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Lipochitooligosaccharides (LCOs) are molecules secreted by rhizobia during the establishment of rhizobia-legume N2-fixing symbiosis. Some recent reports have shown that there are physiological effects on host and non-host rhizobia after LCO treatment. However, the cellular mechanisms underlying these observations are not known. Therefore, the study was aimed at assessing phenotypic changes by measuring photosynthesis, leaf area and dry weight on a non-host plant (tobacco). Our results did not show any significant physiological changes following LCO treatment. We also wanted to explore the molecular basis of changes in the plant cell by looking at gene and protein profiling following LCO treatment in a natural host plant (soybean) using real-time RT-PCR and SDS-PAGE. To do so, a reproducible stimulation method for soybean seed germination by LCO was successfully developed. The results obtained on soybean did not reveal significant differences in gene expression between water and LCO-treated seeds for the genes cdc2, WASI, ICL1 and 14-3-3 studied. In addition, LCO treatment did not change the protein profile compared to the water control in a significant way.

Disa comprises 163 species, 131 of which occur in South Africa (SA). The genus is distributed across winter- and summer-rainfall areas, but few species transverse both climatic regions. Species are therefore regarded as winter-rainfall or summer-rainfall endemics - yet release their seeds in autumn, irrespective of provenance. Disa contributes 40 % of threatened Orchidaceae in SA, with half of the local species requiring conservation initiatives. In vitro seed germination is a potential conservation tool for producing large numbers of genetically diverse plants in relatively short periods. However, only 11 winter-rainfall Disa species are easily germinated ex situ. Studies were therefore undertaken on summer-rainfall taxa, which are ungerminated in vitro, in an effort to define their germination parameters. This thesis describes mechanisms that control germination in Disa and establishes practical propagation methods for seed culture. Two seed types occur in Disa; i) comparatively large, pale and pyriform seeds in members of the D. uniflora sub-c1ade, which populate streamside habitats under conditions of winter-rainfall maxima, and ii) smaller, variously brown and fusiform seeds in the remainder of the genus. Seed morphometrics distinguished seed types, although embryo dimensions were similar. Testa continuity, which is disrupted in the large seeds, also supported separation. Typically, small seeds are ungerminated in vitro, whilst large seeds germinated readily. Increased seed size did not necessarily impart increased germ inability, as several germinable, small-seeded species exist - being winter-rainfall species Attempts to establish in vitro germinability revealed that increased water availability and charcoal supplementation promoted germination in intractable species. The control of germination was therefore proposed as a trade-off between water availability and the presence of phyto-inhibitors - two features typical of seeds exhibiting water-impermeable dormancy. Three germinability categories were recognized; i) easily germinable species, ii) poorly germinable species through media manipulation, and iii) ungerminated species. Germination of immature seed in the absence of media modification was comparable to mature seed germination under modified conditions, providing evidence of the role of an impermeable seed testa in regulating germination. Testa impermeability in mature, small-seeded species was demonstrated using aqueous EVANS' blue dye and was linked to i) testa integrity and ii) increased levels of leachable phenolics (LPC) - which are hydrophobic and phytotoxic. In addition, this research revealed an impervious and elaborate embryo carapace in small seeds. Large-seeded species were highly permeable at dehiscence, with perforated testae and negligible LPC. Germinability was ultimately defined by a significant regression with LPC. Phenolic deposition increased exponentially with increasing seed maturity and reflected decreased permeability and the development of testa colouration. The testa precludes the use of viability stains such as nc and FDA, unless rendered permeable through scarification. This was achieved using NaOCI. Viability and germinability percentages did not correlate well for the small-seeded Disa species, indicating that i) the methods used to break dormancy are inadequate, ii) additional factors may be acting in concert with the testa to regulate germination and iii) that the determination of mature Disa seed viability is ineffective. As an alternative, the germination potential of immature seed was estimated as the ratio between the proportion of embryos stained with TTC and the proportion of seeds permeable to EVANS' blue. Attempts to relieve water-impermeable dormancy in Disa resulted in the formulation of a dual-phase protocol - with the specific aim of increasing water availability to the embryo. Dual-phase cultures comprised a solid, charcoal-rich medium overlaid with a reduced strength, liquid medium fraction of the same type. The solid fraction negated the influence of leached phenols and allowed protocorms to establish polarity, whilst the fluid fraction increased water availability. The dual-phase protocol allowed germination of nine summer-rainfall Disa species, usually in percentages that approximated their estimated germination potential. For the remaining species, germination is controlled by more complex factors. Large seeds are atypical in containing starch, the hydrolysis of which facilitated their rapid, autonomous germination. Small-seeded Disa species stored lipids and proteins and germinable species accumulated starch post-germination. The embryo protoplasts of all species contained appreciable amounts of soluble sugars, irrespective of germinability. However, decreased sucrose and increased fructose correlated significantly with decreased seed germinability. This study provides evidence of the nutritional value of mycotrophy, with endophytes liberating soluble carbohydrate and non-carbohydrate compounds upon lysis. However, few species were germinated symbiotically, suggesting that endophytes isolated from adult roots do not necessarily support germination in the same species. Similar endophytic fungi occur in Australian and Holarctic orchids. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

Dormancy characteristics and optimum conditions for germination of Cleome gynandra seeds have not been explained. Seed storage proteins were extracted, analysed with SDS-PAGE and sequenced. Seed proteins of Cleome were characterised by comparison with those of wild mustard (Brassica kaber). Wild mustard showed seed proteins composed of two α-chains of molecular weight (24-32 kDa) and another two β-chains of 18-22 kDa. The seed proteins of Cleome comprised two α-chain polypeptides of molecular weight (25-30 kDa), two β-chain polypeptides of molecular weight (18-20 kDa) and a smaller β-chain of 13-15 kDa. The storage proteins occurred in the seeds as dimeric complexes of molecular weight 40-65 kDa, which were broken into polypeptide chains of approximately 20 and 30 kDa by the reducing . action of DTT. Comparison with proteins in the proteome library and similarity index further confirmed that the seed proteins of Cleome had similarities with those of wild mustard. Two dimensional SDS-PAGE showed that the two species have nine similar polypeptides and four different ones. Events associated with dormancy release during seed germination still require explanation. Seeds of Cleome are characterised by low germination and there has been no explanation for this. Changes in protein expression during germination of Cleome in the presence or absence of light and at constant or alternating temperatures were examined. The germination of Cleome seeds at 20 degrees C was inhibited by light, but it was improved at 20 degrees C in darkness. There was no photoinhibition when seeds were germinated at constant 30 degrees C or alternating 20/30 degrees C (16 h night and 8 h day) for 10 days. Four proteins were observed to decrease in expression as germination progressed, but remained unchanged during photoinhibition. Photoinhibition was expressed more in seeds that were harvested late, after the pods had turned brown. These seeds showed a fifth, low molecular weight protein (13 kDa) that was absent from the immature seeds and embryos. Photinhibition is a pseudo-dormancy condition during which seed storage proteins are not utilised and the seed coat could partially play a role in it. The temperatures for the germination of Cleome in darkness have been determined. However, prior to this study the effects of temperature, light and pre-germination treatments (chilling, scarification, hydration and germination in the presence of KN0(3) or GA(3) on the germination of the seeds of this species have not been investigated. Seeds were germinated for 10 days and the final count of germination was used to determine seed performance. The highest germination percentage (60% and 80%, for a 2-year old and a l-year old seed lot, respectively) of untreated seeds was achieved when alternating temperatures of 20/30 degrees C (16 h/S h) in the dark or constant 30 degrees C in the dark were used. Among the pre-germination treatments, only scarification (puncturing of seeds at the radicle end) improved germination. Seeds were found to be negatively photoblastic, and the phenomenon was more pronounced when they were germinated at 20 degrees C and 12 h photoperiod or longer. Germination of photoinhibited seeds was, however, improved by treatment with GA(3) It is recommended that the germination of Cleome be undertaken under conditions of darkness and at either alternating 20/30 degrees C or continuous 30 degrees C. Seed lot vigour and seedling vigour are two important seed quality aspects that are used in defining the seed germination process. Seed germination is appropriately characterised by radicle protrusion and the attainment of normal seedling structures. However, the international rules for testing seeds combine radicle protrusion and normal seedling attainment in separating seed germination into the first and final counts. The challenge to a seed analyst testing the germination of a species whose first and final counts are unknown is that there is no statistical guideline to determine these important stages of seed germination. Cauliflower and broccoli, for which the first and final counts are published in the international rules for testing seeds and Cleome, for which there is no data on the first and final counts, were examined to determine the statistical significances of the first and final counts. Analysis of variance, logistic regression, 'broken-stick' regression models and survival analysis procedures were used. Analysis of variance showed that there were no differences between the germination percentages on the fourth, fifth and seventh days of germination. Low and stable standard deviations were recorded when evaluating germination after the fourth day. The germination curves of broccoli and cauliflower did not fit the Gompertz curve but fitted the exponential curve. The broken-stick model 'broke' the cumulative germination curve for the Cleome seed lots into two linear curves that were significantly different, but failed to break those for broccoli and cauliflower. However, this study confirmed the first and final counts for broccoli and cauliflower as determined by the international rules for testing seeds. Broken-stick modelling and life table analyses confirmed the fourth day as being appropriate to determine the first count for Cleome germination. There was no evidence of further seed germination after the seventh day as shown by probability density and hazard rate. It is suggested that for Cleome, the 'first count' and 'final count' be performed on the fourth and seventh day of the germination, respectively. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Inconsistent seed germination poses a problem for efficient seedling production of Echinacea species. Evidence suggests that ethylene can be effective for improving germination in Echinacea species. The objectives of this research were: to develop an ethylene pre-germination treatment that enhances germination in Echinacea species that is retained following drying and storage, and to determine if the ethylene effect on enhanced germination was an important mode of action for dormancy release. Four species of Echinacea (E. purpurea, E. tennesseensis, E. angustifolia and E. simulata) treated with 1-aminocyclopropane-1-carboxylic acid (ACC) or ethephon resulted in faster and generally higher germination. Pre-treatment of seeds with ACC or ethephon followed by drying was as effective as chilling stratification for enhancing germination depending on the species. While ethylene pretreatments did increase germination to some extent depending on species, it was concluded that 60-day stratification alone was a more commercially-viable treatment. Ethylene production or perception was not necessary for germination in untreated or stratified seeds as shown by aminoethoxyvinylglycine (AVG), silver thiosulfate (STS), and 1-methylcyclopropene (MCP) treatments. Both stratification and ACC treatment reduced Echinacea seed sensitivity to ABA and could be a common mechanism for enhanced germination. However, it does not appear that the increased germination seen after stratification was mediated through ethylene production because final germination percentages were generally unchanged following inhibition of ethylene production or action. In contrast, inhibition of ethylene production and perception reduced early 3-day germination suggesting that ethylene was more involved in seed vigor than final germination. It was determined that there is no physiological significance of ethylene for dormancy release in these Echinacea species.

Our research investigated whether priming switchgrass seeds with water or ethephon would increase stand establishment in the field. ‘Alamo’ seed germinated faster and grew taller than ‘Cave-in-Rock.Seeds primed for six days in water or for one day in ethephon 10 mM had the greatest seedling densities. In growth chamber environments seed priming were tested to hasten germination velocity. Seeds primed for two, four or six days in water germinated faster than unprimed seeds. Ethephon treatments reduced overall germination and germination velocity. Accent and Accent Q herbicides containing nicosulfuron are used to control weeds. To test ‘Alamo’ sensitivity to these herbicides, greenhouse evaluations were conducted. Seedlings treated with Accent Q had lower shoot fresh and dry weights than Accent treated seedlings. Seedling atrazine tolerance was examined in a greenhouse study at various growth stages (1, 2 and 4 true leaves). One and two true leaf were more sensitive to herbicide damage than the 4 leaf seedlings. To investigate difference in atrazine tolerance due to differential atrazine metabolism, 14C atrazine metabolism was examined in 1, 2 and 4 leaf ‘Alamo’ seedlings. 24-48 hours after exposure, 4 leaf seedlings metabolized atrazine at a greater rate than 1 and 2 leaf stage seedlings.