FOCUS ON: CONTROLLED CROSSES
RED GRAPEFRUIT
A Mutation Love Story
Introduction
Round, sweet and delicious, Rio Red and Star Ruby grapefruits are popular. In some varieties of pink grapefruit, the color starts to fade over time, but Rio Red and Star Ruby varieties remain a deep red with a tangy sweet taste. What causes this appealing difference in color? Read on to learn about how farmers and scientists maximized mutations to bring these ruby colored treats to our tables.
Yellow or "white" grapefruits were the norm until recently. Red grapefruit is a recent favorite. Pink varieties sometimes lose their color with time. All grapefruits come from the pomelo, a close relative.
Pink and red grapefruit varieties are the result of mutations. What comes to mind when you think about mutations? Are mutations good or bad? How do you feel about mutations being part of your food?
How did plant breeders help create pink grapefruit?

About the Organism
Grapefruit trees are 16 to 20-foot tall trees that live in subtropical climates with warm winters and hot summers. The trees are evergreens with long, dark green leaves.

Flowers develop near the leaves. The grapefruit flowers have white petals outside the plant's reproductive organs. After pollen is transferred onto the stigma and travels down the style to the ovary where the ova are fertilized, a fruit begins to grow.

The fruits have a thick yellow-orange skin. The fleshy white to pink to deep red, depending upon the variety of the tree. Some varieties have fruits with many seeds; others are seedless. The juicy flesh is segmented, like that of an orange and some people peel grapefruits to eat them, while other people slice the fruit in half and scoop out the flesh with a spoon.

What is the grapefruit's real name?
Grapefruit Reproduction
The female reproductive parts are the pistil, with it's sticky stigma opening into the tubular style that connects to the ovary. The male reproductive structure is the stamen, comprised of the thin filament on top of which sits the anther, which contains lots of pollen.
Grapefruit flowers have white petals. The fruit develops in the ovary below the petals.
What makes the fleshy interior of grapefruits different colors? Pigments—chemical structures that reflect certain wavelengths of light—cause the colors we see in nature. Two carotene pigments cause the grapefruit colors: beta-carotene, a precursor of Vitamin A, is responsible for the pink hues and lycopene, a different pigment, causes the darker red hues. Lighter colored grapefruits have less pigment, while pink and red colored grapefruits have more pigment.
Budsports: Naturally Occurring Mutations
Grapefruit, like many other types of citrus, has a somewhat fragile genome and is prone to natural, spontaneous mutations. Sometimes a cell forming a branch undergoes a genetic change. As that cell divides, the genetic mutation continues into the daughter cells. The budsport, or branch that develops from that original mutated cell, is genetically different from the rest of the tree. Some budsports die, others mutate back to the original form, while others include interesting and desirable new traits.
Genetic Mutation
A mutation is a change in an organism's DNA. Mutations can range from base pair changes to duplications of an organism's genome. If the mutation happens in a region of DNA that contains a gene, the protein that gene codes for can be altered. Not all mutations are bad; some mutations are beneficial for an organism and most are neutral. Mutations can increase a population's genetic diversity by bringing new alleles.
Sometimes mutations occur naturally in a branch-forming cell, leading to a whole branch containing a single mutation. The mandarin orange tree (left panel) has a budsport (enlarged in the right panel). The budsport branch has distinctly different fruit than on the rest of the tree; they ripen faster and have a smother rind, higher sugar levels and lower acidity than the fruits on the rest of the tree.
A budsport can be used to make a whole tree by grafting the desired branch onto a host tree, called the rootstock. Grafting is usually done on young rootstock, before it produces fruit. The top of the rootstock is cut off and any rootstock buds are removed if they grow later. A piece of stem with some bark and a bud is cut off the budsport. Growers slice open part of the bark of the rootstock and gently peel away the bark a little, leaving it on the stem, but making space between the bark and the next layer in. Growers then slice some of the bark off the piece of budsport and insert the shaved piece inside the bark of the rootstock. The two pieces are wrapped tightly together and eventually they fuse together. The buds, limbs and fruits that grow are from the tissue of the budsport.
The original grapefruits, natural hybrids of the pomelo (Citrus maxima) and the sweet orange (Citrus sinensis), were described in the mid 1700s as having a white flesh, just like the pomelo. The first grapefruit variety grown in Florida in the mid 1800s, 'Duncan,' eventually produced two different varieties, 'Marsh' and 'Waters.' At different times, trees of both those varieties produced budsports that had interesting traits as a result of natural, spontaneous mutation. Some had hints of pink in the flesh or the membranes of the fruits! In Thompson pink, the mutations resulted in more beta-carotene being produced, adding more red pigment to the fruits. On trees of those first pink-fruiting lines, 'Thompson' and 'Foster' more budsports appeared. In time, some of those budsports developed into new lines of deeper pink grapefruits, including 'Ruby Red' in 1929. Ruby Red's darker pink color was due to more lycopene being produced. Interestingly, fruits that were produced later in the season weren͛t quite as pink, as less lycopene was produced.

Red color occurred as spontaneous mutations in two lineages of grapefruits. Some fruits, like those of Ruby Red, contain red pigment in the flesh. A different lineage, including Reuben Pink, contains pink pigments in the membranes between grapefruit segments.
Think and Apply
Using the data in the table below, compare the beta carotene and lycopene levels of the Marsh White grapefruit to the beta carotene and lycopene levels of the pink or red varieties. What can you infer about the relationship between carotenoids and color of grapefruit flesh?
Answer:
Marsh White has lower beta carotene and lycopene levels (each 0.02 μg per g fresh weight). Carotenoid levels seem to increase as redness increases. Thompson Pink has 1.95 μg per g fresh weight beta carotene and 1.25 μg per g fresh weight lycopene. Ruby Red variety has the most beta carotene (2.77 μg per g fresh weight) and lycopene (1.98 μg per g fresh weight). You can infer that a grapefruit with more carotenoids is going to have a darker colored flesh.
Grapefruits and other citrus species are grown in the climate belts covering subtropical zones where winters are mild and summers are hot. Tropical climate zones near the equator are not good regions for growing citrus often because the hot, wet climates lead to disease problems
Grapefruit are grown in semitropical, tropical and mild temperate regions worldwide where winters are mild. In the US, Florida and Texas produce the most grapefruit, but there are groves in California and Arizona as well. Grapefruit are also grown in Jamaica, Trinidad, the Bahamas, Cuba, Mexico, Chile, Argentina, Israel, Morocco, Cyprus, Turkey, Spain, South Africa, Australia and even India. In China, a close relative of the grapefruit, the pomelo (Citrus maxima) is popular.
How do grapefruits grow?
Think and Apply
The first grapefruits were first described in the 1750s in Barbados. Where would grapefruit fit on the domestication timeline below? How does grapefruit compare to the domestication of other crops?
Answer:
The domestication of grapefruit is very recent compared to that of many of the foods we eat. Grapefruit would be placed near the right end of the timeline.
How Nutritious?

Sweet and flavorful grapefruits are naturally low-calorie foods, with a whole grapefruit providing only approximately 100 Calories. Grapefruits are low in protein and fats, so most of the calories come from sugars. They are also low in sodium and cholesterol. However, grapefruits are a great source of Vitamin C and Vitamin A.

The beta carotene and lycopene in grapefruits are thought to act as antioxidants, or substances that prevent cell damage by removing free radicals (oxidants) in our bodies. Beta-carotene is also a precursor for Vitamin A.

Grapefruits and their close relatives, pomelos, also contain a class of compounds known as furanocoumarins. Plants produce furanocoumarins as defense mechanisms against predators. For humans, however, the furanocoumarins can interfere with the action of various prescription medications, so some medicines contain warnings not to eat grapefruit while taking the medicine.
Vitamin C & A
Vitamin C is a water-soluble vitamin that acts as an antioxidant in our bodies.

Vitamin A is a fat-soluble vitamin that plays an important role in vision, bone growth, reproduction, cell division, and cell differentiation. It helps regulate the immune system and may help fight infections. Vitamin A promotes healthy surface linings of the eyes and the respiratory, urinary, and intestinal tracts, which helps prevent infection.
The Challenge
As grapefruits grew in popularity through the mid 1900s, consumers developed a taste for the pinker, sweeter varieties. Consumers also preferred grapefruits with few or no seeds. In order to grow more of the pink varieties, farmers became interested in growing grapefruits in places beyond Florida, grapefruit's first home in the United States.

Fruit color and seedlessness aren't the only traits that appeal to farmers. Farmers need strong, healthy plants that can resist the plant diseases and pests that damage crops. Another desired trait is hardiness, or resistance to cold, because sudden cold temperatures damage citrus trees. In Florida, freezes happen occasionally, but the grapefruit groves are scattered widely throughout the state, so a freeze usually impacts only some of the groves. In Texas, however, grapefruit production is concentrated in the Rio Grande valley. If a freeze happens there, it can wipe out huge swaths of trees. Growers in both places need trees that can survive despite a little bit of cold every now and again.

These economic concerns – the desirability of red fruit and the need for hardy trees – prompted agricultural scientists to seek varieties with all these positive traits in one plant. Farmers and scientists already knew that grapefruit tissue mutated regularly; budsports show that grapefruit can make pink pigments. How could scientists capitalize on that tendency to mutate and use mutations to develop a hardy grapefruit tree that bears lots of fruit with a consistent deep-red color, appealing taste, and fewer seeds?
The Solution:
Irradiating Seeds to Find Redder Mutations
Plant scientists accepted the challenge of developing delicious, deep-red grapefruits that grow on hardy trees that bear lots of fruit. However, grapefruits have some unusual traits, including polyembryony and nucellar seed formation, that make conventional plant breeding methods impractical. Consequently, scientists needed a different way to try to produce changes in the plants that would lead to the desired traits.

Dr. Richard Hensz of the Texas A&I Citrus Center (now known as the A&M Citrus Center) recognized that natural mutations had created many popular varieties of grapefruit and other citrus plants. He wondered if inducing, or causing, mutations could genetically alter grapefruits so that they met farmers' and consumers' criteria for desirable trees and fruits.
Polyembryony
Polyembryony happens when a fertilized egg develops into two or more embryos.
Nucellar Seed Formation
Seeds are produced after male and female genetic material sexually. Nucellar tissue surrounds the gamete. In some cases, other seeds form from the nucellar tissue and those seeds contain only the genetic material of the parent plant.
Human-caused, or induced, mutations led to the Rio Red grapefruit variety.
Results of Irradiating Grapefruit Seed and Budstock
At most grocery stores today, you can find a wide variety of grapefruits when they're in season. The different varieties have flesh that range from white to pink to red. Two popular varieties, Star Ruby and Rio Red, are the successful results of induced mutations caused by irradiation, although the process was different for each.

Star Ruby variety
In 1959, Hudson grapefruit seeds were irradiated with x-rays and thermal neutrons at Brookhaven National Laboratories. Those seeds were planted, then a year later buds were removed from seedlings and propagated on sour orange rootstock. The fruits, first harvested and evaluated in 1966, were named 'Star Ruby'. Researchers saw generally positive changes in fruit traits, especially the redder fruit. In contrast, the trees had some interesting qualities, but also some concerns. Despite some of the limitations of the trees, the production was a success. "It is a unique tree and the color, texture and quality of the fruit give it marketing advantages over the presently grown Ruby Red grapefruit for both fresh fruit and processing," (Hensz, 1971, p56). The Star Ruby budwood was released to growers in 1971, grown successfully in the US and elsewhere, and the fruits have been accepted by consumers worldwide.
Star Ruby Fruits
  • Seedless (meaning few instead many seeds)
  • Thinner, smoother peel ripens earlier, making shipping easier
  • Fruit three times redder than Ruby Red although the color decreases during the season
  • Firm fruit flesh, making sectioning easier Juice retains its red color, even when canned for a year
Star Ruby Trees
  • Compact, bushy tree structure
  • Trunk cambium is red
  • Some trees revert to seediness; their fruits have lots of seeds
  • Growers reported low or erratic fruit production
  • Farmers have to be very careful about applying herbicides because the leaves are very sensitive and their chlorophyll can be damaged
  • Sensitive to freeze damage, water mold damage & winter chlorosis (reduction of green color in leaves)
Rio Red Variety
In 1963 Ruby Red budstock was irradiated with x-rays and thermal neutrons at Brookhaven National Laboratories. That irradiated budstock was then grown out on sour orange rootstock. Scientists were excited to see that fruit from one of the trees, called 1-48, had deep red flesh. More trees were propagated from tree 1-48, but they were genetically unstable. One tree, however, produced an interesting budsport with an even deeper red fruit. That budsport, named 1-48S, was propagated and 1200 trees were planted. 1-48S became known as 'Rio Red' and budwood was released to growers in 1984. Rio Red has been grown successfully around the world, and the fruits have been accepted by consumers worldwide.
Ruby Red Fruit
  • Seedless (meaning few instead many seeds)
  • Deep red fruit, with good taste
  • Firm fruit flesh, making sectioning easier
  • Juice is a deep red
Rio Red Ruby Trees
  • Trees have a longer, more open structure
  • Trunk cambium is not red
  • Vigorous; not genetically unstable.
  • Sensitive to freeze damage (many trees affected by 1989 Texas freeze)
  • Less sensitive to herbicides, water mold damage & winter chlorosis as Star Ruby
Think and Apply
The new varieties resulting from induced mutations are redder than other grapefruit varieties. How do their pigment levels compare to the pigment levels of other varieties?
Answer:
Induced mutations resulted in big differences in carotenoid levels. The carotenoid levels of Star Ruby and Rio Red are significantly higher than those of the other three varieties! Rio Red has beta carotene levels that are somewhat higher (3.80 μg per g fresh weight) and lycopene levels that are significantly higher (16.19 μg per g fresh weight). Star Ruby, the reddest grapefruit, has the highest beta carotene (7.23 μg per g fresh weight) and lycopene (19.23 μg per g fresh weight)

Conclusion
In nature, spontaneous mutations happen all the time; some mutations have a beneficial effect on the organism, some are harmful, and most are neutral. Mutation creates variation and it is from that variation that plant breeders are able to produce new varieties. Because grapefruit trees grow and reproduce slowly, conventional plant breeding techniques aren't practical. Therefore, inducing mutations with irradiation was a useful technology to improve grapefruit seedlessness and deep red color. Although only two of the many varieties of grapefruit were produced using irradiation, those two varieties possess traits that both consumers and farmers value, making them a valuable commodity worldwide.

Grapefruit aren't alone! Mutation breeding has been used for lots of crops. Radiation breeding, using different kinds of high energy radiation like neutrons, UV radiation, gamma rays or x-rays, has produced useful mutations in varieties of cotton, peanuts, peas, pears, wheat, barley, rice, bananas, cassava, sorghum, wheat, barley and more. Chemical mutagenesis, using ethyl methanesulfonate (EMS), has produced mutations that led to desired traits like improved cooking oil quality from flax and a reduction of certain fats in canola. By providing more variation within a crop variety, mutation breeding is one of the many tools that plant breeders use to try to create crops with desired traits.

Now that you've learned the story of these deep red grapefruit, how do you feel about mutations being part of your food?
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