A hibridação somática via fusão de protoplastos é um método de combinação genética que agrega integralmente os dois genomas genitores, assim como suas respectivas organelas citoplasmáticas, sendo uma ferramenta alternativa aos métodos convencionais de melhoramento. O objetivo deste trabalho foi a obtenção de híbridos somáticos interespecíficos de laranja doce (Citrus sinensis L. Osbeck) \'Pêra\' e \'Westin\' com a tangerina \'Fremont\' (C. clementina hort. ex Tan. x C. reticulata Blanco), \'Thomas\' (C. reticulata Blanco) e \'Nules\' (C. clementina hort. ex Tan), e o tangelo \'Nova\' (C. reticulata Blanco x C. paradisi Macf.) visando à produção de genitores tetraplóides com características agronômicas desejáveis. Como fontes de protoplastos foram utilizados calos embriogênicos de laranja e folhas jovens de tangerina e tangelo, coletadas de plantas cultivadas em casa-de-vegetação. Para o isolamento dos protoplastos, as células de ambas as fontes foram imersas em solução enzimática e incubadas no escuro por 14 horas, sob agitação. Após o isolamento, os protoplastos isolados de calos foram fundidos quimicamente (polietilenoglicol - PEG) com protoplastos não embriogênicos, isolados de mesofilo foliar. Em seguida, os protoplastos foram plaqueados nos meios de cultura líquido BH3, EME e BH3 + EME, e incubados em ausência de luz, sob temperatura de 27 ºC. Após 20 dias de incubação, foram iniciados subcultivos sequenciais para nutrição e redução do potencial osmótico do meio de cultura. Os microcalos desenvolvidos foram transferidos para meio de cultura EME + maltose (13 g.L-1) para a indução da embriogênese somática. Os embriões desenvolvidos foram transferidos para meio de cultura EME + sacarose (25 g.L-1) e em seguida para o meio de cultura 1500 (1,5 g.L-1 de extrato de malte) e finalmente para o meio B+ para desenvolvimento e germinação. As plantas regeneradas foram individualizadas, enraizadas ou microenxertadas e aclimatizadas em casa-devegetação. A confirmação da hibridação somática foi feita por análise morfológica, determinação da ploidia, por citometria de fluxo e análise do DNA com marcadores moleculares do tipo SSR e RAPD. Foram obtidos híbridos somáticos de três combinações (Pêra + Fremont, Pêra + Nules e Pêra + Nova) os quais serão avaliados para utilização diretamente como copa ou como genitor tetraplóide em programas de melhoramento de citros. / Somatic hybridization by protoplasts fusion is a method of genetic combination of two parental genomes, and their cytoplasmic organelles. It is an alternative tool to conventional breeding methods. The objective of this work was to obtain interspecific somatic hybrids of \'Pera\' and \'Westin\' sweet oranges (Citrus sinensis L. Osbeck) with \'Fremont\' (C. clementina hort. ex Tan. x C. reticulata Blanco), \'Thomas\' (C. reticulata Blanco) and \'Nules\' (C. clementina hort. ex Tan) mandarins and Nova tangelo (C. reticulata Blanco x C. paradisi Macf.) to produce tetraploid parents with desirable agronomic characteristics. The sources of protoplasts were sweet orange embryogenic callus and mandarin and tangelo young leaves, collected from plants cultivated in screenhouses. For protoplasts isolation, cells from both sources were immersed in enzyme solution and incubated in the dark for 14 hours (40 rpm). After isolation, the protoplasts isolated from callus were fused chemically (polyethylene glycol - PEG) with non embryogenic protoplasts, isolated from leaf mesophyll. The protoplasts were cultivated in BH3, BH3 + EME and EME liquid culture media and incubated in the dark, under temperature of 27 ºC. After 20 days of incubation, subcultures were initiated in order to reduce the osmotic potential of culture medium. The microcalus developed were transferred to EME medium supplemented with maltose (13 g.L-1) to induce somatic embryogenesis. The developed embryos were transferred into EME + sucrose (25 g.L- 1), culture medium 1500 (1.5 g.L-1 of malt extract) or B+ culture medium for development and germination. The regenerated plants were individually rooted or micrografted, and further acclimatized in screenhouse. Somatic hybridization was confirmed by analysis of leaf morphology, ploidy determination by flow cytometry and molecular analysis by SSR and RAPD markers. Somatic hybrids were obtained of the three combinations (Pera + Fremont, Pêra + Nules and Pêra + Nova) which will be evaluated for direct are as scion or as a tetraploid parent in citrus breeding programs.

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.

Atualmente, o Huanglongbing (HLB), doença associada à bactéria Candidatus Liberibacter spp., é a principal ameaça dos Citrus, não tendo sido encontrado ainda espécies resistentes e tolerantes. O melhoramento genético tradicional apresenta limitações para a obtenção de novas variedades porta-enxerto e copa de citros em decorrência a fatores ligados à biologia do gênero. Na tentativa de sobrepor essas dificuldades, a transformação genética destaca-se por permitir a introdução de genes exógenos, os quais, além de reduzir o período de obtenção de material melhorado geneticamente, poderão conferir resistência a doenças em variedades de interesse agronômico. Desse modo, o objetivo desta pesquisa consistiu no estudo da organogênese in vitro, e na obtenção de plantas transgênicas via Agrobacterium tumefaciens das tangerinas \'Fremont\', \'Thomas\' e \'Nules\', com o gene que codifica o peptídeo antibacteriano atacina A (attA), controlado pelos promotores AtSUC2 e AtPP2, visando a expressão gênica preferencial nos vasos do floema. Adicionalmente, foi avaliada a transformação genética via A. tumefaciens de suspensões celulares de tangerina \'W-Murcott\', de laranja doce \'Hamlin\' e de tangelo \'Page\', e a transformação genética direta via PEG de protoplastos da tangerina \'W-Murcott\', com os fatores de transcrição VvmybA1 e Ruby, dirigidos pelos promotores com expressão preferencial nos tecidos embrionários 6105 e DC3. A eficiência da organogênese in vitro foi influenciada pelo tipo de explante e concentração de BAP. Após os experimentos de transformação genética de segmentos de epicótilo e internodal das tangerinas \'Fremont\', \'Thomas\' e \'Nules\', as plantas regeneradas foram analisadas por PCR, Southern blot e RT-qPCR e confirmadas como transgênicas pela presença e transcrição do gene attA no tecido vascular. A transformação genética de suspensões celulares mostrou-se eficiente com alta produção de antocianina nos embriões somáticos regenerados de tangerina \'W-Murcott\', de laranja doce \'Hamlin\' e de tangelo \'Page\'. A transformação genética direta de protoplastos de tangerina \'W-Murcott\' mostrou-se viável e também foi possível a obtenção de embriões somáticos transgênicos. Os fatores de transcrição VvmybA1 e Ruby se mostraram úteis para detecção visual do material transgênico / Currently, Huanglongbing (HLB), associated to Candidatus Liberibacter spp., is the main threat to the citrus culture. The conventional plant breeding shows limitations to the obtain new varieties of rootstock and scion, due to factors related to the biology of the genus. In attempt to overcome these barriers, genetic engineering is notable for allowing the introduction of foreign genes, which, besides reducing the time to obtain genetically improved material may confer disease resistance in varieties of agronomic interest. Thus, the objective of the research was the study of in vitro organogenesis, and obtain transgenic plants of \'Fremont\', \'Thomas\' and \'Nules\' mandarins via Agrobacterium tumefaciens with the gene encoding the antibacterial peptide attacin A (attA), controlled by the promoters AtSUC2 and AtPP2, aiming to preferential gene expression in phloem. In addition, the genetic transformation of cell suspensions, via A. tumefaciens, of \'W-Murcott\' mandarin, \'Hamlin\' sweet orange and \'Page\' tangelo and the direct genetic transformation, via PEG, of \'W-Murcott\' mandarin protoplasts were evaluated with VvmybA1 and Ruby transcription factors driven by 6105 and DC3 promoters, with preferential expression in embryonic tissues. The in vitro organogenesis of the varieties studied was influenced by the type of explant and BAP concentration. After genetic transformation experiments of epicotyl and internodal segments of \'Fremont\', \'Thomas\' and \'Nules mandarins, regenerated plants were analyzed by PCR, Southern blot and RT-qPCR and confirmed as transgenic by presence and transcription of attA gene. The genetic transformation of cell suspensions was efficient with high anthocyanin production in the somatic embryos regenerated of \'W-Murcott\' mandarin, \'Hamlin\' sweet orange and \'Page\' tangelo. The direct genetic transformation of \'W-Murcott\' mandarin protoplasts revealed to be viable and it was also possible to obtain transgenic somatic embryos. The VvmybA1 and Ruby transcription factors were useful tools for visual detection of transgenic material

Atualmente, o Huanglongbing (HLB), doença associada à bactéria Candidatus Liberibacter spp., é a principal ameaça dos Citrus, não tendo sido encontrado ainda espécies resistentes e tolerantes. O melhoramento genético tradicional apresenta limitações para a obtenção de novas variedades porta-enxerto e copa de citros em decorrência a fatores ligados à biologia do gênero. Na tentativa de sobrepor essas dificuldades, a transformação genética destaca-se por permitir a introdução de genes exógenos, os quais, além de reduzir o período de obtenção de material melhorado geneticamente, poderão conferir resistência a doenças em variedades de interesse agronômico. Desse modo, o objetivo desta pesquisa consistiu no estudo da organogênese in vitro, e na obtenção de plantas transgênicas via Agrobacterium tumefaciens das tangerinas \'Fremont\', \'Thomas\' e \'Nules\', com o gene que codifica o peptídeo antibacteriano atacina A (attA), controlado pelos promotores AtSUC2 e AtPP2, visando a expressão gênica preferencial nos vasos do floema. Adicionalmente, foi avaliada a transformação genética via A. tumefaciens de suspensões celulares de tangerina \'W-Murcott\', de laranja doce \'Hamlin\' e de tangelo \'Page\', e a transformação genética direta via PEG de protoplastos da tangerina \'W-Murcott\', com os fatores de transcrição VvmybA1 e Ruby, dirigidos pelos promotores com expressão preferencial nos tecidos embrionários 6105 e DC3. A eficiência da organogênese in vitro foi influenciada pelo tipo de explante e concentração de BAP. Após os experimentos de transformação genética de segmentos de epicótilo e internodal das tangerinas \'Fremont\', \'Thomas\' e \'Nules\', as plantas regeneradas foram analisadas por PCR, Southern blot e RT-qPCR e confirmadas como transgênicas pela presença e transcrição do gene attA no tecido vascular. A transformação genética de suspensões celulares mostrou-se eficiente com alta produção de antocianina nos embriões somáticos regenerados de tangerina \'W-Murcott\', de laranja doce \'Hamlin\' e de tangelo \'Page\'. A transformação genética direta de protoplastos de tangerina \'W-Murcott\' mostrou-se viável e também foi possível a obtenção de embriões somáticos transgênicos. Os fatores de transcrição VvmybA1 e Ruby se mostraram úteis para detecção visual do material transgênico / Currently, Huanglongbing (HLB), associated to Candidatus Liberibacter spp., is the main threat to the citrus culture. The conventional plant breeding shows limitations to the obtain new varieties of rootstock and scion, due to factors related to the biology of the genus. In attempt to overcome these barriers, genetic engineering is notable for allowing the introduction of foreign genes, which, besides reducing the time to obtain genetically improved material may confer disease resistance in varieties of agronomic interest. Thus, the objective of the research was the study of in vitro organogenesis, and obtain transgenic plants of \'Fremont\', \'Thomas\' and \'Nules\' mandarins via Agrobacterium tumefaciens with the gene encoding the antibacterial peptide attacin A (attA), controlled by the promoters AtSUC2 and AtPP2, aiming to preferential gene expression in phloem. In addition, the genetic transformation of cell suspensions, via A. tumefaciens, of \'W-Murcott\' mandarin, \'Hamlin\' sweet orange and \'Page\' tangelo and the direct genetic transformation, via PEG, of \'W-Murcott\' mandarin protoplasts were evaluated with VvmybA1 and Ruby transcription factors driven by 6105 and DC3 promoters, with preferential expression in embryonic tissues. The in vitro organogenesis of the varieties studied was influenced by the type of explant and BAP concentration. After genetic transformation experiments of epicotyl and internodal segments of \'Fremont\', \'Thomas\' and \'Nules mandarins, regenerated plants were analyzed by PCR, Southern blot and RT-qPCR and confirmed as transgenic by presence and transcription of attA gene. The genetic transformation of cell suspensions was efficient with high anthocyanin production in the somatic embryos regenerated of \'W-Murcott\' mandarin, \'Hamlin\' sweet orange and \'Page\' tangelo. The direct genetic transformation of \'W-Murcott\' mandarin protoplasts revealed to be viable and it was also possible to obtain transgenic somatic embryos. The VvmybA1 and Ruby transcription factors were useful tools for visual detection of transgenic material

Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression by Sanjeev V. Raikar Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×106 g-1FW was obtained when cell suspensions were used as the tissue source, with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×106 g-1FW) of L. corniculatus was achieved from cotyledons also with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm2 for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

Indiana University-Purdue University Indianapolis (IUPUI) / Dehydrins are type II LEA proteins induced in many plants during drought, low temperature, and high salinity to confer stress tolerance. AtERD14 is an Arabidopsis thaliana dehydrin that functions in part of the cold stress pathway. AtERD14 has chaperone-like capabilities that allow it to bind and protect various proteins from dehydration stresses. In order to determine the necessary components for cold induction of AtERD14, AtERD14prom::GFP/GUS and AtERD14prom::AtERD14 in AtERD14 KO constructs were created and stably transformed into A. thaliana. Analysis of the constructs showed the AtERD14 promoter alone was insufficient to respond to cold, and it was necessary to attach the AtERD14 coding region to the promoter to induce a cold response in ERD14. On the other hand, the RD29aprom::GFP/GUS promoter did respond to cold stress, indicating that RD29a does not require its coding region to support an increased amount of reporter activity after cold stress. The protoplast transformation system, while capable of transient expression of introduced constructs in protoplasts, was difficult for use for cold-inducible expression.