SUMMARY Cordia trichotoma, popularly known in Brazil as ‘louro-pardo’, has numerous environmental and timber-related applications. This species is naturally propagated by seeds; however, this form of propagation results in restricted seedling production due to high genetic variability associated with a low degree of forest improvement. Thus, cloning of selected adult trees is an alternative for producing high-quality plants. The aims of this work were to evaluate the emission of epicormic shoots from pruned branches and to define a protocol for the in vitro establishment of C. trichotoma selected adult trees. Propagules used in these experiments were derived from the selection of four C. trichotoma adult trees. The branches were placed in a greenhouse to induce epicormic shoots. Number of shoots according to the climatic season (autumn and spring) and shoot collection time were evaluated. Three sodium hypochlorite concentrations were evaluated for five minutes (C1 = 0.58 % active chlorine, C2 = 1.12 % active chlorine, and C3 = 2.12 % active chlorine) for in vitro establishment. Results indicated that the epicormic shoot technique could be considered viable for shoot emission of C. trichotoma, regardless of the climatic season (autumn or spring). Sodium hypochlorite solution applied for 5 minutes (1.12 % of active chlorine) was effective for the in vitro establishment of nodal segments from epicormic shoots, resulting in 61.3 % of established explants. These results are promising for the cloning of adult trees during the mature stage of development in the field without the need to cut the selected tree.

RESUMEN: Cordia trichotoma, conocida popularmente en Brasil como "louro-pardo", tiene numerosas aplicaciones ambientales y madereras. Esta especie se propaga naturalmente por semillas; sin embargo, esta forma de propagación resulta en una producción restringida de plántulas por la alta variabilidad genética asociada con un bajo grado de mejoramiento forestal. La clonación de árboles adultos seleccionados es una alternativa para producir plantas de alta calidad. El objetivo de este trabajo fue evaluar la emisión de brotes epicórmicos de ramas podadas y definir un protocolo para el establecimiento in vitro de árboles adultos seleccionados. Los propágulos utilizados se derivaron de la selección de cuatro árboles adultos y las ramas se colocaron en un invernadero para inducir brotes epicórmicos. Se evaluó el número de brotes según la época climática (otoño y primavera), el tiempo de recolección de los brotes, y tres concentraciones de hipoclorito de sodio durante cinco minutos (C1 = 0,58 % de cloro activo, C2 = 1,12 % de cloro activo y C3 = 2,12 % de cloro activo) para el establecimiento in vitro. Los resultados indicaron que la técnica de brotes epicórmicos podría considerarse viable para la emisión de brotes independientemente de la estación climática. La solución de hipoclorito de sodio (1,12 % de cloro activo) fue eficaz para el establecimiento in vitro de segmentos nodales de brotes epicórmicos, resultando en un 61,3 % de explantes establecidos. Los resultados son prometedores para la clonación de laureles durante la etapa madura de desarrollo en el campo sin necesidad de cortar el árbol seleccionado.

ABSTRACT Background: We have developed a micropropagation technique methodology to clonal micro-garden formation of Bambusa vulgaris selected adult plant. Collection site (i.e., stock plant cultivated in different environments) and seasonality of shoot collection (i.e., months of the year) effects on in vitro culture were evaluated. Explants that showed normal development were ex vitro rooted in mini-incubator system, and three culture media (WPM, MS and deionized water + agar) supplemented with plant growth regulators (IBA, NAA and BAP) and antibiotic (streptomycin sulphate and culture medium free - control) were evaluated. Micropropagated plants were acclimatized in a shadow house and transferred to a semi-hydroponic system for establishment of a clonal micro-garden. The efficacy of the cloning protocol was determined with genetic fidelity analysis by ISSR molecular markers. Results: Considering all inoculations, 21.9% of nodal segments were in vitro established in nine shoot collections. The rooting percentage was 36.6%, and no interactions were observed between the use of culture medium and antibiotic. Culture medium free antibiotic resulted in 80.0% of survival and 50.0% of adventitious rooting. Micropropagated plants presented adequate growth and adaptation to ex vitro conditions in the clonal micro-garden. Molecular analyses by ISSR markers indicated the absence of genetic variations, and histological analyses revealed normal adventitious root formation originating from meristematic cells. Conclusion: The formation of a clonal micro-garden was demonstrated, proving the feasibility of the tested technique. Our results contribute to the development of a clonal propagation protocol for B. vulgaris selected adult plants.

Resumo Visando fornecer informações que sirvam de base para estudos de melhoramento genético de Eucalyptus foi realizada a análise da diversidade genética usando marcadores ISSR. As espécies estudadas foram Eucalyptus urophylla e Eucalyptus microcorys, ambas com potencial econômico florestal. Os indivíduos estudados pertencem a um teste de espécies e procedências instalado no ano de 1974 e permanecem isentos de tratos silviculturais. Para as análises foram utilizados nove primers ISSR universais. A partir dos resultados avaliou-se a existência de variação intra e interespecífica por meio da porcentagem de polimorfismo, conteúdo de informação polimórfica (PIC) e distância Euclidiana entre indivíduos. A fim de analisar a distância Euclidiana entre os indivíduos foram feitas a análise de coordenadas principais (PCoA) e análise permutacional de dispersão multivariada (PermDisp) seguida pelo teste de Tukey. Observou-se elevada porcentagem de polimorfismo (57,14% para Eucalyptus microcorys e 80,95% para Eucalyptus urophylla). Considerando todos os grupos avaliados, os valores de PIC foram superiores a 0,5 para quatro primers (UBC827, UBC835, UBC841). A variabilidade interna foi significativamente maior na população de Eucalyptus urophylla em relação à Eucalyptus microcorys, o que pode estar associado à maior capacidade do Eucalyptus urophylla em hibridizar naturalmente.

Abstract Here, we deployed a genetic diversity analysis using ISSR markers in two Eucalyptus species, as a fundamental step toward breeding strategies. The selected species were Eucalyptus urophylla and Eucalyptus microcorys, both with economic potential in forestry. The studied individuals belong to a species and the provenance test that was installed in 1974 and until now remains exempt from silvicultural treatments. Nine universal ISSR primers were used in the analyses. Intra and interspecific variation through the polymorphism percentage, polymorphism information content (PIC) and Euclidean distances among individuals were computed. The Euclidean distance among the individuals was used to perform a principal coordinate analysis (PCoA), as well as the permutation analysis of multivariate dispersion (PermDisp), followed by the Tukey’s test. A high percentage of polymorphic bands were detected, with 57.14% for Eucalyptus microcorys and 80.95% for Eucalyptus urophylla. PIC values were higher than 0.5 for four primers (UBC827, UBC835, UBC841, and UBC842). The genetic variability was significantly higher within the Eucalyptus urophylla population than in Eucalyptus microcorys, which may be associated with the greater ability of Eucalyptus urophylla to naturally hybridize, therefore, enabling the introduction of new alleles to their populations.