The germinated embryos were transferred to a banana multiplication medium for generating multiple shoots of each transformation event (Fig. 2E, G and I).
As a shoot multiplication medium which contains charcoal, LPch is generally used for between 28 42 days; growth on this medium would be dependent on the microorganisms present.
Briefly, they were maintained by sub-culturing at 4-week intervals on a shoot multiplication medium, at 22 ± 1°C under cool white fluorescent tubes (55 μmol m-2 s-1), with a 16-h photoperiod.
When shoots regenerated from a cell colony, they were labelled as a series for each transformed cell colony, and transferred to multiplication medium containing 200 mg·l-1 Timentin to suppress A. tumefaciens growth.
> -wrap-foot> Aftexcisinging multiple shoots, when mother explants were transferred to the fresh shoot multiplication medium (MS medium + 1.0 mg L−1 BAP), they proliferated significantly during the next two subcultures and reduced thereafter (Fig. 2).
The bacterial mass was then transferred to the multiplication medium (sugarcane juice or molasses) and incubated at 35 °C, with agitation, till an optical density (540 nm) of about 0.5 was achieved.
The four standard tissue culture media were: shoot initiation medium ½LP5 (Hargreaves and Menzies 2007), shoot multiplication medium LPch (Hargreaves and Menzies 2007), embryogenesis medium EDM6 (Hargreaves et al. 2009) and embryogenesis medium Glitz (Hargreaves et al. 2009).
Yeast inoculum was prepared by separately inoculating two loops of each yeast strain in 125-mL flasks containing 50 mL of the multiplication medium (clarified sugar cane juice or molasses with approximately 4 g 100 mL−1 of total reducing sugars, pH 5.5, as supplied by a local fuel ethanol-producing unit, and sterilized at 120 °C for 20 min).
The multiplication medium consisted of QL macronutrients [ 30] and DKW micronutrients, vitamins and organic compounds [ 31], 3% sucrose and 0.7% agar (HispanLab, S.A).
Putative transgenic plants, from many transformed cell colonies, were transferred back to the multiplication medium containing only Timentin for clonal micropropagation.
