Published in Agron. J. 96:359-368 (2004).
© American Society of Agronomy
677 S. Segoe Rd., Madison, WI 53711 USA
COTTON
In Vitro Analysis of Cotton Pollen Germination
John J. Burke*,
Jeff Velten and
Melvin J. Oliver
USDA-ARS, SPA, Plant Stress and Water Conserv. Lab., Lubbock, TX 79415
* Corresponding author (jburke{at}lbk.ars.usda.gov).
Received for publication June 3, 2002.
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ABSTRACT
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Crops with economically valuable reproductive structures show the greatest negative discrepancy between average and record yields. To alleviate environmental stress–related yield reductions, a better understanding of the relative sensitivities of pollen development, dehiscence, pollen germination, pollen tube growth, fertilization, and subsequent boll development is needed. Progress in identifying the sensitivities of these developmental stress responses has been hampered in part by the lack of a rapid and reliable method of germinating cotton (Gossypium spp.) pollen in vitro. The present study describes the development of an in vitro cotton pollen germination system that provides improved pollen germination levels and pollen tube growth over existing methods. This procedure was used to evaluate the temperature sensitivity of pollen from greenhouse and field-grown cotton. The medium comprises 10% (w/v) agarose (pH 7.6), 25% (w/v) sucrose, 0.52 mM KNO3, 3.06 mM MnSO4, 1.66 mM H3BO3, 0.42 mM MgS04·7H20, and 1.0 µM A3 gibberellic acid. The medium is overlayed with a layer of 1.5% agarose before use. Optimum pollen germination and rapid tube elongation occurred between 28 and 31°C under 80% relative humidity. Decreased pollen germination occurred at temperatures above 37°C, and decreased tube elongation occurred at temperatures above 32°C. Evaluation of pollen, taken at 1400 h from field-grown cotton plants, showed that pollen from flowers exposed to direct sunlight had reduced viability compared with pollen from flowers inside the canopy. This could be attributed to the fact that flowers from field-grown irrigated cotton plants exposed to full sunlight experienced internal temperatures as high as 39°C, well above the 28 to 31°C optimum for pollen germination.
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INTRODUCTION
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CROP SPECIES GROWN throughout the world experience environmental stresses that limit their growth, development, and full expression of their genetic potential for agronomic yield. Comparison of average crop yields with reported record yields has shown that the major crops grown in the USA exhibit annual average yields three- to sevenfold lower than record yields due to unfavorable environmental conditions (Boyer, 1982). Analysis of yields from corn (Zea mays L.), wheat (Triticum aestivum L.), soybean [Glycine max (L.) Merr.], sorghum (Sorghum vulgare L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), potato (Solanum tubersosum L.), and sugar beet (Beta vulgaris L.) revealed that the average yield represented only 22% of the mean record yield. Crops with economically valuable reproductive structures showed the greatest discrepancy between average and record yields. Those crops having marketable vegetative structures exhibited approximately threefold reductions in yield (Boyer, 1982). These data suggest that plants have high productivity potential but are operating well below their genetic potential.
Yield loss might be lessened by identifying and optimizing those plant protective mechanisms that could be used to improve stress-resistant germplasm stocks. One such protective mechanism is acquired thermotolerance, a process postulated to be closely linked to the heat shock response. Plants are frequently exposed to elevated soil and air temperatures resulting in a reduction in their growth, development, and ultimately productivity. Subjecting them to a period of sublethal elevated temperatures induces a transient state of thermotolerance, which raises the injury threshold and protects the plants from subsequent, otherwise lethal, high temperatures. This acquisition of thermotolerance is a complex physiological phenomenon that has been shown to involve at least some heat shock proteins (HSPs) (Vierling, 1991). Although varying in magnitude among plant cultivars, most vegetative tissues exhibit an inducible heat shock response. Germinating pollen, however, has not been found to exhibit the heat shock protein induction pattern upon exposure to elevated sublethal temperatures and concomitantly exhibits rapid losses in viability upon heat exposure (Hopf et al., 1992). This may explain Boyer's observation that crops with economically valuable reproductive structures show the greatest discrepancy between average and record yields (Boyer, 1982).
Modest progress has been achieved in selecting cotton varieties with improved heat tolerance by heat treatment of pollen before pollination, allowing only the more heat-tolerant pollen to be effective in subsequent crosses (Rodriguez-Garay and Barrow, 1988). The process of selecting pollen with improved heat tolerance could be accelerated with a rapid and reliable method of germinating cotton pollen to measure viability across a range of environmental stresses. Current pollen germination techniques include hanging drop culture, sitting drop suspension culture, suspension culture, and surface culture (Alexander and Ganeshan, 1989; Brewbaker and Kwack, 1963; Cheng and Freeling, 1976; Dawkins and Owens, 1993; Egea et al., 1992; Feijo et al., 1995; Frascaroli and Tuberosa, 1993; Mulcahy and Mulcahy, 1988; Pareddy and Petolino, 1992; Pundir, 1972; Shivanna and Rangaswamy, 1992; Shivanna et al., 1991; Singh et al., 1992; Tanaka et al., 1995; Tupy et al., 1983; Vogt et al., 1994; Yistra et al., 1995; Zonia and Tupy, 1995). The hanging drop and sitting drop cultures use only small volumes of germination media and small amounts of pollen and are therefore of limited usefulness in physiological and biochemical studies.
Cotton pollen has proved to be recalcitrant to traditional in vitro germination and pollen tube growth protocols. Kearney and Harrison (1932) described the failure of in vitro techniques and went so far as to use the percentage of pollen grains that burst when placed in weak sugar solutions as a measure of viability. Failures to germinate cotton pollen in vitro drove Iyengar (1938) to dissect cotton pollen tubes from in situ germinated pollen. Bronkers (1961) first described a reliable technique for in vitro cotton pollen germination. Miravalle (1965) has since reported that the pollen tubes grown in this media were short, the cytoplasm was cloudy and granular, and the process required 24 h or longer. In 1972, Taylor described a medium that overcame many of the limitations outlined by Miravalle (1965). Taylor reported rapid pollen germination (2–3 h), more normal-appearing cytoplasm, and longer pollen tubes. Wauford (1979) further improved upon Taylor's medium and averaged 47% germination and 2.6-mm pollen tube lengths. Although Wauford's protocol was an improvement upon Taylor's medium, the 2.6-mm pollen tube length achieved in vitro does not compare with the 20- to 40-mm tube lengths reported in vivo. The most recent in vitro cotton pollen germination report by Barrow (1981) described the use of a hanging drop technique to forcefully eject pollen tube–like structures. Recent findings in our laboratory revealed that the pollen tube–like structures were not tubes but were pollen cytoplasm ejected from the pollen as it osmotically ruptured in a way similar to that reported by Kearney and Harrison (1932).
The present study describes the development of a pollen germination media and technique that provides high pollen germination levels and improved pollen tube growth. In developing the media, it was necessary to evaluate the following variables: temperature, humidity, pH, and C source. The resulting media and technique were then used to evaluate the temperature sensitivity of cotton pollen and has provided insights into plant morphological characteristics that reduce pollen viability in the field.
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MATERIALS AND METHODS
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Plant Growth Conditions
Greenhouse Conditions
Seeds of cotton cultivars Gregg 65, PM2200, PM2156, PM2326, DP90, and SG521 (Gossypium hirsutum L.) and Pima S-7 (Gossypium barbadense L.) were planted into hydroponic rock wool slabs (15 by 90 by 8 cm, width by length by depth) saturated with Peters Professional water soluble fertilizer [0.95 g/L 5-11-26 HYDRO-SOL (Scotts-Sierra Hortic. Prod. Co., Maryville, OH), supplemented with 0.475 g/L calcium nitrate (Ca Hydro Agri North America, Tampa, FL), and 0.238 g/L magnesium sulfate (Scotts-Sierra Hortic. Prod. Co., Maryville, OH)]. Two rock wool slabs per cultivar were planted with three seeds per pad per replication. The rock wool slabs were then placed in greenhouses using a randomized complete block design with five replications. Nutrients were maintained using a nonrecycling hydroponic watering system. Plants were grown at 28 ± 5°C (air temperature) for 120 d under natural light. Other species evaluated include tobacco (Nicotianum tabacum L.) cultivar SR1, corn cultivar DK626, and soybean cultivar Mitchell 450. Six plants for each species were grown under the same conditions as described above.
Field Conditions
Cotton (G. hirsutum L.) cultivar Paymaster 2326RR was planted on 1-m centers on 5 June 2000, in an Acuff sandy loam soil at Lubbock, TX. Standard cultural practices were used throughout the experiment. Irrigation (77 m3 per irrigation) was applied 10 times during the growing season via a center pivot equipped with drop socks.
Pollen Germination Media
The solid germination medium developed during this study consisted of 10% (w/v) agarose, 25% (w/v) sucrose, 0.52 mM KNO3, 3.06 mM MnSO4, 1.66 mM H3BO3, 0.42 mM MgS04·7H20, and 1.0 µM A3 gibberellic acid. The medium pH was brought to 7.6 before adding sucrose and agarose, after which it was liquified by autoclaving and poured into Easy GripTM Petri dishes (35 by 10 mm, Becton Dickinson Labware, Franklin Lakes, NJ), covered, and stored in a refrigerator until needed. Before use, the plates were removed from the refrigerator and brought to room temperature. Any condensation on the Petri dish lids was removed. The medium was overlaid with a 100 µL of hot 1.5% agarose, which was immediately smoothed with a glass rod and allowed to cool for 30 min. As the agarose overlay cooled, flowers were harvested randomly from the six plants within each replicate, the dehisced pollen from these flowers combined into a single sample per replicate, and the sample placed on the surface of the germination medium and allowed to germinate at 80% humidity. The composition of the final medium was based upon the results of experiments evaluating temperature, humidity, pH, and sugar content as described below.
Temperature Optimum for Pollen Germination
The temperature optimum for pollen germination and pollen tube growth was evaluated initially on pollen from greenhouse-grown Gregg 65 cotton. To achieve desired temperature treatments, the bottom of a 35- by 10-mm Petri dish containing the pollen germination medium was placed in an inverted lid containing saturated NH4SO4. The lid containing the Petri dish bottom was positioned on moist 3MM filter paper on the temperature blocks of an electronically controlled eight-position thermal plate system termed CELTEC (Burke and Mahan, 1993). The temperature blocks were set to a range of temperatures from 20 to 43°C. Cotton flowers were harvested randomly from the six plants within each replicate, the dehisced pollen from these flowers combined into a single sample per replicate, and the sample placed on the surface of the germination medium. After the pollen was sprinkled on the surface of the pollen germination media, another Petri dish lid was placed on top so that the edges of the two lids met. The seam between the two lids was wrapped with Parafilm (American National Can, Menasha, WI) and pollen germination and pollen tube growth were evaluated microscopically using an Olympus BX60 microscope (Olympus Optical Co., Tokyo, Japan), and video images from a MTI 3 CCD camera (PAGE-MTI, Michigan City, IN) were recorded. Experiments to determine temperature effects on pollen germination and on pollen tube elongation were performed on PM2156, PM2326, and DP90 cotton. Relative pollen germination and tube length were determined for three replicate samples of each cultivar by evaluation of equal magnification photographs and analyzing tube intersections using a modified line intersect method (Tennant, 1975). Relative pollen tube lengths obtained by this method were adjusted to a per-pollen-grain basis by counting all pollen grains within each photograph, thereby overcoming problems of uneven pollen distribution on germination plates. The three replicate samples for each of the three cultivars were combined, and the percentage germination and relative pollen tube lengths were evaluated for significant differences at the 0.05 level using JMP Version 5.0 statistical discovery software (SAS Inst., Cary, NC).
Humidity Effects on Pollen Germination
The effects of 35, 50, 80, and 100% relative humidity levels on pollen germination and pollen tube growth were evaluated initially on pollen from Gregg 65 cotton grown in the greenhouse. Humidity levels were obtained by using 1.7-L storage containers (Rubbermaid Save and Serve, Rubbermaid, Wooster, OH) containing saturated solutions of (i) CaCl2 providing 35% relative humidity, (ii) CaNO3 providing 53% relative humidity, (iii) NH4SO4 providing 80% relative humidity, and (iv) deionized water providing 100% relative humidity as described by Gawel and Robacker (1986). Cotton flowers were harvested randomly from the six plants within each replicate, the dehisced pollen from these flowers combined into a single sample per replicate, and the sample placed on the surface of the germination medium. Pollen was sprinkled onto the surface of the media and incubated at 28°C for 180 min at the various humidity levels. Pollen germination and pollen tube growth were evaluated microscopically and video images recorded. Relative pollen germination and tube length were determined for five replicate samples by evaluation of equal magnification photographs and analyzing tube intersections using a modified line intersect method (Tennant, 1975). Relative pollen tube lengths obtained by this method were adjusted to a per-pollen-grain basis by counting all pollen grains within each photograph, thereby overcoming problems of uneven pollen distribution on germination plates. The percentage germination and relative pollen tube lengths were evaluated for significant differences at the 0.05 level using JMP Version 5.0 statistical discovery software (SAS Inst., Cary, NC).
pH Requirements for Optimal Pollen Germination
The effect of media pH was evaluated initially on pollen from greenhouse-grown cotton Gregg 65 at pH 6.0, 6.5, 7.0, 7.6, 7.6, and 8.0. Cotton flowers were harvested randomly from the six plants within each replicate, the dehisced pollen from these flowers combined into a single sample per replicate, and the sample placed on the surface of the germination medium. Pollen germination and pollen tube growth were evaluated at 90 min after addition of the pollen to the respective media. Pollen germination and pollen tube growth were evaluated microscopically, and video images were recorded. Relative pollen germination and tube length were determined for five replicate samples by evaluation of equal magnification photographs and analyzing tube intersections using a modified line intersect method (Tennant, 1975). Relative pollen tube lengths obtained by this method were adjusted to a per-pollen-grain basis by counting all pollen grains within each photograph, thereby overcoming problems of uneven pollen distribution on germination plates. The percentage germination and relative pollen tube lengths were evaluated for significant differences at the 0.05 level using JMP Version 5.0 statistical discovery software (SAS Inst., Cary, NC).
Sugar Requirements for Pollen Germination
The effect of the C source in the germination media on pollen germination and pollen tube growth was evaluated on pollen from greenhouse-grown Gregg 65 cotton. Sugar concentrations of 25% (w/v) sucrose, 25% (w/v) maltose, 16% (w/v) glucose, or 16% (w/v) fructose were added to the germination media containing 10% (w/v) agarose, 0.52 mM KNO3, 3.06 mM MnSO4, 1.66 mM H3BO3, 0.42 mM MgS04·7H20, and 1.0 µM A3 gibberellic acid, pH 7.6. Sugar concentrations were chosen to provide similar osmotic pressures in the media. Cotton flowers were harvested randomly from the six plants within each replicate, the dehisced pollen from these flowers combined into a single sample per replicate, and the sample placed on the surface of the germination medium. Pollen was carefully sprinkled on the surface of the respective media and incubated at 28°C at 80% humidity for 180 min. Pollen germination and pollen tube growth were evaluated microscopically, and video images were recorded. Relative pollen germination and tube length were determined for five replicate samples by evaluation of equal magnification photographs and analyzing tube intersections using a modified line intersect method (Tennant, 1975). Relative pollen tube lengths obtained by this method were adjusted to a per-pollen-grain basis by counting all pollen grains within each photograph, thereby overcoming problems of uneven pollen distribution on germination plates. The percentage germination and relative pollen tube lengths were evaluated for significant differences at the 0.05 level using JMP Version 5.0 statistical discovery software (SAS, Cary, NC).
Time Course of Pollen Germination
The time course of pollen germination was evaluated initially on pollen from Gregg 65 plants grown in the greenhouse. Cotton flowers were harvested randomly from the six plants within each replicate, the dehisced pollen from these flowers combined into a single sample per replicate, and the sample placed on the surface of the germination medium. Pollen was germinated at 28°C for 30, 60, 120, 180, and 240 min using the technique and media previously described. Pollen germination and pollen tube growth were evaluated microscopically and video images recorded. Relative pollen germination and tube length were determined for five replicate samples by evaluation of equal magnification photographs and analyzing tube intersections using a modified line intersect method (Tennant, 1975). Relative pollen tube lengths obtained by this method were adjusted to a per-pollen-grain basis by counting all pollen grains within each photograph thereby overcoming problems of uneven pollen distribution on germination plates. The percentage germination and relative pollen tube lengths were evaluated for significant differences at the 0.05 level using JMP Version 5.0 statistical discovery software (SAS Inst., Cary, NC).
Evaluation of Canopy Effects on Pollen Viability of Field-Grown Cotton
Temperatures of anthers of field-grown Paymaster 2326RR cotton flowers from within the canopy (shade) and at the top of the canopy (sun) were measured with both a hand-held Raynger ST30 PRO infrared thermometer (Raytek Corp., Santa Cruz, CA) and an Omega Model HH21 microprocessor thermometer (Omega Eng., Stamford, CT) equipped with a Type-T thermocouple. Six flowers selected at random from full sun or full shade were measured with each thermometer at 1000, 1200, 1400, and 1600 h. Six sun and shade flowers were harvested per replicate at 1400 h, and pollen viability was evaluated by shaking the dehisced pollen onto the surface of germination plates and incubating the pollen at 28°C for 120 min under 80% relative humidity. Three replicates were evaluated for pollen germination and relative pollen tube lengths. Relative pollen germination and tube length were determined for the samples by evaluation of equal magnification photographs and analyzing tube intersections using a modified line intersect method (Tennant, 1975). Relative pollen tube lengths obtained by this method were adjusted to a per-pollen-grain basis by counting all pollen grains within each photograph, thereby overcoming problems of uneven pollen distribution on germination plates. The samples were evaluated for significant differences at the 0.05 level using JMP Version 5.0 statistical discovery software (SAS Inst., Cary, NC).
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RESULTS AND DISCUSSION
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An in vitro pollen germination media is needed to better understand the regulatory mechanisms influencing the directional pollen tube growth and to evaluate abiotic stresses on cotton pollen germination and pollen tube growth. Variables of temperature, humidity, pH, and sugar were evaluated in the development of a reliable media and technique.
Pollen Germination Media
This study describes the development of an in vitro cotton pollen germination system that optimizes pollen germination and pollen tube growth. Using the systems described by Taylor (1972) and Wauford (1979) as a foundation, the new pollen germination system increases the rigidity of the matrix, eliminates the need for some salts and hormones, and describes the microenvironment needed to enhance pollen tube growth. The medium comprises 10% (w/v) agarose (pH 7.6), 25% sucrose, 0.52 mM KNO3, 3.06 mM MnSO4, 1.66 mM H3BO3, 0.42 mM MgS04·7H20, and 1.0 µM A3 gibberellic acid, which is overlayed with a thin layer of 1.5% agarose before use. Initial studies evaluated substituting Wauford's use of 3.5% (w/v) agar in the pollen germination medium with increasing concentrations of agarose: 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, and 10% (w/v). As agarose concentrations increased from 1 to 8%, overall pollen tube length increased, and pollen tube ruptures decreased. Concentrations above 8% resulted in reduced pollen germination and reduced tube elongation. Additional studies found that the use of 100 µL of 1.5% agarose to overlay a 10% agarose basal medium provided enough moisture to enhance germination without rupturing pollen tubes.
Temperature Optimum for Pollen Germination
The temperature effect on pollen germination and pollen tube elongation was evaluated over a range of temperatures from 20 to 43°C. Pollen germination was high across the range of temperatures from 20 to 37°C. The percentage pollen germination declined from a mean of 71% at 37°C to 23% at 40°C, with little germination occurring at 43°C (Fig. 1)
. Pollen tube elongation rate was low at 20°C and increased with increasing temperature up to 28°C. The 28 and 31°C samples exhibited similar pollen tube lengths with significant (0.05 level) declines in tube length observed at 34°C and above (Fig. 1).
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Fig. 1. Photomicrograph of Gregg 65 cotton pollen exposed to 28°C on the pollen germination medium for 180 min. The graphs show the temperature dependence of pollen germination and pollen tube elongation of PM2156, PM2326, and DP90 cotton compared with samples incubated at 28°C. The three replicate samples for each of the three cultivars were combined and the percentage germination and relative pollen tube lengths evaluated. The data show reduced pollen germination at 40°C and above (LSD = 22.89) while pollen tube length was reduced with temperatures of 34°C or above (LSD = 15.201). Error bars represent standard errors.
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The universality of the pollen germination medium among cotton varieties was evaluated by checking pollen germination and pollen tube growth in G. hirsutum L. cv. PM2200, PM2156, PM2326, DP90, SG521 and G. barbadense L. cv. Pima S-7 (Fig. 2)
. Pollen germination and pollen tube growth were observed in all cultivars tested. Although the pollen germination medium was designed especially for cotton, its versatility beyond cotton was confirmed as the medium supported the germination and pollen tube growth of corn (Fig. 3A)
, soybean (Fig. 3B), and tobacco (Fig. 3C).
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Fig. 2. Photomicrographs of PM2200, PM2156, PM2326, DP90, SG521, and Pima S-7 pollen on the pollen germination medium following a 90-min incubation at 28°C. Pollen germination and pollen tube elongation were observed in all cultivars tested.
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Fig. 3. Photomicrographs of (A) corn cultivar DK626 pollen, (B) soybean cultivar Mitchell 450 pollen, and (C) tobacco cultivar SR1 pollen on the solid medium developed for cotton pollen germination. Pollen germination and pollen tube development were apparent in all three species.
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Humidity Effects on Pollen Germination
The effect of humidity levels on pollen germination and pollen tube elongation was evaluated at 35, 50, 80, and 100% relative humidity (Fig. 4)
. Pollen that germinated on media in 35% relative humidity had short pollen tubes located at the interface between the pollen grain and the germination medium. The 50% relative humidity resulted in increased pollen tube length while the best elongation occurred at 80% relative humidity. Although germination levels were high, most pollen tubes remained short as they ruptured when incubated under 100% relative humidity. A range of humidity (50–80%) can be used during pollen germination; however, if humidity levels are too low (35% or less), germination occurs, but only short tubes are observed. If the humidity level is too high (100%), germination occurs, and tubes rupture shortly thereafter.
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Fig. 4. Representative photomicrographs of Gregg 65 cotton pollen incubated at 28°C for 180 min on the pollen germination medium with surrounding relative humidity (RH) levels of 35%, 50 to 80%, and 100%. Pollen tube elongation was reduced at 35%, and pollen tubes ruptured in a 100% RH. Maximum pollen tube elongation occurred between 50 and 80% RH.
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pH Requirements for Optimal Pollen Germination
The effect of media pH on pollen germination and pollen tube elongation was evaluated at 0.5 pH intervals between the biologically significant pH range of 6.0 and 8.0. Pollen germination and pollen tube growth were evaluated at 30, 60, 90, and 120 min after addition of the pollen to the media. Pollen germination and pollen tube elongation were similar across the pH range of 6 to 8 (Fig. 5)
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Fig. 5. Representative photomicrographs of Gregg 65 cotton pollen incubated on pollen germination media having pH values ranging from 8.0 to 6.0. Similar pollen tube elongation and pollen germination were observed across this range of pH values.
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Sugar Requirements for Pollen Germination
Wauford (1979) evaluated the effectiveness of different sugars to support cotton pollen germination and pollen tube growth. No germination was found on fructose, 12% germination on raffinose, and 33% germination on glucose and sucrose. Pollen tube growth was greatest on sucrose, less on raffinose, and only short tubes were observed on glucose.
The effects of using sucrose, maltose, glucose, and fructose as C source were evaluated in the present study. Photomicrographs of cotton pollen incubated for 120 min on a germination media containing sucrose, glucose, maltose, or fructose are shown in Fig. 6
. Maximum germination and pollen tube elongation occurred in the sucrose-containing medium. Pollen placed on glucose and maltose exhibited lower germination percentages and shorter pollen tube lengths than seen with pollen placed on a sucrose medium. Similar to Wauford's findings, no germination or pollen tube growth was observed on media containing fructose as the sole C source.
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Fig. 6. Photomicrographs of cotton pollen incubated for 120 min on a germination media containing (A) sucrose, (B) glucose, (C) maltose, and (D) fructose. Relative pollen tube lengths are provided in the graph. Maximum germination and pollen tube elongation occurred in the sucrose-containing medium.
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Time Course of Pollen Germination
The time course of cotton pollen germination and pollen tube elongation following 30, 60, 120, and 180 min at 28°C was evaluated (Fig. 7)
. Pollen germination and tube initiation were apparent within the first 30 min of incubation on the media. Pollen germination was routinely marked by a single pollen tube arising from the pollen grain. In a small percentage of the sample, pollen grains exhibited the emergence of two tubes. One tube would continue to elongate while the second tube would remain relatively short (two to three pollen diameters in length, Fig. 7 arrows). Pollen tube length increased with increasing incubation time. As incubation times at 28°C extended beyond the 180 min exposure, an increasing number of pollen tube tips would rupture, and a pool of cytoplasm was seen around the tip. The rupturing of the pollen tubes could be reduced by using the incubation temperature of 22°C (Fig. 8A)
. The diagram shown in Fig. 8B shows the path of a single pollen tube's growth during a 5 h incubation of one of the pollen grains shown in Fig. 8A.
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Fig. 7. Representative photomicrographs showing the time course of cotton pollen germination and pollen tube elongation at 28°C. Relative pollen tube lengths determined at 30-min intervals are provided in the graph.
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Fig. 8. (A) Photomicrograph of cotton pollen incubated at 22°C for 5 h, and (B) diagram of the path of pollen tube development of a single pollen grain shown in (A).
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Evaluation of Canopy Effects on Pollen Viability of Field-Grown Cotton
Having developed an effective system for evaluating pollen temperature sensitivity in vitro, we wanted to see if inhibitory temperatures observed in the lab also translated to inhibition of pollen in the field. Cotton anther temperatures were measured within flowers located inside the canopy and on the surface of the canopy of field-grown cotton. Figure 9
shows anther temperatures measured at 1000, 1200, 1400, and 1600 h. Temperatures of flowers exposed to full sunlight increased gradually from 30°C at 1000 h to 43°C by 1600 h. Temperatures of flowers shaded by the canopy tracked air temperatures, maintaining anther temperatures of 22°C at 1000 h to 35°C at 1600 h. Pollen was harvested at 1400 h from shaded flowers (Fig. 10A)
having internal temperatures of 30°C and from flowers exposed to full sun (Fig. 10B) having internal temperatures of 38°C to determine the effects of the elevated temperatures on pollen germination and tube elongation. The harvested pollen was incubated on the pollen germination media at 28°C for 120 min. Greater pollen germination and pollen tube lengths were seen in flowers that were shaded by the canopy than from the flowers exposed to full sunlight and elevated temperatures.
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Fig. 9. Graph of anther temperatures of flowers shaded by the canopy (open bars) and flowers exposed to the full sun (solid bar) from 1000 to 1600 h. Elevated temperatures were seen in the flowers exposed to full sunlight at all measurement times compared with flowers shaded by the canopy.
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Fig. 10. Diagram of a cotton plant illustrating the relative position of flowers measured in the (A) shade and (B) sun at 1400 h. The photomicrographs show pollen incubated on the germination media at 28°C for 120 min of pollen taken from (A) shaded flowers and (B) flowers exposed to full sun. Greater pollen germination and pollen tube length are seen in flowers that were shaded.
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SUMMARY
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This study describes the development of a solid medium for the germination of cotton pollen in vitro and demonstrates its usefulness in the analysis of the temperature sensitivity of pollen from greenhouse and field-grown cotton. The highest percentage germination and most rapid pollen tube elongation occurred at 28°C. Adjustment of incubation temperature to 22°C slowed pollen tube growth but enhanced the overall length of the tubes over extended times. Maintenance of humidity levels between 55 and 80% optimized pollen tube elongation. Lower humidity resulted in short pollen tubes located at the interface between the pollen grain and the solid medium. Humidity levels greater than 80% supported rapid pollen tube elongation; however, pollen tubes ruptured, and a pool of cytoplasm was observed at the tip of the pollen tube if kept in a high-humidity environment. The solid pollen germination medium developed in this study also supported the germination and pollen tube elongation of corn, soybean, and tobacco pollen.
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ACKNOWLEDGMENTS
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The author thanks Jacob Sanchez, Chris Huff, Julie Smith-Morrow, and Julia Veyro for their excellent technical assistance. Mention of a commercial or proprietary product does not constitute an endorsement by the USDA. The USDA offers its programs to all eligible persons regardless of race, color, age, sex, or national origin.
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V. G. KAKANI, K. R. REDDY, S. KOTI, T. P. WALLACE, P. V. V. PRASAD, V. R. REDDY, and D. ZHAO
Differences in in vitro Pollen Germination and Pollen Tube Growth of Cotton Cultivars in Response to High Temperature
Ann. Bot.,
July 1, 2005;
96(1):
59 - 67.
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