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Ice nucleation activity (INA) of plant intrinsic origins is considered to play important roles in plant cold hardiness mechanisms. Yet, only a few studies have addressed the spatial and temporal localization of plant INA, how it is regulated and what its functional roles are. In our previous study ( Kishimoto et al., 2014 ), we revised a test tube method and developed a highly reproducible assay for measuring INA of plant specimens and demonstrated that high INA occurred in the cell wall fraction of wintering bark tissues of blueberry stems and corresponded well to the freezing behavior (extracellular freezing) of the stem bark. Here, we followed precisely seasonal changes in the stem INA of two blueberry cultivars and alterations in the stem INA caused by artificial incubation at various low temperatures. INA of newly developed shoots was low but increased rapidly by July when the stem became seemingly matured, then gradually increased with the maximum in October or early November just before the first autumnal frost. Following the subsequent recurrent frosts, the stem INA gradually decreased. This tendency was consistent between the two cultivars differing in the level of cold hardiness. INA in the stems of September until February was increased by incubation at 0-7 °C whilst decreased by freezing to lower temperatures. The in vitro results corroborate the seasonal changes in the stem INA in the field but the mechanisms remain to be investigated. The highest level of INA (expressed as the median ice nucleation temperature) observed with current year stems (7.5 mm-long) of Woodard in October of 2010-2013 was -0.9 ∼ -1.0 °C when determined with 2 mL assay system (-1.1 ∼ -1.3 °C with 0.5 mL system). This may likely be one of the highest INA of biological origins ever reported.

Constant relative humidity (RH) chambers using phosphoric acid (PA) are useful for controlled desiccation of pollen and in vitro cultured materials for cool-dry storage or cryopreservation (Ishikawa et al., 2005, Ishikawa and Oda, 2008). Here we demonstrate that the chambers are also convenient for accelerated ageing of seeds. Rice seeds 3-11 years old (stored at 8-9°C) were artificially aged for 0-41 days in PA-assisted relative humidity (RH) chambers (RH 93%) placed in an incubator set at 37°C. At designated time periods during ageing, vigour and longevity of the seeds with different storage history were evaluated by monitoring germination timecourse and seedling shoot and root growth. Under these ageing conditions, seeds harvested in 2002 had half germinability (50% longevity) period of 33 days while older seeds (stored for longer periods) showed faster decline in germinability, longer lag time before germination, slower rates of germination and reduced shoot and root growth. Root growth was more sensitively affected by storage and accelerated ageing than shoot growth. PA-assisted system is simple, economical and safer than conventional RH chamber systems using saturated solutions or H2SO4 (Winston and Bates, 1960, Solomon, 1951). Various constant RH are available at desired temperatures using a single compound and convenient for realizing controlled deterioration of seeds at various RH and temperature regimes.

Relative humidity (RH) in constant RH chambers using various concentration of phosphoric acid (PA) was re-determined with a recently developed hygrometer (HygroLog NT2-D equipped with a sensor HygroClip S, ROTRONIC AG), which is superior in accuracy. Newly determined RH-PA concentration profiles were reverse semi-sigmoidal and very similar at 25 and 10°C. Differences in RH values between the present and previous determinations were larger at PA concentrations of 0-50% and 90-100%, primarily due to the property of the hygrometers employed. Accordingly, amendments were made to all the RH values in the previous paper (Ishikawa et al., 2005). This will make the PA-based constant RH system more accurate and reliable and ready to be used for various purposes.

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl2, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds.

� 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 � 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.

Smooth bromegrass (Bromus inermis Leyss) is an extremely cold hardy perennial grass and its cell culture is an excellent system for studying mechanisms of cold hardiness induced by low temperature or abscisic acid (ABA). Agrobacterium tumefaciens-mediated transformation of non-embryogenic bromegrass cultures was attempted. Agrobacterium strain EHA105 carrying a binary vector that contained the neomycin phosphotransferase (NPT II), beta-glucuronidase (GUS) and green fluorescent protein (GFP) genes were co-cultivated for 3 days with bromegrass cells at the late exponential or early stationary growth phase (7–9 days after subculture). These conditions gave optimal transformation efficiency. Putative transformants were identified by selection for geneticin resistance and by examining the calluses using fluorescence microscopy. This allows the elimination of escapes and selection of cells that express the target genes. PCR and Southern blot analyses confirmed the integration of the GUS and GFP genes into the genome of transformed bromegrass cell lines. Transformants with various levels of GUS expression were obtained with a high frequency following Agrobacterium-mediated gene transfer and visual selection by GFP. The successful transformation method described allows reverse genetics approaches for analyzing cold hardiness genes isolated from bromegrass cells.

For successful storage and cryopreservation of recalcitrant (short-lived or desiccation-sensitive) plant seeds, excised seed embryos or pollen, precise control of water content and/or slow drying rates are often crucial. To realize such controlled desiccation, we developed simple and safe constant relative humidity (RH) chambers using a single compound, phosphoric acid (PA). It consisted of a bowl of PA-water solution placed in a hermetically sealed box. With this apparatus, RH ranges of 4-88% were obtained from 100-10% PA solutions, respectively. Similar RH-PA concentration profiles were obtained at 24 and 7°C. Constant RH was ensured at a constant temperature as temperature fluctuations resulted in mirror-image RH oscillations within the chamber. When the temperature was greatly changed within 5-30°C ranges, the chamber regained the same equilibrium RH in 4-8 h. Using this system, typical recalcitrant winter rye pollen was successfully stored under RH50% at 7°C for 10 days. Alginate beads, irrespective of sucrose concentrations (0.7-1.2 M), were desiccated to equilibrium water contents of 11.5-32% (fresh weight basis) under constant RH of 27-78% at 24°C. This system is simple, economical and safer than conventional RH chamber systems using saturated solutions or sulfuric acid. It allows constant RH available at various temperatures and is useful for controlled desiccation of small recalcitrant genetic or in vitro resources for dry-cold or cryogenic storage.

Alginate beads containing axillary buds of in vitro-grown gentian (Gentiana scabra Bunge var. buergeri Maxim.), were successfully cryopreserved following 2 step-preculture with sucrose and desiccation. The optimal preculture conditions were as follows: axillary buds were excised from in vitro-grown gentian plants and precultured on semi-solid Murashige and Skoog (MS) medium containing 0.1 M sucrose for 10 days (25 °C, 16-h photoperiod) (first step). This was followed by incubation on semi-solid MS media containing 0.4 M (1 day) and then 0.7 M sucrose (1 day) (second step). After preculture, the buds were encapsulated in alginate beads and desiccated aseptically on silica gel for 9 h to a water content of 10% (fresh weight basis), followed by immersion in liquid nitrogen (LN). With this protocol, 87% of the gentian buds survived exposure to LN and showed normal development of shoots and roots in vitro and in vivo. Depletion of NH4NO3 in the regeneration medium did not improve survival following desiccation and exposure to LN. The results show that 2 step-preculture with sucrose is effectively applicable in encapsulation–desiccation based cryopreservation of gentian axillary buds. This preculture can replace the conventionally used lengthy cold-hardening treatment and is useful for routine cryopreservation of gentian germplasm.

Tissue-cultured shoot primordia of melon (Cucumis melo L. cv. prince melon) were successfully cryopreserved in liquid nitrogen (LN) using a slow prefreezing method. The highest survival and recovery were obtained with the following procedure. Three week-old shoot primordia clumps were dissected into pieces of 2-3 mm of diameter and precultured in standard medium for 3 days. They were directly soaked in CSP1 cryoprotective solution (10%w/v sucrose, 10%w/v dimethylsulfoxide and 5%w/v glycerol) and incubated at room temperature for 30 min. Samples were ice-inoculated at -8 °C and cooled at a rate of between 0.3 and 1 °C min−1 with a programmable freezer to -30 °C for prefreezing. They were then plunged into LN for storage. After rapid thawing in 40 °C water, the cryoprotective solution was slowly diluted 5 fold in a dropwise manner with 3% sucrose and the shoot primordia were transferred onto regeneration medium. Under optimal conditions, more than 80% of cryopreserved shoot primordia were viable and 50 to 80% regenerated shoots after one month of reculture. Cryopreserved shoot primordia could be used both for reproducing a shoot primordia culture and for regenerating plants.

Background The role of abscisic acid (ABA) as a possible activator of cold acclimation process was postulated since endogenous levels of ABA increase temporarily or constitutively during cold-hardening. Exogenous application of ABA has been known to induce freezing tolerance at ambient temperatures in in vitro systems derived from cold hardy plants. Yet, some cell cultures acquired much greater freezing tolerance by ABA than by cold whilst maintaining active growth. This raises questions about the relationships among ABA, cold acclimation and growth cessation. To address this question, we attempted to 1) determine whether exogenous ABA can confer freezing tolerance in chilling-sensitive rice suspension cells and seedlings, which obviously lack the mechanisms to acquire freezing tolerance in response to cold; 2) characterize this phenomenon by optimizing the conditions and compare with the case of cold hardy bromegrass cells. Results Non-embryogenic suspension cells of rice suffered serious chilling injury when exposed to 4°C. When incubated with ABA at the optimal conditions (0.5-1 g cell inoculum, 75 μM ABA, 25-30°C, 7–10 days), they survived slow freezing (2°C/h) to −9.0 ~ −9.3°C (LT50: 50% killing temperature) while control cells were mostly injured at −3°C (LT50: -0.5 ~ −1.5°C). Ice-inoculation of the cell suspension at −3°C and survival determination by regrowth confirmed that ABA-treated rice cells survived extracellular freezing at −9°C. ABA-induced freezing tolerance did not require any exposure to cold and was best achieved at 25-30°C where the rice cells maintained high growth even in the presence of ABA. ABA treatment also increased tolerance to heat (43°C) as determined by regrowth. ABA-treated cells tended to have more augmented cytoplasm and/or reduced vacuole sizes compared to control cultures with a concomitant increase in osmolarity and a decrease in water content. ABA-treated (2–7 days) in vitro grown seedlings and their leaves survived slow freezing to −3°C with only marginal injury (LT50: -4°C) whereas untreated seedlings were killed at −3°C (LT50: -2°C). Conclusions The results indicate that exogenous ABA can induce some levels of freezing tolerance in chilling-sensitive rice cells and seedlings, probably by eliciting mechanisms different from low temperature-induced cold acclimation.

Somatic embryos of melon (Cucumis melo L.) were cryopreserved in liquid nitrogen (LN2) after controlled desiccation. Desiccation of embryos was carried out slowly under controlled relative humidity (RH) of 50-65 % after preculture with 10mg/l abscisic acid (ABA). Desiccated embryos were immersed directly into LN2. Of the total number of embryos 65 % survived storage in LN2 when desiccated at a RH of 60 %. Viability was affected mainly by the size of the embryos. The water content of the desiccated embryos was 11.8% at RH 60%, which was considered to be reasonably low to avoid freezing injury of the cells. This study demonstrates that the desiccation method enabled the cryopreservation of melon somatic embryos without the use of expensive facilities.

The effects of culture conditions on abscisic acid (ABA)-induced freezing tolerance were determined in smooth bromegrass (Bromus inermis Leyss cv. Manchar) cell suspension cultures. Bromegrass cultures initiated with 2g fr wt of cells achieved maximum freezing tolerances (greater than —32°C) at 25 to 30°C in the presence of 75 to 100^M ABA. High levels of freezing tolerance induced by ABA were correlated with high growth rates at 25 and 30°C. In control cells, incubation at 10°C induced optimum levels of hardiness with minimal growth. Prolonged exposure (6 weeks) of cells to 3°C, with or without ABA, increased freezing tolerance only by several degrees. Exogenous ABA concentrations greater than IOO^M were not inhibitory to growth. Repeated exposure to ABA, however, retarded growth and made the cells tolerant to temperatures below — 40°C. Removal of ABA from the medium resulted in dehardening of the cells both at 25 and 3°C. Nitrogen had a marginal effect on ABA-induced hardening at 25°C, but inhibited age-dependent hardening of control cell cultures. Light had no effect on the freezing tolerance of cultured cells. Addition of 10% sucrose, 30 min prior to freezing, to bromegrass cells treated with ABA for 4 days increased freezing tolerance more than 15°C. These observations are discussed in relation to the contrasting behaviour of the low temperature and photoperiod dependent cold acclimation of plants.