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.

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.).

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.

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.

Trong những năm vừa qua đã có nhiều nghiên cứu tập trung tìm hiểu về vai trò của vi sinh vật trong quá trình tạo trầm hương ở một số loài thực vật thuộc chi Trầm Aquilaria. Dựa trên giả thiết về mối liên hệ giữa vi sinh vật nội cộng sinh trong cây và sự hình thành trầm, chúng tôi đã phân lập và tìm hiểu một sô đặc điểm sinh học của các chủng nấm cộng sinh từ mẫu cây Trầm dó (A. crassna Pierre ex Lecomte) thu tại phía Nam Việt Nam. Bằng phương pháp phân tích đặc điểm hình thái kết hợp với sinh học phân tử DNA barcoding, ít nhất ba nhóm nấm cộng sinh với mẫu thực vật nghiên cứu đã được xác định, đó là các chủng nấm thuộc chi Geotrichum, Fusarium và Colletotrichum của ba họ vi nấm Dipodascaceae, Nectriaceae và Glomerellaceae. Đáng chú ý là chủng nấm Geotrichum candium SHTr1 phân lập được từ phần gỗ tối màu bên trong một mẫu thân cây có mùi hương hoa quả rất đặc trưng và biểu hiện hoạt tính kháng vi khuẩn kiểm định Staphylococcus aureus yếu. Một chủng vi nấm khác là Fusarium verticillioides SHTr3 cũng biểu hiện hoạt tính kháng hai vi khuẩn Gram dương kiểm định là Bacillus subtillis và S. aureus với cùng giá trị MIC là 50 μg.mL-1. Tại nồng độ 200 μg.mL-1, cặn chiết trong dung môi ethyl acetate của hai chủng nấm F. verticillioides SHTr3 và Colletotrichum truncatum SHTrHc7 biểu hiện hoạt tính chống oxy hóa trên hệ DPPH tương đối đáng kể với giá trị SC lần lượt là 53,87 và 71,82%. Kết quả của nghiên cứu đã góp phần làm rõ thêm bức tranh đa dạng của quần xã vi sinh vật cộng sinh với cây Trầm dó A. crassna tại Việt Nam, cũng như cung cấp thêm dữ liệu về hoạt tính sinh học và hé lộ vai trò của các vi sinh vật này trong sự tạo trầm, qua đó đóng góp nhất định cho vấn đề bảo tồn cây gỗ quý này tại Việt Nam.

Culms and leaves of Cymbopogon citratus L. were collected from two regions of Phu Tho province (Thanh Son and Phu Ninh) and used as materials for essential oil extraction. Oils obtained were steam-distilled, analyzed for chemical composition and evaluated for cytotoxic activity against three different cancer cell lines. The GC/MS analysis showed that citral is the major content of the steam-distilled essential oils which was found in the range of 64.15-76.22%. Camphene was found only in culm oils of both regions but it was not detected in the leaf oils. Interestingly, the isomer forms of ocimene present at higher content in the culm oils than in the leaf oils whereas myrcene content in the leaf oils is higher than that in the culm oils. In a cytotoxicity test, four essential oils of culms and leaves of C. citratus from Thanh Son and Phu Ninh showed potent activity against A549 (human lung carcinoma) cell line with the IC50 values ranging from 4.01±0.39 to 6.3±0.54 µg/ml. The essential oils (culms and leaves) from Phu Ninh exhibited moderate effects on the Hela (human cervical adenocarcinoma) cells with the IC50 values of 19.43±1.16 and 42±2.41 µg/ml, respectively. However, they were inactive against the human hepatocellular carcinoma Hep3B cell line. The essential oils from Thanh Son exhibited potent cytotoxic activity against Hela and Hep3B cell lines with the IC50 values ranging from 1.18±0.26 to 8.91±0.32 µg/ml. The results indicated that the essential oils of C. citratus from Thanh Son, Phu Tho could be considered as a promising candidate for the natural sources of anticancer agents.

The thin cell layer culture technique (TCL) has been used for the effective tissue culture of several dozen plants with commercial importance such as field crops (rice, cereals), horticultural commodities (fruits, vegetables, ornamental plants), medicinal plants and herbs (Panax vietnamensis Ha et Grushv.), and even forestry trees (Pinus sp.), and woody fruit plants (Citrus spp., apple). In the present, TCL was used to evaluate the efficiency of shoot regeneration and propagation for purple passion fruit (Passiflora edulis Sims.). The internodes were cut longitudinally (lTCL) and was used as an initial material. The results showed that the shoot regeneration rate and number of shoots from internode-lTCL depended on position of internodes (1, 2, 3, 4 and 5) and the plant growth regulator (BA and NAA). After 8 weeks of culture, internode-lTCL derived from 3rd internode of P. edulis cultured on MS medium supplemented with 1.5 mg/L BA in combination with 1.0 mg/LNAA gave the highest shoot regeneration rate (83.33%), and number of shoots (3.00 shoots/explant). These shoots were cultured on modified MS (MSM) medium supplemented with 1.0 mg/L BA showed higher efficiency of shoot multiplication (3.56 shoots/explant and 6.67 cm height) than the other BA treatments. In addition, the highest rooting rate was 76.67% when cultured on MSM medium containing 2.0 mg/L IBA. The survival rate of plantlets was 83.33% when transferred into greenhouse condition after 10 weeks. The results of this study were the initial success in establishing an effective in vitro regeneration and propagation of Passiflora edulis Sims. through internode-lTCL.