Plant Cell, Tissue and Organ Culture

Supplementary materials for Plant Cell, Tissue and Organ Culture Article tile: Agrobacterium mediated Genetic Transformation of Miscanthus sinensis Authors: Ok-Jin Hwang 1, Mi-Ae Cho 1, Yun-Jeong Han 1, Yong-Min Kim 1, Soo-Hyun Lim 2, Do-Soon Kim 2, Ildoo Hwang 3, and Jeong-Il Kim 1* Affiliations: 1 Department of Biotechnology and Kumho Life Science Laboratory, Chonnam National University, Gwangju 500-757, Korea; 2 Department of Plant Science, Research Institute for Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea; 3 Department of Life Sciences and Biotechnology Research Center, Pohang University of Science and Technology, Pohang 790-784, Korea *Corresponding author: Jeong-Il Kim (kimji@chonnam.ac.kr) 1

Supplementary Table 1 Phenotypic characteristics of 7 germplasms of M. sinensis used in the present study Germplasm code Plant height (cm) No. of Stem (no./100 cm 2 ) Stem thickness (mm) Stem density (g/cm 3 ) SNU-M-022 197.0 3.3 a 15.0 2.9 a 4.19 0.18 a 0.28 0.01 b SNU-M-025 207.0 3.3 a 15.0 2.9 a 5.72 0.11 b 0.20 0.01 a SNU-M-032 275.0 5.8 d 15.3 4.3 a 7.96 0.47 d 0.54 0.04 d SNU-M-034 230.0 5.0 b 14.7 4.4 a 7.42 0.21 cd 0.67 0.01 e SNU-M-037 245.0 2.9 c 12.7 4.3 a 7.98 0.16 d 0.26 0.01 b SNU-M-045 245.0 2.9 c 18.0 3.5 a 5.54 0.15 b 0.36 0.01 c SNU-M-107 240.0 8.7 bc 16.3 4.8 a 6.98 0.09 c 0.37 0.01 c Average values of plant height, stem thickness and density are shown. Phenotypic characteristics were measured in the third year after planting their rhizomes. Means with different letters in each column are significantly different at P < 0.05, using Duncan. 2

Supplementary Table 2 Optimization of conditions for Agrobacterium inoculation and cocultivation Inoculation conditions No. of inoculated calli No. of calli with GFP expression (%) 5.2 150 5 (3.3) ph 5.5 150 2 (1.3) 5.7 150 2 (1.3) Acetosyringone ( M) 0 150 0 (0.0) 200 150 3 (2.0) 400 150 10 (6.7) Co-culture 3 days 150 3 (2.0) period 5 days 150 5 (3.3) The percentage was calculated using the number of transformed calli with GFP signal from all inoculated calli. 3

Supplementary Table 3 Optimized conditions for the Agrobacterium-mediated genetic transformation of M. sinensis Media Callus induction Agrobacterium inoculation Co-cultivation Selection Shoot induction Root induction Composition MS salts and vitamins, 3 mg L -1 2,4-D, 30 g L -1 sucrose, 750 mg L -1 MgCl 2 6H 2 O, 25 mm L-proline, 2 g L -1 Gelrite; ph 5.7 1/2 MS salts and vitamins, 3 mg L -1 2,4-D, 20 g L -1 sucrose, 10 g L -1 glucose, 400 μm acetosyringone; ph 5.2 MS salts and vitamins, 3 mg L -1 2,4-D, 20 g L -1 sucrose, 10 g L -1 glucose, 400 μm acetosyringone, 3 g L -1 Gelrite; ph 5.7 MS salts and vitamins, 3 mg L -1 2,4-D, 30 g L -1 sucrose, 750 mg L -1 MgCl 2 6H 2 O, 25 mm L-proline, 250 mg L -1 cefotaxime, 50 mg L -1 hygromycin (or 5 mg L -1 PPT for herbicide-resistant M. sinensis), 2 g L -1 Gelrite; ph 5.7 MS salts and vitamins, 2 mg L -1 kinetin, 30 g L -1 sucrose, 750 mg L -1 MgCl 2 6H 2 O, 25 mm L-proline, 250 mg L -1 cefotaxime, 30 mg L -1 hygromycin (or 3 mg L -1 PPT for herbicide-resistant M. sinensis), 2 g L -1 Gelrite; ph 5.7 1/2 MS salts and vitamins, 20 g L -1 sucrose, 750 mg L -1 MgCl 2 6H 2 O, 125 mg L -1 cefotaxime, 2 g L -1 Gelrite; ph 5.7 4

a LB T1 HYG P 35S T2 egfp P ubi RB SmaI BamHI HindIII b LB T1 BAR P 35S intron-gus T2 P 35S RB Supplementary Fig. 1 T-DNA regions of the binary vectors used for Agrobacteriummediated genetic transformation of M. sinensis. a pcambia1300 harboring egfp gene. Arrows indicate directions of transcription. HindIII and SmaI were used for the cloning of egfp gene cassette, and BamHI and HindIII restriction sites were used for DNA gel blot analysis. LB, left border; RB, right border; P 35S, CaMV 35S promoter; P ubi, Ubiquitin promoter; HYG, hygromycin resistance gene (i.e., hygromycin phosphotransferase II, HPTII); egfp, enhanced green fluorescent protein gene; T1, CaMV 35S terminator; T2, A. tumefaciens nos gene terminator. b pcambia3301. intron-gus, GUS coding region with a catalase intron insertion; BAR, phosphinothricin acetyltransferase gene.

a Callus induction (%) c Embryogenic callus (%) 100 80 60 40 20 0 50 40 30 20 10 0 ph5.5 ph5.5 ph5.7 3:0.1 3:0.05 3:0.01 3:0 2:0.1 2:0.05 2:0.01 2:0 1:0.1 1:0.05 1:0.01 1:0 2,4-D : BA (mg L -1 ) ph5.7 3:0.1 3:0.05 3:0.01 3:0 2:0.1 2:0.05 2:0.01 2:0 1:0.1 1:0.05 1:0.01 1:0 2,4-D : BA (mgl -1 ) 25 C 25 C b Callus induction (%) d Embryogenic callus (%) 100 80 60 40 20 0 50 40 30 20 10 0 ph5.5 3:0.1 3:0.05 3:0.01 3:0 2:0.1 2:0.05 2:0.01 2:0 1:0.1 1:0.05 1:0.01 1:0 ph5.5 ph5.7 2,4-D : BA (mg L -1 ) ph5.7 28 C 3:0.1 3:0.05 3:0.01 3:0 2:0.1 2:0.05 2:0.01 2:0 1:0.1 1:0.05 1:0.01 1:0 2,4-D : BA (mgl -1 ) 28 C Supplementary Fig. 2 Combinatory effect of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl-adenine (BA), ph and temperature on callus induction or embryogenic callus induction. Callus was induced from mature seeds on callus induction medium containing various 2,4- D : BA combinations and ph (5.5 or 5.7) at 25 C (a and c) or28 C (b and d). Four weeks later, the percentages of callus induction were calculated using the number of induced calli from seeds (a and b). After 8 weeks, the percentages of embryogenic callus induction were calculated using the number of embryogenic calli from all induced calli (c and d). Error bars represent standard errors of three replicates.

Geumo SNU-M-022 SNU-M-025 SNU-M-032 SNU-M-034 SNU-M-037 SNU-M-045 SNU-M-107 Supplementary Fig. 3 Comparisons of plant regeneration potentials in different germplasms. The regeneration potentials of 7 M. sinensis germplasms from Seoul National University (SNU) were compared with the commercially obtained seeds (Geumo). Embryogenic calli were cultured on regeneration medium for 4 weeks under white light condition. Embryogenic calli from different germplasms were differentiated into plantlets. Bar,1cm.

a c e g b d f h Supplementary Fig. 4 GFP expression of transformed calli. a-b Non-transformed calli included as a negative control. c-h Transformed calli with pcambia1300 harboring egfp gene. Embryogenic calli were inoculated with Agrobacterium suspensions, and co-cultured for 5 days on co-cultivation medium. After 6 weeks of incubation on selection medium containing 50 mg L -1 hygromycin, GFP expression on transformed calli (b, d, f, and h) was visualized under UV in dark room by using an Illumatool LT-9000 Bright Light system (Lightools Research, CA). Bar, 5 mm.

a b Supplementary Fig. 5 Comparison of shoot induction between hygromycin resistance and herbicide resistance selection markers. a Hygromycin resistance selection. b Herbicide resistance selection. 6 weeks after transformation, hygromycin-resistant or PPT-resistant calli were transferred onto regeneration medium containing 30 mg L -1 hygromycin or 3 mg L -1 PPT. Differentiation of transformed calli into plantlets were dependent on selectable markers used. Shoot induction on medium containing herbicide was delayed compared with that on medium containing hygromycin. These pictures were taken approximately 6 weeks after incubation on shoot induction medium. Bar, 1 cm.