Culture medium combination and preparation method of loose-phase embryogenic callus of cat bean

Abstract

The application discloses a culture medium combination and a preparation method of loose embryogenic callus of cat's claw bean. The application firstly provides a culture medium combination for inducing loose embryogenic callus of cat's claw bean. Through screening of components of the culture medium, the culture medium combination is used for inducing loose embryogenic callus of cat's claw bean, and the loose embryogenic callus of cat's claw bean with high induction success rate and strong proliferation stability can be obtained. Further, the application provides a preparation method of loose embryogenic callus of cat's claw bean by using the culture medium combination. The preparation method has high success rate in inducing cat's claw bean embryogenic callus, and can establish a cat's claw bean loose embryogenic callus system with stable proliferation. The application not only lays a foundation for cat's claw bean somatic cell mutagenesis and genetic transformation research, but also provides an important theoretical basis and reference for cat's claw bean tissue culture rapid propagation system, in vitro mutagenesis breeding, gene editing and suspension culture.

Description

Technical Field

[0001] This invention belongs to the field of plant culture technology, specifically relating to a culture medium composition and preparation method for loose embryogenic callus of cat's bean. Background Technology

[0002] Cat bean (Ligustrum lucidum) belongs to the genus Ligustrum of the family Fabaceae. Also known as 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 cultivated in Guangxi Zhuang Autonomous Region, Guangdong, Guizhou, Yunnan, Hainan and Jiangxi. Cat's bean is characterized by its drought and poor soil tolerance and strong adaptability. It is widely cultivated in Guangxi Zhuang Autonomous Region, primarily for the extraction of levodopa, a highly effective drug for treating Parkinson's disease and hepatic coma. Extraction of natural levodopa from cat's bean has a long history, and with continuous improvements in extraction technology, the extraction yield has reached as high as 9.54%–9.94%. As the medicinal and feed uses of cat's bean are increasingly developed and utilized, the demand for it will increase significantly. However, because cat's bean is an annual plant, production mainly relies on harvesting seeds for preservation and levodopa extraction. Furthermore, seasonal limitations, low propagation coefficients, and limited yield make it difficult to meet current market demand. Therefore, rapid propagation through tissue culture to increase cat's bean yield is one effective method.

[0003] Establishing a stable loose embryogenic callus system for *Lysimachia foetida* can provide important theoretical basis and reference for rapid propagation systems of *Lysimachia foetida* tissue culture, in vitro mutagenesis breeding, gene editing, and suspension culture. Furthermore, it lays the foundation for research on somatic cell mutagenesis, genetic transformation, medicinal development value, and comprehensive utilization rate of *Lysimachia foetida*. However, there is currently no research on a highly efficient and stable *Lysimachia foetida* loose embryogenic callus system. Hang Ling et al. (2001) studied plant regeneration through *Lysimachia foetida* tissue cell culture and obtained initial embryogenic callus, but the resulting callus was a tightly structured callus, not a loose embryogenic callus. Therefore, providing a culture medium and method for preparing loose embryogenic callus of *Lysimachia foetida* with high induction rate and good proliferation stability is of great significance for establishing a systematic and efficient rapid propagation system of *Lysimachia foetida* tissue culture and a secondary metabolite production system. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a culture medium combination for inducing loose embryogenic callus from cat's bean.

[0005] A second objective of this invention is to provide a method for preparing loose embryogenic callus from cat's bean based on the aforementioned culture medium combination.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention first provides a culture medium combination for inducing loose embryogenic callus from cat's bean, comprising the following culture media:

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

[0009] Culture medium ②: MS medium with 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, and VC 1.0-2.0 mg / L;

[0010] Culture medium ③: MS medium with 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, and VC 2.0-3.0 mg / L.

[0011] In the culture medium combination provided by this invention, culture medium ① is used to induce the germination of cat's bean seeds, culture medium ② is used to induce the formation of embryogenic callus, and culture medium ③ is used to induce the formation of loose embryogenic callus. The types of antioxidants and plant growth regulators added to culture media ①, ②, and ③ were screened. Specifically, the effects of adding activated carbon and vitamin C to culture media ① and ② on seed germination and explant-induced embryogenic callus formation were investigated. The results showed that adding activated carbon and vitamin C to culture medium ① could reduce oxidation during seed germination. Adding only vitamin C to culture medium ② could reduce oxidation during callus induction to a certain extent without affecting the explant's induction into callus. Furthermore, the effects of adding different plant growth regulators to culture media ② and ③ on the induction of embryogenic callus and the acquisition of stable, loosely proliferating embryogenic callus were investigated. The results showed that adding NAA, IBA, and ZT to culture medium ② increased the induction rate, and the callus was yellow-green and in good condition. Adding 6-BA, NAA, and IBA to culture medium ③ resulted in stable callus proliferation, and the callus was yellow-green and loosely proliferating. Therefore, by using the above-mentioned culture medium combination to induce loose embryogenic callus from cat's bean, a loose embryogenic callus from cat's bean with a high induction success rate and strong proliferation stability can be obtained.

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

[0013] Preferably, the culture medium ② is MS medium supplemented with 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, and VC 1.0 mg / L.

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

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

[0017] S1. Obtaining sterile cat bean plants: Rinse, soak, and disinfect cat bean seeds, remove the seed coat, and inoculate them into culture medium ① to induce seed germination until they grow into plants with two pairs of leaves;

[0018] S2. Induction of embryogenic callus from cat's bean: Leaves and / or stem segments of the plant obtained in step S1 were used as explants, and after wound treatment, they were inoculated into culture medium ② and cultured until embryogenic callus was obtained.

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

[0020] In the method described above, this invention investigates the effect of selecting different explants in step S2 to induce embryogenic callus. The results show that when stem segments are used as explants for callus induction, browning occurs, but the retained stem segments generate callus at a faster rate. When shoot tips are used as explants, dense, yellowish-green callus is induced at the wound site. When leaves are used as explants, the callus induced at the wound and vein wound sites is yellowish-green and relatively loose. Therefore, selecting stem segments and leaves as explants is beneficial for inducing embryogenic callus in *Phyllostachys edulis*.

[0021] Furthermore, the leaf is a young leaf.

[0022] Further, the rinsing, soaking, and disinfection method described in step S1 is as follows: rinse the seeds with clean water, then shake with detergent, rinse with running water until no foam remains, then soak the seeds in isothiazolinone solution and rinse with running water; then irradiate the seeds with ultraviolet light, treat with 75% alcohol, rinse with sterile water 2-3 times, disinfect with 0.1% HgCl solution containing Tween 20, and rinse with sterile water 8-10 times.

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

[0024] Isothiazolinone solution and detergent can be used for simple disinfection of seed surfaces. Through exploration of disinfection experiments (UV irradiation, 75% alcohol treatment, and immersion disinfection with 0.5 mL Tween added to 0.1% HgCl solution), it was found that if the disinfection time is insufficient or subsequent disinfection steps are omitted (i.e., simple disinfection of seed surfaces with only isothiazolinone), the contamination rate is greatly increased. If the disinfection time is too long or other disinfection steps are added, it will cause excessive damage to the seeds and increase the mortality rate.

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

[0026] Furthermore, the wound treatment in step S2 involves quickly cutting the explant leaves or stem segments with a scalpel to create wounds. The leaves are cut into 0.8cm × 0.8cm squares and laid flat on the surface of the culture medium ②, and the stem segments are cut into 0.5-0.8cm lengths and laid flat on the surface of the culture medium ②. The rapid cutting of the explants reduces water loss and ensures explant viability.

[0027] Furthermore, the method for cultivating embryogenic callus in step S2 is to cultivate in the dark until the leaves or stem segments curl, and then cultivate under light until callus forms, thereby obtaining preliminary embryogenic callus tissue of the cat bean.

[0028] Furthermore, the embryogenic callus described in step S3 is a loose, green embryogenic callus.

[0029] Furthermore, the callus induction culture described in step S3 involves inoculating embryogenic callus into culture medium ③ and culturing for 10-15 days until the embryogenic callus swells, followed by 3-4 generations of callus subculture.

[0030] Preferably, step S3 involves selecting high-quality callus tissue induced in culture medium ②, removing brown callus and leaves that have not been induced to produce callus, and inoculating loose, green embryogenic callus into culture medium ③ to continue callus induction culture. After about 10 to 15 days, the callus swells, and brown callus and leaves that have not been induced to produce callus are removed. The callus is then inoculated into culture medium ③ to continue callus induction culture. This process is repeated for 3 to 4 generations of callus subculture to obtain stable cat's bean loose embryogenic callus tissue.

[0031] Selecting high-quality callus plays a crucial role in subsequent callus induction and culture. Subculturing the callus not only ensures the preservation of its embryogenic state but also yields high-quality, loosely embryogenic callus tissue with stable genetic characteristics. The loosely embryogenic callus tissue obtained by the method of this invention can be used as a bioreactor for the production of secondary metabolites. Because it easily disperses into single cells, it can be used for liquid suspension culture, improving the production efficiency of secondary metabolites.

[0032] Therefore, this invention also provides loose embryogenic callus from cat's bean prepared by any of the methods described above. The loose embryogenic callus from cat's bean has the characteristics of not losing its callus embryogenic state and being stably inherited.

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

[0034] The present invention also provides the application of the loose embryogenic callus of the 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] This invention first provides a culture medium composition for inducing loose embryogenic callus from *Phragmites australis*. Through component screening, the resulting culture medium composition yields loose embryogenic callus from *Phragmites australis* with high induction success rate and strong proliferation stability. Furthermore, this invention utilizes the aforementioned culture medium composition to provide a method for preparing loose embryogenic callus from *Phragmites australis*. This method achieves a high success rate in inducing embryogenic callus from *Phragmites australis* and can establish a stable proliferation system. This not only lays the foundation for research on somatic cell mutagenesis and genetic transformation of *Phragmites australis*, but also provides important theoretical basis and reference for rapid propagation systems of *Phragmites australis* tissue culture, in vitro mutagenesis breeding, gene editing, and suspension culture. Attached Figure Description

[0037] Figure 1 This is a picture of a cat's bean seed germination plant.

[0038] Figure 2 Image of cat bean germ callus.

[0039] Figure 3 This is a diagram of loose callus tissue from cat beans.

[0040] Figure 4 Figures showing the effects of adding VC and activated charcoal on callus induction; where 1 is the effect of adding VC on callus induction, and 2 is the effect of adding activated charcoal on callus induction.

[0041] Figure 5 Figure 1 shows the effect of different explants on callus induction; 2 shows the effect of stem segment on callus induction; 3 shows the effect of bud tip on callus induction; and 4 shows the effect of young shoot on callus induction. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

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

[0044] Cat bean seeds source: collected from a base in Nanning City, Guangxi Zhuang Autonomous Region.

[0045] Example 1

[0046] A culture medium composition for loose embryogenic callus from cat's bean, comprising the following media:

[0047] Culture medium ① (seed germination induction): MS medium with 7.5 g / L carrageenan, 30 g / L sucrose, 1 mg / L vitamin C, and 0.1% activated carbon;

[0048] Culture medium ② (inducing the formation of preliminary embryogenic callus): MS medium with 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, and VC 1.0 mg / L;

[0049] Culture medium ③ (inducing the formation of loose embryogenic callus): MS medium with 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, and VC 2.0 mg / L.

[0050] Culture medium preparation:

[0051] Using MS as the basic culture medium, different concentrations of plant growth regulators or nutrients were added according to the above culture medium combination formula: 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid (NAA), indolebutyric acid (IBA), sucrose, activated carbon, and carrageenan. The pH was adjusted to 5.8 to 6.0, dispensed into culture flasks, and sterilized in an autoclave at 121°C and 0.1 MPa for 20 minutes. After cooling and solidification, the culture was ready for use.

[0052] If plant growth regulators that cannot be sterilized at high temperatures, such as indoleacetic acid (IAA), vitamin C, or zeatin (ZT), are added, the prepared stock solution should be filtered through a 0.22 μm pore size filter membrane, sterilized at high temperature in the basal culture medium, cooled to 60°C, and then the plant growth regulator should be added. After it cools and solidifies, it can be used for later use.

[0053] Example 2

[0054] A culture medium composition for loose embryogenic callus from cat's bean, comprising the following media:

[0055] Culture medium ①: MS medium with 8.5 g / L carrageenan, 40 g / L sucrose, 0.5 mg / L vitamin C, and 0.08% activated carbon;

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

[0057] Culture medium ③: MS medium with carrageenan 8.5 g / L, sucrose 40 g / L, 6-BA 5.0 mg / L, NAA 0.2 mg / L, IBA 1 mg / L, and 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 composition for loose embryogenic callus from cat's bean, comprising the following media:

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

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

[0063] Culture medium ③: MS medium with 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, and VC 2.5 mg / L.

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

[0065] Example 4

[0066] Cat's bean embryogenic callus was prepared using the culture medium combination described in Example 1. The specific steps are as follows:

[0067] 1. Selection and Detoxification of Cat Bean Seeds

[0068] Collect and harvest mature cat pods with pods attached.

[0069] Gently peel the pods off the collected seeds to obtain grayish-white seeds; rinse with clean water, then treat with detergent on a shaker for 30 minutes, rinse with running water until no foam remains, then soak the seeds in a 100 mg / L isothiazolinone solution for 3 minutes, and rinse with running water for 10 minutes.

[0070] Seeds were placed in a clean bench and irradiated with ultraviolet light for 20 minutes, treated with 75% alcohol for 1 minute, rinsed twice with sterile water, and then sterilized for 9 minutes with 0.5 mL of Tween 20 mixed with 0.1% HgCl solution, followed by rinsing with sterile water 8 to 10 times. The seeds were then dried with filter paper and irradiated with ultraviolet light until the surface was completely dry. After removing the seed coat, the seeds were inoculated onto culture medium ① to induce germination until they developed into plants with two pairs of leaves. The resulting plants are shown below. Figure 1 As shown.

[0071] 2. Induction of feline bean germ-like callus

[0072] Under aseptic conditions, select healthy young leaves, stem segments without axillary buds (all stem segments mentioned below are without axillary buds), and leaves from culture medium ① as explants. Use a sharp scalpel to quickly create wounds on the explants. For example, cut the leaves into squares of 0.8cm × 0.8cm and lay them flat on the surface of culture medium ②. Cut the stem segments into lengths of 0.5-0.8cm and lay them flat on the surface of culture medium ②.

[0073] Dark culture for 7 days until leaves and stem segments curl, followed by light culture for 20 days until callus formation to obtain preliminary embryogenic callus tissue of cat bean (e.g. Figure 2 ).

[0074] 3. Induction of loose embryogenic callus from cat's bean

[0075] Select high-quality callus tissue from medium ②, remove brown callus and leaves that have not been induced to produce callus, and inoculate the looser, green embryogenic callus into medium ③ for continued callus induction culture. After about 10-15 days, the callus will swell. Remove brown callus and leaves that have not been induced to produce callus, and inoculate the remaining callus into medium ③ for continued callus induction culture. Perform 3 to 4 generations of callus subculture to obtain stable, loosely embryogenic callus tissue (such as...). Figure 3 ).

[0076] The optimization of the seed detoxification process revealed that isothiazolinone solution and detergent can be used for simple disinfection of the seed surface. Through exploration of disinfection experiments (UV irradiation, 75% alcohol treatment, and immersion disinfection with 0.5 mL Tween added to 0.1% HgCl solution), it was found that insufficient disinfection time or omission of subsequent disinfection steps (i.e., simple disinfection of the seed surface using only isothiazolinone) significantly increased the contamination rate. Conversely, excessively long disinfection times or the addition of other disinfection steps resulted in excessive seed toxicity and increased mortality.

[0077] Comparative Example 1

[0078] Investigating the effects of adding vitamin C and activated charcoal on the induction of callus in cat soybean embryos.

[0079] Severe oxidation occurred during the induction of callus tissue from cat's bean leaves and stem segments. Furthermore, considering the addition of activated charcoal and vitamin C during seed germination to reduce oxidation, experiments were conducted to investigate the effects of adding vitamin C and activated charcoal to culture media ① and ② on callus induction. The experimental methods were the same as those in Example 4.

[0080] The results showed that adding vitamin C and activated charcoal during seed germination could reduce oxidation, but adding activated charcoal did not improve oxidation during embryogenic callus induction and even inhibited callus formation at explant wounds. Figure 4 In the induction stage of embryogenic callus from cat's bean, adding vitamin C at a concentration of 1.0–2.0 mg / L can reduce oxidation during the induction of embryogenic callus to a certain extent without affecting the induction of explants into embryogenic callus. Figure 4 In Figure 2, the explant wound has swelled and callus tissue has begun to be induced. Therefore, activated charcoal 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] Investigating the effects of different explants on the induction of embryogenic callus in cat beans.

[0083] Young leaves, stem segments without axillary buds (all stem segments mentioned below are stem segments without axillary buds), bud tips, and leaves were selected as explants to conduct experiments on the effects of different explants on callus induction. Specifically, in step 2 of Example 4, different parts of the cat's bean plant were selected as explants, and the remaining steps were the same as in Example 4.

[0084] The results showed that even with the addition of vitamin C for antioxidant treatment, callus induction using stem segments as explants still resulted in browning, and the induced callus was water-soaked and light in color. Figure 5 (1) However, not all stem segments turn brown; the remaining unbrowned stem segments often generate callus faster, so stem segments can still be used as explants for callus culture; using shoot tips as explants to induce callus, dense yellow-green callus is induced at the wound site. Figure 5 2); with leaves ( Figure 5 4) and tender leaves ( Figure 5 In the case of explant-induced callus, the callus tissue induced at the wound and leaf vein wounds is yellowish-green and relatively loose.

[0085] Therefore, stem segments, leaves, and young leaves can be selected as explants for inducing embryogenic callus in cat's bean.

[0086] Comparative Example 3

[0087] Investigating the effects of different plant growth regulators on embryogenic callus induction in cat's bean.

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

[0089] The results showed that the above-mentioned plant growth regulators alone could not induce callus tissue, and the leaves turned yellowish-brown and died. However, the addition of 0.5 to 1.0 mg / L 6-BA caused the explant edges to curl and the wound to show signs of callus induction. Therefore, a combination of plant growth regulators was used in the experiment. As shown in Table 1, when 2,4-D, KT, GA3, and TDZ were added to the culture medium, the induction time of callus tissue was longer, and the callus was grayish-white or yellowish-brown. Some callus turned white and dried, which was not only difficult to subculture and proliferate, but also had a short lifespan and died quickly. When NAA, IBA, and ZT were added, the induction rate was increased, and the callus was yellowish-green and in better condition. Based on the combined effects of 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 are added to the culture medium, the induction rate can reach 90.23%, and the callus state is yellow-green and loose. Using this as material, stable loose embryogenic callus from cat's bean can be screened.

[0090] Table 1. Effects of different plant growth regulators on callus induction in *Pseudolaricisoides*.

[0091]

[0092] Comparative Example 4

[0093] Combinations of different plant growth regulators, such as 6-BA, NAA, 2,4-D, IBA, and ZT, were selected to conduct experiments on 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 experimental steps were the same as in Example 4.

[0094] The results, as shown in Table 2, indicate that while the addition of 2,4-D hormone to the culture medium resulted in a small amount of callus proliferation, the callus tissue became whitish and dry, lacking the loose callus morphology. The addition of NAA, IBA, and ZT hormones caused the callus to become waterlogged, which was detrimental to stable callus proliferation. However, the addition of 3.0 mg / L 6-BA, 0.05 mg / L NAA, and 0.2 mg / L IBA to the culture medium resulted in stable callus proliferation, with the callus exhibiting a yellow-green, loose morphology. The obtained loose embryogenic callus tissue can be used as a bioreactor for the production of secondary metabolites. It can be used in liquid suspension culture to improve the production efficiency of secondary metabolites. This not only lays the foundation for research on somatic cell mutagenesis and genetic transformation in cat's bean but also provides important theoretical basis and reference for rapid propagation systems of cat's bean tissue culture, in vitro mutagenesis breeding, gene editing, and suspension culture.

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

[0096]

[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing loose embryogenic callus from cat's bean, characterized in that, Includes the following steps: S1. Obtaining sterile cat bean plants: Rinse, soak, and disinfect cat bean seeds, remove the seed coat, and inoculate them into culture medium ① to induce seed germination until they grow into plants with two pairs of leaves; S2. Induction of embryogenic callus from cat's bean: Leaves and / or stem segments of the plant obtained in step S1 were used as explants, and after wound treatment, they were inoculated into culture medium ② until embryogenic callus was obtained. S3. Obtain stable cat bean loose embryogenic callus: Select the embryogenic callus obtained in S2 and inoculate it into culture medium ③ to continue callus induction culture, and carry out 3-4 generations of callus subculture to obtain stable cat bean loose embryogenic callus. Culture medium ①: MS medium with 7.5-8.5 g / L carrageenan, 30-40 g / L sucrose, 0.5-1 mg / L vitamin C, and 0.08-0.12% activated carbon; Culture medium ②: MS medium with carrageenan 7.5-8.5 g / L, sucrose 30-40 g / L, 6-BA 0.5-1.0 mg / L, NAA 0.05 mg / L, ZT 0.5 mg / L, and VC 1.0-2.0 mg / L; Culture medium ③: MS medium with 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; The stem segment mentioned in step S2 is a stem segment without axillary buds; The embryogenic callus mentioned in step S3 is a loose, green embryogenic callus; the callus induction culture mentioned in step S3 is to inoculate the embryogenic callus into culture medium ③ and culture it for 10 to 15 days until the embryogenic callus swells, and then perform 3 to 4 generations of callus subculture.

2. The preparation method according to claim 1, characterized in that, The rinsing, soaking, and disinfection method described in step S1 is as follows: rinse the seeds with clean water, then shake with detergent, rinse with running water until there is no foam, then soak the seeds in isothiazolinone solution, and rinse with running water; then irradiate the seeds with ultraviolet light, treat with 75% alcohol, rinse with sterile water 2-3 times, disinfect with 0.1% HgCl solution containing Tween 20, and rinse with sterile water 8-10 times.

3. The application of the loose embryogenic callus of cat's bean prepared by any one of the preparation methods described in claims 1 to 2 in the production of cat's bean secondary metabolites.

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