Norzulaani Khalid

Plant regeneration from embryogenic suspension cultures of Musa acuminata cv. Mas (AA)

Embryogenic callus was established using immature male flower of Musa acuminata cv. Mas. After 5–6 months of culture, embryogenic callus was obtained at 21.75±11.9 from 750 immature male flower clusters with translucent somatic embryos proliferated from the whitish friable callus. It was observed that flower clusters ranging from 4 to 11 responded to form embryogenic callus and out of which 3–10 somatic embryos were formed per flower cluster. Embryogenic callus were obtained at a percentage of 10.00±0.3 on M1 medium initially supplemented with 18 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for 3 months and subsequently transferred to the same media with reduced 2,4-D (9 μM) for the next 2–3 months. Embryos developed into translucent spheres and slightly torpedo shaped embryos in suspension cultures. Plantlets were obtained on medium M4 supplemented with 0.8μM BA, at an average regeneration rate of 13.00±0.58.

Docking of Noncompetitive Inhibitors into Dengue Virus Type 2 Protease: Understanding the Interactions with Allosteric Binding Sites

A group of flavanones and their chalcones, isolated from Boesenbergia rotunda L., were previously reported to show varying degrees of noncompetitive inhibitory activities toward Dengue virus type 2 (Den2) protease. Results obtained from automated docking studies are in agreement with experimental data in which the ligands were shown to bind to sites other than the active site of the protease. The calculated K(i) values are very small, indicating that the ligands bind quite well to the allosteric binding site. Greater inhibition by pinostrobin, compared to the other compounds, can be explained by H-bonding interaction with the backbone carbonyl of Lys74, which is bonded to Asp75 (one of the catalytic triad residues). In addition, structure-activity relationship analysis yields structural information that may be useful for designing more effective therapeutic drugs against dengue virus infections.

Transcripts and MicroRNAs Responding to Salt Stress in Musa acuminata Colla (AAA Group) cv. Berangan Roots

Physiological responses to stress are controlled by expression of a large number of genes, many of which are regulated by microRNAs. Since most banana cultivars are salt-sensitive, improved understanding of genetic regulation of salt induced stress responses in banana can support future crop management and improvement in the face of increasing soil salinity related to irrigation and climate change. In this study we focused on determining miRNA and their targets that respond to NaCl exposure and used transcriptome sequencing of RNA and small RNA from control and NaCl-treated banana roots to assemble a cultivar-specific reference transcriptome and identify orthologous and Musa-specific miRNA responding to salinity. We observed that, banana roots responded to salinity stress with changes in expression for a large number of genes (9.5% of 31,390 expressed unigenes) and reduction in levels of many miRNA, including several novel miRNA and banana-specific miRNA-target pairs. Banana roots expressed a unique set of orthologous and Musa-specific miRNAs of which 59 respond to salt stress in a dose-dependent manner. Gene expression patterns of miRNA compared with those of their predicted mRNA targets indicated that a majority of the differentially expressed miRNAs were down-regulated in response to increased salinity, allowing increased expression of targets involved in diverse biological processes including stress signaling, stress defence, transport, cellular homeostasis, metabolism and other stress-related functions. This study may contribute to the understanding of gene regulation and abiotic stress response of roots and the high-throughput sequencing data sets generated may serve as important resources related to salt tolerance traits for functional genomic studies and genetic improvement in banana.

Production of clonal planting materials from Gracilaria changii and Kappaphycus alvarezii through tissue culture and culture of G. changii explants in airlift photobioreactors

Global demand for seaweed resources has increased due to their emergent use as sources of biopharmaceuticals, nutraceuticals and biofuels. These high-valued products make possible the use of micropropagation techniques that may be more costly than conventional mariculture. This study reports the successful tissue culture of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva and Gracilaria changii (B. Xia & Abbott) Abbott, Zhang and Xia. Callus induction of K. alvarezii was successfully developed following an explant sterilisation protocol. Callus formation and regeneration of K. alvarezii was observed in solidified Provasoli’s enriched seawater medium. Different culture conditions such as agar concentration, growth hormones, nutrients, irradiance and enrichment media were investigated to determine the suitable conditions for explant culture of G. changii. Proliferations of adventitious shoots were induced under the most suitable culture conditions. G. changii explants were successfully cultured in airlift photo-bioreactors, with no decrease in the carbohydrate content in the G. changii explants. This micropropagation technique can provide a useful alternative system for seedling production of economically important seaweeds.

Rapid micropropagation of Boesenbergia rotunda (L.) Mansf. Kulturpfl. (a valuable medicinal plant) from shoot bud explants

A successful protocol was developed for mass propagation of Boesenbergia rotunda (L.) Mansf. Kulturpfl., an important medicinal plant. Numerous shoots were induced from young shoot bud of B. rotunda mature rhizome on Murashige and Skoog (1962) medium supplemented with 30.0 g/l sucrose, 2.0 g/l gelrite, different concentrations of 6-benzylaminopurine (BAP) and a-naphtaleneacetic acid (NAA). Plant medium supplemented with different concentrations of BAP alone or with NAA produced varying degree of multiple shoots. A supplementation of 2.0 mg/l BAP and 0.5 mg/l NAA gave the best result. Ninety percent of the explants induced multiple shoots within 10 to 14 days of inoculation with five maximum numbers of shoots per explant. The numbers of multiple shoots was low during initial subculture but increased after third subculture and were slightly decreased after fourth subculture. Rooting was spontaneous in almost all the treatments after 10 to 14 days of culture. Micropropagated plantlets were successfully acclimatized. Key words : Boesenbergia rotunda, micropropagation, medicinal plant, Zingiberaceae.

Transcriptome profiling shows gene regulation patterns in a flavonoid pathway in response to exogenous phenylalanine in Boesenbergia rotunda cell culture.

BackgroundPanduratin A extracted from Boesenbergia rotunda is a flavonoid reported to possess a range of medicinal indications which include anti-dengue, anti-HIV, anti-cancer, antioxidant and anti-inflammatory properties. Boesenbergia rotunda is a plant from the Zingiberaceae family commonly used as a food ingredient and traditional medicine in Southeast Asia and China. Reports on the health benefits of secondary metabolites extracted from Boesenbergia rotunda over the last few years has resulted in rising demands for panduratin A. However large scale extraction has been hindered by the naturally low abundance of the compound and limited knowledge of its biosynthetic pathway.ResultsTranscriptome sequencing and digital gene expression (DGE) analysis of native and phenylalanine treated Boesenbergia rotunda cell suspension cultures were carried out to elucidate the key genes differentially expressed in the panduratin A biosynthetic pathway. Based on experiments that show increase in panduratin A production after 14 days post treatment with exogenous phenylalanine, an aromatic amino acid derived from the shikimic acid pathway, total RNA of untreated and 14 days post-phenylalanine treated cell suspension cultures were extracted and sequenced using next generation sequencing technology employing an Illumina-Solexa platform. The transcriptome data generated 101, 043 unigenes with 50, 932 (50.41%) successfully annotated in the public protein databases; including 49.93% (50, 447) in the non-redundant (NR) database, 34.63% (34, 989) in Swiss-Prot, 24,07% (24, 316) in Kyoto Encyclopedia of Genes and Genomes (KEGG) and 16.26% (16, 426) in Clusters of Orthologous Groups (COG). Through DGE analysis, we found that 14, 644 unigenes were up-regulated and 14, 379 unigenes down-regulated in response to exogenous phenylalanine treatment. In the phenylpropanoid pathway leading to the proposed panduratin A production, 2 up-regulated phenylalanine ammonia-lyase (PAL), 3 up-regulated 4-coumaroyl:coenzyme A ligase (4CL) and 1 up-regulated chalcone synthase (CHS) were found.ConclusionsThis is the first report of Boesenbergia rotunda de novo transcriptome data that could serve as a reference for gene or enzyme functional studies in the Zingiberaceae family. Although enzymes that are directly involved in the panduratin A biosynthetic pathway were not completely elucidated, the data provides an overall picture of gene regulation patterns leading to panduratin A production.

Enhancement of banana plant regeneration by incorporating a liquid-based embryo development medium for embryogenic cell suspension

Summary Recovering plants from embryogenic cell suspensions is a complex process starting with somatic embryo development which is synchronised on medium lacking plant growth regulators, followed by differentiation and regeneration of plants. An improved method for high frequency recovery of banana plants was investigated by incorporating a liquid-based, embryo-development medium. The highest regeneration rate (approx. 32,000 plants ml–1 settled cell volume) was obtained using this liquid protocol. This is one of the highest scores recorded among published data on plant recovery in Musa spp. A detailed, comparative histological study has been carried out on somatic embryos developed in semi-solid or liquid-based media. Most mature embryos developed from the liquid protocol enabled differentiation and regeneration within 4 months. Over 3,000 plants are currently under field observation to establish the rate of somaclonal variation.

Effects of Selected Physicochemical Parameters on Zerumbone Production of Zingiber zerumbet Smith Cell Suspension Culture

Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement.

Boesenbergia rotunda: From Ethnomedicine to Drug Discovery.

Boesenbergia rotunda is a herb from the Boesenbergia genera under the Zingiberaceae family. B. rotunda is widely found in Asian countries where it is commonly used as a food ingredient and in ethnomedicinal preparations. The popularity of its ethnomedicinal usage has drawn the attention of scientists worldwide to further investigate its medicinal properties. Advancement in drug design and discovery research has led to the development of synthetic drugs from B. rotunda metabolites via bioinformatics and medicinal chemistry studies. Furthermore, with the advent of genomics, transcriptomics, proteomics, and metabolomics, new insights on the biosynthetic pathways of B. rotunda metabolites can be elucidated, enabling researchers to predict the potential bioactive compounds responsible for the medicinal properties of the plant. The vast biological activities exhibited by the compounds obtained from B. rotunda warrant further investigation through studies such as drug discovery, polypharmacology, and drug delivery using nanotechnology.

SIMPLE ONE-MEDIUM FORMULATION REGENERATION OF FINGERROOT (BOESENBERGIA ROTUNDA (L.) MANSF. KULTURPFL.) VIA SOMATIC EMBRYOGENESIS

SummaryMost published protocols necessitate different media formulations for multistep somatic embryogenesis. This study aims to establish a simple but effective formulation for the regeneration of plantlets of the pharmaceutically active Boesenbergia rotunda (L.) Mansf. Kulturpfl, formerly Boesenbergia/Kaempferia pandurata (Schult), to ensure a superior and consistent supply of materials for commercialization purposes. In this study, a single-medium formulation of Murashige and Skoog (MS) supplemented with 13.54μM 2,4-dichlorophenoxyacetic acid (2,4-D) was found to be the only medium out of eight formulations to promote the complete somatic embryogenesis process for the culture of B. rotunda (L.). Callus cultures were initiated from a total of 280 explants of rhizome meristem. The percentage of cultures forming embryogenic callus was 23.3 ±4.3% on this MS medium augmented by 13.54μM 2,4-D. The best plantlet regeneration rate was attained from the first subcultured callus with a mean of 6.6±0.1 plantlets per 1 cm diameter aggregate of callus. Somatic embryogenesis characteristic of monocots was evident from histological studies. The regenerated plantlets have been successfully established in soil.