筑波大学University of Tsukuba博士（農学）Doctor of Philosophy in Agricultural Science2023この博士論文は内容の要約のみの公開（または一部非公開）になっていますdoctoral thesi

Long Thien Tran

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Abstract of Thesis  Affiliation  Doctoral Program in Agricultural Sciences     Degree Programs in Life and Earth Sciences     Graduate School of Science and Technology  Student ID Number 202036023 Name   Long Thien Tran Thesis Title Utilization of iaa9-3 Mutation in Research and Breeding of Seedless Tomatoes （種なしトマトの研究と育種における iaa9-3変異の利用） Abstract of Thesis The environmental challenges, especially global warming, are impairing fruit production in most crops, including tomato (Solanum lycopersicum), one of the most important fruity vegetables worldwide, due to its high nutritional value and economic benefits. Many breeding strategies have been developed to increase the tolerance ability of tomato plants against heat stress. One potential approach is using parthenocarpy, a unique ability enabling plants to produce seedless fruits without pollination and fertilization. In tomatoes, the mechanisms of parthenocarpic regulation were diverse and strongly related to phytohormones. The common phytohormones, including auxin, gibberellin, cytokinin, ethylene, brassinosteroid, and abscisic acid, were evidenced to independently or together promote parthenocarpy in tomatoes. In this study, I conducted multiple experiments on the iaa9-3 mutants to investigate their utilizability for seedless tomato breeding. The iaa9-3 is a mutated allele on the  IAA9 gene, which encodes IAA9- an auxin response inhibitor protein. Through this study, I provide evidence that 1) the iaa9-3 mutants are potential for seedless tomato breeding even though they still carry some limitations that need to be eliminated (Chapter 2); 2) the performance of iaa9-3 mutants can be improved by introducing a pollen avoidant characteristic, stigma exsertion (Chapter 3) and by increasing the leaf complexity (Chapter 4); and 3) the iaa9-3 can be used as a model to identify the new parthenocarpic mutations for future breeding (Chapter 5). In addition, I also proposed directions for future research and utilization of parthenocarpy in seedless tomato breeding (Chapter 6). The detailed findings presented in each chapter are summarized in the following paragraphs. In Chapter 2: “The potential of iaa9-3 in seedless tomato breeding”, I introduced the iaa9-3 mutation into a commercial cultivar CV62 and evaluated its effects on parthenocarpic aspects of segregating mutant lines. The evaluation results illustrated that iaa9-3 mutants have some advantages over other parthenocarpic mutants, including 1) retaining the normal stamen structure and fertile pollen; 2) forming sufficient seeds for self-pollinated propagation; 3) producing a high yield of seedless tomato fruits, over 3,000 g/plant, which is acceptable for commercial varieties grown in the open-field conditions; and 4) increasing the quality of seedless fruits compared to seeded fruits. However, the iaa9-3 mutants also showed some limitations that need to be improved, including 1) producing a significant number of seeded fruits (accounted for 60% of total fruits) under normal growing conditions; and 2) forming simple leaves (potato leaf style) which are concerned to cause the lacking of leaf shade for fruit development. These limitations directed the experiments presented in the following chapters. Chapter 3: “Identification of stigma exsertion regulation and its potential benefits to parthenocarpy in tomato”, points out a practical approach to avoid the first limitation of seeded fruit formation in iaa9-3 mutant plants. I hypothesized the high potential of stigma exsertion, a phenomenon that stigma is at a higher position than anther cone in self-pollination avoidance. The regulation of stigma exsertion and its applicability in tomato breeding has been illustrated through previous research but not fully understood. This research aims to (1) investigate the potential mechanism of stigma exsertion regulation in tomato, and (2) examine the benefits of stigma exsertion to parthenocarpic tomato breeding. Using the Mut-Map strategy (based on the whole genome sequencing analysis), I identified new potential genes controlling the anther length, relative length of pistil and anther cone, or the expression of Style2.1, which directly or indirectly contribute to stigma exsertion phenotype. In addition, from the segregating population derived from a multi-parental crossing system, I successfully isolated one “double mutant” line APi7-4-5 with strong parthenocarpy (98%) and long stigma exsertion (4.16 mm). This line produced 100% (43/43) seedless fruit under greenhouse growing conditions, confirming the benefits of long stigma exsertion on the production of parthenocarpic tomato fruit. The free self-pollination obtained in line APi7-4-5 also suggested that hybrid tomato seeds can be produced safely without emasculation under greenhouse conditions, using long stigma exserted materials. The findings of this work contribute to a better understanding of the regulation of stigma exsertion phenomenon in tomato plants and point out the high potential of using this trait in seedless tomato breeding. In Chapter 4: “Developing iaa9-3 mutants showing high compound leaves”, I mainly conducted breeding activities, including conventional crossing and pedigree selection on segregants from F2 to F4 generations. Using the parental lines of MT-iaa9-3 (donor of parthenocarpy) and Aichi First (donor of complex leaf phenotype), I isolated several parthenocarpic lines producing high compound leaves. This result could not be achieved when introducing iaa9-3 into other genetic backgrounds of Moneymaker and Ponderosa. The result indicated that leaf complexity in the iaa9-3 mutant could be obtained using suitable genetic background (Aichi First). The newly isolated lines significantly improved to other iaa9-3 mutant lines obtained previously, giving them a higher potential for seedless tomato breeding materials. In addition, I used Mut-Map strategy based on NGS to investigate several genes as potential candidates causing the complex leaf phenotype. These genes seem different from previous discoveries about the roles of ARFs transcription activators. The function of these newly found candidate genes has not been validated; further study is needed to investigate their contributions. Using iaa9-3 as a model, I proposed that an ideal parthenocarpic mutant should satisfy the following criteria: (i) producing a high parthenocarpic fruit set ratio without emasculation (as similar as iaa9-3 plus stigma exsertion mutants which can reach over 90%; it is recommended that over 70% might be acceptable for the real cultivation), (ii) showing no/ minor side effects (such as the potato leaf style of iaa9-3 mutants due to the abundance expression of this gene in vegetative tissues; the new parthenocarpic gene(s) should specifically express in fruit tissue only), and (iii) being maintained easily, which is required for both self-propagation and hybrid seed production. I produced parthenocarpic tomato fruits by a unique method using soft X-ray treated pollen. I monitored the expression pattern of all tomato genes using RNA-Seq analysis to identify the most responded genes to X-ray treatment. By that way, I obtained more than 4,300 differently expressed genes after pollination by X-ray pollen. These genes seem to involve different pathways to regulate cell division and expansion to initiate fruit development. Through this experiment, I selected 59 genes as potential candidates for regulating parthenocarpic tomato fruits without limitations of iaa9-3. Further exploring the functions of these candidate genes would enable identifying ideal parthenocarpic genes for breeding highly adaptable and seedless tomato cultivars. These findings are presented in Chapter 5: “Identification of new parthenocarpic candidate genes in tomato” Finally, in Chapter 6, I proposed several directions for future research and utilization of results from this project in seedless tomato breeding, including 1) Validating the function of candidate genes regulating stigma exsertion phenotype and creating the reliable marker(s) (at both phenotypic and molecular levels) to utilize them in combination with parthenocarpic mutations, not only iaa9-3 but also others; 2) Confirming the mechanism regulating the complex leaf phenotype in iaa9-3 mutant plants (derived from Aichi First genetic background); 3) Investigating new parthenocarpic genes from 59 proposed candidates to find the ideal materials for breeding of seedless tomato cultivars; 4) Breeding seedless tomato cultivars using parthenocarpy (from iaa9-3 mutation) in combination with stigma exsertion and compound leaf phenotype. The plant materials derived from this study can be used directly or as donors in the future breeding of seedless tomato varieties; 5) Investigating the new system for producing seedless tomatoes using long stigma exsertion and X-ray-treated pollen. 

種なしトマトの研究と育種におけるiaa9-3変異の利用

https://tsukuba.repo.nii.ac.jp/record/2009352/files/DA10984_abstract.pdf

種なしトマトの研究と育種におけるiaa9-3変異の利用

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