Production and antibacterial properties of flavour and flavonoid compounds from cultured tissues of citrus hystrix D. C. ('Limau Purut')

Analyses of the flavour compounds from the selected parts of flower, seedlings grown in-vitro and callus cultures of Citrus hystrix D.C. were performed using the gas chromatography (GC) technique. The results showed that the major flavour compound obtained from the flower was citronellal. Young seedlings g rown in-vitro on the Murashige and Skoog (MS) basal medium without plant growth regulators did not produce an citronellal. Nevertheless, the quantity of limonene was remarkably higher (101.00 ± 5.24 119/g fwt. tissue) than in the petal and ovary and pollen plus anther. Callus derived from stem treated with 2.0 mg/l (w/v) NAA plus 1 .0 mg/l (w/v) kinetin reached a maximum growth (0.94 ± 0.08 g fwt.lculture) after six weeks of culture. No callus was fou nd from the leaf and peel. Maximum production of flavour compou nds Le. cyclohexanol, limonene and p-pinene were obtained after three and four weeks of culture. Treatment of the C. hystrix stem-derived callus with 1.0 mg/l (w/v) of kinetin showed higher growth (0.42 ± 0.04 g fwt/culture) than all treatment with NAA. Most of the flavour compounds in the callus were found highest after being treated with 5.0 mgll (w/v) of NAA. Addition of various concentrations of salicylic acid (0 to 20.0 mM), yeast extract (0 to 0.5%) and alginate (0 to 0.5%) into the medium, decreased the callus g rowth. However, treatment of stem-derived callus with 0.3% of (w/v) of alginate resulted in higher growth (0.88 ± 0.05 g fwt/culture) than the other treatments. On the other hand, callus treated with yeast extract and salicylic acid were able to syntheses two and six additional compounds respectively compared to the control. Treatment of C. hystrix stem-derived callus with proline and phenylalanine decreased the callus growth but Significantly increased the production of flavour compounds i.e. p-cymene, terpineol, citronellal , citronellol , y-terpene and β-pinene. Analyses of flavonoid compounds from young seedlings, callus and different parts of C. hystrix intact plant such as leaf, flower, stem and fruit was performed by high performance liquid chromatography technique (HPLC). The highest production of flavonoids i.e. naringin (11.66 ± 0.76 mg/g dwt. tissue), rutin (34.63 ± 1.69 mg/g dwt. tissue) and kaempferol (3.01 ± 0.02 mg/g dwt. tissue) was found in the peel; hesperidin (3.21 ± 0.23 mg/g dwt. tissue) in the leaf and quercetin (0.68 ± 0.04 mg/g dwt. tissue) in the whole flower. In the young seedlings, naringin (5.26 ± 0.25 mg/g dwt. tissue), and rutin (0.91 ± 0.03 mg/g dwt. tissue) were found in high concentration in the stem compared to the leaf and root. The naringin and rutin content of stem-derived callus showed the maximum values at 2.29 ±. 0.09 and 0.90 ±. 0.03 mg/g dwt. tissue respectively after six weeks of culture. Treatment of stem-derived callus with 0.3% and 0.5% (w/v) of agarose gave the highest production of naringin and rutin at 12.13 ± 0.07 and 3.09 ± 0.05 mg/g dwt. tissue, respectively. Addition of 2.0 mM phenylalanine into the culture medium also increased the production of naringin (24.05 ±. 1.02 mg/g dwt. tissue) and rutin (3.52 ± 0.12 mg/g dwt. tissue). The essential oils (contains flavour compounds) and the methanolic extracts (contains flavonoid compounds) were obtained from peel, leaf, juice and stem-derived callus. Results showed that most extracts were able to inhibit gram positive and gram negative bacterial growth

Characterization of Differentially Expressed Transcripts of Oil Palm (Elaeis Guineensis Jacq.) Suspension Cells

Oil palm tissue culture through somatic embryogenesis remains a problem because the production cost is still high and the process is very labour-intensive. The average callogenesis rate per ortet is only 20% with embryogenesis rate averages 6%. In this study, oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxin(s). The aim of this study was to understand and identify the underlying factors that are involved in the induction of somatic embryo through suspension cells cultured in different auxin(s) concentrations. Transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin(s) were examined by using suppression subtractive hybridization (SSH). The four different hormone combinations examined were: T1 (0.1 mg/l 2,4-D and 1 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1 mg/l NAA), T3 ( 1 mg/l NAA) and T4(0.4 mg/l 2,4-D). The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. A total of 2019 cDNAs were cloned and isolated from these SSH libraries. Reverse northern analyses showed that 82 clones were isolated from library 1, 64 clones from library 2, 72 clones from library 3 and 76 clones from library 4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consisted of sequences from treatment T1, 8 contigs from treatment T2, 10 contigs from treatment T3 and 13 contigs contain sequences from treatment T4, respectively. Northern analyses of 5 transcripts that were shown to be differentially expressed by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-α,β dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin. The full length cDNA sequence of EgLSD is 1801 bp with 58 bp of 5’ untranslated region and 119 bp of 3’ non coding region. The full length cDNA sequence of EgER6 is 810 bp with 120 bp of 5’ untranslated region and 88 bp of 3’ non coding region. The gene expression of these two candidates was further monitored by real-time quantitative RT-PCR. The transcripts of EgLSD were present throughout cell maturation from 0 day to 25 days with higher increase in ABA treated samples compared to the control without ABA treatment. On the other hand, the transcript level of EgER6 decreased drastically from 1 to 0.0001 for ABA treated samples. The results showed that these two genes respond to ABA during maturation stage in oil palm suspension cultures. The current finding showed that there is a crosstalk between ABA and Ethylene, whereby sugar acts as a switch to control the expression of EgLSD and EgER6. These genes can be used as a marker for somatic embryogenesis of oil palm through cell suspension culture

Agrobacterium-Mediated Transformation of Oil Palm (Elaeis Guineensis Jacq.) Suspension Culture

The life cycle of flowering plants in general can be divided into two growth phases: vegetative and reproductive. The reproductive phase can be subdivided into the development of the inflorescence meristem and floral meristems. Control of flowering and the regulation of plant architecture has been thoroughly investigated in a number of wellstudied dicot plants such as Arabidopsis, Antirrhinum, tomato and tobacco. However, in monocot plants, molecular information related to plant reproduction is still limited. In A rabidopsis , the Terminal Flowering I(TFLI) gene, LEAFY, and the target genes of CONSTANS (CO) including the FLOWERING LOCUS T (FI) and SUPPRESSOR OF OVEREXPRESSION OF COI (SOCI) genes, have a major role in promoting flowering and thus controlling flowering time. To investigate the regulation of meristem identity as well as the control of floral transition in oil palm, we transferred genes pCAMBIA/TFL1, pCAMBIA/JIT60, pCAMBIA/LFY, pGA/LFY and pCAMBIA/SOCl into oil palm embryogenic callus. This present study focuses on the optimization of Agrobacterium-mediated transformation and the analysis of transgenic plants. The objective of this study was also to clone the putative oil palm (OPSOCI and OPLFy) genes into a plasmid binary vector system, so as to transform these genes into oil palm embryogenic callus and to determine their effect on expression driven by a cauliflower mosaic virus (CaMV) 35S promoter in oil palm. The success of gene delivery using Agrobacterium into the plant genome is often based on several factors including temperature used during co-cultivation, the binary vector and promoters used, and the plant genotype itself. The most important factors contributing to the success of T -DNA transfer is the type of plant material used. We used embryogenic suspension cells as starting material for the transformation of oil palm because of the large numbers of totipotent cells found in these cultures

Regeneration of Malaysian Indica Rice (Oryza sativa) Variety Mr232 via Optimised Somatic Embryogenesis System

In vitro studies of indica rice variety MR232 via somatic embryogenesis system was established by using mature seeds. By manipulating various plant growth regulators, the optimum medium was obtained by using MS medium containing 5 mg/L NAA (a-naphthaleneacetic acid) and 1 mg/L 2, 4-D (2,4-dichlorophenoxyacetic acid) without browning effect. This treatment showed higher percentage of callus induction and frequency of embryogenesis at the range of 91-97%, which was further, confirmed with the histology studies. The highest whitish somatic embryos frequency (87%) was initiated by incubating embryogenic calli on media containing 10 mg/L ABA and 9 mg/L gelrite agar for 4 weeks. However, the numbers of regenerated plant on medium containing NAA and that was previously pre-treated with 10 mg/L ABA, 9 mg/L gelrite agar and incubation at 8 weeks was the best treatment for shoots induction with 10 plantlets per 3 gm of somatic embryos

Effect of elicitors on the production of naringin and rutin in leech lime (Citrus hystrix) callus

Treatment of Citrus hystrix callus with various types of elicitors decreased the callus growth as the concentration of elicitors increased. However, callus growth remained relatively constant at 0.05% (w/v) alginate and slightly increased to 1.30 ± 0.04 g dwt./culture at 0.1% (w/v) alginate. Analysis of the flavonoids using high performance liquid chromatography (HPLC) showed that only naringin and rutin were produced. Maximum production of naringin (12.13 ± 0.07 mg/g dwt.) and rutin (3.09 ± 0.05 mg/g dwt.) (p ≤ 0.05) was found in callus treated with 0.5% (w/v) agarose

Analysis of flavour compounds in leech lime (Citrus hystrix) flower and yield improvement in callus

The major flavour compound obtained from Citrus hystrix flower was citronellal. Plantlet grown on a basal Murashige and Skoog (MS) medium without phytohormone do not produce any citronellal. However, the quantity of limonene was remarkably higher (101.62 ± 5.24 μg/g fwt.) in stem than petal (27.30 ± 1.42 μg/g fwt.), ovary (10.76 ± 0.01 μg/g fwt.) and pollen and anther (6.64 ± 0.24 μg/g fwt.). Callus was successfully induced from stem, embryo and petiole on the MS medium supplemented with sucrose (30 g/litre), naphthalene acetic acid (NAA) (2.0 mg/litre) and kinetin (1.0 mg/litre) but only limonene and cyclohexanol have been produced. Treatment of callus derived from stem under different types of light did not increase the number of flavour compounds. Treatment of callus under bright white cool fluorescent light showed the highest production of cyclohexanol (14.1 ± 1.11 μg/g fwt.) and limonene (1.48 ± 0.09 μg/g fwt.) compared to that of other treatments

Effect of NAA, kinetin and three elicitors on the growth and production of flavour compounds from leech lime (Citrus hystrix) calli

Treatment of the leech lime (Citrus hystrix) callus with 1.0 mg/L (w/v) kinetin exhibited the maximum growth (0.415 ± 0.09 g fwt.) compared to all treatments with naphthaleneacetic acid (NAA). Analysis using gas chromatography (GC) showed that treatment with kinetin inhibited the synthesis of flavour compounds. On the other hand, treatment with 5.0 mg/L (w/v) NAA gave the highest production of cyclohexanol (4.16 ± 0.03 μg/g fwt.), p-cymene (5.13 ± 0.98 μg/g fwt.) and limonene (1.83 ± 0.19 μg/g fwt.). Addition of various concentrations of individual elicitors such as yeast extract and agarose into the medium decreased the callus growth. However, treatment of callus with 0.3% (w/v) alginate resulted in higher callus growth (0.88 ± 0.1 g fwt.) compared to control. Among the three different elicitors tested, only treatment with yeast extract was able to increase the number of flavour compounds and two new compounds were synthesised compared to the control. The quantities of flavour compounds produced also varied depending on the concentration of elicitor used

Isolation and characterization of differentially expressed transcripts from the suspension cells of oil palm (Elaeis guineensis Jacq.) in response to different concentration of auxins

Oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxins. In this study, transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin concentrations were examined using suppression subtractive hybridization. Total RNA was first isolated from oil palm suspension cells proliferated in liquid medium with different hormone concentrations for 6 months. Four different hormone combinations: T1 (0.1 mg/l 2,4-D and 1.0 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1.0 mg/l NAA), T3 (1.0 mg/l NAA), and T4 (0.4 mg/l 2,4-D) were used for the treatments. The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. Reverse northern analyses showed that 14.13% of these clones were preferentially expressed in T1, 13.70% in T2, 14.75% in T3, and 15.70% in T4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consist of sequences from treatment T1, 8 contigs were from treatment T2, 10 contigs were contains sequences of treatment T3 and 13 contigs contains sequences of treatment T4. Northern analyses of five transcripts that were shown to be differentially expressed in the oil palm suspension cells by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-α,β-dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin

Persistence of anticancer activity in berry extracts after simulated gastrointestinal digestion and colonic fermentation

Fruit and vegetable consumption is associated at the population level with a protective effect against colorectal cancer. Phenolic compounds, especially abundant in berries, are of interest due to their putative anticancer activity. After consumption, however, phenolic compounds are subject to digestive conditions within the gastrointestinal tract that alter their structures and potentially their function. However, the majority of phenolic compounds are not efficiently absorbed in the small intestine and a substantial portion pass into the colon. We characterized berry extracts (raspberries, strawberries, blackcurrants) produced by in vitro-simulated upper intestinal tract digestion and subsequent fecal fermentation. These extracts and selected individual colonic metabolites were then evaluated for their putative anticancer activities using in vitro models of colorectal cancer, representing the key stages of initiation, promotion and invasion. Over a physiologically-relevant dose range (0–50 µg/ml gallic acid equivalents), the digested and fermented extracts demonstrated significant anti-genotoxic, anti-mutagenic and anti-invasive activity on colonocytes. This work indicates that phenolic compounds from berries undergo considerable structural modifications during their passage through the gastrointestinal tract but their breakdown products and metabolites retain biological activity and can modulate cellular processes associated with colon cancer

Epigallocatechin-3-gallate (EGCG) for Clinical Trials: More Pitfalls than Promises?

Epigallocatechin-3-gallate (EGCG), the main and most significant polyphenol in green tea, has shown numerous health promoting effects acting through different pathways, as antioxidant, anti-inflammatory and anti-atherogenic agent, showing gene expression activity, functioning through growth factor-mediated pathways, the mitogen-activated protein kinase-dependent pathway, the ubiquitin/proteasome degradation pathway, as well as eliciting an amyloid protein remodeling activity. However, epidemiological inferences are sometimes conflicting and in vitro and in vivo studies may seem discrepant. Current knowledge on how to enhance bioavailability could be the answer to some of these issues. Furthermore, dose levels, administration frequency and potential side effects remain to be examined