Construction of Optimal Regeneration System for Chrysanthemum ‘11-C-2’ Stem Segment with Buds

Chrysanthemum morifolium ‘11-C-2’ is a variety of chrysanthemums with high ornamental and tea value, experiencing significant market demand. However, as cultivation areas expand, issues such as viral infection, germplasm degradation, low proliferation coefficient, and slow proliferation rate arise, necessitating the establishment of an efficient in vitro regeneration system. This study, based on the principles of orthogonal experimental design, explored the regeneration system of Chrysanthemum cultivar ‘11-C-2’ using sterile seedlings. The research focused on three key stages: adventitious bud differentiation, rooting culture, and acclimatization–transplantation, employing shoot-bearing stem segments and leaves as explants. The findings indicate that the optimal explant for the Chrysanthemum ‘11-C-2’ sterile seedlings is the shoot-bearing stem segment. The best medium for adventitious bud differentiation was determined to be MS supplemented with 1.5 mg/L 6-BA and 0.5 mg/L NAA. Bud differentiation began on day 17 with a 100% differentiation rate, completing around day 48. The maximum differentiation coefficient reached 87, with an average of 26.67. The adventitious buds were then cultured for rooting in the optimal medium of 1/2 MS supplemented with 0.1 mg/L NAA. Rooting was initiated on day 4 and was completed by day 14, achieving a rooting rate of 97.62%. After a 5-day acclimatization under natural light, the rooted seedlings were transplanted into a growth substrate with a peat-to-vermiculite ratio of 1:2. The plants exhibited optimal growth, with a transplantation survival rate of 100%. The findings provide data support for the efficient large-scale propagation of ‘11-C-2’ and lay the foundation for germplasm preservation and genetic transformation research of tea chrysanthemums.

This study employs orthogonal experimental design principles using bud-carrying stem segments and leaves of tea chrysanthemum '11-C-2' as explants.It investigates the effects of different ratios of four plant growth hormones-6-BA, NAA, 2,4-D, and TDZ-on the proliferation and regeneration of '11-C-2'.The study identifies the optimal rooting medium, explores the best acclimatization period and transplantation substrate for '11-C-2', and provides a theoretical basis for its large-scale rapid propagation and germplasm preservation.It also lays the groundwork for genetic transformation and molecular mechanism research related to tea chrysanthemums.

Optimal Medium for Adventitious Bud Differentiation of '11-C-2'
According to the R and k values of the differentiation coefficient in Appendix A. Table A1, the primary factors influencing the differentiation coefficient of bud-carrying stem segments of '11-C-2' are 6-BA concentration > 2,4-D concentration > NAA concentration > TDZ concentration.The optimal levels for these factors are ranked as A2 > A3 > A1, B3 > B1 > B2, C1 > C2 > C3, D1 > D2 > D3.The effects of different levels of these factors on the differentiation coefficient of '11-C-2' shoot segments are highly significant (p < 0.001).The optimal combination of factors in the experiments for influencing the differentiation coefficient is A2B3C1D2 Table 1, which corresponds to MS + 1.5 mg/L 6-BA + 0.5 mg/L Plants 2024, 13, 2403 3 of 21 NAA + 0.1 mg/L TDZ.However, the range analysis shows the optimal combination as A2B3C1D1, which corresponds to MS + 1.5 mg/L 6-BA + 0.5 mg/L NAA.The differentiation coefficients were compared by Duncan method, with results expressed as "mean ± standard deviation".Different lowercase letters indicate highly significant differences (p < 0.001), but there was no significant difference among the variation at 5% of One-way ANOVA in leaf differentiation coefficient (p = 0.454).The total number of explants per experimental treatment was N = 30.
For the leaves of '11-C-2', the primary factors influencing the differentiation coefficient are NAA concentration > 6-BA concentration > TDZ concentration > 2,4-D concentration.The optimal levels for these factors are ranked as A2 > A1 > A3, B1 > B2 > B3, C1 > C2 > C3, D1 = D2 > D3.However, the effects of these factors at different levels on the differentiation coefficient of '11-C-2' leaves are not significant (p = 0.454).The optimal combination of factors in the experiments for influencing the differentiation coefficient is A1B1C1D1 Table 1, which corresponds to MS + 1.0 mg/L 6-BA + 0.1 mg/L NAA.However, the range analysis shows the optimal combination as A2B1C1D1 or A2B1C1D2.To reduce hormone usage, TDZ is set at the first level of 0 mg/L, corresponding to MS + 1.5 mg/L 6-BA + 0.1 mg/L NAA.The optimal combinations for both bud-carrying stem segments and leaves are not included in the nine treatments listed in Table 1.
According to Table 2, the concentrations of 6-BA, NAA, 2,4-D, and TDZ have a highly significant impact on the differentiation coefficient of bud-carrying stem segments of '11-C-2' (p < 0.001).The analysis of variance results is consistent with the range analysis results, indicating that the optimal medium for adventitious bud differentiation for '11-C-2' bud-carrying stem segments is MS + 1.5 mg/L 6-BA + 0.5 mg/L NAA.However, the concentrations of these four hormones do not show significant differences in their impact on the differentiation coefficient of '11-C-2' leaves (p > 0.05), making it impossible to determine the optimal hormone ratio for the culture medium.According to Appendix A. Table A2, there is a highly significant interaction effect between the concentrations of 6-BA and NAA on the differentiation coefficient of budcarrying stem segments.When the NAA concentration is 0.1 mg/L, the differentiation coefficient decreases as the 6-BA concentration increases.However, when the NAA concentration is 0.3 mg/L or 0.5 mg/L, the differentiation coefficient first increases and then decreases with increasing 6-BA concentration, peaking at 1.5 mg/L 6-BA Figure 1A.When the 6-BA concentration is 1.0 mg/L or 1.5 mg/L, the differentiation coefficient first decreases and then increases with increasing NAA concentration.At 1.5 mg/L 6-BA, the differentiation coefficient increases with increasing NAA concentration, peaking at 0.5 mg/L NAA Figure 1B.However, low concentrations of NAA or high concentrations of 6-BA are not suitable for adventitious bud differentiation in bud-carrying stem segments.According to Appendix A. Table A2, there is a highly significant interaction effect between the concentrations of 6-BA and NAA on the differentiation coefficient of budcarrying stem segments.When the NAA concentration is 0.1 mg/L, the differentiation coefficient decreases as the 6-BA concentration increases.However, when the NAA concentration is 0.3 mg/L or 0.5 mg/L, the differentiation coefficient first increases and then decreases with increasing 6-BA concentration, peaking at 1.5 mg/L 6-BA Figure 1A.When the 6-BA concentration is 1.0 mg/L or 1.5 mg/L, the differentiation coefficient first decreases and then increases with increasing NAA concentration.At 1.5 mg/L 6-BA, the differentiation coefficient increases with increasing NAA concentration, peaking at 0.5 mg/L NAA Figure 1B.However, low concentrations of NAA or high concentrations of 6-BA are not suitable for adventitious bud differentiation in bud-carrying stem segments.In summary, the optimal explant for '11-C-2' is the budded stem segment.Validation of the optimal combination identified in the analysis revealed that when this explant is cultured on the optimal medium for adventitious bud differentiation, which is MS + 1.5 mg/L 6-BA + 0.5 mg/L NAA (combination A2B3C1D1).Callus formation occurs at the cut ends of the stem segments within 9 days.The callus then differentiates into adventitious buds within 17 days.Additionally, axillary buds on the stem segments can directly develop into adventitious buds within 12 days, with a differentiation rate of 100% Figure 2A.Complete differentiation of adventitious buds is achieved within 48 days Figure 2B, with an average differentiation coefficient of 26.67 and a maximum of 87 Figure 2C, significantly higher than combination A2B3C1D2, and the buds show vigorous growth.In summary, the optimal explant for '11-C-2' is the budded stem segment.Validation of the optimal combination identified in the analysis revealed that when this explant is cultured on the optimal medium for adventitious bud differentiation, which is MS + 1.5 mg/L 6-BA + 0.5 mg/L NAA (combination A2B3C1D1).Callus formation occurs at the cut ends of the stem segments within 9 days.The callus then differentiates into adventitious buds within 17 days.Additionally, axillary buds on the stem segments can directly develop into adventitious buds within 12 days, with a differentiation rate of 100% Figure 2A.Complete differentiation of adventitious buds is achieved within 48 days Figure 2B, with an average differentiation coefficient of 26.67 and a maximum of 87 Figure 2C, significantly higher than combination A2B3C1D2, and the buds show vigorous growth.

Optimal Rooting Medium for '11-C-2'
Based on Table 3 and Appendix A. Table A1, the main factors affecting the number of

Optimal Rooting Medium for '11-C-2'
Based on Table 3 and Appendix A. Table A1, the main factors affecting the number of roots and root length in '11-C-2' plantlets are the NAA concentration and the basal medium, with NAA concentration being more significant.The optimal levels for these factors are in the order of A2 > A3 > A1 and B1 > B2 > B3.The effect of different levels of these factors on root number and length is highly significant (p < 0.001).The primary factors affecting the rooting rate of '11-C-2' plantlets are also the NAA concentration and the basal medium, with NAA concentration being more influential.However, the optimal levels for rooting rate are in the order of A3 > A2 > A1 and B1 > B2 > B3.The variation in these factors' levels does not significantly impact the rooting rate (p = 0.943).Therefore, the optimal combination for promoting root development in '11-C-2' plantlets is A2B1, which corresponds to 1/2 MS medium supplemented with 0.1 mg/L NAA.This optimal combination is included in the 9 treatments listed in Table 3.The rooting rate, root number, and root length were compared by Duncan method, with results expressed as "mean ± standard deviation".Differences indicated by different lowercase letters are highly significant (p < 0.001), but there was no significant difference among the variation at 5% of One-way ANOVA in rooting rate (p = 0.943).
Each experimental treatment involved a total of N = 54 plantlets.
According to Table 4, the base medium and NAA concentration have a highly significant effect on the root number and root length of '11-C-2' (p < 0.001), but no significant effect on the rooting rate (p > 0.05).The results of the variance analysis are consistent with the range analysis results, indicating that the optimal rooting medium for '11-C-2' is 1/2 MS + 0.1 mg/L NAA.According to Appendix A. Table A2, there is a highly significant interaction effect between the base medium and NAA concentration on the changes in root number and root length of rooting seedlings.When the NAA concentration is constant, varying the base medium results in a trend where root number and length first increase and then decrease, with the highest root number and length observed with 1/2 MS as the base medium.Conversely, when the base medium is constant, increasing the NAA concentration leads to a decreasing trend in both root number and length, with the maximum root number and length observed at an NAA concentration of 0.1 mg/L Figure 3.
Rooting rate R2 = 0.111 (adjusted R2 = −0.051);Root number R2 = 0.912 (adjusted R2 = 0.896); Ro length R2 = 0.907 (adjusted R2 = 0.890); "***" indicates highly significant difference at the 0.001 lev According to Appendix A. Table A2, there is a highly significant interaction effe between the base medium and NAA concentration on the changes in root number an root length of rooting seedlings.When the NAA concentration is constant, varying th base medium results in a trend where root number and length first increase and then d crease, with the highest root number and length observed with 1/2 MS as the base m dium.Conversely, when the base medium is constant, increasing the NAA concentratio leads to a decreasing trend in both root number and length, with the maximum root num ber and length observed at an NAA concentration of 0.1 mg/L Figure 3.In summary, the optimal rooting medium for '11-C-2' in the experimental treatments is 1/2 MS + 0.1 mg/L NAA.The adventitious buds of '11-C-2' are capable of normal rooting across all nine media tested in Figure 4. Comparative analysis of rooting morphology for representative seedlings from each treatment Figure 5 shows that the optimal treatment has superior root length, root number, and plant condition compared to others.This indicates that a small amount of NAA added to a suitable base medium can promote rooting in '11-C-2', resulting in healthy seedlings, whereas excessive NAA inhibits rooting and slows seedling growth.across all nine media tested in Figure 4. Comparative analysis of rooting morphology for representative seedlings from each treatment Figure 5 shows that the optimal treatment has superior root length, root number, and plant condition compared to others.This indicates that a small amount of NAA added to a suitable base medium can promote rooting in '11-C-2', resulting in healthy seedlings, whereas excessive NAA inhibits rooting and slows seedling growth.is 1/2 MS + 0.1 mg/L NAA.The adventitious buds of '11-C-2' are capable of normal rooting across all nine media tested in Figure 4. Comparative analysis of rooting morphology for representative seedlings from each treatment Figure 5 shows that the optimal treatment has superior root length, root number, and plant condition compared to others.This indicates that a small amount of NAA added to a suitable base medium can promote rooting in '11-C-2', resulting in healthy seedlings, whereas excessive NAA inhibits rooting and slows seedling growth.

Optimal Hardening Time and Transplant Medium for '11-C-2'
According to Appendix A. Table A1, the main factors affecting the height of '11-C-2' plants are hardening time > transplant medium (0-10 d) and transplant medium > hardening time (10-15 d).The optimal levels for these factors are ranked as A3 > A2 > A1 (0-15 d), B3 > B2 > B1 (0 d), and B2 > B3 > B1 (5-15 d).However, the differences in plant height at 0 days are not significant (p = 0.198).Therefore, the optimal combination for influencing the height of '11-C-2' plants in the experimental treatments is A3B2, meaning transplanting into a medium with a 1:2 ratio of peat to perlite after 5 days of hardening results in the best growth.This optimal combination is included in the nine treatments listed in Table 5.The plant heights were compared by Duncan method, with results expressed as "mean ± standard deviation".Different lowercase letters indicate statistically significant differences (p < 0.001).Each experimental treatment involved a total of N = 30 rooted plantlets.
According to Table 6, there are no significant differences in plant height (0 days) due to hardening time and transplant medium.However, these factors have highly significant differences in plant height (5 days) (p < 0.001) and very significant differences (p < 0.01) in plant height (10 days) and (15 days) (p < 0.001).The results of the variance analysis are consistent with those of the range analysis, indicating that the optimal hardening time for '11-C-2' is 5 days and the optimal transplant medium is a 1:2 ratio of peat to perlite.According to Appendix A. Table A2, there is a highly significant interaction effect between hardening time and transplant medium on plant height changes.When the transplant medium ratio is constant, the height of '11-C-2' increases continuously from 0 to 15 days, with the tallest plants observed after 5 days of hardening.Conversely, when the hardening time is constant, varying the transplant medium ratio results in a trend where plant height first increases and then decreases from 0 to 15 days, with the tallest plants observed at a peat-to-perlite ratio of 1:2 in Figure 6.
In summary, the optimal hardening time for '11-C-2' in the experimental treatments is 5 days, and the best transplant medium is a 1:2 ratio of peat to perlite.Rooted '11-C-2' seedlings gradually acclimate to the external environment by slowly increasing the opening of the culture bottles under natural light.Both hardening and transplant survival rates can reach 100%, with the transplantation process completed in about 15 days Figure 7.During the early transplant period (0-10 d), longer hardening times enhance '11-C-2' growth, while in the later period (10-15 d), the transplant medium ratio primarily affects the growth of '11-C-2'.
days, with the tallest plants observed after 5 days of hardening.Conversely, when t hardening time is constant, varying the transplant medium ratio results in a trend whe plant height first increases and then decreases from 0 to 15 days, with the tallest plan observed at a peat-to-perlite ratio of 1:2 in Figure 6.In summary, the optimal hardening time for '11-C-2' in the experimental treatmen is 5 days, and the best transplant medium is a 1:2 ratio of peat to perlite.Rooted '11-C seedlings gradually acclimate to the external environment by slowly increasing the ope ing of the culture bottles under natural light.Both hardening and transplant survival ra can reach 100%, with the transplantation process completed in about 15 days Figure 7.During the early transplant period (0-10 d), longer hardening times enhance '11-C-2' growth, while in the later period (10-15 d), the transplant medium ratio primarily affects the growth of '11-C-2'.

Establishment of '11-C-2' Regeneration System with Sprouted Stem Segments
Based on the results from the stages of shoot regeneration, rooting, and acclimatization, this study has developed an initial regeneration system for tea chrysanthemum '11-C-2' using shoot segments with buds as explants Figure 8.The shoot segments were inoculated onto the optimal medium for adventitious bud differentiation, MS + 1.5 mg/L 6-BA + 0.5 mg/L NAA (Figure 8A).Over 5 days, the shoot segments gradually changed from light green to green.Approximately 2 days later, the basal cut surface of the shoot segments began to swell, and after 1-3 days, a yellow-green callus formed at the base of the swollen segments.After continuing the culture for about 8 days, the callus started to differentiate into adventitious buds.Buds from axillary shoots differentiated directly after 12 days, completing the differentiation process in approximately 48 days Figure 8B, with an average differentiation coefficient of 26.67 and a maximum of 87 Figure 8C.Once the adventitious shoots had reached a length of 2 cm, '11-C-2' adventitious shoots were excised and inoculated in the optimal rooting medium of 1/2 MS + 0.1 mg/L NAA.A concentration

Establishment of '11-C-2' Regeneration System with Sprouted Stem Segments
Based on the results from the stages of shoot regeneration, rooting, and acclimatization, this study has developed an initial regeneration system for tea chrysanthemum '11-C-2' using shoot segments with buds as explants Figure 8.The shoot segments were inoculated onto the optimal medium for adventitious bud differentiation, MS + 1.5 mg/L 6-BA + 0.5 mg/L NAA (Figure 8A).Over 5 days, the shoot segments gradually changed from light green to green.Approximately 2 days later, the basal cut surface of the shoot segments began to swell, and after 1-3 days, a yellow-green callus formed at the base of the swollen segments.After continuing the culture for about 8 days, the callus started to differentiate into adventitious buds.Buds from axillary shoots differentiated directly after 12 days, completing the differentiation process in approximately 48 days Figure 8B, with an average differentiation coefficient of 26.67 and a maximum of 87 Figure 8C.Once the adventitious shoots had reached a length of 2 cm, '11-C-2' adventitious shoots were excised and inoculated in the optimal rooting medium of 1/2 MS + 0.1 mg/L NAA.A concentration of 1 mg/L NAA was employed, and the adventitious shoots began to root successively within four days.The root system was stout and dense in general, with most of the roots exhibiting a yellowish hue and a few displaying a white coloration.The rooting rate was 97.62%, and the rooting culture could be completed within 14 days Figure 8D.The rooted seedlings exhibited a good growth condition in general Figure 8E.Subsequently, the rooted seedlings were gradually opened under natural light for a period of five days.Following this, they were transplanted into the growth substrate, with a mass ratio of charcoal:vermiculite of 1:2.The plants exhibited robust growth, and the survival rate of the transplanting reached 100% in Figure 8F.
of 1 mg/L NAA was employed, and the adventitious shoots began to root successively within four days.The root system was stout and dense in general, with most of the roots exhibiting a yellowish hue and a few displaying a white coloration.The rooting rate was 97.62%, and the rooting culture could be completed within 14 days Figure 8D.The rooted seedlings exhibited a good growth condition in general Figure 8E.Subsequently, the rooted seedlings were gradually opened under natural light for a period of five days.Following this, they were transplanted into the growth substrate, with a mass ratio of charcoal:vermiculite of 1:2.The plants exhibited robust growth, and the survival rate of the transplanting reached 100% in Figure 8F.

Discussion
Plant tissue culture is a crucial technique for conserving endangered species and maintaining plant quality, but it is influenced by various factors such as explant type, medium composition, and plant hormone formulations.The choice of explant is fundamental in plant regeneration.Different explants from the same plant, depending on their growth stage and physiological characteristics, exhibit variations in callus induction, regeneration rate, and regeneration coefficient [60,78,79].For chrysanthemums, explants such as petals, petioles, axillary buds, hypocotyls, and shoot tips can be used [80][81][82][83][84][85].However, shoot segments and leaves are widely used due to their ease of collection and abundance [86,87].In this study, we found that the basal cut of shoot segments with buds form calluses and differentiate into adventitious buds, while axillary buds on the shoot segments directly differentiate into adventitious buds, resulting in a shorter differentiation time and higher coefficient.In contrast, leaf cuttings take longer to form callus and differentiate into adventitious buds, with a lower differentiation coefficient, consistent with previous studies [88,89].
In tissue culture, the medium contains specific ratios of cytokinins and auxins.High concentrations of cytokinins promote adventitious bud formation, high concentrations of auxins favor root formation, and a specific balance of both hormones is optimal for callus formation [79,90].For chrysanthemum species, MS medium is commonly used for adventitious bud differentiation [91], often supplemented with hormones like 6-BA, NAA, KT,

Discussion
Plant tissue culture is a crucial technique for conserving endangered species and maintaining plant quality, but it is influenced by various factors such as explant type, medium composition, and plant hormone formulations.The choice of explant is fundamental in plant regeneration.Different explants from the same plant, depending on their growth stage and physiological characteristics, exhibit variations in callus induction, regeneration rate, and regeneration coefficient [60,78,79].For chrysanthemums, explants such as petals, petioles, axillary buds, hypocotyls, and shoot tips can be used [80][81][82][83][84][85].However, shoot segments and leaves are widely used due to their ease of collection and abundance [86,87].In this study, we found that the basal cut of shoot segments with buds form calluses and differentiate into adventitious buds, while axillary buds on the shoot segments directly differentiate into adventitious buds, resulting in a shorter differentiation time and higher coefficient.In contrast, leaf cuttings take longer to form callus and differentiate into adventitious buds, with a lower differentiation coefficient, consistent with previous studies [88,89].
In tissue culture, the medium contains specific ratios of cytokinins and auxins.High concentrations of cytokinins promote adventitious bud formation, high concentrations of auxins favor root formation, and a specific balance of both hormones is optimal for callus formation [79,90].For chrysanthemum species, MS medium is commonly used for adventitious bud differentiation [91], often supplemented with hormones like 6-BA, NAA, KT, or TDZ to determine the best hormone combination by adjusting the ratio and types of auxins and cytokinins [88,92,93].Established regeneration systems for chrysanthemums typically use 6-BA and NAA to induce adventitious buds [36,[94][95][96].This study found that high concentrations of 6-BA and low concentrations of NAA are not suitable for callus induction and bud differentiation in '11-C-2', consistent with findings that high 6-BA concentrations inhibit bud differentiation in other species like chrysanthemum and Helenium aromaticum [57,93].Additionally, 2,4-D and TDZ are unsuitable for '11-C-2' callus induction and bud differentiation, contrary to results reported by Luo Hong [57].The differentiation times, bud differentiation rates, and differentiation coefficients also vary among different chrysanthemum varieties [34,97].In this study, bud-bearing stem segments of "11-C-2" can differentiate into adventitious buds within 17 days, with a differentiation rate of 100% and a coefficient of 26.7.However, leaf segments take longer to differentiate into adventitious buds, with a differentiation coefficient of only 2. This indicates that, compared to leaf segments, stem segments in some chrysanthemum varieties have a shorter differentiation time and a higher coefficient, making them more suitable as explants.
In chrysanthemum root culture, a common approach is to use 1/2 MS basic medium with low concentrations of NAA to stimulate root growth [87,98,99].However, either omitting NAA or using high concentrations of NAA can also promote root formation in chrysanthemums [57,81].Other hormones like IBA and IAA are also used and can achieve good rooting results [100,101].This study found that a small amount of NAA promotes rooting in '11-C-2' adventitious buds, while either omitting NAA or using excessively high concentrations negatively affects root length, number, and plant growth.This finding is consistent with previous research and suggests that while the optimal rooting medium varies among most chrysanthemum species, some share similar rooting media.
After removing chrysanthemum-rooted seedlings from culture bottles, changes in the growth environment can significantly impact their survival rate.Therefore, acclimation is necessary before transplanting.Studies have shown that seedlings have the highest survival rate after 3 days of acclimation [102], with survival rates decreasing if the duration is shorter or longer [103].In this study, '11-C-2' seedlings achieved a 100% survival rate after 5 days of acclimation, indicating strong adaptability and stress resistance.Suitable acclimation substrates include vermiculite, perlite, and decomposed straw [104], or a peat and coconut coir mix in equal parts [105].By adjusting the ratio of vermiculite to peat, this study found that a 1:2 ratio of peat to vermiculite provided the best growth conditions for the seedlings, with a 100% survival rate.This suggests that seedlings can adapt more quickly to external conditions when grown on a substrate with appropriate aeration and water retention.
In summary, this study demonstrates that shoot tip segments of '11-C-2' are the most suitable explants for adventitious bud differentiation.Using appropriate concentrations of 6-BA and NAA, successful differentiation is achieved.The effects of these hormones on bud differentiation have been clearly defined.Additionally, the study identified the optimal rooting medium from various combinations of basic media and NAA concentrations, enhancing our understanding of the role of media, hormone types, and concentrations in plant tissue culture.Based on orthogonal experiments, the optimal acclimatization duration and transplantation substrate were efficiently determined, adding valuable insights into the acclimatization and transplantation stages for chrysanthemum species.The developed high-efficiency propagation system for '11-C-2' using shoot tip segments integrates results from all three stages.Future work should focus on optimizing this system to improve its stability and efficiency, laying the groundwork for developing genetic transformation systems and gene function validation using '11-C-2'.Furthermore, plant tissue culture can remove certain chrysanthemum viruses, and this study provides a foundation for establishing efficient virus-free seedling propagation techniques for tea chrysanthemum.

Experimental Material
The Chrysanthemum '11-C-2' material used in this experiment was autonomously bred by the Laboratory of Forage and Ornamental Horticulture, Beijing Academy of Agriculture and Forestry Sciences as shown in Figure 9.

Experimental Material
The Chrysanthemum '11-C-2' material used in this experiment was autonomously bred by the Laboratory of Forage and Ornamental Horticulture, Beijing Academy of Agriculture and Forestry Sciences as shown in Figure 9.

Aseptic Seedling Culture
Select healthy '11-C-2' chrysanthemum plants in the vegetative growth stage and cut the stems into segments with two axillary buds each Figure 9C.Immerse the segments in a 3% detergent solution for 15 min, followed by shaking on a shaker for 10 min.Rinse the segments three times with sterile water, then quickly disinfect with 75% alcohol, followed by three more rinses with sterile water.Next, sterilize the segments in a 5% sodium hypochlorite solution for 15 min and rinse them three times with sterile water.Place the sterilized segments on sterile absorbent paper in a sterile Petri dish.In each 250 mL culture bottle, add 50 mL of MS medium without any hormones and inoculate with five segments.After 25 days of aseptic culture, a large number of '11-C-2' chrysanthemum aseptic seedlings are obtained.Culture conditions: 25 °C temperature, 1500 lx light intensity, and photoperiod (16 h light/8 h dark).

Adventitious Bud Differentiation
Using the sterile seedlings of tea chrysanthemum '11-C-2' as experimental materials, the stems were cut into 1 cm segments containing buds, and the leaves were cut into 0.5 cm × 0.5 cm pieces as explants.After undergoing the previously described disinfection and sterilization process, the explants were inoculated onto MS basal medium supplemented with different concentrations of 6-BA, NAA, 2,4-D, and TDZ.Following the principles of orthogonal experimental design, nine different hormone combinations were formulated for adventitious bud differentiation in Appendix A. Table A5.For each experimental group, five 250 mL culture flasks were used, each containing 50 mL of the medium and inoculated with two explants, with three replicates, resulting in a total sample size of N = 30 per group.During the adventitious bud differentiation culture, the state of the explants was monitored.After 54 days of cultivation, the differentiation coefficient of the adventitious buds for '11-C-2' was recorded, and the optimal hormone combination for the differentiation medium was identified.Since the orthogonal test represents all possible

Adventitious Bud Differentiation
Using the sterile seedlings of tea chrysanthemum '11-C-2' as experimental materials, the stems were cut into 1 cm segments containing buds, and the leaves were cut into 0.5 cm × 0.5 cm pieces as explants.After undergoing the previously described disinfection and sterilization process, the explants were inoculated onto MS basal medium supplemented with different concentrations of 6-BA, NAA, 2,4-D, and TDZ.Following the principles of orthogonal experimental design, nine different hormone combinations were formulated for adventitious bud differentiation in Appendix A. Table A5.For each experimental group, five 250 mL culture flasks were used, each containing 50 mL of the medium and inoculated with two explants, with three replicates, resulting in a total sample size of N = 30 per group.During the adventitious bud differentiation culture, the state of the explants was monitored.After 54 days of cultivation, the differentiation coefficient of the adventitious buds for '11-C-2' was recorded, and the optimal hormone combination for the differentiation medium was identified.Since the orthogonal test represents all possible experimental combinations by selecting a subset of representative combinations, if the optimal hormone combination is not included in the initial test, further experiments are required to validate the selected optimal combination to ensure the accuracy of the experimental results.The cultivation conditions were the same as those described in Section 4.2.1.

Rooting Culture
After 54 days of differentiation culture of '11-C-2' adventitious buds Figure 2B, the resulting buds were grown to a height of 2 cm and had developed two leaves.These buds were then separated into individual units using sterile tweezers Figure 2C and transferred to rooting media containing varying concentrations of NAA on different basal media.In accordance with the orthogonal experimental design, nine different rooting media treatments were established in Appendix A. Table A6.Each treatment consisted of three 250 mL culture flasks, each containing 50 mL of medium and inoculated with six adventitious buds.The experiment was repeated three times, yielding a total sample size of N = 54 per group.During the rooting culture, the growth status of the plants and root development were observed.After 14 days of culture, the rooting rate, root length, and the number of roots of '11-C-2' seedlings were recorded.The cultivation conditions were the same as described in Section 4.2.1.

Acclimatization and Transplantation
Under natural light conditions, the caps of the '11-C-2' rooting bottles are gradually opened, first slightly, then halfway, and finally completely, to acclimatize the plants for different durations.After acclimatization, seedlings are carefully removed from the bottles using sterile tweezers.The roots are washed with water to remove the residual medium, and seedlings measuring 3 cm in height with 5 leaves are treated with 50% Carbendazim solution for 2 min.Then, the seedlings are planted into growth soil with varying ratios of peat moss to vermiculite.According to orthogonal experimental design principles, 9 different treatments are set up see Table A7.Each treatment includes 10 planting pots (6 cm × 6 cm × 8 cm), with one plant per pot, and each treatment is repeated three times, resulting in a total of 30 samples per group.Watering is performed every 5 days with 150 mL, and plant growth is observed.Plant height is measured at 0, 5, 10, and 15 days, and survival rates are calculated at 15 days.Cultivation conditions: temperature 25 • C, humidity 50%, with gradually increasing ventilation during acclimatization.

Data Analysis
In this study, the data were organized using Microsoft Excel, and the experimental data were analyzed by ANOVA and simple effects analysis using the SPSS 27.0 software.The multiple comparisons were conducted using Duncan method, and the results were expressed as "mean ± standard deviation".The following formulas were utilized for calculation: Differentiation rate = (Number of explants producing adventitious buds/total number of uncontaminated explants in the same treatment) × 100%; Differentiation coefficient = (Total number of adventitious buds differentiated/Total number of uncontaminated explants in the same treatment) × 100%; Rooting rate = (Number of rooted plantlets/Total number of uncontaminated plantlets in the same treatment) × 100%; Transplant survival rate = (Number of surviving transplants/Total number of transplants) × 100%.

Conclusions
Based on the results from the stages of adventitious bud differentiation, rooting, and acclimatization, this study successfully established a highly efficient propagation system for tea chrysanthemum '11-C-2' using shoot tip segments as explants.The optimal differentiation medium is MS with 1.5 mg/L 6-BA and 0.5 mg/L NAA, while the best rooting medium is 1/2 MS with 0.1 mg/L NAA.The ideal acclimatization period is 5 days, and the most effective transplantation substrate is a peat ratio of 1:2.This system achieves bud differentiation in 48 days with a 100% differentiation rate and an average coefficient of 26.67, reaching up to 87.Rooting is completed in 14 days with a rooting rate of 97.62% and healthy root development.Acclimatization and transplantation are achieved in 20 days with a 100% survival rate and strong plant growth.This method minimizes environmental impacts, enables year-round production, significantly enhances the propagation efficiency of tea chrysanthemum '11-C-2', and shortens the breeding cycle, providing a robust foundation and data support for its large-scale cultivation and application.

Conflicts of Interest:
Author Junjun Yu was employed by the company China United Engineering Corporation Limited.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The peat used is a product from PINDSTRUO, and the vermiculite is white vermiculite from Xinjiang with a particle size of 1-3 mm.The components of the transplanting soil are measured by weight using a balance, with the soil mixture consisting of peat and vermiculite in a specified ratio.And, they do not need high-temperature sterilization treatment, and can be directly used to simulate the field growing environment.

Figure 1 .
Figure 1.Effect of 6-BA (A) and NAA (B) concentrations on the differentiation coefficient of stem segments with buds.

Figure 1 .
Figure 1.Effect of 6-BA (A) and NAA (B) concentrations on the differentiation coefficient of stem segments with buds.

Plants 2024 , 22 Figure 2 .
Figure 2. Illustrates the adventitious bud differentiation of '11-C-2' stem segments.(A) Callus induction and adventitious bud differentiation process using stem segments as explants (Bar = 2 cm); (B) Completed state of adventitious bud differentiation (Bar = 2 cm); (C) Maximum differentiation coefficient of '11-C-2' stem segments (Bar = 2 cm); (a) Formation of callus at the cut end of the budded stem segment; (b) The callus starts to differentiate into adventitious buds; (c) Axillary buds on the stem segment directly differentiate into adventitious buds.

Figure 3 .
Figure 3. Effects of basal medium and NAA concentration on root length and root number.(A) T effect of basic medium on the number of seedlings; (B) Effect of NAA concentration on the numb

Figure 3 .
Figure 3. Effects of basal medium and NAA concentration on root length and root number.(A) The effect of basic medium on the number of seedlings; (B) Effect of NAA concentration on the number of seedling roots; (C) Effect of base medium on root length of seedling; (D) Effect of NAA concentration on root length of seedling.

Figure 5 .
Figure 5. Morphological appearance of tissue-cultured plantlets after 14 days of rooting under different treatments.Bar = 2 cm.

Figure 5 .
Figure 5. Morphological appearance of tissue-cultured plantlets after 14 days of rooting under different treatments.Bar = 2 cm.

Figure 5 .
Figure 5. Morphological appearance of tissue-cultured plantlets after 14 days of rooting under different treatments.Bar = 2 cm.

Figure 6 .
Figure 6.Effects of seedling refining time and transplanting medium on plant height.(A) Effect seedling refining time on plant height (5 d); (B) Effect of transplanting medium on plant height d); (C) Effect of seedling refining time on plant height (10 d); (D) Effect of transplanting medium plant height (10 d); (E) Effect of seedling refining time on plant height (15 d); (F) Effect of transpla ing medium on plant height (15 d).

Figure 6 .
Figure 6.Effects of seedling refining time and transplanting medium on plant height.(A) Effect of seedling refining time on plant height (5 d); (B) Effect of transplanting medium on plant height (5 d); (C) Effect of seedling refining time on plant height (10 d); (D) Effect of transplanting medium on plant height (10 d); (E) Effect of seedling refining time on plant height (15 d); (F) Effect of transplanting medium on plant height (15 d).

4. 2 .
Experimental Method 4.2.1.Aseptic Seedling Culture Select healthy '11-C-2' chrysanthemum plants in the vegetative growth stage and cut the stems into segments with two axillary buds each Figure9C.Immerse the segments in a 3% detergent solution for 15 min, followed by shaking on a shaker for 10 min.Rinse the segments three times with sterile water, then quickly disinfect with 75% alcohol, followed by three more rinses with sterile water.Next, sterilize the segments in a 5% sodium hypochlorite solution for 15 min and rinse them three times with sterile water.Place the sterilized segments on sterile absorbent paper in a sterile Petri dish.In each 250 mL culture bottle, add 50 mL of MS medium without any hormones and inoculate with five segments.After 25 days of aseptic culture, a large number of '11-C-2' chrysanthemum aseptic seedlings are obtained.Culture conditions: 25 • C temperature, 1500 lx light intensity, and photoperiod (16 h light/8 h dark).

Author
Contributions: Q.C. accomplished all the experiments and data analysis; B.P., Y.W., L.C., J.Y., L.W., Y.F. and H.L. offered assistance in some experiments; K.G. and C.H. were responsible for the overall experimental design and manuscript revision.All authors have read and agreed to the published version of the manuscript.Funding: This work was financially supported by the Beijing Natural Science Foundation (6244044) and the Beijing Jin Qiao Engineering Seed Fund (070220052).Data Availability Statement: Data are contained within the article.

Table 5 .
Results of visual analyses using plant height as an indicator.

Table A1 .
Range analysis of each dependent variable in the regeneration process., and k3 are the average values of different levels of influencing factors of each dependent variable in the regeneration process.The R-value is the range of k1, k2, and k3.

Table A4 .
Composition of base medium.The pH range of different base mediums was 5.6~5.8.To prepare the media, weigh 38.4 g of 1/4 MS powder, 39.45 g of 1/2 MS powder, and 41.74 g of MS powder.Dissolve each in 1000 mL of distilled water, adjust the pH to 5.7 using 1 M NaOH or HCl, and then autoclave at 116 • C for 30 min.The basic media used were products from Qingdao Haibo Biotechnology Co., China, with product codes HB8469-8, HB8469-6, and HB8469.

Table A5 .
Orthogonal experimental design for apical meristem differentiation.
All basic media were MS.

Table A6 .
Orthogonal experimental design for rooting culture.
The component of base medium is shown in TableA4.

Table A7 .
Orthogonal experimental design for acclimatization and transplantation.