Juliana Degenhardt-Goldbach

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

Micropropagation of Calophyllum brasiliense (Cambess.) from nodal segments

Micropropagation of Calophyllum brasiliense Cambess. (Clusiaceae) is a way to overcome difficulties in achieving large-scale plant production, given the recalcitrant nature of the seeds, irregular fructification and absence of natural vegetative propagation of the species. Cultures were established using nodal segments 2 cm in length, obtained from 1-2 year old seedlings, maintained in a greenhouse. Mercury chloride and Plant Preservative Mixture™ were used in the surface sterilizing stage, better results being achieved with Plant Preservative Mixture™ incorporation in culture medium, at any concentration. Polyvinylpyrrolidone, activated charcoal, cysteine, ascorbic acid or citric acid were added to the culture medium to avoid oxidation. After 30 days of culture, polyvinylpirrolidone and ascorbic acid gave better results, eliminating oxidation in most explants. For shoot multiplication, benzylaminopurine was used in concentrations of 4.4 and 8.8 µM in Woody Plant Medium, resulting in an average of 4.43 and 4.68 shoots per explant, respectively, after 90 days. Indole-3-butyric acid and α-naphthalene acetic acid were used to induce root formation, reaching a maximum rooting rate of 24% with 20µM α-naphthalene acetic acid. For acclimatization. the rooted plants were transferred to Plantmax® substrate and cultured in a greenhouse, reaching 79% of survival after 30 days and 60% after one year.

ORGANOGÊNESE INDIRETA A PARTIR DE EXPLANTES FOLIARES E MULTIPLICAÇÃO IN VITRO DE BROTAÇÕES DE Eucalyptus benthamii X Eucalyptus dunnii

Os objetivos deste trabalho foram avaliar diferentes meios de cultura na organogenese indireta e na multiplicacao in vitro de brotos de Eucalyptus benthamii x Eucalyptus dunnii. Para organogenese, 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 concentracoes de thidiazuron (0,1 e 0,5 μM) e presenca ou nao de PVP-40 (250 mg L-1). Apos 70 dias de cultivo foram avaliadas as porcentagens de explantes oxidados totalmente, formando calo, produzindo antocianina, formando gema, formando brotacoes e o numero de brotacoes formadas por explante regenerando. No experimento de multiplicacao, brotacoes 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 oxidacao, de explantes apresentando clorose total ou parcial, massa fresca e numero medio 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 organogenese (8,3%). No meio de cultura MS com 1,11 μM de BAP, a taxa de multiplicacao foi maior que nos outros meios, no primeiro e segundo subcultivos (9,28 e 9,24 por mes), nao havendo diferenca entre os tres meios nos demais subcultivos.

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.

A Model for Trans-Kingdom Pathogenicity in Fonsecaea Agents of Human Chromoblastomycosis

The fungal genus Fonsecaea comprises etiological agents of human chromoblastomycosis, a chronic implantation skin disease. The current hypothesis is that patients acquire the infection through an injury from plant material. The present study aimed to evaluate a model of infection in plant and animal hosts to understand the parameters of trans-kingdom pathogenicity. Clinical strains of causative agents of chromoblastomycosis (Fonsecaea pedrosoi and Fonsecaea monophora) were compared with a strain of Fonsecaea erecta isolated from a living plant. The clinical strains of F. monophora and F. pedrosoi remained concentrated near the epidermis, whereas F. erecta colonized deeper plant tissues, resembling an endophytic behavior. In an invertebrate infection model with larvae of a beetle, Tenebrio molitor, F. erecta exhibited the lowest survival rates. However, F. pedrosoi produced dark, spherical to ovoidal cells that resembled muriform cells, the invasive form of human chromoblastomycosis confirming the role of muriform cells as a pathogenic adaptation in animal tissues. An immunologic assay in BALB/c mice demonstrated the high virulence of saprobic species in animal models was subsequently controlled via host higher immune response.

MICROPROPAGAÇÃO DE Pinus tecunumanii

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.

RESGATE VEGETATIVO DE Campomanesia xanthocarpa Mart. ex O. Berg POR ALPORQUIA

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.

Germinação in vitro de sementes e multiplicação de Calophyllum brasiliense

Calophyllum brasiliense is a tree species with limited natural reproduction. In vitro germination may be an alternative for obtaining high-quality seedlings. Seeds were maintained in water before surface disinfestation and compared with control seeds (i.e. not immersed), without differences between treatments. HgCl2 used during surface-disinfestation reduced contamination rates of cultures. Fungal contamination was reduced with fungicide added to culture medium (23 to 6.4%), although bacterial contamination increased (24 to 36%). In another experiment, seeds were immersed in plant preservative mixture (PPMTM) prior to surface disinfestation. By combining immersion for 48 h and 2 mL L-1 in culture medium, contamination was only 6%. Seeds immersion in GA3 prior to surface disinfestation reduced root formation as concentration increased. Germination rate and GSI were reduced, respectively, from 72% and 0.129 (24 h) to 60% and 0.092 (48 h) according to exposure time to GA3. After 90 days in multiplication medium containing benzylaminopurine, average number of shoots per nodal segment was 3.4. In conclusion, in vitro germination of C. brasiliense seeds is feasible in sucrose-free WPM medium and reaches a high contamination-free rate (up to 93.3%). Germinação in vitro de sementes e multiplicação de Calophyllum brasiliense Resumo Calophyllum brasiliense é uma espécie arbórea com sistema de propagação natural limitada. A germinação in vitro pode ser uma alternativa para obtenção de plântulas de qualidade. Sementes foram mantidas em água antes da desinfestação e comparadas com sementes controle (não imersas), sem diferença entre os tratamentos. HgCl2 usado durante a desinfestação reduziu a contaminação das culturas. A contaminação fúngica foi reduzida com fungicida adicionado ao meio (23 para 6,4%), mas a porcentagem de bactérias foi aumentada (24 para 36%). Em outro experimento, as sementes foram imersas em plant preservative mixture (PPMTM) antes da desinfestação. Combinando a imersão por 48 h e 2 mL L-1 no meio de cultura, a contaminação foi de 6%. A imersão das sementes em GA3 antes da desinfestação reduziu a formação de raízes conforme a concentração foi aumentada. A germinação e o IVG foram reduzidos, respectivamente, de 72% e 0,129 (24 h) para 60% e 0,092 (48 h), de acordo com o tempo de exposição a GA3. Após 90 dias em meio de multiplicação contendo benzilaminopurina, o número médio de brotações por segmento nodal foi 3,4. A germinação in vitro de C. brasiliense é viável em meio WPM sem sacarose, com até 93,3% de sobrevivência. ISSN: 1983-2605 (online)