A method for improving the induction rate of sea fennel embryogenic callus in suspending culture

Abstract

The application discloses a method for improving the induction rate of suspensibility culture of sea fennel embryogenic callus and belongs to the field of industrial biotechnology. The method comprises the following steps: after sea fennel is sterilized, the sea fennel is cut into segments and is inoculated into an induction culture medium with a pH value of 5-6, and the segments are cultured under light at a temperature of 20-30 DEG C for 15-30 days, so that sea fennel embryogenic callus is obtained, wherein the induction culture medium is composed of MS basic salt culture medium, 25-35 g / L of sucrose, 8-9 g / L of agar, 0.3-0.5 mg / L of 2,4-D, 0.3-0.6 mg / L of NAA and 1-2 mg / L of KT. The method can realize the induction rate of 75%-94.2% of the suspensibility culture of embryogenic callus, can shorten the induction time, and provides good callus for large-scale culture of sea fennel cells.

Description

Technical Field

[0001] This invention relates to a method for improving the induction rate of embryogenic callus from sea fennel that can be cultured in suspension, and belongs to the field of industrial biotechnology. Background Technology

[0002] sea ​​fennel ( Crithmum maritimum ) originates from the Apiaceae family ( Apiaceae Sea fennel is an edible, salt-tolerant plant native to Europe. It grows in the saline coastal regions of Europe, found on rocks and dunes along the Mediterranean and Atlantic coasts. Salt-tolerant plants and microorganisms are frequently used as natural reservoirs of active molecules for pharmaceutical development. Sea fennel thrives even in nutrient-poor conditions and on eroded reefs thanks to its abundant self-protective bioactive substances, including rich amounts of amino acids, lipids, proteins, vitamins, and bioactive peptides. These active substances counteract the high free radical content caused by high salinity and possess antibacterial, anti-inflammatory, and immune-boosting properties, ensuring its survival in nutrient-poor and extremely saline environments.

[0003] Existing methods for inducing embryogenic callus in sea fennel primarily involve using seedlings, stem segments, leaves, and roots after seed germination as explants. However, seed germination takes at least three months, significantly extending the time required to establish a liquid suspension culture system. Furthermore, the induction rate of existing embryogenic callus is relatively low, and the induced cells exhibit poor suspension culture performance.

[0004] In view of this, this application provides a method for improving the induction rate of embryogenic callus from sea fennel that can be cultured in suspension, in order to overcome the above-mentioned deficiencies. Summary of the Invention

[0005] This invention provides a method for improving the induction rate of embryogenic callus in suspension-cultured sea fennel. Through explant screening, this invention ultimately determines stem segments as callus inducers, directly inducing embryogenic callus in sea fennel without germination. The induction rate of embryogenic callus in suspension-cultured sea fennel reaches 75%-94.2%, with a maximum of 90%. Compared to the highest induction rate of 76% in existing technologies, this represents an improvement of up to 18.2%, shortening the induction time and providing well-prepared callus for large-scale culture of sea fennel cells.

[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for improving the induction rate of embryogenic callus from sea fennel that can be cultured in suspension, comprising the following steps: After sterilizing sea fennel, cut it into sections and inoculate it into an induction medium with a pH of 5-6. Culture it under light at a temperature of 20-30℃ for 15-30 days to obtain sea fennel embryogenic callus. The induction medium is a nutrient solution composed of MS basal salt medium, sucrose 25-35 g / L, agar 8-9 g / L, 2,4-D 0.3-0.5 mg / L, NAA 0.3-0.6 mg / L and KT 1-2 mg / L.

[0007] The principle of this invention is that any explant part of sea fennel can be induced to generate callus, but the efficiency of embryogenic callus that can be cultured in suspension varies significantly. This invention uses stem segments of sea fennel as explants to directly produce callus. By optimizing the induction and culture conditions, the resulting embryogenic callus has a high cell formation rate and strong cell suspension cultureability.

[0008] The induction medium of this invention consists of MS basal salt medium, sucrose, agar, 2,4-D, NAA, and KT. MS basal salt medium provides the basic nutrients for plant cell growth, providing essential inorganic salts and trace elements; sucrose provides the carbon source for plant cell growth; agar acts as a solidifying agent to facilitate surface growth and reproduction of callus cells; 2,4-D acts as an auxin; NAA acts as a growth hormone and promotes the formation of loose embryogenic callus, making it more suitable for suspension culture; KT acts as a kinetin, delaying senescence of detached leaves, inducing bud differentiation, and increasing stomatal aperture. The induction medium of this invention can promote the generation of sea fennel embryogenic callus cells suitable for suspension culture, rapidly and efficiently obtaining loose embryogenic callus cells for suspension culture.

[0009] Based on the above technical solution, the present invention can be further improved as follows.

[0010] Furthermore, the induction medium consists of MS basal salt medium, 30 g / L sucrose, 9 g / L agar, 0.5 mg / L 2,4-D, 0.3 mg / L NAA, and 1 mg / L KT.

[0011] The further beneficial effect of adopting the above-mentioned method is that the induction culture medium with the above-mentioned composition is more conducive to inducing sea fennel explants to form embryogenic callus cells.

[0012] Furthermore, the MS basic salt culture medium aqueous solution comprises: KNO3 1900 mg / L, NH4NO3 1650 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, CaCl2·2H2O 440 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, Na2-EDTA 37.3 mg / L, FeSO4·7H2O It consists of 27.8 mg / L of glycine, 2.0 mg / L of pyridoxine hydrochloride, 0.6 mg / L of pyridoxine hydrochloride, and 0.1 mg / L of thioammonium hydrochloride.

[0013] Furthermore, the pH value of the induction medium is 5.85.

[0014] Furthermore, the temperature is 25°C, and the induction culture time is 18 days.

[0015] Definitions: 2,4-D, or dichlorophenoxyacetic acid, is a representative synthetic plant growth regulator. NAA, or naphthaleneacetic acid, is an auxin analogue that functions similarly to plant auxins.

[0016] KT, or kinetin, is a non-natural cytokinin.

[0017] The beneficial effects of this invention are as follows: This invention, through the screening of explants, ultimately determined that stem segments are used as callus inducing agents, directly inducing embryogenic callus from sea fennel without germination, achieving an induction rate of 75%-94.2% for suspension-cultured embryogenic callus, with a maximum of 90%. Compared with the highest induction rate of 76% in the prior art, this represents an improvement of up to 18.2%, which can shorten the induction time and provide callus in good condition for large-scale culture of sea fennel cells.

[0018] This invention develops an induction medium that promotes the generation of embryogenic callus cells from sea fennel, which can rapidly and efficiently obtain loose embryogenic callus cells for suspension culture.

[0019] The method of this invention is simple, has broad market prospects, and is suitable for industrial production. Attached Figure Description

[0020] Figure 1 For comparison experiment 1, non-embryonic callus cells were used.

[0021] Figure 2 The vitrification callus cells described in Comparative Experiment 2 are used.

[0022] Figure 3 For comparative experiments, embryonic callus cells were used. Detailed Implementation

[0023] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0024] Implementation Example 1: The method for improving the induction rate of embryogenic callus from sea fennel in this embodiment includes the following steps: Ten sea fennel plants were sterilized and cut into segments to obtain 300 stem explants. These explants were inoculated into an induction medium with a pH of 6.0 and cultured at 30°C under light for 30 days to obtain 225 sea fennel embryogenic callus cell clusters. The induction medium consisted of MS basal salt medium, sucrose 35 g / L, agar 9 g / L, 2,4-D 0.5 mg / L, NAA 0.6 mg / L, and KT 2 mg / L. The MS basal salt culture medium consists of the following components: KNO3 1900 mg / L, NH4NO3 1650 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, CaCl2·2H2O 440 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, Na2-EDTA 37.3 mg / L, and FeSO4·7H2O. It consists of 27.8 mg / L of glycine, 2.0 mg / L of pyridoxine hydrochloride, 0.6 mg / L of pyridoxine hydrochloride, and 0.1 mg / L of thioammonium hydrochloride.

[0025] The induction rates of callus cells and embryogenic callus cells were calculated separately. The callus cell induction rate refers to the rate at which explants form callus cells on the induction medium; the embryogenic callus induction rate is the ratio of embryogenic callus cells to the total number of callus cells generated.

[0026] The calculation formula is as follows: Callus induction rate = (Number of callus formation / Number of explants) * 100% Embryogenic callus induction rate = (Number of embryogenic callus / Number of callus generated) * 100% Calculations show that the callus induction rate in this embodiment is 98%, and the embryonic callus induction rate is 76.5%.

[0027] Implementation Example 2: The method for improving the induction rate of embryogenic callus from sea fennel in this embodiment includes the following steps: Ten sea fennel plants were sterilized and cut into segments to obtain 300 stem explants. These explants were inoculated into an induction medium with a pH of 5.85 and cultured at 25°C under light for 30 days to obtain 281 sea fennel embryogenic callus cell clusters. The induction medium consisted of MS basal salt medium, 30 g / L sucrose, 8.5 g / L agar, 0.5 mg / L 2,4-D, 0.3 mg / L NAA, and 1 mg / L KT. The MS basal salt medium consists of: KNO3 1900 mg / L, NH4NO3 1650 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, CaCl2·2H2O 440 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, Na2-EDTA 37.3 mg / L, and FeSO4·7H2O. It consists of 27.8 mg / L of glycine, 2.0 mg / L of pyridoxine hydrochloride, 0.6 mg / L of pyridoxine hydrochloride, and 0.1 mg / L of thioammonium hydrochloride.

[0028] Calculations show that the callus induction rate in this embodiment is 100%, and the average induction rate of embryonic callus is 93.7%.

[0029] Implementation Example 3: The method for improving the induction rate of embryogenic callus from sea fennel in this embodiment includes the following steps: Ten sea fennel plants were sterilized and cut into segments to obtain 300 stem explants. These explants were inoculated into an induction medium with a pH of 5.0 and cultured at 20°C under light for 30 days to obtain 244 sea fennel embryogenic callus cell clusters. The induction medium consisted of MS basal salt medium, 25 g / L sucrose, 8 g / L agar, 0.3 mg / L 2,4-D, 0.3 mg / L NAA, and 1 mg / L KT. The MS basal salt medium consists of: KNO3 1900 mg / L, NH4NO3 1650 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, CaCl2·2H2O 440 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, Na2-EDTA 37.3 mg / L, and FeSO4·7H2O. It consists of 27.8 mg / L of glycine, 2.0 mg / L of pyridoxine hydrochloride, 0.6 mg / L of pyridoxine hydrochloride, and 0.1 mg / L of thioammonium hydrochloride.

[0030] Calculations show that the callus induction rate in this embodiment is 100%, and the embryonic callus induction rate is 81.3%.

[0031] Comparative test In order to obtain better sea fennel embryogenic callus cells for suspension culture, the applicant studied the effects of different explant embryos, leaves and stem segments, as well as different hormone concentrations in the culture medium on callus formation.

[0032] Comparative Experiment 1: Induction of Embryogenic Callus from Sea Fennel Seed Explants Table 1 shows that the embryo induction rate is relatively high when the culture medium contains 2,4-D, NAA, and KT simultaneously. The callus induced under different ratios of 2,4-D, NAA, and KT are basically the same, with most being white, spongy, filamentous, non-embryonic callus (e.g., ...). Figure 1As shown in the image, only a small amount of brightly colored, loosely granular embryogenic callus was induced. While the non-embryonic callus in this state also exhibited a loose structure, microscopic examination revealed elongated cells without a distinct shape, and the callus viability was weak. After subculture, the color of the callus gradually deepened and browning occurred. The experimental results show that only a very small number of embryos were induced to produce embryogenic callus. This callus was generally yellowish-white with a certain metallic luster, and its surface was granular with uniform cells. The induced embryogenic cells grew slowly during suspension culture, and the solution showed large particle aggregation and browning.

[0033] Table 1. Effects of different induction conditions on embryogenic callus induction from seed embryos.

[0034] 1 0 0.3 0 0 0 White flocculent 2 0 0.6 1 0 0 root 3 0 1 2 0 0 root 4 0.3 0.3 0 5.5 65 White flocculent 5 0.3 0.6 1 17.6 100 White flocculent 6 0.3 1 2 18.4 100 White flocculent 7 0.5 0.3 1 26.3 100 White flocculent 8 0.5 0.6 2 10.8 100 White flocculent 9 0.5 1 0 0 100 White flocculent 10 1 0.3 2 8.3 72.2 White flocculent 11 1 0.6 0 0 100 White flocculent 12 1 1 1 0 100 White flocculent

[0035] Comparative Experiment 2: Induction of Embryogenic Callus Cells from Sea Fennel Leaf Explants

[0036] Callus induction using sea fennel leaves took longer than that using stem segments. A small amount of callus appeared on the leaves after 12-14 days, and this callus only appeared and grew at the leaf margins, not inducing callus formation throughout the entire explant. Most of the callus induced from leaves was non-embryonic, white, cottony, with only a small amount induced as embryogenic callus. A water-soaked, transparent, and curled callus, known as vitrified callus, also appeared among the leaf-induced callus. Figure 2 As shown in the figure, the callus tissue exhibits poor growth after subculture.

[0037] Table 2 shows that when the culture medium contains only NAA, callus formation from leaves is almost impossible to induce. However, when the medium contains a higher level of NAA (1.0 mg / L), a small number of leaves can produce callus. The same conclusion holds true when the culture medium contains only 2,4-D. When the culture medium contains both 2,4-D and NAA, the induction rate can reach 100%. The high concentration of KT contributes to the formation of embryogenic callus, presumably by regulating plant cell osmosis. However, when leaves are used as explants, the induction rate of embryogenic callus in suspension culture is also very low, indicating that leaves are not a relatively good explant for inducing embryogenic callus.

[0038] Table 2 Effects of different induction conditions on leaf-induced embryogenic callus

[0039] 1 0 0.3 0 0 0 none 2 0 0.6 1 0 0 none 3 0 1 2 0 36.7 White flocculent 4 0.3 0.3 0 0 83.3 White flocculent 5 0.3 0.6 1 8.7 100 White flocculent 6 0.3 1 2 20 100 transparent particles 7 0.5 0.3 1 38 100 White flocculent 8 0.5 0.6 2 31.2 100 White flocculent 9 0.5 1 0 0 100 White flocculent 10 1 0.3 2 13.3 100 White flocculent 11 1 0.6 0 6.7 100 White flocculent 12 1 1 1 0 100 White flocculent

[0040] Comparative Experiment 3: Induction of Embryogenic Callus Cells from Sea Fennel Stem Segments

[0041] The callus formation time of stem segments is generally 12-15 days. The higher the concentration of hormones 2,4-D and NAA in the culture medium, the earlier the callus formation. The formed callus is mainly a bright yellow, transparent, granular embryogenic callus (such as...). Figure 3 As shown in the figure, the induction rate of embryogenic callus from stem segments is much higher than that from seed embryos and leaves. In particular, with the increase of KT concentration, the generation rate of embryogenic callus cells can be significantly improved. The callus cells formed are mostly transparent granular, relatively loose, and have good cell growth status in liquid suspension culture.

[0042] Table 3 Effects of different induction conditions on embryogenic callus induction from stem segments

[0043] 1 0 0.3 0 0 0 none 2 0 0.6 1 1.4 16.7 none 3 0 1 2 4.5 26.4 White flocculent 4 0.3 0.3 0 26.4 100 White flocculent 5 0.3 0.6 1 35.5 100 Transparent granules 6 0.3 1 2 52.5 100 Transparent granules 7 0.5 0.3 1 94.2 100 Transparent granules 8 0.5 0.6 2 73.6 100 Transparent granules 9 0.5 1 0 36.2 100 White flocculent 10 1 0.3 2 52.1 100 Transparent granules 11 1 0.6 0 45.1 100 White flocculent 12 1 1 1 34.2 100 White flocculent

[0044] The induction results of embryogenic callus varied under different induction conditions. The highest induction rate of embryogenic callus reached 94.2% when 2,4-D was 0.5 mg / L, NAA was 0.3 mg / L, and KT was 1 mg / L. This indicates that 2,4-D has a significantly higher induction effect on embryogenic callus formation than NAA, while KT has the second highest induction effect on embryogenic callus cells, and can also significantly improve the induction efficiency of embryogenic cells.

[0045] Therefore, stem segments are the most suitable in vitro tissue type for inducing embryogenic callus in sea fennel. The optimal induction medium for callus induction consists of MS basal salt medium, 30 g / L sucrose, 8.5 g / L agar, 0.5 mg / L 2,4-D, 0.3 mg / L NAA, and 1 mg / L KT. This invention, through explant screening, ultimately determined that stem segments are the preferred inducing agent for callus induction, directly inducing embryogenic callus in sea fennel. This resulted in an induction rate of over 94% for suspension-cultured embryogenic callus, shortening the induction time and providing well-preserved callus for large-scale culture of sea fennel cells.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the induction rate of embryogenic callus from sea fennel that can be cultured in suspension, characterized in that, Includes the following steps: 1) Sterilize the stem segments of sea fennel to obtain sterilized explants; 2) The sterilized explants obtained in step 1) are subjected to callus induction culture to obtain callus culture; the culture medium used for callus induction culture is: MS basal salt medium, sucrose 25-35 g / L, agar 8-9 g / L, dichlorophenoxyacetic acid 0.5 mg / L, naphthaleneacetic acid 0.3-0.6 mg / L, and kinetin 1-2 mg / L; The MS basal salt medium consists of KNO3 1900 mg / L, NH4NO3 1650 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, CaCl2·2H2O 440 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, Na2-EDTA 37.3 mg / L, and FeSO4·7H2O. It consists of 27.8 mg / L of glycine, 2.0 mg / L of pyridoxine hydrochloride, 0.6 mg / L of pyridoxine hydrochloride, and 0.1 mg / L of thioammonium hydrochloride.

2. The method according to claim 1, characterized in that, The disinfection method in step 1) includes the following steps: after rinsing the sea fennel, it is disinfected with ethanol solution and sodium hypochlorite solution in sequence.

3. The method according to claim 2, characterized in that, The ethanol solution has a volume percentage of 75% and a disinfection time of 30 seconds; the sodium hypochlorite solution has a mass percentage of 4-8% and a disinfection time of 15 minutes.

4. The method according to claim 1, characterized in that, In step 2), the explants are young stem segments, cut into 1-2 cm long segments, and then callus induction culture is performed.

5. The method according to claim 1, characterized in that, The pH of the culture medium in step 2) is 5.8-6.

0.

6. The method according to claim 1 or 5, characterized in that, The conditions for callus induction include: culturing for 20 days at a temperature of 25°C, light intensity, and relative humidity of 65%.

7. The method according to claim 1 or 5, characterized in that, The method is used to culture embryogenic callus from sea fennel.

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