Protoplast Fusion

A meeting place for rose breeders.
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Karl K
Posts: 1327
Joined: Sat Jun 02, 2012 4:49 pm

Protoplast Fusion

Post: # 70842Post Karl K
Wed Jan 08, 2020 1:30 pm

As usual, I found this little tidbit while searching for something unrelated.

Mottley, J., Yokoya, K., Matthews, D., Squirrell, J., Wentworth, J.E. (1996). Protoplast fusion and its potential role in the genetic improvement of roses. Acta Horticulturae 424: 393-397.

The fusion of callus-derived protoplasts from various cultivars of roses (Rosa hybrida 'Frensham') was induced using polyethylene glycol. These protoplasts were fused either with protoplasts from cherry (Prunus avium x pseudocerasus 'Colt') or blackberry (Rubus laciniatus 'Thornless Oregon'). 

Rosa persica x xanthina was self-fused and tetraploid plants were obtained. These tetraploids exhibited unusual morphology, contained a chromosome complement of 28 which is double the diploid number for this hybrid, and had significantly increased guard cell lengths and chloroplast numbers.

Putative somatic hybrid plants involving either 'Frensham' and blackberry, or 'Frensham' and cherry, have also been regenerated. Of the two parents, these plants appeared most like 'Frensham' but with several distinct morphological differences. Root tip squash preparations and RAPD analysis revealed genetic similarities to 'Frensham' but further work to fully characterise these putative hybrids is ongoing.

david zlesak
Posts: 411
Joined: Tue Nov 26, 2013 12:27 pm

Re: Protoplast Fusion

Post: # 70843Post david zlesak
Wed Jan 08, 2020 1:47 pm

Hi Karl!

It would be great if there was more momentum with protoplast fusion in roses and follow up papers on characterizing these putative fusion hybrids, if they still exist. From my memory I think I came across something at some point that questioned if these plants were really fusion hybrids, but I forgot where. Maybe it was at a rose research meeting with some of the authors. I suspect with tissue culture being pretty variety specific and challenging in roses already, that stripping off cell walls and having individual protoplasts to keep from bursting and then regenerate plants from is a a lot to ask of the plant material. A friend did has MS with protoplast isolation and regeneration (goal is fusion) with lilacs and he spent many many hours continually in the lab changing enzymes and monitoring the the process/salt levels so the protoplasts didn't well up and burst. Using cells from embyros definitely had more potential too versus cells from more typical plant tissue of established cultivars. I suspect there is a lot of potential for great new rose hybrids just with rose and rose as well as work with other species for those that are willing to develop the techniques and have the patience to work with protoplasts.

henry kuska
Posts: 1033
Joined: Thu Dec 15, 2011 9:06 pm

Re: Protoplast Fusion

Post: # 70848Post henry kuska
Thu Jan 09, 2020 12:37 am

Use the download available at this link: ... mnA&_iepl=

Posts: 1885
Joined: Wed Dec 31, 1969 7:00 pm

Re: Protoplast Fusion

Post: # 70863Post Don
Sun Jan 12, 2020 9:10 am

What doesn't kill them makes them stronger.

Karl K
Posts: 1327
Joined: Sat Jun 02, 2012 4:49 pm

Re: Protoplast Fusion

Post: # 70864Post Karl K
Sun Jan 12, 2020 4:30 pm

I came across this one while searching for something else.

ISHS Acta Horticulturae 572: XX International Eucarpia Symposium, Section Ornamentals, Strategies for New Ornamentals - Part II (2002)
Authors:  A. Schum, K. Hofmann, R. Felten
Keywords:  protoplast regeneration, blackspot resistance, Rosa wichuraiana, Rosa multiflora, Rosa roxburghii
Abstract: With the intention to exploit new sources of disease resistance in rose breeding, the feasibility of introgression of genes by means of somatic hybridization is evaluated. Protocols have been established for (I) regeneration of protopasts in a variety of rose genotypes, (II) PEG mediated fusion, and (III) preferential regeneration of heterologous fusion products. Protoplasts isolated from non embryogenic source material gave rise to callus in R. canina, R. caudata, R. corymbifera 'Laxa', R. multiflora (two accessions), R. roxburghii, R. spinosissima, R. wichuraiana (two accessions), as well as in R. x hybrida 'Elina' and 'Pariser Charme'. Protoplasts isolated from embryogenic cell suspensions of 'Heckenzauber' and 'Pariser Charme', as well as from non embryogenic suspensions of the hybrid R. persica x R. xanthina were regenerated into plants. In order to suppress sustained cell divisions of non-fused protoplasts or of homologous fusion products, protoplasts were pretreated with either rhodamine 6G (0.1 mmol) or iodoacetate (0.5 – 1 mmol) for 15 minutes as well as with X-rays (300 Gy) at a dose rate of 3 Gy/min. Induced defects are mostly complementary, thus corresponding pretreatment of protoplasts prior to fusion allows preferential regeneration of the heterologous fusion products aimed at. Specific genotypes of different wild species within the genus Rosa were identified to carry resistance genes against Diplocarpon rosae, the causal agent of blackspot (von Malek-Podjaski, 1999). For introgression of resistance into cultivars by means of somatic hybridization, experiments concentrate at the time being on diploid accessions of Rosa multiflora, Rosa wichuraiana and Rosa roxburghii. Putative somatic hybrid callus lines were obtained from 'Heckenzauber' + Rosa wichuraiana or Rosa multiflora as well as from 'Pariser Charme' + Rosa wichuraiana, Rosa multiflora or Rosa roxburghii, respectively. Shoots were regenerated from the combination of 'Pariser Charme' and Rosa wichuraiana. The hybrid character of some selected regenerates was exemplarily confirmed by flow cytometry and AFLP-analysis.

Karl K
Posts: 1327
Joined: Sat Jun 02, 2012 4:49 pm

Re: Protoplast Fusion

Post: # 70865Post Karl K
Sun Jan 12, 2020 6:05 pm

This abstract is not entirely clear, but it seems to suggest that hybrids and cybrids might be obtained between species where the chromosomes of one species are usually expelled.

Anim Biotechnol. 2014 Apr 3;25(2):139-49.
Incompatibility of nucleus and mitochondria causes xenomitochondrial cybrid unviable across human, mouse, and pig cells.
Yu G1, Tian J, Yin J, Li Q, Zhao X.
The nucleus and mitochondria are on correlative dependence; they interact in the process of protein transportation and energy metabolism. The compatibility of nucleus and mitochondria is essential for interspecies somatic cell nuclear transfer (iSCNT) and xenomitochondrial cybrid. In order to test the compatibility of nucleus and mitochondria among human, mouse, and pig cells, we compared the performances of cybrids that fused inter- and intra-species. The ρ0 cells from human and pig cell lines were created as nucleus donors which were transfected with GFP-neo for cell selective system in advance, and mitochondria donor cells were labeled by Mitochondria-RFP. Human and mouse platelets were also used as a mitochondrial donor. Results indicated that all interspecies cybrids declined to die in 2-4 d after the cell fusion in the selection medium, while intraspecies cybrid cells survived and formed stable clones. As a conclusion, the incompatibility between nucleus and mitochondria is the critical factor for the formation of interspecies cybrids.

Karl K
Posts: 1327
Joined: Sat Jun 02, 2012 4:49 pm

Re: Protoplast Fusion

Post: # 70959Post Karl K
Tue Jan 21, 2020 3:24 pm

The previous note is obscure, but I think it suggests one of the mechanisms that isolates some species and genera. Here is some further info.

Science Frontiers, no. 25: Jan-Feb 1983
Up until now, the three genetic systems were thought to be discrete, each going down its own pathway. But chloroplasts genes have now been found inside plant mitochondria, overturning conventional wisdom. To sum it all up, DNA seems promiscuous — no respecter of privacy and breaking down all isolating genetic barriers.
(Ellis, John; "Promiscuous DNA — Chloroplast Genes inside Plant Mitochondria," Nature, 299: 678, 1982.)

Science Frontiers, no. 34: Jul-Aug 1984
It was a surprise when DNA sequences from mitochondria in yeast cells were discovered setting up shop in the nuclear genomes (i.e., the normal genetic endowment of the cell nucleus). Now biologists find that DNA sequences in many species regularly and frequently hop from one genome to another. Genetic material from cell chloroplasts mix with that of the mitochondria and that of the normal nucleus in what seems to be a free-for-all. This genome hopping has earned DNA the adjective "promiscuous."
(Lewin, Roger; "No Genome Barriers to Promiscuous DNA," Science, 224:970, 1984.)

This might explain some odd cases where reciprocal crosses differ greatly in vigor. Or where chromosomes of one species in a cross are eliminated, whether that species is seed parent or pollen parent.

Karl K
Posts: 1327
Joined: Sat Jun 02, 2012 4:49 pm

Re: Protoplast Fusion

Post: # 71023Post Karl K
Thu Jan 30, 2020 1:39 pm

Thinking about this matter, I got to thinking about some research on cytoplasmic inheritance in general that had fascinated me some time back (2001!), but then I dropped the ball. I have some notes on my web page, but didn't get around to posting some of the most meticulous and important of the research.

Michaelis: Interaction between Nucleus and Cytoplasm in Epilobium (1959) ... m1959.html

Then there is another item that I had forgotten entirely.
"... Thus in Vicia Faba a number of plasmatic differences are to be inferred from reciprocal crosses between the subspecies major and minor. Amongst other effects the minor cytoplasm seems to eliminate the F2 segregates which are pure for certain genes from the stock. There are six genes concerned and they are linked in one complex." ... obium.html

The above statement refers to work by Sirks (1931), but I haven't found the original paper, yet. I mention it because it is reminiscent to Hurst's observation on self-seedings of Rosa x micrugosa.
Platyrhodon microphylla, from China and Japan, the so-called Chestnut Rose with cup-shaped fruits covered with fleshy spines, seems more promising in its fertility since I have succeeded in raising the second generation of a cross with Rosa rugosa. The results, however, although extremely interesting from the scientific point of view, are not very promising horticulturally, since the grandparent R. rugosa has been reproduced in facsimile several times, while the others resemble the parent hybrid with strange mutational variations.

Can this cross be made in both directions? Would the F2 progeny from the reciprocal crosses be different?

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