Agriculture Reference
In-Depth Information
cut of the razor blade, tetrazolium solution becomes accessible to the embryo and the internal tissues are
exposed to facilitate interpretation of staining patterns. If seeds are too small to permit consistent slicing
through the embryo, a lateral cut may be made to open the seed coat and further steps taken after staining
to allow viewing of the embryo. For large dicot seeds such as cotton and watermelon in which the embryo
comprises nearly the entire interior of the seed, the seed coat may be removed from the soaked seed, expos-
ing the entire embryo. In Pinus spp., the embryo is located in the center of the seed and is surrounded by the
megagametophyte. The seed coat is permeable to tetrazolium, so it is sliced off-center after staining and the
embryo excised and evaluated. These are only a few examples of preparation techniques that can be applied
to other seeds with similar structures.
Except for hard seeds, tetrazolium solution is able to penetrate the seed coat of legumes and certain
other species, so that dry seeds may be placed directly in the solution with no further preparation. However,
uniformity of staining intensity is enhanced by pre-moistening these seeds in water. It may also be desirable
to remove the seed coat from certain beans and other large-seeded legumes after moistening and before
staining to improve the uniformity and speed of staining.
The following procedures are recommended for preparation of different groups of seeds prior to stain-
ing. Several of these procedures may be necessary for the same seed. Speciic examples of different genera
are given for each of the techniques and procedures presented. These examples were taken from Table 1 of
the ISTA Tetrazolium Handbook (1985) and ISTA Working Sheets on Tetrazolium Testing (2003).
recommended Procedures for Preparation for Staining with Tz Solution:
1. Moistening
a. Moisten slowly between or on moist paper or cloth media, at least until the internal tissues
become fully imbibed [e.g., large-seeded legumes such as Phaseolus and Glycine (Fabaceae)].
b. Moisten by soaking directly in water, or by use of extra moisture within the media. Excessively
dry or aged seeds frequently beneit by slow initial moistening before soaking [ Vicia faba
(Fabaceae)].
c. Prolong the moistening time to provide an opportunity for elongating radicles to crack some of
the seed coat. This aids in the removal of the seed coat [ Carthamus (Asteraceae)].
d. Moistening may not be essential [ Medicago and Melilotus (Fabaceae)].
2. Additional seed coat preparation usually not necessary [ Abies and Pinus (Pinaceae); Brassica
spp. (Brassicaceae); Phaseolus and Glycine (Fabaceae)].
3. Remove hard-seededness by one of several ways
a. Nicking, puncturing or cutting of the seed coat in a non-destructive location such as the back
side of the cotyledon or opposite the hilum [most hard-seeded legumes (Fabaceae)].
b. Filing, sandpapering, or grinding in a non-destructive position [ Chaenomeles (Rosaceae) and
Cassia (Fabaceae)].
c. Cracking the seed coat with a vise, adjustable wrench, hammer, nut cracker, dog nail clipper,
etc. The inner seed coat should be punctured, cut or torn [many hard-seeded nuts and nutlets of
Fagus (Fagaceae) and Juglans (Juglandaceae)].
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