CN114058640A - Efficient agrobacterium-mediated sugarcane genetic transformation method - Google Patents
Efficient agrobacterium-mediated sugarcane genetic transformation method Download PDFInfo
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- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention relates to a high-efficiency agrobacterium-mediated sugarcane genetic transformation method, which takes tender recurrent leaves at the top of a stem of a sugarcane as explants to induce the explant to generate high-quality embryonic callus, and takes the high-quality embryonic callus as an agrobacterium infection receptor material. In the infection process, cell walls of partial plants are digested by cellulase R-10, macerozyme R-10 and pectinase Y-23, and the infected conditions are set at 28 ℃ for dark culture for 7-10 days, so that the conversion efficiency is improved. After 0.01-0.1% herbicide resistant culture for 20-40 days, carrying out differentiation culture to obtain resistant transformed plants, and transplanting. The invention is a transformation method which can efficiently obtain transgenic sugarcane plants, and has low experimental cost and simple method.
Description
Technical Field
The invention relates to the technical field of sugarcane, in particular to an efficient agrobacterium-mediated sugarcane genetic transformation method.
Background
Sugarcane is the most important sugar crop in the world, accounts for more than about 70% of the sugar yield in the world, and is distributed in major tropical and subtropical regions of the world. It is a polyploidy aneuploid plant with highly complex genetic background. Sugarcane is an annual asexual propagation crop, a large number of aneuploids exist in modern sugarcane cultivars, the number of chromosomes is large, the genome structure is complex, the traditional cross breeding technology is long in period, the progeny genome is highly heterozygous, and the character recognition and screening are difficult. In addition, the flowering period is long, the flowering period is not met, part of the sugarcane is difficult to bloom, and the pollen is insufficient or the activity is low, so that part of the excellent sugarcane cannot be hybridized and utilized. The genetic engineering breeding technology is a necessary beneficial supplement of the traditional sugarcane breeding work and is an important breakthrough approach for the genetic improvement of sugarcane varieties starting from molecular technology.
The research on the genetic transformation of sugarcane, which is an important sugar crop and economic crop with high safety level of genetic transformation, has been paid much attention. Transgenic technology advantages include: (1) avoids a large amount of gene recombination caused by variety hybridization; (2) can be directionally improved by introducing exogenous genes into other organisms; (3) greatly shortens the breeding period. In addition, because the sugarcane is not directly eaten, the sugarcane is industrially processed into cane sugar, ethanol and the like which can remove transgenically expressed proteins; sugarcane cultivars are not prone to flowering under natural conditions, and therefore unintended natural crossing due to pollen transmission can be avoided. Thus, sugarcane transgenes are considered to be the transgenic crop with the lowest risk for transgenes (grade I) both domestically and internationally. In addition, because the sugarcane can be propagated asexually, the variation and the character separation in the propagation process are avoided. This property improves the gene stability of transgenic sugarcane, thereby shortening the breeding cycle and reducing the breeding cost. Therefore, the sugarcane transgenic technology has great potential research and commercial value from 2 aspects of transgenic benefit and safety.
The transgenic technology applied to sugarcane at present mainly comprises a gene gun mediated method and an agrobacterium mediated method. In recent years, the transgenic research of sugarcane mainly takes novel disease and insect resistance, cold resistance, drought resistance and high sugar as well as the production of high-added-value products as a bioreactor as the main part, and meanwhile, great progress is made in the detection research of transgenic organisms. However, the transgenosis of sugarcane is still limited by some factors, such as low transformation efficiency, low gene expression efficiency, few researches on the safety of transgenosis, possible legal and social problems, and the like. Establishing a plurality of efficient, rapid and stable genetic transformation systems of sugarcane main cultivars, researching a safe system, developing large-fragment gene and polygene transformation, realizing space-time control expression of genes and the like is a new development trend of sugarcane transgenic technology.
The research of transgenic sugarcane begins in the 80 th 20 th century, early sugarcane transgenic plants are obtained mainly by adopting a gene gun transformation method, but the transformation efficiency is low, chimeras are more, the insertion copy number of exogenous genes is more, the genetic stability is poor, and the transformation cost is high. Since 1998, researchers have more adopted Agrobacterium-mediated method, which has the advantage of low copy number of foreign gene insertion, and can realize transformation of large fragment of DNA.
Disclosure of Invention
The invention aims to provide an efficient agrobacterium-mediated sugarcane genetic transformation method, which greatly improves transformation efficiency and saves cost in the transformation process by improving the transformation method.
In order to solve the technical problems, the invention adopts the following technical scheme:
a high-efficiency agrobacterium-mediated sugarcane genetic transformation method comprises the following steps:
(1) induction of sugarcane embryogenic callus: cutting off the top end of a sugarcane stem from a sugarcane plant, taking a young and tender sugarcane rotor blade, placing the sugarcane rotor blade on a callus induction culture medium, taking the sugarcane rotor blade as a callus culture, culturing the callus culture in a dark environment, selecting a flaxen hard embryonic callus with high activity as an induced callus after 30-60 days, and cutting up the callus culture before agrobacteria is impregnated;
(2) activating agrobacterium: culturing agrobacterium, centrifugally collecting agrobacterium cultured in vitro, and resuspending with a staining culture medium to obtain a staining solution;
(3) and (3) carrying out dip dyeing on the embryogenic callus: collecting the callus cultures obtained in the step (1), putting the callus cultures into the staining solution obtained in the step (2), shaking up gently in a dark environment for 0.5-2.0 hours, removing the agrobacterium suspension, transferring the callus into a culture dish, sucking the redundant agrobacterium suspension by using filter paper, and air-drying in an ultra-clean workbench for 30 minutes until no water exists on the surface;
(4) dip-dyeing culture: transferring the impregnated callus culture obtained in the step (3) into a co-culture medium, and co-culturing for 7-10 days in a dark environment at 28 ℃ to finish the impregnation process;
(5) screening of the stained embryogenic callus: transferring the callus cultures impregnated in the step (4) into a screening culture medium, incubating for 20-40 days in a dark environment at 28 +/-2 ℃ to obtain resistant callus, transferring the resistant callus into a regeneration culture medium, and culturing in an illumination culture box to obtain differentiated buds;
(6) and (3) differentiation and regeneration processes: transferring the buds differentiated in the resistance healed wounds obtained in the step (5) into a rooting culture medium, culturing for 20 days in a light culture box, transferring single buds in each bud into a fresh rooting culture medium, culturing for 15-25 days, and taking the leaves of the resistance seedlings, performing molecular detection analysis, and transplanting into soil.
Further, the method comprises the following steps of; in the step (1), the cross section of the sugarcane verticillium leaf is a 1-2mm slice, the callus culture is cultured in a dark environment at 25-28 ℃, and a fresh callus induction culture medium is replaced every 10-15 days.
Further, the method comprises the following steps of; in the step (2), the agrobacterium is cultured to OD of logarithmic growth phase600The concentration is 0.5-1.0, the dip-dyeing culture medium is suspended by shaking table culture, the shaking table culture is carried out at the temperature of 28 +/-2 ℃ for 0.5-2.0 hours, and 1.0-3.0% of cellulase R-10+0.5-1.0% of isolation enzyme R-10+0.4-1.0% of pectinase Y-23 is added after the shaking table culture.
Further, the method comprises the following steps of; in the step (5), the temperature of the illumination incubator is set to be 28 +/-2 ℃, the illumination intensity is 5000-.
Further, the method comprises the following steps of; in the step (6), the molecular detection analysis method can detect whether the foreign gene has been introduced into the genome of sugarcane by a PCR method from the DNA level and the RNA level.
Further, the method comprises the following steps of; in the step (6), the molecular detection and analysis method can detect the change of the expression level of the exogenous gene by the QRT-PCR method.
Further, the method comprises the following steps of; the tissue induction culture medium comprises: MS +30 g/L sucrose +2 mg/L2, 4-D +8 mg/L carrageenan;
the staining culture medium is as follows: 1/5 MS +30 g/L sucrose +30 g/L glucose +100m mol acetosyringone;
the co-culture medium is as follows: MS +1.0 mg/L2, 4-D +30 g/L sucrose +8g/L carrageenan +200mg/L timentin;
the screening culture medium is as follows: MS +2.0 mg/L2, 4-D +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta;
the regeneration culture medium is as follows: MS +1.0 mg/L6-BA +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta;
the rooting culture medium comprises: MS +2mg/L NAA +8g/L carrageenan + 300mg/L timentin +5g sucrose +0.01-0.1% Basta.
Compared with the prior art, the invention has at least one of the following beneficial effects:
(1) the invention adopts 1.0-3.0% of cellulase R-10+0.5-1.0% of macerozyme R-10+0.4-1.0% of pectinase Y-23 to partially remove cell walls of the surface cells of the embryonic callus, thereby greatly improving the conversion efficiency of the sugarcane.
(2) The implementation of the method also needs to combine high-quality embryogenic callus and proper culture and co-culture time to achieve the purpose of improving the transformation efficiency.
(3) The invention has high transformation efficiency, obtains few false positives and chimeras, is suitable for the research of large-scale transgenic sugarcane, greatly reduces the workload and reduces the cost of transformation research.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1:
an agrobacterium-mediated high-efficiency transgenic sugarcane method comprises the following steps:
(1) induction of sugarcane embryogenic callus: cutting off the top end of a sugarcane stem from a sugarcane plant, taking a young and tender sugarcane rotor blade, wherein the cross section of the sugarcane rotor blade is a1 mm slice, placing the sugarcane rotor blade on a callus induction culture medium, taking the sugarcane rotor blade as a callus culture, culturing the callus culture in a dark environment at 25 ℃, replacing a fresh callus induction culture medium every 10 days, selecting a yellowish hard embryonic callus with high activity as an induced callus after 30 days, and cutting up the callus culture before agrobacterium is infected;
(2) activating agrobacterium: OD of Agrobacterium culture to logarithmic growth phase600The concentration is 0.5, the agrobacterium tumefaciens thalli cultured in vitro are collected centrifugally, and are resuspended by a staining culture medium, and are cultured for 0.5 hour by a shaking table at the temperature of 26 ℃, so as to obtain a staining solution;
(3) and (3) carrying out dip dyeing on the embryogenic callus: collecting the callus cultures obtained in the step (1), putting the callus cultures into the staining solution obtained in the step (2), shaking up gently in a dark environment for 0.5 hour, removing the agrobacterium suspension, transferring the callus into a culture dish, sucking the redundant agrobacterium suspension by using filter paper, and airing for 30 minutes in an ultra-clean workbench until no water exists on the surface;
(4) dip-dyeing culture: transferring the impregnated callus culture obtained in the step (3) into a co-culture medium, and culturing for 7 days in a dark environment at 28 ℃ to finish the impregnation process;
(5) screening of the stained embryogenic callus: transferring the callus cultures impregnated in the step (4) into a screening culture medium, incubating for 20 days in a dark environment at 26 ℃ to obtain resistant callus, transferring the resistant callus into a regeneration culture medium, and culturing for 10 days in an illumination incubator to obtain differentiated buds;
(6) and (3) differentiation and regeneration processes: transferring the buds differentiated in the resistance healed wounds obtained in the step (5) into a rooting culture medium, culturing for 20 days in an illumination culture box, transferring single buds in each bud into a fresh rooting culture medium, culturing for 15 days, taking the leaves of the resistance seedlings, performing molecular detection analysis, and transplanting into soil; by improving the transformation method, the transformation efficiency is greatly improved, and the cost in the transformation process is saved.
Example 2:
on the basis of the embodiment 1, the agrobacterium-mediated efficient sugarcane transgenic method is characterized in that: the method comprises the following steps:
(1) induction of sugarcane embryogenic callus: cutting off the top end of a sugarcane stem from a sugarcane plant, taking a young and tender sugarcane rotor blade, wherein the cross section of the sugarcane rotor blade is a 2mm slice, the sugarcane rotor blade is placed on a callus induction culture medium, the sugarcane rotor blade is a callus culture, the callus culture is cultured in a dark environment at the temperature of 28 ℃, a fresh callus induction culture medium is replaced every 15 days, after 60 days, a yellowish hard embryonic callus with high activity is selected as an induced callus, and the callus culture is cut up before agrobacterium is infected;
(2) activating agrobacterium: OD of Agrobacterium culture to logarithmic growth phase600The concentration is 1.0, the agrobacterium tumefaciens thalli cultured in vitro are collected centrifugally, and are resuspended by a staining culture medium, and are cultured for 2.0 hours by a shaking table at the temperature of 30 ℃, so as to obtain a staining solution;
(3) and (3) carrying out dip dyeing on the embryogenic callus: collecting the callus cultures obtained in the step (1), putting the callus cultures into the staining solution obtained in the step (2), shaking up gently in a dark environment for 2.0 hours, removing the agrobacterium suspension, transferring the callus into a culture dish, sucking the redundant agrobacterium suspension by using filter paper, and airing for 30 minutes in an ultra-clean workbench until no water exists on the surface;
(4) dip-dyeing culture: transferring the impregnated callus culture obtained in the step (3) into a co-culture medium, and culturing for 10 days in a dark environment at 28 ℃ to finish the impregnation process;
(5) screening of the stained embryogenic callus: transferring the callus cultures impregnated in the step (4) into a screening culture medium, incubating for 40 days in a dark environment at the temperature of 30 ℃ to obtain resistant callus, transferring the resistant callus into a regeneration culture medium, and culturing for 30 days in an illumination culture box to obtain differentiated buds;
(6) and (3) differentiation and regeneration processes: transferring the buds differentiated in the resistance healed wounds obtained in the step (5) into a rooting culture medium, culturing for 20 days in a light culture box, transferring single buds in each bud into a fresh rooting culture medium, culturing for 25 days, taking the leaves of the resistance seedlings, performing molecular detection analysis, and transplanting into soil. By improving the transformation method, the transformation efficiency is greatly improved, and the cost in the transformation process is saved.
Example 3:
on the basis of the embodiment 1-2, the agrobacterium-mediated high-efficiency sugarcane transgenic method is characterized in that: the method comprises the following steps:
(1) induction of sugarcane embryogenic callus: cutting off the top end of a sugarcane stem from a sugarcane plant, taking a young and tender sugarcane rotor blade, wherein the cross section of the sugarcane rotor blade is a 2mm slice, the sugarcane rotor blade is placed on a callus induction culture medium, the sugarcane rotor blade is a callus culture, the callus culture is cultured in a dark environment at 26 ℃, a fresh callus induction culture medium is replaced every 12 days, after 45 days, a yellowish hard embryonic callus with high activity is selected as an induced callus, and the callus culture is cut up before agrobacterium is infected;
(2) activating agrobacterium: OD of Agrobacterium culture to logarithmic growth phase600The concentration is 1.0, the agrobacterium tumefaciens thalli cultured in vitro are collected centrifugally, and are resuspended by a staining culture medium, and are cultured for 1.5 hours by a shaking table at the temperature of 28 ℃, so as to obtain a staining solution;
(3) and (3) carrying out dip dyeing on the embryogenic callus: collecting the callus cultures obtained in the step (1), putting the callus cultures into the staining solution obtained in the step (2), shaking up gently in a dark environment for 1.5 hours, removing the agrobacterium suspension, transferring the callus into a culture dish, sucking the redundant agrobacterium suspension by using filter paper, and airing for 30 minutes in an ultra-clean workbench until no water exists on the surface;
(4) dip-dyeing culture: transferring the impregnated callus culture obtained in the step (3) into a co-culture medium, and culturing for 8 days in a dark environment at 28 ℃ to finish the impregnation process;
(5) screening of the stained embryogenic callus: transferring the callus cultures impregnated in the step (4) into a screening culture medium, incubating for 30 days in a dark environment at 28 ℃ to obtain resistant callus, transferring the resistant callus into a regeneration culture medium, and culturing for 20 days in an illumination culture box to obtain differentiated buds;
(6) and (3) differentiation and regeneration processes: transferring the buds differentiated in the resistance healed wounds obtained in the step (5) into a rooting culture medium, culturing for 20 days in a light culture box, transferring single buds in each bud into a fresh rooting culture medium, culturing for 20 days, taking the leaves of the resistance seedlings, performing molecular detection analysis, and transplanting into soil. By improving the transformation method, the transformation efficiency is greatly improved, and the cost in the transformation process is saved.
Example 4:
based on the examples 1-3, in the step (2), 1.0-3.0% cellulase R-10+0.5-1.0% macerozyme R-10+0.4-1.0% pectinase Y-23 is added after shake cultivation; partial cell wall removal treatment is carried out on the surface cells of the embryonic callus, so that the conversion efficiency of the sugarcane is greatly improved.
Example 5:
on the basis of the examples 1-4,
the tissue induction culture medium comprises: MS +30 g/L sucrose +2 mg/L2, 4-D +8 mg/L carrageenan;
the staining culture medium is as follows: 1/5 MS +30 g/L sucrose +30 g/L glucose +100m mol acetosyringone;
the co-culture medium is as follows: MS +1.0 mg/L2, 4-D +30 g/L sucrose +8g/L carrageenan +200mg/L timentin;
the screening culture medium is as follows: MS +2.0 mg/L2, 4-D +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta;
the regeneration culture medium is as follows: MS +1.0 mg/L6-BA +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta;
the rooting culture medium comprises: MS +2mg/L NAA +8g/L carrageenan + 300mg/L timentin +5g sucrose +0.01-0.1% Basta; wherein, Basta is glufosinate-ammonium herbicide, the transformation efficiency is high, the obtained false positives and chimeras are few, the method is suitable for the research of large-scale transgenic sugarcane, the workload is greatly reduced, and the cost of transformation research is reduced.
Example 6:
on the basis of the examples 1-5, the temperature of the illumination incubator is set to be 26 ℃, and the illumination intensity is 5000 Lux; is convenient for culturing in the illumination incubator.
Example 7:
on the basis of the examples 1 to 6, the temperature of the illumination incubator is set to be 30 ℃, and the illumination intensity is 8000 Lux; is convenient for culturing in the illumination incubator.
Example 8:
on the basis of the examples 1 to 7, the temperature of the illumination incubator is set to be 28 ℃, and the illumination intensity is 6500 Lux; is convenient for culturing in the illumination incubator.
Example 9:
on the basis of examples 1-8, the callus cultures were minced, the length of the minced callus cultures being 2 mm; facilitating other operations.
Example 10:
on the basis of examples 1 to 9, in step (6), the method of molecular detection analysis can detect whether a foreign gene has been introduced into the genome of sugarcane by the PCR method from the DNA level and the RNA level; is convenient for molecular detection and analysis and can be transplanted.
Example 11:
on the basis of examples 1 to 10, in step (6), the method of molecular detection analysis can detect the change in the expression level of the foreign gene by the QRT-PCR method; is convenient for molecular detection and analysis and can be transplanted.
Example 12:
on the basis of examples 1 to 11, sugarcane variety ROC22, pCAMBIA3301 plant expression vector containing Bar gene and GFP gene, and Agrobacterium strain EHA105 were used as the study materials. Introduction of vectors into AgrobacteriumAgrobacterium EHA105 of a pCAMBIA3301 plant expression vector containing the Bar gene and GFP gene was prepared, EHA105 competent cells were purchased from TAKARA BAO bioengineering (Dalian) Co., Ltd, and the pCAMBIA3301 vector was introduced into EHA105 according to the procedure of the competent transformation instruction. Culturing the callus; under aseptic environment, cutting tender annular leaves at the tip of the sugarcane stem into round slices of 1-2mm, culturing at 25-28 deg.C for 45 days, and replacing culture medium every 10 days to obtain yellowish embryonic callus with strong meristematic ability. Infecting the embryonic callus with agrobacterium; a single colony of Agrobacterium EHA105 containing the Bar gene and GFP gene was picked, inoculated into 50ml YEP medium (50 mg/L rifampicin, 100mg/L streptomycin sulfate), cultured for 24 hours, and OD was measured600Collecting the thallus at 4 ℃ and 5000rpm of 0.6, resuspending the thallus in 50ml of MS liquid culture medium, adding 50 mu M/L AS, carrying out shake culture at 28 ℃ for 1.5 hours, adding 2.0% of cellulase R-10, 0.5% of eductase R-10 and 0.6% of pectinase Y-23, and obtaining the infection bacterial liquid for transformation. Collecting embryonic callus, cutting, sucking the old culture medium with sterile filter paper, putting the callus into agrobacterium infection solution containing expression vector, and culturing for about 30 minutes in dark with gentle shaking. The agrobacterium suspension is removed. The callus was transferred to a petri dish, excess agrobacterium suspension was blotted dry with filter paper, and air dried in a clean bench for about 30 minutes. The calli were transferred to fresh medium and incubated for 3 days at 22 ℃ in the dark. Infected calli were transferred to (MS +1.0 mg/L2, 4-D +30 g/L sucrose +8g/L carrageenan +200mg/L timentin) co-medium. The cells were cultured at 28 ℃ in the dark for 7 days. 5) Induction of resistant callus emergence infection of the completed callus was then transferred to screening medium (MS +2.0 mg/L2, 4-D +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta), incubated at 28 ℃ in the dark for 35 days, the resistant callus on the dark medium was transferred to regeneration medium (MS +1.0 mg/L6-BA +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta) and cultured in light incubator for 14 days. The differentiated green shoots were transferred to rooting medium (MS +2mg/L NAA +8g/L carrageenan + 300mg/L timentin +5g sucrose +0.01-0.1% Basta) and cultured in a light incubator for 20 days. In each budThe single buds are transferred into a fresh rooting culture medium for 20 days. Taking the leaves of the resistant seedlings for molecular detection and analysis, and transplanting the leaves into soil.
Example 12:
based on examples 1-11, an agrobacterium-mediated high-efficiency transgenic sugarcane method comprises the following steps: sugarcane variety ROC22, pCAMBIA3301 plant expression vector containing Bar gene and GFP gene and Agrobacterium strain EHA105 are used as research materials; introducing the vector into agrobacterium; agrobacterium EHA105 of pCAMBIA3301 plant expression vector containing Bar gene and GFP gene was prepared, EHA105 competent cells were purchased from TAKARA BAO bioengineering (Dalian) Co., Ltd, and pCAMBIA3301 vector was introduced into EHA105 according to the procedure of the competent transformation instruction. Culturing the callus; under aseptic environment, cutting tender annular leaves at the tip of the sugarcane stem into round slices of 1-2mm, culturing at 28 deg.C for 40 days, and replacing culture medium every 10 days to obtain yellowish embryonic callus with strong meristematic ability. Infecting the embryonic callus with agrobacterium; selecting a single agrobacterium EHA105 colony containing a Bar gene and a ScD27.2 gene, inoculating the single agrobacterium EHA105 colony in 50ml of YEP culture medium (50 mg/L rifampicin and 100mg/L streptomycin sulfate), culturing for 36 hours, determining OD600 to be 0.6, collecting the bacteria at 4 ℃ and 5000rpm, resuspending the bacteria in 50ml of MS liquid culture medium, adding 50 mu M/L AS, culturing for 1.5 hours in a shaking table at 28 ℃, adding 2.5% of cellulase R-10, 0.6% of macerase R-10 and 0.5% of pectinase Y-23, and obtaining an infecting bacteria liquid for transformation. Collecting embryonic callus, cutting, putting the callus into agrobacterium infection solution containing expression vector, and culturing in dark with gentle shaking for about 30 minutes. The agrobacterium suspension is removed. The callus was transferred to a petri dish, excess agrobacterium suspension was blotted dry with filter paper, and air dried in a clean bench for about 30 minutes. The calli were transferred to fresh medium and incubated for 3 days at 22 ℃ in the dark. Infected calli were transferred to (MS +1.0 mg/L2, 4-D +30 g/L sucrose +8g/L carrageenan +200mg/L timentin) co-medium. The cells were cultured in the dark at 28 ℃ for 10 days. Inducing the emergence of resistant callus; the infected calli were then transferred to selection medium (MS +2.0 mg/L2, 4-D +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta), incubated at 28 ℃ in the dark for 35 days, and the resistant calli on the dark medium were transferred to regeneration medium (MS +1.0 mg/L6-BA +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta) and cultured in a light incubator for 14 days. The differentiated green shoots were transferred to rooting medium (MS +2mg/L NAA +8g/L carrageenan + 300mg/L timentin +5g sucrose +0.01-0.1% Basta) and cultured in a light incubator for 20 days. The single shoots of each shoot were transferred to fresh rooting medium for 20 days. Taking the leaves of the resistant seedlings for molecular detection and analysis, and transplanting the leaves into soil.
Although the invention has been described herein with reference to a number of illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure and claims of this application. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.
Claims (7)
1. A high-efficiency agrobacterium-mediated sugarcane genetic transformation method is characterized by comprising the following steps: the method comprises the following steps:
(1) induction of sugarcane embryogenic callus: cutting off the top end of a sugarcane stem from a sugarcane plant, taking a young and tender sugarcane rotor blade, placing the sugarcane rotor blade on a callus induction culture medium, taking the sugarcane rotor blade as a callus culture, culturing the callus culture in a dark environment, selecting a flaxen hard embryonic callus with high activity as an induced callus after 30-60 days, and cutting up the callus culture before agrobacteria is impregnated;
(2) activating agrobacterium: culturing agrobacterium, centrifugally collecting agrobacterium cultured in vitro, and resuspending with a staining culture medium to obtain a staining solution;
(3) and (3) carrying out dip dyeing on the embryogenic callus: collecting the callus cultures obtained in the step (1), putting the callus cultures into the staining solution obtained in the step (2), shaking up gently in a dark environment for 0.5-2.0 hours, removing the agrobacterium suspension, transferring the callus into a culture dish, sucking the redundant agrobacterium suspension by using filter paper, and air-drying in an ultra-clean workbench for 30 minutes until no water exists on the surface;
(4) dip-dyeing culture: transferring the impregnated callus culture obtained in the step (3) into a co-culture medium, and co-culturing for 7-10 days in a dark environment at 28 ℃ to finish the impregnation process;
(5) screening of the stained embryogenic callus: transferring the callus cultures impregnated in the step (4) into a screening culture medium, incubating for 20-40 days in a dark environment at 28 +/-2 ℃ to obtain resistant callus, transferring the resistant callus into a regeneration culture medium, and culturing in an illumination culture box to obtain differentiated buds;
(6) and (3) differentiation and regeneration processes: transferring the buds differentiated in the resistance healed wounds obtained in the step (5) into a rooting culture medium, culturing for 20 days in a light culture box, transferring single buds in each bud into a fresh rooting culture medium, culturing for 15-25 days, and taking the leaves of the resistance seedlings, performing molecular detection analysis, and transplanting into soil.
2. The method of claim 1, wherein the method comprises the steps of: in the step (1), the cross section of the sugarcane verticillium leaf is a 1-2mm slice, the callus culture is cultured in a dark environment at 25-28 ℃, and a fresh callus induction culture medium is replaced every 10-15 days.
3. The method of claim 1, wherein the method comprises the steps of: in the step (2), the agrobacterium is cultured to OD of logarithmic growth phase600The concentration is 0.5-1.0, the dip-dyeing culture medium is suspended by shaking table culture, the shaking table culture is carried out at the temperature of 28 +/-2 ℃ for 0.5-2.0 hours, and 1.0-3.0% of cellulase R-10+0.5-1.0% of isolation enzyme R-10+0.4-1.0% of pectinase Y-23 is added after the shaking table culture.
4. The method of claim 1, wherein the method comprises the steps of: in the step (5), the temperature of the illumination incubator is set to be 28 +/-2 ℃, the illumination intensity is 5000-.
5. The method of claim 1, wherein the method comprises the steps of: in the step (6), the molecular detection analysis method can detect whether the foreign gene has been introduced into the genome of sugarcane by a PCR method from the DNA level and the RNA level.
6. The method of claim 1, wherein the method comprises the steps of: in the step (6), the molecular detection and analysis method can detect the change of the expression level of the exogenous gene by the QRT-PCR method.
7. The method of claim 1, wherein the method comprises the steps of: the tissue induction culture medium comprises: MS +30 g/L sucrose +2 mg/L2, 4-D +8 mg/L carrageenan;
the staining culture medium is as follows: 1/5 MS +30 g/L sucrose +30 g/L glucose +100m mol acetosyringone;
the co-culture medium is as follows: MS +1.0 mg/L2, 4-D +30 g/L sucrose +8g/L carrageenan +200mg/L timentin;
the screening culture medium is as follows: MS +2.0 mg/L2, 4-D +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta;
the regeneration culture medium is as follows: MS +1.0 mg/L6-BA +8g/L carrageenan +200mg/L timentin +5g sucrose +0.01-0.1% Basta;
the rooting culture medium comprises: MS +2mg/L NAA +8g/L carrageenan + 300mg/L timentin +5g sucrose +0.01-0.1% Basta.
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