In this communication we evaluate the field performance of two
micropropagated Portuguese carob cultivars (Galhosa and Mulata) throughout the
season, particularly at extreme conditions of light and temperature. Two irrigated
plots were established in the field: 1) micropropagated plants, vs 2) seedlings. During
the first year following transplantation to the field, we followed net photosynthetic
rate, stomatal conductance, transpiration rate, chlorophyll a fluorescence and leaf
contents in chlorophyll, carotenoids and protein. No significant differences were
detected between seedlings and micropropagated plants along the year. However, at
the end of summer, despite irrigation, the photosynthetic rate (NP), the quantum
yield of PSII (fPSII) and the intrinsic efficiency of open PSII reaction centers
(F’v/F’m) declined, concomitantly with the increase of the thermal energy dissipation
at the PSII (NPQ). As the maximal photochemical efficiency of PSII (Fv/Fm) was
maintained high (0.82), these results indicate that regulated thermal dissipation in
light harvesting complexes was promoted in order to avoid photoinhibition. After
the first growth period in the field, data from micropropagated plants did not differ
from seedlings, and those plants showed the characteristic behaviour of plants well
adapted to Mediterranean climates. So, in vitro propagation could be use as a
promising alternative to traditional propagation and establishment of carob
orchards

Correia, Maria João

David, Maria Manuela

Osório, Júlio

Osório, Maria Leonor

Romano, Anabela

Sapientia

    Seasonal Changes in CO2 Assimilation in Leaves of Seedlings and Micropropagated Plants of Carob Tree Established in Field  M.L. Osório, J. Osório, M.M. David, M.J. Correia and A. Romano     The authors aknowledge Acta Horticulturae, a publication of the International Society for Horticulture Science. The definitive version of the article in http://www.actahort.org/ Seasonal Changes in CO2 Assimilation in Leaves of Seedlings and Micropropagated Plants of Carob Tree Established in Field  M.L. Osório1,2, J. Osório2, M.M. David2, M.J. Correia2 and A. Romano1  1-CBME - Centro de Biomedicina Molecular e Estrutural 2-CDCTPV - Centro de Desenvolvimento de Ciências e Tecnologias de Produção Vegetal    Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. Fax: + 351 289 818419   Keywords: Photosynthesis, photoinhibition, photochemistry, survival  Abstract In this communication we evaluate the field performance of two micropropagated Portuguese carob cultivars (Galhosa and Mulata) throughout the season, particularly at extreme conditions of light and temperature. Two irrigated plots were established in the field: 1) micropropagated plants, vs 2) seedlings. During the first year following transplantation to the field, we followed net photosynthetic rate, stomatal conductance, transpiration rate, chlorophyll a fluorescence and leaf contents in chlorophyll, carotenoids and protein. No significant differences were detected between seedlings and micropropagated plants along the year. However, at the end of summer, despite irrigation, the photosynthetic rate (NP), the quantum yield of PSII (φPSII) and the intrinsic efficiency of open PSII reaction centers (F’v/F’m) declined, concomitantly with the increase of the thermal energy dissipation at the PSII (NPQ). As the maximal photochemical efficiency of PSII (Fv/Fm) was maintained high (0.82), these results indicate that regulated thermal dissipation in light harvesting complexes was promoted in order to avoid photoinhibition. After the first growth period in the field, data from micropropagated plants did not differ from seedlings, and those plants showed the characteristic behaviour of plants well adapted to Mediterranean climates.  So, in vitro propagation could be use as a promising alternative to traditional propagation and establishment of carob orchards.  INTRODUCTION Carob tree (Ceratonia siliqua L.), a medium (10-20 m) evergreen polygamo-dioecious tree of slow growth and great longevity, is one of the most economically important tree species of the Mediterranean basin. The use of micropropagated plants would avoid time lasting procedures of grafting, with high rate of unsuccessful, to obtain the desired varieties. Although carob tree (Ceratonia siliqua) is well adapted to Mediterranean climates, micropropagated plants tend to display shade characteristics and suffer from light stress when exposed to natural environmental conditions. In a previous paper we reported on the establishment of a field trial of seedlings and micropropagated plants of two Portuguese carob cultivars (Galhosa and Mulata). After three months, clonal trial has been established with 100% of success and plants showed good photosynthetic performance and growth (Osório et al. 2007). However, the performance of micropropagated plants at the field can be affected throughout the season, since they will be coping with substantial higher light and extreme temperatures which are potential stressful conditions to them. It has been demonstrated that the combination of these factors, that predispose plants to photoinhibition or down-regulation process, contributes to the reduction in carbon assimilation that will further affect the ability of young plants for growth and survival (Chaves et al, 2002). In particular, a CO2 deprivation at the chloroplast level by stomatal closure could enhance the sensitivity of the photosynthetic apparatus to high irradiance (Flexas et al. 1998). Protection mechanisms against excess light are thus an important strategy under Mediterranean conditions and may be achieved by several mechanisms such as the regulated thermal dissipation at the light harvesting complexes (Demmig-Adams and Adams 1996). This photoprotective mechanism competes with photochemistry for the absorbed energy, leading to a decrease in quantum yield of PSII (Genty et al. 1989). Understanding the plant’s potential to acclimate to new environments is of particular importance to predict and improve performance and survival of micropropagated plants during the process of acclimation. The aim of this study was to find out how photosynthetic capacity of micropropagated plants of C. siliqua acclimates to environmental changes along the year. Thus, we provide data to evaluate the field performance of these plants of C. siliqua throughout season, particularly at extreme conditions of light and temperature, highlighting possible differences between micropropagated plants and seedlings.  MATERIAL AND METHODS   Experimental site and plant material  The experiment was carried out in a field trial near Tavira in Algarve at south of Portugal. Two irrigated plots of Ceratonia siliqua (L.) were established: 1) micropropagated plants; 2) seedlings. The study was conducted by means of concurrent gas exchange and chlorophyll fluorescence measurements in sun-exposed leaves. The measurements were carried out, at the middle of the light period, in the youngest fully expanded leaf, sampled from five plants of each set. Physiological performance has been followed throughout the first year after transplantation into field and plants were irrigated twice a week during summer. Plant survival and growth were also evaluated.   Gas exchange and chlorophyll a fluorescence measurements  Gas exchange measurements were performed under natural conditions of photosynthetic photon flux density (PPFD), air temperature, vapour pression deficit (VPD) and CO2 concentration, using a portable Minicuvette System HCM 1000 (Walz, Effeltrich, Germany) in current-year, non-senescent, leaves. Leaf net photosynthetic rate (PN) and stomatal conductance (gs) were calculated according to the equations of von Caemmerer and Farquhar (1981). Chlorophyll a fluorescence measurements were performed in the same leaves using a portable pulse amplitude modulation fluorometer (PAM-2000 system, Walz, Germany). The maximal photochemical efficiency of PSII (Fv/Fm) was estimated from Fo (basal fluorescence) and Fm (maximal fluorescence) which values were taken before dawn. The intrinsic efficiency of open PSII reaction centers (F’v/F’m) was calculated from basal and maximal fluorescence measured under natural irradiance (F’o and F’m) and the quantum yield of PSII in light-adapted leaves (φPSII) was evaluated by the (F’m-Fs)/F’m ratio (Genty et al. 1989). The photochemical quenching (qp), which was used as an estimate of the fraction of open centres, was calculated as: qp= 1-(Fs-F’o)/ (F’m-F’o) (Bilger and Schreiber 1986) and the thermal energy dissipation at the PSII as: NPQ = (Fm/F’m)-1 (Cornic 1994).  Photosynthetic pigments and total soluble protein  Leaf discs were collected and quickly frozen in liquid nitrogen Chl a and b, as well as total carotenoids (xantophylls and carotenes) extraction was performed in dim light by grinding the frozen samples in the mortar with 100% acetone. The levels of pigments were determined using the extinction coefficients and equations predetermined by Lichtenthaler (1987). Soluble proteins were extracted by homogenizing leaf samples with 50 mM HEPES containing 0.1% Triton X-100. Determination of leaf total soluble protein concentration was performed according to Bradford (1976), using the Bio-Rad Protein Assay Dye (Bio-Rad, Hercules, CA) and bovine serum albumin as a standard.  Statistical analysis Statistical analysis and graphic display were performed using SPSS® for Windows (release 15.0, SPSS Inc. Chicago, IL) and SigmaPlot (Version 9, SPSS Inc., Chicago, IL) software packages, respectively. Values shown are mean ± standard error of five replicates. All pairwise comparisons of individual means were done by the Duncan test. Differences were considered significant at P≤0.05.  RESULTS AND DISCUSSION  Survival and Growth Micropropagated plants survived transplanting with 100% of success compared with the 97% of the seedlings and in general, plants showed good growth and uniformity one year after transfer to the field. Micropropagated plants showed higher shoot length and number of branches than seedlings, although these differences were not statistically significant, (Table 1). Bigger increases in shoot length and new branches production were observed in seedlings that were smaller at transplantation (Fig. 1).   Gas Exchange and Chlorophyll Fluorescence  After transplantation (Jun 04), all gas exchange parameters were improved in all plants, particularly net photosynthesis rate (Fig. 2). No significant differences were detected between seedlings and micropropagated plants along the year either in gas exchange or in chlorophyll fluorescence (Fig. 3). However, at the end of summer, despite irrigation, the photosynthetic rate (NP), the quantum yield of PSII (φPSII) and the intrinsic efficiency of open PSII reaction centers (F’v/F’m) declined, concomitantly with the increase of the thermal energy dissipation at the PSII (NPQ) (Fig. 3). As the maximal photochemical efficiency of PSII (Fv/Fm) was maintained high (0.82), these results indicate that regulated thermal dissipation in light harvesting complexes was promoted in order to avoid photoinhibition. This downregulation of photosynthetic activity expected to be quickly reversible following the relief of seasonal summer stress.    Photosynthetic pigments and soluble protein The increase on photosynthetic capacity of micropropagared plants after Nov 04 can be associated with the increase in the concentration of chlorophyll (Fig. 2). Moreover, total soluble protein content decrease in micropropagated plants to values analogous to seedlings and no significant differences were detected after the acclimation period (4 Nov) and during the year, which may suggest that Rubisco, that usually comprises 40-60% of leaf soluble protein, is at least present in comparable quantities in micropropagated and seedlings of carob tree, under the natural growing conditions. Conclusions  This study shows that micropropagated plants of carob tree have excellent field survival. After the first growth period in the field, data from micropropagated plants did not differ from seedlings, and micropropagated plants showed, in the absence of water deficit, the characteristic behaviour of well adapted plants to Mediterranean climates. So, the similar field performance revealed by micropropagated young plants and seedlings, pointing out the use of in vitro propagated plants as promising alternative to traditional propagation and establishment of carob orchards. In fact, this methodology as compared to traditional propagation offer certain advantages for large scale production of carob trees, particularly, avoiding time lasting procedures of grafting and supplying plants genetically identical to the mother plants  ACKNOWLEDGEMENTS We thank financial support by INIAP - project AGRO 770 “Plantation of a clonal field of micropropagated plants of carob tree”. M.L. thanks Foundation for Science and Technology (FCT) for the post-doctoral grant.  Literature Cited Bilger, W. and Schreiber. U., 1986. Energy-dependent quenching of dark level chlorophyll fluorescence in intact leaves. Photosynth.  Res. 10: 303-308. Bradford, M.M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. Chaves, M.M., Pereira, J.S., Maroco, J., Rodrigues, M.L., Ricardo C.P.P., Osório. M.L., Carvalho, I., Faria, T. And Pinheiro, C., 2002. How plants cope with water stress in the field. Photosynthesis and growth. Ann. Bot. 89: 907-916. Cornic, G., 1994. Drought stress and high light effects on leaf photosynthesis. p. 297-313. In: Baker NR, Bowyer JR (Eds.), Photoinhibition of photosynthesis: From Molecular Mechanisms to the Field. BIOS Scientific Publishers, Oxford. Demmig-Adams, B. and Adams, WWIII., 1996. Xanthophyll cycle and light stress in nature: uniform response to excess direct sunlight among higher plant species. Planta 198: 460-470. Flexas, J., Escalona, J.M. and Medrano, H., 1998. Down-regulation of photosynthesis by drought under field conditions in grapevines leaves. Aust. J.  Plant Physiol.  25: 893-900. Genty, B., Briantais, J.M. and Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta  990: 87-92. Lichtenthaler,H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in enzymology 148:349-382.  Osório, M.L., Osório, J., David, M.M. and Romano, A. 2007. Field Performance of seedlings and micropropagated plantlets of carob tree. Acta Hort. 748: 259-264. Von Caemmerer, S. and Farquhar, G.D., 1981. Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376-387.      Tables  Table 1. Main shoot length, trunk diameter and number of branches of micropropagated ‘Mulata and ‘Galhosa’ and seedlings of carob tree one year after transplantation at field.  Plant Type Survival (%) Shoot length (cm) Diameter  (cm) Nº of  branches ‘Mulata’ 100 a 130.4 ± 5.61 a 10.0 ± 0.9 a 22.0 ± 3.4 a ‘Galhosa’ 100 a 143.8 ± 5.68 a 15.5 ± 1.8 a 18.7 ± 3.6 a Seedling 97 a 116.0 ± 14.3 a  12.4 ± 1.5 a 14.8 ± 1.3 a The values shown are means ± SE from five samples. Values followed by the same letter are not significantly different at P≤0.05 (one-way ANOVA).   Figures  Shoot lenght Diameter BranchesIncreases (cm)020406080100120aabaa a bbabaMulata Galhosa Seedling Fig. 1. Increases in shoot length, trunk diameter and number of branches of micropropagated ‘Mulata and ‘Galhosa’ and seedlings of carob tree from transplantation until one year after. The values shown are means ± SE from five samples. Values followed by the same letter are not significantly different at P≤0.05 (one-way ANOVA, Duncan test).  Total carotenoids  (g m-2 )0.000.020.040.060.080.100.120.14data vs G_CARTOT data vs M_CARTOT data vs S_CARTOT g s (mmol m-2  s-1 )050100150NP (mmol m-2  s-1 )0510JUN-04 NOV-04 JUN-05 SEP-05NP/g s (mmol mol-1)050100150abaaaaaaaaaaaaaaaaaaabaaaaaaaaaaaaTotal chlorophyll (g m-2 )0.00.10.20.30.40.50.60.70.8JUN-04 NOV-04 JUN-05Total soluble protein (g m-2 )01234567891011aaaaa bbbbbbaaaaaaaaaaaaaaaaba babGalhosa Mulata Seedling  Fig. 2. Net photosynthesis rate (NP), stomatal conductance (gs), instantaneous water use efficiency NP/gs, chlorophyll, carotenoids and protein of micropropagated plants and seedlings of carob tree. The values shown are means ± SE from five samples. Values followed by the same letter are not significantly different at P≤0.05 (one-way ANOVA, Duncan test).   JUN-04 NOV-04 JUN-05 SEP-05q p0.00.20.40.60.8JUN-04 NOV-04 JUN-05 SEP-05NPQ01234baabaaaaaaaaaaaa aaaaaaaaaΦΦ ΦΦe0.00.10.20.30.40.50.6abbaaaaaaabF'v /F'm0.00.20.40.60.8aabbaaaaaabaabab G alhosa  Mulata  Seedling   Fig. 3. Quantum yield of PSII (φPSII), intrinsic efficiency of open PSII reaction centers (F’v/F’m), photochemical quenching (qp) and non-photochemical quenching (NPQ) of chlorophyll fluorescence of micropropagated plants and seedlings of carob tree The values shown are means ± SE from five samples. Values followed by the same letter are not significantly different at P≤0.05 (one-way ANOVA, Duncan test).           

Seasonal changes in CO2 assimilation in leaves of seedlings and micropropagated plants of Carob tree established in the field

Universidade do Algarve

  
 
 
Seasonal Changes in CO2 Assimilation in Leaves of Seedlings and 
Micropropagated Plants of Carob Tree Established in Field 
 
M.L. Osório, J. Osório, M.M. David, M.J. Correia and A. Romano 
 
 
 
 
The authors aknowledge Acta Horticulturae, a publication of the International Society for 
Horticulture Science. 
The definitive version of the article in http://www.actahort.org/ 
Seasonal Changes in CO2 Assimilation in Leaves of Seedlings and 
Micropropagated Plants of Carob Tree Established in Field 
 
M.L. Osório1,2, J. Osório2, M.M. David2, M.J. Correia2 and A. Romano1  
1-CBME - Centro de Biomedicina Molecular e Estrutural 
2-CDCTPV - Centro de Desenvolvimento de Ciências e Tecnologias de Produção Vegetal    
Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. Fax: + 351 
289 818419  
 
Keywords: Photosynthesis, photoinhibition, photochemistry, survival 
 
Abstract 
In this communication we evaluate the field performance of two 
micropropagated Portuguese carob cultivars (Galhosa and Mulata) throughout the 
season, particularly at extreme conditions of light and temperature. Two irrigated 
plots were established in the field: 1) micropropagated plants, vs 2) seedlings. During 
the first year following transplantation to the field, we followed net photosynthetic 
rate, stomatal conductance, transpiration rate, chlorophyll a fluorescence and leaf 
contents in chlorophyll, carotenoids and protein. No significant differences were 
detected between seedlings and micropropagated plants along the year. However, at 
the end of summer, despite irrigation, the photosynthetic rate (NP), the quantum 
yield of PSII (φPSII) and the intrinsic efficiency of open PSII reaction centers 
(F’v/F’m) declined, concomitantly with the increase of the thermal energy dissipation 
at the PSII (NPQ). As the maximal photochemical efficiency of PSII (Fv/Fm) was 
maintained high (0.82), these results indicate that regulated thermal dissipation in 
light harvesting complexes was promoted in order to avoid photoinhibition. After 
the first growth period in the field, data from micropropagated plants did not differ 
from seedlings, and those plants showed the characteristic behaviour of plants well 
adapted to Mediterranean climates.  So, in vitro propagation could be use as a 
promising alternative to traditional propagation and establishment of carob 
orchards. 
 
INTRODUCTION 
Carob tree (Ceratonia siliqua L.), a medium (10-20 m) evergreen polygamo-
dioecious tree of slow growth and great longevity, is one of the most economically 
important tree species of the Mediterranean basin. The use of micropropagated plants 
would avoid time lasting procedures of grafting, with high rate of unsuccessful, to obtain 
the desired varieties. Although carob tree (Ceratonia siliqua) is well adapted to 
Mediterranean climates, micropropagated plants tend to display shade characteristics and 
suffer from light stress when exposed to natural environmental conditions. In a previous 
paper we reported on the establishment of a field trial of seedlings and micropropagated 
plants of two Portuguese carob cultivars (Galhosa and Mulata). After three months, clonal 
trial has been established with 100% of success and plants showed good photosynthetic 
performance and growth (Osório et al. 2007). However, the performance of 
micropropagated plants at the field can be affected throughout the season, since they will 
be coping with substantial higher light and extreme temperatures which are potential 
stressful conditions to them. It has been demonstrated that the combination of these 
factors, that predispose plants to photoinhibition or down-regulation process, contributes 
to the reduction in carbon assimilation that will further affect the ability of young plants 
for growth and survival (Chaves et al, 2002). In particular, a CO2 deprivation at the 
chloroplast level by stomatal closure could enhance the sensitivity of the photosynthetic 
apparatus to high irradiance (Flexas et al. 1998). Protection mechanisms against excess 
light are thus an important strategy under Mediterranean conditions and may be achieved 
by several mechanisms such as the regulated thermal dissipation at the light harvesting 
complexes (Demmig-Adams and Adams 1996). This photoprotective mechanism 
competes with photochemistry for the absorbed energy, leading to a decrease in quantum 
yield of PSII (Genty et al. 1989). Understanding the plant’s potential to acclimate to new 
environments is of particular importance to predict and improve performance and survival 
of micropropagated plants during the process of acclimation. The aim of this study was to 
find out how photosynthetic capacity of micropropagated plants of C. siliqua acclimates 
to environmental changes along the year. Thus, we provide data to evaluate the field 
performance of these plants of C. siliqua throughout season, particularly at extreme 
conditions of light and temperature, highlighting possible differences between 
micropropagated plants and seedlings. 
 
MATERIAL AND METHODS 
 
 Experimental site and plant material  
The experiment was carried out in a field trial near Tavira in Algarve at south of 
Portugal. Two irrigated plots of Ceratonia siliqua (L.) were established: 1) 
micropropagated plants; 2) seedlings. The study was conducted by means of concurrent 
gas exchange and chlorophyll fluorescence measurements in sun-exposed leaves. The 
measurements were carried out, at the middle of the light period, in the youngest fully 
expanded leaf, sampled from five plants of each set. Physiological performance has been 
followed throughout the first year after transplantation into field and plants were irrigated 
twice a week during summer. Plant survival and growth were also evaluated.  
 
Gas exchange and chlorophyll a fluorescence measurements  
Gas exchange measurements were performed under natural conditions of 
photosynthetic photon flux density (PPFD), air temperature, vapour pression deficit 
(VPD) and CO2 concentration, using a portable Minicuvette System HCM 1000 (Walz, 
Effeltrich, Germany) in current-year, non-senescent, leaves. Leaf net photosynthetic rate 
(PN) and stomatal conductance (gs) were calculated according to the equations of von 
Caemmerer and Farquhar (1981). Chlorophyll a fluorescence measurements were 
performed in the same leaves using a portable pulse amplitude modulation fluorometer 
(PAM-2000 system, Walz, Germany). The maximal photochemical efficiency of PSII 
(Fv/Fm) was estimated from Fo (basal fluorescence)
 
and Fm (maximal fluorescence) which 
values were taken before dawn. The intrinsic efficiency of open PSII reaction centers 
(F’v/F’m) was calculated from basal and maximal fluorescence measured under natural 
irradiance (F’o and F’m) and the quantum yield of PSII in light-adapted leaves (φPSII) was 
evaluated by the (F’m-Fs)/F’m ratio (Genty et al. 1989). The photochemical quenching 
(qp), which was used as an estimate of the fraction of open centres, was calculated as: qp= 
1-(Fs-F’o)/ (F’m-F’o) (Bilger and Schreiber 1986) and the thermal energy dissipation at the 
PSII as: NPQ = (Fm/F’m)-1 (Cornic 1994).  
Photosynthetic pigments and total soluble protein  
Leaf discs were collected and quickly frozen in liquid nitrogen Chl a and b, as 
well as total carotenoids (xantophylls and carotenes) extraction was performed in dim 
light by grinding the frozen samples in the mortar with 100% acetone. The levels of 
pigments were determined using the extinction coefficients and equations predetermined 
by Lichtenthaler (1987). Soluble proteins were extracted by homogenizing leaf samples 
with 50 mM HEPES containing 0.1% Triton X-100. Determination of leaf total soluble 
protein concentration was performed according to Bradford (1976), using the Bio-Rad 
Protein Assay Dye (Bio-Rad, Hercules, CA) and bovine serum albumin as a standard. 
 
Statistical analysis 
Statistical analysis and graphic display were performed using SPSS® for 
Windows (release 15.0, SPSS Inc. Chicago, IL) and SigmaPlot (Version 9, SPSS Inc., 
Chicago, IL) software packages, respectively. Values shown are mean ± standard error of 
five replicates. All pairwise comparisons of individual means were done by the Duncan 
test. Differences were considered significant at P≤0.05. 
 
RESULTS AND DISCUSSION 
 
Survival and Growth 
Micropropagated plants survived transplanting with 100% of success compared 
with the 97% of the seedlings and in general, plants showed good growth and uniformity 
one year after transfer to the field. Micropropagated plants showed higher shoot length 
and number of branches than seedlings, although these differences were not statistically 
significant, (Table 1). Bigger increases in shoot length and new branches production were 
observed in seedlings that were smaller at transplantation (Fig. 1).  
 
Gas Exchange and Chlorophyll Fluorescence  
After transplantation (Jun 04), all gas exchange parameters were improved in all 
plants, particularly net photosynthesis rate (Fig. 2). No significant differences were 
detected between seedlings and micropropagated plants along the year either in gas 
exchange or in chlorophyll fluorescence (Fig. 3). However, at the end of summer, despite 
irrigation, the photosynthetic rate (NP), the quantum yield of PSII (φPSII) and the intrinsic 
efficiency of open PSII reaction centers (F’v/F’m) declined, concomitantly with the 
increase of the thermal energy dissipation at the PSII (NPQ) (Fig. 3). As the maximal 
photochemical efficiency of PSII (Fv/Fm) was maintained high (0.82), these results 
indicate that regulated thermal dissipation in light harvesting complexes was promoted in 
order to avoid photoinhibition. This downregulation of photosynthetic activity expected to 
be quickly reversible following the relief of seasonal summer stress.   
 
Photosynthetic pigments and soluble protein 
The increase on photosynthetic capacity of micropropagared plants after Nov 04 
can be associated with the increase in the concentration of chlorophyll (Fig. 2). Moreover, 
total soluble protein content decrease in micropropagated plants to values analogous to 
seedlings and no significant differences were detected after the acclimation period (4 
Nov) and during the year, which may suggest that Rubisco, that usually comprises 40-
60% of leaf soluble protein, is at least present in comparable quantities in 
micropropagated and seedlings of carob tree, under the natural growing conditions. 
Conclusions 
 This study shows that micropropagated plants of carob tree have excellent field survival. 
After the first growth period in the field, data from micropropagated plants did not differ 
from seedlings, and micropropagated plants showed, in the absence of water deficit, the 
characteristic behaviour of well adapted plants to Mediterranean climates. So, the similar 
field performance revealed by micropropagated young plants and seedlings, pointing out 
the use of in vitro propagated plants as promising alternative to traditional propagation 
and establishment of carob orchards. In fact, this methodology as compared to traditional 
propagation offer certain advantages for large scale production of carob trees, particularly, 
avoiding time lasting procedures of grafting and supplying plants genetically identical to 
the mother plants 
 
ACKNOWLEDGEMENTS 
We thank financial support by INIAP - project AGRO 770 “Plantation of a clonal field of 
micropropagated plants of carob tree”. M.L. thanks Foundation for Science and 
Technology (FCT) for the post-doctoral grant. 
 
Literature Cited 
Bilger, W. and Schreiber. U., 1986. Energy-dependent quenching of dark level 
chlorophyll fluorescence in intact leaves. Photosynth.  Res. 10: 303-308. 
Bradford, M.M., 1976. A rapid and sensitive method for quantification of microgram 
quantities of protein utilising the principle of protein-dye binding. Analytical 
Biochemistry 72: 248-254. 
Chaves, M.M., Pereira, J.S., Maroco, J., Rodrigues, M.L., Ricardo C.P.P., Osório. M.L., 
Carvalho, I., Faria, T. And Pinheiro, C., 2002. How plants cope with water stress in the 
field. Photosynthesis and growth. Ann. Bot. 89: 907-916. 
Cornic, G., 1994. Drought stress and high light effects on leaf photosynthesis. p. 297-313. 
In: Baker NR, Bowyer JR (Eds.), Photoinhibition of photosynthesis: From Molecular 
Mechanisms to the Field. BIOS Scientific Publishers, Oxford. 
Demmig-Adams, B. and Adams, WWIII., 1996. Xanthophyll cycle and light stress in 
nature: uniform response to excess direct sunlight among higher plant species. Planta 
198: 460-470. 
Flexas, J., Escalona, J.M. and Medrano, H., 1998. Down-regulation of photosynthesis by 
drought under field conditions in grapevines leaves. Aust. J.  Plant Physiol.  25: 893-
900. 
Genty, B., Briantais, J.M. and Baker, N.R., 1989. The relationship between the quantum 
yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. 
Biochim. Biophys. Acta  990: 87-92. 
Lichtenthaler,H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic 
biomembranes. Methods in enzymology 148:349-382.  
Osório, M.L., Osório, J., David, M.M. and Romano, A. 2007. Field Performance of 
seedlings and micropropagated plantlets of carob tree. Acta Hort. 748: 259-264. 
Von Caemmerer, S. and Farquhar, G.D., 1981. Some relationships between the 
biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376-387. 
 
 
 
  
Tables 
 
Table 1. Main shoot length, trunk diameter and number of branches of micropropagated 
‘Mulata and ‘Galhosa’ and seedlings of carob tree one year after transplantation at field. 
 
Plant Type Survival 
(%) 
Shoot length 
(cm) 
Diameter  
(cm) 
Nº of  branches 
‘Mulata’ 100 a 130.4 ± 5.61 a 10.0 ± 0.9 a 22.0 ± 3.4 a 
‘Galhosa’ 100 a 143.8 ± 5.68 a 15.5 ± 1.8 a 18.7 ± 3.6 a 
Seedling 97 a 116.0 ± 14.3 a  12.4 ± 1.5 a 14.8 ± 1.3 a 
The values shown are means ± SE from five samples. Values followed by the same letter 
are not significantly different at P≤0.05 (one-way ANOVA). 
 
 
Figures 
 
Shoot lenght Diameter Branches
In
cr
ea
se
s 
(cm
)
0
20
40
60
80
100
120
a
a
b
a
a a b
ba
ba
Mulata Galhosa Seedling
 
Fig. 1. Increases in shoot length, trunk diameter and number of branches of 
micropropagated ‘Mulata and ‘Galhosa’ and seedlings of carob tree from transplantation 
until one year after. The values shown are means ± SE from five samples. Values 
followed by the same letter are not significantly different at P≤0.05 (one-way ANOVA, 
Duncan test). 
 
To
ta
l c
a
ro
te
n
o
id
s 
 
(g 
m
-
2 )
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
data vs G_CARTOT 
data vs M_CARTOT 
data vs S_CARTOT 
g s
 
(m
m
o
l m
-
2  
s-
1 )
0
50
100
150
N
P 
(m
m
o
l m
-
2  
s-
1 )
0
5
10
JUN-04 NOV-04 JUN-05 SEP-05
N
P/
g s
 
(m
m
o
l m
o
l-1
)
0
50
100
150
a
b
a
a
a
a
a
a
a
a
a
a
a
a
a
a
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a
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a
a
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a
a
a
a
a
a
a
a
a
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a
a
To
ta
l c
hl
o
ro
ph
yl
l (g
 
m
-
2 )
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
JUN-04 NOV-04 JUN-05
To
ta
l s
o
lu
bl
e 
pr
o
te
in
 
(g 
m
-
2 )
0
1
2
3
4
5
6
7
8
9
10
11
a
a
a
a
a bb
b
b
b
b
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
ab
a b
ab
Galhosa Mulata Seedling
 
 
Fig. 2. Net photosynthesis rate (NP), stomatal conductance (gs), instantaneous water use 
efficiency NP/gs, chlorophyll, carotenoids and protein of micropropagated plants and 
seedlings of carob tree. The values shown are means ± SE from five samples. Values 
followed by the same letter are not significantly different at P≤0.05 (one-way ANOVA, 
Duncan test). 
 
 
JUN-04 NOV-04 JUN-05 SEP-05
q p
0.0
0.2
0.4
0.6
0.8
JUN-04 NOV-04 JUN-05 SEP-05
N
PQ
0
1
2
3
4
b
a
a
b
a
a
a
a
a
a
a
a
a
a
a
a a
a
a
a
a
a
a
a
a
ΦΦ ΦΦ
e
0.0
0.1
0.2
0.3
0.4
0.5
0.6
a
b
b
a
a
a
a
a
a
a
b
F
'v /F
'm
0.0
0.2
0.4
0.6
0.8
a
a
bb
a
a
a
a
a
a
ba
abab
 G alhosa  Mulata  Seedling 
 
 
Fig. 3. Quantum yield of PSII (φPSII), intrinsic efficiency of open PSII reaction centers 
(F’v/F’m), photochemical quenching (qp) and non-photochemical quenching (NPQ) of 
chlorophyll fluorescence of micropropagated plants and seedlings of carob tree The 
values shown are means ± SE from five samples. Values followed by the same letter are 
not significantly different at P≤0.05 (one-way ANOVA, Duncan test). 
 
 
 
 
 
 
 
 
 
 


https://sapientia.ualg.pt/bitstream/10400.1/1058/1/Osorio%20et%20al_AHort2009_SAP.pdf

Seasonal changes in CO2 assimilation in leaves of seedlings and micropropagated plants of Carob tree established in the field

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Universidade do Algarve