Physiological changes in gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation.

BACKGROUND AND AIMS: Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing. METHODS: In standard two-step preculture, excised gentian axillary buds were incubated for 11 d on MS medium with 0.1 m sucrose at 25 degrees C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0.4 m and 0.7 m sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied. KEY RESULTS: During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0.1 m sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2.4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels. CONCLUSIONS: These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.

Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses.

BACKGROUND AND AIMS: Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival. METHODS: Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments. KEY RESULTS: Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant. CONCLUSIONS: The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.