Dear Megha

Duque (2010) co-cultured leaflets of in vitro grown Medicago truncatula plants with Agrobacterium tumefaciens EHA105 bearing the p35SAdc-Gus constructs.

Also, Lin et al. (2011) developed a new plant transformation vector, pHairyRed, for enabling high throughput, non-destructive selection of Agrobacterium rhizogenes-mediated ‘hairy-root’ transformation. They stated that it can potentially be used to transform a wide range of scientifically and agronomically important plant species, including Glycine max, Phaseolus vulgaris, Arabidopsis thaliana, Nicotiana tabacum, Lotus japonicus, and Medicago truncatula.

pCAMBIA binary vector can be a useful one for Medicago truncatula genetic transformation.

When you search online with keywords 'pCambia' and 'Medicago truncatula', you can see quite a few papers come up.

But, I am interested in how come no transgenic lines obtained from 'pSITE2CA-GFP gateway estination vectors'? Do you have a map of pSITE2CA-GFP. How did you screen your putative transgenic lines? By looking for GFP? By PCR? By Southern analysis?

Hi Megha Gupta,

Ok, I found the similar map of 'pSITE2CA-GFP gateway destination vectors' in the paper below. Seems that the GFP gene they used is not eGFP (enhanced GFP). The weak expression of GFP gene might have hindered your screening for 'transgenic' lines.

Article pSITE Vectors for Stable Integration or Transient Expression...

To test whether this vector can express in Medicago truncatula, you can do a quick 'transient' assay-- Agroinfiltration--to see whether the GFP is expressed well. You can do Agroinfiltration in both Medicago truncatula and tobacco leaves (most used plant material for Agroinfiltration) and compare both. Once inject your Agrobacterium, you should see GFP results on the leaves in 2-3 days.

Thanks Yuan for your suggestions. I have confirmed GFP expression with test gene in Nicotiana using confocal and as well as with PCR. But I am not able to generate callus with agro-cocultivated leaf explants. I will try with pCAMBIA also.

Thanks a lot.

@ Megha Gupta,

How did you do Nicotiana (tobacco) experiment? Stable transformation??

It was a transient expression.

You can be use pORE vector, it is smaller in size and suitable for Agrobacterium transformation.

Hi Megha Gupta,

If you use the original pCambia vectors containing GFP, the expression of that GFP version is weak. They have tested it in rice. They suggested to replace their GFP version with a better GFP version, such as eGFP (enhanced GFP).