Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

An in vitro technique was adapted for screening the ability of Rosa hybrida L. genotypes to form embryogenic callus to elucidate the inheritance of this ability. Filament and leaf petiole explants of modern rose cultivars 'Tournament of Roses' and 'Baby Love' were cultured on somatic embryogenesis induction media and evaluated for the ability to produce embryogenic callus. Cultures of 'Tournament of Roses' produced somatic embryos at a much higher frequency versus 'Baby Love' that produced no embryos. Subsequently, filament explants of eleven 'Tournament of Roses' x 'Baby Love' progeny genotypes were cultured on somatic embryogenesis induction media and evaluated for the ability to undergo somatic embryogenesis. The progeny genotypes produced somatic embryos at varied frequencies. The results obtained indicated that the ability to undergo embryogenesis in Rosa hybrida L. is heritable in an additive fashion with the involvement of more than one gene.

An in vitro technique was adapted for screening the ability of Rosa hybrida L. genotypes to form embryogenic callus to elucidate the inheritance of this ability. Filament and leaf petiole explants of modern rose cultivars 'Tournament of Roses' and 'Baby Love' were cultured on somatic embryogenesis induction media and evaluated for the ability to produce embryogenic callus. Cultures of 'Tournament of Roses' produced somatic embryos at a much higher frequency versus 'Baby Love' that produced no embryos. Subsequently, filament explants of eleven 'Tournament of Roses' x 'Baby Love' progeny genotypes were cultured on somatic embryogenesis induction media and evaluated for the ability to undergo somatic embryogenesis. The progeny genotypes produced somatic embryos at varied frequencies. The results obtained indicated that the ability to undergo embryogenesis in Rosa hybrida L. is heritable in an additive fashion with the involvement of more than one gene.

Micropropagation was investigated in order to develop protocols for rapid mass production of shoots ofSorbus aucuparia and Alnus glutinosa. Removal of apical dominance either physically by pruning the plantlets or chemically by using anti-auxins TIB A (2,3,5-triiodobenzoic acid) and NPA (1-naphthylphthalamic acid) was investigated. There was a 6-fold increase in the number of rooting-ready shoots of S. aucuparia produced by the pruning of plantlets grown in vitro. A. glutinosa however, needed more drastic measures to remove apical dominance and block the endogenous auxin transport. Incorporation of TIBA (3 μm) in the medium produced an initial 8- fold increase in the number of shoots. However, repeated subculture of shoots of Alnus on TIBA containing medium proved detrimental to shoot multiplication. There was 100% rooting of shoots of S. aucuparia on agar solidified medium. The auxin: cytokinin ratio of the multiplication medium played an important role in the rooting ability of shoots. A. glutinosa also had 100% rooting on agar solidified medium. Plants were acclimatised in Baumgartner vessels before transferring to soil. There was 100% rooting and survival of the shoots of A. glutinosa both after transfer to Baumgartner vessels and subsequent transfer to soil. In S. aucuparia the survival rates in Baumgartner vessels was 70% and after transfer to soil was 65%. Direct somatic embryogenesis from zygotic tissue of both S. aucuparia and A. glutinosa was achieved. Embryos of S. aucuparia were produced on medium containing MS salts and vitamins supplemented with 1 μM BAP, 1 μM kinetin, 0.5 μM NAA, 250 mg/L L-glutamine and 500 mg/L casein hydrolysate. A. glutinosa embryos were obtained on medium containing salts and vitamins of Driver and Kuniyuki (1984) supplemented with 3 μM BAP. No auxin was required. Adventitous shoot regeneration from leaves of S.aucuparia was also achieved at a frequency of 40% on medium containing MS salts and vitamins supplemented with 10 μM TDZ and 1 μM NAA. A method for chromosome doubling of S. aucuparia using 15 uM pronamide to treat shoot tips immersed in a semi-solid medium was developed. After 14 days of treatment, 86.5% treated shoots survived and there was 44.5% chromosome doubling of the survivors. The tetraploid shoot had a higher rate of multiplication than the diploid shoots. The involvement of extracellular proteins in direct somatic embryogenesis of Prunus 'Colt' was studied. Changes in the expression of proteins were observed from the first day of transferring the tissue to embryo induction medium. Most changes were seen on the days 28 and 35 when the embryos became visible.

Successful efficient whole cauliflower plant regeneration via somatic embryogenesis from root derived callus tissue was achieved. The research confirmed for the first time the capability of mass production of cauliflower somatic embryos through the indirect pathway. The best callus induction and proliferation was on semi solid Murashige and Skoog (MS) medium supplemented with 2, 4-D at 0.15 mg L-1 and Kinetin at 0.1 mg L-1 and 3% sucrose. The response of different explant types (cotyledon, hypocotyls and root) through callus induction and subsequent culture was determined. The best period for subsequent callus culture was 21 days. Continuous immersion in agitated liquid medium technique was subsequently used for primary somatic embryo production. The culture requirements were empirically optimized including: explants source and size of callus tissue, blending duration, plant growth regulator combinations and concentrations as well as carbohydrate type and concentration. The highest mean number of somatic embryos (30.9) per explant was achieved using root derived embryogenic callus tissue on MS medium provided with IAA 0.05 mgL-1 and Kinetin at 0.5 mgL-1 and 2% sucrose. Somatic embryos were developed and matured on this medium and germinated with the highest percentage (60%) on semi-solid MS medium devoid of growth regulators. The culture conditions that led to the formation of secondary somatic embryos were identified. The presence of activated charcoal in the culture medium had an effect on this process but some abnormality of secondary somatic embryos was observed. Artificial seeds were produced by encapsulating the somatic embryos with a sodium alginate gel (2%) and complexing with calcium chloride (100 mM) for 20 min. The ability of these artificial seed for germination was evaluated using various combinations of plant growth regulators that were either incorporated in the artificial matrix or in the germination semi-solid culture medium. It was confirmed that cauliflower root derived embryogenic callus tissue can be cryopreserved following a preculture-dehydration technique. Following cryopreservation, embryogenic cultures can proliferate in agitated liquid medium, and somatic embryos at the globular stage were formed. Also cold storage at 5 °C in the dark was used successfully to store cauliflower callus tissue for three months without diminution of the competence for somatic embryos formation. This ability for cold storage could have a positive effect in reducing costs and efforts that result from subsequent sub-culture. The encapsulation-dehydration technique was assessed for cryopreservation of somatic embryos but failed to lead to survival of any embryos. Somatic embryos that were produced in this study were able to be well acclimated using a reliable weaning procedure that achieved high rates of survival of plantlets and their subsequent growth to normal plants in the field was assessed. Morphological characteristics of somatic plants compared favourably with zygotic plants but although there was phenotypic similarity, some differences in plant height, curd size and time for curd maturity were observed.

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Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008. / Committee Chair: Dr. Sheldon May; Committee Member: Dr. Donald Doyle; Committee Member: Dr. Gerald Pullman; Committee Member: Dr. James Powers; Committee Member: Dr. Nicholas Hud.

The objectives of this thesis were to trace the developmental pathways of somatic embryos of an abscisic-acid independent line of Larix x eurolepis. to catalogue the phenotypes of mature embryos, to determine critical stages of development and to attempt to increase the number of maturing somatic embryos. The low rate of maturation could not be entirely explained by differences in phenotypes of early embryos, critical stages of development, or the lack of plant growth regulators in the medium. In addition, the shape and epidermal type of the mature embryo did not always determine the type of epicotyl produced, nor did it affect the rooting and mortality rates. Six types of embryonal structures were evident in the aggregates of line 2086: (1) a smooth (SEMLS) or (2) rough (REMLS) embryonal mass subtended by a cylindrical, compact, long suspensor. (3) a rough embryonal mass subtended by a long, loose suspensor (REMLLS). (4) a rough embryonal mass subtended by a suspensor arising from greater than one quarter of the surface area of the embryonal mass (REMST). (5) a rough embryonal mass subtended by a short, compact, cylindrical suspensor (REMSS). and (6) a cluster of meristematic cells which may or may not have single suspensor cells attached (MC). For isolated embryonal structures of all types, to continue development into a nodule or a mature embryo was the least common fate, while proliferation and developmental arrest were more common. In general, the more organized embryonal structure types (SEMLS and REMLS) had higher rates of maturation compared to the other 4 types but the most common fate was still developmental arrest (74% SEMLS. 62% REMLS), followed by proliferation (10% SEMLS. 30% REMLS), and nodule or embryo development (16% SEMLS. 9% REMLS). REMLLS and REMST embryonal structures became developmentally arrested or proliferated (43-47%) while the rate of nodules/mature embryos production was 9-11%. Neither individual REMSS nor MC structures produced any nodules or mature embryos, but REMSS had a lower rate of developmental arrest (81%) and a higher rate of proliferation (19%) than MC (89% and 11% respectively). Embryos at more advanced stages of development were less likely to die, become developmentally arrested or become nodules, but more likely to become mature embryos than embryos at less advanced stages of development. A critical stage of development appeared to be the focal zone stage at the formation of a complete polyphenol band around the basal end of the embryonal mass. At this stage, the majority of immature embryos became mature embryos (61%) while only 3% of the embryos died. 10% became developmentally arrested, and 20% became nodules. The majority of mature somatic embryos were normally proportioned with a smooth epidermis (43%) rather than vitrified (12%). normal with a rough epidermis (12%) or misshapen (smooth or rough. 33%). The shape of the mature embryo was associated with the type of epidermis, with mature somatic embryos with normal proportions more likely to have smooth epidermis (78%) than a rough epidermis (22%) while mature embryos with abnormal proportions were as likely to have a smooth epidermis as a rough epidermis. The shape of the mature embryo was associated with the shape of the epicotyl produced. Normal-smooth, mature embryos were more likely to produce normal-smooth epicotyls (73%) than twin epicotyls (21%), vitrified epicotyls (2%) or misshapen epicotyls (5%) compared to vitrified mature embryos (42% normal-smooth epicotyls, 34% twin epicotyls, 23% vitrified epicotyls, 1% misshapen epicotyls) or misshapen mature embryos (22% normal-smooth epicotyls, 47% twin epicotyls, 7% vitrified epicotyls, 24% misshapen smooth/rough embryos). The number of mature embryos which germinated or died was not associated with either the epidermal quality or the shape of the mature embryo. Few SEMLS or REMLS embryonal structures responded to auxin and cytokinin treatments. There appeared to be a trend towards less developmental arrest and proliferation and more nodules/mature embryos produced on media with no auxin compared to media with 2,4-D and a trend towards more developmental arrest and fewer nodules/mature embryos on media without BA compared to media with BA. Only nodules on media without plant growth regulators produced roots or cotyledons. There was no effect of embryonal structure type (SEMLS or REMLS), or sucrose concentration (58 μM or 174 μM) on the maturation of immature embryos, but on media without ABA, fewer immature embryos proliferated or became developmently arrested and more embryos became nodules or mature embryos than on medium with 6-24 μM ABA. / Graduate

This study focused on induction of somatic embryogenesis of Magnolia spp. and cultivars utilizing leaf and seed (immature and mature) tissues with attempted micropropagation experiments. In a preliminary experiment, direct embryo regeneration was successful in a single leaf tissue of M. ‘Yellow Bird’. After various micropropagation experiments, microshoot proliferation rates decreased. As a result of minimal leaf material, mature seeds were utilized but had contamination issues. Subsequent experiments utilized immature seeds. M. ‘Leonard Messel’ and M. stellata had significantly greater embryo regeneration rates and M. ‘Rosea’, M. stellata, and M. kobus had greater callus induction rates. Woody Plant Medium had significantly greater rates of embryo regeneration as compared to Yellow Poplar medium. Further experimental measures including various collection times of immature seeds are necessary for an efficient regeneration protocol to support potential research utilizing floral-inducing genes to induce rapid breeding cycles for selection of magnolias with diverse floral characteristics.