Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The Australian almond improvement program was initiated in 1997 to develop improved cultivars that are adapted to local conditions and consumer demands. The program combines molecular techniques along with the traditional approach of controlled hybridisation with mass selection. This research project was carried out to assist the Australian almond improvement program in the areas of micropropagation, cryopreservation, and rapid identification of self-incompatibility genotypes of almond. Micropropagation was accomplished successfully for two commercially important almond cultivars ('Nonpareil' 15-1 and 'Ne Plus Ultra') and an almond/peach hybrid rootstock by culturing shoot tips, about 0.7 cm long with 3 - 5 leaves, on appropriate shoot multiplication media. For 'Nonpareil' 15-1, AP medium with 0.049 uM IBA, 3 uM BAP, 0.058 M sucrose, and 0.7% agar at pH 5.7 was effective. MS medium with 0.049 uM IBA, 5 uM BAP, 0.088 M sucrose, and 0.7% agar at pH 5.7 was suitable for 'Ne Plus Ultra'. For the almond/peach hybrid rootstock, MS medium supplemented with 10 uM BAP, 0.088 M sucrose, and 0.7% agar provided the best shoot proliferation. Shoots of the rootstock, about two cm long, readily produced roots after one week in the dark and two weeks in the light on half strength MS medium supplemented with 2.4 uM IBA, 0.088 M sucrose and 0.7% agar at pH 5.7, with 88.0% rooting efficiency. The two almond cultivars did not readily produce roots, but, at about 1.5 cm long, were micrografted successfully onto the rootstock. These micrografted plantlets were acclimatised and transferred to potting mix with 92% survival. Shoot tips of the two almond cultivars and the almond/peach hybrid rootstock were cryopreserved successfully using a one-step vitrification technique. Three-week-old in vitro cultures were cold-hardened at 4°C on multiplication media (Murashige and Skoog for 'Ne Plus Ultra' and the hybrid rootstock; Almehdi and Parfitt for 'Nonpareil' 15-1) for three weeks. Shoot tips, 2 - 2.5 mm long, were excised and precultured for one day at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond cultivars and 60 min for the almond/peach hybrid rootstock, and then stored under liquid nitrogen (LN) for up to 24 months. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄N0₃ or (NH₄)₂S0₄. Shoot regeneration was usually observed within 2 - 3 weeks. Survival of shoots after thawing varied from 56-80% for 'Ne Plus Ultra', 35-53% for 'Nonpareil' 15-1, and 62 - 82% for the almond/peach hybrid rootstock. Non-vitrified shoots that were stored on basal medium at 3.5-5°C showed good survival up to six months, but thereafter survival decreased rapidly. Cryopreservation has considerable potential for long-term storage of almond germplasm, but future research should be aimed at improving the regeneration of 'Nonpareil' 15-1, the most important commercial cultivar grown in Australia. The genetic stability of almond DNA to both in vitro culture and the cryopreservation process was evaluated by comparing the fingerprints of the DNA from the original orchard trees, from the in vitro cultures before and after cryopreservation for up to 24 months, and from plants regenerated from in vitro cultures. The fingerprints were prepared by initially digesting the DNA with two isoschizomer pairs of restriction enzymes, one of each pair being 'methylation sensitive' and the other 'methylation insensitive', followed by amplification of the digested products using randomly amplified polymorphic DNA (RAPD) with six different 10-mer primers. Changes in methylation were found between the original orchard trees and in vitro cultures, and there was also the possibility that some structural changes may have occurred. However, no methylation or structural changes could be attributed to the cryopreservation procedure. Plants regenerated from the in vitro cultures before and after cryopreservation should be monitored carefully in the future for changes in morphology compared to the original trees. Partial genomic and cDNA sequences of the self-incompatibility alleles S1, S2, S7, S8, S9, S10, S23, and Sf were obtained from Prunus dulcis cvs 'Anxaneta' (S2S9), 'Cristomorto' (S1S2), 'Ferragnes' (S1S3), 'Gabaix' (S5S10), 'Ne Plus ultra' (S/S7), 'Nonpareil' 15-1 (S7S8), 'Primorskiy'(S5S9), 'Ramilette' (S6S23), and IRTA Selection 12-2 (SfSf). Total DNA was extracted from leaves, and cDNA was prepared from total RNA extracted from styles. The partial cDNA sequences of the S1 allele from 'Ferragnes', and the S7 and S8 alleles from 'Nonpareil' 15-1 matched those reported in the literature for the alleles Sb, Sc, and Sd respectively. The sequences of the S1, S2, S7, S8, S9, S10, S23, and Sf alleles found in genomic DNA contained introns of 562, 253, 1,530, 2,208, 1,343, 710, 494, and 662 bp respectively, and partial exons of 510, 537, 489, 498, 486, 495, 489, and 543 bp respectively. In addition, one allele of the Australian cultivars, 'Johnston's Prolific' and 'Pierce', was identified and found to have the same sequence as S23 in 'Ramilette', suggesting that this cultivar may have been an early introduction to Australia from Spain. The exon/intron splice junction sites of all alleles followed the GT / AG consensus sequence rule, and the sequences were found to be highly conserved. Both the length and the sequence of each intron was unique, and a technique of identifying the S-alleles of almond was developed based on primers that targetted the intron sequences. The use of these primers has increased the speed, precision, and efficiency with which the incompatilibity genotypes of almond cultivars can be detected, compared to other published techniques. The primers confirmed the S-allele specificities for 26 out of 30 cultivars for which published information is available, and are currently in use in the Australian almond improvement program to identify incompatibility groups in the breeding progeny. Future work should be directed towards obtaining the sequences of the introns for the remaining known S-alleles, S3 to S6, and S11 to S22. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1051244 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Horticulture, 2002

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The Australian almond improvement program was initiated in 1997 to develop improved cultivars that are adapted to local conditions and consumer demands. The program combines molecular techniques along with the traditional approach of controlled hybridisation with mass selection. This research project was carried out to assist the Australian almond improvement program in the areas of micropropagation, cryopreservation, and rapid identification of self-incompatibility genotypes of almond. Micropropagation was accomplished successfully for two commercially important almond cultivars ('Nonpareil' 15-1 and 'Ne Plus Ultra') and an almond/peach hybrid rootstock by culturing shoot tips, about 0.7 cm long with 3 - 5 leaves, on appropriate shoot multiplication media. For 'Nonpareil' 15-1, AP medium with 0.049 uM IBA, 3 uM BAP, 0.058 M sucrose, and 0.7% agar at pH 5.7 was effective. MS medium with 0.049 uM IBA, 5 uM BAP, 0.088 M sucrose, and 0.7% agar at pH 5.7 was suitable for 'Ne Plus Ultra'. For the almond/peach hybrid rootstock, MS medium supplemented with 10 uM BAP, 0.088 M sucrose, and 0.7% agar provided the best shoot proliferation. Shoots of the rootstock, about two cm long, readily produced roots after one week in the dark and two weeks in the light on half strength MS medium supplemented with 2.4 uM IBA, 0.088 M sucrose and 0.7% agar at pH 5.7, with 88.0% rooting efficiency. The two almond cultivars did not readily produce roots, but, at about 1.5 cm long, were micrografted successfully onto the rootstock. These micrografted plantlets were acclimatised and transferred to potting mix with 92% survival. Shoot tips of the two almond cultivars and the almond/peach hybrid rootstock were cryopreserved successfully using a one-step vitrification technique. Three-week-old in vitro cultures were cold-hardened at 4°C on multiplication media (Murashige and Skoog for 'Ne Plus Ultra' and the hybrid rootstock; Almehdi and Parfitt for 'Nonpareil' 15-1) for three weeks. Shoot tips, 2 - 2.5 mm long, were excised and precultured for one day at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond cultivars and 60 min for the almond/peach hybrid rootstock, and then stored under liquid nitrogen (LN) for up to 24 months. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄N0₃ or (NH₄)₂S0₄. Shoot regeneration was usually observed within 2 - 3 weeks. Survival of shoots after thawing varied from 56-80% for 'Ne Plus Ultra', 35-53% for 'Nonpareil' 15-1, and 62 - 82% for the almond/peach hybrid rootstock. Non-vitrified shoots that were stored on basal medium at 3.5-5°C showed good survival up to six months, but thereafter survival decreased rapidly. Cryopreservation has considerable potential for long-term storage of almond germplasm, but future research should be aimed at improving the regeneration of 'Nonpareil' 15-1, the most important commercial cultivar grown in Australia. The genetic stability of almond DNA to both in vitro culture and the cryopreservation process was evaluated by comparing the fingerprints of the DNA from the original orchard trees, from the in vitro cultures before and after cryopreservation for up to 24 months, and from plants regenerated from in vitro cultures. The fingerprints were prepared by initially digesting the DNA with two isoschizomer pairs of restriction enzymes, one of each pair being 'methylation sensitive' and the other 'methylation insensitive', followed by amplification of the digested products using randomly amplified polymorphic DNA (RAPD) with six different 10-mer primers. Changes in methylation were found between the original orchard trees and in vitro cultures, and there was also the possibility that some structural changes may have occurred. However, no methylation or structural changes could be attributed to the cryopreservation procedure. Plants regenerated from the in vitro cultures before and after cryopreservation should be monitored carefully in the future for changes in morphology compared to the original trees. Partial genomic and cDNA sequences of the self-incompatibility alleles S1, S2, S7, S8, S9, S10, S23, and Sf were obtained from Prunus dulcis cvs 'Anxaneta' (S2S9), 'Cristomorto' (S1S2), 'Ferragnes' (S1S3), 'Gabaix' (S5S10), 'Ne Plus ultra' (S/S7), 'Nonpareil' 15-1 (S7S8), 'Primorskiy'(S5S9), 'Ramilette' (S6S23), and IRTA Selection 12-2 (SfSf). Total DNA was extracted from leaves, and cDNA was prepared from total RNA extracted from styles. The partial cDNA sequences of the S1 allele from 'Ferragnes', and the S7 and S8 alleles from 'Nonpareil' 15-1 matched those reported in the literature for the alleles Sb, Sc, and Sd respectively. The sequences of the S1, S2, S7, S8, S9, S10, S23, and Sf alleles found in genomic DNA contained introns of 562, 253, 1,530, 2,208, 1,343, 710, 494, and 662 bp respectively, and partial exons of 510, 537, 489, 498, 486, 495, 489, and 543 bp respectively. In addition, one allele of the Australian cultivars, 'Johnston's Prolific' and 'Pierce', was identified and found to have the same sequence as S23 in 'Ramilette', suggesting that this cultivar may have been an early introduction to Australia from Spain. The exon/intron splice junction sites of all alleles followed the GT / AG consensus sequence rule, and the sequences were found to be highly conserved. Both the length and the sequence of each intron was unique, and a technique of identifying the S-alleles of almond was developed based on primers that targetted the intron sequences. The use of these primers has increased the speed, precision, and efficiency with which the incompatilibity genotypes of almond cultivars can be detected, compared to other published techniques. The primers confirmed the S-allele specificities for 26 out of 30 cultivars for which published information is available, and are currently in use in the Australian almond improvement program to identify incompatibility groups in the breeding progeny. Future work should be directed towards obtaining the sequences of the introns for the remaining known S-alleles, S3 to S6, and S11 to S22. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1051244 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Horticulture, 2002

Thesis (Ph. D.)--Murdoch University, 2006. / Thesis submitted to the Division of Science and Engineering. Includes bibliographical references (p. 191-207).

Thesis (M.S.)--Virginia Polytechnic Institute and State University. / Vita. Abstract. Includes bibliographical references (leaves 55-60). Also available via the Internet.

Thesis (Ph. D.)--University of Wisconsin-Madison, 1944. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Physiological and morphological aspects of acclimatization were studied in cultured tomato (Lycopersicon esculentum Mill.), banana (Musa accuminata L.) and date palm (Phoenix dactyli/era ). The nutrient availability from agar solidified culture medium was determined to establish the nutrient status of the cultured plandets before transfer to ex vitro conditions. Analysis of the plant tissues demonstrated decreasing tissue concentrations of the major elements nitrogen, phosphorus and potassium with decreasing concentration of basal salts in the medium. The effects of agar and increasing sodium concentration in the culture medium was studied in cultured banana plants. Plandets grown on agar solidified medium with increased levels of sodium, exhibited reduced growth and stomatal movement. The use of agar as a solidifying agent was shown to reduce root growth, development and stomatal functioning in these plants. The efficiency of ion and water uptake, and translocation in in vitro and acclimatized tomato plants was assessed using [32P]-orthophosphate and [3H]_ tritiated water. The functional capacity of the root system fOlmed in vitro was established, and assessed following acclimatization treatments at 40% and 80% relative humidity. Comparative studies with tomato seedlings demonstrated reduced efficiency of ion translocation to the shoot in plandets growing in vitro. However, transport to the shoot improved during acclimatization. Ion absorption studies on in vitro and acclimatized palm plants demonstrated phosphate uptake and translocation in both plant types. A detailed examination of the tissue structure through the root/shoot junction and roots of · cultured, acclimatized and seedling tomato plants illustrated differences in the vascular development between the three plant types. However, no major abnormalities were observed which could have accounted for the reduced translocation efficiency in the cultured plants. Increased vascularization present in the root/shoot junction of the cultured plants may increase resistance to the transpiration flow through the region. The type of root system produced in vitro and the root/shoot ratio was manipulated using varying IAA and sucrose treatments. Improved root development and plantlet survival rates were achieved by reduced exposure to IAA during the root initiation phase followed by root elongation on IAA free medium supplemented with sucrose. Acclimatization at low relative humidity (40%) was achieved by producing plandets with balanced root/shoot ratios and a well developed root system.

No description available.

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

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

Components of the South African indigenous flora are disappearing at an alarming rate, due to pressures on land use. The flora is protected by proclamation of reserves and conservation legislation, however these measures can never be wholly successful. For these reasons, methods for propagting Clivia miniata, Gloriosa superba and Sandersonia aurantiaca using in vitro techniques were investigated. The highly sought after Clivia miniata var citrina can be successfully cultured using fruit and floral explants. Use of these explants may limit the number of plants produced in culture due to the seasonal nature of flowering. Gloriosa superba and Sandersonia aurantiaca can be propagated using corm explants, with subsequent in vitro stimulation of cormlet formation. To establish a successful tissue culture procedure an integrated approach to all aspects of the culture is necessary. Sterilization techniques should be empirical and specific for each species and explant. The most critical factor in establishing a culture technique is the choice of a suitable explant. Without a suitable explant the success of the culture procedure may be severely limited. Nutritional and environmental variation may modify the explant response in culture, but initial culture response can be directly related to the origin of the explant, particularly, size, time of the year, age and physiological status. Since the discovery of colchicine in Gloriosa by CLEWER, GREEN and TUTIN (1915) a number of researchers have put forward the idea that Gloriosa would serve as a source of colchicine. The present trend in biochemical production is via artificial synthesis, however many desirable compounds still have to be extracted from plant material for biochemical production. The utilization of plant cells that are cultured in vitro provides a viable alternative to the problems involved in the production of chemical compounds. Levels of colchicine in Gloriosa and Sandersonia are very similar, in the range of ± 0,9%. From evidence presented by BELLET and GAIGNAULT (1985), levels of colchicine in the two study species is much higher than the recorded level (0,62%) of Colchicum. This higher level of the alkaloid makes these two plants a viable source for commercial colchicine production. Levels of colchicine recovered from in vitro grown roots and callus was 10 - 20 times lower than that found in -in -viv-o tissue. Levels of colchicine extracted from plantlets grown in vitro was the same as that normally recorded for parent tissue. Higher levels of colchicine in malformed roots adds to the evidence that differentiation increases colchicine production in Gloriosa tissue in vitro. It has been shown that Gloriosa and Sandersonia tissue can synthesize colchicine in vitro. The extent to which the cells synthetic capacity can be enhanced has yet to be determined. However, research into speedier and more wide ranging methods for metabolite production in culture is receiving attention throughout the world. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1988.