G C A T

T A C G
G C A T
genes
Article
Standardized Genetic Transformation Protocol for
Chrysanthemum cv. ‘Jinba’ with TERMINAL
FLOWER 1 Homolog CmTFL1a
Saba Haider 1 , Yaohui Gao 2 and Yike Gao 1, *
1 National Flower Engineering Research Centre, Beijing Key Laboratory of Ornamental Plants Germplasm
Innovation and Molecular Breeding, College of Landscape Architecture of Beijing Forestry University,
Beijing 100083, China; sabahaider@bjfu.edu.cn
2 Architectural Institute, Inner Mongolia University of Science & Technology, Alding Street No.7,
Kundulun District, Baotou 014010, China; yaohui_gao@163.com
* Correspondence: gaoyk@bjfu.edu.cn; Tel.: +86-138-0102-1804




Received: 29 May 2020; Accepted: 22 July 2020; Published: 28 July 2020


Abstract: Chrysanthemum (Chrysanthemum x morifolium Ramat.) cultivar Jinba is a distinctive

short-day chrysanthemum that can be exploited as a model organism for studying the molecular
mechanism of flowering. The commercial value of Jinba can be increased in global flower markets
by developing its proper regeneration and genetic transformation system. By addressing typical
problems associated with Agrobacterium-mediated transformation in chrysanthemum, that is, low
transformation efficiency and high cultivar specificity, we designed an efficient, stable transformation
system. Here, we identify the features that significantly affect the genetic transformation of Jinba
and standardize its transformation protocol by using CmTFL1a as a transgene. The appropriate
concentrations of various antibiotics (kanamycin, meropenem and carbenicillin) and growth regulators
(6-BA, 2,4-D and NAA) for the genetic transformation were determined to check their effects on in vitro
plant regeneration from leaf segments of Jinba; thus, the transformation protocol was standardized
through Agrobacterium tumefaciens (EHA105). In addition, the presence of the transgene and its stable
expression in CmTFL1a transgenic plants were confirmed by polymerase chain reaction (PCR) analysis.
The CmTFL1a transgene constitutively expressed in the transgenic plants was highly expressed in shoot
apices as compared to stem and leaves. Overexpression of CmTFL1a led to a delay in transition to the
reproductive phase and significantly affected plant morphology. This study will help to understand
the biological phenomenon of TFL1 homolog in chrysanthemum. Moreover, our findings can explore
innovative possibilities for genetic engineering and breeding of other chrysanthemum cultivars.

Keywords: Chrysanthemum morifolium; CmTFL1a; meropenem; transformation efficiency; expression

pattern; transgenic plants

1. Introduction
Chrysanthemum (Chrysanthemum x morifolium Ramat) has the largest floriculture commodity in
China and is a popular traditional flower of esthetic and cultural importance. There is a growing
demand for high quality chrysanthemum flowers and cut flowers in the local market. The plants can
be regenerated from adventitious shoots from various chrysanthemum tissues or calli using in vitro
culture methods [1]. There are various cultivars of chrysanthemum being commercially propagated,
but Jinba holds significance due to its fast-growing habit. Jinba is a short-day chrysanthemum cultivar
with relatively low transformation efficiency compared with other cultivars, making it difficult to
obtain transgenic plants.

Genes 2020, 11, 860; doi:10.3390/genes11080860 www.mdpi.com/journal/genes

Genes 2020, 11, 860 2 of 17

Efficient genetic transformation with the production of various transgenic crop plants is
dependent on several factors, such as gene transfer methods, DNA recombinant technologies
and tissue culture techniques [2–4]. Diverse methods for gene transfer have been developed,
including Agrobacterium, particle bombardment, microinjection, in planta transformation and liposome
encapsulation [5]. Among them, Agrobacterium-mediated transformation has a unique significance
and is being used for the insertion of specific genes into numerous ornamental plant species because
of its authenticity and reliability. Several studies have been reported concerning the evaluation of
Agrobacterium-mediated transformation in chrysanthemum [6–8]. The susceptibility of chrysanthemum
to Agrobacterium-mediated transformation is well established and has been characterized by low
transformation efficiency since 1975 [9,10]. The formation of crown gall in various parts of
Chrysanthemum morifolium due to B6 Agro-infection has been reported to be the main reason behind
this low transformation efficiency. It was reported that regeneration of chrysanthemum is dependent
on genotype, similar to tomato [11] and cucumber [12].
The most important feature that hinders the rapid advancement of genetically modified
chrysanthemums is protocol reliance [13]. Each cultivar has a unique specificity for tissue culture
and genetic manipulation methods. Therefore, no one protocol can be implemented for a wide range
of cultivars. For Jinba, the genetic transformation has usually been performed with A. tumefaciens.
Various factors significantly affect the rate of Agrobacterium-mediated transformation in chrysanthemum
including the plant cultivar, source and size of explant, type of bacterial strain used, kind and
concentration of selective agent used for the selection of genetically modified plants, time period and
temperature of cocultivation and timing of Agrobacterium treatment [14]. A comprehensive evaluation
of these factors is necessary to standardize the genetic transformation protocol for the Jinba cultivar of
chrysanthemum plants.
Flowering is the major developmental phase, comprising of transition from vegetative to
reproductive growth stages [15]. A comprehensive research on TFL1 homologs has revealed
diverse functions such as constitutive expression of CsTFL1 in chrysanthemum that caused delay in
flowering time. Similar results were found in the ecotropic expression of maize TFL1 homologs [16].
Several pathways regulate floral induction and flowering time to optimize adaptation and reproductive
success in higher plants, including vernalization, plant hormone, photoperiodic and autonomous
pathways. Thus, different plant species have developed their optimal approaches for the regulation
of flowering time with environmental cues [15]. Two significant phosphatidylethanolamine-binding
protein (PEBP) genes controlling these pathways are FT (FLOWERING LOCUS T) and TFL1 (TERMINAL
FLOWER 1) [17–19]. TFL1 plays a key role in suppressing flowering by antagonizing the function of
the FT protein, which is responsible for the promotion of flowering [20]. Moreover, it helps in the
formation of inflorescences, maintains vegetative growth and an indeterminate inflorescence [21,22].
Several genes homologous to TFL1 have been identified in many plants. Some of the examples include
Malus domestica [23], Chrysanthemum morifolium [21] and Chrysanthemum Seticuspe f. boreale [24].
This work aims to enhance the number of putative regenerated shoots of Jinba by optimizing the
protocol of Agrobacterium-mediated transformation, to maximize the feasibility of producing transgenic
plants by standardizing media composition, and to examine the effect of CmTFL1a expression on
the flowering of chrysanthemum plants by phenotypic analysis. CmTFL1a was isolated from C.
morifolium Jinbudiao in our lab and its functional role in chrysanthemum was analyzed as mentioned
in [21]. CmTFL1a was inserted into in vitro Jinba plants as a transgene to obtain transgenic lines.
Various parameters responsible for successful transformation were assessed, including cocultivation
period and cocultivation temperature at different intervals, for comprehensive screening. To ensure
that the hormones and antibiotics used could enhance the regeneration capability of explants, various
hormone and selective agent treatments at different concentrations were recorded. These include
6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D),
carbenicillin, meropenem and kanamycin. The regenerated transgenic shoots were kept in rooting
medium for one month then transferred into soil media. As a result, the maximum possibility of
Genes 2020, 11, 860 3 of 17

producing transgenic plants by standardizing the composition of media was achieved. Through
repeated experiments, the combination of 6-BA and 2,4-D was proven to be most appropriate for
producing a greater number of transgenic shoots. At the same time, the removal of agrobacterial
strain was effectively accomplished by using meropenem, as compared to carbenicillin, at a specific
concentration. Phenotypic analysis showed that CmTFL1a, like various CEN/TFL1 genes, promotes
vegetative growth, and negatively regulates the reproductive phase. It was found that overexpression
of CmTFL1a significantly extended vegetative growth, delayed flowering and promoted atypical
inflorescences in transgenic chrysanthemum plants.

2. Materials and Methods

2.1. Plant Material

A cultivar of C. morifolium named Jinba was grown in the greenhouse of Beijing Forestry University
(Beijing, China). The optimal conditions for the photoperiod were maintained along with temperatures
ranging between 18 ◦ C and 25 ◦ C.

2.1.1. Preparation of Surface Sterilization Solution

In order to prepare 4% calcium hypochlorite (Ca(OCl)2 ), 10 g of (Ca(OCl)2 ) was dissolved in
250 mL of autoclaved distilled water and was stored at room temperature.

2.1.2. Preparation of MS Media

For preparing 1 L of medium, the desired quantity of MS (for shoot initiation and multiplication
media) and sucrose was dissolved in 300 mL distilled water following the recipe mentioned in
Section 2.1.3. The required quantity of growth regulators was added in the medium after thawing
followed by the addition of 400 mL of hot distilled water. After constant stirring, the pH was adjusted
to 5.7. In another beaker, agar was added in 300 mL distilled water and was heated until the agar
melted. The two solutions were mixed and stirred well, followed by pouring of medium in test tubes.
Sterilization of the test tubes was performed by autoclaving for 15 min at 121 ◦ C.

2.1.3. In Vitro Propagation of Chrysanthemum Plants

In vitro propagation of Chrysanthemum was performed by taking pedicels as chrysanthemum
explants from greenhouse plants. Pedicels with a diameter of 2–2.5 mm were harvested at the
preflowering stage. The bottom part of the cuttings was removed and only pedicels (3 cm long) were
used without any leaves. They were rinsed in tap water with 2 drops of colorless liquid soap for
3 min and then dried using filter paper. Surface sterilization was performed inside sterile conditions
of a laminar flow hood by soaking the pedicels in a 4% calcium hypochlorite (Ca(OCl)2 ) for 15 min.
Furthermore, the explants were rinsed with sterile distilled water for 2 min followed by changing the
water at least three times. Fragments from the treated explants were prepared by cutting the pedicel
into pieces 8–10 mm long. The semi-cylindrical fragments (one per tube) were incubated on 15 mL
medium for shoot initiation (MS 4.4 g L−1 + Myoinositol 100 mg L−1 + 2 mg L−1 BAP (1 mL stock
solution + IAA (1 mL stock solution) 1 mg L−1 + Sucrose 20 g/L + Agar 8 g L−1 ) at pH 5.7, and were
cultivated at 24 ◦ C, 80 µE m−2 s−1 and 16:8 h day-night photoperiod. Micropropagation was performed
by gently cutting the shoots into microcuttings with one node and were incubated on 10 mL medium
for multiplication (MS 4.4 g L−1 + Sucrose 20 g L−1 + Agar 6.5 g L−1 ) at pH 5.7 Jain and Ochatt [25].

2.2. Plant Expression Vector Construction

Agrobacterium tumefacians strain EHA105 carrying the binary vector pCAMBIA1301 with CsTFL1a
was isolated from C. morifolium Jinbudiao under the control of cauliflower mosaic virus (CaMV) 35s
promotor used for transformation. The full length CmTFL1a cDNA was obtained by PCR using primers
CmTFL1a-F and CmTFL1a-R, whereas the products were purified using a PCR purification kit (AXGEN,
Genes 2020, 11, 860 4 of 17

USA). Cloning of amplified fragments into the pEASY-Blunt vector (TRANSGEN, Beijing, China)
was accomplished and verified by sequencing. A pCAMBIA 1301– pmi- CmTFL1a overexpression
vector was constructed with the Seamless Assembly Cloning Kit (CloneSmarter, Houston, TX, USA).
An Escherichia coli phosphomannose isomerase (PMI) marker gene and mannose selective agent were used
in a mannose selection system for transformation in chrysanthemum. Finally, amplification of CmTFL1a
from pEASY-CmTFL1a with CmTFL1a-pmi-F and CmTFL1a-pmi-R primers was accomplished, followed
by digestion of pCAMBIA 1301- pmi vector with XhoI, leading towards assembling of CmTFL1a into
the plasmid using Seamless Assembly [21]. The experiment was repeated three times to ensure the
best possible results.

2.3. Shoot Regeneration Medium

A total of 18 shoot regeneration media with various concentrations of hormones such as 6-BA,
2,4-D and NAA, and antibiotics such as carbenicillin, meropenem and kanamycin were prepared. Three
distinct concentrations, which are listed in Table 1, were used for each hormone and antibiotic.
The concentrations of the growth regulators 6-BA (0.5 mg L−1 ), 2,4-D (0.2 mg L−1 ) and NAA
(0.1 mg L−1 ) [26]; and those of antibiotics carbenicillin (200 mg L−1 ) [13], meropenem (50 mg
L−1 ) [27] and kanamycin (7 mg L−1 ) [28] were modified and used as controls. The purpose was to
differentiate the impact of these hormones and antibiotics on the regeneration capability of treated
leaves. Three biological replicates were used for each treatment by repeating the experiment thrice.

Table 1. Various concentrations of hormones and antibiotics used in MS medium for chrysanthemum transformation.

Antibiotics
Hormones Concentrations (mg L−1 )
Concentrations (mg L−1 )
6-BA 2,4-D NAA Car Mer Ka
* 0.5 0.1 * 0.1 * 200 45 6
1 * 0.25 0.2 300 * 50 *7
1.5 0.4 0.5 400 55 8
* These concentrations were used as controls; 6-BA: 6-benzylaminopurine; 2,4-D: 2,4-dichlorophenoxyacetic acid;
NAA: naphthaleneacetic acid; Car: carbenicillin; Mer: meropenem; Ka: kanamycin.

2.4. Agrobacterium-Mediated Transformation in Chrysanthemum

Chrysanthemum plants were genetically transformed via A.tumefaciens by following the
unstandardized procedures of Naing et al. [29] with modifications. Leaf explants of Jinba were divided
into small segments of 0.5–1 cm2 and dipped into Agrobacterium suspensions with various optical
densities to determine the optimal optical density of Agrobacterium, which was directly proportional to
the transformation efficiency. The time for suspension was optimized to 10 min. After suspension, the
leaves were dried by blotting with filter paper before being placed on the shoot regeneration medium
(MS + 0.5 mg L−1 6BA + 0.1 mg L−1 NAA as control). Treated explants plated on cocultivation medium
(MS + 0.5 mg L−1 6BA + 0.1 mg L−1 NAA as control) with carbenicillin (400 mg L−1 ) were left for 2
days under dark conditions. Then, the leaves were transferred to selection medium (MS + 6-BA +
NAA + Meropenem + Kanamycin) containing different concentrations of antibiotics. Stock solutions
of these antibiotics were prepared by filter-sterilization at concentrations of 400 mg L−1 , 50 mg L−1 and
30 mg L−1 , respectively. Carbenicillin and meropenem ensure the removal of any residual bacteria,
while kanamycin is a selective agent that promotes callus regeneration and shoot proliferation strength
of the callus generated. Assessment of the factors affecting transformation efficiency was performed by
manipulating different concentrations and combinations of growth regulators and antibiotics (Table 1),
days to coculture and the temperature requirements during cocultivation. Each treatment contained 15
explants with 3 replications, whereas the experiment was conducted thrice.
Genes 2020, 11, 860 5 of 17

2.5. PCR Analysis of Transgenic Plants

Genomic DNA was isolated from the leaves of transgenic and wild-type (WT) plants using a
TIANGEN DNA secure Plant Kit followed by PCR analysis using specific primers and PCR parameters,
as listed in Table 2. Analysis of PCR products was accomplished by gel electrophoresis using 2%
agarose stained with ethidium bromide. The expression of the inserted transgene was analyzed by
quantitative real-time PCR (qRT-PCR). Specific primers for qRT-PCR (Table 2) were designed by the
Primer 5.0 software. The total volume of 20 µL mixture was prepared with 2 µL cDNA template (10X),
0.4 µL of each amplification primer (10 mmol L−1 ) and 10 µL 2x SYBR Green Master Mix (TAKARA,
Japan). In all cases, the standard thermal cyclic conditions were: 50 ◦ C for 2 min, 35 cycles of 95 ◦ C for
10 min, 95 ◦ C for 15 s, 60 ◦ C for 15 s and 72 ◦ C for 10 min. Three biological replicates were analyzed.

Table 2. Primers and polymerase chain reaction (PCR) parameters used for the current study.

Genes Primers PCR Conditions

CmTFL1a-F CCA ACA ACC CAA CTT GTT 98 ◦C for 30 s, followed by undergoing
CmTFL1a
AAA A 25 cycles of 98 ◦ C for 10 s, 60 ◦ C for 30 s
CmTFL1a-R CGA CAG AAT TTG ACC
AAA AG
98 ◦ C for 30 s, followed by undergoing
CmTFL1a-Q CmTFL1a-QF CCT CGT GTT GAT ATC GGT
25 cycles of 98 ◦ C for 10 s, 60 ◦ C for 30 s
CmTFL1a-QR GTA ACA ATC CAG TGA
AGA TGT
95 ◦ C for 2 min, followed by 30 cycles of
Actin-F GGT GTC ATG GTT GGT ATG
Actin 95 ◦ C for 20 s, 57 ◦ C for 40 s, and 72 ◦ C
GGTC
for 1 min
Actin-R CCT CTG TGA GTA GAA CTG
GGTGC 95 ◦ C for 2 min, followed by 30 cycles of
CmTFL1a-pmi CmTFL1a-pmi-F GAT GTT TAG ATA GAG 95 ◦ C for 20 s, 57 ◦ C for 40 s, and 72 ◦ C
ACT CGA GAT GGC AAG ATT AAC ATT for 1 min
AAC TTC GGG TCA
CmTFL1a-pmi-R AGT GTG TAA TAC CTC
TTT CTC CAG TTA TCG TCT GGG AGC
TGT TTC TCT T

All the cultures were placed in a completely randomized design. A total of 18 treatments were
used with three biological replicates. Each replicate dish had 5 explants; i.e., a total of 270 explants
was used for genetic transformation. All the experiments were repeated three times. The data were
analyzed using analysis of variance (ANOVA), and means were compared using Duncan’s multiple
range test (p < 0.05).

3. Results

3.1. Role of the Cocultivation Period in Genetic Transformation Efficiency

Time of cocultivation may vary with the plant and species. The average time of cocultivation
for chrysanthemum plants is two or four days [30,31]. Resistant shoots were not produced when the
treated explants were cocultured for one day. However, the maximum number of putative shoots was
observed when the explants were cocultured for two days in darkness, while the number of resistant
shoots in the explants cocultured for more than two days was reduced, as shown in Figure 1.
Genes 2020, 11, 860 6 of 17
Genes
Genes 2020,
2020, 11,
11, xx FOR
FOR PEER
PEER REVIEW
REVIEW 66 of
of 17
17

Figure 1. Relation
Figure 1. Relation of
of cocultivation
cocultivation period
period with
with shoot
shoot regeneration
regeneration and transformation efficiency
and transformation efficiency of
of
Jinba. Black columns
Jinba. Black columns represent
columns represent shoot regeneration percentage, red columns represent transformation
represent shoot regeneration percentage, red columns represent transformation
efficiency
efficiency percentage.
percentage. Explants
Explants were
Explants were cocultured
cocultured for
for 1,
1, 22 and
and 33 days.
days. Three
Three biological
biological replicates
replicates were
were
used. Error bars
Errorbars
used. Error represent ± SD.
represent± ±
barsrepresent a,
SD.
SD. b, and
a, and
a, b, c
b, andare statistically
c are
c are significant
statistically
statistically at
significant
significant p ≤ 0.05 by
p ≤by
at p ≤at0.05 ANOVA
0.05 on
by ANOVA
ANOVA ranks
on
on ranks
test.
ranks test.
test.

3.2. Relation
3.2. Relation between cocultivation
cocultivation temperature and
and transformation efficiency
efficiency
3.2. Relation between
between cocultivation temperature
temperature and transformation
transformation efficiency
It can be seen in Figure 22that temperature adjustment during cocultivation plays a crucial role in
It can be
be seen
seen in
in Figure
Figure 2 that
that temperature
temperature adjustment
adjustment during
during cocultivation
cocultivation plays
plays aa crucial
crucial role
role
shoot
in regeneration. Explants were cocultivated at various temperature conditions and the maximum
in shoot regeneration. Explants were cocultivated at various temperature conditions and the
shoot regeneration. Explants were cocultivated at various temperature conditions and the
number of putative
maximum shoots was observed when the temperature maintained during the cocultivation
maximum number
number of
of putative
putative shoots
shoots was
was observed
observed when
when the
the temperature
temperature maintained
maintained
◦ C. However, explants cocultured at 24 ◦ C yielded fewer transgenic shoots than those
during
during the
the
period was 22period
cocultivation was
cocultivation period was 22
22 °C.
°C. However,
However, explants
explants cocultured
cocultured at at 24
24 °C
°C yielded
yielded fewer
fewer transgenic
transgenic
produced
shoots 22 ◦ Cproduced
atthose but moreattransgenic shootstransgenic
than those produced at 28 ◦produced
C. In fact,atovergrowth of
shoots than those produced at 22 °C but more transgenic shoots than those produced at 28 °C. In
than 22 °C but more shoots than those 28 °C. In fact,
fact,
bacteria wasof observed
overgrowth
overgrowth of bacteriaafter
bacteria was explants
was observedwere
observed aftertransferred
after explants to the
explants were
were selection to
transferred
transferred medium.
to the
the selection
selection medium.
medium.

Shoot Regenration %
Shoot Regenration %
Transformation Efficiency %
Transformation Efficiency %

a
12 a 12
12 12

10 10
10 10

8 b 8
8 b 8

6 c 6
6 c 6

a
4 a 4
4 4

b b
b b
2 2
2 2

0
0
22ooC 24ooC 28ooC
22 C 24 C 28 C
Co-cultivation
Co-cultivation Temperature
Temperature ((oC)
o
C)

Figure
Figure 2. Effects of
2. Effects
Effects of cocultivation
cocultivation temperature
temperature onon shoot
shoot regeneration
regeneration andand transformation
transformation efficiency
efficiency of
of
Jinba. Black
Black columns
columns represent
represent shoot
shoot regeneration
regeneration percentage, red columns represent transformation
Jinba. Black columns represent shoot regeneration percentage, red columns represent transformation
efficiency percentage. Coculture plates were maintained at 22,at2422, ◦ C. Three biological replicates
and 26 and
efficiency percentage.
percentage. Coculture
Coculture plates
plates were
were maintained
maintained at 22, 24 24 and 26 26 °C.
°C. Three
Three biological
biological
were used.were
replicates Errorused.
bars Error
represent ±represent
SD. a, b, and c are statistically p ≤ 0.05 by
significant atsignificant ANOVA by on
replicates were used. Error bars represent ± SD. a, b, and c are statistically significant at
bars ± SD. a, b, and c are statistically at pp ≤≤ 0.05
0.05 by
ranks test.on ranks test.
ANOVA
ANOVA on ranks test.

3.3.
3.3. Role
Role of
of Various
Various Growth
Growth Regulators
Regulators and
and Antibiotics
Antibiotics on
on Shoot
Shoot Regeneration
Regeneration
Genes 2020, 11, 860 7 of 17

3.3. Role of Various Growth Regulators and Antibiotics on Shoot Regeneration

Minimal quantities of growth regulators and antibiotics that can promote shoot regeneration
and help to destroy nontransgenic cells were investigated in the selection of transgenic plants.
The growth regulators used included 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), and
2,4-dichlorophenoxyacetic acid (2,4-D), whereas the antibiotics used were carbenicillin, kanamycin
and meropenem. The maximum callus induction rate was reached with less browning of leaf discs
when a combination of the hormones 6-BA and 2,4-D at concentrations of 1 mg L−1 and 0.25 mg L−1 ,
respectively, was used in the selection medium. This combination eventually resulted in the maximum
regeneration of putative shoots. Increasing the concentration of 6-BA to 1.5–2 mg L−1 and 2,4-D to
0.3–0.4 mg L−1 resulted in a prominent decrease in the total number of shoots per explant and shoot
regeneration percentage, as shown in Table 3. Furthermore, the effect of 6-BA and NAA combination
on regeneration and genetic transformation percentage of the resistant shoots was examined; 0.8 mg
L−1 NAA with 1 mg L−1 6-BA was the most suitable combination for the maximum number of shoots,
whereas a low concentration range of NAA 0.1–0.4 mg L−1 retarded the growth of the explants, as
shown in Table 4. The comparison shows that the combination of 6-BA and 2,4-D generated a greater
number of regenerated putative shoots than the combination of 6-BA and NAA.

Table 3. Role of 6-benzylaminopurine (6-BA) and 2,4-D hormones combinations in browning rate of
leaves, callus induction rate and the average number of regenerated shoots of Jinba.

Growth Regulators Browning Rate of Callus Induction Average Number of

6-BA 2,4-D Leaves (%) Rate (%) Regenerated Shoots

0.5 0.1 71.33 abx 0.00 e 0

0.5 0.25 78.60 a 1.50 de 0
0.5 0.4 66.43 bc 4.1 de 1f
1 0.1 14.96 f 42.4 b 1.77 e
1 0.25 4.32 g 61.43 a 1.96 a
1 0.4 31.7 e 46.03 b 1.84 b
1.5 0.1 49.53 d 31.83 c 1.62 c
1.5 0.25 62.46 c 5.76 d 1.24 d
1.5 0.4 72.63 ab 0.33 e 0
x Mean totals with different letters are different (p ≤ 0.05) by ANOVA on ranks test.

Table 4. Effects of 6-BA and NAA hormones combinations on browning rate of leaves, callus induction
rate, and the average number of regenerated shoots of Jinba.

Growth Regulators Browning Rate of Callus Induction Average Number of

6-BA NAA Leaves (%) Rate (%) Regenerated Shoots

0.5 0.1 66.46 ex 7.6 cd 1.00 f

0.5 0.4 85.47 b 28.6 b 1.62 b
0.5 0.8 71.43 d 10.53 c 1.13 e
1 0.1 82.8 c 0.00 d 0
1 0.4 72.0 d 10.82 c 1.22 cd
1 0.8 22.93 f 50.73 a 1.75 a
1.5 0.1 70.90 d 13.5 c 1.3 d
1.5 0.4 91.4 a 0.00 d 0
1.5 0.8 87.1 b 13.73 c 1.48 c
x Mean totals with different letters are different (p ≤ 0.05) by ANOVA on ranks test.

In addition to growth regulators, different selective agents were also tested at specific
concentrations. Kanamycin at a concentration of 7 mg L−1 resulted in a significant increase in
callus induction (Figure 3) when used along with carbenicillin at a concentration of 200 mg L−1 , as
shown in Figure 4a,b. Four out of 5 shoots were tested positive in each biological replicate. When the
Genes 2020, 11, 860 8 of 17
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concentration of
concentration of kanamycin
kanamycin was was increased to to 8–10 mgmg L−1−1, shoot regeneration was not significantly
concentration of kanamycin was increased
increased to 8–108–10 mg LL−1,, shoot
shoot regeneration
regeneration was was not
not significantly
significantly
inhibited. However,
inhibited. However, more callus
more callus browning,
browning, followed
followed by by shoot
shoot development
development was was prevalent.
prevalent. By further
By further
further
inhibited. However, more callus browning, followed−1by shoot development was prevalent. By
increasing
increasing thethe kanamycin concentration to 15 mg L−1, poor callus induction was observed and shoots
increasing the kanamycin
kanamycin concentration
concentration to to 15
15 mg
mg LL−1,, poor
poor callus
callus induction
induction waswas observed
observed andand shoots
shoots
could not
could not regenerate even after 4–5 weeks. Eventually, necrosis was predominant when the
could notregenerate
regenerate evenevenafterafter
4–5 weeks. Eventually,
4–5 weeks. necrosisnecrosis
Eventually, was predominant when the kanamycin
was−1 predominant when the
kanamycin concentration
concentration was increased was increased and varied between 15–25
−1 mg L−1 in theAnother
medium. Another
kanamycin concentration wasand varied and
increased between 15–25
varied mg L15–25
between in themgmedium.
L in the medium.antibiotic,
Another
antibiotic,
meropenem, meropenem,
was tested wasalongtested
withalong with carbenicillin
carbenicillin to to
investigate investigate
its effect its
on effect
the on the removal
effective effective
antibiotic, meropenem, was tested along with carbenicillin to investigate its effect on the effective
removal
of of bacterial
bacterial strains from transgenic calli. Although it is uncommonly used for shoot
removal of strains
bacterial from transgenic
strains from calli. Although
transgenic calli.itAlthough
is uncommonly used for shoot
it is uncommonly development,
used for shoot
development,
meropenem gave meropenem
satisfactory gave satisfactory
results, results,
especially when especially when
used at when
a minimalused at a minimal concentration
of 50 mg L−1 .
development, meropenem gave satisfactory results, especially usedconcentration
at a minimal concentration
of
A 50 mg
higher L −1. A higher rate of callus induction was observed followed by a greater number of putative
of 50 mg Lrate
−1. A of callus
higher induction
rate of callus was observed
induction was followed by a greater
observed followed by anumber of putative
greater number shoots
of putative
shoots (Figure
(Figure 4c) with 4c)
fourwith four tested
shoots shootsas tested as positive
positive per per replication.
replication. Fewer Fewer regenerated
regenerated calli andcalli and
shoots
shoots (Figure 4c) with four shoots tested as positive per replication. Fewer regenerated calli and
shoots
were were produced when the concentration of meropenem was increased to 55–60
−1 mg L−1.
shootsproduced
were producedwhen the concentration
when of meropenem
the concentration was increased
of meropenem to 55–60 mg
was increased L . Contrary
to 55–60 mg L−1.
Contrary
to to carbenicillin,
carbenicillin, regenerated regenerated
shoots were shoots were not negatively
not negatively affected by antheincrease in the
Contrary to carbenicillin, regenerated shoots were not affected
negatively by affected
an increaseby in meropenem
an increase in the−1
meropenem concentration.
concentration. From the above-mentioned
From the above-mentioned results,
results, it was results,
determined it was determined that 7 mg L−1
7 mg L−1 kanamycin
thatdetermined
meropenem concentration. From the above-mentioned it was that 7 mgand L
kanamycin
50 mg L −1 and 50
meropenem mg L −1 meropenem were appropriate concentrations for the effective selection of
were appropriate concentrations for the effective selection of resistant transgenic
kanamycin and 50 mg L meropenem were appropriate concentrations for the effective selection of
−1
resistant
Jinba transgenic Jinba plants.
plants.
resistant transgenic Jinba plants.

Figure 3. Developmental stages and screening of CmTFL1a transgenic plants of chrysanthemum cv.
Figure 3. Developmental
Developmental stages
stages and
and screening
screening of
of CmTFL1a
CmTFL1a transgenic
transgenic plants
plants of
of chrysanthemum
chrysanthemum cv.
cv.
Jinba. Callus regeneration after 2 weeks on selection medium containing meropenem (50 mg L−1 −1) and
Jinba. Callus regeneration after 2 weeks on selection
selection medium
medium containing
containing meropenem
meropenem (50
(50 mg
mg LL −1) and
kanamycin (7 mg L−1−1). Induction of resistant adventitious shoots after 3 weeks. Kanamycin resistant
kanamycin (7 mg L−1 ).).Induction
Inductionofofresistant
resistant adventitious
adventitious shoots
shoots after
after 3 weeks. Kanamycin
Kanamycin resistant
shoots with large green leaves after 4 weeks.
shoots with large green leaves after 4 weeks.

Browning Rate % Browning rate %

Callus Induction Rate % Callus Induction %
100 100
90 a 90
a
a
80 80
b
80 80
b 70 70
Callus Induction rate (%)
Browning Rate (%)
Callus Induction Rate %

c a
c
Browning Rate %

60 b 60
d b
60 60
b 50 50

40 d 40
40 40 c
c 30 30
d
20 20
20 20
10 10

0 0
0 0 5 6 7 8
200 300 400 500 Kanamycin (mg/L)
Carbenicillin (mg/L)

(a) (b)
(a) (b)
Figure 4. Cont.
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Browning Rate %
Callus Induction rate %

100 100
a

Callus Induction Rate (%)

80 80
a

Browning Rate (%)

b
60 60
c

b
40 d c 40

d
20 20

0 0
45 50 55 60
Meropenem (mg/ L)

(c)
(c)
Figure
Figure4.4.4.Effects
Figure Effectsof
Effects ofofvarious
various
various antibiotics
antibiotics
antibiotics combinations
combinations
combinations on onbrowning
on browning rate ofrate
browning leafof
rate ofleaf
discsleafdiscs
and discsand
callusand callus
callus
induction
induction
induction
from from leaf
fromof leaf
leaf discs discs of Chrysanthemum
discs of Chrysanthemum
Chrysanthemum Jinba;
Jinba; (a) EffectsJinba; (a) Effects
(a) Effects
of carbenicillin; of
(b)of carbenicillin;
carbenicillin;
Effects (b) Effects
(b) Effects
of kanamycin; ofof
(c) Effects
kanamycin;
kanamycin;
of meropenem. (c)
(c)Effects
Black ofofmeropenem.
Effectscolumns
meropenem. Black
represent Black columns raterepresent
columns
browning represent
(%), browning
browning
red columns rate
rate(%),
represent (%),red columns
redinduction
callus columns
represent
represent callus
callus induction
induction rate
rate (%). Three
(%). Three biological
biological replicates
replicates were
were used.
used. Error
Error bars
bars
rate (%). Three biological replicates were used. Error bars represent ± SD. a, b, c, and d are statisticallyrepresent
represent ±±SD.
SD.
a,significant
b, c, and d
a, b, c, and at are
d are statistically
p ≤ statistically
0.05 by ANOVAsignificant
significantat p ≤ 0.05
at ptest.
on ranks by ANOVA on ranks
≤ 0.05 by ANOVA on ranks test. test.

3.4.
3.4. Recognitionofof
3.4.Recognition
Recognition ofthe
thePresence
the Presenceofof
Presence ofTransgenes
Transgenesinin
Transgenes Jinba
inJinba Transgenic
JinbaTransgenic Plants
TransgenicPlants
Plants
The
The presence
presenceof
Thepresence ofofthe
thetransgene
the transgene
transgene CmTFL1a
CmTFL1a
CmTFL1awaswasdetected
was in transgenic
detected
detected shootsshoots
inintransgenic
transgenic and, with
shoots and,PCR
and, withanalysis,
with PCR
PCR
it was
analysis, shown
analysis,ititwas that
wasshown all putative
shownthatthatall shoots
allputative contained
putativeshoots fragments
shootscontained of
containedfragments 1100 bp
fragmentsofof1100 in length.
1100bp However,
bpininlength. these
length.However,
However,
fragments
these were were
thesefragments
fragments absent in control
wereabsent
absent plants.plants.
inincontrol
control Positive
plants. CmTFL1a
Positive
Positive transgenic
CmTFL1a
CmTFL1a shoots shoots
transgenic
transgenic are shown
shoots arein
are Figure
shown
shown 5.
inin
Moreover,
Figure the outcomes
Figure5.5.Moreover,
Moreover,the of RT-PCR
theoutcomes analysis
outcomesofofRT-PCR exhibited
RT-PCRanalysis constant
analysisexhibited expression
exhibitedconstant of CmTFL1a
constantexpression in all transgenic
expressionofofCmTFL1a
CmTFL1ainin
lines
all #5, #6, #7
alltransgenic
transgenic and#5,
lines
lines #8,#6,
#5, as#7
#6,shown
#7and in
and#8,#8,Figure 6. ininFigure
asasshown
shown Figure6.6.

Figure
Figure5.5.Positive
PositiveCmTFL1a
Positive CmTFL1atransgenic
CmTFL1a transgenicshoots
shootsofofChrysanthemum
Chrysanthemumcultivar
cultivarJinba;
Jinba;(a)
(a)putative
putativeresistant
resistant
shoot
shooton
onrooting
rootingmedium;
medium;(b)
(b)TG:
TG:Transgenic
Transgenicplants,
plants,WT:
WT:Wild
Wildtype
Wild typeplants
plantsproduced
producedinina agreenhouse.
greenhouse.

Figure 6. RT- PCR Analysis for the expression of CsTFL1a in transgenic lines, M: DL 2000 DNA Marker;
Figure
Figure6.6.RT-
RT-PCR
PCRAnalysis
Analysisfor forthe
theexpression
expressionofofCsTFL1a
CsTFL1ainintransgenic
transgeniclines,
lines,M:
M:DLDL2000
2000DNA
DNA
1: positive control, 2: WT, 3: double distilled (dd) H2 O; 4–8: Transgenic resistant plants.
Marker;
Marker;1:1:positive
positivecontrol,
control,2:2:WT,
WT,3:3:double
doubledistilled
distilled(dd) 2O;
(dd)HH2O;4–8:
4–8:Transgenic
Transgenicresistant
resistantplants.
plants.
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3.5. Expression Pattern of CmTFL1a Gene and Comparison of Other Growth Trends in Transgenic
3.5.
3.5. Expression Pattern
Chrysanthemum Plantsof CmTFL1a Gene and Comparison Comparison of of Other
Other Growth
Growth Trends
Trendsin inTransgenic
Transgenic
Chrysanthemum
Chrysanthemum Plants Plants
qRT-PCR analysis was performed to examine the expression pattern of CmTFL1a in Jinba
qRT-PCR
qRT-PCR
transgenic analysis
analysis
plants. was
After wasperformed
performed
confirmation to from
examine
to examine
PCR, theaexpression of 4pattern
the expression
total of CmTFL1a
pattern
transgenic of of
lines in chrysanthemum
Jinba transgenic
CmTFL1a in Jinba
plants.
(CmTFL1a #5, #6, #7, #8) were obtained and were selected to observe the relative expression level#5,
transgenic After confirmation
plants. After from PCR,
confirmation a total
from of
PCR, 4 transgenic
a total of lines
4 of chrysanthemum
transgenic lines of (CmTFL1a
chrysanthemum of
#6, #7,
(CmTFL1a #8) were
#5, #6,obtained
#7, #8) and
were were
obtained selected
and to observe
were selected the relative
to observe
the transgene comprehensively. The results show that CmTFL1a was absent in control WT plants, expression
the relativelevel of the
expression transgene
level of
comprehensively.
the transgene
whereas Theexpressed
highly results show
it wascomprehensively. The results
in shoot CmTFL1a
thatapices,show was
that
followed absent
CmTFL1a in
was
by vegetative control
absent
buds, WTin plants,
control
floral whereas
budsWT andplants, it
in the
was
whereashighlyit expressed
was highly in shoot
expressed apices,
in shootfollowed
apices, by vegetative
followed by buds,
vegetative
stems of CmTFL1a transgenic lines (Figure 7). The highest expression of CmTFL1a in shoot apices is floral buds
buds, and
floral in
budstheandstems
in of
the
CmTFL1a
stems transgenic
relatedoftoCmTFL1a lines (Figure
transgenic
the suppression of lines 7). The highest
(Figure
inflorescence 7). The expression of CmTFL1a
highest expression
development. Besides, in shoot
of CmTFL1a
phenotypic apices
in shootisapices
measurements related
wereis
to the
related suppression
to the of inflorescence
suppression of development.
inflorescence Besides,
development. phenotypic
Besides,
taken for wild-type and all CmTFL1a transgenic lines. Results on phenotyping depicted a rapid measurements
phenotypic were
measurements taken for
were
wild-type
taken
increase forinandthe all
wild-type CmTFL1a
number and alltransgenic
of days CmTFL1a lines.
to floral buds inResults
transgenic on Results
lines.
transgenic phenotyping depicted
on phenotyping
lines, relative to WT plants. a rapidTheincrease
depicted a rapid
floral in
buds
the number
increase
in wild in the of number
days to floral
chrysanthemum buds
of appeared
days inafter
to floral transgenic
buds
142 in lines,
transgenic
days, relative
whereas no to
lines, WT
relative
bud plants.
to WTThe
formation floral
plants.
in CmTFL1aThebuds
floralin buds
wild
transgenic
chrysanthemum
in wild chrysanthemum appeared after
appeared 142 days,
after 142 whereas
days, no
whereas bud formation
no bud
plants was seen at this stage, as shown in Figure 8a. The four transgenic lines produced floral buds in
formationCmTFL1ain transgenic
CmTFL1a plants
transgenic
was
plantsseen
in 151, was at seen
153, this stage,
at this
160 and 154as shown
stage,
days, as in Figure
shown
about 10 to in208a. The8a.
Figure
days four
laterThe transgenic
four
than lines
transgenic
control plantsproduced
lines
(Figure floral
8b,c). buds
produced floralinbuds
151,
153, 160153,
in 151, and160 154anddays,
154about
days,10about
to 2010 daysto 20later
daysthan latercontrol
than plants
control(Figure 8b,c). 8b,c).
plants (Figure
3.0
3.0 CmTFL 1a lines
Control WT
2.5 CmTFL 1a lines
Control WT
of CmTFL1a

2.5 a
of CmTFL1a

a
2.0
2.0 b
Expression

b
1.5 c
Expression

1.5 c
d
1.0
Relative

d
1.0
Relative

e
0.5
e
0.5

0.0
Control WT Stems Leaves Shoot apices Vegetative buds Floral buds
0.0
Control WT Stems Leaves Shoot apices Vegetative buds Floral buds
Figure 7. Relative expression of CmTFL1a in chrysanthemum cv. Jinba. Expression of CmTFL1a in
Figure
Figure 7. Relative expression of
various7.tissues of expression
chrysanthemum of CmTFL1a
CmTFL1a
by qPCR
in
in chrysanthemum
chrysanthemum cv.
cv. Jinba.
analysis. Actin was Jinba. Expression of CmTFL1a in
used as a reference CmTFL1a in
gene. Three
various
various tissues
tissues of chrysanthemum
of chrysanthemum by qPCR analysis. Actin was used as a reference gene. Three
biological replicates were used for by qPCR
each analysis.
sample. used as
Error bars represent a reference
± SD. gene.
a, b, c, d, andThree
e are
biological
biological replicates
replicates were
were used
used for
for each
each sample.
sample. Error bars represent ± ± SD. a, b, c, d, and e are
are
statistically significant at p ≤ 0.05 by ANOVA on ranks test.
statistically
statistically significant at pp ≤≤ 0.05
significant at 0.05 by
by ANOVA
ANOVA on on ranks
ranks test.
test.

(a) (b)
(a) (b)
Figure 8. Cont.
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(c)
(c)
Figure
Figure 8.
8. The
The flowering
The flowering time
flowering time of
time of CmTFL1a
of CmTFL1a transgenic
CmTFL1a transgenic chrysanthemum
transgenic chrysanthemum cultivar cultivar Jinba;
Jinba; (a)
(a) appearance
appearance of of
floral bud
floral bud
floral bud inin wild
in wild chrysanthemum
wild chrysanthemum
chrysanthemum (upper)(upper) after
(upper) after 142
after 142 days
142 days
days in in contrast
in contrast with
contrast with transgenic
with transgenic chrysanthemum
transgenic chrysanthemum
chrysanthemum
(bottom);
(bottom); (b)
(bottom); floral
(b) floral
(b) bud
floral bud formation
formation after
bud formation after 151
after 151 and
151 and 153
and 153 days
153 days in
days transgenic
transgenic lines
in transgenic
in lines #1
lines #1 and
#1 and #2,
and #2, respectively;
#2, respectively;
respectively;
(c)
(c) floral budding period in wild chrysanthemum and in several transgenic
(c) floral budding period in wild chrysanthemum and in several transgenic lines. Three biological
floral budding period in wild chrysanthemum and in several transgenic lines.
lines. Three
Three biological
biological
replicates were
replicates were
replicates used.
wereused. Error
used. Error
Errorbarsbars represent ±
represent±±SD.
barsrepresent SD. a, b, c, d, and
SD.a,a,b,b,c,c,d,d,and
and e are
e are statistically
statistically
e are significant
significant
statistically at pat≤p
significant at ≤≤ 0.05
p0.05 by
0.05
by ANOVA
ANOVA
by ANOVA on
on ranks ranks test.
test.test.
on ranks

To observe
To
To observeand
observe andcompare
and compare
compare growth
growth
growth potential
potential
potentialat the
at production
at the
the production
production of floral
of buds,buds,
of floral
floral plant height,
buds, plant leaf length
plant height,
height, leaf
leaf
and
lengthleaf
andwidth
leaf were
width recorded
were in
recordedboth inWTbothand
WT transgenic
and plants.
transgenic Consequently,
plants. Consequently,
length and leaf width were recorded in both WT and transgenic plants. Consequently, plant heights plant heights
plant and
heights
leaf
and length
and leaf of CmTFL1a
leaf length
length of CmTFL1a
of CmTFL1alineslines
werewere
lines significantly
were increased,
significantly
significantly increased,
increased,in comparison
in comparison
in comparison withwith
withWTWT
WTones (Figure
ones
ones 9).
(Figure
(Figure
The
9). plantplant
9). The
The heightheight
plant of the WT
height of plantWT
of the
the wasplant
WT 22 cm,was
plant while
was 22thecm,
22 highest
cm, whilegenetically
while the
the highest modified
highest chrysanthemum
genetically
genetically modified
modified
plant was
chrysanthemum40 cm tall, as shown in (Figure 10). Among transgenic lines,
chrysanthemum plant was 40 cm tall, as shown in (Figure 10). Among transgenic lines, strain #1
plant was 40 cm tall, as shown in (Figure 10). Among strain
transgenic#1 and #2
lines, plants
strain #1were
and
and
significantly
#2 plants higher,
were while
significantlyplant height
higher, differences
while plant between
height strain #3
differences and #4
between
#2 plants were significantly higher, while plant height differences between strain #3 and #4 were were nonsignificant.
strain #3 and #4 When
were
compared with When
nonsignificant.
nonsignificant. WT plants,
When the leaf
compared
compared length
with
with WT and
WT width
plants,
plants, the of
the alllength
leaf
leaf transgenic
length and lines were
and width
width of all significantly
of all transgenic different.
transgenic lines
lines were
were
These variations
significantly show higher growth potential of transgenic plants in
significantly different. These variations show higher growth potential of transgenic plants in
different. These variations show higher growth comparison
potential of to the
transgenic wild-type
plants in
plants, which
comparison toindicates
the that
wild-type the trans-CmTFL1a
plants, which gene
indicates can promote
that the the growth
trans-CmTFL1a
comparison to the wild-type plants, which indicates that the trans-CmTFL1a gene can promote the of different
gene can tissue
promoteorgans
the
in chrysanthemum.
growth
growth of
of different
different tissue
tissue organs
organs in in chrysanthemum.
chrysanthemum.

Figure
Figure 9. Phenotype
9. Phenotype
Phenotypeof of WT
ofWT
WTandand CmTFL1a
andCmTFL1a
CmTFL1a transgenic
transgenic lines
lines of
of Chrysanthemum.
Chrysanthemum. WT represents wild-
Figure 9. transgenic lines of Chrysanthemum. WTWT represents
represents wild-
wild-type
type plant
type plant while #1-4 are positive CmTFL1a transgenic lines of Jinba.
plant whilewhile #1-4positive
#1-4 are are positive CmTFL1a
CmTFL1a transgenic
transgenic lines
lines of of Jinba.
Jinba.
Genes 2020, 11, 860 12 of 17
Genes 2020, 11, x FOR PEER REVIEW 12 of 17

Figure 10. Growth potential (plant height, leaf length and leaf width in cm) of CmTFL1a
CmTFL1a transgenic
chrysanthemum. Three biological replicates were used. represent ±
used. Error bars represent ± SD. a, b, c, d, and e are
statistically significant at p ≤
≤ 0.05 by ANOVA on ranks test.

4. Discussion
4. Discussion
Recently, unlike conventional breeding, the introduction of target genes for engineering crops has
Recently, unlike conventional breeding, the introduction of target genes for engineering crops
been made possible by genetic transformation techniques that do not require long-term periods of
has been made possible by genetic transformation techniques that do not require long-term periods
selection [32]. However, there have been few reports on crop improvement in chrysanthemum plants
of selection [32]. However, there have been few reports on crop improvement in chrysanthemum
via the Agrobacterium-mediated transformation method [33–35].
plants via the Agrobacterium-mediated transformation method [33–35].
4.1. Comparison between Standardized and Unstandardized Transformation Protocols
4.1. Comparison between Standardized and Unstandardized Transformation Protocols
In this study, we conducted a comparison between the unstandardized [29] and standardized
In this study, we conducted a comparison between the unstandardized [29] and standardized
protocols of Agrobacterium-mediated transformation for the chrysanthemum cultivar Jinba. Data for
protocols of Agrobacterium-mediated transformation for the chrysanthemum cultivar Jinba. Data for
the optimal conditions of all individual parameters were recorded. The transformation of leaf explants
the optimal conditions of all individual parameters were recorded. The transformation of leaf
of Jinba was performed by following both unstandardized and standardized methods. Different
explants of Jinba was performed by following both unstandardized and standardized methods.
media compositions used in Naing et al. [29] were adapted and modified with new concentrations by
Different media compositions used in Naing et al. [29] were adapted and modified with new
following other literature, as mentioned in Section 2.3. The standardized protocol shown in Scheme 1
concentrations by following other literature, as mentioned in Section 2.3. The standardized protocol
exhibited a higher regeneration and transformation percentage of shoots than the unstandardized
shown in Scheme 1 exhibited a higher regeneration and transformation percentage of shoots than the
protocol by using different selective agents at minimal concentration and generating a greater number
unstandardized protocol by using different selective agents at minimal concentration and generating
of positive transgenic plants The salient parameters which were adapted and standardized in the
a greater number of positive transgenic plants The salient parameters which were adapted and
current study were shoot regeneration, cocultivation, selection and rooting media composition, as
standardized in the current study were shoot regeneration, cocultivation, selection and rooting media
well as cocultivation period and cocultivation temperature. Each of these parameters contributed to
composition, as well as cocultivation period and cocultivation temperature. Each of these parameters
the extent of transformation efficiency in this cultivar. Above all, the standardized protocol resulted
contributed to the extent of transformation efficiency in this cultivar. Above all, the standardized
in a greater number of positive transgenic shoots in each replication, as confirmed by PCR analysis.
protocol resulted in a greater number of positive transgenic shoots in each replication, as confirmed
Therefore, the standardization of individual features related to Agrobacterium-mediated transformation
by PCR analysis. Therefore, the standardization of individual features related to Agrobacterium-
is equally imperative for improving the transformation efficiency in chrysanthemum cultivars.
mediated transformation is equally imperative for improving the transformation efficiency in
chrysanthemum cultivars.
Genes 2020, 11, 860 13 of 17
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Scheme 1.1. Standardized protocol for for transformation

transformation inin chrysanthemum
chrysanthemum cv.
cv. Jinba.
Jinba. Regeneration
Regeneration
medium
medium (RM):
(RM):MSMS++ 0.5 mgLL−1
0.5mg −1 6-BA +
+ 0.5 mg LL−1 NAA, selection
−1NAA, selection medium (SM): MS++ 0.5
(SM): MS mgLL−1
0.5 mg −1 6-BA

++ 0.5 mgLL−1 NAA +

0.5 mg
−1
mg LL−1 meropenem +
+ 50 mg
−1
+ 7 mg
−1
mg LL−1 kanamycin, rooting
rooting medium:
medium: MS MS++ 50
50mg
mgLL−1
−1

meropenem + −1
+ 0.1 mg L−1 NAA.
NAA.

4.2. Optimization of Parameters is Crucial for Higher Transformation Efficiency

4.2. Optimization of Parameters is Crucial for Higher Transformation Efficiency
Several studies have focused on the importance of cocultivation duration to improve the
Several studies have focused on the importance of cocultivation duration to improve the
regeneration and transformation percentage of putative shoots [36], whereas it has been reported that
regeneration and transformation percentage of putative shoots [36], whereas it has been reported that
the optimal cocultivation period is dependent on the explant genotype [37]. It was observed that
the optimal cocultivation period is dependent on the explant genotype [37]. It was observed that
coculturing of chrysanthemum (Dendranthema grandiflora) var. Micromargara explants for two days
coculturing of chrysanthemum (Dendranthema grandiflora) var. Micromargara explants for two days
significantly affected their transformation efficiency [36]. A prolonged cocultivation period resulted
significantly affected their transformation efficiency [36]. A prolonged cocultivation period resulted
in the overgrowth of bacteria. Thus, coculturing does not improve the transformation efficiency of
in the overgrowth of bacteria. Thus, coculturing does not improve the transformation efficiency of
plants [28].
plants [28].
The optimization of temperature conditions during cocultivation significantly affects
The optimization of temperature conditions during cocultivation significantly affects
transformation efficiency. The main reason is that the T-DNA transfer machinery works more
transformation efficiency. The main reason is that the T-DNA transfer machinery works more
efficiently at low temperatures. In this study, explants of Jinba exhibited the highest transformation
efficiently at low temperatures. In this study, explants of Jinba exhibited the highest transformation
efficiency when cocultured for 2 days at 22 ◦ C. Our findings were consistent with some other reports
efficiency when cocultured for 2 days at 22 °C. Our findings were consistent with some other reports
that indicated that a temperature of 20 ◦ C for a period of 2–3 days is optimal for orchid plant and A.
that indicated that a temperature of 20 °C for a period of 2–3 days is optimal for orchid plant and A.
tumefaciens cocultivation [38,39]. When the cocultivation temperature in this study was increased from
tumefaciens cocultivation [38,39]. When the cocultivation temperature in this study was increased
23 to 28 ◦ C, the transformation efficiency was very low.
from 23 to 28 °C, the transformation efficiency was very low.
Few reports are available on the effects of various antibiotics on Agrobacterium-mediated
Few reports are available on the effects of various antibiotics on Agrobacterium-mediated
transformation of Chrysanthemum. The exclusion of Agrobacterium from cultures is equally essential.
transformation of Chrysanthemum. The exclusion of Agrobacterium from cultures is equally essential.
The prolonged presence of Agrobacterium strains in cultured explants may decrease the cell proliferation
The prolonged presence of Agrobacterium strains in cultured explants may decrease the cell
rate resulting in the death of cells [30]. There are several antibiotics used to eliminate Agrobacterium cells
proliferation rate resulting in the death of cells [30]. There are several antibiotics used to eliminate
from treated explants, for example, carbenicillin, cefotaxime, timentin and meropenem. Meropenem
Agrobacterium cells from treated explants, for example, carbenicillin, cefotaxime, timentin and
belongs to the carbapenem class of antibiotics that possesses extraordinary in vitro bactericidal activity
meropenem. Meropenem belongs to the carbapenem class of antibiotics that possesses extraordinary
against various microorganisms and is considered novel in removing Agrobacterium strains [40].
in vitro bactericidal activity against various microorganisms and is considered novel in removing
In this study, the authors examined the effects of two bactericidal antibiotics, i.e., meropenem
Agrobacterium strains [40]. In this study, the authors examined the effects of two bactericidal
and carbenicillin, on callus growth. It was found that a higher concentration of carbenicillin,
antibiotics, i.e., meropenem and carbenicillin, on callus growth. It was found that a higher
200 mg L−1 , was required to control the −1 regrowth of Agrobacterium. On the other hand, a much lower
concentration of carbenicillin, 200−1mg L , was required to control the regrowth of Agrobacterium. On
quantity of meropenem, 50 mg L , was required for the same purpose. It was confirmed that minor
the other hand, a much lower quantity of meropenem, 50 mg L−1, was required for the same purpose.
concentrations of meropenem are efficient in eliminating Agrobacterium cells from Phalaenopsis [30,40].
It was confirmed that minor concentrations of meropenem are efficient in eliminating Agrobacterium
cells from Phalaenopsis [30,40]. A higher concentration of carbenicillin may also lead to cell necrosis
Genes 2020, 11, 860 14 of 17

A higher concentration of carbenicillin may also lead to cell necrosis in the culture medium for two
weeks. In contrast, meropenem effectively ceased bacterial growth at lower concentrations without
any phytotoxicity.
For the selection of putative transgenic plants, as well as for improving shoot regeneration and
transformation frequency of Jinba shoots, kanamycin was used at various concentrations, as the
optimal concentration of kanamycin differs for each cultivar. It was reported that 30 mg L−1 kanamycin
was optimal for the selection of C. morifolium var. Vivid Scarlet plants [14]. In addition, kanamycin
prominently reduced shoot regeneration from stem segments of Yamabiko plants at a concentration
of 10 mg L−1 [31]. In the current study, 7 mg L−1 kanamycin exhibited maximum callus induction
followed by the greatest number of putative resistant shoots. However, 7.5–8 mg L−1 kanamycin
completely inhibited callus induction and led to browning of treated explants.

4.3. Chrysanthemum CmTFL1a Delays Flowering and Regulate Growth Potential

TFL1 controls the shoot apical meristem in the vegetative state of a plant [41]. During the juvenile
period, it expresses in shoot apices that continue during floral bud and flower development [42].
The overexpression of the CmTFL1 gene in chrysanthemum significantly delayed flowering [22,24],
which shows that the TFL1 gene has a conservative role in flowering regulation. Besides, the
overexpression of the maize ZEA CENTRORADIALIS (ZCN) gene (belong to the TFL1-like family)
extended the indeterminate growth of the inflorescence meristem, resulting in later floral transition [43].
In apple, the expression of MdTFL1 and MdTFL1a was dominant in apical shoots during vegetative
growth [44]. The TFL1 gene was able to cause a significant delay in flowering and aided in the
regulation of inflorescence architecture in several fruit tree species: citrus (Citrus sinensis) [45], apple
(Malus × domestica) [46] and Japanese apricot (Prunus mume) [47]. Moreover, 35S:: CmTFL1a significantly
improved the growth potential (plant height, leaf length, leaf width) of 35S:: CmTFL1a genetically
modified plants as compared to wild plants. Specifically, an increase in plant height may be related to
a high level of CmTFL1a gene expression in shoot apices, promoting the growth of the apical meristem.
These results were consistent with the findings of [48].

5. Conclusions
In this work, the authors used an optimized method for regeneration and Agrobacterium-mediated
transformation in the chrysanthemum cultivar Jinba. The optimized protocol can lead to a higher rate
of transformation frequency as compared to an unstandardized one. Genetic transformation with A.
tumefaciens by using leaf explants was effective in the construction of a standardized transformation
scheme for Jinba. In summary, this study proposes that the evaluation of ideal conditions is crucial
to increase the success rate of genetic transformation in chrysanthemum. Because of the lower
concentrations and short period time for meropenem application, this method is the simplest as well
as most cost-effective technique. In addition, maximum transgenic lines detected by RT-PCR showed
stable expression of CmTFL1a. We assume that the protocol and conditions enhanced in this study will
be helpful in the genetic engineering of this cultivar with various desirable genes. Furthermore, it will
help to understand the biological phenomenon of the TFL1 homolog in chrysanthemum.

Author Contributions: S.H. conceived the study and wrote the manuscript; Y.G. (Yike Gao) investigated,
supervised the research project and revised the manuscript; Y.G. (Yike Gao) contributed reagents/materials.
Culture establishment, gene transfer process and validation of results were supervised by Y.G. (Yaohui Gao).
Culture establishment, production of transgenic plants, data record and application of statistical techniques
to analyze study data were conducted by S.H. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by National Natural Science Foundation of China (Grant No.31971706).
Acknowledgments: This work was supported by National Natural Science Foundation of China (No.31971706).
Conflicts of Interest: The authors declare that they have no conflict of interest.
Genes 2020, 11, 860 15 of 17

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