CN116746489B - Sweet pepper tissue culture breeding medium and method based on two-step seedling method - Google Patents

Sweet pepper tissue culture breeding medium and method based on two-step seedling method Download PDF

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CN116746489B
CN116746489B CN202310773507.XA CN202310773507A CN116746489B CN 116746489 B CN116746489 B CN 116746489B CN 202310773507 A CN202310773507 A CN 202310773507A CN 116746489 B CN116746489 B CN 116746489B
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sweet pepper
seedling
tissue culture
seedlings
watering
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CN116746489A (en
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陈汉鑫
马馨怡
林艺辉
王锦玲
林秀芳
罗燕华
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ZHANGZHOU INSTITUTE OF AGRICULTURAL SCIENCES
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants

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Abstract

The invention discloses a sweet pepper efficient tissue culture breeding medium and a method based on a two-step seedling method. The invention adopts secondary seedling formation. 1. The GMS culture medium has fast bud growth vigor; basal callus-free; no edema of seedlings occurred. 2. The bud of the invention grows well; longer internodes, large and stretched leaves, greenish color and developed root system at the base. 3. In the traditional tissue culture method, cytokinin is often used in the proliferation process, and is easy to cause seedling variation, so that the cytokinin is avoided as much as possible in order to keep the stable characters of crops such as sweet peppers, and the two-step seedling method just avoids the use of the cytokinin.

Description

Sweet pepper tissue culture breeding medium and method based on two-step seedling method
Technical Field
The invention relates to a sweet pepper tissue culture breeding medium and method based on a two-step seedling method.
Background
Sweet pepper (Capsicum frutescens) is also called as vegetable pepper, and is one of important vegetables supplied annually in China; the fruit and vegetable juice has the highest vitamin content in fruits and vegetables, is rich in carotene, capsanthin, catalpa acid, malic acid, various saccharides, proteins, mineral substances and other nutrient substances, has the effects of improving appetite, promoting digestion, preventing and treating scurvy and the like, and is deeply favored by people.
In recent years, along with the increase of the market price of sweet peppers, the planting area of the sweet peppers is continuously increased, but the planting area still cannot meet the demands of domestic and foreign markets, and especially the seedling supply is not required. At present, artificially cultivated sweet pepper seedlings are mainly bred by seed and grafting, wherein the seed breeding is long in time consumption and high in cost (especially foreign varieties, most of the sweet pepper seedlings are sterile, the seeds depend on import and are high in price), and the emergence speed and the emergence rate are relatively low; the grafting breeding has wound, weak seedling stress resistance, high disease and insect infection rate and low survival rate, and the problems directly affect the mass production process of the sweet pepper industry.
Disclosure of Invention
The invention mainly aims at providing a sweet pepper efficient tissue culture breeding method based on a two-step seedling method.
The technical scheme adopted for solving the technical problems is as follows:
one of the technical proposal is as follows:
a sweet pepper efficient tissue culture breeding culture medium based on a two-step seedling method comprises the following components:
Macroelements of ：KNO3 1150-1250mg/L、NH4NO3 825-875mg/L、KH2PO4 55-65mg/L、MgSO435-40mg/L、Ca(NO3)2 380-420mg/L;
Microelements ：KI 0.80-0.9mg/L、H3BO3 6.0-6.5mg/L、MnSO4·4H2O 22.0-23.0mg/L、ZnSO4·7H2O 8.0-9.0mg/L、Na2MoO4·2H2O 0.20-0.30mg/L、CuSO4·5H2O 0.020-0.030mg/L、CoCl2·6H2O 0.020-0.030mg/L;
Iron salt: feSO 4·7H2O 27.0-28.0mg/L、Na2-EDTA·2H2 O37.0-38.0 mg/L;
Organic components: 95-105mg/L inositol, 0.3-0.7mg/L nicotinic acid, 0.3-0.7mg/L pyridoxine hydrochloride, 0.3-0.7mg/L thiamine hydrochloride and 1.8-2.2mg/L glycine.
Preferably, the culture medium also comprises NAA 0.2-0.4mg/L and sucrose 25-35g/L.
The second technical scheme of the invention is as follows:
A sweet pepper efficient tissue culture breeding method based on a two-step seedling method comprises the following steps:
Step one: screening sweet pepper finished product strains which are vigorous in growth, beautiful in fruits and free of plant diseases and insect pests in a sweet pepper planting base; cutting off sweet pepper fruits before transplanting, removing leaves at the lower ends of branches, then transplanting the sweet pepper finished plant with root soil balls into a plastic big basin together, and placing the plastic big basin into a greenhouse; after the transplanting of the sweet peppers is recovered, taking branches with flower buds, removing the flower buds and leaves on the branches, leaving leaf stalks of 0.8-1.2cm, cleaning, sterilizing and draining water; cutting the sterilized branches into stem segments with the length of 1.5-2.0cm and 1 stem node;
Step two: a GMS medium configured for sweet pepper stem segment induced germination comprising:
The major elements are ：KNO3 1150-1250mg/L、NH4NO3 825-875mg/L、KH2PO4 55-65mg/L、MgSO435-40mg/L、Ca(NO3)2 380-420mg/L;
Microelements ：KI 0.80-0.9mg/L、H3BO3 6.0-6.5mg/L、MnSO4·4H2O 22.0-23.0mg/L、ZnSO4·7H2O 8.0-9.0mg/L、Na2MoO4·2H2O 0.20-0.30mg/L、CuSO4·5H2O0.020-0.030mg/L、CoCl2·6H2O 0.020-0.030mg/L;
Iron salt: feSO 4·7H2O 27.0-28.0mg/L、Na2-EDTA·2H2 O37.0-38.0 mg/L;
Organic components: 95-105mg/L inositol, 0.3-0.7mg/L nicotinic acid, 0.3-0.7mg/L pyridoxine hydrochloride, 0.3-0.7mg/L thiamine hydrochloride and 1.8-2.2mg/L glycine.
Adding NAA 0.2-0.4mg/L, sucrose 25-35g/L and carrageenan 6-10g/L into the culture medium; the pH is 5.5-6.0; inoculating the sterilized stem segments in a culture medium, and culturing: the temperature is 26-28 ℃, the illumination intensity is 2400-2600lx, and the illumination test is 10-15h/d;
Step three: after the sweet pepper seedling grows to 7-8cm, cutting the sweet pepper seedling into stem segments with knots, and inoculating the stem segments into a 1/2GMS culture medium, wherein the culture conditions are unchanged. The preparation method of the 1/2GMS medium comprises the following steps:
Culturing on the basis of GMS, halving the concentration of macroelements, keeping the concentration of microelements, ferric salt and organic components unchanged, and adding IBA 0.5-1.0mg/L, sucrose 15-25g/L, carrageenan 6-10g/L and activated carbon 0.5-1.5g/L; the pH is 5.5-6.0;
Step four: selecting root seedlings of sweet peppers which have no callus at roots, have more than 2 normal root systems, are 4-5cm high, have about 3 leaves, are strong in growth and consistent in growth vigor, and performing adaptive seedling hardening for about 18-25 d; transplanting the plant into a greenhouse after the stems of the plant become hard and the colors of the leaves become green; the substrate used for transplanting is: the turf and vermiculite are prepared according to the volume ratio of (1.8-2.2): 1, and each cubic matrix needs 2.5-3.5kg of calcium magnesium phosphate fertilizer;
Further, in the first step, the stem segment of sweet pepper is disinfected as follows: brushing the material with a soft brush from top to bottom; placing the material into an ultrasonic oscillator, and performing ultrasonic oscillation for 15-25min; then soaking in 70% alcohol for 25s-35s on an ultra-clean workbench, sterilizing for 6-10min with 0.1% mercuric chloride and 2-3 drops of tween-80, washing with sterile water for 4-6 times, washing to remove residual sterilizing liquid, and filtering with sterile filter paper to remove residual water.
In the fourth step, ventilation is gradually increased 5-7 days before the sweet pepper tissue culture seedlings are planted in the field, and the seedbed environment is controlled to be similar to the field environment. The watering amount is gradually reduced 3-5 days before field planting, the adaptability of seedlings is exercised, and the field planting survival rate is improved.
In the fourth step, before the substrate is used, the grass carbon and the vermiculite are firstly mixed and dry-mixed, then the calcium magnesium phosphate fertilizer is put into the substrate and is mixed for 2 times, then the substrate is sprayed with water and is wet-mixed for 2 times, the substrate raw materials are fully mixed and mixed, and then a greenhouse film is used for covering and stewing for 1-3 days.
Further, in the fourth step, after the substrate is piled and stewed, the substrate is transferred into a 72-hole cave dish, the sweet pepper tissue culture seedlings are transplanted well, root fixing water is poured thoroughly, and watering is not carried out in the winter and spring low-temperature period; after the new leaves are extracted, water is properly controlled, and a principle of non-drying and non-watering is adopted.
Further, low-temperature overcast and rainy days are avoided after watering to prevent diseases; watering for 1 time in the morning and evening in summer and autumn in a high-temperature period; the seedling bed is thoroughly watered each time, and the periphery of the seedling bed is uniform, so that dead angles caused by missed watering are prevented; if the seedlings are in overgrowth, temperature control and fertilizer control are adopted, but water cannot be controlled, so that root system damage is avoided.
Compared with the background technology, the technical proposal has the following advantages:
1. the culture medium is suitable for efficient tissue culture and breeding of the sweet pepper by a two-step seedling method; the bud growth vigor induced by the culture medium is fast; basal callus-free; no seedling edema phenomenon occurs; the bud of the invention grows well; longer internodes, large and stretched leaves, greenish color and developed root system at the base.
2.In the traditional tissue culture method, cytokinin is often used in the proliferation process, and is easy to cause seedling variation, so that the cytokinin is avoided as much as possible in order to keep the stable characters of crops such as sweet peppers, and the two-step seedling method just avoids the use of the cytokinin.
3. The two-step seedling method of the invention has the obvious advantages that: in the traditional tissue culture method, due to the addition of cytokinin, the hormone level of the cytokinin needs to be reduced before the later rooting so as to ensure the normal growth of root systems, and the transplanting efficiency is low; the two-step seedling method adopted by the invention can directly root after induction because cytokinin is not added, so that the transplanting of the root seedlings can be immediately carried out in the planting season, and the transplanting efficiency is greatly improved. In other seasons, the root of the root-producing seedling can be cut off, the stem is cut into a plurality of sections, and then the sections are inoculated into a rooting culture medium for continuous reproduction, so that materials are fully utilized, and waste caused by seasons is reduced.
Drawings
The invention is further described below with reference to the drawings and examples.
FIG. 1 is a physical diagram of example 1 in Table 1.
FIG. 2 is a physical diagram of example 2 in Table 1.
FIG. 3 is a physical diagram of example 3 in Table 1.
FIG. 4 is a physical diagram of comparative example 1 in Table 1.
FIG. 5 is a physical diagram of comparative example 2 in Table 1.
FIG. 6 is a physical diagram of comparative example 3 in Table 1.
FIG. 7 is a physical diagram of comparative example 4 in Table 1.
FIG. 8 is a physical diagram of example 1 in Table 2.
FIG. 9 is a physical diagram of example 2 in Table 2.
FIG. 10 is a physical diagram of example 3 in Table 2.
FIG. 11 is a physical diagram of comparative example 1 in Table 2.
FIG. 12 is a physical diagram of comparative example 2 in Table 2.
FIG. 13 is a physical diagram of comparative example 3 in Table 2.
FIG. 14 is a physical diagram of comparative example 4 in Table 2.
Detailed Description
The following examples illustrate the invention further, but do not limit it.
Example 1
Step one: screening sweet pepper finished product strains which are vigorous in growth, beautiful in fruits and free of plant diseases and insect pests in a sweet pepper planting base; cutting off sweet pepper fruits before transplanting, removing leaves at the lower ends of branches, then transplanting the sweet pepper finished plant with root soil balls into a plastic big basin together, and placing the plastic big basin into a greenhouse; after the transplanting of the sweet peppers is recovered, taking the branches with flower buds, removing the flower buds and leaves on the branches, leaving leaf stalks of 0.8-1.2cm, and cleaning. Then sterilizing, and brushing the materials from top to bottom by using a soft brush; placing the material into an ultrasonic oscillator, and performing ultrasonic oscillation for 15min; then soaking in 70% alcohol for 25s on an ultra-clean workbench, sterilizing for 6min with 0.1% mercuric chloride and 2-3 drops of tween-80, washing with sterile water for 4 times, washing to remove residual sterilizing liquid, and filtering with sterile filter paper to remove residual water. Cutting the sterilized branches into stem segments with the length of 1.5-2.0cm and 1 stem node;
Step two: inoculating the sterilized stem segments into a GMS culture medium, and culturing under the condition: the temperature is 26 ℃, the illumination intensity is 2400lx, and the illumination test is 10h/d;
The composition of the GMS medium is:
The major elements are ：KNO3 1150mg/L、NH4NO3 825mg/L、KH2PO4 55mg/L、MgSO4 35mg/L、Ca(NO3)2 380mg/L;
Microelements ：KI 0.80mg/L、H3BO3 6.0mg/L、MnSO4·4H2O 22.0mg/L、ZnSO4·7H2O8.0mg/L、Na2MoO4·2H2O 0.20mg/L、CuSO4·5H2O 0.020mg/L、CoCl2·6H2O 0.020mg/L;
Iron salt: feSO 4·7H2O 27.0mg/L、Na2-EDTA·2H2 O37.0 mg/L;
Organic components: inositol 95mg/L, nicotinic acid 0.3mg/L, pyridoxine hydrochloride 0.3mg/L, thiamine hydrochloride 0.3mg/L, glycine 1.8mg/L.
NAA 0.2mg/L, sucrose 25g/L and carrageenan 6g/L are added into the culture medium; adjusting the pH to 5.5;
step three: after the sweet pepper seedling grows to 7-8cm, cutting the sweet pepper seedling into stem segments with knots, and inoculating the stem segments into a 1/2GMS culture medium, wherein the culture conditions are unchanged.
The preparation method of the 1/2GMS medium comprises the following steps:
Culturing on the basis of GMS, halving the concentration of macroelements, keeping the concentration of microelements, ferric salt and organic components unchanged, and adding IBA 0.5mg/L, sucrose 15g/L, carrageenan 6g/L and activated carbon 0.5g/L; the pH is adjusted to 5.5;
Step four: selecting root seedling with no callus, more than 2 normal root systems, 4-5cm high, 3 leaves, strong growth and uniform growth vigor, and adaptively hardening off about 18 d. And (5) transplanting the plant leaves into a greenhouse after the stems of the plant leaves are hardened and the colors of the plant leaves are changed to green. The substrate used for transplanting is: turf and vermiculite are prepared according to the volume ratio of 1.8:1. Before the use of the matrix, the grass carbon and the vermiculite are mixed and dry-mixed, and each cubic matrix needs 2.5kg of calcium magnesium phosphate fertilizer, the calcium magnesium phosphate fertilizer is put into the matrix and is uniformly mixed for 2 times, then the matrix is sprayed with water and is wet-mixed for 2 times, the raw materials of the matrix are fully and uniformly mixed, and then a greenhouse film is used for covering and stewing for 1 day. After the substrate is piled and stewed, the substrate is transferred into a 72-hole cave dish, the sweet pepper tissue culture seedlings are transplanted, fixed root water is poured thoroughly, and watering is not carried out in the winter and spring low-temperature period; after the new leaves are extracted, water is properly controlled, and a principle of non-drying and non-watering is adopted. Low-temperature overcast and rainy days are avoided after watering to prevent diseases; watering for 1 time in the morning and evening in summer and autumn in a high-temperature period; the seedling bed is thoroughly watered each time, and the periphery of the seedling bed is uniform, so that dead angles caused by missed watering are prevented; if the seedlings are in overgrowth, temperature control and fertilizer control are adopted, but water cannot be controlled, so that root system damage is avoided. Gradually increasing ventilation quantity 5 days before the sweet pepper tissue culture seedlings are planted in the field, and controlling the seedbed environment to be similar to the field environment. The watering amount is gradually reduced 3 days before field planting, the adaptability of seedlings is exercised, and the field planting survival rate is improved.
Example 2
Step one: screening sweet pepper finished product strains which are vigorous in growth, beautiful in fruits and free of plant diseases and insect pests in a sweet pepper planting base; cutting off sweet pepper fruits before transplanting, removing leaves at the lower ends of branches, then transplanting the sweet pepper finished plant with root soil balls into a plastic big basin together, and placing the plastic big basin into a greenhouse; after the transplanting of the sweet peppers is recovered, taking the branches with flower buds, removing the flower buds and leaves on the branches, leaving leaf stalks of 0.8-1.2cm, and cleaning. Then sterilizing, and brushing the materials from top to bottom by using a soft brush; placing the material into an ultrasonic oscillator, and performing ultrasonic oscillation for 20min; then soaking in 70% alcohol for 30s on an ultra-clean workbench, sterilizing for 8min with 0.1% mercuric chloride and 2-3 drops of tween-80, washing with sterile water for 5 times, washing to remove residual sterilizing liquid, and filtering with sterile filter paper to remove residual water. Cutting the sterilized branches into stem segments with the length of 1.5-2.0cm and 1 stem node;
step two: inoculating the sterilized stem segments into a GMS culture medium, and culturing under the condition: the temperature is 27 ℃, the illumination intensity is 2500lx, and the illumination test is 13h/d;
The composition of the GMS medium is:
The major elements are ：KNO3 1200mg/L、NH4NO3 850mg/L、KH2PO4 60mg/L、MgSO4 37mg/L、Ca(NO3)2 400mg/L;
Microelements ：KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L;
Iron salt: feSO 4·7H2O 27.8mg/L、Na2-EDTA·2H2 O37.3 mg/L;
organic components: inositol 100mg/L, nicotinic acid 0.5mg/L, pyridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 0.5mg/L, glycine 2.0mg/L.
NAA 0.3mg/L, sucrose 30g/L and carrageenan 8g/L are added into the culture medium; adjusting the pH to 5.8;
step three: after the sweet pepper seedling grows to 7-8cm, cutting the sweet pepper seedling into stem segments with knots, and inoculating the stem segments into a 1/2GMS culture medium, wherein the culture conditions are unchanged.
The preparation method of the 1/2GMS medium comprises the following steps:
culturing on the basis of GMS, halving the concentration of macroelements, keeping the concentration of microelements, ferric salt and organic components unchanged, and adding IBA 0.7mg/L, sucrose 20g/L, carrageenan 8g/L and activated carbon 1.0g/L; the pH is adjusted to 5.8;
Step four: selecting root seedling with no callus, more than 2 normal root systems, 4-5cm high, 3 leaves, strong growth and uniform growth vigor, and adaptively hardening off about 22 d. And (5) transplanting the plant leaves into a greenhouse after the stems of the plant leaves are hardened and the colors of the plant leaves are changed to green. The substrate used for transplanting is: turf and vermiculite are prepared according to the volume ratio of 2.0:1. Before the use of the matrix, the grass carbon and the vermiculite are mixed and dry-mixed, and each cubic matrix needs 3.0kg of calcium magnesium phosphate fertilizer, the calcium magnesium phosphate fertilizer is put into the matrix and is uniformly mixed for 2 times, then the matrix is sprayed with water and is wet-mixed for 2 times, the raw materials of the matrix are fully and uniformly mixed, and then a greenhouse film is used for covering and stewing for 2 days. After the substrate is piled and stewed, the substrate is transferred into a 72-hole cave dish, the sweet pepper tissue culture seedlings are transplanted, fixed root water is poured thoroughly, and watering is not carried out in the winter and spring low-temperature period; after the new leaves are extracted, water is properly controlled, and a principle of non-drying and non-watering is adopted. Low-temperature overcast and rainy days are avoided after watering to prevent diseases; watering for 1 time in the morning and evening in summer and autumn in a high-temperature period; the seedling bed is thoroughly watered each time, and the periphery of the seedling bed is uniform, so that dead angles caused by missed watering are prevented; if the seedlings are in overgrowth, temperature control and fertilizer control are adopted, but water cannot be controlled, so that root system damage is avoided. Gradually increasing ventilation quantity 6 days before the sweet pepper tissue culture seedlings are planted in the field, and controlling the seedbed environment to be similar to the field environment. The watering amount is gradually reduced 4 days before field planting, the adaptability of seedlings is exercised, and the field planting survival rate is improved.
Example 3
Step one: screening sweet pepper finished product strains which are vigorous in growth, beautiful in fruits and free of plant diseases and insect pests in a sweet pepper planting base; cutting off sweet pepper fruits before transplanting, removing leaves at the lower ends of branches, then transplanting the sweet pepper finished plant with root soil balls into a plastic big basin together, and placing the plastic big basin into a greenhouse; after the transplanting of the sweet peppers is recovered, taking the branches with flower buds, removing the flower buds and leaves on the branches, leaving leaf stalks of 0.8-1.2cm, and cleaning. Then sterilizing, and brushing the materials from top to bottom by using a soft brush; placing the material into an ultrasonic oscillator, and performing ultrasonic oscillation for 25min; then soaking in 70% alcohol for 35s on an ultra-clean workbench, sterilizing for 10min with 0.1% mercuric chloride and 2-3 drops of tween-80, washing with sterile water for 6 times, washing to remove residual sterilizing liquid, and filtering with sterile filter paper to remove residual water. Cutting the sterilized branches into stem segments with the length of 1.5-2.0cm and 1 stem node;
step two: inoculating the sterilized stem segments into a GMS culture medium, and culturing under the condition: the temperature is 28 ℃, the illumination intensity is 2600lx, and the illumination test is 15h/d;
The composition of the GMS medium is:
the major elements are ：KNO3 1250mg/L、NH4NO3 875mg/L、KH2PO4 65mg/L、MgSO4 40mg/L、Ca(NO3)2 420mg/L;
Microelements ：KI 0.90mg/L、H3BO3 6.5mg/L、MnSO4·4H2O 23.0mg/L、ZnSO4·7H2O9.0mg/L、Na2MoO4·2H2O 0.30mg/L、CuSO4·5H2O 0.030mg/L、CoCl2·6H2O 0.030mg/L;
Iron salt: feSO 4·7H2O 28.0mg/L、Na2-EDTA·2H2 O38.0 mg/L;
organic components: 105mg/L inositol, 0.7mg/L nicotinic acid, 0.7mg/L pyridoxine hydrochloride, 0.7mg/L thiamine hydrochloride and 2.2mg/L glycine.
NAA 0.4mg/L, sucrose 35g/L and carrageenan 10g/L are added into the culture medium; adjusting the pH to 6.0;
step three: after the sweet pepper seedling grows to 7-8cm, cutting the sweet pepper seedling into stem segments with knots, and inoculating the stem segments into a 1/2GMS culture medium, wherein the culture conditions are unchanged.
The preparation method of the 1/2GMS medium comprises the following steps:
culturing on the basis of GMS, halving the concentration of macroelements, keeping the concentration of microelements, ferric salt and organic components unchanged, and adding IBA 1.0mg/L, sucrose 25g/L, carrageenan 10g/L and activated carbon 1.5g/L; the pH is adjusted to 6.0;
Step four: selecting root seedling with no callus, more than 2 normal root systems, 4-5cm high, 3 leaves, strong growth and uniform growth vigor, and adaptively hardening off about 25 d. And (5) transplanting the plant leaves into a greenhouse after the stems of the plant leaves are hardened and the colors of the plant leaves are changed to green. The substrate used for transplanting is: turf and vermiculite are prepared according to the volume ratio of 2.2:1. Before the use of the matrix, the grass carbon and the vermiculite are mixed and dry-mixed, and each cubic matrix needs 3.5kg of calcium magnesium phosphate fertilizer, the calcium magnesium phosphate fertilizer is put into the matrix and is mixed uniformly for 2 times, then the matrix is sprayed with water and is wet-mixed for 2 times, the raw materials of the matrix are fully mixed and uniformly mixed, and then a greenhouse film is used for covering and stewing for 3 days. After the substrate is piled and stewed, the substrate is transferred into a 72-hole cave dish, the sweet pepper tissue culture seedlings are transplanted, fixed root water is poured thoroughly, and watering is not carried out in the winter and spring low-temperature period; after the new leaves are extracted, water is properly controlled, and a principle of non-drying and non-watering is adopted. Low-temperature overcast and rainy days are avoided after watering to prevent diseases; watering for 1 time in the morning and evening in summer and autumn in a high-temperature period; the seedling bed is thoroughly watered each time, and the periphery of the seedling bed is uniform, so that dead angles caused by missed watering are prevented; if the seedlings are in overgrowth, temperature control and fertilizer control are adopted, but water cannot be controlled, so that root system damage is avoided. Gradually increasing ventilation quantity 7 days before the sweet pepper tissue culture seedlings are planted in the field, and controlling the seedbed environment to be similar to the field environment. The watering amount is gradually reduced 5 days before field planting, the adaptability of seedlings is exercised, and the field planting survival rate is improved.
Comparative example 1
Example 2 was repeated except that in step two, the existing conventional MS medium was used instead of the GMS medium; in step three, the conventional MS rooting medium is used to replace 1/2GMS medium, and a two-step seedling method is not used, and a conventional tissue culture method of conventional induction-proliferation-rooting is used.
Wherein the formula of the conventional MS culture medium is as follows:
macroelements of ：KNO3 1900mg/L、NH4NO3 1650mg/L、KH2PO4 170mg/L、MgSO4 370mg/L、CaCl2·2H2O 440mg/L;
Microelements ：KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L;
Iron salt: feSO 4·7H2O 27.8mg/L、Na2-EDTA·2H2 O37.3 mg/L;
organic components: inositol 100mg/L, nicotinic acid 0.5mg/L, pyridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 0.5mg/L, glycine 2.0mg/L.
Adding 0.5mg/L, NAA 0.3.3 mg/L of 6-BA, 30g/L of sucrose and 8g/L of carrageenan into the culture medium; the pH was adjusted to 5.8.
The formula of the conventional MS rooting culture medium is as follows:
Taking a conventional MS as a basic culture medium, halving the concentration of macroelements, keeping the concentration of microelements, ferric salt and organic components unchanged, and adding 1.0mg/L IBA, 25g/L sucrose, 10g/L carrageenan and 1.5g/L active carbon; the pH was adjusted to 6.0.
Comparative example 2
Example 2 was repeated except that in step two, the existing conventional MS medium was used instead of the GMS medium; in step three, the conventional MS rooting culture medium is used to replace 1/2GMS culture medium, and a two-step seedling method is used, so that the conventional proliferation step is avoided. Wherein the formula of the conventional MS culture medium is as follows:
macroelements of ：KNO3 1900mg/L、NH4NO3 1650mg/L、KH2PO4 170mg/L、MgSO4 370mg/L、CaCl2·2H2O 440mg/L;
Microelements ：KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L;
Iron salt: feSO 4·7H2O 27.8mg/L、Na2-EDTA·2H2 O37.3 mg/L;
organic components: inositol 100mg/L, nicotinic acid 0.5mg/L, pyridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 0.5mg/L, glycine 2.0mg/L.
Adding 0.5mg/L, NAA 0.3.3 mg/L of 6-BA, 30g/L of sucrose and 8g/L of carrageenan into the culture medium; the pH was adjusted to 5.8.
The formula of the conventional MS rooting culture medium is as follows:
Taking a conventional MS as a basic culture medium, halving the concentration of macroelements, keeping the concentration of microelements, ferric salt and organic components unchanged, and adding 1.0mg/L IBA, 25g/L sucrose, 10g/L carrageenan and 1.5g/L active carbon; the pH was adjusted to 6.0.
Comparative example 3
Example 2 was repeated except that 0.5mg/L of 6-BA was added to the GMS medium in the second and third steps, and the conventional tissue culture method of "induction-proliferation-rooting" was used without using the two-step seedling method.
Comparative example 4
Example 2 was repeated except that 0.5mg/L of 6-BA was added to the GMS medium in the second and third steps, and the two-step seedling method was used without the conventional propagation step.
Table 1 test results
Note that: the square difference analysis results in the list are expressed by letters, different uppercase letters indicate that the difference is extremely significant (P < 0.01), and different lowercase letters indicate that the difference is significant (P < 0.05).
From the experimental results in table 1, it can be seen that: the use of MS medium and GMS medium has little effect on the germination rate, but has a great effect on the growth condition of buds.
As can be seen from the results in Table 2, since both methods were carried out during the test, only auxin (IBA) was added, no mitogen was added, and a single-node stem propagation method was adopted. However, from the results, there was a significant difference in proliferation factors, and the difference in seedling growth conditions was also apparent.
Table 2 test results
Note that: the square difference analysis results in the list are expressed by letters, different uppercase letters indicate that the difference is extremely significant (P < 0.01), and different lowercase letters indicate that the difference is significant (P < 0.05).
1. The invention reduces the content of ammonium nitrogen in the culture medium (1). When the content of ammonium nitrogen in the culture medium is too high, the culture medium has certain poisoning and inhibiting effects, but the culture medium cannot lack ammonium salt, otherwise, the pH value of the culture medium can drift towards the alkaline direction, and the addition of a small amount of ammonium salt in nitrate can prevent the drift; (2) increasing the potassium content. Potassium plays an important role in metabolism of cells, and an increase in the content thereof can improve bud growth. There is a trend towards increasing amounts of potassium in the tissue culture of many plants. (3) increasing the content of phosphorus and calcium. Phosphorus is one of essential elements of plants, and the proper increase of the content of phosphorus in tissue culture is beneficial to the growth of cultures; calcium plays an important role in metabolism of plants and a plurality of physiological and biochemical processes, and the increase of the content of calcium ions is beneficial to the growth of the secondary seedlings. (4) reducing the chlorine content. The excessive amount of chloride ions adversely affects tissues and cells, such as inhibiting differentiation of tissues, etc. Therefore, the reduction of chloride ions is beneficial to the growth of the secondary seedlings.
2. The traditional tissue culture method of the method (1) comprises three steps: induction, proliferation and rooting; in the invention, a two-step seedling method is adopted, namely: and (5) induction and rooting. In the traditional tissue culture method, cytokinin is often used in the proliferation process, and is easy to cause seedling variation, so that the cytokinin is avoided as much as possible in order to keep the stable characters of crops such as sweet peppers, and the two-step seedling method just avoids the use of the cytokinin.
(2) The two-step seedling method of the invention has the obvious advantages that: in the traditional tissue culture method, due to the addition of cytokinin, the hormone level of the cytokinin needs to be reduced before the later rooting so as to ensure the normal growth of root systems, and the transplanting efficiency is low; the two-step seedling method adopted by the invention can directly root after induction because cytokinin is not added, so that the transplanting of the root seedlings can be immediately carried out in the planting season, and the transplanting efficiency is greatly improved. In other seasons, the root of the root-producing seedling can be cut off, the stem is cut into a plurality of sections, and then the sections are inoculated into a rooting culture medium for continuous reproduction, so that materials are fully utilized, and waste caused by seasons is reduced.
The foregoing description is only illustrative of the preferred embodiments of the present invention, and therefore should not be taken as limiting the scope of the invention, for all changes and modifications that come within the meaning and range of equivalency of the claims and specification are therefore intended to be embraced therein.

Claims (6)

1. A sweet pepper tissue culture breeding method based on a two-step seedling method comprises the following steps:
Step one: screening sweet pepper finished product strains which are vigorous in growth, beautiful in fruits and free of plant diseases and insect pests in a sweet pepper planting base; cutting off sweet pepper fruits before transplanting, removing leaves at the lower ends of branches, then transplanting the sweet pepper finished plant with root soil balls into a plastic big basin together, and placing the plastic big basin into a greenhouse; after the transplanting of the sweet peppers is recovered, taking branches with flower buds, removing the flower buds and leaves on the branches, leaving leaf stalks of 0.8-1.2 cm, cleaning, sterilizing and draining water; cutting the sterilized branches into stem segments with lengths of 1.5-2.0 cm and 1 stem node;
step two: a GMS medium for sweet pepper stem-segment induced germination was configured, consisting of the following components:
the macroelements are: KNO3 1150-1250 mg/L, NH4NO3 825-875 mg/L, KH2PO4 55-65 mg/L, mgSO4 35-40 mg/L, ca (NO 3) 2 380-420 mg/L;
Microelements ：KI 0.80-0.9 mg/L、H3BO3 6.0-6.5 mg/L、MnSO4·4H2O 22.0-23.0 mg/L、ZnSO4·7H2O 8.0-9.0 mg/L 、Na2MoO4·2H2O 0.20-0.30 mg/L 、CuSO4·5H2O 0.020-0.030 mg/L、CoCl2·6H2O 0.020-0.030 mg/L;
Iron salt: feSO4.7H2O 27.0-28.0 mg/L, na-EDTA.2H2O 37.0-38.0 mg/L;
Organic components: 95-105 mg/L inositol, 0.3-0.7 mg/L nicotinic acid, 0.3-0.7 mg/L pyridoxine hydrochloride, 0.3-0.7 mg/L thiamine hydrochloride, and 1.8-2.2 mg/L glycine;
Adding NAA 0.2-0.4 mg/L, sucrose 25-35 g/L, carrageenan 6-10 g/L into the culture medium; the pH is 5.5-6.0; inoculating the sterilized stem segments in a culture medium, and culturing: the temperature is 26-28 ℃, the illumination intensity is 2400-2600 lx, and the illumination test is 10-15 h/d;
Step three: cutting sweet pepper seedlings into stem segments with knots after the sweet pepper seedlings grow to 7-8 cm, and inoculating the stem segments into a 1/2GMS culture medium, wherein culture conditions are unchanged; the preparation method of the 1/2GMS medium comprises the following steps:
Taking GMS as a basic culture medium, halving the concentration of macroelements, keeping the concentration of microelements, ferric salt and organic components unchanged, adding 0.5-1.0mg/L IBA, 15-25 g/L sucrose, 6-10 g/L carrageenan and 0.5-1.5g/L active carbon; the pH is 5.5-6.0;
Step four: selecting root-free callus, having more than 2 normal root systems, 4-5 cm pieces high, 3 leaves, strong growth and consistent growth vigor of sweet pepper root seedlings, and performing adaptive seedling hardening 18-25 d; transplanting the plant into a greenhouse after the stems of the plant become hard and the colors of the leaves become green; the substrate used for transplanting is: the turf and vermiculite are prepared according to the volume ratio of (1.8-2.2): 1, and each cubic matrix needs 2.5-3.5 kg of calcium magnesium phosphate fertilizer.
2. The two-step seedling method-based sweet pepper tissue culture breeding method according to claim 1, wherein the method is characterized in that: in the first step, the stem segment of sweet pepper is disinfected as follows: brushing the material with a soft brush from top to bottom; placing the material into an ultrasonic oscillator, and performing ultrasonic oscillation for 15-25 min; then soaking 25-35 s in 70% alcohol on an ultra-clean workbench, sterilizing 6-10 min with 0.1% mercuric chloride and 2-3 drops of tween-80, washing 4-6 times with sterile water, washing residual sterilizing liquid, and filtering residual water with sterile filter paper.
3. The two-step seedling method-based sweet pepper tissue culture breeding method according to claim 1, wherein the method is characterized in that: in the fourth step, ventilation is gradually increased 5-7 days before the sweet pepper tissue culture seedlings are planted in the field, and the seedbed environment is controlled to be similar to the field environment; the watering amount is gradually reduced 3-5 days before field planting, the adaptability of seedlings is exercised, and the field planting survival rate is improved.
4. The two-step seedling method-based sweet pepper tissue culture breeding method according to claim 1, wherein the method is characterized in that: and step four, mixing grass carbon and vermiculite, dry mixing, adding calcium magnesium phosphate fertilizer into the matrix, stirring for 2 times, spraying water to the matrix, wet mixing for 2 times, fully stirring and mixing the raw materials of the matrix, and covering and stewing for 1-3 days by using a greenhouse film.
5. The two-step seedling method-based sweet pepper tissue culture breeding method according to claim 1, wherein the method is characterized in that: step four, after the substrate is piled and stewed, transferring the substrate into a 72-hole cave dish, transplanting the sweet pepper tissue culture seedlings well, and watering thoroughly to fix root water without watering in the low-temperature period of winter and spring; after the new leaves are extracted, water is properly controlled, and a principle of non-drying and non-watering is adopted.
6. The two-step seedling method-based sweet pepper tissue culture breeding method according to claim 5, wherein the method is characterized in that: low-temperature overcast and rainy days are avoided after watering to prevent diseases; watering for 1 time in the morning and evening in summer and autumn in a high-temperature period; the seedling bed is thoroughly watered each time, and the periphery of the seedling bed is uniform, so that dead angles caused by missed watering are prevented; if the seedlings are in overgrowth, temperature control and fertilizer control are adopted, but water cannot be controlled, so that root system damage is avoided.
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