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. 2025 Aug 7;15:28902. doi: 10.1038/s41598-025-14918-9

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

Hassan Abedini Aboksari 1, Pejman Azadi 2,✉, Mohammad Hossein Azimi 3, Sepideh Kalatejari 1, Azam Borzouei 4
PMCID: PMC12332005  PMID: 40775034

Abstract

Micropropagation of Zantedeschia, commonly known as Calla lily, represents a valuable and economically feasible method for plant propagation. However, the success of in vitro cultivation is heavily dependent on the proper formation of tubers, as plants lacking well-developed tubers face reduced survival rates during subsequent transfer and acclimatization stages. This research aimed to investigate the effects of varying concentrations of sucrose, cycocel, and paclobutrazol on the size and production of microtubers in potted Zantedeschia cultivars, specifically Sun Club, Orania, and Zazu, under in vitro conditions. The study was conducted as two separate experiments using a completely randomized design. In the first experiment, the Zantedeschia cultivars were exposed to various concentrations of sucrose (3%, 6%, and 9%) combined with different concentrations of cycocel (0, 150, 200, and 250 mg/l). The second experiment involved the same sucrose concentrations combined with paclobutrazol (0, 0.5, 1.5, and 3 mg/l). The results indicate that Zantedeschia cultivars demonstrate varying responses in survival rates and tuber sizes following acclimatization. The control group exhibited survival rates of 40% for Sun Club, 32% for Orania, and 53% for Zazu, along with corresponding tuber sizes of 23.91 mm, 25.71 mm, and 20.55 mm, respectively. Notably, the application of 6% sucrose in combination with 0.5 mg/L paclobutrazol significantly enhanced survival rates to 86% for Sun Club, 80% for Orania, and 91% for Zazu, while also increasing tuber diameters to 48.72 mm, 50.41 mm, and 44.06 mm, respectively. These findings contribute to develop an efficient micropropagation protocol for the Calla lily by enhancing tuber size to improve survival rates, thereby addressing a critical bottleneck in its propagation.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-14918-9.

Keywords: Calla lily, Growth retardants, Ornamental plant, Plant tissue culture, Tuber formation

Subject terms: Plant biotechnology, Plant sciences, Plant biotechnology, Plant development, Plant hormones

Introduction

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 lily5,9–11comprehensive 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 timeframe8,12.

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 plantlets14,15. 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 organs19–21. 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 studies26–28.

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 condition28,29.

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.

Materials and methods

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.

Table 1.

Different concentrations of sucrose and Cycocel on three studied potted calla Lily cultivars (Sun club, orania, and Zazu).

Treatment (Code) Cultivars Sucrose (3, 6 and 9%) Cycocel (150, 200 and 250 mg/L)
Su.S3.C0 Sun club 3% -
O.S3.C0 Orania 3% -
Z.S3.C0 Zazu 3% -
Su.S6.C0 Sun club 6% -
O.S6.C0 Orania 6% -
Z.S6.C0 Zazu 6% -
Su.S9.C0 Sun club 9% -
O.S9.C0 Orania 9% -
Z.S9.C0 Zazu 9% -
Su.S3.C1 Sun club 3% 150
O.S3.C1 Orania 3% 150
Z.S3.C1 Zazu 3% 150
Su.S6.C1 Sun club 6% 150
O.S6.C1 Orania 6% 150‌
Z.S6.C1 Zazu 6% 150
Su.S9.C1 Sun club 9% 150
O.S9.C1 Orania 9% 150
Z.S9.C1 Zazu 9% 150
Su.S3.C2 Sun club 3% 200
O.S3.C2 Orania 3% 200
Z.S3.C2 Zazu 3% 200
Su.S6.C2 Sun club 6% 200
O.S6.C2 Orania 6% 200
Z.S6.C2 Zazu 6% 200
Su.S9.C2 Sun club 9% 200
O.S9.C2 Orania 9% 200
Z.S9.C2 Zazu 9% 200
Su.S3.C3 Sun club 3% 250
O.S3.C3 Orania 3% 250
Z.S3.C3 Zazu 3% 250
Su.S6.C3 Sun club 6% 250
O.S6.C3 Orania 6% 250
Z.S6.C3 Zazu 6% 250
Su.S9.C3 Sun club 9% 250
O.S9.C3 Orania 9% 250
Z.S9.C3 Zazu 9% 250
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Table 2.

Different concentrations of sucrose and Paclobutrazol on three studied potted calla Lily cultivars (Sun club, orania, and Zazu).

Treatment (Code) Cultivars Sucrose (3, 6 and 9%) Paclobutrazol (0.5, 1.5 and 3 mg/L)
Su.S3.P0 Sun club 3% -
O.S3.P0 Orania 3% -
Z.S3.P0 Zazu 3% -
Su.S6.P0 Sun club 6% -
O.S6.P0 Orania 6% -
Z.S6.P0 Zazu 6% -
Su.S9.P0 Sun club 9% -
O.S9.P0 Orania 9% -
Z.S9.P0 Zazu 9% -
Su.S3.P1 Sun club 3% 0.5
O.S3.P1 Orania 3% 0.5
Z.S3.P1 Zazu 3% 0.5
Su.S6.P1 Sun club 6% 0.5
O.S6.P1 Orania 6% 0.5
Z.S6.P1 Zazu 6% 0.5
Su.S9.P1 Sun club 9% 0.5
O.S9.P1 Orania 9% 0.5
Z.S9.P1 Zazu 9% 0.5
Su.S3.P2 Sun club 3% 1.5
O.S3.P2 Orania 3% 1.5
Z.S3.P2 Zazu 3% 1.5
Su.S6.P2 Sun club 6% 1.5
O.S6.P2 Orania 6% 1.5
Z.S6.P2 Zazu 6% 1.5
Su.S9.P2 Sun club 9% 1.5
O.S9.P2 Orania 9% 1.5
Z.S9.P2 Zazu 9% 1.5
Su.S3.P3 Sun club 3% 3
O.S3.P3 Orania 3% 3
Z.S3.P3 Zazu 3% 3
Su.S6.P3 Sun club 6% 3
O.S6.P3 Orania 6% 3
Z.S6.P3 Zazu 6% 3
Su.S9.P3 Sun club 9% 3
O.S9.P3 Orania 9% 3
Z.S9.P3 Zazu 9% 3
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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:

graphic file with name d33e1410.gif

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.

Results

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.

Table 3.

Analysis of variance of the effect of different calla Lily cultivars (Sun club, Orania and Zazu), application of sucrose and cycocel on plant parameters.

Treatments DF Mean Squares
In vitro After acclimatization (Greenhouse)
Micro tuber diameter Micro tuber weight Plant height Survival rate Tuber diameter Tuber weight Plant height Leaf area
Replication 2 0.09** 0.01** 0.12** 21.19** 5.11* 0.12** 16.66** 10.58NS
Cultivar (Cu) 2 77.57** 1.56** 7.01** 2070.11** 332.77** 9.81** 1.66** 3622.23**
Sucrose (S) 2 146.70** 0.95** 1.38** 3327.25** 206.31** 9.50** 28.11** 1759.60**
Cycocel (C) 3 46.55** 0.20** 17.23** 174.38** 142.92** 2.00** 14.14** 92.08**
Cu×S 4 3.92** 0.11** 4.05** 173.11** 4.75** 0.18** 3.68** 165.89**
Cu×C 9 3.19** 0.01** 0.56** 13.42** 3.28** 0.08** 1.76** 10.89**
Cu×S×C 16 6.22** 0.01** 0.85** 11.95** 2.42* 0.20** 1.59** 23.89**
Error 69 0.15 0.002 0.01 2.12 1.2 0.01 0.28 21.54
CV (%) - 3.52 4.33 0.83 2.59 3.82 2.46 3.66 16.94
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NS: Non significant, **: Significant at 1%, *: Significant at 5%.

Table 4.

Analysis of variance of the effect of different calla Lily cultivars (Sun club, Orania and Zazu), application of sucrose and paclobutrazol on plant parameters.

Treatments DF Mean squares
In vitro After acclimatization (Greenhouse)
Micro tuber diameter Micro tuber weight Plant height Survival rate Tuber diameter Tuber weight Plant height Leaf area
Replication 2 1.09** 0.21** 3.29* 106.78** 10.36** 0.23** 2.04** 37.70**
Cultivar (Cu) 2 23.64** 5.56** 7.98** 1544.73** 549.26** 15.24** 14.39** 3255.93**
Sucrose (S) 2 271.26** 2.09** 2.44** 3594.48** 334.15** 14.45** 7.25** 1452.18**
Paclobutrazol (P) 3 313.77** 5.08** 34.99** 3472.94** 2252.71** 24.65** 8.98** 22.78**
Cu×S 4 44.96** 0.15** 8.15** 214.62** 29.23** 0.75** 2.84** 133.11**
Cu×P 9 1.96** 0.16** 3.05** 14.92** 1.89** 0.43** 0.71** 3.65**
Cu×S×P 16 9.72** 0.03** 2.14* 14.98** 9.13** 0.40** 0.33** 1.80**
Error 69 2.78 0.01 1.12 0.92 0.66 0.02 0.07 0.51
CV (%) - 11.59 7.11 9.35 1.37 2.05 3.31 1.92 2.81
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NS: Non significant, **: Significant at 1%, *: Significant at 5%.

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).

Table 5.

The mean comparison of the interaction effect of different calla Lily cultivars (Sun club, Orania and Zazu) and application of different levels of sucrose and Cycocel on plant parameters (In vitro and after acclimatization condition).

In vitro After acclimatization (Greenhouse)
Treatments Tuber diameter Tuber weight Plant height Survival rate Tuber diameter Tuber weight Plant height Leaf area
(mm) (g) (cm) (%) (mm) (g) (cm) (cm2)
Su.S3.C0 5.67p 0.88o 12.36ij 40.00q 23.91rs 2.58r 13.83ijklmn 27.68ghijkl
O.S3.C0 9.48ml 1.07jkl 12.75fgh 32.00t 25.71opqr 4.45gf 14.72fghi 32.04efghij
Z.S3.C0 7.55o 0.91o 12.53ghi 53.00lm 20.55t 2.33s 13.73ijklmn 19.94lmno
Su.S6.C0 10.21jk 0.97mno 13.86b 61.00fg 27.51klmno 4.12ij 16.62b 41.76abcd
O.S6.C0 12.42efg 1.61b 14.75a 58.00hi 31.22efg 4.56ef 17.75a 48.66a
Z.S6.C0 11.65hi 1.23fgh 14.73a 63.00ef 23.96rs 3.74mn 15.91bcd 37.64bcdef
Su.S9.C0 9.76jjkl 0.98lmno 12.86cd 61.00fg 24.88pqrs 4.09ijk 15.58cdefg 33.65defghi
O.S9.C0 10.18jkl 1.20ghi 12.98c 50.33n 29.74ghij 4.34gh 15.80bcd 41.49abcd
Z.S9.C0 7.45o 0.97mno 12.53ghi 61.00fg 24.67rsq 3.55o 15.63bcdef 19.65lmnop
Su.S3.C1 8.45n 0.92no 12.64efg 43.00p 24.92pqrs 3.37p 13.61ijklm 23.32jklmn
O.S3.C1 9.51klm 1.39cd 12.53ghi 35.00s 27.37mnlo 3.76ml 14.65fghij 25.80hijklm
Z.S3.C1 7.73o 0.89o 12.57fgh 53.33lm 23.09s 3.00q 13.88ijklmn 17.00no
Su.S6.C1 9.86jkl 1.04klm 11.47no 66.00cd 29.82fghij 4.36gh 15.83bcd 35.70cdefg
O.S6.C1 15.74b 1.63b 12.84cd 51.33mn 34.61bc 5.00bc 15.78bcde 43.55abc
Z.S6.C1 12.04fgh 1.28efg 11.39no 68.00bc 26.20nopq 4.01jk 15.83bcd 29.64fghijkl
Su.S9.C1 10.26j 1.01lmn 12.81cd 59.00gh 28.05ijklmn 3.93kl 15.51defgh 29.62ghijk
O.S9.C1 12.65def 1.31def 12.74def 56.00ijk 31.39efg 4.37gh 15.78bcde 38.67bcde
Z.S9.C1 11.02i 1.23fgh 12.97c 66.00cd 26.83mnop 3.58no 14.39ijklm 17.52mnop
Su.S3.C2 7.34o 0.91o 10.84p 46.00o 27.76jklmno 3.68mno 13.66jklmn 19.65lmn
O.S3.C2 12.87de 1.36cde 12.67defg 38.00rg 31.89def 4.34gh 14.59fghijk 25.53 ijklm
Z.S3.C2 9.13m 0.96mno 12.45hi 62.00f 25.93opqr 3.33p 13.20no 17.21 no
Su.S6.C2 14.63c 1.21ghi 11.31o 71.00a 32.54de 5.00bc 14.77efghi 32.40 efghi 
O.S6.C2 18.59a 1.81a 11.86kl 65.33de 38.51a 5.37a 15.77bcde 34.48 defgh
Z.S6.C2 12.46efg 1.38cde 11.36o 72.00a 29.48ghijk 4.12ij 15.63bcdef 17.21no
Su.S9.C2 11.36hi 1.13ijk 12.24j 65.00de 29.74ghij 4.12ij 14.77efghi 23.25 jklmn
O.S9.C2 12.74def 1.43c 11.57mn 56.00ijk 35.73b 4.86cd 14.66fghij 33.65 defghi
Z.S9.C2 12.79def 1.43cde 12.74def 70.00ab 28.29hijklm 4.02jk 14.31ijklm 19.47lmnop
Su.S3.C3 8.41n 0.95mno 10.44q 46.00o 27.76jklmno 3.72mno 13.52lmn 18.24mno
O.S3.C3 12.65def 1.34cde 11.69ml 36.00rs 30.24fgh 4.21hi 12.46o 23.32jklmn
Z.S3.C3 8.45n 0.95mno 11.87k 58.00hi 24.72qrs 3.36p 12.54o 13.44p
Su.S6.C3 12.74def 1.22fghi 10.51q 68.00bc 30.36fgh 4.72de 14.59fghijk 25.80 hijklm
O.S6.C3 13.27d 1.72a 11.49no 56.66hij 33.69cd 5.24a 14.77efghi 30.27 efghijkl
Z.S6.C3 11.81gh 1.36cde 11.85kl 66.00cd 30.38fgh 4.07ijk 15.60bcdef  17.57 mnop
Su.S9.C3 11.45hi 1.16hij 11.42no 62.00f 29.98fghi 4.05ijk 12.47o  18.28 mnop
O.S9.C3 14.07c 1.38cde 10.91p 54.00kl 34.49bc 5.04b 14.66fghij  29.58 ghijk
Z.S9.C3 12.47efg 1.33de 11.94k 65.00de 29.04hijkl 4.06ijk  13.75ijklm  13.53 p
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In each column, means with the similar letters are not significantly different at 5% level of probability using Duncan’s test. Su: Sun club, O: Orania, Z: Zazu, S (3, 6 and 9): Sucrose (3, 6 and 9%), C (0, 1, 2 and 3): Cycocel (0, 150, 200 and 250 mg/L).

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).

Table 6.

The mean comparison of the interaction effect of different calla Lily cultivars (Sun club, orania, Zazu) and application of different levels of sucrose and Paclobutrazol on plant parameters (In vitro and after acclimatization condition).

In vitro After acclimatization (Greenhouse)
Treatments Tuber diameter Tuber weight Plant height Survival rate Tuber diameter Tuber weight Plant height Leaf area
(mm) (g) (cm) (%) (mm) (g) (cm) (cm2)
Su.S3.P0 5.67m 0.88p 12.36gh 40.00q 23.91p 2.58q 13.83hi 27.68hi
O.S3.P0 9.48kl 1.07nop 12.75ef 32.00r 25.71o 4.45n 14.72ef 32.04ef
Z.S3.P0 7.55lm 0.91p 12.58fg 53.00o 20.55q 2.33q 13.73ef 19.94kl
Su.S6.P0 10.21jkl 0.97op 13.86bc 61.00m 27.55op 4.12o 16.62b 41.76ab
O.S6.P0 12.42hijk 1.61hij 14.75a 58.00n 31.22m 4.56mn 17.75a 48.66a
Z.S6.P0 11.65ij 1.23lmn 14.73a 63.00l 23.96p 3.74p 15.91c 37.64cd
Su.S9.P0 9.76jkl 0.98op 12.86def 60.00m 24.88op 4.09o 15.58cd 33.65h
O.S9.P0 10.18jkl 1.20mno 12.98de 50.00p 29.74mn 4.37no 15.80c 41.49b
Z.S9.P0 7.45m 0.97op 12.53fg 61.00m 24.67op 3.55p 15.63cd 19.65kl
Su.S3.P1 12.82hij 1.43jkl 10.32stu 67.00k 38.15j 4.83ml 13.51ijkl 21.02k
O.S3.P1 16.58cdefg 2.17c 11.24qr 54.00o 48.16cd 6.17d 14.34fg 24.71i
Z.S3.P1 11.82ijk 1.62hij 12.53fg 81.00ef 36.94jk 4.76ml 13.32jklm 12.78p
Su.S6.P1 19.26bc 1.83fgh 12.54fg 86.00b 48.72bcd 6.60bc 14.68ef 33.72e
O.S6.P1 23.41a 2.82a 11.55op 80.00f 50.41a 7.37a 16.32b 42.59a
Z.S6.P1 18.79bc 2.07cde 11.19qr 91.00a 44.06ef 5.81e 15.64cd 18.47lm
Su.S9.P1 18.62bcde 1.72ghi 11.55op 82.00ef 44.07ef 6.71b 14.91de 31.33fg
O.S9.P1 16.75defg 2.75a 11.47opq 80.00f 48.93bc 5.61efg 15.34d 39.22c
Z.S9.P1 18.63bcde 2.03cdef 12.18ij 89.00ab 41.55hi 4.89kl 15.06de 18.61l
Su.S3.P2 12.80hij 1.38klm 9.43tuv 65.00k 37.76jk 4.75lm 13.38fg 20.18k
O.S3.P2 14.12fghi 2.11cd 10.73rst 53.00o 48.05cd 5.65ef 14.32fg 23.44j
Z.S3.P2 10.44jkl 1.36klm 12.44g 75.00hi 36.38k 4.37no 13.23jklm 11.75pq
Su.S6.P2 16.72cdefg 1.73ghi 10.33stu 84.00c 46.28d 6.13d 13.01lm 32.06f
O.S6.P2 20.43b 2.70a 10.55st 73.00i 50.08ab 7.11a 14.17gh 40.85b
Z.S6.P2 16.49cdefg 1.92defg 10.67rst 84.00c 43.58efg 5.21hij 14.73ef 16.75n
Su.S9.P2 18.74bcd 1.66h 11.14qr 81.00ef 42.87fgh 5.32ghij 14.22n 30.94fg
O.S9.P2 16.74cdefg 2.64b 10.23stu 76.00gh 48.92bc 6.31cd 13.58ijk 36.37d
Z.S9.P2 17.44bcde 2.02cdef 11.62no 83.00cd 40.92i 5.41fghi 13.64ijk 18.33ml
Su.S3.P3 12.42hijk 1.38klm 9.16uv 61.00m 37.13jk 4.38no 12.88m 18.33ml
O.S3.P3 13.82ghi 1.84efgh 10.51rst 50.00p 46.44d 5.53efg 13.15klm 23.46j
Z.S3.P3 10.41jkl 1.43jkl 11.87klm 70.00j 38.06j 4.56mn 13.72hij 11.21q
Su.S6.P3 15.52defghi 1.62hij 9.01v 81.00ef 47.44cd 6.10d 12.89m 31.38fg
O.S6.P3 19.53bc 2.73a 10.07tuv 77.00g 50.55a 6.81b 13.82hi 41.30b
Z.S6.P3 16.67cdefg 1.93defg 10.42stu 82.00de 43.96e 5.57efg 13.83hi 16.52n
Su.S9.P3 18.23bcde 1.68hi 10.26stu 80.00f 45.03e 5.17ijk 12.04n 30.38gh
O.S9.P3 15.42efgh 2.65ab 9.71tuv 74.00j 47.27d 6.52bc 13.22 jklm 34.60e
Z.S9.P3 17.23bcdef 1.98cdef 10.95uv 80.66ef 42.19ghi 5.50efgh 13.06lm 16.91n
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In each column, means with the similar letters are not significantly different at 5% level of probability using Duncan’s test. Su: Sun club, O: Orania, Z: Zazu, S (3, 6 and 9): Sucrose (3, 6 and 9%), P (0, 1, 2 and 3): Paclobutrazol (0, 0.5, 1.5 and 3 mg/L).

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.

Fig. 1.

Fig. 1

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Comparison of calla lily micro tubers growth in in vitro condition after using paclobutrazol and sucrose. Left: Control treatment (3% sucrose) including Sun Club, Orania and Zazu respectively and Right: 0.5 mg/L paclobutrazol with 6% sucrose treatment including Sun Club, Orania and Zazu respectively. Scale Bar: 20 mm.

Fig. 3.

Fig. 3

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Comparison of calla lily micro tubers growth in the first period of dormancy (three months after acclimatization). Left: Control treatment (3% sucrose) including Sun Club, Orania and Zazu respectively and Right: 0.5 mg./L paclobutrazol with 6% sucrose treatment including Zazu, Sun Club and Orania respectively. Scale bar: 20 mm.

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).

Fig. 2.

Fig. 2

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Comparison of quality of calla lily acclimatized plants after plantlets transfer to ex vitro condition in two months later. Left: control treatment (3% sucrose) including Sun Club, Orania and Zazu respectively and Right: 0.5 mg/L paclobutrazol with 6% sucrose treatment including Zazu, Sun Club and Orania respectively. Scale bar: 6 cm.

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.

Table 7.

Pearson’s correlation matrix for plant parameters of different calla Lily cultivars (Sun club, orania, Zazu) and application of different levels of sucrose and Cycocel on plant parameters (In vitro and after acclimatization condition).

‌ In vitro After acclimatization (Greenhouse)
Tuber diameter Tuber weight Plant height Survival rate Tuber diameter Tuber weight Plant height Leaf area
‌ In vitro Tuber diameter 1
Tuber weight 0.86** 1
Plant height −0.08ns 0.21ns 1
After acclimatization Survival rate 0.36* 0.16ns −0.07ns 1
Tuber diameter 0.84** 0.78** −0.28ns 0.15ns 1
Tuber weight 0.84** 0.74** −0.18ns 0.20ns 0.89** 1
Plant height 0.33* 0.37* 0.47** 0.31ns 0.26ns 0.41* 1
Leaf area 0.36* 0.36* 0.56** −0.05 0.38* 0.52** 0.74** 1
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Levels of significance: *p ≤ 0.05, **p ≤ 0.01, ns: Not significant.

Table 8.

Pearson’s correlation matrix for plant parameters of different varieties (Sun club, orania, Zazu) and application of different levels of sucrose and Paclobutrazol on plant parameters (In vitro and after acclimatization condition).

‌ In vitro After acclimatization (Greenhouse)
Tuber diameter Tuber weight Plant height Survival rate Tuber diameter Tuber weight Plant height Leaf area
‌ In vitro Tuber diameter 1
Tuber weight 0.82** 1
Plant height −0.43* −0.46* 1
After acclimatization Survival rate 0.72** 0.54* −0.32ns 1
Tuber diameter 0.86** 0.87** −0.67** 0.61** 1
Tuber weight 0.88** 0.85** −0.51* 0.66** 0.91** 1
Plant height −0.06ns −0.08ns 0.72** −0.15ns −0.32ns −0.11 1
Leaf area 0.21ns o.34ns 0.24ns −0.14ns 0.06ns 0.28ns 0.61** 1
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Levels of significance: *p ≤ 0.05, **p ≤ 0.01, ns: Not significant.

Discussion

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 acclimatization16,21,33. 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 plant37–39. 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 fluctuations40,38,42. 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 plantlets24,35. 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.

Conclusion

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.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (33.4KB, xlsx)

Author 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.

Data availability

All data generated or analyzed during this study that are published in this article (and its Supplementary Information files) are available.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

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References

  • 1.Wei, Z. et al. Assessing genetic diversity and population differentiation of colored calla Lily (Zantedeschia Hybrid) for an efficient breeding program. Genes8 (7), 168–184 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Xuan, S. et al. Tissue culture of calla Lily (Zantedeschia spreng.): an updated review on the present scenario and future prospects. Int. J. Eex Bot.92 (8), 2413–2428 (2023). [Google Scholar]
  • 3.Gutman, Y. et al. Ecological adaptations influence the susceptibility of plants in the genus Zantedeschia to soft rot pectobacterium spp. Hortic. Res.8 (13), 1–12 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Luo, J. P., Wawrosch, C. & Kopp, B. Enhanced micropropagation of dendrobium huoshanense C.Z. Tang et S.J. Cheng through protocom-like bodies: the effects of cytokinins, carbohydrate source and cold pretreatment. Sci. Hortic.123, 258–262 (2009). [Google Scholar]
  • 5.Hashemidehkordi, E., Mortazavi, S. N. & Azadi, P. An efficient in vitro propagation protocol of pot calla Lily (Zantedeschia spp. Cv. Orania and sun club) via tuber production. Int. J. Hortic. Sci. Technol.8 (4), 343–351 (2021). [Google Scholar]
  • 6.Koech, A. A., Isutsa, D. K. & Wu, Q. Explants hormones and sucrose influence in vitro shoot regeneration and rooting of calla Lily (Zantedeschia albomaculata L. Spreng.) ‘black magic’. J. Agric. Sci. Technol.7, 53–66 (2005). [Google Scholar]
  • 7.Zhang, X. et al. Haploid plant production in Zantedeschia aethiopica ‘hong gan’ using anther culture. Sci. Hortic.129, 335–334 (2011). [Google Scholar]
  • 8.Chen, J. J., Liu, M. C. & Ho, Y. H. Size of in vitro plantlets affects subsequent tuber production of acclimated calla Lily. Hortic. Sci.35, 290–292 (2000). [Google Scholar]
  • 9.Chang, H. S., Charkabarty, D., Hahn, E. J. & Peak, K. Y. Micropropagation of calla Lily (Zantedeschia albomaculata) via in vitro shoot tip proliferation. Vitro Cell. Dev. Biol. Plant.39, 129–134 (2003). [Google Scholar]
  • 10.Kumar, A. & Dogr, A. I. In vitro micropropagation of calla lily: an overview. Ind. J. Pure App Biosci.8 (2), 144–153 (2020). [Google Scholar]
  • 11.El-Gedawey, H. I. M., Rida, M. F. & Gaber, M. K. In vitro micropropagation of Zantedeschia Elliottiana plants. Sci. J. Flowers Ornam.9 (1), 1–11 (2022). [Google Scholar]
  • 12.Welsh, T. E. & Clemens, J. Protected cropping of Zantedeschia tubers and cut flowers in new Zealand. Acta Hort. 319, 335–340 (1992). [Google Scholar]
  • 13.Smeekens, S. Sugar-induced signal transduction in plants. Ann. Rev. Plant. Physiol. Plant. Mol. Biol.51, 49–81 (2000). [DOI] [PubMed] [Google Scholar]
  • 14.Joshi, S. K. & Dhar, U. In vitro propagation from axenic explants of Lilium oxypetalum (D. Don) baker, an endemic bulbous plant of high altitude himalaya. Acta Physiol. Plant.31 (4), 833–838 (2009). [Google Scholar]
  • 15.Bakhshaie, M., Babalar, M., Mirmasoumi, M. & Khalighi, A. Effects of light, sucrose and cytokinins on somatic embryogenesis in lilium ledebourii (Baker) bioss. Via transverse thin cell-layer cultures of bulblet microscales. J. Hortic. Sci.85, 491–496 (2010). [Google Scholar]
  • 16.Askari, N., Visser, R. & De Klerk, G. Growth of Lily bulblets in vitro, a review. Int. J. Hortic. Sci.5 (2), 133–143 (2018). [Google Scholar]
  • 17.Salem, J. & Hassanein, A. M. In vitro propagation, microtuberization, and molekuler characterization of three potato cultivars. Biol. Plant 61, 427–437 (2017). (2017).
  • 18.Yu, W. C., Joyce, P. J., Cameron, D. C. & Cown, B. H. Sucrose utilization during potato microtuber growth in bioreactors. Plant. Cell. Rep.19, 407–413 (2000). [DOI] [PubMed] [Google Scholar]
  • 19.Zhou, S. P., He, Y. K. & Li, S. J. Induction and characterization of in vitro corms of diploid-taro. Plant. Cell. Tiss Organ. Cult.57, 173–178 (1999). [Google Scholar]
  • 20.Kumar, S., Kashyap, M. & Sharma, D. R. Vitro regeneration and bulblet growth from Lily bulbscale explants as affected by retardants, sucrose and irradiance. Biol. Plant.49, 629–632 (2005). [Google Scholar]
  • 21.Sochacki, D., Marciniak, P., Ciesielska, M., Zarod, J. & Sutrisno The Influence of Selected Plant Growth Regulators and Carbohydrates on In Vitro Shoot Multiplication and Bulbing of the Tulip (Tulipa L.). Plants 12(5), 1134 (2023). [DOI] [PMC free article] [PubMed]
  • 22.Sarkar, J. Effect of Paclobutrazol on physiology, growth and yield of sunflower (Helianthus annuus L). Plant Physiol. Rep.28, 231–237 (2023). [Google Scholar]
  • 23.Desta, B. & Amare, G. Paclobutrazol as a plant growth regulator. Chem. Biol. Technol. Agric. 8(1), 1–15 (2021). (2021).
  • 24.Wu, Y. et al. Differential effects of Paclobutrazol on the bulblet growth of Oriental Lily cultured in vitro: growth behavior, carbohydrate metabolism, and antioxidant capacity. J. Plant. Growth Regul.38, 359–372 (2019). [Google Scholar]
  • 25.Iranbakhsh, A. & Ebadi, M. The induction of growth of potato (Solanum tuberosum L.) microtubers (sante cultivar) in response to the different concentrations of 6-benzylaminopurine and sucrose’. Afr. J. Biotechnol.10 (52), 10626–10635 (2011). [Google Scholar]
  • 26.Moreno, D., Berli, F. J., Piccoli, P. N. & Bottini, R. Gibberellins and abscisic acid promote carbon allocation in roots and berries of grapevines. J. Plant. Growth Regul.30, 220–228 (2011). [Google Scholar]
  • 27.Christiaens, A., Pauwels, E., Gobin, B. & Van Labeke, M. Flower differentiation of azalea depends on genotype and not on the use of plant growth regulators. Plant. Growth Regul.75, 245–252 (2015). [Google Scholar]
  • 28.Kumar, S., Kashyap, M. & Sharma, D. R. In vitro regeneration and bulblet growth from Lily bulbscale explants as affected by retardants, sucrose and irradiance. Biol. Plant.49, 629–632 (2005). [Google Scholar]
  • 29.Vreugdenhil, D. Potato biology and biotechnology advances and perspectives. Elsevier. 2td, Oxford, UK (2007).
  • 30.Murashige, T. & Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant.15, 473–497 (1962). [Google Scholar]
  • 31.Simko, I. Effects of kinetin, Paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light. Plant. Growth Regul.12, 23–27 (1993). [Google Scholar]
  • 32.Podwyszynska, M. & Marasek, A. Effects Thidiazuron and Paclobutrazol on regeneration potential of tulip flower stalk explants in vitro and subsequent shoot multiplication. Acta Soc. Bot. Pol.72 (3), 181–190 (2003). [Google Scholar]
  • 33.Nagaraju, V., Bhowmik, G. & Parthasarathy, V. A. Effect of Paclobutrazol and sucrose on in vitro Cormel formation in gladiolus. Acta Bot. Croat. 61 (1), 27–33 (2002). [Google Scholar]
  • 34.Langens-Gerrits, M. M., De Klerk, G. J. M. & Croes, A. Phase change in Lily bulblets regenerated in vitro. Physiol. Plant.119, 590–597 (2003). [Google Scholar]
  • 35.Podwyszyńska, M. The mechanism of in vitro storage organ formation in ornamental geophytes. Floric Ornam. Biotechnol.6, 9–23 (2012). [Google Scholar]
  • 36.Tesfahun, W. & Fatih Yildiz, F. A. Review on: response of crops to Paclobutrazol application. Cogent Food Agric.4 (1), 104–113 (2018). [Google Scholar]
  • 37.Srivastava, M., Kishor, A., Dahuja, A. & Sharma, R. R. Effect of Paclobutrazol and salinity on ion leakage, proline content and activities of antioxidant enzymes in Mango (Mangifera indica L). Sci. Hortic.125, 785–788 (2010). [Google Scholar]
  • 38.Hajihashemi, S. & Ehsanpour, A. A. Influence of exogenously applied Paclobutrazol on some physiological traits and growth of Stevia rebaudiana Bertoni under in vitro drought stress. Biologiae68, 414–420 (2013). [Google Scholar]
  • 39.Pal, S. et al. Paclobutrazol induces tolerance in tomato to deficit irrigation through diversified effects on plant morphology, physiology and metabolism. Sci. Rep.6, 39321 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Soumya, P. R., Kumar, P. & Pal, M. Paclobutrazol: a novel plant growth regulator and multi-stress ameliorant. Ind. J. Plant. Physiol.22, 267–278 (2017). [Google Scholar]
  • 41.Saleem, K. et al. Alleviating drought stress in strawberry plants: unraveling the role of Paclobutrazol as a growth regulator and reducer of oxidative stress induced by reactive oxygen and carbonyl species. J. Plant. Growth Regul.42 (12), 7731–7774 (2023). (2023). [Google Scholar]
  • 42.Sharma, K. D. et al. Effect of Paclobutrazol and Putrescine on antioxidant enzymes activity and nutrients content in salt tolerant citrus rootstock sour orange under sodium chloride stress. J. Plant. Nutr.36, 1765–1779 (2013). [Google Scholar]
  • 43.Roosta, H. R., Nasab, S. V. & Raghami, M. The effect of 6-benzyl amino purine and Cycocel on the production of microtubers in two varieties of potato in vitro. Irn J. Hortic. Sci.46 (1), 141–156 (2015). [Google Scholar]
  • 44.Jo, E. A., Tewari, R. K., Hahn, E. J. & Peak, K. Y. In vitro Sucrose concentration affects growth and acclimatization of Alocasia amazonica plantlets. Plant Cell Tiss. Organ Cult. 96, 307–315 (2009). (2009).
  • 45.Ovono, O. P., Kevers, C. & Dommes, J. Tuber formation and development of Dioscorea cayenensis–D. Rotundata complex. Effect of polyamines. Vitro Cell. Dev. Biol. Plant.46 (1), 81–88 (2010). [Google Scholar]
  • 46.Geigenberger, P. Regulation of sucrose to starch conversion in growing potato tubers. J. Exp. Bot.54 (382), 457–465 (2003). [DOI] [PubMed] [Google Scholar]
  • 47.Grospietsch, M., Stodulkova, E. & Zamecnik, J. Effect of osmotic stress on the dehydration tolerance and cryopreservation of Solanum tuberosum shoot tips. Cryo-Letters20, 339–346 (1999). [Google Scholar]

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