Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression by Sanjeev V. Raikar Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×106 g-1FW was obtained when cell suspensions were used as the tissue source, with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×106 g-1FW) of L. corniculatus was achieved from cotyledons also with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm2 for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

Disputats. Rheinische Friedrick-Wilhelms-Universität, 2001. / Haves kun i elektronisk udg.

Grünland mit hoher Produktivität und Futterqualität bildet die Grundlage der Wiederkäuerernährung. In Grünlandbeständen mit ausbleibender oder geringer Stickstoffdüngung sind Leguminosen unerlässlich für Produktivität und Futterqualität, was auf die Fähigkeit von Leguminosen Luftstickstoff zu binden zurückzuführen ist. Gegenwärtig ist Trifolium repens L. eine der wichtigsten Futterleguminosen im Grünland der gemäßigten Zonen Europas. Es ist allerdings bekannt, dass T. repens eine gute Wasserversorgung benötigt, um einen hohen Ertrag zu erzielen. Verringerte Niederschlagsmengen in der Vegetationsperiode, die unter Klimawandelbedingungen vorausgesagt werden, könnten somit die Ertragsleistung von T. repens verringern. In Zukunft steigt dadurch möglicherweise auch die Bedeutung anderer Futterleguminosen, die besser an trockenere Bedingungen angepasst sind und somit als Alternative für T. repens dienen könnten. Da die Kenntnisse über das agronomische Potenzial solcher möglichen alternativen Leguminosen begrenzt sind, haben wir in dieser Studie fünf vielversprechende und wahrscheinlich besser an Trockenheit angepasste Leguminosen untersucht. Für unsere Versuche haben wir Lotus corniculatus L., L. uliginosus Schkuhr, Medicago lupulina L., M. falcate L. und Onobrychis viciifolia Scop. ausgewählt. In einem ersten Schritt wurde das agronomische Potenzial der Leguminosen im Hinblick auf Etablierung und frühe Ertragsentwicklung mit nicht limitierter Wasserversorgung getestet. Weiterhin wurden der Ertrag und die Ertragsstabilität sowie die Wassernutzung der alternativen Leguminosen bei temporärer Trockenheit untersucht und mit der von T. repens verglichen. Der Einfluss von Trockenstress auf wichtige Futterwert bestimmende Inhaltsstoffe der Leguminosen (Rohprotein, neutrale Detergenzienfasern, saure Detergenzienfasen und wasserlösliche Kohlenhydrate) war überdies Gegenstand der Betrachtungen. Um die oben genannten Parameter zu untersuchen, wurde von 2009 (Einsaatjahr) bis 2011 ein Experiment in Großgefäßen in einer Vegetationshalle durchgeführt. In diesem Versuch wurden alle Leguminosen sowohl in Monokultur als auch in einer praxisüblichen Mischung mit Lolium perenne L. angesät. Im Versuchszeitraum folgten die klimatischen Bedingungen in der Vegetationshalle einem normalen jahreszeitlichen Verlauf, der Frost im Winter und höhere Temperaturen im Sommer umfasste. Der für den Versuch notwendige Trockenstress wurde in drei Aufwüchsen innerhalb von zwei Jahren durch temporären Bewässerungsstopp erzeugt. Dabei wurde im Frühjahr 2010 (April/Mai) ein moderater und im Sommer 2010 (Juli/August) sowie im Frühjahr 2011 (April/Mai) je ein starker Trockenstress induziert. Mit Ausnahme von M. falcata, welches eine verzögerte Anfangsentwicklung zeigte, waren die Keimung und die Etablierung von allen alternativen Leguminosen vergleichbar mit der von T. repens. Die Ertragsleistung von M. lupulina und L. corniculatus in Monokultur war ähnlich hoch wie die von T. repens. In Mischung zeigten beide alternativen Leguminosen zwar Potenzial, aber die Ertragsleistungen waren dennoch geringer als die der T. repens/L. perenne Mischung. In unserem Versuch führte Trockenstress zu verringertem Ertrag und er beeinflusste auch die agronomische Wassernutzungseffizienz (Verhältnis von Ertrag zu Wasserverbrauch). Ausschlaggebend waren dabei die Stärke und die Dauer des Trockenstresses. Besonders starker, aber auch bereits moderater Trockenstress führten bei T. repens zu erheblichen Ertragsverlusten von bis zu 56%. Demgegenüber zeigten vor allem M. lupulina, aber auch L. corniculatus und M. falcata lediglich marginale Ertragsverluste bei moderatem Trockenstress und meist geringere Ertragsverluste als T. repens bei starkem Stress. Die agronomische Wassernutzungseffizienz war bei moderatem Stress verhältnismäßig stabil, wohingegen starker Stress im Vergleich zur Kontrolle meist zu einer geringeren agronomischen Wassernutzungseffizienz führte. Sowohl unter Kontroll- als auch unter Stressbedingungen zeigte speziell M. lupulina in Monokultur eine ähnliche oder sogar eine höhere agronomische Wassernutzugseffizienz als T. repens. Zudem war die agronomische Wassernutzungseffizienz der Mischungen mit M. lupulina, L. corniculatus und M. falcata weniger negativ von starkem Trockenstress betroffen als die Mischung mit T. repens. Dies bestätigte zum einen die Trockenheitsempfindlichkeit von T. repens und zum anderen die bessere Anpassung der alternativen Leguminosen an trockenere Bedingungen. Wir beobachteten, dass eine Änderung in der Stickstofffixierungsleistung der Leguminosen eine gute Erklärungsgröße für Änderungen des Ertrags und der agronomischen Wassernutzungseffizienz darstellt. Hohe Stickstofffixierungsleistung geht dabei meist mit höherem Ertrag und höherer agronomischer Wassernutzungseffizienz einher. Die intrinsische Wassernutzungseffizienz (Verhältnis von assimiliertem CO2 und stomatärer Leitfähigkeit), gemessen als 13C, war ein schlechterer Indikator für die agronomische Wassernutzungseffizienz: Obwohl die intrinsische Wassernutzungseffizienz unter starkem Trockenstress zunahm, sank die agronomische Wassernutzungseffizienz meist ab. Dennoch besitzt die Erhöhung der intrinsischen Wassernutzungseffizienz ein gewisses Potenzial als Anpassung an trockenere Bedingungen Der Trockenstresseffekt auf die Futterqualität war in unserer Studie generell deutlich geringer als der Effekt auf den Ertrag. Besonders moderater Stress hatte wenig Einfluss auf die Futterqualität, während sich die Effekte bei starkem Stress verstärkten. Starker Trockenstress führte meist zu einer Verringerung des Rohprotein- und Fasergehalts (neutrale und saure Detergenzienfasern), wohingegen sich der Gehalt an wasserlöslichen Kohlenhydraten erhöhte. Dies könnte ein Hinweis darauf sein, dass sich die Futterqualität bei Trockenstress sogar verbessert. Nichtsdestotrotz hatten in unserem Versuch die Leguminosenart und die Einsaat als Monokultur oder Mischung einen größeren Einfluss auf die Futterqualität als der Trockenstress. Der Einfluss von Trockenstress auf die Futterqualität ist deshalb bei der Wahl einer geeigneten Futterleguminose weniger von Bedeutung als andere agronomische Eigenschaften. Zusammenfassend ist zu sagen, dass besonders M. lupulina und in geringerem Maße auch L. corniculatus und M. falcata Potenzial als Alternative für T. repens bei Trockenstress zeigen. Nach ausreichender Etablierungszeit entwickeln sich besonders M. lupulina aber auch L. corniculatus und M. falcata stabiler und können sogar höhere Erträge als T. repens bei Trockenstress produzieren. Bezüglich der Futterqualität sind oben genannte alternative Leguminosen ebenfalls vergleichbar mit T. repens.

Comprendre le fonctionnement des écosystèmes est un enjeu crucial, en particulier dans un contexte de changements globaux. Afin de mieux prédire les processus écosystémiques, j’ai testé la précision et les limites des hypothèses du biomass-ratio de Grime (HBMR) et de l’annulation idiosyncratique (HAI), cette dernière étant une hypothèse originale de cette thèse. Pour cela, j’ai appliqué le principe du biomass-ratio aux traits fonctionnels, en employant la méthode des traits agrégés en communauté, pour estimer la réponse globale des espèces en mélange. La décomposition des litières plurispécifiques constitue un bon modèle biologique, pour lequel je me suis posé les questions suivantes : (1) est-ce que l’HBMR prédit bien les taux de décomposition en mélanges plurispécifiques ? ; (2) est-ce que le degré de variabilité de ces taux diminue pour des raisons biologiques avec l’augmentation de la richesse spécifique (RS) des mélanges (HAI) ? ; (3) est-ce que la variabilité des taux entre mélanges diminue quand les conditions abiotiques du site deviennent plus limitantes ? ; (4)considérant que les mélanges plus contrastés fonctionnellement sont susceptibles de développer plus d’interactions, est-ce que la déviation à la prédiction augmente avec la dispersion fonctionnelle des mélanges(« FDis », La liberté & Legendre 2010) ? Cette thèse inclut deux expériences de décomposition en sachets à litières : (1) à Sherbrooke (QC, Canada) avec des microcosmes, impliquant des litières de six espèces d’arbres, décomposant seules et en mélanges et (2) sur trois sites au climat contrasté dans la région de Clermont-Ferrand (France) avec des litières de quatre espèces d’herbacées, décomposant seules et en mélanges. Les résultats montrent des déviations positives et négatives par rapport aux taux prédits, mais l’HBMR décrit bien la réponse moyenne des litières plurispécifiques. Bien que l’HAI ait été rejetée, les résultats montrent une convergence des taux observés vers les taux prédits quand (1) la RS des mélanges augmente, (2)l’échelle spatiale augmente et (3) le climat est plus limitant pour la décomposition. Enfin, malgré des corrélations entre FDis et interactions entre espèces dans les litières, cette relation n’est pas généralisable et l’hypothèse de corrélation positive entre FDis et déviation à l’HBMR a été rejetée. / Understanding ecosystem functioning is a key goal in ecology, especially in the context of global changes. To better predict ecosystem processes, I tested the accuracy and the limits of Grime’s biomass-ratio (BMRH) hypothesis and a novel idiosyncratic annulment (IAH) hypothesis. I applied the biomass-ratio to functional traits, using the community-weighted means (CWM) to estimate the global response of species in mixtures. I studied the decomposition of litter species mixtures as a biological model and asked the following questions : (1) does the BMRH predict well the decomposition rates of mixed species litters ? ; (2) does the degree of variability of these rates decrease with increasing species richness (SR) beyond that expected from purely mathematical causes (IAH) ? ; (3) does the variability of rates between mixtures decrease with less favourable abiotic conditions for decomposition ? ; (4) as more functionally contrasted mixtures are expected to develop more interactions, does the deviation from prediction increase with increasing functional dispersion in mixtures (« FDis », Laliberté & Legendre 2010) ? This study involves two decomposition experiments using litterbags : (1) at Sherbrooke (QC, Canada), in microcosms, involving litters from six tree species, decomposed alone and in mixtures and (2) in three climatically contrasted sites in the region of Clermont-Ferrand (France) with litters from four herbaceous species, decomposed alone and in mixtures. Despite both positive and negative deviations from expectation occurring at all levels of SR, the BMRH well described the average response of mixed species litters. Although I rejected the IAH, the results showed a convergence to the predicted values based on CWM with (1) increasing the SR in mixtures, (2) increasing the spatial scale of the study and (3) a less favourable climate to decomposition. Finally, although there was a correlation between litter interactions and functional divergence, this relationship was not generalizable and I rejected the hypothesis of a positive correlation between FDis and the deviations from BMRH.

Sommaire : Comprendre le fonctionnement des écosystèmes est un enjeu crucial, en particulier dans un contexte de changements globaux. Afin de mieux prédire les processus écosystémiques, j’ai testé la précision et les limites des hypothèses du biomass-ratio de Grime (HBMR) et de l’annulation idiosyncratique (HAI), cette dernière étant une hypothèse originale de cette thèse. Pour cela, j’ai appliqué le principe du biomass-ratio aux traits fonctionnels, en employant la méthode des traits agrégés en communauté, pour estimer la réponse globale des espèces en mélange. La décomposition des litières plurispécifiques constitue un bon modèle biologique, pour lequel je me suis posé les questions suivantes : (1) est-ce que l’HBMR prédit bien les taux de décomposition en mélanges plurispécifiques ? ; (2) est-ce que le degré de variabilité de ces taux diminue pour des raisons biologiques avec l’augmentation de la richesse spécifique (RS) des mélanges (HAI) ? ; (3) est-ce que la variabilité des taux entre mélanges diminue quand les conditions abiotiques du site deviennent plus limitantes ? ; (4) considérant que les mélanges plus contrastés fonctionnellement sont susceptibles de développer plus d’interactions, est-ce que la déviation à la prédiction augmente avec la dispersion fonctionnelle des mélanges (« FDis », Laliberté & Legendre 2010) ? Cette thèse inclut deux expériences de décomposition en sachets à litières : (1) à Sherbrooke (QC, Canada) avec des microcosmes, impliquant des litières de six espèces d’arbres, décomposant seules et en mélanges et (2) sur trois sites au climat contrasté dans la région de Clermont-Ferrand (France) avec des litières de quatre espèces d’herbacées, décomposant seules et en mélanges. Les résultats montrent des déviations positives et négatives par rapport aux taux prédits, mais l’HBMR décrit bien la réponse moyenne des litières plurispécifiques. Bien que l’HAI ait été rejetée, les résultats montrent une convergence des taux observés vers les taux prédits quand (1) la RS des mélanges augmente, (2) l’échelle spatiale augmente et (3) le climat est plus limitant pour la décomposition. Enfin, malgré des corrélations entre FDis et interactions entre espèces dans les litières, cette relation n’est pas généralisable et l’hypothèse de corrélation positive entre FDis et déviation à l’HBMR a été rejetée. // Abstract : Understanding ecosystem functioning is a key goal in ecology, especially in the context of global changes. To better predict ecosystem processes, I tested the accuracy and the limits of Grime’s biomass-ratio (BMRH) hypothesis and a novel idiosyncratic annulment (IAH) hypothesis. I applied the biomass-ratio to functional traits, using the community-weighted means (CWM) to estimate the global response of species in mixtures. I studied the decomposition of litter species mixtures as a biological model and asked the following questions : (1) does the BMRH predict well the decomposition rates of mixed species litters? ; (2) does the degree of variability of these rates decrease with increasing species richness (SR) beyond that expected from purely mathematical causes (IAH)? ; (3) does the variability of rates between mixtures decrease with less favourable abiotic conditions for decomposition? ; (4) as more functionally contrasted mixtures are expected to develop more interactions, does the deviation from prediction increase with increasing functional dispersion in mixtures (« FDis », Laliberté & Legendre 2010)? This study involves two decomposition experiments using litterbags: (1) at Sherbrooke (QC, Canada), in microcosms, involving litters from six tree species, decomposed alone and in mixtures and (2) in three climatically contrasted sites in the region of Clermont-Ferrand (France) with litters from four herbaceous species, decomposed alone and in mixtures. Despite both positive and negative deviations from expectation occurring at all levels of SR, the BMRH well described the average response of mixed species litters. Although I rejected the IAH, the results showed a convergence to the predicted values based on CWM with (1) increasing the SR in mixtures, (2) increasing the spatial scale of the study and (3) a less favourable climate to decomposition. Finally, although there was a correlation between litter interactions and functional divergence, this relationship was not generalizable and I rejected the hypothesis of a positive correlation between FDis and the deviations from BMRH.

Despite the importance of a high white clover (Trifolium repens) content in temperate pastoral systems in terms of livestock performance and nitrogen fixation, the proportion of white clover in grass-clover pastures is often low (<20%). This thesis examined in two experiments whether the white clover content of pastures could be improved by sowing white clover with alternative grass species to diploid perennial ryegrass (Lolium perenne L.). In a pasture experiment, DM production, pasture composition and morphology of grass-clover mixtures was measured over the establishment year (January 2007 to January 2008) where white clover was sown in fine mixtures with diploid perennial ryegrass, tetraploid perennial ryegrass, timothy (Phleum pratense L.) and cocksfoot (Dactylis glomerata L.). Pastures were irrigated and rotationally grazed with on-off grazing with Coopworth ewe hoggets. Total annual DM production of pasture was more than 20% higher in tetraploid (12521 kg DM ha⁻¹) and diploid (11733 kg DM ha⁻¹) perennial ryegrass than timothy (9751 kg DM ha⁻¹) and cocksfoot (9654 kg DM ha⁻¹). However, timothy (5936 kg DM ha⁻¹) and cocksfoot (5311 kg DM ha⁻¹) had more than four times higher white clover annual DM production than tetraploid (1310 kg DM ha⁻¹) and diploid (818 kg DM ha⁻¹) ryegrass. Pasture growth rate at the first three harvests in autumn was significantly greater in tetraploid and diploid ryegrass than timothy and cocksfoot. Timothy and cocksfoot had a higher proportion of white clover than tetraploid and diploid perennial ryegrass throughout the entire year. This was due to more and larger white clover plants in timothy and cocksfoot plots. In a grazing preference experiment, the partial preference of sheep for white clover offered in combination with the same grass species as in the pasture experiment was measured in five grazing tests in May, September, October, November and December 2007. Pastures were sown in January 2007. Paired plots (grass and clover both 4.2 m x 10 m) were grazed by three Coopworth ewe hoggets between 9am and 5pm, and preference was recorded by decline in pasture mass and visual scan sampling for grazing time. Grazing preference for clover was generally low throughout these tests (e.g. average apparent DM intake from clover = 47%; average grazing time from clover = 44%). Several explanations are proposed for this low preference including a high N content and intake rate of the grass relative to the clover. No significant differences were found among the grass treatments in total grass grazing time, total clover grazing time, ruminating time, the proportion of grazing time on clover, selective coefficient for clover and DM intake percentage from clover at any date. There was no significant change in overall sward surface height (SSH) decline among grass treatments throughout all the tests except December 2007 when the overall SSH decline for cocksfoot was significantly lower than the other species. The study indicated that the rapid growth rate of perennial ryegrass in the early phase of pasture establishment, rather than differences in partial preference, was the key factor limiting white clover content in the mixed swards relative to cocksfoot and timothy pastures. It is concluded that high clover-containing pastures capable of delivering high per head performance can be established through the use of slow establishing pasture species such as timothy and cocksfoot.

The Canterbury plains are frequently exposed to summer drought and climate predictions forecast that the severity and frequency of summer drought will increase. The most commonly used pasture grass, perennial ryegrass (Lolium perenne L.), is drought sensitive. One possible method to maintain sward dry matter (DM) production under water stress is to use an alternative grass species such as tall fescue (Festuca arundinacea Schreb.). The objective of this research was to compare summer DM production of monoculture swards of perennial ryegrass and tall fescue under various seasonal drought regimes to study physiological and biochemical drought responses of each species. Data were collected over a period of two-summer seasons, Season One (2006-2007) and Season Two (2007-2008) in an automated rain shelter at Lincoln, Canterbury. Drought treatments included exposure of plants to a spring or autumn drought or a four-weekly "irrigated" drought regime. DM yields of the two species were similar under each watering regime. The control treatment, under non-limiting conditions, has the highest accumulated yield in both Season One and Two for ryegrass (17.1 and 15.7 t DM ha⁻¹) and tall fescue (18.8 and 16.0 t DM ⁻¹) respectively. Spring and autumn drought treatments were similar for the two species in accumulated yield in either season, however the exposure to drought stress returned yields lower than the control. Consistently, the lowest-yielding treatment was the four-weekly irrigated drought, which resulted in an average yield across species in Season One of 10.1 t DM ha⁻¹ and 8.35 t DM ha⁻¹ in Season Two. Growth rates of the swards were calculated using accumulated DM production against accumulated thermal time using a base temperature of 3°C for both species. The control treatments showed a strong linear relationship for both species in both seasons, though Season Two showed a period of approximately 390 °Cd of no growth. Spring growth was similar for all treatments until October when both the spring drought and four-weekly irrigated treatment deviated from the control as water stress commenced. Growth also ceased under autumn drought later in the season. The physiological drought responses between species and among treatments differed. Tall fescue under control conditions had the highest photosynthesis rates of 20.5 µmol CO₂ m⁻² s⁻¹,or 22% higher than ryegrass, whereas the four-weekly irrigated treatment showed no inter-species differences. Differences were also found for other gas exchange parameters. Physiological water use efficiency (phys WUE) in ryegrass was 15% greater than tall fescue in Season Two. Photosynthesis and gas exchange rates against leaf water potential showed declining gas flow in both species across all treatments in response to drying soil conditions and across all irrigation treatments. The osmo-protectant proline was 22% higher in concentration in ryegrass than in tall fescue in Season Two and increased in drought stressed treatments in both seasons. Water stress was found to reduce total chlorophyll concentrations in all treatments and in tall fescue, while little change occurred in the chlorophyll a:b ratio. In conclusion, the findings from this thesis suggest similar DM responses for the two species under drought. The findings suggests that tall fescue performs more as a "water user" under drought conditions, compared with perennial ryegrass, which is more a "water saver." Resonses to the changing environment to a point, before "shuttting up shop" through lower stomatal conductance.

Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

This study consisted of two sheep grazed dryland pasture experiments. Experiment l compared sheep production from 3-year-old cocksfoot based pastures grown in combination with white, Caucasian, subterranean or balansa clover with a ryegrass-white clover pasture and a pure lucerne forage. Sheep liveweight gain per head from each pasture treatment and the pure lucerne stand was recorded in the 2006/07 and 2007/08 seasons. The cocksfoot-subterranean clover pasture provided equal (381 kg LW/ha in 2006) or higher (476 kg LW/ha in 2007) animal production in spring and gave the highest total animal production (646 kg LW/ha) averaged across years of the five grass based pastures. However, total annual liveweight production from lucerne was higher than any grass based pasture mainly due to superior animal production during summer when lucerne provided 42-85% higher animal production than any of the grass based pastures. In Experiment 2, the effect of stocking rate (8.3 (low) and 13.9 (high) ewes + twin lambs/ha) and time of closing in spring on lamb liveweight gain, pasture production and subterranean clover seedling populations was monitored over 2 years for a dryland cocksfoot-subterranean clover and ryegrass-subterranean clover pasture in Canterbury. In both years, twin lambs grew faster (g/head/d) in spring at low (327; 385) than high (253; 285) stocking rate but total liveweight gain/ha (kg/ha/d) was greater at high (7.26; 7.91) than low (5.43; 6.38) stocking rate. Ewes also gained 0.5 and 1.5 kg/head at the low stocking rate in 2006 and 2007 respectively but lost 0.2 kg/head in 2006 and gained 0.3 kg/head at high stocking rate in 2007. Mean subterranean clover seedling populations (per m²) measured in autumn after grazing treatments in the first spring were similar at both low (2850) and high (2500) stocking rate but declined with later closing dates in spring (3850, 2950, 2100 and 1700 at 2, 4, 6, 8 weeks after first visible flower). Seedling populations measured in autumn after grazing treatments in the second spring were also unaffected by stocking rate (low 1290, high 1190) but declined with later closing dates in spring (1470, 1320 and 940 at 3, 5 and 8 weeks after first flowering, respectively). The effect of stocking rate and closing dates in spring on pasture and clover production in the following autumn was similar to the effects on seedling numbers in both years. However, clover production in the following spring was unaffected by stocking rate or closing date in the previous year at the relatively high seedling populations generated by the treatments. This was presumably due to runner growth compensating for lower plant populations in pastures that were closed later in spring. Subterranean clover runner growth in spring may not compensate in a similar manner if seedling numbers in autumn fall below 500/m². Mean annual dry matter production from cocksfoot and ryegrass pastures grown with and without annual clovers pasture production ranged from 6.4 to 12.4 t DM/ha/y but stocking rate (8.3 vs. 13.9 ewes/ha) during spring did not affect annual pasture production. Pastures overdrilled with annual clovers yielded 23-45% more dry matter production than pastures grown without annual clovers. The study confirms the important role of subterranean clover in improving pasture production and liveweight gains of sheep in dryland cocksfoot and ryegrass pastures. Lowering stocking rate from 13.9 to 8.3 ewes/ha was a less effective method of increasing seed production of subterranean clover in dryland pastures although it did lead to increased liveweight gain per head.

Although New Zealand is fortunate in having a climate mild enough to allow some growth of pasture in winter even in the extreme south of the South Island (Duffy, 1971), growth is nevertheless considerably less than in spring and summer. O’Connor et al, (1968) point out that at Lincoln, Canterbury, winter production from a New Zealand Certified Grasslands Ruanui Perennial ryegrass (Lolium perenne c.v.) and New Zealand Certified Grasslands Huia white clover (Trifolium repens c.v.) sward is at best only 8% of the mean total annual dry matter (D.M.) production. A similar seasonal pattern is shown by irrigated pastures (Rickard, 1968), as well as pastures in the milder North Island of New Zealand (O’Connor and Vartha, pers comm.). Stocking of grazing animals is normally related to the growth rate of pastures in the spring, with conserved hay or silage, plus specially grown greenfeed or root crops being fed if necessary to offset the winter feed deficit. However on hill country too steep for tractor cultivation, cropping and conservation are not possible. Animals have to rely upon in situ grazing of native or dominant browntop (Agrostis tenuis) swards which may not have been improved by the aerial introduction of clovers. In an unimproved state, the quality and quantity of the herbage grown on such areas are low, Molloy (1966) have discussed clover introduction into native swards and the notably beneficial result this has on stock thrift, particularly during the winter period. Considerably better growth rates of stock grazing legumes rather than grasses have been noted by many authors including Ulyatt, (1971), and McLean et al (1962); this superiority being due largely to the greater digestible organic matter intake and higher ratio of soluble to insoluble carbohydrate associated with the herbage of legumes (Ulyatt, 1971). Consequently the presence of clover, and the winter activity of this, have an important influence on the productivity of steep hill country during winter. An increase of winter activity is also desirable, but not essential, in clover associated with flat or gently rolling pastures. The main pasture legume used in New Zealand has, in the past, been the white clover cultivar Huia. Whilst since 1945 selection and breeding of ryegrasses has brought about a very considerable improvement in the winter or cool season activity of these (Corkill, 1966), no legume cultivar having an increased level of cool-season activity has been released to commerce in New Zealand over the same period to date. Breeding for increased winter growth has been an objective of the Grasslands Division Plant Breeding Section for many years (Barclay, 1960). Since 1957, breeding and selection work has been proceeding with the objective of increasing the winter growth of the New Zealand Certified Grasslands white clover cultivar – Huia (henceforth to be referred to as Huia) without sacrificing the moderately good summer growth of this strain, (Barclay, 1969). Seed of a promising cultivar selected during the course of this work – New Zealand Grasslands 4700 white clover, (henceforth to be referred to as 4700) became available for testing in 1967. The investigation to be reported here aimed to measure the cool-season activity of 4700 by comparison with that of Huia, at the same time elucidating if possible the factors controlling this growth.