Taac! The first Tea vines, resistant to downy mildew thanks to genetic editing and the application of the molecular scissors Crispr/Cas9, were planted in the open field on September 30 at the San Floriano University Campus in Valpolicella, in the Municipality of San Pietro in Cariano (Vr).

They are the result of the work of the Department of Biotechnology of the University of Verona coordinated by Mario Pezzotti and the university spinoff EdiVite. This is only the second agrarian Tea species to emerge from the closed environment of laboratories in Europe, after the blast-resistant rice developed by the University of Milan and immediately vandalized last June.

Field experimentation only in Italy

The field experimentation was able to start thanks to the first openings of 2023 and the amendment to the agriculture decree which last July extended the authorization for field experimentation of Tea (Italy is so far the only country in Europe to have granted it) until December 2025.

Senator Luca De Carlo, the first signatory of the amendment (together with colleague Giorgio Bergesio), claimed the correctness of a measure in line with the goal of food sovereignty. "Agriculture - he said - should not be the battleground of a sterile ideological opposition." "We need more pragmatism and lightness," reiterated De Carlo, who admitted to always being an admirer of the movie "Il ragazzo di campagna" by Renato Pozzetto ("Taac!").

Time to dig

Time to dig, then: to help the Biotechnology department students in the historic first planting of Tea vines, practically the whole agricultural world was there. Ettore Prandini, president of Coldiretti: "we have historically been averse to GMOs because they are the tool that has concentrated the food resources of the entire planet in the hands of just three multinational corporations, while Tea achieves the opposite result of protecting the distinctiveness and biodiversity of our productions."

Cristiano Fini, president of Cia Agricoltori Italiani: "The crisis of Italian agriculture is a production crisis. Climate change and the spread of diseases like golden flavescence reduce yields and farmers' incomes. Research and innovation are the only hope, but we cannot expect Tea to work miracles." Carlo Piccinini, president of Alleanza delle cooperative agroalimentari: "Europe was the cradle of enlightenment and trust in science, now it is only the den of superstitions. Those who invest in research and innovation win, those who try to chase scientific advances with regulations like the one that distinguishes between category 1 and 2 Tea are destined to lag behind."

Christian Marchesini, president of the Valpolicella Consortium, representing Confagricoltura: "Our territory is anthropized where sensitive varieties like Corvina must face downy mildew, powdery mildew, and esca disease while guaranteeing the highest level of sustainability. We can only save ourselves by investing in research."

Shortly before, Minister Francesco Lollobrigida, despite being in a video connection from the "Terra Madre" event in Turin organized by SlowFood, reiterated the ministry's commitment to new precision biotechnologies: "Italy has always been at the forefront with examples like that of the geneticist Nazareno Strampelli whose valuable work in genetic improvement of wheat has ensured food security for a rapidly growing global population, but it was not without criticism." "We need to work to ensure a future for brilliant agricultural biotechnologists who are currently forced to find work abroad."

Regulatory constraints still not overcome

A strong and widespread consensus that clashes with the need to protect the experimentation site with nets and barbed wire and with the requirement to indicate at the entrance of the vineyard that GMOs are being experimented with.

"The European regulation - Pezzotti admits - which was supposed to free NBTs (New Breeding Techniques) from the suffocating constraints of GMOs is still in a stalemate, but it is not the only legislative limit in Europe that blocks this promising technique." "The current Upov rules for obtaining plant breeders' rights cannot be applied to clones obtained from genome editing that do not show morphological differences but only very small genetic variations."

A limitation that would not allow to reap the rewards of the substantial investments and efforts of researchers. They have undoubtedly achieved a remarkable result: no one expected to see Tea vines in the field so quickly. A step that would allow the collection of data for a quick approval, in case the regulatory issue of the new European regulation is resolved.

"It would be the culmination - Pezzotti recalls - of an effort that started a long time ago: twenty years have passed since we started studying the vine genome." "The success of research projects that led to the first decoding of the genome allowed us to analyze its structure and functions to understand how these findings can be applied to a complex system like that of a tree plant." This has led, on one hand, to the possibility of exploiting MAS, marker-assisted selection in genetic improvement projects through recurrent crossing, and on the other hand, according to Pezzotti, to the possibility of protecting the vines most linked to our territorial viticulture through Tea.

Regeneration from protoplast

The working group of the University of Verona has achieved the goal through regeneration from protoplast, a "DNA-free" method that has been patented by EdiVite.

Only 12 years have passed since Jennifer Doudna and Emmanuelle Charpentier, the two researchers awarded the Nobel Prize in Chemistry, discovered and understood how to use Crispr/Cas9, these molecular scissors capable of intervening at the level of a single nitrogen base, for genetic editing.

"Applying - says Sara Zenoni, professor of Agricultural Genetics at the University of Verona - this system to tree plants, and in particular to the vine, was not easy." The main problem remains being able to regenerate a whole plant from a single cell in which the desired mutation has been induced.

The method patented by Verona involves starting from the somatic cells of the anthers or pistils of the vine flower, a tissue capable of regressing to a meristematic state forming embryogenic calli. These are treated at a very early stage to isolate, once the cell wall has been removed, the individual protoplasts.

The Crispr/Cas9 complex, composed of a protein and RNA, works like a precision molecular scissors and can be "designed" if the exact sequence of the target gene is known. In the model patented by EdiVite, the complex is able to enter, without using Agrobacterium as a vector, into the nucleus of a single protoplast, positioning itself precisely on the DNA chain and cutting it. When the cell intervenes to repair the sequence, mutations are created that effectively alter DNA transcription and silence gene expression, such as protein synthesis.

Susceptibility genes and resistance genes

The gene operated on to obtain the resistant Chardonnay planted in Verona is DMR6, one of the known susceptibility genes to downy mildew. These are genes involved in the interaction mechanism between pathogen and plant, they do not trigger a rapid hypersensitivity reaction as in the case of resistance genes present in Piwi varieties today, but the lack of their expression inhibits the onset of infection. The level of resistance that can be achieved is estimated, in the case of downy mildew, to be about 40%. This percentage could be improved by trying to add resistance genes through cisgenesis, but in this case, with the current regulation proposal, the modification would exceed the 20 nucleotide threshold and fall into the category of type 2 Tea, with the obligation to label them as GMOs.

Meanwhile, science progresses and EdiVite, as Zenoni explains, is already working on Glera and other varieties, as well as on modifying the susceptibility gene Mlo for powdery mildew.

In these latter cases, however, an additional effort is needed to overcome the problem of so-called recalcitrant varieties, all those that currently have an extremely low protoplast regeneration rate.

What is EdiVite

EdiVite is the university spinoff born within the Department of Biotechnology of the University of Verona, with the support of academic partners and private partners from the wine production world. The goal is to produce vines more resistant to pathogens in order to reduce the use of phytosanitary products necessary for vineyard defense. To this end, the Verona startup has developed and patented the application of DNA-free genomic editing in the vine. The first regeneration of a vine from a single cell took place in the San Floriano laboratories in 2019 and this spurred the foundation of the spinoff in 2020. Two years later, in 2022, it was possible to develop the first plant edited with a specific mutation in the DMR6 gene of susceptibility to powdery mildew.

After another two years, EdiVite was the first entity to obtain authorization for the experimentation of edited vines in the field from the Ministry of Environment and Energy Safety with the start of the Vitea1 project.