# Jeff's Foray into PTC [3-10-12 First plates]



## Jeffww (Aug 6, 2010)

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...


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## OverStocked (May 26, 2007)

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.


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## Jeffww (Aug 6, 2010)

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.


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## OverStocked (May 26, 2007)

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


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


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## Jeffww (Aug 6, 2010)

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.


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## plantbrain (Dec 15, 2003)

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


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## Jeffww (Aug 6, 2010)

plantbrain said:


> 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.


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## Jeffww (Aug 6, 2010)

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...


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## OverStocked (May 26, 2007)

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....


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## Jeffww (Aug 6, 2010)

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.


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## Jeffww (Aug 6, 2010)

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.


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## Jeffww (Aug 6, 2010)

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.


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## aman74 (Feb 19, 2007)

Jeffww said:


> 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.


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## Jeffww (Aug 6, 2010)

aman74 said:


> 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.
> 
> ...


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.


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## Jeffww (Aug 6, 2010)

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.


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## jl209 (Mar 2, 2006)

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.


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## aman74 (Feb 19, 2007)

Jeffww said:


> 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)
> ...


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.


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## Jeffww (Aug 6, 2010)

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.


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## aman74 (Feb 19, 2007)

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.


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## Jeffww (Aug 6, 2010)

aman74 said:


> 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.


I just do google searches for tissue culture. Then I came across someone talking about buces.. I'll save that for later. they are _pricey_ plants.


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## gordonrichards (Jun 20, 2009)

Be prepared to lose a number of containers. Glass jars are very good.

-Gordon


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## Jeffww (Aug 6, 2010)

gordonrichards said:


> Be prepared to lose a number of containers. Glass jars are very good.
> 
> -Gordon


I'm going with gladware simply because I have no need for glass. I'm not sterilizing using a pressure cooker. Only a microwave. On top of that I can't get those metal lids in the microwave either lol. I'm hoping I can cut down losses to about 5%...I've done this before once using some stuff I borrowed from school...Out of the 12 tubes I had 10 of them were infected with fungus. This was done without using any form of sterilization other than boiling the agar and tubing them right away...No clean box was used either. Explants were sterilized using only 70% alcohol....once. As you can see it's actually not hard to keep a few things sterile...you just need to be careful.


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## Jeffww (Aug 6, 2010)

Plants will be in tomorrow. That means I'll have to have my grow box all set up then too...Downoi is going straight into my 60P though..I better start increasing the humidity in my bin too...hmm...Anyone know a good substrate for emersed anubias? I'm thinking a mixture of 1 part sand to 2 parts peat/pottingsoil mix...


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## Jeffww (Aug 6, 2010)

Well..plants didn't come today (which sucks) but I was able to go to the store and buy some tetracycline hydrochloride. It's sold by API under the name of T.C. tetracycline and is available at almost any store. 16 dollars gets you 5000mg of it which will last you 10 forevers since you only use a few mg per L of medium. If anyone wants this stuff in the future but doesn't want to pay for the whole packet I'll be happy to send 500mg packets out.


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## aman74 (Feb 19, 2007)

Thanks for the updates!


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## Jeffww (Aug 6, 2010)

We've got pictures: 









My ghetto clean box. The point of the clean box is to restrict air movement and to keep cultures sterile. What I noticed about most clean box designs was that their fronts were completely and utterly exposed. (a box turned sideways. My box limits the entry way as much as possible and makes uninterrupted air movement into the box impossible. On top of that I can just use the draw strings on the trash bag to seal it up after I'm done working. It's also small and mobile so I can throw it in my closet under a grow light when I'm done plating and take it out to do work.










Some rather vital equipment. My tetracycline and my glad ware. at 8 for 2.50 it's a good idea to buy around 2 packs of these. Each one is the perfect size for starting cultures. 









This is for replating cultures when they get big enough...


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## Jeffww (Aug 6, 2010)

So I was doing some experiments with corn starch in the microwave...So far it looks like 3.5g of Starch to 50ml of water is a good starting number...Maybe. I ran out of corn starch and my experiment exploded in the microwave since it overboiled to fast from the bottom up and splattered all over the place like the slime from ghostbusters. 

What I'm most excited about is that after about 45 seconds on high in the microwave the goo actually has a decent amount of clarity, comparable to agar actually!


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## 150EH (Dec 6, 2004)

Number 7 on your list did this with C. nurii, well maybe not exactly how you are going to do it but this is pretty cool and way, way over my head, I was reading everything over to get a clue to what PTC is, I'm starting to get it a little but there more to come, but so far really cool.


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## Jeffww (Aug 6, 2010)

150EH said:


> Number 7 on your list did this with C. nurii, well maybe not exactly how you are going to do it but this is pretty cool and way, way over my head, I was reading everything over to get a clue to what PTC is, I'm starting to get it a little but there more to come, but so far really cool.


It's pretty over my head too. I'm approaching this from a "lab tech" viewpoint. As in only following protocol like an instruction booklet from ikea and ignoring the technical comments. The basic principle is simple:

1. You get a tissue simple from a plant (theoretically from anywhere)
2. Make your medium (Same difficulty as making a cake/instant pancakes) 
3. Put the tissue in the medium 
4. Grow baby grow 

My main barrier is sterilization. One of the reasons I need to keep testing my microwave technique is that starch will 'splode and ruin my microwave if I'm not careful. 

Edit: My anubias will be here on tuesday thanks to gordon richards and I'm about to order some C. Keei.

One comment on the Keei. Out of respect of the person I'm buying it from (looking4roselines) I will *not* mass produce this plant as I will with anubias. I may culture it and sell off a few plantlets but I won't be turning this into the thousands of plants I'm threatening.


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## gordonrichards (Jun 20, 2009)

Good luck with the anubias! Hope it works out well for you. :^)

-Gordon


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## Jeffww (Aug 6, 2010)

My girlfriend (awesome) just ordered my chems for me for my bday. 
9g TC grade Agar 
1g benzylaminopurine
5g Napthaleneacetic acid
10 x 1L packets of MS w/ vitamins 

I can't wait! Chems _and_ keei will get here on thurs or friday. 

Things are going to start getting exciting around here!


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## james0816 (Jun 26, 2008)

Ok..I confess...total noob on this subject...but since this is a lab project it has really sparked my attention.

Can I ask as to what exactly this is?


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## Da Plant Man (Apr 7, 2010)

james0816 said:


> Ok..I confess...total noob on this subject...but since this is a lab project it has really sparked my attention.
> 
> Can I ask as to what exactly this is?


Tissue culturing plants! (I hope I am right, I just skimmed the thread :flick: )

Basically you keep a sterile environment with a simple medium, throw a little piece of plant tissue in it, and watch it grow. You use hormones to promote growth. 'course it is a whole lot more complicated as you can see, but thats it put into laymans terms.


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## aman74 (Feb 19, 2007)

Jeffww said:


> So I was doing some experiments with corn starch in the microwave...So far it looks like 3.5g of Starch to 50ml of water is a good starting number...Maybe. I ran out of corn starch and my experiment exploded in the microwave since it overboiled to fast from the bottom up and splattered all over the place like the slime from ghostbusters.
> 
> What I'm most excited about is that after about 45 seconds on high in the microwave the goo actually has a decent amount of clarity, comparable to agar actually!


So you're using the starch to replace the agar? If so, is that to keep costs down?

I was watching some of those instructional videos and the guy was using an orchid medium which was mostly carbon. He didn't seem to be having any issue boiling it. Can you use something like that or are the mediums very specific to the plant your working with?

It's tough trying to figure out all the finer points of this. I wish there was a book for dummies.


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## Jeffww (Aug 6, 2010)

aman74 said:


> So you're using the starch to replace the agar? If so, is that to keep costs down?
> 
> I was watching some of those instructional videos and the guy was using an orchid medium which was mostly carbon. He didn't seem to be having any issue boiling it. Can you use something like that or are the mediums very specific to the plant your working with?
> 
> It's tough trying to figure out all the finer points of this. I wish there was a book for dummies.


Yep. Just trying to cut corners here. 

Agar is expensive 9g is 4 dollars. And it takes around 6g/L of media. It really accounts for over 70% of the media cost. I've done some reading and apparently the best media is about 50g of plain corn starch (99cents for 450grams) from the grocery store and 1g of agar to 1L of water. 

The issue isn't really boiling...it's hi-speed boiling. When the contents of the culture vessel boils so fast that a bubble of gas forms and blasts the contents of the cup all over the place. This isn't an issue with autoclave or pressure cooker sterilized media since the temperature increase is gradual and the stuff inside can start to convect due to heat differentials so all the bubbles can get out before it builds to critical pressure. 

I would say any gelling medium suggested for general tissue culture is fine. But for the average hobbyist like you or I, cutting costs by about 80% is well worth it. 

source: http://docs.google.com/viewer?a=v&q...oFK5yA&sig=AHIEtbTqOn6oZlf1Bnq3pv_GB2ZfdApDnA


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## Jeffww (Aug 6, 2010)

james0816 said:


> Ok..I confess...total noob on this subject...but since this is a lab project it has really sparked my attention.
> 
> Can I ask as to what exactly this is?


I guess a methlab is technically a "lab" too lol. This is about as ghetto as it gets. There's just lots of number crunching (well not really; just some stoichiometry) before I digest all this data into something useable for everyone. 

edit: To avoid triple post...

Plant list expanded: 

Anubias 'nana' 
Anubias barteri 
Pogostemon helferi (Not sure if I'll culture this in its emersed or submersed form yet) 
Cryptocoryne affinis
Cryptocoryne nurii 
Cryptocoryne keei 
Cryptocoryne cordata 'kr01' 


My practice plants will be the nana and affinis first....After that I'll move on to the big fry.


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## Jeffww (Aug 6, 2010)

Microwave sterilization is tough...so far I've come up with: 

1minute burst on max power (700watt oven I think....) 
15 second burst
10 
10
10
8
8
8
8 
8 
.
.
.

The 5-8 second bursts can go on indefinitely....This should prevent overboiling. 

Here's a quick step by step on starch gel making... Now this *has not* been tested yet on actual tissue. This is what I've come up with
This recipe is for cornstarch alone. Not sure what difference the agar will make. I assume that it'll just be a bit firmer. But the procedure will be the same.
1. To mixing cup add 100ml of water
2. Add 8g of corn starch and mix and make sure starch is well suspended 
3. Add to culture vessel. LOOSELY cap lid. 
4. Microwave (I'm using a 900watt oven, I just found out) 1 minute [*make sure you have a dummy bowl of water in there with your culture vessels, microwaving small amounts of water is bad for your microwave*] swirl it around after the minute is up as cornstarch will settle on the bottom making the top very slimy/thin and the bottom like rubber (not good).
5. Microwave for 20-15 second burst
6. 10 second burst
7. 10 second burst
8. 8 second burst
9. 8 second burst
10. 8 second burst
11. Making sure the lid is not completely sealed, float in ice water to gel quickly. 
Media and container should be sterilized by now. Do not open it until you are back in the clean area. 

Some tips: 
Keep papertowels under your vessels in case they DO overboil (which they will)
Using taller culture vessels allots more time for you 
Don't mess with it too much when it comes out. Otherwise the starch will fail to crystallize.
Speed is the key, the quicker you work the less chance for contamination at some point
Avoid overboiling like the devil. A slick of starch gel is a highway for infection. 











The finished product is very firm (although not as clear as I had hoped). Firm enough to hold a plantlet but also very yielding. 


edit: I've been practicing and after about going through 100grams of starch I've come up with:

Results vary: 900 watt oven used @ 50% strength. Total liquid volume in microwave ~ 2cups 

1. Set oven at 50%
2. Microwave for 3:30 non-stop. At this point the liquid should be nearing boiling temperature. This part is critical 
3. Go for 8-15 second bursts only until you reach 5 minutes. At this point the media should be sterilized with a 88-96% success rate.


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## Jeffww (Aug 6, 2010)

Word of warning over peracetic acid...don't stick your face into a hot container of it. My eyes burn. lol


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## gordonrichards (Jun 20, 2009)

Be mindful of your dirty fingers! Gloves good sir, gloves.


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## Jeffww (Aug 6, 2010)

Received the keei and cordata today. Thanks a bunch yue! Oh and gordon I got your anubias on tuesday. I killed one on accident because I cut it in half and the halves just turned to mush. Lesson learned.

Anyone have tips for me on keeping crypts emersed? These are my first emersed ones and I dropped some serious cash on them so I kinda want them to live lol.


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## Jeffww (Aug 6, 2010)

Got the chemicals too. MS went into the freezer...bagged in a sterilized jar in a bigger bag. Other chems went in my cabinet also double bagged. Fun begins tomorrow...

I'm first going to: 

1- make a batch of 10 small culture cups of full strength media. 
2- Hold on to them for 1-2 weeks and see how well my sterilization process works....
3- If all is well I'll keep going with this process and hopefully I'll be able to run some practice cultures using purple bamboo (fast growing so it will hopefully show if I've got my hormones/media correct very quickly) on the ones that are kept sterile. It'll also help me practice sterilizing tissue. 

I'm also planning on running a few practice tests on sterilizing *submersed* tissue. Apparently it's harder to work with because it tends to be literally coated in a layer of bacteria. For these tests I'm going to try working with submersed staurogyne porto velho.


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## 150EH (Dec 6, 2004)

Da Plant Man said:


> Tissue culturing plants! (I hope I am right, I just skimmed the thread :flick: )
> 
> Basically you keep a sterile environment with a simple medium, throw a little piece of plant tissue in it, and watch it grow. You use hormones to promote growth. 'course it is a whole lot more complicated as you can see, but thats it put into laymans terms.


Youngsters today are way too smart!!!!!!! But you explaination was direct and correct!

It was the pics of clean boxes that brought me in!!

It sounds like it's going pretty good just be careful, the nieghbors may call the Police to report your lab set up, you could be in jail for a week before they figure out it's a Aquatic Plant Lab!


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## Jeffww (Aug 6, 2010)

Wow...lol. Well not much to report back here...I did fill up about 2 pages in my notebook today though..What I learned: 

The behavior of the agar + starch mixture is utterly different. The agar settles out of the solution and gels at the bottom way before the starch can gel....Leaving me with a liter of wasted media and 3 hours gone....

Oh well better luck next time. I have 9L worth of material left....

What needs to be done is: 

Either use starch only at higher conc. ... Agitate very frequently (time consuming and a pain in the ass). 

Also for everyone thinking of ordering chemicals...order the hormone SOLUTIONS. It took me FOREVER to find NaOH. I remember it being common when I was a kid and that was only 10 years ago. But now you can't find just plain lye or Drano with NaOH only ANYWHERE....SO I ended up making a suspension of the BAP instead of an actual stock solution. (No big deal since I'm not going to be storing it) 

My formulation was: 

to 1L of DW:

30g table sugar
55g Starch
1g Agar
4.33g MS 
3mg BAP 
Adjusted to pH 6...maybe (this may have been the problem my pH reading wasn't entirely accurate)
....Sigh back to the drawing board.


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## Jeffww (Aug 6, 2010)

Off to buy some Drain cleaner for the NaOH...best I can find is 20%...and doing calculations that's

20g/100g soln. or 20g/80ml of h2o x 100= 2000g/8L = 1kg /4L or ~ 250g/L A 5M solution 6 times the concentration I need for making stock BAP solution. I need to dilute 10ml into 60ml of water to get more or less 1M NaOH. I'll also buy some HCL at home depot tomorrow while I'm at it.... 


EDIT: SUCCESS! Okay I was being a little impatient. Seems like all I had to do was wait overnight. All of the jars were able to gel all the way! Now it's just ....more waiting


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## Jeffww (Aug 6, 2010)

Some random pics of my emersed set up... 








A. barteri and C. wendtii 'brown' 









A. nana, two varieties, one smaller than the other, S. portovelho and purple bamboo 










C. cordata kr01 










C. keei 










C. nurii 











C. affinis


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## Jeffww (Aug 6, 2010)

So far so good...not obvious fungal contamination yet. This weekend might be the right time to do my first few cultures.


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## Jeffww (Aug 6, 2010)

So I've decided since one of my Keei leaves are starting to fall off (the plant itself is doing great, TONS of pearling, 0 actual melt) I'll take that one and hopefully it'll be a good culture. I'll do at least 2 with purple bamboo and the rest with S. porto velho.


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## 150EH (Dec 6, 2004)

What's going on over there? Any updates or new successes?


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## TactusMortus (Jun 28, 2011)

I am also interested to see how this is progressing.


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## HD Blazingwolf (May 12, 2011)

i second both of these affore mention motions!


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## Jeffww (Aug 6, 2010)

Haven't done much...the media in the jars is just standing there. I've been too busy but I'll definitely get started this weekend!


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## HD Blazingwolf (May 12, 2011)

awesome!!! we all anxiously await your response HAHA


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## Da Plant Man (Apr 7, 2010)

Lemme tell you a story;

I had no idea what PTC was until this thread. I mean, I had seen the links on Ghanzafar Ghori's blog, but never clicked on them. Now after researching like mad, I think I am going to try this. Never done anything 'lab like' before, so this will be a first. We also live on a farm and I can say that our house is 500% non-sterile. Whats the fun if there is no challenge, right?

So now, for my birthday, I am asking for sterile urine cups and awesome agar! I'm just a normal kid  

(And yes, I know I could use gladware, but I want glass cups) 




Jeffww said:


> Off to buy some Drain cleaner for the NaOH...best I can find is 20%...and doing calculations that's
> 
> 20g/100g soln. or 20g/80ml of h2o x 100= 2000g/8L = 1kg /4L or ~ 250g/L A 5M solution 6 times the concentration I need for making stock BAP solution. I need to dilute 10ml into 60ml of water to get more or less 1M NaOH. I'll also buy some HCL at home depot tomorrow while I'm at it....


Pssst, My mother uses this to make soap. Its 100% lye.

http://www.amazon.com/Rooto-Corp-10...FPRK/ref=sr_1_1?ie=UTF8&qid=1320337770&sr=8-1


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## 150EH (Dec 6, 2004)

about a man named Jed.......


Da Plant man, you keep it up and we are going to remove your Da, your just getting too smart. But it's very cool that your interested in this stuff.


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## Da Plant Man (Apr 7, 2010)

This stuff is awesome, I mean who knew you could grew a pineapple plant from just a 1cm piece that you took off of a unripe one? 

If I remove the 'Da', I will have the same smartness, just with a more politically correct screen name :icon_wink


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## Da Plant Man (Apr 7, 2010)

Update? I got the book:  Plants from Test Tubes: An Introduction to Micropropagation for my birthday yesterday. Like 4 chapters in, they gave plans for a lab that can produce like a million plantlets. I am tempted, lol. If you can get the book, get it. Don't get the kindle version, its harder to use for reference. Whats cool is that the people who wrote it, live in washington, so I might get to meet them...Hopefully....


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## Da Plant Man (Apr 7, 2010)

I really want an update.................


Finished the book a couple days ago. Its awesome, except everything is large scale. Like for a couple million plants  plus, it assumes that you will have employees and such 

Oh, and the prices are off. But thats to be expected since the book was written in 1996. pH probe costs $300+  I got mine for like $16


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## 150EH (Dec 6, 2004)

At least the prices are off in a good direction, now tech items are getting cheap.


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## Jeffww (Aug 6, 2010)

So after being unable to work on my project for school related reasons I'm back and I've prepared a fresh batch of media. Spring break is culture week for me. 


EDIT: So I've got all my cultures poured out. This time I just went: 
1L h2o
6.5g agar (made the resulting jelly BEAUTIFULLY clear) 
4.4g MS 
1ml mg/ml BAP 
30g table sugar
nuked for 5 minutes


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## Jeffww (Aug 6, 2010)

Some emersed picture updates





































































I've expanded my collection a little. I have now emersed: 

L. repens
L. lacustris 
L. 'red' 
L. 'rubin' 
Limno sessiflora 
limno repens 
Periscaria kawagoneam 
Myriopyllum mattogrossense 
E. Acicularus 
*Anubias barteri 
Anubias barteri 'nana' *
*Ultricularia gramminifolia *
Hemmianthus micranthemoides 
Purple bamboo 
*Staurogyne portovelho *
Hygrophila araguia 

Soon to add: 
*C. wendtii 'tropica' 
C. wendtii 'gecko' 
C. pondeterfolia *

When my crypts grow out a bit in the submersed tank I'll transfer over: 

*C. affinis 
C. nurrii 
C. parva
C. keei 
C. cordata 'KR01'* 

*Bolded plants are those I'm considering for tissue culture*


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## Jeffww (Aug 6, 2010)

Just showing off my handiwork. As you can see the gel is of much higher clarity. The red markings denote multiplication medium while those without markings are of lower BAP concentrations and are called establishment medium. Later on I will have to make rooting medium but that's a ways off. 



















compared to my first sample batch using part starch part agar mixture. I liked the firmness of the starch mixture better but this has the benefit of easily identifying contamination.


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## Jeffww (Aug 6, 2010)

Good news everyone! So I was digging through some stuff at my house and it turns out I've had one of these: http://www.iqair.com/home-air-purifiers/healthproseries/tech-specs.php sitting around...

99.97% efficiency for .3micron particles HEPA filter....Not bad. Good enough to stop most fungal spores definitely. Feels good to have an asthmatic family with friends in the medical business.


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## keithy (Jun 8, 2010)

Jeffww,
I would like to see you succeed in this because you have put so much effort into it. Also, I believe you have got past the learning curve of the "how to" which most ppl have not. All the best.


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## Jeffww (Aug 6, 2010)

Thanks for the encouragement keithy. So today I've begun culturing...I've excised about 12 or so actively growing tips of anubias barteri and barteri nana. I have about 15 vessels available. The other 3 will be reserved for crypts.


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## zachary908 (Feb 12, 2011)

Hey, Jeff! I got the hygro last night. Thanks again, man!


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## Jeffww (Aug 6, 2010)

No problem. Diversity in the US plant market is important. We have so few plants widely available to hobbyists compared to asia and europe no sense in holding on to a plant without sharing it.


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## mistergreen (Dec 9, 2006)

If you need agar, buy it at bulk on amazon. You can buy 2 pound's worth for $50. It'll save you in the long run and will last a life time.

I have some I can give you at discount.


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## Jeffww (Aug 6, 2010)

Looks like my technique on the microwave wasn't as good this time. I have fungal contamination on 9 of my jars. I think I know the reason since the multiplication jars have no contamination but were made in the same way. The only difference between the two was that they were microwaved for a full minute longer.


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## Jeffww (Aug 6, 2010)

Well...Looks like I need to try again. None of my jars took. The explants were not sterilized properly. However it seems that I've learned a few things: 

1.) Microwave sterilize for at least 6 to 7 minutes. The jars of multiplication media I have are still pristine. 

2.) Paracetic acid is probably insufficient for sterilizing our plants since the runners in crypts and anubias are heavily associated with the soil. 

All in all I learned a good bit and now I have more agar on order and I also bought a new crypt wendtii pot from my LFS to tinker with. This one has tons of runners so I should get a good number of explants off it. 

Also. I have a a few question to anyone who may be reading this. What's your interest in some TC stems? I'm looking to recoup some of my losses so far and I'm wondering if anyone would like to buy a jar of say TC Ludwigia 'red'. I could probably sell each jar for 4-5 dollars and they would each contain on the order of 20-30 small stems.


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## Jeffww (Aug 6, 2010)

Got my new agar from mr.green and I've got new gels plated out. Waiting for a week before I do any explanting so I can see if I've got any contamination...looks good so far though. 

Also I got on order some buces from xue.


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## Jeffww (Aug 6, 2010)

Replated today....I was able to put in 1 plantlet of Buce. 'copper leaf' We'll see where this goes...I personally don't have much confidence since there was some film on some of the other cups...


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