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 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:
1. Assoc. Prof. Dr. Dang Trong Luong;
2. 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
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
1


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.
Structure of the thesis: The main thesis consists of 116 pages
with 25 tables and 29 figures. The thesis consists of 5 chapters:
2


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

3


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.
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.

4

- 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

5


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.

6


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
No Trait
Expression level
1 Plant height
80-150 cm
2 Dissection of plant at the base
Rounded
3 Smell of stem
Yes
4 Length of petioles
Short (3-6 mm)
5 Leaf blade shape

Elliptic-lanceolate
6 Leaf length
From 13 - 16 cm
7 Leaf width
From 2,5 – 3,3 cm
8 Ratio of length/width of leaf
From 3 – 5
9 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
2 - 4 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
4 – 6 tubers.
25 Root length
5 – 10cm
26 Root length at the widest part
2 – 3cm
27 Growth time
Long >10 months
3.1.2. Classification based on morphological traits

3.1.2.1. Classification to genus level

7


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.
3.1.3.2. Analysis of amplified products

8


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

9


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
( 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
ginger are in the same group with the following species: A. guinaensis, A.

10


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 5 minutes for the first time, 15 minutes for the
second time, between the two periods, rinse well with sterile distilled
water at least 3 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 8 weeks of culture).
Growth
Callus
Formula regulators (mg/l) regeneration
Callus morphology
rate (%)
TDZ
2,4D
Some specimens could not
ĐC
0
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
<0,001
To regenerate callus from Bac Kan ginger shoot bud cutting, use a
combination of 0.5 mg/l TDZ and 3 mg/l 2.4D, callus regeneration rate
was 75.56%, callus had a dry, firm and bright white surface. This
research result is consistent with the previous announcement when
studying the propagation of Curcuma kwangsiensis Lindl. and Alpinia
purpurata by callus regeneration method [80, 114].
3.2.3. Regenerating shoot buds from callus
The research results showed that MS medium supplemented with
2.0 mg/l Vitamin B1 and 3.0 mg/l BAP was suitable for the regeneration
13


of shoot buds from callus, the rate of shoot bud burst reached 78.89%,
the rate of shoot bud regeneration reached 9.71 shoot buds/callus.

3.2.4. Multiplication of in vitro shoot buds
3.2.4.1. Effect of BAP and Kin on the ability to regenerate and multiply
shoot buds in vitro
Research results show that the combination use of 2.0 mg/l BAP;
1.0 mg/l Kin and 0.2 mg/l α-NAA increased shoot bud multiplication
rate (reaching 5.98 times) and shoot bud mean height (6.07 cm)
compared with the separate use of 3.5mg/l BAP (shoot bud
multiplication rate reached 5.03 times, average height of shoot buds
reached 4.18cm) according to the study of Trinh Thi Thanh Huong et al
(2014).
3.2.4.2. Effect of coconut water on the ability to regenerate and multiply
shoot buds in vitro
Table 3.9 Effect of coconut water content on shoot multiplication
efficiency
(after 4 weeks of culture)
CT

CT22

CT23
CT24
CT25
CT26
LSD0,05
Pvalue

Coconut
Shoot Number of
Multiplier
water

height leaves/buds Shoot morphology
(times)
(ml/l)
(cm)
(leaves)
Fat buds, dark
0
5,91
5,87
4,67
green stems, good
growth.
Fat buds, dark
100
6,32
5,90
5,00
green stems, good
growth.
Fat buds, dark
150
5,69
5,77
4,33
green stems
Medium bud, light
200
4,71
5,27
3,67

green stem
Small buds, light
250
3,71
4,67
3,33
green stems, some
yellow leaves.
0,14
0,38
0,81
<0,001

<0,001
14

0.0142


Compared with the results of Trinh Thi Thanh Huong (2014), the use
of MS + 30g/l Succrose + 6 g/l agar + 2.0mg/l BAP + 1.0mg/l Kin +
0.2mg/l α-NAA + 100ml/l coconut water improved shoot multiplier, which
increased from 5.03 times to 6.32 times, and the shoots obtained were of
good quality and fat with large leaves and dark green stems and grew well.
Thus, the appropriate content of coconut water to supplement to
the medium is 100ml/l and the suitable medium to multiply Bac Kan
ginger buds in vitro is: MS + 30g/l Succrose + 6 g/l agar + 2.0mg/l BAP
+ 1.0mg/l Kin + 0.2mg/l α-NAA + 100ml/l coconut water.
3.2.5. Regenerating roots to create complete plants in vitro
The results of the study showed that the formula using MS

medium supplemented with 0.6mg/l NAA had the best root
regeneration and complete plant formation with rooting time from 19 to
21 days, the average height/plant was 9.63cm, the average number of
leaves/plant was 5.33 leaves, the average number of roots was 5.51
roots/shoot and the average length of roots was 4.43cm. This research
result is consistent with the previous publication of the author when
researching on propagation of ginger family, especially it confirmed the
research results of Trinh Thi Thanh Huong et al (2014) when
researching on Bac Kan Limestone ginger [19,51,112].

0,2

0,4

1,0

0,8

0,6

0,2

A

0,4

1,0

0,8

0,6

B

Figure 3.15. Complete plant morphology after 6 weeks of culture
A: On ½MS medium (supplemented with NAA content from 0.2 -1.0mg/l)
B: On MS medium (supplemented with NAA content from 0.2 -1.0mg/l)

15


3.2.6. Transfer stage
3.2.6.1. Transfer substrate
On a 100% fine sand substrate, the quality of the plant was
significantly improved. The average plant height, number of leaves and
roots/plant after 15 days reached 9.43 cm; 5.33 leaves/plant; 5.33
roots/plant compared with the average plant height, number of leaves
and roots/plant initially being 9.2 cm, 5 leaves/plant and 5 roots/plant.
By the 30th day, the average height of the plant was 9.67 cm, the
average number of leaves was 5.67 leaves/plant and the average number
of roots per plant was 6.00 roots/plant.
3.2.6.2. Reasearch on physiological and biochemical changes of Bac Kan
ginger in the transfer stage
3.2.6.2.1. Free water, bound water content and dry matter content
3.2.6.2.2. Photosynthetic pigment content
3.2.6.2.3. Chlorophyll fluorescence
3.2.6.2.4. Catalase activity
3.2.7. Process of propagating Bac Kan ginger plants by in vitro thin layer
culture technology
3.2.7.1. Process maps

3.2.7.2. Process Description
* Step 1: Prepare materials
* Step 2: Create callus
* Step 3: Regenerate shoots from callus
* Step 4: Multiply shoots in vitro
* Step 5: Regenerate a complete in vitro plant
* Step 6: Transplant plants in nursery
3.3. Agro-biological characteristics of Bac Kan ginger plants derived
from tissue culture
Bac Kan ginger plants derived from in vitro culture were grown in
Viet Tri City - Phu Tho Province under field conditions to assess agro-

16


biological characteristics: Growth patterns of plant height; Leaf growth
patterns; Tillering patterns; Some factors constituting the productivity.
Planting time: April 2016
3.3.1. Growth patterns of plant height
The research results showed that the growth of plant height of Bac
Kan ginger during the monitoring period was relatively slow. 20 days after
planting, the average height of the plant was 4.39cm. 50 days after
planting, the average height of the plant was 8.88cm (increased by
4.49cm/30 days); 80 days after planting, the average height of the plant is
13.60 cm (increased by 4.72 cm/30 days).
The growth rate of the plant height was highest at 5.41 cm/30 days
(170-200 days after planting) and 4.73 cm/30 days (140 - 170 days after
planting). 290 days after planting, the average plant height was 41.85cm;
320 days after planting, the average plant height was 44.26 cm.
3.3.2. Leaf growth patterns

The research results showed that, corresponding to the growth
speed of the plant height, leaf growth patterns of Bac Kan ginger were
also relatively slow. 20 days after planting, the average number of leaves
was 3.03/stem; 50 days after planting, the average number of leaves
reached 3.59 leaves/setm; Growth rate was 0.56 leaves/30 days. From 50
to 80 days after planting, the growth rate was 0.55 leaves/30 days; 80 110 days after planting, the growth rate was 0.48 leaves/30 days. From
110 days after planting onwards, the speed of leaf growth of Bac Kan
ginger was faster than the first stage. 320 days after planting, the average
number of leaves reached 9.48 leaves/stem.
3.3.3. Tillering patterns
The results showed that, 20 days after planting, the tillering rate
was low, the average number of branches was 1.10 branches/plant,
during this period the plants had to adapt to field environment
conditions, the ability to accumulate nutrients was limited. After 50
days, when the plant started to grow its root and turned green, Bac Kan
17


ginger plant was able to start tillering, the number of branches/plant
reached 1.57 branches and 80 days after planting, it reached 2.23
branches/plant. After 140 days, Bac Kan ginger plant continued tillering
but the tillering rate was lower than the first stage. 320 days after
planting, the average number of branches/plant was 5.34 branches/plant.

3.3.4. Some factors constituting the productivity of Bac Kan ginger
through tissue culture
The research results showed that 320 days after planting, the
average number of roots/clump of Bac Kan ginger plant reached 4.73
roots/clump, the average root length was 5.41cm and the average root
width was 2.26cm. The average individual yield or weight of

roots/clump of Bac Kan ginger was 147.7g/clump.
3.4. Chemical composition of essential oil of Bac Kan ginger
3.4.1. Essential oil extraction results
The essential oil obtained from the specimens of leaves was light
yellow in color with a pleasant aroma. In the Bac Kan natural ginger
specimen, from 400g of fresh leaves, 0.440g of essential oil was obtained,
which was 0.110% concentration (based on the specimen of fresh
material). Regarding the tissue culture Bac Kan ginger specimens, the
content of essential oil obtained from leaves of the specimens grown in
Bac Kan and grown in Phu Tho had the same proportion of essential oil
content, respectively 0.113% and 0.133%, and both were higher than that
of Bac Kan natural ginger specimen. This proves that planting tissue
specimens in two different locations (Bac Kan and Phu Tho) did not
affect the concentration of essential oil (Table 3.17).
Essential oil obtained from the specimens of roots was dark yellow
in color and also had a pleasant aroma similar to that obtained from the
leaves. When compared with the Bac Kan natural ginger specimens,
tissue culture ginger specimens showed a higher concentration of
essential oil obtained from the leaves than from the roots. In the Bac
Kan natural ginger specimens, from 500g of specimens of fresh roots,
18


0.412g of essential oil could be obtained, which was 0.082%
concentration (calculated based on the specimen of fresh material).
Regarding tissue culture Bac Kan ginger specimens, essential oil
obtained from the roots had a much lower concentration than the
essential oil obtained from the leaves (0.075% for specimens of roots
compared to 0.113% for specimens of leaves). (Table 3.17).
3.4.2. Chemical composition of essential oil

By gas chromatography-mass spectrometry analytical method (GCMS), the essential oil obtained from leaves of the Bac Kan natural ginger
(TNBK) specimen was identified to have 14 components (accounting for
94.38%), the tissue culture Bac Kan ginger (CMBK) specimen had 13
components (accounting for 85,16%), the tissue culture Phu Tho ginger
(CMPT) specimen had 18 components (accounting for 84.33%). The
composition of essential oil obtained from Rhizome of TNBK specimen
had 15 components (94.24%), CMBK specimen had 14 components
(85.27%), CMPT specimen had 18 components (88.65%) (Table 3.18 ).
Essential oil obtained from leaves contained the main compound as
decenal (80.78%), followed by decen-1-ol (4.91%), octenal (2.71%),
decanal (1.25%) and 2-decenoic acid (1.03%). Decenal is a major
component of coriander essential oil Coriandrum sativum L., which is
used as a spice and a flavoring agent [84]. Just like the essential oil
obtained from leaves, the essential oil obtained from natural ginger root
constituted the highest content of decenal (80.48%), followed by octenal
(3.01%), 2-decenoic acid (1.69%), decen-1-ol (1.53%) and dodecanal
(1.17%).

19


Table 3.18: Chemical composition of essential oil obtained from
research specimens
Leaf specimen (%)
No

Compound

Root specimen (%)

Molecula
r formula

TNB
K

CMB
K

CMP
T1

CMP
T2

TNB
K

CMB
K

CMP
T1

CMP
T2

-

-

-

-

0,43

-

0,31

2,79

C10H16

2 Octanal <n->

0,36

0,56

0,41

0,59

0,22

1,05

0,32

0,23

C8H16O

3 Cymene <o->

0,67

0,23

0,68

1,18

0,25

0,15

0,38

0,87

C10H14

4 Limonene

-

-

-

0,14

0,12

-

-

0,98

C10H16

5 Cineole 1,8

-

-

0,27

-

0,39

0,22

0,45

1,78

C10H18O

2,71

0,71

1,64

3,42

3,01

0,32

1,36

4,47

C8H14O

7 Borneol

-

-

0,34

-

0,36

-

1,02

0,98

C10H18O

8 Decanal

1,25

0,58

0,76

1,85

0,91

-

0,25

1,00

C10H20O

9 Fenchyl acetate

-

-

2,54

-

2,25

2,11

3,35

5,25

C12H20O2

10 Decenal <2E->

80,78

39,60

47,11

53,48

80,48

22,21

42,52

61,59

C10H18O

11 Decen-1-ol <2E->

4,91

0,53

1,62

2,26

1,53

0,97

1,55

1,13

C10H20O

-

-

1,41

-

0,84

1,16

2,35

2,51

C12H20O2

1,03

21,94

8,60

2,67

1,69

36,29

21,22

2,19

C10H18O2

0,46

16,89

15,90

9,25

0,60

16,11

11,75

2,36

C12H22O2

15 Dodecanal <2E->

0,98

1,14

1,17

-

1,17

0,64

0,94

0,99

C12H22O

16 Decanol <n->

0,58

-

0,78

-

-

-

-

-

C10H22O

0,20

0,17

-

-

-

0,12

-

C9H18

1 Camphene

6 Octenal <2->

12 Bornyl acetate
13 2-Decenoic acid <E->
14

Decenyl Acetate
<2E->

17 Non-1-ene
18 Nonanal

-

0,45

0,25

-

-

0,64

0,25

-

C9H18O

19 Nonanoic acid

-

1,70

-

-

-

1,77

-

-

C9H18O2

20 Decanoic acid

-

0,63

-

-

-

-

0,37

-

C10H20O2

94.38

85,16

84,33

98,21

94,24

85,27

88,65

98,05

Total

Note:

TNBK: The specimen grown naturally in Bac Kan
CMBK: Tissue culture specimen grown in Bac Kan
CMPT1: 01-year-old tissue culture specimen grown in Phu Tho
CMPT2: 02-year-old tissue culture specimen grown in Phu Tho
( - ): N/A

When compared with the essential oil of some other Alpinia species,
the essential oil obtained from Bac Kan natural ginger is of great difference
from the essential oil of these species: while the main components of the
essential oil of Bac Kan natural ginger were decenal, octenal, 2-decenoic
acid, decen-1-ol and dodecanal, the essential oil of Riềng nếp (A. galanga)
20


contained 1.8-cineol, camphor, alpha-fenchyl acetate; essential oil of A.
conchigera contained 1.8-cineol, myrcen and farnesol as the main
components and essential oil of A. tonkinensis contained myrcen, 1,8cineol and camphor [61, 70].
When analyzing the specimen of essential oil obtained from a 2-yearold culture tissue ginger plant grown in Phu Tho, the content of the main
substance Decenal <2E-> increased to 53.48% (for leaf specimen) and
61.59% (for root specimen), which is asymptotic to the content of naturally
grown ginger plants. This also shows that tissue culture plants can
synthesize major substances relatively fully when reaching 2 years of age.
The result is completely consistent with the fact that the time of harvesting
Bac Kan ginger is 2 years old.
3.5. Microbial resistance of essential oil extracted from Bac Kan ginger plant
The antifungal and antibacterial abilities of essential oils obtained
from Bac Kan ginger were tested on 8 strains of microorganisms (Table
3.20). The results of the study showed that specimens of essential oils
collected from natural plants and 1-year-old tissue culture plants cultivated
in Phu Tho and Bac Kan province were resistant to 5-6/8 strains of testing
microorganisms at the inhibitory concentration of below 200mg/ml. Only
the essential oil obtained from a 2-year-old tissue culture plant grown in
Phu Tho province was resistant to 7/8 strains of testing microorganisms.

21

22

8. NCM PT-CR2: Root specimen of 2-year-old Phu Tho tissue culture22
ginger

7. NCM PT-TL2: Leaf specimen of 2-year-old Phu Tho tissue culture ginger

6. NCM PT-CR1: Root specimen of 1-year-old Phu Tho tissue culture ginger

4. NCM BK-CR1: Root specimen of 1-year-old Bac Kan tissue culture ginger 5. NCM PT-TL1: Leaf specimen of 1-year-old Phu Tho tissue
culture ginger

3. NCM BK-TL1: Leaf specimen of 1-year-old Bac Kan tissue culture ginger

2. Bản địa-CR1: Root specimen of 1-year-old Bac Kan indigenous ginger

1. Bản địa-TL1: Leaf specimen of 1-year-old Bac Kan indigenous ginger

Note:

Table 3.20: Antifungal and antibacterial abilities of essential oils obtained from tissue culture Bac Kan ginger


CONCLUSION & RECOMMENDATIONS
CONCLUSION:
1. Identified that Ginger plant in Bac Kan province belongs to
Alpinia genus with the scientific name Alpinia coriandriodora D. Fang.
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.
2. 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. The
phylogenetic trees from matK and ITS combined data block showed that
Bac Kan ginger (Alpinia coriandriodora) has genetic similarity with
species in the same distribution area and has a very close relationship
with some members of Alpinia genus in southern China (Guangxi,
Yunnan, Quang Dong, Hainan) such as A. japonica, A. coriacea and A.
guangdongensis, etc.
3. Established the propagation process of Bac Kan ginger by
regenerating callus from shoot bud cutting. Callus was well developed on
MS medium supplemented with 0.5 mg/l TDZ and 3.0 mg/l 2.4D. MS
medium supplemented with 2.0 mg/l of Vitamin B1 and 3.0 mg/l BAP was
suitable for regeneration of shoots from callus. MS medium supplemented
with 2.0 mg/l BAP, 1.0 mg/l Kin, 0.2 mg/l α-NAA and 100 ml/l coconut
water was suitable for multiplying Bac Kan ginger buds in vitro. MS
medium supplemented with 0.6 mg/l α-NAA had the best root regeneration
and complete plant formation. The transfer stage was carried out on a
100% fine sand substrate.
4. By steam extraction and distillation method, Bac Kan ginger
essential oil from leaves was obtained with efficiency ranging from 0.110%
to 0.133% and the essential oil from root was obtained with efficiency
ranging from 0.075% - 0.082% compared with fresh specimen weight.
23