and 4.00 mg/L, achieved in control conditions respectively. Also, the highest amount of azadirachtin production was 68.27 mg/L, observed in control conditions on the 6th day of treatment. It seems that suitable cell growth, except in some cases, occurred in the absence of elicitors, but azadirachtin accumulation and production were stimulated by nanoparticles treatment. However, the results showed that the CuO nanoparticles caused a decrease in overall azadirachtin accumulation and production in the cells.

Bioaccumulation and Biosorption Activities of Indoor Metal-Tolerant Penicillium simplicissimum for Removal of Toxic Metals

In this study, the bioaccumulation and biosorption of aqueous metals (cadmium, copper, lead, zinc and trivalent chromium) by live and dead cells of Penicillium simplicissimum (isolate 10) were investigated and compared. Removal experiments under varying cell concentrations, pH and initial metal concentrations revealed that live cells generally achieved highest metal removal rates when 0.80 g fresh weight of cells was used, with metal solutions at pH 5 and concentrations of 50–100 mg L−1 (up to 150 and 200 mg L−1 for cadmium and lead, respectively). On the contrary, biosorption of metals by dead cells occurred at maximum rates when as little as 0.15 g dry weight of cells was used, in conditions of pH 5–7 (metal dependent) and lower metal concentration of 50 mg L−1. This comparative analysis suggested that although effective bioremediation by live cells required more cell biomass and preferred the five metals at concentrations of 50–100 mg L−1, the amount of metals removed by live cells were significantly higher (36.2–86.4% for 100 mg L−1 and pH 5) than by dead cells (8.5–58.9%). The results indicated that bioaccumulation is a favourable process that aids in metal removal together with biosorption, and is presumably more effective than the independent process of biosorption alone (by dead cells). Hence, the metal removal potential of P. simplicissimum, especially live cells of this indoor metal-tolerant isolate, may be applied to wastewaters for effective removal within optimum pH range and metal concentrations.

COPPER SULPHATE ELICITATION OF OPTIMIZED SUSPENSION CULTURE OF ANDROGRAPHIS PANICULATA NEES YIELDS UNPRECEDENTED LEVEL OF ANDROGRAPHOLIDE

Concerted efforts have been made to push up andrographolide production through optimization of culture conditions, calli induction, cell growth and elicitation in cell suspension culture of Andrographis paniculata Nees. Optimum callus induction was obtained with cotyledon and hypocotyls of the plant on Skoog and Hilderbrandt (SH) medium containing 2.0 µg/mL 2,4- dichlorophenoxyacetic acid and 0.1µg/mL 6-benzyl amino purine (BAP). Half MS medium containing 20 g/L sucrose and 20 h photoperiod showed highest cells fresh weight (CFW) (17.96 ± 0.06 g/50 mL), growth index (10.95 ± 0.96) and andrographolide yield (4.61±0.688 mg/g DCW). The addition of copper sulphate (500 µM/L), Methyl jasmonate (25 mg/L), Chitin (500 mg/L) or Fungal mycelium (500 mg/L) in separate experiments showed significant increase (p=0.05) in bioproduction of andrographolide to the extent of 29.42±0.31 mg/g DCW, 13.13±0.11mg/g DCW, 19.45±0.68 mg/g DCW and 13.629±1.12 mg/g DCW respectively, copper sulphate thus proved to be the most effective one. The study indicated that a holistic approach involving both culture conditions and elicitation could enhance the overall production of secondary metabolites (andrographolide) appreciably in this herb and possibly in other medicinal plants.

Cell bioreactor culture of Orostachys cartilaginous A. Bor. and involvement of nitric oxide in methyl jasmonate-induced flavonoid synthesis

Orostachys cartilaginous A. Bor. is a high-value medicinal plant, whereas flavonoids are important secondary metabolites. Bioreactor cell culture is an alternative method for the mass production of flavonoids in O. cartilaginous. This study investigated the adaption of culture conditions using bioreactors with different sizes to provide a reference for the pilot-scale culture of O. cartilaginous cells in the future. Results showed that cell fresh and dry weights per culture medium among the balloon-type airlift bioreactors of 3, 5, and 10 L did not change. Moreover, approximately equal amounts of total flavonoids were synthesized in bioreactors with different sizes, indicating that the culture conditions optimized in a bioreactor of certain size can be used in bioreactors of other sizes. This study used methyl jasmonate (MeJA) as an abiotic elicitor to treat 25-day-old O. cartilaginous cells, and an event whether nitric oxide (NO) was involved in flavonoid synthesis in MeJA-induced flavonoid synthesis was investigated to improve flavonoid accumulation. The contents of total flavonoids and flavonoid monomers including quercetin, kaempferide, epicatechin gallate, quercetin-3-O-glucose, and kaempferol-3-rutinoside, were significantly improved by MeJA treatment, reaching the maximum value at 48 h after elicitation. During the MeJA elicitation, NO burst in the early stage and NO content peaked at 6 h. In addition, nitrate reductase (NR) inhibitors of tungstate and glutamine blocked NO generation and inhibited flavonoid synthesis in MeJA-stimulated cells. However, such inhibition of flavonoid synthesis was relieved by a NO donor (sodium nitroprusside), thereby suggesting that NO was involved in MeJA-induced flavonoid synthesis through NR pathway. The present finding has a critical significance for understanding the mechanism of the defense response stimulated by the MeJA elicitor and can provide a new strategy that regulates NO burst and flavonoid synthesis by controlling the NR activity.

New biological trends on cell and callus growth and azadirachtin production in Azadirachta indica.

Azadirachtin is an important secondary metabolite from Azadirachta indica used as a natural biopesticide. This study is the first comprehensive report concerning the influence of plant growth regulators on callus induction, cell suspension growth, and azadirachtin accumulation and production in cell suspension cultures of A. indica. We investigated the effect of plant growth regulators including different types of auxins and cytokinins and their combinations on callus induction, cell suspension growth, and azadirachtin accumulation and production. The highest percentage of callusing (100%) obtained at different combinations of plant growth regulators on MS medium supplemented with 1mg/L picloram and 2mg/L kinetin and the highest fresh weight of callus (264.50mg) was observed in MS medium containing 1.5mg/L NAA and 3mg/L kinetin. In cell suspension cultures, the maximum cell density, SCV, and PCV were 2.44 × 106cells per mL, 97.95%, and 81.46%, respectively, obtained in the MS medium containing 1.5mg/L 2,4-D and 3mg/L zeatin riboside. The highest average growth rate (0.25days) was on MS medium containing 1.5mg/L NAA and 3mg/L zeatin riboside. The MS medium supplemented with 1mg/L picloram and 2mg/L kinetin produced the highest amount of fresh cell weight (493.02g/L), dry cell weight (77.27g/L), azadirachtin accumulation (3.69mg/gDW), and azadirachtin production (285.64mg/L). The results showed that all measured indices had positive correlation with together except FCW and DCW with azadirachtin accumulation.

Factors Affecting Cell Biomass and Flavonoid Production of Ficus deltoidea var. kunstleri in Cell Suspension Culture System

A study was conducted to establish in vitro culture conditions for maximum production of biomass and flavonoid content for Ficus deltoidea var. kunstleri, locally named as Mas Cotek, known to have a wide variety of potential beneficial attributes for human health. Size of initial inoculum, cell aggregate and initial pH value have been suggested to influent content of biomass and flavonoid for cell suspension culture in several plant species. In the present study, leaf explants were cultured by cell suspension culture procedures in MSB5 basal medium supplemented with predetermined supplements of 30 g/L sucrose, 2.75 g/L gelrite, 2 mg/L picloram and 1 mg/L kinetin with continuous agitation of 120 rpm in a standard laboratory environment. Establishment of cell suspension culture was accomplished by culturing resulting callus in different initial fresh weight of cells (0.10, 0.25, 0.50, 1.0, and 2.0 g/25 mL of media) using similar basal medium. The results showed that the highest production of biomass (0.65 g/25 mL of media) was recorded from an initial inoculum size of 2.0 g/25 mL media, whereas the highest flavonoid (3.3 mg RE/g DW) was found in 0.5 g/25 mL of media. Cell suspension fractions classified according to their sizes (500–750 µm, 250–500 µm, and <250 µm). Large cell aggregate size (500–750 µm) cultured at pH 5.75 produced the highest cell biomass (0.28 g/25 mL media) and flavonoid content (3.3 mg RE/g DW). The study had established the optimum conditions for the production of total antioxidant and flavonoid content using DPPH and FRAP assays in cell suspension culture of F. deltoidea var. kunstleri.

Blue Diode and Red He-Ne Lasers Affect the Growth of Anthocyanin Producing Suspension Cells of Apple

Light is an effective factor in cell suspension culture and must be controlled for optimizing cell growth. Growth of anthocyanin producing suspension cells of a red-fleshed genotype of apple (RFA) was assessed in response to blue diode laser (BDL) and red He Ne (RHNL) laser. The suspension cells in L-shaped test tubes were exposed to short-term laser radiation for 20 min in a rotary shaker. The fresh cell weight (FCW), dry cell weight (DCW), cell volume after sedimentation (CVS), cell number (CN) and cell viability (CV) as criteria of cell growth were recorded at 0, 4, 8, 12 days during cell culture. The cell growth was negatively affected in response to BDL and RHNL compared to control and darkness, respectively. The FCW and DCW was enhanced by BDL whereas were not affected by RHNL. Also, only 30.4 mWcm-2 intensity of BDL could increase CVS in RFA cells. Changes in CN were not displayed by RHNL and BDL. BDL more than RHNL decreased CV. Cell death rates observed due to BDL and RHNL were 40.42% and 33.67%, respectively. All these results showed that these lasers had diverse effects on FRA cell growth, however, these cells were more sensitive to BDL than RHNL especially in higher intensities, presumably because of its damage to cell membrane leading to cell death.

Efficient Bioproduction of Mycosporine-2-glycine, Which Functions as Potential Osmoprotectant, using Escherichia coli Cells

Mycosporine-2-glycine (M2G) is known to be synthesized in halotolerant cyanobacterium Aphanothece halophytica. Escherichia coli cells in which the M2G synthetic genes of A. halophytica were introduced could synthesize M2G. Here, we report that M2G producing transformed E. coli cells showed salt tolerance compared to control cells. This result suggested that M2G could function as a potential osmoprotectant in E. coli. To our knowledge, this is the first report presenting the evidence that mycosporine-like amino acid confers salt tolerance on E. coli. Intracellular M2G content in the transformed E. coli cells were varied depending on NaCl concentration with maximum level at 0.75 M. Moreover, intracellular M2G level was affected by a supply of glycine with maximum level at 5 mM. In conclusion, we found that transformed E. coli cells could produce 205 μg of M2G/g fresh weight of cells under the best effective growth condition in this study. Thus, the results obtained here offer the potential for the bioproduction of mycosporine-like amino acids using the genetically engineered E. coli cells.

Optimization of cell growth and bacoside-A production in suspension cultures of Bacopa monnieri (L.) Wettst. using response surface methodology

Plant secondary metabolites have emerged as potential raw materials, which are used in the pharmaceutical, food, chemical, and cosmetic industries. Bacoside-A, a secondary metabolite produced by Bacopa monnieri, is known for its memory-facilitating properties. In recent years, various strategies have been developed to enhance biomass accumulation and synthesis of secondary compounds in cultures. In the present investigation, various factors affecting the production of biomass and bacoside-A in the cell suspension cultures of B. monnieri were optimized using the statistical experimental design approach. Preliminary screening by Plackett–Burman’s design revealed that among the tested factors, glucose, KNO3, KH2PO4, and inoculum density significantly influenced cell growth and bacoside-A production. Furthermore, using response surface methodology (RSM), glucose, KNO3, and KH2PO4 at a concentration of 5.67, 0.313, and 0.29%, respectively, and an inoculum density of 0.66% in basal MS medium were found to be optimal for cell growth and bacoside-A production. After optimization, the biomass yield increased about twofold (from 5.52 to 12.58 g L−1 fresh cell weight) and bacoside-A production about 1.7-fold (5.56 to 9.84 mg g−1 dry weight). The present study results show the successful application of RSM to enhance the production of biomass and accumulation of bacoside-A content in cell suspension cultures of B. monnieri.

Establishment of Callus Induction and Cell Suspension Cultures of Dendrathema Indicum var. Aromaticum a Scented Chrysanthemum

Dendranthema indicum var. aromaticum is an important aroma plant in genus Dendrathema, and the establishment of callus cultures and cell suspension cultures is the basement of further protoplast fusion studies, which make it possible to breed new fragrant chrysanthemum. In this study, the effects of different plant growth regulating substances in different concentrations on callus induction were investigated with stem segments, leaves, petioles as explants. The results showed that the optimal explants were lower stem segments according to the percentage of callus formation, callus hardness, growth potential and shoot differentiation. The optimal induction mediums were MS supplemented with 1.0 mg．l-12.4 D and 0.2 mg．l-1 6-BA. The cell suspension culture system was established by using the subculture calli. The results showed that the suitable inoculum size was 2g and the suitable cell suspension culture medium was MS supplemented with 0.2 mg．l-1 6-BA and 0.5 mg．l-1 2,4-D. The time course of cell growth showed that the greatest cell fresh weight appeared on day 14 and the highest cell viability on day 3.

Chitosan mediated enhancement of hydrolysable tannin in Phyllanthus debilis Klein ex Willd via plant cell suspension culture

Phyllanthus debilis Klein ex Willd. is wild medicinal plant used in the traditional system of medicine. This plant has been actively used for hepatoprotection and to cure many diseases including jaundice and so on; which leads to complete extinction of this particular species. Therefore, the chitosan mediated cost effective cell suspension method has been developed for the production of hydrolysable tannin. The hydrolysable tannins are the main therapeutically active constituents with antioxidant, anticancer, and antimicrobial properties. An in vitro cell suspension culture was optimized by adding chitosan for production of hydrolysable tannin. According to the growth kinetics, a maximum biomass of 4.46±0.06g fresh cell weight and 1.33±0.04g dry cell weight were obtained from the optimal suspension medium consisted of MS medium+0.5mgL−1 BAP+1.5mgL−1 NAA. Chitosan was treated at the stationary phase which leads to the highest accumulation of hydrolysable tannin compared to the untreated control. Hydrolysable tannin was observed and compared using HPLC at the Rt of 4.91 in both chitosan treated and untreated cells. This is the first ever report where use of chitosan has been done to enhance the production of the hydrolysable tannin in P. debilis using cell suspension culture technique.

Optimization of Baicalein and Chrysin Production in Cell Cultures of Oroxylum indicum (L.) Vent.

A phytochemical screening revealed the presence of phytoconstituents like flavonoid, anthraquinone and phenol in the callus and suspension culture of Oroxylum indicum (L.) Vent. (Bignoniaceae). The quantitative estimation using TLC technique reflected the presence of two flavonoids viz. baicalein and chrysin in three fractions (chloroform, ethyl acetate and n-butanol) of callus extract and in intra and extracellular suspension cultures. Protocol was developed for quantification of phytoconstituents by HPLC with methanol: water (70:30) mobile phase and c-18 column in all the three fractions of callus extract. Both baicalein and chrysin flavonoids were there in all the three callus fractions. Maximum amount of baicalein was present in nbutanol fraction of callus while maximum amount of chrysin was present in chloroform fraction. Cell suspension cultures of O. indicum were established in shake flask system to optimize medium formulation for cell growth and flavonoid content. Murashige and Skoog’s basal medium (MS) supplemented with 1 mg/L BAP, 0.5 mg /LNAA , 0.1 mg /L 2,4-D and 1 mg /L AgNO3 was found optimum for growth parameters viz. cell counting, PCV, fresh weight and dry weight. Presence of baicalein and chrysin in intracellular and extracellular fractions of this suspension culture is a successful event of present work.

Establishment and characterization of a Satureja khuzistanica Jamzad (Lamiaceae) cell suspension culture: a new in vitro source of rosmarinic acid.

An in vitro approach to the production of rosmarinic acid (RA), a medicinally important caffeic acid ester, in a cell suspension culture (CSC) of Satureja khuzistanica Jamzad (Lamiaceae) has been investigated for the first time. The CSC was established from friable calli derived from shoot tip explants in Gamborg's B5 liquid medium supplemented with 30g/L sucrose, 20mg/L L-glutamine, 200mg/L casein hydrolysate, 5mg/L benzyladenine (BA) and 1mg/L indole-3-butyric acid (IBA). The effect of nitrogen source (KNO3 and (NH4)2SO4) and their different concentrations on the fresh and dry weight (g/L), as well as RA content (mg/g dry weight) were measured. CSC growth measurements indicated a maximum specific cell growth rate of 1.5/day, a doubling time of 7.6days and a high percentage of cell viability (96.4%) throughout the growth cycle. Maximum cell fresh weight (353.5g/L), dry weight (19.7g/L) and RA production (180.0mg/g) were attained at day 21 of culture. Cell growth and RA content were affected by nitrogen deficiency. Media containing 8.3mM of total nitrogen (¼ of B5 standard medium) led to a minimum cell fresh weight (243.0g/L), dry weight (17.4g/L) and RA content (38.0mg/g) after 21 days. The established CSC provided useful material for further optimization experiments aimed at a large-scale production of RA.