Volume 25, No. 2, 2003

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Additional Symposium Abstracts (free download)

Applicability of Nondestructive Techniques in Seed Research
Carmen Rafaela Carvajal*, J. A. Vozzo, Ramesh Patel, and Asmita Roy
A slightly invasive biopsy procedure allows quantitative analyses while using computerized tomography (CT) for biodensity of morphology, and magnetic resonance imaging (MRI) of mobile proton distributions for comparative physiology. The  supplemental procedures provide a useful comparison of dormant and germinated seed tissues to correlate form, function, and amount of metabolic substrate during treatments and storage trials.

CT uses thin, contiguous, nondestructive slices of single-plane images to identify and quantify area and volume in any tissues within a 5000 Hounsfield unit biodensity range. The data are expressed in either histograms of densities or numerical mapping of the same density units. MRI provides presence and distributions of the hydrogen ion density for bulk water and/or fatty acid molecules.These mobile hydrogen ion distributions are visually displayed by both area and volume (pixel and voxel determinations). The biopsy is a sample extraction through a 1.0- to 3.0-mm bore penetrating the seed coat. Chemical analyses, for example, nitric oxide synthase (NOS), are quantitative for elemental and organic changes in challenged tissue. NOS is a widely reported free radical functioning as a messenger molecule for both inter- and intracellular significance. Although NOS is not definitely shown to affect plant cellular metabolism, it is an assay and can be controlled using both enhancements and inhibitors.
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(pp. 69-77)
Temperate and Tropical Tree Seed Physiology and the Economy of Nature
R.H. Ellis
Difficulties in tree seed supply, dormancy and germination mean that the majority of seed researchers have ignored temperate and especially tropical tree species in their studies. Examples of results from several comparatively large systematic investigations of tree seed dormancy and survival are presented. Although the responses might be regarded as complex, they tend to tally with those for other plant propagules.
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(pp. 78-82)
Seed Research in the United States––and its Future
Miller B.McDonald*
Of all agricultural inputs, seed is the most important for two reasons. First, it is the propagule carrying unique genetics that culminate in optimum crop response to varying environments. Second,

seed is the reproductive unit responsible for ensuring successful stand establishment for most agricultural crops. The current worldwide value of seed is $4 billion (U.S.) and this value is expected to increase to $20 billion (U.S.) by 2010. Successful seed production, therefore, is important and has been the subject of intense focused research in the past. The objective of this presentation is to compare how seed research has changed in the United States (U.S.) from 25 years ago till today and what can be projected ahead for successful seed research programs.
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(pp. 83-91)
Ethephon (2-Chloroethylphosphonic Acid) Combined with Short Prechilling Improves Germination in Stored Beechnuts

Claudine Muller*, Elyane Laroppe
Beechnuts are deeply dormant and require long cold prechilling before they germinate (one to three months). The methodology of pretreatment without medium at controlled moisture content applied before or after storage is the best method to take into account the dormancy and the heterogeneity within a seedlot (consequence of the genetic variability). It allows a very fast and grouped seedling emergence in the nursery. However, this long prechilling often damages older, less vigorous seedlots.

The effect of chemical treatments on dormancy breaking, in particular growth regulators, as a means of shortening the treatment, has been studied. In previous experiments ethephon was shown to strongly stimulate percentage and germination rate of freshly collected beechnuts and to reduce the duration of the cold requirement by half in comparison with the classical pretreatment which needs 4 to 20 weeks. In the present study ethephon + short prechilling (3 weeks) strongly stimulates germination and seedling emergence of beechnuts stored for 3 years at either 7 or 9% moisture content. It avoids the viability loss of less-vigorous beech seeds during longer prechilling (5 weeks) and allows restoring the initial potential of the seedlots. No significant effect of storage moisture content is observed on seedling emergence when seeds are short-prechilled with ethephon. Thus, ethephon used in association with short cold treatment opens new prospects for dormancy breaking particularly in nurseries where short prechilling are preferred.
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(pp. 92-103)
Freezing Response in Scots Pine Seeds as Assessed by DSC and Germination Test
Gunnar Sven Pamuk*, Urban Bergsten, and Philippe Lingois
Responses of Scots pine (Pinus sylvestris L.) seeds to freezing temperatures after incubation for 5 days at 15 °C were examined for dry (7%), 15%, 20%, 25%, and 30% moisture content seeds using differential scanning calorimetry (DSC) to identify critical moisture content at freezing. In a complementary experiment, seeds of the different initial moisture contents were incubated for 2 or 5 days, and subsequently exposed to –5 °C or –20 °C during 2 days or 5 days, and tested for germination (21 days, 20 °C).

In the DSC experiments, seeds with higher moisture content (20%, 25%, and 30%) had significantly wider endothermic peaks (–30.51 J/g, –40.36 J/g, and –47.02 J/g) than seeds with lower moisture contents (15% and dry seeds; –6.94 J/g and –0.65 J/g). Germination capacity ranged from 97% (control) to 58% (30% moisture content, 5 days of incubation, 5 days of freezing at –20 °C). Germination capacity after freezing was significantly higher (81%–83%) for seeds with moisture contents from dry to 20%, compared to seeds with moisture contents between 25%–30% (68%–72%). Seeds exposed to –20 °C for 2 and 5 days had significantly lower germination capacity (72%) than seeds exposed to –5 °C (80%–83%). In  conclusion, freeze damage increased with the combination of high seed moisture and decreasing below 0 °C temperatures, and increased freezing duration. The critical moisture content of Scots pine seeds is between 15%–20% according to DSC and germination tests. To enable autumn seeding, artificial seed coating should restrict water uptake under this critical moisture content during winter and allow for water uptake and germination in spring.
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(pp. 104-109)
Early Growth of Ramon (Brosimum alicastrum Sw.) and Relationships Between Seed Weight and Seedling Size
Early Aníbal Niembro Rocas*
Ramon, Brosimum alicastrum Sw. (Moraceae), is one of the tropical native trees of the Yucatán Peninsula in Mexico with agroforestry potential. Almost one millon ramon seeds are sown in forest nurseries as part of the activities of the National Reforestation Program (PRONARE) in the southeast region of Mexico, however, there are so many gaps of information about the performance of ramon seeds that its propagation has been limited. The objectives of this study were:

(1) to evaluate the emergence rate and early seedling growth of a sample of ramon seeds, the product of free pollination, and (2) to explore the relationships among the seed weight and growth of seedlings 50 days after the sowing date. The variables were: (1) seed weight, (2) emergence rate [days], (3) seedling height ; (4) main root length; (5) stem diameter; (6) number of leaves, (7) stem and leaf fresh weight, (8) root fresh weight, (9) stem and leaf dry weight, (10) root dry weight, (11) sturdiness quotient, and (12) shoot/root ratio. The results of 184 observations for each variable, were subjected to descriptive statistics analysis and simple correlation analysis. The resulting seedlings showed high morphological variability that was  significantly associated with the fresh weight of the seeds. Seed sizing is recommended to maintain standards for ramon nursery production, which can be achieved by seed selection before sowing.
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(pp. 110-123)
Recalcitrant Behavior of Cherrybark Oak Seed: An FT-IR Study of Desiccation Sensitivity in Quercus pagoda Raf. Acorns
Sharon Sowa* and Kristina F. Connor
The recalcitrant behavior of cherrybark oak (Quercus pagoda Raf.) acorns was examined in terms of effects of moisture content on seed storage longevity and (short term) seed germination. Seed samples collected over two consecutive years were fully hydrated, then subjected to drying under ambient conditions of temperature and relative humidity on the lab bench and sampled regularly for moisture determination (gravimetric analysis) and germination (greenhouse conditions).

Fourier transform infrared spectroscopy (FT-IR) was used to follow changes in macromolecular structure as moisture and viability were lost. Transmission spectra were collected on dry and rehydrated samples of separate embryonic axis and cotyledon tissue. Long-term storage longevity was highly dependent on initial acorn moisture content. Germination was also highly dependent on short-term moisture content, and severely declined when seed moisture dropped below 17% (fresh weight basis). FT-IR analyses revealed significant differences in moisture and lipid profiles between embryonic axis and cotyledon tissue during short term drying. A strong absorbance near 1740 cm-1 in cotyledon tissue indicated a high concentration of ester carbonyl groups (storage lipids). Membrane lipid structure exhibited reversible shifts between gel and liquid crystalline phases upon drying and rehydration in both axes and cotyledons (peak frequency and bandwidth near 2850 cm-1); however, reversibility declined as viability was lost. Irreversible changes in protein secondary structure, illustrated by shifts in the amide absorbance near 1650 cm-1, were the most sensitive indicators of viability loss.
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(pp. 124-127)
Effect of Maturation on Seed Germination of Dalbergia cochichinensis
Pierre Le Quang Hung
Germination of Dalbergia cochichinensis Pierre seeds at various stages of maturity (green, yellowish-brown and dark-brown fruits) showed the presence of primary dormancy. The germination capacity (30 °C/20 °C – 16h/8h light/darkness) was highest for seeds from green fruits (after a germination period of 28 days), followed by yellowish-brown and dark-brown fruits. After four weeks of storage, germination of seeds from yellowish-brown fruit was highest, while mean germination rate was lowest from seeds of green fruits.
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(pp. 128-139)
Germination of Horse Chestnut Seeds—Cell Growth and Hormonal Regulation
Natalie V. Obroucheva* and Olga V. Antipova
The course of germination events includes seed dormancy, its release, the preparation for cell elongation in the axes, early germination and postgerminative extension of seedling organs. The specificity of horse chestnut seeds is manifested in

(1) their recalcitrance, (2) dominance of coat-imposed dormancy over embryo dormancy, (3) embryo dormancy breakage by cytokinins only and (4) growth initiation by cell elongation in the embryo axes up to an axis length of 3.0–3.5 cm, confirmed by cell length measurements and the absence of both mitoses and thymidine kinase activity. In horse chestnut seeds, the hormonal regulation differs during the progress from deep dormancy to seedling growth. Embryo dormancy is maintained by abscisic acid and is broken by cytokinins. Cell preparation for elongation proceeds in nondormant seeds and includes an accumulation of endogenous osmotica accompanied by cell wall loosening. The latter occurs via activation of plasmalemma H+-ATPase and extrusion of protons into cell walls, thus acidifying them. Cell elongation after radicle emergence is stimulated by gibberellins. These conclusions are in agreement with the data on dynamics of endogenous abscisic acid and cytokinins.
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(pp. 140-148)
Testing of Endogenous Germination Periodicity in Picea glauca, Pinus contorta and Pinus banksiana Seeds
B.S.P. Wang
Previously published reports indicated that germination of some tree seeds might be controlled by an endogenous germination periodicity. It is most important to confirm whether this phenomenon is true because it could affect laboratory seed testing. This study was designed to test this hypothesis with

white spruce (Picea glauca [Moench] Voss), jack pine (Pinus banksiana Lamb.) and lodgepole pine (Pinus contorta var. latifolia Engelm.) seeds. Results of monthly germination tests over 12 months under laboratory conditions showed there was no consistent trend of endogenous control of germination periodicity in all three species tested. Variations were found in both the rate and total germination of P. glauca and P. contorta but only in the rate of germination of P. banksiana seeds; however, these variations were reduced by an extended photoperiod (16 hours) and almost completely eliminated by a moist chilling treatment.
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(pp. 149-167)
DNA Changes in Naturally and Artificially Aged Longleaf Pine (Pinus palustris Mill.) Seeds
E. L. Tolentino, Jr.,* W.W. Elam, and F. T. Bonner
Total seed DNA of naturally and artificially aged [accelerated aging test (AAT)] longleaf pine seeds was quantified and analyzed. No seeds germinated after 240 hr of AAT. Seeds in the forest condition lost germination after 180 days. Seeds in 4 °C conditions maintained 50% germination in three lots after 360 days. Storage at 30 °C caused significant decline in germination after 360 days.

For artificially aged seeds, DNA followed a low-high-low pattern coinciding respectively to the more viable and non-viable state with peaks at the intermediate aging regimes. Three lots showed increases in DNA after 90 days under forest conditions. Increases are probably due to hyperchromism. DNA was stable for seeds stored in 4 °C and 30 °C conditions. Gel electrophoresis revealed DNA fragmentation after 144 hours of AAT and 90 days of natural forest conditions. Incipient signs of fragmentation were observed in seeds stored at 30 °C.No fragmentation was observed in DNA of seeds stored at 4 °C. Big molecular weight fragments were possibly formed through the crosslinking of liquid peroxidation by-products and DNA for the following treatments: 240 or more hours of AAT, 4 °C after 360 days, and all seeds at 30 °C conditions.DNA fragmentation and crosslinking are presumed critical in non-germination of longleaf pine seeds.Regression analysis revealed differences in the rate of change of DNA in the two aging regimes. The use of AAT to examine aging mechanisms may not be reliable. The high seed moisture contents and the massive fungal infection in AAT are probable reasons.
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(pp. 168-182)
Germination Changes of Picea abies Seeds at Water-based Pretreatments
Eila Tillman-Sutela*, Anu Hilli and Anneli Kauppi
Water-based pretreatments are used to reduce the great variety in germination parameters of conifer seeds. However, treatment results of Norway spruce seeds are frequently unsatisfactory. The aim of this work was to study changes in the germination parameters of spruce seeds during the multiphased pretreatment chain and the impact of seed structures on these changes.

Furthermore we studied the effect of storage on germination indices of pretreated spruce seeds. Seeds were extracted using the ordinary method by sprinkling water on the cones or restricting that amount of water sprinkled on the cones. Cleaned and dried seeds were stored at –3 °C for three months prior to IDS-treatment. The changes in germination parameters were studied using germination tests and radiography. Seed structures were observed using SEM. In general germination parameters of seed lots increased during the pretreatment chain despite the opening of the seed coat, which occurred in fungi-infected seeds already at the extraction phase. The seed coat usually opened from the micropyle almost as far as the edge of the nucellar cap. The IDS-treatment succeeded well, and the germination parameters in the best fractions were 13–28% better than in the seed batches prior to IDS-treatment. The germination indices remained nearly unchanged during one year’s storage at –18 °C. This indicates that the large nucellar cap typical for spruce seeds protected the megagametophyte and the embryo during pretreatment phases and short-term storage. The results also pointed out how pretreatments should be improved to get them better adapted for spruce seeds.
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(pp. 183-190)
Effect of Liquid Nitrogen Storage on Seed Germination of 51 Tree Species
Jill R. Barbour* and Bernard R. Parresol
Two liquid nitrogen storage experiments were performed on 51 tree species. In experiment 1, seeds of 9westerntree species were placed in a liquid nitrogen tank for 3 time periods: 24 hours, 4 weeks, and 222 days. A corresponding control sample accompanied each treatment.

For three species,Calocedrus decurrens, Pinus jefferyi, and Pinus contorta, the germination percent was not significantly different from the controls in any of the liquid nitrogen treatments. Exposure to 24 hours of liquid nitrogen did not affect the germination percent for any of the 9 species compared to their controls. Two species, Abies x shastensis and Picea engelmannii, exhibited a significant negative response to 4 weeks exposure to liquid nitrogen. Four species Abies amabilis, Abies concolor, Pinus monticola, and Pseudotsuga menziesii, exhibited a significant positive response to the 222-day exposure to liquid nitrogen when compared with Control D. Experiment 2 examined the germination response to liquid nitrogen storage after a 24 hour exposure for 42 tree species. The germination percent for nine of the 42 species, Acer rubra, Celtis occidentalis, Lonicera tartarica, Malus prunifolia, Physiocarpus opulifiolius, Pinus banksiana, Pinus clausa, Pinus nigra, and Pinus rigida, was significantly affected by 24 hours exposure to liquid nitrogen. Liquid nitrogen exposure had a negative affect on germination for 7 species and a positive effect for 2 species, Pinus nigra, and Pinus rigida. Only 8 species had enough data to calculate the correlation coefficient between moisture content and germination after exposure to liquid nitrogen. Correlations were significant for 4 species. Two species, Abies fraseri and Liriodendron tulipifera had negative correlations; two species, Pinus ponderosa and Pinus taeda had positive correlations.
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