Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol15.1995:

Tree Physiology 15, 433--438
© 1995 Heron Publishing----Victoria, Canada

Juvenility and maturation in conifers: current concepts
MICHAEL S. GREENWOOD
Department of Forest Ecosystem Science, College of Natural Resources, Forestry and Agriculture, University of Maine, Orono, ME 04469, USA
Received August 10, 1994

Summary Maturation in conifers includes several distinct
and persistent changes in the growth habits of apical meristems.
Despite many studies on maturation in conifers, there are still
many aspects of the process that have not been elucidated. For
example, it is not known why maturation of cotyledon-derived
tissue culture plantlets is rapid, whereas the natural maturation
process is gradual. Also, it is not known whether rejuvenation
occurs as a result of mature cells reverting to the juvenile state,
or whether rejuvenation results from selective multiplication
of cells that have never matured. In this paper, I review the
primary causes of maturation in woody plants, with emphasis
on gene expression and its role in cell determination during the
maturation process. Recent experiments demonstrating both

accelerated maturation and apparent rejuvenation in several
woody species are discussed with reference to several maturation models.
Keywords: apical meristems, cell determination, gene expression, growth, maturation models, tissue culture plantlets.

Introduction
Maturation (also called phase change or ontogenetic aging) in
woody plants has received renewed attention over the last 15
years as a result of efforts to effect either flowering or vegetative propagation of woody plants. These efforts have met with
limited success because vegetative propagation of woody
plants becomes increasingly difficult with age and juvenile
trees can rarely be induced to flower. Thus, a detailed understanding of the maturation process is essential to the development and evaluation of methods for rejuvenating tissue and
enhancing flowering. Maturation is an integral part of the life
cycle of all vascular plants. Four phases of maturation have
been recognized (Greenwood 1987, Poethig 1990): (1) the
embryonic phase, (2) the post-embryonic juvenile vegetative
phase, (3) the adult vegetative phase, and (4) the adult reproductive phase. In woody plants, maturation is associated with
decreased growth rates during phases 2--4 (which often persist
in vegetative propagules), increased plagiotropism, and
changes in reproductive competence, branching characteristics
and foliar morphology. In addition to morphological changes,

there are numerous physiological and biochemical changes
that accompany the transition to the adult phases (see reviews
by Hackett 1985, Zimmerman et al. 1985, Greenwood 1987,
Pierik 1990, Poethig 1990, Haffner et al. 1991, Bonga and von

Aderkas 1993, Greenwood and Hutchinson 1993).
Maturational changes are particularly persistent and difficult
to reverse in conifers, as has been demonstrated in several
species by following the growth of grafted scions from ortets
of different ages for several years. In these studies, maturational characteristics were observed without the confounding
effects of size, because the grafted plants were initially the
same height. In radiata pine (Pinus radiata D. Don), loblolly
pine (Pinus taeda L.), eastern larch (Larix laricina (Du Roi) C.
Koch) and Douglas-fir (Pseudotsuga menziesii (Mirb.)
Franco), height, diameter growth and number of branches per
unit of stem length all decrease with increasing ortet age, with
the most pronounced decline occurring between ages 1 and 4
years (Sweet 1973, Greenwood 1984, Greenwood et al. 1989,
Ritchie and Keeley 1994). The proximity of mature scions to
juvenile rootstocks does not result in rejuvenation, because

some mature characteristics persist several years after grafting.
Thus, these grafting studies demonstrate that maturation results in changes in the growth habits of the apical meristem that
persist even when the mature meristem is re-exposed to physiological conditions associated with a young plant, including
inputs from a juvenile rootstock. Trewavas (1983) argues that
meristems ‘‘behave very much like individual organisms,’’ and
the studies described above show that the mature behavior of
grafted meristems is to some degree autonomous and resists
the effects of changed environmental inputs. Thus, a plant can
be considered a colony of semi-autonomous meristems, simultaneously competing with one another, but serving the whole
plant over time by subtly altering their behavior.

Maturation: meristems change their habits
Maturation involves changes in the habitual behavior of meristems, where habit is defined as a behavior pattern that develops in response to a particular set of physiological inputs. By
definition, habits tend to resist change, but nonetheless can be
altered in response to varying environmental inputs. The earliest maturation event is the polarization of the embryo into roots
and shoots, where each meristem, starting with the same genes,
acquires unique habits adapted to different environments. In
shoots, the meristems continue to develop new habits as the
plant grows. Over time, the meristems of conifers change their
behavior, losing regenerative potential and capacity for vegetative growth, but gaining reproductive competence and more

massive leaves, which are better adapted to the increasing

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GREENWOOD

environmental stresses associated with increased height
(Hutchison et al. 1990). Insect and amphibian metamorphoses
provide spectacular examples of adaptive morphological and
behavioral change in the living world, and occur in response to
environmental and hormonal cues. The causes of maturation in
plants are less well defined.
Attempts to reverse maturation and restore the regenerative
competence associated with the juvenile state have met with
varying degrees of success (Greenwood 1987, Bonga and von
Aderkas 1993). Although regenerative competence has been
temporarily restored by a variety of cultural treatments, other
maturational traits may remain unchanged. Hackett (1985),
Greenwood (1987) and Pierik (1990) all conclude that the
criteria for maturation or rejuvenation must not be confined

only to increased regenerative ability or qualitative observations of increased vigor (e.g., Huang et al. 1992). There are
many maturational characteristics that appear to change independently of one another (Poethig 1990). Also, a temporary
increase in vigor (reinvigoration) may be a response to
changed cultural conditions, rather than true rejuvenation.
Consequently, we cannot assume that the apparent rejuvenation of one trait, like rooting ability, will be accompanied by
long-term rejuvenation of other important traits, like growth
rate.

Maturation: changes in gene expression
If the polarization of roots and shoots is a maturational event,
then maturation is clearly associated with changes in gene
expression. Recently, maturational changes in leaf morphology (heteroblasty) have been used to examine the role of gene
expression in maturation (Poethig 1990). In eastern larch, leaf
weight/leaf area and chlorophyll content increase with maturation, and net photosynthesis also increases as a result of the
increase in chlorophyll content (Greenwood et al. 1989,
Hutchison et al. 1990); however, among individual trees, there
is no correlation between changes in leaf weight/leaf area and
chlorophyll content. In English ivy (Hedera helix L.), leaf
weight/leaf area and net photosynthesis increase with maturation, but chlorophyll content does not change (Bauer and
Bauer 1980). In red spruce (Picea rubens Sarg.), mature needles are much larger than juvenile needles and exhibit less net

photosynthesis per unit area, which is associated with decreased stomatal conductance (Rebbeck et al. 1992).
In both larch and English ivy, a comparison of cDNA libraries made from RNA extracted from juvenile and mature foliage
indicates that the types of polyA-RNA (mRNA) produced are
mostly similar (Hutchison et al. 1988, Hackett et al. 1991);
however, there are some differences in gene expression between juvenile and mature shoots. In both larch and English
ivy, the expression of sequences for the chlorophyll a/b binding
protein (cab) gene decreases with maturation (Hutchison et al.
1990, Woo et al. 1994). Some other exceptions include the
gene for dihydroflavanol reductase (DFR) in English ivy,
where anthocyanin is only produced in the petioles of juvenile
leaves. The lack of anthocyanin in mature petioles is due to the
absence of DFR in the petioles, which in turn is due to lack of

expression of the DFR gene (Murray and Hackett 1991, Murray et al. 1994b). In addition, the proline-rich protein (PRP)
gene is expressed more strongly in mature petioles than in
juvenile petioles, as indicated by a five- to tenfold increase in
the concentrations of PRP mRNA (Woo et al. 1994). Because
the expression of the PRP gene occurs in cells associated with
root regeneration in the petiole, Murray et al. (1994a) propose
that elevated expression of this gene may inhibit root meristem

regeneration. Thus, the available evidence (see also studies
reviewed in Poethig 1990 and Haffner et al. 1991) indicates
that maturation is associated with limited but perhaps significant changes in gene expression.

Maturation: origin of the mature state
Although the time course and stability of maturation have been
characterized in several species, it is very difficult to separate
cause from effect during maturation. Hence, we can only
speculate about the primary causes of maturation, and how
they affect development. Haffner et al. (1991) conclude that
phytohormones regulate maturation and suggest that the ratios
of abscisic acid (ABA), gibberellins (GAs), auxins and cytokinins, rather than their absolute concentrations play a primary
role in causing maturational change. Although phytohormones
probably do affect changes in the maturation state, changes in
their relative concentrations may, like morphological characteristics, be symptoms of a more fundamental driving force.
The finding that exogenously applied GA3 can cause reversion
of mature English ivy to the juvenile state (Robbins 1957) led
to the hypothesis that GA3 is important in the maturation
process. The observation that GA4/7 promotes flowering in
most genera of the Pinaceae suggests that GA promotes maturation in conifers (Zimmerman et al. 1985). However, a good

argument can be made that the opposite is true. The first
flowering observed in young spruces and pines is usually
exclusively female, with the ratio of males to females increasing steadily with age and size, and GA4/7 usually is much more
effective in promoting the production of female strobili than
the production of male strobili (Greenwood and Hutchison
1993). Also, in eastern larch, juvenile scions produce a larger
total number of strobili than mature scions, and most of the
strobili are female. Mature scions of eastern larch are more
responsive to GA4/7 than juvenile scions, but only female
flowering is significantly promoted (Eysteinsson and Greenwood 1993). Therefore, if the production of predominantly
female flowers is associated with an immature habit, then any
treatment that increases the ratio of female to male flowers
must also promote some degree of rejuvenation.
However, if the natural maturation process in English ivy
and conifers depends on changes in GA concentrations, there
must be an underlying mechanism that regulates either the
concentrations of endogenous GA and perhaps other phytohormones, or the ability of the plant to respond to their presence.
The tenet that genes control development, along with the
advent of recombinant DNA methodology, has led to recent
studies on the role of gene expression as a possible primary

cause of maturation. Although genes are likely candidates for

JUVENILITY AND MATURATION IN CONIFERS

the regulation of hormone metabolism or the mediation of
tissue responses to them, and changes in gene expression
during maturation have been recently demonstrated, they too
may be only symptoms, rather than causes, of maturation
itself. At present, we cannot rule out epigenetic factors as the
primary cause of maturation (Bonga 1982, von Aderkas and
Bonga 1993).

Maturation models
Models developed to account for maturation must consider a
variety of causal mechanisms and must account for the maturational phenomena that have been demonstrated experimentally for several woody plant species. Five major considerations are discussed.
(1) The onset of the mature state is usually gradual, but can
be abrupt. For example, grafting studies on loblolly pine and
eastern larch have shown that maturational change is gradual
for most traits, except for plagiotropism and branch frequency
(Greenwood 1984, Greenwood et al. 1989). However, in several conifers, abrupt maturation has been observed immediately following tissue culture propagation from adventitious

buds induced on the cotyledons of mature embryos. The best
example of this so-called accelerated maturation has been
documented in field tests of plantlets and seedlings from the
same half-sib families of loblolly pine, where the cotyledonderived plantlets exhibit slower growth, earlier flowering and
reduced branch number after four growing seasons than the
seedlings (McKeand 1985, Frampton and Isik 1987). Reduced
growth rate by cotyledon-derived plantlets of Virginia pine
(Pinus virginiana Mill.) relative to seedlings has also been
reported (Aimers-Halliday et al. 1991). In addition, Gronroos
et al. (1993) reported that rooted adventitious shoots from
zygotic embryos of lodgepole pine (Pinus contorta Dougl. ex
Loud.) did not grow as fast as seedlings.
Isogenetic plantlets and seedlings have been prepared from
both Douglas-fir (Ritchie et al. 1994) and hybrid larch (Smith,
Weber, Hutchison and Greenwood, unpublished data) by removing a few cotyledons from a new germinant and inducing
buds on them by treatment with the cytokinin benzylaminopurine (BAP). After elongation, the buds were rooted and the
resultant plantlets were then compared with the donor seedlings or cuttings rooted from them. In Douglas-fir, plantlets
and rooted cuttings were obtained from the same clones within
each of six full-sib families and grown for two years in the
field. The plantlets grew significantly less than their rooted

cutting ramets. In a similar study, a comparison of the foliage
of plantlets and seedlings from four genotypes of a Larix ×
eurolepis A. Henry hybrid indicated that plantlet foliage exhibited significantly reduced specific leaf area compared with
seedling foliage (Table 1). In addition, the plantlet foliage
exhibited reduced expression of the cab gene relative to seedling foliage, corresponding to the differences between juvenile
and mature foliage of eastern larch reported by Hutchison et
al. (1990). There were too few plantlets or seedlings to assess
the significance of differences in height or diameter growth.
Whether the accelerated maturation described in these five

435

Table 1. Comparison of maturation characteristics of four pairs of
isogenetic plantlets and seedlings of hybrid larch (Larix × eurolepis).
Cotyledons were excised from 2--3-week-old seedlings without sacrificing the seedlings in January 1990. Shoots were induced on excised
cotyledons cultured on BL medium (McLaughlin and Karnovsky
1989) with either 2.4 g l − 1 Ca(NO3)2 or 1.5 g l −1 glutamine as N source
and exposed to 10 mg l −1 benzyladenine for 14--21 days. Shoots were
transferred to hormone-free WPM medium for elongation and were
rooted on 1/2 strength WPM + 5 mg l −1 indole-3-butyric acid, then
transferred to soil in the same greenhouse where the original seedlings
were growing in March 1990. The isogenetic seedlings and plantlets
were overwintered in an unheated greenhouse, and specific leaf area
and chlorophyll concentration were measured in July 1991, and height
and diameter were measured in November 1991 as described in
Hutchison et al. 1990.
Characteristic

Seedlings

Plantlets

Significance

Chlorophyll, mg gDW− 1
SLA, m2 g−1
Height, cm
Diameter, mm

8.1
0.022
94.1
15.1

9.8
0.016
91.9
16.0

< 0.001
< 0.0001
ns
ns

species is due to the origin of the tissue (cotyledons) or the
result of exposure to cytokinin or some other stress during bud
induction is not known. Cotyledons are determinate organs in
that they only persist for a few weeks and then die. They appear
to retain regenerative potential while alive, but their determinate growth may be associated with the premature onset of
mature characteristics in plants regenerated from them. Mature
foliar characteristics of somatic seedlings of red spruce have
been reported (Nsangou 1994). In addition, Bonga and von
Aderkas (1993) report plagiotropic growth by a European
larch (Larix decidua Mill.) somatic seedling and several of its
ramets that had been regenerated from haploid tissue derived
from the megagametophyte. Although cytokinin and 2,4-D
were used to produce the embryogenic tissue in red spruce,
only 2,4-D was used to induce the embryogenic tissue in
European larch. Although these last two observations are preliminary, they suggest the need to consider the role of tissue
culture stress in the induction of mature growth behavior.
(2) Maturation affects a wide variety of morphological,
physiological and biochemical traits, but these traits appear to
vary independently of one another (examples discussed
above).
(3) Maturational traits are often persistent, and their maintenance is not always a function of tree size or proximity to roots.
Differences in chlorophyll content, leaf weight/leaf area and
xylem morphology among eastern larch scions from trees of
different ages grafted at the same time persist for several years
(Greenwood 1984, Takemoto, Greenwood and Hutchinson,
unpublished data). On the other hand, there is anecdotal evidence that mature morphological characteristics disappear
quickly after cuttings of Populus and Eucalyptus species are
grafted or rooted.
(4) There is evidence that the cells of the apical meristem
itself become determined in some woody plants. Grafted apices from mature plants of Citrus or Sequoiadendron consisting

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GREENWOOD

of only the apical meristem and one to two leaf primordia grow
out into plants that have mature characteristics (Navarro et al.
1975, Monteuuis 1991).
(5) Rejuvenation methods bring about gradual reversion to
the juvenile condition. For example, Huang et al. (1992) report
that a gradual increase in rooting ability occurs during serial
regrafting of shoots from mature trees of Sequoia sempivirens
(D. Don) Endl. However, Monteuuis (1991) reports rejuvenation of a single grafted apex (from a total of 300 attempts) after
only one grafting in Sequoiadendron giganteum (Lindl.)
Buchh.
Both Greenwood and Hutchinson (1993) and Hackett
(1985) contend that maturation involves cellular determination
at some point. But whether the cells within the dome of the
apical meristem themselves are determined is not certain.
Poethig (1990) argues that the transition to the mature state is
‘‘specified by a series of independently regulated, overlapping
programs that modify the expression of a common set of
processes required for shoot growth.’’ He contends that maturation at the level of cell determination is regulated by factors
both intrinsic and extrinsic to the cells of the apical meristem
itself, but the onset of maturational characteristics is initiated
by factors extrinsic to the apical meristem. Thus, the expression of the mature state is a function of both changes in the
apical meristem and changed inputs to that meristem as a result
of its increasing size and complexity. However, serial propagation of Norway spruce (Picea abies (L.) Karst.) prolongs the
juvenile state, but maturation occurs slowly, even though plant
size and complexity have been limited (St. Clair et al. 1985).
Poethig’s model accounts well for the seeming independence
of the expression of different maturation phenomena in the
same plant. But his conclusions are mainly based on evidence
from herbaceous annual plants, and thus may not account for
the persistence of the mature state observed in woody plants.
The possibility that the onset of maturation also requires intrinsic changes in the meristem itself, perhaps driven by the ability
to count cell divisions, must also be considered (e.g., Greenwood and Hutchison 1993). Whatever the nature of the intrinsic change, extrinsic factors, like the physiological
consequences of reaching a critical size, are usually also required for the expression of mature characteristics in normal
plants. In the case of accelerated maturation, very young plantlets appear juvenile, and mature characteristics are not evident
until some vegetative growth has occurred.

Hackett et al. (1992) propose that there are a number of
‘‘switches,’’ either in series or in parallel, or both, that must be
activated for the mature state to occur. Several switches operating in parallel would also account for independent onset of
mature traits. I contend that a simple model involving only one
switch can account for the complex phenomenon of maturation
(Greenwood 1987). The switch could reside within individual
cells in the apical meristem, and once activated would make
that cell mature, so that over time the apex would become a
mosaic with an ever increasing percentage of mature cells.
Expression of mature characteristics would, therefore, be a
function of the ratio of juvenile to mature cells in the apex at a
given time. Rejuvenation would result from conditions that
favor more rapid multiplication of juvenile cells, which would
account for the gradual rejuvenation described by Huang et al.
(1992) and others (see Greenwood 1987). Monteuuis’ (1991)
report of abrupt rejuvenation of a single sequoiadendron shoot
may reflect the presence of an abnormally high number of
juvenile cells in one of the meristems (of 300) that he micrografted. A comparison of three different types of maturation
models in terms of the five characteristics of woody plant
maturation described above is shown in Table 2.
A model that successfully explains the primary cause(s) of
maturation must also account for natural rejuvenation, which
accompanies sexual reproduction. Because plants do not have
germ lines specialized for the production of gametes, sexual
reproduction begins with cells from the somatic plant body.
Rejuvenation may be associated with the formation of gametes, but the finding that adventitious embryos in Citrus arise
from diploid cells of the nucellus suggests that either somatic
cells in the ovule have themselves become rejuvenated, or
some of them have never become mature (see discussions in
Zimmerman et al. 1985, Pierik 1990). Jorgensen (1991) reported that somatic embryos derived from callus originating
from the anthers of Aesculus and Quercus were both diploid
and haploid, suggesting that some of the embryos came from
maternal diploid cells or are the result of subsequent deploidization as described by von Aderkas and Bonga (1993). If the
ability to form somatic embryos is a characteristic of truly
juvenile cells (as suggested by Greenwood 1987), then these
observations provide evidence for the presence of juvenile
cells of unknown origin in mature plant parts. In conifers,
attempts to obtain explants from reproductive tissue that will
regenerate plants have to date been unsuccessful (e.g., Wang et

Table 2. Evaluation of three maturation models in terms of five criteria based on experimental studies of woody plant maturation. The intrinsic
model assumes that cells in the apex become determined in a mature state. The extrinsic model assumes that meristem cells are not determined,
but differentiate into the mature state in the subapical zone in response to inputs external to the apex. The intrinsic--extrinsic model assumes an
interaction between partly determined meristematic cells and external inputs.
Criteria

Intrinsic

Extrinsic

Intrinsic--extrinsic

Rate of onset of maturation
Persistent mature state
Independent onset
Gradual rejuvenation
Meristem cells determined

Gradual
Yes
Yes
Yes
Yes

Gradual or abrupt
Should not persist
Yes
Should be immediate
Not determined

Gradual or abrupt
May or may not persist
Yes
Yes
Yes

JUVENILITY AND MATURATION IN CONIFERS

al. 1991). The existence of juvenile cells in mature plant parts
may be due to in situ rejuvenation or the presence of a mechanism for selectively activating vestigial juvenile cells during
the formation of reproductive structures.
If mature cells, such as those in the petioles of English ivy,
which do not express DFR, possess DNA sequences (perhaps
altered regulatory sequences) that differ from those in corresponding juvenile cells, then the creation of a maturation
marker that can identify individual mature cells is possible.
However, if the differential expression of the DFR gene is due
to an epigenetic factor such as DNA methylation, then the
identification of a marker for mature cells remains difficult.
Acknowledgment
This manuscript is Paper No. 1923 of the Maine Agricultural Experiment Staton.

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