Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l^?1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH₄NO₃ (24.7 mM), 3.0× KNO₃ (56.4 mM), 3.0× MgSO₄ (4.5 mM) and 3.0× KH₂PO₄ (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day^?1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.     

Clonal propagation of Stevia rebaudiana Bertoni by stem-tip culture

Clonal propagation of Stevia rebaudiana has been established by culturing stem-tips with a few leaf primordia on an agar medium supplemented with a high concentration (10 mg/l) of kinetin. Anatomical examination has suggested that these multiple shoots originate from a number of adventitious buds formed on the margin of the leaf. Innumerable shoots can be obtained by repeating the cycle of multiple-shoot formation from a single stem-tip of Stevia. These shoots produce roots when transferred to a medium containing NAA (0.1 mg/l) without kinetin. The regenerated plantlets can be transplanted to soil.     

Structures of the novel diterpene glycosides from Stevia rebaudiana

From the commercial extract of the leaves of Stevia rebaudiana, two new diterpenoid glycosides were isolated besides the known steviol glycosides including stevioside, rebaudiosides A–F, rubusoside, and dulcoside A. The structures of the two new compounds were identified as 13-[(2-O-6-deoxy-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (1), and 13-[(2-O-6-deoxy-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (2), on the basis of extensive NMR and MS spectral data as well as chemical studies.     

Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light

The aim of this study was to determine whether steviol glycoside accumulation is under phytochrome control. The results indicate that Stevia rebaudiana Bertoni plants grown under short-day conditions showed precocious flowering and stagnation of steviol glycoside accumulation. Long night interruption by red LED light stimulated and sustained the vegetative growth as well as the accumulation of steviol glycosides in the leaves. After 7 weeks of treatment, steviol glycoside content was about two-fold higher in LED-treated plants than in the short-day control group. The effects of red LED light were measured both in a greenhouse and in a phytotron, irrespective of cultivar-specific differences. Therefore, it can be concluded that a mid-night interruption by red LED light during short photoperiods provides an easy and inexpensive method to increase vegetative leaf biomass production with an increased steviol glycoside yield.     

Elicitation of thiophene production by cultured hairy roots of Tagetes patula

Hairy roots of Tagetes patula were initiated by infecting the seedlings with Agrobacterium rhizogenes. The hairy roots were grown in liquid medium MS (Murashige and Skoog 1962) supplemented with sucrose. The roots were treated with three different elicitors obtained from mycelial culture of Aspergillus niger, Rhizopus oligosporus and Pencillium notatum. Accumulation of biomass and the production of thiophenes were studied over a period of six weeks in culture. The HPLC separation profile of the thiophenes indicated the presence of several structurally different thiophenes. α-terthienyl being predominent. Maximum production of thiophene was recorded at the end of the fourth week in culture with a content of 0.138 % (w/w on dry weight basis). Treatment of hairy roots with mycelial extract of Aspergillus niger (1.5 % v/v) elicited an increase in thiophene content by 1.6 folds over the control.     

High forskolin production in hairy roots of Coleus forskohlii

Hairy roots of Coleus forskohlii were induced by infection with the Agrobacterium rhizogenes MAFF 03-01724 strain. Growth and forskolin production of two hairy root clones cultured in various liquid media were examined. Hairy root clone B9 grew well in woody plant liquid medium and showed a high forskolin yield (ca. 1.3 mg/ 100 ml flask) after 5 weeks of culture. The time course of growth and forskolin production of the clone B9 cultured in woody plant liquid medium was also examined. Rapid growth started at week 2 and continued until week 5. The highest forskolin yield (ca. 1.6 mg/100 ml flask) was obtained at week 5. Productivity was much higher than that previously reported. Received: 19 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997     

Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni)

Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In the present study, seed germination, stem cuttings and micropropagation were investigated for higher productivity. Fresh seeds showed better germination (25.51–40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg l⁻¹). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg l⁻¹) and gibberellic acid (GA₃; 0.5 mg l⁻¹). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51–40%) and stem cutting (60%).     

Effects of plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on plant growth,stevioside, NPK,and chlorophyll content of Stevia rebaudiana

Stevia rebaudiana Bertoni is a unique medicinal plant which is mostly utilized as a sugar substitute for diabetic patients. In this research, regenerated plantlets of stevia in tissue culture is transferred to pots in greenhouse and inoculated with plant-growth-promoting rhizobacteria (PGPRs) (Bacillus polymixa, Pseudomonas putida, and Azotobacter chroococcum) and arbuscular mycorrhizal fungus (AMF) (Glomus intraradices). The results showed that in comparison to control, inoculation with a single microorganism, significantly increased root and shoot biomass as well as stevioside, chlorophyll, and NPK content in plants. However, such increased effects have been found to be further enhanced significantly due to dual compatible mixtures of inoculants resulting from their strong synergistic relationships among themselves. All growth parameters recorded the highest in 60-days-old plants in the treatment of Glomus+Azotobacter and followed with Glomus+Bacillus and Azotobacter+Pseudomonas treatments, respectively. Furthermore, high-performance liquid chromatography (HPLC) chromatograms revealed that the highest stevioside content have been produced in same treatments. Triple treatments had less positive effects compared to dual inoculations. Probably competence between microorganisms in triple inoculations has reduced their efficiency. Thus, suitable combination of mycorrhizal fungi and PGPR as biotic elicitors can enhance growth and stevioside content in tissue culture-regenerated plantlets of stevia.     

Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway) leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur in most bacteria and in all higher plants. Treatment with the antibiotic fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and lochnericine by Catharanthus roseus hairy root cultures in the exponential growth phase. However, fosmidomycin did not significantly affect alkaloid levels in stationary phase hairy root cultures. Feeding with 1-deoxy-D-xylulose, 10-hydroxygeraniol, or loganin resulted in significant increases in alkaloid production by exponential phase hairy root cultures. These results suggest that the DXP pathway is a major provider of carbon for the monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus hairy roots in the exponential phase.     

Arabidopsis thaliana hairy roots for the production of heterologous proteins

To evaluate the ability of Arabidopsis thaliana hairy roots to produce heterologous proteins, hypocotyls were transformed with Rhizobium rhizogenes harbouring a green fluorescent protein gene (gfp) fused to a plant signal peptide sequence. Hairy root transgenic lines were generated from wild-type or mutant genotypes. A line secreted GFP at 130 mg/l of culture medium. Unlike as was previously found with turnip hairy roots, a His-tag was still attached to approximately 50?% of the protein. Control of the pH and addition of a protease inhibitor to the culture medium resulted in up to 87?% of the GFP retaining the His-tag. A. thaliana hairy roots expressing the human serpina1 (α-1-antitrypsin) gene secreted the protein, which was visible on a PAGE gel. Protein activity in the culture medium was demonstrated using an elastase inhibition assay. A. thaliana hairy roots can now be considered for the production of heterologous proteins, making it possible to mine the numerous genetic resources for enhancing protein production and quality.     

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g^–1 astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l^–1, astragaloside IV 0.9 mg g^–1). Polysaccharide yields were similar amongst the different bioreactors (range 25–32 mg g^–1). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.     

Growth and betacyanin production by hairy roots of Beta vulgaris in airlift bioreactors

Hairy roots of red beet (Beta vulgaris L.) were cultivated in different types of airlift bioreactors (cone, balloon, bulb, drum and column bioreactors of 5 l capacity and containing 3 l of half strength Murashige & Skoog medium). The cone type of airlift bioreactor gave the highest biomass of hairy roots and betacyanin accumulation. Betacyanin accumulation was 27 mg g^–1 dry wt in cultures aerated at 0.3 vvm. Light irradiation of 20 mol m^–2 s^–1 promoted hairy root growth but optimum betacyanin (34 mg g^–1 dry wt) accumulation was with the cultures grown under 60 mol m^–2 s^–1.     

Psoralen production in hairy roots and adventitious roots cultures of Psoralea coryfolia

Psoralea corylifolia is an endangered plant producing various compounds of medical importance. Adventitious roots and hairy roots were induced in cultures prepared from hypocotyl explants. Psoralen content was evaluated in both root types grown either in suspension cultures or on agar solidified medium. Psoralen content was ~3 mg g⁻¹ DW in suspension grown hairy roots being higher than in solid grown hairy roots and in solid and suspension-grown adventitious roots.     

Optimization of indole acetic acid production by isolated bacteria from Stevia rebaudiana rhizosphere and its effects on plant growth

The ability to synthesize Indole-3-acetic acid (IAA) is widely associated with the plant growth promoting rhizobacteria (PGPR). The present work deals with isolation and characterization of such bacteria from the rhizosphere of medicinal plant Stevia rebaudiana and optimization of IAA production from its isolates. The optimization of IAA production was carried out at different pH and temperature with varied carbon and nitrogen sources of culture media. Out of different isolates obtained, three of them were screened as efficient PGPRs on the basis of different plant growth promoting attributes. Isolates CA1001 and CA2004 showed better production of IAA at pH 9 (91.7?µg?ml^?1) and at temperature 37?°C (81.7?µg?ml^?1). Dextrose (1%) was found to be the best carbon source for isolate CA1001 with 104?µg?ml^?1 IAA production. Isolate CA 2004 showed best production of IAA 36?µg?ml^?1 and 34?µg?ml^?1 at 1.5% and 1% Beef extract as nitrogen source respectively. Isolate CA 1001 showed 32?µg?ml^?1 IAA production at 0.5% nicotinic acid concentration. From the current study, CA1001 and CA2004 emerged as noble alternatives for IAA production further which also resulted in root and shoot biomass generation in crop plants, hence can be further used as bio-inoculants for plant growth promotion.     

Contributions to the biotechnological production of sweeteners from stevia rebaudiana bertoni. II. Induction of stevioside accumulation in cell cultures by variation of the nutrient medium and the analysis of small amounts of stevioside

Cell cultures of Stevia rebaudiana in general do not contain stevioside when grown on their basal nutrient medium. No stable and prolonged stevioside accumulation was achieved by varying the medium components. However, in some cases a transient state was observed where more stevioside was synthesized than catabolized. The dynamic behaviour of product synthesis which led to accumulation of stevioside during only a short period was demonstrated. For accurate determination of very small amounts of stevioside, a combined TLC-HPLC method was used [1].