Vietnam Journal of Biotechnology

" Panax vietnamensis Ha et Gmshv., a rare Panax genus of Vietnam, is a well known Vietnamese ginseng (Ngoc Linh Ginseng) for its rich pharmaceutical compositions, most importantly saponin. In order to obtain a stable and saponin-rich biomass of P. vietnamensis, a tissue culture procedure was established. A TLC analysis of saponin composition was also conducted to investigate the presence of saponin in callus, shoot and root biomass. Successful callus induction from leaf and petiole explants was obtained from MS medium n supplemented with 1.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid), 0.2 mg/l TDZ (Thidiazuron) under a photoperiod of 16 h. In the following steps, the optimal auxin and its concenfration, appropriate photoperiod condition as well as callus size that were the best for callus proliferation were investigated. Among the auxins, including 2,4-D, IBA (Indole-3-butyric acid) and NAA (a-Naphthaleneacetic acid), 2,4-D at 1.0 mg/l was found to be the most effective for callus growth. Callus at the size of 0.5 x 0.5 cm grew the best as compared to bigger ones, such as 0.7 x 0.7 cm and 1.0 x 1.0 cm. The effects of phytohormones, sucrose and activated charcoal (AC) on shoot regeneration from callus and shoot proliferation have also been studied. Calli cultured on MS medium supplemented with 1.0 mg/l BA and 1.0 mg/l NAA regenerated more shoots. The suitable « medium for shoot proliferation was MS'/z medium, supplemented with 1.0 mg/l BA, 0.5 mg/l NAA, 50 g/l sucrose and 2.0 mg/l AC. Callus was grown on MS'/2 medium supplemented with 3.0 mg/l NAA to regenerate roots. Root proliferation was obtained on MS'A medium containing 5.0 mg/l NAA. In saponin analysis experiment, thin layer chromatograms show that obtained calli, shoots and roots from the above experiments had ginsenoside-Rgl and majonoside-R2, two main ginsenosides of Vietnamese Ginseng but only roots have ginsenoside-Rbl. These results indicate that Vietnamese Ginseng biomass can be used as a new source for saponin isolation for pharmaceutical and cosmetic industry.

Dendrobium heterocarpum Lindl. is an endangered species which is currently used as ornamental pot plant for its beautiful flowers. An increase in collection for trade or any other purposes may lead to a dramatic decrease in the population of this species, thus becoming rare or endangered species in the near future. In this study, effects of plant growth regulators (BA, NAA, IAA, IBA, TDZ) and natural supplements (carrot, potato, and banana extracts) on protocorm like bodies (PLBs) formation; growth and development of shoot; and root regeneration of D. heterocarpum Lindl. as well as type of substrates on acclimatization and growth of seedlings were investigated. The results showed that PLBs formation was optimal on MS medium supplemented with 2.0 mg/L BA and 1.0 mg/L NAA (7.11 PLBs/explant; PLBs formation percentage of 68.9%) or MS medium supplemented with 1 mg/L TDZ and 0.5 mg/L NAA (7.29 PLBs/explant; PLBs formation percentage of 75.53%). For subculture, MS medium supplemented 1.5 mg/L BA and 60 g/L banana extract (22.40 shoots/explant; shoot length of 2 cm) was the most suitable for shoot regeneration and growth. Additionally, root formation was the most suitable on ½ MS medium supplemented with 1 mg/L NAA (4.4 roots/shoot; root length of 3.12 cm; root formation of 95.56%). Finally, the sufficiently rooted plantlets were transferred to greenhouse for hardening. After 60 days, coconut fiber substrate was the most suitable for seedling growth and development (with survival rate of 97.78%, root number of 5 and shoot length of 3.4 cm). The results of propagation in vitro Dendrobium heterocarpum Lindl. contribute to conservation and sustainable development as well as towards the rapid multiplication of seedlings for commercial commercialization of this wild orchid species.

In vitro culture establishment is one of the most important stages in micropropagation. The disinfectant effectiveness depends on the type of surface disinfectant, concentration and the time treatment. In this initial study, silver nanoparticles (AgNPs) were used as a disinfectant for petioles, flower stalks and stems of Begonia tuberous. In addition, thin cell layer culture (TCL) technique has been applied for the purpose of somatic embryogenesis. The results showed that AgNPs were effective in eliminating infectious microorganisms on B. tuberous explants; which were identified included 4 species of fungi (Fusarium sp., Aspergillus aculeatus, Trichoderma sp. and Penicillium sp.) and 1 species of bacteria (Pseudomonas sp.). At concentrations of 200 ppm and 300 ppm, AgNPs were not only effective in disinfection but also increased the induction rate of somatic embryogenesis in flower stalk TCL explants (approximately 40.00%); a similar effect was observed in stem TCL explants at the same concentration. Meanwhile, for petiole TCL explants, the induction rate of somatic embryogenesis was optimal when using AgNPs at a concentration of 100 - 300 ppm to disinfected the explant. In contrast, at high (400 ppm) or low (50 ppm) concentrations of AgNPs did not play a disinfecting role and stimulated somatic embryogenesis. In addition, explants derived from AgNPs sterilization did not show any abnormalities in somatic embryogenesis with shapes such as globular, heart, torpedo, and cotyledon. AgNPs showed double efficacy in sterilization of explants and improved efficiency of somatic embryogenesis from TCL petioles, flower stalks and stems explants; thus increasing the efficiency micropropagation of B. tuberous.

Surface sterilization is one of the most important steps in preparation of explants for micropropagation, because microbial contaminations present a major challenge to the initiation and maintenance of viable in vitro cultures. Most of popular surface disinfectants are considered as highly toxic influence either directly or indirectly to health and environment. Previous studies have demonstrated that toxicity of nano silver (Ag nanoparticles) can destroy effectively microorganisms but it is safe for human health. So, the silver nanoparticles have been widely used in different fields of life, such as medicine, pharmaceuticals, cosmetics, biology and agriculture. However, reports on the effect of silver nanoparticles for surface sterilization of plant explants are still limited. Nano silver and typical disinfectants were tested for sterilization of African violet (Saintpaulia ionantha H. WENDL.), by varying their concentration and time of exposure. The aim of this study was to examine sterilization capacity and explant morphogenesis when using nano silver in micropropagation. After decontamination step, we evaluated the growth and development of explants in different stages of the micropropagation process of African violet. The results indicated that the treatment using nano silver agent at concentration of 0.05% for 15 minutes was the best for controlling the infection. Nano silver could be used to replace the commonly used decontamination substances without causing adverse effects on plant growth and development. This is the first report on in vitro establishment using nano silver to reduce bacterial infections and the growth and development of African violet (Saintpaulia ionantha H. WENDL.).

Identification of species based on DNA sequences (DNA barcodes) is an aid to a taxonomic classification using morphological characteristics. DNA barcoding uses standard short genomic regions that are universally present in target lineages and has sufficient sequence variation to identify species in the genus. A variety of loci has been suggested as DNA barcodes for plants, including genes and non-coding regions in the nuclear and plastid genomes such as psbA-trnH, matK, rbcL, and ITS. In this study, we evaluated five potential DNA barcodes including 18S, ITS, matK, psbA-trnH, and rbcL for their ability to distinguish between species across our samples in the genus Panax L. Multiple alignments with 41 GenBank sequences selected from 9 species showed that the 18S region had the highest level of average similarity (99.87 %), followed by rbcL, matK, psbA-trnH, and ITS regions with lower levels of average similarities (99.27 %, 98.66 %, 96.82 % and 96.50 %, respectively). The phylogenetic trees showed that four of eleven ginseng samples were Panax vietnamensis Ha et Grushv., and three samples were Panax stipuleanatus with bootstrap values of 100 %. However, sequences of all five screened loci in the last four samples, which were considered as Panax bipinnatifidus based on morphological characteristics, were highly similar to Panax stipuleanatus with the level of sequence similarity reached 99.81 % - 100 %. In addition, our results showed that of five investigated DNA regions, ITS, and psbA-trnH were the most promising barcodes that could identify P. vietnamensis Ha et Grushv. and P. stipuleanatus species within the genus Panax L.

Five species of the family Salamandridae are currently recognized from Vietnam: Paramesotriton deloustali from Lao Cai, Ha Giang, Tuyen Quang, Bac Kan, Quang Ninh and Vinh Phuc provinces; P.guanxiensis from Cao Bang province; Tylototriton asperrimus from Ha Giang and Lao Cai provinces; T.verrucosus from Lai Chau province; and T. vietnamensis from Bac Giang, Phu Tho, and Lang Son provinces. Specimens of Tylototriton species were collected from different localities in Northern Vietnam for phylogenetic analysis. Comparison of 450 base pairs of partial Cytochrome b mitochondrial DNA sequences shows 100% identity between Tylototriton aperrimus from Ha Giang and Lao Cai provinces. The same comparisons show 100% identity between specimens of Tylototriton vietnamensis from Bac Giang and Phu Tho provinces and 99.8% between specimens from Bac Giang and Lang Son provinces. This identity shows little geographic variation of conspecific species. However, the sequences of the T. asperrimus and T.vietnamensis from above localities showed an uncorrected pairwise sequence divergence of 8 - 8.3%. Diagnostic morphological characters of the five Vietnamese species are presented based on examined specimens or literature. Comparison of morphological characters between T. vietnamensis and T. hainanensis showed that the first species differs from the latter by having tip of forelimb reaching to nostril (tip of forelimb reaching to eye in T. hainanensis), snout-vent length shorter than tail length (snout-vent length longer than tail length in T. hainanensis). Furthermore, we provided additional data on the habitat and reproduction of Paramesotriton deloustali, Tylototriton asperrimus, and T. vietnamensis

Contamination of heavy metals (As, Cd, Hg, Pb and Sn) in honeybee products (Apis cerana) from Northern Vietnam is determined. The study was carried out in two main harvesting seasons of beekeeper farms (April and October), over two years (2018-2019). A total of 72 samples from 24 honeybee hives from 8 provinces and one city were collected. The results showed that the quality of three products in nearly all research sites were met the standards in accordance with the national technical regulation on the limits of heavy metal contamination in food of the Vietnamese Health Ministry, except for the pollen and beeswax from HY2 site. The concentration of Pb was most notably value in this study, which was determined at fairly high levels in pollen (3,767 mg/kg) and beeswax (5,840 mg/kg) from HY2 site. This can be a warning for this metal significant contamination in the habitat. Specially, Hg was not detected in most samples or only recorded without significant. For the environmental types, the mean value of As and Sn in all honeybee’s product types in semi-rural area were higher than that in rural area. Thus, the detection of the heavy metals proves that honeybee’s products could be good indicators to detect the environmental contaminants and monitor the habitat quality of a particular area

Genetic diversity plays an important role in diversity conservation at multiple levels and supports to monitor and assess genetic variation. In plants, genetic diversity provides the ability to adapt and respond to environmental conditions that helps plants to survive through changing environments. Genetic diversity analyses based on molecular genetic markers are effective tools for conservation and reintroduction of rare and endangered species. In recent years, the development of various chemical and molecular techniques for studying genetic diversity has received great attention. While biochemical markers are primarily used in the diagnosis of pathogens, DNA markers have been developed and widely applied for identification of species and population based on the genotype of an organism that is more stable and not easily affected by the environmental factors. PCR-based molecular marker tools, such as restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSRs) are used for analysing the difference in the targeted DNA sequences. With the rapid and robust development of genomic sequencing technology it is now possible to obtain and analyse DNA sequences of the whole genome of studied organisms. However, each type of DNA markers has different principles, as well as the pros and cons of specificity. In this article, we review methods and point out DNA markers, which are considered as reliable and widely used tools for the detection of genetic variation. In addition, we present the application of DNA marker in analysing genetic diversity of wild, domestic and medicinal plants, as well as some perspectives on the future of DNA marker’s application in the analysis of genetic diversity.

Despite many advantages over injection vaccines such as cost effectiveness, safety and easy to use, and so on, oral vaccines are negligibly concerned. This is mostly because of the availability of vast surface in the gastro-intestinal tract, thereby requiring lot of antigens which could hamper their potential. To circumvent this issue, a novel strategy for targeting antigens to M cells (microfold cells), a minority of cells located in the small intestine for antigen transportation, is utilized by making a fusion protein comprised of an antigen with an M cell specific ligand. Discovered via biopanning, Co1 peptide is a potential ligand because of its small size (12 amino acids) and having an adjuvant capacity. To develop a monitoring model, we fused GFP (green fluorescent protein) as a monitoring marker with Co1 peptide. Initially, co1-gfp fusion gene was created by overlap extension PCR on GFP-encoded vector backbone, then it was incorporated into an expression vector pET22b before transforming into E. coli DH5α. The recombinant vector was screened by PCR method, sequenced and aligned with designed sequences. The in-frame vector was then introduced into E. coli BL21(DE3) for expression by inducing with 0.5mM IPTG. Fusion protein was purified using Ni-affinity chromatography. The results showed that the fused genes were in-frame cloned and completely matched with the designed sequences. SDS-PAGE and Western blot analyses showed Co1-GFP protein expressed in soluble form and could be purified at one-step elution of 500mM imidazole. The purified fusion protein could emit fluorescent light under UV excitation. Collectively, recombinant Co1-GFP fusion protein was successfully produced and its potential applications need to be warranted.

Passiflora edulis Sims. belonged to the genus Passiflora, is one of the important economic crops of the world as well as Vietnam. Nowadays, the commercial P. edulis is mainly propagated by seeds, cuttings and grafting; however, these methods still have some limitations such as genetic degradation and heterogeneity and the spread of pathogenic viruses. Micro-propagation has been used for clonal breeding and disease-free plant breeding, as well as providing a source of materials for Passiflora breeding. In this study, leaf explants of P. edulis Sims. (2.0-month-old) excised from the in vitro culture of ex vitro axillary buds cut longitudinally and transversally into thin cell layers (lTCL and tTCL) were used as plant materials to evaluate the shoot regeneration. In addition, the effects of explant age and lighting condition on shoot regeneration were also investigated. After 8 weeks of culture, the results showed that shoot regeneration rate (100%) and shoot multiplication coefficient (13.33) of the in vitro leaf-tTCL-4 were higher than those of other treatments and control. The shoot regeneration rate of P. edulis Sims. also varied with the change of explant age. The highest shoot regeneration rate (100%) was obtained from leaf explants of 1.5-month-old shoots after 8 weeks of culture. Moreover, the light (fluorescent lamps with photoperiod of 16 hours/day and lighting intensity of 40 - 45 μmol.m-2.s-1) improved not only morphogenesis rate, but also shoot regeneration rate (100%) of leaf explants after 8 weeks of culture. This study provided a novel method for rapid micro-propagation of P. edulis Sims.