Crop Science 42:651-654 (2002)
© 2002 Crop Science Society of America
NOTES
Anaerobic conditions improve germination of a gibberellic acid deficient rice
Jonathan M. Frantz and
Bruce Bugbee*
Crop Physiology Lab., Dep. of Plants, Soils, and Biometeorology, Utah State Univ., Logan, UT 84322-4820
* Corresponding author (bugbee{at}cc.usu.edu)
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ABSTRACT
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Dwarf plants are useful in research because multiple plants can be grown in a small area. Rice (Oryza sativa L.) is especially important since its relatively simple genome has recently been sequenced. We are characterizing a gibberellic acid (GA) mutant of rice (japonica cv. ‘Shiokari,’ line N-71) that is extremely dwarf (20 cm tall). Unfortunately, this GA mutation is associated with poor germination (70%) under aerobic conditions. Neither exogenous GA nor a dormancy-breaking heat treatment improved germination. However, 95% germination was achieved by germinating the seeds anaerobically, either in a pure N2 environment or submerged in unstirred tap water. The anaerobic conditions appear to break a mild post-harvest dormancy in this rice cultivar.
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INTRODUCTION
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DWARF LINES OF CROP PLANTS are useful in research because they can be grown in confined quarters such as a lab bench or in small growth chambers. The short stature of dwarf lines is typically the result of mutations related to hormone production or perception (Chandler and Robertson, 1999; Hanumappa et al., 1999). Analysis of a dwarf rice line (O. sativa japonica cv. ‘Shiokari’ line N-71) by Honda et al. (1996) revealed accumulations of gibberellic acid-20 (GA20 inactive form) and nondetectable levels of GA1 (active). The disproportionate GA20/GA1 relationship is the result of a mutation in the gene that codes for 3ß-hydroxylase, the enzyme that converts GA20 to GA1 (Kinoshita and Shinbashi, 1982; Mitsunaga et al., 1994). The absence of GA1 results in an extremely short (20 cm tall) rice cultivar that is hereafter referred to in this manuscript as ‘Super Dwarf’. Application of GA3 to seedlings reverses the dwarf growth habit (Mitsunaga et al., 1994). Super Dwarf has poor seedling vigor as characterized by poor germination (
70%), variable germination rates, and uneven stand establishment.
Germination of monocots has been extensively studied (Jones, 1926; Roberts, 1961; Cohn and Hughes, 1986). Ritchie and Gilroy (1998) recently proposed a more detailed model describing the biochemical and molecular factors preceding germination. The model is based on the barley aleurone system and shows GA is perceived on the plasma membrane after imbibition under aerobic conditions. The perception of GA is followed ultimately by the transcription of hydrolytic enzymes including 
-amylase. -Amylase is excreted outside the plasma membrane and breaks down starch into simple sugars. Hanson and Jacobsen (1984) found that -amylase is not synthesized in barley (Hordeum vulgare L.) under anaerobic conditions, even in the presence of GA, and barley seeds fail to germinate. Conversely, rice produces -amylase and can thus germinate under both aerobic and anaerobic conditions; however, enzyme synthesis and germination are delayed under anaerobic conditions (Perata et al., 1992). It is not known if the synthesis of -amylase under anaerobic conditions is mediated by GA, is constitutively activated, or is stimulated by multiple signals (GA in addition to other metabolic products).
Freshly harvested seed of rice and other cereal grains can have low germination caused by post-harvest dormancy, which is referred to as exhibiting "nondeep physiological dormancy" (Hartmann et al., 1997). While fresh seed of japonica cultivars is usually not dormant (Ellis et al., 1983), the low germination percentage of Super Dwarf rice may be the result of nondeep physiological dormancy. This type of dormancy often disappears after dry storage.
Many factors can break dormancy including light, temperature, exogenous applications of GA (Berrie, 1984), nitrate (Cohn et al., 1983), alcohols, carboxylic acids (Cohn et al., 1987), and smoke (Thomas and van Staden, 1995). Roberts (1963)(1965) found post-harvest dormancy of rice was reduced by exposing seed to 3 d of dry heat (50–55°C), although the underlying mechanism is still unknown. The International Rice Research Institute (IRRI) heats all japonica and indica cultivars regardless of their dormancy tendencies to 50°C for 3 d. Our objective was to improve the germination rate and uniformity of Super Dwarf rice.
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Materials and Methods
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Seeds of Super Dwarf rice were obtained from Toshiro Kinoshita, Hokkaido University, Japan. This breeding line originated through mutant selection from Shiokari (Kinoshita and Shinbashi, 1982).
Germination tests were performed in growth chambers (model CEL 25-7-HI, Sherer-Gillet Co., Marshall, MI) set at constant temperature treatments of 20, 23, 27, 30, 33 or 36°C ±0.5°C. Non-green seeds were hand-selected from a single seed lot for all tests. There were three replicates of each treatment, and each replicate consisted of a germination box containing 25 seeds. Seeds were counted twice daily for 10 d, or until there was no change in the number of seeds germinated compared with the previous 24 h. These tests identified an optimum temperature (33°C) at which subsequent tests were performed.
Anaerobic and hypoxic treatments were imposed on Super Dwarf rice, wheat (Triticum aestivum L. cv. USU Apogee), and two cultivars of indica rice (29-Lu-1 and Ai-Nan-Tsao). The parent line Shiokari was not used because high quality seed was not available for the tests. The germination boxes were set up by placing one layer of blotter paper in the bottom and wetting to saturation with tap water. Tap water was used instead of distilled water because in the USA it typically contains at least 1 mM calcium (Ben Jarvis, personal communication) and micronutrients required by germinating seeds. Tap water was injected by syringe through a septum in the lid of the box after purging the head space for 24 h with humidified N2 (anaerobic) or a combination of humidified N2 and atmospheric air (hypoxic, 0.01, 0.02 and 0.04 mol fraction O2).
Many previous studies probably lacked continuous, strict anaerobic conditions for the duration of their trials. Because of this, we have described the details of how we obtained steady-state anaerobic or hypoxic conditions. In our systems, O2 concentration was continuously measured in the head space with an O2 sensor (model O2S, Apogee Instruments, Logan, UT) that was placed inside the box and monitored with a datalogger (CR10T, Campbell Scientific, Logan, UT). O2 leakage into the boxes was detected by the O2 sensors if the gas flow was stopped for more than 30 s. To prevent this leakage, boxes were maintained under a small positive pressure with the humidified gases to ensure that all leaks were from the inside out according to standard gas-exchange methods for open systems. Ultra-high purity N2 was used, which contains <0.5 µmol mol-1 (ppm) O2. This concentration would result in a maximum of 0.6 nmol L-1 O2 at 20°C to 0.42 nmol L-1 O2 at 33°C (0.02–0.013 ppb) dissolved in solution. The continuous O2 measurements allowed us to verify steady-state anaerobic conditions within the boxes for the duration of each trial. For the hypoxic environments, a continuous flow of humidified gas was adjusted as needed to maintain boxes in 0.01, 0.02, or 0.04 mol fraction O2 according to the O2 sensor.
For reference, two visual indicators of O2 status were used. These were root growth in rice and germination in wheat. Rice, germinating under anoxia, produces a coleoptile and no radicle (Perata and Alpi, 1993). In rice that germinates under water, this development permits the coleoptile to elongate above the water line, reach O2, transport O2 to the seed via aerenchyma, and initiate root growth. On the other hand, wheat seeds are often added to anaerobic germination tests as an example of a seed that fails to germinate in the absence of O2 (Alpi and Beevers, 1983; Perata et al., 1992).
The effect of exogenously applied GA on germination was evaluated by adding GA3 to tap water at 50, 100, and 1000 µM and adding the solutions to germination boxes containing Super Dwarf seeds. Heat treatments were evaluated by heating seeds in a forced-air drying oven at 50 ± 1°C for 72 h prior to placement in the germination boxes.
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Results and Discussion
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Super Dwarf rice germinated best at 33°C (Fig. 1)
. The indica cultivars 29-Lu-1 and Ai-Nan-Tsao were much less sensitive to temperature than Super Dwarf and germinated above 90% at all temperatures. In anaerobic conditions, Super Dwarf had higher germination percentages at all temperatures, and reached 95% germination at 33°C. At 0.01 mol fraction O2, germination decreased to 76%, and was 68% at 0.04 mol fraction and 0.209 mol fraction O2 (fully aerobic conditions) (Fig. 2)
. There was no effect of O2 on final germination percentages of indica cultivars. Perata et al. (1992) suggested that indica varieties have lower rates of germination in anoxia than japonica varieties, but reach similar final germination percentages after 4 d in anoxia. We observed a 1-d delay in the start of germination in anoxia when compared to aerobic germination, but no difference between indica or japonica varieties in the start of germination. USU-Apogee wheat did not germinate until the O2 concentration was above 0.01 mol fraction O2. The O2 requirement was likely due to the warm temperatures, as Alpi and Beevers (1983) reported 50% wheat germination in 0.01 mol fraction O2 at 20°C. In our studies, wheat germination was only 65% at 33°C because this temperature is well above optimum for germination. At 17°C, germination of this seed lot was 92% in fully aerobic conditions. Warm temperatures increase respiration rates (Q10 of 2.0) and decrease O2 solubility in solution (30% from 20–35°C). This could make seeds more sensitive to O2 at warmer temperatures.
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Fig. 1. The effect of temperature on aerobic and anaerobic germination of 29-Lu-1, Ai-Nan-Tsao, and Super Dwarf rice. Bars represent ±1 standard error of the mean.
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Fig. 2. Effect of O2 concentration on germination of 29-Lu-1, Ai-Nan-Tsao, Super Dwarf, and USU-Apogee wheat. All tests were conducted at 33°C. Bars represent ±1 standard error of the mean. USU-Apogee wheat was also tested at 17°C in fully aerobic conditions.
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We hypothesized that exogenous application of GA would compensate for the lack of active GA in this mutant line. The difference between the highest rate of GA application (1000 µM) and no GA treatment was not statistically significant based on an ANOVA (Table 1). High application rates of GA should have improved germination. This indicates that GA was either not entering the seeds or that GA by itself is not sufficient to fully break dormancy in the nongerminated seed of this breeding line. The former of the two options is not likely because the seedlings had significant elongation of the coleoptile for up to 10 d after final application of GA when compared to lower GA additions or no GA applications. Seed germination models based on the barley aleurone system include GA signaling as one of the first steps in the biochemical mechanism of seed germination. The germination mechanism in rice has not been studied in such detail. The fact that -amylase is synthesized under anaerobic conditions in rice but not in barley and wheat suggest that the biochemical mechanism for rice germination differs from other cereals. Also, since GA remains at undetectable levels in this breeding line (Honda et al., 1996) yet is able to germinate to 95% anaerobically suggests that -amylase is not activated by GA under anaerobic conditions.
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Table 1. Germination percentages with standard error of the mean of Super Dwarf germinated aerobically with GA3 applied exogenously at different concentrations. An ANOVA indicated no statistical difference (P = 0.092) in germination percentages among concentrations. Seedlings were elongated for two weeks after germination with GA3.
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The heat treatment did not improve germination percentages in this rice in aerobic conditions and reduced germination in the anaerobic treatment (Table 2). Interestingly, the non-heated, aerobically germinated rice had a final germination percentage of 80%. This set of tests was performed on the same seed lot after it had been in dry storage for 10 mo. It is likely that the non-deep physiological dormancy diminished in dry storage. After two years in dry storage, the germination percentage of aerobically germinated seed did not improve further. This is still too low for optimum stand establishment, but suggests some natural dormancy-breaking mechanism. Many forest species require fire to break dormancy while other seeds in the same ecosystems only require exposure to smoke (de Lange and Boucher, 1990). Mechanical disruption of the seed structure (i.e., seed coat, perisperm) may occur at high temperature, as in priming (Oluoch and Welbaum, 1996).
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Table 2. Germination percentages with standard error of the mean of Super Dwarf germinated aerobically or anaerobically with or without a heat treatment of 50°C for 3 d. The heat treatment reduced the germination of the anaerobic treatment and had no effect on the aerobic treatment.
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Fermentation products can break dormancy. Small alcohols and carboxylic acids such as ethanol, pyruvate, and lactate are produced anaerobically and these types of compounds are known to have dormancy-breaking activity (Cohn et al., 1989). The concentrations required for such activity, however, are as much as five orders of magnitude larger than for compounds such as GA when applied exogenously (Cohn et al., 1989).
Our results indicate that Super Dwarf rice has a seed dormancy that can be overcome by germination in anaerobic conditions. The fermentation products that are formed in the anaerobic conditions may break dormancy. Fermentation products or intermediates of glycolysis may activate -amylase under anaerobic conditions. Because Super Dwarf does not contain measurable levels of active GA, it would indicate that GA is not an essential hormone for activating -amylase as it is in the barley aleurone system.
It is not always practical to purge the head space of imbibed seeds with N2 to achieve 95% germination. Good germination can be achieved, however, by germinating seeds under at least 5 cm of unstirred water. While not strictly anaerobic, the amount of O2 dissolved in the water and subsequent diffusion to the seeds is small enough to allow good germination.
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NOTES
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This research was supported by the National Aeronautics and Space Administration Advanced Life Support Program administered by the Johnson Space Center and by the Utah State Agric. Exp. Stn., Utah State Univ. Approved as journal paper no. 7389.
Received for publication May 4, 2001.
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ABSTRACT
INTRODUCTION
