plants
Article
A Highly Efficient Agrobacterium rhizogenes-Mediated Hairy
Root Transformation Method of Idesia polycarpa and the
Generation of Transgenic Plants
Hui Wang 1,2 , Kaimao Cheng 1 , Tongjie Li 1 , Xiaoyu Lan 3 , Li Shen 3 , Huayan Zhao 1, * and Shiyou Lü 1,2, *
Citation: Wang, H.; Cheng, K.; Li, T.;
Lan, X.; Shen, L.; Zhao, H.; Lü, S. A
Highly Efficient Agrobacterium
rhizogenes-Mediated Hairy Root
Transformation Method of Idesia
polycarpa and the Generation of
Transgenic Plants. Plants 2024, 13,
1791. https://doi.org/10.3390/
plants13131791
Academic Editor: Roger Thilmony
Received: 6 May 2024
Revised: 23 June 2024
Accepted: 26 June 2024
Published: 28 June 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Plants 2024, 13, 1791. https://doi.org/10.3390/plants13131791
1 State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University,
Wuhan 430062, China; huiwang@stu.hubu.edu.cn (H.W.); 17764217061@163.com (K.C.);
tongjieli@stu.hubu.edu.cn (T.L.)
2 Hubei Hongshan Laboratory, Wuhan 430070, China
3 Shaanxi Agricultural and Forestry Technology Co., Ltd., Xi’an 710005, China; lxy97@163.com (X.L.);
shenli1963@163.com (L.S.)
* Correspondence: huayanzhao@hubu.edu.cn (H.Z.); shiyoulu@hubu.edu.cn (S.L.); Tel.: +86-27-88663882 (S.L.)
Abstract: Idesia polycarpa is a promising woody oilseed species because of its high oil yield. However,
its use is greatly limited due to the lack of varieties with good qualities; additionally, gene function has
been less studied in this plant because an efficient transformation method has not been established
yet. In this study, we established a rapid and efficient hairy root transformation method by infecting
the whole seedling, the rootless seedling, and the leaf petiole with Agrobacterium rhizogenes using
different infection methods. Among these transformation methods, a higher transformation efficiency
was obtained using the whole seedling, which could reach up to 71.91%. Furthermore, we found that
the seedling age significantly affected the transformation efficiency, either using whole or rootless
seedlings. Additionally, we found that the transgenic roots could regenerate transgenic shoots. Taken
together, our study lays the foundation for future study and for genetically modifying wood traits in
the future.
Keywords: Idesia polycarpa; Agrobacterium rhizogenes; hairy roots; oil-bearing woody plant
1. Introduction
Idesia polycarpa Maxim., a dioecious plant, is a woody oilseed tree primarily found in
Asian countries, including China, Japan, and Korea [1]. Its fruits can be processed to yield
edible vegetable oil [2], which is rich in unsaturated fatty acids (FAs), including palmitoleic
acid (C16:1), oleic acid (C18:1), and linoleic acid (C18:2) [1,3]. This woody plant attracts
significant attention due to its high oil content and nutritional value [4], with potential
applications in healthcare products [4,5], industrial oils [1], cosmetic preparations [6],
landscape gardening [2], etc. However, due to the lack of an efficient regeneration and
transformation method for I. polycarpa, its industrial use and gene function study are
greatly limited.
Various transformation methods have been developed for the delivery of exogenous
genes into plant cells. The most widely used transformation method is mediated by
Agrobacterium tumefaciens. However, this transformation method is time-consuming and
inefficient, since the transgenic plants are usually regenerated from transformed cells
via the tissue culture stage. Moreover, successful Agrobacterium transformation is largely
dependent on species, genotype, and explant type [7]. Particle bombardment and elec-
troporation were also used for gene delivery. However, these methods cannot generate
stably transformed plants; thus, gene function cannot be genetically analyzed. Besides the
above-mentioned transformation methods, Agrobacterium rhizogenes-mediated hairy root
transformation is being widely used for some plants, including soybean [8,9], cotton [10],
https://www.mdpi.com/journal/plants
Plants 2024, 13, 1791 2 of 10

pigeon pea [11,12], cannabis [13], Sphaeralcea angustifolia [14], tea plant [15], citrus [16], Tung
Tree [17], etc. This method involves the integration of Ri plasmid fragments into the host
plant genome by infecting plant leaves or stems. With this approach, the production of
transgenic hairy roots can either be achieved via the traditional tissue culture process or,
more conveniently, circumvent the need for tissue culture altogether, thus quickly regener-
ating the transgenic plants [18–20]. Taken together, this method is simple, time-saving, and
also avoids damaging plant tissues.
Due to the high industry values of I. polycarpa, here, we initially evaluated the ef-
fects of different explant types on hairy root induction, including whole seedlings, root-
less seedlings, and petioles. We also checked the impacts of development stages on the
Agrobacterium-mediated transformation of I. polycarpa, such as non-, semi- and fully ligni-
fied stages. Additionally, we successfully induced transgenic buds from these genetically
modified roots, ultimately yielding transgenic I. polycarpa plants. Our study is of great
value for genetically modifying wood traits and for conducting scientific research on woody
plants in the future.

2. Results
2.1. Establishment of Transformation System Using Whole Seedlings as Explants
In this study, we first used the whole seedling for the transformation, and the strain
used for the transformation was Agrobacterium rhizogenes K599 carrying pFGC-mCHERRY
construct (Figure 1A). The base stem was punched using a syringe, and the bacteria solution
was then applied to the wounding sites (Figure 1B–F). To maintain high humidity levels,
these infected plants were always covered with a transparent plastic lid. After two to three
weeks, a small area of callus emerged around the injection sites of the seedlings. After
another four to five weeks, hairy roots appeared and exhibited a robust growth state later.
Previous studies have shown that hairy roots originate from cortical cells, and the
proportion of these cells decreases with the physiological age of the seedlings [21]; it
is possible that the maturity state of the seedlings might affect the sensitivity of these
cells to Agrobacterium rhizogenes, thereby influencing the transformation efficiency. To
optimize the transformation efficiency, 20-, 30-, 40-, and 100-day-old seedlings were used
for transformation. The induction rates of hairy roots were different among the tested
plants with developmental stages (Table 1). Root induction rates were higher in the younger
explants, including 20-, 30-, and 40-day-old seedlings, while lower in old explants, such
as 100-day-old explants. Moreover, the transformation efficiency varied among plants
at different development stages. The transformation rate of 40-day-old seedlings was
highest (up to 71.91%), while the transformation rate of 100-day-old seedlings was only
zero (Table 1), and that of 20 and 30-day-old seedlings were 49.94% and 58.09%, respectively.
Plants containing transgenic roots account for 59.87% of total transformed plants (Table S1).
The ratio of transgenic roots per seedling ranged between 36.47% and 53.07% (Table S2).
To identify the transgenic roots, we usually checked the fluorescent signals using a
portable fluorescence detection instrument equipped with a red filter, since the construct
carrying mCHERRY was used for this study (Figure 1A), and those successfully transformed
hairy roots exhibited red signals (Figure 1F). To confirm the reliability of the detection
method, we also identified the transgenic roots using other methods, including confocal
microscopy, PCR, and RT-PCR methods. Under confocal microscopy, strong signals were
detected in the transformed roots’ epidermis cells expressing mCHERRY but were not seen
in the non-transformed ones (Figure 1G). We also detected the mCHERRY gene using PCR
analysis. Strong bands were detected in the transgenic roots but were not present in the
non-transformed roots (Figure 1H). Meanwhile, we detected the transgene expression levels
using RT-PCR analysis (Figure 1I). Our results showed that the bands from the mCHERRY
gene were only detected in the transgenic roots, indicating that this gene was expressed in
the transgenic roots. Taken together, these results confirmed the reliability of the detection
method mediated by the portable fluorescent lamp.
 from the mCHERRY gene were only detected in the transgenic roots, indicating that this
Plants 2024, 13, 1791 gene was expressed in the transgenic roots. Taken together, these results confirmed the
3 of 10
reliability of the detection method mediated by the portable fluorescent lamp.

Figure1.1.Agrobacterium
Figure Agrobacteriumrhizogenes-mediated
rhizogenes-mediatedtransformation
transformationprocedure
procedureusingusingwhole
wholeseedlings
seedlingsandand
transgenic
transgenicroot identification.
root (A)(A)
identification. Schematic diagram
Schematic of PFGC-mCHERRY.
diagram of PFGC-mCHERRY. (B–F) Workflow of genera-
(B–F) Workflow of
generation
tion of transgenic
of transgenic roots. In roots.
short, In short, clumps
bacteria bacteriawere
clumps were prepared,
prepared, andclumps
and bacterial bacterial clumps
were were
collected
collected
using using (B).
the needle the Bacteria
needle (B).
clumpsBacteria
wereclumps
applied were
to theapplied
junctiontosites
thebetween
junctionthe
sites betweenand
hypocotyl the
hypocotyl
roots and roots
(C). Bacterial (C). Bacterial
solution solution
was injected wasthe
into injected into the
soil around soil
the around sites
puncture the puncture
(D). Hairy sites (D).
roots
Hairy roots
emerged emerged
around around the
the puncture sitespuncture
(E), whichsites (E), further
were which were further
detected usingdetected using
a portable a portable
fluorescent
fluorescent lamp (F). Bars represent 1 cm. (G) Signals observed in transgenic roots expressing
lamp (F). Bars represent 1 cm. (G) Signals observed in transgenic roots expressing mCHERRY using a
mCHERRY using a confocal laser scanning microscope with an excitation wavelength of 633 nm.
confocal laser scanning microscope with an excitation wavelength of 633 nm. Bars represent 50 µm.
Bars represent 50 µm. (H) PCR analysis of the insertion of mCHERRY into the genome. (I) RT-PCR
(H) PCR analysis
analysis of mCHERRY insertion ofinmCHERRY
of the expression into the and
non-transformant genome. roots.analysis of mCHERRY
(I) RT-PCR
transgenic
expression in non-transformant and transgenic roots.
Table 1. Hairy root induction rates and transformation efficiency of whole seedlings at different
ages.1. Hairy root induction rates and transformation efficiency of whole seedlings at different ages.
Table

Seedling
SeedlingAge
Age Rooting
RootingRates
Rates(%)(%) Transgenic Rates
Transgenic Rates(%)(%)
2020 days
days oldold 96.45
96.45 ± ±1.86
1.86
a a 49.94
49.94 ±± 4.52
4.52bb
3030
days oldold
days 100 ± 0 a
100 ± 0 a 58.09 ±
58.09 ± 4.10
4.10 ab
ab
40 days old 98.25 ± 1.67 a 71.91 ± 5.97 a
100 days old 10.88 ± 1.00 b 0.00 ± 0 c
Note: The data presented are averages ± SD, and statistical analysis was conducted using SPSS software 27 for
t-tests, different lowercase letters indicate that the difference is significant (p < 0.05).
 40 days old 98.25 ± 1.67 a 71.91 ± 5.97 a
100 days old 10.88 ± 1.00 b 0.00 ± 0 c
Plants 2024, 13, 1791
Note: The data presented are averages ± SD, and statistical analysis was conducted using4 SPSS of 10
software 27 for t-tests, different lowercase letters indicate that the difference is significant (p < 0.05).

2.2.
2.2.Establishment
EstablishmentofofTransformation
TransformationSystem
SystemUsing
UsingRootless
RootlessSeedlings
SeedlingsasasExplants
Explants
To
Tofind
findout
outthe
themost
mostsuitable
suitableexplants
explantsforfortransformation,
transformation,we wealso
alsochose
chosethe therootless
rootless
seedlings
seedlingsasasexplants.
explants.The Theprimary
primary roots were
roots removed
were removed at the stem
at the basebase
stem nearnearthe stem–root
the stem–
junctions, and the
root junctions, andcutting sitessites
the cutting of the plantlets
of the were
plantlets dipped
were dipped into
intoK599
K599colony
colonyclumps
clumps
(Figure 2). Hairy roots were induced at 30 days after being dipped, and
(Figure 2). Hairy roots were induced at 30 days after being dipped, and the transgenic the transgenic roots
were checked using the portable fluorescent lamp. Seedlings
roots were checked using the portable fluorescent lamp. Seedlings of differentof different developmental
ages were used in
developmental thiswere
ages study, andinthe
used average
this study, hairy
and therootaverage
induction rate
hairy wasinduction
root 71.35%, andrate
the plants containing transgenic roots accounted for about 32.58%
was 71.35%, and the plants containing transgenic roots accounted for about 32.58% of the total infected
of the
plants (Table S1).
total infected The ratios
plants (TableofS1).
transgenic
The ratios roots
of per seedlingroots
transgenic are between 26.5%are
per seedling andbetween
34.76%
(Table S2). The transformation efficiency of 20-, 30-, 40- and 100-day-old
26.5% and 34.76% (Table S2). The transformation efficiency of 20-, 30-, 40- and 100-day-oldseedlings is 9.72%,
30.00%, 58.22%, and 0.05%, respectively (Table 2). Taken together, these findings
seedlings is 9.72%, 30.00%, 58.22%, and 0.05%, respectively (Table 2). Taken together, these showed
that the seedling
findings showedagethataffects the transformation
the seedling age affects efficiency either using
the transformation whole seedlings
efficiency either usingor
rootless seedlings.
whole seedlings or rootless seedlings.

Figure2.2.Agrobacterium
Figure Agrobacteriumrhizogenes-mediated
rhizogenes-mediatedtransformation
transformationprocedure
procedureusing
usingrootless
rootlessseedlings.
seedlings. In
In
short, bacteria colonies were piled up using a surgical blade (A); meanwhile, the seedling roots were
short, bacteria colonies were piled up using a surgical blade (A); meanwhile, the seedling roots were
removed using a blade (B). The cutting site of the rootless seedling was dipped into the bacterial
removed using a blade (B). The cutting site of the rootless seedling was dipped into the bacterial
clumps (C). The induced hairy roots appeared around the cutting sites (D), and the transgenic roots
clumps (C). The induced hairy roots appeared around the cutting sites (D), and the transgenic roots
were detected using a portable fluorescent lamp (E). Bars represent 1 cm.
were detected using a portable fluorescent lamp (E). Bars represent 1 cm.
Table 2. Adventitious root induction rates and transformation efficiency of rootless seedlings with
Table 2. Adventitious
different ages. root induction rates and transformation efficiency of rootless seedlings with
different ages.
Seedling Age Rooting Rates (%) Transgenic Rates (%)
Seedling Age
20 days old Rooting
97.92Rates
± 2.00(%)
a Transgenic Rates
9.72 ± 5.00 c (%)
2030 days
days oldold 98.33
97.92 ± ±2.00
1.67
aa 30.00 ± 10.00
9.72 ± 5.00 c b
3040
days oldold
days 98.33 ± 1.67
100 ± 0 aa 58.22±± 10.00
30.00 2.50 ab
40100
days old old
days 100 ±
13.6 ± 04.48
a b 58.22 ± 2.50 ca
0.05 ± 0.03
100 days old 13.6 ± 4.48 b 0.05 ± 0.03 c
Note: The data presented are averages ± SD, and statistical analysis was conducted using SPSS
Note: The data presented are averages ± SD, and statistical analysis was conducted using SPSS software 27 for
software 27 for t-tests, different lowercase letters indicate that the difference is significant (p < 0.05).
t-tests, different lowercase letters indicate that the difference is significant (p < 0.05).

2.3.Establishment
2.3. EstablishmentofofTransformation
TransformationSystem
SystemUsing
UsingLeaf
LeafPetioles
PetiolesasasExplants
Explants
Duetoto
Due thethe
factfact
thatthat
many many leaf explants
leaf explants can becan be obtained
obtained fromplant,
from a single a single plant, as
as compared
compared to the rooted or rootless seedlings, we also utilized them
to the rooted or rootless seedlings, we also utilized them as explants for Agrobacterium as explants for
Agrobacterium rhizogenes transformations. To increase leaf viability and
rhizogenes transformations. To increase leaf viability and reduce water evaporation, we first reduce water
evaporation,
removed we first
the distal partsremoved the distal
of the leaves parts ofthe
and reserved theproximal
leaves and reserved
parts with thethe proximal
leaf petiole.
parts with the leaf petiole. The petioles were dipped into the Agrobacterium
The petioles were dipped into the Agrobacterium rhizogenes K599 clumps and then incubated rhizogenes K599
clumps and then incubated in the trays filled with vermiculite (Figure
in the trays filled with vermiculite (Figure 3A,B). The trays were always covered with cling 3A,B). The trays
were always covered with cling film for moisture retention. After
film for moisture retention. After 2–3 weeks, small amounts of white calli appeared around 2–3 weeks, small
amounts
the of white
infection sites. calli
After appeared
6 weeks,around the infection
hairy roots were seen sites. After the
around 6 weeks, hairysites
infection roots
ofwere
leaf
seen around
petioles; the infection
the hairy sites ofrate
root induction leafwas
petioles;
68.25%,theand
hairytheroot
leafinduction rate was
transformation 68.25%,
efficiency
andup
was thetoleaf transformation
53.48% (Figure 3C–E).efficiency was up to 53.48% (Figure 3C–E).
 Plants 2024, 13, 1791 Plants 2024, 13, x FOR PEER REVIEW 5 of 10 5 o

Figure 3. procedure
Figure 3. Transformation Transformation
usingprocedure
leaves asusing leavesand
explants as explants and leaf transformation
leaf transformation efficiency.efficiency.
D) Transformation
(A–D) Transformation procedure of procedure of leaf
leaf explants. Barsexplants. Bars1represent
represent 1 cm.
cm. Briefly, Briefly,ofpetioles
petioles leavesof leaves w
the distal parts removed were dipped into bacterial clumps (A) and then inserted into
with the distal parts removed were dipped into bacterial clumps (A) and then inserted into wet
vermiculites (B). The induced hair roots emerged around the petioles about 6 weeks after infec
vermiculites (B). The induced hair roots emerged around the petioles about 6 weeks after infection (C),
(C), and the fluorescent signals of transgenic roots were detected by the portable fluorescent l
and the fluorescent(D).
signals of transgenic
(E) Leaf rootsefficiency.
transformation were detected by the portableefficiency
The transformation fluorescent
waslamp (D). based o
calculated
(E) Leaf transformation
leaves,efficiency. The transformation
and experimental efficiency
data were repeated was
three calculated
times. The databased
were on 21 leaves,
statistically analyzed u
and experimental SPSS software
data were versionthree
repeated 27 for t-tests,
times. The** indicate
data werestatistical significance
statistically (p <using
analyzed 0.05). SPSS
software version 27 for t-tests, ** indicate statistical significance (p < 0.05).
2.4. Regeneration of Transgenic Shoots from Transgenic Roots
2.4. Regeneration of Transgenic Shootswe
In our study, from Transgenic
noticed Roots
that I.polycarpa roots are prone to generating new sho
In our study, I. polycarpa
without the addition of hormones. are
we noticed that roots prone
Thus, we to cutgenerating new shoots
the transgenic roots carrying
without the addition of hormones.
mCHERRY Thus,
gene into we cut the
segments andtransgenic
placed them rootsincarrying the mCHERRY
vermiculite under strong light
gene into segmentshighand placed them
humidity. After in vermiculite
around 30 days,under strong
shoots light and
sprouted out high
of thehumidity.
transgenic roots
After around 30confirm if the newly
days, shoots sproutedemergedout branches were transgenic,
of the transgenic roots. we Toused the portable
confirm if the fluoresc
lamp to detect
newly emerged branches the fluorescent
were transgenic, signals
we used theinportable
these newly emerged
fluorescent leaves.
lamp The signals w
to detect
detected
the fluorescent signals in in the newly
these leaves emerged
growing leaves.
from the Thetransgenic
signals were rootsdetected
but were in not
the seen in
untransformed
leaves growing from leaves
the transgenic (Figure
roots but 4A,B).
were notTo seen
confirm the untransformed
in the reliability of theleaves
detection meth
(Figure 4A,B). Towe also examined
confirm the transgenic
the reliability shootsmethod,
of the detection using otherwe also methods,
examined including
the conf
microscopy, PCR, and RT-PCR methods. The bands
transgenic shoots using other methods, including confocal microscopy, PCR, and RT-PCR representing the mCHERRY g
were only detected in the transgenic buds, whereas
methods. The bands representing the mCHERRY gene were only detected in the transgenic they were undetectable in the n
transformed buds (Figure 4C,D). These results indicate
buds, whereas they were undetectable in the non-transformed buds (Figure 4C,D). These that the mCHERRY gene
expressed
results indicate that in the transgenic
the mCHERRY gene was buds. Here, a in
expressed total
theof 54 root segments
transgenic buds. Here,wereaused for
study, among which 8 segments successfully generated
total of 54 root segments were used for this study, among which 8 segments successfully new shoots. This result indic
generated new shoots. This result indicates that the transgenic shoots were generatedtissue cult
that the transgenic shoots were generated from transgenic roots that bypass
from transgenic To examine
roots where tissue
that bypass the transgenes
culture. were expressed,
To examine wherewe also checked the were
the transgenes fluorescence i
month-old plants. We found that the fluorescence was detected in all parts of so
expressed, we also checked the fluorescence in 6-month-old plants. We found that the
transgenic plants, including the leaves, stems, and roots (Figure 4E–I). To further ch
fluorescence was detected in all parts of some transgenic plants, including the leaves, stems,
which tissues the signals had accumulated in, we obtained cross-sections of leaves, ste
and roots (Figure 4E–I). To further check which tissues the signals had accumulated in, we
Plants 2024, 13, x FOR PEER REVIEW and roots and found that the signals were almost detected in most tissues of 11(Figure 4F
obtained cross-sections of leaves, stems, and roots and found that the signals were 6almost
Taken together, the method we established enables the possibility of modifying I. polyc
detected in most tissues (Figure 4F–I). Taken together, the method we established enables
fruit traits using gene editing technology.
the possibility of modifying I. polycarpa fruit traits using gene editing technology.

Figure 4. Cont.
Plants 2024, 13, 1791 6 of 10

Figure 4. Transgenic shoot regeneration from transgenic roots and the identification of transgenic
Figure 4. Transgenic shoot regeneration from transgenic roots and the identification of transgenic
seedlings. (A) Shoots regenerating from transgenic roots. The plants were observed in a bright field
seedlings. (A) Shoots regenerating from transgenic roots. The plants were observed in a bright field
(left) and using a portable fluorescent lamp (right). Upper panel, non-transformed plant; lower panel,
(left) and using a portable fluorescent lamp (right). Upper panel, non-transformed plant; lower
plant transformed with 35Spro: mCHERRY. Bars represent 1 cm. (B) Signals detected in transgenic
panel, plant transformed with 35Spro: mCHERRY. Bars represent 1 cm. (B) Signals detected in
shoots and
transgenic untransformed
shoots shoots using
and untransformed a confocal
shoots using laser scanning
a confocal lasermicroscope
scanning with the excitation
microscope with the
wavelength at 633 nm. Bars indicate 20 µm. (C) RT-PCR analysis of mCHERRY
excitation wavelength at 633 nm. Bars indicate 20 µm. (C) RT-PCR analysis of mCHERRY expression expression in non-
intransformant and transgenic
non-transformant shoots. Total
and transgenic RNA
shoots. wasRNA
Total extracted
was from the transgenic
extracted from the leaves. IpACTIN
transgenic leaves.
was used as the internal control. (D) PCR analysis of mCHERRY inserted into the
IpACTIN was used as the internal control. (D) PCR analysis of mCHERRY inserted into the genome. genome. Genomic
DNA was
Genomic DNAextracted from leaves
was extracted fromof untransformed and transformed
leaves of untransformed shoots. (E) Six-month-old
and transformed whole
shoots. (E) Six-month-
old whole transgenic
transgenic plants under plants underfluorescent
a portable a portablelamp.
fluorescent lamp.1 Bars
Bars indicate indicate
cm. (F–I) 1cM. (F–I)
Cross-sections of Cross-
leaf
sections of leaf
blades (F), blades (F),
leaf midveins leaf
(G), midveins
stems (H), and(G),
rootsstems
(I) of (H), and roots
transgenic plants(I)
andofuntransformed
transgenic plants
plantsand
untransformed plants
(WT). Bars in (F–I) (WT). Bars
represent in 200,
20, 50, (F–I)and
represent
100µm. 20, 50, 200, and 100µm.

3.3.Discussion
Discussion
In this study, we successfully obtained the transgenic roots of I. polycarpa using
In this study, we successfully obtained the transgenic roots of I. polycarpa using
Agrobacterium rhizogenes-mediated genetic transformation technology. This method has
Agrobacterium rhizogenes-mediated genetic transformation technology. This method has
been successfully applied to some woody plants as well, since it overcomes many chal-
been successfully applied to some woody plants as well, since it overcomes many
lenges that arise from the Agrobacterium tumefaciens-mediated transformation method,
challenges
includingthat
longarise from the Agrobacterium
transformation periods, rigidtumefaciens-mediated
sterile conditions, thetransformation method,
limitations of species
and genotype, etc. [21]. Moreover, this technology can be flexibly applied to different types
of explants, including leaves, stem segments, seeds, and rootless seedlings [22–25]. In our
study, using three types of explants, including whole seedlings, rootless seedlings, and
leaves, we successfully obtained transgenic roots. Moreover, the transgenic roots could
generate transgenic shoots. Thus, the establishment of the transformation procedure in
I. polycarpa laid a solid foundation for future gene function identification and industrial use.
Root regeneration efficiency is crucial for Agrobacterium rhizogenes-mediated hairy
root transformation. Our research has revealed that this efficiency varies significantly
among explants at different developmental stages (Tables 1 and 2). Either using whole
seedlings or rootless seedlings as explants, the root induction rates of young explants
Plants 2024, 13, 1791 7 of 10

including 20-, 30-, and 40-day-old seedlings were significantly higher than that of 100-day-
old seedlings (Tables 1 and 2), indicating that the root regeneration efficiency is closely
related to the developmental stages. This result is consistent with previous studies [26–28]
that obtained similar results using Arabidopsis leaf petioles as explants [26]. Moreover,
these studies identified that the decreasing root regeneration rates of the older explants
are due to the reduction in the ability to biosynthesize auxin during maturation [26]. The
reduced auxin production ability in older explants is attributed to two possible reasons.
The first reason is due to the ethylene production induced by wounding. In our study, and
previous studies, the explants were wounded either by punching or cutting before transfor-
mation (Figures 1 and 2). Wounding treatments do not only induce auxin accumulation, but
also trigger ethylene production and activate the expression levels of transcription factor
ETHYLENE INSENSITIVE 3 (EIN3) [27]. Ethylene treatment, or the activation of EIN3, is
identified to suppress root regeneration ability. Relative to young explants, the older ones
contain higher EIN3 levels, thus inhibiting root regeneration efficiency by suppressing the
expression of two key genes, WUSCHEL RELATED HOMEOBOX 11 (WOX11)/WOX12
and WOX5/WOX7, which are required for de novo root regeneration [27]. The other
possible reason is related to the regulatory roles of the miR156-SPLs-AP2/ERFs pathway.
Transcription factors AP2/ERFs are identified to promote root regeneration by inducing
auxin biosynthesis but are negatively regulated by SPLs. SPLs are preferentially accumu-
lated in older explants, thereby suppressing root regeneration by repressing the activity of
AP2/ERFs [28]. Besides the above-mentioned two possible reasons, we speculated that the
root regeneration ability of explants with different developmental ages might be closely
associated with the proportion of cambium cells. Those young explants exhibiting higher
root regeneration ability usually contain a higher proportion of cambia cells, whereas the
old ones contain fewer cambia cells. Therefore, the old explants display decreased root
regeneration efficiency.
In our study, we noticed that a higher transformation efficiency was obtained by
30- 40-day-old explants, while the younger (20-day-old seedlings) or older explants
(100-day-old seedings) showed a lower transformation efficiency (Tables 1 and 2). One
possible reason for this is that 20-day-old explants are too vulnerable to withstand the
Agrobacterium infection, though they have similar root induction rates to 30- or 40-day-old
explants. However, the low transformation efficiency of 100-day-old explants might be due
to their low root regeneration ability (Tables 1 and 2).
Our study showed that the transgenic hair roots of I. polycarpa could produce the
shoots, though this conversion of roots to shoots requires a relatively long period of time
(Figure 4). The method we used is similar to the recently reported cut-dip-budding (CDB)
method [20]. Using the CDB method, transformants are successfully obtained from some
other plants, including two herbaceous plants (Taraxacum koksaghyz and Coronilla varia),
a tuberous root plant (sweet potato), and three woody plant species (Ailanthus altissima,
Aralia elata, and Clerodendrum chinense) [20]. Until now, the acquisition of transgenic plants
of I. polycarpa or other plants has been mainly based on the root-suckering capability of
plants. In addition, we found that light plays a crucial role in the conversion of roots
to shoots of I. polycarpa, since the buds of I. polycarpa preferentially often emerge on the
root parts exposed to light. That is consistent with previous studies’ observations that
light plays a key role in shoot apical meristem growth and leaf initiation by regulating
both cytokinin signaling and auxin transport [29,30]. Anyway, the enhancement of bud
regeneration ability in non-sterile environments will facilitate the establishment of genetic
transformation systems in woody plants.

4. Conclusions
Here, we first established the Agrobacterium rhizogenes-mediated transformation meth-
ods of I. polycarpa using whole seedlings, rootless seedlings, or petioles as explants, respec-
tively. Among the different explants, the whole seedlings display a higher transformation
efficiency. We also found that the developmental age has effects on transformation effi-
Plants 2024, 13, 1791 8 of 10

ciency. Finally, we successfully obtained transgenic shoots from transgenic roots. Our study
will be helpful for genetically modifying woody plants in the future.

5. Materials and Methods

5.1. Plant Materials and Growth Conditions
The seeds of I. polycarpa, obtained from Hanzhong, Shaanxi Province, were sown
in nutrient-rich soil in big trays (55 cm × 28 cm). The seedlings were germinated in
Petri dishes for 10 days on wet filter paper and grown in a growth chamber with two
fully expanded true leaves, and were transferred to small individual pots (7 cm × 7 cm).
One I. polycarpa seedling was planted in each pot. The plants grew in a greenhouse with
temperatures set between 22 and 26 ◦ C with a 16 h light/8 h dark photoperiod.

5.2. Preparation of Agrobacterium rhizogenes

The plasmid carrying pFGC-mCHERRY was transformed into the Agrobacterium rhizogenes
strain K599 using the freeze–thaw method. A freshly single colony growing on a solidified
LB medium containing appropriate antibiotics was inoculated into a 3 to 5 mL LB liquid
medium containing kanamycin, rifampicin, and 10 mM calcium chloride. The cultures
were shaken at 28 ◦ C for 12 h. Then, 300 µL of bacterial cultures was plated onto LB agar
plates containing appropriate antibiotics and cultured for another day. The colonies were
piled up for injection. To prepare the Agrobacterium solution for irrigation, the overnight
cultures were diluted in LB media at a 1:1000 ratio and then subcultured for another day
until OD600 ranged between 0.8 and 1.0.

5.3. Agrobacterium rhizogenes Infection

For the I. polycarpa Agrobacterium rhizogenes infection, some simple modifications were
made to the cut-dip-budding (CBD) method [20]. Specifically, the stems near the roots
(about 1–2 cm above the roots) were punched with a syringe, or the primary roots of the
seedlings were cut off, or freshly picked leaves containing petioles were used. Agrobacterium
K599 colonies screening on solidified LB plates containing appropriate antibiotics were
applied to the wound sites. The infected seedlings were planted in a pot filled with wet
sterile vermiculite and irrigated with 3–5 mL of Agrobacterium solution. Finally, the pots
were covered with a transparent lid to maintain high humidity levels, and hairy roots grew
up approximately 5–6 weeks later. PCR analysis, gene expression analysis, and fluorescence
observation were conducted to determine the generation of transgenic roots.

5.4. Regeneration of Transgenic Shoots

The lignified transgenic roots were cut into multiple segments with a length of 5–6 cm.
After that, the segments were vertically inserted into the sterilized vermiculite and incu-
bated in a greenhouse until the shoots emerged. The treated explants were cultured at
22–26 ◦ C with a 16 h light/8 h dark cycle until buds developed from the root cuttings
(approximately 1 month). Putative transgenic buds were then transferred to the potted soil
(nutrient soil: vermiculite = 1:1) and grown under normal growth conditions.

5.5. Fluorescence Observation

The transformed hairy roots were thoroughly cleansed, and the mCHERRY fluorescent
signals were inspected using a portable excitation light source (LUYOR-3410GR) paired
with specific glasses featuring LUV-50A filters. For further analysis, the mCHERRY signal
was detected in intact leaves or freehand sections of leaf blades and midveins, the stems, and
the roots using a confocal fluorescence microscope (LSM980, Zeiss, Oberkochen Germany)
equipped with a 610 nm emission filter, and a 587 nm excitation filter was utilized.

5.6. PCR Analysis

DNA was extracted from transgenic roots or shoots using the CTAB method. PCR am-
plification was performed using 2×Taq Master Mix reagents (Vazyme, Nanjing, China). To
Plants 2024, 13, 1791 9 of 10

detect the inserts of mCHERRY gene fragments in transgenic roots and transgenic shoots, a
PCR was performed using specific primers mCHERRY-F: 5′ -ATGGGAGCCAATGGAGTG-3′
and mCHERRY-R: 5′ -TCAAAACTGGTTCCGGTAC-3′ , respectively [17].

5.7. RT-PCR Analysis

Total RNA was extracted from transgenic roots or shoots using the Trizol reagent
(Simgen, Hangzhou, China). Reverse transcription was carried out using HiScriptII1st Strand
cDNA Synthesis Kit (Vazyme, Nanjing). RT-PCR analysis was performed using the above-
mentioned primers, mCHERRY-F and mCHERRY-R. IpACTIN was used as the internal con-
trol, of which the primers are IpACTIN-qPCR-F: 5′ -AAGACCTACACCAAGCCGAA-3′ and
IpACTIN-qPCR-F: 5′ -CTCCGCACTCAGCATTAGGACA-3′ , respectively.

5.8. Statistical Analysis

All experiments were repeated three times. Each statistical analysis was carried out
using SPASS software 27 for t-tests. We calculated the induction rate (%) of hairy roots
by using the ratio of the number of plants producing hairy roots to the total number of
survived injected plants and separately calculated the induction rate (%) of transgenic hairy
roots based on the ratio of the number of transgenic hairy root plants to the total number of
hairy root plants.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/plants13131791/s1, Table. S1 Average hairy root induction rates
and transformation efficiency from whole seedlings and rootless seedings. Table. S2 Induction of
transgenic roots contained in each seedling from whole seedlings and rootless seedings.
Author Contributions: Conceptualization, S.L. and H.Z.; methodology, H.W., K.C. and T.L.; soft-
ware, H.W.; validation, H.W., X.L. and L.S.; formal analysis, H.W. and K.C.; investigation, S.L.;
resources, X.L. and L.S.; writing—original draft preparation, H.W. and H.Z.; writing—review and
editing, all authors.; supervision, S.L. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the National Key R&D Program of China, grant number
2023YFD2201102.
Data Availability Statement: Data are contained within the article and Supplementary Materials.
Conflicts of Interest: Authors Xiaoyu Lan and Li Shen were employed by the company Shaanxi
Agricultural and Forestry Technology Co., Ltd. The remaining authors declare that the research was
conducted in the absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.

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