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Post by dToad on Feb 5, 2012 9:47:55 GMT -5
I mixed up the initial media: 1/3 MS + .5mg /l kinetin + .5 ml/l PPM + 30gm/l sugar + 7.5gm/l agar C206 + .5 ml/l PPM + 30gm/l sugar + 7.5 gm / l agar for 5 culture vessels each (200ml media), final pH adjusted to 5.5
Two cuttings were taken from the same typical VFT. They were sterilized using Frank Tromble’s technique (rinse, 5 min in 10% bleach + detergent, 3 X sterile water rinse.)
They were divided into 4 explants, and two were placed in each media type.
Now I wait for results.
Note: I've added PPM to the media for the initial division cultures to help kill off any remaining bacteria and fungus on the explants. Future subcultures will be started with known sterile plant material, and therfore will not have the PPM added to the media.
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Post by lloyd on Feb 5, 2012 20:09:06 GMT -5
If you add PPM to the medium, you can also sterilize with 4% PPM in MS. Also if you use PPM, you are possibly hiding mould which just isn't growing. Dividing and planting in non-PPM medium may allow hidden but live spores to contaminate the newer bottles.
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Post by dToad on Mar 13, 2012 7:30:46 GMT -5
Results to Date:
Batch 1 - explants taken 5 Feb C206 - vessel #1 blackend, vessel #2 white at both ends of explant (fungus?) 1/2 MS + Kinetin - vessel #1 blackend, vessel #2 showing several plantlets Note: Both 1/2 MS + Kinetin show some signs of oxidation
Batch 2 - explants taken 10 Feb 3 Vessels C206 + PPM, all still green no sign of plantlet development
Batch 3 - explants taken 14 Feb 3 vessels C206 no PPM, 2 discoloured but not black, one green, no sign of plantlet development
I'll keep all vessels going for at least 10 weeks.
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Post by lloyd on Mar 13, 2012 10:20:38 GMT -5
Congratulations on the plantlets. I'll have to try the 1/3 MS + .5mg /l kinetin someday. After all my explant failures, I came to the conclusion that CP's are super sensitive to phenolic oxidation either by hormones or by an interaction of PPM & hormones. I think I would try C216 which is without the 2iP with or without PPM or a lower concentration of PPM. Also try the de-contamination by PPM if you're using PPM instead of the other sterilization method. Keep the green explant ones and throw the rest out. You might try replating the darkened but not blackened ones.
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Post by dToad on Apr 1, 2012 5:10:01 GMT -5
After nine weeks, only one out of the ten VFT explants has multiplied. The plantlets are only about 3mm tall. I was expecting better, with the plants about 25-40 mm in size.
I've kept the TC vessels in my CP grow environment. I think that this may be a huge factor in the slow growth and the poor multiplication of the explants.
I'll build a TC incubator this week. My references call for an environment of 25°-27°C, with T8 or T12 warm white or GrowLux bulbs on 12" centers, 8" above the shelf. How does this compare with what you are using?
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Post by lloyd on Apr 1, 2012 20:19:48 GMT -5
I don't think the light/temperature is that big a deal. I found CP explants are hard-seeds are pretty "easy". I think you're doing well to get explants going at all.
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Post by dToad on Apr 15, 2012 8:06:20 GMT -5
I started building the TC incubator last Thursday. My back crapped out Friday, so it will be a few more days before I can get back to it.
I have all the components at hand now. The finished incubator will hold 50 baby food jars. I'll line the inside with an emergency blanket (available at Walmart for $4), as mylar does not seem to be available around here. The bottom, and lower portion of the sides will be covered with a cheap mirror for better wear resistance. Walmart had 4" table top fans for $10. I'll use one of these for air movement. A 100W incandescent bulb will provide the heat.
The hardest components to find were the 24" single tube T8 fixture, and bulb. I had to get the local Home Depot to order one in for me, and it was expensive at $40. When I unpacked it, I discovered that the ballast was rated for F32T8, F25T8, and F17T8. I could have bought a 48" fixture for half the price and cut it down to size. All of the local hardware stores carried only the cool white bulbs in the 24" size. Only the local nurseries and aquarium shops carry warm white or grow bulbs. They charge three or more times the price of the cool white bulbs of course.
Reading another board suggests that the optimal temperature for CP TC is 22°C.
I used 7.5 gm/l of Phytotech's A296 agar. I think this was too much, and kept the nutrients from the explants. My scale was a Lucky 7, and I'm not certain of its accuracy. I've got a 100gm / .01gm digital scale coming from the US with a set of calibration weights. I'll experiment a bit to find a sweet spot for the agar. I'll also do some PH testing to find the effect of agar on PH. I'll start with water at a PH of 5.7 for all experiments.
I had hoped to have a bunch of VFTs ready for the flea markets this summer. It looks as though this will be delayed until August at the earliest, if I make it at all this year. Oh well, Murphy strikes, and no plan survives contact with the enemy.
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