Acclimatization of micropropagated plantlets induces an antioxidative burst: a case study with <Emphasis Type="Italic">Ulmus minor</Emphasis> Mill.

In this article, the effects of increased light intensities on antioxidant metabolism during ex vitro establishment of Ulmus minor micropropagated plants are investigated. Three month old in vitro plants were acclimatized to ex vitro conditions in a climate chamber with two different light intensities, 200 μmol m⁻² s⁻¹ (high light, HL) and 100 μmol m⁻² s⁻¹ (low light, LL) during 40 days. Immediately after ex vitro transfer, the increase of both malondialdehyde (MDA) and electrolyte leakage in persistent leaves is indicative of oxidative stress. As the acclimatization continues, an upregulation of the superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) enzyme activities were also observed. Simultaneously, MDA content and membrane permeability stabilized, suggesting that the antioxidant enzymes decrease the deleterious effects of reactive oxygen species (ROS) generation. Unexpectedly, newly formed leaves presented a different pattern of antioxidative profile, with high levels of MDA and membrane leakage and low antioxidant enzyme activity. Despite these differences, both leaf types looked healthy (e.g. greenish, with no necrotic spots) during the whole acclimatization period. The results indicate that micropropagated U. minor plantlets develop an antioxidant enzyme system after ex vitro transfer and that, in general, LL treatment leads to lower oxidative stress. Moreover, new leaves tolerate higher levels of ROS without the need to activate the antioxidative pathway, which suggests that the environment at which leaves are exposed during its formation determinate their ability to tolerate ROS.     

Effect of light irradiations on photosynthetic machinery and antioxidative enzymes during ex vitro acclimatization of Tylophora indica plantlets

Abstract

In the present communication, we studied the effect of light stress on pigment contents, net photosynthetic rate, electrolyte leakage, malondialdehyde concentration and various antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and ascorbate peroxidase (APX) during acclimatization of micropropagated Tylophora indica plantlets. Pigment (Chlorophyll a, b and carotenoids) contents in ex vitro formed leaves were found significantly higher compared to the in vitro formed ones. In vitro plantlets (day 0) exhibited a low photosynthetic activity, but with the emergence of new leaves a significant increase in net photosynthetic rates was observed. Changes in the activity of the antioxidant enzymes system were also observed during the critical days of acclimatization. Plantlets showed increased levels of SOD production, indicating its preventive mechanism of membrane oxidation and damage to biological molecules in high light (HL) acclimatized plantlets. The CAT activity increased at both low lights (LL) and HL during the whole period of acclimatization. Likewise, photoexposure of plantlets at LL and HL showed elevated activity of GR and APX against 0 day plantlets.     

Changes in photosynthetic capacity and antioxidant enzymatic systems in micropropagated <Emphasis Type="Italic">Zingiber officinale</Emphasis> plantlets during their acclimation

Ginger (Zingiber officinale Rosc.) plantlets were propagated in vitro and acclimated under different photosynthetic photon flux densities (60 and 250 μmol m⁻² s⁻¹ = LI and HI, respectively). Increases in chlorophyll (Chl) content and Chl a/b ratio were found under both irradiances. In vitro plantlets (day 0) exhibited a low photosynthesis, but chloroplasts from in vitro leaves contained well developed grana and osmiophillic globules. Photoinhibition in leaves formed in vitro was characterized by decrease of photochemical efficiency and quantum efficiency of photosystem 2 photochemistry in HI treatment during acclimation. The new leaves formed during acclimation in both treatments showed a higher photosynthetic capacity than the leaves formed in vitro. Also activities of antioxidant enzymes of micropropagated ginger plantlets changed during acclimation.     

In vitro growth and single-leaf photosynthetic response of Cymbidium plantlets to super-elevated CO2 under cold cathode fluorescent lamps

To examine the effectiveness of super-elevated (10,000 μmol mol⁻¹) CO₂ enrichment under cold cathode fluorescent lamps (CCFL) for the clonal propagation of Cymbidium, plantlets were cultured on modified Vacin and Went (VW) medium under 0, 3,000 and 10,000 μmol mol⁻¹ CO₂ enrichment and two levels of photosynthetic photon flux density (PPFD, 45 and 75 μmol m⁻² s⁻¹). Under high PPFD, 10,000 μmol mol⁻¹ CO₂ increased root dry weight and promoted shoot growth. In addition, a decrease in photosynthetic capacity and chlorosis at leaf tips were observed. Rubisco activity and stomatal conductance of these plantlets were lower than those of plantlets at 3,000 μmol mol⁻¹ CO₂ under high PPFD, which had a higher photosynthetic capacity. On the other hand, plantlets on Kyoto medium grown in 10,000 μmol mol⁻¹ CO₂ under high PPFD had a higher photosynthetic rate than those on modified VW medium; no chlorosis was observed. Furthermore, growth of plantlets, in particular the roots, was remarkably enhanced. This result indicates that a negative response to super-elevated CO₂ under high PPFD could be improved by altering medium components. Super-elevated CO₂ enrichment of in vitro-cultured Cymbidium could positively affect the efficiency and quality of commercial production of clonal orchid plantlets.     

Improvement of acclimatization of micropropagated grapevine: Photosynthetic competence and carbon allocation

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. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro sucrose concentration affects growth and acclimatization of <Emphasis Type="Italic">Alocasia amazonica</Emphasis> plantlets

Plantlets of Alocasia amazonica were regenerated on the MS medium supplemented with different concentrations (0–9%) of sucrose. An absence of sucrose in the growth medium induced generation of leaves, however, it decreased multiplication. On contrary, sucrose supply of 6% or 9% enhanced multiplication but hampered photoautotrophic growth (generation of leaves). Increasing sucrose supply also increased sugars and starch content and number of stomata and decreased water potential and size of stomata during in vitro growth period. During ex vitro acclimatization, shoot length, root length, leaf number and root number of Alocasia plantlets grown with 3% sucrose, were found to be better among the other studied sucrose concentrations. Under ex vitro acclimatization, number of stomata, contents of various carbohydrates in the leaves were increased but size of stomata decreased with increasing sucrose supply during in vitro growth period. Moreover, water potential of leaves of plantlets, which have been grown with a sucrose concentration other than 3%, was decreased. During in vitro growth, net CO₂ assimilation rate (P_N), transpiration (E), stomatal conductance (g_s) and variable fluorescence to maximum fluorescence ratio (Fv/Fm) were unaffected, however, during acclimatization these were changed and maximum P_N, E, and g_s were observed in the plantlets micropropagated with 3% sucrose. Fv/Fm was decreased severely in the plantlets micropropagated with 6% sucrose during acclimatization. Thus a sucrose concentration of 3% in the medium is appeared to be better among studied concentrations for both in vitro growth and ex vitro acclimatization of A. amazonica plantlets.     

Physiology of Eucalyptus plantlets grown photoautotrophically in a scaled-up vessel

Summary Nodal cuttings of Eucalyptus camaldulensis L. plantlets were cultured photoautotrophically (sugar-free nutrient medium and with enriched CO₂ and high photosynthetic photon flux) in a scaled-up vessel (volume 4.0 liters) under forced ventilation (SV-treatment). After 28 d of culture, physiological aspects of the plantlets were compared with plantlets grown photomixotrophically (20 g l⁻¹ sucrose in the medium) in a Magenta vessel (volume 0.4 liters) under natural ventilation (control). In the SV-treatment net photosynthetic rates were enhanced, normal stomatal closing and opening were observed, and the epicuticular leaf-wax content was significantly higher than the control. The anatomical study showed well-organized palisade and spongy mesophyll layers of SV leaves. The SV-treatment also allowed in vitro acclimatization, and after transplanting ex vitro, the transpiration rate and the percent water loss was lower than those of the control and thus the SV plantlets acclimatized easily ex vitro.     

Cost reduction in the micropropagation of banana by using tubular skylights as source for natural lighting

Summary Daylight instead of artificial light was exploited for the in vitro culture of banana. Tubular skylights rediverted natural light into an interior enelosed room whereby each skylight, available for ca. US$600, could sufficiently illuminate an area of 3–5 m². The maintenance-free system allowed only a minimum of heat transfer and no cooling was necessary. The culture room required no electricity supply and under our conditions savings on costs for electricity of US$6 m⁻² wk⁻¹ were achieved, as compared to a standard growth room equipped with artificial lighting and controlled photoperiod and temperature regimes. Under natural light conditions, micropropagated plantlets were well developed at mean photosynthetic photon flux densities (PPFD) of 5–13 μmol m⁻² s⁻¹ and photoperiods of 9–14 h. Micropropagation rates were either the same or significantly higher than under artificial lighting. Single shoots on rooting medium showed some symptoms of etiolation, yet acclimatization rates averaged 95%. A step-like culture rack. rather than a vertical one, permitted uniform plant growth on all levels. This paper describes an established micropropagation system of low cost and simplicity.     

Effects of photon flux density on the morphology,photosynthesis and growth of a CAM orchid, <Emphasis Type="Italic">Doritaenopsis</Emphasis> during post-micropropagation acclimatization

Micropropagated plantlets are fragile and often lack sufficient vigour to survive the acclimatization shock during transplantation to the soil. Effects of photosynthetic photon flux densities (PPFDs) on growth, photosynthesis and anatomy of micropropagated Doritaenopsis were studied after 4 months of acclimatization in a greenhouse at 25 °C. The plantlets were transferred to three different PPFDs for four months, i.e. low light (175), intermediate light (270) and high light (450 mol m^–2 s^–1). For most of the growth parameters measured i.e. leaf length, leaf area, leaf width, fresh weight, dry weight, chlorophyll (Chl) a/b ratio, were greater for the intermediate light levels after 4 months of acclimatization. The only exception was leaf thickness, which was increased more under high light levels. Results showed that the survival of Doritaenopsis plantlets was greatest (90%) in low light and intermediate light (89%) compared with only (73%) at high light. However, at low light levels, pigment concentrations (chlorophyll a, b and total chlorophyll) were higher. Net CO₂ assimilation (A), stomatal conductance (g) and transpiration (E) were higher in plantlets grown at high level PPFD than at low after 4 months of acclimatization. Photosynthetic efficiency (Fv/Fm) decreased insignificantly; only at mid day for the high light treatment whereas leaf temperature and stomatal closure increased compared to low light. Scanning electron microscopic (SEM) images of leaves from acclimatized plantlets showed an increase in wax formation for the high light grown plantlets compared to those at low light. Microscopic analysis of acclimatized root sections showed highly developed multiseriate-velamen layers and higher root cell activity; while shoots had larger leaf air spaces than those of in vitro grown plantlets. These results suggest that physiological acclimation occurs at the intermediate PPFD (270 mol m^–2 s^–1) in Doritaenopsis compared to treatment at the high light level.     

Photosynthesis and carbon metabolism in plantain (Musa AAB) plantlets growing in temporary immersion bioreactors and during ex vitro acclimatization

Summary The photosynthetic capacity changes and the main enzymatic systems related to carbon metabolism were investigated during the in vitro culture of plantain shoots (Musa AAB cv. CEMSA 3/4) in temporary immersion bioreactors (TIB) and their subsequent acclimatization. The maximal rate of photosynthesis (Pn), transpiration, and the activity of the carbon metabolism enzymes phosphoenolpyruvate carboxylase (PEPC), acid invertase (AI), pyruvate kinase (PK) and sucrose phosphate synthase (SPS) were measured every 7 d during the 21 d of elongation in TIB, and the following 42 d of acclimatization. Sucrose content in the liquid medium and in the leaves was also determined. The most significant changes in plant growth were observed during acclimatization. During the in vitro stage photosynthesis was limited (4–6 μmol CO₂m⁻²s⁻¹); the photosynthetic rate however increases rapidly and significantly as soon as in vitro culture is over during acclimatization. PEPC activity increased during the whole evaluation period. The highest levels were achieved around days 42 and 56. PK and SPS activities were optimal during the first weeks in acclimatization (28–35 d), while AI increased at the beginning of the elongation phase (7 d), and later at the end of the acclimatization (49–63 d). The relationships between morphological parameters, photosynthetic capacity of the plantlets and the carbon metabolism enzymes during both phases of the culture are discussed.     

Photosynthetic and biochemical characterization of in vitro-derived African violet (Saintpaulia ionantha H. Wendl) plants to ex vitro conditions

African violet (Saintpaulia ionantha H. Wendl) is one of the most easily and commonly tissue-cultured ornamental plants. Despite this, there are limited reports on photosynthetic capacity and its impact on the plant quality during acclimatization. Various growth, photosynthetic and biochemical parameters and activities of antioxidant enzymes and dehydrins of micropropagated plants were assessed under three light intensities (35, 70, and 100 µmol m^?2 s^?1 photosynthetic photon flux density – PPFD). Fresh and dry plant biomass, plant height, and leaf area were optimal with high irradiance (70–100 µmol m^?2 s^?1 PPFD). Chlorophyll and carotenoid contents and net photosynthesis were optimal in plants grown under 70 µmol m^?2 s^?1 PPFD. Stomatal resistance, malondialdehyde content, and F_v/F_m values were highest at low light irradiance (35 µmol m^?2 s^?1 PPFD). The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, increased as light irradiance increased, signaling that high light irradiance was an abiotic stress. The accumulation of 55, 33, and 25 kDa dehydrins was observed with all light treatments although the expression levels were highest at 35 µmol m^?2 s^?1 PPFD. Irradiance at 70 µmol m^?2 s^?1 PPFD was suitable for the acclimatization of African violet plants. Both low and high irradiance levels (35 and 100 µmol m^?2 s^?1 PPFD) induced the accumulation of antioxidants and dehydrins in plants which reveals enhanced stress levels and measures to counter it.     

Benzopyrans in <Emphasis Type="Italic">Hypericum polyanthemum</Emphasis> Klotzsch ex Reichardt cultured in vitro

Hypericum polyanthemum Klotzsch ex Reichardt, an endemic species of Southern Brazil, was micropropagated on MS medium supplemented with 1.78 μM BAP. Shoot proliferation and rooting was achieved on hormone-free medium and plantlets survived acclimatization. The bioactive compounds: 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) were quantified in the leaves, stems and roots of propagated and acclimatized plantlets and compared with the field-grown plants. The HPLC analysis revealed that the three benzopyrans are accumulated in the aerial parts and the concentration varied with the age of the plant whereas the roots were capable of accumulating only HP3. Greatest yield of HP1 (7.12 mg/g DW) was quantified in the leaves of the acclimatized plantlets, whereas the flowers of the plants from natural habitat displayed higher amounts of HP2 (11.04 mg/g DW) and HP3 (13.99 mg/g DW).     

Effect of Pb ions on superoxide dismutase and catalase activities in leaves of pea plants grown in high and low irradiance

The role of irradiance on the activity of antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT) was examined in the leaves of Pisum sativum L. plants grown under low (LL) or high (HL) irradiance (PPFD 50 or 600 μmol m⁻² s⁻¹) and exposed after detachment to 5 mM Pb (NO₃)₂ for 24 h. The activities of both enzymes increased in response to LL compared with HL and no effect of Pb ions was observed. Photosystem (PS) 1 and PS 2 activities were also investigated in chloroplasts isolated from these leaves. LL lowered PS 1 electron transport rate and changes in photochemical activity of PS 1 induced by Pb²⁺ were visible only in the chloroplasts isolated from leaves of LL grown plants. PS 2 activity was influenced similarly by Pb ions at both PPFD. This study demonstrates that leaves of HL grown plants were less sensitive to lead toxicity than those from LL grown plants. Changes in electron transport rates were the main factors responsible for the generation of reactive oxygen species in the chloroplasts and as a consequence, in induction of antioxidant enzymes.     

Morpho-anatomical and physiological changes of Indian sandalwood (Santalum album L.) plantlets in ex vitro conditions to support successful acclimatization for plant mass production

Santalum album L. (Indian sandalwood) is an economically important but vulnerable tropical tree species. Cultures were established via direct shoot regeneration from axillary buds on Murashige and Skoog (MS) medium supplemented with 2.5 mg L^?1 6-benzylaminopurine (BAP). The shoots were multiplied using MS medium containing 1.0 mg L^?1 BAP and 0.5 mg L^?1 indole-3 acetic acid and rooted on half strength MS medium containing 1.0 mg L^?1 indole-3 butyric acid. The rooted plantlets were hardened and acclimatized in greenhouse using soilrite® and cocopeat (1:1) mixture. The concentrations of photosynthetic pigments were analyzed and detected less under in vitro conditions (6.05 μg g^?1 FW) as compared to the 4 weeks old hardened (6.91 μg g^?1 FW) and 12 weeks old acclimatized plantlets (7.8 μg g^?1 FW) under greenhouse (ex vitro) environment. The anatomical evaluation of plantlets at subsequent stages of propagation suggested that the in vitro raised plantlets possessed structural abnormalities such as underdeveloped cuticle, unorganized tissue systems, reduced mesophyll tissues, fewer vascular elements and mechanical tissues, and loosely arranged thin walled paranchymatous ground tissues, which were slowly repaired during ex vitro hardening and acclimatization process to validate the developmental adaptation of micropropagated plantlets for maximum survival in the field (98.0% survival rate). The findings could help in the optimization of high-frequency commercial micropropagation of S. album for year-round production, and supply of this economically prominent vulnerable plant species to the farmers and the industries that rely on it.

     

Chlorophyll fluorescence as an indicator of photosynthetic functioning of in vitro grapevine and chestnut plantlets under ex vitro acclimatization

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 mol m^–2 s^–1 after in vitro phases at 50 mol 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 mol m^–2 s^–1. The results taken as a whole suggest that 300 mol 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.     

Mycorrhizal fungi enhancement of growth and gas exchange of micropropagated guava plantlets (Psidium guajava L.) during ex vitro acclimatization and plant establishment

Psidium guajava L.) plantlets was determined during acclimatization and plant establishment. Guava plantlets were asexually propagated through tissue culture and grown in a glasshouse for 18 weeks. Half of the plantlets were inoculated with a mixed endomycorrhiza isolate from Mexico, ZAC-19, containing Glomus diaphanum, G. albidum and G. claroides. Plantlets were fertilized with modified Long Ashton nutrient solution that supplied 11 μg P ml⁻¹. Gas exchange measurements were taken at 2, 4, 8, and 18 weeks after inoculation using a portable photosynthesis system. All micropropagated guava plantlets survived transplant shock. After 6 weeks, mycorrhizal plantlets had greater shoot growth rates and leaf production than non-mycorrhizal plantlets. This also corresponded with increased photosynthetic rates and stomatal conductance of mycorrhizal plants. By 18 weeks, mycorrhizal plantlets had greater shoot length, leaf area, leaf, stem, and root dry mass. However, gas exchange was comparable among treatments, in part because the container size was restricting growth of the larger mycorrhizal plantlets. Non-mycorrhizal plantlets had greater leaf area ratios and specific leaf areas than mycorrhizal plantlets. Increased leaf tissue mineral levels of P, Mg, Cu, and Mo also occurred with mycorrhizal plantlets. Roots of inoculated guava plantlets were heavily colonized with arbuscules, vesicles and endospores. Guava plantlets were highly mycotrophic with a mycorrhizal dependency index of 103%. Accepted: 27 December 1999     

Carbon metabolism in leaves of micropropagated sugarcane during acclimatization phase

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 1,000 μmol m⁻² s⁻¹ and 90% relative humidity during the first 21 d; followed by 2,000 μmol m⁻² s⁻¹ and approximately 80% of relative humidity. All measurements were carried out at the end of rooting phase concomitant with day 0 of acclimatization and at 7-d intervals thereafter (0, 7, 14, 21, 28, 35, 42 d). As the in vitro plantlets were transferred to the acclimatization phase, photosynthesis increased significantly during the first 7 d. 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 21 d, respectively, after 21 d 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 7 d. 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.     

An elite protocol for accelerated quality-cloning in <Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus cv. Sciella

A novel, efficient, and simple protocol was developed on in vitro mass propagation and acclimatization of Gerbera jamesonii Bolus cv. Sciella, an ornamental plant with attractive flowers. Shoot tip was used as the primary explant for in vitro establishment in which Murashige and Skoog (MS) medium supplemented with a low level of NAA (0.5 mg l⁻¹) and BAP (1.5 mg l⁻¹) promoted earliest axillary bud initiation within 5 d in 91.6% of the inoculants. Five axillary buds were initiated from a single explant within 13 d after inoculation. A very high rate of shoot multiplication (14 shoots per inoculated axillary bud) and proliferation was achieved when MS medium was fortified with a relatively higher level of BAP (2 mg l⁻¹) and 60 mg l⁻¹ ADS within 27 d of multiple shoot culture. A maximum number of well-developed roots per plant was observed in MS medium with 0.5 mg l⁻¹ IAA in the next 26 d. In the easy low-cost acclimatization process of 20 d, a combination of sand, soil, cow urine, and tea leaves extract (1:1:1:1; v/v) ensured 95% survival rate. Sixty-one well-acclimatized plants were obtained from a single shoot tip within 86 d. The sustained multiple shoot culture for 15 mo paved the way toward the conservation of genetic resources as well as beneficial economics. The clonal fidelity study of micropropagated and sustained cultured clones using ISSR primers ensured the continuous supply of quality propagules retaining genetic uniformity. The in vitro-generated plants performed better over conventionally propagated plants in the field condition.     

Effect of cytokinins on <Emphasis Type="BoldItalic">in vitro</Emphasis> development of autotrophism and acclimatization of <Emphasis Type="Italic">Annona glabra</Emphasis> L.

The role of cytokinins in the differentiation of the photosynthetic apparatus in micropropagated plants and their effect on the plant’s ability to transition from a heterotrophic to an autotrophic condition during acclimatization was investigated. Annona glabra L. shoots were cultured on woody plant medium supplemented with sucrose and different cytokinins to evaluate leaf tissue for chloroplast development, chloroplast numbers, photosynthetic pigmentation, total photosynthetic potential, and soluble sugar content. Plants were transferred to the rooting medium in the presence or absence of sucrose and then acclimatized. Kinetin and benzyladenine (BAP) stimulated chloroplast differentiation. Inclusion of zeatin in the medium induced the formation of greater numbers of chloroplasts in the leaves, while plants cultivated in the presence of only kinetin and BAP demonstrated greater chlorophyll a and carotenoid content. The use of kinetin and BAP during in vitro culture promoted accumulation of dry matter during the acclimatization phase, especially in plants rooted under autotrophic conditions (without sucrose). Kinetin and BAP promoted development of more leaf area and greater plant survival rates in plant acclimatization on both autotrophic and heterotrophic media. The inhibitory effects of thidiazuron on the differentiation of chloroplasts, accumulation of chlorophyll a, and photosynthetic potential were examined.     

Effects of CO<Subscript>2</Subscript> concentration on acclimatization and physiological responses of two cultivars of carob tree

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.