Epidermal transpiration, ultrastructural characteristics and

PHYSIOLOGIA PLANTARUM 118: 554–561. 2003
Printed in Denmark – all rights reserved
Copyright # Physiologia Plantarum 2003
ISSN 0031-9317
Epidermal transpiration, ultrastructural characteristics and net
photosynthesis of white spruce somatic seedlings in response to
in vitro acclimatization
Mohammed S. Lamhamedia,*, Hélène Chamberlandb and Francine M. Tremblayb
a
Ministe`re des Ressources naturelles, Foreˆt Que´bec, Direction de la recherche forestie`re, 2700, rue Einstein, Sainte-Foy, Que´bec,
Canada G1P 3W8
b
Centre de Recherche en Biologie Forestie`re, Faculte´ de Foresterie et de Ge´omatique, Pavillon Charles-Euge`ne Marchand,
Universite´ Laval, Que´bec, Canada G1K 7P4
*Corresponding author, e-mail: [email protected]
Received 16 August 2002; revised 25 February 2003
Mortality of transplanted somatic seedlings at the stage of
acclimatization is often high and likely due to rapid change in
environmental conditions. To investigate the potential of in
vitro acclimatization of somatic seedlings before soil transfer,
somatic seedlings of white spruce (Picea glauca [Moench]
Voss) were germinated on a liquid medium supplemented
with sucrose. After 6 weeks in germination, sucrose was
omitted from the medium for a supplementary 6 weeks at
which time somatic seedlings were acclimatized in vitro in
their germination tubes before transfer to soil. In vitro
acclimatization of somatic seedlings was realized by transferring the test tubes containing the germinated somatic seedlings
to the greenhouse for 9 days. During this period, the culture
tube lids of acclimatized somatic seedlings were lifted
progressively increasing air exchange between the tube and
the greenhouse whereas, for non-acclimatized somatic
Introduction
The use of somatic embryogenesis as an efficient
commercial technique in forestry is still limited by its
relatively high production costs and low survival rate of
seedlings during ex vitro transplanting. During in vitro
culture, somatic seedlings are grown under unnatural
conditions such as high relative humidity, low light
intensity, relatively constant temperature, large fluctuations
in CO2 and limited space. Due to these highly stressful
conditions, in vitro plantlets can develop some abnormalities in the epidermal wax, stomata and roots leading to an
inability of the plantlet to control excessive epidermal
seedlings the culture tubes were maintained closed during
in vitro acclimatization. In vitro acclimatized somatic
seedlings had higher asymptotic net photosynthesis (Pn) at
light saturation than non-acclimatized seedlings (6 versus
4.5 mmol m2 s1). At the end of the in vitro acclimatization
period, a lower rate of epidermal transpiration was also
observed for acclimatized somatic seedlings (3.85 versus
4.75% h1). Microscopic observations showed that starch
granules were more abundant in needles of acclimatized
somatic seedlings than in non-acclimatized somatic seedlings,
probably as a result of their greater photosynthetic capacity.
Needles from acclimatized somatic seedlings also showed
more epicuticular wax projections than needles from
non-acclimatized somatic seedlings. These structural changes
may help somatic seedlings to restrict epidermal water loss
and stomatal aperture.
transpiration, which is considered to be the main cause of
seedling mortality following transfer to soil (Ziv 1995).
Wardle et al. (1983) observed that high relative humidity
during in vitro growth inhibited surface wax production on
Brassica oleracea L. plantlets.
To improve the rate of survival and physiological
functioning of somatic seedlings, Ziv (1995) suggested
that, for ex vitro acclimatization, the procedures such
as gradual decrease in relative humidity (RH), removal
of sugars and increase of CO2 and light intensity should
be applied during the in vitro acclimatization period.
Different techniques were applied to decrease RH
Abbreviations – ABA, abscisic acid; PPFD, photosynthetic photon flux density; RH, relative humidity; RWC, relative water content.
554
Physiol. Plant. 118, 2003
including uncapping of culture vessels, use of dessicants,
a ventilation system, and permeable membranes (Ripley
and Preece 1986, Tanaka et al. 1992). During ex vitro
acclimatization, somatic seedlings are generally transferred under mist conditions with a gradual decrease in
relative humidity (Khlifi and Tremblay 1995). This type
of acclimatization is still a limiting step in moving
towards large-scale production. Malfunctioning of the
water balance as well as poor development of the
photosynthetic apparatus are the major constraints
when somatic seedlings are transferred to regular greenhouse conditions (Preece and Sutter 1991).
Several thousands of white spruce, black spruce and
hybrid larch somatic seedlings representing several
genotypes have been produced in our laboratory since
1996 using in vitro acclimatization in the greenhouse. Our
hypothesis has been that the period of in vitro acclimatization was sufficient to increase net photosynthesis and induce
development of normal amounts of wax which decrease
epidermal transpiration. In vitro acclimatization consisted
of a gradual transition of somatic seedlings from in vitro
to greenhouse environmental conditions by progressively
uncapping the test tubes during 9 days prior the transfer to
soil. The objectives of this study were: (1) to evaluate the
effect of in vitro acclimatization on survival of somatic
seedlings after transfer to soil; (2) to follow the rate of net
photosynthesis of Picea glauca somatic seedlings during the
period of in vitro acclimatization; (3) to compare light
saturation curves at the end of in vitro acclimatization
between acclimatized and non-acclimatized somatic
seedlings, and (4) to evaluate the effects of in vitro
acclimatization period on epidermal transpiration, epicuticular wax and starch accumulation.
Materials and methods
Plant materials and growth conditions
Embryogenic tissue lines originated from controlled
crosses of selected white spruce genotypes by the Canadian
Forest Service-Quebec Region (Beaulieu 1996). The
embryogenic lines used in this study, G-305, G-369 and
G-362, had been maintained by subculturing every 14 days
on HLM-1 medium (Tremblay 1990) for a maximum of
6 months before maturation. Somatic embryos were
matured according to Isabel et al. (1993) with 45 mM abscisic acid (ABA). After 5 weeks of maturation, normal
mature somatic embryos were transferred onto Sorbarod
TM plugs (Baumgartner, Papier SA, Lausanne, Switzerland)
saturated with liquid Campbell and Durzan’s salts half
strength (Campbell and Durzan 1975) supplemented with
1.5% sucrose (w/v) (Khlifi and Tremblay 1995). Every
2 weeks, fresh liquid medium was added, but after 6 weeks,
the sucrose was removed from the medium.
In vitro acclimatization and growth conditions
Test tubes containing germinated somatic seedlings
showing normal and well-developed epicotyl were transPhysiol. Plant. 118, 2003
ferred to the greenhouse. One-half of the somatic seedlings were submitted to a gradual acclimatization by
letting increasingly greater air exchanges between the
test tubes and the greenhouse during 9 days prior to
transplanting. On the first day, somatic seedlings were
transferred to the greenhouse operated with a day/night
temperature of 25/18 C, a RH of 35–45% and a 17 h
photoperiod. From days 2–5, the culture tube lids were
lifted halfway in order to permit a progressive adaptation
of somatic seedlings to the greenhouse environment.
From days 6–8, the culture tubes were completely
uncapped and somatic seedlings were directly exposed
to greenhouse conditions. On day 9, the somatic seedlings were transplanted to soil as regular seedlings. The
other half of somatic seedlings (non-acclimatized) had
their culture tubes closed during the in vitro acclimatization period were used as a control.
Survival after in vitro acclimatization
For both acclimatized and non-acclimatized seedlings
and for each clone (G-305, G-369 and G-362), more
than 100 somatic seedlings were transferred to
Styroblock1 (Beaver Plastics Ltd, Edmonton,
Canada) cavities (45 cavities per block, 340 cm3 per
cavity) filled with a moistened mixture of peat and
vermiculite (3/1, v/v). After 3 months, the rate of
survival of acclimatized and non-acclimatized somatic
seedlings was evaluated.
Net photosynthesis
Measurements of net photosynthesis (Pn) were carried
out on days 1, 6, 8 and 9 of the period of in vitro
acclimatization using a portable open-mode gas
analyser system with a cylindrical coniferous cuvette
(Model LCA-4; Analytical Development Company,
Hoddesdon, UK). On each sampling day, six somatic
seedlings of white spruce (line G-305) were selected
randomly from both in vitro acclimatized and nonacclimatized somatic seedlings. The total needle area
was measured for each sample used in gas exchange
measurements, as described in details by Lamhamedi
et al. (2000).
At the end of the 9-day in vitro acclimatization, the
relationship between Pn and photosynthetic photon flux
density (PPFD) was determined using the model
described by Hanson et al. (1987). PPFD varied between
0 and 1000 mmol m2 s1 by varying the distance between
the cuvette and light source, and by covering the cuvette
with a black nylon screen or with aluminium paper
(Lamhamedi et al. 1994). The three parameters (B1, B2
and B3) of this model (Eqn 1) were determined using the
non-linear procedure in SAS (SAS Institute Inc., Cary,
NC, USA). A Hougaard’s measure of skewness also was
used to assess linearity of the estimator of each parameter (Drapper and Smith 1981, Ratkowsky 1990) using
multiple measurements that were arbitrarily chosen by
varying photosynthetic photon flux density (PPFD).
555
2
B3
6
Pn ¼ B1 41 1 B1
1 PPFD
B
2
3
7
5
ð1Þ
where B1 is the net photosynthesis at saturation (Pn); B2
is the point of compensation and B3 is the respiration at
zero PPFD.
Light use efficiencies (E: Eqn 2) of the net photosynthesis–
PPFD relation for both acclimatized and non-acclimatized
somatic seedlings were obtained from the first derivative of
the function Pn ¼ f(PPFD) evaluated at PPFD ¼ 0.
E¼
B1
B2
B3
B3
1
log 1 B1
B1
ð2Þ
The contribution of light to limitation of photosynthesis
was calculated for both acclimatized and non-acclimatized
somatic seedlings (Jones 1995) using (Pn,max Pn)/Pn, max,
where Pn,max is the rate of net photosynthesis at light
saturation, calculated as the limit of the function
Pn ¼ f(PPFD), when PPFD tends towards infinity. Pn is
the assimilation rate under non-saturating light calculated
at PPFD ¼ 550 mmol m2 s1 using empirical models
developed for both acclimatized and non-acclimatized
somatic seedlings.
Epidermal transpiration
At the end of the period of in vitro acclimatization,
10 somatic seedlings from both the acclimatized and
non-acclimatized groups were used to estimate epidermal
transpiration. Shoots of somatic seedlings (line G-305)
were cut under water and allowed to fully hydrate
overnight by placing their cut ends in culture tubes
containing distilled water and sealed in plastic bags.
Next morning the shoots were placed into home-made
Plexiglas1 dessicators (56 cm long, 29 cm wide, 46 cm
high). Relative humidity of 85 6 2% was generated
using saturated Na2CO3 salt solution (Rockland 1960,
Bomal and Tremblay 1999). A fan was used to maintain
homogeneity of relative humidity within the dessicator.
The weight loss of each sample was determined
(6 0.1 mg) every 30 min for 10 h. Epidermal transpiration
rates were determined by construction of curves of water
loss versus time (Quisenberry et al. 1982). Theoretically,
these curves should have three distinct phases: (1) an
initial steep linear decline in relative water content
associated with stomatal transpiration; (2) a curvilinear
phase where the effects of closing stomata are important;
and finally (3) a linear portion indicative of epidermal
transpiration. The transition point between the curvilinear and linear regions of the curve was determined
from the change in the slope and the significance of the
coefficient of determination of the linear region, as
suggested by Schulte and Hinckley (1985). The rate of
556
epidermal transpiration was calculated by linear regression as the slope of water-loss over time (Snedecor and
Cochran 1989). Statistical differences in epidermal
transpiration between the two treatments (acclimatized
versus non-acclimatized) were performed using an
analysis of covariance according to Snedecor and
Cochran (1989).
Tissue processing for microscopy
At the end of the period of in vitro acclimatization and
for each clone (G-305, G-369 and G-362), composite
samples (20–30 needles) were collected from acclimatized
and non-acclimatized somatic seedlings. For each clone,
the middle portions of 3–4 needles that had been selected
randomly, were cut in small fragments (2 mm long),
placed in a fixative solution consisting of 3% glutaraldehyde in 100 mM cacodylate buffer, pH 7.2. Samples were
submitted to a gentle vacuum (1/2 atmosphere) for 5 h
then placed overnight at 4 C. After three 20 min washes
in the buffer, they were post-fixed overnight at 4 C in
1% osmium tetroxide containing 2 mM K ferrocyanide
and 6% sucrose in cacodylate buffer. They were thereafter washed with the buffer, dehydrated in a graded
series of ethanol, and embedded in Epon (Polysciences,
Halifax, Canada).
Semi-thin sections were stained with 0.1% toluidine
blue and examined under a Reichert-Jung Polyvar
microscope (Reichert-Jung Polyvar, Vienna, Austria).
Photographs were taken with a Technical Pan Estar
(Kodak, Toronto, Canada) film.
For transmission electron microscopy, ultra-thin sections
were deposited on nickel grids and stained with uranyl acetate and lead citrate. Observations were made with an electron
microscope (JEOL, Model 1200X; Jeol Tokyo, Japan).
For scanning electron microscopy, three needles from
two different genotypes (G-305 and G-369) were fixed
with osmium tetroxide vapours for 48 h and fastened to
specimen holders with double-sided silver tape. They
were thereafter coated with gold (4 1 min.) using
a sputter-coater instrument (Semprep 2; Nanaotech,
Manchester, UK). Observations were carried out with a
scanning electron microscope (JEOL, JSM-35; Jeol). The
structure of the waxes was characterized following the
classification and terminology of plant epicuticular
waxes defined by Barthlott et al. (1998).
Results
Survival after in vitro acclimatization
After 9 days of in vitro acclimatization followed by transfer into soil without supplementary care, 95–100% of
these somatic seedlings (G-305, G-369 and G-362) survived. Furthermore, they resume growth immediately
without any sign of transplanting shock. No sign of
dessication could be observed on the needles after in
vitro acclimatization. Non-acclimatized somatic seedlings
dehydrated rapidly and 100% mortality was observed.
Physiol. Plant. 118, 2003
Fig. 1. Relative water content curves for acclimatized (&) and nonacclimatized (*) Picea glauca somatic seedlings (line G-305) with
the cuticular phase of water loss rate fitted by linear regression
of the data indicated between arrows. Each point is the mean of
10 samples.
Epidermal transpiration
Acclimatized and non-acclimatized somatic seedlings of
Picea glauca showed different water retention curves
(Fig. 1). The rates of water loss (% h1), controlled by
epidermal transpiration, are indicated by the slopes of
regression. Covariance analysis showed that these rates
differed significantly (P 4 0.05) among acclimatized and
non-acclimatized somatic seedlings, with, respectively,
3.85 versus 4.75% h1. In fact, as the needles dehydrated,
acclimatized somatic seedlings showed more effective
restriction of epidermal water loss than those of
non-acclimatized somatic seedlings. After 8 h of
transpiration, the relative water content (RWC) was
higher in acclimatized somatic seedlings.
Fig. 2. Net photosynthesis of acclimatized and non-acclimatized
Picea glauca somatic seedlings (line G-305) during the period of in
vitro-acclimatization. Bars indicate standards errors, with n ¼ 6.
positive values of Pn (Fig. 2). On days 6, 8 and 9,
acclimatized somatic seedlings maintained significantly
higher values of Pn than non-acclimatized seedlings.
This difference in Pn was not attributed to changes in the
root system since the latter was similarly well developed in
both acclimatized and non-acclimatized somatic seedlings.
At the end of in vitro acclimatization, acclimatized
and non-acclimatized somatic seedlings showed different
fitted Pn responses to the PPFDs (Fig. 3). In compararison
with non-acclimatized somatic seedlings, acclimatized
somatic seedlings had a higher asymptotic Pn at saturation
(6 versus 4.5 mmol m2 s1), a lower dark respiration
rate (1.42 versus 1.04 mmol m2 s1) and a higher
compensation point (83 versus 79 mmol m2 s1). In
addition, in vitro acclimatization increased light use
efficiency by 29% in comparison with non-acclimatized
somatic seedlings (0.0190 versus 0.0147).
Net photosynthesis during in vitro acclimatization and
photosynthetic light-response models
Light and electron microscopy
During 9 days of in vitro acclimatization, both acclimatized and non-acclimatized somatic seedlings showed
One of the main differences observed at the light microscope level between leaf samples was the accumulation of
A
4
2
[ (
)]
Pn = 6 1 – 1.2366(1 – PPFD/83)
0
0
200
400
600
800
1000
–2
PPFD
–4
B
6
1200
Net photosynthesis
Net photosynthesis
6
4
2
[ (
)]
Pn = 4.5 1 – 1.2330(1 – PPFD/79)
0
–2
0
200
400
600
800
1000
1200
PPFD
–4
Fig. 3. Photosynthetic PPFD response of data (*) and fitted curves (*) of (A) acclimatized and (B) non-acclimatized Picea glauca somatic
seedlings (lines G-305, G-369 and G-362).
Physiol. Plant. 118, 2003
557
Fig. 4. Cross sections from needles of (A) acclimatized and (B) non-acclimatized somatic seedlings. In (A), mesophyll cells contain numerous
starch grains (arrows). In (B), mesophyll cells are devoid of starch grains and are characterized by a thin layer of cytoplasm along their cell
walls. A thicker cuticle covers the epidermal cells (E) of the acclimatized somatic seedlings (A). Note the thicker epidermal walls in Fig. 4A in
comparison with those in Fig. 4B. N, nucleus; V, vacuole. Scale bar for (A) and (B): 20 mm.
starch grains in mesophyll cells from acclimatized
somatic seedlings (Fig. 4). Starch occurred in chloroplasts as large granules which in many cases appeared
to fill the cell cytoplasm (Fig. 4A, arrows). In contrast, in
non-acclimatized somatic seedlings, starch grains were
rare and mesophyll cells consisted of a thin layer of
cytoplasm with distinct chloroplasts along the cell wall
(Fig. 4B). Another difference observed between acclimatized and non-acclimatized somatic seedlings was an
apparent thickening of the outermost epidermal walls
in the former.
Electron microscope observations further confirmed
the differences in starch distribution between the two
types of samples (Fig. 5). Indeed, needles from acclimatized samples displayed chloroplasts with numerous large
and electron-lucent starch grains (Fig. 5A) whereas those
from non-acclimatized samples showed roundish
chloroplasts that were generally devoid of starch deposits
(Fig. 5b).
Under scanning electron microscopy, wax projections
were much more abundant on needles from acclimatized
somatic seedlings than from non-acclimatized needles
(Fig. 6). On acclimatized somatic seedlings, these projections formed membraneous platelets distributed as
rosettes along the needle surface and forming long
threads over the stomata (Fig. 6A). In contrast, the
needle surface of non-acclimatized samples appeared
smoother with a few thread-like wax projections present
over stomata (Fig. 6B).
Discussion
A 9-day in vitro acclimatization was sufficient to insure
plant survival and growth after transfer to soil. Our
acclimatization protocol induced marked physiological
and anatomical changes in Picea glauca somatic
seedlings (Figs. 1–6). In vitro acclimatized somatic
Fig. 5. Transmission electron
micrographs showing differences
between mesophyll cells from
(A) acclimatized and (B) nonacclimatized somatic seedlings.
In acclimatized somatic seedlings
(A), distinct electron-lucent
starch grains (S) are present in
chloroplasts (C). In contrast,
chloroplasts in non-acclimatized
somatic seedlings (B) are devoid
of starch grains. CW, cell wall; N,
nucleus. Scale bars: 1 mm.
558
Physiol. Plant. 118, 2003
A
B
Fig. 6. Scanning electron micrographs showing wax features on
needles from (A) acclimatized and (B) non-acclimatized somatic
seedlings. Wax projections forming rosettes are more abundant on
(A) acclimatized than on (B) non-acclimatized somatic seedlings.
Numerous wax threads are uniformly distributed over stomata (S) of
the (A) acclimatized needles of somatic seedlings. Scale bar: 10 mm.
seedlings exhibited greater maximum Pn rates, higher
light use efficiency, accumulation of starch granules
and epicuticular wax than non-acclimatized somatic
seedlings. Furthermore, excised shoots of acclimatized
somatic seedlings exhibited a lower epidermal transpiration than non-acclimatized somatic seedlings. The values
of epidermal transpiration are indicative of the
significant effect of in vitro acclimatization on the ability
of somatic seedlings to adapt rapidly to new environmental conditions. It is reasonable to assume that the
lower epidermal transpiration on acclimatized somatic
seedlings resulted from the presence of well-developed
epicuticular waxes which may retard water loss, making
in vitro acclimatized somatic seedlings less susceptible to
water stress after transfer to soil. A second physiological
adaptation observed during in vitro acclimatization
concerns the control of stomatal closure. The value of
RWC at which stomata closed was significantly
increased in in vitro acclimatized somatic seedlings. The
high RWC observed in acclimatized somatic seedlings
(Fig. 1) suggests that the well-developed epicuticular
waxes, shown by electron microscopy (Fig. 6) might
Physiol. Plant. 118, 2003
play an important role in the maintenance of plant
water balance just after transfer to soil from in vitro
conditions to the greenhouse. An insufficient epicuticular
wax formation and its effects on epidermal transpiration
observed for non-acclimatized somatic seedlings was
consistent with observations reported for several species
including mature trees (Baig and Tranquillini 1976,
Wardle et al. 1983, Kozai et al. 1992, Ziv 1995, Kerstiens
1996a, Jenks and Ashworth 1999). However, a recent
study (Lamhamedi et al. 2000) showed that after one
growing season under greenhouse conditions, somatic
and zygotic seedlings of white spruce showed similar
epicuticular wax features.
The photosynthetic ability of both acclimatized and
non-acclimatized somatic seedlings on day 1 suggests
the establishment of autotrophic growth at the end of
germination. The autotrophic growth was established in
response to the removal of sucrose from the medium
6 weeks after beginning the germination of white spruce
somatic embryos. It is well established that the presence
of high concentrations of sucrose in the culture medium
lead to dramatic decreases in photoautotrophic development and to less efficient acclimatization of plants grown
under in vitro conditions (Kozai 1991, Kozai et al. 1992,
Serret and Trillas 2000). Considerably less information is
available regarding the photosynthetic response of coniferous somatic seedlings during in vitro acclimatization.
From our results, it appears that the photosynthetic
performance of acclimatized somatic seedlings can be
viewed as a response to the removal of sucrose and also
to increased PPFD levels at the end of in vitro acclimatization when culture tubes were completely uncapped
(Fig. 2). This agrees with previous results (Evers 1982,
Kozai 1991, Ziv 1995, Kozai et al. 1997) showing that
sucrose levels and light intensity affect net photosynthesis of in vitro plants, lower concentrations of sucrose
being positively correlated with higher photosynthetic
capacity. Evers (1982) measured Pn of Douglas-fir in
vitro plants and found that the in vitro maximum Pn
reached the same levels as reported for trees. In contrast,
for both in vitro acclimatized and non-acclimatized P.
glauca somatic seedlings, the rates of Pn were lower than
those recorded on newly transplanted zygotic seedlings
(Marsden et al. 1996). Low photosynthesis rates have
also been reported for plants under in vitro conditions
(Grout 1988). During the period of in vitro acclimatization, Pn increased significantly (Fig. 2) probably as a
result of improved stomatal function due to the small
vapour pressure deficit gradient between the intercellular
leaf space and the saturated vessel atmosphere (Blanke
and Belcher 1989).
The presence of starch grains in the chloroplasts of
acclimatized somatic seedlings (Figs 4 and 5) can be
directly related to current photosynthesis. Using steadystate labelling with 14C, it was demonstrated that most
starch in chloroplasts of needles of 1-year-old balsam fir
and 26-year-old Douglas-fir was generated from current
photosynthesis (Little 1970, Webb and Kilpatrick 1993).
The positive effect of current net photosynthesis on
559
starch accumulation in mesophyll cells of white spruce in
the present study is consistent with recent studies on
Gardenia jasminoides Ellis and Nicotiana tabacum L. cv.
(Serret and Trillas 2000, Kadleček et al. 2001). Furthermore, current photosynthesis is considered to be the
critical factor in establishing root growth (van den
Driessche 1987).
The progressive uncapping of the culture tubes during
in vitro acclimatization simultaneously induces several
changes in the seedling micro-environment, particularly
the relative humidity, the temperature, the light quality
and quantity and the vapour pressure deficit. From the
results of this study, it seems that the formation of
epicuticular waxes is induced by the decreased of RH
around the seedlings during in vitro acclimatization.
Accumulation of epicuticular waxes can be viewed as a
rapid adaptation of boreal species to harsh environmental
conditions. Grantz (1990) reported that wax deposition on
guard cells and other cells of the stomatal complex was
absent at high humidity, and induced at low humidity.
This accumulation is also attributed to other environmental factors as well as to plant genetics (Vanhinsberg and
Colombo 1990, Kerstiens 1996a, Jenks and Ashworth
1999). Epicuticular waxes increase light reflectance,
reducing the energy absorbed by the needles, and consequently decreasing the temperature of needles (Jenks and
Ashworth 1999). On the other hand, a possible decrease in
needle temperature is also favoured by the ability of
cuticles to absorb water molecules associated with the
polysaccharides of the cuticular membrane (Kerstiens
1996b). Such decreases in temperature at the needle
surface can be advantageous for acclimatized somatic
seedlings by leading to a reduction in the rate of respiration and transpirational losses, and to more active
photosynthesis. In contrast, an increase in needle temperature can arise in non-acclimatized somatic seedlings
without well-developed epicuticular waxes. This situation
results in a high vapour pressure gradient between the
needle surface and the surrounding air, thus increasing
stomatal transpiration and the possibility of water loss
through the poorly developed cuticle.
In vitro acclimatization directly in culture vessels was
found to improve the photosynthetic capacity of somatic
seedlings by influencing the three parameters (B1, B2 and
B3) determined by empirical models. From the results of
empirical models of the relation Pn–PPFD, it appears
that an increase of PPFD at the end of the period of in
vitro acclimatization increases the photosynthetic
capacity of somatic seedlings. Our results are in agreement with those reported by Whish et al. (1992) showing
that a decrease of RH during in vitro culture increased
plant survival after transfer to soil. The protocol
described here for in vitro acclimatization of P. glauca
somatic seedlings allows a greater rate of survival
(. 95%). The same protocol was applied to several
thousands of seedlings in our laboratory with similar
success on P. mariana, P. abies and hybrid larches
(unpublished data). In vitro acclimatization induced
greater accumulation of epicuticular waxes suggesting
560
that these structural changes may be some of the strategies used by seedlings to restrict epidermal water loss and
stomatal aperture. Acclimatization under in vitro conditions
contributes to a reduction in the labour costs associated with
the handling of non-acclimatized seedlings. These results of
in vitro acclimatization will also prove useful for the production of somatic seedlings on a large scale.
Acknowledgements – We thank Dr Jean Beaulieu (Canadian Forest
Center, NRC) for providing the seeds from controlled crosses. We
also thank Martine Blais (CRBF) for the production of all somatic
seedlings, and Barbara Sochanski (DRF, Forêt Québec) for her
advice on experimental designs and statistical analysis. Thanks are
also extended to Dr Hank Margolis and Dr Abdelmalek El Meskaoui for critical reading of the manuscript. This research program
was supported by a grant to FMT from Ministry of Industry Trading, Science and Technology, QC, in partnership with CPPFQ Enr.,
PAMPEV Inc., and BECHEDOR Inc.
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