Spine micromorphology of normal and hyperhydric Mammillaria gracilis Pfeiff. (Cactaceae) shoots.

Artificial conditions of tissue culture affect growth and physiology of crassulacean acid metabolism plants which often results in formation of hyperhydric shoots. In in vitro conditions Mammillaria gracilis Pfeiff. (Cactaceae) growth switches from organized to unorganized way, producing a habituated organogenic callus which simultaneously regenerates morphologically normal as well as altered hyperhydric shoots. In this study, influence of tissue culture conditions on morphology of cactus spines of normal and hyperhydric shoots was investigated. Spines of pot-grown Mammillaria plants and of in vitro regenerated shoots were examined with stereo microscope and scanning electron microscope. The pot-grown plants had 16-17 spines per areole. In vitro grown normal shoots, even though they kept typical shoot morphology, had lower number of spines (11-12) and altered spine morphology. This difference was even more pronounced in spine number (six to seven) and morphology of the hyperhydric shoots. Scanning electron microscopy analysis revealed remarkable differences in micromorphology of spine surface between pot-grown and in vitro grown shoots. Spines of in vitro grown normal shoots showed numerous long trichomes, which were more elongated on spines of the hyperhydric shoots; the corresponding structures on spine surface of pot-grown plants were noticed only as small protrusions. Scanning electron microscopy morphometric studies showed that the spines of pot-grown plants were significantly longer compared to the spines of shoots grown in tissue culture. Moreover, transverse section shape varies from elliptical in pot-grown plants to circular in normal and hyperhydric shoots grown in vitro. Cluster and correspondence analyses performed on the scanning electron microscope obtained results suggest great variability among spines of pot-grown plants. Spines of in vitro grown normal and hyperhydric shoots showed low level of morphological variation among themselves despite the significant difference in shoot morphology.

Isoesterases related to cell differentiation in plant tissue culture.

Normal, habituated and transformed in vitro tissue lines of sugar beet (Beta vulgaris L.), horseradish (Armoracia lapathifolia Gilib.) and potato (Solanum tuberosum L.) were studied with regard to isoesterase patterns. Isoenzymes were separated in gradient gels (5-12%) of polyacrylamide and by isoelectric focussing in pH range 4-9. 1- and 2-naphtylacetate were used as substrates of broad spectrum which cover also esterases (arylesterases and carboxylesterases) reacting with organophosphorous compounds. Distinct isoesterase patterns were noticed in sugar beet normal, habituated and crown gall tumour tissues. Horseradish tumour and teratoma, on the contrary, differed only in one anodic isoenzyme. Even the malformed shoots and unorganised tissue of teratoma had the same patterns. In potato tuber tissue, change in isoesterase pattern, characterised by disappearance of a dominant dark area, was observed during tumour development. The gradient gels gave more stable and reproducible isoenzyme patterns than isoelectric focussing.

Protein parameters of differential gene activation during development and tumorigenesis.

Various models of normal and abnormal developmental systems were addressed to get an insight into molecular parameters of cell differentiation at the level of protein gene products. Electrophoretic analysis of heterogeneous protein mixtures permitted qualitative analysis of developing systems, particularly during organogenesis in mammals, as well as of neoplastic growth in the animal and plant kingdoms. From our earlier findings indicating that the definite protein patterns characteristic of adult organs are acquired long after the adult morphological and histological characteristics of these tissues have developed, it has been repeatedly proven that quantitative changes in whole proteins is not a dependable indicator of cell differentiation.

Peroxidase as a developmental marker in plant tissue culture.

Peroxidase was studied as a developmental marker in pumpkin (Cucurbita pepo L.) callus lines and horse-radish (Armoracia lapathifolia Gilib) transformants. Embryogenic callus lines DE grown on MS medium with 2.4-D and NA-3 grown on medium with NAA and adenine sulfate showed about a 20 times higher enzyme activity than the habituated non-embryogenic line Z5b/T grown on medium without hormones. A rise in peroxidase activity indicated that somatic embryogenesis was triggered in a few habituated tissue cultures. Separated globular embryoids had a manifold lower enzyme activity than the callus from which they originated. SDS-electrophoresis showed distinct polypeptide patterns between the horse-radish leaves and crown galls, but the tumor characteristic protein bands failed to be identified. In horse-radish crown galls and short bushy plants regenerated from hairy roots an enhanced peroxidase activity was registered. Due to its high peroxidase level and abundant biomass production horse-radish transformants should facilitate enzyme production.