Hi,
I have worked with onion epidermal cell transformation for a long time now. Did some successful transformations with high efficiency towards the end of last year and beginning of this year during the winter. Suddenly, the transformation rate started decreasing and now its a disaster; few to no transformation these days.
I have tried by changing different parameters like onions, spermidine, pressure (900-1350, noramlly i use 1100psi), etc.
Recently, I checked with an experiment if my construct is fine and the precipitation is ok. I found out that the constructs could transiently express the protein in maize endosperm but does not seem to work in onions.
Can anyone kindly suggest based on their experience ? How can I get a high transformants as before?
Is still enough Helium gas (enough pressure) in the helium tank? Have you checked that? Is the quality of rupture discs still ok?
Thank you for replying Yuan-Yeu Yau.
The rupture disc ruptures at the correct pressure (1100 psi) and of-course the helium tank meter shows the pressure of 1300psi which 200 psi more than i need.
I have changed the onions several times, do u know what could be the problem.
Thanks in advance.
Desperate to solve this.
Hi Saugat, has there been any changes in either rate chamber comes to vacuum or rate of pressurization as helium tank pressure decreases?
has anyone changed the gap distance between rupture disk and carrier sheet on your gun?
Hi Edward,
The vacuum rate is normal as mentioned in the manual by Bio-rad as well as rate of pressurization is also not a problem. Because the system works for maize endosperm transformation. However, its only the onion transformation thats not working. And also the gap distance between rupture disk and carrier membrane is the same. However, what i see is unusual clumped gold particles in the onion epidermal layer. could gold clumping be a problem?
Thanks in advance
Do you see clumping during your particle preparation? Maybe try spinning at lower rate if you do - particularly on last spin after DNA has bonded. Do you use IPA for carrier sheet sanitization? It might be better than EtOH in terms of uniform deposition of the particles onto carrier sheet.
Hi Edward,
I used water bath sonicator to re suspend the golden particles, although this helped a little bit , there still exists little bit of clumping. Low transformation efficiency is still a major problem. I tried all what you said. Recently, I observed that the gold particles are more on top than penetrated through the onion epidermis. Could it be a problem. Since, this is related to height, vacuum and pressure, these parameters have not been changed and are set at its appropriate points.
Hi Saugat, has anything changed with the manner by which onion tissue is prepared (preculture duration, storage, moisture content)? Maybe this tissue is more sensitive to these initial conditions in terms of bombardment than corn.
Hi Edward, The onion slices are used for shooting. They are not precultured or made any changes. I know the transformation efficiency greatly depends upon the the vitality of the epidermal cells and I take care of this as far as possible. Another factor that is different with the maize endosperm is that the maize endosperm is a germinating tissue and has very active cell division and are relatively much smaller than epidermal cells. Therefore, the chances of hitting the nucleii is greater in maize endosperm cells.
Hi Phillip, thanks for the reply. I have not tried bombarding the other side of the epidermal layer since this is no the general practice (and also its difficult to maintain the vitality of the onion epidermal cells if done). However, I have tried checking the penetration of the gold particles and to me, it looks like the gold particles have not penetrated enough to reach the nucleii. Have any of you had this experience, kindly suggest? Thanks
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology