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 A importância de espécies dos gêneros Eucalyptus e Corymbia vem sendo evidenciada nos programas de silvicultura, principalmente pela adaptação em diversas condições ambientais e pelos múltiplos produtos que podem ser obtidos. Como a seleção e a clonagem de indivíduos superiores geralmente são realizadas na fase adulta, o desenvolvimento e a adequação de técnicas de propagação vegetativa que possibilitem o resgate e o estabelecimento in vitro de espécies desses gêneros são necessários. Dessa forma, o presente estudo teve como objetivo induzir brotos epicórmicos a partir de ramos podados de árvores adultas e promover o estabelecimento in vitro de nove espécies, sendo seis do gênero Eucalyptus e três de Corymbia. O material utilizado para a obtenção dos explantes foi proveniente da seleção de duas matrizes de cada espécie com 44 anos de idade, oriundas da implantação de um teste de espécies e procedências. Foram avaliados o número de gemas e brotos de cada ramo aos 15, 25, 35 e 45 dias após o início do experimento em casa de vegetação. De acordo com os resultados, a indução de brotações epicórmicas a partir de ramos podados foi considerada uma técnica viável e eficiente para obter fontes de propágulos de matrizes adultas de todas as espécies, apresentando um aumento gradual das características avaliadas ao longo do tempo. Além disso, o cultivo in vitro é uma alternativa eficaz para estabelecer propágulos a partir de brotações epicórmicas de árvores adultas da maioria das espécies avaliadas, com estabelecimento de até 62,5%. Contudo, para Corymbia citriodora (Hook.) K.D. Hill & L.A.S Johnson e Eucalyptus tereticornis Smith é necessário o desenvolvimento de mais estudos para viabilizar o estabelecimento in vitro.

ABSTRACT The importance of Eucalyptus and Corymbia has been evident in forestry programs, mainly due to their adaptation to various environmental conditions and the multiple products that can be obtained. As the selection and cloning of superior individuals are usually performed in adulthood, the development and adaptation of the vegetative propagation techniques that enable the rescue and in vitro establishment of species are necessary. Thus, the present study aimed to induce epicormic shoots from pruned branches of adult trees and to promote the in vitro establishment of nine species: six of Eucalyptus and three of Corymbia. The material used to obtain the explants came from the selection of two 44-year-old mother plants of each species, from species, and provenance tests. The number of buds and shoots of each branch was evaluated at 15, 25, 35, and 45 days after the beginning of the experiment in a greenhouse. According to the results, the induction of epicormic shoots from pruned branches was considered a viable and efficient technique to obtain propagule sources from adult mother plants of all species, presenting a gradual increase in the evaluated characteristics over time. Also, in vitro culture is an effective alternative to improve the propagation of the evaluated species, with an establishment of up to 62.5%. However, for Corymbia citriodora (Hook.) K.D. Hill & L.A.S Johnson and Eucalyptus tereticornis Smith it is necessary to develop further studies to enable the in vitro establishment.

ABSTRACT The relevance of Eucalyptus dunnii has been evidenced mainly for its wood quality and cold tolerance among cultivated subtropical eucalypts. However, rooting is a challenge for its propagation, particularly when adult material is involved. This study aimed to assess the mixotrophism on the in vitro elongation and adventitious rooting phases in Eucalyptus dunnii microcutting. The experimental material used was obtained from a ministumps of Eucalyptus dunnii clones. In order to evaluate gas exchange and sucrose supplementation on in vitro elongation and adventitious rooting, the experiment was prepared in a 3×4 factorial arrangements with three forms of sealing (rigid polypropylene caps with no membrane (0/M), with a membrane (1/M), with three membranes (3/M) and four sucrose concentrations (0, 10, 20 and 30 g.L-1). At 30 days in the elongation phase it was evaluated (length, number of shoots per explant, oxidation, bud vigor, pigment content, leaf area and anatomy) and rooting (length, root diameter and rooting). Results show that sucrose should be added in the culture medium for in vitro elongation and can be reduced to concentrations between 10 and 20 g.L-1. In vitro rooting requires the use of 30 g.L-1 of sucrose. The use of flasks with membranes that allow gas exchange is an effective alternative to promote the in vitro elongation and adventitious rooting of Eucalyptus dunnii microcutting.

RESUMO A micropropagação via proliferação de gemas axilares tem sido recomendado para rejuvenescimento/revigoramento de clones selecionados, e consequentemente melhoria no enraizamento de mudas clonais. O sucesso de um protocolo de micropropagação depende da fase de introdução in vitro, visto que as etapas seguintes de multiplicação, alongamento e posterior enraizamento, só podem ser executadas após o estabelecimento de culturas assépticas e com bom vigor vegetativo. Diante disso, o presente estudo teve como objetivo avaliar o efeito da qualidade de luz na introdução in vitro de clones híbridos de Corymbia torelliana x C. citriodora e Corymbia citriodora x C. torelliana pela técnica de micropropagação via proliferação por gemas axilares. As mini-stumps, fornecedoras dos explantes para introdução in vitro, foram conduzidas em minijardim clonal semi hidropônico. Segmentos nodais de três clones de Corymbia torelliana x C. citriodora (TC01, TC02 e TC03) e de um clone de Corymbia citriodora x C. torelliana (CT01) foram coletados, desinfestados e inoculados em meio de cultura JADS, à fim de comparar os efeitos da qualidade da luz de Escuro/lâmpada fluorescente, Lâmpada fluorescente, LEDs branco e LEDs vermelho/azul, e. Aos 30 dias após a inoculação, foram avaliadas as características: porcentagem média de contaminação, oxidação, explantes não reativos, comprimento de brotos e o número médio de brotações por explante maiores que 0,5 cm. Com base nos resultados obtidos, o uso da fonte de luz LEDs vermelho/azul obteve os melhores resultados, para todas as características avaliadas na introdução in vitro.

ABSTRACT Micropropagation via axillary bud proliferation is recommended for rejuvenation or reinvigoration of selected clones, as well as for improving clonal seedlings rooting. The success of a micropropagation protocol depends on the in vitro introduction, since following phases, multiplication, elongation, and rooting can only take place once the aseptic crop with vegetative vigor has been established. This study aims to assess the effect of light on the in vitro introduction of hybrid clones of Corymbia torelliana x C. citriodora e Corymbia citriodora x C. torelliana by the micropropagation technique through proliferation by axillary buds. The mini-stumps, suppliers of explants for in vitro introduction, were conducted in semi-hydroclonal mini-clonal hedge. Nodal segments from three Corymbia torelliana x C. citriodora (TC01, TC02 e TC03) clones and one Corymbia citriodora x C. torelliana (CT01) clone were collected, disinfested and inoculated in JADS culture medium, in order to compare the effects of light quality from a dark/fluorescent lamp, a fluorescent lamp, and white and red/blue LEDs. At 30 days after inoculation, the following characteristics were evaluated: average contamination percentage, oxidation, non-reactive explants, shoot length and average number of shoots per explant greater than 0.5 cm. Gathered data showed that the use of red/blue LED light source obtained the best results in all assessed characteristics in the in vitro introduction.