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