Dear Researchers,
Im trying vacuum infiltration for seed transformation of a recalcitrant plant. I tried different time durations of vacuum infiltration (1 min to 10 min), culture density (OD 600=0.6 to 1.5), infection time (10 m to 1 h) and co-cultivation period (24 h to 6 days). But still im not getting uniform GUS expression in transformed tissue after selection. What should I do to increase the transformation efficiency? Can any experienced persons help me in this regard? Thanks in advance.
A pilot experiment (transient expression assay) was needed to test whether your transformation was effective. If the transient expression result was positive, it means the stable transformation would be possible. In addition, a cutting for the seed to expose the wounding tissue is highly required for a transformation assay.
Maybe try Finer's sonication (SAAT) method, both with and without vacuum infiltration. Then check transients after co culture against controls as Huiquan suggests.
Are you germinating the seeds so that you will have actively growing cells? Have you added acetosyringone to induce the vir operon?
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