o UNIVERSITY OF SCIENCE AND TECHNOLOGY OF HANOI

UNDERGRADUATE SCHOOL

Research and Development

BACHELOR THESIS
By
LÊ HÀ PHƯƠNG
Department of Pharmacological, Medical and Agronomical
Biotechnology

Title:
HAIRY ROOT INDUCTION BY AGROBACTERIUM
RHIZOGENES – MEDIATED TRANSFORMATION IN
PARAMIGNYA TRIMERA FOR ROOT BIOMASS
PRODUCTION

Supervisors: Dr. Lê Thị Vân Anh

USTH PMAB laboratory

Hanoi, September 2019

Table of Contents
ACKNOWLEDGEMENTS ...........................................................................
LIST OF ABBREVIATIONS ........................................................................
LIST OF FIGURES .......................................................................................
ABSTRACT ....................................................................................................
TÓM TẮT .......................................................................................................
I/ INTRODUCTION .................................................................................... 1
1. Paramignya trimera .............................................................................................. 1
2. Hairy roots induced by Agrobacterium rhizogenes mediated genetic transformation
4
II/ OBJECTIVES ......................................................................................... 6
III/ MATERIALS AND METHODS .......................................................... 6
1. Materials ............................................................................................................... 6
2. Methods ................................................................................................................. 7
IV/ RESULTS AND DISCUSSIONS .......................................................... 9
1. In vitro callus and shoot tissues regeneration for genetic transformation ............ 9
1.1. Callus regeneration .................................................................................... 9
1.2. Shoots induction ....................................................................................... 11
1.3. In vitro root induction test ........................................................................ 12
2. In vitro transformation ........................................................................................ 14
2.1. Conditions optimization: Tissue types ..................................................... 14
2.2. Conditions optimization: Bacteria strains ............................................... 14
2.3. Conditions optimization: Time of co-culture .......................................... 14
2.4. Conditions optimization: Antibiotics combinations ................................ 15
3. In planta transformation ..................................................................................... 16
4. Transformation analysis ..................................................................................... 19
V/ CONCLUSIONS AND PERSPECTIVES ............................................ 21
REFERENCES ........................................................................................... 22
ACKNOWLEDGEMENTS

I would like to express my gratitude to those who has given me this chance of internship
and has supported me throughout the process.

First, I sincerely thank USTH, PMAB department and my supervisor Le Thi Van Anh for
having accepted my internship and has provided me with supports and guidance to
complete this dissertation. I especially thank my supervisor for her patience and
understandings.

Second, I am as well grateful for the helps that people in LMI RICE has given me, both
teachers and students there have given me a hand when I needed.

Third, I am so blessed to have my family, my church members and my friends, who have
continuously and steadfastly supported me, both physically, mentally and spiritually
during my internship time.

Finally, and foremostly, I am grateful for all of these that my LORD and GOD, Jesus
Christ, has given me. Without HIM, I would not even be able to attend university in the
first place.

I wish the best regards and blessings to USTH, PMAB departments, LMI RICE and all
students in USTH.

Le Ha Phuong

Hanoi, 20th August 2019

LIST OF ABBREVIATIONS

BP BIOTECHNOLOGY- PHARMACOLOGY
USTH University of Science and Technology of Hanoi
WPM Woody Plant Medium
MS Murashige & Skoog ( medium)
2,4D 2,4 Dichlorophenoxyacetic acid
IBA Indole-3-butyric acid
TDZ Thidiazuron
BAP Benzyl-amino-purine
AS Acetosyringone
LIST OF TABLES

Table 1. Callus formation from cotyledon under different conditions of supplemented

auxin…………………………………………………………………...………………9-10
Table 2. Shoot induction from cotyledon under different conditions supplemented by
BAP and TDZ at concentration of 1 mg/l...........................................................................11
Table 3. Root induction from piece of in vitro shoot in the WPM conditions
supplemented by 2,4D and by 3mg/l IBA (B)…………………………………………...13
Table.4. The efficiency of in planta transformation by the percentage of plants developed
induced roots……………………………………………………………………………...18
LIST OF FIGURES

Figure 1. Seeds, fruits and young saplings of P. trimera………………………………1

Figure 2. Schematic representation of Agropine type Ri plasmid of A. rhizogenes…4
Figure 3. Callus induced from P. trimera cotyledon in the WP medium supplemented by
1.5 mg/l (A) and 2.5 mg/l 2,4D (B) after 2 weeks of culturing..........................................10
Figure 4. Shoot induction from P. trimera seed in the WP medium without (A) and
supplemented by 1mg/l of BAP (B) 2 weeks after germination.…………….………...…11
Figure 5. Root induction from P. trimera piece of in vitro shoot in the WP medium
supplemented by 3mg/l 2,4D (A) and by 3mg/l IBA (B)…...……………………………12
Figure 6. Pink color appeared 1 day after transferring to cefotaxime-supplemented
medium, upper view (A) and lower view (B)…………………………………………….13
Figure 7. Roots of natural plant in soil…………………………………………………..16
Figure 8. Induced roots on plants from the 1st experiments of in planta transformation.17
Figure 9. Induced roots on plants from the 2nd experiment of in planta transformation..18
Figure 10. Induced roots on plants from the 3rd experiment of in planta
transformation…………………………………………………………………………….18
Figure 11. Stable integration of rolC genes was determined by PCR analysis of genomic
DNA extracted from in planta transformed hairy root...............................................………19
ABSTRACT

Paramignya trimera is a Vietnamese native plant belonging to the Rutaceae family

commonly found in Khanh Hoa. It is used as a Vietnamese traditional medicine to treat
hepatitis and has recently become well-known and well- sought for its anti-cancer
bioactivities. Due to being rumored as a panacea by Vietnamese for hepatic cancer, the
natural source of the plant has been overexploited. To date, several researches has been
conducted to confirm its bioactivities on various cancer cell lines, to extract and determine
its bioactive compounds and to clone the plant for preservation and biomass production.
However, the production rates remain in short of demand as conventional cultivated plants
requires from 3 to 5 years to mature and produce biomass with enough bioactive
compounds. Moreover, since the roots are harvested for the highest concentration of
medicinal compounds, the production cycle is inevitably reset after each harvest.
Therefore, this research aims to: i) study the Agrobacterium rizhogenes mediated
transformation for hairy root induction in P. trimera (in vitro and in planta); and to ii)
optimize the condition for in vitro genetic transformation to induce the hairy root. The
procedure for genetic transformation for in planta method had been successfully
implemented, with hairy roots induction confirmed and a transformation efficiency of about
60%. Moreover, although the in vitro transformation was not successful and is still being
studied, the results of 4 optimized conditions could be used for further research on in vitro
micro-propagation or in vitro adventitious root production. Thus, Agrobacterium
rhizogenes – mediated transformation in Paramignya trimera will be a feasible solution for
the hairy root induction in Paramignya trimera.

Key words: Paramignya trimera, Agrobacterium rhizogenes, genetic transformation, hairy

root, root biomass production, in vitro culture
TÓM TẮT

Xáo Tam Phân (Paramignya trimera) thuộc họ cam quýt (Rutaceae), một loài cây bản địa
Việt Nam phân bố ở tỉnh Khánh Hoà là một cây thuốc sử dụng trong y học cổ truyền có tác
dụng giải độc, mát gan, tiêu viêm. Các nhiên cứu gần đây cũng khẳng định Xáo tam phân
là loại cây dược liệu tiềm năng với thành phần hoá học phong phú; có nhiều chức năng sinh học
quan trọng đặc biệt như khả năng gây độc tế bào với nhiều dòng tế bào ung thư, khả năng bảo vệ
gan, khả năng kháng viêm. Tuy nhiên, lượng cây trồng theo phương thức truyền thống không
đáp ứng được nhu cầu sừ dụng do cây thân gỗ, sinh trưởng chậm nên cần đến 3 đến 5 năm
để phát triển và tổng hợp đủ lượng hoạt chất. Ngoài ra, vì sản phẩm thu hoạch là rễ cây,
chu trình sản xuất buộc phải bắt đầu lại sau mỗi đợt thu hoạch. Vì vậy, đề tài này sẽ i)
nghiên cứu khả năng cảm ứng tạo rễ tơ ở xáo tam phân bằng phương pháp chuyển gen sử
dụng Agrobacterium rhizogenes (in vitro và in planta); và ii) tối ưu hoá các điều kiện
chuyển gen in vitro. Quy trình chuyển gene cho phương pháp in planta đã được thực hiện
thành công: rễ tơ được tạo và xác nhận với hiệu suất chuyển gene khoảng 60%. Ngoài ra,
dù phương pháp in vitro chưa thành công và vẫn đang trong quá trình nghiên cứu, kết quả
của tối ưu hóa 4 điều kiện có thể được sử dụng cho các nghiên cứu về nhân giống vô tính
in vitro và sản sinh rễ bất định in vitro. Vì vậy, phương pháp chuyển gen in planta sử dụng
Agrobacterium rhizogenes có thể là một giải pháp hữu hiệu để cảm ứng tạo rễ tơ Xáo tam
phân.

Từ khóa: Paramignya trimera, Agrobacterium rhizogenes, chuyển gen, tạo sinh khối rễ,
nuôi cấy in vitro
I/ INTRODUCTION

1. Paramignya trimera
Belonging to the Paramignya genus of the Rutaceae family (Khan, 2007), in Vietnam, P.
trimera is found in the mountain of Lap Vo, Tay Ninh (Phạm Hoàng Hộ, 2000) and mainly
collected from Hon Heo, Ninh Hoa, Khanh Hoa (V. T. Nguyen, 2017). P. trimera is a
woody climbing shrubs with fragrant yellow to brown wood due to the high contents of
aromatic oils (T. H. Nguyen, 2016; Phạm Hoàng Hộ, 2000). While young saplings have no
spikes, matured plants are covered in long, slightly curved protective spikes. Leaves are
simple, smooth and elongated narrow teardrop or heart shaped (blunt-end, may have
indentation at the tip) (Phạm Hoàng Hộ, 2000). Whites 2mm flowers have 3 sepals and 3
4-mm-petals, 3 separated pistils and 3 ovaries in 1 ovule. Spherical or egg-shaped fruits
measured up to 15mm (Phạm Hoàng Hộ, 2000). Large double-sided, convex and ovate
green seeds.

Figure 1. Seeds, fruits and young saplings of P. trimera

In order to investigate the medicinal activities according to the uses of P. trimera as folk
medicine, several researches had been conducted on different extracts from roots, stems
and leaves of the plant on the cytotoxic, antioxidant, hepatoprotective and alpha-
glucosidase inhibitory bioactivities. In general, methanol extracts and partitioned n-hexane
fractions has demonstrated in vitro cytotoxic effects against 5 different cancer cell lines:
HepG2 (human hepatocellular cancer cells), HTC116 (human colon cancer cells), MDA

1
MB231 (human breast cancer cells), OVCAR-8 (human ovarian cancer cells) and Hela
(human epithelial cervical carcinoma cells) (T. M. K. Nguyen, Pham, & Do, 2013). It has
also been confirmed that methanol extracts and Chloroform fractions of P. trimera roots
and stems possess great hepatoprotective capacity that is comparable to Silymarin, a
commercial hepato-protective medicine, particularly in lowering the concentration of AST
and ALT enzymes, total Cholesterol levels and reducing sizes and symptoms of liver
cirrhosis (Nguyễn et al., 2015; T. H. Nguyen, 2016). Moreover, leaves and roots methanol
extracts have been examined separately, and both displays strong antioxidant capacities,
potent cytotoxicity and anti-proliferative activities that is substantially higher than
gemcitabine against various cancer cell lines; with leaves extracts on pancreatic cancer cells
(MiaCaPa2, BxPc3, and CFPAC1) and roots extracts on 15 cancer cell lines of MiaPaCa-2
(pancreas), HT29 (colon), A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate),
BE2-C (neuroblastoma), MCF-7 (breast), MCF-10A (normal breast), and U87, SJ-G2,
SMA (glioblastoma)Du145 (prostate), BE2-C (neuroblastoma), MCF-7 (breast), MCF-10A
(normal breast), and U87, SJ-G2, SMA (glioblastoma) (V. T. Nguyen, 2017; V. T. Nguyen,
Sakoff, & Scarlett, 2017).
Regarding bioactive phytochemical composition of P. trimera extracts, many compounds
from several pharmacologically active chemical classes, including coumarins and coumarin
glycosides, acridone alkaloids, phenols, flavonoids and chromenes, have been isolated and
assessed. It is revealed that the bioactivities of P. trimera extracts is due to the high contents
of various medicinal secondary metabolites, consisting of a complex mixture of coumarins,
phenolics, flavonoids, chromenes and other minor compound groups.
The major constituent of P. trimera extracts, with 20 compounds identified, is coumarins
and coumarin glycosides, a secondary plants metabolites benzopyrone chemical class
known for a wide ranges of medicinal activities, such as antioxidant, anti-inflammatory,
antibacterial, antifungal, antidiabetic and anticancer (Venugopala, Rashmi, & Odhav,
2013). Ostruthin, the featuring coumarin found in P. trimera roots and stems, proves to be
anti-inflammatory on BV2 microglia cells (Anh et al., 2017), antimycobacterial, antifungal

2
(Venugopala et al., 2013), anti-hyperglycaemic via alpha-glucosidase inhibition (Dang et
al., 2017; M. T. T. Nguyen et al., 2018), cytotoxic against two cell lines of human pancreatic
cancer PANC-1 and PSN-1 in nutrient deprived medium (Anh et al., 2017), as well as
somewhat cytotoxic on hepatic cancer HepG2 (Dương et al., 2016). Similarly, ninhvanin
(8-Methoxyostruthin) is also anti-inflammatory on BV2 microglia cells(Anh et al., 2017),
considerably alpha-glucosidase inhibitory(Dang et al., 2017) and fairly cytotoxic to HepG2
hepatic cancer cell(Dương et al., 2016). Other BV2-microglia-cell-anti-inflammatory
compounds, paratrimerin E and lunvagetin, shows notable alpha-glucosidase inhibitory
activities(Dang et al., 2017); and substantial protection against gastric ulcers in rats caused
by pylorus ligation and aspirin, as well as gastric ulcers in rats and guinea pigs induced by
cold-restraint stress (Goel, Maiti, Manickam, & Ray, 1997), respectively. In addition,
several other coumarins have been tested for anti-inflammatory activities (8-Geranyl-7-
hydroxycoumarinin stems) (Anh et al., 2017) and anti-hyperglycaemic (paratrimerin F,
Scopoletin, Xanhthyletin and Pandanusin A in roots, 5-O-methylanisocoumarin B and
demethylsuberosin in stems) (Dang et al., 2017).
Other phytochemical groups found in P. trimera extracts, such as acridone alkaloids,
phenolics, flavonoids and chromenes and miscellaneous compounds also contribute to the
diverse bioactivities of the extracts. For instance, acridones alkaloids from P. trimera roots
aid in alpha-glucosidase inhibition (Citrusine-I, Glycocitrine-III, Oriciacridone E, 5-
Hydroxynoracronycin, paratrimerin C and D) (Dang et al., 2017) and cytotoxicity against
HepG2 hepatic cancer cell (Citrusine-I, lycocitrine-III, Oriciacridone E and 5-
Hydroxynoracronycin) (Dương et al., 2016). Likewise, some phenolics compounds,
chromenes, flavonoids and other phytochemicals (Methyl 4-hydroxybenzoate, Vanillin,
Vanillic acid, Syringic acid, Paratrimerin G and H; Daedalin A; trans-3,4-dihydroxy- 3,4-
dihydroanofinic acid, liquiritigenin; methyl trans-sinapate, psoralene and (S)-marmesin)
are also capable of noticeably reducing glucose absorption into bloodstream via inhibiting
alpha-glucosidase (Dang et al., 2017a; M. T. T. Nguyen et al., 2018).
To sum up, literature research on P. trimera showed that this is a potential medicinal plant
with rich chemical composition. Multiple important biological functions are available such
3
as cytotoxicity on cancer cell lines, protection over the liver or anti-inflammatory actions.
Therefore, we need further researches to facilitate the medical use of this plant.
2. Hairy roots induced by Agrobacterium rhizogenes mediated genetic transformation
“Hairy roots”, initially a term regarding the excessive growth of a single or clusters of
adventitious roots in dicot plants Moore, Warren, & Strobel, 1979), was first found and
reported in nursery apples (Woodworth, 1892) and later mentioned in a survey of fruit –
diseases in Western New York (Fred Carlton, Fred Maas, & Frank Henry, 1900). The
malformation of roots is small and somewhat hair-like, hence the name “Hairy roots”.

Figure.0.2. Schematic representation of Agropine type Ri plasmid of A. rhizogenes

(Ozyigit, Dogan, & Artam Tarhan, 2013)

The disease is a natural biotransformation caused by Agrobacterium rhizogenes, in which

the copy of T-DNA (termed T-strand) on Ri-plasmid (root inducing plasmid) of the bacteria

4
is integrated into the genomes of infected tissues (Spano, Pomponi, Van Slogteren, &
Tempe, 1982). T-DNA consists of 2 regions: TR on the right containing aux genes
responsible for autonomous auxin production for independent growth on hormone-free
medium, and TL on the left harbouring 4 rol genes (rolA, rolB, rolC and rolD) of the rooting
locus that induce hairy root.(Cardarelli et al., 1987; Sevón & Oksman-Caldentey, 2002).
VirD2 gene on another region of Ri-plasmid, encodes VirD2 protein that plays a role in the
synthesis and transfer of T-strand into infected host cells. (Stachel, Timmerman, &
Zambryski, 1986))
Regarded as an impressive tool of dicot-plant genetic transformation, up to date, there have
been numerous researches on bioactive phytochemicals production in medicinal plants
applying hairy root culture, internationally and domestically. For instances of international
publications, the yield of the anti-malarial compounds sesquiterpene artemisinin in
transformed roots of Artemisia annua is reported to be superior to the whole intact plants
(Weathers, Cheetham, Follansbee, & Teoh, 1994); the transformed hairy roots of Digitalis
lanata heightens in anthraquinones and flavonoids contents (Pradel, Dumke-Lehmann,
Diettrich, & Luckner, 1997); and the hairy root production of puerarin, a medicinal
compound used in both common food and alternative medicines, in Pueraria phaseoloides
increases by about 1.6 times compared to normal plants (Shi & Kintzios, 2003). Another
example, hairy root cultures of Panax vietnamensis (Vietnamese ginseng) resulting in
higher amount of several ginsenosides (L. T. Ha et al., 2016; T. L. Ha et al., 2014), has
shown the potential and success of this technique in both foreign and national contexts.
Other accomplished studies on medicinal plants in Vietnam includes induction and culture
of hairy roots in Platycodon grandiflorum (Phương, Bạch, & Phương, 2016), in Salvia
miltiorrhiza (Ninh, Lê, Lã, Nguyễn, & Nguyễn, 2015) and the designed construction of a
competent hairy root vector that contains rol genes examined on Nicotiana tabacum
(tobacco) (Le, Tran, Pham, Chu, & Duong, 2013).
In overall, the induction and culture of hairy root in plants or medicinal plants specifically,
have been shown to have advantages such as strong growth, no land orientation or
dependence on exogenous growth regulators, hereditary stable and capable of synthesizing
5
secondary metabolites with equal or higher concentrations compared to original plants.
Therefore, it is necessary to study the hairy root induction and culture in P. trimera

II/ OBJECTIVES

In this thesis, the aim is to examine the method and to optimize conditions for
Agrobacterium rhizogenes - mediated transformation to induce hairy root generation on
Paramignya trimera.

III/ MATERIALS AND METHODS

1. Materials
1.1. Plant materials
P. trimera seeds were obtained from Ninh Van, Ninh Hoa, Khanh Hoa, examined and
confirmed to be identical to P. trimera samples from National Institute of Medicinal
Materials.1-month-old saplings used for in planta transformation were obtained by
germinated seeds in soil.
1.2. Agrobacterium rhizogenes strands

2 different strands of A. rhizogenes, K599 and TR175 are obtained from Institute of
Biotechnology via Assoc Prof. Phạm Bích Ngọc.
1.3. PCR and DNA electrophoresis materials and equipment

Primers for 2 genes, RolC and VirD2, are bought from the company of Macrogen, Korea.
DNA templates were extracted from root induced by A. rhizogenes mediated
transformation. DNA root extracts from non-transformed plants were used as the negative
control. The positive control is the cultured A. rhizogenes bacteria.
PCR and electrophoresis kit and machines are from LMI RICE and USTH PMAB
laboratories.
6
 1.4. Media and other chemicals

The basic medium of 2% sucrose Woody Plant medium (WPM) from Duchefa biochemie
was used for all process of in vitro culture.
For regeneration of plant tissues, different kinds of auxin or cytokinin were supplemented
into the basic medium, including:
- Auxin: 2,4D (2,4 Dichlorophenoxyacetic acid), IBA (Indole-3-butyric acid), α-NAA
(α- Naphthaleneacetic acid), IAA ( Idole-3-acetic acid).
- Cytokinin: TDZ (thidiazuron) and BAP (Benzyl-amino-purine)
For in vitro culture of plant tissues undergone transformation, the basic medium was added
either 200µM Acetosyringone (AS) (for co-culture step) or antibiotics (for selection step).
For bacteria culture, the medium is solid and liquid Yeast Mannitol Broth (YMB).
For hydroponic plant culture, medium is a solution of MS/10 added Ridomil fungicide at
2,5g/L at the first month after in planta transformation and 0,25g/L for the rest of the
experiments to cope with root rot by mycorrhizal root fungus.
The hydroponics systems used includes the hydroponic chamber of Institute of
Biotechnology and a self- made system consisting of 3-liter-glass bowls and fish tank
aerators.
2. Methods
2.1. In vitro regeneration of plant tissues for in vitro transformation
Plant tissues and seeds were surface sterilized in 75º ethanol for 5 minutes and 4% NaClO
for 30 minutes before placing in culture medium.
- To obtain in vitro shoots and cotyledons, P. trimera seeds were germinated in solid
basic medium without any supplementation.
- The culture supplemented with one of two different kinds of cytokinin, TDZ
(thidiazuron) and BAP (Benzyl-amino-purine) at different concentrations was used
to germinate multi-shoot seedlings.
- For in vitro callus regeneration, pieces of in vitro cotyledons were cultured in basic
medium supplemented with one of two types of auxins ( 2,4D (2,4
7
 Dichlorophenoxyacetic acid) or IBA (Indole-3-butyric acid)) at the different
concentrations.
- To increase plant materials for transformation, 2 cm-segments of in vitro germinated
shoots were cultured in the basic medium added either 2,4D or IBA at the different
concentrations.
2.2. In vitro A. rhizogenes – mediated transformation
2 different wild type strains of A. rhizogenes, K599 and TR175, were used for
transformation. Bacterial suspension was pre-cultured in 5mL of liquid YMB for 24 hours
at 28ºC in orbital incubator at 120 rpm, then cultured in 35mL of liquid YMB for 24 hours
at the same temperature and orbital speed. The bacterial cells were collected by
centrifugation at 4000 rpm for 10 minutes at 4ºC and suspended in liquid WPM
supplemented with 200µM AS for inoculation (OD600nm = 0.6)
In vitro plant tissues were wounded using a sterile surgical blade then immediately
immerged in bacterial suspension for 30 minutes. Sequentially, the inoculated tissues were
placed on sterile tissue papers for drying before incubating in the co-culture medium in the
dark at 28°C from 24 to 72 hours. After co-culture, the tissues were washed with liquid
WPM added antibiotics (Carbenicillin, Cefotaxime and Streptomycin at different
concentrations), dried by sterile tissue paper and placed in the solid selection medium with
the same concentration of antibiotics in order to eliminate bacterial growth.
2.3. In planta A. rhizogenes – mediated transformation
Roots of intact plants are wounded using sewing or syringe needles, then incubated in
bacterial suspension as described above for about 12 hours. Next, plant roots are washed
with tap water, and transferred to hydroponics system with added antibiotics and fungicide.
2.4. Molecular characterization of hairy roots
To confirm that the induced roots are transformed hairy roots, PCR with the specific
primers following the time and temperature cycle as depicted in the appendix, and DNA
gel electrophoresis are applied.

8
The presence of rolC and VirD2, two genes on bacterial T-DNA, are used to check the T-
DNA integrated into infected plant tissues and the presence of remaining bacteria after
incubation, respectively. Genomic DNA templates from induced and normal roots are
extracted for genetic transformation validation and negative controls following the CTAB
procedure (Stewart & Via, 1993). Cultured bacteria were used as the positive control.
PCR products are analysed by DNA 1% agarose gel in TAE 0.5x buffer electrophoresis for
30 minutes and dyed in EtBr for 1-1,5 hours. The results are observed under UV light in
UV chambers.

IV/ RESULTS AND DISCUSSIONS

1. In vitro callus and shoot tissues regeneration for genetic transformation

Here, we had performed the first experiments to prepare materials for in vitro genetic
transformation and obtain preliminary results without statistical tests.
1.1. Callus regeneration
In order to find out the optimal phytohormone conditions for callus regeneration from P.
trimera cotyledons, 2 types of auxin, 2,4D (2,4 Dichlorophenoxyacetic) and IBA (Indole-
3-butyric acid), were examined in 4 concentrations for each, ranging from 1.0 to 2.5 mg/L.
The results are presented in table 1 and figure 3.
Table 1. Callus formation from cotyledon under different conditions of supplemented auxin

Hormone Time for callus Percentage of sample Morphology of callus

concentration induction (week) having callus
(mg/L) induction (%)
Control 0
2,4D 1.0 0
1.5 3 24 Brown and spongy. 1 week after formation,
become friable and dark brown (Fig.3A)
2.0 2 79 White to light brown and spongy. 3 weeks
after formation, become friable and dark
brown

9
 2.5 2 80 White to light brown and spongy. 3 weeks
after formation, become friable and dark
brown (Fig.3B)
IBA 1.0 0
1.5 3 17 Brown and spongy. 1 week after formation,
become friable and dark brown
2.0 2 70 White to light brown and spongy. 3 weeks
after fonavigrmation, become friable and
dark brown
2.5 2 65 White to light brown and spongy. 3 weeks
after formation, become friable and dark
brown

Figure 3. Callus induced from P. trimera cotyledon in the WP medium supplemented by 1.5 mg/l
(A) and 2.5 mg/l 2,4D (B) after 2 weeks of culturing

From the table above, the concentration of hormones between 2.0 to 2.5 resulted in the
highest viability of calluses. There are little differences between 2,4D and IBA, with 2,4D
proves to be more effective than IBA in terms of callus viability.
However, for both 2,4D and IBA, the induced calluses are only suitable for transformation
in about 3 weeks.
In comparison to the publish research “In vitro propagation of Xao tam phan (Paramignya
trimera (Oliv.) Guill.)” (Tran et al., 2017) that regenerated calluses from matured leaf
segments, callus regeneration from cotyledons appear to be more effective, as in overall,
the time for callus induction in WPM medium added auxin is much shorter, in 2 weeks
instead of over 12 weeks. The percentage of callus – induced cotyledon tissues is also higher
10
than matured leaves, about 65% -80% and 11,7%-46,7%, respectively. Moreover, while a
combination of BA and IBA was required for callus regeneration in matured leaf segments,
it only took either IBA or 2,4D to induce callus on cotyledon pieces. This could be since
cotyledon tissues are much less differentiated than matured leaves, which increases the
chances and reduces time of de-differentiation.
1.2. Shoots induction
Different from callus regeneration which used cotyledon tissues, shoots were obtained
directly from in vitro germinated P. trimera seeds in basic medium. 2 types of cytokinin
were supplemented into WPM in the concentration of 1 mg/L in comparison to natural
conditions. The results are depicted in table 2 and figure 4.

Table 2. Shoot induction from cotyledon under different conditions supplemented by BAP and TDZ at
concentration of 1 mg/l

Hormone Time for

Germination Number of Morphology of seedling (7 days
type germination
percentage (%) shoots/seed after germination)
(1mg/L) (days)
Shoots are thinner, dark green,
wood appears earlier (2-3 weeks
No
2 100 1 after germination).
hormone
Only primary root, white, longer.
(Fig.4.A)
Shoots are bigger, shorter, light
TDZ 2 100 2-4 green. Wood appears much later.
No root or shorter root.
Shoots are bigger, shorter, light
BAP 2 100 3-4 green. Wood appears much later.
No root or shorter root. (Fig.4.B)

11
 Figure 4. Shoot induction from P. trimera seed in the WP medium without (A) and supplemented by
1mg/l of BAP (B) 2 weeks after germination.
Like 2 types of auxin, there is marginal differences between TDZ and BAP, with the later
shows a slightly higher effect. This result is somehow similar to the sprouting effects of
TDZ and BAP examined in the research aimed to regenerate shoots from dormant sprouts
on mature stems of P. trimera (Trương, Nguyễn, Nguyễn, & Trần, 2015), with BAP
performed noticeably better than TDZ. However, there are remarkable differences between
shoots regeneration from seed germination and from dormant sprouts on stem, regarding
shoot morphology, sizes and growth time.
The delayed development of wood in shoots in cytokinin-supplemented medium is likely
due to the proliferation promotion effect of cytokinin which retains cells at non-
differentiated state, which could be beneficial for transformation and root induction.

1.3. In vitro root induction test

Since the plant material available was limited, the explants for genetic transformation were
increased by culturing small pieces of in vitro shoots induced without cytokinin in 2%-

12
sucrose-1%-agar-WPM added either 2,4D and IBA at a high concentration of 3mg/l. This
also tested the rooting ability of in vitro cultured shoot tissues of P. trimera.
The results are shown in table 3 and figure 3.
Table 3: Root induction from piece of in vitro shoot in the WPM conditions supplemented by 2,4D
and by 3mg/l IBA (B)
Hormone (1mg/L) Time for root induction Root induction per Length of root (cm)
(weeks) centage (%)
No hormone
2,4D 3-4 1000 0.6
IBA 2-3 100 3.3

Figure 5. Root induction from P. trimera piece of in vitro shoot in the WP medium supplemented by
3mg/l 2,4D (A) and by 3mg/l IBA (B)

The results show that both auxins induced roots on shoot explants, and IBA is noticeably
more effective.
As mentioned above, the delayed wood development and differentiation of germinated
shoots in cytokinin-supplemented medium could be the reason for the success of root
induction from germinated shoot segments compared to matured shoot segment, as no in
vitro root had been induced in matured shoot segments in the publish research “in vitro
propagation of Xao tam phan (Paramignya trimera (Oliv.) Guill.)” (Tran et al., 2017).

13
Nonetheless, this experiment does not include the survival and growth of root-induced-
shoot segments, which should be further investigate.

2. In vitro transformation
After successfully prepared plant materials (including regenerated calluses, shoots and
cotyledons), genetic transformation was performed according to the method mentioned
above. Four conditions for in vitro Agrobacterium rhizogenes mediated transformation
method were examined for the optimization of hairy root induction are bacteria strains,
time of co-cultivation with bacteria and antibiotic combinations for bacteria elimination
after co-cultivation.

2.1. Conditions optimization: Tissue types

Among three tissue types used in this genetic transformation experiment, cotyledon
appeared to be the most resilient and viable after the antibiotic selection step. In vitro shoot
tissues germinated from both natural and hormone-supplemented media showed no
differences, and both died afterwards. For calluses, the spongy tissues led to difficulty in
bacteria eradication.

2.2. Conditions optimization: Bacteria strains

Apparently, both K599 and TR175 strains of A. rhizogenes have similar performances with
marginal differences. The K599 strains, however, displayed faster and stronger growth on
solid YBM agar medium, even when preserved at 4℃, which will reduce incubation time
for bacteria preparation. However, this could lead to quicker degeneration of the K599 stock
bacteria if not preserved at -80℃ in the long run.

2.3. Conditions optimization: Time of co-culture

Time of co-culture in this experiment ranges from 12h to 72h. It is found that the longer the
co-culture time, the higher the chances of successful genetic transformation. Nevertheless,
the removal of bacteria in the following step will be more challenging as the co-cultivation
time increases. Therefore, the time range of 24h is likely the most effective in both terms.

14
 2.4. Conditions optimization: Antibiotics combinations
Each antibiotic was tested separately to assess antibacterial capacity at several
concentrations. While Cefotaxime demonstrated the best antibacterial capacity, it also
caused the culture medium to change colours from semi-transparent white to dark pink, as
shown in the figure below, ultimately kill off the cultured tissues.

Figure 6. Pink colour appeared 1 day after transferring to cefotaxime-supplemented medium, upper
view (A) and lower view (B)

Carbenicillin and Streptomycin , although only followed up in the second and third ranks,
showed no toxic reactions to cultured tissues. Therefore, a combination of antibiotics was
used to effectively remove bacteria without harming the tissues.
The effective combination of antibiotics currently used includes Cefotaxime (1g/L),
Carbenicillin (1g/L) and Streptomycin (200mg/L).
After the optimization of antibiotic conditions in vitro culture of transformed tissues, we
ran out of seed samples. Therefore, in vitro studies were halted until the following fruiting
seasons of P. trimera in September 2019. The first attempt of in vitro cultured of
transformed tissues (stems and cotyledons) proved unsuccessful.

15
When we retrieve more fruits and seeds sample in September 2019, we continued the in
vitro transformation experiments following a similar-to-the-first- try-procedure. We are
now performing experiments and waiting for the root induction.
3. In planta transformation
In comparison with in vitro transformation method, in planta transformation and
hydroponics culture harvested promising results. The first round of transformed plants
started rooting after 3 months. The photos below were taken after 4-6 months of hydroponic
culture in MS/10 medium (Fig.8). The numbers of roots sprouted and percentage of rooting
plants over the total number of plants undergone transformation method increased along
with cultured time. The induced roots in hydroponic culture are much thicker, less branched
and have slower lignification than regular roots in soil (Fig.7). The type of induced roots
resembles single hairy roots and a broom-knot-like group without the tumour knot.
(Hedgcock, 1908)

Figure 7. Roots of natural plant in soil

16
 Figure 8. Induced roots on plants from the 1st experiments of in planta transformation

The second and third rounds of transformation shows similar results with rooting initiation
time shorten down to 1 month. Root morphology is the same as the first round (Fig.9 &
Fig.10). The percentage of plants developed induced roots over the total number of plants
undergone transformation is shown in table 4. Although the percentage varied considerably
among experiments, the overall efficiency is high. In addition, since some plants took
longer to grow induced roots, the efficiency is more likely to increase along with cultivation
time.
In overall, the longer the hydroponic culture time, the newer induced roots sprouted
(white roots). Later spouted young roots are more in numbers, stronger, thicker and longer
than the first-appeared ones.

17
Table.4. The efficiency of in planta transformation by the percentage of plants developed induced roots
1st experiment 2nd experiment 3rd experiment

˷ 60% 80% 40%

The average efficiency: 60%

Figure 9. Induced roots on plants from the 2nd experiment of in planta transformation

Figure 10. Induced roots on plants from the 3rd experiment of in planta transformation

18
4. Transformation analysis
To investigate the integration of T-DNA, the induced root was sampled and rolC gene
(539bp) localized within T-DNA was sought. Figure 11.A shows that rolC was detected in
both hairy root and bacteria. The normal adventitious roots were used as negative control.
To confirm that the target for PCR amplification of the rolC gene was T-DNA incorporated
into the plan genome, but not DNA contamination from remaining bacteria, PCR analysis
using virD2 primer (338bp) was performed. The absence of the PCR product of VirD2
indicated that transformation had occurred and that hairy roots were bacteria-free. As
shown in the figure 11.B, virD2 was only detected in K599 strains.
A B

Figure 11. Stable integration of rolC genes was determined by PCR analysis of genomic DNA extracted
from in planta transformed hairy root.
A: Detection of rolC (539 bp) in transformed hairy root (Rol +, transformed hairy root), untransformed
roots (Rol -, negative control) and in A.rhizogenese K599 (Rol/K599).
B: Detection of VirD2 in only A.rhizogenese K599; Vir/+, transformed hairy root; Vir/K599, A.rhizogenese
K599; Vir/-, negative control

Literature research on P. trimera showed that this is a potential medicinal plant with rich
bioactive compound as well as bioactivities (V. T. Nguyen, 2017; V. T. Nguyen, Bowyer,
Vuong, Altena, & Scarlett, 2015; Son, 2017). Moreover, the induction and culture of hairy
root medicinal plants have been shown to have advantages such as strong growth, no land

19
orientation or dependence on exogenous growth regulators, hereditary stable and capable
of synthesizing secondary metabolites with equal or higher concentrations compared to
original plants (Guillon, Trémouillaux-Guiller, Pati, Rideau, & Gantet, 2006a). The major
factors that effect on the efficiency including the Agrobacterium strains, the co-culture
conditions, the explant types were reported (Guillon, Trémouillaux-Guiller, Pati, Rideau,
& Gantet, 2006b). In this project, we studied the optimal conditions for hairy root induction
by Agrobacterium rhizogenese mediated transformation by using 2 different
Agrobacterium strains, 3 type of tissues with different time of co-cultivation for in vitro
transformation. Besides, the in-planta transformation was also tested.
The first attempts of in vitro transformation failed as the culture medium changed colours
from semi-transparent white to dark pink after adding cefotaxime that ultimately kill off the
cultured tissues (Fig.6). Antibiotic may negatively influence on the plant tissues, especially
in genetic transformation process as the tissue is more sensitive (Wiebke, Ferreira, Pasquali,
Bodanese-Zanettini, & Droste, 2006). We hypothesized that the pink color may be caused
by the reactions between this antibiotic and one/some metabolites in the plant tissues. To
overcomes, the different combinations of antibiotic were tested, it seems that the effective
combination of antibiotics currently used includes Cefotaxime (1g/L), Carbenicillin (1g/L)
and Streptomycin (200mg/L).
Other factors were tested including the Agrobacterium strains, the time of co-cultivation,
the explant types. It seems the most effective time of co-cultivation is 24 hours to eliminate
the bacteria in the next steps, meanwhile, any significant difference was recognized when
using 2 bacteria strains. However, since we have not yet induced the hairy root by this in
vitro transformation methods, further experiments need to be performed.
In parallel, the in planta transformation was tested in which the intact plant was merged in
the bacteria suspension overnight as the co-cultivation process. In planta Agrobacterium
transformation gives a practical alternative in overcoming challenges that arise from in-
vitro transformation such as meticulous sterilization (Karthikeyan, Shamala, & Wei, 2018)
This technique was widely used to generate transgenic hairy roots in Phaseolus acutifolius,

20
P. vulgaris, P. coccineous, P. lunatus, Vicia hirsuta, V. faba, Medicago
truncatula and Pisum sativum. (Estrada-Navarrete et al., 2006; Vieweg et al., 2004). In
our studies, the hairy roots were successfully induced with the average efficiency of about
60% (Table.4). The PCR result also confirmed the T-DNA was successfully integrated into
the plant genome. This indicated the techniques in-planta transformation can be used to
induce hairy roots in P. trimera with high efficiency. The next step is to test the condition
for hairy root culture to get the root biomass.

V/ CONCLUSIONS AND PERSPECTIVES

1. Conclusions
In this study, the hairy roots were successfully induced by in planta transformation by using
Agrobacterium rhizogenese, K599 and TR175 strains. The efficiency of transformation is
around 60%.
Additionally, although in vitro transformation was not successful, in vitro tissue
regeneration to prepare genetic transformation materials have been preliminary
experimented and the 4 conditions for in vitro transformation have been optimized.
2. Perspectives
The time allowed was not sufficient for the study of root biomass production by hairy root
culture due to the slow growth and development of hydroponics cultured plants and roots.
Therefore, the next step is to examine the conditions for hairy root culture to increase root
biomass production, as well as the medicinal compound contents and bioactivities of
induced hairy roots in comparison to natural roots.
In additional, the results on in vitro culture to produce the in vitro callus, hypocotyl and
cotyledon will be used for further studies such as in vitro micro-propagation or in vitro
adventitious root production. More experiments and repeats on tissue preparation are also
needed.

21
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 APPENDICES

APPENDIX 1
PCR cycles for transformation confirmation

For each test, 35 PCR cycles of 20-25 µL are performed at temperatures and times
according to the figure below.

Figure 1. PCR cycles: temperatures and times

26
 Composition of MS

Micro Elements mg/l

CoCl2.6H2O 0.025

CuSO4.5H2O 0.025

FeNaEDTA 36.70

H3BO3 6.20

KI 0.83

MnSO4.H2O 16.90

Na2MoO4.2H2O 0.25

ZnSO4.7H2O 8.60

Macro Elements mg/l

CaCl2 332.02

KH2PO4 170.00

KNO3 1900.00

MgSO4 180.54

NH4NO3 1650.00

27
Vitamins mg/l

Glycine 2.00

myo-Inositol 100.00

Nicotinic acid 0.50

Pyridoxine HCl 0.50

Thiamine HCl 0.10

Composition of WPM

Micro Elements mg/l

CuSO4.5H2O 0.25

FeNaEDTA 36.70

H3BO3 6.20

MnSO4.H2O 22.30

Na2MoO4.2H2O 0.25

ZnSO4.7H2O 8.60

28
Macro Elements mg/l

CaCl2 72.50

Ca (NO3)2 .4H2O 471.26

KH2PO4 170.00

K2SO4 990.00

MgSO4 180.54

NH4NO3 400.00

Vitamins mg/l

Glycine 2.00

myo-Inositol 100.00

Nicotinic acid 0.50

Pyridoxine HCl 0.50

Thiamine HCl 1.00

29
30
31