Shoot tip and cotyledon explants of Eucalyptus saligna Sm. cultivated on different kanamycin levels

Abstract

An efficient selec tive agent helps to obtain transgenic plant s; 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-1 kanamycin on a callus formation medium and for shoot tip were tested 50, 75, 100, 125 and 150 mg.L-1 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-1 kanamycin must be ideal for the cotyledons selection and 50 mg.L-1kanamycin for shoot tip.

Preliminary results for genetic transformation of shoot tip of Eucalyptus saligna Sm. via Agrobacterium tumefaciens

“…These results were according those found in Eucalyptus saligna cultured on kanamycin after 28 days; these explants produced ca. 2.6 shoots per explant (Silva et al, 2010). Even with the increase of the kanamycin level to the 75 mg.L -1 the explants continued multiplying (ca.…”

“…At the end of the sixth subculture (154 days) the explants did not survived for both treatments (0 and 100 μM acetosyringone). These results are according those found in E. saligna cultured at 50-150 mg.L -1 kanamycin, which at the 84 days the explants begin to die and all explants died in cultures above 140 days (Silva et al, 2010). The protocol for genetic transformation of leaf explants of E. saligna, the explants are selected with 50 mg.L -1 kanamycin; and the selection occurs in ca.…”

“…Cotyledonary explants were selected with kanamycin in ca. three months too (Silva et al, 2010). The time of selection of shoot tips can be an inconvenient (five to six months), however, case the efficiency of the transformation rate was higher than other explants, this explants type can be a good alternative.…”

“…Frost is too severe that eucalypt trees show since burned tip until dead of the aerial part (Selle and Vuaden, 2008). However, genetic transformation can overcome susceptibility to biotic and abiotic stresses (Silva et al, 2010) what can allow the adaptation of this species even in cold climates. Although the risks pointed in the culture of the genetically modified plants, there are several methodologies that guarantee the safe use of these plants (Silva et al, 2007).…”

Methods of asepsis for in vitro establishment and germination of Eucalyptus grandis

“…The process for the establishment of Eucalyptus in vitro using seeds has been laborious mainly due to the size of them and the method of processing that generate a mixture with high amount of inert materials, which may be responsible for the simultaneous inoculation of bacteria and fungi, and in turn delay in the establishment of seed culture medium in vitro. In vitro tissue culture is important to offer high rates of multiplication from segments of tissue, however it is also an efficient tool for obtaining large numbers of individuals free of contaminating sources (Dutra et al, 2009;Hossain et al, 2010;Hung and Trueman, 2010;Silva et al, 2010;Silva et al, 2011). Since the seeds of Eucalyptus are subjected to intense exchange of germplasm, methods of seed cultivation in vitro offer alternatives to mitigate these problems, limiting the spread of pests and diseases such as rust.…”