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京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第20459号 / 人博第809号 / 新制||人||194(附属図書館) / 28||人博||809(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 瀬戸口 浩彰, 教授 加藤 眞, 教授 市岡 孝朗 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

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Fall-sown barley will be increasingly important in the era of climate change due to higher yield potential and efficient use of water resources. Resistance/tolerance to biotic and abiotic stresses will be critical. Low temperature is an abiotic stress of great importance. Resistance to barley stripe rust (incited by Puccinia striifomis f. sp. hordei) and scald (incited by Rhynchosporium secalis) will be important in higher rainfall areas. Simultaneous gene discovery and breeding will accelerate the development of agronomically relevant germplasm. The role of FR-H1 and FR-H2 in low temperature tolerance (LTT) has been well documented. However the question still remains: is LTT due only to FR-H1 and FR-H2 or are there other, undiscovered, determinants of this critical trait? We developed two doubled haploid mapping populations using two lines from the University of Nebraska (NE) with superior cold tolerance and one line from Oregon State University (OR) with good malting quality and disease resistance: NB3437f/OR71 (facultative x facultative) and NB713/OR71 (winter x facultative). Both were genotyped with a custom 384 oligonucleotide pool assay (OPA). QTL analyses were performed for LTT, vernalization sensitivity (VS), and resistance to barley stripe rust and scald. Disease resistance QTL were identified with favorable alleles from both NE and OR germplasm. The role of VRN-H2 in VS was confirmed and a novel alternative winter allele at VRN-H3 was discovered in the Nebraska germplasm. FR-H2 was identified as a determinant of LTT and a new QTL, FR-H3, was discovered on chromosome 1H that accounted for up to 48% of the phenotypic variation in field survival at St. Paul, Minnesota, USA. The discovery of FR-H3 is a significant advancement in barley LTT genetics and will assist in developing the next generation of fall-sown varieties. / Graduation date: 2012

La betterave sucrière est une plante bisannuelle dont le besoin de vernalisation est absolu. Ce processus correspond à l’acquisition de l’aptitude à la montaison et à la floraison et résulte d’une exposition prolongée à de basses températures. La durée de froid requise pour induire la montaison puis la floraison varie suivant les génotypes et reflète leur tolérance à la montaison, qui constitue donc un caractère agronomique essentiel. Cette thèse visait à (i) mettre en évidence un éventuel contrôle épigénétique (méthylation ADN) de l‘induction de la montaison chez des génotypes de betterave sucrière résistants ou sensibles à la montaison, (ii) identifier les séquences ciblées par des remaniements de méthylation de l’ADN et d’expression associés, et (iii) caractériser certaines séquences candidates en vue de leur utilisation comme marqueurs de la montaison. Nos travaux ont montré que l’amplitude et la cinétique des variations de méthylation de l’ADN observées au cours de la vernalisation semblent être des éléments critiques de l’induction et de la tolérance à la montaison. Par une approche ciblée, des séquences dont la méthylation de l’ADN est remaniée ont été identifiées. L’implication dans la transition florale de deux BvRNMT (RNA METHYLTRANSFERASES) et de la méthylation des ARN, tels que l’ARNm de BvFL1, un répresseur floral, a ainsi été mise en évidence chez la betterave sucrière. Enfin, grâce à une approche génomique, un réseau de gènes intégratif incluant la réponse à l’environnement, la signalisation hormonale et l’induction de la floraison a été identifié. La cinétique d’activation de ces gènes définirait le niveau de tolérance à la montaison des différents génotypes de betterave sucrière. / Sugar beet is a biennial plant with an absolute requirement of vernalization, corresponding to the acquisition of the competence to bolt and flower after a prolonged exposure to low temperatures. The cold duration needed to induce bolting and flowering varies depending on the genotypes, reflecting their bolting tolerance, which is an essential agronomic trait. This work aimed at (i) investigating a possible epigenetic control of bolting induction in bolting sensitive and bolting resistant sugar beet genotypes, (ii) identifying sequences targeted by DNA methylation and expression remodeling, and (iii) characterizing candidate sequences which could be used in marked-assisted selection for plant breeding. Our data suggest that the time course and amplitude of DNA methylation variations are critical points for the induction of sugar beet bolting and represent an epigenetic component of the genotypic bolting tolerance. In addition, we identified differentially methylated sequences exhibiting variations of gene-body DNA methylation and expression during cold exposure and/or between genotypes. Among them, two RNA METHYLCYTOSINE TRANSFERASES, in association with RNA methylation such as BvFL1 mRNA, a floral repressor, were shown to play a role in floral transition. Finally, using microarrays we identified an integrative network of genes including response to environment, phytohormone signalling and flowering induction. The activation kinetics of these genes could define the bolting tolerance level of sugar beet genotypes.

The risks and profitability associated with four early planting dates, and three different types of bolting risk reduction strategies, were evaluated for Quebec celery production. The four planting dates studied were April 17 th, April 24th, May 1st and May 8 th. The impacts on yield if plants were harvested after eleven week, twelve, thirteen, and fourteen weeks in the field were investigated. The three risk reduction strategies considered were plants grown from planting until June 2nd with no row covers, under floating row covers of spunbonded polyester (Agryl P17, Plastitech) and under floating row covers made of clear, perforated polyethylene (Plastitech). The results suggest that the floating row covers can help plants reach the required market standards sooner that if no row covers were used from the planting date investigated until June 2nd. At the earliest two planting dates, the results suggest that row covers may significantly suppress bolting and allow greater yields at earlier harvest dates. The results also suggest that row covers may provide an advantage at later planting dates, as they may allow for the plants to reach marketable standards sooner. The results also suggest that there is a 50 percent chance of encountering bolting rate of 30 percent or higher if transplanting plants on April 17th. The row covers reduced the probability that plants will bolt to close to zero at for all four planting dates. The most profitable strategy was determined to be planting on April 17th, growing the plants under row covers made of spunbonded polyester (Agryl P17, Plastitech) from planting until June 2nd and then harvesting after the plants had been in the field for thirteen weeks.

Vernalized celery seedlings (Apium graveolens L. var. Florida 683) were predevernalized under different temperature and light regimes prior to field transplanting. At least 89% of predevernalized plants bolted during both 1999 and 2000 suggesting that the predevernalization treatments were not effective in counteracting the vernalizing stimulus. Predevernalization treatments had no long-term effect on growth and development. Non-vernalized celery seedlings were transplanted on clear polyethylene mulch, thermo-selective polyethylene mulch or no mulch in combination with clear perforated polyethylene mini tunnel, single or double layer of agrotextile mini tunnel or no mini tunnel. Very few plants bolted in 1999. Mulch increased fresh weight and number of celery leaves compared with controls. Plants grown on clear polyethylene mulch had significantly higher mortality rates than other treatments. Agrotextile mini tunnels reduced bolting to 0% in 2000. Mini tunnels, especially a single layer of agrotextile, increased fresh weight, plant height and stem diameter in 2000.

iv The objective of this study was to optimize tissue culture and regeneration parameters of immature inflorescence culture of Triticum aestivum cv. Bezostaja- 01 and Triticum durum cv. Kiziltan-91. The effects of callus age and vernalisation time of explants on regeneration success were evaluated. For determination of optimum vernalisation time of immature inflorescence, plants subjected to 4 &deg / C for 1, 2, 3, 4, and 5 weeks, respectively. Tillers containing immature inflorescences were collected at the same time. Percentage of inflorescence formed tillers over total explants were reached the highest value, 79 %, at 4 weeks cold treated Kiziltan cultivar and, 73 %, at 5 weeks cold treated Bezostaja cultivar. Isolated immature inflorescences were put onto 2mg /L 2,4-dichlorophenoxyacetic acid and picloram containing callus induction medium for Kiziltan and Bezostaja cultures, respectively. Callus induction rate were found to be 100 % for Kiziltan and Bezostaja. These explants were taken to regeneration after 6, 9, 12 and 15 weeks of dark incubation period. The regeneration capacities of calli were determined as shooting percentage and data were collected after 4, 8, 12, and 15 week regeneration period. The highest shooting percentage of 69 %, were obtained from 6 weeks old calli produced from 4 weeks vernalised explants in Kiziltan cultures at the end of 15 weeks regeneration period. However, shooting percentage was 57.2 % for 9 weeks old calli while it decreases to 37.6 % in 12 weeks old calli and 44.2 % in 15 weeks old calli at the end of 15 weeks regeneration period. This showed that prolonged dark incubation period decreased regeneration capacity of the callus. However, there was no significant difference in regeneration capacities of calli produced from Bezostaja immature inflorescence and the highest shooting percentage was obtained from 9 weeks old calli produced from 5 weeks vernalised explants, 27.4 %. Besides regeneration studies, optimization of transformation parameters for winter wheat cultivars Kiziltan and Bezostaja by using Agrobacterium tumefaciens AGLI containing binary vector pALl56 was performed. Transformation efficiencies were determined by monitoring the transient expression of uidA gene via histochemical GUS assay. Three to four weeks old calli were found to be more responsive to Agrobacterium-mediated transformation in Kiziltan cultures. However, four to five weeks old calli were found to be more responsive to Agrobacterium-mediated transformation in Bezostaja cultures. Different transformation protocols were used. It was found that MGL based and MMA based protocols could be used for Bezostaja and Kiziltan transformation, respectively. The highest GUS expression, 84%, was obtained from 28 weeks old calli produced from 5 weeks vernalised explants in Bezostaja cultures.

Finding the alternate biofuel feedstock(s) in addition to and/or replacement of traditional soybean feedstock is necessary to meet the future demand of biofuels. Two field studies were conducted in diverse environments in Texas during 2007-2011 to evaluate the yield, adaptation, and oil content of 4 cool-season crop species (rapeseed, safflower, flax, and camelina). In addition to the evaluation of yield and adaptation in these cool-season crops, two more studies were conducted during 2009-2011 to study flax yield components (field study) and the effect of vernalization and photoperiod on flowering of flax (growth chamber study). Out of two field studies conducted in Texas, the evaluation of four cool-season crops was designed as a randomized complete block with fifty-one genotypes (four species) and three replications in nine locations across the Texas. In addition to the evaluation of cool-season crops, an exclusive replicated study was conducted in flax to evaluate 20 genotypes for the yield, adaptation, and association between yield and its components in three locations in South Texas. Additionally, a growth chamber study was setup as a split-split plot design with twenty genotypes, two vernalized treatments (vernalized and unvernalized), and two photoperiods (10 hours and 14 hours). Spring rapeseed (canola) and safflower were the highest yielding crops with a maximum yield of 1372 kg ha-1 and 1240 kg ha-1, respectively. In South and Central Texas, fall - seeded flax yield averaged 1075 kg ha^-1 with a mean oil content of 38.3%. The flax genotype evaluation in Southeast Texas suggested that all genotypes developed in Texas showed relative cold tolerance compared to genotypes developed in other locations. A cross between Caldwell / Dillman (Texas genotype) was highly adapted to the environments of southeast Texas. Nekoma and York (genotypes developed in North Dakota) yielded well in non-cold years (> -2 degrees C) in College Station. Overall, flax is well adapted to growth in the area surrounding College Station, TX. The results of association of yield and its components in flax suggest that tiller number was the most significant contributing factor (p<0.05) affecting yield of flax in all three locations. However, the effect of tiller number was almost negated by the effect of pods per tiller (compensatory) in two out of three locations. The effect of vernalization and photoperiod on flowering of 20 genotypes of flax suggested that Texas genotypes delayed anthesis for 7 days or more in non-vernalized seedlings. These genotypes also delayed anthesis for 12 days or more in vernalized and short day conditions compared to vernalized and long day conditions. In summary, the spring rapeseed in diverse environments of Texas and fall-planted flax in South Texas showed promising yield and adaptation. Selection for more productive tiller number and intrinsic earliness of flowering to reduce the time of maturation would benefit the flax yields in Southeast Texas. Safflower was widely adapted to Texas and with increased oil content could have potential to the biofuel industry in Texas.