In vitro culture of coffee zygotic embryos for germplasm preservation

Immature zygotic embryos of Coffea arabica L. Cv. Cauvery (Catimor) were cultured on Murashige and Skoog (1962) (MS) medium supplemented with abscisic acid (ABA) at 0, 0.4, 3.8, 18.9, 37.8 and 75.6 μM., L-cystein hydrochloride at 50 mg 1^-1 and sucrose at 3%. Cultures were preserved in parafilm sealed Petri dishes in dark at a temperature of 25 ± 1 °C for up to two years. The preserved embryos were taken out from the media at 6 month intervals in order to test their viability by germination on MS + NAA (0.5 μM) + BA (4.4 μM). On the preservation media devoid of ABA or with a low concentration (0.4 μM) of ABA, the embryos germinated and showed higher mortality with increasing duration of storage. In contrast, the embryos became increasingly dormant with increasing concentrations of ABA and a 74.2% survival was found even after 2 years on medium supplemented with 18.9 μM or 37.8 μM of ABA. The results suggest that embryos can be preserved with a little loss of viability in the presence of ABA even at the normal room temperature (25 + 1 °C) up to two years without any transfer. Application of this technique for germplasm preservation of coffee is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

A rapid effect of applied brassinolide on abscisic acid concentrations in <Emphasis Type="Italic">Brassica napus</Emphasis> leaf tissue subjected to short-term heat stress

Three-week old canola (Brassica napus L.) seedlings grown at 20/16°C (day/night) were subjected to short-term (4 and 8 h) heat stress (45°C) or maintained at a normal temperature of 20°C. Half of the plants under each treatment received a 10⁻⁶ M solution of brassinolide (BL) 1 h prior to beginning the temperature treatments. The concentration (ng/g dry weight) of endogenous abscisic acid (ABA) was subsequently determined in young leaves via the stable isotope dilution method. Applied BL had no effect on endogenous ABA for plants maintained at normal temperatures. However, ABA concentration was significantly elevated by heat stress alone and doubled by heat stress + BL. These results suggest that the well-known enhancement of tolerance to high temperature stress that can be obtained by BL or 24-epi-BL applications may be caused by a brassinosteroid-induced elevation in endogenous ABA concentration.     

Effects of low temperature on growth and non-structural carbohydrates of the imperial bromeliad <Emphasis Type="Italic">Alcantarea imperialis</Emphasis> cultured in vitro

The imperial bromeliad Alcantarea imperialis grows naturally on rocky outcrops (‘inselbergs’) in regions where daily temperatures vary from 5 to 40°C. As carbohydrate metabolism is altered in response to cold, it could lead to reprogramming of the metabolic machinery including the increase in levels of metabolites that function as osmolytes, compatible solutes, or energy sources in order to maintain plant homeostasis. The aim of this study was to evaluate the effects of different temperatures on plant growth and non-structural carbohydrates in plants of A. imperialis adapted to low temperature. Seedlings of A. imperialis were grown in vitro under a 12-h photoperiod with four different day/night temperature cycles: 5/5°C, 15/15°C, 15/30°C (dark/light) and 30/30°C. Plants were also cultivated at 26°C in ex vitro conditions for comparison. The results showed an inverse relationship between temperature and germination time and no differences in the percentage of germination. Plants maintained for 9 months at 15°C presented a reduced number of leaves and roots, and a dry mass four times lower than plants grown at 30°C. Sugar content was higher in plants grown at 15°C than at 30°C. However, the highest amount of total sugar was found in plants growing under warm day/cold night conditions. Myo-inositol, glucose, fructose and sucrose were found predominantly under high temperatures, while under low temperatures, sucrose was apparently replaced by trehalose, raffinose and stachyose. Starch content was highest in plants grown under high temperatures. The lowest starch content was detected under low temperatures, suggesting its conversion into soluble carbohydrates to protect the plants against cold. These results indicated that low temperature retarded growth of A. imperialis and increased sugar levels, mainly trehalose, thus suggesting that these sugar compounds could be involved in cold tolerance.     

Low-temperature effect on enzyme activities involved in sucrose–starch partitioning in salt-stressed and salt-acclimated cotyledons of quinoa (Chenopodium quinoa Willd.) seedlings

The effect of low temperature on growth, sucrose–starch partitioning and related enzymes in salt-stressed and salt-acclimated cotyledons of quinoa (Chenopodium quinoa Willd.) was studied. The growth of cotyledons and growing axes in seedlings grown at 25/20 °C (light/dark) and shifted to 5/5 °C was lower than in those only growing at 25/20 °C (unstressed). However, there were no significant differences between low-temperature control and salt-treated seedlings. The higher activities of sucrose phosphate synthase (SPS, EC 2.4.1.14) and soluble acid invertase (acid INV, EC 3.2.1.25) were observed in salt-stressed cotyledons; however, the highest acid INV activity was observed in unstressed cotyledons. ADP-glucose pyrophosphorylase (ADP-GPPase, EC 2.7.7.27) was higher in unstressed cotyledons than in stressed ones. However, between 0 and 4 days the highest value was observed in salt-stressed cotyledons. The lowest value of ADP-GPPase was observed in salt-acclimated cotyledons. Low temperature also affected sucrose synthase (SuSy, EC 2.4.1.13) activity in salt-treated cotyledons. Sucrose and glucose were higher in salt-stressed cotyledons, but fructose was essentially higher in low-temperature control. Starch was higher in low-temperature control; however, the highest content was observed at 0 day in salt-acclimated cotyledons. Results demonstrated that low temperature induces different responses on sucrose–starch partitioning in salt-stressed and salt-acclimated cotyledons. Data also suggest that in salt-treated cotyledons source–sink relations (SSR) are changed in order to supply soluble sugars and proline for the osmotic adjustment. Relationships between starch formation and SuSy activity are also discussed.     

Effect of salicylic and abscisic acid administered through detached tillers on antioxidant system in developing wheat grains under heat stress

The mechanism imparting thermotolerance by salicylic acid (SA) and abscisic acid (ABA) is still unresolved using either spraying technique or in vitro conditions. Alternative way of studying these effects under near in vivo conditions is through the use of liquid culturing technique. Effects of SA and ABA (100 μM) on antioxidative enzymes, antioxidants and lipid peroxidation were studied in detached tillers of three wheat (Triticum aestivum L.) cultivars PBW 343, C 306 (heat tolerant) and WH 542 (heat susceptible) cultured in a liquid medium. Ears were subjected to heat shock treatment (45°C for 2 h) and then maintained at 25°C for 5 days. Heat shock treatment resulted in increased peroxidase (POD) activity, while superoxide dismutase (SOD) and catalase (CAT) activities were reduced compared to control. The decrease in CAT activity was more significant in susceptible cultivar WH 542. Concomitantly, content of α-tocopherol and lipid peroxides increased in heat-treated wheat ears, whereas contents of total ascorbate level were reduced. Following treatment with SA and ABA, activities of all three antioxidative enzymes increased in correspondence with an increase in ascorbate and α-tocopherol content. Apparently, lipid peroxide content was reduced by SA in heat tolerant cultivars (PBW 343 and C 306) whereas in susceptible cultivar it was decreased by ABA. The up-regulation of the antioxidant system by SA and ABA possibly contributes to better tolerance against heat shock-induced oxidative damage in wheat grains.     

Thermotolerance and antioxidant response induced by heat acclimation in <Emphasis Type="Italic">Freesia</Emphasis> seedlings

Followed a heat acclimation pretreatment, seedlings of Freesia hybrida ‘Shangnong Jinghuanghou’ were exposed to heat stress at 38°C for 6 h treatment and then recovered at 22°C for 72 h to study the impact of heat acclimation (30°C) on thermotolerance under heat stress. The results showed that the pretreated seedlings performed better under heat stress than control. Heat acclimation could slow down the decrease of chlorophyll contents under heat stress and recover better. Higher levels of soluble sugar and proline and slight lower level of soluble protein were observed in pretreated seedlings. After recovery, similar levels of proline and soluble protein were maintained in all seedlings. However, a higher level of soluble sugar was maintained in pretreated seedlings. MDA content and EL showed a stable level in pretreated seedlings while a significant increase in control, followed by a significant decrease after recovery. Significant different responses of SOD, POD, CAT, and APX activities were observed in pretreated seedlings and control. Heat acclimation led to higher activities of these enzymes and a significant response of antioxidant enzyme activities occurred in a time-dependent manner under heat stress. Exposure to high temperature caused a significant increase in SOD and APX activity, and much higher levels in SOD and APX activity were observed in pretreated seedlings compared to control during heat stress. A slight difference in change pattern of POD and CAT activity was presented. The highest activities of POD and CAT were observed at 4 and 6 h of heat stress in pretreated seedlings and control, respectively. After 72 h recovery, the activities of all tested enzymes decreased to similar levels in all seedlings.     

Pretreatment with 5-aminolevulinic acid mitigates heat stress of cucumber leaves

Cucumber seedlings were pretreated with 3 μM 5-aminolevulinic acid (ALA) followed by cultivation at normal (25/18 °C) or high (42/38 °C) day/night temperature to investigate the protective effects of ALA on heat stress in plants. Heat elevated the contents of malondiadehyde (MDA), superoxide radical (O₂ ^.−) and hydrogen peroxide (H₂O₂) in leaves of all plants but less in ALA-pretreated plants. Heat treatment resulted in higher antioxidant enzyme activities and proline and soluble sugar contents and weaker growth inhibition in ALA-pretreated plants than in those treated with heat alone. These results indicate that ALA pretreatment increased the tolerance of seedlings to heat stress.     

Temperature-dependent phosphate solubilization by cold- and pH-tolerant species of Aspergillus isolated from Himalayan soil

Ten species of Aspergillus isolated from soil samples collected from different locations in the Indian Himalayan region have been studied for their growth requirements and tricalcium phosphate solubilization at different temperatures. The Aspergillus species could grow at low temperature and tolerated a wide range of pH. Phosphate solubilization by various Aspergillus species ranged between 374 μg/ml (A. candidus) to 1394 μg/ml (A. niger) at 28°C, 33 μg/ml (A. fumigatus) to 2354 μg/ml (A. niger) at 21°C, 93 μg/ml (A. fumigatus) to 1452 μg/ml (A. niger) at 14°C, and 21 μg/ml (A. wentii) to 83 μg/ml (A. niger) at 9°C. At 21 and 28°C, phosphate solubilization showed a decrease within 4 weeks of incubation, whereas at 9°C and 14°C, it continued further up to 6 weeks of incubation. In general, phosphate solubilization by different Aspergillus species was recorded at a maximum of 28°C or 21°C; biomass production was favored at 21°C or 14°C. Conversely, A. nidulans and A. sydowii exhibited maximum phosphate solubilization at 14°C and produced maximum biomass at 21°C. Data suggest that suboptimal conditions (higher or lower temperature) for fungal growth and biomass production were optimal for the production of metabolites involved in phosphate solubilization. Significant negative correlations were obtained between pH and phosphate solubilization for eight species at 28°C, for seven at 21°C, and for nine at 14°C. Extracellular phosphatase activity was exhibited only in case of A. niger, whreas intracellular phosphatase activity was detected in all species, the maximum being in A. niger. Statistically significant positive or negative correlations were obtained between phosphate solubilization and other parameters in most cases at different temperatures.     

Shoot regeneration from leaf explants of <Emphasis Type="Italic">Brunfelsia calycina</Emphasis>

A protocol for plantlet regeneration through shoot formation was developed for the neotropical shrub Brunfelsia calycina. This shrub is unique in its change in flower color from dark purple to white. Explants from young and mature leaves were incubated on MS medium (pH 5.7, 30 g/l sucrose, 7.5 g/l agar) with various combinations of Indole-3-acetic acid (IAA) and 6-Benzyladenine (BA) under a 16 h photoperiod at a constant temperature of 25°C. Shoot emergence was best at 4.44 μM BA and 2.85 μM IAA for young leaf explants, and at 8.88 μM BA, 2.85 μM IAA for mature leaf explants. When shoots were transferred to MS medium supplemented with 1.23–2.46 μM indole butrytic acid (IBA), they developed roots.     

Peroxidase activity and lignification in soybean root growth-inhibition by juglone

The changes in activities of soluble and cell wall-bound peroxidases and lignin contents in juglone-stressed soybean (Glycine max) seedlings and their relationships with root growth were investigated. Soybean seedlings (3-d-old) were cultivated in nutrient solution supplemented with 0.5 to 25 μM juglone for 24 h. Length and dry mass of roots decreased after 5 to 25 μM juglone treatments. Low juglone concentrations (≤ 1 μM) increased soluble peroxidase activity, while high concentrations (≥ 10 μM) inhibited activities of soluble and cell wall-bound peroxidases. Juglone (≤ 1 μM) did not affect lignin content but highly increased lignification after 5 to 25 μM treatments. Results indicate that lignification may be an important step in root growth reduction of juglone-stressed soybean.     

Early development of germlings of <Emphasis Type="Italic">Sargassum thunbergii</Emphasis> (Fucales,Phaeophyta) under laboratory conditions

Morphology and culture studies on germlings of Sargassum thunbergii (Mertens et Roth) Kuntze were carried out under controlled laboratory conditions. Growth characteristics of these germlings grown under different temperatures (from 10 to 25°C), irradiances (from 9 to 88 μmol photons m⁻² s⁻¹), and under blue and white light conditions are described. The development of embryonic germlings follows the classic “8 nuclei 1 egg” type described for Sargassaceae. Fertilized eggs spent 5–6 h developing into multicellular germlings with abundant rhizoids after fertilization. Under conditions of 20°C, 44 μmol photons m⁻² s⁻¹ and photoperiod of 12 h, young germlings with one or two leaflets reached 2–3 mm in length after 8 weeks. Temperature variations (10, 15, 20, 25°C) under 88 μmol photons m⁻² s⁻¹ significantly influenced the growth rate within the first week, although this effect became less obvious after 8 weeks, especially at 15 and 20°C. Variation in germling growth was highly significant under different irradiances (9, 18, 44, 88 μmol photons m⁻² s⁻¹) at 25°C. Low temperature (10°C) reduced germling growth. Growth of germlings cultured under blue light was lower than in white light. Optimal growth of these germlings occurred at 25°C and 44 μmol photons m⁻² s⁻¹.     

Cryopreservation of horse-chestnut (Aesculus hippocastanum L. ) somatic embryos using three different freezing methods

Cryopreservation of somatic embryos of Aesculus hippocastanum L. cultured on nutritive media containing abscisic acid (ABA) at concentrations of 0.75 μM, 7.5 μM and 75.0 μM was evaluated for three cooling methods: (i) slow freezing with cryoprotectants, (ii) fast freezing with cryoprotectants, and (iii) fast freezing with desiccation techniques. The ‘cryoprotectant’ freezing techniques included the embryo pretreatment on ABA containing medium for 4 days, followed by cryoprotective treatment in liquid medium containing 0.5 M dimethylsulfoxide, 0.5 M glycerol, 1.0 M sucrose, and cooled at slow, and rapid rates. Embryos pretreated on a medium containing 0.75 μM ABA, and cooled to −35 °C at 1°C /min, held for 30 min at this transfer temperature and then immersed in liquid nitrogen (LN) had the best embryo recovery (43%). The ‘desiccation’ method involved an air drying step of similar ABA-pretreated, non-cryoprotected embryos followed by rapid cooling. Embryos precultured on 0.75 μM ABA, then subjected to a 4 h period of air desiccation (water content reduction to 13%) showed about the same level of survival (46%) as found with the ‘cryoprotectant’ slow freezing technique. The air-dry ‘desiccation’ method is easier to apply than the more complicated ‘cryoprotectant’ method. This revised version was published online in June 2006 with corrections to the Cover Date.     

Contents of soluble carbohydrates in yellow lupin seeds maturated at various temperatures

The objective of this paper was to compare the levels of soluble sugars in seeds of yellow lupin cv. Juno matured at different temperatures. The temperature regimes applied were 1). 26 °C for 24 h (high temperature), 2). 24 °C for 12 h and 19 °C for the next 12 h (optimum temperature regime), 3). 26 °C for 16 h and 4 °C for the next 8 h (high-low temperatures). Six soluble carbohydrates (d-galactose, myo-inositol, sucrose, raffinose, stachyose and verbascose) were quantified. Seeds maturing at constant temperature 26 °C accumulated more raffinose (by 100 %) than seeds maturing at optimum temperature regime. Seeds maturing at high temperature accumulated less stachyose and verbascose than those maturing at optimum temperature conditions, the differences being 45 and 24 %, respectively. In seeds maturing at high-low temperature the level of raffinose decreased while the level of stachyose and verbascose increased, compared to those maturing at optimum conditions. The contents of sucrose, d-galactose and myo-inositol in seeds maturing at optimum temperatures was lower than in seeds maturing at both high and high-low temperature regimes. It was shown, that temperature conditions — constant high temperature, or physiologically optimal thermal oscillations (24 °/19 °C) or high-low temperature regime — differently affect the contents of six soluble carbohydrates in maturing seeds of yellow lupin.     

Benzyladenine and low temperature promote phase transition from juvenile to vegetative adult in bulblets of Lilium?×?formolongi ‘White Aga’ cultured in vitro

Scales excised from lily bulblets were cultured on MS medium supplemented with 0.044 or 4.4 μM BA in the dark for 180 days. The culture period was divided into stage 1 (day 0–30), stage 2 (day 31–90) and stage 3 (day 91–180). The scales were cultured at 25°C in stage 1, 25°C or 8°C in stage 2, and 25°C in stage 3. When the scales were cultured on medium with 4.4 μM BA at 25°C for 180 days, bulblets with and without an elongated stem were produced. The percentage of bulblets with elongated stems greatly increased when the scales had been cultured at 8°C in stage 2. On medium with 0.044 μM BA, only bulblets without elongated stems were produced. The diameter of shoot primodia significantly enlarged in bulblets produced on medium with 4.4 μM BA at 8°C in stage 2 and no such enlargement occurred under the other conditions. Nearly square parenchyma cells were observed in the non-elongated shoot primodia in the former bulblets but not in the latter. These cells changed into longitudinally rectangular ones in the internode of elongated stems. Procambium was arranged almost parallel to the shoot axis in the stem of bulblets in the medium with 4.4 μM BA, but not in the medium with 0.044 μM BA.     

Effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii (Cyanophyta)

The effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii were determined. Of the three temperatures tested, 10, 25 and 35°C, the maximal geosmin concentration and geosmin productivity were yielded at 10°C, while the highest chl a production was observed at 25°C. In the studies on light intensity, the maximal geosmin concentration and geosmin productivity were observed at 10 μmol m⁻² s⁻¹, while the highest chl a production was at 20 μmol m⁻² s⁻¹. It was suggested that more geosmin was synthesized with lower chl a demand. Meanwhile, the relative amounts of extra- and intracellular geosmin were investigated. Under optimum growth conditions (20 μmol m⁻² s⁻¹, 25°C; BG-11 medium), the amounts of extracellular geosmin increased as the growth progressed and reached the maximum in the stationary phase, while the intracellular geosmin reached its maximum value in the late exponential phase, and then began to decline. However, under the low temperature (10°C) or light (10 μmol m⁻² s⁻¹) conditions, more intracellular geosmin was synthesized and mainly accumulated in the cells. The proportions of extracellular geosmin were high, to 33.33 and 32.27%, respectively, during the stationary phase at 35°C and 20 μmol m⁻² s⁻¹. It was indicated that low temperature or light could stimulate geosmin production and favor the accumulation of geosmin in cells, while more intracellular geosmin may be released into the medium at higher temperatures or optimum light intensity.     

Growing season temperatures limit growth of loblolly pine (Pinus taeda L.) seedlings across a wide geographic transect

We grew potted loblolly pine (Pinus taeda L.) seedlings from a single provenance under well watered and fertilized conditions at four locations along a 610 km north–south transect that spanned most of the species range to examine how differences in the above-ground environment would affect growth rate, biomass partitioning and gas exchange characteristics. Across the transect there was an 8.7°C difference in average growing season temperature, and temperature proved to be the key environmental factor controlling growth rate. Biomass growth was strongly correlated with differences in mean growing season temperature (R ² = 0.97) and temperature sum (R ² = 0.92), but not with differences in mean daily photosynthetic photon flux density or mean daily vapor pressure deficit. Biomass partitioning between root and shoot was unchanged across sites. There was substantial thermal acclimation of leaf respiration, but not photosynthesis. In mid-summer, leaf respiration rates measured at 25°C ranged from 0.2 μmol m⁻² s⁻¹ in seedlings from the warmest location to 1.1 μmol m⁻² s⁻¹ in seedlings from the coolest site. The greatest biomass growth occurred near the middle of the range, indicating that temperatures were sub- and supra-optimal at the northern and southern ends on the range, respectively. However, in the middle of the range, there was an 18% decrease in biomass increment between two sites, corresponding to 1.4°C increase in mean growing season temperature. This suggests that thermal acclimation was insufficient to compensate for this relatively small increase in temperature.     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.     

Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization

Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase (CotA) from Bacillus sp. HR03 and then expressed it under microaerobic conditions and decreased temperature in order to obtain high amounts of soluble protein. The laccase was purified and its biochemical properties were investigated using three common laccase substrates, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). K _M and k _cat were calculated 535 μM and 127 s⁻¹ for ABTS, 53 μM and 3 s⁻¹ for 2, 6-DMP and 5 μM and 20 s⁻¹ for SGZ when the whole reactions were carried out at room temperature. Laccase activity was also studied when the enzyme was preincubated at 70 and 80°C. With SGZ as the substrate, the activity was increased three-fold after 50 min preincubation at 70°C and 2.4-fold after 10 min preincubation at 80°C. Preincubation of the enzyme in 70°C for 30 min raised the activity four-fold with ABTS as the substrate. Also, l-dopa was used as a substrate. The enzyme was able to oxidize l-dopa with the K _M and k _cat of 1,493 μM and 194 s⁻¹, respectively.     

Effect of Plant Growth Regulators on Flower-Opening of Pharbitis nil

Flower buds of Pharbitis nil (due to open the next morning)cut from plants in the field before noon open very slowly bothin darkness and at a low temperature (20°C), unlike thebuds cut in the evening. On cool cloudy days, even the budscut in the evening open very slowly. Addition of sucrose, mineralnutrients or plant growth regulators other than ABA to the waterin which the cut buds were placed did not promote flower-openingunder such conditions, but addition of ABA (10–100 µM)greatly promoted it. IAA (100 µM) given alone or in combinationwith ABA suppressed floweropening completely. Mature flowerbuds placed in an ABA solution opened even under continuouslight at 25°C just as those kept in darkness without ABA;flower-opening occurred about 12 h after the application ofABA. ABA given to the buds in darkness at 25°C and thatgiven in continuous light at 20°C also advanced the timeof flower-opening. The action mechanism of ABA is discussed. ¹ This paper is dedicated to the memory of Dr. Joji Ashida,the first president of the Japanese Society of Plant Physiologist. (Received October 28, 1982; Accepted January 7, 1983)