Culture method for inhibiting vitrification of tissue culture seedlings

Abstract

The invention belongs to the technical field of plant tissue culture, and discloses a culture method for inhibiting vitrification of tissue culture seedlings, which comprises the following steps: S1, on the basis of an MS basal culture medium, adding mannitol and an MES buffer agent to prepare an anti-vitrification culture medium; s2, adventitious buds of tissue culture seedlings to be cultured are taken and placed in the anti-vitrification culture medium prepared in the step S1 to be cultured, and the culture environment is that the pH value is 5.8-6.2, the culture temperature is 23-27 DEG C, and the illumination intensity is 1000-2000 lx. The method is low in cost, easy and convenient to operate and suitable for large-scale tissue culture seedling raising of German iris and rhododendron, and the vitrification rate is remarkably reduced to 10% or below.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and particularly relates to a culture method for inhibiting vitrification of tissue-cultured seedlings. Background Art

[0002] Vitrification is a unique physiological disorder phenomenon in plant tissue culture, manifested as test-tube seedlings being semi-transparent or water-soaked, with wrinkled, fragile and easily broken leaves, swollen and dwarf stems; anatomically, it is manifested as non-functional stomata, lack of cutin layer, only spongy tissue without palisade tissue. Such seedlings have a high water content in the body, but low dry matter contents such as chlorophyll and protein, and significantly reduced photosynthetic ability and enzyme activity. Vitrified seedlings are difficult to subculture and have extremely low transplanting survival rates. The vitrification of tissue-cultured seedlings is closely related to the imbalance of the medium osmotic pressure, improper hormone ratio and environmental conditions.

[0003] In view of the vitrification of tissue-cultured seedlings, traditional methods alleviate vitrification by adjusting the hormone ratio (such as reducing cytokinin) or increasing the light intensity, but this easily leads to a decrease in the proliferation rate or an increase in cost. Specific medium formulations and culture methods for the vitrification of tissue-cultured seedlings such as Iris germanica and Rhododendron are not disclosed in the existing literature. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a culture method for inhibiting vitrification of tissue-cultured seedlings. The present invention aims to inhibit the vitrification of tissue-cultured seedlings without increasing costs and affecting the proliferation rate.

[0005] To achieve the above purpose, the present invention provides a culture method for inhibiting vitrification of tissue-cultured seedlings, including the following steps:

[0006] S1. Prepare an anti-vitrification medium;

[0007] Preparation process: Based on the MS basal medium, add mannitol and MES buffer to obtain the anti-vitrification medium;

[0008] S2. Place the adventitious buds of the tissue-cultured seedlings to be cultivated in the anti-vitrification medium prepared in step S1 for cultivation.

[0009] Further, in step S1, the MS basal medium: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L.

[0010] Further, in the anti-vitrification medium, the concentration of mannitol is 1 - 2 g / L, and the concentration of MES buffer is 1 - 2 g / L.

[0011] Further, in the anti-vitrification medium, the concentration of mannitol is 1 g / L, and the concentration of MES buffer is 1 g / L.

[0012] Further, in the step S2, the culture environment for adventitious buds of tissue-cultured seedlings is as follows: pH value is 5.8 - 6.2, culture temperature is 23 - 27 °C, and light intensity is 1000 - 2000 lx.

[0013] Further, in the step S2, the culture time of adventitious buds of tissue-cultured seedlings in the anti-vitrification medium is 30 days.

[0014] Further, the tissue-cultured seedlings are tissue-cultured seedlings of Iris germanica or Rhododendron.

[0015] Beneficial effects:

[0016] 1. In the anti-vitrification medium prepared in the present invention, mannitol and MES buffer are added. The concentration of mannitol is 1 - 2 g / L, which is used to adjust the osmotic pressure and inhibit excessive water absorption of cells; the concentration of MES buffer (2-(N-morpholino)ethanesulfonic acid) is 1 - 2 g / L, which is used to stabilize the pH value (5.8 - 6.2) of the medium and reduce metabolic toxicity.

[0017] 2. The method of the present invention can reduce the vitrification rate of tissue-cultured seedlings of Iris germanica and Rhododendron to less than 10%.

[0018] 3. The method of the present invention can improve the robustness of Iris germanica seedlings and Rhododendron seedlings, and the transplanting survival rate is over 95%.

[0019] 4. The culture method of the present invention is simple to operate, without additional equipment investment, and is suitable for large-scale production.

[0020] Other advantages, objectives, and features of the present invention will be described to some extent in the subsequent description, and to some extent, based on the study of the following text, will be obvious to those skilled in the art or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following description. Specific embodiments

[0021] To make the technical solutions, advantages, and objectives of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the attached tables of the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of this application.

[0022] The present invention provides a culture method for inhibiting vitrification of tissue-cultured seedlings, including the following steps:

[0023] S1. Prepare an anti-vitrification medium;

[0024] Preparation process: Based on the MS basal medium, mannitol and MES buffer are added to obtain the anti-vitrification medium;

[0025] Among them, the concentration of mannitol is 1 - 2 g / L, and the concentration of MES buffer is 1 - 2 g / L;

[0026] MS basal medium: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L;

[0027] S2. Take the adventitious buds of the tissue culture seedlings to be cultivated and place them in the anti-vitrification medium prepared in step S1 for cultivation;

[0028] Among them, the cultivation environment of the adventitious buds of the tissue culture seedlings in the anti-vitrification medium is: pH value 5.8 - 6.2, cultivation temperature 23 - 27 °C, light intensity 1000 - 2000 lx; the cultivation time of the adventitious buds of the tissue culture seedlings is 30 days.

[0029] Example 1

[0030] This example provides a cultivation method for inhibiting vitrification of tissue culture seedlings of Iris germanica, including the following steps:

[0031] S1. Based on the MS basal medium, mannitol and MES buffer are added to obtain the anti-vitrification medium;

[0032] S2. Take the adventitious buds of Iris germanica and place them in the anti-vitrification medium prepared in step S1 for cultivation;

[0033] Among them, the cultivation environment of the adventitious buds of Iris germanica in the anti-vitrification medium is: pH value 6, cultivation temperature 25 °C, light intensity 1500 lx, 12 h / d; the cultivation time of the adventitious buds of Iris germanica is 30 days.

[0034] In this example, 8 groups of experiments (medium numbers M0 - M7) were carried out. The basal medium for each group of experiments is MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L. Different concentrations of mannitol and MES are added. 10 clusters of adventitious buds are inoculated in each bottle, with 3 replicates. After 5 weeks, the vitrification rate is counted and the state of the seedlings is observed. See Table 1 for details.

[0035] Table 1 Summary of the cultivation of tissue culture seedlings of Iris germanica

[0036]

[0037] Result analysis: From the experimental data in Table 1, it can be seen that when the concentrations of added mannitol and MES are 1 - 2 g / L, the state of the seedlings is: the seedlings are green and robust. Among them, when the concentrations of added mannitol and MES are 1 g / L, the vitrification rate of tissue-cultured Iris germanica seedlings is only 10.00%. The seedlings have well-developed roots, dark green leaves, and a high survival rate after transplantation.

[0038] Example 2

[0039] This example provides a culture method for inhibiting the vitrification of tissue-cultured Rhododendron seedlings, which includes the following steps:

[0040] S1. Based on the MS basal medium, add mannitol and MES buffer to obtain the anti-vitrification medium.

[0041] S2. Place the adventitious buds of Rhododendron in the anti-vitrification medium prepared in step S1 for culture.

[0042] Among them, the culture environment of the adventitious buds of Rhododendron in the anti-vitrification medium is: pH value 6, culture temperature 25 °C, light intensity 1500 lx, 12 h / d; the culture time of the adventitious buds of Iris germanica is 30 days.

[0043] In this example, 6 groups of experiments (culture medium numbers M0 - M5) were carried out. The basal medium for each group of experiments is MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L. Different concentrations of mannitol and MES were added. 10 clusters of seedlings were inoculated in each bottle, with 3 replicates in total. After 5 weeks, the vitrification rate was counted and the state of the seedlings was observed. The specific results are shown in Table 2.

[0044] Table 2 Summary of the culture situation of tissue-cultured Rhododendron seedlings

[0045]

[0046]

[0047] Result analysis: From the experimental data in Table 2, it can be seen that when the concentrations of added mannitol and MES are 1 - 2 g / L, the state of the seedlings is: the seedlings are green and robust. Among them, when the concentrations of added mannitol and MES are both 1 g / L, and when the added mannitol is 2 g / L and the MES concentration is 0 g / L, the vitrification rate of tissue-cultured Rhododendron seedlings is 0. The seedlings have well-developed roots, dark green leaves, and a high survival rate after transplantation.

[0048] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the present technical solution, and they should all be covered within the protection scope of the present invention.

Claims

1. A cultivation method for inhibiting vitrification of tissue-cultured seedlings, characterized in that, It includes the following steps: S1. Prepare an anti-vitrification culture medium; Preparation process: Based on the MS basal medium, add mannitol and MES buffer to obtain the anti-vitrification culture medium; S2. Take the adventitious buds of the tissue culture seedlings to be cultivated and place them in the anti-vitrification culture medium prepared in step S1 for cultivation.

2. The culture method for inhibiting vitrification of tissue-cultured seedlings according to claim 1, characterized in that: In step S1, the MS basal medium: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L.

3. A cultivation method for inhibiting vitrification of tissue culture seedlings according to claim 2, characterized in that: In the anti-vitrification culture medium, the concentration of mannitol is 1 - 2 g / L, and the concentration of MES buffer is 1 - 2 g / L.

4. A cultivation method for inhibiting vitrification of tissue-cultured seedlings according to claim 3, characterized in that: In the anti-vitrification culture medium, the concentration of mannitol is 1 g / L, and the concentration of MES buffer is 1 g / L.

5. A cultivation method for inhibiting vitrification of tissue culture seedlings according to claim 4, characterized in that: In step S2, the culture environment for the adventitious buds of the tissue culture seedlings is: pH value 5.8 - 6.2, culture temperature 23 - 27 °C, light intensity 1000 - 2000 lx.

6. The culture method for inhibiting vitrification of tissue-cultured seedlings according to claim 5, characterized in that: In step S2, the culture time of the adventitious buds of the tissue culture seedlings in the anti-vitrification culture medium is 30 days.

7. A cultivation method for inhibiting vitrification of tissue culture seedlings according to claim 6, characterized in that: The tissue culture seedlings are tissue culture seedlings of Iris germanica or Rhododendron.

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