CN115644062B

CN115644062B - Culture medium and method for improving induction rate of embryo of difficult-to-embryo Chinese cabbage

Info

Publication number: CN115644062B
Application number: CN202211371891.2A
Authority: CN
Inventors: 张红; 王超楠; 黄志银; 范伟强; 张斌; 刘晓晖
Original assignee: Tianjin Kerun Agricultural Science & Technology Co ltd
Current assignee: Tianjin Kerun Agricultural Science & Technology Co ltd
Priority date: 2022-11-03
Filing date: 2022-11-03
Publication date: 2023-11-17
Anticipated expiration: 2042-11-03
Also published as: CN115644062A

Abstract

The application relates to the technical field of plant tissue culture, in particular to a culture medium for improving embryo induction rate of a difficult-to-embryo Chinese cabbage and a method thereof. Wherein the culture medium of the application is prepared by adding griseofulvin with the concentration of 0.05-2.0mg/L into 1/2NLN culture medium. The scheme provides a method for improving the induction rate of microspore embryo of tested Chinese cabbage by adding the antibiotic griseofulvin into the culture medium for the first time, the method not only can remarkably improve the embryo emergence rate of difficult-to-come-out Chinese cabbage materials and break the limitation of genotype on microspore embryo induction to a certain extent, but also can reduce the pollution rate in the microspore culture process, is beneficial to establishing a Chinese cabbage microspore efficient culture system, and accelerates the innovation of Chinese cabbage germplasm and the breeding process.

Description

Culture medium and method for improving induction rate of embryo of difficult-to-embryo Chinese cabbage

Technical Field

The application relates to the technical field of plant tissue culture, in particular to a culture medium for improving embryo induction rate of a difficult-to-embryo Chinese cabbage and a method thereof.

Background

Chinese cabbage (Brassica rapa L.ssp. Pekinensis) is a two-year old leaf vegetable belonging to Brassica species of Brassicaceae. According to incomplete statistics, the annual sowing area of the Chinese cabbage is approximately 267 kilohms 2, and the Chinese cabbage occupies about 15 percent of the total sowing area of the Chinese vegetables, and is one of the vegetable crops with the largest cultivation area in China. In recent years, along with the change of the vegetable industry mode and the people consumption mode in China, the demands of the market on the bolting-resistant Chinese cabbages suitable for winter and spring cultivation are continuously increased, but the domestically bred bolting-resistant Chinese cabbages have relatively narrow germplasm resources due to relatively late starting of breeding research, and have obvious gaps between the bolting-resistant and commodity properties and foreign varieties, so that the current urgent need is to continuously improve the gathering and utilization consciousness of the bolting-resistant resources of breeders, and strengthen the skill of accelerating the creation and purification of high-quality germplasm resources by applying the efficient breeding technology.

Free microspore culture is a high-efficiency regeneration system developed on the basis of anther culture. The method mainly comprises the steps of culturing haploid microspores, doubling chromosomes, embryogenesis and the like, and a large amount of homozygous inbred line materials with rich genotypes and stable inheritance can be obtained within 1-2 years, so that a more efficient way is provided for new germplasm creation and new variety breeding.

At present, microspore embryo emergence is mainly stimulated by pretreatment and adding additives such as hormone, active carbon and the like with different proportions into a culture medium in the culture process of the brassica microspores, but in actual application, whether microspore embryo induction is successful or not is greatly influenced by genotype. As for the cabbages, researches show that the embryo induction rate and the number of cotyledon embryos of the varieties of the wrung shell type cabbages are superior to those of overlapped and enfolded varieties, the embryo induction rate of microspore embryos of the heat-resistant early-matured varieties of the cabbages in summer and autumn and the late-matured varieties of the winter cabbages is obviously higher than those of the early-matured varieties of the bolting resistance of Yu Chunxing. For varieties which are difficult to be embryo-out, such as bolting resistant types, the embryo-out rate of the microspores is difficult to find by a system or method with obvious effect. The present application has been made in order to overcome the above-mentioned drawbacks.

Disclosure of Invention

The first aim of the application is to provide a culture medium for improving the induction rate of the embryo of the difficult-to-come-out Chinese cabbage, which can effectively reduce the pollution rate in the culture process and improve the induction success rate of the embryo of the test material.

The second aim of the application is to provide a method for improving the induction rate of the embryo of the cabbages difficult to be produced, so as to solve the technical problem that the free microspores of the cabbages of the bolting-resistant type are cultured and difficult to be produced.

The application is realized by the following technical scheme:

in a first aspect, the application provides a culture medium for improving the induction rate of embryo of difficult-to-embryo Chinese cabbage, which is prepared by adding griseofulvin with the concentration of 0.05-2.0mg/L into 1/2NLN culture medium.

Further, in a preferred embodiment of the present application, the concentration of griseofulvin in the medium is 0.1-1.0mg/L.

Further, in a preferred embodiment of the present application, the concentration of griseofulvin in the medium is 0.5mg/L.

In a second aspect, the application provides a method for improving induction rate of embryo of difficult-to-embryo Chinese cabbage, which comprises the steps of picking buds from a primary flowering stage to a full flowering stage, processing to obtain purified microspores, and adding griseofulvin with concentration of 0.05-2.0mg/L into a 1/2NLN culture medium to obtain the culture medium for culturing the microspores.

Further, in a preferred embodiment of the present application, the treatment steps of the flower buds sequentially include disinfection and grinding;

further, in a preferred embodiment of the present application, the treatment step of the flower buds before sterilization further comprises a pre-cooling treatment.

Further, in a preferred embodiment of the present application, the flower bud is a single-core borderline flower bud.

Further, in the preferred embodiment of the present application, the sterilization means includes alcohol sterilization and sodium hypochlorite sterilization;

the grinding method is that the sterilized flower buds are poured into a B5 washing culture medium to be ground and extruded, so that pollen is fully released in the culture medium to obtain microspore solution, and the supernatant is poured out to obtain purified microspores.

Further, in the preferred embodiment of the application, the pre-cooling treatment is carried out at a temperature of 4-8 ℃ for 24-72 hours.

Further, in a preferred embodiment of the present application, the treated purified microspores are subjected to a resuspension dilution with a medium to obtain a purified microspore-containing density of 1X 10 ⁵ -2×10 ⁵ After each/mL of suspension, culture was performed.

Further, in a preferred embodiment of the application, the suspension is subjected to a heat shock treatment at a temperature of 30-35℃for a period of 24-72 hours prior to the culturing.

Compared with the prior art, the application has at least the following technical effects:

the application develops a culture medium for improving the embryo induction rate of the difficult-to-embryo Chinese cabbage, and harmful substances (such as phenols, abscisic acid and the like) fungal interference and the like can be generated due to the increment and growth of cells in the microspore culture process, so that the occurrence of embryos is inhibited. The experiment discusses the application of griseofulvin in microspore culture for the first time, and by adding the antibiotic griseofulvin into a 1/2NLN culture medium, the pollution and poison of harmful substances such as fungi and the like generated in the microspore culture process to microspores are inhibited, so that certain materials which are difficult to produce embryos are free from the restriction of genotypes, and the induction of microspore embryos is facilitated. According to the method for improving the induction rate of the embryo of the difficult-to-embryo Chinese cabbage, provided by the application, the griseofulvin is added into the culture medium, so that the embryo emergence rate of the tested Chinese cabbage can be obviously improved, the pollution rate in the process of culturing the free microspores can be reduced, a more efficient Chinese cabbage free microspore culture system can be established, and the researches on the germplasm innovation and haploid breeding of the Chinese cabbage are accelerated.

Drawings

FIG. 1 is a graph showing the effect of microspores of test materials of different genotypes under the same culture condition in comparative example 1.

In FIG. 1, the embryo of the test material D2 (FIG. A), the material D4 (FIG. C) and the material D8 (FIG. B) is successfully induced by the culture method of the microspores of the Chinese cabbage without adding griseofulvin.

FIG. 2 is a graph showing the effect of the experimental example 5 of the present application on the development of microspores before and after the addition of griseofulvin.

Test material D12 without griseofulvin added to the medium of the left panel of fig. 2 did not induce embryo formation.

The right panel of FIG. 2 shows that the medium supplemented with 1.0mg/L griseofulvin successfully induced the embryo emergence of test material D12.

Detailed Description

Embodiments of the present application will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the application, but are not intended to limit the scope of the application to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.

The embodiment provides a culture medium for improving the induction rate of embryo of difficult-to-embryo Chinese cabbage, which is characterized in that griseofulvin with the concentration of 0.05-2.0mg/L is added into 1/2NLN culture medium;

further preferably, the concentration of griseofulvin is 0.1-1.0mg/L.

Further preferably, the concentration of griseofulvin is 0.5mg/L.

The embodiment provides a method for improving induction rate of difficult-to-embryo Chinese cabbage embryos, which comprises the following steps:

step S1: selecting a bolting-resistant Chinese cabbage variety for planting, picking buds from a primary flowering period to a full flowering period, processing to obtain purified microspores, and culturing the purified microspores, wherein a culture medium is adopted in microspore culture, and the culture medium is a griseofulvin with the concentration of 0.05-2.0mg/L added into a 1/2NLN culture medium;

further preferably, the concentration of griseofulvin is 0.1-1.0mg/L.

Further preferably, the concentration of griseofulvin is 0.5mg/L.

Wherein, during microspore culture, harmful substances (such as phenols, abscisic acid, etc.) fungal interference, etc. can be generated due to proliferation and growth of cells, thereby inhibiting embryo occurrence. Griseofulvin is a chlorine-containing non-polyene antifungal antibiotic obtained from culture broth of Penicillium griseofulvum (Penicillium griseofulvin) by Oxford et al, and is widely used for treating skin and stratum corneum fungal infections. With the intensive research on the metabolism mechanism of griseofulvin, the griseofulvin has obvious inhibition effect on plant fungal diseases. The experiment discusses the application of griseofulvin in microspore culture for the first time, and by adding the antibiotic griseofulvin into a 1/2NLN culture medium, the pollution and poison of harmful substances such as fungi and the like generated in the microspore culture process to microspores are inhibited, so that certain materials which are difficult to produce embryos are free from the restriction of genotypes, and the induction of microspore embryos is facilitated.

Step S2: the picked flower buds are sterilized, ground and filtered in sequence to obtain purified microspores;

further, the sterilization modes comprise alcohol sterilization and sodium hypochlorite sterilization; the disinfection mode is a common disinfection mode, and the disinfection method is simple, safe and convenient to implement.

Further, in the preferred embodiment of the application, the buds are sterilized with 70-75% alcohol for 20-40s and 10-12% sodium hypochlorite solution for 10-12min.

Further, in a preferred embodiment of the present application, the sterilized flower buds are rinsed with sterile water; wherein the disinfecting solution is washed with water to prevent sodium hypochlorite residual liquid from corroding flower buds, and the purpose of the disinfecting solution is sodium hypochlorite which is a chlorine bleaching agent and has corrosiveness

Further, the grinding mode is that the sterilized flower buds are poured into 10-12mL of B5 washing culture medium and ground and extruded to fully release pollen in the culture medium to obtain microspore solution, and the supernatant is poured to obtain purified microspores. The washing culture mediums are multiple, but the embryo yield is higher after the screening of the test screening by using the B5 washing culture medium.

Further, in a preferred embodiment of the present application, after filtration of the microspore solution obtained in the washing medium, the purified microspores are obtained by pouring out the supernatant after centrifugation in a vessel containing the B5 washing medium. The purpose is to remove larger tissue residues by adopting a centrifugal mode, the obtained purified microspores are purer, and the subsequent microspore embryo yield results are more accurate.

Further, in a preferred embodiment of the present application, the sterilization step is preceded by a microscopic examination and pretreatment;

wherein the pre-cooling treatment temperature is 4-8deg.C, and the pre-cooling time is 24-72 hours. Wherein, the picked flower buds are subjected to low-temperature treatment, the development path of gametophytes scheduled by microspores can be effectively interrupted, the development path of sporophytes of the microspores is started, the occurrence frequency of microspore callus or embryos is greatly improved, and the dedifferentiation is most effective after 24-72 hours of environmental precooling treatment at 4-8 ℃. Wherein too long a time will reduce microspore activity.

Further, in a preferred embodiment of the application, the best quality of buds at the single-core border can be screened by microscopic examination. The flower bud selection is the basis and key of microspore culture technology, researches show that the induction rate of microspore embryoid embryos is highest in a single-core side stage, and the length of the flower buds is determined after microscopic examination, so that the accuracy of flower bud selection is improved, the workload is reduced (the subsequent one-to-one microscopic examination step is avoided for screening the flower buds), and the test efficiency is improved.

Further, in a preferred embodiment of the present application, the length of the picked flower buds is 2.5-4mm. The method aims at solving the problems that the time and the labor are inevitably wasted when a plurality of test materials are observed by a microscope to select flower buds, so that the length of the flower buds is the simplest method for selecting flower buds in microspore culture, but the size range of the flower buds of different materials is relatively wide. The scheme is obtained through long-term experiments, and the embryo emergence effect is best when the bud length of the bud sample in the single-core approach period is 2.5-4mm.

Step S3: the purified microspores obtained after step S2 are subjected to resuspension dilution with a medium to obtain a purified microspore-containing culture medium having a density of 1X 10 ⁵ -2×10 ⁵ After each/mL of the suspension, the suspension was packed and cultured.

The culture density of microspores is also an important culture condition affecting embryo induction, and too high and too low microspore concentrations are unfavorable for embryo development. When the density is too high, a large amount of nutrient substances are consumed, and the concentration of the released toxic substances is increased; microspore development has a population effect, and too low a density can also affect microspore embryo development.

Further, in a preferred embodiment of the application, the packaged microspore suspension is sealed, heat shocked at 30-35℃for 24-72 hours, and then transferred to a incubator at 25℃for dark culture. Wherein, the high temperature heat shock can effectively improve the symmetrical division ratio of microspores and the embryogenic capacity of the microspores in the differentiation start of the microspores.

Step S4: after culturing in the dark for 2 weeks in an incubator, counting the number of embryoids visible to the naked eye.

The following describes specific embodiments of the present application in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the application, are not intended to limit the application.

1. Test materials

TABLE 1 Donor plant numbering and designation

Numbering device	Name of the name	Numbering device	Name of the name
				Code	Name	Code	Name
D1	Top-up	D13	CR-Chunjia
				D2	Starlight	D14	CR-Minxi
D3	Green baby	D15	CR-Jin Li
				D4	Strong root 118	D16	CR spring jade
D5	C3	D17	Golden armor
				D6	Tulong (Tulong)	D18	Cold-resistant gold queen
D7	Jingchun CR3	D19	Bright
				D8	Golden tripod CR	D20	Spring of North China
D9	CR Fu will	D21	19 general and exquisite jade
				D10	Degao 69	D22	Exquisite yellow 012
D11	Jinjin Chinese cabbage	D23	Moon shy 156
				D12	CR-Shunmei

TABLE 2 Main reagents

Table 3 experiment apparatus

Name of the name	Company (Corp)
		60mm x 15mm sterile plastic culture dish	Huada Gene technologies Co., ltd
Constant temperature incubator	NINGBO SAIFU EXPERIMENTAL INSTRUMENT Co.,Ltd.
		Beaker	Acciaierie Oqi SpA
Microscope	Nikon Co Ltd
		Vitality analyzer AmphaTMZ32	ZEALQUEST SCIENTIFIC TECHNOLOGY Co.,Ltd.

2. Test verification

The test collection and introduction of 23 varieties with excellent commodity and difficult embryo emergence (bolting resistance), the variety information is shown in table 1, the materials to be tested are sown at a Wu Qing test base of Tianjin vegetable institute in 3 sowing periods of 2021, 12, 29, 1, 15, so as to prolong the optimal collection period of the flower buds, and the flower buds are taken at the end of 2021 and at the end of 3 to 5 months for free microspore culture.

1. The influence of different genotypes on microspore embryo emergence rate is verified, and meanwhile, the method for improving the induction rate of the embryo of the difficult-to-emergence Chinese cabbage is provided, which comprises the following specific steps:

(1) Picking the buds of the Chinese cabbage in sunny weather from the initial flowering period to the full flowering period of the plant;

picking cabbage flower buds, microscopic examination, selecting 30 flower buds in a single-core side period, wherein flower buds with the length of 2.5-4mm can be selected in advance, so that the flower buds which meet the standard can be placed in an environment with the temperature of 4 ℃ for precooling treatment for 24 hours as the standard for picking subsequent flower bud samples.

(2) After precooling treatment, washing the table top with distilled water for several times, irradiating the table top with an ultraviolet sterilizing lamp for 30min, then placing the selected flower buds in a sterile beaker, sterilizing the flower buds with 75% alcohol for 30s and 10% sodium hypochlorite solution for 10min in sequence, and washing the flower buds with sterile water for 3 times and 3-5 min each time.

(3) Pouring 10ml of B5 washing culture medium into a flask, lightly squeezing the mixture by using a sterilized pestle to disperse pollen, filtering the mixture, and transferring the mixture to a centrifuge tube; adding 10ml of B5 culture medium into a centrifuge tube, centrifuging at 1000rpm for 2-3min, and pouring out supernatant; and repeating the centrifugation for 1 time according to the steps, and pouring out the supernatant to obtain a precipitate which is the purified microspores required by the target.

(4) Resuspension of the purified microspores obtained in step (3) with 1/2NLN-13 medium, and adjusting the density of the purified microspore suspension to 1×10 ⁵ -2×10 ⁵ And each mL.

(5) The purified microspore suspension with adjusted density is split into sterile plastic dishes of 60mm by 15mm, sealed with Parafilm, heat-shocked at 32℃for 24h, and then transferred to a dark culture at 25℃with constant temperature.

(6) After 2 weeks of dark culture, the number of embryoids was counted after obtaining macroscopic embryoids. The embryoid numbers were counted 1 time every 7 days for 5 times, and the data shown in Table 4 were obtained.

TABLE 4 embryo emergence of microspores of Chinese cabbage with different genotypes

Genotype of the type	Embryo yield/(embryo bud-1)	Genotype of the type	Embryo yield/(embryo bud-1)
				D1	0d	D13	0d
D2	0.344b	D14	0d
				D3	0d	D15	0d
D4	0.200c	D16	0d
				D5	0d	D17	0d
D6	0d	D18	0d
				D7	0d	D19	0d
D8	0.500a	D20	0d
				D9	0d	D21	0d
D10	0d	D22	0d
				D11	0d	D23	0d
D12	0d

Note that: single factor analysis of variance is carried out by adopting Duncan's new complex polar difference method, and the difference represented by different lowercase letters in the same column reaches a significant difference level

(P<0.05)。

The experimental data confirm that only D2, D4 and D8 varieties of selected bolting-resistant cabbages with different genotypes successfully induce embryo, and the verification experiment proves that the embryo emergence rates of the test materials with different genotypes are obviously different, and the varieties selected in the scheme are varieties difficult to develop embryo. The embryo-out diagram is shown in figure 1

Example 1

The embodiment provides a culture medium for improving the induction rate of embryo of difficult-to-embryo Chinese cabbage, wherein griseofulvin with the concentration of 0.05mg/L is added into 1/2NLN culture medium to obtain culture medium 1.

The embodiment provides a method for improving the induction rate of the embryo of the difficult-to-embryo Chinese cabbage

In this example, 19 parts of cabbage material with difficult embryo emergence except for D2, D4, D8 and D12 were selected as the test materials.

picking cabbage flower buds, microscopic examination, selecting 30 flower buds in a single-core side period, wherein flower buds with the length of 2.5-4mm are selected in advance so as to be used as the standard for picking subsequent flower bud samples, and the flower buds meeting the standard are placed in a 4 ℃ environment for precooling treatment for 24 hours.

(2) The flower buds after precooling treatment are washed by distilled water for several times, the table top is irradiated by an ultraviolet sterilizing lamp for 30min, then the selected flower buds are placed in a sterile beaker, the flower buds are sterilized by 73% alcohol for 35s and 10% sodium hypochlorite solution for 12min in sequence, and the flower buds are washed by sterile water for 3 times each for 5 min.

(3) Pouring 10ml of B5 washing culture medium into a flask, lightly squeezing the mixture by using a sterilized pestle to disperse pollen, filtering the mixture, and transferring the mixture to a centrifuge tube; adding 10ml of B5 culture medium into a centrifuge tube, centrifuging at 1000rpm for 2min, and pouring out supernatant; and repeating the centrifugation for 1 time according to the steps, and pouring out the supernatant to obtain a precipitate which is the purified microspores required by the target.

(4) Resuspension of the purified microspores obtained in step (3) with medium 1, and adjusting the density of the purified microspore suspension to 1X 10 ⁵ -2×10 ⁵ And each mL.

(5) The purified microspore suspension with adjusted density is split into sterile plastic dishes of 60mm by 15mm, sealed with Parafilm, heat-shocked at 32℃for 48h, and then transferred to a dark culture at 25℃with constant temperature.

(6) After a culture period of 2 weeks, counting the number of embryoids after obtaining macroscopic embryoids. Counting the embryoid number 1 time every 7 days, counting 5 times,

example 2

The embodiment provides a culture medium for improving the induction rate of embryo of difficult-to-embryo Chinese cabbage, wherein griseofulvin with the concentration of 0.1mg/L is added into 1/2NLN culture medium to obtain culture medium 2.

The embodiment provides a method for improving the induction rate of the embryo of the difficult-to-embryo Chinese cabbage, and the steps of the method are the same as those of the embodiment 1.

Example 3

The embodiment provides a culture medium for improving the induction rate of embryo of difficult-to-embryo Chinese cabbage, wherein griseofulvin with the concentration of 0.5mg/L is added into 1/2NLN culture medium to obtain culture medium 3.

Example 4

The embodiment provides a culture medium for improving the induction rate of embryo of difficult-to-embryo Chinese cabbage, which is obtained by adding griseofulvin with the concentration of 1.0mg/L into a 1/2NLN culture medium to obtain a culture medium 4.

Example 5

The embodiment provides a culture medium for improving the induction rate of embryo of difficult-to-embryo Chinese cabbage, which is obtained by adding griseofulvin with the concentration of 2mg/L into a 1/2NLN culture medium to obtain a culture medium 5.

Comparative example

The control example provides a method for improving the embryo induction rate of the cabbages difficult to embryo, which is used as test materials, wherein the test materials are the same as those in the example 1, and 19 parts of the cabbages difficult to embryo except for D2, D4, D8 and D12 are selected as the test materials.

The procedure of this control was the same as in example 1, except that the selection of the medium was changed in the (4) th step, and 1/2NLN medium was used.

The statistics of the embryo yield of the induced embryo in the culture medium with different concentrations of griseofulvin in examples 1-5 and comparative example are shown in Table 5

TABLE 5 embryogenesis of microspores with addition of griseofulvin

Table 5

(P<0.05)。

The data in the above table show that under the same culture conditions, compared with a control group without adding griseofulvin in the culture medium, the addition of griseofulvin enables 19 parts of materials difficult to be embryo-derived successfully to induce 16 parts of embryo materials, most of materials in 0.05-2.0mg/L of griseofulvin can induce embryo-derived, and when the concentration of griseofulvin is 0.5mg/L, the embryo-derived rate of 16 parts of materials is highest. From this, it can be seen that the addition of griseofulvin at the appropriate concentration can promote the development of embryoid bodies of the material.

In order to illustrate that the culture medium provided by the application and the culture medium adopted by the application have better embryo-producing effect, the following experiment is specially carried out:

experimental example 1

Wherein the procedure of experimental example was the same as in example 1, the medium selection was different only in the microspore culture stage. Wherein the culture medium is Melatonin (MT) which is a plant hormone added with auxin type in 1/2NLN-13 culture medium, and the concentration gradient is 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L; the statistical results are shown in the following table:

TABLE 6 embryogenesis of microspores with Melatonin (MT) addition

(P<0.05)。

Experimental example 2

Wherein the procedure of experimental example was the same as in example 1, the medium selection was different only in the microspore culture stage. Wherein the culture medium is 1/2NLN culture medium, and the cytokinin type phytohormone is Kinetin (KT) and has concentration gradient of 0.05mg/L, 0.1mg/L, 0.2mg/L, 0.4mg/L, and 0.5mg/L; the statistical results are shown in the following table:

TABLE 7 embryogenesis of microspores with Kinetin (KT) addition

(P<0.05)。

Experimental example 3

Wherein the procedure of experimental example was the same as in example 1, the medium selection was different only in the microspore culture stage. Wherein the culture medium is 1/2NLN culture medium added with sodium butyrate (NaB) as an acetylase inhibitor, and the concentration gradient is 0, 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L; the statistical results are shown in the following table:

TABLE 8 embryogenesis of microspores with sodium butyrate (NaB) addition

(P<0.05)。

Experimental example 4

The procedure of this experimental example was the same as in example 1, except that the medium selection was only in the microspore culture stage. Wherein the culture medium is prepared by adding heavy metal additive silver nitrate (AgNO 3) into 1/2NLN culture medium, and the concentration gradient is 0, 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L; the statistical results are shown in the following table:

TABLE 9 embryogenesis of microspores with silver nitrate (AgNO 3) addition

Note that: single factor analysis of variance was performed using the new complex polar difference method of duncan's, with different lowercase letters in the same column indicating significant level of difference (P < 0.05).

Experimental example 5

The procedure of this experimental example was the same as in example 1, except that the medium selection was only in the microspore culture stage. Wherein the culture medium is 1/2NLN culture medium added with griseofulvin with concentration gradient of 0, 0.05mg/L, 0.1mg/L, 0.5mg/L, 1.0mg/L, and 2.0mg/L; the statistical results are shown in the following table:

TABLE 10 embryogenesis of microspores with griseofulvin addition

Experimental example 6

The procedure of this experimental example was the same as in example 1, except that the medium selection was only in the microspore culture stage. Wherein the culture medium is 1/2NLN culture medium added with antibiotics which are amphotericin B, and the concentration gradient is 0, 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L; the statistical results are shown in the following table:

TABLE 11 embryogenesis of microspores with amphotericin B addition

As can be seen from the above 1-6 experimental examples, the material which is easier to embryo out of D2 shows inhibition effect on embryo out of material 2 after different inducers such as sodium butyrate and silver nitrate are added, and the embryo out of D12 cannot be induced. Some inducers exhibit an enhancing effect on material embryo emergence, such as melatonin and kinetin, but less in embryo emergence rate. The addition of griseofulvin can promote the embryo yield and the embryo yield is higher.

Experimental example 7

Influence on embryo induction rate after adding griseofulvin in different culture medium classes

The procedure of this experimental example was the same as in example 1, except that griseofulvin was added only to a different type of medium selected during the microspore culture stage, and the concentration of griseofulvin was 0.5mg/L. Wherein the culture medium is selected from NLN culture medium and 1/2NLN culture medium

TABLE 12 embryogenesis of microspores with griseofulvin addition for different media categories

From the table, 2 different types of culture mediums added with griseofulvin successfully induce 2 parts of bolting-resistant material to be embryo-out, wherein the induction rate of 1/2NLN culture medium embryo is higher.

Experimental example 8

Influence of different bud picking periods on embryo induction

Under the same conditions as in example 1, D2, D4 and D8 were used as test materials, and the buds were harvested in the early flowering stage, the full flowering stage and the final flowering stage, respectively, and the suitable material-harvesting period was determined.

The initial flowering period refers to 50% of flowers from the first layer of flowers on the main flower stem to the first branch; full bloom refers to the occurrence of flower buds with weaker growth from 50% of flowers on the first branch to the top of most of the lateral branches; the final flowering period refers to the stop of the development of the buds from the occurrence of the weaker buds at the top of most of the lateral branches to the top of the lateral branches. The influence of buds in different periods on embryo induction rate is determined, and the statistical results are shown in the following table:

TABLE 13 influence of bud picking period on the induction rate of microspore embryos of Chinese cabbage

Note that: single factor analysis of variance (a variance) was performed using Duncan's new complex polar method, where the difference was significantly different in the same column with different lower case letters (P < 0.05)

From the table, the embryo induction rate of the bud picking in the early flowering stage is highest, and the difference between the embryo induction rate and the embryo induction rate in the full flowering stage and the embryo induction rate in the final flowering stage reaches a remarkable level. And the second stage is the full bloom stage, and the lowest induction rate is the final bloom stage. The bud picking period has extremely remarkable influence on the induction of microspore embryos of Chinese cabbage, and the initial flowering period is the most suitable bud picking period.

Experimental example 9

Influence of the Low temperature pretreatment time on the embryogenesis of white cabbage

Picking flower buds with proper sizes by taking D2, D4 and D8 as test materials, carrying out low-temperature pretreatment in a refrigerator at 4 ℃, setting 24h, 48h and 72h for 3 treatments, taking non-low-temperature treatment as a control, and analyzing the influence of the low-temperature pretreatment on embryo induction. The statistical results are shown in the following table:

influence of surface 14 Cold treatment time on the Induction Rate of microspore embryo of Chinese cabbage

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As can be seen from the table, the embryoid induction rate of the Chinese cabbage can be improved by performing proper low-temperature pretreatment, the embryoid induction rate is highest by taking 4 ℃ treatment for 24 hours as the most suitable treatment, and the embryoid induction rate begins to decrease after the treatment time exceeds 24 hours, which indicates that the long-time low-temperature treatment has an inhibition effect on the activity of microspores.

Experimental example 10

Influence of high-temperature heat-shock treatment time on embryogenesis of white vegetables

Picking flower buds with proper sizes by taking D2, D4 and D8 as test materials, carrying out high-temperature heat shock treatment in a constant temperature box at 32+/-1 ℃, setting 24 hours, 48 hours and 72 hours for 3 treatments, taking the non-heat shock treatment as a control, and analyzing the influence of the high-temperature heat shock treatment on embryo induction. The statistical results are shown in the following table:

TABLE 15 influence of heat shock treatment time on the induction rate of microspore embryos of Chinese cabbage

As can be seen from the table, the high-temperature pretreatment can improve the embryoid induction rate of the Chinese cabbage, the embryoid induction rate is highest after the treatment is carried out for 48 hours at 32+/-1 ℃, and the embryoid induction rate begins to decrease after the treatment time exceeds 48 hours, which indicates that the long-time high-temperature treatment is unfavorable for embryogenesis of microspores.

Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the application and is not intended to limit the scope of the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A culture medium for improving the embryo induction rate of difficult-to-embryo Chinese cabbage, which is characterized in that griseofulvin with the concentration of 0.05-2.0mg/L is added into a 1/2NLN culture medium; the Chinese cabbage difficult to embryo is CR Fujian, CR-Chunjia, lautful or photophobia 156.

2. The medium of claim 1, wherein the concentration of griseofulvin is 0.1-1.0mg/L.

3. The medium of claim 2, wherein the concentration of griseofulvin is 0.5mg/L.

4. A method for improving induction rate of embryo of difficult-to-come-out Chinese cabbage is characterized in that the method comprises the steps of picking buds from an initial flowering period to a full flowering period, obtaining purified microspores, and culturing the microspores by adopting a culture medium comprising: 1/2NLN+0.05mg/L griseofulvin;

wherein, the processing steps of the flower buds sequentially comprise disinfection and grinding; the step of pre-sterilization treatment of the flower buds further comprises pre-cooling treatment; the pre-cooling treatment temperature is 4 ℃, and the pre-cooling time is 24-48 hours; subjecting the treated purified microspores to resuspension dilution with a culture medium to obtain purified microspores having a density of 1×10 ⁵ -2×10 ⁵ Culturing after each/mL of suspension; carrying out heat shock treatment on the suspension before culturing, wherein the temperature of the heat shock treatment is 32+/-1 ℃ and the time is 24-48 hours;

the variety of the Chinese cabbage difficult to be embryo-removed is starlight or strong root 118.

5. The method for improving embryo induction rate of refractory Chinese cabbage of claim 4, wherein the flower buds are flower buds in a single-core approach period.

6. The method for improving the induction rate of difficult-to-embryo Chinese cabbage embryos according to claim 4, wherein the sterilization mode comprises alcohol sterilization and sodium hypochlorite sterilization;

the grinding mode is that the sterilized flower buds are poured into a B5 washing culture medium to be ground and extruded, so that microspores are released in the culture medium to obtain microspore solution, and the supernatant is poured to obtain purified microspores.