Culture medium composition for loose embryogenic calluses of cat beans and preparation method of culture medium composition for loose embryogenic calluses of cat beans

Abstract

The invention discloses a culture medium composition for loose embryonic calluses of cat beans and a preparation method of the culture medium composition. The invention firstly provides the culture medium combination for inducing the loose embryogenic callus of the cat bean, and the obtained culture medium combination is used for inducing the loose embryogenic callus of the cat bean by screening the components of the culture medium, so that the loose embryogenic callus of the cat bean with high induction success rate and strong proliferation stability can be obtained. Furthermore, the invention provides a preparation method of the loose embryogenic callus of the cat bean by utilizing the culture medium combination, the success rate of inducing the embryogenic callus of the cat bean by the preparation method is high, and meanwhile, a stable-proliferation loose embryogenic callus system of the cat bean can be established; not only can a foundation be laid for research on somatic cell mutagenesis and genetic transformation of the catbean, but also important theoretical basis and reference are provided for a catbean tissue culture rapid propagation system, in-vitro mutagenesis breeding, gene editing and suspension culture.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation, and in particular relates to a culture medium combination and a preparation method for loose embryonic callus of cat bean. Background Art

[0002] Cat bean belongs to the genus Mucuna of the Fabaceae family. It is also known as Mucuna bean (quinoa bean), dog claw bean, dragon claw bean, tiger bean, mouse bean, etc. It is an annual twining herbaceous vine that grows in subtropical regions. It is grown in Guangxi Zhuang Autonomous Region, Guangdong, Guizhou, Yunnan, Hainan and Jiangxi. Cat beans are drought-resistant, barren-resistant and highly adaptable. A large area of ​​cat beans is planted in the Guangxi Zhuang Autonomous Region. They are mainly used to extract L-dopa and produce specific drugs for the treatment of Parkinson's disease and hepatic coma. The extraction of natural L-dopa from cat beans has a history of many years, and with the continuous improvement of L-dopa extraction technology, the extraction amount is as high as 9.54-9.94%. The medicinal value and feed use of cat beans are gradually developed and utilized, and the demand for cat beans will increase significantly. However, because cat beans are annual plants, the production mainly relies on harvesting seeds for seed preservation and L-dopa extraction. Due to seasonal restrictions, low reproduction coefficients and limited cat bean yields, it is difficult to meet the current market demand. Therefore, increasing cat bean yields through tissue culture and rapid propagation is one of the effective ways.

[0003] Establishing a stable loose embryonic callus system of cat bean can provide important theoretical basis and reference for cat bean tissue culture rapid propagation system, in vitro mutagenesis breeding, gene editing and suspension culture. In addition, it also lays the foundation for the research of cat bean somatic cell mutagenesis, genetic transformation, medicinal development value, comprehensive utilization rate, etc. However, there is no research on the loose embryonic callus system of cat bean that can be efficiently induced and stably proliferated. Hang Ling et al. (2001) studied the plant regeneration of Ligusticum chuanxiensis tissue cell culture and obtained the initial embryonic callus, but the embryonic callus prepared was a callus with a tight structure, and no loose embryonic callus was obtained. Therefore, providing a culture medium and method for preparing loose embryonic callus of cat bean with high induction rate and good proliferation stability is of great significance to the establishment of a systematic and efficient cat bean tissue culture rapid propagation system and secondary metabolite production system. Summary of the invention

[0004] The object of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and to provide a culture medium combination for inducing loose embryonic callus of cat bean.

[0005] The second object of the present invention is to provide a method for preparing loose embryonic callus of cat bean based on the culture medium combination.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention first provides a culture medium combination for inducing loose embryonic callus of cat bean, comprising the following culture medium:

[0008] Culture medium ①: MS culture medium plus carrageenan 7.5-8.5 g / L, sucrose 30-40 g / L, VC 0.5-1 mg / L, and activated carbon 0.08-0.12%;

[0009] Culture medium ②: MS culture medium plus carrageenan 7.5-8.5 g / L, sucrose 30-40 g / L, 6-BA 0.5-1.5 mg / L, NAA 0.05-0.5 mg / L, ZT 0.5-1 mg / L, VC 1.0-2.0 mg / L;

[0010] Culture medium ③: MS culture medium plus carrageenan 7.5-8.5 g / L, sucrose 30-40 g / L, 6-BA 3.0-5.0 mg / L, IBA 0.2-1 mg / L, NAA 0.05-0.2 mg / L, VC 2.0-3.0 mg / L.

[0011] In the culture medium combination provided by the present invention, culture medium ① is used to induce germination of cat bean seeds, culture medium ② is used to induce formation of embryonic callus, and culture medium ③ is used to induce formation of loose embryonic callus; by screening the types of antioxidant substances and plant growth regulators added in culture medium ①, ②, and ③, specifically the effects of activated carbon and VC added in culture medium ① and ② on seed germination and explant induction of embryonic callus, the results show that the addition of activated carbon and VC in culture medium ① can reduce oxidation in the seed germination stage, and the addition of only VC in culture medium ② can reduce oxidation in callus induction to a certain extent and will not affect the induction of explants into callus; in addition, the effects of adding different plant growth regulators in culture medium ② and ③ on inducing embryonic callus and obtaining stably proliferating loose embryonic callus are studied, and the results show that when NAA, IBA, and ZT are added to culture medium ②, the induction rate increases, the callus state is yellow-green, and the state is better, and when 6-BA, NAA, and IBA are added to culture medium ③, the callus can stably proliferate, and the callus state is yellow-green and loose. Therefore, by using the above culture medium combination to induce loose embryonic callus tissue of Cat Bean, loose embryonic callus of Cat Bean with high induction success rate and strong proliferation stability can be obtained.

[0012] Furthermore, the culture medium ② is MS culture medium plus 7.5-8.5 g / L carrageenan, 30-40 g / L sucrose, 0.5-1.0 mg / L 6-BA, 0.05 mg / L NAA, 0.5 mg / L ZT, and 1.0-2.0 mg / L VC.

[0013] Preferably, the culture medium ② is MS culture medium plus 7.5 g / L carrageenan, 30 g / L sucrose, 0.5 mg / L 6-BA, 0.05 mg / L NAA, 0.5 mg / L ZT, and 1.0 mg / L VC.

[0014] Furthermore, the culture medium ③ is MS culture medium plus carrageenan 7.5-8.5 g / L, sucrose 30-40 g / L, 6-BA 3.0 mg / L, IBA 0.2 mg / L, NAA 0.05 mg / L, and VC 2.0-3.0 mg / L.

[0015] Preferably, the culture medium ③ is MS culture medium plus 7.5 g / L carrageenan, 30 g / L sucrose, 3.0 mg / L 6-BA, 0.2 mg / L IBA, 0.05 mg / L NAA, and 2.0 mg / L VC.

[0016] The present invention also provides a method for preparing loose embryonic callus of cat bean using any of the above-mentioned culture medium combinations, comprising the following steps:

[0017] S1. Obtaining sterile cat bean plants: Washing, soaking, and disinfecting cat bean seeds, removing the seed coat, and inoculating them on a culture medium ① to induce seed germination to grow into plants with two pairs of leaves;

[0018] S2. Inducing embryonic callus of cat bean: taking leaves and / or stem segments of the plants obtained in step S1 as explants, inoculating them in culture medium ② after wound treatment and culturing them until embryonic callus is obtained;

[0019] S3. Obtain stable loose embryonic callus of cat bean: Select the embryonic callus obtained in S2 and inoculate it into culture medium ③ to continue callus induction culture, carry out 3 to 4 generations of callus subculture, and obtain stable loose embryonic callus of cat bean.

[0020] In the method, the present invention explores the influence of different explants selected in step S2 to induce embryonic callus. The results show that when stem segments are used as explants for callus induction, browning will occur, but the retained stem segments generate callus faster; when bud tips are used as explants to induce callus, compact yellow-green callus is induced at the wound; when leaves are used as explants to induce callus, the callus induced at the wound and vein wound is yellow-green and loose. Therefore, selecting stem segments and leaves as explants is conducive to the induction of embryonic callus of cat beans.

[0021] Furthermore, the leaves are young leaves.

[0022] Furthermore, the method of rinsing, soaking and disinfecting in step S1 is to rinse the seeds with clean water, then shake them with detergent, rinse them with running water until there is no foam, then soak the seeds in an isothiazolinone solution, and rinse them with running water; then the seeds are subjected to ultraviolet irradiation, 75% alcohol treatment, rinsed with sterile water for 2 to 3 times, disinfected with a 0.1% HgCl solution containing Tween 20, and rinsed with sterile water for 8 to 10 times.

[0023] Preferably, the method of rinsing, soaking and disinfecting is to rinse the seeds with clean water, shake them with detergent for 30 minutes, rinse them with running water until there is no foam, soak the seeds in 100 mg / L isothiazolinone solution for 3 minutes, and rinse them with running water for 10 minutes; place the seeds in a clean bench and irradiate them with ultraviolet light for 20 minutes, treat them with 75% alcohol for 1 minute, rinse them with sterile water twice, add 0.5 mL / L Tween 20 to 0.1% HgCl solution for disinfection for 9 minutes, rinse them with sterile water 8 to 10 times, use filter paper to absorb the moisture on the surface of the seeds, and irradiate them with ultraviolet light and blow until there is no moisture on the surface of the seeds.

[0024] Among them, the surface of the seeds can be simply disinfected by using isothiazolinone solution and detergent. Through the exploration of the steps of the disinfection experiment (ultraviolet irradiation, 75% alcohol treatment, 0.5mL Tween added to 0.1% HgCl solution for immersion disinfection), it was found that if the disinfection time is insufficient or the subsequent disinfection steps are omitted (i.e., only the surface of the seeds is simply disinfected with isothiazolinone), the infection rate is greatly improved, and if the disinfection time is too long or other disinfection steps are added, it will cause excessive lethality to the seeds and increase the mortality rate.

[0025] Furthermore, the stem segment in step S2 is a stem segment without axillary buds.

[0026] Furthermore, the wound treatment in step S2 is to use a scalpel to quickly cut the explant leaf or stem segment to form a wound, cut the leaf into a square of 0.8 cm×0.8 cm, and lay it flat on the surface of the culture medium ②, and cut the stem segment into a length of 0.5 to 0.8 cm and lay it flat on the surface of the culture medium ②. Among them, the rapid action when cutting the explant can reduce the water loss rate of the explant to ensure the activity of the explant.

[0027] Furthermore, the method of culturing to obtain embryonic callus in step S2 is to perform dark treatment culture until the leaves or stem segments curl, and then perform light culture until callus is formed to obtain preliminary embryonic callus tissue of cat bean.

[0028] Furthermore, the embryonic callus in step S3 is fluffy, green embryonic callus.

[0029] Furthermore, the callus induction culture in step S3 is to inoculate the embryonic callus into the culture medium ③ and culture it for 10 to 15 days until the embryonic callus swells, and then perform 3 to 4 generations of callus subculture.

[0030] Preferably, step S3 is to select high-quality callus tissue induced in culture medium ②, remove brown callus and leaves without induced callus, inoculate the loose, green embryonic callus into culture medium ③ to continue callus induction culture, and the callus swells after about 10 to 15 days. The brown callus and leaves without induced callus are removed and inoculated into culture medium ③ to continue callus induction culture, and 3 to 4 generations of callus subculture are performed to obtain loose embryonic callus tissue of cat bean with stable growth.

[0031] The selection of high-quality callus plays an important role in the subsequent callus induction culture, and after callus subculture, not only can the embryonic state of the callus be ensured not to be lost, but also high-quality loose embryonic callus with stable inheritance can be obtained. The loose embryonic callus obtained by the method of the present invention is used as a bioreactor for the production of secondary metabolites because it is easy to disperse into single cells and can be used for liquid suspension culture to improve the production efficiency of secondary metabolites.

[0032] Therefore, the present invention also provides loose embryonic callus of cat bean prepared by any of the above methods. The loose embryonic callus of cat bean has the characteristics of not losing the embryonic state of callus and stable inheritance.

[0033] The present invention also provides application of the loose embryonic callus of cat bean in producing cat bean secondary metabolites.

[0034] The present invention also provides application of the loose embryonic callus of cat bean in cat bean breeding or new variety development.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention first provides a culture medium combination for inducing loose embryonic callus of cat bean. By screening the components of the culture medium, the obtained culture medium combination is used to induce loose embryonic callus of cat bean, and a loose embryonic callus of cat bean with high induction success rate and strong proliferation stability can be obtained. Further, the present invention uses the culture medium combination to provide a method for preparing loose embryonic callus of cat bean. The method has a high success rate for inducing embryonic callus of cat bean, and a loose embryonic callus system of cat bean with stable proliferation can be established, which can not only lay a foundation for the research on somatic cell mutagenesis and genetic transformation of cat bean, but also provide important theoretical basis and reference for the tissue culture rapid propagation system, in vitro mutagenesis breeding, gene editing and suspension culture of cat bean. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a picture of cat bean seeds germinating into plants.

[0038] Figure 2 This is a picture of the embryonic callus of cat bean.

[0039] Figure 3 This is a picture of the loose callus tissue of cat bean.

[0040] Figure 4 These are the results of the effects of adding VC and activated carbon on callus induction; among them, 1 is the effect of adding VC on callus induction, and 2 is the effect of adding activated carbon on callus induction.

[0041] Figure 5 The figures are the results of the effects of different explants on callus induction; among them, 1 is the result of the effect of stem segments on callus induction; 2 is the result of the effect of bud tips on callus induction; 3 is the result of the effect of leaves on callus induction; 4 is the result of the effect of tender shoots on callus induction. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] Source of cat bean seeds: collected from the base in Nanning City, Guangxi Zhuang Autonomous Region.

[0045] Example 1

[0046] A culture medium combination for loose embryonic callus of cat bean, comprising the following culture medium:

[0047] Culture medium ① (seed germination induction): MS culture medium plus carrageenan 7.5 g / L, sucrose 30 g / L, VC 1 mg / L, 0.1% activated carbon;

[0048] Culture medium ② (inducing the formation of preliminary embryonic callus): MS culture medium plus carrageenan 7.5 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.05 mg / L, ZT 0.5 mg / L, VC 1.0 mg / L;

[0049] Culture medium ③ (to induce the formation of loose embryonic callus): MS medium plus carrageenan 7.5 g / L, sucrose 30 g / L, 6-BA 3.0 mg / L, NAA 0.05 mg / L, IBA 0.2 mg / L, VC 2.0 mg / L.

[0050] Medium preparation:

[0051] Use MS as the basic culture medium, add different concentrations of plant growth regulators or nutrients according to the above culture medium combination formula: 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid (NAA), indolebutyric acid (IBA), sucrose, activated carbon, carrageenan, adjust the pH to 5.8 to 6.0, divide into culture bottles, put into a high-pressure steam sterilizer at 121°C, 0.1Mpa for sterilization for 20 minutes, and wait for it to cool and solidify for use.

[0052] If plant growth regulators such as indoleacetic acid (IAA), VC, and zeatin (ZT) that cannot be sterilized at high temperatures are added, the prepared mother liquor must be filtered through a 0.22μm pore size filter membrane, and the basal culture medium must be cooled to 60°C after high temperature sterilization. The plant growth regulators are then added and allowed to cool and solidify before use.

[0053] Example 2

[0054] A culture medium combination for loose embryonic callus of cat bean, comprising the following culture medium:

[0055] Culture medium ①: MS culture medium plus carrageenan 8.5 g / L, sucrose 40 g / L, VC 0.5 mg / L, 0.08% activated carbon;

[0056] Culture medium ②: MS culture medium plus carrageenan 8.5 g / L, sucrose 40 g / L, 6-BA 1.5 mg / L, NAA 0.5 mg / L, ZT 1 mg / L, VC 2.0 mg / L;

[0057] Culture medium ③: MS culture medium plus carrageenan 8.5 g / L, sucrose 40 g / L, 6-BA 5.0 mg / L, NAA 0.2 mg / L, IBA 1 mg / L, VC 3.0 mg / L.

[0058] The culture medium was prepared according to the method described in Example 1.

[0059] Example 3

[0060] A culture medium combination for loose embryonic callus of cat bean, comprising the following culture medium:

[0061] Culture medium ①: MS culture medium plus carrageenan 8.0 g / L, sucrose 35 g / L, VC 0.75 mg / L, and 0.12% activated carbon;

[0062] Culture medium ②: MS culture medium plus carrageenan 8.0 g / L, sucrose 35 g / L, 6-BA 1 mg / L, NAA 0.25 mg / L, ZT 0.75 mg / L, VC 1.5 mg / L;

[0063] Culture medium ③: MS culture medium plus carrageenan 8.0 g / L, sucrose 35 g / L, 6-BA 4.0 mg / L, IBA 0.6 mg / L, NAA 0.125 mg / L, VC 2.5 mg / L.

[0064] The culture medium was prepared according to the method described in Example 1.

[0065] Example 4

[0066] The embryonic callus of Psoralea corylifolia was prepared using the culture medium in the culture medium combination described in Example 1, and the specific steps are as follows:

[0067] 1. Selection and detoxification of cat bean seeds

[0068] Collect mature cat pods with pods.

[0069] Gently peel off the pods of the collected seeds to obtain gray-white seeds; rinse with clean water, shake with detergent for 30 minutes, rinse with running water until there is no foam, soak the seeds in 100 mg / L isothiazolinone solution for 3 minutes, and rinse with running water for 10 minutes.

[0070] The seeds were placed in a clean bench and exposed to ultraviolet light for 20 minutes, treated with 75% alcohol for 1 minute, rinsed with sterile water twice, sterilized with 0.1% HgCl solution with 0.5 mL Tween 20 mixed for 9 minutes, and rinsed with sterile water 8 to 10 times; the surface moisture of the seeds was absorbed with filter paper and exposed to ultraviolet light and aired until there was no moisture on the surface of the seeds, the seed coat was removed and inoculated into culture medium ① to induce seed germination until the seeds grew into plants with two pairs of leaves. The resulting plants were as follows Figure 1 shown.

[0071] 2. Induction of embryonic callus of cat bean

[0072] Under sterile conditions, select young leaves, stem segments without axillary buds (the stem segments described below are all stem segments without axillary buds) and leaves with good growth in culture medium ① as explants, and use a sharp scalpel to quickly form wounds on the explants, such as cutting the leaves into 0.8 cm × 0.8 cm squares and spreading them on the surface of culture medium ②, and cutting the stem segments into 0.5-0.8 cm lengths and spreading them on the surface of culture medium ②.

[0073] The cells were cultured in the dark for 7 days until the leaves and stems curled up, and then cultured in the light for 20 days until callus was formed to obtain the initial embryonic callus tissue of cat bean (such as Figure 2 ).

[0074] 3. Induction of loose embryonic callus of Cat Bean

[0075] Select high-quality callus tissue in medium ②, remove brown callus and leaves that have not been induced to callus, inoculate the relatively fluffy, green embryonic callus into medium ③ to continue callus induction culture, and the callus will swell in about 10 to 15 days. Remove the brown callus and leaves that have not been induced to callus, inoculate them into medium ③ to continue callus induction culture, and perform 3 to 4 generations of callus subculture to obtain stable growth of loose embryonic callus tissue of cat bean (such as Figure 3 ).

[0076] Among them, the seed detoxification step was optimized, and the results showed that the surface of the seeds could be simply disinfected using isothiazolinone solution and detergent. Through the exploration of the disinfection experiment (ultraviolet irradiation, 75% alcohol treatment, 0.5mL Tween added to 0.1% HgCl solution for immersion disinfection), it was found that if the disinfection time was insufficient or the subsequent disinfection steps were omitted (i.e., only the surface of the seeds was simply disinfected with isothiazolinone), the infection rate was greatly improved, and if the disinfection time was too long or other disinfection steps were added, the seed lethality would be too great and the mortality rate would increase.

[0077] Comparative Example 1

[0078] To explore the effect of adding VC and activated carbon on the induction of embryonic callus of cat bean

[0079] In the process of inducing callus tissue of cat bean leaves and stem segments, severe oxidation occurred, and combined with the addition of activated carbon and VC during seed germination to reduce oxidation, experiments were conducted to investigate the effects of adding VC and activated carbon to culture medium ① and ② on callus induction. The experimental method was the same as that in Example 4.

[0080] The results showed that adding VC and activated carbon during the seed germination stage could reduce oxidation, but adding activated carbon did not improve the oxidation during embryonic callus induction, and also inhibited the formation of callus at the wound of explants. Figure 4 1; Adding VC in the concentration range of 1.0-2.0 mg / L during the induction stage of embryonic callus of cat bean can reduce oxidation in the induction of embryonic callus to a certain extent and will not affect the induction of explants into embryonic callus. Figure 4 In Figure 2, it can be seen that the wound of the explant has swelled and callus tissue has begun to be induced. Therefore, activated carbon and vitamin C can be added to culture medium ①, while vitamin C can be added to culture medium ②.

[0081] Comparative Example 2

[0082] Study on the effects of different explants on embryonic callus induction of cat bean

[0083] Select young leaves, stem segments without axillary buds (the stem segments described hereinafter are all stem segments without axillary buds), bud tips, and leaves as explants to test the effects of different explants on callus induction, that is, in step 2 of Example 4, different parts of cat bean plants are selected as explants, and the remaining steps are the same as in Example 4.

[0084] The results showed that when stem segments were used as explants for callus induction, browning still occurred even with the addition of VC for antioxidant treatment, and the induced callus was water-soaked and lighter in color ( Figure 5 1), but not all stem segments turn brown. The remaining unbrown stem segments often generate callus faster, so the stem segments can still be used as explants for callus culture. The bud tip is used as an explant to induce callus tissue, and a compact yellow-green callus is induced at the wound ( Figure 5 2); with leaves ( Figure 5 Middle 4) and tender leaves ( Figure 5 3) When inducing callus on explants, the callus tissue induced at the wound and vein wound is yellow-green and in a loose state.

[0085] Therefore, stem segments, leaves and young leaves can be selected as explants for inducing embryonic callus of P. cat bean.

[0086] Comparative Example 3

[0087] Study on the effects of different plant growth regulators on embryonic callus induction of cat bean

[0088] Plant growth regulators such as 6-BA, NAA, 2,4-D, IBA, KT, GA3, ZT, and TDZ were selected to test the effects of different plant growth regulators on callus induction, that is, the plant growth regulators in the culture medium ② were screened. Only some representative combinations of different plant growth regulators are shown below. The test steps are the same as those in Example 4.

[0089] The results showed that through experimental exploration, it was found that the use of the above plant growth regulators alone could not induce callus tissue, and the leaves turned yellow-brown and then died. However, adding 0.5 to 1.0 mg / L concentration of 6-BA made the edges of the explants curl and the wound showed signs of inducing callus tissue. Therefore, the experiment was carried out using a combination of plant growth regulators. As shown in Table 1, when 2,4-D, KT, GA3, and TDZ were added to the culture medium, not only the time for inducing callus tissue was longer, but the callus state was grayish white or yellowish brown, and some whitish and dry callus appeared, which was not only difficult to subculture and proliferate, but also had a shorter maintenance time and died faster. When NAA, IBA, and ZT were added, not only the induction rate increased, but the callus state was yellow-green and in better condition. According to the induction rate and callus state, when 0.5-1.0 mg / L 6-BA, 0.05 mg / L NAA and 0.5 mg / L ZT were added to the culture medium, the induction rate could reach 90.23%, and the callus was yellow-green and loose. This was used as the material to screen the loose embryonic callus of cat bean with stable growth.

[0090] Table 1 Effects of different plant growth regulators on embryonic callus induction of Cat Bean

[0091]

[0092] Comparative Example 4

[0093] A combination of different plant growth regulators such as 6-BA, NAA, 2,4-D, IBA, and ZT was selected to test the effects of different plant growth regulators on the stability of loose callus, that is, the plant growth regulators in culture medium ③ were screened, and the test steps were the same as those in Example 4.

[0094] The results showed that: as shown in Table 2, when 2,4-D hormone was added to the culture medium, although the callus tissue proliferated a small amount, the color was whitish and dry, and it did not have the morphology of loose callus; when NAA, IBA, and ZT were added, the callus tissue showed a water-soaked state, which was not conducive to the stable proliferation of callus tissue; when 3.0 mg / L 6-BA, 0.05 mg / L NAA, and 0.2 mg / L IBA were added to the culture medium, the callus tissue could proliferate stably, and the callus state was yellow-green and loose. The obtained loose embryonic callus tissue was used as a bioreactor for the production of secondary metabolites, which could be used for liquid suspension culture to improve the production efficiency of secondary metabolites, which not only laid the foundation for the study of somatic cell mutagenesis and genetic transformation of cat beans, but also provided important theoretical basis and reference for the tissue culture rapid propagation system, in vitro mutagenesis breeding, gene editing, and suspension culture of cat beans.

[0095] Table 2 Effects of different plant growth regulators on the stability of loose callus

[0096]

[0097] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A culture medium combination for inducing loose embryonic callus of cat bean, characterized in that: The following media are included: Culture medium ①: MS culture medium plus carrageenan 7.5-8.5 g / L, sucrose 30-40 g / L, VC 0.5-1 mg / L, and activated carbon 0.08-0.12%; Culture medium ②: MS culture medium plus carrageenan 7.5-8.5 g / L, sucrose 30-40 g / L, 6-BA 0.5-1.5 mg / L, NAA 0.05-0.5 mg / L, ZT 0.5-1 mg / L, VC 1.0-2.0 mg / L; Culture medium ③: MS culture medium plus carrageenan 7.5-8.5 g / L, sucrose 30-40 g / L, 6-BA 3.0-5.0 mg / L, IBA 0.2-1 mg / L, NAA 0.05-0.2 mg / L, VC 2.0-3.0 mg / L.

2. The culture medium combination according to claim 1, characterized in that: The culture medium ② is MS culture medium plus 7.5-8.5 g / L carrageenan, 30-40 g / L sucrose, 0.5-1.0 mg / L 6-BA, 0.05 mg / L NAA, 0.5 mg / L ZT, and 1.0-2.0 mg / L VC.

3. The culture medium combination according to claim 1, characterized in that: The culture medium ③ is MS culture medium plus 7.5-8.5 g / L carrageenan, 30-40 g / L sucrose, 3.0 mg / L 6-BA, 0.2 mg / L IBA, 0.05 mg / L NAA, and 2.0-3.0 mg / L VC.

4. A method for preparing loose embryonic callus of cat bean based on the culture medium combination according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Obtaining sterile cat bean plants: Washing, soaking, and disinfecting cat bean seeds, removing the seed coat, and inoculating them on a culture medium ① to induce seed germination to grow into plants with two pairs of leaves; S2. Inducing embryonic callus of cat bean: taking leaves and / or stem segments of the plants obtained in step S1 as explants, inoculating them in culture medium ② after wound treatment until embryonic callus is obtained; S3. Obtain stable loose embryonic callus of cat bean: Select the embryonic callus obtained in S2 and inoculate it into culture medium ③ to continue callus induction culture, carry out 3 to 4 generations of callus subculture, and obtain stable loose embryonic callus of cat bean.

5. The preparation method according to claim 4, characterized in that: The method of rinsing, soaking and disinfecting in step S1 is to rinse the seeds with clean water, then shake them with detergent, rinse them with running water until there is no foam, then soak the seeds in an isothiazolinone solution, and rinse them with running water; then the seeds are subjected to ultraviolet irradiation, 75% alcohol treatment, rinsed with sterile water for 2 to 3 times, disinfected with a 0.1% HgCl solution containing Tween 20, and rinsed with sterile water for 8 to 10 times.

6. The preparation method according to claim 4, characterized in that: The stem segment described in step S2 is a stem segment without axillary buds.

7. The preparation method according to claim 4, characterized in that: The embryonic callus in step S3 is fluffy, green embryonic callus.

8. The preparation method according to claim 4, characterized in that: The callus induction culture in step S3 is to inoculate the embryonic callus into the culture medium ③ and culture it for 10 to 15 days until the embryonic callus swells, and then perform 3 to 4 generations of callus subculture.

9. The loose embryonic callus of cat bean prepared by the preparation method according to any one of claims 4 to 8.

10. Use of the loose embryonic callus of cat bean according to claim 9 in producing cat bean secondary metabolites.

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