Thesis (Ph. D.) -- Lincoln University, 2007. / Also available via the World Wide Web.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2004. / Härtill 4 uppsatser.

Three studies applicable to organic management and cultivation of birdsfoot trefoil (BFT, Lotus corniculatus L.) are presented here. The first is a histological analysis of lignification in BFT stems that supports recommendations to harvest BFT at approximately 6 weeks of regrowth, or early bloom. Lignification decreases digestibility and is correlated with high shear force required to break BFT stems. The sixth internodes from the base of 10 BFT and two alfalfa plants were sampled during 15 weeks of midsummer regrowth. The lignified xylary ring reached its maximum radial width by 7.5 weeks of regrowth, and full bloom was reached by 6.5 weeks of regrowth. The second study evaluated establishment of BFT, which competes poorly with weeds and produces low yields during establishment. At an organic, irrigated site in northern Utah the effects of autumn vs. spring seeding, seeding rates of 3, 7 20, and 34 kg pure live seed (PLS) ha-1 and use of a companion crop on 3 years of annual yields and foliar cover of mature stands were determined. First year yields increased linearly with seeding rate (P≤0.05). Use of a companion crop reduced first year yields, and did not reduce weed cover. Autumn seeding of BFT alone, following harvest of a summer crop, is recommended for irrigated production. The final study evaluated BFT establishment on five organic dairy farms in souther Idaho and northern Utah. Participating producers broadcast seeded 4-ha BFT pastures in the fall at a rate of 25 kg PLS ha-1. All farms achieved high BFT density, but only two farms had higher BFT than weed density. These two farms also had high BFT cover the spring following autumn seeding, and their pastures produced 6000 to 7600 kg of dry matter ha-1 by 20 June 2012 and supported grazing for the remainder of the summer. Establishment was enhanced by crop rotation and sprinkler irrigation.

To make use of the information on infestation estimates of forage insect pests obtained by the conventional methods of sampling, it is important to estimate the actual losses taking place in association with definite numbers of the insect. Six month old red clover, alfalfa, and birdsfoot trefoil plants were exposed for a month to spittlebug infestations of 0, 50, 100, and 150 adults. Although the spittlebug mortality was lowest when reared on alfalfa and highest on birdsfoot trefoil, yellowing appeared on alfalfa and birdsfoot trefoil. On red clover there was a consistent increase in protein and nitrogen free extract and a reduction in crude fiber and ash content indicating slight reduction in growth. None of these differences were significant. On alfalfa the loss of xylem sap due to feeding caused a consistent general reduction of proteins, fat and ash content and an increase in dry matter and nitrogen free extract. Of these constituents only the increase in dry matter was significant with an infestation of 150 spittlebugs per cage. No differences in dry matter, proteins and fat were significant. Another test on 4 month old alfalfa with infestation levels of 0, 100, 200 and 300 showed similar consistent patterns but the difference in dry matter was significant only when the infestation was 200 spittlebugs or more. On birdsfoot trefoil there was a consistent increase of dry matter, proteins, and ash and reduction of fat and crude fiber. Only the difference of dry matter and fats was consistent showing that an infestation of 100 or more spittlebugs per sq ft will affect the general growth of the plants. The survival of 1st instar nymphs of the pea aphid (a phloem feeder) was very poor on birdsfoot trefoil. There was a fair amount of survival on red clover and those on alfalfa survived fully. There was no visible symptoms on birdsfoot trefoil and red clover but the alfalfa showed retarded growth. On red clover there was a consistent increase of dry matter, fat, crude fiber and nitrogen free extract but a reduction of proteins. The difference in crude fiber was significant when the infestation was 1000 pea aphid nymphs or more. A consistent reduction of proteins and crude fiber was also recorded on alfalfa but these differences were not significant. On birdsfoot trefoil, there was a decrease in dry matter and an increase in fat and ash content. The difference in ash content was significant when the infestation of pea aphid nymphs was 1000 or more. Potato leafhopper (phloem feeder) injury was very characteristic on birdsfoot trefoil. The terminal 1 to 2 inches of the sterns became wilted and bent down. On alfalfa there was gradual yellowing and the retarded growth was evident. The red clover did not show any characteristic symptoms. Infestations of 20 or more caused a consistent increase in protein, fat, crude fiber and a reduction of nitrogen free extract. On alfalfa there was consistent decrease in dry matter. On birdsfoot trefoil, there was consistent increase in dry matter, fat, and nitrogen free extract but a reduction of crude fiber showing that feeding on birdsfoot trefoil probably retards growth and condensation of carbohydrates to form crude fibers. The increase in fat was significant when the infestation was 40 potato leafhoppers or more. Alfalfa weevil adult feeding caused characteristic stripping of leaves, girdling of stem and drying of the entire plant. Feeding of 10 or more weevils for 18 days on a two month growth of alfalfa after the 1st cutting, cause a consistent reduction of proteins, fat and crude fiber and an increase of dry matter, ash, and nitrogen free extract. Increase of percent dry matter was significant when the infestation of alfalfa weevil adults was 20 or more. In another test, the dry matter, fat and ash gave similar consistent results, when 40, 60, and 80 adults were fed on 4 month old alfalfa for 18 days. Reduction of percent fat was significant when the infestation was 40 or more alfalfa weevil adults. Alfalfa weevil larval feeding showed stripping of the leaves and buds. Infestation of 30 or less, 2nd to 4th instar larvae for 12 days caused a consistent reduction of fat and ash and an increase of dry matter, proteins and crude fiber. The infestation was not high enough to show a significant change in plant constituents. / Ph. D.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2018-06-14T13:19:23Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_tese_natalia_silveira_correa.pdf: 16569 bytes, checksum: ed2405236282a6dfb013ce0c61b1b3aa (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-06-14T20:52:30Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_tese_natalia_silveira_correa.pdf: 16569 bytes, checksum: ed2405236282a6dfb013ce0c61b1b3aa (MD5) / Made available in DSpace on 2018-06-14T20:52:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_tese_natalia_silveira_correa.pdf: 16569 bytes, checksum: ed2405236282a6dfb013ce0c61b1b3aa (MD5) Previous issue date: 2017-06-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O objetivo deste trabalho foi avaliar a viabilidade, o vigor da semente e o crescimento de plantas de cornichão e trevo vermelho, provenientes de áreas da Embrapa Pecuária Sul, submetidas a diferentes doses de herbicidas pré e pós emergentes (diclosulam, bentazona e iodosulfurom-metílico), com a finalidade de identificar os herbicidas seletivos à estas forrageiras. Na primeira etapa do experimento foi avaliado o efeito do herbicida pré-emergente diclosulam no potencial fisiológico das sementes e crescimento inicial das plântulas. As sementes das forrageiras, foram semeadas em substrato previamente umedecido com solução de diclosulam nas concentrações de 0; 0,5; 1,0 e 1,5 vezes a dose registrada do produto pelo fabricante. Na segunda etapa, após o cultivo em casa de vegetação por 107 dias, foi realizada a aplicação dos pósemergentes, por meio de pulverizador costal de pressão constante em condições atmosféricas de 23º C de temperatura e 71% de umidade relativa do ar (UR). Foram utilizadas concentrações do herbicida referentes a zero; 0,5; 1,0 e 1,5 vezes a dose registrada pelo fabricante. Os resultados da primeira etapa, demostraram que as variáveis comprimento da parte aérea, comprimento de raiz e massa seca total foram alteradas negativamente pela exposição às diferentes doses de diclosulam, enquanto o potencial germinativo e a capacidade de emergir não foram prejudicadas, em ambas as espécies estudadas. Na segunda etapa os resultados obtidos mostraram maior sensibilidade das plantas de cornichão ao herbicida bentazona e das plantas de trevo vermelho ao herbicida iodosulfurom-metílico. Portanto é possível concluir que, as diferentes doses de diclosulam não comprometem o potencial germinativo, nem a capacidade de emergir das sementes de cornichão e trevo vermelho, porém, interferem negativamente no seu desenvolvimento. Já com a aplicação dos pósemergentes, o cornichão apresentou sensibilidade ao herbicida bentazona e características de tolerância ao herbicida iodosulfurom-metílico, enquanto o trevo vermelho respondeu de forma contrária, sendo mais sensível a aplicação do iodosulfurom-metílico e tolerante ao bentazona, ativando mecanismos de adaptação e permitindo o desenvolvimento da planta. / The objective of this research was to evaluate viability, seed vigor and growth of birdsfoot trefoil and red clover, plants from Embrapa Pecuária Sul, was submitted to different doses of pre and post emergent herbicides (diclosulam, bentazon and iodosulfuron-methyl), with purpose to identify herbicides selective to these forages. In the first stage of the experiment the effect of the pre-emergent herbicide diclosulam in physiological potential of seeds and initial growth of seedlings was evaluated. The seeds of this forages were sown on a substrate previously moistened with diclosulam solution at concentrations 0; 0,5; 1,0 and 1,5 times the dose registred by manufacturer. In second stage, after the cultivation in the greenhouse for 107 days, the postemergence application was carried out by a costal sprayer with constant pressure at atmospheric conditions of 23 ºC and 71% relative humidity). Herbicide concentrations used were zero; 0,5; 1,0 and 1,5 times the dose registred by manufacturer. The results of the first stage showed that the variables shoot length, root length and total dry mass were negatively altered by exposure to different doses of diclosulam, while germination potential and emergence capacity were not impaired in both species studied. In the second stage, the results obtained showed greater sensitivity of birdsfoot trefoil plants to herbicide bentazon and the red clover plants to iodosulfuron-methyl herbicide. Therefore, it is possible to conclude that, different doses of diclosulam do not compromise germination potential, neither ability to emerge from birdsfoot trefoil and red clover seeds, but negatively interfere in its development. With the post-emergence application, the birdsfoot trefoil showed sensitivity to herbicide bentazon and characteristics of tolerance to herbicide iodosulfuron-methyl, while the red clover responded in a contrary way, being more sensitive the application of the herbicide iodosulfuron-methyl and tolerant to bentazon, activating mechanisms of adaptation and allowing plant development.

Five rotational grazing experiments were carried out at Massey University's Riverside farm, in the Wairarapa, on the East Coast of the Southern North Island, New Zealand, to compare the effects of feeding Lotus corniculatus L. (birdsfoot trefoil; cv. Grasslands Goldie) or perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) dominant pasture upon sheep year round productivity. These studies also investigated under grazing, seasonal and annual net herbage accumulation rate and seasonal dynamics of undisturbed (i.e. non-grazed) net herbage accumulation rate of L. corniculatus relative to that of grass-dominant pasture. Aspects of in vivo digestibility of dry matter (DMD), organic matter (OMD), digestible organic matter in the dry matter (DOMD) and metabolisable energy (ME) concentration of L. corniculatus at different stages of maturity over the spring, summer and autumn were investigated in three indoor digestion trials. 1. Two field experiments (Chapter 2) were conducted during spring to assess the effects of grazing mixed age undrenched ewes on L. corniculatus (n = 50) or pasture (n = 50) and their lambs (mainly twins) on live weight (LW), wool production, faecal nematode egg count (FEC) and dag score. In Experiment 1 (18 October 2000 to 21 January 2001) and Experiment 2 (3 October 2001 to 2 January 2002) both forages were fed ad libitum. Total condensed tannin (CT) concentration in the diet selected was 24 to 27 g CT/kg DM for L. corniculatus and 1.4 to 1.5 g CT/kg DM for pasture. The LW gain, weaning LW and wool production were consistently greater (P < 0.001) for lambs grazing L. corniculatus, in either Experiment 1 (258 vs. 189 g/day; 36.1 vs. 30.1kg; 1.17 vs. 0.98 kg) and in Experiment 2 (247 vs. 162 g/day; 31.8 vs. 24.1kg; 1.17 vs. 0.81 kg), respectively. Ewe and lamb dag scores were strongly and positively correlated with dag weight (P < 0.001) and generally increased with time in sheep grazing pasture, whilst grazing on L. corniculatus consistently reduced dag score. FEC in ewes grazing pasture showed a post-parturient rise (PPR) following lambing, whilst ewes grazing L. corniculatus had a reduced PPR in FEC. Up to day 70, FEC in lambs grazing L. corniculatus was lower than that for lambs grazing pasture, but between day 70 and the end of both experiments (approximately day 90), FEC in lambs grazing L. corniculatus increased to similar values as for pasture-fed lambs. FEC was not correlated with dag score or dag weight in ewes or lambs grazing pasture, but these indices were weakly and positively correlated in ewes and lambs grazing L. corniculatus, suggesting that lowering FEC on L. corniculatus also reduced dag formation. It was concluded that under dryland farming conditions, the use of L. corniculatus during the spring/early summer lactation period can increase lamb LW and wool production, whilst eliminating the need for pre-lambing anthelmintic drenching and probably reducing the amount of insecticide needed to control flystrike. These effects compared to pasture are probably due to higher digestibilty, higher ME concentration, higher voluntary feed intake (VFI), and to the effect of CT in reducing rumen protein degradability and controlling internal parasites in sheep grazing L. corniculatus. The absence of endophyte in L. corniculatus may have also have contributed to these effects. 2. During 2001 and 2002 (Chapter 3), grazing trials from February to November were conducted for 279 days (Experiments 1) and 285 days (Experiment 2), to compare the effects of grazing shorn mixed age Romney ewes in light condition on L. corniculatus versus pasture during the mating period (9 weeks, Experiment 1) and 11 weeks (Experiment 2). In Experiment 2, the length of time (days) that ewes need to graze L. corniculatus before mating to maximise reproductive performance was also investigated. Common objectives in both Experiments were to measure forage feeding effects on ewe wool production and LW of their lambs at weaning. In Experiment 1, groups of ewes (n = 100) were fed on either L. corniculatus or pasture at a herbage allowance of 1.8 kg green DM/ewe/day for the first three weeks of feeding and increased to ad libitum (2.3 kg green DM/ewe/day) during the mating period for two cycles. In Experiment 2, groups of 75 ewes grazed L. corniculatus for 42, 21, 10 and 0 days before a synchronised oestrus, with pasture being grazed for the balance of the 42 days. All L. corniculatus groups continued grazing L. corniculatus for a further 5 weeks. Feed allowance was initially 2.0 kg green DM/ewe/day, increased to 2.3 kg green DM/ewe/day during the mating period over the two cycles. At the end of L. corniculatus feeding in both experiments the groups were combined and grazed on pasture until weaning. Total CT concentration in the diet selected was 18 to 29 g CT/kg DM for L. corniculatus, with only trace amounts in pasture. In Experiment 1 mating ewes on L. corniculatus compared to pasture increased number of lambs born and lambs weaned per ewe lambing by 16 and 32% units respectively (P < 0.05), due to more multiple and less single births (P = 0.06) and to reduced lamb mortality (P < 0.05) between birth and weaning. In Experiment 2, increasing the numbers of days of grazing L. corniculatus before ovulation (0, 10, 21, 42 days) linearly increased ovulation rate (P < 0.05), lambs born and lamb weaned by up to 16% units, but had no effect upon lamb mortality. Mating ewes on L. corniculatus increased wool production (P < 0.01) and fibre length (P < 0.05) in Experiment 1 but not in Experiment 2. Grazing L. corniculatus had no effect on lamb birth weight and only small positive effects on weaning LW. It was concluded that, under commercial dryland farming conditions, the use of L. corniculatus during the mating season in late summer/autumn can be used to increase reproductive efficiency and wool production, with the largest responses in years with exceptionally dry autumn periods. These effects are probably due to the higher digestibility and ME concentration of L. corniculatus than pasture and to the CT in L. corniculatus reducing rumen protein degradability and leading to greater essential amino acid (EAA) absorption from the small intestine. Effects of forage CT upon the uterine microenvironment at the time of conception, implantation and early foetal growth, need to be investigated in future studies. It is also suggested that effects of mating on L. corniculatus upon lamb mortality between birth and weaning should be further investigated with ewe numbers/treatment increased from 100 to 350. 3. During the summer of 2002/2003, another grazing trial (Chapter 4: 95 days) compared the effects of grazing L. corniculatus and pasture on LW and the dynamics of nematode parasite infection in Suffolk x Romney weaned lambs fed ad libitum. Half of the lambs (n = 30) grazing either L. corniculatus or pasture received oral anthelmintic at the start and at monthly intervals (regular-drenched groups), whilst the remaining 30 lambs in each treatment only received oral anthelmintic when mean faecal nematode egg counts (FECs) exceed 1,000 eggs/g wet faeces (trigger-drenched groups), which occurred on day 58 only for both groups. Trigger and regular-drench lambs grazed separate areas. Total CT concentration in the diet selected was 40 to 31 g CT/kg DM for L. corniculatus, with only trace amounts in pasture. Regular-drenched lambs grazing L. corniculatus had significantly higher LW gain (298 g/day) and carcass weight gain (133 g/day) than all the other groups, whilst trigger-drenched lambs grazing L. corniculatus had significantly greater LW gain (228 g/day) and carcass gain (99 g/day) than regular-drenched (200; 66 g/day) and trigger-drenched (187; 63 g/day) lambs grazing pasture. Carcass fatness was significantly lower for trigger-drenched lambs than for regular-drenched lambs, when fed either L. corniculatus or pasture. Dag score was consistently lower for regular-drenched lambs grazing L. corniculatus than pasture; trigger-drenched lambs showed similar effects up to day 48, with no differences between the two groups thereafter. Regular anthelmintic treatment maintained FECs at low values, while parasitised lambs on L. corniculatus tended to have higher FECs than pasture-fed lambs. Relative to trigger-drenched lambs that grazed pasture, grazing trigger-drenched lambs on L. corniculatus had significantly reduced worm burdens of Haemochus contortus, Teladosargia spp., Nematodirus spp. and Cooperia spp. at slaughter, but greater burdens of Trichostrongylus spp., Chabertia ovina, Oesophagostonum spp. and Trichuris ovis were present in L. corniculatus-fed lambs. It was concluded that grazing L. corniculatus under dryland farming conditions compared to pasture can increase LW gain of weaned lambs, whilst reducing reliance on anthelmintic drenches to control parasites. These effects are probably due to increased protein supply from the action of CT enabling the lambs to have a higher LW gain when carrying a parasite burden, and to L. corniculatus better maintaining its high ME value under drought conditions. Using L. corniculatus to finish weaned lambs without anthelmintic drenches for a seven-week period is proposed. 4. A three-year study (Chapter 5; November 2000 to October 2003) was conducted to compare, under grazing conditions, seasonal and annual grazed net herbage accumulation rate and seasonal dynamics of undisturbed (i.e. non-grazed) net herbage accumulation rate of L. corniculatus relative to grass-dominant pasture. Prediction equations to estimate standing DM in L. corniculatus and pasture from the rising plate meter (RPM) and sward surface height were also generated. L. corniculatus and pasture growing in a moderate fertility and low-pH soil (pH 5.35) accumulated similar total herbage masses (24.3 vs. 24.1 t DM/ha) over the 3-year period, with the DM production being greater for L. corniculatus than for pasture during 2000-2001, producing more DM during summer/autumn drought conditions. The net herbage accumulation rate from undisturbed areas of L. corniculatus and pasture were similar in spring, summer and autumn. Seasonal variation in the calibration regressions fitted to estimate herbage mass of L. corniculatus non-destructively, suggested a combination of destructive and nondestructive methods are needed to assess herbage mass. It was concluded that L. corniculatus has the potential to increase the performance of a pasture-based sheep dryland farming system due to its ability to grow in acidic soils, its tolerance of drought conditions during summer/autumn and its seasonality of feed supply. 5. Three digestion experiments involving cryptorchid weaned lambs were conducted for 14 days over the spring, summer and autumn to determine changes in in vivo digestibility of DM, OM, digestible OM in the DM and ME concentration of L. corniculatus at different stages of maturity. In vivo digestibility samples were then used as standards to investigate if the enzymatic in vitro system of Roughan and Holland (1977) could predict OMD and DOMD of CT-containing L. corniculatus. Digestibility of L. corniculatus declined as it matured, but the rate of decline was much less than occurs for temperate grasses and for white clover. It was concluded that the in vitro enzymatic system of Roughan and Holland (1977) can be used to predict OMD and DOMD of L. corniculatus, provided a standard curve involving in vivo data generated with L. corniculatus is used. Using a standard curve with in vivo data from pasture led to bias which increased at lower OMD values. Reasons for the consistent differences between L. corniculatus and pasture standard curves are discussed, including possible effects of residual bound CT in lowering in vitro digestibility. From this series of experiments, this study is the first to report that relative to conventional perennial ryegrass/white clover, mating ewes on L. corniculatus under grazing conditions may reduce post-natal lamb mortality. It is also the first study to show that grazing sheep on L. corniculatus can maintain productivity during spring and summer with reduced dependence on anthelmintic drench input. It is concluded that whole farm modelling, mechanical harvesting and conservation strategies, selection of L. corniculatus germplasm for creeping-type plants more suited to grazing and the integration of new crops containing secondary compounds, such as chicory, should be considered to support major advances in sustainable dryland sheep farming systems.

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.

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.

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.