Method for preserving pear tissue culture seedlings for long time

Abstract

The invention discloses a long-term preservation method for pear tissue culture seedlings. According to the main method, tissue culture seedlings which are just inoculated to a subculture medium are placed in a plant incubator at 10 DEG C to be cultured for 6 months, and the purpose of reducing the subculture frequency is achieved in a low-temperature delayed growth mode. The tissue culture seedlings after low-temperature preservation can be normally subcultured and propagated, so that the method can effectively prolong the subculture period of the pear tissue culture seedlings, and has great significance in long-term preservation of pear germplasm resources in the form of the tissue culture seedlings and genetic transformation and germplasm innovation by utilizing the tissue culture seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of long-term preservation of pear germplasm resources, and particularly relates to a method for long-term low-temperature preservation of pear tissue culture seedlings. Background Art

[0002] Pear (scientific name: Pyrus L.) is a plant of the genus Pyrus in the Rosaceae family. It has high economic and nutritional value and is widely cultivated around the world. Pear originated in China and is the third largest cultivated fruit tree in my country. However, the existing varieties in my country are mainly traditional local varieties, which lack comprehensive quality and cannot meet people's needs. This urgently requires breeders to speed up the cultivation of new varieties. Germplasm resources are the material basis for breeding work, and the country attaches great importance to the preservation and utilization of germplasm resources. Therefore, how to give full play to my country's advantages as one of the origins of pears and better preserve germplasm resources is an important basic work.

[0003] There are two main ways to preserve pear germplasm resources. One is to establish a germplasm resource garden for preservation. However, since pear germplasm resources are abundant and are perennial woody plants, this preservation method will not only occupy a large amount of land resources, but also often require a lot of manpower for fine cultivation management to protect them from natural conditions and pests and diseases; the second is the in vitro preservation of germplasm resources, that is, the tissue culture seedlings obtained by disinfection and primary culture of pear explants are subcultured to preserve germplasm resources in the form of tissue culture seedlings. However, subculture still requires a lot of manpower and time. Not only does it need to be subcultured every 30-40 days, but in this process, bacteria or fungi often contaminate the tissue culture seedlings due to frequent subculture, resulting in the loss of germplasm resources. At the same time, frequent subculture may lead to the variation of somatic asexual lines, thereby leading to the loss of original germplasm.

[0004] There are studies on methods to better preserve germplasm resources by extending the storage time of tissue culture seedlings. The study on the preservation of pear test tube seedling germplasm resources pointed out that the storage time of tissue culture seedlings can be extended by adding PP333 (delaying plant growth) and mannitol (reducing water evaporation in the culture medium), but this method of adding growth inhibitors to the culture medium requires strict control of the concentration, otherwise it will cause deformity and death of the test tube seedlings. Summary of the invention

[0005] The purpose of the present invention is to provide a method for long-term preservation of pear tissue culture seedlings, reduce the number of subcultures, save manpower and material resources, and better preserve pear germplasm resources.

[0006] In order to achieve the above object, the specific technical solution of the present invention is as follows:

[0007] A method for long-term preservation of pear tissue culture seedlings, comprising the following steps:

[0008] (1) Take the tissue culture seedlings of Pear Autumnia and Confrens, and cut the explants used for subculture with a scalpel;

[0009] (2) inoculating the explants cut in step (1) into a corresponding pear tissue culture seedling subculture medium;

[0010] (3) placing the subculture medium containing the explants in step (2) in a plant incubator for 6 months; wherein the incubator temperature is 10±1° C., the photoperiod is 16 / 8 h, and the light intensity is 1300-1800 lx;

[0011] As a preferred embodiment of the present invention, the formula of the subculture medium for the Qiuzi pear tissue culture seedlings in step (2) is: 4.74g / LMS+2.0mg / L6-BA+0.2mg / LIBA+30g / L sucrose+7.5g / L agar; the formula of the subculture medium for the Confrens tissue culture seedlings is: 4.74g / LMS+0.5mg / L6-BA+0.1mg / LIBA+30g / L sucrose+7.5g / L agar; the specific method of medium preparation is: measure 1L of distilled water with a measuring cylinder, pour it into a conical flask and add 4.74g MS and 30g sucrose, stir continuously to make it completely dissolved, add corresponding hormones, adjust the solution pH to 5.8-6.0 with a pH meter and 1M potassium hydroxide aqueous solution, finally add 7.5g agar, put the conical flask containing the unsterilized culture medium into a high-temperature pressure sterilizer, sterilize it at 121°C for 20min, and after sterilization, divide it into tissue culture bottles for cooling.

[0012] As a preferred embodiment of the present invention, the subculture cycle of the Qiuzi Pear and Confrenus tissue culture seedlings used as explant sources in step (1) is 40 days.

[0013] As a preferred embodiment of the present invention, the explants cut in step (1) only retain the stem segments at the growing point and 0.5-1.0 cm below and 2-4 young leaves at the top, and the growing point is alive and has not turned brown or died.

[0014] As a preferred embodiment of the present invention, the hormones used in the Qiuzi pear tissue culture seedling culture medium and the Confrenus tissue culture seedling culture medium in step (2) are prepared in advance and stored in a refrigerator at 4°C. The specific preparation method is: weigh an appropriate amount of hormone powder, dissolve it with a small amount of 1M potassium hydroxide aqueous solution, and add distilled water to make up to 1 mg / ml.

[0015] As a preferred embodiment of the present invention, the explants described in step (2) are inoculated in a manner that the stem base is vertically inserted into the culture medium.

[0016] As a preferred embodiment of the present invention, the incubator temperature in step (3) is 10°C, the photoperiod is 16 / 8h, and the light intensity is 1500lx.

[0017] As a preferred embodiment of the present invention, the method further comprises the following step of restoring the vitality of the tissue culture seedlings: taking the tissue culture seedlings in step (3) out of the incubator, re-cutting and inoculating them in a newly configured subculture medium and culturing them for 40 days.

[0018] As a further preferred embodiment of the present invention, after the tissue culture seedlings are taken out of the incubator in step (3), they need to be re-cut into stem segments 0.5-1.0 cm below the growth point with the help of a scalpel and 2-4 leaves at the top are retained.

[0019] As a further preferred embodiment of the present invention, the tissue culture seedlings in step (3) are re-cut and inoculated into a newly configured subculture medium, and cultured in a plant growth chamber for 40 days, wherein the temperature of the plant growth chamber is 25±1° C. and the photoperiod is 16 / 8 h.

[0020] The present invention adopts the above technical solution to preserve pear tissue culture seedlings, which has the following advantages:

[0021] (1) The components used in the subculture medium are all commonly used components for plant tissue culture. No additional components need to be added to the subculture medium. The method for preparing the subculture medium is simple and the cost is low.

[0022] (2) The plant incubator used in the low-temperature preservation process is a conventional laboratory equipment, and the external conditions of the preservation process can be easily achieved using a plant incubator.

[0023] (3) During low-temperature storage, the activity of bacteria and fungi is reduced, which greatly reduces the possibility of contamination during subculture. The survival rate of tissue culture seedlings can reach 100%

[0024] (4) After cryopreservation, the method for restoring the vitality of tissue culture seedlings is simple. It only requires regular cutting, inoculation on subculture medium and placement in a plant growth room. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The state of Qiuzi pear tissue culture seedlings after cutting

[0026] Figure 2 Qiuzi pear tissue culture seedlings stored at 4°C for 10 days

[0027] Figure 3 Qiuzi pear tissue culture seedlings stored at 4℃ for 1 month

[0028] Figure 4 Qiuzi pear tissue culture seedlings stored at 10℃ for 3 months

[0029] Figure 5 Qiuzi pear tissue culture seedlings stored at 10℃ for 6 months

[0030] Figure 6 After six months of cryopreservation, the Qiuzi pear tissue culture seedlings were cut and subcultured in the plant growth room for 40 days.

[0031] Figure 7 The state of Confluence tissue culture seedlings after cutting

[0032] Figure 8 Confrens tissue culture seedlings stored at 10℃ for 3 months

[0033] Fig. 9 Confrens tissue culture seedlings stored at 10°C for 6 months

[0034] Fig.10 After six months of cryopreservation, the Confrens tissue culture seedlings were recut and subcultured in the plant growth room for 40 days. Specific implementation methods

[0036] In order to more accurately describe the present invention, the present invention is illustrated by the following implementation cases.

[0037] The culture medium used in the present invention is shown in the following table

[0038] Table 1: Formula of subculture medium for Qiuzi pear

[0039] Components content MS medium 4.74g / L 1.0mg / ml 6-BA 2ml / L 1.0mg / ml IBA 200μl / L sucrose 30g / L Agar 7.5g / L

[0040] Table 2: Confrance culture medium formula

[0041] Components content MS medium 4.74g / L 1.0mg / ml 6-BA 0.5ml / L 1.0mg / ml IBA 100μl / L sucrose 30g / L Agar 7.5g / L

[0042] Example 1

[0043] (1) Preparation of Qiuzi Pear Explants

[0044] Take out the Qiuzi pear tissue culture seedlings that have been subcultured for 40 days, and use a sterilized scalpel in a clean bench to remove excess stem segments and leaves, leaving only the stem segments 0.5-1.0 cm below the growth point and 2-4 tender leaves at the top. Figure 1 .

[0045] (2) Inoculation of Qiuzi Pear Explants

[0046] The culture medium was configured according to the formula of the subculture medium for the tissue culture seedlings of Qiuzi Pear shown in Table 1. The specific method of the culture medium configuration is as follows: 1L of distilled water was measured with a measuring cylinder, poured into a conical flask, and 4.74g MS and 30g sucrose were added, and the mixture was stirred continuously to completely dissolve, and the corresponding hormones were added. The pH of the solution was adjusted to 5.8-6.0 with a pH meter and a 1M potassium hydroxide aqueous solution (acidity is conducive to the growth of tissue culture seedlings, and alkalinity is conducive to the coagulation of the culture medium. In the actual operation process, the pH within this range does not affect the subculture and preservation of tissue culture seedlings), and finally 7.5g of agar was added, and the conical flask containing the unsterilized culture medium was placed in a high-temperature autoclave, and sterilized at 121°C for 20min. After the sterilization was completed, it was divided into tissue culture bottles for cooling. After the culture medium was cooled, the Qiuzi Pear explants were vertically inoculated in the subculture medium with the stem base downward.

[0047] (3) Storage of Qiuzi Pear Tissue Culture Seedlings at Low Temperatures of 4°C and 10°C

[0048] The inoculated Qiuzi pear tissue culture seedlings were placed in a plant incubator, and the temperature of the incubator was set to 4℃ and 10℃, the photoperiod was 16 / 8h, and the light intensity was 1500lx. The growth status of the tissue culture seedlings was observed. The results showed that the Qiuzi pear tissue culture seedlings were strongly stressed at 4℃ and rapidly accumulated secondary metabolites in response to adversity, such as anthocyanins. Figure 2 As the storage time at 4°C increased, the vitality of the Qiuzi pear tissue culture seedlings decreased and they died. Figure 3 At 10℃, the Qiuzi pear seedlings did not show obvious stress phenotypes, and their growth rate dropped significantly, which enabled them to achieve the purpose of low-temperature preservation. Figure 4 As shown; the status of the two groups of seedlings at 6 months Figure 5 shown.

[0049] (4) Preparation of Confrenus explants

[0050] The preparation of Confrens explants was the same as that of Akiko Pyrus explants, e.g. Figure 7 .

[0051] (5) Inoculation of Confrenus explants

[0052] The culture medium was prepared and the explants were inoculated according to the formula of the subculture medium for the Confrens tissue culture seedlings shown in Table 2. The preparation method of the culture medium for the Confrens tissue culture seedlings and the method of explant inoculation were the same as those for the Qiuzi pear tissue culture seedlings.

[0053] (6) Storage of Confluence tissue culture seedlings at 10°C

[0054] The inoculated tissue culture seedlings were placed in a plant incubator, the temperature of the incubator was set to 10°C, the photoperiod was 16 / 8h, and the light intensity was 1500lx, and the growth status of the tissue culture seedlings was observed. The results showed that the Confrens tissue culture seedlings did not show obvious stress phenotypes at a low temperature of 10°C, and the growth rate dropped significantly, which could achieve the purpose of low temperature preservation. At 3 months, the status of the Confrens tissue culture seedlings was as follows Figure 8 As shown; the status of the two groups of seedlings at 6 months Fig. 9 shown.

[0055] (7) Restoration of vitality of tissue culture seedlings

[0056] The two tissue culture seedlings of Qiuzi Pear and Confrens, which had been stored at 10°C for 6 months, were taken out, explants were re-prepared and inoculated into the corresponding plant culture medium, and then placed in a plant growth room for 40 days. The temperature in the plant growth room was 25±1°C and the photoperiod was 16 / 8h. The status of the two tissue culture seedlings after 40 days in the plant growth room is as follows Figure 6 , Fig.10 .

Claims

1. A method for long-term preservation of pear tissue culture seedlings, characterized in that: The following steps are involved: (1) Take the tissue culture seedlings of Pear Autumnia and Confrens, and cut the explants used for subculture with a scalpel; (2) inoculating the explants cut in step (1) into a corresponding pear tissue culture seedling subculture medium; (3) The subculture medium containing the explants in step (2) is placed in a plant incubator for up to 6 months; wherein the incubator temperature is 10±1° C., the photoperiod is 16 / 8 h, and the light intensity is 1300 lx-1800 lx.

2. The method for long-term preservation of pear tissue culture seedlings according to claim 1, characterized in that: The formula of the subculture medium for the Qiuzi pear tissue culture seedlings in step (2) is: 4.74g / LMS+2.0mg / L6-BA+0.2mg / LIBA+30g / L sucrose+7.5g / L agar; the formula of the subculture medium for the Confrens tissue culture seedlings is: 4.74g / LMS+0.5mg / L6-BA+0.1mg / LIBA+30g / L sucrose+7.5g / L agar; the specific method of the culture medium configuration is: measure 1L of distilled water with a measuring cylinder, pour it into a conical flask and add 4.74gMS, 30g sucrose, stir continuously to dissolve it completely, add the corresponding hormone, adjust the solution pH to 5.8-6.0 with a pH meter and 1M potassium hydroxide aqueous solution, finally add 7.5g agar, put the conical flask containing the unsterilized culture medium into a high-temperature pressure sterilizer, sterilize it at 121°C for 20min, and after the sterilization is completed, divide it into tissue culture bottles for cooling.

3. The method for long-term preservation of pear tissue culture seedlings according to claim 1, characterized in that: The subculture cycle of the Qiuzi Pear and Confrenus tissue culture seedlings used as explant sources in step (1) is 40 days.

4. The method for long-term preservation of pear tissue culture seedlings according to claim 1, characterized in that: After the explant is cut in step (1), only the stem segment 0.5-1.0 cm below the growth point and 2-4 young leaves at the top are retained, and the growth point is vigorous and has not turned brown or died.

5. The method for long-term preservation of pear tissue culture seedlings as claimed in claim 2, characterized in that: The hormones used in the culture medium for the Qiuzi pear seedlings and the Confrenus seedlings in step (2) are prepared in advance and stored in a refrigerator at 4°C. The specific preparation method is: weigh an appropriate amount of hormone powder, dissolve it with a small amount of 1M potassium hydroxide aqueous solution, and add distilled water to make up to 1 mg / ml.

6. The method for long-term preservation of pear tissue culture seedlings according to claim 1, characterized in that: The explants described in step (2) are inoculated by vertically inserting the stem base into the culture medium.

7. The method for long-term preservation of pear tissue culture seedlings according to claim 1, characterized in that: The incubator temperature in step (3) is 10°C, the photoperiod is 16 / 8h, and the light intensity is 1500lx.

8. The method for long-term preservation of pear tissue culture seedlings according to claim 1, characterized in that: Described method also comprises the following tissue culture seedling vigor recovery step: The tissue culture seedlings in step (3) are taken out of the incubator, recut and inoculated into a newly configured subculture medium and cultured for 40 days.

9. The method for long-term preservation of pear tissue culture seedlings as claimed in claim 8, characterized in that: After the tissue culture seedlings are taken out of the incubator in step (4), they need to be recut into stem segments 0.5-1.0 cm below the growth point with the aid of a scalpel and 2-4 leaves at the top are retained.

10. The method for long-term preservation of pear tissue culture seedlings according to claim 8, characterized in that: The re-cutting in step (4) and inoculation into a newly configured subculture medium are cultured in a plant growth chamber for 40 days, wherein the temperature of the plant growth chamber is 25±1° C. and the photoperiod is 16 / 8 h.

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