CN117736964A - Preparation method and application of licorice protoplast - Google Patents
Preparation method and application of licorice protoplast Download PDFInfo
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- CN117736964A CN117736964A CN202311701106.XA CN202311701106A CN117736964A CN 117736964 A CN117736964 A CN 117736964A CN 202311701106 A CN202311701106 A CN 202311701106A CN 117736964 A CN117736964 A CN 117736964A
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application discloses a preparation method and application of licorice protoplast. In one aspect, the present application provides a method for preparing licorice protoplasts, comprising the steps of: s1, selecting primary cotyledons of liquorice, cutting the primary cotyledons into cotyledon fragments, and inoculating the cotyledons into a callus culture medium for sterile induction culture of callus; s2, picking out the callus, putting the callus into enzymolysis liquid, and carrying out oscillation enzymolysis; s3, filtering the obtained enzymolysis product by using a 100-mesh cell filter screen, centrifuging the obtained filtrate, removing the supernatant, and cleaning the precipitate to obtain the liquorice protoplast. On the other hand, the application also provides a preparation method of the liquorice protoplast, and application of the liquorice protoplast in the fields of improving liquorice varieties, cultivating mutant liquorice plants or cultivating hybrid liquorice plants. The preparation method can be used for efficiently preparing the liquorice protoplast, and has the advantages of higher preparation yield, higher activity of the prepared liquorice protoplast and better quality.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a preparation method and application of licorice protoplast.
Background
The Chinese medicine is a medicine which is collected, processed and prepared according to the guidance of the Chinese traditional medicine theory, illustrates the action mechanism and guides the clinical application. The Chinese medicine is mainly derived from natural medicines and processed products thereof, including plant medicines, animal medicines, mineral medicines, and partial chemical and biological medicines. Since herbs are the most common herb, books containing herbs are called "herbal".
At present, the quality of the artificially planted and cultivated traditional Chinese medicinal materials is degraded due to various reasons, so that the medicinal effect of the traditional Chinese medicinal materials is unstable.
Licorice is a perennial herb of Glycyrrhiza genus of Leguminosae, has the American name of "Zhongzhiwang", is one of the most commonly used bulk medicinal materials, and is also an indispensable important medicinal material in many Chinese medicinal formulas. The medicinal materials of liquorice mainly comprise three types of liquorice, namely uralensis, glycyrrhiza glabra and glycyrrhiza distension, and the drug effects of the three types of liquorice have certain differences. As with many traditional Chinese medicinal materials, glycyrrhrizae radix has the problems of wild plant resource deficiency, quality degradation of artificially planted and cultivated Glycyrrhrizae radix, and unstable drug effect.
Protoplasts are cells from which the cell wall has been removed, a concept of bioengineering, and since there is no barrier to the cell wall, protoplasts are ideal materials for genetic transformation studies. In one aspect, protoplasts can be used to improve traits in plants; on the other hand, the protoplast can be utilized to breed mutant plants with utility value; in a third aspect, the unaffinity barrier of distant crosses can also be overcome by protoplast fusion to obtain valuable filial offspring. However, the prior art has less researches on licorice protoplast, and the preparation method of licorice protoplast described in the prior art document has generally lower yield and lower activity.
Disclosure of Invention
In order to solve at least one of the technical problems, a preparation method capable of efficiently preparing liquorice protoplasts and having higher preparation yield, and the prepared liquorice protoplasts have higher activity and better quality is developed.
In one aspect, the present application provides a method for preparing licorice protoplast, comprising the steps of:
s1, selecting primary cotyledons of Glycyrrhiza glabra, cleaning, sterilizing, shearing the cotyledons into cotyledon fragments, inoculating the cotyledons into a callus culture medium, and carrying out sterile induction culture on the callus for 24-32 days; the callus culture medium contains 4-6 mg/L apple pollen;
s2, picking out the callus of the cotyledon subjected to callus induction culture in the step S1 on a sterile operation table, putting the callus into enzymolysis liquid, and carrying out shaking enzymolysis for 6-6.5 h at a rotating speed of 50-60 rpm to obtain an enzymolysis product; the concentration ratio of each component of the enzymolysis liquid comprises: 25-30 mM PVP, 35-40 mM MES,0.6M mannitol, 1.8% cellulase R10,0.2% educt enzyme, 0.6% pectase, 0.1-0.2% BSA,10mM CaCl 2 The balance being purified water; KH is used for the enzymolysis liquid 2 PO 4 Adjusting the pH to 5.8;
s3, filtering the enzymolysis product obtained in the step S2 by using a 100-mesh cell filter screen, centrifuging the obtained filtrate, removing the supernatant, and cleaning the precipitate to obtain the licorice protoplast.
By adopting the technical scheme, the application designs a preparation method of the liquorice protoplast, which adopts liquorice callus as a raw material, prepares the liquorice protoplast by using specific enzymolysis liquid and enzymolysis technology, has higher preparation yield and higher activity of the prepared liquorice protoplast; the method adopts a specific induction culture mode to culture and proliferate the liquorice callus, and then prepares the liquorice protoplast, so that the preparation time is effectively shortened, and the preparation yield and the activity of the prepared liquorice protoplast are further improved; the licorice protoplast prepared by the application has higher quality and has the capability of culturing regenerated plants.
Optionally, in the step S1, the cotyledons of the primary Glycyrrhiza glabra for 10-12 days are selected, and the cotyledons are sheared into 0.5cm 2 The following cotyledon fragments.
Optionally, in the step S1, the callus culture medium adopts MS culture medium as basic culture medium, and 6-BA hormone, NAA hormone, sucrose and apple pollen are added; in the callus culture medium, the concentration of 6-BA is 0.5-0.8 mg/L, the concentration of NAA is 0.4-0.6 mg/L, and the concentration of sucrose is 20-24 g/L.
Optionally, in the step S1, the aseptic induction culture is performed by inoculating 6-8 g cotyledon fragments in every 100mL of culture medium.
Optionally, in the step S1, the fresh callus culture medium is added to the culture system every 10 days, and the addition amount of the fresh callus culture medium is controlled to be 30-40% of the initial culture medium dosage every 10 days.
Optionally, in the step S1, the aseptic induction culture is performed by first performing light-proof culture for 1 day, and then performing whole-course illumination culture at room temperature.
Optionally, in step S2, before the enzymolysis treatment, the enzymolysis solution needs to be filtered with a 0.22 μm filter membrane.
Optionally, in the step S3, the rotational speed of the centrifugal processing is controlled to 500-600 rpm.
On the other hand, the application also provides a preparation method of the liquorice protoplast, and application of the liquorice protoplast in the fields of improving liquorice varieties, cultivating mutant liquorice plants or cultivating hybrid liquorice plants.
Optionally, the regeneration culture method of the licorice protoplast comprises the following steps:
s4, inoculating the protoplast into a regeneration medium, and inoculating 1X 10 according to 100mL of the regeneration medium 6 ~5×10 7 Seed density of each protoplast, and culturing for 7 days in dark; the regeneration culture medium adopts KM8P culture medium as basic culture medium, and is added with 2,4-D hormone, 6-BA hormone, NAA hormone, glucose and BSA, wherein in the regeneration culture medium, the concentration of the 2,4-D hormone is 0.25mg/L, the concentration of the 6-BA is 1.5mg/L, the concentration of the NAA is 1.0mg/L, the concentration of the glucose is 6.5g/L, and the concentration of the BSA is 0.1%;
s5, transferring the protoplast obtained after the light-shielding culture in the step S4 to illumination culture, and continuing to culture for 7 days;
and S6, supplementing fresh regeneration medium which is equal to the regeneration medium used in the step S4 into the culture system after the 7-day illumination culture in the step S5, and continuing the illumination culture for 30-35 days to obtain the regenerated licorice callus.
By adopting the technical scheme, the application designs a culture method for culturing regeneration plants by adopting the liquorice protoplast prepared by the application, and provides a feasible technical means for improving and researching and developing liquorice varieties and obtaining high-quality liquorice plants.
In summary, the present invention includes at least one of the following beneficial technical effects:
1. the application designs a preparation method of liquorice protoplast, which adopts liquorice callus as a raw material, prepares the liquorice protoplast by using specific enzymolysis liquid and enzymolysis technology, and has higher preparation yield and higher activity of the prepared liquorice protoplast.
2. The method adopts a specific induction culture mode to culture and proliferate the liquorice callus, and then prepares the liquorice protoplast, so that the preparation time is effectively shortened, and the preparation yield and the activity of the prepared liquorice protoplast are further improved.
3. The licorice protoplast prepared by the application has higher quality and has the capability of culturing regenerated plants.
4. The application designs a culture method for culturing regeneration plants by adopting the liquorice protoplasts prepared by the application, and provides a feasible technical means for improving and researching and developing liquorice varieties and obtaining high-quality liquorice plants.
Description of the embodiments
The present application is described in further detail below with reference to examples.
The application designs a preparation method of liquorice protoplast, which comprises the following steps:
s1, selecting primary cotyledons of Glycyrrhiza glabra, cleaning, sterilizing, shearing the cotyledons into cotyledon fragments, inoculating the cotyledons into a callus culture medium, and carrying out sterile induction culture on the callus for 24-32 days; the callus culture medium contains 4-6 mg/L apple pollen;
s2, picking out the callus of the cotyledon subjected to callus induction culture in the step S1 on a sterile operation table, putting the callus into enzymolysis liquid, and carrying out shaking enzymolysis for 6-6.5 h at a rotating speed of 50-60 rpm to obtain an enzymolysis product; the concentration ratio of each component of the enzymolysis liquid comprises: 25PVP of 30mM, MES of 35 to 40mM, mannitol of 0.6M, cellulase R10 of 1.8%, educt enzyme of 0.2%, pectase of 0.6%, BSA of 0.1 to 0.2%, caCl of 10mM 2 The balance being purified water; KH is used for the enzymolysis liquid 2 PO 4 Adjusting the pH to 5.8;
s3, filtering the enzymolysis product obtained in the step S2 by using a 100-mesh cell filter screen, centrifuging the obtained filtrate, removing the supernatant, and cleaning the precipitate to obtain the licorice protoplast.
Compared with the prior art, the method adopts the callus as the preparation material, designs the specific callus culture medium and the culture method, designs the specific enzymolysis liquid and the enzymolysis method, and greatly improves the yield and activity of the protoplast.
The following are examples of the present application, all of which are commercially available.
The cotyledons used in the examples of the present application were obtained in the following manner:
1) Cleaning the Glycyrrhiza glabra seeds with water, then cleaning with 95% ethanol, sterilizing, and planting into shallow soil;
2) After 5 days of planting, the Glycyrrhiza glabra seedlings and cotyledons grow out about 8 days.
The cotyledons used in the examples herein are selected from the group consisting of primary cotyledons after emergence of seedlings.
The protoplast cleaning solution used in the embodiment of the application comprises the following components in concentration ratio: 8% mannitol, 4mM CaCl 2 60mM MgSO 4 KH 30mM 2 PO 4 The balance being purified water.
Example 1
The callus culture medium of this example was configured as follows:
adopting MS culture medium as basic culture medium, adding 6-BA hormone, NAA hormone, sucrose and apple pollen; wherein, the concentration of 6-BA is 0.5mg/L, the concentration of NAA is 0.4mg/L, the concentration of sucrose is 20/L, and the concentration of apple pollen is 6mg/L.
The enzymolysis liquid of this embodiment is prepared as follows:
the concentration ratio of each component comprises: 25mM PVP,35mM0.6M mannitol, 1.8% cellulase R10,0.2% educt enzyme, 0.6% pectase, 0.2% BSA,10mM CaCl 2 The balance being purified water;
KH for enzymolysis liquid 2 PO 4 The pH was adjusted to 5.8 and then sterilized with a 0.22 μm filter.
The preparation method of the licorice protoplast in the embodiment comprises the following steps:
s1, selecting primary cotyledons of Glycyrrhiza glabra, cleaning, sterilizing and cleaning with 95% ethanol, shearing into cotyledon fragments of 1X 1cm, inoculating about 10g of cotyledon fragments into 100mL of callus culture medium, and performing sterile induction culture of callus for 24 days; culturing at room temperature under illumination in a sealed condition.
S2, picking out the callus of the cotyledon subjected to callus induction culture in the step S1 on a sterile operation table, and placing the callus into a triangular flask filled with 100mL of enzymolysis liquid; sealing the triangular flask, placing the triangular flask on a shaking table, and oscillating and hydrolyzing for 6 hours at a speed of 50rpm to obtain an enzymolysis product.
S3, filtering the enzymolysis product obtained in the step S2 by using a 100-mesh cell filter screen, centrifuging the obtained filtrate at 600rpm for 4min, removing the supernatant and collecting the precipitate; washing the collected precipitate with protoplast washing liquid for 1 time to obtain Glycyrrhrizae radix protoplast.
The single cleaning procedure of the step S3 is as follows: soaking and cleaning the precipitate in protoplast cleaning solution for 1min, centrifuging at 500rpm for 5min, removing supernatant, and collecting precipitate.
Example 2
The difference between this example and example 1 is that the ratio of the callus culture medium in this example is different.
The callus culture medium of this example was configured as follows:
adopting MS culture medium as basic culture medium, adding 6-BA hormone, NAA hormone, sucrose and apple pollen; wherein, the concentration of 6-BA is 0.8mg/L, the concentration of NAA is 0.6mg/L, the concentration of sucrose is 24g/L, and the concentration of apple pollen is 4mg/L.
Example 3
The difference between this example and example 1 is that the ratio of the callus culture medium in this example is different.
The callus culture medium of this example was configured as follows:
adopting MS culture medium as basic culture medium, adding 6-BA hormone, NAA hormone, sucrose and apple pollen; wherein, the concentration of 6-BA is 0.6mg/L, the concentration of NAA is 0.5mg/L, the concentration of sucrose is 22g/L, and the concentration of apple pollen is 5.2mg/L.
Example 4
The present embodiment differs from embodiment 3 in the specific process of step S1 in this embodiment.
In step S1 of this example, the cotyledons of primary Glycyrrhiza glabra planted for 12 days were selected, and the cotyledons were cut into 0.5cm pieces 2 Is a cotyledon fragment of (c).
Example 5
The present embodiment differs from embodiment 4 in the specific process of step S1 in this embodiment.
In step S1 of the present example, the culture was carried out at room temperature under light-shielding conditions for 1 day in a sealed manner, and then at room temperature under light irradiation for 31 days in a sealed manner.
Example 6
The present embodiment differs from embodiment 5 in the specific process of step S1 in this embodiment.
In step S1 of this example, 6g of cotyledon pieces were inoculated into 100mL of the callus culture medium, and 30mL of fresh callus culture medium was supplemented every 10 days to the culture system.
Example 7
The present embodiment differs from embodiment 5 in the specific process of step S1 in this embodiment.
In step S1 of this example, 8g of cotyledon pieces were inoculated into 100mL of the callus culture medium, and 40mL of fresh callus culture medium was supplemented every 10 days to the culture system.
Example 8
The difference between this example and example 7 is that the ratio of the enzymatic hydrolysate in this example is different.
The enzymolysis liquid of this embodiment is prepared as follows:
concentration proportioning bag for each componentThe method comprises the following steps: 30mM PVP,40mM MES,0.6M mannitol, 1.8% cellulase R10,0.2% educt enzyme, 0.6% pectase, 0.1% BSA,10mM CaCl 2 The balance being purified water.
Example 9
The difference between this example and example 7 is that the ratio of the enzymatic hydrolysate in this example is different.
The enzymolysis liquid of this embodiment is prepared as follows:
the concentration ratio of each component comprises: 28mM PVP,36mM MES,0.6M mannitol, 1.8% cellulase R10,0.2% educt enzyme, 0.6% pectase, 0.12% BSA,10mM CaCl 2 The balance being purified water.
Example 10
The present embodiment differs from embodiment 9 in the specific process of step S2 in this embodiment.
In step S2 of the embodiment, the enzymolysis is carried out for 6.5 hours at a rotation speed of 60 rpm.
Example 11
The present embodiment differs from embodiment 9 in the specific process of step S2 in this embodiment.
In step S2 of the embodiment, the enzymolysis is carried out for 6 hours at a rotation speed of 60 rpm.
Example 12
The present embodiment differs from embodiment 10 in the specific process of step S3 in this embodiment.
In step S3 of this example, the obtained filtrate was subjected to centrifugation at 400rpm for 6min.
Example 13
The present embodiment differs from embodiment 10 in the specific process of step S3 in this embodiment.
In step S3 of this example, the obtained filtrate was subjected to centrifugation at 650rpm for 4min.
Example 14
The present embodiment differs from embodiment 10 in the specific process of step S3 in this embodiment.
In step S3 of this example, the obtained filtrate was subjected to centrifugation at 350rpm for 8min.
Example 15
The present embodiment differs from embodiment 10 in the specific process of step S3 in this embodiment.
In step S3 of this example, the obtained filtrate was subjected to centrifugation at 460rpm for 8min.
Comparative example 1
The enzymatic hydrolysate of this comparative example was prepared as follows:
the concentration ratio of each component comprises: KH of 0.7M 2 PO 4 Mannitol, 0.7M, cellulase R10, 1.5%, pectase, 10mM CaCl 2 KNO of 100mg/L 3 250mg/L MgSO 4 0.1mM KI,0.1mM CuSO 4 The balance being purified water, pH 5.6.
The preparation method of the licorice protoplast of the comparative example comprises the following steps:
s1, taking cotyledon and hypocotyl of a sterile seedling of the Glycyrrhiza glabra growing for 7d, accurately weighing 10g of the cotyledon and hypocotyl, placing into a sterile triangular flask, adding 150mL of the sterile enzymolysis solution, and sealing the triangular flask mouth for standby.
S2, placing the triangular flask in the step S1 on a shaking table, and carrying out shaking enzymolysis for 14h at a rotating speed of 40rpm to obtain an enzymolysis product.
S3, filtering the enzymolysis product obtained in the step S2 through a 60-mesh cell screen, centrifuging the filtrate at 500rpm for 5min, removing the supernatant, and collecting the precipitate.
S4, washing the precipitate obtained in the step S3 for 1 time to obtain the protoplast of the comparative example.
Comparative example 2
The callus culture medium of this comparative example was prepared as follows:
adopting MS culture medium as basic culture medium, adding 6-BA hormone, NAA hormone and sucrose; wherein, the concentration of 6-BA is 1.5mg/L, the concentration of NAA is 0.5mg/L, and the concentration of sucrose is 30g/L.
The present comparative example differs from example 11 in the process of step S1 of the present comparative example.
In step S1 of this comparative example, the incubation time was 68 days. The calli of this comparative example were cultured once every 14 days, and calli were selected for 5 times of subculture.
Protoplasts obtained in examples 1 to 15 and comparative examples 1 to 2 of the present application were identified.
The yield of protoplasts was measured by a hemocytometer assay and the yield of protoplasts obtained per 1g of isolated licorice tissue was calculated.
The activity of protoplasts was measured by FDA method.
The specific detection results are shown in the following table 1.
Table 1 test results of examples 1 to 15 and comparative examples 1 to 2
| Preparation of protoplast yield (1X 10) from 1g of material 5 Personal computer | Protoplast Activity (%) | |
| Example 1 | 1.56 | 74.6 |
| Example 2 | 1.54 | 75.1 |
| Example 3 | 1.59 | 75.5 |
| Example 4 | 1.68 | 75.2 |
| Example 5 | 1.77 | 76.2 |
| Example 6 | 1.99 | 77.7 |
| Example 7 | 1.97 | 78.0 |
| Example 8 | 1.96 | 77.8 |
| Example 9 | 2.04 | 78.4 |
| Example 10 | 2.14 | 78.2 |
| Example 11 | 2.12 | 78.2 |
| Example 12 | 2.08 | 78.5 |
| Example 13 | 2.16 | 76.8 |
| Example 14 | 2.01 | 78.6 |
| Example 15 | 2.12 | 78.5 |
| Comparative example 1 | 0.48 | 71.2 |
| Comparative example 2 | 0.36 | 69.4 |
As can be seen from the data in Table 1, the preparation method of the present application can effectively obtain licorice protoplast, and the quality of the prepared licorice protoplast is higher than that of comparative examples 1 and 2; the preparation method of the application has the advantages that the yield of the protoplast is higher, the apple pollen is adopted to induce and culture the callus, the culture time can be greatly shortened, the yield is also greatly improved, and compared with comparative example 1 and comparative example 2, the yield is improved by more than 10 times. Therefore, the preparation method of the application has the advantages that the yield of the prepared licorice protoplast is higher, the activity of the prepared licorice protoplast is higher, and the quality is better.
As can be seen from the data in table 1, in examples 1 to 4 of the present application, the yield and activity of example 3 are significantly higher than those of examples 1 and 2, and are significantly higher than those of comparative example 2; the yield of example 4 is significantly higher than that of example 3. Therefore, the specific callus culture medium added with apple pollen can greatly improve the yield and activity of protoplasts, optimize the proportion of the culture medium and further improve the yield and activity after adopting a specific material treatment mode.
By comparing the data in Table 1 with examples 5-7 and example 4, the yield and activity of protoplasts are significantly improved. Therefore, the method of light-shielding culture and light-illumination culture is adopted in the preculture, and fresh culture medium is timely supplemented, so that the culture rate of the callus can be greatly improved, and the quality of the obtained callus is also greatly improved.
Through the data of table 1, the comparison between examples 8-9 and examples 6-7 of the present application can show that after the proportion of the enzymolysis liquid is optimized, the yield and activity of protoplast can be further improved.
By comparing the data in Table 1 with examples 10-11, it can be seen that the specific enzymolysis liquid is adopted, enzymolysis is performed at a speed of 60rpm, the enzymolysis effect is optimal, the yield can be greatly improved, and the activity of protoplast can be kept at a better level; during purification treatment, the centrifugal speed has a great influence on the yield and activity of the protoplast, and the centrifugal effect is optimal when the centrifugal speed is about 450 rpm.
The licorice protoplast prepared by the application can be used in the fields of improving licorice varieties, cultivating mutant licorice plants or cultivating hybrid licorice plants and the like. In the application in the above fields, whether or not licorice protoplasts can be efficiently regenerated is the most critical issue.
The following are examples 16 to 18 of the present application, in which the licorice protoplast prepared in example 1 of the present application was used as a material for regeneration culture.
The regeneration media used in examples 16 to 18 of the present application were prepared as follows:
adopting KM8P culture medium as basic culture medium, adding 2,4-D hormone, 6-BA hormone, NAA hormone, glucose and BSA; wherein, the concentration of 2,4-D hormone is 0.25mg/L, the concentration of 6-BA is 1.5mg/L, the concentration of NAA is 1.0mg/L, the concentration of glucose is 6.5g/L, and the concentration of BSA is 0.1%.
Example 16
The regeneration culture method of the licorice protoplast of the embodiment comprises the following steps:
s4, 1×10 6 Inoculating each protoplast into 100mL regeneration medium, and culturing for 7 days in dark;
s5, transferring the protoplast obtained after the light-shielding culture in the step S4 to illumination culture, and continuing to culture for 7 days;
s6, supplementing fresh regeneration medium which is equal to the regeneration medium used in the step S4 into the culture system after the 7-day illumination culture in the step S5, and continuing illumination culture for 30 days to obtain regenerated licorice callus.
Example 17
The regeneration culture method of the licorice protoplast of the embodiment comprises the following steps:
s4, 1×10 7 Inoculating each protoplast into 100mL regeneration medium, and culturing for 7 days in dark;
s5, transferring the protoplast obtained after the light-shielding culture in the step S4 to illumination culture, and continuing to culture for 7 days;
s6, supplementing fresh regeneration medium which is equal to the regeneration medium used in the step S4 into the culture system after the 7-day illumination culture in the step S5, and continuing the illumination culture for 35 days to obtain the regenerated licorice callus.
Example 18
The regeneration culture method of the licorice protoplast of the embodiment comprises the following steps:
s4, 5×10 7 Inoculating each protoplast into 100mL regeneration medium, and culturing for 7 days in dark;
s5, transferring the protoplast obtained after the light-shielding culture in the step S4 to illumination culture, and continuing to culture for 7 days;
s6, supplementing fresh regeneration medium which is equal to the regeneration medium used in the step S4 into the culture system after the 7-day illumination culture in the step S5, and continuing the illumination culture for 35 days to obtain the regenerated licorice callus.
The cell tissues obtained by culturing in examples 16 to 18 were examined, the number of callus cells was measured, and the division rate was calculated according to the formula of division rate= (number of callus cells/number of initial protoplasts) ×100%. Specific detection results are shown in table 2 below.
TABLE 2 protoplast division rates for examples 16-18
| Protoplast division rate (%) | |
| Example 16 | 62.6 |
| Example 17 | 66.8 |
| Example 18 | 66.2 |
As can be seen from the data in Table 2, the protoplast prepared in example 1 of the present application has good regeneration and division performance, and can be regenerated in a culture medium to obtain licorice callus cells.
It can be seen from the data in table 2 that the regeneration medium and the regeneration culture method designed in the application can obtain higher division rate of regeneration culture of the licorice protoplast.
The foregoing are all preferred embodiments of the present application, and are not intended to limit the scope of the present application in any way, therefore: all equivalent changes in structure, shape and principle of this application should be covered in the protection scope of this application.
Claims (10)
1. A method for preparing licorice protoplast, which is characterized by comprising the following steps:
s1, selecting primary cotyledons of Glycyrrhiza glabra, cleaning, sterilizing, shearing the cotyledons into cotyledon fragments, inoculating the cotyledons into a callus culture medium, and carrying out sterile induction culture on the callus for 24-32 days; the callus culture medium contains 4-6 mg/L apple pollen;
s2, in the absence ofPicking out the callus of the cotyledon subjected to callus induction culture in the step S1 on a fungus operation table, putting the callus into an enzymolysis liquid, and carrying out shaking enzymolysis for 6-6.5 h at a rotating speed of 50-60 rpm to obtain an enzymolysis product; the concentration ratio of each component of the enzymolysis liquid comprises: 25-30 mM PVP, 35-40 mM MES,0.6M mannitol, 1.8% cellulase R10,0.2% educt enzyme, 0.6% pectase, 0.1-0.2% BSA,10mM CaCl 2 The balance being purified water; KH is used for the enzymolysis liquid 2 PO 4 Adjusting the pH to 5.8;
s3, filtering the enzymolysis product obtained in the step S2 by using a 100-mesh cell filter screen, centrifuging the obtained filtrate, removing the supernatant, and cleaning the precipitate to obtain the licorice protoplast.
2. The method according to claim 1, wherein in the step S1, the cotyledons of primary Glycyrrhiza glabra are selected for 10-12 days, and the cotyledons are sheared into 0.5cm 2 The following cotyledon fragments.
3. The method according to claim 1, wherein in the step S1, the callus culture medium is prepared by adding 6-BA hormone, NAA hormone, sucrose and apple pollen to MS culture medium as a basal medium; in the callus culture medium, the concentration of 6-BA is 0.5-0.8 mg/L, the concentration of NAA is 0.4-0.6 mg/L, and the concentration of sucrose is 20-24 g/L.
4. The method according to claim 1, wherein in the step S1, the aseptic induction culture is performed by inoculating 6-8 g cotyledon pieces per 100mL of the culture medium.
5. The method according to claim 1, wherein in step S1, the fresh callus culture medium is added to the culture system every 10 days, and the addition amount of each fresh callus culture medium is controlled to be 30-40% of the initial culture medium.
6. The method according to claim 1, wherein in step S1, the aseptic induction culture is performed by culturing in the dark for 1 day and then culturing in the whole process at room temperature.
7. The method according to claim 1, wherein the enzymatic hydrolysis is performed in step S2 by filtering the enzymatic hydrolysate with a 0.22 μm filter.
8. The method according to claim 1, wherein in the step S3, the rotational speed of the centrifugation is controlled to 500 to 600rpm.
9. A method for preparing a licorice protoplast according to any one of claims 1-8, for use in the field of improving a licorice variety, cultivating a mutant licorice plant, or cultivating a hybrid licorice plant.
10. The use of the method for preparing licorice protoplasts as claimed in claim 9, comprising the step of regenerating and culturing licorice protoplasts, wherein the method comprises the steps of:
s4, inoculating the protoplast into a regeneration medium, and inoculating 1X 10 according to 100mL of the regeneration medium 6 ~5×10 7 Seed density of each protoplast, and culturing for 7 days in dark; the regeneration culture medium adopts KM8P culture medium as basic culture medium, and is added with 2,4-D hormone, 6-BA hormone, NAA hormone, glucose and BSA, wherein in the regeneration culture medium, the concentration of the 2,4-D hormone is 0.25mg/L, the concentration of the 6-BA is 1.5mg/L, the concentration of the NAA is 1.0mg/L, the concentration of the glucose is 6.5g/L, and the concentration of the BSA is 0.1%;
s5, transferring the protoplast obtained after the light-shielding culture in the step S4 to illumination culture, and continuing to culture for 7 days;
and S6, supplementing fresh regeneration medium which is equal to the regeneration medium used in the step S4 into the culture system after the 7-day illumination culture in the step S5, and continuing the illumination culture for 30-35 days to obtain the regenerated licorice callus.
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