CN105917961B - Method for inducing fission of protoplast fusion nucleus in needle mushroom seed and normal formation of fruiting body - Google Patents

Abstract

The invention discloses a method for inducing the nucleus division and normal formation of fruiting bodies of protoplast fusion in needle mushroom seeds, which comprises the following steps: s1, preparing an induction culture medium: mixing testa Tritici and semen gossypii uniformly, adding water, placing into a glass bottle, and sterilizing at high temperature under high pressure; s2, inoculating and culturing: inoculating the cultured needle mushroom fused mother-daughter seeds into an induction culture medium for culture, and splitting the protoplast fused daughter nuclei; s3, inducing fruiting: and (4) fusing the inoculated and cultured needle mushrooms with the mother and son seeds for inducing fruiting to form sporocarp. The invention prepares the induction culture medium, inoculates and cultures and induces the fruiting to make the fused daughter cell nucleus of the flammulina velutipes protoplast form 2 cell nuclei from 1 division, and can normally form the fruiting body.

Description

Method for inducing fission of protoplast fusion nucleus in needle mushroom seed and normal formation of fruiting body

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for inducing the fission of protoplast fusion nuclei in needle mushroom seeds and the normal formation of fruiting bodies.

Background

The common microbial breeding technology mainly comprises the modes of natural separation, mutation screening, cell combination, protoplast fusion, gene introduction, gene disruption and the like, wherein the protoplast fusion refers to a process of fusing protoplasts of two cells with different genetic traits by a manual method so as to obtain a stable recon with the parental genetic traits. The breeding mode can break the species boundary limit of the microorganism, realize the gene recombination of distant strains, enable the transmission of genetic materials to be more complete, and obtain more chances of gene recombination, thereby combining more excellent characters into a single strain. The technology is emphasized by vast domestic and foreign edible fungi as breeding workers, particularly, the research progress of China, Japan and Korea is fast, and the fusants are obtained in seeds, seeds and genera successively, but the obtained fusants mainly comprise heterokaryons with cytoplasm fused and nucleus fused, and the poor stability is the biggest defect of the fusants.

The famous flammulina velutipes, namely, flammulina velutipes, broussonetia papyrifera, pleurotus eryngii, dried mushroom, pleurotus cornucopiae, frozen mushroom, golden mushroom, intelligent mushroom and the like, is known as follows, and English is: "Enoki Mushroom". The botanical name is Flammulina velutiper (Fr.) Sing. Because its stipe is slender and similar to that of gold needle mushroom, it is called gold needle mushroom, belonging to the order Agaricales, white mushroom family needle mushroom, and is a kind of fungi, algae and lichen. The needle mushroom has high medicinal food therapy effect. The selection of good strains of needle mushroom by using a protoplast fusion technology is the key point of the research of workers in the field, but the traditional method has the problem that the fusant cannot produce mushroom after the fusion of needle mushroom protoplasts.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for inducing the nucleus division and normal formation of fruiting bodies of protoplast fusion seeds in needle mushroom seeds.

The purpose of the invention is realized by the following technical scheme: a method for inducing the nucleus division and normal formation of fruiting bodies of protoplast fusion in needle mushroom seeds comprises the following steps:

s1, preparing an induction culture medium: uniformly mixing bran and cottonseed, adding water, filling into a glass bottle, sealing with a cotton plug, and sterilizing at 105-120 ℃ under 0.08-0.12 MPa for 2.5-3.5 h;

s2, inoculating and culturing: selecting needle mushroom fusant strains, transferring the needle mushroom fusant strains to a PDA culture medium, culturing for 8-10 days at 18-25 ℃ to obtain mother seeds, inoculating the mother seeds into the induction culture medium prepared in the step S1, culturing for 30-40 days at 20-25 ℃, and splitting the protoplast fusant nuclei;

s3, inducing fruiting: and (3) placing the inoculated and cultured needle mushroom fused mother-son seeds under the conditions of temperature of 10-18 ℃, relative humidity of 90-95% and illumination intensity of 10-50 Lux for inducing fruiting, and forming sporocarp, wherein the time for inducing fruiting is 50-60 days.

Further, the weight ratio of the bran, the cottonseed and the water in the step S1 is 1: 3-6: 2-4.

The invention has the following advantages: the traditional method has the technical problem that the fusant cannot produce mushroom after the fusion of the flammulina velutipes protoplast, so the key technology of the flammulina velutipes protoplast fusion breeding is to solve the problem that the karyon is formed by inducing the karyon to split, the invention prepares an induction culture medium, inoculates and cultures and induces the mushroom to enable the karyon of the flammulina velutipes protoplast fusion daughter to split from 1 to form 2 karyons and can normally form a sporocarp.

Drawings

FIG. 1 is a schematic diagram of induction of fruiting by fusant;

FIG. 2 is a schematic of the hyphal morphology of the fusions;

FIG. 3 is a schematic diagram showing the hyphal morphology of the fusions after induction.

Detailed Description

The invention is further described with reference to the following figures and examples, without limiting the scope of the invention to the following:

example 1: a method for inducing the nucleus division and normal formation of fruiting bodies of protoplast fusion in needle mushroom seeds comprises the following steps:

s1, preparing an induction culture medium: mixing testa Tritici and semen gossypii uniformly, adding water, placing into a glass bottle, sealing with a cotton plug, and sterilizing at 105 deg.C under 0.08MPa for 2.5 h; the weight ratio of the bran to the cottonseed to the water is 1:3: 2;

s2, inoculating and culturing: selecting needle mushroom fusant strains, transferring the needle mushroom fusant strains to a PDA culture medium, culturing for 8d at 18 ℃ to obtain mother strains, inoculating the mother strains into the induction culture medium prepared in the step S1, culturing for 30d at the temperature of 20 ℃, and splitting the protoplast fusant nuclei;

s3, inducing fruiting: and (3) placing the inoculated and cultured needle mushroom fused mother-son seeds under the conditions of 10 ℃ of temperature, 90% of relative humidity and 10Lux of illumination intensity to induce fruiting, so as to form fruiting bodies, wherein the time for inducing fruiting is 50 d.

Example 2: a method for inducing the nucleus division and normal formation of fruiting bodies of protoplast fusion in needle mushroom seeds comprises the following steps:

s1, preparing an induction culture medium: mixing testa Tritici and semen gossypii uniformly, adding water, placing into a glass bottle, sealing with a cotton plug, and sterilizing at 120 deg.C under 0.12MPa for 3.5 h; the weight ratio of the bran to the cottonseed to the water is 1:6: 4;

s2, inoculating and culturing: selecting excellent flammulina velutipes fusogenic strains, transferring the strains to a PDA culture medium, culturing for 10d at 25 ℃ to obtain mother seeds, inoculating the mother seeds into the induction culture medium prepared in the step S1, culturing for 40d at 25 ℃, and splitting the protoplast fusogenic daughter nuclei;

s3, inducing fruiting: and (3) placing the inoculated and cultured needle mushroom fused mother-son seeds under the conditions of the temperature of 18 ℃, the relative humidity of 95% and the illumination intensity of 50Lux for inducing fruiting, wherein the time for inducing fruiting is 60d, and forming sporocarp.

Example 3: a method for inducing the nucleus division and normal formation of fruiting bodies of protoplast fusion in needle mushroom seeds comprises the following steps:

s1, preparing an induction culture medium: mixing testa Tritici and semen gossypii uniformly, adding water, placing into a glass bottle, sealing with a cotton plug, and sterilizing at 110 deg.C and 0.09MPa for 3 hr; the weight ratio of the bran to the cottonseed to the water is 1:4: 3;

s2, inoculating and culturing: selecting needle mushroom fusant strains, transferring the needle mushroom fusant strains to a PDA culture medium, culturing for 8.5 days at 20 ℃ to obtain mother seeds, inoculating the mother seeds into the induction culture medium prepared in the step S1, culturing for 34 days at 22 ℃, and splitting the protoplast fusant nuclei;

s3, inducing fruiting: and (3) placing the inoculated and cultured needle mushroom fused mother-son seeds under the conditions of the temperature of 13 ℃, the relative humidity of 92% and the illumination intensity of 26Lux for inducing fruiting, and forming fruiting bodies, wherein the time for inducing fruiting is 54 d.

Example 4: a method for inducing the nucleus division and normal formation of fruiting bodies of protoplast fusion in needle mushroom seeds comprises the following steps:

s1, preparing an induction culture medium: mixing testa Tritici and semen gossypii uniformly, adding water, placing into a glass bottle, sealing with a cotton plug, and sterilizing at 115 deg.C under 0.103MPa for 3.2 h; the weight ratio of the bran to the cottonseed to the water is 1:5: 3;

s2, inoculating and culturing: selecting a needle mushroom fusant strain, transferring the needle mushroom fusant strain to a PDA culture medium, culturing for 9.5 days at 22 ℃ to obtain a mother strain, inoculating the mother strain into the induction culture medium prepared in the step S1, culturing for 36 days at 24 ℃, and splitting a protoplast fusant nucleus;

s3, inducing fruiting: and (3) placing the inoculated and cultured needle mushroom fused mother-son seeds under the conditions of 16 ℃, 94% of relative humidity and 42Lux of illumination intensity to induce fruiting, so as to form fruiting bodies, wherein the time for inducing fruiting is 58 d.

The following experiments illustrate the beneficial effects of the present invention:

1. dikaryon strain acquisition experiment

The fruiting bodies induced by fruiting in the examples 1-4 are subjected to tissue isolation culture to obtain new strains, and the isolation method comprises the following steps: the inner tissues of the pileus of the sporocarps are taken and put on a PDA culture medium to be cultured at the temperature of 20-25 ℃ for 8-10 days, and experiments show that the sporocarps of the examples 1-4 can be used for culturing new strains.

2. Strain identification method

The fusant strain cultured by tissue isolation needs to be identified as a double karyon, and the double karyon strain causing fruiting is only required to be identified if the hypha has the locked joint marks and the number of cell nucleuses in the cells is 2.

The identification method comprises the following steps: (1) lock-like joint mark observation

Placing mycelium of the strain subjected to tissue isolation culture in the step 1 on a glass slide, spreading the mycelium, covering the glass slide, observing under a microscope (400 times), and obtaining a double-core body which is in locked combination on the mycelium, namely the fusant with normal fruiting.

(2) Cell nucleus staining observation method

And (3) inserting a sterile cover glass on the outer side of the tissue isolated strain cultured on the culture dish, culturing at 25 ℃, and carrying out microscopic observation after the mycelium grows on the cover glass.

3. Experiment of fusion Induction

The number of fusions and the number of induced fruiting by fusions were counted in examples 1-4, and the results are shown in Table 1 and FIG. 1.

Table 1: protoplast fusion induced fruiting status

Name (R)	Number of fusant	Number of mushrooms (one)	Rate of induction
				Example 1	32	32	100％
Example 2	40	40	100％
				Example 3	38	38	100％
Example 4	35	35	100％

4. Experiment for separating and culturing fusant binuclear bodies

4.1 formula and preparation method of culture medium:

the formula is as follows: 200g of potato, 20g of glucose, 20g of agar and 1000ml of water.

The preparation method comprises the following steps: peeling potato, slicing, placing into a pot, adding 1000ml of water, heating and boiling for 30 minutes, filtering, taking filtrate, adding agar strips into the pot, boiling until the agar strips are completely melted, adding glucose, heating and stirring for melting, then supplementing water to 1000ml, subpackaging the culture medium into test tubes with the loading of 10ml, and plugging with a silica gel plug. Sterilizing in an autoclave at 121 deg.C under 0.103MPa for 30 min, placing on wood strips obliquely to form slant culture medium, and cooling to solidify to obtain slant culture medium.

4.2 strain separation method:

taking the fruiting body formed by induction, cutting internal tissue blocks at the intersection of the pileus and the stipe by using a sterile scalpel, putting the internal tissue blocks on a PDA culture medium, culturing for 5-6 days at 20 ℃, cutting tip strains, transferring the tip strains to the PDA culture medium, culturing for 8-10 days at 20 ℃, taking hyphae, observing the morphological characteristics of the hyphae under a microscope, wherein the hyphae have locked joints and 2 nuclei in cells, and thus obtaining the fusion which is successfully induced, as shown in figures 2 and 3. As can be seen from fig. 3: the fusant hypha morphology is nonclaviform union and only has 1 nucleus, and the fusant hypha morphology after induction has latiform union and 2 nuclei.

Claims (1)

1. A method for inducing the nucleus division and normal formation of fruiting bodies of protoplast fusion in needle mushroom seeds is characterized in that: it comprises the following steps:

s1, preparing an induction medium: uniformly mixing bran and cottonseed, adding water, filling into a glass bottle, sealing with a cotton plug, and sterilizing at 105-120 ℃ under 0.08-0.12 MPa for 2.5-3.5 h; the weight ratio of the bran to the cottonseed to the water is 1: 3-6: 2-4;

s2, inoculating and culturing: selecting needle mushroom fusant strains, transferring the needle mushroom fusant strains to a PDA culture medium, culturing for 8-10 days at 18-25 ℃ to obtain mother seeds, inoculating the mother seeds into the induction culture medium prepared in the step S1, culturing for 30-40 days at 20-25 ℃, and splitting the protoplast fusant nuclei;

s3, inducing fruiting: and (3) placing the inoculated and cultured needle mushroom fused mother-son seeds under the conditions of temperature of 10-18 ℃, relative humidity of 90-95% and illumination intensity of 10-50 Lux for inducing fruiting, and forming sporocarp, wherein the time for inducing fruiting is 50-60 days.

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