Introduction

Grapes have been a significant part of the fruit supply needed by human societies for thousands of years and today it is the most important cultivated fruit in the world both in terms of cultivated area and economic value. Grapevine belongs to the genus Vitis, which is the only edible genus of the Vitaceae family, comprising 11 genera and 600 species1. Siah-e-Samarghandi (Vitis vinifera L.) is one of the major grape cultivars grown in the north and northwest of Fars province, Iran. Siah-e-Samarghandi is a grainy, dark-purple color with medium-to-late ripening time2. This cultivar has high special importance in Iran and Fars province in terms of freshness and high anthocyanin content. Siah-e-Samarghandi grape is cultivated under dry conditions with an average rainfall of 300 to 450 mm in different parts of this province. Due to the fact that in fresh grapes, the appearance of the cluster and the size of the grapes are important. However, there are issues such as the presence of small and large berries, the low quality of the cluster, the limited number of berries in the cluster (caused by berry fall), which reduce the yield of the product3. Different biological activities including natural antioxidants, preservatives, and food fungicides are exhibited by consuming the grape seed extract to prevent food contamination by harmful microorganisms4,5.

Regarding types of compatibility in grape cultivars, most of them are self-compatible and self-pollinated. Cleistogamy is common in grapevine. It is true but there are exceptions although they are not so common and some of them are self-incompatible. There are several factors that influence yield and fruit quality. One of these basic factors is the characteristics of reproductive biology among grape cultivars. The full development of the flower organs and the production of suitable pollen with high germination capacity are the first condition for fertility. The germination ability of pollen grains depends on the cultivar, nutritional conditions and environmental factors, and there is a wide variation in the optimal germination conditions of pollen grains1,6.

The role of potassium on the regulation of osmotic pressure involves three important factors for cell growth, 1- increasing the elasticity of the cell wall 2- the accumulation of soluble substances inside the cell6- The role of potassium in stomata opening/closing, the opening and closing of the stomata depends on the presence of potassium together with an anion in the guard cells of the stomata7.

The use of gibberellic acid in fresh seedless grapes increases the berry size of the berries during fruit set.8.

Considering the high level of grape cultivation, it is very important to find suitable solutions to increase the quality.and pollen treatments of cultivars such as Siah-e-Shiraz and other cultivars, because these treatments have pollen grains with high germination (data not provided) and the placement and germination of pollen grains of these cultivars (healthy pollen grains are a rich source of auxin hormones and GA3) on the stigma of the flower of the Siah-e-Samargandi leads to the stimulation of the growth of the ovary and the synthesis of more of these hormones and the formation of the fruit, as a result of having healthy pollen grains, healthy seeds are formed in the fruit (Fig. 1A-F). The main purpose of this experiment is to investigate the cause of shot berries, as well as the effects of some treatments such as the interaction of gibberellic acid (GA3) with potassium nitrate (KNO3) and cross-pollination in preventing or mitigating this issue in Siah-e-Samarghandi grapes.

Fig. 1
figure 1

The fruitset of pollinated clusters of grape cv. Siah-e-Samarghandi with six pollen grain along with flower diagram were presented above respectively as follow Rishbaba (A), Siah-e-Shiraz (B), Askari (C), Rotabi (D), Atabaki (E), Siah-e- Samarghandi (F)Siah-e-Shiraz.

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Materials and methods

Plant material and growth conditions

This experiment was done in a commercial rainfed vineyard in the village of Kholar, located in the northwest and 35 km northwest of Shiraz city, Iran (with latitude of 290–57/ Nand 52014/ S) during 2 years (2021–2022). This region had a mild and cool climate with an average annual rainfall of 450 mm and clay loamy soil. The distance between the vines was 3.5 m in rows and between the rows 4 m. The area was non-irrigated (rainfed). The collection of plants material complies with relevant institutional, national and international guidelines and legislation and permission was obtained by the commercial orchards had cooperation with Shiraz University for the collection of the plant material.

The stages of experiment

This study was divided into three distinct experiments.

Experiment 1: Interaction of GA3 and KNO3 under open pollination on qualitative traits

Gibberellic acid (GA3) (Sigma product) at 0, 30, 60 and 90 mg/L and potassium nitrate (KNO3) (Sigma product) at 0 and 1.5% as foliar spray after flower opening in open pollination condition. Clusters with open pollination alone were used as control.

Experiment 2: Interaction of GA3 and KNO3 under self-pollination on qualitative traits

GA3 at concentrations of 0, 30, 60 and 90 mg/L KNO3 with concentrations of 0 and 1.5% were used in the conditions of bagging the clusters before the flower opening. In this experiment, four clusters per vine were enclosed in paper bags 10 days before anthesis and kept covered for 10 days post-anthesis to prevent cross-pollination. The bags were then removed, and the clusters were sprayed with the aforementioned treatments. The clusters with bagswere used as control in this experiment. The control was applied without GA3 KNO3 treatments.

The first and second experiments were conducted in factorial design based on randomized complete blocks with four replications.

Experiment 3:Cross-pollination

The third experiment included cross-pollination treatments (controlled pollination) of Siah-e-Samarghandi cultivar with pollen grain from five cultivars (Rotabi, Rishbaba, Askari, Atabaki and Siah-e-Shiraz). The pollen grain of Siah-e-Samarghandi cultivar was used for sel-pollination of this this cultivar and recognized as control in this experiment.

Pollen from these cultivars was applied to four selected clusters on each Siah-e-Samarghandi vine during flowering. Suitable clusters were enclosed in paper bags 1–3 days before anthesis. The flowers of the pollinizer cultivars with 25% of opened flowers were placed inside the bags. After 10 to 14 days of flowering, all bags were removed from the cluster.

Time of measurement

The yield of the vine was measured separately after fruit ripening with TSS ≥ 16 and then eight clusters (including four clusters with bags and others without it) were randomly selected from the four sides of the vine and it was transferred to the Physiology Laboratory of Horticulture Department, School of Agriculture, Shiraz University, Shiraz, Iran to investigate quantitative and qualitative characteristics.

Initial fruit set

By counting the number of flowers (10 days before anthesis) and the number of berries (10 days after flowering) formed, the percentage of fruit set was calculated using the following formula.

Fruit set (%) = (number of berries/ number of flowers) × 100.

Physical characteristics

Berry weight

Berry weight was attained by a digital balance.

Number of seeds per berry

Ten berries were randomly selected from each cluster in the first two experiments, while 50 berries were selected in the third experiment. The number of seeds in each berry was counted, and the average seed number per berry was calculated for each treatment.

Fruit firmness

Firmness of berries was estimated according to the method of9. The measurement was performed using a penetrometer (Effegi type (Bishop) FT 327 firmness tester, Italy).

Biochemical characteristics

TSS, TA and pH

A titration method (0.6 N NaOH) was used to determine titratable acidity (TA), as represented by the percentage of citric acid in the solution3. A refractometer ATAGO, Japan was used to measure TSS3. A digital pH meter was used to measure the pH of the juice at room temperature3.

Phenolic compound

Juice extract was diluted 1,1 with 80% methanol for phenolic compound examination,. Then, 100 μL of alcoholic extract was blended in with 400 μL phosphate cushion and 2.5 mL of Folin reagent (Sigma‐Aldrich). After 1 min, 2 mL of Na2CO3 (7.5%) was added to the mixture, and the sample was kept at 25 °C for 5 min before a spectrophotometer (BioTek Instru-ments, Inc., USA)) was used to measure the absorbance at 760 nm. The results were expressed as mg of gallic acid per 100 g FW using gallic acid as the standard10.

Total anthocyanin

Anthocyanins were assessed by pH differential strategy utilizing two different buffers, 25 mM KCl buffer with a pH of 1.0 and 0.4 M Sodium acetate buffer with a pH of 4.5. Five replicates were taken from each sample after incubation for 15 min at 510 and 700 nm in the two buffers. The total anthocyanin content was determined in accordance with the method of Sabir et al.11.

Antioxidant activity

Determination of antioxidant activity was done by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) method11. Regarding this method, 100 ml juice diluted in the ratio of 1:100 methanol and water. Then, this extract was mixed with 2 ml of 0.1 mM DPPH in methanol. After 30 min, the absorbance of the resulting solution was measured at 517 nm by a Cecil 2010 UV–visible spectrophotometer. The absorption of DPPH radical without extract was regarded as a control. The antioxidant activity was calculated using the following equation,

Antioxidant activity (%) = [1—(A 517 nm sample/A 517 nm control)] × 100.

Statistical analysis

The experiment was conducted based on completely randomized design with three replications (each replication containing four clusters). Data were analyzed by SAS software version 9.1 and the means were compared by Tukey test at 0.05 P level. R software was used to draw clustering heatmap for multivariate analysis.

Results

According to the results, the main effect of year was not significant (p value < 0.05) on measured traits (data not shown), so, the data means of two years were reported.

Experiment 1

Initial fruit set

Based on the results of Table 1, the highest fruit set was seen in GA3 treatment at 30 mg/L alone was 27.26% while the lowest fruitset (16.75%) was attributed to the control. Other treatments, except the KNO3 treatment of 1.5% alone were not significant compared to the control treatment.

Table 1 Interaction of GA3 and KNO3 on quantitative traits under under open pollination.
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Weight and number of seeds per berry

The highest number of seeds per berry was obtained in the KNO3 treatment alone (1.86), which was not significant compared to the control. The lowest one was obtained in 1.5% KNO3 + 90 mg/L GA3 (0.94). GA3 at 30, 60 and 90 mg/L in combination with KNO3 decreased number of seeds per berry by 34, 32 and 47%. By increasing levels of GA3 alone, the number of seeds per berry decreased by 18, 32 and 37.5%, respectively (Table 1).

TSS, TA, pH and TSS/TA

TA and TSS in all treatments were lower than the control treatment (Fig. 2A and B). GA3 treatments alone or with KNO3 increased the pH of fruit juice compared to the control. By increasing the concentrations of GA3, pH value also increased in comparison with control (Fig. 2C).

Fig. 2
figure 2

Interaction of GA3 and KNO3 under open pollination on qualitative traits under under open pollination.

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Antioxidant, phenolic content, anthocyanin

Phenolic content deceased in all treatments. It was seen that the highest and lowest one in KNO3 + GA3 at 30 mg/L (502.03 mg GAE/ 100 g FW) and control (393.25 mg GAE/ 100 g FW), respectively (Fig. 2D).

The highest antioxidant (53.19%) and lowest (16.25%) was obtained with GA3 at 30 mg/L alone (Fig. 2E).

GA3 alone at 30, 60 and 90 mg/L increased fruit anthocyanin by 31, 21 and 26%, respectively (Fig. 2F). GA3 alone at 30 mg/L and control had the highest (872.10 mg/L) and lowest (660.86 mg/L) anthocyanin (Fig. 2F).

Experiment 2

Fruit set

Based on the results of Table 2, the percentage of fruit set in the GA3 treatment at 30 mg/L alone was 37.06% which was significant increase compared to the control (19.94%).

Table 2 Interaction of GA3 and KNO3 on quantitative traits under self pollination.
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Berry weight and number of seeds per berry

According to Table 2, increase in berry weight was seen in GA3 at 30, 60 and 90 mg/L alone, but the increase was higher in GA3 at 30 mg/L. KNO3 alone increased berry weight in comparison with control, but this increase was lower in combination with GA3 at 30, 60 and 90 mg/L. The highest and lowest berry weight was observed in KNO3 + GA3 at 90 mg/L (3.379 g) and control (0.8 g).

TSS, TA, pH

TA in all treatments was lower compared to the control (Fig. 3A). GA3 treatments alone or with KNO3 increased TSS and pH of fruit juice compared to the control (Fig. 3B and C). As the concentration of GA3 increased, both TSS and pH of the fruit juice also increased compared to the control (Fig. 3B and C).

Fig. 3
figure 3

Interaction of GA3 and KNO3 under self-pollination on qualitative traits under self pollination.

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Antioxidant, phenolic content, anthocyanin

According to Fig. 3D, phenolic content deceased in all treatments. The highest value was observed in KNO3 + GA3 at 60 mg/L (476.4 mg GAE/ 100 g FW) (Fig. 3D).

The highest antioxidant (58.24%) and lowest (33.09%) was obtained with GA3 at 30 mg/L alone.

GA3 alone at 30, 60 and 90 mg/L increased fruit anthocyanin by 18, 16, and 11%, respectively (Fig. 3E). GA3 alone at 30 mg/L and control had the highest (839.89 mg/L) and lowest (710.41 mg/L) anthocyanin (Fig. 3F).

Experiment 3

Berry weight and number of seeds per berry

Cross-pollination treatments were significantly different from self-pollination in terms of cluster weight. The highest value of cluster weight was related to Askari cultivar (365 g) and the lowest value was related to the Siah-e-Samarghandi (44.67 g).

According to Table 3, pollination with pollens of Ratobi (25.83 cm) and Rishbaba (24.53 cm) compared to self pollination of Samarghandi (21.32 cm) had a significant difference in terms of cluster length. Pollination with Askari, Atabaki and Siah-e-Shiraz pollens had no significant difference compared to self pollination. Referring to Table 3, it was found that all the treatments were significantly different from self pollination in terms of cluster diameter. The largest cluster diameter was related to the Rotabi treatment (11.12 cm) and the lowest one was related to the self pollination (4.53 cm).

Table 3 Interaction of GA3 and KNO3 on quantitative traits under cross pollination (pollination with pollen of different cultivars).
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The results showed that all the berries were seedless in self-pollination conditions of Siah-e- Samargandi cultivar. The highest number of seeds per berry was obtained in the pollination treatment with Siah-e-Shiraz pollen (2.55) and the lowest one was obtained in self-pollination treatment (Table 3).

The highest berry weight was seen in the pollination with Rotabi, followed by Askari, Siah-e-Shiraz cultivars and the lowest cluster weight (0.76 g) in self-pollination.

The highest fruit set was observed in Siah-e-Shiraz (24.62%) and the lowest one was in Atabaki (14.03%). The effect of all treatments on fruit set, except the Atabaki treatment, was significant compared to self-poolination treatment.

The highest and lowest number of berries in the cluster was observed in cross-pollination with Siah-e-Shiraz cultivar (91.85) and Atabaki (52.35) cultivars, respectively.

TSS, TA and pH

The results of the comparison of the pollination treatments of Siah-e-Samargandi grape on TA showed that none of the treatments had significant differences with the control treatment (Fig. 4A-F). The highest amount of TA was obtained by cross-pollination with Atabaki cultivar (1.11%) (Fig. 4A) and the lowest amount was obtained in the Rotabi treatment (0.75%).

Fig. 4
figure 4figure 4

Box plot of TSS, TA, TSS/TA, pH, total phenolic compound, total anthocyanin content and Antioxidant Capacity in fruit juice of the studied grape under cross pollination (pollination with pollen of different cultivars).

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Cross-pollination with Atabaki treatment with TSS value of 16.93% was significant compared to the self pollination treatment (14.97%). Other treatments were not significant compared to each other and compared to the self-pollination treatment (Fig. 4B).

pH of fruit juice was significant in all treatments compared to the control treatment. The highest (3.51) and lowest pH (3.15) was observed in pollination with Rotabi cultivar and self pollination treatment (Fig. 4C).

Antioxidant, phenolic content, anthocyanin

None of the pollination treatments had a significant effect on the amount of fruit phenol (Fig. 4D).

Antioxidant activity was highest in self-pollination treatment (55.78%), and the lowest ones in the Atabaki pollen (18.88%) and Askari pollen (31.54%). The rest of the treatments were not significant compared to the control treatment.

Rotabi cultivar having the highest amount of anthocyanin (913.43 mg/L) compared to the control treatment (674.08 mg/L). None of pollens Askari, Rishbaba, Atabaki, and Siah-e-Shiraz cultivars were significant compared to self-pollination (Fig. 4).

Multivariate analysis

In grouping the cultivars based on increasing dissimilarity, a hierarchical agglomerative cluster assessment was performed (Fig. 5). The first group (I) included ‘Siah-e-Samarghandi cultivar had the lowest cluster weight. The second cluster (II), which included ‘Atabaki’ and ‘Siah-e-Shiraz’ cultivar, had medium value for cluster weight, high anthocyanin and low values for other traits.

Fig. 5
figure 5

Cluster analysis of grape cultivars based on physical and chemical properties of juice (gradient from low (Red), medium (orange) to high (yellow). Abbreviations, TSS, Total soluble solids, TA, Total Acidity (Titratable Acidity) under cross pollination (pollination with pollen of different cultivars).

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Discussion

The effects of KNO3, GA3 and different types of pollination on the quantitative characteristics of Siah-e-Samarghandi grape

As stated in the results, with the increase in GA3 concentration, the number of seeds per berry decreased. So that the lowest number of seeds per berry was observed for the interaction of KNO3 with GA3 at 90 mg/L (results of the first and second experiment). In relation to the berry weight, the highest berry weight was in the treatment of the interaction of KNO3 with GA3 at 90 mg/L and the lowest one was in the control treatment (the results of the first and second experiments). In the first experiment, the percentage of fruit set in response to GA3 treatments with or without KNO3 increased compared to the control treatment. So that, in the first experiment, the percentage of fruit set was the highest in the treatment of GA3 at 30 mg with or without KNO3, and the control treatment and KNO3 alone had the lowest percentage of fruit set. In the second experiment, the highest percentage of fruit set was related to the GA3 treatment of 30 mg/L without KNO3, and the lowest fruit set was related to the control. It seems that considering the fact that in female cultivars, fruit set in self-pollination is formed due to the stimulation of inactive pollen grains, and on the other hand, this stimulating role of inactive pollen grains can be caused by the effect of gibberellin and auxin present in them, therefore, the external use of acid Gibberellic can play such a role in fruit set. This report is consistent with changes in source-reservoir relationships that affect fruit growth and development. GA3 increases the size and weight of the fruit by moving carbohydrates to the fruit12.

The effects of KNO3, GA3 and different types of pollination on the quality characteristics of Siah-e-Samarghandi grapes

The results of our experiment indicated that increasing the concentration of GA3 with or without combination with KNO3 led to an increase in pH and TSS of fruit juice (second experiment) that was in agreement with the results of Gilani et al.13.

The percentage of total soluble solids (TSS) in GA3 treatment alone decreased with the increase of GA3 concentration from 0 to 90 mg/L. But in the interaction of KNO3 treatment with GA3, TSS percentage increased with the increase of GA3 concentration (experiment II). Potassium reduces the amount of acid in fruits and reacts with tartaric acid to form potassium bitartaric and limits its solubility. In other words, lowering the amount of potassium bitartrate leads to the reduction of tartaric acid and as a result the pH increases14. In the present study, GA3 increased the pH but decreased the percentage of soluble solids compared to other treatments in first experiment. Because GA3 causes cell enlargement and increases water absorption, it reduces TSS. According to the reports of Taleb and Abu Zahra8, treatment with GA3 leads to an increase in TSS percentage of grape fruit which is not consistent with the results of this experiment. The results of the experiment indicate that the percentage of antioxidant activity is the highest in the treatment of 1.5% KNO3 without the presence of GA3, and the lowest in the interaction of KNO3 treatment with 60 mg GA3 (first experiment). .

But in the second experiment, the highest percentage of antioxidant activity is related to the interaction of GA3 treatment of 30 mg/L with 1.5% KNO3, and the lowest is related to the 1.5% KNO3 treatment without the presence of GA3. These results showed that in seeded berries (experiment 1), which are a rich source of GA3, foliar spraying of GA3 leads to a decrease in antioxidant activity. But in seedless berries (second experiment), because the berries are seedless and do not have a rich source of GA3, foliar spraying with GA3 at (30 mg/L) leads to an increase in the percentage of antioxidant activity. It should be noted that potassium can also increase antioxidant activity due to its role and presence in the synthesis and acceleration of the transfer of carbohydrates and enzymes (experiment 1).

In an experiment, Muscat grape clusters were treated with GA3 100 mg/L showed that GA3 successfully induced seedless in the clusters, increased cluster size, and accelerated cluster growth. As a result, it causes premature ripening of seedless berries15.

The amount of anthocyanin in red grapes varies with the cultivar, fruit maturity, cultivation and production area, seasonal conditions and yield. In the interaction of GA3 at 30 mg/L with 1.5% KNO3, the highest value and the control cultivar had the lowest value (experiment 1). In the second experiment, the interaction of GA3 60 mg/L with KNO3 1.5% was the highest and the control treatment had the lowest amount of anthocyanin. The amount of fruit phenol was the highest in the interaction of GA3 treatments with KNO3 of 1.5%, and the reason for this increase can be attributed to the interaction of the effect of KNO3 and GA3 in increasing the amount of phenolic substances16. The results of this experiment were consistent with the report that stated that KNO3 increased anthocyanins and increases the amount of polyphenolic compounds in berries17,18,19,20,21.

The percentage of fruit set was different and significant in response to the pollen of different cultivars compared to the control treatment. So, this percentage was the highest in Shiraz black treatment (24.62%), and it was the lowest in Atabaki pollen (14.03%) and self-pollination treatment (14.10%). According to the positive relationship between the number of seeds and fruit set and size, it can be said that because the Shiraz black cultivar had higher seeds, it also had more fruit set compared to other treatments. It can be concluded that the higher the number of seeds in a berry, the higher the percentage of fruit formation and the lower the percentage of spherical berries. In an experiment, fruit set in the cross-pollination method varied from 30 to 40.6 percent and from 15 to 34.3 percent in the self-pollination method. In this study, cross-pollination led to a significant increase in fruit set, berry weight, number of seeds per berry, percentage of total soluble solids (TSS) and total acid (%TA), in comparison with self-pollination9,18.

The effect of pollen grains of different grape cultivars on the quality characteristics of Siah-e-Samarghandi cultivar

According to the above results, clusters pollinated with pollens of Atabaki cultivar led to an increase in TA. The acid ratio of fruit juice increased in all pollination treatments compared to the control treatment. The percentage of total soluble solids showed the highest increase in the Atabaki treatment, and the lowest increase was related to self-pollination treatment.

The results indicate that the amount of anthocyanin was the highest in the pollination treatment with the Rotabi cultivar and the lowest in the pollination treatment with the pollen of the Siah-e-Samarghandi cultivar (control).

Phenolic substances had the highest amount in Askari pollination treatment and the lowest amount in the cross-pollination treatment with Rotabi cultivar.

Sabir20,22 reported that cross-pollination treatments increased or improved quantitative and qualitative traits such as TA, TSS, sugar-acid ratio and other fruit characteristics. It has been reported that the sources of pollen grains had a significant effect on quantitative traits such as the amount of berry seeds, percentage of fruit set, berry weight and qualitative traits such as fruit juice acid percentage, TSS and TA. It has an Italian cultivar22.

It was reported that the effect of pollen grain on the mentioned characteristics is significant in some crossings and not significant and this issue can be related to the mutual effect of cultivars23. Pollen grains of Atabaki cultivar had highest effect on TSS of Siah-e-Samarghandi cultivar and cross-pollination with Rishbab had the lowest one.

Conclusion

In this experiment, it was found that all the three mentioned experiments led to an increase in the percentage of fruit set, a significant reduction in berry drop, an improvement in cluster morphology and other quantitative and qualitative characteristics of the fruit. Considering the role of KNO3 and GA3 on increasing fruit size and decreasing or not having seeds in grapevine cv. Siah-e- Samarghandi and the positive application of different sources of pollen grains on decreasing shot berry, it was suggested to use KNO3,GA3 and cross-pollination with pollen grains for further studies on this cultivar.