No abstract

The present work examined seven Hypericum species (Н. perforatum, Н. humifusum, Н. kalmianum, Н. annulatum, Н. tomentosum, Н. pulchrum, and H. rumeliacum) produced in vitro and regenerated after cryopreservation. The aim of the study was to assess, by means of light microscopy (LM) and transmission electron microscopy (TEM), the effect of freezing temperature on leaf histological organization and mesophyll chloroplast ultrastructure. Histological analysis showed a negative effect of ultralow temperatures on leaf tissue structure in Н. pulchrum and a positive effect in Н. perforatum. The TEM analysis showed that chloroplasts from ultralow temperature treated H. annulatum, H. tomentosum, and H. rumeliacum had a typically structured internal membrane system without destruction of thylakoid membranes, however, those of Н. humifusum had very high grana, and in Н. perforatum, chloroplast thylakoid destruction occurred. The chloroplast internal membrane system of in vitro cultured control plants and in vitro cultured cryopreserved plants of Н. kalmianum and Н. pulchrum had a specific spatial orientation without any destruction of the membranes. The results show a specific response of each species to these experimental conditions.Additional key words: chloroplast ultrastructure, in vitro culture, leaf histology.

The ultrastructure of leaf mesophyll cells of in vitro cultured Hypericum perforatum L. plants regenerated after cryopreservation was studied. Electron microscopy analysis revealed that the chloroplasts in plants pretreated with abscisic acid and regenerated after cryopreservation were round, with increased amount of starch, rather small volume of the thylakoid system, and destroyed envelope. Plants pretreated with 0.3 M mannitol and cooled at rates of 0.1 or 0.3 °C min -1 possessed chloroplasts with high starch content that resulted in a reduction of a membrane system. However, the pretreatment with 0.3 M mannitol and cooling at a rate of 0.2 °C min -1 was the best as chloroplast ultrastructure resembled the controls regenerated without cryopreservation.

The leaf structure and chloroplast ultrastructure of kidney tea (Orthosiphon stamineus Benth.) was studied in in vitro culture on standard MS medium supplemented with or without plant growth regulators (PGRs). The cytokinin N 6 -benzyladenine (BA) negatively affected the structure of the palisade parenchyma and chloroplast ultrastructure and increased the stomatal frequency of the adaxial epidermis. The auxin indole-3-butyric acid (IBA) did not modify the morphology of regenerated leaf tissues as well as the chloroplast ultrastructure. The effect of both PGRs applied in combination was manifested in well-differentiated mesophyll parenchyma, typical chloroplast ultrastructure and increased stomatal frequency on both leaf surfaces. This protocol can be suggested for further ex vitro propagation.

The ultrastructure of cotton leaves, exhibiting reddening as symptom of physiological disorder, was examined by means of transmission electron microscopy. Osmiophilisation of the membrane compartment was established. Massive agglomerations on the tonoplast in the vacuole of cells under the adaxial epidermis were observed, and were referred to as electron-dense osmiophilic substance, most probably of anthocyanin nature. In chloroplast stroma a zone of low electron density enclosing numerous osmiophilic aggregations of unclear chemical character was differentiated. Fragmentation and severe destruction of thylakoids in chloroplasts of reddening cotton leaves was not detected.