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.

SUMMARY: Few studies have focused on the in vitro multiplication methods for Eucalyptus cloeziana. In this work we developed an indirect organogenesis protocol to obtain micropropagated plants. The interactions of plant growth regulators in juvenile tissues were evaluated. Hypocotyl, cotyledon and root tissues obtained from in vitro germinated seedlings were subjected to combinations of α-naphthaleneacetic acid (NAA), thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D). In vitro callogenesis, adventitious bud induction, shoot elongation, and ex vitro survival and rooting were evaluated. Callogenic structures were observed in all evaluated tissues. Morphogenic characteristics related to the meristematic center was observed. The tissues that presented the callus mass were subcultured in a regeneration culture medium supplemented with 1.0 mg L-1 of 6-benzylaminopurine (BAP). Only hypocotyl and cotyledon induced adventitious bud and shoot elongation. Hypocotyl subjected to 2.0 mg L-1 TDZ had the highest number of elongated shoots. The two tissues (i.e., hypocotyl and cotyledon) combined with thidiazuron were characterized by the ex vitro survival of microcuttings and by adventitious rooting. Results confirmed tissue culture efficiency for microplants production by indirect organogenesis from hypocotyl and cotyledon cultivated with TDZ, and its implementation can be an alternative for forest tree breeding programs of E. cloeziana.

RESUMEN: Pocos estudios se han centrado en los métodos de multiplicación in vitro para Eucalyptus cloeziana. En este trabajo se desarrolló un protocolo de organogénesis indirecta para obtener plantas micropropagadas. Se evaluaron las interacciones de los reguladores del crecimiento en tejidos juveniles. Los tejidos del hipocótilo, cotiledón y raíces obtenidos de plántulas germinadas in vitro se sometieron a combinaciones de ácido α-naftaleneacético (ANA), tidiazurón (TDZ) y ácido 2,4-diclorofenoxiacético (2,4-D). Se evaluaron la callogénesis in vitro, la inducción adventicia de yemas, el alargamiento de brotes, y la sobrevivencia y enraizamiento ex vitro. Se observaron estructuras calogénicas en todos los tejidos evaluados. Se observaron características morfogénicas relacionadas con la formación de centros meristemáticos. Los tejidos que presentaron la masa del callo fueron subcultivados en medio de cultivo de regeneración suplementado con 1,0 mg L-1 de 6-bencilaminopurina (BAP), y solo los tejidos del hipocótilo y cotiledón formaron yemas adventicias y brotes alargados. Los tejidos del hipocótilo sometidos a 2,0 mg L-1 de tidiazuron obtuvieron mayor número de brotes alargados. Los dos tejidos (hipocótilo y cotiledón) combinados con TDZ se caracterizaron por sobrevivencia y el enraizamiento adventicio ex vitro de las microestacas. Los resultados confirmaron la eficiencia del cultivo de tejidos para la producción de microplantas por organogénesis indirecta a partir de tejidos del hipocótilo y cotiledón cultivados con TDZ y su implementación puede ser una alternativa para los programas de mejoramiento forestal de E. cloeziana.

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.