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View Full Version : My home TC lab is open, Finally!


Chironex
10-04-2008, 07:40 PM
:woohoonaner: Just finished setting up a few things and starting my first culture today, an embryo rescue is first on my agenda.

The flow hood is in my photo gallery along with some pics of the recently received starts.

Will continue to post progress reports as more gets going.

xavierdlc61887
10-04-2008, 07:43 PM
nice man hey did u ever send the seeds cause i never got anything...if not well its cool thanks anyway

chong
10-04-2008, 07:53 PM
Congratulations, Scot!!!!

TomK
10-04-2008, 08:19 PM
Very cool. Looking forward to watching your progress as I slowly accumulate my lab. :)

Gabe15
10-04-2008, 08:28 PM
Looks good. Before you start trying to culture anything, I would recommend going through your normal procedure of making your medium and putting it in the vessel, and with the hood on, leave the vessel open in there for about 10min or so, then seal it again. If after a few days you don't see any contamination, you should be good to go. If you do see contamination, then check over your whole setup and methods to try to find out where it is coming from. Even with a professional setup and experience, contamination can be a problem and it would be good to make sure you are at least capable of having a truly sterile environment before trying to TC anything rare.

Chironex
10-04-2008, 08:33 PM
Looks good. Before you start trying to culture anything, I would recommend going through your normal procedure of making your medium and putting it in the vessel, and with the hood on, leave the vessel open in there for about 10min or so, then seal it again. If after a few days you don't see any contamination, you should be good to go. If you do see contamination, then check over your whole setup and methods to try to find out where it is coming from. Even with a professional setup and experience, contamination can be a problem and it would be good to make sure you are at least capable of having a truly sterile environment before trying to TC anything rare.

Good advice, Gabe. Will do. I set up some petri dishes with M&S medium in them just for that purpose since reading your post.

Chironex
10-04-2008, 10:01 PM
OMG, I just roasted some petri dishes. Check out my gallery. Well now I know.

JCDerrick
10-05-2008, 12:30 AM
LOL, I saw those at the top of the forum and was like "Uh oh"

Your set up looks great, I wish I had the time to try my hand at TC's. For now I'll have to settle for the pups.

Chironex
10-05-2008, 06:13 PM
Gabe, the agar is clear so far, so I am hoping to have aseptic conditions in the hood. Wish me luck!!! I just went ahead and did 5 seeds for embryo rescue - man those little buggers are hard to hold and see. Hopefully the embryos weren't mangled too much. We will have to see if anything grows besides contamination. I tried to be sooooo careful about that, but it's a crap shoot.
If they get something going I will post some pics. I placed each GA7 jar into a zip-loc baggie afterwards (done in the hood) so I am hoping to avoid contamination there, too. I hope at least one of them is a good rescue, but they are so difficult to see, much less to slice the seed. The first one, the scalpel blade broke and the seed went flying. I found it, resterilized everything and tried again, but the embryo looked rotten. It was all brown - perhaps oxidized from leaving the seeds in 10% bleach solution overnight! Oops!
How do you hold onto the seeds and cut them? I found it very difficult using forceps and a surgical scalpel. Is there another, easier way to do this? When you do ER, do you use a macroscope to see better? I was thinking of doing that, but after the difficulty cutting the seed, there is no way I can imagine doing this on a macroscope stage without knocking it over. Maybe one of the reverse viewing types with the stage as part of the base????
Anyway, the first cultures are going now. I have a Saba corm that's not doing anything after 3-4 weeks, so I might grab it to TC it. It has one more week to show me signs of life, or it's going under the knife!!!

mskitty38583
10-06-2008, 09:04 AM
congrats scot! im not that ambitious yet.....i want to try growing from seed first but i have a lot more to learn before i even start that. congrats again!!!!!:goteam::goteam::goteam::woohoonaner::birthdaynana:

Tog Tan
10-06-2008, 11:12 AM
Way to go Scot! This is something I have always been dreaming about, now that you are onto to it, its gonna be 'look n learn' from your updates. All the best mate.

MediaHound
10-06-2008, 11:42 AM
Yea, way to go! Sounds awesome!

Bananaman88
10-06-2008, 12:48 PM
Good luck, Scot! Sounds like you're having fun now!

Bob
10-06-2008, 01:14 PM
Sounds really cool, once you have it down the sky's the limit I guess. The petri dishes look like Frankenstein got to them though!

Gabe15
10-10-2008, 03:09 AM
Perhaps you had seen this post awhile back, but this was my first couple attempts at embryo rescue, I also briefly detail some tips on opening the seeds.

http://www.bananas.org/f30/germination-easy-way-3526.html

Chironex
10-10-2008, 03:10 AM
Yes, I saw and learned from it. Thank you Gabe.

Chironex
10-21-2008, 01:50 AM
Just put Manzano and another cold hardy banana into culture. The Manzano was last week, the cold hardy banana this evening. The one this evening was my best apical meristem dissection yet. It was textbook, except the media was still a bit soft when I put it in, so it sunk in a bit deeper than I wanted it, but that means more exposure to the media.
I was going to TC the Saba corm, but as I was lifting it out of the pot, I noticed that roots were growing like crazy and pups were forming, so I left it alone.
I will have some pictures tomorrow in my gallery. My camera isn't that great, so the resolution is so so. But a couple turned out ok.
The Manzano explant is elongating and no signs of contamination.
As for the Embryo rescue, despite being on the wrong media, it looks like there are some changes going on with one of the cultures, so I am optimistic that at least one will take. (cross your fingers!)

Bananaman88
10-21-2008, 11:57 AM
Good luck, Scot. Sounds like it's going well thus far.

kimarj
10-21-2008, 01:25 PM
:goteam::goteam::drum:Congrats!!

Chironex
10-21-2008, 04:48 PM
As promised, here is a picture of the apical meristem, ready to go into the media. To give you some scale, the lid it is sitting in is for a baby food jar.

http://www.bananas.org/gallery/watermark.php?file=14068 (http://www.bananas.org/gallery/showphoto.php?photo=13745&ppuser=2761)

http://www.bananas.org/%5Burl=http://www.bananas.org/gallery/showphoto.php?photo=13745&ppuser=2761%5D%5Bimg%5Dhttp://www.bananas.org/gallery/watermark.php?file=14068%5B/img%5D%5B/url%5D

http://www.bananas.org/%5Burl=http://www.bananas.org/gallery/showphoto.php?photo=13745&ppuser=2761%5D%5Bimg%5Dhttp://www.bananas.org/gallery/watermark.php?file=14068%5B/img%5D%5B/url%5D

Chironex
10-22-2008, 01:22 AM
I have to say, I am having a blast doing this so far. I keep a journal to log all of the details, so if I make a mistake, it is noted. (Like roasting the 2 culture jars in the pressure cooker!!! LOL)

It is amazing to me that that tiny piece of apical meristem can create so many banana plants. After I feel like i have it down pat, I am going to attempt to induce some mutant variegates.

Thank you for all of your kind words and support.

bigdog
10-22-2008, 08:49 AM
Scot, I've found the best way to rescue embryos from Musa seeds is with a small vice, mounted on a table. You just slowly tighten the vice until you hear a crack, then remove the seed from the vice. Find the crack, and pry it open with a scalpel, knife, something sharp. I haven't yet mangled the first embryo with this method.

Regarding contamination...it really is ridicuously easy to grow mold, lol. We did some cultures the other day, and 3 out of our 9 cultures got some contamination. I didn't feel so bad though, seeing the next group get every single one of theirs contaminated! This was with a professional setup, but most of the students had very little or no tc experience. Even with a constant flame, wiping down with ethyl alcohol, 10% bleach soak, rinsing with distilled water many times, and working as close to the hood as possible, contamination is still possible. It only takes one little particle! Good luck with your setup there.

Frank

Chironex
10-23-2008, 05:41 PM
Scot, I've found the best way to rescue embryos from Musa seeds is with a small vice, mounted on a table. You just slowly tighten the vice until you hear a crack, then remove the seed from the vice. Find the crack, and pry it open with a scalpel, knife, something sharp. I haven't yet mangled the first embryo with this method.

Regarding contamination...it really is ridicuously easy to grow mold, lol. We did some cultures the other day, and 3 out of our 9 cultures got some contamination. I didn't feel so bad though, seeing the next group get every single one of theirs contaminated! This was with a professional setup, but most of the students had very little or no tc experience. Even with a constant flame, wiping down with ethyl alcohol, 10% bleach soak, rinsing with distilled water many times, and working as close to the hood as possible, contamination is still possible. It only takes one little particle! Good luck with your setup there.

Frank

Thanks! I think I will buy a C - clamp to do the same thing. That way I can sterilze it in the pressure cooker, wipe it with alcohol and put in in the hood to crack the seeds. I try to do as much under the hood as I can.
So far, only one contaminated out of 7 cultures. It was one of the Ingens embryo rescues, and we know that they seem to be likely to have endocarpic contaminates, no matter how much I sterilize them. But, I will say that out of the 5 I did, 1 or 2 look like there is some promise. The other 2 have no contamination, but are doing nothing, so I may have injured the embryo upon removal. The 5th one was the contaminated.

The Manzano and the California Cold hardy banana cultures appear to be aseptic so far. I will be doing 2 more cultures tomorrow or this weekend.

I appreciate your encouragement.

Richard
10-23-2008, 05:53 PM
Go Scot, go! I see from your avatar that your cortex stem has gone banana.

Chironex
10-23-2008, 05:58 PM
Go Scot, go! I see from your avatar that your cortex stem has gone banana.

The way I see it, if Jimmy Buffett can have Parrotheads, I can have BananaHeads, right?

Chironex
10-23-2008, 08:22 PM
Here is one of 3 pics posted in my gallery of a Manzano TC. It is beginning to shoot now.

http://www.bananas.org/gallery/watermark.php?file=14160&size=1 (http://www.bananas.org/%5Burl=http://www.bananas.org/gallery/showphoto.php?photo=14160&ppuser=2761%5D%5Bimg%5Dhttp://www.bananas.org/gallery/watermark.php?file=14160&size=1%5B/img%5D%5B/url%5D)

Chironex
10-23-2008, 11:03 PM
Here is one that was just put into TC today for one of our members. The surgery went well, and the prognosis is excellent.
http://www.bananas.org/gallery/watermark.php?file=14170 (http://www.bananas.org/gallery/showphoto.php?photo=14170&ppuser=2761)
http://www.bananas.org/gallery/watermark.php?file=14167&size=1 (http://www.bananas.org/gallery/showphoto.php?photo=14167&ppuser=2761)

damaclese
10-24-2008, 10:14 AM
Scot you are so cool dude thanks for sharing this with us congrats looking forwerd to more info and pics keep them coming! from the pics they defanatly look good to me! how are the Engen seeds doing? can't wate till you can Tc those im sure you will have no problims selling them to the Org rember im in line to LOL

harveyc
10-24-2008, 12:20 PM
Scot, thanks for all the photos, comments, etc.

Whey you refer to "California Cold Hardy", are you referring to the one sold by Shawn in Lodi, CA? Or is there something else referred to as that?

Harvey

Chironex
10-24-2008, 02:36 PM
Scot, thanks for all the photos, comments, etc.

Whey you refer to "California Cold Hardy", are you referring to the one sold by Shawn in Lodi, CA? Or is there something else referred to as that?

Harvey

Yes, I believe that is the one. Thanks for your kind remarks.

Bob
10-25-2008, 06:15 AM
Scot you are so cool dude thanks for sharing this with us congrats looking forwerd to more info and pics keep them coming! from the pics they defanatly look good to me! how are the Engen seeds doing? can't wate till you can Tc those im sure you will have no problims selling them to the Org rember im in line to LOL
Ditto for me.

ursula
10-25-2008, 09:30 AM
with all the experience you all have you make me feel very insecure!
cant you discuss technical theatre for a while?
ursula

MediaHound
10-25-2008, 10:34 AM
Good work Scot!

Chironex
10-25-2008, 07:00 PM
Today put 6 E. perrieri embryos into culture along with 1 laterita and 1 itinerans embryo. Also took one ingens seed from germination tray and rescued the embryo onto culture media. Gabe was very helpful while I was doing this and Frank's idea of using a small vise to crack the seed hull worked like a dream! It's the only way to go from now on. Look how small an itinerans seed is and imagine trying to split it without damaging an embryo the size of a pin head! Using the vise, the embryos just fell out, or were easily removed if they were stubborn. Note: this method works best on dry seeds, if soaked, they just split open slightly. Embryos were easier to extract from the dry seeds.

We will see how they look as time passes. No pic's yet, my camera resolution is not that great, so they would be blurred, if visible at all.
Seeds were first sterilized in Physan 20 with some Tween 20 as a surfactant. Then they were rinsed for 20 minutes in 10% bleach water solution. The seeds were then cracked, as aseptically as possible, and put into a quick rinse in 10% bleach solution, rinsed 3 times in sterile water, then placed upright onto M&S media.
I hope to get my hands on some better media for embryo rescue at some point, but this was all that was available for now.
Now we wait...

Chironex
10-25-2008, 07:04 PM
with all the experience you all have you make me feel very insecure!
cant you discuss technical theatre for a while?
ursula

Technical theatre - ummm, project your voice from the diaphram, that's the extent of my knowledge - you got me there. lol

51st state
10-26-2008, 12:59 PM
Looking good there Scot. keep it up.

Once you've got it nailed I'll try get some unusual stuff over to you

Chironex
10-26-2008, 01:12 PM
Kev, that would be fun. Wait until I get more trials in with different media. Some variances occur with different banana varieties. Everything is being logged so that the formulas that work on certain bananas will already be known. Need to get the medium for embryo rescue, as the normal M&S is not as robust.

edd82
11-20-2008, 04:27 AM
Wow that's so cool :-) Good luck with it.

Chironex
12-25-2008, 03:01 PM
Yesterday 8 more ingens embryos were dissected and placed into embryo rescue. Still waiting for chemicals to use with the new medium using modified Nitsch & Nitsch.

The Viente Cohol was sub-divided again into 12 plantlets. The California Cold-hardy banana has not done anything since induction. Can't figure out why, as there does not appear to be any contamination. It just seems to be happy taking its time.

The Manzano culture is also taking its sweet time to do anything. Next cultures I do I will split up using charcoal in the medium and citrate. I have also been doing some reading about alternative media such as straight peat with some hormones. Others are cotton balls, perlite, vermiculite and gellan gum. I got some of each to test the differences in cultures.

Phytotech Labs has a Banana culture medium that also shows some promise, so I am going to set-up some trials with it, too.

The hope is to aggregate banana TC data and construct a "best culture environment" database for each banana type, including Ensete and Musella. Sometimes the strength of the medium including the additives can vary the results dramatically.

There is also an antibiotic that has been shown to induce variegation that remains true to type. This may be a key to use in AeAe culturing.

harveyc
12-26-2008, 12:32 PM
Wow, Scot, you've really got into this! What are your objectives in producing plants? Are these for your own use, to give away to friends and family, or do you plan on selling plants?

While I have no plans to for commercial applications, I thought I might like to learn TC so that I could produce more plants for my own use. For instance, a fast maturing variety like Viente Cohol might work well being grown as an annual so I wouldn't have to worry about cold hardiness. I'm thinking I could TC some plants in the fall or winter and grow them in pots in my greenhouse until spring and hope for ripe fruit by the end of summer or early fall from several plants. I have lots of relatives and friends to share the fruit with.

I'm not sure I have the discipline necessary to carry out the TC process, though. I just need to focus my attention, perhaps.

Thanks for sharing!

Chironex
12-26-2008, 02:02 PM
Thanks Harvey, it's my O.C.D. kicking in. :ha: I don't tend to do anything half-way, it's pedal to the metal - especially when it involves fascinating research. If it interests me, I dive in head first. (Kinda geeky that way, have to know all about it)

There are still spots available for you in March for the Las vegas TC workshop sponsored by Carol Stiff's organization, Kitchen Culture Kits. Let me know if you can attend. We sure would like to have as many attend as possible. This will keep the costs lower for everyone.

What will I do with these plants? I would like to recover some costs, but want to offer them to our members here and perhaps some special ones on eBay. Of course, some will actually be for my enjoyment. When I develop the techniques for culturing variegated banana plants that remain true-to-type, I think auctioning some of them will yield the money to update my homemade lab equipment, provide for more chemical supplies and lab supplies. Some will be held back to be sold exclusively to our members, a benefit of membership. Some will be traded for explant material to further expand the availability of the rarer forms to our members. Keep in mind that everything being done is operating at a pure loss at the moment, all expenses are out of pocket including the cost of getting most of my ex-plant material. As most of you know, I have bid quite a bit for many special banana plants from all over the world. The vision I have is to make these more available to our members.

This is only a hobby for me. I do not intend to become a competitor of the commercial labs - more like a specialist for certain, limited production runs of rare bananas.

I am presently studying the methods of tissue culturing using male and female flowers, which has both advantages and disadvantages. On the positive side, the entire plant is not destroyed, which may lead to more plant culture being made available as someone would not have to sacrifice the entire plant for it to be TC'ed. The downside is that it appears to be a bit more complicated. I hope to make some efforts to learn this procedure in 2009. I will need to get some immature male buds at the proper time. I will try it on my own plants once I get one o flower, which should occur this summer with any luck. Once I get this procedure down pat, I will post a new thread with some photos.

Embryogenic Cell Suspensions are also fascinating, but since they tend to produce less somaclonal variation, it doesn't really appeal to me. It also requires more lab equipment and closer monitoring along the way. Time demands are intensified and that is a luxury that I cannot endure.