CN114711108A - A rapid method for determining the developmental stage of rice microspores - Google Patents

Abstract

The invention relates to the field of agricultural biotechnology, in particular to a method for rapidly determining the developmental stage of rice microspores, which is to rapidly determine the developmental stage of rice microspores through the bending degree of a branch and the color of the glume. The invention provides a method for quickly determining the development period of rice microspores. The most suitable plant state for rice microspore culture can be found through the bending degree of the primary branch and the color of the glume, and the method is simple and easy to identify. The device can effectively avoid dependence on the instrument, improve the efficiency of rice microspore collection and the effectiveness of cultivation, and improve the production benefit.

Description

A rapid method for determining the developmental stage of rice microspores

technical field

The invention relates to the field of agricultural biotechnology, in particular to a method for rapidly determining the developmental stage of rice microspores.

Background technique

Rice is one of the most important food crops in the world. With the great success of the Green Revolution and two major yield breakthroughs in hybrid rice technology, rice yield levels have almost stagnated. Hence the discovery of new genetic sources and innovative breeding selection strategies to develop high-yielding rice varieties to meet the demands of population growth and the challenges of climate change. Double haploid breeding by anther/microspore culture has become a convenient alternative to traditional crop improvement techniques. The advantage of double haploid is that it can fix homozygosity immediately, shorten the breeding cycle, improve selection efficiency, expand genetic variation and Expression of recessive genes suitable for breeding. Microspore culture is an effective method to obtain haploid or double haploid plants. Its advantages lie in single-cell origin, easy gene mutation and transformation, avoiding the influence of anther wall, and higher culture efficiency. It is widely used in plant breeding and basic research. It has a good application prospect.

The embryogenesis of microspores only occurs in a specific period of microspore development, and the best period for mesoembryogenic induction in rice is the middle and late mononuclear stage of microspore development (Mishra and Rao 2016). , but this method is cumbersome and requires high equipment. It is also measured by the method of flag leaf distance on rice (Mayakaduwa and Silva 2017), but the panicle type of rice is panicle, and the cob of the inflorescence is highly branched, The microspore development period of different branches of a panicle is different, and microscopic examination is also required to determine the appropriate period.

However, there is no report on a method to rapidly determine the developmental stage of rice microspores.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the deficiencies of the above methods and provide a method for quickly determining the developmental stage of rice microspores. The present invention finds that the degree of curvature of the primary branch and the color of the glume can determine the developmental stage of the microspores. The degree of curvature of the stem and the color of the glume, which are easily identifiable morphological indicators closely related to the developmental stage of the microspores, are used to obtain materials. In the case of large-scale microspore culture, the efficiency of sampling is improved and the dependence on the instrument is reduced.

In the present invention, materials are obtained when the primary branches of young rice ears have different degrees of curvature (equal to 90°, greater than 90° and less than 180°, equal to 180°) and glume colors (light yellow, yellow-green, green), and observe microspores. During the developmental period, the response of different bending degrees of branch and glume color to microspore culture was analyzed, and the optimal period of rice harvesting was determined to improve the efficiency of harvesting and cultivation.

The first aspect of the present invention provides a method for quickly determining the developmental stage of rice microspores, which is to rapidly determine the developmental stage of rice microspores through the bending degree of the primary branch and the color of the glume.

The method includes the following steps:

(a) Select the young ears of rice from which the flag leaves have been drawn;

(b) Remove the primary stem from the main stem of the ear, pick up the base of the primary stem with tweezers, and divide the primary stem into four categories according to the degree of curvature of the primary stem and the color of the glume (Figure 1):

Class I: Hold it with tweezers and perpendicular to the ground, equal to 90°, the glume color is light yellow; the microspore development period is the mononucleate stage;

Class II: The curvature of the tweezers is between 90° and 180°, and the color of the glume is yellow-green;

Class III: clamped with tweezers and parallel to the ground, the glume color is yellow-green; the microspore development period is 50% of the mononucleate stage and the dinucleate stage;

Class IV: Picked up with tweezers and parallel to the ground, the glume is green; the developmental stage of microspores is most of the binucleate stage.

Further, the rice variety is Nipponbare.

In the second aspect of the present invention, a method for quickly obtaining materials for rice microspores is provided, which is to select the young ears of rice from which the flag leaves have been extracted, remove the primary stem from the main stem of the ear, and use tweezers to pick up the base of the primary stem, Take young spikes with a stalk curvature between 90° and 180° and a yellow-green glume color. The microspore development stage in this period is in the middle and late mononuclear stage, which is more suitable for microspore culture and can form more callus, which is the best period for microspore culture.

A third aspect of the present invention provides a method for rapidly sampling and culturing rice microspores, comprising the following steps:

1) Material selection: select the young ears of rice that have been extracted from flag leaves;

2) pretreatment of young ear: the young ear is placed in a fresh-keeping bag for low temperature treatment;

3) Color classification of primary branch and glume: take the young ear with the curvature of the branch between 90° and 180° and the color of the glume yellow-green;

4) Free microspores: inoculate anthers, spin at high speed, collect microspores free, and pretreat the collected microspores in a suitable solution and temperature;

5) Microspore culture and callus induction: Purify the pretreated microspores, place them in an induction medium for culture, and induce callus formation.

Further, it specifically includes the following steps:

(A) Material sampling and pretreatment of young ears

Take the ear that has been extracted from the flag leaf, keep only one leaf, cut off the leaf, put it in a fresh-keeping bag, and put it in a 5°C refrigerator for low-temperature pretreatment for 10 days;

(B) Primary branch classification

Peel off the young ear of rice from the leaf sheath, cut the primary stem from the main stem, pick up the base of the stem with tweezers, take the stem with a curvature between 90° and 180°, and the color of the glume is yellow-green. young ear;

(C) Anther pretreatment

On a sterile ultra-clean workbench, disinfect with 10% disinfectant for 10 minutes before inoculation, rinse with sterile water 4 to 5 times, receive 20 branches and anthers of about 100 flower buds per test tube (50ml), add 12ml of extract, placed in the refrigerator at 5℃ for 2d;

(D) Microspore dissociation

After the pretreatment of the anthers, use a high-speed disperser on the ultra-clean workbench for ultra ^- speed rotary cutting, and filter the rotary-cut suspension with a 150-mesh sieve. , to collect microspores;

(E) Microspore callus induction

The collected microspores were pretreated with the extract solution at 25°C in the dark for 2 days, and then the microspores were purified and cultured. Wash once with induction medium before culturing, then adjust the microspore density to 1.0×10 ⁵ ·mL ^-1 , take 1.0 mL of microspore suspension and inoculate it in a petri dish (35mm×12mm), repeat 3 times, use Seal with parafilm, 25 ℃, dark culture, 23d, callus formation.

The extract contained 320 mM mannitol, 2 mM CaCl ₂ , 1.6 mM N-Morpholineethane sulfonic acid (MES) and 1 mM colchicine, pH 5.8.

The induction medium was based on N6 medium, supplemented with 175mM sucrose, 1mM 2,4-Dichlorophenoxyacetic acid (2,4-D), 1.6mM MES, 3.4mM glutamine , 4.3mM proline, pH5.8.

The advantages of the present invention are:

1. The present invention provides a method for quickly determining the development period of rice microspores. The optimal microspore sampling period of the material Nipponbare is when the bending degree of the primary branch is greater than 90° and less than 180°, and the color of the glume is expressed as: Yellow-green is the best time for microspore culture.

2. The present invention provides a method for quickly determining the developmental stage of rice microspores. The most suitable plant state for rice microspore culture is found through the bending degree of the branch and the color of the glume. This method is simple and easy to identify.

3. It can effectively avoid the dependence on the instrument, improve the efficiency of rice microspore collection and the effectiveness of cultivation, and improve the production efficiency.

Description of drawings

Fig. 1. The sampling standard of primary shoot and glume color.

Figure 2. Microspore culture callus yield under four types of rice young ears.

Figure 3. Microspore viability under different classifications.

Figure 4. Microspore developmental stages under different classifications.

Figure 5. Callus yield under different classifications.

Detailed ways

The specific embodiments provided by the present invention will be described in detail below with reference to the examples.

Example:

1. Materials

The rice variety Nipponbare (a well-known public rice variety) is planted in the field and managed with normal water and fertilizer.

2. Method

2.1 Material sampling and pretreatment of young ears

Take the ear that has been extracted from the flag leaf, keep only one leaf, cut off the leaf, put it in a fresh-keeping bag, and put it in a 5°C refrigerator for low-temperature pretreatment for 10 days.

2.2 Classification of primary branches

Peel off the young ear of rice from the leaf sheath, cut off the primary stem from the main stem, pick up the base of the stem with tweezers, and divide the primary stem into four categories according to the bending angle and the color of the glume: Ⅰ Class: the primary branch is completely perpendicular to the ground, equal to 90°, the color of the glume is light yellow; Class II: the angle between the primary branch and the ground is greater than 90° and less than 180°, the color of the glume is yellow-green ; Class III: The primary branch is parallel to the ground, equal to 180°, and the color of the glume is yellow-green; Class IV: The primary branch is parallel to the ground, equal to 180°, and the color of the glume is green.

2.3 Anther pretreatment

On a sterile ultra-clean workbench, disinfect with 10% disinfectant for 10 minutes before inoculation, rinse with sterile water 4 to 5 times, receive 20 branches and anthers of about 100 flower buds per test tube (50ml), add 12ml of the extract was placed in the refrigerator at 5°C for 2d treatment.

2.3 Microspore dissociation

After the pretreatment of the anthers, use a high-speed disperser on the ultra-clean workbench for ultra ^- speed rotary cutting, and filter the rotary-cut suspension with a 150-mesh sieve. , to collect microspores.

2.4 Observation of microspore development period

After the microspores were dissociated, take 10 μl of the microspore suspension, add FDA dye, mix well, place in the dark at room temperature for 10 minutes, observe under a fluorescence microscope, and count the viable microspores and all microspores in one field of view.

Take 1 μl of the microspore precipitate, slowly add Carnot’s fixative (alcohol:acetic acid = 3:1), try not to disperse, after fixing for 10 min, remove the supernatant, add 10 μl ddH ₂ O and 10 μl 0.1mg/ml DAPI for staining liquid, mixed evenly, placed in the dark for 10 min at room temperature, and observed under a fluorescence microscope.

2.5 Microspore callus induction

The collected microspores were pretreated with the extract solution at 25°C in the dark for 2 days, and then the microspores were purified and cultured. Wash once with induction medium before culturing, then adjust the microspore density to 1.0×10 ⁵ ·mL ^-1 , take 1.0 mL of microspore suspension and inoculate it in a petri dish (35mm×12mm), repeat 3 times, use Seal with parafilm, 25 ℃, dark culture, 23d, callus formation.

The extract contained 320 mM mannitol, 2 mM CaCl ₂ , 1.6 mM N-Morpholineethane sulfonic acid (MES) and 1 mM colchicine, pH 5.8.

The induction medium was based on N6 medium, supplemented with 175mM sucrose, 1mM 2,4-Dichlorophenoxyacetic acid (2,4-D), 1.6mM MES, 3.4mM glutamine , 4.3mM proline, pH5.8.

Both the extract and induction medium were filter sterilized.

3. Growth statistics and data processing

Microspore Viability: The number of viable microspores in a field of view as a percentage of all microspores.

Callus yield: the amount of callus when the microspores were cultured for 23 days. After the liquid medium was absorbed by the petri dish, it was weighed with an electronic balance and expressed in mg·dish ^-1 ;

The test results were analyzed using Microsoft Excel 2010, DPS v7.05 data processing system.

4. Results and Analysis

See Figure 1 for the standards for the color of primary branch and glume: Class I: Picked up with tweezers and perpendicular to the ground, the glume color is light yellow; Class II: Picked up with tweezers, the curvature is between 90° and 180° , the color of the glume is yellow-green; Class III: It is parallel to the ground when picked up with tweezers, and the color of the glume is yellow-green; Class IV: When it is picked up with tweezers, the color of the glume is green.

According to the different degrees of curvature of the primary branch and the color of the glume, rice young panicles are divided into four types. After low temperature treatment, the vigor of type II microspores is the best, reaching 54.70%. The microspore development stage is in the middle and late mononuclear stage (Table 1). .

Table 1 Identification of microspore viability and developmental stages of different types of rice young panicles after low temperature treatment

See Figure 2 for the callus yields obtained after the young ears of rice were cultured with microspores classified by different degrees of branch curvature and glume color. Type II microspore callus yield was the highest.

In the present invention, the bending degree of the primary branch of the young ear of rice is between 90° and 180°, the color of the glume is yellow-green, and the microspore development stage is in the middle and late stages, which is more suitable for microspore culture and can form more injured tissue.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments, and those skilled in the art can make various equivalents without departing from the spirit of the present invention. Modifications or substitutions of these equivalent modifications or substitutions are all included within the scope defined by the claims of the present application.

Claims (5)

1. a method for quickly determining the development period of rice microspores, it is characterized in that, be by the bending degree and glume color of a branch stem, quickly determine the development period of rice microspores, comprise the following steps: (a) Select the young ears of rice from which the flag leaves have been drawn; (b) Remove the primary stem from the main stem of the ear, pick up the base of the primary stem with tweezers, and divide the primary stem into four categories according to the degree of curvature of the primary stem and the color of the glume: Class I: Hold it with tweezers and perpendicular to the ground, equal to 90°, the glume color is light yellow; the microspore development period is the mononucleate stage; Class II: The curvature of the tweezers is between 90° and 180°, and the color of the glume is yellow-green; Class III: clamped with tweezers and parallel to the ground, the glume color is yellow-green; the microspore development period is 50% of the mononucleate stage and the dinucleate stage; Class IV: Picked up with tweezers and parallel to the ground, the glume is green; the developmental stage of microspores is most of the binucleate stage. 2. The method for rapidly determining the developmental stage of rice microspores according to claim 1, wherein the rice variety is Nipponbare. 3. a method for quickly obtaining materials of rice microspores, is characterized in that, is to choose the young ear of rice that flag leaf has been drawn out, a branch stalk is taken off from the main stalk of the ear, a branch stalk base is picked up with tweezers, and a branch is taken. Stem curvature between 90° and 180°, young spikes with yellow-green glume color. 4. a kind of rice microspore quick sampling and culturing method, is characterized in that, comprises the following steps: 1) Material selection: select the young ears of rice that have been extracted from flag leaves; 2) pretreatment of young ear: the young ear is placed in a fresh-keeping bag for low temperature treatment; 3) Color classification of primary branch and glume: take the young ear with the curvature of the branch between 90° and 180° and the color of the glume yellow-green; 4) Free microspores: inoculate anthers, spin at high speed, collect microspores free, and pretreat the collected microspores in a suitable solution and temperature; 5) Microspore culture and callus induction: Purify the pretreated microspores, place them in an induction medium for culture, and induce callus formation. 5. rice microspore quick sampling and culturing method according to claim 4, is characterized in that, comprises the following steps: (A) Material sampling and pretreatment of young ears Take the ear that has been extracted from the flag leaf, keep only one leaf, cut off the leaf, put it in a fresh-keeping bag, and put it in a 5°C refrigerator for low-temperature pretreatment for 10 days; (B) Primary branch classification Peel off the young ear of rice from the leaf sheath, cut the primary stem from the main stem, pick up the base of the stem with tweezers, take the stem with a curvature between 90° and 180°, and the color of the glume is yellow-green. young ear; (C) Anther pretreatment On a sterile ultra-clean workbench, disinfect with 10% disinfectant for 10 minutes before inoculation, rinse with sterile water for 4 to 5 times, connect 20 primary branches and anthers of about 100 flower buds to each 50ml test tube, add 12ml to extract liquid, placed in the refrigerator at 5°C for 2 days; (D) Microspore dissociation After the pretreatment of the anthers, use a high-speed disperser on the ultra-clean workbench for ultra ^- speed rotary cutting, and filter the rotary-cut suspension with a 150-mesh sieve. , to collect microspores; (E) Microspore callus induction The collected microspores were pretreated with the extract solution at 25℃ for 2 days in the dark, and then the microspores were purified and cultured; before culturing, they were washed once with the induction medium, and then the microspore density was adjusted to 1.0×10 ⁵ ·mL ^-1 , take 1.0 mL of microspore suspension and inoculate it in a petri dish, repeat 3 times, seal with parafilm, 25 ℃, dark culture, 23 days, callus formation; The extract contains mannitol 320 mM, 2 mM CaCl ₂ , 1.6 mM N-morphine ethanesulfonic acid and 1 mM colchicine, pH 5.8; The induction medium was based on N6 medium, supplemented with 175 mM sucrose, 1 mM 2,4-dichlorophenoxyacetic acid, 1.6 mM MES, 3.4 mM glutamine, 4.3 mM proline, pH 5.8.

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