Characterization of new late-spring-frost-tolerant apricot hybrids: physical and biochemical fruit quality attributes, volatile aroma compounds

ABSTRACT: Late spring frosts is one of the most important restricting abiotic stress factors of apricot growing worldwide. In this study; some physical, biochemical fruit quality characteristics and volatile aroma compounds were determined in fresh fruit samples of thirteen late spring frost tolerant apricot hybrids recently obtained from Turkish apricot breeding programme. A wide variation was reported among apricot genotypes in all of the evaluated physical and biochemical fruit characteristics and also volatile aroma compounds.Fruit size varied between 27.2 and 60.5 g, total soluble solids between 13.6 and 17.4 %, total carotene 26.6 and 42.8 (mg.100 g-1), and total phenolics content 354.2 and 673.1 (GAE.100 g-1). A total of 42 flavor components belonging to various flavor compound groups were identified. The main volatile aroma compound groups detected in the assessed apricot genotypes were; aldehydes, ketones, esters, alcohols, terpenes, acids, and other compounds. Among the detected compounds; Hexanal, 2-Hexenal, 1-Hexanol, 2-Hexen-1-ol, Limonene were the most abundant compounds in terms of concentration. Hexanal varied between 55.8 and 528.5 µg.kg-1, and 2-Hexen-1-ol changed between 25.7 and 297.9 µg.kg-1 fresh weight. Correlation analysis revealed significant correlations among some aroma compounds and biochemical fruit quality characteristics. Significant correlations were reported for esters with titratable acidity (r=0.79) and total carotene (r=-0.61) and aldehydes were found as highly correlated with total soluble solids (r=-0.69). The results of the study will be beneficial in terms of food analysis, cultivation, and breeding studies of apricot.


INTRODUCTION
Extreme climatic events such as drought, heat waves and late spring frosts threaten sustainable agricultural production in recent years due to global climate change.In the fruit growing, late spring frost damage possibilities increase when the genotypes bloom and set fruit early (MUFFLER et al., 2016;VITRA et al., 2017).According to climate warming scenarios, it is foreseen that the growth, productivity and geographical spread of many economically important plant species will be restricted (GU et al., 2008;VITASSE et al., 2019).This is undoubtedly true for fruit species whose yield and quality characteristics are shaped by environmental conditions such as climate and soil (LUEDELING, 2012;NAWAZ et al., 2020).
Although, apricot is a deciduous fruit species, and frost occurrence frequency is minimized in the winter, late spring frosts can usually cause serious damages.The main problems of apricot production in the world are extreme winter and late spring frosts, poor adaptation to environmental conditions, plum pox virus disease, drought and global climate change (LEDBETTER, 2008;ASMA, 2011).Apricot, peach and some plum cultivars are often damaged by spring frosts because they bloom earlier than other fruit types.In temperate climates, spring frost is an important environmental factor that limits apricot yields, as a consequence of strong injuries to buds, flowers and developing fruits (BARTOLINI et al., 2006).Late spring frosts frequently occur in Malatya, the world's most important dried apricot production center, and serious economic losses are faced.Product losses till to 50-90% have occurred due to severe frosts as it had been confronted in 2004, 2010 and 2014 late springs (ASMA et al., 2016).
In general, the probability of frost damage differs according to the region, the level and duration of the frost, the cultivars and the physiological situation and phenological phase stage of the trees (GUERRIERO et al., 2006).In order to minimize the economic losses in apricot, many researches have been carried out on physiology (KAyA et al., 2018;DUMANOGLU et al., 2019), molecular features (NAZEMI et al., 2016;SALAZAR et al., 2016) artificial freezing tests (OZTURK et al., 2006;GUNES, 2006) and field studies (AKÇA et al., 2000;GUERRIERO et al., 2006).Obviously, the most effective method to protect from spring frosts is to breed new late-blooming apricot varieties that are cold-resistant or tolerant (DEMIRTAS et al., 2010;DOĞAN, 2018).
Natural components such as dietary fibers, phenolic compounds, organic acids, carotenoids, and sugars are of the important fruit quality elements related to the nutritional value of fruits and vegetables (GÜÇLÜ et al., 2006;AKIN et al., 2008).Thanks to their antioxidant activities these compounds scavenge free radicals and destroy their chain reactions and stabilize unstable oxygen molecules (DAI & MUMPER, 2010;CARBONE et al., 2018).Apricots are also known as a rich source of bioactive compounds, especially for polyphenols and carotenoids (ERDOGAN-ORHAN & KARTAL, 2011).
Being a mixture of various metabolites and a result of special assortment, aroma compounds play a key role in the formation of the special characteristics of many foods and beverages.Besides, these compounds strongly influence the fruit quality variation among species and cultivars.Previous studies revealed that aldehydes, esters, alcohols, terpenes and acids were the main aroma compound groups of apricot fruits, and hexanal, hexyl acetate, (Z)-3-hexenyl acetate, (E)-2-hexenyl acetate, ethanol, 1-hexanol, (Z)-3-hexenol and (E)-2-hexen-1-ol were the main detected compounds (GOKBULUT & KARABULUT, 2012;PINTEA et al., 2020).
Currently, fruit breeders mostly select new cultivars according to external fruit characteristics such as fruit weight, color, appearance and harvest time; organoleptic and nutritional characteristics are of the secondary goals.However, consumers have plenty of fruit and vegetable alternatives on the market, and fruit composition is becoming more and more important.There are very few studies on the phytochemical and aroma compounds of apricot varieties tolerant to abiotic stress conditions such as drought, late spring frosts and heat waves (NAZEMI et al., 2016;KHADIVI-KHUB & KHALILI, 2017).The knowledge about fruit quality characteristics of apricots tolerant to late spring frosts is limited, especially for biochemical attributes and volatile aroma compounds.The comparison of cultivars and obtained hybrids provide valuable support for breeding studies.
Here, the fruit quality, biochemical characteristics and aroma components of trees tolerant to spring late frosts of thirteen promising apricot genotypes obtained from Apricot Breeding Programme launched by İnönü University Apricot Research Centre in 1999.The main objectives of the study is to detect how the fruit quality attributes including physical, biochemical and aroma components change in late spring frost tolerant apricots, and compare the tolerant hybrids in relevant characteristics.

Plant materials and experimental design
The plant materials of this study were 13 apricot hybrids characterized by late spring frost tolerance, high fruit quality, and yield obtained from the "Multi-Purpose Apricot Breeding Project", and a reference cultivar ('Kabaaşı') which was also reported as a late spring frost tolerant cultivar in previous reports (GUNES, 2006).The origin, pedigree and some phenological records of the plant material is presented in table 1. Besides, some organoleptic fruit quality characteristics of the assessed genotypes are presented in table 2.
The research was carried out at the Research and Application Orchards of İnönü University located in Malatya Province of Turkey.Trees of the hybrids evaluated as part of the study were planted in a 3 × 1.5 m and the cultivars in a 5 × 5 m grid, and Zerdali seedlings were used as rootstocks.The trees were subjected to standard cultural practices including drip irrigation and fertigation.There were no significant negative impacts of water stress, nutrient deficiency or pests and diseases that would affect the results, and in order to evaluate environmental impacts the study was performed across two consecutive years (2016 and 2017).All of the observations, measurements and analyses were performed on the fruit samples collected from outer canopy layer of each genotype.

Physical and biochemical fruit quality traits
Collected fruit samples, 25 fruits from each replicate, were evaluated for some physical and biochemical fruit quality traits.Fruit weight (FW) and kernel weight (KW) values were measured using a precision scales (Ohaus PAJ 812 CM 0.01 g, Germany) and expressed in grams, and flesh/kernel rate (F/K) was calculated by division of these values and expressed in percentage.Flesh firmness (FF) was measured by a manual penetrometer (Akyol Gy-3, Turkey) and expressed as kg.cm 2-1 .
As part of biochemical fruit quality attributes, the contents of total soluble solids (TSS), titratable acidity (TA), total carotene (TC), and total phenolics (TP) were determined.TSS and TA percentages were measured on the homogenized fruit juice extracted from the sample fruits.TSS was measured via manual refractometer (Greinorm 0-32 Brix, Germany).TA was detected according to the method reported by HAFFNER & VESTRHEIM (1996) and expressed as the malic acid (%).The maturity index (TSS/TA rate) was calculated by division of these values.
The total amount of TC was determined by modifying the method applied by AKIN et al. (2008).Fruit samples (5 g from each genotype) were mixed with 20 ml of petroleum ether-methanol (90:10) solvent, and homogenized in 13600 rpm for 5 minutes, and mixed with 10 ml diluted water and vortexed for 30 seconds.The mixture than centrifuged in 6000 rpm for 4 minutes, the pellet were mixed with 7.5 ml of petroleum ether-methanol, and homogenized in 13600 rpm for 1 minute, and centrifuged again in 6000 rpm for 4 minutes.The obtained extract was filtered and absorbance values were read at 450 nm (Shimadzu UV-1800, Kyoto, Japan), and TC values were calculated based on the calibration curve obtained using carotene standard prepared in the range of 5-100 ppm and the results are given in milligrams of TC equivalents per 100 g of fruit sample (mg.100g -1 ).
TP value was measured according to Folin-Ciocalteu spectrophotometric method described by SINGLETON et al. (1999) using 2% (w/v) Na 2 CO 3 Table 1 -Origin, pedigree and some phonological records of assessed apricot genotypes.(water) and Folin-Ciocalteu reagent.In this context, 5 g fruit sample were mixed with 25 ml of methanol containing 0.1% HCl and kept in the freezer at -18°C for 24 hours.The mixture (40 μL) was mixed with 200 μL of Folin-Ciocalteu reagent, vortexed for 1 minute, kept in the dark for 5 minutes, mixed with 600 μL of 2% Na 2 CO 3 and the obtained samples were read at 765 nm (Shimadzu UV-1800, Kyoto, Japan) after keeping in the dark for 120 minutes.A calibration curve formed by reading of gallic acid solutions prepared at different concentrations (50-1000 ppm) was used to calculate the results which were expressed as milligrams of gallic acid equivalents (GAE) per 100 g fruit sample (GAE.100g-1 ).

Volatile aroma compounds Sample preparation and SPME conditions
Volatile aroma compound contents of apricot fruit samples were detected by SPME with a Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) (50/30 µm coating thickness; 2 cm length; Supelco, Bellefonte, PA, USA) fiber.A 3 g of the homogenized sample in triplicate was immediately transferred into 15 mL of SPME vials (Supelco, Bellefonte, PA, USA) within 2 min, followed by 10 µL of internal standard containing 81 mg/kg of 2-methyl-3-heptanone (all organic volatile compounds except acids) and 2-methylpentanoic acid (for organic volatile acids; Sigma-Aldrich Co., USA) in methanol as internal standard.Vials were placed on a heater at 40 °C for 30 min to accumulate the volatiles up to head space.Subsequently, fiber was injected in vial to absorb volatile compounds for 30 min.Desorption temperature was 250 °C MS sampler.

GC-MS conditions
Desorption of the extracted volatiles was carried out on a Shimadzu GC-2010 gas chromatography-QP-2010 mass spectrometry system (Shimadzu Corporation, Kyoto, Japan).Separation was achieved with DB-Wax column (60 m × 0.25 mm × 0.25 mm; J&W Scientific, Folsom, CA, USA).The volatile compounds were identified by retention index (RI), using an n-alkane series (C10-C26) under the same conditions.WILEy 8 and NIST 05 mass spectral libraries used to identify peaks.

Statistical analysis
All of the samples were prepared, and all of the measurements and analyses were performed with three replications in a randomized block design experiment.The obtained data were evaluated according to Duncan's Multiple Range Test.Pearson's Correlation Test were applied to analyze correlations among the physical and biochemical fruit quality characteristics and aroma groups.Besides, in order to assess the differences among the volatile aroma compounds of the genotypes, the unweighted pair-group average method (UPGMA) analysis was applied with the squared Euclidean distance.All of the statistical analyses were performed at the significance level of P ≤ 0.05 using 'IBM SPSS Statistics 22 for Windows' software package.

Physical and biochemical fruit quality traits
The physical and biochemical fruit quality of the hybrids and the reference cultivar were evaluated in two consecutive years and the average values of the results obtained in two years are presented in table 3. Significant differences and large variation were obtained among the hybrids and the reference cultivars in all of the assessed fruit quality traits.
When the results of the years were compared, the results of the first study year were significantly higher than that of the second probably because of the cropload differences between the years which was lower in the first year.However, the impact of the years did not change the results when comparing the genotypes.For that reason, the year effect was ignored by pooling the data of 2016 and 2017.The main reason for the differences in the crop-load between years is thought to be related to the occurrence of more severe late spring frosts in 2016 compared to 2017, and the greater frost damage to flowers and fruits in 2016 due to these frosts.While severe frosts such as -5.7 °C and -5.3 °C occurred in 2016, especially during the flowering period, moderate frosts occurred in 2017 (Table 4).
As part of weight parameters; FW, KW, and F/K were measured and evaluated.FW was found as the lowest in '2-216' with 27.2 g and the highest fruit weight was found in '10-06' with the value of 60.5 g.Conversely, the reference cultivar 'Kabaaşı' was the genotype presenting the lowest average KW value with 2.2 g, while '11-35' gave the highest KW value (3.8 g).F/K values varied between 8.1 ('5-16') and 16.9 ('3-42') %.In terms of fruit flesh firmness, it was determined that the assessed hybrids had higher values than the reference cultivar and the genotype '6-74' had the hardest fruits with the value of 8.6 kg.cm 2-1 .On the other hand, the lowest value was obtained in '1-18'.
The highest TSS content was measured in the reference cultivar 'Kabaaşı' (21.5 %), while in the  hybrids the genotype '2-67' was found as having the highest TSS value (17.4 %).The lowest TSS value (11.4 %) was found in '6-74'.The obtained TSS/TA ratio values changed between 7.4 ('5-16') and 19.5 ('Kabaaşı').The maturity index values were reported in accordance with the sensorial fruit taste characteristics of the assessed genotypes described in table 2. The highest TSS/TA value was found in the reference cultivar 'Kabaaşı' and the fruit taste was scored as 'Sweet', while the genotypes '5-16' which was scored as 'Sour' presented the lowest maturity index.
In order to evaluate the bioactive compound potential of the late spring frost tolerant apricot hybrids examined as part of the study, the contents of TC and TP were detected and evaluated (Table 3).The amount of TC in the apricot genotypes varied between 28.2 and 42.8 mg.100g -1 .The lowest and highest TC values were found in '3-42' and '10-06', respectively.In terms of TP, the lowest values found was 354.2 GAE.100g -1 ('3-42), whilr 673.1 GAE.100g -1 ('10-06') was the highest value.

Volatile aroma compounds
Concentrations of the volatile compounds in thirteen apricot genotypes are given in table 5.A total of 42 volatiles were identified in the apricot samples including 10 aldehydes, 5 ketones, 9 esters, 7 alcohols, 5 terpenes, 2 acids and 4 other compounds.

Cluster analysis
The frost tolerant hybrids evaluated in this study were subjected to cluster analysis and classified based on the volatile aroma compound concentrations (Figure 1).According to the results, the assessed hybrids were distributed in three main clusters.The hybrid '6-74' formed the third cluster alone which was especially separated from the other genotypes with high contents of Hexanal, 2-Hexanal; and consequently, total concentrations of Aldehydes.The hybrids '1-18', '3-42', '8-34', and '9-04' constituted the second cluster.
Clarification of interactions among fruit characteristics via correlation analyzes brings useful practical knowledge for consumer food preferences and also for breeding studies aiming those preferences.Most of the correlations on physical and biochemical fruit quality traits presented in this current study were in accordance together with some opposite results and additional correlations reported by the previous studies (CALİSKAN et al., 2012;KARAAT & SERÇE, 2019;ÇUHACI et al., 2021).The variation among the reported results would probably be caused from the genotypic and environmental conditions.As far as we know, this study presents the first report on the correlations among aroma compounds and fruit quality parameters in assessed apricot hybrids.The obtained results indicated some significant correlations for aldehydes (with TSS) and esters (with TA and TC).

CONCLUSION
Late spring frost damages, one of the most restricting factor of apricot growing, can be significantly reduced by breeding tolerant or resistant plant genotypes.Studies on the breeding of new late spring frost tolerant apricot hybrids and analysis of fruit quality characteristics, especially bioactive compounds and aroma volatiles are limited.In this study, promising findings were obtained related to fruit quality characteristics and especially aroma compounds in new apricot genotypes tolerant to late spring frosts.All of the evaluated fruit quality characteristics varied significantly among the late spring frosts tolerant apricot genotypes assessed within the scope of the study.Especially, the hybrids '8-34' and '10-06' were found to be advantageous due to their tolerance and fruit quality characteristics.As a result of the study, the relationships between fruit quality characteristics and aroma components were also examined and a significant correlation was found between aldehydes and ketones  and some biochemical fruit quality characteristics.The results of the study revealed important data regarding the fruit characteristics of apricot genotypes tolerant to spring late frosts.

Table 3 -
Physical and chemical fruit quality properties of apricot cultivars and hybrids subjected to assessments.

Table 2 -
Sensorial fruit quality characterization of apricot cultivars and hybrids subjected to assessments.

Table 4 -
Spring late frosts recorded at the study area in 2016 and 2017.

Table 6 -
Correlations among aroma groups and other assessed fruit quality traits.* .Correlation is significant at the 0.01 level * .Correlation is significant at the 0.05 level.FW: Fruit Weight, KW: Kernel Weight, TSS: Total Soluble Solids, TA: Titratable Acidity, FF: Flesh Firmness, F/K: Flesh/Kernel Rate, TC: Total Carotenoids, TP: Total Phenolics.