Grafting as a way to introduce new genes

General Discussions
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As per this fascinating thread, rootstocks can often share genes with a scion that can be inherited by the scion’s progeny. Which runs counter to what we think we know about genetics, but . . . there it is!

The cool thing is, I just barely read something similar earlier today:

Apparently the rootstock variety can make a significant difference to the flavor of a scion variety, so it does more than just impart size, disease resistance, cold hardiness, etc.

If those changes can be inherited, too . . . wow, that’s interesting.

My suspicion is that those changes are probably epigenetic. I can see it being easy for a rootstock to flip epigenetic switches in a scion, and epigenetics can be inherited.

But you know, whatever the mechanism happens to be . . . let’s take advantage of it! :smiley:

Of course, my first thought was, “Ooh, could I graft onto a Musa basjoo?” Because Musa basjoo is super cold hardy, but sterile. That seems like a cool idea to get some of its genes into a landrace. Unfortunately . . .

Sadly, it looks like plants that have only one cotyledon (such as bananas, lilies, and grasses) don’t have a cambium layer that can be easily attached to another variety through grafting. So banana grafting is probably out.

Still.

Figs, feijoas, and persimmons can all be grafted, and I’m planning to landrace them for greater cold hardiness and drought tolerance (and the usual suspects like flavor too, obviously).

I was planning to overgraft anything healthy and flavor-meh into a variety I like better anyway, but if this means I can do that and still keep those genes somewhat in the population . . . that’s awesome!

Side note: Figs can’t be insect-pollinated in my climate, because we don’t have fig wasps here (they can’t survive lower temperatures than 12 degrees Fahrenheit). But they can be hand-pollinated! And apparently that’s a good idea, even if you aren’t trying to get seeds for planting:

It also raises some interesting questions about citrus breeding. A lot of citrus species are routinely grafted onto (inedible, thorny) trifoliate orange rootstocks because that’s the most cold hardy citrus species. That raises two interesting questions for me:

a) Does this change the flavor of the fruits any? (For the worse?) If so, in places that don’t need the cold hardiness, maybe it would be better to grow seedlings on their own roots instead of purchasing grafted trees.

b) Does this change the cold hardiness of the scions’ offspring any? (For the better?) If so, that implies a person who needs the extra cold hardiness of a grafted tree should definitely start planting any seeds they get from those grafted trees. Maybe some of those seedlings will be able to survive without grafting.

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This is such a fascinating topic, and there is still so much that is unknown.
Studies have shown that exosomes are at least one mechanism for grafts to exchange heritable genetic information. Exosomes are membrane bound vesicles that are secreted by one cell and absorbed by another. They seem to function in intact organisms (in both plants and animals) but have only recently been discovered.
In grafted plants they transmit DNA, RNA, proteins and organelles between the two sides of the graft. Recently germinated seedlings seem to absorb exosomes from across the graft more readily. When a scion forms seeds they also seem to absorb more exosomes, which can sometimes result in Dna or organelles being incorporated into that lineage, theoretically forever.
Mentor grafting can in principle happen between any plants where the flow of sap can be maintained long enough. I read a report on a mentor graft between a legume and a grass from China, but take any research paper with a grain of salt since there is a lot of fraud and fudging in “professional” science.
As to how useful the technique is- well even under ideal conditions only a very small percentage of grafts undergo transformation. There is no practical way to control which genes/organelles are transferred. This means you would probably have to do a very large number of grafts over a long period, then grow out large numbers of scion seedlings to look for useful traits. Doable, but personally I think wide hybridisation gives more meaningful results on small scales and in short time periods. Hand pollination is usually more technically feasible than seedling grafting.

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Oh, interesting! I’ve never heard of exosomes before, and that’s a cool thing to know about. That may explain how all the parts of a multicellular organism can stay tightly connected and work together!

It would also make sense as a mechanism of how a plant can learn to accept a newly introduced piece that has different DNA. Are exosomes seen in animals, as well as plants? I’m wondering if human bodies who’ve received an organ from a donor behave with any similarities to grafted plants.

I completely agree that wide crosses are probably better, because introducing lots of DNA is likely to shake some interesting traits into the population that may be benficial. But of course, you can always do both!

And if you already have a suitable rootstock for some reason (for instance, it came with a fruit tree you bought or inherited from a previous owner of your house), it’s fun to consider how you may be able to use that resource in additional ways.

Hmmm, can mentor grafting be done with a tiny seedling being bark grafted onto a much older tree? If so, I’m thinking something like this may be very interesting:

  • Get a very cold hardy rootstock.
  • Buy a bunch of seeds from varieties of that species (or various species that are compatible with the rootstock) that sound awesome that are almost but not quite hardy to your zone.
  • Start the seeds in pots.
  • Cut off the tops of the seedlings and bark graft them onto the rootstock.

Would that work to mentor graft them? How early does mentor grafting have to be done?

If you tried that with, say, apples, what percentage of the seedlings would be able to survive being turned into scions that early?

If someone plans to make a frankentree covered in their own seedlings anyway, maybe it would be worth learning how to graft very young seedlings to see if they can get that rootstock’s favorable traits more noticeably into the genes?

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You are right about animals also having exosomes. They are implicated in the rejuvenation effects in elderly people getting regular transfusions of young blood (real life vampires, though these ones have false teeth and fat wallets). Organ transplants cause weird personality shifts in some patients (there is a surgeon documenting these fascinating cases) in ways that correlate with the personality of the organ donor (including really weird specific details at times). Maybe exosomes are involved.

Grafting seedlings soon after germination requires its own tricks and techniques that vary with every species and location you try it in. Preventing wilting often requires controlled humidity until the union joins (which in turn requires management of fungal and bacterial pathogens at times). If you took the time to master grafting freshly germinated apple seeds you might be the only person in the world doing it. But if you tried to start doing it with plums you would be almost back to square 1 figuring out the specifics of making it work. Then throw in the complexity of figuring out how each species of rootstock behaves with each scion. Sometimes graft incompatibility doesn’t appear for years in woody species.

An alternative approach might be mixed species tissue culture at a liquid cell culture level. There might be a way to harvest solutions of exosomes from one species (optimised for exosome secretion), then feed them to a separate cell culture under conditions (eg starvation) that facilitate uptake and incorporation. But at this stage you are close in complexity to doing artificial transgenesis.

Another approach could be to use the power of parasitic plants to form graft unions to tiny seedlings. Dodder would be my guess as the best candidate. It could bridge the rootstock to tiny scion while the seedling is still rooted, only cutting it free from its own roots once the parasite is attached. This might be the most convenient way to attach hundreds of seedlings to a mature rootstock (though dodder needs tender tissues to latch on).

I still see mentor grafting as interesting but not especially powerful or practical. There have to be better ways of shot gunning the DNA from one species into a target plant species. This fairly old review paper outlines the technique of adding foreign DNA into pollen used for pollination- Pollen grains as a target for introduction of foreign genes into plants: an assessment - PMC

Following the links to papers that cited this review turned up this one-Transgene expression in cowpea (<i>Vigna unguiculata</i> (L.) Walp.) through <i>Agrobacterium</i> transformation of pollen in flower buds | African Journal of Biotechnology

In this study they applied cultures of Agrobacter to cowpea anthers the night before pollen shedding. When they applied the pollen to pistils most of the pods aborted, but the ones that set gave seeds with a 0.36% rate of transgene expression. This is pretty good for such a simple technique. Agrobacter culture is pretty easy. The plasmids for carrying genes are well known. Inserting new genes into the plasmids is also pretty straight forward (like third year biochem student stuff, assuming they have quality reagents).

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The first thing to understand is that under normal graft conditions, the scion transformation will be about zero, with mostly just “physiological” type effects. Graft hybridization of the seedlings of a scion will likewise be low under normal graft conditions, however its inherently higher than the previous example, perhaps 0-15% novel characters depending on the species among other things. I would follow the technique for the mentor graft or you’re likely wasting time and effort.

It’s true there is still very much unknown about this. There is also a lot of misunderstanding in general about this subject. The research on graft hybridization is very interesting and there are different types of studies with different conditions and results. There are in vitro methods, but the ones I’ve read about all require inserting antibiotic resistance genes prior to making the cell grafts, in order to select out hybrids later, so a non starter for amateur breeders. I honestly wouldn’t worry too much about looking into the molecular mechanisms. The fact is now that there are an ensemble of different ones we know of that potentially are at play. With the level of complexity being so high, a full understanding is still beyond our understanding. Graft hybridization is indeed highly contingent, but it’s not random or unpractical. There are very powerful changes you can make to the phenotype/genotype of plants this way, and you can do it across different families (even grasses) if you follow the right protocol. You end up changing the regulation of thousands of genes among other things. Rather than thinking, “will the particular gene that I want get transferred into the seed” it’s more helpful to think “how can I blend to the degree that I want qualities from the rootstock into the scion and its progeny.” Here is some research done that you can get an idea. Its different from your typical piecemeal discrete thinking about genetic transfer.

Some examples:

Pear wide hybrid seedling mentor grafted on lemon rootstock becomes evergreen. This was a Michurin experiment. This is a 100% success rate int terms of transforming the scion in the direction of the rootstock. It may not be a 100% success rate in terms of making a particular discrete change that you want to make, but since you can effect it by degree and keep changing the direction of variation, then there are a wide open field of possible variations with some predictability. With woody plants of course you don’t need to look for variation in the seedlings since the graft lives many years in the field.

Tomatoes mentor grafted onto goji plants taking on the flavor and polysaccharide profile of goji fruits. Chinese research to make tomatoes more medicinal, this was successful and patents were subsequently filed. This is very similar to Michurin’s graft hybrids of pome fruits.

Vitis vines on shisandra rootstock showing a blending of various morphological characters from the rootstock increasing over the years. Also a 100% success rate in terms of every graft being transformed. The goal was to develop grapes with medicinal qualities of shisandra berries and was achieved. Again this is very similar to Michurin’s observations. This is a recent study with very detailed molecular analysis someone wants to look at it.

There are many more similar examples. There are also examples that more random and chaotic, such as novel phenotypic variation that wasn’t previously present. You find this in many studies, particularly the distant graft mutagenesis ones. But overall its a by degree type of phenomenon, which you can effect with by how long a plant lives grafted, or by how many repeated seedling generation mentor grafts you do, or both.

According to Michurin, the point of developing this method was two fold. Yes of course to introduce new variation not possible with sexual crossing, but moreover as a way of breeding for a small scale that is actually more efficient and less random than conventional fruit tree selection. The work is fairly painstaking, but it’s not the most technical thing either.

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Amazing to hear all the details here. I havent dug into Michelins experiments, and there are indeed a lot of labs in Asia working with the technique so it is hard to keep up with it all.
Secondary metabolites that contribute to medicinal qualities are often produced by endosymbiotic microbes in plants, so it isnt surprising to see that trait being transmitted.
Work on the vine that mimics the shape of leaves in its immediate vicinity (Boquelia) looks like it may be mediated by the vine “borrowing” endosymbiont microbes from its neighbours, and that suggests possibly the microbes are influencing plant morphology.
It is all a gloriously tangled mess. I am keen to be directed to more recent research on mentor grafting if you have any sources to recommend.

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I’ve read a lot of these papers over the years, including the ones cited on the linked thread, and seen no evidence of DNA alteration or durable transmission of genetic changes.

Yes, mRNA absolutely makes it’s way over, much as the COVID vaccines coopt our cellular machinery to manufacture foreign proteins. Yes, epigenetic factors are definitely at play, with demonstrated methylation changes. But no studies that I’ve been able to find show that the changes are durable. That is, when material is sexually propagated and maintained outside of the initiating conditions (such as in the presence of the graft material), that the changes remain over time and generations.

And without that key demonstration, this remains a great area for research and trying to better understand genetic machinery, but a poor way to create new plant varieties for practical use. I would love to find evidence to the contrary.

I agree that there’s some great stuff here for improving grafted stock, though.

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I think you are missing key papers on this front (I miss a lot of them as well- it is impossible to keep up)

Here is an example of confirmed chloroplast transmission between grafts, leading to replacement of the original chloroplast lineage in the recipient plant:
https://www.pnas.org/doi/abs/10.1073/pnas.1114076109

I don’t dispute that epigenetic change, which can potentially revert in the future, are part of the process as well. And to be fair, is even DNA acquisition “permanent” in any meaningful sense since it can always change again in the future.

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That is a good point on chloroplast transmission. I had missed that and will go read the paper you cited.

The issue for producing new varieties isn’t that things can get lost. It’s that no one that I’ve seen has reported proof of transmission of graft-induced traits that isn’t 100% lost again over time and/or generations without the graft present (barring the chloroplast data I’m going to go read about, of course–and presumably if we can transmit chloroplasts, mitochondria are on the table, too? I can’t picture the mechanism here, but will go read).

Edit: It’s right there in the abstract. :grinning: Mitochondria are included, and the mechanism is unknown.

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I suspect endosymbiont microbes are also transmitted permanently (but again endosymbionts can be lost over time since this can be done experimentally with antibiotics in the lab).

Interestingly plant phylogenetics is often based on reporter genes in the chloroplast. If grafting and parasites have been shuffling chloroplasts over time it somewhat muddies the waters. Constructing evolutionary trees based on different reporter genes often yields substantially different results. This is probably the reason why- genomes and organisms are much more porous to information than we previously realised.

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Given the number of crops, including nearly all tree fruits, that are solely propagated through grafting, any routine horizontal transfer calls into question much of what we think we know about those varieties. Is graft compatibility influenced by the initial rootstock for a variety, from which all material flows? For material that is always grafted from seedling to give earlier evaluation, is the graft more influential that previously thought in the traits under assessment? Do we need to track source rootstock provenance, or even worse, a pedigree of rootstocks formerly used, when evaluating scions for new propagations? This just seems like a huge can of worms.

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Links to interesting research:

https://www.jstage.jst.go.jp/article/jplantres1887/74/881-882/74_881-882_480/_pdf?fbclid=IwAR1NtR2aHiEXz3to7kvfhVHIU3E06gwJCd0NMKGjrRoFcIbdTHFUpcO8qEs

This paper by Yagashita in 1961 marks the beginning of Japanese pepper graft hybrid research. From 1961 through the early 2000s, Japanese researchers continued publishing multiple papers on these original lines, as well as creating new experimental lines of pepper graft hybrids. These studies show transmission of characters across the graft union and into seedlings and inherited for multiple generations (decades) afterwards without the need of continued grafting. All of these studies are worth looking at, in one they inoculated the scion with a virus at the same time as mentor grafting it, to interesting results.

Similar results were shown again in Capsicum was more recently:
https://www.sciencedirect.com/science/article/abs/pii/S0304423812002798

https://assets.researchsquare.com/files/rs-2459037/v1/26dc7aa4-682b-4c04-b3bd-9ec31dd91bd7.pdf?c=1678342478
The Vitis/Shisandra graft hybrid is pretty cool. The transformed grape vines continued producing the medicinal Shisandra compounds, as well as having stably transformed morphology (pretty dramatic) even when they were grown subsequently on their own roots. This is the most molecularly detailed work that I’ve seen.

The distant graft mutagenesis studies are worth taking a look at. This work done in China spans multiple decades and involves many different crop species and graft combinations.

There are more studies but these are a good sampling. You can see the work has been done over decades by researchers in different countries.

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Grafting to alter genetics is a very different technique from grafting for propagation. There is no need to worry about cultivar stability of fruit trees and such. You will get cell fusion graft hybrids in all graft unions in the callus tissue, but these won’t have any noticeable effect the grafted plant, they would need to be subcultured to be revealed.

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It’s fun to consider the ensembles of mechanisms going on. It’s definitely a both/and type of situation, and we are so used to zooming in that we sometimes miss the forest. I suspect there is a lot going on in the intersection of exosomes and transposon mobilization (as well as horizontal transposon transfer) in graft hybrids. But whatever the mechanisms, it can be done and changes can be stably inherited.

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Could you link me to this paper? I’m interested in this, but also a bit mad that patented something about this.

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Thulahn,

Search this paper for Lycium and you can find some references. I’ll see if I can dig up the patents, I think there were a couple, but the translation and photos were both terrible.

https://www.researchgate.net/publication/323724247_The_communication_of_endogenous_biomolecules_RNA_DNA_protein_hormone_via_graft_union_might_play_key_roles_in_the_new_traits_formation_of_graft_hybrids

EDIT, link to study on tomato / goji graft:

https://www.sciencedirect.com/science/article/abs/pii/S030442381530234X

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Oh, it’s related to the paper I cited in my article. I’m going to have to start taking some detailed notes. Thank you.