MINIS TRY OF EDUCATION AND
TRAINING

VIET NAM ACADEMY OF SCIENCE
AND TECHNOLOGY

INSITITUTE OF ECOLOGY AND BIOLOGICAL RESOURCES

----------------------------PHAN XUAN BINH MINH

RESEARCH SOME SCIENCE BASIS FOR CONSERVATION,
DEVELOPMENT AND USE SUSTAIN SOME SPECIES OF Anoectochilus
Blume GENUS OF VIETNAM

Major
Code

: Plants
: 9 42 01 11

SUMMARY OF BIOLOGY DOCTORAL THESIS

HA NOI - 2019
1


Thesis completed in: Insititute of Ecology and Biological Resources -Viet Nam
Academy of Science and Technology.

Super visor 1: Assoc. Prof. Dr. Tran Minh Hoi
Super visor 2: Dr. Pham Huong Son

Phan Xuan Binh Minh, Pham Huong Son, Tran Minh Hoi, Nguyen Thi
Van, (2015), Propagation Solutions for Conservation and Development of
Anoectochilus lylei Rolfe ex Downie, Proceeding of the 6 th Nationnal
Scientific Conference on Ecology and Biological Resources, Publisher of
Natural Science and Technology : 695 – 699.
Pham Huong Sơn, Nguyen Thị Lai, Phan Xuan Binh Minh, (2015),
Isolation and simultaneous quantification ofadenosine, narcissin and
roseoside in Anoectochilus roxburghii(Wall.) Wall ex Lindl, Journal of
Chemical, Biological and Physical Sciences 5 (4): 4002 – 4007.
Tran Minh Hoi, Tran Van Thai, Chu Thi Thu Ha, Ha Thi Van Anh, Phan
Xuan Binh Minh, (2016), Flavonoids from Anoectochilus annamensis
and their Anti-inflammatory Activity, Natural Product Communications
11(5): 613 - 614.
Phan Xuan Binh Minh, Pham Huong Son, (2016), Distridution, Habitat
of Five Species of the Genus AnoectochilusBlume in Vietnam,
Proceedings The 12th Asia Pacific Orchid Conference: 110- 116
Phan Xuan Binh Minh, Pham Huong Son, Tran Minh Hoi, (2017),
Liquid Medium for Efficient Micropropagation of Anoectochilus
annamensisAver., Proceeding of the 7 th Nationnal Scientific Conference
on Ecology and Biological Resources, Publisher of Natural Science and
Technology : 1756- 1762.
Phan Xuan Binh Minh, Bui Thị Thanh Phuong, Pham Huong Son,
Nguyen Thị Phuong Lan, Vu Thi Thao, (2018), The effects of linght
emitting diode lighting on growth and development of A. annanesis and
A. roxburghii in vitro cultured shoots, Journal of Biology, 40(1): 32- 38.
DOI: 10.15625/0866-7160/v40nl. 10636

3



It is a scientific basis for the sustainable development of some species in
the Anoectochilus Blume genus in Viet Nam .
4. New points of the thesis
4


- This is a relatively complete and systematic scientific research project
(biological, ecological, distribution characteristics) of 5 valuable Anoectochilus
Blume species in Vietnam (A. annamensis, A. calareus, A. elwesii, A. lylei and A.
setaceus).
- Studying on the chemical composition of 2 species A. setaceus and
Anoectochilus annamensis and determine:
+ 8 compounds from A. Setaceus, including 2 newly added compounds,
adensine and reoside.
+ 6 compounds from A. annamensis, including 1 new compound 4’, 5dihydroxy-3,3’, 7- trimethoxyflavone 4’-O-α-L-rhamnopyranosyl- (1→6)-β-Dglucopyranoside.
- The study identified scientific bases for successful propagating of A.
annamensis, A. lylei and A. setaceus in vitro culture methods from seeds and buds.
- Proposing some initial solutions, feasible to propagate , cultivating for
conservation in relocation conditions (ex situ) combined with conservation in
place (in situ) of 3 species A. annamensis, A. lylei and A. setaceus.
- Investigating, evaluating and collecting some information on the current
status of exploitation, trading and consumption of some species Anoectocilus at 3
National Parks, 2 nature reserve and some areas have forests in some localities.
- Base on the research results, the system has proposed solutions for
effective conservation of species belonging to the Anoectochilus genus.
5. The layout of the thesis
The entire thesis consists of 156 pages, 20 drawings, 49 pictures including
parts:
- Introduction: 3 pages.
- Chapter 1: Overview of research issues: 30 pages

- The results of the study on the use of Anoectochilus species: According
to traditional medicine Anoectochilus is a very valuable medicine in the herb
shops, the medicinal plant is cool and sweet, Antipyretic, cooling blood, good for
lung, dissolving cancer, fostering health, healing organs such as hot liver, lung
disease, Haemorrhage, cough, chest pain, splenalgic, sore throat, Hypertension,
retarded children, kidney failure.
- Medicinal Value.
According to modern medicine, there are many research results indicating
that purl species contain active substances such as: flavonoid, Amino acids have
antibacterial, anti-aging, hypotension, diabetes and paralyzes the mechanism of
strange cell activity. There are currently products and active ingredients of some
Anoectochilus species such as: A. setaceus, A. formosanus… were commercial.
6


The results of the study on on propatating and cultivating: The results of
this study began in the 80s and 90s in China The results showe that the
propagation method is mainly in vitro propagation from seeds in culture medium
with microbial supplement to support the growth and development of
Anoectochilus. In addition, recent studies have used liquid propagation culture
medium systems and LED lighting systems. The cultivating method has two
methods: Cultivating in the greenhouse and cultivating under forest canopy.
1.3 The situation of research on Anoectochilus Blume in Vietnam
The results of research, survey, inventory of species composition have
been implemented by Pham Hoang Ho, Nguyen Thien Tịch, Nguyen Tien Ban
and Averyanov L. since the 90s of the last century.
The research results on chemical composition and medicinal value of
Anoectochilus are very limited. Vu Cong Luan et al (2005) published the results
of propagation and quantitative β- sitosterrol, an active ingredient used to treat
cardiovascular diseases in culture medium plants. After that, author Do Thi Gam

Binh.
2.3 The main research content of the thesis
Morphological characteristics
Anoectochilus in VietNam.

and

classification

of

species

on

Determination of the active ingredient in two species of loài A. setaceus
và A. annamensis.
Studying and investigating the actual situation of exploitation and trade of
species belonging to the genus Anoectochilus. Then giving management and
propaganda solutions for local people to understand the value of Anoectochilus
species and protect them.
Proposing solutions to conserve and sustainably develop rare species
belonging to the Anoectochilus.
+ Solution for management: Bringing noectochilus species to the list of
medicinal plants that need to be protected, proposing reasonable conservation
areas and propaganding in the community.
+ Technical solution: Research on propagation techniques by cultivating
method in vitro, cultivates 3 Anoectochilus species A. annamensis, A. setaceus, A.
lylei serving conservation and sustainable development.
2.4 Research methods

vitro culture techniques such as: Liquid culture culture, using lighting system LED
2.4.5 Method of cultivating ex vitro
9


- Method of cultivating in greenhouse: Study suitable conditions when
cultivating plants in vitro in greenhouse.
- Method of cultivating under forest canopy: Seasonal research, pests,
How to prevent when planting under the forest canopy.
2.4.6 Processing methods and statistics
Methods of processing and analyzing data on software Iristat 5.0
CHAPTER 3: RESEARCH RESULTS AND DISCUSSION
3.1 Morphological characteristics, distribution of Anoectochilus in Vietnam
3.1.1. Morphological characteristics
The genus Anoectochilus are terrestrial, perennial, deciduous, sympodial
herbs with a creeping, above-ground rhizome with wiry roots that look woolly.
The leaves black or gray-black, rarely dark gray-brown with green tint, commonly
dense reticulated with net of silvery-white nerves. The flowers are relatively large,
hairy, velvety, resupinate and arranged in a short spike. Mesochile flangeswith at
least 1–2 long linear fringes 3–5mm long . The dorsal sepal and petals overlap to
form a hood over the column with the lateral sepals spreading apart from each
other. The labellum is relatively large with two sections - an upper "epichile" and
lower "hypochile" separated by a narrow section. The hypochile has a cylindershaped spur containing two large glands and is joined to the epichile with a "claw"
that has spreading teeth or a long fringe. The fruit is a hairy capsule containing a
large number of winged seed. Plants regenerate in nature from buds and seeds.
During the survey period from 2012-2017 at 6 national parks, 5 natural
reservé and 7 points have forests in many different provinces, The team collected
and identified 5 species of the genus Anoectochilus including: A. annamensis
Aver.; A. calcareus Aver.; A. elwesii C.B. Clarke ex Hook.; A. lylei Rolfe ex
Downie0(A. setaceus Wall. ex Lindl..

Ky Anh - Nghe An
Nam Tra My-Quang Nam

A.
annamensis

A.
calcareus

National Park
+
+
+
+
+
+
+
_
Natural reserve
+
_
+
+
Other areas
+
-

A.
elwesii



+

+
+
+
+
+
+

+
+

Notes: + There is distribution in nature; - Do not see distribution in nature.
The above results show that 12 new distribution points have been
recorded for all 5 species of Anoectochilus in Vietnam compared to those of two
authors Nguyen Tien Ban (2005) and Averyanov (2009).

11


3.2. Chemical composition of two Anoectochilus Blume species (A. setaceus
Blume and A. annamensis Aver.)
3.2.1. Isolation and determination of the chemical structure of compounds in
Anoectochilus setaceus
The process of isolation and determination of chemical composition has
identified 8 compounds, There are two compounds first discovered in A. setaceus:
adenosine and roeoside and a major flavonoid compound in the species of the
Anoectochilus genus (isorhamnetin-3-rutinoside).
Table 3.2: List of substances isolated from A. setaceus


4

ARW10.1

3.5-Dihydroxy-3’4’-7-trimethocy
flavone

5

ARW11.4

Adenosine-5'
methyl sunpua

6

ARW12.2

(6S,9S)-Roseoside

7

ARW18.7

Stigmasterol

8

ARW13.7

C18H16O7
M = 344

-

C28H32O16
M = 624

-

C30H36O16
M = 652

Chất mới

5,4'-dihydroxy-3,7,3'trimethoxyflavonone

2

AAE14.18
(trùng với
ARW3.5)

Isorhamnetin 3-Orutinoside

3

AAE14.13

14


6

AAH8.15
Di-(2-ethylhexyl)
phthalate (DEHP)

3.3. Status quo of exploitation, use and trade of species belonging to the genus
Anoectochilus Blume
3.3.1. Status quo of exploitation
In the last 10 years, the demand for purchasing medicinal materials in
general and species of Anoectochilus has increased rapidly. In 11 surveyed areas,
there are bosses buying NTFP and 100% of them have purchased Anoectochilus.
According to the local people in these 11 regions, the exploitation of
Anoectochilus began in the late 90s, developed strongly in the early 2000s when
15


Chinese bosses ordered to buy. Until now, the exploitation and purchasing
continues to take place, but the number and scale are smaller due to the depleted
natural resources.
3.3.2. Status quo of use and trade and management of Anoectochilus Blume
species locally.
At the 18 sample collection sites, people have collected and traded species
belonging to the Anoectochilus genus.
In researching and surveying about understanding and value of using
species belonging to Anoectochilus genus, The research team chose 3 National
Parks, 2 Natural Reserves, 2 other forest sites are known that almost all exploited
Anoectochilus is for sale at a rate of less than 10%. The reason is that most people
as well as managers do not know or know very little about the value of genetic

propagation by in vitro culture method, especially in vitro culture from seeds is
an effective solution for many countries around the world such as: Japan, China,
Thailand ... used. In in vitro propagation there are three main stages: Shoot
induction, Shoot growth, Roor growth.
a. Shoot induction
In research, there are two main contents to find mineral culture medium
formulas, growth regulator and appropriate concentrations for each research
object.
Influence of mineral culture medium and growth regulator on the budding
ability of the A. annamensis, A. lylei
Research results show that the culture medium is suitable for regeneration
of A. annamensis is shoots cultured on MS medium supplemented 1.0 mg/l TDZ.
culture medium of A. lyle grown best in MS medium supplement with 1,5mg/l
BAP.
- Influence of mineral culture medium and growth regulators on germination
ability of A. annamensis, A. lylei, A. setaceus.
Research results of the effects of mineral medium and growth regulators
on the germination ability of three species of Anoectochilus Blume (A.
annamensis, A. lylei, A. setaceus.) is shown in Table 3.8.
Table 3.8: Influence of mineral medium and growth regulators on
germination ability A. annamensis seedlings cultured in vitro.
Concentration
Medium
TDZ (mg/l)

Rate of green sprouts
(%)
17

The average mass

73.2a

1.42b

1.5

65.6b

1.86a

2.0

58.7c

1.93a

LSD0.05

3.1

0.1

0

15.8f

0.46e

0.1


2.7

0.08

0

16.4f

0.47f

0.1

20.5e

0.72e

0.5

29.7d

0.89d

1

66.2a

1.35c

1.5



0.59e

0.5

29.2d

0.72d

1

57.4a

1.07c

1.5

51.8b

1.52b

2.0

48.4c

1.64a

LSD0.05

2.4

Effect of Culture conditions:

The result ofAnoectochilus annamensis grows best in MS Medium
supplemented with 0.5mg/l TDZ after 8 weeks in culture for mean number of
shoots per explants 9.23, Shoots grow well. As for A. lylei after 8 weeks in culture
for mean number of shoots per explants 9.12.
Using liquid medium in Shoot growth not only increases biomass, cost
savings it is also a premise for large-scale seed production processes and research
to create artificial seeds. This method has been applied on many crop objects.
Research results on three different propagating conditions: Solid medium, liquidshaked (80rpm), liquid medium, supplemented with cotton to cultivate 3 species
of A. annamensis, A. lylei, A. setaceus showed that in the liquid-shaked medium,
the mean number of shoots per explants was higher than the solid medium and the
cotton medium in all three Anoectochilus Blume species. Liquid-shaked medium
stimulates the process of shoots and roots to grow stronger and more importantly
the morphology of the tree is not changed. In addition to adapting to the liquidshaked medium conditions of the growing root for the tree stands up-right, the
diameter is larger and the stems are harder. These are also important indicators for
seedlings in vitro. Using liquid medium in In vitro propagation in general and
medicinal plants in particular to increase weight and lower product costs have
been implemented in some countries such as Japan, India ... these studies.
-

Effect of lighting conditions

In Shoot growth The determination of suitable culture medium and
external conditions is crucial to the efficiency of Shoot growth processing. In
nature, most of Anoectochilus are distributed under the evergreen forest canopy
with a canopy cover of 70-80% so these species are suitable for low light.
According to Ma ZQ et al. (2010), A. formosanaus is suitable for 30- 0 µmol / m2
/ s lux ligh. In this study, LED illumination sources are in different wavelengths of
(λ= 430-470 nm; λ= 470-510 nm; λ= 510-560 nm; λ= 560- 600 nm) with light

than those at the concentration other. But giving the best results is still in the
formula 0.5mg / l IBA with the indicators (frequency of shoots forming roots
93.33%, mean number of roots per shoot 2.64; mean leghth of roots 11.60mm). So
the most suitable concentration for A. lylei is NAA at a concentration of 0.5mg / l
with frequency of shoots forming roots 89.72%, mean number of roots per shoot
2.78, mean leghth of roots 10.76mm.
- Effect of culture Medium conditions
From the positive results when using liquid medium in the Shoot
proliferation of three species of Anoectochilus. Further research was carried out
during the root formation on medium formulas MS supplemented 0.5mg/l IBA
and MS medium supplemented 0.5mg/l NAA in different mediums: Solid medium
( supplemented 8g agar/l), cotton medium, liquid medium and Shaked at 60 rpm
for three species A. annamensis, A. lylei, A. setaceus results after 6 weeks of
culture. The above results show that for all three types of Anoectochilus Blume of
21


A. annamensis, A. lylei, A. setaceus of the liquid-shaked medium, the best results
are obtained. Liquid-shaked medium stimulated the formation and development of
roots better than static liquid environment and solid environment, moreover the
liquid-shaked environment also helped the root system to develop more evenly,
more feathers on the roots help the plants stand steadily.
3.4.1.2 Solutions for Culturing Anoectochilus
a. Culturing in greenhouse conditions
The stage of putting in vitro plants into nursery is a very important,
determine the applicability of the entire culture process in vitro to production
practices. This is also the period of many difficulties for transferring plants from
test tubes to nurseries resulting in sudden changes in living conditions from
artificial environments to natural conditions with many difficulties to control
natural factors such as: light, temperature, humidity… tested on organic mulch

At the time of December, plants had low survival rates and plants grew slowly
due to the weather conditions. So the most suitable time for planting
Anoectochilus Blume in the North is March.
- Pests harmful and prevention
As we know, the species of Anoectochilus Blume are very sensitive to
ecological environment conditions, they are suitable to live in a humid and lowlight condition, which is also an appropriate condition for the proliferation of
harmful species. When living in natural conditions they are protected by symbiotic
and reciprocal relationships. So when cultivating Anoectochilus Blume, it is
difficult to avoid effects of harmful organisms.
The studies in Hoa Binh from March 2014 to February 2015 on
Anoectochilus show that there are 4 major pest species: Vermilion spider,
snail, slug, bacterial soft rot. The survey results on the level of harm are
shown in Table 3.20
Tabe 3.20: Damage level of major pests and diseases on Anoectochilus
Blume (March 2014 - February 2015)
Popularity

Test period

Vermilion
spider

Snail

Slug

Bacterial soft rot

3/2013



6/2013

+++

+

-

++

7/2013

+++

+

-

+

8/2013

+++

+

+

+


12/2013

+

++

+

-

1/2015

-

++

+

-

2/2015

-

+

++

+

species

Sapa Medicinal Station
(National Institute of
Medicinal Materials Ministry of Health)

2

A. setaceus Blume and
A. calcareus Aver.

Tam Dao Medicinal
Station (National
Institute of Medicinal
Materials - Ministry of
Health)

3

A. setaceus Blume;

Xuan Lien Nature
reserve

2

A. annamensis Aver.
and A. elwesii

A. setaceus Blume and

garden
area