The development of in vitro study of plant regeneration of Etligera Coccinea from Rhizome and Inflorescence Derived Callus

Tuhau, Etlingera coccinea (Blume) S. Sakai & Nagam, is a unique member of the Zingiberaceae family. Although it has a pungent odor, it is regarded as a delicacy by the Kadasan Dusun community in Sabah. The inner stem and its florescence are eaten and the whole plant is regarded to have importance in traditional medicine. This species grows to about 10m in high, its inflorescence grows separately from its young shoots, and appears directly from ground or either half immersed or buried in the ground. Micropropagation is a rapid propagation technique, but the biggest problem is contamination with fungi and bacteria. A wide range of microorganisms (filamentous fungi and bacteria) have been identified as major contaminants in this research. The contaminants have been introduced with explant, during surface sterilization methods in the laboratory by endophytic bacteria. Meanwhile, Fungus may also arrived with an explant, airborne or enter culture. Rhizomes and stems of Etlingera coccinea were used as explants (5-l0mm width) and were cultured in different types of plant growth regulators with different concentration (NAA, BAP, 2,4-D, TDZ and KIN) under fully light condition. Rhizomes and stems scales rinsed under running tape water for 1 hour were surface sterilized, then soaked in solutions containing ethanol (95%)(v/v) and different concentration of sodium hypochlorite (20%, 10%, 15%) (v/v). During the experiment, fungal contaminants were observed in full treatments. Determined contaminants were identified according to their morphological characteristic. There are also no callus have been induced for all treatments due to fungal contaminants

Production and Biological Activity of Monoterpenes from Flowers and Callus Cultures of Michelia Alba Dc.

Analysis of volatile compounds from the selected flower buds, grown in -vitro and caUus cultures of Michelia alba D.C were performed using the gas chromatography technique with mass spectrometry detector (GC-MS). The results showed that the major volatile compound obtained from the three-selected tlower bud was dihydrocarveol. Petal grown in-vitro on the Murashige and Skoog (MS) basal medimn with different concentrations of plant growth regulators did not produce any dihydrocarveol. But callus grown in the culture medium containing 1.25 mgIL (w/v) ofNAA plus 1.35 mgIL (w/v) ofBAP (T20) and 0.8 mgIL (w/v) ofNAA plus 1.85 mgIL of BAP (T23) produced linalool as a major compounds. On the other hand, callus treated with 1.25 mgIL (w/v) ofNAA plus 0.45 mgIL (w/v) ofBAP (TIO) and T20 (1.25 mgIL (w/v) of NAA plus 1.35 mgIL (w/v) of BAP were able to synthesis cyclohexane (32.96±1.94%) compared to T23 (17.015±1.06%). Callus derived from flower petal of M alba treated with 1.25 mgIL (w/v) ofNAA plus 0.45 mgIL (w/v) of BAP (TlO) gave the higher growth (23.83±1.44 g F.wtI culture; 0.23±0.09 g D.wtlculture) respectively at pH 5.7, 25±2°C and in the complete dark condition after 5 weeks of culture. Treatment of M alba flower petal derived callus with 1.0 mgIL (w/v) NAA plus 1.0 mgIL (w/v) BAP supplied with 150 mgIL (w/v) of casein hydrolysate showed higher growth (0.211±0.02 g D.wtI culture) than all other concentrations of casein hydrolysate. Most of the new volatile compounds in the callus cultures were found after treatment with 150 mgIL (w/v) of casein hydrolysate. Addition of various concentrations of bioelicitors, jasmonic acid (0 to 0.25 mglmL (w/v) yeast extract (0-0.3% (w/v» and pectinase (0-0.25 mglmL (w/v» into the culture medium, decreased the cell growth. However, treatment of flower petal derived callus with (0.25 mglmL (w/v» of pectinase showed the highest growth (0.1l8±0.002 g D.wtlculture) on day 18th compared to other treatment. On the other hand, petal derived callus treated with jasmonic acid and L-phenylalanine decreased the callus growth but significantly increased the production of new volatile compounds i.e. cinnamaldehyde, caryophyllene, nerolidol, cinnamic acid, ocimene, famasene, undecene, germacrene D and also linalool. The essential oils obtained from flower bud showed the insecticidal activities, against Tribolium castaneum adults. The insecticidal property of essential oil obtained from flower and petalderived callus was probably associated with presence of linalool, eugenol and geraniol. Result from seed germination assay also showed that pentane extract of Michelia alba flowers was able to effect the germination of the Brassica nigra L (mustard seeds) tested. The phytotoxicity effect and cytotoxicity action may also be possibly associated with the presence of cineol, limonene and linalool though they are minor components

Total RNA Extraction from the aromatic Phalaenopsis bellina, Endemic in Sabah, Borneo

Phalaenopsis bellina is an attractive orchid due to its unique appearance and distinctive floral fragrance. Many past studies on this plant had focused on the plant at the molecular level; however, it requires sufficient quantities of high-quality P. bellina RNA. RNA is more delicate to manipulate than DNA due to its structural instability and its vulnerability to various secondary metabolites, such as polyphenols and polysaccharides. Therefore, in this study, four RNA isolation methods which are a modified phenol-chloroform method and three commercial kits (Vivantis, Novogene, and Analytik Jena) were used on the leaves and flowers of P. bellina for comparison. The yield and purity of total RNA were determined using spectrophotometry. The results showed that the total RNA isolated using the modified phenol-chloroform method had the highest yield (1223.75 ± 68.51 ng/µL) and purity compared to the three commercial kits with an OD260/280 value of 2.07 and an OD260/230 value of 2.26, respectively. In particular, the isolated RNA did not show any detectable genomic DNA contamination or other impurities. The RNA isolated using the phenol-chloroform method was also evaluated by electrophoresis, reverse transcription, and PCR. The results indicated that the phenol-chloroform method appears to be superior for total RNA extraction. Thus, this developed method is proven to be suitable for the RNA extraction of plants rich in polysaccharides and polyphenols, and amenable for future molecular studies on P. bellina

Molecular phylogeny study of schismatoglottis from different regions in Sabah using internal transcibed spacer region

The Schismatoglottideae tribe, primarily in Borneo, is notably diverse within the Araceae family. It encompasses various rainforest herbs, adapting to terrestrial, lithophytic, and rheophytic habitats. The Schismatoglottis genus, with over 175 species, mostly exclusive to Borneo due to geological preferences, stands out. A study aimed to understand genetic relationships among Schismatoglottis species from Kadamaian-Kinabalu Park, Kionsom Recreational Area, Imbak Canyon Conservation Area, Mensalong Forest Reserve, Danum Valley Conservation Area, Tawau Hills Park, and Mantailang Crocker Range Park. The Internal Transcribed Spacer (ITS) regions of twelve Schismatoglottis species were sequenced. Genomic DNA was extracted from dried-silica-gel leaf tissue using a commercial kit (Promega, USA). ITS region lengths ranged from 528 bp to 708 bp. BLAST analysis accurately identified species within the Schismatoglottis genus (95% to 98% similarity). Phylogenetic analysis revealed a highly supported sister clade, uniting Schismatoglottis porpax species from Mensalong and Kionsom, despite differing elevational habitats. This pioneering study delves into Schismatoglottis phylogenetics, particularly in Sabah, with seven of the twelve species showing potential for new species classification, pending further research

Flower Initiation Pattern, Developmental Stages, and Seed Morphology of Paraphalaenopsis Labukensis P.S. Shim, A. Lamb & C.L. Chan, An Endangered Orchid in Sabah

Background: Paraphalaenopsis labukensis P.S. Shim, A. Lamb & C.L. Chan is a monopodial epiphytic species that can only be found in Sabah. P. labukensis orchids have unique characteristics in that it has a long floral lifespan as compared to other orchid species. The flower developmental pattern of P. labukensis greatly influenced capsule formation and seed maturation. Objective: The present research was conducted to record the initiation of flower initiation, and floral morphology, and to observe the flowering and capsule development, as well as the effect of different capsule ages on asymbiotic seed germination. Methods: A total of three individual plants of P. labukensis were observed. The flowering stages were characterized by quantitative parameters such as length of inflorescence, diameter, and length of buds, the number of flowers produced, and the length of the capsule formed. All the data were recorded through direct observation. Results: Overall, twelve morphological landmark that define each stage of floral development was recorded. Based on the observation, P. labukensis inflorescence was asymmetric and in the shape of a panicle. The number of flowers varied among inflorescences, ranging from 3–5, that blossomed at different times. Furthermore, early capsules appeared 40–90 days after pollination (DAP). Then, 120 DAP of the capsule was selected as the most suitable capsule age for germination as it had reached its maturation period. Conclusion: Identifying the duration of the whole flowering developmental process will aid in the production of capsules to attain a reliable and adequate seed source for in vitro seed germination

Chemical composition of essential oil from Etlingera coccinea (Blume) S. Sakai & Nagam in Kadamaian, Kota Belud, Sabah

Etlingera coccinea (Blume) S. Sakai & Nagam is a member of Zingiberaceae family. It is commonly known as ‘Tuhau’ in Sabah, Malaysia and consumed as a local delicacy and used as a traditional remedy for stomachache, food poisoning, and gastric problems. The plant has been reported to have bioactive properties such as anticancer, antioxidant and antibacterial. Due to the high demand for this bioactive compound in national and international markets, chemical profiling of leaves, stems and rhizomes from E. coccinea was carried out. Eight germplasms were collected from Trail 1 (Kg. Gensurai) and Trail 2 (Kg. Melangkap Noriou) and submitted to the hydrodistillation process to obtain the essential oil before analysing with GC-MS. From the result obtained, a total of 85 compounds were found and 26 of these were terpenoid compounds. There are several classes of compound present in different parts of E. coccinea, such as, monoterpene, sesquiterpene, alcohol, aldehyde, alkane, alkene, ketones, fatty acids derivatives, esters, amines as well as norterpene. Most of the compounds found in E. coccinea are monoterpenes at 18 compounds, followed by alcohols (14 compounds), alkanes and alkenes (12 compounds), sesquiterpenes (8 compounds), aldehydes (7 compounds), ketones, fatty acid derivatives, esters (4 compounds), and lastly, amine and norterpene, one compound each. However, total terpenoids from all plant parts from both sites were less than 50% of total abundance. Only the rhizome part from site 2 showed the highest terpenoid abundance (43.34%). Hence, the identified compounds from the study could be expended for large-scale profiling to obtain higher yields of important constituents

Effect of Drying Techniques on Phytochemical Contents and Biological Activities on Bamboo Leaves as Potential Herbal Tea

Presently, bamboos have gained global attention for their therapeutic potential. However, it is rarely considered for its biological activities due to the limitations of studies, especially in Sabah, Malaysia. Thus, searching for the phytochemical content and biological activities among bamboo leaves has increased demand in certain Asian countries. Six species of bamboos, namely Bambusa vulgaris, B. multiplex, B. tuldoides, Dinochloa sublaevigata, Gigantochloa levis, and Schizostachyum brachycladum, were studied with five drying methods: sun drying, shade drying, microwave drying, oven drying, and freeze-drying. The infused leaves of bamboo extracts were analyzed for their total phenolic (TPC) and flavonoid (TFC) contents. The antioxidant activities were determined using two assays; the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the ferric reducing antioxidant power (FRAP) assays. Toxicity tests of potential bamboo extracts were investigated using the brine shrimp lethality bioassay (BSLB). Bioactive compounds of potential bamboo extracts were determined using liquid chromatography-tandem mass spectrometry (LC-MS). The freeze-drying method exhibited the highest yield for the phytochemical contents and antioxidant activities, excluding B. vulgaris, which is preferable to microwave drying. TPC and TFC results showed a range of 2.69 ± 0.01 – 12.59 ± 0.09 mg GAE/g and 0.77 ± 0.01 – 2.12 ± 0.01 mg QE/g, respectively. The IC50 of DPPH ranged from 2.92 ± 0.01 to 4.73 ± 0.02 μg/mL, showing high radical scavenging activity. FRAP values also significantly differed, ranging from 6.40 ± 0.12 to 36.65 ± 0.09 mg TE/g. Toxicity studies of potential bamboo extracts displayed no toxicity activity against the BSLB based on LC50 analysis. This data may help in utilizing the bamboo leaves as functional food applications to be developed into bamboo tea

Phytochemical content and antioxidant properties of Bornean wild durian from Sabah

Borneo is the centre of diversity of the genus Durio (family: Malvaceae; local name: durian). Durian fruit is known to contain high amounts of the major bioactive compounds (as antioxidants) such as anthocyanins, carotenoids, polyphenols and flavonoids. Two types of wild durian species, namely Durio kinabaluensis Kosterm. & Soegeng (durian tupoloh) and Durio oxleyanus Griff. (durian sukang) were studied. The 80% methanolic extracts of flesh, seed and peel (mesocarp and exocarp) were analysed for antioxidant activities, total phenolic and total flavonoid content. The antioxidant activities were determined using three parameters; 2,2-diphenyl-1-picrylhydrazyl radical assay (DPPH), 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical cation assay, and Ferric reducing antioxidant power assay (FRAP). Durio kinabaluensis mesocarp extract displayed the highest antioxidant properties and total phenolic content. The non-edible parts of both durians (seed and peel) exhibited higher phytochemical contents and antioxidant properties compared to the flesh parts. This data may contribute to the pharmaceutical applications, health benefit information of wild durians and helps in popularising the potential of these fruits in international markets and ultimately protects them from extinction

Untargeted Metabolite Profiling of Wild and In Vitro Propagated Sabah Jewel Orchid Macodes limii J.J. Wood & A.L. Lamb

Macodes limii J.J. Wood & A.L. Lamb is a terrestrial jewel orchid native to Sabah, recognised for its sparkling golden-yellow venations, uniformly distributed on its leaves. Despite its high ornamental value, the exploration of the plant’s medicinal potential remains ambiguous. The current study was conducted to gain a fundamental understanding of the metabolite composition and regulation in M. limii plants from two different growing environments: wild and in vitro cultivation, as well as to analyse their phytochemical contents and antioxidant activity. The metabolite profiling of the M . limii plant extracts through gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has tentatively identified compounds from various classes including sugars, carbohydrates, sugar alcohols, amino acids, organic acids, phenolic derivatives and lipid and lipid-like compounds. Subsequently, the multivariate statistical analysis confirmed the existence of significant metabolite variations across distinct growth environments. Notably, the leaf extract derived from wild-grown plants displayed the highest levels of total phenolic and flavonoid content, contributing significantly to its higher antioxidant activity as measured by the 2,2- diphenyl-1-picrylhydrazyl (DPPH) assay. The discovery has offered a fundamental understanding of the metabolites in M. limii jewel orchids, indicating that in vitro regenerated plants may represent a viable alternative for further investigating their therapeutic potential, thus helping to alleviate the impact on wild populations

Phytochemical screening and antioxidant activity of Tuhau (Etlingera coccinea) rhizome and leaf

The indigenous people of Borneo have historically utilized the wild ginger Etlingera coccinea, locally known as Tuhau, for its medicinal properties. However, most of its medicinal properties are yet to be investigated. Therefore, this study aims to assess the phytochemical profile and antioxidant capacity of Tuhau leaf and rhizome. Phytochemical screening was conducted to assess the presence of flavonoids, glycosides, saponins, proteins and amino acids, tannins, and steroids. Additionally, total phenolic and flavonoid contents were determined. The antioxidant potential of the extracts was evaluated through 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3 ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. Results revealed the presence of flavonoids, glycosides, saponins, proteins and amino acids, tannins, and steroids in the leaf extract. Furthermore, the leaf extract exhibited higher total phenolic (61.57 ± 0.29 mg GAE/g dry extract) and flavonoid contents (30.32 ± 0.42 mg CE/g dry extract) compared to the rhizome extract (33.57 ± 0.29 mg GAE/g dry extract and 9.38 ± 0.02 mg CE/g dry extract, respectively). Moreover, the leaf extract demonstrated significantly superior antioxidant activity (71.48 ± 0.53 %, 28.48 ± 0.14 %, and 2.31 ± 0.03 mM Fe2+/g dry extract) compared to the rhizome extract in DPPH, ABTS, and FRAP assays, respectively. Consequently, Tuhau leaf exhibited promising potential to be utilized in the food industry