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

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

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



- 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

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

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

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


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

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

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

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

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

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

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 (%)



-

-

0,43

-

0,31

2,79

C10H16

2 Octanal <n->

0,36

0,56

0,41

0,59

0,22

1,05

0,32


-

0,14

0,12

-

-

0,98

C10H16

5 Cineole 1,8

-

-

0,27

-

0,39

0,22

0,45


-

0,36

-

1,02

0,98

C10H18O

8 Decanal

1,25

0,58

0,76

1,85

0,91

-

0,25

1,00


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


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


-

-

C10H22O

0,20

0,17

-

-

-

0,12

-

C9H18

1 Camphene

6 Octenal <2->

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


-

-

-

1,77

-

-

C9H18O2

20 Decanoic acid

-

0,63

-

-

-

-

0,37


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

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