ABSTRACT: The tetraploid breeding program of Mr. Norris includes both clonal and seedling treatment with colchicine, an alkaloid found in the autumn crocus, Colchicum autumnale. Clonal treatment involves injecting O.5cc of a 0.4% colchicine solution into the plant fan just above the growing point, concentrating on offsets which would be expected to bloom the following year. Chimeras normally are produced instead of tetraploids, and seed is obtained through crosses of chimera X chimera or chimera X tetraploid. Treatment of seedling iris involves a 12-hour soak in a .075% colchicine solution followed by a 12-hour waiting period in moist vermiculite then a 3-4 day soak in ice water. The seedlings are then planted. Evaluation of ploidy level is made by microscopic examination of stomata size. Chimera or tetraploid cells display a stomata size of 45-50 microns over the long direction, rather than the 35-40 microns normally observed in diploid cells. Pollen size also is an indication of ploidy levels, with diploid pollen averaging lIS microns and tetraploid pollen ranging from 125-150 microns. Chimeras may not be produced until two or three vegetative generations following treatInent. 
Several different methods of producing tetraploid irises have been tried by the writer over a period of time, but for the most part these have been variations of the seedling and clonal treatment. Tetraploids and in most cases chimeras have been produced by both methods. Each has its advantages and disadvantages so the particular situation will determine which will be used. 
As with most colchicine treatment, the best results with the irises are obtained when the plants to be treated are making their most vigorous growth. Here in Kentucky the best growth is made in the fall when the weather starts to cool and there is plenty of moisture in the soil, so this is the preferred time for making the clonal treatment. My method consists of injecting 0.5cc of 0.4% colchicine into the fan just above the growing point. Offsets that would be expected to bloom the next year and that are making good growth are the ones that are injected, several fans to a plant if they are available. Quite a few plants have been given this treatment but only two have bloomed the following year and both had a large percentage of tetraploid pollen. Close watch needs to be kept on the treated plants as the chimeras might not show up for two or three vegetative generations. 
When the new growth starts on the treated plants I usually screen each offset by using a microscope to measure the stomata size. The stomata of the diploids usually measures 35-40 microns over the long diameter while those of a chimera or tetraploid will measure 45-50 microns. "When such a plant is found I plant the chimera portion separately, then when it blooms a further check can be made of the pollen size. Diploid pollen averages 115 microns, but some such as I.fulva may run as low as 90. Tetraploid pollen runs from 125 to 150 microns. 
It is always a good idea to check pollen from blooms of the same plant that have not been treated with colchicine to be sure of the proper size to check for. Due to the shape of the pollen it is difficult to get an accurate measurement on dry pollen, so I sprinkle the pollen on a drop of 4 % acetic acid. (Vinegar is usually 5% acetic acid so I dilute it to get the 4%.) As soon as the pollen hits the acetic acid it becomes rounded so it is easily measured. When plain water is used the pollen continues to take up water and swell so a measurement would have to be made very fast if it is to be accurate. If full-strength vinegar is used the pollen will expand to its full size initially then will begin to shrink. Many of the anthers will have both the diploid- and tetraploid-sized pollen on it and this is easy to see. Some blooms will have anthers with one or two fully tetraploid while the others are diploid. This kind is usually easy to spot as the anthers will be different sizes, and many times the other parts of the bloom will vary in size. 
The real proof comes when the chimera is crossed with a tetraploid. As Mr. Joseph Mertzweiller has shown us, it is possible to get seed from crossing a diploid with a tetraploid, but the chances are very good that more seed will be produced from a chimera crossed with a tetraploid. So far none of my chimera X chimera crosses have produced seed, and what tetraploid seed I have produced came from crossing the chimeras with the tetraploids. I owe Joe a vote of thanks for the plants and seed he has given me, as this no doubt put me several years ahead in the production of new tetraploid plants. It was a big step he took when he produced the first tetraploid Louisianas by crossing the chimeras. 
As stated earlier, two or three vegetative generations may be needed for the chimeras to show up. I did manage to get three generations in one winter and this speeded things up by several years. Three fans were potted up from each of several different clones, then they were brought inside when the weather started to cool off. When a fan is injected with colchicine it stops growing for a while, then will put up leaves that check out as being tetraploid. There will usually be six or seven of these tetraploid-appearing leaves, then the next one will show up as a diploid. At this point a cut is made through the fan, through the growing point if possible, and down into the rhizome. 
After another waiting period, new offsets will start to put out (with luck) at each leaf. The stomata size can be measured and the diploid ones removed. Any that show the tetraploid stomata size are left on the plant until they have established a good root system, then they are removed and potted up separately. If the ones that are repotted revert to diploid they in turn can be split through the crown to promote another generation. At times four or five offsets that check out as diploid will put up, and the rest of the offsets will put out after these first ones are removed. When this happens there is a good chance the last ones to start growing will be tetraploid, as they are slower growers than the diploids. 
Three plants that were given this treatment bloomed this year and had a high percentage of tetraploid pollen. The plants were FULL ECLIPSE, SUN FURY and GOLD RESERVE. I should have had a record crop of tetraploid seed but the verbena moth and their larvae wiped me out before the pods had a chance to mature. 
Colchicine treatment of the seedling irises has changed very little since the book Colchicine was written by Eigsti and Dustin and published back in 1955. They recognized then that the colchicine treatment of the monocots would have to be different from that of the dicots and suggested a 12-hour soak in the colchicine followed by a 12-hour soak in room temperature water. This general treatment is still being used with the colchicine solution being .05% to .075%. Seedlings given this treatment usually show signs of tumorous growth shortly after being planted and are very susceptible to rot when they are allowed to get too wet. Figures given for the production of chimeras or tetraploids run from 3% to 5% of the plants that live. I have made one innovation in my seedling treatment that cuts down on the tumorous growth and allows more seedlings to live following the colchicine treatment without any need for special handling. The total number of chimeras and tetraploids produced from my treated seedlings is somewhat greater but that is probably due to a lower mortality rate. 
I soak the seedlings for 12 hours in a .075% colchicine solution, but instead of putting them in the water soak at this time, they are held in moist vermiculite for an additional 12 hours. The time spent in the vermiculite allows more of the cells to progress to the tetraploid stage. Then instead of putting them in room temperature water they are held for 3-4 days in ice water. The ice water stops all cellular activity while at the same time it leaches the colchicine from the cells. The seedlings resume normal growth shortly after being planted and do not require any special care to prevent rotting. 
The treated seedlings, like the plants from the clonal treatment, will put up &-7 leaves that appear to be tetraploid, but in the majority of cases the next leaves will have reverted to diploid. Chimera or tetraploid plants, if there are any, will show up in future vegetative generations. 
I am by no means satisfied with the small number of chimera seedlings that are produced, and several variations of the colchicine treatment will be tried this fall and winter in an effort to produce a higher percentage of chimeras and plants that are fully tetraploid. 
Samuel N Norris developed an interest in tetraploidy in plants when the tetraploid Crestwood series of Hemerocallis were introduced. Although he spent some time treating daylilies with colchicine, because of heavy activity in the field he decided to devote his attention to other plant varieties. During this period he enjoyed his star achievement, the cross of Iris dichotoma (pardanthopsis dichotoma) X Belamcanda chinensis, seedlings of which were named XPardancanda norrisii - the famed Candy Lilies. Following some preliminary work with Louisiana irises, Norris worked during the next 15-18 years to produce tetraploid arilbred iris. He recently has turned his attention back to Louisiana irises, and his work focuses on the production of new chimeras through dona! and seedling colchicine treatments.
Editor's Note:  This article first appeared in the SLI Newsletter in the December 1991 edition.