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

Abstract

The invention 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 culture medium comprises a callus induction culture medium, a complete dedifferentiation culture medium and a callus subculture multiplication culture medium, an explant of the ferula sinkiang is sequentially subjected to callus induction culture, complete dedifferentiation culture and subculture multiplication culture, the callus of the ferula sinkiang can be efficiently induced, and the survival rate of the ferula sinkiang is increased. The propagation coefficient of the callus is remarkably improved, a large number of healthy and stable calluses are obtained in a short time, the time cost is saved, and technical support for seedling culture and rapid propagation is provided for research on protection and development of ferula asafetida species resources; the ferula sinkiang callus obtained by the culture method can be used as a gene editing receptor, and the breeding process of ferula sinkiang is accelerated through a gene editing technology.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium for Xinjiang Ferula tissue culture, a callus culture method and applications thereof. Background Art

[0002] Xinjiang Ferula (Ferula sinkiangensis), a medicinal plant of the genus Ferula in the Apiaceae family, is endemic to my country. This species is included in the Pharmacopoeia of the People's Republic of China as the main source of medicinal Ferula in my country. With the rapid increase in population, the demand for medicinal Ferula and the changes in the growth environment, the distribution area of ​​Xinjiang Ferula has been reduced from the original 2,000 to 4,000 hectares to less than 1 / 20 of the original. At the same time, the species has problems such as a small population, few flowering plants, and inbreeding in small populations that may lead to rapid population degeneration. For the above reasons, Xinjiang Ferula has been listed as a national second-level key protected wild plant and has been rated as critically endangered (CR) in China's threatened species assessment. Therefore, it is urgently necessary to carry out ex situ conservation research on Xinjiang Ferula, which is in a critical situation.

[0003] Plant tissue culture is a new biotechnology science developed on the basis of plant physiology. Since the outstanding feature of plant tissue culture is rapidity, for some rare plants with low reproduction coefficients, tissue culture can achieve the purpose of efficient and rapid reproduction. At present, tissue culture technology has developed into an important technology for ex situ protection of endangered species and industrial cultivation of seedlings. Some studies have explored the tissue culture and artificial cultivation of Xinjiang Ferula, but since Xinjiang Ferula produces offspring through sexual reproduction, the seeds of this plant are scarce and dormant, and germination often takes a long time. When cultivated under laboratory conditions, its plants are short and difficult to grow true leaves, which limits the ex situ protection, large-scale cultivation and industrial development of this genus.

[0004] At present, there are few studies on callus induction of Ferula xinjiangensis. In existing studies, Ferula xinjiangensis seedlings without true leaves are more conducive to callus production, but the overall callus production rate is low, and the browning problem is serious, and many materials have stagnant growth. This also leads to the fact that this technology has not yet been widely used in the rapid propagation, ex situ protection and large-scale seedling cultivation of Ferula xinjiangensis. Therefore, providing a culture method that can induce a large number of Ferula xinjiangensis callus in a short period of time is of great significance for the research on the protection and development of species resources, and at the same time lays the foundation for the protection of Ferula xinjiangensis germplasm resources and the genetic breeding of excellent traits. Summary of the invention

[0005] The purpose of the present invention is to provide a culture medium for Xinjiang Ferula tissue culture, a callus culture method and application thereof. Xinjiang Ferula explants are cultured using the culture medium provided by the present invention, Xinjiang Ferula callus can be efficiently induced, the proliferation coefficient can be improved, a large number of healthy and stable callus can be obtained in a short period of time, and technical support for seedling cultivation and rapid propagation is provided for the research on the protection and development of Ferula species resources.

[0006] The invention provides a culture medium for Xinjiang Ferula tissue culture, comprising a callus induction culture medium, a complete dedifferentiation culture medium and a callus subculture proliferation culture medium; the callus induction culture medium uses MS liquid culture medium as a basic culture medium and further comprises 2,4-D, sucrose and agar; the concentration of 2,4-D in the callus induction culture medium is 0.5-3.0 mg / L;

[0007] The complete dedifferentiation medium uses MS liquid medium as a basic medium and further 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 proliferation culture medium uses MS liquid culture medium as a basic culture medium and further comprises 2,4-D, NAA, 6-BA, sucrose and agar; the concentration of 2,4-D in the callus subculture proliferation culture 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 5.8-6.0, respectively.

[0010] The present invention also provides application of the culture medium described in the above technical solution in Xinjiang Ferula plant tissue culture.

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

[0012] Inoculating the Xinjiang Ferula explants in a callus induction medium for callus induction culture to obtain Xinjiang Ferula primary callus; inoculating the Xinjiang Ferula primary callus in a complete dedifferentiation medium for complete dedifferentiation culture to obtain Xinjiang Ferula dedifferentiated callus; inoculating the Xinjiang Ferula dedifferentiated callus in a callus subculture proliferation medium for subculture proliferation culture to obtain Xinjiang Ferula callus;

[0013] 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 in the above culture medium.

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

[0015] Preferably, the complete dedifferentiation culture is light-dark alternating culture; 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)°C, and the dark culture temperature is (15±0.5)°C; the complete dedifferentiation culture time is 15 to 30 days.

[0016] Preferably, the subculture proliferation culture is light-dark alternating culture; 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)°C, and the dark culture temperature is (15±0.5)°C; the subculture proliferation culture time is 30 to 35 days.

[0017] Preferably, the Xinjiang Ferula explant comprises the hypocotyl of Xinjiang Ferula.

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

[0019] The present invention also provides application of the culture method described in the above technical solution in Xinjiang Ferula plant tissue culture.

[0020] The invention provides a culture medium for Xinjiang Ferula tissue culture, comprising a callus induction culture medium, a complete dedifferentiation culture medium and a callus subculture proliferation culture medium. The culture medium provided by the invention can efficiently induce Xinjiang Ferula explants into Xinjiang Ferula callus (with an average callus rate of 58.25±4.54%) through suitable callus induction culture medium, complete dedifferentiation culture medium and callus subculture proliferation culture medium, and significantly improve the proliferation coefficient of callus (with an average proliferation coefficient of 13.83±0.83), obtain a large amount of healthy and stable callus in a short period of time, save time cost, and provide technical support for seedling cultivation and rapid propagation for the research on the protection and development of Ferula species resources.

[0021] Furthermore, the Xinjiang Ferula callus obtained by the cultivation method of the present invention can be used as a receptor for gene editing, and the breeding process of Xinjiang Ferula can be accelerated through gene editing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 This is a schematic diagram of Xinjiang Ferula seedlings after germination from seeds;

[0024] Figure 2 This is a schematic diagram of the induction of primary callus from the hypocotyl of Ferula xinjiangensis;

[0025] Figure 3 Schematic diagram of callus tissue obtained from complete dedifferentiation of hypocotyl of Ferula xinjiangensis;

[0026] Figure 4 This is a schematic diagram of the subculture of Ferula xinjiangensis callus for 30 days. DETAILED DESCRIPTION

[0027] The invention provides a culture medium for Xinjiang Ferula tissue culture, comprising a callus induction culture medium, a complete dedifferentiation culture medium and a callus subculture proliferation culture medium; the callus induction culture medium uses MS liquid culture medium as a basic culture medium and further comprises 2,4-D, sucrose and agar; the concentration of 2,4-D in the callus induction culture medium is 0.5-3.0 mg / L;

[0028] The complete dedifferentiation medium uses MS liquid medium as a basic medium and further 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 proliferation culture medium uses MS liquid culture medium as a basic culture medium and further comprises 2,4-D, NAA, 6-BA, sucrose and agar; the concentration of 2,4-D in the callus subculture proliferation culture 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 the present 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 of the present invention 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; the pH of the callus induction medium is preferably 5.8-6.0, further preferably 5.8-5.9, more preferably 5.83. Adding an appropriate concentration of 2,4-D to the callus induction medium of the present invention is beneficial to the induction of primary callus by Xinjiang Ferula.

[0031] In the present invention, the complete dedifferentiation 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 complete dedifferentiation medium of the present invention is 0.5 mg / L; the concentration of 6-BA in the complete dedifferentiation medium is 0.5 mg / L; the concentration of sucrose in the complete dedifferentiation medium is preferably 30 g / L; the concentration of agar in the complete dedifferentiation medium is preferably 6 g / L; the pH of the callus subculture proliferation medium is preferably 5.8 to 6.0, more preferably 5.8 to 5.9, and more preferably 5.83. Adding appropriate concentrations of 2,4-D and 6-BA to the complete dedifferentiation medium of the present invention is conducive to the complete dedifferentiation of primary callus.

[0032] In the present invention, the callus subculture proliferation medium uses MS liquid medium as the basic medium and further 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, and 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, and 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, and 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 complete dedifferentiation medium is preferably 6 g / L; the pH of the callus subculture proliferation medium is 5.8-6.0, further preferably 5.8-5.9, and more preferably 5.87. Adding appropriate concentrations of 2,4-D, NAA and 6-BA to the subculture proliferation medium of the present invention can significantly increase 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 invention also provides application of the culture medium for Ferula tissue culture in Xinjiang Ferula plant tissue culture.

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

[0035] Inoculating the Xinjiang Ferula explants into a callus induction medium for callus induction culture to obtain Xinjiang Ferula primary callus; inoculating the Xinjiang Ferula primary callus into a complete dedifferentiation medium for complete dedifferentiation culture to obtain Xinjiang Ferula dedifferentiated callus; inoculating the Xinjiang Ferula dedifferentiated callus into a callus subculture proliferation medium for subculture proliferation culture to obtain Xinjiang Ferula 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 in the culture medium for Ferula tissue culture.

[0037] The present invention inoculates Xinjiang Ferula explants into a callus induction medium for callus induction culture to obtain Xinjiang Ferula primary callus. In the present invention, the Xinjiang Ferula explant is preferably a hypocotyl, and the hypocotyl is preferably obtained by cutting a Xinjiang Ferula sterile seedling. The present invention selects a hypocotyl as an explant, and the hypocotyl has extremely young meristem cells, which have undergone complete rejuvenation brought by a sexual process and are very easy to be cultured into callus.

[0038] In the present invention, the callus induction culture is preferably first dark induction culture, and then light-dark alternation culture. The time of the dark induction culture of the present invention is preferably 7 to 10 days, and more preferably 7 days; the time of the light-dark alternation culture is preferably 15 to 30 days, and further preferably 18 to 28 days, and more preferably 20 to 25 days; the time of light culture in each cycle of the light-dark alternation culture is preferably 12 hours, and the time of dark culture is preferably 12 hours; the temperature of light culture in the light-dark alternation culture is preferably (25±0.5)°C, and more preferably 25°C; the temperature of dark culture in the light-dark alternation culture is preferably (15±0.5)°C, and more preferably 15°C. The present invention first performs dark induction culture to accelerate the callus initiation speed, which is more conducive to inducing callus, and performing light-dark alternation culture after dark induction culture can increase the callus emergence rate of explants.

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

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

[0041] The invention also provides application of the culture method in Xinjiang Ferula plant tissue culture.

[0042] In order to further illustrate the present invention, the culture method of Xinjiang Ferula callus provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

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

[0044] Example 1

[0045] (1) Preparation of culture medium

[0046] ① Callus induction medium: MS liquid medium is used as the basic medium, and only contains 2,4-D2.5 mg / L, sucrose 30 g / L and agar 6.0 g / L, pH = 5.83, and is sterilized at 121°C for 15 min to obtain callus induction medium.

[0047] ② Complete dedifferentiation medium: MS liquid medium is used as the basic medium, and only contains 2,4-D0.5mg / L, 6-BA 0.5mg / L, sucrose 30g / L and agar 6.0g / L, pH = 5.83, and is sterilized at 121℃ and high temperature and high pressure for 15min to obtain a complete dedifferentiation medium.

[0048] ③ Callus subculture proliferation medium: MS liquid medium is used as the basic medium, and only contains 2,4-D0.5mg / L, NAA0.1mg / L, 6-BA 0.5mg / L, sucrose 30g / L and agar 6.0g / L, pH=5.87, and is sterilized at 121℃ for 15min to obtain callus subculture proliferation medium.

[0049] (2) Explant pretreatment

[0050] After sterilization, the Xinjiang Ferula seeds were placed in wet sand and low-temperature stratified at 4°C for 90 days. After being taken out, they were germinated in a sterile environment to obtain sterile seedlings. Figure 1 The radicle and cotyledon of the sterile seedlings were removed to obtain the hypocotyls, which were cut into 1 cm pieces as explants.

[0051] (3) Callus induction

[0052] The obtained Xinjiang Ferula explants were inoculated into callus induction medium, cultured in darkness for 7 days, and then cultured in light and dark alternation for 25 days until white silky callus was produced, which was the primary callus. Figure 2 In each cycle of the light-dark alternating culture, the light culture is carried out for 12 hours and the dark culture is carried out for 12 hours. The temperature of the light-dark alternating culture is 25° C. for light and 15° C. for darkness.

[0053] (4) Complete callus culture

[0054] The primary callus tissue was inoculated into a completely dedifferentiated medium and cultured in alternating light and dark for 25 days until the white silky callus tissue was completely dedifferentiated to obtain yellow-white, granular, healthy new callus tissue, which was the dedifferentiated callus tissue. Figure 3 In each cycle of the light-dark alternating culture, the light culture is carried out for 12 hours and the dark culture is carried out for 12 hours. The temperature of the light-dark alternating culture is 25° C. for light and 15° C. for darkness.

[0055] (5) Callus subculture

[0056] The dedifferentiated callus was inoculated into the callus subculture proliferation culture and cultured in light and dark alternation for 30 days to obtain a large number of proliferating light yellow-green, granular, and vigorously growing Xinjiang Ferula callus. Figure 4In each cycle of the light-dark alternating culture, the light culture is carried out for 12 hours and the dark culture is carried out for 12 hours. The temperature of 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] Callus formation rate (%) = (number of explants forming callus tissue / total number of inoculated explants) × 100.

[0059] Callus inoculation weight: the measured value of the callus tissue weight inoculated on the culture medium before subculture proliferation begins.

[0060] Increase coefficient = fresh weight after 30 days of subculture / callus inoculation weight.

[0061] Table 1 Example 1 Hypocotyl number, callus rate, callus inoculation weight, proliferation coefficient

[0062] Hypocotyl (pcs) Cure rate (%) Callus inoculation weight (g) Proliferation coefficient Biological replicate 1 28 64.29 0.61 13.05 Biological replicate 2 16 53.33 0.68 14.96 Biological replicate 3 21 57.14 0.35 13.52 average value - 58.25±4.54 - 13.83±0.83

[0063] As shown in Table 1, the hypocotyl callus of Ferula xinjiangensis was successfully induced by plant tissue culture technology, with an average callus rate of (58.25±4.54)%, an average proliferation coefficient of 13.83±0.83, and a light yellow-green robust state of the callus.

[0064] Example 2

[0065] A tissue culture method similar to 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 was generated. The callus rate was (22.22±2.72)% after 30 days.

[0067] Example 3

[0068] A tissue culture method similar to 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 was generated. The callus rate was (26.67±4.71)% after 30 days.

[0070] Comparative Example 1

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

[0072] Observe the callus induction, generate white flocculent callus, and the callus rate is (0.00±0.00)% after 30 days.

[0073] Comparative Example 2

[0074] A tissue culture method similar to 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 was generated. The callus rate was (17.78±2.72)% after 30 days.

[0076] The healing rates of Xinjiang Ferula callus in Examples 1 to 3 and Comparative Examples 1 to 2 were statistically analyzed. The results are shown in Table 2.

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

[0078] Group 2,4-D concentration (mg / L) Cure rate (%) 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] It can be seen from Table 2 that appropriate concentrations of 2,4-D can promote the production of Xinjiang Ferula callus.

[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 was generated. The callus rate was (0±0)% after 30 days.

[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 was generated. The callus rate was (4.44±5.44)% after 30 days.

[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 was generated. The callus rate was (22.22±3.14)% after 30 days.

[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 was generated. The callus rate was (31.11±3.14)% after 30 days.

[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 was generated. The callus rate was (15.56±3.14)% after 30 days.

[0095] According to Examples 1 to 3 and Comparative Examples 1 to 7, the callus induction rates of different explants in different callus induction media were statistically analyzed. The results are shown in Table 3.

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

[0097] 2,4-D concentration (mg / L) Hypocotyl callus rate (%) Cotyledon healing rate (%) 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 the hypocotyl, the callus rate is significantly higher than that of the cotyledon, and the hypocotyl of Ferula foetida is more suitable for inducing callus tissue.

[0099] Example 4

[0100] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture proliferation 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 bright yellow in color. 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 proliferation 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 bright yellow. After 30 days, the callus proliferation coefficient was (10.51±0.22).

[0105] Example 6

[0106] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture proliferation 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 bright yellow. After 30 days, the callus proliferation coefficient was (13.22±0.53).

[0108] Comparative Example 8

[0109] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture proliferation culture 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 yellow-brown in color. 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 proliferation 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 Example 1, except that the concentration of 2,4-D in the callus subculture proliferation 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 yellow-brown in color. 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 proliferation 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 bright yellow in color. After 30 days, the callus proliferation coefficient was (12.45±0.77).

[0120] Comparative Example 12

[0121] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture proliferation 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 Example 1, except that the concentration of 2,4-D in the callus subculture proliferation 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 proliferation 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 bright yellow in color. 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 proliferation 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, and some parts were yellow-brown. After 30 days, the callus proliferation coefficient was (9.16±0.84).

[0132] Comparative Example 16

[0133] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture proliferation 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 proliferation 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 bright yellow in color. After 30 days, the callus proliferation coefficient was (11.00±1.41).

[0138] Comparative Example 18

[0139] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture proliferation 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 yellow-brown in color. After 30 days, the callus proliferation coefficient was (4.39±0.33).

[0141] Comparative Example 19

[0142] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture proliferation 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 bright yellow in color. After 30 days, the callus proliferation coefficient was (9.79±0.81).

[0144] Comparative Example 20

[0145] A tissue culture method similar to Example 1, except that the concentration of 2,4-D in the callus subculture proliferation 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 bright yellow in color. After 30 days, the callus proliferation coefficient was (9.11±0.46).

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

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

[0149]

[0150]

[0151] According to Table 4, NAA can promote the subculture proliferation of Xinjiang Ferula hypocotyl callus, and has an effect on the growth of callus. When NAA is not added to the subculture proliferation medium, the proliferation coefficient decreases, and most of the callus appear browning, dark yellow; under the condition of 2,4-D and 6-BA addition ratio of 1:1 to 1:1.5, the callus grows well and is bright yellow. On this basis, adding 0.1 mg / L NAA can improve the state of callus. This shows that the appropriate concentration of hormone ratio has a certain promoting effect on the subculture proliferation of callus, and the inappropriate hormone ratio has a toxic effect on the callus, and the callus formed is mostly dark yellow or yellow-brown.

[0152] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium for Xinjiang Ferula tissue culture, characterized in that: The invention comprises a callus induction medium, a complete dedifferentiation medium and a callus subculture proliferation medium; the callus induction medium uses MS liquid medium as a basic 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; The complete dedifferentiation medium uses MS liquid medium as a basic medium and further 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; The callus subculture proliferation culture medium uses MS liquid culture medium as a basic culture medium and further comprises 2,4-D, NAA, 6-BA, sucrose and agar; the concentration of 2,4-D in the callus subculture proliferation culture 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.

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 5.8-6.0 respectively.

3. Application of the culture medium according to claim 1 or 2 in Xinjiang Ferula plant tissue culture.

4. A method for culturing Xinjiang Ferula callus, characterized in that: The following steps are involved: Inoculating the Xinjiang Ferula explants in a callus induction medium for callus induction culture to obtain Xinjiang Ferula primary callus; inoculating the Xinjiang Ferula primary callus in a complete dedifferentiation medium for complete dedifferentiation culture to obtain Xinjiang Ferula dedifferentiated callus; inoculating the Xinjiang Ferula dedifferentiated callus in a callus subculture proliferation medium for subculture proliferation culture to obtain Xinjiang Ferula callus; 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 in the culture medium according to claim 1 or 2.

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

6. The culture method according to claim 4, characterized in that: The complete dedifferentiation culture is light-dark alternating culture; 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)°C, and the dark culture temperature is (15±0.5)°C; the complete dedifferentiation culture time is 15 to 30 days.

7. The culture method according to claim 4, characterized in that: The subculture proliferation culture is light-dark alternating culture; 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)°C, and the dark culture temperature is (15±0.5)°C; the subculture proliferation culture time is 30 to 35 days.

8. The culture method according to claim 4, characterized in that: The Xinjiang Ferula explant comprises the hypocotyl of Xinjiang Ferula.

9. The culture method according to claim 8, characterized in that The hypocotyls of the Xinjiang Ferula are taken from the Xinjiang Ferula sterile seedlings.

10. Use of the culture method according to any one of claims 4 to 9 in Xinjiang Ferula plant tissue culture.

Images