Temperature and Photocontrol of Onoclea Spore Germination 1

Germination of Oncwk seblis L. spores is controlied by light and temperature. Temperatures of30 C can induce maximal germination in the dark to a level of 60 to 95% of that induced by a saturating dose of red light (0.38 joules/square meter) providing the spores are placed at the elevated temperature immediately after being sown. Maximum dark germination occurs with a minhmum exposure of 16 to 24 hours at 30 C, suggestn that the temperature treatment is required for the induction of germination rather than for the germination process per se. Interaction of temperature and light for induction of germination shows nonaddWive behavior. Germination induced by light and temperature applied consecutively never exceeded that which could be induced by a saturatin dose of red light alone. Imbibition of the spores at 25 C in the dark for 12 or more hours prior to incubation at 30 C results in a loss of thermosensitivity. Dose response curves for red light induction of germination after varying times of imblbtion at 25 C show no concomitant loss of sensitivity of the spores to red Irradiation. This suggests that the mechanism and/or pathway of thermonuction of germination differs from that of photoinduction. The loss of thermosensitivity as a result of presoaking at 25 C can be prevented if the spores are imbibed at 25 C in osmotic agents such as 03 molar mannitol or 0.1 gram per liter of polyethylene glycol 400 or in 0.08% dimethylsulfoxide or 10 micrograms per miiiter of herbicide SAN 9789 (4chloro-5-(methylamino)-2-(a,a,a-trifluoro-m-tolyl-3-(2H)pyrkdazlnone). The latter two substances are hypothesized to act upon membranes. These results suggest that the degree of hydration and possibly changes in membrane properties play a role in the change in sensitivity of Onocka spores to temperature. Fern spore germination has been shown repeatedly to be stimulated by light (4, 12). Few fern species have spores which are capable of germination in the dark, and, of those which do show dark germination, many require special inductive treatments (12 and ref. cited therein). Germination of spores of Onoclea sensibilis L., the sensitive fern, is enhanced by low dosages of light; red light is the most efficient for enhancement but shorter and longer wavelengths are also effective if high enough dosages are given (15). Reversal of red induced germination with far red light between 700 and 750 nm has not been demonstrated, and, hence, phytochrome has not been definitively identified as the photoreceptor (15). Onoclea spores are also capable of germinating in the dark. Hartt (4) demonstrated that the degree of dark germination is temperature-dependent with maximal germination of 95% occurring at 28 and 29 C. Edwards and Miller (2) have observed dark germination as high as 65% at 25 C when scored by the uptake of acetocarmine. The capacity to germinate in the dark is lost with the aging of the spores without the spores losing their responsiveness to continuous diffuse illumination (4, 12). ' This research was supported by a Faculty Grant-In-Aid from the Grants Committee of Arizona State University. The objective of this study was to determine the relationship between induction of dark germination by high temperatures and the induction of germination by very short exposures to low intensity red radiation. The data presented suggest that the thermoand photocontrol of Onoclea spores is mediated by two different mechanisms. MATERIALS AND METHODS Plant Material and Germination Procedure. Sporophylls of Onoclea sensibilis L. were collected in southeastern Michigan in February, 1975 and the spores were harvested and stored as described previously (15). The light requirements and characteristics for germination were comparable to those reported in Towill and Ikuma (15). Germination was scored 5 days after treatment by the protrusion method. Each experimental treatment was done in duplicate. Counts from two slides of 200 spores/slide from each of the replicate dishes were averaged to yield the per cent germination for a given treatment. Standard deviations of the mean were calculated for each value with N = 4. Temperature Experiments. Temperatures used in this study included 10, 18, 20, 25, 27, 30, 33, and 36 C and were attained using temperature-regulated incubation chambers which fluctuated ±0.5 C from the stated value. Spores which were kept at room temperature were placed in light-tight drawers in a dark room maintained at 25 ± 1 C. Approximately 5 mg of spores were sown on 10 ml of distilled H20 in a 5-cm Petri dish under a dim green safelight. The Petri dishes were placed in light-tight cylinders for transfer to the appropriate temperature. Duration and time of application of the temperature and light treatments are presented under "Results." Experiments were repeated at least three times. The data presented in the figures and tables represent the results from a single experiment, the trends of which are consistent with the data from replicate experiments. Experiments Using Osmotica and Agents Which Affect Membrane Properties. In experiments using osmotic agents or agents which affect membranes, 5 mg of spores were sown on the respective media under dim green light, and the spores maintained at room temperature in the dark for the time indicated in the individual experiment. After incubation in the media, the spores were washed 10 times, each with 10 ml of distilled H20. After the final washing, 10 ml of distilled H20 was added and the dishes treated as described under "Results." SAN 97892 kindly supplied by the research personnel at SandozWander Inc., was Technical 98.8%. DMSO, mannitol, CaCl2 and all other chemicals used were reagent grade. Light Sources. The green safelight source was a 15-w cool-white fluorescent lamp (General Electric F15T8-OW) filtered through two sheets of green Plexiglas (No. 2092 Rohm and Haas). These filters transmit between 490 and 590 nm with maximal transmission of 45% at 535 nm. The red filter was a 15-w cool-white 2 Abbreviations: SAN 9780: 4-chloro-5-(methylamino)-2-(a,a,a-trifluoro-m-tolyl)-3-(2H)pyridazinone; DMSO: dimethylsulfoxide. 116 www.plantphysiol.org on January 31, 2018 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. THERMOAND PHOTOCONTROL ONOCLEA SPORES fluorescent lamp filtered through two layers of red Plexiglas (No. 2423) which transmitted above 600 nm with maximal transmission reached at 660 nm. Light intensities were measured with a YSI-Kettering model 65 radiometer. The intensity of the red light source was 38 mw/M2. RESULTS AND DISCUSSION Effects of Temperature on Dark and Photoinduced Germination. Since temperature can effect not only the degree of dark germination but also the extent of photoinduced germination (14), we compared the effects of various temperatures on the per cent germination of spores maintained constantly in the dark or given a photoinductive treatment. In the first set of experiments spores were maintained at a constant temperature during the period of imbibition and germination; spores receiving the photoinductive treatment were irradiated with 5 min of red light (38 mw/M2) 12 hr after the spores were sown. Figure IA indicates that maximal dark germination occurs at 30 C while the temperature for optimal red induced germination ranges between 27 and 30 C. Although the spores did not germinate at 10, 18, or 20 C, they remained fully viable, germinating to a level of 95% when placed under continuous illumination at room temperature. In contrast, spores maintained at 36 C lost their viability, failing to germinate when placed under continuous illumination. Depending upon the spore batch employed, the degree of dark germination induced by 30 C ranges from 61% (Fig. IA) to 95% of that induced by saturating doses of red light at the same temperature. A minimum of 16 hr at 30 C induces maximal dark germination. Protrusion of the rhizoid through the spore coat does not occur until the 4th day at 30 C. These data reaffirm temperature dependency of dark germination of Onoclea spores as had been demonstrated by Hartt (4) and suggest that 30 C is necessary only for initiation of germination and perhaps for the initial stages of the germination process. Induction of dark germination by elevated temperatures has been reported for several other light-requiring fern spores but the data are too limited to develop any generality between temperature-sensitive dark germination and photoinduction (4, 12 and ref. cited therein). Since temperature influences not only the processes of germination but also the development of photosensitivity (14), a second set of experiments was designed to determine the effect of temperature on the processes succeeding the development of photosensitivity and the photoinductive treatment. Spores were maintained at room temperature in the dark for 12 hr, irradiated with 5 min of red light, and then transferred to various temperatures. Controls were treated similarly with elimination of the light treatment. The data (Fig. 1B) indicate that the optimal temperatures for the processes succeeding photoinduction range from 27 to 30 C. The final percentage of germination induced by red light is about the same whether the spores are soaked at the given temperature or at room temperature prior to irradiation. In contrast, the final percentage of dark germination at a given temperature decreases as a result of presoaking at room temperature (compare Fig. IA with Fig. 1B). This latter observation suggests that Onoclea spores lose their sensitivity to elevated temperatures with presoaking at room temperature. Interaction of Thernoand Photoinduction of Germination. To investigate the interaction of temperature and light on induction of germination, spores were soaked for 2 hr in the dark at room temperature to develop photosensitivity, then irradiated with either a subsaturating or saturating dose of red light (2 and 90 sec, respectively, at 38 mw/M2) and, after irradiation, placed at 30 C for varying periods of time. After the 30 C treatment, spores were transferred to room temperature until germination was scored 5 days later. The results (Fig. 2) indicate that 30 C enhances germination induced by the subsaturating dose of red light but has little effect upon germination induced by the saturating dose. z 0 H-