Polyploid induction of Eucalyptus dunnii Maiden to generate variability in breeding programs

Articles Polyploid induction of Eucalyptus dunnii Maiden to generate variability in breeding programs Inducción de poliploides de Eucalyptus dunnii Maiden para generar variabilidad en programas de mejoramiento genético Indução poliplóide de Eucalyptus dunnii Maiden para gerar variabilidade nos programas de melhoramento genético A. Castillo1  http://orcid.org/0000-0001-7381-3887 V. López1  http://orcid.org/0000-0002-4809-1576 E. Tavares2  http://orcid.org/0000-0002-7667-3928 F. Santiñaque3  http://orcid.org/0000-0002-6943-3037 M. Rizza Dalla1  http://orcid.org/0000-0003-4065-0940 1Instituto Nacional de Investigación Agropecuaria (INIA), Unidad de Biotecnología, Estación Experimental INIA Las Brujas, Canelones, Uruguay. 2Forestal Oriental, Paysandú, Uruguay. 3Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay. Abstract: Eucalyptus dunnii Maiden produces good quality cellulose pulp, showing good frost tolerance. However, in Uruguay, it needs more than six years to reach the reproductive stage. Genome duplication was proposed as a strategy to obtain useful variability. The application of mitosis inhibitors for chromosomal duplication, in explants growing in vitro, was evaluated. Two antimitotic agents were used: colchicine and oryzalin in different concentrations and exposure times, in two types of explants: explants growing in vitro and pre-germinated seeds. The number of chloroplasts was used as a rapid ploidy estimator and confirmed by flow cytometry. For chloroplast count, fluorescein diacetate (fda) applied to in vitro leaves was used for staining. Oryzalin was effective for inducing plant duplication in E. dunnii from in vitro explants. In pre-germinated seeds, both antimitotic agents induced polyploids. The average number of chloroplasts was 5.5 in diploid control plants and more than 7 in tetraploids. Obtained plantlets were successfully cloned in the greenhouse. This is the first report on artificial polyploidy obtained in E. dunnii. Keywords: polyploidy; oryzaline; colchicine; in vitro culture Resumen: La Eucalyptus dunnii Maiden produce pulpa de celulosa de buena calidad, además muestra buena tolerancia a las heladas. Sin embargo, en Uruguay presenta una fase juvenil mayor a los 6 años hasta alcanzar la etapa reproductiva. Como estrategia de mejoramiento genético, se propuso la duplicación del genoma para obtener nueva variabilidad. Se evaluó la aplicación de inhibidores de mitosis para inducir la duplicación cromosómica en explantes creciendo in vitro. Se usaron dos agentes antimitóticos: colchicina y oryzalin, en diferentes concentraciones y tiempos de exposición en dos tipos de explantes: explantes creciendo in vitro y semilla pre-germinada. El número de cloroplastos se usó como estimador rápido de ploidía y se confirmó mediante la citometría de flujo. En el conteo de cloroplastos se usó para la tinción el diacetato de fluoresceína (fda) aplicado en hojas de explantes in vitro. El oryzalin fue efectivo para obtener duplicación en E. dunnii a partir de explantes in vitro. En semillas pregerminadas, ambos agentes antimitóticos generaron poliploides. El número promedio de cloroplastos fue de 5,5 en plantas control diploides y más de 7 en tetraploides. Las plantas obtenidas fueron clonadas con éxito en invernáculo. Este es el primer reporte de poliploides artificiales obtenidos en E. dunnii. Palabras clave: poliploidía; oryzalin; colchicina; cultivo in vitro Resumo: Eucalyptus dunnii Maiden produz celulose de boa qualidade e mostra boa tolerância à geada. Contudo, no Uruguai apresenta uma fase juvenil com mais de 6 anos de idade até atingir a fase reprodutiva. Como estratégia de melhoramento genético, foi proposta a duplicação do genoma para obter uma nova variabilidade. Foi avaliada a aplicação de inibidores da mitose para induzir a duplicação cromossómica em explantes que crescem in vitro. Foram utilizados dois agentes antimitóticos: colchicina e oryzalina em diferentes concentrações e tempos de exposição em dois tipos de explantes: explantes que crescem in vitro e sementes pré-germinadas. O número de cloroplastos foi utilizado como um estimador de ploidia rápida e confirmado por citometria de fluxo. Na contagem de cloroplastos, o diacetato de fluoresceína (fda) aplicado nas folhas in vitro explant foi utilizado para coloração. A oryzalin foi eficaz na obtenção de duplicação em E. dunnii a partir de explantes in vitro. Nas sementes pré-germinadas, ambos os agentes antimitóticos geraram poliploides. O número médio de cloroplastos foi de 5,5 em plantas de controlo diplóide e mais de 7 em tetraplóides. As plantas obtidas foram clonadas com sucesso numa estufa. Este é o primeiro relatório de poliploides artificiais obtido em E. dunnii. Palavras-chave: poliploidia; oryzalin; colchicina; cultura in vitro 1. Introduction The E. dunnii (Maiden) is a diploid species (2n=22) that belongs to the Myrtaceae family1, originally from small populations in Queensland, Australia2, and grows well in temperate climates, showing good tolerance to frost. In Uruguay, eucalyptus plantations represent one of the most important economic activities, mainly due to cellulose pulp production and, to a lesser extent, solid wood production for furniture, and panels and chips manufacture. The most planted species are E. globulus, E. grandis and E. dunnii3. Genetic improvement is essential to obtain genotypes more adapted to our soil and climate conditions. Forest species have problems associated with the long period of juvenility until plants reach maturity and flowering; this is why a breeding program becomes slow, expensive, and complex4. Considering the long reproduction cycle in these species, technologies have been developed to accelerate and shorten the breeding cycles; one of these technologies is polyploid induction. Polyploidy exists in nature and constitutes a way of generating new variability, mainly through the production of non-reduced gametes and interspecific hybridization5, with the potential for almost instant reproductive isolation, in addition to being a proven source of novel adaptation6)(7. Genome duplication is important for variability generation1 since the change in the number of gene copies results in changes in the morphology, structure, and physiology of new individuals8)(9. Different methods have been used to generate polyploid trees, the application of colchicine is among the most used. This compound has shown great effectiveness in the results. Likewise, there are other compounds, such as trifluralin and oryzalin, which have the advantage of being used in lower concentrations and are also specific for plant cells10. Both oryzalin and colchicine work in a similar way, stopping the cell multiplication process. During mitosis, colchicine inhibits microtubule polymerization; this failure in the formation of microtubules stops mitosis before reach (...truncated)