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Extra info for Agrobacterium Protocols, Second Edition: Volume II (Methods in Molecular Biology Vol 344)
10. In some protocols which have been shown to be effective in our hands for certain cultivars, an overnight incubation of the explants in a high hormone medium can be employed at this stage. This is formulated as MS20 plus 10X the growth regulators used in CM, and plates should be sealed, and kept overnight in the dark at 18–22°C prior to inoculation with Agrobacterium the following day and following the protocol as described. 11. Inoculation methods vary between reports. In some cases a heavy inoculum of Agrobacterium is used for a reduced co-cultivation period.
In this paper we describe detailed protocols for the Agrobacterium-mediated transformation of cassava using GSEs. Embryos are produced from explant tissues cultured on semi-solid MS-based callus induction medium (24). Embryos are then induced from callus tissues and directly inoculated with Agrobacterium for transformation. Selection for transformants is typically based on antibiotic resistance conferred by a gene present in the T-DNA. The transformation efficiency using our system ranges between 1 and 5% [defined as the frequency of polymerase chain reaction (PCR) transgene-positive plants per 100 cassava GSEs infected].
At this stage, 5 mL of the culture can be taken and used to make 1 mL glycerol stocks, 650 µL of culture medium to 350 µL of 50% Glycerol (v:v) and the samples flash frozen and stored at −80°C. 3. Inoculation, Co-Cultivation, and Regeneration 1. 5-mm wide excised from 3- to 4-wk old potato stock plants (see Note 9). 2. Explants (up to 30 per plate) are placed into Petri dishes containing 15 mL of MS20 to avoid dessication (see Note 10). 3. 6. per 90-mm Petri dish, dishes sealed with Nescofilm and placed on orbital shaker at 50 rpm for 45 min at 22°C (see Note 11).