Tina, I was pleasantly surprised Desperado Love was a triploid. It is one of my favourite daylilies. It is a joy to work with.

The ATP database lists Desperado Love as both tetraploid and triploid. How was its triploid status determined or by whom? The AHS database lists its ploidy as tetraploid.

Who determined that Lillian's Little Joe and the others are Triploid?

I was wondering the same thing

For these cultivars identified as being triploids:
Chicago Apache, Spacecoast Gator Eye, Spacecoast Starburst, Crystal Pinot, Daring Deception, Strawberry Fields Forever, Moonlit Masquerade

The research reference is:
Ploidy Variation in Hemerocallis spp. and the Implications on Daylily Breeding
C. Zhang, D.M. Cao, X.C. Zhang, L.F. Kang, J.J. Duan, X.L. Ma, G.J. Yan and Y.S. Wang
in Proceedings of the International Conference on Germplasm of Ornamentals, Eds.: Qixiang Zhang and Guijun Yan
Acta Hort. 977, ISHS 2013 pp 197-204

The researchers counted the chromosomes in the cells.
= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
For Garnet Robe identified as being a triploid the reference is:
T. Arisumi, the Daylily Journal Vol 25 March 1971 pages 32-35 "Triploidy as a cause of low fertility in Garnet Robe.
Arisumi counted the chromosomes in the cells.
= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
Arisumi research "Experiments in breeding for triploid daylilies" the Daylily Journal Vol 24 1970 pg 33-37
and "Embryo development and seed set in crosses for triploid daylilies" Botanical Gazette 1973 Vol 134 pg 135-139
= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
For the research that produced VT Spirit:
"Development of triploid daylily (Hemerocallis) germplasm by embryo rescue"
Euphytica October 2009, Volume 169, Issue 3, pp 313-318
Zhiwu Li, Linda Pinkham, N. Faith Campbell, Ana Carolina Espinosa, Rumen Conev
= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
For Celtic Sunrise and Forbidden City identified as being triploids the research reference is:
Ploidy estimation in Hemerocallis species and cultivars by flow cytometry
Hiroyuki Saito, Keiko Mizunashi, Shigefumi Tanaka, Yukiko Adachi, Masaru Nakano
Scientia Horticulturae 97 (2003) 185–192
= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
I cannot help identify the basis for the triploid ploidy in the ATP database.

Some general triploid Hemerocallis research:

Chromosome Numbers in Hemerocallis, with Reference to Triploidy and Secondary Polyploidy
A. B. STOUT
CYTOLOGIA 3:250-259, 1932

Cytological Studies on the Hybrids between Triploid and Diploid Hemerocallis
Michio Matsuoka
Japanese Journal of Breeding 22:168-171, 1972


MEIOSIS IN DIPLOID AND TRIPLOID HEMEROCALLIS
S. O. S. DARK
New Phytologist Volume 31, pages 310–320, 1932

The list I posted, including Lillian's Little Joe, was generated by simply going to the Daylily Database Search page (http://garden.org/plants/group...) and toggling the ploidy box for Triploid (....in other words, I have no idea how it got designated that way, it just came up in a list with the other Trips in the database when I searched). Fred, since you can verify that it is an error you can contact Calif_Sue to alter it, and I have noted it above.

Fantastic Triploid References, Maurice! It is so very nice to see research links that bring new knowledge to the world. I have already put two of the Trips on order for spring (my $5 splurges, yay!), and am very excited to include them in my garden.

Tina Said
Fred, since you can verify that it is an error you can contact Calif_Sue to alter it, and I have noted it above.

I have no way of knowing if it is or it's not, it's not listed in the AHS data base as such.

When a hybridizer crosses a diploid with a diploid they can only assume that all the seedlings are diploids. When they cross a tetraploid with a tetraploid they can only assume that all the seedlings are tetraploids.

Unfortunately, in diploids very rarely a triploid seedling will be produced usually because of 'accidents' during the development of the male and female gametes (pollen and ovules).

In tetraploids there often are more 'accidents' than in diploids during gamete production. A standard tetraploid daylily would have 4 chromosomes for each of 11 sets or 44 chromosomes in total (4 of chromosome A, 4 of chromosome B, and so on to 4 of chromosome J, and 4 of chromosome K). The gametes, for example the pollen would have 2 chromosomes of each of the 11 sets or 22 in total (2 of chromosome A, and so on to 2 of chromosome K).

Unfortunately, due to errors a tetraploid's gametes might not have the correct number of chromosomes (that is, 22) but might have 23, or 24 or 25 or 26 and so on or might have 16, 17, 18, 19, 20, or 21 and so on. A gamete that has 23 chromosomes might have an extra A chromosome or an extra chromosome of any one of the other 10 sets of chromosomes. When two tetraploids are crossed a pollen grain with 17 chromosomes might join an ovule with 16 chromosomes to produce a seedling with 33 chromosomes (a triploid). But those chromosomes might or might not be three of each of the 11 sets. They might be three of some sets, two of other sets and four of some sets.

There is also another way that triploids can be produced by both diploids and tetraploids. Although natural pollinations do not occur extremely frequently in daylilies they do sometimes happen. Natural pollinations happen often in some cultivars (for example Stella de Oro) and less frequently in others. Hand pollinations made by the hybridizer are normally diploid with diploids and tetraploids with tetraploids but natural pollinations may also be diploid x tetraploid or tetraploid x diploid. Rarely those cross-ploidy natural pollinations will be successful and produce a triploid seedling. And even more rarely a triploid seedling might be registered as a diploid or as a tetraploid.

Sometimes a cultivar is purchased that is mislabelled and that is not only a different name but involves a different ploidy. If the misidentified cultivar is used in hybridizing then all the crosses will be with a different ploidy even though the hybridizer believes them to be with the same ploidy. Any seedlings that are produced are likely to be triploids (excepting those from natural pollinations accidentally of the correct ploidy).

The only way to be certain that a seedling is a triploid (or is any other ploidy) is by measuring the amount of its DNA or by counting its chromosomes.

There are a surprising number of different triploid daylilies that have been collected in natural populations in Asia where there are no tetraploid species to help increase the proportion. Although infrequent they do not seem to be extremely rare so they can be expected to occur in hybridizing gardens.

Surely it is possible to find out why some cultivars are recorded as both tetraploid and triploid in the ATP database?

A bit more published research on triploid and cross-ploidy pollinations in daylilies from:
Yike Gao, and Shuying Gao He Qi, 2012. A STUDY ON THE COMPATIBILITY BETWEEN DIFFERENT PLOIDY CULTIVARS OF HEMEROCALLIS AND THE GENETIC RELATIONSHIPS AMONG THEIR HYBRIDS. Acta Hort. (ISHS) 937:547-554

Here are their important methods:

"Flower buds expected to open next day were emasculated and stigmas were
bagged to prevent self- and cross pollination. Diploids were crossed with diploids,
triploids and tetraploids respectively, by means of hand-pollination. One month later, the
number of fruits within every combination was counted and the seed-setting rate
calculated. Upon turning yellow and cracking from top, fruits were collected and number
of seeds counted, where-after the seed-setting rate was calculated as a percentage."

- by "emasculated" they mean the stamens were removed from the flower.

Below are some of their interesting findings:

There were 21 tetraploid x diploid crosses and 3 diploid x tetraploid crosses. A total of 360 flowers were pollinated and 39 pods produced with an average of 2.3 seeds.

There were 4 triploid x diploid crosses (total of 264 flowers pollinated, 5 pods in total, 20 seeds total - very unusually 13 seeds were in one pod and might best be discounted giving 4 pods and 7 seeds) and 2 diploid x triploid crosses (total of 296 flowers pollinated, 6 pods in total, 11 seeds total).

Important reminder: Arisumi found that even when seeds appeared normal the majority did not produce viable seedlings. The seeds produced by this research were not tested for germination so one cannot expect that 360 tetraploid - diploid pollinations would produce 90 seedlings or that 560 diploid-triploid pollinations would produce 18 seedlings. I would estimate that the tetraploid - diploid crosses might have produced 18 seedlings and that the diploid-triploid crosses might have produced 4 seedlings.

I'm sure Sue can see who submitted the info for example for LILLIAN'S LITTLE JOE and maybe find out why that person said it is a triploid.

That is one reason I mainly use the AHS database for info on daylilies and/or the hybridizers website (if info is still available). All the info is submitted by the hybridizer so it should be accurate. Yes there are some errors in the AHS database but for the most part it is correct.

Don't get me wrong, I love the database here especially since we can add our own photos. However, since anyone can add info it can make things confusing. Sue tries to make sure the info is correct but I'm sure she cannot verify everything and assumes people submitting the info knows it is correct. I know Dave is trying to redo/update the database so multiple entries can be submitted by multiple people so it may be less confusing as maybe we will now who/where/why it was submitted kind of thing and different peoples observations will be noted and not just ONE persons. The comment sections are good for people to add this type of info if that is where they add it.

But this is a good example as to what can happen. This is not the first issue to arise from database issues and that is why Dave is trying to add to the database (I assume that is still in the works).

The research is really clear as a pointer for what we see happening in our gardens every day ... for me, with just thirty cultivars blooming last year, already one is confirmed a tet when it is listed in all databases as a dip-only. There is so much variety and experimentation going on to produce new forms that it seems quite natural for seasons, colors, heights, right on down the line, to seem very much in the eye of the beholder, and only scientific observations and instruments can really measure or help confirm the differences, when more certainty is desired. The ATP database is unique in striking a balance between what is advertised, what has been researched, and what is also seen in the garden, with the option for everyone to post detailed comments on each cultivar, including whatever experience they themselves have had with a plant.

Would a microscope, at the very least, be a useful tool to determine ploidy issues? I'm hoping someone knows what magnification is needed for both pollen examination and for looking at chromosomes. I have read that chromosomes are visible by optical microscope at the chromatin/division stage (when they become the X-shape form) but am not sure when or what part/s of a plant might have such activity happening, if at all? Is it only in seeds, or would other parts of the plant continue the division? Would I see 2-, 3-, and 4-legged chromosomes in that X-shape, or just find 22, 33, or 44 X-shapes floating around in any random cell?

I'm so excited ... my top choice for bridge triploid is all set for shipping this spring, and at the very least I feel like I'll be able to check it for trip-pollen producing status by its ripe pollen, once it blooms. I'd really love a way to count chromosome strands of any offspring! And, I'm looking forward to posting pictures of my tet-crossed "dip" seedlings as soon as they bloom, and will be curious to check on the nature of their pollen, too.

Tina good microscopes are expensive. All mine have been more than US$10k. Don't faint - a good-named (reputation) microscope is the one piece of equipment that can appreciate in value.

Yikes! But ... surely that's because you are a professional scientist ....

... i'm reading that dividing cells, fixed and stained, can possibly be read through a very simple light microscope at 66+ magnification, or at least 100+...

...ebay is telling me that used vintage kid's light microscopes (up to 400 magnification) are about $20 ....

... give me hope? or give me your upward adjustments? name brands? (.... fingers crossed....)

To be able to see and count chromosomes requires that a procedure is followed using various chemicals as well as a reasonable microscope. I would guess you could purchase one on e-bay but it would probably need to be able to magnify 400 to 500x and probably cost around $500-$600 or much more.

Colchicine is often used (or at least it was back when I made chromosome squashes) to make chromosomes shorter and thicker and easier to count. Hydrochloric acid, heated for a few minutes is used to make the plant tissue softer and easier to squash so that the chromosomes can be spread apart so that they are easier to count. Chemical stains are used to make the chromosomes visible. Some of these chemicals may have been replaced with other more modern chemicals but the basic process has not changed much.

The root tips are the tissue usually used to count chromosomes although the very early stages of pollen production can also be used. However, those very early stages occur in buds that are very small and it would probably require another different (dissecting) microscope just to be able to see and cut out the necessary tissue.

The published research on daylilies indicates that chromosome preparations for counting are not easy in this species.

"For example, I was unable to get a dip cross from Dixie Land Band its first summer, so I tried some tet crosses with it and got tons of seedlings. Nowhere is it listed as a tet, so it must have been quietly converted and may now be the predominant form that is being sold. It is not the only "dip" cultivar that I've found to be a "closet tet," and since no one keeps track of tet conversions, from now on I am going to routinely cross all unsuccessful "dips" with tets."

There are other possible explanations.

Dixie Land Band may have been produced by a cross of two diploid daylilies. It may have low fertility with diploids but better fertility with tetraploids. That could mean that it is a triploid that occurred naturally. There are no tetraploids registered with Dixie Land Band as a parent; that suggests that it was not converted.

Some of the older cultivars are not many generations removed from having triploids such as Europa as their parents. If one uses triploids as one of the parents in a cross one can produce triploid seedlings. Since there were no tetraploids to test seedlings if a seedling from such a cross was fertile with diploids it would have been registered as a diploid. Then crosses of diploids with the fertile triploid could have produced triploid seedlings that were not fertile with diploids but would have been automatically assumed to be infertile diploids.

There may be quite a few older cultivars, especially those that have low fertility, that are triploids but are registered as diploids. They may be infertile (for all practical purposes) with diploids but have noticeable fertility with modern tetraploids.

In the past, that speculation has been published in the Daylily Journal for older spidery diploid cultivars. ["So where are the seeds already?" by Rosemary Whitacre in the Daylily Journal 46 1991 pages 281-289. Names 'Pinky Green' registered as a diploid as not accepting diploid pollen but accepting tetraploid pollen successfully and both Madrid and 'Beau Soleil' as chimeras. A quote from that article, "The thing about these old spiders that many people do not know is that quite a few are irregular diploids. This means they are not quite the standard 22 chromosome jobs. Wheeler's, Russell's and Carl Milliken's daylilies all have among them irregular polyploids - usually aneuploids - but some are outright triploids or even somewhat irregular spontaneous tets. This is the heritage of Hemerocallis fulva 'Europa'." ]

{Aneuploid means not having an even number of chromosome sets - for daylilies that would mean not having 22, 33 or 44 chromosomes; an aneuploid daylily might have 23 or 21, chromosomes, or it might have 22 chromosomes but made up of nine pairs (18) plus one single (1) and one triple (3) instead of the correct two sets of 11 or 11 pairs.}

A cultivar that is infertile with diploids but fertile with tetraploids may be a tetraploid but it may also be a triploid.

The method for counting chromosomes in daylilies from one of the recent research articles.

"Chromosome number of root tips was counted using conventional squash method
(Yan et al., 1994). Plants were watered in the morning, and root tips of about 1 cm long
were collected around 9:30 am next day. The materials were treated with saturated
solution of p-dichlorobenzene for 5 h, fixed for 22 h in fresh Cannoy’s I fixative, and then
transferred to 70% ethanol for preservation. Root tips were softened with 1 M
hydrochloric acid at 60°C for 8 min and stained for 15 min with basic fucshin before
being squashed between a glass slide and a cover slip. For each genotype, 10 cells with
good chromosomal dispersion were selected, counted, and photographed under the
microscope of 400× magnification."

Yan et al. 1994 is
Yan, G., Ferguson, A.R. and McNeilage, M.A. 1994. Ploidy races in Actinidia chinensis.
Euphytica 78:175-183.

Their method for counting chromosomes in kiwi vine (Actinidia)

"Chromosome spreads were prepared according to a procedure modified from Chen et al. (1982), Geber &
Schweizer (1988) and Zhuang (1990). Five tight young shoot tips (or occasionally ten root tips) were collected and held in saturated aqueous p-dichlorobenzene for 3-4 h. Samples were then fixed in Carnoy's I fixative and left for up to 24 h. After being washed with 0.075 M KCl, the samples were digested in 4% cellulase (Onozuka R-10) + 1% pectinase (Macerozyme R-10), both from Yakult Honsha Co. Ltd.. Japan, in 0.1 M citrate buffer. pH 4.8, at 25° C for 3 h. After being washed in distilled water, the digested samples were refixed in glacial acetic acid:methanol, 1:3, and then smeared on microscope slides prechilled at - 20° C. Slides were dried quickly over an alcohol flame, left overnight and then stained in 2.5% Giemsa (Gurr's Improved Stain R 66, Gurr, BDH) in 0.067 M phosphate buffer, pH 7.2, for 30 min."

So many steps forward, thank you @admmad. I had only found articles about the ubiquitous onion root tip squash method (as well as lily and orchid), and I guess it may be that non-household chemicals are needed in the process to view any plant chromosomes.

But, there is also that more coarse and inexact method outlined to distinguish dip from tet (no mention of trips...might they be sized by having length between dip and tet?) by measuring guard cells in the leaf:

http://www.hostalibrary.org/mi...

Any thoughts on whether this would work well enough to get a general idea about ploidy? If it were, I have seen some digital microscopes (100-800x) that have either measuring software or built-in reticles (at about 10 micron lengths) running anywhere from $30-100, with free shipping and a return option. At the lower-end price, and with no non-household chemicals needed, I might just be tempted to bite. Thoughts?

If you have cultivar X, a diploid and the converted tetraploid version of that cultivar then comparing pollen sizes or guard cell sizes will identify which is diploid and which is tetraploid. A tetraploid cell is approximately twice the volume of the diploid cell which means that any measurement (length, width or height) of the tetraploid cell is about 35% longer than the diploid cell of the same cultivar.

Unfortunately cell size varies between different diploids and it varies between different tetraploids. The result is that some diploids have cells that are larger than some tetraploids (and vice versa some tetraploids have cells that are smaller than some diploids). The two distributions overlap.

The only observations that I know of on cell sizes in different ploidy daylilies did not look at a random sample of cultivars (they only looked at what they grew) and missed looking at both small-flowered diploids and tetraploids and longish-petaled diploids and tetraploids. I have been told that spidery daylilies have cell sizes that are 'different' (I assume from the diploids that were measured). So one can place little reliance on how much the measured cell sizes overlapped. I suspect that cell sizes are not only affected by ploidy but also by other factors - perhaps a general measure of plant size/robustness (perhaps for guard cells by leaf thickness and for pollen by flower, petal, anther thickness, etc).

What is probably more important is that triploid cell sizes will be intermediate between diploids and tetraploids and unfortunately completely overlap with both. A small cell size then might identify a plant as a diploid or triploid (but not perfectly from a tetraploid) while a large cell size might identify a plant as a triploid or tetraploid (but not perfectly from a diploid).

To get to a point where one could identify some cell sizes as diploid or triploid (not being able to identify which ploidy and with some of those being tetraploid - that is, as an example, after the cells of cultivar Y were measured it was possible to say that cultivar Y is probably a diploid or triploid but might be a tetraploid) and other cell sizes as triploid or tetraploid (without being able to identify which ploidy and with some of those being diploid, as an example, after the cells of cultivar Z were measured it probably is a triploid or tetraploid but might be a diploid ) one would probably need to measure the cells of several hundred diploid cultivars and several hundred tetraploid cultivars to build a good frequency distribution of cell sizes.

It may be possible (no guarantee) that triploids have a wider range of cell sizes than diploids or tetraploids. Statistically that would be described as triploids might have a larger variance in cell size than diploids or tetraploids. That is a statistical measure requiring statistical analysis and large numbers of cell measurements of known diploids, triploids and tetraploids to lay the foundation. To identify a cultivar as triploid would then require measurement of a large number of cells and statistical analyses comparing its variance with that of the known diploids and tetraploids. If in analyzing the known diploids and tetraploids one found that their size variances overlapped then one would be no better off than with the original measurement (the average or mean value).