Agrobacterium-Mediated Genetic
Transformation System of Amorpha fruticosa using Callus from the
Cotyledonary Node
Yiteng Zhang1†, Jiali Liu1†, Ailing Zhong1, Ziang Liu2, Kai Wang2, Zhenyu Wang3, Minghui
Li1, XiuFeng Li3 and Qingjie Guan1*
1Key Laboratory of Saline-alkali Vegetation Ecology
Restoration, Ministry of Education, College of Life Sciences, Northeast
Forestry University, Harbin 150040, China
2Forestry Institute, Northeast Forestry University,
Harbin 150040, China
3Northeast Institute of Geography and Agroecology, Key
Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences,
Harbin 150081, China
*For correspondence: guanqingjie@nefu.edu.cn
†Contributed equally to this work and are co-first
authors
Received 15 November 2021; Accepted 25 February 2022; Published 30
March 2022
We used Agrobacterium-mediated
infection of callus induced from the cotyledonary nodes of Amorpha fruticosa L. to study the β-glucuronidase
gene (GUS)-integrated genetic transformation system. Transformed calluses were selected
under 40 mg·L−1 kanamycin, differentiated into resistant
adventitious buds, and developed into transformants. A single copy of gus was integrated in the genome of most T0 generation
plants. Gus chemical staining analysis showed blue color in resistant calluses,
adventitious buds, and the roots and leaves of transformed plants. This
indicated gus overexpression driven by the 35S
promotor and resultant β-glucuronidase activity. The genetic
transformation system in this study could be used to study other functional
genes of A. fruticosa and facilitate transgenic
breeding for strain improvement. © 2022 Friends Science Publishers
Keywords: Amorpha fruticosa
L.; Agrobacterium;
Cotyledonary node; Callus; Genetic transformation
Introduction
Amorpha fruticosa L. (Fabaceae) is
native to North America. It was introduced into China for use as a windbreak,
for soil erosion control, and as an ornamental plant (Wang et al. 2002).
A. fruticosa is not only an important landscape
ecological tree species, but also a commodity tree species with high
economic value. Its extracts contains biologically active substances of
medicinal value. A. fruticosa has been used as
an herbal medicine to treat fever, burns, purulent edema, and eczema in China.
Induction of cell division has been used to study the activity of flavonoids
extracted from A. fruticosa leaves. These
flavonoids have potential value in new drug development (Hovanet
et al. 2015). Fifteen medicinal ingredients including glucopyranoside,
vitexin, and chrysoeriol have been isolated from A.
fruticosa leaves (Cui et al. 2017). Development of new A. fruticosa varieties is normally accomplished using
conventional breeding but transgenic technology provides an alternative means
for genetic improvement of A. fruticosa. Plant genetic transformation is an
important aspect of genetic engineering technology. It is defined as a series
of events starting from the selection of required genes, delivery, integration
into plant cells, expression and finally the production of the whole plant (Choudhury and Rajam 2021). Guan and Luo (2009) developed
a regeneration system for callus induced in the stem segements
of A. fruticosa.
The Agrobacterium-mediated genetic transformation
system has often used the GUS for the genetic analysis. Jefferson et al.
(1987) cloned GUS from Escherichia coli strain K-12.
GUS is commonly used as a reporter gene in plant genetic transformation. β-glucuronidase
is characterized by high stability, wide pH range and
easily detected activity. It catalyzes the X-Gluc
hydrolysis reaction and produces dark blue compounds (which are presented as
blue spots) in plants. This facilitates evaluation of transformation effects or
transformation efficiency (Shimomura et al. 1962).
To develop a basic method for the study of functional genes and new line
development of A. fruticosa, we used Agrobacterium to mediate GUS
transformation. In this research we infected callus induced from cotyledonary
nodes of A. fruticosa to establish an
effective genetic transformation system.
Materials and Methods
Experimental material
Plant materials (A. fruticosa seeds) were gifts of Wu Songquan,
School of Agriculture of Yanbian University (Jilin
Province, China). The bacterial strain used,
Agrobacterium tumefaciens EHA105, carried the pBI121-GUS plasmid were available stored in our
laboratory. Cetyltrimethyl
ammonium bromide (CTAB), deoxynucleoside triphosphate
(dNTP), and Taq polymerase, were purchased from TaKaRa
Biotechnology (Dalian) (Liaoning Province, China). Kana and X-Gluc were purchased from Promega (Beijing, China).
Carbenicillin disodium (Carb) was purchased from Sangon
Biotech (Shanghai, China). The 6-benzylaminopurine (6-BA),
2,4-dichlorophenoxyacetic acid (2,4-D), naphthylacetic
acid (NAA), Kinetin (KT), and 1-(2-chloro-4-pyridyl)-3-phenylurea
(CPPU) were purchased from Nachuan (Harbin, China). Culture
media composition were as follows:
(1) The
medium for callus induction of the cotyledonary node of
A. fruticosa contained MS Medium
(MS) + 6-BA 3.0 mg·L−1 + NAA 1.0 mg·L−1 +
2,4-D 0.5 mg·L−1;
(2) The co-culture
medium was the callus induction medium plus acetosyringone
(AS), containing MS + 6-BA 3.0 mg·L−1 + NAA 1.0 mg·L−1
+ 2,4-D 0.5 mg·L−1 + 20 μmol·L−1 AS;
(3) The callus induction and
screening medium contained MS + 6-BA 3.0 mg·L−1
+ NAA 1.0 mg·L−1 + 2,4-D 0.5 mg·L−1+ 40 mg·L−1
Kana + 500 mg·L−1 Carb;
(4) Screening medium for differentiation of adventitious buds from
callus contained MS + 2 mg·L−1CPPU
+ 2 mg·L−1 KT + 40 mg·L−1 Kana+ 500 mg·L−1
Carb;
(5) The rooting medium
contained 1/2 MS + 0.1 mg·L−1 NAA + 40 mg·L−1
Kana.
Experimental methods
Callus induction in A. fruticose:
Full-size mature seeds were sterilized with 70% (v/v)
alcohol and 5% sodium hypochlorite and inoculated into 1/2
MS medium
for germination. When the two cotyledons unfolded, the
hypocotyl was cut. The separated cotyledons were placed face up on the callus
induction medium, followed by incubation at 23–25°C in a tissue culture chamber
with an illumination intensity of 54 μmol
m−2s−1
and a 14:10 (L:D) photoperiod.
Agrobacterium-mediated transformation: A single colony of Agrobacteria
containing the plasmid of interest (pBI121-GUS) was picked and cultured in 100
mL yeast extract peptone (YEP) medium containing 50 mg·L−1
Kana + 100 mg·L−1 Rifampicin at 28°C and 140 rpm for 48 h.
Once the bacteria were grown to approximately ODλ600 = 0.5
(measured by ultraviolet (UV)-spectrophotometry), AS at a final concentration
of 20 μmol·L−1 and 1:10,000 (v/v) Triton X-100 were added
to the bacterial culture, which was then used to immerse the calli of the cotyledonary nodes. After 10–15 min of
infection, the calli were placed on sterile filter
paper to remove excess bacterial liquid (Guan et al. 2019) and incubated
with the co-culture medium at 25 ± 2°C in darkness for 3 d. Subsequently, the calli were inoculated into the callus induction and
screening medium and grown in the tissue culture chamber (under the same
conditions as previously described). Resistant calli
were inoculated into the adventitious bud differentiation screening medium to
screen the regenerated and transformed buds. The differentiated resistant buds
were rooted and cultured in plant rooting medium to become resistant
regenerated plants. After ventilating for acclimatization, the seedlings in the
culture pots were moved into the plant culture room, the culture conditions
were set as the culture temperature of 25 ± 1°C, photosynthetically active
radiation of 57 µmol/m−2s−1 (cool-white
fluorescent lamps as light resource), and an artificial 10/14 h light/dark
cycle.
DNA extraction and PCR: Genomic DNA extraction was
performed using the CTAB method (Sambrook and Russell 2006). The genomic DNA of the
Kana-resistant plants was extracted and the wild-type A. fruitcosa
genomic DNA was used as a control. DNA template at 1/100 dilution was used to
perform PCR to amplify 5,791–7,747 bp PCR products using GUS-Forward and
GUS-Reverse (GUS-F, R) primers. These were designed according to the pBI121-GUS
sequence to detect the GUS integration. The PCR products were separated by 0.8%
agarose gel electrophoresis.
Southern blot analysis of insert copy number of the transgenic lines: The genomic DNA (10 µg)
of the leaves of GUS transgenic A. fruitcosa
lines #1, #5, #6, #18, and #20 at the T0 generation extracted by CTAB method
was incubated with HindIII/BamHI
restriction enyzmes overnight at 37°C, followed by
separating the cleavage products by 1.2% agarose gel electrophoresis (45 V) for
approximately 9 h. A gel imager was used to detect the enzymatic digestion of
the DNA. After denaturation, membrane transferring, DIG-labeled GUS (573 bp)
probe hybridization (DIG-labeling kit purchased from Roche), membrane washing,
and developing in CDP-StarTM reagent
(Roche), the membranes were placed on the Image Quant LAS 4000 imaging analyzer
(GE Healthcare Life Sciences in Germany) for signal detection (Agrawal et al.
2000).
Northern blot analysis of the
integrated GUS expression in A. fruitcosa at
T0 generation: Total RNA of the GUS transgenic A.
fruitcosa lines #1, #5, #6, #18, #20 at the T0
generation was prepared by the Biozol
one-step method. Five micrograms total RNA was then denatured at 65°C for 10
min and subsequently separated by 1.5% formaldehyde-agarose gel
electrophoresis, followed by transferring the RNA onto a Hybond-N+
nitrocellulose membrane. The RNA was cross-linked
by UV irradiation on the membrane, followed by DIG-GUS DNA probe hybridization
at 50°C for 12 h and developing in CDP-StarTM
reagent before signal detection by LAS 4000 imaging analyzer (Mamiatis et al. 1985).
Histochemical
staining analysis of β-glucuronidase activity in the genetic transformants
under the 35S promoter: GUS histochemical staining was
performed as described by Jefferson (1987) and Sieburth
and Meyerowitz (1997). The materials were first soaked in a buffer containing
100 mM sodium phosphate buffer pH 7.0, 0.5 mmol·L−1 potassium
ferrocyanide, and 0.5 mmol·L−1 potassium ferricyanide. The
materials included the calli induced by transfection
of pBI121-GUS plasmids, the resistant buds on the differentiation screening
medium, and the roots and leaves of resistant regenerated plants. Wild-type
(WT) corresponding tissues were used as control. After rinsing, the GUS
staining solution (50 mmol·L−1 sodium phosphate buffer pH 7.0
containing 0.5 mmol·L−1 K3[Fe(CN)6], 0.5
mmol·L−1 K4[Fe(CN)6], 10 mmol·L−1Na2EDTA,
0.1% (v/v) Triton X-100, 20% methanol, and 0.5mg·mL−1 X-Gluc) was incubated at 37°C overnight and then the staining
was observed.
Results
GUS transgenic A. fruitcosa: line establishment and PCR results
After soaking and infecting the
callus of A. fruticosa cotyledons with
pBI121-GUS, the resistant calli were selected by
induction, and the Kana-resistant buds were selected by adventitious bud
differentiation. The rooting culture was selected to obtain transformed seedlings.
The acclimated seedlings were cultivated in pots and grown into GUS transgenic
lines with GUS overexpression (Fig. 1).
To analyze the 35S-GUS integration of Kana-resistant
regenerated plant lines (T0), the CTAB method was used to randomly
extract the leaf genomic DNA of 13 lines of transgenic resistant plants,
followed by using GUS detection
primers (GUS-F, R) to perform PCR and separating the PCR products by 0.8%
agarose gel electrophoresis. In (Fig. 2), lanes 1, 3, 4 and 6–13 (positive
controls) of the agarose gel show the integrated GUS DNA fragment of approximately 500 bp size; while no target DNA was
detected in lanes WT, 2, and 5. WT was a negative control, and the DNA sample
of the lane1 was the positive
control. The #1, #3, #4, and #6–13 lines of A. fruticosa
had integrated GUS; while the #2 and #5 lines of A. fruticosa had no integrated GUS.
Gene insertion copy number and overexpression in the GUS transgenic A. fruitcosa lines
We subjected 10 µg of genomic DNA
extracted from the leaves of GUS transgenic seedlings by CTAB methods to BamHI/HindIII restriction enzyme
digestion, agarose gel electrophoresis (Fig. 3), membrane transfer, and
DIG-labeling Southern blot analysis. The CDP Star signal of the Southern blot
was detected by LAS 4000. Figure 3B shows that the transgenic lines T0#1, #3
and #5 had a single-band signal, suggesting that a single copy of GUS was
inserted into the genomic DNA of A. fruticosa
in these lines. However, the transgenic lines T0#4 in the Southern blot had a
double-band signal, suggesting that two GUS copies were inserted into the
genomic DNA of A. fruticosa in the T0#4 line.
Most of the GUS transgenic A. fruticosa lines
had a single GUS gene insertion into the genomic DNA. This confirmed that GUS,
mediated by Agrobacterium transformation, was successfully integrated
into the A. fruticosa chromosome. We used
DIG-GUS labeled Northern blot to detect the GUS transgenic A. fruticosa lines in the T0 generation (T0#1, #3, #5).
The CDP-StarTM signal showed that WT had
no expression compared with the GUS overexpressing lines. The GUS transgenic
lines (T0#1, #3, #5) had a single band of hybridization signal, indicating
that the GUS transgenic A. fruticosa lines
at the T0 generation expressed mRNA of the exogenous GUS gene
(Fig. 4). The protein synthesized by the translation of
GUS expression was β-galactosidas and GUS staining was an effective method to
detect β-galactosidase activity.
Activity of β-galactosidase expressed from the 35S promoter
in resistant callus, adventitious buds, and transgenic lines
The Kana-resistant and
non-transgenic calli containing pBI121-GUS plasmid
after the Agrobacterium-mediated infection were subjected to GUS
staining at 37°C overnight. The surface of most transgenic calli
was blue and only a few calli were not stained. Most
of the transgenic calli were stained in blue and the
control calli without Agrobacterium-mediated
infection were yellowish-white (Fig. 5). The transgenic calli
stained blue confirmed the transient expression and successful plant
transformation. The GUS staining analysis of transgenic resistant adventitious
buds and differentiated WT-adventitious buds showed that the transgenic
resistant adventitious buds were stained blue. The WT-adventitious buds were
not stained, indicating that the resistant adventitious buds had GUS transgene
expression and β-galactosidase activity. In addition, GUS staining
of roots and leaves of regenerated plants lines grown by rooting culture of
resistant adventitious buds showed blue color (Fig. 5C and 5D), indicating that
35S-GUS-integrated transgenic plants overexpress GUS. The 35S promoters
triggered an increase in β-galactosidase activity. These results
demonstrate the feasibility of using Agrobacterium-mediated infection of
callus induced from cotyledons of A. fruticosa
to accomplish genetic transformation.
Discussion
Over the years, the
application of genetic transformation in A. fruticosa
has developed steadily, which shows that the plant can successfully carry out
genetic engineering and combine the characteristics of interest. Genetic
engineering technology has enabled efficient genetic transformation systems for
plants. Transformation can be used to analyze gene function in combination with
gene-knockout technology (Yang and Zhou 2005). Although there are many developments in transgenic A fruticosa technology in different countries, it lags
behind many other important crops. Arabidopsis (Clough and Bent 1998) and rice
(Toki et al. 2006) are model plants for molecular biology research
because their genetic transformants are stable. Due to its strong adaptability,
A. fruticosa can grow at minus 40°C and
Fig. 1: Callus infection of the
cotyledonary node of A. fruticosa for the
transformation of regenerated plants. (A) Cotyledonary node-induced calli; (B) Calli differentiated into resistant adventitious
buds; (C) Regenerated lines of resistant adventitious buds from rooting
culture; (D) Transgenic seedlings of the regenerated lines from soil culture
Fig. 2: PCR detection of GUS transgenic
lines. WT represents a negative control with a DNA template from non-transgenic
plant. “+” represents the positive control with plasmid DNA template; lanes
1–13 represent DNA templates from transgenic plant lines
Fig. 3: DIG-GUS labeled Northern blot was used
to detect the GUS transgenic A. fruticosa line
at the T0 generation (T0#1, #3, #4, and #5). The CDP-StarTM
signal showed that WT had no expression and GUS transgenic lines (T0#1, #3, #4,
and #5) had hybridization signals, indicating that the transgenic A. fruticosa lines at the
Fig. 4: Gene expression signal detected by
Northern blot. The #1, #3, and #5 are the numbers of transgenic plants
where the annual
precipitation is only about 200mm. Its ability to resist flooding, salt and
alkali, barren, wind and sand, insects, tobacco and pollution is very rare in
plant populations (Sun et
al. 2021). It is desirable to establish a genetic transformation system for
A. fruticosa. Stable transformation can
transfer integrated genes in successive generations and meet the requirements
of functional genomics and transgenic breeding (Choudhury and Rajam 2021).
Establish an efficient and high-throughput transformation system for A. fruticosa plants, and finally introduce the required
characters into the plants, so as to improve their yield. A genetic
transformation receptor system with efficient and stable regenerative capacity, sensitivity
to selective antibiotics, and sensitivity to Agrobacterium infection is
required for completing gene transfer.
Selection pressure of
Kana (40 mg·L−1) was used to differentiate the calli of the cotyledons of A. fruticosa
infected by Agrobacteria containing GUS into resistant adventitious
buds. Molecular testing revealed the single- and double-copy insertions in the
regenerated plants (Fig. 3). In addition, GUS at the mRNA level was
overexpressed by the 35S promoter. However, the type of integration was
unrelated to the activity of the translated protein (Papadopoulou
et al. 2005). GUS encodes β-glucuronidase, which hydrolyzes
X-gluc and produces a blue color (Lambé et al. 1995). Detection of β-glucuronidase activity in the
transgenic lines reflects the expression of GUS-encoded protein (Yancheva et al. 1994). Recently, it has been
reported that in addition to transforming Agrobacterium strains, there
are many modified bacterial species of plants. Such as Sinorhizobium
meliloti, Mesorhizobium
loti, ensifer adhaerens,
in which S. meliloti can infect monocotyledons
and dicotyledons (Rathore and Mullins 2018). In this research, 35S promoter
driven Gus overexpression and production β-glucuronidase activity
provides a new direction for transgenic breeding of improved strains.
In conclusion, this study achieved
the goal of successful gene editing and stable transformation of A. fruticosa. The scheme of Agrobacterium
mediated genetic
Fig. 5: GUS histochemical staining of the
process of genetic transformation of A. fruticosa
by Agrobacterial infection. (A) GUS transient expression of the
transgenic calli; (B) GUS expression in resistant
adventitious buds; (C and D) GUS expression in the roots and leaves of the
transgenic lines
transformation of A.
fruticosa was optimized. It is expected that a
major breakthrough in the genetic improvement of A. fruticosa
is no longer far away. Therefore, this method can play an important role in the
functional genomics of A. fruticosa gene and
release the real potential of gene editing in the production of improved A. fruticosa varieties.
Conclusion
We used GUS
histochemical staining to detect β-glucuronidase activity in
callus, adventitious buds, and transgenic lines during A. fruticose
transformation and regeneration. The staining verified successful establishment
of a genetic transformation system with efficient and stable regenerative
capacity, sensitivity to selective antibiotics, and sensitivity to Agrobacterium
infection of A. fruticosa.
Acknowledgements
This work was supported
by the Fundamental Research Funds for the Central Universities (No.
2572021DS03), and the College Students’ innovation and Entrepreneurship
Project.
Author Contributions
Qingjie Guan conceived and
designed the study. Yiteng Zhang and Jiali Liu performed the experiments and drafted the
manuscript. Ailing Zhong and Ziang Liu contributed to
the sample measurement and data analysis. XiuFeng Li,
Kai Wang, Zhenyu Wang and Minghui
Li draft revision. All authors read and approved the final manuscript.
Conflicts of Interest
All authors declare no conflict
of interest.
Data Availability
Data presented in this
study will be available on a fair request to the corresponding author
Ethics Approval
Not applicable in this
paper
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