Breeding Strategies for Improving Shelf Life in Tomatoes

Tomato is one of many plants that have evolved an “edible fruit” strategy for seed dispersal.  Mature seed is encased in a fruit designed to be attractive for consumption by fruit eating animals.  Seed dispersal occurs when the consumed seed passes safely through the digestive tract and is deposited with feces on the soil some distance from the mother plant.  In tomato the fruit ripening process involves several steps designed to enhance attractiveness for consumption:  an increase in fruit sugars, acids and flavor-enhancing aromatic compounds that greatly improve tastiness of the fruit; fruit softening to a more edible texture; and obvious fruit pigmentation designed to signal to passing animals that the fruit is fully ripe and ready to eat.  These features were preserved during the domestication of tomato and the more recent development of tomato as one of the world’s most important fruit/vegetable crops.

One of the modern dilemmas in tomato production and breeding relates to managing post-harvest losses associated with the modern agricultural practice of concentrated fruit production in one area and fruit consumption in another place (and time).  Ripe fruit is easy to damage in transit and deteriorates relatively quickly.  Picking mature green (MG) fruit for shipment and gassing with ethylene at a distant delivery point to “ripen” the fruit solved the problem of damage in shipping, but comes with an unfortunate sacrifice in flavor.  As an alternative to this practice plant biologists and tomato breeders have looked at various genetic variants (mutations) in genes controlling the ripening process, and examined how these novel alleles might be deployed in the development of varieties with great flavor and enhanced shelf life.  In this post I’ve tried to summarize the current understanding of this field and share some of our related breeding efforts.

Tomato Fruit Development (from Alba et al., 2005)

 

The Ripening Process

Tomatoes are a climacteric fruit, which means that the plant hormone ethylene is required for fruit ripening.  Ethylene is rapidly produced in tomato fruit at the breaker (BK) stage and drives a series of reactions that together define the fruit ripening process.  During normal ripening there are simultaneous and independent processes that lead to 1) accumulation of sugars, organic acids and volatile organic compounds influencing flavor, 2) conversion of chloroplasts to chromoplasts and the synthesis and accumulation of carotenoid pigments and 3) softening of the fruit.  In a perfect modern tomato, ripening steps 1&2 proceed normally and step 3 proceeds at slow rate – allowing the tomato fruit to keep peak flavor, color and texture for an extended period of time.

ESL, or extended shelf life, is a term describing a collection of traits that together extend the potential time between picking of fully ripe or nearly fully ripe fruit, and the deterioration of fruit quality.  Fruit quality deterioration is usually associated with fruit softness/undesirable texture and fruit rotting.  Taste panels have identified fruit texture as an important determinant in consumer preference, and soft or mealy fruit is a major “turn-off”.  Deterioration in fruit firmness/texture is generally associated with a ripening related spike in polygalacturonase (PG) and other enzymes that degrade fruit cell wall polysaccharides. Thus, a decline in fruit firmness typically coincides with dissolution of the middle lamella and hemicellulosic/pectic cell wall polysaccharides, thereby undermining the polysaccharide network that hold cells together in the fruit pericarp.  FlavrSavr tomato, the commercially unsuccessful GE trait introduced by Calgene in 1985, was designed to specifically suppress PG activity in ripening tomatoes.  Recent research has also implicated cuticle composition and architecture as traits influencing ripening-induced fruit softening (Saladie et al. 2007 and Kosma et al. 2010).  The cuticle has long been implicated as a contributor to fruit strength, and cuticle structure changes during the ripening process.  Kosma et al, show that during the ripening process ESL mutants generally have cuticles with mechanical properties significantly different than the wild type – likely contributing to ESL per se.

It should be noted that independent of the several novel mutant alleles described below, there are significant genetic differences in firmness in tomatoes.  Unfortunately there are a couple of studies that report fruit firmness at harvest is not well correlated with the maintenance of fruit firmness postharvest.  We have found that pericarp thickness, relative to size of the locules, is a heritable trait that significantly impacts firmness per se, and appears in many cases to be associated with improved shelf life (see photos below).  This combination of traits is common in many newer commercial hybrids.

Firm when ripe phenotype

There are several mutations in key structural or regulatory tomato genes that affect the ripening process.  These genes generally either inhibit ethylene synthesis and/or modify ethylene’s downstream effects on specific biochemical processes related to fruit ripening.  To better understand climacteric fruit ripening per se, and to examine the potential utilization of these mutant alleles for delayed ripening/extended shelf life – tomato scientists have characterized several mutant alleles associated with a delayed ripening phenotype.  Several key ripening mutants are described in detail below.

Key genetic mutations affecting tomato fruit ripening

rin = ripening inhibitor.  The RIN gene is a transcription factor that acts as a master regulatory gene controlling numerous genes and pathways associated with tomato fruit ripening.  The rin loss of function mutant is a recessive allele that both represses genes associated with ethylene synthesis and modifies downstream processes associated with the normal ripening process.  Specifically rin modifies expression of other transcription factors associated with fruit ripening (e.g. NOR); prevents normal fruit pigmentation by suppressing synthesis of Phytoene synthase (PSY), the primary enzyme regulating flux into the carotenoid pathway (see Genetic Control of Fruit Color in Tomatoes); suppresses key steps in the accumulation of sugars, organic acids and aromatic compounds associated the improved flavor in ripe tomato fruit; suppress enzymes (e.g. polygalacturonase = “PG”) associated with breakdown of cell wall polysaccharides that lead to ripening-related fruit softening; and modifies cutin and fruit wax content and composition.   The rin/rin homozygote plant produces fruit that never fully ripen and have much firmer fruit with a significantly longer shelf life (see photo below).  The lack of normal color and flavor significantly limits commercial potential of rin/rin plants.  In the heterozygous condition rin/+ plants produce fruit with near normal fruit color and flavor, and shelf life that is intermediate between rin/rin and +/+ (wild type) plants. F1 hybrids with the rin/+ genotype and extended shelf life have been widely commercialized and are a key driver in the recent availability of “vine ripened” tomatoes in grocery stores.  The extended shelf life allows picking at or near the full ripe stage when flavor is near peak, and remaining firm for an extended period of time for shipping to distant locations.

We have been developing and testing new rin/rin inbreds and rin/+ hybrids for the last few years. 

Striped rin/rin cherry

Although rin/rin lines generally have very low fruit sugars, there are differences in sugar levels between rin/rin lines.  The sweetest rin/rin lines generally produce the sweetest rin/+ hybrids, though this is also heavily influenced by the non-rin parent in the hybrid.  Lycopene levels in rin/+ hybrids is a little lower than wild type (orange/red vs dark red), but normal red color can be restored in ogc/ogc crimson types (e.g. Mountain Magic).  Enhanced shelf life in rin/+ hybrids appears to be influenced by rin per se, but also on the genetic background of the rin and wild type parents, specifically those genes influencing fruit firmness.  Ripening is a little slower with rin/+ hybrids, adding perhaps 5-7 days.  We are making great progress on rin/+ hybrids and it appears possible to combine a significant improvement in shelf-life with exceptional flavor in fruit in a wide range of colors, shapes and sizes. 

Fruit at BK +7 stage (7 days after breaker stage in the WT)

                      Wild Type                     rin/rin                        nor/nor

Photo by Martel, 2010

nor = non-ripening.  The NOR gene is an unrelated transcription factor that also serves as a master regulator of fruit ripening in tomato.  The recessive loss of function mutant allele nor has been widely studied.  The nor/nor homozygote has a very similar phenotype to rin/rin, and nor/+ hybrids also have much restored color and flavor with extended shelf-life – though reports in the literature suggest less color and flavor and longer shelf life in nor/+ relative to rin/+.  The specific mechanisms for modification of ripening in nor mutants is less understood than with rin – but like RIN, NOR helps regulate multiple genes and pathways important in tomato fruit ripening.  Commercial nor/+ hybrids have been commercially successful, though probably less so than rin/+.  Note that the next few mutants described here, alc and dfd, are thought to be allelic to nor (i.e. independent NOR mutants) with subtle but significant differences in ESL phenotypes.

alc = alcobaca.  The Spanish tomato landraces Alcobaca, Penjar and Tomàtiga de Ramellet are generally “long keeping” types with much delayed fruit deterioration.  These landraces have been selected for hundreds of years for local adaptation to a dry climate and for fruit that will have acceptable quality for months after harvest.  The photo below shows a typical fall/winter storage strategy employed in the region – fruit are hung in small bunches for medium term storage.  Note the term tomatiga de penjar means tomato for hanging.  There is a single recessive allele “alc” associated with the slow ripening phenotype.  The alc allele is believed to be another mutation at the NOR locus.  Fruit from alc/alc plants have significantly lower levels of endogenous ethylene, suppressed polygalacturonase activity and firmer fruit.  Fruit harvested at the onset of ripening mature to an orange color, and those left on the plant until full ripening have normal red color.  The landraces listed above are all alc/alc and can remain firm for several months, though there is wide variation for this LSL trait within local populations – suggesting alc + other factors are at play.  The ESL trait associated with alc also appears to be subject to the level of water stress during fruit production – with generally enhanced ESL under more arid production conditions.  Hybrids that are heterozygous for alc (alc/+) have shelf life intermediate between +/+ and alc/alc, and may be another interesting candidate gene/allele for an extended shelf life tomato

Alcobaca type tomatoes hung for winter storage

                                                 

Effect of alc on fruit deterioration

dfd = delayed fruit deterioration.  The dfd trait was first found in certain ecotypes growing in the southern Mediterranean.  The literature suggests that dfd is a partially dominant mutant allele of NOR, and may indeed by identical to or a slight variation to alc.  DFD controls cuticle composition and leads to decreased cell water loss, increasing cell turgor (firmness) per se, and decreasing fruit water loss generally during ripening.  Normally as tomato fruit ripen the cuticle weakens and grows less resistant to penetration.  Fruit of dfd plants require significantly more force for cuticle penetration than those from wild type varieties, and do not exhibit a normal progressive weakening of the cuticle during ripening.  Fruit from dfd plants exhibit the normal ripening-induced fruit cell wall breakdown and cell separation typical of wild type, but show substantial swelling of pericarp cells during the ripening that is atypical, with a ~4x increase in cell size vs wild type in ripe fruit, likely related to increased cell turgor.  There is also less fruit water loss in dfd vs wild type ripening fruit – another contributing factor to improved fruit firmness.  Increased cell turgor, decreased fruit moisture loss and increased cuticle strength all appear to be related to changes in cuticle wax content and composition in dfd vs wild type.

 

Unlike rin, and nor, dfd’s affect on fruit firmness/LSL was independent of normal fruit coloration and ripening-related accumulation of sugars and organic acids.  Futhermore dfd/dfd plants maintained firmer fruit without impacting expression of genes, such a PG, involved in ripening induced cell wall degradation (unlike alc).  The dfd mutant appears to represent a very novel approach for ESL that may be used in combination with other ESL traits to enhance shelf life in tomato hybrids or O.P. varieties.

Changes in Fruit Coloration after Breaker Stage

Fruit Shelf Life of Nine LSL Tomato Hybrids (Yogendra et al. 2013)

Nr = never ripe and Gr=green ripe.  These are dominant, gain of function mutations at independent loci, that each results in reduced ethylene responsiveness in tomato fruit tissue.   The ethylene insensitivity in both Gr and Nr have a negative impact on seed germination and seedling vigor and completely prevent normal fruit ripening.  Negative plant and fruit phenotypes prevent any commercial use of these mutant alleles.

Summary

Although the mutant alleles rin, nor and alc generate a somewhat similar ESL phenotype in plants heterozygous for these alleles, rin and nor are independent loci and all three have different modes of action. With all three alleles, extended shelf life is associated with later maturity, and with rin and nor also associated with decreased pigmentation (see photo above).  The mutant alleles of these three genes have a similar effect on extending shelf life, and the maintenance of firmness is due both to the mutant alleles per se, and the background genotype of both the male and female parents.  We have found that a rin/+ genotype in a firm fruited background can extend shelf life for over two weeks.  In such a case a fruit picked fully ripe can stay crisp and firm on the countertop (or in transit to local or distant markets) for at least 14-21 days.  Since several of the key aromatic compounds impacting flavor are directly derived from lycopene and other carotenoid pigments, in theory one might expect that the lower carotenoid pigment content of rin/+ hybrids might lead to lower flavor.  However by selecting ruthlessly for flavor in parent lines, we have been able to identify rin/rin parents that contribute high flavor to rin/+ hybrids. 

It is currently unclear how closely related are the NOR mutants nor, alc and dfd.  The dfd mutant is somewhat of a mystery, perhaps due to a Cornell patent filing on a specific dfd sequence – in the patent they do describe this as a NOR mutant derived from a Mediterranean ecotype.  To complicate matters more a Davis, CA company Arcadia has patented an induced mutation in NOR (reference), which they claim to be an improvement on the naturally occurring nor loss of function mutant.  It is too early to know how similar the Arcadia mutant might be to alc or dfd.

The primary use of extended shelf life (ESL) tomato hybrids will likely be for medium/large size grower (field or protected culture) producing for distant markets.  Picking an ESL hybrid at or just before full ripening (in the marketplace = vine ripened) then packing and shipping, can be a consumer and taste-friendly alternative to the traditional “green and gassed” model.  We think ESL types will also be well suited to smaller producers selling in more local markets.  These types could be picked less frequently, and once picked, be much less prone to post harvest losses.  It appears there may be several different gene/allele options for ESL, with varying efficacy, ease of use, and freedom to operate.  We think ESL will be an increasing important trait for fresh market tomatoes, with perhaps evolving breeding strategies for optimization of the trait.  We will build on our early success with rin, and continue to follow and explore the other options described here.  Our multi-year effort in selecting for fruit firmness and flavor per se is paying off – deployment of rin or one of the NOR mutants will likely require a firm fruit background for optimization of ESL, and a high flavor background will likely be needed to counter the delayed ripening effect of rin/+, nor/+,  or alc hybrids.

Crosses Between Cherry and Beefsteak Tomatoes

Whether a new cross is designed to be a F1 hybrid, which is sold as such commercially, or a breeding start for a program to develop new inbred/O.P. lines, selection of parents is critical.   Most breeders will select parents that complement each other, so that in the F1 (or F8) generation you capture the best characteristics of each parent.

We have now made and tested several dozen crosses between beefsteak and cherry types – hoping to capture some of the exquisite taste of the best cherries (think Sungold) in a larger fruited type.  We have also used early fruiting cherries to get earlier maturity in these hybrids and hybrid-derived Fx progeny.

There are some general patterns that we find interesting:

In crosses between smaller x larger fruit, the fruit size in the F1 generation will be much smaller than the mid-parent mean.  My tomato friend Bill Jeffers told me many years ago that fruit size in hybrids could be estimated using the mean of the square root of the fruit weight of the parents – for example (1oz x 9oz) –-> (√1 + √9)/2=2oz fruit wt for the F1.  I’ve found this to be a pretty good rule of thumb.

   

     In the F2 generation we have found almost the whole range of variation in fruit size, but heavily skewed toward smaller fruit.  The frequency distribution typically looks something like this: 

In this example, again the parents were a 1oz cherry and a 9oz medium beefsteak.  You need to look at a lot of F2 plants to find one that comes close to capturing the size of the large fruited parent.

The frequency distribution for maturity (i.e. earliness in fruit set) is similarly skewed toward the early parent, but in these F2 populations earliness and fruit size are negatively correlated – generally small fruited types are early, larger fruited types are later.   Thankfully we have been able to get earlier large fruited types by selecting primarily for size in the F2 and then for size and earliness in the F3 – again using large populations in both generations.

Our "go-to" cherry parent - derived from Sungold

Much of our recent crossing involves commercial hybrids (e.g. Bella Rosa, Mountain Merit, Panzer and Tasti-Lee) with exceptional disease resistance as the large fruited parent and very flavorful striped cherries as the small fruited parent, so we are also selecting in the F2 and F3 for flavor, stripes, plant health and fruit quality.  So far we are making progress on all fronts.  

Fruit from F5 line derived from cherry x beefsteak cross

Deconstructing Flavor in Tomatoes

I’ll start with this caveat: growing conditions can have a huge influence on taste/flavor in tomatoes.  Fruit harvested early or late in the season is usually inferior in flavor compared with those harvested mid-season; high flavor is often favored with plants in light drought stress; and growing area, soil type/fertility and disease incidence/plant health can significantly effect fruit taste.  Perceived “likeability” is also often confounded with non-flavor fruit quality attributes, such as color, texture and skin thickness.  For example, most folks struggle to see anything that is not red and round as a proper tomato.  We have used blind taste tests (literally – I’m talking blindfolds) to get a non-biased flavor assessment on something like GWR bicolors.  Taking off the blindfolds is fun, and informative.  However, there is a genetic component to flavor per se that is important, and subject to modification through plant breeding.

Knife at the ready

In our tomato breeding program, selection for flavor has always been primary.  We taste several thousand tomato plants in our breeding nurseries each year, and it is generally not possible for us to do anything other than apply a simple subjective selection criterion – select what you think tastes good.  We can manage that in our moderately sized breeding program – large companies cannot.  In an effort to perhaps apply more objective criteria and to design more efficient breeding strategies, we’ve been reading more on what is known about the biochemical and genetic basis for tomato taste and flavor

We have conducted tomato tastings at Frogsleap Farm for the last 5-6 years, and are always surprised with the person-to-person variation in ranking tomatoes for flavor.  As with most things that we eat and drink – taste is personal.  That said, there are trends in what people like in tomato taste/flavor – as illustrated in recent scientific studies by Harry Klee and colleagues at the University of Florida.

For decades it has been known that tomato flavor is primarily influenced by three categories of compounds: sugars, acids and volatile aromatics - and that it is a balance between these compounds that is important, not the just level of an individual component per se.  In the next several paragraphs I’ll try and outline the current understanding of the individual flavor components, how they interact, and the potential implications for tomato breeding.

Yilmaz, 2000 (reference) shows the following typical chemical composition of a tomato fruit:

Constituent	% DM
Fructose	25
Glucose	22
Saccharose	1
Citric acid	9
Malic acid	4
Protein	8
Dicarboxylic amino acid	2
Pectic substances	7
Cellulose	6
Hemicellulose	4
Minerals	8
Lipids	2
Ascorbic acid	0.5
Pigments	0.4
Other amino acids, vitamins, and polyphenols	1
Volatiles	0.1

To further set up this discussion; flavor = taste + aroma.  The tongue recognizes five basic tastes; sweet, sour, bitter, salty and savory - all of which come from non-aromatic/volatile constituents.  There are various aromas in tomatoes, favorable and unfavorable, and these are due to volatile compounds, to which we have receptors in the nose.

Sugars

Glucose and fructose are the major sugars in tomato, with glucose predominating early and fructose increasing with advancing fruit maturity.  In a mature ripe tomato the glucose:fructose ratio is generally about 1:1.  The tomato wild relative S. chmielewskii accumulates sucrose, rather than glucose – the result of a recessive loss-of-function allele of the gene controlling production of the enzyme acid invertase.  This allele and its high sucrose phenotype have been introgressed into commercial tomatoes.  Total sweetness index (TSI) is used to indicate sweetness. The contribution each sugar makes to this parameter is described relative to sucrose, and calculated as: [(1.00 × sucrose) + (0.76 × glucose) + (1.50 × fructose)].  When tomato samples are spiked with any of the three sugars, tasters perceive less overall aroma and less ripe tomato flavor.  Flavor intensity was highest with samples spiked with sucrose or fructose (Baldwin andThompson, 2000).

Total soluble solids, measured as Brix, is the sum of sugars (65%), organic acids (13%) and other minor components, and is intrinsically correlated to sugar content in tomatoes.  Although Brix is also highly correlated to sweetness in various tomato tasting experiments, Tieman, et al. found that specific volatile aromatic compounds enhanced perceived sweetness in their tasting experiments (more on this later).

Sweet (beef x cherry) segregate

Smaller fruited types generally have higher sugar content/Brix.  Tomato breeders at the University of Florida crossed a high sugar cherry tomato with a low sugar large fruited type and evaluated segregating progeny (Georgelis, et al. 2004).  They found a negative correlation between sugar content and fruit size or earliness; higher sugars in indeterminate vs determinate types; and no relationship between sugar content and fruit yield.  Others have reported a negative correlation between sugar content and fruit yield, which better matches our personal experience.  We’ve made a lot of crosses between cherry and beefsteak types.  Can you recover cherry level sweetness in a beefsteak segregate? – no.  We have found some pretty tasty larger fruited lines from such crosses, but they are not sugar bombs.

Photosynthesis is the conversion of CO₂ to sugar.  It is the ultimate source of energy in all the food we eat and in the fossil fuels we burn.  Chlorophyll is a green pigment, stored in plastids (chloroplasts), that is critical to photosynthesis.  In most plants chlorophyll is concentrated in leaves.  Tomato plants have chlorophyll/chloroplasts in leaves, stems and in unripe fruit.  In fact we now know that the chloroplasts in unripe fruit are an important source of sugars for the fruit, and that more chloroplasts generally result in improved fruit quality (Nguven, et al 2014).  Dark green unripe fruit is good for fruit quality.  GLK2 is a transcription factor that controls the formation of chloroplasts in tomato fruit.  The GLK2 wild type gene “U” has a phenotype with darker green unripe fruit and lingering green shoulders in ripe fruit.  The loss-of-function mutant “u” has lighter green unripe fruit and no green shoulders – so called uniform ripening.  The uniform ripening trait has been bred into most commercial tomatoes to facilitate commercial harvest and to present a more uniform red fruit (in grocery stores).   There are a lot of reasons why supermarket tomatoes generally taste bad – and this is one of them (Powell, 2012).   Since all heirloom types contain the wild type allele at this locus, any breeding program for improved taste should insure preservation of the wild type.  This is particularly true for progeny from crosses between heirloom and commercial types.

Green shoulders on non-uniform ripening wild type

Numerous studies have shown that generally, perceived sweetness is the most powerful determinate of “likeability” for taste in tomatoes.  That is certainly supported by our less rigorously designed tasting exercises here, although it is not uncommon for someone to say, “that one is too sweet”.  Sweet and sour - the ying and the yang.

Organic acids

Organic acids are what provide tomatoes the sour/tangy counterbalance to sweet.  The two primary organic acids in tomato are citric acid and malic acid.  Malic acid content is high in developing fruit, but decreases as the fruit matures.  Citric acid level increases somewhat during fruit development and generally represents 65-70% of the organic acids in ripe tomato fruit.  Tieman et al. report that tomato fruit citric acid concentration, when corrected for fructose content, was correlated to flavor intensity in their tasting trials.  Tomato fruit also contain low levels of free amino acids.   Glutamic acid, g-aminobutyric acid, glutamine, and aspartic acid comprise about 80% of the total free amino acids in
tomatoes.  Glutamic acid (glutamate) is concentrated in the gel around the seed and although it is a component of the umami (i.e savory) taste in foods, it seems to be negatively correlated with flavor in fresh tomatoes (Buchell et al., 1999).  

There is significant genetic variation for organic acid content and pH in tomatoes, with pH ranging from 4.1 to 4.8.  Low acid tomatoes are generally bland or one-dimensional, and a combination of high acid and high sugar is generally favorable.  In a classic study at UCD (Stevens et al., 1979, see graph below) tasters found that overall flavor intensity was correlated with both acidity (sourness) and sugars (sweetness), but that acidity (ph or titratable acidity) was most closely related to flavor intensity.  Tomato-like flavor, something we all love, was not strongly correlated with either sugar or acids – and is driven instead by volatile aromatics. 

Stevens, et al., 1979

Volatile aromatic compounds
Over 400 volatile aromatic compounds (AKA volatile organic compounds = VOCs), are found in tomato, and are either derived from carotenoid pigments, fatty acids, phenylalanine or free amino acids (Klee, 2010).  VOCs generally accumulate during fruit ripening.  Volatile compounds can be perceived by humans in two ways – sniffed through the nostrils (smell) or forced up behind the palate after chewing/swallowing (flavor).  It is generally agreed that taste is a complex interaction between smell, flavor, sugar and acids. 

Tieman, et al., 2012 (reference) summarizes the results from tasting panels and the relationship between chemical composition and “likeabiliy” in a broad collection of 152 heirloom varieties compared with samples of store-bought tomatoes.  Levels of reducing sugars (fructose and glucose), organic acids (citrate and malate) and 28 VOCs were measured, and multivariate analysis was used establish relationships between concentration of these compounds and various flavor attributes determined by the tasting panel. Flavor intensity was positively correlated with twelve different VOCs, seven of which were significant after accounting for fructose content.  Sweetness was also positively correlated with twelve VOCs, eight of which overlapped with those enhancing flavor, and three of which were independent predictors of sweetness after accounting for fructose content.  Although other studies have reported volatile induced enhancement of sweet, fruity, sour, bitter, smoky, and salty tastes, this study found the most significant enhancement was to sweetness. 

Tomato plants engineered to lack fatty acid-derived VOCs (the predominant family of VOCs in tomato) scored similarly for “likeability” to those with normal levels of these compounds.  In contrast, plants without carotenoid-derived VOC’s were perceived as significantly less sweet, and with a lower “likeability” score.  Other research finds that the concentration and composition of the various carotenoid pigments in tomatoes is directly related to the concentration of specific carotenoid-derived VOCs.  Carotenoid cleavage dioxgenase (CDD) genes code for various CDD enzymes, which in the chromoplast enable the conversion of pro-lycopene (tangerine), lycopene (red), and β-carotene (orange) to specific VOCs, most of which impart a fruity flavor.  The VOC geranial, derived from lycopene, is one of which Tieman et al. reports enhances sweetness.  In yellow and GWR fruited types the cartenoid pathway is truncated  early (see Genetic control of fruit color intomatoes) and virtually no carotenoid-derived VOCs are produced.   Although I have never tasted a true yellow-fleshed tomato that I found exceptional, there are a handful of GWR fleshed heirlooms and several of our GWR breeding lines that have excellent flavor – which in light of these data, is perplexing.

Green flesh - outstanding flavor

Klee and Giovannoni, 2011 (reference) make an interesting point.  Virtually all of the VOCs that contribute to tomato flavor are derived from a chemical compound essential for the human diet – essential fatty acids, essential amino acids and carotenoid pigments.  For example β-carotene is a precursor for Vitamin A, and it’s VOC derivative β-ionone is a key flavor volatile often associated with an intense fruity flavor.  They suggest an effective co-evolution between plants and animals – plants produce essential nutrients that have a characteristic flavor profile and animals evolve the ability to seek out such products, eat them and thus distribute the seed. 

Tieman et al. used standard genomic tools to segregate the tomato lines in their study into genetically related subgroups.  Their conclusion: “Based on these data, we found no obvious genetic subgroups that could explain liking, sweetness, or tomato flavor intensity. There was no obvious genetic clustering of good versus bad taste when varieties were sorted by chemical composition. These latter data also indicate the chemical complexity of liking, as there is no simple pattern of chemical content that separates high from low consumer liking scores.”  They also found extraordinary genetic variation for concentration of critical VOCs among the tomato lines studied, much more so than for sugar or acid concentrations.  This suggests a rich opportunity for breeding once specific VOC linked molecular markers are identified and deployed to enable efficient selection in large populations.

Conclusions

Wet lab characterization of the various flavor components is expensive and labor intensive.  Deployment of molecular markers associated with various genes that together contribute to perceived tastiness will allow more efficient selection and should help deal with the complexity of this trait.  In the mean time I think we (Frogsleap Farm) are stuck with the olfactory tools Mother Nature has provided, which although subject to individual preferences, can certainly send us in the right direction.  We will continue to combine tasting with objective measurements of soluble solids (Brix).  This literature review provided some new guidance – dark green unripe fruit are favorable (avoid uniform ripening types), high cartenoid pigment content is good, and some pigments probably better than others (e.g. β-carotene).  To the extent that the clear skin mutant allele y generally depresses the carotene pathway (see Genetic control of fruit color in tomatoes), I’m steering toward the yellow skinned wild type.  The green flesh phenotype (gf/gf) provides for an incomplete breakdown and conversion of chloroplasts to chromoplasts, which would seem to lead to a decrease in the cartenoid pigment content vs the wild type Gf (e.g red vs purple).  However, we routinely find our best tasting tomatoes carry the gf phenotype – on this point I am perplexed. 

Winner in 2014 "blind taste test"

We’ll be cautious with determinate and dwarf types which just may not have the leaf area to support maximum sugar accumulation, though I know there is a practical place for both types for some growers.  Nailing flavor in cherry and grape types is relatively simple, it gets harder with larger fruited types – which is where we are concentrating now.  Another challenge is to get great flavor with acceptable fruit yield and high fruit quality (non-splitting, firm flesh and good shelf life).  We are making progress on all fronts.  We will also anxiously follow progress in Harry Klee’s lab at the University of Florida, where they continue to provide a better understanding of flavor in tomatoes, and also create positive energy for challenging the bland tasting stereotype we are routinely presented in grocery stores and restaurants.