INFLUENCE OF ROOTSTOCKS ON THE PRODUCTIVITY AND CHEMICAL COMPOSITION OF PRUNUS DOMESTICA L. FRUITS

The influence of seedling and clonal rootstocks of different spreads on Prunus domestica L. plum fruits quality and productivity of Yaichnaya Sinyaya and Utro varieties was studied. The significant change of productivity and the fruit weight of the varieties under study was to determine under the influence of the rootstock. Depending on the scion-stock combination the plum tree's productivity varied from 7.5 kg/tr. (Utro/140-1) to 15.1 kg/tr. (Yаichnaya Sinyaya /Novinka) at the mean value of 11.5 kg/tr. Medium-growing rootstocks Novinka and OPA-15-2 provided the maximum value of the varieties productivity The significant productivity decrease relatively to seedling rootstock was stated for the combinations with low-growing rootstock 140-1. Soluble solids content in the fruits of Yаichnaya Sinyaya variety is higher than in the fruits of Utro variety, moreover, the highest values were determined on Novinka and OPA-15-2 rootstocks: on 4 – 4.5% higher in comparison with the fruits on the seeding rootstock at average. The rootstock causes less influence on titratable acids. The antioxidant activity of Yаichnaya Sinyaya variety fruits is 30% higher than that of Utro variety fruits on average. The maximum values of antioxidant activity in Yаichnaya Sinyaya variety fruits were fixed on OPA-15-2 rootstock (16.37%), the minimal ones – on Skorospelka Krasnaya rootstocks (14.68%). In the fruits of Utro variety the highest values were stated on OP-23-23 rootstock (13.16%), and the lowest ones – on the seedling rootstock (10.93%). The content of phenolic compounds sum is 60% higher in the fruits of Yаichnaya Sinyaya variety than in Utro variety ones on average. The decrease of the content of phenolic compounds sum was stated in the fruits of Utro variety on all the rootstocks in comparison with the combination Utro/seedling rootstock. The decreasing series of ash elements accumulation (K > P > Ca > Mg > Mo > S > Zn > Si) was determined. The strongest variety differences on total mineral element content were overvalued on medium-growing clonal rootstocks (Novinka, OP-23-23 and OPA-15-2). OPA-15-2 and OP-23-23 rootstocks provided the highest fruit quality on the combination of economic and biochemical parameters.


INTRODUCTION
The popularity of domestic plum (Prunus domestica L.) in different horticultural zones of Russia is connected with ecological plasticity, winter resistance, early maturity, and stable productivity of cultivated varieties (Upadysheva, 2015;Eremin and Brizhinov, 2011). New clonal stocks are used to duplicate new valuable varieties of fruit crops and to create intensive plantations because they favorably influence on adaptivity, early maturity, and productivity of grafted plants (Upadysheva, 2017;Eremin et al., 2000). The spread, the start of fruiting, and the productivity of grafted plants depend on the rootstock (Blazek and Pistekova, 2012; Hatton et al., 2015).
Fruits quality is a genetically associated character for each variety, but in the scientific literature there are data about the significant influence of a rootstock on the weight Seedling rootstocks (seedlings of domestic plum and alucha) and clonal ones of different growing spread  and Novinka and low-growing -140-1, VVA-1) are used for plums cultivation in the Central region of Russia (Eremin, 2000). Many questions devoted to plum varieties propagation and scion-stock combination selection are successfully solved in the world; at the same time, the life length of grafted plants and the harvest quality depending on a variety and a rootstock are not studied enough. The main component of plum fruits' chemical composition is minerals. Among them, K, P, Mg, and Ca are found in big quantities, whereas other minerals such as Fe, B, Cr, Mn, Cu, and Zn are present in much fewer quantities (Cosmulescu et al., 2017). The mineral composition changes and the content of bioactive compounds in fruits Prunus domestica L. depending on the rootstock were practically understudied. In scientific literature, the data about the stock influence on the leaves mineral composition are only presented (Meland, 2010;Milošević and Milošević, 2011;Ionica et al., 2013;Reig et al., 2018). The accurate forecast of plum scion-stock combinations germinability and productivity and the stock influence on the nutrients and bioactive substances accumulation in plum fruits is very important for the output yield of high quality.

Figure 1
The garden of intensive type during blossoming.

Figure 2
The plum fruits: Yaichnaya Sinyaya (on the left) and Utro (on the right).
That is why this work aimed to determine the influence of different growing strength rootstocks on the fruits chemical composition and productivity of the domesticated plum varieties bred by Horticulture. 2 Federal State Budget Scientific Institution «Federal Scientific Selection and Technological Center of Horticulture and Nursery Breeding» (Federal Horticultural Research Center).

Scientific hypothesis
The domesticated plum (Prunus domestica L.) fruits productivity and quality, their biological composition depending on the used rootstocks are not studied enough. We have checked the influence of the strong-growing seedling and medium and low-growing clonal rootstocks on the fruit productivity, quality, and nutritional value of the plum varieties, i.e. Utro and Yaichnaya Sinyaya grown in Moscow region conditions. We supposed that on the base of the field and laboratory experiments the optimal stock for each variety that will provide the high productivity and valuable bioactive substances accumulation in the plum fruit will be found.

Conditions of plant growing
The field researches were held in 2018 -2019 on the experimental Prunus domestica L. plantations, located at laboratory plot of Federal Horticultural Research Center for Breeding, Agrotechnology and Nursery in Moscow region (55° 56′ of North latitude, 37° 64′ East longitude). The plantation overall area was 0.5 ha. The garden of intensive type was set out in 2010 using the scheme 5 x 2.5 m. The soil in the row spacing was black fallow (Figure 1).

Biological material
The fruits of Utro and Yaichnaya Sinyaya varieties on 6 stocks, i.e. strong-growing P. domestica L. seedlings, medium-growing clonal rootstocks Skorospelka Krasnaya The biochemical studies were held in the laboratory of biochemistry and physiology of Federal Horticultural Research Center for Breeding, Agrotechnology, and Nursery.

Chemicals
All chemical substances chosen for the analysis were of an analytical sort and were bought from Sigma Aldrich (USA).

Sample preparation
From representative not less than 500 g fruits probe, 300 g of fruit without stone were prepared. The mass was homogenized using the analytical homogenizer IKAA11 basic (Germany). Then it was extracted by double distilled water (to determine antioxidant activity and phenol compounds sum) and by pure methanol (to study the metabolites composition) and centrifugated at 4000 g (Sigma, Germany) within 10 min. The supernatant was used for measurement purposes. We performed all extractions in triplicate independent samples.

Basic chemical analyses
General biochemical parameters, i.e. soluble solids content (SSC) and total titratable acidity (ТТА). SSC was determined via refractometric method according to (GOST ISO 2173, 2013), the values were expressed in %. ТТА was estimated via the potentiometric method by pH meter HI 2211 HANNA (Germany) via titrating with 10 N. NaOH and expressed in the equivalent of apple acid, % (GOST ISO 750, 2013).

Total phenolic compounds analysis
The total phenolics amount was determined with Folin-Ciocalteu reagent according to the method described by Velioglu et al. (1998). A standard curve with gallic acid was used. Different concentrations of gallic acid were prepared in distilled water, and absorbance was recorded at 750 nm. 100 μL of the diluted sample (1:10) was dissolved in 500 μL of Folin-Ciocalteu reagent and 1000 μL of distilled water. The solutions were mixed and incubated at room temperature for 1 min. After 1 min, 1500 μL of 20% sodium carbonate (Na2CO3) solution was added. The final mixture was shaken and then incubated for 2 h in the dark at room temperature. The absorbance was measured at 750 nm using a Helios Υ UV-vis spectrophotometer and the results are expressed in mg of gallic acid (GEA) calculated on the wet weight of plants.

Тоtal antioxidant capacity
The scavenging activity on the 2, 2-diphenyl-1picrylhydrazyl (DPPH) radical was determined spectrophotometrically according to the method described by Brand-Williams et al. (1995). The principle of the analysis was based on the colour change of DPPH solution from purple to yellow as the radical was quenched by antioxidants. The homogenized leaves were mixed with distilled water and methanol. The samples were put on the shaker Lab-PU-01 (Russia) for 6 hours, and then they were filtered and the antioxidant activity was measured in 10 minutes after interaction between the extract and the reagent. The absorbance was recorded at 517 nm to determine the concentration of the remaining DPPH. All measurements were performed in triplicate. The radicalscavenging activity was calculated as a percentage as follows: The lower absorbance of the reaction mixture indicates a higher level of free radical scavenging activity.

EDS -analysis
The chemical composition of the basic ash components (Mg, Si, P, S, K, Ca, Zn, Mo) was determined by the method of energy dispersive spectrometry (ESD) on the analytical raster electron microscope JEOL JSM 6090 LA according to the methodology (Motyleva, 2018). X-ray microanalysis data are presented in the form of standard protocols which contain the microstructure picture of the sample under study, the table of the data in weighting and atomic correlation, spectra, and histograms рresented in Figure 3.

Мetabolic analysis by gas chromatography-mass spectrometry
The metabolites analysis was fulfilled using the method of gas chromatography-mass spectrometry (GC-MS) via GCMS chromatograph JMS-Q1050GC («JEOL Ltd», Japan). Capillary column DB-5HT (Agilent, USA); length 30 m, inner diameter -0.25 mm, the film thickness -0.52 um, and gas-carrier -helium) was used. The temperature gradient during the analysis was within 40 -280 °С, the injector and interface temperature -250 °С, the ionic source -200 °С. Gas flow in the column was equal to 2.0 mL.min -1 , split-flow injection mode, sample injected in volume 1 -2 mcL of the evaporated extract. The analysis was held for 45 min. The derivation was held using silylation reagent N,O bis (trimethylsilyl) trifluoracetamide (BSTFA) following the method described by Robbins (2003). The substances identification was done according to NIST-5 National Institute of Standards and Technology (USA) retention behavior and mass spectra the scanning range was 33 -900 m/z. The substance identification credibility was within 75 -98%. Number of repeated analyses: 3 Number of experiment replication: 2

Statistical analysis
All the analyses were performed in triplicate. The results were expressed as mean values (n = 3) in standard deviation (SD). Statistical analyses were carried out through the Excel package (Microsoft Excel, v. 2016).

RESULTS AND DISCUSSION
We studied the plum trees' productivity of different scion-stock combinations in the field conditions, determined the fruit average weight, fulfilled the fruits organoleptic and degustation estimation. As a result of our studies, it was stated that during 2018 -2019 the plum tree's productivity was 11.5 kg/tree on average, and depending on scion-stock combination it changed from 7.5 kg/tree (Utro/140-1) to 15.1 kg/tree (Yаichnaya Sinyaya/Novinka). This parameter was above average at the understudied varieties grafted on medium-growing clonal rootstocks Novinka and OPA-15-2. The minimum parameters were stated at the combinations with lowgrowing rootstock 140-1 (Table 1).
The rootstock influence on P. domestica L. productivity is discussed in the papers ( Studied varieties belong to large-fruited ones with fruits weight more than 30 g. The Utro variety's average fruit weight is 31.2 g, but depending on the stock it varied from 29.7 g (seedlings P. domestica L.) to 33.8 g (OP-23-23). The understudies varieties fruits were significantly larger under OP-23-23 rootstock influence. The Utro variety fruit stone was separated from the flesh well, its portion in the fruit weight was 6% on average with variation from 5.7% (Novinka) to 6.5% (seedlings P. domestica L.).
The Yаichnaya Sinyaya variety fruit stone was larger and was near 7% of the fruit weight for all the combinations, it separated from the fruit flesh well. According to the degustation results, the understudied varieties of fruits were characterized with balanced sour-sweet taste and smell (4.2 -4.8 scores). The degustation estimation had  Depending on the variety and the rootstock SSC in the fruits varied from 12.72% (Utro/Skorospelka Krasnaya) to 17.48% (Yaichnaya Sinyaya/Novinka). In the Utro variety fruits on Novinka, OPA-15-2 and 140-1 rootstocks SSC was on 1 -1.3% higher in comparison with the fruits on the seedling (Figure 4). In the Yaichnaya Sinyaya variety fruits, SSC was higher than in the Utro variety ones; SSC maximum values were determined on Novinka, Skorospelka Krasnaya, and OPA-15-2 rootstocks, i.e. on 4 -4.5% higher than in the fruits on the seedling at average.    Our study results confirm the rootstock influence on bioactive substance synthesis in Prunus domestica L fruits. The antioxidant activity of Yaichnaya Sinyaya variety fruits is 30% higher than Utro variety fruits on average ( Table 2). The maximum AA values are determined in Yaichnaya Sinyaya variety fruits on the rootstocks OPA -15-2 (16.37%) and 140-1 (16.58%), and the minimal ones -on Skorospelka Krasnaya rootstock (4.68%). The highest AA values in Utro variety fruits were registered on Skorospelka Krasnaya (13.9%) and ОР-23-23 (13.16%) rootstocks, and the lowest ones -on the seedlings (10.93%). РС content in Yaichnaya Sinyaya variety fruits was 60% higher than in Utro variety ones on average ( Table 2). The maximum PC content was registered in Yaichnaya Sinyaya variety fruits on Skorospelka Krasnaya and 140-1 rootstocks, i.e. 0.94 and 0.91 mg.g -1 of the fruits equivalent of gallic acid relatively, and the minimum PC content were stated on OP-23-23 rootstock, i.e. 0.43 mg.g -1 of the fruits equivalent of gallic acid.
In Utro variety fruits on all the rootstocks, the decrease of PC sum content was stated in comparison with Utro/seedling combination, and on Skorospelka Krasnaya, OPA-15-2, and 140-1 rootstocks the content was twice less.
The mineral substances are an important component of human nutrition, they are a part of enzymes and have antioxidant activity; they are not synthesized in the human organism, but get inside it only with meals. Eight main elements, i.e. Mg, Si, P, S, K, Ca, Zn, Mo, were determined in the plum fruits. K is the main element in the plum fruits. Its content varied from 26.84 mass % (Utro/OP-23-23) to 30.76 mass % (Utro/OPA-15-2) ( Table  3). K content variation coefficient is low (4.88%) that speaks about the insignificant influence of the rootstock on this element accumulation. P content varied within the range from 2.69 mass % (Utro/ ОP-23-23) to 4.34 mass % (Utro/Novinka). P content variation coefficient is 16.7%, which speaks about a stable input of this element in plum fruits.
The portion of Ca in Utro variety fruits is from 1.04 mass % (Utro/OPA-15-2) to 3.29 mass % (Utro/Skorospelka Krasnaya). This element variation coefficient is medium. In Yaichnaya Sinyaya variety fruits K is accumulated on 4% less than K and twice less than Ca than in Utro variety fruits (Table 4). The content of Mg, P, S, K, and Zn on all the rootstocks is higher than on seedlings. Among all the scion-stock combinations we can distinguish the fruits of the following combinations on all the elements sum content: Yaichnaya Sinyaya/OР-23-23 (36.63 mass %), Yaichnaya Sinyaya/140-1 (36.48 mass %), and Yaichnaya Sinyaya/ Skorospelka Krasnaya (38.89 mass %).
Significant differences in mineral elements sum content were determined in the fruits between the varieties and the scion-stock combinations of varieties and rootstocks. The greatest differences in the mineral substances sum content were registered on the medium-growing clonal rootstocks Novinka, OP-232-23, and OPA-15-2.
Higher accumulation of Ca, Mg, and K were determined in the fruits of both varieties on Skorospelka Krasnaya rootstock in comparison with other rootstocks. The decreasing series of the accumulation of the elements in plum fruits is as follows: K > P > Ca > Mg > Mo > S > Zn > Si.
The K, Ca, and P values received by us as a result of this study correlate to the data given in the scientific literature in a proportional relation. The differences for some elements content can be connected with the environmental conditions, such as the soil type, the precipitation depth, and the fertilizers application (Kabata-Pendias, Mukherjee and Trace, 2007).

CONCLUSION
The present study stated the rootstock influence on the plants' productivity and bioactive substances accumulation in Prunus domestica L. fruits. Under the influence of medium-growing rootstocks, i.e. OPA-15-2, Novinka, and OP-23-23, the increase of not only main economic parameters (output yield, the fruit size, and weight, sensor evaluation, SSC and ТТА) but biologically important compounds (antioxidants, metabolites, macro, and microelements) was registered as well. The optimal rootstocks for Yaichnaya Sinyaya and Utro varieties are medium-growing rootstocks OPA-15-2, Novinka, and OP-23-23.