Responses to Drying of Recalcitrant Seeds of
Quercus nigra
L.
F. T. B O N N E R
USDA Forest Serice, Southern Forest Experiment Station, Starkille, Mississippi, USA
Received : 28 September 1995 Accepted : 2 February 1996
It has been suggested that rate of desiccation can influence the expression of recalcitrant behaviour in seeds, thus complicating the task of determining which seeds are truly recalcitrant. The objective of this study was to see if variable rates of desiccation influenced such behaviour in Quercus nigra L., a tree seed known to be recalcitrant.
Acorn moisture content, leachate conductivity, and germination were determined at various times during desiccation at three rates at 27 and 40°C. Moisture contents and germination decreased as the severity of desiccation increased. Leachate conductivity increased slightly but was not a sensitive indicator of loss of viability. The critical (lethal) moisture content for these acorns was 10–15 %, although rehydration within 48 h of reaching this level prevented death in about 25 % of the acorns. At 27°C any desiccation treatment that produced losses of 30–50 mg of moisture per g of acorn dry weight per day should be suitable as a test for recalcitrance in the genus. Apparent physiological or fungal damage at 40°C rules out the higher temperature for such a test.
#1996 Annals of Botany Company
Key words : Quercus nigraL., desiccation rate, temperature, recalcitrant, leachate conductivity, germination, viability.
I N T R O D U C T I O N
Seeds that can be safely dried to moisture contents between 6 and 10 % and stored successfully at low temperatures have been described as ‘ orthodox ’ in storage behaviour, while seeds that cannot be dried to these levels without losing viability have been described as ‘ recalcitrant ’ (Roberts, 1973). While these terms have been characterized as less than ideal choices to describe physiological behaviour of seeds (Berjak, Farrant and Pammenter, 1990), they have been widely accepted and are commonly used by all seed scientists today. Among economically important tree species, seed handling experiences have clearly identified which are orthodox and which are recalcitrant. Temperate recalcitrant species include Castanea (Pritchard and Manger, 1990), Quercus, Aesculus, and some Acer species (Bonner, 1990). Tropical recalcitrant tree species include some Hopea, Shorea, and Dipterocarpus (Tang and Tamari, 1973 ; Yap, 1986 ; Tompsett, 1987), and a number of economically important fruits, such as cocoa, mango, jackfruit, and durian (Chin and Roberts, 1980). For many lesser known species, however, classification of storage behaviour has not been made. The logical way to do this is to desiccate the seeds uniformly and periodically sample for moisture content and germination. If the seeds attain moisture contents of 10 % or less without losing viability, then one concludes that they are orthodox. If viability is lost long before seed moisture reaches this level, then one concludes that they are recalcitrant.
The protocol for a test for recalcitrance may not be so simple, however, when one considers what rate of drying and what drying temperature to use. Test conditions reported in the literature are typically described in terms of
relative humidity and temperature. Some temperatures reported are 2°C (Gosling, 1989), 15 and 20°C (Tompsett, 1984 ; Tompsett and Pritchard, 1993), 25°C (Farrant, Berjak and Pammenter, 1993), and 35°C (Becwar, Stanwood and Roos, 1982). Relative humidities are not always specified, but those reported have ranged from 15 % (Tompsett, 1984) to 50 % (Farrant et al., 1993). Tompsett (1984) concluded that viability loss was independent of drying method for nine species of Araucaria. Pritchard et al. (1995) reported similar results for excised embryos of seven species of Inga. Berjak et al. (1990) took the opposite position, based on studies with excised embryos and intact seeds of Aicennia marina at different stages of differentiation. Additional complications may be seed size (Tompsett and Pritchard, 1993), stage of seed development or maturity (Finch-Savage, 1992 ; Farrant et al., 1993 ; Tompsett and Pritchard, 1993), and chemical nature of the principal food reserves (Tompsett, 1984).
The objective of the present research was to examine loss of seed moisture and viability in intact seeds of the recalcitrant species Quercus nigra L. during desiccation at different temperatures and rates of moisture loss, and to evaluate these responses in relation to methodology for a standardized test for recalcitrance. A secondary objective was to relate loss of viability to electrolyte leakage from intact seeds as they were desiccated.
M A T E R I A L S A N D M E T H O D S
The acorns used in this study were collected from the ground after natural shedding from mature trees in Oktibbeha County, Mississipi (latitude 33°30«N, longitude
88°45«W) in Nov. and Dec. 1992. This collection procedure assured a supply of acorns at full dry weight and complete germination potential (Bonner, 1974). Rotten and insect-infested seeds were removed by water flotation and visual inspection. To minimize any influence of size, the acorns were graded with round-hole metal screens. All acorns used in the study were between 13 and 14 mm in diameter. The acorns were stored moist at 3°C in polyethylene bags with a wall thickness of 0±1 mm (Bonner and Vozzo, 1987) until used in the desiccation experiments.
There were three desiccation tests. In the first, acorns were dried at two temperatures : 27 (room temperature) and 40°C and at three drying rates at each temperature. These rates were designated as fast, medium, and slow. At 27°C medium drying was imposed by spreading the acorns in a single layer on the laboratory benchtop ; fast drying was similar, except that a desk-top fan constantly blew a stream of air over the acorns. Laboratory relative humidity (RH) was 55³5 %. Slow drying was imposed by placing acorns in desiccators at 95 % RH over a saturated salt solution of potassium chloride (KCl). At 40°C fast drying was imposed by spreading acorns in a single layer in a forced-draft oven set at 40°C. Medium drying was achieved in desiccators of Ca(NO
$)#\4H#O (40 % RH) in constant temperature incu-bators at 40°C. Slow drying was imposed in a similar manner in desiccators of KCl (83 % RH). All conditions were replicated three times, although benchtop drying replications took place at the same time at three different locations in the same room.
Acorn samples were drawn at different times during desiccation, depending on the rates of drying and results of preliminary trials. Three control samples were also drawn at the beginning of the treatments. The duration of desiccation varied from 62 h (fast drying at 40°C) to 35 d (slow drying at 27°C). Sampling was designed to produce seven data points for each treatment between full imbibition and death of the seeds. Each sample consisted of 60 acorns. Ten intact acorns were used for determination of moisture content on two subsamples of five each according to Bonner (1981). All moisture contents are reported as a percentage of fresh weight. The remaining 50 acorns were weighed and immersed intact in 100 ml distilled water in 250 ml beakers. The beakers were covered and left in the laboratory at 27°C for 24 h. Two beakers with 100 ml of distilled water, but no acorns, were treated similarly as blanks to determine the base conductivity of the water.
After 24 h, the acorns were strained from the water and conductivity of the water was measured in 100-ml beakers with a YSI Model 32 Conductance Meter. The mean conductivity of the blanks was subtracted from each sample reading, and the result was divided by initial weight of the acorns. Conductivities were expressed as microSiemens per gram of fresh weight (µS g−").
The acorns were prepared for germination tests according to official testing prescriptions of North America (As-sociation of Official Seed Analysts, 1993). In this procedure, the acorns were cut in half with pruning shears. The pericarps were removed from the distal halves of the acorns, which contain the embryonic axes, and these halves were placed on trays of moist cellulose wadding, cut side down.
The trays were placed in germinators for testing at a daily cycle of 30°C for 8 h with lights and 20°C for 16 h in the dark. In a slight departure from official testing rules, germination was counted three or four times per week for a maximum of 28 d, and acorns were considered germinated when their radicles had grown enough to contact the cellulose wadding (approx. 6 mm).
To evaluate desiccation treatments, linear regressions of percent germination (transformed to arcsine) on drying time in days were calculated for all six combinations of drying rate and temperature. Differences between regression slopes within each temperature were tested for significance ac-cording to Freese (1967). Percent germination was also regressed on acorn moisture contents for all conditions. Because these relationships were clearly not all linear, curve-fitting routines (Jandel Scientific, 1991) were used to model these responses. Acorn moisture contents were also regressed on drying time for each of the treatment combinations. These regression equations were then used to calculate the time required to reach 50 and 0 % of initial germination and the acorn moisture contents at this point.
Because leachate data of some samples suggested that values could have been inflated because of one or two rotten acorns, a second desiccation test was carried out with measurements made on individual acorns. Only one tem-perature (27°C) and two drying rates were used. Fast drying consisted of placing acorns on the benchtops with a fan blowing constantly. Slow drying consisted of placing the acorns in a desiccator with anhydrous CaSO% at a 2 : 1 desiccant :acorn volume ratio. At least five samples of 30 acorns each were drawn over a 10-d period. Ten acorns were used for two moisture determinations as in the first test, and 20 were weighed individually and placed in 30 ml test tubes with 20 ml of distilled water. Leachate con-ductivities and germination were measured as before.
The third desiccation test evaluated the ability of Q. nigra acorns to recover from near-lethal desiccation. Acorns were dried on a laboratory benchtop as before at 27°C for 6 d to produce a moisture content close to 15 %, a level suggested by preliminary testing to be the approximate lethal limit for this species. At that point, moisture content was determined on six samples as previously described, and the remaining acorns were sealed in polyethylene bags with 0±1 mm wall thickness at room temperature to prevent further des-iccation. Over the next 7 d, three samples of 50 acorns each were removed on six occasions, imbibed by soaking overnight at 27°C, and tested for germination as before.
R E S U L T S Moisture content
Acorn moisture loss over time during desiccation was generally linear, although the initial loss in the first few days (or hours) was sometimes at a slightly higher rate (Fig. 1). Expressing moisture loss as mg of water per g of acorn dry weight per day (mg gd. wt−"d−") (Table 1), shows that the
effects of desiccation treatments were not as uniform as was hoped for. At 27°C there was a much greater difference in rate of loss between medium and slow treatments (32s. 9)
35 35
5 0
Drying period (days)
Acorn moisture content (%)
25 10 30 25 20 15 5 10 15 20 30 35
Acorn moisture content (%)
10 30 25 20 15 Slow Medium Fast B A Slow Medium Fast
F. 1. Loss of acorn moisture content at 27 (B) and 40 (A)°C with three rates of desiccation. (E—E) slow ; (x— —x) medium ;
(y– – –y) fast. Brackets indicate³s.e. of the means.
T1 . Relatie rates of moisture loss(mg gd. wt−"d−") of intact acorns of Q. nigra at three drying rates and two temperatures. Rates were calculated from beginning and ending mean acorn moisture contents and expressed in mg of
moisture lost per g of acorn dry weight per day
Drying
Drying rates
temperatures (°C) Fast Medium Slow
27 48 32 9
40 61 17 8
than between fast and medium (48s. 32). At 40°C rates of loss were much greater between fast and medium (61s. 17) than between medium and slow (17s. 8). A comparison of regression slopes for moisture content on drying time within each temperature, however, showed that all were signifi-cantly different from each other (P!0±01).
10 100
0 35
Acorn moisture content (% of fresh weight)
Germination (arcsin transformation of %)
50 90 80 70 60 40 30 20 10 30 25 20 15 B 100
Germination (arcsin transformation of %)
50 90 80 70 60 40 30 20 10 A Slow Medium Fast Fast Slow Medium
F. 2. Relationship of germination (arcsin transformation) to acorn moisture content at 27 (B) and 40 (A)°C with the three rates of desiccation. y¯ab}x, where y is germination and x is acorn moisture
content. Symbols are as in Fig. 1.
Germination
As expected for recalcitrant seeds, germination decreased with desiccation at both drying temperatures. Rate of desiccation made little difference at 27°C, but differences among rates were much greater at 40°C (Fig. 2).
A comparison of the times required to reach 0 % and 50 % of initial germination also illustrate the differences among desiccation treatments very clearly. At medium drying, a faster loss of viability at 27 than at 40°C (Fig. 2) was expected because of the higher rate of moisture loss at 27°C : 32 mg gd. wt−"d−"as compared to 17 mg gd. wt−"d−"
for 40°C (Table 1). In the slow drying treatments, rates of moisture loss were practically the same (8 and 9 mg gd. wt.−"
d−"), yet germination was lost much more quickly at 40°C
than at 27°C : 7 d sooner for 50 % loss. This difference suggests a strong effect of temperature on acorn physiology apart from moisture loss. There was also extensive growth
T 2 . Acorn moisture contents (%) at the times that 50 and 0% of original germination was reached in all drying
treatments
Drying
Drying rates
temperature (°C) Fast Medium Slow 50 % germination 27 15±4 14±8 16±6 40 15±3 19±8 23±0 0 % germination 27 11±4 10±5 12±2 40 10±8 15±1 18±8 168 100
Time (hours) at critical moisture content (%)
Germination (%) 48 50 90 80 70 60 40 30 20 10 24 72 96 120 144 0
F. 3. Recovery of acorns after desiccation to 16 % moisture content at 27°C. Germination is shown after increasing intervals of time between final desiccation and rehydration. Vertical bars indicate³s.e.
of the means where larger than the symbols.
of microorganisms at 40°C, which is another likely cause of seed deterioration.
If one wished to produce a ‘ standard ’ test for re-calcitrance, the acorn moisture contents at the times of half and total loss of viability are of primary interest (Table 2). Loss of 50 % viability at moisture contents of 15±4 to 23±0 % and total loss of viability at moisture contents of 10±5 to 18±8 % are consistant with the recalcitrant nature of Q. nigra. At 27°C half of total viability was lost at approxi-mately the same moisture content (14±8–16±6 %) irrespective of drying rate, and the range of acorn moisture at the time of total viability loss was also small (10±5–12±2 %). These data suggest that any of the drying rates tested at 27°C should be equally good for a test for recalcitrance. The slow rate (9 mg gd. wt−"d−") may be physiologically suitable, but
35 d is too long for the purpose of a test. The higher rates of 32–48 mg gd. wt−" d−" should be more suitable. The
medium drying rate at 40°C (17 mg gd. wt−"d−") was also
suitable, but the micro-organism growth present at the higher temperature should probably rule it out as a standard test.
100
Drying period (days)
Germination (%) 2 50 90 80 70 60 40 30 20 10 1 3 4 5 6 0 10 0 Conductivity ( µ S g –1 ) 5 9 8 7 6 4 3 2 1
F. 4. Relationship of germination to leachate conductivity with fast desiccation at 27°C (E—E) and 40°C (x— —x). Vertical bars indicate³s.e. of the means. Solid bars show conductivity at 27°C ;
empty bars are 40°C.
100
Drying period (days)
Germination (%) 10 50 90 80 70 60 40 30 20 10 5 15 0 10 0 Conductivity ( µ S g –1 ) 8 6 4 2
F. 5. Relationship of germination to leachate conductivity with medium desiccation at 27 and 40°C. Symbols are the same as in
Fig. 4.
In the test designed to measure recovery ability following desiccation to critical levels at 27°C, acorn moisture content averaged 16±1(³0±3) % when desiccation was stopped, slightly higher than intended. Germination of immediately reimbibed acorns was 56 %, very close to what would be predicted from Table 2. Viability was quickly lost, however, if reimbibition was delayed (Fig. 3). Within 24 h, ger-mination had dropped below 30 %, and fell below 20 % before 72 h had elapsed.
Leachate conductiity
Leachate conductivity was poorly correlated with loss of viability in the bulk samples. At fast and medium rates of drying, only the medium rate at 40°C produced mean conductivities above 2µS g−"(Figs 4 and 5). Furthermore,
100
Drying period (days)
Germination (%) 10 50 90 80 70 60 40 30 20 10 5 15 20 25 30 0 25 0 Conductivity ( µ S g –1 ) 5 20 15 10 35
F. 6. Relationship of germination to leachate conductivity with slow desiccation at 27 and 40°C. Symbols are the same as in Fig. 4.
already near zero. Mean leachate conductivities reached much higher levels at the slow drying rate at both temperatures (Fig. 6), but these high values were recorded before any major loss of viability at 27°C and after total loss of viability at 40°C.
Measurements with individual acorns also yielded mixed results (Table 3). At the slow rate of drying, conductivity increased from 5±0 to 8±3µS g−" as germination dropped
from 92±5 to 68±3 %, then stayed the same as germination continued to decrease to a minimum of 21±7 %. At the faster rate of drying, conductivity changed very little as ger-mination fell to 13±3 %. A feature common to both bulk and single-acorn measurements was that leachate conductivity was always greater at the slower drying rates.
D I S C U S S I O N
Results of this study strongly suggest that fast to moderate rates of drying at 27°C for 10 d would be suitable for a test for recalcitrance for Quercus. Rates of moisture loss from
T3 . Germination, moisture content, and leachate conductiity fromQ. nigra acorns dried at two rates. Conductiity data were obtained on indiidual acorns and reportedalues(s.e.) are means for that sample period. (n¯40 at 0 days and 60 for
other days)
Slow drying Fast drying
Leachate Leachate
Drying Moisture Germination conductivity Moisture Germination conductivity time (d) content (%) (%) (µS g−") content (%) (%) (µS g−")
0 28±9 92±5 5±0 (0±2) 28±9 92±5 5±0 (0±2) 1 23±2 76±6 7±1 (0±4) 25±3 86±7 6±0 (0±3) 2 22±1 81±7 7±7 (0±7) — — — 3 — — — 21±8 66±7 6±4 (0±3) 4 20±0 68±3 8±3 (0±5) — — — 7 — — — 16±4 31±7 5±6 (0±3) 8 17±5 23±3 8±5 (0±5) — — — 9 — — — 15±7 13±3 5±6 (0±3) 10 16±8 21±7 8±4 (0±5) — — —
intact acorns of approximately 30–50 mg gd. wt−"d−"appear
to be suitable. The data support the conclusions of Tompsett (1984) with Araucaria and Pritchard et al. (1995) with Inga that viability loss is independent of desiccation method or rate, although the temperature effect at 40°C demonstrates that there are limits to this principle. Furthermore, one must always be aware that species differences are crucial and application of these results to any others must be approached with caution.
Results indicated that the higher temperature (40°C) was not suitable for a test for recalcitrant behaviour. There was an apparent detrimental effect of the 40°C temperature separate from desiccation. In accelerated aging studies with Q. nigra at 41°C and 100 % RH, (Blanche et al., 1989), 200 h of aging produced significant acorn deterioration. The decrease in germination was accompanied by a large increase in amino-nitrogen, probably the result of protein degra-dation. The 40°C drying treatments used in this study did not have the 100 % RH of accelerated aging, but the initial conditions were very similar. Effects of micro-organisms were probably a significant factor at 40°C.
Low temperatures, such as Gosling (1989) used, were not tested in the current study, but they would not seem to be suitable for a test for recalcitrance. The objective of Gosling’s test was not to identify recalcitrance, of course, and test conditions probably produced a whole array of different physiological conditions in the acorns. Q. robur acorns needed almost 30 weeks of drying at 2°C to reach moisture contents of 25 %, which is much too slow for a test for recalcitrance.
In the current study, 10–12 % moisture content of intact acorns was calculated to be the critical (lethal) minimum for Q. nigra acorns at 27°C. In earlier tests with this species (Agmata, 1982), 15 % was estimated as the lethal minimum. The difference between 10 and 15 % moisture is not very significant, however, because the last 50 % of viability was lost so rapidly (Fig. 2). Moisture contents of the embryonic axes may be even more important than those of the intact seeds. Unpublished work in our laboratory with Q. nigra suggests that when intact acorn moisture contents are about 15 %, the embryonic axes are actually still hydrated at a
much higher level (30 %). Similar observations have been reported by Berjak et al. (1990) and Tompsett and Pritchard (1993) for other recalcitrant species. It is important to know that when the lethal minimum moisture content is ap-proached, rehydration within a few hours can revive the seeds and prevent total loss of viability. This condition is probably also a function of the ability of the seed coat or other covering structures to restrict movement of moisture to or from the axes.
Tompsett (1984) and Tompsett and Pritchard (1993) identified seed size, stage of development, and principal food reserves as other factors affecting rates of internal desiccation of seeds. Size and stage of development were essentially eliminated as contributing factors in the current study by precautions taken during collection and prep-aration of the acorns. Among Quercus species, the nature of stored food reserves are surely important. The subgenus Erythrobalanus, which includes Q. nigra, has fat as the major acorn food reserve, while food reserves of acorns of the subgenus Lepidobalanus are primarily carbohydrates (Bonner and Vozzo, 1987). Splitting of the pericarp is a major factor in desiccation of Quercus. The dimensional shrinkage of the large cotyledons will often split the pericarp, especially in rapid drying conditions. This leads to much more rapid desiccation and quicker seed death. Species with food reserves that are primarily carbohydrates desiccate quite rapidly and are much more likely to have split pericarps than species with fats as the primary food reserves. The lack of correlation between leachate conductivity and seed death in this study was surprising. The test has proven useful with a number of agricultural and tree species (Perry 1981 ; Bonner, 1991 ; Bonner and Agmata-Paliwal, 1992), but all were orthodox in storage behaviour. Becwar et al. (1982) obtained good correlation between leachate con-ductivities and loss of germination for the recalcitrant seeds of Acer saccharinum, but only when the pericarp was removed before soaking. Leachate conductivities from intact A. saccharinum fruits increased very little as the seeds died from desiccation. Likewise, Sun, Irvine and Leopold (1994) reported good correlation of viability loss with leachate conductivity for Q. rubra, but with squares of cotyledon tissue, not intact acorns.The presence of vascular openings in the cup scar area of Quercus pericarps provides good pathways for exchange of liquids and solutes (Bonner, 1968), however, so the same situation may not occur with this genus. The initial increase in conductivity before significant desiccation was probably due to electrolytes within and beneath the pericarps and may not be connected to loss of viability at all.
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