Z. Opatrný

The role of some exogenous and endogenous factors in the isolation of protoplasts from potato cell cultures and their recovery in cell colonies

Protoplasts isolated from cell suspension cultures of potato were cultured as far as the level of regeneration of substrains. The influence of various factors (age of the cell culture, mode of precultivation, composition of the enzymatic mixture, incubation period) on the efficiency of isolation as well as the viability of protoplasts, has been studied. Various cultivation procedures have been screened and the effect of especially the hormonal composition of the medium on different phases of the regeneration of cell colonies (regeneration of the cell wall, growth and elongation, cell division) evaluated.

Plant recovery from long-term callus cultures and from suspension culture derived protoplasts of Solanum phureja

Summary Protoplasts were isolated from suspension cultures of Solanum phureja and induced to proliferate by culture in modified Shepard and Totten media. Shoot formation from the protoplast derived calluses could be achieved on media containing zeatin only when the protoplasts were cultivated from the beginning on media containing cytokinin and lacking 2,4-D. The shoots regenerated from long-term callus cultures as well as the protoplast derived shoots developed to whole plants after transfer onto media without growth regulators.

L-Tryptophan Aminotransferase and L-Tryptophan Dehydrogenase, Enzymes of IAA Synthesis, in Normal and Tumorous Tobacco Tissue Cultures

Summary The normal BV-N tobacco tissue culture as well as tumor tissue cultures T-24 (octopine positive) and C-58 (nopaline positive) contain active L-tryptophan aminotransferase (TAT) and L-tryptophan dehydrogenase (TDH), which are the primary enzymes of the indolylpyruvate (IPyA) pathway leading to the IAA synthesis in plants. In both cultures the activity of TAT gradually increased during the whole subcultivation period, whereas TDH activity slowly decreased. The activity changes of TAT and TDH neither coincided with changes of the content of L-trp ( El Bahr et al. 1984), nor of IAA. The regulatory mechanism in the IAA synthesis is thought to be based on the low affinity of L-trp to the enzymes (KM are of millimolar range), limiting the endogenous conversion of L-trp to IPyA. The tobacco tumor tissue cultures differed from the normal ones in their higher IAA level, steeper growth rate and in the kinetics of the investigated enzymes. In crown-gall tissues the pH optimum of TAT was 9.2 as compared to 8.5 of normal tissue, the dissociation of the coenzyme PRP is lowered in TAT of tumor tissue, and finally, the stronger activation of TDH by the coenzyme NADPH in comparison to NADH, which was shown by normal tissue, could not be registered in the crown-gall tissue. Normal tissue cultures as well as crown-gall tissues preserve their ability to synthesize auxin by the IPyA pathway characteristic of eukaryotes. Even if the properties of the enzymes TAT and TDH in crown-gall tissues are modified, it does not seem probable that TAT and TDH represent the key enzymes responsible for the strong increase of the IAA content in the tumor strains.

Changes in L-tryptophan and auxin content during the life cycle of normal and tumorous tobacco cell culture

Summary The relationships between L-tryptophan (L-trp) and IAA levels during the sub cultivation period were studied in two tobacco cell cultures, normal and tumorous. In normal culture the content of L-trp was low at the lag phase (1.5 μ g · g −1 f.w.) and it increase gradually in the course of the exponentional phase of growth (up to 3.5 μ g · g −1 f.w.). In the tumorous culture an additional increase of L-trp content was observed during the lag phase (3.25 μ g · g −1 f.w.), then after a decrease in the exponential growth phase (to 1.7 μ g · g −1 f.w.) the L-trp content increased again (to 3.7 μ g · g −1 f.w.). However, the changes of IAA level were similar in both cultures and did not follow changes in L-trp content. IAA formed a distinct maximum toward the end of the exponential phase which was lower in normal culture (70 ng · g −1 f.w.) and higher in the tumorous expiant (125 ng · g −1 f.w.). In both cell cultures the total content of L-trp was roughly proportional to the increase of the mass of the explant during growth, but the total production of IAA in the culture was not. The correlation between the L-trp and IAA levels during sub cultivation period thus could not be proved. We assume that the IAA level is regulated by a system independent of the free L-trp content in the explant.

Regeneration of chlorophyll chimeras from leaf explants ofNicotiana tabacum L.

Chimeral plants with variegated leaf blades were obtained by induction of organogenesis in primocultures of leaf explants ofNicotiana tabacum L. cv. Burley 49, chlorophyll mutation White Seedling. Only green plants regenerated from primocultures of explants taken from dark green leaf areas of the chimeras. The possibility of a multicellular initiation of chimera regeneration from tissue cultures is discussed.

The effect of pre-cultivation of tobacco tissue culture on enzymatic separation of protoplasts from various cell types

A method of enzymatic separation of protoplasts from long-term tissue culture ofNicotiana tabacum L. is described. The efficiency of this method is dependent on conditions of separation and on the portion of meristematic cells in the tissue culture. This portion can be increased by pre-cultivations of the tissue on medium containing suitable concentration of hormones. The knowledge of the micromorphology of the filamentous culture enables us to investigate the course of release of protoplasts from various cell types. A preferential lysis of cell walls was observed between neighbouring cells in filaments and the fusion of their protoplasts was recorded. The preservation of cell walls which are not in a contact with other cells may be a result of the cell wall heterogeneity.

Anther cultures of maize

The presence of synthetic auxins (2,4-D at a concentration as low as of 0.5 to 5.0 mg I-1, NAA at least at 5 mg I-1) in the cultivation medium was essential for the induction of callogenesis in anther cultures ofZea mays L. The application of IAA was ineffective. Kinetin induced bursting, darkening and a rapid anther necrosis, but at an appropriate concentration ratio with 2,4-D it stimulated pollen maturation at the same time.

The cytokinin-like effect of a lowered temperature on the micromorphology ofNicotiana tabacum L.

The lowering of the cultivation temperature allows one to alter the growth intensity and micromorphology of tobacco cell strains specifically. By a long-term low temperature treatment the effect is deepened, by transferring inocula into normal cultivation temperature it is repaired. Both the growth and morphogenic effects of the low temperature correspond to those of cytokinins, exhibiting even the same strain specificity.

Effect of gamma-radiation on the growth of primary and “long-term” tissue cultures (Nicotiana tabacum L.)

Growth of primary (cv. Wisconsin 38) and long-term (cv. Virginia Bright Italia) tissue cultures obtained fromNicotiana tabacum L. pith was affected by exposure to a single dose of gamma-radiation ranging from 1 to 150 krad. The inhibitory effect was proportional to the applied dose. Changes in the degree of inhibition during the subculture interval were ascertained by quantitative evaluation of the growth response. Primary cultures proved to be several times more radiosensitive than long-term cultures, the capacity to recover from radiation damage of the cultures participating in the resulting manifestation of the effect. The possible reasons for these differences are discussed.AbstractRůst primárních (cv. Wisconsin 38) a long-term tkáňových kultur (cv. Virginia Bright Italia), odvozených z dřeněNicotiana tabacum L., byl ovlivněn gama-zářením, aplikovaným jednorázově, v dávkách 1–150 krad. Inhibiční efekt byl úměrný velikosti použité dávky. Kvantitativním hodnocením růstu kultur byly zjištěny změny v rozsahu této inhibice v průběhu subkultivačního intervalu. Primární kultury vykázaly několikanásobně nižší radioresistenci než long-term kultury, přičemž na výsledném projevu ovlivnění se podílela zotavovací schopnost kultur. Uvažovány jsou možné příčiny této rozdílnosti.

Androgenesis in vitro in anther cultures of chlorophyll mutants ofNicotiana tabacum

The course of androgenesisin vitro was investigated with anther cultures of two chlorophyll mutants of Nicotiana tabacum. A different sensitivity to the hormonal composition of the medium was revealed between the cultures White Seedling and Sulfur; the stimulatory effect of kinetin on the frequency of androgenesis was observed only in White Seedling cultures. In addition to green plants, “aurea” (mutation Sulfur) or “albino” (mutation White Seedling) phenotypes also differentiated in both cultures. The possible causes of variability in the participation of green and mutant forms are discussed.AbstractPrůběh androgenese in vitro byl sledovän v prašníkových kulturäch dvou chlorofylových mutantNicotiana tabacum L. Byla zjištěna rozdílná citlivost kultur White Seedling a Sulfur k hormonálnímu složení media; stimulační efekt kinetinu na frekvenci androgenese byl pozorován pouze u kultur White Seedling. V obou kulturách se kromě zelených rostlin diferencovaly též rostliny fenotypu „aurea” (mutace Sulfur) či „albino“ (mutace White Seedling). Možné příčiny variability v zastoupeí zelených a mutantních forem jsou diskutovány.