How’d we “make” a nonbrowning apple?

When my friends find out about my work with Arctic® Apples, invariably one of the first questions they ask is: How’d we do that? That is, how’d we “make” a nonbrowning apple? Here’s what I tell them:

First, a quick biochemistry lesson.  When the cell of a typical apple is ruptured – for example, by biting, slicing or bruising – polyphenol oxidase (PPO) found in one part of the cell mixes with polyphenolics found in another part of the cell. (PPO is a plant enzyme. Polyphenolics are one of the many types of chemical substrates that serve various purposes, including supplying its aroma and flavor.) When PPO and polyphenolics mix, brown-toned melanin is left behind.

Arctic® Apples produce practically no PPO so that enzymatic browning reaction never occurs.  This means Arctic® Apples’ polyphenols aren’t burned up when the apple is bitten, sliced, or otherwise bruised. No chemical reaction, no yucky brown apple left behind. Conventional Vs. ArcticSo how’d we “make” a nonbrowning apple? The small number of genes  (four, to be exact) that control PPO production were identified several years back, when the apple’s genome was mapped. To create a nonbrowning Arctic® version of an existing apple variety, our science team uses gene silencing to turn down the expression of PPO, which virtually eliminates PPO production, so the fruit doesn’t brown. This genetic transformation is aided by modern science tools. (We’ll explain what we mean by “modern science tools” in a later post.)

This transformation takes place in a laboratory in a petri dish, with a small sample of apple tissue. We confirm the genetic transformation was successfully completed before growing the tissue out into a tiny plantlet and eventually moving it to an orchard. (We’ll explain how we confirm the transformation in a later post, too.)

Personally, I was amazed to find out how “simple” this transformation process is. (I put the word in quotes out of deference to the head of our science team, Dr. John Armstrong, who knows best how much hard work and brain power went into making this process look simple to me.)

The end result of all this science is just an apple tree, now with very low PPO production to prevent enzymatic browning in its fruit. Our Arctic® Apple trees grow and behave in the orchard, blossom and bear fruit just like their conventional counterparts. We’ve got almost 10 years of test orchard experience to document that. Arctic® Apples are also compositionally and nutritionally similar to conventional apples, further indicating that lower levels of PPO aren’t consequential to the tree or the fruit. It’s only when one of our apples is bitten, sliced or cut that the Arctic® Apple difference becomes clear.

What role does PPO play in the plant, you might ask? In some plants, PPO plays a defensive role – for example, tomatoes produce high levels of PPO when attacked by pests or pathogens. In contrast, apples produce very low levels of PPO, and only in very young fruit.  Its presence is probably left over from apples of ages ago, playing no role in today’s apples.

I always close my talk with friends with this intriguing sidebar to the story: When eaten by humans, polyphenolics may have health-promoting benefits. For example, phenolics are believed to act as antioxidants, fighting the well-documented damaging effects that oxidation can have on the heart, other organs and throughout the body. Not enough is known yet about phenolics for the health community to suggest a recommended intake amount, as for other vitamins and nutrients such as Vitamin C (the best-known antioxidant), fiber and so on – but they are certainly worth watching!

Comments

  1. Anonymous

    WOW, it is amazing what you can do with biotechnology these days. I was just wondering if you would be able to put up a flow chart so that we can see the process visually? Thanks for the great infomation, it really helped with my assesment. 🙂 

  2. Anonymous

    If PPO can protect the plant and helps it fight off bacteria and pesticides and diseases. Just how different is the ammount of bacteria within the apples or how much gathers when sliced?has there been any trials on that? If so I would like to see those results. we all want an apple that doesnt brown but not at the price of the apple not being able to fight off infections.

    • Joel

      Thanks for your question! Arctic apples have been planted in field trials for over a decade, and there has been a great deal of testing and observation of them by third-party horticultural experts. It has been demonstrated that respond to pests just the same as their conventional counterparts, as well as grow at the same rate, require the same inputs, etc. – the only notable difference is when the fruit itself is cut, bitten or bruised! The full dataset on this is publicly available online here: http://www.aphis.usda.gov/brs/aphisdocs/10_16101p.pdf

      Regarding their ability to resist bacterial/fungal infections once cut – Arctic apples can actually better resist infection compared to conventional apples, since the enzymatic browning reaction typically breaks down cell walls and makes the fruit more susceptible to entry by foreign bodies. That said, for commercial sliced apple products, even Arctic apples would still be given an anti-bacterial/fungal wash, just as conventional slices are, but they would not require anti-browning treatments, which often add an off-taste and are quite expensive.

  3. Anonymous

    I’m curious–if the apple genome was only completely mapped in 2010, allowing researchers to “turn off” the PPO enzyme, then why do you say that Arctic apples have been grown for a decade?

    • Joel

      Thanks for your question! Even though the full apple genome was completed in 2010, the location of the PPO genes that we specifically target was determined much earlier. We were able to determine the proof of concept for nonbrowning apples around 2000, and have had field trials growing in Washington state since 2003, and NY state since 2005!

  4. Andy

    So if PPO is being inhibited, and it contributes to flavor, one can expect at least a difference in flavor between non-GM Grannies and the Arctic varietal?

    • Joel

      Hi Andy, thanks for your question!

      While polyphenol oxidase (PPO) and polyphenolics have similar sounding names, they are two very different things. PPO is an enzyme, and doesn’t contribute directly to flavor (whereas polyphenolics do), but what it DOES do is cause the browning reaction.

      The browning reaction results in the brown-toned melanin you see when an apple is bruised or the flesh is exposed. Since this reaction causes a decrease in the flavor/taste of the apple, in part by “burning up” the polyphenolic content, a sliced/bruised Arctic apple will indeed taste better. However, the first bite of a fresh, unspoiled Arctic Golden would taste just the same as a conventional Golden Delicious.

      So, one way to put it is that Arctic apples taste the same as the conventional counterparts in general, but better retain their flavor and nutrition.

  5. Frank

    In the second paragraph of your article you state that “Polyphenolics are one of the many types of plant-proteins…” Many years have passed since I studied biochemistry, but at that time phenols were not considered proteins, and I doubt if they are today. Or am I wrong about this? 

    • Joel

      Well spotted Frank! You are correct, and technically, a better definition would be to call polyphenolics a group of chemical substrates. The post has been updated to better reflect this.

    • Joel

      Hi and thanks for your question! Yes – we do use nptII (kanamycin resistance) as a marker gene to assist in the transformation process, but there is NO detectable amount of the NPTII protein in Arctic fruit (nor are there any other novel proteins present). We actually have a blog post dedicated to that very topic, “Exploring the marker gene used in Arctic apples” and also cover the subject in our FAQ if you’d like to learn more.

  6. Michaeljwjr

    Has there been any change in the behaviour of bees in the area around these apple trees? Do these Artic Apples beget other arctic apples? As in can I take an arctic apple, plant it, and grow a tree that produces arctic apples? 

    • Joel

      Hi Michael, thanks for your questions!

      Bee behavior around Arctic apple trees is the exact same as as it would be around any apple trees. Arctic trees grow the same way, respond to pests/weather just the same and don’t required any special treatments. Other than the fact that the fruit doesn’t brown when they’re bitten, sliced or bruised they’re just like any other apple.

      Regarding the potential to grow an Arctic apple tree from Arctic apple seeds, you would not be able to do so. Commercially, all apple orchards are now propagated by grafting, but if you planted an Arctic’s seeds, you would be very unlikely to successfully grow an apple tree in the first place, and even if you did, it would not produce Arctic apples.

  7. aeamonaco

    I wouldn’t have imagined that Artic apples could withstand like that. As an artist implicated in the environnement, i’m always on the look out for new information to provide to our followers and especially if this can avoid throwing out food. I wonder if this fruit can be grown in Europe and/or if a procedure could be incorperated insite the tree itself. People wast good food because of the coloration.

    If we can somehow get argriculture to accept a procedure that would keep fruit from browning for at least 24 hours, then the gain would be substantial.

    • Joel

      Actually, Arctic apples do resist browning for much, much longer than 24hrs and this trait in part of the fruit itself rather than from any additives! As you suggest, this benefit can have a very positive impact in reducing food waste throughout the supply chain, which is one of the main reasons we developed nonbrowning apples.

      Regarding Europe, we are focused on bringing Arctic apples to North American markets first, but other markets are certainly possible in the future.

  8. Gary Smith

    cban has a postcard of sorts that claims; “It was genetically modified by inserting a new genetic sequence into the apple cells with genetic material from apple, as well as from a plant virus and two different bacteria”. I have read the executive summary for the APHIS application and understand that Agrobacterium was used to vector the transformation, and is a common practice in GM, but am unclear on the role of Cauliflower Mosaic Virus. I am guessing that cban is being opportunistic by assuming the ignorance of the average consumer on the details of the process and suspect that no non-apple DNA was transferred to the Arctic Apple in the process as suggested in the postcard. Are you able to elucidate on the matter?

    • Joel

      Hi Gary, thanks for your great question!

      First things first: There is no cauliflower mosaic virus (CaMV) in Arctic apples! What we do use is a a small promoter element of CaMV calls CaMV35s, which is compact, well-defined and commonly used in biotechnology. CaMV35s is a promoter that we use in the transformation process so that gene silencing will occur.

      A rough analogy for how promoters work: the transgene we use to silence polyphenol oxidase (PPO) is the music we want to play, and the promoter is the stereo. You could also say promoters are like the capital letter at the start of a sentence, in that they tell the plant when to start reading the gene’s instructions (to produce less PPO).

      With all this said, even if Arctic fruit did contain CaMV it would not be an issue. Consumers regularly consume CaMV on cruciferous vegetables (such as cauliflower).

      Thanks again for your question and check out a similar post, “How we introduce the nonbrowning trait in Arctic apples” for a bit more on how we use tools like Agrobacterium, markers and promoters to assist in the transformation process.

  9. HealthyandCute

    Apples are one of the few fruits that can withstand the environnement. However, once fallen from the tree, they stand to decompose. The beauty of what we can see with Artic Apples is that this process isn’t visible right away. Due to genetic modification in the genome through biotechnology, engeneers have silenced the gene that is responsable for the expression of polyphenol oxidase. And no problem when it comes to decomposing because it will be visible. I beleive this is a major breakthrough that should be applied to other fruits that are brown and still fresh with a good smell just like other products made by nature. I am in the oil industry and what can be done for the apple could propably be done for the flowers keeping them viable longer and allowing us to process them in a more efficient way. @ http://www.biee-group.com/

  10. Anonymous

    Hello,

    can you please post a map or diagram of the construct used for transformation?  or refer me to a website with that information?  (just the CaMV35S::ppo portion).  I’m teaching my biology class about gene regulation via RNAi-silencing, and I think it would be timely to show how fundamental research into gene silencing can lead to applications.

    Thanks!

    • Joel

      Hi there – thanks for your inquiry!

      There are a couple good diagrams on our transformation in an article Chemical & Engineering News did in 2013 on our Arctic apples.

      We also have a few additional diagrams in the petition we submitted to the USDA requesting deregulation that may be of interest.

      Thanks for your interest and please don’t hesitate to let us know if there’s any other info we can provide that would be of help!

  11. Anonymous

    when growing these apples will cross polination come into the picture? when bees are using these trees will they change the genes of other flowers by bringing those genes into other flowers to make them into articapple flowers/other apple trees?

    • Joel

      Hi there – due to the use of biotechnology to introduce the nonbrowning trait into our Arctic® apples, they are indeed considered to be GMO. However, there should be no reason for consumer concern, as Arctic®  apples are likely the most tested apples in existence, and have passed through years of rigorous regulatory review.

  12. Anonymous

    Do you consider your apples to be a Gmo apple? there has been some bad news about gmos and was wondering how this would affect your company. 

    • Joel

      Thanks for your question!

      We’ve addressed cross-pollination directly in a few other posts that should provide some insight on this, most directly here. And, we also have an infographic describing apple propagation in a more general sense here

      Likely the most important point, though, is that even if cross-pollination were to occur, the resulting fruit would not be affected. Just as a Gala pollinating with a Fuji does not turn the Fuji into a Gala, an Arctic apple would not turn other apples into Arctic fruit!

  13. Erika

    I was just wondering if this nonbrowning trait would affect the cooking of these apples by various methods: baking, frying, or stewing. Is the texture of the flesh similar to that of a Golden Delicious or other yellow varietals? When do you estimate these will be available for public consumption in the U.S.?

    • Joel

      Hi Erika, thanks for your questions and apologies for the belated response!

      Arctic® apples would still experience the caramelization of their sugars when cooked using the methods you mention, though obviously there’d be less of a rush to get the apples in the oven, pan, etc. in order to avoid enzymatic browning from the flesh being exposed to the air.

      The “first bite” texture, taste, etc. on an undamaged Arctic® Golden would be the exact same as a conventional Golden Delicious. The only difference would be after the apple is bruised or sliced, and we actually did some taste testing to get consumers’ reactions in that case. 

      Regarding availability, we anticipate having small, test-market quantities of Arctic® apples available in 2017, with increasing amounts of fruit becoming more widely available each year going forward. The first two varieties will be Arctic® Golden and Arctic® Granny, with Arctic® Gala and Arctic® Fuji not far behind!

  14. Karen Loftus

    Do you know if the PPO that you removed from the apples had any other function, and could you provide a list of the phytonutrients / polyphenols that the apples do contain, such as those contained in this quote by G. Mateljan?: “Recent research has shown that apple polyphenols can help prevent spikes in blood sugar through a variety of mechanisms. Flavonoids like quercetin found in apples can inhibit enzymes like alpha-amylase and alpha-glucosidase. Since these enzymes are involved in the breakdown of complex carbohydrates into simple sugars, your blood sugar has fewer simple sugars to deal with when these enzymes are inhibited. In addition, the polyphenols in apple have been shown to lessen absorption of glucose from the digestive tract; to stimulate the beta cells of the pancreas to secrete insulin; and to increase uptake of glucose from the blood via stimulation of insulin receptors. All of these mechanisms triggered by apple polyphenols can make it easier for you to regulate your blood sugar.”

    • Joel

      Hello Karen, thanks for your excellent question!

      While Arctic apples have reduced levels of PPO, this change does not negatively impact the phenolic levels of Arctic fruit. In fact, the browning reaction involves a chemical reaction in which PPO interacts with apples compounds which actually “burns up” phenolic content! So, the nonbrowning trait helps Arctic apples better retain their phenolic levels.

      As for other roles PPO may serve, we wrote a post on that very subject earlier this year that we hope you’ll check out. Please don’t hesitate to let us know if you have any additional questions!

  15. Vicki Vance

    I am trying to understand what kind of silencing transgene you are using.  Is this published work?  Are the details of the construct available?  From the diagram you referred to, it seems that you are using sense versions of each of the four genes (it doesn’t say how much or what part of each gene is used).  In addition, there is no indication that this is a “hairpin” construct (i.e. one designed to make double stranded RNA directly).  So am I correct in assuming that your transgene is a sense transgene that has silenced by chance?  have you done any analysis of the siRNAs produced?

  16. Maria

    Hello, I find all the information is complete and quite educative. But I was wondering if the suppression transgene artificially synthesized?

  17. David Bowne

    People will do anything these days to complain, but when I get good service I will compliment it and this website helped me a lot on a research paper, and I just wanted to thank you.

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