Desiccation and cryopreservation of spruce somatic embryogenic tissue and mature somatic embryos
Date
1997
Authors
Percy, Robin Elizabeth Laird
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Abstract
The effects of drying and cryopreservation on the survival of spruce (Picea glauca and Picea glauca x engelmanni ) embryogenic cultures were investigated. This work was undertaken with the aim of developing a reliable, well-defined technique for the drying and storage of conifer embryogenic tissue and mature somatic embryos. In this procedure, tissue is dried over salt solutions and then frozen directly in liquid nitrogen. Specific objectives were to: (i) characterize water release from embryogenic cultures using a well controlled drying system, (ii) determine whether cultured tissue could be desiccated in dry air to low relative water contents (RWC) without causing fatal damage, (iii) determine whether the tissue, once dried to well-defined water contents, could be frozen directly in liquid nitrogen without the use of cryoprotectants and still remain viable after thawing and rehydration, and (iv) determine a convenient method of assessing viability following drying and/or freezing. Two developmental stages were tested: immature somatic embryos (embryogenic tissue) and mature somatic embryos.
My study has shown that TTC (2,3,5-triphenyltetrazolium chloride) stain is a reliable indicator of viability for spruce embryogenic tissue and mature somatic embryos following drying and freezing. However, the vital stain fluorescein diacetate (FDA) is not suitable for use with embryogenic tissue. TTC slightly over-estimates the viability of mature somatic embryos following freezing. It is recommended that more than one test always be used for estimating viability and that any proxy indicator of viability be used in conjunction with regrowth tests.
Embryogenic tissue initially survived drying to very low RWC ( < 0.025). There was a decline in desiccation tolerance over time that could not be explained by conventional tissue water relations. The decline in tolerance might have been related to changes in the ratio of small, densely cytoplasmic cells to large vacuolate cells (ratio decreased over time) since the cultures had recently come out of cryopreservation when the study began. The drying technique employed reliably dried tissue to known RWC and was rigorously tested as an alternative to the conventional multi-step method for cryostorage. However, embryogenic tissue did not survive direct immersion in liquid nitrogen following drying, regardless of the RWC to which it had been pre-dried. It is concluded that drying alone cannot be used as replacement for the steps used to prepare conifer embryogenic tissue for cryopreservation, and that cryoprotectants likely play roles, in addition to their osmotic effect, in the maintenance of cellular integrity.
Mature somatic embryos, however, survived the removal of virtually all free water, and subsequent freezing in liquid nitrogen. Remarkably, no cryoprotectant additives were required. For conifer somatic embryos, this is the first report of embryos cryopreservation without the application of cryoprotectants and a slow-freezing step. In terms of mature conifer somatic embryos, it is the first report of survival following exposure to liquid nitrogen. The RWC of interior and white spruce somatic embryos at bound water was 0.13-0.14 ( or 0.28 gH2O g-1 fm), similar to the average water content of white and interior spruce zygotic embryos excised from stored dried seed (0.325 gH2O g-1 fm and 0.365 gH2O g-1fm, respectively) .
The highest survival after freezing in liquid nitrogen was in those embryos pre-dried to water potentials of -15 to -20 MPa. The RWC at these water potentials was close to bound water values. There was minimal survival after freezing embryos pretreated at higher water potentials, likely due to intracellular ice formation, and there was no survival of embryos after pretreatment over silica gel, probably as a result of severe mechanical disruption within cells.