Mutating and Breeding Grapevine in a Limited Space Through the Use of Bonsai Techniques

Frithfolk

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Lol, what a mouth-full! So, this is going to be one lengthy post and I will be updating it continuously over the next few years as the breeding program progresses.

First, a bit about myself: I'm just some guy. I'm not a scientist and I have no experience with bonsai. Everything I am about to attempt is theoretical and based on papers I've read over the last few decades. You see, I've been wanting to do something like this for a long time but never committed. Then, today, I bought all of the supplies and decided to jump in head-first.

Well, what is it that I'm wanting to do exactly? I want to breed plants. I want to create new varieties that people have never imagined. However, I live in China in a little box high up in the sky and, as such, cannot just plant a thousand plants our in my yard. My plan to get around this? BONSAI!

So, let's just jump into the fray, shall we?

The variety I will be using is Vitus Amurensis, the Amur Grape. It's very sour but is lacking in tannins and sugar. What I wish to do is to subject it to a treatment of chemical mutagens and selectively trim away normal growth whilst keeping the visually mutated growth. Then, after fruiting begins in a couple years, use the seeds from the fruits that are most desirable for wine making and restart the program. The old plants will be rooted out in the community, sold online, or given to friends. Finally, when I have the characteristics I want, I will induce polyploidism and then backcross to the diploid mother to produce seedless triploid grapes.

Simply put, I want to take these wild grapes and try to make them into a tiny, seedless, tannic, sour, and sweet wine grape.

Here's the process I'm planning on:

The Seeds

grapes.jpg
Grape seeds normally go through a dormancy period before sprouting and to get them to sprout without one requires special treatment.
Here's my planned treatment:

Bathe in distilled water for 12hr.
water-bowl-2328122.jpg

Then, remove and soak in 3% hydrogen peroxide for 24 hours (Approx. 1 mole I believe)
peroxide.jpg

Next, I will remove the seeds and leave them under dripping tap water in a muslin cloth for twelve hours.
Then, I will put them into a bowl of 0.15% giberallic acid, 1% ethyl methanesulfonate, and water for 24 hours

ga.jpgems.jpg

After this, I will remove them, rinse with distilled water and refrigerate them at around 3 Celsius for a week.

fridge.jpg

The Grow Area


For the grow area, I will be using a commercial storage stand. 150*60*200 cm
stand.jpg

The lights will be double row LED 36 watt full spectrum pink-white with an output of 167 umol. Each layer will contain two rows for a total of 334 umol for an area of 120 x 54 cm.

lights.jpg

The seedlings will be potted in 21 count 54x28cm trays. Each plant will have a root growing area of around 6.5 x 6.5 x 5.5 cm.
trays.jpg

They'll be rooted in a mixture of the local dirt and coco peat.
cocobrick.jpg
 

Frithfolk

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The Pruning

I plan to create a dilute solution of ethyl methanesulfonate (0.5%) and apply it by dropper to the axillary buds before each pruning. Then, once the apical bud has been removed, I will allow the axillary buds to grow in and choose which ones to keep and then trim the rest. I am hoping that this regiment will lead to an ever increasing number of mutations in both unexpected and desired ways.
grape-bonsai-tree.jpg
 

Wires_Guy_wires

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You're planning on using possible carcinogens inside your home?
You're not a scientist, but you want to produce multiploid plants.. How do you plan on checking the nucleus numbers?
Grapes can take decades to flower, they pollinate by wind and pollinators, how do you plan on preventing self pollination?
Have you thought about the prevalence of mutations? In other words.. Are you aware that some mutations can be reverted? Or that they might not pass a breeding cycle because the mutations are on different parts in the DNA?
You are aware that backcrossing a polyploid plant to a diploid mother can yield polyploid, diploid, triploid and tetraploid plants?

What I want to understand: why mutate them yourself? And why chemical, with just one or two chemicals that usually affect the same genes in all plants you treat? I would go for random mutations, so either UV-C bursts or some other kind of radiation, viral agents or stuff like thag.

There are hundreds of wildtypes out there to make new hybrids from, which will save you a lot of work.

I'm a big fan of DIY science, but I want you to be careful and realistic before you spend hundreds of hours on something that doesn't get you the results you want.
 

Rivian

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Lets say you apply a mutagen to your grapevine bonsai shoot and a dividing cell mutates genetically or epigenetically to produce the perfect grape, and the whole shoot carries this characteristic forward. Then you still have to wait at least a year after treating any shoot to find out if it worked or not?
Also, seeing that seed tray, you want to take care of over 100 grapevine bonsai?
 

Forsoothe!

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It takes some number of years growing-on before one can characterize the specifics of a given plant, usually 5+ years. Even if you get the characteristic you want, you will also get the rest of the plant, including a lot you don't want. Breeding them out will lead to losing some good characteristics that you will have to chase back in. The reason people do this with a very large number of seedlings, or as in your case a large number of cuttings from which they do heavy culling of a very large percentage is that mother nature will keep dodging & weaving and luck plays a significant role. Tenacity and especially volume wins.
 

Frithfolk

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You're planning on using possible carcinogens inside your home?
You're not a scientist, but you want to produce multiploid plants.. How do you plan on checking the nucleus numbers?
Grapes can take decades to flower, they pollinate by wind and pollinators, how do you plan on preventing self pollination?
Have you thought about the prevalence of mutations? In other words.. Are you aware that some mutations can be reverted? Or that they might not pass a breeding cycle because the mutations are on different parts in the DNA?
You are aware that backcrossing a polyploid plant to a diploid mother can yield polyploid, diploid, triploid and tetraploid plants?

What I want to understand: why mutate them yourself? And why chemical, with just one or two chemicals that usually affect the same genes in all plants you treat? I would go for random mutations, so either UV-C bursts or some other kind of radiation, viral agents or stuff like thag.

There are hundreds of wildtypes out there to make new hybrids from, which will save you a lot of work.

I'm a big fan of DIY science, but I want you to be careful and realistic before you spend hundreds of hours on something that doesn't get you the results you want.
Thank you for the critical and clear-eyed reply. This is just the type of thing I wanted when I signed up. I've thought carefully about all of this, but it's always great to be able to bounce ideas back and forth to see if there were any holes in my analysis. Let me go ahead and jump in with my responses.

1. Yes, EMS is a very dangerous chemical. I fortunately have masks, gloves, and special waste disposal bags and location available already because of the epidemic.
2. I am planning to identify the polyploid plants through phenotypical analysis.
3. The Amur Grape begins fruit production within the second to third year of growth from seed. For the creation of the triploid plants, I will seperate the phenotypically tetraploid plants, break off the flower clusters and smack them against the flowers of the non-treated diploid mothers. The resultant seeds should have a mixture of self-fertilized diploids and also triploids. I will know which ones are triploid after growing out the seeds and seeing if they produce seedless grapes.
4. With a 1% EMS solution, I am expecting 50% seed germination and survival. Of those surviving, the literature suggests between 2-10% will displaying phenotypical mutations. As for how the further application of EMS on the growing nodes will affect this, I am not certain.
5. Yes
6. EMS is a genome-wide mutagen which results in diverse and unpredictable mutations. It is the premier choice in generating diverse novel traits in plants chemically.

I will definitely be doing my utmost to maintain safety throughout. As for time, I have all of the time in the world. :D
 

Frithfolk

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Lets say you apply a mutagen to your grapevine bonsai shoot and a dividing cell mutates genetically or epigenetically to produce the perfect grape, and the whole shoot carries this characteristic forward. Then you still have to wait at least a year after treating any shoot to find out if it worked or not?
Also, seeing that seed tray, you want to take care of over 100 grapevine bonsai?
Yeah, I'll have to wait at-least two years to see if any of the fruit comes out in novel ways.

I'll be growing between 250-300 micro bonzai plants.
 

Frithfolk

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It takes some number of years growing-on before one can characterize the specifics of a given plant, usually 5+ years. Even if you get the characteristic you want, you will also get the rest of the plant, including a lot you don't want. Breeding them out will lead to losing some good characteristics that you will have to chase back in. The reason people do this with a very large number of seedlings, or as in your case a large number of cuttings from which they do heavy culling of a very large percentage is that mother nature will keep dodging & weaving and luck plays a significant role. Tenacity and especially volume wins.
Yep, I'm planning to start with around 250 seedlings but, if everything goes okay the first year, I would like to expand to maybe an additional 500 clones next year from interesting branches.
 

Wires_Guy_wires

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Thank you for the critical and clear-eyed reply. This is just the type of thing I wanted when I signed up. I've thought carefully about all of this, but it's always great to be able to bounce ideas back and forth to see if there were any holes in my analysis. Let me go ahead and jump in with my responses.

1. Yes, EMS is a very dangerous chemical. I fortunately have masks, gloves, and special waste disposal bags and location available already because of the epidemic.
2. I am planning to identify the polyploid plants through phenotypical analysis.
3. The Amur Grape begins fruit production within the second to third year of growth from seed. For the creation of the triploid plants, I will seperate the phenotypically tetraploid plants, break off the flower clusters and smack them against the flowers of the non-treated diploid mothers. The resultant seeds should have a mixture of self-fertilized diploids and also triploids. I will know which ones are triploid after growing out the seeds and seeing if they produce seedless grapes.
4. With a 1% EMS solution, I am expecting 50% seed germination and survival. Of those surviving, the literature suggests between 2-10% will displaying phenotypical mutations. As for how the further application of EMS on the growing nodes will affect this, I am not certain.
5. Yes
6. EMS is a genome-wide mutagen which results in diverse and unpredictable mutations. It is the premier choice in generating diverse novel traits in plants chemically.

I will definitely be doing my utmost to maintain safety throughout. As for time, I have all of the time in the world. :D
1. Consider that this will spread through your room because plants breathe and shed all kinds of molecules. Residual chemicals can last for decades in your home and will end up in your food.
2. Phenotypical analysis in what sense? I've worked with all kinds of ploids, some, but not all of them, express no phenotypical difference even though they were 24N or 1N. Plants are known to revert to 2N too, it's hard to keep polyploidy stable for longer periods of time because there's constant division.
3. Are you sure that it works like that? Crossing a 2N with a 4N doesn't always yield a 3N, the resulting offspring can be all kind of possibilities, even another 4N. Then there's still the stability thing.. A 3N can divide into 2N or even a haploid if the other nucleus proves to be dysfunctional. For breeding lines, we used 3000-6000 polyploids made from existing 'cream of the crop' cultivars to find roughly 15 good specimens. We repeated that selection 40 times a year to produce one stable line in 10 years. These were vegetables with a 2 year lifecycle.
6. You're dealing with a same-batch seeds, of a same age, and mutations can be repaired in some cases. I expect that you're going to find a bunch of the same mutations.

I would go with wildtype breeding, better disease resistance and just as much variation as human induced. And it's also safer for you and anyone living in your house.

If you want to do effective breeding and development, it's good to map dominant and recessive traits. To do this well, you need 4 generations.
To produce a stable crop, you need at least 6 backcrosses in most cases, or take a risk with two parents and produce F1 hybrids, but since grapes get so old, farmers might come and claim your money once the plants revert to a recessive trait that became dominant due to environmental factors.
 

Frithfolk

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Wires, I've taken your worries to heart and have decided on some modifications. First, I will be opening all of the windows in the house during the EMS seed soaking, which will be done in a sealed, and afterward disposed of, container, and, secondly, I will not be doing the node-drip indoors, that is if I do it at all. The half-life of EMS in water is 96 hours, but it is volatile and its half-life in air is 90 days. So, node-drips seem an unprofitable endeavor.

As for your other points on the actual program itself, they are all very informative. However, I am only doing this amateurishly and, as such, would be quite content having done nothing more than created a bunch of little bonsai grapevines.

However, I have one additional detail about producing a stable crop which I omitted before; I am hoping to do an anther culture later on and see if I cannot create some double haploids. However, that won't be till much later. For now, I am just trying to mutate some grape seeds and get them to germinate.

For all interested, there have been a few changes to my process:

First, a look at what has arrived:
IMG_20201121_131341.jpgIMG_20201121_131333.jpgIMG_20201121_131404.jpgIMG_20201121_131346.jpg1606054161148.jpeg1606054162084.jpeg

Well, the man I bought the seeds from wasn't sure if they had already been treated to germinate. So, being worried about over-treating the seeds, I cut back on everything substantially.

I did the water soak for six hours, the peroxide soak for two hours, the running under tap water was kept at twelve hours, and the gibberellic acid treatment was cut down to three hours.

Of note, the gibberellic acid came in an ethanol solution. While the percentage of ethanol wasn't listed, it smelled quite strong; upward of ~60%. As it was 3% gibberellic acid, I diluted with water at a rate of 10ml solution to 190ml water. This left me with my intended 0.15% GA3, but also an unexpectedly high amount of ethanol(I estimate as much as 3%). So, this also influenced my decision to cut back the treatment from a 24 hour soak to only 3 hours.

The seeds are now sitting outside of my window. The nights here are around -3c and the days get up to 6c. I am supposing that being next to the window and embedded in dirt should keep the seeds above freezing. I will leave them there for the next week.

Here are pictures of both the seeds soaking in water and the trays filled with substrate.
IMG_20201121_131818.jpgIMG_20201121_154621.jpg

The seeds had a definite smell of yeast as they were soaking and I fear whether they were removed from after a whole bunch fermentation, a popular wine method for the native species of grape. It would definitely have rendered them non-viable and I wouldn't put it past unscrupulous merchants to do such a thing. However, they all eventually settled in the water which is meant to be a sign of grape seed viability. I may dissect one before the EMS treatment to see if they are well.

Oh, and of further note, I have moved the EMS treatment to AFTER they have been left in the cold for seven days. The reason for this is two-fold. First, I don't know whether the gibberellic acid will react with the EMS in any way. Mr. Wires has given me a new-found appreciation of safety. Further, I have now read that GA3 is in-fact an anti-mutagen that can repair the DNA damage done by EMS if applied after the EMS treatment. As such, I have put the EMS treatment at the very end and have separated them with the seven day chill.
 
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Wires_Guy_wires

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Did you wash the coco bricks before you used them?
I found that some coco bricks from disputable sources are factory-washed with sea water because it's cheaper - and coco grows near the sea so it's also a transporation kind of thing. But it increases the salt content of the coco considerably.

Good to read that you've taken safety into account.

I can give you a broad setup for a breeding programme if you want, I've been doing amateurish breeding for quite some time and I have a standard way of going at things now. That, and I spent quite some time in actual breeding and R&D labs for both plants and animals.

Good luck!
 

Frithfolk

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Did you wash the coco bricks before you used them?
I found that some coco bricks from disputable sources are factory-washed with sea water because it's cheaper - and coco grows near the sea so it's also a transporation kind of thing. But it increases the salt content of the coco considerably.

Good to read that you've taken safety into account.

I can give you a broad setup for a breeding programme if you want, I've been doing amateurish breeding for quite some time and I have a standard way of going at things now. That, and I spent quite some time in actual breeding and R&D labs for both plants and animals.

Good luck!
No, I hadn't. However, the seeds have yet to be planted, they are still being cold stratified, so I most definitely will now that you've said as much.

I would love to read your programme. In-fact, I have been reading a book a day on the general maintenance of potted plants, their trimming and care, and on all types of things of that nature. Your input, especially as one that has worked in a professional setting with regard to plant breeding, is invaluable. Thank you.

Today, the last of the packages arrived and I have assembled the grow house;

stack.jpg

Of-course, the substrate will now be removed and washed before potting once again.

Bonsai, here I come!
 

Bnana

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It might not be necessary to wash it, only if it is too salt. It is quite likely that that is not the case and that would make life easier.
 

Wires_Guy_wires

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It might not be necessary to wash it, only if it is too salt. It is quite likely that that is not the case and that would make life easier.
Here in Europe this isn't an issue anymore since 2009 or so. Before that period it was a real issue and more common than people want to admit, even at renowned soil sources. The coco peat was washed with sea water and then dried in the sun. If one would add water without flushing, the salt concentration would be close to sea water, which is too salty with the wrong salts.

It doesn't do much harm to wash/flush it anyways.
 

NOZZLE HEAD

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Great project!

I would mess with ethyl methanesulfonate, except for the fact that it is fairly hard to acquire where I am.

Any chemical can be handled safely with proper PPE, think the bunkers plutonium is handled in with robot arms and 4 foot thick leaded glass, but I would be reticent to handle EMS inside a dwelling.

I have used oryzalin, for inducing polyploidy, but without noticeable phenotypical variation.

The research I have seen shows that an EMS seed soak is successful in inducing leaf form mutations and dwarfing. Success rates are largely dependent on species.
 

Frithfolk

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In-fact, the peat had a very peculiar smell reminiscent of puer tea and, upon washing, the liquid ran a deep ruby red. So, whether the sodium content was or was not a problem, I think washing was the right choice and, as it has already been done, there's no sense in contemplating its necessity now.

Nozzle, yes, the control of chemical substances is far more lax here in China than in the West. I spent approximately five dollars American to purchase a gram; if you find it possible, I would be willing to assist in your procurement of the substance. However, I do not know whether there'd be controls on such a thing.

As for my project, there's not much to update. I have washed the peat till it ran clear and repotted it. Also, the seeds continue to stratify.
 

Leo in N E Illinois

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EMS - ethyl methanesulfonate - tends to cause point mutations, changes in the DNA. It is not expected to produce a large number of polyploids. If your initial stock is diploid, it will come out of the process, still being diploid. THis is what @NOZZLE HEAD was pointing out

Oryzalin and Colchicine and similar chemicals, cause non-disjuncture of the chromosomes. This results in a high percentage of various polyploids. Many will be aneuploids, one will have to sort the diploids, tetraploids, the higher ploids and the aneuploids. Phenotypic traits like measuring guard cell sizes can work. Measuring guard cells can be done with a simple dissecting scope, as opposed to chromosome counting which requires 1000 x magnification.

Point mutations are rather different than the changes caused by ploidy number changes.
 

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