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.

ABSTRACT We addressed a major challenge in the in vitro clonal propagation of Corymbia citriodora, Eucalyptus urophylla and E. benthamii by using an ex vitro adventitious rooting strategy in a mini-incubator. Mini-incubators were placed in four environments for rooting. A shade house with no fogging system and a greenhouse with no ventilation but with a fogging environment had the best performance in terms of rooting, root growth and survival of microcuttings. Daily recording of the temperature within each mini-incubator in each environment allowed the verification of negative correlations between the maximum average temperature and the survival, adventitious rooting and root growth. The ideal maximum air temperature for the efficient production of clonal plants was 28.4°C (± 5.5°C), and the minimum was 20.3°C (± 6.2°C). E. benthamii was more sensitive to higher temperatures than C. citriodora and E. urophylla. Nevertheless, placing mini-incubators in the shade house with no fogging system resulted in a stable and uniform performance among the three species, with 100.0% survival and 81.4% rooting. Histological sections of the adventitious roots revealed connection with the stem vascular cambium. Therefore, our experimental system demonstrated the potential of mini-incubators coupled with the proper environment to optimize the adventitious rooting performance of microcuttings.

A peroxidação lipídica e alongamento de raiz de Eucalyptus grandis × Eucalyptus camaldulensis foram estudados em resposta às condições de estresse com alumínio (Al), um metal conhecido por limitar a produtividade agrícola em solos ácidos, principalmente devido à redução do crescimento radicular. No Brasil, o híbrido Grancam 1277 (E. grandis × E. camaldulensis) tem sido plantado na região do "Cerrado", local com grande ocorrência de solos ácidos. O presente estudo mostra que o híbrido teve redução de crescimento das raízes e aumento dos níveis de peroxidação lipídica, após 24h de tratamento com 100 µM de Al. No entanto, um possível processo de tolerância começou após este período, uma vez que foram observadas redução na peroxidação lipídica e recuperação de alongamento da raiz após 48h de exposição Al. A compreensão da dinâmica temporal de tolerância ao Al pode ser útil principalmente para selecionar genótipos mais tolerantes e também para identificar genes de interesse para aplicação em bioengenharia.

Lipid peroxidation and root elongation of Eucalyptus grandis × Eucalyptus camaldulensis were studied under stress conditions in response to aluminum (Al), a metal known to limit agricultural productivity in acidic soils primarily due to reduced root elongation. In Brazil, the Grancam 1277 hybrid (E. grandis × E. camaldulensis) has been planted in the "Cerrado", a region of the country with a wide occurrence of acidic soils. The present study demonstrated that the hybrid exhibited root growth reduction and increased levels of lipid peroxidation after 24h of treatment with 100 µM of Al, which was followed by a reduction in lipid peroxidation levels and the recovery of root elongation after 48h of Al exposure, suggesting a rapid response to the early stressful conditions induced by Al. The understanding of the temporal dynamics of Al tolerance may be useful for selecting more tolerant genotypes and for identifying genes of interest for applications in bioengineering.

Objetivou-se micropropagar E. benthamii x E. dunnii, testando concentrações de cloro para a assepsia de explantes, a concentração ótima de benzilaminopurina (BAP) e ácido naftalenoacético (ANA) para a proliferação de gemas e a relação entre BAP e ácido giberélico (GA3) em dois meios de cultura para o alongamento de brotações. Segmentos nodais dos genótipos H12, H19 e H20 foram desinfetados com 0,5; 1,0; 1,5 e 2,0% (v v-1) de cloro. Os explantes foram multiplicados em meio ½MS suplementado com BAP (0; 0,25; 0,50; 0,75 e 1,00 mg L-1) e ANA (0; 0,025; 0,050; 0,075 e 0,100 mg L-1) para produção de gemas, e alongados nos meios MS e ½MS suplementados com BAP (0; 0,05 e 0,10 mg L-1) e GA3 (0; 0,1; 0,2 e 0,3 mg L-1). A combinação de 0,50 mg L-1 de BAP e 0,050 mg L-1 de ANA proporcionou melhor proliferação de gemas para os genótipos H12 e H20. As concentrações de 0,10 e 0,20 mg L-1 de GA3 combinadas com 0,10 mg L-1 de BAP em ½MS, promoveram os melhores resultados no alongamento, para os clones H12 e H20, respectivamente. O enraizamento foi baixo, com média de 12,0% para condições in vitro e 14,4% ex vitro.

This study was designed to micropropagate E. benthamii x E. dunnii, by testing chlorine concentrations for explant asepsis, the optimal concentrations of benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) for bud proliferation, and the ratio between BAP and gibberellic acid (GA3) in two nutrient media for shoot elongation. Nodal segments from H12, H19 and H20 clones were disinfected with 0.5, 1.0, 1.5 and 2.0% (v v-1) of chlorine. Explants were grown on ½MS medium supplemented with BAP (0, 0.25, 0.50, 0.75 and 1.00 mg L-1) and NAA (0, 0.025, 0.050, 0.075 and 0.100 mg L-1) for bud production. They were elongated on MS and ½MS media supplemented with BAP (0, 0.05 and 0.10 mg L-1) and GA3 (0, 0.1; 0.2 and 0.3 mg L-1). The 0.50 mg L-1 BAP and 0.050 mg L-1 NAA combination was optimal for bud proliferation for H12 and H20. GA3 concentrations of 0.10 and 0.20 mg L-1 combined with 0.10 mg L-1 BAP on ½MS resulted in the longest shoots, for H12 and H20, respectively. Regardless of clone, the rooting rate was low, with an average of 12.0% and 14.4% of plants having roots for in vitro and ex vitro conditions, respectively.

O Eucalyptus apresenta grande importância no setor florestal e muitos avanços na área do melhoramento foram conquistados com o advento da biotecnologia. Contudo, alguns genótipos promissores ainda não possuem protocolos de multiplicação com técnicas de clonagem, como a miniestaquia. Objetivou-se avaliar concentrações de IBA na sobrevivência, enraizamento e vigor vegetativo de miniestacas de E. benthamii x E. dunnii, com a determinação da dose de máxima eficiência técnica. Minicepas dos clones H12, H19 e H20 foram manejadas em minijardim clonal em sistema semi-hidropônico. Para o enraizamento, mergulhou-se a porção basal da miniestaca em soluções hidroalcoólicas, cujas concentrações foram: 0, 2.000, 4.000, 6.000 e 8.000 mg L-1 de IBA. O experimento foi conduzido no delineamento inteiramente casualizado, sendo constituído por três clones e cinco concentrações de IBA, com cinco repetições, contendo dez miniestacas por repetição. O IBA influenciou positivamente os processos rizogênicos das miniestacas com resposta diferenciada entre os clones, a qual variou de 30,32 a 55,45% de enraizamento. Para os clones H12 e H19, os incrementos positivos ocorreram até a maior concentração de IBA. Contudo, a faixa situada entre os tratamentos de 4.000 e 6.000 mg L-1 de IBA promoveu os melhores resultados de enraizamento para o clone H20.

Eucalyptus has great importance in the forestry sector and many advances in the area of improvement have been achieved with the advent of biotechnological techniques. However, some promising genotypes still do not have multiplication protocols with cloning techniques, such as minicutting. The study aimed to evaluate IBA concentrations for survival, rooting and vegetative vigor of E. benthamii x E. dunnii minicuttings, and determine the maximum technical efficiency dose. Ministumps H12, H19 and H20 clones were cultivated in a clonal minigarden under a semi-hydroponic system. For rooting, the minicutting basal portion was plunged in hydro-alcoholic solutions, whose concentrations were: 0; 2,000; 4,000; 6,000 and 8,000 mg L-1 of IBA. The experiment was conducted in a completely randomized design, with the factors consisting of three clones and five IBA concentrations, with five replications, containing 10 minicuttings per replication. IBA positively influenced the minicutting rooting processes, with differentiated behavior between the clones, with 30.32 to 55.45% rooting variation. The positives increments occurred until the highest IBA concentration for H12 and H19 clones. However, the range between 4,000 and 6,000 mg L-1 of IBA treatments promoted the best rooting results for the H20 clone.