M.Sc. (Botany) / Please refer to full text to view abstract

De novo shoots, roots, and flower buds were regenerated on thin cell layer explants excised from pedicel tissue of tomato. Direct shoot organogenesis was greatest when media contained 10µM kinetin and 0.001µM indole-3-acetic acid (IAA); however, shoot regeneration was increased in subsequent experiments by substituting 10 µM zeatin or 10 µM benzyladenine for kinetin. Root formation occurred when media contained higher (0.1 and 10µM) auxin concentrations. Flowers were formed on elongated shoots with several leaves when media contained 10µM IAA and 0.1µM kinetin. Competence for de novo shoot morphogenesis was tested on thin cell layers of eleven tomato cultivars. All tomato cultivars formed shoots directly on thin cell layer explants at varying frequencies (29%-63%). The mean number of shoots per explant was greatest for 'Large Red Cherry', 'Ohio 7814' and 'BL 6807', and poorest for 'Campbell 1327' and 'Red Alert'. Active cell divisions were observed in subepidermal cells during the first week of culture, and meristematic centers of dividing cells were evident after 2 weeks. Well developed shoot apices were observed on 50% of the explants 4 weeks after culture initiation. Shoot morphogenesis was compared among tomato plants placed into micropropagation, callus, and thin cell layer tissue culture systems. More shoots were produced on thin cell layer explants than on cotyledon calli, or micropropagated shoot tips. Genetic recombination rates and map distances were compared among hybrid plants grown in the greenhouse and regenerated from the aforementioned tissue culture systems. Increased recombination rates and map distances were detected between the sunny (sy) and baby leaf syndrome (bls) genes on chromosome 3, and between the white virescence (wv) and anthocyanin reduced (are) genes on chromosome 2. The percent change in the former ranged from 4.5%-5.9% for micropropagated shoot tips, 3.7%-8.5% for plants from cotyledon calli and 2.8%-5.9% for plants from thin cell layers. The percent change between the wv and are loci ranged from 4.5%-6.1% for micropropagated shoot tips, and 3.2%-5.0% and 3.9%-5.7% for plants from cotyledon calli and thin cell layers, respectively. Conversely, a decreased map distance was observed between bls and the solanifolia (sf) locus which is more distal to the centromere on the same arm of chromosome 3 as bls. Changes in recombination rates among plants regenerated from tissue culture may result from an influence of the tissue culture process on meiosis of regenerated plants. / Ph. D.

Propagation by tissue culture is effective for many woody ornamental plants, but propagules often become desiccated and die during the acclimatization period. This loss is due in part to stomata that fail to close in response to the reduced humidity outside of the culture environment. KCl was used in in vitro treatments to determine if an additional K supply would improve microshoot stomatal function and water status during acclimatization. The effects of the KCl treatments upon subsequent microshoot rooting and percent fresh weight gain were also evaluated. In preliminary experiments, microshoots of the flame azalea (Rhododendron calendulaceum) were subcultured onto modified Woody Plant Medium amended with a wide range of KCl concentrations for various time periods. It was determined that microshoots did not grow well when cultured at in vitro KCl levels above 50 mM, so treatments were adjusted to 0, 30, and 60 mM KC1, with 9 days of in vitro exposure. After treatment, percent tissue K was determined by atomic absorption spectrophotometry and microshoot water potentials were measured by thermocouple psychrometry. The capacity for the microshoots to resist desiccation after in vitro KCl treatment was determined by percent rooting and fresh weight gain after exposure to dehydration stress, and by gravimetric weight loss of microshoots placed in isopiestic tubes. In addition, microshoots from in vitro KCl treatments were evaluated for percent stomatal closure and water potential during a 38-day acclimatization period. In vitro KCl treatments induced elevated tissue K levels in microshoots and reduced microshoot water potentials, but it could not be shown that these effects specifically enabled the microshoots to resist desiccation. Rooting and percent fresh weight gain were not affected by in vitro treatments, nor was gravimetric weight loss. Microshoot maturation, as a function of days out of culture, had the greatest effect upon increased stomatal function, which, coupled with the onset of rooting, improved microshoot water status. / Master of Science

Call number: LD2668 .T4 1984 B46 / Master of Science

Tissue culture and radicle excision techniques were employed to evaluate salt tolerance in alfalfa (Medicago sativa L.). Plant suspension cultures of either seedling root or shoot origin were studied in media with or without supplemental NaCl (3.54 g liter⁻¹). In most cases, the growth rates of root-derived cultures were stimulated by this low level of supplemental NaCl while most shoot-derived cultures were not stimulated by NaCl. Excised radicles of three populations of alfalfa which possessed widely differing ranges of germination salt tolerance were screened in four salts (NaCl, KCl, Na₂SO₄, and K₂SO₄) at six varying concentrations. As was observed in the tissue culture experiments, low levels of NaCl (7.09 g liter⁻¹) stimulated radicle elongation of all populations as compared to the elongation levels of the control solutions (no supplemental salts). In general, for NaCl, the population that posessed the highest degree of germination salt tolerance (Az-St 1982) also displayed the greatest rates of radicle elongation especially in the highest salt concentrations. Additionally, this population along with the moderately germination salt tolerant population (Az-ST 1979) maintained higher rates of elongation in KCl, K₂SO₄ and Na₂SO₄ than did the control germplasm which has little germination salt tolerance (Mesa Sirsa Control). Examinations of each individual population in all four salts simultaneously, indicated that the sulfate salts reduced radicle elongation to a greater extent than did the chloride salts. Evaluation of both osmotic effects and specific ion effects showed that the specific ion effects attributed to the anions were more detrimental to radicle elongation than were the osmotic effects.

An in vitro system of generating protoplasts from their callus cultures was established. The friable callus was more productive in terms of producing protoplasts than the green compact callus. The concentration of the various cell wall degrading enzymes had an effect on the viability of the protoplasts in the medium. The protoplast system developed from the experiments was stable and could be used for the transformation experiments of Albizia lebek and for other plant improvement practices.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Vendula Damborská Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: The Optimization of the growth of the plant tissue culture of Juniperus virginiana The main goal of this diploma thesis lies in the optimization of cultivation medium composition for the tissue culture of Juniperus virginiana (varieties 'Hetzii', 'Glauca', 'Grey Owl'). The best results of all tested media were achieved by using Schenk and Hildebrandt medium with an addition of α-NAA (3.0 mg.l-1 ), kinetin (0.2 mg.l-1 ) and ascorbic acid (15.0 mg.l-1 ). The cultures were cultivated at the temperature of 25 řC and light period of 16 hours light/8 hours dark. The growth curve for the tissue culture of Juniperus virginiana 'Glauca' was set for 2 different cases: with and without the addition of phenylalanin (biogenetic precursor of phenylpropans biosynthesis). The maximal growth was reached on 35th day of the cultivation in both cases, phenylalanin increases the growth rate by 29.65 %.

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Hana Novotná Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Secondary metabolites of plant tissue culture of Trifolium pratense L. Explant cultures are perspective sources of secondary metabolites. Nevertheless production of flavonoids and isoflavonoids by the suspension culture of Trifolium pratense L. is not high. Elicitation is one of the methods used to enhance the biosynthesis of secondary metabolites. Elicitation induces physiological changes, stimulates defensive or stress-induced reactions in plants and subsequently triggers the synthesis of secondary metabolites. The objective of this study was to observe the influence of two elicitors - abscisic acid and ascorbic acid - on the production of flavonoids and isoflavonoids by the Trifolium pratense L. suspension culture (Sprint variety). The culture was cultivated in Gamborg medium to which 2 mg.l-1 of 2,4- dichlorophenoxyacetic acid and 2 mg.l-1 of 6-benzylaminopurine were added, at the temperature of 25 řC and 16 hours light / 8 hours dark period. The best elicitation effect of abscisic acid on the production of flavonoids and isoflavonoids was observed after a 6-hour application of the highest 500 µmol.l-1...

Call number: LD2668 .T4 1986 R35 / Master of Science / Horticulture, Forestry, and Recreation Resources

A mist reactor was used to study plant growth and development under various environmental conditions towards the production of healthy plantlets ready for soil transplant in one step from inoculation. In addition, a 3D type of cultivation via surface attachment of explants to vertically hanging strips inside the mist reactor was also investigated to maximize productivity with minimal footprint. Using carrot as the model species, pre-embryogenic cell suspensions were successfully spray-inoculated onto hanging poly-L-lysine (PLL)-coated nylon mesh to which they then attached and remained for several weeks while they developed into rooted plantlets. To study single step micropropagation from shoot explants to fully acclimatized plantlets, Artemisia annua was used as the model species. Nodal cuttings of A. annua were inoculated onto PLL-coated mesh strips by briefly immersing the strips in the suspension of nodal cuttings. Investigation of medium, phytohormones, CO2, ventilation level and humidity ensued resulting in selection of a preferred final process that reduced physiological aberrations like hyperhydricity and was time efficient. The nodal cuttings that attached to the strips were first misted with half strength shooting medium for 7 days to develop new shoots. Then the new shoots were misted with the rooting medium supplemented with NAA for 12 days to develop roots. Rooted plantlets were acclimatized in the same rooting medium for 9 days to acquire fully functional stomata prior to planting into soil. Taken together this study suggested that fully developed plantlets ready for planting into soil could be obtained in a single step in a bioreactor from embryogenic cells or from nodal explants.