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Post by Medina on Jul 5, 2011 19:18:16 GMT
tissue culture of Nepenthes growing in pots is difficult, for that I decided to try with it. After many experiments I founded a disinfection method that works for me: First, the explants are shot tips of Nepenthes plants, growing in pots with peat moss. This in one  And this the other  The explants were dissected, washed in detergent, soaked 1 min in ethanol, then 30 min in fungicide solution(trademark Benomil), rinsing with running tap water, then 15 min in 10 % NaOCL followed for 10 min in sterile water. After disinfection, the shot tips were placed in flasks with half strength MS medium. The results are in next photographs:    One month latter, four explants from three different Nepenthes species are growing and there are not signs of biological contaminants. |
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Post by endodoc on Jul 6, 2011 14:19:14 GMT
Medina
Thank you for sharing this information with the forum, a few questions, how much sugar in the media and did you use any PPM?
Thanks
Ed
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Post by Medina on Jul 7, 2011 13:51:27 GMT
Good point, I add 30 g/L of sugar and PPM is difficult to get it in México, I did not use it.
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Post by endodoc on Jul 7, 2011 17:07:35 GMT
Medina
Thank you once again and congratulations
Ed
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Post by coldcoffee on Jul 9, 2011 5:16:21 GMT
Medina, is ppm difficult to get because it can't be imported in?
Great pictures by the way. I will have to try this with my N. ventricosa. Have you tried with Kinetin?
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Post by Medina on Jul 11, 2011 1:41:04 GMT
Yes coldcofee, I do not how to get it. The next step with nepenthes is add growth regulators like BAP and ANA in different concentrations and may be Kinetin too.
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Post by coldcoffee on Jul 11, 2011 7:27:44 GMT
Very cool, have you checked out planttccases.org? It is a wikipedia style site for plant TC protocols. I hope you will consider contributing this to it. Also, have you checked out the home tissue culture group? You can order PPM from them in smaller quantities, but again if importation is an issue that might be a moot point. www.hometissueculture.org/catalogHTCGsupplies.htmBest of luck, I look forward to seeing your progress! |
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Post by Medina on Mar 25, 2012 21:41:27 GMT
Well, here is another shot tip of Nephentes raffelsiana growing in MS medium. Notice the two died leaves with the disinfection treatment.  Here is the same plant with another leave come up  now the plant is bigger than the beginning and show other leave.  |
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Post by lunauta on Apr 10, 2012 19:14:01 GMT
have you pictures of the nepenthes of the first post? still alive?
tks for the info.
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Post by Medina on Jun 21, 2012 20:51:24 GMT
Following with the nep work, the next photograph show N. raflesiana in vitro, from the shoot tip explant, growing in 1/3MS plus BAP 2 mg/L . The plant has more leaves and the lateral buds have begun to grow, they will be cut and put on in a new medium for get more plants.  Continuing with other Nep, I cut other shoot tip from N, sanguinea, next was disinfected and now it is growing in medium 1/3MS.  |
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Post by Medina on Aug 19, 2012 20:03:38 GMT
Continuing with micropropagation of N. raflesiana, the lateral shoots are cut off and were placed in flasks with 1/3MS where they are now growing  |
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coline
Full Member
Life's essence: patience
Posts: 484 
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Post by coline on Jan 2, 2013 4:59:58 GMT
Great tissue culture!, have you already tried to take some of the explants out and grow them as actual plants?
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coline
Full Member
Life's essence: patience
Posts: 484
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Post by coline on May 16, 2013 3:47:05 GMT
I tried the protocol and I may say that it indeed works with an efficiency of 70% or maybe even more , the varieties tried were ventrata and "gentle", both are growing really fast and recovering from dissinfection.
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coline
Full Member
Life's essence: patience
Posts: 484
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Post by coline on Jul 24, 2013 13:32:07 GMT
Well, I realized I never posted some pictures about the success, so, these were the plants just yesterday when I decided to multiply them: Ventrata  'Gentle'  What I did next is that I cut the shoots that each one had and placed them in medium, and they were already tall, I did micro cuttings and plated them in separate jars, so from 4 I had I got now 20 jars. |
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coline
Full Member
Life's essence: patience
Posts: 484
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Post by coline on Apr 5, 2014 0:53:34 GMT
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