CN109169279B - Method for efficiently obtaining regenerated plants by culturing common head cabbage seed pods - Google Patents
Method for efficiently obtaining regenerated plants by culturing common head cabbage seed pods Download PDFInfo
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- CN109169279B CN109169279B CN201811088892.XA CN201811088892A CN109169279B CN 109169279 B CN109169279 B CN 109169279B CN 201811088892 A CN201811088892 A CN 201811088892A CN 109169279 B CN109169279 B CN 109169279B
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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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- 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 method for efficiently obtaining regenerated plants by culturing common head cabbage seed pods, which comprises the steps of carrying out bud stripping and pollination 1-2 d before the common head cabbage seed plants bloom, and taking 8-9 d of seed pods after pollination as explants; inoculating the sterilized culture medium to an induction culture medium, and performing illumination treatment in a culture room to obtain red and white combined light (white light: red light: 3: 1); inducing adventitious buds, and transferring to a proliferation culture medium for subculture proliferation; and finally, transferring the seedlings to a rooting culture medium to induce rooting, thereby obtaining rooted regenerated seedlings. According to the characteristics of the explant, the method for efficiently culturing the common head cabbage seed pod to obtain the regenerated plant is established through different material taking time, culture medium hormone proportion and illumination treatment, and has the advantages of easiness in obtaining the explant, quickness in inducing adventitious buds, high propagation efficiency and the like.
Description
Technical Field
The invention relates to a method for efficiently obtaining a regenerated plant by culturing common head cabbage seed pods, belonging to the technical field of biology.
Background
The establishment of a plant tissue culture regeneration technology system plays an irreplaceable role in the preservation and propagation of some plant breeding materials with important values. The study on tissue culture of cabbage explants at home and abroad takes cotyledons and hypocotyls of aseptic seedlings as explants most. However, the method is not suitable for the preservation and propagation of male sterile lines, self-incompatible lines and materials which are difficult to self-copulate. The axillary buds of the strains in the reproductive growth period are used as explants for tissue culture, so that the method is a relatively high-efficiency and simple asexual propagation method; however, most varieties have difficulty in that no or few axillary buds occur. The number of buds of each healthy common head cabbage seed plant is generally 800-2000 according to varieties and cultivation and management conditions, and materials are rich and easily available. The invention establishes a simpler, more convenient, more efficient and more universal in vitro regeneration technical system by taking the seed pod pollinated in the bud period as an explant, and provides a more applicable method for asexual propagation preservation and propagation of male sterile lines, self-incompatible lines and common head cabbage materials with difficult seed setting.
Disclosure of Invention
The invention provides a method for efficiently culturing common head cabbage seed pods to obtain regenerated plants, which aims to provide a set of efficient and innovative system of the system by adjusting and improving a regeneration technology system through different material taking time, different illumination treatment and hormone proportion of adventitious buds and a subculture multiplication medium.
1. A method for culturing common head cabbage seed pods to obtain regenerated plants efficiently is characterized by comprising the following steps:
1) explant selection
Carrying out bud stripping and pollination on the common head cabbage seed plants 1-2 days before blooming, and taking seed pods 8-9 days after pollination as explants; and sterilizing the explant for 1-2 min by using 75% by volume of ethanol, then sterilizing for 15-20 min by using 7-8% by volume of sodium hypochlorite, and finally washing for 3-4 times by using sterile water.
2) Differentiation Induction
Inoculating the explant to an induction culture medium after sterilization, and culturing in a culture room with the temperature of 25 +/-0.5 ℃ and the daily illumination time of 14h, wherein adventitious buds can be directly induced within 15-20 days;
the induction culture medium is MS +0.1mg/L NAA +1.0 mg/L6-BA + 6-7 g/L agar + 25-30 g cane sugar, and the pH value is 5.8;
the illumination is as follows: the number ratio of the red light lamps to the white light lamps is 1: 3;
3) subculture multiplication
Transferring the induced single buds or cluster buds (cutting the cluster buds into single buds) to a subculture multiplication medium, transferring once in 25-30 days, and multiplying the buds by about 220 times after 3-4 subcultures; the light culture environment during the period is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;
the subculture multiplication medium is MS +0.1mg/L NAA +0.8 mg/L6-BA + 6-7 g/L agar + 25-30 g sucrose, and the pH value is 5.8;
4) root induction
When the sprouts grow to 3-4 cm high, transferring the sprouts to a rooting culture medium, and inducing the sprouts to root for 15-20 days to obtain rooted seedlings; the light culture environment during the period is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;
the rooting medium is MS +0.1mg/L NAA +0.1mg/L IBA + 7-8 g/L agar + 20-25 g sucrose, and the pH value is 5.8;
5) transplanting and field planting
After hardening off the rooted seedlings at normal room temperature for 24 hours, transplanting the rooted seedlings into a plug tray with 50 holes for growth, wherein the matrix is a cruciferous vegetable seedling culture matrix; and 6-8 true leaves are planted in the field after the seedlings grow.
Has the advantages that:
(1) the number of explants used is abundant. The explant is a seed pod pollinated in the bud period, and each robust common head cabbage seed plant generally has 800-2000 buds. Therefore, explants are readily available;
(2) adventitious buds are formed early. By optimizing culture conditions such as different material taking time, culture medium formula, illumination treatment and the like, the adventitious bud can be directly induced about 15 days at the earliest. In the isolated regeneration system of common head cabbage, the induction of adventitious bud is required to be 40-50 days (Zhengzi pine, a tissue culture method for improving the isolated regeneration efficiency of common head cabbage, CN 103125398A; Zhaenghui, etc., a propagation method for maintaining the RGMS male sterile line of common head cabbage, CN 101564009A).
(3) The regeneration frequency of the invention is about 90 percent, the number of single buds contained in the directly induced cluster buds is about 7, and the regeneration frequency and the differentiation frequency are both higher, thus the invention is an efficient and convenient in vitro regeneration technical method.
Drawings
FIG. 1 shows the formation of adventitious buds at an early stage.
FIG. 2 the adventitious bud later stage is formed.
FIG. 3 regenerated plantlets.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention is realized in such a way that the method for culturing the common head cabbage seed pod to efficiently obtain the regeneration plant comprises the following steps:
1) explant selection
Carrying out bud stripping and pollination on the common head cabbage seed plants 1-2 days before blooming, and taking seed pods 8-9 days after pollination as explants; and sterilizing the explant for 1-2 min by using 75% of ethanol by volume fraction, sterilizing for 15-20 min by using 7-8% of sodium hypochlorite by volume fraction, and finally washing for 3-4 times by using sterile water.
In the embodiment of the invention, the explant sampling treatment comprises the following steps: and (5) taking seed pods of 5d, 7d, 8d, 9d, 10d and 12d after pollination for culturing. The result shows that the regeneration rate of 5d and 7d seed pods after pollination is low when the seed pods are cultured, the regeneration rate of 10d and 12d after pollination is not much different from that of 8d and 9d after pollination, but the adventitious bud induction rate of 10d and 12d after pollination is low, and the seed pods 8-9 d after pollination are screened out to be used as explants.
2) Differentiation Induction
Inoculating the explant to a culture medium of MS +0.1mg/L NAA +1.0 mg/L6-BA + 6-7 g/L agar + 25-30 g sucrose and pH5.8, wherein the temperature of the culture chamber is 25 +/-0.5 ℃, and the illumination is white light: the red light is 3:1, the illumination time is 14 hours per day, 4-5 days of pods basically begin to expand, 7-10 days of pods begin to grow adventitious buds, and 15-20 days of pods can directly induce the adventitious buds;
in an embodiment of the present invention, wherein:
A. differentiation induction medium was prepared by adding different concentrations of 6-BA and NAA on the basis of MS solid medium as follows: the concentrations of 6-BA are 1.0mg/L, 2.0mg/L and 3.0mg/L respectively; concentration of NAA is 0.1mg/L, 0.2mg/L and 0.3mg/L respectively;
TABLE 1NAA and 6-BA test treatment
B. In the embodiment of the present invention, 2 treatments of B1 (white light) and B2 (white light: red light: 3:1) were adopted as the light treatment;
single-factor and multi-factor tests are respectively carried out on the A (hormone treatment) and the B (light treatment), and the test results show that the hormone combination concentration is higher, more calluses are formed, but the adventitious buds formed directly are low; the regeneration rate under the A1 treatment is not obviously different from that under the A1 treatment, but the A1 treatment can directly form adventitious buds and has the highest adventitious bud induction rate. Under B2 (white light: red light: 3:1), adventitious buds formed early; taken together with the results of the experimental data, the optimal culture conditions for differentiation induction were the combination of a1+ B2, i.e.: MS +0.1mg/L NAA +1.0 mg/L6-BA, white light: red light is 3:1, the regeneration frequency is averagely 90%, and the single buds contained in the directly induced cluster buds are averagely 7.
3) Subculture multiplication
Cutting the induced cluster buds into single buds, transferring the single buds to a culture medium of MS +0.1mg/L NAA +0.8 mg/L6-BA + 6-7 g/L agar + 25-30 g of cane sugar and pH5.8, transferring for one time for 25-30 days, and carrying out 3-4 subcultures to proliferate the buds by about 220 times; the light culture environment during the period is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;
in the present example, we reduced the 6-BA concentration of the subculture multiplication medium according to the concentration of the optimum hormone ratio in the above differentiation induction. The subculture multiplication medium is prepared by adding 6-BA and NAA with different concentrations on the basis of MS solid medium and treating as follows: the concentrations of 6-BA are 0.6mg/L, 0.8mg/L and 1.0mg/L respectively; concentration of NAA is 0.1mg/L and 0.2mg/L respectively;
TABLE 2NAA and 6-BA test treatments
Test results show that under the treatment of C2, the regeneration and proliferation coefficient of the adventitious bud is averagely 4.0, the bud grows robustly, and no root is generated; the other proliferation coefficients are low, or the growth of adventitious buds is slow; the optimal medium was selected as the C2 combination, i.e.: MS +0.1mg/L NAA +0.8 mg/L6-BA
4) Root induction
When the sprouts grow to 3-4 cm high, transferring the sprouts to a culture medium of MS +0.1mg/L NAA +0.1mg/L IBA + 7-8 g/L agar + 20-25 g sucrose and pH5.8, and inducing the sprouts to root for 15-20 days to obtain rooted seedlings (as shown in figure 3); the light culture environment during the period is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;
5) transplanting and field planting
After hardening off the rooted seedlings at normal room temperature for 24 hours, transplanting the rooted seedlings into a plug tray with 50 holes for growth, wherein the matrix is a cruciferous vegetable seedling culture matrix; and 6-8 true leaves are planted in the field after the seedlings grow.
In conclusion, the common head cabbage is selected to be subjected to bud stripping and pollination 1-2 d before blooming, seed pods 8-9 d after pollination are taken as explants, the explants are inoculated to an adventitious bud induction culture medium with the pH value of 5.8 and containing MS +0.1mg/L NAA +1.0 mg/L6-BA + 6-7 g/L agar + 25-30 g sucrose, and the illumination treatment is red-white combined light (white light: red is 3: 1); after adventitious buds are formed at the edges of the leaves, the adventitious buds are cut and transferred to a multiplication culture medium of MS +0.1mg/L NAA +0.8 mg/L6-BA + 6-7 g/L agar + 25-30 g sucrose and pH5.8 for culture; when the sprouts grow to 3-4 cm high, transferring the sprouts to a culture medium of MS +0.1mg/L NAA +0.1mg/L IBA + 7-8 g/L agar + 20-25 g sucrose and pH5.8 to induce rooting, and obtaining regenerated seedlings. By the technical system, the adventitious buds are early formed, the regeneration frequency is about 90 percent, and the number of single buds contained in the directly induced cluster buds is about 7.
Claims (2)
1. A method for culturing common head cabbage seed pods to obtain regenerated plants efficiently is characterized by comprising the following steps:
(1) explant selection
Carrying out bud stripping and pollination on common head cabbage seeds 1-2 days before blooming, taking seed pods 8-9 days after pollination as explants, and carrying out sterilization treatment;
(2) differentiation Induction
Inoculating the explant to an induction culture medium after sterilization, and culturing in a culture room at the temperature of 25 +/-0.5 ℃ for 14 hours per day to induce and generate adventitious buds;
the illumination conditions of the culture are as follows: the number ratio of the red light lamps to the white light lamps is 1: 3;
the induction culture medium is as follows: MS +0.1mg/L NAA +1.0 mg/L6-BA + 6-7 g/L agar + 25-30 g/L sucrose, pH5.8;
(3) subculture multiplication
Transferring the induced single buds or cluster buds to a subculture multiplication medium, and transferring for one time in 25-30 days, wherein the illumination culture environment is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;
the subculture multiplication medium comprises: MS +0.1mg/L NAA +0.8 mg/L6-BA + 6-7 g/L agar + 25-30 g/L sucrose, pH5.8;
(4) root induction
When the sprouts grow to 3-4 cm high, transferring the sprouts to a rooting culture medium, wherein the illumination culture environment is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;
the rooting medium is MS +0.1mg/L NAA +0.1mg/L IBA + 7-8 g/L agar + 20-25 g/L sucrose, and the pH value is 5.8;
(5) transplanting and field planting
Hardening the rooted seedlings at room temperature for 24 hours, and transplanting the rooted seedlings into a plug for growth, wherein the substrate is a cruciferous vegetable seedling culture substrate; and 6-8 true leaves are planted in the field after the seedlings grow.
2. The method for efficiently obtaining regenerated plants by culturing common head cabbage seed pods according to claim 1, wherein the sterilization treatment in the step (1) is as follows:
and sterilizing the explant for 1-2 min by using 75% of ethanol in volume fraction, then sterilizing for 15-20 min by using 7-8% of sodium hypochlorite in volume fraction, and finally washing with sterile water.
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| CN101473791A (en) * | 2009-02-11 | 2009-07-08 | 西北农林科技大学 | Breeding method for maintaining spring property of inbred line in spring common head cabbage breeding |
| CN101564009A (en) * | 2009-06-03 | 2009-10-28 | 西北农林科技大学 | Propagation method for keeping cabbage RGMS male sterile line |
| CN102805035A (en) * | 2012-08-28 | 2012-12-05 | 邢台市蔬菜种子公司 | Common head cabbage tissue culture method |
| CN103125398A (en) * | 2013-03-20 | 2013-06-05 | 江苏省江蔬种苗科技有限公司 | Tissue culture method for improving in-vitro regeneration efficiency of common head cabbage |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101473791A (en) * | 2009-02-11 | 2009-07-08 | 西北农林科技大学 | Breeding method for maintaining spring property of inbred line in spring common head cabbage breeding |
| CN101564009A (en) * | 2009-06-03 | 2009-10-28 | 西北农林科技大学 | Propagation method for keeping cabbage RGMS male sterile line |
| CN102805035A (en) * | 2012-08-28 | 2012-12-05 | 邢台市蔬菜种子公司 | Common head cabbage tissue culture method |
| CN103125398A (en) * | 2013-03-20 | 2013-06-05 | 江苏省江蔬种苗科技有限公司 | Tissue culture method for improving in-vitro regeneration efficiency of common head cabbage |
Non-Patent Citations (1)
| Title |
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| LED光源对结球甘蓝（Brassica oleracea var. Capitatal）不定芽再生的影响;郑子松等;《天津农学院学报》;20130630;第20卷(第2期);第1.2.3节、第2.2节 * |
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