Exploiting induced genetic diversity through using mutagenesis is particularly important in giant miscanthus (Miscanthus x giganteus; Mxg) due to its restricted genetic variability. Experiments were conducted to develop an efficient in vitro propagation protocol for Mxg, induce mutations in Mxg using a chemical mutagen, and select Mxg in vitro for heat tolerance. To optimize in vitro propagation of Mxg, five explant types [i.e. immature inflorescences, shoot apex (in vitro), shoot apex (greenhouse), leaf explants (in vitro), and leaf explants (greenhouse)] were tested on five media. Shoot forming calli from immature inflorescences, an excellent source of explant in Mxg, grown in media with 13.6 microM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.44 microM 6-benzylaminopurine (BA) resulted in greatest shoot regeneration rate. Optimization of explant and callus type and media resulted in efficient in vitro proliferation of Mxg and the developed protocol was utilized in consecutive experiments of mutation induction and in vitro selection of Mxg for heat tolerance. Immature inflorescence explants (1-2 mm) were treated with 0.6%, 1.2%, and 1.8% of ethyl methanesulfonate (EMS) whereas the calli (1-2 mm3) were treated with 1.2%, 2.4%, and 3.6% of EMS for 90 min. Results of inter simple sequence repeat PCR analysis revealed polymorphisms indicating presence of genetic differences in Mxg putative mutants. In vitro callus cultures (mutagen treated and non-treated) of Mxg subjected to temperature treatments of 45±2°C for 12 hrs or 40±2°C for 7 days were selected for heat tolerance. Assessment results of electrolyte leakage and photosystem II (PS II) efficiency tests indicated a significant difference in percent membrane damage among Mxg clonal lines whereas PSII was weakly affected by the heat stress. The results suggest that in vitro derived Mxg clonal lines may be utilized for further studies of Mxg heat tolerance in developing potential Mxg ecotypes to adapt to different thermal environments. These studies provided the first investigation of in vitro induced mutagenesis in Mxg using a chemical mutagen. Genetic analysis results presented in this study indicates the potential use of developed Mxg putative mutants in future research programs, although significant morphological alterations were not observed during preliminary screening in the greenhouse.

Anthracnose, caused by <i>Colletotrichum truncatum</i>, is a major disease of lentil (<i>Lens culinaris</i> Medik.) on the Canadian prairies. Resistance to the more virulent race Ct0 of the pathogen is extremely rare within the <i>L. culinaris</i> gene pool thus resistance is being introgressed from <i>Lens ervoides</i>. The overall hypothesis of this project was that resistance derived from <i>Lens ervoides</i> accession L-01-827A would provide uniquely low levels of resistance to <i>Colletotrichum truncatum</i>. Individual studies were performed to: i) test field-resistance of interspecific lines; ii) develop a cutting-method to generate replicated phenotypic data on single plants; iii) study the deterioration of resistance between the juvenile phase (JP) and adult phases (AP) in the line CDC Redberry; and iv) examine genetic control of resistance in a susceptible and partially resistant background. Field evaluations of <i>L. culinaris x L. ervoides</i> lines indicated resistance genes from the wild species will provide unprecedented levels of disease control and heritability of the trait was estimated to be moderate. Single plant phenotyping of resistance is unreliable, thus the feasibility of using clonal propagation of individual plants to generate replicated ratings was evaluated. Results showed consistency in segregation ratios between cutting and seedling-derived plants of the same population, thus the method was utilized for testing of genetic control of segregating populations. CDC Redberry showed varying disease levels depending on the age of the plants with resistance acquisition in the JP that decreased as the plants proceeded through the AP. The F₁, F₂ and F_2:3 generations of two introgression populations were tested for resistance. Resistance from <i>L. ervoides</i> to both races of <i>C. truncatum</i> appeared to be due to the same gene(s) or from the same linkage block in both populations. Models for genetic control were consistent between F₂ and F_2:3 generations, however were different between the populations depending on whether the interspecific line was crossed into a susceptible or partially resistant <i>L. culinaris</i> background. However duplicate recessive epistasis seemed to control susceptibility in the susceptible background and when JP resistance was not a factor. There were significant differences between the JP and AP on more than a third of the F₂s tested supporting different resistance gene action based on growth phase. Resistance in the JP seemed to be due to dominant and recessive epistasis. It was postulated that the the populations may have had segregation distortion commonly found in interspecific populations, thus the previously described genotypes for cotyledon colour and albino plants were used to test the hypothesis of segregation distortion. Segregation of these traits were found to be similarly distorted to previously reported interspecific <i>L. culinaris x L. ervoides</i> populations supporting the hypothesis that continued segregation distortion was found in the introgression populations. Overall, it was found that resistance derived from <i>L. ervoides</i> accession L-01-827A is a highly effective source for the lentil breeding program, however the result suggest more than one backcross to <i>L. culinaris</i> be necessary to properly integrate the resistance genes to eventually obtain fully fertile, adapted lentil cultivars.

Anthracnose, caused by <i>Colletotrichum truncatum</i>, is a major disease of lentil (<i>Lens culinaris</i> Medik.) on the Canadian prairies. Resistance to the more virulent race Ct0 of the pathogen is extremely rare within the <i>L. culinaris</i> gene pool thus resistance is being introgressed from <i>Lens ervoides</i>. The overall hypothesis of this project was that resistance derived from <i>Lens ervoides</i> accession L-01-827A would provide uniquely low levels of resistance to <i>Colletotrichum truncatum</i>. Individual studies were performed to: i) test field-resistance of interspecific lines; ii) develop a cutting-method to generate replicated phenotypic data on single plants; iii) study the deterioration of resistance between the juvenile phase (JP) and adult phases (AP) in the line CDC Redberry; and iv) examine genetic control of resistance in a susceptible and partially resistant background. Field evaluations of <i>L. culinaris x L. ervoides</i> lines indicated resistance genes from the wild species will provide unprecedented levels of disease control and heritability of the trait was estimated to be moderate. Single plant phenotyping of resistance is unreliable, thus the feasibility of using clonal propagation of individual plants to generate replicated ratings was evaluated. Results showed consistency in segregation ratios between cutting and seedling-derived plants of the same population, thus the method was utilized for testing of genetic control of segregating populations. CDC Redberry showed varying disease levels depending on the age of the plants with resistance acquisition in the JP that decreased as the plants proceeded through the AP. The F₁, F₂ and F_2:3 generations of two introgression populations were tested for resistance. Resistance from <i>L. ervoides</i> to both races of <i>C. truncatum</i> appeared to be due to the same gene(s) or from the same linkage block in both populations. Models for genetic control were consistent between F₂ and F_2:3 generations, however were different between the populations depending on whether the interspecific line was crossed into a susceptible or partially resistant <i>L. culinaris</i> background. However duplicate recessive epistasis seemed to control susceptibility in the susceptible background and when JP resistance was not a factor. There were significant differences between the JP and AP on more than a third of the F₂s tested supporting different resistance gene action based on growth phase. Resistance in the JP seemed to be due to dominant and recessive epistasis. It was postulated that the the populations may have had segregation distortion commonly found in interspecific populations, thus the previously described genotypes for cotyledon colour and albino plants were used to test the hypothesis of segregation distortion. Segregation of these traits were found to be similarly distorted to previously reported interspecific <i>L. culinaris x L. ervoides</i> populations supporting the hypothesis that continued segregation distortion was found in the introgression populations. Overall, it was found that resistance derived from <i>L. ervoides</i> accession L-01-827A is a highly effective source for the lentil breeding program, however the result suggest more than one backcross to <i>L. culinaris</i> be necessary to properly integrate the resistance genes to eventually obtain fully fertile, adapted lentil cultivars.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 17, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. Corinne Valdivia. Includes bibliographical references.

A jabuticabeira (Plinia sp.) é uma espécie de difícil propagação vegetativa e um protocolo eficiente para tal ainda não foi definido. O objetivo deste trabalho foi investigar a eficiência de técnicas de propagação vegetativa da espécie e desenvolver um protocolo eficiente para desinfestação e estabelecimento inicial de explantes in vitro. Testou-se o potencial de enraizamento de estacas lenhosas de P. cauliflora, utilizando quatro concentrações de ácido indolbutírico (AIB) (0, 2000, 4000 e 6000 mg L-1) e dois procedimentos (corte vertical e anelamento da estaca); e o potencial de enraizamento de estacas apicais herbáceas de P. cauliflora, utilizando cinco concentrações de AIB (0, 2000, 4000, 6000 e 8000 mg L-1) e em duas épocas de implantação (outubro e dezembro). O percentual de enraizamento das estacas foi avaliado após 180 dias da implantação dos experimentos. Foi testada também a compatibilidade de enxertia de três espécies de jabuticabeira (P. cauliflora, P. trunciflora, P. jaboticaba) sobre porta-enxertos de P. cauliflora, em duas épocas de implantação (maio e agosto). Avaliou-se o percentual de enxertos brotados e o número e tamanho de brotos, após 90 dias da implantação. Para alporquia, foram testados dois diâmetros de ramo (1,0-1,5 cm e 2,0-2,5 cm) e duas larguras do anelamento (1,5 cm e 3,0 cm), na espécie P. cauliflora. Avaliou-se o percentual de enraizamento e o número e tamanho de raízes, após 180 dias da implantação do experimento. Testou-se também o período de imersão (5, 10 e 15 minutos) em hipoclorito de sódio a 1,25% no estabelecimento in vitro de explantes caulinares e radiculares de seedlings de P. trunciflora. Avaliou-se o percentual de contaminação e o número de brotos e folhas dos explantes, após 45 dias de incubação. Observouse que o enraizamento de estacas lenhosas é dependente da aplicação de AIB, sendo que o maior percentual de enraizamento (50%) foi obtido na maior concentração de AIB (6000 mg L-1) conjugada com o corte vertical. Para as estacas herbáceas, o enraizamento foi baixo (máximo de 10%). Entretanto, há o potencial de enraizamento e, por isso, ajustes na técnica devem ser testados para maximizá-lo. A enxertia e a alporquia são técnicas recomendáveis para propagação da jabuticabeira, pois proporcionam alto percentual de formação de mudas. Há compatibilidade aparente entre as três espécies enxertadas sobre P. cauliflora. A utilização de garfos retirados de plantas em frutificação deve ser evitada, pois ocorre inibição da brotação posterior dos enxertos. Na alporquia, ramos de diâmetro superior a 2,0 cm, proporcionam enraizamento de 87,5% e maior número e tamanho de raízes, em relação a ramos de menor diâmetro. A utilização do menor tempo de imersão (cinco minutos) em hipoclorito de sódio 1,25% é eficiente para desinfestação dos explantes caulinares de seedlings de jabuticabeira e permite seu estabelecimento inicial in vitro, proporcionando o desenvolvimento de brotações. / The jabuticaba tree is a specie of difficult vegetative propagation and an efficient protocol has not been defined yet. The aim of this work was to test the efficiency of vegetative propagation techniques for jabuticaba tree and develop an efficient protocol for disinfection and initial establishment of in vitro explants. It was tested the rooting potential of wood cutting of P. cauliflora, utilizing four concentrations of Indolbutiric Acid - IBA (0, 2000 , 4000 and 6000 mg L-1) and two procedures (cross section and cuttings girdling); and the rooting potential of softwood terminal cuttings of P. cauliflora, utilizing five concentrations of IBA (0, 2000, 4000, 6000 and 8000 mg L-1) in two periods of implantation (October and December 2007). The rooting potential of cuttings was evaluated after 180 days of the beginning of the experiments. It was also tested the compatibility of grafting of three species of jabuticaba tree (P. cauliflora, P. trunciflora, P. jaboticaba) on rootstocks of P. cauliflora, and two periods (May and August). It was evaluated the survival percentage of grafting, number and size of shoots, after 90 days of the beginning of the experiment. For air layering techniques, it was tested two diameters of branch (1.0-1.5 cm and 2.0-2.5 cm) and two widths of girdling (1.5 cm and 3.0 cm) in P. cauliflora. It was evaluated the rooting percentage, and number and size of roots, 180 days after the beginning of the experiment. It was also tested the period of immersion (5, 10, 15 minutes) in 1,25% sodium hypochlorite solution in the in vitro establishment of shoot and root of the seedlings explants of P. trunciflora. After 45 days of incubation it was evaluated the percentage of contamination and number of shoots and leaves in each explant. It was observed that rooting of wood cutting is dependent of application of IBA, so the biggest rooting percentage (50%) was obtained in biggest concentration of IBA (6000mg L-1) associated with cross section. For the softwood terminal cuttings, the rooting was small (maximum of 10%). Meantime, exist the potential of rooting, and, changes of technique must be tested for maximization. The grafting and the air layering techniques are recommended for jabuticaba tree propagations, because this techniques provide high percentage of plants formation. There is visible compatibility between the three species grafted on rootstocks of P. cauliflora. The utilization of grafts collected from plants in fructification should be avoided, because they reduce the percentage of plants establishment. In air layering techniques, branches with diameter of 2.0 cm provides higher percentage of rooting and the biggest number and size of roots in relation of branches with small diameter (1.0 to 1.5 cm). The utilization of less time in immersion (five minutes) in 1,25% sodium hypochlorite solution is efficient for shoots explants disinfestations of jabuticaba tree seedlings and allows the initial in vitro establishment, providing development of shootings.

A identificação de metodologias eficientes para essa escolha de genitores em programas de melhoramento tem recebido grande atenção dos pesquisadores. O cruzamento dialélico é muito utilizado com esta finalidade; entretanto, à medida que o número de genótipos a serem testados aumenta, sua utilidade torna-se progressivamente inviável. Alternativas como medidas de dissimilaridade (baseadas em dados morfológicos, moleculares e genealógicos) têm sido propostas. Deste modo, o objetivo deste trabalho foi o de estimar a capacidade geral (CGC) e especifica (CEC) de combinação, heterose e depressão endogâmica, nas gerações F1 e F2, através de cruzamento dialélico de dez genótipos de trigo, bem como avaliar o relacionamento entre genitores e o desempenho de seus híbridos, com as medidas de dissimilaridade anteriormente citadas. Dez genitores elite foram cruzados de forma dialélica, desconsiderando os recíprocos. Os resultados obtidos revelaram uma maior participação dos efeitos aditivos na expressão genotípica das constituições utilizadas. Os parentais FUNDACEP 50, Pampeano, BRS 208 e Abalone foram os que apresentaram os maiores valores de CGC para rendimento de grãos, evidenciando o potencial destes genótipos para utilização em cruzamentos a fim de obter progênies superiores. Também, as combinações envolvendo os genótipos BRS Guamirim x BRS 208, UTF 0506 x BRS Timbaúva, BRS 208 x Abalone, CD 115 x Abalone e BRS Timbaúva x Abalone, apresentaram elevado grau de heterose e baixa perda de vigor para os caracteres de maior importância agronômica. A heterose revelada pelo F1 não garante populações segregantes promissoras, pois em muitos casos não ocorreu associação da heterose do F1 com desempenho dos bulks das gerações F2. As estimativas das correlações entre as matrizes de dissimilaridade permitiram observar a concordância existente entre as matrizes de dissimilaridade Morfológica+AFLP x Molecular AFLP (0,466) e entre Morfológica+AFLP x genealógica (0,42). A distancia genealógica foi positivamente associada com os caracteres massa de 100 grãos e peso de grãos por planta nas gerações F1 (0,67 e 0,62, respectivamente) e F2 (0,62 e 0,59, respectivamente), o que lhe confere moderada confiabilidade em substituir os cruzamentos dialélicos na escolha dos genitores. Em contrapartida, as medidas de distancia morfológica e combinada (molecular AFLP+morfológica), não foram associadas com o desempenho dos híbridos, em ambas as gerações avaliadas. / The identification of efficient methodologies for the choice of parents in breeding programs has received great attention from researchers. The diallel cross is widely used with this purpose, however, as the number of genotypes to be tested increases, its utility becomes increasingly unviable. Alternatives such as measures of dissimilarity (based on morphological dates, molecular and genealogical) have been proposed. Thus, the objective of this study was to estimate the general (CGC) and specific (CEC) of combining , heterosis and inbreeding depression in F1 and F2 generations, through diallel of ten wheat genotypes and to assess the relationship between parents and the performance of their hybrids, with the dissimilarity measures described above.Ten elite parents were crossed in a diallel form, disregarding the reciprocal. The results revealed a greater participation of additive effects on phenotypic expression of the constitutions used. The parents FUNDACEP 50, Pampeano, Abalone and BRS 208 presented the highest values of CGC for grain yield, demonstrating the potential of these genotypes for use in crosses to promote superior progenies.Also, the combinations involving the genotypes BRS Guamirim x BRS 208, UTF 0506 x Timbaúva BRS, BRS 208 x Abalone, CD 115 x Abalone and BRS Timbaúva x Abalone, showed a high degree of heterosis and low inbreeding for characters of greatest agronomy importance. Heterosis revealed by F1 does not warrant segregating populations, because in many cases there was no association with heterosis of F1 and with performance of bulks on F2 generations. Estimates of correlations between the dissimilarity matrices allowed to observe the correlation between the dissimilarity matrices Morophological + AFLP x Molecular AFLP (0.466) and between Morophological + AFLP x pedrigree (0.427). The genealogical distance was positively associated with weight of 100 grains and grain weight per plant in F1 generation (0.67 and 0.62, respectively) and F2 (0.62 and 0.59, respectively), it gives moderate reliability to replace the diallel in the choice of parents. In contrast, the measures of morphological distance and combined (morphological + molecular AFLP), were not associated with the performance of hybrids, in both generations avaliable.

A identificação de metodologias eficientes para essa escolha de genitores em programas de melhoramento tem recebido grande atenção dos pesquisadores. O cruzamento dialélico é muito utilizado com esta finalidade; entretanto, à medida que o número de genótipos a serem testados aumenta, sua utilidade torna-se progressivamente inviável. Alternativas como medidas de dissimilaridade (baseadas em dados morfológicos, moleculares e genealógicos) têm sido propostas. Deste modo, o objetivo deste trabalho foi o de estimar a capacidade geral (CGC) e especifica (CEC) de combinação, heterose e depressão endogâmica, nas gerações F1 e F2, através de cruzamento dialélico de dez genótipos de trigo, bem como avaliar o relacionamento entre genitores e o desempenho de seus híbridos, com as medidas de dissimilaridade anteriormente citadas. Dez genitores elite foram cruzados de forma dialélica, desconsiderando os recíprocos. Os resultados obtidos revelaram uma maior participação dos efeitos aditivos na expressão genotípica das constituições utilizadas. Os parentais FUNDACEP 50, Pampeano, BRS 208 e Abalone foram os que apresentaram os maiores valores de CGC para rendimento de grãos, evidenciando o potencial destes genótipos para utilização em cruzamentos a fim de obter progênies superiores. Também, as combinações envolvendo os genótipos BRS Guamirim x BRS 208, UTF 0506 x BRS Timbaúva, BRS 208 x Abalone, CD 115 x Abalone e BRS Timbaúva x Abalone, apresentaram elevado grau de heterose e baixa perda de vigor para os caracteres de maior importância agronômica. A heterose revelada pelo F1 não garante populações segregantes promissoras, pois em muitos casos não ocorreu associação da heterose do F1 com desempenho dos bulks das gerações F2. As estimativas das correlações entre as matrizes de dissimilaridade permitiram observar a concordância existente entre as matrizes de dissimilaridade Morfológica+AFLP x Molecular AFLP (0,466) e entre Morfológica+AFLP x genealógica (0,42). A distancia genealógica foi positivamente associada com os caracteres massa de 100 grãos e peso de grãos por planta nas gerações F1 (0,67 e 0,62, respectivamente) e F2 (0,62 e 0,59, respectivamente), o que lhe confere moderada confiabilidade em substituir os cruzamentos dialélicos na escolha dos genitores. Em contrapartida, as medidas de distancia morfológica e combinada (molecular AFLP+morfológica), não foram associadas com o desempenho dos híbridos, em ambas as gerações avaliadas. / The identification of efficient methodologies for the choice of parents in breeding programs has received great attention from researchers. The diallel cross is widely used with this purpose, however, as the number of genotypes to be tested increases, its utility becomes increasingly unviable. Alternatives such as measures of dissimilarity (based on morphological dates, molecular and genealogical) have been proposed. Thus, the objective of this study was to estimate the general (CGC) and specific (CEC) of combining , heterosis and inbreeding depression in F1 and F2 generations, through diallel of ten wheat genotypes and to assess the relationship between parents and the performance of their hybrids, with the dissimilarity measures described above.Ten elite parents were crossed in a diallel form, disregarding the reciprocal. The results revealed a greater participation of additive effects on phenotypic expression of the constitutions used. The parents FUNDACEP 50, Pampeano, Abalone and BRS 208 presented the highest values of CGC for grain yield, demonstrating the potential of these genotypes for use in crosses to promote superior progenies.Also, the combinations involving the genotypes BRS Guamirim x BRS 208, UTF 0506 x Timbaúva BRS, BRS 208 x Abalone, CD 115 x Abalone and BRS Timbaúva x Abalone, showed a high degree of heterosis and low inbreeding for characters of greatest agronomy importance. Heterosis revealed by F1 does not warrant segregating populations, because in many cases there was no association with heterosis of F1 and with performance of bulks on F2 generations. Estimates of correlations between the dissimilarity matrices allowed to observe the correlation between the dissimilarity matrices Morophological + AFLP x Molecular AFLP (0.466) and between Morophological + AFLP x pedrigree (0.427). The genealogical distance was positively associated with weight of 100 grains and grain weight per plant in F1 generation (0.67 and 0.62, respectively) and F2 (0.62 and 0.59, respectively), it gives moderate reliability to replace the diallel in the choice of parents. In contrast, the measures of morphological distance and combined (morphological + molecular AFLP), were not associated with the performance of hybrids, in both generations avaliable.

The NDSU EarlyGEM or the Early Germplasm Enhancement of Maize (Zea maize L.) is a long term incorporation program designed to increase the genetic diversity of short season hybrids. Starting in 1999, exotic GEM breeding crosses derived from temperate accessions: BR52051, CH05015; tropical accessions: SCR01, CUBA17, FS8B; and tropical hybrid DKB844 along with late checks: B73, Mo17, and Iowa Stiff Stalk Synthetic (BSSS), were adapted to short-seasons and incorporated via a modified backcross (BC) procedure. This study was designed to assess the genetic diversity in exotic derived BC1:S1 lines and their competitive potential as sources of new and unique hybrids. Useful genetic diversity was evaluated with testers belonging to opposite heterotic groups, LH176 representing a non stiff stalk and TR3026 x TR2040 a stiff stalk testers and were tested in five North Dakota environments over two years (2009 and 2010). All the traits showed highly significant (P<0.01) differences across genotypes except root and stalk lodging. Among 236 experimental testcrosses, 64 were statistically not different (LSD, 0.05) to industry hybrids for grain yield. BC derived lines from BR52051, CHO5015, DKB844 showed diverse alleles for low grain moisture (below 87 relative maturity days) at harvest and high grain yield. SCR01, BR52051, CHO5015 and CUBA117 derived lines produced hybrids with high grain oil (4. 9% vs. 4.1%) and grain protein (10.4% vs. 9.1%) contents compared to top checks. The results showed that the exotic incorporations are the sources of unique new alleles for early maturing maize not present in existing US germplasms (e.g. B73, Mo17, and BSSS). Even though each exotic cross was unique to integrate diverse alleles, utilizing multiple unique exotic crosses for incorporation showed large variation for specific traits. Phenotypic correlations of traits showed grain moisture played the most important role for short season hybrid development. Exotic incorporation through NDSU EarlyGEM has shown a new way of breeding early maturing maize keeping the breeding program open and genetic diversity high.

CRISPR/Cas has been developed for targeted mutagenesis in diverse species, including plants. However, precise genome editing via homology-directed repair (HDR) is inefficient in plants, limiting our ability to make large deletions or insertions in the plant genomes. Prime editing increases the control over the desired editing and allows the precise introduction of all types of mutations, including insertion, deletions, and all possible base conversions, albeit at low efficiencies. Here, we designed a dual prime editing system to generate large deletions and precise insertions of sequences by repairing template complementarity. We coupled dual pegRNA with Cas9 nickase (nCas9) to generate deletions and insertions. In another modality, we used dual pegRNA with wild-type Cas9 to generate double-stranded breaks to improve the editing at the targeted sites. We tested dual pegRNAs to delete the last exon in OsCCD7, delete the microRNA targeted sequence in OsIPA, and insert the T7 promoter in the 3'UTR of OsALS. Our results showed a high frequency of targeted insertion of the T7 promoter sequence in the 3'UTR of OsALS with wtCas9 and nCas9. Sanger sequencing analysis showed partial deletions at the targeted locus. Further improvements in the designs of pegRNAs will increase the precise genome insertions and deletions in plants.

Plant hormones and their gene regulatory networks orchestrate a diverse array of metabolic and physiological changes crucial for growth, development, and environmental responses. Targeting the engineering of hormone signaling networks holds promise for enhancing plant health, crop productivity, and vigor. However, these networks are intricate, featuring negative feedback loops, extensive interconnections between pathways, pleiotropy, and overlapping gene expression. These complexities pose challenges in identifying candidate genes and parsing apart their isolated functions that could be strategically engineered to achieve desired plant phenotypes. Integration of comparative evolution, synthetic biology, and expression analysis facilitates the deconstruction of these networks. Through systems biology approaches data dimensionality can be reduced, enabling the attribution of specific phenotypes to associated genes. Here, I reviewed how the employment of these above-mentioned approaches can aid in the identification of candidate genes involved the regulation of growth and development within specific tissues, and how through synthetic biology we can explore the sequence-function space of candidate genes and their pathway modules. Candidate genes identified through this process can be evaluated through comparative evolutionary approaches, and efficiently tested in synthetic systems for engineering of their molecular functionalities in a high-throughput manner. Here, as a case study, I employ a systems biology approach to identify tissue-specific candidate genes within the auxin regulatory network in soybean shoot development. This method aims to minimize pleiotropy and off-target effects by utilizing expression analysis tissue-specificity score and principal component analysis. I primarily, focused on three pivotal components of the nuclear auxin signaling pathway: Aux/IAA transcriptional repressors, ARF transcription factors, and TIR1/AFB auxin receptors. These components collectively modulate auxin signaling, influencing various growth and environmental responses. I identified genes within the three pivotal components of auxin signaling involved in early shoot architecture development, which has advantages from weed suppression to yield in soybean cultivation. I used a yeast chassis to investigate the function of pleiotropic auxin receptors, which primarily regulate Aux/IAA levels and orchestrate transcriptional changes in response to auxin. I explored whether these receptors modulate auxin response in a concerted fashion, as they are generally not tissue specific. Here, I reported that auxin receptors interact in an epistatic manner to modulate auxin response. This case of study serves as a foundation in engineering plant genotype-phenotype via auxin signaling. / Doctor of Philosophy / Plant hormones are essential for controlling various processes that drive plant growth, development, and responses to the environment. Scientists are exploring ways to engineer the networks that regulate these hormones to improve plant health, boost crop yields, and enhance plant strength. However, these networks are complex, with many interacting parts, making it difficult to identify which genes to modify to achieve specific outcomes in plants. To tackle this challenge, researchers use a combination of approaches, including studying how these networks have evolved, analyzing large amounts of biological data, and using synthetic biology to test and refine their findings. By breaking down the complexity of these networks, they can link specific genes to particular plant traits. Once these genes–trait links are identified, they can be further tested and engineered to optimize plant characteristics. In this study, I focused on the auxin hormone, which plays a key role in numerous aspects of plant growth including soybean shoot development and Arabidopsis root development. I looked at three main components of the auxin regulatory network: Aux/IAA proteins (which act as repressors), ARF transcription factors (which control gene expression), and TIR1/AFB receptors (which detect auxin levels). These components work together to regulate how plants grow and respond to their environment. I identified key genes within these main auxin components that are important for early development of soybean shoots and minimizes off-target effects. This can help improve soybean farming by enhancing weed control and enhancing crop yields Using a synthetic biology yeast system, I studied the function of TIR1/AFB auxin receptors and how this family of receptors interact to perceive auxin and control the levels of Aux/IAA proteins, consequently controlling the plant's growth in response to auxin. I found that auxin receptors work in concert in a way that reduces their overall effect on the plants response to auxin. This research lays the groundwork for future efforts to engineer plant traits by modifying the auxin signaling pathway, which could lead to improved crop performance and resilience.