Nghiên cứu biểu hiện gen GmCHI liên quan đến tổng hợp flavonoid và cảm ứng tạo rễ tơ ở cây thổ nhân sâm (talinum paniculatum) tt tiếng anh

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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,

from which select lines of plants with high flavonoid content. However,
there have been no studies on the application of this method to improve
flavonoid content; but the application of plant biotechnology such as
gene transfer and cultivation plant cell and tissue in vitro in medicinal
plants has been paid attention to and highly effective in the reception of
bioactive substances, including flavonoids.

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In plants, flavonoids are synthesised by the phenylpropanoid
biosynthesis pathway, converts the amino acid L-phenylalanine (L-Phe)
into 4-coumaroyl CoA (or one thiol ester with the presence of a 4hydroxycinnamate). There are many enzymes involved in the synthesis
of flavonoids pathway such as phenylalanine ammonia-lyase, cinnamate
4-hydroxylase, 4-Coumarate CoA ligase, chalcone synthase, chalcone
isomerase ... In particular, Chalcone isomerase (CHI) is the key enzyme
in flavanone biosynthesis that catalyses the intramolecular cyclization of
bicyclic chalcones (i.e., naringenin chalcone) into tricyclic (S)flavanones (i.e., naringenin). The synthesis of naringenin, the first
flavanone, directs the flavonoid pathway to the synthesis of other
flavanones, flavonols and anthocyanins. Thus, it is of great interest to
improve its flavonoid content by overexpressing the CHI gene that
encodes the key enzyme of flavonoid synthesis, and the GmCHI gene
encoding chalcone isomerase, a key enzyme in metabolizing synthetic
flavonoid in soybeans, was chosen.
In addition, T. paniculatum is a plant with tuberous roots, many
secondary compounds concentrated in the roots, including flavonoids.

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

in the transgenic T. paniculatum lines in the T1 generation.
(iii) Study on establisment of hairy root lines in T. paniculatum plants
via Agrobacterium rhizogenes.
4. New contributions of the dissertation
The dissertation is a systematic study, from the identification of T.
paniculatum samples collected in some localities to create the initial
material source for in vitro culture to transfer the structure of carrying
GmCHI gene into T. paniculatum and analysis of soybean GmCHI gene
expression in transgenic T. paniculatum plants.
The main results are as follows:
1) Identifying 5 samples of T. paniculatum collected in 5 localities in
Vietnam belong to T. paniculatum species, Talinum genus, Portulacaceae
family.
2) For the first time, the GmCHI gene isolated from soybean cultivars was
successfully expressed in the T. paniculatum and created two transgenic
T. paniculatum lines with higher flavonoid content than the wild-type
plants.
3) Create the 5 hairy root lines from the T. paniculatum to serve as a
material for selecting the hairy root lines with high pharmaceutical
content.
5. The scientific and practical significance of the dissertation
The results of the dissertation are of scientific and practical
significance in the approach to improve flavonoid content by
overexpressing the CHI gene that encodes the key enzyme of flavonoid
synthesis and creating the hairy root lines in the T. paniculatum.
Scientific significance, the research results of the dissertation will
be the basis for applying the technique of creating hairy roots and

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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

gastric ulcers. Currently, there is no published research on the flavonoid
content of T. paniculatum plants. However, it has been determined that
the species in the genus Talinum have very low flavonoid content (about
0,897 mg/g fresh leaves) (Afolabi và cs, 2014).
Flavonoids are synthesised by the phenylpropanoid biosynthesis
pathway and chalcone isomerase (CHI) is the key enzyme in flavanone
biosynthesis that catalyses the intramolecular cyclization of bicyclic
chalcones (i.e., naringenin chalcone) into tricyclic (S)-flavanones (i.e.,

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naringenin). To improve the content of bioactive compounds in T.
paniculatum (including flavonoids), until now, studies on improving the
content of bioactive compounds including flavonoids in T. paniculatum
have mainly focused towards increasing the biomass of cells and hair
roots. Zhao et al. (2009) proposed to select appropriate materials and
concentrations of growth-promoting substances to maximize the
formation of calli and buds, the rooting proportion, and the survival rate
of seedlings in a greenhouse. Muhallilin et al. (2013) studied the effect
of auxin-type plant growth regulators (IAA, NAA, IBA and 2.4-D) at
various concentrations (1 mg/l, 2 mg/l and 3 mg/l) for root induction on
leaf explants of T. paniculatum. While Yosephine et al. (2012) studied
the effect of aeration and inoculum density on biomass and saponin
content of T. paniculatum Gaertn. hairy roots in a balloon-type bubble
bioreactor by transforming a leaf sample of T. paniculatum with A.

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

the basis of the morphological characteristics of roots, tubers, stems,
leaves, flowers, fruits, seeds, and providing materials for morphological
and molecular biology analysis.
The A. rhizogenes ATTC 15834 were provided by the Department
of Plant Cell Technology, Vietnam Institute of Biotechnology, Vietnam
Academy of Science and Technology.
The Agrobacterium tumefaciens (A. tumefaciens) CV58 carrying
the 35S-CHI-cmyc construct in a pCB301 transgenic plasmid were
provided by the Department of Modern Biology & Biological Education,
Thai Nguyen University of Education.
2.2. CHEMICALS, EQUIPMENTS AND RESEARCH LOCATIONS
The chemicals used in the study are bought from worldwide
famous firms like Fermentas, Bio-Neer, Invitrogen, Trizol Reagents,
Maxima® First Strand cDNA Synthesis. The experiments were carried
out on the modern equipments of the Genetic Engineering Department,
Faculty of Biology, College of Education - Thai Nguyen University, and
Laboratory of Gene Technology, Department of Applied DNA
Technology and Department of Plant Cell Technology, Institute of
Biotechnology, Vietnam Academy of Science and Technology.
2.3. RESEARCH METHODS
2.3.1. The identification of T. paniculatum plants was done according to
the method suggested by Pham Hoang Ho (1999), Do Tat Loi (2004),
search on website and
molecular classification methods based on some DNA barcodes such as
ITS region, three partial sequences of matK, rpoC1 and rpoB genes.
2.3.2. In vitro culture methods: (1) Sterilization methods seeds; in vitro
method of multiple shoot in T. paniculatum; (3) Culture method to create
hairy roots in T. paniculatum plants.
2.3.3. Agrobacterium-mediated transformation via T. paniculatum
cotyledonary nodes and regeneration and selection of transgenic T.

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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

and flowers (Figure 3.1). Tuberous roots of T. paniculatum are cylindrical
with many small roots (Figure 3.1 B). The stems are upright and divided
into several branches (Figure 3.1 A). The leaves are staggered, generally
oval, ovate-oblong, or egg back shaped; thick, glossy with wavy veins,
without hairs (Figure 3.1 C). The flowers of the plants have five reddish
purple wings, two sepals, more than ten stamens, and a spherical ovary
(Figure 3.1 D). The fruits are small, and the ripe fruit is ash gray in color
(Figure 3.1 E). The seeds are very small, slightly flat, and black (Figure
3.1 F)

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Figure 3.1. T. paniculatum plant. A: T. paniculatum plant; B: Tuberous
roots; C: branch, leaves; D: bud and flowers; E: fruits; F: seeds.
After comparing the morphological characteristics observed in the
samples of T. paniculatum with the characteristics of T. paniculatum
according to the description of Pham Hoang Ho (1999), Do Tat Loi
(2004) and also looking up />shows samples of BG, TN1, TN2, HT, QN T. paniculatum were
determined to belong to T. paniculatum species, Talinum genus,
Portulacaceae family.
However, if the plant is in its growing stage without flowers, it is
easy to confuse it with the same species of T. triangulare. In addition,
the classification of T. paniculatum encounters obstacles if the plants
have been completely or partially processed. As a result, it is necessary
to use an extra method and criterion for the classification. The

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

Figure 3.5. PCR analysis of partial
sequences of matK. M: Marker 1 kb;
1: matK-TN1, 2: matK-TN2, 3: matKBG, 4: matK-HT, 5: matK-QN

3.1.2.2. Characteristics of the partial sequences of matK
The results of testing PCR products by electrophoresis on 1%
agarose gel with 1 kb DNA ladder are shown in figure 3.5. The results
showed that the PCR products of all samples obtained a DNA band of
about 800 bp in size, which was similar to the predicted size of the
partial sequences of matK. The results of the sequencing indicated an
matK segment of 808 bp in size. Using the BLAST tool in NCBI, the
matK sequences isolated from five T. paniculatumin samples (matKTN1, matK-TN2, matK-BG, matK-HT, matK-QN) were 99 %
homologous to the three matK sequences of T. paniculatumin GenBank,
which had an accession number AY015274, KY952520, GQ434150;
Thus, it can be concluded that the partial sequences of matK isolated
from the five T. paniculatumin samples is the partial sequences of matK
of T. paniculatum species.
In addition to the sequence of ITS regions (inside the nucleus) and
the matK gene segment (chloroplast gene), we sequenced two
chloroplast gene segments rpoC1 and rpoB. Two partial sequences of
rpoC1 and rpoB genes isolated from T. paniculatum plants are 595 bp
and 518 bp in length, respectively. Using the BLAST tool in NCBI, the
two partial sequences of rpoC1 and rpoB genes isolated from five T.

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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

3.2.1.1. The results of the optimal conditions for sterilization seeds
Table 3.3 shows that the seeds sterilized with 70% alcohol for 1
minute and then with 60 % bleach for 10 minutes resulted in the highest
efficiency (the rate of uninfected bottles is 92.23 %, the rate of
germinated seeds reaches 91.55 %, the buds grow well).

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Table 3.3. Effect of 60 % bleach and 0.1 % HgCl2 on seeds germination
rate after 10 days of culture (n = 30)
Sterilizatio
Rate of
Rate of
Size of
Morphological of
n time
uninfecte germinatin seedling
seedling
(minutes)
d bottles
g seeds
after 10
(%)
(%)
days (cm)

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

compared
of
number
quality
n
of shoots/
to the
shoot
of leaves/
(mg/l)
samples
control
(cm)
Shoot
After 2 weeks
1,5
1,68a
136,58
0,87a
4,74b
F, NG
After 4 weeks
1,5
1,78a
132,83
2,88c
6,14a
F, NG
Note: The value in each column with the same accompanying letters
shows no difference with p < 0.05. F, NG: Fat, normal green.

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

(cm)
leaves/
(mg/l)
samples
plants (%)
shoot
After 2 weeks
2,0
3,04d
218,7
0,87a
5,22c F, NG
After 4 weeks
2,0
3,24c
216,00
2,88b
6,52a F, NG
Note: The value in each column with the same accompanying letters
shows no difference with p < 0.05. F, NG: Fat, normal green.
Table 3.4 shows that the MS medium supplemented with 2 mg/l
BAP is suitable for shoot emergence and bud growth from the lateral
shoot bud, the number of shoots/samples reached 3.04 (after 2 weeks)
and 3.24 (after 4 weeks). Comparison of results in table 3.4 and table 3.5
shows the generation and shoot regeneration in the lateral shoot bud
explants more effectively than the generation and shoot regeneration in
cotyledonary explants at the same BAP concentration. Thus, 2 mg/l BAP
is the appropriate growth stimulant to create multiple shoots from the
lateral shoot bud T. paniculatum plant.
The effect of IAA and NAA on in vitro rooting ability

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.

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Table 3.7 shows that the MS medium supplemented with 0,5 mg/l
IAA is suitable for rooting from the lateral shoot bud, the number of
shoots/samples reached 3.04 (after 2 weeks) and 3.24 (after 4 weeks).
MS medium supplemented with 0.5 mg/l IAA gives the highest
rooting rate of 80.17 %, reflect increases of 2.66 fold (after 2 weeks) and
98.12 % reflect increases of 1.09 fold (after 4 weeks) compared to the
control. Thus, 0.5 mg/l IAA is the appropriate in vitro rooting ability of
T.paniculatum.
Effect of NAA on in vitro rooting ability of T. paniculatum in
table 3.8 shows that MS medium supplemented with 0.5 mg/l NAA
gives the highest rooting rate of 58.33 %, reflect increases of 1.93 fold
(after 2 weeks) and 94.36 % reflect increases of 1.05 fold (after 4 weeks)
compared to the control. Thus, 0.5 mg/l NAA is the appropriate in vitro
rooting ability of T. paniculatum.
Bảng 3.8. Effect of NAA on in vitro rooting ability of T. paniculatum
(n=30)
The rate of
The number
Root
NAA concentration
shoots creates
of roots
length (cm)

(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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Bảng 3.9. The effect of multiple shoot from cotyledons and lateral shoot
bud after A. tumefaciens infection (n=150)
The
Height of
The number Shoots
number of
Materials
shoot
of leaves/
quality
shoots/
(cm)
shoot
samples
After 6 weeks
Cotyledons
3,02b
1,34a
5,21b
Fat
a
a
Lateral shoot bud
1,40
1,13
3,43a
Thin
Note: The value in each column with the same accompanying letters

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

transgenic experiment, we cultivated two control batches called DC0 and
DC1. All samples in DC0 died as expected, while in DC1, 35 plants
survived in the greenhouse.

Figure 3.10. Transformation and regeneration of transgenic T.
paniculatum plants. A: seeds of T. paniculatum; B: germination in
germination medium (GM); C: co-cultivation in co-cultivation medium
(CCM) in the dark; D: inducing shoot generation; E: multiple shoot
regeneration after 3 weeks; G, H: rooting and fully rooted plantlets in
rooting medium (RM); I: seedling substratum; K: plants in greenhouse.
Table 3.10. The results of transformation of the structure carrying the
GmCHI gene through A. tumefaciens infection
Numbe
Number
Numb
Controls
r of
Number
of plants
Total
er of
and
samples of shoots
grown in
sampl
plants
experime
produci produci
the
es

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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Note: DC0 contained non-transgenic samples cultured in medium
supplemented with selection antibiotics, and DC1 contained nontransgenic samples cultured in medium without selection antibiotics
3.2.3. The results of analyse transgenic T. paniculatum plants
3.2.3.1. Verifying the integration of the GmCHI gene into T.
paniculatum genomes in the T0 generation
The results of testing the transgenic T. paniculatum by PCR
The results of PCR analysis with the specific primers CHI-NcoIF/CHI- NotI-R in figure 3.11 obtained a single DNA fragment with a
size of about 0.66 kb corresponding to GmCHI gene size transferred to
the T. paniculatum in 8 plants (T0-2.1, T0-2.2, T0-4, T0-7, T0-10, T0-12,
T0-14, and T0-16).
1. The results of testing the transgenic T. paniculatum
by Southern blot
Eight PCR-positive plants (T0- 2.1; T0- 2.2; T0- 4; T0- 7; T0- 10;
T0- 12; T0- 14; T0- 16) and wild-type plants were subjected to Southern
Blot analysis to determine whether the transgene was integrated into the
transgenic plant genomes. Figure 3.12 shows that DNA bands occurred
in 6 transgenic plants (T0-2.1, T0-2.2, T0-4, T0-7, T0-10, and T0-14),
while there was no band in T0-12, T0-16 and wild-type plants. T0-7
showed 2 DNA bands corresponding to 2 copies, and the remaining
lines, T0-2.1, T0-2.2, T0-4, T0-10, and T0-14, had only 1 copy. The
transformation frequency of the CHI transgene at this stage was 5/730 =
0.68%. The growth and development of the transgenic plants with
positive Southern blot hybridization results were evaluated, and their
next generations were subjected to protein expression analysis.

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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

(Figure 3.13). As seen in figure 3.13, we detected a band of
approximately 25 kDa corresponding to the molecular weight of the
recombinant CHI protein in 2 T1-generation lines, T1- 2.2 and T1- 10,
while there was no band in the T1-4, T1-14 and wild-type lanes,
suggesting that the CHI transgene was inherited from the T0 generation
to the T1 generation in 2 transgenic lines, T1-2.2 and T1-10, and
translated into recombinant CHI protein. Thus, the transformation
frequency of the CHI gene in this period was 0.27 % (2/730).

Figure 3.13. Western blot
analysis for recombinant CHI
expression in 4 transgenic T.

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Figure 3.14. ELISA determined the
recombinant CHI protein content in
the two transgenic T. paniculatum


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paniculatum lines in the T1 lines (T1-2.2 and T1-10) and nongeneration and non-transgenic transgenic plants (WT – wild type)
plants
The contents of the recombinant CHI protein in two transgenic
lines, T1-2.2 and T1-10, and wild-type plants were analysed by ELISA
(Figure 3.14). The recombinant CHI protein contents in T1-2.2 and T1-10
were 6.14 µg.mg-1 and 4.29 µg.mg-1, respectively. This result demonstrated

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

content (approximately 2.74 mg/g), an increase of 480.70 % compared to
that of wild-type plants. These results illustrated that overexpression of the
CHI gene in two transgenic T. paniculatum lines T1-2.2 and T1-10
effectively increased the flavonoid content in transgenic plants.

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3.2.4. Discuss the results of transformation and creation of transgenic T.
paniculatum lines
The current research directions are mainly focused on cultivation
in vitro to increase biomass, and there is no research work that establishes an
effective gene transfer method to improve the content of bioactive
compounds in T. paniculatum, including flavonoids. In this study, we
overexpressed chalcone isomerase (CHI), a key enzyme in the flavonoid
biosynthesis pathway, in order to increase the total flavonoid content in
transgenic T. paniculatum. We isolated the CHI gene from soybean and
transferred it into T. paniculatum via A. tumefaciens following a method
previously described by Olhoft et al. (2006). From a total of 730 samples, 28
plants belonging to 18 transgenic lines survived. In this study, we
demonstrated that overexpression of the CHI gene isolated from soybean
remarkably increased the total flavonoid content in transgenic T.
paniculatum plants. We have successfully generated two transgenic lines,
T1-2.2 and T1-10, that contain 4.24 mg.g-1 and 2.74 mg.g-1 of flavonoid,
respectively, which reflect increases of 7.4-fold and 4.8-fold, respectively,
compared to that in wild-type plants. Moreover, the CHI transgene was

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

3.3.1.1. Investigate suitable materials to create hairy roots in T.
paniculatum plants
The results of investigate suitable materials to create hairy roots in T.
paniculatum plants are shown in table 3.12 and figure 3.16.
Table 3.12. The results of investigate suitable materials to create hairy roots
in T. paniculatum plants (n=150, after 4 weeks)
Materials
The rate of samples
The number
Root length
creates roots
of
(cm)
(%)
roots/samples
Leaves
65,9c
3,45c
3,25c
Lateral shoot
55,6a
1,89a
1,59a
bud
Cotyledons
58,2b
2,32b
1,82b
Note: The value in each column with the same accompanying letters shows
no difference with p < 0.05.

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

concentration AS
The
The
rate of Concen rate of
sample tration sample
OD600
s
AS
s
creates (μmol/l creates
roots
)
roots
(%)
(%)
0,2
23,42a
50
43,23b
0,4
34,56c
75
47,32c
e
0,6
65,9
100
65,9d
0,8
43,24d

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

Coculture
time
(day)

The rate
of
samples
creates
roots
(%)

1
2
3
4
5

36,12d
65,9e
23,34c
14,12b
4,12a

Note: The value in each column with the same accompanying letters shows
no difference with p < 0.05.
3.3.1.3. Study to determine the bactericidal threshold of cefotaxime
The results of determining the bactericidal threshold of cefotaxime in
table 3.14 show that the optimal cefotaxime concentration of bactericidal is
500 mg/l for the rate of uninfected implant disk is 93.76 % and the rate of
creating hairy roots is 65,9 %. This result is consistent with the study of

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

band with clear clarity at position 520 bp (same position as the rolC
positive control) and no DNA band at position 338 bp of VirD2 gene
(Figure 3.17 B); in contrast to positive control wells, negative control
and non-transgenic root control (unidentified) wells had no bands in
locations 338 bp and 520 bp.

A
B
Figure 3.17. Electrophoresis image for PCR product of rolC gene (A)
and virD2 gene segment (B)
3.3.1.5. Effect of state of the MS medium on hairy roots growth of
T.paniculatum
In the three types of state of the liquid MS medium solid, semi-solid,
liquid, hairy roots on liquid culture for shaking with the highest growth rate
(4.11 g fresh weight), followed by semi-solid medium (3,02 g fresh weight)
and finally solid medium (2.12 g fresh weight) increased 7.47; 5.49 and 3.85
fold compared to that in original root volume after 4 weeks of culture (Table
3.15). Thus, in state of the liquid MS medium without growth regulator,
shaking culture conditions are suitable for hairy roots growth. The image
showing the results of cultivating hairy roots in T.paniculatum is shown in
figure 3.18.
Table 3.15. Effect of state of the MS medium on hairy roots growth of T.
paniculatum

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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)

7,47
5,49
3,85

0,34b
0,23a
0,18a

Note: The value in each column with the same accompanying letters
shows no difference with p ≤ 0.05.

Figure 3.18. Image of adventitious hairy roots induction and culture of T.
paniculatum roots
3.3.2. Discuss the results of creation of hairy roots lines in T.
paniculatum
The culture of hairy root biomass by A. rhizogenes to acquire
secondary compounds with biological activity is an effective solution that
can overcome the limitations of traditional breeding methods and methods
of growing cell biomass. For T.paniculatum, study on hairy roots and
application of techniques for increasing hairy root biomass were published
by Yosephine et al (2012). In Vietnam, the study of creating the roots of
hairy roots from plants and especially in the T. paniculatum plant is very
new. This study showed the results of production / establishment of hairy
root lines in vitro of T. paniculatum through Agrobacterium rhizogenes. Of
the three types of materials that infect by A. rhizogenes (cotyledon, stem,
leaf tissue), leaf tissue is a suitable material for transforming and inducing
hairy roots. Density of bacteria corresponding to OD600 value=0.6;
concentration AS 100 μmol/l; Infection time of 10 minutes; 2 days of coculture; cefotaxime concentrations of 500 mg/l are suitable conditions for
inducing hairy roots from leaf tissue. In state of the liquid MS medium

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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

CHI protein was expressed successfully in two transgenic T. paniculatum
lines of T1-2.2 and T1-10 in the T1 generation with contents of 6.14 µg/mg
and 4.29 µg/mg, respectively.
Two transgenic T. paniculatum lines of T1-2.2 and T1-10, that
contain 4.24 mg/g and 2.74 mg/g of flavonoid, respectively, which reflect
increases of 7.4-fold and 4.8-fold, respectively, compared to that in wildtype plants.
1.3. Leaf tissue is a suitable material for transforming and inducing hairy
roots in T. paniculatum. Density of bacteria corresponding to OD600
value=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. In state of the liquid MS

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medium without growth regulator, shaking culture conditions are suitable
for hairy roots growth in T. paniculatum.
2. Recommendations
2.1. Continue to analyse and evaluate the two transgenic T. paniculatum
lines (T1-2.2 and T1-10) in T2 and T3 generations to select the transgenic T.
paniculatum lines with high and stable flavonoid content.
2.2. Continue to analyse and compare the flavonoid content between the
hairy roots lines and non-transgenic roots T. paniculatum.

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