The overall aim of this PhD study was to develop protocols to improve the efficiency of eucalypt controlled pollinations (CPs) in order to make it more cost-effective for forestry companies to perform them on a commercial scale on small-flowered species. To achieve this, three research areas were explored, namely pollen handling, breeding systems and controlled pollination technique. Study species were Eucalyptus grandis, E. dunnii, E. smithii, E. nitens, E. urophylla and E. macarthurii. The first specific aim of the study was to identify a suitable liquid in vitro germination medium for reliably testing pollen viability of all six study species. Six levels of sucrose [0, 10, 20, 30, 40 and 50% (w/v)] were tested, both with (0.15 mg l-1) and without boric acid. The optimal sucrose concentration was found to be 30% (w/v), with boric acid stimulating pollen tube growth. A second aim was to determine temperatures suitable for the short-, medium- and long-term storage of E. smithii, E. nitens and E. grandis pollen. Pollen samples were stored at room (25oC), refrigerator (4oC), freezer (-10oC) and liquid nitrogen (-196oC) temperatures, and pollen viability tested every two months over a 12-month period. There was a rapid decline in the germination of pollen stored at 25oC, while temperatures cooler than 4oC appeared to maintain pollen viability for the duration of the 12-month study. Recommendations were thus to use a refrigerator for short-term (< 2 months), a freezer for medium-term (up to 10 months) and cryopreservation for longer-term storage. In the second part of the study, breeding systems of E. urophylla and E. grandis were examined by studying pollen-tube growth in the style after single-donor self- and cross-pollinations. Results showed that, in addition to both species exhibiting reduced seed yields following self-pollination, pollen tubes from self-pollen took significantly longer than those from cross-pollen to grow to the base of the style. This suggested the presence of both late-acting self-incompatibility and cryptic self-incompatibility (CSI) as possible mechanisms responsible for outcrossing in these two species. In a follow-up study, the siring ability of self- and cross-pollen was examined after single- and mixed-donor pollinations were performed on E. grandis. Once again, single-donor cross-pollinations resulted in a significantly higher number of seeds compared to self-pollinations. In addition, microsatellite molecular markers revealed that 100% of the progeny from mixed (self + outcross) pollinations were outcrossed, confirming the competitive advantage of cross-pollen. To date, CSI has never been associated with Eucalyptus, making this the first study to suggest its presence in the genus. For the final study area, three CP-techniques were compared, namely the Conventional method, One Stop Pollination (OSP) and Artificially Induced Protogyny (AIP), in E. grandis, E. smithii and E. macarthurii maternal parents. Although the AIP technique produced the highest seed yields in all three species, it also resulted in high self- and foreign-pollen contamination (determined using microsatellite markers). This necessitated exploration of different methods of isolating the pollinated flower, and this led to the identification of a novel method which uses sodium alginate gel. Flowers hand-pollinated and isolated with sodium alginate produced progeny that were 100% outcrossed with the applied pollen, confirming the superiority of this innovative isolation technique compared to the currently used exclusion bag. Sodium alginate isolation also increased the efficiency of CPs as the gel was naturally shed, removing the need for operators to return to the tree to remove the isolation material, and thereby reducing the cost per seed. Application of these results should make commercial CP-seed production of small-flowered eucalypts a practical reality. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

The spreading of the invasive plant Garden Lupine (Lupinus polyphyllus) has become a matter of national importance in Sweden, due to it posing a threat to native plant and pollinator diversity. The effective attraction of bumblebees (Bombus spp.) as pollinators facilitates the production of large numbers of seeds, which are key to the Garden Lupine’s success. Possible self-pollination could also provide a competitive edge for the plant. The objective of this study was to study the relationships between Garden Lupine color morphs, pollinator attraction and seeding. Inflorescences of three color morphs were studied during the flowering period, and bumblebee behavior was observed on the site. After seeding, any produced seeds were collected and analyzed, as well as experimentally germinated to provide insight into their viability. A subset of inflorescences of each color morph was prevented access to pollinators, to study potential self-pollination effects. Bumblebees preferred blue flowers over pink, but no difference in pollination between the color morphs was found. Flower color did not affect seed production or seed morphology. Self-pollinated inflorescences produced fewer seeds than those with access to pollinators, but no difference in seed morphology or germinative success between the pollination methods could be established. The results suggest that seed production and germination are less dependent on pollination than expected. The ability to germinate through self-pollination provides insight into the invasive potential of Garden Lupine, suggesting that further studies are needed to successfully counteract its spread. / Den invasiva växten blomsterlupin (Lupinus polyphyllus) har på senare år blivit en nationell angelägenhet i Sverige, där den hotar mångfalden av inhemska växter och pollinatörer. Blomsterlupinens framgångsrika tilldragning av framförallt humlor (Bombus spp.) som pollinatörer möjliggör det stora antalet frön som den producerar, vilket är nyckeln till dess invasiva etablering. Eventuell förmåga till självpollinering kan också utgöra en konkurrensfördel. Målet med studien var att utforska relationen mellan blomsterlupinens färgmorfer, pollinering samt fröbildning. Blomställningar av tre färgmorfer studerades under blomningsperioden. Humlornas beteende observerades också under perioden vid lupinlokalen. Efter frösättning samlades alla producerade frön upp och analyserades, varefter ett frögroningsexperiment utfördes för att belysa frönas grobarhet. I ett fältexperiment nekades en delmängd av blomställningarna tillgång till pollinatörer, för att studera eventuell självpollinering och dess effekter. Humlorna föredrog blåa blommor före rosa, men ingen skillnad i pollinering mellan färgmorferna kunde fastställas. Blommornas färg hade ingen effekt på fröproduktion eller -morfologi. Självpollinerade blomställningar producerade färre frön överlag än de med tillgång till pollinatörer, men ingen skillnad i frömorfologi eller grobarhet mellan pollineringsmetoderna kunde påvisas. Resultaten antyder att fröproduktion och frögroning hos blomsterlupin är beroende av pollinering i mindre grad än förväntat. Förmågan att gro genom självpollinering belyser blomsterlupinens invasiva potential, och antyder att fler studier behövs för att framgångsrikt motverka dess spridning.

Phenotypic plasticity plays an important role in organisms’ adaptability to environmental change such as global warming caused by greenhouse-gas emissions. One plastic response to increased temperatures is for organisms to shift their phenology. It is of great concern that the phenologies of interacting species, such as plants and pollinators, may be shifting at different rates, causing temporal mismatches, which for plants can lead to unsuccessful reproduction. The “reproductive assurance hypothesis” states that plants capable of self-pollination should be under high selection to employ this as their main reproductive strategy in the event of pollinator scarcity to ensure reproduction, and consequently invest less in attracting pollinators. This study examines how soil warming in the Hengill geothermal area in Iceland affects the flowering phenology, reproductive strategy and investment in attractiveness to pollinators in the self-compatible herb Cerastium fontanum (Caryophyllaceae), when grown in a common garden in Stockholm, Sweden. Previous research showed that C. fontanum from warmed soils flowered earlier in situ than plants from colder soils, and later when grown in a common environment. In this study, C. fontanum plants collected along a temperature gradient followed the same counter-gradient pattern, where plants from warmer soils flowered later than plants from colder soils. Soil temperature at site of origin positively affected flower number but had no effect on flower size, seed production from autogamous self-pollination or visitation rate. Based on my findings it does not appear that C. fontanum, despite having an earlier flowering phenology in situ, is under any selection to alter its reproductive strategy or investment in attractiveness to pollinators when grown in a common temperature, and therefore it seems unlikely that plants are experiencing a temporal mismatch with insect pollinators. However, it would be worthwhile to conduct a similar experiment in Iceland to better understand how an earlier flowering affects pollination systems.

<p>Amur honeysuckle (<i>Lonicera maackii</i>) is a shrub endemic to East Asia which has become invasive and nearly ubiquitous to the forest ecosystems of eastern North America. Through its extended growing season, competitive ability, and potential allelopathy, Amur honeysuckle alters native herbaceous-layer plant communities and inhibits the regeneration of native tree species. As such, it is representative of a range of invasive shrub species imported from East Asia. My thesis contributes to questions fundamental to the understanding this and other invasive shrubs: 1) How do species become invasive? 2) How can the invasions of the species be effectively controlled? 3) How does the ecosystem respond to treatment of the invasive species?</p> <p>First, I examined the role of self-compatibility within Amur honeysuckle. I compared the berry production, seed production, and germination rates between closed-pollinated and open-pollinated flowering branches of Amur honeysuckle individuals across multiple types of invasions (heavy, light, and sprouting). I found that Amur honeysuckle not only possesses the ability to self-pollinate, but that it can produce viable self-pollinated seed sets. This ability may help explain how Amur honeysuckle able to invade isolated forest patches far removed from the main invasion.</p> <p>Second, I evaluated the effectiveness of a novel herbicide adjuvant in reducing the amount of herbicide needed in the ‘cut-stump’ method of controlling Amur honeysuckle. Combining various concentrations of the most common herbicide in the world, glyphosate, with concentrations of cellulases derived from fungi, 2XL, I examined whether the cellulases improved the effectiveness of glyphosate, potentially by increasing glyphosate movement into the vascular tissue of Amur honeysuckle through the degradation of cell walls. While 2XL was not an effective adjuvant, glyphosate concentrations of less than half the recommended dosages were equally effective as higher concentrations in preventing sprouting of treated stumps. The ineffectiveness of 2XL may imply a need for protein-mitigated diffusion of glyphosate across cell walls and into the vascular tissue of the plant, which would be inhibited by the breakdown of cell walls.</p> Finally, I tested how deeper intensities of mulching-head treatments affected the sprouting response of Amur honeysuckle and the response of the herbaceous-layer plant community after treatment. I found a negative relationship between the volume of sprouting Amur honeysuckle and increasing depth of mulching-head treatment. Additionally, increasing mulching-head intensities were correlated with increased herbaceous layer diversity and conservation value as represented by Floristic Quality Index. Mulching-head treatments are a promising tool in controlling heavy invasions of non-native shrubs.