Changes in photosynthetic activity,pigment composition,electrolyte leakage,lipid peroxidation,and antioxidant enzymes during ex vitro establishment of micropropagated <Emphasis Type="Italic">Rauvolfia tetraphylla</Emphasis> plantlets

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) μmol m⁻²s⁻¹ photosynthetic PPFD for 4 weeks 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.     

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.     

Stability and activity of rubisco in chestnut plantlets transferred to <Emphasis Type="Italic">ex vitro</Emphasis> conditions under elevated CO<Subscript>2</Subscript>

Summary Heterotrophic plantlets obtained by in vitro propagation are biochemically different compared to autotrophic plantlets. When heterotrophic plantlets are transferred to ex vitro conditions, higher irradiance levels are generally applied. Irradiance levels higher than those used in vitro lead to oxidative stress symptoms, that can be counteracted by CO₂ concentrations above normal. We analyzed the stability and activity of Rubisco and leaf-soluble sugars and starch contents in chestnut plantlets transferred from in vitro to ex vitro conditions under four treatments obtained by associating two irradiances of 150 (low light, LL) and 300 (high light, HL) μmolm⁻²s⁻¹, respectively three and six times in vitro irradiance, with two CO₂ levels of 350 (low CO₂, LCO₂) and 700 (high CO₂, HCO₂) μll⁻¹. In in vitro plantlets it was possible to immunodetect apparent products of degradation of Rubisco large subunit (LSU). In ex vitro plantlets, these degradation products were no longer dtected except under LL associated with LCO₂. The decrease in soluble sugars and starch in plantlets under HL HCO₂ gave an indication of a faster acquisition of autotrophic characteristics. However, under the same treatment, a down-regulation of Rubisco activity was observed. From the results taken as a whole, two aspects seem to be confirmed: HL HCO₂ is more efficient in inducing an autotrophic behavior in chestnut ex vitro plantlets; actively growing systems as ex vitro plantlets reflect the down-regulation of Rubisco by HCO₂ without accumulation of carbohydrates.     

Photosynthetic parameters of Ulmus minor plantlets affected by irradiance during acclimatization

In order to set up large-scale acclimatization protocols of micropropagated plants, an in-depth knowledge of their physiological responses during in vitro to ex vitro transfer is required. This work describes the photosynthetic performance of Ulmus minor micropropagated plants during acclimatization at high irradiance (HI; 200 ± 20 μmol m^?2 s^?1 or low irradiance (LI; 100 ± 20 μmol m^?2 s^?1). During this experiment, leaf pigment content, chlorophyll a fluorescence, gas exchange, stomata morphology, the activity of the Calvin cycle enzymes and saccharides were measured in persistent and new leaves. The results indicated that HI induces a higher photosynthetic performance compared to LI. Therefore, plants acclimatized under HI are likely to survive better after field transfer.     

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.     

Thidiazuron-induced high-frequency direct shoot organogenesis of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L.

Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l⁻¹ activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 μmol m⁻² s⁻¹). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m⁻² s⁻¹ and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 μmol m⁻² s⁻¹. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m⁻² s⁻¹) tested. Intercellular CO₂ concentration (C _i) and the ratio of intercellular CO₂ concentration to ambient CO₂ (C _i/C _a) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.     

Production of reactive oxygen species and development of antioxidative systems during <Emphasis Type="Italic">in vitro</Emphasis> growth and <Emphasis Type="Italic">ex vitro</Emphasis> transfer

Ex vitro transfer is often stressful for in vitro grown plantlets. Water stress and photoinhibition, often accompanying the acclimatization of in vitro grown plantlets to ex vitro conditions, are probably the main factors promoting production of reactive oxygen species (ROS) and in consequence oxidative stress. The extent of the damaging effects of ROS depends on the effectiveness of the antioxidative systems which include low molecular mass antioxidants (ascorbate, glutathione, tocopherols, carotenoids, phenols) and antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase). This review is focused on ROS production and development of antioxidative system during in vitro growth and their further changes during ex vitro transfer.     

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.     

Morphological evaluation and antioxidant activity of <Emphasis Type="Italic">in vitro</Emphasis>- and <Emphasis Type="Italic">in vivo</Emphasis>-derived <Emphasis Type="Italic">Echinacea purpurea</Emphasis> plants

An effective in vitro protocol for rapid clonal propagation of Echinacea purpurea (L.) Moench through tissue culture was described. The in vitro propagation procedure consisted of four stages: 1) an initial stage - obtaining seedlings on Murashige and Skoog (MS) basal medium with 0.1 mg L⁻¹ 6-benzylaminopurine, 0.1 mg L⁻¹ α-naphthalene acetic acid and 0.2 mg L⁻¹ gibberellic acid; 2) a propagation stage — shoot formation on MS medium supplemented with 1 mg L⁻¹ 6-benzylaminopurine alone resulted in 9.8 shoots per explant and in combination with 0.1 mg L⁻¹ α-naphthalene acetic acid resulted in 16.2 shoots per explant; 3) rooting stage — shoot rooting on half strength MS medium with 0.1 mg L⁻¹ indole-3-butyric acid resulted in 90% rooted microplants; 4) ex vitro acclimatization of plants. The mix of peat and perlite was the most suitable planting substrate for hardening and ensured high survival frequency of propagated plants. Significant higher levels were observed regarding water-soluble and lipid-soluble antioxidant capacities (expressed as equivalents of ascorbate and α-tocopherol) and total pnenols content in extracts of Echinaceae flowers derived from in vitro propagated plants and adapted to field conditions in comparison with traditionally cultivated plants.     

Effect of abscisic acid on photosynthetic parameters during <Emphasis Type="Italic">ex vitro</Emphasis> transfer of micropropagated tobacco plantlets

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.     

Effect of irradiance during acclimatization on content of proline and phytohormones in micropropagated Ulmus minor

This study aimed to investigate the effects of irradiance on plant growth and content of proline and phytohormones during ex vitro acclimatization of micropropagated Ulmus minor plants. In vitro rooted plants were acclimatized to ex vitro conditions in a climate chamber with two irradiances, 200 μmol m^?2 s^?1 (high irradiance, HI) and 100 μmol m^?2 s^?1 (low irradiance, LI) for 40 d. Immediately after the ex vitro transfer, the plants experienced a water deficit [wilting leaves with the reduced relative water content (RWC)], but following the experiment, the recovery of the RWC was more pronounced in the HI treatment. Also, the content of proline, ABA, and JA-Ile were higher in HI treatment. Growth analyses revealed that HI improved growth and biomass production.     

Improvement of <Emphasis Type="Italic">ex vitro</Emphasis> transfer of tobacco plantlets by addition of abscisic acid to the last subculture

Tobacco (Nicotiana tabacum L.) plantlets were grown on Murashige and Skoog medium in ventilated Magenta boxes and for the last subculture 10 μM ABA was added to the medium. After three weeks plantlets were transferred into pots with Perlite moistened with water and grown in controlled conditions (16-h photoperiod, day/night temperature 25/20 °C, air humidity about 45 %) either under low or high irradiance of 150 (LI) and 700 (HI) μmol m⁻² s⁻¹, respectively. Content of endogenous ABA was 271.7 pmol g⁻¹(f.m.) in ABA treated plantlets, while in control plantlets it was only 53.3 pmol g⁻¹(f.m.). After ex vitro transfer, stomatal conductance and transpiration rate decreased considerably in comparison with in vitro grown plantlets and remained lower also 7 d after ex vitro transfer, especially in ABA-treated plants and so wilting of plants was practically eliminated. Net photosynthetic rate also decreased 1 d after ex vitro transfer but after 7 d it was mostly higher than that of in vitro grown plantlets. Water use efficiency significantly increased in ABA-treated plants. Chlorophyll a+b content did not change immediately after ex vitro transfer, nevertheless, after 7 d chlorophyll content was higher in ABA-treated plants. Pool of xanthophyll cycle pigments (XCP) and the degree of their deepoxidation (DEPS), which are connected with harmless dissipation of light energy, increased under high irradiance. Contents of XCP and ABA precursors (neoxanthin and violaxanthin) were lower in ABA-treated plants than in control plants indicating less stress in these plants. Most chlorophyll a fluorescence parameters did not change considerably after ex vitro transfer and so the photoinhibition was not observed even under HI. Slight increase in non-photochemical quenching under HI in ABA-treated plants suggested their better photoprotection. Thus application of ABA to the last subculture can improve acclimatization of in vitro grown plants to ex vitro conditions     

Leaf anatomy of <Emphasis Type="Italic">Cynara scolymus</Emphasis> L. in successive micropropagation stages

Summary Leaf structure along the successive stages of Early French artichoke Cynara scolymus L. micropropagation was characterized using light and transmission electron microscopy. The mesophyll presents disorganized spongy and palisade parenchyma with large intercellular spaces and a few small chloroplasts in the leaves of plants cultured in vitro. In addition, both epidermal surfaces of such leaves invariably show a cell wall of the same thickness with a very thin cuticle and open stomata. In the root differentiation stage in vitro, structural changes take place in the leaves that are favorable for survival in the acclimatization stage: conspicuous cuticle, greater cell wall thickness, functional stomata, better mesophyll organization, developed vascular bundles, and the presence of sclerenchymatous tissue are observed. These features found in later in vitro stages are maintained in the following ex vitro stages, some becoming more evident. Our results demonstrate that the structural changes required to ensure appropriate acclimatization of micropropagated artichoke plants begin at the root differentiation stage, which can reduce in vivo acclimatization time and achieve greater survival of transferred plants.     

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.     

Humid incubation period and plantlet age influence acclimatization and establishment of micropropagated grapes

Summary In vitro rooted grape (Vitis vinifera L.) plantlets (4 or 8 wk from culturing microcuttings) were plantedex vitro in polythene sachets (24×12 cm) filled to one-third their height with planting mixture. The sachets were misted, closed, and incubated at ambient temperature (25–30°C) under 16 h photoperiod (40–50 μ E·m^−2.·sec⁻¹) for 1,2, or 3 wk before opening. Maximum establishment with no or minimum damage toin vitro formed leaves was obtained with 3 wk of closed sachet incubation in both age groups. Opening the sachet at 2 wk from planting resulted in marginal scorching of lower leaves and some reduction in establishment and vigor. Opening at 1 wk led to severe leaf scorching and significant reduction in establisment particularly in 4-wk-oldin vitro plantlets while growth was more affected in 8-wk-old ones. Relative humidity ofin vitro culture vessel was 68–75% while closed sachets had RH values of 45–77% depending on length of incubation and ambient RH. Diurnal variation in RH of sachet in relation to ambient RH was the major factor that facilitated acclimatization rather than the overall fall in RH during the period of closed incubation. Satisfactory acclimatization of plantlets to withstand the open sachet RH (50–55%) by 3 wk and the ambient RH (30–40%) by 4 wk was achieved. Monitoring water loss from detached leaves of plantlets showed a significant reduction between the date of planting and Week 3, and again between Weeks 3 and 4. Comparison of growthin vitro andex vitro suggested that shifting toex vitro earlier was more beneficial. This observation was confirmed by transferring 3-, 4-, and 5-wk-old plantlets fromin vitro rooting medium toex vitro and recording the growth at 8 wk fromin vitro culturing when 3 wkin vitro plus 5 wkex vitro combination showed maximum vigor. The leftover stumps after subculturing of 1–4-mo.-old stock cultures could also be effectively used forex vitro establishment.     

Micropropagation of <Emphasis Type="Italic">Zingiber rubens</Emphasis> and assessment of genetic stability through RAPD and ISSR markers

Protocol was developed for high frequency in vitro multiplication of an endemic species, Zingiber rubens Roxb. The sprouted buds of the rhizomes were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 0.5–5.0 mg dm⁻³), indole-3-acetic acid (IAA; 0.5–2.0 mg dm⁻³), kinetin (KIN; 1.0–3.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0.5–1.0 mg dm⁻³) and adenine sulphate (ADS; 80–100 mg dm⁻³). MS basal medium supplemented with 3 mg dm⁻³ BA and 0.5 mg dm⁻³ IAA was optimum for shoot elongation. The elongated shoots (1–2 cm) were transferred to multiplication medium containing 2 mg dm⁻³ BA, 1 mg dm⁻³ IAA and 100 mg dm⁻³ ADS. The multiplication rate remained unchanged in subsequent subcultures. Upon ex vitro transfer, 85 % of plants survived. Genetic stability of micropropagated clones were periodically evaluated at an interval of 6 months up to 30 months in culture using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis and genetic uniformity in all regenerants was confirmed.     

Chlorophyll fluorescence in micropropagated <Emphasis Type="Italic">Rhododendron ponticum</Emphasis> subsp. <Emphasis Type="Italic">baeticum</Emphasis> plants in response to different irradiances

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 (ETR_max) and light saturation coefficient (E_k) than plants grown under low irradiance (LL). The capacity of tolerance to photoinhibition (as assessed by the response of photochemical quenching, q_p) varied as follows: HL > IL > LL. Thermal energy dissipation (q_N) was also affected by growth irradiance, with higher saturating values being observed in HL plants. Light-response curves suggested a gradual replacement of q_p by q_N 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 (F_v/F_m) was observed in LL plants, indicating the occurrence of chronic photoinhibition. In contrary, the decrease in F_v/F_m was quickly reverted in HL plants, pointing to a reversible photoinhibition.     

In vitro propagation of Typhonium flagelliforme (Lodd) blume

Summary An in vitro culture system was developed for Typhonium flagelliforme using buds from the rhizomes. The mineral salts of four media were tested. These were Murashige and Skoog (MS), Nitsch and Nitsch (NN), Gamborg B5 (GB5) and White (W) of which MS medium was found to be the best medium for in vitro culture of T. flagelliforme. The addition of as low as 0.1 mg l⁻¹ (0.54 μM) α-naphthalene acetic acid (NAA) with the presence or absence of N⁶-benzyladenine (BA) in the MS medium caused abnormal shoot formation. The best medium for maximizing shoot number combined with normal complete plantlets from each bud was MS medium supplemented with 0.3 mg l⁻¹ (1.33 μM) BA and 0.5 mg l⁻¹ (2.46 μM) indole-3-butyric acid (IBA). The best acclimatization process was to transfer the normal plantlets, with all the leaves removed, into sand plus coconut husks substrate (1∶1) and placed in intermittent water mists house or shaded plant house with 50% light exclusion. Ninety two percent of the plantlets survived using this acclimatization method.