Hi everyone,
Can you suggest a best protocol to bombard a gene on soft petal (such as Lilies)? What is the best shooting pressure and distance? Also, I have a problem with tissues browning and eventually cell death happened after bombardment. Any suggestion? Thank you.
Hi H.N.,
1. Unless the bombardment parameters for the same Lilies/tissues you are doing have been studied & published and you can use it as a guide, it would be best to do some bombardment experiments to figure out the parameters for your own experiment purpose first. Different tissues may need different parameters (especially you are using soft petals). Let's say you have 2 'shooting distances' and 2 'shooting pressures" want to consider, then 4 sets of experiments can be set up to see the results. You can first try the gus gene to see the result.
2. The 'browning' might be due to high concentration of selection agent (or antibiotic) in the medium. For us, before bombardment experiments, we usually conduct a separate experiment by placing 'non-transgenic' tissue on medium plates containing a series of concentrations of selection agent (such as hygromycin) to evaluate which concentration is the best one for our 'putative transformants' selection.
Good luck on your research,
Some tissue death is to be expected, especially at the center of the bombarded area. You could try conditions used for onion epidermal peels (a delicate sample type) as a starting point and modify the protocol from there.
If you're not set on doing particle bombardment you could try transient expression by Agro. infiltration. It may result in less tissue damage.
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