Silva, A. L. L et al. 1
J. Biotec. Biodivers. v. 1, N.1: pp. 1-5, Nov. 2010
Vol. 1, N. 1: pp. 1-5, November 2010
Special Number
Journal of Biotechnology
and Biodiversity
Shoot tip and cotyledon explants of Eucalyptus saligna Sm. cultivated on different kanamycin levels
André Luís Lopes da Silva , Yohana de Oliveira , Jefferson da Luz Costa , Elisângela Masetto , Clarissa de Souza Mudry , Eduardo Andrea Lemus Erasmo and Gessiel Newton Scheidt
Departamento de Engenharia de Bioprocessos e Biotecnologia; Universidade Federal do Paraná; 81531-970; Curitiba – PR – Brasil. Departamento de Fitotecnia e Fitosanidade; Universidade Federal do Paraná; 80035-050; Curitiba – PR – Brasil. Departamento de Ciências Agrárias e Tecnológicas; Universidade Federal do Tocantins; 77402-970; Gurupi – TO – Brasil.
ABSTRACT
An efficient selective agent helps to obtain transgenic plants; the most used selective agent for genetic transformation of Eucalypt is kanamycin. The aim of this research was to determine ideal dose of kanamycin in shoot tip and cotyledon explants of E. saligna for use as selective agent in genetic transformation. Two experiments were carried out, first in order to test effects of the kanamycin in cotyledons and second, in order to test effects of the kanamycin in shoot tip. In the cotyledons experiment were tested 0, 12.5, 25, 37.5 and 50 mg.L kanamycin on a callus formation medium and for shoot tip were tested 50, 75, 100, 125 and 150 mg.L kanamycin on a multiplication medium. Cotyledons can be selected more quickly than shoot tip, moreover needs lower kanamycin levels than shoot tip, what results more speed and economy during genetic transformation process. It is probable that levels lower than 12.5 mg.L kanamycin must be ideal for the cotyledons selection and 50 mg.L kanamycin for shoot tip.
Key words: Eucalypt, woody plant, genetic transformation
INTRODUCTION
Eucalypt is the most cultivated woody species around the world and Brazil is the second larger producer, several species are cultivated to wood, cellulose and paper (Alfenas et al., 2004). Eucalyptus saligna is relatively adapted in all Brazilian territory (Ferraz and Coutinho, 1984), however, this species is no much tolerant to cold (Le Roux and Van Staden, 1991), and it is susceptible to frost (Ferraz and Coutinho, 1984). The largest restriction to eucalypt culture in the south of Brazil is due to cold and to the incidence of frosts (Silva et al., 2009). Frost is too severe that eucalypt trees show since burned tip until dead of the aerial part (Selle and Vuaden, 2008). Genetic transformation can overcome susceptibility to biotic and abiotic stresses. However, for an efficient protocol of genetic transformation, several factors are involved. One of them is use of an efficient selective agent,
which allows in vitro regeneration of transgenic
plants and eliminating non transgenic plants, this is possible using a tolerance gene to the selective agent, such as NPTII gene (i.e. it codes an enzyme called neomycin phosphotransferase II) that inactivate antibiotics of aminoglycosides class (e.g. gentamicin, kanamycin, neomycin, and others) . Nevertheless, in Eucalyptus the kanamycin effects is very variable and it is dependent of the species and clone (Gonzales et al., 2001).
The aim of this research was to determine ideal dose of kanamycin in shoot tip and cotyledon explants of E. saligna for use as selective agent in genetic transformation.
MATERIAL AND METHODS
Explants source
In order to test effects of the kanamycin in cotyledons, these explants were obtained through in vitro germination. Seeds of Eucalyptus saligna
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Author for correspondence: clonageinvitro@yahoo.com.br