Yohana de Oliveira

Selective agent and A. tumefaciens overgrowth-control antibiotics in Eucalyptus camaldulensis Cotiledonary culture

The objectives of the present work were to establish the minimal lethal dose of the selective agent to determine the type and concentration of appropriate antibiotics for the elimination of Agrobacterium tumefaciens inoculated explants, without interfering with the regenerative potential of the E. camaldulensis cotyledonary explants. Non-transformed explants were cultivated in medium supplemented with kanamycin. The results showed that the antibiotic was suitable for the selection of transformed cells in the concentration of 9 mg L-1 as it inhibited the growth of non-transformed cells. Cotyledons infected with A. tumefaciens were cultivated in MS N/2 medium supplemented with BAP, ANA, Km and cefotaxime or AugmentinO . The highest average of regenerated shoots by explant (5,4) was observed in the presence of 300 mg L-1 of AugmentinO /15 days, followed by 150 mg L-1/15 days and 100 mg L-1/30 days.

Efeito dos ácidos naftaleno acético e indolilbutírico no enraizamento de estacas de jambolão [Syzygium cumini (L.) Skeels]

Jambul usually propagates by seeds, which causes variability in the descendant plants and represents a problem in the formation of commercial orchards. The development of a protocol for vegetative propagation by cuttings would enable the reproduction of all features of the Mother plant, uniformity in populations and easy propagation. The aim of this work was to evaluate the effect of naphthaleneacetic acid (NAA) and indolebutyric acid (IBA) on rooting of jambul cuttings. Twelve-cm-long cuttings from the median region of branches were prepared through bevel cut in the base and right cut above the last axillary bud, keeping one pair of halved leaves. Cutting bases were immersed for 10s in aqueous solutions containing NAA or IBA at 0, 500, 1000 and 1500 mg L-1 concentrations. Plastic trays containing medium sand were used in the planting. The cuttings were kept in a greenhouse under intermittent nebulization and, at 120 days after planting, the following variables were evaluated: percentage of rooted, with calluses, alive (not-rooted and without calluses) and dead cuttings; length of the three largest roots (cm); and number of roots per cutting. The best rooting was observed by using 1000 mg L-1 of both tested plant growth regulators. Rooting percentage was slightly higher under NAA relative to IBA.

Pré-aclimatização in Vitro de abacaxi-ornamental

O abacaxi ornamental (Ananas bracteatus Schult. f.) e bastante utilizado em composicoes paisagisticas para delimitar areas ou canteiros. A micropropagacao permite obter altas taxas de multiplicacao, podendo ser uma alternativa viavel e vantajosa de propagacao vegetativa, visto que a demanda por esta especie vem aumentando, tanto no mercado nacional quanto no internacional. Neste trabalho objetivou-se avaliar a influencia de diferentes irradiâncias e concentracoes de sacarose durante o enraizamento in vitro, sobre a sobrevivencia das mudas na fase de aclimatizacao. Para tanto, foram testadas diferentes concentracoes de sacarose (0, 10, 20 e 30 g L-1) no meio de enraizamento, bem como duas irradiâncias (22,9 e 66,9 mmol m-2 s-1) em sala de crescimento. O melhor resultado em termos de enraizamento foi obtido no meio de cultura contendo 30 g L-1 de sacarose sob irradiância de 66,9 mmol m-2 s-1. Apos a fase de enraizamento in vitro, as plantas foram transferidas para casa de vegetacao e mantidas sob nebulizacao intermitente por 60 dias. Os resultados de sobrevivencia aos 30 e 60 dias nao diferiram estatisticamente para os dois niveis de irradiância testados. Entretanto, para concentracoes de sacarose, a testemunha apresentou sobrevivencia inferior (50%) diferindo estatisticamente dos demais tratamentos que foram semelhantes entre si (95 a 100%).

Indirect organogenesis from leaf explants of Eucalyptus benthamii x Eucalyptus dunnii and shoot multiplication

Background In Brazil, especially in the Southern region, stresses caused by cold and eventual frost are those that exert the most negative effect on the productivity of Eucalyptus spp. The genetic transformation techniques may contribute to forestry improvement programs in order to obtain genotypes expressing new interesting characteristics. They allow shortening the long breeding cycles and avoiding manipulation of adult trees. Their efficiency depends on establishment of regeneration procedures that allow the development of shoots from the transformed tissues. E. benthamiix E. dunnii hybrids have shown superiority to their parents concerning growth and frost tolerance [1], but no information about their in vitro organogenesis has been reported in the literature. The objective of this study was to evaluate the effect of some factors of culture medium on indirect organogenesis and shoot multiplication of anE. benthammi x E. dunnii clone. Methods In vitroestablished shoots,provided by EMBRAPA-Florestas (Colombo, PR, Brazil), were used as explant source. Cultures were maintained under white fluorescent tubes providing a photon flux density (PFD) of approximately 20 μmol m -2 s -1 , a 16-h photoperiod and a temperature of 25±2 °C. The cultures were performed in glass flasks containing 25 mL MS [2] medium supplemented with 1.11µM BA and sealed with rigid polypropylene caps. For the indirect organogenesis, leaves were excised from shoots at the petiole base, split into two halves and inoculated in culture media. The cultures were done in Petri dishes kept in a growth chamber in the dark throughout the experiment. The statistical design was performed in a factorial scheme (2:2:2) and a comparison was done between two culture media (MS-N/2, with half concentration of potassium and ammonium nitrates, and JADS [3] with 0.1 µM NAA, with and without PVP-40 (250 mg L -1 ) and two TDZ concentrations (0.1 and 0.5 µM). After 70 days, the percentages of explants forming callus, oxidized explants, explants producing anthocyanin, explants forming buds and shoots, and the number of shoots per explant were evaluated. For the multiplication test, the statistical design was performed in a factorial scheme (3:2) with three culture media (MS, WPM [4] and JADS, with 1.11µM BA) and two subcultures (28 and 56 days after the initial culture period). The analyzed variables for each subculture were: percentage of oxidation, of explants showing chlorosis, fresh weight and number of shoots. Results and discussion Regarding the oxidation, the higher rates (100%) were observed on JADS medium in presence or absence of PVP-40 and on MS-N/2 medium (68.3%) in presence of PVP-40. However, the JADS medium showed the highest percentage of callus formation (83.3%). In MS-N/2 medium the highest percentage of callus formation (55%) was found in the presence of PVP-40 and 0.5µM

Substratos, concentrações de ácido indolbutírico e tipos de miniestacas no enraizamento de melaleuca (Melaleuca alternifolia Cheel)

Melaleuca alternifolia tem como produto principal o oleo essencial extraido das folhas devido as propriedades antifungicas e antibacterianas. Pouco se tem relatado sobre a propagacao desta especie, sendo a miniestaquia uma alternativa para a propagacao vegetativa de clones superiores visando a implantacao de campo de producao. Este trabalho teve como objetivo avaliar o efeito de diferentes substratos, concentracoes de AIB, e tipo de miniestaca, no enraizamento de Melaleuca alternifolia. No primeiro experimento foram testados os substratos, areia de granulometria media, Plantmax HT®, Golden-Mix® e vermiculita. No segundo experimento foram avaliadas diferentes concentracoes de AIB (0, 500, 1000 e 2000 mg L-1), em dois tipos de miniestacas (apical e mediana). As miniestacas foram confeccionadas com 5 cm de comprimento, mantidas em casa de vegetacao com nebulizacao intermitente, e, apos 45 dias do plantio, foram avaliadas as porcentagens de miniestacas enraizadas, com calos e nao responsivas, o numero de raizes formadas por miniestaca e o comprimento das tres maiores raizes (cm). O substrato Golden-Mix® e as miniestacas coletadas da porcao apical do ramo submetidas ao tratamento com 500 mg L-1 de AIB apresentaram maior porcentagem de enraizamento e melhor qualidade do sistema radicial.

Use of kanamycin for selection of Eucalyptus saligna genetically transformed plants

Background Several factors may affect the genetic transformation efficiency of woody species. One factor is the use of an efficient selective agent that inhibits the development of non-transformed cells and just allows the development of transformed tissues. The most used selection agent is the neomycin phosphotransferase II (NPTII) gene, which confers resistance to aminoglycoside antibiotics kanamycin, neomycin and G-418 [1]. The selective agent concentration in culture medium may have influence on shoot regeneration and high concentrations may promote adverse effects on organogenic potential [2]. The kanamycin effects in Eucalyptus are variable and depend on the species and genotypes [3]. The purpose of this study was to evaluate the effect of kanamycin concentration on transformation efficiency for Eucalyptus saligna cotyledons after co-culture with Agrobacterium tumefaciens.

In vitro shoot organogenesis from Eucalyptus sp. leaf explants

Background In vitro organogenesis is one of the key techniques associated with genetic transformation, as it determines the successful regeneration of transgenic plants after co-cultivation with bacteria. Therefore, the development of efficient regeneration protocols is the most critical step in developing genetic transformation. Protocols have been developed for several species of eucalyptus. In most of the studies cotyledon, hypocotyl and leaf segments of plants cultivated in vitro are used as explants [1]. Several eucalyptus functional genomics projects have used Populus species as a model for it counts with well established regeneration and transformation protocols. However, the use of Eucalyptus clones as model plants could be more adequate, if clones with high regeneration rates as those obtained for Populus could be found. The aim of this study was to evaluate several variables in the in vitro organogenesis from leaves of an Eucalyptus sp. clone maintained in vitro at Embrapa Forestry.