Summary The effects of supplemental mass pollination (SMP) were studied in two Pinus sylvestris seed orchards differing in pollen production. Pollen was dusted over the whole tree during the period of peak female receptivity using a pressurized backpack sprayer. The success of SMP was assessed by means of allozyme markers. In the orchard with high pollen production, detectable differences in SMP success rate were found between clones, but the success rate was not influenced by the number of pollinations per day. The average estimated success rate of SMP was 19%. In the orchard with low pollen production, no significant differences in SMP suc-cess rate were found between years (22 versus 34%) or between clones. The SMP success rate in the low pollen production orchard varied between 14 and 69%.
Keywords: isozymes, pollen production, Scots pine, seed or-chard management.
Introduction
Based on height growth, the average genetic superiority of first-generation plus-tree seed orchards of Pinus sylvestris L. in Sweden is estimated to be between 6 and 8% compared with unselected stand material (Danell 1991, 1993). However, the genetic potential of seed orchards has not been entirely ex-ploited mainly because it has not yet been possible to eliminate pollen contamination from unselected sources outside the or-chards (e.g., Savolainen 1991, Di-Giovanni and Kevan 1991). Estimates of pollen contamination in P. sylvestris seed or-chards range between 17 and 74% (El-Kassaby et al. 1989, Harju and Muona 1989, Yazdani and Lindgren 1991, Wang et al. 1991, Paule 1991). Pollen contamination affects the growth, quality and hardiness of the seedling output. Serious pollen contamination in southern orchards producing seed intended for northern regions with a harsh climate makes the seed crops unsuitable for the intended forest regeneration area (Lestander and Lindgren 1985, Andersson and Westin 1990, Savolainen 1991). Beside pollen contamination, differences in random mating and gamete production capacity within and among clones may affect the genetic quality of the output from seed
orchards (e.g., Eriksson et al. 1973, Jonsson et al. 1976, Bhu-mibhamon 1978, Chung 1981). Thus, there is scope to develop methods that facilitate a higher recovery of the potential ge-netic gain of seed orchards.
In seed orchards of Pinus taeda L. and Pseudotsuga menzi-esii (Mirb.) Franco, having limited pollen supply, supplemen-tary mass pollination (SMP) (Bridgwater and Trew 1981), defined as the broadcast application of pollen to unisolated female strobili, has been used to increase the yield of sound seeds (see review by Bridgwater et al. 1993). The success rate of SMP has been estimated by using pollen with unique bio-chemical marker alleles (Wheeler and Jech 1985, Yazdani et al. 1986). Based on this technique, high success rates of SMP have been reported for seed orchards of P. menziesii (Wheeler and Jech 1985), P. taeda (Blush 1987) and P. sylvestris (Eriksson et al. 1994). The latter investigation showed average success rates between 66 and 84% when individual strobili were pollinated; however, in an operational study, in which whole grafts were pollinated, the success rate declined to 7--26%.
Besides competition by pollen from other sources, the suc-cess rate of SMP may be influenced by other factors including the prevailing environmental conditions at flowering and the number of occasions a graft must be pollinated in order to achieve a satisfactorily high success rate. Eriksson et al. (1994) concluded that, for a single strobilus, timing rather than the number of pollinations is critical but, because of the gradual maturation of female strobili, whole grafts need to be polli-nated more than once a day during the flowering period. Bridgwater et al. (1993) concluded that a single pollination at peak female receptivity is enough to achieve fairly high suc-cess rates in P. taeda. However, differences in sucsuc-cess rates among clones have been reported for P. taeda (Bridgwater and Williams 1983, Blush 1987) and P. menziesii (Wheeler and Jech 1985) that can be explained mainly by differences in flower phenology in relation to main pollen shedding. Bridg-water et al. (1993) concluded that pollination of clones that flower before maximum pollen flight provides the best oppor-tunity for successful SMP, but did not exclude the possibility of successful use of SMP for clones that flower during
maxi-Effects of supplemental mass pollination (SMP) in a young and a
mature seed orchard of Pinus sylvestris
U. ERIKSSON,
1G. JANSSON,
1R. YAZDANI
2and L. WILHELMSSON
11 Forestry Research Institute, Glunten, S-751 83 Uppsala, Sweden
2 Department of Forest Genetics, The Swedish University of Agricultural Sciences, P.O. Box 7027, S-750 07 Uppsala, Sweden
Received May 31, 1994
mum pollen flight. Differences in SMP success rate between clones could also be expected for P. sylvestris because there are large variations in flower phenology among and within clones of this species. Furthermore, because the course of flowering varies between years as a result of differing weather conditions during the flowering period (Sarvas 1962, Jonsson et al. 1976), annual variations in flowering phenology could also influence SMP success rate. Therefore, we have tested the hypothesis that the success rate of SMP in P. sylvestris seed orchards is influenced by clone, weather and frequency of application.
Materials and methods Experiment 1
Experiment 1 was carried out in the spring of 1987 in a mature P. sylvestris seed orchard with high pollen production (Ta-ble 1). Trees were pollinated for 8 days between May 28 and June 4, 1987. A mix with equal portions of pollen from two clones not cultivated in the orchard, designated A and B, was used. The clones were chosen because they had rare isozyme markers. Clone A is heterozygous at the LAP-B locus with the genotype B1/B2, and clone B is heterozygous for the GOT-B locus with the genotype B1/B3. B1 is the rare allele at both loci.
Four grafts from each of three mother clones (C--E) were each subjected to four pollination treatments as follows: (i) wind-pollinated control (Control), (ii) SMP of whole trees once per day during the period of peak receptivity, (iii) SMP of whole trees up to three times per day during the period of peak receptivity, and (iv) SMP of whole trees up to six times per day during the period of peak receptivity.
Experiment 2
Experiment 2 was carried out during both 1987 and 1988 in a P. sylvestris seed orchard with low pollen production (Table 1). Supplemental mass pollination was carried out during 11 days in both 1987 and 1988 (June 15 to June 26, 1987, and June 1
to June 12, 1988). Pollen from one clone, not represented in the orchard, was used in both years. The pollen clone (F) is heterozygous at the GOT-B locus with the genotype B1/B2, where B1 is the rare allele. Nine randomly selected clones (G--O), not represented in Experiment 1, were used as mother clones. Two individual grafts were sampled per clone, one for each treatment. The same grafts and clones were pollinated both years. The two pollination treatments in Experiment 2 were: (i) wind-pollinated control (Control), and (ii) SMP of whole trees twice a day during the period of peak receptivity.
Application of pollen
In both experiments, the pollen mix was dusted over clusters of receptive, unisolated female strobili throughout the whole graft in an attempt to pollinate as many receptive strobili as possible. Pollen was delivered to the strobili from a pollination wand activated by compressed air from a tube mounted on a modified backpack sprayer (Eriksson et al. 1994). About 3 ml of pollen was dusted over each graft in each pollination. The SMP was started when approximately 20% of the female strobili on a graft were judged to be receptive, according to the classification system presented by Jonsson et al. (1976). No pollinations were done during precipitation. The intention was to pollinate each graft for 3--5 days during the period of peak female strobilus receptivity. As a consequence of frequent precipitation, which slowed maturation of female strobili, the pollination period was extended to 4 to 8 days per graft in Experiment 1. For the same reason, the pollination period in Experiment 2 was extended to about 11 days in 1987 and to between 7 and 11 days in 1988.
Weather and amount of pollen
For both experiments, temperatures were estimated as the daily mean temperatures recorded by the Swedish Meteorological and Hydrological Institute. For Experiment 1, we used the data collected at Station 9240 Arvika, situated 40 km southwest of the orchard. The station closest to the orchard in Experiment 2 was Station 6311 Skagsudde, situated 40 km north of the orchard. The occurrence of precipitation was observed daily during the experimental periods in both experiments. The amount of pollen in the air during the experimental periods was assessed with a pollen-catching device (Sarvas 1962).
Pollen handling, cone collection and seed extraction
In 1986, pollen was extracted under controlled temperature and humidity conditions as described by Eriksson (1993). The pollen lots were stored in sealed glass jars at −20 °C. All cones on the treated grafts were collected in the autumn of 1988 (Experiments 1 and 2) and 1989 (Experiment 2). Seeds were extracted and stored at −4 °C until isozyme analyses were done. Numbers of empty seed and filled seeds per cone were determined for all treatments. Two clones in Experiment 2 produced no seed after the pollinations in 1987, and one clone produced no seed after the pollinations in 1988. These clones were excluded from subsequent analyses.
Table 1. Description of the seed orchards.
Characteristic Experiment 1 Experiment 2
Name 493 Askerud 123 Klocke Latitude 59°53′ N 62°54′ N Longitude 13°10′ E 18°16′ E
Altitude 80 m 75 m
Area 14 ha 16 ha
Year of establishment 1966--1969 1968--1972 Spacing 5 × 5 m 5.6 × 5.6 m Mean height (approx.) 5 m 3.5 m
No. of clones 43 60
Soil texture Clay Silt Seed yield, 1979--19881 52 kg ha−1 3.6 kg ha−1 Pollen production, 19872 40 kg ha−1
--Pollen production, 19882 39 kg ha−1 0.5 kg ha−1
Marker detection
The analyses of SMP success rate were carried out by isozyme separation and starch gel electrophoresis. In Experiment 1, two enzymes, leucine aminopeptidase (LAP) (Rudin 1977) and glutamate oxalate transaminase (GOT) (Rudin 1975), were analyzed in the diploid embryo and in the haploid megagame-tophyte. One enzyme (GOT) was analyzed in Experiment 2. With one exception, 100 seeds were analyzed per clone and treatment in Experiment 1 (only 40 seeds were available for clone D in treatment 3P). In Experiment 2, with a few excep-tions, about 100 seeds per clone and treatment were used in the subsequent studies.
The genotypes of the mother clones in the orchard with high pollen production were unknown at the time of the experiment. In a multilocus analysis of all clones in the orchard, mother clone E appeared to have the same rare allele (B1) in the GOT system as father clone B.
Statistical analysis
Statistical analyses of frequencies were performed on the logit transformations of y (Ashton 1972), as described by Eriksson et al. (1994). Briefly, the observations y were the observed proportions of SMP success rates and empty seed frequencies on each graft. In both experiments, the empirical logits lij of the responses in the treatment × clone subclasses were obtained as:
lij= ln
Because the logit transformations are undefined when the observed and adjusted yij values are 0 or 1, values of yij equal to 0 or 1 were excluded from the subsequent analysis (Harville and Mee 1984).
The observed values of LAP B1 and GOT B1 frequencies were adjusted for the contribution from the background pollen to the marker allele frequency by means of Equation 2. This equation assumes that the background contributions of LAP B1 and GOT B1 are proportional to the part not fertilized by the SMP pollen:
aij=(oij− cj)/(1 − cj), (2)
where aij is the adjusted observed frequency of LAP B1 or GOT B1 in the seed from treatment i and clone j, oij is the corresponding observed LAP B1 or GOT B1 frequency, and cj is the observed allele frequency for the pooled control data. Values of a less than 0 are unrealistic and have therefore been given the value 0.
In Experiment 1, the total contribution rates from SMP were computed as:
yij=(aijLAPB1+ aijGOT B1), (3)
where yij is the success rate of SMP pollen fertilization for a particular treatment × clone subclass, and aijLAPB1 and aijGOTB1
are the observed rates adjusted according to Equation 2.
In Experiment 2, the total contribution rates from SMP were computed as:
yij= aijGOT B1, (4)
where yij is the success rate of SMP pollen fertilization for a particular clone × year subclass, and aijGOTB1 is the observed
rate adjusted according to Equation 2.
The statistical analyses of the logit values were performed with the weighted least squares procedure available in the GLM procedure of the SAS program (SAS Institute Inc., Cary, NC). The following model was used for Experiment 1:
lij=µ+ ti+ sj+ eij, (5)
where lij is the logit value of success rate in subclass ij, µ is the overall mean, ti is the fixed effect of the ith treatment where i = 1, 2 or 3, sj is the fixed effect of the jth clone where j = 1, 2 or 3, and eij is the random residual effect of ijth observed logit value assumed to be individually and independently distrib-uted (IID) (0, π2/3). The weight used for the ijth observation was:
nij× yij(1 − yij),
where nij is the number of observations in the ijth class. The following model was used for Experiment 2:
lij=µ+ si+ uj+ eij, (6)
where lij is the logit values of success rate in subclass ij, µ is the overall mean, si is the fixed effect of the ith clone where i = 1, ..., 8, uj is the fixed effect of the jth year where j = 1 or 2, and eij is the random residual effect of the ijth observed logit value assumed IID (0, π2/3). The weight, used for each obser-vation in the least squares equation, was the same as in Equa-tion 5.
The estimated responses in both experiments were back-transformed from the underlying scale to the visible frequency scale by means of Equation 7:
p^ = 1
(1 + e_r^
) ,
(7)
where p^ is the estimated frequency corresponding to a least-squares mean r^ measured in the logit scale.
Because all father clones were heterozygous for the locus with the rare marker allele, the estimated frequencies p^ were multiplied by 2. In both experiments, the open-pollinated con-trols were included as one of the treatments in the analyses of empty seed frequency.
The ordinary least squares procedure of the SAS software package was used to analyze numbers of filled seeds per cone. The following model was used for Experiment 1:
where zij is the number of filled seeds per cone in subclasses ij, µ is the overall mean, ti is the fixed effect of the ith treatment where i = 1, ..., 4, sj is the fixed effect of the jth clone where j = 1, 2 or 3, and eij is the random residual effect of ijth observed value assumed IID (0, σe2). The following model was used for
Experiment 2:
zijk=µ+ ti+ sj+ uk+ eijk, (9)
where zijk is the number of filled seeds per cone in the sub-classes ijk, µ is the overall mean, ti is the fixed effect of the ith treatment where i = 1 or 2, sj is the fixed effect of the jth clone where j = 1, ..., 8, uk is the fixed effect of the kth year where k = 1 or 2, and eijk is the random residual effect of ijkth observed value assumed IID (0, σe2). The wind-pollinated controls were
included as a treatment in the analyses of the number of filled seeds per cone.
Results Experiment 1
From May 29 to May 31, the weather was dry and sunny, but turned rainy from June 1 to the end of the experimental period (Figure 1). The average daily mean temperature was about 10 °C, and only small amounts of pollen were trapped during
the experimental period. The main pollen shedding in the orchard started on June 4 (Figure 1).
The results from the ANOVAs of SMP success rate, empty seed frequency and number of filled seeds per cone are pre-sented in Table 2. The number of pollinations per day (pollina-tion treatments) did not significantly affect the success rate. However, significant differences in success rate were found between clones. The pollination treatments had a significant effect on empty seed frequency, but there was no significant difference in empty seed frequency among clones. There were no significant effects of either pollen treatment or clone on number of filled seeds per cone. Estimated least squares means of SMP success rate, empty seed frequency and number of filled seeds per cone for treatments and clones are presented in Table 3.
Experiment 2
During the 1987 experimental period, the weather was mostly rainy and cold with an average daily mean temperature of about 10 °C (Figure 2). Two small peaks of pollen in the air were detected (Figure 2). Unfortunately, the device for pollen catching was defective from June 24, 1987, to the end of the experimental period, but another device for pollen catching in another part of the orchard indicated that there were only small amounts of pollen in the air during this time.
Table 2. Analyses of variance for success rate, empty seed frequency and number of filled seeds per cone in Experiments 1 and 2.
Source of variation Success rate Empty seed frequency Number of filled seeds per cone
df MS F-value P df MS F-value P df MS F-value P
Experiment 1
Treatment 2 1.48 9.32 0.052 3 229.1 27.93 < 0.001 3 11.2 0.67 0.6 Clone 2 3.15 19.81 0.019 2 9.5 1.16 0.37 2 71.4 4.28 0.07
Error 3 0.16 6 8.2 6 16.7
Experiment 2
Year 1 8.10 1.60 0.27 1 37.3 0.83 0.37 1 204.1 16.4 < 0.001 Treatment -- -- -- -- 1 21.0 0.47 0.50 1 5.5 0.4 0.52 Clone 7 3.15 1.64 0.33 2 253.2 5.62 0.001 2 140.1 11.3 < 0.001
Error 4 5.05 6 45.0 6 12.4
During the first half of the 1988 experimental period, the weather was rainy and cold with an average daily mean tem-perature of about 8 °C (Figure 3). The weather during the latter part of the period was mostly sunny with only a few showers and an average daily mean temperature of about 12 °C. Small amounts of pollen in the air were found before June 8 when the
main pollen shedding started (Figure 3).
The ANOVAs for SMP success rate, empty seed frequency and number of filled seeds per cone are shown in Table 2. No significant effects, for either years or clones, were found in the analysis of success rate. Significant differences in empty seed frequency were found between clones, but not between years
Table 3. Success rate of SMP, empty seed frequency and number of filled seeds per cone for treatments in Experiment 1. The frequency and standard error range for success rate and empty seed frequency are expressed as back-transformed (probability scale) values and estimated least squares means. The number of filled seeds per cone are estimated least squares means.
Success rate Empty seed frequency Number of filled seeds per cone
Frequency Error range Frequency Error range
Treatment-wise
Max 1 SMP per day 0.16 0.15--0.17 0.22 0.20--0.24 16.74 Max 3 SMP per day 0.24 0.22--0.26 0.36 0.33--0.40 12.88 Max 6 SMP per day 0.18 0.17--0.19 0.21 0.19--0.24 13.81
Control -- -- 0.13 0.11--0.15 16.50
Average 0.19 0.22 14.98
Clone-wise
Clone C 0.26 0.24--0.27 0.27 0.23--0.31 11.16 Clone D 0.11 0.10--0.13 0.18 0.14--0.22 14.26 Clone E 0.23 0.22--0.25 0.22 0.21--0.23 19.52
Average 0.19 0.22 15.34
Figure 2. Daily mean temperatures (dashed line) recorded by the Swedish Meterological and Hydrological Institute at their station 9311 Skagsudde, situated 40 km north of the orchard, and the amounts of pollen in the air (solid line), as-sessed with a pollen catching device, in Experiment 2, 1987. The bar shows the pe-riod of female strobili receptivity on the treated grafts, i.e., the experimental period.
or pollination treatments. The difference in number of filled seeds per cone was significant between years but not between pollination treatments. Furthermore, clones had significantly different numbers of filled seeds per cone. Estimated least squares means of SMP success rate, empty seed frequency and number of filled seeds per cone for years, treatments and clones are presented in Table 4.
Discussion
Success rate and number of pollinations per day
Eriksson et al. (1994) concluded that a single SMP of a P. sylvestris strobilus is enough to achieve a high success rate, provided that the strobilus is at peak receptivity when polli-nated, whereas whole trees should be pollinated more than once a day, because not all female strobili on a tree reach peak receptivity at the same time (Sarvas 1962). However, we found that increasing the number of pollinations per day caused only a small, nonsignificant increase in SMP success rate in Experi-ment 1. The absence of pollination treatExperi-ment effects in our study may be associated with the slow maturation of female strobili as a result of the cold and rainy weather. Thus, only a limited number of new unpollinated strobili may have become receptive between pollinations. If so, this suggests that SMP of a graft should be carried out relatively more often during a hot day than during a cold day.
Success rate and weather conditions
The average SMP success rate in Experiment 1 was higher than that reported for a comparable study in 1986 (Eriksson et al. 1994). One explanation could be that the weather conditions differed between 1986 and 1987. In contrast to 1987, the weather in 1986 was mostly sunny with little or no precipita-tion. The finding that there was no significant difference in SMP success rate between years in Experiment 2 does not
exclude the possibility that there could be differences between years with more variable weather conditions. Even though the weather in 1988 improved during the second part of the experi-mental period, both flowering periods studied in Experiment 2 were rainy and cold. Frequent precipitation and high humidity could have contributed to SMP success rate in two ways. First, precipitation reduces the amount of competing pollen in the air by washing it to the ground. Sarvas (1962) showed that abun-dant precipitation during the flowering period destroys as much as 50% of the total annual pollen catch. Andersson (1955) and Eklund-Ehrenberg and Simak (1957) also observed that humid weather conditions prevent pollen from shedding in P. sylvestris. Second, rain drops could serve as agents in the transport of pollen from the bract scales to the micropyle. In a study of the pollination mechanism in P. taeda, it was con-cluded that the force of falling raindrops can cause the move-ment of pollen from the micropylar arms to the micropyle (Brown and Bridgwater 1987). In P. taeda, Greenwood (1986) found that the transfer of pollen through the micropyle to the nucellus is carried out by either precipitation or the pollen drop, whichever appears first. Sweet et al. (1992) studied artificial pollination of Pinus radiata D. Don and found signifi-cantly more pollen grains in the micropyles after pollinations with pollen suspended in water compared with dry pollina-tions. Sarvas (1962) claims that rain water has little chance of penetrating between the bud scales in P. sylvestris; however, Eklund-Ehrenberg and Simak (1957) reported that already captured pollen grains were washed from the strobili when pollinated P. sylvestris strobili were rinsed with water. They concluded that rain not only prevents pollen shedding, but it also reduces the possibility of already captured pollen reaching the pollen chamber. Furthermore, both Sarvas (1962) and Ek-lund-Ehrenberg and Simak (1957) reported that pollen vigor is rapidly lost when pollen is exposed to humid conditions.
Table 4. Success rate of SMP, empty seed frequency and number of filled seeds per cone for years and clones in Experiment 2. The frequency and standard error range for success rate and empty seed frequency are expressed as back-transformed (probability scale) values and estimated least squares means. The number of filled seeds per cone are estimated least squares means.
Success rate Empty seed frequency Number of filled seeds per cone
Frequency Error range Frequency Error range
Year-wise
1987 0.34 0.27--0.44 0.40 0.38--0.44 9.68 1988 0.22 0.16--0.29 0.38 0.36--0.39 15.08
Average 0.27 0.39 12.38
Clone-wise
Clone G 0.34 0.22--0.50 0.36 0.32--0.40 5.45 Clone H 0.22 0.14--0.34 0.31 0.29--0.33 20.83 Clone I 0.42 0.31--0.57 0.34 0.30--0.37 10.05 Clone J 0.69 0.55--0.85 0.44 0.39--0.50 11.23 Clone K 0.18 0.08--0.39 0.59 0.54--0.64 12.30 Clone L 0.24 0.13--0.42 0.43 0.39--0.46 11.85 Clone M 0.14 0.05--0.37 0.34 0.30--0.38 6.05 Clone N 0.20 0.10--0.37 0.31 0.29--0.33 21.25
Clonal differences in SMP success rate
In Experiment 1, differences in SMP success rate among clones cannot be explained by differences in the timing of peak receptivity because we observed only marginal differences between the grafts in time of peak receptivity. Furthermore, all pollinations were carried out before the main pollen release occurred. Another explanation could be that preferential fertili-zation occurred and the degree to which it did so varied among clones.
Empty seed frequency and number of filled seeds per cone
Compared with the control trees, all SMP-treated trees in Experiment 1 had a higher empty seed frequency. Because formation of empty seed in the Pinus genera always requires the presence of a germinating pollen grain in the ovule (McWilliam 1959, Sarvas 1962, Plym-Forshell 1974, Owens et al. 1981), these findings suggest that the applied pollen germinated but was not able to complete the fertilization proc-ess as succproc-essfully as pollen from competing sources. In Ex-periment 2, the empty seed frequency in the control and SMP-treated trees was similar, indicating that the pollen sup-plied in Experiment 2 was of similar vigor to that of the competing wind-borne pollen. We conclude that pollen used in SMP must be highly viable to compete successfully with wind-borne pollen (Bridgwater et al. 1993). Other factors that could cause formation of empty seeds in P. sylvestris are homozygosity of sublethal genes causing degeneration of the embryo or disturbances during embryo development induced by climate or poor nutrition (e.g., Sarvas 1962, Plym-Forshell 1974). The latter is the most probable explanation for the difference in number of filled seeds observed between years in Experiment 2.
Success rate and pollen production in the seed orchard
Although SMP success rate was higher in the orchard with low pollen production than in the orchard with high pollen tion (cf. Tables 2 and 3), the effects of orchard pollen produc-tion may explain only some of this difference. First, almost all of the pollinations in the study were carried out before the time for main pollen shedding. Moreover, the small amount of pollen shed during the experimental period was probably washed away by the frequent rains. Second, the grafts were taller in the orchard with high pollen production than in the orchard with low pollen production, and therefore, female strobili at the top of the taller grafts were more difficult to detect than those at the top of the smaller grafts. This interpre-tation supports the idea that relatively small, densely spaced grafts offer the best prospects of successful SMP of P. sylvestris. Finally, if the amount of seed orchard and back-ground pollen was insufficient for an adequate pollination in the orchard with low pollen production, the number of filled seeds per cone would have been higher in SMP-treated trees than in control trees. It is unlikely that the supply of pollen from within and outside the orchard was limited during the flowering period. Hadders (1973) showed that seed production in a young seed orchard was normal despite the emasculation of all male strobili in the orchard. Thus, we conclude that
pollen was not a limiting factor in determining seed yields in this study.
Supplemental mass pollination is a practical means for im-proving the genetic gain both in young and in mature P. sylvestris seed orchards. However, to improve the gain fur-ther as well as to reduce the cost per harvested seed, it will be necessary to combine the use of SMP on selected clones with flower stimulation techniques. New propagation methods will also have to be developed if complete control of the parentage is to be achieved in mass propagation of improved P. sylvestris.
Acknowledgments
The authors thank Anders Ramström, Mats Eriksson, Roger Granbom and Inga-Britt Carlsson for skillful assistance. We are also grateful to Prof. Öje Danell for fruitful discussions and good advice, as well as to Prof. Gösta Eriksson for valuable comments on the manuscript. Finan-cial support was provided by the Swedish Forestry Research Founda-tion (SSFf) and by the research Committee of the Swedish NaFounda-tional Board of Forest Research.
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