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Plant Cell, Tissue and Organ Culture (PCTOC) https://doi.org/10.1007/s11240-018-1388-7

RESEARCH NOTE

Study on factors influencing transformation efficiency in Pinus massoniana using Agrobacterium tumefaciens

Samaneh Sadat Maleki1  · Kourosh Mohammadi1 · Kong Shu Ji1

Received: 4 July 2017 / Accepted: 1 February 2018 © Springer Science+Business Media B.V., part of Springer Nature 2018

AbstractGenetic manipulation of gymnosperms using Agrobacterium—mediated system, a powerful field in forest biotechnology, has a potential to accelerate forest tree improvements with desired attributes. However, most conifers such as pine have been considered recalcitrant to genetic transformation. The present study reports the successful production of transgenic Pinus massoniana from zygotic embryos using Agrobacterium tumefaciens harboring the pBI121:CslA2 binary vector. Various independent parameters were tested for their effects on transformation efficiency in P. massoniana. According to our results, combination of Agrobacterium density at OD600 of 0.5, cold treatment of Agrobacterium suspension at 4 °C for approximately 5 h, inoculation period of 5 h, and the addition of 100 µM acetosyringone in co-cultivation medium significantly enhanced the transformation efficiency. The stable integration of CslA2 gene into the genome of putative transgenic plants was confirmed by polymerase chain reaction and Southern blot hybridization, and the expression levels were determined using quantitative reverse transcription-PCR. Our optimized transformation procedure could provide an opportunity for transferring economi-cally important genes into P. massoniana and other conifer species as well.

Keywords Efficient transformation system · Pinus massoniana · Agrobacterium tumefaciens · Molecular analysis

Abbreviations2,4-D 2,4-Dichlorophenoxy acetic acid6-BA BenzylaminopurineNAA Naphthalene acetic acidAS AcetosyringoneOD Optical density at 600nmNPTII Neomycin phosphotransferase

Introduction

Masson pine (Pinus massoniana Lamb.) is an evergreen gymnosperm- native to central and southern China, and is a significant forest tree species. The genus Pinus is the most economically and ecologically important plant group in the world. Pine wood is widely used in production of fiber and solid wood (Zhang et al. 2012). Genetic manipulation of conifers provides great opportunities to improve pest and herbicide resistance, and growth rate (Sarmast 2016) as well as modify quality traits within a shorter time compared to conventional breeding (Grant et al. 2015). Agrobacterium tumefaciens, a T-DNA mediated gene transfer system, is an attractive method for foreign gene integration into plant cells due to simplicity, low copy number, and inexpen-sive transfer of large DNA segment (Hou et al. 2016). The Agrobacterium-mediated transient transformation method in plants is influenced by several factors, including bacte-rial cell density (Nyaboga et al. 2015), co-cultivation tem-perature (Sainger et al. 2015), plant growth regulators (Li et al. 2017), plant species and genotype (Song et al. 2013), acetosyringone (Li et al. 2015), and infection time (Dutta et al. 2013). There have been several reports on the trans-formation system in conifers via Agrobacterium. Somatic

Communicated by Sergio J. Ochatt.

* Kong Shu Ji ksji@njfu.edu.cn

Samaneh Sadat Maleki smaleki97@yahoo.com

Kourosh Mohammadi kgenetics.njfu@yahoo.com

1 Co-Innovation Center for Sustainable Forestry in Southern China, The key Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China

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embryogenesis propagation has been well established in woody plants, which are characterized by long life cycles, rather than traditional breeding (Isah 2016). In some reports, zygotic embryos have been used as target materials in the transformation of P. strobus (Levee et al. 1999; Tang et al. 2007), Picea abies and P. taeda (Wenck et al. 1999), P. taeda L. (Tang et al. 2001), P. radiata (Cerda et al. 2002; Grace et al. 2005), P. pinaster Portuguese (Tereso et al. 2006), P. kesiya Royle ex Gord (Malabadi et al. 2008), P. pinaster Ait (Alvarez and Ordás 2013), and P. elliottii Engelm (Tang et al. 2014). Until now, there has been no previous report of the transfer of a foreign gene into the genome of P. mas-soniana using the Agrobacterium-mediated transformation system.

The present study aimed to generate transgenic P. mas-soniana plants using A. tumefaciens strain EHA105 which overexpresses the CslA2 gene, by assessing various param-eters that influence transformation efficiency, including the density of Agrobacterium infective suspension, cold treat-ment of Agrobacterium suspension, Agrobacterium infection time, and concentration of acetosyringone. This transforma-tion system offers an opportunity to transfer economically important genes into P. massoniana.

Materials and methods

Plant materials

Dry mature seeds (collected from the National Masson Pine Seeds Base, Forestry Science Institute, Xinyi City, Guang-dong Province, China) extracted from cones were placed in plastic bags at 4 °C prior to transformation. The seeds were surface cleansed in 2% NaClO (w/v) for 12 min followed by rinsing three times in sterile water. Then, the seeds were

washed in running tap water for 1 h and soaked in water overnight. The seed coat was carefully removed under sterile conditions. The embryos were then excised using a sterilized scalpel. Subsequently, the embryos were washed with 0.2% (v/v) HgCl2 for 2 min and then immersed in 70% (v/v) etha-nol for 1 min. Finally, the explants were rinsed five times with sterile water, placed in pre-culture DCR medium, and kept in the dark at 24 ± 1 °C (Table 1). After 1 week, mature zygotic embryos were collected for Agrobacterium infection.

Construction of the plant expression vector

The open reading frame sequence containing a 1605-bp nucleotide DNA fragment of the CslA2 gene was inserted in vector pBI121 after digestion with BamHI and SacI restriction enzymes. The resulting plasmid was introduced into competent Agrobacterium tumefaciens strain, EHA105 (Hood et al. 1993), using the freeze–thaw method (Holsters et al. 1978). In this construct, expression of the CslA2 was driven by the cauliflower mosaic virus (CaMV) 35S pro-moter. The structure of the T-DNA region of the expression vector pBI121 is described in Fig. 1.

Transformation and regeneration of P. massoniana

Agrobacterium containing the binary vector, pBI121:CslA2, was grown overnight in a dark incubator at 28 °C in liquid Luria Bertani medium containing 50 mg/L Rifampicin and 50 mg/L Kanamycin with vigorous agitation. The bacterial cells were centrifuged (5000×g, 4 °C, 10 min), and the pel-let was diluted in liquid inoculation DCR medium (Table 1) with different densities of Agrobacterium (OD600 nm = 0.1, 0.3, 0.5, and 0.7). Cold treatment was applied to the Agro-bacterium suspension at 0, 4, 8 and 10 °C for 5 h in a tem-perature-controlled refrigerated incubator prior to embryo

Table 1 Composition of Pinus massoniana media

Media Composition Duration

Pre-culture DCR medium, 4 mg/L kinetin, 8 mg/L 2,4 dichlorophenoxyacetic acid (2,4-D), 4 mg/L 6-benzylaminopurine (BA), 30 g/L sucrose and 7 g/L agar

7 days

Liquid inoculation DCR medium, 4 mg/L kinetin, 2 mg/L 6-benzylaminopurine (BA), 30 g/L sucrose, 1 mg/L Thiamine HCl, 0.5 mg/L Pyridoxine HCl, 0.5 mg/L Nicotinic acid, 100 mg/L Vitamin C, 2 mg/L Glycin, 200 mg/L Myo-Inositol, 200 µM acetosyringone

5 h

Co-cultivation DCR medium, 4 mg/L kinetin, 2 mg/L 6-benzylaminopurine (BA), 30 g/L sucrose, 1 mg/L Thiamine HCl, 0.5 mg/L Pyridoxine HCl, 0.5 mg/L Nicotinic acid, 100 mg/L Vitamin C, 2 mg/L Glycin, 200 mg/L Myo-Inositol, acetosyringone, 7 g/L agar

3 days

Liquid wash DCR medium, 500 mg/L Timentin 15 minSelection DCR medium, 4 mg/L kinetin, 2 mg/L 6-benzylaminopurine (BA), 30 g/L sucrose, 1 mg/L Thiamine HCl,

0.5 mg/L Pyridoxine HCl, 0.5 mg/L Nicotinic acid, 100 mg/L Vitamin C, 2 mg/L Glycin, 200 mg/L Myo-Inositol, 7 g/L agar, 200 mg/L Timentin, 35 mg/L kanamycin

8–12 weeks

Shoot induction DCR medium, 2 mg/L 2,4 D, 1.0 mg/L 6-BA and 35 mg/L kanamycin 6–8 weeksRoot induction DCR medium, 1 mg/L NAA 5 weeks

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infection. For Agro-infection of the explants, 7-day-old pre-cultured embryos were immersed in the Agrobacterium suspensions for 1, 3, 5 and 8 h. Afterward, the embryos were transferred to the DCR co-cultivation medium (Table 1) with various acetosyringone (AS) concentrations (0, 25, 50 or 100 µM) in the dark at 28 °C for 3 days. Then, the infected embryos were rinsed three times with a wash medium for 15 min to eliminate the Agrobacterium, followed by exten-sive washing with double-distilled water. The explants then were gently blotted dry on sterile filter paper to drain off excess bacteria before transferring to the plates containing a selection medium (Table 1). After 4 weeks in darkness, the resistant calli were transferred to fresh medium. All surviv-ing calli from the transformation experiment after a third round of selection were transferred to a shoot induction DCR medium (Table 1). After 6 weeks of culture, the regener-ated shoots that were sub-cultured weekly were separated from the tissues and transferred to DCR rooting medium (Table 1). Plants with well-developed roots were potted in soil after 5 weeks. The pH of all media was adjusted to 5.8 prior to autoclaving. All cultures were placed at 24 ± 1 °C in a culture room. In this experiment, the transformation efficiency was estimated as the percentage of independently transformed explants of the total number of explants.

Genomic DNA extraction, PCR and southern blot analysis

We used the CTAB method as described previously (Poreb-ski et al. 1997) to extract total genomic DNA from needle tissues of transformed and untransformed plants for PCR amplification and Southern blot hybridization. Purify DNA concentrations were measured by a BioDrop spectrophotom-eter (1000–1100 ng/µL). PCR amplification was performed with two sets of primers: CaMV 35S (F:5′-CTA ACC CAC AGA TGG TTA GAG AGG CT-3′ and R:5′-GCG TCA TCC CTT ACG TCA GTG-3′) to isolate a 736-bp fragment of the promoter and virG (F:5′-TTG GTT CGC ATT TTC GTC ATCCG-3′ and R:5′-CAA GAA ACT CTC TCG TAC TAA

ACG GCTT-3′) to isolate a 473-bp fragment. The PCR was carried out for 30 cycles under the following conditions: 95 °C for 3 min, 95 °C for 30 s, 60 °C for 30 s, 72 °C for 90 s, and a final extension at 72 °C for 10 min. The PCR products were visualized by gel 1% agarose. In Southern blot analysis, the genomic DNA of the transgenic plants was digested with BamHI and SacI (Takara, Japan), separated on a 0.8% agarose gel at 25 V overnight then blotted on a nylon membrane. The membrane was hybridized with DIG-labeled 404-bp CaMV 35S probe using the primers F:5′-GGC CAT GGA GTC AAA GAT TCA AAT AGA-3′ and R:5′- AAC GTC TTC TTT TTC CAC GAT GCT C-3′.

RNA extraction and real‑time‑PCR

Total RNA was isolated from needle tissues of wild-type and transformed P. massoniana plants using RNA prep pure Plant Kit (Polysaccharides and Polyphenolics-rich) (Tian-gen Biotech, Beijing, China) following the manufacturer’s instruction. The extracted RNA samples were used for the reverse transcription process to synthesize first-strand cDNA by Prime Script 1st Strand cDNA Synthesis Kit (Takara, Dalian, China) according to the manufacturer’s instructions. Expression levels of pmCslA2 were analyzed by the 2−ΔΔCT method using Applied Biosynthesis quantitative Real-time PCR system (Applied Biosystems, USA) and the SYBR Green PCR Master Mix. The primers used in real-time RT-PCR experiment were as follows: CslA2, F:5′-TTT ATC CCA TGG TCT TAG TGC AGA TACC-3′ and R:5′-AAT GAT AAC CCG ATT TGA AGG CCA TGA-3′ to amplify a 110-bp ampli-con of the pmCslA2 cDNA and the β-actin, F:5′-CCA CGA AAC TAC TTA CAA CTC CAT C-3′ and R:5′-GGG CTG TGA TTT CCT TGC TC-3′ for normalization expression.

Statistical analysis

Statistical differences were considered significant when the confidence intervals showed no overlap of the mean values with an error value of 0.05 using One-way ANOVA with

Fig. 1 Linear plasmid vector pBI121:CslA2 indicating: a promoter (35Spro, cauliflower mosaic virus 35S promoter), two terminators (nosTer, terminator from the nopaline synthase gene), nptII (coding

region of the neomycin phosphotransferase gene) and T-DNA borders (LB left border; RB right border)

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Duncan’s test. Each experiment was repeated independently for three times with around 30 explants.

Results

The present study describes a procedure to establish an Agrobacterium -mediated transformation method for P. mas-soniana. This is the first successful report of transferring a gene into the P. massoniana genome. Figure 2 shows an overview of the steps used in Agrobacterium-mediated trans-formation and regeneration procedure in Pinus. The devel-opment of an efficient plant transformation methodology is extremely important to genetic manipulation in vitro. High transformation efficiency was obtained by applying vari-ous independent factors such as Agrobacterium suspension concentration, cold treatment of Agrobacterium suspension, Agrobacterium infection time, and concentration of acetosy-ringone in P. massoniana.

The development and regeneration of zygotic embryos into plants after transformation occurred within 20–24 weeks of culture. Figure 3a shows the mature embryo prior to Agrobacterium infection. The transformed calli (Fig. 3b), which were obtained from embryos inoculated with Agro-bacterium and incubated in callus induction medium after optimization, were white in color and had relatively soft tissues. Callus induction and growth were conducted in the dark. Putative transgenic shoots regenerated 6–8 weeks after the resistant calli were transferred to shoot induction medium (Fig. 3c). Shoot regeneration was performed under a 16/8 h light/dark. The shoots were placed in root induction

medium. Root induction and development occurred in the presence of NAA. Both auxin type and concentration influ-ence root initiation (Zhu et al. 2010). The regenerated trans-formed plantlets of masson pine were transferred to soil and allowed to acclimatize in a greenhouse (Fig. 3d).

Effects of A. tumefaciens density on P. massoniana transformation

Suspension cultures of the Agrobacterium at a wavelength of 600 nm with different optical densities (0.1, 0.3, 0.5, or 0.7) were performed in this experiment. The highest transforma-tion efficiency (25.4%) was achieved after infecting with an agrobacterial suspension at OD600 of 0.5. The transforma-tion efficiencies using suspensions with an OD600 of 0.1, 0.3 and 0.7, was 6, 14 and 19.78% respectively (Fig. 4a). These findings indicate that there are significant differences in transformation efficiency among various OD treatments.

Effects of cold treatment of Agrobacterium suspension on P. massoniana transformation

Cold treatment of Agrobacterium (Zhang et al. 2013) prior to infection the embryos in the transformation reduced callus browning and death, thereby improving efficiency. Some of the transformed calli without cold treatment of Agrobac-terium infective suspension turned reddish-brown in color. The highest transformation efficiency (47.9%) was observed after cold-treatment of the Agrobacterium suspension at 4 °C for 5 h (Fig. 4b).

Remove seeds fromP.massoniana cones Remove seed coat

Pre-cultureAgrobacteriumInfection Co-cultivationEmbryo excision

Wash the embryos

Callus induction medium

Shooting mediumRooting mediumTransformed plantConfirmation of transgenic plant

Fig. 2 An overview the main steps for Agrobacterium transformation and regeneration of Pinus massoniana

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Effects of immersion time on P. massoniana transformation

Various infection times was tested for (1, 3, 5 and 8 h) were assessed in this study. Figure 4c, shows the effect of immer-sion time on transformation efficiency of P. massoniana embryos. The infected embryonic explants were exhibited a high percentage of transformation (35.6%) after infecting with Agrobacterium suspension for 5 h, whereas, the longer period of inoculation reduced the survival rates of embryos after co-cultivation with Agrobacterium, thus resulting in low transformation efficiency (19.54%). These results indi-cate that the time of inoculation has a significant effect of transfer of T-DNA from Agrobacterium to the plant cells.

Effects of acetosyringone on P. massoniana transformation

Acetosyringone is a phenolic inducer of the virulence genes (vir) and is secreted by wounded tobacco cells (Stachel et al. 1985). Transformation efficiency was tested for the co-cultivation period using different concentrations of ace-tosyringone (0, 25, 50 and 100 µM). As shown in Fig. 4d, the

frequency level of transformation was significantly improved to a maximum of 59.75% with increasing the AS concentra-tions from 0 to 100 µM. This finding is in agreement with the work on other Pinus species (P. strobus and P. kesiya Royle ex Gord.) using 100 µM acetosyringtone (Levee et al. 1999; Malabadi et al. 2008).

Analysis of variance (ANOVA) showed highly significant p values (< 0.0001) for 3 degree of freedom in different fac-tors that used for transformation. A summary of statistical analysis is presented in Table 2.

Molecular analysis of the transgene in transformed P. massoniana lines

The presence of the transgene was detected in the recipi-ent plants by PCR using specific primers. Amplification of the 736-bp of CaMV 35S and 473-bp of the virulence gene VirG were initially tested in a total of 67 independent trans-genic lines. Figure 5 shows the presence of CaMV 35S and absence of VirG due to clarify genetic transformation but not due to any contamination of Agrobacterium in five ran-domly transgenic plants. No amplification was observed in the sample obtained from untransformed tissues. For further

Fig. 3 Transformation and regeneration of transformed masson pine plants. a Zygotic embryo (Scale bar 1 mm). b Transformed callus (Scale bar 1 cm). c Transgenic shoots (Scale bar 1.2 cm). d Accli-mated transgenic plant in the greenhouse (bar 2.0 cm)

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analysis, the quantitative real-time PCR (qRT-PCR) was per-formed to investigate the level of pmCslA2 expression in the transgenic lines. The results indicated that the expression level of the pmCslA2 increased 1.97, 4.32, and 2.39 fold higher than that in the wild-type (Fig. 6).

Southern blot hybridization was used for estimating the transgene copy number in three randomly selected trans-genic plants. Southern blot analyses presented 1–3 copies in most transgenic plants, confirming the stable integration of

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Fig. 4 Factors affecting the transformation efficiency of P. massoni-ana. a Effect of Agrobacterium density on transformation efficiency (%) at 0.1, 0.3, 0.5, and 0.7 OD600. b Effects of cold treatment of Agrobacterium suspension on transformation efficiency (%) at 0, 4, 8 and 10 °C. c Effects of Agrobacterium infection duration on transfor-

mation efficiency (%) for 1, 3, 5 and 8 h. d Effect of AS concentra-tions at 0, 25, 50 and 100 µM on transformation efficiency (%). Each treatment consisted of 25–35 embryos and experiments were repli-cated three times

Table 2 The summary statistics of the effect of different factors on transformation efficiency

Factor Variance F statistic p value

Optical density OD600 58.376 83.13 < 0.0001Agrobacterium cold treatment 305.6324 99.18 < 0.0001Agrobacterium infection time 64.5429 44.84 < 0.0001Acetosyringone concentration 220.804 37.75 < 0.0001

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genetic material into the genome. The non-transformed con-trol plants did not exhibit any hybridization signals (Fig. 7).

Discussion

Modern agricultural biotechnology has facilitated the pro-duction of genetically engineered plants in absence of a sex-ual partner as well as in the optimization of transformation procedure in generating of economically important trans-genic plants with high efficiency. In present study, the open reading frame of the PtaCslA2 gene from P. taeda under the control of CaMV 35S promoter and NPTII gene as selection marker was cloned in a pBI121 vector and overexpressed in P. massoniana using an efficient Agrobacterium-mediated transformation system. Zygotic embryos were used as excel-lent explant source for the generation of transgenic lines. Studies on DNA transfer using the A. tumefaciens-mediated transformation protocol has a great effect on improvement of woody plant species (Tang et al. 2004; Malabadi and Nata-raja 2007; Choudhury et al. 2014).

The production and regeneration of transgenic conifers by somatic embryogenesis, is a little known as a routine technique in plant laboratory (Tang and Newton 2005). However, considerable research efforts on efficiency are still required. In this study, the effects of several independ-ent factors influencing the efficiency of foreign gene trans-fer into P. massoniana embryogenic cells were monitored using A. tumefaciens-mediated transformation. These fac-tors included bacterial density, cold treatment of bacterial suspension, infection duration, and AS concentration. The concentration of Agrobacterium refers to its growth condi-tions and the Agrobacterium during logarithmic growth phase has been shown to have an optimal density of higher infection ability (Li et al. 2017). The results of the present study demonstrate that, the Agrobacterium concentration and infection time significantly influenced transformation

Fig. 5 PCR detection of T-DNA sequences. Lane 1: 250 -bpll Generay DNA marker, Lane 2: untransformed P. massoniana, Lanes 3–7: DNA from P. mas-soniana transformed, Lane 8: Positive control for CaMV 35S and virG isolated from the binary vector pBI121 CamV35S (736 bp)

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Fig. 6 Real time analysis of CslA2 expression in transgenic Pinus. β-actin gene was used as a reference for normalization. WT: wild type P. massoniana; Line 1, 2, 3: transgenic lines. Error bars represent means ± SD (n = 3)

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Fig. 7 Southern blot analysis of genomic DNA isolated from P. massoniana needles, digested with BamHI and SacI and hybridized with a CaMV 35S probe. Lane 1: Dig labeled DNA Marker; Lane 2: Plasmid: pBI121:CslA2 as a positive control; Lane 3: non-transgenic control plant; Lanes 4, 5 and 6: three transgenic lines digested with BamHI; Lane 7, 8 and 9 the same three transgenic lines but digested with SacI

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efficiency, similar to that observed in other transformation systems (Dutt and Grosser 2009, Maheshwari and Koval-chuk 2016). Increased Agrobacterium concentration and long infection time could lead to bacterial overgrowth and induction of hypersensitive necrotic responses in explants, thereby resulting in low transformation efficiency (Kuta and Tripathi 2005; Ombori et  al. 2013). Furthermore, using a low density Agrobacterium suspension and short infection time usually resulted in weak infection ability, which in turn induces low transformation efficiency. Con-sequently, attention to Agrobacterium density and infection duration in development of transformation systems is criti-cal. Our results suggest that, the Agrobacterium infective suspensions with an OD600 of 0.5 and infection period of 5 h are the optimum for transformation. Tang et al. (2014) showed that the highest transformation efficiency of slash pine embryos was achieved using an immersion time of 50 s and infecting with A. tumefaciens at an OD600 of 0.9. These observations indicate that the different Pinus spe-cies benefit from various Agrobacterium concentration and infection durations.

Acetosyringone has been employed as a chemical inducer of the vir gene of Agrobacterium (Stachel et al. 1985; Fortin et al. 1992) to increase the efficiency of trans-formation. Acetosyringone levels up to 200 µM are not considered to be toxic to Agrobacterium (Stachel et al. 1985). As shown in Fig. 4d, the transgenic plants were obtained without the addition of acetosyringone, although their transformation efficiencies were low. Among the evaluated levels of acetosyringone, using a 100 µM con-centration resulted in the highest transformation efficiency (59.75%). Similarly, an enhancement in the efficiency of transformation in wheat has been observed using 100 µM acetosyringone (Rashid et al. 2010). Interestingly, cold treatment of the infective Agrobacterium suspension improved callus induction and also transformation effi-ciency by preventing callus browning. Furthermore, the cold treatment improves somatic embryo development and germination of Citrus jambhiri Lush (Singh 2014). Our observations demonstrate that the cold treatment at lowest temperature (0 °C), was inhibitory to callus formation, and the transformation efficiency of P. massoniana using 4 °C is higher than that using 8 or 10 °C.

In summary, the overall findings of the present study have clearly demonstrated the production of stable transformed P. massoniana plants via Agrobacterium-mediated gene trans-fer. This efficient protocol can be potentially used in studies on gene function as well as in plantation forestry.

Acknowledgements This study was supported by “The Thirteenth 5-year Plan” National Key Research Project “Pinus massoniana sus-tainable management technology research on Pinus massoniana”,and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Author contributions SM and KJ designed the experiments. SM con-ducted the experiments and collated the results. KM performed the statistical analysis and prepared the graphs. Drafting of manuscript and reading and final approval of the version to be published; SM, KM and KJ.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

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