Preliminary results of in-vitro laboratory propagation of barberry (Berberis L.)

. The genus Berberis vulgaris ( Berberis L.) is currently found in tropical, subtropical, temperate, and cold climate zones. There is little doubt that Southeast Asia is where the majority of Berberis vulgaris' interspecies biodiversity is found. Additionally, species are widely distributed, mostly in hilly areas, and, in terms of moisture requirements, they are mesophytic and xeromesophytic plants. In this article, experiments on in-vitro propagation of Berberis vulgaris ( Berberis L.) seedlings were carried out in laboratory conditions . During the experiments, the time of sterilization of the explants obtained from the Berberis vulgaris plant was determined. Different concentrations of hormones and auxins were used in several nutrient mediums for growing in artificial nutrient medium. The results depicted that the highest rate was observed in the variant, where 2ml.BAP+0.5NAA+30g sucrose+7g accounted for 87% of plant rooting rate. It was observed that the rooting rate was slightly higher in the control variant of WPM-Woody plant medium-nutrient medium than the control variant of the MS nutrient medium, which was 17%.


Introduction
The current distribution of the genus Berberis vulgaris (Berberis L. ) covers tropical, subtropical, temperate and cold zones. Clearly, the interspecies biodiversity of Berberis vulgaris is mainly located in Southeast Asia (provinces of China-Sichuan, Yunnan, and Southeast Tibet, the mountains of Central Asia and the western sea coast of South America). Furthermore, species are widespread mainly in mountainous regions, they are mesophytic and xeromesophytic plants in relation to moisture [1].
Paleobotanical researchers A.N. Krishtofovich and T.N. Baykovskaya found traces of Berberis vulgaris leaves in the Lower Sarmatian deposits in the area of Russia known as Krinka. E.F. Kutuzkina Armavir reported that traces of Berberis vulgaris leaves were similar to Berberis vilgaris L. in ancient Sarmatian deposits . In 1933, geologist A.Ya.
Petrosyants investigated fossils in a Tertiary salt bed in Kyrgyzstan. According to E.F. Kutuzkina, the remains of this plant are deciduous trees-shrubs of the northern slope, which grew in the Kochkor depression [2][3][4][5].
Afterwards, V.S. Kornilova [3] found about 60 plant traces, and identified them as Picea, Juniperus, Populus, and Berberis species. Evidently, berberis vulgaris species are widely distributed in North, Central and South America, Europe (Caucasus and Crimea regions together) and Central Asia, and accordingly, they appeared in the Cretaceous period before the separation of the Gondwana continent. In the Berberis L. series, the oldest species belong to the Sinenses df Vulgares series, and the first series includes 45 species, which are mainly from East and Central Asia and it was distributed in North America and the Far East and the Caucasus. Whereas, the second series includes 15 species, which were distributed in Europe, the Caucasus, Iran, the Far East and Central Asia [1,5].
Scientific work of De Candolle (De Candolle, 1821) plays a major role in the scientific study of the Berberidaceae family. In the research, more than 29 types of berberis vulgaris were investigated. Eurasian and South cypresses are well distinguished from each other by obvious biomorphological features: the flowers of South American cypresses are dark fiery, and the leaves are evergreen. The flowers of Eurasian cycads are lemon-yellow, and the fruits are bluish-black, red-pink, and they are mainly deciduous species [4][5][6].
In 1905, S.K. Schneider [5 ] proposed the use of a new natural system in order to improve the systematics of the genus Berberis L. He was the first to divide all the species included in the genus Berberis L. into two botanical-geographical groups: Septentrionales S.K. Schneider (European, Asian and North American species) and Australes S.K. Schneider (South American species) groups. The first group includes all Eurasian species, 2 North American species. The main cypress species in this group are in the north, and only 3 cypress species grow naturally south of the equator (2 in East Africa and one in Indonesia). Similarly, K.Z. Ismailova [8] studied the naturally distributed Berberis integerrima and Berberis nummularia Berberis vulgaris species in the forest phytocenoses formed on the lower terrace of the Zarafshan River in the Zarafshan Reserve, and the research revealed many natural red hybrids in the territory of this 2352-hectare and 45-km-long reserve along the river basin. Clearly, fruit color was ranged in color from purple to dark purple [7,8].
Noteworthy, berberis vulgaris species prevent soil erosion on hillsides with their lateral roots, and grow freely on rocky and eroded hillsides. Berberis vulgaris species grow in nature as mesophytic, sometimes xeromesophytic plants, but most of them are distributed in mountainous areas, mostly on northern mountain slopes, especially in well-supplied places with moisture near streams [9,10]. In fact, mountain conditions have a unique effect on the biology of Berberis vulgaris species, which makes it difficult to understand the variability and geographical characteristics of their seeds, their formation and germination.
Central Asian Berberis vulgaris species have been used by the local population for their fruits and roots. They are also cultivated as an ornamental medicinal shrub. Its fruits are rich in biologically active substances and vitamins, therefore, it is used as a blood-stopping agent in scientific medicine. In the following years V. Oblonga, B. integgerima, B. nummularia, valuable species of Berberis vulgaris are being cultivated as medicinal plants in industrial plantations [8]. This research aims at further investigation of berberis vulgaris towards unrevealing variabilities, formation and germination process of Berberis L.

Methods and materials
In this research, for in vitro cultivation of cells and tissues isolated from plants, nutrient mediums with the composition created by T. Murasiga and F. Skuga were used. Furthermore, sterilization of initial plant materials was carried out according to the method of R. G. Butenko [5], and statistical analysis of the research results was undertaken using the dispersion method of A.B. Dospehov [7], where Microsoft Excel was employed in statistical calculation.
During the experiment, medicinal and ornamental berberis (Berberis L) plant was cultivated in vitro. In Table 1, the method recommended by R. G. Butenko [5] was used to sterilize the explants obtained for the experiment. First, donor seedlings of Berberis L. were selected for the experiment . In the selection of donor seedlings, attention was paid to all aspects of the plants, seedlings with + indicators were taken as donors. For the experiment, 100 explants for each variant were prepared from the meristem of seedlings. To sterilize the prepared explants, they were first immersed in 70% ethyl alcohol for 20 seconds and sterilized with 3-5-7% sodium hypochlorite and 7% hydrogen peroxide for different minutes ( Table 1).
The main purpose of our experimental work was to directly root the young shoots of the Berberis vulgaris (Berberis L) plant. In order to root Berberis vulgaris (Berberis L) explants in artificial nutrient medium, Murasige and Skuga (MS) and WPM-Woody plant mediumnutrient medium were experimented with different concentrations of plant hormones cytokinin and auxins. The air temperature in the special room where the explants were planted was kept at 22-23 °C , relative air humidity at 63-65% for 14-20 days, and 14 days after planting the explants, rooting processes started in the explants.

Results and Discussion
According to the results of sterilization, survival of explants after 10 minutes of sterilization in 0.5% solution of sodium hypochlorite was recorded as 88%, which was the highest indicator compared to other experimental variants (Table 1). It was found that a high result was achieved in the variants where different concentrations of cytokinins and auxins were applied to the nutrient medium of Berberis L. explants in Murasige and Skuga (MS). In the experiment, the lowest indicator was observed in the control variant without hormones (Figure 1). Currently, there are several nutrient media that differ from each other in terms of composition. Nutrient media for cell tissue growth, all macroelements (nitrogen, phosphorus, potassium, calcium, magnesium, sulfur) and microelements (boron, manganese, zinc, copper, molybdenum), which are necessary for good plant growth, and vitamins, carbohydrates, phytohormones or their synthetic analogues should be stored. T.Murasiga, F.Skuga and WPM-Woody plant medium were mainly used in 1962 to grow explants of Berberis vulgaris bush in vitro. It was reported that BAP (benzylaminopurine), NSK (naphthalene acetic acid), Auxin 2.4-D (diclofenoxy acetic acid) were used in these nutrient media. In the control variant, MS nutrient medium itself was used, followed by MS+ 1ml.BAP+30g sucrose+7g in the second variant, 2ml.BAP+0.5.NAA+30g sucrose+7g in the third variant, 1ml.BAP+2ml 2.4D+30gr sucrose+7gr in the fourth variant and 1.5ml.BAP+2ml,2.4D+30g sucrose+7gr in the fifth variant (Table 2).
Furthermore, in the case of WPM-Woody plant medium-food environment, the same method was used, accordingly, concentration of BAP (benzylaminopurine), NSK (naphthalene acetic acid), Auxin 2.4-D (diclofenoxy acetic acid) were used. Clearly, in the control variant, WPM-Woody plant medium-nutrition medium without hormones was used, followed by 2ml.BAP+0.5ml.NAA+30g sucrose+7g in the second variant, 1ml.BAP+0.5ml.NAA+30g sucrose+ 7g in the third variant, 2ml.BAP+0.5ml 2.4D+30gr    It was reported that rooting rate of explants planted in MS-nutrient medium without hormones was 7 percent. Noteworthy, the highest rate was observed in the variant, where 2ml.BAP+0.5NAA+30g sucrose+7g, accounted for 87% of plant rooting rate. However, in the rest of variant, the rooting rate of explants were around 23-68%. It was observed that the rooting rate was slightly higher in the control variant of WPM-Woody plant mediumnutrient medium than the control variant of the MS nutrient medium, which was 17%. Noteworthy, the highest result of the rooting rate was found in the variant of WPM-Woody plant medium-nutrient, where 1.5ml.BAP+2ml 2.4D+30g sucrose+7g was applied, and it was 61%. In the rest of variants, rooting rate was ranged in between 41%-54% ( Figure 2).

Conclusion
It was found that a high result was achieved in the variants where different concentrations of cytokinins and auxins were applied to the nutrient medium of Berberis L. explants in Murasige and Skuga (MS). In the experiment, the lowest indicator was observed in the control variant without hormones.
It was reported that rooting rate of explants planted in MS-nutrient medium without hormones was 7 percent. Noteworthy, the highest rate was observed in the variant, where 2ml.BAP+0.5NAA+30g sucrose+7g., accounted for 87% of plant rooting rate.
It was observed that the rooting rate was slightly higher in the control variant of WPM-Woody plant medium-nutrient medium than the control variant of the MS nutrient medium, which was 17%.