I want to transform Arabidopsis with pTF101.1 binary vector which has a bar resistance gene. Normally people have used this vector for crops such as soybean and maize, not frequently been used for Arabidopsis flower dip transformation. Can we use it for Arabidopsis?
Thanks!!
Hi Sachin,
As long as the T-DNA contains parts that work in Arabidopsis (promoter, terminator, etc), you should be fine. Some backbones work better than others (I have not tried this one), but if your goal is to obtain transgenics (and not e.g. create a population of insertional mutants with high efficiency) you should be OK.
As a side comment, do not use commercial formulations of ammonium glufosinate if you are going to select in plates. If you do, use the pure form (ammonium glufosinate, phosphinothricin) or you might experience seed germination problems. On the other hand, if you sow your seeds on soil and let them germinate and open the cotyledons, you can water them with 150 mg/L of Basta. After approx 2-3 weeks you will see the transformants growing green and developing true leaves, whilst the non-transformed ones yellow down and do not develop the second pair of true leaves.
Additionally, if you do not know this method and feel like giving it a try, the 'floral inoculating protocol' works like a charm. I especially liked the fact that it is 'easy' and you do not need to grow big volumes of bacteria and wash afterwards.
https://www.jstage.jst.go.jp/article/plantbiotechnology/27/4/27_4_349/_article
Cheers,
Ruben
https://www.jstage.jst.go.jp/article/plantbiotechnology/27/4/27_4_349/_article
Hi,
@Grégoire. I had seen the protocol with agro on plates but never tried it... interestingly, that matches the observation that I can use liquid cultures kept in the fridge for the second transformation. I find it a bit puzzling though (I did my PhD in agro-mediated transformation), since the optimal temperature for activation of genes vir is 28C, and the optimal for T-pilus formation is around 18C. That would mean that the bacteria literally wake up in contact with the plant, instead of being pre-induced as in standard protocols.
Good luck with the tranformation, it's great to share useful info in this place.
Best,
Ruben
Thanks to all of you to answer my questions, it is quite enlightening. I will go ahead and transform Arabidopsis with pTF101.1 with floral inoculation protocol, for the first time. Thanks once again.
Hi,
@Grégoire. My usual procedure is to grow an O/N culture in 10 ml, then transform the next day in the afternoon at about 5pm (lights go off at 11pm, 16h/8h light/dark at 22C). I keep the culture in the fridge and 4-5 days later I put it in a 28C shaker at about 10am, then transform in the afternoon. I transform 3 plants grown each one in a small pot (about 5cm diameter, 6 cm height). I have not done experiments to compare transformation efficiency with fresh/fridge bacteria, but usually I get 20-40 lines per inoculated plant.
Oops... I have just realised that a few days ago I wrote a detailed description on this thread, and it seems that I never pressed the 'Add' button! :S
To tell the long story short, I use the simple inoculating solution (sucrose and silwet). I spin down the ago and resuspend in 300 ul of solution. This is enough for 3 plants, and instead of pipetting a drop on the meristems as described on the paper, I 'wash' the meristem pipetting in and out. Then I shade the plants with a tray and retire it the day after.
The plants I have used so far are virtually of any age. Once I even used plants that had some mildiu or thrip infection (sometimes I have to push forward), and to my surprise I got many transformants.
As to the agro, I do not measure the OD600. I simply spin down and resuspend.
I did a PhD in genetic transformation and developed a protocol to transform cork oak somatic embryos from scratch. I studied OD, cocultivation time, stage of the bacterial culture, light and temperature during the coculture, stage of the somatic embryos... and all these factors influenced the transformation efficiency. For arabidopsis I have seen a lot of protocols where people do not care about OD, development, etc...actually when you grow agro densely on plates the physiology of the bacteria can change dramatically and the bacteria become filamentous!
To me this means that floral transformation is a method extremely robust....
Cheers,
Ruben
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