Arabidopsis is one of the easiest to transform. I have transformed soybean and pennycress as well. They were doable, just takes a little more effort. Other friends appear to make corn transformation look pretty easy as well.
Hi Thanks for such a informational video. I was wondering do you maintain a specific OD (or you check OD of overnight grown culture) when you dissolve pellet into infiltration buffer?
Observing the OD is not necessary. My overnight cultures are passed log phase. A wide range of ODs can yield transgenic plants. The stage of the plant is quite critical.
It helps the bacterial suspension better enter the flower and get to the ovule be breaking the surface tension of water. Silwet L-77 is a nonionic detergent.
Hello. How do you select transgenic plants in this method? Does this technique require PCR, gel electrophoresis, or DNA sequencing to evaluate the plants and validate the efficiency of transformation?
Basta, hygromycin, or kanamycin selection can be employed based of the construct used. It is best practice to check your putative transgenics out with PCR, however, all of those three markers listed have a very low rate of false positives if selection is carried out correctly.
You would have to be able to grow recombinant Agrobacterium to do transformation at home. Please note, in the USA making transgenic organisms is regulated. Growing Agrobacterium requires an autoclave (pressure cooker) to sterilize media, growth media, a shaker, glassware, antibiotics, petri dishes, etc. You would also probably have to transform Agrobacterium. In short, this all could be performed in a modest lab. I suggest you visit your local college and work with them, as this would be the most straightforward way to transform plants.
Thanks. There are published procedures for many plants that you could look up. However, transformation of many plants will require a sterile laminar flow hood to work in.
@@AqleemAbbas I have a print protocol here with more details that you are askng about patharkar.com/2018/11/transforming-arabidopsis-by-agrobacterium-floral-dip
Hello Sir, what is the use of infecting leaves with agrobacterium? are you generating mutants in your experiment or doing some other studies? because to generate mutants just need to infect buds, i guess.
If you look closely in the video I am dipping the floral buds and they are the target of the transformation process. Some of the seeds from this dipped plant will be transgenic.
5% sucrose + 0.05% Silwet L-77. You can find a print protocol with all the details on my website. patharkar.com/2018/11/transforming-arabidopsis-by-agrobacterium-floral-dip
Find a print protocol and more details for this video at patharkar.com/2018/11/transforming-arabidopsis-by-agrobacterium-floral-dip Have questions? Leave us a comment.
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A specialist keeping it simple. Binary vector innoculum says quite something.
Thanks
Hi, Rahul, thank you for video. What do you think about in planta transformation of another Plant species?
Arabidopsis is one of the easiest to transform. I have transformed soybean and pennycress as well. They were doable, just takes a little more effort. Other friends appear to make corn transformation look pretty easy as well.
Hi Thanks for such a informational video. I was wondering do you maintain a specific OD (or you check OD of overnight grown culture) when you dissolve pellet into infiltration buffer?
Observing the OD is not necessary. My overnight cultures are passed log phase. A wide range of ODs can yield transgenic plants. The stage of the plant is quite critical.
i have a Question. Function of Silwet L-77 in this experiment. Thanks you.❤
It helps the bacterial suspension better enter the flower and get to the ovule be breaking the surface tension of water. Silwet L-77 is a nonionic detergent.
Did the bacteria still have plasmid that promote tumor when you insert the modifiction plasmid to them ??
Tumor causing genes have been removed from this disarmed stain.
Hello. How do you select transgenic plants in this method? Does this technique require PCR, gel electrophoresis, or DNA sequencing to evaluate the plants and validate the efficiency of transformation?
Basta, hygromycin, or kanamycin selection can be employed based of the construct used. It is best practice to check your putative transgenics out with PCR, however, all of those three markers listed have a very low rate of false positives if selection is carried out correctly.
I forgot to add Silwet before dipping. Is there any chance I will get transformants or should I start again?
The chance is greatly reduced but there is a slight possibility of getting a transgenic. I recommend doing dipping again with Silwet.
Thank you so much sir
can you give some ideas about how we can do like this experiment in our home using home available materials or chemicals
You would have to be able to grow recombinant Agrobacterium to do transformation at home. Please note, in the USA making transgenic organisms is regulated. Growing Agrobacterium requires an autoclave (pressure cooker) to sterilize media, growth media, a shaker, glassware, antibiotics, petri dishes, etc. You would also probably have to transform Agrobacterium. In short, this all could be performed in a modest lab. I suggest you visit your local college and work with them, as this would be the most straightforward way to transform plants.
Excellent video. How would one go about figuring out how to do this for other types of plant?
Thanks. There are published procedures for many plants that you could look up. However, transformation of many plants will require a sterile laminar flow hood to work in.
@@RahulPatharkarGreat. What is solution that you dissolve pilet? Which stage of plant you take? Why floral parts?
@@AqleemAbbas I have a print protocol here with more details that you are askng about patharkar.com/2018/11/transforming-arabidopsis-by-agrobacterium-floral-dip
Hello. Can i reference this video for my presentation about the method?
Sure
@@RahulPatharkar thank you
Hello Sir, what is the use of infecting leaves with agrobacterium? are you generating mutants in your experiment or doing some other studies? because to generate mutants just need to infect buds, i guess.
If you look closely in the video I am dipping the floral buds and they are the target of the transformation process. Some of the seeds from this dipped plant will be transgenic.
What are you adding to the Bacterial Pellet
I assume you're talking about the "Silwet L-77". Everything is in the protocol in the video description
5% sucrose with 0.05% Silwet L-77.
That is correct, please follow the written protocol.
@@RahulPatharkar Sir, are the sucrose and Silwet reconstituted in MilliQ or the media in which agrobacterium is grown for floral dip?
@@hemanthravishankar3287 MilliQ water.
I couldn't catch your words...5 percent sucrose + what? Sir...could u tell me
5% sucrose + 0.05% Silwet L-77. You can find a print protocol with all the details on my website. patharkar.com/2018/11/transforming-arabidopsis-by-agrobacterium-floral-dip
Find a print protocol and more details for this video at patharkar.com/2018/11/transforming-arabidopsis-by-agrobacterium-floral-dip
Have questions? Leave us a comment.
The link is not available
Link is not available.
@@rafaelfonsecabenevenuto9234 The domain changed to patharkar.com. Please try the link above now that I have corrected it.
I wish if the audio was better
Unfortunately, the lab equipment makes a lot of background noise that cannot be turned off.
@@RahulPatharkar A clip-on microphone positioned closer to the mouth should pick up more voice and less noise. Still, this was interesting to watch.
Thanks Rahul
You are welcome. All the best.
Thank you very much!
You're welcome!
Hello, I'd like to know if you have an Instagram account? Thanks in advance and best regards Andreas
I don't have one.
@@RahulPatharkar ok, do u have any other social media platform,website? Ps- do you have anything for sale? Thanks in advance and best regards, 🌱
@@andreaslinden3262 I do not have anything for sale. I do have a website: patharkar.com