David Z... what does this mean?

Hi David,

Thankyou for posting the link to your pollen diametre paper in the maximowicziana thread. It was very timely as my caffiene treated laevigata will hopefully bloom in about a month and I’ve decided to test for doubling this way instead of by root-tip squash incase I got incomplete doubling The plant is doing really well but for some strange reason I’m having trouble striking it. Could that be a side-effect of the treatment?

Anyway, the real reason for this post is to ask you about something in the paper. Table 3 on pg 7 includes this data:

Triploid - RADrazz (Knockout) - predicted ploidy of gametophytic material x4 - predicted ploidy of sporophytic material x8 - mean pollen diametre 46.8±5.2µm.

There were a few other x4, such as ‘Leonie Lamesch’ (and x3… e.g. ‘Incognito’, ‘Robusta’ and ‘JP Connell’), pollens reported from triploids as well. I was wondering what this means as I’m having trouble getting my head around it? Is this why ‘Knockout’ has relatively low fertility or is this just what you meant when you said that some cultivars seemed to have unusually large pollen that made prediction difficult or inaccurate?

It means apply 5 times as much pollen of that variety onto a seed parent that sets and germinates well, lol.

Hi Simon!!

That just means Knock Out has very large pollen. If a person just had the pollen diameter to estimate the ploidy of Knock Out, the large size (46.8uM) would be comparable/within the expected range of pollen from a normal 8x rose. So, the sporophytic ploidy (that of the normal plant) would be predicted to be 8x and the gametophytic ploidy (that of an egg or pollen nucleus) would be 4x. Typically the sporophytic ploidy prediction is just twice that of the gametophytic (except for Caninae section roses). So, what this shows is that with our complicated modern roses, meiosis is goofy and there is limited predictive value for pollen diameter for especially triploids. Triploids as a group though have greater standard deviations, or variability in pollen diameter than diploids or tetraploids. Also if one looks at the relative percent aborted pollen triploids have more aborted pollen. In studies of Leen Leus and others, additionally, even though pollen may look plump under the microscope, there is typically a lower rate of pollen from triploids that can germinate from germination assays in sugar water solutions.

Knock Out does seem to have a lot less pollen in general than other roses. Additionally, the rate of aborted pollen is high. Much of that larger stainable pollen probably is unreduced and has three sets of chromosomes in it. Of course there is variable sizes and ones that would have 1x or 2x in them. It seems like those are the ones likely contributing to viable offspring. For instance, Home Run is triploid and Knock Out is the male parent. The female parent is a cross of City of San Francisco and Baby Love. BL is tetraploid and likely CSF too. So, likely KO may have just contributed 1x to the triploid Home Run. Sometimes plants can do a little “genomic editing” after fertilization and kick out some extra chromosomes.

When Knock Out is used as a female (open pollinated or in controlled crosses with tetraploids), it seems like most of the offspring are a mix of triploid and tetraploids.

Hopefully this helps clarify things. The predicted values are just based on pollen diameter compared to a scale derived from data in the earlier table and in this case does not predict ploidy well.

Thankyou, David. I figured this is what you meant :slight_smile: So, would you recommend using pollen diametre as an indicator of ploidy in my caffienated laevigata? I have the control plant and the treated plant. If I can get pollen from both and compare them might that give some indication of doubling?

HI Simon!

Yes!! I think comparing pollen diameter from your original and treated plants would be a very very good comparison. Your original plant would make a great control to compare with. You can also compare stomate size for evidence of doubling in meristem layer one (pollen checks layer two). You can take a slide, put a drop of superglue on it and press a leaf underside into it. Let it set up to the point where you can peel the leaf back off, but so the imprint stays nicely there. You can then look at the imprints under the microscope and measure the guard cells around the stomates and overall cell sizes.

I hope you successfully doubled your plant!!

Thanks, David. I’ve been doing the stomata activity with kids for near 20 years (next July)… only I use nail polish. I figured the thin film might distort when removing it from the back of the leaf and mess up the measurements. Pressing it into superglue on a slide would fix this! I remember seeing a version of this using acetate sheets and some kind of solvent as well but never tried it because the nail polish works so well with kids.

[quote=SimonV]

It was very timely as my caffiene treated laevigata will hopefully bloom in about a month and I’ve decided to test for doubling this way instead of by root-tip squash incase I got incomplete doubling[/quote]

Simon,

I’m surprised that you are trying caffeine to double the chromosome number. The reports I’ve read suggest that caffeine is useful for reducing chromosome number by forcing an extra cell division … except Peña, et al., who observed cell fusion.

Karl

Peña, et al. Bimeiosis induced by caffeine. Chromosoma 83(2): 241-248. (1981)

A 0.1% caffeine solution has been injected into plants of rye at various stages of spike development. Cytokinesis was inhibited in the germ line, and the resulting binucleate cells underwent bimeiosis. Nuclear fusions occurred during cell divisions of the germ line, giving rise to mononucleate tetraploid PMCs which showed 14 bivalents instead of the expected up to 7 quadrivalents. A decrease in chiasma frequency was also noted.

Schlegel R, Pardee AB. Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science. 1986 Jun 6;232(4755):1264-6.

Caffeine was shown to induce mitotic events in mammalian cells before DNA replication (S phase) was completed. Synchronized BHK cells that were arrested in early S phase underwent premature chromosome condensation, nuclear envelope breakdown, morphological “rounding up,” and mitosis-specific phosphoprotein synthesis when they were exposed to caffeine. These mitotic responses occurred only after the cells had entered S phase and only while DNA synthesis was inhibited by more than 70 percent. Inhibitors of protein synthesis blocked these caffeine-induced events, while inhibitors of RNA synthesis had little effect. These results suggest that caffeine induces the translation or stabilizes the protein product (or products) of mitosis-related RNA that accumulates in S-phase cells when DNA replication is suppressed. The ability to chemically manipulate the onset of mitosis should be useful for studying the regulation of this event in mammalian cells.

Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 452 (1) (2000) pp. 67-72

Inducing somatic meiosis-like reduction at high frequency by caffeine in root-tip cells of Vicia faba

Yihua Chen, Lihua Zhang, Yihua Zhou, Yuxuan Geng and Zhenghua Chen

Abstract: Germinated seeds of Vicia faba were treated in caffeine solutions of different concentration for different durations to establish the inducing system of somatic meiosis-like reduction. The highest frequency of somatic meiosis-like reduction could reach up to 54.0% by treating the root tips in 70 mmol/l caffeine solution for 2 h and restoring for 24 h. Two types of somatic meiosis-like reduction were observed. One was reductional grouping, in which the chromosomes in a cell usually separated into two groups, and the role of spindle fibers did not show. The other type was somatic meiosis, which was analogous to meiosis presenting in gametogenesis, and chromosome paring and chiasmata were visualized.

This is why:

Chromosome doubling of haploids of common wheat with caffeine

Julian Thomas, Qin Chen, Neil Howes


Genome, 1997, 40(4): 552-558, 10.1139/g97-072

Abstract

Treatment of dividing plant cells with caffeine inhibits their cytokinesis, thereby inducing the formation of binucleate cells that contain polyploid nuclei. This study was undertaken to determine whether caffeine treatments would induce chromosome doubling and seed set in haploids of common wheat (Triticum aestivum L. em. Thell.) through the further development of diploid nuclei. Ten sterile wheat haploid plants, obtained through the agency of corn (Zea mays L.) pollination, were multiplied by vegetative propagation (subdivision of well-tillered plants) to produce about 50 crowns per haploid. Washed and trimmed crowns were treated with 0.3, 1.0, 3.0, and 10.0?g?L?1 caffeine for 3, 6, 12, and 24?h. While treatment with 0.3?g?L?1 caffeine did not restore fertility, higher concentrations of caffeine resulted in pollen shedding and substantial seed set compared with untreated controls. Many combinations of caffeine concentration and duration produced comparable results; however, treatment with 3?g?L?1 for 24?h was the most effective caffeine treatment on the basis of the number of seeds recovered, as well as the size and incidence of fertile sectors. Compared with a standard colchicine treatment (3-h immersion in 2?g?L?1 colchicine dissolved in 0.5% dimethyl sulfoxide), all caffeine treatments produced fewer seeds, principally because colchicine-doubled sectors were often large, while caffeine-doubled sectors were numerous but generally small. In conclusion, caffeine produced useful numbers of seeds for all clones; gliadin banding patterns and chromosome counts indicated that genetic and cytogenetic stability of the doubling process were equal or superior for caffeine compared with colchicine.Key words: chromosome doubling, haploid, caffeine, colchicine,

I did have the full article but cannot locate it now.