Tissue culture and rapid propagation method for primula tibetica and application of tissue culture and rapid propagation method

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a primula tibetica tissue culture rapid propagation method and application. The method comprises the following steps: drying newly harvested primula tibetica seeds in the shade, sequentially treating the seeds with 75% alcohol and 2% sodium hypochlorite, inoculating the seeds into a seed germination culture medium for culturing, shearing terminal bud parts of plants as explants, inoculating the explants into a cluster bud induction culture medium for culturing for 21-35 days, shearing the induced cluster buds into single buds, inoculating the single buds into a subculture multiplication culture medium for culturing for 45-55 days, and carrying out subculture on the single buds to obtain primula tibetica var. Tibetica var. Tibetica var. Tibetica var. Tibetica var. Tibetica var. Tibetica var. And cutting the multiple shoots subjected to subculture multiplication into small sections with 1-2 buds, inoculating the small sections into a rooting culture medium according to polarity, and culturing for 20-30 days. According to the method disclosed by the invention, the average multiplication coefficient of primary bud induction is 4.4-4.6, the rooting rate is 83.33-93.33%, and the average number of adventitious roots is more than 10.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically a method and application for rapid propagation of Primula spp. tissue culture. Background Technology

[0002] *Primula sinensis* Lour., a perennial herbaceous plant belonging to the genus *Primula* in the family Primulaceae, is endemic to China, native to Shaanxi, Hubei, Sichuan, Guizhou, and southern Gansu. It is prized for its attractive plant form, pale blue-purple or rose-red flowers, and blooming during the winter and spring (December to March of the following year), a period when few other flowers bloom, thus possessing a long history of cultivation and significant ornamental value. Furthermore, the entire plant can be used medicinally, demonstrating potential medicinal value. Currently, wild resources of *Primula sinensis* face serious threats. According to the *China Biodiversity Red List - Higher Plants (2020)* published by the Ministry of Ecology and Environment in 2023, its protection status has been assessed as "Endangered" (EN). The criteria B1ab(iii) upon which this assessment is based indicate that its main threats include: an extremely narrow and fragmented distribution range (EOO), and the continued degradation or loss of its specific shady and damp limestone crevices habitat. As a species endemic to China, its wild populations are entirely dependent on the fragile local ecosystems within the country, making their stability highly vulnerable. Traditional seed and division propagation methods are inefficient, and over-reliance on wild resources will exacerbate their depletion.

[0003] Plant tissue culture technology has significant advantages in this regard, enabling the rapid propagation of large numbers of genetically consistent seedlings in a short period of time. However, as a unique and endangered species endemic to China, *Primula tibetica* lacks a complete and publicly available methodology specifically designed for efficient sterilization of explants, induction of shoot clusters, and rooting culture. Its optimal culture medium formulation and conditions remain unknown.

[0004] Existing technologies have yielded numerous methods for rapid in vitro propagation of different genus *Primula*, such as: Publication No. CN101766121A, entitled "A Method for Anther Culture of *Primula simonii*"; Publication No. CN101855995A, entitled "Tissue Culture Propagation Method of *Primula chuandongensis*"; Publication No. CN103004609A, entitled "Tissue Culture Method of *Primula baihuaensis*"; and Publication No. CN103404444A, entitled "*Primula yingyangensis*". Rapid propagation method of spring tissue culture; Publication No.: CN101011033A, Name: Rapid propagation method of Primula jaffreyana by tissue culture; Publication No.: CN101011034A, Name: Rapid propagation method of Primula jaffreyana by tissue culture; Publication No.: CN101015280A, Name: Rapid propagation method of Primula jaffreyana by tissue culture; Publication No.: CN101015279A, Name: Rapid propagation method of Primula jaffreyana by tissue culture; Zhou Guoxing. Tissue culture and rapid propagation of Primula jaffreyana King [D]. Tibet University [2026-01-21]. DOI:CNKI:CDMD:2.1015.953368. Analysis of existing in vitro culture techniques for Primula revealed that tissue culture conditions vary greatly among different species, and existing formulas for certain species cannot be directly applied to Primula tibetica. Furthermore, its endangered status and unique habitat make it difficult to obtain and stably establish a sterile culture system.

[0005] Therefore, developing efficient and sustainable artificial breeding techniques is crucial for the protection and utilization of Tibetan Primrose, and is an effective way to balance its endangered status protection with sustainable resource utilization. Summary of the Invention

[0006] The purpose of this invention is to provide a highly efficient and stable tissue culture rapid propagation system, so as to provide reliable technical support for its germplasm resource preservation, population restoration and industrial development, which is of positive significance for protecting my country's unique biodiversity and horticultural resources.

[0007] To achieve the above objectives, the technical solution adopted by this invention is: a method for rapid propagation of Primula tibetica through tissue culture, comprising the following steps:

[0008] (1) Seed disinfection: The newly harvested seeds of Primula spp. were dried in the shade and used as experimental materials. They were disinfected with 75% alcohol for 20-30 seconds, rinsed with sterile water 4-5 times, and then the explants were treated with 2% sodium hypochlorite and rinsed with sterile water 4-5 times.

[0009] (2) Obtaining sterile seedlings: After sterilization, the seeds are inoculated in seed germination medium and cultured for 25 to 35 days;

[0010] The seed germination medium formula is: 1 / 2 MS + sucrose 15.0-25.0 g / L, pH=5.6-5.8;

[0011] (3) Explant acquisition: The terminal bud of the plant is cut off as an explant. The terminal bud should contain a complete growth point and a pair of fully unfolded young leaves on the periphery.

[0012] (4) Primary shoot induction: The above-mentioned apical buds were inoculated into shoot induction medium and cultured for 21-35 days;

[0013] The formulation of the primary shoot induction medium is as follows: 2B5 + 6-BA 1.0-2.0 mg / L + NAA 0.2 mg / L + sucrose 25.0-30.0 g / L + agar 6 g / L, pH=5.6-5.8;

[0014] (5) Subculture of bud clusters: Cut the induced bud clusters into single buds and inoculate them into subculture culture medium for 45-55 days;

[0015] The formula for the bud proliferation medium is: 2B5 + 6-BA 1.0-2.0 mg / L + NAA 0.2 mg / L + sucrose 25.0-30.0 g / L + agar 6 g / L, pH=5.6-5.8;

[0016] (6) Rooting culture of clustered buds: Cut the subcultured clustered buds into small segments with 1 to 2 buds, and inoculate them into the rooting medium according to polarity and culture for 21 to 28 days;

[0017] The rooting medium is formulated as follows: 1-2B5 + NAA 0.1 mg / L + sucrose 10.0-20.0 g / L, pH=5.6-5.8.

[0018] Furthermore, in steps (2), (4), (5), and (6), the culture conditions are: a temperature of 22±2℃, a light intensity of 1500~2000Lx, and a light-dark alternation environment with 10~12h of sunlight.

[0019] Furthermore, in step (2), the seed germination medium is formulated as follows: 1 / 2 MS + 20.0 g / L sucrose.

[0020] Furthermore, in step (4), the formulation of the primary shoot induction medium is: 2B5 + 6-BA 2.0 mg / L + NAA 0.2 mg / L + sucrose 25.0 g / L + agar 6 g / L.

[0021] Furthermore, in step (6), the rooting medium is formulated as follows: B5 + NAA 0.1 mg / L + sucrose 15.0 g / L.

[0022] The beneficial technical effects of this invention are:

[0023] (1) This invention provides a stable and reproducible method for the efficient and rapid propagation of Primula tibetica. Using this method, efficient cultivation can be achieved, with an average proliferation coefficient of 4.4 to 4.6 for the first generation of bud-inducing clumps and a rooting rate of 83.33% to 93.33%. The root system is well-developed, with an average of more than 10 adventitious roots. This provides a reliable technical solution and data foundation for the industrialization and promotion of Primula tibetica.

[0024] (2) This invention uses clonal propagation induced by clustered buds, which can stably maintain the excellent genetic traits of the maternal parent. The established rapid propagation system can not only provide high-quality seedlings for the construction of the Tibetan Primrose "in vitro germplasm bank" and the large-scale restoration of wild populations, effectively alleviating the dependence on wild resources; but also lays a key technical foundation for its large-scale commercial seedling production and subsequent genetic improvement research, which has dual significance for the protection and sustainable utilization of Tibetan Primrose germplasm resources. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The morphology of explants and shoot clusters in a representative treatment combination at five weeks in Example 1 (MS vs. 2B5 medium).

[0027] Figure 2 This is a comparison of the phenotypes of single-bud proliferation in subculture of Example 2.

[0028] Figure 3 This is the rooting induction sample from Example 3, after 3 weeks.

[0029] Figure 4 This is the case of induction of primary clustered shoots for 35 days in Example 4.

[0030] Figure 5 This is the result of subculture proliferation culture for 55 days in Example 4.

[0031] Figure 6 This is the result of rooting culture for 28 days in Example 4.

[0032] Figure 7This is the case of inducing the first generation of clustered shoots for 35 days in Example 5.

[0033] Figure 8 This is the result of subculture proliferation culture for 55 days in Example 5.

[0034] Figure 9 This is the rooting result of Example 6 after 42 days of rooting culture.

[0035] Figure 10 This is the growth of Primula vulgaris after 42 days of rooting culture in Example 6. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1

[0038] Primary cluster bud induction:

[0039] This embodiment aims to illustrate the preferred culture medium composition for inducing shoot clusters of Primula vulgaris provided by the present invention and its application effects, and to demonstrate its superior effect compared with other conventional formulations.

[0040] 1. Method

[0041] The preferred culture medium composition of this invention is as follows: 2B5 as the basal medium, supplemented with 1–2 mg / L of 6-BA and 0.2 mg / L of NAA, and containing 25–30 g / L sucrose and 6 g / L agar, with the pH adjusted to 5.6–5.8. In use, explants from the terminal buds of Primula vulgaris are inoculated into this medium and cultured at 24°C under 1500–2000 Lx light and 12 h of light-dark alternation. After 5 weeks, the induction rate reached 100%, and the average proliferation coefficient was 4.4–4.6.

[0042] To verify the effectiveness of the formulation, a comparative experiment was conducted. The experiment employed a three-factor full factorial design, with factors including culture medium type (MS, 2B5), 6-BA concentration (1, 2, 3 mg / L), and NAA concentration (0, 0.2, 0.4 mg / L), resulting in a total of 18 treatment combinations. To control system errors and the microenvironment gradient within the culture rack, the experiment used a combination of complete randomization (inoculation according to run order) and spatial randomized block design. Specifically, five replicates of each treatment combination were randomly and evenly distributed across five spatial blocks within the culture rack, ensuring that each block contained one replicate of each treatment combination (randomized block design, see Table 1).

[0043] Culture conditions and methods: The culture was carried out at a temperature of 24℃, a light intensity of 1500 Lx, and a daily light exposure of 12 h. The number of clustered shoots in each culture bottle was counted at 3 and 5 weeks of culture.

[0044] Table 1. Randomized block design for screening primary shoot cluster culture media

[0045] Standard sequence Run sequence culture medium 6-BA (mg / L) NAA (mg / L) Processing combined identifiers 87 1 2B5 2 0.4 2B5-2-0.4 27 2 MS 3 0.4 MS-3-0.4 33 3 2B5 2 0.4 2B5-2-0.4 ... ... ... ... ... ... 16 88 2B5 3 0 2B5-3-0 68 89 2B5 2 0.2 2B5-2-0.2 64 90 2B5 1 0 2B5-1-0

[0046] 2. Observation and Statistics

[0047] During the cultivation period, observations and statistics were conducted at weeks 3 and 5. At week 3, the number of visible buds (≥ 2 mm in length) was counted and classified into three levels: Level 0 (none), Level 1 (< 3), and Level 2 (≥ 3). At week 5, the actual number of buds (statistical criteria are shown in Table 2 notes) was counted, and morphological changes in the explants were recorded. Their health status was scored (0-4 levels, scoring criteria are shown in Table 2 notes). Relevant data and typical conditions are shown in Tables 2 and 5, respectively. Figure 1 Finally, statistical analysis was used to verify the optimal culture medium formulation for inducing shoot clusters.

[0048] 3. Results

[0049] At week 3 of culture, sequential logistic regression analysis showed that none of the factors and their interactions had a significant effect on shoot induction (P > 0.05). At week 5, generalized factor regression analysis showed that NAA concentration had a highly significant effect on the number of induced shoots (P < 0.001), and there was a significant interaction with the culture medium type (P < 0.05); after removing outliers, the interaction between 6-BA and NAA was also significant (P = 0.034). Further analysis of variance and multiple comparisons showed highly significant differences in the number of induced shoots among different treatment combinations (P < 0.001). The combination (2B5-2-0.2) had the highest number of induced shoots (mean 4.6), followed by the combination (2B5-1-0.2) (mean 4.4), both significantly better than all other treatments. The overall induction levels of all treatments under MS medium were low.

[0050] Table 2. Descriptive statistics of screening treatment combinations for primary shoot clusters in culture medium.

[0051]

[0052] Table Notes:

[0053] I. Statistical criteria for 5-week-old clustered buds: Only buds with intact morphology and new leaf length ≥ 6 mm were selected for statistical analysis.

[0054] II, N: Biological replication number.

[0055] III. Data in the same column marked with the same capital letter indicate that the difference is not significant at the P < 0.05 level according to Tukey's multiple comparison test.

[0056] IV. Scoring criteria for explant morphology: 1. No obvious changes, maintaining the state at the time of inoculation; 2. Slight chlorosis or slight wrinkling of leaf margins, but most tissues are healthy; 3. Obvious wrinkling and curling, or partial browning and yellowing; 4. Severe wrinkling, browning, transparency (water-soaked), or complete death.

[0057] 5. Conclusion

[0058] This embodiment verifies the comprehensive advantages of the primary shoot induction medium composition provided by the present invention. The composition uses 2B5 as the basal medium and contains 1-2 mg / L 6-BA and 0.2 mg / L NAA. Results show that this composition can simultaneously achieve high induction rates and high-quality regenerated seedlings. Specifically, NAA is the key factor in inducing shoot clusters in this scheme, with the most significant effect at a concentration of 0.2 mg / L, and it needs to work synergistically with 6-BA to improve efficiency. Regarding explant health, the 2B5 medium used in this scheme is significantly superior to the conventional MS medium: by week 5, the explants in the 2B5 medium ( Figure 1 The leaves of the FJ plant were robust and dark green, with a morphological health score close to 0; while the explants in the MS medium ( Figure 1 The degree of wrinkling of AE was higher, and the rate of deformity of new leaves was generally increased (Table 2). This confirms that the combination of (2B5 + 6-BA 1~2 mg / L + NAA 0.2 mg / L) described in this invention has the comprehensive advantages of high efficiency and high quality in inducing primary shoots of Primula spp., providing a reliable solution for the rapid tissue culture propagation of this species.

[0059] Example 2

[0060] Clustered bud proliferation culture:

[0061] This embodiment aims to illustrate that by using the preferred culture medium composition of the present invention to subculture the buds of Primula spp., robust seedlings with good budding characteristics can be stably obtained.

[0062] 1. Method

[0063] The robust and uniformly sized bud clusters of Primula spp. induced in Example 1 were used as explants. To investigate the applicability of culture media that performed well in the induction stage to the proliferation stage, the three culture media with the highest bud cluster induction rates in Example 1 (denoted as proliferation media A, B, and C) were selected for subculture proliferation:

[0064] Formula A: 2B5 + 6-BA 1 mg / L + NAA 0.2 mg / L;

[0065] Formula B: 2B5 + 6-BA 2 mg / L + NAA 0.2 mg / L;

[0066] Formula C: 2B5 + 6-BA 3 mg / L + NAA 0.4 mg / L;

[0067] Each formulation was used as one treatment, and each treatment was repeated five times, with three single shoot clusters inoculated into each bottle. The culture conditions were the same as in Example 1 (temperature 24°C, light intensity 1500–2000 Lx, 12 h light per day). The culture was continued for approximately 8 weeks, and the proliferation and growth of shoot clusters in each treatment were systematically observed and recorded.

[0068] 2. Results

[0069] During and at the end of the cultivation period ( Figure 2 Morphological observations showed that, within the same 8-week culture period, there were significant differences in robustness, leaf development, and clustering characteristics among the different formulations of the shoots. Among them, the shoots subcultured using formulations A and B (i.e., a combination of 1–2 mg / L 6-BA and 0.2 mg / L NAA) exhibited the most robust plants, with well-developed, dark green leaves, and a greater number of compact shoots, demonstrating significantly better overall performance than the formulation C treatment group.

[0070] 3. Conclusion

[0071] This embodiment demonstrates that the optimal culture medium composition (2B5 + 6-BA 1-2 mg / L + NAA 0.2 mg / L) provided by the present invention performs excellently in subculture and can continuously and stably obtain robust proliferating seedlings, providing a reliable solution for the large-scale rapid propagation of tissue culture seedlings of this species.

[0072] Example 3

[0073] Clumped bud rooting culture:

[0074] This embodiment verifies, through comparative experiments, the significant advantages of the preferred rooting culture medium formula (B5~2B5 + NAA0.1 mg / L + sucrose10~20g / L) provided by the present invention in promoting the rooting quality and efficiency of Primula spp. tissue culture seedlings.

[0075] 1. Method

[0076] A two-factor full factorial design was employed, with factors including culture medium type (2B5, B5) and NAA concentration (0.0, 0.1, 0.2 mg / L), resulting in six treatment combinations, each with 10 replicates, for a total of 60 experimental units. Each unit was inoculated with three shoot clusters. The inoculation order strictly followed the randomized run sequence generated by Minitab22, and all culture flasks were placed completely randomly on the tissue culture racks after inoculation. To control for variations in the microenvironment, a randomized block design was used, dividing the culture racks into 12 blocks (rows) to ensure that the 10 replicates of each treatment combination were randomly distributed across different blocks. Rooting was observed at 10 days and 3 weeks post-inoculation, and the data were analyzed using a generalized factor regression model.

[0077] Table 3 Randomized block design for screening rooting media

[0078] Standard sequence Run sequence culture medium NAA (mg / L) Processing combined identifiers 6 1 B5 0.2 B5-0.2 18 2 B5 0.2 B5-0.2 37 3 2B5 0 2B5-0 ... ... ... ... ... 35 58 B5 0.1 B5-0.1 58 59 B5 0 B5-0 45 60 2B5 0.2 2B5-0.2

[0079] Table 4. Descriptive statistics of screening treatment combinations for rooting culture medium

[0080]

[0081] Table Notes:

[0082] I, N: Biological replication number.

[0083] II. Plants / bottle: The standard for "successful rooting" is the growth of healthy roots with a length of ≥5mm. The number of successfully rooted plants in each replicate is counted, and the average number of rooted plants in each treatment group is calculated.

[0084] III. Data in the same column marked with the same capital letter indicate that the difference is not significant at the P < 0.05 level according to Tukey's multiple comparison test.

[0085] 2. Results

[0086] Generalized factor regression analysis showed that, 10 days post-inoculation, both the culture medium type and NAA concentration had a highly significant effect on rooting (culture medium: P = 0.010; NAA: P < 0.001). Tukey's multiple comparisons (Table 4) further indicated that, without NAA, the B5 medium treatment (B5-0.0) produced the highest number of roots (mean 1.5), and was significantly different from all treatments with added NAA (0.1 or 0.2 mg / L) (groups labeled A vs. B); while the 2B5 medium (2B5-0.0, mean 0.9) performed second best without NAA. It was also found that, at this stage, the addition of NAA (0.1 or 0.2 mg / L), regardless of the culture medium used, significantly inhibited rooting.

[0087] However, this early difference did not persist. By week 3 of cultivation, the generalized factor regression model was no longer significant (P = 0.160), and the Tukey test also confirmed that there was no significant difference in the number of roots among all treatment combinations (all were grouped into the same group D), indicating that the early inhibitory effect disappeared over time. Morphological observation ( Figure 3 The results showed that the roots of the treatments without NAA (2B5-0.0 and B5-0.0) were thin, few, and messy; while the treatments with NAA (especially 0.1 mg / L) had a large root biomass (average number of adventitious roots >10), short and thick roots that radiated outward from the center, and a more regular overall arrangement, showing better growth potential.

[0088] 3. Conclusion

[0089] This embodiment verifies the excellent effect of the rooting medium composition provided by the present invention, which comprises 1-2B5 basal medium, 0.1 mg / L NAA, and 10-20 g / L sucrose. The results show that after 3 weeks of culture using this composition, explants can efficiently form high-quality roots. Specifically, when using 2B5 medium, the average number of rooted plants reached 2.8 per bottle, with a rooting rate of 93.33%; when using B5 medium, the average number of rooted plants reached 2.5 per bottle, with a rooting rate of 83.33%. The roots obtained in the above embodiments all exhibit significant characteristics of large biomass, short and thick morphology, and a radially regular arrangement, with a significantly better overall effect than the control group without NAA. This fully demonstrates the key role of 0.1 mg / L NAA in improving root quality and rooting efficiency in the composition of the present invention, and the high applicability of 1-2B5 medium within the framework of this scheme.

[0090] Example 4

[0091] A rapid propagation method for Primula tsao-ko tissue culture, comprising the following steps:

[0092] (1) Seed disinfection: The newly harvested seeds of Primula spp. were dried in the shade and used as experimental materials. They were disinfected with 75% alcohol for 20 seconds, rinsed with sterile water 4 times, and then the explants were treated with 2% sodium hypochlorite and rinsed with sterile water 4 times.

[0093] (2) Obtaining sterile seedlings: The sterilized seeds were inoculated in seed germination medium (formulation: 1 / 2 MS + 15.0 g / L sucrose, pH=5.6) and cultured for 25 days under alternating light and dark conditions with a temperature of 22±2℃, a light intensity of 1500 Lx, and 12 h of sunlight.

[0094] (3) Explant acquisition: The terminal bud of the plant is cut off as an explant. The terminal bud should contain a complete growth point and a pair of fully unfolded young leaves on the periphery.

[0095] (4) Primary shoot induction: The above-mentioned apical buds were inoculated into shoot induction medium (formulation: 2B5 + 6-BA 2.0 mg / L + NAA 0.2 mg / L + sucrose 30.0 g / L + agar 6 g / L, pH=5.6), and cultured for 35 days under alternating light and dark conditions with a temperature of 22±2℃, a light intensity of 1500 Lx, and 12 h of daylight. The results are as follows. Figure 4 As shown.

[0096] (5) Subculture of bud clusters: The induced bud clusters were cut into single buds and inoculated into a subculture culture medium (2B5 + 6-BA 2.0 mg / L + NAA 0.2 mg / L + sucrose 25.0 g / L + agar 6 g / L, pH=5.6). The medium was cultured for 55 days under alternating light and dark conditions at a temperature of 22±2℃, a light intensity of 1500 Lx, and 12 h of daylight. The culture results are as follows. Figure 5 As shown.

[0097] (6) Rooting culture of clustered shoots: The subcultured clustered shoots were cut into small segments with 1-2 buds each, and inoculated into rooting medium (formulation: B5 + NAA 0.1 mg / L + sucrose 15.0 g / L) according to polarity. The culture was carried out for 28 days under alternating light and dark conditions at a temperature of 22±2℃, a light intensity of 1500 Lx, and 12 h of daylight. The culture results are as follows. Figure 6 As shown.

[0098] Example 5

[0099] A rapid propagation method for Primula tsao-ko tissue culture, comprising the following steps:

[0100] (1) Seed disinfection: The newly harvested seeds of Primula spp. were dried in the shade and used as experimental materials. They were disinfected with 75% alcohol for 25 seconds, rinsed with sterile water 5 times, and then the explants were treated with 2% sodium hypochlorite and rinsed with sterile water 5 times.

[0101] (2) Obtaining sterile seedlings: After sterilization, the seeds were inoculated into seed germination medium (formulation: 1 / 2 MS + 20.0 g / L sucrose, pH=5.7) and cultured for 30 days under alternating light and dark conditions with a temperature of 22±2℃, a light intensity of 2000 Lx, and 10 h of sunlight.

[0102] (3) Explant acquisition: The terminal bud of the plant is cut off as an explant. The terminal bud should contain a complete growth point and a pair of fully unfolded young leaves on the periphery.

[0103] (4) Primary shoot induction: The above-mentioned apical buds were inoculated into shoot induction medium (2B5 + 6-BA 1.0 mg / L + NAA 0.2 mg / L + sucrose 30.0 g / L + agar 6 g / L, pH=5.7), and cultured for 35 days under alternating light and dark conditions with a temperature of 22±2℃, a light intensity of 2000 Lx, and 10 h of daylight. The culture results are as follows. Figure 7 As shown.

[0104] (5) Subculture of bud clusters: The induced bud clusters were cut into single buds and inoculated into a subculture culture medium (formulation: 2B5 + 6-BA 1.0 mg / L + NAA 0.2 mg / L + sucrose 30 g / L + agar 6 g / L, pH=5.7). The medium was cultured for 55 days under alternating light and dark conditions at a temperature of 22±2℃, a light intensity of 2000 Lx, and 10 h of daylight. The culture results are as follows. Figure 8 As shown.

[0105] (6) Rooting culture of clustered shoots: The subcultured clustered shoots were cut into small segments with 1 to 2 buds and inoculated into rooting medium (formulation: 2B5 + NAA 0.1 mg / L + sucrose 15 g / L, pH=5.7) according to polarity. The medium was cultured for 21 days under alternating light and dark conditions with a temperature of 22±2℃, a light intensity of 2000 Lx, and 10 h of sunlight.

[0106] Example 6

[0107] A rapid propagation method for Primula tsao-ko tissue culture, comprising the following steps:

[0108] (1) Seed disinfection: The newly harvested seeds of Primula spp. were dried in the shade and used as experimental materials. They were disinfected with 75% alcohol for 30 seconds, rinsed with sterile water 5 times, and then the explants were treated with 2% sodium hypochlorite and rinsed with sterile water 5 times.

[0109] (2) Acquisition of sterile seedlings: The sterilized seeds were inoculated in seed germination medium (formulation: 1 / 2 MS + 25.0 g / L sucrose, pH=5.8) and cultured for 35 days under alternating light and dark conditions with a temperature of 22±2℃, a light intensity of 1500 Lx, and 12 h of sunlight.

[0110] (3) Explant acquisition: The terminal bud of the plant is cut off as an explant. The terminal bud should contain a complete growth point and a pair of fully unfolded young leaves on the periphery.

[0111] (4) Primary shoot induction: The above-mentioned apical buds were inoculated into shoot induction medium (formulation: 2B5 + 6-BA 2.0 mg / L + NAA 0.2 mg / L + sucrose 30.0 g / L + agar 6 g / L, pH=5.8), and cultured for 35 days under alternating light and dark conditions with a temperature of 22±2℃, a light intensity of 2000 Lx, and 12 h of sunlight.

[0112] (5) Subculture of bud clusters: The induced bud clusters were cut into single buds and inoculated into subculture culture medium (formulation: 2B5 + 6-BA 1.0 mg / L + NAA 0.2 mg / L + sucrose 30.0 g / L + agar 6 g / L, pH=5.8). The medium was cultured for 45 days under alternating light and dark conditions with a temperature of 22±2℃, a light intensity of 2000 Lx, and 12 h of sunlight.

[0113] (6) Rooting culture of clustered shoots: The subcultured clustered shoots were cut into small segments with 1 to 2 buds and inoculated into rooting medium (formulation: 1.5B5 + NAA 0.1 mg / L + sucrose 20.0 g / L, pH=5.8) according to polarity. The temperature was 22±2℃, the light intensity was 2000Lx, and the light intensity was 12h. The culture was carried out under alternating light and dark conditions.

[0114] To clarify the upper limit of the optimal transplanting period, this example observed rooted seedlings cultured for 42 days. Their root systems maintained their initial radiating branching morphology and were healthy. However, the main problem was that as the culture time increased, the overall plant size significantly increased, and the leaves, whose growth was restricted in the sealed culture container, became brittle and easily subjected to mechanical damage or breakage upon transplanting. Figure 10 Meanwhile, multiple radial taproots continue to grow linearly, leading to severe physical interweaving, layering, and space competition within the limited cultivation space, significantly increasing the overall complexity of the root system. Figure 9 In contrast, seedlings cultivated for 21-28 days have moderately sized plants, robust leaves that are not easily damaged, and their radiating root systems are of moderate length, spatially independent, and easily separable. Figure 3 , 6 This is the ideal form to ensure transplanting efficiency and survival rate. Therefore, limiting the transplanting time window to 21-28 days is the best balance between ensuring the robustness of the plant and root system, maintaining the ideal spatial configuration, and maximizing the feasibility of transplanting operations.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for rapid propagation of Primula vulgaris through tissue culture, characterized by the following steps: include: (1) Seed disinfection: The newly harvested seeds of Primula spp. were dried in the shade and used as experimental materials. They were disinfected with 75% alcohol for 20-30 seconds, rinsed with sterile water 4-5 times, and then the explants were treated with 2% sodium hypochlorite and rinsed with sterile water 4-5 times. (2) Obtaining sterile seedlings: After sterilization, the seeds are inoculated in seed germination medium and cultured for 25 to 35 days; The seed germination medium formula is: 1 / 2 MS + sucrose 15.0-25.0 g / L, pH=5.6-5.8; (3) Explant acquisition: The terminal bud of the plant is cut off as an explant. The terminal bud should contain a complete growth point and a pair of fully unfolded young leaves on the periphery. (4) Primary shoot induction: The above-mentioned apical buds were inoculated into shoot induction medium and cultured for 21 to 35 days; The formulation of the primary shoot induction medium is as follows: 2B5 + 6-BA 1.0-2.0 mg / L + NAA 0.2 mg / L + sucrose 25.0-30.0 g / L + agar 6 g / L, pH=5.6-5.8; (5) Subculture of bud clusters: Cut the induced bud clusters into single buds and inoculate them into subculture culture medium for 45-55 days; The formula for the bud proliferation medium is: 2B5 + 6-BA 1.0-2.0 mg / L + NAA 0.2 mg / L + sucrose 25.0-30.0 g / L + agar 6 g / L, pH=5.6-5.8; (6) Rooting culture of clustered buds: Cut the subcultured clustered buds into small segments with 1 to 2 buds, and inoculate them into the rooting medium according to polarity and culture for 21 to 28 days; The rooting medium is formulated as follows: 1-2B5 + NAA 0.1 mg / L + sucrose 10.0-20.0 g / L, pH=5.6-5.

8.

2. The method according to claim 1, characterized in that, In steps (2), (4), (5), and (6), the culture conditions are: temperature of 22±2℃, light intensity of 1500~2000Lx, and alternating light and dark conditions with 10~12h of sunlight.

3. The method according to claim 1, characterized in that, In step (2), the seed germination medium is formulated as follows: 1 / 2 MS + 20.0 g / L sucrose.

4. The method according to claim 1, characterized in that, In step (4), the formulation of the primary shoot induction medium is: 2B5 + 6-BA 2.0 mg / L + NAA 0.2 mg / L + sucrose 25.0 g / L + agar 6 g / L.

5. The method according to claim 1, characterized in that, In step (6), the rooting medium is formulated as follows: B5 + NAA 0.1 mg / L + sucrose 15.0 g / L.

6. The application of the method according to any one of claims 1-2 in the rapid propagation of Primula stenoptera tissue culture.

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