Acclimatization of Micropropagated Plants to Ex Vitro Conditions

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

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.     

The physiology of ex vitro pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> L. Merr. var MD-2) as CAM or C3 is regulated by the environmental conditions

Many plant species grown under in vitro controlled conditions can be used as models for the study of physiological processes. Adult pineapple can display CAM physiology while in vitro it functions as a C3 plant. Ex vitro Ananas comosus has plastic morphology and physiology, both easy to modify from C3 to CAM by changing the environmental conditions. The yield of survival for a rentable propagation protocol of pineapple is closely related with the C3/CAM shift and the associated physiological characteristics. In the present work, ex vitro pineapple plants were divided in two sets and subjected to C3 and CAM-inducing environmental conditions, determined by light intensity and relative humidity, respectively, 40 μmol m⁻² s⁻¹/85% and 260 μmol m⁻² s⁻¹/50%. The results demonstrated that the stress imposed by the environmental conditions switched pineapple plants from C3 to CAM behavior. Comparing to CAM induced, C3-induced pineapple plants showed substandard growth parameters and morphological leaf characteristics but a better rooting process and a higher ABA production, a phenotype closer to adult plants, which are expected to produce fruits in a normal production cycle. We conclude that the upholding of these characteristics is conditioned by low light intensity plus high relative humidity, especially during the first 8 weeks of ex vitro growth. It is expected that the better understanding of pineapple acclimatization will contribute to the design of a protocol to apply as a rentable tool in the pineapple agronomic industry.     

Structural and ultrastructural differences between field,micropropagated and acclimated leaves and stems of two Leucospermum cultivars (Proteaceae)

The anatomy of field, in vitro and acclimatized shoots (leaves and stems) of two cultivars of Leucospermum (L. cordifolium ‘Flame Spike’ and L. ‘Tango’) was compared using light, scanning and transmission electron microscopy. Field plants showed several scleromorphic anatomical structures related to excess solar radiation such as: cuticle thickness, subepidermal collenchyma and sclerenchyma. Furthermore, a large quantity of phenolic deposits present in the cell lumen of various tissues is also a scleromorphic feature. The special conditions during in vitro culture result in plantlets with abnormal morphology and anatomy. These disorders are associated with the gaseous environment in the culture vessels, low irradiance in the incubation chamber and the addition of sucrose, nutrients and growth regulators to the culture medium. After transfer from in vitro to ex vitro conditions, substantial changes in leaf and stem anatomy were observed, above all in cuticle thickness, epidermal characteristics (stomatal and trichome index, and stomatal and pore size), differentiation of leaf mesophyll, chloroplast structure, and amount and localization of phenolic deposits. These changes allowed the plants to adapt to the new environmental conditions. The study of anatomical features of in vitro shoots facilitated adapting the acclimation protocol to predict which plantlet would survive the critical acclimation stage.

     

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.     

Anatomy and morphology of photinia (Photinia × fraseri Dress) in vitro plants inoculated with rhizobacteria

Photinia × fraseri Dress (photinia) is a woody plant with high ornamental value. The anatomy and morphology of micropropagated photinia inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense and Azotobacter chroococcum, in combination with pulses of 49.2 μM indole-3-butyric acid during rhizogenesis, were characterized using light and electron microscopy. Leaves of inoculated in vitro plants showed better development than those subjected to auxin control only. All inoculated treatments, independent of the bacterial strain used, had leaves with two layers of palisade parenchyma, a thick cuticle and linear unicellular trichomes. There was no proliferation of undifferentiated tissue in any treatment and the plants showed shoot–root vascular connections. Ex vitro leaves and in vitro plants inoculated with Azospirillum brasilense Cd and Azotobacter chroococcum 42 had large stomata with elliptic aperture radially surrounded by small stomata on the abaxial foliar surface. In addition, plants of these treatments had a large root hair zone over the root surface. Bacteria were only observed on surfaces of root hairs. The results suggest that the structural changes induced by bacterial inoculation of photinia in vitro plants could lead to better adaptation to ex vitro conditions after transplanting.     

Structural differences between hyperhydric and normal in vitro shoots of <Emphasis Type="Italic">Handroanthus impetiginosus</Emphasis> (Mart. ex DC) Mattos (Bignoniaceae)

The anatomy of normal and hyperhydric shoots (leaves and stems) of in vitro Handroanthus impetiginosus was compared using light microscopy, scanning electron microscopy, and transmission electron microscopy. In contrast to normal shoots, hyperhydric shoots presented numerous anatomical abnormalities at the proliferation stage. Disorganized cortex, epidermal holes, epidermal discontinuity, collapsed cells, and other structural characteristics were observed in hyperhydric shoots. So, by using anatomical analysis of in vitro H. impetiginosus shoots at the proliferation stage, we can predict which plants will survive the rhizogenesis and acclimatization stages.     

In vitro propagation of three <Emphasis Type="Italic">Agave</Emphasis> species used for liquor distillation and three for landscape

In vitro bud clusters of Calathea orbifolia (Linden) Kennedy were obtained and subcultured in semi-solid (agar) medium and temporary immersion system (TIS) for 12 weeks. Uniform young plants were selected and transferred to soilless mix in a growth chamber for ex vitro acclimatization during 35 days, followed by growing in a shaded greenhouse for 65 days. Comparison of in vitro leaf anatomy, ex vitro photosynthetic behaviors and growth was made between two cultural systems. Plants in TIS produced thicker leaf chlorenchyma and aquiferous parenchyma, lower stomatal frequency and more epicuticular wax than did those in semi-solid medium. Plants from semi-solid medium had consistently lower leaf Fv/Fm values than plants from TIS. Leaf Fv/Fm value in plants from TIS decreased to 0.65 at day 7 after transfer and increased soon up to 0.76 thereafter. In contrast, leaf Fv/Fm value in plants from semi-solid medium reduced to 0.27 at day 7 after transfer and increased slowly up to 0.68 at day 35. During ex vitro acclimatization, plants in TIS had substantial higher photosynthetic rates than plants in semi-solid medium. Plants from TIS had subsequent higher leaf area, fresh and dry weights than plants from semi-solid medium.     

Artichoke Leaf Morphology and Surface Features in Different Micropropagation Stages

Artichoke (Cynara scolymus L.) leaf size and shape, glandular and covering trichomes, stomatal density, stomata shape, pore area and epicuticular waxes during micropropagation stages were studied by scanning electron microscopy (SEM) and morphometric analysis with the aim to improve the survival rate after transfer to greenhouse conditions. Leaves from in vitro shoots at the proliferation stage showed a spatular shape, ring-shaped stomata, a large number of glandular trichomes and juvenile covering hairs, but failed to show any epicuticular waxes. Leaves from in vitro plants at the root elongation stage showed a lanceolated elliptic shape with a serrated border, elliptical stomata, decreased pore area percentage, stomatal density, and mature covering trichomes. One week after transfer to ex vitro conditions, epicuticular waxes appeared on the leaf surface and stomata and pore area were smaller as compared to in vitro plants. Artichoke acclimatization may be improved by hormonal stimulation of root development, since useful morphological changes on leaves occurred during root elongation.     

Substitution of benzyladenine with meta-topolin during shoot multiplication increases acclimatization of difficult- and easy-to-acclimatize sea oats (Uniola paniculata L.) genotypes

Benzyladenine (BA) is the only cytokinin to effectively induce shoot multiplication in vitro between genotypes of the important dune grass species Uniola paniculata (sea oats). However, a significant genotype-specific negative carryover effect of BA on ex vitro acclimatization has been observed. In the present study, the effects of multiplication media supplemented with meta-topolin (mT), a BA-analog, BA or no plant growth regulator, were compared on in vitro multiplication, rooting and ex vitro acclimatization using easy- and difficult-to-acclimatize sea oats genotypes. Both genotypes exhibited similar in vitro shoot dry weight, number of harvestable shoots and percent rooting when cultured under standard conditions (with 2.2 μM BA) or with an equimolar concentration of mT. In addition, both genotypes exhibited similar ex vitro leaf length and shoot production under these two culture conditions. However, ex vitro acclimatization of rooted microcuttings of the difficult-to-acclimatize genotype significantly increased when produced on shoot multiplication medium containing mT rather than BA. Meta-topolin concentrations 10 μM or greater were inhibitory to in vitro rooting and acclimatization ex vitro of both genotypes. Nevertheless, survival of the difficult-to-acclimatize genotype was significantly greater when cultured in the presence of 2.2 μM–30 μM mT, compared to 2.2 μM BA. Therefore, a potential solution to overcome the detrimental BA carryover effect on ex vitro survival in sea oats is the substitution of BA with 2.2 μM mT for Stage II shoot multiplication. Use of mT may provide an efficient method to ensure in vitro propagation of a large number of diverse sea oats genotypes for dune restoration.     

Large-scale field acclimatization of Olea maderensis micropropagated plants: morphological and physiological survey

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.     

Using LED lighting in somatic embryogenesis and micropropagation of an elite sugarcane variety and its effect on redox metabolism during acclimatization

This study investigated the ability of a light emitting diode (LED) to induce somatic embryogenesis (SE), shoot multiplication, and rooting of sugarcane (RB98710). We also accessed the effects on acclimatization. MS medium was used for all stages and was supplemented with different concentrations of growth regulators according to the culture stage. The material was maintained in a growth room under fluorescent (FL) or LED (82?% red, 18?% blue) lighting after rooting plants were acclimatized. We conducted both biometric and biochemical analyses before and after acclimatization. The LED conditions favored the formation of callus; however, the FL was more efficient at plant regeneration. A histological analysis showed the formation of somatic embryos occurred through direct and indirect pathways. The plants obtained through SE and grown under LED had a higher multiplication rate over six subcultures. Shoots rooted in both light sources, but the number of shoots and the weight gain of the roots were higher under LED. The malondialdehyde (MDA) level did not differ among treatments. Our results indicate the SE induction phase should be conducted under FL and the remaining micropropagation process should be performed using LED. After acclimatization the plants grown under LED did not change the SOD and CAT activities during the first 5 days, which suggests there was no acclimatization impact. The H₂O₂ and MDA values observed do not suggest damage to membranes. There was better development, lower water loss, and higher survival rate in plants from in vitro culture under LED conditions when compared to FL.     

Stomatal Morphology during Acclimatization of Tobacco Plantlets to ex vitro Conditions

Image analysis was used in studying stomatal morphology during acclimatization of tobacco plantlets to ex vitro conditions, 45 d after transfer leaf area was 15 times, and total number of stomata per leaf four times increased. During acclimatization stomatal density was decreased considerably on both leaf sides, and was compensated by an increase in stomatal sizes, e.g., in stomatal length and in stomatal area (both guard cells and pore). Elongation of stomata was increased indicating that the originally circular stomata of in vitro plantlets were changed into elliptical ones in ex vitro acclimatized plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Study of the effects of foliar application of ABA during acclimatization

This study aims to elucidate the effects of abscisic acid (ABA) foliar application on plant hardening during ex vitro acclimatization using a forest model species Ulmus minor L. Plant leaves were sprayed with ABA (0, 50 and 100 μM) immediately after ex vitro transfer and twice a week for the first 3 weeks of acclimatization. After this period, parameters related with photosynthesis, hormone levels and oxidative stress were measured to assess plant performance. The results demonstrated that ABA foliar application alleviates the negative shock of ex vitro acclimatization since it reduces the water loss through transpiration, relieving the risk of wilting. Moreover, ABA promoted net CO₂ assimilation rate (P _N ) and plant dry mater accumulation. ABA treatment increased the antioxidant battery during acclimatization, with more effective results at the concentration of 50 μM ABA. Also, flow cytometry data support that cytosolic compounds, which may increase in response to 50 μM ABA, could also protect DNA from oxidative damage. We propose here that ABA foliar application (immediately after ex vitro transfer), by preventing water loss, enhancing photosynthesis efficiency and the activity of antioxidant enzymes, improves the plants hardening and ability to deal with the ex vitro stresses.     

The effects of in vitro and ex vitro root initiation on subsequent microcutting root quality in three woody plants

In vitro- and ex vitro-rooted microcuttings of Acer rubrum L. Red Sunset, Betula nigra L., and Malux x- domestica Borkh McIntosh were distinguished by several important anatomical and morphological properties which continued to regulate both root system and whole plant quality in later stages of production. In vitro microcuttings formed adventitious roots in greater number and more quickly than ex vitro microcuttings. Roots produced in vitro were characterized by extremely enlarged cortical cells and, consequently, had a much greater diameter than ex vitro roots. However, the vascular system of in vitro roots was underdeveloped (primary vascular tissues only) as compared to ex vitro roots, which produced vascular cambium and secondary growth during the same early stage of production. At least 50% of the post-transplant in vitro adventitious roots either died immediately, or temporarily persisted during acclimatization without producing any further growth. For the surviving in vitro-produced roots, the cortex partially collapsed after transplant, and new root extensions with ex vitro-like structure were produced. Only then did the in vitro portion of the root begin to form secondary vascular tissues. Shoots from in vitro treatments continued to grow vigorously during adventitious root initiation and during acclimatization, so that the plants were significantly taller and had a greater shoot area than those receiving comparable ex vitro rooting treatment. In vitro rooting led to a horizontal root morphology which continued to distinguish these treatments from ex vitro rooted plants during later stages of production, when anatomical differences in the roots could no longer be detected.Abbreviations BA benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron - WPM woody plant medium     

Telomere length in Agave tequilana Weber plants during the in vitro to ex vitro transition

Acclimatization ex vitro is a key stage of the micropropagation process, in which the vitro plants leave the sterile, high humidity environment in which they originated and form new leaves and roots, during which they suffer different types of stress. Changes in the telomere length (shortening and lengthening) have been associated with age, the development of tissue, loss of cell replication and the ability of regeneration in different plant species. However, the genetic and biological factors that are involved in the process of shortening of telomeres across the ageing of plant species are still unknown. In this study, we used terminal restriction fragments (TRF) to examine the changes of telomere length during the in vitro to ex vitro transition in vitro plants of Agave tequilana, and their relationships with age in plants grown in commercial plantations. The results showed that in vitro grown plants present the longest telomeres and that a shortening occurs during the first 6 months of ex vitro acclimatization, (compared to the plantlets that were kept in vitro). A lengthening of the telomeres was observed in the acclimatized 1-year-old plants and that this was maintained in 2 and 3-year-old plants. We also observed TRF variations in the different tissues (leaves, stems and roots) of acclimatized plants. In field plants, we did not observe any important changes in the length of the telomeres. We suggest that agaves have a mechanism that maintains telomere length at the non-critical stages during development.

     

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.     

Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)

Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.     

Rooting affects the photosystem II activity: in vitro and ex vitro studies on energy hybrid sorrel

Rumex tianschanicus × Rumex patientia is a high-biomass-yielding plant suitable for fuel and biogas production. The protocol of the hybrid sorrel micropropagation was used to study the changes in the photosystem II (PSII) activity as well as to analyse the ultrastructure of the chloroplasts. The lowest effective PSII quantum yield [Y(II)] and an apparent electron transport rate of PSII [ETR(II)] were observed for adventitious shoots that had been regenerated in vitro, before rooting. These fluorescence parameters were higher and similar for both the leaves of the same adventitious shoots that had been rooted under in vitro conditions and for the shoots that had been acclimated and grown in ex vitro conditions. The analysis indicated that the PSII activity strongly depends on the formation of properly functioning roots and that in vitro or ex vitro culture conditions are, at least to some degree, less important. TEM analysis revealed that chloroplasts from plants rooted in vitro were sufficiently mature and acclimatization processes have less impact on their development. This is the first report concerning the analysis of PSII activity and the ultrastructure of the chloroplasts at all of the stages of micropropagation, i.e. adventitious shoot formation in vitro, rooting in vitro and acclimation to ex vitro conditions. It strongly indicated that rooting under in vitro conditions, rather than the acclimation to ex vitro conditions, plays a key role in the development of a completely functional photosynthetic apparatus in hybrid sorrel.     

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.