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INTRODUCTION

1. Introduction
Talinum paniculatum is a herbaceous plant known for its high
medicinal value. Studies on the chemical composition of Talinum
paniculatum show that in leaves and roots contain bioactive substances
such as alkaloid, flavonoid, saponin, tannin, phytosterol, phytol; the
content of phytol is very high (69,32 %). Since ancient time, T.
paniculatum has been used in traditional medicine, especially in the
treatment of type 2 diabetes, dermatitis, gastrointestinal disorders,
physiological weakness and reproductive disorders. Galactogue in leaves
has anti-inflammatory effects, during the maternity has increased the use
in stimulation of lactation, and has the ability to treat these ulcers. The
roots of T. paniculatum are used to promote fertility and treat
gynecological diseases such as menstrual cycle abnormalities... Steroid
saponin in the roots of Talinum paniculatum has the effect of preventing
and treating arteriosclerosis, and is also a raw material to synthesize sex
hormones.
Flavonoids play important roles in human health such as having
an antioxidant effect, having hepatoprotective activities, antiinflammatory, anticancer, antibacterial... However, no studies have been
conducted to acquire flavonoids in T. paniculatum because of the
flavonoid content of species in the Talinum genus, including T.
paniculatum very low. The problem is how to improve the content of
flavonoids of the Talinum genus in general and T. paniculatum in
particular to be able to be used in public health care.
So far, there have been a number of major approaches applied to
medicinal plants to increase flavonoid content. It is to use selective
methods from populations or experimental hybrid or mutant mutations,

Therefore a method has been proposed for enhancing flavonoid content
in Talinum paniculatum plants by applying tissue culture technique to
produce the hairy roots to enhance biomass. When plant tissue (leaves,
lateral shoot bud, cotyledons ...) is infected with Agrobacterium
rhizogenes, T-DNA in Ri-plasmid structure carries rol genes and auxincoding genes of IAA type to be transferred into plant tissue.
Simultaneous expression of rol genes and auxin synthesis genes will
result in a pattern of hairy roots in plant tissue that are infected with A.
rhizogenes.
For the above reasons, we conducted the research entitled “A
study on the expression of GmCHI gene involved in flavonoid
synthesis and hairy root induction of Talinum paniculatum plants”.
2. Aims of the study
Creating GmCHI transgenic T. paniculatum lines has higher
flavonoid content than wild-type plants and determine suitable
conditions in induction of hairy roots in vitro of T. paniculatum.

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3. Objectives of the study
(i) Study on identification of T. paniculatum samples collected in some
localities by comparative morphological method, combined with
molecular classification methods based on some DNA barcodes (ITS
region; partial sequences of matK, rpoB, rpoC1).
(ii) Study on GmCHI gene transfer and creation of transgenic T.
paniculatum lines. Analysis of the recombinant CHI protein expression


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transgenic plants to improve the pharmaceutical content in T.
paniculatum and some other medicinal plants.
Practical significance, the hairy roots and the transgenic T.
paniculatum lines provide materials for selecting T. paniculatum
varieties with high flavonoid content. The results of the study have
opened up the future prospects for applying to create hairy roots and
overexpression techniques in improving pharmaceutical content in
medicinal plants.
6. Dissertation structure
The dissertation has 129 pages (including appendix) and is divided into
chapters and sections: Introduction (04 pages); Chapter 1: Literature
Review (37 pages); Chapter 2: Materials and Research Methods (16
pages); Chapter 3: Results and Discussion (41 pages); Conclusions and
Recommendations (02 page); Publications related to the dissertation (03
page); References (14 pages); Appendix (12 pages). The dissertation has
16 tables, 33 figures and 131 references.
Chapter 1. LITERATURE REVIEW
The dissertation has 131 references, including 6 references in
Vietnamese; 125 references in English to summarize the relevant
content, including: (1) Studies on in vitro culture of Talinum
paniculatum plants; (2) Flavonoids and the pathway of flavonoid
biosynthesis in plants, (3) CHI and expression of gene encoding CHI.
Talinum paniculatum (T. paniculatum) plants contain flavonoids
and saponins, which have strong antioxidant properties and are used in
the treatment of numerous diseases, such as inflammation, allergies, and

rhizogenes. In Vietnam, there have been no published reports on creating
and cloning hairy root lines in Talinum paniculatum plants. Other the
effective approach to enhance the flavonoid content in T. paniculatum
plants is overexpression of the chalcone isomerase gene in the
phenylpropanoid biosynthesis pathway. In the world, there have been a
number of studies on overexpression of CHI genes in tomato plants
(Muir et al., 2001), tobacco (Li et al., 2006), peony (Lin et al., 2014)...
Results of total flavonoid, flavonol and anthocyanin content increased
many fold compared to non-transgenic control plants. Currently, there is
no research work that transferred CHI gene into T. paniculatum.Thus,
the application of technology to improve flavonoid content of T.
paniculatum by overexpressing the CHI gene that encodes the key
enzyme of flavonoid synthesis should be considered and focused on
research.
Chapter 2. MATERIALS AND RESEARCH METHODS
2.1. RESEARCH MATERIALS
Seeds and samples of T. paniculatum collected from September
2015 to March 2016 in 5 localities: Tan Yen district, Bac Giang province
(BG); Thai Nguyen city (TN1); Dai Tu district, Thai Nguyên province

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(TN2); Son Tay town, Ha Noi capital (HT); Hoanh Bo district, Quang
Ninh province (QN).
The seeds of T. paniculatum were cultivated and analyzed on


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paniculatum plants were performed followed the method previously
described by Olhoft et al. (2006).
2.3.4. Methods of analysing transgenic plants: Determine the presence
and incorporation of GmCHI gene into the host genomes of transgenic T.
paniculatum plants using PCR and Southern blot of Southern (1975).
Analysis of recombinant CHI protein expression in transgenic plants by
Western blot and ELISA was performed according to the method of Sun
et al. (2006). Determination of total flavonoid content in T. paniculatum
by absorption spectroscopy method were performed followed the
method previously described by Kalita et al. (2013).
2.3.5. Methods of analysis and data processing: The data in the study
were statistically processed by SPSS software to determine average
values, variance, standard deviation, average sample error.
Chapter 3. RESULTS AND DISCUSSION
3.1. RESULTS OF IDENTIFYING OF T. PANICULATUM SAMPLES
3.1.1. Morphological characteristics of T. paniculatum samples
collected in some localities
The results of comparing the five T. paniculatum samples collected from
the localities (Tan Yen district, Bac Giang province; Thai Nguyen city;
Dai Tu district, Thai Nguyên province; Son Tay town, Ha Noi capital;
Hoanh Bo district, Quang Ninh province) show that samples of T.
paniculatum are similar in morphology, including roots, stems, leaves

DNA barcoding method can be used to accurately identify the T.
paniculatum samples without confusion with other herbs.
3.1.2. Characteristics of the ITS region and partial sequences of
matK
3.1.2.1. Characteristics of the ITS region
The results of testing PCR products by electrophoresis on 1%
agarose gel with 1 kb DNA ladder are shown in figure 3.2. The results
showed that the PCR products of all samples obtained a DNA band of
about 600 bp in size, which was similar to the predicted size of the ITS
region.
The results of the sequencing indicated an ITS segment of 643 bp
in size. Using the BLAST tool in NCBI, the ITS sequences isolated from
five T. paniculatumin samples (ITS-TN1, ITS-TN2, ITS-BG, ITS-HT,
ITS-QN) were 99 % homologous to the three ITS sequences of T.
paniculatumin GenBank, which had an accession number JF508608,

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EU410357 and L78094; Thus, it can be concluded that the ITS region
isolated from the five T. paniculatumin samples is the ITS region of T.
paniculatum species.

Figure 3.2. PCR analysis of ITS
region. M : Marker 1 kb; 1: ITSTN1, 2: ITS-TN2, 3: ITS-BG, 4:
ITS-HT, 5: ITS-QN

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paniculatumin samples are the partial sequences of rpoC1 and rpoB
genes of T. paniculatum species.
3.1.3. Discuss the results of identifying T. paniculatum samples
Based on morphological characteristics, T. paniculatum samples
were identified with characteristics of nutritional organ, reproductive
organs and similar to the description characteristics of T. paniculatum
according to Pham Hoang Ho (1999), Do Tat Loi (2004). However, if the
plant is in its growing stage without flowers, it is easy to confuse it with
the same species of T. triangulare, therefore, it is not possible to identify
these T. paniculatum samples from the same species or different species.
As a result, it is necessary to use an extra method and criterion for the
classification. The DNA barcoding method with the ITS region and three
partial sequences of matK, rpoC1, rpoB genes can be used to accurately
identify the T. paniculatum samples without confusion with other herbs.
ITS region and three partial sequences of matK, rpoC1 and rpoB genes
isolated from T. paniculatum plants are 643 bp, 808, 595 and 518 bp in
length, respectively. Using the BLAST tool in NCBI, the three partial
sequences of matK, rpoC1 and rpoB isolated from five T. paniculatumin
samples were 97 %, 99 %, 99 % homologous to the chloroplast gene
sequences of T. paniculatum due to Liu et al. (2018) solve the sequence
which had an accession number. Based on the combination of the
characteristics of morphology and nucleotide sequences of ITS region,
matK, rpoC1 and rpoB genes, the T. paniculatum samples collected in
some northern provinces of Vietnam were determined to belong to T.
paniculatum species, Talinum genus, Portulacaceae family.
3.2. GENERATE TRANSGENIC T. paniculatum LINES GmCHI
3.2.1. The develoment in vitro regeneration system for gene transfer
in T. paniculatum plants

Effect of 60 % bleach on rate of germinating seeds
10
92,23c
91,55c
1,58b
Fat, normal green
Effect of 0,1 % HgCl2 on rate of germinating seeds
5
91,25b
82,26c
1,39c
Fat, normal green
Note: The value in each column with the same accompanying letters
shows no difference with p < 0.05.
3.2.1.2. The results of in vitro multiple shoot regeneration and rooting
in T. paniculatum
Effect of BAP on the generation and shoot regeneration in
cotyledonary explants
Table 3.4 shows that the MS medium supplemented with 1,5 mg/l
BAP is suitable for shoot emergence and bud growth from the
cotyledons, the number of shoots/samples reached 1.68 (after 2 weeks)
and 1.78 (after 4 weeks).
Bảng 3.4. Effect of BAP on the generation and shoot regeneration in
cotyledonary explants (n=30)
BAP
The
%
Height The
Shoots
concentratio number

Effect of BAP, the combination of BAP and IBA on the generation and
shoot regeneration in the lateral shoot bud explants
Results of analyzing the effect of BAP on the generation and
shoot regeneration in the lateral shoot bud explants are shown in table
3.5.

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Bảng 3.5. Effect of BAP on the generation and shoot regeneration in the
lateral shoot bud explants (n=30)
Increase
The
BAP
The
compared
Height
numbe Shoots
concentratio number of
quality
to
of shoot
r of
n
shoots/
wild-type

of T.paniculatum
The effect of IAA and NAA on in vitro rooting ability of T.
paniculatum is shown in table 3.7.
Table 3.7. Effect of IAA on in vitro rooting ability of T. paniculatum
(n=30)
The rate of
IAA
The number
shoots creates
Root length
concentration
of roots
roots
(cm)
(mg/l)
/ shoots
(%)
After 2 weeks
0,5
80,17d
5,13d
0,92b
After 4 weeks
0,5
98,12d
13,23d
3,79c
Note: The value in each column with the same accompanying letters
shows no difference with p < 0.05.


(mg/l)
roots (%)
/shoots
After 2 weeks
0,5
58,33e
3,21c
0,31a
After 4 weeks
0,5
94,36c
10,43c
2,79c
Note: The value in each column with the same accompanying letters
shows no difference with p < 0.05.
Comparison of the rate of shoots creates roots and the number of
roots/shoots at the same time of the two optimal concentrations of 0.5
mg/l IAA and 0.5 mg/l NAA showed IAA is more effective than NAA.
Thus, the optimal root stimulant in T. paniculatum is 0.5 mg/l IAA.
3.2.2. Transformation of GmCHI gene and regeneration of
transgenic T. paniculatum
3.2.2.1. The results of survey of transgenic materials via A. tumefaciens
The results of multiple shoot from cotyledons and lateral shoot
bud after A. tumefaciens infection are shown in table 3.9 and figure 3.9.

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shows no difference with p < 0.05.
The results of table 3.9 and figure 3.9 show that the effect of
multiple shoot from cotyledons after A. tumefaciens infection reflect
increases of 2.15 fold (after 6 week) compared to the lateral shoot bud.
At the same time shoots are produced from cotyledons with height,
number of leaves, and quality of shoots better than shoots created from
lateral shoot bud. Thus, cotyledons are suitable materials to create
multiple shoot for gene transfer in T. paniculatum plants.

Hình 3.9. The effect of multiple shoot from cotyledons and lateral shoot bud
after A. tumefaciens infection
A, B: The generation and shoot regeneration in cotyledonary explants
through A. tumefaciens infection after 4 weeks and 6 weeks. C, D: The
generation and shoot regeneration in lateral shoot bud through A.
tumefaciens infection after 4 weeks and 6 weeks.
3.2.2.2. Transformation of the structure carrying the GmCHI gene and
regeneration of transgenic T. paniculatum
The results of transformation of the structure carrying the GmCHI
gene T. paniculatum through A. tumefaciens infection via cotyledonary
nodes are shown in table 3.10 and figure 3.10. Table 3.10 showns that from
a total of 730 samples, we obtained 18 CHI transgenic plant lines,

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accounting for 2.46% of the initial samples. In parallel with the CHI

growi
nts
ng
ng roots
greenho
ng
shoots
use
ĐC0
40
0
0
0
0
ĐC1
40
30
68
40
35
Experime
730
200
63
43
28
nts 3
times

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Figure 3.11. The presence of the
CHI gene from transgenic T.
paniculatum lines in the T0
generation identified by PCR
using the specific primers CHINcoI-F/CHI- NotI-R

Figure 3.12. The integration of the
CHI gene into the T. paniculatum
genome determined by Southern
blot from PCR-positive transgenic
T. paniculatum plants with probe
segment CHI labelled with biotin

3.2.3.2. Analysis of the recombinant CHI protein expression in the
transgenic T. paniculatum lines in the T1 generation
All 6 transgenic plants grew and developed normally and could
produce flowers and fruits. However, only the seeds of 4 plants
germinated and developed into T1 generation plants, namely, T1-2.2,
T1-4, T1-10, and T1-14. The leaves from these T1 generation transgenic
lines were used to analyse the expression of recombinant CHI protein.
Total protein extracted from the leaves of transgenic plants was
denatured and run on 10% SDS-PAGE and analysed by Western blot

that CHI protein was overexpressed in these two transgenic T. paniculatum
lines.

3.2.3.3. Determination of total flavonoid content in the T1 generation
transgenic T. paniculatum lines
Samples including leaves, stems and roots from the two
transgenic plants (T1- 2.2; T1- 10) and wild-type plants were used to
analyse the total flavonoid content (Table 3.11).
Table 3.11. Total flavonoid content of the two transgenic T. paniculatum
lines T1-2.2 and T1-10 and non-transgenic plants
Increase compared to
Total flavonoid
Samples
non-transgenic plants
content (mg/g)
(%)
Wild-type plants
0,57a
100
T1- 2.2
4,24c
743,86
T1- 10
2,74b
480,70
Note: The value in each column with the same accompanying letters
shows no difference with p < 0.05.
Table 3.11 show that T1-2.2 had the highest flavonoid content
(approximately 4.24 mg/g), an increase of 743.86 % compared to that of
wild-type plants (approximately 0.57 mg/g). T1-10 had lower flavonoid

inherited from the T0 to the T1 generation and stably expressed, suggesting
that we have obtained two stable transgenic lines in which the transgenes
would be passed through generations. Previously, Li et al. (2006) transferred
the CHI gene isolated from Saussurea medusa (Asteraceae) into transgenic
tobacco plants, leading to an increase in the total flavonoid content 5-fold
greater than that in non-transgenic plants. Transferring the Ps-CHI1 gene
into tobacco (Nicotiana tabacum L.) via Agrobacterium to obtain transgenic
plants in the T1 generation caused a 3-fold increase in the flavonoid and
flavone content compared to that in the wild type. Overexpression of the
CHI gene isolated from petunia in tomato (Muir et al., 2001) has generated
transgenic tomatoes with a flavonoid content 78-fold higher than that in wild
type… These studies confirmed the efficiency of transferring the CHI gene
isolated from one species into another species to increase the contents of
flavones and isoflavones in transgenic plants.
In addition to the approach to improve flavonoid content in T.
paniculatum plants by overexpression of the CHI gene, the technology for

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establismenting of hairy root lines is the research direction to increase in
vitro biomass to increase the contents of flavonoids in T. paniculatum plants.
3.3. ESTABLISMENT OF HAIRY ROOT LINES IN T. paniculatum
PLANTS
3.3.1. The results of establisment of hairy root lines in T. paniculatum
plants


Hình 3.16. Investigate suitable materials to create hairy roots in T.
paniculatum plants after 4 weeks A. rhizogenes infection.
The results of table 3.12 and figure 3.16 shown that in the three
types of materials that infect by A. rhizogenes (cotyledon, lateral shoot
bud, leaf tissue), leaf tissue is a suitable material for the highest rate of
hairy roots 65.9 % (after 4 weeks), the lowest is the lateral shoot bud for
the rate of hairy roots of 55.6% (after 4 weeks). Thus, leaf tissue is a

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suitable material for transforming and inducing hairy roots in T.
paniculatum plants.
3.3.1.2. Effect of density of bacteria, concentration AS, infection time, coculture time on the effect of creating hairy roots from leaf tissue in T.
paniculatum plants
Density of bacteria corresponding to OD600=0.6; concentration AS 100
μmol/l; infection time of 10 minutes; 2 days of co-culture; cefotaxime
concentrations of 500 mg/l are suitable conditions for inducing hairy roots
from leaf tissue (65,9 %) (Table 3.13).
Table 3.13. Effect of density of bacteria, concentration AS, infection time,
co-culture time on the effect of creating hairy roots from leaf tissue in T.
paniculatum plants (n=150, after 4 weeks)
Effect of density
Effect of
of bacteria

125
45,14c
1,0
29,43b
150
40,10a

Effect of infection
time
The
rate of
Infection sample
time
s
(minute) creates
roots
(%)
5
45,23d
10
65,9e
15
40,07c
20
34,12b
25
12,51a

Effect of co-culture
time

Yosephine et al (2015).
Table 3.14. Determine the bactericidal threshold of cefotaxime after 4 weeks
Concentration
The rate of
The rate of samples
cefotaxime (mg/l)
uninfected implant
creates roots (%)
disk (%)

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500

93,76d

65,9d

Note: The value in each column with the same accompanying letters
shows no difference with p ≤ 0.05.
3.3.1.4. Analysis of hairy roots carried rolC gene by PCR
Results of electrophoresis of PCR products of two pairs primers
of rolC and VirD2 genes showed that two partial sequences of rolC and
VirD2 are 0.5 kb and 0.3 kb in positive control wells, respectively (pRi
plasmid 15834); the wells running PCR products of the hairy root lines
(lines 2, 3, 6, 7, 8) (Figure 3.17 A) all have the presence of a single DNA

State of the
medium

Original
root
weight (g)

Liquid
Semi-solid
Solid

0,55
0,55
0,55

Fresh root
weight after
4 weeks of
culture (g)
4,11c
3,02 b
2,12a

Increases in
root weight
(fold)

Dry root
weight
(g)


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without growth regulator, shaking culture conditions are suitable for hairy
roots growth. The 5 obtained hairy root lines (2, 3, 6, 7, 8) were confirmed
by the presence of rolC gene and absence of virD2 gene through PCR.
However, in order to use these T. paniculatum roots in producing flavonoids
in particular and secondary metabolites in general, it is necessary to
continue the study, comparing the content of pharmaceutical substances
between the roots of hairy roots with The roots of natural ginseng plants.
CONCLUSION AND RECOMMENDATIONS
1. Conclusion
1.1. The collected T. paniculatum samples in some localities were
determined to belong to T. paniculatum species, Talinum genus,
Portulacaceae family by comparative morphology method combined with
DNA barcode analysis.
1.2. The cotyledonary and the lateral shoot bud explants are suitable
material to create multiple shoots in T. paniculatum. The MS medium
supplemented with 50 ml/l coconut water + 1,5 mg/l BAP is the suitable for
shoot emergence and growth from axillary cotyledons. The MS medium
supplemented with 50 ml/l coconut water + 2.0 mg/l BAP is the suitable for
shoot emergence and growth from the lateral shoot bud explants.
The cotyledons are suitable materials to create multiple shoot for
gene transfer in T. paniculatum plants. From a total of 730 samples, 28
GmCHI transgenic plants were survived in the greenhouse. Recombinant