Culture medium and method for constructing Chinese cabbage high-frequency regeneration system

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a culture medium and a method for constructing a Chinese cabbage high-frequency regeneration system. According to the Chinese cabbage material 'Y677P-3' cotyledons with stems and the in-vitro culture high-frequency regeneration method, the cotyledons with stems of aseptic seedlings of Chinese cabbages are used as explants for in-vitro culture and are induced into buds on a differential culture medium, and then the buds are transferred to a rooting culture medium for seedling formation. Wherein the differential culture medium comprises the following main components: an MS basic culture medium, 0.5 mg / L NAA (naphthylacetic acid), 4mg / L 6-BA (6-benzylamino adenine), 4.5 mg / L LAgNO3, 30g / L cane sugar and 8g / L agar. According to the Chinese cabbage in-vitro culture high-frequency regeneration method provided by the invention, the culture medium with a specific ratio is adopted, so that the differentiation frequency of the explant can be up to 100%, the regeneration period is short, and a stable new receptor material is provided for Chinese cabbage transgenic breeding research.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to a culture medium and a method for constructing a high-frequency regeneration system of Chinese cabbage. Background Art

[0002] Chinese cabbage (Brassica rapa L.ssp.pekinensis) belongs to the vegetable crop of the genus Brassica of the cruciferous family. It is widely cultivated in my country. Traditional breeding methods are mainly used for genetic improvement. In recent years, plant genetic engineering has developed rapidly, and transgenic technology has also been gradually applied to Chinese cabbage breeding. However, Chinese cabbage belongs to AA genome crops, and the in vitro regeneration ability of plants is relatively low. Although there are some reports of successful transformation, it is affected by genotype, and most Chinese cabbage varieties are difficult to regenerate, and the transformation success rate is relatively low, which has hindered the development and application of Chinese cabbage genetic engineering. Therefore, setting up an efficient and stable Chinese cabbage regeneration system is an important prerequisite for carrying out its genetic transformation research.

[0003] At present, domestic and foreign scholars have done a lot of work in the establishment of the regeneration system of Chinese cabbage crops, and have made some progress. It is found that factors such as genotype, explant type, hormone type and concentration combination ratio, and AgNO3 (silver nitrate) concentration are extremely important factors affecting the regeneration research of Brassica plants. The regeneration systems reported by previous researchers have not been able to increase the regeneration rate of Chinese cabbage to more than 95%. The present invention explores the factors that affect the in vitro regeneration of Chinese cabbage and establishes a high-frequency regeneration system. This transformation system will lay the foundation for the molecular breeding of Chinese cabbage and the study of gene function. Summary of the invention

[0004] The invention aims to provide a method for in vitro tissue culture of Chinese cabbage, which adopts a specific culture medium to improve the regeneration rate of Chinese cabbage.

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

[0006] In one aspect, the present invention provides a medium for in vitro tissue culture of Chinese cabbage, the medium comprising an induction medium, the components of the induction medium in terms of final concentration being: MS+30 g / L sucrose+8 g / L agar+0.5 mg / L NAA+3-6 mg / L AgNO3+2-6 mg / L 6-BA;

[0007] The culture medium includes a rooting culture medium, and the components of the rooting culture medium in terms of final concentration are: MS+30 g / L sucrose+8 g / L agar+0.5 mg / L NAA.

[0008] Preferably, the components of the induction medium in terms of final concentration are: MS+30 g / L sucrose+8 g / L agar+0.5 mg / L NAA+4.5 mg / LAgNO3+4 mg / L 6-BA, pH=5.8.

[0009] On the other hand, the present invention also provides a method for in vitro tissue culture of Chinese cabbage, comprising the following steps:

[0010] S1: Place the sterilized Chinese cabbage seeds in a sterile culture dish to culture and obtain sterile seedlings;

[0011] S2: Select the sterile seedlings cultured in step S1 for 4-5 days, cut the petiole cotyledons below the growth point as explants, inoculate them into induction medium for culture, and obtain adventitious buds;

[0012] S3: The adventitious buds obtained in step S2 are transferred to a rooting medium for rooting culture to obtain tissue culture seedlings.

[0013] Preferably, the length of the petiole of the petiole cotyledon described in step S2 is 1-2 mm.

[0014] Preferably, the height of the adventitious buds described in step S2 is 2-3 cm.

[0015] Preferably, the material of the Chinese cabbage seeds described in step S1 is "Y677P-3".

[0016] Preferably, the culture environment in steps S1, S2 and S3 is 25°C and the light intensity is 16h / d.

[0017] Compared with the prior art, the beneficial effects are:

[0018] 1) The present invention uses the petiole cotyledons of Chinese cabbage "Y677P-3" as materials, studies the effects of different explant types, seedling ages, AgNO3 concentrations and 6-BA concentrations on the regeneration rate of adventitious buds, establishes a high-frequency regeneration system for Chinese cabbage, and provides stable new receptor materials for transgenic breeding research of Chinese cabbage.

[0019] 2) The present invention adopts a culture medium with a specific ratio, so that the explant differentiation frequency can reach up to 100%, and the regeneration period is short. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1The figures show the differentiation of regenerated buds from different types of explants of Chinese cabbage material "Y677P-3"; A: differentiation of regenerated buds from cotyledon-cotyledon petiole explants; B: differentiation of regenerated buds from hypocotyl explants.

[0022] Figure 2 This is the differentiation of regenerated adventitious buds of the Chinese cabbage material "Y677P-3" at different seedling ages; A: seedling age 4 days; B: seedling age 5 days; C: seedling age 6 days; D: seedling age 7 days.

[0023] Figure 3 This is the differentiation of regenerated adventitious buds of the Chinese cabbage material "Y677P-3" under different AgNO3 concentration treatment conditions; A: 0mg / L AgNO3; B: 3mg / L AgNO3; C: 4.5mg / L AgNO3; D: 6mg / LAgNO3.

[0024] Figure 4 This is the differentiation of regenerated adventitious buds of the Chinese cabbage material "Y677P-3" under different 6-BA concentration treatment conditions; A represents 0 mg / L 6-BA; B represents 2 mg / L 6-BA; C represents 4 mg / L 6-BA; and D represents 6 mg / L 6-BA. DETAILED DESCRIPTION

[0025] On the one hand, the present invention provides a culture medium for in vitro tissue culture of Chinese cabbage, the culture medium comprising an induction culture medium, the components of which are calculated by final concentration: MS+30g / L sucrose+8g / L agar+0.5mg / L NAA+3-6mg / LAgNO3+2-6mg / L 6-BA; and a rooting culture medium, the components of which are calculated by final concentration: MS+30g / L sucrose+8g / L agar+0.5mg / L NAA.

[0026] Among them, MS medium is a commercially available product;

[0027] Wherein, AgNO3 is preferably: 3, 4.5, 6 mg / L, and most preferably 4.5 mg / L;

[0028] The 6-BA content is preferably 2, 4, or 6 mg / L, and most preferably 4 mg / L.

[0029] Furthermore, the components of the induction medium in terms of final concentration are: MS+30g / L sucrose+8g / L agar+0.5mg / L NAA+4.5mg / LAgNO3+4mg / L 6-BA, pH=5.8.

[0030] In another aspect, the present invention provides a method for in vitro tissue culture of Chinese cabbage, comprising the following steps:

[0031] S1: Place the sterilized Chinese cabbage seeds in a sterile culture dish to culture and obtain sterile seedlings;

[0032] S2: Select the sterile seedlings cultured in step S1 for 4-5 days, cut the petiole cotyledons below the growth point as explants, inoculate them into induction medium for culture, and obtain adventitious buds;

[0033] S3: The adventitious buds obtained in step S2 are transferred to a rooting medium for rooting culture to obtain tissue culture seedlings.

[0034] The seedling age in step S2 is preferably 4 days.

[0035] Furthermore, the length of the petiole of the petiole cotyledon described in step S2 is 1-2 mm.

[0036] Furthermore, the culture method comprises the induction medium according to claim 1 and the rooting medium according to claim 2.

[0037] Furthermore, the material of the Chinese cabbage seeds described in step S1 is "Y677P-3".

[0038] Furthermore, the culture environment in steps S1, S2 and S3 is 25°C and the light intensity is 16h / d.

[0039] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0041] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0042] Example 1

[0043] Effects of explant types on differentiation of regenerated adventitious buds

[0044] 1. Material: The material of Chinese cabbage seeds is "Y677P-3"

[0045] 2. Test methods

[0046] 2.1 Obtaining sterile seedlings

[0047] Select 30-40 seeds / bottle (full seeds, no wormholes) and put them in a 50ml centrifuge tube. Wash them with 75% ethanol for 2 minutes on a clean bench, then add about 30ml 0.1% mercuric chloride, shake for 8 minutes, and finally wash them with sterile water 3 times, 3 minutes / time. Put a large filter paper in the culture dish, pour the cleaned seeds on the filter paper, dry them, and evenly sow them in the culture medium. Place them evenly, seal them, write the date, mark them, and place them on the light culture rack in the tissue culture room for 4-5 days;

[0048] 2.2 Effects of different explant types on the differentiation of regenerated adventitious buds

[0049] The hypocotyls (the hypocotyls of the sterile seedlings with a length of about 5 mm) and petioled cotyledons (the sterile seedlings were carefully and quickly cut from the cotyledon petioles to ensure that the growth point was cut off and the cotyledon petioles were 1-2 mm) of the sterile seedlings 4 days after sowing were selected as explants and inoculated into the optimal bud induction medium (MS + 30 g / L sucrose + 8 g / L agar + 0.5 mg / L NAA + 4.5 mg / L AgNO3 + 4 mg / L6-BA, pH = 5.8) at 25 ° C and 16 h / d of light for adventitious bud induction. Each explant was repeated 30 times, and the test was repeated three times. The formation of adventitious buds was observed and recorded within three weeks.

[0050] 3. Results and Analysis

[0051] The results are shown in Table 1. It can be seen from Table 1 that the difference in the differentiation rate of regenerated adventitious buds of the two types of explants reached a significant level (P<0.05), among which the adventitious bud differentiation rate of petiole cotyledon explants was the highest. In three repetitions, the regeneration rate was 100% in two of them, and the average regeneration rate could reach 98.9%. In the three repetitions of hypocotyl explants, only one hypocotyl differentiated into regenerated buds, and the adventitious bud differentiation rate was only 2.2%. After three weeks of observation, it was found that the wound of petiole cotyledon explants began to gradually swell and form callus tissue around 3 days, regeneration bud points appeared around 7 days, and more than 98% of the explants had differentiated into regenerated buds around 12 days ( Figure 1 A), the hypocotyl explants began to swell after 7 days of culture, and more than 97% of the explants formed large callus tissues around 20 days, and no regenerated buds were produced ( Figure 1 B).

[0052] Table 1 Effect of explant type on adventitious bud differentiation of Chinese cabbage material “Y677P-3”

[0053]

[0054] Example 2

[0055] Effect of Seedling Age on Differentiation of Adventitious Buds

[0056] 1. Materials: Same as Example 1

[0057] 2. Test methods

[0058] 2.1 Obtaining sterile seedlings: Same as Example 1;

[0059] 2.2 Effect of seedling age on differentiation of regenerated adventitious buds

[0060] The seedling age was screened by using the Chinese cabbage material "Y677P-3". From the date of sowing, sterile seedlings were cultured for 4, 5, 6, and 7 days, respectively. The petiole cotyledons were cut and placed on the optimal bud induction medium at 25°C and 16h / d of light for adventitious bud induction. Each explant was repeated 30 times, and the experiment was repeated three times. The formation of adventitious buds was observed and recorded within three weeks.

[0061] 3. Results and Analysis

[0062] The results are shown in Table 2. It can be seen from Table 2 that the differentiation of adventitious buds of explants with four different growth times was significantly different (P<0.05). For the same number of explants, the number of adventitious buds differentiated in the explants within 4-5 days of seedling age ranged from 20 to 30, and the average differentiation rate of adventitious buds ranged from 97.8% to 98.9%. Among them, the average differentiation rate of adventitious buds of explants with a seedling age of 4 days was 98.9%, which was the highest, while the average differentiation rate of adventitious buds of explants with a seedling age of 7 days was 5.6%, which was the lowest. The results are as follows Figure 2 The results showed that the age of the seedlings affected the in vitro regeneration of Chinese cabbage, but the older the seedlings, the better the differentiation rate of regenerated buds.

[0063] Table 2 Effect of seedling age on adventitious bud differentiation of Chinese cabbage material “Y677P-3”

[0064]

[0065]

[0066] Example 3

[0067] Effect of AgNO3 concentration on differentiation of regenerated adventitious buds

[0068] 1. Materials: Same as Example 1

[0069] 2. Test methods

[0070] 2.1 Obtaining sterile seedlings: Same as Example 1;

[0071] 2.2 Effect of AgNO3 concentration on the differentiation of regenerated adventitious buds

[0072] The AgNO3 concentration was set to 0, 3, 4.5, and 6 mg / L, and the regeneration of explants was observed after 20 days (Table 3).

[0073] 3. Results and Analysis

[0074] The results are shown in Table 3. It can be seen from Table 3 that the differentiation rates of adventitious buds under the four concentration treatments are significantly different. When the AgNO3 concentration is 4.5 mg / L, the explants show the highest regeneration rate of 97.7%. When AgNO3 is not added, all petiole cotyledons take root, no regenerated buds are produced, and the explant regeneration rate is 0. When the AgNO3 concentration is 6 mg / L, the regeneration rate drops significantly, indicating that AgNO3 has a greater effect on the differentiation of adventitious buds. The results are as follows Figure 3 Therefore, the AgNO3 concentration was determined to be 4.5 mg / L.

[0075] Table 3 Effects of different AgNO3 concentrations on the differentiation of adventitious buds of Chinese cabbage material “Y677P-3”

[0076]

[0077] Example 4

[0078] Effects of 6-BA concentration on adventitious bud differentiation

[0079] 1. Materials: Same as Example 1

[0080] 2. Test methods

[0081] 2.1 Obtaining sterile seedlings: Same as Example 1;

[0082] Effect of 2.26-BA concentration on adventitious bud differentiation

[0083] The 6-BA concentration was set to 0, 2, 4, and 6 mg / L, and the explant regeneration was counted after 20 days (Table 4).

[0084] 3. Results and Analysis

[0085] The results are shown in Table 4. It can be seen from Table 4 that when 6-BA was not added, the induction effect of NAA alone caused the explants to root in large quantities, and the adventitious bud differentiation rate was 0. When the 6-BA addition concentrations were 2, 4, and 6 mg / L, the regeneration of the explant adventitious buds was good. When the addition concentration reached 4 mg / L, the adventitious bud differentiation rate was the highest, reaching 97.8%. Therefore, the optimal concentration of 6-BA was set to 4 mg / L. The results are shown in Figure 4 shown.

[0086] From this we can see that the concentration of exogenous hormones promotes the growth of regenerated buds, but the higher the concentration is not necessarily better. The higher the concentration, the lower the differentiation rate of regenerated buds may be. Therefore, the concentration of exogenous hormones also has a great influence on the in vitro regeneration system of Chinese cabbage.

[0087] Table 4 Effects of different 6-BA concentrations on the differentiation of adventitious buds in Chinese cabbage material “Y677P-3”

[0088]

[0089] In summary, the present invention establishes an optimal bud induction medium (MS+30g / L sucrose+8g / L agar+0.5mg / L NAA+4.5mg / LAgNO3+4mg / L 6-BA, pH=5.8) by exploring the factors that affect the in vitro regeneration of Chinese cabbage. Under this system, it is found that the highest differentiation rate of regenerated adventitious buds of the Chinese cabbage material "Y677P-3" can reach 100%. The transformation system and transformation materials will lay a foundation for the research on molecular breeding and gene function of Chinese cabbage.

[0090] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A medium for in vitro tissue culture of Chinese cabbage, characterized in that: The culture medium includes an induction culture medium, and the components of the induction culture medium are: MS+30g / L sucrose+8g / L agar+0.5mg / L NAA+3-6mg / LAgNO3+2-6mg / L 6-BA according to the final concentration.

2. A medium for in vitro tissue culture of Chinese cabbage, characterized in that: The culture medium includes a rooting culture medium, and the components of the rooting culture medium in terms of final concentration are: MS+30 g / L sucrose+8 g / L agar+0.5 mg / L NAA.

3. The culture medium according to claim 1, characterized in that The components of the induction medium in terms of final concentration are: MS+30 g / L sucrose+8 g / L agar+0.5 mg / L NAA+4.5 mg / L AgNO3+4 mg / L 6-BA, pH=5.

8.

4. A method for in vitro tissue culture of Chinese cabbage, characterized in that: The culture method comprises the following steps: S1: Place the sterilized Chinese cabbage seeds in a sterile culture dish to culture and obtain sterile seedlings; S2: Select the sterile seedlings cultured in step S1 for 4-5 days, cut the petiole cotyledons below the growth point as explants, inoculate them into induction medium for culture, and obtain adventitious buds; S3: The adventitious buds obtained in step S2 are transferred to a rooting medium for rooting culture to obtain tissue culture seedlings.

5. The Chinese cabbage in vitro tissue culture method according to claim 4, characterized in that: The length of the petiole of the petiole cotyledon described in step S2 is 1-2 mm.

6. The Chinese cabbage in vitro tissue culture method according to claim 4, characterized in that: The height of the adventitious buds described in step S2 is 2-3 cm.

7. The Chinese cabbage in vitro tissue culture method according to claim 4, characterized in that: The culture method comprises the induction medium according to claim 1 and the rooting medium according to claim 2.

8. The Chinese cabbage in vitro tissue culture method according to claim 4, characterized in that: The material of the Chinese cabbage seeds described in step S1 is "Y677P-3".

9. The Chinese cabbage in vitro tissue culture method according to claim 4, characterized in that: The culture environment in steps S1, S2 and S3 is 25°C and the light intensity is 16h / d.

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