興大園藝 (Horticulture NCHU) 42 (4) : 81 – 97 2017 -81- Transformation of OsMADS14 (AP1-like) Gene into Erycina pusilla Chanon Lapjit 1) I-Chun Pan 2) Liang-Jwu Chen 3) Meng-Jiau Tseng 4) Key word: Erycina pusilla, OsMADS14 (AP1), Gene Transformation Summary Erycina pusilla is a fast growing epiphytic Oncidiinae species. MADS-box gene involved in regulating early-flowering and alteration of floral organ. In this study, the rice gene OsMADS14 (AP1-like) driven by Oncidium AP1 promoter (p1301-AP1-MADS14) was transferred into the PLBs of the Erycina pusilla via Agrobacterium-mediated gene transformation. The regenerated plantlets were selected with 5~10 ppm hygromycin and GUS histochemical staining. The regenerated plantlets were confirmed by PCR and RT-PCR. Results indicated that the OsMADS14 gene was present in the genome of transformed Erycina pusilla plants and expressed its mRNA. Changes in the leaf and plant morphology, flower shape, blooming time, and multiple flower stalks were found in the OsMADS14 transformed Erycina pusilla plants. This is the first report on the ectopic expression of MADS-box gene in Erycina pusilla using an efficient and sophisticated gene transformation technology. Introduction The Orchidaceae has great diversity in floral morphology and a rich array of species. It comprises the largest family of flowering plants (Chase et al., 2003). The extraordinary variety 1) Ph.D. of Department of Horticulture, National Chung Hsing University. Current Address: Department of Plant Science and Agricultural Resources, Khon Kaen University, Thailand. 2) Assistant Professor, Department of Horticulture, National Chung Hsing University. 3) Professor, Institute of Molecular Biology, National Chung Hsing University. 4) Professor, Department of Horticulture, National Chung Hsing University. Corresponding author. -82- of orchid floral features and appearances ensure a large consumer demand for orchids. Oncidium, a genus in subtribe Oncidiinae, is a popular and important cut flower (Pan et al., 2012). This orchid needs about 3 years to reach sexual maturity under natural conditions, and its flowering being precisely regulated by temperature (Chen and Hsu, 2003). To develop new commercial species with competitive advantageous traits such as shorter vegetative stage or tropical growth of Oncidium species continues to be a pressing need (Pan et al., 2012). Erycina pusilla is a fast to growing epiphytic orchid with a relatively low chromosome number (2n = 6) and small genome size (1.5 pg per 1C nucleus). It had been suggested as a model species for orchid functional genomics and genetic analyses and short juvenility (Chiu and Chang, 2011). Taxonomy and chloroplast genome analyses place Erycina pusilla in the Oncidiinae family close to Oncidium 'Gower Ramsey' (Pan et al., 2012). Orchids are among the most important plants in flower markets around the world. A few research on the MADS box genes and flower development have been reported in orchids (Lu et al., 1993; Hsu et al., 2003). The ABCDE model predicted the interaction of five classes of MADS box genes in regulating flower development in many plant species. The involvement of these MADS box genes in floral transition and initiation was further evidenced by ectopic expression of these genes in transgenic plants (Thiruvengadam and Yang. 2009). Ectopic expression of AP1 or its orthologues from heterologous plants produced an extremely early-flowering phenotype (Mandel and Yanofsky, 1995). Similar phenotypes were also observed in 35S::AP3/35S::PI and 35S::SEP3 transgenic plants (Pelaz et al., 2000; Honma and Goto, 2001). In addition to causing sepal and petal conversion into carpel and stamen like structures, ectopic expression of AG or its orthologues from heterologous plants also produced an early flowering phenotype (Mizukami and Ma, 1997; Bradley et al., 1993). In this study, we attempted to transform the Erycina pusilla utilizing OsMADS14 (AP1-like gene), a gene involved in regulating early-flowering and alteration of floral organs, to produce novel and/or new flower type of Erycina pusilla floras with high marketing value. Materials and Methods 1. Plant materials Erycina pusilla plants were purchased from Flower Space Orchids Company, Changhua, Taiwan. The seed capsules after self-pollination were collected and used as experimental material. 2. Induction of protocorm-like bodies (PLBs) and proliferation The preparation of Erycina pusilla PLBs and proliferation were performed as previously -83- described (Lapjit et al., 2015). After 6 months of induction, protocorm-like bodies (PLBs) formed from embryoid bodies in vitro were used as explants for Agrobacterium-mediated genetic transformation. 3. Plasmids and genes Agrobacterium tumefaciens strains GV3103 containing p1301-AP1-MADS14 were used for genetic transformation of Erycina pusilla PLBs. The plasmid p1301-AP1-MADS14 (Fig. 1A) contains rice OsMADS14 (AP1-like) gene driven by AP1 promoter, an intron-interrupted - glucuronidase (gusA) gene, and the hptII gene conferring resistance to hygromycin. Rice OsMADS14 (AP1-like) gene was provided by Dr. Liang-Jwu Chen, Institute of Molecular Biology, National Chung Hsing University. The plasmid p1301-AP1-MADS14 was constructed by Dr. Menq-Jiau Tseng’s laboratory. The sequences of the OsMADS14 primers were as follows: MADS14-1, 5'-ATGGGCGGGGCAAGGTGCA-3'; MADS14-2, 5'-TTAGCCGTTGATGTGGC TC -3'. 4. Agrobacterium-mediated gene transformation A single colony of Agrobacterium tumefaciens containing p1301-AP1-MADS14 was transferred into 5 ml LB medium (Luria Bertani Broth (Miller) 25 g/l and agar 8 g/l) containing 100 mg/l kanamycin and incubated at 28℃ for 2 days. The bacterial culture was then transferred into 20 ml LB medium containing 100 mg/l kanamycin and incubated at 28℃ for 2 days. About 4 hour before transformation, 200 mM acetosyringone (AS) was added to the bacterial culture. The Agrobacterium tumefaciens culture (A600 = 0.8 - 1.0) was centrifuged at 8,000 ×g for 10 min and the pellet was resuspended in 20 ml 1/4 MS medium. The PLBs were immersed in Agrobacterium cell suspension for 20 minutes and gently shaken on rotary shaker at 80 rpm in incubator at 28℃ to ensure that the entire PLBs is fully submerged for bacterial adherence onto PLBs. The PLBs were blotted dry on sterile paper and co-cultured on 1/4 MS medium plates without charcoal, but with 200 mM AS and incubated at 28℃ in darkness for 3 days. The infected PLBs were washed three time with 1/4MS medium containing sucrose 20 g/l, 1.1 g MS vitamins and 1 g/l tryptone at pH 5.2 and then after autoclave added 200 mg/l cefotaxime to prevent Agrobacterium tumefaciens overgrowth and then transferred to 1/4MS medium supplemented with sucrose 20 g/l, 1.1 g MS vitamins, 1 g/l tryptone and 8 g agar at pH 5.2 and 150 mg/l cefotaxime. The PLBs were cultured with a 16 h photoperiod (120 µmol m-2 s-1) at 25℃. Light was provided by cool white fluorescent lamps with an intensity of 120 µmol m-2 s-1. The PLBs were subcultured every 2 weeks for 1 month. After 1 month the transformed PLBs were transferred to a selection medium with sucrose 20 g/l, 1.1 g MS vitamins, 1 g/l tryptone, 8 g agar at pH 5.2 and 150 mg/l cefotaxime, 5 mg/l hygromycin for selecting putative transformants. The newly formed -84- green PLBs were further subcultured to fresh selection medium every 2 weeks for 2 months. After 2 months the PLBs were subcultured in 1/4MS medium without hygromycin for root formation. Regenerated plants about 1 cm in height with five to six leaves and three to four roots were eventually transferred to CN1 medium containing sucrose 20 g/l, 2.2 g MS vitamins, 1 g/l tryptone, 170 mg/l Na．H2PO4．H2O, 100 mg/l myo-Inositol, 200 ml coconut water, 1 g/l charcoal and 8 g/l agar at pH 5.2, after 3 months regenerated plants about 2-3 cm in height with 6-8 leaves and 4-8 roots were eventually transferred to pots containing sphagnum moss and acclimatized under greenhouse conditions. 5. Screening and selection of transgenic Erycina pusilla plants All transgenic plants selected on selection medium with sucrose 20 g/l, 1.1 g MS vitamins, 1 g/l tryptone, 8 g/l agar, 150 mg/l cefotaxime and 5 mg/l hygromycin at pH 5.2 for selecting putative transformants. To confirm the integration of the transgenes in the transgenic orchids positive for GUS histochemical staining (0.1 M Na-phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 1 mM X-Gluc, 0.5 mM K-ferrocyanide, 0.5 mM K-ferricyanide, pH 7.0) 6. Molecular analysis of transgenic Erycina pusilla plants The DNA extraction kit, Plasmid Miniprep Purification Kit (GMbiolab Co., Ltd. Taichung, Taiwan) was used to extract the genomic DNA from the sample. The extraction method was based on the protocol provided by the manufacturer. All amplification products stained with loading dye were separated on 1% (w/v) agarose gel. 7. Statistical Analysis Data were analyzed using one-way analysis of variance (ANOVA) using statistic version 8 and mean separation was conducted using Least Significant Difference test at p ≤ 0.05. Results 1. Verifying Plasmid Identity The genetic map of p1301-AP1-MADS14 (Fig. 1A), and digestion by restriction enzymes (Fig. 1B) are shown in Figure 1. The p1301-AP1-MADS14 was digested by BamHI/KpnI to produce 747 bp DNA fragment which was correspond to OsMADS14 gene, and by XbaI/KpnI to produce 1.0 bp fragment. The results of restriction-enzymes digestion indicated that p1301-AP1- MADS14 plasmid, contained the target genes in the right way and was ready for the transformation. -85- (A) (B) Fig. 1. The genetic map of p1301-AP1-MADS14 (A), and digestion by restriction enzymes (B).