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Tetraploidy Conversion

Topics:

    1.     Solution Calculations and Preparation
    2.     Oryzalin Conversion of Lilies
    3.     Monocots vs. Dicots
    4.     Three  methods of attempting colchicine conversion on bulbs
    5.     Colchicine Concentrations
    6.     An Easy and Effective Method of Colchicine Treatment
    7.     Other Colchicine techniques for Iris
    8.     Other Surflan Techniques
    9.     Polyploid Conversion of Daffodils
    10.     Other Chemicals
Sites of Interest:

    Chromosomal Numbers. Translocations And Their Consequences
    Terms: polyploidy by multiple of chromosome sets
    Polyploidy: A Discussion
    Speciation by Polyploidy
    Abstract: Single-Copy Sequence Evolution, Polyploidy and Phylogeny Reconstruction.
    Abstract: "Evolution in Dryopteris: The Role of Hybrids in Polyploid Speciation"
    Abstract: Mutation Breeding: Theory and Practical Applications

Books:
    Cytotaxonomy of the Liliaceae and Allied Families

This FAQ was created and is maintained by Beth Matney. Last changed Februrary 25 ,2000.



Oryzalin Conversion of Lilies:

Date: Wed, 26 Feb 1997
reprinted from:  Lilium-L

Cooking Up Tetraploids in the Kitchen
Arthur Evans, Arkansas
(NALS Yearbook 1993 No. 46, pg 10-12)

My wife, Crow, is very good about sharing the kitchen with my non-cooking projects. You know, ordinary things like brewing beer, sterilizing soil in the oven, making nutrient medium for embryo cultures, "nuking" critter-infested bulbs in the microwave, and storing an unreasonable number of lily bulbs in the refrigerator. She does raise an eyebrow, however, when I start playing with poisonous chemicals in the food-prep area. That's one reason I decided to switch from colchicine to oryzalin for my conversion attempts as per Dr. Jaap van Tuyl's article in the 1990 NALS Yearbook. Oryzalin is alleged to be much less toxic to people than colchicine. By the same token, it is supposedly more effective in interrupting normal mitosis in rapidly dividing lily cells which can result in converting a diploid lily scale into a tetraploid scale bulblet.

It works. I've done it. Here's how.

Oryzalin is sold by Dow-Elanco as a pre-emergence herbicide under the name Surflan AS. It comes in one gallon jugs of 40% active ingredient at a cost of about $100 per gallon. I got mine from Hummert Co. Inc., the well-known horticultural supply company. Since Dr. Van Tuyl advises the .005% strength treatment solution, I calculate that one gallon would make a fair-sized swimming pool of treatment solution, more than enough even for an obsessive-compulsive gardener like me. I might even use some of it for the purpose stated on the label. Seriously though, if my math is correct, .5 ml, of 40% oryzalin dissolved in 4 liters of water makes an approximately .005% oryzalin treatment solution. That sounds weak, but it is strong enough. Don't be tempted to make it stronger. In fact, next time I try a batch of conversions, I will make it half strength. It burns some scales so severely that they never form bulblets.

The oryzalin is hard to dissolve in water. I pre-dissolve .5 ml (easy to measure with an injection syringe from the drugstore) in .5 cup of warm isopropyl alcohol or vodka to which I have added 2 tablespoons of 99% DMSO (dimethyl sulfoxide) from the veterinary drug shelf of my local farmers' supply store. The DMSO is a strong organic solvent known to enhance the absorption of drugs through skin. It smells awful, and it couldn't be good for you, more than enough reason to wear latex rubber examination gloves and a moisture-resistant face mask. Your family dentist will be glad to give you plenty of these. Gradually add warm water in increments to the oryzalin and solvent mixture in a clean milk jug with violent agitation and incantation. The incantation is optional, but the looks on the faces of the grandchildren are priceless. I presume this solution is fairly stable, but I make up a new batch after treating 2 batches of scales.

Use scales which have started to show a tiny ridge or bump of rapidly dividing cells or "callus" around the broken edge at the base of the scale. If you incubate scales in the moist sphagnum in a zip-lock bag at 80ºF it takes from 1-4 weeks to get to this stage of development, Don't wait until the callus develops a tiny bulblet. The more tissue the drug has to penetrate in order to reach the actively dividing meristematic cells, the less likely you will be to get a completely convened tetra bulblet. If a bulblet arises from a mixture of converted and non-converted cells we call it a mixoploid. These plants revert to diploid status since diploid cells grow and multiply faster than tetra cells and tend to take over completely over time.

Now, for the actual treatment, bear in mind that the drug only works on cells that are actively dividing, so it makes sense that you want to use a temperature high enough to stimulate rapid cell division and a time long enough to intercept lots of divisions. I don't know what the optimum temperature or time is for most lilies. It may vary with the genetic background of the cultivar you are working with. What I have done is soak scales in converted quart jars in the hot water heater closet which maintains about 85ºF for 6 hours. Make sure your jars, scales, and solution are all close to your chosen treatment temperature before you start. Due to the amount of burning of the scales in previous treatments, I may reduce treatment temperature to 75ºF or 80ºF next time.

After treatment, rinse scales with several changes of fresh water to get rid of as much of the drug as possible. Handle treated scales only with gloved hands. Be a fanatic about cleaning all the equipment and food preparation surfaces as soon as you have placed scales into labelled plastic bags of slightly moist sphagnum or vermiculite. I use a Sharpie pen to label the bag with the cultivar name, date of scaling, date of oryzalin treatment, and any variation from the standard treatment. This year I used Zip-lock vegetable bags with ventilation holes. All bags then go into a garbage bag with loosely closed top to keep the small bags from drying out. The big bag then gets incubated at 75ºF to 80ºF from October or November until bulblets are well-formed, usually about the end of February or March. At that time the bulblets are vernalized, simulating winter chill, for 3 months in a refrigerator set at 35ºF.

When the vernalization is complete, I inspect the bulblets individually, looking for physical characteristics which may indicate conversions. Most bulblets will look quite ordinary because they are still unconverted. If you said the aforementioned incantation correctly, however, you may find a few (5%-10% at best) bulblets which have obviously shorter, thicker scales which stand apart from each other at 90 degrees or more. I plant these bulblets in large pots and protect them from all harm. A high percentage of these odd-looking bulblets will soon show the large stomates (.005 inches long) on the underside of the leaf which indicate conversion to tetraploid status. Ordinary looking bulblets are planted in rows in the garden. Their stomates are measured in mid-summer to pick out any tetras that slipped by in earlier inspection. I don't have much space so I don't grow the diploids to maturity. When large enough to bloom, test the converted tetras both ways in crosses with fertile seed-grown tetras. There will be variations in the fertility between different conversions of the same cultivar. Due to the low number of successful conversions in each batch, I would certainly advise that you start with at least 100 clean, healthy scales of each cultivar. If you have large, blooming-size bulbs to start with, you can usually harvest 10-20 usable scales from each one. Scaled cores can be replanted to grow again.

In dozens of attempts I was only able to convert one cultivar, old 1b pink 'Gypsy' with colchicine. In the fall of 1993 I attempted to convert 8 cultivars with oryzalin and I believe I have succeeded in converting 4 of them. They are 'Yellow Star', 'Connecticut Star', 'Shirley', and 'Nutmegger'. These have not been tested for fertility with tetras or chromosome-counted, so I will have to wait for these tests to confirm what my stomate measurements and observations of other plant characteristics lead me to believe.

In the fall of 1994 I treated about 20 cultivars and seedlings. The scales are producing fair crop of bulblets, a few of which look like valuable tetraploid breeding prospects. I would be interested in corresponding with and assisting anyone who wants to experiment with oryzalin conversion of lilies. No doubt the method I use can be improved. If we pool our data, we might learn a lot.

POSTSCRIPT.

As of 12/95 few of the bulblets from the conversion attempts of last fall have sprouted, probably due to insufficient vernalization time. I will chill bulblets for 3 months next time. Of the cultivars which did sprout, stomate measurements of 'Allegra', 'Nepal' and 'Henry VIII' indicate conversions. Hopefully, more cultivars from the 1994 attempts will sprout in the spring of 1996 and show large stomates as well.

One of the 1993 conversions, 'Connecticut Star', did bloom this summer Its pollen was used on several tetra Asiatics, including tetra 'Nutmegger' and some of my own seedlings. All produced good seed, another indication of conversion.

I believe there will always be a place for excellent diploid lilies. Many of the best  garden genotypes are still in the diploid gene pool. Getting those superior genes into the polyploid gene pool is a worthwhile goal for amateurs and pros alike. Some of these diploids can be used with tetras in 4 X 2 crosses if they produce significant amounts of pollen. Others may produce a few triploid embryos when pollinated by tetras in 2 X 4 crosses. Many diploids however, refuse to participate in such liberal nonsense as  crossing with polyploids. In such a predicament, the only solution is to convert the diploid to tetraploid status. There is much to do, and though it is time-consuming, it is not too difficult for kitchen technology. Hopefully, your spouse will be as understanding as mine has been.

23 Feb 1997 posted to Lilium-L:

Oryzalin conversion:  Yes, I would reduce or eliminate the DMSO and alcohol or as an alterative, reduce the oryzalin to .003%.  Exposure time (soaking) about the same or longer, temperature about the same.  These changes are proposed due to the degree of burning seen on callus after treatment.  Also, some people don't wait until they see a tiny ridge of callus before they treat scales.  Dr. Robert Griesbach,my friend and mentor, treats scales within a few days of scaling.  His theory is that by intercepting the earliest cell divisions not yet visible to the eye, it is more likely to result in complete conversions rather than the partial conversions we call mixoploids or chimeras.  I suspect it would be a help if we knew how long the drug persists in the scale tissue.  Some scales (Journey's End) failed to make bulblets for me for almost a year, until I chilled them 3 months and brought them back to warm room temperature.  Would they still have had enough oryzalin in the scales to affect cell division if I had treated them almost a year before?  That may be an exaggerated example, but when used as a pre-emergence herbicide, Surflan is supposed to be effective for up to 3 months.  We use it at a tiny fraction of the herbicide concentration, so I presume that half-life, so to speak, is much less.  Perhaps Kathy Andersen, organic chemist lilyite, daffodilian, chrysanthimuse, llamabilist, could help us here. Go for it, Kathy!

 Dr. Griesbach treats germinating seeds and claims reasonable success in doing so.  I look forwared to him writing an article for the NALS yearbook on his experiments.  Naked eye, but considering my advancing  age and decadence (not to mention decrepitude) magnification would probably help.  Back lighting of course, is more important than magnification.

23 Feb 1997 posted to Lilium-L:

Oryzalin affects cell division somewhat like colchicine does, but more efficiently and with less toxicity to the user.  It is available as a pre-emergence herbicide called Surflan-AS  (Dow Chemical Co.)  which is 40% oryzalin.  I have advocated for some years that we should enlarge and enrich the tetraploid gene pool by converting the best garden lily genotypes to tetra forms.  This is possible with a bit of work by amateurs everywhere.

Wed, 20 Jan 1999 private correspondence:

I've learned a bit more about conversion since the article was printed, however. Dr. Jaap van Tuyl,a leader in Lilium hybridizing and conversion, has changed part of his technique from the one I mentioned. He now advises starting the oryzalin soak immediately after scaling rather than waiting until callus tissue starts to form on the broken edge of the scale. He claims that this modification produces fewer partial conversions or mixoploids and more pure conversions in which all somatic cells are 48 chromosome tetraploid conversions.

Arthur

Alternate Solution Calculations:
40% active ingredients is 40 grams surflan/100 ml solution

To make 1% surflan, you'd dilute 1 ml of 40% solution in 39 ml water.
To make 0.01% surflan, you'd dilute 1 ml of 40% solution in 3999 ml water  (essentially 4 liters -- the error is insignificant)
To make 0.005% surflan, 0.5 ml in 4 liters of water (1 ml in 8 ml water) is correct.

(me, myself and I, I'd use 0.1 ml surflan in 800 ml of water for 0.05% )

Kay Lancaster



Monocots vs. Dicots:

Date:    Sat, 16 Jan 1999
From:    "James W. Waddick" <jim-jim@SWBELL.NET>
reprinted from: Alpine-L Digest

"Can anyone tell me if there is anyone who has treated sprouting monocot seedlings with colchicine to induce chromosome doubling? Fairly straight forward with dicots  - treating the seedlings just as the first true leaf emerges. How & when would you go about it with the monos?"

Dear John;
        Exactly the same- at least with iris. Just as the seeds are cracking open or the cotyledon emerging soak in the appropriate concentration for the appropriate length of time and expect -hope that- most seedlings die. The ones that survive are likely to be tetraploids.  I suspect most other monocots are similar.

James W. Waddick
Kansas City MO   USA



Three  methods of attempting colchicine conversion on bulbs:

Date:    Sun, 17 Jan 1999
From:    "Stephen J. Vinisky" <stevev@EUROPA.COM>
reprinted from: Alpine-L Digest

I have heard of three other methods of attempting colchicine conversion on bulbs.

1) Dr. Harold Koopowitz of UC Irvine soaks twin scaled segments in a petri dish prior to putting the twin scales in a warming cupboard.

2) The late Jack Romine, former American Daffodil Society President, was discouraged with the high percentage of non viable seed when sprouted seed was treated. He began using a syringe to inject colchicine between the scales of full grown bulbs just above the basal plate. The bulbs were sectioned horizontally at first to determine where a growth point ( which ultimately becomes an offset ) was forming. Losses were still high. Later on he stopped sectioning and injected between each scale which is a tedious, delicate business.

3) Dr. Bob Carr of Ocala, Florida converts emerging flower buds which are hooked up to three hypodermic needles and tubing which drips colchicine into the forming anthers within the bud. The colchicine is mixed with blue food coloring. Three weeks prior to bloom the drip is discontinued. The blue dye gives an immediate visual indication if the needles were inserted correctly. Blue green styles indicate that the needles and colchicine were at least in the right location. Potentially converted pollen can then be checked under a microscope. Converted pollen is available in the year which conversion was attempted unlike the normal wait required to grow many bulbs on from seed to maiden flowering.

Three very different and creative approaches to the problem. As has been pointed out, colchicine is a highly toxic poison.

Stephen J. Vinisky
Sherwood, Oregon   USA



Colchicine Concentrations:

Date:    Sun, 17 Jan 1999
From:    John Weagle & Ken Shannik/HalifaxNS <InsigneGdn@AOL.COM>
reprinted from: Alpine-L Digest

To quote Augie Kehr (article in the Journal Magnolia Society Vol20#2), noted Magnolia breeder who has induced polyploidy in Magnolias :  "Colchicine usually comes as a powder.  ... make a 1% solution by dissolving 100milligrams in 100cc distilled water. The final solution .. use 1cc of solution to 19cc of water for a .25% solution. ...To this 5 drops of non-phytotoxic wetting agent. and 5 drops DMSO dimethyl sulfoxide which .. magically speeds up cell penetration and absorption manifold times."   The latter chemical DMSO is extremely hazardous as well and makes this whole brew wickedly dangerous - DMSO is NOT available in Canada. DMSO was not used in the concoction to induce hexaploidy (114 chromosomes) in our Magnolia hybrid  - its doubled chromosomes were glaringly apparent almost immediately, crinkled extremely thick leaves & very slow growth. One drop is placed in the centre growing tip just as first true leaves emerge & humidity kept as high as possible as drying will increase the concentration and death may result. Sometimes the cotyledons will make no true leaves for a year or more   - watch these!

John Weagle Z6
Halifax Nova Scotia CANADA



NOTE: Solution Calculation Error in the above paragraph:
    1% solution would be 1 GRAM of colchicine powder in 100 ml of water.  (1 cc = 1 ml)

100 mg is 0.1 grams, so 100 mg/100 ml water is 0.1% colchicine.

1 cc of a 1% solution + 19 ml water would give you the same concentration as 5 ml 1cc solution + 95 ml water, or 5% of the original 1%
concentration, or 0.05% solution of colchicine

To make a 0.25% solution, which is 1/4 of 1%, you'd use 1 ml 1% solution + 3 ml water.

(The 1 cc of 0.1% colchicine + 19 ml water recipe gives you 0.005% solution of colchicine)

If I had to make a 0.25% solution of colchicine from scratch, and had a scale capable of weighing 100 mg accurately, I'd use 100 mg colchicine in 400 ml of water, and forget about the intermediate dilutions.

I'm not sure what concentration of colchicine is recommended or what he intends here.

Kay Lancaster



Date:   21/01/99
From:    (August E Kehr)
To:     John Weagle & Ken Shannik/HalifaxNS <InsigneGdn@AOL.COM>

Dear John,
        The stock solution of aqueous colchicine is made by dissolving 1(one) gram of colchicine powder in 100 c.c. of water to make a 1.0 % solution. The stock solution is then diluted by adding 1cc of stock to 19 cc water. The amounts you quoted in Magnolia Journal 20. No.2 is incorrect. To my knowledge no one has previously called this error to my attention, and there was no erratum published.  In fact this is the first time the error was ever called to my attention..The report cited is 15 years old, and it indicates that few people have read the article very critically, including myself.  I do not know at this juncture what figures were in the original manuscript, but I must admit I had never noticed it myself. The final figure in the original article did have the correct final concentration of .25%.
        In addition I have since learned that some germinating plantlets may be injured by a "non phytotoxic" wetting solution, so in later reports that concentration has been drastically cut to 0.1 drop.  This concentration seems to be adequate and is made by diluting one drop of the wetting solution in 10 drops of water, and then using one drop of the diluted wetting agent solution. In other words non phytotoxic wetting agent CAN be toxic. It is better to err in the direction of too little than too much.
        I regret the error in the published report if it caused anyone any inconvenience. However, because no one has noted the error previously, it indicates how little interest there was in the paper. However, I am humbled by the error and hope it did not cause any undue problems. I do recall the original manuscript was originally one single article, but it was divided into two parts by the Editor.
        I believe that a 1% concentration would be very harmful to magnolia buds, and could readily kill the buds in an environment that is kept at near 100% relative humidity and about 70 degrees temperature.

Augie



An Easy and Effective Method of Colchicine Treatment

August E. Kehr, 240 Tranquility Place, Hendersonville, NC 28739 USA

Although colchicine treatment of plants to induce polyploidy has been known for over 50 years, relatively few polyploid forms have been developed. The conventional method has consisted of germinating seeds on filter paper or blotting paper, then, when germination is under way, treating the seeds with an aqueous solution of colchicine of varying concentrations and for varying periods of time.. This treatment affects all parts of the seedling as well as the growing point, although the growing point is the only target and the only part that must be affected if the plant is to continue growing with an increased number of chromosomes. A major fault of that method is that the roots are affected and commonly grow badly or not at all. Thus, the method is ineffective from a practical standpoint, Furthermore, it is difficult to plant the treated seeds from the filter paper to the growing media- As a consequence the incidence of success by this method is from extremely low to none at all. As a result few polyploid plants are produced that will grow on to maturity. Thus the past accomplishments of the production of polyploids have been quite unsatisfactory. As an example, the development of commercially acceptable yellow flowered camellias has not progressed because no one has successfully produced a fertile polyploid F1 hybrid with the yellow species that has been available for about 30 years.

The new method described here radically increases the chances of success. In actuality, with large-seeded plants, such as magnolias, one can produce polyploids with almost 100% success. I have over 50 polyploid magnolia trees induced by this method.

First, it is necessary to make a stock solution. For convenient storage I use 1.0 gram of colchicine in 200 cc water (about 3.25 oz) to make a 0.5% stock solution. This stock solution will keep for years, if need be, in the refrigerator and can be diluted accurately without the need for analytical scales. A tiny crystal of paradichlorobenzene (moth crystals) placed in the stock solution will prevent any contamination from molds or bacteria arid has no deleterious effect on the colchicine. In fact, the moth crystal enhances the effect of the colchicine.

The stock solution is diluted by adding 180 cc of water to 20 cc of stock solution, to which should then be added 1 cc of dimethyl sulfoxide (commonly called DMSO), and one tenth drop of surfactant (as used for herbicides or in dish washing fluid). One tenth of a drop is made by diluting a drop of the surfactant with ten drops of water, then using one drop of the resulting solution. Young seedlings are very sensitive to the surfactants and the leaves are damaged or even killed by too great a concentration. The DMSO drastically increases the permeability of cell walls and thereby improves the penetration of the colchicine into the plant tissues and thus improves its action on the dividing cells. The surfactant 'wets' the surface of the cells and likewise improves the passage of the chemical into the plant tissues. Thus, the two chemicals increase the overall effectiveness of the treatment. Be sure not to get the chemicals on your hands, face, or eyes. In case you do so, wash with warm water at once.

Seeds are planted in the normal way in the normal substrate. When the cotyledons of the germinating seedlings are well developed, and just before the first true leaves appear, the colchicine mixture of colchicine, DMSO, and surfactant is misted in a fine mist using a good atomizer, until a small amount collects in the crevices between the two cotyledons. The seedlings are misted daily for 7-10 days until the true leaves are plainly visible.

The method is most effective when the seedlings are grown in a shallow box-like structure (8-10 inches or 25-30 cm high), covered by glass or polythene to maintain a relative humidity near 100%. Put the container or box under cool white fluorescent lights for 18 hours per day and maintain a temperature of 24-27 degrees C (75-80 degrees F). The high humidity will keep the aqueous solution from drying out and the tiny seedlings moist for most of the 24 hours of the day.

The improved method maintains normal and healthy root growth and avoids the difficulty of transplanting wet seed from filter paper. Of course, polyploid plants derived by this method have normal (not doubled) roots with normal (not doubled) chromosome numbers, and so one must propagate from the polyploid shoots or by seed from polyploid shoots. Seedlings grown from polyploid shoots when selfed, or cross-pollinated with other plants with polyploid shoots, will produce 100% polyploid offspring. Likewise, such offspring will have all their roots, as well as their shoots 100% polyploid.

Perhaps the most difficult problem for the inexperienced person is how to recognize polyploid seedlings. In general, the cotyledons and new leaves are darker green in color and 'fatter' than unaffected seedlings. Also, the first true leaves may occasionally become abnormal or ragged in growth. The new leaves of doubled plantlets are broader than the undoubled and heavier in texture. Under the microscope, the cells of the polyplolds are twice the volume and about 25% larger in diameter when compared with undoubled cells.

Colchicine in crystalline form is expensive and is sold in small quantities from 100 mg to about 5 gm per bottle. I have found it impossible to get all the chemical from small bottles. Consequently, I now buy the desired quantity and dilute all the contents of the bottle at one time to make stock solution. In this way two objectives are met: an accurate stock solution is made up without the use of scales to weigh out the yellow colored powder, and none of the expensive chemical is wasted.

Using the method described I treated seed of the mysterious red maximum rhododendron from near Mt. Mitchell and obtained 30-35 polyploid plants. These I have given to friends and I hope that in future years some superior clones will arise that may merit naming.

In conclusion, it is surprising to me that [in] over half a century no one seems to have come up with this simple method. How was it possible to have missed it for so long?



Other Colchicine techniques for Iris:

Date:    Sun, 17 Jan 1999
From:    "James W. Waddick" <jim-jim@SWBELL.NET>
Subject: Colchicine and MORE
reprinted from: Alpine-L Digest

Steve Vinisky:
>I have heard of three other methods of attempting colchicine conversion on bulbs.

Steve is inciteful in mentioning these alternate methods of inducing tetraploidy with Colchicine. Here's some others for Iris:

        1. A mature fan is cut to the rhizome in summer and a small 'cup' is scooped out the rhizome. Dilute colchicine solution is kept in the cup for the desired period-keeping it full. Evenmtually new shoots may emerge fully tetraploid.

        2. Following pollenation, a hypodermic with cochicine is inserted in the base of the pedicel and appropriate cochicine is injected as the seed pod develops. Some of these sedds will produce tetraploid plants.

        Colchicine is not the only answer. Some folks use the very available herbicide 'Treflan' to induce tetraploidy. The cultivar Ajuga "Catlin's Giant' is supposedly a tetraploid that appeared at the edge of a large patch treated with Treflan, The central plants were all killed, but a single crown on the edge developed into this giant cv.

        There are a number of other chemical agents used to induce tetraploidy, variegation, flower doubly and other mutations. Handling any potentially mutagenic chemical has inherent dangers - beware.

        Any other chemical mad scientists trying other treatments?

James W. Waddick
Kansas City MO  USA



Other Surflan Techniques:

Date:    Mon, 18 Jan 1999
From:    Tony Avent <tony@PLANTDEL.COM>
Subject: Re: bulbs and colchicine
reprinted from: Alpine-L Digest

John:

        With all the talk of tetraploidy inducements, and the great dangers involved with colchicine, let me pass along a much safer method.  Instead of using colchicine, researchers have found that Surflan pre-emergent herbicide has the same effect as colchicine.  Researchers tell me that the mutation rate on plants is higher and much lower in humans.

        Dip 5-7 day old seedlings in a solution of Surflan (High Rate is 1:100, while a low rate is 1:200).  This is using the standard store shelf solution to start.  The seedlings should remain in solution for 24 hrs. After this, they should be thoroughly rinsed for 3-4 hours under running water.  Then plant and wait.  While 50% should die, most of the rest will be tetraploid.

        We have had excellent success with this method.  Among the tets so far include ligularia, rohdea, and hosta. I hope this is of use.

Tony Avent
Raleigh, NC   USA

Tue, 19 Jan 1999 reprinted from: Alpine-L Digest:

        Tetraploids are reportedly induced by the entire group of pre-emergent herbicides that include both Treflan and Surflan.  I'll have to rely on some more scientific types to explain how this group works.  A article titled "Drugs with Colchicine-like Effects that Specifically Disassemble Plant but Not Animal Microtubules" outlines the scientific stuff.  This was published in The Annals of the NY Academy of Sciences, 466:767, 784 (1986) by A.S. Bajer and J. Mole-Bajer Univ. of Oregon.

        To date, there are three hostas on the market now that are Treflan induce tetraploids, H. 'Patriot', H. 'Night Before Christmas', and H. 'Grand Tiara'.   It is my understanding that the ploidy levels have been confirmed by Ben Zonneveld of Leiden, Netherlands.

Tony Avent

Solution Calculation:
High Rate of 1:100 using standard store shelf solution (40%):
40% active ingredients is 40 grams surflan/100 ml solution or 0.4 grams per 1 ml solution
1:100 would be 1ml solution + 99 ml water, or
0.4 grams Surflan/100ml, or 0.4% Surflan Solution

Beth Matney



Polyploid Conversion of Daffodils:

Date: Tue, 16 Sep 1997 13:26:31 -0700
From: Harold Koopowitz <hkoopowi@uci.edu>
Subject: Polyploidy Conversion
reprinted from: Daffodil list

Just a short note to update the group on our project to try and convert some of the intersectional sterile hybrids to fertile polyploids.

Elise Havens sent us about 12 bulbs of each of the following cultivars. Swift Arrow, Dainty Miss, Bell Song and Oryx. I added a lot of bulbs of N. jonquilla. Two of my undergraduates then reduced all the Haven's bulbs to twin scales and the jonquilla to quarter bulbs. They were soaked in Cleary's to prevent fungal infections and then  incubated in my office at about 70F until the first appearances of bulblets could be discerned between the scales.  Except for some control groups all the twin scales were then soaked in Oryzalin for 48 hours and then washed for two days. Scales were then partially dried and returned to plastic bags where they contniue to be incubated. The bulblets are now growing quite well. Oryzalin is a herbicide that probably works like colchicine but is supposed to be somewhat more effective in converting plant cells to polyploids. We will probably grow the bulblets for another 10 days and then refrigerate them for a few weeks before planting out in flats in the greenhouse. Hopefully we will be able to recognize the polyploids by their leaves before they reach flowering size.

We will probably try to do some late twin scaling if I can get  Hawera and Erlicheer from the local nurseries. Both of these have I think great potential not only for breeding but in their own right as polyploids.  We tried to twin scale "uncured bulbs" of Erlicheer right after digging them during the summer but lost all of the scales to bacterial rot. We had used Cleary's as the fungicide but bacteria seemed to get them. There is a report of a converted fertile Hawera, called Thawera, in Tasmania.

Harold Koopowitz
Irvine, CA   USA

Tue, 19 Jan 1999 Addendum (private post):

It is now spring in 1999. Bulblets were plants in tubs in the greenhouse with relatively few emerging the first year 1997-98.  But there were few losses the bulbs just rooted and formed underground. They were harvested in the late summer of 1998 and then planted out in the ground. Some were planted,  in California but most were sent to a friend in Oregon during the Fall of 1998. By January 1999 some of the bulblets have emerged. It is still too early to know how effective our treatments have been but several plants of N. jonquilla and one Erlicheer look promising. Many have still to emerge.

Harold



Other Chemicals:

Date:    Tue, 19 Jan 1999 19:23:10 -0800
From:    Kay Lancaster <kay@FERN.COM>
Subject: Re: Tetraploidy conversion
reprinted from: Gardens list

PDB, paradichlorobenzene, and beta hydroxyquinoline (that should be the Greek B with the tail, not the word "beta") may also cause polyploidy. Effects and concentrations generally have to be figured out for each plant.

See Tiwari, S. 1983.  Cytological effects of colchicine, paradichlorobenzene and B-hydroxyquinoline on root tops of lentils. LENS, Lentil Experimental News Service Newsletter, v 10(1): 22-24

Sarbhoy, RK.  1980.  Effect of paradichlorobenzene on the somatic chromosomes and mitosis of Lens esculenta (L.) Moench.   Cytologia 45(3):381-388

I've not use B hydroxyquinoline, but I have used PDB on lots of preps for chromosome cytology slides.

(oh yeah... REALLY watch out for DMSO.  Methylethylbadstuff, as George Shirley would say.  I wouldn't think of handling it except in a fume hood and with double gloves.  Too good a solvent.  I had a housemate in grad school... chemist who worked with the stuff: Kidney damage from soap film on the skin.  "There are old chemists, and bold chemists, but no old, bold, chemists.~ )

Kay