Ultrastructure of the vascular cambium of Pinus ponderosa seedlings preserved by cryofixation and cryosubstitution.

摘要

Current knowledge of cambial ultrastructure is the result of careful use of chemical fixation. Cryofixation is virtually instantaneous and when used in combination with cryosubstitution, the tissues are not exposed to fixative chemicals, osmotic gradients or pH stress so ultrastructural detail is more accurately preserved. Given the unique structure of fusiform cambial cells, these factors make cryofixation and substitution (CFS) particularly appropriate for electron microscopy of the cambium. To my knowledge, this is the first time CFS has been used to study cambial cell structure and division. With successful CFS of cambial cells, the tonoplast was smooth and unbroken, and the plasma membrane was tightly appressed to the cell wall with no wrinkles, undulations or folds. Electron dense bodies were not observed between the plasma membrane and the cell wall. Microtubules were present on nuclear membrane in contact with the nuclear pores, at the primary cell walls of the cambial cells, and at the developing secondary cell walls of xylem and phloem cells. Axially oriented microfilament bundles traversed the cytoplasm and were seen in association with the nuclear membrane. CFS preserved ultrastructural features of fusiform cambial cell division that were not retained with chemical fixation. Spindle microtubules separated daughter chromosomes diagonally across the radial width of the fusiform cells. Microtubules were present between the separating chromosomes at anaphase, and may have given rise to the microtubules of the phragmoplast. The cell partitioning membrane (cell plate) was assembled in the phragmoplast by microtubule arrays that organised numerous vesicles at the phragmoplast midline. The vesicles fused to form tubulo-vesicular networks at the trailing edge of the phragmoplast. Consolidation of these networks into complete cell membranes took place in the thin peripheral cytoplasm trailing the phragmoplast. Where the new membrane merged with the old, the parent plasmalemma was loosened from the cell wall and the membranes joined via a short tubulo-vesicular network.