Potato (Solanum tuberosum L.) is one of the most important vegetatively propagated crops worldwide, ranking fourth after wheat, rice and maize in economic and nutritional importance and is classified as the most significant dicotyledonous tuber crop (World Book, Potato, 2000). It belongs to the family Solanaceae and originates from South America. Due to its wide economic and nutritional significance, potato has been a major focus of numerous studies in plant biotechnology. The success of these biotechnological applications largely depends on the development of efficient tissue culture systems that enable plant regeneration from cultured cells and tissues. Scientific studies have shown that the response of cultured plant explants is often genotype-specific, which necessitates the establishment of precise regeneration protocols tailored for each cultivar (Campos et al., 2016; Tara, et al., 2017; Yadav and Sticklen, 1995). Traditionally, potato is propagated by planting sprouts or “eyes” present on seed tubers. However, in vitro micropropagation provides an efficient means of producing large numbers of clonal, disease-free plantlets within a short period under controlled aseptic conditions (Copeland, 1982; Espinoza et al., 1986). In vitro propagation using vegetative buds has proven to be more reliable for maintaining the genetic integrity of clonal germplasm (Wang and Hu, 1982). Sharma et al., (2023) reported that the highest shoot induction percentage (100%) and an average of 3.75 shoots per explant were achieved from culturing potato (Solanum tuberosum L.) cv. Kufri Sindhuri shoot buds on MS medium supplemented with 1.5 mg L^-1 BA and 0.1 mg L^-1 NAA. Chandana et al., (2024) successfully cultured shoots of potato (Solanum tuberosum L.) cv. Kufri Surya on MS medium provided with 3.0 mg L^-1 BAP and 5.0 mg L^-1 Kin, achieving the highest buds induction rate (93.0%), with a mean of 3.47 shoots explant^-1, maximum shoot length (7.17 cm) and the highest leaves number (10.72 leaves explant^-1) after 30 days of culture (initiation stage). The highest multiplication rate was obtained with a mean of 9.30 shoots explant^-1 and maximum shoot length (7.20 cm) after 21 days of culture (multiplication stage). Similarly, Silva et al., (2024) investigated the effect of different concentrations of BAP and Zeatin on the micropropagation of potato (Solanum tuberosum L.) cv. Cecilia. They reported that nodal explants cultured on MS medium complemented with 0.3 or 0.4 mg L^-1 BAP achieved the highest shoots number (2.5 shoots explant^-1) with shoot lengths of 3.2 and 3.1 cm and leaves numbers of 5.7 and 8.2 leaves explant^-1, respectively, after 21 days of culture. Meanwhile, culturing on MS medium with 0.05 or 0.1 mg L^-1 Zeatin resulted in 2.2 and 2.4 shoots explant^-1, with shoot lengths of 4.9 and 5.3 cm and leaves number of 7.9 and 7.3 leaves explant^-1, respectively, after 21 days. Eid et al., (2025) obtained the highest shoots number (8.0 shoots explant^-1) and shoot length (6 cm) by culturing potato shoots on MS medium supplemented with 1.0 mg L^-1 BAP and 0.5 mg L^-1 Kin for 25 days. Bandyopadhyay et al., (2025) found that the highest rate of shoot formation was obtained when potato ( Solanum tuberosum L.) cv. Pilak shoot buds were cultured on MS medium supplemented with 1.5 mg L^-1 zeatin. For rooting, Othman et al., (2016) reported that culturing potato shoots derived from tissue culture on MS medium enriched with 1.0 mg L^-1 IBA combined with 0.5 mg L^-1 NAA resulted in the highest mean roots number (16.80 roots explant^-1) and root length (9.46 cm) in cv. Lady Balfour, while cv. Bellini achieved the highest mean root number (18.20 roots explant^-1) and root length (9.64 cm) after four weeks of culture on the same medium. Hajare et al., (2021) recorded the highest average root count 21.12 roots per shoot and root length 7.87 cm for the Solanum tuberosum L. Gudiene cultivar when explants were cultured on MS medium augmented with 1.0 mg L^-1 IBA and 0.5 mg L^-1 IAA. Molla et al., (2022) achieved 100% rooting by culturing potato buds on MS medium supplemented with 1.0 mg L^-1 IBA, recording the highest mean roots number (22.35 roots explant^-1) and root initiation within 5-6 days after 28 days of culture. Xhulaj and Gixhari (2018) demonstrated differential acclimatization responses in potato plantlets derived from cv. Excuisita and Bergerac, where only the plantlets of cv. Excuisita survived after acclimatization in a pre-prepared soil-sand mixture in the greenhouse and successfully produced minitubers with an average weight of 10-12 g.
       
The present study aims to produce minitubers of potato (Solanum tuberosum L.) cv. Arizona Class A from the multiplication of vegetative buds derived from dormancy-broken mother tubers.

This study was carried out in the Laboratory of Cells and Plant Tissue Culture at Department of Horticulture and Landscape Design, Collage of Agriculture and Forestry, Mosul University, during the period from February 2024 to January 2025. This experiment was conducted using vegetative buds excised from seed tubers of potato (Solanum tuberosum L.) cv. Arizona Class A. The tubers were stored in darkness at 23-24oC for two months to break dormancy and promote sprout initiation and elongation. Sprouts of 2-3 cm in length were carefully excised from the tubers using a sharp, sterile scalpel. The explants were first rinsed under running tap water for 10-15 minutes to remove soil particles and surface microbes. They were then washed in a commercial detergent solution (“Zahi,” locally available) for 5 minutes with continuous agitation, followed by thorough rinsing under running tap water. For surface sterilization, the explants were transferred to a laminar airflow cabinet and treated with 7% (v/v) commercial sodium hypochlorite (NaOCl) solution for 20 minutes with constant agitation. The sterilization solution was prepared from a household bleach containing 6% NaOCl and the sterilization step was enhanced using a vacuum pump to improve penetration. After sterilization, the explants were rinsed three times with sterile distilled water, each rinse lasting 5 minutes, as described by Kassab Bashi  et al. (2023). The sterilized sprouts were trimmed to 2 cm in length and cultured on Murashige and Skoog (MS) basal medium with full salt strength, 3% sucrose and 6 g L-1 agar, fortified with either BA at concentrations of 0.0, 0.25, 0.5, 1.0 and 2.0 mg L^-1 or Zeatin at concentrations of 0.0, 0.25, 0.5, 0.75 and 1.0 mg L^-1 for shoot initiation and multiplication. For rooting, buds were cultured on MS medium augmented with IBA at concentrations of 0.0, 0.25, 0.5, 0.75 and 1.0 mg L^-1, The pH was adjusted to 5.7±1 Then, they were distributed into bottles, with each bottle containing 40 ml and having a capacity of 210 ml, The bottle openings were covered with aluminum foil. Following a daily photoperiod of 16 hours of light followed by 8 hours of darkness, the cultures were transferred to the incubation chamber, which was maintained at a temperature of 24±2^oC and a light intensity of 3000 lux. The plantlets produced from the multiplication experiments were acclimatized under greenhouse conditions at the College of Agricultural Engineering Sciences, Duhok. The plantlets were transplanted into plastic seedling trays (55 x 30 cm) with 18 cells, each cell 9 cm deep, filled with peat moss as the growth substrate. The trays were initially covered with a transparent plastic lid to maintain high relative humidity during the early stages of acclimatization (Fig 1A). On the day following transplantation, the plantlets were irrigated by foliar spraying with quarter-strength MS nutrient solution, applied every two days for a total of five sprays. Approximately one week after transplanting, the plastic covers were removed and the plants were subsequently treated with foliar sprays of the commercial nutrient solution Trazex at 1 g L^-1, applied twice weekly (Fig 1B). Minitubers were harvested after the foliage had senesced and the tubers were collected from the plants (Fig 1C-E). The experiment was laid out in a completely randomized design (CRD) and treatment means were compared using Duncan’s multiple range test at a 0.05 probability level. Each treatment was replicated ten times, with each replicate consisting of a single explant (Al-Rawi and Khalaf-Allah, 1980).

Table 1 shows that the buds cultured on MS medium supplemented with 1.0 mg L^-1 BA produced the highest mean number of shoots (4.5 shoots per explant) after four weeks of culture. In contrast, the treatment with 2.0 mg L^-1 BA showed a clear superiority during the multiplication stage after an additional four weeks, reaching the highest mean shoots number of 17.2 shoots per explant, surpassing all other concentrations used (Fig 2 A,B). This same treatment (2.0 mg L^-1 BA) also gave the greatest shoots length (5.15 cm per shoot) and the highest mean leaves number (12.3 leaves per shoot). The table also shows that there were no significant differences in the number of stolons among the treatments; however, the highest mean stolons number (9.7 stolons per explant) was recorded at 1.0 mg L^-1 BA.


       
From the data presented in (Table 2), it is observed that buds cultured on MS medium equipped with 0.5 mg L^-1 Zeatin produced the highest mean number of shoots (2.0 shoots per explant) after four weeks of culture. In contrast, the treatments with 0.5, 0.75 and 1.0 mg L^-1 Zeatin were significantly superior to the other treatments after an additional four weeks of subculture, recording mean shoots number of 4.5, 5.3 and 5.0 shoots per explant, respectively. The table also shows that the treatment with 1.0 mg L^-1 Zeatin produced the highest mean shoot length (3.45 cm) and the highest mean number of stolons (5.8 stolons per plant) as shown in (Fig 2C, D), whereas no significant differences were observed in the mean number of leaves among all treatments.


       
The results presented in (Table 1 and 2) indicate that the use of both BA and Zeatin, which are plant growth regulators belonging to the cytokinin family, is responsible for increasing the number of lateral shoots up to the optimal concentration, thereby suppressing apical dominance. The gradual increase in Zeatin concentration led to a reduction in apical dominance, resulting in an increase in shoot number and a significant superiority of certain treatments (Hopkins and Hüner, 2004; Wasfi, 1995). The physiological effect of cytokinins is explained by their role in promoting cell division and elongation, which in turn enhances growth characteristics, as well as their influence on protein and nucleic acid synthesis. This is reflected in the noticeable increases in shoot length, shoot number and leaf number achieved by the treatments (Hartmann et al., 2002). The current study results exceeded those reported by Asma et al., (2001), with the highest number of shoots reaching 17.2 per plantlet at 2.0 mg/L BA, compared to 14 shoots per plantlet in the previous study.
       
Table 3 illustrates the effect of different concentrations of IBA on the rooting stage of potato (Solanum tuberosum L.) cv. Arizona Class A buds cultured on full-strength MS medium. The results showed that the treatment with 0.5 mg L^-1 IBA achieved the highest rooting percentage (90%), which was significantly superior to the other treatments except for the 0.75 mg L^-1 IBA treatment. Both 0.5 and 0.75 mg L^-1 IBA treatments were significantly superior in terms of root number and root length compared to the other treatments. Meanwhile, the treatment with 0.75 mg L^-1 IBA was significantly superior in terms of the mean shoots number (8.9 shoots per explant) this treatment in true produced the highest number of stolons reaching (30.7 stolons per explant) (Fig 2E, F). One possible explanation for these results is that IBA, as an auxin-type hormone, plays a crucial role in stimulating roots growth by enhancing cell division and elongation in root differentiation zones, thus promoting the formation of adventitious roots. The addition of growth regulators enhances overall root biomass and mean root length up to the optimal concentration; however, further increases in growth regulator concentrations have an inhibitory effect (Muhammad and Al-Rayes, 1982).



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The acclimatization and minituber production stages
 
It is observed from (Table 4 and 5) that all BA and Zeatin treatments resulted in 100% minitubers formation, indicating the success of the acclimatization process and the induction of minitubers. The treatment with 0.5 mg L^-1 BA recorded the highest mean shoot number (13.0 shoots per plant), the longest mean shoot length (12.7 cm) and the highest mean leaf number (74.1 leaves per shoot). This treatment also produced the highest mean number of minitubers (12.2 minitubers per plant), which was significantly superior to the other treatments. This superiority is attributed to the effective role of the growth regulator BA in stimulating cell division and enhancing the biosynthesis of tubers (Aksenova et al., 2012). The highest mean tuber weight and volume were recorded in the control treatment, reaching 1.375 g and 1.945 mL, respectively however, it did not differ significantly from most of the other treatments. In contrast, the different concentrations of Zeatin showed no significant differences in mean shoot number, shoot length, or leaf number of the longest shoot. However, the treatment with 0.75 mg L^-1 Zeatin produced the highest mean number of minitubers (12.1 minitubers per plant), while the highest mean tuber weight was recorded in the control (0.812 g) and the 1.0 mg L^-1  Zeatin treatment (0.780 g). The highest mean tuber volume (1.795 mL) was observed in the control treatment. This response may be attributed to the fact that Zeatin, as a naturally occurring cytokinin, plays an important role in promoting cell division, thereby contributing to tuber formation.

Tissue culture technology demonstrated high efficiency in the propagation of the potato cultivar Arizona Class A. The use of 2.0 mg L^-1 BA was superior in increasing the number of shoots, while 0.5 mg L^-1 IBA achieved a rooting percentage of 90%. Moreover, 0.5 mg L^-1 BA and 0.75 mg L^-1 Zeatin resulted in the highest mean number of minitubers, 12.2 and 12.1 minitubers per plant, respectively. This approach represents a reliable method for producing pure, high-quality potato seedlings.

The present study was supported by the University of Mosul, College of Agriculture and Forestry, Department of Horticulture and Landscape Design.
 
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