Summary of philosophy doctoral dissertation on biology: Regeneration and micropropagation of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) using thin cell layer technique
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MINISTRY OF EDUCATION
AND TRAINING
VIETNAM ACADEMY OF
SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLGY
-----------------------------
VU THI HIEN
REGENERATION AND MICROPROPAGATION
OF Panax vietnamensis Ha et Grushv.
USING THIN CELL LAYER TECHNOLOGY
Major: Plant Physiology
Code: 9.42.01.12
SUMMARY OF PHILOSOPHY DOCTORAL DISSERTATION
ON BIOLOGY
Ho Chi Minh City - 2018
The work was realized in Graduate University of Science and
Technology, Vietnam Academy of Science and Technology
Advisor 1: Prof. Duong Tan Nhut, Ph.D.
Advisor 2: Thai Xuan Du, Ph.D.
Reviewer 1:...................................................................................
Reviewer 2:...................................................................................
Reviewer 3:...................................................................................
The thesis will be evaluated by doctoral committee at Graduate
University of Science and Technology, Vietnam Academy of
Science and Technology on ………..2018
The thesis is available at:
- Library of Graduate University of Science and Technology
- National Library of Vietnam
1
INTRODUCTION
1. The necessity of the dissertation
Ngoc Linh ginseng is a Vietnamese endemic ginseng with the
scientific name Panax vietnamensis Ha et Grushv. Since discovered in
1973, it can be said that Ngoc Linh ginseng is one of the most important
medicinal plants. Many reports indicated that Ngoc Linh ginseng has not
only the pharmacological characteristics of a Ginseng, but also the
individual characteristics such as anti-stress, decrease of depression and
anxiety, stimulation of the immune system, resistance to cytotoxic toxins,
antioxidant in vitro and in vivo, etc. The success of propagation of Ngoc
Linh ginseng is still limited because this species is only grown on Ngoc
Linh mountain. To harvest gingseng roots, the propagation period lasts 6 to
7 years to store enough bioactivities. Our thesis entitled "Regeneration and
micropropagation of Ngoc Linh ginseng (Panax vietnamensis Ha et
Grushv.) using thin cell layer technique" has been carried out. The aim of
this study is to obtain a number of vigorous plantlets with and high quality
roots and tubers, especially, they are well adapted to the natural conditions,
thereby contributing to preserving this precious medicinal plant.
2. Objective
The objective of the study was to find the explant resources, the type
and concentration of plant growth regulators (PGRs), as well as the in vitro
culture conditions suitable for different morphogenesis processes - callus
induction, direct embryogenesis, shoot and root formation, etc. The growth
and development of in vitro Ngoc Linh ginseng derived from thin cell layer
(TCL) was examined in Quang Nam to assess the adaptability of in vitro
Ngoc Linh plantlets in its natural territory, compared to those growing in
Bidoup - Nui Ba National Park, Lam Dong.
3. The contents of the thesis
3.1. Research on the morphogenesis from different explant resources
3.2. Research on growth and subsequent development of plantlets in vitro
in different ecological conditions
3.3. Qualitative and quantitative saponin in plant in vitro and at nursery.
CHAPTER I
OVERVIEW
The thesis has consulted 34 Vietnamese documents and 94 English
documents and 2 internet documents; (1) Introduction to ginseng; (2) Ngoc
Linh ginseng (Panax vietnamensis Ha et Grushv.); (3) cell culture
technology; (4) factors influencing morphogenesis; (5) plant growth
regulators; (6) the role of light in the regeneration, growth and development
of plants; (7) The plant regeneration.
2
CHAPTER II
RESEARCH OBJECTIVE AND METHODS
2.1. Materials
2.1.1. Plant materials
Explant source for morphogenesis: including leaf, petiole and rhizome
of 3-month old Ngoc Linh ginseng in vitro plants; Explants grew under
different ex vitro conditions: Ngoc Linh ginseng intact plants with rhizomes
and leaves, about 3 cm in height; explant sources for determining bioactive
agents: in vitro plantlets, 6-month old, 1-year old and 2-years old plants.
2.1.2. Equipment - tools, standard chemicals and solvents
Light intensity meter LI-250A Light meter; microscope; Equipment
used in HPLC analysis (High Performance Liquid Chromatography, Rg1,
Rb1, MR2). Chloroform: methanol: water (65:35:10).
2.2. Research Methods
2.2.1. Plant morphogenesis method
2.2.2. Plant morphology and microscope observation method
2.2.4. Saponin content analysis method
2.2.4.1. Thin layer chromatography method
2.2.4.2. High Performance Liquid Chromatography (HPLC)
2.3. Research establishment methods
2.3.1. Content 1: Researching the morphogenesis from different explant
sources
2.3.1.1. Evaluating the effect of single PGRs on morphogenesis of leaf
explant tTCL_L under light and dark conditions
2.3.1.2. Evaluating the effect of single PGRs on morphogenesis of petiole
explant tTCL_L under light and dark conditions.
2.3.1.3. Evaluating the effect of single PGRs on morphogenesis of lTCL_C
petiole explant under light and dark conditions
2.3.1.4. Evaluating the effect of single PGRs on morphogenesis of TTCL_R
rhizome explant under light and dark conditions
2.3.1.5. Evaluating the effect of the combination of auxin and cytokinin on
the morphogenesis of the leaf explant tTCL_L under light and dark
conditions.
2.3.1.6. Evaluating the effect of the combination of auxin and cytokinin on
the morphogenesis of the tTCL_C petiole explant under light and dark
conditions.
2.3.1.7. Evaluating the effect of the combination of auxin and cytokinin on
the morphogenesis of the lTCL_C petiole explant under light and dark
conditions.
3
2.3.1.8. Evaluating the effect of the combination of auxin and cytokinin on
the morphogenesis of the rhizome explant tTCL_R under light and dark
conditions.
2.3.1.9. Morphological anatomy
2.3.1.10. Develop Ngoc Linh plantlets from somatic embryos
2.3.2. Research on growth and subsequent development of in vitro
plantlets under different ecological conditions
2.3.2.1. Research on the growth and development of Ngoc Linh ginseng
cultured in vitro grown in Quang Nam
2.3.2.2. Research on the growth and development of Ngoc Linh ginseng
cultured in vitro grown in the Heaven Gate of Bidoup - Nui Ba National
Park (Lam Dong)
2.3.3. Content 3: Qualitative and quantitative saponin in ginseng in vitro
and in complete ginseng at nursery stage.
2.3.3.1. Determine the content of saponin in Ngoc Linh ginseng in vitro, 6
month ginseng, 1 year and 2 year old seedlings planted in Quang Nam.
2.3.3.2. Quantification of saponins in Ngoc Linh ginseng in vitro, 6 month
ginseng, 1 year and 2 year old trees were planted in Quang Nam.
2.4. Statistics
The experiment was completely randomized (CDR). The mean of the
follow-up indices among the treatment formulas was analyzed by ANOVA
method, then compared with the Ducan test at confidence level P <0.05
using SPSS 16.0 software. [58].
2.5. Culture conditions
2.5.1. In vitro condition
2.5.2. Ex vitro condition
2.6. Location and time of the experiment
CHAPTER III
RESULTS AND DISCUSSIONS
3.1. RESULTS
3.1.1. Research on the morphogenesis from different explant sources
3.1.1.1. Effect of single PGRs on morphogenesis of leaf explant tTCL_L
under light and dark conditions
The in vitro leaf explants of TTCL_L were inoculated in culture
medium. After 10 weeks of culture, results were observed as shown in
Table 3.1; 3.2 and 3.1; 3.2.
4
Table 3.1. Effect of single PGRs on morphogenesis of leaf explant tTCL_L
in photoperiod of 16 hours/day
Embryo
genesis
(%)
Callogenes
is (%)
Root
formation
(%)
Root
numbe
r
0
0e
0d
0e
0e
0.01
0.05
0.1
0.2
0.5
1.0
0.1
0.2
0.5
1.0
2.0
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0.1
0e
0d
0e
0e
0.2
43.3 d
0d
0e
0e
0.5
70.6 b
44.4 c
0e
0e
2,4D
1.0
86.3 a
97.7 a
42.1 b
2.86 a
2,4D
2.0
49.6 c
76.6 b
9.9 d
1.06 c
0.1
0e
0d
0e
0e
0.2
0e
0d
0e
0e
0.5
0e
0d
0e
0e
NA
A
1.0
70.0 b
74.4 b
26.6 c
0.64 d
NA
A
2.0
89.0 a
97.7 a
63.3 a
2.59 b
PGRs
(mg/l)
Cont
rol
TDZ
TDZ
TDZ
TDZ
TDZ
TDZ
BA
BA
BA
BA
BA
2,4D
2,4D
2,4D
NA
A
NA
A
NA
A
Remarks
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Survive but no induction
Survive but no induction
Survive but no induction
Survive but no induction
Survive but no induction
Necrosis
A few global embryos
A few global and heart
shaped embryos
White,
brown,
and
compact callus. Embryos
cluster
White,
brown,
and
compact
callus.
Few
embryos
Necrosis
Necrosis
Necrosis
heart,
global
shaped
embryos. Few short roots.
Dark brown callus
Heart, global, cotyledon
and
torpedoe
shaped
embryos
Short white roots, dark
brown callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
5
Table 3.2. Effect of single PGRs on morphogenesis of leaf explant tTCL_L
in dark conditions
Control
TDZ
TDZ
TDZ
TDZ
TDZ
TDZ
0
0.01
0.05
0.1
0.2
0.5
1.0
0f
0f
0f
0f
0f
0f
0f
0d
0d
0d
0d
0d
0d
0d
Root
formation
(%)
0d
0d
0d
0d
0d
0d
0d
BA
0.1
0f
0d
0d
0d
BA
0.2
0f
0d
0d
0d
BA
0.5
0f
0d
0d
0d
BA
1.0
0f
0d
0d
0d
BA
2.0
0f
0d
0d
0d
2,4-D
0.1
37.6 e
0d
0d
0d
2,4-D
0.2
84.3 b
39.9 b
0d
0d
2,4-D
0.5
71.6 c
96.6 a
73.3 b
2.74b
2,4-D
1.0
92.0 a
98.8 a
79.9 a
2.87a
2,4-D
2.0
47.3 d
97.7 a
57.7 c
2.63c
NAA
NAA
NAA
NAA
0.1
0.2
0.5
1.0
0f
0f
0f
0f
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
NAA
2.0
0f
13.3 c
0d
0d
PGRs
(mg/l)
Embryogenesis
(%)
Callogenesis
(%)
Root
number
0d
0d
0d
0d
0d
0d
0d
Remarks
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Survive. no
development
Survive. no
development
Survive. no
development
Survive, no
development
Survive, no
development
Global
and
heart shaped
embryos
Global, heart
shaped, and
cotyledon
embryos
Global, heart
shaped, and
cotyledon
embryos;
white root
Global, heart
shaped, and
cotyledon
embryos;
white root
Yellow and
some while
callus;
few
embryo, white
root
Necrosis
Necrosis
Necrosis
Necrosis
yellow callus
at leaf egles
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
6
3.1.1.2. Effect of single PGRs on morphogenesis of petiole explant tTCL_C
under light and dark conditions.
After 10 weeks culturing, the morphogenicity indicators are
presented in Table 3.3 and Figure 3.3.
Table 3.3. Effect of single PGRs on morphogenesis of petiole explant
tTCL_C at photoperiod of 16 hours/day and in dark.
Embryogeneis
(%)
PGRs
(mg/l)
Callogeneis
(%)
light
Root
formation
(%)
Dark
Root
number
Contro
l
TDZ
TDZ
TDZ
TDZ
TDZ
TDZ
BA
BA
BA
BA
BA
2,4-D
2,4-D
0
0e
0e
0d
0d
0.01
0.05
0.1
0.2
0.5
1.0
0.1
0.2
0.5
1.0
2.0
0.1
0.2
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
2,4-D
0.5
0e
83.3 a
0d
0d
2,4-D
1.0
16.6 d
63.3 b
0d
0d
2,4-D
NAA
NAA
NAA
2.0
0.1
0.2
0.5
63.3 c
0e
0e
0e
33.3 d
0e
0e
0e
0d
0d
0d
31.3 c
0d
0d
0d
2.0 c
NAA
1.0
86.6 a
0e
75.5 b
6.4 b
NAA
2.0
76.6 b
46.6 c
89.9 a
15.5 a
Remarks
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Soft yellowish
callus
Soft yellowish
callus
Brown callus
Necrosis
Necrosis
White short root
Black callus.
Some white
lateral root
Yellowish
callus. A lot of
white lateral
root
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
3.1.1.3. Effect of single PGRs on morphogenesis of lTCL_C petiole explant
in light and dark conditions
The indicators recorded after 10 weeks of culture are shown in
tables 3.4, 3.5 and 3.4, 3.5.
7
Table 3.4. Effect of single PGRs on morphogenesis of lTCL_C petiole
explant at photoperiod of 16 hours/day.
Embryogenesis
(%)
Callogenesis
(%)
0e
0e
0e
0e
0f
0c
0e
0e
0f
0c
TDZ
TDZ
TDZ
TDZ
BA
BA
BA
BA
BA
2,4-D
0
0.0
1
0.0
5
0.1
0.2
0.5
1.0
0.1
0.2
0.5
1.0
2.0
0.1
Root
formation
(%)
0f
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
2,4-D
0.2
23 d
24.4 d
25.6 d
0.8 c
2,4-D
0.5
59.6 c
89.9 b
79.9 a
4.7 ab
2,4-D
1.0
86.3 a
97.7 a
85.5 a
6.2 a
2,4-D
2.0
69.6 b
98.8 a
71 b
4.0 b
NAA
NAA
NAA
0.1
0.2
0.5
0e
0e
0e
0e
0e
0e
0f
0f
0f
0c
0c
0c
NAA
1.0
58.6 c
84.4 c
10.6 e
3.1 b
NAA
2.0
84.0 a
96.6 a
37.7 c
4.0 b
PGRs
(mg/l)
Control
TDZ
TDZ
Root
number
0c
Remarks
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Few embryos. Black
compact callus
Cluster of global,
heart shaped and
cotyledon embryos
Yellow
compact
callus.
Green
elongated root
Many global, heart,
torpedo shaped and
cotyledon embryos
Brown
callus.
Yellowish roots
Heart and global
shaped embryos only
at leaf edges
Brown compact callus
Few transparent white
root
Necrosis
Necrosis
Necrosis
Large amount of
callus. Purple embryo
cluster
milky white heart and
global
shaped
embryos.
Little brown calli
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
8
Table 3.5. Effect of single PGRs on morphogenesis of lTCL_C petiole
explant in dark.
PGRs
(mg/l)
Embryogenesis
(%)
Callogenesis
(%)
Root
formation
(%)
Root
number
Contr
ol
TDZ
TDZ
TDZ
TDZ
TDZ
TDZ
BA
BA
BA
BA
BA
0
0c
0e
0g
0f
0.01
0.05
0.1
0.2
0.5
1.0
0.1
0.2
0.5
1.0
2.0
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0e
0g
0g
0g
0g
0g
0g
0g
0g
0g
0g
0g
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
2,4-D
0.1
0c
46.6 d
11.0 f
0.34 ef
2,4-D
0.2
0c
67.7 c
35.5 de
0.91 d
2,4-D
0.5
49.6 b
84.4 b
41.1 cd
0.51 e
2,4-D
1.0
69.6 a
94.4 a
46.6 c
1.87 c
2,4-D
NAA
NAA
2.0
0.1
0.2
0c
0c
0c
0e
0e
0e
NAA
0.5
0c
0e
0g
0g
0g
31.1 e
0f
0f
0f
0.94 d
NAA
1.0
0c
45.5 d
61.1 b
6.09 b
NAA
2.0
0c
81 b
94.4a
19.2 a
Remarks
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Embryogenic
calli.
Few
yellow roots
Large amount
of yellow and
white
calli.
White root
Few
global
embryos.
Brown callus.
Few
white
roots
White soft calli.
Few embryos.
Transparent
white root
Necrosis
Necrosis
Necrosis
Few
white
roots
Little
yellow
callus.
Milky
white
short roots.
Little
yellow
callus
Many
milky
white
short
roots.
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
9
3.1.1.4. Effect of the combination of auxin and cytokinin on the
morphogenesis of the rhizome explant tTCL_R in light and dark conditions.
After 10 weeks, we observe and record the indicators, the results are
shown in tables 3.6, 3.7 and Figures 3.6 and 3.7.
Table 3.6. Effect of the combination of auxin and cytokinin on the
morphogenesis of the rhizome explant tTCL_R at photoperiod of 16
hours/day.
PGRs
(mg/l)
Embryogenesis
(%)
Callogenesis
(%)
Root
formation
(%)
Root
number
Contr
ol
TDZ
TDZ
TDZ
TDZ
TDZ
TDZ
BA
BA
BA
BA
BA
0
0g
0f
0c
0c
0.01
0.05
0.1
0.2
0.5
1.0
0.1
0.2
0.5
1.0
2.0
0g
0g
0g
0g
0g
0g
0g
0g
0g
0g
0g
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
2,4-D
0.1
0g
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
75.5 c
0c
0c
2,4-D
0.2
31.0 f
87.7 b
0c
0c
2,4-D
0.5
57.3 d
89.9 b
0c
0c
2,4-D
1.0
79.6 b
97.7 a
0c
0c
2,4-D
2.0
91.0 a
88.8 b
0c
0c
NAA
0.1
0g
0f
0c
0c
NAA
0.2
71.0 c
0f
0c
0c
Remarks
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Little white
callus
Large
amount
white callus. Few
global
shaped
embryos.
Large amount of
brown calli.
Cluster of global,
and heart shaped
embryos
Large amount of
dark brown calli.
Global,
heart
shaped
and
cotyledon
embryos.
Large amount of
dark brown calli.
Many
global,
heart shaped and
cotyledon
embryos.
Necrosis
Embryo shaped
global, heart,
cotyledon,
torpedo
10
NAA
0.5
70.6 c
0f
0c
0c
NAA
1.0
48.6 e
44.4 e
74.4 b
5.09 b
NAA
2.0
47.3 e
67.7 d
83.3 a
9.24 a
Global, heart,
torpedo shaped
and cotyledon
embryos.
Global and heart
shaped embryos.
Few white roots
Global shaped
and cotyledon
embryos.
Yellowish callus.
Short white roots.
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.7. Effect of the combination of auxin and cytokinin on the
morphogenesis of the rhizome explant tTCL_R in dark.
Control
TDZ
TDZ
TDZ
TDZ
TDZ
TDZ
BA
BA
BA
BA
BA
0
0.01
0.05
0.1
0.2
0.5
1.0
0.1
0.2
0.5
1.0
2.0
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
Root
formation
(%)
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
0d
2,4-D
0.1
56.3 b
41.1 c
0d
0c
2,4-D
0.2
84.0 a
61 b
24.4 c
0.93 b
2,4-D
0.5
46.3 c
91 a
27.7 c
0.69 bc
2,4-D
1.0
0d
95.5 a
44.4 b
0.58 bc
2,4-D
2.0
0d
31 d
0d
0c
NAA
0.1
0d
0f
PGRs
(mg/l)
NAA
0.2
Embryogenesis
(%)
Callogenesis
(%)
0d
0f
Root
number
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0c
0d
0c
28.2 c
1.28 b
Remarks
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Global shaped
embryos.
Yellowish
callus.
Global,
heart
shaped,
and
cotyledon
embryos.
Yellow callus
Few embryo
Large amount
of yellow calli
Brown compact
callus
Few white roots
Yellow callus
Explants turned
yellow
Short
white
roots
11
NAA
0.5
0d
24.4 e
NAA
1.0
0d
0f
NAA
2.0
0d
0f
48.8 b
52.2 b
0.59 bc
1.37 b
98.8 a
21.7 a
Few roots
Few
yellow
short roots
Many
white
roots and lateral
roots
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
3.1.1.5. Effect of the combination of auxin and cytokinin on the
morphogenesis of the leaf explants tTCL_L in light and dark conditions
The results obtained after 10 weeks of culture are shown in tables
3.8, 3.9, 3.10, 3.11, 3.12, 3.13; Figures 3.8, 3.9, 3.10.
Table 3.8. Effect of the combination of 2,4-D and BA on the
morphogenesis of the leaf explants tTCL_L at photoperiod of 16 hours/day.
PGRs
(mg/l)
Morphogenesis
Remarks
2,4-D
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
BA
0.1
0.2
0.5
1.0
2.0
1.0
1.0
1.0
Callogenesis (%)
100 a
100 a
100 a
93.3 b
90 c
47.7 f
60.0 e
80.0 d
2.0
1.0
100 a
White green compact callus
Many greenis white callus
Little yellow and milky white soft callus
Little yellowis green compact callus
Light yellow soft callus
Little yellowish brown callus
Little green and brown compact callus
Little brown compact callus
Large amount of yellow and milky white
soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.9. Effect of the combination of 2,4-D and BA on the
morphogenesis of the leaf explants tTCL_L in dark
PGRs
Morphogenesis
(mg/l)
Remarks
2,4-D
BA
Callogenesis
(%)
1.0
1.0
0.1
0.2
100 a
100 a
Root
formation
(%)
27.3 a
25.5 b
1.0
0.5
100 a
0c
1.0
1.0
0.1
1.0
2.0
1.0
100 a
93.3 b
73.3 d
0c
0c
0c
Little yellow and milky white soft callus
Large amount of milky white soft callus
Large amount of transparent and milky
white soft callus
White and milky white soft callus
Brownish yellow soft callus
Brownish yellow compact callus
12
0.2
1.0
80.0 c
0c
0.5
2.0
1.0
1.0
93.3 b
93.3 b
0c
0c
Little brownish yellow and transparent
compact callus
White and milky white soft callus
White and brownish red soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.10. Effect of the combination of 2,4-D and TDZ on the
morphogenesis of the leaf explants tTCL_L at photoperiod of 16 hours/day.
PGRs
Morphogenesis
(mg/l)
Remarks
2,4-D
TDZ
1.0
1.0
0.01
0.05
Callogenesis
(%)
100 a
100 a
1.0
0.1
100 a
1.0
1.0
1.0
0.1
0.2
0.5
2.0
0.2
0.5
1.0
0.2
0.2
0.2
0.2
100 a
100 a
100 a
80 b
80 b
100 a
73.3 c
White and yellow soft callus
Greenish white and brownish yellow compact callus
Large amount of greenish white and redish yellow
compact callus
Little greenish white and yellow soft callus
Little greenish white and redish yellow compact callus
White and yellow soft callus
Little greenish white and redish yellow compact callus
Little green compact callus
White and brown soft callus
Brownish yellow soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.11. Effect of the combination of 2,4-D and TDZ on the explants
tTCL_L in dark.
PGRs
(mg/l)
2,4-D
1.0
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
2.0
Morphogenesis
TDZ
0.01
0.05
0.1
0.2
0.5
1.0
0.2
0.2
0.2
0.2
Callogenesis (%)
100 a
100 a
100 a
100 a
100 a
86.6 c
80.0 d
93.3 b
93.3 b
0e
Morphogenesis of the leaf Remarks
Brownish yellow soft callus
Little yellow and milky white soft callus
Large amount of milky white soft callus
Little white and brownish yellow soft callus
Transparent white and milky white soft callus
Little milky white and brownish yellow soft callus
Little transparent white and brown soft callus
White and brown soft callus
Little milky white and brownish yellow soft callus
Necrosis
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
13
Table 3.12. Effect of the combination of NAA and BA on the
morphogenesis of the leaf explants tTCL_L at photoperiod of 16 hours/day.
PGRs
(mg/l)
NAA
BA
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
2.0
0.1
0.2
0.5
1.0
2.0
1.0
1.0
1.0
1.0
Morphogenesi
s
Callogenesis
(%)
13.3 c
0d
0d
33.3 b
0d
0d
0d
0d
60 a
Remarks
Little green compact callus
Necrosis
Necrosis
Little brown compact callus
Survival but no development
Necrosis
Necrosis
Necrosis
Little green compact callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.13. Effect of the combination of NAA and BA on the
morphogenesis of the leaf explants tTCL_L in dark.
PGRs
(mg/l)
Morphogenesis
NAA
BA
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
2.0
0.1
0.2
0.5
1.0
2.0
1.0
1.0
1.0
1.0
Callogenesis
(%)
40 e
33.3 f
53.3d
93.3 b
13.3 g
0h
0h
90.0 c
100 a
Remarks
Little transparent white and brownish yellow callus
Little brown compact callus
Little brown compact callus
White and brownish yellow soft callus
Little brownish yellow compact callus
Survival but no development
Necrosis
Little greenish white and brownish yellow soft callus
White and brown soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
3.1.1.6. Effect of the combination of auxin and cytokinin on the
morphogenesis of the tTCL_C petiole explant in light and dark conditions.
After 10 weeks of culture, the results were observed, recorded and
compared the effect of each pair of 2,4-D in combination with BA, 2,4-D in
combination with TDZ and BA in combination with NAA. present in tables
3.14, 3.15, 3.16, 3.17, 3.18, 3.19 and Figures 3.11, 3.12, 3.13.
14
Table 3.14. Effect of the combination of 2,4-D and BA on the
morphogenesis of the tTCL_C petiole explant at photoperiod of 16
hours/day
PGRs
(mg/l)
Morphogenesis
2,4-D
BA
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
2.0
0.1
0.2
0.5
1.0
2.0
1.0
1.0
1.0
1.0
Callogenesis
(%)
98.8 a
97.7 a
98.8 a
97.7 a
74.4 c
96.6 a
98.8 a
98.8 a
86.6 b
Remarks
Little green and brownish yellow soft callus
Yellowish white soft callus
Yellow soft callus
Milky white, green, and brown compact callus
Little light yellow compact callus
Little green compact callus
Green and white compact callus
Large amount of green and purple compact callus
Little yellow soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.15. Effect of the combination of 2,4-D and BA on the
morphogenesis of the tTCL_C petiole explant in dark.
PGRs
(mg/l)
Morphogenesis
2,4-D
BA
1.0
1.0
1.0
1.0
1.0
0.2
0.5
2.0
0.1
0.2
0.5
1.0
2.0
1.0
1.0
1.0
Callogenesis
(%)
97.7 a
98.8 a
97.7 a
97.7 a
98.8 a
96.6 a
97.7 a
96.6 a
Remarks
Light yellow soft callus
Large amount of yellow and milky white soft callus
Milky white and light yellow soft callus
Little light brown and light yellow soft callus
Low light brown and light yellow soft callus
Yellow brown soft callus
Milky white and light yellow soft callus
Little yellow soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.16. Effect of the combination of 2,4-D and TDZ on the
morphogenesis of the tTCL_C petiole explant at photoperiod of 16
hours/day
PGRs
(mg/l)
Morphogenesis
2,4-D
TDZ
1.0
1.0
1.0
1.0
1.0
0.01
0.05
0.1
0.2
0.5
Callogenesis
(%)
100 a
100 a
100 a
100 a
100 a
Remarks
Green and white soft callus
Dark yellow soft callus
Large amount of yellow and milky white soft callus
Little yellow and milky white soft callus
Little green, brown, and white soft callus
15
1.0
0.1
0.2
0.5
2.0
1.0
0.2
0.2
0.2
0.2
100 a
100 a
86.6 b
100 a
0c
Little yellow and milky white soft callus
Small green compact callus
Little green, and brownish yellow compact callus
Little yellow and milky white soft callus
Necrosis
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.17. Effect of the combination of 2,4-D and TDZ on the
morphogenesis of the tTCL_C petiole explant in dark.
PGRs
(mg/l)
Morphogenesis
2,4-D
TDZ
1.0
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
2.0
0.01
0.05
0.1
0.2
0.5
1.0
0.2
0.2
0.2
0.2
Callogenesis
(%)
100 a
100 a
100 a
100 a
100 a
100 a
93.3 c
93.3 c
79.9 c
100 a
Remarks
Transparent white and brownish yellow callus
Brownish yellow soft callus
Large amount of yellow and milky white soft callus
Transparent yellow soft callus
Brownish redish and yellow soft callus
Brownish red soft callus
Little brown compact callus
Little yellow soft callus
White and yellow soft callus
Little milky white soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.18. Effect of the combination of NAA and BA on the
morphogenesis of the tTCL_C petiole explant at photoperiod of 16
hours/day.
PGRs (mg/l)
NAA
BA
1.0
0.1
1.0
0.2
1.0
0.5
1.0
1.0
1.0
2.0
0.1
1.0
0.2
1.0
0.5
1.0
2.0
1.0
Morphogenesis
Callogenesis (%)
100 a
100 a
100 a
100 a
100 a
0c
0c
0c
86.6 b
Remarks
Necrosis
Necrosis
Large amount of yellow and white compact callus
Yellow and white compact callus
Brownish yellow compact callus
Necrosis
Necrosis
Necrosis
Little brown compact callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
16
Table 3.19. Effect of the combination of NAA and BA on the
morphogenesis of the tTCL_C petiole explant in dark.
PGRs
(mg/l)
Morphogenesis
NAA
BA
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
2.0
0.1
0.2
0.5
1.0
2.0
1.0
1.0
1.0
1.0
Callogenesis
(%)
100 a
100 a
100 a
100 a
100 a
0b
100 a
100 a
100 a
Remarks
Very little brownish yellow soft callus
Little yellow and milky white soft callus
Large amount dark yellow soft callus
Large amount dark yellow soft callus
Large amount dark yellow soft callus
Necrosis
Large amount dark yellow soft callus
Large amount dark yellow soft callus
Large amount dark yellow soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
3.1.1.7. Effect of the combination of auxin and cytokinin on the
morphogenesis of the lTCL_C petiole explants in light and dark conditions.
The results recorded after 10 weeks of culture shown in tables 3.20 to 3.25
and Figures 3.14, 3.15, 3.16.
Table 3.20. Effect of the combination of 2,4-D and BA on the
morphogenesis of the lTCL_C petiole explants at photoperiod of 16
hours/day
PGRs (mg/l)
Morphogenesis
Embryogene
sis (%)
Remarks
0.1
0.2
0.5
1.0
2.0
1.0
1.0
1.0
Callog
enesis
(%)
100 a
93.3 b
86.6 c
66.7 d
13.3 e
86.6 c
100 a
93.3 b
24.4 c
48.8 a
31.0 b
0d
0d
0d
0d
0d
1.0
100 a
0d
Little brownish red, green and yellow compact callus
Red and milky white soft callus
Little white, green and red callus
Red, brownish yellow, and light green callus
Little light yellow, and red callus
Very tittle callus
Little red brown compact callus
Green, and yellow callus on cut surface
Little yellow and milky white soft callus on explant
surface and two cut surfaces
2,4D
BA
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
2.0
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
17
Table 3.21. Effect of the combination of 2.4-D and BA on the
morphogenesis of the lTCL_C petiole explants in dark
PGRs
(mg/l)
Morphogenesis
Shoot
Callogen
formation
esis (%)
(%)
100 a
0b
100 a
21.0a
100 a
0b
100 a
0b
100 a
0b
46.7 d
0b
2,4D
BA
1.0
1.0
1.0
1.0
1.0
0.1
0.1
0.2
0.5
1.0
2.0
1.0
0.2
1.0
80.0 c
0b
0.5
1.0
93.3 b
0b
2.0
1.0
100 a
0b
Remarks
Brownish yellow, and milky white soft callus
Large amount of milky white soft callus
Milky white soft callus
Little yellow and milky white soft callus
Light yellow soft callus
Little brownish red compact callus
Little yellow and brown compact callus on surface and two
cut explants
Milk white soft callus on explant surface and two cut
surfaces
Little yellow and brown soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.22. Effect of the combination of 2.4-D and TDZ on the
morphogenesis of the lTCL_C petiole explants at photoperiod of 16
hours/day
PGRs (mg/l)
Morphogenesis
2,4-D
TDZ
Callogenesis
(%)
1.0
0.01
86.6 c
1.0
0.05
93.3 b
1.0
0.1
93.3 b
1.0
0.2
100 a
1.0
1.0
0.1
0.2
0.5
2.0
0.5
1.0
0.2
0.2
0.2
0.2
100 a
93.3 b
73.3 d
73.3 d
93.3 b
0e
Remarks
Little milky white, and transparent white soft callus on one
cut surface
Little milky white soft callus on explant surface and two cut
surfaces
Greenish white compact callus
Large amount of milky white soft callus on explant surface
and two cut surfaces
Greenish white compact callus on one cut surface
Brownish red and milky white soft callus on two cut surfaces
Little green compact callus on one cut surface
Little milky white soft callus on one cut surface
Large amount of milky white soft callus on one cut surface
Necrosis
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
18
Table 3.23. Effect of the combination of 2.4-D and TDZ on the
morphogenesis of the lTCL_C petiole explants in dark
PGRs (mg/l)
Morphogenesis
2,4-D
TDZ
Callogenesis (%)
1.0
0.01
93.3 b
1.0
0.05
100 a
1.0
0.1
100 a
1.0
0.2
100 a
1.0
0.5
93.3 b
1.0
1.0
86.6 c
0.1
0.2
0e
0.2
0.2
80.0 d
0.5
0.2
100 a
2.0
0.2
86.6 c
Remarks
Milky white, and brown soft callus on one cut surface
Little yellow and milky white soft callus on one cut
surface
Large amount of transparent and milky white soft callus
on two cut surfaces
Milky white and brownish yellow soft callus
Large amount of yellow and milky white soft callus on
one cut surface
Little milky white and yellow soft callus on two cut
surfaces
Survival but no development
White and brownish yellow soft callus on explant
surface and two cut surfaces
White and brownish yellow soft callus on explant
surface and two cut surfaces
Little brownish yellow soft callus on one cut surfaces
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.24. Effect of the combination of NAA and BA on the
morphogenesis of the lTCL_C petiole explants at photoperiod of 16
hours/day
PGRs (mg/l)
Morphogenesis
Remarks
NAA
BA
Callogenesis
(%)
1.0
0.1
60.0 c
Little brownish green compact callus on two cut surfaces
1.0
0.2
53.3 d
Little brownish green compact callus on one cut surface
1.0
0.5
13.3 f
Little brown compact callus
1.0
1.0
46.6 e
Little brown compact callus on two cut surfaces
1.0
2.0
80.0 b
Little green and brown callus on explant surface and two
cut surfaces
0.1
1.0
0g
Necrosis
0.2
1.0
0g
Necrosis
0.5
1.0
0g
2.0
1.0
93.3 a
Necrosis
Little white and brown callus on explant surface and one
cut surfaces
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
19
Table 3.25. Effect of the combination of NAA and BA on the
morphogenesis of the lTCL_C petiole explants in dark
PGRs (mg/l)
Morphogenesis
NAA
BA
Callog
enesis
(%)
1.0
0.1
1.0
Adventitious root
Remarks
Root
formation
(%)
Number
of roots
86.6 c
80.0 b
1.1 b
0.2
100 a
93.3 a
3.9 a
1.0
0.5
93.3 b
7.7 c
0c
1.0
1.0
86.6 c
0d
0c
1.0
2.0
86.6 c
0d
0c
0.1
1.0
0d
0d
0c
Brownish yellow soft callus on one cut
surface
Necrosis
0.2
1.0
0d
0d
0c
Necrosis
0.5
1.0
0d
0d
0c
2.0
1.0
100 a
0d
0c
Survival but no development
Little milky white soft callus on explant
surface and two cut surfaces
Long white root
Large amount of brownish yellow soft
callus on explant surface and one cut
surface. Long white root
Little yellow and brown soft callus on
explant surface and two cut surfaces
Long white root
Little yellow and white soft callus
Long white root
Little yellow soft callus on one cut surface
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
3.1.1.8. Effect of the combination of auxin and cytokinin on the
morphogenesis of the tTCL_R rhizome explants in light and dark
conditions.
After 10 culture weeks, results are shown in Tables 3.26 to 3.31 and
Figures 3.17, 3.18, 3.19.
Table 3.26. Effect of the combination of 2.4-D and BA on the
morphogenesis of the tTCL_R rhizome explants at photoperiod of 16
hours/day
PGRs (mg/l)
2,4-D
BA
1.0
0.1
1.0
0.2
1.0
0.5
Morphogenesis
Callogenesis (%)
100 a
100 a
100 a
Remarks
Large amount of white and brownish yellow compact
callus
Large amount of white and brownish yellow compact
callus
Large amount of milky white soft callus
20
1.0
1.0
0.1
0.2
0.5
2.0
1.0
2.0
1.0
1.0
1.0
1.0
100 a
100 a
34.4 e
66.6 c
50 d
72.2 b
Large amount of milky white soft callus
Little light yellow and greenish white soft callus
Little brown compact callus
Little brown compact callus
Little brownish yellow soft callus
Little milky white and brownish yellow compact callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.27. Effect of the combination of 2.4-D and BA on the
morphogenesis of the tTCL_R rhizome explants in dark.
PGRs (mg/l)
2,4-D
BA
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
2.0
0.1
0.2
0.5
1.0
2.0
1.0
1.0
1.0
1.0
Morphogenesi
s
Callogenesis
(%)
100 a
100 a
100 a
100 a
100 a
13,3 e
73.3 d
83.3 c
86.6 b
Remarks
Large amount of white and yellow compact callus
Large amount of milky white and yellow compact callus
Large amount of milky white friable callus
Large amount of milky white friable callus
Little milky white and brownish yellow friable callus
Little yellow compact callus
Little brown compact callus
Little milky white and brownish yellow compact callus
Little milky white and yellow compact callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.28. Effect of the combination of 2.4-D and TDZ on the
morphogenesis of the tTCL_R rhizome explants at photoperiod of 16
hours/day
PGRs (mg/l)
2,4-D
1.0
1.0
1.0
1.0
1.0
1.0
Morphogenesis
Callogenesis Shoot
TDZ
(%)
formation
(%)
0.01
100 a
0b
0.05
100 a
0b
0.1
100 a
0b
0.2
100 a
0b
0.5
86.6 b
0b
1.0
86.6 b
0b
0.1
0.2
33.3 d
42 a
0.2
0.2
33.3 d
42 a
0.5
2.0
0.2
0.2
86.7 b
80.0 c
0b
0b
Remarks
White callus
Little white and brownish yellow soft callus
Large amount of greenish white callus
Little yellow and milky white soft callus
White and brown soft callus
Brownish yellow and greenish white soft callus
Some black explants, few greenish white shoots with 3
leaves per shoot
Some black explants, few greenish white shoots with 3
leaves per shoot
Milky white friable callus with brown explant edges
Milky white and yellow compact callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
21
Table 3.29. Effect of the combination of 2.4-D and TDZ on the
morphogenesis of the tTCL_R rhizome explants in dark
PGRs (mg/l)
2,4-D
TDZ
1.0
1.0
1.0
1.0
1.0
1.0
0.1
0.2
0.5
2.0
0.01
0.05
0.1
0.2
0.5
1.0
0.2
0.2
0.2
0.2
Morphogenesis
Callogenesis
(%)
100 a
100 a
100 a
100 a
93.3 b
80.0 c
33.3 d
80.0 c
93.3 b
93.3 b
Remarks
Large amount of milky white and yellow soft callus
Large amount of yellow and milky white soft callus
Milky white and yellow soft callus
Brown yellow soft callus
Little white and brownish yellow soft callus
Little white and brownish yellow soft callus
Little yellow compact callus
Little yellow soft callus
Large amount of yellow and milky white soft callus
Little milky white soft callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.30. Effect of the combination of NAA and BA on the
morphogenesis of the tTCL_R rhizome explants at photoperiod of 16
hours/day
PGRs (mg/l)
NAA
BA
1.0
0.1
Morphogenesis
Callogenesis (%)
100 a
1.0
0.2
86.6 b
1.0
1.0
1.0
0.1
0.2
0.5
2.0
0.5
1.0
2.0
1.0
1.0
1.0
1.0
86.6 b
80.0 c
50 e
0f
0f
0f
73.3 d
Remarks
Large amount of greenish white callus
Large amount of yellow, green, and white compact callus
at edges of explants
Little green compact callus
Little brownish green compact callus
Little black and white compact callus
Necrosis
Necrosis
Necrosis
Little greenish yellow compact callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
Table 3.31. Effect of the combination of NAA and BA on the
morphogenesis of the tTCL_R rhizome explants in dark
PGRs (mg/l)
NAA
BA
1.0
0.1
1.0
0.2
1.0
0.5
1.0
1.0
1.0
2.0
0.1
1.0
0.2
1.0
0.5
1.0
2.0
1.0
Morphogenesis
Callogenesis (%)
100 a
86.7 c
80.0 d
80.0 d
73.3 e
0g
0g
33.3 f
93.3 b
Remarks
Little yellow and white soft callus
Little brown soft callus
Little white soft callus with brown edges
Little white, yellow, brown soft callus
Yellow and brown callus
Necrosis
Necrosis
Little dark brown callus
Yellow and brown compact callus
* Different letters within a column indicate significant differences at
P<0.05 by Duncan’s multiple range tests.
22
3.1.1.9. Observation of morphological change of Ngoc Linh ginseng
somatic embryo
The results of anatomy and imaging of cotyledonnic developmental
shapes show that the cotyledon obtained from the treatments did not differ
in morphology and that it existed in the major forms of spherical, haunted
and dicotyledonous.
3.1.1.10. Create a complete and functional plant from somatic embryo
After 8 weeks of culture, all plants induced tubers, leaves uniformly
developed, morphologically normal and structured similar to those grown
in the wild.
3.1.2. Study the growth and development of seedlings in vitro in different
ecological conditions
3.1.2.1. The growth and development of Ngoc Linh ginseng cultured in
vitro was planted in Quang Nam
Table 3.33. Survival, growth and development of Ngoc Linh ginseng in
vitro at nursery stage in Ngoc Linh mountain area, Quang Nam province
Evaluation time
6 months after planting
12 months after planting
18 months after planting
24 months after planting
Survival rate
(%)
93
91
90
90
Germination
rate (%)
66
78
87
87
New rhizome
rate (%)
20
45
70
100
Note: The percentage of new leaf growth and tuber formation is based on
the survival rate of the plant
3.1.2.2. Research on the growth and development of Ngoc Linh ginseng
cultured in vitro in the Gate Area of Heaven, Bi Doup Mountain National
Park, Lam Dong province
Table 3.34. Survival and growth performance of Ngoc Linh ginseng in
vitro at the Heaven Gate of Bidoup Mountain National Park - Lam Dong
province
Evaluation time
6 months after planting
12 months after planting
18 months after planting
24 months after planting
Survival rate
(%)
70
60
50
50
Germination rate
(%)
30
40
50
50
New rhizome
rate (%)
0
20
30
50
Note: The percentage of new leaf growth and tuber formation is based on
the survival rate of the plant
23
3.1.3. Quantification of saponins in vitro and ex vitro in Ngoc Linh
ginseng
3.1.3.1. Quantification of saponins in vitro in Ngoc Linh ginseng after 6
months, 12 months and 24 months
The thin layer chromatographic analysis revealed that samples of
Ngoc Linh ginseng were derived from thin cell layer culture (in vitro
ginseng, 6-month-old ginseng, 1-year-old and 2-year-old ginseng) There
are three saponins Rg1, Rb1, MR2 when the plates run on chloroform:
methanol: water = 65:35:10. This proves that in vitro and ex vitro cultures
of Ngoc Linh ginseng have full presence of all three types of Rg1, Rb1,
MR2. Thus, the qualitative results show that the samples contained
saponins.
3.1.3.2. Quantification of saponins in vitro Ngoc Linh ginseng after 6
months, 12 months and 24 months
Table 3.35. Saponin content of in vitro and ex vitro Ngoc Linh ginseng
Samples
1 (in vitro plants)
Rg1 (%)
0.68
Rb1 (%)
0.51
MR2 (%)
0.97
Total (%)
2.17
2 (6-month-old plants)
0.11
0.11
0.47
0.70
3 (1-year-old plants)
0.96
0.85
0.87
2.69
4 (2-year-old plants)
1.02
1.74
0.77
3.54
CONCLUSION AND RECOMMENDATION
1. Conclusion
The leaf sample tTCL_L gave the highest embryogenesis (89.6%, 29
cotyledonary emgryos per explant) when explants were cultured on
medium supplemented with 2 mg/l NAA under 16-hour light conditions.
lTCL_C, tTCL_L were cultured in 1.0 and 2.0 mg/l 2,4-D
supplementation in light and dark conditions for the highest callogenesis
rate (97,98%).
tTCL_R cultured on medium supplemented with 2 mg/l NAA in dark
conditions resulted in rooting rate of 98.8% and 21.7 roots / sample.
The highest shoot regeneration rate of 42% was found when tTCL_R
was cultured on medium supplemented with 0.2 mg/l TDZ in combination
with 0.2 mg/l 2,4-D under light conditions.
tTCL_L, tTCL_C and lTCL_C, and tTCL_R were cultured on medium
supplemented with 2,4-D and BA or TDZ at different concentrations under
light conditions and in the darkness gave high callogenesis rate.
The embryos obtained in all treatments showed not to be different in
they are in global, heart, torpedo shapes and dicotyledons.
