blue rose - hacking

“… Cocioba works as a scientific consultant in the Bio Art Lab at the School of Visual Arts in Manhattan. He and the lab’s director, Suzanne Anker, are now trying to create the “holy grail” of plant hacking: a blue rose.”

[quote. It’s also hard to imagine people getting super-stoked about one][/quote](A blue rose, that is). I guess he hasn’t been following this forum much!

Back at the dawn of the biotech age I was discussing the ‘blue rose’ concept with some post-docs at Cornell and we concluded, at the time ('83ish), that the easiest approach to getting a blue rose would be shotgun cloning. That is exactly what Mr. Cocioba is doing now. It’s impossible to tell what his source genetics are but petunias, violets and cornflowers are all prospective dna donors.

The technique is, however, dependent on successfully regenerating plants from the cloned tissue and that was a bigger stumbling block than the actual shotgun technique. Fast forward to now and there are a couple of successful regeneration protocols on the books, one by Katherine Kamo at USDA comes to mind.

I guess he hasn’t been following this forum much!

Another fellow at the conference representing a Japanese outfit was very interested in creating not blue roses but, rather, blue chrysanthemums. Apparently the Asian market for chrysanthemums dwarf’s that for roses. I suppose we could check on that because blue mums were accomplished by 2013. To my knowledge Suntori, who owns Florigene, has still not started to market their psorta-blue roses.

Very interesting. One of these days, it will work. At least I can hope!

If only it were so simple. Cornflowers are colored by cyanin, a pigment already common in roses. I have read different explanations for how the pigment is made to appear blue, so I can’t comment on the possibility of getting the blue into roses.

Some “blue” violets are pigmented by violanin, a glucoside of delphinidin. However, the presence of delphinidin derivatives does not ensure that a true blue color will result.

The following link is to a paper on petunias. The normal color is due to hyperacidification of the vacuoles. Mutations of 5 genes each reduced the acidification to varying degrees, resulting in “blue” flowers. So, even if the petunidin could be brought into roses, the result would not be very blue unless the rose vacuoles were also alkalinized to some degree.

While we wait for the lab gang to splice something, there are still three routes to “blue” that have not yet been thoroughly exploited.

  1. Rosacyanins: Two have been identified in ‘Blue Moon’ and ‘Mme Violet’. These are half-siblings, sharing the parent ‘Sterling Silver’, which possibly carries the same pigments.
  2. Anthocyanic vacuolar inclusions: Cyanin bound to proteins is carried into the vacuole. The longer these last, the bluer the flowers appear. As the AVIs break down, the color shifts towards the red.
  3. Co-pigmentation: Cyanin complexes with a co-pigment. The color tends to become more “blue” with age.

It should be possible to combine 2 and 3, so that the AVI blue would shift to a co-pigment blue.

As for the rosacyanins, it might be possible to “purify” the color, enhancing the influence of the rosacyanins while reducing the anthocyanins.

If only it were so simple.

That’s the point here. Elegant engineering methods have already encountered all of the barriers you list. Shotgun cloning is the opposite of elegant, basically a brute force injection of dna fragments into naked somatic protoplasts followed by plant regenration and crossed fingers.

I found an earlier reference to “blue” coloring that is not of the usual anthocyanin type:
Arisumi: Rosa Pigments (1963, 1964)
Another line of approach to the so-called blue roses have been substantiated by “Grey Pearl” which was introduced in 1944. The pigment participated showed complete peculiarities in their chemical behaviours, as they could not been extracted either by the hydrochloric acid or the petroleum ether. Repeated treatments with these solvents could dissolve out the ordinary anthocyanin and carotenoid from the petals of “Grey Pearl”, so that the remnant has shown the beautiful bluish tinge. But it was rather lavender and far from the true blueness. From the offsprings of “Grey Pearl” we have reached to “Sterling Silver”, which is considered to be the most successful performance of this colour range, but there is no discrimination between the intact petals of “Sterling Silver” and those of “Grey Pearl”, from which co-existing anthocyanin and carotenoid were fully removed. Thus we may conclude that the endeavours to produce true blue rose have been directed to eliminate the contaminative pigments from “Grey Pearl”, which disturb the effect of the above undefined lavender pigment.

I got to thinking about this again and it reminded me of something possibly similar.

I have read some reports of “impossible” hybrids that are overwhelmingly maternal, but seem to have taken at least a little something from the putative pollen parent(s). Some of these are probable or possible “partial hybrids”, which result from normal pollination followed by the loss of all or most of the paternal chromosomes. Sometimes only fragments from the pollen chromosomes are incorporated in the maternal chromosomes.

In other cases, as some of us have speculated, the foreign pollen tubes die. Then, bits of DNA are absorbed or otherwise carried into the ovary. This sort of thing is known to happen when bacteria digest foreign DNA, and insert segments into their own chromosomes.

And this item indicates that foreign DNA is taken up by pollen tubes.

“Exogenously supplied DNA increased length growth of pollen tubes, which were studied because they have similar elongated and polarized growth as root hairs.”

These are not proofs that foreign DNA can be partially digested and then incorporated into a new embryo. Still, I have read of some odd cases that really need a satisfactory explanation.

Back in the dark ages, around 1990 or so, Chinese scientists use “the pollen tube pathway” to transform plants with naked DNA. I believe they were successful with cotton and some other crops. A post-doctoral fellow in our department worked in my lab for a time trying to do this for alfalfa, which is a rather different thing than cotton. He did not have success. But he went to University of Wisconsin for another position and then got involved in a biotech company that had reasonable success at other things. That is to say, he was very competent, but the method did not work in alfalfa for whatever reason.

The field of transformation underwent rapid changes in those days and infection with Agrobacterium carrying genes of interest came to dominate most everything. That was especially so for brassicas, like Arabidopsis, and various cabbages where a simple floral dip method was evolved. Application of a mild vacuum with flowers immersed in a culture containing the bacteria, then quick release of the vacuum allows infiltration of the liquid into the flower parts. Somewhere along the way, before or after pollination, the seed sometimes gets invaded by the Ti plasmid of the bacterium. DNA pieces carried by the plasmid get incorporated into the DNA of the plant (embryo of seed). If the seeds are planted on a medium containing an antibiotic to which the plasmid carries resistance, you can kill all the seedlings that don’t have the plasmid in their genome, while the ones with it survive. Then you can grow the plants and get another generation of seed. From these you can select for the trait you want, maybe (if you have a way to identify what you are looking for, such as a certain enzyme to which you have made an antibody). Overall, this is really easy compared to a lot of other ways of trying to do things. Even first year graduate students have success rates that are reasonable.

Of course these species of plants have a generation time of 6 weeks to 6 months which is considerably faster than most roses. So progress in genetic engineering of these is a lot quicker, which is why arabidopsis is a model system. That you can grow a whole plant in a large test tube is another important feature. That they like growing under ordinary fluorescent shoplights is another.

Probably someone could use the floral dip method on roses, but I don’t know of any publications on this. I would use a plant like R multiflora nana which blooms quickly, germinates quickly.

Probably someone could use the floral dip method on roses

This is equivalent to shotgunning but even easier. Grind up blue petunias and strip out the dna and there’s your stew. Generate, grow up and screen tens of thousands of seedlings, job done. I’m thinking one, maybe two semesters…

What I was thinking of is a bit more refined than grind and grow. First you get Agrobacterium, cut and clone in a gene of interest, say the cluster that synthesizes petunidin in petunias. Then floral dip the roses, get seed, grow seed, select a marker like hygromycin resistance. Generally under 100 clones is enough to get one that has good expression levels. The real problems come when you over-express the pathway proteins. MicroRNA regulation was discovered exactly that way. Only it was introducing extra genes for colors into petunias, from petunias that was the challenge. Instead of superbright petunias they got white, or striped ones. The Nobel prize actually should have gone to a plant biochemist, David Baulcomb, not to a couple of medical types, He understood what caused the suppression of colors, before they did it in animals.

If we had a resistance gene for RRD, or BS, we could clone it in via Agro, if floral dip works. That is the thing we’ve not yet tested, so far as I am aware. Only the regulatory hurdles keep us from doing it. Maybe we could crowd-source funding from Trump supporters in exchange for shares in the product if they jump the hurdles for us. Let’s make a deal. Are you familiar with irony?

I’ve been reviewing the basis for blue color in a variety of plants, and have found a wide variety of distinct approaches. Delphinidin in roses may be a fun thing to consider, but getting from there to an actual blue color is not a simple thing.

In the meantime, I’ve also been looking at the rare pigment that have already been identified in roses. For example, Mikanagi, et al. (2000) found cyanidin 3-rutinoside and peonidin 3-rutinoside in ‘Arthur Hillier’ (Rosa macrophylla x R. moyesii) among others. I googled a while and was pleasantly surprised to find that these two pigments are responsible for the lilac to purple shades of Anthurium amnicola.

The authors note that the cyanidin 3-rutinoside is magenta, while the peonidin 3-rutinoside is pink. They suggest (without direct evidence) that, “the lavender to purple color is probably influenced by copigmentation and pH of plant tissues.”

Roses have a diverse assortment of potential co-pigments, so it should not be difficult to find one or more that nudge these rutinosides towards new shades of lilac through purple roses.

Furthermore, some of the existing “blue” roses (e.g., ‘Rhapsody in Blue’ and ‘L’Evêque’) gain their bluish tint from AVIs (vacuolar anthocyanic inclusions).

I can’t predict any particular result, but it would be interesting to see whether a cyanidin 3-rutinoside would get a similar blue-ward color shift when wrapped up in AVIs.

Arisumi (1963) suggested another possible route to “blue” roses:
“In stock, Matthiola incana, the shift towards the bluer shade appeared to originate from the acylation of anthocyanin molecule. If this situation is true, the ρ-coumaroyl cyanin might offer the third clue to so-called blue roses.”

This pigment is found in Rosa willmottiae, R. sweginzowii, R. moyesii, and others.

I scanned Table 4 from Mikanagi, et al. (2000) showing numerous species and some garden roses along with their pigments.

Another step towards blue.

Novel compound contained in blue rose
US 20120011771 A1

[0005] Since rosacyanins have a cyanidin backbone in a portion of their structure, there the possibility that they are synthesized based on cyanidin, a common precursor with cyanidin or an analog of cyanidin. However, since this remains to be only speculation, what types of substances are actually used as precursors and what types of pathways are used in synthesis have yet to be clearly determined.
[0006] On the other hand, delphinidin is synthesized instead of a portion of the cyanidin in roses in which flavonoid 3′,5′-hydroxylase gene is expressed as a result of genetic recombination as previously described. If the aforementioned hypothesis regarding the rosacyanin synthesis pathway, namely that rosacyanin is synthesized by using cyanidin as a precursor, is correct, then rosacyanin would not be synthesized in these genetically modified roses in which cyanidin serving as precursor is essentially absent.
[0007] When the inventors of the present invention conducted an analysis to obtain findings regarding rosacyanin synthesis using the aforementioned genetically modified roses that hardly contain any cyanidin or have a considerably decreased cyanidin content in comparison with a host as described in Patent Document 1 or Patent Document 2, contrary to expectations, a novel compound was found to be present having a chemical structure that clearly differed from that of rosacyanins inherently possessed by roses. Moreover, this novel compound was clearly determined to be uniquely present in roses in which flavonoid 3′,5′-hydroxylase gene was expressed by genetic recombination, thereby leading to completion of the present invention.

It is worth noting that Arisumi (1963) found that after removing the anthocyanins and carotenes from ‘Grey Pearl’, a color remained that looked like that of ‘Sterling Silver’. It may be that the “blue rose” mentioned in the patent might give rise to even “bluer” varieties by breeding out the excess anthocyanins, including delphinidin glucosides.

It is well known that anthocyanin pigments are produced by a series of enzyme-mediated transformations. Loss or defect of one such enzyme can result in the loss of one or more pigments. For instance, Bateson (1909) crossed two white-flowered sweet peas (from different strains) and raised a batch of seedlings with two-toned purple flowers similar to the wild Lathyrus odoratus.

Le Grice (1968) made a case for the involvement of Rosa foetida and the Pernetianas in the development of “blue” roses.

The argument seemed to make sense so long as one assumed that the lilac-tones were due to co-pigments from the yellow roses that altered the color of cyanin. But with the discovery of rosacyanins, the argument seemed to fail because no such pigment is present in R. foetida. Clearly something else is at work if this species made any real contribution.

R. foetida bicolor is colored by peonin over the yellow ground color. In the normal ‘Lutea’ form, the red pigment is absent. Why?

Suppose that the enzyme being suppressed in the petals of the ‘Lutea’ form (but active in ‘Bicolor’) is the one that attaches glucose molecules to the peonidin backbone. If this is the case, then varieties like ‘Sterling Silver’ combine this suppression of glucose transferase (from Foetida) with rosacyanin synthesis from a different source. The rosacyanin colors are more pronounced for two reasons: (1) they are not masked by cyanidin glucosides, and (2) there is more of the cyanidin backbone available to synthesize rosacyanins rather than cyanidin glucodes.

Rosa hracziana Tamamsch. was described by S. G. Tamamshyan in 1994 from the right bank of the River Razdan in Aparan floristic region of Armenia. The species is distinguished by carnose, dark red, stoutish and quite long fruit-stalks.

Chemistry of Natural Compounds, Vol. 47, No. 1, 2011 [Russian original No. 1, January-February, 2011]
A. R. Novruzov and L. A. Shamsizade

Thus, cyanidin-3-glucoside, cyanidin-3,5-diglucoside, delphinidin-3-glucoside, and delphinidin-3,5-diglucoside were identified for the first time in total anthocyanins from fruit pulp of R. hracziana using chromatography, spectroscopy, total and stepwise hydrolysis, oxidation by H2O2, and comparison with authentic samples. All components with the exception of delphinidin-3,5-diglucoside were found in R. spinosissima anthocyanidins.

The monoglucosides of cyanidin and delphinidin dominated quantitatively the anthocyanin complex from fruit of both Rosa species.

This is not exactly hacking, but it could be a clue for breeders.

The Anthocyanin Pigments of Plants p. 282 (2014)
By Muriel Wheldale Onslow

Gawalowsky, A., ‘Künstliche Blatt- und Blütenfärbungen,’ Wiener landw. Zeit., 1911, LXI, p. 616.
Author finds that treatment of soil with sodium orthophosphate gives a deep red or blue-violet colour to petals of Rosa centifolia; also to Malva tinctoria a deep brown-red. Potassium carbonate causes development of anthocyanin in Lactuca sativa.

This may explain what Schmidt wrote about ‘Veilchenblau’:

Everybody’s Magazine 24: 746-757 (1911)
The Quest of the Perfect Rose
Franklin Clarkin

“Veilchenblau,” wrote Herr Schmidt, "is a direct seedling of the ‘Crimson Rambler,’ not cultivated by fructification with another kind. By culture of several years, the new kind has rested constant. There have been no dosings with chemicals.

Needless to say, such chemical treatments do not affect inheritance. But they can give some indication of hidden potentials. If a group of Centifolias (or other pink roses) are given the sodium orthophosphate soil treatment, those that produce blue-violet flowers are more likely transmit this potential to their offspring than others that turn deep red.