SCIENTIFIC DEVELOPEMENT OF THE TICAR EVENT
Dr. Fernando Bravo Almonacid (CONICET)
Diseases caused by viruses affect the entire potato plant. They are transmitted vegetatively (by the use of diseased seed tubers), mechanically (by contact between leaves, operators or machinery) and, mainly, by aphid vectors (insects that transmit these viruses) (see FIGURE 1 (b)). There are no antiviral products for the control of plant viruses. The only means of control are agricultural management, the use of virus-free seeds and the use of insecticides to eliminate aphids (vectors).
FIGURE 1: Electron microscopy shows the particles of the PVY virus (a) and to the right the aphid vectors (b).
FIGURE 2: symptoms of PVY infection in leaves (a) and tubers (b).
FIGURE 3: Structure of Potato Virus Y (PVY)
An efficient way to introduce resistance to viruses is conventional genetic improvement. This is based on the cross between individuals of the same species that show different characteristics, and then select those specimens that present the virtues sought by the breeder. In the case of the potato, this is a slow and complicated procedure, given the genetic characteristics of the species. In addition, the Spunta variety (more than 90% of the production for fresh consumption in Argentina) produces very little botanical seed, which makes crossings even more difficult.
Resistance to viruses by genetic engineering
Biotechnological approaches are the most effective in introducing resistance to plant viruses. The technologies for this exist since 1986 and have been continuously perfected since then. In Argentina, the genetic transformation of the potato is available from 1989-90 at the INGEBI-CONICET (Research Institute in Genetic Engineering and Molecular Biology). In 1999 CONICET signed an agreement with Tecnoplant SA for the development of potato plants of the Spunta variety resistant to PVY.
Resistance mechanism
The plants have a wide range of defense devices against plant pathogens, but they do not have an immune system like that of animals, based on antibodies. In the case of plant viruses, the defense mechanism is highly selective and is based on recognizing the presence of the DNA or RNA of the invading virus and eliminating it. Each cell of the plant has this mechanism. When a virus enters the plant, a race starts between the virus and these defense mechanisms. Depending on who wins this race, a state of high resistance or immunity can be achieved. If the pathogen wins the race, the plant will get sick.
The technology applied in our case consists in permanently establishing this defense mechanism by directing resistance specifically against the PVY virus, a process similar to what a vaccine produces in the case of humans or animals, which activates and prepares the immune system to the defense against a certain pathogen.
The post-transcriptional silencing or PTGS (of English; Post-Transcriptional Gene Silencing), the modification introduced by genetic engineering in Spunta TICAR, affects only the regulation of a defensive mechanism of the plant itself and does not introduce any new characteristic in it, except the resistance to this particular virus. It is a process that allows the degradation of a specific RNA, in this case the PVY virus RNA.
FIGURE 4: Operation of the Spunta virus defense system (right) vs. TICAR (left).
Posttranscriptional silencing, a natural defense mechanism of plants against viruses
As seen in FIGURE 4 (left), when the PVY virus infects an TICAR plant it encounters the defense mechanism already established, since the plant produces double stranded RNA (dsRNA) that has homology to the virus sequence and is cut by the enzyme DICER into small fragments called siRNA. These siRNAs are the guide for a degradation complex (RISC) to eliminate RNA that has homology to these siRNAs, that is, the PVY virus. Instead, as seen in FIGURE 4 (right), when PVY infects a Spunta potato plant, the virus or its RNA can enter a cell and multiply to give rise to new viruses, consequently, the plant will get sick, since it could not trigger the defense mechanism (PTGS) in time. This mechanism has been used to achieve papaya plants resistant to the PRSV virus. These plants are grown and marketed since 1998 in Hawaii, and also approved in Canada and Japan. China also produces and markets PRSV resistant papayas since 2006. More recently, the same mechanism has been used to obtain apples and potatoes that do not suffer from browning, thanks to the silencing of the enzyme polyphenol oxidase, events already approved for commercialization in the United States and Canada. Also in Brazil, EMBRAPA, developed bean plants resistant to the BGMV virus, and obtained approval for commercialization.
Simplot Plant Sciences, the world's second frozen pre-fried cane industry, uses the same technology applied at Spunta TICAR and has obtained approval for its “Innate” brand potato in some key foreign markets, including Japan, and has several other applications of access to the foreign market pending. Simplot obtained approval to sell its first Russet Burbank generation in Japan during the month of August 2017. The first generation of Innate Burbanks, Atlantics and Ranger Russets - all grown to resist bruising, as well as avoid rusting after cutting and possessing Low levels of acrylamide, which is a potentially harmful chemical generated during the frying of some starchy foods - was approved last spring in Australia and New Zealand. The company has also requested the approval of the first generation in China, South Korea, Taiwan, Malaysia, Singapore and Mexico, and plans to apply soon in the Philippines. Simplot has already received approval from the second generation of Innate varieties in Canada and the US.
In the US, Simplot recently sold out its inventory of fresh Innate potatoes, marketed under the Russet white label, for the third consecutive year. Innate was distributed in approximately 4,000 supermarkets in 40 states. Retailers and food service professionals have been willing to pay a premium for Innate potatoes, because they avoid bruising, even at the end of the season when the problem is widespread.
Development of TICAR Spunta, field trials, environmental safety and food safety. Commercial release
Once obtained in the INGEBI laboratory, a large number of events potentially resistant to PVY (more than 400) were cloned in vitro to obtain microplantulas. Each of them was a clone with potential resistance to PVY. The seedlings were acclimatized and challenge tests were carried out with the virus in greenhouses (discarding 295 of these clones) and then in the field (discarding another 83 clones), which allowed to select the clones of better behavior against the disease and were led to new trials in the field, in several locations, to conduct performance and performance studies in production conditions.
These studies were carried out under strict controls on all processes by the National Directorate of Agricultural Biotechnology, under the former Ministry of Agribusiness of the Nation, and especially the monitoring and permanent evaluation of CONABIA [1].
In collaboration, INGEBI-CONICET and Tecnoplant staff carried out the field trials that allowed us to select two promising events, resistant to the virus and move towards commercial liberation. Successive greenhouse selections and nine field trials in four different locations in the country during 1998 and 2001 (see FIGURE 5) were defining the most promising clones until reaching TIC-AR 233-5, which was selected to enter Phase II of CONABIA, in which it was necessary to complete a very important series of requirements and regulations, demonstrating the environmental biosecurity of the event.
FIGURE 4: Operation of the Spunta virus defense system (right) vs. TICAR (left).
The chosen event was immune to PVY in trials during which 85% of Spunta control plants were infected. All trials were authorized and approved by CONABIA. The resistance to the virus was confirmed in several subsequent tests in which the absence of infection in the TIC-AR plants could be verified, while the Spunta control plants showed different degrees of infection.
In parallel, the analyzes of food aptitude of the TIC-AR 233-5 variety were performed. Different phenotypic and biochemical characteristics were analyzed that demonstrated that the composition and nutritional value of the TIC-AR 233-5 variety are identical to those of the original Spunta variety.
To increase the environmental safety levels of our Spunta TICAR event, gene flow tests were carried out in the field, which allowed us to verify the absence of horizontal transfer to related species and other potato varieties. All this information was published in an article, Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina - Bravo Amonacid, F., et al in Transgenic Research.
Additionally, the entire genome of the TIC-AR 233-5 variety was sequenced. This genomic sequence was compared with the reference genome of the potato, which allowed to confirm the consistency of the modifications made, verify the place where the insertion occurred and ensure the non-occurrence of other unwanted modifications.
During 2014, the Agricultural Markets Department of the former Ministry of Agribusiness of the Nation made a positive market impact report. Also, before the arrival of a new government in 2017, a new impact evaluation was carried out on the market, reaching the same conclusions as those obtained in 2014.
On April 29, 2015, CONABIA approves the TICAR transgene event, thus culminating with the fulfillment of its demanding requirements.
On October 6, 2015, Resolution 399/2015 of the former Ministry of Agriculture, Livestock and Fisheries of the Nation was published in the Official Gazette of the Republic of Argentina, approving the commercialization of the TICAR transgenic event, subject to the presentation of additional information required by SENASA. This information was completed and by July 2018, SENASA approved its demanding requests for information and analysis, which guarantee the food safety of the event for consumption.
On August 8, 2018, by Resolution 65/2018 the former Ministry of Agribusiness of the Nation releases for its commercialization the transgenic event of potato resistant to PVY Spunta TICAR 233-5.