Exploring the marker gene used in Arctic® apples

As you can read about in our FAQ section, the process required to transform a conventional apple to an Arctic® variety necessitates the use of a marker gene that makes the plant tissue resistant to the antibiotic kanamycin. This process requires a doctorate in molecular biology to fully comprehend, so we turn to our head scientist John Armstrong, who just happens to have these credentials. 

Should people worry that the insertion of our marker gene may add a new protein to Arctic® apples? The simple answer is no, as there are no proteins expressed in Arctic® apples that aren’t in conventional apples. You may ask how that’s possible considering we use a marker gene which produces a protein (NPTII) that makes the plant tissue resistant to the antibiotic kanamycin. Transgenic event

Here comes the molecular biology lesson though, so stay with me! We start our project with tiny pieces of apple leaf tissue, grown in small glass jars. The kanamycin-resistance gene is attached to the apple genes that turn off the enzyme that initiates apple browning. Together these are added to the leaf tissue. If the kanamycin-resistance gene is expressed at a high enough level to make the leaves resistant to antibiotics, it proves the plant’s transformation was successful (since the gene sequence that prevents browning is attached to the marker gene, we know it has transferred too). This also demonstrates that the protein is present in the leaf tissue at this point, but this does not carry over into the apples themselves.

Through our Arctic® apple field trials, we produced a lot of apples that were tested for many different things. Many of these apples were sent to special testing facilities that specialize in detecting proteins using a technique called Enzyme-Linked Immunosorbent Assay (ELISA). Through these tests, which are able to detect 1/10th of a part per billion, it was determined that mature Arctic® apples do NOT have a detectable amount the protein NPTII. To give an example of how sophisticated and accurate these tests are, they would be able to detect a penny in amongst between 100-250 ton coal-sized railcars!

Better still, even if there was a detectable amount, there is still nothing to worry about as it has been determined that “there is no potential toxicity from this protein” and on top of that, it would be broken down in digestion almost immediately. So, the key message here is that there is no need to worry about the marker gene we use. Arctic® apples have proven to be just as safe as conventional apples!


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Comments

  1. Anonymous

    You said it wasn’t found in the fruit, but was it present elseware, such as the leaves? I’m more worried about bacteria gaining resistance from absorbing the genes from decaying plant matter. 

    • Joel

      Correct – as mentioned above, we introduce resistance to the antibiotic kanamycin into the leaf tissue in order to determine if the nonbrowning trait was successfully accepted.

      There is no need for concern, however, as the antibiotic resistance gene was derived from naturally occuring soil bacterium which is extremely common across the planet. In essence, one square foot of typical soil has more of this antibiotic resistance than an entire Arctic apple tree’s worth of leaves.

  2. Jeff Leonard

    John, could you tell me more about the promoters being used to drive nptII and the RNAi construct? I assume they are different so that you have tissue specific expression.

    • Joel

      Hi Jeff – thanks for your question!

      There is a detailed explanation of the promoters we’ve used in Arctic apple transformation in our petition for deregulation we submitted to the USDA. If you want to have a look at pages 35-36 in particular, there are overviews of their elements, and a visual representation as well. 

      Hope that helps, just let us know if there’s any other questions we can help answer!

    • Joel Brooks

      Hi Scott,

      Great question with a somewhat complicated answer!

      The type of gene suppression used to develop Arctic apple cultivars is currently known as RNA interference (RNAi). While scientists were trying to understand the process since its discovery in 1986, it has been variously known as antisense suppression, co-suppression, post-transcription gene silencing (PTGS), transcription gene silencing (TGS) and virus-induced gene silencing (VIGS) until it was realized that all of these processes were describing the same phenomenon. RNA interference is a natural cellular process used to control gene expression during development and in defense against virus and transposons.

      Thanks,
      Joel

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