Nghiên cứu xác định tên khoa học, quy trình nhân giống in vitro và hoạt tính sinh học của tinh dầu cây gừng bản địa ở bắc kạn (gừng đá) tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES *** - VU XUAN DUONG RESEARCH TO IDENTIFY THE SCIENTIFIC NAME, IN VITRO PROPAGATION PROCESS AND BIOLOGICAL ACTIVITY OF ESSENTIAL OIL FROM INDIGENOUS GINGER IN BAC KAN (GỪNG ĐÁ) Specialization: Biotechnology Code number: 9420201 SUMMARY OF AGRICULTURAL DOCTORAL THESIS Hanoi, 2019 The dissertation has been completed at: Vietnam Academy of Agricultural Sciences Scientific supervisors: Assoc Prof Dr Dang Trong Luong; Dr Do Tuan Khiem Reviewer 1:………………………………………… Reviewer 2: ………………………………………… Reviewer 3: ………………………………………… The dissertation will be defended at the Dissertation committee at the National level Vietnam Academy of Agricultural Sciences, on Date ….month… year The full text of the dissertation can be found in the following libraries: - The National Library of Vietnam; - The Library of Vietnam Academy of Agricultural Sciences INTRODUCTION Necessity of the research subject The indigenous ginger plant in Bac Kan (Gừng đá) is classified as a rare and valuable plant that needs to be preserved under Decision No.80/2005/QD-BNN dated December 5, 2005 by the Ministry of Agriculture and Rural Development Limestone ginger is a native species of specialties of Bac Kan province and is distributed mainly in Liem Thuy and Xuan Duong communes of Na Ri district, Bac Kan province In recent years, the demand for Limestone ginger of local people and tourists coming to Bac Kan has increased, especially on holidays, and the price often ranges from 600 thousand to one million VND/1kg of fresh gingers This led to the depleted exploitation of naturally grown Limestone gingers Despite its high economic efficiency, Limestone ginger plants are grown mainly based on experience in small and scattered areas The propagation and preservation of the plants are still according to the experience of the local people so the ginger roots are not guaranteed in terms of quality with multiple infections and hence, gradually degenerated Moreover, the scientific name of this species has not been identified yet Due to the morphological traits of the plant with many characteristics similar to ordinary ginger (Zingiber officinarum Roscoe), it is now classified in the genus Ginger (Zingiber), of which the scientific name in Decision No.80/2005/QD-BNN is “Zingiber sp.” In order to create a database of biological traits for identification, initially identify the chemical composition and biological activity of the essential oil obtained from indigenous ginger plants in Bac Kan, and at the same time, develop the process of rapid propagation from shoot bud cutting to apply to the creation of disease-free seed sources for farmers, we decided to choose the topic: “Research to identify the scientific name, in vitro propagation process and biological activity of essential oil from indigenous ginger in Bac Kan (Gừng Đá)” Objectives of the thesis To identify species, biological traits, chemical composition, and bioactive activities of essential oil and to develop propagation technical process using tissue culture techniques from shoot bud cutting to contribute to the conservation and development of genetic sources of native ginger plants in Bac Kan Scientific significance and practical significance of the topic 3.1 Scientific significance Providing scientific data on biological traits and propagation ability by tissue culture technology of Bac Kan ginger, which is the basis for preserving and developing valuable genetic resources; The thesis results are reference materials for research and training 3.2 Practical significance The results of the topic contribute to elucidating the taxonomic position of Bac Kan ginger plants, thereby opening directions for further studies; The propagation process by tissue culture technology is applied in practice to create a large number of uniform and disease-free planlets, creating favorable conditions for expanding the scale of development of Bac Kan ginger, contributing to poverty alleviation for mountain people Contributions of the thesis The thesis is the first project to systematically research on the taxonomic position and identification of Bac Kan ginger based on morphological traits and sequences of genes ITS (Internal Transcribed Spacer) and matK (the gene encoding maturaseK) Developing the technical process of propagating the Bac Kan ginger gene sources by plant tissue culture technology by regenerating callus from shoot bud cutting, contributing to the creation of a large number of uniform and disease-free planlets, creating favorable conditions for expanding the scale of development of Bac Kan ginger, contributing to poverty alleviation for mountain people Structure of the thesis: The main thesis consists of 116 pages with 25 tables and 29 figures The thesis consists of chapters: Introduction (3 pages); Literature review (32 pages); Materials, contents and research methodologies (19 pages); Results and discussion (60 pages); Conclusion and recommendations (2 pages) The thesis references 118 documents including 35 Vietnamese and 83 English documents CHAPTER LITERATURE REVIEW The literature review presents a general overview of the Ginger family (Zingiberaceae Lindl.) and the situation of research on biodiversity and taxonomy of the ginger family, thereby shows that the study of taxonomy of species in the ginger family continues to discover new species for science or record the distribution of species in the local area and different countries This is the basis for further research and improvement of the taxonomic system of the Ginger family, paving the way for further studies, in order to exploit the value of use and conservation of valuable species in this family As for Bac Kan ginger, there is currently no research to identify the exact scientific name of the species Therefore, a thorough research is needed to identify the scientific name of the species and its taxonomic position in the Ginger family based on morphological traits analysis and biotechnology application in sequencing ITS-rADN region (Internal transcribed spacer) and matK gene (genes encoding maturaseK) region In particular, the studies of Kress et al., (2002, 2005) were analyzed as the basis for this study Analyze the achieved results in the application of tissue culture technology in the propagation of ginger family in the world and in Vietnam In which, focus on analyzing the research of Trinh Thi Thanh Huong et al (2014) which is the basis for carrying out this research The literature review also mentions a number of studies on the chemical composition of essential oil of the ginger family, thereby showing that the research on essential oil of the ginger family is still limited, especially for species distributed in nature and there is almost no studies on the chemical composition of essential oil of tissue culture plants compared with naturally grown plants Therefore, systematic studies are needed to assess the effect of plant propagation by tissue culture on the chemical composition of essential oil, thereby providing solutions for efficient extraction and use of plants producing essential oil CHAPTER MATERIALS, CONTENTS AND RESEARCH METHODOLOGIES 2.1 Subject and scope of the study - Research subject: Bac Kan native ginger plant (Gừng đá) - Scope of the study: The study was conducted on ginger specimens collected in Bac Kan province The topic focuses on identifying the taxonomic position of Bac Kan ginger by morphological analysis and molecular markers of matK and ITS gene sequences Analyzing chemical composition, biological activity of essential oil and developing propagation process by tissue culture technology from shoot bud cutting 2.2 Research materials - The specimens were naturally grown plants (wild specimens) collected in Liem Thuy and Xuan Duong communes of Na Ri district, Bac Kan province, from 846 - 862m above sea level The total number of specimens used for morphological analysis and sequencing of ITS gene and matK gene was 06 - Materials used to study in vitro propagation process were newly regenerated shoots from healthy and disease-free mother plants Shoot size was from - 8cm 2.3 Research contents - Content 1: Identification of indigenous ginger species in Bac Kan based on analysis of morphological traits and sequencing of ITS and matK gene segments - Content 2: Completing the technical process of propagating Bac Kan ginger plants by in vitro thin layer culture technology - Content 3: Agro-biological characteristics of Bac Kan ginger plants derived from tissue culture - Content 4: Determination of chemical composition and biological activity of Bac Kan ginger essential oil 2.4 Research methodologies 2.4.1 Classification method based on morphological traits Using the comparative morphological methodology, referencing the monographs of Pham Hoang Ho (2000) [20], Nguyen Quoc Binh (2011) [6] and Journal of Tropical and Subtropical Botany [48] 2.4.2 Plant classification method based on genetic sequence 2.4.2.1 Total DNA extraction method 2.4.2.2 Electrophoresis testing total DNA extraction results 2.4.2.3 DNA quality check on agarose gel 2.4.2.4 DNA quality check by spectrophotometer 2.4.2.5 DNA amplification by PCR 2.4.2.6 PCR product sequencing 2.4.2.7 Sequence editing 2.4.2.8 Building a phylogenetic tree 2.4.3 Tissue culture methods 2.4.3.1 Reasearch on clean in vitro specimen creation 2.4.3.2 Reasearch on callus regeneration 2.4.3.3 Reasearch on regeneration of shoot buds from callus 2.4.3.4 Reasearch on quick shoot buds producing 2.4.3.5 Reasearch on complete seedlings creation 2.4.3.6 Reasearch on plant hardening and transfer substrate 2.4.3.7 Reasearch on physiological and biochemical changes of in vitro plant in the transfer stage 2.4.4 Characteristics of growth and development of in vitro native plants 2.4.5 Biochemical analysis method Chưng cất thu tinh dầu phương pháp lơi nước Việc phân tích định tính thực hệ thống thiết bị sắc ký khí phổ ký liên hợp GC/MS hãng Agilent Technologies HP 6890N Distillation of essential oil is conducted by steam extraction method The qualitative analysis was performed on the gas chromatography–mass spectrometry (GC/MS) systems of Agilent Technologies HP 6890N 2.4.6 Method of analyzing antimicrobial activity Antimicrobial activity was conducted to assess the antibiotic ability of the extracted specimens on 96-well microtitre microplates according to the method of Vander Bergher and Vlietlinck (1991) and McKane & Kandel (1996) 2.5 Research location and duration The study of morphological analysis was conducted at Vietnam National Museum of Nature - 18 Hoang Quoc Viet - Hanoi The specimens were sent to the Chinese Academy of Sciences, Guangzhou, China Implementation time: from 2/2016 to 6/2017 The study of specific gene segment sequencing was done at the Institute of Biotechnology - Vietnam Academy of Science and Technology Implementation time: from 01/2017 - 7/2017 In vitro propagation studies were conducted at the Agricultural Genetics Institute - Vietnam Academy of Agricultural Sciences; Hung Vuong University - Viet Tri City - Phu Tho Province Implementation time: from 2/2016 to 6/2017 The study of biochemical analysis and antibiotic activity were conducted at the Institute of Natural Products Chemistry - Vietnam Academy of Science and Technology Duration: from 7/2017 - 8/2018 The study of assesment of the growth and development abilities of tissue culture Bac Kan ginger was conducted in Phu Tho and Na Ri district - Bac Kan province Implementation time: from 4/2016 - 8/2018 CHAPTER RESULTS AND DISCUSSION 3.1 Taxonomy of Bac Kan ginger plant 3.1.1 Biological traits of Bac Kan ginger plant 3.1.1.1 Some agronomic and morlogical traits Table 3.1: Some typical traits of Bac Kan ginger No Trait Expression level Plant height 80-150 cm Dissection of plant at the base Rounded Smell of stem Yes Length of petioles Short (3-6 mm) Leaf blade shape Elliptic-lanceolate Leaf length From 13 - 16 cm Leaf width From 2,5 – 3,3 cm Ratio of length/width of leaf From – Leaf color Dark green 10 Leaf stripe Yes 11 Leaf margin Pubescent 12 Leaf margin color Dark green 13 Leaf smell Yes 14 Flowering frequency Once a year 15 Number of flowers/cincinnus - flowers 16 Flower structure Inflorescence 17 Flower color Sepia, yellow 18 Root shape Branching 19 Root size Small (200g) 20 Root skin color Sepia 21 Root flesh colour in the center Gray 22 Secondary color in the root flesh Yellow 23 Yield of root/cluster (kg) 150 – 300g 24 Number of tubers on cluster – tubers 25 Root length – 10cm 26 Root length at the widest part – 3cm 27 Growth time Long >10 months 3.1.2 Classification based on morphological traits 3.1.2.1 Classification to genus level According to the morphological analysis results compared to the classification key of Nguyen Quoc Binh (2011), Bac Kan ginger plant has traits such as inflorescence on the top, stem with leaves, dimpled shaped pistil, spherical fruits, which are the characteristics of the plants of the genus Alpinia (Alpinia) to distinguish them from other genera of the Ginger family (Zingiberaceae) [6] Based on that, we identified the plant named Limestone ginger in Bac Kan to be in the genus Alpinia (Alpinia), which is one of the new valuable findings because previously it was commonly called "Limestone ginger" and understood as a plant of the genus Ginger (Zingiber) 3.1.2.2 Classification to species level After identifying that the genus source of Bac Kan ginger belongs to Alpinia genus (Alpinia), we used classification key to species of Alpinia genus (Alpinia) in Vietnam [8] However, the trait description shows that this species did not match with any of the species so far reported from Vietnam On this basis, we continued to examine neighboring areas (in China), used the plant species list and species identification key of species of China's Alpinia genus (Alpinia) [48] and compared to near species, the results identified Bac Kan ginger with the scientific name Alpinia coriandriodora D Fang with typical traits: typical aroma and deciduous This is an additional species to the Vietnamese flora, increasing the total number of known species of the genus Alpinia Roxb in Vietnam to 34 species [18] This plant was published by D Fang in the journal Acta Phytotax Sine 16 (4): 79 1978 [57] 3.1.3 Classification of Bac Kan ginger based on the ITS and matK gene sequences 3.1.3.1 Total DNA extraction Extracted DNA of research specimens The results showed that the A260/A280 indexes of all specimens ranged from 1.8-2.0, proving that the total DNA obtained is guaranteed for future experiments 3.1.3.2 Analysis of amplified products zerumbet, A mutica, A polyantha, A mutan, A blepharocalyx, A rugosa, A.calcarata, A macrlure, A guangdongensis and A.japonica 3.1.3.5 Classification of Bac Kan ginger based on the combination of ITS and matK genes The phylogenetic tree results generated by a single data block such as matK and ITS are similar, but they show that results from a single data block are unclear and support levels are low Therefore, we used phylogenetic trees from matK and ITS combined data block to identify the phylogenetic relationship of Alpinia and the position of Alpinia coriandriodora (Figure 3.10) The analysis of combined molecular data by the maximum likelihood (ML) and Bayesian Inference (BI) method strongly supported that Alpinia is not a nonmonophyletic group with a very high support index (BS: 100%, PP: 1.0) (Figure 3.10) The phylogenetic tree distinguished six distinct branches of Alpinia, which is completely consistent with the research by Kress et al., (2005) [77] Among them, Bac Kan ginger (Alpinia coriandriodora) was recorded as a member of Alpinia genus with the phylogenetic position in branch VI (Figure 3.10) The analysis also shows that Bac Kan ginger (Alpinia coriandriodora) has a very close relationship with some Alpinia members in southern China (Guangxi, Yunnan, Quang Dong, Hainan) such as A japonica , A coriacea and A guangdongensis, etc This result shows that A coriandriodora has genetic similarities with species in the same distribution area Moreover, this result is a solid basis to confirm the addition of Alpinia coriandriodora to the Vietnamese flora [51] 11 Figure 3.10 Tree diagram of the genetic relationship between species of A coriandriodora and species in the Alpinia genus based on the combined data block of matK and ITS (A), and the phylogenetic position of Alpinia coriandriodora in Alpinia (B) genus The ML and PP support indexes of BI analysis are presented on the branches "-" indicates support index of lower than 50% Red: Position of A coriandriodora; Green: species belonging to the same taxonomic branch as A coriandriodora) 12 3.2 Propagating Bac Kan ginger plants by in vitro thin layer culture technology 3.2.1 Creating in vitro clean specimen The effective sterilization formula for specimens of young shoot buds of Bac Kan ginger is dual disinfection with 2.5% NaOCl and 0.5ml Tween20 solution in minutes for the first time, 15 minutes for the second time, between the two periods, rinse well with sterile distilled water at least times 3.2.2 Regenerating callus from shoot bud cutting Table 3.6 Effect of combination of TDZ and 2.4D on callus regeneration of shoot bud cutting (after weeks of culture) Growth Callus Formula regulators (mg/l) regeneration Callus morphology rate (%) TDZ 2,4D Some specimens could not ĐC 0 32,22 regenerate callus and turned black CT8 0,5 1,0 34,44 Callus surface was dry, CT9 0,5 2,0 44,44 firm and bright white CT10 0,5 3,0 75,56 Some specimens could not CT11 0,5 4,0 54,44 regenerate callus and turned black LSD0,05 5,05 P.value