Master of Science / Genetics Interdepartmental Program / Eduard D. Akhunov / The CRISPR/Cas9-based genome editing system holds a great promise to accelerate wheat improvement by helping us to understand the molecular basis of agronomic traits and providing means to modify genes controlling these traits. CRISPR/Cas9 is based on a synthetic guide-RNA (gRNA) that can guide Cas9 nuclease to specific targets in the genome and create double strand breaks (DSB). The DSB are repaired through the error-prone non-homologous end joining process causing insertions or deletions that may result in loss-of-function mutations. Here, we have developed an effective wheat genome editing pipeline. We used next-generation sequencing (NGS) data to estimate genome editing efficiency of many gRNAs using the wheat protoplast assay and choose the most efficient gRNAs for plant transformation. We successfully applied this pipeline to five wheat orthologs of the rice yield component genes that have been identified previously. We obtained edited plants for all these genes and validated the effect of the mutations in TaGW7 on wheat traits, which showed trends similar to those in rice. These results suggest that transferring discoveries made in rice to improve wheat is a feasible and effective strategy to accelerate breeding.

It is becoming increasingly important to improve the yield of seed crops to feed an expanding population and, more recently, to cope with additional strains on food-oriented agriculture posed by biofuel production and global climate change. One strategy to increase yield is to increase seed size. However in nature, due to the limited resources of the mother plant, an enlarged seed size is often associated with a concomitant reduction in seed number. Using the model plant Arabidopsis thaliana, a biotechnological approach to increase seed size through the modification of the triploid endosperm or the ovule integuments was shown to be a viable strategy to improve seed yield. Targeted over-expression of the ANT transcription factor specifically within the endosperm significantly increased seed size without negative effects on fertility. Overcoming compromised fertility in arf2 mutants established that ARF2 and the integuments are important factors in determining seed size and yield. To ensure agronomic relevance, both the gross yield and the harvest index (HI, ratio of seed yield to biological yield) were used to assess the impact of traits, such as increased integument size, introduced into Arabidopsis. To uncover novel regulators of seed size and further develop the current understanding of seed development, second-site mutations were induced in the auxin response factor 2 (arf2) mutant, which produces large seeds due to extra cell division in the ovule integuments. The ARABIDOPSIS CRINKLY 4 (ACR4) gene was identified as a suppressor of the arf2 seed phenotype. In arf2-8 acr4 double mutants, an additive effect on cell proliferation was observed, indicating that ACR4 affects the seed coat through a developmental pathway independent of ARF2. Natural variation present in Arabidopsis was used to study seed yield and its components. Considerable variation in seed size, yield and HI was revealed. Significantly, high seed weight was not associated with high yield or high HI. In contrast, high seed number and reduced plant stature were revealed as important components of high yield and yield efficiency. Additionally, ERECTA was identified as a potential ‘Green Revolution’ gene. Seed size in Arabidopsis was shown to be an extremely plastic trait in response to alterations in the post-flowering source-sink ratio, increasing by 35.0 – 66.2% in the ecotypes tested. Furthermore, a difference in seed weight between restricted (high source-sink ratio) and unrestricted (low source-sink ratio) pollinations was first observed remarkably early in seed development. However, reducing seed number by restricting pollination did not substantially alter integument or endosperm development in order to facilitate increased nutrient uptake.

Made available in DSpace on 2016-12-12T20:12:29Z (GMT). No. of bitstreams: 1 PGEF14MA017.pdf: 394745 bytes, checksum: c1827ef1d01e5069a7ca04ba452e1da7 (MD5) Previous issue date: 2014-06-26 / Proposals for the determination of seed quality through analysis of digital images of seeds and seedlings have been used in order to obtain objective information in a relatively short period. However, its effectiveness depends mainly on the type and adequacy of the procedures. Thus, the aim of this work to: 1) evaluate the efficiency of the software ENVI EX and QUANTUM GIS in the measurement of seed of Pinus taeda, replacing the conventional method; 2) determine the influence of size, assessed by image analysis, the physiological quality of seeds of Eucalyptus saligna, Eucalyptus and Pinus taeda dunnii.; 3) verify the efficiency of the software ENVI EX and QUANTUM GIS in the classification of seed vigor through analysis of digital images of seedlings of Eucalyptus saligna and E. dunnii, replacing the conventional method. To meet the objectives 1 and 2, the seeds were arranged on a graph paper and then photographed and analyzed using the ENVI EX and Quantum GIS programs, for the classification of seeds. The seeds of Pinus taeda, before being photographed, were measured with the aid of a digital caliper (conventionally), for comparison with the results obtained by the software. Then the seeds were germinated, separated by size classes, plus control (unrated). To meet the objective 3, the length of normal seedlings obtained in a germination test with appropriate methodology by species was evaluated through analysis of digital images using the software ENVI EX and Quantum GIS, and a conventional (manual) . With the results obtained, it was possible to verify that the software ENVI EX and Quantum GIS are efficient to measure the seeds of Pinus taeda. The size of the seeds of E. saligna and E. dunnii influences on physiological quality, with higher seed medium and large to small. This influence is not observed in seeds of P. taeda. In addition, you can use the software cited in the measurement of seedlings of Eucalyptus saligna and Eucalyptus dunnii, replacing the conventional method / Propostas para a determinação da qualidade de sementes por meio de análise de imagens digitais de sementes e plântulas têm sido utilizadas visando à obtenção de informações objetivas, em período relativamente curto. No entanto, sua eficiência depende, principalmente, da espécie e de adequações nas metodologias. Desta forma, objetivou-se com esse trabalho: 1) avaliar a eficiência dos softwares ENVI EX e QUANTUM GIS na mensuração de sementes de Pinus taeda, em substituição ao método convencional; 2) determinar a influência do tamanho, avaliado por meio da análise de imagens, na qualidade fisiológica de sementes de Eucalyptus saligna, Eucalyptus dunnii e Pinus taeda.; 3) verificar a eficiência dos softwares ENVI EX e QUANTUM GIS na classificação do vigor de sementes por meio da análise das imagens digitais de plântulas de Eucalyptus saligna e E. dunnii, em substituição ao método convencional. Para atender aos objetivos 1 e 2, as sementes foram dispostas sobre um papel milimetrado e, em seguida, fotografadas e analisadas por meio dos programas ENVI EX e Quantum GIS, para a classificação das sementes. As sementes de Pinus taeda, antes de serem fotografadas, foram mensuradas com o auxílio de um paquímetro digital (forma convencional), para posterior comparação com os resultados obtidos pelos softwares. Em seguida, as sementes foram colocadas para germinar, separadas por classes de tamanho, além da testemunha (sem classificação). Para atender ao objetivo 3, o comprimento de plântulas normais, obtidas em teste de germinação realizado com metodologia adequada por espécie, foi avaliado por meio da análise das imagens digitais, utilizando os softwares ENVI EX e Quantum GIS, e de forma convencional (manual). Com os resultados obtidos, foi possível verificar que os softwares ENVI EX e QUANTUM GIS são eficientes para a mensuração das sementes de Pinus taeda. O tamanho das sementes de E. dunnii e E. saligna influencia na sua qualidade fisiológica, sendo sementes médias e grandes superiores às pequenas. Essa influência não é verificada em sementes de P. taeda. Além disso, é possível utilizar os softwares citados na mensuração de plântulas de Eucalyptus dunnii e Eucalyptus saligna, em substituição ao método convencional