Development of an encapsulation-vitrification protocol for Rubia akane (nakai) hairy roots: a comparison with non-encapsulation.

BACKGROUND: A comparison of different cryopreservation techniques should be based on the characteristics of both the methodology and the material in question using an optimized procedure. OBJECTIVE: This study aimed at developing an encapsulation-vitrification procedure for hairy roots of Rubia akane using alternative loading and vitrification solutions, based on the existing optimized droplet-vitrification procedure. MATERIALS AND METHODS: Encapsulated roots were first precultured in liquid medium with 10% sucrose for 3 days, then with 17.5 % sucrose for 1 day, after which they were osmoprotected with solution C6-40 % (20 % glycerol + 20 % sucrose) for 50 min, cryoprotected with solution A3-90 % (37.5 % glycerol + 15 % DMSO + 15 % EG + 22.5 % sucrose, w/v) on ice for 40 min, cooled and warmed in 2 ml cryovials, and unloaded in 35% sucrose solution for 60 min. RESULTS: Through the application of this procedure to aged-clustered roots, up to 97.5 % post-cryopreservation regeneration was observed. In our previous study, droplet-vitrification of hairy roots of R. akane resulted in 83.8 % post-rewarming regeneration following preculture with 10 % sucrose for 2 days and 17.5 % sucrose for 4-5 h, and osmoprotection with solution C4-35 % (17.5 % glycerol + 17.5 % sucrose) for 30 min, and cryoprotection with solution A3-70 % (29.2 % glycerol + 11.7 % DMSO + 11.7% EG + 17.4% sucrose, w/v) on ice for 20 min. In the present study, higher post-cryopreservation regeneration was observed by using a higher concentration of vitrification solution (A3-70 % → A3-90 %, B5-80 % → B1-100 %) and/or a longer cryoprotection duration (A3-70 % at room temperature (RT) for 8 min → 15-30 min, on ice for 20 min → 40-80 min; B5-80 % for 15 min → 30-60 min). CONCLUSION: Even though encapsulation provided some degree of protection from the cytotoxicity of vitrification solutions to cytotoxicity-sensitive R. akane hairy roots, an overall higher post-cryopreservation regrowth was obtained using the droplet-vitrification procedure under optimized conditions. This result implies that this sensitive material was not sufficiently cryoprotected, and thus, rapid cooling and warming using foil strips was more efficient than cryopreservation of encapsulated samples.

Development of vitrification protocol in Rubia akane (nakai) hairy roots using a systematic approach.

BACKGROUND: A solution-based vitrification protocol is a process of sequentially changing-solutions from which both influx of cryoprotectants (loading) and efflux of water (dehydration) were accomplished before cryo-exposure. Hence, we need to properly control the concentration /composition of the cryoprotectant solutions. OBJECTIVE: The study was, using a systematic approach, to develop a protocol for Rubia akane hairy roots, a very sensitive material to cytotoxicity of vitrification solutions. METHODS: Due to the poor response of 10-year in vitro maintained R. akane hairy roots to already established cryopreservation protocols, the following sets of experiments were designed: 1) combinational effect of preculture, osmoprotection and cryoprotection with PVS2-based (A3-70%) and PVS3-based (B5-80%) vitrification solutions; 2) different cooling/warming rates and warming temperature; 3) varying unloading solutions (25%, 35%and 45% sucrose) and durations (7 min and 30 min) with or without changing the unloading solutions. RESULTS: Preculture and osmoprotection treatments were necessary to acquire cytotoxicity tolerance in both vitrification solutions tested and osmoprotection treatment was more critical, especially in B5-80%. A sequential osmoprotection treatment (C10-50%) following conventional osmoprotection (C4-35%) was needed to increase the post-cryopreservation regrowth. Aluminum foil strips were superior to cryovials, but the warming temperature tested (20 degree C and 40 degree C) did not affect post-cryopreservation recovery. In the unloading procedure, a longer duration (30 min) with a higher sucrose solution (S-45%) was harmful, possibly due to osmotic stress. CONCLUSION: R. akane hairy roots are very sensitive to cytotoxicity (both osmotic stress and chemical toxicity) and thus a proper process (preculture, osmoprotection, cryoprotection and unloading) is necessary for higher post-cryopreservation recovery.

Expression profiles of calcium-dependent protein kinase genes (CDPK1-14) in Agrobacterium rhizogenes pRiA4-transformed calli of Rubia cordifolia under temperature- and salt-induced stresses.

Agrobacterium rhizogenes genetically transform plant cells naturally via horizontal gene transfer by the introduction of T-DNA from the Ri plasmid into genomic DNA to create favorable conditions for successful colonization. An intriguing feature of pRiA4-transformed cells is their recently discovered enhanced tolerance to abiotic stress stimuli and activation of antioxidant enzyme expression. The mechanism by which A. rhizogenes modulates the defense responses of transformed cells remains unclear. It has been established that calcium-dependent protein kinase (CDPK) genes mediate crosstalk of signaling pathways in plants, and these genes have been implicated in biotic and abiotic stress signaling. In this study, we identified fourteen CDPK genes from Rubia cordifolia and examined their expression in aerial plant organs as well as in non-transformed and A. rhizogenes A4-transformed calli. Expression of RcCDPK4, RcCDPK5, RcCDPK7, and RcCDPK10 was 1.2- to 3.9-fold higher in pRiA4-transformed cells than in non-transformed cells, whereas expression of RcCDPK1, RcCDPK9, RcCDPK11, and RcCDPK14 was 1.2- to 1.9-fold lower. Agrobacterium transformation substantially modified the transcriptional responses of specific RcCDPK isoforms in pRiA4-transformed cells under conditions of temperature- and salinity-induced stress. On the basis of the results, we suggest that A. rhizogenes T-DNA genes exert their diverse biological functions by altering the expression of various CDPK genes.

Effects of lovastatin, clomazone and methyl jasmonate treatment on the accumulation of purpurin and mollugin in cell suspension cultures of Rubia cordifolia.

AIM: To determine the IPP origin of the naphthoquinones (NQs) in Rubia cordifolia, and to evaluate the effects of methyl jasmonate (MeJA) treatment, MEP, and MVA pathway inhibitor treatment on the accumulation of anthraquinones (AQs) and NQs in cell suspension cultures of R. cordifolia. METHODS: Cell suspension cultures of R. cordifolia were established. Specific inhibitors (lovastatin and clomazone) and MeJA were supplied to the media, respectively. Treated cells were sampled every three days. Content determination of purpurin (AQs) and mollugin (NQs) were carried out using RP-HPLC. The yield of the two compounds was compared with the DMSO-supplied group and the possible mechanism was discussed. RESULTS: Lovastatin treatment increased the yield of purpurin and mollugin significantly. Clomazone treatment resulted in a remarkable decrease of both compounds. In the MeJA-treated cells, the purpurin yield increased, meanwhile, the mollugin yield decreased compared with control. CONCLUSION: The IPP origin of mollugin in R. cordifolia cell suspension cultures was likely from the MEP pathway. To explain the different effects of MeJA on AQs and NQs accumulation, studies on the regulation and expression of the genes, especially after prenylation of 1,4-dihydroxy-2-naphthoic acid should be conducted.

The rolB gene suppresses reactive oxygen species in transformed plant cells through the sustained activation of antioxidant defense.

The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.

Evaluation of analgesic and anti-inflammatory activity of Diospyros Cordifolia Extract

Abstract: In this study we evaluated the analgesic and anti- inflammatory activities of the methanol extract of stem bark of Diospyros cordifolia (MEDC) Roxb. The analgesic effects of the stem bark of the plant was assessed in mice using the tail-flick method while carrageenan, histamine and dextran induced paw oedema was used to study the antiinflammatory effects in rats. The MEDC exhibited significant (p<0.01) analgesic effects comparable to the reference drug diclofenac sodium. MEDC also was evaluated for its anti-inflammatory potential against carrageenan, histamine and dextran induced rat paw edema. The methanol extract (25 and 50 mg / kg body weight) exhibited significant (p<0.01) activity against all phlogistic agents used in a dose dependent manner. All these effects were compared with reference drug phenylbutazone (100 mg/kg body weight)

Decreased ROS level and activation of antioxidant gene expression in Agrobacterium rhizogenes pRiA4-transformed calli of Rubia cordifolia.

Microbe-plant interactions often lead to a decrease in the reactive oxygen species (ROS) level of plant cells, which allows pathogen survival through the suppression of plant immune responses. In the present investigation, we tested whether transformation of Rubia cordifolia cells by Agrobacterium rhizogenes had a similar effect. We isolated partial cDNA sequences of ascorbate peroxidase, catalase and Cu/Zn superoxide dismutase genes (RcApx1, RcApx2, RcApx3, RcCAT1, RcCAT2, RcCSD1, RcCSD2 and RcCSD3) from plant tissues, as well as pRiA4-transformed and normal calli of Rubia cordifolia, and studied their expression by real-time PCR. Transcription profiling revealed that ascorbate peroxidase (RcApx1) and Cu/Zn superoxide dismutase (RcCSD1) were the most abundant transcripts present in both plant tissues and non-transformed calli. Catalase genes were weakly expressed in these samples. The pRiA4-transformed calli showed enhanced expression of several genes encoding ROS-detoxifying enzymes. Confocal microscopy imaging revealed decreased ROS level in pRiA4-transformed calli compared to the control. These results demonstrate that A. rhizogenes, like other plant pathogens, uses a strategy aimed at decreasing ROS levels in host cells through the general upregulation of its antioxidant genes.

Suppression of reactive oxygen species and enhanced stress tolerance in Rubia cordifolia cells expressing the rolC oncogene.

It is known that expression of the Agrobacterium rhizogenes rolC gene in transformed plant cells causes defense-like reactions, such as increased phytoalexin production and expression of pathogenesis-related proteins. In the present study, we examined whether this phenomenon is associated with increased production of reactive oxygen species (ROS). Single-cell assays based on confocal microscopy and fluorogenic dyes (2,7-dichlorofluorescein diacetate and dihydrorhodamine 123) showed reduced steady-state levels of ROS in rolC-expressing Rubia cordifolia cells as compared with normal cells. Paraquat, a ROS inducer, caused significant ROS elevation in normal cells but had little effect on rolC-transformed cells. Likewise, ROS elevation triggered by a light stress was suppressed in transformed cells. Our results indicate that the rolC gene acts as a ROS suppressor in unstressed cells and its expression prevents stress-induced ROS elevations. We detected a two- to threefold increase in tolerance of rolC-transformed cells to salt, heat, and cold treatments. Simultaneously, rolC-transformed cells maintained permanently active defensive status, as found by measuring isochorismate synthase gene expression and anthraquinone production. Thus, the oncogene provoked multiple effects in which ROS production and phytoalexin production were clearly dissociated.

Dynamics and localization of H2O2 production in elicited plant cells.

H(2)O(2) produced in plant cells plays a dual role. In addition to its antimicrobial effect, it also acts as a secondary messenger initiating and modulating responses of plants exposed to unfavorable external signals. A suspension culture of Rubia tinctorum cells challenged with elicitors was used as a model system to investigate H(2)O(2) formation. Cellular H(2)O(2) was measured by a modified titanium(IV) method, while that in the medium was detected with scopoletin fluorescence. Localization of H(2)O(2) production at the ultrastructural level was carried out by the CeCl(3) reaction. A fungal elicitor induced H(2)O(2) production with transient maxima, the first of which appeared 4 min after treatment. Three subsequent maxima appeared in the cells up to 48 h after treatment. Exposure of cells to exogenous jasmonic acid and salicylic acid also changed the H(2)O(2) concentration maxima over 48 h; however, their timing was slightly shifted. Fungal-elicitor, jasmonic acid, and salicylic acid treatments had different effects on the H(2)O(2) concentration in the medium. Ultrastructural investigations revealed that electron-dense precipitates were present at the plasmalemma and in some nearby vesicular cytoplasmic structures 30 min after treatment. Later samples showed cytochemical-precipitate accumulation in the cell walls. These deposits appeared to be local and independent of the direction of the external signal. We could not detect the presence of H(2)O(2) in peroxisomes, mitochondria, plastids, or the central vacuolar space. Electron energy loss spectroscopy investigations distinguished between the cerium-containing precipitates and other electrondense particles, thereby proving that H(2)O(2) generation occurs locally.

Effects of Ca(2+) channel blockers and protein kinase/phosphatase inhibitors on growth and anthraquinone production in Rubia cordifolia callus cultures transformed by the rolB and rolC genes.

The transformation of Rubia cordifolia L. cells by the 35S- rolB and 35S- rolC genes of Agrobacterium rhizogenes caused a growth inhibition of the resulting cultures and an induction of the biosynthesis of anthraquinone-type phytoalexins. Inhibitor studies revealed a striking difference between the rolC- and rolB-gene-transformed cultures in their sensitivity to verapamil, an L-type Ca(2+) channel blocker. The rolC culture possessed a 2-fold lowered resistance to the inhibitor than the normal culture, while the rolB culture was 4-fold more resistant to the treatment. Additionally, growth of the rolC culture was totally inhibited when the culture was grown in Ca(2+)-free medium, whereas growth of the rolB culture was reduced by less than half. We interpreted these results as evidence for a lack of calcium homeostasis in both transgenic cultures. Anthraquinone (AQ) production was not inhibited in the normal or transformed cultures by the Ca(2+) channel blockers verapamil and LaCl(3), or by diphenylene iodonium, an inhibitor of NADPH oxidase, or by the protein kinase inhibitor staurosporine. These results indicate that the induction of AQ production in non-transgenic and transgenic cultures does not proceed through the activation of the common Ca(2+)-dependent NADPH oxidase pathway that mediates signal transduction between an elicitor-receptor complex via transcriptional activation of defense genes. Okadaic acid and cantharidin, inhibitors of protein phosphatases 1 and 2A, caused an increase in AQ production in transgenic cultures. Okadaic acid stimulated AQ accumulation in the non-transformed culture, whereas cantharidin had no effect. These results show that different phosphatases are involved in AQ synthesis in normal and transgenic cultures of R. cordifolia.

Increase in anthraquinone content in Rubia cordifolia cells transformed by rol genes does not involve activation of the NADPH oxidase signaling pathway.

It has been reported that rol plant oncogenes located in Ri-plasmids of Agrobacterium rhizogenes activated synthesis of secondary metabolites in the transformed plant cells. The activator mechanism is still unknown. In this work, we studied whether the NADPH oxidase-signaling pathway, which regulates the synthesis of defense metabolites in plants, is involved in the activator function of the rol genes. It was demonstrated that the transformation of Rubia cordifolia cells by the rolB and rolC genes caused an induction of biosynthesis of anthraquinone-type phytoalexins. Inhibition studies revealed a striking difference between the rolC and rolB transformed cultures in their sensitivity to Ca2+ channel blockers and calcium deficiency. The rolC culture displayed lowered resistance to the inhibitors compared to the non-transformed culture, while the rolB culture was more resistant to the treatment. The assumption was made that the oncogenic potential of rol genes is realized through the alteration of calcium balance in the plant cells. Anthraquinone production was not inhibited in the non-transformed and transformed cultures by Ca2+ channel blockers, as well as by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor staurosporine. These results indicate that the induction of anthraquinone production in transgenic cultures does not involve the activation of Ca2+-dependent NADPH oxidase pathway.