Standardized Genetic Transformation Protocol for Chrysanthemum Cv

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; [email protected] 2 Architectural Institute, Inner Mongolia University of Science & Technology, Alding Street No.7, Kundulun District, Baotou 014010, China; [email protected] * Correspondence: [email protected]; 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 1 1 1 medium for shoot initiation (MS 4.4 g L− + Myoinositol 100 mg L− + 2 mg L− BAP (1 mL stock 1 1 solution + IAA (1 mL stock solution) 1 mg L− + Sucrose 20 g/L + Agar 8 g L− ) at pH 5.7, and were 2 1 cultivated at 24 ◦C, 80 µE m− s− 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 1 1 1 for multiplication (MS 4.4 g L− + Sucrose 20 g L− + Agar 6.5 g L− ) 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 Table1, were used for each hormone and antibiotic. 1 1 The concentrations of the growth regulators 6-BA (0.5 mg L− ), 2,4-D (0.2 mg L− ) and NAA 1 1 (0.1 mg L− )[26]; and those of antibiotics carbenicillin (200 mg L− )[13], meropenem (50 mg 1 1 L− )[27] and kanamycin (7 mg L− )[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.

1 Antibiotics Hormones Concentrations (mg L− ) 1 Concentrations (mg L− ) 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 1 1 (MS + 0.5 mg L− 6BA + 0.1 mg L− NAA as control). Treated explants plated on cocultivation medium 1 1 1 (MS + 0.5 mg L− 6BA + 0.1 mg L− NAA as control) with carbenicillin (400 mg L− ) 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 1 1 of these antibiotics were prepared by filter-sterilization at concentrations of 400 mg L− , 50 mg L− and 1 30 mg L− , 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 (Table1), 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 Table2. 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 (Table2) were designed by the Primer 5.0 software. The total volume of 20 µL mixture was prepared with 2 µL cDNA template (10X), 1 0.4 µL of each amplification primer (10 mmol L− ) 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 Figure1. Genes 2020, 11, 860 6 of 17 Genes 2020, 11, x FOR PEER REVIEW 6 of 17

Figure 1. Relation of cocultivation period with shoot re regenerationgeneration and transformation efficiency efficiency of Jinba. Black Black columns columns represent represent shoot shoot regeneration regeneration percentage, red columns represent transformation efficiencyefficiency percentage. Explants Explants were were cocultured for 1, 2 and 3 days. Three biol biologicalogical replicates were used. Error Error bars bars represent represent ± SD.SD. a, b, a, and b, and c are c arestatistica statisticallylly significant significant at p ≤ at 0.05p by0.05 ANOVA by ANOVA on ranks on ± ≤ test.ranks test.

3.2. Relation between cocultivation temperature and transformation eﬃciency 3.2. Relation between cocultivation temperature and transformation efficiency It can be seen in Figure2 that temperature adjustment during cocultivation plays a crucial role in It can be seen in Figure 2 that temperature adjustment during cocultivation plays a crucial role shoot regeneration. Explants were cocultivated at various temperature conditions and the maximum in shoot regeneration. Explants were cocultivated at various temperature conditions and the number of putative shoots was observed when the temperature maintained during the cocultivation maximum number of putative shoots was observed when the temperature maintained during the period was 22 C. However, explants cocultured at 24 C yielded fewer transgenic shoots than those cocultivation period◦ was 22 °C. However, explants cocultured◦ at 24 °C yielded fewer transgenic produced at 22 C but more transgenic shoots than those produced at 28 C. In fact, overgrowth of shoots than those◦ produced at 22 °C but more transgenic shoots than those ◦produced at 28 °C. In fact, bacteria was observed after explants were transferred to the selection medium. overgrowth of bacteria was observed after explants were transferred to the selection 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

bb bb 2 2 2 2

0 0 22oC 24oC 28oC 22oC 24oC 28oC Co-cultivation Temperature (oC) Co-cultivation Temperature (oC) Figure 2. Figure 2. EffectsEffects of cocultivation temperature on shoot re regenerationgeneration and transformation efficiency efficiency of Jinba. Black columns represent shoot 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, 24 and 26 C. Three biological replicates efficiency percentage. Coculture plates were maintained at 22, 24 and◦ 26 °C. Three biological werereplicates used. were Error used. bars E representrror bars representSD. a, b, and± SD. c area, b, statistically and c are statistically significant atsignificantp 0.05 by at ANOVAp ≤ 0.05 by on replicates were used. Error bars± represent ± SD. a, b, and c are statistically significant≤ at p ≤ 0.05 by ranks test. ANOVA on ranks test.

3.3. Role of Various Growth Regulators and Antibiotics on Shoot 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 1 1 when a combination of the hormones 6-BA and 2,4-D at concentrations of 1 mg L− and 0.25 mg L− , respectively, was used in the selection medium. This combination eventually resulted in the maximum 1 regeneration of putative shoots. Increasing the concentration of 6-BA to 1.5–2 mg L− and 2,4-D to 1 0.3–0.4 mg L− resulted in a prominent decrease in the total number of shoots per explant and shoot regeneration percentage, as shown in Table3. Furthermore, the e ﬀect of 6-BA and NAA combination on regeneration and genetic transformation percentage of the resistant shoots was examined; 0.8 mg 1 1 L− NAA with 1 mg L− 6-BA was the most suitable combination for the maximum number of shoots, 1 whereas a low concentration range of NAA 0.1–0.4 mg L− retarded the growth of the explants, as shown in Table4. 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 1 f 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 1 concentrations. Kanamycin at a concentration of 7 mg L− resulted in a significant increase in 1 callus induction (Figure3) when used along with carbenicillin at a concentration of 200 mg L − , as shown in Figure4a,b. Four out of 5 shoots were tested positive in each biological replicate. When the Genes 2020, 11, 860 8 of 17 Genes 2020, 11, x FOR PEER REVIEW 8 of 17 Genes 2020, 11, x FOR PEER REVIEW 8 of 17

concentration of kanamycin was increased to 8–10 mg L−11, shoot regeneration was not significantly concentration of kanamycin was increased to 8–108–10 mgmg LL−−1,, shoot shoot regeneration regeneration was was not significantly significantly inhibited. However, more callus browning, followed by shoot development was prevalent. By further inhibited. However, However, more callus browning, followed by shoot development was prevalent. By further further increasing the kanamycin concentration to 15 mg L−11, poor callus induction was observed and shoots increasing the kanamycin concentrationconcentration toto 1515 mgmg LL−−1,, poor poor callus callus induction induction was was observed observed and and shoots shoots could not regenerate even after 4–5 weeks. Eventually, necrosis was predominant when the could notnot regenerate regenerate even even after after 4–5 weeks.4–5 weeks. Eventually, Eventually, necrosis necrosis was predominant was predominant when the kanamycinwhen the kanamycin concentration was increased and varied between 151 –25 mg L−1 in the medium. Another kanamycinconcentration concentration was increased was and increased varied and between varied 15–25 between mg L 15–25− in themg medium. L−1 in the Anothermedium. antibiotic, Another antibiotic, meropenem, was tested along with carbenicillin to investigate its effect on the effective antibiotic,meropenem, meropenem, was tested was along tested with along carbenicillin with carbenicillin to investigate to investigate its effect its on effect the e ffonective the effective removal removal of bacterial strains from transgenic calli. Although it is uncommonly used for shoot removalof bacterial of strainsbacterial from strains transgenic from calli.transgenic Although calli. itAlthough is uncommonly it is uncommonly used for shoot used development, for shoot development, meropenem gave satisfactory results, especially when used at a minimal concentration1 development,meropenem gave meropenem satisfactory gave results, satisfactory especially results, when especially used at when a minimal used concentrationat a minimal concentration of 50 mg L− . of 50 mg L−1. A higher rate of callus induction was observed followed by a greater number of putative Aof 50 higher mg L rate−1. A ofhigher callus rate induction of callus wasinduction observed was followedobserved followed by a greater by a numbergreater number of putative of putative shoots shoots (Figure 4c) with four shoots tested as positive per replication. Fewer regenerated calli and (Figureshoots (Figure4c) with 4c) four with shoots four testedshoots as tested positive as positive per replication. per replication. Fewer regeneratedFewer regenerated calli and calli shoots and shoots were produced when the concentration of meropenem was increased to 55–1 60 mg L−1. wereshoots produced were produced when the when concentration the concentration of meropenem of meropenem was increased was increased to 55–60 mgto 55–60 L− . Contrary mg L−1. Contrary to carbenicillin, regenerated shoots were not negatively affected by an increase in the Contraryto carbenicillin, to carbenicillin, regenerated regenerated shoots were shoots not negativelywere not anegativelyffected by affected an increase by inan theincrease meropenem in the meropenem concentration. From the above-mentioned results, it was determined1 that 7 mg L−1 meropenemconcentration. concentration. From the above-mentioned From the above-mentio results, itned was results, determined it was that determined 7 mg L− kanamycin that 7 mg andL−1 kanamycin1 and 50 mg L−1 meropenem were appropriate concentrations for the effective selection of kanamycin50 mg L− meropenem and 50 mg wereL−1 meropenem appropriate were concentrations appropriate for concentrations the effective selection for the ofeffective resistant selection transgenic of resistant transgenic Jinba plants. resistantJinba plants. transgenic Jinba plants.

Figure 3. Developmental stages and screening of CmTFL1a transgenic plants of chrysanthemum cv. Figure 3. Developmental stages stages and and screening screening of of CmTFL1aCmTFL1a transgenictransgenic plants plants of of chrysanthemum chrysanthemum cv. cv. Jinba. Callus regeneration after 2 weeks on selection medium containing meropenem (50 mg L−1) and Jinba. Callus Callus regeneration afterafter 2 weeks on selection mediummedium containingcontaining meropenemmeropenem (50(50 mgmg LL−11) and kanamycin (7 mg L−1). Induction of resistant adventitious shoots after 3 weeks. Kanamycin resistant− kanamycin (7 mg L −11).). Induction Induction of of resistant resistant adventitious adventitious shoots shoots after after 3 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 Ra te % Callus Induction % 100 100 90 90 a a a 80 80 b 80 80 b

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B C d 20 20

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

FigureFigure 4. 4. 4. Effects EEffectsffects ofof of variousvarious various antibiotics antibiotics antibiotics combinations combinations combinations on browningon on browning browning rate ofrate rate leaf of discs of leaf leaf and discs discs callus and and induction callus callus inductionfrominduction leaf discsfrom from of leaf leafChrysanthemum discs discs of of ChrysanthemumChrysanthemum Jinba; (a)Effects Jinba;JinbaJinba; of carbenicillin; ;( a ()a )Effects Effects (bof )E of carbenicillin; ff carbenicillin;ects of kanamycin; (b(b) )Effects Effects (c)Eff ectsof of kanamycin;ofkkanamycin;anamycin; meropenem. ( c()c )Effects BlackEffects columnsof of meropenem. mmeropenem.eropenem represent .Black Black browning columns columns rate represent (%),represent red columnsbrowning browning represent rate rate (%), (%), callus red red inductioncolumns columns representraterepresent (%). callus Three callus induction biological induction replicatesrate rate (%). (%). Three wereThree used.biological biological Error replicates barsreplicates represent were were used. used.SD. a,Error Error b, c, bars andbars represent d represent are statistically ± ±±SD. SD. ± a,significanta, b, b, c, c, and and d atd are arep statistically statistically0.05 by ANOVA significant significant on ranks at at p p≤ test. ≤0.05 0.05 by by ANOVA ANOVA on on ranks ranks test. test. ≤ 3.4.3.4. Recognition Recognition of of the the Presence Presence of of Transgenes Transgenes in inin Jinba JinbaJinba Transgenic TransgenicTransgenic Plants PlantsPlants TheThe presence presence presence of of thethe the transgene transgene transgeneCmTFL1a CmTFL1a CmTFL1awas was was detected detected detected in transgenic in in transgenic transgenic shoots shoots shootsand, with and, andand, PCR ,with with analysis, PCR PCR analysis,itanalysisanalysis, was shown ,it it was was thatshown shown all that putativethat all all putative putative shoots shoots contained shoots co containedcontainedntained fragments fragments fragments of 1100 of of bp1100 1100 in bp length. bp in in length. lengthlength. However, .However, HHowever,owever, these thesefragmentsthese fragments fragments were were absent were absent absent in control in in control cocontrol plants.ntrol plants. plants. Positive Positive PositiveCmTFL1a CmTFL1a CmTFL1atransgenic transgenic transgenic shoots shoots are shoots shown are are inshown shown Figure in in5 . FigureMoreover,Figure 5. 5. Moreover, Moreover, the outcomes the the outcomes ofoutcomes RT-PCR of of analysis RT-PCR RTRT-PCR-PCR exhibited analysis analysis constant exhibited exhibited expression constant constant ofexpression expressionCmTFL1a ofin of CmTFL1a all CmTFL1a transgenic in in alllinesall transgenic transgenic #5, #6, #7lines lines and #5, #5, #8, #6, #6, as #7 shown#7 and and #8, in#8#8, Figureas, as shown shown6. in in Figure Figure 6. 66. .

FigureFigure 5. 5. Positive PositivePositive CmTFL1a CmTFL1a transgenic transgenictransgenic shoots shoots shoots of of of Chrysanthemum Chrysanthemum cultivar cultivar Jinba; JinbaJinba; (;a ( )a putative) putative resistant resistant shootshoot on on rooting rooting medium; medium; (b ( b) TG:) TG: TG: Transgenic Transgenic Transgenic plants, plants, WT: WT: Wild Wild type typetype plants plants produced produced in in a a greenhouse. greenhouse. greenhouse.

Figure 6. RT- PCR Analysis for the expression of CsTFL1a in transgenic lines, M: DL 2000 DNA Marker; FigureFigure 6. 6. RT- RTRT- -PCR PCR Analysis Analysis for for the the expression expression of of CsTFL1a CsTFL1a in in transgenic transgenic lin lines,lines,es, M: M: DL DL 2000 2000 DNA DNA 1: positive control, 2: WT, 3: double distilled (dd) H2O; 4–8: Transgenic resistant plants. Marker;Marker; 1: 1: positive positive control, control, 2: 2: WT, WT, 3: 3: double double distilled distilled (dd) ((dd)dd H) H2O;2O; 4–8: 44–8:–8 Transgenic: Transgenic resistant resistant plants. plantsplants. . GenesGenes2020 2020, ,11 11,, 860 x FOR PEER REVIEW 10 ofof 1717 Genes 2020, 11, x FOR PEER REVIEW 10 of 17 3.5. Expression Pattern of CmTFL1a Gene and Comparison of Other Growth Trends in Transgenic 3.5.3.5.Chrysanthemum Expression Pattern Plants of CmTFL1a Gene and Comparison ofof OtherOther GrowthGrowth TrendsTrends inin TransgenicTransgenic ChrysanthemumChrysanthemum PlantsPlants qRT-PCR analysis was performed to examine the expression pattern of CmTFL1a in Jinba CmTFL1a transgenicqRT-PCR plants. analysis analysis After was was confirmation performed performed to from examine to examine PCR, the a expression totalthe expressionof 4 pattern transgenic pattern of lines of ofCmTFL1ain chrysanthemum Jinba transgenic in Jinba CmTFL1a plants.transgenic(CmTFL1a After #5,plants. confirmation #6, #7, After #8) wereconfirmation from obtained PCR, afrom and total werePCR, of 4 transgenic selecteda total ofto linesobserve4 transgenic of chrysanthemumthe relative lines of expression chrysanthemum ( level#5, of #6,(theCmTFL1a #7, transgene #8) were#5, #6,comprehensively. obtained #7, #8) were and wereobtained The selected results and towere show observe selected that the CmTFL1a to relative observe was expression the absent relative levelin expressioncontrol of the WT transgene level plants, of CmTFL1a comprehensively.thewhereas transgene it was comprehensively. highly The expressed results show Thein shoot results that apices, show fo thatllowedwas CmTFL1a absentby vegetative inwas control absent buds, WT in floral control plants, buds WT whereas and plants, in the it waswhereasstems highly of itCmTFL1a was expressed highly transgenic inexpressed shoot lines apices, in shoot(Figure followed apices, 7). The byfo llowedhighest vegetative by expression vegetative buds, floral of buds, CmTFL1a buds floral and in buds in shoot the and stemsapices in the of is CmTFL1a CmTFL1a stemsrelated of to CmTFL1atransgenic the suppression transgenic lines (Figure of linesinflorescence7 ).(Figure The highest 7). devel The expressionopment. highest expressionBesides, of phenotypic of CmTFL1ain shoot measurements in apices shoot is apices related were is torelatedtaken the suppressionfor to thewild-type suppression of inflorescenceand all of CmTFL1ainflorescence development. transgenic devel Besides,opment. lines. Results phenotypicBesides, on phenotypic phenotyping measurements measurements depicted were taken a rapidwere for CmTFL1a wild-typetakenincrease for in andwild-type the allnumber and of daysalltransgenic CmTFL1a to floral buds lines.transgenic in Results transgenic lines. on Results phenotypinglines, relative on phenotyping depictedto WT plants. a rapiddepicted The increase floral a rapid buds in theincreasein wild number chrysanthemumin the of number days to floralof appeareddays buds to floral in after transgenic buds 142 indays, transgenic lines, whereas relative lines, no tobud relative WT formation plants. to WT The inplants. CmTFL1a floral The buds floral transgenic in buds wild CmTFL1a chrysanthemuminplants wild waschrysanthemum seen at appeared this stage, appeared after as 142shown after days, 14in 2whereasFigure days, whereas8a. no The bud four no formation bud transgenic formation in lines in producedCmTFL1atransgenic floraltransgenic plants buds wasplantsin 151, seen was 153, at seen this160 andstage,at this 154 as stage, days, shown asabout shown in Figure 10 toin 820 Figurea. days The 8a. fourlater The transgenicthan four control transgenic lines plants produced lines (Figure produced floral 8b,c).buds floral in buds 151, 153,in 151, 160 153, and 160 154 and days, 154 about days, 10 about to 20 10 days to 20 later days than later control than plantscontrol (Figure plants 8(Figureb,c). 8b,c). 3.0

3.0 CmTFL 1a lines Control WT 2.5 CmTFL 1a lines Control WT 2.5 a a 2.0 CmTFL1a 2.0 b CmTFL1a b 1.5 c

1.5 c d

1.0 d 1.0

Relative Expression of Relative 0.5 e Relative Expression of Relative 0.5 e

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 7. Relative expression of CmTFL1a in chrysanthemum cv. Jinba. Expression of CmTFL1a in Figurevarious 7. tissues Relative of expressionchrysanthemum of CmTFL1a by qPCR in chrysanthemumanalysis. Actin wascv. Jinba.used asExpression a reference of CmTFL1agene. Three in various tissues of chrysanthemum by qPCR analysis. Actin was used as a reference gene. Three variousbiological tissues replicates of chrysanthemum were used for byeach qPCR sample. analysis. Error Actin bars wasrepres usedent as± SD.a re a,ference b, c, d,gene. and Three e are biological replicates werewere usedused forfor eacheach sample.sample. Error bars repres representent ± SD. a, b, c, d, and e areare statistically significant at p ≤ 0.05 by ANOVA on ranks test. ± statistically signiﬁcant at p 0.05 by ANOVA on ranks test. statistically significant at p ≤≤ 0.05 by ANOVA on ranks test.

(a) (b) (a) (b) Figure 8. Cont. Genes 2020,, 11,, 860x FOR PEER REVIEW 1111 of of 17

(c)

Figure 8. TheThe flowering flowering time of CmTFL1aCmTFL1a transgenictransgenic chrysanthemum chrysanthemum cultivar Jinba; ( a) appearance of floralfloral bud in wild chrysanthemum (upper) after 142 days in contrast with transgenic chrysanthemum (bottom); ( b) floralfloral bud formation after 151 and 153 days in transgenic lines #1 and #2, respectively; (c) floralfloral buddingbudding periodperiod inin wildwild chrysanthemumchrysanthemum andand inin severalseveral transgenictransgenic lines.lines. Three biological replicates werewere used.used. ErrorError bars bars represent represent ±SD. SD. a, a, b, b, c, c, d, d, and and e aree are statistically statistically significant significant at pat p0.05 ≤ 0.05 by replicates were used. Error bars represent± ± SD. a, b, c, d, and e are statistically significant at≤ p ≤ 0.05 ANOVAby ANOVA on rankson ranks test. test.

To observeobserve and and compare compare growth growth potential potential at theat the production production of floral of floral buds, bu plantds, height,plant height, leaf length leaf andlength leaf and width leaf width were recorded were recorded in both in WT both and WT transgenic and transgenic plants. plants. Consequently, Consequently, plant plant heights heights and leafand lengthleaf length of CmTFL1a of CmTFL1alines lines were were significantly significantly increased, increased, incomparison in comparison with with WT WT ones ones (Figure (Figure9). The9). The plant plant height height of the WTof the plant WT was plant 22 cm, was while 22 the cm, highest while genetically the highest modified genetically chrysanthemum modified plantchrysanthemum was 40 cm tall,plant as was shown 40 cm in tall, (Figure as shown 10). Among in (Figure transgenic 10). Among lines, transgenic strain #1 andlines, #2 strain plants #1 were and significantly#2 plants were higher, significantly while plant higher, height while differences plant betweenheight differences strain #3 and between #4 were strain nonsignificant. #3 and #4 Whenwere comparednonsignificant. with When WT plants, compared the leaf with length WT andplants, width the ofleaf all length transgenic and width lines wereof all significantlytransgenic lines diff erent.were Thesesignificantly variations different. show higherThese growthvariations potential show ofhi transgenicgher growth plants potential in comparison of transgenic to the wild-typeplants in plants,comparison which to indicates the wild-type that the plants, trans- CmTFL1awhich indicatesgene can that promote the trans- theCmTFL1a growth of gene different can tissuepromote organs the ingrowth chrysanthemum. of different tissue organs in chrysanthemum.

Figure 9. Phenotype of WT and CmTFL1a transgenic lines of Chrysanthemum. WT represents wild- Figure 9.9. Phenotype ofof WT WT and andCmTFL1a CmTFL1atransgenic transgenic lines lines of Chrysanthemum.of Chrysanthemum. WT WT represents represents wild-type wild- type plant while #1-4 are positive CmTFL1a transgenic lines of Jinba. planttype plant while while #1-4 are#1-4 positive are positiveCmTFL1a CmTFL1atransgenic transgenic lines oflines Jinba. of 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 transgenictransgenic chrysanthemum. Three biolog biologicalical replicates were used. Error bars represent ± SD. a, b, c, d, and e are ± statistically significantsignificant 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 Scheme1 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 Genes 2020, 11, x FOR PEER REVIEW 13 of 17

Scheme 1.1. Standardized protocol forfor transformationtransformation inin chrysanthemumchrysanthemum cv.cv. Jinba.Jinba. RegenerationRegeneration 1 1 1 medium (RM):(RM): MSMS+ + 0.50.5 mgmg LL−−1 6-BA ++ 0.5 mg LL−−1NAA,NAA, selection selection medium (SM):(SM): MSMS+ + 0.50.5 mgmg LL−−1 6-BA 1 1 1 1 ++ 0.50.5 mgmg LL−−1 NAA ++ 50 mgmg LL−−1 meropenem ++ 7 mg LL−−1 kanamycin, rooting medium:medium: MSMS+ + 5050 mgmgL L−−1 1 meropenem + 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 regrowth of Agrobacterium. On the other hand, a much lower concentration− of carbenicillin, 200 mg L−1, was required to control the regrowth of Agrobacterium. On quantity of meropenem, 50 mg L 1, was required for the same purpose. It was confirmed that minor the other hand, a much lower quan− tity 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 1 optimal concentration of kanamycin diﬀers for each cultivar. It was reported that 30 mg L− 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 1 1 of 10 mg L− [31]. In the current study, 7 mg L− kanamycin exhibited maximum callus induction 1 followed by the greatest number of putative resistant shoots. However, 7.5–8 mg L− 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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