Improving the Rooting Capacity of StemCuttings of Virginia Pine by Severe Stumping

of Parent Trees

Christopher L. Rosier, Smurfit-Stone Container Corporation, Fernandina Beach, FL32024; John Frampton, Department of Forestry and Environmental Resources, NorthCarolina State University, Raleigh, NC 27695; Barry Goldfarb, Department of Forestryand Environmental Resources, North Carolina State University, Raleigh, NC 27695;Frank A. Blazich, Department of Horticultural Science, North Carolina State University,Raleigh, NC 27695; and Farrell C. Wise, MeadWestvaco Corp., Summerville, SC 29484(former).

ABSTRACT: Two experiments were conducted to develop a protocol for rooting stem cuttings from5-year-old Virginia pine (Pinus virginiana Mill.). The first experiment tested the effects of stumpingtreatment (none and 1⁄4, 1⁄2, and 3⁄4 original height remaining), auxin type [indole-3-butyric acid or1-napthaleneacetic acid (NAA)], and auxin concentration (0, 2, 4, 6, 8, or 12 mM) on adventitious rootformation. Stumping refers to the process of cutting back the stem combined with partial crown removal.The second experiment quantified the effects of crown position on the rooting of stem cuttings from stumpedand nonstumped trees. Rooting percentage increased and percentage of mortality decreased as the severityof the stumping treatment increased. Auxin concentration significantly affected every rooting trait exceptroot angle. Overall, cuttings from trees stumped to 1⁄4 original height and treated with 4 mM NAA rootedin higher percentages (74%) and produced more primary roots (5.5) with greater total root lengths (601mm) than the other treatments. The original crown position, particularly height, significantly affectedrooting percentage. Primary needle length significantly increased as the severity of the stumping treatmentincreased and was positively correlated with rooting capacity. South. J. Appl. For. 30(4):172–181.

Key Words: Christmas trees, indole-3-butyric acid, 1-naphthaleneacetic acid, cloning, Pinus virginiana.

Virginia pine (Pinus virginiana Mill.) is one of the mostcommonly grown Christmas tree species in the Piedmontand Coastal Plains of the Southeast. It is a desirable Christ-mas tree species for several reasons, including rapid growth(3–6 years to harvest), short needles, good branch structurefor holding ornaments, and a pleasant pine aroma (Framp-

ton and Isik 2004). However, Virginia pine has severalsignificant problems, including poor form, large phenotypictree-to-tree variation, and extreme susceptibility to the Nan-tucket pine tip moth (Rhyacionia frustrana Comstock). Be-cause of these and other problems, Christmas tree growersoften are only able to market approximately 50% of theVirginia pines planted (Frampton and Isik 2004).

Recently, there has been increasing interest in propagat-ing Virginia pine asexually by the use of rooted stemcuttings to exploit clones with desirable Christmas treecharacteristics. Asexual propagation would provide a wayto capture benefits from cloning, including increased ge-netic gain and uniformity (Zobel and Talbert 1984) forimportant Christmas tree traits. Other potential benefitsinclude the ability to combine desirable characteristics, suchas rapid growth, uniform and full crown density, and tipmoth resistance. Previous research has shown that the retailvalue of Virginia pine as a Christmas tree is affected by

NOTE: John Frampton can be reached at (919) 515-7580; Fax: (919)515-8149; john_frampton@ncsu.edu. This research was fundedjointly by the North Carolina Agricultural Research Service(NCARS) (Raleigh, NC) via the Christmas Genetics Program,by the Golden Leaf Foundation, and by the Eastern NorthCarolina Christmas Tree Growers Association. Use of tradenames in this report does not imply endorsement by theNCARS of the products named or criticism of similar ones notmentioned. Special thanks are extended to Jimmy Prince, SteveWarren, and the entire staff at the NC State University Horti-cultural Crops Research Station (Clinton, NC), for help inestablishing and maintaining the research trial from which thecuttings were collected. Manuscript received August 10, 2005,accepted June 20, 2006. Copyright © 2006 by the Society ofAmerican Foresters.

172 SJAF 30(4) 2006

characteristics that are under strong genetic control (Framp-ton and Isik 2004, Knoth et al. 2002), as are other traits,including height, stem straightness, density of foliage, sym-metry, and branches per whorl (Belanger and Bramlett1975, Meier and Goggans 1977). Because of the shortrotation and high market value of Christmas trees relative toforest trees and the large phenotypic variation that currentlyexists, genetic improvement of Virginia pine for desirableChristmas tree characteristics can be justified.

Little research involving propagation of Virginia pine bystem cuttings has been conducted. The issues that have beeninvestigated include the effects of auxin treatment (Holifieldet al. 1991, Rosier 2003, Snow and May 1962), time ofcollection (Brown et al. 1991, Holifield et al. 1991, Rosier2003, Snow and May 1962), crown position (Snow and May1962), genotype (Brown et al. 1991), and photoperiod dur-ing rooting (Snow and May 1962). However, one of themajor limitations in the effective use of vegetative propa-gation for Christmas tree production and forest regenerationis maturation (Zobel and Talbert 1984), i.e., physiologicaland morphological changes that occur with increases in treeage (Greenwood 1984). In many conifer species, maturationincreases the time for root initiation to occur, decreasesrooting capacity, and decreases growth rate of cuttingsfollowing rooting (Zobel and Talbert 1984). Younger treeswill often root readily, but as the tree matures, rootingbecomes increasingly difficult (Hartmann et al. 2002, Zobeland Talbert 1984).

Another problem often complicating the rooting of co-nifer cuttings is the influence of crown position (Hartmannet al. 2002). Cuttings collected from the lower crown tend toroot at higher percentages because they are physiologicallymore juvenile than cuttings collected from branches higherin the crown (Zobel and Talbert 1984). These differencesbecome much more pronounced as stock plants mature. Forexample, using stem cuttings of 8- and 9-year-old Virginiapine collected in March, Snow and May (1962) found that18% from the lower crown rooted, whereas, only 1 percentfrom the upper crown rooted.

Hedging, or maintaining a plant at a fixed height, is animportant stock plant management technique that maintainsjuvenility, reduces position effects, increases shoot production,allows for easier cutting collection, and reduces strobili pro-duction (Hartmann et al. 2002). Previous research with loblollypine (Pinus taeda L.) (Cooney 1999), radiata pine (Pinusradiata Don) (Bolstad and Libby 1982, Fielding 1954, Libbyet al. 1972, Menzies and Klomp 1988), Douglas-fir [Pseudot-suga menziesii (Mirb.) Franco] (Black 1972), and Norwayspruce (Picea abies L.) (Bentzer 1993) has demonstrated thatcontinuous hedging of a stock plant increases cutting produc-tion while preventing or delaying maturation.

At the time of selection for Christmas tree quality, typ-ically 4–5 years old, Virginia pine does not produce juve-nile shoots with a high rooting capacity. Although previousresearch has shown that continuous hedging of juvenilestock plants maintains production of juvenile shoots, it isnot known whether stumping older trees of Virginia pine

will induce juvenile shoots that root better than cuttingscollected from intact trees. Therefore, two experiments wereconducted to examine the possibility of increasing produc-tion and rooting of vertically oriented (nonplagiotropic)shoots from older trees of Virginia pine of the same agestumped to various heights. For convenience, we refer to theprocess of cutting back the stem combined with partialcrown removal as stumping. Experiment 1 tested the effectof stumping treatments, auxin type [indole-3-butyric acid(IBA) or 1-napthaleneacetic acid (NAA)], and auxin con-centrations on shoot production and subsequent rootingcapacity. Five auxin concentrations (2–12 mM) of eachauxin type and a 0 mM IBA control were tested on cuttingsgenerated from intact (nonstumped) trees and from treesstumped to 1⁄4, 1⁄2, and 3⁄4 original height. Experiment 2tested the effects of stumping treatments and crown positionon the rooting capacity of stem cuttings of Virginia pine.

Materials and MethodsField Design and Cutting Collection

The stock plants (study trees) were part of a 5-year-oldprogeny test located at the North Carolina State UniversityHorticultural Crops Research Station, Clinton, North Caro-lina (Frampton and Isik 2004). The stock plants had beengrown from seed and cultured as Christmas trees (Johnson1991). Before treatments were applied, total height wasmeasured for each study tree, and soil and foliage sampleswere collected for mineral nutrient analyses. Analyses re-vealed deficiencies in nitrogen and potassium. In May 2001,a soluble solution of Peters 20-20-20 fertilizer was appliedto the base of each tree at a rate of 36.7 kg � ha�1.

Stumping treatments were applied to the primary axesMar. 16–17, 2001. Once the primary axes had been stumped,the previous year’s growth and all remaining terminal budswere removed from the remaining branches. For Experiment1,144 trees were selected from the border rows of the progenytest and were arranged into eight linear blocks to account forwithin-site variation. Genetic identity of the border trees hadnot been maintained but they were from a mixture of open-pollinated families being evaluated in the test. Each blockcontained four control (nonstumped) trees, six trees stumped to1⁄4 height, four trees stumped to 1⁄2 height, and four treesstumped to 3⁄4 height. (More trees were included in the mostsevere stumping treatment to ensure that enough cuttings wereproduced for this treatment.) For Experiment 2, four additionaltrees (one per treatment) were treated in seven of the eightblocks, for a total of 28 study trees.

Softwood cuttings were collected on June 4 (Experiment1) and June 6–7 (Experiment 2), 2001. In Experiment 1, 45cuttings were collected from all trees except those stumpedto 1⁄4 height, from which 25 cuttings each were collected,since more trees had been stumped. In Experiment 2, theexact position of 50 cuttings per tree was determined bymeasuring the distance from the stem, height from theground, and degrees from north. In Experiment 2, alongwith measuring the exact position of each of the cuttings,the length of the most proximal primary needle of eachcutting was also measured to the nearest 1 mm. After

SJAF 30(4) 2006 173

collection, the cuttings were immediately wrapped in moistpaper towels; transported to Raleigh, North Carolina, on ice;and stored overnight at 4° C. All cuttings were set (insertedinto the rooting medium) the day after collection.

Auxin PreparationIBA and NAA were tested for their ability to increase

adventitious rooting and enhance root development in Ex-periment 1. No auxin (a control) and five IBA and NAAconcentrations (2, 4, 6, 8, or 12 mM) were examined. InExperiment 2, all cuttings were treated with 4 mM IBA. Theauxin solutions were prepared by dissolving a knownamount of the free acid of each auxin in 70% isopropylalcohol and then diluting the solution with deionized waterto make a 50% isopropyl alcohol-water solution. Solutionswere stored at 4° C in an opaque bottle and used within 2days of preparation.

Test Design and Rooting EnvironmentThe experiments were conducted in a propagation green-

house located at the Horticulture Field Laboratory, Raleigh,North Carolina. Before preparation and setting, the cuttingsfrom different trees within the same stumping treatmentused in Experiment 1 were pooled and thoroughly mixed.The pooled cuttings were collected from 48 trees for thestumped-to-1⁄4-height treatment and 32 trees for the othertreatments. In both experiments, the cuttings were recutbasipetally to a length of 9 cm, and auxin was applied for 3seconds to the basal 1.5 cm of each cutting. Cuttings for thecontrol treatment in Experiment 1 were dipped into a 50percent isopropyl alcohol/deionized water solution. Needleswere not removed before or after the auxin was adminis-tered. The auxin-treated cuttings were air-dried for a mini-mum of 15 min before setting to a depth of 3 cm into amedium of 3 horticultural perlite:2 peat (by volume).

During the rooting period, the cuttings were subjected tointermittent mist using a Grower Junior (McConkey Co.,Sumner, WA) irrigation boom. Frequency of mist applica-tion was controlled according to relative humidity (RH) andtime of day. As RH in the greenhouse decreased, the fre-quency of mist application increased; however, frequencyof mist application was less during the night, decreasingfrom an average of one mist cycle every 6 min during theday to an average of one mist cycle every 60 min at night.Flow rate was held constant during the first 3 weeks at 0.06 lm�2 per mist cycle, and at 0.05 l m�2 per mist cycle for the9 remaining weeks. Average day and night temperatures inthe greenhouse were 26.1 � 2° C and 20.5 � 2° C, respec-tively. All cuttings were rooted under ambient light condi-tions; i.e., natural seasonal photoperiod and irradiance.

The experimental design for rooting Experiment 1 was arandomized complete block. There were seven blocks de-signed to account for environmental variation within thegreenhouse. Each block contained eight cuttings per treat-ment per block, for a total of 2,464 cuttings. The entirestudy was surrounded by a border row. The experimentaldesign for Experiment 2 was a completely randomizeddesign with a total of 1,400 cuttings. In both studies, rootingdata were recorded after 16 weeks in the greenhouse.

Rooting success was measured by calculating rooting per-centage, percentage of mortality, number of primary roots,total root length, root symmetry, and root angle. The latter fourtraits were assessed only for cuttings that rooted. All analyseswere conducted using plot means (n � 8). A cutting wasconsidered rooted if a minimum of one primary root �1 mmin length was present. Root systems were considered symmet-rical when the angle between two roots was more than 135degrees, using the stem center as the vertex (Frampton et al.1999). Systems with three or more roots were symmetrical ifthe angle between the two roots that were farthest apart was�135 degrees. Cuttings with one root were scored as havingasymmetrical root systems. Root angle was a rooting charac-teristic developed to determine the percentage of geotropicroots of a cutting. Root angle was the percentage of primaryroots that formed an angle of �60 degrees with an extension ofthe cutting’s stem.

Statistical Analysis for Experiment 1Analyses of variance (ANOVAs) were conducted using

the General Linear Model procedure of the Statistical Anal-ysis System version 8.1 (SAS Institute, Inc., 1999). Stump-ing, block, auxin, concentration, concentration squared, andall of the two- and three-way interactions not involvingblock were sources of variation in Experiment 1. Concen-tration and its squared term were treated as continuousvariables in the ANOVAs. Potential transformations of boththe dependent and independent variables were exploredusing previously described techniques (Rosier et al. 2004).

Despite using the most appropriate data transformationsand including squared terms for concentration, the least-squares regression approach failed to adequately define therelationship between concentration and the variables as-sessed. Therefore, nonlinear regression techniques wereused by regressing each dependent variable on treatmentmeans, concentration or auxin type by concentration, usingthe NLIN procedure of the Statistical Analysis SystemVersion 8.1 (SAS Institute, Inc., 1999). The double expo-nential model (Rawlings et al. 1998), modified by twoconstants (A and B), was used, as follows:

Trait ��1(B � e���2 � Concentration)�e���1 � Concentration))

�A��1 � �2��(1)

where e � base of natural logarithm; �1, �2, A, and B areregression parameters; and Concentration � auxin concen-tration in mM.Statistical Analysis for Experiment 2.—Cutting collectionpositions were converted to a three-dimensional (x, y, z)coordinate system. The x-axis ran from �/2 to 3�/2 radians(east to west), the y-axis from 0 to � radians (north tosouth), and the z-axis was the cutting height from the treebase. The x, y, and z values for each tree were standardizedto a mean of 0 and a SD of 1 to compare the cutting positionamong trees within the same stumping treatments using thefollowing equation:

S � �d � d� �/std, (2)

174 SJAF 30(4) 2006

where S � standardized distance; d � measured distance; d�

� mean measured distance of all cuttings on a tree; andstd � SD of measured distance of all cuttings on a tree.

Discriminant analysis using full and reduced models wasused to identify variables important in classifying rootedand nonrooted cuttings. Ten dependent variables were used:primary needle length, x, y, z, x2, y2, z2, xy, xz, and yz.Within each stumping treatment, stepwise techniques usingforward selection and allowing variables to enter the modelat P � 0.15 were conducted. A full model was also used forcomparison. In addition, a full model was run for each of theeight individual trees within each stumping treatment.Squared canonical correlations that represent the percentageof variation in the dependent variable discriminated by theset of independent variables were used to compare models.

Using both a linear and quadratic term, mean tree pri-mary needle lengths were regressed on each rooting re-sponse variable. Treatment least square means for primaryneedle length, rooting percentage, and total root length wereseparated using pairwise t-tests with Tukey-Kramer experi-mentwise error rate adjustments (Kramer 1956).

ResultsExperiment 1

ANOVAs and transformations of the independent anddependent variables for all rooting traits are presented inTable 1. Since no significant differences were identified forroot angle, this trait is not further mentioned.

Rooting PercentageThe main effects of stumping, block, concentration, and

concentration squared were significant (Table 1). Signifi-cant interactions were detected between concentration andauxin, between concentration squared and auxin, and be-tween concentration squared and stumping (Table 1). As theseverity of the stumping treatment increased, rooting per-centages increased; 27, 50, 63, and 64% for nonstumpedcontrols and trees stumped to 3⁄4, 1⁄2, and 1⁄4 original height,respectively. With the exception of the control trees, whenNAA was applied at increasing concentrations to the basesof the cuttings, rooting percentage reached a peak and theneither leveled off or decreased slightly (Figure 1). When

IBA was applied to the bases of the cuttings, the highestpredicted rooting percentage (73%) occurred where treeshad been stumped to 1⁄2 original height and treated with 2mM. Using the predicted optima from the nonlinear regres-sion analysis, the greatest rooting percentage for cuttingsfrom trees stumped to 1⁄4 and 3⁄4 original height (60 and59%, respectively), occurred when 3.5 and 5 mM IBA,respectively, were applied. In the nonstumped control trees,predicted rooting percentage increased steadily from 14% inthe 0 mM IBA–treated cuttings to 36% at 12 mM IBA.When NAA was applied to the base of the cuttings, thehighest predicted rooting percentage (76%) using the non-linear regression analysis occurred when trees werestumped to 1⁄4 original height and the cuttings were treatedwith 4 mM (Figure 1). In the remaining stumping treat-ments, the greatest rooting percentage for cuttings fromtrees stumped to 1⁄2 and 3⁄4 original height (70 and 65%,respectively), occurred when 4 and 3 mM IBA, respec-tively, were applied. When NAA was applied to cuttingsfrom the nonstumped trees, the rooting percentage in-creased from 14% when no auxin was applied to 31% at12 mM.

Percentage of MortalityThe main effects of block, concentration, and concentra-

tion squared were significant (Table 1). When auxin wasapplied, percentage of mortality decreased from the controlto 2 mM and then increased with increases in auxin con-centration up to 12 mM (Figure 2). The highest predictedmortality (23%) occurred for cuttings treated with 12 mMauxin.

Number of Primary RootsThe main effects of block, concentration, and concentra-

tion squared were significant (Table 1). Significant interac-tions were also detected between concentration and auxinand between concentration squared and auxin. Using non-linear regression analysis, the predicted number of primaryroots increased with increases in auxin concentration, re-gardless of the type of auxin applied (Figure 3A). WhenIBA was applied, the predicted number of primary roots

Table 1. Analysis of variance values for traits assessed in a rooting trial of stem cuttings of Virginia pine from5-year-old stock plants.

Rooting (%) Mortality (%) Number of primary roots Total root length Root symmetry Root angle

y transformation Arcsine square root Square root Square root Square root None Nonex transformation None Log Log None Log LogStumping 0.0004 0.5909 0.8047 0.6646 0.5219 0.2903Replication �0.0001 0.0008 �0.0001 �0.0001 0.0012 0.5644Auxin 0.2091 0.3919 0.2705 0.0295 0.7377 0.9852Stumping Auxin 0.4290 0.7686 0.5254 0.5954 0.4978 0.7206Ca 0.0003 0.0494 0.0003 0.0045 0.0155 0.4975C2 �0.0001 0.0029 0.0009 0.0767 0.0059 0.4563C Stumping 0.9558 0.7557 0.8575 0.7546 0.5445 0.1561C2 Stumping 0.0069 0.7300 0.6935 0.5044 0.4974 0.1325C Auxin 0.0382 0.3923 0.0318 0.0038 0.2662 0.6683C2 Auxin 0.0167 0.5431 0.0393 0.0070 0.3274 0.7618C Stumping Auxin 0.4809 0.5351 0.2764 0.4811 0.5087 0.6009C2 Stumping Auxin 0.4748 0.3507 0.2880 0.4956 0.6894 0.5848

a C, auxin concentration.

SJAF 30(4) 2006 175

produced increased only slightly, from 2.5 in 0 mM IBA-treated cuttings to 3.1 for cuttings treated with 12 mM.When NAA was applied, however, the predicted number ofprimary roots produced increased dramatically from 2.5 inthe 0 mM IBA-treated cuttings to 5.5 at 12 mM.

Total Root LengthThe main effects of block, auxin, and concentration were

significant (Table 1). Significant interactions were detectedbetween concentration and auxin and between concentrationsquared and auxin (Table 1). Regardless of the auxin used,

predicted total root length increased with increases in auxinconcentration (Figure 3B). When IBA was applied, pre-dicted total root lengths increased slightly from 266 mm inthe 0 mM IBA-treated cuttings to 429 mm at 12 mM. WhenNAA was applied, predicted total root lengths increaseddramatically from 266 mm in the 0 mM IBA control to 614mm at 12 mM.

Root SymmetryThe main effects of block, concentration, and concentra-

tion squared were significant (Table 1). Across all other

Figure 1. Effect of stumping and indole-3-butyric acid (IBA) (A) or napthaleneacetic acid (NAA) (B) on rooting percentage of stemcuttings of Virginia pine collected in 2001. R2 values for IBA equations were 0.93, 0.99, 0.86, and 0.93 for stumped to 1⁄4, 1⁄2, and 3⁄4height and nonstumped treatments, respectively. R2 values for NAA equations were 0.90, 0.92, 0.97, and 0.90 for stumped to 1⁄4, 1⁄2,and 3⁄4 height and nonstumped treatments, respectively.

176 SJAF 30(4) 2006

treatments, the frequency of symmetrical roots increasedwith increasing concentrations from 19% in the 0 mMIBA-treated cuttings to 33% at 12 mM (Figure 4).

Experiment 2Effect of Stumping Height and Crown Position

Results from the stepwise discriminant analysis to clas-sify rooted and nonrooted cuttings on the basis of theircrown position and primary needle length for each stumpingtreatment are presented in Table 2. In general, as the sever-ity of the stumping treatment increased, length of the pri-mary needle, rooting percentage, and total root length in-creased significantly. All stumping treatments producedcuttings that generated greater total root lengths than thosefrom nonstumped trees. The squared canonical correlationsin the discriminate analyses represent the percentage ofvariation in the dependent variable (percent rooting) dis-criminated by the set of independent variables (primaryneedle length and collection position coordinates). Whencompared with the position traits, primary needle lengthconsistently explained more of the variation: 8.1, 33.8, 11.3,and 4.8% for trees stumped to 1⁄4, 1⁄2, and 3⁄4 height and thenonstumped control, respectively. The stumping treatmentmeans for primary needle length, rooting percentage, andtotal root length are also presented in Table 2.

For the effect of crown position, height (z) consistentlyexplained more of the variation in crown position forstumped stock plants. The partial squared canonical corre-lations for height were 0.46, 0.01, and 0.03 when stockplants were stumped to 1⁄4, 1⁄2, and 3⁄4 original height, re-spectively (Table 2). In the nonstumped stock plants, dis-

tance from the main stem accounted for all of the variationdetected, with a total squared canonical correlation of 0.15.

Using the full model as a comparison to explain thevariation, the squared canonical correlations were 0.15,0.36, 0.18, and 0.16 for trees stumped to 1⁄4, 1⁄2, and 3⁄4height and nonstumped controls, respectively. Using the fullmodel on individual trees, the mean R2 values were 0.39,0.56, 0.39, and 0.41 for trees stumped to 1⁄4, 1⁄2, and 3⁄4height and nonstumped control, respectively, whereas theirR2 value ranges were 0.19–0.64, 0.26–0.77, 0.12–0.67,and 0.21–0.54, respectively.

Primary Needle LengthPercentage of rooting and total root length were the only

two traits assessed that had a significant relationship withprimary needle length. Predicted rooting percentage contin-ued to increase with increases in primary needle length from0% at 0.3 cm to 70% at 4.0 cm (Figure 5A). Predicted totalroot lengths increased with increases in primary needlelength from 0 mm when no primary needle was present to309 mm when the primary needle length was 2.8 mm,before decreasing slightly with increases in primary needlelength (Figure 5B).

DiscussionEffects of Stumping and Crown Position

In the present study using stumping, as in previousresearch in pine species (Pinus spp. L.) using hedging,positive effects on rooting were observed (Bolstad andLibby 1982, Cooney 1999, Fielding 1954, Libby et al. 1972,

Figure 2. Effect of auxin (mean of indole-3-butyric acid and napthaleneacetic acid) concentration on mortality of stem cuttings ofVirginia pine collected in 2001 (R2 � 0.96).

SJAF 30(4) 2006 177

Menzies and Klomp 1988). Rooting of cuttings increased,and mortality decreased significantly, as the severity of thestumping treatments increased. Although not significantlydifferent, primary root production and total root length weregreater in cuttings collected from the most intensivelystumped trees (data not presented).

The stumping treatments used in this study stimulateddevelopment of shoots with dramatically altered mor-

phology that developed from fascicular buds producedwhen the trees were younger (Figure 6). Primary needlelength, a characteristic associated with juvenility in pinespecies (Frampton 1987, Haines et al. 1992, Cooney1999, Masri 2000), was found to significantly decreasewith stumping height and was positively correlated withrooting percentage and total root length (Figure 5). Itconsistently explained more of the variation in rooting

Figure 3. Effect of auxin type and concentration on number of primary roots (A) and total root length (B) of stem cuttings of Virginiapine collected in 2001. R2 values for number of primary roots were 0.98 and 0.94 for indole-3-butyric acid (IBA) and napthaleneaceticacid (NAA), respectively. R2 values for total root length were 0.90 and 0.97 for IBA and NAA, respectively.

178 SJAF 30(4) 2006

percentage than did position, regardless of stumpingheight (Table 2). These data confirm that primary needlelength may be used as an indicator of juvenility andsubsequent rooting capacity.

The crown position from which the cuttings werecollected correlated highly with rooting percentage forcuttings collected from most individual 5-year-old Vir-ginia pine trees stumped to various heights. In somecases, the amount of variation explained within a singletree was as high as 77%; however, the large amount of

tree-to-tree variation reduced the average relationship foreach stumping treatment. The height from which thecuttings were collected was found to be the most impor-tant position variable. As the distance from the base ofthe tree increased, rooting percentages decreased, sup-porting the concept that the maturity of the meristemsproducing the shoots increases as the distance from thebase of the tree increases. The nonstumped control treat-ment was the exception, perhaps because too few cuttingsactually rooted to detect a trend.

Figure 4. Effect of auxin concentration on root symmetry of stem cuttings of Virginia pine. Root symmetry is averaged over allstumping heights, and auxin concentration responses are averaged over indole-3-butyric acid and napthaleneacetic acid (R2 � 0.93).

Table 2. Results of discriminant analysis for crown position effects on rooting of stem cuttings of Virginia pine.Standardized distances from the base of the donor tree are denoted x, y, and z. Means are also presented for primaryneedle length, rooting percentage, and total root length.

Stumpingtreatment

Mean primary needlelength (mm)

Mean rooting(%)

Mean total rootlength (mm) Variables

Squared canonicalcorrelation 100

Partial Total

1/4 Height 3.44a 69a 303a PNLd 8.07 14.41Z 4.63

(3.19–3.94)e (30–100) (175–519) X2 0.01Y2 1.38

1/2 Height 3.24a 63a,b 300a PNL 33.84 35.72Z 0.93

(2.88–3.42) (22–94) (207–540) YZ 0.84Z2 1.11

3/4 Height 2.31b 57b 275a PNL 11.26 15.93Z 2.76

(1.29–2.58) (44–84) (152–469) X2 1.82XY 0.76

Nonstumpedcontrol

0.82c 17c 154c PNL 4.82 14.84

Y2 3.89(0.69–0.99) (2–34) (55–306) X2 3.88

X 2.29XY 0.89

a,b,c Values followed by different lowercase letters are significantly different at P � 0.05.d PNL � Primary needle length.e Range of mean tree values.

SJAF 30(4) 2006 179

Auxin Type and Concentration EffectsRelative to IBA, NAA increased rooting percentage,

number of primary roots, and total root length for stemcuttings collected from stumped trees of Virginia pine. Onthe other hand, when cuttings were collected from non-stumped control trees, no significant differences (P �0.923) in rooting percentage were detected for auxin typeover the range of concentrations assessed. The lack of

influence of auxin type on rooting percentage agrees withprevious research with Virginia pine that used a larger rangeof auxin concentrations (0–60 mM) and cuttings from threestages of development (softwood, semi-hardwood, andhardwood) from control trees of a similar age (Rosier et al.2004). Lack of response to auxin type in the mature cuttingscollected from the nonstumped controls and their dissimilarresponse to concentration increases may be related to a lossof competence in responding to auxin or to endogenouslevels of auxin.

Auxin concentration significantly affected every rootingtrait assessed except root angle (Table 1). Inclusion of awide range of concentrations resulted in a clear depiction ofthe trends. Rooting percentage increased and mortality de-creased with increases in auxin concentration up to anoptimum concentration before steadily decreasing (Figures1 and 2). The number of primary roots and total root lengthcontinued to increase with increases in auxin concentration.Depending on the auxin type, these traits either showed littlechange (IBA) or increased steadily with increases in con-centration (NAA). Regardless of stumping height or type ofauxin, primary root production and total root lengths werepositively related and exhibited similar patterns as the con-centration of auxin increased. Root symmetry, not affectedsignificantly by auxin type, also increased steadily withincreases in auxin concentration and paralleled primary rootproduction. This may be expected since a greater number ofprimary roots increases the likelihood that symmetry willoccur.

Higher concentrations of auxin, especially NAA, af-fected the origin of root initiation on the stem. In cuttingstreated with 0–4 mM auxin, root primordia appeared toemerge from the callus formed below the cut end of the

Figure 5. Relationship between primary needle length and rooting percentage (A) and total root length (B) for stem cuttings from5-year-old Virginia pine trees subjected to four stumping treatments. R2 values for rooting percentage and total root length were 0.52and 0.28, respectively.

Figure 6. Five-year-old intact Virginia pine tree (A) and a typ-ical cutting taken from the tree (B) compared with a similar treestumped to 1⁄4 its original height (C) and a typical cutting takenfrom the tree (D).

180 SJAF 30(4) 2006

stem, although this was not determined microscopically.This phenomenon only occurred in cuttings from trees thathad been stumped, especially the trees that had beenstumped to 1⁄4 and 1⁄2 original heights. At concentrations 4mM NAA and 8 mM IBA, the bases of the cuttings fromstumped trees appeared necrotic. Following tissue dieback,callus formation was slight, occurring just above thewounded tissue. Subsequent adventitious root formationconsisted of numerous roots measuring a few millimeters inlength that originated above the callus.

Higher concentrations of exogenously applied auxin notonly affected the origin of root initiation on the stem butalso detrimentally affected rooting percentage and increasedcutting mortality. When IBA was applied, regardless of theconcentration, minimal basal tissue necrosis was observed,and rooting percentages generally reached an optimum be-fore decreasing slightly. However, when 8 mM NAA wasapplied, the tissue at the base of the cuttings became ne-crotic, leading to reduced callus formation and, in somecases, mortality.

Although its results are not presented herein, a studyinvestigating combinations of auxin types was conductedsimultaneously with the current study (Rosier 2003). Fourcombinations of two concentrations (2 and 4 mM) of bothIBA and NAA were tested in a factorial arrangement. Gen-erally, these combinations produced negative effects on therooting traits assessed relative to the use of a single auxin.Auxin was detrimental when 4 mM was applied in anycombination. Future research should address combinationsof auxin concentrations lower than those evaluated.

ApplicationOften, when progeny tests in tree improvement programs

reach an age to appropriately evaluate performance forselection, maturation prevents effective vegetative propaga-tion, creating a dilemma for implementation of clonal for-estry. This problem has been circumvented in the NorthCarolina State University Christmas Tree Genetics Programby applying results from this study. Virginia pine selectionswere made in progeny tests at age 5 (from seed) on the basisof economic assessments by three Christmas tree growers(Frampton and Isik 2004), and select trees were then graftedin a greenhouse. Stumping treatments were applied to boththe original field ortets and the grafted ramets. The resultingjuvenile shoots were collected and rooted to produce stockplants for additional cycles of propagation. Thus, elite ge-netic selections are being multiplied rapidly to soon provideChristmas tree growers with clonal planting stock. A similarapproach may be feasible for forestry regeneration pro-grams of other southern pine species.

Literature CitedBELANGER, R.P., AND D.L. BRAMLETT. 1975. Virginia pine as a Christmas

tree. USDA For. Ser. Res. Note SE-223. SE For. Experiment Station,Asheville, NC. 4 p.

BENTZER, B.G. 1993. Strategies of clonal forestry with Norway spruce. P.120–138 in Clonal Forestry II: Conservation and application, Ahuja,M.R., and W.J. Libby (eds.). Springer-Verlag, Berlin, Germany. 240 p.

BLACK, D.K. 1972. The influence of shoot origin on the rooting ofDouglas-fir cuttings. Int. Plant Prop. Soc. Comb. Proc. 22:142–167.

BOLSTAD, P.V., AND W.J. LIBBY. 1982. Comparisons of radiata pinecuttings of hedge and tree-form origin after seven growing seasons.Silvae Genetica 31:9–13.

BROWN, G.F., Q. HOLIFIELD, J.L. FORD-LOGAN, AND G.S. FOSTER. 1991.Clonal variation in rooting ability of Virginia pine. P. 187–194 in Proc.21st South. For. Tree Impr. Conf., Univ. Tenn. and TN Division ofForestry, Knoxville, TN. 342 p.

COONEY, B. 1999. Effects of family, shearing height, pruning intensity andtime of shoot origin on shoot morphology and rooting of loblolly pinestem cuttings. MSc thesis. NC State Univ., Raleigh, NC. 83 p.

FIELDING, J.M. 1954. Methods of raising Monterey pine from cuttings in theopen nursery. Austr. For. and Timber Bureau. 32. Canberra, NewZealand. 29 p.

FRAMPTON, J., AND F. ISIK. 2004. Genetic improvement of Pinus virginianafor Christmas tree production. For. Genet. 11(2):137–147.

FRAMPTON, L.J. JR. 1987. Rooting of fasicular shoots from mature loblollypine. P. 57–64 in Phytotron report. NC State University, Raleigh, NC.138 p.

FRAMPTON, L.J. JR., B. GOLDFARB, S.E. SURLES, AND C.C. LAMBETH. 1999.Nursery rooting and growth of loblolly pine cuttings: Effects of rootingsolution and full-sib family. South. J. Appl. For. 23(2):108–115.

GREENWOOD, M.S. 1984. Phase change in loblolly pine: Shootdevelopments as a function of age. Physiol. Plant. 61(3):518–522.

HAINES, R.J., T.R. COPLEY, J.R. HUTH, AND M.R. NESTER. 1992. Shootselection and the rooting and field performance of tropical pinecuttings. For. Sci. 38(1):95–101.

HARTMANN, H.T., D.E. KESTER, F.T. DAVIS JR., AND R.L. GENEVE. 2002.Hartmann and Kester’s plant propagation: Principles and practices.7th Ed. Prentice Hall, Upper Saddle River, NJ. 880 p.

HOLIFIELD, Q., J.L. FORD-LOGAN, G.S. FOSTER, AND G.F. BROWN. 1991.Efficiency of hormonal treatments for the propagation of Virginia pineby cuttings. P. 182–186 in Proc. 21st South. For. Tree Impr. Conf.,Univ. Tenn. and TN Division of Forestry, Knoxville, TN. 342 p.

JOHNSON, J.E. 1991. Christmas tree production manual. Virginia Coop.Ext. Serv., VA Polytech. Inst. and State Univ., Pub. 420-075.Blacksburg, VA. 212 p.

KNOTH, J., J. FRAMPTON, AND R. MOODY. 2002. Genetic improvement ofVirginia pine planting stock in South Carolina. HortTechnology12(4):675–678.

KRAMER, C.Y. 1956. Extension of multiple range tests to group means withunequal numbers of replications. Biometrics 12(3):307–310.

LIBBY, W.J., A.G. BROWN, AND J.M. FIELDING. 1972. Effects of hedgingradiata pine on production, rooting, and early growth of cuttings.N.Z. J. For. Sci. 2(2):263–283.

MASRI, R. 2000. Effects of hedging and serial propagation on morphology,rooting, and growth of loblolly pine (Pinus taeda L.) cuttings. PhDdissertation, NC State Univ., Raleigh, NC. 196 p.

MEIER, R.J., AND J.F. GOGGANS. 1977. Heritabilites of height, diameter andspecific gravity of young Virginia pine. For. Sci. 23(4):450–456.

MENZIES, J., AND P. KLOMP. 1988. Effects of parent age on growth and formof cuttings, P. 18–41 in Workshop on growing radiata pine fromcuttings, Menzies, M.I., et al. (eds.). N. Z. For. Res. Inst. Bull. No. 135.Rotorua, New Zealand.

RAWLINGS, J.O., S.G. PANTULA, AND D.A. DICKEY. 1998. Appliedregression analysis: A research tool. 2nd Ed. Spring-Verlag, NewYork, NY. 553 p.

ROSIER, C.L. 2003. Factors affecting the rooting of Fraser fir (Abiesfraseri) and Virginia pine (Pinus virginiana) stem cuttings. MSc thesis,NC State Univ., Raleigh, NC. 169 p.

ROSIER, C.L., J. FRAMPTON, B. GOLDFARB, F.C. WISE, AND F. BLAZICH.2004. Growth stage, auxin type, and concentration influence rooting ofVirginia pine stem cuttings. HortScience 39(6):1392–1396.

SAS INSTITUTE, INC. 1999. SAS/STAT User’s Guide, Version 6, 4th Ed.,Vols. 1 and 2. SAS Institute, Inc., Cary, NC.

SNOW, A.G., AND C. MAY. 1962. Rooting of Virginia pine cuttings. J. For.60(4):257–258.

ZOBEL, B., AND J. TALBERT. 1984. Applied forest tree improvement. JohnWiley and Sons, Inc., New York. 505 p.

SJAF 30(4) 2006 181