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Grain protein content (GPC) in wheat has been found to be regulated by the gene NAM-B1. Homologues to the NAM-B1 gene have been found in barley, HvNAM-1 and HvNAM-2. Previous studies have found that base mutations in the NAM-1 gene at base position 544 might have an impact on GPC. Previous studies also found that landrace of barley showed higher GPC than cultivated barley, indicating that plant improvement might have affected base mutations and therefore GPC. I wanted to study if there are any nutritional differences in Nordic barley and if those differences might correlate with haplotypes. Comparisons of barley varieties from four Nordic countries, and two varieties from the US used as low and high GPC controls, did not show any significant differences depending on their origin country and no differences regarding plant improvement status between the countries. When sequencing Nordic barley varieties, five haplotypes were found for the gene HvNAM-1, and two haplotypes for the gene HvNAM-2. A low polymorphism for both genes indicate a strong natural selection for the consensus haplotype which might be preferable for Nordic climate with a short growing season and cold temperatures. Even though it is not clear what is the cause of the low polymorphism in Nordic barley varieties, they showed a generally higher nutrient content than barley varieties of the high GPC and may be suitable for breeding for a yield with a high nutrient content.

Kiwifruit are of huge economic importance for New Zealand representing 29 percent of total horticultural exports. Fruit size is the biggest determinant of what consumers are willing to pay, and there is also a positive relationship between consumer preference for flavour and percentage dry matter. The two main cultivars exported from New Zealand are Actinidia chinensis ‘Hort 16A’ (gold kiwifruit) and A. deliciosa ‘Hayward’ (green kiwifruit). Under current commercial practice the only product allowed for use on kiwifruit to increase fruit size in New Zealand is Benefit®. Benefit® has been shown to induce different results when applied to A. chinensis and A. deliciosa, whereas synthetic plant growth regulators such as the cytokinin-like substance N-(2- chloro-4-pyridyl)-N’-phenylurea (CPPU) have been found to promote similar increases in fresh weight of fruit in both cultivars. Final fruit size is determined by both cell division and cell enlargement. It was been shown that fresh weight can be increased in both of the major Actinidia cultivars even though their physiology differs. Hormonal control of fruit size in relation to cell division and cell enlargement phases of fruit growth was studied in both A. chinensis and A. deliciosa. CPPU was applied to both cultivars in a growth response experiment where fruit were collected throughout the growing season. The objective of this experiment was to create growth curves, to compare and contrast the effect on A. chinensis and A. deliciosa, and to provide material for hormone analysis. Application of CPPU was found to significantly increase the fresh weight of both A. chinensis and A. deliciosa fruit (46.98 and 31.34 g increases respectively), and alter the ratio of inner and outer pericarps of A. chinensis fruit. CPPU and Benefit® were applied individually and together to both cultivars. It was found that only A. chinesis fruit were affected by the application of Benefit®; fresh weight was increased by 26.38 g, and percentage dry matter was significantly reduced. There was a statistically significant (p < 0.05) interaction between CPPU and Benefit® when applied to A. chinensis. 3,5,6-trichloro-2-pyridyloxyacetic acid (3,5,6-TPA) was applied to A. deliciosa on two application dates at three concentrations and was found to decrease fresh weight of fruit, but significantly increase percentage dry matter regardless of application date or concentration. Lastly CPPU and 1-naphthalene acetic acid (NAA) were applied to A. deliciosa at two application dates and in all combinations. Application date affected the response to both a low concentration of CPPU and NAA. A synergistic interaction was observed when CPPU was applied early plus NAA late (CPPU early (4.53 g increase) plus NAA late (13.29 g) < CPPU early plus NAA late (33.85 g). Finally endogenous hormone content was studied. Methods were developed and tested for the simultaneous analysis of both indole-3-acetic acid (IAA) and cytokinins. Freeze dried fruit were purified using Waters Sep-pak® cartridges and Oasis® columns then IAA was quantified by high pressure liquid chromatography. Preliminary results indicate a correlation between application of CPPU and endogenous IAA, high concentrations of IAA correlated well with periods of rapid fruit growth particularly for CPPU treated fruit.

Les furocoumarines sont des composés phénoliques impliqués dans la défense contre les herbivores. Ces molécules sont majoritairement décrites dans quatre familles botaniques, notamment les Rutaceae, dont font partie les agrumes. Ces molécules sont phototoxiques ce qui peut poser des problèmes pour leur utilisation comme par exemple en cosmétique ou en phytothérapie. D’autre part, en cas d’ingestion par exemple via la consommation de jus de certains agrumes, elles ont responsables de l’inhibition d’enzymes de détoxication comme le CYP3A4 humain. Cela peut conduire à des surdosages médicamenteux connus sous le nom d’Effet Pomelo. Ce travail de thèse a consisté à réfléchir et à développer, des outils qui permettront de générer de manière ciblée des variétés de pomelo qui ne produisent plus de furocoumarines. Nous avons abordé l’ensemble des étapes essentielles pour la mise en place d’une stratégie global : i) des méthodes reproductibles ont été développées pour la production de protoplastes et de cultures cellulaires de pomelo Star Ruby ; ii) des conditions de transformation de protoplastes par électroporation ont également été mises au point ; iii) finalement, pour inhiber de manière spécifique la voie de biosynthèse des furocoumarines, nous avons choisi de mettre en œuvre une approche d’édition de génome en utilisant une méthodologie CRISPR/Cas9. La mise au point de la méthode a été réalisée avec un gène codant pour une umbelliferone 6-dimethylallyl transférase. Les résultats obtenus indiquent que la stratégie est envisageable. Pour renforcer la stratégie CRISPR/Cas9, nous avons mis en œuvre une démarche d’identification de gènes cibles additionnels. En utilisant une approche de data mining de bases de données génomiques et transcriptomiques nous avons identifié 18 séquences candidates, potentiellement impliquées dans la voie de biosynthèse des furocoumarines. L’expression hétérologue des protéines correspondantes et leur caractérisation fonctionnelle a permis de montrer que CYP706J12 est en mesure de métaboliser l’hérniarine, une coumarine. Ce résultat apporte des éléments pour émettre des hypothèses sur l’évolution convergente de la synthèse des coumarines et des furocoumarines chez les végétaux supérieurs. / Furanocoumarins are phenolic compounds involved in defense against herbivores. These molecules are mainly described in four botanical families. Rutaceae, one of those families, includes Citrus species. Furanocoumarins are phototoxic compounds, which can be problematic for their use in cosmetics or in phytotherapy. Furanocoumarin ingestion via citrus juice consumption, may inhibit human enzymes of detoxification, such as human CYP3A4. This can lead to drug overdoses known as the “Grapefruit Juice Effect”. This work consisted in the development of tools that will allow to generate new varieties of pomelo that no longer produce furanocoumarins by targeted genome edition. We have covered the essential steps for the implementation of a global strategy: i) reproducible methods have been developed for the production of protoplasts and cell cultures of Star Ruby grapefruit; ii) conditions for protoplast transformation by electroporation have also been developed; iii) finally, to specifically inhibit the furanocoumarin biosynthetic pathway, we chose to implement a genome editing approach using a CRISPR / Cas9 methodology. The development of the method was carried out with a gene encoding umbelliferon 6-dimethylallyltransferase. The results obtained indicate that the strategy is feasible. To strengthen the CRISPR / Cas9 strategy, we implemented a method to identify additional target genes. Using a data mining approach of available genomic and transcriptomic databases we identified 18 candidate sequences potentially involved in the furanocoumarin biosynthetic pathway. Heterologous expression of the corresponding proteins and their functional characterization made it possible to show that CYP706J12 is able to metabolize herniarin (a coumarin). This result provides elements to hypothesize about the convergent evolution of coumarin and furanocoumarin synthesis in higher plants.

A number of morphological, floral, rhizome, root and herbage characteristics were studied in order to describe genetic variation and environmental responses in T. ambiguum. From each of six lines, 30 genotypes were clonally progagated into five diverse edaphic and altitudinal sites and grown for one season. Comparisons of growth and form were also made between T. ambiguum, T. repens cv. 'Grasslands Huia' and Lotus pedunculatus cv. 'Grasslands Maku'. The polyploid lines were found to have larger leaves than the diploids while all the lines of T. ambiguum had larger leaves than did white clover. Tetraploid lines had longer petioles and were taller and more erect than the diploids or hexaploids. Floral initiation was found to become later as ploidy level increased but the tetraploid lines exhibited a very large variation in flowering date. The cultivar Treeline was found to produce the most herbage under all conditions although not significantly more than cultivar Prairie or C.P.I. 57353. However, as nodulation was not studied it was not possible to determine whether variety differences were due to root nodulating ability or some other genetically determined parameter. None of the T. ambiguum varieties produced as much herbage as cv. Huia or cv. Maku at any site. However, all the T. ambiguum lines performed relatively better under harsher conditions. Because a large proportion of T. ambiguum was below ground the best T. ambiguum line, cultivar Treeiine, produced equivalent total plant dry weight to cv. Huia and cv. Maku at 1200 m. a.s.l., the high altitude site. Cultivar Prairie was found to have the highest proportion of rhizomes to total plant mass bu because cv. Treeline had higher total plant dry weight both cultivars produced equivalent mean rhizome dry weight. The number of rhizomes, number of daughter plants and rhizome dry weight were all highly correlated and these three characteristics showed similar trends among varieties. Rhizome length was found to increase with ploidy level, as did rhizome internode length. However, the number of nodes was found to be higher in the diploids than in the polyploids. Cultivar Treeline was found to have a high proportion of branching nodes on its rhizomes while C.P.I. 57353 and cv. Summit had the least. Rhizome production was restricted in the Wakanui silt loam soil of high bulk density. However, herbage growth and rhizome branching was increased, probably because of the higher fertility. It was shown using factor analysis on genotypic correlations, that rhizome characteristics and herbage yields were relatively independently inherited. However, morphological characters tended to be related to herbage yields. The polyploid varieties were found to be more genetically variable than the diploids . Broad sense heritabilities were calculated for all parameters measured, and in general, morphological characteristics had higher values than agronomic characteristics. As the genetic variation within each line was higher than the variation among lines, it is apparent that selection within lines should result in the largest gains. Therefore, comparison of the mean performance of the presently highly variable lines is relatively uninformative. By sacrificing some genetic diversity, large gains could be made in performance. It is suggested that cv. Prairie be used as the basis for selecting a highly rhizomatous cultivar while cv. Treeline could be used in the selection of a cultivar with higher herbage production suitable for high country conditions. In a second trial, an established stand of cv. Treeline produced up to 13250 kg ha⁻¹ for one season under good growing conditions. The management required to produce this amount of herbage was to irrigate and cut to ground level every two months. The growth rate was considerably depressed when cut monthly. It was also shown that root and rhizome yield reached 12600 kg ha⁻¹, indicating a massive reserve of assimilates, particularly useful for surviving periods of stress. Seed yields were found to be adequate, reaching levels equivalent to 500-700 kg ha⁻¹. These results were discussed in relation to earlier observations on T. ambiguum by workers in Russia, Australia, U.S.A. and New Zealand. Suggestions were made for further genetic and agronomic testing.

Some aspects of the presence of systemic endophytic fungi in agriculturally important New Zealand grasses were studied in relation to plant breeding. Seedling resistance to adult Argentine stem weevil feeding in perennial ryegrass, Italian ryegrass and tall fescue was found to be related to the presence of their respective Acremonium endophytes in the seed rather than to plant genetic resistance. In addition a study of perennial ryegrass revealed that this resistance was independent of endophyte viability. The seedling resistance conferred by the endophyte of Italian ryegrass was found to be beneficial for field establishment. This endophyte differs from that in perennial ryegrass and tall fescue in that it does not confer resistance to Argentine stem weevil on mature plants, but only on seedlings. The extent of plant genetic seedling tolerance to adult Argentine stem weevil feeding was limited to broad inter-specific differences, with tall fescue more tolerant than perennial ryegrass and both of these more tolerant than Italian ryegrass. This ranking corresponds with previous observations on feeding preference on mature plants. A study of factors affecting the concentration of endophyte mycelia in infected seed of perennial ryegrass revealed that plant genetic factors had little effect. The major factors studied were: 1) the endophyte concentration in the maternal parent plant directly influenced the endophyte concentration in the seed. 2) nitrogen fertilizer applications to a seed crop reduced the concentration of mycelia in the seed, with earlier applications having a greater effect. 3) application of the fungicide propiconazole (Tilt) to a seed crop reduced the endophyte concentration in the seed. 4) the endophyte concentration in the seed was found to directly influence the endophyte concentration in seedlings, six month old plants and that of seed harvested from a first year seed crop. As there have been no previous reports of tetraploid perennial ryegrass cultivars with endophyte an experiment was conducted to determine if these could be developed by the standard procedure of colchicine treatment. The results revealed that endophyte was retained following colchicine treatment.

This thesis is presented in the form of a series of papers some of which have already been published. It is prefaced by a review of research on the agronomy and physiology of wheat yield in New Zealand, and concluded with a general discussion, Other relevant papers in which the author was a contributor may be found in the Appendix. / Between 1972 and 1976 a series of field experiments were conducted to investigate the factors limiting grain yield in wheat (Triticum aestivum L). Spike population restricted yield when it was below about 500 spikes/m² but once it exceeded 700/m² grain yield declined due to a reduction in the number of grains per spikelet. Irrigation, high sowing rate, and nitrogen fertiliser generally increased spike number but the effects on the number of grains/spikelet were variable, apparently depending on processes related to leaf area index. The semi-dwarf Karamu outyielded New Zealand wheats when spring-sown because it produced more spikelets/spike and more grains/ spikelet than the other cultivars. The superior grain set of Karamu was because more of the distal florets within each spikelet produced grain than in the other cultivars. The results are discussed in relation to cultural practices and plant breeding.

Variables for the accelerated ageing (AA) test, methods for reducing fungal contamination during the AA test, using the conductivity test as a vigour test, the effect of seed size on seed vigour and the relationship between laboratory test results and field perfonnance in selected Brassica spp were investigated. In the first experiment, three seed lots of turnip rape hybrid (B. rapa x campestris), turnip (B. campestris) and forage rape (B. napus); and seven seed lots of Asian rape (B. napus), six seed lots of Asian kale (B. oleraceae var. alboglabra L.) and five seed lots of choisum (B. rapa var. pekinensis) with germinations above 90% were aged at two different temperatures (41 and 42°C ± 0.3°C) and three ageing times (24, 48 and 72 ± 15 minutes). The second experiment was divided into three sections. In the first, the same seed lots and species were aged at one temperature (41°C) and time (72 h), but either 40 ml of saturated salts; KCl (83%RH), NaCl (76%RH), NaBr (55%RH); or distilled water (96%RH) were used as the ageing solutions. In the second, one turnip rape hyprid seed lot was aged at three temperatures (41, 42 and 45°C) and two times (72 and 96h), again using the three saturated salts and distilled water as ageing solutions. In the third, three turnip rape hybrid seed lots and three Asian kale seed lots were surface sterilised (1 % sodium hypochlorite) prior to ageing at one temperature (41°C) and time (72 h). In the third experiment, the same species and seed lots used in experiment one at their original seed moisture content (SMC) were tested for conductivity after soaking in deionised water for 4, 8, 12, 16, 20 and 24 h. They were then re-tested after the SMC had been adjusted to 8.5%. In the fourth experiment, three seed lots of forage rape and three seed lots of Asian kale were graded into three seed size categories; large (retained on a 2.0 mm screen), medium (retained on a 1.7 mm screen) and small (passed through a 1.7 mm screen). Graded seeds were then tested for standard germination, AA (41°C/48 h) and conductivity (measured at 16 and 24 h). In the final experiment, the relationships between laboratory tests for the six species (each consisting of three seed lots), field emergence from three sowings, and cold room emergence were evaluated. Both time and temperature influenced post-AA germination. Increasing the ageing period from 48 to 72 hours at 41°C, and 24 to 48 hours at 42°C resulted in decreased mean germination percentage for all species but not always clear separation of seed lots. While there were sometimes few differences between ageing at 41°C and 42°C, the former is preferred because it is already the temperature used for other species. For Asian rape, choisum and turnip, the previously recommended testing conditions of 41°C/72 h provided good seed lot separation, but for Asian kale and turnip rape hybrid, AA testing at 41°C/48 h provided better results. Seed moisture content after ageing ranged from 29-37% depending on species. Fungal growth on seeds during the ageing period appeared to reduce post-ageing germination in some seed lots . Substituting saturated salts for distilled water did not stress seed lots in the AA test, due to the lowered RH%, the exception being seed lots 1210 and 1296. For forage and Asian species, seed lot germination mostly remained above 90% when aged for 72 h at lowered RH%. Increasing the ageing duration from 72 to 96 hours resulted in some decreases in post-AA germination but no clear separation of seed lots. Surface sterilising the seeds prior to the AA test resulted in a lower incidence of contaminant fungi which was associated with a lower percentage of abnormal seedlings. The conductivity test was mostly able to identify vigour differences among forage and Asian vegetable brassica seed lots. Differences in conductivity readings were observed among seed lots in all species. Increasing the period of imbibition resulted in increased conductivity from most seed lots but radicle emergence occurred after 16-20 h of imbibition. Variation was observed in the time to reach 95% maximum of the imbibition curve for most species. Conductivity readings at 16 h would avoid possible influences of radicle emergence on results. Adjusting the SMC to 8.5% resulted in reduced variation in conductivity among replicates of seed lots, due to a reduction in imbibition damage. Seed size had a significant effect on both post-AA germination and conductivity results. In forage rape, large size seeds had higher post-AA germination cf. medium cf. small size seeds. In Asian kale, large size seeds had higher post-AA germination compared with small size seeds. For both forage rape and Asian kale, large size seeds had lower conductivity readings cf. small size seeds. The correlation analyses demonstrated significant relationships between AA testing and field emergence parameters (percentage emergence, emergence index and emergence rate). Significant relationships were also observed between conductivity testing and these field emergence parameters. Based on the correlation analysis, AA testing at 41°C/48 hand/or 42°C/48 h could be recommended to be used as an AA test for turnip and Asian rape; and 41°C/48 hand/or 41°C/72 h for Asian kale and choisum. Based on the correlation analysis, conductivity testing at 16 h can be used to predict the field emergence potential of forage and Asian vegetable seed lots. Vigour tests were consistently able to provide better indicators of field perfonnance than the standard germination test, although these relationships did vary with the different field sowings.

The permanent pasture based on a grass-clover association and utilised in situ by grazing stock is the basis of New Zealand's chief primary industries. Although white clover is the main legume species used for this purpose its production and persistence in the drier eastern areas of the South Island often leaves much to be desired. In such situations subterranean clover may provide a useful alternative. Subterranean clover has been a very useful species in the past and will probably continue to play an important role in the future, provided the most climatically adapted cultivars are grown. The aim of the field trials was to evaluate the productive performance of several subterranean clover cultivars in Canterbury. Because of the big influence of seed yield on subsequent production and persistence in annuals, this aspect was also investigated. The subterranean clover cultivars Geraldton, Yarloop, Woogenellup, Clare, Mt. Barker and Tallarook were evaluated under cutting in swards at two sites in Canterbury.