Method for tissue culture subculture rooting and seedling formation of baeckea frutescens
By combining in-bottle seedling strengthening and rooting culture medium, the problems of low rooting rate and low survival rate of Pinus thunbergii seedlings were solved, achieving efficient cultivation of Pinus thunbergii seedlings with significantly improved rooting rate and survival rate, and a suitable cycle.
Patent Information
- Application Number
- CN202510079959.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-18
- Publication Date
- 2025-12-16
Smart Images
Figure BDA0005248299070000031 
Figure BDA0005248299070000041 
Figure BDA0005248299070000042
Abstract
Description
Technical Field
[0001] This invention belongs to the field of asexual propagation technology of seedlings, and specifically relates to a method for tissue culture subculture and rooting of Pinus thunbergii seedlings. Background Technology
[0002] Baeckea frutescens, a perennial shrub or small tree belonging to the genus Baeckea in the family Myrtaceae, is also known as broom branch, iron broom, stone pine grass, and fragrant firewood. It is mainly distributed in Hainan, Guangdong, Guangxi, Fujian, Zhejiang, and Jiangxi provinces. With its well-developed root system, strong reproductive capacity, and tolerance to poor soil and drought, Baeckea frutescens is a pioneer species for windbreak and sand fixation, soil and water conservation, and forest stand restoration. Traditionally used for broom making, its cultivation and processing have also led to its significant economic value in the handicrafts, pharmaceutical, and daily chemical industries. Baeckea frutescens branches are woven into garden fences and exported to international markets in Europe, America, and the Middle East. It can also be further processed to extract basalt oil, which is used in the fragrance and pharmaceutical industries and has air-purifying, heat-clearing, detoxifying, insecticidal, and antipruritic effects. The leaves can also be made into natural and environmentally friendly mosquito coils with good mosquito-repellent properties.
[0003] As the economic and medicinal value of *Pinus argentea* becomes increasingly prominent, the demand is also growing daily. Expanding the artificial cultivation of *Pinus argentea* is a pressing issue that needs to be addressed to meet the demand for raw materials. Currently, the main methods for cultivating *Pinus argentea* seedlings are cuttings and sowing. In nature, *Pinus argentea* seeds are small, have a low germination rate, and produce weak seedlings that are difficult to manage later. Cuttings also have a low rooting rate and require very high water management. Both methods suffer from low survival rates, low propagation coefficients, and long cycles, making it difficult to meet market demand for seedling production. Summary of the Invention
[0004] This invention provides a method for subculture rooting and seedling production of *Pinus argentea* through tissue culture. The method improves the rooting rate and transplant survival rate of *Pinus argentea* tissue culture seedlings by promoting rooting of strong seedlings in bottles. It solves the problems of low germination and rooting rates, low survival rates, and long cycles in the current main cultivation methods of *Pinus argentea* seedlings, which make it difficult to meet the market demand for seedling production.
[0005] The objective of this invention is achieved through the following technical solution:
[0006] A method for subculturing and rooting seedlings from tissue culture of *Pinus arborescens* includes the following steps:
[0007] 1) The subcultured seedlings of *Pinus argentea* were inoculated into a seedling strengthening medium to obtain strong seedlings. The seedling strengthening medium formula includes: MS + sucrose content 20-35 g / L + agar 5-6 g / L;
[0008] 2) Strong seedlings are inoculated into rooting medium to obtain rooted seedlings. The rooting medium formula is: 1 / 2 MS + IBA content 1-3 mg / L + NAA content 1-2 mg / L + sucrose content 25-35 g / L;
[0009] 3) Transplant the rooted seedlings outdoors to harden them off, and plant the bottle seedlings in a mixed substrate of nutrient soil and yellow soil for cultivation, and spray water to keep them moist.
[0010] Preferably, the seedling culture medium formula in step 1) is: MS + IBA content 2mg / L + NAA content 1.5mg / L + sucrose content 35g / L + agar 5-6g / L.
[0011] Preferably, the culture temperature in step 1) is 25-28℃, the light intensity is 1900-2100 lx, the light cycle is 12h / d, and the culture time is 30 days.
[0012] Preferably, the rooting medium formula in step 2) is: 1 / 2 MS + IBA content 2 mg / L + NAA content 1.5 mg / L + sucrose content 35 g / L.
[0013] Preferably, the culture temperature in step 2) is 25–28°C, the light intensity is 1900–2100 lx, the light cycle is 12 h / d, and the culture time is 60 days.
[0014] Preferably, the outdoor hardening-off process described in step 3) includes 2 days of hardening-off with the lid closed and 3 days of hardening-off with the lid open.
[0015] Preferably, the mass ratio of nutrient soil to yellow core soil in step 3) is 1:3.
[0016] Preferably, the relative humidity for water spraying in step 3) is 70-80%, and the culture temperature is 25±2℃.
[0017] The *Pinus arborescens* seedlings of this invention, after 30 days of cultivation in a seedling-strengthening medium, showed increased stem length and thickness. After 60 days of cultivation in a rooting medium, the rooting rate of the *Pinus arborescens* seedlings reached 80%–100%. The tissue-cultured seedlings had stems longer than 5 cm and more than 6 adventitious roots, exceeding the average root length by 1.5 cm. After 30 days of outdoor hardening-off cultivation, the transplant survival rate reached 90.15%. Attached Figure Description
[0018] Figure 1 Gangsong's successor training;
[0019] Figure 2 Orthogonal experimental results on rooting of *Pinus thunbergii* tissue culture;
[0020] Figure 3 Rooting situation in actual production;
[0021] Figure 4 Transplanting in actual production;
[0022] Figure 5 The survival rate of transplanted seedlings in actual production. Detailed Implementation
[0023] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited to the scope shown in the embodiments. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, after reading the contents of this invention, those skilled in the art can make various modifications to the present invention, and these equivalent changes also fall within the scope defined by the appended claims.
[0024] The materials used in this invention are: MS culture medium (purchased from PhytoTechnology Laboratories, USA); IBA (indolebutyric acid), NAA (naphthaleneacetic acid); and nutrient soil (organic matter + humic acid ≥ 55%, total porosity: 70-80%, pH value 5.5-6.5%, total nutrients 3.5-4%, purchased from Keliang Agricultural Technology Co., Ltd.)
[0025] Example 1
[0026] 1) Inoculate the subcultured pine seedlings into a seedling strengthening medium to obtain strong seedlings (e.g. Figure 1 As shown), the seedling culture medium formula is: MS + sucrose content 30g / L + agar 5g / L, without adding any growth hormones. The culture conditions include: temperature 26℃, light intensity 2000 lx, photoperiod 12h / d, culture for 30d.
[0027] 2) Strong seedlings were inoculated into rooting medium to obtain rooted seedlings. Rooting medium was prepared according to the orthogonal design scheme in Table 1 and cultured at a temperature of 26℃, a light intensity of 2050 lx, a photoperiod of 12 h / d, and a culture time of 60 days. Stem segment growth and rooting were observed and statistically analyzed (results are detailed in Table 2 and...). Figure 2 and Figure 3 );
[0028] 3) Transplant healthy, vigorous seedlings (over 5cm tall) with robust root systems outdoors. Harden them off under natural light for 2 days, then allow them to harden off for another 3 days with the bottle caps removed. Remove the seedlings from the culture bottles, wash off the root culture medium, and plant them in a non-woven bag containing a mixture of nutrient soil and yellow soil (1:3 mass ratio). Maintain a relative humidity of 75% and a temperature of 25℃. Cover with a thin film to retain moisture. Transplant after 30 days (e.g., ...). Figure 4 (As shown) and the survival rate was calculated (see Table 2 for details). Figure 5 ).
[0029] Table 1 Orthogonal Design Table
[0030]
[0031]
[0032] Table 2 Results of stem segment growth, rooting, and transplant survival rate
[0033]
[0034] The table shows that group 4 had the most roots, with a rooting rate of 100%. After hardening off and transplanting, the survival rate was 90.15%. Therefore, the optimal formula for the rooting medium is 1 / 2 MS + IBA 2 mg / L + NAA 1.5 mg / L + sucrose 35 g / L.
[0035] Example 2
[0036] 1) The subcultured seedlings of Pinus massoniana were inoculated into a seedling strengthening medium to obtain strong seedlings. The seedling strengthening medium was formulated as follows: MS + sucrose 30g / L + agar 5g / L, without adding any growth hormones. The culture conditions included: temperature of 26℃, light intensity of 2000 lx, photoperiod of 12h / d, and culture for 30 days.
[0037] 2) Strong seedlings were inoculated into rooting medium to obtain rooted seedlings. The rooting medium consisted of 1 / 2 MS medium with 2 mg / L IBA, 1.5 mg / L NAA, and 35 g / L sucrose. The culture temperature was 26℃, and the light intensity was set to groups 1-5 sequentially at 1900 lx, 1950 lx, 2000 lx, 2050 lx, and 2100 lx. The photoperiod was 12 h / d, and the culture time was 60 days. Rooting was observed and statistically analyzed. With the increase of light intensity, the corresponding rooting rates were 90.2%, 94.4%, 96.3%, 100%, and 100%, respectively.
[0038] 3) Seedlings with a height of over 5cm and good growth and vigorous root development from the above 5 different light groups were transplanted outdoors and placed under natural light for 2 days to harden off. After hardening off for another 3 days with the bottle caps removed, the seedlings were removed from the culture bottles, the culture medium was washed off the roots, and they were planted in non-woven bags containing a mixture of nutrient soil and yellow soil in a mass ratio of 1:3. The bags were kept moist with water at a relative humidity of 75% and a temperature of 25℃. The bags were covered with a thin film to retain moisture. After 30 days, the seedlings were transplanted and the survival rate was recorded. The transplant survival rates for groups 1-5 were 82.37%, 85.14%, 86.42%, 90.15%, and 88.26%, respectively.
[0039] Example 3
[0040] 1) The subcultured seedlings of Pinus massoniana were inoculated into a seedling strengthening medium to obtain strong seedlings. The seedling strengthening medium was formulated as follows: MS + 20 g / L sucrose + 5 g / L agar, without adding any growth hormones. The culture conditions included: 25℃, light intensity of 1900 lx, photoperiod of 12 h / d, and culture time of 30 days.
[0041] 2) Strong seedlings were inoculated into rooting medium to obtain rooted seedlings. The rooting medium was 1 / 2 MS with IBA content of 1 mg / L, NAA content of 1 mg / L, and sucrose content of 25 g / L. The culture temperature was 25℃, the light intensity was 1900 lx, the photoperiod was 12 h / d, and the culture time was 60 days. The rooting rate was 88.6%.
[0042] 3) Transplant healthy seedlings with a height of 5cm or more and vigorous root growth to the outdoors and harden them off under natural light for 2 days. After hardening off for another 3 days with the bottle caps off, remove the seedlings from the culture bottles, wash off the culture medium from the roots, and plant them in non-woven bags containing a mixture of nutrient soil and yellow soil (mass ratio of nutrient soil to yellow soil is 1:3). Spray water to maintain a relative humidity of 70% and a temperature of 23℃. Cover with a thin film to maintain moisture. After 30 days, the survival rate after transplanting is 80.73%.
[0043] Example 4
[0044] 1) The subcultured seedlings of Pinus massoniana were inoculated into a seedling strengthening medium to obtain strong seedlings. The seedling strengthening medium was formulated as follows: MS + sucrose 35g / L + agar 6g / L, without adding any growth hormones. The culture conditions included: 28℃, light intensity of 2100 lx, photoperiod of 12h / d, and culture time of 30 days.
[0045] 2) Strong seedlings were inoculated into rooting medium to obtain rooted seedlings. The rooting medium was 1 / 2 MS with IBA content of 3 mg / L, NAA content of 2 mg / L, and sucrose content of 30 g / L. The culture temperature was 28℃, the light intensity was 2100 lx, the photoperiod was 12 h / d, and the culture time was 60 days. The rooting rate was 92.8%.
[0046] 3) Transplant healthy seedlings with a height of 5cm or more and vigorous root growth to the outdoors and harden them off under natural light for 2 days. After hardening off for another 3 days with the bottle caps off, remove the seedlings from the culture bottles, wash off the culture medium from the roots, and plant them in non-woven bags containing a mixture of nutrient soil and yellow soil (mass ratio of nutrient soil to yellow soil is 1:3). Spray water to maintain a relative humidity of 80% and a temperature of 27℃. Cover with a thin film to maintain moisture. After 30 days, the survival rate after transplanting is 84.19%.
[0047] Example 5
[0048] 1) The subcultured seedlings of *Pinus thunbergii* were inoculated into a seedling strengthening medium to obtain strong seedlings. The seedling strengthening medium was formulated as follows: MS + IBA 2 mg / L + NAA 1.5 mg / L + sucrose 35 g / L + agar 5 g / L. The culture conditions included: 25℃, light intensity 1900 lx, photoperiod 12 h / d, and culture time 30 days.
[0049] 2) Strong seedlings were inoculated into rooting medium to obtain rooted seedlings. The rooting medium was 1 / 2 MS with IBA content of 1 mg / L, NAA content of 1 mg / L, and sucrose content of 25 g / L. The culture temperature was 25℃, the light intensity was 1900 lx, the photoperiod was 12 h / d, and the culture time was 60 days. The rooting rate was 90.5%.
[0050] 3) Transplant healthy seedlings with a height of 5cm or more and vigorous root growth to the outdoors and harden them off under natural light for 2 days. After hardening off for another 3 days with the bottle caps off, remove the seedlings from the culture bottles, wash off the culture medium from the roots, and plant them in non-woven bags containing a mixture of nutrient soil and yellow soil (mass ratio of nutrient soil to yellow soil is 1:3). Spray water to maintain a relative humidity of 70% and a temperature of 23℃. Cover with a thin film to maintain moisture. After 30 days, the survival rate after transplanting is 86.67%.
[0051] This invention produces seedlings of Pinus thunbergii with high rooting rate, high survival rate, and suitable growth cycle, making it suitable for market promotion.
Claims
1. A method for subculturing and rooting seedlings from tissue culture of *Pinus arborescens*, characterized in that, The method includes the following steps: 1) The subcultured seedlings of *Pinus argentea* were inoculated into a seedling strengthening medium to obtain strong seedlings. The seedling strengthening medium formula includes: MS + sucrose content 20-35 g / L + agar 5-6 g / L; 2) Strong seedlings are inoculated into rooting medium to obtain rooted seedlings. The rooting medium formula is: 1 / 2 MS + IBA content 1-3 mg / L + NAA content 1-2 mg / L + sucrose content 25-35 g / L; 3) Transplant the rooted seedlings outdoors to harden them off, and plant the bottle seedlings in a mixed substrate of nutrient soil and yellow soil for cultivation, and spray water to keep them moist.
2. The method for subculturing and rooting seedlings of *Pinus thunbergii* according to claim 1, characterized in that, The seedling culture medium formula for step 1) is: MS + IBA content 2mg / L + NAA content 1.5mg / L + sucrose content 35g / L + agar 5-6g / L.
3. The method for subculturing and rooting seedlings of *Pinus thunbergii* according to claim 1 or 2, characterized in that, The cultivation temperature in step 1) is 25-28℃, the light intensity is 1900~2100 lx, the light cycle is 12h / d, and the cultivation time is 30 days.
4. The method for subculturing and rooting seedlings of *Pinus thunbergii* according to claim 1, characterized in that, Step 2) The rooting medium formula is: 1 / 2 MS + IBA content 2 mg / L + NAA content 1.5 mg / L + sucrose content 35 g / L.
5. The method for subculturing and rooting seedlings of *Pinus thunbergii* according to claim 1 or 4, characterized in that, Step 2) The culture temperature is 25-28℃, the light intensity is 1900-2100 lx, the light cycle is 12h / d, and the culture time is 60 days.
6. The method for subculturing and rooting seedlings of *Pinus thunbergii* according to claim 1, characterized in that, Step 3) describes outdoor hardening-off, which includes 2 days of hardening-off with the lid closed and 3 days of hardening-off with the lid open.
7. The method for subculturing and rooting seedlings of *Pinus thunbergii* according to claim 1, characterized in that, The mass ratio of the nutrient soil and yellow core soil mentioned in step 3) is 1:
3.
8. The method for subculturing and rooting seedlings of *Pinus thunbergii* according to claims 6-7, characterized in that, Step 3) involves spraying water to maintain a relative humidity of 70-80% and a culture temperature of 25±2℃.