Tissue Culture Techniques

Media Preparation

1. Organize your work before you start. This will help eliminate wasted time. Determine the quantity of each medium and the amount of plant growth regulator needed for each experiment are determined before mixing medium. Make slightly more medium than is required. A media check off list is helpful in making medium to keep track of ingredients.

2. Remove media stock solutions and plant growth regulator stock solutions to be used from the refrigerator. Make up fresh stock solutions if adequate quantities are not available or if solutions have precipitated.

3. Plant growth regulators stock solutions are prepared biweekly and stored at 4º C in the dark. Cytokinins (thidiazuron and BA) are dissolved in approximately KOH prior the addition of deionized water. Gibberellins and auxins (2,4-D, IBA and NAA) are dissolved in 100% ethanol prior the addition of water.

4. Volumetric flasks are filled ½ to ¾ full with deionized water. Proper aliquots of stock solution and plant growth regulators are added and each item is marked on the check off list as used. Sucrose, 30 g 1-1, is added and stirred until dissolved. Flasks are brought to volume with deionized water and emptied into a larger beaker or Erlenmeyer flask.

5. The pH meter is calibrated with 7.0 and 4.0 pH buffers prior to adjusting medium pH. The medium pH is adjusted to 5.8 with 1 N KOH or HC1.

6. Sigma agar, 1 g 1-1, is added to the medium unless otherwise noted. The medium is placed on high heat and stirred until the agar dissolves. The medium appears translucent when the agar is in solution. CAUTION: there is only a slight difference between the temperature at which agar goes into solution and boils over. DO NOT LEAVE MEDIUM UNATTENDED. Do not heat in volumetric flasks. If the medium boils over, wait until the hot plates have cooled and clean off the hot plates with soapy water if necessary.

7. Proper aliquots are poured into culture vessels; test tubes contain 15 ml medium, glass baby food jars contain 25 ml and GA7s contain 50 ml. Culture vessels are capped and placed in the autoclave for 20 minutes at 121º C and 1 kg cm-2.

8. Autoclaved medium is stored on the clean medium racks in the laboratory. Avoid storing medium for more than 2 weeks, it is better to make fresh medium for successful, repeatable experiments. 9. Put away equipment, throw away trash, rinse glassware and leave the lab CLEANER than it was when you started. Special preparation for medium containing antibiotics and bialaphos

Medium containing filtered sterilized ingredients (antibiotics and bialaphos) is prepared differently since autoclaving may destroy antibiotic or balaphos activity.

1. Antibiotics and bialaphos are dissolved in water or phosphate buffer (pH 7.0) as indicated. Stock solutions are filtered sterilized through a 22 µm filter disk attached to a syringe into sterile glass baby food jars under sterile conditions in a laminar flow hood. Filtered sterilized stock solutions are stored in sterile baby food jars at 0º C in the dark.

2. Medium preparation is the same as above except after the addition of agar, the medium is directly placed into Erlenmeyer flasks and autoclaved for 20 minutes. Do not fill Erlenmeyer flasks more than 2/3 to 3/4 full with medium.

3. Medium is removed from the autoclave. When the temperature reaches approximately 60º C, filtered sterilized ingredients are added to the medium under sterile conditions in a laminar flow hood. After mixing, the medium is poured into sterile culture vessels.

4. Medium is stored at 4ºC in the dark for no longer than 2 weeks. Laminar Flow Hood Instructions

1. Organize your work before you start. This will help eliminate wasted time. Prepare media ahead, have plenty of sterile distilled water (if needed) and have all instruments, plant material(s) and/or cultures to be used assembled.

2. Hands and arms are thoroughly washed with an antibacterial soap prior to working in the hood and are occasionally sprayed with 70% ethanol while working in the hood. Note, alcohol will dry skin, therefore you may want to use hand cream when finished.

3. Ethanol (70%) is used to disinfest the laminar flow hood, culture containers, instruments and anything that is placed into the hood. WARNING: ethanol is VERY FLAMMABLE, so be careful around flame or Bacti-cinerators. Fire extinguishers are located near the hoods. Know the fire extinguisher location and how to use the fire extinguisher before beginning use in the laminar flow hood. If the fire extinguisher is used, clean up the mess afterwards.

4. Turn on laminar flow hood (if not already on) and spray all interior surfaces with ethanol. Place Bacti-cinearators and other electronic equipment (if used) into the hood. Avoid spraying electronic instruments with ethanol. Thoroughly spray instruments (i.e. forceps, scalpels), containers or anything else that will go into the hood with ethanol. Place sprayed items into the hood.

5. Forceps, scalpels and scissors are sterilized in Bacti-cinerators or a Steri-matic sterilizer for approximately 20 to 30 seconds. Bacti-cinerators should be glowing red inside before using. Also, make sure alcohol has evaporated before placing instruments into Bacti-incinerators. Instruments are placed on a disinfected stainless steel test tube rack for cooling. Sterilization of instruments is performed after each use to help ensure that cross contamination from other cultures would be prevented.

6. Plant material is dissected on a glass plate. The glass plate is sprayed with 70% ethanol and wiped with a clean Kimwipe prior to dissection. Do not wipe with Kimwipes alone, Kimwipes are not sterile and must be wetted with alcohol.

7. Equipment and culture vessels are placed in a manner as to assure a clear wide path between the working area and the filter surface. All tissue culture procedures, (i.e. explant initiation and transfers) are performed as close to the back of the hood as possible. Working near the hood edge is avoided. The closer you work to the outside hood edge, the greater the chance that you may contaminate your cultures.

8. Avoid talking, singing or coughing into the hood. Avoid touching items (and yourself, such as scratching your nose) that have not been sprayed with alcohol. If you are sick (cold or flu) avoid working in the hood if possible. This often lead to contaminated cultures.

9. To avoid contamination, do not rest instruments on the hood surface or contact any other surfaces once instruments are sterile. Work as quickly and efficiently as possible. 10.Once finished, clean up the hood and surrounding areas. Put away equipment, throw away trash, wash glassware and leave the lab CLEANER than it was when you started. If you poured media in the hood, clean up spilled media from the hood’s surface. Murashige and Skoog Stock Solution Preparation

These stock solutions are grouped together based on their compatibility. Nutrients in each stock solution are weighed, dissolved with deionized water and brought to one liter final volume.

Final Organics concentration Dispense 10 ml stock per liter medium mg 1-1

Myo-inositol 10.0 grams 100.0 Glycine 0.2 2.0 Nicoltinic acid 0.05 0.5 Pyridoxine HC1 0.05 0.5 Thiamine HC1 0.01 0.1

Phosphates Dispense 10 ml stock per liter medium

KH2PO4 17.0 170.0 H3B03 0.62 6.2 KI 0.083 0.83 Na2Mo04 ●2H2O 0.025 0.25 CoC12 ● 6H2O 0.0025 0.025

Nitrates Dispense 20 ml stock per liter medium

KNO3 95.0 1900.0 NH4NO3 82.5 1650.0

Calcium Dispense 20 ml stock per liter medium

CaC12 22.0 440.0

Sulphates Dispense 20 ml stock per liter medium

MgSO4 ● 7H2O 18.5 370.0 MnSO4 ● 7H2O 1.115 16.9 ZnSO4 ● 7H2O 0.43 8.6 CuSO4 ● 5H2O 0.00125 0.025

Iron Dispense 20 ml stock per liter medium

Na2-EDTA 1.865 37.3 FeSO4 1.39 27.8