Glowing Plant March update
Here's March's update:
- We are still working to debug the latest issues with the Lux plants.
- We have fixed the challenges with the Fluc v2 plants.
- We are expanding the maker kit beta tester program so anyone can join
- We are still planning for a non-accredited investor fundraise launching around May 16th
Autoluminescent plant Lux v1
We are still in the process of regenerating the plant. The first batch of regenerants didn't glow stably, although we did find one weird plant which was glowing a little but which then stopped glowing which was a very strange and unexplained result.
We've updated our hypotheses for what is going on based on the results from the last month, in particularly the lack of success from any of the regenerated plants. These are the three most likely reasons for the challenges we are facing:
1. The most likely scenario is premature removal of selection pressure leading to false positives and seedlings that do not include the six lux genes. In order to speed up the regeneration process and shorten the time to getting seeds we removed the selection pressure after 2 month. We did this because the plants grow much bigger much faster once selection pressure is removed. It seems to be working but the academic literature suggests keeping the plants under selection pressure till the roots have formed so this may have been a mistake. Now we are keeping the plants longer under selection and this has reduced the number of regenerants dramatically, which supports the hypothesis. This longer selection does lead to slower growth delaying our progress and means we don't have clear results yet. We are waiting for the next batch of plants to root before running the assay to see how well they glow.
2. The second possible issue is caused by the low transformation efficiency of co-bombardment with a large construct: We have chosen to do co-bombardments (lux and marker on constructs) to ease the marker removal step (as we don't want to spread herbicide resistant genes into the environment). Under this hypothesis, only a portion of the selected regenerants would have received the lux construct reducing the efficiency. With our Fluc plants that proportion was 50%, but with the larger Lux construct we expect that proportion to fall even lower. Starting last week, we have been testing using a single construct for bombardment, which means almost all of the regenerants selected should glow. If this works we will have an additional challenging step to remove the selection marker as we won't be able to out-cross the marker as it will be co-inserted with the glowing genes. We plan to explore the CRISPR/Cas9 system to achieve this if we have to.
3. The final possibility is gene silencing of the construct. It's possible that the anomalous plant which glowed briefly for a few days is evidence to support gene silencing. The promoters we are using are also used by the plant for essential life functions, so silencing should kill the plant (in theory) and hence this shouldn't happen. Gene silencing is poorly understood though and not many groups have created transgenic plants with this many genes inserted so there's plenty of uncertainty here. Generating a large number of independent transformation events is one way to address silencing issues as the problem may be more or less sever depending where in the genome the construct is integrated. In the meantime, we have also performed a floral dip to transform Arabidopsis with the lux construct to test if silencing is happening there (this is a much faster regeneration process).
Fluc v2 plants
We discussed the germination problems in the last update.Our hypothesis about anther sterility was correct and now we have nicely germinated T2 lines growing in our growth tent. These plants will be tested for luminosity and marker presence next week. Once we find one that glows nicely without a marker sequence, we will be ready to grow that one to make it's own seeds and ship out like the Fluc v1 plants - so this is back on track.
We shipped the first maker kit to a school this week which is exciting, looking forward to hearing about the kids reaction. Our revised permit guidance document also seems to be working better and one beta tester got the permit approved within a month which is awesome. The kit is still in beta, but we are now ready to roll out to anyone who wants to be a beta tester. If you backed the maker kit and would like to be a beta tester (and aren't already part of the program) then please get in touch with us.
Title III fundraise
We are still planning a public equity crowdfunding campaign. This is provisionally scheduled to launch on May 16th. More on that in a future update.
All the best,
the glowing plant team.