# Tissue Culture Doubts



## kshitij

Hello everyone,
I am searching everywhere for information reagrding the tissue culture of aquatic plants...it was great to see many posts in APC reagrding the tissue culture...
I have some doubts specificly reagrding the tissue culture of aquatic plants :
1. How to sterilze the ex-plant ?
2. Which part is most suitable ex-plant ?
3. Multiplication medium & hormones per optimum rate ?
4. Rooting medium ?
5. Subculture period ?

Also it is my request to all members that if anyone has any sucessful protocols regarding any aquatic plants ....plz do share with me as it will guide me to the next level


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## edwardn

What you are asking would be probably covered by Plant Propagation in Vitro - probably a 4 hrs. university course....

Look for articles on the subject on Internet - that would be my best advise.


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## Ghazanfar Ghori

1. How to sterilze the ex-plant ?
You want to use emersed grown plants, since they will be easier to sterilize. How you sterilize them exactly depends on the type of plant, the part of the plant you're trying to sterilize and the size of the part of the plant you're trying to sterilize. There is no black/white answer to this - but there are a few general guidelines. 
1-2 seconds in alcohol followed by
10 minutes in 10% bleach
followed by 2 x rinses of 5 minutes each in sterile water.


2. Which part is most suitable ex-plant ?
Depends on the plant. Stem fragments, leaf fragments, the growing tip (meristem) are common parts used.

3. Multiplication medium & hormones per optimum rate ?
Depends on the type of plant. If you cannot find the protocol online, you must use trial and error to determine this.

4. Rooting medium ?
See above.

5. Subculture period ?
See above.


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## kshitij

@edwardn:
yup...a deep course. But wanted to know frm the experience from the hobbyists.

@hazanfar Ghori :
Thanks for the information .
For the exact thing , i want to propogate Staurogyne sp. 
Another thing , can you give me any links where i can find some protocols online, this shall really help me a lot bro.
How many plants have you tried propogating via tissue culture ??


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## Ghazanfar Ghori

I've cultured Staurogyne sp. though I must say, TC is only worth the effort for slow growing / rare / hard to propagate otherwise type plants. 

In a nutshell,

Staurogyne sp. 
Sterilize stem fragments that have atleast 1 node, minus the leaves for 10 mins in 10% bleach, followed by a 5 minute rinse in distilled water.

Initiate invitro using
Standard MS at standard concentration, with 
30g of sucrose / liter
1 ml/l PPM
1 ml of 1 mg/l BAP
pH adjusted to 5.7
6.5-7g of Agar / l


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## kshitij

"TC is only worth the effort for slow growing / rare / hard to propagate otherwise type plants"
I completely agree with you .....

Coming on to the topic, have you obtained good results with this ??
I mean did you get good growth ? 

My main aim is to start TC for the following plants and propogate them in numbers  :
Staurogyne sp. 
Anubias rare species.
Echinodorus Rubin
Pogostemon Helferi

First i want to TC these then the more rare varities later....

Thanks again Ghazanfar Ghori for your response


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## SueNH

There is a lot of tissue culture done for the garden plants, hostas, daylilies and iris. Try looking up those.

There is also a lot of questions on whether it makes a true to form clone. You hear a lot of complaints about the plant not being quite right. Most I suspect are mislabling problems from the giant production houses that do it though rather than sports created by the tissue culturing.

A product called BAP is most commonly used.


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## cah925

I'm taking a micropropagation class like edwardn suggested. I'm lucky enough to have Dr Kane as my professor. He gave a speech on the subject at last year's AGA convention. Dr Kane hooked me up with a micropropagation scholarship and is giving me free reign in the lab to experiment with any plants I choose. I'm currently working on Erio cinereum. I'm looking to use this method for hard to find or hard/slow propagating plants also.
It really is about trial and error with most plants. I'm using 4 different Stage 1 mediums with various cytokinin levels to get the Erio to grow. I'm using 2 different types of tissues as well. The buds should develop into new plants as long as they don't get contaminated. I'm also trying to get pieces of leaves to propagate through non-zygotic embryogenesis. This process will take much longer as the cells have to de-differentiate, then form a meristemoid for plant development.
One problem with tissue culture is possible callus development usually formed due to high auxin:cytokinin levels in the medium. With callus comes undetermined genetic variability. All plants appear to develop correctly through the various stages, but the problems are not apparent until the plants begin to grow after transplant. We were told a story in class today about a banana farm in Jamaica. This farm ordered several thousand plants from a lab. Calluses formed in culture, but the lab did not handle the callus correctly. Everything was fine until the plants were planted in the ground. Some plants were distorted or stunted, others did not fruit, some just died from unknown causes.


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## kshitij

@SueNH:
Had a look with the garden plants , including sugarcane, actually my friend is more into this he understands it more perfectly but both of us are now looking for some solution 

@cah925:
"I'm taking a micropropagation class"  Even i should join you because i need some knowledge regarding TC .
Good to hear that you are working with Dr.Kane , please do keep us updated on Erio cinerum . By chance if you need any cinerum's do let me know i will send them 









Once the protocol is established ,i bet everything will go out perfectly.


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## Zapins

Wow, yes finally. This is the thread I've been waiting for. I bought some supplies a few months ago, failed at setting up a successful culture (contaminated). I'll have questions soon, just sleepy now.


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## cah925

k****ij said:


> "I'm taking a micropropagation class"  Even i should join you because i need some knowledge regarding TC .
> Good to hear that you are working with Dr.Kane , please do keep us updated on Erio cinerum . By chance if you need any cinerum's do let me know i will send them


pm sent



k****ij said:


> Once the protocol is established ,i bet everything will go out perfectly.


Developing protocol on something that hasn't been documented (at least publicly) is very challenging. Part of this process with Dr Kane is doing research on plants in the same genus to help develop a plan using various hormones. Finding info like this is also challenging and I'm having to resort to looking at how other non-related aquatics are propagated. The Erio is for a class project, but I now have an ongoing relationship with Dr Kane to use the lab at my disposal to work on other plants as well. We are both hoping to get a research article published on my work with aquatic micropropagation.


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## kshitij

PM replied.I will send them for you [smilie=n: [ no $ required ]

"Developing protocol on something that hasn't been documented (at least publicly) " - PM me 
Very nice to see you working hard ....I hope to learn something and looking forward to see your published article.......


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## Chuukus

Are you guys using 1mg of PPM in 50mg of water to disenfect explants and using PPM in the media. Or are you guys just using a Bleach or Alcahol to disinfect the plants. The last time I made my batches without PPM and had almost 80% contamination. The person I learned from doesnt use PPM but he told me I should have more luck with it.


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## Ghazanfar Ghori

PPM works nice. I use it in all my media, and a tad in the final rinse.


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## Chuukus

Thank you for sharing your protocall for Stargoune sp thats verry nice of you.


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## kshitij

You are right chuukus , very kind of him 
I shall surely give it a try and post my experience along with pics....
Thanks again Ghazanfar Ghori .......


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## Seattle_Aquarist

Hi All,

Ghazanfar is amazing! He visited us at GSAS for our January '09 meeting and did an absolutely excellent presentation on crypts with emphsis on emersed culture. I regularly visit his blog, he has become quite the TC expert it seems! Thank you Ghazanfar for sharing your experiences with us!


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## 954baby

Nice thread, when I get some free time I'd love to try some TC. Any recommendations on where I can go or books I can buy to get started? too bad I just graduated, I would have loved to take a class on TC.


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## Chuukus

954baby said:


> Nice thread, when I get some free time I'd love to try some TC. Any recommendations on where I can go or books I can buy to get started? too bad I just graduated, I would have loved to take a class on TC.


Here is a place to buy pretty much everything you need to get started http://www.kitchenculturekit.com/Catalog.htm there are other places also but I think your better off buying from here. I didnt and payed rediculious shipping cost. Go on you tube and do a search for tissue culture there is a guy that has a series of videos on the subject. Some advice take your tme learn as much as you can before you jump right in. This is all about trial and error dont get discouraged if you dont get results the first time and rememmber contamination is the enemy. Im no expert but if you have questions PM me and Ill gladly help if I can. Good luck!


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## Ghazanfar Ghori

Hey no problem! I'm happy to share info!
Check out this vid I made of Lagenandra meeboldi invitro
http://kryptokoryne.aquaticscape.com/2010/02/28/lagenandra-meeboldii-video/


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## Chuukus

Thanks for the inspiration Ghazanfar!


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## Chuukus

Ghazanfar Ghori said:


> I've cultured Staurogyne sp. though I must say, TC is only worth the effort for slow growing / rare / hard to propagate otherwise type plants.
> 
> In a nutshell,
> 
> Staurogyne sp.
> Sterilize stem fragments that have atleast 1 node, minus the leaves for 10 mins in 10% bleach, followed by a 5 minute rinse in distilled water.
> 
> Initiate invitro using
> Standard MS at standard concentration, with
> 30g of sucrose / liter
> 1 ml/l PPM
> 1 ml of 1 mg/l BAP
> pH adjusted to 5.7
> 6.5-7g of Agar / l


So there is no 2nd stage for rooting Stargoune sp. Just keep multiplying until your satisfied with the amount of plants you have then they can go straight into the tank. But the plants have to be in the emersed form to start the tissue culture? Do you culture all plants in there emersed form? Sorry for asking so many questions


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## Ghazanfar Ghori

Stage 2 is multiplication, Stage 3 would be rooting - and there wouldn't be much of a need to do it with Staurogyne sp. They can go straight into the tank.

Emersed form plants are generally easier to sterilize than submersed grown ones so its advisable to start with that.


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## fleshgear

i have tried tc 3 times now, with no sucess yet. the first time i tried using too many different types of plants. there was one echinodorus rose that started to grow but then like the rest they filled up with funky colors of mold.

the second time i only tried cryptocoryne wendtii. no success it molded within a week.

the third time which is still going. i tried a piece of anubias and crypt again. all but three has mold.

i have setup a control jar with every setup and i cut the agar and run my twezers just too see where the contaminats came from. the first two times were fine with no mold. this last time there is a tiny little spot of mold where i made a cut.

as for steralizing the plants i have tried (the first 2 times) a five min rinse and scrub with tap water, 10 min bath in 10% bleach, 5 min bath in 70% alcohol, 2 min bath in hydrogen peroxide, and rinse in distiled water.

the third time was a 20% bleach mix. and i also cut off all of the outside of the plant before i did all of the baths. i thought i would kill the plants for sure, but i was also hoping to kill the mold.

all of these plants were submersed, i have not tried any emersed plants yet. i have also tried african violets with every attempt. the violets are usually the last to get mold.

i have not tried bap or ppm


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## Chuukus

From what I understand PPM is not so harsh on the plant tissue as bleach or alcahol. I have read that 1ml of PPM in 49ml of water is a good mix to disenfect plants you can even soak the parts over night I would use bleach first rinse alcalol second rinse and PPM last final rinse. This is what I plan to do this weekend. Like you my first few times I had lots of contamination just recieved 50ml of PPM so well se how it goes.


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## fleshgear

does any one know where i can get ppm in Canada?
i found a few places from the States but the shipping is more than the ppm


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## Ghazanfar Ghori

Did you try getting the manufacturer to ship it directly?


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## fleshgear

who is the manufacture?

is it plant cell technology?


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## Ghazanfar Ghori

Yep. Plant cell tech.


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## hedson_25

i hope this help


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## Ghazanfar Ghori

Here's a pic of Staurogyne sp. in tissue culture.


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## Seattle_Aquarist

Hi Ghazanfar,

Nice healthly looking plant you have there!


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## Ghazanfar Ghori

Thanks! Pretty easy to grow, invitro and otherwise.


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## Chuukus

Ghazanfar Ghori said:


> Thanks! Pretty easy to grow, invitro and otherwise.


I have some thats two weeks in but cant see any growth So far no contamination thats a good sighn. Same thing with Anubias Petite I keep trying with no sucsess. How many weeks old is the jar in the picture?


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## fleshgear

my last attempt had all of them contaiminated. except for one, it was a crypt. but there is no growth. i killed the plant.


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## Ghazanfar Ghori

Chuukus said:


> I have some thats two weeks in but cant see any growth So far no contamination thats a good sighn. Same thing with Anubias Petite I keep trying with no sucsess. How many weeks old is the jar in the picture?


Well, thats a hard question to answer. I started this last year, but just as an experiment to see if I could sterilize it without killing it. Never intended to really grow it much. So, it sat there, in the same jar, for a year. Nutrients ran out. The plant shriveled down to almost nothing. Three weeks ago, I gave it a new jar. It was its birthday after all. It liked its birthday present.


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## Ghazanfar Ghori

fleshgear said:


> my last attempt had all of them contaiminated. except for one, it was a crypt. but there is no growth. i killed the plant.


The most difficult part - kill everything off the plant, but the plant itself. Patience and keeping careful logs helps you narrow down the sweet spot.


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## kshitij

Hello all,
sry for the late comeback.....

@Ghazanfar Ghori :

:hail:


I am not into TC by myself much, so half knowledge but i am tyring to learn....We tried the follwoing plants :
1.Anubias nana(3 diffrernt tubes) - Contaminated.
2.Staurogyne sp.(2 different tubes) - Contaminated.
3.Hydrophilla compacta species - Contaminated.

Now my staurogyne sp. will arrive soon and we shall try the protocol suggested by Ghazanfar Ghori.


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## Ghazanfar Ghori

Grow the plants emersed in clean environment first - that'll help reduce risk of contamination.


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## kshitij

I have set the above Staurogyne sp. picture are my desktop background, will give me inpiration everytime i see it


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## Ghazanfar Ghori

Lol!


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## doubleott05

when they are in the jars when do you need to start adding light?


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## cah925

Generally, light starts immediately after you put the cultures in jars. There are some exceptions like potato micro tubers.


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## dabrybry

Saving to view later


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## Seattle_Aquarist

Hi dabrybry,

Also check out Ghazanfar's blog for some additional pointers. GSAS invited Ghazanfar to Seattle a few years ago and he did an outstanding presentation on emersed grown crypts and touched on what he was doing with tissue culture. AGA has a video of a tissue culture workshop at their 2010 convention.


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