B52 . . .

[BigBella]

Here is a pleasant surprise from the back of the shelf: a B52 culture that began as a late-season flower stalk back in August of 2011. It was grown in a simple media without any plant growth regulators (hormones) whatsoever; and I forgot about it until I was cleaning up this morning.

To give an idea of some of the advantages of tissue culture -- there is a second photo of a plant (about the size of a quarter), also produced from a flower talk (still visible in the shot), and of a similar age . . .


Dionaea muscipula cv. "B52" 15 February 2012

 

[amphirion]

sweeeeeeet.

 

[kulamauiman]

now if I could just get to prevent over sterilizing my explants to death....

 

[dionae]

Try using PPM to sterilize them kulamauiman.

 

[BigBella]

now if I could just get to prevent over sterilizing my explants to death....

Hey, it's PPM all of the way. I simply pinched off the flower stalk with my ratty, nasty fingernails and tossed it into an 8% solution of PPM to 1:3 MS (without sugar or pH adjustment); no bleach; no hydrogen peroxide; no muss, no fuss . . .

 

[richjam1986]

I've tried using clipped flower stalks as cuttings, no sucess yet.Your second picture gives me a better idea how to do it (thanks ) Would like to try TC. Maybe one of these days...

 

[thez_yo]

Look at those manly, beastly fangernails

You gonna make a huge VFT arrangement minibog to show off all the pretty plants you got? Hey did anything happen to those stalks I sent you - I don't remember. I do have other flower stalks right now that I can't bend over to pinch off so I think you should come over and take them off my plants in person

 

[mmlr38]

PPM is an excellent biocide though I've not had as much luck with it yet as I have with the traditional bleach method...most likely because of the amount of time spent working with each.

 

[kulamauiman]

PPM is an excellent biocide though I've not had as much luck with it yet as I have with the traditional bleach method...most likely because of the amount of time spent working with each.

I was going to try the peracetic acid again. Seemed like less hassles I did get nice well decontaminated capensis explants. Just over cleaned them....

 

[Peatmoss]

So cool! I have gotta try TC, it boggles my mind how fast the plants grow.

 

[jimscott]

So cool! I have gotta try TC, it boggles my mind how fast the plants grow.

My thoughts exactly!

 

[BigBella]

PPM is an excellent biocide though I've not had as much luck with it yet as I have with the traditional bleach method...most likely because of the amount of time spent working with each.

While I still use bleach solutions from time to time, especially with some "explants" grown outside, I generally reserve the PPM to sterilize sensitive seeds, such as those of Nepenthes; and, as a matter of fact, it was some of that leftover solution that I had used on the B52 flower stalk. I can generally re-use the PPM solution about six times (filtered after each use), before it declines or loses its efficacy . . .

 

[kulamauiman]

could one use dichloroisocyanuric acid in pace of bleach with similar times?

 

[BigBella]

could one use dichloroisocyanuric acid in pace of bleach with similar times?

Dichlor is very similar in action to bleach, and I have known some orchid growers who have used it on seed to some success; though I do not know of the preferred concentration or duration . . .

 

[DroseraBug]

Nice Work.

I use traditional bleach methods with explants and seed of several different carnivorous plant families. Would like to here more about your ppm methodology. For me, it is cost/benefit. If I have to use ppm (more expensive when you rule in concentration) to sterilize something I'm interested in propagating, yet, I can get comparable results with something as cheap as bleach or peroxide, I'll stick with traditional kitchen micropropagation. I do use ppm but minimally.

I'm definitely interested in hearing more about this because from my research, have not read defined methods (i.e. concentrations of ppm and methods used that work in tissue sterilizatinon with explants or tougher seed to sterilize such as Heliamphora/Nepenthes/Sarracenia).

Could you give a recommended concentration of ppm to use when sterilizing vft or pitcher plant seed?
Thanks for posting.

 

[BigBella]

Thanks . . .

Quite to the contrary, I have found PPM (Plant Preservative Mixture) economical to use for a number of reasons -- chiefly its very predictable success in the establishment of TC cultures, with no issues (in my experience) with either vitrification or phenolic bleeding when used with cuttings: an all-too-common occurrence with bleaching, hydrogen peroxide, and peracetic acid sterilization practices. Also, the lion's share of failures I have seen with those pursuing Nepenthes culture -- especially for the first time -- have always stemmed from over-bleaching. I cannot tell you the number of photos and queries sent my way in the last couple of years -- shots of all-too sterile cultures with very dead translucent seed; and considering the work I have done with some expensive material for clients over the years, PPM has proved to be highly predictable and consistent. There had been some discussion -- more lip-flapping than anything else -- that its use (either in media or simply as a disinfectant) could inhibit germination; but I have not found that to be the case (http://icps.proboards.com/index.cgi?board=tissue&action=display&thread=5073).

I currently use four and eight percent solutions of PPM (though I have experimented with others), mixed with thirty percent MS (regardless of what your final media will be) -- just the salts dissolved in distilled water -- no sucrose, PGRs (plant growth regulators), or pH adjustment. I generally make small batches, usually not exceeding 50 ml at a time (so only 2 ml of PPM is used for 4% and 4 ml for 8%). Both solutions can be reused upwards of six times (the mix filtered between use and kept refrigerated) For highland Nepenthes, four to six hours in 4% percent seems to be effective; for lowlanders, twelve hours or more in 8% percent (the seed typically being far more contaminated by algal and mold issues than their highland counterparts -- even if freshly-collected).

For Dionaea, Sarracenia, or Heliamphora seed (or leaves or rhizomes), I would treat them as though highland Nepenthes.

As an aside, my 2011 contamination rate with that methodology was 1.05% -- and there were hundreds of cultures . . .

 

[mmlr38]

While I still use bleach solutions from time to time, especially with some "explants" grown outside, I generally reserve the PPM to sterilize sensitive seeds, such as those of Nepenthes; and, as a matter of fact, it was some of that leftover solution that I had used on the B52 flower stalk. I can generally re-use the PPM solution about six times (filtered after each use), before it declines or loses its efficacy . . .

Right. PPM does seem to be very good for sterilization of sensitive seeds. It would probably work quite well for Venus fly trap flower stalks too since they're fairly easily cleaned and not super sensitive to over sterilization. But with leaves off flytraps in my greenhouse, I haven't had as much luck using PPM as opposed to bleach. But I've worked far more with bleach, have a method that works quite well for me, so I don't have the need to experiment with PPM to get something that works.

Quite to the contrary, I have found PPM (Plant Preservative Mixture) economical to use for a number of reasons -- chiefly its very predictable success in the establishment of TC cultures, with no issues (in my experience) with either vitrification or phenolic bleeding when used with cuttings: an all-too-common occurrence with bleaching, hydrogen peroxide, and peracetic acid sterilization practices.

Really? I've found that PPM creates more phenolic bleeding on Dionaea leaves than does bleach. But phenolic bleeding is fairly easily managed by replating/moving tissue around in the vessel.

 

[BigBella]

I've found that PPM creates more phenolic bleeding on Dionaea leaves than does bleach. But phenolic bleeding is fairly easily managed by replating/moving tissue around in the vessel.

That hasn't been my experience; and I do realize that phenolic bleeding can be managed -- but I am of the school of thought that, once a culture is plated, I don't want to have to deal with it for a few months. If bleach works consistently for you, by all means use it; it is just that I had the leftover PPM solution from seed sterilization on hand in the fridge on hand.

I still use the 10:1 water to bleach solution or peracetic acid for initial sterilization of lowland Nepenthes seed, which are notoriously difficult to clean. Even after being "nuked" with Clorox, I will occasionally have to "rescue" a culture a few weeks down the line . . .

 

[flasker]

In TC, I prefer 5 gram/liter NaDCC (Sodium DiChloroisoCyanurate) in 10 - 15 minutes in most cases for seeds and explants. In some cases, it doesn't work well with thick seed cover so I use bleach. Never try PPM so can't tell but I use Physan 20 for very sensitive explants. Will try with Nep when I have some viable seeds.

 

[BigBella]

Here are a couple of shots -- about five weeks out -- from the "flasking" of a late January Dionaea flower stalk, illustrating the "callus stage" of micro-propagation (TC), basically a wad of undifferentiated tissue, only now beginning to resemble leaves. The central dark mass in the first photo is the stalk itself. This was another culture that I had all but ignored -- and only noticed it this morning over coffee . . .

Dionaea muscipula cv. "B52" 2 March 2012