Establishment of tamarix micropaniculata plant tissue culture regeneration system
By using the seeds of *Tamarix spicata* as explants, the tissue culture method has solved the problems of excessive damage to the tree and environmental dependence caused by softwood cuttings. It has achieved efficient and stable cultivation and propagation of *Tamarix spicata*, supports gene function research, and is suitable for intensive production.
Patent Information
- Application Number
- CN202511300403.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2025-10-31
AI Technical Summary
In existing technologies, the cultivation of Tamarix spp. mainly relies on softwood cuttings, which is highly damaging to the tree and greatly affected by changes in the external environment. The lack of tissue culture methods results in low propagation speed and efficiency, making it difficult to achieve stable cultivation and gene function research.
Using Tamarix spicata seeds as explants, tissue culture was carried out by removing hairs, disinfecting, and inoculating them in a specific culture medium, including root induction and adventitious shoot induction. Using MS medium and different concentrations of plant growth regulators, a regeneration system with high seed germination rate and low contamination rate was achieved.
It has enabled efficient and stable cultivation and propagation of Tamarix spp., reduced the demand for manpower and material resources, provided technical support for genetic research, and is suitable for intensive production and germplasm resource preservation.
Smart Images
Figure CN120858875A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant tissue culture of Tamarix chinensis, specifically relating to a method for regeneration of plant tissue culture using Tamarix chinensis seeds as explants. Background Technology
[0002] *Tamarix leptostachya*, a shrub belonging to the genus *Tamarix* in the family Tamaricaceae, possesses all the tolerance and resistance of other species in the genus. It can grow normally in low-lying wetlands, moderately to severely saline-alkali land, arid areas, and sandy soils, making it a top choice for windbreak and sand fixation, afforestation, soil and water conservation, and saline-alkali land improvement and restoration. *Tamarix leptostachya* has a beautiful and full shape, and its flowering period is from mid-June to late July. The flowers are brightly colored, including pink, reddish-pink, red, and purple, making it highly ornamental and thus an excellent tree species for afforestation in desert saline-alkali soils.
[0003] Propagating *Tamarix spp.* through large-scale, continuous collection of old branches can damage the tree itself. Therefore, current cultivation of *Tamarix spp.* primarily relies on establishing clonal scion nurseries. Research reports indicate that using current-year shoots for softwood cuttings yields a survival rate of 69.0%, while container seedlings transplanted from softwood cuttings achieve a survival rate of 94.0%. Currently, no research has been found on the aseptic cultivation of *Tamarix spp.* through tissue culture. This invention establishes a *Tamarix spp.* plant tissue culture regeneration system. By sterilizing and inducing differentiation of *Tamarix spp.* seeds, complete plants and adventitious buds are obtained. This system eliminates the adverse effects of changes in the external natural environment, facilitating stable cultivation and propagation of *Tamarix spp.*, preserving germplasm resources, and cultivating superior new varieties. Tissue culture can improve the propagation rate and growth efficiency of Tamarix spp., enabling subsequent exploration of key gene functions through gene transduction. This approach aligns with the needs of modern forestry development and provides technical convenience for future research on the stress resistance mechanisms and gene functions of Tamarix spp. through genetic engineering and molecular biology, demonstrating broad application prospects. Summary of the Invention
[0004] The purpose of this invention is to provide a plant tissue culture regeneration method using Tamarix chinensis seeds as explants, providing important technical support for cultivating aseptic tissue culture seedlings of Tamarix chinensis through tissue culture.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] The establishment of a tissue culture regeneration system for *Tamarix spicata* includes the following steps:
[0007] 1) Hair removal: Gently rub off the long, soft hairs on the seeds, and sift them 3-5 times through a 40-mesh sieve to remove as many impurities as possible. In a clean bench sterilized with ultraviolet light, place the Tamarix chinensis seeds into a sterile 2 ml centrifuge tube, and rinse the seeds 3-5 times with sterile water using a pipette;
[0008] 2) Disinfection: After cleaning, the seeds are first treated with 75% ethanol for 10-15 seconds for surface disinfection, and then rinsed with sterile water 2-3 times. Then, they are treated with 1% sodium hypochlorite solution for 20-25 seconds and rinsed with sterile water 5-6 times to obtain disinfected seeds.
[0009] 3) Rooting induction: Sterilized Tamarix chinensis seeds were inoculated into rooting induction medium at 25℃, with a light exposure of 16 h followed by an 8 h dark period, and a light intensity of 120 μmol / m². 2 / s; The preparation method of the rooting induction medium is as follows: 22.5 g / L sucrose is added to MS medium, the pH value of the medium is adjusted to 5.8-6.0, 6 g / L agar is added, and the medium is sterilized at 121℃ for 20 min by high temperature and high pressure to obtain the basic medium. Then, 1-naphthaleneacetic acid (NAA) is added to the final concentration of 0.05 mg / L.
[0010] 4) Adventitious bud induction: Disinfected Tamarix chinensis seeds were inoculated into adventitious bud induction medium. The culture conditions and the preparation method of the basic medium were the same as above. 6-Benzylaminopurine (6-BA) was then added to a final concentration of 1.0 mg / L.
[0011] This invention provides a method for inducing Tamarix spp. seeds to develop into complete plants and differentiate adventitious buds through tissue culture by removing the seed hairs and inoculating them onto a culture medium under sterile conditions. This method reduces the contamination rate to 0% and achieves a germination rate of 85%, resulting in a high germination rate and low contamination rate. This method is helpful for the rapid and large-scale cultivation of Tamarix spp.
[0012] The beneficial effects of this invention are as follows:
[0013] 1. Existing propagation techniques for *Tamarix sibirica* primarily rely on seed propagation to establish scion nurseries and softwood cuttings, which are highly susceptible to changes in external environmental temperature. Currently, there is no method for cultivating *Tamarix sibirica* using plant tissue culture. This invention utilizes *Tamarix sibirica* seeds to cultivate complete plants and induce adventitious bud differentiation through explant sterilization. This allows seedlings to grow in an artificially provided culture medium and environment, facilitating management and highly intensive production. Compared to traditional methods, this eliminates the need for weeding, watering, fertilizing, and pest and disease control, saving manpower, material resources, and land resources.
[0014] 2. The disinfection method in this invention enables the seed germination rate to reach 85% and the contamination rate to be reduced to 0%. The disinfection operation is simple and quick, with low technical requirements and a low contamination rate, making it suitable for application.
[0015] 3. This invention screened out suitable induction media for the development of Tamarix chinensis seeds into complete plants and the differentiation of adventitious buds. In the combination of MS medium and different concentrations of plant growth regulators 6-BA and NAA, the seeds developed into complete plants and showed the best rooting performance in the rooting induction medium with 0.05 mg / L NAA added alone; the seeds of Tamarix chinensis showed the best effect in differentiating adventitious buds in the adventitious bud induction medium with 1.0 mg / L 6-BA added alone. Attached Figure Description
[0016] Figure 1 Adventitious buds induced by 2.0 mg / L 6-BA for seed differentiation of Tamarix chinensis.
[0017] Figure 2 Adventitious buds induced by 2.0 mg / L 6-BA for seed differentiation of Tamarix chinensis.
[0018] Figure 3 0.05 mg / L NAA was used to induce rooting of Tamarix chinensis seeds into complete plants.
[0019] Figure 4 Adventitious buds induced by 1.0 mg / L 6-BA for seed differentiation of Tamarix chinensis.
[0020] Figure 5 Adventitious buds induced by 1.0 mg / L 6-BA for seed differentiation of Tamarix chinensis. Detailed Implementation
[0021] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:
[0022] Example 1: Selection of Sodium Hypochlorite Mass Fraction
[0023] This embodiment investigated the effects of different mass fractions of sodium hypochlorite on the disinfection effect and germination rate of Tamarix chinensis seeds. The seeds were disinfected using sodium hypochlorite with mass fractions of 1%, 3%, 5%, and 10%, respectively.
[0024] Table 1. Effects of different mass fractions of sodium hypochlorite on seed disinfection efficacy and germination rate.
[0025] Table 1 shows that seeds germinated after disinfection with 1% and 3% sodium hypochlorite, but all seeds died after disinfection with 5% and 10% sodium hypochlorite. The germination rate was 85% after disinfection with 1% sodium hypochlorite, but dropped to 14.3% after disinfection with 3% sodium hypochlorite, indicating that the amount of available chlorine significantly affects the germination rate. Although both 1% and 3% sodium hypochlorite can thoroughly disinfect seeds, the germination rate is higher with 1% sodium hypochlorite. Therefore, using 1% sodium hypochlorite is more suitable for disinfecting Tamarix chinensis seeds.
[0026] Example 2: Selection of Disinfection Time
[0027] This embodiment investigated the effects of different disinfection times on the disinfection effect and germination rate of Tamarix chinensis seeds. The seeds were disinfected with 1% sodium hypochlorite for 20-25 seconds, 40-45 seconds and 1 minute, respectively.
[0028] Table 2. Effects of different disinfection times on seed disinfection efficacy and germination rate.
[0029] Table 2 shows that all three disinfection times achieved good disinfection results with low contamination rates, but the duration of disinfection significantly affected seed germination rates. After 20-25 seconds of disinfection, the germination rate was 85%, but after 40-45 seconds, the germination rate dropped drastically to 6.7%, and all seeds died after 1 minute of disinfection. Based on these results, a disinfection time of 20-25 seconds is more suitable for disinfecting Tamarix chinensis seeds.
[0030] Example 3: Selection of Adventitious Bud Induction Culture Medium
[0031] This study investigated the effect of combined concentrations of 6-BA and NAA plant growth regulators on adventitious shoot induction from Tamarix chinensis seeds. The basal culture medium consisted of MS medium + 22.5 g / L sucrose + 6 g / L agar. The proliferation coefficient was calculated as the number of regenerated shoots per explant divided by the number of germinations.
[0032] Table 3 Effects of different combinations of plant growth regulator concentrations on adventitious bud differentiation
[0033] Table 3 shows that the plant growth regulator concentration combination with the highest adventitious bud proliferation coefficient in Example 3 was the addition of 2.0 mg / L 6-BA alone, with a proliferation coefficient of 7.7. However, from a long-term growth perspective, 2.0 mg / L 6-BA leads to excessive adventitious bud differentiation, making it difficult to differentiate into normal plants (e.g., ...). Figure 1 , Figure 2Secondly, from highest to lowest, the addition of 0.5, 0.05, 0.1, and 0.3 mg / L NAA alone resulted in proliferation coefficients of 5, 4.7, 4.3, and 3.4, respectively. Although the proliferation coefficients were high, the seeds had well-developed root systems and tended to grow directly into complete plants rather than differentiate into adventitious buds (e.g., Figure 3 Taking into account the adventitious bud differentiation status, proliferation coefficient, germination rate, and induction rate, the optimal concentration for inducing adventitious bud differentiation in *Tamarix chinensis* seeds was selected as 1.0 mg / L 6-BA alone (e.g., ...). Figure 4 , Figure 5 ).
[0034] Example 4: Selection of Rooting Induction Culture Medium
[0035] As shown in Example 3, the addition of 0.05, 0.1, 0.3 and 0.5 mg / L NAA alone can induce seeds to root and develop into complete plants. Therefore, this example investigated the effects of these NAA hormone concentrations on the induction of rooting and development of Tamarix chinensis seeds. The basal culture medium was MS medium + 22.5 g / L sucrose + 6 g / L agar.
[0036] Table 4. Plant development under different NAA concentrations
[0037] As shown in the previous embodiment, all four NAA hormone concentrations in this embodiment could induce *Tamarix spp.* seeds to develop into complete plants, but the growth status varied significantly. In this embodiment, NAA had a greater impact on the development of *Tamarix spp.* stem segments and roots. Considering the seed germination rate and induction rate in Example 3, and the plant growth status and root development in this embodiment, 0.05 mg / L NAA was selected as the optimal concentration for inducing *Tamarix spp.* seeds to root and develop into complete plants.
[0038] Example 5
[0039] The establishment of a tissue culture regeneration system for *Tamarix spicata* includes the following steps:
[0040] 1) Hair removal: Gently rub off the long, soft hairs on the seeds, and sift them three times through a 40-mesh sieve to remove as many impurities as possible. In a clean bench sterilized with ultraviolet light, place the Tamarix chinensis seeds into a sterile 2 ml centrifuge tube, and rinse the seeds three times with sterile water using a pipette;
[0041] 2) Disinfection: After cleaning, the seeds are first treated with 75% ethanol for 12 seconds for surface disinfection, and then rinsed 3 times with sterile water. Then, they are treated with 1% sodium hypochlorite solution for 20 seconds and rinsed 5 times with sterile water to obtain disinfected seeds.
[0042] 3) Rooting induction: Sterilized Tamarix chinensis seeds were inoculated into rooting induction medium at 25℃, with a light exposure of 16 h followed by an 8 h dark period, and a light intensity of 120 μmol / m². 2 / s; The preparation method of the rooting induction medium is as follows: 22.5 g / L sucrose is added to MS medium, the pH value of the medium is adjusted to 5.8, 6 g / L agar is added, and the medium is sterilized at 121℃ for 20 min by high temperature and high pressure to obtain the basic medium. Then, 1-naphthaleneacetic acid (NAA) is added to the final concentration of 0.05 mg / L.
[0043] 4) Adventitious bud induction: Disinfected Tamarix chinensis seeds were inoculated into adventitious bud induction medium. The culture conditions and the preparation method of the basic medium were the same as above. 6-Benzylaminopurine (6-BA) was then added to a final concentration of 1.0 mg / L.
[0044] Example 6
[0045] The establishment of a tissue culture regeneration system for *Tamarix spicata* includes the following steps:
[0046] 1) Hair removal: Gently rub off the long, soft hairs on the seeds, and sift them 5 times through a 40-mesh sieve to remove as many impurities as possible. In a clean bench sterilized with ultraviolet light, place the Tamarix chinensis seeds into a sterile 2 ml centrifuge tube, and rinse the seeds 4 times with sterile water using a pipette;
[0047] 2) Disinfection: After cleaning, the seeds are first treated with 75% ethanol for 15 seconds for surface disinfection, and then rinsed 3 times with sterile water. Then, they are treated with 1% sodium hypochlorite solution for 25 seconds and rinsed 6 times with sterile water to obtain disinfected seeds.
[0048] 3) Rooting induction: Sterilized Tamarix chinensis seeds were inoculated into rooting induction medium at 25℃, with a light exposure of 16 h followed by an 8 h dark period, and a light intensity of 120 μmol / m². 2 / s; The preparation method of the rooting induction medium is as follows: 22.5 g / L sucrose is added to MS medium, the pH value of the medium is adjusted to 5.9, 6 g / L agar is added, and the medium is sterilized at 121℃ for 20 min by high temperature and high pressure to obtain the basic medium. Then, 1-naphthaleneacetic acid (NAA) is added to the final concentration of 0.05 mg / L.
[0049] 4) Adventitious bud induction: Disinfected Tamarix chinensis seeds were inoculated into adventitious bud induction medium. The culture conditions and the preparation method of the basic medium were the same as above. 6-Benzylaminopurine (6-BA) was then added to a final concentration of 1.0 mg / L.
[0050] The embodiments listed above are merely examples of the better disinfection and induction effects of the present invention, and are not limited to the above embodiments. Any extensions and modifications made within the scope of the present invention should fall within the protection and coverage of the present invention.
Claims
1. Establishment of a tissue culture regeneration system for Tamarix chinensis, Its characteristics include the following steps: 1) Hair removal: Gently rub off the long, soft hairs on the seeds, and sift them 3-5 times through a 40-mesh sieve to remove as many impurities as possible. In a clean bench sterilized with ultraviolet light, place the Tamarix chinensis seeds into a sterile 2 ml centrifuge tube, and rinse the seeds 3-5 times with sterile water using a pipette; 2) Disinfection: After cleaning, the seeds are first treated with 75% ethanol for 10-15 seconds for surface disinfection, and then rinsed with sterile water 2-3 times. Then, they are treated with 1% sodium hypochlorite solution for 20-25 seconds and rinsed with sterile water 5-6 times to obtain disinfected seeds. 3) Rooting induction: Sterilized Tamarix chinensis seeds were inoculated into rooting induction medium at 25℃, with a light exposure of 16 h followed by an 8 h dark period, and a light intensity of 120 μmol / m². 2 / s; The preparation method of the rooting induction medium is as follows: 22.5 g / L sucrose is added to MS medium, the pH value of the medium is adjusted to 5.8-6.0, 6 g / L agar is added, and the medium is sterilized at 121℃ for 20 min by high temperature and high pressure to obtain the basic medium. Then, 1-naphthaleneacetic acid (NAA) is added to the final concentration of 0.05 mg / L. 4) Adventitious bud induction: Disinfected Tamarix chinensis seeds were inoculated into adventitious bud induction medium. The culture conditions and the preparation method of the basic medium were the same as above. 6-Benzylaminopurine (6-BA) was then added to a final concentration of 1.0 mg / L.
2. The establishment of a tissue culture regeneration system for *Tamarix chinensis* according to claim 1, characterized in that... Step 1) Gently rub off the long, soft hairs from the seeds and sift them 3-5 times through a 40-mesh sieve to remove as many impurities as possible.
3. The establishment of a tissue culture regeneration system for *Tamarix chinensis* according to claim 1, characterized in that... In steps 1) and 2), gently tap and rinse the seeds using a 1 mL pipette throughout the process.
4. The establishment of a tissue culture regeneration system for *Tamarix chinensis* according to claim 1, characterized in that... Step 2) Treat the seeds with 75% ethanol for 10-15 seconds for surface disinfection, and then rinse with sterile water 2-3 times.
5. The establishment of a tissue culture regeneration system for *Tamarix chinensis* according to claim 1, characterized in that... In step 2), treat the seeds with a 1% sodium hypochlorite solution for 20-25 seconds and wash them with sterile water 5-6 times.
6. The establishment of a tissue culture regeneration system for *Tamarix chinensis* according to claim 1, characterized in that... The rooting induction medium in step 3) is: MS + 22.5 g / L sucrose + 6 g / L agar + 0.05 mg / L NAA.
7. The establishment of a tissue culture regeneration system for *Tamarix chinensis* according to claim 1, characterized in that... In step 4), the adventitious bud induction medium is MS + 22.5 g / L sucrose + 6 g / L agar + 1.0 mg / L 6-BA.