CN115720851A - Sugarcane somatic embryo and induction method thereof - Google Patents
Sugarcane somatic embryo and induction method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of somatic embryo induction. The invention provides a sugarcane somatic embryo and an induction method thereof, comprising the following steps: 1) Culturing the sugarcane cells in a DEM culture medium for 11-17 days; (2) Transferring the sugarcane cells to a DEM culture medium containing G418 for culturing for 8-12 days, then subculturing for 8-12 days in the DEM culture medium containing G418, and screening the survival sugarcane cells; (3) Transferring the survival sugarcane cells to NB culture medium containing G418 for screening and culturing for 8-12 days, and subculturing to NB culture medium containing G418 for screening for 8-12 days to obtain sugarcane somatic embryos. According to the invention, through exploration and optimization of the induction conditions of the direct somatic embryos of the sugarcane, the adaptability of different sugarcane varieties to a culture medium is found to be different, the culture conditions of the direct somatic embryos of the sugarcane are optimized, and transgenic sugarcane plants can be obtained in a shorter period.
Description
Technical Field
The invention relates to the technical field of induction of somatic embryos, in particular to a sugarcane somatic embryo and an induction method thereof.
Background
Sugarcane is the most important sugar crop in China, and the genetic transformation of the sugarcane is usually realized through indirect somatic embryogenesis, particularly through a procedure of dedifferentiation of immature leaves at the top of the sugarcane, callus and redifferentiation. The genetic transformation scheme using sugarcane embryogenic callus as the transformation receptor material needs a longer plant tissue culture period, can cause the increase of somaclonal variation after long-term culture, is not beneficial to screening after transformation, and the transformation efficiency of the method is very low, so the improvement of sugarcane genetic transformation should focus on optimizing a tissue culture system or searching for a more efficient sugarcane genetic transformation receptor material, and the main aim is to improve the transformation efficiency and the screening efficiency and further shorten the sugarcane transgenic period. At present, sugarcane embryogenic callus is still mainly used as a transformation receptor in domestic sugarcane transgenosis, so that the domestic sugarcane transgenosis is slow in progress, a new scheme based on direct somatic embryogenesis needs to be developed and optimized for local varieties, the domestic sugarcane cultivation process is accelerated, and technical support is provided for research on sugarcane and sugarcane pathogenic bacteria functional genes.
Disclosure of Invention
The invention aims to provide a sugarcane somatic embryo and an induction method thereof, and provides technical support for research of sugarcane and sugarcane pathogenic bacteria function genes.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for inducing sugarcane somatic embryos, which comprises the following steps:
(1) Culturing the sugarcane cells in a DEM culture medium for 11-17 days;
(2) Transferring the sugarcane cells to a DEM culture medium containing G418 for culturing for 8-12 days, then subculturing for 8-12 days in the DEM culture medium containing G418, and screening the survival sugarcane cells;
(3) Transferring the survival sugarcane cells to NB culture medium containing G418 for screening and culturing for 8-12 days, and subculturing to NB culture medium containing G418 for screening for 8-12 days to obtain sugarcane somatic embryos.
Preferably, the temperature of the cultivation in the step (1) is 24 to 28 ℃, and the light intensity is 25 to 35 [ mu ] mol m -2 s -1 The illumination time is 10-14 h/d.
Preferably, the concentration of G418 in step (2) and step (3) is independently 25 to 35mg/L.
Preferably, the sugarcane is ROC22, medium sugarcane No.1 or CP88-1762; the sugarcane cells in the step (1) are cells in immature leaves at the top ends of the sugarcanes, and the sugarcane cells in the step (1) are transgenic sugarcane cells.
Preferably, the preparation method of the transgenic sugarcane cell comprises the following steps:
(1) Combining a target gene with a pBWA (V) BU vector to obtain a HIGS interference vector;
(2) Transforming the HIGS interference vector into competent escherichia coli, and extracting a plasmid after induction expression;
(3) Transforming the plasmid into sugarcane cells to obtain transgenic sugarcane somatic cells;
the nucleotide sequence of the target gene is shown as SEQ ID NO.1, and the sequence of the HIGS interference vector is shown as SEQ ID NO. 2.
The invention also provides a sugarcane somatic embryo obtained by the induction method.
The invention also provides a using method of the sugarcane somatic embryo, which comprises the following steps:
(1) Transferring the sugarcane somatic embryos to a SG culture medium containing G418 for culture to obtain sugarcane seedlings;
(2) And (4) hardening the sugarcane seedlings, and transplanting the hardened sugarcane seedlings into a culture medium to obtain the sugarcane seedlings.
Preferably, the concentration of G418 in the SG medium in step (1) is 15 to 25mg/L.
Preferably, the root length of the sugarcane seedlings in the step (1) is 2-3 cm.
Preferably, the culture medium in the step (2) comprises the following raw materials in parts by volume: 8-10 parts of vermiculite and 8-10 parts of nutrient soil.
The invention provides a sugarcane somatic embryo and an induction method thereof, comprising the following steps: 1) Culturing the sugarcane cells in a DEM culture medium for 11-17 days; (2) Transferring the sugarcane cells to a DEM culture medium containing G418 for culturing for 8-12 days, then subculturing for 8-12 days in the DEM culture medium containing G418, and screening the survival sugarcane cells; (3) Transferring the survival sugarcane cells to NB culture medium containing G418 for screening and culturing for 8-12 days, and subculturing to NB culture medium containing G418 for screening for 8-12 days to obtain sugarcane somatic embryos. According to the invention, through exploration and optimization of the induction conditions of the direct somatic embryos of the sugarcane, the adaptability of different sugarcane varieties to a culture medium is found to be different, the culture conditions of the direct somatic embryos of the sugarcane are optimized, and transgenic sugarcane plants can be obtained in a shorter period.
Drawings
FIG. 1 shows the growth state of different varieties cultured on DEM medium for seven days;
FIG. 2 shows the growth state of different varieties cultured on DEM medium for twelve days;
FIG. 3 shows the acceptor material at different incubation times;
FIG. 4 shows the effect of rooting, screening, culturing and hardening seedlings;
FIG. 5 shows the growth of different regions of sugarcane No.1 on DEM medium.
Detailed Description
The invention provides a method for inducing sugarcane somatic embryos, which comprises the following steps:
(1) Culturing the sugarcane cells in a DEM culture medium for 11-17 days;
(2) Transferring the sugarcane cells to a DEM culture medium containing G418 for culturing for 8-12 days, then subculturing for 8-12 days in the DEM culture medium containing G418, and screening the survival sugarcane cells;
(3) Transferring the survival sugarcane cells to NB culture medium containing G418 for screening and culturing for 8-12 days, and subculturing to NB culture medium containing G418 for screening for 8-12 days to obtain sugarcane somatic embryos.
In the present invention, the time period of the culture in step (1) is preferably 14 days.
In the present invention, the time for the culture in step (2) is preferably 10 days.
In the present invention, the time for the culture in step (3) is preferably 10 days.
In the present invention, the temperature for the culture in step (1) is preferably 24 to 28 ℃, and more preferably 26 ℃.
In the present invention, the light intensity for the cultivation in the step (1) is preferably 25 to 35. Mu. Mol m -2 s -1 More preferably 30. Mu. Mol m -2 s -1 。
In the present invention, the light irradiation time for the culture in the step (1) is preferably 10 to 14h/d, and more preferably 12h/d.
In the present invention, the concentration of G418 in step (2) and step (3) is preferably 25 to 35mg/L, and more preferably 30mg/L, independently.
In the present invention, the sugarcane is preferably ROC22, sugarcane No.1 or CP88-1762.
In the present invention, the sugarcane cell in step (1) is preferably a cell in an immature leaf at the top of sugarcane.
In the present invention, the sugar cane cell in step (1) is preferably a transgenic sugar cane cell.
In the present invention, the method for producing the transgenic sugarcane cell is preferably:
(1) Combining a target gene with a pBWA (V) BU vector to obtain a HIGS interference vector;
(2) Transforming the HIGS interference vector into competent escherichia coli, and extracting a plasmid after induction expression;
(3) Transforming the plasmid into sugarcane cells to obtain transgenic sugarcane somatic cells;
the nucleotide sequence of the target gene is shown as SEQ ID NO.1, and the sequence of the HIGS interference vector is shown as SEQ ID NO. 2.
In the present invention, the pBWA (V) BU vector in step (1) is preferably cleaved with the restriction endonuclease Eco 31I enzyme.
In the present invention, the binding in step (1) is preferably performed by ligation using T4 DNA ligase.
In the present invention, the Escherichia coli in the step (2) is preferably Escherichia coli Top10.
In the present invention, the extraction of the plasmid in step (2) is preferably carried out after the growth of a single colony of E.coli.
In the present invention, the method for transformation described in step (3) is preferably a gene gun-mediated transformation method.
The invention also provides a sugarcane somatic embryo obtained by the induction method.
The invention also provides a using method of the sugarcane somatic embryo, which comprises the following steps:
(1) Transferring the sugarcane somatic embryos to a SG culture medium containing G418 for culture to obtain sugarcane seedlings;
(2) And (4) hardening the sugarcane seedlings, and transplanting the hardened sugarcane seedlings into a culture medium to obtain the sugarcane seedlings.
In the present invention, the concentration of G418 in the SG medium in step (1) is preferably 15 to 25mg/L, and more preferably 20mg/L.
In the present invention, the root length of the sugarcane seedling in step (1) is preferably 2 to 3cm, and more preferably 2.5cm.
In the present invention, the culture medium in step (2) preferably comprises the following raw materials in parts by volume: 8-10 parts of vermiculite and 8-10 parts of nutrient soil, and further preferably 9 parts of vermiculite and 9 parts of nutrient soil.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The test plants: sugarcane No.1, relatively tip rot resistant sugarcane cultivar ROC22 and cultivars CP88-1762 in the sugarcane cultivars susceptible to tip rot are provided by the subject group and are all planted in the specimen garden of Guangxi university.
Example 1
(1) Combining a target gene with a nucleotide sequence of SEQ ID NO.1 with a pBWA (V) BU vector to obtain a HIGS interference vector shown in SEQ ID NO. 2;
(2) Transforming the HIGS interference vector into competent escherichia coli Top10, and extracting a plasmid after induction expression;
(3) Transforming the plasmid into sugarcane cells to obtain transgenic sugarcane somatic cells;
(4) Culturing the transgenic sugarcane somatic cells in DEM culture medium for 11 days (temperature 28 ℃, light intensity 35 mu mol m) -2 s -1 The illumination time is 10 h/d);
(5) Transferring the sugarcane cells into DEM culture medium containing 25mg/L of G418 for culturing for 8 days, then subculturing for 12 days in DEM culture medium containing 35mg/L of G418, and screening the survival sugarcane cells;
(6) Transferring the survival sugarcane cells to NB culture medium containing 25mg/L G418 for screening culture for 12 days, and subculturing to NB culture medium containing 35mg/L G418 for screening for 8 days to obtain sugarcane somatic embryos;
(7) Transferring the sugarcane somatic embryos to SG culture medium containing 15mg/L G418 for culture to obtain sugarcane seedlings with root length of 2 cm;
(8) When the root of the sugarcane seedling grows to 2cm, unscrewing a bottle cap of a culture bottle for culture for 2d, then half-opening the bottle cap for culture for 2d, finally removing the bottle cap, moving the bottle cap to the outside of a culture chamber for hardening the seedling for 2d, then taking out the seedling, washing the attached culture medium with running water, transplanting the seedling to a small flowerpot (the volume ratio of vermiculite to nutrient soil is 8, mixing and then carrying out high-pressure sterilization), spraying MS inorganic salt solution diluted by 10 times at regular time, spraying a proper amount of eight percent of carbendazim in time if fungal pollution occurs, covering an organic glass cover for moisture preservation, and removing the organic glass cover after one week to allow the sugarcane seedling to continue to grow under the natural illumination condition.
Example 2
(1) Combining a target gene with a nucleotide sequence of SEQ ID NO.1 with a pBWA (V) BU vector to obtain a HIGS interference vector shown in SEQ ID NO. 2;
(2) Transforming the HIGS interference vector into competent escherichia coli Top10, and extracting a plasmid after induction expression;
(3) Transforming the plasmid into sugarcane cells to obtain transgenic sugarcane somatic cells;
(4) Culturing the transgenic sugarcane somatic cells in DEM medium for 17 days (temperature 24 ℃, illumination intensity 25 mu mol m) -2 s -1 The illumination time is 14 h/d);
(5) Transferring the sugarcane cells into DEM culture medium containing 35mg/L of G418 for culturing for 12 days, then subculturing for 8 days in DEM culture medium containing 25mg/L of G418, and screening the survival sugarcane cells;
(6) Transferring the survival sugarcane cells to NB culture medium containing 35mg/L G418 for screening and culturing for 8 days, and subculturing to NB culture medium containing 25mg/L G418 for screening for 12 days to obtain sugarcane somatic embryos;
(7) Transferring the sugarcane somatic embryos to an SG culture medium containing 25mg/L G418 for culture to obtain sugarcane seedlings with root lengths of 3 cm;
(8) When the root of the sugarcane seedling grows to 3cm, unscrewing a bottle cap of a culture bottle for culture for 2d, then half-opening the bottle cap for culture for 2d, finally removing the bottle cap, moving the bottle cap to the outside of a culture chamber for hardening the seedling for 2d, then taking out the seedling, washing the attached culture medium with running water, transplanting the seedling to a small flowerpot (the volume ratio of vermiculite to nutrient soil is 10, mixing and then carrying out high-pressure sterilization), spraying MS inorganic salt solution diluted by 10 times at regular time, spraying a proper amount of eight percent of carbendazim in time if fungal pollution occurs, covering an organic glass cover for moisture preservation, and removing the organic glass cover after one week to allow the sugarcane seedling to continue to grow under the natural illumination condition.
Example 3
(1) Combining a target gene with a nucleotide sequence of SEQ ID NO.1 with a pBWA (V) BU vector to obtain a HIGS interference vector shown in SEQ ID NO. 2;
(2) Transforming the HIGS interference vector into competent escherichia coli Top10, and extracting a plasmid after induction expression;
(3) Transforming the plasmid into sugarcane cells to obtain transgenic sugarcane somatic cells;
(4) Culturing the transgenic sugarcane somatic cells in DEM medium for 14 days (temperature 26 ℃, illumination intensity 30 mu mol m) -2 s -1 The illumination time is 12 h/d);
(5) Transferring the sugarcane cells to DEM culture medium containing 30mg/L of G418 for culturing for 10 days, then carrying out subculture on the DEM culture medium containing 30mg/L of G418 for 10 days, and screening the survival sugarcane cells;
(6) Transferring the survival sugarcane cells to NB medium containing 30mg/L of G418 for screening and culturing for 10 days, and subculturing the survival sugarcane cells to NB medium containing 30mg/L of G418 for screening for 10 days to obtain sugarcane somatic embryos;
(7) Transferring the sugarcane somatic embryos to SG culture medium containing 20mg/L G418 for culture to obtain sugarcane seedlings with root length of 2.5 cm;
(8) When the root of the sugarcane seedling grows to 2.5cm, unscrewing a bottle cap of a culture bottle for culture for 2d, then half-opening the bottle cap for culture for 2d, finally removing the bottle cap, moving the bottle cap to the outside of the culture chamber for hardening the seedling for 2d, then taking out the seedling, washing the attached culture medium with running water, transplanting the seedling into a small flowerpot (the volume ratio of vermiculite to nutrient soil is 1:1, mixing and then carrying out high-pressure sterilization), spraying MS inorganic salt solution diluted by 10 times at regular time, spraying a proper amount of one eighth of carbendazim in time if fungal pollution occurs, covering an organic glass cover for moisture preservation, and removing the organic glass cover after one week to enable the organic glass to continue to grow under the natural illumination condition.
Test example 1
The top central lobe cut sections of three varieties including ROC22, chinese sugarcane No.1 and CP88-1762 are taken to be simultaneously subjected to direct somatic embryo induction on a DEM culture medium, the change of the section of the sugarcane of different varieties contacted with the culture medium can be obviously observed in the seventh day of culture, as shown in figure 1, the bottom of the material of the ROC22 and the Chinese sugarcane No.1 is browned, the nutrition absorption of the material is influenced, the growth state is poor, and the growth state of CP88-1762 is good. On the twelfth day of culture, as shown in FIG. 2, ROC22 and sugarcane No.1 could not induce somatic embryos as expected, while CP88-176 could induce better-conditioned embryos for genetic transformation at expected times, indicating that different sugarcane varieties require different culture media for direct induction of somatic embryos.
Test example 2
Taking the sugarcane No.1 to perform direct somatic embryo induction generation culture on a DEM culture medium to obtain materials which are cultured for 11 days, 14 days and 17 days (figure 3), and it can be seen that the somatic embryos directly induced from the 11 th day are fewer in number but in a good state, and the somatic embryos induced from the 14 th day and the 17 th day are significantly increased in number but in a poor yellow state.
Test example 3
The seedlings screened in example 3 were transferred to SG medium containing 20mg/L G418 for rooting culture and subcultured once after ten days. During the rooting screening culture, it can be seen that some seedlings are dead after necrosis of the basal part and dead after rooting, and some seedlings are high in normal rooting plants (FIG. 4A). After two cycles of rooting screening, the material is transferred to a rooting medium without screening pressure for culture, subculture is carried out once every 15 days, when resistant seedlings grow roots of about 2.5cm (figure 4B), the resistant seedlings are transplanted into nutrient soil to be cultured in an artificial climate incubator for 4 weeks (figure 4C), and then the resistant seedlings are transplanted into cultivated soil to grow under natural light.
Test example 4
A subarea is arranged every 1cm from the base of the growing point at the top end of the sugarcane No.1, and is divided into four areas which are respectively named as areas I, II, III and IV from bottom to top. The cultivation was carried out under the same cultivation conditions, and their growth state was recorded by photographing until the twelfth day (FIG. 5). It can be seen from the figure that the growth state is good and the sugarcane cutting segment with the region IV of direct somatic embryo can be induced more quickly, and then the regions II and III, the region I nearest to the stem tip of the sugarcane grows slowly and the state of the induced somatic embryo is poor
As can be seen from the above examples, the present invention provides a sugarcane somatic embryo and an induction method thereof, comprising the following steps: 1) Culturing the sugarcane cells in a DEM culture medium for 11-17 days; (2) Transferring the sugarcane cells to a DEM culture medium containing G418 for culturing for 8-12 days, then subculturing for 8-12 days in the DEM culture medium containing G418, and screening the survival sugarcane cells; (3) Transferring the survival sugarcane cells to an NB culture medium containing G418 for screening and culturing for 8-12 days, and subculturing and transferring the survival sugarcane cells to the NB culture medium containing G418 for screening for 8-12 days to obtain sugarcane somatic embryos. According to the invention, through exploration and optimization of the induction conditions of the direct somatic embryos of the sugarcane, the adaptability of different sugarcane varieties to a culture medium is found to be different, the culture conditions of the direct somatic embryos of the sugarcane are optimized, and transgenic sugarcane plants can be obtained in a shorter period.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for inducing sugarcane somatic embryos, which is characterized by comprising the following steps:
(1) Culturing the sugarcane cells in a DEM culture medium for 11-17 days;
(2) Transferring the sugarcane cells to a DEM culture medium containing G418 for culturing for 8-12 days, then subculturing for 8-12 days in the DEM culture medium containing G418, and screening the survival sugarcane cells;
(3) Transferring the survival sugarcane cells to NB culture medium containing G418 for screening and culturing for 8-12 days, and subculturing to NB culture medium containing G418 for screening for 8-12 days to obtain sugarcane somatic embryos.
2. The induction method according to claim 1, wherein the temperature of said culture in the step (1) is 24 to 28 ℃ and the intensity of light is 25 to 35 μmol m -2 s -1 The illumination time is 10-14 h/d.
3. The induction method according to claim 2, wherein the concentration of G418 in step (2) and step (3) is 25 to 35mg/L independently.
4. The induction method according to claim 3, wherein said sugarcane is ROC22, sugarcane No.1 or CP88-1762; the sugarcane cells in the step (1) are cells in immature leaves at the top ends of the sugarcanes, and the sugarcane cells in the step (1) are transgenic sugarcane cells.
5. The induction method according to claim 4, wherein the transgenic sugarcane cell is prepared by:
(1) Combining a target gene with a pBWA (V) BU vector to obtain a HIGS interference vector;
(2) Transforming the HIGS interference vector into competent escherichia coli, and extracting a plasmid after induction expression;
(3) Transforming the plasmid into sugarcane cells to obtain transgenic sugarcane somatic cells;
the nucleotide sequence of the target gene is shown as SEQ ID NO.1, and the sequence of the HIGS interference vector is shown as SEQ ID NO. 2.
6. Sugarcane somatic embryos obtained by the induction method according to any one of claims 1 to 5.
7. The method of using the sugarcane somatic embryo of claim 6, comprising the steps of:
(1) Transferring the sugarcane somatic embryos to a SG culture medium containing G418 for culture to obtain sugarcane seedlings;
(2) And (4) hardening the sugarcane seedlings, and transplanting the sugarcane seedlings into a culture medium to obtain the sugarcane seedlings.
8. The use method according to claim 7, wherein the concentration of G418 in the SG medium in step (1) is 15-25 mg/L.
9. The use method of claim 8, wherein the root length of the young sugarcane seedlings in the step (1) is 2-3 cm.
10. The use method according to claim 9, wherein the culture medium in step (2) comprises the following raw materials in parts by volume: 8-10 parts of vermiculite and 8-10 parts of nutrient soil.
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| CN116491421A (en) * | 2023-05-26 | 2023-07-28 | 广西大学 | Gene gun-mediated sugarcane embryo genetic transformation method |
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