New fine aroma varieties of hops ( Humulus lupulus L.) Saaz Brilliant, Saaz Comfort, Saaz Shine and Mimosa

Four new aroma hop varieties – Saaz Brilliant, Saaz Comfort, Saaz Shine and Mimosa – were registered in the Czech Republic in 2019. All of the new hop varieties have significantly higher yields than the traditional Saaz aroma variety. Saaz Comfort has the significantly highest content of alpha acids (5.59 %) whereas Mimosa has the significantly lowest content of alpha acids (1.90 %). The Saaz Comfort and Saaz Brilliant varieties show a variability of alpha acid content below 20 %. The other hop varieties, Saaz Shine and Mimosa, as well as Saaz, have a variability of alpha acid content above 25 %. Mimosa has the significantly highest content of beta acids (6.07 %). Mimosa shows the highest average cohumulone content (29.29 % rel.) whereas Saaz Comfort has the lowest cohumulone content (18.04 % rel.). Saaz Comfort and Saaz Shine have the highest average contents of hop oils (0.84 % w. and 0.75 % w., respectively). The Saaz, Saaz Shine and Saaz Brilliant varieties show the significantly highest farnesene contents (13.47 % rel., 12.50 % rel. and 12.38 % rel., respectively), which are higher than those of Saaz Comfort and Mimosa.


Introduction
Currently, the Hop Research Institute in Žatec has registered 20 hop varieties. The first clonal selection dates back to 1853. It was done in the Auscha population by Kryštof Semš from Vrbice near Roudnice, who performed the first positive selection in his vegetation. Associate professor Karel Osvald was the founder of modern methods of hop breeding based on clonal selection in the original regional vegetations. He started applying clonal selection in 1927. His longtime efforts in Czech hop growing resulted in three clones (Fric, 1992), which were named after him (Osvald's clone 31, Osvald's clone 72 and Osvald's clone 114). These clones currently take up 90 % of the total hop growing area in the Czech Republic. In the 1960s, hop hybridization (i.e. crossbreeding) was introduced to hop breeding. In 1994, Bor and Sládek were the first registered Czech varieties resulting from hybridization. In 1996, the new Premiant variety was registered. Thanks to its better performance parameters, it replaced the Bor variety. In 2001, the first Czech bittering hop variety (Agnus) was registered. It shows an alpha acid content of 10 % (Nesvadba et al., 2002). In the years 2004 to 2017, the Hop Research Institute in Žatec registered an additional 8 hop varieties Nesvadba et al., 2017), namely Harmonie, Rubín, Vital (developed for biomedical applications as well) , Kazbek, Bohemie, Saaz Late, Gaia and Boomerang. In 2019, the Saaz Brilliant, Saaz Comfort, Saaz Shine and Mimosa aroma hop varieties were registered (Nesvadba et al., 2019). Last but not least, the breeding of hops for low-trellis production (Nesvadba V., 2016) and the registration of the first Czech varieties of hops for low-trellis production (Country, Jazz and Blues) in the years 2018 and 2019 need to be mentioned. In the past, breeding was aimed at first at productivity, typical aroma and higher alpha acid contents and tolerance to diseases (Wirowskij, 1980). Many important characteristics are based on a polygenic way (Nesvadba at al., 1999). Only several characteristics are monogenic, e.g. dwarfness, which is used for breeding process aimed at hop varieties suitable for low trellises (Darby, 2001). Hop is a dioecious plant and just female plants bear cones. It is the reason why male plants enter the breeding process as an unknown pollinator (Neve, 1981). Therefore, it is very important to test also males . Testing breeding is important because it brings knowledge about characteristics given to progeny by male plants. Chemical analyses aimed at contents of hop resins and essential oils help to enrich this knowledge as well . Hop varieties can be divided into aroma, bitter and others (IHGC hop variety list 2018). As hop plants are grown at the same place minimally for ten years, emphasis is on stability of yield and content of alpha acids. All the registered hop varieties are followed within maintaining breeding, where stability and uniformity are assessed.
Hop breeding in the Czech Republic mostly focuses on aroma hops. The Saaz fine aroma variety is the best-known Czech hop variety around the world. The breeding of aroma hops dates back at least 160 years. It gives preference to aroma features, including the aroma of hop cones, the balanced ratio of alpha/beta acids and the positive impact on beer quality. The breeding of aroma hops is based on the Saaz variety. In recent years, foreign varieties or even wild hops have been used as well (Nesvadba et al., 2018). In 1995, a new program for breeding aroma hop varieties with Saaz in their origin was launched. It resulted in the registration of three new varieties (Saaz Brilliant, Saaz Comfort and Saaz Shine) that show numerous features identical with Saaz. Another registered variety from this program is Mimosa, which is different. All new varieties are currently being grown in pilot conditions. Brewing tests and test brews are under way in brewery operations (Nesvadba et al., 2020).
Performance stability is a very important parameter for hop growing and beer brewing. Therefore, it is necessary to test the new hop varieties over a longer period of time and in different locations. Profitability of hop growing for at least 15 years is important for hop growers. Stability of the content and composition of hop oils and resins are key for breweries. New varieties are being tested in different hop growing locations.

Material and methods
The creation of hop varieties is based on the hybridization method. Seeds from suitable parent components are sown in a greenhouse and young plants are tested in terms of their resistance to Pseudoperonospora humuli and Sphaerotheca humuli. Resistant and tolerant plants are planted in a breeding hop field. In the second year of cultivation, the best genotypes are selected for the second breeding stage. Upon a five-year evaluation, the best genotypes are propagated and planted three times in a testing nursery and later on as part of field and zoning experiments. At that stage, features important for hop growing and beer brewing are evaluated in greater detail. The best genotypes are submitted for registration experiments. New hop varieties are evaluated in breeding nurseries, field experiments and piloting areas. Mother plants are always original. Therefore, the performance of some hop varieties can be lower than that of virus-free plants (Nesvadba, 1999). At the moment, each variety is being monitored in a maintenance breeding program including a minimum of 40 plants. Every year, 10 mother plants are evaluated. Each mother plant is evaluated in terms of morphological features with regard being paid to any deviations from the uniformity of the particular hop variety.
The following is evaluated for each variety: hop yield, content and composition of hop resins (EBC 7.4 -Krofta K., 2008), content and composition of hop oils (based on liquid chromatography). Each plant is harvested separately. An experimental Volf picking machine is used for hop picking. Yield is shown in kg of fresh hops per plant (hereinafter: kg/plant). The conversion of hop yield is based on the number of plants per hectare, which amounts to 2,900 plants at a spacing of 1.14 x 3.00 m. The coefficient of dry matter in fresh hops and dry hops is 4. The following statistics were prepared: average (A), median (Med) and standard deviation (s). Relative amount of variability is used to compare a set with different levels. Resulting variability amounts are dimensionless numbers (mostly in %). This makes it possible to compare the variability of statistical features differing in measure units. Coefficient of variation (CV), showing the extent of variability in %, was used for data processing. The t-test was applied to determine and prove the difference between hop varieties. The difference of sets was determined on the basis of significance level (), which shows the probability of difference of the tested sets (MELOUN, 1994). For example, if the significance level is determined as  = 0.01, it means there is a 99% probability that the sets under review are different.
Evaluation was performed in the following locations: 1. Ohře river basin -Rybňany and Stekník. There are alluvial soils in this location. Hop fields are irrigated. 2. Rakovník region -Nesuchyně and Chrášťany. There are brown soils in this location. It is a drier area with no irrigation. 3. Údolí zlatého potoka ("Golden Creek Valley") -Očihov. Permian red soils at the Blšanka creek, no irrigation. 4. Auscha region -Radovesice. Alluvial brown soils. Table 1 shows that the Saaz Shine and Saaz Comfort varieties have the highest average yields (2.16 t/ha and 2.11 t/ha, respectively). The benchmark variety Saaz has the lowest yield (1.65 t/ha). Based on the t-test, Saaz Shine, Saaz Comfort, Mimosa and Saaz Brilliant have higher yields than Saaz with a probability of 99 %. No statistical difference was determined between other varieties in terms of hop yield. Saaz Comfort has the lowest variability of hop yield (33.39 %), whereas Saaz Shine has the highest (43.94 %). The values of minimum and maximum yields have a broad range, which is due to the conversion of harvested plants per hectare. However, the results suggest that the new varieties can generate very high yields in good conditions. The Saaz Brilliant variety has an average content of alpha acids of 3.77 % with a variability of 18.95 % ( Table 2). The variability suggests that year and location contribute nearly 20 % to the content of alpha acids. The median is 3.69 %. The highest content of alpha acids was determined in 2017 in Chrášťany (Rakovník district) and amounted to 5.36 %. A content of alpha acids exceeding 5 % was determined in four additional locations in Očihov in 2014 (5.14 % w.) and in Rybňany in 2009 and 2010 (5.09 % w. and 5.10 % w., respectively). In contrast, the lowest content of alpha acids (2.38 % w.) was found in 2015 in Nesuchyně (Rakovník district). Out of 64 samples, 7 samples had an alpha acid content below 3 % w. in Rybňany in the years 2013 to 2017 and one sample in Nesuchyně in 2015. The average content of beta acids is 2.75 % w. and the median is 2.80 % w. Variability is almost at the same level as that of alpha acid content, amounting to 19.26 %. The lowest content of beta acids (1.60 % w.) was determined in Nesuchyně in 2015 and the highest content of beta acids (4.03 %) in Očihov in 2014. The average and median values of the alpha/beta ratio are almost identical, amounting to 1.39 and 1.38, respectively. Variability is 17.53 %. The lowest alpha/beta acid ratio was found in Očihov in 2014 (0.97) and the highest in Očihov in 2015 (2.22). Such a high ratio was exceptional since the second highest alpha/beta acid ratio was 1.87 and was determined in Rybňany in 2015. The alpha/beta acid ratio is influenced by the year because in 2015 the average alpha/beta ratio was 1.67 (11 samples) and in 2014 it amounted to 0.98 (12 samples  Unfortunately, in the years 2017 to 2019 the highest content of alpha acids was 5.44 % w. and the average in the years 2017 to 2019 was only 4.56 %, which is 1.03 % w. less than the average since 2005. The average content of alpha acids is likely to be at 4.5 % in the hop growing practice. The average content of beta acids is 5.67 % w. and the median amounts to 5.51 % w. Variability of the beta acid content is 22.64 %. The lowest content of beta acids of 3.14 % w. was determined in the same sample as the lowest content of alpha acids (in Rybňany in 2018) and amounted to 3.14 % w. The highest content of beta acids of 8.57 % w. % was in 2014 (Očihov). This sample shows the lowest content of alpha acids (4.50 % w.), and therefore the alpha/beta ratio is as low as 0.5. The average and median of the alpha/beta ratio are nearly at the same level (1.02 and 1.05, respectively). Variability is 22.64 %. The lowest alpha/beta ratio was determined in Očihov in 2014 (0.50). In contrast, the highest ratio was in Rybňany in 2011 (1.58). The variety is characterized by a balanced alpha/beta ratio.

Chart 1: Content of alpha and beta acids in the Saaz Shine variety in 2019
The Mimosa variety has an average content of alpha acids of 1.90 % w. The median amounts to 1.75 % w. Content variability is 42.41 % ( Table 5). The highest content of alpha acids was determined at the beginning of the breeding process, i.e. upon a selection from descendants after hybridization. A high content of alpha acids was found in the years 2008 (3.80 % w.), 2009 (4.84 % w.) and 2010 (4.80 % w.). After 2011, the highest content of alpha acids was determined in Rybňany in 2015 and amounted to 2.74 %. It was probably due to a virus or viroid infection, which will be the subject of further research. After 2011, the average content of alpha acids was 1.74 % w., thus reducing the variability to 28.38 %. The Mimosa variety is characterized by a low content of alpha acids. Out of 47 samples, 13 samples have an alpha acid content below 1 % w. The lowest content of alpha acids (0.82 % w.) was determined in Rybňany in 2018. The average content of alpha acids is 6.07 and the median is higher, namely 6.12 % w. The content of beta acids has a low variability (17.76 %). The content of beta acids is not influenced by the age of the plant in the same way as the content of alpha acids since the content of beta acids was 5.79 -7.82 % w. in the years 2008 to 2010. The highest content of beta acids was found in Rybňany in 2015 (8.44 % w.). In contrast, the lowest content of beta acids was determined in Rybňany in 2019 (4.34 % w.). Mimosa is characterized by a low alpha/beta ratio. The average is 0.31, the median is 0.28 and variability 39.10 %. The highest ratio (0.8) was found in samples from the years 2009 and 2010, which have a high content of alpha acids and beta acids (5.76 % w. and 5.80 % w., respectively). Samples with an alpha/beta ratio below 0.2 also have a high content of beta acids, namely 4.34 -6.33 % w. The average content of cohumulone is 29.29 % rel., the median is 29.40 and variability is 7.15 %. The lowest content of cohumulone of 21.20 % rel. determined in Rybňany in 2014 was exceptional because other samples have a cohumulone content above 25 % rel. The highest content was found in Rybňany in 2017, namely 34.20 % rel. The average colupulone content is 55.98 % rel., the median is 56.20 % rel. and variability is very low (3.54 %). The lowest colupulone content was found in a sample from Rybňany in 2018 (50.10 % rel.) and the highest colupulone content was in a sample from Rybňany in 2010 (59.40 % rel.). The average and median of the xanthohumol content are almost identical (0.34 % w. and 0.33 % w., respectively) and those of DMX content are identical (0.06 % hm.). Content variability is very different. With respect to xanthohumol content it amounts to 16.71 % and for DMX content it is 41.50 %. The highest contents of xanthohumol (0.53 % w). and DMX (0.12 % w.) were determined in Rybňany in 2015. The lowest contents of xanthohumol (0.24 % w.) and DMX (0.03 % w.) were found in 2018. Chart 2 shows average contents of alpha and beta acids of the hop varieties under review compared to the standard variety Saaz. With a probability of 99%, the Saaz Comfort variety has a higher content of alpha acids (5.59 % w.) than the other varieties. In contrast, with a probability of 99%, the Mimosa variety has a significantly lower content of alpha acids (1.90 % w.). No statistical difference could be determined between the Saaz Brilliant, Saaz Shine and Saaz varieties. The Mimosa variety has a significantly higher content of beta acids compared to the Saaz Comfort variety with a 90 % probability and compared to the Saaz, Saaz Shine and Saaz Brilliant varieties with a 99% probability. The Saaz Comfort variety has a significantly higher content of beta acids than Saaz, Saaz Shine and Saaz Brilliant with a 99 % probability. Saaz has a significantly higher content of beta acids than Saaz Shine and Saaz Brilliant with a 99% probability. No statistically significant difference in the content of beta acids was determined between Saaz Shine and Saaz Briliant. Average alpha/beta ratios of Saaz Brilliant ( Chart 4 shows that Mimosa has the highest average content of cohumolone (29.29 % rel.) and Saaz Comfort the lowest content of cohumulone (18.04 % rel.). For all of the varieties, except for Saaz Brilliant and Saaz Shine, a significant difference in cohumulone was determined compared to Saaz with a 99% probability. The lowest variability of the cohumulone content was determined in Saaz Shine (6.84 %), Mimosa (7.15 %) and Saaz (7.25 %). Only Saaz Comfort has a variability of the cohumulone content above 10 %.
Chart 4: Average content of cohumulone and its variability in the new aroma varieties and the benchmark variety Saaz The Saaz Brilliant variety has the significantly lowest content of xanthohumol compared to the other varieties (Chart 5) with a 99 % probability. Saaz has a significantly lower xanthohumol content with respect to Saaz Shine with a 99% probability and with respect to Saaz Comfort and Mimosa with a 98 % probability. No statistical significance of the difference in the xanthohumol content was determined between the Saaz Shine, Saaz Comfort and Mimosa varieties. With a probability of 99%, Saaz Comfort has the significantly highest DMX content compared to the other varieties. No significant difference in the DMX content was established between Saaz and Mimosa. However, both varieties have a significantly higher DMX content than Saaz Brilliant and Saaz Shine with a probability of 99 %. No significant difference was established between Saaz Brilliant and Saaz Shine. The xanthohumol/alpha ratio is very interesting. The ratio of xanthohumol and alpha acid content is also crucial. It states how many grams of xanthohumol are contained in 100 g of alpha acids. Breweries hop beer according to the content of alpha acids. Mimosa has the highest xanthohumol/alpha ratio, namely 19.6 (Chart 6). When 100 g of alpha acids are used per 1000 l of beer for hopping, this variety contains 12 g of xanthohumol. Almost identical xanthohumol/alpha ratios were found in Saaz Shine (10.2) and Saaz (9.2). The lowest xanthohumol/alpha ratios were determined in Saaz Comfort (6.1) and Saaz Brilliant (5.7).

Conclusion
The results show quantitative and qualitative parameters of the new hop varieties. In conclusion, the results can be compared to the Saaz variety on the basis of statistical significance. Table 7 shows higher or lower significance of the difference between the aroma hop varieties under review and the benchmark variety (Saaz). Saaz Brilliant has identical parameters as Saaz in terms of the content of alpha acids, cohumulone content, the content of hop oils and myrcene, caryophyllene and farnesene contents. The Saaz Shine variety has numerous identical parameters as well, including hop yield, cohumulone content and the contents of myrcene, farnesene, humulene and selinenes. The Saaz Comfort and Mimosa varieties have only one identical parameter (caryophyllene content and DMX content, respectively). It is important for hop growing that all of the new varieties have significantly higher hop yields. The Saaz Comfort variety has a significantly higher content of alpha acids and together with the Mimosa variety significantly higher contents of beta acids than Saaz. All varieties except Saaz Brilliant have significantly higher hop oil contents. To conclude, the newly registered varieties are not identical with Saaz. However, Saaz is substantially represented in their origin, and therefore "Saaz" is part of their names: Saaz Brilliant, Saaz Comfort and Saaz Shine. The Mimosa variety is considerably different from Saaz and its brewing applications differ as well. In conclusion, it is necessary to mention that the results are merely partial results. They are based on evaluations performed for 15 years (12 years for Mimosa) but the samples were taken in six locations only.