Plant Responses to Different Growth Regulators (Hormones) in Tissue Culture

Transcription

1 Plant Responses to Different Growth Regulators (Hormones) in Tissue Culture (A Classroom Exercise) Plant growth regulators (sometimes referred to as plant hormones) induce various responses in plant pieces grown in the tissue culture environment Auxins (such as naphthalene acetic acid [NAA], indolebutyric acid [IAA], indoleacetic acid [IBA] ) tend to induce root growth Cytokinins (such as benzylaminopurine [BAP], kinetin, zeatin) tend to induce shoot growth When combinations of auxins and cytokinins are included in the plant medium, callus growth (a proliferation of unorganized cells), roots, shoots, and/or sometimes somatic embryos (embryos which develop from vegetative cells) will develop. This will depend on the species that is cultured, and the concentration and type of "hormone" that is used. For further information on this process, see: and Laboratory Exercise The following experiment was designed to demonstrate the effects that plant growth regulators can have on plant growth in tissue culture. These were designed to be used with a "Kitchen Culture Kit" customized kit and the manual, Plant Tissue Culture for the Classroom and Home (Stiff, 1998). This was designed for 4 lab sections. Each lab section has 6 teams. The following media will be prepared with different concentrations of the two plant growth regulators, BAP and NAA (others can also be substituted or added to the experiment): 57

2 Suggested Hormone Treatments Treatment/Hormone BAP/liter NAA/liter mg mg 4 2 mg 2 mg 5 2 mg 0.1 mg mg 2 mg Students will collect two African violet leaves per team. Disinfest and culture according to the Manual. See: http;// Observe cultures weekly, and compare your observations with the other teams and lab sections. A suggested format is at the end of this section. 58

3 Plant Responses/Hormones... Preparation Instructions for the Instructor! Have your students go on a "scavenger" hunt to find the materials needed for these experiments.! They will need to find: " 120 baby food jars (short or tall oz, with covers) " forceps (6) " kitchen knife (6) " 70% alcohol " bleach " dish detergent " vinegar " Tums or baking soda " 1 tablespoon measuring spoon " florists tape (try Michael's or Walmart) " pressure cooker or microwave " food coloring (optional) " dinner or salad plates (6) " pint jars for disinfecting plant leaves (18) " African violet plants (healthy) " quart jars (6) " table sugar (sucrose) " plastic box or other as recommended in the manual " protective clothing (goggles, gloves, dust mask, apron, shoes)! Prepare media: " Label 6 quart jars as 1, 2, 3,4, 5, 6 " To each jar, add - 4 cups water - 1 package MS basal medium - 2 tablespoons sugar - 1 vial of PPM (vial = 1 ml) 59

4 " Stir to mix " Add 2 mg BAP (1 vial) to jar #2, #4, #5 " Add 2 mg NAA (1 vial) to jar #3, #4, #6 " Add 0.1 mg BAP (specially marked vial) to jar #6 " Add 0.1 mg NAA (specially marked vial) to jar #5 " OPTIONAL: ADD FOOD COLORING TO DISTINGUISH MEDIA TYPES: - #2 - one drop blue - #3 - one drop red - #4 - one drop green - #5 - one drop yellow - #1 and #6 - no colors (or use 1 drop red + 1 drop yellow for #6) " Mix all well. Adjust ph of each medium to " Add 3 tablespoons media to each baby food jar + 1/16 tsp agar (use level Baskin-Robbins pink spoon or Gerber baby spoon). " Cap and process in the pressure cooker for minutes. " Remove from cooker, swirl to mix, and allow to cool.! Distribute 5 jars to each team. Culture as directed above. Record observations. 60

5 Suggested Format for Recording Observations No. Date Days in culture Swelling? Shoots or Roots? Callus? Contamination Other Observations? 61