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.

Acclimatization of Micropropagated Plants to Ex Vitro Conditions

The primary issues regarding the lack of protocol reproducibility among laboratories are environmental factors. Light (quantity and particularly quality), is one of those main factors, and studies seldom present the spectral quality of the light sources used. With the advent of light-emitting diode (LED) technology, impressive progress has been made in environmental controls and morphogenetic responses, as directed by the light used in the culture shelves. A wide array of LED lights with different spectra are currently available and light is important in large-scale propagation, especially liquid bioreactor systems. LED technology continues to evolve rapidly and has created additional possibilities. This laboratory has dedicated extensive efforts to implement photoautotrophic propagation, and light is a key component of the system. This review presents relevant topics on the influence of light in various plant tissue culture-based techniques.

Light quality in plant tissue culture: does it matter?

The effects of ozone treatment (200 ppb, 3 h) on the accumulation of main secondary metabolites have been investigated in Melissa officinalis (lemon balm) aseptic shoot cultures in order to evaluate the biotechnological application of this gas for improving the yield of secondary metabolites of medicinal plants During the treatment, we found (1) an activation of enzymes involved in phenolic metabolism [as confirmed by the increase of shikimate dehydrogenase, phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase activities (about twofold higher than controls)], (2) a development of cellular barriers with a higher degree of polymerization of monolignols [as indicated by the increase of lignin (+23 % compared to controls)], (3) an accumulation of phenolic compounds, in particular rosmarinic acid (about fourfold compared to control plants cultivated in filtered air) and (4) an increase of antioxidant capacity (as documented by the improved 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity) The effect of ozone as elicitor of the production of secondary metabolites in lemon balm shoot cultures was dependent on the specific regime, the time of exposure and the concentration of the stressor After the end of the treatment, we found cell death and hydrogen peroxide (H2O2) deposition concomitant with a prolonged superoxide anion-generation, suggesting that a transient oxidative burst had occurred

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Ozone-elicited secondary metabolites in shoot cultures of Melissa officinalis L.

In the past, members of the Cactaceae family were mostly propagated by seeds or vegetatively by cuttings and grafting. Seeds, however, do not guarantee genetic stability and, in the case of some cacti, seeds are difficult to obtain, their germination rates are low, or they need to be stored for a long time in specific light and temperature conditions. Traditional vegetative reproduction in vivo, on the other hand, may be less efficient insofar as a limited number of plants can be obtained. Major issues associated with traditional propagation methods may be solved by the use of plant tissue culture. Today micropropagation techniques are applied in order to produce large numbers of new high-quality plants in a relatively short time and space. This is especially important for endangered and desirable species such as cacti. This paper discusses the achievements, current state and future prospects of cactus micropropagation methods. We also provide an overview of cactus multiplication by areole activa...

Micropropagation of Cacti—a Review

This work reports the leaf morphology of six grapevine genotypes, five belonging to Vitis vinifera and one to Vitis ripari a. Earlier studies on these genotypes showed different levels of susceptibility to grapevine downy mildew ( Plasmopara viticola ). The aim of this work was to detect differences between the leaf morphology of these cultivars at the macro- and microscopic levels, and to characterize morphological traits which could be associated with susceptibility and resistance to downy mildew. An ampelographic description of each genotype was used to develop a scheme illustrating the characteristic leaf morphology. The density and morphology of the trichomes and the stomatal index was assessed by means of microscopical techniques. Distinct macro and microscope differences among the genotypes were seen. No clear relation between ampelographic characteristics and susceptibility to downy mildew was observed. The two cultivars that in earlier studies were found to be the least susceptible to downy mildew were the most similar in terms of their spongy mesophyll. Both showed very little or no wax on the abaxial surface of their leaves.

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Macro- and microscopic leaf characteristics of six grapevine genotypes ( Vitis spp.) with different susceptibilities to grapevine downy mildew

To improve photosynthesis and growth of grapevines cultivated in vitro (Seyval blanc and SO 4) effects of light intensity, spectral irradiance and CO 2 concentration on stomatal behaviour, CO 2 fixation and photorespiration were studied. Stomata were shown to respond to changes of light intensity but, unlike photosynthesis, their reactions were delayed and stomatal closure was incomplete in the dark. In contrast, alterations of the CO 2 concentration in the headspace (50-2200 ppm) did not cause stomatal reactions. Photosynthesis vs, light intensity relationships indicated lower light compensation points, higher quantum yield and higher rates of light-saturated photosynthesis with ''Fluora'' lamps (maximal spectral irradiance al 460 and 680 nm) compared to ''projector'' lamps (maximal spectral irradiance at 620 nm). Photosynthesis vs. intercellular CO 2 concentration relationships indicated varietal differences, the carboxylation efficiency and rates of photosynthesis at CO 2 saturation being distinctly higher in the more vigorous variety SO 4 compared to Seyval blanc. Under the usual light conditions of our in vitro culture (50-60 µmol quanta · m -2 s -1 , Fluora) the headspace CO 2 concentration ranged from 145 to 155 ppm while at the end of a 10-hour dark period it increased to values >3000 ppm. Rates of photorespiration were high (>50 % of photosynthesis) due to the relative low CO 2 concentrations and, presumably, due to elevated O 2 concentrations in the headspace. It is concluded that the often observed low rates of photosynthesis of in vitro plantlets are mainly due to low light intensity and CO 2 concentration in the headspace, the latter depending on the low rates of gas diffusion between ambient air and headspace.

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H. Düring

Papers

Stomatal behaviour, photosynthesis and photorespiration of in vitro -grown grapevines: Effects of light and CO 2