Cut of the crop High-yields of non-browning eggplant via genome editing

Recent technological advances are making it possible to quickly breed bountiful and desirable crops. Dr Martin Kottackal and colleagues at the Khalifa Center for Genetic Engineering and Biotechnology (KCGEB) are making strides in this area. A leading institute in the United Arab Emirates, the KCGEB is under The Presidential Court and In an uncut eggplant, the PPO enzyme and polyphenols do not interact, and the fruit remains white on the inside. But when the fruit is cut or damaged, cell compartments are disrupted and release the polyphenols and PPO, which then undergo oxidation. Enzymatic browning can also increase when the fruit has been physically damaged, for example, during harvest and transportation, resulting in food wastage. Plant breeders have therefore aimed to produce eggplants that do not undergo enzymatic browning.

In an uncut eggplant, the PPO enzyme and polyphenols do not interact, and the fruit remains white on the inside. But when the fruit is cut or damaged, cell compartments are disrupted and release the polyphenols and PPO, which then undergo oxidation. Enzymatic browning can also increase when the fruit has been physically damaged, for example, during harvest and transportation, resulting in food wastage. Plant breeders have therefore aimed to produce eggplants that do not undergo enzymatic browning.

TO BREED OR NOT TO BREED?
TRAIT IS THE QUESTION Plant breeding allows the selection of crops with desirable traits (qualities) such as large fruit size, or drought resistance. A plant with a desirable trait is crosspollinated with another lacking it -to reap the benefits of both the original plants in a new hybrid variety with the positive characteristics selectively bred for. Over successive generations, plant hybrids have been produced with desirable traits that make them valuable to producers and consumers.
This form of traditional plant breeding has led to the development of modern varieties of several food crops consumed today, including wheat, rice, and tomatoes. Plant breeding has played a crucial role in agricultural developmentbut how does selectively breeding traits at the molecular level work?
TRAITS: IT'S ALL IN THE DNA Traits (also known as phenotype or appearance), such as colour, shape, size, resistance to disease, and number of seeds, are controlled by genes, a set of instructions present in every cell's DNA. The expression of a gene changes it stepwise into proteins, which govern the phenotype. A single gene can control

Cut of the crop
High-yields of non-browning eggplant via genome editing Eggplant is cultivated globally -but its production is fraught with issues affecting the crop's overall yield and post-harvest properties. The fruit can be less appealing when damaged or cut due to the enzymatic browning of tissues. Cultivating high-yielding yet non-browning eggplants is an area of ongoing research at the Khalifa Center for Genetic Engineering and Biotechnology, UAE. The researchers have improved the fruit's properties by genome editing a key enzyme, polyphenol oxidase. As a result of tweaking a single gene, eggplants produce more fruit that is also non-browning. different trait attributes by producing various forms of the same protein, a phenomenon known as pleiotropy.
During reproduction, and when cells divide, natural changes are introduced into DNA sequences as DNA is copied. The cells sometimes repair these gene mutations, but at other times, they do not. Instead, they are carried through to the next generation -resulting in an altered trait. Mutations give rise to variations in any species, resulting in different colours, behaviour, and survival of its progeny.
Plant breeders have more recently used chemicals and irradiation techniques to induce random mutations in seeds that alter the function of specific proteins, therefore, altering traits. By altering genes in different plant varieties, this form of plant breeding has given rise to the vast selection of crops with desirable characteristics that we consume today. However, this method of plant breeding is a long process, requiring at least six generations of selectively bred plants before the final variety with desirable characteristics is produced. Moreover, the mutations can be randomly introduced throughout the plant.
PRECISION BREEDING USING GENOME EDITING Now, new tools have been identified over the last decade that dramatically reduce the time needed for breeding crops with superior characteristics, introducing desirable traits quickly and precisely by more accurately controlling the desired genetic mutations.
Techniques and tools have been developed to alter the sequences of genes at specific locations in a plant's genome (complete set of genetic material in its DNA). The most well-known tool, called CRISPR-Cas9, uses a concept naturally occurring in bacteria. It involves molecular components that can locate target sequences in a genome and cut the DNA at specific locations. Natural repair mechanisms in the plant cells would usually repair these changes successfully, but sometimes the plant fails to, and a mutation is introduced.
Specific genes can be targeted using the method to be edited to introduce precise mutations. The introduced mutation would be identical to one that could have arisen from traditional or mutational plant breeding, except that there is only a single precise mutation, instead of several thousand random ones. Genome editing significantly shortens the time required for selective plant breeding from roughly a decade to only a few years. Remarkably, scientists have also developed methods by which no foreign DNA is left behind in the edited plant, only the desired mutation. This method of precision breeding has led to several research studies in recent years that demonstrate its efficiency in rapidly improving plant traits.
Professor Amiri, Director of Khalifa Center explains, 'Gene editing has a high potential for practical applications in agriculture, and contributes to global food security. The science of gene editing -a faster strategy than standard breedingis making waves in agriculture by improving the traits of interest in a precise way.

EGGPLANT: WHAT'S IN A NAME?
Also known as aubergine, eggplant is a fruit crop and member of the Solanaceae family. The purple colouration of eggplant is caused by the compound anthocyanin, a powerful antioxidant that can remove harmful free radicals from the body. It's also high in fibre, making it good for digestion. Eggplant breeding across the world aims to generate varieties that can better resist or tolerate different pests, pathogens, and environmental stresses, as well as improve post-harvest organoleptic qualities (its sensory appeal -eg, taste, texture, and smell).

LOCATION, OXIDATION, DISCOLOURATION!
One unappealing property of eggplant is its susceptibility to browning when cut or damaged, affecting its flavour and nutritional properties. These undesirable traits also reduce the fruit's post-harvest value. This phenomenon -known as enzymatic browning -is governed by the activity of a plant enzyme called polyphenol oxidase (PPO). This enzyme combines polyphenols and oxygen (when the inside of the fruit is exposed to air) in an oxidation reaction, producing a brown colour.

Non-edited
Genome-edited: petite eggplant with more fruits (front view)

Personal Response
What do you think will be the impact of your research and how could other crops be genome edited to produce desirable traits?
Browning damages fruits and vegetables and contributes to food waste. Non-browning retains the sensory-nutritional profile and food quality of fruits and vegetables. Our research has the potential to increase market value of crops as non-browning enhances people's visual perception and appetite making the crop more acceptable to consumers. Non-browning after slicing locks its flavour and nutritional value. The yield increase (over 10% of control) contributes to the overall production of eggplant. The development of genome-edited crops for desirable traits mainly contributes to food security by increasing the yield, providing safe food, and reducing food waste.  ) and long-lasting yield of fruits in the edited plants. This is the first ever trial of genome-edited eggplant in field conditions. With the knowledge that PPO regulates the degree of browning in cut fruits, the research team targeted one of the genes encoding this enzyme, PPO2, in eggplants. In particular, they targeted this enzyme's copper ion A binding site (a conserved region across PPO genes in different species). The researchers used the CRISPR-Cas9 genome-editing system to mutate the corresponding location on the gene.
The study confirmed that no random mutations were introduced into the plants, and gene-edited eggplants that contained mutated PPO2 were identified. Surprisingly, the researchers discovered that mutation of this specific region of PPO2 resulted in multiple phenotypes in the progeny across generations. These included early seed germination, higher yield of eggplants, fewer seeds, decreased activity of the PPO enzyme, and reduced or no browning of the cut fruits harvested.

NEXT-GENERATION EGGPLANTS
The discovery of the pleiotropic nature of this gene is novel and of immense benefit to a plant breeder, given that different traits can be enhanced by mutating the same target. This ongoing research is now focused on field trials of the same edited crop. In field conditions, the initial results have been promising so far, and show high yields compared to non-edited crops.
Generating new varieties of eggplant that can be grown in the field without any foreign DNA -yet altered to produce higher quantities and nonbrowning fruits -is the aim of ongoing research by the KCGEB researchers. The discovery of the hidden pleiotropic nature of PPO2 will certainly pave the way forward for accelerating plant breeding to produce desirable traits such as high yield.

CRISPR-Cas9-based editing of an eggplant's enzyme reduces enzymatic browning and results in increased and
longer-lasting yields of fruits.
reinforce food security, especially in countries with less arable land.'

EGGPLANT EDITING
Genome editing has been used as a tool recently to alter the enzymatic browning of cut eggplant fruits. As part of ongoing research, Kottackal and colleagues published results from their recent study to show how CRISPR-Cas9-based editing The image shows the browning of juice from nonedited eggplant (left), with little browning of juice from the edited eggplant (two tubes on right).
In their research, Kottackal and colleagues have altered the enzymatic browning in cut eggplant fruits using genome editing.