Method for promoting stem elongation and adventitious bud regeneration of hibiscus mutabilis
By using a culture medium with a specific hormone ratio on the stem segments and leaf discs of Hibiscus mutabilis, stem elongation and adventitious bud regeneration were promoted, solving the problem of low propagation efficiency of Hibiscus mutabilis and achieving efficient tissue culture and genetic transformation support.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-27
- Publication Date
- 2026-03-24
AI Technical Summary
The existing technology for Hibiscus mutabilis has a low propagation coefficient, slow growth, low bud induction regeneration efficiency, and lacks a suitable hormone ratio scheme, which limits its large-scale production and genetic transformation research.
Elongation of *Hibiscus mutabilis* stem segments was promoted by using an elongation medium supplemented with 5.0–20.0 mg/L GA3 and 0.5 mg/L IAA in MS basal medium, and adventitious bud regeneration was induced by using a regeneration medium supplemented with 1.0 mg/L 6-BA and 0.05–0.1 mg/L NAA in MS basal medium.
It significantly improved the tissue culture efficiency of Hibiscus mutabilis, with an average stem growth of 21.77 mm, a growth rate of 0.54 mm/day, a bud regeneration rate of up to 95.83%, and a propagation coefficient of 679.02%, thus solving the key technical bottleneck of regeneration difficulties.
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Figure CN121713854A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for promoting stem elongation and adventitious bud regeneration in Hibiscus mutabilis. Background Technology
[0002] Hibiscus syriacus (Crossostephium chinense), also known as jade hibiscus, Qi'ai, thousand-year-old hibiscus, and fragrant chrysanthemum, is an evergreen subshrub belonging to the genus Crossostephium in the family Asteraceae. It has many branches covered with soft hairs; leaves are clustered at the branch tips, narrowly oblanceolate or narrowly spatulate, densely covered with grayish soft hairs on both sides; the flower heads are disc-shaped, forming a leafy raceme, with bisexual disc flowers; the achenes are angular, with lacerated scales at the apex. Hibiscus syriacus prefers a sunny, warm, and humid climate, is heat-tolerant and drought-tolerant, but intolerant of shade, waterlogging, and cold; it thrives best in loose soil rich in organic matter.
[0003] Chrysanthemum indicum possesses significant ornamental and medicinal value. Its plant is a rare "white-leaved plant," with an elegant appearance, widely used in home decoration and urban greening, especially in South China. Simultaneously, as a traditional Chinese medicine, the entire plant and roots of Chrysanthemum indicum can be used medicinally. It is pungent, slightly bitter, and warm in nature, possessing the effects of dispelling wind and dampness, reducing swelling and detoxifying, and relieving exterior syndromes and cold. It is commonly used to treat colds due to wind-cold, rheumatic pain, stomachache, infantile convulsions, diabetes, and carbuncles and boils. Modern pharmacological studies have shown that its extracts and various active ingredients (such as tansyrinx compounds) have significant inhibitory effects on α-glucosidase and can promote insulin secretion, demonstrating good hypoglycemic potential and providing important evidence for the development of diabetes drugs.
[0004] Currently, the main propagation methods for Hibiscus mutabilis are sowing and cutting. However, these two methods are limited by season, climate, and mother plant resources, resulting in low propagation coefficients that are difficult to meet the needs of large-scale production and medicinal resource development. Although preliminary explorations of tissue culture technology have been conducted, existing systems have significant shortcomings: in conventional MS medium, Hibiscus mutabilis grows slowly, plants are stunted, bud regeneration efficiency is low, and there is a lack of suitable hormone ratio schemes, severely restricting its propagation efficiency and genetic transformation research. Furthermore, the genetic transformation technology for Hibiscus mutabilis is weak, making it difficult to improve traits or deeply analyze the synthesis mechanism of its medicinal components through genetic engineering, thus limiting its further application in the fields of medicine and ornamental plants. Summary of the Invention
[0005] To address the aforementioned problems in the prior art, the technical problem to be solved by this application is to provide a method for promoting the elongation of hibiscus stem segments and the regeneration of adventitious buds.
[0006] To solve the above-mentioned technical problems, the technical solution of this application is as follows:
[0007] A method for promoting stem elongation of Hibiscus mutabilis includes: inoculating the top of a stem segment of Hibiscus mutabilis as an explant into an elongation medium for culture; wherein the elongation medium is MS basal medium supplemented with 5.0~20.0 mg / L GA3 and 0.5 mg / L IAA.
[0008] In some embodiments, the elongation medium further comprises 30 g / L sucrose and 7 g / L agar.
[0009] In some embodiments, the pH of the elongation medium is 5.8 ± 0.2.
[0010] A method for inducing adventitious bud regeneration from leaf discs of Hibiscus mutabilis includes: inoculating explants from leaf discs of Hibiscus mutabilis into a regeneration medium for culture; wherein the regeneration medium is MS basal medium supplemented with 1.0 mg / L 6-BA and 0.05~0.1 mg / L NAA.
[0011] In some embodiments, the regeneration medium further comprises 30 g / L sucrose and 7 g / L agar.
[0012] In some embodiments, the pH of the regeneration medium is 5.8 ± 0.2.
[0013] A rapid tissue culture propagation method for Hibiscus mutabilis includes the following steps:
[0014] (1) Using the method described above, the stem segments of Hibiscus mutabilis were elongated to obtain sterile seedlings;
[0015] (2) Take the leaves of the sterile seedlings obtained in step (1), prepare leaf discs, and use the method described above to induce adventitious bud regeneration to obtain regenerated plants.
[0016] A culture medium for stem elongation of Hibiscus mutabilis consists of MS basal medium supplemented with 5.0 mg / L GA3, 0.5 mg / L IAA, 30 g / L sucrose and 7 g / L agar, at a pH of 5.8 ± 0.2.
[0017] A culture medium for inducing the regeneration of adventitious buds from the leaf discs of Hibiscus mutabilis consists of MS basal medium supplemented with 1.0 mg / L 6-BA, 0.08 mg / L NAA, 30 g / L sucrose, and 7 g / L agar, at a pH of 5.8 ± 0.2.
[0018] The application of the culture medium described therein in improving the transgenic efficiency of Hibiscus mutabilis.
[0019] Compared with the prior art, the beneficial effects of this application are as follows:
[0020] This invention provides a highly efficient tissue culture protocol specifically for Hibiscus mutabilis, with significant beneficial effects: First, the optimal hormone ratio for promoting stem elongation (MS + 5.0 mg·L⁻¹) is specifically screened. -1 GA3+0.5 mg·L -1 IAA (Induced Aging of Adventitious Buds) resulted in an average stem segment growth of 21.77 mm and a growth rate of 0.54 mm / day, effectively obtaining robust mother seedlings and laying the foundation for subsequent experiments. Secondly, a highly efficient adventitious bud regeneration system for Hibiscus mutabilis was established for the first time, with the optimal regeneration medium (MS + 1.0 mg·L⁻¹). -1 6-BA + 0.08 mg·L -1 The adventitious bud regeneration rate of NAA reached 95.83%, and the propagation coefficient reached 679.02, solving the key technical bottleneck of its difficult regeneration. The whole method significantly improved the tissue culture efficiency of Hibiscus mutabilis and provided strong technical support for its germplasm resource preservation, rapid commercial propagation and transgenic breeding. Attached Figure Description
[0021] Figure 1 Figure showing the effect of different concentrations of growth regulators on the plant height of Hibiscus mutabilis;
[0022] Figure 2 The figure shows the effect of different growth regulators on the regeneration of adventitious buds of Hibiscus mutabilis. Detailed Implementation
[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Alternatively, they may be carried out according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0024] Example 1
[0025] The *Hibiscus mutabilis* chrysanthemum used in this experiment was a strain harvested from the Baguazhou Chrysanthemum Garden in Nanjing, Jiangsu Province. After explant sterilization, sterile seedlings were obtained. The explant sterilization method was as follows: 3-5cm stem tips of *Hibiscus mutabilis* chrysanthemum* were taken from the Baguazhou base on a sunny midday and kept fresh in an ice box. During the experiment, excess leaves were removed, leaving only the main stem segment. The surface dust was brushed off, and a small amount of detergent was added to a beaker and washed for 10 minutes. The beaker was then rinsed with running tap water for 30 minutes. In a clean bench, the beaker was sterilized with 75% alcohol for 1 minute, the alcohol was discarded, and 20 mL of 15% hydrogen peroxide was added and sterilized for 8 minutes. The residual liquid was discarded, and the beaker was washed three times with sterile water for 5 minutes each time. The water was blotted dry on filter paper, and the beakers were placed in prepared MS medium and cultured for one month to obtain sterile seedlings. The culture medium was prepared as follows: MS medium (without sucrose and agar) (purchased from Qingdao Haibo Biotechnology Co., Ltd.) was used as the basic medium, with 30 g·L⁻¹ sucrose (SUC) added. -1 and agar (A) 7 g·L -1 The pH of the culture medium was adjusted to 5.8±0.2 with 1 M NaOH and HCl, and then autoclaved at 116 ℃ for 30 min. A measured amount of plant growth regulator was added in a clean bench; GA3 and IAA were both manufactured by Beijing Solarbio Technology Co., Ltd. To improve the stem elongation of *Hibiscus mutabilis*, four hormone formulations were used in this experiment (see Table 1).
[0026] Table 1. Different ratios of plant growth regulators
[0027]
[0028] Using 30-day-old sterile seedlings of *Hibiscus mutabilis* as explants, 0.5cm ± 0.2cm top stem segments were taken in a clean bench, and the exact length was measured using calipers. These segments were then inoculated onto culture media containing different concentrations of growth regulators and cultured under conditions of 2000-4000 Lux light, 25℃ temperature, 40-60% humidity, and a light / dark ratio of 16 / 8 h. Two seedlings were inoculated onto each culture medium, with three replicates. After 40 days, the height growth was recorded, and the growth amount and growth rate were calculated using the following formula:
[0029] Growth = Plant height after 40 days - Inoculated plant height;
[0030] Growth rate = (Plant height after 40 days - Inoculated plant height) / Culture time.
[0031] The statistical results are shown in Table 2:
[0032] Table 2. Effects of different concentrations of growth regulators on the plant height of *Hibiscus mutabilis*.
[0033]
[0034] Note: The Duncan test was used for analysis. p < 0.05 indicates a statistically significant difference.
[0035] The short stature of Hibiscus mutabilis severely restricts its propagation and transgenic efficiency. Therefore, increasing plant height and producing robust seedlings are prerequisites for successful transgenic results. This experiment used four different ratios of GA3 and IAA culture media to screen for the optimal hormone ratios for promoting the growth and strengthening of Hibiscus mutabilis. Figure 1 As shown in Table 2, although the plant height was slightly increased in the culture medium with only IAA compared to the culture medium without GA3, the difference was not significant. However, the plant height was significantly different when both GA3 and IAA were added compared to the culture medium without GA3. Furthermore, the addition of 5.0 mg·L⁻¹ resulted in a greater difference in plant height. -1 GA3 with 0.5 mg·L -1 IAA (isolated arborvitae) showed the largest average growth, reaching 21.77 mm. Therefore, the most suitable elongation medium for Hibiscus mutabilis was MS + 5.0 mg·L⁻¹. -1 GA3+0.5 mg·L -1 IAA + 30 g·L -1 sucrose + 7g·L -1 Agar.
[0036] Example 2
[0037] Leaf disc adventitious bud regeneration is fundamental to stable genetic transformation. To screen for the most suitable culture media for inducing adventitious bud regeneration in Hibiscus mutabilis, this experiment used six MS media containing different concentrations of 6-BA and NAA ratios (Table 3). Young leaves from sterile Hibiscus mutabilis seedlings that had grown for 35-45 days were cut into 3 mm × 3 mm leaf discs in a clean bench and inoculated onto the four culture media. Sixteen leaf discs were placed in each dish, and the experiment was repeated three times. The light intensity was 2000-4000 Lux, the culture temperature was 25℃, and the culture period was 45 days. The number of adventitious buds was counted, and the adventitious bud regeneration rate and propagation coefficient were calculated using the following formulas:
[0038] Adventitious bud regeneration rate = (Number of explants with regenerated adventitious buds / Total number of inoculated explants) × 100%;
[0039] Propagation coefficient = (Number of regenerated adventitious buds - Number of leaves of inoculated explant) / Number of leaves of inoculated explant × 100%.
[0040] Table 3. Different ratios of plant growth regulators
[0041]
[0042] In the regeneration process of Chrysanthemum plants, the type, concentration, and solubility of growth regulators play a crucial role. By screening media containing six different concentrations of 6-BA and NAA, the most suitable medium for shoot regeneration was determined. Figure 2 As shown in Table 4, *Hibiscus mutabilis* can differentiate shoots on regeneration media with different hormone concentrations, but the differentiation effect varies greatly depending on the hormone ratio. Specifically, the leaf disc differentiation was best achieved on L3 (MS + 6-BA 1.0 mg·L⁻¹). -1 +NAA 0.08 mg·L -1 The induction effect was best in the culture medium, which was reflected in the highest adventitious bud regeneration rate and propagation coefficient of the leaf disc. The adventitious bud regeneration rate was 95.83% and the propagation coefficient was as high as 679.02%, which was significantly higher than that of other growth regulator concentration treatments. Moreover, the growth rate of adventitious buds was significantly higher than that of leaf discs in other culture media at the same period.
[0043] Table 4. Effects of different plant growth regulator ratios on the regeneration of Hibiscus mutabilis.
[0044]
[0045] Note: The Duncan test was used for analysis. p < 0.05 indicates a statistically significant difference.
[0046] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.
Claims
1. A method for promoting stem elongation of hibiscus, characterized in that, The stem tips of Hibiscus mutabilis were used as explants and cultured in elongation medium; the elongation medium was MS basal medium supplemented with 5.0~20.0 mg / L GA3 and 0.5 mg / L IAA.
2. The method according to claim 1, characterized in that, The elongation medium also contains 30 g / L sucrose and 7 g / L agar.
3. The method according to claim 1 or 2, characterized in that, The pH of the elongation medium is 5.8 ± 0.
2.
4. A method for inducing adventitious bud regeneration in the leaf disc of Hibiscus mutabilis, characterized in that, Leaf disc explants of Hibiscus mutabilis were inoculated into a regeneration medium for culture; the regeneration medium was MS basal medium supplemented with 1.0 mg / L 6-BA and 0.05~0.1 mg / L NAA.
5. The method according to claim 4, characterized in that, The regeneration medium also contains 30 g / L sucrose and 7 g / L agar.
6. The method according to claim 4 or 5, characterized in that, The pH of the regeneration medium is 5.8 ± 0.
2.
7. A method for rapid tissue culture propagation of Hibiscus mutabilis, characterized in that, Includes the following steps: (1) Using the method described in any one of claims 1-3, elongation culture is performed on the stem segments of Hibiscus mutabilis to obtain sterile seedlings; (2) Take the leaves of the sterile seedlings obtained in step (1), prepare leaf discs, and induce adventitious bud regeneration using the method described in any one of claims 4-6 to obtain regenerated plants.
8. A culture medium for the elongation of stem segments of Hibiscus mutabilis, characterized in that, It is prepared by adding 5.0 mg / L GA3, 0.5 mg / L IAA, 30 g / L sucrose and 7 g / L agar to MS basal medium, with a pH of 5.8 ± 0.
2.
9. The application of the culture medium according to claim 8 in improving the transgenic efficiency of Hibiscus mutabilis.
10. A culture medium for inducing the regeneration of adventitious buds from the leaf disc of Hibiscus mutabilis, characterized in that, The solution was prepared by adding 1.0 mg / L 6-BA, 0.08 mg / L NAA, 30 g / L sucrose and 7 g / L agar to MS basal medium, with a pH of 5.8 ± 0.2.