Paulownia species especially Paulownia tomentosa and its hybrids, have received growing attention in the world because they grow rapidly, yield high biomass and can be used in various ecological and industrial applications, including timber production, carbon sequestration and agroforestry systems. Paulownia has these features, which make it a strategic species in the context of sustainable forestry and large-scale afforestation initiatives (Akyol and Gurel, 2020; Smerea et al., 2024). Both its significance and the lack of large-scale propagation of Paulownia using conventional techniques, e.g. seeds and stem cuttings, are due to low germination rates, seasonality and low uniformity of planting material. In this respect, plant tissue culture techniques provide an effective and reliable approach for rapid clonal propagation of elite genotypes on a large-scale under controlled conditions (George et al., 2008; Rout and Mohapatra, 2006; Hesami et al., 2020).
       
Previous studies have demonstrated the successful application of in vitro propagation techniques across different plant species. For instance, direct plant regeneration using shoot tip explants has been effectively achieved in sugarcane, highlighting the potential of tissue culture for rapid mass multiplication (Kaur and Kapoor, 2017). Similarly, efficient in vitro propagation protocols have been reported for aquatic and ornamental species, confirming the broad applicability of culture media optimization and explant selection in micropropagation systems (Nguyen et al., 2021; Kundu et al., 2024).
       
Past research has already established that the in vitro propagation of woody plants is highly dependent on the nature and concentration of plant growth regulators during different phases of development. BEN belongs to the family of cytokinins that facilitate shoot development and multiplication and IBA and IAA are auxins responsible to induce roots and root development (Bonga and von Aderkas, 2019; Ibrahim et al., 2022). Yet, when use of auxin is likely to cause basal callus development that can decrease root quality and the success of the procession of acclimatization.
       
Recent progress in micropropagation of Paulownia focused on optimizing culture media composition and hormonal balance to promote shoot emergence and root system structure (Smerea et al., 2024). However, there are still problems with root quality and ex vitro survival, especially under hormone-induced rooting conditions.
       
Along with the scientific significance, the study helps to solve urgent environmental issues in Iraq, where the priorities in the country are afforestation as a measure to fight desertification and growing dust storms. An efficient and scalable micropropagation protocol developed with Paulownia can thus be useful in the sustainable rehabilitation of the environment. The objective of the present paper was to evaluate the efficacy of the medium in regard to shoot development, proliferation, rooting and acclimatization by evaluating the responses of a variety of regulatory agents for plant growth and media for culture.

Implementation plan and experimental duration
 
The experimental work was implemented over a period of approximately one year in the Plant Tissue Culture Laboratories of the Agricultural Research Center under controlled laboratory and greenhouse conditions.
 
Explant preparation and plant material
 
The source of explants was healthy Timothy donor plants that were 2-3 years old. Surface pollutants were removed by excising actively growing shoot tips and thoroughly washing them under running tap water. Surface sterilization was conducted under aseptic conditions for 7-10 minutes using 0.1% (w/v) mercuric chloride (HgCl₂). Afterward, four rinses with sterile distilled water were administered. Murashige and Skoog, (1962) (MS) basal medium was employed to cultivate the explants. Prior to autoclaving at 121°C and 1.04 kg cm^-2 for 20 minutes, the pH of all media was changed to 5.7.
 
Establishment stage
 
A mixture of MS media with IAA (indole-3-acetic acid) was used for the culture of the explants and benzyladenine (BA) at concentrations of 0.0, 0.5 and 1.0 mg L^-1 in different combinations. The cultures were kept at 25±2°C and exposed to light for 16 hours a day. It was noted after 30 days how many shoots emerged, how tall the plant was, how many leaves it had, how many nodes it had and what kind of roots it had.
 
Shoot multiplication stage
 
Established shoots were transferred to different media formulations: hormone-free MS, MS + 0.5 mg L^-1 BA + 2 mg L^-1 BA, ¾ MS + 0.1 mg L^-1 IBA, MS +¾ MS + 2 mg L^-1 kinetin  and 2 mg L^-1 BA .
       
In a separate experiment, MS media, including 0.3 mg L^-1 NAA and 0.5 mg L-1 GA₃, was augmented with BA at concentrations of 3.0 ,2.0, 1.0,  and 0.0 mg L^-1. Growth metrics were documented after a duration of 30 days.
 
Rooting stage
 
Root induction was evaluated using IAA or IBA separately at concentrations of 3.0 ,2.0, 1.0 and 0.0 mg L^-1. number of roots, Rooting percentage and root length, were assessed after four weeks.
       
Due to callus-mediated rooting observed in auxin-treated media (Fig 1), an additional rooting experiment was conducted using sterile distilled water. Root development was recorded weekly for four weeks.


 
Acclimatization
 
Rooted plantlets were gently removed, washed free of agar and relocated to plastic containers filled with 1:1 mixture of peat moss and sandy soil. Plants were grown under greenhouse conditions and the survival percentage was recorded (Fig 2).


 
Statistical analysis and experimental design
 
All trials were conducted using a fully randomized design (CRD). Data were presented as standard error ± mean. Mean comparisons were conducted utilizing Duncan’s multiple range examination at P≤0.05.

Shoot establishment
 
MS medium augmented with 0.5 mg L^-1 IAA in conjunction with either 0.5 or 1.0 mg L^-1 BA resulted in 100% shoot emergence. Although hormone-free MS produced the tallest shoots, BA at 1.0 mg L^-1 significantly enhanced qualitative traits including leaf and node numbers (Table 1, Fig 3). This confirms the synergistic role of auxin-cytokinin balance in activating axillary buds of woody species, as reported by Hesami et al., (2020).




 
Shoot multiplication
 
Significant differences were observed among multiplication media (Table 2). MS + 0.5 mg L^-1 BA + 0.1 mg L^-1 IBA yielded the highest shoot number and vegetative growth, whereas rooting was completely inhibited, reflecting cytokinin dominance.Supplementation of MS medium with GA₃ and NAA shown that 2.0 mg L^-1 BA yielded the most sprout proliferation (6 shoots per explant; Table 3, Fig 4). Higher BA concentrations reduced growth, likely due to hormonal stress and ethylene accumulation, in agreement with George et al., (2008) and Bonga and von Aderkas (2019).






 
Rooting and acclimatization
 
Although IAA and IBA achieved high rooting percentages (Table 4 and 5), roots frequently originated from basal callus, which adversely affected acclimatization. In contrast, rooting in sterile distilled water produced well-organized roots with continuous vascular connections, achieving 100% rooting and superior acclimatization success (Table 6, Fig 5). This supports the concept of hormone-free final rooting stages to enhance plantlet quality (Bonga, 2024).

A micropropagation protocol of for Paulownia was optimized and it will support large-scale propagation and afforestation programs.

The authors would like to express their sincere gratitude to the Agricultural Research Center for providing laboratory facilities and technical support for conducing the experiments in the Plant Tissue Culture Laboratories.

The authors declare that there is no conflict of interest.