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Carob trees (Ceratonia siliqua L.) regenerated in vitro can acclimatize successfully to match the field performance of seed-derived plants
Abstract
The use of in vitro regenerated plants in forestry and orchard depends ultimately on the development of efficient transplantation protocols, ensuring high survival rates and successful establishment under field conditions. We tested the performance of micropropagated carob trees (Ceratonia siliqua L.) throughout the acclimatization process in terms of survival, growth and physiological traits, including field comparisons with seed-derived and mother plants. The field trial was 100 % successful, i.e. we found no major differences between micropropagated, seed-derived and mother plants in terms of growth rate, height, number of leaves, photosynthetic efficiency, chlorophyll fluorescence, chlorophyll content and soluble protein content, although these parameters changed significantly during acclimatization. Stomatal conductance (g
s) was reduced by fourfold when plants were transferred from in vitro culture to the growth chamber, thus preventing uncontrolled wilting. The photosynthetic rate (P
N) was relatively low in vitro, in the growth chamber and the greenhouse, but increased to match seed-derived and mother plants in the field. The chlorophyll a/b ratio in leaves from in vitro and growth chamber plants was typical of shade plants (2.1) but became more characteristic of sun plants in the subsequent acclimatization stages (3.1–3.5). The maximum efficiency of photosystem II (F
v/F
m) remained mostly constant at ~0.80 throughout acclimatization, as is typical for healthy, non-stressed plants. We conclude that our micropropagation and acclimatization protocols provide a suitable alternative to traditional mass propagation techniques for the carob tree.
... Las condiciones físicas típicas de propagación como son una alta humedad, iluminación no adecuada, niveles bajos de CO2, altos niveles de carbono en el medio y la presencia de RCV generan que plantas obtenidas mediante el CTV tengan varias deficiencias anatómicas, fisiológicas y morfológicas que impiden su correcta transición al medio ex vitro (Laforge et al., 1991;Kane, 2011;Osório et al., 2012). Sin embargo es necesario mencionar que las estructuras con estas deficiencias también pueden servir como reservorio de nutrientes para las nuevas estructuras, por lo cual cualquier intento de mejorar las reservas de las plantas in vitro debería mejorar la eficiencia de aclimatización (Laforge et al., 1991;Kane, 2011;Osório et al., 2012). ...
... Las condiciones físicas típicas de propagación como son una alta humedad, iluminación no adecuada, niveles bajos de CO2, altos niveles de carbono en el medio y la presencia de RCV generan que plantas obtenidas mediante el CTV tengan varias deficiencias anatómicas, fisiológicas y morfológicas que impiden su correcta transición al medio ex vitro (Laforge et al., 1991;Kane, 2011;Osório et al., 2012). Sin embargo es necesario mencionar que las estructuras con estas deficiencias también pueden servir como reservorio de nutrientes para las nuevas estructuras, por lo cual cualquier intento de mejorar las reservas de las plantas in vitro debería mejorar la eficiencia de aclimatización (Laforge et al., 1991;Kane, 2011;Osório et al., 2012). La aclimatización es una etapa crucial para poder superar el estrés fisiológico impuesto por las condiciones in vitro y para poder garantizar el desarrollo de nuevas estructuras autótrofas que sean capaces de soportar las condiciones "normales" encontradas en el exterior (Pospíšilova et al., 1999;Carvalho y Amâncio, 2002). ...
... Las plantas transferidas desde el ambiente in vitro a condiciones de invernadero o de campo experimentan una rápida pérdida de agua debido al ineficiente control de la transpiración (Osório et al., 2012). Para superar estas limitaciones, generalmente, las plantas son transferidas a sustrato esterilizado y bien drenado, son mantenidas a una alta humedad ambiental, luz reducida y una temperatura de 20-27°C, también pueden ser transferidas a un ambiente con mayor cantidad de CO2 (Carvalho y Amâncio, 2002;Kane 2011). ...
Astrophytum asterias es una cactácea endémica de Nuevo León y Tamaulipas en México, y del sur de Texas en Estados Unidos, está considerada como vulnerable por la IUCN, se encuentra en el Apéndice I de la CITES y está listada como en peligro de extinción por la NOM-059-SEMARNAT-2010, por la destrucción de su hábitat, la colecta ilegal y limitantes biológicas como un lento crecimiento, una lenta maduración sexual, una obligada xenogamia, y una baja calidad en la polinización. Por lo cual son necesarias medidas que amortigüen el descenso de sus poblaciones naturales; una alternativa que ha resultado efectiva en distintas especies amenazadas, como las cactáceas, es el Cultivo de Tejidos Vegetales. En el presente estudio fue posible establecer las condiciones experimentales que permitieron la desinfección de semillas, la germinación in vitro y la posterior regeneración in vitro de A. asterias. Explantes (apicales, hipocótilos y raíces) obtenidos de 33 plántulas germinadas in vitro fueron sembradas en frascos con medio MS (Murashige y Skoog, 1962) adicionado con BAP/ANA 2/0.5 mg/l; KIN/ANA 2/0.5 mg/l y sin reguladores de crecimiento vegetal (CONTROL); con 11 réplicas por tratamiento. Las respuestas a los 12 meses de iniciada la inducción fueron: a) Formación de callo de color verdoso a marrón principalmente friable, pero en ocasiones compacto de color blanco o hialino, b) Respuesta organogénica principalmente de tipo indirecta a través de callo friable en el caso de los explantes ápice e hipocótilos, pero en ocasiones directa en el caso de raíces en el tratamiento control. La presencia de RCV no fue necesaria para el surgimiento de brotes, sin embargo al estar presentes aceleraban la aparición y el crecimiento de estos. La menor cantidad de brotes en explantes de tipo apical (22 brotes) y la aparición abundante de callo previa a la organogénesis, sugiere que A.asterias es una especie con una alta concentración endógena de auxinas. En el tratamiento CONTROL se obtuvo el mayor número de brotes con un total de 82, seguido del tratamiento BAP/ANA con 74 brotes. El explante con una mayor cantidad de brotes totales a través de los tres tratamientos fue el explante hipocótilo con 101 brotes. El explante que regeneró una mayor cantidad de brotes por tratamiento fue el explante hipocótilo del tratamiento control con 60 brotes seguido del explante hipocótilo del tratamiento BAP/ANA con 41 brotes. Los brotes más consolidados se individualizaron y se subcultivaron a frascos con medio MS a los que se les cambiaron las tapas plásticas por películas plásticas con un filtro de 0.02 μm, con el objetivo de reducir la hiperhidratación. Los resultados obtenidos en el presente estudio aportan al conocimiento y permiten observar el establecimiento de un método efectivo, pero mejorable, para la regeneración y conservación de A. asterias.
... The endosperm of carob seeds produces a gum (carob bean gum) rich in galactomannans, used as a food additive [3]. In addition, the seed coat and pod pulp are used in the food industry to produce locust bean gum (LBG) and natural antioxidants [11,12,40]. Carob pods are also used in the pharmaceutical and cosmetic industries [2,32,40] and could provide an economical source of bioethanol in the future, particularly in arid regions [40,51,65]. ...
... In addition, the seed coat and pod pulp are used in the food industry to produce locust bean gum (LBG) and natural antioxidants [11,12,40]. Carob pods are also used in the pharmaceutical and cosmetic industries [2,32,40] and could provide an economical source of bioethanol in the future, particularly in arid regions [40,51,65]. This gum is used as a thickener, stabilizer, binder and gelling agent in the food industry and in several technical applications, including the manufacture of chemicals, paper and cosmetics and the pharmaceutical industry [33]. ...
... In addition, the seed coat and pod pulp are used in the food industry to produce locust bean gum (LBG) and natural antioxidants [11,12,40]. Carob pods are also used in the pharmaceutical and cosmetic industries [2,32,40] and could provide an economical source of bioethanol in the future, particularly in arid regions [40,51,65]. This gum is used as a thickener, stabilizer, binder and gelling agent in the food industry and in several technical applications, including the manufacture of chemicals, paper and cosmetics and the pharmaceutical industry [33]. ...
Ceratonia siliqua L. is a slow growing evergreen tree of the family Fabaceae used for the rehabilitation of marginal and submarginal dry areas of the Mediterranean basin due to it's resistant to drought and salt tolerance. In this study, the effects of different basal media (Woody Plant Medium and Murashige and Skoog medium), explant types (cotyledon and hypocotyl) and growth regulators (BA, Kinetin and NAA) on in vitro callus formation, differentiation of callus to shoot and root formation were investigated. High frequencies of caullogenesis were obtained and the best medium for callus induction was WPM supplemented with 1.0 mg L-1 BA + 0.5 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA and 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA for hypocotyl explants and 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA; 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA and 1 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA for cotyledon explants. Callus induction was more readily obtained from hypocotyl explants than cotyledon explants. It was determined that explant types as significant on shoot formation, statistically. The shoot ratio was obtained from cotyledon explants in WPM as 10%. The best regeneration was obtained from cotyledon explants placed on WPM (30%) instead of MS medium.
... Leaves must now produce a photoassimilate which they formerly imported from the culture medium. This requirement involves modifying the photosynthetic apparatus to maintain its efficiency under a varying light energy load and to alleviate the damaging effects of environmental extremes (Os orio et al., 2012;Savitch et al., 2000). The success of in vitro propagation as a source of material for reforestation or for the establishment of commercial plantations and orchards therefore depends on efficient transplantation protocols that ensure high survival rates and allow the micropropagated plants to become established in their new environment (Hazarika, 2006). ...
... The success of in vitro propagation as a source of material for reforestation or for the establishment of commercial plantations and orchards therefore depends on efficient transplantation protocols that ensure high survival rates and allow the micropropagated plants to become established in their new environment (Hazarika, 2006). Field assessment of gas exchange will provide information about stomatal control of net photosynthetic rate, transpiration and water-use efficiency (Durkovi c et al., 2016;Os orio et al., 2012). The photosynthetic apparatus is a conservative element within the plant cell, and its reaction centres have low specific characterization, but the features (size, pigment composition) of the antenna complexes are highly variable and specific to different groups of plant species (Kirova et al., 2009). ...
... Leaves must now produce a photoassimilate which they formerly imported from the culture medium. This requirement involves modifying the photosynthetic apparatus to maintain its efficiency under a varying light energy load and to alleviate the damaging effects of environmental extremes ( Os orio et al., 2012;Savitch et al., 2000). The success of in vitro propagation as a source of material for reforestation or for the establishment of commercial plantations and orchards therefore depends on efficient transplantation protocols that ensure high survival rates and allow the micropropagated plants to become established in their new environment ( Hazarika, 2006). ...
Micropropagated plants experience significant stress from rapid water loss when they are transferred from an in vitro culture to either greenhouse or field conditions. This is caused both by inefficient stomatal control of transpiration and the change to a higher light intensity and lower humidity. Understanding the physiological, vascular and biomechanical processes that allow micropropagated plants to modify their phenotype in response to environmental conditions can help to improve both field performance and plant survival. To identify changes between the hybrid poplar [Populus tremula × (Populus × canescens)] plants propagated from in vitro tissue culture and those from root cuttings, we assessed leaf performance for any differences in leaf growth, photosynthetic and vascular traits, and also nanomechanical properties of the tracheary element cell walls. The micropropagated plants showed significantly higher values for leaf area, leaf length, leaf width and leaf dry mass. The greater leaf area and leaf size dimensions resulted from the higher transpiration rate recorded for this stock type. Also, the micropropagated plants reached higher values for chlorophyll a fluorescence parameters and for the nanomechanical dissipation energy of tracheary element cell walls which may indicate a higher damping capacity within the primary xylem tissue under abiotic stress conditions. The performance of the plants propagated from root cuttings was superior for instantaneous water-use efficiency which signifies a higher acclimation capacity to stressful conditions during a severe drought particularly for this stock type. Similarities were found among the majority of the examined leaf traits for both vegetative plant origins including leaf mass per area, stomatal conductance, net photosynthetic rate, hydraulic axial conductivity, indicators of leaf midrib vascular architecture, as well as for the majority of cell wall nanomechanical traits. This research revealed that there were no drawbacks in the leaf physiological performance which could be attributed to the micropropagated plants of fast growing hybrid poplar.
... root dysfunctions, stomatal and gas exchange disorders), which may impact carbon metabolism and ultimately plant growth. In particular, typical in vitro propagation conditions such as high humidity, low illumination and CO 2 levels, high carbon source levels and the presence of growth regulators, provide minimal stress and optimum conditions for shoot/plant multiplication, but may result in the development of morphologically, anatomically and physiologically abnormal plantlets (Os orio et al., 2012;Dias et al., 2013). The heterotrophism promoted during in vitro conditions and the poorly developed mechanisms to control water loss (e.g. ...
... Physiological performance of micropropagated plants has been followed in several species, such as Tuberaria major (Os orio et al., 2013), Olea maderensis (Santos et al., 2003) and Ceratonia siliqua (Os orio et al., 2012). In a few cases, differences in the performance of micropropagated plants were identified, but after a period of adaptation to the natural environment performances were mostly identical to those from plants originating from seed. ...
Pinus elliottii var. elliottii is a pine species with enormous economic value particularly for timber and resin industries, and is subject of high pressure for genetic improvement and cloning elite genotypes. We have recently developed a robust micropropagation protocol for this species. Plantlets performance needs to be evaluated to validate this protocol for further mass propagation. Micropropagated plantlets and seed-derived plants with similar age and shoot length were compared regarding photosynthesis, carbohydrates and pigments content, water status, DNA content and cell cycle dynamics. Micropropagated plantlets had an overall physiological performance similar to seed-derived plants. In particular, except for the transpiration rate (E), CO2 assimilation rate (A) and total soluble sugars (TSS) content, no major differences between plantlets and seddlings in terms of relative water content (RWC), chlorophyll a fluorescence and pigments content were found. Genetic fidelity analyses support that the micropropagation protocol neither induce DNA content changes nor alterations in cell cycle dynamics.
... In accordance with our findings, other authors found decreased F v /F m and photosynthetic index (PI) in cucumber plants exposed to red light (19,20,27). Osório et al. (2012) also state low F v /F m for photosynthesis, photochemical efficiency and photoinhibition studies. ...
This research studied growth and yield of Syagrus schizophylla, an extinction-endangered ornamental palm, grown under five light gradients. The treatments were: G1 - PAR=1234.10 μmol photons m-2 s-1, G2 - PAR=913.16 μmol photons m-2 s-1, G3 - PAR=666.34 μmol photons m-2 s-1, G4 - PAR=419.56 μmol photons m-2 s-1 and G5 - PAR=534.77 μmol photons m-2s-1. Before the experiment and at three, five and seven months of treatment, growth (plant height, collar diameter, number of leaves, petiole length, leaf length and width), gas exchange, chlorophyll a, and leaf green color intensity were assessed. The highest net photosynthetic rates were observed in plants under G2, G3 and G4. Values of maximum quantum efficiency (Fv/Fm) over 0.75 were observed under G2. At seven months, estimated SPAD values were 36 in G2 plants and 32 in G1 plants. According to the Dickson quality index (DQI), presented the highest growth and development rates. We conclude that G2 suits plants to be transplanted into the field, whereas G3 would be best for plants grown under shade environments.
Highlights:
• Syagrus schizophylla is an extinction endangered ornamental palm.
• One layer of Red Chromatinet® 50% should be recommended for the first year of growth of schizophylla plants to be later transplanted to the field.
• Two Red Chromatinet® 50% layers would be best for plants meant to grow under shade environments.
• The highest net photosynthetic rates were observed in plants under G2, G3 and G4.
... Literature highlights several culture media formulations differing in mineral composition being selected for in vitro plant propagation. Murashige and Skoog basal medium (MS, 1962), initially developed for optimal growth of tobacco calli, remains the most used composition in the case of many species (George and De Klerk 2008) including C. siliqua (Naghmouchi et al. 2008;Osório et al. 2012;Radi et al. 2013;Shahzad et al. 2017). However, this medium is not necessarily optimal for all plant species (Pierik 1997;Niedz and Evens 2007). ...
La présente invention fourni un nouveau milieu nutritif, développé pour la première fois pour la micropropagation in vitro de caroubier. Le milieu nutritif développé contient les éléments essentiels pour la croissance des vitroplants de caroubier àdes concentrations appropriées à cette espèce. La présente invention fourni également un procédé simple, rapide et économique pour la micropropagation de caroubier en utilisant le milieu de culture liquide et la technique d'enracinement ex vitro. Ce procédé constitue un outil important qui pourra faciliter la multiplication en masse de vitroplants de qualité chez le caroubier afin de répondre à la demande mondiale croissante en matière de cette espèce.
... Literature highlights several culture media formulations differing in mineral composition being selected for in vitro plant propagation. Murashige and Skoog basal medium (MS, 1962), initially developed for optimal growth of tobacco calli, remains the most used composition in the case of many species (George and De Klerk 2008) including C. siliqua (Naghmouchi et al. 2008;Osório et al. 2012;Radi et al. 2013;Shahzad et al. 2017). However, this medium is not necessarily optimal for all plant species (Pierik 1997;Niedz and Evens 2007). ...
A new basal culture medium was developed and tested using a rapid and efficient protocol of in vitro axillary shoot bud proliferation of Ceratonia siliqua L., an important Mediterranean Fabaceae plant species. In a first experiment, the new formulated ‘LA’ mineral composition significantly improved shoot growth and proliferation as compared with Murashige and Skoog medium (MS, 1962) in both solid and liquid culture media. However, the liquid culture system proved to be the most suitable for shoot induction, shoot length (about fourfold higher), and multiplication rate (about two-fold higher), the difference being significant. The measured growth and proliferation parameters were further improved when LA mineral composition was optimized, in a second experiment. The highest multiplication rate (6.3) was achieved during the second subculture using the optimized ‘LAC’ medium. Noticeably, hyperhydricity and shoot-tip necrosis symptoms were absent in both formulated LA and LAC compositions when using the liquid culture system. In vitro rooting in solid medium showed 41.7 to 46.3% response on a solid medium which was more suitable than the liquid culture system, the difference being significant. In contrast, pretreated microcuttings with 3 μM IBA (indole-3-butyric acid) were successfully rooted ex vitro, showing significantly higher response (91.7%), average root number (8.3), and root length (31.5 mm). The plantlets were successfully acclimatized showing more than 90% survivability and normal morphology. The present study is a first cost-effective protocol for carob micropropagation combining the use of the newly formulated LAC basal medium, a liquid culture system, and ex vitro rooting.
... The pods are used amongst others as animal fodder with nutrition similar to barley and outing oats, chocolate replacement, and as industrial gum agent (Karababa and Coşkuner, 2013;Shepperd, 2008). Because of the many uses and increased demand, experimentation has been undertaken in the multiplication of this tree (Osório et al., 2012). The first step in this process is seed germination. ...
The pods of Ceratonia siliqua L. (Fabaceae) have taken foot in the ever-growing diet industry as chocolate replacements. The demand for the production of pods has subsequently increased and the upscaling of production is required. The horticultural practices to achieve such results are not as easily achieved with the carob tree as is commonly known. This is because C. siliqua is a slow growing tree, with different genders. Only the female trees produce the pods of interest. Another obstacle to overcome is seed dormancy that is attributed to its seemingly impervious seed coat. In this study, there were four treatments: the first seeds were treated with boiling water (100°C) for 30 min, the second group was chemically scarified by sulphuric acid for 30 min, the third treatment mechanically scarified until the green endocarp was visible, and last was the untreated control group. All treatments were then subjected to a 24 h imbibition period in distilled water and incubated at 25°C, with three different photoperiods: 8 h light, 12 h light or complete darkness. The germination percentages achieved showed that mechanical scarification had the highest germination percentage (90%), followed by the sulphuric acid (36%) and boiled water (24%) treatments. Photoperiod did not affect germination. Thus, in order to propagate C. siliqua successfully, mechanical scarification of the seed is very important to ensure a high germination percentage of the seeds if the crop is to be commercialized at a large scale.
... Understanding the oxidative stress status of micropropagated plants during acclimatization can be of great importance to improve survival rates and aid successful establishment under natural conditions. Moreover, the comparison between in vitro-produced plants and field-grown plants is useful in this type of research (Osório et al. 2012;Osório et al. 2013). Although there are several studies addressing the ex vitro performance of in vitrogrown plants, only a few have focused on the oxidative response throughout the acclimatization process. ...
Plantago algarbiensis Samp. and Plantago almogravensis Franco are species endemic to Portugal at risk of global extinction. The aim of this study was to investigate the ex vitro performance of micropropagated P. algarbiensis and P. almogravensis plants in terms of survival, relative water content (RWC), photosynthetic pigment contents, H2O2 accumulation, activities of superoxide dismutase (SOD) and catalase (CAT), lipid peroxidation, and soluble protein content, in comparison with wild-grown plants. Relatively high survival rates and RWC values during the acclimatization process were observed for both species. In P. algarbiensis, the pigment content increased when plantlets were transferred to ex vitro conditions, indicating enhanced light absorption capacity. No significant alterations in H2O2 content, CAT activity, or lipid peroxidation level were observed during acclimatization, but the protein content decreased in plants at the end of the growth chamber and greenhouse stages. When P. almogravensis plantlets were transferred to the ex vitro environment, decreases in the H2O2 content were observed that correlated to increased CAT activity and SOD maintenance, which lead to decreased lipid peroxidation and protein content. It was concluded that micropropagated P. algarbiensis and P. almogravensis plants were able to manage the oxidative stress induced by the in vitro environment and to perform well under ex vitro conditions.
... This may have been a strategy for avoiding photoinhibitory damage to the photosynthetic apparatus, considering that the plants did not show F v /F m values lower 0.75 (Maxwell and Johnson 2000). F v /F m is a sensitive indicator of changes in photosynthesis, photochemical efficiency, and in particular, photoinhibition (Osório et al. 2012). S. coronata plants showed lower qP values, but only on the 12th day of the stress treatment, which is precisely when marked reductions in the g s and A were observed. ...
Key message
The in vitro culture of zygotic embryos is an efficient alternative to overcome dormancy of
Syagrus coronata
and young plants showed high resilience during drought and rehydration.
Abstract
A method for obtaining plants from the cultivation of zygotic embryos of the species Syagrus coronata (Mart.) Becc. was evaluated, and rates of contamination, oxidation, germination and normal plants and the use of pre-acclimatization treatments involving modifications of culture vessel lids were assessed. Anatomical descriptions of the embryos and plants during the initial stages of development were also performed. Additionally, the mechanisms of drought tolerance, water relations, gas exchange, chlorophyll fluorescence and biochemical parameters from the plants were evaluated during drought and recovery. In the first week of in vitro culture, we observed the intumescense of the embryo, suggesting that dormancy is not related to maturity level. Anatomically, the embryonic axis was located at the embryo’s proximal region, while the distal region contained the haustorium. The use of Y3 medium with activated charcoal or lauric acid resulted in the highest number of plants, and the pre-acclimatization treatment (one 95 mm2 opening in the lid) allowed for a 100 % survival of the plants under ex vitro conditions. The plants adjusted their metabolic rates during water deficit by maintaining their leaf water concentrations, robust stomatal control and high rates of non-photochemical energy dissipation. Moreover, during recovery, the plants rapidly reached their photosynthetic capacities. This behavior indicates that the use of in vitro culture does not compromise the plants’ performance, which was indicated by the high resiliencies they displayed during the experimental period.
... The development of instruments capable of imaging Chl fluorescence provided a powerful tool to identify spatial heterogeneity of leaf photosynthetic performance, and offered new possibilities to understand the operation and regulation of photosynthesis (Baker, 2008;Gorbe and Calatayud, 2012). This rapid non-destructive method has been used to assess the effects of other abiotic stresses on photosynthetic efficiency (Calatayud et al., 2006;Osório et al., 2011Osório et al., , 2012Osório et al., , 2013Martins et al., 2013). The use of Chl fluorescence imaging to quantify the degree of photosynthetic leaf heterogeneity related with Fe nutrition seems obvious, but to our knowledge this is the first report of such a study. ...
Bare-root transplants of strawberry (Fragaria × ananassa Duch. cv ‘Diamond’) were grown in a Hoagland's nutrient solution with or without Fe. Forty two days after Fe deprivation, recovery was induced by addition of 10 μM of Fe (Fe-EDDHA) to the Fe(−) nutrient solution. Total leaf chlorophyll concentration in young leaves decreased progressively with time in Fe-deprived plants, and before Fe resupply it was only 6% of that of Fe(+) control plants. Spatio-temporal changes on photosynthetic efficiency were monitored by imaging chlorophyll a fluorescence in four areas of interest (AOIs) located on the midrib and on interveinal mesophyll areas of leaf blades. Chlorophyll fluorescence images (Fv/Fm, ϕII, NPQ, qp) showed a large spatial variation, particularly at day 42, with greater values in midrib areas. Temporal changes were also observed in all measured parameters along the experimental period, but the onset and intensity of impact was clearly different between parameters. Maximal efficiency of PSII (Fv/Fm) was the last parameter to be affected, being the effects visible only in plants that had lost over 90% of their chlorophyll (day 42). In contrast, actual efficiency of PSII (ϕII) and photochemical quenching (qp) were affected earlier on, showing noticeable changes by day 20, when chlorophyll concentration had declined by 38%. Decreases in ϕII were balanced by increases in quantum yield of non-regulated energy dissipation (ϕNO). Fluorescence parameters, with the exception of ϕII and Fe content, recovered within eight days following Fe resupply to values found in Fe(+) plants. The results of this study indicate that: (i) Chl fluorescence imaging is a useful technique to evaluate Fe deficiency (ii) Fe stress generates spatio-temporal heterogeneity in fluorescence response (iii) ϕII measured in interveinal mesophyll areas could be used as an early and fast indicator of Fe deficiency and could be applied for fertilization management.
This datasheet on Ceratonia siliqua covers Identity, Overview, Associated Diseases, Pests or Pathogens, Distribution, Dispersal, Biology & Ecology, Environmental Requirements, Uses, Management, Genetics and Breeding, Food Quality, Economics, Further Information.
Low survival rate, poor adaptation to ex-vitro environments, and time required for hardening the plants to cope with fluctuated environments of field trial are identified as major barriers in this technology. In present study, iso-osmotic adjustment in the culture medium using sucrose and/or mannitol was applied to the in-vitro cloning of Curcuma zedoaria (white turmeric) plants, which were transferred to ex-vitro conditions and subsequently cultivated in the greenhouse conditions prior to harvest after 9 months. During both in-vitro and ex-vitro development of plant, growth and physiological traits under 3% sucrose (Suc) + 2.5% mannitol (Man) were lower than those in control (3% Suc; conventional tissue culture). Interestingly, pseudostem height and root length in acclimatized plantlets under 3% Suc + 2.5% Man were sharply dropped by 60.13% and 92.37% over control, respectively, resulting in a decrease in the ex-vitro adaptation by 56.27% and 33.33% over the control. A positive relationship between reduction of net photosynthetic rate (P n ) and sucrose concentration in the leaf tissues was evidently observed. Remarkably, the morphological and physiological traits of aboveground and underground parameters of acclimatized plantlets under 3% Suc + 2.5% Man were maximized over control, leading to high yield of curcuminoids (229.4 mg plant − 1 ) in the dry rhizome (31 g plant − 1 ) when cultivated under greenhouse microenvironments for 9 months. Based on this investigation, we propose that plantlets of C. zedoaria micropropagated using 3% Suc + 2.5% Man can readily acclimatize under ex-vitro conditions and subsequently develop as healthy plants with compact and uniform size.
Ceratonia siliqua L. is a Mediterranean medicinal plant traditionally cultivated for its ethnopharmacological benefits, such as antidiarrheal, antidiabetic, enhance acetylcholine, antioxidant, antiatherosclerotic, and for its possible anti-neurodegenerative potential. The aim of the present study was to evaluate the chemical composition, as well as the cognitive-enhancing, anxiolytic, and antioxidant activities of the aqueous extract from C. siliqua (CsAE) leaves against 6-hydroxydopamine (6-OHDA) zebrafish Parkinson’s disease (PD) model. CsAE (0.1, 0.3, and 1 mg/L) was administered by immersion to zebrafish (Danio rerio) for eight consecutive days and one hour before each behavioral test of each day, while 6-OHDA (250 µM) treatment was supplied one day before the novel tank diving test (NTT). Qualitative and quantitative analyses were performed by the ultra-high-performance liquid chromatography (UHPLC) analysis. The memory performance was evaluated through the NTT and Y-maze tests. Additionally, the in vitro and in vivo antioxidant status and acetylcholinesterase (AChE) activity was also assessed. Our finds demonstrated that CsAE presented positive antioxidant and anti-AChE activities, which contributed to the improvement of cognitive function in the 6-OHDA zebrafish PD model.
The efficient maintenance of the activity of excised branches is the powerful guarantee to accurately determine gas exchange flux between the detached branches of tall trees and the atmosphere. In this study, the net photosynthetic rate (NPR) of the excised branches and branches in situ were measured simultaneously by using two photosynthetic instruments to characterize the activity of the excised branches of Phyllostachys nigra. The ratio of normalized NPR of excised branches to NPR in situ was used to assess the photosynthetic activity of detached branches. Based on photosynthetic activity, an optimal hydroponics protocol for maintaining activity of excised P. nigra branches was presented: 1/8 times the concentration of Gamborg B5 vitamin mixture with pH = 6. Under the best cultivation protocol, photosynthetic activity of excised P. nigra branches could be maintained more than 90% within 6 hr in the light intensity range of 200–2000 μmol/(m ² ·sec) and temperature range of 13.4–28.7°C. The nitrogen dioxide (NO 2 ) flux differences between in situ and in vitro branches and the atmosphere were compared using double dynamic chambers. Based on the maintenance method of excised branches, the NO 2 exchange flux between the excised P. nigra branches and the atmosphere (from −1.01 to −2.72 nmol/(m ² ·sec) was basically consistent with between the branches in situ and the atmosphere (from −1.12 to −3.16 nmol/(m ² sec)) within 6 hr. Therefore, this study provided a feasible protocol for in vitro measurement of gas exchange between tall trees and the atmosphere for a period of time.
Prolific and rapid in vitro plant organogenesis via direct regeneration has been obtained from axenic seedling-derived petiole and leaf explants of Ficus religiosa in Murashige and Skoog (MS) medium containing different concentrations of cytokinins in combination with indole-3-butyric acid (IBA). MS medium with 1.5 mg/l 6-benzylaminopurine plus 0.15 mg/l IBA produced the highest shoot induction frequency with an average of 6.26 and 10.13 shoots per leaf and petiole explants, respectively. After 4 weeks, the highest root formation frequency (96.7%), root number (5.73), and root length (4.76 cm) were with MS medium containing 2.0 mg/l IBA plus 0.1 mg/l α-naphthalene acetic acid. In addition, the effect of four sodium nitroprusside (SNP) treatments on acclimatization was also studied. Highest morphological traits such as survival rates, fresh and dry root weights as well as antioxidant enzymatic activities such as superoxide dismutase, peroxidase, and catalase was achieved with 125 ppm SNP. The α-amino acid, proline, content was highest with this treatment while the highest H2O2 (hydrogen peroxide) was in the controls. This study introduces a cost-effective, prolific, and efficient in vitro multiplication system to supply pharmaceutical and ornamental needs. It is the first report of an in vitro organogenesis protocol for F. religiosa by direct regeneration through axenic seedling-derived petiole and leaf explants, which can be efficiently employed for the utilization of active biomolecules.
Key message
The article describes refined regeneration process in Ceratonia siliqua using different plant growth regulators along with antioxidant, SEM and IRGA analysis to understand the developmental behaviour of the plantlets.
Abstract
The present study describes a simplified seed germination process in Ceratonia siliqua L. and gives a comparative analysis of hormonal supply for enhanced adventitious shoot regeneration through aseptic seedling-derived cotyledonary nodes. The axillary bud induction and multiplication was greatly influenced by the concentration and type of cytokinin viz. 6-benzylaminopurine (BA), meta-Topolin (mT), Kinetin (Kn) and Thiadiazuron (TDZ). Adenine-based cytokinins, BA and mT, augmented in Murashige and Skoog medium, were proved to be more responsive. The medium containing 10 µM BA produced a maximum of 9.1 ± 3.0 shoots/ explant. A combination of optimal concentration of BA with NAA (0.5 µM) however, resulted into a significant production of 28.10 ± 0.14 shoots per CN. Half-strength MS + IBA (10 µM) in the presence of light ensured more number of root formation while half-strength MS + IBA (5.0 µM) and stimulation in the dark for 1 week ensured better root growth (4.98 ± 0.08 cm). Plantlets were successfully acclimatized in soilrite™, showing 65% survival. The work is supported with the studies based on antioxidant enzyme activity as well as on net photosynthetic rate and its related attributes in comprehension with SEM analysis of leaves showing stomatal micromorphology during three stages: in vitro, during acclimatization and in net house conditions. The studies during acclimatization reflect better understanding of the stabilization of micropropagated plantlets to the environmental conditions.
Tissue culturing is widely used for the clonal propagation of several plant species. However, high mortality rates may occur during the acclimatization phase due to the sudden, dramatic changes in light levels. This study evaluated the effect of light level during the acclimatization of Neoregelia concentrica (Vellozo) LB Smith using chlorophyll a fluorescence and growth analysis. N. concentrica plants were multiplied and rooted in vitro on Murashige and Skoog medium and then transferred to plastic trays with individual cells containing vermiculite. The treatments consisted of the ex vitro cultivation of plants under light levels of 30, 50, 70, and full sunlight (100% light) for 28 d. Chlorophyll a fluorescence measurements were taken at 0 (in vitro), 7, 14, 21, and 28 d of acclimatization, and growth trait analyses were made at 28 d. The high irradiance under full sunlight (100% light) induced photoinhibition and reduced the growth rate. Plants grown under partial light (30, 50, and 70%) were less inhibited. A 50% reduction in light level during N. concentrica acclimatization produced rapid plant recovery and higher growth rates than full sunlight.
Tuberaria major (Willk.) P. Silva and Rozeira is a critically-endangered rock rose species endemic to Portugal. Because the species needs to be preserved, this study evaluated the morphological and physiological traits of micropropagated T. major plants during acclimatization and field transfer. There were no significant differences between wild and micropropagated plants in the field, although the latter underwent significant changes during acclimatization. Leaf pubescence and leaf mass per area increased during acclimatization whereas the chlorophyll content and chlorophyll/carotenoid ratio declined to eventually match those of wild plants. Stomatal conductance (gs) and transpiration rates (E) also declined substantially during acclimatization, thus preventing uncontrolled wilting. Photosynthetic rate (PN) was initially negative but increased during the later stages of acclimatization. Maximum quantum yield of PSII (Fv/Fm) remained constant at 0.78–0.85, showing that the plants were healthy and unstressed. PSII quantum efficiency (ϕPSII) was initially low but increased during acclimatization along with photosynthetic performance as the energy partitioning in PSII was adjusted. This was balanced by the decline in non-regulated energy dissipation (ϕNO) from an initially high value. Electrolyte leakage and malondialdehyde content remained constant at similar levels in both groups of plants, but H2O2 levels were higher in the field, perhaps indicating the early induction of antioxidant defense systems. The present study shows that T. major has enough phenotypic plasticity to adapt to changing environments and that the procedure described herein can be used for the restoration and preservation of this species.
The importance of both stomatal closure and reduced carboxylation efficiency
on the photo- synthesis decline in response to long term water stress was
previously measured in field-grown grapevines. Here we address the question of
whether water stress affects the photochemical capacity of leaves, measuring
gas-exchange rates and chlorophyll fluorescence under drought and moderate
irrigat- ion at intervals through the summer season during three consecutive
years.
We conclude that usually water stress does not induce photoinhibition in
field-grown grapevines, even when stomatal conductance and photosynthesis are
reduced to very low values. Moreover, down-regulat- ion of photochemical
reactions is low, leading to a general pattern of photosynthetic response to
drought consistent in large reductions of stomatal conductance
(g), followed by a consistent decrease of
CO2 assimilation (A) but with a
much lower effect on electron transport rate (ETR). In consequence, the
intrinsic water-use efficiency (A/g) increased, as well
as the ratio ETR/A. It is suggested that increased
electron transport to alternative pathways, such as photorespiration,
prevented further down-regulation of ETR under drought conditions.
These results are in agreement with our previous reports for potted plants.
However it is clear that, under field conditions with a much more slowly
developed water stress, ETR reductions are more attenuated than in potted
plants, reducing their incidence in carbon assimilation, which seems to be
mainly regulated by stomatal closure.
A field test of wych elm (Ulmus glabra Huds.) trees micropropagated in tissue culture was established to compare their photosynthetic characteristics with those from control grafted scions. Measurements were made at the sixth growing season after planting. The results showed similarities between the two stock types in terms of height, diameter at breast height, and leaf morphology. Grafted trees, which were markedly less rejuvenated than micropropagated trees, performed better in many of the light absorption and leaf gas exchange attributes including maximum CO2 assimilation rate at saturating photosynthetic photon flux density (Pmax), apparent quantum yield of photosynthesis (a), transpiration rate (E), and stomatal conductance (gs). In the grafts, the direct effect of photosynthesis could be clearly identified by a significantly lower specific leaf area and a higher content of leaf dry mass. Chlorophyll a fluorescence parameters showed nonstressed growth patterns in both stock types. Rates of the maximal photochemical efficiency of photosystem II (Fv/Fm), potential electron capacity (‘area’), and variable-to-initial fluorescence ratio (Fv/F0) were higher in micropropagated elms. Findings from this study indicate that micropropagated elm stock is ready for testing on a larger scale.
This study reports the effects of light availability during the acclimatization phase on photosynthetic characteristics of micropropagated plantlets of grapevine (Vitis vinifera L.) and of a chestnut hybrid (Castanea sativa × C. crenata). The plantlets were acclimatized for 4 weeks (grapevine) or 6 weeks (chestnut), under two irradiance treatments, 150 and 300 µmol m-2 s-1 after in vitro phases at 50 µmol m-2 s-1. For both treatments and both species, leaves formed during acclimatization (so-called `new leaves') showed higher photosynthetic capacity than the leaves formed in vitro either under heterotrophic or during acclimatization (so-called `persistent leaves'), although lower than leaves of young potted plants (so-called `greenhouse leaves'). In grapevine, unlike chestnut, net photosynthesis and biomass production increased significantly with increased light availability. Several parameters associated with chlorophyll a fluorescence indicated photoinhibition symptoms in chestnut leaves growing at 300 µmol m-2 s-1. The results taken as a whole suggest that 300 µmol m-2 s-1 is the upper threshold for acclimatization of chestnut although grapevine showed a better response than chestnut to an increase in light.
Publisher Summary This chapter presents detailed information on chlorophylls and carotenoids to give practical directions toward their quantitative isolation and determination in extracts from leaves, chloroplasts, thylakoid particles, and pigment proteins. The chapter focuses on the spectral characteristics and absorption coefficients of chlorophylls, pheophytins, and carotenoids, which are the basis for establishing equations to quantitatively determine them. Therefore, the specific absorption coefficients of the pigments are re-evaluated. This is achieved by using a two-beam spectrophotometer of the new generation, which allows programmed automatic recording and printing out of the proper wavelengths and absorbancy values. Several procedures have been developed for the separation of the photosynthetic pigments, including column (CC), paper (PC), and thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). All chloroplast carotenoids exhibit a typical absorption spectrum that is characterized by three absorption maxima (violaxanthin, neoxanthin) or two maxima with one shoulder (lutein and β-carotene) in the blue spectral region.
The possibility of utilising chopped and deseeded carob pods (kibbles) as a source of polyphenolic antioxidants was examined by performing extractions with various solvent systems, in order to evaluate and optimize the conditions for the recovery of polyphenols. Maximum quantities of polyphenolic components were found in 80 % acetone extracts, as evaluated by measuring total polyphenol and total flavanol content. By contrast, ethyl ace-tate was inefficient in extracting polyphenols. The assessment of the antioxidant potency of carob pod extracts employing two characteristic in vitro models showed that carobs con-tain polyphenols with appreciable antiradical and reducing properties. The values obtained were compared to the data on red wines and pure polyphenolic antioxidants.
Little is known about physiological changes that occur with micropropagated chile ancho pepper (Capsicum annuum L. cv. San Luis) plantlets during acclimatization. Plantlets were transferred to ex vitro conditions to study selected physiological changes and growth performance during acclimatization and post-acclimatization.
The physiology of the plantlets was characterized by measuring leaf gas exchange and water status. Plant growth was determined
by assessing plant height, leaf number, total leaf area, relative growth rate (RGR), and leaf, root, and stem dry matter (DM).
Chile pepper plantlets became acclimatized within 6 days after transplantation. During this period, physiological adjustments
occurred, which were critical for plantlet survival. After initial ex vitro transplanting, plantlets experienced water deficit [leaf wilting and reduced relative water content (RWC)], which corresponded
with reduced stomatal conductance (g
s) and transpiration (E), and an increase in stomatal resistance (r
s). Thus, leaf stomata that developed in vitro were functional ex vitro. Because of this stomatal control, plantlets minimized transplant shock, recovered and survived. Prior to transplanting,
plantlets were photomixotrophic, as indicated by low photosynthetic rates (A). During acclimatization, RWC, g
s, E, and A were significantly lower two days after transplanting. However, within 6 days after transplanting, plantlets recovered and
became photoautotrophic – attaining high A, g
s, and E. Water use efficiency was initially low during the first days after transplanting, but increased dramatically at the end
of the acclimatization period in part due to increased A. The stabilization and improvement of plantlet water status and gas exchange during acclimatization and post-acclimatization
closely correlated with increased plantlet growth.
Micropropagation allows large-scale plant multiplication and germplasm preservation, representing an added value in forest
breeding strategies to combat desertification and/or protect endangered species. We developed a large-scale micropropagation
protocol of Olea maderensis (a native endangered wild olive of Madeira Archipelago) using OMG medium (rich in Fe, Mg and Mn) supplemented with zeatin
for elongation and with NAA for rooting. We now describe the performance of micropropagated plants during five-period field
acclimatization: (1) in vitro, (2) growth-cabinet, (3) greenhouse, (4) open-greenhouse, and (5) field mountain in Porto Santo
Island. One hundred OG4 plants were acclimatized, showing >95% surviving rates. During acclimatization, several physiological
parameters were evaluated; water content remained higher in in vitro/greenhouse conditions, decreased in field leaves. Soluble
protein contents decreased during the first acclimatization periods increasing thereafter. Membrane permeability slightly
increased during the field acclimatization. Chlorophylls content increased in in vitro leaves, while during acclimatization,
mostly chl b decreased, increasing chl a/chl b ratio. F
0 decreased in first acclimatization periods, increasing thereafter, while the other parameters (F
v; F
m; F
v/F
m) decreased. Nutrient contents decreased in plants transferred to poor field soil conditions, reaching values similar to mother
plant leaves. Overall, with the exception of PSII fluorescence, field acclimatized plants had similar values to mother plants,
showing a good adjustment to stressful field conditions. This protocol is being used in large-scale micropropagation within
a reforestation program, and is an example of R&D technologies with immediate application on protection of endangered ecosystems.
Carob tree (Cerafoma siliqua L.) micropropagated shoots were rooted on half-strength Murashige and Skoog medium, supplemented with different types and concentrations of sugars, in order to determine the effects of sugar composition and concentration on in vitro rooting and in vivo establishment of the plantlets. Among the various sugars tested, the best rooting response was obtained with 145 mM sucrose, both in terms of rooting frequency and index of rooting. The use of filter-sterilized rather that autoclaved fructose increased root number and root length. Sugar treatment during rooting slightly influenced plantlet survival and growth during acclimatization. A reduction in the glucose concentration during rooting was beneficial for plantlet acclimatization.
Grapevine plantlets multiplied in vitro were acclimatized at 40 or 90 μmol m−2 s−1 photon flux density for 12 or 16 h per day, respectively. In the high-light regime a decrease in total chlorophyll and an
increase in chlorophyll a/chlorophyll b ratio occurred. However, at high-light intensity lower photosynthetic capacities and higher apparent photosynthesis were
measured than at the low-light regime. In leaves expanded during acclimatization, the light compensation point was higher
in plantlets under high-light while quantum yield was higher in low-light conditions. High-light also gave rise to an increase
in carbohydrate concentration. As a whole, the results suggest that high-light increases carbon assimilation and growth although
with a low investment in the photosynthetic apparatus.
An in vitro propagation protocol based on axillary bud proliferation has been developed for mature female trees of Ceratonia siliqua L. `Galhosa' and `Mulata'. Browning and contaminants were the major obstacles for culture establishment. Shoot culture initiation was greatly influenced by explanting season, with the highest survival percentage observed in spring. The cultivar, cytokinin type and concentration were the most important factors affecting shoot multiplication. The best multiple-shoot response was obtained with `Mulata' on Murashige and Skoog medium supplemented with 4.44 M 6-benzyladenine or 4.56 M zeatin. Rooting was achieved on growth-regulator-free medium after basal dipping of shoots in indole-3-butyric acid (4.9 mM). Plantlets were successfully acclimatized (80–85%) under high relative humidity and then moved to the glasshouse. A field trial was established to follow their agronomic behaviour.
The aim of the study was to establish whether the quantity and the quality of light affect growth and development of Withania somnifera plantlets. We have studied growth and histo-physiological parameters [stomatal characteristics, chloroplastic pigments concentrations,
photosynthesis, and transpiration (E)] of W. somnifera plantlets regenerated under various light intensities, or monochromatic light or under a mixture of two colors of light in
tissue culture conditions. Plantlets grown under a photon flux density (PFD) of 30μmolm-2s-1 showed greater growth and development than those raised under other PFDs. Chlorophylls and carotenoids, numbers of stomata,
rate of photosynthesis (PN) and transpiration (E), stomatal conductance (gs), and water use efficiency (WUE) increased with increasing the PFD up to 60μmolm-2s-1. Light quality also affected plantlets growth and physiology. Highest growth was observed under fluorescent and in a mixture
of blue and red light. Very few stomata were developed in any of the monochromatic light but under fluorescent or under a
mixture of two colors stomatal numbers increased. Similarly, gs, E, PN, and WUE were also higher under fluorescent light and under a mixture of red and blue light. Regressional analysis showed
a linear relationship between PN (r
2=70) and gs and between E (r
2=0.95) and gs. In conclusion, both the quality and the quantity of light affect growth of plantlets, development of stomata and physiological
responses differently depending on the intensity and the wavelength of light.
The activity of the main enzymes related to the sucrose metabolism, photosynthesis, and sucrose concentration were studied
in sugarcane (Saccharum spp hybrid) plantlets. Acclimatization was developed in two steps. (1) Light intensity of 1,000μmol m−2 s−1 and 90% relative humidity during the first 21d; followed by 2,000μmol m−2 s−1 and approximately 80% of relative humidity. All measurements were carried out at the end of rooting phase concomitant with
day0 of acclimatization and at 7-d intervals thereafter (0, 7, 14, 21, 28, 35, 42d). As the in vitro plantlets were transferred to the acclimatization phase, photosynthesis increased significantly during the first 7d. After
this period, the increase was constant with only a small but nonsignificant decline after being transferred to the uncontrolled
external conditions. The activity of the sucrose synthase began to show a decrease, starting from day 7, and was related to
the changes that began to happen in these plants from its adaptation to new ex vitro conditions. Due to the increase of fresh weight favored by the high light intensity and lower relative humidity, an increase
of the sucrose phosphate synthase activity was observed. The maximum activity of the acid and neutral invertases was reached
at 14 and 21d, respectively, after 21d of acclimatization period. There was a marked tendency for the activity of both enzymes
to decrease. The sucrose content was decreased only in the first 7d. The metabolism of sugarcane plantlets seemed to be susceptible
to the environmental changes during the acclimatization phase but did not contribute to inhibitory factors for normal development.
The effects of photosynthetic photon flux density (PPFD) on antioxidant metabolism and photosynthetic properties in leaves
during ex vitro establishment of micropropagated Rauvolfia tetraphylla plantlets were investigated. In vitro-propagated plantlets were acclimatized at either 50 (Low-light=LL) or 300 (High-light=HL)
μmolm−2s−1 photosynthetic PPFD for 4weeks under controlled conditions. Increases in chlorophyll (Chl) a, b and carotenoid levels were observed in plantlets acclimatized at both light intensities. At transplantation, micropropagated
plantlets were not photosynthetically active, but the net photosynthetic rate increased in newly formed leaves over time during
acclimatization. The observed differences in pigment contents and photosynthetic rates suggested adaptation of plantlets from
heterotrophic to autotrophic mode of nutrition during acclimatization. Changes in activities of antioxidant enzymes were also
observed during acclimatization. Superoxide dismutase activity increased in plantlets acclimatized at HL intensities. Likewise,
changes in activity of catalase and ascorbate peroxidase were also detected. These observed changes reflected the ability
of plants in developing an antioxidant enzymatic defense system aiding in survival against oxidative stress and in reducing
release of free radicals.
Winter wheat (Triticum aestivum L cv. Monopol) plants grown under either control (20˚C, 250 PFD), low temperature (5˚C, 250 PFD) or high light conditions (20˚C, 800 PFD) were compared in order to assess the roles of the Calvin cycle and the Mehler reaction in the differential sensitivity to chronic photoinhibition. Despite similar photosynthetic responses to irradiance, the partial pressure of CO2 [p(CO2)] and photoinhibition, photosynthetic acclimation to cold temperature appears to be quite distinct from acclimation to high light. First, the lower ratio of Rubisco oxygenation/Rubisco carboxylation and the reduced effects of p(CO2) on number of electrons per mole of CO2 fixed in cold-acclimated compared to high light-grown wheat indicate that photorespiration is differentially suppressed in cold-acclimated Monopol. Second, inhibition of the Calvin cycle by glyceraldehyde during photo-inhibition indicated that the sensitivity of high light-acclimated Monopol to photoinhibition was more dependent on Rubisco activity than the sensitivity of cold-acclimated plants to photoinhibition. Third, cold-acclimated Monopol exhibited higher electron transport rates in the presence of either ambient CO2, 2 kPa O2 or N2, 2 kPa O2 (either 77% or 68%, respectively) relative to controls compared to high light-acclimated plants exposed to similar gaseous environments (either 57% or 38%, respectively). Last, the activation state of NADP–malate dehydrogenase indicated that the stroma is highly reduced during cold acclimation relative to either controls or high light-grown Monopol. Thus, in cold-acclimated wheat, the Mehler reaction appears to play an important role while photorespiration plays a minimal role in mitigating the sensitivity to photoinhibition. In contrast, both photorespiration and the Mehler reac-tion appear to mitigate the sensitivity to photoinhibition in high light-grown Monopol. This is consistent with the differential sensitivity to methylviologen and the differential SOD activity observed between cold-acclimated and high light-grown Monopol.
This work aimed to evaluate the phytochemical content and to determine the antioxidant and cytotoxic activities of methanol extracts of the carob tree (Ceratonia siliqua L.) germ flour. The extracts were rich in phenolic compounds, had considerable antioxidant activity, and reduced the viability of cervical (HeLa) cancer cells. The chemical content and the biological activities of the extracts were significantly affected by gender and cultivar. Female cultivar Galhosa had the highest levels of phenolic compounds, and the highest antioxidant activity. Extracts from the hermaphrodite trees and from the female cultivars Galhosa and Costela/Canela exhibited the highest cytotoxic activity. The most abundant compound was theophylline. The phenolic content was correlated to both antioxidant and cytotoxic activities. Our findings provide new knowledge about the health implications of consuming food supplemented with carob germ flour.
In common with many other higher plant species, Arabidopsis undergoes photosynthetic acclimation, altering the composition of the photosynthetic apparatus in response to fluctuations in its growth environment. The changes in photosynthetic function that result from acclimation can be detected in a noninvasive manner by monitoring chlorophyll (Chl) fluorescence. This technique has been used to develop a screen that enables the rapid identification of plants defective at ACCLIMATION OF PHOTOSYNTHESIS TO THE ENVIRONMENT (APE) loci. The application of this screen to a population of T-DNA-transformed Arabidopsis has successfully led to the identification of a number of mutant lines with altered Chl fluorescence characteristics. Analysis of photosynthesis and pigment composition in leaves from three such mutants showed that they had altered acclimation responses to the growth light environment, each having a distinct acclimation-defective phenotype, demonstrating that screening for mutants using Chl fluorescence is a viable strategy for the investigation of acclimation. Sequencing of the genomic DNA flanking the T-DNA elements showed that in the ape1 mutant, a gene was disrupted that encodes a protein of unknown function but that appears to be specific to photosynthetic organisms, whereas the ape2 mutant carries an insertion in the region of the TPT gene encoding the chloroplast inner envelope triose phosphate/phosphate translocator.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Chlorophyll fluorescence has been widely used in laboratory studies in understanding both the mechanism of photosynthesis itself and the mechanisms by which a range of environmental factors alter photosynthetic capacity. The measurement of chlorophyll fluorescence is both non-destructive and non-invasive, and thus has considerable potential for use in the field situation. Applications range simply from a means of rapidly identifying injury to leaves in the absence of visible symptoms to a detailed analysis of causes of change in photosynthetic capacity. This paper introduces the topic of chlorophyll fluorescence, its interpretation and its application in field studies, giving particular attention to interpretation and measurement of fluorescence induction kinetics and to the application of recently developed modulated light fluorimeters. Three commercial fluorimeters designed for field applications were compared: (1) Plant Stress Meter, BioMonitor AB, Sweden; (2) MFMS, Hansatech Ltd, UK; (3) PAM 101, H. Walz, Federal Republic of Germany. The structure and potential of each are briefly reviewed. It was beyond the scope of this comparison to examine the full potential of each instrument but measurements of the widely used parameter F<sub>v</sub>/F<sub>max</sub> were made with each on: (1) Triticum aestivum L. leaves treated with the herbicide Atrazine; and (2) Picea abies (L.) Karst. samples collected from sites which were known to receive different levels of ambient air pollution. In both experiments, the results obtained from the three fluorimeters showed good agreement. The relative merits of each instrument to field applications are discussed.
A new type of modulation fluorometer was used in the study of energy-dependent chlorophyll fluorescence quenching (qE) in intact leaves. Under conditions of strong energization of the thylakoid membrane (high light intensity, absence of CO2) not only variable fluorescence, FV, but also dark-level fluorescence, FO, was quenched, leading to definition of a quenching coefficient, qO. Information on qO was shown to be essential for correct determination of photochemical (qQ) and energy dependent quenching (qE) by the saturation pulse method. The relationship between qE and qO was analysed over a range of light intensities at steady state conditions. qE was found to consist of two components, the second of which is linearly correlated with qO. qO and the second component of qE are interpreted to reflect the state 1 - state 2 shift caused by LHC II phosphorylation.
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.
Photon yields of oxygen evolution at saturating CO2 were determined for 44 species of vascular plants, representing widely diverse taxa, habitats, life forms and growth conditions. The photonyield values on the basis of absorbed light (fa) were remarkably constant among plants possessing the same pathway of photosynthetic CO2 fixation, provided the plants had not been subjected to environmental stress. The mean fa value ±SE for 37 C3 species was 0.106±0.001 O2·photon-1. The five C4 species exhibited lower photon yields and greater variation than the C3 species (fa=0.0692±0.004). The fa values for the two Crassulaceanacid-metabolism species were similar to those of C3 species. Leaf chlorophyll content had little influence on fa over the range found in normal, healthy leaves. Chlorophyll fluorescence characteristics at 77 K were determined for the same leaves as used for the photon-yield measurements. Considerable variation in fluorescence emission both at 692 nm and at 734 nm, was found 1) among the different species; 2) between the upper and lower surfaces of the same leaves; and 3) between sun and shade leaves of the same species. By contrast, the ratio of variable to maximum fluorescence emission at 692 nm (Fv/FM, 692) remained remarkably constant (The mean value for the C3 species was 0.832±0.004). High-light treatments of shade leaves resulted in a reduction in both fa and the Fv/FM, 692 ratio. The extent of the reductions increased with time of exposure to bright light. A linear relationship was obtained when fa was plotted against Fv/FM, 692. The results show that determinations of the photon yield of O2 evolution and the Fv/FM, 692 ratio can serve as excellent quantitative measures of photoinhibition of overall photosynthetic energy-conversion system and of photochemistry of photosystem II, respectively. This is especially valuable in field work where it is often impossible to obtain appropriate controls.
Acclimatizing, hardening-off, or conditioning plantlets from the in vitro to the ambient environment can be a challenge that may result in death or damage to a large percentage of micropropagated plants. When grown in the high humidity, low light environment often encountered in vitro, leaves have altered anatomy; poor cuticle development, with low amounts of epicuticular waxes; and the stomatal apparatus functions poorly with stomata remaining open, even in darkness or in stressful conditions. Gradually acclimatizing the plantlets to the ex vitro environment using mist, fog, or high humidity combined with shading will reduce losses. Light levels can be gradually raised and relative humidity gradually lowered as the plantlets grow new leaves and their shoots elongate. Eventually, plants will thrive in ambient conditions. Soil additives, such as mycorrhizal fungi combined careful attention to sanitation will also aid survival.
Keywords:
acclimatization;
microshoots;
greenhouse-grown;
microcuttings;
rooting
There is now potential to estimate photosystem II (PSII) activity in vivo from chlorophyll fluorescence measurements and thus gauge PSII activity per CO2 fixed. A measure of the quantum yield of photosystem II, ΦII (electron/photon absorbed by PSII), can be obtained in leaves under steady-state conditions in the light using a modulated fluorescence system. The rate of electron transport from PSII equals ΦII times incident light intensity times the fraction of incident light absorbed by PSII. In C4 plants, there is a linear relationship between PSII activity and CO2 fixation, since there are no other major sinks for electrons; thus measurements of quantum yield of PSII may be used to estimate rates of photosynthesis in C4 species. In C3 plants, both CO2 fixation and photorespiration are major sinks for electrons from PSII (a minimum of 4 electrons are required per CO2, or per O2 reacting with RuBP). The rates of PSII activity associated with photosynthesis in C3 plants, based on estimates of the rates of carboxylation (vo) and oxygenation (vo) at various levels of CO2 and O2, largely account for the PSII activity determined from fluorescence measurements. Thus, in C3 plants, the partitioning of electron flow between photosynthesis and photorespiration can be evaluated from analysis of fluorescence and CO2 fixation.
Diurnal time course of the water relations of Ceratonia siliqua, Quercus coccifera, Pistacia terebinthus and Olea oleaster was studied on healthy and degraded sites for getting a suitable parameter describing the water stress impact on vegetation, and selecting the species suitable for reforestation. Out of the species used C. siliqua, showed high maximum stomatal conductance (g L), with high relative water content (RWC) under field conditions, and relatively constant minimum (midday) water potential (C min) and pre-dawn water potential (C pd 's). O. oleaster plants appeared to be unable to prevent dehydration inspite of consistent decrease of stomatal conductance (g L), when subjected to increasing water stress. Stomatal closure in fact, was not sufficient to prevent water loss and relative water content (RWC) dropped to about 70%. P. terebinthus species is water spender. Under water shortage condition inspite of complete closure of stomata (stomatal conductance dropped to 0.08 s cm À1), relative water content showed variable values. Q. coccifera typically showed high stomatal conductance activity and also high relative water content (average 83%) on healthy site, but on the degraded site it reduced stomatal conductance, thus maintaining high RWCs (over 80%) and preventing leaf water potential (C L) to drop to critical values.
The genetic diversity of 15 carob (Ceratonia siliqua L.) cultivars located in an experimental field from Algarve (Portugal) was evaluated over 7 years using 12 fruit and seed phenotypic characters, in order to characterize carob cultivars. The values of morphological traits obtained by cultivar were compared with those from other countries of the Mediterranean basin. Statistically significant differences were found between cultivars for all characters which were examined, what indicates a high genetic diversity. The relationship among these characters was analyzed by principal component analysis (PCA) resulting in the separation of these cultivars classed in four groups (clusters I–IV) and in four ungrouped cultivars. A three dimension of the model was found to be significant and explained 74.5% of the total variation, in which the first component accounting for 34.6% of the total variation is dominated by fruit characters, while the second component is dominated by seed characters. Cultivars plotted on the left-lower quadrant on the space determined by principal components 1 and 2 are characterized by fruits with high seed yield more appropriated for industrial rentability. The correlation analyses established by cultivar provided a specific understanding about the way how fruit and seed characteristics correlate within each cultivar. This approach can be useful for the development of a breeding programme, aiming to increase the seed yield, seed thickness, individual and total seed weight by fruit, characteristics that are determinant to improve the industrial exploitation of carob. # 2007 Published by Elsevier B.V.
This study reports survival and physiological responses of micropropagated Ceratonia siliqua L. cvs. Galhosa and Mulata plants during ex vitro acclimatization under ambient (AC; 330 mol mol–1) or elevated (EC; 810 mol mol–1) CO2 concentration and a photosynthetic photon flux density of 125 mol m–2 s–1. CO2 enrichment during acclimatization did not improve survival rate that was around 80 % for both treatments. Eight weeks after ex vitro transplantation, photosynthetic capacity and apparent quantum yield in acclimatized leaves were higher in comparison with those in in vitro-grown leaves, without any significant difference between CO2 treatments. Chlorophyll content increased after acclimatization. However, EC led to a decrease in the total amount of chlorophyll in new leaves of both cultivars, compared to those grown at AC. Soluble sugars and starch contents were not markedly affected by growth EC, although starch had significantly increased after transfer to ex vitro conditions. EC induced an increase in the stem elongation and in the effective life of leaves, and a decrease in the number of new leaves.
An efficient, rapid and large scale propagation of a multipurpose herb, Ocimum basilicum through in vitro culture of nodal segments with axillary buds from mature plants has been accomplished. Among the cytokinins,
6-benzyladenine (BA), thidiazuron (TDZ), kinetin (Kin) and 2-isopentenyl adenine (2-iP) tested as supplements to Murashige
and Skoog (MS) medium, 5.0μM BA was optimum in inducing bud break. The highest rate of shoot multiplication was achieved
on half-strength MS medium supplemented with 2.5μM BA and 0.5μM indole-3-acetic acid (IAA) combination. The shoots regenerated
from TDZ supplemented medium when subcultured to hormone-free MS medium considerably increased the rate of shoot multiplication
and shoot length by the end of third subculture. For rooting, MS medium supplemented with 1.0μM indole-3-butyric acid (IBA)
proved to be better than that supplemented with IAA or α-naphthalene acetic acid (NAA). The in vitro raised plantlets with
well developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse
with 90% survival rate. Chlorophyll a and b, carotenoids and net photosynthetic rate were measured in leaves during ex vitro
acclimatization at 0, 7, 14, 21 and 28days. Firstly these parameters showed a decreasing trend but subsequently increased
after 7days of acclimatization. These findings indicate that the adaptation of micropropagated plants to ex vitro conditions
is more extended in time than generally accepted.
The special conditions during in vitro culture result in the formation of plantlets of abnormal morphology, anatomy and physiology. After ex vitro transfer, these plantlets might easily be impaired by sudden changes in environmental conditions, and so need a period of
acclimatization to correct the abnormalities. This review is focused upon contemporary information on the changes in leaf
structure, water relations and photosynthesis during acclimatization of plantlets to ex vitro conditions. It also describes some ways of improving plant survival and for the speeding up of acclimatization.
The aim of this research was to determine whether exogenous abscisic acid (ABA) applied immediately after ex vitro transfer of in vitro grown plants can improve their acclimatization. Tobacco (Nicotiana tabacum L.) plantlets were transferred into pots with Perlite initially moistened either by water or 50 µM ABA solution and they
were grown under low (LI) or high (HI) irradiance of 150 and 700 µmol m−2 s−1, respectively. Endogenous content of ABA in tobacco leaves increased considerably after ABA application and even more in
plants grown under HI. Stomatal conductance, transpiration rate and net photosynthetic rate decreased considerably 1 d after
ex vitro transfer and increased thereafter. The gas exchange parameters were further decreased by ABA application and so wilting of
these plants was limited. Chlorophyll (a+b) and β-carotene contents were higher in ABA-treated plants, but the content of xanthophyll cycle pigments was not increased.
However, the degree of xanthophyll cycle pigments deepoxidation was decreased what also suggested less stress in ABA-treated
plants. No dramatic changes in most chlorophyll a fluorescence parameters after ex vitro transfer suggested that the plants did not suffer from restriction of electron transport or photosystem damage.
The aim of this study was to investigate acclimation of micropropagated plants of Rhododendron ponticum subsp. baeticum to different irradiances and recovery after exposure to high irradiance. Plants grown under high (HL) or intermediate (IL)
irradiances displayed higher values of maximum electron transport rate (ETRmax) and light saturation coefficient (Ek) than plants grown under low irradiance (LL). The capacity of tolerance to photoinhibition (as assessed by the response of
photochemical quenching, qp) varied as follows: HL > IL > LL. Thermal energy dissipation (qN) was also affected by growth irradiance, with higher saturating values being observed in HL plants. Light-response curves
suggested a gradual replacement of qp by qN with increasing irradiance. Following exposure to irradiance higher than 1500 μmol m−2 s−1, a prolonged reduction of the maximal photochemical efficiency of PS 2 (Fv/Fm) was observed in LL plants, indicating the occurrence of chronic photoinhibition. In contrary, the decrease in Fv/Fm was quickly reverted in HL plants, pointing to a reversible photoinhibition.
Additional key wordselectron transport-photochemical quenching-photoinhibition-thermal energy dissipation
There is a growing interest worldwide to find out new and cheap carbohydrate sources for production of bioethanol. In this context, carob pod (Ceratonia siliqua) is proposed as an economical source for bioethanol production, especially, in arid regions. The carob tree is an evergreen shrub native to the Mediterranean region, cultivated for its edible seed pods and it is currently being reemphasised as an alternative in dryland areas, because no carbon-enriched lands are necessary. In this work, the global process of ethanol production from carob pod was studied. In a first stage, aqueous extraction of sugars from the pod was conducted, achieving very high yields (>99%) in a short period of time. The process was followed by acid or alkaline hydrolysis of washed pod at different operating conditions, the best results (R = 38.20%) being reached with sulphuric acid (2% v/v) at 90 °C, using a L/S (liquid/solid) ratio of 7.5 and shaking at 700 rpm for 420 min. After that, fermentation of hydrolysates were tested at 30 °C, 125 rpm, 200 g/L of sugars and 15 g/L of yeast with three different kinds of yeasts. In these conditions a maximum of 95 g/L of ethanol was obtained after 24 h. Finally, the distillation and dehydration of water–bioethanol mixtures was analyzed using the chemical process simulation software CHEMCAD with the aim of estimate the energy requirements of the process.
Diurnal time course of gas exchange rates and chlorophyll fluorescence were measured in situ in attached leaves of nectarine trees (Prunus persica L. Batsch, var. Silver King) subjected to different water availabilities under summer conditions in central Portugal. CO2 assimilation rate (An) and stomatal conductance (gs) of well-watered trees decreased along the day in response to high temperature and vapour pressure deficit. Soil water deficit increased the sensitivity of leaf gas exchange to summer atmospheric conditions: An and gs exhibited important midday depressions under water shortage. In spite of a very good correlation between the decline in An and reductions in gs observed over the course of the day, intercellular CO2 concentration became higher in the late afternoon. During the day, the quantum yield of PSII electron transport in the light (Φe), the electron transport rate (ETR), the intrinsic efficiency of open PSII reaction centers (F'v/F'm), and the photochemical quenching (qp) and non-photochemical quenching (NPQ) of chlorophyll fluorescence remained constant in well-watered trees, in spite of some decrease in stomatal conductance in the afternoon. Water stress induced after midday a large, but reversible, decrease of Φe, F'v/F'm and ETR, and an increase in NPQ. Simultaneously, an increase in ETR/A was observed. Because water stress led to a reduction in the size of the pools of electron acceptors after midday, as indicated by the decrease in qp, the contribution of thermal de-excitation at PSII (given by NPQ) in the protection against photoinhibition became more important in stressed trees. The increase in ETR/A suggests that in water stressed plants the excitation energy in the photosynthetic apparatus is partially diverted to the photosynthetic reduction of O2, via photorespiration, Mehler-peroxidase reaction or the water-water cycle.
The special conditions during in vitro culture results in the formation of plantlets of abnormal morphology, anatomy and physiology. Tissue culture conditions that promote rapid growth and multiplication of shoots often results in the formation of structurally and physiologically abnormal plants. They are often characterized by poor photosynthetic efficiency, malfunctioning of stomata and a marked decrease in epicuticular wax. Qualitatively also, the waxes present on the surface of the leaves of in vitro cultured plants may vary. The conditions under which most laboratories done tissue culture is high relative humidity and low light, no supplemental CO2, high sucrose and nutrient containing medium may contribute to a phenotype that cannot survive the environmental conditions when directly placed in a greenhouse or field. Understanding these abnormalities is a prerequisite to develop efficient transplantation protocols. The present review summaries the major abnormalities in in vitro culture of plants and also highlight the current and developing methods that are satisfactory for acclimatization of in vitro cultured plantlets.
The importance of photoprotective strategies for Photosystem (PS) II function under all light regimes was determined from the content of functional PS II, measured by repetitive flash yield of oxygen evolution, in leaves of pea (Pisum sativum L.) grown under 50 (low light), 250 (medium light), and 650 (high light) μmol photons m−2 s−1. The modulation of PS II functionality in vivo was induced in 1.1% CO2 after infiltration of leaves with water (control), nigericin (a lipophilic uncoupler) or dithiothreitol (DTT, inhibitor of violaxanthin to zeaxanthin conversion) through the cut petioles of leaves of 22–24 day-old plants. In all nigericin-treated pea leaves, photoinactivation of PS II was greatly increased with increasing photon exposure (mol photons m−2). This nigericin-induced increase of photoinactivation of PS II was greater than that induced by DDT in medium- and high-light pea leaves, but comparable in low-light peas. In low-light peas during steady-state photosynthesis, the development of non-photochemical quenching of chlorophyll fluorescence (NPQ, measured as ) was lowest, accompanying the highest reduction state of PS II (measured by photochemical quenching, qP). Further, the susceptibility of PS II to light stress, estimated as [Park et al., Plant Cell Physiol., 36 (1995) 1163–1167], was highest in low-light peas. The decline of chlorophyll fluorescence from maximum (Fm) to steady-state level during induction phase of chlorophyll fluorescence was retarded by nigericin treatment, with a greater inhibitory effect in high- and medium- than low-light pea leaves. From these results, we suggest that the greater susceptibility of low-light peas to light stress than medium- and high-light peas is ascribed to lower capacities for both the utilization of absorbed light by photosynthesis and for non-radiative dissipation of absorbed light as heat.
Measurements of the quantum yields of chlorophyll fluorescence and CO2 assimilation for a number of plant species exposed to changing light intensity and atmospheric CO2 concentrations and during induction of photosynthesis are used to examine the relationship between fluorescence quenching parameters and the quantum yield of non-cyclic electron transport. Over a wide range of physiological conditions the quantum yield of non-cyclic electron transport was found to be directly proportional to the product of the photochemical fluorescence quenching (qQ) and the efficiency of excitation capture by open Photosystem II (PS II) reaction centres (Fv/Fm). A simple fluorescence parameter, ΔφF/φFm, which is defined by the difference in fluorescence yield at maximal φFm, and steady-state φFs, divided by φFm, can be used routinely to estimate changes in the quantum yield of non-cyclic electron transport. It is demonstrated that both the concentration of open PS II reaction centres and the efficiency of excitation capture by these centres will determine the quantum yield of non-cyclic electron transport in vivo and that deactivation of excitation within PS II complexes by non-photochemical processes must influence the quantum yield of non-cyclic electron transport.
The levels of two subunits of chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), total soluble proteins, carbon and leaf nitrogen content, and photosynthetic pigments in various plants (avocado, oak, olive, and strawberry) grown in vitro and ex vitro were analysed. Compared to ex vitro grown plants, micropropagated avocado, oak, and strawberry showed a markable decrease in large subunit Rubisco. However, the small subunit only decreased in strawberry and oak. Contrary to this, olive did not reveal any difference in the level of either subunit. The C/N ratio increased significantly in in vitro grown plants, except in the case of olive, where an opposite behaviour was found. Leaf chlorophyll concentration on unit mass basis was higher in all the in vitro plants than in those of greenhouse- grown plants. Only avocado plantlets showed a statistically significant decrease in total soluble proteins. Further, overall data suggest that in vitro cultural conditions have a species-specific influence on large and small subunits of Rubisco, independent of the protein, chlorophyll, or nitrogen level.
During the first days after transplanting micropropagated plants to greenhouse conditions, in vitro leaves are the only source to cover metabolic demands and to sustain plants’ adaptation and regrowth. However, the way these leaves act can differ depending on plant species and in vitro conditions. Here, we describe two main groups. In Calathea the in vitro leaves function as storage organs, from which the accumulated reserves (glucose, fructose) are consumed during the first days after transfer, until new leaves appear; these in vitro leaves never become fully autotrophic. On the contrary, in Spathiphyllum in vitro leaves are photosynthetically competent and normal source-sink relations are observed. Any surplus in photoassimilates at the end of the photoperiod is converted into starch. Three weeks after transfer, those leaves start to senesce and newly developed leaves become the main source of carbohydrates. In both plant species, higher photosynthetic activities are measured once new leaves are fully developed.
Extracts from fruit pulps of six female cultivars and two hermaphrodite Portuguese carob trees [(Ceratonia siliqua L., Fabaceae)] exhibited strong antioxidant activity and were rich in phenolic compounds. The extracts decreased the viability of different human cancer cell lines on a dose- and time-dependent manner. Gender and cultivar significantly influenced the chemical content and the biological activities of the extracts. Extracts from hermaphrodite trees had a higher content of phenolic compounds, and exhibited higher antioxidant and cytotoxic activities. Among females, cv. Aida had the highest radical scavenging activity and total content of phenolics, Mulata the highest capacity to inhibit lipid oxidation and Gasparinha the strongest cytotoxic activity on HeLa cells. The decrease in cell viability was associated with apoptosis on HeLa and MDA-MB-231 lines. (+)-Catechin and gallic acid (GA) were the main compounds identified in the extracts, and GA contributed to the antioxidant activity. Our results show that the antioxidant and cytotoxic activities of carob tree fruit pulps are strongly influenced by gender and cultivar, and provide new knowledge about the advantages of hermaphrodite trees over female cultivars, namely, as a source of compounds with biological interest, which may represent an increase of their agronomic interest.
Carob has been widely grown in the Mediterranean region for a long time. It has been regarded as only a forest tree and has been neglected for other economical benefits. However, in recent years, this fruit has gained attention for several applications. As petroleum has become depleted, renewable energy production has started to gain attention all over the world; including the production of ethanol from underutilized agricultural products such as carob. In this project, the optimum extraction conditions were determined for the carob fruit by using the response surface design method. The obtained extract was utilized for production of ethanol by using suspended Saccharomyces cerevisiae fermentation. The effect of various fermentation parameters such as pH, media content and inoculum size were evaluated for ethanol fermentation in carob extract. Also, in order to determine economically appropriate nitrogen sources, four different nitrogen sources were evaluated. The optimum extraction condition for carob extract was determined to be 80 degrees C, 2h in 1:4 dilution rate (fruit: water ratio) according to the result of response surface analysis (115.3g/L). When the fermentation with pH at 5.5 was applied, the final ethanol concentration and production rates were 42.6g/L and 3.37 g/L/h, respectively, which were higher than using an uncontrolled pH. Among inoculum sizes of 1%, 3%, and 5%, 3% was determined as the best inoculum size. The maximum production rate and final ethanol concentration were 3.48 g/L/h and 44.51%, respectively, with an alternative nitrogen source of meat-bone meal. Overall, this study suggested that carob extract can be utilized for production of ethanol in order to meet the demands of renewable energy.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Plants cultured in vitro suffer from several physiological and biochemical impairments due to the artificial conditions of growth, namely the composition of the heterotrophic media. Upon transfer to ex vitro, the higher irradiances, compared to in vitro, can lead to oxidative stress symptoms, which can be counteracted by CO2 concentrations above atmospheric levels. Here we analyse the stability of Rubisco in in vitro grapevine plantlets, and after transfer to ex vitro under four acclimatization treatments: low irradiance (LL, 150 micromol m(-2)s(-1)) and high irradiance (HL, 300 micromol m(-2)s(-1)) in association with CO2 concentrations of 350 (LCO2) and 700 (HCO2) microL L(-1). Proteins were separated with SDS polyacrylamide gel electrophoresis and two-dimensional electrophoresis and Rubisco degradation peptides were analysed by immunoblotting with anti-LSU antibodies. These degradation products were present in the leaves of plantlets under both in vitro and ex vitro treatments. Under LCO2 they were maintained for almost all of the 28 days of the acclimatization period, while becoming scarcely detected after 14 days under HCO2 and after 7 days when HCO2 was associated with HL. These results appear to confirm the counteraction of HCO2 concentrations over the oxidative stress eventually caused by HL. The patterns of soluble sugars in acclimatizing leaves under HLHCO2 also gave an indication of a faster acquisition of autotrophic characteristics.
Improvement of acclimatization of micropropagated grapevine: photosynthetic competence and carbon allocation Evaluation of fruit and seed diversity and characterization of carob (Ceratonia siliqua L.) cultivars in Algarve region
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Micropropagation of the mediterranean tree Ceratonia siliqua L Eco-physiological behavior of some mediterranean plants as suitable candidates for reclamation of degraded areas
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A, Barros S, Martins-Louçã MA (2002) Micropropagation of the mediterranean tree Ceratonia siliqua L. Plant Cell Tissue Organ Cult 68:35–41 Sakcali MS, Ozturk M (2004) Eco-physiological behavior of some mediterranean plants as suitable candidates for reclamation of degraded areas. J Arid Environ 57:1–13
Ethanol production from carob extract by using Saccharomyces cerevi-siae Photosyn-thesis and carbon metabolism in leaves formed prior and during ex vitro acclimatization of micropropagated plants
- I Bialka Kl
- A Demirci
- Karhan
I, Bialka KL, Demirci A, Karhan M (2010) Ethanol production from carob extract by using Saccharomyces cerevi-siae. Bioresource Technol 101:5290–5296 Van Huylenbroeck JM, Piqueras A, Debergh PC (1998) Photosyn-thesis and carbon metabolism in leaves formed prior and during ex vitro acclimatization of micropropagated plants. Plant Sci 134:21–30
Carob tree. Ceratonia siliqua L. Institute of Plant Genetics and Crop Plant Research
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- K Mitrakos