To select for transformed plants, sterilized seeds were suspended in 0.1% sterile agarose and plated on kanamycin selection plates at a density of approximately 3000 seeds per 150 × 15 mm2 plate, cold-treated for 2 days, and then grown for 7–10 days in a controlled environment at 24°C under 23 h light 50–100 μEinsteins m-2 s-1. Selection plates contained 1/2X MS medium (Sigma Chemicals #M-5519), 0.8% agar (Sigma Chemicals #A-1296), 50 μg ml-1 kanamycin monosulfate. In some experiments, Petri plates and lids were sealed with surgical tape for the first week of growth. Excess moisture during growth was removed by briefly opening the plates and shaking moisture off the lid. Transformants were identified as kanamycin-resistant seedlings that produced green leaves and well-established roots within the selective medium. Some transformants were grown to maturity by transplanting, preferably after the development of 3–5 adult leaves, into heavily moistened potting soil.
2007-02-10
10:12:53
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1 answers
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mike
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