Gabe15

For most Musa seeds, if they are fresh or soaked in water for a few days prior to cutting, the seeds will cut easily with a scalpel. If they are dry, I use tweezers to hold down the seed while I spear the seed next to the micropyle with the tip of the scalpel, once its in deep, I press down on one side to form a crack in the seed and it usually splits nicely and the embryo either falls out or is easily dug out.

For Ensete, Musella, Musa ingens and other thick shelled species, I haven't found a good reliable way yet, but Frank seems to have a good idea with the vice clamp, I bet that would make opening those types really easy.

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Growing bananas in Colorado, Washington, Hawaii since 2004. Commercial banana farmer, 200+ varieties.

mrbungalow

Well,

Tried this yesterday, and now some more or less sterile embryos are lying on banana multiplication medium.

In sikkimensis I couldn't even find the embryos, all that came out of the seeds was white powder. So no sikkimensis are tested this way.

A little more easy with ensete ventricosum. There I could find the embryos pretty easy. I guess time will show if it works.

How long should I give them?

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Gabe15

You should know whether they are growing or not in 2-4days. They will first begin to swell and after a week or so you may start to see the beginning of a leaves and a root. Can you post some pictures of your cultures?

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Growing bananas in Colorado, Washington, Hawaii since 2004. Commercial banana farmer, 200+ varieties.

microfarmer

Do you have these on a heat cycle like normal seed germination, or a steady setting? I'm thinking about 85 degrees?

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My new hero...

mrbungalow

I keep them in constant 27 degrees C, as I've heard this is the ideal temp. for cell-activity in most musa species. I wouild think this is the naked embryo and doesn't contain the other seed structures that inhibit germination. I doubt changing temps is neither necessary or ideal for getting cells in an embryo to start dividing. (Gabe might know more)

Gabe, will post pics soon! Holding my breath to see what will happen!

Regards
Erlend

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Gabe15

As long as they are not in a cold room, they will be fine, temperature does not matter much, just keep them somewhat warm.

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Growing bananas in Colorado, Washington, Hawaii since 2004. Commercial banana farmer, 200+ varieties.

marksbananas

I ma after some advice what inside the seed is the embryo ?

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Just Bananas about Bananas

Chironex

Frank, how do you keep contamination out of the embryo when using this method? Do you sterilize the vise first? Is it inside of the hood? I have a small wooden vise that is used for seal stamp carving (Chinese seals) if I can find it, I may use that under the hood since it is very small, already has a V groove to hold the seed, and uses a small screw to keep from over-cracking the seed. I would think a small C clamp might also be good since it can be sterilized easily. I might also get some fine tipped needlenose pliers to pull apart the seed hull pieces once they crack. Another idea would be to use a rotary tool (Dremel) with a fine saw blade - this might be where the vise would come in handy.

So far no contamination in the 5 embryo rescues I have going. Looks like the embryos are swelling some, but they have only been in culture since 10/5.

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Scot

Chironex

I never thought of using the spear technique, I have been trying to use the scalpel like a steak knife. I will have to try your method, but I also think the seed cracking vise has some merit. Thanks Frank and Gabe for the ideas.

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Scot

Chironex

Well, the ingens embryo has contamination on the latest one that I attempted to rescue. I threw caution to the wind, opened the jar and sprayed it with Physan 20 to see if I can halt the contamination. We will see if that works.
I am going to wait on any further embryo rescue attempts until I can make some Nitsch & Nitsch medium modified for embryo rescue specifically. I am also going to use sterile coconut water and activated charcoal to see if I can get the endogenous contaminants out of the embryos. Could sterilize with silver nitrate too, might try it.

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Scot

Chironex

The second trial is underway. I prepared the modified N&N medium with only 2 exceptions, first I added PPM and secondly, I used sucrose in place of maltose.
The biggest challenge I have is getting embryos out intact, those guys are tiny! The seeds I have are quite fresh, too, so it may be that the embryos are not fully mature. I have read something about embryo maturation. From what I recall, this explains why some of the older seeds actually germinate and fresh ones do not. This is contrary to what I understood, but I only scanned the article, so don't take this for gospel.
This medium is quite dark due to the activated charcoal used in preparation. I will post some photos in the gallery.
Keep your fingers crossed!

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Scot

Gabe15

The other day I put in embryos of M. sikkimensis, M. nagensium, M. cheesmanii, M. 'Darjeeling Giant', M. 'Helen's Hybrid' and Ensete perrieri. I aimed for 5 of each but some of them were bad, damaged or lost during excision. I am using normal MS for the time being until the N&N ingredients come in. No germination yet, but its only been 3 days. I've had them start to grow after 2 days and take as long as 7. It seems that if they do not begin showing even small signs of growth after about 1 week then they might not be viable, this however is just based my own (few) observations.

Since almost all of these species have very thick and hard seed coats, I used the pliers on a leatherman to crack them open. It worked very except that some are so hard my hand would hurt after a while and I would have to take a break!

With enough practice I find it easy to find the embryos because they are always a different color from the endosperm, usually an off-white to slightly yellowish color and shaped like a mushroom.

Some embryos actually do well with being placed into TC media while immature. In some hybrid experiments (I know in particular with papaya), embryos would not mature properly so the seeds could not be planted. However the immature embryos could be rescued and would grow in vitro.

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Growing bananas in Colorado, Washington, Hawaii since 2004. Commercial banana farmer, 200+ varieties.

griphuz

So is the idea that one embryo gives one plant, or do you want to devide the growint cells afterwards before producing plants?

And do you sterilise the embryo's before putting them in the culture?
Kind regards,
Remko.

Chironex

First we get the embryos growing, then they are divided once they start developing shoots. At that point, they are like normal TC plants.

I sterilize the seed surfaces first, then crack them open. The embryos are supposed to be sterile while inside the seed. I do as much of this under the hood as possible to minimize the chances of contamination. Sometimes you win, sometimes you lose.

I was not pleased with the first jar of embryos, so I did another set of them in another jar. This time I was pleased with the embryos I was able to extract. I do not recommend soaking them as this seems to make it more difficult to dissect the embryos. It seems easier to find them and get them out if the seeds are dry.

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Scot

Richard

Ah! Exactly the information I need about my papaya seeds.

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Back in business at plantsthatproduce.com

Chironex

Just put the rest of my E. perrieri seed embryos onto the new medium this evening. These are much easier to find than ingens embryos. Big difference when they are dry versus soaked! Once the water hydrates the endosperm, I found it very difficult to locate the embryos, even though I knew where they were supposed to be.
I believe I had 12 E. perrieri embryos that made it to culture. I am sure Gabe has not mentioned how many go flying across the room when you crack them sometimes! (hahaha!) I probably have 4 embryos in carpet medium now! Not holding much hope for their survival, although the carpet does have plenty of dirt, who knows!
I will try to post some pics, but my camera does not have quite the resolution that Gabes has.
Well, back to the TC's, I have 16 plantlets to divide, but first I need to prepare more medium and fill some jars. Guess I will be here for awhile.

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Scot

griphuz

Nice Scot!

So you get 1 plant out of 1 embryo usually, OR you have to invoke shoot/sucker formation in a special medium or something?
Is that more difficult with Ensetes compared to Musas (because Ensete normally doesn't do so as a plant)?
Kind regards,
Remko.

Gabe15

In my trials, depending on the species and how much hormone is in the medium, sometimes they will germinate normally or sometimes they will form many shoots right away. Either way, depending on the level of hormones you put into the medium you can control how much they sucker. I have germinated embryos that could be transferred directly to soil but I prefer to subculture them and create more plants before planting them in soil in case anything happens to them. It's always good to have backups.

I haven't had much experience tissue culturing Ensete (just do to availability), however when I got an E. glaucum embryo to germinate in vitro, it grew very strangely and I could never regenerate any plants from it. I grew some E. ventricosum from meristem tip culture for a short period of time and they seemed to grow normally but I would have to try more to really compare them. The tissue culture process can be made very difficult from the differing habits of different varieties in culture. Some banana varieties are very easy and "behave" well while other varieties are very slow, grow out of control, or grow in strange and hard to deal with patterns.

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Growing bananas in Colorado, Washington, Hawaii since 2004. Commercial banana farmer, 200+ varieties.

Chironex

Gabe, I have some protocols for TC'ing Ensete that I came across in research articles. Let me know if you want them.

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Scot

Chironex

The embryo rescues continue with Ensete and Musa. I am experiencing a lot of what seems to be endogenous contamination. Next I will try sterilization using 1% silver nitrate instead of bleach solution. Dr Frederic Bakry has sent a research article about his procedure and reports little to no contamination using this technique. Another Musa researcher has also corroborated this finding.
Some are still in good shape and a few appear to be doing something, but it is still a bit soon to expect significant changes in the embryos.

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Scot