Crop Science 40:968-970 (2000)
© 2000 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Estimation of Pollen Viability in Grain Sorghum
M.R. Tuinstra and
J. Wedel
Kansas State Univ., Dep. of Agronomy, Manhattan, KS 66506 USA
mtuinstra{at}bear.agron.ksu.edu
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ABSTRACT
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Male fertility of crop plants is a function of pollen production and viability. Differences in pollen production can be evaluated by simple observation, but pollen viability is more difficult to quantify. The objective of this study was to develop an in vitro pollen germination assay for sorghum [Sorghum bicolor (L.) Moench]. In experiments evaluating common germination media substrates, large differences in germination were observed in response to changing concentrations of sucrose, boric acid, and calcium nitrate in agar-based media. In vitro germination was maximized following incubation on agar supplemented with 0.9 M sucrose, 2.43 mM boric acid, and 2.12 mM calcium nitrate. The effect of incubation temperature was also significant. Pollen germination was not affected by changes in temperature between 20 and 40°C, but germination was significantly reduced at 10°C.
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INTRODUCTION
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MALE FERTILITY OF CEREAL CROPS is a function of pollen production and viability and can be strongly influenced by the environment (Brooking, 1979; Herrero and Johnson, 1980; Shivanna et al., 1991; Khatun and Flowers, 1995). Problems with male fertility can result in reduced seed set and yield potential in grain and seed production fields. In sorghum, problems with male fertility also can contribute to problems with ergot infection, Claviceps africana Freder., Mantle & De Milliano or Claviceps sorghi McRae. Ergot is a fungal pathogen that infects unfertilized ovaries. First identified in India in 1917 and Kenya in 1924, ergot until recently was characterized as an Old World disease (Frederickson and Mantle, 1988; Frederickson et al., 1991). However, a recent ergot epidemic (C. africana) has led to the spread of the disease through most of the sorghum-producing areas in North and South America and Australia. Fungicides effective against ergot have been identified; however, pollen-based management to minimize the window of opportunity for ergot infection generally is viewed as the most effective strategy for controlling this disease (Bandyopadhyay et al., 1998).
Male fertility of sorghum varies from day to day under normal field conditions. Adverse environmental conditions can result in reduced pollen fertility and has been shown to predispose sorghum to ergot infection (Bandyopadhyayet al., 1998). Research has indicated that variations in temperature, humidity, and cloud cover can influence pollen production and viability (Artschwager and McGuire, 1949; Brooking, 1979; McLaren and Wehner, 1992). Cold temperature stress prior to flowering appears to reduce pollen viability during anthesis by disrupting meiosis during early stages of microsporogenesis (Brooking, 1979). The physiological effects of high temperature stress, humidity, and cloud cover on male fertility of sorghum are not as well characterized.
Pollen viability can be quantified by several different procedures. Under field conditions, pollen viability can be tested by evaluating seed set on self- or cross-pollinated florets. This particular measurement may indicate the presence or absence of fertile pollen; however, quantifying the absolute level of pollen viability using this procedure can be difficult. Under laboratory conditions, viability can be quantified more precisely by simple assays: evaluation of pollen germination in vivo (Sun et al., 1991) or in vitro (Artschwager and McGuire, 1949; Lansac et al., 1994), use of vital stains including aniline blue to detect callose in pollen tubes or iodine to determine starch content (Brooking, 1979; Heslop-Harrison et al., 1984), or measurement of enzymatic activity (Heslop-Harrison et al., 1984). In vitro pollen germination generally is believed to provide the best estimate of pollen viability in vivo (Stone et al., 1995).
Given the current threat of ergot to sorghum production around the world, an efficient pollen viability assay is needed to assess differences in male fertility among commercial sorghum hybrids and breeding lines. The objective of this research project was to develop a rapid, field-based assay for pollen viability in sorghum. Several researchers have reported in vitro germination of sorghum pollen in distilled water or in sucrose solutions; however, the development of an agar-based assay should provide greater flexibility for use under field conditions (Ayyangar and Rao, 1931; Stephens and Quinby, 1934; Artschwager and McGuire, 1949; Lansac et al., 1994). Our preliminary attempts to germinate sorghum pollen grains on water or sucrose-based agar media were unsuccessful or resulted in pollen grains with poorly developed pollen tubes. The specific research objectives of this project were (i) to identify in vitro conditions optimal for germination of sorghum pollen on an agar-based medium, and (ii) to analyze the effects of incubation temperature on pollen germination.
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Materials and methods
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Pollen Germination Assay
A factorial experiment design was used to evaluate the effects of sucrose, boric acid, and calcium nitrate on sorghum pollen germination. Experiments were conducted in tissue culture plates (15 by 100 mm) containing a 1% (w/v) agar medium supplemented with different concentrations of the three reagents in all combinations. The effect of sucrose was initially tested at five concentrations including 0.5, 0.7, 0.9, 1.1, and 1.3 M. Boric acid was tested at three levels including 0.81, 1.62, and 2.43 mM (50, 100, and 150 mg/L respectively). Calcium nitrate tetrahydrate was tested at three levels including 0.42, 1.27, and 2.12 mM (100, 300, and 500 mg/L). The pollen source for these studies was a genetically heterogeneous plant population representing hundreds of different sorghum accessions and breeding lines. This population was grown in a greenhouse at Kansas State University at Manhattan, KS, between November 1997 and June 1998. Plants were grown in 11.3-L pots, and natural lighting was augmented with high pressure sodium and metal halide lamps programmed for a 12-h photoperiod. Fresh pollen was collected and bulked each day as needed from at least 10 different genotypes between 0800 and 0900 h. Bulk pollen was sampled immediately after collection (<5 min) using a fine, camel-hair brush to dispense pollen from a pollinating bag onto the germination media. Plates were incubated in the dark for 4 h at 25°C. Pollen grains were evaluated for germination with a light microscope at 100x magnification. Germination was quantified as the percentage of germinated pollen grains per 100 evaluated. Pollen grains were considered germinated when the pollen tube length was greater than the diameter of the pollen grain. The factorial experiment was blocked in time with five replications following a randomized complete block design.
A second study was designed to evaluate a wider range of concentrations for each germination medium substrate. In these experiments, individual substrate concentrations were varied while others were held constant at near optimum concentrations as indicated in the first experiment (1% agar, 0.9 M sucrose, 2.43 mM boric acid, 2.12 mM calcium nitrate). Sucrose effects on pollen germination were evaluated at six treatment levels (0, 0.5, 0.7, 0.9, 1.1, and 1.3 M); boric acid was evaluated at six treatment levels (0, 0.81, 1.62, 2.43, 3.23, 4.04 mM); and calcium nitrate was evaluated at six treatment levels (0, 0.42, 1.27, 2.12, 2.96, 3.81 mM). These experiments were blocked in time with six replications following a randomized complete block design.
Effect of Incubation Temperature on Pollen Germination
The effect of incubation temperatures of 10, 20, 30, and 40°C on pollen germination was evaluated. The experiment was blocked in time with ten replications following a randomized complete block design. Tissue culture plates containing optimal germination medium (1% agar, 0.9 M sucrose, 2.43 mM boric acid, 2.12 mM calcium nitrate) were prepared and stored at 4°C until needed. At 0800 h, plates for a given replication were removed from cold storage and equilibrated at the appropriate temperature treatments for 60 min prior to collecting pollen. At 0900 h, bulk pollen was collected and immediately sown on plates representing each temperature treatment. Plates were incubated at the designated temperature for 4 h, and pollen germination was quantified as previously described.
Statistical Analysis
Each experiment was conducted as a randomized complete block design. Block effects were considered random, and treatment main effects and interactions were evaluated as fixed effects. The statistical analysis was conducted by the PROC GLM procedure in Statistical Analysis Systems (SAS). The Error Mean Square was used to test each of the treatment sources of variation based on expected mean squares for fixed treatment effects as described in Steele and Torrie (1980). Differences among treatment main effects were evaluated for significance by the least significant difference (LSD0.05).
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Results and discussion
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Pollen Germination
Analysis of variance of the initial factorial experiment indicated highly significant differences in pollen germination associated with the sucrose and calcium nitrate treatments (Table 1)
. Pollen germination was greatest at intermediate sucrose concentrations, and improved with increasing calcium nitrate concentration. Pollen germination was highest on germination medium containing 0.9 M sucrose, 2.43 mM boric acid, and 2.12 mM calcium nitrate. Germination also was good at lower sucrose concentrations (0.5–0.7 M); however, a greater percentage of pollen grains burst under these conditions. Differences in pollen germination associated with boric acid treatment were not significant given the narrow range of boric acid concentrations that were evaluated in this experiment (Table 1). Significant interactions were identified between sucrose and calcium nitrate and between sucrose and boric acid treatments (Table 1). Visual assessment of the data suggested that these factors had synergistic effects on pollen germination at near optimum concentrations.
The results of experiments to optimize individual substrate concentrations were consistent with outcome of the initial factorial experiment (Table 2)
. As expected, variation in sucrose concentration had a large impact on sorghum pollen germination. Very little pollen germination was observed at low or excessively high sucrose concentrations. The highest levels of pollen germination were observed on germination medium containing between 0.5 and 0.9 M sucrose. Variation among sucrose treatment effects appeared to be associated with changes in osmotic potential in the medium. A significant response in pollen germination was identified in the analysis of a broad range of boric acid treatments including very low concentrations. At low boric acid concentrations, pollen germination was roughly one-third of that observed under optimum conditions. Pollen germination was maximized on germination medium containing 2.43 mM boric acid. A large response in pollen germination was associated with changes in calcium nitrate concentration. Pollen germination was highest at intermediate concentrations on germination medium containing between 1.27 and 2.96 mM calcium nitrate. Pollen germination was inhibited at excessively high concentrations of calcium nitrate. This pattern of calcium induced pollen germination was consistent with the results of experiments in other species (Steer and Steer, 1989). On the basis of the results of these experiments, sorghum pollen germination should be optimized by evaluation on agar medium containing 0.9 M sucrose, 2.43 mM boric acid, and 2.12 mM calcium nitrate.
Recent reports have indicated that sorghum pollen can be germinated in water and suggest that germination may be an autonomous response that does not require supplements (Lansac et al., 1994). These findings are not supported by the results of this study in which sorghum pollen germination was shown to be strongly dependent on the addition of sucrose, boric acid, and calcium nitrate to agar-based germination medium. Similar results have also been shown in other studies. In many species, pollen germination is dependent on the addition of certain key substrates such as calcium nitrate to the germination media (Steer and Steer, 1989).
Effects of Incubation Temperature on Pollen Germination
Pollen germination was evaluated over a range of temperatures that might be commonly encountered under field conditions (Table 3) . No significant differences were detected at incubation temperatures ranging between 20 and 40°C, but trends in the data suggest that pollen germination may be inhibited at excessively high temperatures. Cold temperature stress negatively influenced pollen germination. Very little pollen germination was observed following incubation at 10°C. Pollen tubes of grains germinated at 10°C appeared to be shorter and less well developed after 4 h than pollen tubes evaluated at higher incubation temperatures.
Given this temperature profile, pollen germination is not likely to be influenced by changing temperatures under normal field conditions. Evaluation of pollen germination should be possible following incubation at ambient temperature, thereby simplifying the assay procedure. These results of incubation temperature response contrast with the results of earlier studies in sorghum described by Ayyangar and Rao (1931) in which pollen germination was reported to increase as incubation temperature decreased.
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NOTES
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Contribution No. 99-212-J from the Kansas Agric. Exp. Stn.
Received for publication May 27, 1999.
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REFERENCES
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ABSTRACT
INTRODUCTION
