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1 ABSTRACT Charles University in Prague Faculty of pharmacy in Hradec Králové Department of pharmacognosy Student: Václav Předota Supervisor: PharmDr. Marie Kašparová, PhD. Title of diploma thesis: Plant tissue culture of Juniperus virginiana The derivation of callus cultures from leaves of Juniperus virginiana (varieties Hetzii, Glauca and Grey Owl) and determination of their growth curves were studied in this work. The cultures were cultivated at the temperature of 25 řC and light period of 16 hours light/8 hours dark on the Schenk and Hildebrandt medium with the addition of 3.0 mg.l-1 α-NAA and 0.2 mg. l-1 kinetin. It is clear from the growth curves, that all three varieties reached their maximum in growth on 35th day of the cultivation. The best results were achieved by variety Glauca.

The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro. Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable. When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ?? 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ?? 10)% total soluble protein in the hairy roots. Inoculum virus concentration and the medium in which infection was performed affected the virus yield and the percentage of inoculated cultures that accumulated virus. When cultures were inoculated using 9.0 μg mL-1 TMV, virus accumulated in the biomass to 4.2 ?? 0.60 mg g-1 dry weight. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor. N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ?? 140)-fold less infectious than TMV as measured by local lesion assays. Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium. Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.

Leaf discs of Saintpaulia ionantha x confusa hybrids (African Violets) were cultured and transferred between hormonal free medium (MS basal medium) and shoot-inducing medium (SIM) to determine whether there is a window of competence in shoot regeneration. The results showed that cultured cells were not responsive to shoot-inducing signals (i.e. not competent) until 3-5 days after explant isolation but the ability to regenerate shoots was not lost in surviving cells/tissues cultured on basal medium. Light microscopic observations found that first periclinal divisions of epidermal cells occurred at 3-5 days on SIM. Meristemoids were then formed from the derivatives of the original epidermal cells. It is proposed that cellular competence for shoot regeneration is acquired in culture. The pre-competent period consists of a resting phase and a "dedifferentiation" phase in which epidermal cells divide periclinally to form "dedifferentiated" cells which are the true target cells for shoot induction. Mutagenic treatments and propagation of a chimeral African Violet cultivar were carried out to study cell origin of adventitious shoots in African Violet leaf culture. The results suggest that adventitious shoots may originate from either multiple cells or single cells. The multiple cell origin is in contrast to the hypothesis of exclusively single cell origin for adventitious shoots proposed in several studies. Several factors were studied to identify the source of variations in shoot regeneration among individual explants and to define conditions favourable for shoot regeneration in African Violet in vitro culture. These factors include donor plant growth temperature, the presence of light and the quality of light in culture, the role of chlorophyll in cultured tissues, position of explants in leaves, age of explant materials, source and type of explants, wounding and leaf disc orientation on media. It was found that the presence of light in culture, age of leaf explants, source (in vitro cultured vs pot plant) and type (leaf vs petiole) of explants, wounding and leaf disc orientation on media all had a statistically significant effect on shoot production.

The responses of the tepary bean (Phaseolus acutifolius) to in vitro tissue culture systems were documented. Tests were conducted to identify the optimal auxin and cytokinin combinations required for optimal callus growth. Regeneration experiments were conducted to: (1) determine the effect of explant source and age on regeneration, (2) effect of callus age on regeneration, (3) the cultivation status of the explant source, and (4) the effects of nutritional additives on somatic embryogenesis. The callus was easily induced and maintained in all hormonal medias except those containing IAA and 6BA. The results for regeneration were most promising from cultures derived from immature cotyledon tissue. Ammonium Chloride, glutamine and Absisic acid appeared to have little affect on embryogenesis, however the addition of kinetin enabled the embryos to develop to the torpedo stage. Callus age and cultivative status of explant source had no effects on plantlet regeneration.

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