What you probably have is a chimera (more correctly, a 'graft chimera'). At the graft union, a shoot will occasionally start growing which contains cells from both plants. Since the tomato is actually the seed container only and not the result of a fertilization, it also contains both cell types.

Unfortunately, this does not affect the sex cells, although both types may be present, and the chimera does not propagate into the seedlings. The only way you can maintain it is vegetatively, and even then some shoots will revert to one or the other of the two types on occasion.

George

I expect that Geoforce's explanation is correct. I did not find any information about 'Speckled Candle' in a Google search but I do have a speculation which offers a second possibility.

The second possibility depends on whether 'Speckled Candle' has a red fruit with yellow speckles (or yellow fruit with red speckles or streaks, splashes, dots, etc) of a particular sort. If such speckles are due to a transposon (a jumping gene) then 'Roman Candle' was a particular type of sport of 'Speckled Candle' in which a transposon 'jumped' out of one gene (or into a gene) and thus activated or deactivated that gene. Roman Candle would have a stabilized transposon that no longer 'jumped'. The stresses and changed environment of the graft might have caused the transposon to become reactivated and start to 'jump' at high frequencies again. As such the unusual fruit would be a type of reversion back to 'Speckled Candle'.

Streaked/spotted colours are often (but not always) due to transposons. And transposons are the cause of some mutations.

I did find this information:

Speckled Roman striped (red) : Developed by SSE member John Swenson as a result of a stabilized cross of Antique Roman and Banana Legs. Gorgeous 3" wide by 5" long fruits with jagged orange and yellow stripes. Meaty, great tomato taste, ideal for processing. Very productive, few seeds. Still throws an occasional yellow striped fruit. Indeterminate, 85 days from transplant.

Roman Candle (yellow) : Spectacular smooth iridescent yellow fruits that are 2" wide by 4" long. Very meaty with nice flavor. One of the few pure-yellow banana- shaped fruits available to gardeners. Great for making salsa base or tomato sauce. Originated as a sport from John Swensons beautiful Speckled Roman, both decended from Antique Roman. Indeterminate, 85-90 days

I think the interesting points here are the 'stabilized cross' and the 'orange with yellow stripes' versus what appears to be a red with yellow spots that you have. It is my suspicion that a transposon is involved.

Well...
I think I have a chemeric hybrid.
The tomatoes I harvested are distinct from Roman Candle and Speckled Roman.

They appear from pictures to be longish,
whereas my speckled tomatoes are squat.

Well, I have heard that seedlings of intergeneric grafted plants usually segeregate as one parent or another.

I think I remember this is because the chromosomes can't pear up properly with the other.

However,
this is a tomato and tomato graft. They're the same species. So my assumption is that the genes are able to more easily mix up. (Sorry for my layman's terms...)

I've read a few interesting links from Karl King (the one with that rude person). Well,

I don't really know what to say.
I'm not sure that if I grow seedlings from this Chimera, I won't be sure that it's an actual self fertilization of the Chimera, or pollen from the other parent transfered to the be.

It's so late, I wonder if it is possible to root the chimera and try to save it for the next spring? If I grow the chimera next year, I can make sure there aren't any external factors (outside pollination) and prove that this maybe one other way to hybridize...

I'm sorry...
I forgot the link:

Tomato is a perennial and as long as it does not experience frost will continue to grow; you should be able to dig the plant up with its roots and pot it and grow it in the house or other frost-free location until next year.

I don't know what sort of links Karl King may have referenced so I would have to read them in full to be able to make any conclusions but since they seem to reference research on graft-induced genetic changes and since at least one reference to such changes suggests that they are due to transposons or retrotransposons I still think that transposons are involved in some manner in your speckled tomato.

Below is the description of graft chimeras from one web-site:
"Graft chimaera (frequently called 'graft hybrids') are cellular hybrids between representatives of two or more named genera. They are a rare phenomenon arising exclusively in cultivation where two species, frequently of two distinct genera, are grafted together. The mingling of tissues belonging to the stock and scion through grafting in a nonsexual manner. The chimaera come up as a branch or shoot from the point of union which contains tissues of both species, resulting in a arrangement of both species being expressed in the shoot. Such shoots can be vegetatively propagated and cultivated."

Graft chimeras are branches that have cells of both species but these two different types of cells do not fuse; they remain separate. Chimeras can be of different sorts, periclinal, sectorial, etc. Often they may simply involve the three layers that make up most plant organs (leaves, flowers, etc). In those chimeras each layer may be from a different species (or in the case of your tomato would be from a different cultivar).

Since the cells do not fuse then the chromosomes of the two different species (or tomato cultivars) never have to align. That is why the seeds derived from such chimeras produce one or the other or both of the original plants involved in the graft and not intermediates or mixtures of their characteristics. The chromosomes need to align when a sexual hybrid between species is created and this differs from grafts.

Of the three tissue layers, two are involved in producing ovules and pollen and that is one reason why a graft chimera may produce offspring of both types (if one layer is made up of cells of one of the graft participants and the other layer is made up of cells of the other graft participant).

In the reference to the graft-induced genetic changes the molecular mechanism for this to occur (which is unusual and may be rare) has been suggested as transposons or retrotransposons (forms of jumping genes).

The interesting thing about transposons is that they cause mutations and variegation. Both of these may be present in your red fruit with yellow spots/streaks. I agree that your fruit is not 'Speckled Roman'.

I'm talking about this link that I've attached from a previous discussion that I found after making this post.

It's a very argument for both sides (and rather fiesty).

The link that Karl King attaches is very interesting indeed.

Again,
seeds are available to people who are interested in them. From the smaller fruit of course...

Thank you for the reference, Touchstone.

It seems that the research quoted may be from the 1940s and earlier and may be associated with Lysenko. Unfortunately, research of that type was probably not repeated by independent researchers using normal research techniques (accepted practices) and generally has not been accepted as verified by geneticists.

One of the problems with any experiment/research is that there are nearly always many possible answers and explanations for the results. The design of the experiment must control or eliminate as many of those potential explanations as possible leaving only one. Without being able to read all the details of the original research and knowing that such research has been criticised from the time that it was discussed (in Lysenko's book for example) I would not have any confidence that the experiments were designed properly or analysed correctly. Apparently, one of the criticisms has been that the experiments had no controls, were not statistically analyzed and reported anecdotal observations.

There has been more recent similar research published by Chinese researchers. I have read the abstracts of this research and the suggested method that could cause their "graft-induced genetic changes" is transposons or retrotransposons.

Again, I do not know but have not found any verified repetitions of the Chinese research. And depending on the exact design of their experiments there may not have been any graft-induced genetic changes in their work either.

Below is a link to the full details of one of the more recent experiments. In the discussion the researchers suggest that transposons may be involved in their graft-induced genetic changes.

However, I see little justification for their claim that the genetic changes they observed were due to transmission of any genetic changes from the sweet potato to the mungbean. The alternate explanation is that due to only 20% survival of their grafts and then continued self-pollination of the mungbeans they merely selected individuals from a genetically mixed population of mungbean and the selfing made that naturally occurring genetic variation homozygous and visible. To eliminate that as a possibility they would need to begin by using a highly inbred line of mungbean that was verified to be highly homozygous.

Although mungbean naturally self-pollinates, accessions can be genetically variable and inbred lines are generated and used in research.

Genetic transformation is used routinely in laboratories with many different techniques successfully introducing foreign DNA into plant cells. And messenger RNA has been shown to be transported in plants in the phloem. It is quite possible that grafted individuals might be altered genetically.

Well, it is for certain that a chimera has been produced. While it's not extra ordinary, and again, I only did this graft for space issues... it's good to have stumbled on this subject.

I'm rooting the chimeras.

The next year I will grow them and make sure that there are no other tomatoes near by.

If seeds from the self pollinated fruit from this chimera show variation othar than that being Amish Paste or Roman Candle, then perhaps we may have stumbled a new way to hybridize?

Actually,
I have no idea how significant this will be.
I just think it's cool.

I've read about orange/ tangerine chimeras making seedlings that are either orange or tangerine, but never mixed.

Yet, the article from Karl King gives an exception.
Since my tomato chimeric hybrid is not the extraordinary potato/tomato or the nightshade/tomato type,

perhaps there will be seedlings that will not show this segration.

But it makes me wonder.
I do hybridize roses, and most of them are grafted on 'Dr. Huey'. Yet, there has been no seedling I've raised that has shown Dr. Huey characteristics.

But maybe this maybe for different reasons.
Roses are woody. Tomatoes are not. and I've never seen a rose chimera.

Well,
this is something more of us needs to experiment.

If seeds from the self pollinated fruit from this chimera show variation othar than that being Amish Paste or Roman Candle, then perhaps we may have stumbled a new way to hybridize?

The problem you will be faced with is how to compare. You cannot objectively compare the seedlings with photos or descriptions of Amish Paste or Roman Candle because where and how plants are grown can affect how they look. To make valid conclusions you would need to grow several plants from seeds from Amish Paste and several plants from seed from Roman Candle as well as several plants from seed from the chimeric tomato.

And then you would need to take objective measurements of the characteristics of all three groups of plants.

Objectively, you would need to measure the same characteristic on several different fruits from several different plants in each group.

If the characteristic that interests you the most is the red fruit with yellow variegation then objectively you would need to grow many Roman Candle plants to make certain it does not produce such fruits without being grafted. Since Roman Candle is a sport of Speckled Roman and Speckled Roman occasionally produces fruit with yellow stripes it is entirely possible that Roman Candle can revert to produce a fruit with yellow stripes without grafting. Bear in mind that although Roman Candle is a sport of Speckled Roman more than colour changed; both size and shape also apparently changed (from the descriptions quoted above).

Since Speckled Roman appears to have a high sporting rate it is quite possible that its own sport, Roman Candle also has a high sporting rate, again without any need of grafting and with multiple differences rather than just fruit colour.

I just think it's cool.

I do also.

I do hybridize roses, and most of them are grafted on 'Dr. Huey'. Yet, there has been no seedling I've raised that has shown Dr. Huey characteristics.

But maybe this maybe for different reasons.
Roses are woody. Tomatoes are not. and I've never seen a rose chimera.

That is an excellent point. I would not expect the woodiness to affect the transportation of molecules from one part of the grafted plant to another in general. But different plant species may have different abilities to transport molecules in their phloem.

Most roses are grafted to specific root stocks as are dwarf fruit trees. Yet one does not hear of any unusual transfer of characteristics from the graft stock to their seedlings. I would think that indicates the rarity of any similar transmission.

I'm going to attempt this again.
But this time with two very distinct varaities.

Perhaps some kind of yellow beefstake with bumpy shoulders,
and a red or puple cherry type with smooth shoulders.

This is something I am interested doing again just to experiment.

Touchstone,

Rose chimeras are possible, just rare. Darwin discussed one that combined the Tea rose 'Devoniensis' and Rosa banksiae. I know of another that is currently growing in Belgium, combining an orange Polyantha with a Dog Rose (possibily R. laxa Hort., but I don't recall off hand). The owner has promised me a picture, but I'm still waiting. He says it has orange flowers like the Poly, but the plant generally resembles the other parent.

Vegetative hybrids have a long history, and are frequently confused with chimeras. In fact, chimeras have been known to give rise to vegetative hybrids. Laburnocytisus adami is a chimera that frequently reverts to the two species. As a chimera it is sterile even when grown near the species (according to Darwin). But the reversions are fertile.

Darwin raised seedlings from the Laburnum reversion and all the seedlings were purely of that species -- except for a couple that had remarkably long clusters of flowers, very unlike the species. Rev. William Herbert, Darwin's friend, also raised seedlings of a similar reversion. His also were like the Laburnum -- except for those with purple flower stalks, again very unlike the species.

Lucien Daniel (French) experimented with vegetative hybridization in the late 19th century. He grafted flower stems of tender kohlrabi onto hardy cabbages, then bagged the flowers to prevent cross-pollination. Seedlings were kohlrabis, but somewhat modified and much hardier.

Other French researchers continued the work. If you can read French check out the "IV Conférence internationale de génétique", Paris 1911 (pub. 1913). There is an extensive report with illustrations, mostly of Brassicas and their kin grafted in various combinations. Someday I'll get it scanned and send it off to one of my French-speaking friends to translate.

Skipping over the various workers in the former Soviet Union, we come to Frankel in Israel who found that cytoplasmic male sterility in petunias could be transferred from a scion to seedlings of the stock plant, 'Rosy Morn'. The stock plants were not themselves modified, so this was unlikely to have involved an infection. And only around 30% of the seedlings were male sterile, though this varied with the seasons. The plants stopped producing male sterile offspring soon after the scions were removed.

Rick (US) used Frankel's male sterile petunia on tomatoes, but could not get the sterility to transfer.

I read a report over 20 years ago, I think it was from an American source but I can't find it, of mature apple varieties top grafted onto apple seedlings. Some of the varieties were able to force the seedlings to adopt a specific root habit -- either fibrous or taproot, according to the variety. Ungrafted seedlings from the same batch of seeds were variable as to root habit.

After the root habit had been established, the researchers removed the scions, allowing the stocks to produce trunks of their own free from foreign influence. The root habits were retained. Root cuttings from the trees also preserved the "acquired" root growth habit.

I don't recall any mention of seedlings from the modified trees, so I can't say that the obviously modified "heredity" of the seedlings could be passed along to seedlings.

The reported effects of vegetative hybridization are not always obvious. Certainly we cannot expect anything like what we get from ordinary cross-breeding. The results would be more like partial hybrids. For example, Michurin reported that the Siberian Crabapple (Malus baccata) should never be used as a rootstock for apple trees that are to be used for breeding. The trees themselves were not modified noticeably, but seedlings from them had too many "wild" qualities of the fruit. This could be retested by anyone interested in raising bad apple seedlings. But as Frankel found with petunias, the transfer seems to be influenced by weather. Michurin lived in central Russia. A proper repetition of the experiment should be in a similarly harsh climate.

Also, it is a good idea to test the same clone, grafted and own-root. If there is an influence it will be found by comparing the two crops of seedlings, not by comparing them to the stock. If 'Doctor Huey' is influencing the seedlings of scions grafted to it, I would not expect anything so blatant as a direct transfer of the harsh purple-red flower color.

If I were trying to find an influence I'd start with a seedling rose -- the younger the better. I'd bud it to 'Dr Huey' and keep the original plant growing. Then I'd collect a large number of seeds from each, raise the seedlings and compare them as groups.

I hope everyone is aware that cell fusion can occur in plants with no assistance from humans. And cells can undergo spontaneous reduction division (meiosis). Thus, it may happen that cells of the two parents meeting at the graft union fuse, then do a reduction division to get back to their original states -- almost -- with just a smidgen of foreign DNA left in each daughter cell.

I read a report on seed grafts -- I think it was a slice of maize embryo inserted into a wheat embryo. After the graft had healed, some of the cells were found to have an intermediate chromosome number, suggesting that cell fusion and reduction division had occurred. (BTW, spontaneous reduction division can follow colchicine-induced chromosome doubling. Thus, shoots may form that differ significantly from the parent plant but have the same chromosome number.)

When your tomato/tomato chimera (if it is) sets seed it is likely that some offspring will be ordinary sexual hybrids of Amish Paste and Roman Candle. If nothing weird or interesting is happening, then the ova and pollen will be of the "pure" parental types. Even if you bag the flowers so they are forced to self pollinate, some of the pollen may be Amish Paste and some of the ova Roman Candle.

Karl

If I were trying to find an influence I'd start with a seedling rose -- the younger the better. I'd bud it to 'Dr Huey' and keep the original plant growing. Then I'd collect a large number of seeds from each, raise the seedlings and compare them as groups.

Because this method removes the genetic variability potentially present between scions (the buds) it eliminates the possibility that graft survival is genetically based and that the scions that take are a selected population of the original. However, because the rose seedling is likely to be heterozygous at many loci (genes) any differences found between the two groups of seedlings may still have alternate explanations. For example, the graft environment might preferentially alter the production/survival of gametes (on the basis of genotype) in the scion. That would not involve changes to genes, nuclear or cytoplasmic, but would still rely on genetically-based selection.

Another possible interpretation for genetic differences between the two groups of seedlings would be that the graft environment alters/increases the rates of spontaneous mutations in the scion.

Designing an experiment to eliminate potential explanations leaving only one is often very difficult. The alternative may be to eliminate as many as possible and then collect as much information as possible to eliminate other alternatves. In the case of a possibly rare effect that is likely to resources beyond the home environment.

Admmad,

"Another possible interpretation for genetic differences between the two groups of seedlings would be that the graft environment alters/increases the rates of spontaneous mutations in the scion."

Yes, that is a good possibility and reason enough to make such tests. There is even the possibility of differences in cross-over frequency and distribution. Yet more good reasons!

One of the worst aspects of Mendelian experiments (early 20th century) was that the researchers combined data from different crosses, different generations, different years and different habitats -- in order to get the "good" numbers they were determined to find.

Another book I'd like to read (in translation) was published by de Mol in 1921. He studied hyacinths and their chromosomes, and concluded that many of the variations were a direct result of breeding with plants that had been forced to bloom out of season.
De l'existence de varietes heteroploides de l'hyacinthus orientais L. dans les cultures hollandaises. Willem Eduard de Mol.

Reproduction is a complex process, as I'm sure you know. In addition to the mechanics of flower production, there is genomic reprogramming, crossovers, specialized processing of male and female gametes, etc. Also, gametophytes (pollen tubes and egg sacs) are organisms with properties of their own. Changing the environment (temperature, chemistry, photoperiod, etc.) can alter the competition among gametophytes. And because gametophytes employ thousands of the same genes used by sporophytes (the flowering plants), there is bound to be an effect.

The point of graft-hybridization experiments is to induce variation, preferably variation that is not commonly found in plants raised from seed.

Karl

While I take many of the old papers cited by Karl with a grain of salt (as I do some modern papers), I have no doubt that certain things affecting phenotype do move across graft unions.

However, I think the possibility that this is a sport is much greater, and another reasonably large possibility is that what you got wasn't what it was sold to you as. These things happen all the time.

I'm going with sport. I also think it's likely that this is a gain-of-function mutation (reverting back to the standard red color). In which case, a selfed population ought to segregate.

The point of graft-hybridization experiments is to induce variation, preferably variation that is not commonly found in plants raised from seed.

Whenever an idea is not mainstream several questions come to mind:

What is the frequency of such observations?

How consistent are they within the same species and how common are they in other species?

How often have they been replicated by others?

In the case of graft-hybridization there is the added question:

Is the variability induced any different than that induced by other genetic methods?

With any research report there are always questions that relate to the methods used and the conclusions drawn and whether other explanations are possible that are more likely to be the cause of the observations than those suggested by the researchers.

As an example, for pepper graft-hybrids the Japanese researchers report that transposons or retro-transposons are possible candidates for the cause of genetic changes. Transposon mutagenesis is currently used in mainstream genetics and it is possible that the genetic changes are the same sort as are present in mainstream genetic work.

Frankel's findings with the transmission of cytoplasmic male sterility (CMS) in Petunia is interesting in many respects. CMS is used in producing F1 hybrid seed and five or six generations of backcrossing are required to move CMS into new strains. Graft-induced genetic changes would save resources and increase profitable returns for companies in the hybrid seed business. Yet little research is available indicating that there is (or has been) interest in this topic. A search of the literature finds at least 11 reports that reference Frankel's research or are referenced within those reports. Frankel, Edwardson & Corbett, and Bianchi, all working on Petunia reported positive observations. Everett, and van Marrewijk, both working on Petunia reported negative results. Sands, Rhoades, Lacadena, McWhirter, Rick, and Gabelman working with other species all reported negative results.

van Marrewijk worked with a number of Petunia strains, including 'Rosy Morn' obtained from Frankel and was unable to repeat Frankel's findings. From a short summary of van Marrewijk's thesis the critical conclusion was drawn that other than Frankel's original 1956 report, none of the other positive results in Petunia eliminated alternative causes of the observations.

There has been research on cytoplasmic male sterility and it has been found to be related to mitochondria and their genetic systems.

Elakazal wrote:
"While I take many of the old papers cited by Karl with a grain of salt (as I do some modern papers), I have no doubt that certain things affecting phenotype do move across graft unions."

One of the things I like most about old papers is that the authors had prejudices and biases of their own -- different from those of modern writers. Darwin and Herbert, for instance, were able to report on their anomalous seedlings from Laburnum-reversions because the prejudice against such things existed but was not yet a dominant paradigm.

Similarly, in the early 19th century Girou de Buzareingues was free to write about what we now call "segregation distortion" without offending Mendelists -- because there were no Mendelists to offend.

More recent researchers tend to fall back on the overused concept of "gene mutation" whenever things do not the way they like. How many partial hybrids have been dismissed as "purely maternal" plus a mutation? Nowadays partial hybrids have been accepted -- DNA evidence supports them -- but not everyone is entirely happy about them.

Admmad wrote:
"Whenever an idea is not mainstream ..."

I guess that's the safe approach. Never consider what the mainstream has not yet embraced. Likely as not the mainstream will eventually be proved wrong, but until then one can avoid being despised along with the "cranks". And when the cranks take over, new converts can begin despising and condeming other heretics who refuse to accept the mainstream opinion whole-heartedly.

Izhar and Frankel (1971) found that pH and its apparent influence on callase activity differed between fertile and sterile petunias. Whether this involved midochondria is another question, possibily already answered.

Boeshore et. al, (1983) wrote, "Similarly, we found no clear relationship between the mitochondrial DNA restriction patterns of male fertile somatic hybrids and those of the male fertile parent. In view of this finding and the evidence that cytoplasmic male sterility and fertility segregate in somatic hybrids of petunia (Izhar et al. 1983) the nature of cytoplasmic male sterility in petunia differs significantly from cytoplasmic male sterility in tobacco (Gerstel 1980)."

The fact that CMS is related to mitochondria in some species does not prove that the same will be true for other species. And the fact that CMS is not always transmitted by grafting is not evidence that it is never transmitted by grafting.

By the way, Frankel (1962) wrote, "The original male sterile line was kindly furnished by W. Atlee Burpee Co."

And if you get to a library before I do, you might check on 'Graft induced transmission of cytoplasmic male-sterility in alfalfa' by TE Thompson, JD Axtell - J Hered, 1978.

Karl

Frankel, R, 1962. Further evidence of graft induced transmission to progeny of cytoplasmic male sterility in Petunia. Genetics 47:641-646.

Boeshore, M, et. al. 1983. Novel composition of mitochondrial genomes in Petunia somatic hybrids derived from cytoplasmic male sterile and fertile plants. Molecular Genetics and Genomics 190(3): 459-467.

Izhar, S and R Frankel. 1971. Mechanism of male sterility in Petunia: The relationship between pH, callase activity in the anthers, and the breakdown of the microsporogenesis. Theoretical and Applied Genetics. 41(3): 104-108.

The fact that CMS is related to mitochondria in some species does not prove that the same will be true for other species.

Science does not prove theories or hypotheses; it only disproves them and when it does they must be replaced or modified. If they are modified to the point of being cumbersome and over-complicated they are typically replaced.

Cytoplasmic male sterility in tobacco is apparently due to a deletion of the nad7 gene in the mitochondrial DNA. One of the fascinating findings on mitochondria is that they exist as populations within many individuals. In the ancestors (progenitors) of the cytoplasmic male sterile lines two populations (or more) of mitochondria are found, the most prevalent is normal and the abnormal is present at what is termed substoichiometric amounts. In the cytoplasmic male sterile offspring the proportions are reversed. And in reversions (not due to restorer genes) the proportions are again reversed. ["Organization and Expression of the Mitochondrial Genome in the Nicotiana sylvestris CMSII Mutant" Lelandais et al Genetics 150: 873882 1998]

Cytoplasmic male sterility in Petunia seems associated with mitochondria and their DNA although a different gene appears involved ["The Male Sterility-Associated pcf Gene and the Normal atp9-1 Gene in Petunia Are Located on Different Mitochondrial DNA Molecules" O. Folkerts and M. R. Hanson Genetics 129: 885-895 1991]

The following quote is from research that examined a protein involved in restoring the fertility of CMS Petunia [A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants, Bentolila et al, PNAS 99:1088710892 2002]
"Cytoplasmic male sterility (CMS), a maternally inherited condition in which a plant is unable to produce functional pollen, has been observed in numerous species (1). Mitochondrial defects have been shown to be responsible for all of the CMS characterized so far. The regions whose expression is associated with CMS contain unusual ORFs that are often chimeric in structure and frequently cotranscribed with conventional mitochondrial genes (2)."

The safe approach is to always question all published reports, but unfortunately many of us are human and we only question those reports that are unexpected. However, questioning something we read is to be encouraged not denied. Those reports that conducted the research with proper controls, provide full details of their methods, collect appropriate objective data and analyze it appropriately, have valid and sufficient controls... in other words conduct science using validated procedures will enable the reader to accept the findings with some confidence. However, even such reports will produce debate if other independent researchers are unable to repeat the observations.

The finding that different populations of mitochondria are present in the fertile progenitors to cytoplasmic male sterile stocks, and that within those CMS stocks there are again different populations of mitochondria suggests that any research pre-dating the ability to measure sub-stoichiometric populations of mtDNA and identifying the DNA sequences within those populations will always be open to the alternate explanation of selection in a graft environment, that is grafting would be simply one way to trigger the change in proportions of the different mitochondria. Unfortunately, from the published attempts at replicating Frankel's findings in both Petunia and other species, it appears that it is not a very reliable or very successful method for doing so.

Admmad,

"Unfortunately, from the published attempts at replicating Frankel's findings in both Petunia and other species, it appears that it is not a very reliable or very successful method for doing so."

I take it that you are not accusing Frankel of being a fraud or a fool. If some others have been unable to replicate his results ... well, no telling where the fault lies. I haven't tried converting 'Rosy Morn' or 'Black Prince' to male sterile forms. I just mentioned the research as an instance of graft transmission of an hereditary trait.

But getting back to basics, we have another case that appears similar. According to Darlington (Evolution of Genetic Systems, 1958):

"... susceptibility to nodule bacteria is cytoplasmically inherited in clover and characteristic for each species. But seedlings of Phaseolus vulgaris are susceptible to P. lunatus bacteria if their parent has been grafted on roots of P. lunatus. "

The problem is the same. A trait that presumably is inherited cytoplasmically may still be graft-transmissible.

Whether there is any commercial value in transferring bacterial susceptibility seems irrelevant at this point. The fact that it can happen is interesting enough -- although CMS would be easier for amateurs to observe.

Even easier to observe are the changes in color, form and flavor that have been reported in vegetative hybrids of various members of the Solanaceae: including tomatoes, eggplants and peppers.

You wrote, "...questioning something we read is to be encouraged not denied." I agree. In addition I think everyone who is interested in such things should conduct experiments of their own. We may hope that they also make use of adequate controls, and learn to distinguish artifacts from real evidence.

For example, this thread began with an apparent chimera of Amish Paste and Roman Candle. If the two varieties are "pure" -- as tomatoes go -- then we could make predictions about the seedlings from the odd fruit.

If the fruit is a chimera (and the flower was self-pollinated), the seeds may give rise to three distinct types of plants: "pure" Amish Paste, "pure" Roman Candle, and F1 hybrids of Amish Paste and Roman Candle.

However, if something more interesting is going on, we may expect a more diverse batch of segregating seedlings -- possibily approaching results that would not be expected until the F2 if sexual hybrids were involved.

It doesn't really matter at this point whether transposons, mitochondria, viruses, plasmagenes or some as-yet-unidentified phenomena are involved. Get the facts first, worry about theory later on. Give all those college students something to write their theses about.

Some really interesting research can be conducted by amateurs, even without using the latest turbocharged gene sequencer.

Karl

But getting back to basics, we have another case that appears similar. According to Darlington (Evolution of Genetic Systems, 1958)

I am sure that we could take each example, of the many that were published many years ago, and follow the trail of more recently published research that provided useful information for determining if the original conclusions were reasonable. We would probably learn a great deal about many interesting subjects. But it would be time-consuming and from my viewpoint I am handicapped by having poor access to libraries with older research journals. I expect that time would be more efficiently used by examining the original research papers and determining whether accepted scientific practices were used and thus their conclusions warranted.

I am going to continue with Frankel's research because although I do not think he was a fraud or a fool I do consider that his conclusions were unwarranted. It is clear from his published comments that he knew exactly where his experimental design failed to eliminate alternative explanations.

His 1956 paper was considered by van Marrewijk as having shown an effect of the grafting so I will use it as the basis for my comments. From a quick examination of recent research published by the Chinese and Japanese groups on graft-induced genetic changes I expect that, in general, similar comments could be made in relation to many of the papers published on similar subjects.

Frankel's paper gives no indication that he randomized the growing of any of his plant material and thus any systematic environmental differences present were not eliminated from affecting his observations. It is normal practice to have a randomized design to account for such potential effects.

Nor did he provide any description of how he classified plants as sterile versus fertile. If there was any subjectivity involved in that classification then 'blind' methods would be needed to eliminate any researcher biases (that is the person classifying the plants would not know which plant was from the experimental group and which was from the control group).

And most seriously he had no control for the effect of grafting. The description in the paper indicated that the effect of grafting was severe both on the scions and on the seeds produced from them. He needed a group that was fertile scion grafted to fertile stock to be able to compare and eliminate the effects of maternal environment on the offspring. These are known in plant species and can be short-term or long-term.

If he had a control group he would then have had to analyze the differences in fertility between the two groups to show that the experimental treatment had a significant effect.

His research material was open-pollinated, genetically variable and thus permitted alternative explanations involving selection, etc.

There was no description of how he chose the plants to provide the scions, was one plant used for all scions? were a number of plants chosen at random used? and so on.

He clearly understood that his design allowed alternative explanations as he states:
"For several reasons these data do not permit extensive speculation about the nature of this cytoplasmic sterility." and he went on to mention the high heterozygosity, the lack of large numbers of offspring, the possibility of distorted segregation ratios due to high seedling mortality.

In my opinion his conclusions are unwarranted and although he thought, "There is a remote possibility, for instance, that nutritional deficiency, which might have been induced by grafting, might cause a disturbance in cytoplasmic
enzyme activity in such a way as to lead to an increase or decrease of sterility-determining entities of the cytoplasm." I consider that is the most likely explanation (omitting the specification of enzyme activity).

With hindsight, from what is known now about cytoplasmic male sterility and mitochondria, the most likely explanation is that his fertile stock had individuals with substoichiometric mitochondrial variants present and that the stress induced by grafting caused a change in proportions. That type of explanation would also help explain why the results of replicating the experiment in Petunia could fail - that is, they would fail if the fertile stock chosen did not harbour the mitochondrial variant and they would fail if the grafting was easily successful and did not provide high levels of 'stress'.

If 'Rosy Morn' has spontaneously produced cytoplasmic male sterile individuals then that would be evidence that it contains individuals with substoichiometric (one variant DNA copy per every, eg 100-200 cells) quantities of the variant mitochondria.

It is intersting to note that a report (Bentolila, et al)quoted "In Petunia, a single dominant nuclear gene termed Rf confers fertility to lines carrying the only known CMS cytoplasm in this genus (16)."

Reference 16 is a paper by Izhar, et al and Bentolila, et al thank Izhar for providing Petunia stocks; Izhar worked with Frankel. Apparently none of the cytoplasmic male sterile lines from grafted individuals are available from Frankel's work or the other research groups.

It doesn't really matter at this point whether transposons, mitochondria, viruses, plasmagenes or some as-yet-unidentified phenomena are involved.

Although for Touchstone, as the graft has already been made and a variant found this does apply, in general I cannot agree that it does not matter.

For example, if one is interested in genetic variation caused by a transposon then it would be easier and more efficient to begin with a stock that has an active transposon than to graft a stabilized strain in the hopes of reactivating the transposon and producing interesting genetic variants.

The question returns to "does grafting cause any changes that are different from those that can be caused by alternative methods?" and "can those alternative methods be more practical/effective/efficient than grafting?"

If the cytoplasmic male sterility history (Frankel) is a general example I would conclude that the answer to those two questions, in order, is no and yes.

"... susceptibility to nodule bacteria is cytoplasmically inherited in clover and characteristic for each species. But seedlings of Phaseolus vulgaris are susceptible to P. lunatus bacteria if their parent has been grafted on roots of P. lunatus. "

Although I am not able to read the original research on this grafting work with Phaseolus perhaps these more recent papers provide information that might help explain the older results [for those interested:-)].

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specicity, Dasharath P. Lohar and Kathryn A. VandenBosch, Journal of Experimental Botany, 56(416):16431650, 2005.

Recognition of Leguminous Hosts by a Promiscuous Rhizobium Strain, S. SHANTHARAM AND PETER P. WONG, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 43:677-685, 1982.

Nodulation of Pole Bean (Phaseolus vulgaris L.) by Rhizobium Species of Two Cross-Inoculation Groups, ARYA K. BAL, S. SHANTHARAM, AND PETER P. WONG, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 44:965-971, 1982.

"If 'Rosy Morn' has spontaneously produced cytoplasmic male sterile individuals then that would be evidence that it contains individuals with substoichiometric (one variant DNA copy per every, eg 100-200 cells) quantities of the variant mitochondria."

As Frankel noted, 'Rosy Morn' does produce the occasional male sterile seedling without benefit of grafting. The frequency increased abruptly when the male sterile clone was grafted to the stock plant, then dropped again when the grafted shoot was removed. Usually. In his 1962 paper he discussed a plant that continued to produce male sterile offspring long after the male sterile shoot was removed.

"Although for Touchstone, as the graft has already been made and a variant found this does apply, in general I cannot agree that it does not matter."

The point I was trying to make is that amateurs should not be put off because they lack the latest shiny gadgets and technical jargon. There are so many alternative explanations that experts can go on arguing for decades. That's fine. But while they debate the old research we can go on finding more cases for them to examine.

Segregation distortion is a very interesting phenomenon and could easily apply to organelles. Consider the green/white variegated geraniums (Pelargonium). Yes, the plants are chimeras (in regards to chloroplast distribution). But they can be raised from seed because chloroplasts are present in the pollen. So, pollinating a green variety by a variegated will give a proportion of variegated offspring. The chloroplasts sort themselves out -- greens ending up in the inner layer, whites in the outer -- producing more chimeras.

Why do the white and the green chloroplasts sort themselves in this way? What conditions favor the multiplication of one type over the other in different tissues?

I have no information on the effects of grafting on geraniums. I mention the case only to suggest that something provided by the grafted scion might favor the increase of a rare mitochondrial variant -- if that is what is involved. Would a scion with variant mitochondria favor the multiplication of a similar variant in the stock? I see no immediate objection. And could some change in sap chemistry provoke segregation distortion during the production of ova and pollen? I can't say yes or no.

Then there is the possibility that changes in sap chemistry may induce changes in gene expression. For example, two specimens may differ in sap pH or metal ion concentration. Either condition might alter gene expression and reveal hidden potentials that could segregate and become fixed (or canalized).

Much of the research on graft transmission of traits in tomatoes, eggplants, peppers, petunias, etc. deals with traits that are already present in the species, though not in the particular varieties. If a white tomato is grafted to a red one, red fruited seedlings from the white are not really novel. They are reversions. The same is true for differences in cluster size and leaf shape, or differences in capsaicin production in peppers.

Now we get into a philosophical problem that seems to be at the heart of opposition to vegetative "hybridization". What do we mean by "transmission"?

In the older mendelian view, transmission of heredity meant the movement of definite units called "genes". Everything else moved from the genes outwards. Now we know that this is not always true. Healthy cells from human ovaries can be transformed into cancer cells by cultivating them in a semi-rigid matrix. No mutagens are required. And cancerous prostate cells can be transformed into normal cells (non-cancerous, anyway) by exposing them to ECGC, a polyphenol found in green tea. The "heredity" (expression) of cells can be altered by environmental conditions.

It has been learned that environmental conditions present at the time of embryo formation can establish alterations in phenotype that persist through maturity, in the Norway spruce:

The maternal temperature during zygotic embryogenesis influences the adaptive properties of Norway spruce progenies; a "memory" involving DNA methylation and differential transcription of phytochrome genes?
http://www.treebiotech2003.norrnod.se/s3_o.htm

It is at least conceivable that, in some cases, grafting of dissimilar parents could alter conditions surrounding the embryos enough to change gene activation patterns that would persist through the lives of some of the plants, and then to segregate (apparently) in the next generation. The genetic potential might rarely or never be exposed but for the influence of the other grafted part.

This would not be "transmission", in the sense that mendelists use the word, from scion to stock (or vice versa) because no "genetic material" was exchanged. But there still could be transmission of "epigenetic information".

The most eye-catching instances are those where a trait present in one part appears to have been transmitted to the other. These are also the cases that are most alarming to mendelists. But we should be alert to modifications of all kinds. This is why we should keep the original plants that provide stocks and scions for grafting. To compare progeny of each grafted plant to its ungrafted original.

Vegetative "hybridization" is imprecise at best. But so is zapping a plant with ionizing radiation or dosing it with chemical mutagens. At least amateurs do not face health risks when grafting tomatoes, and the variations produced are less likely to be harmful to the plants. Sometimes useful variants may be found that exhibit qualities not found in the seed parent's species.

If anyone should find a genuinely new trait in seedlings from a grafted plant -- such as a new pigment, or capsaicin in a tomato -- I'd like to hear about it. But if it's just an existing pigment expressed in a different tissue or different concentration I wouldn't be so surprised.

But while they debate the old research we can go on finding more cases for them to examine.

It does not matter how many times poorly designed research is replicated, it is still poorly designed and the conclusions unwarranted.

Are there any examples of appropriately designed analyzed/etc. research reports in which alternative explanations are eliminated, that conclude that grafting induced genetic changes, and therefore those conclusions are warranted?

Critics can always raise objections and offer alternative explanations to discredit research that challenges their prejudices. One way to observe this is to do a little research on your own that does not fit neatly into "mainstream" preconceptions. Then you will see how many armchair critics are anxious to attack you.

Case in point: in 1884 Prof. Millardet published the results of his work with strawberry hybrids. In 1927, Mangelsdorf and East savaged the professor's work because it did not conform entirely to their own work. In one case, Millardet pollinated a white-fruited Fragaria vesca (2X) by a red fruited form of F. chiloensis (8X). One of the seedlings seemed to be a "pure" vesca -- except that it bore red fruit and was pollen sterile. Mangelsdorf and East would have none of it. They suggested that Millardet had been fooled by stray pollen, stray seeds or a stray runner. Pretty insulting, I think.

Then in 1934 East had a problem of his own. He had pollinated a white-fruited Fragaria vesca by a red-fruited F. virginiana (8X). One of his seedlings looked like F. vesca except that it had red fruit and the anthers failed to dehisce.

East knew beyond personal doubt that his odd plant was not a simple reversion or "mutant" because it differed in minor characters from the strain of F. vesca he was using. It took him three years of study to find one trait that was never found in that species, but was present in the octoploid pollen parent.

Interestingly, while East cited Millardet's work, he neglected to mention his own role in the previous "critical analysis" which has made Millardet look the fool.

It could be difficult to "prove" vegetative "hybridization" to a confirmed geneticist. Until recently, almost all geneticists believed (devoutly) that any DNA not involved in gene-coding was mere "junk". Now we know that microRNAs inhabit those genetic wastelands, and that microRNAs are very important in determining the developmental program.

http://www.plantcell.org/cgi/content/short/tpc.016238v1
Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes (2003)

Here's another:

Nature 431, 356 - 363 (16 September 2004)
RNA silencing in plants
David Baulcombe
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK

"There are at least three RNA silencing pathways for silencing specific genes in plants. In these pathways, silencing signals can be amplified and transmitted between cells, and may even be self-regulated by feedback mechanisms. Diverse biological roles of these pathways have been established, including defence against viruses, regulation of gene expression and the condensation of chromatin into heterochromatin. We are now in a good position to investigate the full extent of this functional diversity in genetic and epigenetic mechanisms of genome control."

That's the outline of it: RNAi transmitted between stock and scion, then the state (silenced or heterochromatic) retained by feedback.

No "genes" would be transmitted -- which is why I am sceptical that a tomato/pepper graft could result in a "hot" tomato. Changes in development and gene regulation, on the other hand, would not require any GENETIC changes (heterochromatization, methylation) that could not be attributed to some other hypothetical cause by a devout critic.

Critics can always raise objections and offer alternative explanations to discredit research that challenges their prejudices.

Raising objections and offering alternative more probable explanations is part of science. It is a necessary part, perhaps even vital, in separating possible explanations from plausible explanations and hopefully finding the real explanations. And it should happen in all circumstances. When done logically and appropriately it points the way to new tests that distinguish between alternative explanations. After such series of experiments the critics would be faced with offering alternative explanations that have no known scientific validity and thus are unwarranted in themselves.

An example. A researcher designs an experiment with all the appropriate controls and the results lead to a speculative explanation. Criticisms suggest alternative explanations, perhaps some of which the researcher was not aware of. A new experiment is designed to account/control for those criticisms and the results rule out those alternative explanations. Perhaps there is yet one more cycle of this. At the end of the third set of experiments the speculative original explanation is left. Criticisms now must suggest explanations that are as implausible or even more implausible than the researcher's and are not effective. At this point, if not before, the speculative explanation would be used as the basis of new experimental designs for testing its predictive value. And as long as the observations based on such experiments continued to be in accordance with the explanation/theory it would stand. When a set of observations disproved the theory it would be modified or replaced as necessary.

With any research there are always replications whether published or not. There are always 'mavericks' looking for the unusual, unexpected, rare to exploit. And they have the benefit of knowing where the potential pitfalls are in analyzing the previous research and can modify the experiments to account for the valid criticisms. In due time, even without publication, the research that produces unexpected results but that cannot be replicated becomes ignored.

It may be important to note that even when an original finding is not considered to be explained correctly, by the majority of researchers, in time, when those explanations are in fact correct they become accepted or even very important. McClintock's 'jumping genes' being one example of that.

It could be difficult to "prove" vegetative "hybridization" to a confirmed geneticist.

Science does not work that way. No number of positive confirmations of a hypothesis can ever prove that the hypothesis is correct, yet one validated replicated disproof indicates that the hypothesis is incorrect. The point is that research on any topic must show that explanations derived from theory are wrong to disprove that theory - they can never prove the theory with positive results. Equally it must show that the explanation the researcher considers correct fits all the observations (from the design of the experiment) while the alternatives do not. But that does not prove the researchers explanation either.

The researcher who designs a preliminary experiment (as a quick test) with less than perfect controls, etc expects to have to redo the experiment (if the results are interesting) with all the necessary common accepted objective methodologies in place to resolve the questions and doubts which should be present in everyone's mind based on less than adequate preliminary observations.

RNAi transmitted between stock and scion, then the state (silenced or heterochromatic) retained by feedback.

The postulating of another possible explanation does not help. When the design of the research allows any alternative explanations then the objective, logical result is that the simplest explanation is chosen not the most complex. When the simplest explanation is known to occur and to be common it becomes even more heavily favoured as the correct explanation. Especially if the researcher's conclusion is speculative.

In offering another speculative alternative explanation the end result would be that the researcher would have to redesign the experiment to rule out other alternatives while supplying results that fit the new explanation. Without such observations the new alternative, like any other speculative explanation becomes relegated to the unlikely, or lacking in evidence.

There are accepted practices for designing experiments. When those methodologies are not present in an experiment not only the conclusions but the observations themselves can become suspect. A researcher's conclusions can always be questioned and criticised even without replication of the experiment. In properly designed experiments the observations cannot be questioned without replicating the experiments.

Alternative explanations are easy to produce; it is the logical, objective disproving of those alternatives that allows a field to choose the alternative that is not disproven and move forward and so on. If the workers in a field are unwilling to work within the methods of science it is easy to understand why their observations might be ignored.

It sometimes happens that a phenomenon cannot be scientifically validated because there are no available means for identifying precisely what is going on. Or it may be that the "scientists" of the day are not yet capable of understanding.

As O. F. Cook wrote in 1906 (The Vital Fabric of Descent):

"The organism may be described, for some purposes, as a machine, but it is no mere corn-sheller or steam engine, and there is no assurance that we have, as yet, even a basis of conjecture regarding the principles on which it is constructed, or the ultimate nature of the materials of which it is made. What the mechanism does, however, is a very practical and pressing question which need not be postponed on account of any lack of agreement in general theories, if, indeed, the workings of the device do not afford the best clue to an understanding of its structure."

We should not wait around until academic and corporate scientists give us permission to perform our own experiments. The workings of heredity are available for study by anyone. Many people drive cars without grasping the principles of the internal combustion engine. And how many people using computers today know the first thing about microprocessor architecture?

The phenomenon of post-transcriptional gene silencing (PTGS) in plants is much the same as RNA interference (RNAi) in animals and "quelling" in fungi. It is an aspect of heredity that does not involve structural changes to the DNA. I am not postulating. There is an abundance of research from around the world.

For anyone who is still interested, here is a brief review with links to other research.

Kalantidis K (2004) Grafting the Way to the Systemic Silencing Signal in Plants. PLoS Biol 2(8): e224

Among other things, Kalantidis informs us that the PTGS signal generally moves from carbon source to carbon sink. That is, in a sprouting potato the carbon (as carbohydrates) moves upward from the tuber to the shoots, but later in the season the direction will be reversed as carbohydrates produced in the leaves move downwards to the growing tubers. The PTGS signal, if present, will move accordingly.

Furthermore, the developmental stage of the tissue plays a major role: young tissue is more susceptible to acquired silencing than developmentally mature tissue of the same genotype.

Interestingly enough, techniques devised by Ivan Michurin in the late 19th century agree very nicely with the most recent discoveries. He insisted that the influence moves more readily from old and stable plants into young ones. And when he grafted a young scion to an older stock, he would remove the leaves from the scion -- converting it from carbon source to carbon sink. He knew nothing of RNA and PTGS, of course, but he knew from his own experiments that the techniques worked.

Did "vegetative hybridization" work before the modern discovery of post-transcriptional gene silencing? Did apples fall before Newton "discovered" gravity?

It is nice that scientific theory has finally caught up with practice -- after a century of vehement denials. We now know more about mechanics, which will go a long ways towards improving the old practice. But it is wrong to deny that Charles Darwin, Lucien Daniel and Ivan Michurin (among others) got there first.

In fact, it might be useful to have a second look at some other old observations, such as Naudin's report of somatic segregation in species hybrids. If the two parents differ in the state (silenced or unsilenced) of a particular gene, it would not be surprising to find that both copies become silenced in some tissues, and both unsilenced in others. This would look like the same kind of segregation that he observed among seedlings from the hybrids.

Further information on this phenomenon can be found by searching Google Scholar for the words: graft transformation plants

Karl

It sometimes happens that a phenomenon cannot be scientifically validated because there are no available means for identifying precisely what is going on.

That happens repeatedly throughout scientific research. It is unrelated to the question of 'vegetative hybridization' and whether the reports of its existence are valid scientific research.

The phenomenon of post-transcriptional gene silencing (PTGS) in plants is much the same as RNA interference (RNAi) in animals and "quelling" in fungi. It is an aspect of heredity that does not involve structural changes to the DNA. I am not postulating. There is an abundance of research from around the world.

You are not postulating the existence of PTGS but you are postulating that it explains the findings of 'vegetative hybridization'. And that is perfectly reasonable. The qualifier is that it is speculation without any evidence to suggest that PTGS is involved. It might be but it might equally have nothing to do with it.

The question is much more basic than attempting to find an explanation for the findings of 'vegetative hybridization'. That is, are the findings valid? Does the research actually show what the researchers claims it shows?

The only way to answer such questions is to examine the research and determine if the observations deny alternate explanations. Were the standard operating procedures for scientific research followed. If the research uses proper controls, randomization, analysis and eliminates other explanations through its design (all completely possible and relatively simple - they are part of the routine of scientific research) then that leaves the transfer of something between stock and scion that effects the genetic information somehow.

However, when controls are lacking, alternative explanations are present, etc (scientific methodology is not followed) then the conclusion that 'vegetative hybridization' has caused any genetic change in the scion in any manner is unwarranted and has not been shown.

Designing research to effectively show that 'vegetative hybridization' actually may have an effect can be done - are there any published reports that have done so?

Hi guys--
I didn't forget this... I've planted the seeds today. And I will try to replicate the experiment again.

I bought today seeds of Green Zebra (green round indeterminate with stripes). And I also bought Roma (paste type, red, no stripes and determinate plant).

I bought Roma specifically because I know how it looks and tastes by heart. Any variation, and I'll know it.

Anyways-- Pics coming soon during summer. (CA has had VERY rainy weather...)

I found this thread very interesting.

Did you get any additional information from your first Chimera of Amish Paste x Roman Candle graft?