Sargassum fusiforme was collected from Dongtou, Zhejiang province, during the harvesting season and brought to the laboratory within 2 h in a 4°C cryopreservation box. The surface of S. fusiforme was cleaned with brushes and tweezers to remove attached stray algae and plankton, rinsed with filtered and sterilized natural seawater. Healthy and uniformly sized algal organisms were selected for temporary incubation in a light-illuminated incubator (light incubator, Yanghui, Ningbo, China) for 7 days. The experimental conditions were as follows: temperature was 19°C, light intensity was 90 µmol photons·m^-2 ·s^-1, light:dark photoperiod (L:D) was 12 h:12 h, salinity of the culture solution was 26‰, and the culture medium was supplemented with 200 µmol·L^-1 NaNO₃ and 20 µmol·L^-1 NaH₂PO₄. The culture solution was aerated continuously using an air pump (ACO-9730, Haili, Guangzhou, China).

After 7 days of domestication, healthy stem tips (2–4 cm in length), the lower end of the stem base (4–5 cm from the base), and filamentous holdfasts of S. fusiforme were selected and rinsed three times in sterile seawater. The seaweed was treated with a solution of 0.38% NaClO and 0.5% KI in a 1:1 volume ratio for 2 min, sterilized with sterile water, and rinsed. The samples were then cut into approximately 1.5 cm segments using a scalpel and cultured on modified Provassoli’s enriched seawater (PES) solid medium with different concentrations of UIZ (Macklin, Guangzhou, China). The tissue was cultivated at a temperature of 19°C, with a light intensity of 60 μmol photons·m^-2 ·s^-1 and L:D = 12 h:12 h. The solid medium was changed every month to prevent browning of the tissue (Kerrison et al., 2015; Uji et al., 2015; Muhamad et al., 2018; Xu et al., 2022a).

The experiment comprised of two phases. During the first phase, S. fusiforme explants were cultured in solid medium containing UIZ. A cross-design was used to divide the culture samples into 12 groups based on the source site of the explants (proximal stem tip, proximal stem base, and filamentous holdfasts) and concentration of UIZ (0, 3, 5, and 7 μM). The culture samples were exposed to a light intensity of 90 μmol photons ·m^-2 ·s^-1. During the second stage of culture, the S. fusiforme segments were transferred into modified PES liquid medium on days 14 (first batch), 17 (second batch), 20 (third batch), and 52 (fourth batch) after the first stage of culture. The callus-like/adventitious bud induction rate, callus-like/adventitious bud numbers per explant, relative growth rate (RGR), photosynthetic activity and morphology of S. fusiforme tissue culture were recorded. The liquid PES medium was replaced every three days during incubation.

Morphological changes in S. fusiforme explants were documented using a Sony camera (SONY a6000, Tokyo, Japan) after each medium change. The numbers of callus-like or adventitious buds in each explant segment were recorded. The morphology and cellular structure of the characteristic explants were observed at every 3 days using a stereomicroscope (Leica M80, Wetzlar, Germany) and an orthostatic microscope (Leica DM2700 P, Wetzlar, Germany).

The callus-like/adventitious bud induction rate and callus-like/adventitious bud numbers per explant of S. fusiforme were counted every 3 days during the incubation period. The specific formulas were as follows:

The RGR was calculated by determining the weight of the explants and regenerated juveniles at the time of medium change, using the following formula:

where W₀ is the initial weight of the explant (g), W_t is the weight of the explant and regenerated juveniles on day t (g), and t is the number of culture (days).

The physiological state of regenerating S. fusiforme juveniles was determined by measuring their maximum quantum yield (Fv/Fm) and maximum relative electron transfer rate (rETRm) using Junior-Pam (Walz, Effeltrich, Germany). S. fusiforme explants together with regenerated juveniles were dark-adapted for 30 min, and the Fv/Fm and rETRm in the range of 0 – 820 μmol photons m^-2 ·s^-1 were determined, where Fv = Fm-F₀, Fv is the variable fluorescence, Fm is the maximum fluorescence after dark-adaptation, and F₀ is the minimum fluorescence after dark-adaptation. The rapid light curve was fitted according to the formula described by Platt et al. (1980):

The soluble carbohydrate (SC) content was determined using the anthrone method developed by Ershadi et al. (2015). Approximately 0.1 g of tissue culture from S. fusiforme juveniles was crushed in liquid nitrogen; 1 mL of distilled water was added during the crushing process, and then 2 mL of MgCO₃ suspension was added. The samples were fixed to 10 mL with distilled water and centrifuged in a freezing centrifuge at 10277×g for 10 min. The supernatant (1 mL) was transferred into a test tube and mixed with 2 mL of distilled water, followed by addition of 10 mL of anthrone reagent. The mixture was shaken continuously until gas escaped and then heated in a boiling water bath at 100°C for 5 min. After the mixture cooled, the absorbance was measured at 620 nm using a UV spectrophotometer with distilled water as a reference. The SC content was calculated from the standard curve of absorbance values(mg·g^-1).

The soluble protein (SP) content was determined using Coomassie Brilliant Blue G-250 dye as described by Bradford (1976). Approximately 0.1 g of S. fusiforme juveniles was ground in liquid nitrogen in a mortar, suspended in 10 mL 0.1 mol/L phosphate buffer (pH = 6.8) and centrifuged at 10277×g for 10 min for 10 min at 4°C in an ultra-high-speed freezer centrifuge. The supernatant (1 mL) was collected and mixed with 4 mL of Coomassie Brilliant blue G-250 staining solution. The mixture was allowed stand for 3 min at 25°C and the absorbance measured at 595 nm. For the control group, 1 mL of PBS was mixed with 4 mL of Coomassie Brilliant Blue G-250 staining solution. The mixture was incubated at 25°C for 3 min before absorbance was recorded using a UV spectrophotometer. The SP content was calculated using a bovine serum protein standard curve (mg·g^-1).

The mannitol content was determined as described by Chen et al. (2020). A dried juvenile S. fusiforme sample (0.1 g) was collected from a tissue culture source. The sample was milled using liquid nitrogen, and 30mL of HCl with a mass fraction of 1% was added. After the mixture was stirred for 4 min in a 100°C water bath, the supernatant was collected and diluted 100 times. The diluted solution (1 mL) was added to a centrifuge tube. Sodium periodate (1mL) of was then added, mixed well, then it was placed at 25°C for 15 min. 2mL of 0.1% rhamnose was added, mixed thoroughly, and left to wait for 1 min. Nash reagent (4.0 mL) was added and mixed thoroughly. The mixture was heated in a constant temperature water bath at 53°C for 15 min and cooled to room temperature after the mixture changed color. Finally, the absorbance of mannitol was measured at 413 nm using distilled water as a blank. The standard curve of mannitol was drawn using the absorbance a value as the vertical coordinate and the concentration as the horizontal coordinate(mg·g^-1).

All data were preliminarily processed using Excel (Microsoft, Redmond, WA, USA) and subjected to analysis of variance chi-square (F-test), one-way analysis of variance, and two-way analysis of variance using SPSS 25.0 software (SPSS, Inc., Chicago, IL, USA), with the significance level set at P<0.05. Graphs were drawn using origin 2023 (OriginLab, Northampton, MA, USA), and experimental results are expressed as the mean ± standard deviation.

Figure 1 shows the morphological changes that occurred during the regeneration of different parts of S. fusiforme on days 34 and 62 under various batch and UIZ concentration treatments. When using a shorter time to induce callus-like tissues/adventitious buds on solid medium, and higher concentrations of UIZ, S. fusiforme was less likely to germinate and sprout compared with low concentration group. In the first bath culture, regenerating buds were induced from S. fusiforme filamentous holdfasts, and a few stem tips were produced; production occurred only under conditions of low UIZ content in the medium (0, 3, and 5 UIZ). As the induction time in solid medium was increased, S. fusiforme filamentous holdfasts, stem tips and stem bases were induced with different degrees of callus-like tissues or adventitious buds. The most pronounced morphological changes in S. fusiforme explants were observed in the 3 μM UIZ-cultured filamentous holdfasts, in which a large number of leaves, cylindrical protuberances and a few holdfasts were induced, whereas only cylindrical protuberances were induced at the stem tip and stem base. Most filamentous holdfasts and a few stem tips appeared as callus-like tissues/adventitious buds in the first stage of culture for approximately 9 days, whereas the stem base appeared after at least 30 days of cultivation in solid medium. Furthermore, in this method, adventitious buds continued to regenerate into gas vesicles or leaves after isolation ( Figure 1 ). After initial morphological optimization screening, we cultured the tissue-derived 3 μM UIZ-activated S. fusiforme germlines for a longer period and continued this culture until the next breeding season ( Figure 2 ).

Changes in the explant cells were periodically observed during the culture period using a stereomicroscope and a Leica microscope. Explants treated with UIZ exhibited four distinct morphological structures, whereas regenerating buds formed directly from explants that were not treated with UIZ. The four types involves irregularly structured fragile mulberry embryo-like tissue ( Figures 3A–C ), hard spherical protuberances ( Figures 3D–F ), a colorless or light-yellow filamentous texture ( Figures 3G–I ), and direct regeneration of adventitious buds through the tips or cuts of the S. fusiforme filamentous holdfasts ( Figures 3J–L ). Depending on the site of explant germination and its structure, callus-like tissue can form a hyaline or yellowish bulge or callus through the medullary region at the center of the S. fusiforme holdfast ( Figure 3C ), or regenerative tissue can form through epidermal cells ( Figures 3E, F ).

The cross-sectional structure of S. fusiforme holdfast cells was arranged from outside to inside as follows: epidermis, subepidermis, exodermis, endodermis, and medulla in the middle. The epidermal cells were small, whereas the cortical cells increased in size from the outermost to the innermost layer. Medullary cells were the smallest, and the intercellular boundaries were well-defined ( Figure 4 ). The outer epidermal cells of S. fusiforme holdfasts showed a polygonal outline and were darkest in color, as they are rich in pigments and starch grains. The pith, which has denser cells and fewer starch grains than other regions of S. fusiforme, exhibited an intermediate morphology. In contrast, the medullary zone of S. fusiforme adventitious buds were not necessarily located at the mid-end of the morphology. Morphogenesis was induced in the epidermis and subepidermis of earlier tissues ( Figures 4B, D, F ). The diameter of the nascent buds cells was smaller than that of the filamentous holdfasts of mature S. fusiforme ( Figure 4 ).

In both stages of culture, the callus-like/adventitious bud induction rate and callus-like/adventitious buds numbers per explant were significantly affected by the UIZ concentration at the S. fusiforme explant site (p< 0.05, Figure 5 ). Addition of low concentrations of UIZ significantly increased the callus-like/adventitious bud numbers per explant and induction rate of S. fusiforme near the stem tips. The induction rate of filamentous holdfasts treated with 3 μM UIZ was 100%. Additionally, 27.43 ± 4.58 callus-like/adventitious buds were produced per explant segment (p< 0.05, Figure 5 ). The number of callus-like or adventitious buds per explant was higher in all parts of the explant under the 3 μM UIZ treatment condition compared to that observed under other UIZ concentrations ( Figure 6B ). Furthermore, there was a significant difference in the number of callus-like/adventitious bud per explant between the first batch of the earliest liquid medium transfer and the second, third, and fourth batches of the culture(p< 0.05, Figure 6D ).This result suggests that a minimum of 17 days of cultivation in solid medium is necessary. Batch 4 produced 7.19 ± 3.13 adventitious shoots/callus-like tissues per branch segment (p< 0.05, Figure 6D ). Batch 4 as the group treated with 3 μM UIZ and with the longest solid induction time showed the highest germination rate, indicating that solid medium was beneficial for inducing adventitious buds/callus-like tissues ( Figure 6 ).

Changes in the length of S. fusiforme were significantly affected by different concentrations of UIZ ( Figure 7 ). The greatest effect was observed at 3 μM UIZ (p< 0.001, Figure 7B ). The length of regenerated juveniles varied among the different explant sites of S. fusiforme. Additionally, changes in S. fusiforme’s filamentous holdfasts were greater than those in the stem tips (p< 0.05). The 3 μM UIZ treated filamentous holdfasts provided lengths of up to 3.43 ± 0.39 cm (p< 0.05).

We observed significant differences in the RGR of S. fusiforme based on variations in the culture site, UIZ concentration, and transfer medium (p< 0.05, Figure 8 ). A longer induction time in solid culture resulted in a higher RGR. However, it is necessary to culture the sample in liquid medium for 17days to achieve proliferative culture ( Figure 8 ). Significant variations in the RGR were observed among different parts of S. fusiforme explants. Filamentous holdfasts exhibited the highest RGR of approximately 3.05% when cultured in 3μM UIZ (p< 0.05). The RGR trend in batch 4 of cultured S. fusiforme explants differed from that of the first three batches because the last batch had a long induction time in solid medium and insufficient time for the proliferation of regenerating buds in liquid medium ( Figure 9 ).

Different batches, explant locations, and UIZ concentrations played influenced the Fv/Fm, rETRm and fast light curves of S. fusiforme juveniles. The effect of the S. fusiforme explant site and UIZ concentration in culture batches 1 and 2 did not significantly affect Fv/Fm (P > 0.05, Figure 10A ), whereas the effect on rETRm was significant up to a maximum of 39.82 ± 3.86 (P< 0.05, Figures 10D, E ). The Fv/Fm, rETRm and rapid light curve disparity of S. fusiforme varied significantly in batch 4 according to the explant location and UIZ concentration, with the lowest Fv/Fm was 0.356 ± 0.04 and lowest rETRm was 8.98 ± 0.79 at the stem tip of S. fusiforme in the presence of UIZ compared to in the absence of UIZ (P< 0.05, Figure 10 ).

During the culture process, the concentration of UIZ significantly affected the SC and SP, which increased with increasing UIZ concentrations, The SC contents in the 3, 5 and 7 μM UIZ treatment groups were 105%, 115% and 163% of the control, respectively, with the highest SC content observed in culture containing 7 μM UIZ (P< 0.05, Figure 11A ). The SP contents in the 3, 5, and 7 μM UIZ groups were 124%, 179%, and 276.2% of the control, respectively. The SP content was significantly affected by 5 and 7 μM UIZ treatment (P< 0.05, Figure 11B ). As the concentration of UIZ was increased, the mannitol content was 59%, 65%, and 70% of the control. Exogenously added UIZ significantly reduced the mannitol content compared to that in the control group. However, there were no significant differences among the samples treated with different concentrations of UIZ. The most significant change in the mannitol content of S. fusiforme tissue culture-derived juveniles was observed under the treatment with 3 μM UIZ (P< 0.05, Figure 11C ).