Summary We assessed the effects of nitrogen, drought and gibberellin A4/7 on sexual expression of 2- and 3-year-old jack pine (Pinus banksiana L.) seedlings grown in either a 1/3 peat/sand mix or a 3/1 peat/perlite mix. The seedlings were either watered daily (well-watered treatment) or droughted by withholding water from July 13 to August 24 (drought treat-ment). Half of the plants in each irrigation treatment were sprayed with 200 mg l−1 gibberellin A4/7 at weekly intervals from June 28 to August 24; the remaining plants were not sprayed. Each gibberellin treatment was split into three sub-plots, and each subplot was supplied with nitrogen in the nutrient solution at 3, 100 or 300 mg l−1 from June 27 to September 11. The drought treatment increased pollen strobi-lus production, whereas seed strobistrobi-lus production was higher in well-watered trees than in drought-treated trees in the first year after treatment, but not in the subsequent year. Gibberellin A4/7 promoted seed strobilus production but not pollen strobilus production in the first year after treatment but had no effect in the second year. Among the nitrogen treatments, the largest proportion of trees bearing pollen strobili was in the 3 mg l−1 N treatment. Nitrogen supply did not affect seed strobilus production in the first year after treatment, but in the following year, the proportion of trees with seed strobili was higher in the 3 mg l−1 N treatment than in the 100 and 300 mg l−1 N treatments. Growing medium had no detectable effect on pol-len strobilus production, but in the year following treatment, more trees produced seed strobili in peat/sand than in peat/per-lite.
Keywords: drought, gibberellin, nitrogen, Pinus banksiana, pollen strobili, seed strobili.
Introduction
Some 3-year-old jack pine (Pinus banksiana L.) seedlings treated with gibberellin A4/7 produce pollen and seed strobili (Cecich 1983, Ho and Hak 1994), but few seedlings younger than 3 years produce seed strobili and none produce pollen strobili (Rudolph 1979, Cecich 1983).
Environmental conditions influence sex expression in plants. In particular, pollen strobilus production tends to be increased by nitrogen and water deficiencies (Heslop-Harrison 1972, Charnov 1982, Chailakhyan and Khrianin 1986). For
example, Pinus radiata D. Don cuttings produce pollen strobili after 2 years of growth when subjected to nitrogen deficiency (Sweet and Will 1965). We have observed that nitrogen defi-ciency promotes pollen strobilus production in 5-year-old jack pine seedlings (Fogal et al. 1994), and Riemenschneider (1985) reported that short-term soil water deficits enhance seed strobilus production in jack pine seedlings.
The objective of this study was to test the hypothesis that low nitrogen supply and low water supply accelerate pollen strobilus production in jack pine seedlings less than 3 years old.
Materials and methods
Plant material and treatments
In January 1988, seeds from eight half-sib jack pine families were germinated in 65-ml containers filled with peat/perlite mix (3/1). Seedlings were grown in a greenhouse (20 °C, 16-h daylength, extended with sodium vapor lamps) and watered daily. The plants were fertilized weekly with standard nutrient solution containing 100, 15 and 45 mg l−1, respectively, of N, P and K. In April, the seedlings were transplanted to 4-l containers. In September, seedlings were transferred to a poly-ethylene-covered structure following transplanting to 25-l con-tainers of which half were filled with a 3/1 peat/perlite mix and half were filled with a 1/3 peat/sand mix. In January 1989, at age 11 months, seedlings began to produce seed strobili. At the end of February, artificial extension of daylength was termi-nated and the photoperiod reduced by 2 h per week to 8 h, the nutrient solution was changed to 50, 30 and 90 mg l−1, respec-tively, of N, P and K (hardening nutrient solution), and night-time temperature was reduced to 1 °C with the result that, by the beginning of May, seedlings had received 581 h below 10 °C and 153 h below 5 °C. On May 3, 1989, the seedlings were moved to two polyethylene-covered cold-frame shelters. The polyethylene sheets on two sides of each cold-frame shelter were raised to a height of 1 m to provide free air movement. The average daily temperature recorded within the seedling crowns at four locations within the shelters during the treatment period was 19.2 ± 2 °C. At the time the treatments began, seedling heights averaged 87 ± 5 cm.
Sexual expression in container-grown jack pine seedlings
W. H. FOGAL,
1S. M. LOPUSHANSKI,
1S. J. COLEMAN,
1H. O. SCHOOLEY
1and M. S. WOLYNETZ
21 Petawawa National Forestry Institute, Natural Resources Canada, Forestry Sector, Chalk River, Ontario K0J 1J0, Canada
2 Western Region, Research Branch, Agriculture and Agri-food Canada, Ottawa, Ontario K1A 0C6, Canada
Received June 22, 1994
Tree Physiology 15, 439--442
Experimental design
In each cold-frame shelter, seedlings were arranged in 24 rows with each row containing one seedling from each of the eight half-sib families. Half of the seedlings in each shelter were irrigated daily, and the remainder were drought treated by withholding water from July 13 until August 24, 1989; the standard nutrient supply was maintained during this period (Carlson 1983). Each water treatment plot was split into two subplots to test the effects of exogenously applied gibberellin. At 1-week intervals from June 25 until August 31, 1989, GA4/7 was applied as a foliar spray. The spray contained 200 mg GA4/7 l−1 (200 mg GA4/7 in 20 ml of solvent (Abbott Laborato-ries, Chicago, IL, USA) and 1 g of Aromox C-12/w made up to a final volume of 1 liter with deionized water). During spray operations, polyethylene curtains were erected between treat-ment plots to prevent spray from drifting. Each gibberellin treatment plot was split into three subplots to test the effects of three N supplies (low-N = 3 mg l−1, medium-N = 100 mg l−1, and high-N = 300 mg l−1). Nitrogen was supplied in the nutrient solution as NH4NO3. The pH of the nutrient solutions was adjusted to 5.5--6.0 with HCl.
Predawn xylem water potentials were determined on cur-rent-year needles of a selected tree from each half-sib family in the 100 mg l−1 N treatment combinations. Measurements were made with a pressure chamber (Model 3005, Soil Mois-ture Corp., Santa Barbara, CA, USA) at 2- to 5-day intervals between July 14 and August 24, 1989 (Ritchie and Hinckley 1975). For the period, mean xylem water potentials for well-watered and drought-treated seedlings were −0.85 ± 0.04 and
−2.32 ± 0.13 MPa, respectively, in peat/sand and −0.83 ± 0.03 and −1.67 ± 0.19 Mpa, respectively, in peat/perlite.
Post-treatment dormancy conditions and strobilus counts
On September 11, 1989, the growing medium in each pot was washed with deionized water, and the seedlings were then fertilized once per week for 3 weeks with the hardening nutri-ent solution.
On November 27, 1989, the trees in one cold-frame shelter were moved to a greenhouse providing culture conditions that shortened the winter dormancy period. The remainder of the seedlings were overwintered outside by removing the polyeth-ylene cover from the second cold-frame shelter. To shorten the winter dormancy period, the day/night temperature in the greenhouse was gradually raised from 10/1 to 20/10 °C, the photoperiod was increased at weekly intervals to a 16-h pho-toperiod by December 18, 1989, and the seedlings were wa-tered daily and fertilized weekly with standard nutrient solution. Seed strobili and clusters of pollen strobili, first appearing in January, were counted weekly until February 20, 1990, when a short dormancy period was initiated to arrest growth and induce bud set. Supplemental lighting was stopped, the nutrient solution was changed to the hardening solution, and the day/night temperature was reduced to 10/1 °C. Trees were moved back to the cold-frame shelter on May 22, 1990. Polyethylene covers were replaced on both shelter frames, and all trees were watered daily and fertilized weekly with standard nutrient solution by means of drip
irriga-tion from June 1 to September 30, 1990. Seed strobili and clusters of pollen strobili were counted on the trees that had been overwintered outside in late June 1990. At the end of September 1990, all trees were again given the hardening nutrient solution in preparation for overwintering. At the end of November 1990, the polyethylene covers were removed from both cold-frame shelters, and all of the trees were over-wintered outside. In mid-May 1991, daily irrigation and weekly fertilization with standard nutrient solution recom-menced and continued throughout the 1991 growing season.
Pollen and seed strobili were counted on all trees that re-ceived the shortened dormancy period in January--February 1990 and June 1991, and strobili were counted on all trees that were overwintered outside in June 1990 and 1991.
Statistical analyses
For trees receiving the normal dormancy period and for trees receiving the short dormancy period, the data on number of seed and pollen strobili per tree were not normally distributed. Therefore, the trees were given binomial labels for the pres-ence (1) or abspres-ence (0) of seed or pollen strobili, and the number of strobilus-bearing trees per row was used in a split-plot ANOVA to test for interactions between and among treat-ments (Harvey 1982). The only significant interaction was a second-order interaction among water supply, GA4/7 treatment and nitrogen supply for pollen strobilus counts made in Janu-ary 1990. We used chi-square tests to examine the main effect of each treatment variable on the percentage of trees with seed and pollen strobili by pooling all other treatments.
Results and discussion
Water supply
The drought treatment had no effect on pollen strobilus pro-duction in either assessment year (Table 1), but slightly de-creased both the production of seed strobili (3.5 ± 0.5 versus 4.3 ± 0.5 seed strobili per tree) and the proportion of seedlings producing seed strobili (66 versus 80%) in the January 1990 assessment. Similar results have been reported for other coni-fers including Picea engelmannii (Parry) (Ross 1985) and Tsuga heterophylla (Raf.) Sarg. (Brix and Poortlock 1881). However, Riemenschneider (1985) found that three short peri-ods of drought stress stimulated seed strobilus production in jack pine seedlings.
Gibberellin A4/7
Gibberellin A4/7 foliar spray had no effect on production of pollen strobili in either assessment year (Table 1). The GA4/7 spray enhanced seed strobilus production in the first assess-ment year on trees subjected to the shortened dormant period (4.5 ± 0.5 versus 3.3 ± 0.5 seed strobili per tree) and on trees receiving a natural dormant period (8.3 ± 0.7 versus 3.4 ± 0.4 seed strobili per tree). The proportions of untreated and GA4/7 -treated seedlings bearing seed strobili were 66 and 80%, re-spectively, for seedlings subjected to the shortened dormant period, and 73 and 96%, respectively, for seedlings that
ceived a natural dormant period. The GA4/7 treatment had no effects on seed strobilus production in 1991. Pollen strobilus production is also less sensitive to GA4/7 treatment than seed strobilus production in lodgepole pine (Pinus contorta Dougl.) (Wheeler et al. 1980). The poor response to the GA4/7 treatment was associated with the juvenility of the seedlings rather than with the timing of the applications (Cecich 1983, Ho and Hak 1994). It is well known that the ability of exogenous GA4/7 to elicit sexual responses in pines increases with tree age (Green-wood 1981).
Nitrogen supply
Production of pollen strobili was enhanced by the low-N treat-ment in both assesstreat-ment years in trees having a short dormant period and in trees having a natural dormant period (Tables 1 and 2). For trees that received a short dormant period, the proportion producing pollen strobili decreased with increasing nitrogen supply in January 1990 and 1991. In January 1990, 32% of the low-N trees produced pollen strobili, and the average number of pollen strobilus clusters per tree was 1.5 ± 0.5; the corresponding values for June 1991 were 70% and 9.2
± 1.8, respectively. The proportions of low-N trees having a natural dormant period that produced pollen strobili in June 1990 and 1991 were 23 and 71% with an average of 1.2 ± 0.4 and 25.2 ± 4.4 clusters per tree, respectively.
Nitrogen supply had no influence on seed strobilus produc-tion in 1990 (Tables 1 and 2). In 1991, the proporproduc-tion of trees producing seed strobili was slightly higher in the 3 mg l−1 N treatment than in the other N treatments for trees having a normal dormant period. The proportions of low-N, medium-N
and high-N trees producing seed strobili were 90, 72 and 75%, respectively, and the corresponding values for seed strobili per tree were 7.9 ± 0.8, 7.7 ± 1.1 and 9.9 ± 1.6, respectively.
Growing medium
Pollen strobilus production was not influenced by growing medium, whereas seed strobilus production was affected in 1990, but not in 1991 (Table 1). In January 1990, following the short dormancy, 80% of trees in peat/sand versus 66% in peat/perlite produced seed strobili with an average of 4.5 ± 0.5 and 3.4 ± 0.4 seed strobili per tree, respectively. Following natural dormancy, seed strobili were present on 87% of trees in peat/sand and 76% of trees in peat/perlite with an average of 5.6 ± 0.5 and 6.1 ± 0.7 stobili per tree, respectively.
Conclusions
Neither drought nor GA4/7 treatments were effective in accel-erating male sexual expression in very young jack pine seed-lings. Seed strobilus production was suppressed by drought, whereas it was promoted by GA4/7 . Male sexual expression in container-grown jack pine seedlings was accelerated by reduc-ing the availability of nitrogen. Under N-deficient conditions, one third of the seedlings produced pollen strobili in less than 2 years from seed, and 71% of the seedlings produced pollen strobili in less than 3 years from seed. In contrast, nitrogen deficiency had no effect on seed strobilus production in 2-year-old seedlings and resulted in only a small enhancement in 3-year-old seedlings, indicating that the nitrogen requirements for expression of pollen and seed strobilus development differ, Table 1. Chi-square tests for effects of water supply, GA4/7, nutrient supply and growing medium on percentage of trees with seed and pollen strobili.
Assessment time Treatment Pollen strobili Seed strobili
χ2 P χ2 P
Shortened dormancy
January 1990 Drought 3.725 0.054 5.169 0.023
GA4/7 2.253 0.133 6.752 0.009
Nitrogen 29.892 < 0.001 1.635 0.441
Medium 3.725 0.054 5.169 0.023
Normal dormancy
June 1990 Drought 1.337 0.248 3.574 0.059
GA4/7 0.481 0.488 11.580 0.001
Nitrogen 15.719 < 0.001 0.071 0.965
Medium 0.053 0.817 5.147 0.023
Shortened dormancy
June 1991 Drought 2.562 0.109 0.865 0.352
GA4/7 1.021 0.312 0.243 0.622
Nitrogen 6.621 0.037 2.478 0.290
Medium 2.562 0.109 0.865 0.352
Normal dormancy
June 1991 Drought 1.768 0.184 0.084 0.772
GA4/7 0.077 0.781 0.046 0.830
Nitrogen 13.317 0.001 9.394 0.009
Medium 0.505 0.477 2.818 0.093
making it difficult to accelerate pollen and seed strobilus production simultaneously.
Acknowledgments
We are grateful to G.I. McIntye and A.M. Nanka for providing critical background information for the study and M.L. Anderson for technical assistance.
References
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Post-treatment conditions Assessment time Nitrogen treatment (mg l−1)
3 100 300
Pollen strobilus production
Shortened dormancy January 1990 32 a1 8 b 0 c
(1.5 ± 0.6) (0.3 ± 0.2) (0.2 ± 0.2)
Normal dormancy June 1990 23 a 6 b 3 b
(1.2 ± 0.4) (0.1 ± 0.1) (0.1 ± 0.1)
Shortened dormancy June 1991 70 a 55 b 48 b
(9.2 ± 1.7) (6.4 ± 1.1) (4.1 ± 1.6)
Normal dormancy June 1991 71 a 48 ab 39 b
(25.2 ± 4.4) (16.5 ± 4.1) (7.3 ± 2.8)
Seed strobilus production
Shortened dormancy January 1990 75 a 77 a 67 a
(5.6 ± 0.7) (6.5 ± 0.8) (5.4 ± 0.7)
Normal dormancy June 1990 83 a 83 a 81 a
(3.8 ± 0.5) (4.2 ± 0.7) (3.8 ± 0.6)
Shortened dormancy June 1991 83 a 92 a 87 a
(9.8 ± 1.3) (10.9 ± 1.4) (8.7 ± 1.1)
Normal dormancy June 1991 90 a 72 b 75 b
(7.9 ± 0.8) (7.7 ± 1.1) (9.9 ± 1.6)
1 Percentages followed by different letters within a row are significantly different according to χ2 tests (P < 0.5).
