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0 Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6
Received for publication December 8, 1998. Accepted for publication January 14, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: blueberry • Ericaceae • fruit • germination • highbush • outcross • pollen • seed • selfing • Vaccinium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Snow, 1986
; Levin, 1990
; Spira et al., 1992
), but in some cases insufficient pollen is deposited (Bierzychudek, 1981
; Snow, 1982
). Pollen requirements for optimal fruit yield are not known, although few studies of cultivated crops have investigated pollen deposition by pollinators (Danka, Lang, and Gupton, 1993
; Carre et al., 1994
; Cane, Schiffhauer, and Kervin, 1996
). It is important to determine pollen requirements of cultivated plant species, since seed production and fruit yield are affected by the source and quantity of pollen (Ter-Avanesian, 1978
; Bertin, 1990
). In general, the number of pollen grains required for optimal fertilization exceeds the number of ovules since not every pollen grain is successful at fertilizing an ovule, but too much surplus pollen may reduce the success of individual pollen grains by pollen tube attrition and physical blockage. Pollination research with known numbers of pollen grains and with a known pollen source is needed to determine the precise amount of pollen and varietal sources required for maximal seed and fruit quality.
The amount and source of pollen required for adequate pollination of any highbush blueberry Vaccinium corymbosum L. (Ericaceae) cultivars are not known, even though it is an important crop in North America and depends on insect pollination, commonly through rented honey bee colonies. However, low yields remain common because of inadequate pollen transfer (Brewer, Dobson, and Nelson, 1969
; and reviewed in McGregor, 1976
; Free, 1993
) and because pollination from the same or closely related varieties sometimes results in low fruit set, sterile seeds, and small fruit (Hancock and Siefker, 1982
; Czesnik, Bounous, and Gioffre, 1989
; Free, 1993
).
Pollen source is thought to be an important consideration for obtaining maximum blueberry production. Pollen transferred between varieties can increase yields of blueberries compared to varietal selfing (Free, 1993
) by producing more seeds (Harrison, Luby, and Ascher, 1994
), heavier and larger berries (Lang and Danka, 1991
; Harrison, Luby, and Ascher, 1993
), greater fruit set (El-Agamy, Sherman, and Lyrene, 1981
), and earlier ripening larger berries (Lyrene, 1989
). Generally, pollen from more distantly related blueberry cultivars produces heavier berries (Gupton, 1984
) with increased seed number (Hellman and Moore, 1983
; Gupton and Spiers, 1994
) than does pollen from more closely related types, although this effect depends on pollen donor (Vander Kloet and Tosh, 1984
; Rabaey and Luby, 1988
). Thus the source of pollen is an important variable to consider in order to improve fruit mass and ripening time.
Similarly, sufficient stigmatic pollen deposition is considered important to maximize yield of other fruits. Generally, higher but not excessive numbers of pollen grains are beneficial for optimal seed and fruit production since pollen competition (Mulcahy and Mulcahy, 1987
), ovule and seed abortion (Stephenson, Winsor, and Davis, 1986
), pollen tube attrition (Smith-Huerta, 1997
), and physical blockage of pollen grains (Snow, 1986
) limit the number of successful fertilizations and seeds set. In some species, more seeds and fruit and more vigorous seedlings are produced when surplus pollen and pollen from mixed sources cause pollen tube competition to occur in the style (Snow, 1986
; Bertin, 1990
; Marshall, 1991
), but not in every study (Snow, 1990
). Some studies have found that pollen tubes grow at different rates (Walsh and Charlesworth, 1992
; Johnston, 1993
; Snow and Spira, 1993
) and compete to fertilize ovules, and this pollen competition results in ovule fertilization by the faster pollen tubes. In zucchini squash, this results in better quality seeds and fruit, since faster pollen tubes are more successful at fertilizing ovules (Davis, Stephenson, and Winsor, 1987
).
However, few studies of pollen quantity and source define pollen loads in precise terms (Ter-Avanesian, 1978
), but rather compare "large to small" loads instead of the exact number and source of pollen grains transferred. Also, research on pollen transfer either focuses on pollen competition and progeny fitness without examining seed abundance or fruit characteristics important for crop yields, or concentrates on yield parameters but ignores the exact pollen loads deposited and progeny fitness. We examined 90 studies in the botanical literature that investigated a variety of subjects related to pollen load and source and were unable to find any studies that precisely quantified the number and source of pollen grains transferred in experimental tests, assessed progeny fitness in relationship to pollen competition, and investigated yield parameters for fruits produced under varying treatments of load size and source (Dogterom, unpublished observations).
The objective of the current research was to determine the number and variety of pollen tetrads required by individual highbush blueberry flowers for maximum seed number, fruit set, fruit mass, and minimum days to ripen and to examine progeny fitness based on seed germination. This research was part of a broader study to determine the best managed bee pollinator of highbush blueberry, cultivar ‘Bluecrop’. Our specific objectives were to: (1) determine the maximum number of ‘Patriot’ cultivar pollen tetrads for cross-pollinating the cultivar ‘Bluecrop’; (2) examine the effect of outcrossing (‘Patriot’) and selfing with pollen from ‘Bluecrop’ and/or ‘Patriot’; and (3) determine the size and fertility of ‘Bluecrop’ seeds.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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50% of plantings in western North America are ‘Bluecrop’ (Moore, 1993
). ‘Patriot’ was chosen as the pollen donor since it is grown by commercial growers and has a different parentage than ‘Bluecrop’. Both ‘Bluecrop’ and ‘Patriot’ are northern tetraploid highbush cultivars (Levi and Rowland, 1997
), sharing different proportions of genetic contributions from five cultivars, with ‘Patriot’ containing the genetic contributions from two additional cultivars (Hancock and Siefker, 1982
).
Ovule number
The number of ovules and carpels of ‘Bluecrop’ flowers was determined by dissecting 90 ovaries (nine or ten from each of ten ‘Bluecrop’ plants). These were fixed for 24 h in FPA solution (40% formalin, concentrated propionic acid, 50% ethanol; 5:5:90 by volume) and stored in 70% ethanol. Ovaries were dissected in 70% ethanol with two fine forceps on a black-wax dissection dish under a 32x magnification, and number of ovules and carpels were counted.
Pollination with outcross pollen loads
One, 5-yr-old ‘Patriot’ and nine 3-yr-old ‘Bluecrop’ plants were obtained from Casino Tropical Plants Ltd., Surrey, British Columbia on 13 February 1996. They were potted in separate 25 cm diameter pots with a 50:50 peat-sawdust mixture, placed into a greenhouse with daytime temperatures set at 22°C, and under natural and artificial light (12:12 L:D), fertilized twice monthly with 15–30–15 (N, P, K) flowering plant fertilizer plus slow release fertilizer (16–10–10, 180-d release), and watered twice a day for 30 min. Bloom began after 2 wk.
In our study, pollen loads were chosen to incorporate both the maximum number of tetrads that could be loaded onto a blueberry stigma (300 pollen tetrads; Parrie and Lang, 1992
) and the number of microspores required for optimal seed production (generally between one and five pollen grains to one seed; Bertin, 1990
; Spira et al., 1992
). In blueberries there are four microspores per pollen tetrad (Stushnoff and Palser, 1969
) and in Epilobium canum (Onagraceae) the ratio of microspores to seeds produced is 4:1 (Snow, 1986
). If this ratio holds for Vaccinium spp., 100 pollen tetrads or 400 microspores are required for the average blueberry flower with 100 ovules, presuming that all ovules can be fertilized and matured.
Nearly mature closed ‘Bluecrop’ flowers were emasculated prior to anthesis by first removing the corolla and then the stamens with two fine forceps, leaving the style attached to the ovary. Emasculated flowers were marked individually with a colored thread and tag around the base of the flower and left to mature for 40–48 h. Treatments consisted of transferring 10, 25, 125 or 300 ‘Patriot’ pollen tetrads to 22 randomly assigned emasculated flowers as they opened on a given day. These numbers fall within the range of pollen loads delivered by bees visiting blueberry flowers in the field (M. Dogterom, unpublished data). The means ±1 SE tetrads actually deposited in the above treatments were: 9.72 ± 0.5, 25.0 ± 0.5, 126.5 ± 1.5, and 302.3 ± 2.0 tetrads. Open ‘Patriot’ flowers were rolled between thumb and forefinger, and the released pollen collected onto a coverslip with a black ink dot marked on the undersurface of the coverslip to improve visibility. The coverslip was glued to one corner of a microscope slide to ease handling of the coverslip. Pollen on each coverslip was removed with the head of an insect pin under a dissecting microscope, leaving pollen over the black dot. This pollen was counted and transferred by gently lowering the flower and touching the stigma onto the pollen tetrads, with the aid of a 3x head held magnifier. Pollen tetrads left on the slide were counted under a dissecting microscope, and the process was repeated until the required number of tetrads were loaded onto each stigma. This technique named the black-dot-on-slide technique is a new innovation. Pollen transfer always took <1 h. Each replicate consisting of all four treatments was located on the same or an adjacent branch of a plant. Fruit was picked when blueberries were blue in color, and weighed and stored at 4°C, until seeds were counted and sorted to type (large, small, and flat) by squashing individual berries onto filter paper in a petri dish.
Pollination with outcross, self, and mixed pollen loads
Four-year old ‘Bluecrop’ (17 plants from Gaskin Farms Ltd., Coquitlam, British Columbia) and 6-yr-old ‘Patriot’ plants (three from Casino Tropical Plants Ltd. Surrey, British Columbia) were placed into a greenhouse with diurnal temperatures set at 22°C (min-max 14.5°–34°C). In all 1997 experiments, ‘Bluecrop’ emasculated flowers were left to mature for 20–24 h (Parrie and Lang, 1992
) to shorten experimental protocol, rather than 40–48 h as in the 1996 experiments. Treatments were: (1) 25 ‘Bluecrop’ tetrads (SELF-25); (2) 25 ‘Patriot’ tetrads (CROSS-25); (3) 125 ‘Bluecrop’ tetrads (SELF-125); (4) 125 ‘Patriot’ tetrads (CROSS-125); and (5) 63 ‘Bluecrop’ tetrads plus 63 ‘Patriot’ tetrads (SELF-CROSS-125). The means ±1 SE of tetrads actually deposited were: 24.1 ± 0.3; 25.5 ± 0.4; 123.8 ± 0.5; 125.5 ± 0.4; 125.2 ± 0.8 tetrads.
Time interval between pollination events, using outcross pollen
This experiment compared one pollen load of 70 tetrads to two pollen loads of 35 tetrads, applied 3–4 h apart (range = 165–225 min, N = 14). The means ±1 SE of tetrads actually deposited in these treatments were 70.5 ± 0.6, and 69.6 ± 0.7 tetrads. Five maternal ‘Bluecrop’ plants were pollinated with pollen from one ‘Patriot’ plant on 3 or 4 April 1996.
One-day vs. 3-d outcross pollen loads
This experiment examined the effect of 125 pollen tetrads from 1-d- and 3-d-old ‘Patriot’ flowers (one plant) on ‘Bluecrop’ fruit and seed production (five plants). One-day-old flowers were flowers that had been open for a minimum of 24 h. Twenty-five replicates were completed between 14 and 20 April 1997. The means ±1 SE tetrads actually deposited in these treatments were 124.9 ± 0.5 and 124.7 ± 0.5 tetrads.
Characterization of seed types
Seed size was measured using a calibrated ocular micrometer, and seeds were sorted into three groups: large and dark brown (N = 107), small and golden brown (N = 68) and flat (N = 113). A separate random sample of seed types, large (N = 259), small (N = 302), and flat (N = 123) seeds were placed individually on a well-watered peat pellet adjacent to a color pin coded for treatment and incubated at 70% humidity, 12:12 light regime, daytime temperature of 24°–30°C and nighttime temperature of 3°–4°C. Each seed type was represented on each of ten trays, and location within trays was randomly assigned. Seeds were examined once weekly for germination for 3 mo.
Pollen viability on agar plates
Pollen viability was tested between 20 March and 14 April 1997 using nutrient agar medium in 100-mm petri dishes (Stushnoff and Feliciano, 1968
; Lang and Parrie, 1992
). Pollen from 0- to 5-d-old open flowers was released at a low density onto an agar plate by rolling the flower between the thumb and forefinger. The number of pollen tubes produced by 100 tetrads was counted after incubation at 20°C for 1 and 2 d.
Pollen tube growth in the style from 25 outcross tetrads
We examined pollen tube production from 25 pollen tetrads taken from one ‘Patriot’ plant and placed on stigmas of five ‘Bluecrop’ plants on 20–21 April 1997. Styles were removed from flowers 72 h after pollination when pollen tube growth was complete (Stushnoff and Palser, 1969
; El-Agamy, Sherman, and Lyrene, 1982
) and fixed in FPA solution. The KOH-aniline blue fluorescence technique (Martin, 1959
; Kearns and Inouye, 1993
; Hood and Shew, 1996
) was used to count the number of pollen tubes in each style. Styles were fixed in FPA solution for 24 h, stored in 70% ethanol, washed in running water for 30 min, softened in warm 8 mol/L NaOH for 3 h on a hot-plate, washed in tap water for 1 h, stained for 30 min in 0.1% aniline blue in 0.1 mol/L potassium acetate (pH 6.9), and washed in tap water for 1 h. Preparations were mounted on glycerol on glass slides, the preparation was covered and squashed by pressing down on the coverslip, and tubes observed through an epifluorescence microscope equipped with a filter set (maximum transmission 365 nm). The fluorochrome portion of the analine blue dye (Color Index number 42755) binds to plant glucans and polysaccharides that are present in pollen tubes but not present in stylar tissue.
Statistics
Except where otherwise stated, data were transformed to obtain normality and analyzed using the GLM procedure (SAS, 1985
) followed by Ryan's Q test for differences between means (Day and Quinn, 1989
). Differences between mean percentages were analyzed using Fisher's exact test, and differences between mean length and width of the three seed types were analyzed using a Kruskal–Wallis test with PROC NPAR1WAY (chi-square approximation) (SAS, 1985
). In vitro pollen tube germination data were analyzed using the paired-difference t test (PROC UNIVARIATE Procedure in SAS [1985]
). Bonferroni corrections were made to P values and are shown as corrected values. In all cases 
= 0.05 unless noted otherwise.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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6 carpels) and 17.7 ± 0.2 ovules per carpel.
Pollination with outcross pollen loads
The number of large seeds increased with pollen load of 300 tetrads. But unlike seed number, fruit mass increased significantly as pollen load increased from ten and 25 to 125 pollen tetrads (Fig. 1), but did not increase when tetrad number was increased to 300. Conversely, the number of days to ripen decreased with increased pollen load; it took longer to ripen fruit from the low pollen treatments of ten and 25 than for the high pollen treatments of 125 and 300. At pollen loads of ten or 25 tetrads, it took 7–13 d longer for fruit to ripen than at 125 or 300 tetrads, an important result for growers as earlier fruit and late fruit are more valuable than midseason fruit. Fruit set was not significantly different between treatments.
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2 = 250.18, df = 2, P = 0.0001) and width (2 = 241.11, df = 2, P = 0.0001). "Large" seeds were plump (length = 1.70 ± 0.02 mm; width = 1.05 ± 0.01 mm) and dark brown in color; "small" seeds were not quite as plump (length = 1.02 ± 0.01 mm; width = 0.68 ± 0.01 mm) and were a golden brown in color; "flat" seeds were flattened (length = 0.40 ± 0.01 mm; width = 0.28 ± 0.01 mm) and pale in color. Germination occurred in 220 out of 259 large seeds (85%), but none of the 302 small and 123 flat seeds germinated. Pollen load sizes of ten, 25, 125, and 300 tetrads did not affect mean days to seed germination (P = 0.278; Fig. 5). However, there is weak evidence that there were differences between treatments for percentage seed germination (P = 0.077), with treatment differences between ten and 125 (P = 0.034) and ten and 300 (P = 0.039) load sizes. There was a treatment response of pollen tetrad number on large seed length (P = 0.04) between treatments 25 and 300 (P = 0.028), but not for large seed width (P = 0.204).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our method of transferring a known number of pollen tetrads using the black-dot-on-slide technique provided more detailed information on floral pollen requirements for fruit set than is found in the literature. Other methods of transferring pollen have been used in pollination studies, but pollen deposition has been characterized only as "low," "high," or "excess." These methods could not determine specific pollen requirements for flowers since the exact number of pollen grains deposited were not known (Bertin, 1990
; Harrison, Luby, and Ascher, 1993
).
Seed production response to pollen source and quantity
Reproductive fitness can be measured by the number of fertile seeds and their quality. The quality and number of seeds in turn affect fruit quality and ultimately determine the dispersal of offspring. The largest number of seeds were produced from 300 outcrossed pollen tetrads, which also is the maximum load possible on highbush (Parrie and Lang, 1992
). In 1996, 48 large seeds per ‘Bluecrop’ fruit were produced per flower, which agrees well with a mean seed number for ‘Bluecrop’ reported in other studies in which excess pollen was used for pollination (47 seeds/fruit in Eaton [1967]
; 40.5 in MacKenzie [1997]
; and 46 in Moore, Reynolds, and Brown [1972]
, but 26.7 in Krebs and Hancock [1988]
, and 63 in Darrow [1958]
). Higher seed set may be possible with tetrads from cultivars with larger stigmatic surface areas, since pollen adsorption to the stigma is limited by at least the surface area. Also, a nonrelated outcross pollen may improve successful fertilization and increase seed number closer to ovule number of 100, although there are no reports in the literature of highbush blueberry seed number greater than a mean of 63 seeds (Darrow, 1958
). Thus, only about half of the ovules produce seed. Pollen tube attrition, physical blockage, ovule abortion, seed abortion, or some other factors may reduce seed production to less than ovule capacity.
The mean number of large seeds produced per fruit from the 125 cross pollen treatment was half the mean in 1997 of a similar treatment in 1996, possibly due to a change in methodology between years. Stigmata left for 24 h after emasculation in 1997, rather than 48 h in 1996, may have been less mature and had insufficient stigma fluid for adequate adsorption of all tetrads (Moore, 1964
; Young and Sherman, 1978
). Maternal environmental effects (Stephenson, 1981
; Roach and Wulff, 1987
) and physiological condition of pollen-producing plants (Stephenson et al., 1992
) also can influence the performance and fate of pollen grains.
Microspore availability for fertilization
In this study, stigmatic loading with 125 tetrads or 500 microspores is 5x the number of ovules in highbush blueberry flowers. Since the ratio of microspores to seed, for seed production is 1–5:1, respectively (Snow, 1986
; Bertin, 1990
; Spira et al., 1992
) the number of tetrads would appear more than adequate to fertilize 100 ovules. However, the dramatic increase of seed production with 300 tetrads loaded onto receptive stigma may indicate that some microspores are not available for fertilization because these are not viable. In vivo pollen viability was examined in this study, and we found three to four pollen tubes grew from each tetrad down the style, since the majority of styles produced more than 85 pollen tubes from 25 tetrads. Thus 125 tetrads or 500 microspores would be adequate for 100 ovules. It is possible that the larger number of microspores available when 300 tetrads (1200 microspores) are deposited stimulates fertilization and seed set more than if 125 tetrads or 500 microspores are deposited onto the stigma. The higher ratio of microspores to seeds required for maximum highbush blueberry seed production is not known, although the viability may be lower than in plants where the ratio of microspores to seeds is reported as 1–5:1.
In vitro pollen tube growth, which potentially could be used to identify microspore viability, did not confirm in vivo results since only one to two pollen tubes per tetrad were produced in our in vitro experiment, in agreement with one study (Brewer and Dobson, 1969
) but fewer pollen tubes than in other studies (Goldy and Lyrene, 1983
; Lang and Parrie, 1992
). Variation in pollen tube production could be due to differences between varieties (Goldy and Lyrene, 1983
; Lang and Parrie, 1992
), or the subtle chemical nuances of the artificial medium (Mazer, 1987
) at certain temperatures (Stern and Gazit, 1998
). In vitro pollen tube production may be used as a rough indicator of pollen viability, although it may be an inaccurate predictor of in vivo pollen tube growth down the style (Mazer, 1987
).
Seed quality response to pollen source
The significant effect of a larger pollen load on improved percentage germination of large seeds between treatments 10 and 125 and treatments 10 and 300 (Fig. 1) agrees with the relationship found between increased number of pollen grains deposited onto a stigma and the improved performance of resulting progeny in some plants (Mulcahy and Mulcahy, 1987
; Palmer and Zimmerman, 1994
; Johannsson and Stephenson, 1997
). It is indicated in the literature that pollen tubes that grow at different rates compete to fertilize ovules and the faster tubes fertilize the ovules (Johnston, 1993
; Snow and Spira, 1993
; Davis, Stephenson, and Winsor, 1987
). However, days to germination in our study were not affected by pollen load size and thus does not appear to be related to fitness. Increased sample size might improve the strength of this relationship, but these data do suggest that pollen transfer is related to at least one fitness characteristic in highbush blueberry, percentage germination.
Fruit quality response to pollen source
The pollen load of 125 ‘Patriot’ tetrads gave the greatest fruit mass and the least days to ripen of highbush blueberry cultivar ‘Bluecrop’. Additional tetrads up to 300 did not increase fruit mass or decrease ripening. Decreased ripening time is an important result for grower, as early fruit is more valuable than late fruit. Loads of ten or 25 tetrads were not sufficient to maximize fruit characteristics, but we did not determine the precise point between 25 and 125 tetrads where fruit equality would reach its maximum level. Conceivably, this threshold could occur anywhere in this range, although the optimal level likely is closer to 125 than 25 because 1–5 microspores are needed to fertilize each ovule that is present in the ovary (Bertin, 1990
; Spira et al., 1992
).
‘Bluecrop’ selfing with low and high pollen loads resulted in 40 and 64% fruit set respectively, similar to results in another study (52 and 56% in Knight and Scott, 1964
). Fruit set improved from 40% for selfing with 25 tetrads to 76 and 80% with outcross tetrads of 125 mixed self/outcross and 125 outcross pollen loads respectively, as in other outcrossing studies (Knight and Scott, 1964
; Krebs and Hancock, 1988
). Similarly, fruit mass was increased from 1.05 g for selfing with 25 tetrads to 1.6 and 1.7 g for 125 mixed and outcross tetrads, respectively. Thus, 25 tetrad selfing sets fewer fruit that weigh less than fruits resulting from 125 self, mixed, or outcross loads.
Stigmatic loading techniques between years
The mean number of large seeds produced per fruit from the 125 cross pollen treatment was half the mean in 1997 of a similar treatment in 1996, possibly due to a change in methodology between years. Stigmas left for 24 h after emasculation in 1997, rather than 48 h in 1996, may have been less mature and had insufficient stigma fluid for adequate adsorption of all tetrads (Moore, 1964
; Young and Sherman, 1978
). Maternal environmental effects (Stephenson, 1981
; Roach and Wulff, 1987
) and perhaps physiological condition of pollen-producing plants (Stephenson et al., 1992
) also can influence the performance and fate of pollen grains.
Seed characterization
Characterization of seed types by length and width is important as only large seeds are fertile and indicate pollination success. Although large (fertile), medium (not fertile), and collapsed (not fertile) seeds have been described histologically (Bell, 1957
), seeds are described only qualitatively in most studies (Harrison, Luby, and Ascher, 1994
; MacKenzie, 1997
). Precise characterization of "large fertile seeds" with length and/or width measurements should be used to standardize the definition of seed production in blueberry pollination studies.
Response to sequential loading of pollen tetrads
Sequential pollen loading with pollen from the same source did not affect fruit quality in my study, although in Hibiscus moscheutos L. the timing of sequential pollen loads determined which pollen grains fertilized the ovules (Spira, Snow, and Puterbaugh, 1996
). Outcross pollen in a mixed load may stimulate the less compatible selfing tetrads (Mulcahy and Mulcahy, 1986
; Marshall et al., 1996
). If outcross pollen is beneficial to fruit production and size, it would be important to have pollinator varieties within average flying distance of bee pollinators during a given foraging trip. In my study, there was no significant difference between fruit quality from self, mixed, and outcross loads of 125 tetrads but outcross pollen did produce significantly more fertilized seeds than did self pollen (Fig. 1). Bees enhance fruit production by transferring more pollen, whether self or outcross, thus increasing fruit quality. Knowledge of varietal compatibility is important since blueberry seed set can be reduced when self pollen is present together with outcross pollen (Harrison, Luby, and Ascher, 1994
).
Relationship between pollen load and pollinator availability
Our results indicate that 125 tetrads of 50–100% outcross pollen 
3 d old are sufficient for optimal fruit quality. Thus, pollinator density would be optimal if blueberry flowers received 125 tetrads per flower during bloom. The length of time that stigmata are receptive to pollen grains (7 d; Moore, 1964
) and pollen grains remain viable (3 d; this study) prolongs the time that pollinators can visit flowers. Prolonged pollen viability and stigma receptivity would be important if pollinators are scarce, since it would improve the possibility of increasing plant fitness by producing fertile seeds.
The large number of microspores required for maximal seed production is an indication that sexual selection takes place between flower bloom and seed development. It is not known whether sexual selection occurs during pollination, after pollination and before fertilization, during fertilization, or after fertilization. If these large number of microspores are required because of pollen and ovule incompatibilities, more compatible cultivars would reduce the need for these excessive numbers of microspores.
Two examples from related research we have conducted demonstrate the impact these types of precise pollen transfer studies can have on pollination-related crop production analyses (unpublished data). First, we determined that considerably fewer than 125 pollen tetrads were found on stigmas of cultivated highbush blueberries in the field after one pollinator visit, suggesting that more than one pollinator visit is required for maximum levels of fruit set and size and that growers need to provide adequate pollinators to reach this level of stigma loading. Second, we were able to relate single pollen transfer data to bee species and found that an individual bumble bee is equivalent to four honey bees on a per visit basis. We were then able to extend this analysis to determine the relative economic costs and benefits of renting bumble versus honey bees for blueberry pollination and found honey bees to be far superior to bumble bees economically, in spite of the better per-bee efficacy of bumble bees because there are so many more honey bees than bumble bees in a colony. However, under certain conditions such as the presence of alternate forage, the numerical advantage of honey bees is diminished by attracting bees away from the crop. Nevertheless, these examples indicate the horticultural significance of precise pollen load and cultivar studies and suggest that essential knowledge of pollen requirements for crop production can be a powerful tool to enhance fruit and vegetable set, size, time to ripen, and perhaps other economically important crop characteristics.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bertin, R. I. 1990 Effects of pollination intensity in Campsis radicans. American Journal of Botany 77: 178–187.[CrossRef][ISI]
Bierzychudek, P. 1981 Pollinator limitation of plant reproductive effort. American Naturalist 117: 838–840.[CrossRef][ISI]
Brewer, J. W., and R. C. Dobson. 1969 Pollen analysis of two highbush blueberry varieties Vaccinium corymbosum. Journal of American Society for Horticultural Science 94: 251–252.
———, ———, and J. W. Nelson. 1969 Effects of increased pollinator levels on production of the highbush blueberry Vaccinium corymbosum. Journal of Economic Entomology 62: 815–818.
Cane, J. H., D. Schiffhauer, and L. J. Kervin. 1996 Pollination, foraging, and nesting ecology of the leafcutting bee Megachile (Delomegachile) addenda (Hymenoptera: Megachilidae) on cranberry beds. Annals of the Entomological Society of America 89: 361–367.
Carre, S., I. Badenhausser, J. N. Tasei, J. Le Guen, and J. Mesquida. 1994 Pollen deposition by Bombus terrestris L. between male-fertile and male-sterile plants in Vicia faba L. Apidologie 25: 338–349.[CrossRef][ISI]
Czesnik, E., G. Bounous, and D. Gioffre. 1989 A survey of self-incompatibility in highbush blueberry Vaccinium corymbosum L. Acta Horticulturae 241: 56–63.
Danka, R. G., G. A. Lang, and C. L. Gupton. 1993 Honey bee (Hymenoptera: Apidae) visits and pollen source effects on fruiting of ‘gulfcoast’ southern highbush blueberry. Journal of Economic Entomology 86: 131–136.[ISI]
Darrow, G. M. 1958 Seed number in blueberry fruits. American Society for Horticultural Science 72: 212–215.
Davis, L. E., A. G. Stephenson, and J. A. Winsor. 1987 Pollen competition improves performance and reproductive output of the common zucchini squash under field conditions. Journal of American Society for Horticultural Science 112: 712–716.
Day, R W., and G. P. Quinn. 1989 Comparisons of treatments after an analyses of variance in ecology. Ecological Monographs 59: 433–463.[CrossRef][ISI]
Eaton, G. W. 1967 The relationship between seed number and berry weight in open-pollinated highbush blueberries. HortScience 2: 14–15.
El-Agamy, S. Z. A., W. B. Sherman, and P. M. Lyrene. 1981 Fruit set and seed number from self- and cross-pollinated highbush (4x) and rabbiteye (6x) blueberries. Journal of American Society for Horticultural Science 106: 443–445.
———, ———, and ———. 1982 Pollen incompatibility in blueberries (Vaccinium spp.). Journal of Palynology 18: 103–112.
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