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DEVELOPMENT AND EVALUATION OF A FREEZE‐ETCH, FREEZE‐FRACTURE TECHNIQUE APPLIED TO DEVELOPING WHEAT ENDOSPERM
Author(s) -
Bechtel D. B.,
Barnett B. D.
Publication year - 1986
Publication title -
american journal of botany
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.218
H-Index - 151
eISSN - 1537-2197
pISSN - 0002-9122
DOI - 10.1002/j.1537-2197.1986.tb12124.x
Subject(s) - endosperm , glycerol , caryopsis , liquid nitrogen , paraformaldehyde , materials science , biology , botany , chemistry , biochemistry , poaceae , organic chemistry
A technique was developed and evaluated whereby differentiating wheat endosperm tissue could be processed for the freeze‐etch, freeze‐fracture technique without the use of chemical fixatives. Field grown, developing hard red winter wheat (cv. Newton) caryopses were infiltrated with glycerol prior to freezing in liquid nitrogen‐cooled Freon‐22. Frozen samples were placed in cryogenic vials and stored in liquid nitrogen until needed. Freeze‐fracture was carried out under standard conditions. Evaluation of replicas from glycerol‐imbibed endosperm was made by comparing them to replicas obtained from freshly frozen untreated endosperm, and endosperm that had been prefixed in glutaraldehyde and paraformaldehyde. Other evaluations were made by comparing replicas of glycerol‐treated endosperm to thin sections obtained from wheat that had been imbibed with glycerol, fixed, dehydrated, and infiltrated and embedded in epoxy resin for routine electron microscopy. The results indicate that if the unfixed glycerol‐treated wheat endosperm is handled carefully, replicas can be obtained which show few artifacts due to glycerol and freezing. Typical artifacts such as vesiculation of RER, ice crystal damage, production of fusion intermediates, and membrane particle segregation can be nearly eliminated when this technique is applied to developing wheat endosperm.