A method for preparing and suspension culturing of blackcurrant callus

Abstract

The application discloses a kind of preparation and suspension culture of blackland callus method, belong to the field of industrial biotechnology.It includes the following steps: after sterilization, take flower stem, access pH value is 5-7 in induction medium, under the condition of temperature is 20-30 DEG C and no light, induction culture 15-21 days, namely obtain blackland callus, wherein, the induction medium is made of MS basic medium, sucrose 25-35g / L, agar 6-7g / L, 6-BA 1.0-2mg / L, NAA 0.5-1.5mg / L, 2, 4-D 0.3-1.0mg / L and coconut water 0.5-1.5g / L composition.Under aseptic conditions, clamp the growth state of good blackland embryogenic callus 30g, inoculation is carried out in 200mL / 500mL pH value is 6.0 in MS liquid medium, temperature 25 DEG C, 110 rpm full light oscillation culture, subculture period 7 days, when subculture, the mixture of cell and culture fluid is mixed with fresh culture fluid according to 1:1 volume ratio, after equal division, continue to culture, namely obtain blackland suspension cell, wherein, the liquid medium is made of MS basic medium, sucrose 30g / L, 6-BA 1.5mg / L, NAA 0.5mg / L, 2, 4-D 0.5mg / L and coconut water 1.5g / L composition.The callus out of the rate of 88.6% is realized, and the induction rate of embryogenic callus that can be suspended culture is 68.6%-91.8%, and good callus state is provided for large-scale culture blackland cell.

Description

Technical Field

[0001] This invention relates to a method for preparing and culturing black orchid callus cells, belonging to the field of industrial biotechnology. Background Technology

[0002] Black orchid (Cymbidium kanran) is a herbaceous plant belonging to the genus Cymbidium in the family Orchidaceae. It has a beautiful appearance and is rich in vitamins, aldehydes, and hydroxy acids, making it highly valuable for both its ornamental and economic purposes.

[0003] Black orchid plants have demanding cultivation requirements and are easily affected by climate and pests, making large-scale acquisition difficult. To alleviate the scarcity of plant resources and address the high market demand for black orchids, plant callus cell culture technology has significant application potential. This technology can utilize the totipotency of callus cells to regenerate plants or produce active metabolites. The prerequisite for this technology is the preparation of black orchid callus cells and the establishment of a liquid suspension culture system. However, there are few reports on the preparation of black orchid callus cells.

[0004] The selection of explants is fundamental to the successful preparation of callus cells. Currently, most callus cell preparations utilize plant leaves, stem segments, and flowers. Different explants exhibit significant differences in cell division capacity and regeneration potential, directly impacting callus formation. Studies have shown that plant stamens contain abundant active substances and are less susceptible to bacterial contamination, making them a superior explant for callus induction. However, research on using black orchid stamens for callus induction is limited.

[0005] In view of this, this application provides a method for preparing and culturing black orchid callus cells to overcome the above-mentioned deficiencies. Summary of the Invention

[0006] This invention provides a method for preparing and culturing black orchid callus cells. Through screening explants, this invention ultimately determined that flower stamens are the inducing agents for callus tissue. The callus emergence rate was 88.6%, and the induction rate of embryogenic callus tissue that can be cultured in suspension was 68.6%-92.2%, providing well-preserved callus tissue for large-scale culture of black orchid cells.

[0007] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for preparing and culturing black orchid callus cells, comprising the following steps:

[0008] After disinfecting the black orchid plants, explants from different parts were taken and inoculated into an induction medium with a pH of 5-7. The plants were then induced and cultured in the dark (without light) at a temperature of 20-30℃ for 15-21 days to obtain black orchid embryogenic callus. The induction medium consisted of MS basal salt 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.

[0009] The principle of this invention is that the callus emergence rate varies depending on the explant used to induce callus formation in *Orchidonia spp.*, not all explants can induce callus, and the efficiency of embryogenic callus formation in suspension culture also differs. This invention uses *Orchidonia spp.*, selects inducing explants, and optimizes induction culture conditions to achieve a high callus emergence rate, a high rate of embryogenic callus formation, and strong cell suspension cultureability.

[0010] The induction medium of this invention comprises MS basal salt medium, sucrose, agar, 6-BA, NAA, 2,4-D, and coconut water. MS basal salt medium provides the inorganic salts and nutrients necessary for plant cell growth; sucrose provides the carbon source for plant cell growth; agar acts as a solidifying agent to allow callus cells to grow and multiply on the surface; 6-BA is used as a mitotic hormone; NAA is used as a growth hormone; 2,4-D is used as a growth hormone; and coconut water promotes plant cell growth and aids in the production of embryogenic callus cells. The induction medium of this invention can induce the formation of black orchid callus cells, yielding loosely packed embryogenic callus cells and establishing a liquid suspension culture system.

[0011] Based on the above technical solution, the present invention can be further improved as follows.

[0012] Furthermore, the induced explants were selected from the stamens of black orchids.

[0013] Furthermore, the induction medium consists of MS basal salt 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.

[0014] The further beneficial effect of using the above-mentioned composition is that the induction culture medium is more conducive to inducing the formation of embryogenic callus cells from black orchid explants.

[0015] Furthermore, the pH value of the induction medium is 6, the temperature is 25°C, and the induction culture time is 18 days.

[0016] Furthermore, the culture medium for the suspension culture of black orchid cells consists of MS basal salt medium, 30 g / L sucrose, 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.

[0017] Furthermore, the wet and dry weights of the suspended black orchid cells reached their peak on day 7.

[0018] Glossary

[0019] 6-BA, or 6-benzylaminopurine, is a plant growth regulator that is safe for humans and animals.

[0020] NAA, or naphthaleneacetic acid, is an auxin analogue that functions similarly to plant auxins.

[0021] 2,4-D, or 2,4-dichlorophenoxyacetic acid, is an auxin analog that promotes callus formation.

[0022] The beneficial effects of this invention are:

[0023] (1) This invention ultimately determined to use flower stamens as the inducing agent for callus by screening explants. The callus emergence rate was 88.6%, and the induction rate of embryogenic callus that can be cultured in suspension was 68.6%-92.2%, providing callus in good condition for large-scale culture of black orchid cells.

[0024] (2) The present invention developed an induction medium for the generation of black orchid embryogenic callus cells in suspension culture, obtained loose embryogenic callus cells, and established a suspension liquid culture system for cells.

[0025] (3) The method of the present invention is simple, has broad market prospects, and is suitable for industrial production. Attached Figure Description

[0026] Figure 1 Black Orchid Suspension Cells

[0027] Figure 2 Growth curve of black orchid suspension cells

[0028] Figure 3 For comparison experiment 2, non-embryonic callus cells.

[0029] Figure 4 For comparative experiments, non-embryonic callus cells were used. Detailed Implementation

[0030] 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.

[0031] Implementation Example 1:

[0032] The method for preparing black orchid callus in this embodiment includes the following steps:

[0033] After sterilizing the black orchid plants, the flower stamens were removed, yielding 276 flower stamen explants. These were inoculated into an induction medium with a pH of 5.0 and cultured at 20°C in complete darkness for 21 days, resulting in 190 black orchid embryogenic callus cell clusters. The induction medium consisted of MS basal salt medium, 25 g / L sucrose, 6 g / L agar, 1.5 mg / L 6-BA, 1.0 mg / L NAA, 1.0 mg / L 2,4-D, and 0.5 g / L coconut water.

[0034] 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.

[0035] The calculation formula is as follows:

[0036]

[0037]

[0038] Calculations show that the callus induction rate in this embodiment is 100%, and the embryonic callus induction rate is 69%.

[0039] Implementation Example 2:

[0040] The method for preparing black orchid callus in this embodiment includes the following steps:

[0041] After sterilizing the black orchid plants, the flower stamens were removed, yielding 276 flower stamen explants. These explants were inoculated into an induction medium with a pH of 6.0 and cultured at 25°C in the dark for 18 days, resulting in 248 black orchid embryogenic callus cell clusters. The induction medium consisted of MS basal salt medium, 30 g / L sucrose, 6.5 g / L agar, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L 2,4-D, and 1.5 g / L coconut water.

[0042] Calculations show that the callus induction rate in this embodiment is 100%, and the average induction rate of embryonic callus is 90%.

[0043] Implementation Example 3:

[0044] The method for preparing black orchid callus in this embodiment includes the following steps:

[0045] After sterilizing the black orchid plants, the flower stamens were removed, yielding 276 flower stamen explants. These explants were inoculated into an induction medium with a pH of 7.0 and cultured at 30°C in the dark for 15 days, resulting in 207 black orchid embryogenic callus cell clusters. The induction medium consisted of MS basal salt medium, 35 g / L sucrose, 7 g / L agar, 2.0 mg / L 6-BA, 1.5 mg / L NAA, 0.3 mg / L 2,4-D, and 1.0 g / L coconut water.

[0046] Calculations show that the callus induction rate in this embodiment is 100%, and the embryonic callus induction rate is 75.2%.

[0047] Implementation Example 4:

[0048] The method for culturing black orchid suspension cells in this embodiment includes the following steps:

[0049] Under aseptic conditions, 30g of well-grown black orchid embryogenic callus was collected and inoculated into 200 mL / 500 mL of MS liquid medium at pH 6.0. The liquid medium consisted of MS basal salt medium, 30 g / L sucrose, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L 2,4-D, and 1.5 g / L coconut water. The cells were cultured at 25°C under full light with shaking at 110 rpm for 7 days. For subculturing, the mixture of cells and culture medium was mixed with fresh culture medium at a 1:1 volume ratio, aliquoted, and cultured further to obtain black orchid suspension cells (e.g., ...). Figure 1 (As shown). The growth curve of black orchid cells is as follows. Figure 2 As shown, both wet and dry weights initially showed a significant increase followed by a decrease, peaking on day 7.

[0050] Comparative test

[0051] In order to obtain better black orchid embryogenic callus cells for suspension culture, the applicant studied the effects of different explant leaves, petals and stamens, as well as different hormones and concentrations in the culture medium on callus formation.

[0052] Comparative Experiment 1: Induction of Embryogenic Callus Cells from Black Orchid Leaves

[0053] Callus induction was performed on leaves of black orchid, and it was found that callus formation was extremely difficult to occur on the leaves under different induction conditions.

[0054] Comparative Experiment 2: Induction of Embryogenic Callus Cells Using Black Orchid Petals

[0055] Using black orchid petals as explants, a small amount of callus tissue appeared on the petals within 16-20 days. Callus tissue on the leaves only appeared and grew at the leaf margins, and the entire explant could not be induced to form callus tissue. Most of the callus tissue was white, cottony, non-embryonic (e.g., ...). Figure 3 As shown in the figure, 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 distinct shapes, and the callus viability was weak. After subculture, the color of the callus gradually deepened and browning occurred. When the culture medium did not contain mitogens, almost no callus was induced from the petals, especially after the auxin concentration increased, the petal explants browned and died. The experimental results indicate that coconut water can improve the callus emergence rate of petal explants to some extent. The experimental results show that only a few petals were induced to produce embryogenic callus. The callus was generally pale yellow with a certain metallic luster, and the surface was granular with uniform cells. The induced embryogenic cells grew slowly during suspension culture, and the solution showed large particle aggregation and browning. This indicates that petals are not a relatively good explant for inducing embryogenic callus.

[0056] Table 1. Effects of different induction conditions on petal-induced embryogenic callus

[0057] serial 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 granules 6 1.0 0.5 0.5 1.5 87.5 68.2 Transparent granules 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 granules 9 1.5 0.5 0.3 1.5 77.7 55.9 Transparent granules 10 1.5 0.5 0.5 1.0 75.1 53.7 Transparent granules 11 1.5 1.0 1.0 0.5 72.5 52.5 Transparent granules 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 granules 16 2 1.5 0.3 1.0 70.6 54.7 Transparent granules

[0058] Comparative Experiment 3: Induction of Embryogenic Callus from Black Orchid Stamen Explants

[0059] Table 2 shows that the callus induction rate was low when the culture medium did not contain 6-BA. The induction rate of the flower stamens was higher when the culture medium contained 6-BA, NAA, and 2,4-D simultaneously, presumably because the flower stamens had stronger regenerative capacity and vigor. The callus emergence time for the flower stamens was generally 12-15 days. A close ratio of mitogens and growth hormones in the culture medium was more conducive to callus formation. Before callus formation, callus slowly formed at the tip of the flower stamen and then expanded outwards. The formed callus was mainly a bright yellow, transparent, granular embryogenic callus (e.g., ...). Figure 4 (As shown). With increasing coconut water concentration, the generation rate of embryogenic callus cells can be significantly improved. The resulting callus cells are mostly transparent granular and relatively loose, and their cell growth is good in liquid suspension culture.

[0060] Table 2. Effects of different induction conditions on embryogenic callus induced by flower stamens.

[0061] serial 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 granules 6 1.0 0.5 0.5 1.5 100 93.1 Transparent granules 7 1.0 1.0 0.3 0 86.2 65.3 Transparent granules 8 1.0 1.5 0.5 0.5 100 77.5 Transparent granules 9 1.5 0.5 0.3 1.5 100 68.9 Transparent granules 10 1.5 0.5 0.5 1.0 100 83.6 Transparent granules 11 1.5 1.0 1.0 0.5 100 71.4 Transparent granules 12 1.5 1.5 0.5 0 78.9 73.2 Transparent granules 13 2 0.5 0.5 0.5 84.4 77.3 Transparent granules 14 2 0.5 1.0 0 80.1 70.5 Transparent granules 15 2 1.0 0.5 1.5 100 78.4 Transparent granules 16 2 1.5 0.3 1.0 100 76.8 Transparent granules

[0062] Therefore, flower stamens are the most suitable in vitro tissue type for inducing callus in *Orchidica pubescens*. The optimal induction medium for callus induction consists of MS basal salt medium, 30 g / L sucrose, 6.5 g / L agar, 1.5 mg / L 6-BA, 0.5 mg / L NAA, 0.3 mg / L 2,4-D, and 1.5 g / L coconut water. This invention, through explant screening, ultimately determined that flower petals are the preferred inducing agent for callus induction, achieving an induction rate of over 90% for embryogenic callus. This shortens the induction time and provides well-preserved callus for large-scale culture of *Orchidica pubescens* cells.

[0063] 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 of preparing a black currant callus, characterized by, The method comprises the following steps: After sterilization of the Heilanjian, the pistil is taken and inoculated into an induction medium with pH value of 5-7, and is induced to culture for 15-21 days under the condition of temperature of 20-30 DEG C and no light, so that Heilanjian callus is obtained, wherein the induction medium is composed of MS basic salt 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. A method for suspension culture of Heberia lanata callus, characterized by, The method comprises the following steps: Under sterile conditions, 30 g of the embryogenic callus of Heilanjian in good growth state is clamped and inoculated into 200 mL / 500 mL of MS liquid medium with pH value of 6.0, and is cultured at temperature of 25 DEG C and 110 rpm full light oscillation, the subculture cycle is 7 days, when subcultured, the mixture of cells and culture solution is mixed with fresh culture solution at volume ratio of 1:1, and after being evenly divided, the culture is continued, so that Heilanjian suspension cells are obtained, wherein the liquid medium is composed of MS basic salt medium, sucrose 30 g / L, 6-BA 1.5 mg / L, NAA 0.5 mg / L, 2,4-D 0.5 mg / L and coconut water 1.5 g / L.

3. The method of claim 1, wherein the Heberberix thibetanus callus is prepared by the steps of: The induction medium is composed of MS basic medium, sucrose 30 g / L, agar 6.5 g / L, 6-BA 1.5 mg / L, NAA 0.5 mg / L, 2,4-D 0.5 mg / L and coconut water 1.5 g / L.

4. The method according to claim 1 or 3, characterized in that, The pH value of the induction medium is 6, the temperature is 25 DEG C, and the induction culture time is 18 days.

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