Date palms (Phoenix dactylifera L.) and papayas (carica papaya L.) are two commercially important plantation crops. Their economic potential in South Africa and worldwide is increasing. However, due to disease, pests and socio-economic reasons, planting material is in short supply. Micropropagation provides a method for rapidly propagating selected superior cultivars for commercial and environmental interests. A satisfactory process for the regeneration of elite cultivars should result in individuals phenotypically and genetically identical to the explant from which they were derived. However, due to somaclonal variation generated during in vitro culture, the true-to-typeness is questionable. For this reason a southern African survey for off-types on date palms produced using somatic embryogenesis was conducted. Plant growth variations such as leaf variegation, seedless fruit, broad leaves, compact growth habit and parthenocarpic fruit were recorded and possible explanations for each phenomenon given. Factors influencing the date palm initiation process such as decontaminating agents, plant growth regulators, explant type and nurse cultures were investigated. A double decontamination process with 2.6% and 1.3% sodium hypochlorite was most effective at reducing contamination. Alternative plant growth regulators, TIBA and NAA were ineffective as a substitute to 2,4-D for somatic embryogenesis. The size of the explant and "nurse cultures" played an important role in explant growth and initiating callogenesis. A "nurse culture" reduced the time in culture significantly. The problem areas in the three commercial tissue culture techniques used for date palms were outlined. In the second part of the study, factors influencing initiation, multiplication and rooting of papaya were determined. Presoaking with antibiotic, Rifampicin, and various fungicides had a positive effect on decontaminating papaya explants, while Bronocide™ had little effect. Various methods and materials were used to optimize papaya multiplication and rooting in vitro. The growth and multiplication of papaya was optimal at 50 g l ¯¹ sucrose. Gelling agent, Gelrite, increased multiplication rates significantly but had a negative effect on overall growth causing plants to become vitrified. The addition of activated charcoal reduced vitrification but also reduced multiplication rate. Activated charcoal greatly improved overall growth of papaya and reduced leaf senescence. No vitrification was observed in multiplying papaya cultures where agar and Gelrite combinations were used, but multiplication rate was reduced compared to cultures grown on Gelrite alone. callus removal from the bases of papaya at subculturing reduced multiplication rate and influenced elongation, growth and leaf senescence. Lower concentrations magar and Gelrite improved rooting percentages, but did not provide good support. Damaged roots and lower rooting percentages were observed on plantlets treated with IBA for four weeks compared to those exposed for only two days. A one hour pulse with a higher concentration (5 mg l ¯¹) of IBA greatly improved rooting percentage and further eliminated a second subculture onto an IBA-free medium after two days. Good, strong roots with root hairs were produced on vermiculite medium containing equal volumes of DS salts and vitamins. Modified lids with cotton-wool plugs also reduced leaf abscission. In vitro grafting using stericrepe proved impractical, while grafting in vitro unrooted papaya plants onto ex vitro seedlings was more successful, using wedge and slant grafts. Grafts sealed with pegs and Parafilm™ were less effective. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

Influence de la micro-propagation par embryogenèse somatique sur la variation somaclonale chez le caféier (Coffea arabica): évaluation des changements au niveau phénotypique, cytologique, génétique et épigénique. La variation somaclonale (VS) est une préoccupation majeure de tous les systèmes de micropropagation. Elle est décrite comme un changement phénotypique présent chez les vitroplants et pourrait être générée par une large gamme de mécanismes génétique et épigénétiques. Des hybrides de Coffea arabica hautement productifs sont distribuées sous forme clonale par embryogenèse somatique (ES) en Meso-Amérique. L’objectif de ce travail chez le caféier est d’évaluer la conformité génétique des plants multipliés par ES et de comprendre les mécanismes impliqués dans les SV. Nous avons évalué les variations dans les plantes régénérées au niveau phénotypique, cytologique, génétique (mutations/AFLPs, transposition génétique/S-SAP) et épigénétique (méthylation/MSAP) en utilisant deux approches complémentaires. Tout d’abord, nous avons étudié chez deux hybrides des conditions de cultures industrielles supposées peu mutagènes i.e. une courte période de prolifération (6 mois) et faible apporte en auxine (0-1.4 µM 2,4-D). Deux systèmes de prolifération i.e. l’embryogenèse secondaire et les suspensions embryogènes seront comparés, le dernier étant plus productif et économique. Les analyses moléculaires AFLP et MSAP sur 145 somaplants montrent que les polymorphismes génétique et épigénétique entre plantes mères et somaplants sont extrêmement réduits, i.e. dans l’intervalle 0-0,003% et 0,07-0,18% respectivement, sans différence significative entre les systèmes de prolifération. Pour les deux hybrides testés, des observations phénotypiques massives en pépinière et au champ ont révélé de très faibles niveaux de VS (0,9% pour 800.000 plantes). Des analyses cytologiques ont mis en évidence des nombres de chromosomes anormaux (41-43, 45) chez la plupart des variants et des nombres normaux (44) chez les plants ayant un phénotype normal. Des conditions expérimentales a priori mutagènes ont également été appliquées en utilisant des périodes de prolifération prolongées (4, 12 et 27 mois) chez trois lignées embryogènes indépendantes de la variété Caturra en présence de concentrations élevées en régulateurs de croissance (4.5 μM 2,4-D, 17.8 μM 6-BA), afin de comprendre les mécanismes liés vieillissement des cultures interviennent sur les VS. L’étude des 180 somaplants régénérés a montré que le temps de prolifération affecte fortement la fréquence de VS et d’une manière hautement similaire pour les 3 lignées embryogènes. Aucun variant n’a été trouvé après 4 mois de prolifération alors que 30% et 94% de variants phénotypiques ont été caractérisés chez les plants issus de cultures de 12 et 27 mois, respectivement. Quels que soient l’âge de culture et la lignée embryogène, aucun polymorphisme n’a été trouvé chez les 124 somaplants et un très faible nombre des changements des méthylation avec les MSAP (0,049-0,087%). Cependant, de façon similaire aux somaplants produits en conditions industrielles, les variants phénotypiques montrent systématiquement des nombres de chromosomes anormaux (41-43) et les plants normaux le nombre de chromosomes attendu. Ce travail montre que l’ES s’appuyant sur des suspensions embryogènes peut garantir une propagation conforme des variétés sélectionnées de C. arabica. Il démontre également l’importance de l’âge des cultures sur l’apparition de VS et donc le caractère non aléatoire du phénomène. Les changements génétiques et épigénétiques sont particulièrement limités durant l’ES. Le principal changement chez la plupart des variants est l’aneuploidie, ce qui montre que les aberrations mitotiques jouent un rôle majeur dans les VS chez le caféier. / Influence of micropropagation through somatic embryogenesis on somaclonal variation in coffee (Coffea arabica): assessment of variations at the phenotypical, cytological, genetic and epigenetic level Somaclonal variation (SV) is a major concern in all micropropagation systems. It is described as the phenotypic variation displayed in in vitro-derived regenerants and it is believed to be originated from a large array of genetic and epigenetic mechanisms. Highly productive Coffea arabica hybrids are clonally disseminated in Meso-American region through somatic embryogenesis (SE). The objective of the present work in coffee is to evaluate the trueness-to-type of SE and to understand the mechanisms involved in SV. We assessed the variations in the propagated plants at the phenotypic, cytogenetic, genetic (mutations/AFLP, genetic transposition/S-SAP) and epigenetic (methylation/MSAP) level by using two complementary approaches. First, with 2 hybrids we studied industrial culture conditions expected to be weakly mutagenic thanks to the combined use of short term proliferation period (6 months) and low auxin supply (0-1.4 µM 2,4-D). Two proliferation systems i.e. secondary embryogenesis and embryogenic suspensions were compared, the latter being more productive and economic. AFLP and MSAP molecular analyses on 145 somatic seedlings showed that genetic and epigenetic polymorphisms between mother plants and emblings were extremely low, i.e. ranges of 0–0.003% and 0.07–0.18% respectively, with no significant difference between the proliferation systems. For the two hybrids tested, massive phenotypic observations in nursery and field plots showed very low levels of SV (0.9% from 800,000 plants). Cytological analysis showed abnormal chromosome numbers (41-43, 45) in most of coffee somaclonal variants and normal numbers (44) in phenotypically normal plants. Stressful experimental conditions were also applied by using extended proliferation periods (4, 12 and 27 months) for three independent embryogenic lines established for the Caturra var. in presence of high growth regulator concentrations (4.5 μM 2,4-D, 17.8 μM 6-BA) to understand the mechanisms of culture ageing on SV. The proliferation time strongly affected the SV frequency among the 180 regenerated plants and in a highly similar way with the three embryogenic lines. No variant was found after 4 months proliferation although 30% and 94% phenotypic variants were observed in plants derived from 12 and 27 month-old cultures, respectively. Regardless the culture age and the embryogenic line, no polymorphisms were found in the 124 plants analyzed and very limited methylation changes with MSAP markers (0.049-0.087%). However, similarly to plants derived from industrial conditions, phenotypic variants systematically showed abnormal chromosome numbers and normal plants systematically showed normal numbers. This work showed that SE based on embryogenic suspensions is reliable for true-to-type propagation of selected C. arabica varieties. It also demonstrated the importance of culture age on SV and hence the non random nature of this phenomenon. The genetic and epigenetic alterations are particularly limited during SE. The main change in most of phenotypic variants was aneuploidy showing that mitotic aberrations play a major role in SV in coffee.

Wheat (Triticum aestivum L.) zygotic embryogenesis occurs in a dynamically regulated ovular environment, and in ovulohormones regulate embryogenic processes. Levels of ABA, IAA, and the cytokinins Z, ZR, DHZ, DHZR, iP, and iPA were studied in developing wheat kernels from anthesis to maturity . High cytokinin and low IAA and ABA levels were associated with the early stage of embryo formation and active tissue histodifferentiation. Following histodifferentiation, cytokinin levels declined while IAA accumulated throughout the stage of active grain growth and then declined with grain maturity. ABA levels increased at the soft-dough developmental stage and through to grain maturity. Endogenous +ABA levels in developing wheat grains treated with fluridone, which indirectly blocks ABA synthesis, declined at the soft-dough stage. As a result, mature desiccated fluridone-treated kernels exhibited little dormancy. However, fluridone-treated kernels were not viviparous, suggesting a strong caryopsis-embryo interaction in maintaining embryogenically competent tissues. Induction of embryogenically competent wheat callus cultures was highly variable between genotypes and pre-initiation environments. Genotypic and environmental influences altered endogenous hormone levels and affected embryogenic competence. Establishment of competent embryo explants for somatic embryo induction was favored by a high cytokinin-to-auxin ratio and very low ABA levels throughout histodifferentiation (around 4 to 8 DPA). Similar to embryos forming within the caryopses, competent callus cultures had a high cytokinin (Z)-to-auxin ratio at 7 DPI. Increased frequency of embryogenic cultures was achieved when embryo explants were excised during a narrow window of low hormone levels. Wheat line and pre-initiation environment affected this window. Simulation in vitro of the in ovulo wheat kernel environment improved zygotic embryogenesis in vitro . Embryos exposed to physiologically normal ABA levels and low 0 2 tensions of 2.5 mM (7%) most closely approached morphological and physiological normalcy. The culmination of these studies clearly defines windows of embryo development for explant excision, associated roles of plant hormones in embryogenesis, and in ovulo hormone levels that vastly improve the frequency of successful embryogenesis when simulated in in vitro culture systems.

Research Doctorate - Doctor of Philosophy (PhD) / Somatic embryogenesis (SE) can be induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but not in wild type Jemalong. Wild-type Jemalong will only form callus in the presence of auxin and cytokinin and both Jemalong and 2HA will form roots in response to auxin alone. The F2 analysis of 2HA X Jemalong crosses suggest that a single gene may open the way to SE but additional genes are required to maximise the process. Auxin and cytokinin are required for 2-3 weeks for SE and for de novo root formation auxin is essential for about one week. Abscisic acid (ABA) and ethylene, both stress related hormones, enhance SE induction but inhibit callus and de novo root formation. The WUSCHEL (WUS) gene was investigated in M. truncatula (Mt) and identified by the similarity with Arabidopsis WUS in amino acid sequence, phylogeny, promoter element patterns, and expression patterns in planta. MtWUS is induced by cytokinin after 24-48 h in embryogenic cultures and maximum expression occurs after 1 week which coincides with totipotent stem cell induction. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localises to the embryo and corresponds to the onset of MtCLV3 expression. RNAi studies show that MtWUS expression is essential for callus and somatic embryo production. There is evidence based on the presence of MtWUS promoter binding sites that MtWUS is required for the induction of MtSERF1 which appears to have a key role in the signalling required for SE induced in 2HA. MtWOX5, as for MtWUS, was identified by similarity to Arabidopsis WOX5 based on amino acid sequence, phylogeny, promoter element patterns, and expression patterns in planta. MtWOX5 expresses in the auxin induced root primordium and root meristem and appears to be involved in pluripotent stem cell induction. GA suppresses the MtWOX5 expression in the root apex and suppresses the root primordium induction, consistent with the importance of MtWOX5 in in vitro root formation. The evidence is discussed that the homeotic genes MtWUS and MtWOX5 are “hijacked for stem cell induction which is key to somatic embryo and de novo root induction. In relation to SE, a key role for WUS in the signalling involved in induction is discussed and a model developed.

Triticum aestivum is of major importance both nutritionally and economically globally. Traditional breeding mechanisms have been unsuccessful at keeping pace with the increasing demand for better yielding and more resilient wheat varieties. The introduction of foreign genes into systems has provided a new tool for crop improvement, but has been difficult to apply to elite wheat varieties mainly as result of their recalcitrance to prerequisite tissue culture. Investigations here demonstrate that modification of the ammonium nitrate content in direct somatic embryogenesis induction medium can increase the number of primary embryos produced by over two fold in the elite hard red wheat cultivar Superb. The number of primary embryos which were capable of transitioning into shoot development also increased by two fold. Biolistic transformation efficiency was also improved when targeted scutellar tissue was exposed to elevated ammonium nitrate levels. / x, 81 leaves : ill. ; 29 cm.

Blue-flowered Conospermum are endemic to Western Australia, and show great potential as cut flowers. Propagation from cuttings or seed proved difficult, and root initiation in vitro is problematic. This thesis examines the floral biology of the species and the possibility of using somatic embryogenesis to overcome propagation problems. A survey of explant tissue types for C. eatoniae and C. caeruleum was carried out to identify tissue that could be induced into embryogenic pathways. Vegetative, semi-floral and floral buds were initiated into culture from February to June, but were found unsuitable for embryogenesis, producing shoots, callus or dying in culture. Leaves from in vitro leaf cultures formed callus in the presence of 2,4-D and BAP, but were unable to differentiate into embryos in the presence of a variety of growth regulator combinations and concentrations. Immature zygotes died in culture. Direct embryogenesis and/or embryogenic callus was observed on mature zygotes of the species C. caeruleum, C. spectabile, C. dorrienii and C. brownii, and somatic embryos were maintained in culture for up to 18 months for C. caeruleum. Maturation and germination of somatic embryos proved difficult; treatments of cold, ABA, desiccation or mannitol did not induce maturation. It appears that developmental pathways in Conospermum are well defined and are difficult to alter in vitro. It was concluded that somatic embryogenesis has limited commercial potential in these species. Conospermum species have an active pollination mechanism where the style is held in a state of tension when the flower opens. When pressure is applied at the base of the style by an insect, the style flicks downwards, striking the insect pollinator and releasing pollen from the anther in a single dusty mass. However, the breeding systems of blue-flowered Conospermum have not previously been well explored. Flowers on a C. eatoniae inflorescence opened from the basal end upwards acropetally, with the terminal two or three buds never opening. Fruit and seed set occurred only from the basal one to three buds. Isolation of C. eatoniae and C. amoenum flowers showed they were unable to self-pollinate in the absence of insect pollinators. Experiments to determine the timing of the peak of stigmatic receptiveness were inconclusive. Pollen germinated and penetrated the stigma 0 ¡V 6 days after anther dehiscence. Pollen loads on the stigma did not relate to the number of pollen tubes observed down the style. Controlled pollinations of cultivated C. eatoniae at a field station using self and cross pollen, revealed compatibility with a range of pollen genotypes, as pollen tubes were observed extending down the style. However, late-acting incompatibility could not be ruled out as controlled crosses failed to set any seed as flowers were shed from the bush. DNA analysis of open pollinated C. eatoniae seed progeny from two plants from a field station and two plants in natural bushland revealed very different pollination habits. Plants from the field station showed no outcrossing, with progeny closely resembling the maternal parent, whereas plants from the wild population showed outcrossing with several different paternal parents. These results suggest self-pollinated seed can be reliably obtained in a plantation situation using stands of ramets of the same clone. Alternatively, assuming that the required insect pollinators are present in a cultivated stand, it should be possible to obtain cross pollinated seed by surrounding the maternal plant with the desired paternal parent. Unusual pollen behaviour was observed for many blue-flowered species, a white-flowered species of Conospermum, and close relative, Synaphea petiolaris. Up to three pollen tubes emerged from the triporate pollen in vitro, and at rates of up to 55 ƒÝms-1. This rate was maintained for only 2 s but is greater than 20 times faster than reported in the literature for any species, in vitro or in vivo. Pollen with multiple tubes was also observed on the stigma in vivo in C. amoenum flowers. Changing the osmotic pressure of the germination medium by altering sucrose concentration influenced the number of tubes to emerge from the pollen grain; generally the number of tubes decreased as sucrose increased. However, the rate of tube growth was unaffected. The addition of calcium channel blockers to the germination medium had no effect on Conospermum growth rate, nor did they eliminate pulses of tube growth. Observation of Conospermum pollen ultrastructure revealed similarities to Gramineae pollen. The tube cytoplasm was packed with vesicles filled with material of similar electron density to the cell wall. Few golgi were identified, and the apical end of the tube contained these vesicles, smaller secretory vesicles and mitochondria. This is atypical of the tip, which is normally free of large vesicles. Distinct zones in the cytoplasm were not identified, which is similar to Gramineae. Like the grasses, Conospermum appears to pre-manufacture cell wall material and store it in vesicles ready for rapid germination and extension. A biological function of multiple pollen tube emergence with such rapid growth was not elucidated. This research has shown Conospermum to be a complex and very interesting genus. Further investigation into the remarkable growth of multiple pollen tubes would enhance our knowledge of the biological processes involved in tube growth and the process of fast wall formation. The potential benefits to the cut flower industry of commercialising some of these species warrants further effort to find an efficient method of propagation. Introduction into horticulture may be the only means by which these threatened species will survive.

Thesis (Ph.D.)--University of Melbourne, School of Forest and Ecosystem Science, 2006. / Typescript. Includes bibliographical references (leaves 184-216).

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

No description available.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also called phytic acid, is ubiquitous in eukaryotic cells and the most abundant inositol phosphate derivative. Loblolly pine (LP, Pinus taeda) constitutes the primary commercial species in the southern forest of U.S. Somatic embryogenesis (SE) is an effective technique to maintain the desirable genetic composition of the progeny and to accomplish the efficiency of propagation. SE can also serve as a tool for study of plant development. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). In LP SE, FG tissue is absent in the embryogenic tissue culture. We found that extracts from early-stage FG stimulate growth and multiplication of early-stage somatic embryos, whereas FG water extracts from late stage contain substance(s) inhibitory to early-stage somatic embryo growth (DeSilva et al., 2007). We now present the isolation and identification of the inhibitory substance as InsP6 by means of water extraction, two gel filtrations and two ion exchange FPLC chromatographies. The results represent the first complete structural characterization of InsP6 from a natural product using LC/MS, LC/MS/MS, exact MS, 1D- and 2D-NMR analyses. We also report that there is a good correlation between the amount of InsP6 purified from FG tissue (1.3 nmoles per full-term FG) and the amount of InsP6 which inhibits somatic embryo growth. This novel approach of isolating and characterizing InsP6 from plant tissue, and investigating its role on SE can allow us to improve SE technology by circumventing current bottleneck, to elucidate enigmatic functions of InsP6 in plants, and most importantly, to utilize this molecule properly.