Post by blackboxstar on Oct 3, 2011 2:09:55 GMT
Yesterday I attempted to Tissue Culture for the first time. Here are the methods I used. I would love feedback and I will post updates as things begin to succeed or fail.
Preparation of Materials
Wash and dry baby food jars. Add one drop of food coloring to each jar.
Gather together all of the materials that you will use that need to be sterilized. Place near the pressure cooker.
Media Preparation
1/8 c. table sugar
1 3/8 c. distilled water
1/8 c. stock solution (miracid diluted 1/4 t to 6 c. distilled water)
1/2 inositol tablet
1/4 vitamin tablet with thiamine (Great value one a day for women)
1 T agar
1/4 mL ppm (in the end the concentration of PPM is .5 mL per L or .5%)
Combine sugar, water, stock solution, inositol, vitamin, and ppm. Stir until combined. Test pH and adjust with baking soda or vinegar until pH is 5.6-5.8.
Bring mixture almost to a boil and add the agar. Stir until melted and dispense into baby food jars (roughly 50 mL per jar)
Cap with Phyto-caps and place in pressure cooker. Toss in equipment for sterilization (forceps, wire loop, beakers, etc) Process at 15 PSI for 15 minutes.
Seed Sterilization
We are working with six different seed species. I numbered them.
1. Cephalotus
2. S. alabamenses
3. S. leucophylla
4. D. menziesii
5. D. binata
Each species was separated into three batches and bagged in emptied out teabag bags. Then, for the sake of knowing which seeds were which, I used a sharpie to label them with the species number.
I choose to use colors of media to differentiate between sterilization methods in the final product. Each species would have two jars of seeds for each of the three methods for an end total of six jars per species.
I choose these three methods after reading the book Plants from Test Tubes and the postings of several carnivorous plants propagators from the various corners of the internet. They each came recommended and hopefully they will increase my odds of succeeding with each species.
Red:
10 min. in 20% bleach 80% distilled water with two drops of dawn liquid dish detergent
15 min. in boiled distilled water.
Yellow:
5 min in 50% Isopropyl Alcohol with 2 drops of dawn liquid dish detergent.
3 min. in 10% bleach 90% distilled water
1 1/2 min. in H2O2
Blue:
Quick dip in 50% Isopropyl Alcohol
15 min. 1% bleach 99% distilled water
rinse in boiled distilled water
Divide the 15 packets of seeds into three batches each containing one of each number (1,2,3,4, and 5) Safety pin them together and process.
Sowing
I worked inside of a ten gallon tank that had been soaked in bleach water and then wiped down with Isopropyl Alcohol and turned on its side. I placed a garbage bag (rubbed down with Isopropyl Alcohol) in front of the opening whenever I was not using it.
I wore a basic face mask to limit the air movement from my breath.
I dipped my tools (forceps, wire loop, wire hook) in isopropyl alcohol and then flamed them off with a sterno can style alcohol burner. Then I dipped the hot metal into the agar in hopes that some agar would stick to the metal and make it stickier for grabbing seed (no such luck). Eventually I managed to sow all of the seeds.
After finishing all of the jars I realized that i still had 5 unused jars. I sterilized some extra Ping seeds using red method. And took two tissue samples each from S. purpurea and S. minor and attempted to sterilize them thusly:
5 min. washing in water with dawn
20 min. rinsing in lukewarm tap water (changed often)
Quick dip in 50% Isopropyl Alcohol
3 min. 3% H2O2 (agitated)
6 min. 20% bleach with 2 drops of Dawn (agitated)
5 min. boiled distilled water
Notes
I found that the agar was rather hard, I plan to use less agar next time. Perhaps I will experiment with the agar and find concentrations that work best.
What do you think?
Preparation of Materials
Wash and dry baby food jars. Add one drop of food coloring to each jar.
Gather together all of the materials that you will use that need to be sterilized. Place near the pressure cooker.
Media Preparation
1/8 c. table sugar
1 3/8 c. distilled water
1/8 c. stock solution (miracid diluted 1/4 t to 6 c. distilled water)
1/2 inositol tablet
1/4 vitamin tablet with thiamine (Great value one a day for women)
1 T agar
1/4 mL ppm (in the end the concentration of PPM is .5 mL per L or .5%)
Combine sugar, water, stock solution, inositol, vitamin, and ppm. Stir until combined. Test pH and adjust with baking soda or vinegar until pH is 5.6-5.8.
Bring mixture almost to a boil and add the agar. Stir until melted and dispense into baby food jars (roughly 50 mL per jar)
Cap with Phyto-caps and place in pressure cooker. Toss in equipment for sterilization (forceps, wire loop, beakers, etc) Process at 15 PSI for 15 minutes.
Seed Sterilization
We are working with six different seed species. I numbered them.
1. Cephalotus
2. S. alabamenses
3. S. leucophylla
4. D. menziesii
5. D. binata
Each species was separated into three batches and bagged in emptied out teabag bags. Then, for the sake of knowing which seeds were which, I used a sharpie to label them with the species number.
I choose to use colors of media to differentiate between sterilization methods in the final product. Each species would have two jars of seeds for each of the three methods for an end total of six jars per species.
I choose these three methods after reading the book Plants from Test Tubes and the postings of several carnivorous plants propagators from the various corners of the internet. They each came recommended and hopefully they will increase my odds of succeeding with each species.
Red:
10 min. in 20% bleach 80% distilled water with two drops of dawn liquid dish detergent
15 min. in boiled distilled water.
Yellow:
5 min in 50% Isopropyl Alcohol with 2 drops of dawn liquid dish detergent.
3 min. in 10% bleach 90% distilled water
1 1/2 min. in H2O2
Blue:
Quick dip in 50% Isopropyl Alcohol
15 min. 1% bleach 99% distilled water
rinse in boiled distilled water
Divide the 15 packets of seeds into three batches each containing one of each number (1,2,3,4, and 5) Safety pin them together and process.
Sowing
I worked inside of a ten gallon tank that had been soaked in bleach water and then wiped down with Isopropyl Alcohol and turned on its side. I placed a garbage bag (rubbed down with Isopropyl Alcohol) in front of the opening whenever I was not using it.
I wore a basic face mask to limit the air movement from my breath.
I dipped my tools (forceps, wire loop, wire hook) in isopropyl alcohol and then flamed them off with a sterno can style alcohol burner. Then I dipped the hot metal into the agar in hopes that some agar would stick to the metal and make it stickier for grabbing seed (no such luck). Eventually I managed to sow all of the seeds.
After finishing all of the jars I realized that i still had 5 unused jars. I sterilized some extra Ping seeds using red method. And took two tissue samples each from S. purpurea and S. minor and attempted to sterilize them thusly:
5 min. washing in water with dawn
20 min. rinsing in lukewarm tap water (changed often)
Quick dip in 50% Isopropyl Alcohol
3 min. 3% H2O2 (agitated)
6 min. 20% bleach with 2 drops of Dawn (agitated)
5 min. boiled distilled water
Notes
I found that the agar was rather hard, I plan to use less agar next time. Perhaps I will experiment with the agar and find concentrations that work best.
What do you think?
