Preparation and suspension culture method of black orchid callus cells

Abstract

The invention discloses a preparation and suspension culture method of black orchid callus cells, and belongs to the technical field of industrial biology. The method comprises the following steps that after the black orchid is disinfected, stamens are taken and inoculated into an induction culture medium with the pH value being 5-7, induction culture is conducted for 15-21 days under the condition that the temperature is 20-30 DEG C and no light exists, the black orchid callus is obtained, and the induction culture medium is composed of an MS basic salt culture medium, 25-35 g / L of saccharose, 6-7 g / L of agar, 1.0-2 mg / L of 6-BA, 0.5-1.5 mg / L of NAA, 0.3-1.0 mg / L of 2, 4-D and 0.5-1.5 g / L of coconut water. According to the method, the callus yield of the callus is 88.6%, the induction rate of the embryogenic callus capable of being cultured in a suspension manner is 68.6%-91.8%, and the callus with a good state is provided for large-scale culture of black cymbidium cells.

Description

Technical Field

[0001] The present invention relates to a method for preparing and suspension-culturing callus cells of Cymbidium kanran, belonging to the field of industrial biotechnology. Background Art

[0002] Cymbidium kanran is a herbaceous plant of the genus Cymbidium in the Orchidaceae family. It has a beautiful appearance, is rich in vitamins, aldehydes and hydroxy acids, and has high ornamental and economic values.

[0003] The Cymbidium kanran plants have strict requirements for culture conditions, are easily affected by climate and pests and diseases, and are difficult to obtain on a large scale. To alleviate the shortage of plant resources and solve the problems such as the large market demand for Cymbidium kanran, the plant callus cell culture technology has good application potential. This technology can utilize the totipotency of callus cells to regenerate plants or produce bioactive metabolites. The prerequisite for this technology is to prepare Cymbidium kanran callus cells and establish a liquid suspension culture system. However, there are few relevant reports on the preparation of Cymbidium kanran callus cells.

[0004] The selection of explants is the basis for successfully preparing callus cells. At present, most of the preparations of callus cells select the leaves, stem segments and flowers of plants. There are obvious differences in the cell division ability and regeneration potential of different explants, which directly affect the generation of callus. Research shows that the pistil of plants contains more bioactive substances and is not easily contaminated by bacteria, and it is a good explant for callus induction. However, there are few studies on using the pistil of Cymbidium kanran to induce callus.

[0005] In view of this, the present application provides a method for preparing and suspension-culturing Cymbidium kanran callus cells to make up for the above deficiencies. Summary of the Invention

[0006] The present invention provides a method for preparing and suspension-culturing Cymbidium kanran callus cells. By screening the explants, the present invention finally determines to use the pistil as the inducer of callus. The callus induction rate is 88.6%, and the induction rate of embryogenic callus that can be suspension-cultured is 68.6%-92.2%, providing callus with good state for large-scale culture of Cymbidium kanran cells.

[0007] The technical solution for the present invention to solve the above technical problems is as follows: A method for preparing and suspension-culturing Cymbidium kanran callus cells, comprising the following steps: After disinfecting the Heilan plants, explants from different parts are taken and inoculated into an induction medium with a pH value of 5 - 7. They are induced and cultured for 15 - 21 days under the condition of dark culture (without light) at a temperature of 20 - 30 °C, and then the embryogenic callus of Heilan is obtained. Among them, the induction medium is a nutrient aqueous solution composed of MS basal salt medium, 25 - 35 g / L of sucrose, 6 - 7 g / L of agar, 1.0 - 2 mg / L of 6 - BA, 0.5 - 1.5 mg / L of NAA, 0.3 - 1.0 mg / L of 2,4 - D, and 0.5 - 1.5 g / L of coconut water.

[0008] The principle of the present invention is as follows: The callus induction rates of any explants of Heilan plants are different. Not all explants can induce callus, and the efficiencies of embryogenic callus that can be suspension - cultured in the induced callus are also different. The present invention uses Heilan plants, and by screening and inducing explants and optimizing the induction culture conditions, the callus induction rate of Heilan callus cells is high, the formation rate of embryogenic callus cells is high, and the suspension - culture property of the cells is strong.

[0009] The induction medium of the present invention is composed of MS basal salt medium, sucrose, agar, 6 - BA, NAA, 2,4 - D, and coconut water. Among them, the MS basal salt medium provides the inorganic salts and nutrient elements necessary for the growth of plant cells; sucrose provides the carbon source for the growth of plant cells; agar is used as a coagulant so that the callus cells can grow and reproduce on the surface layer; 6 - BA is used as a splitting hormone; NAA is used as a growth hormone; 2,4 - D is used as a growth hormone; coconut water can promote the growth of plant cells and help the generation of embryogenic callus cells. The induction medium of the present invention can induce the formation of Heilan callus cells, obtain loose embryogenic callus cells, and establish a liquid suspension culture system.

[0010] On the basis of the above - mentioned technical solutions, the present invention can be further improved as follows.

[0011] Further, the induced explant selected is the stamen of Heilan.

[0012] Further, the induction medium is composed of MS basal salt medium, 30 g / L of sucrose, 6.5 g / L of agar, 1.5 mg / L of 6 - BA, 0.5 mg / L of NAA, 0.5 mg / L of 2,4 - D, and 1.5 g / L of coconut water.

[0013] The beneficial effect of adopting the above is that: with the above composition of the induction medium, it is more conducive to inducing the formation of embryogenic callus cells from Heilan explants.

[0014] Further, the pH value of the induction medium is 6, the temperature is 25 °C, and the induction culture time is 18 days.

[0015] Furthermore, the medium for the suspension culture of Dendrobium officinale cells consists of MS basal salt medium, 30 g / L of sucrose, 1.5 mg / L of 6-BA, 0.5 mg / L of NAA, 0.5 mg / L of 2,4-D, and 1.5 g / L of coconut water.

[0016] Furthermore, the wet weight and dry weight of the suspended Dendrobium officinale cells reach their peaks on the 7th day.

[0017] Glossary 6-BA, namely 6-benzylaminopurine, is a plant growth regulator that is safe for humans and livestock.

[0018] NAA, namely naphthaleneacetic acid, is an auxin analog with a similar effect to plant auxin.

[0019] 2,4-D, namely 2,4-dichlorophenoxyacetic acid, is an auxin analog that promotes the formation of callus.

[0020] The beneficial effects of the present invention are as follows: (1) By screening the explants, the present invention finally determines to use the stamen as the inducer of callus. The callus induction rate is 88.6%, and the induction rate of embryogenic callus that can be suspended cultured is 68.6% - 92.2%, providing well-conditioned callus for large-scale culture of Dendrobium officinale cells.

[0021] (2) The present invention develops an induction medium for generating embryogenic suspension-culturable callus cells of Dendrobium officinale, obtains loose embryogenic callus cells, and establishes a suspension liquid culture system for the cells.

[0022] (3) The method of the present invention is simple, has a broad market prospect, and is suitable for industrial production. Brief Description of the Drawings

[0023] Figure 1 For Dendrobium officinale suspension cells Figure 2 For the growth curve of Dendrobium officinale suspension cells Figure 3 For non-embryonic callus cells in Comparative Experiment 2.

[0024] Figure 4 For embryonic callus cells in Comparative Experiment 3. Detailed Embodiments

[0025] The principles and features of the present invention will be 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.

[0026] Example 1: The preparation method of the callus of Cymbidium erythraeum in this embodiment includes the following steps: After disinfecting the Cymbidium erythraeum plants, take the flower pistils to obtain 276 flower pistil explants, inoculate them into an induction medium with a pH value of 5.0, and induce and culture them for 21 days under the conditions of a temperature of 20°C and no light, and 190 callus cell clusters of Cymbidium erythraeum embryogenic callus are obtained. Among them, the induction medium is composed of MS basal salt medium, 25 g / L of sucrose, 6 g / L of agar, 1.5 mg / L of 6-BA, 1.0 mg / L of NAA, 1.0 mg / L of 2,4-D, and 0.5 g / L of coconut water.

[0027] Calculate the induction rate of callus cells and the induction rate of embryogenic callus cells respectively. Among them, the callus cell induction rate refers to the formation rate of callus cells of the explants on the induction medium; the embryogenic callus cell induction rate is the ratio of embryogenic callus cells in the total amount of callus cells generated.

[0028] The calculation formula is as follows:

[0029]

[0030] After calculation, the induction rate of callus cells in this embodiment is 100%, and the induction rate of embryogenic callus cells is 69%.

[0031] Example 2: The preparation method of the callus of Cymbidium erythraeum in this embodiment includes the following steps: After disinfecting the Cymbidium erythraeum plants, take the flower pistils to obtain 276 flower pistil explants, inoculate them into an induction medium with a pH value of 6.0, and induce and culture them for 18 days under the conditions of a temperature of 25°C and no light, and 248 callus cell clusters of Cymbidium erythraeum embryogenic callus are obtained. Among them, the induction medium is composed of MS basal salt medium, 30 g / L of sucrose, 6.5 g / L of agar, 1.0 mg / L of 6-BA, 0.5 mg / L of NAA, 0.5 mg / L of 2,4-D, and 1.5 g / L of coconut water.

[0032] After calculation, the induction rate of callus cells in this embodiment is 100%, and the average induction rate of embryogenic callus cells is 90%.

[0033] Example 3: The preparation method of the callus of Cymbidium erythraeum in this embodiment includes the following steps: After disinfecting the Black Orchid plants, take the flower stamens to obtain 276 stamen explants, and inoculate them into an induction medium with a pH value of 7.0. Induce and culture them for 15 days under the conditions of a temperature of 30°C and no light, and 207 Black Orchid embryogenic callus cell clusters are obtained. Among them, the induction medium consists of MS basal salt medium, 35 g / L of sucrose, 7 g / L of agar, 2.0 mg / L of 6-BA, 1.5 mg / L of NAA, 0.3 mg / L of 2,4-D, and 1.0 g / L of coconut water.

[0034] After calculation, the induction rate of callus cells in this example is 100%, and the induction rate of embryogenic callus cells is 75.2%.

[0035] Example 4: The culture method of Black Orchid suspension cells in this example includes the following steps: Under sterile conditions, clamp 30 g of well-growing Black Orchid embryogenic callus and inoculate it into a 200 mL / 500 mL MS liquid medium with a pH value of 6.0. Among them, the liquid medium consists of MS basal salt medium, 30 g / L of sucrose, 1.0 mg / L of 6-BA, 0.5 mg / L of NAA, 0.5 mg / L of 2,4-D, and 1.5 g / L of coconut water. The temperature is 25°C, and it is cultured with full light oscillation at 110 rpm, and the subculture period is 7 days. When subculturing, mix the mixture of cells and culture medium with fresh culture medium at a volume ratio of 1:1, divide it evenly and continue to culture, and Black Orchid suspension cells are obtained (as Figure 1 shown). The growth curve of Black Orchid cells is as Figure 2 shown. Overall, both the wet weight and dry weight first show a trend of rising significantly and then decreasing, reaching the peak on the 7th day.

[0036] Comparative experiment In order to obtain better Black Orchid embryogenic callus cells for suspension culture, the applicant studied the effects of different explants (leaves, petals, and stamens) and different hormones and concentrations in the medium on the formation of callus.

[0037] Comparative experiment 1: Induce embryogenic callus cells with Black Orchid leaves Use the leaves of Black Orchid to induce callus cells, and it is found that it is extremely difficult for the leaves to form callus under different induction conditions.

[0038] Comparative experiment 2: Induce embryogenic callus cells with Black Orchid petals Using Black Orchid petals as explants, a small amount of callus appears on the petals after 16 - 20 days, and the callus of the leaves only appears and grows at the edge of the leaves, and cannot induce the entire explant to form callus. Most of them are white cottony non-embryogenic callus (as Figure 3As shown, only a small amount of embryogenic callus with bright colors was induced. Although the non-embryogenic callus in this state also showed loose structure, microscopic examination showed that they were all long strip-shaped cells without obvious shape and the vitality of the callus was weak. After subculture, the color of the callus gradually deepened and browning occurred. When the medium did not contain cytokinin, basically no callus was induced from the petals. Especially after the concentration of auxin increased, the petal explants would turn brown and die. From the experimental results, coconut water could, to a certain extent, improve the callus induction rate of petal explants. It can be seen from the experimental results that only a few petals were induced to form embryogenic callus. The callus generally showed light yellow, with a certain metallic luster, the surface of the callus was granular, and the cells were uniform. The induced embryogenic cells grew slowly during suspension culture, and large particles aggregated and browning occurred in the solution. It indicated that petals were not relatively good explants for inducing embryogenic callus.

[0039] Table 1 Effects of Different Induction Conditions on the Induction of Embryogenic Callus from Petals Number 6-BA (mg / L) NAA (mg / L) 2,4-D (mg / L) Coconut water (g / L) Callus induction rate (%) Embryogenic callus induction rate (%) Callus state 1 0 0.5 0.5 0 0 0 None 2 0 0.5 0.3 0.5 0 0 None 3 0 1.0 0.5 1.0 0 0 None 4 0 1.5 1.0 1.5 0 0 None 5 1.0 0.5 1.0 1.0 74.2 53.9 Transparent granular 6 1.0 0.5 0.5 1.5 87.5 68.2 Transparent granular 7 1.0 1.0 0.3 0 71.7 40.4 White flocculent 8 1.0 1.5 0.5 0.5 73.9 52.1 Transparent granular 9 1.5 0.5 0.3 1.5 77.7 55.9 Transparent granular 10 1.5 0.5 0.5 1.0 75.1 53.7 Transparent granular 11 1.5 1.0 1.0 0.5 72.5 52.5 Transparent granular 12 1.5 1.5 0.5 0 68.8 30.3 White flocculent 13 2 0.5 0.5 0.5 69.3 32.8 White flocculent 14 2 0.5 1.0 0 67.4 24.5 White flocculent 15 2 1.0 0.5 1.5 73.8 56.6 Transparent granular 16 2 1.5 0.3 1.0 70.6 54.7 Transparent granular Comparative Experiment 3 Induction of Embryogenic Callus with the Explants of Black Orchid Stamens As can be seen from Table 2, when 6-BA was not contained in the medium, the callus induction rate was relatively low. When 6-BA, NAA, and 2,4-D were simultaneously contained in the medium, the induction rate of stamens was relatively high. It was speculated that this might be because the regeneration ability and vitality of stamens were relatively strong. The callus induction time of stamens was generally 12 - 15 days. When the ratio of cytokinin to auxin in the medium was close, it was more conducive to the generation of stamen callus. Before the formation of callus, callus first slowly formed at the tip of the stamen and then grew outward. The formed callus was mainly embryogenic callus with bright yellow, transparent, and granular surface (as Figure 4 shown). With the increase in the concentration of coconut water, the generation rate of embryogenic callus cells could be significantly improved. The formed callus cells mostly showed transparent granular shape, were relatively loose, and had a good cell growth state in liquid suspension culture.

[0040] Table 2 Effects of Different Induction Conditions on the Induction of Embryogenic Callus from Stamens Number 6-BA (mg / L) NAA (mg / L) 2,4-D (mg / L) Coconut water (g / L) Callus induction rate (%) Embryogenic callus induction rate (%) Callus state 1 0 0.5 0.5 0 13.8 1.7 White flocculent 2 0 0.5 0.3 0.5 20.6 2.6 White flocculent 3 0 1.0 0.5 1.0 26.8 4.6 White flocculent 4 0 1.5 1.0 1.5 36.2 15.1 White flocculent 5 1.0 0.5 1.0 1.0 100 80.3 Transparent granular 6 1.0 0.5 0.5 1.5 100 93.1 Transparent granular 7 1.0 1.0 0.3 0 86.2 65.3 Transparent granular 8 1.0 1.5 0.5 0.5 100 77.5 Transparent granular 9 1.5 0.5 0.3 1.5 100 68.9 Transparent granular 10 1.5 0.5 0.5 1.0 100 83.6 Transparent granular 11 1.5 1.0 1.0 0.5 100 71.4 Transparent granular 12 1.5 1.5 0.5 0 78.9 73.2 Transparent granular 13 2 0.5 0.5 0.5 84.4 77.3 Transparent granular 14 2 0.5 1.0 0 80.1 70.5 Transparent granular 15 2 1.0 0.5 1.5 100 78.4 Transparent granular 16 2 1.5 0.3 1.0 100 76.8 Transparent granular It can be seen that the flower stamen is the most suitable in vitro tissue type for inducing callus of Cymbidium erythraeum Lindl. Among them, the best induction medium for inducing callus is composed of MS basal salt medium, 30 g / L of sucrose, 6.5 g / L of agar, 1.5 mg / L of 6-BA, 0.5 mg / L of NAA, 0.3 mg / L of 2,4-D and 1.5 g / L of coconut water. Through the screening of explants, the present invention finally determines to use petals as the inducer of callus, and the induction rate of embryogenic callus reaches more than 90%, which can shorten the induction time and provide well-conditioned callus for large-scale culture of Cymbidium erythraeum Lindl. cells.

[0041] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing and suspending callus cells of black orchid, characterized in that: The steps include: After disinfecting the black orchid, taking the stamens, inoculating them into an induction culture medium with a pH value of 5-7, and inducing and culturing them for 15-21 days at a temperature of 20-30° C. and without light, so as to obtain black orchid callus tissue, wherein the induction culture medium consists of MS basal salt culture medium, sucrose 25-35 g / L, agar 6-7 g / L, 6-BA 1.0-2 mg / L, NAA 0.5-1.5 mg / L, 2,4-D 0.3-1.0 mg / L and coconut water 0.5-1.5 g / L.

2. The method for preparing black orchid callus according to claim 1, characterized in that: The induction medium is a nutrient aqueous solution consisting of MS basic medium: 30 g / L sucrose, 6.5 g / L agar, 1.5 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L 2,4-D, and 1.5 g / L coconut water.

3. The method for preparing and suspending callus cells of black orchid according to claim 1 or 2, characterized in that: The pH value of the induction culture medium is 6, the temperature is 25° C., and the induction culture time is 18 days.

4. The method for suspension culture of black orchid callus according to claim 1 or 2, characterized in that: The culture subculture period of the suspension culture is 7 days.

5. The method for suspension culture of black orchid callus according to claim 1 or 2, characterized in that: Used to culture black orchid callus cells.

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