Plants & Gardens 1: 241-246 (1945)
L. F. Randolph
Condensed from the Amer. Iris Soc. Bull., May, 1945

ONE of the problems encountered in the growing and cross-breeding of garden varieties of bearded iris is the inconsistency and reluctance with which the seeds germinate. After maturing, seeds normally undergo a period of dormancy during which they will not germinate. The period of dormancy for iris seed persists from several months to many years. In the mild southern climate of the United States some of the seeds harvested in August and planted by November will germinate in February or March. Farther north a month or two more are required.

The proportion of seed that germinates the first year is extremely variable and usually small. Seed that does not germinate the first spring may do so the next year, or the next, or in 5 to 15 years. First year germinations of new crosses have been as low as 10 per cent, although the average in my garden from 1938 to 1942 was 35 per cent.

Seed Dormancy

Breaking the dormancy of iris seed by artificial means is not easy. Conditions which cause other seeds to germinate have little or no effect upon iris seeds which, even after many years, will retain their ability to germinate (viability) but fail to do so under ordinary conditions.

Attempts to improve the germination of iris seed, aside from the embryo culture technique, have not been very successful. Seeds were chipped, chilled before planting, or planted immediately after harvest. Others were subjected to near-freezing temperatures for several weeks, the end of the seed chipped to remove the cap of tissue covering the root portion of the embryo, chipped and subjected to increased oxygen pressure, or soaked in running water. The average rate of germination after treatments never exceeded 64 per cent. A combination treatment of soaking chipped, chilled seeds in running water was the most effective treatment.

It has been demonstrated that iris seed dormancy is not caused by the dormant condition of the embryo itself, for embryos removed experimentally from the seed and cultured on a sterile nutrient medium began to grow almost immediately and soon became normal, healthy seedlings.*

* Apparently there is some growth inhibiting substance present in the stored food in the seed which retards growth of the embryo. Embryos did not germinate and grow well unless the food storage tissue was completely detached. As mentioned above, attempts at ending the seed dormancy, which were really attempts to destroy this inhibiting substance, did not succeed. RANDOLPH and COX, Proc. Amer. Soc. Hort. Science, 43: 299, 1943.

The story of embryo culture technique, as this procedure of removing embryos is called, began in Germany about 40 years ago. Today it is a standard procedure for obtaining seedlings that are difficult or impossible to germinate.

Embryo Culture Technique

The experiments of Hannig (1900), Dietrich and Laibach (1925), and Werkmeister (1936) are German landmarks in the development of this technique. In this country Tukey was the first to employ it on an extensive scale, in the production of sweet cherry seedlings, about 1932. The method is useful in the study of various kinds of botanical problems, as well as of value to the plant breeder and the analyzer of the causes of dormancy in seeds.

The embryo is removed from the seed with a dissecting needle which is sterile

Randolph and Cox described a procedure for culturing iris embryos in 1943. This has since been modified into a simpler and more reliable method, requiring a minimum of special apparatus or facilities. Germicidal solutions are used in connection with removing embryos from seed instead of special sterile chambers or a special transfer room. And a method of growing the seedling plants has now been perfected, which practically eliminates losses from diseases such as damping-off.

The embryo culture technique involves simply the transfer of the embryos from the seed to culture bottles containing sterile nutrient agar.*

*Agar is a clear jelly-like medium, used for culturing the embryos, and is prepared by heating seven grams of agar with one liter of distilled water (ordinary tap water may be used if it is not heavily laden with chemical purificants) until it is completely dispersed. To this is added 20 grams of sucrose (table sugar) and 5 milliliters of each of the stock solutions A and B. The amount of agar recommended is just sufficient to cause the culture solution, when cooled to room temperature, to form a solid medium having the consistency of soft gelatine. The hot mixture is distributed to culture bottles and after being sterilized and allowed to cool is ready for use.

A convenient type of culture bottle is approximately one ounce in size, wide-mouthed, screw capped, such as those manufactured by the Armstrong Cork Co., Lancaster, Pa. Black phenol formaldehyde plastic caps are the most satisfactory after first being boiled in a strong soap solution to prevent leaching of substances injurious to embryos during steam sterilization.

The bottles should be filled with the hot culture solution to a depth of approximately one-half inch. A liter of solution is sufficient for approximately 125 one-ounce bottles. The culture solution should not be allowed to come in contact with the neck of the bottle, if it does, contaminations may result. After the solution has been placed in the bottles they are capped and then sterilized in an autoclave or pressure cooker at fifteen pounds pressure for 20 minutes. The caps should not be screwed down tightly until the bottles have been sterilized and permitted to cool. After removal from the sterilizer, the bottles should be protected from air currents with a covering of paper to prevent excessive condensation of water on the inner surface of the bottles as they cool. They may be stored for several weeks or longer until ready for use if they are protected from dust and excessive evaporation of the agar medium.

At favorable temperatures the embryo soon begins to enlarge and within 2 or 3 weeks grows into a seedling with well developed roots and leaves. It is then transplanted to soil and grown to maturity in the usual manner. The essential steps are few in number and relatively easy to perform, although care must be exercised. The following directions are of interest to those who wish to utilize he embryo culture technique for the production of iris seedlings.

An excised embryo is transferred to a sterile culture bottle

The Work Room

Any well lighted room reasonably free from bacteria and mold spores is suitable for the transfer of the embryos from the seed to culture bottles. The possibility of contamination by air-borne spores may be reduced by spraying the prospective work room with a one per cent water solution of carbolic acid to which several drops of a wetting agent (such as turgitol) have been added for each pint of solution.

Preparation of Nutrient Culture Medium

This is a more or less technical laboratory problem, and is included in the footnote at the bottom of the page for those with the scientific urge, who will want to know.*

* It is convenient to combine most of the various ingredients of the culture medium n two concentrated stock solutions from which the desired quantity of nutrient solution may be prepared as needed.

Solution A
Calcium Nitrate — Ca(NO2) 2 = -4H2O 23.6 gms.
Potassium Nitrate — KNO2 8.5 gms.
Potassium Chloride — KCl 6.5 gms.
Distilled Water 500 ml.
Solution B
Ferrous Sulfate — FeSO47H2O 0.2 gms.
Calgon — (NaPO3)n 1.0 gms.
Magnesium Sulfate — MgSO47H2O 3.6 gms.
Distilled Water 500 ml.

The salts comprising solution A are readily soluble in water and form a solution that is stable at ordinary temperatures. Solution B is prepared by dissolving the ferrous sulfate and magnesium sulfate in 250 milliliters of distilled water. If either the Calgon or ferrous sulfate is added to an aqueous solution of the other salt an insoluble precipitate is formed. Solution B ordinarily is stable at room temperature but as a precaution against the formation of an insoluble precipitate of iron phosphate it should be stored in a refrigerator at near-freezing temperatures. Chemicals of chemically pure grade should be used in making up these solutions. With the exception of Calgon, which is the trade name of a sodium hexametaphosphate product manufactured by Calgon, Inc., Pittsburgh, Pa., they are readily obtainable from chemical supply houses. Calgon forms a soluble complex with both iron and calcium which remains in solution indefinitely and produces a clear nutrient medium ordinarily used for similar purposes. It eliminates the difficulty of retaining in solution the small amount of iron necessary for optimum growth.

Removing Embryo from the Seed

The surface of the dry seed should be sterilized for one to two hours in a concentrated water solution of calcium hypochlorite, to each pint of which a few drops of a wetting agent such as turgitol have been added. The commercial bleaching agent, Chlorox, diluted with approximately an equal volume of water, also may be used for sterilizing the seed.

Culture bottles contain a sterile ungerminated iris seed (left) and seedlings aged six to ten days,
developed from the embryos which have been excised

After they have been surface sterilized the seeds are soaked in sterile or boiled water, changing the water daily for 3 to 5 days to soften the food storage tissues of the seed and facilitate the removal of the embryo. Freshly harvested seeds removed from ripe pods before they begin to open do not need to be sterilized and ordinarily require no preliminary soaking in water. If the dry seeds are not surface sterilized, and are soaked in ordinary tap water, bacterial contamination is likely to occur. Iris embryos rarely survive more than a few days on culture media if contaminating bacteria are present.

The soaked seeds are then immersed in a solution of 50 per cent alcohol in preparation for the removal of the embryos. As the embryos are being removed the razor blade used in opening the seed and the fingers of the operator that come in contact with the seed should be dipped frequently in the alcohol solution, the germicidal properties of which are effective in reducing contamination. Immersion in 50 per cent alcohol for longer than three to four hours is injurious to the embryos of intact seed, and the alcohol should not be permitted to come in direct contact with the embryos for more than a few moments.

Removal of the embryo is achieved in the following manner. A longitudinal cut is made in the seed directly toward, but not quite to, the central region occupied by the embryo. In making the cut it should be started near the scar where the seed was attached to the pod, and extended backward and around the seed to the opposite side. The seed may then be separated into approximately equal halves, exposing the embryo, as indicated in the figure which shows the embryo being removed with a bent dissecting needle. Before removing the embryo from the seed, the needle is dipped in 95 per cent alcohol, flamed, with a single passage through the flame of an alcohol lamp, then dipped in a hexylresorcinol solution, S.T. 37 diluted with an equal volume of water. This antiseptic solution, prepared by Sharp and Dohme, Philadelphia, Pa., and commonly available at drug stores, is not harmful to the iris embryos and as an aid in handling them has proved to be an effective germicide. Also, the embryos adhere to the moist needle more readily than they do to a dry needle. The use of 95 per cent alcohol and flaming to sterilize the needle, in addition to the use of the hexylresorcinol solution, is chiefly precautionary.

In transferring the embryos to the culture bottle, it is advisable to hold the bottle in a horizontal position to prevent the entrance of air-borne germs while the cap is removed. After the embryos have been placed on the surface of the nutrient agar in the bottle the cap should be screwed down firmly, but not too tightly, as an exchange of air within the bottle is essential for the growth of the seedling. Ordinarily two seedlings are placed in each bottle. If one becomes diseased or a contamination develops on the surface of the agar the unaffected embryo may be transferred to a fresh culture bottle.

Growing the Embryo into a Seedling Plant

The embryos are cultured first in darkness at a temperature of 28 to 30 degrees Centigrade (82 to 86 degrees Fahrenheit) for 3 to 5 days, and then transferred to weak daylight or artificial light equivalent in intensity to the light from a north window, for an additional 3 to 5 days. Thereafter, the culture bottles containing the young seedlings should he transferred to the stronger light of a shaded greenhouse or cold frame from which direct sunlight is excluded and in which a temperature ranging from about 65 to 85 degrees F. is maintained. Within two or three weeks after the embryos are planted in the bottles the young seedlings ordinarily have well developed roots and at least two or three seedling leaves. They are then ready to he transplanted from the bottles to soil.

After the young seedlings have been transferred from the culture bottles to soil, either in flats or in seedling beds, they should he protected from sunlight and rapid evaporation for several days. A layer of sifted sphagnum peat approximately an inch in depth is distributed over the surface of the soil in which the seedlings are to be planted, to conserve moisture and inhibit the growth of disease-producing organisms. This is important, and neither soil nor fertilizer should be mixed with this surface layer of peat, or it may no longer protect the seedlings. After the seedlings have become established in the soil they may be handled in the same manner as ordinary seedlings that have been grown directly from seed.

Pot culture of iris seedlings from excised embryos. Seedlings grown in the greenhouse over winter from seed harvested
in August and September may bloom the following spring after being transplanted to the garden.

Iris seedlings produced from excised embryos make excellent growth under greenhouse conditions during the winter months at Ithaca, N.Y. In this latitude night temperatures of 60 to 65 degrees and day temperatures of 65 to 75 degrees F. are most favorable for normal seedling growth, and supplemental lighting is not required, either to increase the intensity of daylight illumination or to extend the photoperiod.

If greenhouse facilities are not available for growing the seedlings during the winter months, the seed may be stored over winter and the embryos cultured in the early spring when the seedlings may be grown in cold frames and then transplanted directly to the garden. The embryos of seed that has been in dry storage for a period of months or even years may be cultured as successfully as the embryos of freshly harvested seed. In fact vigorous, healthy seedlings have been obtained by culturing the embryos of seed that had been stored at room temperature for twelve years.

The procedure outlined above is the result of four years of experimentation and tests to determine optimum conditions for the growth of the excised embryos and young plants. By growing seed in field conditions and in the greenhouse the cycle from seed to flowering was reduced to less than a year, in contrast to the two or three years normally required to obtain bloom. Tests are now in progress to determine the conditions under which the seedlings should be  grown in order to obtain bloom from a  higher proportion of the plants the first year.

Enthusiastic hybridizers will not overlook the advantages of the embryo culture technique. Since it is possible to obtain successive generations of iris at yearly intervals, more rapid progress can be made in the development of new varieties. Of significance, too, is the fact that seedlings can be obtained from crosses that are difficult to make or that produce but few viable seeds or seeds that refuse to germinate.

Many important varieties of iris originated from such crosses: the famous variety Dominion, for example, and the ancestors of our modern tetraploid tall bearded iris. The true intermediate irises which are hybrids between early-blooming dwarfs and later-blooming tall bearded sorts, also the hybrid William Mohr and all of its first generation hybrids are relatively sterile and produce but few seeds. Reproduction of the: would have met with much less success under ordinary circumstances.

Recognition of the need for an effective method of breaking the dormancy of the seed and obtaining bloom from seedlings in a shorter period of time than normal resulted in these experiments on embryo culture. It was especially desirable to obtain a higher per cent of germination from crosses that were| difficult to make or that produced few viable seed. Undoubtedly, future experience will suggest various modifications, but in its present form the embryo culture technique is a practical, reliable method of speeding seedling production in iris, and removing the uncertainty and delay caused by the prolonged dormancy of the seed.

Hetero-Fertilization / Endosperm Failure