A method for culturing red callus of tea tree

Abstract

The present invention discloses a method for rapidly obtaining uniform red callus from tea plants using leaves from Anji white tea tissue culture seedlings. Using the leaves of virus-free tissue culture seedlings from Anji white tea as explants, callus was induced using MS medium containing 6-BA and IBA, achieving a high-frequency induction rate of 99.23%. The primary callus was transferred to dark conditions and induced with TDZ and IBA to obtain white callus. The white callus was then transferred to light conditions to obtain uniform red callus. This method reduces the step of explant disinfection, simplifies the operation, and provides a new method for rapidly extracting anthocyanins from tea plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for culturing red callus of tea trees. Background Art

[0002] Red anthocyanins not only have strong coloring ability, but also have the effects of lowering fat, reducing weight, and anti-oxidation. They are often used as additives to develop functional foods, feeds, and cosmetics. Extracting anthocyanins from the red tissues of natural plants is a safe and effective measure, but this production method is limited by the season. The use of tissue culture technology to preserve red tissues can continuously provide raw materials for extracting anthocyanins. The explants used in conventional tissue culture methods are derived from nature and usually contain fungi, bacteria, and viruses. The operation is complicated, the survival rate is low, and the red callus obtained is uneven. Therefore, developing a new tissue culture method that is simple, easy to operate, and has a fast callus proliferation rate is an important measure to achieve a continuous supply of raw materials for extracting anthocyanins. Summary of the Invention

[0003] The purpose of the present invention is to provide a simple, easy-to-operate and rapid method for obtaining red callus tissue of tea trees, thereby realizing large-scale propagation of red callus tissue of tea trees.

[0004] The present invention provides a method for rapid propagation of red callus of Anji white tea, comprising the following steps:

[0005] A. Explant selection: young leaves of sterile and virus-free tissue culture seedlings of Anji white tea were selected as explants;

[0006] B. inducing callus tissue, inoculating the explant obtained in step A into an induction medium containing PVP to induce callus tissue;

[0007] C. Callus proliferation culture: inoculate the callus tissue induced in step B into a proliferation medium containing PVP, culture in the dark at 25-28° C., and obtain a large amount of callus tissue.

[0008] D. Red callus culture: The callus tissue obtained in step C is moved to a light-irradiated environment to obtain red callus tissue.

[0009] Preferably, the leaves in step A are leaves of sterile and virus-free tissue culture seedlings.

[0010] Preferably, the concentration of the antioxidant PVP in step B is 1.8 g / L -1 .

[0011] Preferably, the induction medium in step B is MS+6-BA4.0 mg·L -1 +IBA 1.5mg·L -1 +PVP1.8g·L -1 + sucrose 30g·L-1 + agar 8g·L -1 , pH 5.8, was used to induce callus tissue.

[0012] Preferably, the concentration of the antioxidant PVP in step C is 1.8 g / L -1 .

[0013] Preferably, the proliferation medium in step C is MS+TDZ 2.0 mg·L -1 +IBA0.2mg·L -1 +PVP 1.8g·L -1 + sucrose 30 gL -1 + agar 8·gL -1 , pH 5.8, used for callus proliferation culture.

[0014] Preferably, the culture conditions in step D are all light culture for 16 hours per day, with a light intensity of 90 μmol·m -2 ·s -1 Under the following conditions, the temperature is 25-28°C; incubate in the dark for 8 hours, the temperature is 23-25°C, and the relative humidity is 70%-85%.

[0015] The present invention provides a method for culturing and rapidly propagating red callus from Anji white tea, including induction and proliferation culture, eliminating the need for explant disinfection. This technology enables rapid and efficient propagation of red callus from Anji white tea, providing a large amount of high-quality raw material for extracting anthocyanins and materials for inducing the cultivation of new tea varieties. Therefore, it is highly practical and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the callus tissue of Anji white tea. DETAILED DESCRIPTION

[0017] The technical solutions provided by the present invention are described in detail below with reference to the embodiments. It should be understood that the protection scope of the present invention is not limited by the specific implementation methods.

[0018] Example 1

[0019] (1) Explant selection

[0020] The young leaves of sterile and virus-free tissue culture seedlings of Anji white tea were selected as explants.

[0021] (2) Induction of callus

[0022] The young leaves of the tissue culture seedlings were inoculated into callus induction medium for culture. The induction medium was MS + 6-BA 4.0 mg·L -1 +IBA 1.5mg·L -1 +PVP 1.8g·L-1 + sucrose 30g·L -1 + agar 8g·L -1 , pH 5.8, after culturing for 20 days, granular green callus tissue was dedifferentiated from the leaf edge, and the callus tissue induced for 30 days was selected for proliferation culture.

[0023] (3) Callus proliferation culture

[0024] The induced callus tissue was selected and placed on a proliferation culture medium for proliferation culture. The culture medium was MS + TDZ 2.0 mg·L -1 +PVP 1.8g·L -1 + sucrose 30g·L -1 + agar 8g·L -1 , pH 5.8, and a large amount of white callus was obtained after 20 days of dark culture.

[0025] (4) Induction of red callus

[0026] The white callus tissue was transferred to culture under light to obtain red callus tissue with uniform color.

[0027] The culture conditions in step (4) were 16 h of illumination per day, with a light intensity of 90 μmol·m -2 ·s -1 Under the following conditions, the temperature is 25-28°C; incubate in the dark for 8 hours, the temperature is 23-25°C, and the relative humidity is 70%-85%.

[0028] Example 2

[0029] (1) Explant selection

[0030] The young leaves of sterile and virus-free tissue culture seedlings of Anji white tea were selected as explants.

[0031] (2) Induction of callus

[0032] The young leaves of the tissue culture seedlings were inoculated into callus induction medium for culture. The induction medium was MS + 6-BA 4.0 mg·L -1 +IBA 1.5mg·L -1 +PVP 1.8g·L -1 + sucrose 30g·L -1 + agar 8g·L -1 , pH 5.8, after 20 days of culture, granular green callus tissue was dedifferentiated from the leaf edge, and the callus tissue induced 30 days later was selected for proliferation culture (Figure A).

[0033] (3) Callus proliferation culture

[0034] The induced callus tissue was selected and placed on a proliferation culture medium for proliferation culture. The culture medium was MS + TDZ 2.0 mg·L -1 +IBA0.2mg·L -1 +PVP 1.8g·L -1 + sucrose 30 g L -1 + agar 8·g L -1 , pH 5.8, and a large amount of white callus tissue was obtained after 20 days of dark culture (Figure B).

[0035] (4) Induction of red callus

[0036] The white callus was transferred to a light-cultured environment to obtain red callus with uniform color (Figure C).

[0037] The culture conditions in step (4) were 16 h of illumination per day, with a light intensity of 90 μmol·m -2 ·s -1 Under the following conditions, the temperature is 25-28°C; incubate in the dark for 8 hours, the temperature is 23-25°C, and the relative humidity is 70%-85%.

[0038] Comparative Example 1

[0039] A tissue culture method similar to Example 1, except that the proliferation medium is MS + TDZ 0.5 mg·L -1 +PVP1.8g·L -1 + sucrose 30g·L -1 + agar 8g·L -1 .

[0040] Comparative Example 2

[0041] A tissue culture method similar to Example 1, except that the proliferation medium is MS + TDZ 1.0 mg·L -1 +PVP 1.8g·L -1 + sucrose 30g·L -1 + agar 8g·L -1 .

[0042] Comparative Example 3

[0043] A tissue culture method similar to Example 2, except that the proliferation medium is MS + TDZ 2.0 mg·L -1 +IAA0.2mg·L -1 +PVP 1.8g·L -1 + sucrose 30 g L -1 + agar 8·gL -1 .

[0044] Comparative Example 4

[0045] A tissue culture method similar to Example 2, except that the proliferation medium is MS + TDZ 2.0 mg·L -1 +NAA0.2mg·L -1 +PVP 1.8g·L -1 + sucrose 30 g L -1 + agar 8·gL -1 .

[0046] Comparative Example 5

[0047] A tissue culture method similar to Example 1, except that the proliferation culture conditions are light culture for 16 hours and the light intensity is 90 μmol·m -2 ·s -1 Under the following conditions, the temperature is 25-28℃; incubate in the dark for 8h at a temperature of 23-25℃.

[0048] Comparative Example 6

[0049] A tissue culture method similar to Example 1, except that the proliferation medium is MS + TDZ 0.5 mg·L -1 +PVP1.8g·L -1 + sucrose 30g·L -1 + agar 8g·L -1 The proliferation culture conditions were light culture for 16 h, light intensity 90 μmol·m -2 ·s -1 Under the following conditions, the temperature is 25-28℃; incubate in the dark for 8h at a temperature of 23-25℃.

[0050] Comparative Example 7

[0051] A tissue culture method similar to Example 1, except that the proliferation medium is MS + TDZ 1.0 mg·L -1 +PVP1.8g·L -1 + sucrose 30g·L -1 + agar 8g·L -1 The proliferation culture conditions were light culture for 16 h, light intensity 90 μmol·m -2 ·s -1 Under the following conditions, the temperature is 25-28℃; incubate in the dark for 8h at a temperature of 23-25℃.

[0052] Comparative Example 8

[0053] A tissue culture method similar to Example 2, except that the proliferation culture conditions are light culture for 16 hours and the light intensity is 90 μmol·m -2 ·s -1Under the following conditions, the temperature is 25-28℃; incubate in the dark for 8h at a temperature of 23-25℃.

[0054] Comparative Example 9

[0055] A tissue culture method similar to Example 2, except that the proliferation medium is MS + TDZ 2.0 mg·L -1 +IAA0.2mg·L -1 +PVP 1.8g·L -1 + sucrose 30g·L -1 + agar 8g·L -1 The proliferation culture conditions were light culture for 16 h, light intensity 90 μmol·m -2 ·s -1 Under the following conditions, the temperature is 25-28℃; incubate in the dark for 8h at a temperature of 23-25℃.

[0056] Comparative Example 10

[0057] A tissue culture method similar to Example 2, except that the proliferation medium is MS + TDZ 2.0 mg·L -1 +NAA0.2mg·L -1 +PVP 1.8g·L -1 + sucrose 30g·L -1 + agar 8g·L -1 The proliferation culture conditions were light culture for 16 h, light intensity 90 μmol·m -2 ·s -1 Under the following conditions, the temperature is 25-28℃; incubate in the dark for 8h at a temperature of 23-25℃.

[0058] The results of observation and statistics on the use of explants to induce callus and promote callus proliferation in Examples 1-2 and Comparative Examples 1-10 showed that Example 2 had the best effect. The callus induced by IBA and 6-BA during primary culture under light conditions was relatively loose and green in color, with an induction rate of 99.43% ( Figure 1 Middle Figure A). The callus tissue on the proliferation culture under dark conditions appears white ( Figure 1 Figure B and Table 1), TDZ-induced callus tissue has a loose texture (Table 1); TDZ and IBA-induced callus tissue proliferation culture has a high proliferation multiple, loose texture, and white color. When the callus tissue obtained under dark conditions is transferred to light conditions, the white callus tissue turns red in about 10 days, and the color is relatively uniform ( Figure 1 C and Table 1); the color of callus tissues under constant light induction by TDZ, TDZ / IBA, TDZ / IAA, and TDZ / NAA were red and white, red and green, red and green, and green with a reddish tint, respectively, and the proliferation rate was low (Table 1).

[0059] Table 1 Red callus of Anji white tea

[0060]

[0061] The invention provides a method for culturing red tea callus using sterile, virus-free tissue culture seedling leaves, eliminating the need for explant disinfection and simplifying the process. Tea callus proliferates rapidly in the dark, and the red callus obtained by transferring the proliferated callus to light produces a more uniform color. Combined with a culture medium and culture conditions selected for the induction and propagation of Anji white tea callus, this method allows for the rapid propagation of red Anji white tea callus, providing a large amount of high-quality raw material for the rapid extraction of tea anthocyanins.

[0062] The above describes specific embodiments of the present invention. However, they are not intended to limit the present invention. Anyone familiar with the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for inducing red callus and proliferation using Anjibai tea leaves, characterized in that: The following steps are involved: A. Explant selection: Select young leaves of sterile and virus-free tissue culture seedlings of Anji white tea as explants; B. Inducing callus tissue: inoculate the explant obtained in step A into an induction medium containing PVP. After 20 days of induction, granular green callus tissue is differentiated. After 30 days of induction, the callus tissue is used for proliferation culture. C. Callus proliferation culture: The callus tissue induced in step B was inoculated into a proliferation medium containing PVP and cultured in the dark at 25-28°C for 20 days to obtain a large amount of white callus tissue; the proliferation medium was MS + TDZ 2.0 mg·L -1 + IBA0.2 mg·L -1 + PVP 1.8 g·L -1 + sucrose 30g·L -1 + agar 8g·L -1 , pH 5.8; D. Deep red callus culture: The white callus obtained in step C is transferred to a light-treated area for culture. After 10 days, a deep red callus with uniform color is obtained.

2. The method according to claim 1, wherein: The leaves in step A are young leaves of well-grown tissue culture seedlings.

3. The method according to claim 1, wherein: The concentration of antioxidant PVP in step B was 1.8 g·L -1 .

4. The method according to claim 1, wherein: The induction medium in step B is MS + 6-BA 4.0 mg·L -1 + IBA 1.5 mg·L -1 + PVP 1.8 g·L -1 + sucrose 30 g·L -1 + agar 8 g·L -1 , pH is 5.

8.

5. The method according to claim 1, wherein: The culture conditions in step D were all light-treated for 16 h per day, with a light intensity of 90 μmol·m -2 ·s -1 Under the following conditions, the temperature is 25-28℃; incubate in the dark for 8 h, the temperature is 23-25℃, and the relative humidity is 70%-85%.

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