阅读说明：本技术 一种通过热处理方式来提高cenh3介导的单倍体诱导率的方法 (Method for improving CENH3 mediated haploid induction rate through heat treatment mode ) 是由 金春莲 王继华 李帆 杨春梅 于 2021-07-01 设计创作，主要内容包括：本发明公开了一种通过热处理方式来提高CENH3介导的单倍体诱导效率的方法,具体涉及生物育种领域。所述方法通过植株培养、单倍体杂交、热处理和幼苗鉴别的步骤最终获得想要的单倍体种子。本发明使用单倍体株系杂交后进行热处理,可以提高杂交后代单倍体植株比率,且该促进能力不受限于单倍体诱导株系的类型,那么这一非生物技术手段将会对CENH3介导的单倍体诱导生物技术在植物育种中的推广起到极大作用。(The invention discloses a method for improving CENH 3-mediated haploid induction efficiency by a heat treatment mode, and particularly relates to the field of biological breeding. The method finally obtains the desired haploid seeds through the steps of plant culture, haploid hybridization, heat treatment and seedling identification. The invention uses haploid strains to be crossed and then carries out heat treatment, can improve the rate of the haploid plants of the filial generation, and the promotion capability is not limited by the type of the haploid inducing strains, so that the non-biotechnology means can play a great role in popularizing the CENH 3-mediated haploid inducing biotechnology in plant breeding.)

1. A method for increasing the induction rate of CENH 3-mediated haploids by means of heat treatment, comprising the steps of:

step one, plant cultivation

Selecting seeds of a tailshap haploid female parent strain to perform plant culture on an MS solid culture medium, after dark culture at 4 ℃ for two days, transferring to 22 ℃, culturing in a 16-hour illumination/8-hour dark culture room for 7 days, taking well-grown seedlings to transfer to soil, and culturing in a 16-hour illumination/8-hour dark plant culture room at 22 ℃ until bolting;

step two, haploid induction hybridization

Taking a haploid inducing line which is 10 days after bolting as a female parent material, taking a Col-0 wild type as a male parent material, removing petals and stamens of flowers one day before the female parent plant is opened by using forceps, picking the male parent flower, removing stigma of the male flower by gentle sweeping, and pollinating, wherein each plant pollinates 5-10 flowers;

step three, heat treatment

Carrying out heat treatment culture on the female parent plant after pollination, transferring the female parent plant to a 16-hour illumination/8-hour dark plant culture room at the temperature of 22 ℃ after the heat treatment culture, and continuously culturing until seeds are mature to obtain F1 generation;

step four, identification

And (3) inoculating the F1 generation seeds into an MS solid culture medium for culture, and detecting the ploidy of the seedlings by a flow cytometer to determine the haploid plants.

2. The method for improving CENH3 mediated haploid induction rate by heat treatment according to claim 1, wherein in the first step, the tailswap haploid female parent is transformed into a CENH3 vector with H3.3 substituted at the nitrogen terminal of the protein in a CENH3 function-deficient mutant, so that a tailswap strain can be obtained.

3. The method for improving the haploid inductivity mediated by CENH3 through the heat treatment mode as claimed in claim 1, wherein in the step one, the MS solid medium comprises 2.21g/L MS salt (Labconsult), 10g/L sucrose, 0.5 g/L2-morpholine ethanesulfonic acid, 100mg/L inositol and 8g/L plant agar, and the pH of the medium is 5.6.

4. The method for increasing the induction rate of CENH3 mediated haploid by means of heat treatment according to claim 1, wherein in the third step, the heat treatment is carried out under the conditions of 30 ℃ per day/25 ℃ per night, 16 hours of light for the day and 8 hours of dark for the night.

5. The method for increasing the induction rate of CENH 3-mediated haploids by heat treatment according to claim 1, wherein in the step three, the heat treatment is performed for 7 days.

6. The method for increasing CENH 3-mediated haploid inductivity through heat treatment as claimed in claim 1, wherein in the step three, the seeds of the plants under the same treatment condition of the same harvest, haploid inducer line are collected and preserved uniformly.

7. The method for improving CENH3 mediated haploid induction rate by heat treatment as claimed in claim 1, wherein in the fourth step, the method for detecting seed ploidy by flow cytometry is to take the leaf of the plant cultured from F1 generation seed and add 300. mu.L of bufferCutting, adding 900 μ L of the buffer solution, mixing, and usingFiltering with a 40 μm cell filter screen to obtain filtrate; mu.L of the filtrate was taken and 50. mu.L of 0.5mg/mL propidium iodide was added, vortexed, and cell ploidy was determined by FACSVerse flow cytometer, data from BD FACSeite^TMAnd (6) analyzing software.

8. The method for improving CENH 3-mediated haploid inductivity through heat treatment as claimed in claim 7, wherein the leaves of the plant cultured from F1 generation seeds are the leaves of the plant cultured for 3 weeks.

9. The method for improving CENH3 mediated haploid inductivity through thermal processing means as claimed in claim 7, wherein the buffer comprises 45mM magnesium chloride, 20mM 3-morpholine propanesulfonic acid, 30mM sodium citrate and 0.1% (v/v) polyethylene glycol octyl phenyl ether.

Technical Field

The invention relates to the field of biological breeding, in particular to a method for improving CENH3 mediated haploid inductivity through a heat treatment mode.

Background

The haploid induction technology can bypass the process of generating an inbred line by multi-generation inbreeding in traditional breeding, shorten the breeding period of plants and improve the efficiency of cross breeding. In the past decades, scientists and breeding workers have devoted themselves to the study of artificially inducing haploids, and have established in vitro male and female gamete regeneration induction systems, pollen ray induction systems, interspecific crossing systems, and in vivo haploid induction systems of induced lines (haploid indexers).

It has been reported that in Arabidopsis, a haploid inducer line can be obtained by editing the centromere-specific Histone CENH3(Centromeric Histone 3specific variant). By transferring a CENH3 vector in which the nitrogen terminal of the protein is replaced by H3.3 into a CENH3 function-deficient mutant, a tailshapp strain can be obtained, which is crossed with an Arabidopsis thaliana Col-0 wild type, and the progeny produces about 1/3 haploid, 1/3 aneuploid and 1/3 diploid. In addition, the point mutant obtained by editing the carboxyl terminal of the Arabidopsis CENH3 also has haploid inductivity, and the haploid inductivity obtained by crossing with the wild type is from 2% to 12%. Due to the conservation of CENH3 in eukaryotes, the feasibility of editing the gene to obtain haploid inducer lines was also extensively examined in other crops. But is only successfully reported in corn and wheat at present, and the haploid induction efficiency is only 3.6% (corn) and 7% (wheat).

The research shows that the haploid induction rate is different in different CENH3 editing systems, and the haploid induction efficiency of a crop haploid strain is far lower than that of Arabidopsis, so that the application of the haploid induction technology in plant breeding production is greatly limited.

Disclosure of Invention

Therefore, the invention provides a method for improving the CENH3 mediated haploid inductivity by a heat treatment mode, and aims to solve the problems of low haploid inductivity, limited application and the like in the prior art.

In order to achieve the above purpose, the invention provides the following technical scheme:

according to the invention, the method for improving the CENH3 mediated haploid induction rate by the heat treatment mode comprises the following steps:

step one, plant cultivation

Selecting seeds of a tailshap haploid female parent strain to perform plant culture on an MS solid culture medium, after dark culture at 4 ℃ for two days, transferring to 22 ℃, culturing in a 16-hour illumination/8-hour dark culture room for 7 days, taking well-grown seedlings to transfer to soil, and culturing in a 16-hour illumination/8-hour dark plant culture room at 22 ℃ until bolting;

step two, haploid induction hybridization

Taking a haploid inducing strain line 10 days after bolting as a female parent material, taking a wild type of the strain as a male parent material, removing petal stamens of flowers one day before the female parent plant opens by using forceps, picking the male parent flowers, removing stigma of the flowers and pollinating, and pollinating 5-10 flowers of each plant;

step three, heat treatment

Carrying out heat treatment culture on the female parent plant after pollination, transferring the female parent plant to a 16-hour illumination/8-hour dark plant culture room at the temperature of 22 ℃ after the heat treatment culture, and continuously culturing until seeds are mature to obtain F1 generation;

step four, identification

And (3) inoculating the F1 generation seeds into an MS solid culture medium for culture, detecting the ploidy of the seeds by a flow cytometer, and selecting the needed haploid plants.

Further, in the first step, the tailswap haploid female parent is transferred into a CENH3 vector with a protein nitrogen end replaced by H3.3 in a CENH3 function-deficient mutant, and thus a tailswap strain can be obtained.

Further, in the first step, the MS solid medium comprises 2.21g/L of MS salt, 10g/L of sucrose, 0.5g/L of 2-morpholine ethanesulfonic acid, 100mg/L of inositol and 8g/L of plant agar, and the pH value of the medium is 5.6.

Further, in the third step, the conditions of the heat treatment culture are that the daily temperature is 30 ℃/the night temperature is 25 ℃, the daily temperature time is 16 hours of illumination, and the night temperature time is 8 hours of darkness.

Further, in the third step, the time for the heat treatment culture is 7 days.

Further, in the third step, the seed harvesting is carried out uniformly by the seeds of the plants under the same treatment condition of the same haploid inducing line.

Further, in the fourth step, the method for detecting the ploidy of the seeds by the flow cytometer comprises the steps of taking leaves of plants cultured by F1 generation seeds, adding 300 mu L of buffer solution, cutting, adding 900 mu L of buffer solution, uniformly mixing, and usingFiltering with a 40 μm cell filter screen to obtain filtrate; mu.L of the filtrate was taken and 50. mu.L of 0.5mg/mL propidium iodide was added, vortexed, and cell ploidy was determined by FACSVerse flow cytometer, data from BD FACSeite^TMAnd (6) analyzing software.

Furthermore, the leaves of the plant cultured by taking the F1 generation seeds are the leaves of the plant cultured for 3 weeks.

Further, the buffer contained 45mM magnesium chloride, 20mM 3-morpholinopropanesulfonic acid, 30mM sodium citrate and 0.1% (v/v) polyethylene glycol octylphenyl ether.

The invention has the following advantages:

the invention can improve the haploid rate of haploid induced hybrid progeny by carrying out heat treatment after hybridizing haploid strains, and the promoting capability is not limited by the type of the haploid induced strains, so that the efficiency of haploid induction on non-model plants such as rice, corn and other crops can be promoted by the non-biological technical means, and the popularization of the CENH 3-mediated haploid induced biotechnology in plant breeding can be greatly facilitated.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 shows the ploidy of a plant detected by a flow cytometer provided in example 1 of the present invention; wherein, the abscissa is the size of the surface area of the cell nucleus, and the ordinate is that different peaks of the cell number detected by the flow cytometer represent haploid cells, diploid cells, tetraploid cells, octaploid cells and hexadecimal cells from left to right, wherein 2 diploid cells, 4 tetraploid cells, 8 hexaploid cells and 16 hexadecimal cells are sequentially arranged from left to right in the graph A; in the diagram B, 1 haploid, 2 diploid, 4 tetraploid and 8 octaploid are shown from left to right.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Col-0 wild-type male parent seeds were cenh3-tailswap and G83E female parent material seeds purchased from NASC seed Bank obtained from UC Davis laboratories

MS salt Labconsult

Example 1A method for increasing the induction rate of CENH 3-mediated haploids by means of a heat treatment

1. Plant cultivation

The arabidopsis seeds are sown in an MS solid culture medium (MS salt is 2.21g/L, sucrose is 10g/L, 2-morpholine ethanesulfonic acid is 0.5g/L, inositol is 100mg/L and plant agar is 8g/L, pH is 5.6) for culture, after dark culture at 4 ℃, the seeds are transferred to 22 ℃, 16-hour light is used for culture in an 8-hour dark culture room for 7 days, well-grown seedlings are taken to be transferred to soil, and the seeds are cultured in a 16-hour light is used for culture in an 8-hour dark plant culture room at 22 ℃.

2. Haploid induced hybridization

Two arabidopsis haploid induction lines cenh3-tailswap and G83E which are 10 days after bolting are used as female parent materials, and an arabidopsis Col-0 wild type is used as a male parent material. Removing the stamens of the petals of the flowers one day before the female parent plant is opened by using forceps, picking the male parent flowers and lightly sweeping off the stigma of the male flowers for pollination. Each plant pollinates 5-10 flowers.

3. Thermal treatment

Culturing the pollinated female parent plant for 7 days under the temperature condition of 30 ℃ day/25 ℃ night (16h day/8 h night) and the same illumination condition for heat treatment, then transferring back to 22 ℃, and continuously culturing under the conditions of 16h illumination/8 h dark until the seeds are mature for seed collection to obtain F1 generation seeds, and uniformly collecting and storing the seeds of the plants under the same treatment condition of the same haploid inducing strain.

4. Ploidy identification of filial generation

The hybridized F1 generations were individually sown in MS solid medium under the culture conditions described in 1. The ploidy of the plants was verified by flow cytometry, by taking a leaf of the plant about 3 weeks, adding 300. mu.L of a buffer (containing 45mM magnesium chloride, 20mM 3-morpholinopropanesulfonic acid, 30mM sodium citrate and 0.1% (v/v) polyoxyethylene octylphenyl ether), chopping with a razor blade, adding 900. mu.L of the above buffer, mixing, and mixing with waterFiltering with a 40-micron cell filter screen, and taking 400-micron samples for detecting ploidy by a flow cytometer. As shown in fig. 1, the abscissa is the size of the surface area of the cell nucleus, and the ordinate is the peak of different cell numbers detected by the flow cytometer from left to right represents the haploid cell, the diploid cell, the tetraploid cell, the octaploid cell and the 16-ploid cell, wherein 2 diploid, 4 tetraploid, 8 octaploid and 16 sixteen-ploid are sequentially arranged from left to right in the diagram a; in the diagram B, 1 haploid, 2 diploid, 4 tetraploid and 8 octaploid are shown from left to right. A is diploid plant without haploid cells, B is haploid plant,a haploid cell peak can be identified. That is, haploid cells are not present in diploid plants, whereas haploid cell peaks can be identified in haploid plants. Add 50. mu.L, 0.5mg/mL propidium iodide to the sample, vortex, and measure cell ploidy using a FACSVerse flow cytometer, data from BD FACSuite^TMAnd (6) analyzing software.

Example 2A method for increasing the induction rate of CENH 3-mediated haploid by varying the duration of heat treatment

1. Plant cultivation

The arabidopsis seeds are sown in an MS solid culture medium (MS salt is 2.21g/L, sucrose is 10g/L, 2-morpholine ethanesulfonic acid is 0.5g/L, inositol is 100mg/L and plant agar is 8g/L, pH is 5.6) for culture, after dark culture at 4 ℃, the seeds are transferred to 22 ℃, 16-hour light is used for culture in an 8-hour dark culture room for 7 days, well-grown seedlings are taken to be transferred to soil, and the seeds are cultured in a 16-hour light is used for culture in an 8-hour dark plant culture room at 22 ℃.

2. Haploid induced hybridization

Two arabidopsis haploid induction lines cenh3-tailswap and G83E which are 10 days after bolting are used as female parent materials, and an arabidopsis Col-0 wild type is used as a male parent material. Removing the stamens of the petals of the flowers one day before the female parent plant is opened by using forceps, picking the male parent flowers and lightly sweeping off the stigma of the male flowers for pollination. Each plant pollinates 5-10 flowers.

3. Thermal treatment

Dividing the female parent plants of the pollinated cenh3-tailswap into 5 groups according to different time points of heat treatment,

the first group is subjected to heat treatment on the day of pollination, namely, the heat treatment is carried out on the day 0, the temperature is taken back to 22 ℃ after the heat treatment is carried out for 3 days, and the culture is carried out under 16-hour illumination/8-hour darkness;

the second group is cultured in a 16-hour illumination/8-hour dark culture room at 22 ℃ for 1 day and then is subjected to heat treatment for 3 days, namely the heat treatment is carried out on the 1 st day, and the second group is taken back to 22 ℃ after the heat treatment for 3 days and is cultured in 16-hour illumination/8-hour dark;

the third group is cultured in a 16-hour illumination/8-hour dark culture room for 2 days at 22 ℃ and then is thermally treated for 3 days, namely the thermal treatment is carried out on the 2 nd day, the culture is carried out after the thermal treatment is carried out for 3 days and then is taken back to 22 ℃ and cultured in 16-hour illumination/8-hour dark;

performing 22 ℃ and 16-hour illumination/8-hour dark culture room culture for 3 days, performing heat treatment for 3 days, namely performing heat treatment on the 3 rd day, taking back to 22 ℃ after the heat treatment for 3 days, and performing 16-hour illumination/8-hour dark culture;

the fifth group is cultured in a 16-hour light/8-hour dark culture room at 22 ℃ for 4 days and then is subjected to heat treatment for 3 days, namely the heat treatment is carried out on the 4 th day, and the fifth group is taken back to 22 ℃ after the heat treatment for 3 days and is cultured in 16-hour light/8-hour dark;

the conditions of the above heat treatment were light irradiation temperature conditions of 30 ℃ day/25 ℃ night (16h day/8 h night).

And (3) after the heat treatment, taking back all the female parent plants subjected to heat treatment to 22 ℃, and culturing in 16-hour light/8-hour dark until the seeds are mature to obtain F1 generation seeds, wherein the seeds of the plants of the same haploid induction strain under the same treatment condition are collected and stored uniformly.

4. Ploidy identification of filial generation

The hybridized F1 generations were individually sown in MS solid medium under the culture conditions described in 1. The ploidy of the plants was verified by flow cytometry, by taking a leaf of the plant about 3 weeks, adding 300. mu.L of a buffer (containing 45mM magnesium chloride, 20mM 3-morpholinopropanesulfonic acid, 30mM sodium citrate and 0.1% (v/v) polyoxyethylene octylphenyl ether), chopping with a razor blade, adding 900. mu.L of the above buffer, mixing, and mixing with waterFiltering with a 40-micron cell filter screen, and taking 400-micron samples for detecting ploidy by a flow cytometer. Add 50. mu.L, 0.5mg/mL propidium iodide to the sample, vortex, and measure cell ploidy using a FACSVerse flow cytometer, data from BD FACSuite^TMAnd (6) analyzing software.

Comparative example

1. Plant cultivation

The arabidopsis seeds are sown in an MS solid culture medium (MS salt is 2.21g/L, sucrose is 10g/L, 2-morpholine ethanesulfonic acid is 0.5g/L, inositol is 100mg/L and plant agar is 8g/L, pH is 5.6) for culture, after dark culture at 4 ℃, the seeds are transferred to 22 ℃, 16-hour light is used for culture in an 8-hour dark culture room for 7 days, well-grown seedlings are taken to be transferred to soil, and the seeds are cultured in a 16-hour light is used for culture in an 8-hour dark plant culture room at 22 ℃.

2. Haploid induced hybridization

Two arabidopsis haploid induction lines cenh-tailswap and G83E which are 10 days after bolting are used as female parent materials, and an arabidopsis Col-0 wild type is used as a male parent material. Removing the stamens of the petals of the flowers one day before the female parent plant is opened by using forceps, picking the male parent flowers and lightly sweeping off the stigma of the male flowers for pollination. Each plant pollinates 5-10 flowers.

3. Thermal treatment

And (3) culturing the female parent plants after pollination in a 16-hour illumination/8-hour dark plant culture room at 22 ℃ until the seeds are mature, harvesting to obtain F1 generation seeds, and uniformly harvesting and storing the seeds of the plants under the same treatment condition of the same haploid induction strain.

4. Ploidy identification of filial generation

The hybridized F1 generations were individually sown in MS solid medium under the culture conditions described in 1. The ploidy of the plants was verified by flow cytometry, by taking a leaf of the plant about 3 weeks, adding 300. mu.L of a buffer (containing 45mM magnesium chloride, 20mM 3-morpholinopropanesulfonic acid, 30mM sodium citrate and 0.1% (v/v) polyoxyethylene octylphenyl ether), chopping with a razor blade, adding 900. mu.L of the above buffer, mixing, and mixing with waterFiltering with a 40-micron cell filter screen, and taking 400-micron samples for detecting ploidy by a flow cytometer. Add 50. mu.L, 0.5mg/mL propidium iodide to the sample, vortex, and measure cell ploidy using a FACSVerse flow cytometer, data from BD FACSuite^TMAnd (6) analyzing software.

Experimental example 1 Heat treatment after fertilization stress improved haploid induction efficiency of Arabidopsis haploid induction line

Through the ploidy identification of hybrid offspring, the experimental results are shown in Table 1, in the control example, the haploid induction efficiency of cenh3-tailswap is 42.5%, and in example 1, after 7 days of heat treatment, the haploid induction efficiency of the induced strain is improved to 96.4%.

Similarly, in the control example, the haploid induction efficiency of G83E was 5.2%, whereas the induction efficiency of this strain increased to 62.5% after 7 days of heat treatment in example 1.

TABLE 1 haploid Induction efficiency of control and Heat-treated haploid lines

Experimental example 2 Effect of the number of days of Heat treatment after pollination on haploid Induction Rate

In example 2, a preliminary experiment was performed before the experiment at the heat treatment time point, and the heat treatment conditions were tested, and it was found that the haploid induction efficiency can reach 100% when the cenh3-tailswap hybrid is heat-treated for 3 days, and the amount of 0DAP haploid plants shown in table 2 can reach 100%. After heat treatment at different time points, the time points for the best heat treatment were selected, and as shown in table 2, heat treatment was performed after pollination for different Days (DAP), and the germination rate and haploid induction efficiency of the seeds of generation F1 were obtained to be different. Wherein the seed germination rate is increased and then decreased along with the delay of the heat treatment days, and the haploid induction efficiency is decreased along with the delay of the heat treatment time point. The optimal treatment scheme is that the seed germination rate and the haploid induction efficiency are combined, heat treatment is carried out for 3 days after pollination is carried out for 2 days, the seed germination rate is high, more offspring can be obtained, and the haploid induction efficiency is over 90%.

TABLE 2 Effect of different heat treatment time points on the induction efficiency of cenh3-tailswap haploids

In conclusion, the method of the invention is used for inducing the haploid after heat treatment, so that the germination rate of the seeds can be improved, and the induction rate of the haploid can also be improved.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.