I don't think the marker gene will impact the transformation efficiency. Both are in the same T-DNA cassette.

You will see some reduction in transformation efficiency with increased size. So your 10kb plasmids are probably going to be somewhat reduced relative to a 3kb control plasmid for example. I doubt GUS or GFP will differ to any significant degree. However often you may have a DNA prep that just works better than another one.

Michael J. Benedik Yuan-Yeu Yau Thank you very much sir for your answers,

So what about that :

After transformation: PCR detection/ GUS assays/GFP fluorescence, so all results should be the same results or can not be?

For example, after transformation, if do detection by PCR using the GUS/GFP specific primers then the overall transformation efficiency

Overall Transformation efficiency 28.82 % including the GUS and GFP PCR positive

PCR GUS-positive efficiency 40.00 %

PCR GFP-positive efficiency 17.10 %

But when did the GUS assays from those PCR GUS positive then

GUS assays efficiency 81.70 % (not all 100% showed the GUS blue stains)

But when did the GFP fluorescent from those PCR GFP positive then

GFP fluorescence efficiency 56.67 %

1- My question is that if those with PCR positive must have the same results (100%) while doing GUS assays or GFP fluorescent? or can not be 100%?

2-Why the pCAMBIA1301 with GUS reporter showed higher transformation efficiency than pCAMBIA1302 with GFP? ( even all the conditions during the experiment same with the same materials).

Ok, so this is a complex question. There are a few different factors going on

1. transformation efficiency. In theory this will be the same for your two vectors but that's not always the case (slight variation due to experiment to experiment differences). Did you transform side by side every time? If not, it can be experimental variation

2. unless this is floral dip you are also going to have regeneration differences

3. transgene detection (PCR), this just lets you know if the gene is present, not the expression level (and often only part of the construct is detected, you aren't checking for the entire cassette)

4. transgene expression (GFP or GUS) you can have plants that have the transgene (most or all of it) present but you do not detect expression

why? well, you need a threshold level of protein activity to see GUS, and you need a certain amount of GFP protein expressed to detect fluorescence

You can also get insertion into silenced areas of the genome

So, there are lots of intermediate steps in the process of calculating overall transformation efficiency. My suggestion is to present all the data (as you did earlier) to provide a more complete picture of each step in the process

Often, unless someone is working on optimizing this process or has a breakthrough on a recalcitrant species they are just happy to get a handful of good transformed plants to work with

Hope this helps!

Amy Klocko Thank you very much Dear Professor for your detailed answer