Plant Cell Growth Curve Materials

A well-established plant suspension cell line. The following procedures are based on the cultivation of tobacco cells. If another cell line is chosen, the medium can be modified. 

Murashige and Skoog salt mixture (Gibco), 2,4-dichlophenoxy-acetic acid (2,4-D), KH2P04, inositol, thiamine HC1, and sucrose to make medium 

Prepare modified Linsmaier and Skoog medium (Linsmaier and Skoog, 1965) which contains 4.3 g/L of Murashige and Skoog salt mixture, 0.2 mg/L 2,4-D, 0.18 g/L KH2P04, 0.1 g/L inositol, 1 mg/L thiamine EICL, and 30 g/L sucrose. Adjust pH to 5.8. with 1 N KOH and dispense the medium into two 125 mL Erlenmeyer flasks so that each flask contains 30 mL each. 

Plug the Erlenmeyer flasks and autoclave them at 15 to 18 psi for 20 minutes. 

Inoculate the flask containing 30 mL of medium with 1.5 mL of seven-day-old suspension culture and put it in a shaker incubator (150 rpm) at 27°C. Keep the cells dark by blocking the glass window of the incubator. 

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