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How the steps are done

Each step on a protocol card names a technique. This page says how to carry it out, in N. C. Deno’s own words from Seed Germination Theory and Practice (2nd edition, 1993), with the page each passage is on.

Find a step

Deno does not describe these, so this page says nothing about them: Separate the seed from its case; Smoke treatment; Sow and cover. Each card’s “Show the original text” has what its source said.

Reading a card

Verified and Derived say how a card was checked against its source; the About page explains both. Temperatures are in °F unless you switch units. Almost every protocol from Deno uses just two temperatures, and three months at each. He says why:

Temperature is a major variable in seed germination. Virtually all of the experiments were conducted at either 40 or 70 deg. F. These two temperatures were chosen first for convenience. The temperature of 70 is the temperature at which most rooms are kept, and 40 is the temperature at which refrigerators are generally set. These two temperatures also roughly correspond to the temperatures of the winter and summer seasons.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 9

Nearly all the work was conducted using periods of three months at one temperature followed by a shift to the other temperature. This led to a series of alternating cycles each of three months duration.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 10

Moist stratification: the paper-towel method

Cold and warm moist stratification are time spent moist at 40 °F4 °C (a refrigerator) or 70 °F21 °C (room temperature). Deno did it in folded paper towels inside thin sandwich bags:

A perforated section of paper towel is torn off and folded in half three times in alternating directions to give a rectangular pad 2.5 x 4.5 inches. The name of the species and any other information is written on the outside of the pad with the Pilot marker. The final (third) fold is opened, and the towel is moistened with water. The seeds are sprinkled on the moist open pad. The third fold is closed and the whole placed in a Baggie. Fold the Baggie several times so that evaporation of water from the towel is inhibited, yet leaving ample access to air to insure aerobic conditions.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 13

It is important to use high wet strength paper towels, and the plain white type are preferred. Also a special warning against Ziploc bags and other polyethylene bags that are of thicker polyethylene film and are constructed to seal tightly.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 14

The paper towels remain moist for periods of months and only need to be remoistened at periods of two months or longer. The towels are inspected and the germinated seeds counted at least once every seven days and as often as each day if germination is occurring at a rapid rate. If there is debris or other material that starts to rot, the seeds are transferred to new moist towel. Dead seeds rot in month or two and are removed. It was usually necessary to transfer ungerminated seeds to a new towel about every three months as even the ScotTowels ultimately disintegrate.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 14

Light

A card that says to sow on the surface means the seed needs light. Deno’s light experiments:

The photoexperiments were started in the same way as the basic procedure except that the seeds were placed on top of the 2.5 x 4.5 inch pad of moist paper towel. Each moist pad was placed in its own Baggie, and the Baggie was folded several times as in the basic procedure. These were placed at a distance of eight inches under two fluorescent lights. These lights were daylight type, four foot long, and forty watts each. The lights were timed to provide alternating twelve hours of light and twelve hours of dark.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 14

Outdoors, and alternating temperatures

The outdoor conditions simply consisted of placing the polyethylene bags containing the towels and seeds outdoors in a shed. The only important precaution is to prevent any direct sunlight from striking the polyethylene bags as that would lead to overheating.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 14

Oscillating temperatures were required for germination by some species, and this behavior was totally unexpected (Chapters 10 and 19). It was found that some species would germinate under outdoor conditions, but not under the alternating three month cycles at 40 and 70.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 11

Gibberellic acid (GA-3)

After opening the third fold and moistening the towel, two inserts were made before sowing the seeds. The first was a 3 x 3 inch piece of polyethylene cut from a Baggie. The second was a small piece (2.5 x 2.5 inches) of ScotTowel folded three times to give a rectangular pad 0.5 x 1.0 inches. This is moistened and placed on top of the polyethylene. The seeds are placed on top of this inner pad and one cubic millimeter of 95% GA-3 is sprinkled on top of the seeds. This amount of GA-3 is about the amount that can be balanced on the 0.5 mm tip of a toothpick using a type of toothpick that is pointed at both ends. The whole is folded once and placed inside a Baggie as in the basic procedure.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 15

The size of the inner pad is such that it takes about six drops of water to moisten it and with the one cubic millimeter of GA-3 this works out to be a concentration of about 1000 ppm.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 15

Should treatment with gibberelic acid-3 (GA-3) be one of my procedures? The answer is an absolute yes, however, it is a method of last resort since there is often the problem that the seedlings are weak and etiolated and have difficulty surviving.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 60

Seeds in fruits and berries

Seeds in fruits and berries generally contain germination inhibitors in the pulp and these seeds were always washed and cleaned. The abbreviation WC is used to indicate that the seeds were washed and cleaned. Some fruits such as those of Euonymus contain inhibitors that are oil soluble rather than the usual water soluble, and such seeds must be washed with detergents.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 16

Hard seed coats

Many treatments have been recommended for such seed, but the only treatment that gives reliable and reproducible (and usually dramatic) effects is to physically produce a hole in the seed coat.

Such holes in the seed coats were made in several ways. Large seeds could be rubbed against a file or sandpaper until the endosperm showed. It is advisable to make this hole away from the embryo to avoid physical injury. With bean shaped seeds of Fabaceae, this means making the hole on the convex edge of the seed.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 16

Soaking in sulfuric acid has often been recommended for seeds with impervious seed coats, but I advise against it. First of all the temperatures, concentrations of sulfuric acid, and times of exposure all have to be worked out for each separate species. More important sulfuric acid is dangerous to use.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 16

Soaking

Soaking seeds in water has often been recommended. Of course seeds in fruits usually need to have the inhibitors removed by washing (Chapter 9), but that is something different than proposing that soaking alone is favorable. … In general there was no advantage in the longer periods of soaking. It is concluded that the supposed favorable effects of soaking are largely a myth.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 12

Dry storage

Some seeds require a period of dry storage to destroy inhibitors, and these are termed D-70 germinators (Chapter 5). … With these D-70 germinators there are opposing processes in dry storage. One is destroying inhibitors and favoring germination and the other is the dieing in dry storage which is unfavorable to germination. There will be an optimum time or range of times for the dry storage.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 10

After germination: growing the seedlings on

From Deno’s chapter on growing plants from seeds.

Sowing in paper towels is the method of choice for species with multistep and extended germinations. … The seedlings are treated like small plants and can either be planted directly outdoors or in pots. … The paper towels should be placed upright so that the root extends vertically along the face of the towel.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 56

For the seedlings of many species, growing the seedling in pots enclosed in a polyethylene bag is virtually a necessity. I call this technique bagging. … The Baggie is closed at the top with a wire Twistem, but not so tightly as to totally seal the bag since aerobic conditions must be maintained. … Before sowing the seeds, it is important to surface sterilize (ref. 37) the medium by pouring boiling water over the medium three times allowing to drain each time. Allow the pot and medium to cool, sow the seeds on the surface, and enclose the pot and contents in the Baggie.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 56–57

Pots enclosed in a Baggie must always be kept under fluorescent lights and never exposed to direct sunlight. … Suffice to say that direct sunlight will heat up the Baggies and cook the seedlings even in time periods as short as thirty minutes.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 57

The medium used most frequently here is composed of equal parts peat moss, leaf mold, and Perlite.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 59

Sowing in open ground is convenient for other species where the seedlings need cool conditions for growth. Examples are Actaei, Asarum, Chionodoxa, Eranthis, Hepatica, Puschkinia, Trillium, and Tulipa.

N. C. Deno, Seed Germination Theory and Practice, 2nd ed. (1993), p. 55

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