MATERIALS AND METHODS

Pomegranate varieties: The research was conducted in the first and second quarters of 2022 at the Transgenomics and Tissue Culture Laboratory of the Center of Genomics and Bioinformatics of Uzbekistan Academy of Sciences. Experimental varieties-August, Kazake-anar, Achikh-dona and Tuyatish were selected from the collection available at the Surkhandarya Research Station of the Scientific Research Institute of Horticulture, Viticulture and Winemaking named after M. Mirzayev.

Plant materials and surface sterilization: Explants were selected from August, Kazake-anar, Achikh-dona and Tuyatish varieties of 2 years old pomegranate grown in greenhouse conditions (Fig. 1a-b). Terminal and lateral bud explants of the various samples, selected for the study, were collected in special containers (Phyto Technology Lab. USA). The explants were cleaned of excess parts in a laboratory condition and kept in a fungicide solution (fundazol 0.02%) for 1 min and under running water for 10 min.

Surface sterilization was performed in a laminar box (HF safe LC. China) under aseptic conditions. The effect level of several chemical compounds for surface sterilization of explants at different time intervals was studied (Table 1). In all variants of the experiment (Control, A₁ and A₂), 50 explants were selected for surface sterilization. Because burning was observed in some explants as a result of the effect of sterilization agents, observations and statistical analyses were made on 40 explants.

Nutrient medium and its content: Two different nutrient media MS and WPM were used in the study (Table 1 and 2). The composition of the medium was prepared according to literature data^20,21. The pH value of the prepared nutrient medium was adjusted to 5.8 using 0.1 N NaOH solution. Nutrient media were divided into 1000 mL heat-resistant conical flasks and sterilized in an autoclave at 120°C under a pressure of 0.75-1.0 atm for 20 min. The sterile medium was equally divided by 50 mL into 500 mL bottles.

Explant regeneration (bud proliferation): The stage of regeneration (proliferation) of explants was carried out on the basis of various concentrations and combinations of three different phytohormones in two various nutrient media (1.0 mg L^–1 BAР+0.1 mg L^–1 NAA and 1.0 mg L^–1 BAР+0.1 mg L^–1 NAA+0.5 mg L^–1 GA₃) (Table 2). The 0.5 cm part of the explants was inserted into the nutrient medium and placed in an upright position. Explants were kept in the dark for the 1st week of cultivation, then grown under a white lamp light at the condition of room temperature 25±1°C and humidity 85±5%, photoperiod 16/8. From the 28th to the 30th day of the observations, the microshoots formed in the explants, depending on the size (higher than 2.0 cm), were transplanted to a new nutrient medium and grown under the above photoperiod conditions. The explants by 30 pieces were selected for each nutrient medium to study shoot organogenesis in explants.

Root formation and rooting development in microshoots (rhizogenesis): Microshoots were grown in a culture medium of 14 000 mg L^–1 sucrose, 100 mg L^–1 myo-inositol and 800 mg L^–1 agar-agars. The supramolecular complex in 0.15 and 0.17 mg L^–1 doses was used as an inducer²² for rhizogenesis (Table 3). The explants were kept in the nutrient medium containing the supramolecular complex preparation until they were transplanted from the rhizogenesis stage to the non-sterile (substrate) conditions, that is, re-subculture was not performed.

Fig. 1(a-b):

Initial material, (a) Two-year-old seedlings of experimental varieties and (b) Selected explant

In the 5th week of cultivation, the rooting percentage, root number and length of each microshoot were quantitatively evaluated. In all cases, 30 microshoots were selected for each nutrient medium containing supramolecular complex preparation.

Transplantation and adaptation of microplates to sterile (soil) conditions: Microplants with morphologically proper root systems were transplanted to biohumus+coconut husk+sand (2: 1: 1) substrate. The substrate sterilized in an autoclave was placed in 8×7 cm plastic containers and microplants were planted there. To maintain high humidity, plastic containers were closed with special light-transmitting lids and kept for 20 days in the conditions of an artificial climate station (phytotron) with relative humidity (70-75%), temperature (25±2°C), under 16/8 light and dark photoperiodic cycle. Taking into account a high level of transpiration due to the relative infirmity of microplants, the slow development of leaf tissue cells and the wide axil of leaves, special lids were opened, artificially moistened and closed at certain time intervals. After 20 days, the acclimatized microplants were planted on a substrate of field soil+sand (1: 1) distributed in 18×18 cm disposable polyethylene containers and transferred to greenhouse conditions. The survival rate of microplants was analyzed on the 30th day after transplanting into the greenhouse.

Data analysis and measurements: The experiment for the stage of the proliferation of explants was carried out using four cultivars, two different nutrient media (MS and WPM) and different concentrations of three types of phytohormones and was statistically evaluated. Significant differences were illustrated by the time of shoot formation, number and length of shoots for explants of each variety as a result of the effect of phytohormone concentrations. The stage of rhizogenesis in microsystems was carried out without phytohormones using the supramolecular complex. Data were analyzed using the ANOVA program.

RESULTS AND DISCUSSION

Surface sterilization stage: In this experiment, the bacterial and fungal infestation of explants was studied and various concentrations of sterilization agents were used. The A₁ variant (5 min H₂O₂ (3%)+15 sec C₂H₅OH (96%)+15 min NaClO (2%)+Twin-20+H₂O) with explants free from fungal infestation did not reveal significant differences compared to the control (5 min H₂O₂ (3%)+10 sec C₂H₅OH (96%)+10 min NaClO (2%)+Twin-20+H₂O). Similar results were observed in the explants of the A₁ variant which were free from bacterial damage.

In experimental option A₂ (5 min H₂O₂ (3%)+20 sec C₂H₅OH (96%)+20 min NaClO (2%)+Twin-20+H₂O) a significant difference was observed relative to the control option, i.e., the percentage of explants free of fungal and bacterial infestation in all experimental varieties showed a high index. In the August and Tuyatish varieties a bacterial infestation, while, in the Achikh-dona variety a fungal infestation was not observed. There was not a significant difference between the A₁ option and the control option (Table 4).

The HgCl₂ and NaClO are the most widely used surface sterilant in the microclonal in vitro reproduction of pomegranate plants. In this study, surface sterilization of explants was effectively performed and achieved a 65% survival rate of explants in sterilization of pomegranate leaf axil buds using a combination of NaClO and Na merthiolate for 20 min. The results obtained in that work²³ were higher than others. Mulaei et al.²⁴ achieved the highest rate (90.58%) of survival of axillary buds using 0.1% HgCl₂ solution for 10 min. Similar results were obtained in our work. In our work, the most optimal choice for surface sterilization of pomegranate explants was the A₂ variant.

Nutrient medium: There were no significant differences between experimental varieties and nutrient media (MS and WPM).

However, the plants grown in the WPM medium differed morphologically, possibly due to the number and length of buds resulting from bud formation time. Further, both the MS and WPM nutrient media were used for explant rhizogenesis (Table 3). For micropropagation of pomegranate mostly full-strength and half-strength MS, WPM and B5 nutrient media were used¹⁵. In this work, we aimed at selecting the optimal culture media for local pomegranate genotypes.

Effect of growth regulators on regeneration of explants (proliferation of buds): The explants that survived during the surface sterilization stage were transplanted into a cytokinin medium from the 2nd week of cultivation. In explants with terminal and lateral buds, the shoot regeneration was formed after 14 days. The number and length of shoots were checked in the 6th week of cultivation. Explants grown in MS₂ (1.0 mg L^–1 BAP+0.1 mg L^–1 NAA+0.5 mg L^–1 GA₃) medium were not morphologically significantly different from explants grown in MS₁ (BAP 1.0 mg L^–1+NAA 0.1 mg L^–1) medium. The difference was on shoot formation day which was observed only in explants of the August variety (Fig. 2 and 3(a-h)).

Verma et al.²⁵ studied the effect of subculture on microbuds proliferation induced by callus induction of leaf explants of pomegranate variety Kandhari Kabuli in solidified MS medium supplemented with 9.0 μM BAP, 2.5 μM kinetin and 1.5 μM GA₃. They found enhanced number (I-1.20, II-3.24, III-4.07 and IV-3.92) and length (I-1.16, II-2.12, III-2.91 and IV-2.70) of microshoots. Besides, the authors showed shoot organogenesis by the addition of 10 μM BAP and 2.5 μM NAA to MS nutrient medium that produced adventitious shoots (42.95%) from explants. Parmar et al.²⁶ showed a higher rate (68.21%) of direct organogenesis from hypocotyl segments of pomegranate variety Kandhari Kabuli using different concentrations of plant growth regulators BAP (1.0-2.5 mg L^–1) and NAA (0.5-1.5 mg L^–1) in MS medium and observed the formation of 3.18 shoots per explant. The results obtained in this work were in correspondence with these above discussed data. Our results are also compatible with previous protocols in other varieties of the pomegranate, where the auxins such as BAP and NAA or IAA had a significant morphogenetic reaction in MS nutrient medium^27-29. Shoot elongation depends on the synergistic effect of cytokinin and GA₃³⁰. But in our study, no significant changes were observed in the effect of GA₃ on the length of the shoot. It may be because of not enough concentration of GA₃.

No significant differences were observed in experimental explants in WPM₂ (1.0 mg L^–1 BAР+0.1 mg L^–1 NAA+0.5 mg L^–1 GA₃) nutrient medium compared to the explants grown in WPM₁ (BAР 1.0 mg L^–1+NAA 0.1 mg L^–1) nutrient medium. The shoot formation in explants of August and Achikh-dona varieties, grown in the WPM₂ nutrient medium, was several days earlier compared to the WPM₁ nutrient medium. The length of explants formed as a result of the organogenesis of shoots in the WPM₂ nutrient medium was of a higher level in August and Kazake-anar varieties compared to explants in the WPM₁ nutrient medium. No significant differences were observed in Achik-dona and Tuyatish varieties (Fig. 2 and 4(a-h)).

The addition of GA₃ to MS₂ and WPM₂ nutrient media resulted in relatively wide nodes in explants. It did not cause significant differences in the explants. El-Agamy et al.³¹ investigated the optimum nutrient medium by propagating Manfalouty and Nab El-Gamal varieties of pomegranate on three different nutrient media: MS, Nitsch & Nitsch and WPM. In their case, the tallest microshoots were grown in (average 5.10-4.58 cm) WPM medium. The number and length of microshoots in this work correspond to their results.

ValizadehKaji et al.⁹ studied Iranian pomegranate varieties Malas Saveh and Yousef Khani in basal WPM and MS nutrient media using different plant growth regulators. Plants grown in WPM nutrient medium had higher shoot proliferation (51.42±32.81), shoot length (2.15±1.31) and the number of leaves (6.97±3.66) compared to MS nutrient media. In this work, micro shoots grown in WPM nutrient medium were slightly morphologically different compared to microshoots in MS medium. However, there was no significant difference between microshoots.

Effect of supramolecular complex on rhizogenesis of microshoots: The rhizogenesis of explants was studied in different concentrations of MS and WPM nutrient media containing the supramolecular complex. The addition of 0.15 mg L^–1 concentration of the complex to Rhizo-MS₁ nutrient medium resulted in an average root number of 1.36-2.14 and an average length of 1.50-2.19 cm in explants. This indicator was 2-3 times higher than the control. As a result of the addition of 0.17 mg L^–1 of the complex to the Rhizo-MS₂ nutrient medium, the number and length of roots in the explants of the experimental varieties were 3-5 times higher than in the control (Fig. 5(a-h)) and 2-2.5 times higher than the explants grown in the Rhizo-MS₁ nutrient medium. Thus, significant differences were determined among these variants. The results of the explants grown in the WPM nutrient medium were similar with those in the MS nutrient medium.

The addition of 0.17 mg L^–1 of the supramolecular complex to the WPM₂ nutrition media increased the root length 3-5 times compared to control (Fig. 6(a-h)).

Fig. 2(a-h):

Effects of phytohormones with different compositions and concentrations on shoot organogenesis of some pomegranate cultivars in MS and WPM nutrient media. MS1 and WPM1 media contained 1.0 mg L–1 BAP+0.1 mg L–1 NAA, MS2 and WPM2 media contained 1.0 mg L–1 BAP+0.1 mg L–1 NAA+0.5 mg L–1 GA3 *p<0.5 in all cases except for August (0.520) and Kazake-anar (1.00) varieties in MS and Achikh-dona (0.866) variety in WPM. Error bars mean standard deviation

In Rhizo-MS₂ and Rhizo-WPM₂ nutrient media containing 0.17 mg L^–1 of the complex, the rhizogenesis stage showed the highest rate in all varieties. Due to the lack of addition of growth regulators to the control variant, rhizogenesis in these explants was of low level: The average number of roots made 0.23-0.33 and average length was 0.11-0.18 cm.

Fig. 3(a-h):

Shoot growth and development in explants of experimental cultivars in MS1 and MS2 nutrient media, (a, e) August, (b, f) Kazake-anar, (c, g) Achikh-dona and (d, h) Tuyatish

Fig. 4(a-h):

Shoot growth and development of explants of experimental cultivars in WPM1 and WPM2 nutrient media, (a, e) August, (b, f) Kazake-anar, (c, g) Achikh-dona and (d, h) Tuyatish

Bachake et al.³² found out that root formation and development in microshoots grown in WPM nutrient medium required less days compared to microshoots grown in MS nutrient medium. The addition of 1.0 mg L^–1 NAA to WPM medium was found to increase the number of primary roots per microshoot (5.0) and the percentage of rooting (76%) compared to other concentrations.

Dessoky et al.³³ studied and achieved the root formation and development of Taify and Yemeni varieties of pomegranate, grown in Taif Province of Saudi Arabia, by adding 1.0 mg L^–1 NAA and 2.0 mg L^–1 IBA to MS nutrient medium in vitro.

Fig. 5(a-h):

Effect of supramolecular complex preparation (0.17 mg L–1) on rhizogenesis of explants of experimental varieties, (a, e) August, (b, f) Kazake-anar, (c, g) Achikh-dona and (d, h) Tuyatish

Fig. 6(a-h):

Effect of supramolecular complex preparation (0.17 mg L–1) on rhizogenesis of explants of experimental varieties, (a, e) August, (b, f) Kazake-anar, (c, g) Achikh-dona and (d, h) Tuyatish

The results in the varieties were 83.3 and 79.6%, respectively. These auxin compounds were the most effective in terms of shoot number and root length per explant.

Singh et al.³⁴ studied rooting and development of cotyledon and nodal explants of Ganesh pomegranate in half and full-strength MS medium and white’s plant nutrient medium with different concentrations of NAA (0.1, 0.2, 0.5 and 0.8 mg L^–1) and supplemented with 200 mg L^–1 activated carbon under in vitro condition. Rhizogenesis in microshoots resulting from the regeneration of explants showed the highest root number (4.17) and root length (3 and 87 cm). These results explain various approaches can result in optimum outcomes in different varieties.

Fig. 7(a-h):

Effect of different concentrations of the supramolecular complex drug on the rhizogenesis of some pomegranate varieties in MS and WPM nutrient media. The MS1 and WPM1 contained 0.15 mg L–1 of the supramolecular complex, MS2 and WPM2 contained 0.17 mg L–1 of the supramolecular complex *p<0.005 in all cases. Error bars mean standard deviation

Patil et al.³⁵ reported that MS medium containing 0.5 mg L^–1 NAA and 0.5 mg L^–1 IBA showed the highest rooting rate. The highest mean rooting response was recorded in MS medium containing 0.5 mg L^–1 NAA (97%) or 0.5 mg L^–1 IBA (97%). Root length was recorded from 0.3 to 3.4 cm in a nutrient medium containing IBA and 1.3-3.2 cm in a medium containing NAA. However, thick root formation was observed in a medium containing 0.5 mg L^–1 IBA. Current results were in agreement with these outcomes that the roots of all the explants of the experimental cultivars grown in the MS medium were thicker than those grown in the WPM medium (Fig. 7(a-h)).

No significant differences were observed in the rhizogenesis of the explants grown in nutrient media (Rhizo-MS₂, Rhizo-WPM₂). However, explants grown in the WPM₂ medium differed in root system thinness compared to explants grown in the Rhizo-MS₂ medium (Fig. 6). The cuticular layer was found well developed in thin roots³⁶.

Fig. 8(a-b):

Adaptation of experimental varieties to non-sterile conditions, (a) Phytotron and (b) Greenhouse

The development of the cuticular layer had a positive effect on the growth and development of microplants in non-sterile conditions. This can be linked with plant genotypes that require further research. The development of Kazake-anar and Tuyatish varieties was relatively slow. Based on the conducted studies and literature analysis, it was assumed that the varieties require different nutrient media or auxins. However, it was found that the growth and development of varieties are not low and this may be related to the genotype of the plant^18,37.

Our group has been carrying out a scientific project on in vitro propagation of local varieties of pomegranate (Punica granatum L.) and the development of healthy and disease-free varieties. In the course of the project, various nutrient media were tested for in vitro reproduction of local genotypes. Various studies on quantitative changes and additions to all components of nutrient media are being conducted and growth and development phases in explants are being studied. Several types of plant hormones (cytokinin and auxin) have been tested and positive results have been achieved. However, imported plant hormones cost expensive. Therefore, we developed a strategy of using plant hormone substitutes such as the supramolecular complex of glycyrrhizic and salicylic acid that lower the cost of developed seedlings several times. The cost efficiency of the used supramolecular complex was about 45 times greater than classical approach that utilizes phytohormones. This approach would enable to lower the expenses and thus lead the rooting of microshoots of pomegranate varieties into practice.

Transplantation of microplants to non-sterile soil condition and their adaptation: Microplants showed a survival rate of 95% in biohumus+coconut husk+sand (2: 1: 1) substrate. Successfully acclimatized plants were transplanted to a substrate of field soil+sand (1: 1) and grown in a greenhouse for 30 days (Fig. 8(a-b)). There was no death of plants in greenhouse conditions.

The microplants grown in the greenhouse conditions were morphologically different compared to the phytotron conditions, i.e., the leaves were large and wide, the intensity of greenness was high, the stems were branched, etc. Such a difference in the growth and development of microplants may be due to the change of climatic factors from artificial to natural, the expansion of the growing area and the implementation of additional agrotechnical measures such as tillage, fertilization and irrigation.

CONCLUSION

In this study, the effect of phytohormones on the regeneration of explants during the in vitro propagation of some local pomegranate genotypes was investigated and the optimal amount of glycyrrhizin and salicylic acid supramolecular complex on the rhizogenesis of microshoots was determined. The addition of the 0.17 mg L^–1 supramolecular complex to the nutritional medium (Rhizo-MS₂ and Rhizo-WPM₂) resulted in 3-5 times higher root number and length in explants compared to the control. About 45 times greater cost efficiency of the complex promotes using it in the practice of rooting while developing microshoots.

SIGNIFICANCE STATEMENT

We developed in vitro propagation of local varieties of pomegranate (Punica granatum L.) varieties of Uzbekistan. The selected surface sterilants in this study reduced the bacterial and fungal damage of explants of the experimental varieties by several times compared to the control. The optimal effect of the type and concentration of phytohormones on the regeneration of explants was determined. The effect of the supramolecular complex of glycyrrhizic and salicylic acids on the rhizogenesis of microshoots was established. The addition of a 0.17 mg L^–1 dose of the supramolecular complex to MS and WPM nutrient media showed several times greater levels of rhizogenesis of microshoots compared to the control.