Abstract Ilex paraguariensis (yerba mate) is a native species from South America and is a rich source of bioactive compounds. There is a lack of research efforts on the phytochemical investigation of callus culture from this species. In the present study, an effort was made to optimize callus culture conditions and to identify secondary compounds. Calli were induced from 10 genotypes using leaf explants and the best genotype was selected to evaluate the effects of cytokinin types and concentrations on callus induction and biomass accumulation. The best genotype and cytokinin treatment were used to conduct one last experiment with sucrose concentrations in culture media and its effects on calli biomass, antioxidant activity and secondary compounds accumulation. Callus initiation was genotype dependent, and the 6-156-6 line had the best response. Zeatin supplemented medium showed higher callus induction rate (82%) and higher biomass accumulation after 120 days (328.2 mg). Higher biomass and secondary compounds accumulation were observed for calli on 3% sucrose medium. Antioxidant activity was not affected by sucrose concentrations. Yerba mate callus culture allowed the accumulation of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, theobromine and caffeine.

ABSTRACT. Plinia peruviana is a species that is native to Brazil and is important due to the taste and medicinal properties of its fruits. Young leaves and split mature seeds were used as explants to initiate somatic embryogenesis to obtain a large number of plants in a short period of time. Leaf discs were cultured in MS medium containing various concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid) or picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid). In the case of the mature seeds, various concentrations of glutamine, 2,4-D and a combination of auxin and BAP (6-benzylaminopurine) were tested for somatic embryogenesis induction. For somatic embryo maturation, several concentrations of PEG 6000 (polyethylene glycol; up to 90 g L-1) were tested. After 60 days of culture using leaf discs, callus formation occurred in all treatments, with the highest averages obtained with 10 μM 2,4-D. However, these calluses did not form somatic embryos. For the cultured seeds, the best treatment was the MS medium with 1,000 mg L-1 glutamine and 10 μM 2,4-D without BAP. The supplementation of 60 g L-1 PEG 6000 was sufficient to promote the maturation of the somatic embryos. Histological analyses of the calluses that were formed from leaf discs showed nonembryogenic characteristics. In contrast, the calluses that originated from mature seeds had small and round cells with little vacuolation, which are characteristics of embryogenic structures.

RESUMO A guabiroba (Campomanesia xanthocarpa) é uma espécie arbórea nativa do Brasil e tem sua distribuição de Minas Gerais até o Rio Grande do Sul, ocorrendo também na Argentina (Misiones), Paraguai e Bolívia. Pode ser usada em paisagismo, pomares domésticos ou para repovoar áreas de proteção ambiental. Seu fruto é bastante apreciado para a produção de sucos, geleias, sorvetes, licores, ou mesmo para consumo in natura. No entanto, existem poucas informações sobre a sua propagação vegetativa. Este trabalho teve por objetivo avaliar o emprego da alporquia como técnica de resgate vegetativo de Campomanesia xanthocarpa. Foram utilizadas 10 plantas-matrizes de Campomanesia xanthocarpa, das quais, ramos jovens, com circunferência entre 3 e 5 cm, foram selecionados para retirada de um anel de casca de aproximadamente 2,0 cm de largura com o auxílio de um estilete a distâncias aproximadas de 30 cm acima da inserção dos ramos no caule. Em cada ferimento foram adicionadas diferentes concentrações de ácido indolbutírico (AIB) (0, 500, 1000 e 2000 mg Kg-1AIB) veiculadas em pasta de vaselina. Em seguida, a região foi envolvida com substrato vermiculita e plástico transparente. Após um ano da confecção dos alporques, foram avaliados: a taxa de sobrevivência, o percentual de calejamento, o percentual de enraizamento e o número de raízes por alporque. A técnica de alporquia em Campomanesia xanthocarpa, utilizando o regulador vegetal AIB não foi eficiente, pois não promoveu o enraizamento dos ramos.

ABSTRACT Guabiroba (Campomanesi axanthocarpa) is an arboreal species native from Brazil, and can be found from Minas Gerais to Rio Grande do Sul state, besides Argentina (Missiones), Paraguay and Bolivia. It can be used for paisagism, orchards, farms, afforestation and environmental recovery. Its fruit is appreciated in the production of juice, jelly, ice cream, liqueur or even in natura. However, there is little information about its propagation. This work aimed to evaluate an air layering method with the intention to study vegetal rescue techniques of Campomanesia xanthocarpa. Ten stock plants of C. xanthocarpa were used for air layering, their young branches, with circunference range 3 - 5 cm, were obtained peridemic rings with approximately 2.0 cm width using a knife, at a distance of 30 cm above the intersection branch. In each ring was added a different concentration of indolebutyricacid (IBA) (0, 500, 1000 e 2000 mgKg-1) diluted in vaseline paste. Then, the region was wrapped with vermiculite substrate and transparent plastic. After one year of layering test, surviving percentage, callus percentage, rooting percentage and the number of roots per branch were evaluated. The air layering technique in Campomanesia xanthocarpa using plant growth regulator IBA was not efficient, because it did not promote the rooting of branches.

RESUMO Pinus tecunumanii é uma das espécies tropicais mais importantes do gênero Pinus no Brasil. Este trabalho teve por objetivo desenvolver um protocolo de micropropagação para Pinus tecunumanii a partir de gemas apicais de plantas jovens mantidas em casa de vegetação. Para a assepsia foi avaliado o efeito de 0,05 ou 0,1% de cloreto de mercúrio em combinação com 0 ou 1 g L-1 de fungicida e NaOCl. O efeito de 2 ou 4 µM BA (6-benzil adenina) foi avaliado na multiplicação in vitro ao longo de três subcultivos. O efeito da adição de 1,5 g L-1 de carvão ativado foi avaliado no meio de alongamento. Para o enraizamento, o efeito dos sais WV5 meia força e de 20 g L-1 de sacarose foi avaliado em meio suplementado com 2.68 µM ácido naftaleno acético (ANA) e 0.44 BA. A assepsia dos explantes com 0,05% de cloreto de mercúrio, hipoclorito de sódio, com ou sem fungicida foi eficiente no estabelecimento in vitro de até 83,3% dos explantes. Em meio suplementado com 2 ou 4 µM BA ocorreu multiplicação de brotações adventícias e após o terceiro subcultivo, 70,83% dos explantes formaram novos brotos em meio suplementado com 2 µM BA, com uma média de 3,7 novos brotos por explante. Em meio suplementado com carvão ativado foi observado alongamento em 59,76% dos explantes, enquanto no meio sem carvão 33,33% dos explantes alongaram. Foi observado enraizamento em 14,8% dos explantes em meio suplementado com sais WV5.

ABSTRACT Pinus tecunumanii is one of the most important tropical species of Pinus in Brazil. This work aimed to develop a micropropagation protocol for Pinus tecunumanii from the apical shoots of young plants grown in a greenhouse. To optimise the aseptic treatment protocol, the effect of 0.05 or 0.1% mercuric chloride in combination with 0 or 1 g L-1 fungicide and NaOCl was evaluated. Next, the effect of 2 or 4 µM BA (6-benzyl adenine) on in vitro multiplication was evaluated over three subcultures. Supplementation with 1.5 g L-1 activated charcoal was tested for elongation. For rooting, the effects of WV5 salts (at half strength) and 20 g L-1 sucrose were evaluated on media supplemented with 2.68 µM naphthalene-acetic acid and 0.44 µM BA. Aseptic treatment of explants with 0.05% mercuric chloride and sodium hypochlorite (with or without fungicide) was effective to establish 83.3% of explants in vitro. On media supplemented with 2 or 4 µM BA, shoot multiplication was promoted. After the third subculture on medium supplemented with 2 µM BA, 70.83% of the explants exhibited new shoots with an average of 3.7 new shoots per explant. On medium containing activated charcoal, elongation was observed in 59.76% of the explants, while on medium without activated charcoal only 33.33% elongated. Rooting was observed in 14.8% of explants on medium supplemented with WV5 salts.

RESUMO Esse trabalho teve como objetivo avaliar o efeito de vários fatores que podem afetar a transformação genética de explantes cotiledonares de Eucalyptus saligna mediante co-cultura com a cepa EHA105 de Agrobacterium tumefaciens. Foi utilizado o vector pBI121 carregando o gene repórter gus sob controle do promotor 35SCaMV. Foi estudado o efeito dos fatores a seguir: pré-cultura dos explantes, uso de diferentes antibióticos e presença de acetosiringona (AS) nos meios de co-cultura. Uma solução contendo ácido ascórbico (250mg.L-1), ácido cítrico (25mg.L-1) e PVP-40 (1g.L-1) foi utilizada no momento da excisão dos explantes. A pré-cultura dos explantes antes da co-cultura com a bactéria foi de 4 dias, em meio de cultura MS suplementado de 4,4µM BAP e 2,7ìM ANA. Após a co-cultura três concentrações de canamicina (12,5; 25 e 50mg.L-1) combinadas com 300mg.L-1 de Augmentin® no meio de cultura foram testadas. O efeito do Augmentin® (300mg.L-1) nos explantes foi comparado ao da cefotaxima (500mg.L-1), em meio contendo 50mg.L-1de Km. Foi concluído que Augmentin® estimula a organogênese, em comparação com a cefotaxima, que a concentração de 12,5mg.L-1 de Km permite a seleção de explantes transformados com o gene gus e, finalmente, a adição de AS (50ìM) aos meios de co-cultura líquido e sólido tem efeito positivo sobre a expressão do gene gus. O uso de antioxidante não é necessário e a pré-cultura dos explantes antes da inoculação com A. tumefaciens não tem efeito na regeneração de gemas nem na expressão do gene gus. A eficiência de transformação foi de 1,5%.

ABSTRACT This study aimed to evaluate the effect of factors that may affect the genetic transformation of cotiledonary explants of Eucalyptus saligna mediated by EHA105 strain of Agrobacterium tumefaciens. The vector pBI121 carrying gus gene under control of 35S CaMV promoter was used. The effect of the following factors was evaluated: explant pre-culture, use of different antibiotics and presence of acetosyringone (AS) in co-culture media. An antioxidant solution was also used during excision, containing ascorbic acid (250mg.L-1), citric acid (25mg.L-1) and PVP-40 (1g.L-1). Pre-culture of the explants before the co-culture with bacteria was done over a 4-day period in MS culture medium supplemented with 4.4µM BAP and 2.7ìM NAA. After theco-culture period, three concentrations of kanamycin (12.5;25 and 50mg.L-1) combined with 300mg.L-1 Augmentin® in the culture medium were tested The influence of the antibiotic was also evaluated by keeping the explants in a medium containing 50mg.L-1 Km and 300mg.L-1 Augmentin® or 500mg.L-1 cefotaxime. It was concluded that Augmentin® stimulates organogenesis, that a Km concentration of 12.5mg.L-1 allows selection of explants transformed with gus gene and, finally, the addition of AS (50ìM) to the liquid and solid co-culture media has a positive effect on gus gene expression. Moreover, the use of an antioxidant solution during cotyledon excision is dispensable and the pre-culture of the explants has no effect on bud regeneration or gus gene expression. A transformation efficiency of 1.5% was reached.

RESUMO Os objetivos deste trabalho foram avaliar diferentes meios de cultura na organogênese indireta e na multiplicação in vitro de brotos de Eucalyptus benthamii x Eucalyptus dunnii. Para organogênese, explantes foliares foram excisados no sentido transversal e cultivados in vitro, sendo os seguintes fatores testados: dois meios de cultura (MS N/2 e JADS) adicionados de 0,1 µM de ANA, duas concentrações de thidiazuron (0,1 e 0,5 μM) e presença ou não de PVP-40 (250 mg L-1). Após 70 dias de cultivo foram avaliadas as porcentagens de explantes oxidados totalmente, formando calo, produzindo antocianina, formando gema, formando brotações e o número de brotações formadas por explante regenerando. No experimento de multiplicação, brotações isoladas foram cultivadas em meio MS, JADS e WPM, adicionados de 1,11 μM de BAP. Foram realizados quatro subcultivos a cada 28 dias e em cada subcultivo foram avaliados: a porcentagem de oxidação, de explantes apresentando clorose total ou parcial, massa fresca e número médio de brotos por explante. O meio de cultura MS N/2 suplementado com 0,1 µM de ANA, 0,5 µM de TDZ e PVP-40 promoveu a maior taxa de organogênese (8,3%). No meio de cultura MS com 1,11 μM de BAP, a taxa de multiplicação foi maior que nos outros meios, no primeiro e segundo subcultivos (9,28 e 9,24 por mês), não havendo diferença entre os três meios nos demais subcultivos.

ABSTRACT The aims of this research were to evaluate different culture media for indirect organogenesis and shoot multiplication of Eucalyptus benthamii x Eucalyptus dunnii. For organogenesis, leaf explants were used to test the following treatments: two culture media (MS N/2 and JADS) supplemented with 0.1 µM 1-naphthaleneacetic acid (NAA) and thidiazuron (TDZ) (0.1 or 0.5 μM), with or without PVP- 40 (250 mg L-1). The percentage of oxidized explants, callus forming explants, explants with anthocyanin, buds, shoots and the shoot number per explant were evaluated. In the multiplication experiment, isolated shoots were cultivated in MS, JADS and WPM media, all supplemented with 1.11 μM BAP. Four subcultures were carried out every 28 days. In every subculture the explant oxidation, partial or total leaf chlorosis, fresh mass and mean number of shoot per explant were evaluated. The MS N/2 medium supplemented with 0.1 µM NAA and 0.5 µM TDZ promoted the highest rate of organogenesis (8.3%) and the culture media MS supplemented with 1.11 μM BAP the multiplication rate was higher than in the other media, in the first and the second subcultures (9.28 and 9.24, respectively), without differences between the three media in the following subcultures.