CN111727876B

CN111727876B - Cross breeding method for shiitake mushrooms

Info

Publication number: CN111727876B
Application number: CN202010734440.5A
Authority: CN
Inventors: 柳凤玉; 梁希才; 刘海军
Original assignee: Pingquan Xicai Application Mycology Technology Development Co ltd
Current assignee: Pingquan Xicai Application Mycology Technology Development Co ltd
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2021-09-03
Anticipated expiration: 2040-07-27
Also published as: CN111727876A

Abstract

The invention relates to the technical field of edible fungus hybridization, and particularly discloses a mushroom hybridization breeding method. The mushroom crossbreeding method specifically comprises the steps of grinding fruiting body tissues of two parent mushroom strains for crossbreeding together, adding 0.3-0.5 wt% of NaCl solution after grinding, stirring to form paste, inoculating the paste into a culture medium for culture, and screening new mycelia obtained by culture to obtain a hybrid variety. The mushroom crossbreeding method does not need special experimental environment, equipment and technicians, has simple operation method, short period and high success rate, can simply, conveniently and quickly breed and domesticate a new mushroom crossbred variety with the advantages of two parents, quickly promotes the development of mushroom industry, optimizes germplasm resources, promotes the yield and the efficiency of the industry and has extremely high application value.

Description

Cross breeding method for shiitake mushrooms

Technical Field

The invention relates to the technical field of edible fungus hybridization, in particular to a mushroom hybridization breeding method.

Background

Lentinus edodes belongs to Basidiomycetes, Agaricales, Tricholomataceae and Lentinus edodes, originates from China, is the second largest mushroom in the world, and is also a rare edible fungus which is popular in China for a long time. The first cultivation of the mushrooms in China has been over 800 years of history so far, and the yield of the mushrooms is the first in the world.

The growth process of the shiitake mushrooms specifically comprises three main stages, namely a first stage: the spores germinate into single-cell multi-core hyphae at certain temperature and humidity, and then a diaphragm is generated to form the multi-cell single-core hyphae; and a second stage: when the mononuclear hyphae grow to a certain stage, two different sexual mononuclear hyphae generate bulges at the part close to the mononuclear hyphae, protoplasm is fused together to become multi-cell binuclear hyphae, and then locking combination is carried out; and a third stage: when the binuclear hyphae after the locking combination grows to a certain physiological stage, a very dense hypha tissue is formed under a proper condition to form primordia of the fruiting body, and the primordia further develop into mushroom buds and finally develop into the fruiting body. Under natural conditions, it takes about 8-12 months or more to complete the whole growth process. After the artificial cultivation by using the wood chips, the whole growth process is shortened to 3-5 months, but the growth period is still longer.

At present, the variety of the lentinus edodes is various all over the country, but the work of breeding and oriented cultivation of excellent strains is less, and the reserved varieties are deficient, although the lentinus edodes strain breeding, domestication and the like are carried out in domestic colleges and universities, scientific research institutions and scientific and technological enterprises, most of the cultivation methods adopt protoplast fusion, molecular marking, mutagenesis, multi-spore and single spore hybridization and other means to carry out the breeding of the lentinus edodes strain, and the methods have the defects of complex operation process (for example, the new species is cultivated by adopting a single spore hybridization means, the complex operations of single spore separation, single spore hypha identification, single spore hypha pairing and the like are needed, the strain is easily infected in the operation process, the hybridization success rate is low), the breeding period is long, the success rate is low, and the like, so that the cultivation speed of the excellent lentinus edodes in China is low, and the reserved varieties are few.

Disclosure of Invention

Aiming at the problems of long breeding period, low success probability and the like of the conventional shiitake variety breeding method, the invention provides a shiitake cross breeding method.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

a crossbreeding method of Lentinus edodes comprises grinding fruiting body tissues of two parent Lentinus edodes strains for hybridization, adding 0.3-0.5 wt% NaCl solution, stirring to form paste, inoculating the paste into culture medium, culturing, and screening to obtain hybrid strain.

Compared with the prior art, the mushroom crossbreeding method provided by the invention directly grinds and crushes the sporophores of the two separated parents, and inoculates the crushed sporophores of the two parents into a culture medium under the protection of NaCl solution with specific concentration as a stabilizer after mixing, so that the crossbreeds of the two parents can be obtained under proper culture conditions. The mushroom crossbreeding method does not need special experimental environment, equipment and technicians, has simple operation method, short period and high success rate, can simply, conveniently and quickly breed and domesticate a new mushroom crossbred variety with the advantages of two parents, quickly promotes the development of mushroom industry, optimizes germplasm resources, promotes the yield and the efficiency of the industry and has extremely high application value.

Preferably, sterilized quartz sand with the mass 0.4-0.6 times of the fruiting body tissue is added in the grinding process.

The addition of the quartz sand can lead the grinding of the sporocarp to be more sufficient, thereby improving the grinding efficiency and further improving the bacteria yield of new hybrid hypha.

Preferably, the concentration of the NaCl solution is 0.4 wt%.

The above-mentioned preferred 0.4 wt% NaCl solution as a protective solution can improve the integrity and stability of the ground cells.

Preferably, the added mass of the NaCl solution is 0.8-1.2 times of the total mass of the fruit body tissue.

Preferably, the fruit body tissue is ground using a mortar which is sterilized and frozen in this order. The mortar frozen by the liquid nitrogen can reduce the temperature of the fruit body tissue, so that the fruit body tissue forms powder or fine and fast shape in the grinding process, thereby ensuring more sufficient grinding. The sterilizing method can be performed by using alcohol lamp flame.

Preferably, the formula of the culture medium is as follows: potato at 220g/L, glucose at 15-25g/L, KH₂PO₄2-4g/L，MgSO₄·7H₂0.4-0.6g/L of O, 18-22g/L of agar and natural pH.

Preferably, the temperature of the culture is 24-26 ℃.

Preferably, the new hyphae are screened using an antagonism assay.

Drawings

FIG. 1 is a photograph of antagonistic lines of ZJ1 and 168 strains in the antagonistic test of example 1 of the present invention;

FIG. 2 is a photograph of antagonistic lines of ZJ1 and 0912 strains in the antagonism assay of example 1 of the present invention;

FIG. 3 is a photograph of an antagonistic line of 168 strain as a control in the antagonistic test of example 1 of the present invention;

FIG. 4 is a photograph of the antagonism line on 0912 as a control in the antagonism assay of example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

1.1 materials

Parent lentinus edodes strain: the common varieties of the mushroom in northern areas are 168 and 0912.

Mother culture medium: 200g/L of potato, 20g/L of glucose and KH₂PO₄3g/L，MgSO₄·7H₂0.5 g/L of O, 20g/L of agar and natural pH.

Culture medium of cultivar: 78% of wood chips, 20% of wheat bran, 1% of gypsum, 1% of brown sugar, 55-60% of water content and natural pH.

Mushroom bag culture medium: 79% of wood chips, 20% of wheat bran, 1% of gypsum, 55-60% of water content and natural pH.

1.2 tools

An ultra-clean workbench, a scalpel, a mortar, an autoclave, tweezers, a seed receiving spoon, sterile water, a biochemical incubator and a plate culture dish.

1.3 methods

The first tide of shiitake mushroom 168 and shiitake mushroom 0912 produced by a shiitake mushroom production test mushroom stick of applied mushroom science and technology development limited company in Pingquan city are selected, and shiitake mushroom 168 and shiitake mushroom 0912 strains which are short in handle, large in cover, not easy to open and good in quality are selected as parent materials.

Placing the selected fruiting bodies of the strains of shiitake mushroom 168 and shiitake mushroom 0912 in a clean bench, sterilizing the surfaces of the fruiting bodies on an alcohol lamp flame, cutting off mushroom curtains and curling edges by using an operating blade, taking mushroom fold parts and meat parts with the depth of 0.5cm, placing the mushroom fold parts and the meat parts in a mortar subjected to alcohol lamp flame sterilization and freezing, controlling the total amount of two fruiting body tissue blocks to be 10g (the fruiting bodies of shiitake mushroom 168 is 5g, and the fruiting bodies of shiitake mushroom 0912 is 5g), then adding sterilized quartz sand into the mixture, starting grinding, grinding the tissue blocks into powder, adding 10g of 0.4 wt% NaCl solution into the mixture, uniformly stirring the mixture to form paste, transferring the paste to the center of a flat plate (mother culture medium) by using an inoculation spoon, placing the plate in a biochemical incubator at 25 ℃ for culture, and performing 3 times of rotary tube culture after hypha grow. And when the hypha growth circle reaches 2-5cm, timely transferring to a new plate culture medium for continuous culture, and primarily screening according to the growth density, color, growth speed and morphological characteristics of hypha to obtain 24 strains.

1.4 screening

Respectively carrying out antagonism tests on 24 different strains obtained by primary screening and original parent strains of shiitake mushroom 168 and shiitake mushroom 0912, respectively inoculating the 24 strains obtained by cross screening and the parents into a plate of a mother seed culture medium at the same time, carrying out constant temperature culture at 25 ℃, judging whether the filial generation is different from the two parents by observing whether an antagonism line exists between the filial generation and the parent strains in the plate, and further judging whether the cross strain is successfully crossed.

According to antagonism tests, 13 of 24 strains (ZJ1-ZJ13) have obvious antagonism lines with parents, taking ZJ1 as an example, the antagonism line between ZJ1 and parent mushroom 168 is shown in figure 1, and the antagonism line between ZJ1 and parent mushroom 0912 is shown in figure 2. The results of the antagonistic test of the control group shiitake mushroom 168 and the antagonistic test of the control group shiitake mushroom 0912 are shown in fig. 3 and fig. 4, respectively, using the parental shiitake mushroom 168 and the shiitake mushroom 0912 as the control tests. It is evident from FIGS. 1-2 that the ZJ1 hybrid strain produced a clear line of antagonism with both parental strains, whereas neither of FIGS. 3 and 4 produced a line of antagonism, demonstrating that the ZJ1-ZJ24 strains are successful hybrids.

Example 2

Selecting and culturing ZJ1, ZJ2, ZJ3, ZJ4, ZJ5 and ZJ6 in 13 strains, inoculating mycelia of ZJ1, ZJ2, ZJ3, ZJ4, ZJ5 and ZJ6 into a mother culture medium, and culturing at 25 ℃ to obtain mother culture mycelia.

Inoculating the mother strain mycelia into culture medium of cultivar, culturing at 25 deg.C and humidity of 70%, and collecting the cultivar mycelia when the mycelia are covered with the cultivar culture medium.

A polyethylene corner-folded bag with a bag size of 17cm × 60cm × 0.005cm is adopted, 1150g of mushroom bag culture medium is filled in each bag, and the mushroom bag is sterilized under normal pressure (100 ℃, kept for 24 hours and taken out of the furnace). Inoculating hypha of the culture strain when the temperature of the culture medium in the center of the culture bag is lower than 20 ℃, and culturing in a spawn running room with the temperature of 25 ℃ and the relative humidity of air of 70%. The fungus bag is full of hypha and burred with large holes after the fungus bag grows into a tumor-shaped object, and the fungus growing period is set to be 120 days. When the 'information mushroom' appears, putting the mushroom on shelf and entering fruiting period management, and the fruiting management after putting the mushroom on shelf is carried out according to a conventional method. Two

parental strains

168 and 0912 were set simultaneously as controls.

The fruiting conditions of different mushroom strains are monitored, and the monitoring results are shown in table 1:

TABLE 1 fruiting status of Lentinus edodes strains

As can be seen from Table 1, ZJ1-ZJ6 were all higher in anti-contamination ability than the control 168 strain and 0912 strain. The fruiting rate of ZJ1-6 is higher than that of the parent strains 168 and 0912, and the hybrid superiority of the new strain is shown.

The growth and development time, the fruiting body characters and the yield of different lentinus edodes strains are monitored, and the monitoring results are shown in table 2:

table 2 growth and development time, fruiting body characteristics and yield of the strains

As can be seen from Table 2, the full-bag times of the initial strains of different strains are not much different, but are within 58-65 days, but the differentiation time from the initial strain to the primordium is much different, and the primordium differentiation time of all the new hybrid strains is between the parental strains 168(150 days) and 0912(86 days), and is within 108-; compared with the control strains 168 and 0912, the hybrid new strain has small difference in the characteristics, color, diameter, pileus thickness and the like of pileus, but is superior to the control strains. The generation form of the fungus bag mushroom buds is closer to that of the 168 strains with long fungus age, and the fungus bag mushroom buds are single-grown and have less appearance, thereby establishing the advantages of large fruiting body and large single mushroom weight of the new hybrid strain. The differences in biological conversion rate are obvious, the new hybrid strain can reach 92.0%, the 168 strain with longer age is 11.4% higher, the 0912 strain with shorter age is 15.2% higher, and the hybrid advantages are fully embodied.

The cultivation test results show that significant difference exists between the new hybrid strain and the parent strain, the new hybrid strain is determined to be the new strain, and the feasibility of the hybrid method is proved.

In conclusion, the mushroom crossbreeding method disclosed by the invention can realize parent crossbreeding with high success rate only by using simple operation tools such as a scalpel, a mortar, an autoclave, tweezers, a seed inoculating spoon and the like in an ultra-clean workbench without special equipment and technicians with special skills, is simple in operation method, short in period and high in success rate, can simply, conveniently and quickly domesticate a new mushroom crossbreed variety with the advantages of two parents, can quickly promote the development of mushroom industry, optimize germplasm resources and promote the yield and efficiency of the industry, and has extremely high application value.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A crossbreeding method of mushroom is characterized in that: grinding the fruiting body tissues of two parent mushroom strains for hybridization together, adding 0.3-0.5 wt% NaCl solution after grinding, stirring to form paste, inoculating the paste into a culture medium for culture, and screening new mycelia obtained by culture to obtain hybrid varieties;

adding sterilized quartz sand with the mass 0.4-0.6 times of that of the sporocarp tissues in the grinding process;

the added mass of the NaCl solution is 0.8-1.2 times of the total mass of the sporocarp tissues;

grinding the sporocarp tissues by using a mortar which is sterilized and frozen in sequence;

the formula of the culture medium is as follows: potato at 220g/L, glucose at 15-25g/L, KH₂PO₄ 2-4g/L，MgSO₄·7H₂0.4-0.6g/L of O, 18-22g/L of agar and natural pH.

2. The crossbreeding method of shiitake mushroom according to claim 1, characterized in that: the concentration of the NaCl solution was 0.4 wt%.

3. The crossbreeding method of shiitake mushroom according to claim 1, characterized in that: the temperature of the culture is 24-26 ℃.

4. The crossbreeding method of shiitake mushroom according to claim 1, characterized in that: screening the new hyphae by adopting an antagonistic test.