CN113115710A - Culture medium for inducing cut flower chrysanthemum petal callus and application - Google Patents
Culture medium for inducing cut flower chrysanthemum petal callus and application Download PDFInfo
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- CN113115710A CN113115710A CN202110582337.8A CN202110582337A CN113115710A CN 113115710 A CN113115710 A CN 113115710A CN 202110582337 A CN202110582337 A CN 202110582337A CN 113115710 A CN113115710 A CN 113115710A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
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Abstract
The invention provides a culture medium for inducing cut flower chrysanthemum petal callus and application thereof, which consists of 6-benzyladenine, naphthylacetic acid, MS, cane sugar and plant gel. The plant growth regulator with high selective valence ratio of the invention adds NAA and 6-BA with specific concentration into the culture medium, and the prepared culture medium not only effectively increases the callus inductivity of the petals, but also improves the weight coefficient of the callus. The cut chrysanthemum related to the invention has wide variety, and the cut chrysanthemum varieties of different varieties are classified and discussed, and the optimal callus induction culture medium of each variety is screened out. The culture medium can obviously improve the induction rate and the weight coefficient of the petal callus of the cut chrysanthemum varieties, well solves the problems of low induction rate and slow callus growth of the petal callus, lays a foundation for callus differentiation in the next step, and also provides a good transgenic material.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium for inducing cut flower chrysanthemum petal callus and application thereof.
Background
Chrysanthemum morifolium (Chrysanthemum morifolium) is perennial root herb of Compositae Chrysanthemum, is rich in variety and various in flower color, is one of ten famous flowers in China and four cut flowers in the world, and has various values of appreciation, edibility, medicinal use and the like. The chrysanthemum is usually bred by cutting, but the breeding efficiency is low, the limitation of climate conditions is large, and certain rare chrysanthemum varieties and new varieties with insufficient female parent sources cannot meet the market demand. The tissue culture technology can obviously improve the proliferation efficiency, accelerate the propagation speed, realize the large-scale production of the chrysanthemum and have extremely high economic and social benefits.
Hill establishes a chrysanthemum regeneration system by taking bud tips as explants for the first time in 1968, and different chrysanthemum varieties by taking petioles, leaves, stem segments, flower organs and the like as the explants have been established so far. Compared with other explants, the petals have less virus, convenient and fast material acquisition and low disinfection difficulty, and the petal has higher variation rate of new plants generated by callus, thus being one of the important means for breeding new species of chrysanthemum. Meanwhile, the tissue culture technology of chrysanthemum plays an important role in the field of chrysanthemum transgenic molecular breeding, and the research and optimization of a chrysanthemum tissue culture regeneration system are the premise and guarantee for carrying out chrysanthemum scientific research.
The genetic background of the chrysanthemum is complex, and the regeneration capacity of the same explant of different chrysanthemum varieties is different. However, the existing chrysanthemum petal callus induction method still has the problems of slow callus growth, low callus induction rate, no classification discussion of culture medium according to genotype, and the like.
Disclosure of Invention
The invention provides a culture medium for callus induction of cut chrysanthemum petals, which can obviously improve the induction rate and the weight coefficient of the cut chrysanthemum callus, and can classify and discuss the cut chrysanthemum of different varieties and screen out the optimal callus induction culture medium of each variety.
The culture medium for inducing the cut flower chrysanthemum petal callus provided by the invention consists of 6-benzyladenine (6-BA) of 0.5-2.0mg/L, Naphthalene Acetic Acid (NAA) of 0.5-2.0mg/L, MS of 4.43g/L, sucrose of 30g/L and plant gel of 3g/L, is an MS culture medium (Murashige & Skoog basic Salt mix) and has a pH value of 5.8.
In the induction culture medium, NAA is an auxin analogue in a plant growth regulator, can promote cell division and expansion, and has obvious effect on the induction and growth of callus; 6-BA is an artificially synthesized cytokinin, and has the characteristics of high efficiency, stability, low price and the like. According to the invention, the 2 hormones are added into a culture medium according to a specific proportion (4.43g/L MS +30g/L sucrose +3g/L plant gel) to prepare 4 callus induction culture media, when tissue culture is respectively carried out on cut flower chrysanthemum petal explants of different varieties, the callus induction rate and weight coefficient of the cut flower chrysanthemum can be obviously improved, and the variety coverage rate is greater than that of the chrysanthemum varieties reported in the prior art.
Preferably, the medium comprising 0.5 mg/L6-BA +2.0mg/L NAA +4.43g/L MS +30g/L sucrose +3g/L plant gel is an optimum medium for callus induction by petals of cut-flower chrysanthemum varieties such as 'flame', 'Buddha Ruige', 'Roman holiday' and 'Jiuyue'.
Preferably, in the culture medium, the culture medium with the components of 1.0 mg/L6-BA +2.0mg/L NAA +4.43g/L MS +30g/L sucrose +3g/L plant gel is the most suitable culture medium for inducing callus by the petals of the cut chrysanthemum 'maiden powder'.
Preferably, the culture medium with the components of 2.0 mg/L6-BA +0.5mg/L NAA +4.43g/L MS +30g/L sucrose +3g/L plant gel is the optimal culture medium for inducing callus by cut chrysanthemum 'field', 'Redox yellow', 'tamari powder', 'Majit', 'Hongdaite' and 'village music' petals.
Preferably, the culture medium with the components of 2.0 mg/L6-BA +1.0mg/L NAA +4.43g/L MS +30g/L sucrose +3g/L plant gel is the most suitable culture medium for inducing callus by cut-flower chrysanthemum ' Plumbum ' Pushan ', ' rhododendron ' and ' Lvjingling ' petals.
The invention also aims to provide a culture method for inducing the cut flower chrysanthemum petals to generate callus, which is realized by the following steps:
(1) placing the petals in a beaker filled with warm water at 25 ℃, dripping 3 drops of Tween-80, standing for 30min, and then placing under running water to wash for 1 hour, wherein the running water is continuously shaken during the period to wash off the adhesive on the surface of the material;
(2) rapidly washing the petal with 70% alcohol on a clean bench for 1 time, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride for sterilizing for 5min, and washing with sterile water for 5 times;
(3) taking out the sterilized petals, drying with sterilized filter paper, cutting the middle parts of the petals into 5mm × 5mm, inoculating into callus induction culture medium, and inoculating 5 petals per bottle;
(4) the callus induction culture conditions are that the culture temperature is 25 +/-1 ℃, the illumination intensity is 2000Lx, the illumination is 16h every day, and the darkness is 8 h.
The invention further aims to provide an application of the culture medium for inducing the chrysanthemum flower petal callus in the generation of the chrysanthemum flower petal callus.
Compared with the prior art, the invention has the beneficial effects that: (1) the plant growth regulator with high selective valence ratio adds NAA and 6-BA with specific concentration into the culture medium, and the prepared culture medium not only effectively increases the callus induction rate of the petals, but also improves the weight coefficient of the callus. (2) The cut chrysanthemum related to the invention has wide variety, and the cut chrysanthemum of different varieties is classified and discussed, and the optimal callus induction culture medium of each variety is screened out. The invention carries out classification research on the cut chrysanthemum varieties, screens out the most suitable callus induction culture medium of each variety, can obviously improve the petal callus induction rate and the weight coefficient of 15 cut chrysanthemum varieties, well solves the problems of low petal callus induction rate and slow callus growth, and lays a foundation for callus differentiation in the next step.
Drawings
FIG. 1 shows callus formation from petals after 30 days of culture in example 1 of the present invention.
FIG. 2 shows callus formation from petals after 30 days of culture in example 2 of the present invention.
FIG. 3 shows callus formation from petals after 30 days of culture in example 3 of the present invention.
FIG. 4 shows callus formation from petals after 30 days of culture in example 4 of the present invention.
FIG. 5 is a table of the varieties of cut chrysanthemum.
Detailed Description
In the following examples, all of the cut chrysanthemum flowers were purchased from Zhejiang Phoenix city.
Example 1
1. Test materials: cut chrysanthemum 'flame', 'florie', 'roman holiday', 'september nine'.
2. Test medium: 4.43g/L MS +30g/L sucrose +3g/L plant gel +0.5-2.0 mg/L6-BA +0.5-2.0mg/L NAA, pH 5.8. The cut flower chrysanthemum petal callus induction culture mediums 1, 2, 3 and 4 have hormone formulations shown in Table 1.
3. The test method comprises the following steps:
material sterilization and inoculation: placing petal in a beaker containing 25 deg.C warm water, dripping 3 drops of Tween-80, and standing for 30 min; sealing the cup mouth with gauze, placing under running water for washing for 1 hour, continuously shaking during the washing, and washing off the adhered substances on the surface of the material; transferring the petals into a sterilized beaker on a clean bench by using tweezers, adding 70% alcohol solution for 5s, and washing with sterile water for 3 times; adding 0.1% mercuric chloride, soaking for 5min, and washing with sterile water for 5 times; taking out the sterilized petals, drying with sterilized filter paper, cutting the middle part of the petals into 5mmx 5mm, inoculating into callus induction culture medium, and inoculating 5 petals per bottle.
Culture conditions and index determination: the culture temperature is 25 + -1 deg.C, the illumination intensity is 2000Lx, the illumination is 16h and the darkness is 8h every day. Each treatment treated 15 petals, set in triplicate. After 30 days of culture, counting the number of petals and the weight of the callus, and calculating the average callus induction rate and the average callus weight coefficient.
The average callus induction rate is the number of petals growing out of the callus/total number of petals.
The average callus weight coefficient ═ sigma callus weight/number of petals of growing callus.
Statistical analysis: analysis of variance was performed using SPSS 24.0 software, and multiple comparisons were performed using the LSD method, with α being 0.05.
4. And (3) test results:
in this example, the callus induction rates of the petals in culture medium No. 1 (4.43g/L MS +30g/L sucrose +3g/L phytogel +0.5 mg/L6-BA +2.0mg/L NAA) were all 100%, and the callus weights were the greatest, indicating that culture medium No. 1 was the most suitable for callus induction by cut chrysanthemum, ` flame `, ` flory `, ` Roman holiday `, ` September `.
TABLE 1 cut flower Chrysanthemum petal callus induction culture medium hormone ratio
| Medium numbering | 6-BA(mg/L) | NAA(mg/L) |
| 1 | 0.5 | 2.0 |
| 2 | 1.0 | 2.0 |
| 3 | 2.0 | 0.5 |
| 4 | 2.0 | 1.0 |
Note: the minimal medium is 4.43g/L MS +30g/L sucrose +3g/L plant gel.
TABLE 2 influence of different hormone concentration ratios on the callus induction rate of different varieties of cut-flower chrysanthemum
| No. 1 culture medium | No. 2 culture medium | No. 3 culture medium | No. 4 medium | |
| Flame | 100.00 | 93.33 | 100.00 | 100.00 |
| Buddha Rui Ge | 100.00 | 93.33 | 83.33 | 100.00 |
| Roman holiday | 100.00 | 100.00 | 93.33 | 90.00 |
| Nine months and nine | 100.00 | 96.67 | 100.00 | 100.00 |
TABLE 3 influence of different hormone concentration ratios on the calli weight coefficients of different varieties of cut-flower chrysanthemum
| No. 1 culture medium | No. 2 culture medium | No. 3 culture medium | No. 4 medium | |
| Flame | 0.18±0.0089a | 0.063±0.0037c | 0.064±0.029c | 0.083±0.0038b |
| Buddha Rui Ge | 0.041±0.0007a | 0.034±0.0009 | 0.027±0.0012c | 0.036±0.0014b |
| Roman holiday | 0.055±0.0035a | 0.046±0.0022b | 0.045±0.0018b | 0.045±0.0040b |
| Nine months and nine | 0.069±0.0018a | 0.037±0.0009b | 0.067±0.0024a | 0.063±0.0024a |
Example 2
1. Test materials: cut chrysanthemum 'maiden powder'.
2. Test medium: the same as example 1;
3. the test method comprises the following steps: the same as in example 1.
4. And (3) test results:
in this example, the callus induction rate of the petals in the culture medium No. 2 (4.43g/L MS +30g/L sucrose +3g/L plant gel +1.0 mg/L6-BA +2.0mg/L NAA) was 100%, and the callus weight was the largest, indicating that the culture medium No. 2 was the most suitable culture medium for callus induction by the cut chrysanthemum 'girl powder' petals.
TABLE 4 influence of different hormone concentration ratios on the callus induction rate of different varieties of cut chrysanthemum
| No. 1 culture medium | No. 2 culture medium | No. 3 culture medium | No. 4 medium | |
| Girl powder | 66.67 | 100.00 | 93.33 | 86.67 |
TABLE 5 influence of different hormone concentration ratios on the calli weight coefficients of different varieties of cut-flower chrysanthemum
| No. 1 culture medium | No. 2 culture medium | No. 3 culture medium | No. 4 medium | |
| Girl powder | 0.023±0.0009c | 0.036±0.0011a | 0.030±0.0021b | 0.025±0.0005c |
Example 3
1. Test materials: cut chrysanthemum ' field ' Ruidostat yellow ', tame powder ', Majit ', Hongdaite ' and country music '.
2. Test medium: the same as in example 1.
3. The test method comprises the following steps: the same as in example 1.
4. And (3) test results:
in this example, the callus induction rate of the petals in culture medium No. 3 (4.43g/L MS +30g/L sucrose +3g/L phytogel +2.0 mg/L6-BA +0.5mg/L NAA) was 100%, and the callus weight was the largest, indicating that culture medium No. 3 was the most suitable for callus induction by petals of cut chrysanthemum 'wild', 'Redotti yellow', 'Tanry powder', 'Majit', 'Hongdaite', 'county music'.
TABLE 6 influence of different hormone concentration ratios on the callus induction rate of different varieties of cut-flower chrysanthemum
| No. 1 culture medium | No. 2 culture medium | No. 3 culture medium | No. 4 medium | |
| Open field | 73.33 | 83.33 | 100.00 | 86.67 |
| Ruidostat yellow | 90.00 | 100.00 | 100.00 | 100.00 |
| Tanshii powder | 100.00 | 100.00 | 100.00 | 100.00 |
| Majit | 93.33 | 100.00 | 100.00 | 100.00 |
| Hongdan special medicine | 93.33 | 100.00 | 100.00 | 100.00 |
| Country music | 100.00 | 100.00 | 100.00 | 100.00 |
TABLE 7 influence of different hormone concentration ratios on the calli weight coefficients of different varieties of cut-flower chrysanthemum
Example 4
1. Test materials: cut chrysanthemum 'minidante', 'purpu mountain', 'rhododendron' and 'green fairy'.
2. Test medium: the same as in example 1.
3. The test method comprises the following steps: the same as in example 1.
4. And (3) test results:
in this example, the callus induction rates of the petals in the 4 media were all 100%, and the callus weight was the greatest in the No. 4 medium (4.43g/L MS +30g/L sucrose +3g/L phytogel +2.0 mg/L6-BA +1.0mg/L NAA), indicating that the No. 4 medium was the most suitable medium for the petals of cut chrysanthemum 'minidante', 'pu shan', 'rhododendron' and 'luo jingling' to induce callus.
TABLE 8 influence of different hormone concentration ratios on the callus induction rate of different varieties of cut-flower chrysanthemum
| No. 1 culture medium | No. 2 culture medium | No. 3 culture medium | No. 4 medium | |
| Pindan specially for treating psoriasis | 100.00 | 100.00 | 100.00 | 100.00 |
| Pushan mountain | 100.00 | 100.00 | 100.00 | 100.00 |
| Du Juan | 100.00 | 100.00 | 100.00 | 100.00 |
| Green fairy | 100.00 | 100.00 | 100.00 | 100.00 |
TABLE 9 influence of different hormone concentration ratios on calli weight coefficients of different varieties of cut chrysanthemum
| No. 1 culture medium | No. 2 culture medium | No. 3 culture medium | No. 4 medium | |
| Pindan specially for treating psoriasis | 0.085±0.0052ab | 0.076±0.0033bc | 0.070±0.0018c | 0.094±0.0031a |
| Pushan mountain | 0.15±0.0057b | 0.085±0.0021d | 0.11±0.0062c | 0.17±0.0039a |
| Du Juan | 0.20±0.0060b | 0.13±0.0025c | 0.14±0.0049c | 0.26±0.013a |
| Green fairy | 0.10±0.0041b | 0.094±0.0069b | 0.11±0.0037b | 0.12±0.004a |
Claims (11)
1. The culture medium for inducing the cut flower chrysanthemum petal callus is characterized by consisting of 0.5-2.0 mg/L6-benzyladenine, 0.5-2.0mg/L naphthylacetic acid, 4.43g/L MS, 30g/L sucrose and 3g/L plant gel, and the pH value is 5.8.
2. The culture medium of claim 1, wherein the culture medium is 0.5 mg/L6-BA +2.0mg/LNAA +4.43g/L MS +30g/L sucrose +3g/L plant gel, pH 5.8.
3. The culture medium of claim 1, wherein the culture medium is 1.0 mg/L6-BA +2.0mg/LNAA +4.43g/L MS +30g/L sucrose +3g/L plant gel, pH 5.8.
4. The culture medium of claim 1, wherein the culture medium is 2.0 mg/L6-BA +0.5mg/LNAA +4.43g/L MS +30g/L sucrose +3g/L plant gel, pH 5.8.
5. The culture medium of claim 1, wherein the culture medium consists of 2.0 mg/L6-BA +1.0mg/LNAA +4.43g/L MS +30g/L sucrose +3g/L plant gel at pH 5.8.
6. Use of a culture medium according to any one of claims 1 to 5 for inducing the formation of chrysanthemum petal callus.
7. Use according to claim 6, characterized in that the medium according to claim 2 is used for callus induction in the petals of the 'flame', 'Buddha regia', 'roman holiday', 'September ninth' cut chrysanthemum variety.
8. Use according to claim 6, wherein the medium according to claim 3 is used for callus induction by petals of cut chrysanthemum 'maiden's powder.
9. Use according to claim 6, characterized in that the medium according to claim 4 is used in the callus induction by the petals of cut chrysanthemum 'field', 'Redotestem yellow', 'Tanry flour', 'Majit', 'Red Dante', 'Country music'.
10. Use according to claim 6, characterized in that the medium according to claim 5 is used in the callus induction of cut-flower chrysanthemum 'Plutella', 'Pushan', 'Rhododendron', 'Lvjining' petals.
11. The use according to any one of claims 6 to 10, wherein the callus induction culture conditions for the use are a culture temperature of 25 ± 1 ℃, a light intensity of 2000Lx, 16h light per day and 8h dark.
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