E. MAGINI
E. MAGINI is Professor at the Istituto di Selvicoltura Florence, Italy. Part I by R. MORANDINI was published in Unasylva, Volume 16, Number 4.
II. Seed treatments, storage, testing and transport
INSECTS and fungi may destroy seed in storage and therefore biotic enemies must be controlled as effectively as possible. As a rule, indirect control, by maintaining, during storage, temperature and humidity conditions that may inhibit mold growth and insect activity, gives satisfactory results when seed is extracted in kilns and kept in disinfected air-tight containers.
For seed which does not pass through the kiln or is not stored in sealed containers, seed treatment by chemicals may occasionally be necessary, even if the storage method adopted is the most suitable, in order to reduce the injury due to fungal or insect attack.
In any case the receptacles in which seed is to be stored must be cleaned before use and treated with strong fungicides and insecticides which should be removed or allowed to evaporate freely after producing their effect. Formaldehyde solutions are often used as fungicides while by far the commonest insecticide is carbon bisulphide. The storage room must be kept very clean, and it is advisable to apply fumigants at the end of each storage season.
A number of chemicals are in use. Some of them injure seeds, particularly if they are not sufficiently dry. Such chemicals should be applied only to receptacles or storage rooms. Others do not affect seed germination, provided that concentration and methods recommended by the manufacturers are followed. These chemicals may be used to treat seed as well as containers.
Formaldehyde was one of the first chemicals used rather generally for disinfection purposes. The resulting adequate control of disease has never been questioned, but there is evidence that it impairs the quality of the seed treated.
Some organic compounds containing mercury (such as ceresan, semesan, uspulun) have been in use for some time with varying results. There are now available new organic compounds which seem to be free from toxic effects. Insecticides such as carbon bisulphide, methyl bromide, paradiclorobenzene are recommended for forest tree seeds by Baldwin (1955).
Generally speaking, it seems that insect infestation of a number of seeds can be eliminated by several different chemicals, considerably within the limits of safety for seed themselves, while the margin of safety for chemical treatment for fungal control is not so great. However, before carrying out large-scale seed treatment with a specific chemical, it is necessary to know the results of storage experiments or practices for the species concerned.
Some fumigants are poisonous and a gas mask is indispensable. Several types of sprayers are available, consisting of a tank with a capacity up to 5 gallons or more, a pump and a discharge device.
Fog sprayers based on the principle are now on the market, with a capacity of 2 liters (2 quarts) or more. The tiny particles generated by aerosol bombs, which discharge at the opening of a valve, have the highest penetration power. This type of sprayer is therefore recommended.
The aim of adequate storage is to keep the seed maintaining the highest germinative capacity as long as possible. Seed storage for a shorter or longer period is frequently a practical necessity connected with programs of artificial regeneration and afforestation.
The ideal would be to sow fresh seed immediately after extraction and cleaning but in practice this is not always possible or convenient. To reduce the dangers from rodents or to increase germination and survival, sowing must be done at the most favorable season. In temperate regions seeds are often extracted in the autumn and kept until the following spring. On the other hand, many major forest species produce seed crops only irregularly, for example, Pinus taeda, Pinus sylvestris, and Picea abies, which have a good crop every three to five years or more. For these species the seed collected in good years must be stored sometimes for several years in order to have a supply through the years of little or no crop.
Knowledge of storing methods will facilitate the building up of seed stocks in good seed years, when seed cost is low and seed quality is high. Seed storage may be of varying duration. It generally tends to increase with the length of the interval between seed years, but it is strongly dependent on the storage characteristics of the species concerned and with the seed cost.
For some species storage is a simple matter (for example, for many seeds of leguminous trees and pines); for others, storage is difficult or very difficult, such as for willows, poplars, elms, okoumé which lose their germinative capacity completely in one to two months, when stored in ordinary conditions. As a rule, there is no economic advantage in storing large, sensitive, and inexpensive seeds of some hardwoods for a period exceeding three to four months, while it may be convenient to keep the small and valuable seeds of many conifers for more than two or three years. In particular, cold storage, requiring expensive equipment and installation should be limited to seeds with a moisture content below 10 to 12 percent of their weight, provided that their behavior is known, and should never be used for seeds which are inexpensive and easy to obtain.
However, it should be kept in mind that fully ripened seeds have a greater chance of remaining viable for long periods, and that seed of high initial viability is more resistant in storage. In practice, therefore, only good quality seed should be stored for long periods.
Generally, seeds keep best at low moisture content and low temperatures, but there are many exceptions to this rule. Some seeds keep satisfactorily at ordinary temperatures, and some are injured if their moisture content is slightly reduced. In considering the problems of adequate storage it should be clearly borne in mind what happens to the seeds after ripening, and what causes their loss of germination capacity. Several factors affect the longevity and keeping qualities of seeds.
Longevity is determined by morphological and physiological features of the seed, as well as by seed processing methods and storage conditions. An excellent review of storage problems and methods for the seeds of temperate forest tree species was made by Holmes and Buszewicz (1958).
The major dangers involved in seed storage and largely responsible for deterioration in the keeping qualities of seeds may be classified in the following two categories:
(a) damage by outside agents: pests and disease,
(b) rate of destructive metabolism, i.e., rate of respiration.
The mold growth is greatly reduced when temperature or relative humidity is low. Mold activity is almost completely inhibited at seed moisture contents of 6 to 10 percent and temperatures near 0°C. Fungicides, such as formalin or organomercury compounds, have often been advocated but they cannot be generally recommended as they can be harmful to seeds, causing abnormal germination. As a rule, good regulation of temperature and humidity during storing is the more suitable means of control.
Most of the insects damage only those seeds which were infested before storing. Fumigation and other control methods are however suitable in some cases, above all for the species whose seeds are subject to infestation by weevils (Carya, Castanea, Quercus, Leguminosae) and whose extraction does not require high temperatures. For the seeds which are extracted in the sun or in kilns at temperatures above 40 to 42°C, fumigation is generally unnecessary, as nearly all insects are killed at these temperatures.
Respiration causes consumption of reserve material, energy release and production of CO2 and water through oxidation of carbohydrates and fats. Continuing respiration may result in a complete exhaustion of reserve materials. On the other hand, in default of atmospheric oxygen, anaerobic respiration may occur. At ordinary temperatures it can cause the complete deterioration of some large seeds of hardwoods.
The respiration rate decreases with decreased moisture content and temperature, so that generally the life span of the seed is largely dependent on temperature and humidity. In artificial storage, respiration may be considerably reduced by keeping both temperature and humidity at a low level.
If either is too high, reduction of the oxygen supply may favor retention of germinative capacity. Air-sealed storage is beneficial also from this point of view. More effective in reducing respiration rate can be:
(a) storage under vacuum or reduced air pressure, which is difficult and expensive in practice,
(b) storage in an atmosphere where oxygen has been substituted by other gases, such as CO2, or N.
PRELIMINARY OPERATIONS (SEED DRYING)
For seeds which must be stored at low moisture content, it is necessary to adjust the seed moisture, especially when sealed storage is chosen. For many seeds the suitable moisture content, at the beginning of the storage period, is less than 10-12 percent and it is usually obtained in the kilns where cones are opened.
Some species that do not reach the required level or do not go through the kiln (for example, okoumé Abies) should be pre-dried. Abies cones do not go through the kilns because the cone opens by itself. The seed therefore usually has a higher moisture content to begin with.
Seeds can be dried in several ways. The commonest are those of drying the seed in the sun or in a warm room, or in an extraction kiln. The first gives good results only when and where the weather conditions are favorable, otherwise it often fails. The second method takes a great deal of space and time and is not always successful. The last method is generally preferable particularly when there is a large amount of seed to be dried.
Forced draft kilns are more suitable for this purpose, as they allow accurate and safe drying of the seed, which is exposed to a large volume of air at a rather low temperature. High temperatures are particularly dangerous for seeds of high moisture content (Barton, 1935). Generally, drying temperatures should not exceed 40°C. The present trend is toward lower temperatures and greater air-flows, in order to ensure safety in drying (Holmes and Buszewicz 1958).
Chemical drying, using desiccating agents such as CaO, silica gel, sulphuric acid or anhydrous Ca Cl2, may be usefully applied for small lots of valuable seed.
DRY STORAGE METHODS
The optimum condition for storing all seeds that withstand considerable desiccation, is, probably, adequate drying followed by sealed storage in the absence of O., and at low temperature (Crocker 1938). Never-theless, these conditions may be unnecessary or impossible in practice.
Drying without temperature and humidity control
This is the simplest and oldest method. The seeds are usually stored in heaps, or in single layers, in sacks or boxes. The seeds must be protected from rodents and stored in cool and ventilated cellars or in special storage buildings (storing sheds) in which the temperature, though not controlled, shows a gradual rise and fall with the season. Seeds of many species, including some Leguminosae, some pines and eucalypts, may be kept satisfactorily for at least six months in this type of storage, provided the air is sufficiently dry. Picea excelsa and Larix decidua seeds may also be kept in jute sacks left standing or in single layers. Big stacks should be avoided. For good storage, seed usually needs to be frequently stirred.
Although this method in certain circumstances can give fairly good results, it is not sufficiently safe and cannot be recommended either for valuable seeds or for long-term storage. Some exceptions are several leguminous species (Acacia and Robinia in particular) which can normally be stored in this way for at least 10 years.
If the air humidity is high, seed may decay rapidly. Seed storage in cool, damp cellars, in open containers, as commonly practiced by many nurserymen in the past, is the worst possible type of storage.
Dry storage in sealed containers
Sealing is the cheapest way to ensure constant moisture conditions. If, when placed in the containers, the seed has a low moisture content, the initial moisture remains unchanged during the storage period.
The containers are usually kept in unheated rooms or cellars. To reduce seed moisture, some desiccating agents may be used (H2SO4, CaCl2,). Dehumidification is obtained to a certain degree by the use of silica gel (for instance with okoumé in Africa). This method gives good results with several species of the following genera: Betula, Ulmus, Picea, Pinus, Pseudotsuga, and for Gleditsia and Caragana. It is not to be recommended for the Southern Pine group of species nor for the five-needled pines.
The initial seed moisture content largely determines the success of storage. It must be low, but excessive drying may be harmful. At uncontrolled temperatures the seed is more sensitive to moisture content than at constant low temperatures.
As a rule for a period of storage exceeding one year the following method is more suitable.
Dry, sealed, cold storage
Conditions of controlled temperature and controlled moisture are best for many species over a long period. The temperature may be around freezing point or just below. In practice, temperatures in the range from 0° to 5°C are considered adequate for most purposes, but for some species long-term storage requires temperatures below freezing point. It is usually very costly to maintain temperature below - 15°C and there is also the risk of a sudden increase of the temperature as a consequence of an accident in the freezing units.
A great number of species may be kept for long periods at temperatures of 3° to 5°C, such as Abies spp. (for Abies alba a temperature below freezing point is advisable), Acer, Cedrus, Fraxinus, Larix, Picea, Sorbus, Pinus, and Pseudotsuga. This is probably the best method also for the expensive seeds of many eucalypts.
Temperatures below freezing are chiefly used for seeds which quickly lose their viability under ordinary conditions, such as those of willows, poplars and elms, or when storage is for special purposes. The control of moisture content is very important both for storage at temperatures below or above freezing point.
Baldwin (1955) reported the following examples of recommended moisture contents for cold storage: Pinus, 7 to 9 percent; Abies, 11 percent; Picea, 6 to 7 percent; Ulmus, 3 to 7 percent; Thuja, 8 percent; Betula, 1 to 5 percent; Eucalyptus, 7 to 9 percent.
Good results have been obtained for Abies alba with a moisture content of 9 to 10 percent; for Fraxinus of 7 to 10 percent; for Chamaecyparis obtusa and Cryptomeria japonica of 4 to 8 percent. For Pseudotsuga the seed moisture content should not exceed about 7 percent.
At Nancy, this method (temperature of 2° to 4°C; seed moisture content between 9 and 11 percent) has been recently applied with success to Abies cephalonica, A. nordmanniana, A. grandis, Larix europaea, Picea excelsa, Pinus laricio (var. calabrica, corsicana and pallasiana), P. pinaster, P. sylvestris and Pseudotsuga douglasii (Bouvarel P. and Lemoine M. 1958). It was observed that it is preferable to place the seed in hermetically sealed containers as soon as possible after seed extraction and that the seed should be used as soon as possible after removing the containers from the cold room or chamber.
Okoumé seeds have been kept fairly well for more than 10 months with a moisture content of 12 to 13 percent, at a temperature of 2° to 5°C (Gauchette 1958).
To avoid any seed deterioration caused by condensation of moisture on the cold seeds, the seeds stored in sealed containers should be brought to room temperature before being opened.
MOIST STORAGE METHODS
Several seeds do not tolerate drying under ordinary conditions, and must generally be stored with a high moisture content. On the other hand, many seeds which can be stored dry may benefit from a cold moist storage to promote after ripening and hasten germination.
Storage on or in the ground
Seeds are stored, generally, mixed or stratified with moist sand, peat or other porous materials, in heaps on the ground, in shallow pits in well-drained soils, or in layers in ventilated sheds.
The choice of the technique depends on local circumstances as well as on the requirements of the seed concerned, but the aim is the same in all cases: the maintenance of constantly moist and cool conditions, combined with a good aeration to avoid overheating. Heaps must be covered with litter and with a layer of sand or soil. The necessary aeration may be insured by bundles of straw or twigs inserted into the heaps.
Below-ground stratification is made in rodent-proof pits. Sometimes seeds are enclosed in screened containers.
The shed designed by Allemann (Vincent G. 1955) provides sufficient ventilation and defence from rodents and ensures at the same time almost uniform temperatures and facilities for inspection and control of seed moisture content. The seeds are stored in layers of 15 to 30 centimeters. Allemann's sheds are well known and widely used in Europe.
The foregoing methods of moist storage are suitable only through the winter months in cool or cold localities, the main dangers being mold and pregermination, although the latter does not always amount to actual damage.
All these methods are employed with variable results almost exclusively for the large-seeded hardwoods, such as Aesculus, Castanea, Carya, Fagus, Juglans and Quercus.
Moist, cold storage
This method implies controlled low temperatures, just above freezing or, less commonly, below freezing.
Moisture is generally maintained at the required level by adding a moist media (sand or peat) to the seed, or (more rarely), by controlling the relative humidity of the air in the storage chamber. The latter type of control is, in principle, preferable, but often too expensive.
Best results have been obtained with some large seeded species, such as Quercus robur, Q. petraea, Fagus sylvatica and Araucaria excelsa.
Subfreezing temperatures may injure seeds, if their moisture content is too high. A uniform temperature of 2° to 4°C is generally recommended. This method can be applied to hasten germination of a great number of species.
Storage in running water
Seeds are kept in moving and well-aerated water. They are often enclosed in screened containers which if necessary, can be anchored or loaded to prevent shifting.
This primitive method is occasionally used in several countries, but it cannot be generally recommended. It may be useful for keeping large seeds of hardwoods during winter.
OTHER METHODS
Although the dry and moist storage methods mentioned above are the commonest, other methods are occasionally employed, especially for small lots.
A method which should be noted although used exclusively for large seeds to prevent loss of moisture is that of dipping the seed in paraffin, or spraying it with latex spray, and then packing in a soft medium. The paraffin technique is ineffective for okoumé seeds kept at room temperature, but it has proved successful with other species, for example, Araucaria imbricata.
FIGURE 17. - The conditioning unit
A. 15-inch fan
B. 1,000 watt finned heater
C. Trough
D. Overflow
E. 1,000 watt strip type heater
F. Fan motor
G. ½-inch inset fitted with ballcock to maintain water level
FIGURE 18. - Seed containers: on the left is a suitable type for both keeping and chipping
Gas exchange is reduced by keeping the seed in airtight containers where air has been replaced by nitrogen gas, inexpensive and easy to obtain in metal bottles from liquid air plants. Research along these lines is being done in Cameroon with several seeds of tropical species.
Regular seed silos could be used for the dry storage of some species. Adequate air circulation is assured by using containers with sulphuric acid or lime which must be frequently renewed.
It is likely that seeds can be stored in dry conditions and at a constant equilibrium moisture by some conditioning units, without any effort to control the temperature.
A simple conditioning unit has been devised in Australia to maintain timber at a definite constant moisture level (Figure 17). The principle involved might be applied to dry storage of seeds.
A room 6.4 × 2.3 and 2.75 meters high (21 × 7.5 × 9 feet) can be air-conditioned by two units. Air circulation is provided by a 38-centimeter (15-inch) propeller, and ¼ hp single-phase electric motor placed in each conditioning unit; heating of the air is done in this lay-out by 1 kilowatt finned electric heater. Humidification is supplied by a 21 × 46 centimeter (8 × 18 inch) water trough in the bottom of the unit, kept at constant level and with a 1 kilowatt immersion heater to make the water evaporate. A humidistat keeps the moisture content constant by raising air temperature if moisture rises or, if moisture drops, adjusting it by heating water and thus humidifying the air.
EFFECTIVENESS OF COED STORAGE
Some significant examples are given in forestry literature of the effectiveness of cold storage. The very sensitive seeds of Ulmus americana and Populus sieboldii have been stored for several years below freezing point. Ulmus americana, sealed at - 4°C with a moisture content of 2 to 3 percent, maintained viability for 15 years, while Populus sieboldii (moisture content 6 percent, temperature - 15°C) kept well over a desiccating agent for 5 years. Sensitive seed of Abies balsamea sealed at 1° to 3.5°C with a moisture content of 8 percent kept its viability for 5 years.
Some more easily stored seeds such as those of Pinus sylvestris, P. ponderosa, and P. radiata, at temperatures above freezing (4° to 6°C), maintained their viability for periods of from 10 to 21 years, when closed in airtight containers. Generally speaking, cold storage methods are the more effective ones.
STORING UNFAMILIAR SEEDS
Some clues may be provided by natural methods of storage, but they do not give a sufficient basis for successful storage practices. In fact, many species require strictly specific moisture or temperature conditions during storage. The importance of carrying out exploratory tests must be emphasized, since they are the only way of discovering the best methods of large-scale storage.
SEED CONTAINERS
Containers may vary in shape, construction and type, depending on whether they are to be used for shipping or only for storage. A type suitable for both purposes is that made of 26-gauge steel sheet electrolitically coated with zinc, with a 7.5-centimeter (3-inch) neck and a screw tap (Figure 18). Cans for storage only are made of tin plate lacquered inside, soldered throughout. For small amounts of seed glass jars are frequently used, while for research purposes plastic receptacles are often employed. In the case of most seeds, the containers must be airtight.
Containers made of galvanized steel sheet usually have a capacity ranging from 30 to 120 liters (0.85 to 1.4 bushels). The use of cylindrical rather than square or rectangular containers entails waste of about 20 percent of the storage space but the gaps left between the containers lead to better air circulation and consequently a more uniform storage temperature.
CABINETS AND CHAMBERS FOR COED STORAGE
When seeds are stored in sealed containers, the control of air humidity inside the cabinet or room is not important provided that the containers in cold storage are kept sealed.
When, however, the seed is not kept in airtight containers, the air humidity should be controlled. This implies a constant temperature level, humidifying and dehumidifying equipment controlled by a humidistat or a wet and dry bulb controller. While humidifying may be accomplished relatively simply and cheaply, expensive equipment is necessary for dehumidifying.
In moist cold storage this expensive equipment can generally be avoided by using special devices. In dry cold storage it is not necessary if the seed is kept airtight.
Some simple conditioning units, devised to bring timber to a definite constant moisture content, do not seem suitable for seed cold storage, as the temperature cannot be maintained at a constant level.
Whenever seed amounts are small and sealed containers are adopted, normal refrigerators may be safely used, especially if a fan is added to make the inside temperature as uniform as possible on condition that they can provide a good temperature control.
Cabinets are usually made for a capacity from 120 to 140 liters (4 to 6 cubic feet) up to 1,200 liters (42 cubic feet). The biggest ones are generally for seed laboratory purposes, as they can be used both for storage and for prechilling. There are several types of cabinet on the market. Some, for example combined climatic and low temperature cabinets, provide a good control both of temperature and air humidity, but they are very expensive.
Cabinets are made for different ranges of temperatures. The range for humidity is often only from 40 percent to 95 percent ± 3 percent; when the humidity is under control the cabinets can generally operate at a temperature as low as 2°C (35°F) or 5°C (41°F), but not at temperatures below freezing. The commonest types are without humidity control and can insure temperatures as low as about 2°C (35°F); others, temperatures as low as - 40°C a (- 40°F) or nearly - 84°C (- 120°F).
Some cabinets can supply cold temperatures, under or above freezing, as well as temperatures up to about 50°C (120°F) with a plus or minus 0.5°F tolerance, and may serve for different uses.
It is possible to have two temperatures on alternating cycles using time switches, an arrangement which may be very useful for research on cold storage and seed stratification.
Many cabinets have double doors of which at least the internal pair is of glass, thus permitting observation of the seeds in the cabinet without changing conditions within the compartment. Special models are constructed with double sheets of glass on top, so that ultraviolet light can be used, if needed, for special study. An interesting model operating with 1 hp Freon 12 compressors, and a 750 watt air heater is used by the Forestry Division in Melbourne (Figure 19).
Moist cold storage can be obtained, without air humidity control, by mixing seeds with moist media and placing the mixture first in open containers and then inside the cabinet. Ample drainage must be provided below to take off any excessive water that may be added during storage.
For large quantities of seed, as in seed extractory plants, refrigerated chambers or rooms are needed.
Where large quantities are concerned, air humidity conditioning is avoided as far as possible, for economic reasons, by adopting air-tight storage for seeds which tolerate drying and special devices for those which do not.
Large-scale cold moist storage is often done by interspersing layers of seed with moist media in wooden barrels or metal drums, the bottom of which contain several holes for drainage.
However, the simultaneous control both of temperature and humidity could be obtained by expensive air-conditioning installations.
As a rule large-scale seed storage is done in rooms where only the temperature is under strict control.
The Forestry Commission of the United Kingdom has a 20,000-kilogram (45,000-pound) storage area, with three rooms of 6.6 × 3.3 × 3 meters (22 × 11 × 10 feet); all insulated by a 10-centimeter (4-inch) cork board. Two rooms are kept at 2°C (35.6°F), and one at - 5°C (23°F). The French forest experiment station has three cold rooms which are kept at 2° to 4°C (35.60 to 39.2°F).
In Japan, cold storage or prechilling rooms are made in a two-storied 5.5 × 5.5 meters (18 × 18 feet) chamber where, in the upper story, ice and snow are packed in March, and covered with a 30-centimeter (1-foot) layer of sawdust. In this way temperature stays at 1° to 2°C. The stove is used for Abies marriana. This simple method may give good results in relatively cold localities, but does not ensure a constant temperature throughout the year. Therefore even where it is practicable it can be recommended only for seed stimulation during one to two months.
The capacity of the cold chamber depends on the amount of seed to be stored and on the number of species or lots; it is also related to the shape of receptacles and to the method of storage, particularly whether it is dry or moist. At Nancy, France, it has been found that when handling many lots the chamber should not be larger than 24 to 40 cubic meters, in which case it should be limited to two or three species. In the smaller type a capacity of 60 kilograms per cubic meter of seed with a specific weight of 0.4 is estimated.
FIGURE 19. - Cabinets for cold storage manufactured in Australia
These figures refer to seed kept in sealed containers. In general it is better not to have a room higher than 2 to 3 meters with good insulation. In the temperate zone of the northern hemisphere the chamber should be built with thick walls and a northern exposure. Automatic defrosting avoids interrupting storage to defrost.
The compressor power required depends on the difference between inside and outside temperatures, on the specific heat of seeds (usually about 0.5), and on the frequency and volume moved in and out. At Nancy, for a movement of 200 kilograms per day and differences of temperature of 20° to 23°C, the power required is estimated to be 50 FH per cubic meter where the refrigeration is expressed in FH, that is, in negative calories or frigories (= F) per hour (= H).
Seed in a chamber should not be stockpiled, but kept in containers piled on shelves 10 centimeters above the floor, away from the walls. Where there is sufficient room cylindrical types of container are preferable. Air circulation within the room should be provided by a fan to obtain a uniform temperature.
LABELING
Before storage each lot of seed should be properly labeled and numbered to avoid mixing seed of different origins. Each time seed is removed note should be taken of its use, destination and person responsible for its removal. Proper account and registration of movement of seed should be made by means of duplicate slips, kept in some centralized control station. Packaging and labeling for shipment has been thoroughly described by Baldwin (1955).
Seed origin and quality certificates should be as those recommended by FAO 1951 Conference (see also FAO forest tree seed directory 1961).
FIGURE 20. - Cylinder for seed scarification
Several species are prone to delayed germination even when environmental conditions are suitable. Such seeds require special treatment to hasten germination, either for testing purposes or in connection with sowing in the nursery or in the field. An interesting review of the problem involved can be found in Baldwin (1942), and in Crocker and Barton (1953).
The main causes of seed dormancy have been identified as:
(a) a seed coat impermeable to water and oxygen;
(b) a dormant embryo.
In some species there is a double dormancy caused by the combination of hard coat and internal dormancy
Pretreatment for hard-coated seeds
Scarification
The aim is to thin down or abrade the coat of the seed so as to permit water absorption and gaseous exchange, and so cause seed germination. Scarification may be done by hand, particularly for laboratory purposes, or by use of special mechanical devices. Several types of these have already been described. One type blows the seed against an abrasive surface, usually concave; another, used for Juniperus virginiana, consists of a metal drum, lined inside with sand paper; it is half filled with seed and revolved at 20 rpm.
The United States Forest Service has developed a model, which is a galvanized iron (28-gauge) cylinder, made in two halves, to permit loading and unloading, lined with No. 2 ½ Grade 30 E silicon carbide sand paper, glued with casein. In the bottom half a strip of wire 16 mesh is soldered, to allow dust to filter through. The sides of the cylinder can be of 1 inch wood. Disks on a 3/4 inch steel shaft rotate inside; the discs are of l/4 inch wood, also lined with sand paper, although solid carborundum grinding wheels might be used instead. Disks are 4 inches apart, and with a ½ inch clearance from the sides of the cylinder. They rotate at a speed of 500 to 900 rpm, loading half of the capacity of the cylinder with seed, which in this way can be satisfactorily treated in few minutes, depending on the seed, which should be tested in any case. A 60 centimeter (24-inch) 28-centimeter (11-inch) diameter cylinder can handle loads of 4.5 kilograms (10 pounds) (Figure 20; see also F. 327823 in the Woody-plant seed manual (United States Department of Agriculture, Forest Service, 1948).
Scarification is successful with many seeds of Leguminosae species, such as those of Cercis, Colutea, Gleditsia, and Robinia. Other seeds may also benefit from scarification (for example, Crataegus species).
Other treatments
These usually take the form of acid treatment or soaking in water. Acid treatment is normally carried out with concentrated sulphuric acid and is applied to several Leguminosae species. The results are not always good, especially when treatment is on a large scale. Sulphuric acid should be handled with the greatest care and treated seed must be thoroughly drained and washed. Best results are obtained if exploratory tests are first made on the seed lot to determine the optimum period of immersion.
For large-scale treatment the equipment required consists of suitable acid containers (wood or earthenware preferred), and wire containers and screens for handling, draining and washing the seeds. The best technique is described in detail in the Woody-plant seed manual.
Soaking in water at different temperatures and for different lengths of time can be more or less effective with several seeds. Hot water treatment is frequently recommended for seeds of legumes, but it is advisable to make preliminary tests to avoid damaging the seed. No special material or equipment is needed.
Large-scale treatment with a 2 percent solution of caustic soda from a few minutes to some hours, according to species, was found successful with some seeds of legumes (Soster 1955). This treatment, however, requires many precautions, as caustic soda may endanger the operators.
Chipping off a small portion (l square millimeter) of the testa at the cotyledon end, and then soaking for three to six hours has proved the safest method to induce a quick germination in several kinds of legume seed, for seed testing purposes. With Quercus spp. the same result may be obtained by soaking the seed for 48 hours, and successively by cutting off one third at the scar end of the seed and removing testa.
PRETREATMENT FOB OVERCOMING INTERNAL DORMANCY
Although some attempts have been made to break dormancy by means of chemicals, the large-scale treatment most widely used is cold stratification. A few species are treated by warm followed by cold stratification. Cold stratification is not needed for all species, and its usefulness for a particular seed lot can only be established by comparative tests. Its use, and the results obtained, depend to a large extent on the personal experience of the nurseryman and on the equipment at his disposal.
Recommended periods of treatment differ widely with the species but, in large-scale practice, the best length of time is generally from 30 to 45 days for pines and several other species. A lengthening of the period may result in seed heating, even if seed is frequently inspected and repacked. Anyway, a few species require a period larger than the one above-mentioned (for example, Carpinus betulus, Crataegus spp., several Juniperus spp., Pinus cembra, Rosa spp.). The temperatures recommended are usually between 0° and 5°C (32° and 41°F).
Though the aims of cold stratification are different from those of cold moist storage, processes and equipments are often very similar. A refrigerator, cold room or ice house is needed, and it must be possible to control temperature within the abovementioned range or up to 10°C (50°F). Seeds should be either uniformly mixed or placed in alternate layers with moist media, inside suitable containers.
For testing purposes seeds are usually spread on moist blotting paper or comparable substratum, in flats or small portable germinators, and these are placed in the refrigerator or cold room. At the end of the stratification or prechilling period flats or small germinators may be placed in germinating chambers or rooms without disturbing the seeds. Otherwise, treated seedsmust be removed and placed in Jacobsen tanks or other standard germinators.
Cold stratification is beneficial not only for seeds which do not tolerate drying but for many other species, such as firs, sitka spruce, maples, ashes, some pines (for example, Pinus cembra, P. attenuata, P. strobus) and linden.
Warm followed by cold stratification is less frequent but it has proved to be effective for a considerable number of species of certain genera (for example, Carpinus, Pstrya, Malus, Viburnum).
Some seeds require a moist cold pretreatment followed by a period at higher temperature, after which a second period at low temperature is necessary to break dormancy.
For seeds of the two latter categories, cabinets ensuring a wide range of temperature are the most suitable, not only for research purposes but also for treating small lots of seeds.
SEED DRESSING, PELLETING, SEED COATING
In the pelleting process the surface is coated with some inert material to which chemicals of various kinds may be added.
Some of the advantages claimed for pelleting are:
1. The incorporation of fertilizer material in the pellet furnishes the young seedlings with needed nourishment.2. Plant growth regulators or stimulants may promote rooting or hasten emergence of the seedlings.
3. Fungicides and insecticides are more effective when in direct contact with the seeds.
4. Seed may be protected against rodents by adding unpalatable, repellent or poisonous substances.
5. Small seeds become larger and heavier which makes for greater ease in sowing and occasionally permits sowing from airplanes.
Before using any particular type of pelleting, however, it would be wise to determine the effect of the coating on the species of seeds to be used. Seeds are sometimes pelleted to repel birds, rodents, pests, and to control disease but seldom for the addition of nutrients. Promising results have been obtained with repellents in the United States of America.
The seed is moistened with a sticker or pelleting slurry, to which enough treating powder is added to dry it, usually in a volume relationship of one part of slurry to four parts of powder. The thickness of the coating is regulated by the amount of slurry in proportion to the amount of seed.
Latex (for example, Dow 512 R), methylcellulose, or Hydrol (for example, C-13 HCP, Flintkote) emulsions are used as stickers. For mechanical pelleting, a cement mixer powered by an electric motor can be used.
It is recommended that about 11.5 kilograms (25 pounds) of seed be treated at a time. Seed should be placed in a small concrete mixer. For 45 kilograms (100 pounds) of seed 5 to 10 liters (5 to 10 quarts) of latex solution (one part of latex to nine parts of water are generally used).
Arasan (tetramethylthiuram disulfide) 75 or Arasan 50 and Endrin are often employed as fungicide. They also seem to have a repellent effect on birds and rodents. Only one needs to be used in the relationship of at least 1 to 1.5 kilograms (2 to 3 pounds) to 45 kilograms (100 pounds) of seed, but Endrin can be blended with Arasan (for example, one part of Endrin to two of Arasan). Effectiveness of the blend as a bird and rodent repellent is increased by the addition of anthraquinone or aluminium powder, flaked. The shining quality of aluminium flakes makes them particularly effective against birds: 225 grams (½ pound) are sufficient for 45 kilograms (100 pounds) of seed.
The equipment needed includes, besides a cement mixer or something equivalent, scales and measures, cans for mixing slurry, screens or trays for treated seed, goggles, duster gloves and dust respirators. The total mixing time should not exceed four minutes since prolonged agitation damages seeds or chips off the pelleted coat.
For large amounts of seed, some of the special equipment for dressing seeds of agricultural crops could be used or small units for forest tree seeds could be constructed on the same principle. Some of these machines are combined "short wet" and "dry" method dressers. Others operate only as slurry dressers. The best machines are designed to work quickly and uniformly and the spreading of dust of the dressing compound, which is irritating or poisonous to the operator. They have a synchronized cycle of weighing seeds and applying chemicals automatically.
Seed may be tested for a great variety of characteristics, the ultimate purpose being to determine its value for sowing. Seeds are usually tested for genuineness, purity, viability, moisture content, weight of 1,000 seeds, and for health conditions.
International rules for seed testing were adopted at the meeting of the International Seed Testing Association in Dublin, Ireland, in 1953. Successively the rules were revised at the Paris meeting in 1956, and the Oslo meeting on 10 July, 1959. At the Oslo meeting, many rules concerning forest seed testing were modified or enlarged, particularly to give more detailed prescriptions for a greater number of individual tree species (International rules for seed testing, Proceedings of the International Seed Testing Association, Volume 24, Number 3, Wageningen, 1959 and FAO forest tree seed directory, 1961).
These rules became effective in the Northern Hemisphere on 1 July 1960, and in the Southern Hemisphere on 1 January 1961. All earlier international rules have been canceled.
A standard procedure for the determination of seed quality is of great importance not only for research but also for the seed trade.
The equipment used in a forest seed laboratory partly reproduces on a smaller scale the conventional production equipment and tools used for the exclusive purpose of making the required tests of quality.
SAMPLING
Every effort should be made to draw from a lot of seed a bulk sample, in such a way that the sample represents, as far as possible, the lot concerned.
Similarly, in taking from the bulk sample the working sample, that is, the reduced sample for all the tests to be carried out by the seed laboratory, great care is needed to obtain a working sample representative of the material sent in for analysis.
Seed triers and mechanical mixers and dividers are often used to draw a representative bulk sample from a lot of seed as the first step consists in taking out a number of small subsamples from different parts of the lot, and the second in mixing and dividing the drawn subsamples to reduce the whole to a convenient size.
There are on the market several types of sampling devices for seeds of agricultural crops which can often be used for some forest tree seeds. Sampling sticks of different sizes, made of heavy seamless brass or aluminium, are very common. They generally consist of a hollow tube, pointed at one end, with a number of slots in the side. Another tube, placed inside the first, can be revolved to close the holes. The stick is thrust into the mass with the holes closed. Then these are opened, permitting the seed to run in through the holes. When enough seed has been taken, the holes are closed by revolving the inner tube. The stick, finally, is withdrawn from the seed mass and emptied. Triers commonly known as "thief" triers, with a single open slot, should be avoided, as they damage seeds.
The sampling stick used in Sweden for forest tree seeds is a tube with evenly spaced rectangular holes which has another perforated tube inside (Figure 21).
It is claimed that exact samples are obtained with a seed sampling can: a stream of seed comes out of the upper container and falls into the lower one. A sampling cup collects samples from this stream, at regular intervals.
Various kinds of seed dividers of which the sampling can is one example, have been devised to aid in reducing the drawn subsamples to the suitable size for the bulk sample, as well as to obtain the working sample from the bulk sample. The common principle is to divide the seeds into a number of different streams (from 16 to 36) which are alternately collected to get two equal parts.
FIGURE 21. - Sampling stick made in Sweden (Photo, Statens Skogsforkninginstitut)
FIGURE 22. - Seed divider with 18
A simple divider consists of a hopper with 16 to 20 channels or ducts in which alternate ones lead to opposite sides, a frame to hold the hopper and two receiving pans. In using this divider, seeds are poured in at approximately equal rates along the entire length of the hopper. This can be made with the aid of a flat-bottoned scoop of proportionate size (Figure 22).
In some precision dividers, division and mixing are carried out by the centrifugal action of a motor-driven revolving disk under the hopper. In the Boerner sampler, described by Baldwin (1942) in which seed flows by gravity only, the numerous separate streams merge into two equally divided streams. Various samplers of the Boerner type or based on the same principle, are on the market and are used for forest tree seeds. Figure 23 shows an extractor sample used in Japan.
Dividers operating on centrifugal principle should not be used for seed sensitive to vibration as are those of Abies species.
GENUINENESS TEST
To determine whether seeds are of the species stated on label or not, a good collection of seeds is needed, especially if the tester is not sufficiently experienced. Au appropriate analytical key for seed identification is useful and should always be to hand in a seed station.
Seed laboratories usually build up seed herbariums and seed display sets, for demonstration and comparative tests. Nevertheless, sometimes seed identification can be made only through seedling identification, as in the case of some Quercus species, so that a seedling herbarium and an analytical key for seedling identification are of great help.
The determination of genuineness of type, variety and strain can seldom be achieved by direct inspection. It is usually necessary to carry out growing tests whose success will depend very much on the knowledge and experience of the seed laboratory staff.
PURITY TEST
The object of the purity analysis is to determine by weight the percentage of pure seed. Separation of true seed from other components may be done entirely by hand or partly by hand and partly mechanically.
Hand separation of impurities is always laborious. It is made with the aid of spatulas, knife blades and forceps. A special spatula manufactured for seed purity analysis, is used in Italy. It consists of a slender steel rod, almost 3 millimeters in diameter, 20 centimeters long, which is bent to a right angle at 1 centimeter from the lower end. The upper end is thrust into a cone-shaped wooden stem, while the bent part becomes progressively thinner and wider. This spatula is held in the hand like a pen.
Mechanical aids can be sieves and blowing tubes. An assortment of small sieves can help in eliminating many impurities. Blowing tubes are effective in separating the light chaff, provided that the air current can be accurately controlled.
Sampling tables and seed sample selection boards are often used in seed laboratories. The first are simple boards where uncleaned samples of seed are analyzed. Seed sample selection boards, also called purity desks, are found in several models using a diaphanoscope arrangement which permits an easier separation of certain impurities; similarly the illuminated purity boards and the black light lamps. More frequently employed than diaphanoscope and black light devices are lenses of various sizes and powers. Combined magnifier and fluorescent light may, when mounted upon a flexible bracket, prove very useful for the analyst.
GERMINATION QUALITY TESTS
The aim in testing seed for germination quality is to provide an indication of the percentage of seeds that may be expected to produce plants. Seed viability can be determined by germination tests or estimated by indirect methods (for example, cutting test or biochemical tests).
Germination tests
Germination tests must be carried out under controlled conditions. Temperature and moisture should be as near as possible to the optimum for the species concerned.
For several species light conditions are also very important.
Germination tests should be carried out in conformity with the rules adopted by the International Seed Testing Association (ISTA) in 1959. The prescribed methods of testing forest tree seeds for germination concern more than 40 genera and in particular almost 70 species. For half of the species and for half of the genera considered special treatment for dormancy is recommended, including:
(a) prechilling at 3 to 5°C or 1 to 5°C;
(b) moist stratification;
(c) pericarp removal;
(d) soaking in water or concentrated sulphuric acid;
(e) piercing seed or chipping or filing off a fragment of testa;
(f) cutting off a third of the seed at the scar end.
For each species or genus details are given about the best pretreatment. ISTA rules also prescribe the substrate to be used, the temperature and light conditions, as well as the days for the first count and for the final one.
Germinators and germination chambers
Many types of germinator for testing tree seeds have been devised. All kinds however depend for their effectiveness upon the control of temperature, moisture, air and light. Any apparatus which can assure a good control of such conditions is adequate for germination tests but any apparatus adopted by a seed station should conform to the requisites of ISTA.
Some types of large germinator, made to contain a number of germination beds, such as Jacobsen or Rodewald types, may give a simultaneous control over all the environmental conditions affecting germination. They are much used for forest tree seeds.
Small or portable individual germinators are also in use (Figure 24). These are adequate, so long as they give a good control of moisture and aeration, allow light to enter, and are placed for use in germinating ovens (called also closed or germination chambers), or incubators where temperature (Figure 25) or both temperature and light are constantly under control. Very often these small germinators are not considered true germinators but only seed boxes or trays, while germination chambers are looked upon as such.
A special portable germinator has proved useful for arid zones. It consists of a 15 × 25 × 10 centimeter (6 × 10 × 4 inch) permeable, porous sandy-loam box or tray, which is filled with sterilized sand, and dipped in another larger tray containing water (Figure 26). Sometimes room germinators or combination room-chamber germinators are used. A detailed description of Jacobsen and Rodewald apparatus can be found in Rohmeder (1938), Baldwin (1942, 1955), Toumey and Korstina (1947), and in the Proceedings of the International Seed Testing Association 1959.
For germinating chambers and combined room and chamber-germinators information may be found in Baldwin (1955), as well as in the abovementioned Proceedings.
Moisture is provided either by irrigation of the medium where the seed is placed or by means of a wick dipped into a water reservoir, which is placed below it and whose level is maintained constant, or else by graduating the relative humidity around the seed.
Irrigation, especially if it must be repeated at frequent intervals, should be avoided, although it is often used, particularly for sand. However, the initial amount of water that should be added to sand depends on the characteristics of the sand and seed to be tested. As a rule, sand should be moistened to 50 to 60 percent of its water-holding capacity.
An automatic water supply through capillarity is preferable and the wick system is often adopted. The Jacobsen type is based on the wick system. In the Rodewald apparatus sand is moistened through wicks reaching the water bath. In order to maintain a high air humidity, each seed bed is covered by glass lids, filtration cups or perforated lids. Bell jars or funnels are provided with an aperture, in which a flock of cotton may be inserted to allow ventilation without undue evaporation and penetration of pathogenous agencies, otherwise the whole apparatus could be covered with a glass plate at an angle of 150° to 160° to prevent the condensing water from soaking the seed beds. Such a lid should not be completely closed so that enough air is provided and the condensation reduced.
It may be desirable to keep enough water in the germination chambers to supply a relative humidity of approximately 90 to 95 percent. Some germination chambers have humidifying units.
Jacobsen and Rodewald apparatus are usually designed to operate at a temperature from 20°C (68°F) to 30°C (86°F). Cabinets are constructed for several ranges of temperature. They most commonly range between 10° and 45°C (50° and 113°F); other ranges are between 25° and 50°C (77 and 122°F); 5° and 50°C (41 and 122°F); or 32° and 120°F (0° and 49°C).
Various types of germinator or germinating chamber usually work at a rather wide range of temperature, but not below room temperature. As the required temperature is very often between 20° and 30°C (68° to 86°F) many of them can be satisfactorily used whenever room temperature does not exceed the working temperature. Modern germination chambers have provision for both heating and cooling by electricity, so that work may continue at the prescribed temperature in hot months or in hot climates.
Good incubators made originally for other purposes may be employed for seed testing with some modifications, providing them, for example, with fluorescent light or with special trays. Others are suitable for testing seed in darkness. Some of those cabinets described for cold storage that work at a wide range of temperatures may serve also as germinating chambers, and are particularly useful for seeds requiring a low temperature for germination.
ISTA prescribes the alternation of two temperatures, e.g., 16 hours at about 20°C and eight hours at about 30°C, for many tree species. (Quick alternation is often obtained, for instance with the Jacobsen apparatus, by changing the water or by allowing a cool water flow through coils or pipes until the lower temperature is reached.
Some cabinets permit a quick alternation as the water jacket may be used for cooling them, by allowing a slow flow of cool water through the jacket.
Other germinating cabinets, in which the changeover is gradual, as cooling down and heating up require a certain amount of time, can operate automatically at predetermined intervals at two different temperatures. These cabinets have a synchronizing attachment with a second thermostat and a time switch.
The alternation of temperature may be obtained, although somewhat laboriously, by transferring portable germinators or germination trays from a cabinet adjusted to the lower temperature to another adjusted to the higher, or from one compartment to another, inside the same cabinet. Some cabinets, in fact, have a cold and a warm compartment, or several compartments at different temperatures. Sometimes room temperature is sufficiently reliable, especially if the room is insulated and underground.
Air conditioners may be used, if necessary, to keep a room at the desired temperature all the year round. If the temperature of the entire room is kept as low as the lowest temperature required, several germination chambers or germinators may be placed in this room and heated individually to maintain the various temperatures wanted.
Choice of the media on which the seed is placed for germination depends on the equipment, the species, the working conditions and the experience of the operator. The media generally preferred are suitable paper materials (such as filter paper, germination blotters, or paper toweling free of toxic chemicals) and sand, but many others are employed, such as porous plates, peat, agar-agar or soil. Sand and paper may be sterilized in ovens.
Silica-gel promises to be suitable for several forest tree seeds, because it holds the moisture better than agar-agar, even though it may be selective to certain species. The gel is prepared according to Winogradsky's formula. Its use for routine tests is not to be recommended as it has not yet been sufficiently studied.
Filter paper, heavy box-paper, or any other absorbent paper may be linked with a filter paper or cotton wick dipped into water, or placed on top of a layer of sand. Sand as germination substratum is preferred for species that have a longer germinative period, for instance, for Rosa sp. Pinus caribaea, P. elliotti, and P. palustris, or with larger seeds (for example, with Pinus pinea, Quercus spp.).
Blotters and folded towels are used for between paper tests. Although this type of test should not be recommended for forest tree seed, generally in Morocco some eucalypt germination is done between paper with good results. Often germination tests are carried out on a 150 × 60 centimeter tray, 4 centimeter high, (60 × 24 × 1.4 inch) filled with a 2-centimeter (0.8-inch) layer of sand kept moist by vaporization. A heavy piece of box-paper is laid on top of it after soaking. A weighed amount (1 to 5 grams) of small seeds, is put inside and covered by the second lap of the paper, until germination. Several kinds of filter paper and germination paper are on the market.
The seed trays are usually of glass, or as mentioned above, of porous sand-loam mixture, porcelain, or clay, stainless steel or plastic material. The walls of the water tanks, in the Jacobsen or in Rodewald apparatus, are of metal (stainless steel, zinc, brass or red copper). Brass or red copper plates (1 millimeter) must be lined internally with tin or with a bituminous coat.
Light, for seed requiring it, can be either natural or artificial. The light intensity should be about 750 to 1250 lux. If germinators are placed near a wide window, this intensity is easily reached during the day, but it must be kept in mind that the germinators should not receive the sun rays directly.
Artificial light may be provided by a set of fluorescent tubes, placed over the germinators or inside the germinating chambers. To control the light intensity a device (better if self-contained) should be used, to switch on or off a number of tubes.
Counting equipment
Two kinds of seed counting devices are frequently used: counting boards and vacuum counters.
Counting boards are useful only for seed of a regular shape and relatively large, such as those of P. cembra, Tilia spp., and some legumes. Boards should be approximately the size of the germination bed. The counter consists of two boards, a stationary top board and a thin solid board below, which serves as a false bottom. In operating the counter, a quantity of seed is placed at the closed corner, the plate tilted and moved so that some seed goes into each perforation. The plate is then placed on the substratum and the bottom is withdrawn, the seed falling regularly spaced. In some types there is a second set of perforations in the bottom board. By slipping the top section about one centimeter, the seed falls upon the germination bed.
In connection with tests on soil trays, a simple counting perforated board can be employed. The board is first placed over the tray. After placing the seed on the board, this is lifted leaving the seed on the tray, where they are pressed down by the board itself (Figure 27). Seed is pressed more evenly and faster by this method.
Vacuum counters may be effective with seeds of various size and shape. For very small and irregularly shaped seeds some difficulties may occur. Mechanical counters are not suitable for very large seeds, such as many nuts.
A vacuum counting device has three essential parts:
(a) a vacuum system including pipes;
(b) counting heads;
(c) a quick release valve.
A small house vacuum cleaner (Figure 28) may be used, but special power units are generally preferable (¼ to ½ hp electric motors). For central installation with piping to several rooms in a building 1 ½ hp units are convenient.
Perforated heads with evenly spaced holes (generally 100) to house the seed, hold the seed by suction, to be released and placed on germination beds. Counting plates or heads are of a square or circular form, according to the shape of the germination bed employed. Spacement and diameter of holes should be proportionate to the seed size. Heads can be of plastic, chromium, brass and aluminium. The face plate should be relatively opaque and of a color which contrasts with the seeds being counted, thus making it easier to check that all holes are properly filled. A good valve is needed to allow a quick release of seed as well as to avoid sucking up too large a proportion of the lightest seeds.
Indirect methods of testing seed for germination quality
Several methods have been developed. The oldest is the simple cutting test, which is unreliable, specially for conifers, and small-seeded hardwoods. For cutting tests only razor blades, penknives, scalpels and bistouries are generally needed. A hand lens or reading glass may be helpful. For a rough preliminary estimate, this test may prove useful.
Biochemical tests are more widely used. Tetrazolium test is admitted by ISTA as an official test to estimate the viability of the following kinds of seeds: Carpinus spp., Crataegus spp., Fraxinus spp., Juniperus spp., Malus spp., Pinus cembra, Prunus spp., Pyrus, spp., Rosa spp., Taxus spp., Tilia spp. Fresh seeds at the end of the germination period should be preferably tested by the tetrazolium method. A 1 percent solution of 2, 3, 5-triphenyltetrazolium chloride or bromide is used for this method. During this treatment the preparations must be kept in darkness at 30°C. Equipment consists, besides chemicals (distilled water and tetrazolium salts) and glassware (small bottles, Petri dishes, etc.), of small snippers, dissecting needles, razor blades and fine scissors. Small incubators or precision ovens, such as those employed for paraffin embedding are useful. A black surface may be helpful in judging properly the staining of embryo and endosperm. Binocular or stereoscopic microscopes should be kept handy. For testing Taxus seed, a vacuum is also necessary.
This equipment permits a quicker determination (few minutes) of the germinative capacity equivalent, under a vacuum, at constant temperature of 45°C (113°F). Seeds are previously split, by a splitting machine, to expose the embryo to the tetrazolium solution. The equipment operates electrically using 30 watts, and a timer indicates the end of the reaction.
X-ray analysis of seed can give some interesting information about embryo position and development and about certain characteristics of the endosperm, but does not distinguish between viable and nonviable seeds, as physiological changes of the seed can seldom be detected. After impregnation with salts of heavy cations (for example, BaCl2,) seed viability, according to Gustafsson and Simak (1958), can be detected on an X-ray film. The X-ray technique has been developed in Sweden and is very rapid for some forest seeds. At present it must be considered valuable for special purposes, but not for official tests. The technique, which is being investigated in many countries, requires a generator of Grenz rays.
In Sweden an X-ray tube is used operating at 10 kilowatts (kW), with current of 18 milliamperes. At Nancy, the X-ray apparatus can be used with a voltage from 7 (small seeds) to 16 kilowatts (large seeds), the best films being obtained when the current is of 60 to 90 milleamperes (mA), and the distance between tube and seed is of 26 to 60 centimeters (about 1 to 2 feet). An X-ray test is claimed to be more reliable than cutting test and easier for interpretation (Bouvarel and Lemoine, 1959).
DETERMINATION OF MOISTURE CONTENT
According to the ISTA rules the moisture content of forest tree seeds should be tested by the air-oven method either at 130°C (e.g., Alnus spp., Pinus spp., Prunus spp. and Pyrus spp.) or at 105°C (e.g., Ceratonia siliqua). For seeds for which even the 105°C method is unsuitable as a consequence of extremely volatile constituents, the Toluene Distillation method is recommended (e.g., Abies spp. and Picea spp.). Coarse grinding is necessary in case of large seeds of legumes, such as Ceratonia siliqua.
The abovementioned methods are applicable to seeds with a moisture of 18 percent (seeds of legumes, 20 percent) or lower, calculated on wet basis. Seeds having a higher moisture content, should be predried at a relatively low temperature.
The equipment needed for seed moisture determination by air-oven methods consists in balances with an accuracy of at least 1 milligram, dishes of noncorrosive metal with rounded bottom corner and a flat bottom, fitted with a cover which fits as snugly as possible, electrically heated ovens, desiccators with blue-colored silica gel or activated alumina as desiccants, and grinding mills.
The length of the drying period at 130°C should be 60 minutes, at 105°C it should be 16 hours. The technique concerning air-oven methods is described in detail in the ISTA rules, 1959. For the Toluene Distillation method see: Official methods of analysis of the Association of Official Agricultural Chemists, published in Washington, 1955. This test needs: a 250-milliliter distilling flask, distilling tube receiver and a straight-tube Liebig condenser.
Ovens
Several types can be used and are on the market. The commonest are the "free ventilating" ovens, but forced-draught semiautomatic ovens are often used. In this latter type, moisture percentage can be read directly on the dial. Infrared, automatic moisture testers make moisture tests in a very short time. As a rule, they cannot be recommended for official tests, but only for quick, rough estimates.
For official tests ovens with good natural ventilation and automatic temperature control must be employed which enable the temperature after heating to remain within the limits of 130° ± 3°C or of 105° ± 2°C.
Some ovens give a complete precise control of temperature from room temperature to 200°C, with an accuracy of 2°C, others for short-term changes in temperature, and may operate up to 300°C. Air circulation in the latter is provided by a centrifugal fan, so that differences between temperature at the center and any other point is of 1°C at 110°C. Accuracy can be ± 1°C, with fan.
The types with air circulation are more suitable for sterilization purposes and in such cases the degree of accuracy may be lower. Several types are available.
WEIGHT DETERMINATIONS
In a seed laboratory it is often necessary to determine weight. Weighing is done in order to determine:
(a) the weight of seed samples;
(b) the weight of pure seed, other crop seed, weed seed and inert matter (purity analysis);
(c) the seed moisture content by air-oven methods;
(d) the weight of 1,000 seeds:
(e) the dosage of chemicals.
Weight of 1,000 seeds
This should be determined by weighing in grams four or more replicates of one hundred seeds each. The results should be computed to different numbers of decimal places according to the weight.
Balances and scales
A seed laboratory must be equipped with balances and scales of various type and accuracy, according to their use. Balances used to determine moisture content must have an accuracy of at least 1 milligram, as already mentioned, and must belong to the analytical type. These may also be used for the dosage of chemicals.
For purity tests and to determine the weight of 1,000 seeds it is advisable to have available balances or scales with two or three different grades of accuracy. The most sensitive balance should be adopted for the lightest seeds, which require minimum weights for purity analysis inferior or equal to 10 grams (for example, Alnus spp., some pines, Picea spp., Populus and Salix spp.). The least sensitive ones should be used with seeds that have minimum weights for purity test of 100 grams or more (for example, several Abies species, Araucaria spp., Acer spp., Carpinus spp., Castanea spp., Cedrus spp., Fagus spp., some pines, Quercus spp, etc.).
As a rule, it may be assumed that for the purity test balances must be used with an accuracy of at least 1 milligram for the seeds belonging to the first above-mentioned category, while for the heaviest seeds balances with a sensitivity of 10 to 25 milligrams are sufficient.
A great variety of balances and scales is available on the market. Precision balances with a capacity of 200 grams, and a sensitivity of 1/20 milligram, which must not take weights from 1/10 to 100 milligrams are very helpful for several different uses.
Among the balances of medium precision which are to be recommended, are the semiautomatic ones, with a capacity of 500 or 1,000 grams, and a sensitivity of 10 or 25 milligrams, which do not take weights up to 2 or 5 grams.
At Nancy, France, the Vivancyl balance which is produced in France by five co-operating manufacturers of precision equipment is used. This is of the type with a single plate suspended by a thread of gold: weight determinations can be obtained very quickly. Its sensitivity is of 1/10 milligram.
Volume-weight: bushel-weight or hectoliter- weight
ISTA rules prescribe that these determinations be made with the aid of a standardized apparatus. Basically, a correct apparatus consists of a precision filling device, an accurately adjusted standard dry measure, and an accurate scale. As a seed laboratory generally has at its disposal several scales, it may be adequate to obtain only one or two precision filling hoppers with stand and as many tester buckets having the desired capacity.
The filling hopper is strictly indispensable because large errors will result if buckets are filled directly by hand or by scooping. The hopper is funnel-shaped and has a sliding gate valve for instant release of seed at the bottom. A heavy base can make the hopper more stable. The hopper must be adjusted to proper standard height above the bucket (generally 5 centimeters).
Standardized ¼-or 1-liter buckets should be used to determine hectoliterweight. For the determination of bushel-weight, 1 pint or 1 quart buckets are recommended, Buckets, as well as filling hoppers, are made of heavy noncorrosive metal, generally of solid brass.
The general methods imply a macroscopic as well as a microscopic examination. Upon receipt, seed can be macroscopically inspected for the presence of alterations due to pathogenic organisms. Particular attention should be given, for Abies, Larix, Pseudotsuga, Quercus and Castanea spp., etc., to seeds showing holes which may contain living insects.
The microscopic examination is of the utmost importance. As a rule the presence of disease organisms is ascertained after a suitable treatment of the seed. Fungus spores or hyphae, may be removed by vigorously shaking 100 seeds or more in a test tube containing water and a wetting agent, or alcohol. The liquid is then either centrifuged or evaporated to a few drops, which are examined under magnification.
Sometimes it is necessary to supplement this procedure by cultural methods involving the use of agar. To identify insects it may be necessary to rear them in glass-jars with fine wire-netting. Examinations must be also made during or at the end of the germination test.
A basic list of disease organisms and pests of forest tree seeds is quoted in the International rules for seed testing 1959. Certificates regarding the health conditions should conform such rules. The essential equipment for health tests consists of one stereoscopic microscope, one or two precision microscopes (at least, 500 power), one centrifugal, and one or two incubators.
The latter should not be placed in the same room where germinators are located. A store of glassware, including Petri dishes, test tubes, glass-jars is also necessary. Agar must be handy, as cultural substratum.
FORMS FOB RECORDING TESTS DATA
The forms for recording data of the tests should pro vide for all important data concerning the sample under examination. Space must be assigned for the results of each type of test, to allow for retests. A satisfactory form for seed test report is given in Baldwin (1942; pages 204 and 205).
Germination chambers and incubators should be disinfected at least once a year, by a 10 percent formaldehyde solution. All openings and fissures must be closed, the latter, if necessary, with adhesive tape. After 24 or 48 hours vessels are removed and vapors are allowed to escape freely before the chamber is used again.
Portable germinators and seed trays may be disinfected with alcohol or they may be put into a dry-oven. Germination mats made of cotton must be boiled after use or sterilized in autoclave.
Other germination substrata which are used only once (filter paper or sand) are generally disinfected in dry-ovens. As a rule, seed is not disinfected because seeds which decay in a clean germinator are generally of poor quality. A number of experiments carried out with different methods of disinfecting seed just before or during germination have shown no reliable improvement in germination percentage.
Petri dishes, test tubes and glass jars must be sterilized in an autoclave when used for rearing disease organisms and pests. The room where germinators are placed and its equipment must be kept very clean. Only certain specific cleaning and polishing materials may be used. Portable vacuum cleaners are recommended to keep the room clean. They are available in many sizes and types and may also be used as blowers. Rated from 40 to 175 cubic feet per minute (12,000 to 26,000 linear feet per minute) they blow dry or hot air (13/PF) for drying purposes, drawing from 260 to 890 watts.
Seed transport and shipment require some care to avoid any molding or overheating of the seed. Seed should be shipped in proper containers which are sufficiently resistant, and give protection against damp.
Linen bags are commonly used: a double envelope ensures greater protection. Metal containers are now used for long-distance shipping. The sizes commonly used contain from 1 to 20 kilograms of seed. Larger containers may be used for storage only.
A bibliography and a list of manufacturers of the equipment mentioned in this paper may be obtained gratis on request from the Forestry and Forest; Products Division, FAO, Viale delle Terme di Caracalla, Rome, Italy.
