[Jeffww]

Just about to trade some plants for some downoi and various anubias so I can get this project underway. I still have to save up for the necessary chemicals though but I'm just going to put this thread here since I'll also be using this as an emersed set up journal too (need a place to hold my plants and all)...

So on the way is:
Various anubias
Downoi

On shopping list is:
Murashige and Skoog w/ gamborg vitamins
NAPHTHALENEACETIC ACID, POTASSIUM SALT, K-NAA
6-BENZYLAMINOPURINE (BA)


Sources:
1. http://namcub.accela-labs.com/pdf/C...ve gelling agent for plant tissue culture.pdf
2. http://www-pub.iaea.org/mtcd/publications/pdf/te_1384_web.pdf
3.http://omnisterra.com/botany/cp/slides/tc/tc.htm
4. http://www.oup.com/uk/orc/bin/9780199282616/ch02.pdf
5. http://journal.ui.ac.id/upload/artikel/04_Invitro culture_Revisi_Zulkarnain.PDF (references a member of the pogostemon genus)
6.http://www.phytotechlab.com/pdf/SIVBMay2004.pdf Talks about antimicrobials Important
7. http://kryptokoryne.aquaticscape.com/2010/03/29/cryptocoryne-tissue-culture-micropropagation-part-2/ A source thanks to the hard work of one of the members on this site.
8. http://www.flytrapcare.com/phpBB3/peracetic-acid-in-sterilization-now-with-success-t3426-15.html Paracetic acid...sounds like hot stuff.
9. http://docs.google.com/viewer?a=v&q...oFK5yA&sig=AHIEtbTqOn6oZlf1Bnq3pv_GB2ZfdApDnA A good article again on starch + water for gelling...This is my biggest cost cutting cheat. Agar is hella expensive.
10. http://www.flytrapcare.com/phpBB3/post103154.html#p103154 some protips from experienced people
Plants have been ordered!
11. http://www.scribd.com/doc/35365880/Plant-Tissue-Culture-for-Home-Gardeners a very clear and concise how-to guide.
12. www.apctt.org/publications/tm_dec_tissue.pdf a flow chart for us in here along with some info on Crypt propogation...I think I have plenty of source now. Time to start culturing!
13. www.phytotechlab.com/pdf/TissueCultureMediaComposition.pdf

 

[Jeffww]

Some good sources added!

Non numbered sources:

Rapid in vitro multiplication of the aquatic angiosperm, Anubias barteri var. undulata
Li-Chun Huang, Yung-Hui Chang and Yu-Li Chang

Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan

Accepted 24 June 1993. ; Available online 25 June 2003.

Abstract
A protocol was established for rapid in vitro miltiplicatin of Anubias barteri Engler var. undulata. By employing very small tips of actively growing lateral shoots as explants, aseptic cultures were produced without nutrient medium addenda of antibiotics of fungicides. Quiescent buds produced only infected cultures. Cultures were initiated and rapid shoot multiplication was attained in a medium containing Murashige and Skoog salts, 3% sucrose, 0.8% Sigma Type A agar and in mg 1−1: 10 thiamine HCl, 10 pyridoxine HCl, 5 nicotinic acid, 2 glycine, 100 i-inositol, 0.3 BA (N6-benzyladenine), 0.01 thiadiazuron and 0.1 NAA (1-naphthaleneacetic acid). Shoots were rooted in small clusters, in a second medium lacking cytokinins. The rooted shoots were readily established as aquarium or greenhouse plants. Based on a steady rate of five-fold proliferation of shoots per month, attained after 7 months following explanting, the projected annual yield of clonal plants by this method is 106 plants per explant. As is commonly observed among many amphibious species, terrestrially and aquatically grown Anubias plants from tissue culture displayed land and submerged forms of foliar morphology.

Aquatic Botany
Volume 47, Issue 1 , January 1994, Pages 77-83
*********
Michael E Kane Corresponding Author Contact Information, Greg L Davis2, Dennis B McConnell, Jennifer A Gargiulo
Purchase
Environmental Horticulture Department, P.O. Box 110670, University of Florida, Gainesville, FL 32611-0670, USA

Received 5 October 1998; Accepted 14 January 1999. Available online 24 March 1999.
Abstract

Procedures for in vitro establishment, axillary shoot proliferation and plantlet acclimatization of the aquatic plant, Cryptocoryne wendtii De Wit. were determined. Surface-sterilized rhizome shoot tips were established on a basal medium (BM) consisting of Murashige–Skoog mineral salts, 0.56 mM myo-inositol, 1.2 μM thiamine–HCL and 87.6 mM sucrose supplemented with 2.2 μM N6-benzyladenine (BA) and 0.57 μM indole-3-acetic acid (IAA) and solidified with 0.8% TC agar. Effects of basal medium supplementation with factorial combinations of BA (0–25 μM) and IAA (0–10 μM) on axillary shoot proliferation from single-node explants were determined after 28 days. Maximum axillary shoot proliferation (sevenfold increase) occurred on medium supplemented with 20 μM BA alone. Excellent microcutting rooting (100%) was achieved by direct sticking microcuttings in Metro Mix 500 soilless planting medium. Greenhouse acclimatization of rooted microcuttings was 100%. High-quality salable plants were produced within eight weeks post-transplant.

Keywords: Araceae; Aquarium plants; Growth regulators; Micropropagation; Plant tissue culture; Shoot culture

Pretty good...

 

[OverStocked]

I've been wanting to get into this and have a few books on it that haven't been touched yet. Nice info you have here.

 

[Jeffww]

What books did you get? I'm trying to make this a budget project so I'll probably try to find them at a library or something...I'm looking for an article I found that talked about callus transferring and explant isolation right now...I'll put that up in a bit.

Found it: http://www.oup.com/uk/orc/bin/9780199282616/ch02.pdf (Added to source list as well)

I'd like everyone to note that the ferts used in MS can be duplicated using miller's microplex or CSM (to a lesser degree). So a dedicated medium is not necessarily required but IMO the money spent on MS is well worth the time saved and you are assured quality.

 

[OverStocked]

I'll look at what they are when I get home!


wait, this is one:
http://www.amazon.com/gp/product/0881923613

 

[Jeffww]

Thanks. I'll chalk that up next to my marine fish breeding guide on my book list....

Also more sources updated...Last set for a good while too.

 

[plantbrain]

Jeff, you might just consider contacting Mike Kane at UF, before he retires!

 

[Jeffww]

Jeff, you might just consider contacting Mike Kane at UF, before he retires!

Will do. Hopefully he'll be nice and share his info with me.

 

[Jeffww]

Well..since I don't really want to pay for the ONE dollar per ml of plant preservative mixture....I'm going to go with some cheap: Tetracycline hydrochloride Although it's not as broad spectrum it's much more broad than most available antibacterials and it's available at most fish stores. If not I'll try using erythromycin or kanamycin...

 

[OverStocked]

Let us know how it works. What I'd like to see is if you try this, a step by step documentation of it. Hell, I'd be willing to sponsor it in some way.

I want to see how to do this but am not sure I have to time to commit to figure it all out on my own....

 

[Jeffww]

That's the plan. Don't worry. I've more or less all the resources at hand for this project...it's not even particularly costly:

Salt bases + Hormones: 40USD
Antimicrobials: 10 USD
Plants: Free
Culture containers: 10USD (gladware from dollar store)
Emersed Set up: 5 dollars (humidity dome + shoplight I already have)

All the tools are the ones I use for keeping my aquarium, no need for a pressure cooker since I'll be doing flash boiling with a microwave to sterilize....When I get the project started this winter I'll convert all those nasty numbers into something simpler like mass/volume.

 

[Jeffww]

Picked up a sheet of rockwool, humidity dome and tray today at the local hydroponics store. I asked if they had plant cloning stuff and they said a guy came in right before me and bought them out of their stuff....I guess I'll order online.

Tomorrow I'm going to go pick up some bigger cfl bulbs..And then some corn starch to test out its gelling-ness.

 

[Jeffww]

Quick question. Do you guys think 2 x 26 watt cfl is enough light for these guys? An emersed set up...over a 20x11 tray.

 

[aman74]

Picked up a sheet of rockwool, humidity dome and tray today at the local hydroponics store. I asked if they had plant cloning stuff and they said a guy came in right before me and bought them out of their stuff....I guess I'll order online.

Tomorrow I'm going to go pick up some bigger cfl bulbs..And then some corn starch to test out its gelling-ness.

A couple questions:

-what other stuff did you need to buy? I'm trying to make a list as well and I saw you had most of the stuff already, so was wondering what else was needed.

-PTC is plant tissue culture?

-you're talking about micropropogation right?

Thanks for all the links! Most are probably over my head and I'm just trying to gather more practical info on how to go about doing this as well.

 

[Jeffww]

A couple questions:

-what other stuff did you need to buy? I'm trying to make a list as well and I saw you had most of the stuff already, so was wondering what else was needed.

-PTC is plant tissue culture?

-you're talking about micropropogation right?

Thanks for all the links! Most are probably over my head and I'm just trying to gather more practical info on how to go about doing this as well.

My materials so far:
Pinsettes/tweezers
1ml pipettes
Lots of Distilled water (go ahead and just spend a tenner on 9 gallon bottles of the stuff)
A microwave (they say pressure cooker is better, but that's money I don't have to spend)
Hormones (listed above, those are the two big ones)
Medium (MS medium w/ vitamins)
Gelling agent ( this can be a lot of things. Agar is the most common, next is stuff like gelcarin, carrageenan. Since I'm poor I'm using corn starch)
A clean box- This can be an aquarium turned on its side with plastic sheets over the front to keep things moderately clean in. I'm using a rubbermaide tote with a desk lamp in it. Everything in the clean box is for clean box use only and non-sterile things aren't allowed in (unless you really have to)
Sterile gloves
A few misters containing your choice of disinfectant (I'm planning on using paracetic acid which is made by 100ml of hot vinegar + 400 ml of H2O2 3%)
Your growing chamber/box. This can easily be inside your rubbermaid tote/clean box depending on how big it is. In fact it's probably better to keep all your cultures in there until you're ready to transfer them to a regular pot.
Antimicrobials - a popular one is called "plant preservative mixture" it's a broad spectrum anti microbial used in the medium itself to keep infections to a minimum. I can't really afford it so I'm going to use tetracycline hydrochloride or kanamycin for just antibacterial protection and make sure my technique is spot on for the rest.
Some ziplock bags...
Some cheap tupper ware that's microwave or heat safe. (gladware is fine)
Razor blades for isolating the explant

PTC is plant tissue culture and it's the same as micropropagation. The whole business isn't too complicated. I'm just making it more complicated to save some bucks. You can get everything you need for a marked up price but with a lot less thinking/ghettoness involved.

My goals for this project are:
1. Have a step by step guide to PTC with all materials and "trade secrets" involved. Total transparency.
2. Sustain some rare crypts for the rest of us
3. Get some $$$ to get started on my reef tank. If I can get the predicted 1000 or so anubias plants then I would say I'm pretty well set....lol.

 

[Jeffww]

Got my cheapo clean box built...it could be bigger but I don't have much room to work with anyways so it's good enough. Probably plenty of space to run 6-8 cultures at once in larger gladware and maybe 10-20 on baby food jars.

 

[jl209]

Awesome I've read about this but I could never wrap my head around it completely. From what I've read its a PITA to get a completely sterile culture. Goodluck I really hope you're successful.

 

[aman74]

My materials so far:
Pinsettes/tweezers
1ml pipettes
Lots of Distilled water (go ahead and just spend a tenner on 9 gallon bottles of the stuff)
A microwave (they say pressure cooker is better, but that's money I don't have to spend)
Hormones (listed above, those are the two big ones)
Medium (MS medium w/ vitamins)
Gelling agent ( this can be a lot of things. Agar is the most common, next is stuff like gelcarin, carrageenan. Since I'm poor I'm using corn starch)
A clean box- This can be an aquarium turned on its side with plastic sheets over the front to keep things moderately clean in. I'm using a rubbermaide tote with a desk lamp in it. Everything in the clean box is for clean box use only and non-sterile things aren't allowed in (unless you really have to)
Sterile gloves
A few misters containing your choice of disinfectant (I'm planning on using paracetic acid which is made by 100ml of hot vinegar + 400 ml of H2O2 3%)
Your growing chamber/box. This can easily be inside your rubbermaid tote/clean box depending on how big it is. In fact it's probably better to keep all your cultures in there until you're ready to transfer them to a regular pot.
Antimicrobials - a popular one is called "plant preservative mixture" it's a broad spectrum anti microbial used in the medium itself to keep infections to a minimum. I can't really afford it so I'm going to use tetracycline hydrochloride or kanamycin for just antibacterial protection and make sure my technique is spot on for the rest.
Some ziplock bags...
Some cheap tupper ware that's microwave or heat safe. (gladware is fine)
Razor blades for isolating the explant

PTC is plant tissue culture and it's the same as micropropagation. The whole business isn't too complicated. I'm just making it more complicated to save some bucks. You can get everything you need for a marked up price but with a lot less thinking/ghettoness involved.

My goals for this project are:
1. Have a step by step guide to PTC with all materials and "trade secrets" involved. Total transparency.
2. Sustain some rare crypts for the rest of us
3. Get some $$$ to get started on my reef tank. If I can get the predicted 1000 or so anubias plants then I would say I'm pretty well set....lol.

Thanks very much, your goals are very similar to mine.

A guide would be great, so I'll look forward to that from you if all goes well. I feel many of us would give this a go if a little of the smoke was cleared away and the process demystified a bit.

I saw an older post that I put in my subscribed threads, but it's going to take awhile to find it now because it was old and I can't remember what time frame it was from. There was a poster there, can't remember if it was the OP or not, he said he was going to attempt this and just needed to get the specific info for each species of plant. He said that was the hardest part as each plant responds differently to the agents used in the process. He reported back that he got the info for a whole bunch of submersed species, but never gave specifics or any links or anything. Did you happen to come across this info anywhere?

For anyone else looking into this, check out the OP's link number 7 and also be sure to look at the bottom of the article for 3 more great links.

That hometissueculture site does seem pretty pricey for the convenience, but support seems good and they are also selling instructional DVD's. They're also taking donations for research, so I'm a little confused as to whether they are non-profit or not?

If you happen to be willing to guide me a bit, feel free to send me a PM. No obligation of course, but if you're so inclined that would be cool.

 

[Jeffww]

I'll be happy to help...Just need to get the ball rolling on my side first. My plants were delayed. I'll have them in wednesday....I just need to wait until november really...so that my friends will go birthday shopping for me lol.

I just thought about another plant that is just BEGGING to be cultured: beucephalandras.

 

[aman74]

Cool man, keep us posted. It will be awhile for me as well. Where did you hear about beucephalandras? Just wondering where you're going for your info. If you can recommend any forums or sites that have info or guides that would be cool too.