Ex vitro rooting method for cherry tissue culture seedlings

Abstract

The invention relates to the technical field of cherry cultivation, in particular to an ex-vitro rooting method for cherry tissue culture seedlings. The invention provides an ex-vitro rooting method for cherry tissue culture seedlings, which comprises the following steps: (1) pretreatment of the tissue culture seedlings: taking out the cherry tissue culture seedlings growing for 7 weeks from a culture bottle, washing the cherry tissue culture seedlings with sterile water, removing calluses and tissue culture substrates, cutting the cherry tissue culture seedlings into single seedlings with the plant height of 2-3cm, picking off 2-4 leaflets at the base parts of the single seedlings, and carrying out rooting treatment on the single seedlings; then soaking the base part of the single seedling in a rooting accelerant; (2) cutting and rooting: cutting the pretreated cherry tissue culture seedlings into a rooting medium for culturing until the cherry tissue culture seedlings root, wherein the insertion depth is 0.6-1.2 cm, and the plant interval is 3-4 cm. The method can improve the rooting rate and the survival rate of the cherry tissue culture seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of cherry cultivation, and in particular to an out-of-bottle rooting method for cherry tissue culture seedlings. Background Art

[0002] Plant tissue culture technology is an asexual reproduction technology with broad application value and development prospects. It is widely used in large-scale in vitro rapid propagation of flowers and fruit trees. With the continuous advancement of science and technology and the deepening of research, this technology will play a greater role in agricultural production, breeding, plant physiology and biochemistry research and other fields.

[0003] Cherry (Prunus pseudocerasus Lindl.) belongs to the Rosaceae, Prunus subfamily, and Prunus genus. It is a tree or shrub. Cherry likes light, warmth, moisture, and fertilizer. It is suitable for growing in sandy loam or loam, and grows better in soil with deep soil layer, loose soil, and good ventilation. It often grows in sunny places on hillsides or ditches, with an altitude of 300 to 600 meters. At the same time, in the field of plant tissue culture, cherry is recognized as a type of plant that is difficult to propagate through tissue culture and root outside the bottle.

[0004] The rooting of cherry tissue culture seedlings is a key link in cherry tissue culture technology. Traditionally, the rooting of cherry tissue culture seedlings mainly relies on the in-bottle rooting method, that is, in a test tube or other closed container, using a specific rooting medium to induce the rooting of tissue culture seedlings. This method is time-consuming and labor-intensive, with high production costs, low rooting rate, and even lower survival rate. At present, the rooting rate of cherries using this method is only 70-80%, and the survival rate is only 20-30%.

[0005] Therefore, it is particularly important to provide a rooting method for cherry tissue culture seedlings which is easy to operate, has low production cost and improves both the rooting rate and the survival rate. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a method for rooting cherry tissue culture seedlings outside a bottle, so as to solve the problems in the prior art that the rooting process of cherry tissue culture seedlings is time-consuming and labor-intensive, has high production costs, low rooting rate and low survival rate.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for rooting cherry tissue culture seedlings outside a bottle, the method comprising the following steps:

[0009] (1) Pretreatment of tissue culture seedlings: Take out the 7-week-old cherry tissue culture seedlings from the culture bottle, rinse them with sterile water, remove the callus tissue and tissue culture medium, cut them into single seedlings with a height of 2-3 cm, remove 2-4 leaflets at the base of the single seedlings, and then soak the base of the single seedlings in a rooting promoter;

[0010] (2) Cutting rooting: The pretreated cherry tissue culture seedlings are cut into the rooting medium and cultured until they take root. The insertion depth is 0.6-1.2 cm and the plant spacing is 3-4 cm.

[0011] Preferably, the rooting promoter comprises cherry exocysts, gibberellic acid and quercetin.

[0012] Preferably, the rooting medium includes cherry exocysts, peat, peat soil, and slag.

[0013] Preferably, the mass ratio of cherry exovesicles, gibberellic acid and quercetin in the rooting promoter is 0.5-1:2-4:7-10.

[0014] Preferably, the mass ratio of cherry exocysts, peat, peat soil and slag in the rooting medium is 0.5-1.5:1:1:1.

[0015] In a second aspect, the present invention provides a combined rooting system for extra-bottle rooting of cherry tissue culture seedlings, the combined rooting system comprising a rooting promoter and a rooting matrix, the rooting promoter comprising cherry exovesicles, gibberellic acid and quercetin, and the rooting matrix comprising cherry exovesicles, peat, peat soil and slag.

[0016] In the present invention, the preparation method of cherry exovesicles is as follows:

[0017] ① Add 250 mL of sterilized PBS to 250 g of fresh cherries, crush them with a crusher, and centrifuge them at 4°C, 3000×g for 10 min to remove larger cell fragments and impurities, and take the colorless or light yellow transparent solution in the middle layer;

[0018] ② Centrifuge the yellow transparent solution obtained in step ① at 4°C, 6000×g for 25 min, and take the supernatant;

[0019] ③ The supernatant obtained in step ② was centrifuged at 4°C, 80,000×g for 25 min, and then the precipitate was resuspended in 3 mL of sterile PBS and centrifuged at 4°C, 100,000×g for 30 min. The precipitate was resuspended in 3 mL of sterile PBS again to obtain cherry extracellular vesicles.

[0020] It should be noted that in the prior art, rooting agents and rooting substrates are used to root cherry tissue culture seedlings outside the bottle. For example, the rooting agents used in the prior art include: indoleacetic acid, chlorogenic acid and boron; the rooting substrates include: peat and perlite. For another example, the rooting substrates used in the prior art include: peat soil, rice husk charcoal, river sand, perlite, vermiculite or rock wool. However, due to the barrier effect of the plant body, the role that the rooting agents and rooting substrates used in the prior art can play when used is often limited.

[0021] Plant extracellular vesicles are nanoscale vesicles secreted by plant cells. They contain 90% of the active ingredients in plants and are the concentrated essence of plant effective ingredients. They have a phospholipid bilayer structure and contain substances such as DNA, small RNA and proteins. They can carry these biological molecules to participate in processes such as intercellular communication, material exchange and signal transmission.

[0022] In a third aspect, there is provided an application of the combined rooting system of the present invention in improving the rooting rate and / or survival rate of cherry tissue culture seedlings.

[0023] In a fourth aspect, a cherry tissue culture seedling is provided, and rooting is performed ex-bottle according to the ex-bottle rooting method of the present invention.

[0024] A fifth aspect provides application of the ex-bottle rooting method of the present invention in ex-bottle rooting of cherry tissue culture seedlings.

[0025] In a sixth aspect, there is provided an application of the ex-bottle rooting method of the present invention in improving the rooting rate and / or survival rate of cherry tissue culture seedlings.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] Compared with the traditional in-bottle rooting method, the in-bottle rooting method of cherry tissue culture seedlings provided by the present invention has the following significant technical effects:

[0028] (1) Easy operation: It avoids the complicated operation and frequent transfer steps in the rooting process in the bottle, simplifying the production process.

[0029] (2) Reduce costs: Reduce the amount of culture medium used and the number of disinfection treatments, thereby reducing production costs.

[0030] (3) Improve rooting rate and survival rate: By optimizing the rooting matrix, the use of rooting promoters, and environmental control conditions, the rooting rate and survival rate of cherry tissue culture seedlings were significantly improved.

[0031] The present invention has been concluded through a large number of experimental screenings that the combined use of the rooting promoter including cherry exovesicles, gibberellic acid and quercetin and the rooting matrix including cherry exovesicles, peat, peat soil and slag of the present invention can help improve the rooting rate and survival rate of cherry tissue culture seedlings. During the experimental screening process, the inventors conducted a large number of experimental screenings. The inventors tried to replace cherry exovesicles, gibberellic acid, quercetin, peat, peat soil and slag with other substances for experiments, and found that the technical scheme after the replacement was not effective in improving the rooting rate and survival rate of cherry tissue culture seedlings; the inventors tried to replace the cherry exovesicles with cherry extracts, and found that the technical scheme after the replacement was also generally effective in improving the rooting rate and survival rate of cherry tissue culture seedlings. The inventors speculate that cherry exovesicles, as tiny vesicles secreted by cells, may contain bioactive substances that are beneficial to the rooting of cherry tissue culture seedlings, such as growth factors, signal molecules, etc. These substances play an important role in intercellular communication, signal transmission, etc. The addition of cherry exovesicles may help provide a more suitable growth environment for cherry tissue culture seedlings, thereby promoting the growth and development of their root system. On the one hand, when cherry exovesicles, gibberellic acid and quercetin are used in combination, they may provide a more optimized growth environment for cherry tissue culture seedlings through a certain effect, thereby promoting the growth and development of the root system, and improving the rooting rate and survival rate. On the other hand, when cherry exovesicles are used in combination with peat, peat soil and slag, they can jointly provide a stable, breathable, water-retaining and nutrient-rich growth environment for the root system of cherry tissue culture seedlings. This environment helps to promote the growth and development of the root system and improve the rooting rate and survival rate of cherry tissue culture seedlings.

[0032] In summary, the combination of the rooting promoter and the rooting matrix of the present invention provides a more optimized and suitable growth environment for the cherry tissue culture seedlings, thereby promoting the growth and development of the root system thereof, and improving the rooting rate and survival rate. This combination method not only helps to solve the problem of the difficulty of rooting of the cherry tissue culture seedlings, but also provides a new technical approach for the large-scale production and promotion of cherries. In addition, by optimizing the quality of each substance in the rooting promoter and the rooting matrix of the present invention, the growth and development of the root system of the cherry tissue culture seedlings can be further promoted, and the rooting rate and survival rate can be improved. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] This embodiment provides a method for rooting cherry tissue culture seedlings outside the bottle, and the method comprises the following steps:

[0036] Pretreatment of tissue culture seedlings: Take out the 7-week-old cherry tissue culture seedlings from the culture bottle, rinse them with sterile water, remove the callus tissue and tissue culture matrix, cut them into single seedlings with a height of 2.5 cm, remove 2-4 leaflets at the base of the single seedlings, and then place the base of the single seedlings in rooting promoter and soak them for 7 minutes.

[0037] Prepare rooting medium: Mix cherry exocysts, peat, peat soil and slag in a mass ratio of 0.5:1:1:1, divide them into plug trays, and water them to keep them moist to improve the moisture retention performance.

[0038] Cutting rooting: Cut the pretreated cherry tissue culture seedlings into the prepared rooting medium with an insertion depth of 0.6-1.2cm and a plant spacing of 4cm.

[0039] Environmental control: Place the cherry tissue culture seedlings after cutting in a rooting room with a light duration of 12-16h / d, a light intensity of 1500-3000Lx, a temperature of 20-25°C, and a humidity of 70-90%; the culture time is 60d.

[0040] Management after rooting: Check the rooting situation regularly, remove sick and weak seedlings in time, and after the root system develops well, carry out seedling hardening treatment to gradually adapt to the external environment.

[0041] in,

[0042] The rooting promoter includes cherry exovesicles, gibberellic acid and quercetin; the mass ratio of cherry exovesicles, gibberellic acid and quercetin in the rooting promoter is 0.5:2:7;

[0043] Gibberellic acid was purchased from Tai'an Jiangzhou Biotechnology Co., Ltd., CAS No. 77-06-5;

[0044] Quercetin was purchased from Shanghai Kanglang Biotechnology Co., Ltd. with a CAS number of 6151-25-3.

[0045] Example 2

[0046] This embodiment provides a method for rooting cherry tissue culture seedlings outside the bottle, and the method comprises the following steps:

[0047] Pretreatment of tissue culture seedlings: Take out the 7-week-old cherry tissue culture seedlings from the culture bottle, rinse them with sterile water, remove the callus tissue and tissue culture matrix, cut them into single seedlings with a height of 3 cm, remove 2-4 leaflets at the base of the single seedlings, and then place the base of the single seedlings in rooting promoter and soak them for 10 minutes.

[0048] Prepare rooting medium: Mix cherry exocysts, peat, peat soil and slag in a mass ratio of 1.5:1:1:1, divide them into plug trays, and water them to keep them moist to improve the moisture retention performance.

[0049] Cutting rooting: Cut the pretreated cherry tissue culture seedlings into the prepared rooting medium with an insertion depth of 0.6-1.2cm and a plant spacing of 3cm.

[0050] Environmental control: Place the cherry tissue culture seedlings after cutting in a rooting room with a light duration of 12-16h / d, a light intensity of 1500-3000Lx, a temperature of 20-25°C, and a humidity of 70-90%; the culture time is 60d.

[0051] Management after rooting: Check the rooting situation regularly, remove sick and weak seedlings in time, and after the root system develops well, carry out seedling hardening treatment to gradually adapt to the external environment.

[0052] in,

[0053] The rooting promoter comprises cherry exovesicles, gibberellic acid and quercetin; the mass ratio of cherry exovesicles, gibberellic acid and quercetin in the rooting promoter is 1:4:10;

[0054] Gibberellic acid was purchased from Tai'an Jiangzhou Biotechnology Co., Ltd., CAS No. 77-06-5;

[0055] Quercetin was purchased from Shanghai Kanglang Biotechnology Co., Ltd. with a CAS number of 6151-25-3.

[0056] Example 3

[0057] This embodiment provides a method for rooting cherry tissue culture seedlings outside the bottle, and the method comprises the following steps:

[0058] Pretreatment of tissue culture seedlings: Take out the 7-week-old cherry tissue culture seedlings from the culture bottle, rinse them with sterile water, remove the callus tissue and tissue culture matrix, cut them into single seedlings with a height of 3 cm, remove 2-4 leaflets at the base of the single seedlings, and then place the base of the single seedlings in a rooting promoter and soak them for 8 minutes.

[0059] Prepare rooting medium: Mix cherry exocysts, peat, peat soil and slag in a mass ratio of 0.8:1:1:1, divide them into plug trays, and water them to keep them moist to improve the moisture retention performance.

[0060] Cutting rooting: Cut the pretreated cherry tissue culture seedlings into the prepared rooting medium with an insertion depth of 0.6-1.2cm and a plant spacing of 4cm.

[0061] Environmental control: Place the cherry tissue culture seedlings after cutting in a rooting room with a light duration of 12-16h / d, a light intensity of 1500-3000Lx, a temperature of 20-25°C, and a humidity of 70-90%; the culture time is 60d.

[0062] Management after rooting: Check the rooting situation regularly, remove sick and weak seedlings in time, and after the root system develops well, carry out seedling hardening treatment to gradually adapt to the external environment.

[0063] in,

[0064] The rooting promoter comprises cherry exovesicles, gibberellic acid and quercetin; the mass ratio of cherry exovesicles, gibberellic acid and quercetin in the rooting promoter is 1:3:9;

[0065] Gibberellic acid was purchased from Tai'an Jiangzhou Biotechnology Co., Ltd., CAS No. 77-06-5;

[0066] Quercetin was purchased from Shanghai Kanglang Biotechnology Co., Ltd. with a CAS number of 6151-25-3.

[0067] Comparative Example 1

[0068] Compared with Example 3, no cherry extracellular vesicles are added to the rooting promoter, and the rest is the same as Example 3.

[0069] Comparative Example 2

[0070] Compared with Example 3, no cherry extracellular vesicles were added to the rooting medium, and the rest was the same as Example 3.

[0071] Comparative Example 3

[0072] Compared with Example 3, the cherry extracellular vesicles in the rooting promoter are replaced with cherry extract, and the rest is the same as Example 3; wherein, the cherries are soaked in ethanol and stirred to dissolve the target component in the ethanol to obtain the cherry extract.

[0073] Effect verification

[0074] Rooting rate test method:

[0075] The rooting rate was calculated 60 days after the tissue culture seedlings were inserted into the substrate. The rooting rate refers to the ratio of the number of tissue culture seedlings that have taken root to the total number of tissue culture seedlings inoculated. This indicator reflects the ability of tissue culture seedlings to take root under specific conditions. The calculation formula is: rooting rate = (number of tissue culture seedlings that have taken root / total number of tissue culture seedlings) × 100%.

[0076] Survival test method:

[0077] Observe the tissue culture seedlings 60 days after they are inserted into the substrate and record the growth status of each tissue culture seedling; judge whether they survive based on their growth status. Usually, surviving tissue culture seedlings should show normal growth signs, such as new leaves growing and a well-developed root system. The survival rate reflects the adaptability and survival rate of the tissue culture seedlings after planting. The calculation formula is: survival rate = (number of surviving tissue culture seedlings / number of rooted tissue culture seedlings) × 100%.

[0078] The results are shown in Tables 1 and 2.

[0079] Table 1 Rooting rate of cherry tissue culture seedlings

[0080] Treatment Group 60d rooting rate / % Example 1 94 Example 2 96 Example 3 98 Comparative Example 1 43 Comparative Example 2 46 Comparative Example 3 52

[0081] Table 2 Survival rate of cherry tissue culture seedlings

[0082] Treatment Group Survival rate / % Example 1 100 Example 2 100 Example 3 100 Comparative Example 1 60 Comparative Example 2 65 Comparative Example 3 62

[0083] The rooting rate of cherry tissue culture seedlings and the survival rate of cherry tissue culture seedlings are used to evaluate the rooting of cherry tissue culture seedlings outside the bottle. It can be seen from Table 1 and Table 2 that the rooting rate and survival rate of cherry tissue culture seedlings can be significantly improved by the treatment methods in Examples 1-3, while Comparative Examples 1-3 cannot improve the rooting rate and survival rate of cherry tissue culture seedlings due to the lack of effective methods or unfavorable conditions. Moreover, the rooting rate of cherry tissue culture seedlings obtained by the treatment method in Example 3 is higher than that of Examples 1 and 2, and is significantly higher than that of each comparative example. These experimental data show that the rooting rate and survival rate of cherry tissue culture seedlings can be effectively improved by the specific treatment method of the present invention.

[0084] It can be seen from the effect verification test that the combined use of the rooting promoter including cherry exovesicles, gibberellic acid and quercetin of the present invention and the rooting matrix including cherry exovesicles, peat, peat soil and slag of the present invention, this combined rooting system can help improve the rooting rate and survival rate of cherry tissue culture seedlings. During the experimental screening process, the inventors also conducted a large number of experimental screenings. The inventors tried to replace cherry exovesicles, gibberellic acid, quercetin, peat, peat soil and slag with other substances for experiments, and found that the replaced technical solution was not effective in improving the rooting rate and survival rate of cherry tissue culture seedlings; the inventors tried to replace cherry exovesicles with cherry extracts, and found that the replaced technical solution was also generally effective in improving the rooting rate and survival rate of cherry tissue culture seedlings. The inventors speculate that cherry exovesicles, as tiny vesicles secreted by cells, may contain bioactive substances that are beneficial to the rooting of cherry tissue culture seedlings, such as growth factors, signal molecules, etc. These substances play an important role in intercellular communication, signal transmission, etc. The addition of cherry exovesicles may help provide a more suitable growth environment for cherry tissue culture seedlings, thereby promoting the growth and development of their root system. On the one hand, when cherry exovesicles, gibberellic acid and quercetin are used in combination, they may provide a more optimized growth environment for cherry tissue culture seedlings through a certain effect, thereby promoting the growth and development of the root system, and improving the rooting rate and survival rate. On the other hand, when cherry exovesicles are used in combination with peat, peat soil and slag, they can jointly provide a stable, breathable, water-retaining and nutrient-rich growth environment for the root system of cherry tissue culture seedlings. This environment helps to promote the growth and development of the root system and improve the rooting rate and survival rate of cherry tissue culture seedlings.

[0085] In summary, the combination of the rooting promoter and the rooting matrix of the present invention provides a more optimized and suitable growth environment for the cherry tissue culture seedlings, thereby promoting the growth and development of the root system thereof, and improving the rooting rate and survival rate. This combination method not only helps to solve the problem of the difficulty of rooting of the cherry tissue culture seedlings, but also provides a new technical approach for the large-scale production and promotion of cherries. In addition, by optimizing the quality of each substance in the rooting promoter and the rooting matrix of the present invention, the growth and development of the root system of the cherry tissue culture seedlings can be further promoted, and the rooting rate and survival rate can be improved.

[0086] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A method for rooting cherry tissue culture seedlings outside a bottle, characterized in that: The method for rooting outside the bottle comprises the following steps: (1) Pretreatment of tissue culture seedlings: Take out the 7-week-old cherry tissue culture seedlings from the culture bottle, rinse them with sterile water, remove the callus tissue and tissue culture medium, cut them into single seedlings with a height of 2-3 cm, remove 2-4 leaflets at the base of the single seedlings, and then soak the base of the single seedlings in a rooting promoter; (2) Cutting rooting: The pretreated cherry tissue culture seedlings are cut into the rooting medium and cultured until they take root. The insertion depth is 0.6-1.2 cm and the plant spacing is 3-4 cm.

2. The method for rooting outside a bottle according to claim 1, characterized in that: The rooting promoter includes cherry exocysts, gibberellic acid and quercetin.

3. The method for rooting outside a bottle according to claim 1, characterized in that: The rooting medium includes cherry exocysts, peat, peat soil and slag.

4. The method for rooting outside a bottle according to claim 2, characterized in that: The mass ratio of cherry exovesicles, gibberellic acid and quercetin in the rooting promoter is 0.5-1:2-4:7-10.

5. The method for rooting outside a bottle according to claim 3, characterized in that: The mass ratio of cherry exocysts, peat, peat soil and slag in the rooting medium is 0.5-1.5:1:1:

1.

6. A combined rooting system for cherry tissue culture seedlings outside the bottle, characterized in that: The combined rooting system comprises a rooting promoter and a rooting matrix, wherein the rooting promoter comprises cherry exocysts, gibberellic acid and quercetin, and the rooting matrix comprises cherry exocysts, peat, peat soil and slag.

7. Use of the combined rooting system according to claim 6 in improving the rooting rate and / or survival rate of cherry tissue culture seedlings.

8. Cherry tissue culture seedlings, characterized in that, Carry out ex-bottle rooting according to the ex-bottle rooting method according to any one of claims 1 to 5.

9. Use of the ex-bottle rooting method according to any one of claims 1 to 5 in ex-bottle rooting of cherry tissue culture seedlings.

10. Use of the ex-bottle rooting method according to any one of claims 1 to 5 in improving the rooting rate and / or survival rate of cherry tissue culture seedlings.