Efficient Agrobacterium-Mediated Transformation of the Commercial Hybrid Poplar Populus Alba × Populus glandulosa Uyeki

Transgenic technology is a powerful tool for gene functional characterization, and poplar is a model system for genetic transformation of perennial woody plants. However, the poplar genetic transformation system is limited to a number of model genotypes. Herein, we developed a transformation system based on efficient Agrobacterium-mediated transformation for the hybrid poplar Populus Alba × Populus glandulosa Uyeki, which is a fast-growing poplar species that is suitably grown in the northern part of China. Importantly, we optimized many independent factors and showed that the transformation efficiency was improved significantly using juvenile leaf explants. Explants were infected by an Agrobacterium suspension with the OD600 = 0.6 for 15 min and then co-cultured in dark conditions for 3 days. Using the improved transformation system, we obtained the transgenic poplar with overexpression of β-glucuronidase (GUS) via direct organogenesis without callus induction. Furthermore, we analyzed the GUS gene in the transgenic poplars using PCR, qRT-PCR, and GUS staining. These analyses revealed that the GUS gene was efficiently transformed, and it exhibited various expression levels. Taken together, these results represent a simple, fast, and efficient transformation system of hybrid poplar plants. Our findings may facilitate future studies of gene functions in perennial woody plants and tree breeding via transgenic technology assisted design.


Introduction
Poplar is an important group of cultivated tree species used in both ecological and economic plantations. Its popularity is largely due to its adaptability to diverse environmental conditions and its capacity for rapid growth [1]. Tree breeding by transgenic technology assisted design is the major technique for improving varieties of poplar with superior hereditary characteristics. This technique can overcome the difficulties associated with the breeding of woody plants, which require many years to produce progeny [2]. The genetic transformation system can introduce foreign genes into plants for understanding and controlling plant gene expression, which provide a powerful tool for analyzing the function of genes and the functional characterization of gene products. Then the in-depth knowledge of gene function is effective for making more purposeful and systematic breeding programs directed at transgenic technology assisted breeding of poplar.

Optimization of the Transformation Procedure
To establish the transformation system of P. alba × P. glandulosa, we optimize several operations in the transformation procedure, including the age of leaf explants ( Figure 1A), the growth stage of bacterium ( Figure 1B), Agrobacterium infection duration ( Figure 1C), and co-culture period ( Figure 1D). The transformation efficiency was significantly increased with juvenile leaf explants, but the frequency of shoot induction was simultaneously decreased ( Figure 1A). The transformation efficiency was significantly increased when Agrobacterium infection with an OD 600 of 0.6, and the transformation efficiency was decreased when the growth time of Agrobacterium was excessively long or short ( Figure 1B). The highest transformation efficiency was obtained with an Agrobacterium incubation time of 15 min ( Figure 1C). The transformation efficiency was not significantly different with different co-culture periods, but the shoot induction efficiency significantly decreased with the extension of the co-culture period ( Figure 1D).

Simple and Fast Transformation System
A simple and fast Agrobacterium-mediated transformation system was developed for P. alba × P. glandulosausing leaf explants and the following protocol: 2 days of pre-culture (Figure 2A), 3 days of co-culture, 4 to 5 weeks of shoot induction ( Figure 2B,C), and 4 to 5 weeks for root induction ( Figure 2D,E) and seedlings development ( Figure 2F). In addition, to determinate the stability of kanamycin-resistance in regenerated poplar plants, the regenerated poplar plants were subcultured on M4 for kanamycin-resistance plants propagation and showed that 96% of the explants from regenerated poplar plants developed into whole poplar plants. Furthermore, to ensure that the regenerated poplar plants were free from Agrobacterium, we subcultured the regenerated poplar plants on M1 for plant propagation free from kanamycin and showed no Agrobacterium occurred on the culture medium or plants in the whole process of subculture. During transformation experiments, a total of 115 leaf segments of P. alba × P. glandulosa were infected with Agrobacterium, 63 shoots were induced cultured on shoot-inducting medium M3, 39 independent kanamycin-resistant plants were obtained cultured on root-inducting medium M4, and 37 positive transgenic poplar lines were finally obtained after being subcultured on M4. The results indicate that the transformation frequency of the system was 32.18%.

Positive Plant Identification by PCR Analysis
To determine whether the transgenic poplar plants were GUS-positive, we selected 20 transgenic poplar plants and isolated the genomic DNA from leaves for PCR analysis. The 35S promoter and part of the GUS gene were PCR-amplified using a specific primer pair and the expected band size of 680 bp, the actin gene that existing in poplar was used as an internal control. The bands were observed at the expected size from all of the samples, but they were not observed for the wild-type poplar plant ( Figure 3A). These results showed that all 20 poplar plants are transgenic plants. To ensure that the regenerated poplar plants were free from Agrobacterium, we used the tzs gene that does not belong to the T-DNA region in Agrobacterium strain as a positive control. The bands of tzs gene were observed from Agrobacterium strain, but they were not observed in all of the poplar plant samples, including the wild-type poplar plant and all of the transgenic poplar plant. ( Figure 3A).

Quantitative Assay of GUS Expression by qRT-PCR
To analyze the GUS expression level of different lines, we isolated the genomic RNA from the randomly selected leaves of the seven positive transgenic poplar plants and wild-type plants. We then used qRT-PCR to analyze the relative expression of GUS. The expression of GUS varied from 10 times higher to 427 times higher than the level of wild-type plants. This result confirmed that the expression of GUS differed among the various transgenic lines ( Figure 3D).

β-Glucuronidase Histochemical Assays
We further stained the leaves of the seven poplar plants and wild-type plants that were analyzed via qRT-PCR. The GUS staining of leaf explants of transgenic poplars showed various levels of intensity, which suggests that the expression of GUS was different in various transgenic poplar plants ( Figure 3B). In addition, we stained different explants of the transgenic poplar line 6, including terminal bud, leaf, juvenile stem, adult stem, and root. All of the explants showed positive GUS staining ( Figure 3C).

Discussion
Genetic transformation is a powerful tool in plant molecular biology and functional genomics research. Since the first successful poplar transformation was established [3], poplars have been transformed for genetic improvement and gene function research far more than any other forest tree species [7,27]. A large number of factors affect the transformation efficiency and the regeneration during the process of poplar genetic transformation, including the explant source, the growth stage of bacterium, co-culture time period, and Agrobacterium infection duration. Juvenile sources of explant tissues have superior regeneration potential compared with other sources due to their lower lignin content [17]. Moreover, Agrobacterium concentration and infection time significantly affect the transformation efficiency [15,17]. In this study, we analyzed the poplar transformation efficiency by varying the developmental stage of leaf explants, Agrobacterium concentrations, the duration for the co-culture period, and duration of Agrobacterium infection. These results indicated that the development stage of leaf explants was the main influencing factor for transformation efficiency of P. alba × P. glandulosa. In addition, the best results for P. alba × P. glandulosa genetic transformation were obtained using juvenile leaf explants that were dipped into Agrobacterium suspension (OD 600 = 0.6) for 15 min and then co-cultured in dark conditions for 3 days (Figure 1).
A major problem for the poplar transformation system is inducing shoots from transformed cells, and there are two major approaches for shoot induction in most of the existing poplar transformation systems. One method is inducing cell division and callus growth on the cut surface of poplar explants infected with Agrobacterium and then inducing shoots from induced calluses [22]. Callus induction is a time-consuming and complex operation, and methods that include the callus phase may lead to plants that differ from the mother plant due to somaclonal variations [28]. Another method is directly inducing shoots from the cut poplar explants infected with Agrobacterium [18,29]. In the present study, we developed a fast poplar transformation system for P. alba × P. glandulosa via direct organogenesis without callus induction (Figure 2). The whole genetic transformation process was completed within 3 months. In addition, the regenerated poplar plants were subcultured on M4 and M1, respectively, showing that the kanamycin-resistance transgenic poplar plants were subcultured steady and the transgenic poplar plants were free from Agrobacterium contamination. These results suggested that the Agrobacterium-mediated transformation regeneration system was adopted as the fast and effective for Populus. alba × P. glandulosa. Nonetheless, the transformation system of P. alba × P. glandulosa exhibits a relatively higher transformation frequency at 32.18%.
In plant genetic transformation, the transgenic tissues will form chimeric shoots that contain transgenic and non-transgenic sections with a certain probability [30,31]. PCR is a powerful and direct tool for detecting a target gene that has been inserted into the genome, and it is routinely used in the identification of transgenic plants [18]. In the present study, transgenic poplar plants that subcultured two times were detected by the previously optimized vector-specific genomic PCR, This indicates that the GUS gene was inserted into the poplar genome successfully in the 20 transgenic poplar lines. ( Figure 3A).
Transgene expression is usually correlated to the transgene copy number that was inserted into the host plants genome [32,33]. The qRT-PCR method has been validated for accurate estimation of transgene expression in various transgenic plants [34]. The current results showed various expressions of GUS in the seven samples. These levels are significantly higher than levels for wild-type plants, revealing that this transformation system can transform the exogenous gene into P. alba × P. glandulosa efficiently, and increased the gene expression significantly ( Figure 3D). In addition, the GUS histochemical analysis showed that the high expression of the GUS gene led to high staining intensity for different transgenic poplar plants and different explants, indicating that the staining intensity was consistent with the GUS gene expression ( Figure 3B,C). Together, these results provide strong evidence that transformation systems are efficient for P. alba × P. glandulosa. Additionally, the transformed exogenous gene is suitable to achieve high expression levels of the target gene and protein.

Plant Materials and Growth Conditions
Explants were collected from P. alba × P. glandulosa plants, sterilized with 0.2% NaClO for 15 min, rinsed three times with sterile water, and transferred to the culture bottle. Seedlings of poplar (P. alba × P. glandulosa) were cultured on M1 (Table 1) and subjected to a 16-h light:8-h dark photoperiod with 500 to 600 µmol m −2 s −1 of photosynthetically active radiation. Plants were kept at 25 • C during the day and at 22 • C at night. Table 1. Composition of media for cultivation, transformation, selection, and regeneration of hybrid poplar P. alba × P. glandulosa.

Agrobacterium Culture and Transformation Procedure
We constructed the 35S:GUS vector and transformed to Agrobacterium tumefaciens GV3101 [35]. A single colony of Agrobacterium tumefaciens was grown in LB medium (containing 50 mg/L kanamycin and 50 mg/L rifampicin) overnight at 28 • C with shaking until the culture density reached an OD 600 of 0.6. The Agrobacterium tumefaciens cells were harvested by centrifugation at 5000 rpm for 10 min. The cells were then suspended with 1/2 MS solution (pH 5.8 to 6.0) containing 5% (w/v) sucrose and acetosyringone (100 µM) as the transformation solution.
For the whole process of genetic transformation, the explants were cultured on Murashige and Skoog medium with different supplementary components, as shown in Table 1. The leaf explants excised from 3-week-old plantlets were cut with three wounds perpendicular to main veins and cultured on M1 for 2 days. The cut leaves were incubated with the suspended Agrobacterium tumefaciens for 15 min with slow shaking every 5 min. The cut leaves were blotted with sterile filter paper to remove the excess bacteria and then cultured on M2 in dark conditions for 3 days. The co-cultivated leaves were washed 2 times in sterilized distilled water for 5 min, blotted with sterile filter paper to remove the excess water, and transferred to M3 for shoot induction. The co-cultivated leaves were transferred to fresh M3 every 10 days, and the growth conditions were similar to the growth conditions of the P. alba × P. glandulosa plants. The shoots grew to 1 to 2 cm after 4 weeks, and they were then cut off and transferred to M4. In this medium, the roots formed and the whole poplar plants were developed. The transgenic poplar plants were grown on M1 for 3 weeks to ensure that plants were free from Agrobacterium contamination.

Plant Genomic DNA Isolation and PCR Analysis
The genomic DNA was isolated from leaves of transgenic and wild-type poplar plants via the cetyltrimethylammonium bromide (CTAB) procedure described previously, with some modifications [36]. For PCR analyses, a forward primer (5 -CCTCTGCCGACAGTGGTC-3 ) was designed from the 35S promoter, and a reverse primer (5 -CATCGGCTTCAAATGGCGTATAGC-3 ) was designed from GUS. For an internal control, actin gene that existing in poplar was used [37], and its primers were forward 5 -AAGGTTGTTGCACCACCAGA-3 and reverse 5 -AACACACAGTCCATCACCGC-3 .

Quantitative Analysis of GUS Expression by qRT-PCR
Whole seedlings of poplar were collected and stored in liquid nitrogen.
The total RNA of these plants was isolated using an RNA Isolation Kit (TIANGEN, Beijing, China). Reverse transcription was performed using the First-Strand cDNA Synthesis Kit (TIANGEN, Beijing, China). qRT-PCR was performed using the SYBR Green Mix (TAKARA) in an optical 96-well plate. The primers for GUS were forward 5 -CTGTGGAATTGATCAGCGTTGGTGG-3 and reverse 5 -AAGACTTCGCGCTGATACCAGACG-3 . For an internal control, 18SrRNA was used, and its primers were forward 5 -CGAAGACGATCAGATACCGTCCTA-3 and reverse 5 -TTTCTCATAAGGTGCTGGCGGAGT-3 . The initial denaturing time was 94 • C for 30 s, followed by 40 cycles at 95 • C for 15 s, 60 • C for 15 s, and 72 • C for 15 s, with a final extension at 72 • C for 10 min.

Conclusions
In summary, we optimized many factors in the genetic transformation process of P. alba × P. glandulosa, and the transformation efficiency was improved significantly. We developed a simple and fast poplar transformation system for P. alba × P. glandulosa with directly induced shoots. Furthermore, the PCR, qRT-PCR, and GUS staining results showed that the transformation systems are efficient for P. alba × P. glandulosa with a 32.2% transformation frequency.