In vitro introduction of Prunus persica (L.) Batsch ‘Dosto yni y̕

. The article discusses the introduction in vitro , selection of the nutrient medium and plant growth regulator effect on the microshoot development of the perspective fruit crop Prunus persica ‘ Dostoyni y̕ . Murashige and Skoog (MS), Gamborg and Eveleg (B5), Blaydes (BL), McCown (WPM) media supplemented with 0.5 mg/l 6-BAP, 0.1 mg/l IBA; 1.0 mg/l 6-BAP, 0.025 mg/l IBA; 0.48 mg/l KIN, 0.1 mg/l IBA; 0.96 mg/l KIN, 0.025 mg/l IBA and succinic acid were used in our research. According to obtained results, the most optimal season for the introduction of P. persica ‘ Dostoini y̕ into in vitro culture was summer. The use of alcohol, and chlorine-containing sterilizing agents in combination with antibiotics (or without them) provides aseptic material. BL and B5 media supplemented with 0.5 mg/l 6-BAP and 0.1 mg/l IBA, as well as 1.0 mg/l 6-BAP and 0.025 mg/l IBA promote microshoot development in vitro . Succinic acid in the culture medium at concentration of 5 and 10 g/l contributes to an additional increase in the multiplication rate.


Introduction
Fruit crops are grown throughout the world mainly as a source of food.The peach (Prunus persica (L.) Batsch.) is one of the most valuable woody plants.Its fruits are the most important component of the human diet since they contain sugars, vitamins, microelements, organic acids, enzymes, and other biologically active substances, which serve as an indispensable part of high-quality nutrition [1].Propagation via seedlings or through regrafting (mainly budding) are traditional ways for peach reproduction [2].
Clonal micropropagation is one of the most effective ways to obtain plant material in significant volumes in a short period [3,4].Micropropagation technologies are known and well-developed, but their improving is necessary for efficiency increase [5].At the same time, the use of in vitro technology, in particular clonal micropropagation, is associated with several problems, which in turn affect the experiment's efficiency and productivity.Explant selection and introduction in vitro onto nutrient medium are some of these problems [3,6].

Results and discussions
The development of a clonal micropropagation protocol is a process consisting of a number of steps.The main ones are the stage of introduction in vitro, selection of the nutrient medium, and growth regulator concentrations that provide the maximum percentage of microshoot formation.

Introduction in vitro
One of the main steps for obtaining a normally developing culture in vitro is sterilization.Therefore, in our studies, various sterilants and their combinations were used during two growing seasons to prevent plant material infection.According to the data obtained, in 2022 (summer period), the percentage of explants without contamination was quite high (Table 1).In 2023, the introduction into culture in vitro was carried out during active flowering and formation of green mass (April-May).The number of sterile explants was relatively low.The use of antibiotics only at the stage of sterilization was ineffective.Their additional introduction into nutrient media reduced contamination and increased the percentage of sterile explants (47%).
Currently, there are a number of sterilizing methods for various explants of fruit and berry crops [10,11].When choosing sterilizing agents, their concentration, combination, exposure duration, the nature of contamination and the type of explant are taken into account [6].In woody plants, in particular in peach, endophytic spore-forming and nonspore-forming bacteria are found in tissues [12], which, getting into in vitro conditions along with the material, develop, causing contamination.Also, the season of the year when selection of material takes place is of great importance under introduction to in vitro culture [13].
We have shown the greatest applicability of the apical parts of the shoots of P. persica cv.'Dostoyniy̕ , selected in the summer and sterilized with alcohol-, and chlorine-containing agents in combination with antibiotics or without them.At an earlier time, antibiotics should be introduced into the culture media to eliminate fungal and/or bacterial infection.

Selection of the nutrient medium for peach microshoot development
It is known that in vitro morphogenesis depends on many factors such as material genotype, type of explant, its orientation, light intensity, temperature, gas composition, and nutrient medium components, including growth regulators [14][15][16].An important task is the selection of the nutrient media for growth supporting introduced into in vitro culture explants.For the cultivation of fruit and berry crops, Murashige and Skoog, Nitsch, Hamborg and Eveleg (B5), Andersen, Lloyd and McCown media are often recommended [17].In our studies, shoot apical parts and nodal segments of P. persica 'Dostoyniy̕ were cultivated on nutrient media B5, MS, WPM, BL (Table 2) supplemented with 0.5 mg/l BAP and 0.1 mg/l IBA; as well as 1.0 mg/l BAP and 0.025 mg/l IBA.According to the obtained results, an in vitro growth and development of P. persica 'Dostoyniy̕ microshoots was observed on all media.Morphogenetic processes in explants from the apical parts occurred on the 8 th d after in vitro introduction, and the nodal segments formed leaves on the 14 th d.The largest number of adventitious microshoots was formed on the BL medium.Microshoots were characterized by smaller size and decreased leaf quantity (Table 2).The highest multiplication rate was observed on the BL medium with 1.0 mg/l 6-BAP, four microshoots per explant could form (Fig. 1).The multiplication index was 1.35 microshoots per explant.A small percentage of hyperhydrated material (up to 10%) and microshoots with callus (20%) was noticed.Reducing 6-BAP concentration to 0.5 mg/l in the Blaydes medium decreased the multiplication rate, explants (almost 3 times) as well as the number of microshoots with callus by half.The replacement of 6-BAP on the kinetin at equimolar concentrations in our experiments reduces the explant hyperhydricity but significantly increases the microshoot percentage with callus (Table 3).

The succinic acid effect on the adventitious microshoot formation
A well-known methodological technique in plant physiology is the use of organic acids as growth regulators [18,19] which participate in the main physiological processes.Nutrient media including malic, citric, and other organic acids are known in the literature [20].Succinic acid is a well-known growth regulator that at high concentrations (10-50 g/l) has a cytokinin-like effect [21].Therefore, its influence on the micropropagation of hard-topropagate crops is of particular interest.As a nonspecific growth stimulator, succinic acid (in the form of potassium succinate) was added to the BL medium at concentrations of 1.0, 5.0, and 10.0 g/l (0.008, 0.04, and 0.08 M).According to our results, the presence of the succinic acid in the BL media had a positive effect on the formation and development of P. persica ʻDostoyniyʼ microshoots, compared with the control (BL medium without succinic acid).The introduction of 1.0 and 5.0 g/l of succinic acid increased the multiplication coefficient and reduced hyperhydratation and callus formation (Table 4).The introduction of 10.0 g/l of succinic acid had a positive effect on the microshoot formation and its number per explant.

Conclusion
According to obtained results, the most optimal season for the introduction of P. persica 'Dostoyniy̕ into in vitro culture was summer.The use of alcohol-, and chlorine-containing sterilizing agents in combination with antibiotics (or without them) provides aseptic material.Blaydes' and Gamborg-Eveleg's media supplemented with 0.5 mg/l 6-BAP and 0.1 mg/l IBA, as well as 1.0 mg/l 6-BAP and 0.025 mg/l IBA promote microshoot development in vitro.Succinic acid introduction at a concentration of 5 and 10 g/l contributes to an additional increase in the multiplication coefficient.

Acknowledgements
The study was carried out within the framework of the State Task No. FNNS-2022-0010 of the FSFIS "NBG-NSC".

Table 1 .
Sterilization method influence on the peach cv.'Dostoyniy̕ explant survival

Table 2 .
In vitro microshoot formation of P. persica cv.ʻDostoyniyʼ on different culture media

Table 3 .
Growth regulators' influence in the BL medium on adventitious microshoots' formation of P. persica cv.ʻDostoyniyʼ in vitro

Table 4 .
Succinic acid influence on morphogenesis and morphometric parameters of in vitro cultivated P. persica ʻDostoyniyʼ explants