Abstract We investigated changes in physiological
characteristics of micropropagated juvenile avocado
(Persea americana Mill.) cultured in three different
su-crose concentrations (5, 30 and 50 g/l) for 4 weeks and
subsequently acclimatized to ex vitro conditions. No
sig-nificant differences in foliar concentrations of total
chlo-rophylls, N
mass, flavonoids and total soluble proteins
were observed during in vitro growth or after
acclimati-zation. However, ex vitro transfer exerted multiple
ef-fects on foliar ratios of chlorophyll a to b, total
chloro-phylls to carotenoids and stem N and C/N ratio. Both
varying sucrose concentrations during in vitro growth
and ex vitro transfer, affected the concentration of
rub-isco subunits independent of other photosynthetic
com-ponents, viz. total chlorophylls, foliar N and total soluble
proteins. These results suggest that preconditioning
juve-nile avocado microplants at different concentrations of
sucrose modify several physiological parameters during
ex vitro acclimatization, like concentrations of
caroteno-ids and rubisco, which can improve vigour of plants in
autotrophic conditions.
Keywords Chlorophylls · C/N ratio · Persea americana ·
Rubisco · Soluble proteins
Introduction
Avocado (Persea americana Mill.), a dicotyledonous
woody species, is one of the major fruit crops of the
tro-pics and subtrotro-pics. Due to its high economic
impor-tance, this species is the subject of intensive studies from
micropropagation to genetic transformation
(Pliego-Alfaro 1988; Cruz-Hernandez et al. 1998). Various
at-tempts have been made to multiply avocado through
tis-sue culture techniques although this species has certain
limitations such as in vitro rooting and ex vitro survival
(Pliego-Alfaro 1988; Barceló-Muñoz et al. 1999). The
high mortality rate generally observed in woody plants
after ex vitro transfer is mainly associated with
physio-logical changes during the heterotrophic to autotrophic
transition. Such changes have been extensively studied
and well characterized in herbaceous plants and also in a
few woody plants (Zacchini and Morini 1998; Van
Huylenbroeck et al. 2000). In most cases, the first 4 weeks
after ex vitro transfer are critical for survival and
plant-lets encounter an intensive transplantation stress mainly
due to sudden exposure to excess light and low air
hu-midity. However, some species are able to overcome this
transitory shock within a short period of time and thus
the rate of recovery is strictly species-specific. In
avoca-do, the mortality rate of tissue-cultured plantlets was
high during the first 4 weeks (Barceló-Muñoz 1995).
Different approaches have been employed for
obtain-ing high survival rates followobtain-ing acclimatization to ex
vitro conditions. Among these, changes in concentrations
of sucrose during in vitro growth, imposed alone or in
combination with other abiotic factors such as light
and/or CO
2level, have been proven to be beneficial for
some plants. In woody plants, several investigators have
shown the influences of culture media sucrose
concen-trations on morphological changes (Nobre et al. 2000),
yet the associated physiological changes have scarcely
been considered (Tay et al. 2000). In the case of
avoca-do, survival rate is slightly greater at high sucrose
con-centration (e.g. 30 g/l) (De La Viña et al. 1999). In that
work, in vitro leaves showed significant changes in
se-lected parameters like rubisco concentration and
photo-synthetic rate as a result of the culture conditions, but no
data on the characteristics of the developing ex vitro
leaves were obtained for comparison.
The present work examines the effect of different in
vitro sucrose concentrations on several physiological
pa-A. Premkumar (✉
) · F. Pliego-Alfaro · M.A. QuesadaJ.A. Mercado
Dept. Biología Vegetal, Universidad de Málaga, 29071, Malaga, Spain
e-mail: [email protected]
Tel.: +34-952-132007, Fax: +34-952-131944
A. Barceló-Muñoz
Centro de Investigación y Formación Agraria, Cortijo de la Cruz s/n, 29140 Churriana, Malaga, Spain DOI 10.1007/s00468-002-0188-0
O R I G I N A L A R T I C L E
Albert Premkumar · Araceli Barceló-Muñoz
Fernando Pliego-Alfaro · Miguel A. Quesada
José A. Mercado
Influences of exogenous sucrose on juvenile avocado during
in vitro cultivation and subsequent ex vitro acclimatization
Received: 6 November 2001 / Accepted: 27 March 2002 / Published online: 22 August 2002 © Springer-Verlag 2002
rameters both during in vitro cultivation and after
trans-fer to ex vitro autotrophic conditions.
Materials and methods
Plant material
In vitro shoots were obtained from a stock of juvenile avocado (Persea americana Mill.), derived from a single seed of the avoca-do cv. Gvaram 13 germinated in vitro (Barceló-Muñoz et al. 1990). Shoots were maintained in active proliferation in a modi-fied MS medium (Murashige and Skoog 1962) with the N45 K macroelement formulation (Margara 1984) and supplemented with 4.4 µM benzyladenine. The medium was solidified with 6 g l–1 A1296 agar (Sigma, Mo., USA). Standard cultural conditions were 25±1°C and a 16 h photoperiod under 40 µmol m–2 s–1irradiance provided by Sylvania Grolux lamps.
In vitro rooting of shoots and sucrose treatments
For rooting, the procedure of Barceló-Muñoz et al. (1990) was used. In general, in vitro shoots (1–1.5 cm long) coming from the proliferation medium were cultured for 3 days in 5 ml of MS me-dium with macroelements at ×1/3 and supplemented with 4.92 µM indol-3-butyric acid. During this period shoots were maintained with agitation in a rollordrum (5 rpm). After this phase, shoots were transferred to auxin-free solid medium containing three dif-ferent concentrations of sucrose (5, 30 and 50 g l–1). In all cases, activated charcoal 8 g l–1(Sigma, USA) was incorporated. Shoots were cultured in these conditions for 4 weeks.
Ex vitro acclimatization
After 4 weeks of in vitro growth, rooted shoots were taken out of culture tubes without damaging the root system. Shoots were washed several times with distilled water to remove traces of cul-ture media on root surfaces. All tissue-culcul-tured plantlets were planted in biodegradable paper pots containing Vermimix (De Baat Spc Mix 20/80, Coevorden, The Netherlands) and kept inside an acclimatization unit that was completely covered with transpar-ent polythene sheets. Plantlets were watered sufficitranspar-ently to avoid drying of the substrate and ×1/4 strength MS (M-5519, Sigma, USA) including macro- and micronutrient solution was supplied once a week. Relative humidity and temperature were 87±1% and 29±2°C during the day and 89±1% and 20±1°C during the night. The whole unit was kept inside a growth cabinet (Ibercex, ASL, Spain) and plantlets were allowed to grow in these conditions for 10 days. Thereafter, humidity was gradually decreased over 10 days according to Marín and Gella (1987) by gradually expos-ing plants to growth cabinet conditions and finally the polythene sheet was removed. The photon flux density at the plant top was 150 µmol m–2s–1with a photoperiod of 16 h.
Sampling
For each sucrose treatment, 60 culture tubes were maintained and in vitro samples were collected after 4 weeks of growth. Fully ex-panded leaves, healthy and greenish in appearance, were collected from 8–10 in vitro plantlets (4 weeks old) and pooled. From this pool three independent samples were taken for each analysis. For ex vitro sampling, leaves initiated during in vitro growth and ex-panded considerably during the ex vitro phase were chosen. Leaves from the middle region of ex vitro plants were chosen after 10 days of full exposure to growth cabinet conditions. Other sam-pling details were as indicated for in vitro samples.
Pigment analyses
Photosynthetic pigments were extracted from leaves in 80% pre-chilled acetone and centrifuged twice at 5,000 g for 15 min. The concentration of pigments in the supernatant was determined spectrophotometrically at 646 and 663 nm for chlorophylls and at 470 nm for carotenoids and calculated per unit fresh mass basis using the equations of Lichtenthaler and Wellburn (1983).
Water-soluble flavonoids were extracted from leaves following the method of Mirecki and Teramura (1984). A known amount of leaf tissue (50 mg) cut into small sections was immersed in acidified methanol (79:20:1 v/v, methanol: water: HCl) and the total contents were estimated from the absorbance of leaf extract at 305 nm after 6 h dark incubation at 4°C and expressed as A305g–1FW.
For all pigment analyses, a UV Visible Spectrophotometer (Shimadzu, UV-1603) was used.
Carbon and nitrogen determination
Leaf and stem materials were dried at 90°C for 3 days and finely powdered. Carbon and nitrogen content of samples were estimated using a Perkin Elmer 2400 CHN elemental analyser.
Quantification of leaf soluble proteins
Total soluble proteins were extracted using the method described by Laukkanen et al. (1997). After pulverizing in liquid nitrogen, the leaf tissue (250 mg) was homogenized in 2 ml of extraction buffer [50 mM Tris-HCl pH 8.6, 20 mM KCl, 10 mM MgCl2and 1.5% (w/v) PVP]. The homogenate was centrifuged at 13,000 g for 15 min and protein concentrations were determined according to Bradford (1976).
Total phenolics determination
Total phenolic concentrations in leaf tissues were determined by the Folin-Ciocalteu method (Singleton and Rossi 1965). Leaf sam-ples (50 mg) were extracted with 2 ml of 50% aqueous methanol. An aliquot (50 µl) of methanolic leaf extract was mixed with 0.475 ml of 0.25 N Folin-Ciocalteu (Panreac, Barcelona, Spain) reagent and after 3 min at room temperature 0.475 ml of 1 M Na2CO3was added. This was vigorously vortexed and allowed to react for 1 h at room temperature. The absorbance of the solution was measured at 724 nm and a standard curve was created using known concentrations of 4-coumaric acid. The total phenolics in leaf tissues were expressed as P-coumaric acid equivalents (CAE) per unit fresh mass basis.
SDS-PAGE
Leaf proteins were extracted as described by Premkumar et al. (2001). Briefly, 250 mg of leaf tissue was homogenized on ice with 3 ml of urea-SDS lysis buffer (4 M urea, 6.25 mM Tris-HCl, pH 6.0, 2% SDS, 2% β-mercaptoethanol). The homogenate was filtered through a single layer of Miracloth (Calbiochem, USA) and proteins were precipitated with trichloroacetic acid (10% final concentration). After centrifuging, the pellets were rinsed repeat-edly with pre-chilled acetone until the pellet turned colourless. Proteins were subsequently solubilised in 2% (w/v) SDS and quantified according to Bradford (1976) with bovine serum albu-min (Sigma) as a standard. SDS-PAGE analysis of proteins fol-lowed Laemmli (1970) using a vertical slab gel (1.5 mm thick, Biorad Mini Protean II) with 4% stacking and 12% resolving gel. Extracted samples were incubated with sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 10% β-mercaptoethanol, 20% glycerol and 0.03% bromophenol blue) at 100°C for 2 min before being loaded onto the gel. Each lane was loaded with equal amounts of protein (15 µg). After electrophoresis, proteins were visualized by
staining gels with Coomassie Brilliant blue-R 250 in 40% metha-nol and 10% acetic acid. Protein profiles from various treatments were confirmed by three separate extractions and gels. A represen-tative gel is shown in the Results section. The sensitivity and pre-cision of SDS-PAGE to changes in levels of rubisco subunits were tested. A strong correlation between extract dilution and rubisco band intensity in SDS-PAGE was found, both using purified solu-bilised spinach rubisco and crude leaf extracts of olive and avoca-do. Furthermore, the recovery of added rubisco when spiking leaf extracts of various plants (avocado, olive and tobacco) with known amounts of purified spinach rubisco was also confirmed. The relative band intensities of rubisco subunits in each lane were analysed densitometrically by a computer-assisted program (area scan method using the Scion Image Program, Scion, USA).
Statistical analyses
ANOVA was used to determine the presence of significant differ-ences due to sucrose treatments in the parameters analysed. The differences were assessed at the 5% level by a Tukey HSD test and all data were tested for homogeneity of variances (Cochran C, Hartley, Bartlett) prior to analysis. Significant differences between in vitro and ex vitro cultural conditions for each of the compo-nents analysed were determined by a Student’s t-test at the 5% level.
Results and discussion
Photosynthetic pigments
In vitro cultivation of juvenile avocado microshoots with
different sucrose concentrations for 4 weeks did not
sig-nificantly affect concentrations of total chlorophylls
ei-ther before or after ex vitro transfer (Table 1), in contrast
to earlier studies on some micropropagated woody plants
(Rival et al. 1997) and several herbaceous plants (Van
Huylenbroeck et al. 2000) which showed a general
in-crease in total chlorophylls. However, the transition from
heterotrophic to autotrophic condition strongly
influ-enced the relative proportions of chlorophyll a and b. A
statistically significant increase in the chlorophyll a/b
ra-tio was observed after ex vitro transfer. This resulted
from a significant increase in chlorophyll a content
par-alleled by a reduction in chlorophyll b content,
particu-larly at the two lower sucrose concentrations. In the case
of chlorophyll b concentration, the pooled data showed
no significant differences, despite a consistent trend. A
considerably lower ratio of chlorophyll a to b in in vitro
grown plants (5 and 30 g/l), similar to that observed for
shade-type leaves (Larcher 1995), suggests an
acc-limative response of avocado plantlets to low irradiance
(40 µmol m
–2s
–1) during in vitro cultivation. A similar
physiological response has also been shown in several
micropropagated plants (Chaves 1994) as well as in
some woody species acclimated to shade environments
(Grassi and Minotta 2000).
As with total chlorophyll, sucrose treatments exerted
no significant effect on carotenoid concentrations during
in vitro or ex vitro growth. However, carotenoid
concen-trations increased significantly as a result of ex vitro
transfer. The average ratio of total chlorophyll to
caro-tenoids shifted from 4.5 (in vitro growth) to 2.6 during
acclimatization. The greater concentration of foliar
caro-tenoids compared to total chlorophylls has been shown
in a few micropropagated herbaceous plants (Van
Huylenbroeck et al. 2000) as well as in several woody
species (Munne-Bosch and Alegre 2000) and has been
regarded as an efficient photoprotective response against
high-light-induced production of triplet state
chloro-phylls and singlet oxygen (Larson 1995). Similar to
these woody plants, progressively acclimatizing
micro-propagated avocado plantlets, regardless of sucrose
treat-ments, displayed higher concentrations of foliar
caro-tenoids following sudden excessive irradiances after in
vitro growth.
In conclusion, ex vitro transfer modifies the light
ab-sorption mechanism of leaves as reflected by differences
in pigment composition. However, minor effects of
su-crose treatments were observed in pigment
concentra-tions.
Carbon, nitrogen and C/N ratio
Minor differences were observed in the carbon
concentra-tions per unit dry mass of leaf and stem tissues of plants
grown with different sucrose treatments and after ex vitro
Table 1 Changes in the concentration of photosynthetic pigments (mg g–1FW) in leaves of juvenile avocado grown under three dif-ferent sucrose concentrations (5, 30 and 50 g/l) during in vitro growth and subsequent ex vitro transfer. Values are means of three replicates. No significant sucrose effect was detected by ANOVA. Statistical differences between in vitro and ex vitro cultural condi-tions for each mean value of the components analysed were deter-mined by Student’s t-testsPigments Sucrose (g/l) In vitro Ex vitro P
Total chlorophyll 5 2.9±0.3 2.8±0.7
30 3.2±1.4 4.1±1.2
50 3.0±0.3 3.8±1.2
Mean 3.0 3.6 n.s.
Chl a 5 1.7±0.2 2.2±0.5
30 1.8±0.8 3.3±0.9
50 1.7±0.2 2.5±0.6
Mean 1.7 2.7 **
Chl b 5 1.1±0.1 0.5±0.1
30 1.3±0.5 0.8±0.3
50 1.2±0.06 1.2±0.6
Mean 1.2 0.8 n.s.
Chl a/b 5 1.4 4.0
30 1.4 4.0
50 1.4 2.4
Mean 1.4 3.5 **
Carotenoids 5 0.6±0.1 1.0±0.3
30 0.7±0.3 1.5±0.5
50 0.6±0.1 1.5±0.5
Mean 0.7 1.4 **
Chl/carotenoids 5 4.4 2.7
30 4.5 2.6
50 4.5 2.5
Mean 4.5 2.6 **
*Significant at P=0.05, **significant at P=0.01, n.s. not signifi-cant
transfer. While significant, carbon increments in
acclima-tized leaves were slight (Table 2). Nitrogen
concentra-tions were more variable and affected by treatments.
Su-crose treatments significantly reduced nitrogen
concen-tration of ex vitro leaves at the two higher sucrose
con-centrations. As a result, the C/N ratio of these two
su-crose treatments increased. Lower nitrogen
concentra-tions cannot be explained as a result of nitrogen
deficien-cies because all plants received the same nutrient supply
and there were no visible symptoms of nutrient
deficien-cy. Although concentration of total nitrogen declined,
concentration of total soluble proteins was unchanged
(Table 3). Nitrogen contents of in vitro leaves were
high-ly variable and no conclusion about the effect of sucrose
on nitrogen metabolism could be drawn. Similarly, no
significant effect of sucrose treatments was found in stem
nitrogen contents, in either in vitro or ex vitro samples.
However, the C/N was higher in ex vitro stems derived
from cultures containing 30 and 50 g/l of sucrose, as
ob-served for ex vitro leaves. Data on C/N ratio in both
leaves and stems followed a trend very similar to stem N.
In many tree species light quality (Aphalo and Lehto
1997) and light quantity (Roux et al. 2001) strongly
in-fluence foliar N, an important factor in limiting
radia-tion-use efficiency and leaf photosynthetic capacity
(Ishida et al. 2000). Individual comparisons between in
vitro and ex vitro samples from each sucrose treatment
revealed that exposure of avocado plantlets to sudden
in-creases in irradiance did not alter total leaf nitrogen, but
decreased stem N in ex vitro acclimatizing plants. This
trend was observed for 30 and 50 g/l sucrose treatments
and confirmed by t-test analysis of pooled data of in
vi-tro and ex vivi-tro samples (Table 2). Like foliar N, stem N
concentration was also used to assess various traits of the
stem such as N sufficiency (Adjctcy and Campbell 1998)
and quality (Wheeler and Center 1997).
The overall data presented in this study suggest that
the ratio between C and N assimilation rates, reflected in
the C/N ratio (Ishida et al. 1999), was significantly
influ-enced by changes in sucrose concentration, as well as by
transfer to ex vitro growth, particularly in stem tissue.
This may be related to an increase in structural carbon
during ex vitro development.
Other foliar components
Secondary metabolites in leaves, mainly phenolic
sub-stances, play a major role depending on plant
tissue/spe-cies during developmental processes and have been
con-sidered essential for plant fitness (Harborne 1982).
These components respond dynamically to biotic and/or
abiotic stresses (Dixon and Pariva 1995) and function as
internal physiological regulators and endogenous
phyto-chemical defence phyto-chemicals (Stafford 1991). In the
pres-ent study, analysis of pooled data showed that concpres-entra-
concentra-tions of total phenolics in avocado tended to increase
during ex vitro acclimatization, particularly in 30 and
50 g/l treatments (Table 3). The greater C/N ratio in
these plants seems indicative of high availability of
car-bon skeleton and a lower nitrogen content would
en-hance the secondary metabolic pathways. Additionally,
accumulation of phenolic substances was earlier
demon-strated as a general defensive response to excessive
visi-ble light (Appel 1993).
Leaf flavonoids analysed using either absolute data or
pooled data of all sucrose treatments (P=0.93) did not
re-spond to either changes in sucrose concentrations or in
vitro and ex vitro cultural conditions (Table 3). This is in
sharp contrast to recent findings of Stojakowska and
Malarz (2000) who showed a close correlation between
optimal flavonoids production and media sucrose
con-centration.
Rubisco components
Both SDS-PAGE and densitometric analyses showed
prominent changes in the concentrations of rubisco
sub-units as results of both sucrose treatments and ex vitro
transfer (Figs. 1, 2). Concentrations of both subunits of
rubisco declined consistently with increasing
concentra-tions of sucrose in in vitro grown avocado plantlets, as
previously reported (De La Viña et al. 1999). However,
Table 2 Changes in the concentrations of carbon and nitrogen on a dry weight basis (% mg–1 DW), and C/N ratio in leaves and shoots of juvenile avocado grown under three different sucrose concentrations (5, 30 and 50 g/l) during in vitro growth and subse-quent ex vitro transfer. Values are means of three replicates. Statis-tical differences among sucrose treatments for in vitro and ex vitro growth conditions were analysed by ANOVA and means with dif-ferent letters indicate significant differences by Tukey test at the 5% level. Statistical differences between in vitro and ex vitro cul-tural conditions were determined by Student’s t-testsElements Sucrose (g/l) In vitro Ex vitro P
Leaf C 5 39.6±1.06 40.7±0.4
30 37.4±1.4 40.0±2.0
50 38.1±1.9 41.6±2.4
Mean 38.3 40.7 **
Leaf N 5 2.2±0.3a 2.4±0.04a n.s.
30 1.8±0.2a 1.9±0.1b n.s.
50 3.4±1.5a 1.9±0.2b n.s.
Leaf C/N 5 18.2±2.2a 16.9±0.4b n.s.
30 20.3±2.7a 21.5±0.1ab n.s.
50 11.3±6.5a 21.8±0.2a n.s.
Stem C 5 35.4±0.6 38.7±0.6
30 37.6±1.1 39.2±1.5
50 37.0±1.1 36.9±1.7
Mean 36.7 38.3 n.s.
Stem N 5 4.0±0.6 3.8±0.2
30 3.5±1.0 1.9±0.1
50 3.4±0.8 2.1±0.5
Mean 3.6 2.6 *
Stem C/N 5 8.8±1.2a 10.0±0.1b n.s.
30 10.6±2.9a 20.4±0.2a *
50 10.7±2.6a 17.4±2.8ab *
*Significant at P=0.05; **significant at P=0.01, n.s. not signifi-cant
this pattern was completely reversed after transplantation
to ex vitro conditions.
A negative relationship was found between leaf N
content and rubisco level in plants grown ex vitro, i.e.
al-though plants from both 30 and 50 g/l showed a
statisti-cally significant reduction in leaf N, rubisco
concentra-tions were remarkably high. The present study, as well as
our earlier findings on the contents of rubisco and leaf N
in various micropropagated plants (Premkumar et al.
2001), strongly supports the suggestion of Warren et al.
(2000) that woody and sclerophyllous species do not
necessarily show positive correlation between foliar N
concentration and rubisco content.
Accumulation of carbohydrates in foliage, under
vari-ous growth conditions, is frequently suggested to be a
reason for reduction in the concentration of rubisco and
not as the result of foliar N content alone (Sims et al.
1998). This would not be the case for avocado. In fact, in
vitro plants did not show differences in sucrose and
starch concentrations and the acclimatized plants from
30 and 50 g/l treatments showed even greater
concentra-tions of starch (unpublished data), although the leaves
also accumulated more rubisco. Therefore, the changes
in rubisco in avocado could not be easily related to leaf
N or levels of endogenous sucrose and starch, although
external sucrose exerted a negative effect.
Besides leaf N, changes in rubisco protein
concentra-tion have been shown to be linked to relative changes in
other photosynthetic components, namely total
chloro-phylls and total soluble proteins in several species under
various experimental conditions (Oksanen and Saleem
1999; Valjakka et al. 1999). In contrast to these, avocado
plantlets exhibited large changes in rubisco without
simi-lar changes in the above-mentioned photosynthetic
com-ponents. In addition, determination of the rates of CO
2 Table 3 Changes in the concentration of total phenolics (mgp-coumaric acid equivalents g–1FW), flavonoids (A
300g–1FW) and total soluble proteins (TSP, mg g–1FW) in leaves of juvenile avo-cado grown in three different sucrose concentrations (5, 30 and 50 g/l) during in vitro growth and subsequent ex vitro transfer. Values are mean of three replicates. No significant sucrose effect was detected by ANOVA. Statistical differences between in vitro and ex vitro cultural conditions for each mean value of the compo-nents analysed were determined by Student’s t-tests
Components Sucrose (g/l) In vitro Ex vitro P
TSP 5 g/l 11.87±3.15 8.71±2.07
30 g/l 9.39±3.65 10.37±3.03 50 g/l 7.07±4.35 7.64±1.42
Mean 9.44 8.91 n.s.
Total phenols 5 g/l 37.38±7.29 40.44±11.15 30 g/l 33.95±6.99 48.35±11.57 50 g/l 38.57±4.30 49.98±9.13
Mean 36.63 46.26 *
Flavonoids 5 g/l 0.184±0.06 0.179±0.02 30 g/l 0.191±0.06 0.167±0.02 50 g/l 0.216±0.10 0.253±0.07
Mean 0.197 0.200 n.s.
*Significant at P=0.05, n.s. not significant
Fig. 1 Changes in SDS-PAGE profiles for total leaf polypeptides
extracted from the leaves of juvenile avocado grown under three different sucrose concentrations (5, 30 and 50 g/l) during in vitro growth and subsequent ex vitro transfer. Arrows on the left side in-dicate large (top) and small (bottom) subunits of rubisco and ar-rowheads on the right side indicate molecular mass standards
Fig. 2 Changes in relative contents of large (A) and small (B)
subunits of rubisco determined by densitometric scanning of elec-trophoretic profiles of whole leaf proteins extracted from in vitro (open bar) and ex vitro acclimatized (shaded bar) plants. The val-ues are means of three independent scan valval-ues and differences among means indicated by different letters are statistically signifi-cant at P=0.05 by Tukey HSD test. Capital and small letters were used for in vitro and ex vitro plants, respectively. For other details see Materials and methods
fixation in the same juvenile avocado plants showed that
the maximum rate of net photosynthesis was not affected
by changes in sucrose concentrations (5 and 30 g/l) (De
La Viña et al. 1999). Thus, increased concentrations of
rubisco subunits after ex vitro transfer appear to be a
storage pool of reduced nitrogen available for growth. In
fact, a dual role for this protein, as a catalyst in
carboxyl-ation of CO
2and as a major storage protein, has been
previously suggested (Huffaker 1982).
In summary, short-term acclimatization seems
direct-ly related to the ability of in vitro plantlets to adapt their
photosynthetic pigments to the suddenly imposed
auto-trophic condition. Although previous studies support an
effect of exogenous sucrose applied during the in vitro
growth phase on photosynthetic pigments, this does not
apply to avocado plantlets where changes in absorption
equipment seem unaffected by the sucrose supply during
the in vitro phase. However, sucrose treatments exert a
significant effect on the carbon–nitrogen balance of the
acclimatizing avocado plants. The plants from the higher
sucrose treatments displayed a higher concentration of
non-structural carbohydrates and changes in the reduced
nitrogen pool, with a relative higher content of rubisco
protein. These modifications could be positive for the
subsequent growth of the acclimatized plantlets. In fact,
although not strictly evaluated in the present study, the
vigour of the plants from the 30 and 50 g/l sucrose
treat-ments was clearly higher than in plants treated with 5 g/l.
Acknowledgements Financial aid in the form of a postdoctoral fellowship to A.P. by the Ministerio de Educación y Ciencias (Spain), (Estancias Temporales de Científicos y Tecnólogos Extranjeros en España) is gratefully acknowledged. Financial support was obtained from research project INIA-SC98–042.References
Adjctcy JA, Campbell LC (1998) Plant tissue nitrate and total shoot nitrogen as tools for the determination of nitrogen suffi-ciency in wheat. Appl Plant Sci 12:33–35
Aphalo JP, Lehto T (1997) Effects of light quality on growth and N accumulation in birch seedlings. Tree Physiol 17:125–132 Appel HM (1993) Phenolics in ecological interactions: the
impor-tance of oxidation. J Chem Ecol 19:1521–1552
Barceló-Muñoz A (1995) Micropropagación de aguacate (Persea
americana Mill). Aspectos histológicos del proceso de
vitri-ficación. PhD thesis, University of Malaga, Spain
Barceló-Muñoz A, Pliego-Alfaro F, Barea JM (1990) Micro-propagación de aguacate (Persea americana Mill.) en fase juvenil. Acta Hortic 1:503–506
Barceló-Muñoz A, Encina CL, Simón-Pérez E, Pliego-Alfaro F (1999) Micropropagation of adult avocado. Plant Cell Tissue Organ Cult 58:11–17
Bradford MM (1976) A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chaves MM (1994) Environmental constraints to photosynthesis in ex vitro plants. In: Lumsden PJ, Nicholas JR, Davies WJ (eds) Physiology, growth and development of plants in culture. Kluwer Academic, Dordrecht, pp 1–18
Cruz-Hernandez A, Litz MG, Witjaksono (1998) Agrobacterium
tumefaciens – mediated transformation of embryogenic
avoca-do cultures and regeneration of somatic embryos. Plant Cell Rep 17:497–503
De La Viña G, Pliego-Alfaro F, Driscoll SP, Mitchell V, Parry MA, Lawlor DW (1999) Effects of CO2and sugars on photo-synthesis and composition of avocado leaves grown in vitro. Plant Physiol Biochem 37:1–9
Dixon RA, Pariva NL (1995) Stess-induced phenylpropanoid metabolism. Plant Cell 7:1058–1097
Grassi G, Minotta G (2000) Influence of nutrient supply in shade-sun acclimation of Picea abies seedlings. Effects on foliar morphology, photosynthetic performance and growth. Tree Physiol 20:645–652
Harborne JB (1982) Introduction to ecological biochemistry. Academic Press, New York
Huffaker RC (1982) Biochemistry and physiology of leaf proteins. In: Boulter D, Parthier B (eds) Nucleic acids and proteins in plants. I. Structure, biochemistry and physiology of proteins. Encyclopaedia of plant physiology, New Series, vol 14. Springer, Berlin Heidelberg New York, pp 370–400
Ishida A, Toma T, Marjenah (1999) Limitation of leaf carbon gain by stomatal and photochemical processes in the top canopy of
Macaranga conifera, a tropical pioneer tree. Tree Physiol
19:467–473
Ishida A, Toma T, Mori S, Marjenah (2000) Effects of foliar nitrogen and water deficits on the carbon economy of Shorea
smithiana Sym. seedlings. Biotropica 32:351–358
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685 Larcher W (1995) Physiological plant ecology. Springer, Berlin
Heidelberg New York
Larson RA (1995) Plant defenses against oxidative stress. Arch Insect-Biochem Physiol 29:175–186
Laukkanen H, Julkunen-Tiitto R, Hohtola A (1997) Effect of different nitrogen nutrients on the viability, protein synthesis and tannin production of Scots pine callus. Physiol Plant 100:982–988
Lichtenthaler HK, Wellburn AR (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in differ-ent solvdiffer-ents. Biochem Soc Trans 11:591–592
Margara J (1984) Bases de la multiplication végétative. Inra, Versailles, Paris
Marin JA, Gella R (1987) Acclimatization of the micropropagated cherry rootstock ‘Masto de Montañana’ (Prunus cerasus L.). Acta Hortic 212:603–606
Mirecki RM, Teramura AH (1984) Effects of ultraviolet-B irradi-ance on soybean. V. The dependence of plant sensitivity on the photosynthetic photon flux density during and after leaf expansion. Plant Physiol 74:475–480
Munne-Bosch S, Alegre L (2000) The significance of beta-caro-tene, alpha-tocopherol and the xanthophyll cycle in droughted
Melissa officinalis plants. Aust J Plant Physiol 27:139–146
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497
Nobre J, Santos C, Romano A (2000) Micropropagation of the Mediterranean species Viburnum tinus. Plant Cell Tissue Organ Cult 60:75–78
Oksanen E, Saleem A (1999) Ozone exposure results in various carryover effects and prolonged reduction in biomass in birch (Betula pendula Roth). Plant Cell Environ 22:1401–1411 Pliego-Alfaro F (1988) Development of an in-vitro rooting
bioas-say using juvenile-phase stem cuttings of Persea americana Mill. J Hortic Sci 63:295–301
Premkumar A, Mercado JA, Quesada MA (2001) Effects of in vitro tissue culture conditions and acclimatization on the contents of rubisco, leaf soluble proteins, photosynthetic pigments, and C/N ratio. J Plant Physiol 158:835–840
Rival A, Beule T, Lavergne D, Nato A, Havaux M, Puard M (1997) Development of photosynthetic characteristics in oil palm during in vitro micropropagation. J Plant Physiol 150:520–527
Roux XL, Walcroft AS, Daudet FA, Sinoquet H, Chaves MM, Rodrigues A, Osorio L (2001) Photosynthetic light acclimation in peach leaves: importance of changes in mass: area ratio,
nitrogen concentration, and leaf nitrogen partitioning. Tree Physiol 21:377–386
Sims DA, Luo Y, Seemann JR (1998) Comparison of photosyn-thetic acclimation to elevated CO2and limited nitrogen supply in soybean. Plant Cell Environ 21:945–952
Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. J Enol Vitic 16:144–158
Stafford HA (1991) Flavonoid evolution: an enzymatic approach. Plant Physiol 96:680–685
Stojakowska A, Malarz J (2000) Flavonoid production in trans-formed root cultures of Scutellaria baicalensis. J Plant Physiol 156:121–125
Tay BS, Ong BL, Kumar PP (2000) Effect of varying CO2 and light levels on growth of Hedyotis and sugarcane shoot cultures. In Vitro Cell Dev Biol Plant 36:118–124
Valjakka M, Luomala EM, Kangasjarvi J, Vapaavuori E (1999) Expression of photosynthesis and senescence related genes
during leaf development and senescence in silver birch (Betula
pendula) seedlings. Physiol Plant 106:302–310
Van Huylenbroeck JM, Piqueras A, Debergh PC (2000) The evolution of photosynthetic capacity and the antioxidant enzy-matic system during acclimatization of micropropagated
Calathea plants. Plant Sci 155:59–66
Warren CR, Chen Z, Adams MA (2000) Effect of N source on concentration of Rubisco in Eucalyptus diversicolor, as measured by capillary electrophoresis. Physiol Plant 110: 52–58
Wheeler GS, Center TD (1997) Growth and development of the biological control agent Bagous hydrillae as influenced by hydrilla (Hydrilla verticillata) stem. Biol Control 8:52–57 Zacchini MS, Morini S (1998) Stomatal functioning in relation to
leaf age in in vitro-grown plum shoots. Plant Cell Rep 18: 292–296
