Seed lot, nursery, and bud dormancy effects on root electrolyte leakage of Douglas-fir ( Pseudotsuga menziesii ) seedlings

Seed lot, nursery, and bud dormancy effects onroot electrolyte leakage of Douglas-fir(Pseudotsuga menziesii) seedlings

Raymund S. Folk, Steven C. Grossnickle, Paige Axelrood, and Dave Trotter

Abstract: The effects of seed lot, nursery culture, and seedling bud dormancy status on root electrolyte leakage (REL)of Douglas-fir (Pseudotsuga menziesii(Mirb.) Franco) seedlings were assessed to determine if these factors should beconsidered when interpreting REL for seedling quality. The relationships of REL to survival, net photosynthesis (Pn),stomatal conductance (gwv), mid-day shoot water potential (Ψmid), root growth capacity (RGC), and relative heightgrowth were determined for each factor. Nursery culture had no effect on the relationship between REL and all othermeasured attributes. Seed lot affected the relationship between REL andPn, Ψmid, and survival. However, critical REL(i.e., lowest value associated with detectable root damage) and PS80 REL (i.e., level associated with an 80% probabilityfor survival) were similar between seed lots. Bud dormancy status affected the relationship between REL and survival,RGC, and relative height growth. Control levels of REL, critical REL, and PS80 REL decreased as the number of daysrequired for 50% terminal bud break declined. Thus, terminal bud dormancy status must be known before REL can beused to assess seedling quality. If the bud dormancy status of Douglas-fir populations is known, then critical and PS80

REL levels may be useful as indices of root damage.

Résumé: Afin de déterminer s’il fallait tenir compte de certains facteurs lors de l’interprétation de la libération desélectrolytes des racines (LER) pour déterminer la qualité des semis, les auteurs ont évalué l’influence du lot desemences, de la méthode culturale et de l’état de dormance des bourgeons de semis de sapin de Douglas (Pseudotsugamenziesii(Mirb.) Franco) sur la LER. Les relations entre la LER et la survie, la photosynthèse nette (Pn), laconductance stomatale (gwv), le potentiel hydrique des tiges au milieu de la journée (Ψmid), la capacité de croissanceracinaire (CCR) et la croissance en hauteur relative ont été établies pour chaque facteur. La méthode culturale n’a pasinfluencé la relation entre la LER et les autres variables. Le lot de semences a influencé les relations entre la LER etPn, Ψmid et la survie. Cependant, la LER critique (c.-à-d. la valeur la plus faible associée à la possibilité de détecterdes dommages aux racines) et la LER PS80 (c.-à-d. la valeur correspondant à une probabilité de survie de 80%) étaientsimilaires entre les lots de semence. L’état de dormance des bourgeons a influencé la relation entre la LER et la survie,la CCR et la croissance en hauteur relative. Les valeurs témoins de la LER, de la LER critique et de la LER PS80

diminuaient à mesure que le nombre de jours requis pour un débourrement de 50% des bourgeons terminaux diminuait.Par conséquent, l’état de dormance des bourgeons terminaux doit être connu avant que la LER puisse être utilisée pourdéterminer la qualité des semis. Si l’état de dormance des bourgeons des populations de sapin de Douglas est connu,alors les valeurs de la LER critique et de la LER PS80 peuvent être utiles comme indices de dommages racinaires.

[Traduit par la Rédaction] Folk et al. 1281

Introduction

Damage to the root systems of coniferous nursery stock isa major concern to both seedling producers and forest man-agers. Studies have shown that root damage can result in re-duced field performance and survival of newly plantedconifer seedlings (Coutts 1981; Lindström 1986a, 1986b;Tabbush 1986; Bigras 1997). Seedling root systems can bedamaged in the nursery by various abiotic agents, such asfreezing temperatures (Bigras and D’Aoust 1992; Coleman

et al. 1992; Simpson 1993; Colombo 1994; Sutinen et al.1996), high temperatures (Langerud et al. 1991; DeYoe etal. 1986), root desiccation (Feret et al. 1985; Deans et al.1990; McKay and White 1997), improper frozen storage(Mason and McKay 1990; McKay and Mason 1991; McKay1992, 1993; Lindström and Mattsson 1994), rough handling(Kauppi 1984; Tabbush 1986; Nelson 1991; McKay et al.1993; Paterson 1993), and biotic agents, such as pathogenicFusarium and Cylindrocarpon species (Dumroese et al.1993). Damage may not always be visually apparent, mak-ing it important that conifer stock be routinely tested.

Determining root damage for stock quality assessmentpurposes requires two important tools: (i) a test that can de-tect and quantify damage and (ii ) a model that facilitates in-terpretation of test results for forecasting seedling survivalpotential and performance potential (defined as inherent per-formance potential by Grossnickle and Folk (1993) and Folkand Grossnickle (1997)). Root electrolyte leakage (REL) hasbeen used as a test to detect and quantify root damage in

Can. J. For. Res.29: 1269–1281 (1999) © 1999 NRC Canada

1269

Received September 15, 1998. Accepted March 25, 1999.

R.S. Folk,1 S.C. Grossnickle, and P. Axelrood.ForestBiotechnology Centre, B.C. Research Inc., 3650 WesbrookMall, Vancouver, BC V6S 2L2, Canada.D. Trotter. Nursery Extension Service, Ministry of Forests,14275 - 96th Avenue, Surrey, BC V3V 7Z2, Canada.

1Corresponding author. e-mail: [email protected]

I:\cjfr\cjfr29\cjfr-08\X99-084.vpThursday, September 09, 1999 2:31:55 PM

Color profile: DisabledComposite Default screen

conifer seedlings following cold storage (Mason and McKay1990; McKay 1992, 1993), after rough handling and desic-cation (McKay et al. 1993; McKay and White 1997), and af-ter exposure to freezing temperatures (Bigras and D’Aoust1992; Coleman et al. 1992; McKay 1993; Bigras and Calmé1994; Colombo 1994; Bigras 1997). In all these studies,REL increased as apparent root damage increased.

Other studies have shown that shifts in REL (i.e., compar-isons of two or more measures before or after root damage)are a good index to quantify changes in root system integrity(McKay et al. 1993; Bigras 1997). Such shifts in REL havebeen related to changes in seedling survival and perfor-mance potential (i.e., height growth under favourable grow-ing conditions) (McKay and Mason 1991; McKay 1992;McKay et al. 1993; Bigras 1997). However, single measuresof REL may be less informative of root system integritysince REL may vary due to factors other than root damage.McKay (1993, 1994) showed that REL decreased inDouglas-fir (Pseudotsuga menziesii(Mirb.) Franco) seed-lings from October to January. Such a change in REL duringfall acclimation may be related to changes in root systemtolerance to lifting (McKay 1994) but may also be relatedsolely to changes in root properties that alter leakage pat-terns during fall acclimation. Thus, REL of undamaged rootsystems may also change during the fall acclimation period.Some studies have also shown REL to differ between vari-ous conifer species (McKay 1992, 1993), while others havesuggested that REL may vary because of nursery environ-ment and cultural practices (McKay 1992). Furthermore,root freezing tolerance has been shown to vary among seedsources of a number of conifer species (Lindström and Ny-ström 1987), suggesting that REL may also vary with seedsource. Currently, it is unclear whether the relationship be-tween REL and seedling quality changes when populationschange in bud dormancy status or are produced from differ-ent nursery practices and seed sources.

Few attempts have been made to quantify the effect ofseed source, nursery culture, and dormancy factors on REL.Differences caused by these factors must be quantified andmodels developed that account for their effects on REL andits relationship to seedling quality. Such models would facil-itate better forecasts of seedling survival potential and per-formance potential for use in operational seedling qualityprograms. This study determined the effects of seed lot,nursery culture, and bud dormancy status on the relationshipbetween REL and seedling quality for 1-year-old Douglas-fircontainer seedlings at the time of fall lifting for frozen storage.

Materials and methods

Two experiments were conducted to evaluate the effect of vari-ous factors on the relationship between REL and seedling quality.The assessment of nursery culture and seed lot effects (experimentone) was conducted during mid-December 1994, and the assess-ment of bud dormancy (experiment two), during October 1995through January 1996.

Plant material and tested factors

Experiment one: effect of nursery culture and seed lotTwo coastal Douglas-fir (Pseudotsuga menziesii(Mirb.) Franco)

seed lots (S) (SL6514, 612 m elevation, 49°15′N, 123°53′W; and

SL6282, 225 m elevation, 50°30′N, 125°30′W) were grown at twooperational nurseries (N) located in southwestern British Columbiaduring 1994. This resulted in four nursery – seed lot combinationsor two seed lot and two nursery seedling groups. Seedlings weregrown at both nurseries as a 1+0 spring-plant stock-type (i.e., grown1 year in the nursery, lifted, and frozen-stored in December forspring planting) in 415B Styroblock® containers (530 seedlings/mand 93 cm3 root volume). Each nursery grew seedlings under dif-ferent cultural regimes, but both seed lots were grown under simi-lar regimes within each nursery (Table 1). Seedlings were removedfrom the nurseries in mid-December 1994 at a time when Douglas-fir would normally be lifted for placement into frozen storage.

To ensure morphological uniformity, 50 seedlings were mea-sured for height and diameter to determine the mean and standarddeviation of each nursery – seed lot combination. Seedlings (n =300) were then selected from each combination that were within±0.5SDs from the mean height and diameter (Table 1).

Experiment two: effect of bud dormancy statusSeedlings (SL6514, as described for experiment one) were sown

on March 21, 1995, grown under standard operational cultural pro-cedures for producing a spring-plant 1+0 container (415B) stocktype in nursery 2 (Table 1) but received two short-day treatments;the first on July 14 and the second on August 15, 1995. Seedlingswere removed from the nursery in early October 1995 and main-tained outdoors at BC Research Inc. stock quality testing labora-tory. Seedlings were screened to ensure morphological uniformity,as described for experiment one (Table 1), then divided into fourgroups, each to be tested at a different time (October 15, Novem-ber 15, December 15, 1995, and January 15, 1996) or bud dor-mancy stage. Stage of bud dormancy was determined by thepresence of a terminal bud and by the number of days for 50% ofseedlings (n = 24) to break bud (DBB50) under optimum environ-mental conditions (controlled environment room at 40% relativehumidity, 22 ± 1.5°C air temperature, 18 h light : 6 h dark photo-period at 600µmol·m–2·s–1) (Ritchie 1984). Thus, one level ofDBB50 was attributed to each sample month.

Development of root damageFor both experiments, seedlings were kept at the BC Research

Inc. outdoor nursery compound, where they were watered and fer-tilized biweekly (75 ppm N of Peters® Conifer Finisher Special,4:25:35 N:P:K) until needed for measurement. Before the studycommenced, seedlings in both experiments appeared healthy, androot damage was not visually evident. Seedlings were broughtfrom the nursery and placed in large plastic pots (to protect theroot systems from drying out) and allowed to acclimate to theabove-described controlled environment room for a 48-h period.Seedlings, with root plugs intact, were placed in an aerated hydro-ponic system at a water temperature of 57 ± 0.5°C to achieve con-trolled levels of root damage. This temperature was determinedduring preliminary trials. Root systems were exposed to a series ofincubation times (heat times) at 57°C to create a range of rootdamage conditions. Heat times wereT = 0, 5, 8, 12, 26, 45, and60 min for experiment one, andT = 0, 2, 6, 12, 20, and 45 min forexperiment two. Twenty-four seedlings were used for each heattime (i.e., 168 for each nursery – seed lot combination in experi-ment one and 144 for each stage of bud dormancy in experimenttwo). After removal of seedlings from the heated hydroponic sys-tem, roots were placed in water (22°C) for 5 min to cool and pre-vent further damage. The root plugs remained relatively intactduring the heating treatment.

Experimental designA randomly selected subset of seedlings from each heat time

(n = 8 in experiment one andn = 12 in experiment two) and test

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group was used for root electrolyte leakage (REL) measurements(described below). Afterwards, seedlings from each experiment(n = 24 per heat time for each nursery, seed lot, and stage of dor-mancy) were potted in 4-L pots (three seedlings per pot) with agrowing medium of sifted peat. Seedlings measured for REL wereindividually tagged, and care was taken to ensure that one of threeseedlings in each pot was a tagged seedling. Seedlings were placedon light tables in the above-described controlled environment roomin a randomized design containing all of the heat treatment expo-sure times and test groups, and kept well watered. Each taggedseedling was measured for shoot water potential, gas exchange,and root growth capacity (described below) so that each attributewas related to results obtained from other testing protocols.

Measured attributes

Root electrolyte leakageRoot electrolyte leakage (REL) was determined after application

of root heat treatments and before seedlings were potted. Rootsamples were collected from eight seedlings in experiment one and12 seedlings in experiment two from each heat time. This resultedin a total of 56 seedlings per nursery – seed lot combination in ex-periment one and 72 seedlings per stage of bud dormancy in exper-iment two. Since the root plug remained relatively intact during theheat treatment, a section (1.0 × 3.0 cm, 0.5 cm depth) of roots andmedium could be excised from one side of the root plug. Eachsample was no more than 10% of the whole root system. Roots>2 mm in diameter were removed from the sample, while the re-maining roots (150–300 mg fresh mass) were washed and rinsed indeionized water and transferred to glass culture tubes containing16 mL of deionized water. Tubes were then stoppered and placedon a 100-rpm shaker at 20°C for 20 h. Conductivity of the solutionin each tube was measured after incubation (leakage conductivity,Cleak). Tubes were then placed in a 95°C water bath for 30 min toinduce maximum tissue injury and conductivity was remeasuredafter samples had cooled to 20°C (total conductivity,Ctotal).

Measured conductivity levels were use to calculate root electro-lyte leakage (REL) (i.e., relative conductivity of roots) using thefollowing formula: REL = Cleak/Ctotal. Examination ofCtotal indi-cated that total electrolytes remained relatively stable for each heattime and decreased only when severe root damage occurred (i.e.,survival <15%) (unpublished results). Thus, some electrolytesleaked out of the severely damaged root systems before root sys-tems could be measured by the root electrolyte leakage test. Conse-quently, REL levels for severely damaged roots were found to belower than those reported elsewhere (McKay and Mason 1991;McKay 1992; Bigras 1997). Premeasurement loss of electrolyteswas avoided in these studies because REL was determined imme-diately after damage and seedlings were not subjected to irrigation.

The underestimation of REL for severely damaged seedlings inthis study was not an important factor for two reasons. First, thispremeasurement leakage was indicative of the electrolyte leakagethat may occur during irrigation of damaged seedlings in the nurs-ery and before seedlings are measured for possible root damage inthe laboratory. Thus, REL in this study may be more indicative oflevels associated with severely damaged seedlings under opera-tional conditions. Second, this study is concerned with the range ofREL associated with critical changes in seedling quality (i.e., RELlevels distinguishing between damaged and healthy root systems,and changes in seedling performance). In this range (<0.35),premeasurement leakage was not detectable.

Shoot water potential (experiment one)Mid-day shoot water potential (Ψmid) was determined after

7 days in a controlled environment room on seedlings previouslymeasured for REL (n = 8 per heat time and nursery – seed lot com-bination) and heat treated for 0, 12, 26, and 45 min. Seedlings

© 1999 NRC Canada

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from the 60 min heat time were dead and excluded from measure-ment. Measurements ofΨmid were conducted 5–7 h after the startof the photoperiod. Lateral branches were excised, and their shootwater potential was determined using a pressure chamber (SoilMoisture Corp. model 3005).

Gas exchange (experiment one)Net photosynthesis (Pn) and stomatal conductance (gwv) were

determined with a LI-6200 (LI-COR Inc., Lincoln, Neb.) gasexchange system and a 0.25-L chamber (LI-6200-13) on eightseedlings per heat time and nursery – seed lot combination. Mea-surements were conducted 2 h after the start of the photoperiod onthe same day thatΨmid measurements were taken and only on seed-lings measured forΨmid and REL. At the end of the trial (8 weeks),or sooner if seedling mortality occurred, needles used for gas ex-change measurement were collected, and their surface area was de-termined with LI-3000 area meter. Gas exchange measurementswere recalculated to represent total needle surface area.

Root growth capacity (experiment one and two)Root growth capacity (RGC) was determined by counting the

number of new white roots≥0.5 cm in length after 14 days in theabove described environmental conditions (Ritchie 1985; Folk andGrossnickle 1997). Only seedlings (n = 24) in heat times with sur-viving seedlings at 14 days (T ≤ 26 min for experiment one) andwith terminal bud set (T < 20 min for experiment two) were mea-sured. The RGC of seedlings previously measured for REL werenoted separately for comparison with nonsampled seedlings to de-termine the effect of the REL sample procedure (i.e., 10% root re-moval) on subsequent seedling performance. No differences weredetected (p > 0.05, t test) between the RGC of REL sampled andnon-REL sampled seedlings.

Relative height growth (experiment two)Seedlings were grown in the above-described controlled-

environment room after application of root temperature treatments.Relative height growth was determined by measuring the length ofthe new shoot leader and the length of the preexisting shoot andcalculated as the ratio of new shoot leader length to the initialshoot length. Since bud set was a prerequisite to subsequent leaderelongation, relative height growth could only be determined forseedlings that had formed a terminal bud at the time of the heattreatment (i.e., October excluded). After 8 weeks (i.e., at the end ofshoot elongation), relative height growth was measured on surviv-ing seedlings (n = 24 or less per heat time,T < 20 min).

Survival (experiment one and two)Eight weeks after application of the heat treatments, each seed-

ling used for REL measurement was categorized as either alive (as-signed a value of 1) or dead (0) (n = 8 in experiment one andn =12 in experiment two). Thus, the survival of each seedling previ-ously sampled for REL was determined. Percent survival was alsodetermined and based on all seedlings (n = 24) in each heat-timetreatment group, nursery – seed lot combination and stage of buddormancy. Percent survival was used to compare actual levels withthose predicted by root damage models (described below). Survivalrates were similar (within 8%) between REL-sampled and non-REL-sampled seedlings, within each heat time, nursery – seed lotcombination, and stage of bud dormancy. This indicated that mea-sured performance attributes of REL-sampled seedlings were alsorepresentative of non-REL-sampled seedlings.

Data analysis

REL response to tested factorsData analysis was conducted as a 2 × 2 × 7factorial design for

experiment one. Analysis of variance assessed the random effects

of nursery (N at two levels), and seed lot (S at two levels), and thefixed effect of heat-time (T at seven levels) on REL, where eachseedling represented an individual REL level. Appropriate MSEterms were used to determine significance of fixed (T) and random(N or S) factors (Wilkinson et al. 1996).

For experiment two, data analysis was conducted as a 4 × 6 fac-torial design. Analysis of variance assessed the fixed effects of ter-minal bud dormancy status (DBB50 at four levels) and heat time (Tat six levels) on REL, where each seedling represented an individ-ual REL level. For this analysis, the categorical variable of DBB50was set to 100 for the first measurement period (October 15) whenseedlings had not yet initiated a terminal bud.

For both experiments, a two step analysis strategy for factorialexperiments with a control was employed (Stehman and Meredith1995). For the heat-time factor,T = 0 min was the control leveland T > 0 min were the active levels (Stehman and Meredith1995). The heat treatment was used as a tool to develop a range ofroot damage or REL levels, and thus, the specific response of RELto heat time (T) and potential interactions of seed lot, nursery, andDBB50 with heat time, were not deemed to be important for thestudy of the response of REL to seed lot, nursery, and DBB50.

Mean and standard errors of REL were calculated for each heattime and treatment combination. Critical REL was defined as thelowest level of REL significantly different from the heat-time con-trol level (T = 0 min). Critical REL was calculated for each nurs-ery – seed lot combination, and DBB50 level, using a one-wayanalysis of variance (factor = heat time), and determining Dun-nett’s critical significance level (d′) (DeHayes and Williams 1989):

d D′ = 2MSE df/

where df is the degree of freedom for the tested factor, MSE is themean square of the error, andD is a value extracted from aDunnett’s table. At test (p ≤ 0.05) was used to determine differ-ences between nursery or seed lot main effects, and Tukey’s meanseparation test was used to determine differences in DBB50 RELmeans, for each heat-time level.

REL relationship to performance potentialAnalysis of covariance, including a test for homogeneity of

slopes (covariate interactions), determined differences in the rela-tionships (slopes) ofPn, gwv, Ψmid, and RGC (performance poten-tial) to REL (covariate) for each seed lot and nursery factorcombination in experiment one. This was followed by linear re-gression analysis to examine these relationships. Regression analy-sis was conducted on individual seedling measurements (n = 32 forPn, gwv, andΨmid; andn = 40 for RGC) for each nursery – seed lotcombination.

For experiment two, means and SEs of RGC and relative heightgrowth were calculated for each heat time and stage of bud dor-mancy (n = 24). Pearson’s correlation coefficient and associatedBonferroni probabilities determined the relationship of the heat-time means of REL with the heat-time means of RGC and relativeheight growth. Correlations were determined for each seedlinggroup with bud set at the time of heat treatment (i.e., for three lev-els of DBB50, November, December, and January).

REL relationship to survival potentialThe effect of nursery culture, seed lot, and bud dormancy status

(DBB50)) on survival were examined using multivariate logisticanalysis and maximum likelihood estimation. Analysis was con-ducted on seedlings (n = 56 for each nursery – seed lot combina-tion, or n = 112 from each seed lot or nursery in experiment one,and n = 72 for each level of bud dormancy in experiment two)measured for REL and categorized as alive or dead after 8 weeks(i.e., each seedling associated with a single REL value and survivalcategory: alive = 1, or dead = 0). All possible interactions were

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included in the first logistic analysis and then excluded from suc-cessive analyses if not significant (p > 0.05).

Based on the above analysis, logistic regression models weredeveloped to examine the relationships between REL and the prob-ability for survival (PS) for each significant (p < 0.05) factor or in-teraction. These models were used to determine the REL associatedwith an 80% probability of survival (PS80). The PS80 was chosenas a benchmark for stock adjudication to allow for a 20% probabil-ity of mortality associated with factors other than root damage.

Results

Response to heat treatmentsMean REL increased (Tables 2 and 3) withT, indicating

that the heat treatments produced a range of REL (T, p ≤0.001) for each nursery – seed lot combination and level ofbud dormancy. Differences in REL were detected betweennursery – seed lot combinations and stages of bud dormancy,within specific heat times. Control or initial levels (T =0 min) of REL also varied between 0.087 and 0.131 in ex-periment one (Table 2), and 0.098 and 0.146 in experimenttwo (Table 3). Differences in REL between nursery – seedlot combinations, or stages of bud dormancy, within activelevels (T > 0 min; see Stehman and Meredith (1995)) of heattime were not the result of differences in initial levels (T =0 min), as indicated by interaction contrasts (i.e., heat-time ×nursery, seed lot, or stage of dormancy) between initial andactive levels (Stehman and Meredith 1995). Thus, differ-ences in the REL response to the heat treatment, amongnursery – seed lot combinations or stages of bud dormancy,occurred during the heating process.

Experiment one: nursery and seed lot effects

REL and performance potentialRoot electrolyte leakage was closely related to perfor-

mance potential for all nursery – seed lot combinations. As

REL increased,Pn, gwv, Ψmid (Fig. 1) and RGC (Fig. 2) de-creased linearly (covariate REL,p ≤ 0.001). Nursery culturehad no effect (N, p > 0.05:N × REL, p > 0.05) on these fourattributes, but mean levels ofPn, gwv, andΨmid were differ-ent (p ≤ 0.001) for the two seed lots. The rate of change inPn (S × REL, p = 0.057;S × N × REL, p = 0.929) andgwv(S× REL, p = 0.409;S× N × REL, p = 0.661) with changesin REL were similar between the two seed lots, but theΨmidresponse was different (S × REL; p = 0.005) for each seedlot. Linear regression models between REL andΨmid(Fig. 1) indicated thatΨmid decreased at a slower rate perunit of REL in seed lot SL6514, compared to SL6282 (i.e.,slopes –2.48 and –5.36, respectively). This accounted forgreater (p ≤ 0.001) meanPn and gwv over a range of REL,for seed lot SL6514 compared with SL6282 (Fig. 1).

The relationship between RGC and REL was different foreach nursery – seed lot combination (S × N × REL; p =0.05), but mean RGC was not different for either factor (SorN; p > 0.05). In general, RGC had a poor relationship toREL for each nursery – seed lot combination (Fig. 2). Linearregression models had very steep slopes, but regression co-efficients were low (r2 ≤ 0.32), and low levels of REL(<0.15) were associated with RGC levels ranging from 0 to>45 roots.

REL and survival potentialSeedling survival was related to changes in REL (or root

damage) (REL,p ≤ 0.001). Both nursery culture and seed lotfactors had no direct effect (i.e., logistic analysis indicatedS, p = 0.189;N, p = 0.308) on the overall probability of aseedling surviving (i.e., PS = 1) across the range of mea-sured REL, but the PS and REL relationship was differentfor each seed lot (S × REL; p = 0.045). Logistic models ofthe main effects of seed lot indicated a greater change in PSper unit change of REL for both nursery populations ofseed lot SL6282, compared with SL6514 (Fig. 3). This was

© 1999 NRC Canada

Folk et al. 1273

Heat treatment(min at 57°C)

Seed lot SL6282 Seed lot SL6514

REL Survival (%) REL Survival (%)

Nursery 10 0.108 (0.008)ab 100 0.110 (0.007)ab 1005 0.163 (0.022)b 100 0.201 (0.012)ab 1008 0.126 (0.010)b 95 0.193 (0.019)a 90

12 0.164 (0.012)ab 100 0.197 (0.024)ab 10026 0.216 (0.026)a 90 0.209 (0.016)a 6545 0.328 (0.021)b 0 0.462 (0.032)a 060 0.459 (0.043)c 0 0.554 (0.036)bc 0Nursery 20 0.087 (0.005)b 100 0.131 (0.009)a 1005 0.155 (0.010)b 100 0.223 (0.013)a 1008 0.179 (0.017)ab 100 0.188 (0.011)a 100

12 0.162 (0.006)b 100 0.235 (0.027)a 9526 0.186 (0.015)a 95 0.237 (0.014)a 10045 0.373 (0.025)ab 0 0.462 (0.026)a 060 0.650 (0.031)ab 0 0.692 (0.027)a 0

Note: Values are means with SE given in parentheses. Values with different letters are significantlydifferent (Tukey’s mean separation test,α = 0.05) than others within each heat-exposure time.

Table 2. Root electrolyte leakage (REL) and survival of two Douglas-fir seed lotsproduced at two nurseries and exposed to 57°C root temperature for various lengths oftime in experiment one.



© 1999 NRC Canada

1274 Can. J. For. Res. Vol. 29, 1999

evident in the slopes of the linear function embedded in thelogistic models developed for each nursery – seed lot combi-nation (Fig. 3), and for each seed lot (Fig. 4).

Experiment two: effects of bud dormancy status

Terminal bud dormancy statusDuring the first measurement period (October 15), a ter-

minal bud was not yet visible on most seedlings. By the nextmeasurement period (November 15), all seedlings had set aterminal bud and had a DBB50 of 29 days. For the final twomeasurement periods (December 15 and January 15), DBB50decreased to 18 and 16 days, respectively.

REL and performance potentialRoot growth capacity and relative height growth indicatedH

ea

ttr

ea

tme

nt

(min

at

57

°C)

No

term

ina

lb

ud

DB

B 50

=2

9D

BB 5

0=

18

DB

B 50

=1

6

RE

LS

urv

iva

l(%

)R

EL

Su

rviv

al

(%)

RE

LS

urv

iva

l(%

)R

EL

Su

rviv

al

(%)

00

.12

9(0

.00

7)ab

10

00

.14

6(0

.00

9)a

10

00

.09

8(0

.00

5)c

10

00

.10

2(0

.00

8)bc

96

20

.29

6(0

.01

5)a

92

0.2

96

(0.0

14

)a9

60

.22

2(0

.03

1)ab

78

0.1

80

(0.0

16

)b8

36

0.2

50

(0.0

19

)b8

70

.36

4(0

.02

0)a

96

0.1

75

(0.0

11)c

92

0.2

23

(0.0

09

)bc

37

12

0.3

20

(0.0

16

)b7

10

.51

6(0

.01

7)a

12

0.3

53

(0.0

20

)b1

40

.25

1(0

.00

9)c

37

20

0.5

02

(0.0

43

)b8

0.6

26

(0.0

24

)a0

0.5

43

(0.0

30

)ab

00

.49

4(0

.00

9)b

04

50

.73

1(0

.01

7)b

00

.69

3(0

.02

0)a

00

.80

4(0

.01

2)a

00

.46

0(0

.01

9)b

0

Not

e:S

eedl

ings

wer

eex

pose

dto

57°C

root

tem

pera

ture

for

vario

usle

ngth

sof

time.

Val

ues

are

mea

nsw

ithS

Egi

ven

inpa

rent

hese

s.V

alue

sw

ithdi

ffere

ntle

tter

sac

ross

all

bud

dorm

ancy

stag

esar

esi

gnifi

cant

lydi

ffere

nt(T

ukey

’sm

ean

sepa

ratio

nte

st,

α=

0.05

)th

anot

hers

with

inea

chhe

atex

posu

retim

e.

Tabl

e3.

Ro

ot

ele

ctro

lyte

lea

kag

e(R

EL

)a

nd

surv

iva

lo

fD

ou

gla

s-fir

see

dlin

gs

(SL

65

14

)w

ithn

ote

rmin

al

bu

d(O

cto

be

r)o

rre

qu

irin

g2

9(N

ove

mb

er)

,1

8(D

ece

mb

er)

,o

r1

6(J

an

ua

ry)

da

ysfo

r5

0%

of

the

po

pu

latio

nto

bre

ak

the

irte

rmin

al

bu

ds

(DB

B5

0)in

exp

eri

me

nt

2.

0

1

2

3

4

5

6

Pn

(µm

ol·

m-2·s

-1)

y = 5.25 - 12.32x; r2 = 0.67SL6282

SL6514 y = 5.39 - 9.42x; r2 = 0.66

10

20

30

40

50

60

gw

v(m

mo

l·m

-2·s

-1)

y = 54 - 114x; r2 = 0.67SL6282

SL6514 y = 63 - 113x; r2 = 0.67

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Root Electrolyte Leakage

-2.5

-2.0

-1.5

-1.0

-0.5

ψ mid

(MP

a)

y = -0.211 - 5.36x; r2 = 0.66

y = -0.531 - 2.48x; r2 = 0.38

SL6282

SL6514

Fig. 1. Relationship of root electrolyte leakage with netphotosynthesis (Pn), stomatal conductance (gwv), and shoot waterpotential (Ψmid) of Douglas-fir seedlings in experiment one. Eachpoint represents a seedling measurement. Linear regressionmodels are given for each seed lot.



that the relationship between REL and seedling performancepotential was variable. As REL increased, RGC and relativeheight growth (Fig. 5) decreased in each stage of bud dor-mancy (DBB50 = 29, 18, and 16). Trends in the means ofthese parameters also indicated that RGC and relative heightgrowth had greater decreases per unit of REL as DBB50decreased. Thus, performance potential tended to decreasewith increasing REL, and this decrease occurred over asmaller range in REL as bud dormancy status changed dur-ing fall acclimation (as DBB50 decreased).

REL and survival potentialBoth the stage of bud dormancy (DBB50, p = 0.007) and

level of apparent root damage (REL,p ≤ 0.001) affected theoverall probability of a seedling surviving (i.e., probabilityof alive category 1) (Table 3). The way PS related to RELwas also different for various levels of bud dormancy(DBB50 × REL; p = 0.032). Survival probability responded

© 1999 NRC Canada

Folk et al. 1275

05

1015

20253035404550 Nursery 1 – SL6282

y = 32.5 - 113.4x; r2 = 0.19

Nu

mb

er

of N

ew R

oo

ts >

0.5

cm

0

5

10

15

20

25

30

35

40

45

50 Nursery 2 – SL6282

y = 65.6 - 290.8x; r2 = 0.29

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7


05

101520253035404550 Nursery 2 – SL6514

y = 41.3 - 144.9x; r2 = 0.32

05

101520253035404550 Nursery 1 – SL6514

y = 48.4 - 162.6x; r2 = 0.21

Fig. 2. The relationship between root electrolyte leakage and thenumber of new roots≥0.5 cm produced after 14 days (rootgrowth capacity) for Douglas-fir seedlings in experiment one.Each point represents a seedling. Linear regression models arepresented for each nursery – seed lot combination.

0.0

1.0

0.00.10.20.30.40.50.60.70.80.91.0Nursery 1 – SL6282

e(8.1 - 42.0x)

1 + e(8.1 - 42.0x)PS =

0.0

1.0

0.00.10.20.30.40.50.60.70.80.91.0Nursery 1 – SL6514

e(3.9 - 17.0x)

1 + e(3.9 - 17.0x)PS =

0.0

1.0

0.00.10.20.30.40.50.60.70.80.91.0Nursery 2 – SL6282

e(5.1 - 31.7x)

1 + e(5.1 - 331.7x)PS =

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0


0.0

1.0

0.00.10.20.30.40.50.60.70.80.91.0Nursery 2 – SL6514

e(4.5 - 18.8x)

1 + e(4.5 - 18.8x)PS =

Aliv

e C

ate

go

ry

Pro

ba

bili

ty o

f Su

rviv

al (

PS

)

Fig. 3. The relationship between root electrolyte leakage and thealive category (alive = 1, dead = 0) for Douglas-fir seedlings inexperiment one. Each point represents a seedling. Survival wasassessed 8 weeks after heat treatments were conducted. Logisticregression models, derived by maximum likelihood estimation,are for each nursery – seed lot combination, and describe theprobability of the alive category = 1 (PS) per unit of REL.



to REL in a similar manner for October (without bud-set)and November (DBB50 = 29 days) seedlings (Fig. 6). How-ever, the highest REL level marking a decrease in PS be-came progressively lower from November to January (i.e.,as DBB50 decreased from 29 to 16 days) (Fig. 6). Further-more, levels of PS predicted by these four models were sim-ilar to actual survival (%) for each stage of dormancy(Fig. 7).

Experiment one and two: critical and PS80 REL levelsMeans for critical REL levels were similar (t test, p >

0.05) between the two seed lots (Table 4). However, criticalREL decreased as DBB50 decreased within the single seedlot tested during fall–winter acclimation. In a similar man-ner, the REL values associated with PS80 were also similarbetween the two seed lots but decreased as DBB50 decreasedwithin the single seed lot tested during the fall–winter accli-mation period (Table 4).

Discussion

REL response versus seedling quality responseThis study indicated that changes in REL within a seed-

ling population are a good index of changes in root systemintegrity, but absolute levels of REL must be interpretedwith respect to other non-root-damaging factors. In agree-ment with other studies (McKay and Mason 1991; McKay1992; McKay et al. 1993; Bigras 1997), this study foundthat increases in REL were related to decreases in survivaland height growth. This study also showed that REL was re-lated to changes in performance attributes (i.e.,Pn, gwv,Ψmid, and RGC) of surviving 1+0 Douglas-fir seedlings, in-dicating that REL is also a sensitive indicator of changes inperformance potential and physiological processes amongdamaged seedlings that appear visually healthy. This sensi-tivity facilitates the use of REL as a tool for seedling qualityassessment. However, certain absolute levels of REL (e.g.,

© 1999 NRC Canada

1276 Can. J. For. Res. Vol. 29, 1999

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0


0.0

1.0

Aliv

e C

ate

go

ry

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Pro

ba

bili

ty o

f Su

rviv

al (

PS

)

1 + e(6.2 - 35.1x)e(6.2 - 35.1x)

PS =SL6282

PS80level

1 + e(4.1 - 17.6x)e(4.1 - 17.6x)

PS =SL6514

Fig. 4. The relationship between root electrolyte leakage and the alive category (alive = 1, dead = 0) for seedlings from two Douglas-fir seed lots in experiment one. Each point represents a seedling. Logistic regression models, derived by maximum likelihoodestimation, are given for each seed lot (two nurseries combined), and describe the probability of the alive category = 1 (PS) per unitof REL.

Date Seed lot No. DBB50 Critical REL PS80

Experiment 1December 15, 1994 SL6282 na 0.18 0.14December 15, 1994 SL6514 na 0.20 0.15Experiment 2October 15, 1995 SL6514 No bud set 0.24 0.31November 15, 1995 SL6514 29 0.24 0.35December 15, 1995 SL6514 18 0.20 0.21January 15, 1996 SL6514 16 0.17 0.15

Note: Bud dormancy was determined by the presence of a terminal bud, or the number of daysrequired for 50% of the population to flush their terminal buds (DBB50). na, not applicable.

Table 4. Critical root electrolyte leakage (REL) (i.e., lowest level of REL significantlydifferent from the control REL atT = 0 min) and REL levels associated with an 80%probability of the alive category = 1 (i.e., alive = 1,dead = 0) (PS80) from logisticregression models for two Douglas-fir seed lots (SL6282 and SL6514) and one seed lot atfour stages of terminal bud dormancy.



0.15–0.25) will sometimes be related to root damage andother times related to healthy root systems. Knowing whenthese levels indicate damage requires an understanding ofwhat and how other factors affect REL. Thus, the use ofREL in operational seedling quality programs requires thatseedling quality adjudication be conducted with the knowl-edge that factors, other than root damage, may influence therelationship between REL and seedling quality.

Nursery culture and seed lot effects on RELNursery culture had no effect on the relationships between

REL and PS or performance potential (Pn, gwv, Ψmid, RGC),and was not deemed to be an important factor affecting therelationship between REL and seedling quality. Thus, theREL test may be robust enough to measure across various

container nursery programs. However, McKay (1992) con-jectured that the relationship between REL and survival mayvary in bare-root seedlings according to nursery practices.This conjecture cannot be totally ruled out for containerseedlings. In this study, only two nurseries were studied, andnursery culture may not have differed enough to affect therelationship between seedling quality and REL. This rela-tionship may be affected by other nursery cultural practicesthat were not tested.

In contrast, the use of REL to forecast survival and perfor-mance potential of stock suspected of having root damagerequires seed lot based models. BothΨmid and seedling sur-vival probability declined at a faster rate per unit of REL forseed lot SL6282 compared with SL6514. This suggested thatsimilar REL levels between the two seed lots were associated

© 1999 NRC Canada

Folk et al. 1277

0

10

20

30

40

50

60

70

80

90

100

Nu

mb

er

of N

ew R

oo

ts0

.5 c

m≥

(R = -0.94, p = 0.056)DBB50 = 29

DBB50

= 18

DBB50

= 16

(R = -0.93, p = 0.072)

(R = -0.98, p = 0.017)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Re

lativ

e H

eig

ht G

row

th (

cm·c

m-1)

(R = -0.96, p = 0.045)DBB50

= 29

DBB50 = 18

DBB50

= 16

(R = -0.783, p = 0.215)

(R = -0.93, p = 0.067)


Fig. 5. The correlations of heat-time means ± SE of root electrolyte leakage (n = 8) with the heat-time means ± SE of the number ofnew roots≥0.5 cm long after 14 days (n = 24) and relative height growth (n = 24) for Douglas-fir seedlings in experiment two. Linesare provided for qualitative observation of trends. Seedling dormancy status was measured as the number of days for 50% of seedlingsto flush their terminal buds (DBB50). Levels of DBB50 = 29, 18, and 16 correspond to the November, December, and Januarymeasurement periods.R, Pearson’s correlation coefficient;p, Bonferroni probability.



with different levels of root damage in each. Thus, the rela-tionship of REL with seedling performance potential andsurvival potential may differ between seed lots, and interpre-tation of absolute REL levels for seedling adjudication musttake this into account.

Bud dormancy and RELBud dormancy status affected the relationship between

REL and Douglas-fir seedling survival potential and perfor-mance potential. Low to moderate levels of REL (i.e.,<0.35) were associated with high probabilities of survival(PS > 0.8) in the November (DBB50 = 29) seedlings but as-sociated with low probabilities of survival (PS < 0.2) in theDecember and January (DBB50 ≤ 18) seedlings. The lowestREL values associated with negligible, or zero, RGC andrelative height growth were also greater for the Novemberseedlings than for the December and January seedlings.Thus, the status of bud dormancy must be known to deter-mine both survival and performance potential of Douglas-fircontainer seedlings from REL.

Greater control REL levels in October and November,compared with December and January, suggested that seed-lings that are actively growing, or with a high DBB50 (i.e.,≈29 days), may be less tolerant to lifting-induced root dam-age than seedlings with lower DBB50 (i.e., ≤18 days).McKay (1994) showed that REL of undercut and wrenchedbareroot Douglas-fir seedlings gradually decreased from21% in mid-November to 12% in February (equivalent to0.21 and 0.12 REL in this study), and seedlings with highprestorage levels had low tolerance to cold storage. How-ever, critical REL and PS80 REL levels measured in thisstudy indicated that high levels of REL in early fall may notbe related to root damage. Critical and PS80 levels were alsogreater in October and November, compared to the Decem-ber and January, and tended to decrease with decreasingDBB50. This indicated that greater levels of REL are re-quired before root damage becomes detectable in activelygrowing or early fall acclimatized (DBB50 = 29) Douglas-firseedlings, compared with seedlings with lower days to budbreak (DBB50 ≤ 18). Thus, high levels of REL (e.g., 0.30) inearly fall-lifted Douglas-fir seedlings may not be indicativeof root damage.

It is unclear why high REL levels (e.g., 0.30) are not in-dicative of root damage in actively growing or early fall ac-climatized Douglas-fir seedlings. Greater REL levels may berelated to phenological changes in membrane leakage ormay be related to the ability of seedlings to recover fromdamage. Changes in plant cell membranes have been shownto be directly involved in cold acclimation (Steponkus1984). Furthermore, Zhao et al. (1995) showed that post-freezing root plasma membrane associated enzyme activitywas enhanced when jack pine (Pinus banksianaLamb.)seedlings were exposed to a fall environmental regime (i.e.,8 h light : 16 h dark photoperiod at 5°C for 4 weeks). Theyconjectured that enhanced plasma-membrane reductase ac-tivity may increase synthesis of lignin, a repair mechanismagainst mechanical injury of cell walls. Further work is re-quired to determine if fall root electrolyte leakage patternsare related to phenological changes in root plasma mem-brane properties.

REL and evaluation of seedling qualityRoot electrolyte leakage is a material attribute that can be

used for assessing functional integrity (i.e., root system in-tegrity) and maximum performance potential of container-grown Douglas-fir seedlings. However, it provides little in-formation about stress resistance or tolerance for field

© 1999 NRC Canada

1278 Can. J. For. Res. Vol. 29, 1999

0

1

0.00.10.20.30.40.50.60.70.80.91.0October

Before bud set

e(7.9 - 21.2x)

1 + e(7.9 - 21.2x)PS =

0

1

0.00.10.20.30.40.50.60.70.80.91.0

November

DBB50

= 29

e(8.0 - 18.6x)

1 + e(8.0 - 18.6x)PS =

0

1

0.00.10.20.30.40.50.60.70.80.91.0

December

DBB50

= 18

e(5.9 - 21.0x)

1 + e(5.9 - 21.0x)PS =

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0


0

1

0.00.10.20.30.40.50.60.70.80.91.0

January

DBB50

= 16

e(6.1 - 30.5x)

1 + e(6.1 - 30.5x)PS =

Aliv

e C

ate

go

ry

Pro

ba

bili

ty o

f Su

rviv

al (

PS

)

Fig. 6. The relationship between root electrolyte leakage (REL)and the alive category (alive = 1, dead = 0) for Douglas-firseedlings in experiment two. Survival was assessed after 8weeks, and each point represents a seedling. Logistic regressionmodels were derived by maximum likelihood estimation, anddescribe the probability (PS) of the alive category = 1 per unitof REL. Models are given for seedlings measured over fourstages of bud dormancy (i.e., determined by the presence of aterminal bud, or the number of days for 50% of seedlings toflush their terminal bud (DBB50))



performance potential assessment of undamaged seedlings(Folk and Grossnickle 1997). This is why the relationshipbetween REL and field performance of bare-root Douglas-firhas been shown to differ according to field-site environmen-tal conditions (McKay and White 1997). Seedlings with lowREL (undamaged stock) and high survival potential mayvary considerably in actual field performance, depending onhow field site environmental conditions allow performancepotential to be expressed (Folk and Grossnickle 1997).

The utilization of REL as a test for seedling quality alsorequires an understanding of the relationship between RELand survival and performance potential. Root electrolyteleakage levels in undamaged seedlings (i.e., not heat treated,T = 0 min) varied among seedlings tested in both experi-ments. Seedlings had greater REL in November (DBB50 =29) than in December and January (DBB50 ≤ 18) (experi-ment two), and seed lot SL6514 had greater REL thanSL6282 (in nursery 2, experiment one). This indicated thatbaseline or background levels may vary in healthy seedlings.

Fluctuations in baseline REL levels are problematic fordetermining root damage and forecasting seedling survivaland performance potential. The calculation and expressionof REL is different than electrolyte leakage levels of foliartissue used to determine frost tolerance (i.e., freeze-inducedelectrolyte leakage; Glerum 1985; Burr et al. 1990). Forfreeze-induced electrolyte-leakage determination, tissuedamage (i.e., by freezing) occurs during test freezing, andleakage levels can be determined for both undamaged con-trols (i.e., baseline levels) and frost-damaged samples(Glerum 1985; Burr et al. 1990). Results of freeze-inducedelectrolyte-leakage testing are often expressed as an index ofinjury that accounts for fluctuations in baseline levels (Flintet al. 1967). In this study, theT = 0 min could have beenused as a control for calculating an index of injury for rootdamage. However, under operational conditions the REL testwill lack a control for determining baseline levels. Thus,REL will vary in root samples that arrive at a laboratory for

testing during the fall acclimation period, and it will be un-clear whether high levels reflect root damage that occurredprior to testing or reflect high baseline levels resulting fromother non-damaging factors. There is the need to interpretREL with respect to factors that may influence electrolyteleakage (i.e.,Cleak in the REL calculation).

In a similar manner, a need exists to develop standardREL testing protocol that account for the possibility ofhypoxia, bacterial growth, and metabolic changes in cellwalls during the incubation period. During the incubationperiod, excised root samples may deplete the supply of dis-solved O2 in the sample solution, and a 16- to 24-h incuba-tion period may result in bacterial growth or changes to rootcell leakage patterns. Such factors may indiscriminately in-fluence root cell leakage. Also, the rate of electrolyte leak-age may be influenced by the tissue composition (e.g.,young roots vs. old roots). However, an incubation period of16–24 h has been used often (Bigras and Calmé 1994;Bigras 1997; McKay 1992, 1993, 1994; McKay and Mason1991; McKay and White 1997) and may be required for afull expression of baseline and damage-induced leakage lev-els. Further work is required to standardize the REL testingprotocol as it relates to incubation times versus changes incell physiology and bacterial contamination.

Specific levels of REL may be useful as universal indicesof the presence of damage in various seed lots. The REL as-sociated with the lowest level of detectable root damage(Dunnett’s critical level), and the REL associated with PS80were similar between the two seed lots. This suggested thatcritical and PS80 REL values could be used as a universal in-dex to determine if root damage has, or has not, occurred indifferent Douglas-fir seed lots during fall acclimation. Thus,critical REL and PS80 indices can provide information aboutthe presence of root damage (Grossnickle and Folk 1993),but information about the degree to which root system func-tional integrity has been compromised may require furthertesting with root growth capacity or net photosynthesis tests

© 1999 NRC Canada

Folk et al. 1279

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0


0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0P

rob

ab

ility

of S

urv

iva

l (P

S)

0

10

20

30

40

50

60

70

80

90

100

Act

ua

l Su

rviv

al (

%)

No bud set

DBB50 = 29

DBB50 = 18

DBB50 = 16

PS80 level

Fig. 7. The relationship between root electrolyte leakage (mean ± SE) and actual percent survival of Douglas-fir seedlings inexperiment two. Survival was assessed after 8 weeks, and each point represents the percentage of seedlings (n = 24 from each heat-treatment time) surviving from a range of root damage levels within each heat time and stage of bud dormancy. Bud dormancy statuswas determined by the presence of a terminal bud, or the number of days for 50% of seedlings to flush their terminal bud (DBB50).See Fig. 6 for a description of the logistic regression models.



(Folk and Grossnickle 1997). Alternatively, seed lot basedroot damage assessment models may be required. The lattersolution may be impractical in some reforestation programs.

Interpretation of critical and PS80 levels must also be con-ducted with regard to terminal bud dormancy status. How-ever, if stock is lifted and assessed only when seedlings havereached a minimum DBB50 level during fall acclimation,separate models may not be required for different stages ofbud dormancy. A benchmark of 0.15 may be appropriate forstock adjudication of container Douglas-fir seedlings thathave reached minimum DBB50 in the fall. This value isgreater than the highest REL measured for control seedlingsin this study and is equal to the PS80 REL determined for theseedlings with the lowest recorded DBB50 in this study (i.e.,DBB50 = 16). Furthermore, a linear regression model of per-cent REL and percent survival of bare-root Douglas-fir, re-ported by McKay and Mason (1991), gives a predictedsurvival rate of 79.3% for an REL = 15% (i.e., 15% equal tothe REL fraction of 0.15 reported here). All but one of theirDouglas-fir populations had greater than 80% survival atREL <15% (McKay and Mason 1991), indicating that maxi-mum survival of outplanted Douglas-fir was associated withthis benchmark. This is in agreement with the PS80 associ-ated with 0.15 REL in this study.

Conclusions

The following conclusions can be drawn from the findingsof this study. First, REL can be used for assessing functionalintegrity (i.e., survival potential) and performance potentialof container-grown Douglas-fir seedlings. Second, baselineor background REL levels may vary in healthy seedlings.Third, the two nursery cultural regimes in this study had noeffect on the relationship between REL and seedling quality.Fourth, the relationship between seedling quality and RELdiffered according to seed lot. However, critical and PS80REL values could be used as a universal index to determineif root damage has, or has not, occurred in differentDouglas-fir seed lots during fall acclimation. Fifth, interpre-tation of critical and PS80 levels must be conducted withregard to terminal bud dormancy status. Sixth, a benchmarkof 0.15 may be appropriate for stock adjudication of con-tainerized Douglas-fir seedlings that have reached minimumDBB50 in the fall.

These findings indicate that REL must not be taken as anindependent seedling quality measurement. In addition, theinterpretation of REL must consider the phenological stateof measured seedlings as well as the potential for geneticvariation between seed sources.

Acknowledgments

This study was funded by a direct contract administeredby the Nursery Extension Service of the British ColumbiaMinistry of Forests. Technical assistance was provided bySteven Storch and Reed Radley of the Forest BiotechnologyCentre, BC Research Inc., and by staff of the Nursery Exten-sion Service. The authors thank Dr. Valery Le May at theFaculty of Forestry, University of British Columbia, for hersuggestions on how to best describe the in-depth statisticalapproach used in this study.

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Seed lot, nursery, and bud dormancy effects on root electrolyte leakage of Douglas-fir ( Pseudotsuga menziesii ) seedlings