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TURFGRASS SCIENCE

Estimation of Pollen Viability, Shedding Pattern, and Longevity of Creeping Bentgrass on Artificial Media

^a Dep. of Horticulture, 257 Horticulture Hall, Iowa State Univ., Ames, IA 50011
^b The Scotts Co., 14111 Scottslawn Rd., Marysville, OH 43041

^* Corresponding author (sfei{at}iastate.edu).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
An estimation of pollen viability is needed to determine pollen daily shedding pattern and longevity. Both parameters provide valuable information for plant breeding and contribute to the risk assessment of pollen-producing transgenic crops. Pollen viability of ‘Crenshaw’ creeping bentgrass (Agrostis stolonifera L.) was estimated through pollen germination on media containing sucrose (1-alpha-D-glucopyranosyl-2-beta-D-fructofranoside, at 0.25, 0.5, 1.0 M), H₃BO₃ (1.0, 2.0, or 4.0 mM), and CaCl₂ (1.0, 2.0, or 4.0 mM). The highest pollen germination percentage was obtained on a medium containing 1.0 M sucrose, 1.0 mM H₃BO₃, and 2.0 mM CaCl₂. Concentrations of all three medium components had significant effects on pollen germination. No two-way or three-way interactions among sucrose, H₃BO₃, and CaCl₂ were observed. A high sucrose concentration of 1.0 M severely inhibited pollen tube growth, causing shorter and thicker pollen tubes. Pollen-tube length of pollen germinated on medium containing 1.0 M sucrose averaged 137 µm, while those germinated on medium containing 0.5 M sucrose averaged 248 µm. The daily shedding pattern of pollen of Crenshaw creeping bentgrass was determined by collecting and germinating pollen from 0800 through 1700 h at 1-h intervals. The results indicated there were two peaks of pollen viability, with the first one occurring at 0900 h and the second peak at 1400 h. The longevity of Crenshaw and ‘Penncross’ pollen was assessed by storing the pollen in a desiccator at 21°C and a relative humidity of 64 to 66%. Our results showed that creeping bentgrass pollen lost viability dramatically within the first 1.5 h of storage and lost viability completely after 3 h of storage.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
GENETIC TRANSFORMATION has proven to be an effective tool to enable the rapid introduction of desirable traits into plants. However, the potential for pollen-mediated gene flow from transgenic plants into wild relatives, the persistence of potential hybrids, and the ecological consequences should be thoroughly assessed and closely monitored. Creeping bentgrass is an important turfgrass grown extensively on golf course greens and fairways in the temperate regions of North America. A variety of creeping bentgrass will likely be the first transgenic turfgrass species to be commercialized because of the availability of an efficient genetic transformation system (Zhong et al., 1993; Hartman et al., 1994).

The breeding behavior of creeping bentgrass is wind-facilitated open pollination. Therefore, pollen are carriers of transgenes and will enable gene flow to occur when wild relatives are present to receive pollen. An efficient and reproducible protocol for estimating viability would enable the daily pollen shedding pattern to be determined. Pollen longevity may also be studied, which will facilitate the estimation of how far viable pollen can potentially travel. This information may then be used to better develop guidelines to reduce the potential of pollen-mediated gene flow to or from transgenic crops in fields where pollen production is involved. Furthermore, an estimation of pollen viability and longevity will provide valuable information to plant breeders when hybridization is involved.

Seed set data may be used to estimate pollen viability. However, these data merely indicate the presence or absence of fertile pollen, or at most provide the relative number or percentage of viable pollen among treatments. The true level of pollen viability cannot be determined with this data.

Estimations of pollen viability with various stains, including aniline blue, iodine, or 1,2,3-triphenyl tetrazolium chloride (Brooking, 1979; Heslop-Harrison et al., 1984; Mulugeta et al., 1994) have been reported. However, pollen staining and viability is not always positively correlated (Mulugeta et al., 1994). Pollen viability has been reliably estimated with artificial medium in a number of grass species including ryegrass (Lolium spp.) (Ahloowalla, 1973) and Kentucky bluegrass (Poa pratensis L.; Teare et al., 1970), but not as yet with creeping bentgrass. Sucrose (Bair and Loomis, 1941; DeBruyn, 1966a, b), H₃BO₃ (DeBruyn, 1966a, b), and Ca ions (Cook and Walden, 1967) are three of the most common nutrient components employed when formulating an artificial medium for in vitro pollen germination.

The objectives of this study were (i) to develop an optimal medium for estimating the viability of creeping bentgrass pollen; (ii) to document the daily shedding pattern of creeping bentgrass pollen, and (iii) to estimate the longevity of creeping bentgrass pollen with an optimal pollen germination medium.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Creeping bentgrass plants from the cultivars Crenshaw and Penncross were established from vegetative stolon nodes during September 2000 and grown in Jiffy Pellets (Jiffy Products, Batavia, IL; 42-mm diameter) in Oregon. Plants of all genotypes in Jiffy pots were transplanted during September 2000 into 18-cm (7-inch) plastic pots containing a potting mixture of 90% peat moss and 10% perlite by volume. Plants were maintained under natural daylength and temperature environment in a cold frame greenhouse for floral induction during winter and spring. Irrigation was provided to prevent drought stress. Fertilization was performed with Miracle Gro (Scotts Co., Marysville, OH; 15:30:15 N:P:K) to avoid visible nutrient deficiencies. Forty-six plants of 16 clones of Crenshaw and 46 plants of 17 clones of Penncross were shipped to Iowa State University at Ames, IA, during a period from February to May in 2001. Plants were maintained in a research greenhouse supplemented with incandescent light to extend daylength to 16 h, were irrigated twice a week, and fertilized with Peters Professional fertilizer (Scotts Co.; 9:45:15) once a week. Preventative fungicides were applied when necessary. The temperature inside the greenhouse was maintained at 21°C.

A factorial experiment involving three sucrose concentrations (0.1, 0.5, and 1.0 M), three H₃BO₃ concentrations (1.0, 2.0, or 4.0 mM), and three CaCl₂ concentrations (1.0, 2.0, or 4.0 mM), a total of 27 different germination media, was performed. All chemicals were dissolved in deionized distilled water. Phytogel (Sigma, St. Louis, MO) was added at 3 g L^-1 to all media and dissolved on a hot plate. Medium was dispensed into 100- x 15-mm Petri plates.

To screen for the best medium for pollen germination, pollen from plants of a single clone (Clone 8; the clone number was randomly assigned) of Crenshaw were collected with a Petri plate (100 x 15 mm) and were immediately placed on various germination media at room temperature for germination. After 30 min of germination, plates were moved to a 4°C cooler to prevent further pollen tube growth and to facilitate counting at a later time. Pollen with a pollen tube longer than its diameter is considered germinated (Tuinstra and Wedel, 2000). At least nine fields of each plate were examined with a Nikon (Melville, NY) upright microscope to count at least 300 pollen grains.

To determine the effect of the sucrose concentration on pollen tube growth, pollen grains collected from Clone 7 of Crenshaw at 1000, 1100, 1200, and 1300 h, respectively, were germinated on media containing 1.0 mM H₃BO₃, 1.0 mM CaCl₂, and with either 0.5 or 1.0 M sucrose. After 5 h of germination, the pollen tube length of each of 30 germinating pollen grains from each treatment was measured with a micrometer mounted on the eyepiece of the microscope.

To determine the daily pollen shedding pattern, pollen were collected from Clone 9 of Crenshaw creeping bentgrass from 0800 through 1700 h at 1-h intervals. A pollen germination test was performed on the optimal germination medium at room temperature.

To determine pollen longevity, pollen were collected from Clone 5 of Penncross and Clone 9 of Crenshaw between 1000 and 1100 h. A portion of pollen grains from each collection was immediately placed on the optimal germination medium to determine the original pollen germination rate. The rest of the pollen was stored in a desiccator. The relative humidity within the desiccator was adjusted to 64 to 66% with a saturated solution of NaNO₂ (Hong et al., 1999). The desiccator was sealed with silicon gel and kept in a Percival (Perry, IA) incubator at 21°C. Stored pollen were removed from the desiccator every 20 min and germinated on the optimal germination medium at room temperature to determine the pollen germination rate across a period of time until pollen lost viability completely.

A randomized experiment with three replications was performed. Data on pollen germination percentage for medium screening was transformed by arcsin transformation and analyzed with a PROC GLM procedure of SAS (PC Version 8.0, SAS Institute, Cary, NC). Least significant difference mean separation was performed to separate means of the main effects of different concentrations of sucrose, H₃BO₃, and CaCl₂ on pollen germination. Pollen tube comparison was performed by LSD.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Effect of Medium Components on Pollen Germination
Normal floral development was observed in all plants, although the number of inflorescences produced varied considerably from clone to clone. Because of this variation, only a few clones had sufficient amounts of pollen grains for experimentation at any given time.

Pollen of Clone 8 from Crenshaw started to germinate 5 min after contact with the germination medium. Of all the 27 different media tested, the highest mean pollen germination percentage (23 ± 11.8%) was obtained on the medium containing 1.0 M sucrose, 1.0 mM H₃BO₃, and 2.0 mM CaCl₂. Analysis of variance indicates that the concentration of three medium components, sucrose, H₃BO₃, and CaCl₂, all had a significant effect on pollen germination rate (Table 1). Pollen germinated on media containing 1.0 M sucrose had significantly higher germination rate than pollen germinated on media containing either 0.5 or 0.25 M sucrose. Pollen germination rate increased as sucrose concentration increased (Table 2). No pollen bursting was observed in our study, even at the lowest sucrose concentration. It was reported in grain sorghum (Tuinstra and Wedel, 2000) that pollen tended to burst at lower sucrose concentrations, possibly because of a lowered osmotic potential. The lowest sucrose concentration (0.25 M) in our experiment may not be low enough to cause this to happen.

At the beginning of the experiment, all media were autoclaved to minimize the time needed for medium preparation. However, no pollen germination was observed on autoclaved medium regardless of the concentrations of all three medium components. A pairwise comparison of autoclaved vs. nonautoclaved medium with the same source pollen indicated only one out of 4771 pollen grains germinated on autoclaved medium containing 0.5 M sucrose, 1.0 mM H₃BO₃, and 2.0 mM CaCl₂; the nonautoclaved medium yielded an average pollen germination rate of 17.5%. It was reported that sucrose in tissue culture medium is hydrolyzed into glucose and fructose during the autoclaving process (Pan and Van-Staden, 1999) and, therefore, hydrolysis of sucrose during autoclaving could be a contributing factor to the poor pollen germination response observed. The hydrolysis of sucrose leads to an increase of osmotic potential; however, the negative effect of autoclaving on pollen germination rate unlikely results from a change in osmotic potential. Even with a complete hydrolysis after autoclaving, a medium containing 0.5 mol sucrose would yield 0.5 mol each of glucose and fructose, which would have the same osmotic potential as in a medium containing 1.0 mol sucrose. In fact, media containing 1.0 M sucrose yielded the highest pollen germination rate in this experiment. Besides the conversion of disaccharide into oligosaccharides, we speculate that the high temperature and high pressure during autoclaving may have caused elements that are essential for pollen germination to become unavailable or some inhibitive elements or compounds have been produced during the autoclaving process.

Although 1.0 M sucrose yielded the highest pollen germination rate, pollen tube growth was severely inhibited on media containing 1.0 M sucrose compared with those germinated on medium containing either 0.5 or 0.25 M sucrose. Figure 1 shows the effect of sucrose concentration on pollen tube growth 5 h after germination. Regardless of the pollen collection time, pollen germinated on media containing 0.5 M sucrose had a significantly higher average pollen tube length than those germinated on medium containing 1.0 M sucrose. Pollen germinated on medium containing 1.0 M sucrose appeared to have short and thick pollen tubes instead of long and slender tubes observed on media containing either 0.25 or 0.5 M sucrose (Fig. 2A,B).

View larger version (38K): [in this window] [in a new window]   Fig. 1. The effect of sucrose concentration on pollen tube length (average of 30 germinating pollen for each treatment). Pollen grains were collected from Clone 7 of ‘Crenshaw’ bentgrass at 1000, 1100, 1200, and 1300 h, respectively. Columns within each pair having a same letter are not significantly different from each other at = 0.05. Bars indicate SE.

View larger version (62K): [in this window] [in a new window]   Fig. 2. Germination of pollen from Clone 7 of ‘Crenshaw’ on medium containing (A) 0.5 M or (B) 1.0 M sucrose. The rest of the medium components are the same with 1 mM H3BO3, 2 mM CaCl2, and 0.3% (by weight) Phytogel. Note the short and thick pollen tubes in (A) vs. the long and slender pollen tube in (B). A germinating pollen of Clone 5 of ‘Penncross’ with double pollen tubes on a medium with 0.5 M sucrose, 1.0 mM H3BO3, 2.0 mM CaCl2, and 0.3% (by weight) Phytogel (C).

Interestingly, we observed germinating pollen grains with double pollen tubes arising from a single germination pore (Fig. 2C) or germinating pollen grains with bifurcated pollen tubes. A similar observation has been reported in ryegrass (Lolium spp.) (Ahloowalla, 1973). Although no data are available on the frequency, the overall occurrence of double tubes or bifurcated tubes was rare in our study. Whether this occurs in vivo, and if it does, what function it has is not known. However, it is quite possible that chemicals in artificial media may have caused this abnormality to occur. It was nearly impossible to establish the correlation between the occurrence of double tubes and the chemical composition in various media, if such correlation exists, because of the extremely low frequency of this phenomenon.

Daily Pollen Shedding Pattern
Data in Fig. 3 shows pollen viability of Clone 9 from Crenshaw bentgrass across a period of 10 h with hourly intervals during the daytime. As shown, the pollen germination rate reached the highest average percentage of 59.8% at 0900 h, it then declined to the lowest percentage of 10.6% at 1200 h before reaching another peak at 1400 h, when the pollen germination rate reached 49.3%. Pollen germination rate then dropped to near zero at 1700 h. Teare et al. (1970) reported that Kentucky bluegrass pollen viability remained high before 0730 h, but started to drop dramatically from 80 to 90% to near zero at 0900 h. However, the temperature in that study was not controlled and increased temperature as the day progressed could have caused the dramatic reduction of pollen viability.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Pollen germination percentage across a period of 10 h during the day from 0800 h to 1700 h. Pollen grains were collected from Clone 9 of ‘Crenshaw’ creeping bentgrass and germinated at room temperatures on the medium containing 1.0 M sucrose, 1.0 mM H3BO3, 2.0 mM CaCl2, and 0.3% (by weight) Phytogel. Bars indicate SE.

Pollen Longevity
Figure 4 shows pollen longevity of Clone 5 from Penncross and Clone 9 from Crenshaw creeping bentgrass. Immediately before storage, pollen germination rate of Penncross was close to 80%. After 1 h of storage, the pollen germination rate of Penncross remained high at 74%. After an additional 0.5 h of storage, however, it started to drop dramatically to less than one fourth of its initial germination rate. After 3 h of storage, the pollen germination rate dropped to zero. Unlike pollen of Clone 5 from Penncross, the initial germination rate of pollen grains from Clone 9 of Crenshaw was low (16.2%). The germination rate dropped to 2.5% after 40 min of storage and no pollen germination was observed after 2 h and 20 min of storage.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Longevity of pollen grains collected from Clone 5 of ‘Penncross’ and Clone 9 of ‘Crenshaw’ between 1000 and 1100 h. Pollen were stored in a sealed desiccator with a relative humidity of 64 to 66% at 21°C. Pollen were removed from the desiccator every 20 min and germinated on the medium containing 1.0 M sucrose, 1.0 mM H3BO3, 2.0 mM CaCl2, and 0.3% (by weight) Phytogel at room temperatures. Bars indicate SE.

The results reported here will allow us to compare pollen longevity between transgenic and conventional creeping bentgrass, an important aspect of risk assessment of transgenic crops involving pollen production. Although our results were obtained under a controlled environment, the temperature inside the greenhouse where creeping bentgrass plants were grown and both the temperature and humidity for pollen storage were set to mimic the weather conditions of the Willamette Valley in Oregon for the month of June and early July, when anthesis of most of the creeping bentgrass cultivars occurs. A large portion of the creeping bentgrass seeds produced in the USA occurs in the Willamette Valley. Seed formation of creeping bentgrass is primarily through wind-facilitated open pollination. Both interspecific and intergenic hybridizations between creeping bentgrass and its wild relatives have been reported (Davies, 1953; Wipff and Fricker, 2001; Belanger et al., 2003). The results obtained in this study may prove helpful in the development of future guidelines for the establishment of isolation distances for seed production fields to reduce the potential for gene flow to or from both conventional and transgenic creeping bentgrasses.

In conclusion, we have formulated an efficient pollen germination medium for estimation of creeping bentgrass pollen viability. With this optimized solid medium we were able to obtain as high as 90% germination rate on some creeping bentgrass genotypes (data not shown). Furthermore, information obtained in this study regarding the pattern of creeping bentgrass pollen shed and pollen longevity of creeping bentgrass should provide valuable information for creeping bentgrass breeding and risk assessment of transgenic creeping bentgrass.

	ACKNOWLEDGMENTS

 
The authors wish to thank Dr. Nick Christians of Iowa State University and Dr. Terry Stone of the Monsanto Companyfor critically reading the manuscript. Our thanks are also extended to Kristen Kubik, who helped to collect the data. The authors also wish to thank Dr. Bruce Branham and an anonymous reviewer for their constructive comments.

Received for publication July 31, 2002.

	REFERENCES

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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (7) Citing Articles via Google Scholar Google Scholar Articles by Fei, S. Articles by Nelson, E. Search for Related Content PubMed Articles by Fei, S. Articles by Nelson, E. Agricola Articles by Fei, S. Articles by Nelson, E. Related Collections Turfgrass Crop Cytology Crop Genetics