Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolites Regulated in Treated Cancer Cell Lines

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints for cancer prevention. Rice callus suspension cultures(RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluate the cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilutions of the A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced side effects.

Factors affecting the accumulation of 9-methoxycanthin-6-one in callus cultures of Eurycoma longifolia

A study was conducted to improve 9-methoxycanthin-6-one productivity （potential anti-tumour compound） from callus cultures of Emycorna longifolia （Tongkat All）. Several factors affecting 9-methoxycanthin-6-one production in callus cultures such as different medium compositions and physical factors were investigated and analyzed. Results show that a higher production of 9-methoxycanthin-6-one （3.84 mg-g-1 DW （Dry Weight）） is obtained from callus cultured in 1/4 MS basal media. At fructose of 2% （w/v）, the production of 9-methoxycanthin-6-one （4.59 mgg-1DW） is promoted to gain the highest yield, compared to other carbon sources tested. The addition of 2.0-mg·L^-1 dicamba also increases 9-methoxycanthin-6-one production （12.3 mg·g^-1 DW）. Higher production of 9-methoxycanthin-6-one was obtained at pH 5.5 （1.53 mg·g^-1 DW）. Production of 9-methoxycanthin-6-one （2.34 mg-g^-1 DW） in callus cultures is also increased when the medium is added with 1×10 ^-1μM phenylalanine. This study suggests that the successful production of 9-methoxycanthin-6-one in vitro cultures has a potential in large-scale production using bioreactor technology.

Anticancer Activities of Plant Secondary Metabolites:Rice Callus Suspension Culture as a New Paradigm

Plant natural products including alkaloids,polyphenols,terpenoids and flavonoids have been reported to exert anticancer activity by targeting various metabolic pathways.The biological pathways regulated by plant products can serve as novel drug targets.Plant natural compounds or their derivatives used for cancer treatment and some novel plant-based compounds which are used in clinical trials were discussed.Callus suspension culture with secondary metabolites can provide a continuous source of plant pharmaceuticals without time and space limitations.Previous research has shown that rice callus suspension culture can kill>95%cancer cells with no significant effect on the growth of normal cells.The role of candidate genes and metabolites which are likely to be involved in the process and their potential to serve as anticancer and anti-inflammatory agents were discussed.Large scale production of plant callus suspension culture and its constituents can be achieved using elicitors which enhance specific secondary metabolites combined with bioprocess technology.

Establishment of Rhodiola quadrifida Hairy Roots and Callus Culture to Produce Bioactive Compounds

Rhodiola quadrifida is a rare mountain medicinal plant whose root extracts are used in traditional Chinese medicine as a hemostatic,antitussive,and tonic in the treatment of gynecological diseases.The aim of the study was to obtain R.quadrifida cultures at different degrees of differentiation in vitro and compare their growth characteristics and the content of salidroside and rosavin.Hairy roots were obtained by incubating cotyledons and hypocotyls in a suspension of Agrobacterium rhizogenes strain A4.The presence of the rolB and rolC genes was proven by polymerase chain reaction.The obtained roots were cultivated in Murashige-Skoog medium(MS).Calluses were obtained from the hairy roots in MS medium with the addition of hormones:3 mg/L 2,4 D and 0.5 mg/L BAP.The presence of the main secondary metabolites of R.quadrifida,salidroside and rosavin,in calluses and salidroside in hairy roots by HPLC/MS was confirmed.The content of salidroside in callus culture was significantly higher than in hairy roots,0.158 and 0.047%,respectively.The content of rosavin in callus culture was 0.07%.The content of rosavin and salidroside in callus culture was close to the level of these substances in the rhizomes of R.quadrifida plants growing in vivo,making this culture promising for its possible biotechnological use.

Influence of Abiotic Elicitors on Accumulation of Thymol in Callus Cultures of Origanum vulgare L.

Callus cultures of Origanum vulgare L. were established from leaf discus on Murashige and Skoog （MS） medium containing different levels of growth regulators, i.e., 2,4-Dichlorophenoxyacetic acid （2,4-D）, Naphthalene acetic acid （NAA）, Benzyl Adenine （BA） and Kinetin （Kn） and incubated under dark condition. Callus tissues were employed to study the influence of abiotic elicitors on the production of thymol. Constant weights of callus （300 mg） were cultured on accumulation medium treated separately with each one of elicitors used （50 g/L sucrose, 200 mg/L NaC1 and 50 or 100 mg/L proline）. The fresh and dry weights of callus were recorded after six weeks. The result indicated that maximum production of fresh and dry callus weight were 1,014 mg and 46.20 mg respectively achieved at 0.5 mg/L 2,4-D and 3 mg/L BA adding to the medium. Dry callus tissues were extracted with 70% methanol and analyzed by HPLC to determine the concentrations of thymol. The addition of abiotic elicitors to MS medium caused significant reduction in fresh weight of callus compared with control treatment. The concentration of thymol in the callus cultured on control treatment was 146.6 ppm. The data showed that 50 or 100 mg/L proline produced the highest yield of thymol 181.48 ppm and 174.58 ppm respectively, followed by sucrose 162.9 ppm, whereas the treatment with NaCI caused reduction in thymol concentration to percentage of 50.56% compared with the control.

Identification of key genes and active anti-inflammatory ingredients in Panax medicinal plants by climate-regulated callus culture combined with gene-component-efficacy gray correlation analysis

Objective:We aimed to establish a novel strategy for identifying key genes and active anti-inflammatory ingredients in Panax medicinal plants.Methods:First,fresh roots of 2-year-old Panax plants,including P.ginseng C.A.Mey.,P.quinquefolium L.,P.notoginseng(Burk.)F.H.Chen,P.japonicus C.A.Mey.,P.japonicus Mey.var.major(Burk.)C.Y.Wu et K.M.Feng,were selected as explants,and callus formation was induced under three experimental temperatures(17,24,and 30℃).Second,high-performance liquid chromatography-mass spectrometry was used to analyze the saponin content of the callus.Nitric oxide reduction efficacy was used for“component-efficacy”gray correlation analysis to find the active anti-inflammatory ingredients.Quantitative reverse-transcription polymerase chain reaction(qRT-PCR)was used to determine the inflammatory factors and verify the active ingredients’anti-inflammatory effects.Finally,qRT-PCR was used to detect the expression of key genes in the callus,and“gene-component”gray correlation analysis was used to examine the relationships between the regulatory pathway of the genes and the components.Results:Among the three experimental temperatures(17,24,and 30℃),the lowest temperature(17℃)is the most suitable for generating Panax callus.Lower-latitude native Panax notoginseng is more adaptable under high culture temperatures(24℃and 30℃)than other Panax plants.The ginsenoside contents of the callus of P.notoginseng and P.japonicus were the highest under similar climate conditions(17℃).Major anti-inflammatory components were G-Rh1,G-Rb1,G-Rg3,and G-Rh6/FloralGKa.CYP76A47 contributed to the accumulation of anti-inflammatory components.Conclusions:This study provides a strategy for the gene-component-efficacy correlational study of multi-component,multi-functional,and multi-purpose plants of the same genus.

Effects of Cerium on Accumulation of Anthocyanins and Expression of Anthocyanin Biosynthetic Genes in Potato Cell Tissue Cultures

The effects of Ce （Ⅳ） on callus growth, anthocyanin content, and expression of anthocyanin biosynthetic genes in callus suspension cultures of Solanum tuberosum cv. Chieftain were studied by the measurement of fresh weight, spectrophotometric assays, and semiquantitative RT-PCR. The results indicate that 0.1 mmol·L^- 1 Ce （ Ⅳ ） can promote callus growth, increase the accumulation of anthocyanins, and enhance the expression of five anthocyanin biosynthetic genes （ CHS, F3H, F3＇5＇H, DFR, and 3 GT） most efficiently. At high concentrations of 1 mmol·L^- 1, Ce （Ⅳ） partially inhibits callus growth and at 2 mmol· L^-1 eventually lends to cell death. The results show that Ce（ Ⅳ ） can induce the expression of anthocyanin biosynthetic genes to produce and accumulate anthocyanins and increase the yield of anthocyanins.

Ethanol Metabolism in Calluses of Several Selected Plant Species on Two Typical Plant-Growth-Regulator Balanced Media

For investigation on the characteristics of ethanol metabolism in tissues of different plant species, calluses from eight selected plant species were cultured on medium supplemented with ethanol in tightly sealed culture flasks. Changes of the ethanol level were detected by gas chromatography. During the culture period, the calluses of tobacco, potato and petunia were, able to catabolize exogenous ethanol, resulting in the prominent decline of the ethanol level in the medium. The calluses of melon and peanut were also able to catabolize thanol but with lower efficiency. The other three calluses of carrot, soybean and rice did not catabolize ethanol but instead produced small to large amount of ethanol, resulting in the increase of the ethanol level in the media. It was also found that changing the balance between auxin and cytokinin could influence only the ethanol metabolism efficiency but could not change the metabolism patterns on ethanol of the cultured calluses. It can be concluded that, ethanol metabolism pattern of calluses in cultures is an innate physiological characteristic of the respective plant species.

Isolation and Characterization of Transcriptionally Active Ty1-copia Retrotransposons in Fragaria × ananassa

One possible mechanism suggested for somaclonal variation is the activation of transposable elements. The activation of retrotransposons by stresses and external changes is commonly observed in plants. In previous study, we isolated the reverse transcriptase （RT） gene sequences of Ty 1-copia retrotransposons from tissue culture strawberry （Fragaria x ananassa） plant, but not the transcriptionally active sequence. For further understanding the relationship between retrotransposon and somaclonal varation, in this study, we isolated the transcriptionally active RT gene sequences from strawberry plants subjected to different abiotic stresses. These retrotransposons were activated by spraying strawberry leaves with 2 mmol L^-1 salicylic acid （SA）, 50 mmol L^-1 methyl jasmonate （MeJA）, 50 mmol L^-1 abscisic acid （ABA）, 50 mmol L^-1 2,4- dichlorophenoxyacetic acid （2,4-D） or by inducing callus growth in 2 types of MS media： first medium supplemented with 0.5 mg L^-1 6-benzylaminopurine （6-BA）, 0.5 mg L^-1 gibberellic acid （GA3）, 1.0 mg L^-1 thidiazuron （TDZ）, and 0.1 mg L^-1 2,4-D, and the second medium supplemented with 0.5 mg L^-1 6-BA, 0.5 mg L^-1 GA3, 2.0 mg L^-1 TDZ, and 0.02 mg L^-1 indole butyric acid （1BA）. Analysis of gene sequences of 17 RTs revealed that none of them contained stop codons and/or indels disrupting the reading frame. These different stress-origin transcriptionally active RTs were remarkably similar to each other- FATEXP2-8 and FATEYS9-7 showed 100% sequence identity. Analysis of pylogenetic of these transcriptionally active RTs and the RT sequences from genome showed that there were close phylogenetic relationships of most of the transcriptionally active RTs. The results of this study have contributed to the background information necessary for future studies for evaluating the relationship between retrotransposons and somaclonal variation.

Effects of CeCl_3 and LaCl_3 on callus and root induction and the physical response of tobacco tissue culture

La3＋ and Ce3＋ have positive effects on plant growth and production. Although it is well known that rare earth elements promote cell growth. The biological effects of La^（3＋） and Ce^（3＋） on callus, shoot and root induction in tobacco are still unclear. The relationships among callus induction, rooting, enzyme activities and stomatal characteristics in tobacco are unknown. The objectives of this study were to identify the relationships between the induction of calluses, shoots, roots, stomata and enzyme activities. The induction percentages of calluses, buds, roots were recorded at 5,10,15, 20 and 25 days after La^（3＋） and Ce^（3＋） treatments. Peroxidase isoenzyme activity was determined by electrophoresis. The characteristics of the stomata were observed under an optical microscope. Our results show that low concentrations of Ce^（3＋）（〈15 mg/L） result in increases in the induction percentages of calluses,buds and roots, but La^（3＋）（〉5 mg/L） inhibits the induction of calluses, buds and roots. There are more peroxidase isoenzyme bands in Ce^（3＋） treatments than in La^（3＋） treatments. This is consistent with the induction percentages of calluses,buds and roots in Ce^（3＋） and La^（3＋） treatments. High enzyme activities may promote the induction of calluses, buds and roots. The stomata area and stomata number of leaves are significantly different between La^（3＋） treatments and Ce^（3＋） treatments. La^（3＋） improves the stomata area and number. Based on these results, we speculate that La^（3＋） may promote the development of the photosynthetic system. Ce^（3＋）may promote tobacco growth and rooting by improving enzyme activities.