Journal: Plant Molecular Biology

Title: ptxD gene in combination with phosphite serves as a highly effective selection system to generate transgenic cotton (Gossypium hirsutum L.)

Journal: Plant Molecular Biology Authors: Devendra Pandeya1, LeAnne M. Campbell1, Damar L. Lopez-Arredondo2, Eugenia Nunes3, Madhusudhana R. Janga1, Luis Herrera-Estrella4, and Keerti S. Rathore1,5* Affiliations: 1Institute for Plant Genomics & Biotechnology, Texas A&M University, College Station, TX, 77843- 2123, USA; 2StelaGenomics Mexico, S de RL de CV, Av. Camino Real de Guanajuato s/n, 36821 Irapuato, Guanajuato, México; 3Faculty of Science, The University of Porto, Porto, Portugal; 4Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, México; 5Dept. of Soil & Crop Sciences, Texas A&M University, College Station, TX, 77843-2474, USA. *Corresponding author: Keerti S. Rathore Institute for Plant Genomics & Biotechnology Dept. of Soil & Crop Sciences Texas A&M University College Station TX, 77843-2123, USA Email: [email protected] Phone: (979) 862 4795 Fax: (979) 862 3414

Online Resource 2

Culture Steps and Media to Select for bar Gene Expressing Events Composition of Culture Media Used to Obtain Transgenic Events Based on PPT Selection

1. P1-AS: 4.31 g/L MS salts (PhytoTechnology Laboratories, #M524), 100 mg/L myo-inositol, 0.4 mg/L

thiamine HCl, 5 mg/L N6-(2-isopentenyl)adenine (2iP), 0.1 mg/L α-naphthaleneacetic acid (NAA), 3%

glucose, 1 g/L MgCl26H2O, pH 5.8, 0.2% phytagel, 50 M acetosyringone (filter-sterilized) (Sigma-

Aldrich, Cat. #D134406).

2. P1-c4P6: 4.31 g/L MS salts (PhytoTechnology Laboratories, #M524), 100 mg/L myo-inositol, 0.4 mg/L

thiamine HCl, 5 mg/L 2iP, 0.1 mg/L NAA, 3% glucose, 1 g/L MgCl26H2O, pH 5.8, 0.2% phytagel, 400

mg/L carbenicillin (filter-sterilized), 6 mg/L phosphinothricin (filter-sterilized).

3. P7-c4P6: 4.31 g/L MS salts (PhytoTechnology Laboratories, #M524), 100 mg/L myo-inositol, 0.4 mg/L

thiamine HCl, 0.1 mg/L 2iP, 5 mg/L NAA, 3% glucose, 1 g/L MgCl26H2O, pH 5.8, 0.2% phytagel, 400

mg/L carbenicillin (filter-sterilized), 6 mg/L phosphinothricin (filter-sterilized).

4. MSEm: 2.68 g/L modified MS salts (PhytoTechnology Laboratories, #M571), 100 mg/L myo-inositol, 1

mg/L nicotinic acid, 10 mg/L thiamine HCl, 1 mg/L pyridoxine HCl, 1.9 g/L KNO 3, 825 mg/L NH4NO3, 2

g/L Glutamine, 500 mg/L Aspargine, 3% glucose, pH 5.8, 0.8% noble agar.

5. EG3: 2.16 g/L MS salts (PhytoTechnology Laboratories, #M524), 0.5% glucose, 100 mg/L myo-inositol,

0.4 mg/L thiamine HCl, 0.01 mg/L NAA, pH 6.5, 0.2% phytagel.

6. MS3: 2.16 g/L MS salts (PhytoTechnology Laboratories, #M524), 0.5% glucose, 0.14 mg/L thiamine HCl,

0.1 mg/L pyridoxine HCl, 0.1 mg/L nicotinic acid, pH 5.8, 0.08% phytagel, 0.6% BactoTM agar.

Culture Steps and Media to Select for ptxD Gene Expressing Events Composition of Media Used to Obtain Transgenic Events Based on Phophite Selection

1. P1-AS: 4.31 g/L MS salts (PhytoTechnology Laboratories, #M524), 100 mg/L myo-inositol, 0.4 mg/L

thiamine HCl, 5 mg/L 2iP, 0.1 mg/L NAA, 3% glucose, 1 g/L MgCl 26H2O, pH 5.8, 0.2% phytagel, 50 M

acetosyringone (filter-sterilized) (Sigma-Aldrich, Cat. #D134406)

2. P1-c4Phi: MS-Phi (1650 mg/L NH4NO3, 6.2 mg/L H3BO3, 332.2 mg/L CaCl2, 0.025 mg/L CoCl2.6H2O, 0.025

mg/L CuSO4.5H2O, 37.26 mg/L Na2-EDTA, 27.8 mg/L FeSO4.7H2O, 180.7 mg/L MgSO4, 16.9 mg/L

MnSO4.H2O, 0.25 mg/L Na2MoO4.2H2O, 0.83 mg/L KI, 1900 mg/L KNO3, 198 mg/L K2HPO3, 8.6 mg/L

ZnSO4), 100 mg/L myo-inositol, 0.4 mg/L thiamine HCl, 5 mg/L 2iP, 0.1 mg/L NAA, 3% glucose, 1 g/L

MgCl26H2O, pH 5.8, 0.2% phytagel, 400 mg/L carbenicillin (filter-sterilized).

3. P7-c4Phi: MS-Phi (1650 mg/L NH4NO3, 6.2 mg/L H3BO3, 332.2 mg/L CaCl2, 0.025 mg/L CoCl2.6H2O,

0.025 mg/L CuSO4.5H2O, 37.26 mg/L Na2-EDTA, 27.8 mg/L FeSO4.7H2O, 180.7 mg/L MgSO4, 16.9 mg/L

MnSO4.H2O, 0.25 mg/L Na2MoO4.2H2O, 0.83 mg/L KI, 1900 mg/L KNO3, 198 mg/L K2HPO3, 8.6 mg/L

ZnSO4), 100 mg/L myo-inositol, 0.4 mg/L thiamine HCl, 0.1 mg/L 2iP, 5 mg/L NAA, 3% glucose, 1 g/L