Culture method of Primula spp. callus in Sikkim

Abstract

This invention relates to plant tissue culture and discloses a method for culturing callus tissue of Primula sikkiens. The steps include: (1) inducing callus tissue: leaves, roots, or stems of aseptic seedlings of Primula sikkiens are inoculated into an induction medium to obtain callus tissue of Primula sikkiens; (2) proliferation culture: the callus tissue of Primula sikkiens is inoculated into a proliferation medium and cultured for 25-60 days. This invention, by selecting a culture medium and plant growth hormones, uses aseptic seedlings to induce callus tissue and then proliferates it to obtain embryogenic callus tissue with high proliferation rate, short growth cycle, and rich polyphenols and flavonoids. It can proliferate in large quantities, providing a material supply foundation for the commercial application of Primula sikkiens in the pharmaceutical, food, or cosmetic industries.

Description

Technical Field

[0001] This invention belongs to the technical field of plant tissue culture methods, specifically relating to a method for culturing callus tissue of Primula sikimi. Background Technology

[0002] Primroses are world-renowned ornamental flowers, prized for their diverse colors and high ornamental value. Along with rhododendrons, gentians, and meconopsis, they are considered the four major alpine flowers. There are approximately 500 species worldwide. China boasts a wide variety of primroses, primarily found in Yunnan and southern Tibet. However, most of these resources remain wild, making collection and research difficult, and many species await further development and utilization.

[0003] Primula sikkimensis, also known as bell-shaped primrose, is a plant belonging to the genus Primula in the family Primulaceae. It has yellow, bell-shaped flowers that cluster at the top of the flower stalk. It grows in forest edges, wetlands, marshes, meadows, and ditches at altitudes of 3200-4400 meters, and is endemic to western Sichuan, northwestern Yunnan, and Tibet in my country. It is endemic to the Hengduan Mountains and the eastern Himalayas. Primula sikkimensis is also a common Tibetan medicine, primarily containing flavonoids such as quercetin, kaempferol, and myricetin. It is one of the original plants used in the Tibetan medicine "Xiangzhe Sebao," mainly for clearing heat, reducing swelling, stopping diarrhea, and promoting blood circulation.

[0004] Currently, there is a lot of research on the chemical composition, pharmacological effects and genetic variations of Primula sikkiensis both at home and abroad, but there is less research on its application in plant tissue culture and aseptic propagation.

[0005] Jia Yin et al. (CN111492979A) induced callus tissue in *Primula forbesii*, but the formulations of the induction and proliferation media differed for different species of *Primula*. Zhang Qixiang et al. (CN101965796) invented a rapid propagation method for *Primula saxatilis*, which involves inducing callus tissue from the leaves and petioles of clustered buds, and then allowing the callus tissue to differentiate into adventitious buds. This method was mainly aimed at obtaining aseptic seedlings quickly, but the callus induction rate did not exceed 30%, and the proliferation rate of the callus tissue was not studied.

[0006] Deng Pingping (Study on Tissue Culture and Polyploid Induction of Six Primula Species, 2006) induced callus tissue from leaves of Primula sikkiensis, but the induction rate was very low, reaching a maximum of only 16.5%. Furthermore, the induced callus tissue was either dense or loosely transparent and brown, making further subculture or proliferation difficult. Moreover, Primula sikkiensis is currently still a wild species, and there has been no large-scale commercial introduction and cultivation in China.

[0007] Therefore, there is a need to develop a cultivation method that can rapidly obtain large quantities of Primula tinctoria callus to meet the raw material requirements for pharmaceutical, edible, or daily chemical products. Summary of the Invention

[0008] This invention aims to provide a method for culturing Primula sigilis callus.

[0009] The method for culturing Primula callus from Sikkim includes the following steps:

[0010] (1) Induction of callus: leaves, roots or stems of aseptic seedlings of Primula sikinensis are inoculated into induction medium to obtain callus of Primula sikinensis; preferably, the culture time is 10-25 days.

[0011] (2) Proliferation culture: The callus tissue of Primula sikinensis was inoculated into the proliferation culture medium and cultured for 25-60 days.

[0012] In step (1), the induction medium is a 1 / 2 MS medium containing 1-5 mg / L 2,4-dichlorophenoxyacetic acid and 1-3 mg / L α-naphthylacetic acid, or a 1 / 2 MS medium containing 1-5 mg / L 2,4-dichlorophenoxyacetic acid and 1-3 mg / L kinetin, or a B5 medium containing 0.5-2 mg / L 2,4-dichlorophenoxyacetic acid and 0.5-2 mg / L α-naphthylacetic acid. It also contains 25-35 g / L sucrose. Preferably, it also contains 3-3.5 g / L gellan gum.

[0013] The pH of the induction medium is 5.6-6, preferably 5.7-5.8.

[0014] More preferably, the induction medium is a 1 / 2 MS medium containing 1-5 mg / L 2,4-dichlorophenoxyacetic acid and 1-2 mg / L α-naphthylacetic acid, or a 1 / 2 MS medium containing 3-5 mg / L 2,4-dichlorophenoxyacetic acid and 1.5-2.5 mg / L kinetin, or a B5 medium containing 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid and 0.5-1.5 mg / L α-naphthylacetic acid.

[0015] In a preferred embodiment of the invention, the induction medium is a 1 / 2 MS medium containing 1-4 mg / L 2,4-dichlorophenoxyacetic acid and 1-2 mg / L α-naphthylacetic acid, or a 1 / 2 MS medium containing 4 mg / L 2,4-dichlorophenoxyacetic acid and 2 mg / L kinetin, or a B5 medium containing 1 mg / L 2,4-dichlorophenoxyacetic acid and 1 mg / L α-naphthylacetic acid. The induction medium also contains 30 g / L sucrose and 3-3.5 g / L gellan gum.

[0016] Preferably, in step (1), the roots of the aseptic seedlings of Primula sikinensis are inoculated into the induction culture medium to obtain callus tissue of Primula sikinensis.

[0017] In step (2), the proliferation culture medium is:

[0018] (a) 1 / 2 MS medium containing 1-7 mg / L 2,4-dichlorophenoxyacetic acid and 0.3-4 mg / L kinetin; or,

[0019] (b); SH medium or B5 medium containing 2-6 mg / L 2,4-dichlorophenoxyacetic acid and 1-4 mg / L kinetin; or,

[0020] (c) SH medium or B5 medium containing 0.5–2 mg / L 6-benzylaminopurine (6BA) and 0.3–1.5 mg / L α-naphthaleneacetic acid; or,

[0021] (d) SH medium or B5 medium containing 0.3-1.5 mg / L thiazuron (TDZ) and 0.1-0.5 mg / L α-naphthaleneacetic acid (NAA).

[0022] The proliferation medium also contains 25-35 g / L sucrose. Preferably, it also contains 3-3.5 g / L gellan gum. The pH of the proliferation medium is 5.6-6, preferably 5.7-5.8.

[0023] Preferably, in combination (a), when using 1 / MS medium, the content of 2,4-dichlorophenoxyacetic acid is 2-6 mg / L and the content of kinetin is 0.5-3 mg / L; when using SH medium or B5 medium, the content of 2,4-dichlorophenoxyacetic acid is 3.5-4.5 mg / L and the content of kinetin is 2-3 mg / L; more preferably, when using 1 / MS medium, the content of 2,4-dichlorophenoxyacetic acid is 2-5 mg / L and the content of kinetin is 0.5-3 mg / L.

[0024] Preferably, in combination (b), the content of 2,4-dichlorophenoxyacetic acid is 3.5-4.5 mg / L, and the content of kinetin is 2-3 mg / L;

[0025] Preferably, in combination (c), the content of 6-benzylaminopurine is 0.8-1.2 mg / L and the content of α-naphthaleneacetic acid is 0.5-1 mg / L; furthermore, when using SH medium, the content of 6-benzylaminopurine is 0.8-1.2 mg / L and the content of α-naphthaleneacetic acid is 0.3-0.6 mg / L; when using B5 medium, the content of 6-benzylaminopurine is 0.8-1.2 mg / L and the content of α-naphthaleneacetic acid is 0.8-1.2 mg / L.

[0026] In combination (d), the content of thiabendazole was 0.3-0.6 mg / L and the content of α-naphthaleneacetic acid was 0.2-0.4 mg / L.

[0027] When using culture medium of combination (a), the culture time is 50-60 days. When using culture medium of combinations (b), (c), or (d), the culture time is 28-35 days.

[0028] In a preferred embodiment of the present invention, the proliferation medium in step (2) is: 1 / 2 MS medium containing 1-6 mg / L 2,4-dichlorophenoxyacetic acid and 0.5-2 mg / L kinetin; or SH medium or B5 medium containing 4 mg / L 2,4-dichlorophenoxyacetic acid and 3 mg / L kinetin; or SH medium containing 1 mg / L 6-benzylaminopurine and 0.5 mg / L α-naphthylacetic acid; or B5 medium containing 1 mg / L 6-benzylaminopurine and 1 mg / L α-naphthylacetic acid (NAA); or SH medium or B5 medium containing 0.5 mg / L thidiazuron and 0.3 mg / L α-naphthylacetic acid. The proliferation medium also contains 25-35 g / L sucrose, preferably 30 g / L. Preferably, it also contains 3-3.5 g / L gellan gum.

[0029] Using the above-mentioned culture medium, the proliferation coefficient can reach over 2.5.

[0030] The cultivation conditions for steps (1) and (2) are: temperature 20±1℃, humidity 50%~70%, light exposure 10-15 hours per day, and light intensity 1500Lx~2000Lx. Preferably, the light exposure time is 12 hours per day.

[0031] In step (1), the sterile seedlings are obtained by the following method: Primula sikkimica seeds are treated with gibberellin and then sterilized before being inoculated onto a sterile aqueous medium. After germination, the seeds are transferred to a culture medium for further cultivation. The culture medium is 2 / 3 MS medium. Preferably, it contains 25-35 g / L sucrose. Preferably, it also contains 3-3.5 g / L gellan gum.

[0032] Preferably, the seeds are treated with 10-50 mg / L gibberellin for 18-36 hours.

[0033] The above method can quickly obtain a large amount of callus tissue from Primula sikkimensis, and tests have shown that it contains abundant polyphenols and flavonoids.

[0034] This method produces Primula sikkieri callus with a high proliferation coefficient and short cycle, resulting in well-formed Primula sikkieri callus. Furthermore, it contains high levels of active ingredients such as total polyphenols and total flavonoids, meeting the requirements of the pharmaceutical, food, and cosmetic industries.

[0035] This invention utilizes plant tissue cell technology. First, seeds of *Primula sikinensis* from Tibet are germinated to establish a sterile propagation system. Then, sterile seedlings are selected for callus induction, resulting in embryogenic callus tissue with high proliferation rate, short growth cycle, and abundant polyphenols and flavonoids. This tissue can proliferate in large quantities, providing a material supply foundation for the commercial application of *Primula sikinensis* in the pharmaceutical, food, and cosmetic industries. The callus tissue obtained using this method is safe and non-toxic, with a short cycle, rapid propagation, simple operation, and low cost. Furthermore, it protects wild primrose resources from destruction, promoting the rational utilization and sustainable development of resources. Moreover, the controllable culture conditions of this method facilitate the acquisition of stable-quality *Primula sikinensis* resources, demonstrating promising application prospects. Attached Figure Description

[0036] Figure 1 The images show aseptic seedlings and callus of Primula sikkimica, where A represents aseptic seedlings of Primula sikkimica, B represents callus induced from roots, and C represents proliferating callus. Detailed Implementation

[0037] Culture medium preparation and culture conditions

[0038] Take one of the following culture media (1 / 2 MS, MS, SH, B5, Heller, WPM, and White) and mix it with different plant growth regulators: α-naphthaleneacetic acid (NAA), 6-benzylaminopurine (6-BA), 2,4-dichlorophenoxyacetic acid (2,4-D), cytokinin (Meta-Topolin, mT), thidiazuron (TDZ), and kinetin (KT). Prepare the culture medium according to a certain ratio, add 30 g / L sucrose and 3-3.5 g / L gellan gum, adjust the pH to 5.7-5.8, and sterilize in an autoclave at 121℃ for 20 min for later use.

[0039] All plant explants were cultured under the following conditions: temperature 20±1℃, humidity 50%~70%, 12 hours of light per day, and light intensity 1500Lx~2000Lx.

[0040] Example 1 Seed disinfection and germination

[0041] Seeds of wild Primula sibiricum were treated with 10-50 mg / L gibberellin for 24 hours, then sterilized under aseptic conditions with 70% ethanol for 30 seconds, rinsed three times with sterile water, and then sterilized with 10% sodium hypochlorite solution containing 1-2 drops of Tween 20 for 12-24 minutes. After rinsing 3-5 times with sterile water, the seeds were inoculated onto sterile, water-soaked cotton. Seeds germinated after 11 days. Once two leaves had emerged, the seeds were transferred to 2 / 3 MS medium (containing 30 g / L sucrose and 3-3.5 g / L gellan gum) for further culture. Sterile seedlings were obtained after 5-15 days of growth. Figure 1 A.

[0042] Example 2: Induction of callus tissue

[0043] In a clean bench, using a sterile scalpel, the aseptic seedlings of Primula sinensis (Sikkim) were removed. Figure 1 A) Leaves, stems, and roots were inoculated into different pre-designed culture media, each supplemented with 30 g / L sucrose and 3-3.5 g / L gellan gum. After 20-30 days of culture, the induction was observed and the callus induction rate was calculated. The induction rate was calculated as follows:

[0044] Callus induction rate = (Number of inducible explants / Number of inoculated explants) × 100%

[0045] The results are shown in Table 1. Callus tissue could be induced in media 1-3 and 14, with induction rates exceeding 20%. Media 2 had the highest induction rate, reaching 55%. The callus tissue obtained through root induction is shown in Table 1. Figure 1 B.

[0046] That is, 1 / 2MS + 2,4-D 4.0 mg / L + KT 2.0 mg / L, or 1 / 2MS or B5 + 2,4-D 1.0~4.0 mg / L + NAA 1.0~2.0 mg / L can be used as induction medium.

[0047] Table 1. Induction of callus tissue in *Prunus cerasifera*

[0048] 1 1 / 2MS+2,4-D 4.0mg / L+KT 2.0mg / L 33％ 2 1 / 2MS+2,4-D 1.0mg / L+NAA1.0mg / L 55％ 3 1 / 2MS+2,4-D 4.0mg / L+NAA2.0mg / L 27％ 4 1 / 2 MS + NAA 1.0 mg / L + TDZ 2.0 mg / L 12％ 5 1 / 2 MS + NAA 0.3 mg / L + TDZ 3.0 mg / L 0％ 6 1 / 2MS + TDZ 2.0 mg / L + NAA 0.5 mg / L 0％ 7 MS + 2,4-D 1.0 mg / L + NAA 1.0 mg / L 13％ 8 MS + 2,4-D 4.0 mg / L + NAA 2.0 mg / L 16％ 9 MS + 2,4-D 4.0 mg / L + KT 2.0 mg / L 4％ 10 MS + TDZ 2.0 mg / L + NAA 0.5 mg / L 0％ 11 MS + TDZ 2.0 mg / L + NAA 1.0 mg / L 0％ 12 MS + TDZ 3.0 mg / L + NAA 0.3 mg / L 0％ 13 B5 + TDZ 2.0 mg / L + NAA 0.5 mg / L 0％ 14 Vitamin B5 + 2,4-D 1.0 mg / L + NAA 1.0 mg / L 22％ 15 Vitamin B5 + 2,4-D 4.0 mg / L + NAA 2.0 mg / L 12％ 16 B5 + TDZ 2.0 mg / L + NAA 1.0 mg / L 2％ 17 B5 + TDZ 3.0 mg / L + NAA 0.3 mg / L 7％

[0049] Example 3: Screening and proliferation of embryogenic callus (1)

[0050] Take the callus tissue induced from the roots of sterile seedlings in Example 2 above. Figure 1 B) Subculture was performed, and well-grown, uniformly morphologically homogeneous callus tissues were selected and inoculated into 1 / 2 MS medium containing different concentrations of 2,4-D and KT. Each medium was supplemented with 30 g / L sucrose and 3-3.5 g / L gellan gum. Three flasks were inoculated for each treatment. After 52 days of culture, cell status was observed and the proliferation coefficient was calculated. The calculation method was as follows:

[0051] Proliferation coefficient = Weight of callus after proliferation / Weight of callus before proliferation

[0052] The results are shown in Table 2. Under 1 / 2 MS and different concentrations of 2,4-D and KT combinations, the proliferation coefficient of Primula callus ranged from 2.4 to 2.9. The callus was yellow, soft, and viscous, with a long proliferation cycle.

[0053] Table 2. Effects of different culture medium formulations on the growth of Primula callus.

[0054]

[0055] Example 4: Screening and proliferation of embryogenic callus culture medium (2)

[0056] To screen suitable basal culture media for the growth of Primula sikinensis, callus tissues with good growth and uniform morphology from Example 2 were inoculated into 1 / 2 MS, MS, SH, B5, Heller, WPM, and White media, respectively, supplemented with 2,4-D 4.0 mg / L, KT 3.0 mg / L, sucrose 30 g / L, and gellan gum 3-3.5 g / L. Three bottles were inoculated into each medium, and cell growth was observed and proliferation coefficients were calculated after 30 days of culture. The results are shown in Table 3: the proliferation coefficients of SH and B5 media were higher, at 2.9 and 2.8, respectively. Compared with Example 2, the proliferation cycle of callus tissues was shortened from 52 days to 30 days, and the soft and pliable texture was improved.

[0057] Table 3. Effects of different basal culture media on the growth of Primula callus.

[0058]

[0059] The next step will be to select SH or B5 basal culture medium in combination with other plant growth regulators for callus screening experiments.

[0060] Example 5: Screening and proliferation of embryogenic callus culture medium (3)

[0061] SH or B5 basal medium was selected, supplemented with different concentrations of plant growth regulators 6-BA and NAA, sucrose 30 g / L, and gellan gum 3-3.5 g / L. Callus proliferation experiments were conducted, with three bottles inoculated into each medium. After 30 days of culture, cell growth was observed and the proliferation coefficient was calculated. The results are shown in Table 4.

[0062] The results showed that the proliferation rate of culture medium No. 3 (B5 + 6-BA 1.0 mg / L + NAA 1.0 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L) was the highest, followed by culture medium No. 2 (SH + 6-BA 1.0 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L). The callus tissue was in a brittle state and could be further subcultured.

[0063] Table 4. Effects of different culture medium formulations on the growth of Primula sigilis callus.

[0064]

[0065] Example 6: Screening and proliferation of embryogenic callus (4)

[0066] To further screen callus tissues in good condition and with high proliferation rates, SH or B5 basal media were selected, supplemented with different concentrations of plant growth regulators TDZ, NAA, and mT, along with 30 g / L sucrose and 3-3.5 g / L gellan gum. Three bottles were inoculated into each medium, and cell growth was observed and proliferation coefficients were calculated after 30 days of culture. The results are shown in Table 5: Medium 1 and 4, i.e., B5 + TDZ 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L and SH + TDZ 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L and SH + TDZ 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L, showed higher proliferation rates, with a proliferation coefficient of 2.6 for both, and the callus tissue was yellow (…). Figure 1 C), in relatively good condition.

[0067] In summary, the proliferation culture medium formulas for Primula sikkimiensis can be selected as follows: B5 or SH + 2,4-D 4.0 mg / L + KT 3.0 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L; B5 or SH + 6-BA 1.0 mg / L + NAA 0.5-1.0 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L; B5 or SH + TDZ 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L. The proliferation coefficients of all these formulas can reach above 2.5, and the resulting callus tissue is in good condition.

[0068] Table 5. Effects of different culture medium formulations on the growth of Primula sigilis callus.

[0069]

[0070] Example 6: Extraction and Detection of Active Ingredients from Callus Tissue

[0071] (1) Culture and extraction of callus tissue

[0072] Roots of sterile Primula sikinensis seedlings were inoculated into sterile medium containing 1 / 2 MS + 2,4-D 1.0 mg / L + NAA 1.0 mg / L + 30 g / L sucrose + 3-3.5 g / L gellan gum for induction. After 20 days of culture, callus tissue was taken and subcultured and proliferated in the medium with a proliferation coefficient of 2.5 or higher as described in Examples 3-5 for 30 days.

[0073] 10g of wet Primula tinkinensis callus was weighed, pulverized, and then extracted with 75% ethanol (material-to-liquid ratio approximately 1:5). The mixture was ultrasonically extracted (500W) for 20 minutes, filtered, and the filtrate was collected. The residue was extracted twice more with solvent. Finally, the filtrates were combined, and the total polyphenol and total flavonoid content of the extract was tested.

[0074] (2) Method for determining total polyphenols

[0075] Standard curve: Gallic acid 0.33 mg / mL solution was successively diluted to concentration gradients of 0, 0.0052, 0.0103, 0.0206, 0.0413, 0.0825, 0.1650, and 0.3300 mg / mL. 100 μL of Primula callus extract was added to 500 μL of 10% Folin-Ciocalteu reagent, mixed thoroughly, and incubated for 5 min (not exceeding 8 min). Then, 400 μL of 7.5% Na₂CO₃ was added, mixed thoroughly, and incubated at room temperature for 60 min. Finally, 100 μL was measured at 765 nm using a microplate reader. The total polyphenol content of Primula callus was calculated based on the standard curve.

[0076] (3) Determination of total flavonoids

[0077] Standard solution: A 0.2315 mg / mL ethanol solution of quercetin was prepared and diluted sequentially to a concentration gradient of 2-10 times. Take 300 μL of Primula callus extract, first add 50 μL of 5% NaNO2, mix, and incubate at room temperature for 6 min; then add 50 μL of 10% Al(NO3)3, mix, and incubate at room temperature for 6 min; finally add 500 μL of 4% NaOH and 140 μL of water, mix, and incubate at room temperature for 15 min. Take 100 μL of the mixture and measure the OD value at 500 nm. Calculate the total flavonoid content of Primula callus based on the standard curve.

[0078] Table 6. Content of active ingredients in Primula sinensis callus extract from Sikkim

[0079] Sikkim Primrose Callus 99±4 135±5

[0080] The results are shown in Table 6. The total polyphenol content of Primula sikinensis callus was 99±4 μg / g fresh weight, and the total flavonoid content was 135±5 μg / g fresh weight.

[0081] In summary, the callus tissue of Primula sikkiens contains abundant polyphenols and flavonoids, which can rapidly proliferate and replace the plant to meet the needs of industries such as medicine, food, and cosmetics.

Claims

1. A method for culturing Primula sinensis callus, characterized by the following steps: include: (1) Induction of callus: The roots of aseptic seedlings of Primula sikinensis were inoculated into the induction medium to obtain callus of Primula sikinensis; (2) Proliferation culture: The callus tissue of Primula sikinensis was inoculated into the proliferation culture medium and cultured for 25-60 days; In step (1), the induction medium consists of: 1 / 2 MS medium or B5 medium + 1.0-4.0 mg / L 2,4-dichlorophenoxyacetic acid + 1.0-2.0 mg / L α-naphthaleneacetic acid + 30 g / L sucrose + 3-3.5 g / L gellan gum, or 1 / 2 MS medium + 4.0 mg / L 2,4-dichlorophenoxyacetic acid + 2.0 mg / L kinetin + 30 g / L sucrose + 3-3.5 g / L gellan gum; In step (2), the composition of the proliferation medium is as follows: SH or B5 medium + 2,4-dichlorophenoxyacetic acid 4.0 mg / L + kinetin 3.0 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L, or SH or B5 medium + 6-benzylaminopurine 1.0 mg / L + naphthaleneacetic acid 0.5-1.0 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L, or SH or B5 medium + thidiazuron 0.5 mg / L + naphthaleneacetic acid 0.3 mg / L + sucrose 30 g / L + gellan gum 3-3.5 g / L.

2. The method for culturing Primula sikinensis callus according to claim 1, characterized in that, The culture time for step (2) is 28-35 days.

3. The method for culturing Primula sikinensis callus according to claim 1, characterized in that, The cultivation conditions for steps (1) and (2) are: temperature 20±1℃, humidity 50%~70%, light exposure 10-15 hours per day, and light intensity 1500 Lx~2000 Lx.

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