Distribution of 9-methoxycanthin-6-one from the intact plant parts and callus cultures of Eurycoma longifolia (Tongkat Ali)

Study was carried out to determine 9-methoxycanthin-6-one distribution in intact plants and callus cultures of Eurycoma longifolia. Qualitative analysis using TLC revealed that the compound 9-methoxycanthin-6-one was present in leaves, petioles, stem, rachis, tap roots, fibrous roots, cotyledons and embryo of the in vivo plants. The quantitative analysis using HPLC showed that the highest concentration of 9-methoxycanthin-6-one content was found in tap roots (4.10 mg.g -1 dry weight (DW) followed by fibrous roots (3.91 mg.g -1 DW), rachis (2.10 mg.g -1 DW), cotyledons (1.44 mg.g -1 DW) and embryo (0.84 mg.g -1 DW). Petioles stem and leaves had relatively low concentrations compared to other intact plant parts, with 0.15 mg.g -1 DW, 0.12 mg.g -1 DW and 0.08 mg.g -1 DW, respectively. Compound 9-methoxycanthin-6-one was also present in callus tissues derived from different explants. The highest concentration was detected in fibrous root-derived callus (7.12 mg.g -1 DW tissues), followed by stem-derived callus (4.18 mg.g -1 DW tissues), leaf-derived callus (2.17 mg.g -1 DW tissues), embryo-derived callus (2.03 mg.g -1 DW tissues), rachis-derived callus (1.25 mg.g -1 DW tissues), tap root-derived callus (0.96 mg.g -1 DW tissues), petiole-derived callus (0.61 mg.g -1 DW tissues) and cotyledon-derived callus (0.18 mg.g -1 DW tissues). From the comparison between the data of using callus tissues and intact plant parts, it has shown that the higher concentration of 9-methoxycanthin-6-one of more than 73.7 % was detected in callus tissues

Optimization of suitable auxin application in a recalcitrant woody forest plant of Eurycoma longifolia (Tongkat Ali) for callus induction

A study was carried out to determine and optimize suitable auxin for callus induction in Eurycoma longifolia. The induction of callus cultures using leaf, petiole, rachis, stem, tap root, fibrous root, cotyledon and embryo segments were successfully achieved by using various auxins such as 2,4-D, IAA, NAA, picloram and dicamba. The cultures were observed daily for one month. The overall results from the experiments showed that the presence of 2,4-D was the most suitable auxin for the highest callus induction and growth performance rate by using various explants types of E. longifolia. The callus initiation in leaf explant was the highest using 1.0 mgL-1 of 2,4-D  (81.67%), followed by picloram (78.33% at 3.0 mgL-1), dicamba (73.33% at 2.0 mgL-1) and NAA (61.67% at 4.0 mgL-1). Auxin, 2,4-D and picloram at 4.0 mgL-1 were found to be the most effective for callus induction (78.33%) using petiole explant. It was followed by using dicamba (73.33% at 2.0 mgL-1), NAA (73.33% at 3.0 mgL-1) and IAA (61.67 % at 3.0 mgL-1), respectively. In addition, the percentage of callus induction from rachis was obtained at highest rate by using picloram (86.67 % at 4.0 mgL-1), followed by 2,4-D (81.67% at 4.0 mgL-1), dicamba (78.33% at 2.0 mgL-1), NAA (58.33% at 2.0 mgL-1) and IAA (45% at 3.0 mgL-1). Meanwhile, for stem explant, it has been noticed that highest callus induction at 88.33% could be obtained from medium containing 2,4-D at 2.0 mgL-1, followed by picloram (80% at 5.0 mgL-1), dicamba (85% at 5.0 mgL-1), NAA (76.67% at 1.0 mgL-1) and IAA (63.33% at 4.0 mgL-1). For tap root explant, the highest amount of callus was formed at 3.0 mgL-1 2,4-D and 1.0 mgL-1 picloram (81.67%), followed by dicamba (78.33% at 1.0 mgL-1). For fibrous root explant, the highest callus was produced in 2,4-D at 4.0 mgL-1 (86.67%), picloram (78.33% at 1.0 mgL-1) and dicamba (78.33% at 2.0 mgL-1). The highest scoring of callus induction from cotyledon was determined by using 2,4-D at 4.0 mgL-1 (85%), followed by picloram (83.33% at 1.0 mgL-1), dicamba (78.33% at 2.0 mgL-1), NAA (56.67% at 1.0 mgL-1) and IAA (36.67% at 3.0 mgL-1). Finally, the highest scoring for callus induction from embryo was produced in 2, 4-D and picloram at 2.0 and 5.0 mgL-1 (both 85%) followed by NAA and IAA 83.33% at 1.0 and 4.0 mgL-1, respectively, and dicamba, 81.67% at 5.0 mgL-1. The percentages of callus induction  using various types of explants were found to be increased significantly by using selected types of auxins in E. longifolia plants.Key words: Eurycoma longifolia, Callus induction, auxin

Comparative Analysis of Lycorine in Wild Plant and Callus Culture Samples of Hymenocallis littoralis by HPLC-UV Method

The Hymenocallis littoralis, an ornamental and medicinal plant, had been traditionally used for wound healing. In the present study, an analytical method using HPLC with ultraviolet detection was developed for the quantification of lycorine in the extracts of different parts of wild plant and tissue culture samples of H. littoralis.The separationwas achieved using a reversed-phase column. The method was found to be accurate, repeatable, and sensitive for the quantification of minute amount of lycorine present in the samples. The highest lycorine content was found in the bulb extract (2.54 ± 0.0

Histology and scanning electron microscopy observations of cryopreserved protocorm-like bodies of Dendrobium sonia-28

The genus Dendrobium possesses horticultural importance. Dendrobium sonia-28 is an important ornamental orchid in the flower industry. Cryopreservation is a favoured long-term storage method for orchids with propagation problems. Protocorm-like bodies (PLBs) of Dendrobium sonia-28 were cryopreserved using the vitrification technique. Histology and scanning electron microscopy (SEM) observations were conducted on stock, non-cryopreserved (control), and cryopreserved PLBs of Dendrobium sonia-28 to detect cryoinjuries resulting from the vitrification protocol. Histological observations of control PLBs indicated that the preculture, osmoprotection, and dehydration steps were not physically damaging to the PLBs. Histological and SEM analyses of cryopreserved PLBs indicated that the freezing and thawing cycles inflicted damages on the parenchymatic regions of the PLBs. Only embryogenic cells survived the treatment. Scanning electron microscopy studies of the control and cryopreserved PLBs indicated that both osmotic and freezing injuries occurred only in the interior regions of the PLBs

A Study on the Use of Organic Additives on the Protocorm-like Bodies (PLBS) Growth of Phalaenopsis violacea Orchid

The potential of different concentrations of various banana cultivars extracts, papaya extract, tomato extract and coconut water (0, 10, 20, 30%) to the control (without carbon source and plant growth regulator free medium) for reliable proliferation of Phalenopsis violacea protocorm-like bodies (PLB) under in vitro condition. The results indicated that organic extracts were taken up from the media as shown by the increased in percentage of PLB proliferation rate. Maximum growth of PLB was obtained in half-strength Murashige and Skoog (MS) semi-solid medium supplemented with 10% of Mas (AA) banana pulp extract. However, it has been noticed that at a higher concentrations of organic extract tend to be inhibitory to the PLB proliferation which could be due to the osmotic stress. PLBs grown in half-strength MS supplemented with papaya extract had the lowest PLB proliferation rate among the different organic extracts. It can be concluded that, adding Mas (AA) banana pulp extract at concentration of 10% can be a potential replacement of sucrose in the media. Therefore, the short time length of in vitro culture and its high efficiency makes addition of suitable organic extracts well suited for mass propagation of Phalenopsis violacea orchid

A study on the use of organic additives on the protocorm-like bodies (PLBS) growth of Phalaenopsis violacea orchid

The potential of different concentrations of various banana cultivars extracts, papaya extract, tomato extract and coconut water (0, 10, 20, 30%) to the control (without carbon source and plant growth regulator free medium) for reliable proliferation of Phalenopsis violacea protocorm-like bodies (PLB) under in vitro condition. The results indicated that organic extracts were taken up from the media as shown by the increased in percentage of PLB proliferation rate. Maximum growth of PLB was obtained in half-strength Murashige and Skoog (MS) semi-solid medium supplemented with 10% of Mas (AA) banana pulp extract. However, it has been noticed that at a higher concentrations of organic extract tend to be inhibitory to the PLB proliferation which could be due to the osmotic stress. PLBs grown in half-strength MS supplemented with papaya extract had the lowest PLB proliferation rate among the different organic extracts. It can be concluded that, adding Mas (AA) banana pulp extract at concentration of 10% can be a potential replacement of sucrose in the media. Therefore, the short time length of in vitro culture and its high efficiency makes addition of suitable organic extracts well suited for mass propagation of Phalenopsis violacea orchid

Cryopreservation of PLBs of Brassidium Fly Away Using Encapsulation-Dehydration Techniqu

In vitro grown protocorm-like bodies (PLBs) of Brassidium Fly Away orchid hybrid were cryopreserved using encapsulation- dehydration technique. The viability of the cryopreserved cells was determined by 2,3,5-triphenyltetrazolium chloride (TTC) assay. For the preculture treatment, the PLBs were excised into two standard sizes of 1-2 and 4-5 mm and were precultured on half-strength Murashige and Skoog (MS) semi solid medium supplemented with diff erent concentrations of sucrose (0, 0.2, 0.4, 0.6, 0.8 and 1.0M). The PLBs size 4-5 mm and 0.6 M sucrose concentration was selected based on highest viability obtained in TTC assay. The PLBs were encapsulated for 30 minutes using 3% (w/v) liquid sodium alginate medium supplemented with 0.4M sucrose and 0.1M calcium chloride and osmoprotected in 0.75M sucrose solution for 24 hours at 25°C. The beads were then dehydrated using 50g heat-sterilised silica gel for four hours, cryopreserved for 24 hours, thawed in a 40±2°C water bath for 90 seconds, and regenerated in semi-solid half-strength. Biochemical analyses were conducted and the cryopreserved PLBs had produced lower content of chlorophyll while the highest specifi c peroxidase activity was observed in cryopreserved PLBs

Optimisation of transient green fluorescent protein (GFP) gene expression in Phalaenopsis Violacea Orchid mediated by Agrobacterium Tumefaciens-mediated transformation system

Numerous transformation factors were successfully optimised to develop a reliable and highly efficient Agrobacterium-mediated transformation into the protocorm-like bodies (PLBs) of Phalaenopsis violacea. The optimisation of factors influencing stable transformation efficiency in new species is very important as it can reduce the costs in labor and materials in the future. Hypervirulent Agrobacterium tumefaciens strains, EHA 101 and 105, harboring the pCAMBIA 1304 plasmid which contains gusA gene and gfp gene as the reporter markers, were used for transformation study. Transient gfp gene expression was used to evaluate the efficiency of T-DNA delivery in transformants due to its simple, non-destructive and cell autonomous procedure. Agrobacterium strain EHA 105 was proved to be better in transforming the targeted PLBs than EHA 101, based on the notably high transient expression of gfp gene in all the parameters tested. Different temperatures during cocultivation period, the concentration of L-cysteine, calcium (CaCl2) and silver nitrate (AgNO3) in cocultivation medium as well as pH and light and dark conditions during co-cultivation period were identified to be major factors in enhancing the percentage of transient gfp gene expression. Increased T-DNA delivery efficiencies were obtained when P. violacea PLBs were co-cultivated with Agrobacterium tumefaciens strain EHA 105 in half-strength MS medium supplemented with 5% of banana Mas extract containing 200 mg.L-1 L-cysteine, 60μM silver nitrate, without calcium, adjusted to pH 5.5 and incubated in the dark at 24°C. The results from transient transformation of PLBs suggested that Agrobacterium-mediated transfer of T-DNA to the naturally recalcitrant P. violacea is feasible and is highly efficient. Consequently, by combining the best treatments, an efficient and reproducible Agrobacterium-mediated transformation protocol could be continued to facilitate the insertion of any desirable traits for the production of transgenic Phalaenopsis violacea orchid

Effects of Droplet-Vitrification Cryopreservation Based on Physiological and Antioxidant Enzyme Activities of Brassidium Shooting Star Orchid

Protocorm-like bodies (PLBs) of Brassidium Shooting Star orchid were successfully cryopreserved using droplet-vitrification method. Vitrification based cryopreservation protocol is comprised of preculture, osmoprotection, cryoprotection, cooling, rewarming, and growth recovery and each and every step contributes to the achievement of successful cryopreservation. In order to reveal the lethal and nonlethal damage produced by cryopreservation, histological observation, scanning electron microscopy (SEM), and biochemical analysis were carried out in both cryopreserved and noncryopreserved PLBs of Brassidium Shooting Star orchid comparing with the control PLBs stock culture. Histological and scanning electron microscopy analyses displayed structural changes in cryopreserved PLBs due to the impact of cryoinjury during exposure to liquid nitrogen. Total soluble protein significantly increased throughout the dehydration process and the highest value was achieved when PLBs were stored in liquid nitrogen. Ascorbate peroxidase (APX) and catalase (CAT) showed the highest enzyme activities in both dehydration and cryostorage treatments indicating that stress level of PLBs was high during these stages

Cytotoxic Potential on Breast Cancer Cells Using Selected Forest Species Found in Malaysia

In vitro studies were carried out to evaluate the cytotoxic potential of three selected forest herbaceous species: Tectaria singaporeana (TS), Blechnum orientale (BO) and Tacca integrifolia (TCI). Methanol/methylene chloride extracts of three plant parts viz. leaves, roots and stems were assessed for their cytotoxic potential against human breast cancer cells (MCF-7wt.). Screening of these extracts was done using the microculture, followed by tetrazolium assay after a period of 72 h. There were significant differences between different parts of plants and dilution levels in terms of cytotoxicity, with roots and concentration of 100 μg mL-1 showing the highest cell mortality of 19.58 and 36.59%, respectively. However, the leaves and the stems of all three plant species did not induce any cytotoxic activity on the cells. Overall, the most promising material (IC50 <100 μg mL-1) were the methanolic extracts from the roots of all three plants. Tectaria singaporeana showed the highest cytotoxic potential with an IC50 value of 28.57 ± 11.74 μg mL-1 followed by Blechnum orientale, 32.07 ± 7.85 μg mL-1 and Tacca integrifolia, 95.03 ± 17.49 μg mL-1. From this study, the extracts of these plants may prove to be useful in cancer treatment and prevention