CN108703074B - Temporary preservation and asexual propagation method of cabbage male sterile plant - Google Patents
Temporary preservation and asexual propagation method of cabbage male sterile plant Download PDFInfo
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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
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- A01G2/10—Vegetative propagation by means of cuttings
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- A—HUMAN NECESSITIES
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- 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 temporary preservation and asexual propagation method of a cabbage male sterile plant, which comprises the following steps: (1) destroying the apical dominance of the male sterile plant of the cabbage, and promoting the axillary buds to grow out from the external root, stem and leaf marks of the mother plant; (2) buckling axillary buds from external rhizomes and ensuring that larger axillary buds have a rhizome medulla part tissue; (3) carrying out asexual propagation on the larger axillary buds with the medullary tissues of the rhizomes in a cuttage mode to obtain a new generation of plants; carrying out vegetative propagation on the smaller axillary buds by adopting a tissue culture mode to obtain rooted seedlings, and transplanting the rooted seedlings to open field for cultivation after training. According to the temporary preservation and vegetative propagation method of the male sterile plants of the cabbages, the axillary buds are promoted to grow out from the outer root, stem and leaf marks of the mother plants by breaking the apical dominance, the vegetative propagation is carried out on the grown larger axillary buds in a cutting mode, and the cutting survival rate is high; for smaller axillary buds, the tissue culture method is adopted for vegetative propagation, the optimal culture medium for axillary bud rooting is optimized, and the regeneration rate is high.
Description
Technical Field
The invention relates to a method for preserving and propagating vegetable male sterile plants, in particular to a method for temporarily preserving and asexually propagating cabbage male sterile plants, belonging to the field of temporary preservation and propagation of cabbage male sterile plants.
Background
Cabbage has significant heterosis, first filial generation (F)1) The trait or traits are superior to those of the parents or a parent. The utilization of heterosis can not only improve the yield and quality of cabbage varieties, but also improve the disease resistance and stress resistance of cabbage varieties, and has become the main research direction of the current Chinese cabbage variety breeding. The male sterile line is used for variety breeding and hybrid seed production, so that the heterosis utilization range can be enlarged, the parent reproduction is easy, and the artificial emasculation is avoided, thereby saving a large amount of manpower and material resources, reducing the seed production cost, improving the seed quality, andis favorable for preventing the loss of germplasm resources and protecting the intellectual property of breeding workers. In the 21 st century, particularly after China came into the world, the cabbage breeding market competes fiercely, some foreign excellent varieties occupy the market of cabbage producing areas in China, some domestic varieties obviously lack competitiveness in aspects of quality, resistance, growth period and the like, and gradually quit the market. A vegetable and flower research institute of Chinese academy of agricultural sciences carries out a series of researches for introducing a cabbage male sterile source, and materials such as radish cytoplasmic sterility, black mustard cytoplasmic sterility, improved radish cytoplasmic sterility and the like are introduced in sequence to be hybridized and continuously backcrossed and transferred with different types of cabbage inbred lines. 6 portions of newly improved Ogu CMS material such as CMSR3625 and CMSR3629 are introduced from the United states in 1998 years such as Fangzhiyuan, more than 30 portions of cabbage inbred lines and more than 20 portions of broccoli inbred lines are used for transformation and multi-generation backcross is carried out, backcross offspring transformed from CMSR3625 and CMSR3629 show that leaves are not yellowed at low temperature, sterile flowers are normally opened, pistil is normal and fructification is good, and the application prospect is good, so that a new variety of sterile line seed production represented by 'Zhonggan 21' is cultivated, and good economic and social benefits are obtained (Fangzhiyuan, Sungtian culture, Liuyumei, and the like, research on male sterility of several types of cabbages and utilization of dominant sterile line [ J]Chinese vegetable, 2001, 1 (6): 20. liuyumei, Fangzhiyuan, Sunpian, etc. the main way for obtaining male sterility of cruciferous crops and the utilization thereof, Chinese vegetables, 2002 (2): 52-55.).
The male sterile source (plant) is a key material for breeding a male sterile line, and the source of the male sterile source (plant) is obtained by natural variation; artificial physical and chemical mutagenesis; distant hybridization; selfing and hybridizing the variety; introduction from home and abroad (Wangzaijia et al, general theory of vegetable breeding, third edition, Chinese agriculture Press, 128-. Since no pollen is available on its own for selfing propagation and maintenance, the problem of temporary preservation of the original male sterile plant is first solved before the male sterile line and the maintainer line are developed.
At present, the temporary preservation method of the crop male sterile plant comprises the following steps:
1. selfing, which is mainly suitable for partial sterile and functional sterile plants to be ineffective for 100 percent of sterile plants; 2. selfing is carried out after hybridization, F1 after hybridization with fertile sister plants of the same variety is carried out, and then selfing is carried out to expect that sterile plants with a certain proportion appear in F2, so that the time is long and the workload is large. 3. The sterile plant obtained by distant hybridization can be backcrossed with parents, BC1 disappears for the male sterility controlled by invisible genes, and the sterile plant is expected to be separated by selfing, which is tedious and risky. 4. Vegetative propagation is most effective and convenient for vegetable crops like cabbage that are susceptible to vegetative propagation. Not only can temporarily keep the male sterility, but also can ensure that other horticultural traits are not separated.
In the flowering and breeding process of cruciferous vegetables such as cabbages, the occurrence frequency of sterile plants is not low, and after the sterile plants are mutated out, how to breed and store temporarily is a big problem, a long time is needed from the sterile plants to the sterile lines and maintainer lines, and if the sterile plants are not kept, the breeding work is abandoned. The breeding mother plant of the cabbage can be known whether to be fertile or not after blooming, and at the moment, different from the vegetative growth stage, the vegetative propagation regeneration rate is lower, the cutting survival rate of leaves and axillary buds is low, and needs to be improved.
Disclosure of Invention
The invention mainly aims to break the bottleneck of vegetative propagation of sterile flowering cabbage parent plants and provides a method for temporarily storing and asexually propagating characteristics of male sterile plants of cabbages, wherein the asexual propagation regeneration rate is high by adopting the method.
The above object of the present invention is achieved by the following technical solutions:
a temporary preservation and asexual propagation method of a cabbage male sterile plant comprises the following steps:
(1) destroying the apical dominance of the male sterile plant of the cabbage, and promoting the axillary buds to grow out from the external root, stem and leaf marks of the mother plant;
(2) buckling axillary buds from big to small from outer rhizome in 2-3 batches and ensuring that the bigger axillary buds are all provided with a rhizome medulla part tissue;
(3) obtaining a new generation of plants by using larger axillary buds in a cuttage mode; carrying out vegetative propagation on the smaller axillary buds by adopting a tissue culture mode to obtain rooted seedlings, and transplanting the rooted seedlings to open field for cultivation after training.
The method for destroying the apical dominance of the male sterile plants of the cabbages in the step (1) is preferably as follows: when the cabbage shoots and blooms, after a male sterile plant is found, the plant is adjusted for several times, and the opened trunk flower is removed firstly; removing flowers of the first-level lateral branches and the second-level lateral branches in sequence, and reserving a plurality of leaves for photosynthetic growth; after 7-10 days, the buds of the third-level lateral branches begin to appear and are removed in time.
The method for promoting the axillary buds to grow from the external root, stem and leaf marks of the mother plant in the step (1) is preferably as follows: removing residual leaves at 1/3 lower part of the stock plant to expose outer rhizome and leaf marks, spraying water for keeping moisture, and shading with black plastic for 3-5 days.
The larger axillary bud in the step (2) or the step (3) means that the leaf length is more than 3.0 cm; the smaller axillary bud in the step (3) means that the leaf length is less than 3.0 cm.
The cuttage mode in the step (3) comprises the following steps: dipping the base part of the axillary bud with 2000mg/L indolebutyric acid, and cutting into a field soil matrix or river sand matrix which is sterilized at the high temperature of 100 ℃ for 45 minutes; sun shading in sunny days, and spraying water in the morning and evening every day to keep the matrix wet; after 7-10 days, the axillary buds begin to sprout and root, and new leaves grow out, the sun shade is gradually removed, the illumination time is prolonged, and the water spraying frequency is reduced; if the plant is river sand matrix, the plant is transplanted to normal field soil, and then is normally managed, and a new generation (through stage development) of plants is grown.
The tissue culture mode in the step (3) comprises the following steps: (a) sterilizing the axillary bud explants; (b) inoculating the sterilized axillary bud explants to an axillary bud rooting culture medium for culture to obtain rooted seedlings.
Wherein the method for sterilizing the axillary bud explants in step (a) comprises the following steps: soaking in 75% alcohol for 30s for surface sterilization, and washing with sterile water for 1-2 times; then sterilizing with 0.1% mercuric chloride or sodium hypochlorite (concentration 10% -40%). Sterilizing for 5-7 minutes if 0.1% mercuric chloride is used, sterilizing for 10-15 minutes if 10% -40% sodium hypochlorite is used, and washing for 3-5 times with sterile water for 2-3 minutes each time.
The composition of the axillary bud rooting culture medium has direct influence on the rooting rateIn order to screen a proper axillary bud rooting culture medium, the composition and the dosage of the axillary bud rooting culture medium are screened. The invention selects the basic components of MS culture medium, adds growth regulating substances with different concentrations, 3 percent of sucrose, 0.8 percent of agar, 5.8 to 6.0 of pH value, sterilizes under the pressure of 0.9 to 1.1kg/cm2Keeping for 15-20 minutes, and using after the culture medium is cooled and solidified. Hormone addition was designed using an orthogonal assay: l is16(45) Four growth regulating substances of 6-BA (A factor), IAA (B factor), NAA (C factor) and 2,4-D (D factor) are selected, four levels of each substance are selected, and the four levels are 16 levels in total, and the three levels are repeated. 6-BA is 0, 2.0, 2.5 and 3.0mg/L respectively; IAA is 0, 0.02, 0.2 and 2.0mg/L respectively; NAA is 0, 0.05, 0.2 and 0.5mg/L respectively; 2,4-D is 0, 0.2, 0.5 and 1.0mg/L respectively; after cabbage plant materials (self is a male sterile plant) taking W14 as a screening culture medium are inoculated into the culture medium, observation and statistics are carried out every 7-10 days, at least three times of experiment treatment are carried out, at least 30 explants are repeated every time, and secondary culture samples are carried out every 15-20 days. And subculturing when the new bud grows to 1.5cm high. Transferring the soil into the soil after the developed root system grows out. The final screening result shows that the optimal culture medium for axillary bud rooting is as follows:
MS +6-BA3.0mg/L + IAA0.02mg/L + NAA0.5mg/L + sucrose 3% + agar 0.8%; the pH value is 5.8-6.0.
The rooting seedling training mode in the step (3) comprises the following steps: removing a mycoderm, placing the tissue culture chamber in a tissue culture room, adding a little sterile water into a triangular flask, exercising for 2-3 days, taking out a single seedling, cleaning a culture medium carried on a root, planting the seedling into mixed nutrient soil of vermiculite and grass carbon ash (the ratio of vermiculite to grass carbon ash is 2: 1), and then carrying out +/-2 ℃ culture at the temperature; the seedlings are hardened for 10-15 days under the conditions of 16-hour illumination and 1500-3000Lx illumination intensity every day.
According to the temporary preservation and asexual propagation method of the male sterile plants of the cabbages, the axillary buds are promoted to grow from the outer root, stem and leaf marks of the mother plants by breaking the apical dominance, and the grown larger axillary buds are propagated in a cutting mode, so that the survival rate is high; for smaller axillary buds, the tissue culture method is adopted for vegetative propagation, the optimal culture medium for axillary bud rooting is optimized, and the tissue culture seedling has high rooting rate and high regeneration rate.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 method for temporary preservation and asexual propagation of male sterile plants of Brassica oleracea
1. Cabbage flowering branch treatment
Processing temporarily discovered flowering cabbage original male sterile plants.
1.1 when the cabbage shoots and blooms, and a male sterile plant is found, adjusting the plant for several times, and removing the opened trunk flower firstly; the flowers of the first-level lateral branch and the second-level lateral branch are removed in sequence, and some leaves are left for photosynthetic growth. After 7 days, buds of the third-level lateral branches begin to appear and are removed in time.
1.2 at 1/3 parts below the stock plant, the residual leaves were removed to expose the outer rhizome leaves, water was sprayed to keep moisture, and the plants were shaded with black plastic for 3 days.
1.3 because the superiority of the upper top of the flowering branch is damaged, a part of axillary buds grow gradually from the outer rhizome and leaf marks of the mother plant in about 10 days, and about three batches of axillary buds grow from large to small at the outer rhizome and leaf marks of the mother plant.
2 separation of axillary buds of external rhizomes of flowering stock
Using a knife sterilized by 75% alcohol to buckle axillary buds from big to small from outer rhizome in 3 batches, ensuring that each big axillary bud has a rhizome medulla tissue and is suitable for cutting and rooting; the smaller axillary buds must be rooted by tissue culture (axillary buds with a leaf length of 3.0cm or more are directly cut, and axillary buds with a leaf length of 3.0cm or less are cultured in a medium).
3 axillary bud rooting culture
3.1 axillary buds (leaf length more than 3.0 cm) are cultured by cutting and rooted
Dipping the base part of the axillary bud with 2000mg/L indolebutyric acid, and cutting into a field substrate sterilized at the high temperature of 100 ℃ for 45 minutes; sun shading in sunny days, and spraying water in the morning and evening every day to keep the matrix wet. After 7 days, axillary buds begin to sprout and root, and new leaves grow out, the sun is gradually removed, the illumination time is prolonged, and the water spraying frequency is reduced. And (4) carrying out normal management later, and growing into a new generation (through stage development) of plants.
3.2 axillary buds (leaf length less than 3.0 cm) in vitro culture and rooting
Sterilizing axillary bud explants: soaking in 75% alcohol for 30s for surface sterilization, and washing with sterile water for 2 times; sterilizing with 0.1% mercuric chloride for 5-7 min, washing with sterile water for 3 times (3 min each)
② axillary bud rooting culture: inoculating the sterilized axillary bud explants to an axillary bud rooting culture medium for rooting culture: MS + BA3.0mg/L + IAA0.02mg/L + NAA0.5mg/L sucrose 3% + agar 0.8% (pH 5.8-6.0); subculture was carried out every 15-20 days. And subculturing when the new bud grows to 1.5cm high. Transferring the soil into the soil after the developed root system grows out.
Thirdly, training the healthy and strong seedlings with roots obtained by in-bottle rooting culture: pretreating materials, removing a mycoderm, placing in a tissue culture room, adding a little sterile water into a triangular flask, exercising for 2-3 days, taking out a single seedling, cleaning a culture medium carried on a root, planting the seedling into mixed nutrient soil of vermiculite and plant ash (vermiculite: plant ash is 2: 1), and then, at the temperature of 25 +/-2 ℃; performing seedling exercising treatment for 10 days under the conditions of 16-hour illumination and illumination intensity of 1500-; after developed root systems grow out, the seedlings are moved into a nutrition pot for continuous culture, and the tissue culture seedlings are moved into the ground for cultivation after the seedling delay in the nutrition pot is finished.
Example 2 method for temporary preservation and asexual propagation of male sterile plants of Brassica oleracea
1. Cabbage flowering branch treatment
Processing temporarily discovered flowering cabbage original male sterile plants.
1.1 when the cabbage shoots and blooms, and a male sterile plant is found, adjusting the plant for several times, and removing the opened trunk flower firstly; the flowers of the first-level lateral branch and the second-level lateral branch are removed in sequence, and some leaves are left for photosynthetic growth. After 10 days, buds of the third-level lateral branches begin to appear and are removed in time.
1.2 at 1/3 parts below the stock plant, removing the residual leaves to expose the outer rhizome and leaf marks, spraying water to keep moisture, and shading with black plastic for 3-5 days.
1.3 because the superiority of the upper top of the flowering branch is damaged, a part of axillary buds grow gradually from the outer rhizome and leaf marks of the mother plant in about 10 days, and about three batches of axillary buds grow from large to small at the outer rhizome and leaf marks of the mother plant.
2 separation of axillary buds of external rhizomes of flowering stock
Using a knife sterilized by 75% alcohol to buckle axillary buds from big to small from outer rhizome in 3 batches, ensuring that each big axillary bud has a rhizome medulla tissue and is suitable for cutting and rooting; the smaller axillary buds must be rooted by tissue culture (axillary buds with a leaf length of 3.0cm or more are directly cut, and axillary buds with a leaf length of 3.0cm or less are cultured in a medium).
3 axillary bud rooting culture
3.1 axillary buds (leaf length more than 3.0 cm) are cultured by cutting and rooted
Dipping the base of axillary buds with 2000mg/L indolebutyric acid, and cutting into a field matrix sterilized at 100 ℃ for 45 minutes. Sun shading in sunny days, and spraying water in the morning and evening every day to keep the matrix wet. After 10 days, axillary buds begin to sprout and root, and new leaves grow out, the sun is gradually removed, the illumination time is prolonged, and the water spraying frequency is reduced. And (4) carrying out normal management later, and growing into a new generation (through stage development) of plants.
3.2 axillary buds (leaf length less than 3.0 cm) in vitro culture and rooting
Sterilizing axillary bud explants: soaking in 75% alcohol for 30s for surface sterilization, and washing with sterile water for 1-2 times; sterilizing with 10-40% sodium hypochlorite for 10-15 min, washing with sterile water for 5 times (3 min each time)
② axillary bud rooting culture: inoculating the sterilized axillary bud explants to an axillary bud rooting culture medium for rooting culture: MS + BA3.0mg/L + IAA0.02mg/L + NAA0.5mg/L sucrose 3% + agar 0.8% (pH 5.8-6.0); subculture was carried out every 15-20 days. And subculturing when the new bud grows to 1.5cm high. Transferring the soil into the soil after the developed root system grows out.
Thirdly, training the healthy and strong seedlings with roots obtained by in-bottle rooting culture: pretreating the materials, removing the mycoderm, placing in a tissue culture room, adding a little sterile water into a triangular flask, taking out for 3 days, taking out single seedling, cleaning the culture medium on the root, and planting the seedling in the mixed nutrient soil of vermiculite and plant ash
(vermiculite: peat ash 2: 1) and then at a temperature of 25 ℃ ± 2; performing seedling exercising treatment for 15 days under the conditions of 16-hour illumination and illumination intensity of 1500-; after developed root systems grow out, the seedlings are moved into a nutrition pot for continuous culture, and the tissue culture seedlings are moved into the ground for cultivation after the seedling delay in the nutrition pot is finished.
EXAMPLE 3 method for temporary preservation and asexual propagation of Male sterile plants of Brassica oleracea
1. Cabbage flowering branch treatment
Processing temporarily discovered flowering cabbage original male sterile plants.
1.1 when the cabbage shoots and blooms, and a male sterile plant is found, adjusting the plant for several times, and removing the opened trunk flower firstly; the flowers of the first-level lateral branch and the second-level lateral branch are removed in sequence, and some leaves are left for photosynthetic growth. After 8 days, buds of the third-level lateral branches begin to appear and are removed in time.
1.2 at 1/3 parts below the stock plant, removing the residual leaves to expose the outer rhizome and leaf marks, spraying water to keep moisture, and shading with black plastic for 3-5 days.
1.3 because the superiority of the upper top of the flowering branch is damaged, a part of axillary buds grow gradually from the outer rhizome and leaf marks of the mother plant in about 10 days, and about three batches of axillary buds grow from large to small at the outer rhizome and leaf marks of the mother plant.
2 separation of axillary buds of external rhizomes of flowering stock
Using a knife sterilized by 75% alcohol to buckle axillary buds from big to small from outer rhizome in 2 batches, ensuring that each big axillary bud has a rhizome medulla tissue and is suitable for cutting and rooting; the smaller axillary buds must be rooted by tissue culture (axillary buds with a leaf length of 3.0cm or more are directly cut, and axillary buds with a leaf length of 3.0cm or less are cultured in a medium).
3 axillary bud rooting culture
3.1 axillary buds (leaf length more than 3.0 cm) are cultured by cutting and rooted
Dipping the base of axillary buds with 2000mg/L indolebutyric acid, and cutting into river sand substrate sterilized at 100 ℃ for 45 minutes. Sun shading in sunny days, and spraying water in the morning and evening every day to keep the matrix wet. After 7-10 days, the axillary buds begin to sprout and take root, and after new leaves grow, the sun shade is gradually removed, the illumination time is prolonged, the water spraying frequency is reduced, the seedlings are transplanted into normal field soil, and then normal management is carried out, so that new generation (through stage development) plants are grown.
3.2 axillary buds (leaf length less than 3.0 cm) in vitro culture and rooting
Sterilizing axillary bud explants: soaking in 75% alcohol for 30s for surface sterilization, and washing with sterile water for 1-2 times; sterilizing with 0.1% mercuric chloride for 7 min, washing with sterile water for 4 times (3 min each)
② axillary bud rooting culture: inoculating the sterilized axillary bud explants to an axillary bud rooting culture medium for rooting culture: MS + BA3.0mg/L + IAA0.02mg/L + NAA0.5mg/L sucrose 3% + agar 0.8% (pH 5.8-6.0); subculture was carried out every 15-20 days. And subculturing when the new bud grows to 1.5cm high. Transferring the soil into the soil after the developed root system grows out.
Thirdly, training the healthy and strong seedlings with roots obtained by in-bottle rooting culture: pretreating materials, removing a mycoderm, placing in a tissue culture room, adding a little sterile water into a triangular flask, exercising for 2-3 days, taking out a single seedling, cleaning a culture medium carried on a root, planting the seedling into mixed nutrient soil of vermiculite and plant ash (vermiculite: plant ash is 2: 1), and then, at the temperature of 25 +/-2 ℃; performing seedling exercising treatment for 10-15 days under the conditions of 16-hour illumination and illumination intensity of 1500-; after developed root systems grow out, the seedlings are moved into a nutrition pot for continuous culture, and the tissue culture seedlings are moved into the ground for cultivation after the seedling delay in the nutrition pot is finished.
Test example 1 axillary bud rooting medium screening test
Selecting basic components of MS culture medium, adding growth regulating substances with different concentrations, 3% of sucrose, 0.8% of agar, 5.8-6.0 of pH value, sterilizing under the pressure of 0.9-1.1 kg/cm2Keeping for 15-20 minutes, and using after the culture medium is cooled and solidified.
Hormone addition was designed using an orthogonal assay: l is16(45) Four growth regulating substances of 6-BA (A factor), IAA (B factor), NAA (C factor) and 2,4-D (D factor) are selected, four levels of each substance are selected, and the four levels are 16 levels in total, and the three levels are repeated. 6-BA is 0, 2.0, 2.5 and 3.0mg/L respectively; IAA is 0, 0.02, 0.2 and 2.0mg/L respectively; NAA is 0, 0.05, 0.2 and 0.5mg/L respectively; 2,4-D is 0, 0.2, 0.5 and 1.0mg/L respectively; after cabbage plant materials (self is a male sterile plant) taking W14 as a screening culture medium are inoculated into the culture medium, observation and statistics are carried out every 7-10 days, at least three times of experiment treatment are carried out, at least 30 explants are repeated every time, and secondary culture samples are carried out every 15-20 days. And subculturing when the new bud grows to 1.5cm high. Transferring the soil into the soil after the developed root system grows out. The results are shown in Table 1.
TABLE 1 screening test results of axillary bud rooting medium
The number of the expanded tissues is equal to the number of the expanded explants/total number of the explants inoculated multiplied by 100 percent
The callus incidence rate is the callus number/explant number multiplied by 100%
Adventitious bud differentiation rate: the number of explants which bud out/total number of explants cultured is multiplied by 100%
Average germination rate: total number of sprouts/number of sprouts explants
Elongation of sprout: number of extended shoots/total number of accessed shoots X100%
Mean multiple shoot number/explant
Rooting rate: the number of the rooted aseptic seedlings/the total number of the transplanted aseptic seedlings is multiplied by 100 percent
Average root number: the total rooting amount/the number of the rooted aseptic seedlings is multiplied by 100 percent
The rooting rate is the number of rooted buds/number of cultured buds multiplied by 100%
As can be seen from the test results in Table 1, of the 16 treatment combinations, the 15 th treatment combination A4B2C4D1The axillary buds have the best regeneration effect, and the external applicationThe plant body has the fastest expansion speed, the expansion degree is 89 percent, and the expansion degree is obviously higher than that of other treatment combinations; the rooting rate is 79%, the rooting rate is also obviously higher than that of other treatment combinations, the comprehensive score is 80.11%, and the comprehensive score is at least 11.78% higher than that of other treatment combinations; therefore, the experiment determines that the optimal culture medium for axillary bud rooting is as follows:
MS +6-BA3.0mg/L + IAA0.02mg/L + NAA0.5mg/L + sucrose 3% + agar 0.8%; the pH value is 5.8-6.0.
Claims (6)
1. A temporary preservation and asexual propagation method of a cabbage male sterile plant is characterized by comprising the following steps: (1) destroying the apical dominance of the male sterile plant of the cabbage, and promoting the axillary buds to grow out from the external root, stem and leaf marks of the mother plant; (2) buckling axillary buds from large to small from outer rhizome in 2-3 batches to ensure that the larger axillary buds have a rhizome medulla tissue; (3) carrying out asexual propagation on the larger axillary buds with the medullary tissues of the rhizomes in a cuttage mode to obtain a new generation of plants; carrying out vegetative propagation on the smaller axillary buds by adopting a tissue culture mode to obtain rooted seedlings, and transplanting the rooted seedlings to open field for cultivation after domestication; the larger axillary bud in the step (2) or the step (3) means that the leaf length is more than 3.0 cm; the smaller axillary bud in the step (3) means that the leaf length is less than 3.0 cm; the cuttage mode in the step (3) comprises the following steps: dipping the base part of the axillary bud with 2000mg/L indolebutyric acid, and cutting the axillary bud into a sterilized field soil matrix or river sand matrix; sun shading in sunny days, and spraying water in the morning and evening every day to keep the matrix wet; after 7-10 days, the axillary buds begin to sprout and root, and new leaves grow out, the sun shade is gradually removed, the illumination time is prolonged, and the water spraying frequency is reduced; if the river sand matrix is to be transplanted into normal field soil, normal management is carried out later, and a new generation of plants are grown; the tissue culture mode in the step (3) comprises the following steps: (a) sterilizing the axillary bud explants; (b) inoculating the sterilized axillary bud explants to an axillary bud rooting culture medium for culture to obtain rooted seedlings; wherein the axillary bud rooting culture medium comprises: MS +6-BA3.0mg/L + IAA0.02mg/L + NAA0.5mg/L + sucrose 3% + agar 0·8 percent; pH value of 5·8-6·0。
2. The temporary storage and asexual propagation method according to claim 1, wherein the method for disrupting the apical dominance of a cabbage male sterile plant in step (1) is: when the cabbage shoots and blooms, after a male sterile plant is found, adjusting the plant, removing flowers from a main stem, sequentially removing flowers of a first-level lateral branch and flowers of a second-level lateral branch, and reserving certain leaves for photosynthetic growth; after 7-10 days of treatment, if the buds of the third-level lateral branches begin to appear, the buds are removed in time.
3. The temporary storage and asexual propagation method according to claim 1, wherein the method for promoting the axillary buds to grow from the external root, stem and leaf marks of the mother plant in the step (1) comprises the following steps: removing residual leaves at 1/3 position below the stock plant to expose the outer rhizome and leaf marks, spraying water for keeping moisture, and shading with black plastic for 3-5 days.
4. The temporary storage and vegetative propagation method according to claim 1, wherein the sterilizing of the axillary bud explants in step (a) comprises: soaking in 75% alcohol for 30s for surface sterilization, and washing with sterile water; then 0.1 percent mercuric chloride or sodium hypochlorite with the concentration of 10 to 40 percent is used for sterilization, and finally, sterile water is used for washing.
5. The temporary storage and asexual propagation method according to claim 4, characterized in that if sterilized with 0.1% mercuric chloride, the sterilization time is 5 to 7 minutes; if 10-40% sodium hypochlorite is used for sterilization, the sterilization time is 10-15 minutes, and the sterile water is washed for 3-5 times, 2-3 minutes each time.
6. The temporary storage and asexual propagation method according to claim 1, wherein the rooted seedling domestication mode in the step (3) comprises: removing a mycoderm, placing the tissue culture chamber in a tissue culture room, adding sterile water into a triangular flask, exercising for 2-3 days, taking out a single seedling, cleaning a culture medium carried on roots, planting the seedling into mixed nutrient soil of vermiculite and plant ash, and exercising for 10-15 days under the conditions of illumination for 16 hours every day at the temperature of 25 +/-2 ℃ and the illumination intensity of 1500-3000 Lx.
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| CN103380699A (en) * | 2013-08-06 | 2013-11-06 | 镇江瑞繁农艺有限公司 | Method for cutting and seedling raising of cauliflower male sterility line cluster buds |
| CN103416203A (en) * | 2013-08-09 | 2013-12-04 | 镇江瑞繁农艺有限公司 | Method for axillary bud cutting seedling of common head cabbage |
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