Periodic Reporting for period 1 - MeloCRISP (Implementation of CRISPR/Cas9 technology in melon to edit fruit ripening and CMV resistant genes)
Okres sprawozdawczy: 2018-05-15 do 2020-05-14
Worldwide, up to 40% of vegetables and fruits are lost after harvest, which is translated into an economic cost estimated in 4 billion EUR for the EU. The main causes of these loses are mechanical, physiological, pathological, and environmental factors. Among them is fruit ripening, which is a biological process that affects post-harvest durability of fleshy fruits. During ripening the fruit suffers several metabolic changes which affect its softness, colour, flavour and sweetness turning into an attractive fruit for the consumers during a narrow time window.
Melon is a crop of high economic importance in Spain, since it is the main melon producer in the EU and the first world exporter.
In this scenario, the MeloCRISP project proposed to progress our current understanding on the genomic control underlying fruit ripening using melon as a model species.
We studied the role of previously identified candidate genes involved in the regulation of fruit ripening by using the latest technologies in the field of gene editing: ¨CRISPR/Cas9¨. Target genes were edited and the role of the genes was studied by comparison of the fruit ripening behavior of the edited and non-edited plants.
Overall, MeloCRISP is a multidisciplinary project in which we studied the genetic control of a trait that affects fruit quality and production. To our knowledge, this is the first time that the CRISPR technology has been implemented on genes involved in agronomically important traits in melon. We have obtained edited plants that validate the role of the target genes in fruit ripening. These results will become a valuable tool for melon breeding programs, which will be able to implement CRISPR/Cas9 technology, and will end up having an impact on the producers and the consumers.
Melon is a crop of high economic importance in Spain, since it is the main melon producer in the EU and the first world exporter.
In this scenario, the MeloCRISP project proposed to progress our current understanding on the genomic control underlying fruit ripening using melon as a model species.
We studied the role of previously identified candidate genes involved in the regulation of fruit ripening by using the latest technologies in the field of gene editing: ¨CRISPR/Cas9¨. Target genes were edited and the role of the genes was studied by comparison of the fruit ripening behavior of the edited and non-edited plants.
Overall, MeloCRISP is a multidisciplinary project in which we studied the genetic control of a trait that affects fruit quality and production. To our knowledge, this is the first time that the CRISPR technology has been implemented on genes involved in agronomically important traits in melon. We have obtained edited plants that validate the role of the target genes in fruit ripening. These results will become a valuable tool for melon breeding programs, which will be able to implement CRISPR/Cas9 technology, and will end up having an impact on the producers and the consumers.
In order to understand the role of candidate genes related to fruit ripening, we have used the latest technology in gene editing: ¨CRISPR/Cas9¨.
We used the ´knockout gene strategy´ in melon, which in addition of being an important economic crop, became a good model to study this trait since it contains genotypes from both major groups of ripening behavior. These groups are climacteric fruits, in which ripening is characterized by a burst of ethylene; and non-climacteric fruits, in which ethylene production does not increase during the ripening process.
Ethylene is a plant hormone which regulates many processes of the plant, being one of them fruit ripening. In climacteric fruits, the increase of ethylene triggers plant metabolism adaptations that lead to changes in fruit colour, flavour and texture.
In the MeloCRISP project, we studied two ripening candidate genes in a climacteric genetic background (cantalupensis melon type ´Vedrantais´). The knockout of these genes by CRISPR /Cas9 resulted in several edited plants for each of the two genes. The selected edited plants were pollinated to produce seeds and the second generation of the edited plants was grown under glasshouse conditions for evaluation of their ripening behavior in comparison with non-edited climacteric plants. To better understand the role of these genes, we daily monitored the plants by measuring the ethylene production in attached fruits (as an indicator of climacteric ripening process) as well as several ripening associated traits such as aroma production, external color change, abscission layer formation in the pedicel of the fruit, the firmness, and the harvest time (date when the fruit abscised from the plant).
The edited plants showed an earlier ripening profile and a different profile of fruit ripening and ethylene production compared to climacteric not edited plants. For one of the target genes, ethylene production was higher compared to the control plants whereas for the other gene no changes were observed on the quantity of the hormone. However, for both edited plants, ethylene production started earlier than in the control plants revealing the role of these genes in the regulation of the ethylene production. The final results showed that both genes have a role in the ripening process affecting the initiation of the ripening process but without affecting other important traits such as firmness.
A scientific article with these results is currently being drafted and it is expected to be published in an open access peer-reviewed journal. The project background, concept and some preliminary results have also been communicated to non-specialized audience, including workshops for high-school students.
We used the ´knockout gene strategy´ in melon, which in addition of being an important economic crop, became a good model to study this trait since it contains genotypes from both major groups of ripening behavior. These groups are climacteric fruits, in which ripening is characterized by a burst of ethylene; and non-climacteric fruits, in which ethylene production does not increase during the ripening process.
Ethylene is a plant hormone which regulates many processes of the plant, being one of them fruit ripening. In climacteric fruits, the increase of ethylene triggers plant metabolism adaptations that lead to changes in fruit colour, flavour and texture.
In the MeloCRISP project, we studied two ripening candidate genes in a climacteric genetic background (cantalupensis melon type ´Vedrantais´). The knockout of these genes by CRISPR /Cas9 resulted in several edited plants for each of the two genes. The selected edited plants were pollinated to produce seeds and the second generation of the edited plants was grown under glasshouse conditions for evaluation of their ripening behavior in comparison with non-edited climacteric plants. To better understand the role of these genes, we daily monitored the plants by measuring the ethylene production in attached fruits (as an indicator of climacteric ripening process) as well as several ripening associated traits such as aroma production, external color change, abscission layer formation in the pedicel of the fruit, the firmness, and the harvest time (date when the fruit abscised from the plant).
The edited plants showed an earlier ripening profile and a different profile of fruit ripening and ethylene production compared to climacteric not edited plants. For one of the target genes, ethylene production was higher compared to the control plants whereas for the other gene no changes were observed on the quantity of the hormone. However, for both edited plants, ethylene production started earlier than in the control plants revealing the role of these genes in the regulation of the ethylene production. The final results showed that both genes have a role in the ripening process affecting the initiation of the ripening process but without affecting other important traits such as firmness.
A scientific article with these results is currently being drafted and it is expected to be published in an open access peer-reviewed journal. The project background, concept and some preliminary results have also been communicated to non-specialized audience, including workshops for high-school students.
To our knowledge, this is the first time that genes involved in agronomic important traits are edited using the CRISPR technology in melon. Our results suggest that both candidate genes used in this study contribute to the regulation of a complex trait such as fruit ripening.
The implementation of the CRISPR technology in this species and the inherence of the editions to the following generations is of high interest and a valuable resource not only for researchers but also for breeders. We expect that our results could be transferred to the seed market and breeding programs, even though nowadays CRISPR technology is still considered as GMO in Europe and thus it is not possible to transfer it yet to the market.
The implementation of the CRISPR technology in this species and the inherence of the editions to the following generations is of high interest and a valuable resource not only for researchers but also for breeders. We expect that our results could be transferred to the seed market and breeding programs, even though nowadays CRISPR technology is still considered as GMO in Europe and thus it is not possible to transfer it yet to the market.