Silva, A. L. L et al. 2

J. Biotec. Biodivers. v. 1, N.1: pp. 1-5, Nov. 2010

Sm. were disinfected with ethanol 70 % during 3 min., and after in NaOCl 6% (with 5 drops Tween 20 per 100 mL) for 30 min., and after washed three times with distilled and autoclavated water. Seeds were sowed on MS half strength medium (Murashige and Skoog, 1962) with 20 g.L sucrose and solidified with 7 g.L agar.

In order to test effects of the kanamycin in shoot tip, shoot clusters of E. saligna were obtained through indirect organogenesis (Dibax, 2007), these clusters were donor of shoot tip; these clusters were multiplicated on M medium, which consisted of MS medium, 30 g.L sucrose, 1.11 μM BAP (6-benzylaminopurine) and solidified with 7 g.L agar.

Tolerance to kanamycin in cotyledon explants Cotyledons from the seedlings (15 days old) were excised and cultivated on MS medium supplemented with 2.7 μM NAA (naphthaleneacetic acid) and 4.4 μM BAP, 30 g.L sucrose and solidified with 7 g.L agar. The treatments were: 0, 12.5, 25, 37.5 and 50 mg.L kanamycin. Explants were maintained in dark until 15 days, after they were placed at 5 μM m s . Explants were subcultured each 28 days. Callus formation percentage and survival percentage were evaluated at 28, 56 and 84 days of in vitro culture.

Tolerance to kanamycin in shoot tip

Shoot tip (5 mm heigth and without lateral shoots) were removed of shoot clusters of E. saligna and cultivated on M medium supplemented with different kanamycin levels (50, 75, 100, 125 and 150 mg.L ). Explants were subcultured each 28 days for the same media. The shoot number and survival percentage were evaluated at 28, 56, 84, 112, 140 and 168 days of in vitro culture. The explants were kept in a growth room with a light intensity of 0.18 μ M m s with a 16 h photoperiod.

Culture conditions and statistical analysis Kanamycin was microfiltered and supplemented to

the media after autoclaving. All media had their pH adjusted to 5.8 and were autoclaved at 1 atm and 121 ºC for 20 min. The cultures were kept at 25 ± 2 ºC. The experimental design was a complete randomized with five replicates of five explants. The data was submitted in a normality analysis for the Lilliefors´s test and, analysis of variance followed by regression analysis at the

level of 5% of error. All analysis were done

following the procedures of the software GENES (Cruz, 2001). Variables from counting were transformed to x 0 . 5 and variables from percentage were transformed to arcsin x / 100 .

RESULTS AND DISCUSSION

Tolerance to kanamycin in cotyledon explants During all experimental period the callus formation occurred only in the absence of kanamycin, and all explants alive, formed callus (ca. 80%). Similar results were found in E. saligna which 85% of explants have formed callus (Dibax, 2007). After 30 days of in vitro culture, survival of cotyledons varying from 70 to 80%. Similar results were observed in cotyledons of Eucalyptus grandis , which levels of kanamycin, 50 and 100 mg.L induced ca. 90% of survival after 30 days of culture (González, 2002).

In Eucalyptus globulus , level of 100 mg.L kanamycin allows an efficient selection for transformed callus (Serrano et al., 1996), nevertheless, in Eucalyptus camaldulensis , level of 20 mg.L kanamycin inhibits significativaly callus formation (Ho et al., 1998).

Callus formed shoots at third month, ca. 15%. However, just 65% of callus was alive at the end of 84 days; they have a browned appearance, probably for oxidation occurrence. In spite of the low light intensity (5 μM m s ) oxidation occurred, however it is probable that, in case of the light intensity more elevate than it, these explants possibly were dead before third month. Surviving explants were drastic affected at the end of third month due to oxidation.

The levels from 12.5 to 50 mg.L were sufficiently to kill all explants in a period of three months (Fig. 1).

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0.0 12.5 25.0

37.5 50.0

Kanamycin (mg.L )

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