I. Tichá

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.

Importance of in vitro pretreatment for ex vitro acclimatization and growth

Abstract The influence of in vitro pretreatment on the physiology and growth of tobacco (Nicotiana tabacum L. cv. Samsun) plantlets during ex vitro transfer and acclimatization was studied. Nodal cuttings were cultured on solid Murashige-Skoog medium with 3% sucrose or without sucrose, and in low (60 μmol m−2 s−1) or high (200 μmol m−2 s−1) photosynthetic photon flux densities. 35 days old plantlets were transferred to soil. After a 20-days-period of acclimatization in the greenhouse the plants were transferred to the open air. The distinctive effects of the different in vitro culture conditions on pigments and photosynthetic parameters disappeared during greenhouse acclimatization. Both the transfers were accompanied by a transient increase in starch content and the transfer to open air by a transient decrease in maximum photochemical efficiency of Photosystem II. In the open air, photosynthetic capacity and starch content started to increase with only small effects of the different pretreatments. Pretreatment had a pronounced effect on growth under these conditions. Growth was highest in plants originally grown with 3% sucrose under high irradiance, and lowest in plants originally grown without sucrose under high irradiance (plantlets suffering from photoinhibition in vitro). Thus, photomixotrophic in vitro culture at elevated photon flux density is most suitable for later ex vitro development of the plants.

Impact of in vitro Cultivation Conditions on Stress Responses and on Changes in Thylakoid Membrane Proteins and Pigments of Tobacco during ex vitro Acclimation

Four physiologically and phenotypically diversified tobacco (Nicotiana tabacum L. cv. Samsun) plantlet variants had been generated by cultivation on media either lacking or containing sucrose (0 and 3 %, m/v) under two different photon flux densities (PFD), 50 µmol m−2 s−1 (LL) and 200 µmol m−2 s−1 (HL). Plantlets were transferred into soil without any pre-acclimation and grown either under PFD of 200 µmol m−2 s−1 or 700 µmol m−2 s−1. Sucrose feeding in vitro resulted in reduced degree and duration of wilting after transfer. The highest readiness for ex vitro acclimation was found in 3 % HL plants, in which changes of photosynthetic apparatus and stress responses were the smallest. On the contrary, the steepest decline of Fv/Fm ratio on the first day after transplantation, doubled chlorophyll content and almost tripled D1/LHC 2 ratio after 7 d of ex vitro growth under 700 µmol m−2 s−1 characterized 0 % HL plants, which had suffered chronic photoinhibition in vitro. Remarkably high abscisic acid content at the end of in vitro cultivation and during acclimation as well as increased synthesis of both D1 and LHC 2 proteins even at the end of analyzed acclimation period were found only in 0 % LL plants. Increase of D1/LHC 2 ratio and chlorophyll contents demonstrate that in vitro developed leaves of all plant variants are able to acclimate to new environment. The most surprising result in the whole study is the drop of D1 protein synthesis in all plants on the 3rd day. Five times decline of photoprotection level of xanthophylls in plants after ex vitro transfer into the same PFD showed stress character of in vitro cultures.

Photosynthesis and photoprotection inNicotiana tabacumL.in vitro-grown plantlets

Nicotiana tabacum L. plantlets were cultured in vitro photoautotrophically (0% sucrose) and photomixotrophically (3% or 5% sucrose) at two irradiances (80 or 380 mumol m-2 s-1) with the aim of investigating the effect of these culture conditions on photosynthetic parameters and on protective systems against excess excitation energy. In plantlets grown photoautotrophically under higher irradiance photoinhibition was demonstrated. These plantlets had a decreased chlorophyll (Chl) a + b content and Chl a/b ratio, an increased content of xanthrophyll cycle pigments and a higher deepoxidation state, a decreased maximum photochemical efficiency of photosystem II (PS II) and actual photochemical efficiency of PS II, and an increased non-photochemical quenching. In the photoautotrophically grown plantlets and those photomixotrophically grown with 3% sucrose, the increase of growth irradiance from 80 to 380 mumol m-2 s-1 stimulated the activities of ascorbate-glutathione cycle enzymes with the exception of ascorbate peroxidase. Ascorbate peroxidase activity was not affected by the increase in growth irradiance but a significant decrease with increasing sucrose concentration was evident. The higher concentration of sucrose in the medium (5%) in combination with the higher irradiance inhibited photosynthesis (decrease in Chl a + b content and net photosynthetic rate) but no significant changes in activities of ascorbate-glutathione cycle enzymes were found. These results suggest that exogenous sucrose added to the medium improved high irradiance and oxidative stress resistance of the plantlets but the effect of sucrose is concentration dependent.

Ex Vitro Phenotype Stability is Affected by In Vitro Cultivation

Plant phenotype stability during ex vitro growth, one of the main requirements of plant micropropagation, was tested on tobacco. Plants cultivated in vitro in the presence of 3 % sucrose under photon flux density (PFD) of 200 μmol m−2 s−1 (3 % HL plants) showed the best growth and photosynthetic parameters in the course of 7-day acclimation. However, significant change in phenotype of these plants appeared under a decrease in PFD to 50 μmol m−2 s−1 during further ex vitro growth (in the period of 7th – 17th day). Much higher internodia elongation was found in 3 % HL plants in comparison with plants grown in vitro on sucrose media under PFD of 50 μmol m−2 s−1 (3 % LL) or without sucrose either under PFD of 50 μmol m−2 s−1 or 200 μmol m−2 s−1 (0 % LL, 0 % HL). It can be presumed that 3 % HL plants show permanent demand for high PFD. Neither ABA or chlorophyll contents nor de novo thylakoid membrane synthesis were related to the morphogenic effect of low PFD. Changeable contents of hexoses in leaves of 3 % HL and 3 % LL plants were in no direct correlation to the elongated growth.

Growth at high CO2 affects the chloroplast number but not the photosynthetic efficiency of photoautotrophicMarchantia polymorpha culture cells

Photoautotrophic suspension cells of Marchantia polymorpha were grown at gas phase CO2 concentrations of 0.4% and 2.0%. At the higher CO2 concentration the chloroplast shape appeared to be modified and the cells had about 70% more chloroplasts per cell. Differences in chlorophyll content per cell were much less pronounced, indicating a reduction in chlorophyll content per chloroplast. Also the cell size was affected by the CO2 concentration, and our data suggest that it was about 37% lower in high CO2 grown cells than in low CO2 grown cells. The capacity and the efficiency of photosynthetic oxygen evolution on a chlorophyll basis and the photosystem II chlorophyll fluorescence parameters were almost identical in both cell types. Immunodection showed that also the ratio of light harvesting complex II antenna proteins and ribulose 1,5 bisphosphate carboxylase/oxygenase were unaltered. These data indicate that the chloroplast density within photoautotrophic culture cells may be regulated independently of their photosynthetic efficiency.

Insertion Profiles in Stomatal Density and Sizes in Nicotiana Tabacum L. Plantlets

Tobacco (Nicotiana tabacum L. cv. Samsun) plantlets were cultured in vitro on Murashige-Skoog medium photoautotrophically (without sucrose) or photomixotrophically (with 3 % sucrose) under two irradiances [70 or 230 µmol m−2 s−1]. Significant differences in stomatal density and sizes in leaves of different insertion levels (3rd, 5th and 7th leaves from bottom) in photomixotrophic plantlets but not in photoautotrophic ones were found after 35 d of culture. Stomatal density was higher in upper leaves and on abaxial leaf side. Higher irradiance enhanced stomatal density in photoautotrophic plantlets. Stomatal sizes decreased with leaf insertion level but no significant differences between leaf sides were found. Abaxial stomata were more circular than the adaxial ones. In photomixotrophic plantlets stomata tended to be more elongated in the 3rd and the 5th leaves, whereas stomatal elongation in photoautotrophic plantlets was similar in all leaves.

Acclimatization of Micropropagated Tobacco Plantlets

Micropropagated plantlets are grown conventionally under conditions of low irradiance (30–70 μmol photons m-2 s-1), high relative air humidity, variable CO2 concentration (from 2% to compensation concentration) and presence of carbohydrates in the medium (1, 2). The consequences of these conditions may be changes in plant anatomy (e. g., stomata, cuticle — for review see 3) and physiology (e. g., low photosynthetic ability — for review see 2). However, photosynthetic ability and starch accumulation of plants in vitro may favour acclimatization to ex vitro environment (1, 4). Acclimatization of plants after transfer to ex vitro environment is considered for the final, but often critical stage of micropropagation of many plant species. Thus, the success of micropropagation is considerably dependent on in vitro growth. The aim of this study was to clarify whether the presence of sucrose in the medium during in vitro culture affects the longterm development under ex vitro conditions.

Excess irradiance causes early symptoms of senescence during leaf expansion in photoautotrophically in vitro grown tobacco plants

Photosynthetic parameters, growth, and pigment contents were determined during expansion of the fourth leaf of in vitro photoautotrophically cultured Nicotiana tabacum L. plants at three irradiances [photosynthetically active radiation (400–700 nm): low, LI 60 µmol m−2 s−1; middle, MI 180 µmol m−2 s−1; and high, HI 270 µmol m−2 s−1]. During leaf expansion, several symptoms usually accompanying leaf senescence appeared very early in HI and then in MI plants. Symptoms of senescence in developing leaves were: decreasing chlorophyll (Chl) a+b content and Chl a/b ratio, decreasing both maximum (FV/FM) and actual (ΦPS2) photochemical efficiency of photosystem 2, and increasing non-photochemical quenching. Nevertheless, net photosynthetic oxygen evolution rate (PN) did not decrease consistently with decrease in Chl content, but exhibited a typical ontogenetic course with gradual increase. PN reached its maximum before full leaf expansion and then tended to decline. Thus excess irradiance during in vitro cultivation did not cause early start of leaf senescence, but impaired photosynthetic performance and Chl content in leaves and changed their typical ontogenetic course.

Leaf anatomy during leaf development of photoautotrophically in vitro-grown tobacco plants as affected by growth irradiance

Tobacco (Nicotiana tabacum L.) plants were cultured in vitro photoautotrophically at three levels of irradiance (PAR 400–700 nm): low (LI, 60 µmol m−2 s−1), middle (MI, 180 µmol m−2 s−1) and high (HI, 270 µmol m−2 s−1). Anatomy of the fourth leaf from bottom was followed during leaf development. In HI and MI plants, leaf area expansion started earlier as compared to LI plants, and both HI and MI plants developed some adaptations of sun species: leaves were thicker with higher proportion of palisade parenchyma to spongy parenchyma tissue. Furthermore, in HI and MI plants palisade and spongy parenchyma cells were larger and relative abundance of chloroplasts in parenchyma cells measured as chloroplasts cross-sectional area in the cell was lower than in LI plants. During leaf growth, chloroplasts crosssectional area in both palisade and spongy parenchyma cells in all treatments considerably decreased and finally it occupied only about 5 to 8 % of the cell cross-sectional area. Thus, leaf anatomy of photoautotrophically in vitro cultured plants showed a similar response to growth irradiance as in vivo grown plants, however, the formation of chloroplasts and therefore of photosynthetic apparatus was strongly impaired.