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) ttta

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES ---***---VU XUAN DUONG RESEARCH TO IDENTIFY THE SCIENTIFIC

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

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

1 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 )”

2 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

3 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

4 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

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 1 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 2 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 6 - 8cm

- 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 bằng phương pháp lôi cuốn hơi nước Việc phân tích định tính được thực hiện trên hệ thống thiết bị sắc ký khí và phổ ký liên hợp GC/MS của 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 3 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

2 Dissection of plant at the base Rounded

8 Ratio of length/width of leaf From 3 – 5

15 Number of flowers/cincinnus 2 - 4 flowers

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 4 – 6 tubers

26 Root length at the widest part 2 – 3cm

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 6 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

The results of electrophoresis showed that the PCR product of the

matK primer pair was about 900bp, the size of the PCR product of the

ITS primer pair was about 800bp, which is consistent with the theoretical size

3.1.3.3 Classification of Bac Kan ginger based on the ITS gene

sequence

3.1.3.3.1 ITS gene sequencing results

Successfully solved 06 new ITS sequences and registered on Genbank with the codes: MN227653, MN227654, MN227655, MN227656, MN227657 and MN227658 (Appendix 3)

After the modification and removal of all ITS genomic vacancies, the ginger specimens obtained had nucleotide sequence similarity of ITS genome from 99 - 100% and length of 698bp There are 02 nucleotide positions with differences between specimens, regarding nucleotide position 597, while specimens of GD01LT, GD02LT and GD01XD are type T nucleotides, the remaining specimens of GD03LT, GD02XD, GD03XD are type C nucleotides; regarding nucleotide position 605, while the GD03LT specimens are type T nucleotides, the remaining specimens are type C The differences between the nucleotides in the aforementioned positions may indicate a genetic diversity among Bac Kan ginger plants, which is a very important basis for research to select superior lines, crossbreed, and select high-yield breeds

The six ITS gene sequences of the research specimens were

compared to the ITS sequences of the genus Alpinia (taxid: 94326)

published on NCBI using the Blast nucleotide tool The results showed that the species with the ITS sequence at the highest level of similarity with the

ITS sequence of Bac Kan ginger specimens include: Alpinia chinensis (EU909426.1), Alpinia japonica (EU909427.1), Alpinia officinarum (EU909422 1), Alpinia pumila, Alpinia nieuwenhuizii

3.1.3.3.2 Building a phylogenetic tree by ITS gene sequence

The phylogenetic tree of Bac Kan ginger specimens by ITS gene indicator (Figure 3.7) was constructed from Maximum Likelihood

method with a value of boostrap value 1000, showing that 06 specimens

of Bac Kan ginger were classified in the same group as: Alpinia

coriacea, A pumila, A japonica, A polyantha, A intermedia, A stachyodes, A maclurei, A suishaensis, A guangdongensis This result

is quite appropriate because in fact, the above species have the same

distribution area as A coriandriodora of southern China

3.1.3.4 Classification of Bac Kan ginger based on the matK gene sequence 3.1.3.4.1 matK gene sequencing results

Successfully solved the sequence for 06 new matK gene sequences

and registered on Genbank with the codes: MN335320, MN335321, MN335322, MN335323, MN335324, MN335325 (Appendix 4) After

the modification and removal of all vacancies in matK gene region, the

ginger specimens obtained (GD01LT, GD02LT, GD03LT, GD01XD, GD02XD, GD03XD) had nucleotide sequence similarity of 100% and length of 700bp

The six matK gene sequences of the study specimens were compared to the matK sequences of the genus Alpinia (taxid: 94326)

published on NCBI using the Blast nucleotide tool

(http://blast.ncbi.nlm.nih.gov/ Blast.cgi) The results showed that: the species with matK sequence at the highest level of similarity with matK sequence of Bac Kan ginger specimens include: Alpinia zerumbet, A

mutica, A kwangsiensis, A hainanensis, A uraiensis, A uraiensis, A shimadae, A formosana, A japonica, A japonica, A chinensis, A calcarata, A oxyphylla

3.1.3.4.2 Building a phylogenetic tree by matK gene sequences

Based on the matK gene sequence obtained from specimens of Bac Kan ginger and the published database of matK gene sequences of Alpinia genera, we have built a tree diagram of genetic relationships between A

coriandriodora species and species in the genus Alpinia based on the

combined data block of matK gene region by the Maximum Likelihood

method (Figure 3.9) The tree diagram shows that specimens of Bac Kan

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]