CN102726300A - Anther culture method of cauliflower - Google Patents

Abstract

The invention relates to an anther culture method of cauliflower, belonging to biology technical field. The method includes taking anther of cauliflower as material, inoculating under sterile condition, performing anther induced callus culture, performing callus differentiation culture, and performing differentiated seedling rooting culture, to realize culture of new cauliflower seedlings. Anther culture can keep stable parent purity, and accelerate selection effect of hybrid progeny. The method has good application value for accelerating culture of excellent cauliflower variety.

Description

A kind of anther cultivation method of cauliflower

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for cultivating cauliflower anthers.

Background technique

Cauliflower ( Brassica oleracea Var. botrytis ), also known as cauliflower, is one of the main varieties planted in autumn and winter in China. Because of its low crude fiber and high nutritional value, it is deeply loved by consumers. By crossing different ecotype varieties, new cauliflower varieties can be bred, which has strong heterosis. However, cauliflower is a cross-pollinated crop of the Brassicaceae family, which suffers from serious self-infertility decline, and the selection of self-incompatible lines is difficult, and the variety depends on population heterozygosity to maintain stability. And breeding hybrids is difficult. Through anther culture, the purity of the parents can be kept stable, and the selection effect of the hybrid progeny can be accelerated. It has very good application value for accelerating the cultivation of fine cabbage varieties.

Contents of the invention

The object of the present invention is to provide a method for cultivating anthers of cauliflower.

The present invention provides a cauliflower anther culture method, the culture method comprising the following steps: (1) anther collection and disinfection treatment; (2) anther callus culture; (3) callus differentiation culture; (4) ) rooting culture of differentiated seedlings; (5) seedling hardening, artificial planting and transplanting.

Described cultivation method specifically comprises the following steps:

(1) Collection and disinfection of anthers

①Anther collection: In the morning on a sunny day, take flower buds with full anthers with a length of 3mm, and check under the microscope to confirm that the pollen development at this time is in the uninucleate stage or in the late stage of uninucleate;

② Disinfection treatment of anthers: After the flower buds are soaked in 70% alcohol for 30 seconds, they are disinfected with 0.1% mercury liter solution for 300 minutes, and then rinsed with sterile water for 5 times;

(2) Anther callus culture

①Callus induction medium: basal medium E3+BA0.5mg/L+NAA0.1 mg/L,

The medium preparation method was operated according to the routine.

②Inoculation: Detoxify the flower buds, remove the petals with a dissecting needle or tweezers under aseptic conditions, hold the filaments with tweezers, take out the anthers, and inoculate the anthers directly and evenly on the callus induction medium, each 200ml tissue culture bottle Connect 3 anthers;

③Cultivation: After inoculation, culture in the dark at 25±1°C for 7 days, and then cultivate in light for 12 hours a day, with a light intensity of 800 lux and a relative humidity of 65%;

(3) Callus differentiation culture

① Differentiation medium: MS+0.5-1mg/L 6-BA + 0.5 mg/L NAA;

②The callus mass grows larger, and gradually turns green, and then transferred to the differentiation medium for cultivation;

③Cultivation conditions: temperature 25°C, humidity 70%, light intensity 1000-1500LX;

(4) Rooting culture of differentiated seedlings

① Rooting medium: 1/2MS+0.1 mg/L NAA+activated carbon 0.2%;

②When the seedling grows to 3-4 cm high and grows 3 leaves, it is inserted into the rooting medium for cultivation;

③Cultivation conditions: temperature 25°C, humidity 70%, light intensity 1500-2000LX;

(5) Fake planting seedlings

① Seedling hardening: When the root of the seedling grows out and the length reaches 2cm, open the bottle cap, the ambient temperature is 25°C, the humidity is controlled at 85%, the light intensity is 2000LX, and the seedling is hardened for 3 to 5 days;

②False planting: After the seedlings are hardened, move them to small plastic trays for seedling cultivation. After the planting, the ambient temperature is 25°C, the humidity is controlled at 90%, and the light intensity is 2000LX. After survival, they are moved to the field for cultivation.

After 3-5 generations of anther culture, the cauliflower progeny tends to be stable, which can be used as the material of the hybrid parent of cauliflower breeding.

The invention has the advantages that the purity of the parents can be kept stable through anther culture, and the selection effect of the hybrid progeny can be accelerated. It has very good application value for accelerating the cultivation of fine cabbage varieties.

Description of drawings

Figure 1 is a picture of anther callus.

Figure 2 is callus differentiation into seedlings.

Figure 3 is the rooting culture.

Fig. 4 is a fake seedling.

Detailed ways

Example 1

1. Collection and disinfection of anthers

(1) Anther collection: In the morning on a sunny day, flower buds with full anthers with a length of about 3 mm were taken as experimental materials, and microscopic examination confirmed that the pollen development at this time was in the uninucleate stage or late uninucleate stage.

(2) Disinfection treatment of anthers: After the flower buds are soaked in 70% alcohol for 30 seconds, they are disinfected with 0.1% mercuric chloride solution for 300 minutes, and then rinsed with sterile water for 5 times.

2. Inoculation and culture of anther callus

(1) Induced callus medium: E3*+BA0.5mg/L+NAA0.1mg/L.

The medium preparation method was operated according to the routine.

(2) Inoculation: detoxify the flower buds, remove the petals with a dissecting needle or tweezers under sterile conditions, hold the filaments with tweezers, and take out the anthers. The anthers were directly and evenly inoculated on the callus induction medium (filaments and shriveled anthers were removed), and 3 anthers were inoculated in each bottle (200 tissue culture bottles).

(3) Cultivation: After inoculation, culture in the dark at 25±1°C for 7 days, and then cultivate in light for 12 hours a day, with a light intensity of 800 lux and a relative humidity of about 65%. After 30-40 days of culture, the anther callus can be seen, as shown in Figure 1.

3. Callus differentiation culture

(1) Differentiation medium: MS+0.5-1mg/L 6-BA+0.5 mg/L NAA.

(2) The callus pieces grew larger and gradually turned green, and then transferred to the differentiation medium, and each bottle received 2 callus pieces.

(3) Culture conditions: temperature 25°C, humidity 70%, light intensity 1000-1500LX, culture for 15-20 days, callus differentiates into seedlings, as shown in Figure 2.

4. Rooting culture

(1) Rooting medium: 1/2MS+0.1 mg/L NAA+activated carbon 0.2%.

(2) When the seedlings grow to a height of 3-4 cm and grow 3 leaves, they are transferred to the rooting medium for cultivation.

(3) Culture conditions: temperature 25°C, humidity 70%, light intensity 1500-2000LX, culture for 10-15 days, the root system is developed, as shown in Figure 3.

5. Fake seedlings

(1) Seedling hardening: When the base of the seedling grows roots and the length reaches 2cm, open the bottle cap. The ambient temperature is 25°C, the humidity is controlled at about 85%, and the light intensity is about 2000LX. Harden seedlings for 3 to 5 days.

(2) False planting: After the seedlings are hardened, move them to small plastic trays for seedling cultivation. The soil for seedling cultivation must be sterilized. After the false planting, the ambient temperature is 25°C, the humidity is controlled at 90%, and the light intensity is 2000LX. Plant seedlings, as shown in Figure 4. After surviving, move it to the field for cultivation.

6. Cultivation of cauliflower hybrid parents: After 3 to 5 generations of anther culture, the offspring of cauliflower tend to be stable, which can be used as materials for cauliflower breeding hybrid parents.

Attached E3 medium:

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Claims (2)

1. A method for cultivating anthers of cauliflower, characterized in that: the method for cultivating comprises the following steps: (1) collection and disinfection of anthers; (2) culture of anther callus; (3) differentiation and cultivation of callus; (4) rooting culture of differentiated seedlings; (5) seedling hardening, artificial planting, and transplanting. 2. the anther cultivation method of cauliflower according to claim 1, is characterized in that, described cultivation method specifically comprises the following steps: (1) Collection and disinfection of anthers ①Anther collection: In the morning on a sunny day, take flower buds with full anthers with a length of 3mm, and check under the microscope to confirm that the pollen development at this time is in the uninucleate stage or in the late stage of uninucleate; ② Disinfection treatment of anthers: After the flower buds are soaked in 70% alcohol for 30 seconds, they are disinfected with 0.1% mercury liter solution for 300 minutes, and then rinsed with sterile water for 5 times; (2) Anther callus culture ①Callus induction medium: basal medium E3+BA0.5mg/L+NAA0.1 mg/L, The medium preparation method is operated according to the routine; ②Inoculation: Detoxify the flower buds, remove the petals with a dissecting needle or tweezers under aseptic conditions, hold the filaments with tweezers, take out the anthers, and inoculate the anthers directly and evenly on the callus induction medium, each 200ml tissue culture bottle Connect 3 anthers; ③Cultivation: After inoculation, culture in the dark at 25±1°C for 7 days, and then cultivate in light for 12 hours a day, with a light intensity of 800 lux and a relative humidity of 65%; (3) Callus differentiation culture ① Differentiation medium: MS+0.5-1mg/L 6-BA + 0.5 mg/L NAA; ②The callus mass grows larger, and gradually turns green, and then transferred to the differentiation medium for cultivation; ③Cultivation conditions: temperature 25°C, humidity 70%, light intensity 1000-1500LX; (4) Rooting culture of differentiated seedlings ① Rooting medium: 1/2MS+0.1 mg/L NAA+activated carbon 0.2%; ②When the seedling grows to 3-4 cm high and grows 3 leaves, it is inserted into the rooting medium for cultivation; ③Cultivation conditions: temperature 25°C, humidity 70%, light intensity 1500-2000LX; (5) Fake planting seedlings ① Seedling hardening: When the root of the seedling grows out and the length reaches 2cm, open the bottle cap, the ambient temperature is 25°C, the humidity is controlled at 85%, the light intensity is 2000LX, and the seedling is hardened for 3 to 5 days; ②False planting: After the seedlings are hardened, move them to small plastic trays for seedling cultivation. After the planting, the ambient temperature is 25°C, the humidity is controlled at 90%, and the light intensity is 2000LX. After survival, they are moved to the field for cultivation.

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