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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.47 No.6 pp.249-254
DOI : https://doi.org/10.14397/jals.2013.47.6.249

Improvement of Agrobacterium-Mediated Transformation of Medicinal Mushrooms

Hee-Sung Park2*, Jang-Won Choi1
2Department of Biotechnology, Catholic University of Daegu, Gyungsan 712-702, Korea
1Department of Bioindustry, Daegu University, Gyungsan 712-714, Korea
Received: OCT. 2. 2013, Revised: NOV. 28. 2013, Accepted: NOV. 29. 2013

Abstract

Wounding to the mycelia of five mushroom species caused them to be susceptible toAgrobacterium-mediated transformation. The high transformation rate indicated that the woundsgenerated by mechanical means were a highly conclusive for agroinfiltration. Some transformants ofGanoderma lucidum were distinctive from the wild type in their morphology and antioxidativeactivity.

 

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I. Introduction

 Mushrooms have long been recognized as a powerful resource associated with health, vigor and long life (Wasser & Weis, 1999). Numerous bioactive compounds have already been identified but a plethora of compounds still await discovery. In order to exploit the biosynthetic potential of medicinal mushrooms, studies of functional genomics has the potential of being highly informative. However, DNA delivery into these mushrooms may be a challenge, and a practical transformation method should be outlined in advance (Meyer, 2008).

 Agrobacterium tumefaciens has a natural capability to transfer its T-DNA into plant genomes. It has also been developed to transform yeast (Bundock et al., 1995) or fungi (de Groot et al., 1998), or even human cells (Kunik et al., 2001). Despite success in some fungi, transformation efficiency has always posed a challenge. At this point, no general rule can be applied to predict the effectiveness of the Agrobacterium-mediated transformation (AMT) technique and therefore, the need for developing an optimized AMT method independently for the fungus of interest persists. 

 To transfer the T-DNA fragment into the plant genome, the wounding of plant cells has been recognized as a critical prerequisite. In some instances, wounds deliberately generated in vitro by mechanical means can facilitate the T-DNA transfer regardless of the susceptibility or recalcitrance of the host plants (Singh & Chawla, 1999; Flores Solís et al., 2003; Kim et al., 2007). In this work, we assessed mechanical wounding, and herein describe its significance for achieving a high frequency of mushroom transformation mediated by Agrobacterium.

II. Materials and Methods

2.1 Mycelia growth

 Ganoderma lucidum (KACC42231), Lentinula edodes (KACC42378), Grifola frondosa (KACC50027), Schizophyllum commune (KACC43373), and Lyophyllum decastes (KACC41766) were obtained from the Korean Agricultural Culture Collection. Mycelia growth was maintained in potato dextrose agar (PDA) or broth (PDB) medium at 25℃.

2.2 Mycelia Transformation

 To transform the mycelia, pCambia1300 (CAMBIA, Australia) carrying genes coding for hygromycin phosphotransferase (hph) under the CaMV35S promoter and neomycin phosphotransferase was introduced into A. tumefaciens LBA4404. Agrobacterium cells were grown (18 h, 28℃, 180 rpm) in Luria-Bertani (LB) broth containing antibiotics (50 mg/mL of kanamycin and 50 mg/mL of streptomycin), collected by centrifugation (2 min, 10,000 rpm) and then resuspended in MS medium at pH 5.7 (Murashige and Skoog, 1962) to a final density of OD600=0.5. Fungal discs (5 mm in diameter) prepared from the PDA culture using a steel loop were mixed with an equal volume of bacterial suspension. Agroinfiltration was performed for 5 min under vacuum followed by co-cultivation (22℃, 2 day). Subsequently, fungal discs were transferred to the selective PDA medium containing hygromycin and 250 mg/mL of cefotaxime and incubated (10-14 day, 25℃). In a wounding experiment, the fungal discs mixed with an equal volume of aluminum oxide particles (60 μm in average size) in a 50 mL tube were vigorously shaken up and down for 2-3 min before agroinfiltration and subsequent co-cultivation (Kim et al., 2007).

2.3 PCR and Southern hybridization

 To isolate genomic DNA, the mycelia mat on PDA was collected, ground in LN2 to a fine powder and then extracted by the CTAB method (Doyle & Doyle, 1987). To confirm DNA delivery, PCR at 94℃, 5min: 35×(94℃, 30s; 65℃, 30s; 72℃, 1min): 72℃, 3min was performed using primers (forward, 5'-GTCGAGA AGTTTCTGATCGA-3', and reverse, 5'-GCTGCATCA TCGAAATTGCC-3') designed to amplify an 830 bp DNA fragment derived from the internal region of the hph gene. For Southern blot hybridization, 10 mg of genomic DNA was digested with EcoRI and separated on a 1% agarose gel prior to transfer to a Nytran membrane for hybridization with the hph gene probe. The 830 bp DNA fragment, PCR-amplified from the pCambia1300 plasmid DNA, was labeled with a Bright-Star Psoralen-biotin nonradioisotopic labeling kit (Life Technologies, Calsbad, CA). Hybridization was carried out in buffer containing 5xSSC, 0.1% SDS, and 5% dextran sulfate for 24 h at 60oC. Hybridization signals were detected using the Bright-Star detection system (Life Technologies, Calsbad, CA).

2.4 Total phenolic content and DPPH scavenging activity

 Mycelia mats from a 50 mL flask culture with shaking (25℃, 5-10 day, 150 rpm) were collected onto a Whatman filter paper, thoroughly washed with distilled water and then dried at 60℃ to a constant weight. Dried fungal mass mixed in methanol (1 g/10 mL) was disrupted using a homogenizer to be extracted by shaking (2 day, 22℃, 150 rpm). The extracts were collected by centrifugation (13,000 rpm, 20 min) and the methanolic extracts were evaporated to dryness under vacuum. Samples were dissolved in methanol and filtered through a 0.2 μm filter disc. Total phenolic content was determined using Folin-Ciocalteu reagent using gallic acid as the standard (Ikawa et al., 2003). It was expressed as mg GAE/g dried fungal mass. DPPH scavenging activity assay was performed using the microplate reader. A mixture of the extract solution (30 μL) and methanolic solution (270 μL) containing 60 μM DPPH radicals was left to stand in the dark for 30 min, and the absorption was measured at 515 nm.

III. Results and Discussion

 Minimum concentrations of hygromycin that completely inhibited the mycelia growth was determined to be 70, 30, 50, 70 and 50 μg/mL, respectively, for G. lucidum, L. edodes, G. frondosa, S. commune and L. decastes.

 Transformation results are shown in Fig. 1A. No transformants were obtained if intact fungal discs from G. lucidum, L. edodes, G. frondosa and L. decastes were used. This was probably due to their natural property to be highly recalcitrant to AMT. In contrast, S. commune was revealed to be susceptible to AMT and the transformation frequency [(No. of transformed fungal discs/No. of fungal discs tested)x100] was 1-2% on average for at least three independent experiments using 100 fungal discs for each experiment. The use of 200 μM acetosyringone-induced Agrobacterium cells (de Groot et al., 1998) exerted little improvement for the recalcitrant fungal species. However, it was quite beneficial for S. commune and resulted in 5-7 fold increase. In the wounding experiment, the fungal discs, once wounded by aluminum oxide particles, developed many transformants. The average of transformation frequencies of triplicate experiments were 32, 17, 10, 56 and 15%, respectively, for G. lucidum, L. edodes, G. frondosa, S. commune and L. decastes. AMT was previously described for G. lucidum but the efficiency was not clearly indicated for the construct of the proinsulin gene driven by the CaMV35S promoter (Ni et al., 2007). The efficiency of AMT for G. frondosa (Hatoh et al., 2013) could not reasonably be compared to this study because fungal cell suspension was used instead of fungal discs. Protoplast-mediated transformation has been available for L. edodes (Kuo & Huang, 2008) and S. commune (van Peer et al., 2009), whereas particle bombardment has been reported for L. descates (Sunagawa et al., 2007). Fungal discs prepared from the margin of the transformants were subsequently subjected to two more rounds of screening. 

Fig. 1. Transformation of medicinal mushrooms with Agrobacterium. Transformants are shown from the experiment using intact fungal discs (Intact) or acetosyringone-induced Agrobacterium (AS), or fungal discs with mechanical wounds (Wounds) (A). T-DNA transfer was confirmed by Southern blot hybridization (B). P1 and P2 are serially diluted pCambia1300 plasmids digested with EcoRI. Nt and T1-T6 represent non-transformant and transformants of G. lucidum, respectively. M indicates DNA size marker.

 In Southern blot hybridization, the integrated hph gene was successfully detected in the EcoRI-digested genomic DNA (Fig. 1B). Also, the fact that most of hybridization signals were detected as a single band suggests that T-DNA integration might have occurred in general into one locus of the genome.

 Many of the transformants showed different morphologies of which some are presented in Fig. 2A. This could probably be attributable to the T-DNA insertion-mediated metabolic changes that manifested as an altered morphology. Transformants of G. lucidum were randomly chosen and the transferred hph gene was confirmed by PCR. The presence of the 0.83 kb DNA fragments was clearly established in all of the transformants. No PCR products were detected from the non-transformants (Fig. 2B).

Fig. 2. Distinctive features of transformants of G. lucidum. Distinctive morphologies developed in some transformants of G. lucidum (A). Transformants (1-30) and non-transformant (Nt) were confirmed for T-DNA transfer by PCR (B). M indicates DNA size marker. DPPH scavenging activity and total phenolic content were compared in transformants 1-30 (designated GT101 - GT130) and Nt (C). In the analysis of DPPH scavenging activity and total phenolic content, values determined in transformants are expressed as fold increase relative to the value determined in Nt. The experiment was carried out in triplicate and averaged.

 Further investigation was performed to determine whether they displayed changes in bioactive potential. In the comparative analysis of DPPH radical scavenging activity, many of 30 randomly chosen transformants were distinctive, to some extent, from the non-transformant (Fig. 2C). For example, transformants GT108, GT119, GT123, GT128, GT129 and GT130 showed stronger DPPH scavenging activity. All of them were determined to have total phenolic content higher than the wild type (0.32 ± 0.1 mg GAE/g dried fungal mass) indicating total phenolic content in a positive correlation with the strength of DPPH scavenging activity. Among the protective actions in biological systems, phenolic compounds are well known to exhibit antioxidant activity.

 AMT in plants was intensively investigated to develop a variety of beneficial tools including wounding agents (Opabode, 2006). Here, putative wounds of the mycelia deliberately generated by vortexing with aluminum oxide particles made them susceptible to AMT. Susceptibility of S. commune could be further improved by wounding. Agrobacterium was proven to be a useful tool to transform many fungal species, including Ascomycetes, Basidiomycetes, Zygomycetes, and even fungus-like protists. However, routine use of it is frequently hampered by low efficiency of transformation. Here we present a simple protocol to transform mycelia of five mushrooms using Agrobacterium without the use of any complicated reagents or media. Our approach, involving wounding of the cells to be transformed, might make possible the successful transformation of other fungal species that are resistant to Agrobacterium-mediated transformation.

 T-DNA has the mutagenic capability to generate loss or gain of function in the targeted organism (Radhamony et al., 2005). If a large enough population of transformants is generated, chance will favor the probability of finding a transgenic line expressing a noble or strengthened bioactivity of interest. The wounding-aided AMT protocol developed in this study would provide a functional prompt for exploring the bioactive potential of medicinal mushrooms.

IV. Acknowledgment

 This study was supported by the Technology Development Program for Food, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Project No.: 111157-03-2).

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