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Crissytal

What is and what should never be
I've been playing around with tissue culture for the last couple months. It's an interesting hobby, I really enjoy it. So far I have had little success with explants. Either I get contamination or I kill the tissue. I haven't found the happy medium between the two yet.

All these are grown from seed on 1/2 MS, no hormones.

Drosophyllum lusitanicum
IMG_5036.jpg


S. purpurea
IMG_5038.jpg


D. regia
IMG_5040.jpg


For fun, Siam Ruby banana meristem (1/2 MS, 5mL/BAP per liter) This media didn't gel properly due to the hormones. When using hormones, use more agar!
IMG_5042.jpg


My setup, I've been at it for awhile. These are from all different dates. Not a whole lot is happening yet.
IMG_5043.jpg


Anyone whom is interested in TC, read. Read any and all information that you can find. Some of the information will be good, some won't. Due to bad information, the first two batches of media that I made failed completely because it didn't gel correctly. Some things will work well for you while others will not. Also, start with seed. There's nothing more disappointing than doing all the work, waiting a week to three and start to see contamination.

The next experiment in getting explants to work is starting the leaf cuttings in distilled water (Pinguicula and Drosera in particular) and using the material that sprouts off of it. In theory this should be cleaner than using a leaf right off the plant. Only time will tell. I have some plantlets forming from roots in bags with moist Sphagnum. I'm in the process of trying this.

Crystal
 
Hooray! That's very encouraging. I've been sweet talking some of the bio professors around my campus lately - I'm hoping that I can sit in on some TC labs if I play my cards right. Best luck!
~Joe
 
Very nice Crystal! Now I remember you saying some time ago that you will try TC, but I see you really got started big:) I totally agree with what you say about starting TC. I started with leaf explants myself, what made the beginning even more disappointing, since the seeds are much easier to put to work on TC. But the spirit is not to give up on the very first failure. Patience and knowledge is the key.Now I think I got the hang of it at least as far as sundews are in play. It is really trial-error approach that works here. Each species needs slightly different media and sterilization protocol, what might work for some will kill other. It is very interesting and I am glad you enjoy experimenting with this! Good luck and I wish you lots of success! Please, let me draw your attention to this new website my friend designed purely for CP-TC experiments. He got it up and running just recently, so there is not a whole lot of info right now, but later it will be a great help for TC enthusiasts: http://www.labflytrap.com/forum/index.php enjoy! :)
 
Outstanding, you're looking very professional there Crystal...

Back in the 70's when i was just a wee bit on the wild side I did a little TC'n.
However, my focus was more fungal in nature..

Go ask Alice,
:0o:
 
/\ lol!!

But looking good Crystal! I've been meaning to bug you for an update but those seedlings are looking mighty fine!
 
Joe: I would certainly try to sweet talk those professors. That would be a great resource to have. Those are resources that I don't have unfortunately. Thanks and good luck to you as well!

klasac: Thank you! :) Yes, I'm serious about getting it to work. I think I've made it through the most discouraging stuff. I have some stuff growing now, so I'm hooked! I tried to start with explants too. None of them made it. I'm still experimenting with it. So far all I've been able to grow from tissue is U. bilboa from stolon cuttings (and the bananas go figure). It's better than nothing though! I agree, patience and knowledge are the major keys in having success. I went through 3-4 batches before I finally got it right. There's so much stuff that's not mentioned in literature that you just have to figure out for yourself. It really is trial-error which I can see putting a lot of people off on the first couple of tries. Thank you! I wish you the best of luck as well. Thanks for the link! I actually just emailed Michal about some of his plants he has in TC. I was a little disappointed to find out he uses seeds. I was hoping for some tips on getting explants into TC. Are you still experimenting with it? Also, do you happen to know a good time frame to move seedlings into media with hormones to increase multiplication?

Av:
When men on the chessboard
Get up and tell you where to go
And you've just had some kind of mushroom
And your mind is moving slow
Go ask Alice
I think she'll know

JP: Thanks! I'm still at it. I've come a long way in the past couple of months. It's okay, as you can see I'm not around on facebook very much. I'm lucky if I log in once a week LOL. FB just isn't my thing :).
 
Awesome setup. You and I have the same grow rack. Of course, mine's down in the basement in pieces and unused at the moment, but when I'm able to get everything back together I'll probably try TC as well.

Congrats on your strikes. :)
 
Veronis: Thanks! Yes, I'm using the popular Lowes rack that used to be cheap. They aren't too cheap anymore if you don't catch them on sale. You should get yours back together and put it to use! Racks don't stay empty long around here ;). You should try TC. It's a very fun hobby.
 
i know nothing about this stuff so pardon my intrusion but the setup u have looks like a drug lab :-))

Ps.. i wach a lot of cops, csi, etc :)
 
  • #10
Yes, Cristal, there is so much stuff you will not find in the literature and sometimes is vital for the success in TC. Michal does have most of his plants in vitro from seeds, but not all of them. I think about 30% comes from direct organogenesis. As far as I am concerned, I still try to find propagation protocols using other material than seeds. The reason is simple- some seeds are very hard to get. If you know how to propagate leaf/flower stalk/root explants you have unlimited amount of plant material at hand:)
You asked about the perfect time to deflask seedlings onto subcultre hormonal medium. Well, I put most of my TC seedlings or plants directly in regular substrate. The optimal time varies. For example, I got seeds from this mother plant of ascendens
DSCF0016-3.jpg

When she produced decent amount of fertile seeds in the summer, I thought of using TC instead of regular sowing to propagate this plant quickly (I love this species:) ). They sprouted for me in 10 days,but some time later the seedling started dying due to overheating in my absence. When I returned I had to act promptly to save last 5 last seedlings (out of 40). So I had to deflask them prematurely. Out of 5 I think 4 will survive:
DSCF0003-14.jpg


With d. burmannii I could keep the seedlings maxinum of 2,5months in the jar, then it was too crowded and I had to deflask them, they look happy now:
DSCF0007-13.jpg


Drosera anglica I propagate from leaf explants now I know how to do it, so it is easy. Even WITHOUT hormones I got multiplication ratio of 14 new plants per leave. After 2 months in TC i deflasked the rootless plants on substrate where they rooted spontanneously:
DSCF0005-6.jpg


But of course, I always keep a back-up sterile material in subculture. You can do this at anytime, as long as the size of the plant allows it.
For example, part of burmanni seedlings I put on hormonal medium into subculture. So far (20 days) I see no propagation via morphogenesis, the plants keep growing and flowering as in ordinary substrate:
DSCF0008-1.jpg


On the other hand, plantlets from leaf explants of d. anglica, put onto hormonal medium, started to form adventitious buds on leaves again. I subcultured 5 plants. With the thought in mind that hormones increase propagation ratio per leave, I should have this jar soon filled with plants:
DSCF0001.jpg


So the best way to make TC is to start from seeds. It is easier. When you have grown seedlings, you can use their sterile tissue for subcultures via other parts of the plant, with no neccessity of annoying sterilization! Since some seeds are hard for me to get (or sprout), I keep on trying other parts. I recently managed to initiate flower stalk of d.venusta and leaves of d.rotundifolia and d. collinsiae 'Faryland'. I keep on trying it is fun:)
 
  • #11
Carnivorous Blake has had some experience with TC. So has Jason Koch.
 
  • #12
Drew: The government probably thinks I do too ha. I had to order some lye recently to break down some hormones. I'm sure I've been flagged.

klasac: Wow! Thank you for all of the detailed pictures and information! That's why I am so interested in TCing explants, I have almost an unlimited amount of tissue while seed is more difficult to come by. I just did some P. 'Titan' flower stalks. So far so good. It's only been a few days though. If I can only ever figure tissue out. Thanks for the deflasking information. I'd probably be better off letting the seedlings grow and get the tissue from them instead of moving the entire seedling. At least that's what I'm thinking at this point.

Beautiful ascendens! I was lucky enough to get ahold of some seeds. I have a few popping up now (not invitro yet). Good luck with your remaining 4/5 seedlings. I hope they do well for you! The D. burmannii and D. anglica have done amazing. Good luck with your experiments! I will continue to experiment as well.

Jim: Thanks!
 
  • #13
Thanx, Crystal!
Of course, you dont need to use the whole sterile seedling/grown plant. I only like to do it this way, because you can almost immediately see, how the actual whole plant reacts to the medium with hormones (flawless growth vs. slow growth or etiolated leaves, etc)...and, at the same time, the propagation is in progress :) If the plant complains about the hormones you see it and next time use other mixture/ratio/hormone type.
Thanx for the compliments on ascendens, it is an old big plant and produces seeds for me:)
I recently got my hands on ascendens seeds, the red form, which is the biggest of all. Cant wait to see the plants sprout.
Good luck on your seedlings and plants! :)
 
  • #14
Hi Crystal! (and others, of course)! I got some new CP material sprouting:
localised D. spatulata seedlings (no hormones):
DSCF0003-1.jpg


d.nidiformis seedlings (no hormones):
DSCF0009-1.jpg


d.collinsiae 'Faryland' (2 leaf explants on media with NAA and Kinetin) , swollen and producing minute adventitious buds:
DSCF0019.jpg


I am particularly happy for the former one, since leafs are hard to make work sometimes. Will report back with more such as d.tokaiensis, d. affinis and others:)
 
  • #15
Hey klasac!

Very nicely done!! So many germinations! Looks like everything is doing very well for you. This might be a silly question, what happens if seed are started on media with hormones? Every reference I have found simply says not to with no reason. Does it inhibit germination?

Congrats on the explant success! I haven't figured it out yet. Lately I've been getting the material sterile, but also killing the explants. Back to the drawing board lol. What is your procedure on the D. collinsiae leaves if you don't mine me asking? Did you do anything ahead of time, like fungus treatment or anything before beginning the sterilizing procedure?

Here are some of my updates (sorry for the picture quailty, taking pictures of seedlings inside jars isn't easy LOL):

D. collinsae
collinsae.jpg


D. felix
felix.jpg


VFT seedling
vftseedling.jpg


Drosophyllum
dewypine.jpg


Gemmae to see if it could be done:
D. roseana
roseana.jpg


D. pulchella x occidentalis
pulchellaxocc.jpg


I'm looking forward to your updates on D. tokaiensis, D. affinis and your others!
 
  • #16
very cool to see both of your tc projects. I'm starting to get jealous...
 
  • #17
Very nice pictures, Crystal!
I see you try everything, even gemmae:) Well not everything is going well for me, but some things do work out fine:)
Hormones are generally not recommended for TC seed germination (other than GA3 for seeds needing stratification). I only make some experiments with hormones and 'weedy drosera seeds', to see the effect on germination time and also further growth. Using hormones for seed germination (mainly cytokinins) can cause undesirable growth defects.
For leaves, I use hormones most of the cases. NAA and BAP or kinetin in low ratio. D. collinsiae i found is kind of easy on TC. I see you have seedling plant in TC , so later you can use its tissue on hormonal media without troubling yourself with sterilization protocol! :) Good luck, it looks great so far! :)
 
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