CN114711108A - Method for rapidly determining development period of rice microspore - Google Patents
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- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- A01G22/22—Rice
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- A—HUMAN NECESSITIES
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention relates to the technical field of agricultural biology, in particular to a method for quickly determining the development period of rice microspores. The invention provides a method for rapidly determining the development period of rice microspores, which finds an optimum plant state for culturing the rice microspores through the bending degree and glume color of a branch stem at one time, and is simple and easy to identify. Can effectively avoid the dependence on instruments, improve the efficiency of obtaining the materials of the rice microspores and the effectiveness of culture, and improve the production benefit.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to a method for rapidly determining the development period of rice microspores.
Background
Rice is one of the most important grain crops in the world. With the great success of the green revolution and the breakthrough of the hybrid rice technology with two times of heavy mass production, the yield level of rice is almost in a standstill. Therefore, new gene sources and innovative breeding selection strategies are discovered, and high-yield rice varieties are developed to meet the demands of population growth and the challenges of climate change. Doubled haploid breeding through anther/microspore culture has become a convenient substitute of a traditional crop improvement technology, and doubled haploids have the advantages of being capable of fixing homozygous immediately, shortening the breeding period, improving the selection efficiency, expanding genetic variation and being suitable for expression of recessive genes for breeding. Microspore culture is an effective method for obtaining haploid or double haploid plants, has the advantages of single cell origin, easy gene mutation and transformation, avoidance of influence of anther wall, higher culture efficiency and the like, and has good application prospect in plant breeding and basic research.
Embryogenesis of microspores only occurs in a specific period of microspore development, the optimal period of embryogenic induction in rice is the middle and late mononuclear stage of microspore development (Mishra and Rao 2016), the identification method is a multi-purpose microscopic examination method, but the method is complicated and has high requirements on instruments, flag leaf distance is also used for measuring on rice (Mayakaduwa and Silva 2017), but the panicle type of rice is a cone inflorescence, the cob of the inflorescence is highly branched, the microspore development periods of different branches of one panicle are different, and the microscopic examination is also needed for determining the proper period.
However, a method for rapidly determining the development period of rice microspores is not reported at present.
Disclosure of Invention
The invention aims to overcome the defects of the methods and provide a method for rapidly determining the development period of rice microspores.
According to the method, materials are taken when primary branches of young rice ears have different bending degrees (equal to 90 degrees, more than 90 degrees and less than 180 degrees, and equal to 180 degrees) and glume colors (light yellow, yellow green and green), the development period of microspores is observed, the reaction of the different bending degrees of the primary branches and the glume colors to microspore culture is analyzed, the optimal material taking period of rice is determined, and the material taking efficiency and the culture efficiency are improved.
The invention provides a method for rapidly determining the development period of rice microspores, which is to rapidly determine the development period of the rice microspores according to the bending degree and glume color of a branch stem.
The method comprises the following steps:
(a) selecting young ears of rice with extracted flag leaves;
(b) taking the primary branch off the spike main stem, clamping the base of the primary branch by using forceps, and classifying the primary branch into four types according to the bending degree and glume color of the primary branch (figure 1):
class I: the mixture is clamped by a pair of tweezers to be vertical to the ground, the angle is equal to 90 degrees, and the glume is light yellow; the microspore development period is the mononuclear period;
class II: clamping by using tweezers, wherein the bending degree is between 90 degrees and 180 degrees, and the glumes are yellow green; the microspore development period is the middle and late mononuclear period and a small amount of dinuclear period;
class III: the glumes are clamped by tweezers to be parallel to the ground, and the glumes are yellow green; the development period of the microspore is respectively 50% of the mononuclear period and the dinuclear period;
class IV: the glumes are clamped by tweezers to be parallel to the ground, and the glumes are green in color; the microspore development stage is the majority of the binuclear stage.
Further, the rice variety is Nipponbare.
The invention provides a method for quickly taking rice microspores, which comprises the steps of selecting young rice ears with extracted flag leaves, taking off primary branches from main branches of the ears, clamping the base parts of the primary branches by using forceps, and taking young rice ears with branch bending degrees between 90 degrees and 180 degrees and greenish yellow glume colors. The microspore development period in the period is in the middle and late stage of the mononuclear, is more suitable for microspore culture, can form more callus, and is the best period for microspore culture.
The third aspect of the invention provides a method for quickly taking and culturing rice microspores, which comprises the following steps:
1) material taking: selecting young ears of rice with extracted flag leaves;
2) pre-treating young ears: placing the young ears in a fresh-keeping bag for low-temperature treatment;
3) classifying the primary branch and glume colors: taking young spikes with the branch and stem bending degree between 90 degrees and 180 degrees and yellow-green glumes;
4) free microspore: inoculating anther, performing high-speed rotary cutting, dissociating and collecting microspores, and pretreating the collected microspores in a proper solution at a proper temperature;
5) microspore culture and callus induction: purifying the pretreated microspore, culturing in an induction culture medium, and inducing callus.
Further, the method specifically comprises the following steps:
(A) taking materials and pretreating young ears
Taking the ears from which the flag leaves are extracted, only keeping one leaf, cutting off the leaves, putting into a fresh-keeping bag, and putting into a refrigerator at 5 ℃ for low-temperature pretreatment for 10 days;
(B) primary branch and stem classification
Stripping the young ears of the rice from leaf sheaths, cutting off primary branches from the main stems, clamping the base parts of the branches by using forceps, and taking the young ears with the bending degree of the branches between 90 degrees and 180 degrees and yellow-green glumes;
(C) anther pretreatment
Sterilizing with 10% disinfectant for 10min before inoculation on a sterile super-clean workbench, washing with sterile water for 4-5 times, inoculating 20 branches and stalks to each test tube (50ml), adding anther of about 100 buds, adding 12ml of the extractive solution, and placing in a refrigerator at 5 deg.C for 2 d;
(D) microspore dissociation
After the anther pretreatment is finished, the anther is cut in a super-speed rotary manner on a super-clean workbench by using a high-speed disperser, the rotary-cut suspension is filtered by using a 150-mesh screen, and the filtrate is filtered at 700 r.min-1Centrifuging at low speed for 5min, repeating for 3 times, and collecting microspore;
(E) microspore callus induction
The collected microspores are pretreated for 2 days in the dark at 25 ℃ by using an extracting solution and then purified and cultured. The microspore density was adjusted to 1.0X 10 after 1 wash with induction medium before cultivation5each.mL-11.0mL of microspore suspension was inoculated into a petri dish (35 mm. times.12 mm) and 3 replicates were sealed with a sealing membrane and cultured in the dark at 25 ℃ for 23 days to form callus.
The extract contained 320mM mannitol and 2mM CaCl21.6mM N-morphine ethane sulfonic acid (MES) and 1mM colchicine, pH 5.8.
The induction medium was N6 medium as a minimal medium, and 175mM sucrose, 1mM 2,4-Dichlorophenoxyacetic acid (2, 4-dichlorphenoxyacetic acid, 2,4-D), 1.6mM MES, 3.4mM glutamine, 4.3mM proline, pH 5.8 were added.
The invention has the advantages that:
1. the invention provides a method for rapidly determining the development period of rice microspores, wherein the optimal microspore drawing period of a material Nipponbare is the optimal period for culturing the microspores when the bending degree of a first-level branch is more than 90 degrees and less than 180 degrees, and the glume color is yellow green.
2. The invention provides a method for rapidly determining the development period of rice microspores, which finds an optimum plant state for culturing the rice microspores through the bending degree and glume color of a branch stem at one time, and is simple and easy to identify.
3. Can effectively avoid the dependence on instruments, improve the efficiency of obtaining the materials of the rice microspores and the effectiveness of culture, and improve the production benefit.
Drawings
FIG. 1 shows the standard of the color of the primary branch and glume.
FIG. 2 shows the yield of microspore-cultured callus under four types of young ears of rice.
FIG. 3 microspore viability under different classifications.
FIG. 4. microspore development stages under different classifications.
FIG. 5. callus production under different classifications.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example (b):
1. material
The rice variety Nipponbare (a well-known public rice variety) is planted in a field and is subjected to normal water and fertilizer management.
2. Method of producing a composite material
2.1 obtaining materials and pretreating young ears
Taking the ear from which the flag leaf is extracted, only keeping one leaf, cutting off the leaf, putting into a fresh-keeping bag, and putting into a refrigerator at 5 ℃ for low-temperature pretreatment for 10 days.
2.2 Primary Classification of branches and stalks
Stripping young ears of rice from leaf sheaths, cutting off primary branches from main branches, clamping the base parts of the branches by using forceps, and dividing the primary branches into four types according to the bending angle and the color of glumes: class I: the primary branches are completely vertical to the ground and equal to 90 degrees, and the glumes are light yellow; class II: the angle between the primary branch and the ground is more than 90 degrees and less than 180 degrees, and the glumes are yellow-green in color; class III: the primary branches are parallel to the ground and equal to 180 degrees, and the glumes are yellow-green in color; the fourth type: the primary branches are parallel to the ground and equal to 180 degrees, and the glumes are green in color.
2.3 anther pretreatment
Sterilizing with 10% disinfectant for 10min before inoculation on a sterile super clean workbench, washing with sterile water for 4-5 times, inoculating 20 branches and stalks to each test tube (50ml), adding anther of about 100 buds, adding 12ml of the extractive solution, and placing in a refrigerator at 5 deg.C for 2 d.
2.3 microspore dissociation
After the anther pretreatment is finished, the anther is cut in a super-speed rotary manner on a super-clean workbench by using a high-speed disperser, the rotary-cut suspension is filtered by using a 150-mesh screen, and the filtrate is filtered at 700 r.min-1And centrifuging at low speed for 5min, repeating for 3 times, and collecting microspores.
2.4 Observation of microspore development stages
After the microspores are dissociated, taking 10 mu l of microspore suspension, adding FDA dye solution, mixing uniformly, standing for 10min in the dark at normal temperature, observing under a fluorescence microscope, and counting viable microspores and all microspores under one visual field respectively.
Collecting 1 μ l microspore precipitate, slowly adding Carnoy fixing solution (ethanol: acetic acid ═ 3:1), keeping for 10min, removing supernatant, adding 10 μ l ddH2O and 10. mu.l of 0.1mg/ml DAPI staining solution, mixing well, standing in the dark for 10min at normal temperature, and observing under a fluorescence microscope.
2.5 microspore callus Induction
The collected microspores are pretreated by the extract in the dark at the temperature of 25 ℃ for 2 days and then purified and cultured. The microspore density was adjusted to 1.0X 10 after 1 wash with induction medium before cultivation5each.mL-11.0mL of microspore suspension was inoculated into a petri dish (35 mm. times.12 mm) and 3 replicates were sealed with a sealing membrane and cultured in the dark at 25 ℃ for 23 days to form callus.
The extract contained 320mM mannitol and 2mM CaCl21.6mM N-morphine ethane sulfonic acid (MES) and 1mM colchicine, pH 5.8.
The induction medium was N6 medium as a minimal medium, and 175mM sucrose, 1mM 2,4-Dichlorophenoxyacetic acid (2, 4-dichlorphenoxyacetic acid, 2,4-D), 1.6mM MES, 3.4mM glutamine, 4.3mM proline, pH 5.8 were added.
Filtering and sterilizing the extract and the induction culture medium.
3. Growth statistics index and data processing
Microspore viability: the number of viable microspores in one field is a percentage of the number of all microspores.
Callus yield: the amount of callus was measured at 23 days after microspore culture by weighing the culture dish with an electronic balance after sucking up the liquid medium, and measuring the amount of callus in mg/dish-1Represents;
the results of the test were analyzed using Microsoft Excel 2010, DPS v version 7.05 data processing system.
4. Results and analysis
The standard of the primary branch and glume color is shown in figure 1: class I: the glumes are clamped by tweezers to be vertical to the ground, and the glumes are light yellow; class II: clamping by using tweezers, wherein the bending degree is between 90 degrees and 180 degrees, and the glumes are yellow green; class III: the glumes are clamped by tweezers to be parallel to the ground, and the glumes are yellow green; class IV: the ground is parallel to the ground by clamping with tweezers, and the glume is green in color.
The rice young ear is divided into four types according to different bending degrees of branches and stalks of the rice young ear and the color of glumes, the activity of the II type microspore after low-temperature treatment is the best, reaches 54.70 percent, and the microspore is in the middle and late stage of mononuclear at the development stage (Table 1).
TABLE 1 identification of the different types of microspore viability and development stage of young ears of rice after low-temperature treatment
The yield of the callus obtained after the young ears of the rice are cultured by microspores with different bending degrees of branches and stalks and classified glume colors at one time is shown in figure 2. The microspore callus of the II type has the highest yield.
In the invention, the bending degree of the primary branch of the young rice spike is between 90 degrees and 180 degrees, the glume color is yellow green, and the microspore development period is in the middle and late stages, so that the young rice spike is more suitable for microspore culture and can form more callus.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (5)
1. A method for rapidly determining the development period of rice microspores is characterized in that the development period of the rice microspores is rapidly determined through the bending degree and the glume color of a branch, and comprises the following steps:
(a) selecting young ears of rice with extracted flag leaves;
(b) taking the primary branch off the spike main stem, clamping the base of the primary branch by using forceps, and dividing the primary branch into four types according to the bending degree and glume color of the primary branch:
class I: the mixture is clamped by a pair of tweezers to be vertical to the ground, the angle is equal to 90 degrees, and the glume is light yellow; the microspore development period is the mononuclear period;
class II: clamping by using tweezers, wherein the bending degree is between 90 degrees and 180 degrees, and the glumes are yellow green; the microspore development period is the middle and late mononuclear period and a small amount of dinuclear period;
class III: the glumes are clamped by tweezers to be parallel to the ground, and the glumes are yellow green; the development period of the microspore is 50 percent of that of the mononuclear stage and the dinuclear stage respectively;
class IV: the glumes are clamped by tweezers to be parallel to the ground, and the glumes are green; the microspore development stage is the majority of the binuclear stage.
2. The method of claim 1, wherein the rice cultivar is Nipponbare.
3. A method for quickly taking microspores of rice is characterized in that young ears of rice with flag leaves extracted are selected, primary branches are taken down from main stems of the ears, bases of the primary branches are clamped by forceps, and young ears with the bending degree of 90-180 degrees and greenish yellow glume colors are taken.
4. A method for quickly taking and culturing microspores of rice is characterized by comprising the following steps:
1) material taking: selecting young ears of rice with flag leaves extracted;
2) pre-treating young ears: placing the young ears in a fresh-keeping bag for low-temperature treatment;
3) classifying the primary branch and glume colors: taking young spikes with the branch and stem bending degree between 90 degrees and 180 degrees and yellow-green glumes;
4) free microspore: inoculating anther, rotary-cutting at high speed, dissociating and collecting microspores, and pretreating the collected microspores in a proper solution at a proper temperature;
5) microspore culture and callus induction: purifying the pretreated microspore, culturing in an induction culture medium, and inducing callus.
5. The method for rapidly taking and culturing the microspores of rice as claimed in claim 4, which comprises the following steps:
(A) taking materials and pretreating young ears
Taking the ears from which the flag leaves are extracted, only keeping one leaf, cutting off the leaves, putting into a fresh-keeping bag, and putting into a refrigerator at 5 ℃ for low-temperature pretreatment for 10 days;
(B) primary branch and stem classification
Stripping the young ears of the rice from leaf sheaths, cutting off primary branches from the main stems, clamping the base parts of the branches by using forceps, and taking the young ears with the bending degree of the branches between 90 degrees and 180 degrees and yellow-green glumes;
(C) anther pretreatment
Sterilizing with 10% disinfectant for 10min before inoculation on a sterile super-clean workbench, washing with sterile water for 4-5 times, inoculating 20 branches and stalks into each 50ml test tube, adding 12ml of extractive solution into anther of about 100 buds, and placing in a refrigerator at 5 deg.C for 2 d;
(D) microspore dissociation
After the anther pretreatment is finished, the anther is cut in a super-speed rotary manner on a super-clean workbench by using a high-speed disperser, the rotary-cut suspension is filtered by using a 150-mesh screen, and the filtrate is filtered at 700 r.min-1Centrifuging at low speed for 5min, repeating for 3 times, and collecting microspore;
(E) microspore callus induction
Pretreating collected microspores for 2 days at 25 ℃ in the dark by using an extracting solution, and then purifying and culturing the microspores; the microspore density was adjusted to 1.0X 10 after 1 wash with induction medium before cultivation5each.mL-1Inoculating 1.0mL of microspore suspension into a culture dish, repeating for 3 times, sealing by using a sealing film, carrying out dark culture at 25 ℃, and forming callus after 23 days;
the extract contained 320mM of mannitol,2mM CaCl21.6mM N-morphinanesulfonic acid and 1mM colchicine, pH 5.8;
the induction medium was N6 medium as a minimal medium, and 175mM sucrose, 1mM 2,4-dichlorophenoxyacetic acid, 1.6mM MES, 3.4mM glutamine, 4.3mM proline, pH 5.8 were added.
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