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 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 ?M de ANA, duas concentrações de thidiazuron (0,1 e 0,5 ?M) 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 ?M 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 ?M de ANA, 0,5 ?M de TDZ e PVP-40 promoveu a maior taxa de organogênese (8,3%). No meio de cultura MS com 1,11 ?M 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

Organogenesis and Agrobacterium tumefaciens-mediated transformation of Eucalyptus saligna with P5CS gene

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ1-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants

Efficient Regeneration of Eucalyptus urophylla x Eucalyptus grandis from Stem Segment

The aim of the present study was to establish an efficient regeneration system for the hybrid E. urophylla x E. grandis by means of organogenesis. Stem segments from seedlings were used as explants and cultured in a modified Murashige and Skoog medium (MS), supplemented with 13.2 µM N-phenyl-N'-[6-(2-chlorobenzothiazol)-yl] urea (PBU) and 0.285 µM indole-3-acetic acid (IAA). PBU was a useful growth regulator. After cultivating for 5 d, 96% explants formed callus. After 30 d, the calli obtained were transferred to MS medium containing different combinations of 6-benzyladenine (BA) and naphthalene acetic acid (NAA). Compared with other growth regulator combinations, PBU stimulated more vigorous calli and restrained browning. In addition, a large percentage (91.3%) of the calli induced by PBU showed adventitious buds formation. Shoot elongation was then stimulated on half-strength MS mineral salts medium supplemented with 6.6 µM PBU and 0.285 µM IAA for 20 d. For rooting, the elongated shoots were cultivated on root induction medium containing 2.46 µM indole-3-butyric acid (IBA). Plantlets were then successfully transplanted to a greenhouse. This procedure represented an efficient way of E. urophylla x E. grandis plant regeneration

Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis dehn and histological study of organogenesis in vitro

The present work aimed at regenerating plants of Eucalyptus camaldulensis from the cotyledonary explants and describing the anatomy of the tissues during callogenesis and organogenesis processes, in order to determine the origin of the buds. The cotyledonary leaves of E. camaldulensis were cultured in Murashige and Skoog (MS), WPM and JADS media supplemented with 2.7 µM NAA and 4.44 µM BAP. The best results for bud regeneration were obtained on MS and WPM media (57.5 and 55% of calluses formed buds, respectively). Shoot elongation and rooting (80%) were obtained on MS/2 medium (with half-strength salt concentration) with 0.2% activated charcoal. Acclimatization was performed in the growth chamber for 48 h and then the plants were transferred to a soil:vermiculite mixture and cultured in a greenhouse. Histological studies revealed that the callogenesis initiated in palisade parenchyma cells and that the adventitious buds were formed from the calluses, indicating indirect organogenesis.<br>Este trabalho teve como objetivo a obtenção de plantas de Eucalyptus camaldulensis a partir de folhas cotiledonares e o estudo da anatomia dos tecidos durante a calogênese e organogênese para determinar a origem das gemas. Folhas cotiledonares foram cultivadas em meios de cultura MS, WPM e JADS suplementados com 2,7 µM de ANA e 4,44 µM de BAP. Os melhores resultados para a regeneração de gemas foram obtidos com os meios MS e WPM. Para o alongamento e enraizamento, o meio de cultura MS/2 contendo 0,2% de carvão ativado apresentou-se eficiente para ambas as etapas. A aclimatização foi realizada mediante a abertura dos frascos na sala de crescimento por 48 horas, seguido da transferência para casa-de-vegetação com nebulização intermitente. Estudos histológicos foram conduzidos e revelaram que a calogênese teve início nas células do parênquima paliçádico e que as gemas adventícias formaram-se a partir dos calos, indicando a organogênese indireta