Fungus for cultivating vaccinium vitis vinifera seedlings and application

Abstract

The invention discloses a fungus for cultivating vaccinium vitis vinifera seedlings and application, and particularly relates to the field of tissue culture. The method aims at solving the problems that at present, red bean and cowberry cultivation conditions are harsh, time and labor are wasted, and large-batch production is not facilitated. The method comprises the following steps: inoculating sterile cowberry seeds into a culture dish taking filter paper as a medium; preparing the separated growth-promoting fungi into a fungicide; after huckleberry tissue culture seedlings grow root hairs and a four-leaf stage, the fungicide is inoculated to roots, and after the fungicide is colonized, the tissue culture seedlings are taken out for standby application; uniformly mixing the sterilized flower soil and vermiculite according to a ratio of 2: 1, putting the mixture into the sterilized flowerpot, and watering sufficiently; inoculating the fungicide to the roots of cowberry seedlings and then planting the cowberry seedlings into flowerpots; and spraying water on the leaves every day to keep the leaves wet, watering once every 2 days, and watering once every 14 days with a Hoagland's nutrient solution. By means of the method, the cultivation period of vaccinium jumble can be shortened, growth of vaccinium jumble seedlings is promoted, large-scale production is facilitated, and the method is suitable for application and popularization.

Description

Technical Field

[0001] The invention belongs to the field of tissue culture, and in particular relates to a fungus for cultivating blueberry seedlings and an application thereof. Background Art

[0002] Vaccinium vitis-idaea L. is an evergreen shrub of the genus Vaccinium in the family Ericaceae, mainly distributed in Greater and Lesser Khingan Mountains and Changbai Mountain in my country. The fruit is rich in a variety of complex phenolic compounds, with anti-inflammatory, antioxidant and anti-cancer effects. It is a berry species with important commercial value and great commercial development potential. However, compared with blueberry and other Vaccinium plants, the commercial development of Vaccinium vitis-idaea has been slow, mainly due to its strict requirements on soil conditions and high difficulty in cultivation.

[0003] Although tissue culture technology for blueberry and other Vaccinium plants has made some progress, tissue rapid propagation technology can be used to quickly prepare sterile tissue culture seedlings and achieve large-scale cultivation. However, the tissue culture technology of red bean bilberry is not yet mature, and there are problems such as complex culture medium screening, high hormone regulation cost and low survival rate of seedlings, which makes it difficult to meet the needs of large-scale production. A more targeted method is needed to shorten the cultivation cycle of red bean bilberry and achieve the purpose of promoting the growth and environmental adaptability of red bean bilberry.

[0004] Mycorrhizal fungi, especially ericoid mycorrhizal fungi, as potential symbiotic partners of blueberry, can significantly promote its root development, improve nutrient absorption and environmental adaptability, and provide important biological support for solving the problem of blueberry cultivation. Therefore, combining the symbiotic effect of mycorrhizal fungi with tissue culture technology to develop an efficient, simple, and environmentally friendly blueberry cultivation method is expected to significantly improve the cultivation efficiency of blueberry, shorten the breeding cycle, and enhance its adaptability, thereby realizing large-scale planting and commercial development. The development of an efficient, environmentally friendly, and targeted blueberry tissue culture technology will provide technical support for the large-scale planting and commercial development of blueberry, and has important application value and broad market prospects. Summary of the invention

[0005] The invention aims to solve the problems that the survival rate of transplanted tissue culture seedlings of Vaccinium plant is low, the cultivation conditions of Vaccinium vitis are harsh, time-consuming and labor-intensive, and it is not suitable for mass production, and provides a method for systematically cultivating Vaccinium vitis tissue culture seedlings and its application.

[0006] The invention discloses a fungus for cultivating blueberry seedlings. The fungus is Lachnum pygmaeumZL6, which is deposited in China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. The deposit date is December 16, 2024, and the deposit number is CCTCC No: M 20242833.

[0007] The invention discloses an application of fungi for cultivating blueberry seedlings. The fungi is made into a bacterial agent for cultivating blueberry seedlings.

[0008] Furthermore, the method for cultivating blueberry seedlings is as follows:

[0009] 1) taking sterile blueberry seeds and inoculating them into a culture medium, culturing them until the blueberry seeds germinate, and setting aside for use;

[0010] 2) inoculating the fungus Lachnum pygmaeum ZL6 into PDA medium, culturing, and diluting with sterile water to obtain a fungal inoculum;

[0011] 3) After the red bean bilberry seedlings cultured in step 1) are inoculated into 1 / 2MS solid culture medium for further cultivation, the fungal agent in step 2) is inoculated into the roots of the red bean bilberry, and then the roots are transferred to the cultivation medium for cultivation, thereby completing the cultivation of the red bean bilberry seedlings.

[0012] Furthermore, the culture medium described in step 1) is a filter paper culture medium.

[0013] Furthermore, the culturing in step 1) until the blueberry seeds germinate is carried out under the conditions of a photoperiod of 16 h light / 8 h dark and a temperature of 23±2°C.

[0014] Furthermore, in step 2), the culture conditions of the fungus Lachnum pygmaeum ZL6 are 25°C±2°C, 150 rpm, and cultured in a constant temperature shaking incubator for 7 to 14 days.

[0015] Furthermore, in step 2), the sterile water dilution is to dilute the cultured fungus Lachnum pygmaeum ZL6 to 1×10 7 ~1×10 9 cfu.

[0016] Furthermore, the components of the PDA culture medium are 200 g of potato, 20 g of glucose, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, and 1000 mL of distilled water.

[0017] Furthermore, in step 2), the blueberry seedlings cultured to the 4-leaf stage in step 1) are inoculated into 1 / 2MS solid culture medium.

[0018] Furthermore, in step 3), the blueberry seedlings cultured in step 1) are inoculated into 1 / 2MS solid culture medium and cultured for further 30 days.

[0019] The present invention has the following beneficial effects:

[0020] The present invention selects the most suitable medium affecting the germination of red bean bilberry, that is, a filter paper medium, by screening the seed germination medium, and then re-cultivates the red bean bilberry by a mycorrhizal seedling method, thereby shortening the cultivation period of the red bean bilberry, promoting the growth of the red bean bilberry and improving the survival rate. No surfactant, protective agent or cross-linking agent is used in the cultivation process of the red bean bilberry, so the red bean bilberry is environmentally friendly, low in cost and simple to operate; the fungal agent prepared by the present invention has the ability to dissolve inorganic phosphorus, secrete iron carriers and produce IAA, is easy to store and can be expanded for production; the method for cultivating red bean bilberry tissue culture seedlings designed by the present invention solves the problem of the long tissue culture period of the genus Vaccinium, saves time and saves cost, thereby achieving the best economic benefit. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a screening diagram of the germination medium for blueberry seeds in Example 1;

[0022] Figure 2 This is a diagram of the plant growth promotion characteristics of the applied fungicide;

[0023] Figure 3 This is a characteristic diagram of colonization of the bacterial agent prepared in Example 1 on the roots of blueberries;

[0024] Figure 4 This is a diagram showing the effect of the bacterial agent prepared in Example 1 on the plant height and root length of blueberry;

[0025] Figure 5 This is a diagram showing the effect of the bacterial agent prepared in Example 1 on the weight of blueberry plants;

[0026] Figure 6 This is a diagram showing the effects of the bacterial agent prepared in Example 1 on the physiological indicators of blueberry leaves;

[0027] Figure 7 This is a diagram of the cultivation status of blueberries after inoculation with the bacterial agent in Example 1. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0029] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0030] Embodiment 1:

[0031] A method for systematically cultivating blueberry tissue culture seedlings is carried out according to the following steps:

[0032] 1. Obtaining sterile red bean bilberry seeds: Select a certain number of bilberry seeds for surface disinfection, treat with 75% ethanol for 30 to 45 seconds, treat with 3.25% sodium hypochlorite solution for 2 to 5 minutes, rinse with sterile water for 3 times, and finally add 1 mL of sterile water, seal, and stand at 4°C for 48 hours to obtain sterile red bean bilberry seeds;

[0033] 2. Selection of germination medium for red bean bilberry: Inoculate sterile red bean bilberry seeds into 1 / 2MS, 1 / 2MS+10mg / L gibberellin and filter paper medium, place the culture dish in a light culture room (photoperiod is 16h light / 8h dark, temperature is 23±2℃), and record the germination time of red bean bilberry;

[0034] Preparation of fungal inoculant: The fungus Lachnum pygmaeum ZL6 was inoculated into 200 mL PDA medium containing glass beads and cultured in a constant temperature shaking incubator at 25°C and 150 rpm for 7 days. The fungus was diluted to 1×10 8 cfu, that is, the preparation of fungal inoculants is completed;

[0035] 4. Cultivation of potted blueberry seedlings: The blueberries cultured to the 4-leaf stage in step 2 are inoculated into tissue culture bottles containing 1 / 2MS solid culture medium and continue to be cultivated for 30 days; the Lachnum pygmaeum ZL6 fungal agent in step 3 is inoculated into the roots of sterile blueberries, and after the mycelium is colonized, the seedlings are transferred to the cultivation medium and cultured in a light culture room, watered every 2 days, and watered with Hoagland's nutrient solution once every 14 days. After one month, the data of blueberries such as plant height, root length, and leaf physiological indicators are recorded.

[0036] The fungus Lachnum pygmaeum ZL6 was isolated and screened from the root tissue of Vaccinium pygmaeum in Daxinganling, and was deposited in the China Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. The deposit date is December 16, 2024, and the deposit number is CCTCC No: M 20242833.

[0037] (1) Screening of seed germination medium:

[0038] The sterile red bean bilberry seeds prepared in this example were evenly dispersed in 1 / 2MS, 1 / 2MS+10mg·L -1The culture dish was placed in a light culture room (with a photoperiod of 16 h light / 8 h dark and a temperature of 23 ± 2 °C) in a medium containing gibberellin and filter paper. Figure 1 As shown, the red bean bilberry seeds germinated first in the filter paper medium, followed by 1 / 2MS + 10mg L -1 Gibberellic acid medium.

[0039] (2) Analysis of plant growth-promoting properties of strains:

[0040] The fungal strain prepared in this example was inoculated into an iron-free medium, an inorganic phosphorus liquid medium, and an IAA liquid medium, respectively, and cultured at 25°C and 150 rpm for analysis of the plant growth-promoting properties of the strain. Figure 2 As shown, it was proved that the strain had the ability to dissolve inorganic phosphorus, secrete siderophores and produce IAA during its growth.

[0041] (3) Growth characteristics of Vaccinium vitis-idaea after mycorrhizal seedling cultivation

[0042] The fungal strain prepared in this example was inoculated into the roots of sterile red bean blueberry tissue culture seedlings. The root morphology after colonization was as follows: Figure 3 As shown, after colonization, the strain was immediately transferred to the cultivation medium for cultivation. In order to facilitate the observation of the growth characteristics of the mycorrhizal seedlings of Vaccinium vitis-idaea (experimental group), a treatment of non-mycorrhized Vaccinium vitis-idaea seedlings (control group) was established. The average plant height, fresh weight and leaf physiological indicators (leaf chlorophyll content, soluble sugar content and leaf carotenoid content) of Vaccinium vitis-idaea seedlings under different treatments are shown in Figure 4 , Figure 5 and Figure 6 It can be seen that the red bean bilberry seedlings treated with mycorrhiza grew faster and had higher leaf physiological indicators. Figure 7 It can be seen that the method provided by the present invention is better, with higher plant height, heavier fresh weight, better leaf development, and can further promote the growth of red bean blueberry seedlings.

Claims

1. A fungus for cultivating blueberry seedlings, characterized in that The fungus is Lachnum pygmaeumZL6, which is deposited in the China Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is December 16, 2024, and the deposit number is CCTCC No: M 20242833.

2. The use of a fungus for cultivating blueberry seedlings as claimed in claim 1, characterized in that The fungus is made into a bacterial agent for cultivating red bean blueberry seedlings.

3. The use according to claim 2, characterized in that The method for cultivating blueberry seedlings is as follows: 1) taking sterile blueberry seeds and inoculating them into a culture medium, culturing them until the blueberry seeds germinate, and setting aside for use; 2) inoculating the fungus Lachnum pygmaeum ZL6 into PDA medium, culturing, and diluting with sterile water to obtain a fungal inoculum; 3) After the red bean bilberry seedlings cultured in step 1) are inoculated into 1 / 2MS solid culture medium for further cultivation, the fungal agent in step 2) is inoculated into the roots of the red bean bilberry, and then the roots are transferred to the cultivation medium for cultivation, thereby completing the cultivation of the red bean bilberry seedlings.

4. The use according to claim 3, characterized in that The culture medium described in step 1) is a filter paper culture medium.

5. The use according to claim 3, characterized in that The step 1) of culturing the blueberry seeds until they germinate is carried out under the conditions of a photoperiod of 16 h light / 8 h dark and a temperature of 23±2°C.

6. The use according to claim 3, characterized in that In step 2), the culture conditions of the fungus Lachnum pygmaeum ZL6 are 25°C±2°C, 150 rpm, in a constant temperature shaking incubator for 7 to 14 days.

7. The use according to claim 3, characterized in that In step 2), the sterile water dilution is to dilute the cultured fungus Lachnum pygmaeum ZL6 to 1×10 7 ~1×10 9 cfu.

8. The use according to claim 3, characterized in that The components of PDA culture medium are 200 g potato, 20 g glucose, 3 g potassium dihydrogen phosphate, 1.5 g magnesium sulfate, and 1000 mL distilled water.

9. The use according to claim 3, characterized in that In step 2), the red bean blueberry seedlings cultured to the 4-leaf stage in step 1) are inoculated into 1 / 2MS solid culture medium.

10. The use according to claim 3, characterized in that In step 3), the blueberry seedlings cultured in step 1) are inoculated into 1 / 2MS solid culture medium and cultured for 30 days.

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