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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.

Water-saving rice-winter crop rotation systems were repeated for 4 cycles from 2000 to 2004 in an urban area, Nishitokyo, Japan, to assess the effects of water-saving (i.e. non-flooded vs. flooded) on grain yield of rice (Oryza sativa L.) and chemical constituents of percolating water. The effects of pre-rice winter cropping compared with fallow on rice yield were also examined. The pre-cultivated crops were wheat (Triticum aestivum L.), italian ryegrass (Lolium multiflorum Lam.) or spinach (Spinacea oleracea L.) with their above-ground parts removed, chinese milk vetch (Astragalus sinicus L.) or rapeseed (Brassica napus L.) with their above-ground parts incorporated before rice transplanting. Neither winter cropping effects nor its interaction with water-saving were significant for rice yield, although the yield after rapeseed incorporation tended to be 9 % higher than that after fallow. In 2001, 2003 and 2004, when more than 70% of irrigation water was saved in the non-flooded trial, average yield in non-flooded trial was 58 % of flooded trial, but water productivity increased (from 0.10 to 0.16 kg m-3). Among the 3 years, yield in non-flooded trial was highest in 2004 when the amounts of irrigation and total water supply was larger, the frequency of dry spells was the lowest, and 2 seedlings were transplanted per hill. The nitrate and nitrite concentrations in the percolating water were far below the environmental standard values by WHO. The study showed that incorporation of winter crops had no negative effects on water-saving rice production at least for the first 4 years, and that under extreme water-saving, irrigation and planting methods could minimize yield reduction.

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.

We noted that ammonia nitrogen was not adsorbed by the cultivated layers of highly permeable paddy fields during the initial fertilization period, but reached the lower layers relatively early. In our study, we considered an exponential equation from an aqua-environmental perspective with the goal of obtaining good growth of rice plants in order to estimate the concentrations and integrated volume of ammonia nitrogen accompanying paddy percolation. Using this exponential equation, we were able to derive a relation between time and concentrations of paddy percolation water, and hypothesized that if percolation rates were less than 10 mm/day, percolation would have no effect on rice growth, while simultaneously helping to maintain the good water quality of the extra-paddy environment. We also clarified the differences between the potential ammonia nitrogen adsorption volume derived from the CEC value and the integrated amount of ammonia nitrogen water in soil, and considered the causes from the perspectives of solute movement and water movement.

A simple and efficient procedure for the transient permeabilization of cultured cells has been developed for the incorporation of a high molecular weight substance, fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (FITC-IgG, 150 kDa) into Nicotiana tabacum BY-2 cells grown in suspension cultures. The cells were treated with cell permeabilization solutions (CPS) comprising varying concentrations of glycerol (Gly), sucrose (Suc), ethylene glycol (EG) and dimethyl sulfoxide (DMSO). When the cells were permeabilized by treating with CPS-3 (Gly:Suc:EG:DMSO=20:5:20:5, w/v%) containing FITC-IgG for 15 s at room temperature followed by washing with ice-cold 1 M sucrose and modified LS medium, 76% of the cells incorporated FITC-IgG and 78% of the cells remained viable according to fluorescein diacetate (FDA) staining. Most treated cells retained intact internal structure and the incorporated FITC-IgG was detected in cytoplasm and in the nuclear region. Cells of different ages were studied. Four day-old cells were found suitable for the incorporation of FITC-IgG and cell survival. Inclusion of 10 mM CaCl 2 in CPS-3 improved both FITC-IgG uptake and cell survival. The regrowth of treated cultures lagged for the first 3 days, then followed a growth pattern similar to the untreated controls.

Axillary buds of in vitro-grown gentian ( Gentiana scabra Bunge var. buergeri Maxim.) were successfully desiccated and cryopreserved using a novel two-step preculture method: preculturing axillary buds excised from in vitro-grown gentian plants on Murashige and Skoog (MS) semi-solid medium containing 0.1 M sucrose for 8–13 days at 25°C under a 16 h-photoperiod (first step) and the subsequent short period incubations on 0.4 M sucrose (MS agar medium) for 1 day followed by 0.7 M sucrose (MS agar medium) for 1 day (second step). After preculturing, the buds were air-dried down to about 10% water content (fresh weight basis) without encapsulation, and then immersed in liquid nitrogen (LN). With this cryogenic protocol, we could obtain 78–90% of survival of axillary buds following desiccation and LN exposure. Most of the buds that survived showed normal shoot and root development. The most important part of this protocol was the preculture which circumvents the conventionally used lengthy cold-hardening treatment. The results show that induction of desiccation tolerance by preculturing was specific to a few species of sugars used in the medium and not merely caused by osmotic effects. The efficient desiccation–cryopreservation protocol shown here using the new preculture method appears promising for routinely cryopreserving gentian germplasm.

Cryopreservation of a non-embryogenic bromegrass suspension culture was attempted using a slow prefreezing method (two-step method) and a vitrification method. We examined 17 solutions as cryoprotectants for the slow prefreezing method. The cryoprotectant (CSP1) comprising sucrose, dimethylsulfoxide (DMSO) and glycerol (10, 10, 5%, w/v) gave the best survival (> 80%) of bromegrass cells exposed to liquid nitrogen (LN) as determined by regrowth and fluorescent diacetate (FDA) staining. The optimum conditions were: exposure time to CSP1 (30 min), cooling rates (0.3–0.5°C/min) following ice-inoculation at −8°C, prefreezing temperature (−30 to −40°C). This protocol worked well when glass vials were used as containers and not plastic cryotubes. With CSP1, the slow dropwise addition of the protectant to the cells on ice that has frequently been used could be circumvented; direct addition of CSP1 to the cells at 25°C did not affect the survival. However, dilution of CSP1 by 5-fold with 3% sucrose following a freeze-thaw had to be done slowly (taking 15 min). The same cryoprotectant (CSP1) was also effective as a pretreatment for cryopreservation of bromegrass cells by vitrification. Following direct treatment with CSP1 at 25°C for 30 min, cells were incubated in a vitrification solution, PVS2 (Sakai et al., 1990) for about 2–4 min at 25°C prior to submersion in LN. This procedure gave 30–40% survival of bromegrass cells based on regrowth and FDA staining. Without the CSP1 pretreatment, bromegrass cells did not survive LN exposure by vitrification with PVS2. Preculture with abscisic acid or with high osmoticum (0.3–0.7 M sucrose) could not be used as a substitute for pretreatment with CSP1.

Several viability assays were compared to determine the most sensitive and appropriate method for estimating the freezing, heat and salt tolerance of Bromus inermis Leyss cells cultured with or without 75 μM abscisic acid (ABA) for 4–7 days at 25°C. The sensitivity and reliability of individual viability tests depended on the type of stress applied and degree of injury. Regrowth, amino acid (AA) leakage and fluorescein diacetate (FDA) staining all gave comparable estimates of freezing tolerance. Triphenyl-tetrazolium chloride (TTC) reduction assays slightly overestimated freezing tolerance, but was most convenient. Polypeptide leakage from freeze-thawed cells, as determined by SDS-polyacrylamide electrophoresis (SDS-PAGE), revealed that protein leakage only occurred in ABA-treated cells frozen to −21°C or colder in contrast to control cells which leaked proteins following freezing to −5°C. The most sensitive test for assessing heat tolerance was regrowth, followed by FDA staining, when TTC. TTC tests overestimated heat tolerance compared with the other tests. The degree of overestimation was greater for heat tolerance estimates than for freezing tolerance estimates. It was partially improved by washing the cells prior to TTC assays. AA leakage tests were not appropriate for assessing heat tolerance, due to the erroneously high values of A280 readings obtained. For estimating salt tolerance, TTC assays were most convenient and were in close agreement with regrowth measurements whereas FDA staining tended to overestimate it. In general, while regrowth was most sensitive and reliable, TTC was most convenient. All viability assays consistently showed that ABA induced cross-adaptation to freezing, heat and salt stresses in bromegrass cells without a prior exposure to any of these stresses.

Summary The growth, freezing tolerance and two-dimensional protein profiles of bromegrass (Bromus inermisLeyss) cell cultures were investigated following repeated exposure to 75 μM abscisic acid (ABA) for 5 weeks. Following prolonged (36 d) exposure to ABA, freezing tolerance was enhanced and fresh-mass gain was reduced compared to a 10-d ABA treatment. Also, fifteen additional ABA-responsive proteins were detected after 5 weeks of ABA treatment that were not detected in cells treated for 10 d. In addition, intensity changes occurred in several ABA-responsive proteins that were detected after 10-d incubation periods. Two polypeptides (26 and 29 kD) initially responsive to ABA decreased over 5 weeks of treatment. Transfer of ABA-treated cells to culture media lacking ABA for 14 d resulted in a 22°C decrease in freezing tolerance and increased growth, but there was no detectable change in the ABA-responsive polypeptides. Abscisic acid treatment at 3°C increased freezing tolerance at an accelerated rate and to a greater level than 3°C alone, but less so than ABA treatments at 25°C. However, the same ABA-responsive polypeptides detected at 25°C were present in cells treated with ABA at 3°C. These observations suggest that synthesis and accumulation of ABA-responsive polypeptides are only one component involved in freezing tolerance and other factors must be involved.

Summary Leaves of Trachycarpus fortunei from palms growing in warm temperate regions of Europe and Japan as outdoor ornamentals were cooled down to low subfreezing temperatures, and the process of ice formation was followed using differential thermal analysis (DTA). Inactivation of photosynthetic function was determined by in vivo chlorophyll fluorescence, and the progress of injuries during freezing was assayed by viability tests after thawing. Ultrastructure of mesophyll cells was investigated by transmission electron microscopy. Silica incrustation of cell walls was documented by means of qualitative energy dispersive X-ray analysis (EDAX) on samples of Trachycarpus fortunei, Washingtonia filifera, and of the bamboo Pseudosasa japonica. DTA revealed typical freezing patterns, with a high temperature exotherm at about -6 to -8°C and a broad low temperature exotherm rising from about -13 to -14°C. As soon as the tissues froze, characteristic changes appeared in the kinetics of in vivo fluorescence. No irreversible inactivation of photosynthesis or damage was detectable at temperatures above appearance of the low exotherm. Within a short time after attainment of the exotherm peak, the leaf segments were found to be lethally damaged. Both of in vivo fluorescence and viability tests clearly indicated that Trachycarpus leaves remain in the persistent supercooled state down to quite low temperatures. The tensile state of water at threshold supercooling temperatures of Trachycarpus leaves was calculated according to the equation of Rajashekar and Burke (1982) and found to be about -14 MPa. An explanation for the permanent maintenance of such a large displacement from thermodynamic equilibrium could be that silica incrustation, as revealed by EDXA, favours supercooling by increasing the rigidity of the cell walls and/or resistance to the growth of ice crystals.

1H nuclear magnetic resonance (NMR) micro-imaging was used to study the freezing behaviour of wintering flower buds of Rhododendron japonicum (A. Gray) Suringer. Amulti-slice multi- echo pulse sequence was used to acquire images at different subfreezing temperatures. The images obtained predominantly reflected the density of mobile (i.e. non-ice) protons mainly from unfrozen water. By comparing these images taken at various subfreezing temperatures, we could determine which tissues produced high temperature exotherms and low temperature exotherms in differential thermal analyses. In flower buds of the cold-hardy R. japonicum, typical extra-organ freezing was successfully imaged. The scales readily froze at –7°C but some florets remained supercooled even at –21°C. The size of the supercooled florets was reduced with decreasing temperature which indicated a gradual decrease in floret water content. With decreasing temperature, there was a gradual decrease in the signal intensity of the flower bud axis including the peduncle and immature pith tissues, which implies either dehydration or partial freezing of these tissues. Deep supercooling in the entire mature pith tissues was also clearly visible in these images. Due to its non-invasive nature, NMR micro-imaging is a useful tool for studying freezing behaviours in various plant tissues, especially for imaging organised or harmonised freezing in complex organs as well as for clarifying the diversity and mechanisms involved in freezing behaviours.