Assessment of PTXD gene as alternative selectable marker for Agrobacterium-mediated maize transformation

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1 Nahampun et al. ssessment of PTXD gene as alternative selectable marker for grobacterium-mediated maize transformation Hartinio N Nahampun 1,2, Damar Lopez-rredondo 3, Xing Xu 1,2,+, Luis Herrera-Estrella 4, Kan Wang 2,* 1 Interdepartmental Plant Biology Major, Iowa State University, mes, I US 2 Department of gronomy, Iowa State University, mes, I , US 3 StelaGenomics México, S de RL de CV, v. Camino Real de Guanajuato s/n, 36821, Irapuato, Guanajuato, México. 4 Laboratorio Nacional de Genómica para la Biodiversidad, Unidad de Genómica vanzada del Centro de Investigación y de Estudios vanzados del Instituto Politécnico Nacional, Km 9.6 carretera Irapuato-León, 36500, Irapuato, Guanajuato, México. + Current address: Zhejiang University, Hangzhou, China *To whom correspondence should be addressed: kanwang@iastate.edu Supplemental Materials Media composition Media composition for maize transformation was prepared as described by Frame et al. (2011), with a slight modifications for phosphite (Phi) selection. Similar to kill curve, N6 and MS salts for Phi selection did not contain phosphate (Pi) as specially formulated by PhytoTechnology Laboratories (Overland Park, KS). Complete media formulations for Hi II and B104 are presented in Table S1. Basic media composition is as follow: N6S: 4 g/l N6 salts; 1 ml/l N6 vitamin stock; 1.5 mg/l 2, 4-D; 0.7 g/l L-proline; 30 g/l sucrose; 0.5 g/l 2-(4-morpholino)-ethanesulfonic acid (MES); 5 mm silver nitrate; 100 mg/l cefotaxime; 100 mg/l vancomycin; 8 g/l purified agar; ph 5.8. N6S-P: 3.59 g/l N6 salts without phosphate; 1 ml/l N6 vitamin stock; 1.5 mg/l 2,4-D; 0.7 g/l L-proline; 30 g/l sucrose; 0.5 g/l MES; 0.5 mm potassium phosphite; 100 mg/l cefotaxime; 100 mg/l vancomycin; 5 mm silver nitrate; 8 g/l purified agar; ph 5.8. Supplemental document 1

2 Nahampun et al. MSS: 4.43 g/l MS salts; 1 ml/l modified MS vitamin stock; 0.5 ml/l dicamba; 0.7 g/l L- proline; 0.5 g/l MES; 100 mg/l casein hydrolysate; 100 mg/l myo-inositol; 30 g/l sucrose; 2.3 g/l Gelrite; 2 mg/l bialaphos; 88 mm silver nitrate; 250 mg/l carbenicillin; ph 5.8. MS-P: 4.3 g/l MS salts without phosphate; 1 ml/l modified MS vitamin stock; 0.5 ml/l dicamba; 0.7 g/l L-proline; 0.5 g/l MES; 100 mg/l casein hydrolysate; 100 mg/l myo-inositol; 30 g/l sucrose; 2.3 g/l Gelrite; 88 mm silver nitrate; 250 mg/l carbenicillin; ph 5.8. Supplemental Results Hi II and B104 kill curve kill curve experiment was designed to determine the optimal Phi concentration to use for selection to develop a system using the ptxd selection marker gene for the transformation selection. Maize immature zygotic embryos (IZEs) were aseptically dissected from ears of Hi II and B104. pproximately 25 IZEs/plate were cultured on the media without Pi and containing different concentrations of Phi. One control plate (N6S or MSS media) was prepared for each genotype. Hi II IZEs were grown on N6S media without Pi (Table S1) containing 0, 0.25, 0.5, 1, 1.5, or 2 mm of Phi. IZEs of B104 were grown on MSS media without Pi and containing 0, 0.5, 1.5, 2.5, 3 or 3.5 mm of Phi. Plates were cultured in the dark at 28 C for 2-4 weeks and callus initiation and growth was observed. Phi tolerance test Two events of transgenic Hi II callus were chosen from group, B and C. Non-transgenic Hi II callus was used as control. Calli were subcultured in six different media: N6S media; N6S with 1.5 mg/l bialaphos; N6S without Pi (N6S -P ) with 0 mm Phi; N6S -P with 1.25 mm Phi; N6S - P with 2.5 mm Phi; and N6S -P with 5 mm Phi. Plates were cultured in the dark at 28 C for 2 weeks and callus initiation and growth was observed. Supplemental Figure legends Fig. S1 Kill curve of Hi-II maize and B104 on media containing varying concentrations of phosphite (Phi). Immature zygotic embryos (IZEs) were cultured on media without phosphate and Supplemental document 2

3 Nahampun et al. containing varying concentrations of Phi. () Hi II IZEs on 0-2 mm Phi, (B) B104 IZEs on mm Phi, Control, plates on regular media containing phosphate and no phosphite. Fig. S2 Morphology of Hi II embryogenic type II callus on media containing different selection agents. Putative transgenic callus were transferred onto first selection 2 (S2) media for 2 weeks. () two putative transgenic events (-7 and -8) selected on bialaphos; (B) three putative transgenic events (B-5, B-6, and B-7) selected with Phi; (C) three putative transgenic events (C-4, C-5 and C-6) selected with Phi+Pi. Fig. S3 Morphology of B104 embryogenic type I callus on media containing different selection agents. () Putative transgenic callus selected on bialaphos after 2 weeks transfer onto the first selection 2 (S2) media; putative transgenic callus selected on (B) Phi and (C) Phi+Pi after 4 weeks transfer onto the first S2 media. Red circle indicates Type I callus. Fig. S4 DN PCR analysis of Hi II and B104 putative transgenic callus events. () Hi II putative transgenic events selected on media containing bialaphos (group ), phosphite (group B), and phosphite supplemented with phosphate (group C). Twenty two representative events were analyzed for the presence of the ptxd (1 Kb) and bar (~400 bp) genes. (B) B104 putative transgenic events generated from bialaphos (group D), Phi (group G) or Phi+Pi (Group H) selection systems. Data from 34 putative transgenic events. M, 100 bp ladder; P, plasmid expressing ptxd; WT, nontransgenic B73 maize; neg, H2O negative control. Fig. S5 Standard curve for qpcr Fig. S6 Transcript analysis of ptxd gene expression in T0 transgenic Hi-II maize plants. Reverse transcriptase analysis showed ptxd gene expression in all transgenic events selected with bialaphos (group ) and phosphite (group B&C); actin transcript is housekeeping gene control. M, molecular marker ladder; P, plasmid expressing ptxd; WT, non-transgenic B73 maize; neg, H2O negative control. Supplemental document 3

4 Nahampun et al. Table S1. Media composition for maize Hi II and B104 transformation using bialaphos or Phi as selective agents (modified from Frame et al., 2011) Supplemental Literatures Frame, B., Main, M., Schick, R., Wang, K. Genetic transformation using maize immature zygotic embryos. In: Plant Embryo Culture: Methods and Protocols. E. Yeung and T.. Thorpe (eds), Springer Science and Business Media, LLC. pp (2011). Supplemental document 4

5 Table S1. Media composition for maize Hi II and B104 transformation using bialaphos or Phi as selective agents (modified based on Frame et al., 2011). Genotypes Hi II B104 Selection treatment groups B C B, C B, C B, C D E, G F, H D E, F G, H D G, H Media Infection liquid (Hinf) Cocultivation (Hcc) Resting (N6S) (HS1) (HS1B-P) (HS1C-P) Selection 2 (HS2) Selection 2 (HS2BC) Pre-regeneration (PR) Pre-regeneration (PR-P) Regeneration 1 (R1) Regeneration 1 (R1-P) Regeneration 2 (R2) Infection (Binf) Cocultivation (Bcc) Resting (MSS) (BS1D) (BS1EG-P) (BS1FH-P) Selection 2 (BS2) Selection 2 (BS2EF-P) Selection 2 (BS2GH-P) Regeneration 1 (R1) Regeneration 1 (R1-P) Ingredients 4 g/l N6 salts, 1 ml/l N6 vitamin stock, 1.5 mg/l 2,4-D, 0.7 g/l L-proline, 68.4 g/l sucrose, 36 g/l glucose, ph 5.2. dd 100 µm acetosyringone after autoclaving. 4 g/l N6 salts, 1 ml/l N6 vitamin stock, 1.5 mg/l 2, 4-D, 0.7 g/l L-proline, 30 g/l sucrose, ph 5.8, 3 g/l Gelrite. dd 5 mm silver nitrate, 100 mm acetosyringone and 300 mg/l L- cysteine after autoclaving. 4 g/l N6 salts, 1 ml/l N6 vitamin stock, 1.5 mg/l 2, 4-D, 0.7 g/l L-proline, 30 g/l sucrose, 0.5 g/l 2-(4-morpholino)-ethanesulfonic acid (MES), ph 5.8, 8 g/l purified agar. dd 5 mm silver nitrate, 100 mg/l cefotaxime, 100 mg/l vancomycin after autoclaving. N6S, 1.5 mg/l bialaphos N6S-P (3.59 g/l N6 salts minus phosphorus) and no bialaphos, 0.5 mm KH 2 PO 3 N6S-P (3.59 g/l N6 salts minus phosphorus) and no bialaphos, 0.5 mm KH 2 PO 3, 0.05 mm KH 2 PO 4 N6S, 3 mg/l bialaphos N6S-P (3.59 g/l N6 salts minus phosphorus) and no bialaphos, 1.25 mm KH 2 PO g/l MS Salts, 1 ml/l MS* vitamin stock, 100 mg/l myo-inositol, 0.25 ml/l 2,4-D, 30 g/l sucrose, ph 5.8, 3 g/l gelrite. dd 2 mg/l bialaphos and 100 mg/l cefotaxime after autoclaving. PR-P (4.3 g/l MS salts minus phosphorus) and no bialaphos, 1.25 mm KH 2 PO g/l MS salts, 1 ml/l MS* vitamin stock, 100 mg/l myo-inositol, 60 g/l sucrose, ph 5.8, 3 g/l Gelrite. dd 3 mg/l bialaphos and 100 mg/l cefotaxime after autoclaving. R1 (4.3 g/l MS salts minus phosphorus) and no bialaphos, 1.25 mm KH 2 PO g/l MS salts, 1 ml/l MS* vitamin stock, 100 mg/l myo-inositol, 30 g/l sucrose, ph 5.8, 3 g/l Gelrite. dd 100 mg/l cefotaxime after autoclaving g/l MS salts, 1 ml/l MS* vitamin stock, 0.5 ml/l dicamba, 0.7 g/l L-proline, 68.4 g/l sucrose, 36 g/l glucose, ph 5.2. dd 100 mm acetosyringone after autoclaving g/l MS salts, 1 ml/l MS* vitamin stock, 0.5 ml/l dicamba, 0.7 g/l L-proline, 100 mg/l casein hydrolysate, 100 mg/l myoinositol, 30 g/l sucrose, ph 5.8, 2.3 g/l Gelrite. dd 88 mm silver nitrate, 100 mm acetosyringone, and 300 mg/l L-cysteine after autoclaving g/l MS salts, 1 ml/l MS* vitamin stock, 0.5 ml/l dicamba, 0.7 g/l L-proline, 0.5 g/l MES, 100 mg/l casein hydrolysate, 100 mg/l myo-inositol, 30 g/l sucrose, ph 5.8, 2.3 g/l Gelrite. dd 2 mg/l bialaphos, 88 mm silver nitrate, and 250 mg/l carbenicillin after autoclaving. MSS, 2 mg/l bialaphos MSS-P (4.3 g/l MS salts minus phosphorus) and no bialaphos, 1.25 mm KH 2 PO 3 MSS-P (4.3 g/l MS salts minus phosphorus) and no bialaphos, 1.25 mm KH 2 PO 3, 0.05 mm KH 2 PO 4 MSS, 6 mg/l bialaphos MSS-P (4.3 g/l MS salts minus phosphorus) and no bialaphos, 2 mm KH 2 PO 3 MSS-P (4.3 g/l MS salts minus phosphorus) and no bialaphos, 1.25 mm KH 2 PO g/l MS salts, 1 ml/l MS* vitamin stock, 100 mg/l myo-inositol, 60 g/l sucrose, ph 5.8, 3 g/l Gelrite. dd 3 mg/l bialaphos and 100 mg/l cefotaxime after autoclaving. R1-P (4.3 g/l MS salts minus phosphorus) and no bialaphos, 1.25 mm KH 2 PO 3 Regeneration g/l MS salts, 1 ml/l MS* vitamin stock, 100 mg/l myo-inositol, 30 g/l sucrose, ph 5.8, 3 g/l Gelrite. dd 100 mg/l cefotaxime after autoclaving. (R2) MS* vitamin stock: modified MS vitamins contain higher thiamine HCl and lower nicotinic acid concentrations compared to the standard MS vitamins (Frame et al., 2011).

6 Figure S1 0 mm Phi 0.25 mm Phi 0.5 mm Phi Control 1.0 mm Phi 1.5 mm Phi 2.0 mm Phi B 0 mm Phi 0.5 mm Phi 1.5 mm Phi Control 2.5 mm Phi 3.0 mm Phi 3.5 mm Phi

7 Figure S B-7 B-6 B-5 C-4 C-5 C-6

8 Figure S3 1 cm B 1 cm C 1 cm 1 cm

9 Figure S4. Hi II events ptxd (1 Kbp) bar (400 bp) M P WT neg M P WT Group M P WT neg M P WT Group B M P WT neg M P WT Group C B. B104 events ptxd (300 bp) 1D 2D M P WT bar (400 bp) 1D 2D M p WT 2D 3D 4D M P WT D 3D M p WT M P WT 4D M p 3D D D M p WT WT M P WT 3G-1 4G-1 3H-1 4H H M p WT 3G-1 4G-1 3H-1

10 Figure S Ct value y = x R² = Log concentration

11 Figure S6 Group 1000 bp M P WT neg ptxd (~300 bp) actin (~) Group B M P WT neg ptxd (~300 bp) 1000 bp actin (~) Group C M P WT neg ptxd (~300 bp) 1000 bp actin (~)

Agrobacterium-mediated transformation of Hi II immature zygotic embryos and recovery of transgenic maize plants

Agrobacterium-mediated transformation of Hi II immature zygotic embryos and recovery of transgenic maize plants Frame, BR, Shou, H, Chikwamba, R, Zhang, Z, Xiang, C, Fonger, T, Pegg, S-E, Li, B, Nettleton,