Drought-tolerant hop genotypes – A promising solution to the problem of climate change for the hop industry?

Climate change can negatively influence hop yield and its quality. Cultivation areas limited by the prohibition of using water from small water sources are most at risk. In order to prevent these areas from completely disappearing in the future, planting hops with varieties or genotypes tolerant to drought could be a promising solution. Therefore, Czech hop breeding targets drought-tolerant genotypes with high crop yield and stability of quantitative and qualitative parameters which will also be well-usable in the Czech brewing industry. Twelve promising genotypes (5165 (Uran), 5194, 5304, 5348, 5398, 5432, 5461, 5464, 5465, 5646, 5669, and 5693) were selected after measurement of their physiological parameters and their basic chemical and sensorial properties were determined. Simultaneously, they were assessed from the point of view of brewing quality using sensory and chemical analysis using a brewing test. For this purpose, experimental beer using identical decoction single-hopped technology, where a hop aroma was highlighted using hopping in a whirlpool was prepared. The results show that three genotypes belong to the category of bitter varieties (5165, 5194, and 5304), whereas the other are genotypes exhibiting the character of aromatic varieties. From a grower’s point of view, genotypes 5165 (Uran), 5194, 5348, 5398, and 5461 show a very high yield. The overall impression of all beer samples is very good, bitterness is fine in most samples and hop aroma is mostly hoppy, herbal, woody, spicy, and in some cases also fruity (5304, 5461, 5465, 5646, and 5693).


Introduction
Czech hops are unique crops that are highly exportable and take part in foreign trade of the Czech Republic.However, climate change, especially the lack of moisture, has reduced hop yields in the Czech Republic in recent years (Nesvadba et al., 2021;Straková et al., 2020;Mozny et al., 2009).Therefore, the growing areas that have a possibility of irrigation are at an advantage, but its use is often limited by the prohibition of using water from small water sources.Hop cultivation areas without any irrigation at all may soon disappear, and even might stop being used for agricultural purposes.This problem could be solved by planting hops with varieties or genotypes tolerant to drought.The use of cultivated areas will remain preserved, there will be no erosion threat to the agricultural soil fund or increased consumption of surface water for irrigation.Hence, the diversity and stability of landscape cultures can be preserved.
Czech hop breeding reflects on this problem and currently focuses on breeding new drought-tolerant genotypes with high crop yield and stability of quantitative and qualitative parameters, which means the content of valuable compounds such as bitter acids and hop oils.Simultaneously, the ongoing project QK21010136 "Application of new varieties and genotypes of drought-resistant hops to cultivation and brewing practice" supported by the Ministry of Agriculture of the Czech Republic emphasizes the properties of hops from the point of view of suitability of its use for production of both traditional Czech lager and new interesting types of beer that today's consumer demands.
Czech hop breeding has a long tradition.Originally, it was associated with the cultivation of aromatic hop varieties.Saaz (semi-early red bine hop) has been for centuries considered as aromatic hop varieties with the best quality in the world.The founder of modern breeding methods using clonal selection in the original regional stands was Karel Osvald, who was involved in clone selection from 1927 when 150 clones were selected.Finally, three Oswald clones, clone 114, clone 31, and clone 72 were chosen for planting by the leading growers themselves (Vent 1999;Osvald, 1931;Osvald, 1929).These clones were officially recognized in 1946 and their cultivation was allowed in 1952.Currently, these clones, well-known as Saaz variety, are still grown on 90% of the total area on which hops are grown in the Czech Republic.The variety Saaz has a fine aroma with a typical pure hop aroma and herbal and floral background that ensures excellent taste of traditional Pilsner Lager (Nesvadba et al., 2020).
Later, in the 1960s, a crossing method for hop breeding began to be used.Bor and Sládek were the first two hybrid cultivars registered in the Czech Republic in 1994 (Rigr et al., 1997) and Premiant was registered in 1996.These aromatic varieties have a lower content of alpha acids and a balanced ratio of alpha and beta-acids ranging from 0.8 to 1.5 (Nesvadba et al., 2022a).
Later, after year 2000, bitter varieties Agnus, Vital and Rubín were registered followed by Gaia and Boomerang in 2017 (Nesvadba et al., 2017).
The hop-crossing method is still used for breeding new hop varieties in the Czech Republic.Concerning the brewing industry in the Czech Republic, hop breeding has always focused on varieties suitable for lager-type beers, for which other hop varieties Harmonie, Saaz Late, Saaz Special, Bohemie, and Kazbek were registered in 2004-2010.
Since 2012, new hop genotypes with a high intensity of a specific flavor have been bred using this method (Nesvadba et al., 2018).The basis of such breeding is a collection of genetic resources of hops which is part of the National Program for the Conservation and Utilization of Plant Genetic Resources and Agrobiodiversity (Charvátová et al., 2017).The collection includes all the world's varieties of hops as well as wild hops, which began to be used in breeding due to their specific aroma.The most commonly used varieties are Kazbek, Columbus, Cascade, and wild hops from North America.
The study aims to test drought resistance of 12 promising genotypes selected from original 70 ones after crossing, which meet chemical and sensory quality parameters in order to be well-saleable in the market.Therefore, promising selected genotypes were grown and characterized using sensory and chemical analysis.Since the genotypes are completely new and no information about their brewing properties exists, they were tested using identical decoction single-hopped technology, where a hop aroma was highlighted using hopping in a whirlpool.Twelve experimental beers from new varieties with the breeding designation 5165 (Uran), 5194, 5304, 5348, 5398, 5432, 5461, 5464, 5465, 5646, 5669, and 5693 were assessed from the point of view of brewing quality using sensory and chemical analysis.

A method of selecting drought-tolerant hop genotypes
The selected 70 breeding genotypes were propagated for greenhouse trials at the same time in order to keep the plants in the same growth phase.For the purpose of measuring physiological parameters of hops using the LCpro SD device (ADC Bioscientific, Ltd., Great Britain), 5 plants of each genotype were propagated and planted in 5 L pots and a standard growing substrate.Subsequently, three plants in the most similar growth phase and condition were selected to measure the physiological parameters.An initial measurement of all genotypes took place on irrigated plants that were not stressed by drought.Each leaf was measured for 8 minutes, of which the first 3 minutes were the stabilization of values after closing the leaf into the measuring chamber, and only the values measured between the 4 th and 8 th minute are considered as measurement results.The conditions in the measuring chamber were set at a temperature of 30 °C with a photosynthetic photon flux density (PPFD) of 650 µmol/m 2 /s.After the initial measurement, their irrigation was always terminated and an effect of drought stress on the visual and physiological manifestations of the plants was subsequently monitored.
After 10 days of the first measurement, the same plants were measured again.The rate of photosynthesis and transpiration was determined after the action of water and also partially of thermal stress when the plants could not compensate for the thermal stress by cooling down due to irrigation (greenhouse conditions).
Thanks to the measured and calculated parameters, but also the visual scoring of the genotypes, it is possible to assess which genotypes show better/worse performance parameters from the point of view of photosynthetic activity after water stress (including comparison with values during irrigation) and thus have potential for application in times of significant weather fluctuations, mainly due to uneven rainfall and longer tropical periods (Nesvadba et al., 2022b).
Based on the preliminary results, 12 hop genotypes that show high or moderate drought resistance were selected.The varieties Sládek and Premiant were chosen as controls.

Visual evaluation (V) after 10 days of stress
1 -negligible growth retardation; slightly yellowish lower leaves; minimal drought symptoms; 2 -slowed growth; plants showed yellowish lower leaves; fast regeneration after watering; 3 -wilted leaves; lower leaves dried up; stopped growth; the plant regenerated and continued to grow after watering; 4 -completely dried up plants; no regeneration after watering.

Brewing technology
Experimental samples of beer were prepared in a pilot scale (Pacovské strojírny, Czech Republic) research brewhouse with a maximum capacity of 50 L using the recipe as described below.The grist composition for each brew was 10 kg of Pale Ale malt from the Czech malthouse Benešov (extract-dry basis 81.6%, color 5.2 EBC).The volume of cold wort was approx.50 L.A decoction mashing regime was used with the mash-in temperature of 35 °C and a rest of 10 min, followed by heating to 52 °C with the temperature gradient of 0.8 °C/min and a rest of 30 min.This was followed by heating to 63 °C with a rest of 20 minutes and separation of mash.Then, the mash was heated to 72 °C with a temperature gradient of 0.8 °C/min and rest to ideal saccharification.This was followed by heating the mash to boil with a temperature gradient of 1.3 °C/min and the duration of boiling of 15 min.This was followed by mixing the mash and the rest of the brew together to a mash-out temperature of 77 °C.A standardized lautering process was controlled according to the clarity and pressure difference below and above the false bottom.The maximum turbidity of sweet wort was set to 30 units EBC and the last running to 60 units EBC.The volume of sweet wort before boiling was 65 L. The atmospheric wort boiling duration was 75 min.Then, beer was hopped using hop pellets three times, the 1 st dosage (40%) at the beginning, the 2 nd dosage (35%) in the 20 th minute of a boil, and the 3 rd dosage (25%) 15 minutes before the end of the boil.Individual doses calculated based on alpha acid content are given in Table 1.The next dosage of 60 g was added into a whirlpool.The target bitterness in beer was 30 BU.
All beer batches were fermented identically at 10 °C with Fermentis W34/70.Maturation took place in a lager cellar at 3-4 °C ± 0.5 °C for 21 days.
Finally, the beer was filtered on a plate filter with S10N filter plates (Hobra Školník, Broumov) and bottled without oxygen access.

Chemical analysis of hops and beer
The determination of bitter acids and hop oils was performed according to EBC 75 and the method of Krofta (2003).

Sensory analysis of hops
An evaluation of the aroma hops was carried out organoleptically.An evaluation of dry cones within 14 days after the harvest was done using the sense of smell where only the dominant aroma was assessed.Hops were dried immediately after harvesting at a maximum temperature of 54 °C.A basic profile of beer together with a hop was evaluated.The basic profile included fullness, intensity of bitterness, bitterness culmination (after 15 s), bitterness aftertaste (after 40 s), bitterness character, astringency, sweetness and sourness evaluated on a scale from 0 to 5, where 0 is none and 5 is maximal sense.The overall impression of beer was rated on a scale of 1 to 9, where 1 was the best beer.
For the evaluation of hop aroma, description analysis for the main aroma and background aromas was used.Descriptors such as hoppy, fruity, citrussy, flowery, resinous, woody, herbal, and spicy were used for this purpose.

3.1.2
Aromatic genotypes 5348 -was obtained by selection of the varieties of the dwarf English variety First Golg, which was freely pol-linated in a breeding hop house in 2007.The genotype has a pleasant hoppy aroma.The yield of 2.5 t/ha was achieved under experimental conditions.5398 -was obtained by backcrossing within the progeny of genotypes 5194 (female) and Czech aromatic varieties Sládek and Harmonie (male) in 2009.The genotype has a weak hoppy aroma with spicy in the background.The yield of 2.7 t/ha was achieved under experimental conditions.
5432 -was obtained by crossing selection of the progeny of European varieties with a high content of alpha acids (female) and Saaz (male) in 2010.The genotype has a fine hoppy aroma.The yield of 2.1 t/ha was achieved under experimental conditions.5461 -was obtained by crossing selection of an offspring of American varieties with a high content of alpha acids (female) and Czech aromatic varieties Premiant and Harmonie (male) in 2011.The genotype has a sharp spicy aroma.The yield of 2.6 t/ha was achieved under experimental conditions.

-was obtained by selection of an offspring of bitter American and European varieties by crossing in 2011.
The genotype has a spicy and herbal aroma.The yield of 2.3 t/ha was achieved under experimental conditions.5465 -was obtained by backcrossing within the progeny to the Sládek (female) variety and the Sládek and Harmonie varieties (male) in 2011.The genotype has a sharp hop aroma.The yield of 2.2 t/ha was achieved under experimental conditions.5646 -was obtained by crossing selection of the progeny of the Kazbek variety and bitter American and European varieties and Saaz (male) in 2013.The genotype has a fruity aroma.The yield of 2.1 t/ha was achieved under experimental conditions.5669 -was obtained by selection of an offspring of the Kazbek and Fuggle varieties by crossing in 2013.The genotype has a fruity aroma with woody and piney in the background.The yield of 2.4 t/ha was achieved under experimental conditions.5693 -was obtained by selection of an offspring of the Kazbek and Fuggle varieties by crossing in crossing.The aroma is fruity and woody, and piney in the background.The yield of 2.4 t/ha was achieved under experimental conditions.

Drought tolerance of selected genotypes
The drought tolerance of selected genotypes was assessed by a visual assessment (V ranging from 1 to 4) and measurement of instantaneous photosynthetic efficiency of water use (WUEi -water-use efficiency, Kirhham, 2005).Results are given in Table 2, where the tested genotypes are ranked according to the highest instantaneous photosynthetic efficiency of water use after the ending of irrigation and after 10 days of water stress.The control varieties Sládek and Premiant are included.
As shown in Table 2, genotypes 5432, 5348, 5465, 5646, 5461, 5464, and 5165 (Uran) demonstrate the best visual evaluation after water stress (V=2).Simultaneously, they had the highest value of WUEi (ranging from 5.40 to 4.03).Therefore, these genotypes can be said to show high tolerance to drought.Genotypes 5398, 5304, 5194, 5669, and 5693 show medium resistance according to a visual and WUEi assessment.Finally, genotypes 5669 and 5693 show a significant decrease in WUEi after 10 days of stress causing the stopped growth (V=3).The worst results were obtained for the reference varieties Sládek and Premiant which were completely dried up after 10 days of stress.

Chemical analysis of selected hop genotypes
The content of bitter acids is given in Table 3.The genotypes 5165 (Uran), 5194, and 5304 belong to a group of bitter varieties due to a high concentration of alpha-acids which is 12.30, 9.53, and 11.63% wt., respectively, also similar to bitter varieties such as Agnus, Rubín, and Vital, whose concentrations are on average higher than 10% wt.The ratio of alpha/beta acids is higher than 2, genotype 5304 even has 3.26.Genotype 5398 has an interesting acids ratio, namely 0.59.
The other genotypes have a lower content of alpha acids (ranging from 3.09 to 8.39% wt.), thus, they belong to aromatic varieties.Genotypes 5646 and 5669 are distinguished from the others by a high content of cohumulone (34.00 and 41.70% rel.) and colupulone (58.00 and 66.90% rel.), respectively.For such varieties, a higher yield of iso-alpha acids during wort boiling can be assumed, as was demonstrated in previous studies.First who described this phenomenon was Rigby (1972), followed by Ono et al. (1984) and Irwin et al. (1985).They demonstrated that during wort boiling the relative amount of formed isocohumulone was significantly higher in comparison with isohumulone and isoadhumulone.Moreover, a relative amount of isocohumulone lost during fermentation is lower than that of isohumulone and isoadhumulone.Next, Irwin et al. (1985) published that cohumulone is better utilized than humulone or adhumulone, probably due to higher losses of humulone and adhumulone in a kettle and of isohumulone and isoadhumulone in a fermenter (Irwin et al., 1985;Ono et al., 1984;Rigby, 1972).
And finally, Jaskula et al. (2008) and Protsenko et al. (2020) determined this phenomenon using a detailed kinetic study.
The total content of hop oils and the content of main specific hop oils such as myrcene, caryophyllene, farnesene, humulene, and selinene group are given in Table 4.Most genotypes have a low content of hop oils with the exception of genotypes 5465, 5165 (Uran), and 5348, which have concentrations of hop oils 2.06, 1.78, and 1.54% wt., respectively.Furthermore, the variability of specific hop oils among the varieties is high.Genotype 5165 (Uran) has the highest ratio of myrcene (24.30% rel.), genotypes 5693 and 5348 have the highest concentration of caryophyllene (13.10% rel.) and humulene (24.70 and 20.20% rel.).Humulene is characteristic also for genotype 5432 (22.70% rel.).Only genotype 5165 (Uran), has a significant concentration of

Chemical analysis of beer
The basic chemical characteristics of experimental beers are given in Table 5.The dispersion of values of original gravity ranging from 10.44 to 12.23% wt. and related parameters corresponds to possibilities of an experimental brewhouse.

Sensory analysis of hop aroma in beer samples
Results of sensory analysis of experimental beers, are given in Table 6 and Table 7.The fullness, sweetness and sourness are very similar among all samples.Slight differences are noticeable in the bitterness profile, where the highest culmination can be found in genotype 5194 (3.8) and the lowest in genotype 5464 (2.9).This genotype also has nearly the lowest after-taste (1.9).Genotype 5194 has, together with a high culmination intensity, the highest bitterness character (3.0), meaning middle-harsh.The other beers have very fine to fine character.Altogether, the overall impression of all samples is very good, their values are ranging from 2.7 to 3.5.The highest aroma intensity was determined in samples 5165 (Uran), and 5194, the best aroma pleasantness was recorded in samples 5646, 5465, and 5165 (Uran).A detailed hop aroma is described in Table 7.

Conclusion
An application of drought-tolerant hop varieties has a long-term effect.Drought-tolerant hop varieties will increase efficiency and productivity, and thus competitiveness in the Czech and world markets for hop growers who lack water for cultivation.
One-off pilot experiments cannot draw a definite conclusion about the use of varieties in brewing in the future.As only a limited amount of hops were available, only one type of beer was prepared, namely lager.However, all the experimental beers were evaluated very positively and from this it can be concluded that they will be usable in the brewing industry.
Further, of the 12 hop genotypes tested, 5 genotypes (5165 (Uran), 5304, 5432, 5461, and 5465) were accepted in the CISTA (Central Institute for Supervising and Testing in Agriculture of the Czech Republic) registration tests in 2022.The best genotypes can be registered as the first Czech drought-tolerant hop varieties as early as 2025.The very promising genotype 5165 (Uran) is currently grown on an area of 0.5 ha and is being tested in a number of industrial breweries and microbreweries.
Currently, all genotypes are tested on two semi-operational areas.The first location is without irrigation in a dry area in Nesuchyně in Rakovník region (GPS 50.4717842N, 13.4068403E).Quantitative and qualitative marks of tolerant genotypes of hops in a dry area are monitored and compared here, with a comparison to a control area with sufficient water intake and irrigation.This semi-operational area is located in Rybňany in Žatec region by the river Ohře (GPS 50.3497122N, 13.5701819E).

Acknowledgement
This study was supported by the Ministry of Agriculture of the Czech Republic using a project No. QK21010136.The cooperation of co-authors was maintained by the

Table 1
Individual hop doses calculated based on alpha acid content.

3.1 Characterization of selected genotypes (breeding, aroma, yield)
vest.The yield of hops is 2.3 t/ha.

Table 2
Visual assessment (V) and measurement of instantaneous photosynthetic efficiency of water use (WUEi) of selected genotypes tested for drought resistance.

Table 3
Content of hop resins in tested genotypes.

Table 4
Content of basic hop oils in tested genotypes

Table 5
Basic chemical parameters of experimental beers OG -original gravity; Alc % vol.-alcohol by volume; Alc % wt.-alcohol by weight; Extract app -apparent extract; Extract real -real extract; Ferm app -apparent fermentation; Ferm real -real fermentation.

Table 6
Sensory profile of experimental beers Uncertainty is 0.5 support of the Technology Agency of the Czech Republic within the project TE02000177 "Center for Innovative Utilization and Strengthening of the Competitiveness of Czech Brewing Raw Materials and Products".