A method for maintaining vitality of long-term subculture poplar tissue culture seedlings by using shuttle culture and its application

Abstract

The present invention discloses a method for maintaining the vitality of long-term subculture poplar tissue culture seedlings by shuttle culture, and belongs to the technical field of plant tissue culture. The present invention provides a method for maintaining the vitality of long-term subculture poplar tissue culture seedlings by shuttle culture, the method comprising first placing the long-term subculture poplar tissue culture seedlings in a rooting subculture medium for subculture three times, then transferring them to a double-layer rejuvenation medium for rejuvenation culture once, and finally transferring them to a rooting medium for rooting culture. The cultivation process of alternating rooting subculture and rejuvenation culture of the poplar tissue culture seedlings disclosed in the present invention solves the problem of growth decline of long-term subculture poplar tissue culture seedlings. The tissue culture seedlings cultivated by the method provided by the present invention root faster, grow more robustly, and have better quality than those cultivated by conventional subculture methods, laying a good foundation for genetic transformation, factory-based seedling cultivation and promotion of poplars.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method and application of maintaining vitality of long-term subculture poplar tissue culture seedlings by using shuttle culture. Background Art

[0002] Poplars belong to the genus Populus (Populus L.) of the Salicaceae family. There are five branches of poplars: white poplar, green poplar, black poplar, euphratica and large-leaf poplar. At present, species of all major branches of poplars can be rapidly propagated by tissue culture. However, compared with white poplar species, other species, especially black poplar species, will grow slowly, have browning leaf tips, and have difficulty taking root after three generations of subculture, making it difficult to subculture for a long time. At present, the main method of obtaining sterile seedlings after continuous disinfection of seedlings cultivated in the field is to maintain the growth vitality of poplar tissue seedlings, which is time-consuming and labor-intensive, and greatly affects the efficiency of poplar tissue culture and rapid propagation and the process of genetic transformation.

[0003] The main reasons for the aging of poplar tissue culture seedlings are as follows: First, the characteristics of the tree species. Generally speaking, the rooting rate of poplar in greenhouses and fields is low, but the tissue culture performance is excellent, it is easy to root, and it can still maintain a high vitality under long-term subculture. On the contrary, the rooting rate of cuttings is high, but the vitality is easy to decline under tissue culture conditions. From the explant to the tissue culture, rooting difficulties occur after 3 generations of subculture, the leaf tips turn brown and curl, the leaves become smaller, the growth is slow, and even yellowing and death occur; second, the tissue culture conditions. The subculture medium for poplar is a medium based on the MS basic medium with the addition of rooting-related plant hormones, while the subculture medium for black poplar is a medium based on the WPM medium with the addition of rooting-related plant hormones. If the black poplar is changed to an MS-based medium, it will age rapidly; third, the physiological state of the explant when it is sampled for tissue culture. If the explants are taken and disinfected for tissue culture in the peak growth season, the tissue culture seedlings grow robustly, while the explants taken near the dormant period have slow growth and are very easy to age.

[0004] Therefore, how to provide a method for maintaining the vitality of long-term subcultured poplar tissue culture seedlings is of great significance for solving the problem of growth decline of long-term subcultured poplar tissue culture seedlings. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application of maintaining the vitality of long-term subculture poplar tissue culture seedlings by shuttle culture, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A culture medium for maintaining the vitality of long-term subculture poplar tissue culture seedlings, the culture medium comprising a rooting subculture medium and a double-layer rejuvenation medium; the components of the rooting subculture medium are: WPM+0.02mg / L IBA+25g sucrose+5.0g / L agar; the double-layer rejuvenation medium is divided into an upper and lower layer; the components of the upper culture medium are: MS+30g sucrose+5.0g / L agar+0.005mg / L IAA; the components of the lower culture medium are: MS+30g sucrose+5.0g / L agar+0.001mg / LGA3.

[0008] Furthermore, the pH of the rooting subculture medium and the double-layer rejuvenation medium are both 5.80.

[0009] Furthermore, the culture conditions of the rooting subculture medium and the double-layer rejuvenation medium are: the day / night photoperiod is 16h / 8h, the light intensity is 3000Lux, and the day / night temperature is 28°C / 26°C.

[0010] Furthermore, the culture medium also includes a rooting culture medium, and the components of the rooting culture medium are: WPM+0.01 mg / LIBA+25 g sucrose+5.0 mg / L agar.

[0011] Furthermore, the pH of the rooting medium is 5.80; the culture conditions of the rooting medium are: the day / night photoperiod is 16h / 8h, the light intensity is 3000Lux, and the day / night temperature is 28°C / 26°C.

[0012] Technical Solution 2: A method for maintaining the vitality of long-term subcultured poplar tissue culture seedlings using shuttle culture, the method comprising first placing the long-term subcultured poplar tissue culture seedlings in a rooting subculture medium for subculture three times, then transferring them to a double-layer rejuvenation medium for rejuvenation culture once, and finally transferring them to a rooting medium for rooting culture.

[0013] Furthermore, the components of the rooting subculture medium are: WPM+0.02mg / L IBA+25g sucrose+5.0g / L agar; the double-layer rejuvenation medium is divided into upper and lower layers; the components of the upper culture medium are: MS+30g sucrose+5.0g / L agar+0.005mg / L IAA; the components of the lower culture medium are: MS+30g sucrose+5.0g / L agar+0.001mg / L GA3; the components of the rooting medium are: WPM+0.01mg / L IBA+25g sucrose+5.0mg / L agar.

[0014] Furthermore, the pH of the rooting subculture medium, the double-layer rejuvenation medium and the rooting medium are all 5.80.

[0015] Furthermore, the culture conditions of the rooting subculture medium, the double-layer rejuvenation medium and the rooting medium are: the day / night photoperiod is 16h / 8h, the light intensity is 3000Lux, and the day / night temperature is 28°C / 26°C.

[0016] Technical solution three: Application of the culture medium in preventing aging of long-term subcultured poplar tissue culture seedlings and maintaining vitality of long-term subcultured poplar tissue culture seedlings.

[0017] The present invention discloses the following technical effects:

[0018] The experimental results of the present invention show that in the shuttle culture mode of three subcultures and one rejuvenation, the long-term subcultured poplar tissue culture seedlings grow robustly and do not show aging phenomenon, and the cultivation mode is easy to operate. By adding only a trace amount of plant hormones to the double-layer rejuvenation medium of the present invention, the IBA content in the rooting medium is halved, and the method provided by the present invention can reduce the cost of long-term subculture preservation of tissue culture plant materials.

[0019] According to the present invention Figure 1 and 2 It can be obtained that the tissue culture seedlings obtained by the shuttle culture of the present invention solve the problem of growth decline caused by long-term subculture. The tissue culture seedlings obtained by the method provided by the present invention have faster rooting, stronger growth and better quality than those cultivated by the conventional subculture method, thus laying a good foundation for the genetic transformation, factory-based seedling cultivation and promotion of poplar. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 Comparison of rooting conditions of tissue culture seedlings of Bofeng No.3 Poplar without shuttle culture (A) and after shuttle culture (B);

[0022] Figure 2 Comparison of the growth of the tissue culture seedlings of Bofeng No. 3 Poplar without shuttle culture (A) and after shuttle culture (B). DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] Shuttle culture of tissue culture seedlings of Bofeng No.3 Populus by long-term subculture

[0030] 1. Rooting and subculture of tissue culture seedlings

[0031] In a clean bench, the leafy stem tip with a tip of 3 cm of the long-term subcultured Bofeng No. 3 poplar tissue culture seedling was cut and transferred to a rooting subculture medium for culture for about 25 days, until the first subculture of the tissue culture seedling 1 with a seedling height of 5 cm and a root length of about 10 cm was obtained; the leafy stem tip with a tip of 3 cm of the tissue culture seedling 1 was transferred to a newly configured rooting subculture medium for culture for about 25 days, and the second subculture of the tissue culture seedling 2 with a seedling height of 5 cm and a root length of about 10 cm was obtained; the leafy stem tip with a tip of 3 cm of the tissue culture seedling 2 was transferred to a newly configured rooting subculture medium for culture for about 25 days, and the third subculture of the tissue culture seedling 3 with a seedling height of 5 cm and a root length of about 10 cm was obtained.

[0032] The formula of rooting subculture medium is: WPM+0.02mg / L IBA+25g sucrose+5.0g / L agar (pH 5.80); the preparation method is: sterilize the culture medium at 120℃ high pressure for 15min, then cool it to 40℃, and divide it into sterile 400ml plastic tissue culture bottles, about 50ml per bottle; the culture conditions are: the day / night photoperiod is 16h / 8h, the light intensity is 3000Lux, and the day / night temperature is 28℃ / 26℃.

[0033] 2. Rejuvenation and rooting of tissue culture seedlings

[0034] The tissue culture seedlings 3 obtained in step 1 are placed on a clean bench, the stem tips above the fifth leaf from the root are cut off, and the roots of the tissue culture seedlings 3 are cut off, leaving 1 cm of old roots, and then the tissue culture seedlings 3 with the stem tips and part of the roots cut off are transferred to a double-layer rejuvenation medium for rejuvenation culture. After culturing for 10 days, when the axillary buds grown from the bases of the fourth and fifth leaves are about 2 cm in length, they are cut off from the base and transferred to a rooting medium for rooting culture.

[0035] The culture conditions for rooting culture and double-layer rejuvenation culture are: the day / night photoperiod is 16h / 8h, the light intensity is 3000Lux, and the day / night temperature is 28℃ / 26℃; the rooting culture medium composition is: WPM+0.01mg / L IBA+25g sucrose+5.0mg / L agar (pH 5.80); the double-layer rejuvenation culture medium is divided into two layers, both 1cm thick, the upper culture medium composition is: MS+30g sucrose+5.0g / L agar+0.005mg / L IAA (pH 5.80), the lower culture medium composition is: MS+30g sucrose+5.0g / L agar+0.001mg / L GA3 (pH 5.80); the preparation method of the double-layer rejuvenation medium is as follows: cool the lower culture medium sterilized at 120°C for 15 minutes to about 40°C, add filter-sterilized GA3 to a final concentration of 0.001 mg / L, divide it into 400 ml plastic tissue culture bottles, place it in a cool place for 1 day, and after the culture medium is completely solidified, cool the upper culture medium sterilized at 120°C for 15 minutes to about 40°C, divide it into tissue culture bottles containing the lower culture medium, and obtain the double-layer rejuvenation medium.

[0036] The rooting conditions of the tissue culture seedlings after the shuttle culture treatment (the mode of rooting subculture three times followed by rejuvenation culture in Example 1 of the present invention) are as follows: Figure 1 As shown in B, the growth of tissue culture seedlings after shuttle culture treatment is as follows Figure 2 As shown in B. The present invention measured the seedling height, rooting rate and root length of 30 Bofeng No. 3 Poplar tissue culture seedlings treated with shuttle culture (Table 1).

[0037] Comparative Example 1

[0038] Detailed culture method of tissue culture seedlings without shuttle culture treatment (conventional subculture): cut the leafy stem tip of the Bofeng No. 3 Poplar tissue culture seedlings at the tip 3 cm in the clean bench, transfer to the rooting subculture medium, and subculture once every 25 days. The tissue culture seedlings in Comparative Example 1 are conventionally subcultured Bofeng No. 3 Poplar tissue culture seedlings.

[0039] The formula of rooting subculture medium is: WPM+0.02mg / L IBA+25g sucrose+5.0g / L agar (pH 5.80); the preparation method is: sterilize the culture medium at 120℃ high pressure for 15min, then cool it to 40℃, and divide it into sterile 400ml plastic tissue culture bottles, about 50ml per bottle; the culture conditions are: 16h / 8h (day / night) photoperiod, light intensity 3000Lux, temperature 28℃ / 26℃ (day / night).

[0040] The present invention measured the seedling height, rooting rate and root length of 30 conventionally subcultured Bofeng No. 3 Poplar tissue culture seedlings (Table 1).

[0041] By comparison, it was found that the Bofeng No. 3 Poplar tissue culture seedlings treated with shuttle culture grew faster and stronger than the conventionally subcultured tissue culture seedlings. Specifically, the rooting rate, root length and seedling height were significantly higher than those of the conventionally subcultured tissue culture seedlings, and the fibrous roots were well developed.

[0042] Table 1 Growth of tissue culture seedlings of Bofeng No. 3 Poplar after shuttle culture and conventional subculture

[0043]

[0044]

[0045] Figure 1 Bofeng No. 3 was not cultured by shuttle ( Figure 1 Comparison of rooting of tissue culture seedlings treated with shuttle culture in Example 1 ( Figure 1 B) in; Figure 2 Bofeng No. 3 was not cultured by shuttle ( Figure 2 A) and the growth of tissue culture seedlings treated with shuttle culture in Example 1 ( Figure 2 B) According to Figure 1 and 2 It can be seen that the tissue culture seedlings obtained by the shuttle culture of the present invention solve the problem of growth decline caused by long-term subculture.

[0046] Compared with conventional subculture methods, the tissue culture seedlings obtained by the method provided by the present invention have faster rooting, stronger growth and better quality, thus laying a good foundation for genetic transformation, factory-based seedling cultivation and promotion of poplars.

[0047] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A culture medium for maintaining the vitality of long-term subculture poplar tissue culture seedlings, characterized in that: The culture medium includes a rooting subculture medium, a double-layer rejuvenation medium and a rooting medium; The components of the rooting subculture medium are: WPM+0.02mg / L IBA+25g / L sucrose+5.0g / L agar; The double-layer rejuvenation medium is divided into two layers, the upper layer medium components are: MS + 30g / L sucrose + 5.0g / L agar + 0.005mg / L IAA; the lower layer medium components are: MS + 30g / L sucrose + 5.0g / L agar + 0.001mg / L GA3; The rooting medium comprises: WPM+0.01 mg / L IBA+25 g / L sucrose+5.0 g / L agar; The pH of the rooting subculture medium, the double-layer rejuvenation medium and the rooting medium are all 5.80; The culture conditions of the rooting subculture medium, the double-layer rejuvenation medium and the rooting medium are: the day / night photoperiod is 16h / 8h, the light intensity is 3000Lux, and the day / night temperature is 28°C / 26°C.

2. A method for maintaining the vitality of long-term subculture poplar tissue culture seedlings using shuttle culture, characterized in that: The method comprises placing the long-term subcultured poplar tissue culture seedlings in a rooting subculture medium for subculture three times, then transferring to a double-layer rejuvenation medium for rejuvenation culture once, and finally transferring to a rooting medium for rooting culture; the components of the rooting subculture medium are: WPM+0.02mg / L IBA+25g / L sucrose+5.0g / L agar; the double-layer rejuvenation medium is divided into an upper and lower layer; the components of the upper layer medium are: MS+30g / L sucrose+5.0g / L agar+0.005mg / L IAA; the components of the lower layer medium are: MS+30g / L sucrose+5.0g / L agar+0.001mg / L GA3; the components of the rooting medium are: WPM+0.01mg / L IBA+25g / L sucrose+5.0g / L agar; The pH of the rooting subculture medium, the double-layer rejuvenation medium and the rooting medium are all 5.80; The culture conditions of the rooting subculture medium, the double-layer rejuvenation medium and the rooting medium are: the day / night photoperiod is 16h / 8h, the light intensity is 3000Lux, and the day / night temperature is 28°C / 26°C.

3. Use of the culture medium according to claim 1 in preventing aging of long-term subcultured poplar tissue culture seedlings and maintaining the vitality of long-term subcultured poplar tissue culture seedlings.

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