A method for screening high-yield Morchella hybrid strains

Abstract

A method for screening high-yield Morchella hybrid strains comprises inoculating the Morchella hybrid strains into 90 mm diameter circular culture dishes containing 20 mL of starch solid culture medium. A 0.5 cm diameter activated mycelium disc is seeded into each dish and cultured in constant temperature incubators at 5°C, 10°C, and 15°C, respectively. Streaking is performed on the 7th, 5th, and 3rd day of incubation, and the mycelial diameters at different temperatures are measured. 1% Lugol's iodine solution is then added dropwise, and the size and transparency of the starch hydrolysis zone surrounding the colony are observed. The hydrolysis zone is then crossed using the cross-streaking method to measure the hydrolysis zone size. Finally, the ratio of the hydrolysis zone diameter (D) to the strain colony diameter (d) is calculated. The criteria for judging the high yield of hybrid strains at each temperature are: hybrid strains with colony diameter greater than 36.24 mm and D / d ratio greater than 0.7 at 5°C; hybrid strains with colony diameter greater than 35.84 mm and D / d ratio greater than 0.66 at 10°C; hybrid strains with colony diameter greater than 33.45 mm and D / d ratio greater than 0.6 at 15°C.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus breeding, and particularly relates to a method for screening a yield-super-parent Morchella hybrid strain. Background Art

[0002] Edible fungi require a carbon source for growth, and most rely on cellulose and lignin as their primary carbon sources. Therefore, providing sufficient carbon sources during the cultivation process is crucial to effectively promote the growth and development of edible fungi. The main sources of carbon for morels include organic matter in the soil and grass meal, sawdust, and organic fertilizer added to the culture medium. However, research has found that soluble starch is the optimal carbon source for morels. Exogenous nutrient packs are the most important source of nutrients in morel production and a key factor in determining morel yield. In actual morel production, wheat grains have been used as the primary component of exogenous nutrient packs. During the cultivation process, the amylopectin and amylose in the exogenous nutrient packs are largely consumed. Studies have shown that a higher proportion of wheat in the exogenous nutrient pack formulation accelerates mycelial growth, shortens the fruiting cycle, and thus increases yield and nutrient conversion.

[0003] In recent years, researchers have begun to shift from domesticating wild species to hybrid breeding in response to market demand, aiming to cultivate new, high-yielding and stable morel varieties. However, morel cultivation relies heavily on natural climate conditions and can only be cultivated once a year, resulting in slow breeding progress and a slow replacement of varieties. Summary of the Invention

[0004] The object of the present invention is to provide a method for screening a yield-superparent Morchella hybrid strain.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a method for screening a high-yield Morchella hybrid strain, comprising the following steps:

[0006] Step 1: The hybrid strain and the parent morel strain were inoculated into culture dishes containing starch solid medium, and each culture dish was inoculated with an activated mycelium disc with a diameter of 0.4-0.6 cm. The dishes were placed in a constant temperature incubator at 4-6°C, 9-11°C, and 14-16°C for dark culture.

[0007] Step 2: Using the cross-streaking method, streak the hybrid strain and the parent cultured at 4-6°C on the 6th to 8th day, streak the hybrid strain and the parent cultured at 9-11°C on the 4.5th to 5.5th day, and streak the hybrid strain and the parent cultured at 14-16°C on the 2.8th to 3.2th day. Measure the mycelial diameter under different temperature conditions, which is calculated as d;

[0008] Step 3: Add Lugol's iodine solution to each culture dish, observe the size and transparency of the starch hydrolysis zone around the colony, and use the cross-line method to mark the hydrolysis zone and measure the diameter of the hydrolysis zone, which is calculated as D;

[0009] Step 4: Calculate the ratio of the hydrolysis zone diameter D to the colony diameter d;

[0010] Step 5: Screen hybrid strains with colony diameters greater than 36.24 mm and D / d ratios greater than 0.7 at 4-6°C;

[0011] Step 6: Screen hybrid strains with colony diameters greater than 35.84 mm and D / d ratios greater than 0.66 at 9-11°C;

[0012] Step 7: Screen hybrid strains with colony diameters greater than 33.45 mm and D / d ratios greater than 0.6 at 14-16°C;

[0013] Step 8: Using the colony diameter and D / d ratio of the two parents as screening criteria, screen out hybrid strains whose colony diameters at different temperatures are larger than those of the two parents and whose D / d ratios are also larger than those of the two parents. This means that the hybrid strain is considered to have the potential to exceed the yield of the parents.

[0014] The preferred technical solution is: the Morchella hybrid strain and the parent are respectively inoculated into a circular culture dish with a diameter of 90 mm and containing 20 mL of starch solid culture medium.

[0015] The preferred technical solution is: the mass fraction of Lugol's iodine solution is 0.1%, and the added amount is 5 mL.

[0016] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0017] The present invention has the advantages of high screening efficiency and high prediction success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Comparison of colony diameter and D / d ratio of hybrid strains of Morchella liumei at different temperatures. Figure 1 A, B, and C are under the conditions of 5℃, 10℃, and 15℃ respectively.

[0019] Figure 2 The results of starch staining for the initial screening of Morchella liumei hybrid strains at different temperatures. Figures A, B, and C are the initial screening of strains at 5°C, 10°C, and 15°C, respectively. DETAILED DESCRIPTION

[0020] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in these embodiments.

[0021] See also Figure 1-2 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no technical substantive significance. Any modification of the structure, change in the proportional relationship or adjustment of the size. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0022] Starch culture medium: 0.5 g beef extract, 1 g peptone, 0.5 g sodium chloride, 0.2 g soluble starch, 100 mL water, 1.5-2.0 g agar; pH 7.0-7.2; sterilization at 115°C, 0.1 MPa, 20 min.

[0023] Example 1: A method for screening high-yield Morchella hybrid strains

[0024] A method for screening a high-yield Morchella hybrid strain comprises the following steps:

[0025] Step 1: The 150 hybrid strain parents of Morchella lilii, Morchella M4 and Morchella M6 obtained were inoculated into 90 mm diameter circular culture dishes containing 20 mL starch solid culture medium, and each dish was inoculated with a 0.5 cm diameter activated mycelium disc and placed in a constant temperature incubator at 5°C, 10°C and 15°C for dark culture.

[0026] Breeding new morel varieties requires both maternal and paternal parent stock. Hybrid morel strains are derived from crossing the maternal and paternal parent stocks. The 150 "Six Sister" hybrid morel strains were obtained by crossing Morchella M4 and Morchella M6.

[0027] Step 2: Use the cross-streaking method to streak the hybrid strain cultured at 5°C on the 7th day, the hybrid strain cultured at 10°C on the 5th day, and the hybrid strain cultured at 15°C on the 3rd day, and measure the mycelial diameters under different temperature conditions.

[0028] Step 3: Add 1% Lugol's iodine solution to each plate, observe the size and transparency of the starch hydrolysis zone around the colony, and use the cross-line method to mark the hydrolysis zone and measure the size of the hydrolysis zone.

[0029] Step 4: Calculate the ratio of the hydrolysis zone diameter (D) to the strain colony diameter (d), take the average of three repetitions, and determine the starch degradation ability of the hybrid strain at each temperature, thereby preliminarily screening the hybrid strain based on starch utilization ability.

[0030] Step 5: The colony values ​​and D / d ratios of the hybrid strain and the parent at 5°C, 10°C and 15°C, e.g. Figure 1 shown.

[0031] Depend on Figure 1 It can be seen that the starch utilization capacity of the six-sister Morchella hybrid strains differed significantly compared with the parent Morchella M4 and Morchella M6 strains. Some hybrid strains had large D / d ratios but small colony diameters, while others had large colony diameters but small D / d ratios. Therefore, screening for hybrid strains with large colony diameters and a large ratio of hydrolysis zone diameter (D) to colony diameter (d) was more practical. Using the parent Morchella M4 and Morchella M6 strains as screening criteria, hybrid strains with colony diameters greater than 36.24 mm and D / d ratios greater than 0.7 at 5°C were selected; hybrid strains with colony diameters greater than 35.84 mm and D / d ratios greater than 0.66 at 10°C were selected; and hybrid strains with colony diameters greater than 33.45 mm and D / d ratios greater than 0.6 at 15°C were selected. At 5°C, a total of 7 hybrid strains, including H4, H6, H12, H14, H16, H31, and H37, were screened; at 10°C, a total of 5 hybrid strains, including H8, H28, H32, H34, and H48, were screened; at 15°C, a total of 4 hybrid strains, including H5, H10, H11, and H13, were screened (Table 1).

[0032] Table 1 Preliminary screening results of Morchella liumei hybrid strains with strong starch degradation ability

[0033]

[0034]

[0035] Note: Data are expressed as mean ± standard deviation (n = 10).

[0036] From Table 1 and Figure 2 The hybrid strains screened at different temperatures all showed distinct circular hydrolysis zones. The starch staining results indicate that the six-sister Morchella hybrid strain can effectively degrade starch and also demonstrate that the strains possess strong starch degradation capabilities at 5°C, 10°C, and 15°C, respectively.

[0037] Table 2 Number of mature ascocarps of Morchella

[0038]

[0039] Depend on Figure 2 As shown in Table 2, with the exception of H31, all parent and hybrid strains of Morchella liumei produced fruit normally. Compared to their parents, with the exception of H5 and H37, the hybrid strains produced more ascocarps than both parents. H4, H10, H11, H16, H27, H28, H32, and H34 also produced far more ascocarps than their parents, demonstrating significant hybrid vigor.

[0040] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A method for screening a Morchella hybrid strain having a yield exceeding that of a Morchella parent, characterized in that: The following steps are involved: Step 1: The hybrid strain and the parent morel strain were inoculated into culture dishes containing starch solid medium, and each culture dish was inoculated with an activated mycelium disc with a diameter of 0.4-0.6 cm. The dishes were placed in a constant temperature incubator at 4-6°C, 9-11°C, and 14-16°C for dark culture. Step 2: Using the cross-streaking method, streak the hybrid strain and the parent cultured at 4-6°C on the 6th to 8th day, streak the hybrid strain and the parent cultured at 9-11°C on the 4.5th to 5.5th day, and streak the hybrid strain and the parent cultured at 14-16°C on the 2.8th to 3.2th day. Measure the mycelial diameter under different temperature conditions, which is calculated as d; Step 3: Add Lugol's iodine solution to each culture dish, observe the size and transparency of the starch hydrolysis zone around the colony, and use the cross-line method to mark the hydrolysis zone and measure the diameter of the hydrolysis zone, which is calculated as D; Step 4: Calculate the ratio of the hydrolysis zone diameter D to the colony diameter d; Step 5: Screen hybrid strains with colony diameters greater than 36.24 mm and D / d ratios greater than 0.7 at 4-6°C; Step 6: Screen hybrid strains with colony diameters greater than 35.84 mm and D / d ratios greater than 0.66 at 9-11°C; Step 7: Screen hybrid strains with colony diameters greater than 33.45 mm and D / d ratios greater than 0.6 at 14-16°C; Step 8: Using the colony diameter and D / d ratio of the two parents as screening criteria, screen out hybrid strains whose colony diameters at different temperatures are larger than those of the two parents and whose D / d ratios are also larger than those of the two parents. This means that the hybrid strain is considered to have the potential to exceed the yield of the parents.

2. The method for screening a Morchella hybrid strain having a yield exceeding that of a Morchella parent according to claim 1, wherein: The Morchella hybrid strain and the parent were inoculated into a circular culture dish with a diameter of 90 mm containing 20 mL of starch solid culture medium.

3. The method for screening a Morchella hybrid strain having a yield exceeding that of a Morchella parent according to claim 1, wherein: The mass fraction of Lugol's iodine solution is 0.1%, and the amount added is 5 mL.

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