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Article

An Effective Agrobacterium-Mediated Transient Transformation System for Studying the Lead-Tolerance Genes in Hydrangea

by
Rong Cong
,
Liang Shi
and
Bing Zhao
*
College of Landscape Architecture and Arts, Northwest A&F University, Xianyang 712100, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(3), 320; https://doi.org/10.3390/horticulturae11030320
Submission received: 4 February 2025 / Revised: 7 March 2025 / Accepted: 10 March 2025 / Published: 14 March 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Abstract

:
Soil lead (Pb) contamination is a severe environmental issue. Hydrangea, with high ornamental value, shows strong tolerance to the heavy metal Pb. Discovery of the gene(s) determining Pb resistance has been hindered by the lack of a stabilized and efficient genetic transformation system. Agrobacterium-mediated transient transformation overcomes the drawbacks of stabilized genetic transformation, such as long cycle, low efficiency, and high cost. In this study, an Agrobacterium-mediated method was adopted. The percentage of leaves that turned blue after GUS histochemical staining among the total number of infected leaves was used to represent the transient transformation efficiency. The effects of receptor material growth state (leaf age), Agrobacterium concentration, infection time, acetosyringone (Ace) concentration, negative pressure intensity, and co-culture time on the transient transformation efficiency of Hydrangea villosa Rehd. were investigated. Consequently, an efficient transient transformation system harboring the GUS reporter gene with a transient transformation efficiency as high as 100% was successfully established. Using this system, we successfully verified the Pb tolerance of HmPAT1, HmPIF1, and HmZAT7, proving the effectiveness of the transient transformation system. This transient transformation technology will help to discover new Pb-tolerant genes, provide new molecular targets for the development of Pb-resistant Hydrangea, and provide a potential phytoremediation strategy for the treatment of heavy metal pollution in soil.

1. Introduction

Soil heavy metal pollution poses a global challenge, urgently demanding innovative and sustainable solutions. Despite remarkable progress in key heavy metal pollution control technologies, practical applications are still hampered by various issues. Physical remediation methods, characterized by high intensity engineering and high costs, are only suitable for small scale remediation. Chemical remediation methods, on the other hand, tend to cause secondary pollution and are limited to in situ treatment [1]. Phytoremediation, which utilizes green plants to remove or immobilize soil heavy metals, represents an eco-friendly, safe, and efficient approach for soil heavy metal pollution management [2]. This method not only directly mitigates heavy metal pollution but also confers additional advantages, including biodiversity conservation, ecosystem balance restoration and maintenance, and ecological environment improvement [3]. Among the many heavy metal-resistant plants, garden ornamental plants have extremely high ornamental value and can largely avoid heavy metals from entering the food web. They play an irreplaceable role in improving the quality of the ecological environment. Therefore, they have an extremely high practical value in the remediation of heavy metal pollution in soil [4].
Hydrangea is a landscape ornamental shrub in the family Saxifragaceae. Its blossoms have considerable esthetic value and are frequently grown in gardens because of their many types, rich floral colors, extended flowering period, and wide distribution. Additionally, studies have shown that Hydrangea can resist and accumulate various heavy metals, such as lead (Pb), zinc (Zn), and cadmium (Cd), owing to its rapid growth, large biomass, and well-developed root structure. This makes Hydrangea an excellent material for the phytoremediation of heavy metals [5,6]. Studies on heavy metal resistance in Hydrangea have progressed from physiological mechanisms and tolerance evaluations [7] to transcriptomics, proteomics, metabolomics, and combined multiomics [8,9,10]. In the early stage of the research group, when comparing the transcriptome of the low Pb-tolerant variety Hydrangea macrophylla ‘Monalisa’ and the high Pb-tolerant variety H. macrophylla ‘Jiacheng’ following Pb stress, three resistance genes—HmPAT1, HmPIF1, and HmZAT7—were found to be overexpressed in ‘Jiacheng’ [10]. The findings implicated these three genes in Pb tolerance. In a previous study, all three genes demonstrated abiotic stress tolerance in several species. PAT1 which encodes a scarecrow-like protein, a member of the GRAS family, contributes to plant stress tolerance through phytochrome and hormone signaling, as well as phloem induction [11]. In Vitis amurensis Rupr., overexpression of VaPAT1 can enhance cold resistance in grapes by promoting jasmonic acid biosynthesis [12], and the PAT1 subfamily protein CaGRAS1 positively regulates drought resistance in Capsicum annuum by regulating abscisic acid (ABA) signaling [13]. Phytochrome interacting factor 1 (PIF1) encodes a light-inducible nuclear basic helix–loop–helix (bHLH) protein, which is one of the members of the bHLH protein family. This protein enhances plant resistance by promoting the expression of genes linked to protective proteins, balancing penetration, activating the reactive oxygen species (ROS) removal system, and participating in the transmission of ABA signals. A variety of plants, including Zea mays L. [14], Myrothamnus flabellifolia Welw. [15], Ipomoea batatas (L.) Lam. [16] have demonstrated the tolerance of this gene to salt and drought. Furthermore, the zinc finger protein ZAT7 has been confirmed by taking part in the hydrogen peroxide (H2O2) signaling pathway in response to plant oxidative stress events. For instance, AtZAT7 enhances the cold and salt resistance of Arabidopsis thaliana (L.) Heynh. [17,18], and transcription group analysis of Triticum aestivum L. varieties indicated that ZAT7 may confer aluminum-resistant characteristics by improving the ROS tolerance of plants [19]. However, research on the Pb tolerance functions of these three genes in Hydrangea macrophylla (Thunb.) Ser. has not been reported yet. Therefore, studying the roles of genes linked to heavy metal resistance in Hydrangea is extremely important.
Agrobacterium tumefaciens is a common tool for stabilized transformation. In recent years, research on Agrobacterium-mediated plant transient transformation systems has attracted growing attention [20]. Compared with stabilized genetic transformation, plant transient transformation does not rely on chromosomal integration of heterologous DNA [21]. It is simpler, more efficient, faster, features high level expression, and it is gene-type independent and safe [22]. This technology has been widely used. Yin et al. established a transient transformation system for Lotus japonicus L. for plant gene function studies [22]. Li et al. developed one for Artemisia annua L. for promoter-activity and transcriptional-activation assays [23]. Zhang et al. set up a system for Spinacia oleracea L. for subcellular localization studies [24]. Xian et al. established a system for Carthamus tinctorius L. to study flavonoid biosynthesis [25]. Now, it is applied not only to herbaceous plants but also to woody ones. For example, Guan et al. established a system for Paeonia lactiflora Pall. and verified the resistance of PlGPAT, PlDHN2, and PlHD-Zip to low/high temperature and drought [26]. Wen et al. developed a system for Malus sieversii (Ledeb.) M. Roem. to screen for disease-resistant genes against Valsa mali Miyabe et Yamada [27]. Han et al. conducted subcellular localization, transient overexpression, and protein interaction experiments on Ginkgo biloba L. genes through its transient transformation system [28]. Transient transformation is a powerful tool for studying gene functions in plants without stabilized genetic transformation systems.
Agrobacterium-mediated stabilized genetic transformation systems were developed for the leaf disks of H. macrophylla ‘hyd1’ [29] and H. macrophylla ‘Blaumeise’ [30], respectively. However, both studies had some issues, such as a low positive rate of transgenic plants, technical complexity, and long duration. Additionally, no study has reported the functional identification of Hydrangea genes. Most previous and ongoing national and international research on the functions of heavy metal resistance genes in Hydrangea involve heterologous expression of model plants, typically tobacco, A. thaliana, and yeast [31,32]. The absence of an effective transient transformation system has hindered the discovery of a molecular regulatory mechanism for Pb tolerance in Hydrangea. Therefore, the roles of heavy metal resistance genes in Hydrangea requires the establishment of an effective Agrobacterium-mediated transient transformation method.
Agrobacterium-mediated transient transformation transfers exogenous genes into plant cells, involving basic steps like explant preparation, Agrobacterium preparation, explant infection by Agrobacterium, and co-culturing [33]. System efficiency is affected by factors such as receptor material growth state, Agrobacterium concentration, infection time, acetosyringone (Ace) concentration, negative pressure intensity, and co-culture time [34]. The growth state of plant materials mainly affects the tightness of the arrangement of leaf cell tissues. Smaller intercellular spaces can lead to a decrease in the transient transformation efficiency [22]. The concentration of Agrobacterium and the infection time affect the viability of Agrobacterium on one hand and determine the number of Agrobacterium entering the cells on the other hand [26]. Ace is a commonly used active phenolic inducer in transient transformation. An appropriate amount of Ace can induce the expression of the Vir genes carried by the Ti plasmid, enhancing the infection and transformation ability of Agrobacterium [35,36]. Negative pressure creates tiny wounds on the plant body, causing the plant to produce phenolic compounds, thereby increasing the transient transformation efficiency [22]. An appropriate co-culture time is beneficial for the transfer of T-DNA to the tissue, enabling the transient overexpression of genes [37].
To address the lag in functional studies of Hydrangea genes due to the lack of an efficient transformation system, in this study, we selected H. villosa as the transient transformation plant material which our research group has established a perfect tissue culture system (the tissue culture method is the same as Hydrangea longipes Franch. [38]). Using the Agrobacterium-mediated method, we aimed to establish an efficient transient transformation system with the β-glucuronidase (GUS) reporter gene. By controlling a single variable, we screened for the growth state of the plants, Agrobacterium concentration, infection time, Ace concentration, negative pressure intensity, and co-culture time, thus establishing and optimizing an efficient transient transformation system for H. villosa. With this system, we studied the functions of three lead-tolerance genes (HmPAT1, HmPIF1, and HmZAT7) in homologous plants by evaluating the relative expression level changes in these genes in transiently transformed leaves and measuring their biochemical parameters. The transient transformation system we established provides a simple and efficient method for the functional study of heavy metal resistance genes in Hydrangea and offers a reference for establishing and optimizing transient transformation systems in other non-model plants.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

In this study, tissue culture seedlings of H. villosa cultivated in the tissue culture room of Northwest A&F University were used as plant materials. The tissue-cultured seedlings were germinated from seeds according to the method previously established by our research group [38]. The stem segments of the tissue culture seedlings were subcultured in MS medium (MS + 30 g·L−1 sucrose + 7 g·L−1 agar + 0.8 mg·L−1 6-BA + 0.2 mg·L−1 IBA, pH = 5.8), and cultured in the tissue culture room with a light intensity of 70 μmol m−2 s−1 at 25 °C, 16/8 h, light/night cycle. Subsequent experiments involved cultivating the seedlings for 2 months, 4 months, and 6 months, respectively.

2.2. Transformation and Growth Conditions of Agrobacterium

The plant overexpression vector used in this study is pBI12 (Figure 1), which contains the β-glucuronidase (GUS) gene. The expression of the GUS gene is controlled by the Cauliflower mosaic virus (CaMV) 35S promoter and the NOS terminator. Moreover, this plasmid is resistant to kanamycin (Kana). The pBI121 plasmid has been used to establish the transient expression in plants such as Pyrus betulifolia Bunge. [39] and Iris foetidissima L. [40].
The plasmids were transferred to Agrobacterium GV3101 (Sangon Biotech, Shanghai, China) using the freeze–thaw method. A total of 200 μL of competent cells were spread onto the YEP solid medium containing 50 mg·L−1 Kan and 10 mg·L−1 Rif, and cultivated 2~3 d in a 28 °C in a constant temperature incubator. A single bacterial colony was then picked and transferred to 25 mL of the YEP liquid medium containing 50 mg·L−1 Kan and 10 mg·L−1 Rif, followed by shaking culture at 28 °C and 200 rpm for 48 h until the OD600 reached 0.4~0.6.

2.3. Establishing and Optimizing the Transient Transformation System

The optimal transient transformation conditions were screened sequentially in the order of leaf age, resuspended Agrobacterium concentration, Agrobacterium infection time, Ace concentration in the resuspension, number of negative pressure treatments, and co-culture time. The optimal conditions obtained from each step were applied to the screening process of the next influencing factor. The initial infection conditions were as follows: the bacterial solution concentration was OD600 = 0.8, the infection time was 40 min, the Ace concentration in the resuspension was 300 μM, the number of negative pressure treatments was 0, and the co-culture time was 2 days.
The Agrobacterium solution was successfully converted to the pBI121-GUS empty carrier and shaking culture to OD600 was 0.4~0.6, then centrifuged at 5000 rpm for 20 min. The resuspension (MS + 1 mM MES + 2 mM MgCl2 + 100, 200, 300 μM Ace + 30 g·L−1 sucrose, pH = 5.8) was added to the collected cells, and the cells were resuspended at OD600 of 0.4, 0.8, 1.2, 28 °C, 100 rpm for 2 h. The 2-month, 4-month, and 6-month-old leaves of H. villosa were completely immersed in the resuspended Agrobacterium solution and infected for 20 min, 40 min, and 60 min, respectively. Then, the leaves and bacterial solution were transferred to a50 mL sterile syringe, sealed at the injection port, and subjected to negative pressure treatment by drawing the syringe 0, 5, and 10 times, each for 5 cm. After that, the leaves were rinsed three times with sterile water and the bacterial solution on the leaves surface was dried with filter paper, and the leaves were flatted up on the culture medium (MS + 30 g·L−1 sucrose + 7 g·L−1 agar + 0.8 mg·L−1 6-BA + 0.2 mg·L−1 IBA + 10 μM Ace, pH = 5.8), and co-cultured for 1, 2, and 3 days at 25 °C in the dark. Each treatment was repeated three times, with 15 leaves in each group. In this experiment, a single factor was used as a single variable, and other factors were used as the standard. By observing and counting the percentage of leaves that turn blue after GUS staining, the transient transformation system was established and optimized. The trial flow is summarized in Figure 2.

2.4. β-Glucuronidase Tissue Chemical Stain Detection of Transient Transformation Gene Expression

Given that the product of the GUS gene, β-glucuronidase is capable of catalyzing the decomposition of 5-bromo-4-chloro-3-indolyl-β-D-glucuronide(X-Gluc) into a blue-colored product, a GUS staining kit (Coolaber, Beijing, China) was employed to conduct staining on the co-cultured leaves of H. villosa. For each treatment, a total of 15 leaves were utilized. The leaves were immersed in the GUS staining solution, which contained X-Gluc, and then incubated at a constant temperature of 28 °C in the dark for a duration of 24 h. Subsequently, the leaves underwent a decolorization process. Initially, they were treated with 75% ethanol, followed by a transfer to 95% ethanol. The 95% ethanol was refreshed 5 times daily until the chlorophyll in the leaves was completely removed. The appearance of a blue color in the leaves indicated the successful expression of the GUS gene, signifying the success of transient transformation. The transformation efficiency was calculated using the following formula: Transformation efficiency (%) = (number of blue-stained leaves/total number of leaves) × 100%. Each experimental group was set up with three biological replicates.

2.5. qRT-PCR Analysis of Pb-Tolerant Gene Screen

Pb(NO3)2 was added to ultrapure water to prepare aqueous solutions with Pb2+ concentrations of 100, 500, and 1000 mg·L−1, respectively. Each solution was then transferred into a beaker and diluted to a final volume of 1 L. The ‘Jiacheng’ plants were transplanted into the lead solutions with corresponding concentrations, while those treated with ultrapure water served as the control group. Each ‘Jiacheng’ plant was fixed in a beaker using foam, with one plant per beaker. One-third of the roots of the plants were kept above the water surface to ensure normal root respiration. Ultrapure water was added at regular intervals every day to maintain the volume at the 1 L mark. The experiment lasted for 48 h, and the light and temperature settings during the experiment were the same as those described in Section 2.1. Three biological replicates were set up for each experimental group.
Total RNA was extracted from the leaves of ‘Jiacheng’ under different concentrations of Pb(NO3)2 solution using RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (TianGen, Beijing, China), and the RNA was reverse-transcribed into cDNA by 1st Strand cDNA Synthesis Kit (OneStep gDNA Removal) (Aidlab, Beijing, China). Taking the cDNA as the template, with the HmPAT1-q-F/R, HmPIF1-q-F/R, HmZAT7-q-F/R as the primers, the qRT-PCR analysis was performed. The circulation parameters were as follows: 94 °C for 3 min, then 40 cycles: 94 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s; after the end of the cycle 95 °C for 15 s, 60 °C for 1 min, 1 °C, 95 °C for 15 s. The samples were normalized with 18s gene as internal reference, and all primers used in qRT-PCR are shown in Table S1. Calculating relative expression of gene by 2−ΔΔCT method [41]. Three biological replicates were set up for each treatment.

2.6. Plasmid Construction and Transient Transformation of Plant Acquisition

The pBI121-GUS empty plasmid was double digested with Xbal and Smal (Takara, Kyoto, Japan), and the CDS region (Remove the stop codon) of the Pb-resistance gene of H. macrophylla ‘Jiacheng’ was cloned using PrimeSTAR® Max DNA Polymerase (Takara, Japan) and the target gene primers with homologous arms. The vector and the target gene fragment were recovered using the PCR product purification and recovery kit (HLINGENE, Shanghai, China). NovoRec® plus One step PCR Cloning Kit (Novoprotein, Suzhou, China) was used to transform the ligation product into DH5α (Sangon Biotech, Shanghai, China) by homologous recombination to construct the recombinant plasmid pBI121-HmPAT1-GUS/pBI121-HmPIF1-GUS/pBI121-HmZAT7-GUS. In order to ensure that there was no base mutation in the gene during the ligation process, the E. coli bacterial solution of the positive clone was sent to Aoke (Xi’an, China) Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the pBI121-HmPAT1-GUS/pBI121-HmPIF1-GUS/pBI121-HmZAT7-GUS overexpression vector was successfully constructed. All primers used in PCR are shown in Table S2.
The overexpression plasmids pBI121-HmPAT1-GUS/pBI121-HmPIF1-GUS/pBI121-HmZAT7-GUS constructed in the previous step and the empty vector pBI121-GUS were transformed into Agrobacterium by the freeze–thaw method. After PCR verification of the bacteria, the Agrobacterium colonies with correct bands were collected. According to the established transient transformation system, the collected Agrobacterium was resuspended with a resuspension solution containing 300 μM Ace to an OD600 of 0.8 and cultured at 100 rpm and 28 °C for 2 h. Then, in a super-clean bench, the leaves of 2-month-old H. villosa tissue-cultured seedlings were immersed and infected for 20 min. The bacterial liquid and leaves were transferred together into a 50 mL syringe, and negative pressure was applied 5 times with the needle sealed (each time the syringe was pushed 5 cm). The leaves were washed 5 times with sterile water, and the liquid on the leaf surface was blotted dry with sterile filter paper. The leaves were placed on the medium with the abaxial side facing up and co-cultured in the dark for 3 days. The leaves with the pBI121-GUS empty vector were used as the control group. In each treatment group, 30 leaves were randomly selected for GUS staining, and the success of transient transformation was judged according to the staining results. The remaining transient blades were used for subsequent experiments.

2.7. Pb Stess Transient Transgenic H. villosa Leaves

After co-culture, 5 mL of Pb(NO3)2 aqueous solution of 0 and 1 M was added to the medium, respectively, and the leaves were stressed at 24 °C for 4h under normal light (at least 10 leaves per sample when sampled). The tissue was then frozen in liquid nitrogen and stored at −80 °C.

2.8. Transient Expression Analysis of Three Pb-Resistant Genes

Total RNA was extracted from the leaves transiently transformed after Pb stress and reverse-transcribed into cDNA. The cDNA was used as a template for qRT-PCR analysis using HmPAT1-q-F/R, HmPIF1-q-F/R, HmZAT7-q-F/R as primers. The method was the same as above.

2.9. Measurement of Biochemical Parameters

The transgenic leaves after Pb stress were collected, and the biochemical parameters were measured. The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were determined according to the method of reference in [42]. The content of ascorbic acid (AsA) was determined by referring to the method from [43]. The content of malonaldehyde (MDA) was assessed following the thiobarbituric acid (TBA) method [5]. The acidic ninhydrin colorimetric method was used to assay the proline (Pro) content [44]. Anthrone colorimetry was employed to measure soluble sugar (SS) content [45]. All experiments were conducted with three biological replicates, each containing at least 10 transient transgenic leaves.

2.10. Statistical Analysis

Data were processed using Microsoft Excel 2016 and statistically analyzed with GraphPad Prism 9.5 for plotting. All experiments had three biological replicates. One-way analysis of variance (ANOVA) and Student’s t-test were used to assess the statistical significance between different treatments. Significance levels were marked with asterisks (* p < 0.05; ** p < 0.01). Data are shown as mean ± SE.

3. Results

3.1. Effects of Leaf Age on Transient Transformation Efficiency of H. villosa

We selected leaves of H. villosa subcultured tissue-cultured seedlings with leaf ages of 2, 4, and 6 months and used Agrobacterium containing the pBI121-GUS carrier for transient transformation. After GUS staining, the leaves of 2- and 4-month-old seedlings turned blue, and the leaf age of 2-month-old had the highest transformation efficiency of 64.44% (Figure 3A). However, the transformation efficiency of 6-month-old leaves was 0%, and the leaves were yellow and wilted, indicating that the GUS expression efficiency decreased with the increase in leaf age (Figure 4A). Therefore, 2-month-old leaves of H. villosa were used in subsequent experiments.

3.2. Effects of Agrobacterium Concentration on Transient Transformation Efficiency of H. villosa

To study the effect of concentration of Agrobacterium for infection on the transient transformation efficiency of H. villosa leaves, three concentrations were used in this experiment. The optical density at 600 nm (OD600) were 0.4, 0.8, and 1.2, respectively (Figure 3B). When the OD600 increased from 0.4 to 0.8, the transformation efficiency increased significantly to 64.44%, which was 1.64 times higher than the OD600 of 0.4. However, when the OD600 was 1.2, the activity of Agrobacterium was reduced, causing significant damage to plants. The leaves were yellowing and wilted (Figure 4B), and the transformation efficiency was significantly reduced to 0%. These results showed that the optimal concentration of Agrobacterium to infection for the transient transformation of H. villosa was 0.8.

3.3. Effects of Infection Time on Transient Transformation Efficiency of H. villosa

The infection time of Agrobacterium is also an important factor affecting the transient transformation efficiency of plants. In this experiment, the transient transformation efficiency at the three infection times of 20, 40, and 60 min were studied. The results are shown in Figure 3C. The highest transient transformation efficiency was 84.44% when the infection time was 20 min, which was significantly higher than the others. The increase in infection time gradually decreased the transient transformation efficiency. The transformation efficiency of leaves infected for 40 min decreased by 20% when compared with those at 20 min. The lowest transformation efficiency was only 48.89% after 60 min of infection, and the leaves showed wilting. After co-culture, GUS staining, decolorization, and leaf browning were observed (Figure 4C). We concluded that the optimal Agrobacterium infection time for the transient transformation of H. villosa was 20 min.

3.4. Effects of Ace Concentration on Transient Transformation Efficiency of H. villosa

Addition of an appropriate amount of Ace to the resuspension could increase the transient transformation efficiency of H. villosa. This test had four concentrations of Ace, 100 μM, 200 μM, 300 μM, and 400 μM, respectively, and the transient transformation efficiency gradually increased with the increasing Ace concentration (Figure 3D). When the concentration in the resuspension was 100 μM, the transformation efficiency was the lowest at 62.22%. At the Ace concentration of 300 μM, the transformation efficiency was the highest at 84.44%, and the leaves appeared to be in good condition. The transient transformation efficiency decreased to 64.44% at the 400 μM Ace concentration, and the high concentration resulted in damaged leaves and reduced transformation efficiency (Figure 4D). Therefore, the optimal Ace concentration of the resuspension in the transient transformation system was 300 μM.

3.5. Effects of Negative Pressure on Transient Transformation Efficiency of H. villosa

Three different pressure intensities were set up to further optimize the transient transformation system (Figure 3E). The transient transformation efficiency was 84.44% in the absence of negative pressure. After negative pressure was applied, the transient transformation efficiency increased significantly. When the negative pressure intensity was applied 5 and 10 times, the transformation efficiency reached 95.56%. Although the GUS staining results showed that some leaves were bluer under 10 times negative pressure compared to 5 times, the higher negative pressure caused mechanical damage to the plants (Figure 4E). At the same transient transformation efficiency, leaves subjected to the negative pressure 5 times were found to be more suitable than those subjected to 10 times negative pressure. Thus, the optimal negative pressure intensity for the H. villosa transient transformation system was 5 times.

3.6. Effects of Co-Culture Time on Transient Transformation Efficiency of H. villosa

This experiment set up three gradients of co-culture time: 1 d, 2 d, and 3 d. As the co-culture time increased from 1 to 2 to 3 d (Figure 3F), the transformation efficiency increased significantly. When co-cultured for 1 d, the transient transformation efficiency was as low as 60%, and the GUS staining was shallow (Figure 4F). After 3 d of co-culture, the transient transformation efficiency reached 100%, which was 1.67 times that after 1 d. Therefore, 3 d was the optimal co-culture time for the transient transformation system of H. villosa.

3.7. Identification of Pb Stress Resistance Genes in H. macrophylla

RNA-seq data analysis of high- and low-Pb-tolerant H. macrophylla under Pb stress in previous studies revealed that some transcripts were highly expressed under Pb stress [10]. We further verified the expression patterns of HmPAT1, HmPIF1, and HmZAT7 under different concentrations of Pb stress through quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The results showed that the relative expression levels of the three genes increased with increasing Pb concentration. These findings preliminarily demonstrate that the three selected genes have the function of responding to Pb stress (Figure 5).

3.8. Transient Overexpression of Three Pb-Resistant Genes in H. villosa

The construction of the pBI121-HmPAT1-GUS/pBI121-HmPIF1-GUS/pBI121-HmZAT7-GUS vector is shown as Figure 6A–C. The constructed pBI121-HmPAT1-GUS/pBI121-HmPIF1-GUS/pBI121-HmZAT7-GUS was transformed into the Agrobacterium-competent GV3101 using the freeze–thaw method. Single colonies were picked and cultured at 28 °C until the OD600 was 0.4~0.6, and the bacterial solution was identified by PCR. Products of approximately 1170 bp, 1425 bp, and 441 bp were amplified from the Agrobacterium suspensions of the three different recombinant plasmids, respectively (Figure 6D). The products were all of the expected size, indicating that the plant overexpression recombinant plasmids pBI121-HmPAT1-GUS/pBI121-HmPIF1-GUS/pBI121-HmZAT7-GUS and Agrobacterium transformations were successful.
The successfully constructed pBI121-HmPAT1-GUS/pBI121-HmPIF1-GUS/pBI121-HmZAT7-GUS overexpression plasmids were used to transform leaves of H. villosa using the Agrobacterium-mediated transient transformation method. pBI121-GUS empty vector-transformed leaves were used as the blank control group. The transient leaves were stained with the GUS staining solution (Figure 6E), and the results showed that all the transgenic leaves turned blue, indicating that the transient transformation system of H. villosa successfully transformed the three Pb resistance genes into leaves.

3.9. qRT-PCR Analysis of Three Pb-Resistant Genes Transiently Overexpressed in H. villosa Under Pb Stress

To verify the feasibility of the established transient transformation system, we obtained the leaves of H. villosa, transiently transformed with three Pb-resistant genes, and then detected their expression under Pb stress through qRT-PCR (Figure 7). Under normal growth conditions, the expression of genes in the leaves that were transiently transformed with Pb resistance genes was significantly higher than that in the control group, indicating that the genes were overexpressed in H. villosa. After 4 h of treatment with 1M Pb(NO3)2 solution, the relative expression of genes in the leaves increased. The relative expression levels of Pb-resistant genes in the leaves of the transiently transformed Pb-resistant genes group were all significantly higher than those in the pBI121-GUS group. Therefore, HmPAT1, HmPIF1, and HmZAT7 were successfully transiently overexpressed in H. villosa leaves and manifested resistance to Pb stress by increasing their relative expression levels.

3.10. Analysis of Biochemical Parameters in H. villosa Transiently Overexpressing Three Pb-Resistant Genes Under Pb Stress

To further verify the function of the HmPAT1, HmPIF1, and HmZAT7 Pb-resistance genes, the activities of three main antioxidant enzymes—superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)—and the contents of ascorbic acid (ASA), malondialdehyde (MDA), proline (Pro), and soluble sugar SS in the leaves of H. villosa with transient overexpression of these three genes under Pb stress were determined. pBI121-GUS was taken as the control group. Under Pb-free conditions, there were no significant differences in the activities of SOD, POD, and CAT between the pBI121-GUS group and the Pb-tolerant gene transformed plants. After 4 h of Pb stress treatment, except for the SOD activities in the pBI121-GUS group, which decreased with Pb treatment, the activities of SOD, POD, and CAT in the remaining leaves increased. The activities of these enzymes in the leaves transiently transformed with the three Pb-tolerant genes were significantly higher than those in the control leaves (pBI121-GUS) (Figure 8A–C). According to the determination results of antioxidant substances (Figure 8D), there was no significant difference in the AsA content in the leaves during normal growth. After Pb stress, the AsA content in the leaves overexpressing the three Pb-tolerant genes increased and was significantly higher than that in the pBI121-GUS group. The analysis of oxidative damage indicators showed that under normal growth conditions there was no significant difference in the MDA content in each group of leaves. After Pb stress, the MDA content in the leaves increased, and the MDA content in the leaves overexpressing the Pb-tolerant genes was significantly lower than that in the pBI121-GUS group (Figure 8E). The analysis of the contents of organic osmotic adjustment substances showed that there was no significant difference in the Pro and SS contents in the leaves without Pb stress (Figure 8F,G). After Pb stress treatment, the Pro and SS contents in the four groups of leaves increased, and the Pro and SS contents in the leaves transiently expressing the three Pb-tolerant genes were significantly higher than those in the pBI121-GUS group.
The above results proved that the transient transformation system was successfully used to transfer the Pb-tolerant genes of ‘Jiacheng’ into leaves of H. villosa and exert their function of resisting heavy metal Pb stress, indicating that the transient transformation system of H. villosa established in this study can be used to study the heavy metal tolerance functions of Hydrangea genes.

4. Discussion

Stabilized and transient genetic transformation are commonly used methods to express exogenous genes in plants. Compared with stabilized genetic transformation, transient genetic transformation is a molecular technology that can introduce target genes into target cells and express them rapidly in a short time. The approach does not involve plant regeneration, does not affect the host genome, and can simultaneously test the function of multiple proteins. Therefore, it exhibits the characteristics of a short cycle and high transformation efficiency [46].
Agrobacterium-mediated transient transformation systems are widely used for the functional analysis of exogenous genes in plants. Their transformation efficiency of these systems is affected by multiple parameters [47]. Agrobacterium-mediated transient transformation of plants is inversely proportional to the age of plant tissues [48]. Several studies have found that with the growth and development of plants, the arrangement of leaf cells becomes closer, the intercellular and compartmentalized spaces become smaller, and the thickness of leaves increases. These changes hinder the penetration and diffusion of Agrobacterium solution into the leaves, thus limiting the transformation potential of tissues [22,49]. In this study, the leaves of tissue-cultured seedlings of H. villosa, after two months of subculture, were selected for transient transformation. At this time, the leaves were young and the leaf area was moderate, which was beneficial to the infection of Agrobacterium, and the transient transformation efficiency was the highest.
The concentration and infection time of Agrobacterium were shown to be crucial in its conversion [50]. Screening the bacterial concentration and infection time showed that when the leaves were infiltrated with Agrobacterium solution (OD600 0.8 for 20 min), the number of positive transient leaves with blue GUS staining was highest. At this point, the leaves grew well and could be used in subsequent experiments. A low concentration of Agrobacterium or too short an infection time could not guarantee that the leaves are completely infected, thereby reducing the transient transformation efficiency. In contrast, too high a concentration of Agrobacterium or too long an infection time could also lead to decreased transformation efficiency and even to browning and wilting of the leaves. This is because high concentrations of Agrobacterium may inhibit the respiration of receptor cells, cause tissue damage or cell death, and even result in the death of bacterial cells owing to competition [51,52]. The optimal Agrobacterium concentration and infection time for the transient expression systems in P. lactiflora [26], Pinus tabuliformis Carrière. [53], and Dendrobium catenatum Lindl. [54] showed similar patterns.
Several studies have reported that phenolic inducers enhance the ability of Agrobacterium to infect and transform host plants [33]. Phenolic compounds directly affect the activation of VirA, thereby inducing the expression of Vir carried by the Ti plasmid. Appropriate accumulation can improve Agrobacterium infection and transformation [35,36]. Ace is an active phenolic compound commonly used in transient transformations. The optimal Ace concentrations for different plant transient transformation systems differed. The highest transformation efficiency was obtained when the Ace concentration in the transient transformation solution of M. sieversii was 250 μM [27], and the optimal Ace concentration in the leaves of Camellia Sinensis (L.) Kuntze. was 150 μM [55]. The transient transformation rate of H. villosa was the highest when 300 μM Ace was added to Agrobacterium resuspensions. However, when the concentration exceeded this level, the transformation efficiency decreased, which may be due to the toxicity of Ace to explants, which is consistent with a study on other plants, such as C. tinctorius [25].
The injured parts of dicotyledonous plants secrete phenolic compounds, such as Ace, which are natural signaling molecules produced by damaged plant cells [56]. Negative pressure and other methods are commonly used to create small wounds on plant tissue surfaces. The release of phenolic substances helps Agrobacterium infect and transform the explants. However, excessive intensity leads to excessive damage in plant tissues, resulting in tissue necrosis and bacterial overgrowth, which affects gene expression. With the increase in vacuum infiltration pressure and time, cell viability, survival rate, and transient transformation efficiency of L. japonicus decreased [22]. Similarly, the optimal negative pressure intensity of P. lactiflora was found to be 10 times, and the transient transformation efficiency decreased under insufficient negative pressure or excessive negative pressure, which led to browning and death of plants [26]. In this study, the leaves of H. villosa were subjected to negative pressure treatment. Negative pressure increased the transient transformation efficiency, and the transformation efficiency at a negative pressure of five times was the same as the efficiency at 10 times. However, the fundamental principle of negative pressure was to damage the plants. Therefore, to reduce the threat of mechanical damage to plants, a negative pressure of five times was selected as the optimal negative pressure intensity.
Co-culture is a key step in the transient transformation method in which T-DNA is transferred and gene expression is initiated [37]. An adequate co-culture time facilitates the transfer of T-DNA to the injured tissues. Nevertheless, the absence of antibiotics in the medium results in excessive Agrobacterium development and damage to plant tissue. Previous studies have shown that the optimal co-culture time for different plants varies. The transient expression of exogenous genes in Cinnamomum camphora (L.) Presl. was reportedly strongest at 48 h and disappeared after 72 h [37]. The expression level of the GUS gene was highest after 7 d of infection in Camptotheca acuminata Decne. leaves [57], and the optimal co-culture time for transient transformation of P. betulifolia leaves was 4 d [39]. In this study, the transient transformation efficiency reached 100% after 3 d of co-culture, marking the establishment of Agrobacterium-mediated transient transformation system of H. villosa.
Woody plants are characterized by high heterozygosity, long breeding times, and low transformation efficiencies. It is not possible to quickly obtain a large number of transgenic plants, and there are still many woody plants whose regeneration process is difficult and genetic improvement is limited [58]. An instant transformation technology is particularly important for Hydrangea, which lacks efficient and stabilized genetic transformation systems, thus being conducive to related research on promoting the genetic function of Hydrangea. Transient transformation systems have been used to study anti-heavy metal genes. Using the transient transformation system of Tamarix hispida Willd., it was found that ThDRE1A plays an important role in Cd accumulation through the ABA and ETH antagonism [59]. The instantaneous expression of OsTIP1;2 in Nicotiana benthamiana increased the activity of enzyme and non-enzyme antioxidants, indicating that this gene has a potential role in reducing As-induced oxidative stress [60]. ZmMTP11 [61] and ZmPIP2;5 [62] were identified in a yeast heterologous transient transformation system with Pb tolerance and promoted Pb accumulation. Except for a few plants that have a transient transformation system of origin, research on heavy metal resistance genes in other plants has mostly used model plants and yeast cells for heterologous expression.
In this study, three stress-resistant genes (HmPAT1, HmPIF1, and HmZAT7), which have been identified under abiotic stress in other species, were screened by analyzing differentially expressed genes in the transcriptomes of high- and low-Pb-tolerant H. macrophylla varieties. (The original RNA sequencing data are stored in the NCBI SRA database, project number PRJNA1021983). Subsequently, the qRT-PCR technique was used to measure changes in the relative expression levels of HmPAT1, HmPIF1, and HmZAT7 genes in ‘Jiacheng’ under different concentrations of Pb stress. The results showed that the relative expression levels of these three genes in ‘Jiacheng’ increased, indicating that all of them responded to Pb stress. When there was no lead stress, the relative expression levels of the corresponding genes in the leaves of H. villosa with the target genes transferred were significantly higher than those in the pBI121-GUS group, indicating the successful transient transformation. The qRT-PCR results after Pb stress treatment indicated that the relative expression levels of Pb-tolerant genes in transgenic plants were higher than those in the non-Pb-treated group. Among them, the homologous genes PAT1, PIF1, and ZAT7 in the leaves of H. villosa in the pBI121-GUS group also responded to Pb stress. After comparing the nucleotide sequences of the homologous genes between H. villosa and ‘Jiacheng’, a high degree of homology was found (Figures S1–S3). However, after Pb stress, the expression levels of the corresponding Pb-tolerant genes in the leaves of H. villosa with overexpressed HmPAT1, HmPIF1, and HmZAT7 were significantly higher than those in the pBI121-GUS group. This suggests that the HmPAT1, HmPIF1, and HmZAT7 genes respond to lead stress. Further detection of the stress-resistant biochemical indices revealed that after lead stress treatment, the content of malondialdehyde (MDA) in the leaves with the overexpression of HmPAT1, HmPIF1, and HmZAT7 genes was significantly lower than that in the plants with the empty vector control. This indicates that the overexpression of these three genes can alleviate the membrane lipid peroxidation caused by lead stress in the leaves of H. villosa. Meanwhile, compared with the pBI121-GUS group, the leaves with the transient overexpression of HmPAT1, HmPIF1, and HmZAT7 genes under lead stress exhibited higher activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and a higher content of ascorbic acid (AsA). This suggests that HmPAT1, HmPIF1, and HmZAT7 can enhance the activities of antioxidant enzymes and the contents of antioxidant substances to scavenge the accumulation of reactive oxygen species (ROS) caused by lead stress and alleviate the harm of peroxidation. The contents of proline and soluble sugar were also significantly higher than those in the plants with the empty vector control, indicating that these three genes can also maintain the osmotic potential through changes in the contents of osmotic adjustment substances and reduce the cellular damage caused by lead stress. These findings suggest that HmPAT1, HmPIF1, and HmZAT7 can improve the lead tolerance of H. villosa by regulating the activities of antioxidant enzymes, the contents of non-enzymatic antioxidants, and osmotic adjustment substances. All of the above results demonstrated that HmPAT1, HmPIF1, and HmZAT7 were lead-tolerant genes, which played crucial roles in Hydrangea’s response to lead stress. This conclusion was consistent with the gene expression patterns in the previous Hydrangea transcriptome (PRJNA1021983) and the qRT-PCR results showing that HmPAT1, HmPIF1, and HmZAT7 in ‘Jiacheng’ were up-regulated in response to lead stress at different time points under lead stress. These findings strongly validated the lead-tolerance functions of HmPAT1, HmPIF1, and HmZAT7.

5. Conclusions

We developed an effective Agrobacterium-mediated transient transformation system for H. villosa. Agrobacterium tumefaciens was resuspended in a 300 μM Ace-containing solution to an OD600 of 0.8 and then used to infect two-month-old leaves for 20 min. Following five rounds of negative pressure infiltration with a syringe, the samples were co-cultivated in the dark for 3 days. At this point, transient transformation efficiency was at its highest, and leaf condition was optimal. Using this method, three stress-resistant genes (HmPAT1, HmPIF1, and HmZAT7) were successfully overexpressed in H. villosa leaves. Analysis of stress resistance indices in transgenic plants after Pb stress demonstrated that overexpression of these three genes in H. villosa resulted in superior tolerance to Pb, indicating that the transient transformation system established in this study was effective and could be applied to the transient overexpression and functional analysis of Hydrangea. The data of transient transformation systems and heavy metal resistance genes of Hydrangea sheds light on the response mechanism of plants and offers new molecular targets for the development of Pb-resistant plants and a plant repair method for dealing with soil heavy metal pollution.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11030320/s1. Table S1: Primer information for quantitative real-time PCR. Table S2: Primer information for PCR. Figure S1: Alignment of HmPAT1 and its homologous sequences in H. villosa. Figure S2: Alignment of HmPIF1 and its homologous sequences in H. villosa. Figure S3: Alignment of HmZAT7 and its homologous sequences in H. villosa.

Author Contributions

Conceptualization, B.Z. and R.C.; methodology, R.C.; software, R.C.; validation, R.C. and L.S.; resources, B.Z.; writing—original draft preparation, R.C.; writing—review and editing, B.Z.; visualization, R.C. and L.S.; supervision, B.Z.; project administration, B.Z.; funding acquisition, B.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the General Project of the Key Research and Development Plan of Shaanxi Province (2024NC-YBXM-236).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. The HmPAT1/PIF1/ZAT7 CDS sequences were derived from the transcriptome sequencing data. The original RNA sequencing data were stored in the NCBI SRA database, project number PRJNA1021983.

Conflicts of Interest

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Map of plant overexpression vector pBI121. The * indicates that this site is blocked by Dca—methylation.
Figure 1. Map of plant overexpression vector pBI121. The * indicates that this site is blocked by Dca—methylation.
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Figure 2. Flow chart of the establishment of Agrobacterium-mediated transient transformation system of H. villosa.
Figure 2. Flow chart of the establishment of Agrobacterium-mediated transient transformation system of H. villosa.
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Figure 3. Screening of influencing factors of instantaneous transformation system. (A) Leaf age. (B) Agrobacterium concentration. (C) Infection time. (D) Acetosyringgone concentration. (E) Negative pressure. (F) Co-culture time. Error bars indicate standard deviation of three replicates. To optimize the individual parameter, each experiment contained 15 samples, and each experiment had 3 replications. The bar indicates a standard error (SE) of three replicates. Asterisks display the level of significance. * p < 0.05, ** p < 0.01, ns p > 0.05 (ANOVA).
Figure 3. Screening of influencing factors of instantaneous transformation system. (A) Leaf age. (B) Agrobacterium concentration. (C) Infection time. (D) Acetosyringgone concentration. (E) Negative pressure. (F) Co-culture time. Error bars indicate standard deviation of three replicates. To optimize the individual parameter, each experiment contained 15 samples, and each experiment had 3 replications. The bar indicates a standard error (SE) of three replicates. Asterisks display the level of significance. * p < 0.05, ** p < 0.01, ns p > 0.05 (ANOVA).
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Figure 4. GUS staining results of transient transgenic H. villosa leaves. (A) Leaf age. (B) Agrobacterium concentration (OD600). (C) Infection time. (D) Acetosyringgone concentration. (E) Negative pressure. (F) Co-culture time. The experiments were repeated three times with similar results.
Figure 4. GUS staining results of transient transgenic H. villosa leaves. (A) Leaf age. (B) Agrobacterium concentration (OD600). (C) Infection time. (D) Acetosyringgone concentration. (E) Negative pressure. (F) Co-culture time. The experiments were repeated three times with similar results.
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Figure 5. Relative expression analysis of three stress-resistant genes in H. macrophylla ‘Jiacheng’ under Pb stress. (A) HmPAT1 relative expression. (B) HmPIF1 relative expression. (C) HmZAT7 relative expression. The bar indicates a standard error (SE) of three replicates. Asterisks display the level of significance. * p < 0.05, ** p < 0.01, ns p > 0.05 (ANOVA).
Figure 5. Relative expression analysis of three stress-resistant genes in H. macrophylla ‘Jiacheng’ under Pb stress. (A) HmPAT1 relative expression. (B) HmPIF1 relative expression. (C) HmZAT7 relative expression. The bar indicates a standard error (SE) of three replicates. Asterisks display the level of significance. * p < 0.05, ** p < 0.01, ns p > 0.05 (ANOVA).
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Figure 6. Transient overexpression of three Pb-resistant genes in H. villosa. (A) Schematic diagram of the recombinant plant overexpression vector pBI121-HmPAT1-GUS. Note: the red-colored part represents the target gene sequence, which is the same as in (B,C). (B) Schematic diagram of the recombinant plant overexpression vector pBI121-HmPIF1-GUS. (C) Schematic diagram of the recombinant plant overexpression vector pBI121-HmZAT7-GUS. (D) Electropherogram of Agrobacterium PCR product containing recombinant expression. Note: M1–3 are DNA markers 2000 bp; 1–4 are pBI121-HmPAT1-GUS/pBI121-HmPIF1-GUS/pBI121-HmZAT7-GUS/pBI121-GUS Agrobacterium PCR bands. (E) GUS staining of transient transgenic H. villosa leaves.
Figure 6. Transient overexpression of three Pb-resistant genes in H. villosa. (A) Schematic diagram of the recombinant plant overexpression vector pBI121-HmPAT1-GUS. Note: the red-colored part represents the target gene sequence, which is the same as in (B,C). (B) Schematic diagram of the recombinant plant overexpression vector pBI121-HmPIF1-GUS. (C) Schematic diagram of the recombinant plant overexpression vector pBI121-HmZAT7-GUS. (D) Electropherogram of Agrobacterium PCR product containing recombinant expression. Note: M1–3 are DNA markers 2000 bp; 1–4 are pBI121-HmPAT1-GUS/pBI121-HmPIF1-GUS/pBI121-HmZAT7-GUS/pBI121-GUS Agrobacterium PCR bands. (E) GUS staining of transient transgenic H. villosa leaves.
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Figure 7. Transient expression analysis of Hydrangea Pb-tolerant genes under Pb stress. (A) HmPAT1 relative expression. (B) HmPIF1 relative expression. (C) HmZAT7 relative expression. The bar indicates a standard error (SE) of three replicates. To optimize the individual parameter, each experiment contained at least 10 samples, and each experiment had 3 replications. Asterisks display the level of significance. * p < 0.05, ** p < 0.01, ns p > 0.05 (ANOVA).
Figure 7. Transient expression analysis of Hydrangea Pb-tolerant genes under Pb stress. (A) HmPAT1 relative expression. (B) HmPIF1 relative expression. (C) HmZAT7 relative expression. The bar indicates a standard error (SE) of three replicates. To optimize the individual parameter, each experiment contained at least 10 samples, and each experiment had 3 replications. Asterisks display the level of significance. * p < 0.05, ** p < 0.01, ns p > 0.05 (ANOVA).
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Figure 8. Analysis of physiological indices in H. villosa leaves with transient overexpression of stress Pb-resistance genes. (A) SOD activity. (B) POD activity. (C) CAT activity. (D) AsA content. (E) MDA content. (F) Proline content. (G) Soluble sugar content. The bar indicates a standard error (SE) of three replicates. To optimize the individual parameter, each experiment contained at least 10 samples, and each experiment had 3 replications. Asterisks display the level of significance. * p < 0.05, ** p < 0.01, ns p > 0.05 (Student’s t-test, using pBI121 as the control group for each gene).
Figure 8. Analysis of physiological indices in H. villosa leaves with transient overexpression of stress Pb-resistance genes. (A) SOD activity. (B) POD activity. (C) CAT activity. (D) AsA content. (E) MDA content. (F) Proline content. (G) Soluble sugar content. The bar indicates a standard error (SE) of three replicates. To optimize the individual parameter, each experiment contained at least 10 samples, and each experiment had 3 replications. Asterisks display the level of significance. * p < 0.05, ** p < 0.01, ns p > 0.05 (Student’s t-test, using pBI121 as the control group for each gene).
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Cong, R.; Shi, L.; Zhao, B. An Effective Agrobacterium-Mediated Transient Transformation System for Studying the Lead-Tolerance Genes in Hydrangea. Horticulturae 2025, 11, 320. https://doi.org/10.3390/horticulturae11030320

AMA Style

Cong R, Shi L, Zhao B. An Effective Agrobacterium-Mediated Transient Transformation System for Studying the Lead-Tolerance Genes in Hydrangea. Horticulturae. 2025; 11(3):320. https://doi.org/10.3390/horticulturae11030320

Chicago/Turabian Style

Cong, Rong, Liang Shi, and Bing Zhao. 2025. "An Effective Agrobacterium-Mediated Transient Transformation System for Studying the Lead-Tolerance Genes in Hydrangea" Horticulturae 11, no. 3: 320. https://doi.org/10.3390/horticulturae11030320

APA Style

Cong, R., Shi, L., & Zhao, B. (2025). An Effective Agrobacterium-Mediated Transient Transformation System for Studying the Lead-Tolerance Genes in Hydrangea. Horticulturae, 11(3), 320. https://doi.org/10.3390/horticulturae11030320

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