Simple method to isolate vacuoles and protoplasts for patch-clamp experiments

It was not possible to obtain protoplasts or vacuoles from the thallus of the liverwortConocephalum conicum by applying cell-wall-degrading enzymes. Therefore, a surgical method was developed to isolate protoplasts and vacuoles. A thallus was plasmolyzed and cut. The few protoplasts along the cutting edge that were not destroyed emerged from the edge under deplasmolysis and became thus accessible for a patch pipette. Whereas under slightly hypoosmolar conditions the emerging protoplast remained largely intact, more hypoosmolar conditions gave rise to isolated vacuoles. This method to isolate protoplasts and vacuoles could also be applied to other plant tissues like leaves ofArabidopsis thaliana. Patch-clamp measurements were performed with isolated vacuoles and excised tonoplast patches. A slowly activating vacuolar channel inC. conicum displayed the characteristic features of higher-plant slowly activating vacuolar channels.Abbreviations AP action potential - SV channel slowly activating vacuolar channel     

Conocephalum salebrosum (Marchantiopsida, Conocephalaceae) – a new Holarctic liverwort species

Detailed analysis of 270 specimens of Conocephalum conicum (L.) Dumort., mainly from the POZW Herbarium revealed some diagnostic differences between two cryptic species originally detected on the basis of isozyme studies. Several diagnostic characters were found in the structure of the archegoniophore, sporophyte and sterile thallus. The most reliable diagnostic features are size and structure of stomatal apparatus of the archegoniophore ‘heads’, type of junction of the air chamber walls with epidermal cells, and the thallus surface details. On the basis of morphological and anatomical diagnostic characters, two formerly cryptic species are recognized following the rules of formal taxonomy. The lectotype of C. conicum preserved in FI was found to possess characteristics of cryptic species C. conicum- species L. Accordingly, the species previously named as C. conicum–species L must bear the name Conocephalum conicum (L.) Dumort. No published name was related to C. conicum – species S, which is therefore described as a new species: Conocephalum salebrosum Szweykowski, Buczkowska &; Odrzykoski. Both species are fully described and their diagnostic characters are illustrated. Distribution maps for both species are given.