Specific germination requirements are set forth in table 2 to which the following paragraphs (a), (b), and (c) are applicable.
(a) Definitions and explainations applicable to table 2--(1) Duration of tests. The following deviations are permitted from the specified duration of tests: Any test may be terminated prior to the number of days listed under ``Final count'' if the miximum germination of the sample has then been determined. The number of days stated for the first count is approximate and a deviatioon of 1 to 3 days is permitted. If at the time of the prescribed test period the seedlings are not sufficiently developed for positive evaluation, it is possible to extend the time of the test period two additional days. (Also, see paragraph (a)(5) of this section and 201.57.)
(1) Duration of tests. The following deviations are permitted from the specified duration of tests: Any test may be terminated prior to the number of days listed under ``Final count'' if the miximum germination of the sample has then been determined. The number of days stated for the first count is approximate and a deviatioon of 1 to 3 days is permitted. If at the time of the prescribed test period the seedlings are not sufficiently developed for positive evaluation, it is possible to extend the time of the test period two additional days. (Also, see paragraph (a)(5) of this section and 201.57.)
(2) Light. Cool white fluorescent light shall be provided where light is required in table 2. The light intensity shall be 75 to 125 foot-candles (750-1,250 lux). (The light intensity for nondormant seed and during seedling development may be as low as 25 foot-candles to enable the essential structures to be evaluated with greater certainty.) The seeds shall be illuminated for at least 8 hours every 24 hours except when transferred to a low temperature germinator during the weekend. When seeds are germinated at alternating temperatures they shall be illuminated during high temperature periods. Seeds for which light is prescribed shall be germinated on top of the substratum except for ryegrass fluorescence tests.
(3) Moisture-on-dry-side. This term means that the moistened substratum should be pressed against a dry absorbent surface such as a dry paper towel or blotter to remove excess moisture. The moisture content thus obtained should be maintained throughout the germination test period.
(4) Potassium nitrate (KNO3). These terms mean a two-tenths (0.2) percent solution of potassium nitrate (KNO3) shall be used in moistening the substratum. Such solution is prepared by dissolving 2 grams of KNO3 in 1,000 ml. of distilled water. The grade of the potassium nitrate shall meet A.C.S. specifications.
(5) Prechill. The term ``prechill'' means a cold, moist treatment applied to seeds to overcome dormancy prior to the germination test. The prechill method varies among kinds, but is usually performed by holding imbibed seeds at a low temperature for a specified period of time. The prechill period is not included in the duration of tests given in table 2, unless otherwise specified.
(6) Predry. The term ``predry'' means to place the seed in a shallow layer at a temperature of 35 to 40 C. for a period of 5 to 7 days, with provisions for circulation of the air.
(7) Substrata (Kinds). The symbols used for substrata are: B= between blottersTB= top of blottersT= paper toweling, used either as folded towel tests or as roll towel tests in horizontal or vertical positionS= sand or soil where soil is an artificial planting mix of shredded peat moss, vermiculite, and perliteTS= top of sand or soilP= covered Petri dishes: with two layers of blotters; with one layer of absorbent cotton; with five layers of paper toweling; with three thicknesses of filter paper; or with sand or soilC= creped cellulose paper wadding (0.3-inch thick Kimpak or equivalent) covered with a single thickness of blotter through which holes are punched for the seed that are pressed for about one-half their thickness into the paper waddingTC= on top of creped cellulose paper without a blotterRB= blotters with raised covers, prepared by folding up the edges of the blotter to form a good support for the upper fold which serves as a cover, preventing the top from making direct contact with the seeds.
(8) Temperature. A single numeral indicates a constant temperature. Two numerals separated by a dash indicate an alternation of temperature, the test to be held at the first temperature for approximately 16 hours and at the second temperature for approximately 8 hours per day. The temperature shall be determined at the substratum level and shall be as uniform as possible throughout the germination chamber. (A sharp alternation of temperature, such as obtained by hand transfer, may be beneficial in breaking dormancy.) If tests are not subjected to alternating temperatures over weekends and on holidays, they are to be held at the first-mentioned temperature during this time. In cases where two temperatures are indicated (separated by a semicolon) the first temperature shall be regarded as the regular method and the second as an alternate method.
(9) Paper substrata must be free of chemicals toxic to germinating seed and seedling growth. If root injury occurs from toxicity of a paper substratum or from the use of potassium nitrate, retests shall be made on soil or on a substratum moistened with water.
(10) Ethephon. This term means a 29 parts per million (0.0029 percent) solution of ethephon [(2-chloroethyl) phosphonic acid] which shall be used to moisten the substratum. This solution is prepared by mixing 0.6 ml of a stock solution with 5,000 ml of distilled water. The stock solution contains 24 grams of active material per 100 ml of propylene glycol or two pounds of active material per gallon. A solution which is five times this concentration (5x29 ppm) may be used for extremely dormant seeds, provided seeds are transferred to substratum moistened with water after 1 to 3 days.
(11) Ethylene. This term means that five (5) ml of ethylene gas per cubic foot (176.57 ml/m\3\) of germinator space is injected into a germinator in which peanut seeds in moist rolled towels have been placed. Following injection of the ethylene, the germinator is kept closed until the first count (5 days). If the germinator door is opened for the purpose of checking or rewetting the samples, another injection of ethylene at the same rate shall be made.
(b) Special procedures and alternate methods for germination referred to in table 2--(1) Alyceclover; swollen seeds. At the conclusion of the 21-day test period, carefully pierce the seed coat with a sharp instrument and continue the test for 5 additional days. Alternate method: The swollen seeds may be placed at 20 C for 48 hours and then at 35 C for 3 additional days.
(1) Alyceclover; swollen seeds. At the conclusion of the 21-day test period, carefully pierce the seed coat with a sharp instrument and continue the test for 5 additional days. Alternate method: The swollen seeds may be placed at 20 C for 48 hours and then at 35 C for 3 additional days.
(2) Bahiagrass; removal of glumes. On all varieties except ``Pensacola,'' remove the enclosing structures (glumes, lemma, and palea) from the caryopsis with the aid of a sharp scalpel. If the seed is fresh or dormant, lightly scratch the surface of the caryopsis.
(3) Beet, Swiss chard; preparation of seed for test. Before the seeds are placed on the germination substratum, they shall be soaked in water for 2 hours, using at least 250 ml of water per 100 seeds, then washed in running water and the excess water blotted off. The temperature of the soaking and washing water should be no lower than 20 C. Samples producing excessive discoloration of the hypocotyl or root should be retested in soil or by washing in running water for 3 hours and testing on ``Kimpak,'' keeping the seed covered with slightly moist blotters. Sugar beets may require 16 hours soaking in water at 25 C, followed by rinsing and then drying for 2 hours at room temperature.
(4) Buffelgrass; alternate method for dormant seed. The caryopses shall be removed from the fascicles and placed on blotters moistened with a 0.2 percent solution of KNO3, in petri dishes. The seeds from a fascicle should be arranged so they will not be confused with seeds from other fascicles during the test. The seeds are then prechilled at 5 C for 7 days and tested at 30 C in light for 21 additional days. Firm ungerminated seeds remaining at the conclusion of the test should be scratched lightly and left in test for 7 additional days.
(5) Cotton (Gossypium spp.); dormant samples. Samples of cottonseed which do not respond to the usual method should be placed in a closed container with water and shaken until the lint is thoroughly wet. The excess moisture should then be blotted off.
(6) Endive (Cichorium endivia); dormant samples. Add about \1/8\ inch of tap water at the beginning of the test and remove excess water after 24 hours.
(7) Green needlegrass; two test methods as prescribed in table 2 shall be used on each sample:
(i) For method 1, acid scarify 400 seeds for 10 minutes in concentrated sulfuric acid (95 to 98 percent H2 SO4). Rinse seeds and dry on blotters for 16 hours, then place seeds on blotters moistened with a solution of 0.055 percent (500 ppm gibberellic acid GA3) and 0.46 percent (3,000 ppm) thiram and germinate 14 days.
(ii) For method 2, plant 400 seeds on blotters moistened with a 0.2 percent solution of KNO3 and germinate 14 days. Refer to Sec. 201.57a(c).
(iii) Report the results of method 2 as the percentage germination. If the number in method 2 is less than method 1, subtract the results of method 2 from method 1 and report the difference as dormant seed.
(8) Rescue grass (Bromus catharticus); dormant samples. Wash for 48 hours in running water, or soak for 48 hours, changing the water and rinsing each morning and night.
(9) Rice (Oryza sativa)--Alternate method. Plant the seeds in moist sand. On the seventh day of the test add water to a depth of one-fourth inch above the sand level and leave for the remainder of the test. Only a final count is made. Dormant seeds: Presoak 24 to 48 hours in 40 C. water. For deeply dormant seeds, presoak 24 hours in 1,000 p.p.m. ethylene chlorohydrin or 5 percent solution of sodium hypochlorite (clorox at bottle strength).
(10) Ryegrass; fluorescence test. The germination test for fluorescence of ryegrass shall be conducted in light [not to exceed 100 foot candles (1,076 lux)] with white filter paper as the substratum. The white filter paper should be nontoxic to the roots of ryegrass and of a texture that will resist penetration of ryegrass roots. Distilled or deionized water shall be used to moisten the filter paper. The test shall be conducted in a manner that will prevent the contact of roots of different seedlings. Roots of some seedlings produce fluorescent lines on white filter paper when viewed under ultraviolet light. First counts shall not be made before the eighth day; at that time remove only normal fluorescent seedlings. Evaluation of fluorescence shall be made under F15T8-BLB or comparable ultraviolet tubes in an area where light from other sources is excluded. If there are over 75 percent normal fluorescent seedlings present at the time of the first count, break the contact of the roots of the nonfluorescent seedlings from the substratum and reread the fluorescence at the time of the final count. At the final count, lift each remaining seedling, observing the path of each root since sometimes faint fluorescence will show on the substratum as the root is lifted. Abnormal seedlings and dead seeds are not evaluated for fluorescence. See Sec. 201.58a(a).
(11) Trifolium, Medicago, Melilotus, and Vicia faba; temperature requirements. A temperature of 18 C. is desirable for Trifolium spp., Medicago spp., Melilotus spp., and Vicia faba.
(12) Garden bean; use of calcium nitrate. If hypocotyl collar rot is observed on seedlings, the sample involved shall be retested using a 0.3 to 0.6 percent solution of calcium nitrate (CaNO3) to moisten the substratum.
(13) Fourwing Saltbush (Atriplex canscens); preparation of seed for test. DE-wing seeds and soak for 2 hours in 3 leters of water after which rince with approximately 3 leters of distilled water. Remove excess water, air dry for 7 days at room temperature, then test for germination as indicated in Table 2.
(c) Procedures for coated seed. (1) Germination tests on coated seed shall be conducted in accordance with methods in paragraphs (a) and (b) of this section. However, kinds for which soaking or washing is specified in paragraph (b) shall not be soaked or washed in the case of coated seed.
(1) Germination tests on coated seed shall be conducted in accordance with methods in paragraphs (a) and (b) of this section. However, kinds for which soaking or washing is specified in paragraph (b) shall not be soaked or washed in the case of coated seed.
(i) Coated seed units shall be placed on the substratum in the condition in which they are received without rinsing, soaking, or any other pretreatment.
(ii) Coated seed units in mixtures which are color coded or can otherwise be separated by kinds shall be germinated as separate kinds without removing the coating material.
(iii) Coated seed units in mixtures which cannot be separated by kinds without removing the coating material shall be de-coated and germinated as separate kinds. The coating material shall be removed in a manner that will not affect the germination capacity of the seeds.
(2) The moisture level of the substratum is important. It may depend on the water-absorbing capacity of the coating material. A retest may be necessary before satisfactory germination of the sample is achieved.
(3) Phytotoxic symptoms may be evident when germinating coated seeds in paper substrata. In such cases a retest in sand or soil may be necessary.
Table 2--Germination Requirements for Indicated Kinds----------------------------------------------------------------------------------------------------------------
Additional directions
First Final ---------------------------------
Name of seed Substrata Temperature (C) count count Specific Fresh and
days days requirements dormant seed----------------------------------------------------------------------------------------------------------------
AGRICULTURAL SEEDAgrotricum.................. B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days.Alfalfa..................... B, T, S 20.............. 4 \1\7 See ] (b)(11)..Alfilaria................... B, T 20-30........... 3 14 Clip seeds.....Alyceclover................. B, T 35.............. 4 \1\21 See ] (b)(1)
for swollen
seeds.Bahiagrass:
Var. Pensacola.......... P, S 20-35........... 7 28 Light; see ] See Sec.
(b)(2). 201.57a
(2). 201.57a
All other vars.......... P 30-35........... 3 21 Light; remove Scratch
glumes; see ] caryopses;
(b)(2). KNO3; see Sec.
(2). KNO3; see Sec.
201.57aBarley...................... B, T, S 20; 15.......... 4 7 ............... Prechill 5 days
at 5 or 10 C
or predry
Barrelclover................ B, T 20.............. 4 \1\14 Remove seeds
from bur; see
] (b)(11).Bean:
Adzuki.................. B, T, S 20-30........... 4 \1\10
Field................... B, T, S, TC 20-30; 25....... 5 \1\8
Mung.................... B, T, S 20-30........... 3 \1\7Beet, field................. B, T, S 20-30........... 3 14 See ] (b)(3)...Beet, sugar................. B, T, S 20-30; 20....... 3 10 See ] (b)(3)...Beggarweed, Florida......... B, T 30.............. 5 \1\28Bentgrass:
Colonial................ P 15-30; 10-30; 15- 7 28 Light; KNO3.... Prechill at 5
25. or 10 C for 7
days.
Creeping................ P 15-30; 10-30; 15- 7 28 Light; KNO3.... Prechill at 5
25. or 10 C for 7
days.
Velvet.................. P 15-25; 20-30.... 7 21 Light; KNO3....Bermudagrass................ P 20-35........... 7 21 Light; KNO3;
see ] (a)(9).Bermudagrass, giant......... P 20-35........... 7 21 Light; KNO3; Prechill at 10
see ] (a)(9). C for 7 days
and then test
at 20-35 C;
continue tests
of hulled seed
for 14 days
and of
unhulled seed
for 21 daysBluegrass:
Annual.................. P 20-30........... 7 21 Light..........
Bulbous................. P, S 10.............. 10 35 KNO3 or soil... Prechill all
samples at 5 C
for 7 days.
Canada.................. P 15-25; 15-30.... 10 28 Light; KNO3.... 10-30 C.
Glaucantha.............. P 15-25; 15-30.... 10 28 Light; KNO3....
Kentucky................ P 15-25; 15-30.... 10 28 Light; KNO3.... Prechill at 10
C for 5 days.
Nevada.................. P 20-30........... 7 21 Light; KNO3....
Rough................... P 20-30........... 7 21 Light..........
Texas................... P 20-30........... 7 28 Light; KNO3.... Prechill at 5 C
for 2 weeks.
Wood.................... P 20-30........... 7 28 Light..........Bluejoint................... TB, P 15-25........... 10 21 Light and KNO3 Prechill at 5 C
optional. for 5 daysBluestem:
Big..................... P, TS 20-30........... 7 14 Light; KNO3.... Prechill at 5 C
for 2 weeks;
see Sec.
201.57a.
Little.................. P, TS 20-30........... 7 14 Light; KNO3.... Prechill at 5 C
for 2 weeks;
see Sec.
201.57a.
Sand.................... P, TS 20-30........... 7 14 Light; KNO3.... Prechill at 5 C
for 2 weeks;
see Sec.
201.57a.
Yellow.................. P, TS 20-30........... 5 14 Light; KNO3.... Prechill at 5 C
for 2 weeks;
see Sec.
201.57a.Bottlebrush-squirreltail.... P, B 20; 15.......... 10 14 ............... See Sec.
201.57a.Brome:
Field................... P, TB 15-25; 20-30.... 6 14 Light.......... Prechill at 10
C for 5 days.
Meadow.................. B, T, TB 20-30........... 6 14 Light optional.
Mountain................ P 20-30........... 6 14 Light..........
Smooth.................. P, B, TB 20-30........... 6 14 Light optional. Prechill at 5
or 10 C for 5
days, then
test at 30 C
for 9
additional
days.Broomcorn................... B, T, S 20-30........... 3 10Buckwheat................... B, T 20-30........... 3 6Buffalograss:
(Burs).................. P,TB,TS 20-35........... 7 14 Light;KNO3..... Prechill at 5 C
for 2 weeks;
See Sec.
201.57a.
(Caryopses)............. P 20-35........... 5 14 Light;KNO3.....Buffelgrass................. S 30.............. 7 28 Light; press See ] (b)(4);
fascicles into see Sec.
well-packed 201.57a.
soil and
prechill at 5
C for 7 days.
Burclover, California....... B, T 20.............. 4 \1\ 14 Remove seeds
from bur; see
] (b)(11).Burclover, spotted.......... B, T 20.............. 4 \1\ 14 Remove seeds
from bur; see
] (b)(11).Burnet, littler............. B, T 15.............. 5 14Buttonclover................ B, T 20.............. 4 \1\ 10 See ] (b)(11).. 15 C.Canarygrass................. B, T 20-30........... 3 7Canarygrass, reed........... P 20-30........... 5 21 Light; KNO3....Carpetgrass................. P 20-35........... 10 21 Light.......... KNO3.Castorbean.................. T, S 20-30........... 7 14 Remove caruncle
if mold
interferes
with test.Chess, soft................. P 20-30........... 7 14 Light.......... Prechill at 5
or 10 C for 7
days.Chickpea.................... T,S 20-30........... 3 \1\ 17Clover:
Alsike.................. B, T, S 20.............. 3 \1\ 7 See ] (b)(11).. 15 C.
Arrowleaf............... B, T 20; 15.......... 4 \1\ 14 See ] (b)(11)..
Berseem................. B, T, S 20.............. 3 \1\ 7 See ] (b)(11).. 15 C.
Cluster................. B, T 20.............. 4 \1\ 10 See ] (b)(11).. 15 C.
Crimson................. B, T, S 20.............. 4 \1\ 7 See ] (b)(11).. 15 C.
Kenya................... B, T, S 20.............. 3 17
Ladino.................. B, T, S 20.............. 3 \1\ 7 See ] (b)(11).. 15 C.
Lappa................... B, T 20.............. 3 \1\ 7 See ] (b)(11).. 15 C.
Large hop............... B, T 20.............. 4 \1\ 14 See ] (b)(11).. 15 C.
Persian................. B, T 20.............. 3 \1\ 7 See ] (b)(11).. 15 C.
Red..................... B, T, S 20.............. 4 \1\ 7 See ] (b)(11).. 15 C.
Rose.................... B, T 20.............. 4 \1\ 10 See ] (b)(11).. 15 C.
Small hop............... B, T 20.............. 4 \1\ 14 See ] (b)(11).. 15 C.
Strawberry.............. B, T 20.............. 3 \1\ 7 See ] (b)(11).. 15 C.
Sub..................... B, T 20.............. 4 \1\ 14 See ] (b)(11).. 15 C.
White................... B, T, S 20.............. 3 \1\ 7 See ] (b)(11).. 15 C.Corn:
Field................... B, T, S, TC 20-30; 25....... 4 7
Pop..................... B, T, S, TC 20-30; 25....... 4 7Cotton...................... B, T, S 20-30; 30....... 4 \1\ 12 Test by
alternate
method; see ]
(b)(5).Cowpea...................... B, T, S 20-30........... 5 \1\ 8Crambe...................... T,B 20;25........... 4 7 ............... KNO3Crested dogtail............. P 20-30........... 10 21 Light.......... Prechill at 5
(5).Cowpea...................... B, T, S 20-30........... 5 \1\ 8Crambe...................... T,B 20;25........... 4 7 ............... KNO3Crested dogtail............. P 20-30........... 10 21 Light.......... Prechill at 5
or 10 C for 3
days.Crotalaria:
Lance................... B, T, S 20-30........... 4 \1\ 10
Showy................... B, T, S 20-30........... 4 \1\ 10
Slenderleaf............. B, T, S 20-30........... 4 \1\ 10
Striped................. B, T, S 20-30........... 4 \1\ 10
Sunn.................... B, T, S 20-30........... 4 \1\ 10Crownvetch.................. B,T,TB,S 20.............. 7 \1\ 14Dallisgrass................. P 20-35........... 7 21 Light; KNO3....Dichondra................... B, T 20-30........... 7 \1\ 28Drop seed, sand............. P 5-35; 15-35..... 5 14 Light; KNO3.... Prechill at 5 C
for 4 weeks;
see Sec.
201.57a.Emmer....................... B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days or
predry.Fescue:
Chewings................ P 15-25........... 7 21 Light and KNO3 Prechill at 5
optional. or 10 C for 5
days.
Hair.................... P 10-25........... 10 28 KNO3...........
Hard.................... P 15-25........... 7 21 Light and KNO3
optional.
Meadow.................. P 15-25; 20-30.... 5 14 Light and KNO3
optional.
Red..................... P 15-25........... 7 21 Light and KNO3
optional.
Sheep................... P 15-25........... 7 21 Light and KNO3
optional.
Tall.................... P 15-25; 20-30.... 5 14 Light and KNO3 Prechill at 5
optional. or 10 C for 5
days and test
for 21 days.Flatpea..................... T 15-25;20........ 14 \1\ 28Flax........................ B, T, S 20-30........... 3 7Foxtail, creeping........... P 15-30........... 7 21 Light;KNO3.....Foxtail, meadow............. P 20-30........... 7 14 Light..........
Galletagrass................ P, B 20; 25; 20-30... 4 10 ............... See Sec.
201.57aGrama:
Blue.................... P, TB 20-30........... 7 14 Light.......... KNO3; see Sec.
201.57a.
Side-oats............... P 15-30........... 7 14 Light; KNO3.... See Sec.
201.57a.Guar........................ B, T, S 30; 20-30....... 5 \1\ 14Guineagrass................. P 15-35........... 10 28 Light; KNO3
optional.Hardinggrass................ P 10-30........... 7 28 Light.......... KNO3.
Alternate method........ P 15-25........... 7 14 Light; presoak
at 15 C for 24
hrs.Hemp........................ B, T 20-30........... 3 7Indiangrass, yellow......... P, TS 20-30........... 7 14 Light; KNO3.... Prechill at 5 C
for 2 weeks;
see Sec.
201.57a.Indigo, hairy............... B, T 20-30........... 5 \1\ 14Japanese lawngrass.......... P 35-20........... 10 28 Light; KNO3....Johnsongrass................ P 20-35........... 7 35 Light.......... KNO3; see Sec.
201.57a.Kenaf....................... T, B 20-30........... 4 \1\ 8Kochia, forage.............. P 20.............. 4 14 ............... See Sec.
201.57a.Kudzu....................... B, T 20-30........... 5 \1\ 14Lentil...................... B, T 20.............. 5 \1\ 10Lespedeza:
Korean.................. B, T, S 20-35........... 5 \1\ 14
Sericea................. B, T, S 20-35........... 7 \1\ 21
Siberian................ B, T, S 20-35........... 7 \1\ 21
Striate................. B, T, S 20-35........... 7 \1\ 14Lovegrass, sand............. P 20-30........... 5 14 Light; KNO3.... Prechill at 5
or 10 C for 6
weeks; see
Sec. 201.57a.Lovegrass, weeping.......... P 20-35........... 5 14 Light.......... KNO3; see Sec.
201.57a.Lupine:
Blue.................... B, T, S 20.............. 4 \1\ 10
White................... B, T 20.............. 3 \1\ 10
Yellow.................. B, T 20.............. 7 \1\ 10Manilagrass................. P 35-20........... 10 28 Light; KNO3....Medic, black................ B, T, S 20.............. 4 \1\ 7 See ] (b)(11)..Milkvetch................... B, T 20.............. 6 \1\ 14
Alternate method........ B, TB, T 15-25........... 10 \1\ 21Millet:
Browntop................ B, P, T 20-30; 30....... 4 14 Light and KNO3 Predry at 35 or
optional. 40 C for 7
days and test
at 30 C.
Alternate method...... B, P, T 5-35............ 4 14 Light; KNO3....
Foxtail................. B, T 15-30; 20-30.... 4 10
Japanese................ B, T 20-30........... 4 10
Pearl................... B, T 20-30........... 3 7
Proso................... B, T 20-30........... 3 7Molassesgrass............... P 20-30........... 7 21 Light..........Mustard:
Black................... P 20-30........... 3 7 Light.......... KNO3 and
prechill at 10
C for 3 days.
India................... P 20-30........... 3 7 Light.......... Prechill at 10
C for 7 days
and test for 5
days; KNO3.
White................... P 20-30........... 3 5 Light..........Napiergrass................. B, T 20-30........... 3 10Needlegrass, green:
Method 1................ P 15-30........... 7 14 H2 SO4,GA3 and
thiram; dark;
see ] (b)(7).
Method 2................ P 15-30........... 7 14 KNO3; dark; see
(b)(7).Oat......................... B, T, S 20; 15.......... 5 10 Prechill at 5
(7).Oat......................... B, T, S 20; 15.......... 5 10 Prechill at 5
or 10 C for 5
days and test
for 7 days or
predry and
test for 10
days..Oatgrass, tall.............. P 20-30........... 6 14 Light..........Orchardgrass................ P, TS 15-25........... 7 21 Light; Prechill at 5
germination or 10 C for 7
more rapid on days.
soil.
Panicgrass, blue............ P, TS 20-30........... 7 28 Light..........Panicgrass, green........... P 15-35........... 10 28 Light; KNO3
optional.Pea, field.................. B, T, S 20.............. 3 \1\8Peanut...................... B, T, S 20-30; 25....... 5 \1\10 Remove shells.. Ethephon or
ethylene; see
] (a) (10) and
(11).Rape:
Annual.................. B, T 20-30........... 3 7
Bird.................... P 20-30........... 3 10 Light.......... KNO3.
Turnip.................. B, T 20-30........... 3 7
Winter.................. B, T 20-30........... 3 7Redtop...................... P, TB 20-30........... 5 10 Light.......... KNO3.Rescuegrass................. P, S 10-30........... 7 28 Light; see ] In soil at 15
(b)(8) for C.
(8) for C.
alternate
method.Rhodesgrass................. P 20-30........... 6 14 Light; KNO3....Rice........................ T, S 20-30; 30....... 5 14 See ] (b)(9) Presoak; see ]
for alternate (b)(9).
method.Ricegrass, Indian........... P 15.............. 7 42 ............... Prechill at 5 C
for 4 weeks
and test for
21 additional
days; see Sec.
201.57a.
Alternate method........ S 5-15; 15; 15-25. 7 28 ............... Dark; prechill
in soil at 5 C
for 4 weeks;
see Sec.
201.57a.Roughpea.................... B, T 20.............. 7 \1\14Rye......................... B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days or
predry.Rye, mountain............... B, T 20; 15.......... 4 7 ............... See Sec.
201.57a.Ryegrass:
Annual.................. P, TB 15-25........... 5 14 Light optional; Light; KNO3;
see ] (b)(10) prechill at 5
for or 10 C for 5
fluorescence days and test
test. at 15-25 C; if
still dormant
prechill for 3
days and
continue test
at 15-25 C an
additional 4
days.
Intermediate............ P, TB 15-25........... 7 14 Light.......... KNO3 and
prechill at 5
or 10 C for 5
days and test
at 15-25 C; if
still dormant
rechill for 3
days and
continue test
at 15-25 C an
additional 4
days.
Perennial............... P, TB 15-25........... 5 14 Light optional; Light; KNO3;
see ] (b)(10) prechill at 5
for or 10 C for 5
fluorescence days and test
test. at 15-25 C; if
still dormant
rechill for 3
days and
continue test
at 15-25 C an
additional 4
days.
Wimmera................. P, TB 15-25; 20-30.... 5 14 Light optional. Light; KNO3;
prechill at 5
or 10 C for 5
days and test
at 15-25 C; if
still dormant
rechill for 3
days and
continue test
at 15-25 C an
additional 4
days.Safflower................... P, B, T, S 15; 20.......... 4 14 Light at 15 C..Sagewort, Louisiana......... P 15-25........... 7 14 Light..........Sainfoin.................... B, T 20-30........... 4 \1\ 14Saltbush, fourwing.......... B 20.............. 5 14 See ] (b)(13).. Prechill at 5 C
for 7 days.
Alternate method........ B 15.............. ....... 21Sesame...................... B, T, TB 20-30........... 3 6Sesbania.................... B, T 20-30........... 5 \1\ 7Smilo....................... P 20-30........... 7 42 Light.......... Prechill at 5 C
for 2 weeks;
see Sec.
201.57a.
Sorghum..................... B, T, S 20-30........... 4 10 ............... Prechill grain
vars. at 5 or
10 C for 5
days; test
sweet vars. at
30-45 C,
maintaining 45
C for 2-4
hours per day.Sorghum almum............... T, S 20-35; 15-35.... 5 21 ............... Prechill at 5 C
for 5 days; on
the 10th day
of test, clip
or pierce the
distal end of
ungerminated
seeds.Sorghum-sudangrass.......... B, T, S 20-30; 25....... 4 10 ............... Prechill at 5
or 10 C for 5
days.Sorgrass \2\................ B, T, S 15-35; 20-35.... 5 21 ............... Prechill at 5
or 10 C for 7
days.Sourclover.................. B, T 20.............. 3 \1\ 14 See ] (b)(11)..Soybean..................... B, T, S, TC 20-30; 25....... 5 \1\ 8Spelt....................... B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days, or
predry.Sudangrass.................. B, T, S 20-30; 15-30.... 4 10 ............... Prechill at 10
C for 5 days.Sunflower................... T,B 20.............. 4 7Sweetclover:
White................... B, T, S 20.............. 4 \1\ 7 See ] (b)(11)..
Yellow.................. B, T, S 20.............. 4 \1\ 7 See ] (b)(11)..Sweet vernalgrass........... P 20-30........... 6 14 Light..........Sweetvetch, northern........ B, TB, T 15-25; 20....... 14 \1\ 28Switchgrass................. P, TS 15-30........... 7 14 Light; KNO3.... Prechill at 5 C
for 2 weeks;
see Sec.
201.57a.Timothy..................... P, TB 15-25; 20-30.... 5 10 Light; see ] KNO3 and
(a)(9). prechill at 5
(9). prechill at 5
or 10 C for 5
days.Timothy, turf............... P, TB 15-25; 20-30.... 5 10 Light.......... KNO3 and
prechill at 5
or 10 C for 5
days.Tobacco..................... P, TB 20-30........... 7 14 Light..........Trefoil:
Big..................... B, T 20.............. 5 \1\ 12
Birdsfoot............... B, P, T 20.............. 5 \1\ 12Triticale................... B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days, or
predry.Vaseygrass.................. P 20-35........... 7 21 Light.......... KNO3.Veldtgrass.................. P 10-30........... 7 28 Light.......... See Sec.
201.57a.Velvetbean.................. B, T, S, C 20-30........... 3 \1\ 14Velvetgrass................. P 20-30........... 6 14 Light..........Vetch:
Common.................. B, T 20.............. 5 \1\ 10
Hairy................... B, T 20.............. 5 \1\ 14
Hungarian............... B, T 20.............. 5 \1\ 10
Monantha................ B, T 20.............. 5 \1\ 10
Narrowleaf.............. B, T 20.............. 5 \1\ 14
Purple.................. B, T 20.............. 5 \1\ 10
Woollypod............... B, T 20.............. 5 \1\ 14 ............... Prechill at 10
C for 5 days,
test at 15 C.Wheat:
Common.................. B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days, or
predry.
Club.................... B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days, or
predry.
Durum................... B, T, S 20; 15.......... 4 10 ............... Prechill at 5
or 10 C for 5
days, or
predry.
Polish.................. B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days, or
predry.
Poulard................. B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days, or
predry.Wheat Agrotricum............ B, T, S 20; 15.......... 4 7 ............... Prechill at 5
or 10 C for 5
days, or
predry.Wheatgrass:
Beardless............... P, TB 15-25........... 7 14 Light and KNO3 KNO3 and
optional. prechill at 5
or 10 C for 7
days.
Fairway crested......... P, TB 15-25; 20-30.... 5 14 Light and KNO3 KNO3 and
optional. prechill at 5
or 10 C for 7
days.
Standard crested........ P, TB 15-25; 20-30.... 5 14 Light and KNO3 KNO3 and
optional. prechill at 5
or 10 C for 7
days.
Intermediate............ P 15-25........... 5 28 Light and KNO3 KNO3 and
optional. prechill at 5
or 10 C for 7
days.
Alternate method...... P 20-30........... 5 28 Light..........
Pubescent............... P 15-25........... 5 28 Light and KNO3 KNO3 and
optional. prechill at 5
or 10 C for 7
days.
Alternate method...... P 20-30........... 5 28 Light..........
Siberian................ P, TB 15-25........... 7 14 Light and KNO3 KNO3 and
optional. prechill at 5
or 10 C for 7
days.
Slender................. P, TB 15-25; 10-30.... 5 14 Light and KNO3 Prechill at 5
optional. or 10 C for 5
days; if still
dormant on the
10th day,
rechill 2
days, then
place at 20-30
C for 4 days.
Streambank.............. P, TB 15-25........... 5 14 Light and KNO3 Prechill at 5
optional. or 10 C for 5
days.
Tall.................... P 15-25........... 5 21 Light and KNO3 Prechill at 5
optional. or 10 C for 5
days.
Alternate method...... P 20-30........... 5 21 Light.......... Prechill at 5
or 10 C for 5
days.
Western................. B, P, T 15-30........... 7 28 Dark........... KNO3 or soil;
see Sec.
201.57a.Wildrye:
Basin................... P 15-25........... 10 21 ............... See Sec.
201.57a.
Canada.................. P 15-30........... 7 21 Light.......... Prechill at 5 C
for 2 weeks.
Russian................. P 20-30........... 5 14 Light.......... Prechill at 5
or 10 C for 5
days.
VEGETABLE SEEDArtichoke................... B, T 20-30........... 7 21Asparagus................... B, T, S 20-30........... 7 21Asparagusbean............... B, T, S 20-30........... 5 \1\ 8Bean:
Garden.................. B, T, S, TC 20-30; 25....... None \1\ 8 ............... See ] (b)(12).
Lima.................... B, T, C, S 20-30........... 5 \1\ 9
Runner.................. B, T, S 20-30........... 5 \1\ 9Beet........................ B, T, S 20-30........... 3 14 See ] (b)(3)...Broadbean................... S, C 20.............. 4 \1\ 14 See ] (b)(11).. Prechill at 10
C for 3 days.Broccoli.................... B, P, T 20-30........... 3 10 ............... Prechill at 5
or 10 C for 3
days; KNO3 and
light.Brussels sprouts............ B, P, T 20-30........... 3 10 Prechill at 5
or 10 C for 3
days; KNO3 and
light.Burdock, great.............. B, T 20-30........... 7 14Cabbage..................... B, P, T 20-30........... 3 10 ............... Prechill at 5
or 10 C for 3
days; KNO3 and
light.Cabbage, Chinese............ B, T 20-30........... 3 7
Cabbage, tronchuda.......... B, P 20-30........... 3 10 ............... Prechill at 5
or 10 C for 3
days; KNO3 and
light.Cardoon..................... B, T 20-30........... 7 21Carrot...................... B, T 20-30........... 6 14Cauliflower................. B, P, T 20-30........... 3 10 ............... Prechill at 5
or 10 C for 3
days; KNO3 and
lightCeleriac.................... P 5-25; 20........ 10 21 Light; see ]
(a)(9).Celery...................... P 15-25; 20....... 10 21 Light; see ]
(9).Celery...................... P 15-25; 20....... 10 21 Light; see ]
(a)(9).Chard, Swiss................ B, T, S 20-30........... 3 14 See ] (b)(3)...Chicory..................... P, TS 20-30........... 5 14 Light; KNO3 or
(9).Chard, Swiss................ B, T, S 20-30........... 3 14 See ] (b)(3)...Chicory..................... P, TS 20-30........... 5 14 Light; KNO3 or
soil; see ]
(a)(9).
(9).
Chives...................... B, T 20.............. 6 14Citron...................... B, T 20-30........... 7 14 Soak seeds 6 Test at 30 C.
hrs.Collards.................... B, P, T 20-30........... 3 10 ............... Prechill at 5
or 10 C for 3
days; KNO3 and
light .Corn, sweet................. B, T, S, TC 20-30; 25....... 4 7Cornsalad................... B, T 15.............. 7 28 Test at 10 C...Cowpea...................... B, T, S 20-30........... 5 \1\ 8Cress:
Garden.................. B, P, T 15.............. 4 10 ............... Light.
Upland.................. P, TB 20-35........... 4 7 Light; KNO3....
Water................... P 20-30........... 4 14 Light..........Cucumber.................... B, T, S 20-30........... 3 7 Keep substratum
on dry side;
see ] (a)(3).Dandelion................... P, TB 20-30........... 7 21 Light; see ]
(a)(9).Dill........................ B, T 20-30........... 7 21Eggplant.................... P, TB, RB, T 20-30........... 7 14 Light; KNO3....Endive...................... P, TS 20-30........... 5 14 Light; KNO3 or See ] (b)(6).
(9).Dill........................ B, T 20-30........... 7 21Eggplant.................... P, TB, RB, T 20-30........... 7 14 Light; KNO3....Endive...................... P, TS 20-30........... 5 14 Light; KNO3 or See ] (b)(6).
soil.Gherkin, West India......... B, T, S 20-30........... 3 7 Test at 30 C...Kale........................ B, P, T 20-30........... 3 10 ............... Prechill at 5
or 10 C for 3
days; KNO3 and
light.Kale, Chinese............... B, P, T 20-30........... 3 10 ............... Prechill at 5
or 10 C for 3
days; KNO3 and
light.Kale, Siberian.............. B, P, T 20-30; 20....... 3 7Kohlrabi.................... B, P, T 20-30........... 3 10 ............... Prechill at 5
or 10 C for 3
days; KNO3 and
light.Leek........................ B, T 20.............. 6 14Lettuce..................... P 20.............. None 7 Light.......... Prechill at 10
C for 3 days
or test at 15
C.Melon....................... B, T, S 20-30........... 4 10 Keep substratum
on dry side;
see ] (a)(3).Mustard, India.............. P 20-30........... 3 7 Light.......... Prechill at 10
C for 7 days
and test for 5
additional
days; KNO3.Mustard, spinach............ B, T 20-30........... 3 7Okra........................ B, T 20-30........... 4 \1\ 14Onion....................... B, T 20.............. 6 10
Alternate method........ S 20.............. 6 12Onion, Welsh................ B, T 20.............. 6 10Pak-choi.................... B, T 20-30........... 3 7Parsley..................... B, T, TS 20-30........... 11 28Parsnip..................... B, T, TS 20-30........... 6 28Pea......................... B, T,S 20.............. 5 \1\ 8Pepper...................... TB, RB, T 20-30........... 6 14 ............... Light and KNO3.Pumpkin..................... B, T, S 20-30........... 4 7 Keep substratum
on dry side;
see ] (a)(3).Radish...................... B, T 20.............. 4 6Rhubarb..................... TB, TS 20-30........... 7 21 Light..........Rutabaga.................... B, T 20-30........... 3 14Sage........................ B, T, S 20-30........... 5 14Salsify..................... B, T 15.............. 5 10 Prechill at 10
C for 3 days..Savory, summer.............. B, T 20-30........... 5 21Sorrel...................... P, TB, TS 20-30........... 3 14 Light.......... Test at 15 C.Soybean..................... B, T, S, TC 20-30; 25....... 5 \1\ 8Spinach..................... TB, T 15;10........... 7 21 Keep substratum
on dry side;
see ] (a)(3).Spinach, New Zealand........ T 15; 20.......... 5 21 Soak fruits On 21st day
overnight (16 scrape fruits
hrs), air dry and test for 7
7 hrs; plant additional
in very wet days.
towels; do not
rewater unless
later counts
exhibit drying
out.Alternate method............ B, T 15.............. 5 21 Remove pulp
from basal end
of fruit.
Squash...................... B, T, S 20-30........... 4 7 Keep substratum
on dry side;
see ] (a)(3).Tomato...................... B, P, RB, T 20-30........... 5 14 ............... Light; KNO3.Tomato, husk................ P, TB 20-30........... 7 28 Light; KNO3....Turnip...................... B, T 20-30........... 3 7Watermelon.................. B, T, S 20-30; 25....... 4 14 Keep substratum Test at 30 C.
on dry side;
see ] (a)(3).----------------------------------------------------------------------------------------------------------------\1\ Hard seeds may be present. (See Sec. 201.57)\2\ Rhizomatous derivatives of a johnsongrass sorghum cross or a johnsongrass sudangrass cross. [20 FR 7928, Oct. 21, 1955]
Editorial Note: For Federal Register citations affecting Sec. 201.58, see the List of CFR Sections Affected, which appears in the Finding Aids section of the printed volume and at www.fdsys.gov.
examinations in the administration of the act Sec. 201.58a Indistinguishable seeds.
When the identification of the kind, variety, or type of seed or determination that seed is hybrid is not possible by seed characteristics, identification may be based upon the seedling, growing plant or mature plant characteristics according to such authentic information as is available.
(a) Ryegrass. In determining the pure seed percentage of perennial ryegrass and annual ryegrass, 400 seeds shall be grown on white filter paper and the number of fluorescent seedlings determined under ultraviolet light at the end of the germination period (see Sec. 201.58(b)(10)).
(1) Fluorescence results are to be determined as test fluorescence level (TFL) to two decimal places as follows:[GRAPHIC] [TIFF OMITTED] TR14DE94.001
(2) The percentage of perennial ryegrass is calculated as follows:
[GRAPHIC] [TIFF OMITTED] TR14DE94.002
where VFL=Variety fluorescence level.
(3) Using results from the above formula, the percentage of annual ryegrass is calculated as follows: % Annual Ryegrass = % Pure Ryegrass-% Perennial Ryegrass
(4) If the test fluorescence level (TFL) of a perennial ryegrass is equal to or less than the variety fluorescence level (VFL) described for the variety, all pure ryegrass is considered to be perennial ryegrass and the formula is not applied.
(5) If the test fluorescence level (TFL) of an annual ryegrass is equal to or greater than the variety fluorescence level (VFL) described for the variety, all pure ryegrass is considered to be annual ryegrass and the formula is not applied.
(6) A list of variety fluorescence level (VFL) descriptions for perennial ryegrass varieties which are more than 0 percent fluorescent and annual ryegrass varieties which are less than 100 percent fluorescent is maintained and published by the National Grass Variety Review Board of the Association of Official Seed Certifying Agencies (AOSCA). If the variety being tested is not stated or the fluorescence level has not been described, the fluorescence level shall be considered to be 0 percent for perennial ryegrass and 100 percent for annual ryegrass. Both VFL (annual) and VFL (perennial) values must always be entered in the formula. If a perennial ryegrass variety is being tested, the VFL (annual) value is 100 percent. If an annual ryegrass variety is being tested, the VFL (perennial) value is 0 percent. For blends the fluorescence level shall be interpolated according to the portion of each variety claimed to be present.
(b) Sweetclover. To determine the presence of yellow sweetclover in samples of white sweetclover, at least 400 seeds shall be subjected to the chemical test as follows:
(1) Preparation of test solution: Add 3 grams of cupric sulfate (CuSO4) to 30 ml of household ammonia (NH4 OH, approximately 4.8 percent) in a stoppered bottle to form tetraamminecopper sulfate ([Cu(NH3)4]SO4) solution used for this test. After mixing, a light blue precipitate of cupric hydroxide (Cu(OH)2) should form. If no precipitate forms, add additional CuSO4 until a precipitate appears. Since the strength of household ammonia can vary, formation of a precipitate indicates that a complete reaction has taken place between CuSO4 and NH4 OH; otherwise fumes from excess ammonium hydroxide may cause eye irritation.
(2) Preparation of seeds: To insure imbibition, scratch, prick, or otherwise scarify the seed coats of the sweetclover seeds being tested. Soak seeds in water for 2 to 5 hours in a glass container.
(3) Chemical reaction: When seeds have imbibed, remove excess water and add enough test solution to cover the seeds. Seeds coats of yellow sweetclover will begin to stain dark brown to black; seed coats of white sweetclover will be olive or yellow-green. Make the separation within 20 minutes, since the seed coats of white sweetclover will eventually turn black also.
(4) Calculation of results: Count the number of seeds which stain dark brown or black and divide by the total number of seeds tested; multiply by the pure seed percentage for Melilotus spp.; the result is the percentage of yellow sweetclover in the sample. The percentage of white sweetclover is found by subtracting the percentage of yellow sweetclover from the percentage of Melilotus spp. pure seed.
(c) Wheat. In determining varietal purity, the phenol test may be used. From the pure seed sample count four replicates of 100 seeds each. Soak the seed in distilled water for 16 hours; then flush with tap water and remove the excess water from the surface of the seeds. Place two layers of filter paper in a container and moisten with a 1 percent phenol (C6 H5 OH) solution. Place the seed, palea side down, on the two layers of filter paper and cover the container. A preliminary observation may be made at 2 hours. At 4 hours, record the number of seeds in each of the following color categories:
(1) Ivory.
(2) Fawn.
(3) Light Brown.
(4) Brown.
(5) Brown Black.
(d) Soybean. In determining the varietal purity, the peroxidase test may be used. Remove and place the dry seed coat from seeds into individual test tubes or suitable containers. Add 10 drops (0.5-1.0 ml) of 0.5 percent guaiacol (C7 H8 O2) to each test tube. After waiting 10 minutes add one drop (about 0.1 ml) of 0.1 percent hydrogen peroxide (H2 O2). One minute after adding hydrogen peroxide, record the seed coat as peroxidase positive (high peroxidase activity) indicated by a reddish-brown solution or peroxidase negative (low peroxidase activity) indicated by a colorless solution in the test tube. Various sample sizes may be used for this test. Test results shall include the sample size tested.
(e) Oat. In determining the varietal purity, the fluorescence test may be used. Place at least 400 seeds on a black background under a F15T8-BLB or comparable ultraviolet tube(s) in an area where light from other sources is excluded. Seeds are considered fluorescent if the lemma or palea fluoresce or appear light in color. ``Partially fluorescent'' seeds shall be considered fluorescent. Seeds are considered nonfluorescent if the lemma and palea do not fluoresce and appear dark in color under the ultraviolet light. [59 FR 64514, Dec. 14, 1994]
Editorial Note: For Federal Register citations affecting Sec. 201.58a, see the List of CFR Sections Affected, which appears in the Finding Aids section of the printed volume and at www.fdsys.gov. Sec. 201.58b Origin.
The presence of incidental weed seeds, foreign matter, or any other existing circumstances shall be considered in determining the origin of seed. [5 FR 35, Jan. 4, 1940. Redesignated at 20 FR 7940, Oct. 21, 1955] Sec. 201.58c Detection of captan, mercury, or thiram on seed.
The bioassay method may be used according to the procedure given in Association of Official Seed Analysts, Handbook No. 26, ``Microbiological Assay of Fungicide-treated Seeds'', May 1964. [38 FR 12733, May 15, 1973] Sec. 201.58d Fungal endophyte test.
A fungal endophyte test may be used to determine the amount of fungal endophyte (Acremonium spp.) in certain grasses.
(a) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the presence of fungal endophyte:
(1) Prepare a 1 percent aqueous aniline blue solution by dissolving 1 gram aniline blue in 100 ml distilled water.
(2) Prepare the endophyte staining solution of one part of 1 percent aniline blue solution with 2 parts of 85 percent lactic acid (C3 H6 O3).
(3) Use stain as-is or dilute with water if staining is too dark.
(b) Procedure for determining levels of fungal endophyte in grass seed:
(1) Take a sub-sample of seed (1 gram is sufficient) from the pure seed portion of the kind under consideration.
(2) Digest seed at room temperature for 12-16 hours in a 5 percent sodium hydroxide (NaOH) solution or other temperature/time combination resulting in adequate seed softening.
(3) Rinse thoroughly in running tap water.
(4) De-glume seeds and place on a microscope slide in a drop of endophyte staining solution. Slightly crush the seeds. Use caution to prevent carryover hyphae of fungal endophyte from one seed to another.
(5) Place coverglass on seed and apply gentle pressure.
(6) Examine with compound microscope at 100-400x magnification, scoring a seed as positive if any identifiable hyphae are present.
(7) Various sample sizes may be used for this test. Precision changes with sample size; therefore, the test results must include the sample size tested.
(c) Procedure for determining levels of fungal endophyte in seedlings from seed samples suspected to contain fungal endophyte:
(1) Select seeds at random and germinate.
(2) Examine seedlings from the sample germinated after growing for a minimum of 48 days.
(3) Remove the outermost sheath from the seedling. Tissue should have no obvious discoloration from saprophytes and should have as little chlorophyll as possible.
(4) Isolate a longitudinal section of leaf sheath approximately 3-5 mm in width.
(5) Place the section on a microscope slide with the epidermis side down.
(6) Stain immediately with the endophyte staining solution as prepared in paragraph (a) (2) and (3) of this section. Allow dye to remain at least 15 seconds but no more than one minute.
(7) Blot off the excess dye with tissue paper. Sections should remain on the slide, but may adhere to the tissue paper; if so, remove and place in proper position on the slide.
(8) Place a coverglass on the sections and flood with water.
(9) Proceed with evaluation as described in paragraph (b) (6) and (7) of this section. [59 FR 64515, Dec. 14, 1994]
tolerances