Assessment of Reproductive Morphology, Cytology, and Metabolic Profiling of Indigenous and Under-exploited Species, Zingiber barbatum (Wall.) from Myanmar

概要

Myanmar possesses great biodiversity due to its broad geographical location, climatic condition and ethnic diversity. The varied climatic conditions and topography of Myanmar enable the growth of plenty of temperate, tropical and subtropical species. Diversity of the ethnic tribes with their own cultures, traditions and preferences on consumption of plant species also influence the ways of plant cultivation and production systems. Myanmar plant genetic resources are still under-explored due to the low investment for exploration and conservation initiatives. This study was carried out to provide information on the assessment of Zingiber barbatum species from Myanmar, which is one of the less-studied species to date.

　The Zingiberaceae is a monocotyledonous family of perennial, mostly rhizomatous herbs. The family comprises species valued as aromatic and spice crops and as having medicinal value. The members of Zingiberaceae are used in the ethnic medicine of Myanmar due to their healing properties, both dry and fresh. The genus Zingiber comprises 144 species of which 37 species have been reported from Myanmar. Taxonomically, Zingiber is classified into four sections based on their inflorescence habits. Most Zingiber species have medicinal value due to their biological activity. Despite wide use as a food, a spice, a dietary supplement, and a traditional remedy only several species are well studied.

　The current study focused on the assessment and characterization of the underexploited medicinal species Zingiber barbatum Wall. endemic to Myanmar and broadly used as a traditional herbal remedy in Myanmar. Z. barbatum belongs to the section Cryptanthium Horan. of the genus Zingiber. It is an indigenous medicinal species and endemic only to Myanmar. Myanmar is considered the center of its diversification. The rhizome is a valuable part of the plant and has applications as a healing anti-inflammatory and analgesic in the ethnomedicine of Myanmar. A few studies on morphological and genetic diversity characterization have revealed a high degree of variability among Z. barbatum genotypes originating from Myanmar. It is a species reported as one of the most troublesome taxa due to very variable morphological features.

　There is still ambiguity that occurs in the characterization of Z. barbatum species due to the lack of comprehensive investigations. Studies on the cytology and phytochemistry of Z. barbatum are absent. Characterization of flower biology is limited to a few old reports and is not sufficiently comprehensive. Zingiber herbaria specimens are not enough for the study of flower biology due to the complexity of characterizing their flowers as they are delicate and ephemeral, and liable to rot while exsiccating in the bracts. Given this fact, a substantial part of this study encompassed the assessment and characterization of reproductive morphology, cytology, and characterization of the volatile organic compounds in the Z. barbatum species. The study was carried out using the Z. barbatum plant collection available in the Gene Research Center of the University of Tsukuba (GRC UT) (Tsukuba, Japan). The plant materials were obtained during a field exploration of plant genetic resources in Myanmar. The Z. barbatum genotypes introduced to the collection were recorded as an accession with an appropriate identification code number and maintained as a living collection in the greenhouse of the GRC UT.

　Two Z. barbatum genotypes, designated as accessions ZO113 and ZO223, were assessed to characterize the reproductive morphology. Accession ZO113 was obtained from the Nay Pyi Taw region and accession ZO223 was obtained from the Mandalay region of Myanmar. The study was conducted based on the conventional method of observation, evaluation, and morphological description of the inflorescence habits and flowers. The minimum quantitative and qualitative parameters were applied for assessment and characterization. The dissection of flowers carried out to characterize and to describe the morphology of the flowers. The study does not include statistical analysis due to the absence of replications for each flowering plant. Two qualitative parameters were different between the two Z. barbatum genotypes. These differences included the phenotypic variations with regards to the shape of inflorescence during growth and development. Accession ZO113 formed a conical shape inflorescence with the acuminate apex at the stage of emergence, which was gradually changed to an ovate-oblong with an obtuse-acute apex at the blossom stage, and to a wide-fusiform with an acute apex at the final stage of flowering. Accession ZO223 formed an elliptic shape inflorescence on a short peduncle, which also changed gradually into an ovate- oblong with an obtuse-acute apex at the blossom stage, and to a fusiform with an acuminate apex at the final stage of flowering. The variation of the central labellum lobe which is characterized as its bifurcation on the middle and the presence of pinkish dots at the base was observed in accession ZO113. It can be assumed that the observed phenotypic variation between the two Z. barbatum genotypes might be due to genetic divergence driven by reproductive isolation, or as a result of collected mutations due to continuous re-creation of the asexual form.

　The non-targeted method was applied to evaluate and characterize the volatile organic compounds in the rhizome of six Z. barbatum accessions from Myanmar. The volatile organic compounds (VOCs) were identified by the application of gas chromatography combined with time- of-flight-mass spectrometry (GC-TOF-MS). The adjusted mass spectra obtained by the hierarchical multi-curve resolution (H-MCR) method were matched against the reference mass spectra in different libraries for the peak annotation and identification. In total, 81 VOCs were identified in the profile of Z. barbatum consisting mainly of monoterpene (21%) and sesquiterpene (30%) hydrocarbons. Twenty-four identified VOCs were significantly different between six Z. barbatum accessions based on Tukey's HSD test. The hierarchical cluster analysis resulted in clear between- groups separation based on 81 identified VOCs forming two clusters. Cluster I comprised accessions ZO63 and ZO160 from the Bago region, while accessions ZO105 and ZO223 from the Mandalay region and ZO191 and ZO217 from Shan state were clustered together in Cluster II. Four accessions collected from Shan state (ZO191 and ZO217) and the Mandalay region (ZO105 and ZO223) of Myanmar had similar VOC profiles in comparison. VOC composition of two accessions (ZO63 and ZO160) from the Bago region were different in 14 compounds compared to the other four accessions. Although all evaluated accessions were grown in uniform conditions (in the field of the GRC UT) at the same altitude and under the same ecological conditions, they possessed variation in their VOC composition. This chemical diversity can possibly be attributed to "plant memory", which is possibly facilitated by biotic and abiotic stresses that a plant faced in its natural environment in the past.

　The DNA content, genome size, and ploidy level in Z. barbatum species have not been reported to date. Cytological assessment was carried out using twenty accessions of Z. barbatum and one accession of Z. officinale as a reference standard. Estimation of the nuclear DNA content (2C- value) was analyzed by the application of the flow cytometry method. Two accessions (ZO208 and ZO189) showed significant differences in nuclear DNA content based on Tukey's HSD test. The 2C-values among the assessed Z barbatum accessions ranged from 2.90 ± 0.26 pg (ZO208) up to 5.98 ± 0.05 pg (ZO189), with an average value of 5.04 ± 0.22 pg. The estimation of the genome size showed that Z. barbatum accessions have a relatively small genome size (1C<3.5 pg) which is a generic characteristic of the family Zingiberaceae. Assessment of the inferred ploidy levels revealed that all evaluated accessions were triploid except two accessions, which were characterized as a diploid (ZO208) and tetraploid (ZO189). In perspective, the sequenced genome analysis can be suitable to confirm and confront the cytological findings for a better understanding of the mechanisms involved in genome size increment and evolution in Z. barbatum.

　Z. barbatum genotypes from Myanmar displayed variation despite the limited number of accessions that were compared for the specific objectives. The current study provides comprehensive information regarding flower biology, cytological status, and volatile metabolite composition of the Z. barbatum species. The considerable observed variation regarding each objective for the studied Z. barbatum species might be the result of human impact, natural selection, a long re-creation of the asexual reproduction and traditional cultivation approaches as has been reported for other economically important crops from Myanmar. In perspective, future studies on Z. barbatum genetic diversity in Myanmar need to increase with the purpose of exploration and exploitation. Moreover, conservation measures and programs need to be developed both at the regional and the country levels for better sustainable utilization of Z. barbatum genetic resources in Myanmar. Information about this study could be considered in conservation and sustainable utilization programs for indigenous Z. barbatum species due to its ethnobotanical and medicinal value in Myanmar. The study could be useful in taxonomic, systematic, phytochemical, and genetic diversity studies of this underexploited species.