experimenting with graft / over-graft to introduce new DNA into existing trees

Hi everyone

I'm going down a rabbit hole researching over-grafting as a method of introducing new DNA traits into existing stock; just as a thought experiment.

It's how we've gotten the trees we have now; selective breeding. Grafting was always just to clone the parent plant onto rootstock to get the same tree right ?

Well as mentioned, i came across some old books in the local library (no i didn't go to the library, they announced they were closing certain sections of the library down and downscaling and had books for sale, so i stuck my nose in and grabbed a few interest books)

So anyway i stumbled across a few pages of interest (William Gilbert: Plant Grafting and the Grand Analogy) that mentions that old hands in rome had grafted their Syzygium rose or crab apple onto a an inedible Quince tree that may be the origin of the modern apple. Obviously this has since been debunked, as Malus sieversii is from Kazakhstan

Which then lead me here
https://orchardnotes.com/research/ancient-roman-fruit-varieties/apples-summary-conclusions/


Which then got me thinking, DNA !

That landed me on this research paper.
https://pmc.ncbi.nlm.nih.gov/articles/PMC3628911/

So the old brain got to work ... could i, in theory; take a tree with an existing graft and rootstock that i know has good fruit; graft a scion from a seed grown seedling onto the good tree, and have at least part of the traits of that good tree transferred to the new tree's seed DNA at which point i'd have a hybrid tree without having to cross pollinate the tree.

My thought process, is that rather than planting the new seedling in the ground and waiting 4-6 years to get it to fruiting and then having the risk of cross pollinating cider species trees into my currently good seed crosses i have now; I'd skip the messy part and try encourage the good traits out of the already existing traits into the new scion wood and then try skip the risk of getting cider apples, as an example.

(sorry apple on the brain, just made pie)

I have only bearly started the experiment, and i see it as a multi decade project.

And yes I do realize this isn't a new concept at all; but this is the first time i've heard of someone mentioning persistent DNA transfer between rootstock and scion as a thing, and then to find out that "hey, we messed with this mRNA thing on plants and discovered it may possibly be worth investigation" makes me optimistic i may be able to harvest "repeatable traits" from the seeds going forward if i were to say plant these in a greenhouse in isolation and expand from there.

The idea being if i don't need to plant it with my other food forest plantings, and i can over-graft them, then perhaps i can scale what i was trying to do (make a set of unique tree varieties) and sort of cement that by grafting just the new wood and crossing those, and then introduce new grafted trees into that environment instead; and collect those seeds seperately, so on and so forth.

Also, if anyone else had stumbled across this before and had any useful happy accidents of other species that have been hetero-grafted together that really shouldn't be that might help point me in a direction to expand my mad scientist brain, it would be appreciated.

Well, I have never even heard of this (gene transfer from root stock to scion - let alone heritable gene transfer).  To be honest, I'm going to have to spend more time reading the PLOS paper you referenced (now, safely downloaded to my hard drive).

So, nothing further to add, for my part, except to say that this does indeed appear to be quite the rabbit hole!

well written

Kevin Olson wrote:Well, I have never even heard of this (gene transfer from root stock to scion - let alone heritable gene transfer).  To be honest, I'm going to have to spend more time reading the PLOS paper you referenced (now, safely downloaded to my hard drive).

So, nothing further to add, for my part, except to say that this does indeed appear to be quite the rabbit hole!

Welcome to the rabbit hole my friend, sorry i dragged you in with me !!

Interesting. The PLosOne paper shows changed in the modification of DNA by methylation, whic is 'epigenetics' rather than a change in DNA sequence.  The methylation patterns are heritable, but can change, and they can affect gene expression; not what genes are present, but rather which ones are expressed.

Douglas Campbell wrote:Interesting. The PLosOne paper shows changed in the modification of DNA by methylation, whic is 'epigenetics' rather than a change in DNA sequence.  The methylation patterns are heritable, but can change, and they can affect gene expression; not what genes are present, but rather which ones are expressed.

Yeah I am very new to the specifics of this whole process. My research into this topic is a month old but this isn't a new concept to me since we've had signs of this behavior in seeds but i didn't think it would also extend to the physical hardwood grafts.

I'm still in the rabbit hole, when i come out the other end with a much firmer grasp on what is happening and can better explain it I'd be interested in continuously supplying a list of our experiments; possibly with a graft compatibility list and a list of trait genetic specific grafts

For anyone who is new to the idea of what we are talking about, let me try explain it. Maybe Douglas can correct anything if i misrepresent it.

The DNA according to this research paper, suggests that the DNA of a plant is sort of like a row of light switches that is turned on or off. But what they are suggesting is that when we put one graft scion onto a rootstock, we can flip some of the switches half way. They may switch off on their own or switch on fully (as in the trait become heritable in seeds or cuttings).

My research here will be looking into whether we can push it on and leave it on generationally without genetic modification but by breeding those traits into the plant.

It's not a new concept and has been done before. But this paper suggests that indeed may be confirmed scientific for the first time that all plants are capable of it not just seeds and not necessarily plants from the same species or genus.

It gets better. Here is an article about the creation of a brand-new allopolyploid species by grafting. Basically the researchers grafted two species of tobacco to each other and then cultured the cells from the graft union. There was some high-tech stuff in there, but I don't really see why you couldn't do basically the same thing with only a knife and a piece of string. Assuming, of course, that you could convince those allopolyploid cells in the graft union to form a shoot...

Eino Kenttä wrote:It gets better. Here is an article about the creation of a brand-new allopolyploid species by grafting. Basically the researchers grafted two species of tobacco to each other and then cultured the cells from the graft union. There was some high-tech stuff in there, but I don't really see why you couldn't do basically the same thing with only a knife and a piece of string. Assuming, of course, that you could convince those allopolyploid cells in the graft union to form a shoot...

Good find !

I love that this thread has already merged into a mini research project so quickly  

If we could find a good, reliable method for producing graft-induced allopolyploids, it would open up some extremely interesting possibilities. Since I live in a cold climate, I imagine "merging" warmer-climate crops with hardier wild relatives. The one I've been thinking about the most is olives. The olive tree is graft compatible with ash (Fraxinus sp.) according to something I read... I'm planning to try this at some point. Very low probability of success, probably, but still...

(In case you couldn't tell, here's a fellow mad scientist )

Eino Kenttä wrote:If we could find a good, reliable method for producing graft-induced allopolyploids, it would open up some extremely interesting possibilities. Since I live in a cold climate, I imagine "merging" warmer-climate crops with hardier wild relatives. The one I've been thinking about the most is olives. The olive tree is graft compatible with ash (Fraxinus sp.) according to something I read... I'm planning to try this at some point. Very low probability of success, probably, but still...

(In case you couldn't tell, here's a fellow mad scientist )

Hey i like to share my borderline insanity with others. Happy to share.

As for your suggestion of getting cold climate native rootstock to graft more sub tropical or tropical varieties onto; A month ago i would have said that it's impossible. Now i am in the "If you have access to 10 rootstock and you have the means to do it; I'd say 1 in 10 odds of something happening is more than likely". And the problem here is that; if you had even one graft take and got leaves on it, I'd call it a success even if it doesn't fruit. Because that means something took. At that point over-grafting practices kick in and i say; if one graft took ... try graft onto that graft.

Eventually i don't see why you couldn't push the plant over the line.

And now i think to myself, okay that gives us maybe one tree.
How would we then replicate that with fewer and fewer grafts between "we got fruit that matches the parent plant" and that native unrelated tree.

Is is easier at that point to use seed and see if it is acclimatized / adapted ?

How long does it take ?

How many cycles of grafting will be needed ?

Can we "tell" that what we got is the same as the parent plant, or have we created something new ?

Still points to investigate.

So for anyone wondering, currently I am working on trying to start with just creating new varieties of fruits (apples, pears, etc.)

In light of this new research, i am also toying with the idea of things that don't do well outdoors here; maybe :

Now, i understand overlord google says its impossible. But There IS nothing that says, if i put enough other grafts between point A and point B that i couldn't eventually get to this point. Figuring out the right combinations of grafts to get from family A to family B is the important part of this process, and even more complex is the jump between genus. So while, yes; you can't just simply graft family A to family B (i do understand this), we can theoretically by grafting a franken-plant of multiple over-grafts get to that point. We only need to match the flow of the xylem and phloem walls to jump between families, which is about getting the cambium to match up.

overlord google :
To successfully graft between different tree families, it's crucial to match the cambium layer of the scion (the part being grafted onto) and the rootstock (the part that will form the roots). This alignment ensures the xylem and phloem, the vascular tissues responsible for transporting water and nutrients, can connect and allow the graft to thrive. While grafting within the same family or genus is generally more successful, it's still possible to graft between different families with careful attention to detail and matching the cambium.

Grafting between different plant genera is generally less successful than grafting within the same genus. While some inter-genus grafts can be successful, particularly within the same family, they are less likely to form a strong and lasting union compared to grafts between closely related species or even different varieties within the same genus


---

coffee over-grafted on carob = cold climate coffee
maybe inga edulis is more suitable as an inter-graft ?
there are not a lot of 'edibles' in the Rubiaceae family, would definitely need to branch outside that family of plants.

cacao over-grafted on carob = sub tropical cacao
The entire Theobroma genus is tropical = unusable as a rootstock

finding some adaptable orchid that we can graft vanilla orchid onto to try get a more cold tolerant vanilla variety

apparently the material needs to match the circulatory system of the wood

list is endless. this is my starting point.

I already note that in Australia, they classify coffee as a weed given its invasive nature in that climate. As someone in the permaculture realm and drinks a lot of coffee; sign me up. Would love it if it became a weed here, am i intentionally going to create invasive plants; maybe. But I am also of the belief that if we can adapt plants and create new varieties and even new species; we should be trying.

Greenhouse sanctuary for the win !

Our food system is fragile and i don't believe that research should stop just because science has taken over. I for one don't believe in lab modified organisms and lab grown food as being natural. I'll take invasive and weedy over the science lab experiment any day.

https://rayon-de-serre.com/blogen/botanical-terms-taxonomy/

Colchicine, an extract of a certain type of crocus, is used to encourage allopolyploidy.  Well, it's probably synthesized, these days.  I don't know the details, but it can facilitate the creation of plant hybrids which would not otherwise be viable.

Interestingly, colchicine is also used to control gout.

Kevin Olson wrote:Colchicine, an extract of a certain type of crocus, is used to encourage allopolyploidy.  Well, it's probably synthesized, these days.  I don't know the details, but it can facilitate the creation of plant hybrids which would not otherwise be viable.

Interestingly, colchicine is also used to control gout.

Thanks for the info Kevin, very interesting clue !

I'd be keen to figure out how its made and maybe find a better basis for why the solution works. It may also be the case it is simply switching again flipping the switches as the tissue heals, it is being absorbed and then the plant is triggering and inheriting those traits. Which reminds me of willow water being used for rooting hormone.

Unfortunately it is not available in any commercially available products i can find yet.
I see mention that it's only available by prescription or being used in research labs.

While Colchicum autumnale does grow here, i do not see the concentrations from raw plant material anywhere yet. I'll do more research on the medications page and see if there are any clues that may lead to a dosage and then work from there. May be a situation where it's still being researched for use on plant matter.


Excellent and interesting additional link and clue. I'll paste it here since is buried at the bottom of the wiki page.

Wikipedia :

Colchicine is used in plant breeding by inducing polyploidy in plant cells to produce new or improved varieties, strains, and cultivars.[20] When used to induce polyploidy in plants, colchicine cream is usually applied to a growth point of the plant, such as an apical tip, shoot, or sucker. Seeds can be presoaked in a colchicine solution before planting. Since chromosome segregation is driven by microtubules, colchicine alters cellular division by inhibiting chromosome segregation during mitosis; half the resulting daughter cells, therefore, contain no chromosomes, while the other half contains double the usual number of chromosomes (i.e., tetraploid instead of diploid), and lead to cell nuclei with double the usual number of chromosomes (i.e., tetraploid instead of diploid).[20] While this would be fatal in most higher animal cells, in plant cells, it is not only usually well-tolerated, but also frequently results in larger, hardier, faster-growing, and in general more desirable plants than the normally diploid parents. For this reason, this type of genetic manipulation is frequently used in breeding plants commercially.[20]

When such a tetraploid plant is crossed with a diploid plant, the triploid offspring are usually sterile (unable to produce fertile seeds or spores), although many triploids can be propagated vegetatively. Growers of annual triploid plants not readily propagated vegetatively cannot produce a second-generation crop from the seeds (if any) of the triploid crop and need to buy triploid seed from a supplier each year. Many sterile triploid plants, including some trees and shrubs, are becoming increasingly valued in horticulture and landscaping because they do not become invasive species and do not drop undesirable fruit and seed litter. In certain species, colchicine-induced triploidy has been used to create "seedless" fruit, such as seedless watermelons (Citrullus lanatus). Since most triploids do not produce pollen themselves, such plants usually require cross-pollination with a diploid parent to induce seedless fruit production.

The ability of colchicine to induce polyploidy can be also exploited to render infertile hybrids fertile, for example in breeding triticale (× Triticosecale) from wheat (Triticum spp.) and rye (Secale cereale). Wheat is typically tetraploid and rye diploid, with their triploid hybrid infertile; treatment of triploid triticale with colchicine gives fertile hexaploid triticale.[80]


Research paper time !
https://pmc.ncbi.nlm.nih.gov/articles/PMC10607973/

Plant tissue culture isn't hard, it's just tedious. Outside of general research, it's done a fair bit with orchids. There's several videos on YouTube showing how.

It'd be significantly faster than hoping noticeable modified cells end up somewhere you can use for scions or in the reproductive parts of a flower (and not triploid or tetraploid.)

K Kaba wrote:Plant tissue culture isn't hard, it's just tedious. Outside of general research, it's done a fair bit with orchids. There's several videos on YouTube showing how.

It'd be significantly faster than hoping noticeable modified cells end up somewhere you can use for scions or in the reproductive parts of a flower (and not triploid or tetraploid.)

IF you like Genetic Engineering sure.

Protoplast fusion is a technique that combines the genetic material of two different cells by first removing their cell walls to create protoplasts, then fusing them together. This process allows for the creation of somatic hybrids, which are new plant varieties that can possess desirable traits from both parent cells, including those not obtainable through traditional sexual hybridization

Literally the first article when you google Protoplast fusion

Protoplast Fusion: A Gateway to Genetic Engineering in Plants
https://plantcelltechnology.com/blogs/blog/protoplast-fusion-a-gateway-to-genetic-engineering-in-plants?srsltid=AfmBOop-H5TQFHpVkvufgbHLN9qIpdKrzoxAJ4IjDD09tm56jJZeDMzU


We're not talking about one for one cloning of plants here, we're talking about new varieties and cultivars and natural acclimatization of plants using grafting.

K Kaba wrote:Plant tissue culture isn't hard, it's just tedious. Outside of general research, it's done a fair bit with orchids. There's several videos on YouTube showing how.

It'd be significantly faster than hoping noticeable modified cells end up somewhere you can use for scions or in the reproductive parts of a flower (and not triploid or tetraploid.)

I realize you saw the part where i was talking about using carob as a root stock and grafting it and are trying to be helpful at speeding the process along, but i think you missed the fundamental part of the experiment using grafting. I'm not looking to MAKE the carob accept the DNA of the cacao or vise versa in a lab setting, I'm trying encourage the carob to take on traits of the cacao to make something that will grow in a cooler climate that maybe tastes like cacao, but is a unique and distinct plant on its own.

Protoplast Fusion completely negates all natural processes and forces one plant to join with another; and I can't guarantee the outcome since i don't have a lab.

Now when it comes to natural acclimatization using grafting, that might be where you'd use Protoplast Fusion; but again it's genetic engineering the hell out of the thing to make it do what you want it to. If we use the example of helping the olive tree acclimatize using ash, what you're proposing would be a disastrous combination without proper testing and equipment. If the dominant resulting DNA was an olive tree but it had all the characteristics of an ash tree; if the allelopathic got into the olives some allelochemicals can be beneficial, others can be toxic or even carcinogenic to humans

Quite literally wrote in the title. "experimenting with graft / over-graft to introduce new DNA into existing trees"

[Edit : This post is in error, it was referring to horizontal movement of hormones between the grafts not in relation to position of the graft. Don't research while tired ]

Okay so what i have read so far suggests that in a homogeous graft we graft so the flow is vertical from bottom to top ( ie. standard graft), while a hetero-graft is done on the horizontal where we are introducing the flow in the horizontal side branches where the sap flow is less prominant and the hormones are being forced towards the fruiting branches.

Still trying to figure out whether the branches need to be pointing upward where the flow is toward branch development or downward to encourage fruiting.

Nevermind !!!

Very much got this one wrong, this is why a graphic is useful

https://www.researchgate.net/figure/Applications-of-heterografting-to-uncover-mobile-molecules-a-Homografting-is-performed_fig1_331525599


We attach the "bad" scion onto the "good" rootstock not the other way around.
Ohh boy, need more coffee for this one. Maybe alcohol lol.

So, we'd encourage it to fruit while attached to the Donor plant; i was thinking we attach the donor to the plant we want to add the trait to.

But then the next image over "Genetic Graft" suggests what i thought would be the case ?

Is it just a case of we do one before the other ? Need more research.

My brain hurts.

Marshall Ashworth wrote:Nevermind !!!

Very much got this one wrong, this is why a graphic is useful

https://www.researchgate.net/figure/Applications-of-heterografting-to-uncover-mobile-molecules-a-Homografting-is-performed_fig1_331525599


We attach the "bad" scion onto the "good" rootstock not the other way around.
Ohh boy, need more coffee for this one. Maybe alcohol lol.

So, we'd encourage it to fruit while attached to the Donor plant; i was thinking we attach the donor to the plant we want to add the trait to.

But then the next image over "Genetic Graft" suggests what i thought would be the case ?

Is it just a case of we do one before the other ? Need more research.

My brain hurts.

A heterograft (or xenograft) uses material from a different species, while a genetic graft (or homograft/allograft) uses material from the same species.

Epigenetics, as Micheal wrote, can switch on, or off, genes that are in the genome, but it does not add new genes.
So the scion may develop differently than the source plant.

List of successful hetero grafting (common)
For anyone wanting to know what already is grafted using this technique.

Stone Fruit:
Grafting peaches, plums, and cherries onto each other is generally successful, with some variations in compatibility depending on the specific varieties.
Pears and Apples:
Some pear and apple varieties can be grafted onto each other, especially when using "wild" pear rootstocks for apple grafts.
Citrus:
Certain citrus species can be grafted onto each other, but compatibility can vary significantly.
Other examples:
Other successful heterografts include grafting hawthorn (various species), medlar, and nashi (Asian pear) onto European pear

Citrus: Sweet orange 'Newhall' scions grafted onto Poncirus trifoliate population or C. junos Siebold ex Tanaka rootstocks.
Melons and Squash: Oriental melon scions grafted onto squash rootstocks.
Tomatoes and Peppers: Tomato scions grafted onto pepper rootstocks or vice versa.
Eggplants: 'Ecavi' eggplant scions grafted onto S. torvum or tomato 'Emperador RZ' rootstocks.
Chrysanthemums: Chrysanthemum scions grafted onto Artemisia rootstocks.
Vegetables: Grafting is used with various vegetables like tomatoes, peppers, cucumbers, and melons to enhance disease resistance and other traits


Additional Resources :

Britannica Overview of Horticultural Propagation practices and history
https://www.britannica.com/science/horticulture/Horticultural-education-and-research

Compatible_Graft_Establishment_in_Fruit_Trees_and_Its_Potential_Markers : https://www.researchgate.net/publication/362865073_Compatible_Graft_Establishment_in_Fruit_Trees_and_Its_Potential_Markers

GRAFTING COMPATIBILITY, SCION GROWTH, AND FUSARIUM
WILT DISEASE INCIDENCE OF INTRASPECIFIC GRAFTED TOMATO
https://bibliotekanauki.pl/articles/55789071.pdf

My project focuses on extending the research into cross species (since it seems since the early 1900s we've stopped really researching this)

I'm still practicing by creating new varieties until i hammer down the grafting part of the exercise; still a little rusty since the time to do so comes around but once a year.
And the more the trees grow, the more spare cuttings wood is available. So the older the trees get, the more fun can be had !

Since the discovery of cloning we seem to have lost all interest in natural processes and seem to be leaping head first into genetic engineering, which i really don't like since it seems we are hellbent on making nature our slave. I'd much rather in the permaculture realm, focus on working with rather than against nature myself.