Paclobutrazol and sucrose boost tuber size and survival rate of micro propagated Zantedeschia spp

Calla lily (Zantedeschia spp.), a genus belonging to the family Araceae, exhibits a variety of cultivars with a wide range of colors. It is highly valued and cultivated as a popular ornamental plant in different regions worldwide¹. Calla lily holds the fifteenth rank among the top 25 flowers in the Dutch floral market and has become one of the most in-demand bulb flowers in the world in recent years². Commercial propagation of Zantedeschia through tuber division is skilled labor-intensive and poses a risk of spreading soft rot (Pectobacterium), resulting in significant economic losses for producers during propagation, forcing, and planting stages³. On the other hand, the production of Zantedeschia from seeds is time-consuming due to allogamy and the genetic impurity of existing cultivars, resulting in considerable genetic diversity among seed-derived plants⁴. In vitro culture has been successful for large-scale and commercial plant propagation⁵. This method is suitable for the regeneration and breeding of valuable plants. Utilizing this technique is an efficient way to propagate uniform plants in bulk in the shortest possible time. Tissue culture is an effective approach for the propagation of tuberous plants with various commercial and breeding objectives⁶. Therefore, the development of commercial in vitro propagation techniques could prove valuable and economically viable⁷.

The formation of micro tubers in tuberous plants like Calla lily is a crucial final step in tissue culture plant production and successful acclimatization. According to reports, tissue-cultured plants that lack proper tubers or have not formed tubers have little chance of survival in the subsequent transfer and acclimatization stages⁸. Although there have been some reports on the micropropagation and in vitro culture of tuberous Calla lily^5,9,10,11comprehensive studies on in vitro tuber development, as well as their quantitative and qualitative characteristics during and following the acclimatization phase, remain lacking.

Previous studies have reported that Calla lily plants regenerated through tissue culture require approximately three years to progress from the acclimatization stage to flowering, following successful establishment in ex vitro conditions. However, this timeframe is influenced by the size of the tubers, which ideally should attain a diameter of 3 cm (initiate flowering size) to 6 cm (commercial flowering size). The application of hormonal compounds under in vitro conditions can impact the tuber size and consequently either shorten or lengthen the aforementioned timeframe^8,12.

Sucrose as a carbon source is the most commonly used carbohydrate in plant tissue culture media¹³. According to research, sucrose is one of the components in plant tissue culture media that can have a positive effect on tuber propagation and growth in vitro. Reports have suggested that a high sucrose concentration in the culture medium can lead to accelerated growth and micro tuber production in tissue-cultured plantlets^14,15. Following these findings reports on Lilium in vitro propagation have shown that the inclusion of sucrose in the growth medium led to an enhancement in tuber size in plantlets¹⁶. Furthermore, a separate study indicated that elevating the sucrose concentration in the proliferation medium beyond the typical 3% resulted in increased growth and tuberization rates¹⁷. The result of Yu et al.¹⁸ shows that, sucrose was identified as an ideal carbon sucrose in plant tissue culture media, exerting a more pronounced influence on the growth of microtubers when compared to alternative carbon sucrose. The researchers identified sucrose availability as a primary factor influencing microtuber size.

Application of plant growth retardants, including cycocel and paclobutrazol in some geophyte species such as diploid-taro, Lilium, Tulip has been correlated with an enhancement in underground storage organs^19,20,21. These compounds are also known as anti-gibberellins²² because they inhibit the function of the gibberellin hormone in the plant, preventing vegetative growth and causing nutrients to accumulate in the storage organ, which increases the size of the tubers²³.

A study has shown that the in vitro application of growth retardants promotes bud differentiation and enhances microtuber formation²⁴. Additionally, experimental results indicate that combining hormonal treatments with sucrose significantly improves microtuber induction under in vitro conditions²⁵. Paclobutrazol has also been widely used in ornamental plant cultivation to stimulate tuber development, with proven effectiveness in both in vitro and greenhouse settings across several studies^26,27,28.

Moreover, the effectiveness of cycocel, used alone or in combination with sucrose, has been investigated in several research on promoting tuber growth within in vitro condition^28,29.

This study was conducted to investigate the impact of different concentrations of sucrose, cycocel, and paclobutrazol on the initiation of microtubers in some commercial cultivars of Calla lily (Zantedeschia spp.). It is worth noting that, to the best of our knowledge this research represents the first documented attempt to perform such experiments under in vitro conditions using Calla lily plantlets, starting prior to their transfer to the rooting medium and continuing throughout the subsequent acclimatization phase. The primary objective of this study was to expedite the tuber growth process, which typically takes approximately three years from plant transfer to the commercial maturity of the tubers. Additionally, this study aimed to enhance the production of high-quality tubers while minimizing the time required, which may subsequently lower cost requirements.

Plant material

The current research was conducted in the Plant Tissue Culture Laboratory of the National Institute of Flowers and Ornamental Plants of Iran in Mahallat. For the experiment, tissue-cultured plantlets of the Sun Club, Orania, and Zazu commercial cultivars were used, which were uniform in terms of regeneration time, subculture frequency, and size under completely identical conditions. To produce plantlets for this study, tubers from three calla lily cultivars (Zantedeschia spp. Sun Club, Orania and Zazu) were sourced from sande group company. The tubers were cut into 2 cm² pieces, washed, and sterilized through a sequence of treatments: a 35-minute hot water bath at 45^°C, followed by 30 s in 70% ethanol and 10 min in 1% sodium hypochlorite, with three rinses in sterilized distilled water. The explants were subsequently placed on Murashige and Skoog (MS) medium²⁸ without hormones for 30 days to encourage establishment, after which shoots were transferred to a proliferation medium containing 2.5 mg/L 6-benzylaminopurine, 1.5 mg/L kinetin, and 3% sucrose⁵.

Tuber growth medium and growth conditions

The experiment began 20 days before the introduction of plantlets to the rooting medium and 40 days prior to their transfer for acclimatization. At the initiation of the experiment, the plantlets measured approximately 8 to 10 centimeters in height, with the basal part of the stem being about 2 millimeters in diameter, and each plantlet possessed two leaves. The plantlets were cultured in the Murashige and Skoog (MS) medium²⁸ supplemented at all stages with 7 g/dm³ agar along with the treatments applied, as listed in Tables 1 and 2. Their pH was adjusted to 5.7 solutions prior to autoclaving. After the addition of growth regulators, the media were sterilized by autoclaving for 20 min at a temperature of 121 °C. Post-treatment, the plantlets were kept in a growth chamber under light conditions of 16 h of illumination at 60 µmol/m²/s provided by white fluorescent Lucca 20 W LED SMD lamps at a temperature of 23 ± 2 °C for a duration of 20 days.

Rooting and acclimatization

After the specified period had elapsed, the plantlets were transferred to MS medium³⁰supplemented with 1 mg/L of indole butyric acid (IBA). Twenty days later, for the purpose of acclimatization, the rooted plants were removed from the rooting medium by distilled water and transplanted into seedling pots containing a volumetric mix of cocopeat, perlite, and peat moss in a 1:1:1 ratio (V/V). For acclimatization, the plants were maintained in a greenhouse with humidity set at 85 ± 10% and a temperature of 25 ± 2 °C.

Post acclimatization and parameters evaluation

After plant establishment, the relative humidity was gradually reduced to 60%, beginning on the tenth day. The plants were maintained in an acclimatization greenhouse for 30 days. Subsequently, they were transferred to a growth greenhouse with a controlled temperature regime of 26–28 °C during the day and 21–23 °C at night, under a constant relative humidity of 60%. In this research, traits such as tuber size and weight, plant height, petiole diameter (under in vitro conditions and after acclimatization), survival rate, and leaf area (in greenhouse conditions) were evaluated. Tuber diameter, plant height, and microtuber weight were measured with a digital scale, leaf area with a leaf area meter, and the survival rate was calculated using the following formula:

Experimental design and statistical analysis

The research was conducted as two separate factorial experiments. The first experiment comprised three factors: sucrose at levels of 3%, 6%, and 9%; cycocel from Zhengzhou Nongda Biochemical Co. at concentrations of 0, 150, 200, and 250 mg/L, and three Zantedeschia cultivars (Sun Club, Orania, and Zazu). The second experiment investigated the sucrose levels and cultivars mentioned earlier, along with different concentrations of paclobutrazol from Duchefa Biochemie at 0.5, 1.5, and 3 mg/L. Both experiments followed a completely randomized design, with 36 treatments in total for each experiment. Each treatment was replicated three times, with 16 plantlets in each replicate. Statistical analysis of the data was performed using SAS software version 9.4, and mean comparisons were conducted using Duncan’s multiple range test.

The results of the analysis of variance (Tables 3 and 4) revealed a significant three-way interaction among the studied factors—cultivar, sucrose, and the tuber growth regulators paclobutrazol and cycocel—in both experiments. As a result, the subsequent discussion will focus specifically on the interactive effects of these three factors on the observed responses.

Effect of plant growth retardant Cycocel

Different Calla lily cultivars exhibited varying rates of successful acclimatization under identical conditions, as indicated by the data in Table 5. The Zazu cultivar exhibited the highest rate of successful acclimatization at 53%, followed by the Sun Club and Orania cultivars with rates of 40% and 32%, respectively. Among the various treatments and cultivars, the treatment combining 6% sucrose with 200 mg/L of cycocel (Z.S₆.C₂) demonstrated the highest percentage of acclimatization, averaging at 72% (Table 5). Notably, this specific treatment also yielded the highest acclimatization percentages for the Sun Club and Orania cultivars, reaching 71% and 65%, respectively, surpassing other treatments and the control (3% sucrose) for these cultivars (Table 5).

Based on the results from the Table 5, the treatment with 6% sucrose and 200 mg/L cycocel exhibited the highest effect on the size and weight of tubers in vitro and post-acclimatization across the studied calla lily cultivars. Overall, the O.S₆.C₂ treatment (Orania cultivar with 6% sucrose and 200 mg/L cycocel) had the most significant influence on tuber size and weight, measuring 18.59 mm and weighing 1.81 g in vitro, and measuring 38.53 mm and weighing 5.37 g post-acclimatization.

The factors and treatments applied in this study had significant effects on the height and leaf area of plants under in vitro conditions and post-acclimatization, as indicated by Tables 3 and 5. When comparing different levels of sucrose alone and in combination with various concentrations of the tuber growth regulator cycocel, no significant differences were observed in treatments with the same levels of sucrose but different cycocel concentrations. The Orania cultivar treated with 6% sucrose had the largest leaf area, measuring 48.66 cm² (Table 5). Leaf area index is an important factor for plant growth and quality, and faster leaf development post-acclimatization can contribute to improved plant growth quality, especially considering the inactivity of stomata in tissue-cultured plants. Based on the results, 6% sucrose had the most significant impact on the leaf area across all three cultivars (Table 5).

The cultivar type and the application of cycocel were identified as the most influential factors affecting plant height. Among the cultivars tested, ‘Orania’ exhibited the greatest height, reaching 12.57 cm in vitro and 14.72 cm after acclimatization under uniform growth conditions without additional treatments (Table 5). Notably, an inverse relationship was observed between cycocel concentration and plant height across all cultivars. For example, in the ‘Sunclub’ cultivar, plant height decreased from 12.36 cm in the control group (no cycocel) to 10.44 cm when treated with 250 mg/L cycocel during in vitro culture. Additionally, the treatment combining 6% sucrose without cycocel application (Z.S₆.C₀, S.S₆.C₀, O.S₆.C₀) resulted in the maximum plant height for all three cultivars, both in vitro (13.86 cm, 14.75 cm, and 14.73 cm for Zazu, Sun Club, and Orania, respectively) and after acclimatization (41.76 cm, 48.66 cm, and 37.64 cm, respectively) (Table 5).

Overall, based on the results obtained and the comparison of treatment effects on the measured traits, the application of 6% sucrose in combination with 200 mg/L cycocel (Z.S₆.C₂, S.S₆.C₂, O.S₆.C₂) produced the most desirable effect on plant height across all three cultivars. This positive effect was observed under both in vitro conditions and during acclimatization, with the same trend maintained in acclimatized plants. Furthermore, plants producing larger tubers with higher concentrations of cycocel and sucrose exhibited reduced in vitro height, and a similar trend in plant height was observed during acclimatization. This may be attributed to the growth-retarding effects of cycocel (Table 5). Typically, plantlets with larger tuber size and favorable indices related to tuber quality, such as tuber diameter and tuber weight, are more likely to acclimatize and survive in subsequent growing seasons. Additionally, adequate leaf area and growth height are also important factors contributing to the successful acclimatization and survival of the plantlets. Under the specified experimental conditions, it was observed that all cultivars subjected to a treatment consisting of 6% sucrose and 200 mg/L cycocel exhibited more favorable traits in terms of plant height and tuber characteristics (tuber diameter and tuber weight) both during in vitro culture and the subsequent acclimatization phase (Table 5).

The effect of plant growth retardant Paclobutrazol

In this experiment, the plant growth retardant paclobutrazol was used in place of cycocel to evaluate its effects in combination with other experimental treatments on the measured physiological and growth traits. According to Table 6, the three calla lily cultivars exhibited varying acclimatization percentages under uniform conditions. Among the different levels of sucrose treatments (3%, 6%, and 9%), the application of 6% sucrose combined with paclobutrazol had the most significant effect on the acclimatization percentage of the Zantedeschia cultivars Sun Club, Orania, and Zazu. The average acclimatization percentages for these cultivars were 61%, 58%, and 63%, respectively, compared to the various treatment levels (Table 6). Notably, the Orania cultivar demonstrated a significant increase in acclimatization percentage when treated with paclobutrazol and 6% sucrose. The acclimatization percentage reached 80% when 0.5 mg/L paclobutrazol was used along with 6% sucrose, while the use of 6% sucrose alone resulted in a acclimatization of 58% of plantlets (Table 6).

Among all the treatments and cultivars, the treatment with 6% sucrose and 0.5 mg/L paclobutrazol (Z.S₆.C₂) yielded the highest acclimatization percentage with an average of 91% in Zazu cultivar (Table 6). Additionally, this treatment led to the highest acclimatization percentages for the Sun Club and Orania cultivars, with values of 86% and 80%, respectively, across both the first and second experiments (Table 6). The results presented in Table 6 demonstrate that the treatment combining 6% sucrose and 0.5 mg/L paclobutrazol had a superior effect on the all three cultivars. This effect was evident in the size and weight of the tubers in vitro. The average sizes of the tubers were 19.26 mm, 23.41 mm, and 18.79 mm, with corresponding weights of 1.83 g, 2.82 g, and 2.07 g, in Sun Club, Orania, and Zazu cultivars respectively. After acclimatization, the same treatment resulted in tubers with average sizes of 48.72 mm, 50.41 mm, and 44.06 mm, and weights of 6.60 g, 7.37 g, and 5.81 g, respectively (Figs. 1 and 3). These results were significantly higher than those obtained from the control treatment, which produced tubers with average sizes of 5.65 mm, 9.48 mm, and 7.55 mm, and weights of 0.88 g, 1.07 g, and 0.91 g, in Sun Club, Orania, and Zazu cultivars respectively. Furthermore, this specific treatment combination even outperformed the best-performing treatment from the first experiment with application of sucrose combined with cycocel (O.S₆.C₂), as indicated in Tables 5 and 6.

Comparison of various paclobutrazol concentrations with cycocel treatments (Tables 5 and 6) revealed that paclobutrazol applications resulted in improved tuber size- and weight-related traits under in vitro conditions. The Orania cultivar, when treated with O.S₆.P₁ (6% sucrose and 0.5 mg/l paclobutrazol), exhibited a significant increase in tuber size and weight, with average values of 23.41 mm and 2.82 g in vitro, and 50.41 mm and 9.48 g post acclimatization. This represented a substantial improvement over the performance of the Orania cultivar with treatment O.S₆.C₂, which had been the optimal treatment in the first experiment (application of sucrose combined with cycocel) for these traits (18.59 mm and 1.81 g in vitro, and 38.53 mm and 5.37 g post acclimatization), as indicated in Tables 5 and 6. Table 6 also highlights significant differences in these traits among the different cultivars, even under the same treatment conditions. This emphasizes the distinct responses of different cultivars to consistent hormone and sucrose levels. Hence, it is crucial to conduct further meticulous investigations that take into account the specific hormone types and concentrations.

The application of paclobutrazol treatments resulted in reduced plant height and leaf area compared to the control samples (Table 6). The height of different cultivars exhibited significant variations both in vitro and after acclimatization, with more pronounced differences observed during the acclimatization phase. As mentioned earlier, the 6% sucrose treatment had the most significant effect on plant height, leading to taller growth (Table 6).

Regarding leaf area, the data in Table 6 revealed a significant reduction when any level of paclobutrazol was applied alongside 3% sucrose, compared to sucrose alone. However, closer examination showed no marked difference in leaf area among the three concentrations of paclobutrazol (0.5, 1.5, and 3 mg/L) used with 3% sucrose (Table 6). The Orania cultivar exhibited the largest leaf area, while the Zazu cultivar had the smallest. Across all three cultivars, the application of 6% sucrose resulted in the highest leaf area (Sun Club: 41.76 cm²Orania: 48.66 cm² and Zazu: 37.64 cm² (Table 6). The trend with the application of different paclobutrazol levels closely paralleled that of paclobutrazol at a 3% sucrose level, but the leaf area with these treatments was larger than with 3% alone (Table 6). The Orania cultivar with 6% sucrose exhibited the largest leaf area (48.66 cm², and noticeable differences in leaf areas were observed with the application of three paclobutrazol levels (0.5, 1.5, and 3 mg/L) (42.59 cm²40.85 cm² and 41.30 cm² respectively) compared to the treatment with 6% sucrose alone (Table 6). However, no significant differences were observed among the different paclobutrazol levels.

Furthermore, our observations indicated that plants treated with paclobutrazol and sucrose exhibited superior quality during the growth period following acclimatization compared to the control group. This enhancement in plant quality was most pronounced at the end of the first growth period, just prior to the onset of dormancy. The improved quality may be attributed to the combined effects of these treatments on increasing microtuber size and reducing the sensitivity of acclimatized plants to environmental stressors (Fig. 2).

The trend of leaf area reduction in the Sun Club cultivar with the applied treatments was similar to that of the Orania cultivar. However, a significant increase was observed in the Zazu cultivar with the application of 6% sucrose (sucrose). The leaf area increased from 19.94 cm² for the 3% sucrose treatment to 37.64 cm² with the application of 6% sucrose. The reduction and difference in leaf area between the different levels of Paclobutrazol in the Zazu cultivar were similar to the other two cultivars. Based on the findings presented in Tables 5 and 6, Paclobutrazol had a slightly more pronounced influence on the reduction of leaf area compared to cycocel.

Different concentrations of Paclobutrazol (0.5, 1.5, and 3 mg/L) combined with varying levels of sucrose led to a reduction in height for all three cultivars under in vitro conditions compared to the application of varying concentrations of sucrose alone (Table 6). In the first experiment, the treatment with 6% sucrose had the most significant influence on the height of tissue-cultured plantlets (Sun Club: 41.76 cm, Orania: 48.66 cm, and Zazu: 37.94 cm), with the Orania cultivar achieving the greatest height among the three cultivars. Comparing the different concentrations of Paclobutrazol in the experimental treatments, it is apparent that an increase in concentration did not result in significant changes in plant height (Table 6). However, a notable point is the comparison of Paclobutrazol treatments with cycocel in the first experiment, which demonstrated a substantial reduction in plant height with Paclobutrazol compared to cycocel during the acclimatization conditions (Tables 5 and 6).

The formation, sizing, and growth of tubers in tissue-cultured Zantedeschia are critical stages spanning from acclimatization to commercial maturation. The application of Paclobutrazol has been shown to significantly enhance tuber development, making it a recommended practice in the cultivation of tissue-cultured Zantedeschia. Compared to Cycocel treatments, Paclobutrazol demonstrated superior efficacy in increasing tuber size, underscoring its practical value in this context. Notably, despite the observed reduction in plant height associated with Paclobutrazol application, tuber size was consistently enhanced. The most pronounced effect was recorded in plants treated with 0.5 mg/L Paclobutrazol in combination with 6% sucrose, across all three cultivars (Table 6). Under post-acclimatization conditions, the combination of Paclobutrazol and 3% sucrose did not significantly affect plant height when compared to the use of sucrose alone. However, at higher sucrose concentrations (6% and 9%), varying levels of Paclobutrazol led to a more marked reduction in plant height, which was concurrently associated with increased tuber size (Table 6).

We conducted correlation analysis on the data and found significant positive and negative correlations between certain traits especially in the second experiments (Tables 7 and 8). In the first experiment, we didn’t observe any significant correlation between the survival rate and traits related to tuber and vegetative organs, except for the size of the tubers under in vitro conditions, which showed a significant correlation with the survival rate at a 5% level (r = 0.36 ≤ 0.05). However, in the second experiment, we found several significant correlations, particularly at a 1% level. There were significant correlations between the survival rate and tuber-related traits (diameter and weight) under both in vitro (diameter: r = 0.72 ≤ 0.01, weight: r = 0.56 ≤ 0.05) and ex vitro (diameter: r = 0.61 ≤ 0.01, weight: r = 0.66 ≤ 0.01) conditions. We also observed a significant negative correlation between the plant height under in vitro conditions and tuber growth under ex vitro conditions (diameter: r= −0.67 ≤ 0.01, weight: r= −0.51 ≤ 0.05). These findings indicate that the combination of Paclobutrazol treatment with sucrose had a greater impact on increasing tuber diameter and weight, while reducing vegetative growth and plantlet height.

Different cultivars demonstrated diverse responses to specific treatments at given concentrations. According to Sikmo³¹plant cultivars can exhibit varied responses to the application of plant growth retardants. Therefore, it is important to investigate and determine the optimal concentration for each cultivar or to identify a cultivar that performs best under the given conditions. A study on different tulip cultivars under in vitro conditions with varying concentrations of Paclobutrazol revealed discrepancies in underground organs (bulbs) production performance among the cultivars³².

Previous studies indicate that larger tuber sizes of in vitro cultured plantlets at the time of transfer for acclimatization significantly increase the likelihood of successful acclimatization^16,21,33. Additionally, the tubers will enter the maturation phase within a shorter time frame and require a reduced growth period³⁴. As the results of this research indicate, cycocel and Paclobutrazol can be effective in in vitro conditions when microtuber formation is essential. One reason these plant growth retardants lead to an increase in tuber size is due to their anti-gibberellin properties³⁵. By inhibiting vegetative growth in vitro, these compounds promote the accumulation of nutrients and carbohydrates in the subterranean organs, which results in an increase in tuber size³⁶. It has also been reported that the application of the plant growth retardant Paclobutrazol can enhance the production and activity of certain enzymes and antioxidants, such as peroxidase, which leads to increased resistance to environmental stresses for the plant^37,38,39. This response is significant for tissue-cultured plantlets due to their sensitivity during the acclimatization period and could be a reason for the improved acclimatization success and survival of plants treated with Paclobutrazol. Paclobutrazol anti-stress property induces water conservation within the plant, reducing moisture stress and increasing survival rates in plants under dry conditions and environmental fluctuations^40,38,42. This factor is critical in the acclimatization stages of tissue-cultured plants.

As cited in the literature, Paclobutrazol belongs to the triazole family and acts as a highly active systemic fungicide, leading to increased plant resistance to fungal pathogens²³. Protecting plants from pathogens in vitro and during acclimatization is a fundamental factor in ensuring survival and a successful transition from in vitro production to acclimatization. Traits affected by the use of Paclobutrazol include the development of plants with reduced height. However, the resulting plants are more robust, with thicker leaf tissue and a higher percentage of cuticle⁴². The factors discussed could be among the effective influences on the outcomes observed in Zantedeschia cultivars treated with Paclobutrazol, ultimately leading to an increase in tuber size and acclimatization in in vitro cultured plantlets^24,35. Based on the presented findings, the treatment involving cycocel showed improvements in acclimatization, tuber performance, and other measured traits compared to the control group (3% sucrose). However, treatments incorporating Paclobutrazol demonstrated even better performance than those with cycocel. This is consistent with previous experiments that reported increased tuberization and indices related to micro tuber formation in potato with the application of cycocel under in vitro conditions⁴³. Additionally, studies have shown that cycocel application resulted in enhanced tuberization, reduced vegetative growth, and decreased plant height under in vitro conditions²⁹.

Sucrose at 6% concentration led to an increase in microtuber formation in in vitro cultured plantlets. Carbohydrates are the primary products of photosynthesis, and their accumulation is an indicator of tuber enlargement. Among various carbohydrates, sugars, mainly sucrose, play a central role in plant growth¹³. Based on numerous studies, sucrose at concentrations higher than the typical 3% in culture media leads to an increase in size and weight of tubers in geophytes¹³. Jo et al.⁴⁴ described the increase in tuber size at high sucrose concentrations as plants being placed under stress. This stress shifts plant behavior towards maturation, which results in tuber formation. This is why the highest number of tubers was obtained when using sucrose concentrations of 6 and 9%. Lower concentrations of sucrose did not significantly impact tuberization and delayed the process, ultimately producing fewer tubers⁴⁵. sucrose plays a dual role in the development of microtubers⁴⁶. This carbon source is readily absorbed and easily converted into starch, which accelerates the tuberization process. The use of growth retardants in combination with sucrose had a more pronounced effect on acclimatization indices and tuber growth. The application of Paclobutrazol improves carbohydrate accumulation in bulbous plants. It is believed that Paclobutrazol influences the physiological effects of sugars and potentially modulates their metabolism, translocation, and activity within different plant organs. Therefore, the combined application of these compounds can synergistically affect tuber growth⁴⁷.

Furthermore, Paclobutrazol by disrupting the gibberellin biosynthesis pathway, resulting in the translocation and accumulation of carbohydrates in the above-ground parts of plants.

Studies have demonstrated that the incorporation of sugar compounds with paclobutrazol significantly enhances the proliferation of tulips, a geophyte species, resulting in improved bulblet development and accelerated maturation²¹. In gladiolus, paclobutrazol application has been shown to stimulate cormel formation from explants and enhance cormel development in plantlets during the proliferation stage. The combination of paclobutrazol with sugar compounds further promotes corm production³³. Additionally, research has indicated that paclobutrazol, when used in conjunction with sucrose-enriched media, suppresses excessive vegetative growth while promoting an increase in the size of underground storage organs.

Previous reports have highlighted that larger tubers in plantlets contribute to improved survival rates, successful acclimatization, and attainment of commercial maturity. However, it is important to consider that excessive concentrations of Paclobutrazol, coupled with smaller leaf sizes, may pose physiological challenges during subsequent growth phases leading to maturity. Therefore, determining the optimal concentration of plant hormones is vital for achieving desirable outcomes.

In conclusion, the application of 0.5 mg/L paclobutrazol in combination with 6% sucrose significantly enhanced acclimatization success, as well as tuber size and weight, in Calla lily cultivars. Effective microtuber formation is a critical factor in the successful acclimatization of tissue-cultured plantlets, as insufficient tuberization can impede commercial production and result in suboptimal plant quality. Paclobutrazol not only promotes accelerated tuber development following plantlet transfer to ex vitro conditions but also facilitates the attainment of marketable tuber size within a shorter period. This study demonstrates that incorporating paclobutrazol with sucrose during the final in vitro growth phase serves as an effective strategy for improving micropropagation efficiency in Calla lilies. The protocol optimizes plant production timelines and has the potential to reduce production costs. Moreover, it minimizes the loss of high-value tissue-cultured plantlets during the acclimatization phase, thereby enhancing overall economic viability. Further research is warranted to elucidate the molecular mechanisms underlying the observed effects, which could provide deeper insights into the regulation of tuberization and stress adaptation in this species.

Research permission

Hereby, I, Pejman Azadi, declare that all the necessary permits have been obtained to carry out the research activities of this project in the National Institute of Flowers and Ornamental Plants of Iran in Mahallat.

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Authors and Affiliations

1. Department of Horticultural Science and Agronomy, Science and Research Branch, Islamic Azad University, Tehran, Iran

   Hassan Abedini Aboksari & Sepideh Kalatejari

2. Department of Genetic Engineering and Biosafety, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

   Pejman Azadi

3. Ornamental Plants Research Center (OPRC), Horticultural Sciences Research Institute (HSRI), Agricultural Research, Education and Extension Organization (AREEO), Mahallat, Iran

   Mohammad Hossein Azimi

4. Agriculture Research School, Nuclear Science and Technology Research Institute, PO Box 31485-498, Karaj, Iran

   Azam Borzouei

Contributions

A. Hassan abedini Aboksari: Practical work lab and green house.wrote the main manuscript.phD studentB.Dr. Pejman azadi: Project Manager, wrote the main manuscript, C. Dr.Mohammad Hossein Azimi, Scientific expert of the project, statistic analysisD.Dr.Sepideh Kalatejari : Scientific expert of the project. Tissue culture Lab.E.Dr.Azam Borzouei: Scientific expert of the project. Lab.

Corresponding author

Correspondence to Pejman Azadi.

Competing interests

The authors declare no competing interests.

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