Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol14.1994:
Tree Physiology 14, 1261-1275
© 1994 Heron Publishing----Victoria, Canada
Maturation in Douglas-fir: II. Maturation characteristics of
genetically matched Douglas-fir seedlings, rooted cuttings and
tissue culture plantlets during and after 5 years of field growth
GARY A. RITCHIE,1 STEVEN D. DUKE2 and ROGER TIMMIS2
1
Weyerhaeuser Company, G.R. Staebler Forest Resources Research Center, 505 North Pearl Street,
Centralia, WA 98531, USA
2
Weyerhaeuser Company Technology Center, Federal Way, Tacoma, WA 98003, USA
Received February 23, 1994
Summary
Seedlings, rooted cuttings from juvenile stock plants, and cotyledon-derived tissue culture plantlets were
propagated from several coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) full-sib families so
that the rooted cuttings and plantlets were clonally identical. The stock types (seedlings, rooted cuttings
and plantlets) were planted in the field in spring 1987. In fall 1991, after five complete growing seasons,
the plants were measured and these values compared to maturation ‘‘markers’’ identified for Douglas-fir
in the companion paper (Ritchie and Keeley 1994). Nodal branch lengths and nodal branch diameters
decreased in the order seedlings > rooted cuttings > plantlets. The decreases were about 21% for nodal
branch lengths and 24% for nodal branch diameters. Seedlings carried significantly more total branches
(nodal + internodal) than the other two stock types. Height growth was similar for the three stock types,
but plantlet height increment was beginning to decrease during the fourth year. We conclude that
vegetative propagules of Douglas-fir exhibited traits of mature trees. These were particularly marked in
the cotyledon-derived plantlets.
Keywords: juvenility, clonal forestry, tissue culture, organogenesis, Pseudotsuga menziesii.
Introduction
Cloning of forest trees offers an approach for exploiting nonadditive, as well as
additive, genetic variance and for increasing crop uniformity (Zobel 1992, Talbert et
al. 1993). Several successful cloning programs have been developed with species in
which mature individuals produce juvenile stump or root sprouts (Ritchie 1994).
With such species, it is possible to evaluate the phenotypic or genotypic potential of
a mature individual, then to replicate it from the propagated juvenile tissue that it
produces. Poplars, (Populus spp.), willows (Salix spp.) and several eucalypt (Eucalyptus spp.) species have been exploited in this manner (Brandão 1984, Zsuffa 1984,
Leakey 1987).
However, with species that do not produce easily obtainable juvenile tissue,
cloning is difficult and expensive. It requires long-term storage of juvenile clonal
ramets while genetically identical ramets are field tested. Following testing, the
selected, stored clones must be repropagated and bulked up to commercial quantities
for field planting. In vitro propagation, through either organogenesis or more recently, somatic embryogenesis, offers considerable promise for cloning tree species
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RITCHIE, DUKE AND TIMMIS
(Dunstan 1988). In such systems, juvenile clone banks may be maintained at low
temperatures (tissue culture plantlets) or cryogenically (somatic embryos) (Gupta et
al. 1993) at relatively low cost during the test period.
Because of the potential of organogenesis for cloning conifer species (Durzan and
Campbell 1974, McKeand and Weir 1984), many tissue culture programs were
initiated during the early 1970s. As the plantlets in these tests began to grow, they
often exhibited mature morphological characteristics in the field even though they
had been cultured from juvenile explants (e.g., McKeand 1985).
Field observations of coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco)
plantlets derived from cotyledon culture, which were growing on several test sites in
western Oregon, prompted Ritchie and Long (1986) to speculate that these trees
might be exhibiting mature characteristics. Many of the plantlets appeared to have
shorter and fewer branches, narrower branch angles, more bluish-green foliage and
coarser needles than seedling controls. However, it was not possible to determine
unequivocally that these plantlets were exhibiting mature characteristics because the
morphological attributes of maturation in Douglas-fir had not been established.
We therefore initiated a study in which an array of putative morphological maturation ‘‘markers’’ in coastal Douglas-fir was screened in an attempt to identify those
that could be used in the field (Ritchie and Keeley 1994). In the present paper, we
report on the use of these markers to evaluate genetically matched, field-planted
Douglas-fir vegetative propagules from tissue culture and rooted cuttings. We also
present information on propagation characteristics and field performance of these
propagule types.
Methods
Douglas-fir seedlings, rooted cuttings and plantlets were produced from seeds of the
same full-sib families. Both of the vegetative propagule types were produced from
the same clones. Following laboratory and greenhouse propagation, the plants were
grown in a bareroot nursery for one year before planting on a forest site. Performance
and morphological development were examined after 2 and 5 years. The morphological traits were then compared to the maturation markers defined for Douglas-fir
by Ritchie and Keeley (1994).
Genetic material
Six full-sib families were selected (Table 1) to represent primarily top performing
crosses with one low performer (1073 × 1077) included for comparison. Seeds from
these crosses were produced at a Weyerhaeuser Company seed orchard (Rochester,
WA) in 1983 specifically for tissue culture studies.
Seed and stratification
On November 1, 1984, seeds of each family were surface sterilized with 10% Clorox
for 7 min, rinsed in tap water for 4 h, soaked in water for 24 h, blotted dry, then placed
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1263
Table 1. Families from which seedlings and clones were propagated.
Female parentMale parentEight-year rank
(of 40 families)
11001037 4
10871088 5
10041049 6
1064108210
in plastic bags and held at 5 °C for stratification. Bag tops were left open to permit
air and water vapor exchange, and bags were manually massaged frequently to
prevent rot.
Tissue culture propagation
After eight weeks in stratification, about 20 seeds per family were sown in a
peat/vermiculite (1/1 v/v) mix in individual 100-mm2 plastic pots and placed in a
controlled environment chamber (23 ± 1 °C, 16-h photoperiod, mixture of incandescent and cool white fluorescent lamps ~120 µmol m −2 s −1) for germination. After the
cotyledons had fully expanded (4 to 5 weeks), a portion of the cotyledon rosette was
excised and cultured on bud induction medium as described by Timmis et al. (1992).
This procedure left at least three individual more-or-less evenly spaced cotyledons
on each germinant to sustain it as a viable seedling. When the epicotyl was about
5 cm long (May 1985), the germinants were grown on in Centralia, WA as stock plant
cutting donors (see below).
The cultured cotyledon explants were transferred to bud elongation medium and
subcultured as described by Pullman and Timmis (1991). When sufficient shoots had
developed to ensure that 50 good quality shoots were available for each clone, the
shoots were given an in vitro root induction treatment (Pullman and Timmis 1991).
The shoots were extracted carefully from the culture jars, dipped basally in a
commercial auxin formulation and set in plastic trays (260 × 530 mm × 60 mm deep)
containing a rooting medium of 1/1 (v/v) sphagnum peat and coarse horticultural
grade perlite. The rooting trays were placed in a walk-in growth room in Centralia,
WA where temperature was about 18 °C, relative humidity was maintained at 85%+
with high pressure propagating fog, and a 16-h photoperiod was provided by sodium
vapor lights (intensity varied across the room from about 150 to 300 µmol m −2 s −1).
By February 1986, most rooting had occurred. Rooted shoots (plantlets) were then
transferred to plastic Progeny Cells (Ray Leach, 40 mm diameter × 200 mm deep)
and placed in a cool, shaded greenhouse for acclimatization. Small plastic bags were
draped over the tops of the shoots after transfer and were removed after 2 or 3 weeks.
Following this, the plantlets were grown as seedlings, fertilized every other week
with half-strength Peters 20-20-20 (N,P,K) and watered as needed. In late May, they
were transplanted to Mima Nursery, Olympia, WA. At this time, clonal effects (e.g.,
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RITCHIE, DUKE AND TIMMIS
size, foliage color, branch angle, numbers of buds, etc.) were readily observed. Some
of the clones exhibited a plagiotropic habit. Plantlets were grown for the remainder
of the season, overwintered, and then grown for an additional year. They were lifted
for field planting in March 1987. By then, plagiotropism had virtually disappeared.
Rooted cutting propagation
Soon after the seedling stock plants (less three or four cotyledons) were delivered to
Centralia (May 1985), they were transplanted to 4-l plastic pots containing three parts
forestry mix (peat/vermiculite) and one part coarse perlite plus 20 g of Osmocote
17-6-10 (N,P,K) + micronutrients per pot. They were placed in greenhouses, hand
watered as needed, and grown in an 18-h photoperiod until mid-October. During the
growing period they were pinched back monthly to stimulate branch proliferation.
They were held in an unheated greenhouse until December 1985, when cuttings were
harvested from each plant and rooted under the same conditions used to root plantlets
(described above). After rooting, the rooted cuttings were transferred to Progeny
Cells, acclimated, and grown in a greenhouse until May, when they were planted in
the nursery beside the plantlets. Clonal effects were observed in the rooted cuttings,
but there was much more within-clone variability in the rooted cuttings than in the
plantlets. Plagiotropism was common in many clones, but it disappeared after the
nursery growing phase was completed.
Seedling propagation
Seeds for seedling controls, which were available for Families 10, 11 and 40 only,
were stratified at 4 °C for 6 weeks in spring 1985. They were sown on April 9 in
Progeny Cells in the greenhouse in a 1/1 (v/v) mix of peat and vermiculite. Seedlings
emerged in May and were grown until late summer. They were watered as needed
with a half-strength solution of Peters 20-20-20 (N,P,K). Seedlings were overwintered in the greenhouse, transplanted to the nursery in May 1986, and lifted with the
other stock types in March 1987. This process produced exceptionally large stock,
which averaged about 600 mm in height in contrast to the rooted cuttings (~400 mm)
and plantlets (~350 mm).
Unfortunately, not all clones responded similarly to propagation by either tissue
culture or rooting cuttings (Table 2), and families and clones that produced large
numbers of cuttings sometimes produced only a few plantlets (e.g., Family 5) and
vice versa (e.g., Family 11). Much of this variation was a reflection of large
differences among families and clones in rootability. Cuttings gave higher rooting
than plantlets in 23 of 29 clones, but there was no relationship (r2 = 0.02) between
rootability as cuttings and rootability as plantlets on a clonal basis (Figure 1). There
were not enough plants in Family 40 to permit its inclusion in the test.
Field planting
The seedlings, rooted cuttings and plantlets were planted in March 1987 on a
northwest-facing site (130 m elevation) on a hillside in the Clemons Tree Farm south
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1265
Table 2. Summary of propagation results by family and clone. Only those clones with at least 15 plants
of each type were used in the field test.
FamilyClonesNumber ofNumber ofNumber ofUsed inNumber of
rankcuttingsplantletsseedlingstestplots
4126 1 0
229 4
34819Yes4
44137Yes7
5116 1 0
219 2
3 9 0
417 4
514 1
6145 9 0
24020Yes5
34540Yes4
4 8 6
548 6
10121 653
220 1
31720Yes3
42117Yes3
52243Yes4
111 75348
21345
3 839
41970Yes3
of Porter, WA. The soil series is Lytell and the site index is 130--140 (Douglas-fir
height in feet at 50 years). A second growth Douglas-fir stand had been harvested
from the site one year earlier. The site had been burned in preparation for planting
and was fenced to prevent browsing.
The site was laid out in paired plots with one row of five trees of each stock type
constituting a plot. Each clone was replicated with a minimum of three plots, so at
least 15 good quality plantlets and rooted cuttings were required for each clone.
Where no seedlings were available, only cuttings and plantlets were used. For
planting, a tree had to be at least 250 mm tall and 6 mm in diameter at the base of the
stem, with a well developed root system. Of the 29 clones propagated, only eight met
these criteria (Table 2).
Trees were planted at a 1 × 1 m spacing. Plots were arrayed randomly across the
site. Filler trees were planted where planting spots existed that did not contain a test
1266
RITCHIE, DUKE AND TIMMIS
Figure 1. Rooting of plantlets and branch cuttings in a common rooting environment. Each point
represents a clonal mean. Clones varied in numbers of individuals (Table 2).
tree, so that there was a tree at every 1 × 1 m grid intersect across the site. The surplus
clonal material was planted on the test site in clonal demonstration blocks.
Data collection
Height and basal stem diameter were measured after the first (1987) and second
(1988) growing seasons. In March--April 1991, after five growing seasons, detailed
measurements were made on each tree (Ritchie and Keeley 1994) including:
(1) height above the ground of the terminal bud scar for each of the 1987--1991
growing seasons, (2) stem diameter (outside bark) in the middle of each internode
for each of the 1987--1990 increments, (3) number of nodal branches (NB) at each
of the 1987--1990 nodes, (4) length (LNB) and diameter (DNB) of the longest nodal
branch at each of the above nodes, (5) the number of internodal branches (INB) for
each of the 1987--1991 internodal segments, and (6) in early May 1992, each tree
crown was assessed for average bud activity based on a five point scale: 1 = dormant,
2 = swelling, 3 = breaking with bud scales parted, 4 = shoot beginning to elongate,
and 5 = shoot partially elongated. The data from (1) above were used to reconstruct
height growth curves. In March 1992, needle chlorophyll concentrations were also
determined (Ritchie and Keeley 1994).
Data analysis
The design structure was a split-plot, and the whole-plot structure was a completely
randomized design. Whole plots were defined by clone, with at least three plots per
clone. Subplots were defined by stock types (seedlings, rooted cuttings or plantlets).
Stock type was considered a fixed effect, and clone was considered random, because
the clones originated from seeds selected essentially at random from full-sib families.
Furthermore, clones were chosen as representative of a wider range of clones and
were not, in themselves, of particular interest. Error terms were calculated for each
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1267
effect using the expected mean squares. Individual means were compared by Fisher’s
Least Significant Difference (LSD) procedure (Milliken and Johnson 1984). The
analysis was run with SAS (SAS Institute, Cary, NC) general linear models (GLM)
procedure.
Results
Height and diameter
After 2 years in the field, most of the 165 test trees were growing vigorously. The
initial differences in height among stock types (STs) were maintained after 2 years,
but there were no differences among STs in annual height growth during 1988 (data
not shown) or 1989 (Figure 2).
Even after 5 years, survival was 100% for all STs. Although there was strong
evidence of height differences among STs for each year (Table 3), these differences
reflected a combination of differences in initial heights and slight differences in
Figure 2. 1989 Height increment of genetically matched Douglas-fir seedlings, rooted cuttings and
plantlets growing on a field site. Vertical bars are ± 1 SE calculated on plot means.
Table 3. P-values from ANOVA on effects of stock type (ST) and clone (CL) on total stem height and
internodal diameter for five successive annual growth increments.
YearStock typeCloneST × CL
HeightDiameterHeightDiameterHeightDiameter
19910.0002--0.0852--0.0485--
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RITCHIE, DUKE AND TIMMIS
growth rate, which were not statistically significant until after five growing seasons
(Figure 3). Clonal (CL) differences in height were significant for the 1987 and 1988
growing seasons, but not thereafter. There were ST × CL interactions in height during
four of the five years.
There were no clonal differences in stem diameter at any internode, and there were
no ST × CL interactions (Table 3). However, there were ST effects on stem diameter
at every internode. The differences were always in the order seedlings > rooted
cuttings > plantlets and were greatest at the oldest, most basal portion of the stem
(i.e., 1987; Figure 4). Absolute differences decreased as stem diameters decreased
with increasing height in the crowns. Proportional difference also decreased with
height in the tree. For example, the difference in diameter between STs at the 1987
Figure 3. Height growth of genetically matched Douglas-fir seedlings, rooted cuttings and plantlets on a
field site. Each line is pooled across families (seedlings) or clones (rooted cuttings and plantlets). Mean
separation at 0.05 by Fisher’s LSD test.
Figure 4. Internodal stem diameters of genetically matched Douglas-fir seedlings, rooted cuttings and
plantlets growing on a field site. Mean separation at 0.05 by Fisher’s LSD test.
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1269
internode was about 26% in contrast to a 21% difference at the 1990 internode. This
suggests that ST effect on stem diameter decreased with tree age.
Branch characteristics
There were ST effects on many branch characteristics at most nodal locations
(Table 4). The ST effects on length and diameter of nodal branches were particularly
marked. Effects of ST on numbers of nodal and internodal branches were significant
in the older internodes (1988, 1989), but decreased substantially in the more recently
formed nodes and internodes.
Effects on LNB and DNB were similar to those for stem diameter. Nodal branch
length increased in the order plantlets < rooted cuttings < seedlings (Figure 5a). The
absolute differences decreased from the bottom to the top of the trees, but in a relative
sense, they did not change appreciably. Branch diameters increased in the same
direction (Figure 5b). At the 1988 and 1990 internodes, branch diameters of rooted
cuttings and plantlets were similar, but were substantially smaller than those of
seedlings. Across the four nodes, branches of rooted cuttings were 12 to 21% thinner
and branches of plantlets were 18 to 25% thinner than seedling branches. The ST ×
CL interactions were generally not significant; exceptions were NB for 1990 (P =
0.0306) and 1987 (P = 0.0133), and INB for 1988 (P = 0.0202) and 1987 (P =
0.0040).
On a total tree basis, all STs had the same number of nodal branches (Table 5);
however, seedlings had significantly more internodal branches and carried about 9%
more branches in total than vegetative propagules.
Bud and foliage characteristics
Of the bud and foliar traits assessed, only bud condition in May differed significantly
among STs (Table 6).
Discussion
The three stock types exhibited similar height growth patterns throughout the 5-year
period, but because of differences in initial height, seedlings were taller than rooted
Table 4. P-values from ANOVA on effects of stock type and clone on numbers of nodal branches (NB),
length of longest nodal branch (LNB), diameter of longest nodal branch (DNB) and numbers of
internodal branches (INB) for four successive annual growth increments.
YearStock typeClone
NBLNBDNBINBNBLNBDNBINB
19900.02790.00390.12490.16070.01930.00070.29090.0706
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RITCHIE, DUKE AND TIMMIS
Figure 5. Length (a) and diameter (b) of nodal branches at different positions in the crowns of Douglas-fir
trees of three stock types. Mean separation at 0.05 by Fisher’s LSD test.
Table 5. Total numbers of branches on genetically matched seedlings, rooted cuttings and tissue culture
plantlets after five growing seasons in the field. Means pooled across eight clones. Mean separation at
0.05 with Fisher’s LSD test.
Stock typeTotal nodalTotal internodalTotal branches
branchesbranches
Seedling21a37a58a
Rooted cutting23a31b53b
Plantlet21a29b51b
cuttings, and rooted cuttings were taller than plantlets. Internodal stem diameters
varied in the order seedlings > rooted cuttings > plantlets for each nodal section.
Clonal effects on height and diameter were generally small and inconsistent. Stock
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1271
Table 6. Bud and foliage characteristics of genetically matched seedlings, rooted cuttings and tissue
culture plantlets during the fifth growing season in the field. Data are pooled across eight clones. Mean
separation at 0.05 with Fisher’s LSD test.
Stock typeMay budNeedle dryChl a contentChl b contentTotal ChlChl a/Chl b
conditionweight (mg)(mg g −1)(mg g −1)(mg g −1)
Seedlings4.07ab6.01.690.732.422.29
Root cuttings4.55a5.51.810.782.592.32
Plantlets3.65b5.71.90.822.722.31
type had a large and consistent effect on nodal branch length and diameter, and both
varied in the order seedlings > rooted cuttings > plantlets. There were clonal effects
on branch length, but not branch number. Total number of branches also varied
significantly in the order seedlings > rooted cuttings > plantlets. Foliar and bud traits
were not affected by stock type or clone, except that May bud activity tended to be
greater in seedlings than in rooted cuttings or plantlets.
To determine whether Douglas-fir plantlets propagated in vitro and rooted cuttings
from juvenile stock plants exhibit signs of early maturation, we overlayed the
morphological characteristics of this plant material across known morphological
maturation markers, then tested for goodness of fit.
Ritchie and Keeley (1994), working with 6-year-old Douglas-fir grafts originating
from juvenile (2 years from seed) and mature (10 years from seed) scions, found that
three traits associated with maturation could be used for diagnostic purposes: (1)
internodal stem diameters were about 30% greater in juvenile trees across all whorls,
(2) the length of the longest nodal branches in juvenile trees was 22 to 35% greater
than that in mature trees, depending on whorl position, and (3) the diameter of these
nodal branches was from 11 to 49% greater in juvenile trees than in mature trees,
depending on whorl position. Numbers of branches, foliage traits, and height growth
patterns were confounded with other factors and of limited diagnostic value.
In the present study, stem diameters significantly decreased in the order seedlings
> rooted cuttings > plantlets (Table 3, Figure 4). The percent decrease from seedling
to plantlet ranged between 21 and 26%, which was only slightly less than the
differences between juvenile and mature grafts in the earlier study. Rooted cuttings
were always roughly intermediate between seedlings and plantlets. Stock type effects
on nodal branch length were highly significant (Table 4). Seedling LNB was consistently about 13% greater than rooted cutting LNB and 21% greater than plantlet LNB
across all whorls (Figure 5a). This is within the range demonstrated for the juvenile
versus mature grafts. Similarly, stock type effects on branch diameter were also
highly significant across all whorls (except 1990) (Table 4). Across whorls, differences between seedlings and rooted cuttings averaged about 17%, and between
seedlings and plantlets, about 24% (Figure 5b). Branch diameters of rooted cuttings
were similar to those of plantlets.
These comparisons confirm that, under the test conditions, vegetative propagules
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RITCHIE, DUKE AND TIMMIS
of Douglas-fir exhibited morphological traits indicative of ontogenetic aging. Furthermore, expression of these traits persisted in the field for at least 5 years.
Douglas-fir plantlets established in coastal Oregon (Ritchie and Long 1986) continue
to exhibit similar traits after 12 years in the field (see also Gupta et al. 1991).
Physiological tests of rooted cuttings have shown that they cold acclimate earlier in
winter, deacclimate later in spring, and attain a more intense state of endodormancy
than genetically matched seedlings (Ritchie et al. 1992). These traits may also be
associated with maturation.
To determine whether the observed morphological differences among stock types
resulted from physiological aging per se or from differences in initial stock type
sizes, we conducted an analysis of covariance with initial height as a covariate. The
ANOVA F-tests for stock type differences gave small P-values with the covariate
model, although the significance generally decreased (Table 7). For example, the
P-values for stock type differences in 1991 height were 0.0002 without adjusting for
the covariate and 0.02 after adjusting. Unfortunately, the imbalance in the study made
the adjusted stock type means nonestimable, so a complete analysis of covariance
was not possible, but we speculate that the size differences among stock types are not
entirely an artifact of differences in initial plant size.
A more definitive experimental design would include matching vegetative and
seedling propagules both genetically and morphologically at the time of planting, and
adding another stock type, namely, propagules derived from rooted cuttings taken
from seedlings at the hypocotyl stage (Frampton and Isik 1987). Such cuttings root
within 2--3 weeks, and should be very similar to seedlings.
There are several possible mechanisms to account for the observed early maturation in vegetative propagules. (1) Prolonged photoperiod extension----long days
imposed on stock plant cutting donors can hasten maturation (Bongarten and Hanover 1985, Lascoux et al. 1993). (2) Water stress----low water potentials imposed on
Table 7. Comparison of analysis of variance P-values and analysis of covariance P-values with initial
plant height as covariate. HT = height, DIAM = stem diameter, LNB = length of longest nodal branch,
DNB = diameter of longest nodal branch.
Variable
Analysis of covariance
1991 HT0.02090.0002
1990 HT0.06350.0002
1990 DIAM0.00110.0001
1989 HT0.03040.0001
1989 DIAM0.00200.0001
1989 LNB0.02910.0009
1989 DNB0.01550.0001
1988 HT0.00630.0001
1988 DIAM0.00170.0001
Analysis of variance
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1273
cuttings or plantlets during rooting can promote maturation in several species
(reviewed by Hackett 1985). Tissue culture plantlets have a thin leaf cuticle and
exhibit poor stomatal control (Sutter and Langhans 1982, Donnelly and Vidaver
1984). Smith (D. Smith, cited in McKeand 1985) observed that radiata pine (P. radiata D. Don) plantlets that received little ex vitro stress remained juvenile. (3) Root
architecture----roots produced by loblolly pine (P. taeda L.) plantlets are inefficient
at nutrient uptake and may also be inefficient at synthesizing growth regulators
(McKeand and Allen 1984). Incomplete vascular connections between root and stem
tissues in micropropagated trees may lead to inadequate supplies of root-produced
growth regulators (Timmis et al. 1992). Either might promote early maturation.
Abnormal root development might also lead to increased water stress; however,
Anderson et al. (1992), using a reciprocal grafting approach, demonstrated that
accelerated maturation is induced by shoots, not root systems, of loblolly pine
plantlets. (4) Tissue of origin----in at least two reports, micropropagules originating
as cotyledon-derived adventitious buds exhibited mature characteristics (Monteuuis
and Dumas 1992, Frampton and Foster 1993). In contrast, loblolly pine plants
micropropagated from axillary or fasicular buds of greenhouse-grown seedlings did
not show early maturation after 5 years in the field (L.J. Frampton, personal communication). Frampton and Foster (1993) suggest that early maturation in this species
is an artifact of the cotyledon system and does not necessarily result from the
propagation method. Because the cotyledon itself is a determinate organ, it may give
rise to plants that reflect its programmed senescence (M.S. Greenwood, personal
communication).
Use of vegetative propagation to hasten maturation could be of practical value.
Improvements in stem form, including smaller and fewer branches, reduced stem
taper, and thinner bark (Spencer 1987), have been achieved in radiata pine by
propagating from slightly mature (two years from seed) ortets (Whiteman et al.
1990). Douglas-fir might be amenable to similar manipulation.
Acknowledgments
We thank Drs. R.W. Stonecypher and C.B. Talbert for help with the experimental field design, and Drs.
M.S. Greenwood and L.J. Frampton for reviewing an earlier version of this manuscript. Patty A. Ward
and James Keeley provided valuable technical assistance throughout the study. Karen Paige supervised
plantlet production. Patricia Crank performed chlorophyll analyses and Gary Helland has maintained the
test site since it was established. This research and its publication were supported by Weyerhaeuser
Company.
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© 1994 Heron Publishing----Victoria, Canada
Maturation in Douglas-fir: II. Maturation characteristics of
genetically matched Douglas-fir seedlings, rooted cuttings and
tissue culture plantlets during and after 5 years of field growth
GARY A. RITCHIE,1 STEVEN D. DUKE2 and ROGER TIMMIS2
1
Weyerhaeuser Company, G.R. Staebler Forest Resources Research Center, 505 North Pearl Street,
Centralia, WA 98531, USA
2
Weyerhaeuser Company Technology Center, Federal Way, Tacoma, WA 98003, USA
Received February 23, 1994
Summary
Seedlings, rooted cuttings from juvenile stock plants, and cotyledon-derived tissue culture plantlets were
propagated from several coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) full-sib families so
that the rooted cuttings and plantlets were clonally identical. The stock types (seedlings, rooted cuttings
and plantlets) were planted in the field in spring 1987. In fall 1991, after five complete growing seasons,
the plants were measured and these values compared to maturation ‘‘markers’’ identified for Douglas-fir
in the companion paper (Ritchie and Keeley 1994). Nodal branch lengths and nodal branch diameters
decreased in the order seedlings > rooted cuttings > plantlets. The decreases were about 21% for nodal
branch lengths and 24% for nodal branch diameters. Seedlings carried significantly more total branches
(nodal + internodal) than the other two stock types. Height growth was similar for the three stock types,
but plantlet height increment was beginning to decrease during the fourth year. We conclude that
vegetative propagules of Douglas-fir exhibited traits of mature trees. These were particularly marked in
the cotyledon-derived plantlets.
Keywords: juvenility, clonal forestry, tissue culture, organogenesis, Pseudotsuga menziesii.
Introduction
Cloning of forest trees offers an approach for exploiting nonadditive, as well as
additive, genetic variance and for increasing crop uniformity (Zobel 1992, Talbert et
al. 1993). Several successful cloning programs have been developed with species in
which mature individuals produce juvenile stump or root sprouts (Ritchie 1994).
With such species, it is possible to evaluate the phenotypic or genotypic potential of
a mature individual, then to replicate it from the propagated juvenile tissue that it
produces. Poplars, (Populus spp.), willows (Salix spp.) and several eucalypt (Eucalyptus spp.) species have been exploited in this manner (Brandão 1984, Zsuffa 1984,
Leakey 1987).
However, with species that do not produce easily obtainable juvenile tissue,
cloning is difficult and expensive. It requires long-term storage of juvenile clonal
ramets while genetically identical ramets are field tested. Following testing, the
selected, stored clones must be repropagated and bulked up to commercial quantities
for field planting. In vitro propagation, through either organogenesis or more recently, somatic embryogenesis, offers considerable promise for cloning tree species
1262
RITCHIE, DUKE AND TIMMIS
(Dunstan 1988). In such systems, juvenile clone banks may be maintained at low
temperatures (tissue culture plantlets) or cryogenically (somatic embryos) (Gupta et
al. 1993) at relatively low cost during the test period.
Because of the potential of organogenesis for cloning conifer species (Durzan and
Campbell 1974, McKeand and Weir 1984), many tissue culture programs were
initiated during the early 1970s. As the plantlets in these tests began to grow, they
often exhibited mature morphological characteristics in the field even though they
had been cultured from juvenile explants (e.g., McKeand 1985).
Field observations of coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco)
plantlets derived from cotyledon culture, which were growing on several test sites in
western Oregon, prompted Ritchie and Long (1986) to speculate that these trees
might be exhibiting mature characteristics. Many of the plantlets appeared to have
shorter and fewer branches, narrower branch angles, more bluish-green foliage and
coarser needles than seedling controls. However, it was not possible to determine
unequivocally that these plantlets were exhibiting mature characteristics because the
morphological attributes of maturation in Douglas-fir had not been established.
We therefore initiated a study in which an array of putative morphological maturation ‘‘markers’’ in coastal Douglas-fir was screened in an attempt to identify those
that could be used in the field (Ritchie and Keeley 1994). In the present paper, we
report on the use of these markers to evaluate genetically matched, field-planted
Douglas-fir vegetative propagules from tissue culture and rooted cuttings. We also
present information on propagation characteristics and field performance of these
propagule types.
Methods
Douglas-fir seedlings, rooted cuttings and plantlets were produced from seeds of the
same full-sib families. Both of the vegetative propagule types were produced from
the same clones. Following laboratory and greenhouse propagation, the plants were
grown in a bareroot nursery for one year before planting on a forest site. Performance
and morphological development were examined after 2 and 5 years. The morphological traits were then compared to the maturation markers defined for Douglas-fir
by Ritchie and Keeley (1994).
Genetic material
Six full-sib families were selected (Table 1) to represent primarily top performing
crosses with one low performer (1073 × 1077) included for comparison. Seeds from
these crosses were produced at a Weyerhaeuser Company seed orchard (Rochester,
WA) in 1983 specifically for tissue culture studies.
Seed and stratification
On November 1, 1984, seeds of each family were surface sterilized with 10% Clorox
for 7 min, rinsed in tap water for 4 h, soaked in water for 24 h, blotted dry, then placed
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1263
Table 1. Families from which seedlings and clones were propagated.
Female parentMale parentEight-year rank
(of 40 families)
11001037 4
10871088 5
10041049 6
1064108210
in plastic bags and held at 5 °C for stratification. Bag tops were left open to permit
air and water vapor exchange, and bags were manually massaged frequently to
prevent rot.
Tissue culture propagation
After eight weeks in stratification, about 20 seeds per family were sown in a
peat/vermiculite (1/1 v/v) mix in individual 100-mm2 plastic pots and placed in a
controlled environment chamber (23 ± 1 °C, 16-h photoperiod, mixture of incandescent and cool white fluorescent lamps ~120 µmol m −2 s −1) for germination. After the
cotyledons had fully expanded (4 to 5 weeks), a portion of the cotyledon rosette was
excised and cultured on bud induction medium as described by Timmis et al. (1992).
This procedure left at least three individual more-or-less evenly spaced cotyledons
on each germinant to sustain it as a viable seedling. When the epicotyl was about
5 cm long (May 1985), the germinants were grown on in Centralia, WA as stock plant
cutting donors (see below).
The cultured cotyledon explants were transferred to bud elongation medium and
subcultured as described by Pullman and Timmis (1991). When sufficient shoots had
developed to ensure that 50 good quality shoots were available for each clone, the
shoots were given an in vitro root induction treatment (Pullman and Timmis 1991).
The shoots were extracted carefully from the culture jars, dipped basally in a
commercial auxin formulation and set in plastic trays (260 × 530 mm × 60 mm deep)
containing a rooting medium of 1/1 (v/v) sphagnum peat and coarse horticultural
grade perlite. The rooting trays were placed in a walk-in growth room in Centralia,
WA where temperature was about 18 °C, relative humidity was maintained at 85%+
with high pressure propagating fog, and a 16-h photoperiod was provided by sodium
vapor lights (intensity varied across the room from about 150 to 300 µmol m −2 s −1).
By February 1986, most rooting had occurred. Rooted shoots (plantlets) were then
transferred to plastic Progeny Cells (Ray Leach, 40 mm diameter × 200 mm deep)
and placed in a cool, shaded greenhouse for acclimatization. Small plastic bags were
draped over the tops of the shoots after transfer and were removed after 2 or 3 weeks.
Following this, the plantlets were grown as seedlings, fertilized every other week
with half-strength Peters 20-20-20 (N,P,K) and watered as needed. In late May, they
were transplanted to Mima Nursery, Olympia, WA. At this time, clonal effects (e.g.,
1264
RITCHIE, DUKE AND TIMMIS
size, foliage color, branch angle, numbers of buds, etc.) were readily observed. Some
of the clones exhibited a plagiotropic habit. Plantlets were grown for the remainder
of the season, overwintered, and then grown for an additional year. They were lifted
for field planting in March 1987. By then, plagiotropism had virtually disappeared.
Rooted cutting propagation
Soon after the seedling stock plants (less three or four cotyledons) were delivered to
Centralia (May 1985), they were transplanted to 4-l plastic pots containing three parts
forestry mix (peat/vermiculite) and one part coarse perlite plus 20 g of Osmocote
17-6-10 (N,P,K) + micronutrients per pot. They were placed in greenhouses, hand
watered as needed, and grown in an 18-h photoperiod until mid-October. During the
growing period they were pinched back monthly to stimulate branch proliferation.
They were held in an unheated greenhouse until December 1985, when cuttings were
harvested from each plant and rooted under the same conditions used to root plantlets
(described above). After rooting, the rooted cuttings were transferred to Progeny
Cells, acclimated, and grown in a greenhouse until May, when they were planted in
the nursery beside the plantlets. Clonal effects were observed in the rooted cuttings,
but there was much more within-clone variability in the rooted cuttings than in the
plantlets. Plagiotropism was common in many clones, but it disappeared after the
nursery growing phase was completed.
Seedling propagation
Seeds for seedling controls, which were available for Families 10, 11 and 40 only,
were stratified at 4 °C for 6 weeks in spring 1985. They were sown on April 9 in
Progeny Cells in the greenhouse in a 1/1 (v/v) mix of peat and vermiculite. Seedlings
emerged in May and were grown until late summer. They were watered as needed
with a half-strength solution of Peters 20-20-20 (N,P,K). Seedlings were overwintered in the greenhouse, transplanted to the nursery in May 1986, and lifted with the
other stock types in March 1987. This process produced exceptionally large stock,
which averaged about 600 mm in height in contrast to the rooted cuttings (~400 mm)
and plantlets (~350 mm).
Unfortunately, not all clones responded similarly to propagation by either tissue
culture or rooting cuttings (Table 2), and families and clones that produced large
numbers of cuttings sometimes produced only a few plantlets (e.g., Family 5) and
vice versa (e.g., Family 11). Much of this variation was a reflection of large
differences among families and clones in rootability. Cuttings gave higher rooting
than plantlets in 23 of 29 clones, but there was no relationship (r2 = 0.02) between
rootability as cuttings and rootability as plantlets on a clonal basis (Figure 1). There
were not enough plants in Family 40 to permit its inclusion in the test.
Field planting
The seedlings, rooted cuttings and plantlets were planted in March 1987 on a
northwest-facing site (130 m elevation) on a hillside in the Clemons Tree Farm south
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1265
Table 2. Summary of propagation results by family and clone. Only those clones with at least 15 plants
of each type were used in the field test.
FamilyClonesNumber ofNumber ofNumber ofUsed inNumber of
rankcuttingsplantletsseedlingstestplots
4126 1 0
229 4
34819Yes4
44137Yes7
5116 1 0
219 2
3 9 0
417 4
514 1
6145 9 0
24020Yes5
34540Yes4
4 8 6
548 6
10121 653
220 1
31720Yes3
42117Yes3
52243Yes4
111 75348
21345
3 839
41970Yes3
of Porter, WA. The soil series is Lytell and the site index is 130--140 (Douglas-fir
height in feet at 50 years). A second growth Douglas-fir stand had been harvested
from the site one year earlier. The site had been burned in preparation for planting
and was fenced to prevent browsing.
The site was laid out in paired plots with one row of five trees of each stock type
constituting a plot. Each clone was replicated with a minimum of three plots, so at
least 15 good quality plantlets and rooted cuttings were required for each clone.
Where no seedlings were available, only cuttings and plantlets were used. For
planting, a tree had to be at least 250 mm tall and 6 mm in diameter at the base of the
stem, with a well developed root system. Of the 29 clones propagated, only eight met
these criteria (Table 2).
Trees were planted at a 1 × 1 m spacing. Plots were arrayed randomly across the
site. Filler trees were planted where planting spots existed that did not contain a test
1266
RITCHIE, DUKE AND TIMMIS
Figure 1. Rooting of plantlets and branch cuttings in a common rooting environment. Each point
represents a clonal mean. Clones varied in numbers of individuals (Table 2).
tree, so that there was a tree at every 1 × 1 m grid intersect across the site. The surplus
clonal material was planted on the test site in clonal demonstration blocks.
Data collection
Height and basal stem diameter were measured after the first (1987) and second
(1988) growing seasons. In March--April 1991, after five growing seasons, detailed
measurements were made on each tree (Ritchie and Keeley 1994) including:
(1) height above the ground of the terminal bud scar for each of the 1987--1991
growing seasons, (2) stem diameter (outside bark) in the middle of each internode
for each of the 1987--1990 increments, (3) number of nodal branches (NB) at each
of the 1987--1990 nodes, (4) length (LNB) and diameter (DNB) of the longest nodal
branch at each of the above nodes, (5) the number of internodal branches (INB) for
each of the 1987--1991 internodal segments, and (6) in early May 1992, each tree
crown was assessed for average bud activity based on a five point scale: 1 = dormant,
2 = swelling, 3 = breaking with bud scales parted, 4 = shoot beginning to elongate,
and 5 = shoot partially elongated. The data from (1) above were used to reconstruct
height growth curves. In March 1992, needle chlorophyll concentrations were also
determined (Ritchie and Keeley 1994).
Data analysis
The design structure was a split-plot, and the whole-plot structure was a completely
randomized design. Whole plots were defined by clone, with at least three plots per
clone. Subplots were defined by stock types (seedlings, rooted cuttings or plantlets).
Stock type was considered a fixed effect, and clone was considered random, because
the clones originated from seeds selected essentially at random from full-sib families.
Furthermore, clones were chosen as representative of a wider range of clones and
were not, in themselves, of particular interest. Error terms were calculated for each
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1267
effect using the expected mean squares. Individual means were compared by Fisher’s
Least Significant Difference (LSD) procedure (Milliken and Johnson 1984). The
analysis was run with SAS (SAS Institute, Cary, NC) general linear models (GLM)
procedure.
Results
Height and diameter
After 2 years in the field, most of the 165 test trees were growing vigorously. The
initial differences in height among stock types (STs) were maintained after 2 years,
but there were no differences among STs in annual height growth during 1988 (data
not shown) or 1989 (Figure 2).
Even after 5 years, survival was 100% for all STs. Although there was strong
evidence of height differences among STs for each year (Table 3), these differences
reflected a combination of differences in initial heights and slight differences in
Figure 2. 1989 Height increment of genetically matched Douglas-fir seedlings, rooted cuttings and
plantlets growing on a field site. Vertical bars are ± 1 SE calculated on plot means.
Table 3. P-values from ANOVA on effects of stock type (ST) and clone (CL) on total stem height and
internodal diameter for five successive annual growth increments.
YearStock typeCloneST × CL
HeightDiameterHeightDiameterHeightDiameter
19910.0002--0.0852--0.0485--
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RITCHIE, DUKE AND TIMMIS
growth rate, which were not statistically significant until after five growing seasons
(Figure 3). Clonal (CL) differences in height were significant for the 1987 and 1988
growing seasons, but not thereafter. There were ST × CL interactions in height during
four of the five years.
There were no clonal differences in stem diameter at any internode, and there were
no ST × CL interactions (Table 3). However, there were ST effects on stem diameter
at every internode. The differences were always in the order seedlings > rooted
cuttings > plantlets and were greatest at the oldest, most basal portion of the stem
(i.e., 1987; Figure 4). Absolute differences decreased as stem diameters decreased
with increasing height in the crowns. Proportional difference also decreased with
height in the tree. For example, the difference in diameter between STs at the 1987
Figure 3. Height growth of genetically matched Douglas-fir seedlings, rooted cuttings and plantlets on a
field site. Each line is pooled across families (seedlings) or clones (rooted cuttings and plantlets). Mean
separation at 0.05 by Fisher’s LSD test.
Figure 4. Internodal stem diameters of genetically matched Douglas-fir seedlings, rooted cuttings and
plantlets growing on a field site. Mean separation at 0.05 by Fisher’s LSD test.
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1269
internode was about 26% in contrast to a 21% difference at the 1990 internode. This
suggests that ST effect on stem diameter decreased with tree age.
Branch characteristics
There were ST effects on many branch characteristics at most nodal locations
(Table 4). The ST effects on length and diameter of nodal branches were particularly
marked. Effects of ST on numbers of nodal and internodal branches were significant
in the older internodes (1988, 1989), but decreased substantially in the more recently
formed nodes and internodes.
Effects on LNB and DNB were similar to those for stem diameter. Nodal branch
length increased in the order plantlets < rooted cuttings < seedlings (Figure 5a). The
absolute differences decreased from the bottom to the top of the trees, but in a relative
sense, they did not change appreciably. Branch diameters increased in the same
direction (Figure 5b). At the 1988 and 1990 internodes, branch diameters of rooted
cuttings and plantlets were similar, but were substantially smaller than those of
seedlings. Across the four nodes, branches of rooted cuttings were 12 to 21% thinner
and branches of plantlets were 18 to 25% thinner than seedling branches. The ST ×
CL interactions were generally not significant; exceptions were NB for 1990 (P =
0.0306) and 1987 (P = 0.0133), and INB for 1988 (P = 0.0202) and 1987 (P =
0.0040).
On a total tree basis, all STs had the same number of nodal branches (Table 5);
however, seedlings had significantly more internodal branches and carried about 9%
more branches in total than vegetative propagules.
Bud and foliage characteristics
Of the bud and foliar traits assessed, only bud condition in May differed significantly
among STs (Table 6).
Discussion
The three stock types exhibited similar height growth patterns throughout the 5-year
period, but because of differences in initial height, seedlings were taller than rooted
Table 4. P-values from ANOVA on effects of stock type and clone on numbers of nodal branches (NB),
length of longest nodal branch (LNB), diameter of longest nodal branch (DNB) and numbers of
internodal branches (INB) for four successive annual growth increments.
YearStock typeClone
NBLNBDNBINBNBLNBDNBINB
19900.02790.00390.12490.16070.01930.00070.29090.0706
1270
RITCHIE, DUKE AND TIMMIS
Figure 5. Length (a) and diameter (b) of nodal branches at different positions in the crowns of Douglas-fir
trees of three stock types. Mean separation at 0.05 by Fisher’s LSD test.
Table 5. Total numbers of branches on genetically matched seedlings, rooted cuttings and tissue culture
plantlets after five growing seasons in the field. Means pooled across eight clones. Mean separation at
0.05 with Fisher’s LSD test.
Stock typeTotal nodalTotal internodalTotal branches
branchesbranches
Seedling21a37a58a
Rooted cutting23a31b53b
Plantlet21a29b51b
cuttings, and rooted cuttings were taller than plantlets. Internodal stem diameters
varied in the order seedlings > rooted cuttings > plantlets for each nodal section.
Clonal effects on height and diameter were generally small and inconsistent. Stock
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1271
Table 6. Bud and foliage characteristics of genetically matched seedlings, rooted cuttings and tissue
culture plantlets during the fifth growing season in the field. Data are pooled across eight clones. Mean
separation at 0.05 with Fisher’s LSD test.
Stock typeMay budNeedle dryChl a contentChl b contentTotal ChlChl a/Chl b
conditionweight (mg)(mg g −1)(mg g −1)(mg g −1)
Seedlings4.07ab6.01.690.732.422.29
Root cuttings4.55a5.51.810.782.592.32
Plantlets3.65b5.71.90.822.722.31
type had a large and consistent effect on nodal branch length and diameter, and both
varied in the order seedlings > rooted cuttings > plantlets. There were clonal effects
on branch length, but not branch number. Total number of branches also varied
significantly in the order seedlings > rooted cuttings > plantlets. Foliar and bud traits
were not affected by stock type or clone, except that May bud activity tended to be
greater in seedlings than in rooted cuttings or plantlets.
To determine whether Douglas-fir plantlets propagated in vitro and rooted cuttings
from juvenile stock plants exhibit signs of early maturation, we overlayed the
morphological characteristics of this plant material across known morphological
maturation markers, then tested for goodness of fit.
Ritchie and Keeley (1994), working with 6-year-old Douglas-fir grafts originating
from juvenile (2 years from seed) and mature (10 years from seed) scions, found that
three traits associated with maturation could be used for diagnostic purposes: (1)
internodal stem diameters were about 30% greater in juvenile trees across all whorls,
(2) the length of the longest nodal branches in juvenile trees was 22 to 35% greater
than that in mature trees, depending on whorl position, and (3) the diameter of these
nodal branches was from 11 to 49% greater in juvenile trees than in mature trees,
depending on whorl position. Numbers of branches, foliage traits, and height growth
patterns were confounded with other factors and of limited diagnostic value.
In the present study, stem diameters significantly decreased in the order seedlings
> rooted cuttings > plantlets (Table 3, Figure 4). The percent decrease from seedling
to plantlet ranged between 21 and 26%, which was only slightly less than the
differences between juvenile and mature grafts in the earlier study. Rooted cuttings
were always roughly intermediate between seedlings and plantlets. Stock type effects
on nodal branch length were highly significant (Table 4). Seedling LNB was consistently about 13% greater than rooted cutting LNB and 21% greater than plantlet LNB
across all whorls (Figure 5a). This is within the range demonstrated for the juvenile
versus mature grafts. Similarly, stock type effects on branch diameter were also
highly significant across all whorls (except 1990) (Table 4). Across whorls, differences between seedlings and rooted cuttings averaged about 17%, and between
seedlings and plantlets, about 24% (Figure 5b). Branch diameters of rooted cuttings
were similar to those of plantlets.
These comparisons confirm that, under the test conditions, vegetative propagules
1272
RITCHIE, DUKE AND TIMMIS
of Douglas-fir exhibited morphological traits indicative of ontogenetic aging. Furthermore, expression of these traits persisted in the field for at least 5 years.
Douglas-fir plantlets established in coastal Oregon (Ritchie and Long 1986) continue
to exhibit similar traits after 12 years in the field (see also Gupta et al. 1991).
Physiological tests of rooted cuttings have shown that they cold acclimate earlier in
winter, deacclimate later in spring, and attain a more intense state of endodormancy
than genetically matched seedlings (Ritchie et al. 1992). These traits may also be
associated with maturation.
To determine whether the observed morphological differences among stock types
resulted from physiological aging per se or from differences in initial stock type
sizes, we conducted an analysis of covariance with initial height as a covariate. The
ANOVA F-tests for stock type differences gave small P-values with the covariate
model, although the significance generally decreased (Table 7). For example, the
P-values for stock type differences in 1991 height were 0.0002 without adjusting for
the covariate and 0.02 after adjusting. Unfortunately, the imbalance in the study made
the adjusted stock type means nonestimable, so a complete analysis of covariance
was not possible, but we speculate that the size differences among stock types are not
entirely an artifact of differences in initial plant size.
A more definitive experimental design would include matching vegetative and
seedling propagules both genetically and morphologically at the time of planting, and
adding another stock type, namely, propagules derived from rooted cuttings taken
from seedlings at the hypocotyl stage (Frampton and Isik 1987). Such cuttings root
within 2--3 weeks, and should be very similar to seedlings.
There are several possible mechanisms to account for the observed early maturation in vegetative propagules. (1) Prolonged photoperiod extension----long days
imposed on stock plant cutting donors can hasten maturation (Bongarten and Hanover 1985, Lascoux et al. 1993). (2) Water stress----low water potentials imposed on
Table 7. Comparison of analysis of variance P-values and analysis of covariance P-values with initial
plant height as covariate. HT = height, DIAM = stem diameter, LNB = length of longest nodal branch,
DNB = diameter of longest nodal branch.
Variable
Analysis of covariance
1991 HT0.02090.0002
1990 HT0.06350.0002
1990 DIAM0.00110.0001
1989 HT0.03040.0001
1989 DIAM0.00200.0001
1989 LNB0.02910.0009
1989 DNB0.01550.0001
1988 HT0.00630.0001
1988 DIAM0.00170.0001
Analysis of variance
MATURATION OF DOUGLAS-FIR VEGETATIVE PROPAGULES
1273
cuttings or plantlets during rooting can promote maturation in several species
(reviewed by Hackett 1985). Tissue culture plantlets have a thin leaf cuticle and
exhibit poor stomatal control (Sutter and Langhans 1982, Donnelly and Vidaver
1984). Smith (D. Smith, cited in McKeand 1985) observed that radiata pine (P. radiata D. Don) plantlets that received little ex vitro stress remained juvenile. (3) Root
architecture----roots produced by loblolly pine (P. taeda L.) plantlets are inefficient
at nutrient uptake and may also be inefficient at synthesizing growth regulators
(McKeand and Allen 1984). Incomplete vascular connections between root and stem
tissues in micropropagated trees may lead to inadequate supplies of root-produced
growth regulators (Timmis et al. 1992). Either might promote early maturation.
Abnormal root development might also lead to increased water stress; however,
Anderson et al. (1992), using a reciprocal grafting approach, demonstrated that
accelerated maturation is induced by shoots, not root systems, of loblolly pine
plantlets. (4) Tissue of origin----in at least two reports, micropropagules originating
as cotyledon-derived adventitious buds exhibited mature characteristics (Monteuuis
and Dumas 1992, Frampton and Foster 1993). In contrast, loblolly pine plants
micropropagated from axillary or fasicular buds of greenhouse-grown seedlings did
not show early maturation after 5 years in the field (L.J. Frampton, personal communication). Frampton and Foster (1993) suggest that early maturation in this species
is an artifact of the cotyledon system and does not necessarily result from the
propagation method. Because the cotyledon itself is a determinate organ, it may give
rise to plants that reflect its programmed senescence (M.S. Greenwood, personal
communication).
Use of vegetative propagation to hasten maturation could be of practical value.
Improvements in stem form, including smaller and fewer branches, reduced stem
taper, and thinner bark (Spencer 1987), have been achieved in radiata pine by
propagating from slightly mature (two years from seed) ortets (Whiteman et al.
1990). Douglas-fir might be amenable to similar manipulation.
Acknowledgments
We thank Drs. R.W. Stonecypher and C.B. Talbert for help with the experimental field design, and Drs.
M.S. Greenwood and L.J. Frampton for reviewing an earlier version of this manuscript. Patty A. Ward
and James Keeley provided valuable technical assistance throughout the study. Karen Paige supervised
plantlet production. Patricia Crank performed chlorophyll analyses and Gary Helland has maintained the
test site since it was established. This research and its publication were supported by Weyerhaeuser
Company.
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