The medicinal plants of the subgenus Protanthium (genus Kaempferia, family Zingiberaceae) in Thailand and Laos. The assessment committee has unanimously approved this thesis, submitted by Miss Penjun Meechonkit, in partial fulfillment of the requirements for the Doctor of Philosophy Health Sciences at Mahasarakham University. Mahasarakham University has given approval to accept this thesis in partial fulfillment of the requirements for the Doctor of Philosophy Health Sciences.
Their location included tropical areas of Africa, India, Southeast Asia with the reported center of distribution in Thailand. This study aimed to study the taxonomy, palynology and molecular information of the subgenus Protanthium (genus Kaempferia, family Zingiberaceae) in Thailand and Laos. The phylogenetic tree of 9 taxa found in Thailand and Laos was also constructed, and the results confirmed the morphological determination.
INTRODUCTION
- Background
- Research Objectives
- Scope of Research
- Research sites
- The benefits expected
To study the taxonomy of the subgenus Protanthium (genus Kaempferia, family Zingiberaceae) in Thailand and Laos. To study the palynology using scanning electron microscopy of the subgenus Protanthium (genus Kaempferia, family Zingiberaceae) in Thailand and Laos. To study the molecular information of the subgenus Protanthium (genus Kaempferia, family Zingiberaceae) in Thailand and Laos.
To get the data of taxonomy of the subgenus Protanthium (genus Kaempferia, family Zingiberaceae) in Thailand and Laos. To obtain the data of palynology of the subgenus Protanthium (genus Kaempferia, family Zingiberaceae) in Thailand and Laos. To obtain the data of molecular information of the subgenus Protanthium (genus Kaempferia, family Zingiberaceae) in Thailand and Laos.
LITERATURE REVIEW
Plant Taxonomy
In the early period of discovery, the genus Kaempferia was reported for 15 species (Larsen, 1996; Sirirugsa, 1992). According to the report of the discovery of plants in the genus in Laos presented in the book titled Checklist of the vascular plants of Lao PDR, it revealed that there are 8 species (Newman et al., 2007). Subsequently, a report revealed that 5 new species were found in the genus Kaempferia of the world Kampferia champasakensis (Picheansoonthon & Koonterm, 2008) K.
Palynology
The result showed that their pollen divided into two shapes is a spherical shape and an ellipsoidal shape.
Molecular study
Molecular study includes the study of genetic markers using DNA fingerprints such as RAPD and AFLP, these methods will give more accurate taxonomic classification results than the DNA base sequence method. As for plants in the Zingiberaceae family, many articles in research reports in the past and present have reported the results of their taxonomic classification, and most articles tend to only present the DNA base sequencing method for taxonomic classification. This research aimed to study the DNA base sequence of a part called the Internal Transcribed Spacer (ITS) on the chromosome and a part of the gene called Maturase K (matK) on the chloroplast, using a number of plant samples.
Furthermore, many research projects aimed to classify samples of plants into many classes, such as research by Ngamriabsakul et al (2004), who used a base sequence of DNA from part of the ITS and trnL-F of a chloroplast for genus classification within a tribe Zingibereae, tribe Zingibereae classified into 2 genera are Curcuma and Hedychium. The base sequence of DNA was also used for other parts to study some genera of plants; for example, a genus Alpinia (Kunthonluxamee, 2008), a genus Hedychium (Wongsuwan, 2010) and a genus Kaempferia (Techaprasan et al., 2010). Regarding DNA fingerprinting research, a RAPD technique was used for a species classification of 5 plant species i.
TAXONOMY
Taxonomic Methodology
Taxonomic treatment of the genus Kaempferia L
Labellum usually deeply bilobed, mostly obovate, usually white or purple, sometimes marked with a different color.
Key to the examples of the subgenus Protanthium (genus Kaempferia, family
Desciptions of Kaempferia in Thailand and Laos
Inflorescences produced towards the end of the dry season from the leafless rhizomes, peduncle 0.5-3 cm long. Inflorescences produced towards the end of the dry season from the leafless rhizomes, peduncle 1.1-3.3 cm long, hairy. Inflorescences produced towards the end of the dry season from the leafless rhizomes, stem 0.6-3.7 cm long, glabrous.
Inflorescences produced at the end of the dry season from leafless rhizomes, peduncles 0.9-3.4 cm long, hairy. Lateral staminodes; M: labellum; N and O: Anthers and anther ridges (N: front view, O: side view); P: Ovary, stylodial gland and lower part of style; Question: tubular fruit of persistent calyx. Inflorescences are produced at the end of the dry season from rhizomes without leaves, peduncles 4-5 cm long, hairy.
Inflorescences are formed towards the end of the dry season from leafless roots, peduncle 0.5-1.4 cm long. Inflorescences, formed towards the end of the dry season from non-leafy roots, petiole subsilent to 1 cm long, glabrous. Inflorescences formed towards the end of the dry season from non-leafy roots, peduncle 0.7-3.1 cm long, hairy.
Inflorescences produced towards the end of the dry season from the leafless rhizomes, peduncle 0.3-8.4 cm long, hairy. H and I: Lateral staminodes; J: Label; K: Anthers and anthers; L: Ovary, stylodial glands and lower part of style;. Inflorescences produced towards the end of the dry season from the leafless rhizomes, peduncle 0.5-1 cm long, hairy.
Inflorescences formed towards the end of the dry season from bare roots, peduncle 1.1-3 cm long, hairy. Dorsal coronal lobe; H and I: lateral coronal lobes; J and K: lateral staminodes; L: Labellum; M: Anthers and anther ridges; N: ovary, stylodial glands and lower part of stylode; A: Fruit with obstinate calyx tube;. Inflorescences, formed towards the end of the dry period from bare roots, bloom at night, peduncle 1.2-3.2 cm long, glabrous.
PALYNOLOGY
Method and material
Scanning electron microscope (SEM)
As mentioned above, the results showed that all pollen grains of the medicinal plants in the subgenus Protanthium, genus Kaempferia have subspheroidal shapes and smooth exine.
MOLECULAR STUDIES
Methodology and Materials
For the salting out effect, the tube was mixed by vortex and then incubated on ice for 15 min (or up to 60 min for highest efficiency). For phase separation, one volume of chloroform:isoamyl alcohol (24:1, v/v) was added to the tube, mixed by vigorous shaking until the emulsion formed, and then centrifuged at 16,000 × g for 15 min at room temperature. The upper aqueous phase was transferred to the new 1.5 ml microcentrifuge tube without interrupting the organic phase, and then added to 2 volumes of absolute ethanol at room temperature to precipitate the DNA.
The mixture was mixed by gentle inversion, and then the DNA pellet was collected by centrifugation at 16,000 × g for 15 min at room temperature. After centrifugation, the supernatant was carefully discarded and the DNA pellet was washed with 500 µl of 80% ethanol. To obtain the DNA sequence of the ITS region for each sample, previously designed forward and reverse primers that are specific for this nuclear region (White et al., 1990) were used in this study.
For the DNA sequencing process, the universal primer sequences, pBluescript KS and pBluescript SK, were additionally 5'-end flanked to the original forward and reverse primer sequences, respectively. Before amplification, each gDNA sample was diluted with TE buffer to a concentration of 100 ng/µl and then used as a template. The reaction mixture was then vortexed, briefly centrifuged, and then placed in a programmable DNA thermal cycler (Amplitronyx ATC401, Nyx Technik).
To assess the quality of isolated gDNA samples and amplified PCR products, the DNA samples were analyzed with gel electrophoresis approach. To analyze the DNA, the gDNA or PCR product sample was mixed with 1 μl of 6X Gel Loading Dye, Blue (NEB, catalog no. B7021S) and then loaded into 1% (w/v) agarose gel, along with 0.25 ng of total mass of 2-Log DNA Ladder (NEB, catalog no. N3200S) as molecular weight standard. After receiving the DNA sequencing results from each sample, the DNA sequence obtained from 3′-end sequencing was reverse-complemented, and then pairwise aligned with 5′-end sequencing result of the same sample using the Clustal.
For phylogenetic clustering, the DNA sequences of the ITS region of all selected taxa were multiply aligned by the Multiple Alignment using Fast Fourier Transform program (The European Bioinformatics Institute, n.d. a) with “localpair” FFTS setting.
Results from molecular study
Subsequently, the multiple sequence alignment was manually edited and revised using the program BioEdit version 7.2.5 (Hall, 1999). The alignment was then tested to find out for a best nucleotide substitution model of Maximum Likelihood method using the MEGA program version 7.0.21 (Kumar et al., 2016). The best model will be indicated, in which the model with the lowest Bayesian Information Criterion (BIC) score is the model that exhibits the maximum posterior probability and is considered to best describe the substitution pattern (Posada and Buckley, 2004).
Once the best model was indicated, phylogeny construction was performed based on the maximum likelihood statistical method with the following parameters; Tamura 3-parameter substitution model (Kumar et al., 2016), between-site levels of a gamma distribution with 5 levels of discrete gamma categories, treatment of gaps/missing data using all sites, and bootstrap reliability testing with 1000 replications. A pair of universal primers, KS_ITS1 and SK_ITS4, was chosen to amplify the conserved ITS region on the chromosome. After generating the PCR product for each plant accession, all PCR reactions were checked for amplified fragments by gel electrophoresis.
The result from the PCR amplification generated from the ITS region of several stocks is shown in Figure 44. After the results from the DNA sequence were obtained, the results from both pBluescript KS and pBluescript SK reactions were ligated to finally obtain the complete DNA sequence -'s. of the ITS region for each membership. Multiple alignment was then tested to find the best substitution model to maximize accuracy in hierarchical clustering of the data.
The total 24 nucleotide substitution models were tested using the MEGA program and the result is shown in Table 2. From the result, the model with the lowest BIC value was Tamura 3-parameter in terms of gamma distribution, which is used to explain evolutionary rates among websites (T92+G). Therefore, this nucleotide substitution model was used to construct the phylogenetic tree with the Maximum Likelihood method.
Within the clade of the family Zingiberaceae, 5 species are grouped separately apart from all Kaempferia rotunda groups as cladistics, in which, K.
CONCLUSION AND DISCUSSION
Taxonomy
In detail, the plane of the staminode and labellum of the flower was perpendicular in Kaempferia rotunda while that of K. The characteristics of the pseudostem in which it is well developed with the straight leaves of K.
Palynology
Molecular studies
The use of the PCR-RFLP technique with DNA in the ITS Region for the identification of Plants of the genus Alpinia (Zingeberaceae). Pollen morphology of the family Zingiberaceae in China- Pollen types and their significance in taxonomy. A study on the morphology, chromosome and pollen of the family Zingiberaceae in Phu Phan National Park.
Genetic variation of Kaempferia (Zingiberaceae) in Thailand based on chloroplast DNA sequences (psbA-trnH and pet A-psb J). Essential oil chemical constituents and RAPD fingerprints of Curcuma and Kaempferia plants in Thailand.
