CN113575409B - Method for improving CENH3 mediated haploid induction rate through heat treatment mode - Google Patents
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Abstract
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.
Description
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 which hybridizes with the Arabidopsis thaliana Col-0 wild type and whose progeny produce about 1/3 haploid, 1/3 aneuploid and 1/3 diploid can be obtained. 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 using
Filtering 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 FACSeiteTMAnd (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, the proportions, the sizes, and the like shown in the specification are only used for matching with the contents disclosed in the specification, so that those skilled in the art can understand and read the present invention, and do not limit the conditions for implementing the present invention, so that the present invention has no technical essence, and any modifications of the structures, changes of the proportion relation, or adjustments of the sizes, should still fall within the scope of the technical contents disclosed in the present invention without affecting the efficacy and the achievable purpose of the present invention.
FIG. 1 shows the ploidy of a plant detected by a flow cytometer provided in example 1 of the present invention; the X-ray fluorescence quantitative analysis method comprises the following steps of A, detecting a cell number, wherein the abscissa is the size of the surface area of a cell nucleus, and the ordinate is the value of the cell number, detected by a flow cytometer, of different peaks representing 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 a 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 specific embodiments, and other advantages and benefits of the present invention will become apparent to those skilled in the art from the following disclosure. 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 plant is identified by a flow cytometer, a leaf of the plant about 3 weeks is taken and added with 30mu.L of buffer (buffer containing 45mM magnesium chloride, 20mM 3-morpholinopropanesulfonic acid, 30mM sodium citrate and 0.1% (v/v) polyethylene glycol octylphenyl ether) was chopped with a razor blade, 900. mu.L of the above buffer was added, mixed well, and then mixed with
Filtering 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 plants, haploid cells do not exist, B is haploid plants, and haploid cell peaks 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 FACSuiteTMAnd (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 plant is identified by a flow cytometer, and one of the plants about 3 weeks is takenSlicing leaves, adding 300 μ L buffer (containing 45mM magnesium chloride, 20mM 3-morpholine propanesulfonic acid, 30mM sodium citrate and 0.1% (v/v) polyethylene glycol octyl phenyl ether), cutting with a knife, adding 900 μ L buffer, mixing, and adding
Filtering 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 FACSuiteTMAnd (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 plant is identified by a flow cytometer, and one plant of about 3 weeks is takenAdding 300 μ L buffer (containing 45mM magnesium chloride, 20mM 3-morpholine propanesulfonic acid, 30mM sodium citrate and 0.1% (v/v) polyethylene glycol octyl phenyl ether) into the leaf, cutting with a knife, adding 900 μ L buffer, mixing, and adding
Filtering 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 FACSuiteTMAnd (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 progeny, 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 haploid induction efficiency of cenh3-tailswap
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.
Claims (6)
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 arabidopsis thaliana of the tailswap haploid female parent strain to perform plant culture on an MS solid culture medium, performing dark culture at 4 ℃ for two days, then 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 22 ℃, 16-hour illumination/8-hour dark plant culture room 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 a pair of tweezers, 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
Inoculating the F1 generation seeds into an MS solid culture medium for culture, and detecting the ploidy of seedlings by a flow cytometer to determine haploid plants;
in the first step, the tailshap haploid female parent is obtained by transferring a CENH3 vector with a protein nitrogen end replaced by H3.3 in a CENH3 function-loss mutant;
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;
in the third step, the time of the heat treatment culture is 7 days.
2. 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 is composed of 2.21g/L MS salt, 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.
3. The method for increasing CENH 3-mediated haploid inductivity through heat treatment as claimed in claim 1, wherein in the step three, the harvest refers to the unified harvest and preservation of plant seeds under the same treatment condition of the same haploid inducer line.
4. The method for improving the haploid inductivity mediated by CENH3 through the heat treatment mode as claimed in claim 1, wherein in the fourth step, the method for detecting the ploidy of seedlings through the flow cytometry is to take the leaves of the plants cultured from F1 generation seeds, add 300 μ L of the buffer solution, cut up, add 900 μ L of the buffer solution, mix, and use Falcon to induce haploid induction rate® Filtering with a cell filter screen of 40 mu m to obtain filtrate; mu.L of the filtrate was taken and 50. mu.L of propidium iodide 0.5mg/mL was added, vortexed, and cell ploidy was determined by FACSEverse flow cytometer, and the data was analyzed by BD FACSEite ™ cell-mass software.
5. The method for improving CENH 3-mediated haploid inductivity through heat treatment as claimed in claim 4, wherein the leaves of the plant cultured from F1 generation seeds are the leaves of the plant cultured for 3 weeks.
6. The method for increasing CENH3 mediated haploid inducer rate by means of heat treatment according to claim 4 characterised in that the buffer consists of 45mM magnesium chloride, 20mM 3-morpholinopropanesulfonic acid, 30mM sodium citrate and 0.1% v/v polyethylene glycol octylphenyl ether.
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