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-   Tissue Culturing & Other Propagation Techniques of Banana Plants (http://www.bananas.org/f260/)
-   -   Tissue Culture (http://www.bananas.org/f260/tissue-culture-4755.html)

51st state 06-06-2008 10:36 AM

Tissue Culture
 
following on from the Ensete Perrieri thread... Is there sufficient demand from .org members to jointly put some of the more unusual species into TC?

I seem to remember a number did this with Siam Ruby a while back. maybe we could create 'wishlists' and see what level of demand is out there.
I know that there are specialists out there doing this but it would be a great way for .org to spread some species out more widely across the globe.
any thoughts? :2722:

Gabe15 06-06-2008 11:02 AM

Re: Tissue Culture
 
Iʻm working on it right now, I have a whole setup in my house ready to go, Iʻm just waiting for my media to come in and hopefully I can start producing some. I already have much of my collection in Hawaii in TC already (including some very unusual species, and many plants not yet available on the market), when I get back there I plan to start selling plants by January hopefully.

Chironex 06-06-2008 11:03 AM

Re: Tissue Culture
 
Kev, I couldn't agree more. I am reading up on TC technique and will be able to do it in a few months or less. I was discussing this with Gabe too. It will definitely help the banana world and I want to be part of that.
Once I get set-up to where I know what I am doing, I will be offering this service to our membership. I am not sure just yet, but I think that taking the tissue from the explant can be done without causing too much injury. From what I have read, tissue is taken from the meristem, which is either growing root tips or in shoots. So, it shouldn't be that invasive as to cause unrecoverable injury to the source plant (explant).
Just need to get a comfort level on my own stock first. Then Dr Frankenstein will be ready to party!!!!

AltadenaGarden 06-06-2008 11:51 AM

Re: Tissue Culture
 
Would anyone like to post a good link about tissue culture for bananas. I am not looking to do it but am interested in how it works.

chong 06-06-2008 12:38 PM

Re: Tissue Culture
 
Quote:

Originally Posted by AltadenaGarden (Post 39536)
Would anyone like to post a good link about tissue culture for bananas. I am not looking to do it but am interested in how it works.

Here's one from one of our members, kgbenson:
Micropropagation of Musa

Richard 06-06-2008 01:42 PM

Re: Tissue Culture
 
It turns out that different generas of plants require different kinds of tissue culture. In other words, some plants are more troublesome than others. Here's a paper I came across while researching the origins of some pineapple guava varieties:

ScienceDirect - Scientia Horticulturae : Improvements in somatic embryogenesis protocol in Feijoa (Acca sellowiana (Berg) Burret): Induction, conversion and synthetic seeds

Mark Hall 06-07-2008 03:11 AM

Re: Tissue Culture
 
Chong, Thanks for the link. Very interesting reading. I did think there would be more pictures after the meristem was removed showing various stages of growth.

damaclese 06-07-2008 09:07 AM

Re: Tissue Culture
 
Quote:

Originally Posted by Gabe15 (Post 39526)
Iʻm working on it right now, I have a whole setup in my house ready to go, Iʻm just waiting for my media to come in and hopefully I can start producing some. I already have much of my collection in Hawaii in TC already (including some very unusual species, and many plants not yet available on the market), when I get back there I plan to start selling plants by January hopefully.

Gabe Scot and i are looking for a Banana called Mysor its appears to be vary drought and heat tolerant if we got one do you think any one on the org would be interested in Tc of them?

Chironex 06-07-2008 09:16 AM

Re: Tissue Culture
 
Pauly, I forgot to mention to you that I have made arrangements for 2 Mysore and have another contact for more if needed. PM me with your address if you want and I can have them sent to you before I get there.

Richard 06-07-2008 10:08 AM

Re: Tissue Culture
 
I have heard from other Musa (fruiting) growers and read a few articles indicating that Tc of Musa usually produces a new cultivar of the plant and less often a plant that is completely true to form of the parent. What are your experiences with this?

Chironex 06-07-2008 10:50 AM

I am not an expert yet, but this contradicts what I have read. The clone produced through micropropagation is an identical copy of the parent (as are all other plantlets multiplied from the same explant.) The only possible way that this can be deviated is through contamination or mutagens deliberately applied to the culture.

Gabe15 06-07-2008 11:45 AM

Re: Tissue Culture
 
Quote:

Originally Posted by damaclese (Post 39631)
Gabe Scot and i are looking for a Banana called Mysor its appears to be vary drought and heat tolerant if we got one do you think any one on the org would be interested in Tc of them?

Mysore is a very common banana, it shouldnʻt be too hard to find. Going Bananas has them. Other common names for it is Pisang Ceylon and Misi Luki, different accessions may vary slightly but they are all the same basic cultivar. If theres that much interest in it, I could look into TCʻn it later on.

Quote:

Originally Posted by Chironex (Post 39654)
I am not an expert yet, but this contradicts what I have read. The clone produced through micropropagation is an identical copy of the parent (as are all other plantlets multiplied from the same explant.) The only possible way that this can be deviated is through contamination or mutagens deliberately applied to the culture.

Quote:

Originally Posted by Richard (Post 39648)
I have heard from other Musa (fruiting) growers and read a few articles indicating that Tc of Musa usually produces a new cultivar of the plant and less often a plant that is completely true to form of the parent. What are your experiences with this?

This is not true, though there is some truth in it. Bananas can and do indeed mutate during micropropagation, however precautions can be taken to lower the rate of mutation. In the field, bananas mutate naturally, this is the reason we have so many cultivars and different varieties in the same groups. In micropropagation, you are generally dealing with many plants at a time (I produce about 300-400 explants in 3hrs, you can see it adds up), and forcing them to grow and proliferate rather quickly. Usually, in a very small number of your explants, there is some mutation. To reduce this, in our lab, we will only subculture a plant 5 times after initiation (this results in about 4,000 explants per original plant). If we run out of explants for that variety, and have tissue subcultured all of the lines 5 times, we will then go collect a new plant from the field, preferably one that was not grown from tissue culture or if not available, has been in the ground for at least 2 years. If a lab continues to reuse the same material over and over, eventually the mutation rate would be unreasonably high and you will see many "off-types". Some mutations can be beneficial (such as dwarfing or increased yield), but most often they are not. Even with these precautions, from time to time a farmer will find an off-type in their field, but tissue culture still has many advantages over traditional propagation.

Richard 06-07-2008 11:52 AM

Re: Tissue Culture
 
Thanks Gabe for that excellent clarification.

D_&_T 06-07-2008 05:39 PM

Re: Tissue Culture
 
Here is a link another member posted a while back.

Kitchen Culture Kits - Intro Page

Gabe15 06-07-2008 07:57 PM

Re: Tissue Culture
 
Quote:

Originally Posted by Mark Hall (Post 39601)
Chong, Thanks for the link. Very interesting reading. I did think there would be more pictures after the meristem was removed showing various stages of growth.

Iʻll try to find some pictures, but in the meantime, I made up this diagram showing basically what happens after the initiation (when tissue culture becomes micropropagation). You will come across inconsistencies in actual practice, such as dead explants, contamination, varying degrees of proliferation, size of regenerated meristems and duration between meristem regenerations. But its generally what happens, or at least the way I do it is.

NOTE: The plants do not actually get smaller and smaller, I just only had a finite working space to show the examples.

mm4birds 06-07-2008 08:47 PM

Re: Tissue Culture
 
good luck on this project. hopefully it will make it easier to obtain great plants for our own collections and prevent some of the rarer plants becoming extinct.:nanablowskisses:

Richard 06-07-2008 08:49 PM

Re: Tissue Culture
 
I received the copy of "Plants From Test Tubes" by Kyte and Kleyn today and started reading it. So far the writing style and information content is excellent.

Chironex 06-09-2008 10:54 PM

Re: Tissue Culture
 
Richard I got the same book from Amazon last week and I'm reading it too. I have also made contact with the Doctor that runs the Kitchen Culture website. She has sent me some more info as well as given my email to her associate who specializes in bananas. I must have downloaded a gazillion pages of info on TC already. Now if i could just find the time to read all of it. Burned through a fresh ink cartridge already.

Richard 06-09-2008 11:34 PM

Re: Tissue Culture
 
Quote:

Originally Posted by Chironex (Post 40008)
Richard I got the same book from Amazon last week and I'm reading it too. I have also made contact with the Doctor that runs the Kitchen Culture website. She has sent me some more info as well as given my email to her associate who specializes in bananas. I must have downloaded a gazillion pages of info on TC already. Now if i could just find the time to read all of it. Burned through a fresh ink cartridge already.

:) I would take all those pages you printed from the internet and throw them into the paper recycling. :) No really, some of them probably are worthwhile, but for any research in just about any subject: first read a thorough, well-rated book like the one we have. 2nd, if the book does not contain what is referred to as "survey of the literature", then find one and read further on topics as needed.

Also I would caution that reading the "how to" of Tiissue Culture is a bit like reading how to be a master cabinet maker. I expect it is going to take me a lot of patience and practice, including going through the amateur, apprentice, and journeyman phases.

sandy0225 06-10-2008 06:07 AM

Re: Tissue Culture
 
Yes, I've been on the home tissueculture webgroup for quite some time now >2 years, and I've learned a lot just from what others are posting. It seems the biggest issue is getting the right chemical blend for propagation of particular plants, they do recommend certain chemicals for bananas, but getting them "dialed in" can be a challenge. Also the biggest problem is contamination. Everything has to be sterile and kept that way. Even the smallest particle of dust contains thousands of different contaminants from bacteria to fungus.
I haven't actually tried tc yet because of lack of time issues, maybe this winter. I said that last year too, though.
I did get a cloning machine though, that sounded very do-able to me. I'm supposed to get it today.

Chironex 06-10-2008 06:29 AM

Re: Tissue Culture
 
Quote:

Originally Posted by sandy0225 (Post 40028)
I said that last year too, though.
I did get a cloning machine though, that sounded very do-able to me. I'm supposed to get it today.

A cloning "machine"? Do you have a link to it? I would be interested to see it. What does it do?

musa_monkey 06-10-2008 06:59 AM

Re: Tissue Culture
 
This maybe ?

Hydroponics Cloning Machines - Plantlighting Hydroponics & Grow Lights

MediaHound 06-10-2008 09:38 AM

Re: Tissue Culture
 
Looks like this thread is really blowing wind into some sails!!
I'm elated to see all the interest everyone has into tissue culturing and bringing some of the more rare bananas into production. I have always had an interest in TC'ing but have never taken the initiative. Perhaps watching the success of our members will help convince me to find the time and space and set it up properly.

Quote:

Originally Posted by Gabe15 (Post 39687)
Iʻll try to find some pictures, but in the meantime, I made up this diagram showing basically what happens after the initiation (when tissue culture becomes micropropagation). You will come across inconsistencies in actual practice, such as dead explants, contamination, varying degrees of proliferation, size of regenerated meristems and duration between meristem regenerations. But its generally what happens, or at least the way I do it is.

NOTE: The plants do not actually get smaller and smaller, I just only had a finite working space to show the examples.

This is great! Thanks for taking the time to create it.
BTW, I copied it over into the banana diagrams category in the gallery.

Thread stuck by the way, everyone, so we can use it as a general reference and a place to point people to.

We do have a page in the wiki about tissue culturing, it's here:
Info:Tissue Culturing - Bananas Wiki
It's fleshing out, but these things take time. Feel free to add to it, we can use both this thread and the page in the wiki as a springboard for information.

Cheers

Dean W. 06-10-2008 11:15 AM

Re: Tissue Culture
 
I want to keep track of this thread. I have an Ensete perreri that might be TC material.

Chironex 06-28-2008 02:06 PM

Re: Tissue Culture
 
Dean, keep in mind that TC'ing a banana requires the original pseudostem to be destroyed. We dissect the apical meristem from the base of the pseudostem. You might be able to re-grow from the corm.
There is always a risk of contamination in TC work, however small the percentage (Less than 2% hopefully), so always keep a back-up, just in case. I have several Ensete perrieri seeds in germination, so if I get a decent germination rate, I plan to TC one of them.

Richard 06-28-2008 03:24 PM

Re: Tissue Culture
 
I thought that banana TC has been successfully initiated from growth tips and cambium material in rhizomes. Is this true and just not a preferred practice, or ?

Chironex 07-03-2008 04:57 PM

Re: Tissue Culture
 
I thought all might be interested to know that I have been emailing the guy who created Little Prince. It is a sport that was created in TC from 4 mutants. It is patented as a variety, so TC'ing it will require permission. I will invite him too join our forum.

mskitty38583 07-04-2008 12:23 AM

Re: Tissue Culture
 
that would be awesome scot!

Chironex 07-04-2008 10:43 AM

Re: Tissue Culture
 
Update: He just emailed me that he will be joining as soon as he gets his computer back online. Apparently a Microsoft security update has locked him out of his desktops and he is having it repaired.
Bottom line is he will be joining us soon. His name is Randy, but I don't have his last name.
It will be exciting to include him among our membership. It will also be helpful to anyone who develops their own sport and seeks to have it patented, from someone who knows the ropes.

Micronana 08-07-2008 03:26 AM

Re: Tissue Culture
 
Hi New member here,
The diagram from Gabe 15 was brilliant, I am microing musa basjoo, dwarf cavendish and ensete v. maurelii (although the last 2 are new to me, and only just initiated).
I have never tried being quite so brutal with them, I usually just cut them down the middle. Might try quartering some in future.
By the way, I've recently read that the ensete don't produce pups spontaniously. Mine in culture have already started sprouting! I'll try damaging a few anyway to see the results. Has any one got any experience of them?

Thanks Dawn

Gabe15 08-07-2008 07:35 AM

Re: Tissue Culture
 
Quote:

Originally Posted by Micronana (Post 47792)
Hi New member here,
The diagram from Gabe 15 was brilliant, I am microing musa basjoo, dwarf cavendish and ensete v. maurelii (although the last 2 are new to me, and only just initiated).
I have never tried being quite so brutal with them, I usually just cut them down the middle. Might try quartering some in future.
By the way, I've recently read that the ensete don't produce pups spontaniously. Mine in culture have already started sprouting! I'll try damaging a few anyway to see the results. Has any one got any experience of them?

Thanks Dawn

Hello and welcome Dawn,
Ensete do not naturally produce pups in the ground, but in culture they will proliferate like normal with the benzyladenine in the media.

51st state 08-10-2008 04:15 PM

Re: Tissue Culture
 
Hi Dawn
welcome aboard the best banana site in the world!!!!!!!!!!!!!

great to see you trying TC'ing some nana's. If you can pull it off I've got a few species you might want to have a go at

Mark Hall 08-10-2008 05:14 PM

Re: Tissue Culture
 
Me too! Welcome by the way:waving: Where are you in the UK?

Micronana 08-19-2008 04:48 AM

Re: Tissue Culture
 
Hi 51st State
I'd be interested to hear what varieties you've got going

and Mark Hall
I'm located near Bognor Regis on the south coast.

Work to do - ho hum

Dawn:bed:

51st state 08-20-2008 11:46 AM

Re: Tissue Culture
 
Hi Dawn
see my profile, which lists the species/varieties I'm growing

kgbenson 08-24-2008 04:28 PM

Re: Tissue Culture
 
Quote:

Originally Posted by Mark Hall (Post 39601)
Chong, Thanks for the link. Very interesting reading. I did think there would be more pictures after the meristem was removed showing various stages of growth.


Yeah - I just never got around to taking many or to posting them. Lemmie look back into some of my files and if I can find them I will try to get them on there.
Keith

griphuz 08-28-2008 09:58 AM

Re: Tissue Culture
 
Maybe this article is usefull for the Ensete's?
http://www.barc.ernet.in/webpages/le.../200210-24.pdf
Regards,
Remko.

Chironex 09-01-2008 12:35 AM

Re: Tissue Culture
 
I know that there are some of us who are doing micropropagation. I would like to see if there is any interest in combining our orders from various suppliers to buy in larger quantities. As an alternative, we could approach vendors for a Bananas.org membership discount and buy at wholesale pricing.

We could do the same with Agri-Starts, Phytotech Labs, etc. All they could do is say no. I just ordered more TC culture jars and noticed a huge discount if ordering by the hundred lot rather than 25 lot. Savings is almost 20% in just this example. Larger quantities could be greater obviously. M&S media is 12-20% lower in quantity.

Anyone interested? Let's talk about it and see if there is enough interest. We have numbers of members into this or interested in TC. Let's make use of it - a nice membership benefit in my opinion.

damaclese 09-01-2008 10:40 AM

Re: Tissue Culture
 
wow! the institute for advanced banana research funded and supported by bananas.org. Interesting concept! Scot every time I read one of your threads the possibilities just explode in my mind.!!!

51st state 09-02-2008 04:33 PM

Re: Tissue Culture
 
agree with all that. got to be a way of having members subscribe to wanting a particular species/variety before you TC it so that you can gauge demand and have customers ready and waiting before you start. Not sure how people like agristarts would react to 'hobbyists' clubbing together though... I'm in

damaclese 09-03-2008 09:22 AM

Re: Tissue Culture
 
well market demand i say if agrastarts wants to keep are business then they should cut down the minimum order to accommodate hobbyist but i really don't think they are all that interested in small orders or they would have adapted already? besides i think we are all capable of taking care of each others Banana needs. I'm sure if we took and inventory of all the plants and supply's that every one on the org had we would find that theres more then adequate stocks to keep every body happy for years!

rohsen 11-09-2008 06:29 PM

Re: Tissue Culture
 
2 Attachment(s)
hello there you all im from amsterdam i make aliving tissue culturing plants and would like to share the following files with you, for anyone trying to micropropagate banana it is essential information!!!

here goes...

Chironex 11-09-2008 08:57 PM

Re: Tissue Culture
 
rohsen, thank you for sending these protocols and propagation media formulations. Do you have a recommendation list of which medium to use for best results on different types of banana plants? Do you have a method for preparing the medium?

rohsen 11-10-2008 02:32 AM

Re: Tissue Culture
 
as im just getting started on musa spp im not sure yet, i do know some cultivars respond well to different formulations, banana is known as an easy species! what i usually do and i advice you the same is check the references given with the media, you can google this reference and see what comes up. if i cant find info i just set up trials using different media, i make my choices depending on what ingredients i have available what growth regulators (hormones or plant growth regulators PGR's for short such as 6-BAP GA3 IBA IAA NAA) required and the price of growth regulators. as far as media preparation goes i usualy buy pre-formulated media such as MS , MS gamborg, Rugini olive medium etc. only rarely do i formulate the media myself. (its a hastle) I only use agar in the initiation of cultures to screen for contaminants, after initiation i use liquid media in a temporary immersion system. any advice on media for musa basjoo ? im looking into propagating this species im also interested in ensete ventricosum

regards
Dave

in my gallerie pictures of my modest lab

Chironex 11-10-2008 03:31 AM

Re: Tissue Culture
 
Dave, I am also just beginning to tissue culture bananas. I use pre-measured M&S medium and add BAP for initiation and shoot proliferation. For rooting, NAA is commonly used. I have not yet tried Basjoo or E. ventricosum. I have 3 varieties in culture now as well as trials of 3 different bananas in embryo rescue. For these I want to make a modified N&N medium which is reportedly more robust for embryo rescue.
Photos of my laminar flow hood are in my gallery, too.

Gabe15 11-10-2008 04:51 AM

Re: Tissue Culture
 
Some banana varieties are very slow in the normal media (such as Lakatan, Fe'i bananas, some Callimusa and Australimusa species), so to help them grow I use full strength MS (instead of the normal 1/2 strength), and its also been recommended to use 30g/L sucrose, currently we are using only 20g/L for normal micropropagation. There is also an issue with different varieties reacting adversely to BA, some varieties proliferate too much and go through some type of dedifferentiation to the point where I cannot regenerate usable explants from the tissue (perhaps it is possible with somatic embryogenesis, but that would be a whole different project...), so for these bananas that have reacted poorly to the BA (E. glaucum and Fe'i varieties), I will experiment with using little or no PGRs, especially upon initiation.

rohsen 11-10-2008 07:24 AM

Re: Tissue Culture
 
2 Attachment(s)
First of all nice to meet you guys!! Gabe, for species proliferating too fast you may want to consider using a growth retardant such as Paclobutrazol. im attaching a publication on use of this and other substances with musa. you'll find it an interesting read! as for embryogenesis .. not so much a different project as you think, very do-able especially if you have a protocol and media formulations.....! which are , naturally , attached!

I dont see to much here on temporary immersion systems, and if there ever was one technique for making loads of musa ....
I have used it succesfully with papaya pineapple and other species.

regards

Dave

Gabe15 11-10-2008 12:22 PM

Re: Tissue Culture
 
Quote:

Originally Posted by rohsen (Post 56839)
First of all nice to meet you guys!! Gabe, for species proliferating too fast you may want to consider using a growth retardant such as Paclobutrazol. im attaching a publication on use of this and other substances with musa. you'll find it an interesting read! as for embryogenesis .. not so much a different project as you think, very do-able especially if you have a protocol and media formulations.....! which are , naturally , attached!

I dont see to much here on temporary immersion systems, and if there ever was one technique for making loads of musa ....
I have used it succesfully with papaya pineapple and other species.

regards

Dave

Thanks for the papers Dave. I would love to try all of these things out one day, but I've been so busy that I don't have time to be making different media for side project this semester, I just use what our lab produces for normal micropropagation for the time being. Hopefully I will have more free time next semester and can experiment more with different media for things like embryo rescue (I use the normal microprop. media which works sometimes, but not always great), somatic embryogenesis, and these hard to culture Musa.

Chironex 11-10-2008 02:40 PM

Re: Tissue Culture
 
Dave, I have read some about liquid media, but from everything I have read, it seems more expensive in terms of equipment, for the hobbyist like me. Is this the same process as developing callus culture and so on? Is there an inexpensive way to set-up this process for a home lab?
Thanks for the research articles.

rohsen 11-10-2008 06:53 PM

Re: Tissue Culture
 
1 Attachment(s)
Scot, indeed the initial cost will be higher than when using babyjars plastic jars or other disposables, however the results are amazing, and after the initial layout the returns wil justify the expense, in some species the multiplication rates will improve by 500% !!! for commercial labs these numbers are .... well you can imagine. added to this are less handling, no use of agar, and especialy in temporary immersion systems you get partially aclimatized and sturdier plants, every time you immerse the plants in liquid media you also renew the air in the culture vessel wich has considerable benefits. I distribute a TIS system in europ and south america wich includes an airpump culture vessels, the works. for 40 1 liter vessels including pump airfilters and all you need. would set you back at least 2500 euros, you would than be able to produce 10s of thousands of plants. so this is for pros, however it is possible to build a set yourself using lowcost options.
attached a paper listing lowcostoptions for developing countrys. and since a home TC lab, and indeed many commercial labs, needs to watch spending it will have some interesting options.

basicly if you can micropropagate conventionally...you can use bioreactors. you would first propagate the old way, and then seed your bioreactor with the product after you established your cultures in agar. see it as the next step towards lots and lots of plants

hope you enjoy the papers,

regards

Dave (who has no university education whatsoever started out as a hobyist)

ps. in this publication you will find in the chapter BIOREACTORS AS A LOW COST OPTION FOR TISSUE CULTURE a discription on how to build a very cheap aparatus that wil absolutly do the trick. ofcourse if anyone cares to try i will happily assist!!


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