Culture medium for tissue culture of ferula sinkiangensis, callus culture method and application thereof

Abstract

The application belongs to the technical field of plant tissue culture, and particularly relates to a culture medium for tissue culture of Ferula sinkiangensis, a callus culture method and application thereof. The application provides a culture medium for tissue culture of Ferula sinkiangensis, including a callus induction culture medium, a complete dedifferentiation culture medium and a callus subculture and proliferation culture medium. The explant of Ferula sinkiangensis is sequentially subjected to callus induction culture, complete dedifferentiation culture and subculture and proliferation culture, so that the callus of Ferula sinkiangensis can be efficiently induced, the proliferation coefficient of the callus is significantly improved, a large amount of healthy and stable callus is obtained in a short period of time, time cost is saved, technical support for seedling raising and rapid propagation is provided for the research on protection and development of Ferula species resources, and the callus of Ferula sinkiangensis obtained by the culture method can be used as a receptor for gene editing, so that the breeding process of Ferula sinkiangensis is accelerated through the gene editing technology.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a culture medium for Ferula assa-foetida tissue culture in Xinjiang, a callus culture method, and their applications. Background Technology

[0002] Ferula sinkiangensis, a medicinal plant belonging to the genus Ferula in the family Apiaceae, is endemic to my country. As a major source of medicinal Ferula in my country, it is listed in the *Pharmacopoeia of the People's Republic of China*. Due to rapid population growth, increased demand for medicinal Ferula, and changes in its growing environment, the distribution area of ​​Ferula sinkiangensis has shrunk from 2,000–4,000 hectares to less than 1 / 20 of its original size. Furthermore, the species suffers from low population density, few flowering plants, and the possibility of inbreeding within small populations leading to rapid population decline. For these reasons, Ferula sinkiangensis has been listed as a Class II National Key Protected Wild Plant and is classified as Critically Endangered (CR) in China's Threatened Species Assessment. Therefore, ex-situ conservation research on this endangered species is urgently needed.

[0003] Plant tissue culture is a newly emerging biotechnology science developed based on plant physiology. Due to its rapid propagation, it can achieve efficient and rapid reproduction of some rare plants with low propagation coefficients. Currently, tissue culture technology has become an important technique for the ex-situ conservation of endangered species and the propagation of seedlings for industrial cultivation. Some studies have explored the tissue culture and artificial cultivation of *Ferula asperata* from Xinjiang. However, because *Ferula asperata* reproduces sexually, its seeds are scarce and dormant, germination often takes a long time, and under laboratory conditions, the plants are small and do not easily develop true leaves, limiting the ex-situ conservation, large-scale cultivation, and industrial development of this genus.

[0004] Currently, research on callus induction in *Ferula assa-foetida* from Xinjiang is limited. Existing studies show that seedlings without true leaves are more conducive to callus formation, but the overall callus rate is low, and browning is a serious problem, with many materials exhibiting growth stagnation. This has prevented the widespread application of this technique in the rapid propagation, ex-situ conservation, and large-scale seedling production of *Ferula assa-foetida* from Xinjiang. Therefore, providing a method for inducing large quantities of *Ferula assa-foetida* callus in a short period is of great significance for research on the conservation and development of species resources, and lays the foundation for the protection of *Ferula assa-foetida* germplasm resources and the genetic breeding of superior traits. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium for tissue culture of *Ferula assa-foetida* from Xinjiang, a method for callus culture, and their applications. Using the culture medium provided by this invention to culture *Ferula assa-foetida* explants can efficiently induce *Ferula assa-foetida* callus, increase the proliferation coefficient, and obtain a large amount of healthy and stable callus in a short period of time. This provides technical support for seedling cultivation and rapid propagation in research on the protection and development of *Ferula assa-foetida* species resources.

[0006] This invention provides a culture medium for Ferula assa-foetida tissue culture in Xinjiang, comprising a callus induction medium, a complete dedifferentiation medium, and a callus subculture proliferation medium; the callus induction medium is based on MS liquid medium and further comprises 2,4-D, sucrose, and agar; the concentration of 2,4-D in the callus induction medium is 0.5–3.0 mg / L.

[0007] The complete dedifferentiation medium is based on MS liquid medium and also includes 2,4-D, 6-BA, sucrose and agar; the concentration of 2,4-D in the complete dedifferentiation medium is 0.5 mg / L and the concentration of 6-BA is 0.5 mg / L.

[0008] The callus subculture and proliferation medium is based on MS liquid medium and also includes 2,4-D, NAA, 6-BA, sucrose and agar; the concentration of 2,4-D in the callus subculture and proliferation medium is 0.1-1.0 mg / L, the concentration of NAA is 0.1-0.5 mg / L, and the concentration of 6-BA is 0.5-2.0 mg / L.

[0009] Preferably, the pH values ​​of the callus induction medium, the complete dedifferentiation medium, and the callus subculture proliferation medium are 5.8 to 6.0, respectively.

[0010] This invention also provides the application of the culture medium described in the above technical solution in the tissue culture of Ferula assa-foetida in Xinjiang.

[0011] This invention also provides a method for culturing Ferula callus from Xinjiang, comprising the following steps:

[0012] Explants of *Ferula asperata* from Xinjiang were inoculated into callus induction medium for callus induction culture to obtain primary *Ferula asperata* callus; the primary *Ferula asperata* callus was inoculated into completely dedifferentiated medium for complete dedifferentiation culture to obtain dedifferentiated *Ferula asperata* callus; the dedifferentiated *Ferula asperata* callus was inoculated into callus subculture and proliferation medium for subculture and proliferation culture to obtain *Ferula asperata* callus.

[0013] The callus induction medium, complete dedifferentiation medium, and callus subculture proliferation medium are the callus induction medium, complete dedifferentiation medium, and callus subculture proliferation medium mentioned above.

[0014] Preferably, the callus induction culture is performed by first performing dark induction culture and then performing light-dark alternating culture; the dark induction culture time is 7-10 days; the light-dark alternating culture time is 15-30 days; in each cycle of the light-dark alternating culture, the light culture time is 12 hours and the dark culture time is 12 hours; the light culture temperature is (25±0.5)℃ and the dark culture temperature is (15±0.5)℃.

[0015] Preferably, the complete dedifferentiation culture is a light-dark alternating culture; the light culture time in each cycle of the light-dark alternating culture is 12h and the dark culture time is 12h; the temperature of the light culture is (25±0.5)℃ and the temperature of the dark culture is (15±0.5)℃; the complete dedifferentiation culture time is 15 to 30 days.

[0016] Preferably, the subculture is a light-dark alternating culture; the light culture time in each cycle of the light-dark alternating culture is 12h and the dark culture time is 12h; the temperature of the light culture is (25±0.5)℃ and the temperature of the dark culture is (15±0.5)℃; the subculture time is 30-35 days.

[0017] Preferably, the explant of Ferula asperata from Xinjiang includes the hypocotyl of Ferula asperata from Xinjiang.

[0018] Preferably, the hypocotyl of the Xinjiang Ferula aspera is taken from aseptic seedlings of Xinjiang Ferula aspera.

[0019] This invention also provides the application of the culture method described above in the tissue culture of Ferula assa-foetida in Xinjiang.

[0020] This invention provides a culture medium for the tissue culture of *Ferula assa-foetida* from Xinjiang, comprising a callus induction medium, a complete dedifferentiation medium, and a callus subculture and proliferation medium. The culture medium provided by this invention, through suitable callus induction, complete dedifferentiation, and subculture and proliferation media, can efficiently induce *Ferula assa-foetida* explants into *Ferula assa-foetida* callus (average callus emergence rate 58.25±4.54%), and significantly improve the callus proliferation coefficient (average proliferation coefficient 13.83±0.83). It allows for the rapid acquisition of large quantities of healthy and stable callus, saving time and costs, and providing technical support for seedling cultivation and rapid propagation in research on the protection and development of *Ferula assa-foetida* species resources.

[0021] Furthermore, the Xinjiang Ferula callus obtained by the culture method of this invention can be used as a recipient for gene editing, thereby accelerating the breeding process of Xinjiang Ferula through gene editing technology. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0023] Figure 1 A schematic diagram of Ferula assa-foetida seedlings germinating from seeds in Xinjiang;

[0024] Figure 2 A schematic diagram of primary callus induced by hypocotyl of Ferula assa-foetida from Xinjiang;

[0025] Figure 3 A schematic diagram of callus obtained from the complete dedifferentiation of hypocotyls of Ferula asperata from Xinjiang.

[0026] Figure 4 A schematic diagram of 30 days of subculture of Ferula callus from Xinjiang. Detailed Implementation

[0027] This invention provides a culture medium for Ferula assa-foetida tissue culture in Xinjiang, comprising a callus induction medium, a complete dedifferentiation medium, and a callus subculture proliferation medium; the callus induction medium is based on MS liquid medium and further comprises 2,4-D, sucrose, and agar; the concentration of 2,4-D in the callus induction medium is 0.5–3.0 mg / L.

[0028] The complete dedifferentiation medium is based on MS liquid medium and also includes 2,4-D, 6-BA, sucrose and agar; the concentration of 2,4-D in the complete dedifferentiation medium is 0.5 mg / L and the concentration of 6-BA is 0.5 mg / L.

[0029] The callus subculture and proliferation medium is based on MS liquid medium and also includes 2,4-D, NAA, 6-BA, sucrose and agar; the concentration of 2,4-D in the callus subculture and proliferation medium is 0.1-1.0 mg / L, the concentration of NAA is 0.1-0.5 mg / L, and the concentration of 6-BA is 0.5-2.0 mg / L.

[0030] In this invention, the callus induction medium is based on MS liquid medium and further includes 2,4-D, sucrose, and agar. The concentration of 2,4-D in the callus induction medium is 0.5–3.0 mg / L, preferably 1.5–3.0 mg / L, more preferably 2.5 mg / L; the concentration of sucrose in the callus induction medium is preferably 30 g / L; the concentration of agar in the callus induction medium is preferably 6 g / L; and the pH of the callus induction medium is preferably 5.8–6.0, more preferably 5.8–5.9, and more preferably 5.83. Adding a suitable concentration of 2,4-D to the callus induction medium of this invention is beneficial for inducing primary callus formation from Xinjiang Ferula assa-foetida.

[0031] In this invention, the completely dedifferentiated medium is based on MS liquid medium and further includes 2,4-D, 6-BA, sucrose, and agar. The concentration of 2,4-D in the completely dedifferentiated medium is 0.5 mg / L; the concentration of 6-BA in the completely dedifferentiated medium is 0.5 mg / L; the concentration of sucrose in the completely dedifferentiated medium is preferably 30 g / L; the concentration of agar in the completely dedifferentiated medium is preferably 6 g / L; the pH of the callus subculture proliferation medium is preferably 5.8–6.0, more preferably 5.8–5.9, and even more preferably 5.83. Adding appropriate concentrations of 2,4-D and 6-BA to the completely dedifferentiated medium of this invention is beneficial for the complete dedifferentiation of primary callus.

[0032] In this invention, the callus subculture and proliferation medium is based on MS liquid medium and also includes 2,4-D, NAA, 6-BA, sucrose and agar. The concentration of 2,4-D in the callus subculture proliferation medium of the present invention is 0.1-1.0 mg / L, preferably 0.2-0.8 mg / L, more preferably 0.5 mg / L; the concentration of NAA in the callus subculture proliferation medium is 0.1-0.5 mg / L, preferably 0.1-0.3 mg / L, more preferably 0.1 mg / L; the concentration of 6-BA in the callus subculture proliferation medium is 0.5-2.0 mg / L, preferably 0.5-1.5 mg / L, more preferably 0.5-1.0 mg / L; the concentration of sucrose in the callus subculture proliferation medium is preferably 30 g / L; the concentration of agar in the completely dedifferentiated medium is preferably 6 g / L; the pH of the callus subculture proliferation medium is 5.8-6.0, more preferably 5.8-5.9, more preferably 5.87. The addition of appropriate concentrations of 2,4-D, NAA, and 6-BA to the subculture proliferation medium of this invention can significantly improve the proliferation coefficient of callus tissue, and obtain a large amount of healthy and stable callus tissue in a short period of time.

[0033] The present invention also provides the application of the above-mentioned culture medium for Ferula assa-foetida tissue culture in the tissue culture of Ferula assa-foetida plants in Xinjiang.

[0034] This invention also provides a method for culturing Ferula callus from Xinjiang, comprising the following steps:

[0035] Explants of *Ferula asperata* from Xinjiang were inoculated into callus induction medium to induce callus culture and obtain primary *Ferula asperata* callus; the primary *Ferula asperata* callus was inoculated into completely dedifferentiated medium to obtain dedifferentiated *Ferula asperata* callus; the dedifferentiated *Ferula asperata* callus was inoculated into callus subculture and proliferation medium to obtain *Ferula asperata* callus.

[0036] The callus induction medium, complete dedifferentiation medium, and callus subculture proliferation medium are the callus induction medium, complete dedifferentiation medium, and callus subculture proliferation medium mentioned above in the culture media used for Ferula assa-foetida tissue culture.

[0037] This invention involves inoculating explants of *Ferula assa-foetida* from Xinjiang onto a callus induction medium to induce callus culture and obtain primary callus tissue from *Ferula assa-foetida*. In this invention, the explants are preferably hypocotyls, which are preferably obtained by cutting sterile *Ferula assa-foetida* seedlings. The use of hypocotyls as explants in this invention is advantageous because hypocotyls possess extremely young meristematic cells. These cells have undergone complete rejuvenation through sexual reproduction and are readily cultured into callus tissue.

[0038] In this invention, the callus induction culture is preferably performed by first performing dark induction culture, followed by light-dark alternating culture. The dark induction culture time is preferably 7–10 days, more preferably 7 days; the light-dark alternating culture time is preferably 15–30 days, further preferably 18–28 days, more preferably 20–25 days; the light culture time in each cycle of the light-dark alternating culture is preferably 12 hours, and the dark culture time is preferably 12 hours; the light culture temperature in the light-dark alternating culture is preferably (25±0.5)℃, more preferably 25℃; the dark culture temperature in the light-dark alternating culture is preferably (15±0.5)℃, more preferably 15℃. Performing dark induction culture first accelerates the callus initiation speed and is more conducive to callus induction; performing light-dark alternating culture after dark induction culture can improve the explant callus emergence rate.

[0039] After obtaining primary callus tissue from *Ferula assa-foetida* in Xinjiang, this invention inoculates the primary callus tissue from Xinjiang into a completely dedifferentiated medium for complete dedifferentiation culture to obtain dedifferentiated callus tissue from Xinjiang *Ferula assa-foetida*. In this invention, the complete dedifferentiation culture is preferably a light-dark alternating culture; the light culture time in each cycle of the light-dark alternating culture is preferably 12 hours, and the dark culture time is preferably 12 hours; the light culture temperature in the light-dark alternating culture is preferably (25±0.5)℃, more preferably 25℃; the dark culture temperature in the light-dark alternating culture is preferably (15±0.5)℃, more preferably 15℃; the complete dedifferentiation culture time is preferably 15–30 days, more preferably 18–28 days, and more preferably 20–25 days.

[0040] After obtaining the dedifferentiated callus tissue of *Ferula assa-foetida* from Xinjiang, this invention inoculates the *Ferula assa-foetida* dedifferentiated callus tissue into a callus subculture medium for subculture to obtain *Ferula assa-foetida* callus tissue. In this invention, the subculture is preferably a light-dark alternating culture; the light culture time in each cycle of the light-dark alternating culture is preferably 12 hours, and the dark culture time is preferably 12 hours; the light temperature in the light-dark alternating culture is preferably (25±0.5)℃, more preferably 25℃; the dark culture temperature in the light-dark alternating culture is preferably (15±0.5)℃, more preferably 15℃; the subculture time is preferably 30–35 days, more preferably 30 days.

[0041] This invention also provides the application of the above-mentioned culture method in tissue culture of Ferula assa-foetida in Xinjiang.

[0042] To further illustrate the present invention, the method for culturing Xinjiang Ferula callus provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0043] The explants used in the embodiments and comparative examples of this invention were all taken from the same batch of sterile seedlings.

[0044] Example 1

[0045] (1) Preparation of culture medium

[0046] ①Callus induction medium: MS liquid medium was used as the base medium, and it also contained 2,4-D 2.5 mg / L, sucrose 30 g / L and agar 6.0 g / L, pH=5.83, and was sterilized at 121℃ for 15 min by high temperature and high pressure to obtain the callus induction medium.

[0047] ② Complete dedifferentiation medium: Based on MS liquid medium, it contains only 0.5 mg / L 2,4-D, 0.5 mg / L 6-BA, 30 g / L sucrose and 6.0 g / L agar, pH=5.83, and is autoclaved at 121℃ for 15 min to obtain the complete dedifferentiation medium.

[0048] ③ Callus subculture and proliferation medium: MS liquid medium was used as the base medium, and it also contained 0.5 mg / L 2,4-D, 0.1 mg / L NAA, 0.5 mg / L 6-BA, 30 g / L sucrose and 6.0 g / L agar, pH=5.87, and was sterilized at 121℃ for 15 min by high temperature and high pressure to obtain the callus subculture and proliferation medium.

[0049] (2) Explant pretreatment

[0050] After detoxification, Xinjiang Ferula seeds were placed in moist sand and stratified at 4℃ for 90 days. After stratification, the seeds were removed and germinated in a sterile environment to obtain sterile seedlings. Figure 1 As shown. The hypocotyl was obtained by removing the radicle and cotyledons of the sterile seedling, and then cut into 1cm pieces to serve as explants.

[0051] (3) Callus induction

[0052] The obtained explants of *Ferula assa-foetida* from Xinjiang were inoculated onto callus induction medium and cultured in the dark for 7 days, followed by alternating light and dark culture for 25 days until white, fibrous callus tissue was produced, which is the primary callus tissue. Figure 2 As shown. In each cycle of the light-dark alternating culture, the light culture lasts for 12 hours and the dark culture lasts for 12 hours. The temperature for the light-dark alternating culture is 25°C for light and 15°C for darkness.

[0053] (4) Complete decomposition and culture of callus

[0054] Primary callus tissue was inoculated onto a completely dedifferentiated medium and cultured in alternating light and dark for 25 days until the white, fibrous callus tissue completely dedifferentiated, yielding new, yellowish-white, granular, and healthy callus tissue, which is the dedifferentiated callus tissue. Figure 3 As shown. In each cycle of the light-dark alternating culture, the light culture lasts for 12 hours and the dark culture lasts for 12 hours. The temperature for the light-dark alternating culture is 25°C for light and 15°C for darkness.

[0055] (5) Callus subculture

[0056] Dedifferentiated callus tissue was inoculated onto callus tissue subculture and cultured in alternating light and dark conditions for 30 days, resulting in a large number of proliferating, pale yellow-green, granular, and vigorously growing Xinjiang Ferula callus tissues. Figure 4As shown. In each cycle of the light-dark alternating culture, the light culture lasts for 12 hours and the dark culture lasts for 12 hours. The temperature for the light-dark alternating culture is 25°C for light and 15°C for darkness.

[0057] Three biological replicates were performed, and the cure rate and proliferation coefficient were calculated. The results are shown in Table 1.

[0058] Healing rate (%) = (Number of explant pieces that form callus / Total number of inoculated explant pieces) × 100.

[0059] Callus inoculation weight: The weight of callus tissue inoculated onto the culture medium before the start of subculture proliferation.

[0060] Value-added coefficient = Fresh weight after 30 days of subculture / Callus inoculation weight.

[0061] Table 1. Number of hypocotyls, callus emergence rate, callus inoculation weight, and proliferation coefficient in Example 1

[0062] Biological Repetition 1 28 64.29 0.61 13.05 Biological Repetition 2 16 53.33 0.68 14.96 Biological repeat 3 21 57.14 0.35 13.52 average value - 58.25±4.54 - 13.83±0.83

[0063] As shown in Table 1, plant tissue culture technology was used to successfully induce hypocotyl callus of Ferula assa-foetida from Xinjiang. The average callus rate was (58.25±4.54)%, the average proliferation coefficient was 13.83±0.83, and the callus was a healthy, pale yellow-green color.

[0064] Example 2

[0065] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus induction medium is 0.5 mg / L.

[0066] The callus induction was observed, and white, flocculent callus tissue was generated. After 30 days, the callus rate was calculated to be (22.22±2.72)%.

[0067] Example 3

[0068] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus induction medium is 1.5 mg / L.

[0069] The callus induction was observed, and white, flocculent callus tissue was generated. After 30 days, the callus rate was calculated to be (26.67±4.71)%.

[0070] Comparative Example 1

[0071] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus induction medium is 0 mg / L.

[0072] The callus induction was observed, and white, flocculent callus tissue was formed. After 30 days, the callus rate was calculated to be (0.00±0.00)%.

[0073] Comparative Example 2

[0074] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus induction medium is 3.5 mg / L.

[0075] The callus induction was observed, and white, flocculent callus tissue was generated. After 30 days, the callus rate was calculated to be (17.78±2.72)%.

[0076] The callus formation rate of Xinjiang Ferula assa-foetida in Examples 1-3 and Comparative Examples 1-2 was statistically analyzed, and the results are shown in Table 2.

[0077] Table 2. Effect of 2,4-D concentration in callus induction medium on callus emergence rate.

[0078] Example 1 2.5 58.25±4.54 Example 2 0.5 22.22±2.72 Example 3 1.5 26.67±4.71 Comparative Example 1 0 0.00±0.00 Comparative Example 2 3.5 17.78±2.72

[0079] As shown in Table 2, appropriate concentrations of 2,4-D can promote the production of callus tissue from Ferula assa-foetida in Xinjiang.

[0080] Comparative Example 3

[0081] A tissue culture method similar to Comparative Example 1, except that cotyledons are used as explants.

[0082] The callus induction was observed, and loose, white, flocculent callus tissue was generated. After 30 days, the callus rate was calculated to be (0±0)%.

[0083] Comparative Example 4

[0084] A tissue culture method similar to Example 2, except that cotyledons are used as explants.

[0085] The callus induction was observed, and loose, white, flocculent callus tissue was generated. After 30 days, the callus rate was calculated to be (4.44±5.44)%.

[0086] Comparative Example 5

[0087] A tissue culture method similar to Example 3, except that cotyledons are used as explants.

[0088] The callus induction was observed, and loose, white, flocculent callus tissue was generated. After 30 days, the callus rate was calculated to be (22.22±3.14)%.

[0089] Comparative Example 6

[0090] A tissue culture method similar to Example 1, except that cotyledons are used as explants.

[0091] The callus induction was observed, and loose, white, flocculent callus tissue was generated. After 30 days, the callus rate was calculated to be (31.11±3.14)%.

[0092] Comparative Example 7

[0093] A tissue culture method similar to Comparative Example 2, except that cotyledons are used as explants.

[0094] The callus induction was observed, and loose, white, flocculent callus tissue was generated. After 30 days, the callus rate was calculated to be (15.56±3.14)%.

[0095] The callus emergence rate of different explants in different callus induction media was statistically analyzed according to Examples 1-3 and Comparative Examples 1-7. The results are shown in Table 3.

[0096] Table 3. Effect of explant type on healing rate

[0097] 2.5 58.25±4.54 31.11±3.14 0.5 22.22±2.72 4.44±5.44 1.5 26.67±4.71 22.22±3.14 0 0.00±0.00 0.00±0.00 3.5 17.78±2.72 15.56±3.14

[0098] According to the data in Table 3, when the explant is a hypocotyl, the callus formation rate is significantly higher than that of the cotyledon. The hypocotyl of Ferula ferox is more suitable for inducing callus tissue.

[0099] Example 4

[0100] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 0.1 mg / L, the concentration of NAA is 1.0 mg / L, and the concentration of 6-BA is 1.5 mg / L.

[0101] The callus proliferation was observed. The callus was light yellow. After 30 days, the callus proliferation coefficient was (11.06±0.79).

[0102] Example 5

[0103] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 1.0 mg / L, the concentration of NAA is 0.1 mg / L, and the concentration of 6-BA is 1.0 mg / L.

[0104] The callus proliferation was observed. The callus was light yellow. After 30 days, the callus proliferation coefficient was (10.51±0.22).

[0105] Example 6

[0106] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 1.0 mg / L, the concentration of NAA is 0.5 mg / L, and the concentration of 6-BA is 0.5 mg / L.

[0107] The callus proliferation was observed. The callus was light yellow. After 30 days, the callus proliferation coefficient was (13.22±0.53).

[0108] Comparative Example 8

[0109] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture is 0 mg / L, the concentration of NAA is 0.1 mg / L, and the concentration of 6-BA is 1.5 mg / L.

[0110] The callus proliferation was observed. The callus was yellowish-brown. After 30 days, the callus proliferation coefficient was (3.73±0.17).

[0111] Comparative Example 9

[0112] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 0 mg / L, the concentration of NAA is 0.1 mg / L, and the concentration of 6-BA is 2.0 mg / L.

[0113] The callus proliferation was observed. The callus was dark yellow. After 30 days, the callus proliferation coefficient was (11.74±0.32).

[0114] Comparative Example 10

[0115] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 0 mg / L, the concentration of NAA is 0.5 mg / L, and the concentration of 6-BA is 1.5 mg / L.

[0116] The callus proliferation was observed. The callus was yellowish-brown. After 30 days, the callus proliferation coefficient was (2.34±0.14).

[0117] Comparative Example 11

[0118] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 0 mg / L, the concentration of NAA is 1.0 mg / L, and the concentration of 6-BA is 1.0 mg / L.

[0119] The callus proliferation was observed. The callus was light yellow. After 30 days, the callus proliferation coefficient was (12.45±0.77).

[0120] Comparative Example 12

[0121] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 0.5 mg / L, the concentration of NAA is 0 mg / L, and the concentration of 6-BA is 1.5 mg / L.

[0122] The callus proliferation was observed. The callus was dark yellow. After 30 days, the callus proliferation coefficient was (6.01±0.34).

[0123] Comparative Example 13

[0124] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 0.5 mg / L, the concentration of NAA is 0 mg / L, and the concentration of 6-BA is 1.0 mg / L.

[0125] The callus proliferation was observed. The callus was dark yellow. After 30 days, the callus proliferation coefficient was (7.21±0.16).

[0126] Comparative Example 14

[0127] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 1.0 mg / L, the concentration of NAA is 0 mg / L, and the concentration of 6-BA is 1.0 mg / L.

[0128] The callus proliferation was observed. The callus was light yellow. After 30 days, the callus proliferation coefficient was (8.40±0.11).

[0129] Comparative Example 15

[0130] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 1.0 mg / L, the concentration of NAA is 0 mg / L, and the concentration of 6-BA is 1.5 mg / L.

[0131] The callus proliferation was observed. The callus was light yellow, with some parts turning yellowish-brown. After 30 days, the callus proliferation coefficient was (9.16±0.84).

[0132] Comparative Example 16

[0133] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 1.0 mg / L, the concentration of NAA is 0 mg / L, and the concentration of 6-BA is 2.0 mg / L.

[0134] The callus proliferation was observed. The callus was dark yellow. After 30 days, the callus proliferation coefficient was (7.18±0.57).

[0135] Comparative Example 17

[0136] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 0.1 mg / L, the concentration of NAA is 0.1 mg / L, and the concentration of 6-BA is 0 mg / L.

[0137] The callus proliferation was observed. The callus was light yellow. After 30 days, the callus proliferation coefficient was (11.00±1.41).

[0138] Comparative Example 18

[0139] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 0.5 mg / L, the concentration of NAA is 0.5 mg / L, and the concentration of 6-BA is 0 mg / L.

[0140] The callus proliferation was observed. The callus was yellowish-brown. After 30 days, the callus proliferation coefficient was (4.39±0.33).

[0141] Comparative Example 19

[0142] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 1.0 mg / L, the concentration of NAA is 1.0 mg / L, and the concentration of 6-BA is 0 mg / L.

[0143] The callus proliferation was observed. The callus was light yellow. After 30 days, the callus proliferation coefficient was (9.79±0.81).

[0144] Comparative Example 20

[0145] A tissue culture method similar to that in Example 1, except that the concentration of 2,4-D in the callus subculture culture medium is 0.5 mg / L, the concentration of NAA is 1.0 mg / L, and the concentration of 6-BA is 2.0 mg / L.

[0146] The callus proliferation was observed. The callus was light yellow. After 30 days, the callus proliferation coefficient was (9.11±0.46).

[0147] The proliferation coefficients of Examples 1, 4-6 and Comparative Examples 9-20 were statistically analyzed, and the results are shown in Table 4.

[0148] Table 4. Effect of hormone concentration in subculture proliferation medium on proliferation coefficient

[0149]

[0150]

[0151] Table 4 shows that NAA can promote the subculture proliferation of hypocotyl callus from *Ferula assa-foetida* in Xinjiang and affects the growth status of the callus. Without NAA in the subculture medium, the proliferation coefficient decreases, and most callus tissues show browning, turning a deep yellow color. Under conditions where the ratio of 2,4-D to 6-BA is 1:1 to 1:1.5, the callus growth is better, and the callus is a light yellow color. Adding 0.1 mg / L of NAA to this condition can improve the callus condition. This indicates that an appropriate hormone concentration has a certain promoting effect on the subculture proliferation of callus, while an unsuitable hormone concentration has a toxic effect on the callus, resulting in callus tissue that is mostly deep yellow or yellowish-brown.

[0152] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium for tissue culture of Ferula assa-foetida in Xinjiang, characterized in that, It includes callus induction medium, complete dedifferentiation medium, and callus subculture proliferation medium; the callus induction medium is composed of MS liquid medium, 2,4-D, sucrose, and agar; the concentration of 2,4-D in the callus induction medium is 0.5~3.0 mg / L; The complete dedifferentiation medium consists of MS liquid medium, 2,4-D, 6-BA, sucrose, and agar; the concentration of 2,4-D in the complete dedifferentiation medium is 0.5 mg / L, and the concentration of 6-BA is 0.5 mg / L. The callus subculture and proliferation medium consists of MS liquid medium, 2,4-D, NAA, 6-BA, sucrose, and agar; the concentration of 2,4-D in the callus subculture and proliferation medium is 0.1~1.0 mg / L, the concentration of NAA is 0.1~0.5 mg / L, and the concentration of 6-BA is 0.5~2.0 mg / L. The explants used in the tissue culture of *Ferula assa-foetida* from Xinjiang were hypocotyls of *Ferula assa-foetida* from Xinjiang.

2. The culture medium according to claim 1, characterized in that, The pH values ​​of the callus induction medium, the complete dedifferentiation medium, and the callus subculture proliferation medium are 5.8 to 6.0, respectively.

3. The application of the culture medium according to claim 1 or 2 in the tissue culture of Ferula assa-foetida in Xinjiang.

4. A method for culturing Ferula assa-foetida callus from Xinjiang, characterized in that, Includes the following steps: Explants of *Ferula asperata* from Xinjiang were inoculated into callus induction medium for callus induction culture to obtain primary *Ferula asperata* callus; the primary *Ferula asperata* callus was inoculated into completely dedifferentiated medium for complete dedifferentiation culture to obtain dedifferentiated *Ferula asperata* callus; the dedifferentiated *Ferula asperata* callus was inoculated into callus subculture and proliferation medium for subculture and proliferation culture to obtain *Ferula asperata* callus. The callus induction medium, the complete dedifferentiation medium, and the callus subculture proliferation medium are the callus induction medium, the complete dedifferentiation medium, and the callus subculture proliferation medium described in claim 1 or 2. The explants of *Ferula assa-foetida* from Xinjiang are hypocotyls of *Ferula assa-foetida* from Xinjiang.

5. The cultivation method according to claim 4, characterized in that, The callus induction culture consists of first dark induction culture, followed by light-dark alternating culture; the dark induction culture time is 7-10 days; the light-dark alternating culture time is 15-30 days; in each cycle of the light-dark alternating culture, the light culture time is 12 hours and the dark culture time is 12 hours; the light culture temperature is (25±0.5)℃ and the dark culture temperature is (15±0.5)℃.

6. The cultivation method according to claim 4, characterized in that, The complete dedifferentiation culture is a light-dark alternating culture; in each cycle of the light-dark alternating culture, the light culture time is 12h and the dark culture time is 12h; the temperature of the light culture is (25±0.5)℃ and the temperature of the dark culture is (15±0.5)℃; the complete dedifferentiation culture time is 15~30d.

7. The cultivation method according to claim 4, characterized in that, The subculture is a light-dark alternating culture; in each cycle of the light-dark alternating culture, the light culture time is 12h and the dark culture time is 12h; the temperature of the light culture is (25±0.5)℃ and the temperature of the dark culture is (15±0.5)℃; the subculture is carried out for 30~35 days.

8. The cultivation method according to claim 4, characterized in that, The hypocotyl of the Xinjiang Ferula aspera was obtained from sterile seedlings of Xinjiang Ferula aspera.

9. The application of the culture method according to any one of claims 4 to 8 in the tissue culture of Ferula assa-foetida in Xinjiang.

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