Lab 03

Control of organogenesis in cultures of Nicotianatabacum

Objective: To observe the effects of various levels of hormones on tobacco explants.

Protocol:

  1. You will be given a container with tissue from one or more sterile tobacco seedling and plates of the sterile medium you made in the first lab (eight plates total).
  1. Using sterile technique, and a sterile 1 mL or 250 uL pipette tip, punch holes from the tobacco leaf (approx. 18), avoiding the large veins in the leaf as much as possible. Make sure that all of the edges of the explants are cut. Make sure that tissue remains wet with MSO at all times.
  1. Using sterile technique, and a scalpel and forceps, experiment with cutting the tobacco leaves into other shaped (squares/triangles) about 1-2 cm2(approx. 3-5 pieces), avoiding the large veins in the leaf as much as possible. Make sure that all of the edges of the explants are cut. Make sure that tissue remains wet with MSO at all times.
  1. Make a few sections where one or more edges of the tobacco explant are uncut approx. 3-5 pieces). Make sure that tissue remains wet with MSO at all times.
  1. Put 3-5 explants, abaxial side up, into each plate (do not crowd) and seal the plates with Parafilm. Label each plate with your name, the date, and the experiment description.
  1. Experiment with placing some explants abaxial side downwards. Seal plates and label. All of these explants, as well as the experimentally cut explants should only be placed on the BA medium. One BA plate with normally cut explants will be placed in the dark.
  1. Examine your explants at weekly intervals and record your observations.

Contents of your 8 plates

Control / IAA / IAA/BA / BA
round explants / round explants / round explants / round explants
round/ dark
round/ upside down
square or triangle
uncut edges