Valuable agronomic traits of chufa ( Cyperus esculentus L.) accessions from the VIR collection: methods of preparing nodules for long-term storage

germination by 15–16%. When stored under low positive (+4°C) or negative temperatures (–18°C and –196°C), their germination percentage remained unchanged. The results of our research show that the chufa collection accessions manifest signi f icant genetic diversity in their bio-chemical composition. Chufa accessions have been selected for a set of valuable agronomic features. The selected accessions can be used as source material in breeding programs. A technique was developed for measuring germination percentage, and preparation for long-term storage was carried out. The optimal age of the shoots from chufa nodules to assess their germination energy was found to be four days, with eleven days for germination percentage assessment. It was shown that three-year storage of chufa nodules at +20°C caused a decrease in their germination by 15–16%. When stored at low positive (+4°C) or negative temperatures (–18°C and –196°C), their germination percentage remained unchanged.


Introduction
Cyperus esculentus L. is a perennial herbaceous plant of the Cyperaceae family, which naturally grows in the Mediterranean countries and in Africa, along the banks of the Nile. The nodules of this plant are considered one of the earliest food sources known to mankind. It is recognized that they were cultivated by the ancient Egyptians, starting from 5000 BC (Allouh et al., 2015). The C. esculentus nodules are commonly known by several names, such as chufa, ground almonds, and tiger nut. Chufa is cultivated in some European countries: the Netherlands, Switzerland, Germany, Poland, Hungary, as well as in some African countries: Mali, Benin and Ivory Coast, as an oilseed and nut-bearing plant. It is grown on an industrial scale in Spain.
In agriculture, it is cultivated as an annual plant. C. esculentus is the only cultivated species of the genus Cyperus. Its valuable nutritional qualities are determined by the content of proteins, oil, carbohydrates and micronutrients in the nodules (Nizova, Kon'kova, 2008;Codina-Torrella et al., 2014). Micronutrients include potassium, phosphorus, magnesium, calcium, sodium, iron, zinc, copper, and vitamin C (Suleiman et al., 2018). Chufa oil is used for food purposes. Oleic acid dominates in the oil composition of chufa: its content is comparable to that in olive oil (Arafat et al., 2009). Chufa oil also contains considerable amounts of essential fatty acids, phytosterols, phospholipids, fat-soluble vitamins, and tocopherols (Ezeh et al., 2014;Shakhova et al., 2014). According to the data produced by M. A. Allouh et al. (2015), chufa consumption increases the level of testosterone in human blood. The experiments by C. Imo et al. (2019) showed that even ethanol extracts from chufa did not produce an expressed negative effect on animal liver function. Using chufa as a natural preservative in confectionery, bakery, meat and other food industries is considered quite promising (Kuznetsova et al., 2019;Glotova et al., 2010). Studying the possibility of isolating such natural bioactive compounds with their subsequent use as preservatives to prolong the shelf life of natural products is a promising trend for the development of food industry. Whole-ground chufa nodules were reported to have an extensive shelf life, despite their significant oil content. The research performed by I. V. Bobreneva and A. A. Baioumy (2019) ascertained the possibility of using chufa as a dietary supplement for preventing and normalizing cardiovascular diseases and confirmed its potential for the food industry in increasing the shelf life and nutritional value of various types of meat products. The optimal amount of chufa in the composition of meat products is 5% (Bobreneva, Baioumy, 2019).
The carbohydrate composition in chufa favors its use in the diet of patients suffering from metabolic syndrome, including diabetes mellitus (Sabiu et al., 2017). Due to its unique properties, chufa is an excellent object for inclusion in the list of Biological and Technical Life Support Systems as the main link that regenerates food, water and air in closed space systems (Motorin et al., 2009;Shklavtsova et al., 2014).
Chufa is also widely used for producing the traditional Spanish drink Horchata de chufa (Butova et al., 2019). It is a white-colored refreshing drink with a pleasant taste. The production of horchata has great economic importance for the food industry in Spain, especially for Valencia, the main region of chufa cultivation. About 90% of the chufa harvest is used for making horchata, with an annual consumption of up to 50 million liters. Horchata production has an estimated retail value of 60 million euros per year (Roselló-Soto et al., 2018a;Roselló-Soto et al., 2018b).
Analyzing physical and chemical properties of chufa oil, such as density, viscosity, or acid, iodine and cetane values, shows that chufa oil can be used for biodiesel production (Sidohounde et al., 2018). Chufa flowers rarely, forming small seeds, so it is propagated vegetatively, with nodules developed on underground shoots from special swellings, in the end of the tillering stage. Considering great practical interest in this crop, the problem of its preservation in plant genebanks is very important. Plant genetic resources are preserved in genebanks usually as seeds, but in the case of chufa it is quite difficult to obtain seeds, especially under northern latitudes, so it seemed relevant to explore the possibility of chufa germplasm conservation in the form of nodules, as the propagules of chufa plants. With this in view, the purpose of this work was to study valuable agronomic characteristics of chufa nodules and develop methods for determining nodule germination and preparation for long-term storage under the conditions of a genebank, including the analysis of the impact of different storage temperatures on germination.

Materials and methods
Eighteen chufa accessions of various origin from the VIR collection served as the target material for the study. The field study was conducted in 2010-2012 in the environments of Krasnodar Territory, Russian Federation (Kuban Experiment Station of VIR). Protein and oil content was measured according to the guidelines on the methods of quality control and safety of bioactive food additives (R 4.1.1672-03…, 2004). The fatty acid composition analysis was performed in accordance with standard methods (IUPAC…, 1979;GOST R 512677-2006…, 2007. The composition of fatty acid methyl esters (FAME) was studied using the CARLO ERBA STRUMENTAZIONE IIRGCS 5300 chromatograph (Agilent Technologies, USA). Vitamin E content was measured in accordance with the guidelines on the methods of analyzing the quality and safety of food products (Skurikhin, Tutelyan, 1987).
To assess germination, the nodules were sprouted up in wet paper rolls in a thermostat at a temperature of +25°C. Methods for determining germination percentage basically conformed to those prescribed for large seeds in the Interstate Standards (GOST 12038-84…, 2016). Moisture content in the nodules was measured according to the Interstate Standards (GOST 12041-82…, 2004). Prior to their placement for storage, the nodules were dried according to the technique recommended for the FAO genebanks (Genebank Standards…, 2014) in the drying room at a temperature of +18…+20°C and a relative humidity (RH) of 10-12%. Then, the nodules were hermetically packed in vacuum-laminated foil bags, 25 nodules per bag. Since there is currently no method for storing chufa nodules, sample packages (several packages for each variant) were stored under different temperatures (+20°C, +4°C, -18°C, and -196°C).
The analysis showed that oil content in chufa accessions varied from 13.1 to 21.06 g/100 g, and protein content from 6 to 10% (Table 2). The following accessions were identified for the highest oil content: k-13 (Germany) with 20.5 g/100g of the product, k-21 (Belarus) with 21.01 g/100 g, and k-11 (Mali) with 21.06 g/100 g. Protein content in chufa nodules is higher than in starchy root crops, such as cassava or sweet potato, being comparable with that in cereals, such as rice and sorghum (Suleiman et al., 2018). The following accessions were identified as the best in terms of protein content in nodules: k-18 (Ivory Coast) with 6.75%, k-11 (Mali) with 6.76%, and k-9 (Bulgaria) with 6.79%. Chufa oil contains large amount of vitamin E: according to our data, the range of its variability was 19.23 to 35.23 mg/100 g of oil.
Nutritional and physical properties of oil largely depend on the amount and composition of tocopherols (vitamin E)bioactive compounds that increase the oil's nutritional value. Moreover, tocopherols are natural inhibitors of highly non-limiting fatty acids and other easily oxidizable substances (Ezeh et al., 2014). The following accessions were identified for their high total vitamin E content: k-7 (Poland) with 35.23 mg/100 g, k-11 (Mali) with 26.54 mg/100 g, and k-19 (Ivory Coast) with 26.35 mg/100 g. The fatty acid composition of chufa oil is characterized by high content of oleic acid. In the course of our research, k-20 (Ukraine) was identified for the highest content of oleic acid in oil (71.29%). Palmitic acid content in the studied accessions varied from 12.86% to 14.53%, stearic acid from 2.83% to 5.78%, linoleic acid from 9.72% to 11.93%, linolenic acid from 0.15% to 0.29%, and arachidic acid from 0.39% to 0.80% (Table 3).
Individual accessions were selected according to a set of valuable agronomic traits: k-7 (Poland) for its yield, 1000 nodule weight, oleic and linoleic acid contents, high content of vitamin E, and low SFA content; k-9 (Bulgaria) for its 1000 nodule weight, and high protein content; k-11 (Mali) for  (2), 2021 • N. G. KON'KOVA • G. F. SAFINA high oil, protein, vitamin E and oleic acid contents; k-12 (Benin) for earliness, yield, and oleic acid content; and k-13 (Germany) for oleic acid and MUFA content. Our research showed that the oil, protein and total vitamin E contents were different in accessions of African and European origin (Codina-Torrella et al. 2014), which was confirmed by the studies where the metabolomic method had been applied to reveal differences in the geographical origin of chufa (Rubert et al., 2018). In European accessions, the oil content ranged from 17.21 to 21.05 g/100 g of the product, protein from 6.53% to 11.21%, and vitamin E from 22.34 to 35.23 mg/100 g of oil. African accessions contained 13.21 to 20.06 g/100 g of oil, and 5.32% to 7.04% of protein, while their vitamin E level varied from 23.85 to 26.54 mg/100 g of oil. No significant differences in the composition of fatty acids were found between the two groups of accessions. The nodule sprouting technique was tested on one of the studied accessions: k-14 in the VIR catalogue (Ivory Coast). During the process of germination, the emergence of shoots and roots was observed. Germination energy was assessed on the shoots and roots that reached the nodule size. Assessment of germination percentage took into account robust shoots (from one to three), twice or more times longer than the nodule, with well-developed adventitious roots (Figure 1, 2).
Germination percentage was assessed on the 7th and 11th days. The results showed that seven days were not enough to assess germination, because new full-fledged seedlings continued to appear after that. The optimal time for determining the germination energy was found to be 4 days, and germination percentage 11 days. The initial values of germination energy and percentage were 20.0 ± 2.5% and 80.0 ± 2.5%, respectively. After measuring the germination percentage, the seedlings were planted into pots with soil for greenhouses (turf: 91.7%, limestone flour: 7.1%, NPK fertilizer: 1.2%) and left for further growth to make sure that they would grow into full-fledged plants (Figure 3).
Then the plantlets were transplanted into the open ground. The analysis of yield was made in the end of the growing season (Figure 4).
Nodule preservation experiments were carried out using the example of k-14, an accession from Ivory Coast. One of the requirements for successful long-term storage of plant germplasm is low moisture content. Before the start of the experi-ments, nodules had moisture content of 5.4%. For storage, the nodules were dried in a drying room at a temperature of +18…+20°C and a relative humidity (RH) of 10-12%. In the process of drying they were periodically weighed and their moisture content was measured. After drying in a drying room for three months, the weight of the nodules became constant at 4.2-4.4% moisture content. Germination percentage before and after drying remained unchanged (80 ± 2.5%). The nodules prepared in this way were hermetically packed in vacuum-laminated foil bags and stored for a long time at different temperatures: +20°C, +4°C, -18°C, and -196°C (in liquid nitrogen) for further research. After three-year storage under different temperature conditions, their germination energy and germination percentage were tested. The experiments were performed in four replications. It was shown that the germination energy, regardless of the storage option, averaged 4.5 ± 2.9%. Germination percentage of nodules stored at +20°C decreased by approximately 15-16%. Under low positive (+4°C) and negative temperatures (-18°C and -196°C), germination percentage remained unchanged (Figure 5). A decrease in germination energy in all storage options may be explained by the drying of nodules before placement for storage and, accordingly, by the slowdown of the initial stage in their germination process.
Thus, even short-term storage of chufa nodules at room temperature adversely affects their germination. Further experiments should demonstrate what time schedules and tem-  peratures are most suitable for the long-term storage of chufa nodules. Currently, one of the urgent problems for both Europe and Russia is to improve nutrition patterns of the population and increase the share of natural and environmentally friendly products in the daily diet (Konarev et al., 2019;Sabo et al., 2018). The main problem of such products is their short shelf life, while the use of chemical preservatives not only negates the benefits of a natural product, but also harms the human organism. This is especially typical for perishable products. To solve this problem and increase the shelf life of natural products, it is promising to use plants with natural antioxidant potential, such as chufa. The main components in the antioxidant system of chufa nodules are tocopherols, mainly γ-tocopherol and α-tocopherol (Yeboah et al., 2012). The use of chufa accessions with a high content of vitamin E (k-7, k-11 and k-19) is promising for the development of this trend in the food industry.
The key factors for successful long-term storage of chufa nodules are humidity and temperature patterns. High moisture and high drying temperatures reduce the content of protein, oil and fiber in chufa nodules (Omale et al., 2020).
A comprehensive study of C. esculentus requires performing molecular genetic studies aimed at obtaining maximum sizes of chufa nodules in order to optimize the processing technology. It is also necessary to develop a biotechnological method for producing a natural preservative from chufa and conduct medical research into the effect of chufa products on carbohydrate and lipid metabolism among patients with diabetes, metabolic syndrome, and obesity.

Conclusion
The results of our research show that the chufa collection accessions manifest significant genetic diversity in their biochemical composition. Chufa accessions have been selected for a set of valuable agronomic features. The selected accessions can be used as source material in breeding programs.
A technique was developed for measuring germination percentage, and preparation for long-term storage was carried out. The optimal age of the shoots from chufa nodules to assess their germination energy was found to be four days, with eleven days for germination percentage assessment. It was shown that three-year storage of chufa nodules at +20°C caused a decrease in their germination by 15-16%. When stored at low positive (+4°C) or negative temperatures (-18°C and -196°C), their germination percentage remained unchanged.
The research was performed within the framework of the State Task according to the theme plan of VIR, Project No. 0662-2019-0001 "The collection of oil and fiber crops at VIR: maintenance, study, and genetic diversity expansion".