Experiment 4 : Transformation Protocol for Tobacco (Nicotiana tabacum L. cv Samsun)

Transcription

1 Experiment 4 : Transformation Protocol for Tobacco (Nicotiana tabacum L. cv Samsun) Plant Biotechnology Resource & Outreach Center, Michigan State University Materials: 1. In vito-cultured, wild type tobacco plant (4-8 week old) 2. Agorbacterium strains Ach5 and LBA4404:pBI Tobacco regeneration medium (liquid and agar plate) Tools: Petr-dishes (00 mm x 15mm); Petri dishes with sterile filter paper; Forceps; Surgical blades (#10) and handles; 50-ml corning tube; parafilm; biohazard bags; permanent markers. Time-line: Streak out bacterial stock onto LB agar medium containing appropriate antibiotic 2-3 days 28 C Pick up and culture a single colony in liquid YEP (or LB) medium with the antibiotic. 2-3 days 28 C Co cultivation on regeneration medium (RM) 4 days in dark Selection on Selection medium: RM mg/l timentin mg/l Kanamycin (If nptii is the selectable marker) Subculture every 3 weeks Plantlets onto MS medium mg/l timentin mg/l Kanamycin 3 weeks Transfer plants to the glasshouse 12 weeks Harvest seeds Transformation Protocol: 1

2 Plant material: In vitro plants of Nicotiana tabacum L. cv Samsun Agrobacterium strain and plasmid: LBA4404: pbi121 Day 1 Prepare bacteria: Culture a single colony in liquid YEP mg/l Kanamycin and shaking at 300 rpm for 2 days at 28 C in the dark. Day 3 Transformation: Explants preparation: Take healthy fully expanded leaves from 4-5 week old tissue culture grown tobacco plant Cut into cm squares (or can use a cork borer, which is about 1.0 cm diameter). Transfer the leaf pieces into a 50-ml corning tube, which contains about 1 ml liquid RM. Bacterial suspension: Collect bacterial cells by a centrifuge at 2,500 xg for 1 min. Discard the liquid and suspend the bacterial pellet using liquid RM. Dilute the suspension to an O.D.600 of Infection: Transfer bacterium suspension to the corning tube with explants Incubate the leaf explants with bacterium at 28C with a gentle shaking at 100 rpm for 30 min. Pour off the bacterium suspension Transfer the explants, 20-30/petri-dish, to the plates with RM agar-medium covered with sterile filter paper (or onto two layers of sterile filter paper soaked with 3.5 ml liquid RM). Co-cultivation: 2

3 Co-cultivation was carried out at 25C in the dark for 4 days. Washing: Following co-cultivation, transfer the explants into a 50-ml corning tube. Washing the explants with liquid RM mg/l timentin for 3 min Rinse the explants with liquid RM twice (3 min/time). Selection and subculture: Blot dry the explants on sterile filter-paper Transfer the explants (abaxial surface of the explants in contact with the medium), 10 pieces/dish, to the agar plates with selection RM Culture the explants at 25C in the dark for 2 weeks followed by a culture at 16-h photoperiod Subculture the explants to fresh selection medium at a 3-week interval Plantlet formation: Transfer the whole explants together with the shoots to selection rooting-medium (MS mg/l Timentin mg/l Kanamycin). Culture the shoots at a 16-hr photoperiod for 3 weeks Transfer the rooted plants to soil in the greenhouse 3

4 Media and Solutions for Tobacco Transformation LB and YEB Media LB and YEB media Sink's Lab Date: LB YEB Bacto-tryptone 10 g Bacto-yeast extract 1 g NaCl 10 g Beef extract 1.8% Bacto-agar 18 g 18 g (agar plate) Peptone Sucrose PH Volume 1 L 1 L Autoclave 20 min 20 min YEP medium: 1 L Yeast extract 10 g Bacto Peptone 10 g NaCl 1.8% Bacto-agar 18 g ph=7.0 All antibiotics should be added when the temperature is lower than 60C Keep poured plates for 2 days at room temperature to visualise any contamination, then store at 4 C. 4

5 Regeneration Medium: (See the attached recipe) MS salts (products of Sigma or Phytotechnology Lab.) B5 Vitamins 30 g/l sucrose BA 2.0 mg/l NAA 0.2 mg/l ph 5.6 Bacto-agar 8 g/l for solid Autoclave For selection RM, when ready to pour (at about 60C) add: Timentin (300mg/mL stock) 1 ml/l= 300 mg/l (or use Cefotaxime) Kanamycin (100 mg/ml stock) 1 ml/l= 100 mg/l BAP (1mg/ml) (6-Benzylaminopurine) Add 1N KOH drop wise to 100 mg BAP until dissolved. Make up to 100mL with Milli- Q H2O Store 4 C NAA (1mg/ml) (Naphthalene acetic acid) Add 1N KOH drops to 100 mg NAA until dissolved. Make up to 100mL with Milli-Q H2O Store 4 C Timentin (300 mg/ml) Add 10 ml sterile Milli-Q H2O to 3g Timentin Filter sterilization through Millipore 0.22 μm Store -20 C Kanamycin Monosulfate (100 mg/l) Add 10 ml sterile Milli-Q H2O to 1g Timentin Filter sterilization through Millipore 0.22 μm Store -20 C 5