Method for obtaining double haploid pepper plant through free microspore culture

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for obtaining double haploid pepper plants through free microspore culture. The method specifically comprises the following steps: (1) performing low-temperature pretreatment on chili flower buds, and performing disinfection; (2) mixing the disinfected flower buds with an extracting solution to obtain a suspension, and dissociating and collecting microspores; the extracting solution comprises mannitol, calcium chloride and a buffer solution; (3) putting the microspores obtained in the step (2) into an induction culture medium for induction culture to obtain calluses; and (4) placing the callus obtained in the step (3) in a differential culture medium for differential culture to obtain the pepper seedlings. According to the method disclosed by the invention, calluses and embryos are prevented from being formed in anther somatic cells, the calluses instead of embryoids are formed through microspore culture, regenerated plants are formed through redifferentiation of the calluses, and a new method is provided for pepper haploid breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for obtaining double haploid pepper plants through free microspore culture. Background Art

[0002] Pepper is a widely grown crop around the world and can be used as vegetables, sauces, ingredients, food colorants and spicy agents, or in the preparation of medicines. At present, people mainly use the method of continuous selfing to purify parents in hybrid vigor breeding. Therefore, it usually takes 5 to 6 generations or even more of continuous selfing and selection to cultivate excellent inbred lines from heterozygous breeding materials. The main advantage of double haploids in plant breeding is that they can quickly achieve complete homozygosity, and the required genotype is fixed in one generation, reducing the time and cost of variety or inbred line development. In vitro androgenesis through anther or free microspore culture is the fastest way and most effective system to achieve homozygosity and produce pure lines. Compared with anther culture, free microspore culture can avoid the formation of callus and embryos from anther body tissue. Free microspores can directly contact the culture medium, which accelerates the optimization of culture conditions; in addition, the high level of spontaneous chromosome doubling in free microspore culture is another advantage of free microspore culture.

[0003] At present, the best method for haploid breeding of pepper is to use a two-step culture method to produce high-quality embryos and plant regeneration through free microspore culture, but the culture steps are too cumbersome and easily increase the probability of contamination. In this study, we obtained callus tissue through free microspore culture, and the callus tissue differentiated into double haploid pepper plants, providing a new method for haploid breeding of pepper. Summary of the invention

[0004] The object of the present invention is to provide a method for obtaining double haploid pepper plants through free microspore culture.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for obtaining double haploid pepper plants through free microspore culture, comprising the following steps:

[0007] (1) pre-treating pepper buds at low temperature and then sterilizing them;

[0008] (2) mixing the sterilized flower buds with an extract to obtain a suspension, and freeing and collecting microspores; the extract comprises mannitol, calcium chloride and a buffer solution;

[0009] (3) placing the microspores obtained in step (2) in an induction medium for induction culture to obtain callus tissue;

[0010] (4) placing the callus obtained in step (3) on a differentiation medium for differentiation culture to obtain pepper seedlings.

[0011] Preferably, the temperature of the low-temperature pretreatment in step (1) is 3 to 5° C. and the time is 1 to 3 days.

[0012] Preferably, in step (2), the mass volume ratio of the flower bud to the extract is 0.6 g:13-18 mL; the concentration of mannitol in the extract is 50-70 g / L; the concentration of calcium chloride is 1.0-1.2 g / L; the concentration of the buffer is 0.9-1.0 g / L; and the buffer is MES.

[0013] Preferably, the method for freeing in step (2) is: the suspension is rotary cut, filtered to obtain a filtrate, and centrifuged 2 to 4 times to collect the microspores.

[0014] Preferably, the rotation speed of the peeling is 3000-5000 rpm, the time is 5-10 s, and the peeling is performed 2-3 times.

[0015] Preferably, the centrifugal speed is 600-800 rpm and the time is 4-6 min.

[0016] Preferably, the induction medium in step (3) is: 1 / 2N6+85-95 g / L maltose+0.8-1.2 mg / L 2,4-D+0.4-0.6 mg / L KT+0.9-1.0 g / LMES, PH=5.7-5.9.

[0017] Preferably, the induction culture method in step (3) is: first pre-treat in the dark at 32-34°C for 3-4 days, and then culture in the dark at 25-28°C for 28-42 days.

[0018] Preferably, the differentiation medium in step (4) is: 1 / 2MS + 25-35 g / L sucrose + 5-7 g / L agar, pH = 5.7-5.9.

[0019] The invention also provides application of the method in cultivating double haploid pepper varieties.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention first performs a pretreatment of 33°C in darkness during induction culture, also called heat shock treatment. Under normal conditions, microspores develop into gametophytes and eventually form mature pollen grains. Heat shock treatment (including the low temperature treatment in step (1)) is a stress process that can prevent microspores from developing into mature pollen grains and promote microspores to enter sporophytes and develop into callus. The purpose of low temperature pretreatment is to artificially change the internal state of microspores, thereby inducing microspores to develop into sporophytes.

[0022] The present invention provides a method for obtaining callus tissue by culturing pepper free microspores, and redifferentiating the callus tissue into a double haploid pepper plant. The development period of pepper free microspores is identified, and the microspores are freed and induced to form callus tissue. The callus is differentiated into pepper seedlings. After ploidy identification, the natural doubling frequency of the method of the present invention can reach 44%, while the natural doubling frequency of the regenerated plant obtained by the two-step culture method is only 16.3%.

[0023] The extracting solution of the present invention is selected to be mannitol, calcium chloride and MES. After the microspores are freed from the anthers, the extracting solution can provide a stable osmotic pressure for the microspores, thereby maintaining the activity of the microspores.

[0024] The method of the invention avoids the formation of callus and embryo from anther cells, forms callus instead of embryoid through microspore culture, and redifferentiation of callus forms regenerated plants, thus providing a new method for pepper haploid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 These are the morphological characteristics of flower buds and anthers at different developmental stages and the corresponding microspore developmental stages of Experimental Example 1.

[0027] Figure 2 The process of callus formation in pepper microspore culture (A) Microspores that swelled and began to divide 3 days after heat shock; (B) Multicellular structure formed after 12 days of culture (scale bar is 125μm); (C) Pepper microspore callus.

[0028] Figure 3 Differentiation of pepper callus into seedlings and ploidy identification (A): buds formed by differentiation of microspore callus; (B): regeneration of pepper seedlings from free microspore culture; (C): double haploid pepper plants; (D): flow cytometry identification of double haploid pepper plants; (E): haploid pepper plants; (F): flow cytometry identification of haploid pepper plants. DETAILED DESCRIPTION

[0029] The present invention provides a method for obtaining double haploid pepper plants through free microspore culture, comprising the following steps:

[0030] (1) pre-treating pepper buds at low temperature and then sterilizing them;

[0031] (2) mixing the sterilized flower buds with an extract to obtain a suspension, and freeing and collecting microspores; the extract comprises mannitol, calcium chloride and a buffer solution;

[0032] (3) placing the microspores obtained in step (2) in an induction medium for induction culture to obtain callus tissue;

[0033] (4) placing the callus obtained in step (3) on a differentiation medium for differentiation culture to obtain pepper seedlings.

[0034] In the present invention, the temperature of the low-temperature pretreatment in step (1) is 3-5°C and the time is 1-3 days; preferably, the temperature of the low-temperature pretreatment is 4°C and the time is 2 days.

[0035] In the present invention, the mass volume ratio of the flower buds to the extract in step (2) is 0.6 g: 13-18 mL; preferably 0.6 g: 14-17 mL; more preferably 0.6 g: 15 mL

[0036] In the present invention, the concentration of mannitol in the extract is 50-70 g / L; the concentration of calcium chloride is 1.0-1.2 g / L; the concentration of the buffer is 0.9-1.0 g / L; preferably, the concentration of mannitol in the extract is 54-66 g / L; the concentration of calcium chloride is 1.1 g / L; the concentration of the buffer is 0.976 g / L; further preferably, the concentration of mannitol in the extract is 60 g / L; the concentration of calcium chloride is 1.1 g / L; the concentration of the buffer is 0.976 g / L.

[0037] In the present invention, the buffer is MES.

[0038] In the present invention, the method for freeing in step (2) is: after rotary cutting of the suspension, filtering to obtain the filtrate, centrifuging 2 to 4 times, and collecting the microspores; preferably 3 times.

[0039] In the present invention, the rotation speed of the peeling is 3000-5000 rpm, the time is 5-10 s, and the peeling is performed 2-3 times; preferably, the rotation speed of the peeling is 3400-4600 rpm, the time is 6-9 s, and the peeling is performed 3 times; further preferably, the rotation speed of the peeling is 600 rpm, the time is 8 s, and the peeling is performed 3 times.

[0040] In the present invention, the centrifugal speed is 600-800 rpm, and the time is 4-6 min; preferably, the centrifugal speed is 640-760 rpm, and the time is 5 min; further preferably, the centrifugal speed is 680-720 rpm, and the time is 5 min; more preferably, the centrifugal speed is 700 rpm, and the time is 5 min.

[0041] In the present invention, the induction medium in step (3) is: 1 / 2N6 + 85-95 g / L maltose + 0.8-1.2 mg / L 2,4-D + 0.4-0.6 mg / L KT + 0.9-1.0 g / L MES, PH = 5.7-5.9; preferably 1 / 2N6 + 87-93 g / L maltose + 0.9-1.1 mg / L 2,4-D + 0.5 mg / L KT + 0.976 g / L MES, PH = 5.8; further preferably 1 / 2N6 + 89-91 g / L maltose + 1 mg / L 2,4-D + 0.5 mg / L KT + 0.976 g / L MES, PH = 5.8; more preferably 1 / 2N6 + 90 g / L maltose + 1 mg / L 2,4-D+0.5mg / LKT+0.976g / LMES, PH=5.8.

[0042] In the present invention, the induction culture method in step (3) is: first pre-treat in the dark at 32-34°C for 3-4 days, and then culture in the dark at 25-28°C for 28-42 days; preferably, pre-treat in the dark at 33°C for 3 days, and then culture in the dark at 26-27°C for 30-40 days; further preferably, pre-treat in the dark at 33°C for 3 days, and then culture in the dark at 26°C for 35 days.

[0043] In the present invention, the differentiation medium in step (4) is: 1 / 2MS + 25-35g / L sucrose + 5-7g / L agar, PH = 5.7-5.9; preferably 1 / 2MS + 27-33g / L sucrose + 6g / L agar, PH = 5.7-5.9; further preferably 1 / 2MS + 30g / L sucrose + 6g / L agar, PH = 5.8.

[0044] The invention also provides application of the method in cultivating double haploid pepper varieties.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1

[0047] 1. Materials

[0048] The excellent pepper variety Hangjiao No. 1 was selected as the test material and purchased from the market.

[0049] 2. Methods

[0050] 2.1 Material planting

[0051] Donor plants are grown in greenhouses and water and fertilizer management is routine;

[0052] 2.2 Bud selection

[0053] At the early flowering stage of pepper, flower buds of different sizes were selected, and the free microspores were stained with DAPI to determine the appearance characteristics of the mononuclear buds. Oversized flower buds were removed in time to extend the flowering period of the plant.

[0054] 2.3 Bud pretreatment: The buds at the appropriate time are placed in a 3℃ refrigerator for 1 day for later use

[0055] 2.4 Disinfection of flower buds: The flower buds that have been treated at low temperature are washed in sterile water for 10 minutes, disinfected with 9% sodium hypochlorite for 10 minutes, washed 3 times with sterile water, disinfected with 9% sodium hypochlorite for 4 minutes, and washed 3 times with sterile water.

[0056] 2.5 Free microspores: About 0.6 g of 20 sterile flower buds were placed in a 50 mL centrifuge tube, 15 mL of extract (50 g / L mannitol + 1.0 g / L CaCl2 + 0.9 g / LMES, pH 5.7, filtered and sterilized), and ultra-high speed rotary cutting was performed on a clean bench using a high-speed disperser (the rotary cutting speed was 3000 rpm, the time was 5 s, and the rotary cutting was repeated twice). The rotary cutting suspension was filtered through a 200 mesh screen, and the filtrate was centrifuged at 600 r / min. -1 , centrifuge at low speed for 4 minutes, repeat twice, and collect microspores.

[0057] 2.6 Microspore callus induction

[0058] The collected pepper microspores were placed in 1 mL of induction medium (1 / 2N6 + 80 g / L maltose, 1 mg / L 2,4-D + 0.4 mg / L KT + 0.9 g / L MES, pH = 5.8, filter sterilized) and the density was adjusted to 1.0 × 10 5 1.0 mL of microspore suspension was inoculated into a culture dish with a diameter of 35 mm, repeated 3 times, sealed with sealing film, pretreated in the dark at 32°C for 3 days, and then the culture dish was placed in the dark at 26°C for 28 days to observe the microspore division and callus formation.

[0059] 2.7 Microspore callus differentiation

[0060] The formed callus tissue was placed on differentiation medium (1 / 2MS + 25g / L sucrose + 5g / L agar, pH = 5.8) and differentiated until it grew into a complete seedling.

[0061] 2.8 Identification of regenerated seedlings: The ploidy of the regenerated seedlings was identified using flow cytometry.

[0062] Example 2

[0063] 1. Materials

[0064] The excellent pepper variety Hangjiao No. 1 was selected as the test material and purchased from the market.

[0065] 2. Methods

[0066] 2.1 Material planting

[0067] Donor plants are grown in greenhouses and water and fertilizer management is routine;

[0068] 2.2 Bud selection

[0069] At the early flowering stage of pepper, flower buds of different sizes were selected, and the free microspores were stained with DAPI to determine the appearance characteristics of the mononuclear buds. Oversized flower buds were removed in time to extend the flowering period of the plant.

[0070] 2.3 Bud pretreatment: The buds at the appropriate time are placed in a 5℃ refrigerator for 3 days for later use

[0071] 2.4 Disinfection of flower buds: The flower buds that have been treated at low temperature are washed in sterile water for 12 minutes, disinfected with 10% sodium hypochlorite for 12 minutes, washed 5 times with sterile water, disinfected with 10% sodium hypochlorite for 6 minutes, and washed 5 times with sterile water.

[0072] 2.5 Free microspores: About 0.6 g of 20 sterile flower buds were placed in a 50 mL centrifuge tube, 15 mL of extract (70 g / L mannitol + 1.2 g / L CaCl2 + 1 g / LMES, pH 5.8, filtered and sterilized), and ultra-high speed rotary cutting was performed on a clean bench using a high-speed disperser (the rotary cutting speed was 5000 rpm, the time was 10 s, and the rotary cutting was performed 3 times). The rotary cutting suspension was filtered through a 300 mesh screen, and the filtrate was centrifuged at 800 r / min. -1 , centrifuge at low speed for 6 minutes, repeat 3 times, and collect microspores.

[0073] 2.6 Microspore callus induction

[0074] The collected pepper microspores were placed in 1 mL of induction medium (1 / 2N6 + 90 g / L maltose, 1.1 mg / L 2,4-D + 0.6 mg / L KT + 1 g / LMES, pH = 5.8, filter sterilized) and the density was adjusted to 1.0 × 10 5 / mL, take 1.0mL of microspore suspension and inoculate it into a culture dish with a diameter of 35mm, repeat 3 times, seal it with sealing film, pretreat it in the dark at 34℃ for 3d, then place the culture dish in the dark at 27℃ for 35d, and observe the microspore division and callus formation.

[0075] 2.7 Microspore callus differentiation

[0076] The formed callus tissue was placed on differentiation medium (1 / 2MS + 35 g / L sucrose + 7 g / L agar, pH = 5.8) and differentiated until it grew into a complete seedling.

[0077] 2.8 Identification of regenerated seedlings: The ploidy of the regenerated seedlings was identified using flow cytometry.

[0078] Example 3

[0079] 1. Materials

[0080] The excellent pepper variety Hangjiao No. 1 was selected as the test material and purchased from the market.

[0081] 2. Methods

[0082] 2.1 Material planting

[0083] Donor plants are grown in greenhouses and water and fertilizer management is routine;

[0084] 2.2 Bud selection

[0085] At the early flowering stage of pepper, flower buds of different sizes were selected, and the free microspores were stained with DAPI to determine the appearance characteristics of the mononuclear buds. Oversized flower buds were removed in time to extend the flowering period of the plant.

[0086] 2.3 Bud pretreatment: The buds at the appropriate time are placed in a 4℃ refrigerator for 2 days for later use

[0087] 2.4 Disinfection of flower buds: The flower buds that have been treated at low temperature are washed in sterile water for 10 minutes, disinfected with 10% sodium hypochlorite for 10 minutes, washed 4 times with sterile water, disinfected with 10% sodium hypochlorite for 5 minutes, and washed 4 times with sterile water.

[0088] 2.5 Free microspores: About 0.6 g of 20 sterile flower buds were placed in a 50 mL centrifuge tube, and 15 mL of extract (60 g / L mannitol + 1.1 g / L CaCl2 + 0.976 g / LMES, pH 5.8, filtered and sterilized) was added. The microspores were cut by high-speed dispersing on a clean bench (the speed of the microspore was 4000 rpm, the time was 9 s, and the microspores were cut 3 times). The suspension was filtered through a 300-mesh screen and the filtrate was centrifuged at 700 r / min. -1 , centrifuge at low speed for 5 minutes, repeat 3 times, and collect microspores.

[0089] 2.6 Microspore callus induction

[0090] The collected pepper microspores were inducing medium (1 / 2N6 + 90 g / L maltose, 1 mg / L 2,4-D + 0.5 mg / L KT + 0.976 g / LMES, pH = 5.8, filtered and sterilized) to a density of 1.0 × 10 51.0 mL of microspore suspension was inoculated into a culture dish with a diameter of 35 mm, repeated 3 times, sealed with sealing film, pretreated in the dark at 33 °C for 3 days, and then the culture dish was placed in the dark at 26 °C for 42 days to observe the microspore division and callus formation.

[0091] 2.7 Microspore callus differentiation

[0092] The formed callus tissue was placed on differentiation medium (1 / 2MS+30g / L sucrose+6g / L agar, pH=5.8) and differentiated until it grew into complete seedlings.

[0093] 2.8 Identification of regenerated seedlings: The ploidy of the regenerated seedlings was identified using flow cytometry.

[0094] Experimental Example 1

[0095] Verify the optimal time to pick pepper buds.

[0096] The flower buds at different developmental stages were divided into four types ( Figure 1 ), the first type is that the corolla is wrapped in the calyx, the second type is that the corolla and the calyx are about the same length, the third type is that the corolla exceeds the calyx by about 20%, and the fourth type is that the corolla exceeds the calyx by about 50%. The anthers of different types of buds were picked out and found that the anthers of these four types of buds were light yellow in color, most of the anthers were light yellow with a small amount of purple on the top, the anthers were purple throughout, and the anthers were purple throughout and continued to grow. The anthers of different types of buds were freed from microspores and then stained with DAPI for observation. The microspores of the first type were all in the mononuclear stage; most of the microspores of the second type were in the mononuclear stage, and a small amount was in the dinuclear stage, that is, the mononuclear side stage; most of the microspores of the third type entered the dinuclear stage, and a small amount was in the mononuclear stage; the microspores of the fourth type were all in the dinuclear stage. The microspores are in the mononuclear side stage, which is the appropriate period for microspore culture, that is, the size of the second type of buds is suitable for sampling. The apparent characteristics of this period are that the corolla is approximately the same length as the calyx, and the anthers are mostly light yellow with a small amount of purple at the tips.

[0097] Experimental Example 2

[0098] Example 3 During the induction of microspore callus, after the microspores were pretreated (heat-shocked) at 33°C in the dark for 3 days, it was observed that some of the microspores began to swell and divide ( Figure 2 A), after 12 days of dark culture at 26℃, obvious multicellular structures can be seen ( Figure 2 B), continue to culture and eventually form callus ( Figure 2C). When the microspores were not subjected to heat shock treatment and directly cultured in the dark at 26°C, the microspores would only swell but not divide, and no callus was formed in the end. Therefore, heat shock at 33°C for 3 days is a necessary condition for initiating the division of pepper microspores and forming callus. The yield of the formed callus was counted, and the average yield of the formed callus per dish was 79.07±2.98mg.

[0099] Experimental Example 3

[0100] Example 3 In the differentiation of microspore callus, the formed callus was placed on a differentiation medium for differentiation. After 4 to 6 weeks of culture, pepper seedlings were obtained. A total of 16 regenerated seedlings were obtained, of which 9 were haploid and 7 were diploid, with a natural doubling frequency of about 44% ( Figure 3 ).

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for obtaining double haploid pepper plants by free microspore culture, characterized in that: The steps include: (1) pre-treating pepper buds at low temperature and then sterilizing them; (2) mixing the sterilized flower buds with an extract to obtain a suspension, and freeing and collecting microspores; the extract comprises mannitol, calcium chloride and a buffer solution; (3) placing the microspores obtained in step (2) in an induction medium for induction culture to obtain callus tissue; (4) placing the callus obtained in step (3) on a differentiation medium for differentiation culture to obtain pepper seedlings.

2. The method according to claim 1, characterized in that: The temperature of the low-temperature pretreatment in step (1) is 3 to 5° C. and the time is 1 to 3 days.

3. The method according to claim 1, characterized in that In step (2), the mass volume ratio of the flower bud to the extract is 0.6 g:13-18 mL; the concentration of mannitol in the extract is 50-70 g / L; the concentration of calcium chloride is 1.0-1.2 g / L; the concentration of the buffer is 0.9-1.0 g / L; and the buffer is MES.

4. The method according to claim 1, characterized in that: The method for releasing the microspores in step (2) is as follows: the suspension is rotary cut, filtered to obtain the filtrate, and centrifuged 2 to 4 times to collect the microspores.

5. The method according to claim 4, characterized in that The rotation speed of the peeling is 3000-5000 rpm, the time is 5-10 s, and the peeling is performed 2-3 times.

6. The method according to claim 4, characterized in that The centrifugal speed is 600-800 rpm and the time is 4-6 minutes.

7. The method according to claim 1, characterized in that The induction medium in step (3) is: 1 / 2N6+85-95 g / L maltose+0.8-1.2 mg / L 2,4-D+0.4-0.6 mg / L KT+0.9-1.0 g / L MES, pH=5.7-5.

9.

8. The method according to claim 1, characterized in that The induction culture method in step (3) is: first pre-treat in the dark at 32-34° C. for 3-4 days, and then culture in the dark at 25-28° C. for 28-42 days.

9. The method according to claim 1, characterized in that: The differentiation medium in step (4) is: 1 / 2MS + 25-35 g / L sucrose + 5-7 g / L agar, pH = 5.7-5.

9.

10. Use of the method according to any one of claims 1 to 9 in breeding double haploid pepper varieties.

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