Growing Giant Sequoias From Seed - Trial 2

So, we tried to grow giant sequoias (Sequoiadendron giganteum) from seed indoors this past winter, with pretty poor results.  Although we had about a 10% germination rate, 80% of the trees that germinated ultimately died (without clear cause).  This is a very difficult tree to grow, but persistence is key.

We are again trying to grow sequoias from seed, but this time we are trying to correct our mistakes.  We used regular tap water last time to cold stratify the seeds - but this may have lead to "damping off" or fungal disease.  This time we committed to distilled water.  We tried to grow the sequoias inside, during the winter, under artificial light.  This time we will plant and keep the trees outside even during winter (but in a cold frame if necessary).

So, to start off, we acquired 2000 more sequoia seeds from Myseeds.co, our preferred site.  We expect again about a 10% germination rate, so perhaps around 200 trees.  We plan to distribute these trees across the country for growth with the Waterboxx in a few years.

First, assemble all needed materials.  To stratify (prepare seeds for germination), you will need to following materials:

• A clean paper plate
• Two clean paper towel squares
• A clean sealable kitchen bag
• Sequoia seeds (at least 10 seeds for every desired tree)
• Distilled water (which won't have any fungal spores which can kill young saplings)

What is needed to cold stratify the seeds - paper plate, two paper towels, sealing kitchen bag, seeds, and distilled water

First, wet one of the paper towels with the distilled water.  Lie this paper towel flat on the paper plate.  Then, carefully spread the (small) sequoia seeds on the wet paper towel, as evenly as possible.

Spread the sequoia seeds on the paper towel wetted with the distilled water - do this with clean hands only

Next, take the second paper towel and wet it with the distilled water.  Lay this carefully over the paper plate now covered with sequoia seeds.  If some seeds get pushed off the plate, I would pick them up and put on the plate - remember that each seed may grow into a tree that will store a lifetime of carbon emissions and live for millenia.

Our two thousand covered sequoia seeds (you probably don't need so many seeds unless you have many acres you wish to plant - expect 1 tree per 10 seeds)

Next, we need to make the large paper towel fit in the plastic bag.  We folded the corners of the paper towels in to fit it on the paper plate.  We then slid the paper plate into the bag, labeled it with the date we plan to open it (one month later).

We plan to plant the seeds into their germination site - cone-tainers - in about one month.  After about one to two years - when the trees reach about 12-16 inches in height, the sequoias will be ready to plant.  We will plant the sequoias with the Groasis Waterboxx during our annual spring sequoia donation and planting, or distribute the trees to our customers.

We will update this post with our progress and next steps in early May.

You can visit our main website (or see our previous sequoia results) We would love to hear your comments below.

How Does Nature Harvest Dew

Nature understands something that humans have only recently begun to grasp - that when there is no rain, there is still water available in the air - water in the form of dew or condensation.  There are several species of animal on three different continents that have learned to harvest this dew and live almost exclusively off of it.

Perhaps the most impressive dew drinker is Moloch horridus, or the Australian Thorny Devil.  This animal is lives in an environment (the Australian Outback) with very little rain.   However, in the desert, because of large swings in temperature between day and night, there is often some dew on the ground in the morning.  Most of this dew is immediately evaporated after sunrise.  However, the Thorny Devil is able to use the "horns" or spikes on its body to collect this dew instead of allowing it to gather on the ground.  The Thorny Devil then channels this moisture to its mouth with special channels evolved just for this purpose.

This same method has evolved on the other side of the world by an unrelated lizard, the state reptile of Texas, the Texas Horned Lizard,  or Phyrnsoma cornutum. The Texas Horned Lizard doesn't rely on dew so much as it does on rain drops after they hit the ground and splinter into much smaller particles.  These particles are caught by the horns on this animal and channeled to its mouth as well, using one way capillary channels.

The African Pygmy Mouse, Mus minutoides also exploits the same effect by piling small stones outside its den at night.  These stones collect condensation (perhaps partly from the mouse's breath) which the mice then drink in the morning.

Our favorite dew harvester, however, is the Fogstand Beetle of the Namib Desert, Stenocara Gracilipes.  This beetle inhabits a desert with less than one inch of rain per year!  How does it survive in such an arid environment?   They will stand on little ridges of sand in the desert when the morning dew rolls in and angle their bodies to 45%.   These beetles have microscopic water loving (hydrophilic) spikes that collect dew, and water moving (hydrophobic) troughs that direct the collected water to their mouths.  These beetles can drink twelve percent of their body weight in water each day using this method!

Is there any way that humans could harvest this dew, not to drink, but to grow food and trees?  Yes, there is.  It is called the Groasis Waterboxx PlantCocoon®, or Waterboxx for short.

The Waterboxx has taken the best insights nature has to offer, including the water loving spikes and water moving troughs and angled top, to collect dew in dry areas.  This dew is then funnels into a reservoir where it is stored by later use by the plant growing inside the Waterboxx.  The dew is slowly released by wicks into the soil. Rare rainfall can completely replenish the 15 L (almost 4 gallon) reservoir of the Waterboxx.  In this way, we can start trees and grow garden plants without watering.

The Waterboxx with tomatoes - you see condensation on the rim of the Waterboxx (where there are no ridges) but none on the ridges as there are microscopic bumps or pyramids there that collect and then funnel water down to a reservoir.  

The Waterboxx may even be able to recycle some of the water transpired (or, simplistically, 'sweated') out by the plant at night.  We haven't yet proven this, but plants release a great deal of water through special pores mostly on the underside of lids (called stomata) at night, and on still nights, it is likely at least some of this water settles on the lid of the Waterboxx and is collected.

A schematic cut away view of the Waterboxx - dew is collected on the tan lid, sent down the siphons (shown here in red), stored in the green reservoir, and slowly distributed to the roots of the growing plant via a wick.  Photo from Groasis.com

The Waterboxx has been used all across the world but its use is just catching on here in the United States.  People are finding that they can grow some vegetables with the Waterboxx without ever adding water, and start trees without any water after planting.  Even better. because the trees develop deeper roots with the Waterboxx, the tree is then far more likely to survive subsequent droughts, even when the Waterboxx is removed and reused.

If you would like like to know more about the Waterboxx or see results of using it, visit our main website, www.dewharvest.com.

The Experimenting With Mycorrhizae (Helpful Root Fungus)

Mycorrhizae (from ancient Greek "mycos" meaning fungus and "riza" meaning roots") are beneficial fungi for growing plant roots. Roots are only able to absorb water and nutrients from the soil that they are in contact with (called the rhizosphere in scientific parlance - a great word in our opinion that we will subsequently overuse).  You (the gardener) generally want the largest rhizosphere possible for your plants, especially in dry climates or places with poor soil.  However,.the goal of the plant is to grow above ground and produce seeds for propagation of the species.  This leads to an challenge and an opportunity - how do you expand the "rhizosphere" while allowing the plant to focus on photosynthesis and fruiting?

Luckily, nature has a solution for us - mycorrhizae.  Mycorrhizal fungi, just like all fungi, cannot grow without getting an food source (they are heterotrophic, like animals).  For this they need the roots of plants to provide them with sugars.  In exchange, the mycorrhizae greatly expand the surface area of the of the "roots" by attaching and allowing the roots to collect water and nutrients from more numerous fungal filaments.  This is seen below - with the corn root appearing much larger than the mycorrhizal fungal root (meaning the corn gets a larger rhizosphere).

A microscopic view of an arbuscular mycorrhizal fungus growing on a corn root. The round bodies are spores, and the threadlike filaments are hyphae. The substance coating them is glomalin, revealed by a green dye tagged to an antibody against glomalin.
Photo by Sara Wright - courtesy of USDA, public domain

Just as roots are not generally considered when gardening, root fungus is thought about even less.  We first heard of mycorrhizae when we discovered the Waterboxx, a brilliant invention to harvest dew and rain water to grow trees and other plants in the desert.  We were not sure of how effective these fungi would be in helping roots until we saw the experiments of our friend Bill McNeese, an expert gardener in the near desert in Southern California.  After his results, we decided we needed to try out mycorrhizae in a controlled experiment, to see how much they improved growth.

Although we didn't have the resources for a large experiment, we decided to plant two peat pots of our garden vegetable seeds indoors, one with mycorrhizal fungi, one without.  We would then try to keep all other variables constant, including light (from overhead grow lights), as well as water and space for the plants.  You can see our results below

Mycorrhiza planting on left, Non-mycorrhiza on right

Kellog's Breakfast heirloom tomato grown with mycorrhizae (left) and without (right) with same amount of light, water, and soil.  Clearly the peat pot with the mycorrhizae has a much higher germination rate and faster growth.  Photo taken on 3/16/16

Carnival peppers, with mycorrhiza added on left and none on right - again clear germination and growth advantage of the mycorrhiza added group

Bell peppers, with mycorrhiza added on left and none added on right - again, the mycorrhizal group had better germination and growth, although not quite as pronounced as the Kellog's Breakfast tomatoes and the Carnival peppers.

Amadeo eggplant, with mycorrhiza on left and none on right.  We are not sure why germination rate is higher with the non-mycorrhizal group for this plant only.

In all but one of the experiments, the seeds with mycorrhizal fungus germinated better and grew faster than those without.  We are not sure why the eggplant did not grow better with mycorrhizae - we will be testing if this is true across all species of eggplant with later experiments with Japanese and white eggplants (check back often).

Updates: April 3, 2016

Amadeo Eggplant - left with mycorrhizae, right without - this is our only plant that hasn't done better with mycorrhizae

Tomatillo with mycorrhizae on the left and without on the right - the tomatilloes with mycorrizae have a significantly higher average height 

Bell pepper - with mycorrhizae on the left and without on the right - the pepper with the helpful fungus is clearly much larger overall

Carnival pepper, grown with mycorrhizal fungus on the left and without on the right - the pepper with mycorrhizae is about 20% larger overall

We used "Mykos" brand Rhizophagus intraradices available on Amazon here.  We used only a very small amount of mycorrhiza (we used a forceps/tweezers to grab about 1/2 inch of mycorhizzal granules between the two parts of the tweezers).  We believed that the mycorrhizae would of course proliferate on their own, and there was no sense putting down more mycorrhiza than what could immediately surround the new plant roots.  As the mycorrhizae are somewhat expensive, this also allows us to conserve resources.  

We plan to plant all of these plants in our garden using the Groasis Waterboxx.  After planting and Waterboxx set up, we will not water them again for the entire growing season.  Between the Waterboxx collecting dew and rainwater, and funneling it to the plant roots, and the mycorrhizae increasing the rhizosphere, we expect these plants to do excellent without any manual watering, and produce large numbers of fruits and vegetables.  

We will continue to update this post with other vegetable plant mycorrhizae experiment results.  If you would like to know more about mycorrhizae (from an academic source), see here.  If you would like to know more about the Waterboxx and how you can garden without ongoing watering, see here. 

Feel free to contact us with any questions by leaving a comment below.

Growing Giant Sequoia Trees From Seed

Giant sequoia trees (Sequoiadendron giganteum) are the largest living things on Earth.   They can live for thousands of years, reach almost three hundred feet in height, resist droughts and forest fires. The single largest sequoia tree now living, General Sherman, has sequestered over a lifetime of carbon emissions by the average American.   What is more, giant sequoias can grow throughout much of the world in temperate regions, including most of the continental United States, so long as they have sufficient water (around 30 inches or 762 mm each year).  Sequoias are common in Britain, but are also in mainland Europe, New Zealand, Australia, Japan, Canada, and almost every state in the U.S.

Establishing sequoias is very difficult, and with trial and error you can expect years of frustration, even if you buy saplings rather than seeds. Even with saplings we didn't have any success establishing sequoias outside in the Midwest until we started using the Waterboxx PlantCocoon to grow sequoias for the first few years.  Since starting to use the Waterboxx PlantCocoon, we have had 100% success, detailed here and here.

As sequoias proved such fun to plant and are so beneficial to the environment, we wanted to know if we could grow sequoia trees from seed.  We had tried this before, but had very poor germination rates (around ~1%), and many of our trees that did germinate soon died.  We were determined to try again, but to follow the best advice available for growing these seeds.

Sequoia seeds are tiny - here is an average sized one on a fingertip before planting.  It is hardly believable that these become the largest living things on Earth.

We bought 500 giant sequoia seeds from MySeeds.co, on Amazon.com here (or more cheaply from their website here).  Sequoia seeds need a very specific process mimicking their natural environment to germinate (including a wet "fall" and cold "winter"), so we tried to replicate that in as short a period as possible.  To start, we laid our seeds on a paper towel and moistened them with water (distilled water for best results as it doesn't contain mold).

Seeds on July 16, 2015, right after getting them in the mail.  Our biggest problem was having the patience to not plant before "hardening" for a month in the fridge.  

We covered these seeds with another moist paper towel, and them put them on a portion of a paper plate.  We then put these in a clean, sealed plastic bag.  This simulated our wet "fall", in order to reawaken the seeds.  For our winter, we placed this plastic bag in the vegetable crisper in the fridge for 30 days.

After 30 days, we removed our seeds.  We started with 500 seeds, but given our poor germination rate before, we didn't expect most to produce anything.  We took about half of these seeds to be planted.  We set up a Cone-tainer rack filled with 98 soil holding cone-tainers (both available here).  We filled these full of potting soil.  For about half of the cone-tainers, we also added some vermiculite, which is excellent at holding moisture.  We then took the very small seeds, and added them to the top of the soil mixture.  For half of the cone-tainers, we used only one seed, and for the other half we used three to four.  We pushed the seeds down slightly into the soil, but we did not bury the seeds.

Our 98 Cone-tainers in a tray, with our seeds just planted, on August 18, 2015.  The vermiculite containing Cone-tainers are white on top.

We waited about two weeks, but didn't see any of the promised germination.  We thought that perhaps nights were getting too cool (sometimes into the upper 50s Fahrenheit), so we put a cold frame we had previously built over the sequoias seeds.

Within two days, we started to see germination of our tiny trees.  We did our best to keep the tiny seedling moist without over watering.

Tiny sequoia trees, just growing from seed on August 31, 2015

We had about 25% germination in our first round. We wanted to have a giant Sequoia tree growing from each Cone-Tainer, so we planted more seeds in each Cone-Tainer that didn't have one germinate.

We did have a few Cone-Tainers with more than two sequoias germinate. We wanted didn't want competition to hurt both sequoias, so we removed the smaller sequoia seedling so the larger could continue to grow unabated.  When we removed the smaller sequoia, what we found was astounding (to us, at least).  Giant sequoias send down a true tap root!   This is incredible, as many trees just send out shallow, lateral, fibrous roots.  This true tap root means sequoias can tap deep sources or water (like water held in capillary channels) as well as underground aquifers.  This means that sequoias will be able to withstand droughts very well.  This only makes sense as many sequoias have lived for three millennia, through many droughts, in California.

A tiny sequoia sapling, a little over a month (9/26/15) after planting, with a taproot over three times the length of the trunk.  These tap roots enable sequoias to survive very long periods without rain.

We are growing these sequoias from seed in Central Indiana, which has harsh winters, so we decided to move our saplings inside to a window sill over the Fall and Winter and provide a little artificial light to speed up growth.  There is a chance this may disrupt the seasonal rhythm of the plant, but we judged this risk as lower than the risk from the freezing.

The sequoias inside (all moved close to get the most light) on October 3, 2015.  We have had about a 33% germination rate so far - not bad for this very difficult to start tree.

We are very impressed that the eventual structure (and beautiful red trunk) has already begun to become evident.

A sequoia about 5 weeks old - we hope this sequoia is 10 inches tall by spring to it can be planted outside with the Waterboxx PlantCocoon.

At this point (October 6, 2015), we still have about 70% of our Cone-tainers without any sequoia seedlings.  This means our germination and survival rate has been somewhere around 15% (because we planted about two seeds per Cone-tainer, on average).  We want each Cone-tainer to have one sequoia, so we planted the most of the remaining seeds into the empty Cone-tainers.  We did save a few seeds just in case none germinated in some Cone-tainers.  

A sequoia at about 10 weeks - again perhaps doubled in size over the past 5 weeks.

By late November, we have planted all 500 seeds and have 50 living sequoia seedlings, for a germination rate of 10%.  As this is our second planting, and we had a germination and early survival rate of 0% previously, we are well pleased.  We are supplementing sunlight with full spectrum CFL light (purchased before full spectrum LEDs were available), and see the smaller sequoias grow ~5% per day - a very healthy growth rate indeed.

Our sequoia seedlings 4 months and 11 days after planting.  We still have 48 living sequoias, with two more lost to damping off.  Our tallest tree is about 3.5 inches, which should put us in range of the desired 10 inches by April with our continued artificial light.

Right now (late March 2016) we have had greatly decreased survival in the late spring.  We asked our friend Joe Welker of Giant-Sequoia.com why this was, and he believed it was because we grew indoors.  We are down to only 10 sequoias, hardly satisfying,

Our sequoias on March 29, 2016 - only 10 left.  We will try continue growing these but try again with a new crop of seeds in a few weeks - outdoors.  

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We plan to start a new crop in outdoors in early June.  The things we will change with this next crop:
1. We will plant using larger size cone-tainers
2. We will plant outdoors
3. We will use only distilled water initially to prevent fungal diseases causing "damping off"
4.  We will keep the sequoias outside all winter (but in a cold frame to prevent desiccation from the wind).

You can see our next trial (this time with 2000 sequoia seeds) here.


We hope to see these sequoias grow to the point they can be transplanted outdoors with the Waterboxx PlantCocoon®.  They will need to be about 10-15 inches tall at that time.  We are growing these sequoias for donation to a few growing partners in the South and Midwest - we will post those plantings online when pictures are available.

Our greater hope is to see giant sequoias planted on public and private property throughout the United States and rest of the world.  This tree grows so large, so fast, and lives so long, that it may be one of the few affordable ways to decrease carbon dioxide in the atmosphere, counteracting the problems caused by excessive carbon dioxide.

We will continue to update this post with our sequoia from seed progress.  We would love to hear your comments below.

If you would like to plant sequoias you already have outside, with the Waterboxx, you can buy a Waterboxx PlantCocoon here.

Plant Paw-Paw - Indiana Banana, America's First Fruit Tree

History can be capricious.  The phrase "American as apple pie" has entered the lexicon of most  Americans.  This is unfair.  The apple tree is derived from wild ancestors in Central Asia and Europe and is not truly an American fruit.  This is of course the American way - adopting and adapting ideas and foods from around the world.  However, most people in this country, who have tried everything from apple butter to apple pie, have never tried true American native fruit - the Paw Paw.

The Paw Paw tree, also known as 'Indiana Banana', Asiminia triloba, is the largest fruit native to the U.S.  It is rich in vitamin and energy content, good tasting, and grows in all or part of 26 states.  Paw paw is a valuable fruit in that it has all 20 essential amino acids or building blocks of protein.  Paw paw also has more vitamin C than banana (twice as much) or apples (three times as much).  It has more potassium than apples (3x) and orange (2x) and almost as much as bananas.  It also has markedly more calcium, magnesium, and iron than these other three fruits.

The paw paw fruit on the vine, from USDA

Paw paw fruit does not transport well fresh, and is only a peak taste for a few days.  It is for this reason primarily that it has never been commercialized.  When eaten fresh off the tree, however, the paw paw has a flavor that is something of a cross between banana, pineapple and mango.  Paw paw fruit can be substituted for banana in most recipes.   

Range of the Paw Paw Tree - most of the Eastern United States (from USGS)

Paw paw is relatively disease and insect resistant.  It is recommended that you buy grafted trees if you want sooner fruit production - our preferred source is Stark Brothers.   According to Sheri Crabtree, a paw paw expert at Kentucky State University, "Pawpaws do have a strong taproot and can be difficult to dig and transplant".  This tap root needs to be kept moist at almost all times, which requires near constant watering. This makes watering them almost daily essential right after planting.  This is not feasible for most home owners, however.  There is a device that may help, called the Groasis Waterboxx PlantCocoon or Waterboxx for short.

The Waterboxx collects dew and rain, stores it in a four gallon reservoir, and slowly releases it to the roots beneath the growing tree.  It also prevents evaporation of soil moisture - allowing a "water column" to form immediately beneath the Waterboxx.  Tap roots are induced to grow straight down in this water column until the tree is well established.  The Waterboxx can then be removed and reused again.  This is all explained in the video below:

We hope to see our natural botanic heritage more appreciated in the future.  We hope you will consider planting a paw paw tree or three.  If you want to try planting with the Waterboxx, it is available here.  

We would love to read your comments below.

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, but have more normal fruit color and flavor than either rin/+ or nor/+, and thus seems to be another interesting candidate gene/all ele for the extended shelf life/excellent flavor combination.

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

Davis EFS F2 segregate 

EFS – extended field storage.  Several new processing type tomato hybrids contain the extended field storage (EFS) trait, which allows for a longer window for field harvest, creating more flexibility for tomato processers.  The alc allele (or perhaps a related NOR mutant) may to be at least partially responsible for this modified ripening phenotype.  While driving near Davis, California in early September 2014, I stopped to pick up a couple of tomatoes that had fallen off a truck on the way to processing.  They had bright crimson flesh and a rich tomato flavor.  In a F2 growout in 2015 we found one F2 plant that appeared never to fully ripen on the vine, but had a bright pink center (see photo).  This combination of a lack of obvious pigmentation on the fruit surface with bright lycopene pigmentation of the fruit pericarp seems atypical of all the ripening mutants described above, and remains a mystery.  We presume this plant to be homozygous for one or more recessive ripening mutants and made several F1 crosses to elite FLF breeding lines.  F2 progeny from winter growouts will be evaluated in 2016.  This was one of the oddest discoveries in our 2015 nurseries and I expect we will learn quite a bit more next year.  In my literature review for this paper I found a one sentence reference to a long keeping variety that appeared to ripen from the “inside out” – perhaps a related phenomenon?

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, they are independent loci and 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 alc, dfd  - and possibly EFS.  EFS is now widely deployed in commercial processing hybrids grown in California, though the ESL phenotype and mode of action appear to be treated as trade secrets.  The dfd mutant is also 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, dfd or EFS.

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.