The genes that save basil from disease

The head of the Laboratory for the Genetics of Plant Diseases at Bar-Ilan University, the winner of the Israel Prize Professor Yigal Cohen and the chief geneticist at the laboratory, Dr. Yariv Ben Naim and the members of the Genesis Seed Company who succeeded in improving the edible basil varieties with a gene for resistance to hop disease from inedible wild varieties and their normal hybridization With cultured plants creates sterile offspring

The next time you eat ravioli in pesto sauce, know that this pleasure is almost denied you. A severe fungal disease causes serious damage to basil all over the world, making it inedible and uneconomical for farmers.

The hop blight (Peronospora belbahrii) appeared a decade ago in Switzerland and since then has erupted as a global epidemic and is found everywhere where sweet basil (Italian basil) is grown. It appeared in Israel for the first time in 2011. The symptoms of the disease were manifested in leaf deformation, chlorosis (yellowing), the appearance of black spores on the underside of the leaves and in advanced stages death and leaf fall. Researchers at Bar Ilan University and the Genesis Seed Company are now reporting that they have succeeded in developing a strain resistant to the fungus that causes the disease without the use of genetic engineering. Bar Ilan University is commercializing the discovery through BIRAD Company for Research and Development Ltd. (Bar Ilan University's knowledge commercialization company).

The head of the laboratory for the genetics of plant diseases at Bar-Ilan University, Israel Prize laureate Professor Yigal Cohen and the chief geneticist at the laboratory, Dr. Yariv Ben Naim, explain in an interview with the Hidan website that the basil plant is an important agricultural branch in Israel, which makes up about 50% of the basket of spices for export estimated at 70 million euros per year. "The Israeli basil is considered high quality, it has a good aroma, texture, color and a long shelf life."

"The disease is spreading all over the world," says Yigal Cohen, "when it arrived in Israel, farmers lost a lot of money, in the destruction of crops and dozens of shipments that were rejected because when they arrived at their destination it turned out to be black. Since my laboratory deals with this type of disease, we set out to define the problem and look for several directions of work to eradicate it, one in the short term - sprays and suitable agrotechnical treatments, and in the long term we looked for a way to provide the plant with genetic resistance to the disease. As for the sprays, here there is a limitation that the regulator forbids the farmer to spray spice plants on a wide scale and rightly so. We would like to reduce their use for the sake of public health."

Genetic resistance means that certain plants or varieties have unique genes that do not allow the fungus to develop on it. When the fungus tries to penetrate the plant tissue, the genes activate a number of cellular and molecular mechanisms, which suppress the fungus and do not allow it to establish itself in the plant.

The first thing we did was to scan all the commercial varieties of basil (Ocimum basilicum) in the world. Unfortunately, we did not find a single variety whose genetic variation gave it such resistance that we could use it for cultivation purposes. Even in the scan of wild types of this species, we did not find any resistance and therefore, we ordered from the world gene banks all the species they hold belonging to the genus Ocimum, to which the cultivated basil also belongs. Out of hundreds of wild types we were able to locate individual types that were resistant to the disease. We were very happy. We raised them, multiplied them, but these types belonging to other species were different in aroma, taste and texture from the basil that consumers are familiar with. The next step was to try and transfer the resistance genes from the wild types to the cultivated basil. But it should be remembered that these are distant "relatives" of the basil and unfortunately only a few have created a hybrid with the cultivated plant.
Dr. Yariv Ben Naim, geneticist and plant disease researcher, explains that even when the hybridization process was successful, additional difficulties were discovered. "We crossed the resistant varieties with the cultured variety but in all cases we got sterile offspring, such as a mule which is the sterile offspring of a horse and a horse. This happens because when distant species are crossed, the division of the mother cell into gametes (gametes) is prevented due to the imbalance in the number of chromosomes. Therefore, in order to get caged plants, it is necessary to repeat the process of pairing the parents every time."

Although gametes are not normally formed in intersex hybrids, there are exceptional cases in which gametes are formed, but those with an unbalanced chromosomal load. The best known example in humans is Down syndrome. In this case, the zygote (embryo) is formed from the union of one normal gamete (23 chromosomes) and a second gamete that was formed asymmetrically and has an extra chromosome (24 chromosomes). Usually when there is a situation of excess or lack of chromosomes the fetus does not develop and is aborted. Only in a small number of cases, for example in Down's syndrome, does a fetus develop, but this pays a price for the genetic error. In order to get basil plants that are indeed fertile, we have developed a method in which we fertilize the caged flowers in a forced way with the pollen of the culture species, and after 4-6 days the flowers are cut off, the fertilized eggs are extracted and they are grown in petri dishes on special growing media that include hormones and nutrients. The extraction process is done at an early stage to prevent miscarriage. But here, it turns out that in the genus Ocimum there is another phenomenon that complicates matters further, polyploidy - a phenomenon in which there is no single genome like we humans have, but a situation in which one cell has two, three and even four subgenomes. This phenomenon makes the improvement process difficult and is known and known in wheat, corn and potatoes.
In the extraction process, tens of thousands of eggs were fertilized and 115 mature plants were obtained from them. Despite the low success rate, it turned out that the gene for resistance is dominant and is found in two subgenomes. From those 115 plants we managed to reach a first generation that was both resistant and fertile. However, the commercial quality was poor and we had to cross these plants with the cultivars while keeping the gene for resistance on only one subgenome. In cooperation with the chemistry department of the university, we succeeded after several generations in getting a fertile basil, absolutely resistant and with a quality aroma. Our method makes it possible to transfer any trait from the wild species and enrich the genetic pool of the basil. Already today we are successfully working on the transfer of additional genes for resistance.
The Genesis Seed Company was interested in the intellectual property created by the researchers and began trading the resistant varieties with Bar Ilan University's BIRAD Company.
Dr. Yitzhak Nir, the owner and CEO of "Genesis Seeds" and Dr. Arnon Brand, of Genesis Seeds explain how they got into the picture.
Dr. Nir: "Genesis Seeds is a private company established in the Negev 20 years ago with the goal of creating an agricultural industry that also includes intellectual property, and at the same time we started producing seeds for the world market. The company is engaged in the cultivation and production of vegetable seeds, spices and flowers. All the growing processes and the production of the seeds are done only in Israel using the approved methods for environmentally friendly organic crops. The company markets to the seed companies only seeds produced by it."
Basil seeds make up about a third of the company's turnover and it has a great reputation for supplying high quality basil seeds. Most of the varieties marketed by the company over the years were cultivated in collaboration with Prof. Nativ Dodai, director of the spice plant department at the Volcani Institute. In 2016, the collaboration between Genesis Seeds and Bar-Ilan University began in imparting hop resistance to cultivated basil varieties. For us, this is a breakthrough on a global scale that will help basil growers deal with the hop disease. This is a rapid course of cultivation and implementation of the research results. In the fall of 2017, we produced the foundation seeds for the breeders at the Genesis Seed Research Center in Bashlim, and already in the spring of 2018, the production areas of the first enclosure 'Prospera' (F1) were planted in an organic area of ​​100 dunams in the Jezreel Valley. The growth was very successful and was collected during the month of September. After cleaning, we expect about 10 tons of seeds intended for distribution in Israel and the world market. During 2018-7, samples were sent to a large number of customers and good results were obtained that confirmed the quality of the variety and its full resistance to the hop disease.

invent a new plant
Dr. Brand explains the uniqueness in cultivating the strain developed at Bar-Ilan University. "What Bar-Ilan and we did was actually to invent a plant with new properties, when the natural genetic variation does not exist. The Bar-Ilan team searched hundreds of cultivars and relatives of cultivated basil, but resistance was not found. This trait had to be reinvented in cultivated basil through crossbreeding with resistant wild species from Africa. We had to overcome a barrier that made it very difficult for the possibility of natural hybridization between the resistant species and the cultured species. They registered a patent for the method and the resistance gene, while we registered the variety in the registration of breeders' rights in Europe, and in the process of registration in the USA. In the coming year we intend to trade several more new varieties suitable for the European and American markets. Experimental seeds were sent to several countries such as Australia, Germany, the Netherlands, the USA and Italy - the "Basil capital of the world". The results speak for themselves with very positive feedback. Most of the world was completely skeptical about resistance against hops because previous attempts to introduce "tolerant" varieties did not provide resistance to the disease. This is the first time that farmers from all over the world see complete resistance without marks on the produce."

Dr. Francis Shalit, VP of business development of Birad said: "Birad (the implementation company of Bar-Ilan University) is proud to use the knowledge and accumulated experience of Prof. Yigal Cohen, a world-renowned expert in pathopathology at Bar-Ilan University and winner of the Israel Prize for Agricultural Research , to benefit agriculture (and farmers) in Israel and the whole world. His extraordinary developments have helped and are helping to improve global problems that afflict the world of agriculture - plant diseases caused by pests. There is a thought to perfect the unique method of Cohen and his laboratory team that led to the development of basil varieties that are also resistant to other crops."

Based on the knowledge and genetic background discovered, the "Genesis Seeds" company began to market the new basil varieties under the name 'Prospera' (a combination of the name of the disease Peronospora and prosperity), and they represent an unprecedented breakthrough in terms of resistance against the hop plague. These varieties are in the testing process in Israel and around the world and are being tested for suitability to the various breeding methods. These days, the Genesis company began to produce the seeds in organic conditions on a commercial scale to market them in Israel and around the world. The plants were planted at the company's production farm in the Jezreel Valley, and the seeds will be produced at the company's production plant in Ashlim in the Negev.