Influence of different factors on in vitro multiplication and rooting of three local Juglans regia L. genotypes in Uzbekistan

The Persian walnut ( Juglans regia L.) is one of the most lucrative and widely distributed nut crops. It is appreciated as a forestry and ornamental tree in addition to its benefits as a fruit crop. Although Central Asian countries, especially Uzbekistan, are among the origins of the Persian walnut; they are not considered as top industrial producers of walnuts. Uzbekistan posse - sses a wide range of walnut genetic resources and as a result of conducted research, several promising, fruitful, early - harvesting varieties and forms have been selected. The aim of this study is to optimize microclonal in vitro propagation of selected Uzbekistan local varieties and forms by evaluating concentrations of different plant growth regulators and genotype on multiplication and rooting stages. As mother plants, 2 forms and one variety were selected: the Ideal variety, 'Form PDM23' and 'Form 202YaKT'. О n the proli - feration stage, the growth rate of walnut microshoots on basal medium Driver and Kuniyuki Walnut Medium (DKW) with different concentrations of 6 - benzylaminopurine (BAP) and Indole - 3 - butyric acid (IBA) (0.01 mg/L) was studied. The rooting stage was assessed in half strength macronutrient DKW medium containing different IBA concentrations (0.0, 2.0, 4.0 and 6.0 mg/L). The Ideal variety and 'Form PDM23' performed best in DKW medium supplied with 1.5 mg/L BAP and 0.01 mg/L IBA, whereas 'Form 202YaKT' performed best in DKW medium supplemented with 1.0 mg/L BAP and 0.01 mg/ L IBA for the proliferation stage. For all genotypes, 6.0 mg/L IBA provided the best rooting results.


Introduction
Persian walnut (Juglans regia L.) is considered as one of the most valuable plants, with all of its parts having economic value and growing primarily in temperate climate.It is widely utilized as a forestry and decorative tree, in addition to its benefits as a fruit crop.Central Asia, particularly Uzbekistan, is one of the centers of origin of walnut (1,2).Vast walnut forests have long existed on the slopes of the Karjontog, Ugom, Piskom, Chatkal, Nurota and Hisar mountain ranges and dense walnut groves could be found in the Bostanlik district mountain ranges (3).Despite this, essentially no scientific research has been conducted in the fields of walnut cultivation technique, disease and pest management and seed breeding in Uzbekistan.Some scientific research has been carried out in walnut forests, but only in the form of expeditions and monitoring.Practical and innovative research on cultured walnut growing have not been conducted, except from some small walnut groves, walnut orchards have not been established on large areas (4).However, in recent years, orchards have been established more than 15000 ha in Jizzakh, Samarkand and other regions (5).In these orchards, saplings of walnut varieties, which are world-famous for their productivity, rapid growth and valuable fruit that meet world standards, are being planted and their introduction features are being studied.In particular, in Jomboy, Bulungur, Payarik districts of Samarkand region, orchards were established based on California Chandler variety and Paradox cl.Vlach rootstocks.However, in these saplings, cases of late autumn and early spring frost damage and crown gall infection were high.However, late autumn and early spring frost damage and crown gall infection were severe in these saplings.This suggests that establishing walnut orchards on the basis promising local varieties and forms would be more effective (5).
Central Asian countries including Uzbekistan has an advantage in developing new varieties of walnut, since it has an unlimited set of genetic resources for breeding.They differ not only in good quality fruits and annual sustainable abundant fruiting, but also in resistance to pests and diseases, early spring frosts, extremely low winter temperatures, soil droughts and a variety of other benefits.Hundreds of forms have been identified in forests and household plots over the years that are appropriate for developing excellent varieties that are superior in quality to those cultivated in the rest of the world (6,7).Some of them were already registered at the Research Institute of Horticulture, Viticulture and Winemaking (RIHVW, Tashkent, Uzbekistan), stored in the uterine garden, but not used for growing varietal seedlings (7,8).Оver the past 25 years, more than 100 forms of walnut have selected with high economically valuable qualities at the Research Institute of Forestry of Uzbekistan (RIFU).These forms need further research in order to use them on industrial scale (9).
In the walnut nursery industry, many various techniques of propagation are used to develop completed propagated trees for orchard planting, including sexual propagation, micropropagation, cuttings, budding and grafting.But, only tissue culture could provide for the production of large quantities of certified materials in a short period of time while keeping the genetic uniqueness of propagated clones (10).Despite the fact that several authors have reported micropropagation procedures for distinct genotypes (11,12), walnuts are considered recalcitrant to tissue culture, making in vitro propagation of newly discovered genotypes challenging (13,14).Furthermore, the low reproducibility of micropropagation protocols is another essential aspect that makes the reproduction of some varieties a more complex process, pushing them to adapt genotype-to-genotype and laboratory-to-laboratory.In addition, since the results of walnut micropropagation are strongly reliant on genotype, using the same procedure under various conditions, even if carried out by the same staff, may result in significant variances (15,16).For this reason, the aim of this study was to examine the effect of different plant hormone concentrations and genotype on the process of microclonal propagation by selecting different promising local varieties and forms for the establishment of industrial orchards.

General in vitro culture conditions
The formulations of DKW (11) and MS (17)

In vitro introduction
In vitro introductions were performed during 2022.Taking into account that the introduction stage is one of the critical stages, in order to increase the efficiency, the trees selected as mother plants were first treated with CuSО 4 *5 H2О in February.Then, in April, it was treated with fungicide and in May, the introduction material was taken.Materials were individually labelled and transferred to the laboratory within 12 h after being collected.Cuttings were washed profusely in tap water for 1 h, then washed in household soap and washed again to remove the soap residues.Then, they were washed in the carbendazim fungicide solution (code 378674, Sigma-Aldrich), for 10 min, in the solution of streptomycin as a bactericide (1 g/L) for 12 min, in ethanol (C2H5ОH) for 2 min and in AgNО 3 solution (0.2%) for 1 min.After each process, the samples were rinsed 3 times in distilled water.All procedures were performed in a sterile laminar hood.Explants were individually inoculated in glass test tubes (150×20 mm) containing 10 mL MS culture medium supplemented with 2 mg/L isopentenyl adenine (2iP) and maintained under the standard photoperiod conditions.Contaminated or dead explants as well as those with profuse phenolic releasing were counted and discarded (14).

Multiplication
Cultures were inoculated in polypropylene containers (LA-Rectangular, 90/180 (A), Lab Associates, Bosschendijk, Netherlands).For in vitro multiplication, DKW formulation supplemented with BAP (0.0, 1.0, 1.5, 2.0 mg/L) and IBA (0.01 mg/L) concentrations was utilized.Ten explants were inoculated in 100 mL of culture medium each vessel.During the 4 th week, subcultures were demonstrated.At the end of subculture, the number and the length of the microshoots, the number of leaves was evaluated for each treatment (18).

Rooting
Cultures were incubated during one week in the darkness in a culture medium with macronutrients of DKW reduced to 50% and different concentration (0.0, 2.0, 4.0, 6.0 mg/L) of IBA.Following that, microshoots were transferred to the expression sub-stage in the same culture medium with vermiculite, without IB nor agar for a 2 week.After 3 weeks, the number of rooted microshoots and the length of the roots were counted, and the general state of the rooted microshoots was recorded (16).

Experimental design and statistical data processing
The statistical significance of data was verified using an analysis of variance of the Microsoft Excel 2013 package.
The least significant difference (LSD) test (P=0.05)was used to conduct mean comparisons.For all experiments, a randomized model was used.The container was the basic experimental unit; thus, the average of the containers was utilized as individual data for analysis.For all trials, each treatment was composed of at least 3 experimental units.
During the proliferation stage, at least 3 subcultures were assessed, with each subculture considered a repetition.

Results and Discussion
The establishment is a crucial stage in the process of microclonal propagation of walnut and microbial contamination, phenol releasing, darkening of explants arе the main problems (10,19).Various contaminations have a great impact on the successful implementation of this process (18,19), especially when the somatic organs of trees grown in the field are used as initiation material.Additionally, each plant contains microorganisms as endo-and ectosymbionts, each genotype has its own microbiome and it is noted that this stage also depends on the genotype (20,21).Similar results were obtained at this stage when DKW or MS medium were utilized (10,19).However, in most cases better result is observed when MS is used (13,14).Therefore, MS medium supplemented with 2 mg/L 2iP was used for initiation in this study.Then, losses due to contamination and the number of successfully established microcuttings were determined depending on the genotype (Fig. 2).In our study, we have found that the highest loss in the number of initiation material is due to bacterial (33%) and fungal contamination (23%) was observed in Form 202YaKT, while it was 2-fold (16%) and 3-fold (8%) less in Ideal variety respectively.However, as a result of phenolization, 36% of Ideal variety and 31% of 'Form PDM23' microcuttings died.The rate of successfully established microcuttings was slightly higher in Ideal variety (40%) than, 'Form PDM23' (36%), but in 'Form 202YaKT' it was almost 2 times lower (22%).Results of several studies was consistent with our findings (22)(23)(24).
After 4 weeks of subculture, shoot number and callus size were investigated and they were different depending on the genotype.The shoot number of explants of Ideal variety (3.1) and 'Form PDM23' (2.9) showed similar results, but it was lower in 'Form 202YaKT' (2.2).Callus size showed the smallest value in 'Form 202YaKT' (1.4) in contrast to Ideal variety (2.01) and 'Form PDM23' (2.09) (Table 1).A similar study compared the performance of Chandler, Hartley and Z60 genotypes and their results were compatible with our study (25).Furthermore, shoot length, shoot number and callus size were studied to determine the relationship between plant hormone treatment (BAP) and genotypes.Results showed that, these parameters increased in all genotypes with increasing hormone concentration (Fig. 3).When the concentration of BAP was 2.0 mg/L, the average shoot length was 4.5 cm and 4.3 cm in the Ideal variety and 'Form PDM23' and the shoot number was 2.8 and 3.5 respectively.However, at this concentration, callus size in both genotypes showed a high index, 4.3 cm and 4.5 cm respectively.Also, yellowish leaves, vitrification and weakening of the main stem were observed (Fig. 4).At 1.5 mg/l concentration of BAP, callus size was significantly smaller in both genotypes, namely, 3.2 cm and 2.5 cm respectively.At 1.5 mg/L and 2.0 mg/L concentrations of BAP, there was no significant difference in shoot length and number on explants of Ideal variety and 'Form PDM23'.In addition, callus size was smaller and the overall condition of seedlings was good at 1.5 mg/L.For these 2 genotypes, 1.5 mg/ L concentration of BAP was found to be optimal in the proliferation stage.In Form 202YaKT, BAP at 1.0, 1.5 and 2.0 mg/L concentrations did not significantly differ in shoot length (respectively, 2.8 cm, 3.2 cm, 3.2 cm) and number (respectively, 1.8, 2.1, 2.2), however, with increasing concentration, callus size also increased (2.5 cm, 3.5 cm, 3.8 cm).Therefore, BAP concentration of 1.0 mg/L was found to be optimal for Form 202YaKT.
Cell division and growth can be controlled in the proliferation step by the application of several cytokinins.Among different cytokinins, benzylaminopurine (BAP), zeatin, kinetin, isopentenyl adenine (2iP) and thidiazuron (TDZ) are the most extensively utilized in walnut micropropagation (26)(27)(28).Significant changes in the studied factors were noted in this study when explants were treated with BAP.Shoot length, number and leaf number increased with increasing hormone concentration, which is similar to the results reported previously (29)(30)(31).This supports our findings and indicates importance of cytokinins in walnut proliferation.
The success of rooting stage is determined by the induction method, the concentration of 3-indole butyric acid (IBA), the light and the quality of the explant (32,33).For the rooting stage, high-quality, well-grown, 2-3 cm explants were chosen.After 3 weeks, the %, number and length of roots were determined (Tablе 2).There was a significant difference between the studied indicators in the control and hormonally treated variants, that is, they increased with the increase in the concentration of IBA.When the concentration of IBA was 6.0 mg/L, the % of rooting reached the highest level in all genotypes (58.3%, 63.2% and 45.7% respectively).At this concentration, the number of roots also increased 2-fold compared to the control in all 3 genotypes (4.2, 4.1, 3.5 respectively).The root length was the highest in 'Form PDM23' (4.4 cm), while Ideal variety and 'Form 202YaKT' showed a similar result (3.4 cm 3.1 cm).The results showed that the average rooting % was the highest in 'Form PDM23' (41.2%), followed by Ideal (37.8%) and 'Form 202YaKT' (32.4%).In addition, when the average number of roots for each genotype was calculated, the Ideal variety showed a higher result (3.6), in contrast to 'Form PDM23' (3.3) and 'Form 202YaKT' (3.1) (Fig. 5).
According to the results of this study, it can be concluded that IBA (6 mg/L) is the best hormonal treatment for the rooting stage of the walnut genotypes mentioned above.In a similar experiment, when the concentration of IBA exceeded this value, a decrease in the % and number of rooting was observed (25).In addition, when IBA was used, the rooting % was higher than when NAA was used (32,34).The influence of the genotype at the rooting stage has been studied in various experiments and corresponds to the results of this study (23,32).

Conclusion
Persian walnut (Juglans regia L.) has economical and nutritional importance through worldwide, as well as Uzbekistan.Therefore, studying properties of different genotypes and creating protocols of their micropropagation would give a chance to utilize them in an industrial scale.The findings of our study revealed that Ideal variety and 'Form PDM23' performed better than 'Form 202YaKT' in the examined factors during the proliferation and rooting stages.BAP 1.5 mg/L was found to be optimal for Ideal variety and 'Form PDM23' for the proliferation stage, while 'Form 202YaKT' performed best in BAP 1.0 mg/L concentration.At the rooting stage, 6.0 mg/L IBA showed the best results for all genotypes.These local Juglans regia L. genotypes, especially the Ideal variety and 'Form PDM23', can be microclonally propagated on an industrial scale and orchards can be created based on them.

Fig. 2 .
Fig. 2. Results of in vitro introduction on different genotypes.
were used as culture media.DKW was prepared from stock solutions and for MS a dry powder (code 5519, Sigma Aldrich; St. Louis, MО, USA) was used.The pH was adjusted to 5.7 with NaОH (0.1 N); afterward the culture media were sterilized by autoclaving during 20 min at 121 °C.All processes performed in Bogbon, SAG-Agro In vitro laboratory (SAG Agro Lab, Samarkand, Uzbekistan).

Table 1 .
Assessment of studied factors on different genotypes Values in each column represent means ± SE. n = 20; shoots of explant means with different letters are significantly different at P < 0.05.

Table 2 .
Assessment of studied factors under different indole butyric acid (IBA) levels on rooting Values in each column represent means ± SE. n = 30; shoots of explant means with different letters are significantly different at P < 0.05.