Protocol Discussion

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polyakov

 
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Protocol Discussion

by polyakov » Tue Apr 18, 2017 8:02 pm

My sterilization protocol which I have used for conventional seed sowing. I wonder if this is not an overkill and would be still adequate for TC/aseptic germination?

In this batch I am going to start some Sundew seeds, passion fruit seeds, Heliamphora juvenile pitchers and vft.

1. Soaking in 5g/L Cane Sugar for 24-30 hours, I know Cane Sugaris better than refined white sugar (at least for people)
2. Rubbing alcohol which I have in 70% and 90% concentration Which one works best for you?
3. Tap water rinse
4. Soaking for 2-5 minutes in Sodium Hypochlorite diluted to 0.50-0.53% (branded as North Bright Bleach). For Nepenthes seeds I watch for color change, so time varies greatly. What's the trick with explants and other seeds?
5. Sterile water one minute soak
6. 2-3 minutes soak in Hydrogen Peroxide 3% or 5% What concentration would work best?
7. Sterile water one minute soak

For some bizarre reasons Carrageenan does not stratify enough for me! This is why I have to try Agar as gelling agent.

Formula: 1.5g/L MS (33%) + 30/L Cane Sugar + 7g/l Agar

I am planning to mix these all at once in distilled water in a beaker, heating at the same time to let Agar dissolve. Adjust Ph, stir well, wait five minutes, see if Ph is stable, then autoclave.

As for Ph adjusting I am planning to use two solutions that I prepared from powders which I got with my Ph meter.

Should I add components to the solution one after another instead? Also, I am questioning if I should heat it at all before autoclaving?

Thanks.
Best wishes,
Andrew

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