After several weeks, the leaf pieces are dying on the antibiotic media while the candidate transgenic chloroplast-transformed platelets have arisen (see arrows). These platelets will be tested for the presence of the trait gene and resistance to insects.
It starts small, with tiny plantlets growing from the vestiges of a bio-blasted leaf.
But there’s big promise in those plantlets.
The mere fact of their growth shows that the chloroplast has been transformed with the new DNA material. At this stage, the plantlets are nurtured and tended with care because we want to be sure they contain the target trait.
Jeffrey Staub, Ph.D., a TechAccel science advancement program manager, is working with partners at the Danforth Center for Plant Science on a project nicknamed “Biosprey.” In previous installments, we’ve reviewed the project goals and the use of the gene gun to get to this point. To recap:
- The goal of the project is an RNAi-based biopesticide. We started with a model insect target of the tobacco hornworm, which also explains why we’re working with tobacco plants.
- So far, we’ve identified the genetic trait we want to express in the plant, and we’ve “shot” that trait into tobacco chloroplast with the gene gun. The resulting plantlets are now growing in a special medium.
“The next step,” Jeff explains, “is a molecular assay to examine the new plants to see if they really are what they should be.”
The molecular assay uses another technique, called PCR, the Polymerase Chain Reaction assay, to confirm that the RNAi gene has been inserted into all of the chloroplasts in the plant. Since plant cells have lots of chloroplasts, the RNAi is at a heavy dose that will be helpful in killing those pesky insects.
Now that we have evidence from the assay showing the trait we expect – the ability to disrupt RNA in the insect’s gut – we will move to the next stage.
Up next is a life-or-death trial for the young transformed plants and the tobacco hornworms.
In tobacco chloroplast transformation, the initial transformed plantlets typically do not have the transgenic DNA in all of their chloroplasts (called heteroplasmy). Using a single piece of leaf from the initial transformed plant, we can regenerate another set of plantlets on a special medium (shown here). Those new plantlets, termed subclones of the original, will typically then have the transgenic DNA in all of their chloroplasts (called homoplasmy).
This is part 3 in our brief series taking a dive inside one of TechAccel’s science advancement projects. Earlier blogs explained the project and some of the technology in use.
