A suspension culture method for proliferating embryonic callus of cat bean and its application

Abstract

The present invention discloses a suspension culture method for the proliferation of cat bean embryonic callus and its application. First, a culture medium combination for the proliferation of cat bean embryonic callus is provided, wherein the culture medium components are screened to be suitable for the suspension culture of cat bean embryonic callus cell proliferation. Based on the culture medium combination, a method for the suspension culture of cat bean cells is provided. The provided suspension culture method for the proliferation of cat bean embryonic callus can establish a stable cat bean embryonic callus suspension cell line. The present invention can provide stable materials for the research of cat bean somatic cell embryonic differentiation, maturation, hybridization and fusion, and germplasm resource preservation, and provides an important theoretical basis and reference for the development of improved varieties, new varieties, and secondary metabolism.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and more specifically relates to a suspension culture method for proliferating embryonic callus of Lycopersicon esculentum and an application thereof. Background Art

[0002] Cat beans, also known as quinoa beans, dog claw beans, dragon claw beans, tiger beans, and mouse beans, are annual, twining, herbaceous vines that grow in subtropical regions. They are cultivated in Guangxi, Guangdong, Guizhou, Yunnan, Hainan, and Jiangxi. Their drought and barrenness tolerance and strong adaptability make them well-suited for cultivation in karst desertification areas. They are cultivated in Baise and Hechi, Guangxi, and other typical karst landforms.

[0003] Cat beans are rich in L-dopa (L-dopa), with a L-dopa content of 6-9% per bean. It is an important bioactive substance in living organisms and a key intermediate in the biochemical metabolic pathway from L-tyrosine to catechol or melanin. It is a clinically effective drug for treating Parkinson's disease-related paralysis in the elderly. Cat bean seeds are currently the primary raw material for L-dopa extraction. However, as cat bean is an annual plant, production primarily relies on harvesting seeds for seed preservation and L-dopa extraction. However, due to various issues such as limited cat bean production, it is difficult to meet current market demand.

[0004] There are not many studies on the use of artificial means to achieve short-term production gains in cat beans through cell suspension culture. Hang Ling et al. (2001) studied plant regeneration from tissue cell culture of lily beans and obtained early embryonic callus, but the proliferation was slow and could not be used for industrialization. Plant cell suspension culture has the advantages of fast growth, controllable environmental and nutritional requirements, and can also develop and utilize secondary metabolites, achieving significant proliferation, which is the only way to achieve industrialization. However, there are currently no reports on how to establish efficient and stable cat bean embryonic callus suspension culture. Therefore, whether a suspension culture method for the proliferation of cat bean embryonic callus cells can be provided is an urgent problem that those skilled in the art need to solve. Summary of the Invention

[0005] The object of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a culture medium combination for the proliferation of embryonic callus of Cat Bean.

[0006] The second object of the present invention is to provide a suspension culture method for the proliferation of embryonic callus of Cat Bean.

[0007] The third object of the present invention is to provide a cat bean embryonic callus suspension cell line obtained by cultured using the suspension culture method.

[0008] The fourth object of the present invention is to provide an application of the cat bean embryonic callus suspension cell line.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] The present invention first provides a culture medium combination for the proliferation of embryonic callus of cat bean, comprising the following culture medium:

[0011] Culture medium ①: MS culture medium plus carrageenan 7.5-8.8 g / L, sucrose 20-30 g / L, and VC 0.5-1.5 mg / L;

[0012] Culture medium ②: MS medium plus carrageenan 7.5-9.5 g / L, sucrose 15-30 g / L, 6-BA 0.5-1.5 mg / L, NAA 0.05-0.5 mg / L;

[0013] Culture medium ③: MS medium plus carrageenan 7.5-10 g / L, sucrose 10-35 g / L, 6-BA 1.0-3.0 mg / L, NAA 0.05-1.0 mg / L, VC 0.5-1.0 mg / L;

[0014] Culture medium ④: 1 / 2MS culture medium plus sucrose 10-15 g / L, 6-BA 3.0-4.5 mg / L, NAA 0.05-0.8 mg / L, and VC 0.5-2.0 mg / L.

[0015] The 1 / 2MS culture medium contains half of the macroelements in the MS culture medium formula.

[0016] The culture medium combination for the proliferation of cat bean embryonic callus provided by the present invention can establish a cat bean seed germination system according to the culture medium 1 formula and maintain a stable germination rate; according to the culture medium 2 formula, it plays an important role in inducing the culture of cat bean embryonic callus, and other formulas cannot induce callus tissue or the induced callus tissue is white and does not show an embryonic state; according to the culture medium 3 formula, it plays an important role in maintaining the embryonic callus state and increasing callus proliferation, and adding VC is to improve its antioxidant capacity; by screening the basal culture medium and the added hormones in the culture medium 4, it is obtained that the cultured cat bean callus tissue maintains the optimal state for a longer time and has better vitality, while other basal culture media cannot achieve this effect. Replacing NAA with other hormones will result in poor effect, short suspension culture maintenance time and suspension culture browning and blackening phenomenon, inability to maintain cell activity, and failure to establish the system; therefore, according to the culture medium 4 formula, a cat bean suspension culture cell line can be well established, and a cat bean embryonic callus suspension cell line with good tissue state is obtained. In summary, the culture medium combination provided by the present invention is suitable for suspension culture of embryonic callus proliferation of cat bean, and can establish efficient and stable suspension culture of embryonic callus tissue of cat bean.

[0017] Furthermore, the amount of NAA added to the culture medium ④ is 0.05-0.1 mg / L.

[0018] Preferably, the culture medium combination for the proliferation of the embryonic callus of Cat Bean comprises the following culture medium:

[0019] Culture medium ①: MS medium plus carrageenan 7.5 g / L, sucrose 30 g / L, VC 0.5 mg / L;

[0020] Culture medium ②: MS medium plus carrageenan 7.5 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.05 mg / L;

[0021] Culture medium ③: MS medium plus carrageenan 7.5 g / L, sucrose 30 g / L, 6-BA 3.0 mg / L, NAA 0.05 mg / L, VC 0.5 mg / L;

[0022] Culture medium ④: 1 / 2MS culture medium plus sucrose 15 g / L, 6-BA 3.0 mg / L, NAA 0.05 mg / L, and VC 0.5 mg / L.

[0023] The present invention also provides a suspension culture method for proliferating embryonic callus of cat bean, comprising the following steps:

[0024] S1. Cat bean seed disinfection: The seeds were rinsed, soaked, disinfected, and the seed coat was removed and inoculated in the above-mentioned medium ① to induce seed germination to grow into plants with two pairs of leaves;

[0025] S2. Induction of embryonic callus from cat bean: The leaves of the plants obtained in S1 were wounded and inoculated in the above-mentioned culture medium ② to obtain embryonic callus;

[0026] S3. Screening of embryonic callus suspension culture initial material of cat bean: The embryonic callus obtained from S2 was inoculated into the above-mentioned medium ③ for proliferation culture, and then 3 to 4 generations of callus subculture screening to obtain embryonic callus suspension culture initial material;

[0027] S4 cat bean embryonic callus suspension primary culture: The embryonic callus suspension culture initial material obtained in S3 was transferred to the above-mentioned medium ④ for suspension culture, and embryonic callus suspension was obtained after screening for 3 to 4 generations;

[0028] S5. Cat bean embryonic callus cell suspension proliferation culture: The embryonic callus suspension obtained in S4 is precipitated, and the deposited embryonic tissue is added to the above-mentioned culture medium ④ for suspension culture. The cultured deposited embryonic tissue is inoculated into the above-mentioned culture medium ④ for subculture proliferation and suspension shaking culture to obtain the cat bean embryonic callus suspension cell line.

[0029] Among them, the present invention is in the screening of cat bean embryonic callus suspension culture initial material, and different callus subcultures are carried out on embryonic callus of different generations and are screened. Studies have shown that the embryonic callus tissue obtained by too little or too much callus subculture is used as the primary suspension culture material, and the callus tissue state in subsequent culture is poor and tends to die, or the resulting suspension tissue produces less levodopa content and poor tissue activity. Only 3 to 4 generations of embryonic callus have good tissue state in subsequent culture, and the levodopa content it produces is higher and the activity is better. Therefore, the suspension culture method for cat bean embryonic callus proliferation provided by the present invention can establish a stable cat bean embryonic callus suspension cell line, and the cell line can provide a large raw material for molecular research such as cell fusion and transgenic research and secondary metabolism research. The research on secondary metabolites can provide more theoretical basis and reference for levodopa research.

[0030] Furthermore, the seeds described in step S1 are collected from mature cat pods with pods, and the pods are gently peeled off to obtain grayish-white seeds.

[0031] Furthermore, the rinsing, soaking and disinfection in step S1 are: rinsing with running water, then shaking with detergent, rinsing with running water until there is no foam, treating with 75% alcohol, rinsing with sterile water, disinfecting with 0.1% HgCl solution for 8-10 minutes, and rinsing with sterile water.

[0032] Among them, if the disinfection time in step S1 is insufficient or the disinfection step is omitted, the infection rate will be 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.

[0033] Preferably, the rinsing, soaking and disinfection in step S1 are: washing with detergent shaker for 30 minutes, rinsing with running water until there is no foam, treating with 75% alcohol for 1 minute, rinsing with sterile water twice, disinfecting with 0.1% HgCl solution for 9 minutes, and rinsing with sterile water 8 to 10 times.

[0034] Furthermore, the removal of the seed coat in step S1 needs to be performed in a sterile environment.

[0035] Furthermore, the method of inducing the seeds to germinate and grow into plants with two pairs of leaves in step S1 is to culture the seeds in the dark until they germinate and then switch to light culture.

[0036] Furthermore, the wounding treatment in step S2 involves selecting healthy leaves and stems from the plants obtained in step S1, quickly injuring the leaves and stem segments using a scalpel, cutting the leaves into 0.8 cm x 0.8 cm squares, and laying them flat on the surface of culture medium ②. The stem segments are cut into 0.8 cm lengths and laid flat on the surface of culture medium ②. Rapid explant cutting reduces water loss and ensures explant viability.

[0037] Furthermore, the culture in step S2 is dark-treated culture until the leaves curl, and then light-treated culture until callus is formed to obtain embryonic callus.

[0038] Furthermore, the embryogenic callus in step S3 is loose, fast-growing, and vigorous. The selected high-quality embryogenic callus plays an important role in the subsequent suspension culture, and callus subculture can ensure that the embryonic state of the callus is not lost.

[0039] Furthermore, the proliferation culture condition in step S3 is culture under a light environment at a temperature of 23 to 27°C.

[0040] Furthermore, the suspension culture conditions in steps S4 and S5 are shaking culture in a light environment, a rotation speed of 120 r / min, and a temperature of 23-27°C.

[0041] Preferably, step S4 comprises adding the embryogenic callus selected in step S3 to culture medium ④ (solid-to-liquid ratio (g / mL) of 1:100), culturing with shaking at a speed of 120 r / min, a temperature of 25°C, and light for 15 days; removing a small amount of supernatant and small pieces of brown granular tissue, adding fresh culture medium, and culturing for 5 days, and further screening for three generations to obtain an embryogenic callus suspension. After three generations of screening and culture, the callus mass can be fully broken up, and inferior callus tissue can be screened out to leave callus tissue with strong vitality and good adaptability, thereby providing original cells for obtaining a better suspension cell line.

[0042] Preferably, step S5 is to remove the brownish-white precipitate and a small amount of supernatant from the mesoembryonic callus suspension obtained in S4, take the remaining viscous tissue, add fresh culture medium ④ (the volume ratio of viscous tissue to culture medium ④ is 1:4) for subculture proliferation suspension culture, and culture with shaking at a speed of 120 r / min, a temperature of 25°C and light environment for 7 to 10 days, then add fresh culture medium ④ and continue subculture proliferation suspension shaking culture for 5 days to obtain the cat bean embryonic callus suspension cell line.

[0043] The present invention also provides a cat bean embryonic callus suspension cell line cultured by any of the above suspension culture methods.

[0044] The present invention also provides application of the cat bean embryonic callus suspension cell line in the development of new cat bean varieties.

[0045] Furthermore, the cat bean embryonic callus suspension cell line can be used in the breeding of cat bean varieties.

[0046] The present invention also provides use of the cat bean embryonic callus suspension cell line in preparing cat bean secondary metabolites.

[0047] Furthermore, the secondary metabolite is levodopa.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The present invention provides a method for suspension culture of cat bean cells. First, a culture medium combination for the proliferation of cat bean embryonic callus is provided, and the culture medium components are screened to be suitable for suspension culture of cat bean embryonic callus cells. Based on the culture medium combination, a method for suspension culture of cat bean cells is provided. The provided suspension culture method for the proliferation of cat bean embryonic callus can establish a stable cat bean embryonic callus suspension cell line. The present invention can provide stable materials for research on somatic cell embryonic differentiation, maturation, hybrid fusion, and germplasm resource conservation of cat bean, and provide an important theoretical basis and reference for the development of improved varieties, new varieties, and secondary metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the formula diagram of MS culture medium.

[0051] Figure 2 This is a picture of the cat bean seed germination plant provided by the present invention.

[0052] Figure 3 This is a diagram of the embryonic callus of Cat Bean provided by the present invention.

[0053] Figure 4 This is a diagram of the embryonic callus proliferation of cat bean provided by the present invention.

[0054] Figure 5 This is a diagram of the cell suspension of cat beans after primary culture provided by the present invention.

[0055] Figure 6 This is a diagram of a balanced embryogenic callus suspension cell line of Cat Bean provided by the present invention.

[0056] Figure 7 Figures 1 and 2 show the results of suspension culture of embryonic callus tissues of different generations as primary suspension culture materials; 1 shows the results of suspension culture of embryonic callus tissues of the first and second generations, 2 shows the results of suspension culture of embryonic callus tissues of the third and fourth generations, and 3 shows the results of suspension culture of embryonic callus tissues of the fifth generation and subsequent generations.

[0057] Figure 8 This is the effect of culture medium containing 2,4-D hormone on embryonic callus suspension culture. DETAILED DESCRIPTION

[0058] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples 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 the art.

[0059] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0060] Source of cat bean seeds: Planted in Nanning City, Guangxi Zhuang Autonomous Region.

[0061] Example 1

[0062] This embodiment provides a culture medium combination for the proliferation of embryonic callus of cat bean, comprising the following culture medium:

[0063] Culture medium ①: MS medium plus carrageenan 7.5 g / L, sucrose 30 g / L, VC 0.5 mg / L;

[0064] Culture medium ②: MS medium plus carrageenan 7.5 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.05 mg / L;

[0065] Culture medium ③: MS medium plus carrageenan 7.5 g / L, sucrose 30 g / L, 6-BA 3.0 mg / L, NAA 0.05 mg / L, VC 0.5 mg / L;

[0066] Culture medium ④: 1 / 2MS culture medium plus sucrose 15 g / L, 6-BA 3.0 mg / L, NAA 0.05 mg / L, and VC 0.5 mg / L.

[0067] The MS culture medium formula is as follows Figure 1 As shown, 1 / 2MS medium is a medium in which the macroelements in the MS medium formula are reduced by half.

[0068] Example 2

[0069] This embodiment provides a culture medium combination for the proliferation of embryonic callus of cat bean, comprising the following culture medium:

[0070] Culture medium ①: MS medium plus carrageenan 8.8 g / L, sucrose 30 g / L, and VC 1.5 mg / L;

[0071] Culture medium ②: MS medium plus carrageenan 9.5 g / L, sucrose 15 g / L, 6-BA 1.5 mg / L, NAA 0.5 mg / L;

[0072] Culture medium ③: MS medium plus carrageenan 10 g / L, sucrose 10 g / L, 6-BA 1.0 mg / L, NAA 1.0 mg / L, VC 1.0 mg / L;

[0073] Culture medium ④: 1 / 2MS culture medium plus sucrose 10 g / L, 6-BA 4.5 mg / L, NAA 0.8 mg / L, and VC 2.0 mg / L.

[0074] The formula of MS medium and 1 / 2MS medium is the same as that in Example 1.

[0075] Example 3

[0076] This embodiment provides a culture medium combination for the proliferation of embryonic callus of cat bean, comprising the following culture medium:

[0077] Culture medium ①: MS medium plus carrageenan 7.5 g / L, sucrose 30 g / L, VC 0.5 mg / L;

[0078] Culture medium ②: MS medium plus carrageenan 7.5 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.05 mg / L;

[0079] Culture medium ③: MS medium plus carrageenan 7.5 g / L, sucrose 35 g / L, 6-BA 3.0 mg / L, NAA 0.05 mg / L, VC 0.5 mg / L;

[0080] Culture medium ④: 1 / 2MS culture medium plus sucrose 15 g / L, 6-BA 3.0 mg / L, NAA 0.05 mg / L, and VC 0.5 mg / L.

[0081] The formula of MS medium and 1 / 2MS medium is the same as that in Example 1.

[0082] Example 4

[0083] This embodiment provides a suspension culture method for the proliferation of embryonic callus of cat bean, which uses the culture medium combination for the proliferation of embryonic callus of cat bean described in Example 1 to perform the suspension culture of embryonic callus of cat bean. The method specifically comprises the following steps:

[0084] (1) Disinfection of cat bean seeds

[0085] Mature cat pea pods with pods were collected and gently peeled to obtain gray-white seeds; after rinsing with clean water, the seeds were shaken in detergent for 30 minutes, rinsed with running water until there was no foam, the 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, disinfected with 0.1% HgCl solution for 9 minutes, and rinsed with sterile water 8 to 10 times; after peeling the seed coat, the seeds were inoculated on culture medium ① to induce seed germination until they grew into plants with two pairs of leaves.

[0086] (2) Induction of embryonic callus of cat bean

[0087] Select well-growing leaves and stems from medium ①. Use a sharp scalpel to quickly incise wounds on the leaves and stem segments. Cut the leaves into 0.8 cm x 0.8 cm squares and lay them flat on the surface of medium ②. Cut the stem segments into 0.8 cm lengths and lay them flat on the surface of medium ②. Culture in the dark for 7 days until the leaves curl. Then, culture in the light until callus forms and obtain embryonic callus.

[0088] (3) Screening of initial materials for embryonic callus suspension culture of cat bean

[0089] Select the embryonic calli in culture medium ② that are loose, fast-growing and vigorous as the initial material for callus proliferation, inoculate them into culture medium ③ for proliferation culture, and obtain the initial material for embryonic callus suspension primary culture after 3 to 4 generations of callus subculture screening.

[0090] (4) Primary suspension culture of embryonic callus of cat bean

[0091] 1.0 g of selected high-quality callus was added to 100 mL of culture medium ④ and cultured with shaking at a speed of 120 r / min, a temperature of 25°C and light environment for 15 days; a small amount of supernatant and small pieces of brown granular tissue were removed, and fresh culture medium was added to culture for 5 days. After further screening for 3 generations, embryonic callus suspension was obtained.

[0092] (5) Suspension proliferation culture of embryonic callus cells of cat bean

[0093] After removing the brown-white precipitate and a small amount of supernatant from the embryonic callus suspension in step (4), 20 mL of the remaining viscous tissue was taken and added to 80 mL of fresh culture medium ④ for subculture proliferation suspension culture. The culture was shaken at a speed of 120 r / min, a temperature of 25°C and light environment for 7 to 10 days, and then 50 mL of fresh culture medium ④ was added to continue subculture proliferation suspension shaking culture for 5 days to obtain a cat bean embryonic callus suspension cell line.

[0094] In the above steps, step (1) and step (2) of dedifferentiation are both initially dark cultured for about 7 days. When germination or leaves begin to curl, they are switched to light culture. The rest are all light culture, and the culture temperature is 25℃±2℃.

[0095] The results are as follows Figures 2 to 6 As shown, in step (1), the seeds are induced to germinate by the culture medium ① until they grow into plants with two pairs of leaves as shown in FIG. Figure 2 As shown, the embryonic callus obtained in step (2) is as follows Figure 3 As shown, the initial material of embryonic callus suspension primary culture obtained by screening in step (3) is as follows Figure 4 As shown, the embryonic callus suspension after primary culture in step (4) is as follows Figure 5 As shown, the embryonic callus suspension cell line of cat bean obtained in step (5) is as follows Figure 6 Furthermore, the culture medium combinations provided in Examples 2 and 3 can also be used to obtain embryogenic callus suspension cell lines of Psoralea corylifolia with good vitality.

[0096] Comparative Example 1

[0097] To explore the effects of the disinfection steps and different disinfection times in step (1) of cat bean seed disinfection in Example 4 on the bacterial infection rate and seed activity, the specific experimental method is as follows:

[0098] (1) Step (1) Seed disinfection: only thiazolinone solution, detergent and 75% alcohol are used for preliminary disinfection; the remaining steps are the same as those in Example 4.

[0099] (2) During the seed disinfection process of step (1), the disinfection time of 0.1% HgCl solution was set to 0 min, 3 min, 5 min, 8 min, 9 min, 10 min, and 12 min for the experiment; the remaining steps were the same as in Example 4.

[0100] The results show that:

[0101] (1) Isothiazolinone solution can quickly inhibit the growth of microorganisms and has a strong inhibitory and killing effect. It has the characteristics of no residue, safe operation, and low cost of use. Detergent and 75% alcohol can simply disinfect the surface of seeds. However, if only thiazolinone solution, detergent and 75% alcohol are used for preliminary disinfection, the seeds in culture medium ① will gradually become infected with mold three days after inoculation, and the mold infection rate can reach 100%;

[0102] (2) After 5 days of inoculation, the seeds began to be infected with mold and bacteria when the treatment time was 3 minutes and 5 minutes, with a mold rate of 50% and a bacteria rate of 65%. The infection rate was significantly reduced when the treatment time was 12 minutes, but the seed germination rate dropped to 60% 7 to 10 days after inoculation. The infection was significantly improved when the treatment time was 8 minutes and 10 minutes, with a mold rate of 5% and a bacteria rate of 10%. Therefore, the disinfection time of 0.1% HgCl solution can be 8 to 10 minutes. Among them, the seed germination rate of the treatment time of 10 minutes was lower than that of the treatment time of 8 minutes. Therefore, the treatment time was changed to 9 minutes and the test was conducted again, which showed that the seed germination rate was improved.

[0103] In summary, through the exploration of the steps and disinfection time of the disinfection experiment, if the subsequent disinfection steps are omitted or the disinfection time is insufficient, the infection rate will reach more than 84%. 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.

[0104] Comparative Example 2

[0105] To investigate the effect of selecting embryogenic calli of different generations in step (3) of Example 4 and transferring them to medium ④ for suspension culture, the specific experimental method is as follows:

[0106] Cat bean embryonic callus suspension primary culture: select 1.0 g of first-generation, second-generation, third-generation, fourth-generation, and fifth-generation embryonic callus tissues, transfer them into 250 mL triangular flasks, add 100 mL of culture medium ④, and culture with shaking for 15 days at a speed of 120 r / min and a temperature of 25 ° C under light conditions to obtain embryonic callus tissue that is preferably suitable for primary suspension culture; the remaining steps are the same as in Example 4.

[0107] The results showed that when the first and second generation embryonic calli were used as primary suspension culture materials, the calli began to turn brown and yellow in the suspension culture medium after 4 to 5 days, and the calli tended to die. In addition, the embryonic state of the first generation embryonic calli was not as good as that of the other subcultures ( Figure 7-1); When embryonic callus of the fifth generation or later generations was used as the primary suspension culture material, the color of the suspension tissue turned dark green in the middle stage of culture and did not turn bright green, and its activity was also poor ( Figure 7-3 ), the content of levodopa produced was low, the content of levodopa in the product was 0.19%; while the color of the tissue after the proliferation of the third and fourth generation embryonic callus was bright green, the content of levodopa produced was high, the content of levodopa was 2.2%, and the activity was better ( Figure 7-2 ).

[0108] Comparative Example 3

[0109] To investigate the effect of the setting of the basal culture medium in culture medium ④ in step (4) of Example 4 on the primary suspension culture of embryonic callus of cat bean, based on the hormone level of culture medium ③, 6 liquid basal culture media were added for comparative experiments: MS, 1 / 2MS, B5, N6, WPN, and White, and the rest were the same as in Example 4.

[0110] Results showed that calli cultured in liquid media based on MS and 1 / 2MS exhibited a greening and growth pattern during incubation. Calli cultured in media based on B5, N6, WPN, and White exhibited similar initial patterns, but none of these patterns occurred after acclimation to the liquid environment. Calli maintained a brownish-yellow coloration, gradually turning brown until callus death. Comparing the MS and 1 / 2MS basal media, the 1 / 2MS group exhibited better calli quality than the MS group, maintaining this optimal state for a shorter period. Suspension cultures in the MS group entered the late stage more quickly, but cell viability and quality declined over time. Therefore, the nutrient composition of 1 / 2MS is more suitable for suspension culture of cat bean calli, and 1 / 2MS should be selected as the basal medium for suspension culture.

[0111] Comparative Example 4

[0112] To explore the effects of different hormone types and concentrations in the culture medium ④ in step (4) of Example 4 on the primary suspension culture of cat bean embryonic callus, a comparative experiment was conducted using 1 / 2MS as the basal medium, with the addition of 5 different hormones and liquid culture media of different concentrations: NAA (0.05, 0.1) mg / L, IBA (0.05, 0.1) mg / L, KT (0.5, 1.0) mg / L, 2,4-D (0.3, 0.8) mg / L, GA3 (1.0, 3.0) mg / L, and additional 6-BA 3.0 mg / L + sucrose 15 g / L + VC 0.5 mg / L, and the rest was the same as Example 4.

[0113] The results showed that when different concentrations of plant hormones were added to the 1 / 2MS basal liquid medium, the proliferation of cat bean embryonic callus was different. The experimental results showed that when KT and GA3 were added, there was no obvious proliferation in the late culture period and the callus color did not change significantly to green, and it quickly turned brown. When 2,4-D was added, there was proliferation, but the cell clusters became severely browned during the linear growth period. It is possible that 2,4-D is beneficial to cell proliferation but not to the maintenance of embryonic state (see Figure 8 ); When NAA and IBA were added, both cells were in a proliferation state, but when IBA was added, it was found that the proliferation entered the decline phase faster after reaching the peak, while after NAA was added, it entered the decline phase faster after reaching the linear growth phase, and could return to the delayed phase and logarithmic growth phase after further subculture; compared with 0.05mg / L NAA and 0.1mg / L NAA, the suspension state was not much different, but after reaching the linear growth phase, it entered the slowing phase faster with the increase of concentration and the growth coefficient was lower.

[0114] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0115] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A suspension culture method for the proliferation of embryonic callus of Cat Bean, characterized in that: The following steps are involved: S1. Sterilization of cat bean seeds: The seeds were rinsed, soaked, sterilized, and the seed coats were removed before inoculation into culture medium ① to induce seed germination and growth into plants with two pairs of leaves; S2. Induction of embryonic callus from cat bean: Wound the leaves of the plants obtained in S1 and inoculate them on culture medium ② in the dark until the leaves curl, then culture them in the light until callus forms to obtain embryonic callus; S3. Screening of initial materials for embryonic callus suspension culture of cat beans: Select embryonic callus with loose, fast-growing and vigorous growth in S2 and inoculate them into medium ③ for proliferation culture, and then perform 3-4 generations of callus subculture screening to obtain initial materials for embryonic callus suspension culture; S4. Primary suspension culture of embryonic callus of cat bean: The initial embryonic callus suspension culture material obtained in S3 was transferred to medium ④ for suspension culture. After 3 to 4 generations of screening, embryonic callus suspension was obtained; S5. Cat bean embryonic callus cell suspension proliferation culture: The embryonic callus suspension obtained in S4 was precipitated, and the deposited embryonic tissue was added to the culture medium ④ for suspension culture. The cultured embryonic tissue was then inoculated into the culture medium ④ of claim 1 for subculture proliferation and suspension shaking culture to obtain a cat bean embryonic callus suspension cell line; Culture medium ①: MS culture medium plus carrageenan 7.5-8.8 g / L, sucrose 20-30 g / L, and VC 0.5-1.5 mg / L; Culture medium ②: MS medium plus carrageenan 7.5-9.5 g / L, sucrose 15-30 g / L, 6-BA 0.5-1.5 mg / L, NAA 0.05-0.5 mg / L; Culture medium ③: MS medium plus carrageenan 7.5-10 g / L, sucrose 10-35 g / L, 6-BA 1.0-3.0 mg / L, NAA 0.05-1.0 mg / L, VC 0.5-1.0 mg / L; Culture medium ④: 1 / 2MS culture medium plus sucrose 10-15 g / L, 6-BA 3.0-4.5 mg / L, NAA 0.05-0.8 mg / L, and VC 0.5-2.0 mg / L.

2. The suspension culture method according to claim 1, characterized in that: The rinsing, soaking and disinfection in step S1 are: rinsing with running water, shaking with detergent, rinsing with running water until there is no foam, treating with 75% alcohol, rinsing with sterile water, disinfecting with 0.1% HgCl solution for 8-10 minutes, and rinsing with sterile water.

3. The suspension culture method according to claim 1, characterized in that: The proliferation culture in step S3 is carried out under a light environment at a temperature of 23 to 27°C.

4. The suspension culture method according to claim 1, characterized in that: The suspension culture conditions in steps S4 and S5 are shaking culture under a light environment, a rotation speed of 120 r / min, and a temperature of 23-27°C.

5. Application of the suspension culture method according to any one of claims 1 to 4 in the development of cat bean varieties.

6. Use of the suspension culture method according to any one of claims 1 to 4 in preparing secondary metabolites of cat beans.

Images