How to Interpret the Test for the Presence of Genetically Engineered Traits in Papaya Seeds

By Martha Crouch, Ph.D., mcrouch@bluemarble.net

The genetically engineered papayas being grown in Hawaii ('SunUp' and 'Rainbow' varieties) are designed to be resistant to papaya ringspot virus.  These varieties have genes that direct the synthesis of three new proteins:  1) the coat protein of the ringspot virus, which causes the papaya to defend itself against further viral infection, 2) a bacterial enzyme that gives the papaya cells resistance to certain antibiotics, which is used in the tissue culture process during development of genetically engineered crops, and 3) a bacterial enzyme, beta-glucuronidase,  that can be used to tell whether cells or plants have been genetically engineered.

These three genes are engineered close to one another on the DNA, and almost always enter the papaya chromosome together during the genetic engineering process.  Thus testing for any one of the proteins will indicate the presence of the other two.

The test we are using  (called the GUS test or assay) detects the beta-glucuronidase protein.  Plants such as papaya do not naturally have this enzyme, so if we detect the protein, we can assume that the papaya is genetically engineered. 

How does the test work?
In this test, we soak papaya tissues in a solution containing a blue dye bonded to glucuronic acid.  The dye is designated as 'X', and the glucuronic acid is abbreviated 'gluc'-thus, it is called 'X-gluc', and rhymes with 'duke'.

X-gluc is actually a colorless liquid.  If the gluc bond is cleaved, releasing the dye, a blue color is formed.  The enzyme beta-glucuronidase can cleave the bond.  Thus, if the tissues soaked in the colorless X-gluc solution have not been genetically engineered, the tissues will remain colorless.  However, if the tissues have been genetically engineered, they will contain the enzyme, which will cleave the gluc away from the dye, and the tissues will turn blue.

What parts of the papaya plant are tested, and why?
In order to see the blue color, it is best to test papaya tissues that are very pale.  Also, the tissues should be young and actively making proteins.

The embryo dissected from inside the seed has been shown to work well in this test.   The black seed coat seems to inhibit the formation of color, so it is removed.  Also, very young leaves from the top of the tree can be used.

The tissues are wounded a bit in the X-gluc solution, to be sure that the dye gets inside of the cells.

Also, the tissues are examined for blue color within a few hours.  If it sits long enough for bacteria grow in the solution, the bacteria might make beta-glucuronidase and cause blue color to form, whether or not the tissues are genetically engineered.

Embryos from an engineered variety, such as 'SunUp' are always included in each test, to show that blue color develops if the enzyme is present (positive control).  Embryos from a previously tested, non-GE papaya are also included, to show that the solution remains colorless if the enzyme is absent (negative control).

What does it mean if the papaya tissues being tested do NOT turn blue?
If your papaya tissues do not turn blue, they do not have the beta-glucuronidase enzyme, and thus probably do not have the virus coat protein gene and antibiotic resistance gene either. 

If you tested embryos from several seeds, and none of them turned blue, you can be fairly certain that the parent tree is free from genetic engineering.  If the parent was genetically engineered, it would carry the engineered gene on at least one of the chromosomes, and at least half of the embryos would inherit it. 

Rarely, the genes may be present but not making the enzyme.  Or, in very rare cases, the enzyme gene may have become separated from the other two genes.  If you need to be absolutely certain that the tree is not genetically engineered, you would have to do a direct test for the presence of the specific DNA sequences in the gene.

What does it mean if the papaya tissues being tested DO turn blue?
If the tissues turn blue (and the negative control does not), then you can assume that the tissues contain the genetically engineered enzyme, and therefore also the genes for the viral coat protein and antibiotic resistance.

If you are testing embryos, and all of them turn blue, the fruit itself and thus the whole tree is probably genetically engineered.  To confirm this, also test a very young leaf.  It should also turn blue.

If some fraction of the embryos turns blue, either the tree is engineered, and the gene is segregating in the offspring, or there is cross-pollination of a non-engineered tree by engineered pollen.  Cross-pollination is more likely if the fruit is from a female tree rather than from a hermaphrodite.  Again, testing a leaf will help distinguish between the possibilities.

'SunUp' has two copies of the engineered gene, one on each of the affected chromosomes.  All of the embryos in seeds from 'SunUp' will get one engineered gene from the mother tree, and all will turn blue in this test.

'Rainbow' is a hybrid, and has only one copy of the engineered gene.  Only half of the embryos in seeds from 'Rainbow' will get the engineered gene from the mother tree.  Depending on where the pollen comes from, half to all of embryos in 'Rainbow' fruits will turn blue (half if the pollen all comes from a non-engineered plant, three-quarters if it self-pollinates, and all if it is pollinated by only 'SunUp' pollen.)

However, if you have a non-engineered tree and you see blue embryos, they must have come from cross-pollination, and any percentage is possible.

What about using the seeds from the tree with colorless embryos to start GE-free seedlings?
Unfortunately, this test is not very good at detecting low levels of cross-pollination by genetically engineered papayas.  There are many fruits on a tree, and hundreds of seeds in each fruit, and each seed was fertilized by a different pollen grain. 

To get GE-free seeds for planting, it is a good idea to use hermaphroditic flowers from a tested tree, and to bag them, so that they self-pollinate.
 

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