Jamorn Somana

Sap Phytochemical Compositions of Some Bananas in Thailand

Banana sap has some special properties relating to various phenomena such as browning of fruits after harvesting, permanent staining of cloth and fibers, and antioxidant and antibleeding properties. Analysis of banana sap using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) indicated the presence of phenolic and aromatic amino compounds of interest due to their special properties. With the online positive electrospray ionization mode (ESI), the possible structures of specific compounds were determined from the fragmentation patterns of each particular ion appearing in the mass spectra. The major compounds revealed from the sap of banana accessions, namely, Musa balbisiana , Musa laterita , Musa ornata , and Musa acuminata , and some cultivars were apigenin glycosides, myricetin glycoside, myricetin-3-O-rutinoside, naringenin glycosides, kaempferol-3-O-rutinoside, quercetin-3-O-rutinoside, dopamine, and N-acetylserotonin. The results indicated that there was a variety of phenolic and aromatic amino contents in many banana species. These compounds were reported to relate with biological activities. Moreover, the identities of these phytochemical compositions may be used as markers for banana diet, the assessment of physiochemical status, or the classification of banana clones.

Anthocyanin Composition of Wild Bananas in Thailand

Anthocyanins were isolated from male bracts of 10 wild species of bananas (Musa spp. and Ensete spp.) distributed in Thailand. Six major anthocyanin pigments were identified by high performance liquid chromatography (HPLC), mass spectrometry (MS), and tandem mass spectrometry (MS/MS). They are delphinidin-3-rutinoside (m/z 611.2), cyanidin-3-rutinoside (m/z 595.8), petunidin-3-rutinoside (m/z 624.9), pelargonidin-3-rutinoside (m/z 579.4), peonidin-3-rutinoside (m/z 608.7), and malvidin-3-rutinoside (m/z 638.8). On the basis of the types of pigment present, the wild bananas can be divided into 5 groups. The first group comprises M. itinerans, Musa sp. one, Musa sp. two, and M. acuminata accessions, which contain almost or all anthocyanin pigments except for pelargonidin-3-rutinoside, including both nonmethylated and methylated anthocyanins. The second group, M. acuminata subsp. truncata, contains only malvidin-3-rutinoside while the third group, M. coccinea, contains cyanidin-3-rutinoside and pelargonidin-3-rutinoside. The forth group, M. acuminata yellow bract and E. glaucum do not appear to contain any anthocyanin pigment. The fifth group consists of M. balbisiana, M. velutina, M. laterita, and E. superbum which contain only nonmethylated anthocyanin, delphinidin-3-rutinoside, and cyanidin-3-rutinoside. Total anthocyanin content in the analyzed bracts ranged from 0-119.70 mg/100 g bract fresh weight. The differences in the type of anthocyanin and variation in the amounts present indicate that wild bananas show biochemical diversity, which may be useful for identifying specific groups of bananas or for clarifying the evolution of flavonoid metabolism in each banana group.

Musa nanensis, a New Banana (Musaceae) Species from Northern Thailand

Abstract A new species of banana (Musaceae), Musa nanensis Swangpol & Traiperm, from Nan, Thailand, is described and illustrated. Based on vegetative features, M. nanensis could be superficially categorized as related to M. laterita; however, it possesses several unique floral characters from the rest of the genus Musa, especially its six tepals and anthers, each fused at the base. A key to banana species of northern Thailand, based on morphology, is provided. The plant was found in a single location and is threatened with extinction due to heavy deforestation in the region.

Development of SSR markers from Musa balbisiana for genetic diversity analysis among Thai bananas

Bananas in Thailand have been surveyed by our team to be at least 140 cultivars in the plantations, 10 wild species and, 4 introduced species. To characterize the genetic relationship of species and cultivars, a set of novel SSR markers was developed. Totaling 53 clones containing SSR motifs were isolated from SSR-enriched library of wild Musa balbisiana Colla ‘Tani’ (BB). Selected positive clones were used to design 28 primer pairs for amplification of 12 wild and 82 cultivar accessions with genome designations AA, AB, AAA, AAB, ABB, and BBB. These SSR markers loci were homology searched to the banana genomes to map their locations. The seven-sets multiplex PCR approach using four fluorescent-labeled universal primers were utilized for cost effectiveness. Capillary fragment analysis yielded the accurate size of amplicons for evaluation of particular patterns for each cultivar. Phylogram and Structure analysis presented the specific genotype of genome groups (A and B genotypes, polyploid hybrid genomes) and cultivar groups. By A:B specific alleles ratio, accurate genome designations of hybrids can be determined. Additionally, a marker, characterized to be partial plastid ycf2 gene, indicated the maternal identification of hybrid cultivars. One SSR marker was also preliminary tested with some wild species and advised to be the candidate fingerprinting marker for species identification. In conclusion, SSR marker sets developed here proved their exploitation in detailed identity and relationship of cultivated bananas, which would be useful for genetic conservation and ongoing breeding programs in Thailand and other areas.

Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana

An enzymatic method for specific determination of stevioside content was established. Recombinant β-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at β-1,2-glycosidic bond to yield rubusoside and glucose. Coupling of this enzyme with glucose oxidase and peroxidase allowed for quantitation of stevioside content in Stevia samples by using a colorimetric-based approach. The series of reactions for stevioside determination can be completed within 1 h at 37 °C. Stevioside determination using the enzymatic assay strongly correlated with results obtained from HPLC quantitation (r2 = 0.9629, n = 16). The percentages of coefficient variation (CV) of within day (n = 12) and between days (n = 12) assays were lower than 5%, and accuracy ranges were 95-105%. This analysis demonstrates that the enzymatic method developed in this study is specific, easy to perform, accurate, and yields reproducible results.

The plasmid vectors, pBS2ndd and pBS3ndd, for versatile cloning with low background in Escherichia coli

For decades, diverse plasmid vectors have been continuously developed for molecular cloning of DNA fragment in the bacterial host cell Escherichia coli. Even with deliberate performances in vector preparation, the cloning approaches still face inevitable background colonies, or false positive clones, that may be arisen from intact or self-ligated plasmid molecules. To assist in such problem, two plasmids, pBS2ndd and pBS3ndd, which resistant to ampicillin and kanamycin respectively, were developed in this study as more advantageous cloning vector. The plasmids carry ndd, a lethal gene from bacteriophage T4 coding for nucleoid disruption protein that binds to the host chromosome and progressively kill the cell. The deadly toxicity of Ndd inhibits host cells that obtain intact or ndd-religated vector from growing, which results in low background and dramatically reduces the effort for selection of recombinants. Moreover, their identical multiple cloning site was designed to support various cloning strategies. Digestion of plasmids with XcmI allows for in vitro T/A ligation, while with EcoRV permits blunt-end ligation, with capability of blue-white colony screening. In vivo homologous recombination cloning is also utilizable by amplification of insert fragments using primers containing homology arms and transformation into capable E. coli strains. To demonstrate their advantages, the plasmids were used to clone PCR product samples for DNA sequencing with low-background and versatile cloning strategies. Such rapid and cost-effective cloning procedures are also proposed here. Finally, the cloning for protein expression with blue-white selection was also possible using egfp as a model regulated by lac and T7 promoters on the plasmid or other build-in promoters with the insert.Graphical Abstract

Three novel mutations in α-galactosidase gene involving in galactomannan degradation in endosperm of curd coconut

The deficiency of α-galactosidase activity in coconut endosperm has been reported to cause a disability to hydrolyze oligogalactomannan in endosperm resulting in curd coconut phenotype. However, neither the α-galactosidase encoding gene in coconut nor the mutation type has been identified and characterized in normal and curd coconuts. In this study, cDNA and genomic DNA encoding α-galactosidase gene alleles from a normal and two curd coconuts were successfully cloned and characterized. The deduced amino acid of wild type α-galactosidase contains 398 amino acid residues with a 17 N-terminal amino acids signal peptide sequence. Three mutant alleles, the first 19-amino acids from 67 to 85 (ADALVSTGLARLGYQYVNL) deletion with S137R and the second R216T, were identified from curd coconut plant no.1 while the third P250R was identified from curd coconut plant no. 10. All mutations of α-galactosidase gene were confirmed by the analysis of parental genomic DNA from normal and curd coconuts. Heterologous expression in Komagataella phaffii (Pichia pastoris) indicated that recombinant P250R, R216T and 19-amino acids deletion-S137R mutant proteins showed no α-galactosidase activity. Only the recombinant wild-type protein was able to detect for α-galactosidase activity. These results are in accordance with the no detection of α-galactosidase activity in developing curd coconut endosperms by tissue staining. While, the accumulation of enzyme activity was present in the solid endosperm of normal coconut. The full-length cDNA and parental genomic DNA sequences encoding α-galactosidase in normal coconut as well as identified curd coconut mutant alleles are reported in Genbank accession no. KJ957156 and KM001681-3. Transcription level of the α-galactosidase gene in mature curd coconut endosperm was at least 20 times higher than normal. In conclusion, absence of α-galactosidase activity caused by gene mutations associates with an accumulation of oligogalactomannan in endosperms, resulting in curd coconut phenotype.