I shouldn’t have to say this, but there are currently no genetically engineered tomatoes on the market. For a short time in the 1990s Calgene sold the Flavr Savr tomato in California grocery stores, but they weren’t able make a profit doing so, so they stopped. The poor taste of most tomatoes for sale in the grocery store today is purely the result of conventional breeding (my post on the subject and Mat_kinase’s).

I shouldn’t have to say this, but there are currently no genetically engineered tomatoes on the market. For a short time in the 1990s Calgene sold the Flavr Savr tomato in California grocery stores, but they weren’t able make a profit doing so, so they stopped. The poor taste of most tomatoes for sale in the grocery store today is purely the result of conventional breeding (my post on the subject and Mat_kinase’s).

Before getting into the how, let’s talk about why this research is important. According to Enhancement of fruit shelf life by suppressing N-glycan processing enzymes in this week’s PNAS, post-harvest fruit and vegetable softening is a big problem, with losses accounting for almost 50% of all produce in developing countries. India, the country that funded the research, and the world’s 2nd largest fruit and vegetable producer, loses 35-40% of produce to softening.

Squished tomato by limaoscarjuliet via Flickr.

We all know that post-consumer food waste is a big problem, and we can alleviate this somewhat in our homes and by choosing restaurants that try to reduce waste. But there isn’t much we can do about pre-consumer waste – from grain that rots in the silo due to fungus to tomatoes that rot in transit due to ripening. By reducing pre-consumer food waste, we can reduce the number of acres needed to produce the same amount of food. In India, preventing all fruit and vegetable softening would be like reducing the amount of land needed to grow fruits and vegetables by 35-40%!

So, how could that softening be prevented?

Researchers have been working for a long time on different parts of the ripening and spoiling process, trying to find ways to slow it down. Nothing has been really effective in getting produce to last longer, and we’ve ended up with produce that is more bland than it used to be, especially when it comes to tomatoes. In short, neither breeding nor genetic engineering has been successful… until now.

In Enhancement of fruit shelf life by suppressing N-glycan processing enzymes, Meli* and fellow researchers found two enzymes that contribute to fruit softening. The enzymes are α-mannosidase and β-D-N-acetylhexosaminidase, α-Man and β-Hex for short. Both of these enzymes break the glycosidic bonds between carbohydrates, as well as between carbohydrate and noncarbohydrate. The role of these enzymes in ripening and softening is to help break down the cell walls that keep the fruit firm. If the enzymes are stopped from breaking down the cell walls, the tomato stays fresh!

Meli and fellow researchers turned off the genes that code for these two enzymes α-Man and β-Hex with biotechnology, but they didn’t use any whole genes from tomatoes or any other species. Instead, they used some pieces of the tomato α-Man and β-Hex genes. These gene fragments are transcribed into RNA under control of the constitutive (always on) CaMV 35S promoter. They then twist and bind with themselves, resulting in double stranded RNA, which activate the RNA interference mechanism that plants and other organisms naturally use to combat double stranded RNA viruses.

The results are pretty striking, as you can see from these pictures. The control tomatoes were unappealingly wrinkly by 20 days, and rotten by 45 days. The tomatoes with α-Man or β-Hex turned off were still firm even at 45 days.

Control and experimental tomatoes over time, from the PNAS article "Enhancement of fruit shelf life by suppressing N-glycan processing enzymes" by Meli, et. al.

RNAi can be used just about any time you want to turn off a gene – it’s even being tested for human use to help combat genetic diseases. For an overview of RNAi that’s a little more detailed than the picture below, check out the RNA Interference interactive video by Nature Reviews (via ERV. Note: the video wouldn’t play on my Mac in Firefox but worked great in Safari).

Overview of RNAi from Huntington's Outreach Project for Education, at Stanford.

The researchers used Agrobacterium to carry the DNA sequences into very young tomato plants, along with a marker gene for kanamycin resistance. Biotech plants can be made without markers but it’s much easier to use them, and there is no risk (for more on antibiotic resistance markers, see GMO Compass).

This work, as far as I can tell, is funded purely by the Indian government – not by private corporations. Specifically, it is funded by the Department of Biotechnology which is part of the Ministry of Science and Technology. They have some pretty impressive goals, as listed in the Plant Biotechnology section, including:

• Genetic engineering and molecular biology tools for forest tree improvement including reduction of generation time, production of horticultural and plantation crops with desired characteristics.
• Transgenics for improved yield, stress tolerance, balanced nutrition, keeping quality of flowers, fruits and vegetables, better nutrient and water utilization capacity should be produced.
• Cataloguing of accessions of wild and land races to study genetic diversity for resolving taxonomic problems.

Tomatoes at Union Square by Lindsay Beyerstein via Flickr.

One of the biggest arguments against biotechnology is that it has been under corporate control. Unfortunately, that’s been true in the United States, where publicly funded research in agriculture has been all but ended. Happily, that’s not the case in India and China. These governments are researching biotech traits for the benefit of their farmers, not for the benefit of shareholders.

If this biotech trait is available royalty-free, then it will presumably be available for breeding by small seed companies and by farmers. I’m imagining beautiful genetically-diverse heirloom tomatoes that have this amazing ability to stay firm on your counter well past the tomato growing season. This means that fewer tomatoes will need to be shipped around the world, and that more can be grown locally. I hope to see some long-lasting tomatoes in my CSA share soon!

* You may have noticed that I usually use the name of the first author rather than the name of the last author when I’m referring to a peer-reviewed paper. In biology-related papers, the first author is the graduate student, or sometimes post-doctoral researcher, who did most if not all of the labwork and writes most if not all of the paper.The last author is the PI (Primary Investigator), who generally provides guidance, helps with experimental design, and edits the paper. The authors in the middle are usually other grad students and their PIs who helped with the project. While all of the authors usually have put in a lot of time and effort, it’s that first author who worked the hardest, and I like to recognize that.

Meli V, Ghosh S, Prabha T, Chakraborty N, Chakraborty S, & Datta A. (2010). Enhancement of fruit shelf life by suppressing N-glycan processing enzymes Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0909329107

Potentially promiscuous pollen from corn tassels by circulating via Flickr.

Many people, including me, are concerned about potential harm to crop biodiversity from gene flow. Most people’s concern focuses on transgenics. There is a certain probability, albeit small, that transgenes will end up in the progeny of non-transgenic plants, weedy relatives of the crop, or wild relatives that grow nearby due to pollen flow. Transgenes can also be moved from place to place by accidental or purposeful movement of seeds.

How much transgene flow is actually happening is a subject of some controversy, but what about gene flow between non-transgenic plants?

There is potential for problems whenever plants that aren’t supposed to cross stray from their intended mates. Some things to think about include how gene flow happens at the field and genetic levels and what characteristics of the genes themselves can affect permanence of contaminating genes once they get into a variety they shouldn’t be in.

Gene flow with transgenes can help us to think about gene flow of “regular” genes

In the 2004 paper Gene Flow from Cultivated Rice (Oryza sativa) to its Weedy and Wild Relatives, Li Juan Chen showed that a marker gene “flowed” in their test field from transgenic cultivated rice  to weedy rice at rates between 0.011 and 0.046 % and to wild rice at rates between 1.21 and 2.19 %. The marker gene Chen used is called bar, which is easy to screen for because it makes plants resistant to the antibiotic and herbicide biaphalos. Just spray the progeny and you’ll know if they’ve got the gene. Chen confirmed presence of the bar gene with PCR. These rates seem pretty low, but rice is mostly a self-pollinator, and the pollen is very short lived. If out-cross rates in rice reach 2.19 % we could expect to see rates even higher in other species. This tells us that transgenes can be passed to weeds, but also, more broadly, tells us that any gene can be passed from cultivated rice to weed rice.

Gene flow could be a problem in the opposite direction as well. In the 2009 paper Gene flow from weedy red rice (Oryza sativa L.) to cultivated rice and fitness of hybrids, Vinod Shivrain showed that progeny of a cross between weedy red rice and cultivated rice were more successful if their mother was the cultivated plant. These hybrid grains can fall back to the field on accident or be collected and planted the following year with the regular seed. Either way, the rice farmer now has rice plants that don’t have all of the desired characteristics of the cultivated rice. The plants will have at least some genes from the weedy rice that could help it out compete the desired rice plants but produce less grain. This paper shows that gene flow from weeds to crops can happen, and that it can be a problem.

Maize, unlike rice, is a promiscuous out-crosser. The pollen is heavy and still fairly short lived, so mostly pollinates plants that are nearby, but wind-carried pollen and stray seed can carry transgenes away from their intended fields. The story of transgenes in landraces of maize is summed up beautifully in the 2007 paper Gene flow from transgenic maize to landraces in Mexico: An analysis (pdf). Kristin Mercer tells us that research on the subject has had mixed results. Transgenes likely do exist in landraces in Mexico, but the extent of the “contamination” is not as wide as some researchers have proposed. Some of Kristen’s other research focuses on how crop alleles move in wild sunflower populations. The sum of her research is that we can expect gene flow back and forth between any compatible plants: wild, weedy, cultivated, transgenic, landrace.

Gene flow’s effect on biodiversity

Image of corn plant by University of Nebraska Lincoln, adapted by Anastasia Bodnar. All other images in this post by Anastasia Bodnar.

Understanding the impact of gene flow on biodiversity (or more appropriately, crop diversity) requires some understanding of what happens at the genetic level. I like to sit down and draw pictures to help me think about genetics. I hope it helps some Biofortified readers as well!

The image to the right shows two hypothetical varieties of corn. On the left is a modern inbred variety. All the plants are identical. There is no or low genetic variability within the inbred, because there is only one version of each gene present in the variety. On the right is a landrace or heirloom variety. All the plants are different from each other to some degree. There is high genetic variability within the landrace because there can be many versions of each gene present in the variety.

Below  is a (very) simplified view of what happens at the chromosomal level when an inbred is crossed with a landrace (in a hypothetical crop with one chromosome). Note: a hybrid or even an open pollinated variety could be substituted for inbred here, it was just easier to use an inbred. Similarly, a wild variety could contaminate a landrace. One landrace can contaminate another. One inbred could contaminate another. Weedy relatives can contaminate crops. Crops can contaminate wild varieties… you get the idea.

In the inbred (red), the two sister chromatids for each chromosome are identical to each other. There is only one version of each gene in the inbred, also known as two copies of the same allele. In the landrace (blue), the two sister chromatids are different from each other. For each gene in the landrace, there can be two different alleles. The different shades of blue indicate different alleles for some genes on the sister chromatids.

Imagine a situation where a field with the inbred is right next to a field with the landrace. Pollen will flow between the fields (to some degree – depending on weather conditions, pollen size, and tons of other factors). If the inbred and the landrace are crossed (whether pollen from the inbred fertilizes the landrace or vice versa), each of the offspring will have about half of the genetic information from the inbred and half from the landrace. Since the two chromatids are the same for the inbred, none of the information from the inbred is lost in any individual. Since the two chromatids in the landrace individual are different, each of the offspring only receive half of the genetic information from the landrace.

When those offspring make gametes, recombination often occurs which results in chromatids that contain some alleles from each grandparent. Crossing over, shown here, is one type of recombination. If those gametes then combine with the inbred, their progeny will only have about 1/4 of its genes from the landrace grandparent.

Genetic diversity can be lost in certain situations. For example, if a farmer growing a landrace finds plants in the field that have positive traits, the farmer will choose to plant those seeds for the next year. If those beneficial traits are due to genes from the inbred, the farmer could effectively select for plants with one or more genes with the inbred and against plants that don’t contain any genes from the inbred. If pollen from the inbred is reintroduced year after year, the farmer could plant seeds from those plants that contain more and more alleles from the inbred variety, and alleles from the landrace could be lost over time.

On the other hand, if the farmer chooses seeds from plants that look more like the landrace, then alleles from the inbred could be lost fairly quickly. If pollen or seeds from the inbred are introduced infrequently, the landrace would maintain a low level of alleles from the inbred, with those alleles eventually disapearing.

Of course there are many situations in between, and those depend greatly on what effect each gene or allele has on the plants they have contaminated.

Once it’s in there, how long will it stay?

Transgene or not, wild or cultivated, all of the genetic material goes into a big mixing pot to be stirred by random mating and natural selection in the case of wild plants or by breeding and artificial selection in the case of cultivated plants. One of Kristen’s points in Gene flow from transgenic maize to landraces in Mexico: An analysis (pdf) is that the permanence of transgenes in a non-transgenic population depends a lot on what the transgene is exactly, and the same idea applies to non-transgenic alleles.

To break it down: Any given transgene or any allele of a gene can have one of three effects on the plant: positive, neutral, and negative. The effect depends on what plant the allele is contaminating and what trait is conferred by the allele. Finally, how long a contaminating allele stays in a population depends on all of these factors.

Positive

Some alleles would be beneficial in almost any situation. Herbivore resistance, including genetically engineered Bt toxin and increased expression of non-transgenic chemical defenses, would help both cultivated and non-cultivated plants escape damage from susceptible herbivores. These types of transgenes and alleles would be likely to persist in any population they contaminated. These would definitely be bad traits to have in weeds. They could be desirable in a landrace from a farmer’s point of view.

Neutral

A gene that increases the size and number of fruits produced by a plant is desirable from an agricultural perspective, but could have a negative effect a wild plant, because the plant would have less resources to devote to other needs like herbivore defense and drought tolerance. These types of alleles will not persist in a wild population, but could persist in a landrace if it is seen as desirable to farmers.

Negative

Alleles or genes that are specific for certain farming systems won’t persist in wild populations, weeds, or landraces unless they are exposed to those farming conditions. These include genetically engineered genes like glyphosate tolerance and the non-transgenic allele for Clearfield tolerance. If these alleles or genes contaminate a population but that population is never sprayed with the chemical, there is no selection pressure to keep the trait.

Of course these are just three examples of different traits and there are thousands if not millions of traits out there that might have different effects, but you get the idea.

Every day, pollen blows and seed is moved. Every day, genes and alleles are transferred from one plant population to another, no matter if they are transgenes or not. Those naughty plants just won’t keep to themselves! If we are truly concerned about gene flow, we really should be considering gene flow from all sources, not just transgenic crops.

Chen LJ, Lee DS, Song ZP, Suh HS, & Lu BR (2004). Gene flow from cultivated rice (Oryza sativa) to its weedy and wild relatives. Annals of botany, 93 (1), 67-73 PMID: 14602665

Shivrain VK, Burgos NR, Gealy DR, Sales MA, & Smith KL (2009). Gene flow from weedy red rice (Oryza sativa L.) to cultivated rice and fitness of hybrids. Pest management science, 65 (10), 1124-9 PMID: 19530257

Mercer, K., & Wainright, J. (2008). Gene flow from transgenic maize to landraces in Mexico: An analysis Agriculture, Ecosystems & Environment, 123 (1-3), 109-115 DOI: 10.1016/j.agee.2007.05.007

Eating locally is great, but since apples only ripen once per year, and they spoil relatively fast, that means we only have fresh apples for a short time each year. That’s too bad, since apples are a wonderful crunchy snack loved by kids and adults that provide health benefits from their fiber and antioxidants.

Shipping the apples from another place (like New Zealand) extends the time that apples are available, but shipping in refrigerated containers is expensive and results in greenhouse gas emissions, and we all know that those apples from far away just don’t taste as good as local ones.

Scab Resistant Selection RS103-130. Image from "Organic Production of a New Australian-bred Scab Resistant Apple in Queensland, Australia" by Middleton, et. al

There might be a way to have local apples available for a much longer time, as well as to have apples shipped in that use less energy and less pesticides!

After more than 20 years of work, researchers in Australia have developed apples that are resistant to black spot aka apple scab, a fungus that destroys fruit and leaves. The scab resistant line, called RS103-130, also stays fresh and crunchy much longer than typical apple lines. They achieved this through some initial crosses with a crabapple species followed by years of selective breeding. The crabapple provided RS103-130 with the Vf gene complex, which has been previously used to produce transgenic scab-resistant apples, which I’ll describe in more detail shortly. You can find the Australian patent for RS103-130 at FreePatentsOnline.

In 2005 and 2006, comparison experiments showed RS103-130 to have many benefits over Galaxy, a typical non-resistant cultivar (see chart below). According to Middleton, et. al, RS103-130 has off white flesh and medium texture, is crisp, sweet, low-acid, and juicy, with a mild flavor.

Chart from "Organic Production of a New Australian-bred Scab Resistant Apple in Queensland, Australia" by Middleton, et. al.

Because of all of these benefits and the reduced pesticides needed, organic apple growers in Australia are very interested in RS103-130. I wasn’t able to find any information on whether RS103-130 has been commercialized yet, or on how long it might be before I can try them. Apparently something happened with RS103-130 lately, because stories appeared in The Independent and in the New York Daily News last week. Neither of the stories say what prompted the coverage, nor does Treehugger, which picked up on the 1st two. If you know what’s new with these apples, please comment!

My first question upon reading these articles was: why has it taken twenty years?! Selective breeding can be painstaking, especially when you’re talking trees. There is a faster way…

The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety showed that the Vf gene can be inserted with biotechnology into apple varieties (in this case, the gene was inserted by Agrobacterium tumefaciens into the Gala apple cultivar). In the introduction of this paper from 2003, Belfanti et. al point out that:

the transfer of these genes by classical breeding to cultivated apples is difficult because of the long juvenile phase, self-incompatibility, and the impossibility of exactly reproducing the heterozygous state of cultivated varieties. Starting from the wild species Malus floribunda 821 carrying the Vf gene, breeders have developed several scab-resistant apple cvs. (2), but not one has met with commercial success. Indeed, when compared with such commercially popular cvs. as Golden Delicious and Gala, the main horticultural and fruit-quality traits of these scab-resistant cvs. are notably different and undoubtedly less acceptable.

Using biotechnology, the researchers were able to confer scab resistance in one generation. In this paper, the authors don’t mention any increase in lifespan for the fresh apples – I’ll look on Web of Science for more info tomorrow. I do appreciate that the authors are hopeful for the future of apple biotech.

The cloning of an apple scab resistance gene represents the basis for further investigation of the resistance mechanism. It also represents a step toward a gene therapy (restoring resistance where lost) of the scab-susceptible cvs. that currently dominate the apple industry. This strategy will allow the transfer of resistance from a wild apple species to any commercial apple genotype while maintaining the horticultural and fruit-quality traits growers and consumers prize most. It may also be possible to achieve greater resistance durability by the simultaneous transfer of several resistance genes from wild apple species. Going one step further, it may be possible to use apple promoters and novel techniques that, by eliminating selective marker genes (38, 39), generate transgenic varieties without any foreign genes and, hence, may make genetically modified plants more acceptable to growers and consumers alike.

I’m particularly interested that Balfanti et. al mentioned cisgenics, although they didn’t use the term. There is potential to insert genes like Vf into many varieties of apples, meaning that cultivars developed for specific microclimates may be quickly made resistant to scab (and potentially given a longer shelf life) without any loss of their other traits. This is a good example of how biotechnology and breeding can have the same results – get a gene into a cultivar – although one takes much longer than the other.