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(American Journal of Botany. 2002;89:248-252.)
© 2002 Botanical Society of America, Inc.

Reproductive Biology

Collection and storage of pollen from Salix (Salicaceae)1

Richard F. Kopp2,5, Charles A. Maynard3, Patricia Rocha de Niella4, Lawrence B. Smart2 and Lawrence P. Abrahamson2,3

2State University of New York College of Environmental Science and Forestry, Faculty of Environmental and Forest Biology, Syracuse, New York 13210 USA; 3State University of New York College of Environmental Science and Forestry, Faculty of Forestry, Syracuse, New York 13210 USA; 4Misiones National University, School of Forestry Science, Eldorado-Misiones, Argentina

Received for publication May 1, 2001. Accepted for publication August 9, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Genetic improvement of willows through traditional breeding can be facilitated by pollen collection and storage so that female flower receptivity need not be synchronized with pollen shed for breeding. Two experiments were completed to test the effectiveness of various organic solvents for willow pollen collection. In the first experiment, seven pollen collection treatments and an untreated control were tested with two willow clones. The other experiment tested three treatments that showed promise in the initial experiment and an untreated control with eight willow clones. Toluene and carbon tetrachloride were effective for pollen extraction, with average pollen germination percentages that were >15%, but both chemicals reduced pollen viability by 10–20% compared with an untreated control based on in vitro germination tests. Pollen extracted with carbon tetrachloride or toluene was successfully used in controlled pollination, and >100 new families were produced with this technique. Pollen viability remained high after 18 mo of storage at –20°C. Based on our results, toluene is the preferred solvent for future willow pollen extractions because it is as effective as carbon tetrachloride, is not a known carcinogen, and is less expensive.

Key Words: breeding • germination • pollination • Salicaceae • Salix • toluene • willow


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Salix tree improvement projects are in progress around the world because willows are used as a feedstock for bioenergy and bio-based products, stream bank stabilization, nutrient filters, and phytoremediation (Christersson, 1986 ; Perttu and Kowalik, 1997 ). Willow breeders may collect plant material from various Salix species in distant geographic regions and plant it in a single location to use in breeding programs. Differing flowering phenologies have been observed among willow species, which may have evolved to restrict natural hybridization (Mosseler and Papadopol, 1989 ), and must be considered for controlled breeding. Flowering of clones used for controlled breeding can be synchronized by bringing flower-bearing shoots into a controlled environment at appropriate times but, if pollen could be collected and stored, synchronizing would be unnecessary and breeding options would be increased.

Pollen collection and storage is practiced in European willow breeding programs (Åhman and Larsson, 1994 ; Lindegaard and Barker, 1997 ), but protocols used for willow pollen extraction and storage were not described. Detailed pollen management protocols have been developed for Populus (Stanton and Villar, 1996 ), but the techniques are not appropriate for Salix. Populus spp. are wind pollinated (Braatne, Rood, and Heilman, 1996 ), and the pollen is easier to handle than willow pollen. Salix spp. are generally insect pollinated (Newsholme, 1992 ) and have adhesive substances on their pollen. These substances need to be removed from willow pollen upon collection to facilitate its use in controlled pollination. Development of a protocol for extraction and long-term storage of willow pollen would benefit willow breeders.

The objective of this study was to develop a protocol for the extraction of willow pollen that was effective for controlled pollinations and yielded pollen that was easily handled and remained viable through long-term storage. Previous research showed that carbon tetrachloride was effective for willow pollen collection (P. Rocha de Niella, State University of New York College of Environmental Science and Forestry, unpublished data), but we sought an alternative that was less expensive and had a lower degree of toxicity. Three experiments were completed: the first to identify several solvents that appeared promising with a small number of willow clones, a second to confirm the effectiveness of selected solvents with a larger number of clones, and the third to determine the duration that solvent-extracted pollen could be stored and still retain its viability. The hypothesis was that a nonpolar solvent could be identified, which was effective for extraction of willow pollen, providing pollen that was easily handled and retained viability through storage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Two separate experiments were completed to identify organic solvents that can be used for willow pollen extraction. In experiment 1, seven solvents, acetonitrile, carbon tetrachloride, chloroform, dichloromethane, ethyl acetate, ethyl ether, and toluene (Table 1), were used to extract pollen from Salix eriocephala (clone S646) and S. miyabeana (clone Sx64). In experiment 2, three solvents identified as having potential in experiment 1 (carbon tetrachloride, ethyl acetate, and toluene) were used to extract pollen from six clones of S. eriocephala (95022, S19, S185, S287, S546, and S646) and two S. purpurea clones (94002 and Pur34).


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  		Table 1. Solvents used to extract pollen from Salix and their health effects on laboratory rats.1

 
In experiment 1, shoots bearing flower buds were collected during early winter and the bases were placed in tap water in a greenhouse where temperature was maintained between 15° and 27°C. Natural light was supplemented with high intensity sodium vapor lamps such that shoots were exposed to 16 h of light per day. Flowers were harvested when flower buds broke and most anthers were shedding pollen. For each solvent treatment, ten flowers were removed from shoots with forceps and placed in a 100-mL glass beaker. Solvent was poured over the flowers until they were submerged (~25 mL); then beakers were gently agitated for 60 sec to remove pollen from the anthers. Solvents were used in a fume hood and caution was used to avoid solvent inhalation and dermal contact. Pollen was separated from the solvent by vacuum filtration through Whatman #42 filter paper (Whatman International, Maidstone, UK).

Pollen was dried on the filter paper for ~2 min in a fume hood and then brushed from the filter paper into sterile 1.5-mL microfuge tubes using a sterile camel's hair brush. Samples were stored at –20°C for 2–5 d until germination tests were completed. As a control, untreated flowers were harvested and stored in beakers sealed with laboratory film along with the other samples at –20°C. The experiment was replicated three times by completing extractions on three separate days. The protocol for experiment 2 was identical to that for experiment 1, except that shoots bearing flower buds were collected during mid-winter.

Pollen germination tests were completed using medium consisting of 10% (m/v) sucrose and 0.7% (m/v) agar, pH 5.0, which was sterilized by heating to 121°C at 0.1 MPa for 15 min and cooled at room temperature. For each sample, ~1 cm3 of solidified medium was scooped with a sterile spatula and placed on a microscope slide. Pollen was applied to the medium by dipping a sterile, disposable, plastic pipette tip into the microfuge tube containing pollen and then gently rolling it across the agar block. For the control treatment, pollen was applied by removing pollen-bearing anthers from flowers using forceps and gently touching the anthers to the medium. Slides were placed on moist paper towels in sealed plastic containers at room temperature. After 4 h of incubation, the number of pollen grains that germinated from a randomly selected sample of at least 100 pollen grains was counted for each treatment and replication. A pollen grain was considered to have germinated if, when viewed at 400x magnification, a pollen tube was visible and had a length that was equal to or greater than the diameter of the pollen grain (Rajora and Zsuffa, 1986 ).

The experimental design for both experiments was a randomized complete block with the three extraction and germination test days serving as blocks. In both experiments, a block consisted of pollen samples from every clone that were collected with each solvent during a day. Pollen samples from each clone were collected three times over a period of 1 wk. Germination tests for all samples in a block were completed on the same day with one batch of germination medium. Batches of germination media were prepared immediately prior to use.

Data were analyzed by analyses of variance ({alpha} = 0.05) with the generalized linear modeling procedure in the Statistical Analysis System (SAS, 1997 ) using the following model:

(1)
where yjdq is the observed value for an individual pollen sample; µ is the overall mean; Bj is the effect of block j, j = 1–3; Sd is the effect of solvent, d = 1–8 in experiment 1 and d = 1–4 in experiment 2; Cq is the effect of clone, q = 1–2 in experiment 1 and q = 1–8 in experiment 2; (S x C)dq is the interaction effect of solvent d and clone q; and ejdq is random error. The Bonferroni mean separation test was used to test for differences among treatment means ({alpha} = 0.05) (Kirk, 1982 ). Analyses were completed with and without inverse sine transformation.

The viability of pollen extracted from S. eriocephala and S. lucida clones with carbon tetrachloride was tested after 6, 12, or 18 mo of storage in microfuge tubes at –20°C. Stored pollen was removed from the freezer, and the percent germination in vitro was determined. Germination percentages of stored pollen were compared with freshly collected pollen, which was tested at time zero. The germination medium in this study was made fresh at each test date and consisted of 15% (m/v) sucrose and 0.7% (m/v) agar, plus 150 mg/L boric acid, pH 5.0. Pollen that had been extracted using carbon tetrachloride and stored for 12 mo was also used for controlled pollinations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Pollen germination percentages in experiment 1 were significantly different among solvents (Table 2). The average pollen germination rate in the untreated control was 25.2% (Fig. 1). Germination percentages of pollen extracted with toluene and carbon tetrachloride were lower than the untreated control (16.0 and 14.7%, respectively), but differences between these two treatments and the control were not significant (Fig. 1). Pollen extracted with acetonitrile, ethyl ether, chloroform, or dichloroethane had germination percentages <5%. Pollen extracted from S. miyabeana clone Sx64 and S. eriocephala clone S646 had similar germination percentages, and no clone x solvent treatment interaction was detected.


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  		Table 2. Analysis of variance for in vitro pollen germination percentage after extraction with various organic solvents

 


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  		Fig. 1. Germination percentages of pollen from S. eriocephala (clone S646) and S. miyabeana (clone Sx64) extracted with solvents and untreated controls in experiment 1. Germination percentages of the two clones were averaged for each solvent treatment. Letters indicate groupings based on Bonferroni t tests ({alpha} = 0.05)

 
Pollen germination percentages in experiment 2 significantly differed by solvents and clones (Table 2). The average germination percentage in the untreated control was 39.3% (Fig. 2). The average germination percentages of pollen extracted with toluene and carbon tetrachloride were similar (21.2 and 17.3%, respectively), while the average germination percentage of pollen extracted with ethyl acetate was lower (7.8%) (Fig. 2). There was a wide range in pollen germination percentages among different clones with a high of 42.8% (clone 94002) down to 3.2% (clone S646) (Fig. 3). No clone x solvent treatment interaction was detected. Inverse sine transformation of the germination percentage data had little effect on probability values in experiments 1 and 2.



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  		Fig. 2. Germination percentages of pollen from S. purpurea (clones 94002 and Pur34) or S. eriocephala (clones 95022, S287, S19, S546, S185, and S646) extracted with solvents and untreated controls for experiment 2. Germination percentages from eight clones were averaged for each solvent treatment. Letters indicate groupings based on Bonferroni t tests ({alpha} = 0.05)

 


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  		Fig. 3. Germination percentages of pollen from S. purpurea (clones 94002 and Pur34) or S. eriocephala (clones 95022, S287, S19, S546, S185, and S646) extracted with solvents in experiment 2. Pollen germination percentages of pollen extracted with three different solvents (toluene, carbon tetrachloride, and ethyl acetate) and the untreated control were averaged for each clone. Vertical bars indicate standard errors

 
Viability of S. eriocephala and S. lucida pollen that was extracted with carbon tetrachloride was reduced after 18 mo of storage at –20°C relative to samples that were not stored, decreasing from 48% germination to 38%. Most of the decline in viability occurred during the first 6 mo of storage (data not shown). Controlled pollination with pollen that was stored for 12 mo resulted in seed set, which was comparable to that obtained using fresh pollen. Intra- and interspecific pollinations attempted using pollen extracted with either carbon tetrachloride or toluene resulted in successful fertilization and production of viable willow seedlings from >100 controlled crosses during the years 1998 through 2000, with success rates exceeding 75%. No controlled pollinations were attempted using pollen extracted with ethyl acetate, acetonitrile, ethyl ether, chloroform, or dichloromethane.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Two of the solvents tested in this study, toluene and carbon tetrachloride, were effective for extraction of pollen from willow based on in vitro pollen germination assays and controlled pollinations. Pollen extracted using carbon tetrachloride or toluene had a nonadhesive, powdery consistency and was easy to use in controlled pollination. Both solvents reduced pollen germination percentages by nearly 10% in experiment 1 and 20% in experiment 2, compared with pollen that was stored without contacting organic solvent. Large variation in pollen germination percentage was observed among clones in this study, but the basis for these differences was not investigated. Pollen germination percentages in this study were lower than those reported for pollen that was never stored or in contact with solvents, for which germination percentages were consistently 70% or higher (Mosseler, 1989 ).

The pollen germination medium and growth conditions used in this experiment were not optimized, so it is likely that the percentage of viable pollen is higher than the germination percentage observed in this study. Our pollen germination assay does allow relative comparisons of treatments and clones. A pollen germination medium containing sucrose, boron, calcium, magnesium, and potassium was effective for germination of pollen from many plant species, and calcium was essential (Brewbaker and Kwack, 1963 ). A pollen germination medium containing 0.6% (m/v) agar, 5% (m/v) sucrose, and 0.001% (m/v) H3BO4 was used successfully with fresh willow pollen (Mosseler, 1989 ). Preliminary tests using media without calcium or boron and with three sucrose concentrations indicated that sucrose concentration was important for germination (data not shown). The pH of the medium used in the experiments described here (5.0) was determined to be effective for willows (data not shown) and may have been close to optimal. Germination of pollen from 13 forest species was significantly affected by medium pH in the range of 2.6–5.6, and pollen tube elongation of Populus tremuloides was inhibited when medium pH was 4.0 or less (Cox, 1983 ). Germination percentages of pollen from four hardwood species was higher at pH 5.0 than at lower pH, but pH above 5.0 was not tested (VanRyn, Jacobson, and Lassoie, 1986 ). A second factor that could have influenced pollen germination percentages was the amount of pollen applied to the medium. A strong effect of pollen grain population density on in vitro pollen germination was observed for many plant species, with poor germination observed when densities were low (Brewbaker and Kwack, 1963 ). Pollen grain density was not controlled in this experiment.

The effectiveness of toluene for pollen extraction from willow species other than those tested should be confirmed, but it was used successfully with all the clones tested in this study, representing four species of Salix. Clone x solvent interaction was not observed in either experiment. Pollen extraction and controlled pollinations completed during 1998–2000 confirmed that toluene was effective for pollen extraction with >50 willow clones, including clones of S. alba and S. viminalis (data not shown).

A possible effect of extracting willow pollen with an organic solvent is that it may widen the range of possible interspecific hybridizations. Treating pollen with organic solvents prevented incompatibility reactions among hybrid poplar species, permitting successful matings among species that were not previously possible (Willing and Pryor, 1976 ). Treating pollen with organic solvents was suggested as a way to eliminate crossing barriers among willows (Stott, 1984 ). Proteins on the surface of pollen grains that are involved in the pollen–stigma interaction have been identified in Brassica oleracea (Stephenson et al., 1997 ), and lipids involved in restricting interspecific and self-pollination were identified in Arabidopsis thaliana (Hulskamp et al., 1995 ). Presumably, willows have similar compounds on their pollen that perform the same function. If the solvent used to extract willow pollen alters or removes these recognition compounds, crosses between species that normally do not hybridize may be possible. Interspecific pollen–pistil relationships have been studied in willows, but pollen recognition compounds were not investigated (Mosseler, 1989 ).

The technique described in this study provides ease of handling stored pollen and eliminates the need to synchronize male and female flowering times, which allows controlled pollinations to be completed much more easily than by using fresh male flowers. Pollen extraction with toluene and subsequent storage may reduce viability when compared with pollen that did not contact organic solvent. However, crosses using pollen samples with only 10% viable pollen yielded large numbers of seeds (data not shown). Toluene was selected as the solvent to use for future willow pollen extractions, because it is equally effective and less expensive than carbon tetrachloride. Also, it is not known to be carcinogenic as is carbon tetrachloride.


   FOOTNOTES
 
1 The authors thank the agencies that financially supported this research, including the New York State Energy Research and Development Authority; the United States Department of Energy Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725; and the United States Department of Agriculture. Back

5 Author for reprint requests (rfkopp{at}syr.edu ). Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Åhman I. S. Larsson 1994 Genetic improvement of willow (Salix) as a source of bioenergy. Norwegian Journal of Agricultural Science Supplement 18: 47-56

Braatne J. H. S. B. Rood P. E. Heilman 1996 Life history, ecology, and conservation of riparian cottonwoods in North America. In R. F. Stettler, H. D. Bradshaw, Jr., P. E. Heilman, and T. M. Hinckley [eds.], Biology of Populus and its implications for management and conservation, part I, 57–85. NRC Research Press, National Research Council of Canada, Ottawa, Ontario, Canada

Brewbaker J. L. B. H. Kwack 1963 The essential role of calcium ion in pollen germination and pollen tube growth. American Journal of Botany 50: 859-865[CrossRef][ISI]

Christersson L. 1986 High technology biomass production by Salix clones on a sandy soil in southern Sweden. Tree Physiology 2: 261-272[Medline]

Cox R. M. 1983 Sensitivity of forest plant reproduction to long range transported air pollutants: in vitro sensitivity of pollen to simulated acid rain. New Phytologist 95: 269-276[CrossRef][ISI]

Hulskamp M. S. D. Kopczak T. F. Horejsi B. K. Kihl R. E. Pruitt 1995 Identification of genes required for pollen-stigma recognition in Arabidopsis thaliana. Plant Journal for Cell and Molecular Biology 8: 703-714

Kirk R. E. 1982 Experimental design, 2nd ed. Brooks/Cole Publishing, Monterey, California, USA

Lindegaard K. N. J. H. A. Barker 1997 Breeding willows for biomass. Aspects of Applied Biology 49: 155-162

Mosseler A. 1989 Interspecific pollen-pistil incongruity in Salix. Canadian Journal of Forest Research 19: 1161-1168

Mosseler A. C. S. Papadopol 1989 Seasonal isolation as a reproductive barrier among sympatric Salix species. Canadian Journal of Botany 67: 2563-2570

Newsholme C. 1992 Willows: the genus Salix. Timber Press, Portland, Oregon, USA

Perttu K. L. P. J. Kowalik 1997 Salix vegetation filters for purification of waters and soils. Biomass and Bioenergy 12: 9-19[CrossRef]

Rajora O. P. L. Zsuffa 1986 Pollen viability of some Populus species as indicated by in vitro pollen germination and tetrazolium chloride staining. Canadian Journal of Botany 64: 1086-1088

SAS. 1997 SAS user's guide: basics, version 7. SAS Institute, Cary, North Carolina, USA

Stanton B. J. M. Villar 1996 Controlled reproduction in Populus. In R. F. Stettler, H. D. Bradshaw, Jr., P. E. Heilman, and T. M. Hinckley [eds.], Biology of Populus and its implications for management and conservation, part I, 113–138. NRC Research Press, National Research Council of Canada, Ottawa, Ontario, Canada

Stephenson A. G. J. Doughty S. Dixon C. Elleman S. Hiscok H. G. Dickinson 1997 The male determinant of self-incompatibility in Brassica oleracea is located in the pollen coating. Plant Journal for Cell and Molecular Biology 12: 1351-1359

Stott K. G. 1984 Improving the biomass potential of willow by selection and breeding. In K. Perttu [ed.], Ecology and management of forest biomass production systems, vol. 15, 233–260. Swedish University of Agricultural Science, Uppsala, Sweden

VanRyn D. M. J. S. Jacobson J. P. Lassoie 1986 Effects of acidity on in vitro pollen germination and tube elongation in four hardwood species. Canadian Journal of Forest Research 16: 397-400[CrossRef]

Willing R. R. L. D. Pryor 1976 Interspecific hybridization in poplar. Theoretical and Applied Genetics 47: 141-151[CrossRef][ISI]





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Right arrow Articles by Kopp, R. F.
Right arrow Articles by Abrahamson, L. P.
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