M. Ondřej

In vitro culture of crown gall and hairy root tumors ofAtropa Belladonna: Differentiation and alkaloid production

Crown galls and hairy roots were induced inin vitro cultivated seedlings ofAtropa belladonna by differentAgrobacterium tumefaciens andA. rhizogenes strains. During furtherin vitro cultivation, tumors and hairy roots showed high differentiation ability which persisted for at least one year ofin vitro culture. Only root cultures, induced by Ri plasmid A4, synthesized detectable amounts of alkaloids.

Transgenic tobacco plants with T-DNA phytohormone synthesis genes

Agrobacterium tumefaciens binary vectors carrying kanamycin resistance gene and either C58 T-DNA gene 4 for cytokinin synthesis or genes 1 and 2 for auxin synthesis were constructed and used for transformation of a short-day tobacco Maryland Mammoth. Kanamycin resistant plants were regenerated from a small fraction of transformed tissue and the presence of T-DNA in their genome was verified by Southern blotting. The level of endogenous cytokinin in plants transgenic for gene 4 and the level of endogenous IAA in those transgenic for genes 1 and 2 increased by more than 100 %. A number of morphological characteristics distinguish them from untransformed controls.

Mitotic cycle kinetics of root meristems of isolated barley embryos and intact seedlings. Labelling of nuclei by3H- thymidine and its cytogenetic consequences

The duration of the mitotic cycle and its individual phases was estimated in root meristems of isolated barley embryos and intact barley seedlings by means of pulse labelling with3H-thymidine and construction of labelled mitoses curve. The duration of the whole mitotic cycle in the cell population of root meristems of isolated barley embryos cultivated in the aerated liquid complete medium is 12.2 h. The mitotic cycle time of root meristems of intact barley seedlings, oultived in Petri dishes on wet blotting paper is 9.2 h. Most of root meristem cells belong to the fraction of rapidly proliferating cells, but this fraction exerts a high degree of variability by itself. Pulse treatment by3H-thymidine in our experimental conditions (74 kBq ml-1 - or 2 μCi ml-1, exposure 0.5 h) did not induoe any chromosomal aberrations in unlabelled cells and only a very low frequency of chromosomal aberrations in labelled cells. Measuring the cell population kinetics by pulse labelling with3H-thymidine can be used simultaneously with the study of induction of ohromosomal aberrations by mutagens.

Differentiation ofPetunia hybrida tissues transformed byAgrobacterium rhizogenes andAgrobacterium tumefaciens

Petunia hybrida plants were inoculated with differentAgrobacterium rhizogenes andA. tumefaciens strains and developed tumors were further cultivatedin vitro. Transformed flowering plants differentiated from tumors induced byA. rhizogenes strains 8196 and TRIOL Transformed but non-rooted plants developed also from tumors incited byA. tumefaciens T37. Cultures of roots transformed byA. rhizogenes strain 15834 did not show increased incidence of chromosomal aberrations in anaphases in comparison with untransformecl control. Permanent growth of isolated untransformedPetunia roots was not induced by addition of IAA into the medium.

The effects of methyl methanesulphonate on mitosis and chromosomes of excised barley embryos

Excised barley embryos were treated with methyl methanesulphonate and cultivatedin vitro in water or in a nutrient medium. The alkylating compound induced a high frequency of chromosome aberrations observed at anaphase and metaphase and depressed the mitotic division in the root-tips of excised embryos.

Analysis of Progeny of Arabidopsis thaliana Plants Regenerated from Crown Gall Tumors

Summary Seeds of Arabidopsis thaliana plants, differentiated from nopaline and octopine crown galls, were harvested and sown on agar medium in bulk. Approximately 5% of the seedlings of both types showed an abnormal phenotype (dwarf type of growth and/or curled leaves). When their calli were subcultured on medium without growth regulators and cultivated in the light, 47 clones expressed opine synthesizing activities which became detectable after 2–5 subcultivations. Seed progeny of a single nopaline teratoma was tested separately. The differentiated floral axes were completly or partly sterile due to the alternations in floral development. In F 1 and F 2 generations, NpDH activity was found in some of the plants; undifferentiated calli appeared occasionally on roots and stems. These calli showed NpDH activity and they prove the transmission of tumor markers to the F 2 generation.

Comparison of hairy root and crown gall tumors ofArabidopsis thaliana

The phenotype appearance ofArabidopsis thaliana hairy roots and crown galls their teratomas and regenerated plants were compared. Several differences were found, which correlate with T-DNA differences between Ti and Ri plasmids.

Growth requirements ofArabidopsis thaliana crown galls

Crown galls induced onArabidopsis thaliana plants by octopine or nopaline strains ofAgrobacterium tumefaciens were grownin vitro on different media. Dark growth of all tumor tissues was strictly hormone-dependent. In contrast, hormonal autonomy was observed in the light where crown gall calli readily differentiated into teratomas and (sometimes fertile) plants. Differentiating tissues always grew more vigorously than subtended calli. The growth of transformed calli was stimulated by vitamins and partly inhibited by growth regulators in concentrations used for the maintenance of untransformed calli. Crown gall calli, teratomas and sometimes regenerated plants were shown to express lysopine or nopaline dehydrogenase activities.

The growth of isolated barley embryos cultivated under different conditions

Isolated barley embryos were cultivated aseptically in three different complete media. The growth of the primary root of each embryo was measured during four days of cultivation. Embryos were cultivated on three different consistencies of media: on agar plates, on cellulose tissue moistened with the medium and in liquid shaken cultures. The last way of cultivation yielded the highest degree of the growth of roots. Optimum combination of conditions in shaken liquid medium was selected on the basis of systematic study. The growth of roots of isolated embryos under appropriate conditions approaches that of roots of seedlings cultived on moistened blotting paper.

Partial synchronization of cell division in root meristem induced by 5-aminouracil

Abstract5-aminouracil induces a partial synchronization of mitoses in barley, onion and garlic root tips. The highest degree of synchronization has been achieved in garlic where the mitotic index reached the value of about 36%, while in onion and barley the values equalled about 20%. The concentration causing the maximal synchronization in barley (400–750 ppm) was many times higher than in garlic (62.5 ppm) and onion (100 ppm). The occurrence of micronuclei was evaluated in garlic, under the conditions when synchronization was maximal. It was increased nearly tenfold as compared with the control.