Luận án nghiên cứu cơ sở khoa học bảo tồn nguồn gen các loài thuộc chi lan kim tuyến (anoectochilus blume) tại tỉnh thanh hoátt

1

MINISTRY OF
MINISTRY OF
EDUCATION AND
AGRICULTURE AND RURAL
TRAINING
DEVELOPMENT
VIETNAM NATIONAL UNIVERSITY OF FORESTRY

NGUYEN TRONG QUYEN

RESEARCH ON SCIENTIFIC BASIS OF GENETIC
RESOURCES CONSERVATION OF Anoectochilus Blume
SPECIES IN THANH HOA PROVINCE
Major: Forest resource management
Code: 9620211

SUMMARY OF PHD DISSERTATION

HANOI, 2022


2
Research work is completed at: Vietnam National University of
Forestry Trường Đại học Lâm nghiệp Việt Nam

Scientific supervisors:

Scientific supervisor 1: Prof. Dr. HOANG VAN SAM
Scientific supervisor 2: Assoc. Prof. Dr. BUI VAN THANG

Chương
Reviewer 1:
Reviewer 2:
Reviewer 3:
The defense will be taken in front of the Institutional Board of
Dissertation Evaluation at: Vietnam National University of
Forestry
At:

time, Date

January

Year 2022T

hn
The dissertation can be found in the libraries:
National Library; Vietnam National University of Forestry Library


3

INTRODUCTION
1. Rationale of the study
The genus Anoectochilus Blume belongs to the family
Orchidaceae, is a valuable plant genus for medical treatment of many
diseases, for examples chest pain, abdominal pain, diabetes,
nephritis, fever, high blood pressure, paralysis positive, liver
disorders… (Nguyen Tien Ban, 2005) [6]. Traditional Chinese
medicine used these plants to treat diabetes, dissolve tumors, reduce

blood lipase and cure hepatitis (Do Tat Loi, 2004) [27]. In decree
06/2019/ND-CP, 3 species of this genus are classified in group IA (A.
setaceus Blume, A.acalcaratus Aver, A.calcareus Aver) and the other
species are in group IIA[12]. The genus Anoectochilus Blume is
facing the risk of habitat loss, degradation, being used as food by
animals, and indiscriminate exploitation leading to a decline in
natural genetic resources. Zhang et al., 2013) [102]. In Thanh Hoa,
many forest ranges in some National Parks and Nature Reserves
could not control the illegal exploitation of Anoectochilus species and
they do not know exactly how many Anoectochilus species in the
area under their management (later confirmed by Phan Xuan Binh
Minh, 2019)[28].
Due to those reasons, the topic “Research on scientific basis
of genetic resources conservation of Anoectochilus Blume species in
Thanh Hoa province” was choosen.
2. Objectives of the study
Providing a scientific foundamental for conservation and
development of Anoectochilus Blume species (Orchidaceae) in
Vietnam in general and in Thanh Hoa in particular;


4

Chapter 1
LITERATURE REVIEW
1.1. The species diversity of Anoectochilus Blume
China is the country with the earliest and most research on
the genus Anoectochilus. According to the Chinese Botanical Book
(2009), [58] there are about 30 species in the genus Anoectochilus
Blume. Chuan Gao (2009) [56] mentioned that the genus

Anoectochilus Blume has about 40 species. Summary of species of
the genus Anoectochilus Blume from the website of the Botanical
List [65], there are 50 species in the world.
In Vietnam, according to Pham Hoang Ho (2000) [19], there
are 6 species in the genus Anoectochilus Blume. Nguyen Thien Tich
(2001) [40] concluded that there are 10 species in Vietnam. Leonid
V. Averyanov and L. Averyanov (2003) [1] mentioned that Vietnam
has 13 species. Nguyen Tien Ban (2005) [6], Vietnam has 12 species.
L.Averyanov et al (2008) [48], again announced that there are only
07 species in Vietnam.
1.2. Research on genetic resources conservation of Anoectochilus
Blume species
1.3. Research on medicinal value and medicinal properties of
species of the genus Anoectochilus Blume
1.4.1. Techniques for propagation
1.4.2. Techniques for planting of Anoectochilus formosanus
Hayata
1.4.3. Genetic research
1.5. Overview of conservation and information on special-use
forests with species distribution of Anoectochilus Blume in Thanh
Hoa province


5
Chapter 2
CONTENTS, LOCATION AND METHODS OF STUDY
2.1. Study contents
- The diversity of species composition of the genus Anoectochilus
Blume in Thanh Hoa.
- Systematize information on morphology, ecology, distribution,

conservation value, key table to identify species of the genus
Anoectochilus Blume in Thanh Hoa.
- Genetic characteristics of A.formosanus.
- In vitro propagation techniques, planting A.formosanus under the
forest canopy and planting in net houses; Analysis and evaluation of
total flavonoid content.
- Proposing solutions for conservation of species of the genus
Anoectochilus Blume in Thanh Hoa
2.2. Location of the study
In 11 special-use forests in Thanh Hoa province, Vietnam National
University of Forestry, Department of Analytical Chemistry, Institute
of Medicinal Materials.
2.3. Study methods
2.3.1. Method of inheriting documents
2.3.2. Professional solution
2.3.3. Interview method
2.3.4. Field investigation and data collection methods
2.3.5. Distribution map construction method
2.3.6. Genetic characteristics of A.formosanus
- Method of extraction of total DNA, Method of analyzing genetic
diversity by RAPD technique


6
- Nucleotide sequencing method
- Methods of analyzing barcoded DNA data
2.3.7. Chemical analytical methods
2.3.7.1. Machines and equipments
2.3.7.2. Solvents and chemicals
2.3.7.3. Implementation process

a) Thin layer chromatography for qualitative analysis
b) Quantification of total flavonoids by UV-Vis spectrophotometric
method
2.3.8. Experimental method
2.3.8.1. In vitro propagation
* Determine the sample sterilization formula
* Rapid multiplication of shoots
* Planting on different substrates
2.3.8.2. Planting techniques of Anoectochilus formosanus Hayata
a) Planting techniques in net houses
- Seedlings: Propagated by tissue culture method.
b) Planting techniques under the forest canopy
- Seedlings: Propagated by tissue culture method.
2.3.8.3. Methods of measuring growth, monitoring pests and diseases
in experiments
2.3.9. Statistical analyses
Statistical analyses were carried out using the software SPSS (version
20.0) and Excel.


7
Chapter 3
RESULTS AND DISCUSSIONS
3.1. The diversity of species composition of the genus
Anoectochilus Blume in Thanh Hoa
3.1.1. Results of the investigation and assessment of species
diversity
The research results showed that 05 species are distributed in
Thanh Hoa province, including: Anoectochilus calcareus Aver),
Anoectochilus elwesii (C.B.Clarke ex Hook.f.) King & Pantl),

Anoectochilus setaceus Blume, Anoectochilus annamensis Aver and
Anoectochilus formosanus Hayata. In which, the species of
Anoectochilus formosanus Hayata is added to the flora of Vietnam.
Table 3.1 Coordinates with recorded species of the genus
Anoectochilus Blume in Thanh Hoa
Research
areas

Species

Pu Hu
Nature
Reserve

A. setaceus
Blume

Pu Luong
Nature
Reserve

A. calcareus
Aver
A. elwesii
King &
Pantl
A. setaceus
Blume

Xuan

Lien
Nature
Reserve

A.annamens
is Aver.
A. setaceus
Blume

Results
Rege
Coordinate
nera
(VN2000)
ted
tree
488.612/
1
2.268.132
488.361/
2.265.715
510.755/
2
2.269.585

Matur
e tree

Total
numbe

r of
trees

5

6

960

IIIa2

2

2

995

IIIa2

3

5

650

IIIb

2

2

800

IIIb

2

6

8

1385

IIIb

1

3

4

1386

IIIb

2

2

300

G-N

1

1

300

G-N

523.978/
2.260.621
508.497/
2.265.422
498.916/
2.208.824
520.912/
2.199.581
520.870/
2.199.629

Mean
elevatio
n (m)

Forest
state

8

Nam
Dong rare
and
precious
gymnospe
rms
conservati
on area

A. calcareus
Aver
A.elwesii
King &
Pantl
A. setaceus
Blume
A. calcareus
Aver
A.formosan
us Hayata

498.508/
2.208.381

2

2

1400

IIIb

488.264/
2.246.352

3

3

980

IIIb

1

1

1

1065

IIIb

1

3

4

1100

IIIab

1

2

3

1200

IIIb

5

5

1215

IIIb, N-

488.883/
2.246.342
488.370/
2.245.893
487.695/
2.245.861
487.521/

2.247.615

IIIb

A. annamensis
Aver

A. formosanus
Hayata

Ben En National Park
Cuc Phuong National Park (in
2
Thanh Hoa ony)
3
Xuan Lien Nature Reserve
4
Pu Luong Nature Reserve
5
Pu Hu Nature Reserve
6
Tam Quy Nature Reserve
Nam Dong rare and precious
7
gymnosperms conservation area
Ham Rong cultural and historical relic
8
site
Lam Kinh cultural and historical relic
9

site
Truong Le cultural and historical relic
10
site
11 Ba Trieu historical relic site

A. setaceus
Blume

1

Special-use forests

A. elwesii King
& Pantl

No.

A.calcareus
Aver

3.1.2. Anoectochilus Blume species distribution
Table 3.1 Anoectochilus Blume species distribution Anoectochilus Blume
in Thanh Hoa

N

N

N

N

N

N

N

N

N

N

Y
Y
N
N

N
Y
N
N

Y
Y
Y
N

Y
N
N
N

N
N
N
N

Y

Y

Y

N

Y

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

* Note: Y natural distribution; N unnatural distribution


9
3.2. Species morphology characteristics
3.2.1. Anoectochilus annamensis Aver

Figure 3.1 A.annamensis Aver was collected and cultivated in the study
area of Van Xuan commune, Thanh Hoa province

Figure 3.2 Natural distribution of Anoectochilus annamensis Aver in
Thanh Hoa province
3.2.2. Anoectochilus calcareus Aver

Figure 3.3 A.calcareus. gene source in Thanh Hoa

Figure 3.4 Natural distribution of the species A.calcareus


10
3.2.3. Anoectochilus elwesii (C.B.Clarke ex Hook.f.) King & Pantl

Figure 3.5 A. elwesii gene source in Thanh Hoa

Figure 3.6 Natural distribution of the species A.elwesii) in Thanh Hoa
3.2.4. Anoectochilus formosanus Hayata
* Morphology characteristics:

Figure 3.7 Anoectochilus formosanus Hayata) natural gene source in
Nam Dong rare and precious gymnosperms conservation area

Figure 3.8 Distribution of the species A.formosanus Hayata


11
3.2.5. Anoectochilus setaceus Blume

Figure 3.9 Anoectochilus setaceus Blume

Figure 3.10 Natural distribution of the species Anoectochilus setaceus
Blume) in Thanh Hoa
3.2.2. Scientific basis for quick search to identify some species of
the genus Anoectochilus Blume in Thanh Hoa province
Table 3.2 Quick lookup to identify some species of the genus
Anoectochilus Blume in Thanh Hoa province
No.
Species
Quick lookup to identify
Upper surface of leave is reddish brown,
Anoectochilus
covered with fine hairs, the lower surface is
1
annamensis Aver lighter in color; prominent reticulate
venation, usually with 5 base veins
Egg-shaped leaves; leaf blade thick, hairy;
Anoectochilus
2
dark green, purpleish green, dark green or
calcareus Aver
light brown. Mature trees have large size

Purplish-brown leave, upper surface darker
than lower one, smooth and hairless; Petals
Anoectochilus
3
are red or Brown dots; white lip, cover has
elwesii
6 tassels on each side, cover has 2 red
stripes
Anoectochilus
Upper surface of leave is dark green, lower
4 formosanus
surface is reddish brown; the veins are
Hayata
silvery-white, sometimes the middle veins


12

5

are pale yellow; lip margin and tassels are
yellow.
Leaves are green to dark green or reddish
brown; veins are yellow or pink; flower
stalk has 5-8 tassels, tassels are white.

Anoectochilus
setaceus Blume

3.3. Genetic diversity of the species Anoectochilus formosanus

Hayata in Thanh Hoa province
3.3.1. Analysis of genetic diversity of the species Anoectochilus
formosanus Hayata by RAPD directive
3.3.1.1. Analysis of DNA polymorrphism
Table 3.3 Sampling area of A.formosanus
Research areas
Date

1/9/2020
5/9/2020

Recorded and collected result
Total
Regen
Mature
numbe
erated
tree
r of
tree
trees

Areas

Coordinate
(VN2000)

Nam Dong
mountain
Nam Dong

mountain

X 487.695
Y 2.245.861
X 487.514
Y 2.247.609

0

2

2

0

8

8

Table 3.4: Analysis of RAPD primer polymorphism in the study with 10
samples of A.formosanus
No.

Primer

PIC

Total
number of
DNA

segments

1
2
3
4
5
6
7
8
9
10

CP03
CP04
CP06
CP08
CP09
CP10
CP11
CP13
CP15
OPB10

0,08
0,19
0,13
0,22
0,15
0,25

0,22
0,44
0,42
0,35

7
6
7
5
5
4
8
4
3
5

Number of
polymorphic
DNA
segments
2
5
4
4
2
2
5
3
2
4

Percentage
of
polymorphic
DNA
segments
28,6
83,3
57,1
80,0
40,0
50,0
62,5
75,0
66,7
80,0


13
11
12
13
14

OPB18
OPD11
RA46
RA142
Tổng TB

0,44
0,30
0,45
0,21
0,27

3
10
7
6
80

2
8
6
4
53

66,7
80,0
85,7
66,7
66,3

3.3.1.2. Analysis of genetic relationships among 10 samples of
A.formosanus
Table 3.5 Genetic similarity coefficient when compared by pair of 10
samples of A.formosanus
KT1
KT2

KT3
KT4
KT5
KT6
KT7
KT8
KT9
KT10

KT1
1
0.654
0.765
0.802
0.691
0.790
0.765
0.716
0.654
0.753

KT2

KT3

KT4

KT5

KT6

KT7

KT8

KT9

KT10

1.000
0.765
0.753
0.716
0.765
0.765
0.741
0.753
0.753

1.000
0.790
0.802
0.802
0.753
0.802
0.840
0.815

1.000
0.691

0.864
0.864
0.765
0.704
0.901

1.000
0.704
0.654
0.704
0.864
0.691

1.000
0.827
0.827
0.741
0.914

1.000
0.753
0.667
0.840

1.000
0.691
0.790

1.000
0.753

1

Figure 3.11 Tree diagram showing the genetic relationships of 10
samples of A.A.formosanus
3.3.2. Analysis of some barcoded DNA sequences of A.formosanus
3.3.2.1. Results of cloning gene matK, rbcL and ITS2 sequences


14

M 1

M 1

M 1

a

b
c
a) Nucleotide sequence of the matK
0 gene

3.3.2.2. Sequence analysis of barcoded DNA fragments

Figure 3.12 Phylogenetic tree between species A.formosanus and 6
others based on the comparison of nucleotide sequences of the matK
gene published on the NCBI International Gene Bank
Table 3.6 Abnormal nucleotide positions in the matK gene sequence

of A.formosanus with 7 gene codes published in the NCBI
International Gene Bank
Abnormal nucleotide positions
Speciesi
A.
formosanus
Hayata
nghiên cứu
A.
formosanus
A. roxburghii
A.
koshunensis
A.
albolineatus
A. lylei
A. elatus
L. discolor

ID in
NCBI

MW6786
27
EU79751
3.1
KY96670
8.1
EU79751
2.1

JN166018
.1
JN166019
.1
KU68709
8.1
AJ543911
.1

1
1
4

1
8
2

2
3
7

2
4
8

2
5
8

2

6
6

2
9
7

3
3
8

3
6
1

4
1
0

4
3
9

5
8
8

6
4
7

6
6
8

6
7
7

6
8
3

7
0
6

7
1
2

A

T

T

G

C

A

T

A

T

T

C

G

A

C

T

T

G

G

A

T

T

G

C

A

T

A

T

T

T

G

A

C

T

T

G

G

A

T

T

G

C

A

T

A

T

T

C

A

A

C

T

T

A

G

A

T

T

G

C

A

T

_

T

T

C

A

A

C

T

T

A

G

T

T

T

T

C

A

T

A

G

T

C

A

G

C

T

T

G

T

T

T

T

G

C

A

T

A

G

T

C

A

G

C

T

T

T

G

T

T

T

T

C

A

T

A

G

T

C

A

G

C

T

T

G

T

T

G

C

G

T

G

C

A

G

G

C

A

A

A

C

A

G

G


15
b) Nucleotide sequence of the rbcL gene

Figure 3.13 Phylogenetic tree between species A.formosanus and other
orchid species based on comparison of rbcL gene nucleotide sequences
published on NCBI International Gene Bank
Table 3.7 Abnormal nucleotide positions in the rbcL gene sequence of
A.formosanus with 5 gene codes published in the NCBI International
Gene Bank
Species
A.formosanus
A. emeiensis
A. elatus
A. qeniculatus
A. lanceolatus
L. discolor

ID in NCBI
MW678628
LC057212.1

KU687104.1
JN166036.1
MK451850.1
MH749095.1

c) Nucleotide sequence of the ITS2 gene

Abnormal nucleotide
positions
161
G
G
G
G
A
A

383
A
A
C
C
C
C


16
Figure 3.14 Phylogenetic tree between species A.formosanus and 4
other orchid species based on comparison of ITS2 gene nucleotide
sequences published on NCBI International Gene Bank

Table 3.8 Abnormal nucleotide positions in the ITS2 gene sequence of
A.formosanus with 4 gene codes published in the NCBI International
Gene Bank
No.
1
2
3
4
5

Species

ID

A.formosanus
nghiên cứu
A.formosanus
A. albolineatus
A. lylei
A.koshunensis

Abnormal nucleotide positions
10
112
221
231
273

MW663932

-

G

C

T

G

GQ396668.1
JN166058.1
JN166060.1
EU700340.1

A
A
A
A

T
T
T
T

C
T
C
C

T
A
T
T

G
G
G
A

3.4. In vitro propagation techniques
Table 3.9 Sampling location of A.formosanus in Nam Dong rare and
precious gymnosperms conservation area (Thanh Hoa)
Reasearch areas
Date

15/7/2017
16/7/2017
17/7/2017

Areas
Nam
Dong
Nam
Dong
Nam
Dong

Results
Regenerated

tree

Mature
tree

Total
number
of trees

5

32

37

486.898/2.246.200

6

46

52

486.753/2.246.324

8

53

61

Coordinate
(VN2000)
487.229/2.246.024

3.4.1. Sample sterilization and shoot regeneration
Table 3.10 Effect of sterilization time on clean explants and shoot
regeneration of A.formosanus
Fomular
KT1
KT2
KT3
KT4
Sig.

Time
(minute)
3
5
7
9

Number of
Clean sample
clean samples
percentages
regenerated
(%)
buds
0

0
13,33
4
46,67
14
73,33
3
0,0001

Percentage of clean
samples regenerated
buds (%)
0
13,33
46,67
10,00
0,0001


17
3.4.2. Fast multiplication bud
* Effect of mineral environment on shoot multiplication:
Table 3.11 Effect of mineral environment on shoot multiplication of
A.formosanus
Culture
medium

Percentage of
Average
Number of

multi-bud number of buds promising
samples (%)
/sample
buds

Average height of
promising buds
(cm)

1/2MS

80,00

3,10 ± 0,19b

2,15 ± 0,08b

2,17 ± 0,07a

MS
Knud*
Sig.

93,33
66,67
0,036

5,31 ± 0,16a
2,33 ± 0,14c
0,0001

3,54 ± 0,12a
1,31 ± 0,12c
0,0001

1,88 ± 0,08ab
1,54 ± 0,14b
0,001

1/2MS

MS

Knud

Figure 3.15 A.formosanus shoot clusters on 1/2MS, MS and Knud culture
media after 8 weeks
* Effect of phytohormones shoot multiplication:
Table 3.12 Effect of combinations of phytohormones on shoot
multiplication of A.formosanus
Phytohormone
(mg/l)

Number of buds/sample
Percentage
of samples
Average
Average
Formula
producing

number of
BAP Kinetin NAA multiple number of buds/
promising
sample
shoots (%)
buds/sample
ĐC
SM1
SM2
SM3
SM4
Sig.

0,5
0,5
0,7
0,7

0,3
0,3
0,2
0,2

0,3
0,1
0,3
0,1

43,33
100

100
100
100

1,47 ± 0,1e
4,69 ± 0,17d
5,610 ± 0,14c
6,27 ± 0,17b
8,75 ± 0,14a
0,0001

1,29 ± 0,09d
2,15 ± 0,07c
3,28 ± 0,08b
3,51 ± 0,07b
5,04 ± 0,07a
0,0001

Average
height of
promising
buds (cm)
3,05 ± 0,27a
2,18 ± 0,09b
2,30 ± 0,13b
2,39 ± 0,13b
2,35 ± 0,11b
0,022

18

a

b

d

c

Figure 3.16 A.formosanus buds on different media after 8 weeks of
culture (a- SM1, b-SM2, c-SM3, d- SM4)
* Results of creating complete plants from shoots in vitro:
Table 3.13 Rooting ability of A.formosanus buds in vitro
Formula
R0
R1
R2
R3
R4
R5
R6
Sig.

Culture
medium

NAA
(mg/l)

MS
1/2MS
1/2MS
1/2MS
MS
MS
MS

0,5
1,0
1,5
0,5
1,0
1,5

a

Percentage
Average
of buds
number of
taking root
roots /budss
(%)
100
1,24 ± 0,12e
100
2,19 ± 0,08d
100
2,49 ± 0,10bcd

100
2,61 ± 0,14bc
100
2,33 ± 0,13cd
100
2,83 ± 0,08b
100
3,64 ± 0,08a
0,0001

Average root
length (cm)
0,76 ± 0,06e
1,19 ± 0,04d
1,34 ± 0,07cd
1,68 ± 0,07b
1,32 ± 0,07cd
1,56 ± 0,10bc
2,07 ± 0,12a
0,0001

b

Figure 3.17 Completed A.fomorsanus plants on different media: a- R3 (6
weeks), b- R6 (6 weeks)
3.4.3. Effect of type of media on the growth of tissue


19
Table 3.14: Effects of different types of subtrates on the viability and

growth of A.fomorsanus after 8 weeks of crowning
Substrate

Alive Growth height
Diameter (mm)
ratio (%)
(cm)

Tree characteristics

66,7%

4,634 ± 0,03c

1,748 ± 0,0016b Thin tree, light green leaves,

Coir

83,3%

4,838 ± 0,06b

1,869 ± 0,0014b Thick tree, green leaves, big
leaves

Dried
moss

96,7%

4,9203 ± 0,04a 1,923 ± 0,0026a Thick, tall tree, big dark green
leaves

Golden
sand

small

Sig.

0,01

0,001

a

0,005

b

c

Figure 3.18 A.formosanus tissue tree grown on different substrates after
8 weeks of crowning (a- Golden sand, b-Coconut coir, c- Dried moss).
3.5. Planting techniques of Anoectochilus
3.5.1. Planting techniques of A.formosanus in net houses
3.5.1.1. Effect of substrate on growth of A.formosanus in net house
Table 3.15: Effect of type of substrate on growth of A.formosanus after 12
weeks of growing in net houses
Alive

CTTN
Dried moss
Coir
Mix
Ferns
Golden sand
Sig.

percentage
(%)
93,3

Height (cm)

Diameter (mm)

90
76,7
56,7
66,7

5,3042±0,2390a
5,1028±0,1375ab
4,8628±0,2203ab
4,7246±0,2261b
4,3016±0,0159b

1,8031±0,0087a
1,7558±0,0384ab
1,7168±0,0266bc

1,6844±0,0031bc
1,6049±0,0366c

0,041

0,031

0,004


20

a

b

d

c

e

Figure 3.19 A.formosanus on different substrates
a- Golden sand, b-Coir, c-Dried moss, d- Ferns, e- Mix
3.5.1.2. Effect of planting density on the growth of A. formosanus
Table 3.16: Effect of density on growth of A.formosanus
CTTN
MĐ (5x5cm)
MĐ (10x10cm)
MĐ (15x15cm)

Sig

Alive
percentage
(%)
90,0
83,3
86,7

Height (cm)

Diameter (mm)

5,2610±0,2170
5,0459±0,2834
4,8836±0,1814
0,5458

1,9616±0,0368
1,8408±0,0353
1,8175±0,0797
0,2152

3.5.1.3. Effects of some foliar fertilizers on the growth and
development of A. formosanus
Table 3.17 Effect of fertilizers on the growth of A.formosanus
CTTN
VINASUBOR fertilizer
Grow More fertilizer
HVP(NPK30-10-10)

fertilizer
N-P-K fertilizer
Sample
Sig

Alive
Diameter (mm)
percentage
Height (cm)
(%)
96,7
5,9207±0,0366a 1,9500±0,0111a
83,3
5,2763±0,0722b 1,8997±0,0415a
80,0

5,2677±0,2596b 1,8363±0,0326ab

80,0
76,7

5,2497±0,1083b 1,7680±0,0531b
4,6120±0,2784c 1,7653±0,0369b
0.007
0.022

From the above results, it shows that A. fomorsanus is suitable for
bio-organic fertilizer application.
3.5.1.4. Effect of season on growth of A.formosanus in net houses

21
Table 3.18 Effect of season on growth of A.formosanus after 12 weeks of
growing in net houses
Season
Spring
Summer
Autumn
Winter
Sig.

Alive
percentage (%)
90,0
73,3
90,0
80,0
0,217

Height (cm)

Diameter (mm)

5,7320±0,2007a
5,1903±0,0381a
5,2194±0,0326ab
5,4392±0,0295b
0,021

1,8826±0,0301a

1,9069±0,0084a
1,8056±0,0036b
1,7667±0,0088b
0,001

3.5.1.5. Results of growing A. formosanus in net houses
Table 3.19 Growth of A.formosanus after 12 months of growing in net
houses
Month
1st month
2nd month
3rd month
4th month
5th month
6th month
7th month
8th month
9th month
10th month
11st month
12nd month
Sig.

a

Alive
percentage
(%)
93,3
90,0

87,7
87,7
86,7
86,7
86,7
83,3
83,3
83,3
83,3
83,3
0,999

Height (cm)

Diameter (mm)

4,320±0,2627a
4,980±0,2879ab
5,467±0,2973b
5,693±0,3002bc
6,360±0,2551cd
6,743±0,1622de
7,067±0,2381de
7,490±0,2082e
8,393±0,2019f
9,160±0,2212g
11,023±0,2660h
12,907±0,3154i
3,07E-17

1,680±0,0603a
1,743±0,0517ab
1,830±0,0503ab
1,890±0,0651bc
1,990±0,0603cd
2,083±0,0467de
2,173±0,0371ef
2,243±0,0722fg
2,373±0,0384g
2,540±0,0458h
2,683±0,0384i
2,827±0,0376k
4,80E-14

b

c

Average fresh
weight
(g/plant)
0,78 g

3,07 g/plant

d

Figure 3.20 A.formosanus in net house (a: seedlings ;b,c, d: 12 month
age plants)

22
3.5.1.6. Pests and diseases of A.formosanus grown in net house
Table 3.20 Harmful insect components of A.formosanus
Component
Thrips
Snails
Grasshopper

ID
+
+

Frequency
Very few (<5% frequency)
Few (6-20% frequency)
Few (6-20% frequency)

c

b

a

Figure 3.21 Harmful insects:a- Thrips, b-Snails,c-Grasshopper
Illness situation:
Table 3.21 Disease components of A.formosanus
Components
Anthracnose on leaves

ID
+

Explain
<10% of plants with uncommon disease

Body rot disease

+

<10% of plants with uncommon disease

a

b

Figure 3.22 Diseases of A.formosanus a- Anthracnose on leaves, b - Body rot disease
Table 3.22 Harmful wild animals
Animal
Mouse
Squirrel

ID
+++
+

Explain
Common (25– 50% number of encounters)
Rare (6-20% number of encounters)

3.5.1.7. Quantitative analysis of total flavonoids in samples of
A.formosanus grown in net house.


23
Table 3.23 Quantitative results of total flavonoids in 12-month-old
A.formosanus samples grown in net house
Medicinal
Sample
KTNT

Total mass

weight
(g)*2,253

Abs

of
concentrate
198,9
(mg)

0,552
0,567
0,561

Content
(%)
1,769

1,825
1,803

Average

1,798 ± 1,295

3.5.2. Planting techniques of A.formosanus under the forest canopy
3.5.2.1. Growth of A.formosanus planted under some forest canopy
Table 3.24 Growth of A.formosanus after 12 weeks of planting under the
forest canopy
Alive

Forest

percentage

Acacia forest
Bamboo forest
Natural wood forest
Natural wood-bamboo forest
Sig.

a

(%)
80,0
86,7
90,0
96,7

0,071

b

Height (cm)

Diameter (mm)

5,3268±0,0406a
5,5736±0,1325ab
5,8626±0,1153b
5,9120±0,0855b
0,011

1,8970±0,0255
1,8592±0,0242
1,8399±0,0087
1,8569±0,0282
0,403

c

d

Figure 3.23 A.formosanus after 12 weeks of planting under the forest
canopy ( a- Acacia forest, b- Bamboo forest, c- Natural wood forest, dNatural wood-bamboo forest)

3.5.2.2. Experimental results of A.formosanus growth under the canopy of
a mixed wood-bamboo natural forest

24
Table 3.25 Experimental results of A.formosanus growth under the
canopy of a mixed wood-bamboo natural forest
Month

Alive
percentage
(%)

Height (cm)

Diameter (mm)

Average
biomass
(g/plant)

1st month

86,7

3,4633±0,2080a

1,6600±0,0603a

Seedling
0,77 g

2nd month

83,3

3,9933±0,2245ab

1,7267±0,0533ab

3rd month

83,3

4,3900±0,2454b

1,8067±0,0484abc

4th month

83,3

4,5567±0,2367bc

1,8700±0,0651bc

5th month

80

5,0800±0,2022cd

1,9633±0,0617cd

6th month

80

5,3833±0,1277de

2,0567±0,0484de

7th month

76,7

5,6300±0,1900de

2,1467±0,0393ef

8th month

76,7

5,9667±0,1618e

2,2200±0,0702fg

9th month

73,3

6,6500±0,1563f

2,3467±0,0353g

10th month

73,3

7,2533±0,1749g

2,5133±0,0426h

11st month

73,3

8,8100±0,2100h

2,6567±0,0410hi

12nd month

73,3

10,3033±0,2551i

2,7933±0,0348i

Sig

3,08E

0,999

-17

5,34E

2,91g/plant

-14

3.5.1.3. Pests and diseases of A. formosanus under forest canopy
Table 3.26 Situation of pests and animals that damage A.formosanus
trees under the forest canopy
Component
The tree was bitten close to
the root

ID
-

Expalin
very few (<5% number of
encounters)

3.5.1.4. Results of analysis of total flavonoid content
Table 3.27 Results of analysis of total flavonoid content
Sample
A. formosanus

Sample order

Weight

Content (%)

M1
M2

2.345
2.317

0,977
1,825


25

2

3

4
5
6

Nam Dong
rare and
precious
gymnosperm

s
conservation
area

Is there a natural distribution of
Anoectochilus in the area?
How many species of
Anoectochilus are there?

Pu Hu
Nature
Reserve

1

Interview content

Pu Luong
Nature
Reserve

No.

Xuan Lien
Nature
Reserve

3.6. Proposing solutions for conservation and development of
species of the genus Anoectochilus Blume in Thanh Hoa
3.6.1. Assessment of major threats to species of the genus

Anoectochilus Blume in Thanh Hoa province
Table 3.28 Summary of interview information

Yes

Yes

Yes

Yes

A. annamensis A.calcareus Genetal called Genetal called
Aver
Aver
Anoectochilus Anoectochilus
Regular,
Regular,
In the area, do people go to the Regular, August
August August Regular, August forest to collect orchids or not, - December of
December December of December of the
what month is the time of
the lunar
of the lunar
the lunar
lunar calendar
collection?
calendar
calendar
calendar
Uprooting both Uprooting Uprooting both

What is the method of
Uprooting both big
big and small both big and big and small
harvesting?
and small trees
trees
small trees
trees
Harvested output in the period
0,4
0,5
0,2
0,5
2000-2010 (per capita/day)?
Harvested output in the period
0,1
0,2
0,1
0,2
2011-2020 (per capita/day)?
Number of cases of illegal mining
of Anoectochilus handled in
0
0
0
0
accordance with the law