Selection of transgenic flax plants is facilitated by spectinomycin

Abstract

Regeneration of transformed flax shoots after inoculation with Agrobacterium tumefaciens carrying a binary vector with either a neomycin phosphotransferase (nptII) gene and a [3-glucuronidase (GUS) reporter gene or a spectinomycin resistance gene was examined. Hypocotyls from 4-day-old seedlings were inoculated with either of the two A. tum@ciens strains. Selection and regeneration were achieved on a medium containing 0.1 ~xM thidiazuron, 0.01 ~M napthalene acetic acid, 100 mg1-1 kanamycin sulphate or spectinomycin sulphate and 300 mg1-1 cefotaxime. Use of different neomycins for the selection of transformed tissues did select transformed calli but not transformed shoots either directly or via a callus phase. Selection based on spectinomycin resistance allowed the growth of transformed shoots. Transgenic shoots were rooted on a medium containing 100 mg1-1 spectinomycin sulphate. Integration of the spectinomycin resistance gene into the flax genome was confirmed by Southern blot hybridizations and spectinomycin resistance was shown to be inherited as a dominant Mendelian trait. Therefore, spectinomycin resistance is more suitable for genetic engineering of flax than aminoglycoside resistance.

Somatic embryogenesis and the effect of particle bombardment on banana Maçã regeneration

“…ABB group), with geneticin, allowed transgenic embryo transformation but interfered with embryo germination. This inhibitory effect has also been previously reported in other crops (Yao et al, 1995;Bretagnesagnard & Chupeau, 1996). Shock waves and sound waves, promoted by the biobalistic equipment, can cause damage to the explant target at the cellular level, as membranes disrupt, thus interfering with the regenerative process (Russel et al, 1992).…”

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in an important Cavendish banana cv. Robusta (AAA)

“…Transformation efficiency could have been further enhanced if embryo germination was done in non-selective medium. There are reports suggesting that antibiotics allow formation of transgenic embryos but interfere with embryo germination (Yao et al 1995;Bretagnesagnard and Chupeau 1996). Hence, the use of subsequent germination medium with reduced levels or removal of hygromycin promises to enhance the regeneration frequency of transgenic plants.…”

Transgenic flax/linseed (Linum usitatissimum L.) - expectations and reality

“…The first transgenic flax cells were created in 1983 (Hepburn et al 1983), the transformation was performed via Agrobacterium tumefaciens. The first attempts aimed at transfer of whole unmodified T-DNA of A. tumefaciens and A. rhizogenes were later changed for integration of model prokaryotic genes (Basiran et al 1987;Dong & McHughen 1993;Mlynárová et al 1994;Bretagne-Sagnard & Chupeau 1996) and of specific genes coding economically important traits (Jordan & McHughen 1988;McHughen 1989;McHughen & Holm 1995) (and others as described in Table 1) into flax genome. Flax/ linseed transformation has already been reviewed in a number of journal papers or book chapters McHughen 2000;Preťová et al 2007;Pavelek et al 2012;Badere 2014), nevertheless, our review deals with GM flax in more detail and to a greater extent, trying to provide a thorough summary of this topic.…”

“…Also the antibiotic spectinomycin was successfully applied in the selection of transformed flax plants (Bretagne-Sagnard & Chupeau 1996). In order to meet the requirement for using non-antibiotic resistance genes for the production of transgenic plants, the phosphomannose isomerase gene was used as an alternative selectable marker for flax transformation (Lamblin et al 2007).…”