RESEARCH ARTICLE Amino Acid and Secondary Metabolite Production in Embryogenic and Non- Embryogenic Callus of Fingerroot Ginger (Boesenbergia rotunda) Theresa Lee Mei Ng1,2, Rezaul Karim1,3, Yew Seong Tan1, Huey Fang Teh2, Asma Dazni Danial2, Li Sim Ho2, Norzulaani Khalid1, David Ross Appleton2, Jennifer Ann Harikrishna1* 1 Centre for Research in Biotechnology for Agriculture & Institute of Biological Sciences, Faculty Science, a11111 University of Malaya, 50603 Kuala Lumpur, Malaysia, 2 Sime Darby Technology Centre, 1st Floor Block B, UPM-MTDC Technology Centre III, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia, 3 Department of Botany, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi 6205, Bangladesh * [email protected] OPEN ACCESS Abstract Citation: Ng TLM, Karim R, Tan YS, Teh HF, Danial AD, Ho LS, et al. (2016) Amino Acid and Secondary Interest in the medicinal properties of secondary metabolites of Boesenbergia rotunda (fin- Metabolite Production in Embryogenic and Non- gerroot ginger) has led to investigations into tissue culture of this plant. In this study, we pro- Embryogenic Callus of Fingerroot Ginger filed its primary and secondary metabolites, as well as hormones of embryogenic and non- (Boesenbergia rotunda). PLoS ONE 11(6): e0156714. doi:10.1371/journal.pone.0156714 embryogenic (dry and watery) callus and shoot base, Ultra Performance Liquid Chromatog- raphy-Mass Spectrometry together with histological characterization. Metabolite profiling Editor: Mohana Krishna Reddy Mudiam, Indian Institute of Chemical Technology, INDIA showed relatively higher levels of glutamine, arginine and lysine in embryogenic callus than in dry and watery calli, while shoot base tissue showed an intermediate level of primary Received: February 5, 2016 metabolites. For the five secondary metabolites analyzed (ie. panduratin, pinocembrin, Accepted: May 18, 2016 pinostrobin, cardamonin and alpinetin), shoot base had the highest concentrations, followed Published: June 3, 2016 by watery, dry and embryogenic calli. Furthermore, intracellular auxin levels were found to Copyright: © 2016 Ng et al. This is an open access decrease from dry to watery calli, followed by shoot base and finally embryogenic calli. Our article distributed under the terms of the Creative morphological observations showed the presence of fibrils on the cell surface of embryo- Commons Attribution License, which permits genic callus while diphenylboric acid 2-aminoethylester staining indicated the presence of unrestricted use, distribution, and reproduction in any medium, provided the original author and source are flavonoids in both dry and embryogenic calli. Periodic acid-Schiff staining showed that credited. shoot base and dry and embryogenic calli contained starch reserves while none were found Data Availability Statement: All relevant data are in watery callus. This study identified several primary metabolites that could be used as within the paper. markers of embryogenic cells in B. rotunda, while secondary metabolite analysis indicated Funding: This work was supported by High Impact that biosynthesis pathways of these important metabolites may not be active in callus and Research Chancellory Grant UM.C/625/1/HIR/ embryogenic tissue. MOHE/SCI/19 from the University of Malaya. Also, the authors would like to acknowledge Bright Spark Programme of the University of Malaya for supporting Rezaul Karim. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS ONE | DOI:10.1371/journal.pone.0156714 June 3, 2016 1/19 Metabolite Profiling of Embryogenic and Non-Embryogenic Callus of Boesenbergia rotunda Competing Interests: The authors have declared 1. Introduction that no competing interests exist. Boesenbergia rotunda is a member of the Zingiberaceae family. This monocotyledon plant is commonly called fingerroot, Chinese keys or Chinese ginger and is used in food, flavourings and traditional medicines [1]. Several flavonoids and chalcone derivatives have been isolated from extracts of B. rotunda, including pinocembrin, pinostrobin, alpinetin, panduratin, carda- monin, quercetin and kaempferol [2]. These compounds are reported to have various biologi- cal effects. Kirana et al. reported that a concentration of 9.0 μg.mL-1 panduratin A completely inhibited the growth of MCF-7 human breast cancer cells and HT-29 human colon adenocarci- noma cells [3]. Pinostrobin extracted from the rhizomes of B. rotunda has been reported to show anti-microbial [4], anti-ulcer [5], anti-viral [6] and anti-tumor [7] activity. Rhizomes and other parts of the plant have also been used to investigate the various biological activities. Jing et al. compared its anti-proliferative effect against five cancer cell lines using extracts from rhi- zomes, leaves and stems from different Boesenbergia species [8]. They found that extracts from the rhizome of B. rotunda gave the most promising results in cytotoxic activity for all five can- cer cell lines. B. rotunda’s various beneficial effects have spurred extensive study of tissue culture of this plant. Mass propagation via tissue culture not only saves time but also yields clonal plant mate- rials that can be manipulated through culture conditions or genetic engineering to produce desirable metabolites. It has long been known that biomass yields and metabolite production are influenced by physiology and plant development. Specifically in B. rotunda, biomass yield has been enhanced via manipulation of physical cell culture conditions [9] and usage of various concentrations of plant growth regulators such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyl amino purine (6-BAP), which have been applied to increase the rates of cell prolifera- tion and somatic embryogenesis [10–12]. Manipulation of cell culture conditions to increase embryogenesis rates can be expected to affect metabolism and thus might also have an impact on the production of specific metabolites of interest. One way to investigate cell metabolism in relation to embryogenesis is through omics technologies such as genomics, transcriptomics, proteomics and metabolomics in order to observe the underlying molecular changes during tissue culture [13–16]. We characterized the shoot base, embryogenic and non-embryogenic calli (dry and watery) of B. rotunda using metabolite profiling to probe the underlying biochemical processes associated with embryo- genesis. Targeted metabolites in various tissue types, including primary and secondary metabo- lites as well as hormones were analyzed using Ultra Performance Liquid Chromatography– Mass Spectrometry (UPLC-MS). Furthermore, to relate biochemistry to morphology, micros- copy analyses were performed on the callus and shoot base. 2. Materials and Methods 2.1 Ethics statement The conduct of this research was approved by the grant management committee of the Univer- sity of Malaya, headed by the Director of Institute of Research Management and Monitoring, Professor Noorsaadah Abdul Rahman ([email protected]). This study did not involve the use of any human, animal and endangered or protected plant species as materials and the study did not include any field study or site study. 2.2 Plant source B. rotunda rhizomes were purchased from a commercial herb farm in Pahang, Malaysia and propagated in the laboratory to generate all sample materials. Initially, the plants were washed PLOS ONE | DOI:10.1371/journal.pone.0156714 June 3, 2016 2/19 Metabolite Profiling of Embryogenic and Non-Embryogenic Callus of Boesenbergia rotunda thoroughly under running tap water for 10 min, then air dried for 30 min before insertion into black polybags to promote sprouting. Samples were sprayed with water every day to induce growth of shoots. Newly formed shoots of less than 5 cm length were harvested for subsequent culture and analysis. Concurrently, additional shoots were allowed to grow to a length of 10 cm and were harvested as 5 cm long shoot samples which we labeled as T1: 1–5 cm portion of the shoot and T2: 6–10 cm portion of the shoot. 2.3 Establishment of tissue culture callus Callus materials were established in three steps: sterilization, explant preparation and callus induction. First, shoots were collected and cleaned thoroughly with tap water. Next, the leaves of the outer layer were removed and the exposed tissues were sterilized with 20% Clorox and Tween-20 for 10 min. Next, the tissues were washed with 95% ethanol followed by thrice rins- ing with deionized water. The sterilized tissue was dried on a clean filter paper. Then, a 1 mm cross-section from the shoot base (SB) tissue, including the shoot meristem, was cut and placed into callus induction media comprising a Murashige and Skoog base supplemented with 1 mg.L-1 α-napthaleneacetic acid (NAA), 1 mg.L-1 indole-3-acetic acid (IAA), 30 g.L-1 sucrose 1 and 2 g.L-1 Gelrite (Sigma Aldrich, Missouri, United States). The callus that formed was 1 transferred to a propagation medium containing 30 g.L-1 sucrose, 2 g.L-1 Gelrite and various concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) as follows; for dry callus (DC) (4 mg.L-1), for embryogenic callus (EC) (3 mg.L-1) and for watery callus (WC) (1 mg.L-1)[10, 12]. Enrichment of embryogenic cells from embryogenic callus was performed by sieving embryogenic calli through a 425 μm stainless steel sieve prior to extraction of metabolites. 2.4 Metabolite extraction protocols Primary and secondary metabolites. Rhizome, shoot tissue and