|
Post by flytrapcare.com on Jan 18, 2009 16:52:39 GMT -5
Hey hb, I too have some Nep seeds that I want to try soon. What is AHick's N1 formulation? I am guessing that it's different than 1/2 MS? Also, what will be your sterilization technique?
|
|
|
Post by hackerberry on Jan 18, 2009 21:41:59 GMT -5
Hi ftc, AJ Hick has developed this N1 formula for germinating Nep seeds in TC. members.cox.net/osp/osp/media/media.html. Nep seeds are tricky to sterilize but I read a couple of ideas that might work. One thing is you need to keep an eye on the seeds when you soak them in 10% bleach, once the seed coat gets a little lighter then you have to rinse it right away. hb
|
|
|
Post by lloyd on Jan 18, 2009 21:45:27 GMT -5
I like soaking in 4%PPM in MS for 4 hours to "sterilize".
|
|
|
Post by flytrapcare.com on Jan 21, 2009 10:46:13 GMT -5
Thanks for the link HB. I'll look into getting some of that. I'll also try to use 10% bleach for sterilization. Any idea about how long it takes for the seed coat to start turning?
lloyd, what is MS? Is that murashige & skoog? If so, I don't understand the technique. I understand soaking them in 4% PPm for 4 hours and then transferring them to MS media. Is that what you were saying?
|
|
|
Post by hackerberry on Jan 21, 2009 11:29:47 GMT -5
Hi ftc,
Some seeds will lighten their coat in a matter of seconds and some may take at least 5 minutes. But I would'nt try soaking them for 10 minutes. I will surely soak them overnight in 2% PPM.
|
|
|
Post by lloyd on Jan 21, 2009 14:17:25 GMT -5
I make a solution (10cc to save on PPM) of 4%PPM in full strength Murashige & Skoog. I soak seeds in it for 4 hours, then pop them right into the medium without rinsing. I also soak them in 250ppm Gibberellic Acid with a drop of Tween 20 for 24 hours before the decontamination.
|
|
|
Post by hackerberry on Feb 12, 2009 17:37:41 GMT -5
Lloyd, just a recap of your nep seed sterilization. How's the germination rate and did you get any contamination? I'm getting around 100 N. Viking seeds and don't want to put them into waste. Thanks!
hb
|
|
|
Post by lloyd on Feb 12, 2009 18:07:34 GMT -5
I'm having zero contamination so far with the PPM technique above. My only contamination is from "unshelled" VFT seeds, AFTER germination, which I am convinced is from systemic moulds inside the seed coat. Even then, after transferring from the contaminated bottle, I'm getting zero contamination. Note, I'm using 0.15% PPM in the medium. No nep germination yet.
|
|
|
Post by hackerberry on Feb 12, 2009 22:08:50 GMT -5
So here's an update after a month and after a replate. 1/2 MS + Vitamins for the media. The Gardenia Jasmine Explant VFTxDVG hb
|
|
|
Post by dvg on Feb 12, 2009 23:06:05 GMT -5
Wow! Those are incredible. And an explant too! You are getting to be very, very good at this tc stuff. Your vft's have a wee bit of variation in them? I checked again and one of the seed stalks I sent you came off of a vft clone from CZ Plants. Great work with those!
|
|
|
Post by lloyd on Feb 12, 2009 23:13:59 GMT -5
Great work with the seeds and particularly the explants. What sterilization techniques and medium additives did you use for the explants?
|
|
|
Post by hackerberry on Feb 12, 2009 23:33:02 GMT -5
dvg - Again, thanks for the kind words. I thought you're gonna try TC? And yes, those are the only bottles that I took pictures of but there are lots of vft variations. Big traps, pure red vfts, etc. We will see again in a month hehe. What clones are the vft from czplants? lloyd, the additives are the ones that you taught me. 1/2 MS, Sugar and I added 1/100 Murashige and Skoog vitamins. Sterilized in 20% bleach for 7 minutes and then directly sowing to medium. Got that technique from a friend who is a biochemist back home. Chat pays off hehe. hb
|
|
|
Post by hackerberry on Feb 13, 2009 8:12:35 GMT -5
Checked this morning and some of the 50+ cobra plant seeds are sprouting in TC. Thanks Flytrap for the seeds!
Hb
|
|
|
Post by flytrapcare.com on Feb 13, 2009 13:04:38 GMT -5
Hey HB,
Nicely done with all of the seeds and explants. Very impressive. I just made second run at TC on Tuesday. I think it went really well. I used explants from Dionaea and Dionaea seed and some sarr seed. I got the media consistency and pH perfect this time, so I was excited about that. I also went much easier on the explants compared to how I sterilized them in my first TC attempt. Currently, all of the tissue is still green, so that's encouraging. I'm guessing that my contamination rate will be pretty high, but I'm hopefully that I'll get at least a few to take without contamination.
QUESTION: How old are those seedlings in your photos?
I'm thinking that I need to replate the seeds that I sowed back in November. I think that I might have gotten the media too firm and for that reason, the seedlings are growing too slowly. I'm trying to compare the size of my seedlings to the size of yours in the photo and mine are minuscule in comparison. Mine are still teeny and they've been in vitro for nearly 4 months. Actually, the VFT seed just started germinating a couple weeks ago and they aren't growing much.
QUESTION: How do you replate? I'm concerned with damaging the seedling. What did you use to move them from one jar to another? Do you just gently pick them up with forceps?
|
|
|
Post by lloyd on Feb 13, 2009 13:49:20 GMT -5
I do the sterile stuff for replating even though I use PPM. Wash my hands with the alcohol gel stuff, spray the glovebox with alcohol, flame the forceps 3 times with alcohol. Then I put the forceps in a flask of alcohol, the old bottle and the new bottles in the glove box. Then I spray again. I open the new and old bottle. Take the forceps out of the alchol, flick the alcohol off. Then I very gently pick up the seedling and sink it into the new agar so it's a third into the agar.
|
|