Callus Suspension Research Articles

In vitro production and distribution of flavonoids in Glycyrrhiza uralensis Fisch.

Glycyrrhiza uralensis Fisch. is known as a common Chinese medicinal herb used to harmonize the effects of other ingredients in most Chinese herbal prescriptions. The rapid production of flavonoids in vitro remains unknown in G. uralensis Fisch. To investigate the in vitro adventitious root regeneration and flavonoid accumulation characteristics in G. uralensis for restrictions on collecting wild plants, suspension cultural and freezing microtomy with histochemical assays were carried out. We reported that multiple adventitious roots were initiated from hypocotyls and stems of G. uralensis. Indole-3-butyric acid (IBA) was more conducive than NAA (1-naphthaleneacetic acid) in inducing G. uralensis adventitious roots, but the addition of 6-BA (6-benzylaminopurine) and KT (kinetin) suppressed the formation of adventitious roots. While the concentration of IBA was 1.0mgL-1, the flavonoid content and yield were the highest at 19.96mgg-1 and 1.23mgg-1, respectively. The optimum medium for adventitious root induction was 1/4-strength Murashige and Skoog's medium containing 0.1mgL-1 IBA. The content of flavonoids in adventitious roots and apicals cultured in vitro was higher than that in suspension callus, reaching 3.87 times the callus flavonoid content. The histochemical localization of flavonoids showed that G. uralensis flavonoids mainly distributed in the epidermal parenchyma cells of the callus outer layers and gradually accumulated in cell wall and cell gaps of the epidermis and endodermis of adventitious roots along with the primary growth of adventitious roots, indicating that there were no flavonoids in the roots at the early stage of adventitious roots formation. The results showed that calli inducing adventitious roots and apicals for 30days obtained the highest yield of flavonoid, indicating effective production for flavonoids instead of wild culture. AlCl3 ethanol solution was better than NaOH aqueous solution in terms of chromogenic and localization effects. We concluded that the highest yield of flavonoid and effective production for flavonoid instead of wild culture could be obtained from calli inducing adventitious roots and apicals.

Anti-inflammatory Effects of Northern Highbush Blueberry Extract on an In Vitro Inflammatory Bowel Disease Model

Blueberry anthocyanins have the ability to efficiently reach the GI tract and exhibit a broad range of biochemical effects. In the context of inflammatory bowel disease (IBD), they remain a promising complement to current IBD treatments. Here, we investigated the anti-inflammatory and antioxidant capabilities of Highbush blueberries in-vitro on two normal colon epithelial cell lines, NCM 356 and CCD 841 CoN using fluorescent microscopy and flow cytometry following stimulation with a pro-inflammatory cytokine cocktail. Treatment with blueberry extract revealed a significant decrease in nuclear and cytoplasmic generated reactive oxygen species (ROS) compared to controls. Additionally, the blueberry extract increased cell viability following treatment with the pro-inflammatory cytokine cocktail. A comparison with previous report on rice callus suspension culture (RCSC) revealed opposing trend with reference to the levels of nuclear and cytoplasmic ROS. It is likely that blueberry extract and RCSC employ different players and pathways to mitigate inflammation.

Lignin biosynthesis genes play critical roles in the adaptation of Arabidopsis plants to high-salt stress

ABSTRACTSalinity is a major abiotic stressor that limits the growth, development, and reproduction of plants. Our previous metabolic analysis of high salt-adapted callus suspension cell cultures from Arabidopsis roots indicated that physical reinforcement of the cell wall is an important step in adaptation to saline conditions. Compared to normal cells, salt-adapted cells exhibit an increased lignin content and thickened cell wall. In this study, we investigated not only the lignin biosynthesis gene expression patterns in salt-adapted cells, but also the effects of a loss-of-function of CCoAOMT1, which plays a critical role in the lignin biosynthesis pathway, on plant responses to high-salt stress. Quantitative real-time PCR analysis revealed higher mRNA levels of genes involved in lignin biosynthesis, including CCoAOMT1, 4CL1, 4CL2, COMT, PAL1, PAL2, and AtPrx52, in salt-adapted cells relative to normal cells, which suggests activation of the lignin biosynthesis pathway in salt-adapted cells. Moreover, plants harboring the CCoAOMT1 mutants, ccoaomt1-1 and ccoaomt1-2, were phenotypically hypersensitive to salt stress. Our study has provided molecular and genetic evidence indicating the importance of enhanced lignin accumulation in the plant cell wall during the responses to salt stress.

Optimization of noni callus induction and establishment of callus suspension system

The aim of the study was to obtain the secondary metabolites in the stem segment of noni and to establish genetic transformation system. The stem segments (no axillary buds) of noni were used as explants to induce the callus, and then to establish the cell suspension system. The factors affecting callus induction and cell suspension were studied. The results showed that the optimal culture medium for induction was MS with 1.0 mg/L 6-Benzylaminopurine (6-BA) and 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and the optimum culture medium for suspension was MS with 1.0 mg/L 6-BA and 0.1 mg/L 2,4-D, 3% sucrose and the pH of 5.85, with the initial inoculation amount of 37.5 g/L, and the speed of 110 r/min and 25±2 °C applying darkness culture. The suspension cells grew well and showed the maximum growth rate. The growth curve of the suspension cells from the stem segment of noni was in "S-typed" trend, and it should be transformed to the fresh medium between 12 and 20 d. During the culture, the pH of the culture medium decreased and then slowly increased, and the optimum pH for the suspension cells culture of callus from noni's stem segments was 4.5-5.0. In this study, the stable cell suspension system of the stem segment of noni was successfully established.

Analysis of phenolics in Calligonum polygonoides in vitro cultured roots

Background: Calligonum polygonoides L. subsp. Comosum (L’Her.) Sosk. is an endangered plant species belonging to family Polygonaceae. Although the plant is rich in phytoconstituents and has multipurpose medicinal applications, but in vitro root culture studies and phytochemical investigations of these cultures are rare. Objectives: To establish in vitro root, callus and cell suspension cultures from in vitro germinated fruits of C. polygonoides to investigate the production of phenolics through root, callus and cell suspension cultures and attempt to enhance cell capacity to accumulate phenolics. Materials and Methods: Modified Murashige and Skoog medium supplemented with 1 mg l-1 indole-3-butyric acid was used to establish the root culture. Elicitation of cell suspension culture was performed using salicylic acid and yeast extract. The phenolic compounds in root, callus and cell suspension cultures were evaluated using reversed phase high performance liquid chromatography technique. Results: The unorganized cell suspension culture contained fewer amounts of phenolic compounds than the differentiated roots tissue. Elicitation produced quantitative reprogramming of phenolic content. Conclusion: The present study provides a chance to improve secondary metabolite yield from this valuable natural plant.

Anti-inflammatory and immune-modulating effects of rice callus suspension culture (RCSC) and bioactive fractions in an in vitro inflammatory bowel disease model

BackgroundRice callus suspension culture (RCSC) has been shown to exhibit potent antiproliferative activity in multiple cancer cell lines. RCSC and its bioactive compounds can fill the need for drugs with no side effects. Hypothesis/PurposeThe anti-inflammatory potential of RCSC and its bioactive fractions on normal colon epithelial cell lines, was investigated. Study designThree cell lines, InEpC, NCM356 and CCD841-CoN were treated with proinflammatory cytokines followed by RCSC. Cytoplasmic and nuclear ROS were assayed with fluorescent microscopy and flow cytometer. Expression analysis of immune-related genes was performed in RCSC-treated cell lines. RCSC was fractionated using column chromatography and HPLC. Pooled fractions 10–18 was used to test for antiproliferative activity using colon adenocarcinoma cell line, SW620 and anti-inflammatory activity using CCD841-CoN. Mass spectrometric analysis was performed to identify candidate compounds in four fractions. ResultsRCSC treatment showed differential effects with higher cytoplasmic ROS levels in NCM356 and CCD841-CoN and lower ROS levels in InEpC. Nuclear generated ROS levels increased in all three treated cell lines. Flow cytometry analysis of propidium iodide stained cells indicated mitigation of cell death caused by inflammation in RCSC treated groups in both NCM356 and CCD841-CoN. Genes encoding transcription factors and cytokines were differentially regulated in NCM356 and CCD841-CoN cell lines treated with RCSC which provided insights into possible pathways. Analysis of pooled fractions 10–18 by HPLC identified 8 peaks. Cell viability assay with fractions 10–18 using SW620 showed that the number of viable cells were greatly reduced which was similar to 6X and 33X RCSC with very little effect on normal cells which similar to 1X RCSC. RCSC fractions increased nuclear and cytoplasmic ROS vs. both untreated and inflammatory control. Analysis of four fractions by mass spectrometry identified 4-deoxyphloridzin, 5′-methoxycurcumin, piceid and lupeol as candidate compounds which are likely to be responsible for the antiproliferative, anti-inflammatory and immune-regulating properties of RCSC. ConclusionRCSC and its fractions showed anti-inflammatory activity on inflamed colon epithelial cells. Downstream target candidate genes which are likely to mediate RCSC effects were identified. Candidate compounds responsible for the antiproliferative and anti-inflammatory activity of RCSC and its fractions provide possible drug targets.

Metabolic Adjustment of Arabidopsis Root Suspension Cells During Adaptation to Salt Stress and Mitotic Stress Memory.

Sessile plants reprogram their metabolic and developmental processes during adaptation to prolonged environmental stresses. To understand the molecular mechanisms underlying adaptation of plant cells to saline stress, we established callus suspension cell cultures from Arabidopsis roots adapted to high salt for an extended period of time. Adapted cells exhibit enhanced salt tolerance compared with control cells. Moreover, acquired salt tolerance is maintained even after the stress is relieved, indicating the existence of a memory of acquired salt tolerance during mitotic cell divisions, known as mitotic stress memory. Metabolite profiling using 1H-nuclear magnetic resonance (NMR) spectroscopy revealed metabolic discrimination between control, salt-adapted and stress-memory cells. Compared with control cells, salt-adapted cells accumulated higher levels of sugars, amino acids and intermediary metabolites in the shikimate pathway, such as coniferin. Moreover, adapted cells acquired thicker cell walls with higher lignin contents, suggesting the importance of adjustments of physical properties during adaptation to elevated saline conditions. When stress-memory cells were reverted to normal growth conditions, the levels of metabolites again readjusted. Whereas most of the metabolic changes reverted to levels intermediate between salt-adapted and control cells, the amounts of sugars, alanine, γ-aminobutyric acid and acetate further increased in stress-memory cells, supporting a view of their roles in mitotic stress memory. Our results provide insights into the metabolic adjustment of plant root cells during adaptation to saline conditions as well as pointing to the function of mitotic memory in acquired salt tolerance.

Phytochemical characteristics of callus suspension culture of Helicteres angustifolia L. and its in vitro antioxidant, antidiabetic and immunomodulatory activities

Helicteres angustifolia L. (H.angustifolia) is an important medicinal plant. This study investigated the phytochemical characteristics of callus suspension cultures of H.angustifolia and its bioactive functions, such as antioxidant, antidiabetic and immunomodulatory activities. Results demonstrated that the cultures were rich in phenolic compounds, flavonoids, terpenoids, saponins and triterpenoids. Compared to the equivalent wild roots, the cultures yielded larger amounts of total phenolics (17.75 mg GAE g−1), flavonoids (56.15 mg RE g−1), saponins (29.21 mg OAE g−1) and polysaccharides (13.69 mg g−1), about 1.62-, 1.90-, 1.14- and 3.41-fold higher than those from the equivalent wild roots, respectively. Furthermore, as the valuable target product from in vitro culture, phenolic compounds were subjected to HPLC analysis, reflecting that some representative phenolic compounds fluctuated significantly under the cultivation conditions. Additionally, bioactive assays showed that the extract of callus suspension cultures (CE) possessed strong antioxidant activities, and rat maltase (IC50 = 13.29 mg mL−1) and sucrase (IC50 = 13.76 mg mL−1) inhibitory activities. As for the immunomodulatory activity, CE could stimulate the proliferation of macrophages (121.93 ± 1.76%) and the production of nitric oxide (200% of the control) and phagocytic activity (178% of the control) at 6.25 μg mL−1. Moreover, CE could mitigate DOX-induced macrophages damage to some extent. In conclusion, the callus suspension culture of H.angustifolia is a potential source for bioactive phytochemical compounds and functional foods.

Early Responses to Severe Drought Stress in the Arabidopsis thaliana Cell Suspension Culture Proteome.

Abiotic stresses are considered the most deleterious factor affecting growth and development of plants worldwide. Such stresses are largely unavoidable and trigger adaptive responses affecting different cellular processes and target different compartments. Shotgun proteomic and mass spectrometry-based approaches offer an opportunity to elucidate the response of the proteome to abiotic stresses. In this study, the severe drought or water-deficit response in Arabidopsis thaliana was mimicked by treating cell suspension callus with 40% polyethylene glycol for 10 and 30 min. Resulting data demonstrated that 310 proteins were differentially expressed in response to this treatment with a strict ±2.0-fold change. Over-representation was observed in the gene ontology categories of ‘ribosome’ and its related functions as well as ‘oxidative phosphorylation’, indicating both structural and functional drought responses at the cellular level. Proteins in the category ‘endocytosis’ also show significant enrichment and this is consistent with increased active transport and recycling of membrane proteins in response to abiotic stress. This is supported by the particularly pronounced enrichment in proteins of the endosomal sorting complexes that are required for membrane remodelling. Taken together, the findings point to rapid and complex physiological and structural changes essential for survival in response to sudden severe drought stress.

Different glyceollin synthesis-related metabolic content and gene expressions in soybean callus suspension cultures and cotyledon tissues induced by alginate oligosaccharides

Abstract Glyceollins are a group of inducible soybean phytoalexins. The aim of the present study was to investigate the mechanism of glyceollin production in callus suspension cultures (CS) and cotyledon tissues elicited with alginate oligosaccharides (AOS) at 1%, 2%, and 3% concentrations that were applied for three days. HPLC analysis of the extracts from 2% AOS-elicited callus suspension cultures (ECS) revealed that glyceollins content was 85.3-fold and 8.2-fold higher than that in unelicited controls and elicited cotyledon tissues (ECT), respectively. UPLC-Q-Exactive Orbitrap MS analysis showed that the 2% AOS induction resulted in elevated levels of metabolites, the intermediary products of glyceollin biosynthesis, such as daidzein, glycitein, genistein, naringenin, glycinol, glyceollidin I/II, and glyceollin I, II, and III in ECS and ECT. Glyceollin I was prominent among the three glyceollin isomers in ECS, whereas isomer II and III were dominant in the synthetic route for glyceollins in ECT. qRT-PCR analysis of related genes showed that UGT, CYP93A1, G2DT, and G4DT displayed a direct effect on glyceollin synthesis. Our results suggested that 2% AOS-induced CS produced glyceollins in higher content compared to ECT by altering metabolite content and gene expression. Elicitation of CS is thus a promising technology for glyceollin synthesis extensively.

High yield production of folates from soybean callus cultures in response to elicitors.

Soybean (Indian variety, JS 335) callus growth and its folate content was sustained on Murashige and Skoog (MS) medium supplemented with optimized levels of auxins and cytokinins. Callus cultures exhibited moderate production of total folate. Callus growth was stimulated with highest biomass content of 21.3g/L DW (24days culture) on medium supplemented with 1.5mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.1mg/L Kinetin (Kn). Total folate production was enhanced by an increase in indole-3-acetic acid (IAA) from 0.5 to 1.0mg/L and decreased thereafter up to 1.5mg/L. 2,4-D at 1.5mg/L repressed the callus growth and also folate production. A concentration of 1.0mg/L IAA and 0.2mg/L Kn elicited folate production to a maximum total folate content of 323.82µg/100g DW. The results indicate that optimized medium for callus suspension cultures conceivably are applied for scale-up studies in bioreactors.

Enhanced production of vanillin flavour metabolites by precursor feeding in cell suspension cultures of Decalepis hamiltonii Wight & Arn., in shake flask culture

The flavour rich tuberous roots of Decalepis hamiltonii are known for its edible and medicinal use and have become endangered due to commercial over-exploitation. Besides 2-Hydroxy-4-methoxy benzaldehyde (2H4MB), other flavour metabolites in tuberous roots include vanillin, 4-Methoxy Cinnamic acid derivatives, aromatic alcohols etc. So far, there are no reports on the pathway of 2H4MB biosynthesis nor there is an organized work on biotransformation using normal and cell suspension cultures for obtaining these metabolites using precursors. The main aim of the study is to develop a method for enhanced production of flavour attributing metabolites through ferulic acid (FA) feeding to the D. hamiltonii callus culture medium. Biomass of D. hamiltonii cell suspension cultures was maximum (200.38±1.56g/l) by 4th week. Maximum production of 2H4MB was recorded on 4th week (0.08±0.01mg/100g dry weight) as quantified by HPLC. Addition of 0.1-1.5mM ferulic acid as precursor in the culture medium showed significant (p<0.001) effect on suspension cultures biomass and respective phenylpropanoid metabolites content and 2H4MB accumulation. The maximum accumulation of vanillin, 2H4MB, vanillic acid, ferulic acid were of 0.1±0.02mg/100g, 0.44±0.01mg/100g, 0.52±0.04mg/100g, 0.18±0.02mg/100g DW respectively in 4weeks of cultured cells supplemented with 1mM ferulic acid as a precursor. The results indicate that, substantial increase in the levels of flavour metabolites in D. hamiltonii callus suspension culture was achieved. This would be having implications in biosynthesis of respective vanilla flavour attributing metabolites at very high levels for their large scale production.

Growth, lipid peroxidation, organic solutes, and anti-oxidative enzyme content in drought-stressed date palm embryogenic callus suspension induced by polyethylene glycol

Polyethylene glycol (PEG) can be used in somatic embryogenesis to enhance embryogenic development and improve the success of in vitro culture but PEG also causes osmotic stress in developing embryos. The effects of PEG on embryo growth and development in date palm cell suspension culture and associated antioxidant enzyme activities were evaluated. Callus maintained on MS basal media was transferred to regeneration liquid media supplemented with increasing levels (0–20%) of polyethylene glycol 6000 (PEG) to induce osmotic stress. The degree of embryogenic callus formation, its fresh weight, and the percentage of normal embryo callus shapes were increased with an increase in the level of PEG up to 10%. Total soluble protein (TSP), proline, glycine betaine (GB), total soluble phenol (TSPh), total sugars (TS), and total soluble organic acids (TOA) also increased whereas superoxide dismutase (SOD) activity decreased in response to PEG supplementation. Raising the PEG level increased malondialdehyde (MDA) concentration up to 10% PEG and thereafter decreased. Glutathione reductase (GR) and catalase (CAT) activities decreased at the highest levels of PEG. The proportion of normal embryo developmental shapes were about 50% compared with 20% abnormal shapes at optimum levels of PEG. Proliferation of somatic embryos was influenced by their developmental shapes. Cv. Samani accumulated more organic solutes compared with cv. Sewi in both control and stress inducing media. In contrast, lipid peroxidation, GR, SOD, and CAT activities were significantly higher in cv. Sewi than in cv. Samani indicating that the cv. Samani had the ability to tolerate a higher level of osmotic stress compared to cv. Sewi due to the enhanced osmotic re-balancing within its tissues.

Capsicum spp in vitro liquid cell suspensions: A useful system for the production of capsaicinoids and polyphenols

Capsicum are among the most extensively cultivated and consumed plant species in the world, because of their unique pungency, aroma and colour. The typical burning sensation caused by chili peppers is due to the occurrence of a group of alkaloids named capsaicinoids. In the present study, the production of solid callus and cell suspensions from hypocotyl explants of three different chili pepper cultivars (Capsicum annuum L. cv. Mazzolino, Capsicum chinense Jacq. cv. Naga Morich and Pimenta de Neyde), was optimised. In addition, C. chinense cv. Naga Morich cell suspensions were supplemented with biotic elicitors (methyl-jasmonate and chitosan) and with precursors and intermediates of capsaicin biosynthesis (vanillin, phenylalanine and valine), and both cells and media were analysed for capsaicinoid, polyphenol, flavonoid contents and for antioxidant activity. This is the first report regarding capsaicinoid elicitation with pure chitosan and with a combination of precursors of both phenylpropanoid and valine pathways. Overall, the highest capsaicinoid levels were detected in cell extracts from cultures treated with 10 μM methyl-jasmonate and with a combination of phenylalanine and valine amino acids (100 μM each). The present results confirm the possibility of using hypocotyl chili pepper cell suspensions to produce high amounts of health beneficial metabolites.

Growth and accumulation of flavan-3-ol in Camellia sinensis through callus culture and suspension culture method

Growth and accumulation of flavan-3-ol in Camellia sinensis through callus culture and suspension culture method

Rice callus suspension culture inhibits growth of cell lines of multiple cancer types and induces apoptosis in lung cancer cell line.

BackgroundCancer is one of the leading cause of mortality. Even though efficient drugs are being produced to treat cancer, conventional medicines are costly and have adverse effects. As a result, alternative treatments are being tried due to their low cost and little or no adverse effects. Our previous study identified one such alternative in rice callus suspension culture (RCSC) which was more efficient than Taxol® and Etoposide, in reducing the viability of human colon and renal cancer cells in culture with minimal or no effect on a normal cell line.MethodsIn this study, we tested the effect of RCSC by studying the dynamics of lactate dehydrogenase (LDH) in lung cancer cell lines (NCI-H460 and A549), breast cancer cell lines (MDA-MB-231 and MCF-7) and colorectal cancer cell lines (SW620 and Caco-2) as well as their normal-prototypes. Complementary analysis for evaluating membrane integrity was performed by estimating LDH release in non-lysed cells and cell viability with WST-1 assay. Fluorescence microscopy with stains targeting nucleus and cell membrane as well as caspase 3/7 and Annexin V assays were performed. Real-time quantitative RT-PCR was performed to evaluate expression of 92 genes associated with molecular mechanisms of cancer in RCSC treated ling cancer cell line, NCI-H460 and its normal prototype, MRC-5. High performance liquid chromatography (HPLC) was used to collect RCSC fractions, which were evaluated on NCI-H460 for their anti-cancer activity.ResultsLower dilutions of RCSC showed maximum reduction in total LDH indicating reduced viability in majority of the cancer cell lines tested with minimal or no effect on normal cell lines compared to the control. Complementary analysis based on LDH release in non-lysed cells and WST-1 assay mostly supported total LDH results. RCSC showed the best effect on the lung non-small carcinoma cell line, NCI-H460. Fluorescence microscopy analyses suggested apoptosis as the most likely event in NCI-H460 treated with RCSC. Gene expression analysis identified significant upregulation of cJUN, NF-κB2 and ITGA2B in NCI-H460 which resulted most likely in the arrest of cell cycle progression and induction of apoptotic process. Further, HPLC-derived RCSC fractions were less effective in reducing cell viability than whole RCSC suggesting that a holistic approach of using RCSC is a better approach in inhibiting cancer cell proliferation.ConclusionsRCSC was found to be an effective anti-cancer agent on cell lines of multiple cancer types with the best effect on lung cancer cell lines. A possible mechanism for the anticancer activity of RCSC is through induction of apoptosis as observed in the lung cancer cell line, NCI-H460.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1423-3) contains supplementary material, which is available to authorized users.

Optimization of methyl jasmonate and β-cyclodextrin for enhanced production of taraxerol and taraxasterol in (Taraxacum officinale Weber) cultures

ContextTaraxacum officinale Weber (TO) commonly known as “dandelion”, is a tropical Asian medicinal plant which contains taraxasterol (TX) and taraxerol (TA) in its roots, which are reported to be commercially important anticancer compounds. ObjectiveThe main objective of the present study was to evaluate the increase in yield of TX and TA through elicitation by addition of abiotic elictors like methyl jasmonate (MJ) and β-cyclodextrin (CD), to the root callus suspension cultures of TO. Materials and methodsThe root callus suspension was maintained on Murashige and Skoog's (MS) medium MS + IAA + BA + 2, 4-D (0.5 ppm + 1 ppm + 0.5 ppm). The concentrations of the abiotic elicitors MJ and CD were optimized using central composite design (CCD) and quantification of TA and TX in elicited cultures was done by High Performance Liquid Chromatography (HPLC) analysis. ResultIt was observed that MJ at a concentration of 0.2 mM showed good increase in content of TX to 0.032% w/w and at concentrations 0.05 mM, 0.1 mM and 0.2 mM showed similar increase in TA content to 0.018% w/w, whereas CD at the concentration of 25 mM showed highest increase in TX content to 0.036% w/w and at the concentrations of 25 mM, 50 mM showed increase in TA content to 0.023% w/w as compared to the plant root (PR) which showed content of TX as 0.0299% w/w and TA as 0.0169% w/w. Discussion and conclusionFrom the present investigation it was concluded that out of the two abiotic elicitors MJ and CD, CD was found to be more effective to increase TA and TX content in Dandelion cell cultures.

TDZ and 4-CPPU Induce Embryogenic Response on Scalps of Recalcitrant East African Highland Banana

Breeding of banana conventionally is very difficult due to high levels of sterility arising from aneuploidy. Transformation of East African highland banana (EA-AAA banana) has been limited by difficulties in induction of embryogenic callus and/or embryogenic cell suspension (ECS), a requirement for efficient genetic transformation. In this study N-phenyl-N’-1,2,3-thidiazol-5-ylurea (TDZ) and N-(2-chloro-4-pyridyl)-N’-phenylurea (4-CPPU) that are highly active in regulating morphogenesis in tissue culture of many plant species were tested for enhancing embryogenic callus induction in recalcitrant EA-AAA banana through scalps. In vitro derived shoot-tips of banana cultivars Mbwazirume, Mpologoma, Nakabululu, Nakinyika and Nfuuka were subcultured on various combinations of TDZ and 4-CPPU till scalps were formed. In all the cultivars, ideal scalps for callus induction were achieved in over 50% of the shoot-tip cultures within four subculture cycles in the medium containing equal proportions of 9–13 µM of TDZ and 4-CPPU, and 26 µM of each. Embryogenic callus was formed on 2.5–20% of the scalp cultures depending on cultivar and, TDZ and 4-CPPU combination. Cultivars Nakinyika, Nakabululu, Mbwazirume and Nfuuka developed regenerable embryogenic callus. Of the plantlets regenerated 5 out of 12 and 4 out of 5 plantlets of cultivars Nakinyika and Mbwazirume, respectively, developed normal roots and were weaned onto soil. These results suggest that TDZ and 4-CPPU increases embryogenic response from scalps of EA-AAA banana which can enhance their genetic transformation.

Effects of plant growth regulators, carbon sources and pH values on callus induction in Aquilaria malaccensis leaf explants and characteristics of the resultant calli

The endangered tropical tree, Aquilaria malaccensis, produces agarwood for use in fragrance and medicines. Efforts are currently underway to produce valuable agarwood compoundsn tissue culture. The purpose of this study was to develop an optimal growth medium, specifically, the best hormone combination for callus suspension culture. Using nursery-grown A. malaccensis, sterilized leaf explants were first incubated on basic Murashige and Skoog (MS) gel medium containing 15g/L sucrose and at pH 5.7. Different auxin types including 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-butyric acid (IBA), were tested at various concentrations (0.55, 1.1 and 1.65 μM) using the basic medium. Leaf explants were incubated for 30 days in the dark. Callus induced by 1.1 μM NAA had the highest biomass dry weight (DW) of 17.3 mg; however the callus was of a compact type. This auxin concentration was then combined with either 6-benzylaminopurine (BAP) or kinetin at 0.55, 1.1, 2.2 or 3.3 μM to induce growth of friable callus. The 1.1μM NAA + 2.2μM BAP combination produced friable callus with the highest biomass (93.3mg DW). When testing the different carbon sources and pHs, sucrose at 15g/L and pH at 5.7 yielded highest biomasses at 87.7mg and 83mg DW, respectively. Microscopic observations revealed the arrangement of the friable cells as loosely packed with relatively large cells, while for the compact callus, the cells were small and densely packed. We concluded that MS medium containing 15 g/L sucrose, 1.1 μM NAA + 2.2 μM BAP hormone combination, and a pH of 5.7 was highly effective for inducing friable callus from leaf explants of A. malaccensis for the purpose of establishing cell suspension culture.

Production of monoclonal antibody against FimA protein from Porphyromonas gingivalis in rice cell suspension culture

Major fimbriae (FimA) of Porphyromonas gingivalis play an important role in the adherence of the bacterium to oral surfaces, making them a target for the development of a vaccine against periodontal disease. In our previous study, monoclonal antibodies to FimA were expressed in tobacco plants using the agroinfiltration method. In this report, for easy purification, monoclonal antibodies to FimA were produced and accumulated in rice callus suspension culture. The accumulated antibodies were purified by protein G-affinity chromatography. The plant-produced monoclonal antibodies inhibited the binding of P. gingivalis to saliva-coated hydroxyapatite beads, as well as the invasion of oral epithelial KB cells due to the bacterium. The antibodies enhanced the intracellular killing of P. gingivalis by polymorphonuclear neutrophils. These results suggest that FimA-specific monoclonal antibodies produced in a rice suspension culture were easily purified and biologically active against P. gingivalis, and thus may be used for passive immunization to prevent P. gingivalis-induced periodontal disease.