Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps

Abstract

The factors influencing transfer of an intron - containing β-glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of β-glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of β-glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of β-glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.

Stable integration of mgfp5 transgenes following Agrobacterium-mediated transformation in Boesenbergia rotunda cell suspension culture

“…However, some plant species require a longer period of co-cultivation; for example, in Vanda orchid, explants co-cultivated for 4 days produced the highest transient gusA expression, compared to those co-cultivated for 1, 2 and 3 days (Gnasekaran et al 2014). Similar findings were reported by De Bondt et al (1994), where a co-cultivation period of 4 days yielded a higher transformation efficiency in apple. Co-cultivation period is also dependent on the Agrobacterium strain used.…”

IN VITRO REGENERATION AND TRANSFORMATION OF APPLE (Malus domestica Borkh.) ROOTSTOCK MALLING 7 USING RICE CHITINASE GENE

“…On the contrary, many reports showed that the infection time and temperature are genotype dependent, while gentle shaking of leaves for different durations at room temperature was preferred to obtain maximum transformation efficiency in M. domestica cv. 'Jonagold' and rootstock MM106 respectively (De Bondt et al, 1994;Sharma et al, 2012 (George et al, 2008). Among the developed putative transformed regenerants, few can be chimeras i.e.…”

Applications of Biotechnology in Kiwifruit (Actinidia)

“…Both A281 and its nononcogenic derivative, EHA105, have the Ti-plasmid pTiBo542 in a C58 chromosomal background (Hood et al, 1993;1986), and have been shown to be superior in gene transfer in other plant species, e.g. apple (Bondt et al, 1994). However, high rates of callus formation do not necessarily mean high efficiency of transgenic plant production, and Wang et al (2007) also found differences in shoot regeneration related to whether cocultivation had been with strains harbouring an oncogenic Ti plasmid (A281) or a nononcogenic Ti plasmid (EHA105).…”