Genetic transformation method of excellent sugarcane variety Liucheng 1541

Abstract

The invention provides a genetic transformation method of an excellent sugarcane variety Liucheng 1541, and belongs to the technical field of plant genetic engineering.The genetic transformation method comprises the steps that leaf tissues of the sugarcane variety Liucheng 1541 are taken and subjected to induction culture on a callus induction culture medium containing 2, 4-dichlorphenoxyacetic acid and 6-benzylaminopurine; the method comprises the following steps: infecting embryogenic calluses with agrobacterium, co-culturing, carrying out recovery culture, transferring the recovered calluses to a screening culture medium containing glufosinate-ammonium, carrying out screening culture, inoculating the screened resistant embryogenic calluses to a differential culture medium containing glufosinate-ammonium, carrying out differentiation culture, and carrying out rooting culture to obtain the glufosinate-ammonium resistant embryogenic callus. The transgenic plant of the Liucheng 1541 is obtained. According to the method, the embryogenic callus induction rate is increased, the problem of callus browning in the variety Liucheng 1541 tissue culture process is solved, the glufosinate-ammonium screening concentration of the embryogenic callus is determined, and a Liucheng 1541 genetic transformation system is determined.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to a genetic transformation method of a fine sugarcane variety Liucheng 1541. BACKGROUND

[0002] Liucheng 1541, as one of the new sugarcane varieties promoted in China's sugarcane industry, is bred by Guangxi Liuzhou Sugarcane Research Institute and belongs to a high-yield, high-quality and multi-resistant sugarcane cultivation variety. The variety has wide adaptability, strong growth vigor and good ratooning ability, and is suitable for planting in the main sugarcane producing areas in the south. Liucheng 1541 has shown good field resistance in the planting practices in Guangxi, Yunnan, Guangdong and other places, and has certain resistance advantages to major viral diseases such as sugarcane mosaic virus (SCMV). In terms of sugar accumulation, Liucheng 1541 has high sugar content and good sugar yield, and is widely used in sugar production, playing a positive role in promoting the development of regional sugarcane industry. As of 2024, the demonstration area of Liucheng 1541 has exceeded 20,000 mu in multiple regions, and has a wide application prospect. However, the adaptability of the variety is generally poor when facing complex environmental stress (pests, weeds), and it is not resistant to pests and herbicides, which are two short board traits. When facing biological hazards such as pests and weeds, the traditional control method is chemical pesticide control and manual weeding, which has problems of environmental pollution, drug resistance and high cost. Although there are studies on transgenic sugarcane with resistance to pests and herbicides, there is no report on the transformation of double-pest-resistant genes for Liucheng 1541. There are difficulties in the current research on the genetic transformation of Liucheng 1541, mainly in the browning of callus in the process of in vitro tissue culture, which limits the molecular genetic improvement and application of the variety. SUMMARY

[0003] To solve the above problems, the present application provides a genetic transformation method of a fine sugarcane variety Liucheng 1541.

[0004] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows: A genetic transformation method of a fine sugarcane variety Liucheng 1541, the genetic transformation method is to take the leaf tissue of the sugarcane variety Liucheng 1541 for induction culture on a callus induction culture medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine, the obtained embryogenic callus is infected with Agrobacterium, and then co-cultured, recovered and cultured, the recovered callus is transferred to a screening culture medium containing glufosinate for screening culture, and then the resistant embryogenic callus after screening is inoculated on a differentiation culture medium containing glufosinate for differentiation culture, and then rooted culture is performed to obtain transgenic plants of Liucheng 1541.

[0005] Furthermore, the concentration of 2,4-dichlorophenoxyacetic acid in the callus induction medium is 1.0-2.5 mg / L, and the concentration of 6-benzylaminopurine is 0.2-0.8 mg / L.

[0006] Furthermore, the callus induction medium is prepared by adding 1.0-2.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.8 mg / L 6-benzylaminopurine, 20-35 g / L sucrose, 7-8 g / L agar and 40-50 mg / L activated carbon to MS medium, with a pH of 5.6-5.8.

[0007] Furthermore, the concentration of glufosinate in the screening medium was 1–3 mg / L; The concentration of glufosinate in the differentiation medium is 1~3 mg / L.

[0008] Furthermore, the screening medium is MS medium supplemented with 1.0-2.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.8 mg / L 6-benzylaminopurine, 200-300 mg / L timentin, 1-3 mg / L glufosinate, 20-35 g / L sucrose, 7-8 g / L agar, and 40-50 mg / L activated carbon, with a pH of 5.6-5.8; The differentiation medium is MS medium supplemented with 200~300 mg / L timentin, 1~3 mg / L glufosinate, 0.5~1 mg / L 6-benzylaminopurine, 20~35 g / L sucrose, 7~8 g / L agar and 40~50 mg / L activated carbon, with a pH of 5.6~5.8.

[0009] Further, the Agrobacterium used for infection is Bar and Cry9Cb Agrobacterium containing genes or Bar 、 Cry9Cb and Vip3Aa11 Agrobacterium of genes; in, Bar The gene is shown in SEQ ID NO: 1, Cry9Cb The gene is shown in SEQ ID NO: 2, Vip3Aa11 The gene is shown in SEQ ID NO:3.

[0010] Further, including Bar and Cry9Cb Agrobacterium tumefaciens is a gene-producing Bar 、 Cry9Cb Gene, construction of plant expression vector Bar + Cry9Cb , and then use plant expression vector Bar + Cry9Cb was transformed into Agrobacterium strain EHA105 to obtain; Contains Bar 、 Cry9Cb and Vip3Aa11 Agrobacterium tumefaciens is a gene-producing Bar 、 Cry9Cb 、 Vip3Aa11 Gene, construction of plant expression vector Bar + Cry9Cb + Vip3Aa11 , and then use plant expression vector Bar + Cry9Cb + Vip3Aa11 The product was transformed into Agrobacterium tumefaciens strain EHA105.

[0011] Furthermore, the embryonic callus is infected with Agrobacterium by picking the embryonic callus, adding a starting liquid culture medium, and warming it; filtering out the starting liquid culture medium and then adding the corresponding infection liquid of Agrobacterium, and ultrasonically shaking it; filtering out the corresponding infection liquid of Agrobacterium, and then adding the corresponding infection liquid of Agrobacterium again, vacuuming it, and finally shaking it to complete the infection; The corresponding infection solution of Agrobacterium is prepared by streaking Agrobacterium onto YEP solid medium containing 50 mg / L kanamycin and inverting the medium to culture; picking a single colony and adding it to YEP liquid medium and shaking it overnight; aspirating the bacterial solution and spreading it onto YEP solid medium containing 5 mg / L rifampicin and 50 mg / L kanamycin and inverting the medium to culture; using the starting liquid medium, pipetting the resuspended bacteria on the medium and shaking the medium to obtain the corresponding infection solution; The starting liquid culture medium is 1 / 5 macronutrient MS macronutrient medium supplemented with 25-35 g / L sucrose, 25-35 g / L glucose, and 100-200 μM acetosyringone, with a pH of 5.3-5.5.

[0012] Furthermore, the genetic transformation method comprises the following specific steps: Embryogenic callus induction: Young leaf tissue of the sugarcane variety Liucheng 1541 was selected as callus explants and placed on a callus induction medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine for induction culture. The callus induction medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine was replaced twice during the induction process. The callus was subcultured three times to obtain embryonic callus. Transgenic plant cultivation: Pick embryonic callus, add starter liquid culture medium, and incubate; filter out the starter liquid culture medium and then add the corresponding infection liquid of Agrobacterium, and shake with ultrasound; filter out the corresponding infection liquid of Agrobacterium, add the corresponding infection liquid of Agrobacterium again, vacuumize, and finally shake and culture; The infected embryogenic callus was transferred to co-cultivation medium and co-cultivated in the dark; The co-cultured callus tissue was transferred to a solid recovery medium and the culture was resumed; The recovered callus tissue was transferred to a screening medium containing glufosinate-ammonium and cultured for screening; The selected resistant embryogenic callus was inoculated on a differentiation medium containing glufosinate ammonium, cultured in the dark and then cultured in the light, and young shoots were differentiated on the surface of the callus; The young shoots were transferred to a new differentiation medium containing glufosinate ammonium for secondary differentiation cultivation; When the cells differentiate into seedlings, they are individually transferred to a rooting medium and cultured until the root system is fully grown, thereby obtaining transgenic plants of Liucheng 1541.

[0013] Furthermore, the co-culture medium is B5 medium supplemented with 1.0-3.0 mg / L 2,4-dichlorophenoxyacetic acid, 100-300 μmol / L acetosyringone, 20-30 g / L sucrose, and 7-8 g / L agar, with a pH of 5.6-5.8; Solid recovery medium is MS medium supplemented with 1.0-2.0 mg / L 2,4-dichlorophenoxyacetic acid, 200-300 mg / L timentin, 20-30 g / L sucrose, 7-8 g / L agar, and 40-50 mg / L activated carbon, with a pH of 5.6-5.8; The rooting medium is MS medium supplemented with 200~300 mg / L timentin, 1~2 mg / L glufosinate, 1~2 mg / L naphthaleneacetic acid, 20~35 g / L sucrose, 7~8 g / L agar and 40~50 mg / L activated carbon, with a pH of 5.6~5.8.

[0014] The beneficial effects of the genetic transformation method of the superior sugarcane variety Liucheng 1541 of the present invention are: The present invention explores the induction status of different concentrations of 2,4-dichlorophenoxyacetic acid on the variety Liucheng 1541, and simultaneously analyzes the regulatory effect of different culture medium additives on anti-browning in callus tissue culture. The results show that a 2.0 mg / L 2,4-dichlorophenoxyacetic acid concentration has the best induction effect, and the induction effects between different concentrations show significant differences; in a culture medium supplemented with 6-benzylaminopurine, the browning of the callus tissue is alleviated, solving the problem of callus tissue browning during the tissue culture of the variety Liucheng 1541; at the same time, through a glufosinate tolerance test, the appropriate glufosinate concentration range for maintaining screening pressure during the differentiation stage of sugarcane callus tissue is 1-3 mg / L, among which the screening efficiency reaches 93.3% and the conversion efficiency is 15.9% at 2 mg / L glufosinate; and using a plant expression vector Bar + Cry9Cb and plant expression vectors Bar + Cry9Cb + Vip3Aa11 Liucheng 1541 was genetically transformed by Agrobacterium-mediated method, and a transgenic line of Liucheng 1541 with herbicide and insect resistance was obtained. The present invention shows through resistance detection that the Liucheng 1541 transgenic strain obtained by the genetic transformation method of the present invention has significant herbicide resistance and insect resistance. In the glufosinate tolerance experiment, the transgenic strain grew normally when sprayed with 0.2% glufosinate. When the concentration was increased to 0.4%, the growth of the plant leaves was slightly inhibited, and the strain grew slowly. When sprayed with 0.6% glufosinate, the leaf edges showed slight yellowing. After spraying 0.8% glufosinate herbicide, the leaf edges showed obvious yellowing and the leaves began to curl. When the non-transgenic strain was sprayed with a glufosinate concentration of 0.2%, the leaves showed obvious loss of green and curling. When sprayed with 0.4% glufosinate, the growth of the plant was severely hindered, the leaves completely lost green and wilted. When the concentration was increased to 0.6%, the plant completely withered and died. The insect resistance experiment showed that the Liucheng 1541 transgenic plants obtained by the genetic transformation method of the present invention were all insect-resistant, and Bar + Cry9Cb + Vip3Aa11 The insecticidal activity of positive plants was significantly better than Bar + Cry9Cb Positive plants, after feeding for 10 days, Bar + Cry9Cb The weight of stem borers in the positive plants was Bar + Cry9Cb + Vip3Aa11 3.39 times that of the positive plants; The present invention systematically screens and determines the optimal 2,4-dichlorophenoxyacetic acid concentration for callus induction in Liucheng 1541, thereby increasing the embryonic callus induction rate and laying the foundation for efficient genetic transformation. The invention also tests the lethal concentration gradient of glufosinate ammonium to clarify the concentration range for continuous screening of embryonic callus. Furthermore, the invention establishes a genetic transformation screening system for Liucheng 1541 by combining screening efficiency and transformation efficiency. This invention is the first to Cry9Cb and Vip3Aa11 The combination of bivalent insect-resistant genes into sugarcane is similar to that of single genes. Cry9Cb Comparison of insect-resistant genes confirmed that their synergistic effect significantly inhibited the growth of stem borers; The genetically modified sugarcane Liucheng 1541 of the present invention is simultaneously polymerized Cry9Cb and Vip3Aa11 These two different types of insect-resistant genes, Cry protein causes osmotic pressure imbalance by destroying the insect midgut epithelial cell membrane, while Vip protein triggers cell apoptosis by binding to specific receptors. This dual action mechanism not only shortens the transgenic breeding cycle, but also expands the coverage of the insecticide spectrum, especially showing a broader spectrum of prevention and control effects on lepidopteran pests; in addition, the synergistic effect of multiple virulence factors of the two proteins can effectively delay the development of resistance of field pests and greatly improve the stability and durability of insect-resistant traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the induction rate of callus tissue in Example 1 of the present invention at different 2,4-dichlorophenoxyacetic acid concentrations; wherein 2,4-D is 2,4-dichlorophenoxyacetic acid; Figure 2 The state of the callus in Example 2 of the present invention; wherein, Figure A shows browning of the callus, Figure B shows browning of the embryonic callus, Figure C shows non-browning of the callus, and Figure D shows non-browning of the embryonic callus; Figure 3 is the anti-browning rate of the callus in Example 2 of the present invention; wherein, no addition represents a culture medium without addition, activated carbon represents a culture medium with activated carbon added, proline represents a culture medium with proline added, PVP represents a culture medium with PVP added, and 6BA represents a culture medium with 6-benzylaminopurine added; Figure 4 This is the optimal screening concentration screening result of glufosinate-ammonium in Example 3 of the present invention; Figure 5 This is the sugarcane genetic transformation process in Example 4 of the present invention; wherein, Figure A is a section of young sugarcane tissue, Figure B is callus culture, Figure C is embryonic callus, Figure D is recovery culture; Figure E is differentiation culture; and Figure F is rooting culture. Figure 6 Plant expression vector in Example 4 of the present invention Bar + Cry9Cb ; Figure 7 Plant expression vector in Example 4 of the present invention Bar + Cry9Cb + Vip3Aa11 ; Figure 8 Cultivation of resistant plants in Example 4 of the present invention; wherein, Figure A shows hardening of resistant plants, and Figure B shows transplanting of resistant plants into soil; Figure 9 Part of Example 4 of the present invention Bar + Cry9Cb + Vip3Aa11 PCR test results of some transgenic plants; Figure A is the screening marker gene Bar Test results, Figure B is the insect-resistant gene Cry9Cb Test results, Figure C is the insect-resistant gene Vip3Aa11 Test results; M represents DNA marker ladder, + represents plasmid vector, - represents non-transgenic plants, 1~21 represent parts Bar + Cry9Cb + Vip3Aa11 Resistant plants; Figure 10 Part of Example 4 of the present invention Bar + Cry9Cb PCR test results of some transgenic plants; Figure A is the screening marker Bar Genetic testing results, Figure B is the insect-resistant gene Cry9Cb Test results; M represents DNA marker ladder, + represents plasmid vector, - represents non-transgenic plants, 1~21 represent parts Bar + Cry9Cb Transgenic plants; Figure 11 Some positive plant screening markers in Example 4 of the present invention Bar Genetic test strip test results; Figure 12 The herbicide resistance test results of transgenic plants with positive test strips in Example 4 of the present invention; Figure 13 The results of the non-GMO herbicide resistance test in Example 4 of the present invention; Figure 14 Average daily weight change of larvae fed with transgenic plants that tested positive in Example 4 of the present invention; wherein CK represents non-transgenic sugarcane, Bar + Cry9Cb represent Bar + Cry9Cb Positive plants, Bar + Cry9Cb + Vip3Aa11 represent Bar + Cry9Cb + Vip3Aa11 Positive plants; Figure 15 The growth status of larvae at different stages in Example 4 of the present invention; Figure A shows the growth status after feeding non-transgenic sugarcane mixed feed for 3 days, and Figure B shows the growth status after feeding Bar + Cry9Cb The growth status of positive plants after 3 days of mixed feed. Figure C shows the feeding Bar + Cry9Cb + Vip3Aa11 The growth status of positive plants after 3 days of feeding mixed feed, Figure D shows the growth status after 10 days of feeding non-transgenic sugarcane mixed feed, and Figure E shows the growth status after feeding Bar + Cry9Cb The growth status of positive plants after 10 days of mixed feed. Figure F shows the feeding Bar + Cry9Cb + Vip3Aa11 The growth status of positive plants after 10 days of mixed feed, Figure G shows the growth status after 25 days of feeding non-transgenic sugarcane mixed feed, and Figure H shows the growth status after feeding Bar + Cry9Cb The growth status of positive plants after 25 days of mixed feed. Figure I shows the feeding Bar + Cry9Cb + Vip3Aa11 Growth status of positive plants after 25 days of mixed feed. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention are described clearly and completely below. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments to facilitate understanding by those skilled in the art.

[0017] Example 1 Screening of callus induction conditions for the superior sugarcane variety Liucheng 1541 This example is a callus induction condition screening process for an excellent sugarcane variety Liucheng 1541, specifically as follows: Healthy, pest-free sugarcane tail sheaths about 40 cm long were selected from the field, and tender leaf tissues at the top growth point were used as callus explants. The callus explants were first washed in sterile water, then immersed in 75% ethanol for 10 min for disinfection. After disinfection, they were thoroughly washed again in sterile water, and the residual water on the surface was absorbed with sterile filter paper. The leaves were peeled off again, and then 0.1 cm thin slices were cut into 0.1 cm slices and placed in 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L and 2.5 mg / L ethanol, respectively. g / L of 2,4-dichlorophenoxyacetic acid callus induction medium; 10-13 explant slices were inoculated in each 90mm diameter culture dish medium, 5 dishes were inoculated for each 2,4-dichlorophenoxyacetic acid concentration, 5 dishes were used as a replicate, 3 replicates were set for each concentration, and dark culture was carried out in a constant temperature incubator at 28°C; callus tissue was obtained after 15 days, and the culture medium was replaced once. Each dish of callus tissue was expanded and inoculated into the culture medium with the same 2,4-dichlorophenoxyacetic acid concentration for subculture under the same culture conditions. A total of three subcultures were performed, with a total culture cycle of 45 days, to obtain embryonic callus tissue. The callus tissue that was subcultured three times in a row was photographed and recorded, and the number of callus tissue blocks and embryonic callus tissue blocks were counted, and the callus tissue induction rate and embryonic callus tissue induction rate were calculated. The results are as follows Figure 1 shown.

[0018] The callus induction medium with different 2,4-dichlorophenoxyacetic acid concentrations is specifically prepared by adding corresponding concentrations of 2,4-dichlorophenoxyacetic acid, 0.5 mg / L 6-benzylaminopurine, 30 g / L sucrose, 8 g / L agar and 50 mg / L activated carbon to MS medium, with a pH of 5.8.

[0019] Callus induction rate (%) = (number of callus-producing blocks / number of inoculated explant blocks) × 100%; Embryogenic callus induction rate (%) = (number of embryonic callus blocks / number of callus blocks) × 100%.

[0020] The above experiments show that, with the same number of explants inoculated, a higher value indicates a higher callus induction rate, which in turn indicates better callus quality, indicating that the appropriate concentration of 2,4-dichlorophenoxyacetic acid was used. The callus induction rates at 0.5 mg / L were 22.94 ± 1.69%, 1.0 mg / L was 28.86 ± 1.72%, 1.5 mg / L was 35.76 ± 2.08%, 2.0 mg / L was 44.38 ± 3.33%, and 2.5 mg / L was 35.17 ± 3.06%. The embryogenic callus induction rates were 33.04±1.69% at 0.5 mg / L, 42.67±1.69% at 1.0 mg / L, 59.63±1.72% at 1.5 mg / L, 68.96±0.98% at 2.0 mg / L, and 49.17±3.06% at 2.5 mg / L. These results indicate that both callus and embryogenic callus showed distinct trends across treatment concentrations. The 2 mg / L treatment was the most effective, significantly outperforming the other concentrations, while the 0.5 mg / L treatment had the worst effect. In summary, the optimal concentration of 2,4-dichlorophenoxyacetic acid for callus induction in the superior sugarcane variety Liucheng 1541 ranges from 1.0 to 2.5 mg / L, with 2.0 mg / L providing the best induction effect.

[0021] Example 2 Screening of browning resistance conditions for the superior sugarcane variety Liucheng 1541 This example describes a screening process for browning resistance in a superior sugarcane variety, Liucheng 1541. Specifically, young sugarcane leaf slices were inoculated into different culture media (replacing the callus induction medium with different 2,4-dichlorophenoxyacetic acid concentrations in Example 1). Thirteen slices were inoculated into each culture dish, with five dishes forming one replicate, and three replicates were set. Embryogenic calli were then cultivated according to the method described in Example 1. The formulas for the different culture media are shown in the following table: Table 1 Formulas of different culture media in the anti-browning process

[0022] Among them, browning rate (%) = (number of browned callus pieces / number of inoculated explant pieces) × 100%.

[0023] During the subculture process of sugarcane callus, browning phenomenon affects the proliferation ability of Liucheng 1541 callus. Under the condition of no supplementary culture medium, it is difficult to induce embryonic callus of this variety, and it is easy to die during the subculture process ( Figure 2 In addition, the embryonic callus formed often had a water-soaked brown appearance ( Figure 2In contrast, in the medium supplemented with 6-benzylaminopurine, the callus of Liucheng 1541 showed a healthy light yellow color ( Figure 2 C in the figure). During the subsequent subculture process, these calli gradually developed into embryonic calli with obvious granularity and good quality ( Figure 2 The differentiation and transformation efficiency of this type of embryonic callus are relatively ideal.

[0024] The experimental results showed that the five different anti-browning media had significant differences in their inhibitory effects on callus browning ( Figure 3 Among them, the browning rate of the medium without additives was the highest (60.92±0.97%), while the browning rates of the medium supplemented with activated carbon, proline, and PVP were 56.38±1.07%, 54.78±1.55%, and 50.42±1.37%, respectively, all showing a certain anti-browning effect. The medium supplemented with 6-benzylaminopurine had the lowest browning rate (36.80±2.04%), and the callus tissue growth state in this group was the best. The data show that the addition of 6-benzylaminopurine can effectively inhibit browning and promote healthy callus growth during the callus subculture process.

[0025] Example 3 Screening of Glufosinate Concentration in Callus of Superior Sugarcane Variety Liucheng 1541 This example describes a glufosinate concentration screening process for callus of an excellent sugarcane variety, Liucheng 1541. Specifically, high-quality embryonic callus was inoculated onto a callus induction medium containing different glufosinate screening doses (0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, and 5 mg / L) and subcultured for 15 days at room temperature in the dark. The callus was then transferred to a differentiation medium containing the same glufosinate concentration and cultured for 20 days at 30°C under 14 h light / 10 h dark conditions. The callus differentiation status was observed, and the callus differentiation results were shown in FIG. Figure 4 .

[0026] Among them, the callus induction medium containing different glufosinate screening doses is MS medium supplemented with 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L 6-benzylaminopurine, 300 mg / L timentin, corresponding concentrations of glufosinate, 30 g / L sucrose, 8 g / L agar and 50 mg / L activated carbon, with a pH of 5.8; The differentiation medium with the same glufosinate concentration was prepared by adding 300 mg / L timentin, the corresponding concentration of glufosinate, 0.5 mg / L 6-benzylaminopurine, 30 g / L sucrose, 8 g / L agar and 50 mg / L activated carbon to MS medium, with a pH of 5.8.

[0027] It can be seen that at a concentration of 0 mg / L glufosinate, the callus tissue was not inhibited and a large number of young shoots were differentiated ( Figure 4 Figure A in Figure 1); At a concentration of 1 mg / L glufosinate, the callus tissue was slightly inhibited, but it could still differentiate into obvious young shoots ( Figure 4 Figure B); At a concentration of 2 mg / L glufosinate, callus differentiation was significantly inhibited, with only a small number of young shoots appearing ( Figure 4 Figure C). At a concentration of 3 mg / L glufosinate, callus tissue could hardly differentiate into shoots ( Figure 4 At 4mg / L and 5mg / L glufosinate concentrations, no sprouts were visible in the callus tissue ( Figure 4 In summary, the optimal glufosinate concentration during the genetic transformation of the superior sugarcane variety Liucheng 1541 was 1-3 mg / L.

[0028] Example 4 Genetic transformation method of an excellent sugarcane variety Liucheng 1541 This embodiment provides a genetic transformation method for an elite sugarcane variety Liucheng 1541, comprising the following specific steps: S1. Embryogenic callus induction The tail sheath of a healthy and pest-free sugarcane variety Liucheng 1541 was selected from the field. The young leaf tissue at the top growth point was used as the callus explant. The callus explant was first washed in sterile water, then immersed in 75% ethanol for 10 minutes for disinfection. After disinfection, it was washed again in sterile water. The residual water on the surface was absorbed with sterile filter paper, and the leaves were peeled again. Then, 0.1 cm slices were cut into thin slices and placed on callus induction medium for primary callus induction culture. Figure 5 As shown in Figure A of . The callus induction medium is MS medium supplemented with 1.0-2.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.8 mg / L 6-benzylaminopurine, 20-35 g / L sucrose, 7-8 g / L agar, and 40-50 mg / L activated carbon, with a pH of 5.6-5.8. In this embodiment, the callus induction medium is specifically MS medium supplemented with 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L 6-benzylaminopurine, 30 g / L sucrose, 8 g / L agar, and 50 mg / L activated carbon, with a pH of 5.8.

[0029] After 15 days of induction, the thin slice explant twisted and expanded, the core leaf quickly elongated to form a tower-like structure, and the edge differentiated into a uniformly distributed light yellow loose cell mass, namely callus tissue. Figure 5 As shown in Figure B. After 15 days of secondary subculture in callus induction medium, the explants were surrounded by pale yellow callus. The callus was divided into small pieces and inoculated into callus induction medium again for the third subculture 15 days later. Figure 5 As shown in Figure C, pale yellow granular embryonic callus grew at the edge of the callus.

[0030] S2. Preparation of Agrobacterium infection solution use Bar 、 Cry9Cb 、 Vip3Aa11 Gene, according to Figure 6 and Figure 7 Plant expression vectors were constructed using conventional methods. Bar + Cry9Cb and plant expression vectors Bar + Cry9Cb + Vip3Aa11 ;in, Bar The gene is shown in SEQ ID NO: 1, Cry9Cb The gene is shown in SEQ ID NO: 2, Vip3Aa11 The gene is shown in SEQ ID NO:3.

[0031] Using plant expression vectors Bar + Cry9Cb ( Figure 6 ) was transformed into Agrobacterium strain EHA105 to obtain Bar + Cry9Cb Agrobacterium strains for plant expression vectors (i.e., strains Bar + Cry9Cb ).

[0032] Using plant expression vectors Bar + Cry9Cb + Vip3Aa11 ( Figure 7 ) was transformed into Agrobacterium strain EHA105 to obtain Bar + Cry9Cb + Vip3Aa11 Agrobacterium strains for plant expression vectors (i.e., strains Bar + Cry9Cb + Vip3Aa11 ).

[0033] The strain Bar + Cry9Cb Streak on YEP solid medium containing 50 mg / L kanamycin, invert and culture at 28℃ for 48 hours; pick a single colony, add 5 mL of YEP liquid medium, shake at 28℃ overnight; take 1 mL of bacterial liquid, spread on YEP solid medium containing 5 mg / mL rifampicin and 50 mg / mL kanamycin, and invert and culture at 28℃ for 2 days; use 100 mL of liquid culture medium to start the bacteria on the medium, pipette and resuspend them, and shake at 28℃ and 200 r / min for 2 hours until the OD 600 =0.6, that is, the strain is obtained Bar + Cry9CbThe corresponding infection solution. The priming liquid culture medium is 1 / 5 macronutrient MS medium supplemented with 25-35 g / L sucrose, 25-35 g / L glucose, and 100-200 μM acetosyringone, with a pH of 5.3-5.5. In this embodiment, the priming liquid culture medium is 1 / 5 macronutrient MS medium supplemented with 30 g / L sucrose, 30 g / L glucose, and 100 μM acetosyringone, with a pH of 5.4.

[0034] The same Agrobacterium infection solution preparation method was used, using strain Bar + Cry9Cb + Vip3Aa11 Preparation of strains Bar + Cry9Cb + Vip3Aa11 The corresponding infection fluid.

[0035] S3. Transgenic plant cultivation Select high-quality embryogenic callus tissue and blow it on sterile filter paper for 1~2 hours until the surface shrinks slightly. Then add 45℃ preheated starter liquid culture medium and warm it at 45℃ for 5 minutes. After filtering out the starter liquid culture medium, add Bar + Cry9Cb + Vip3Aa11 The corresponding infection solution was ultrasonically shaken for 2 minutes; filtered Bar + Cry9Cb + Vip3Aa11 The corresponding infection solution is added again Bar + Cry9Cb + Vip3Aa11 The corresponding infection solution was vacuumed for 5 minutes, and finally shaken at 80 rpm for 10 minutes. The infected embryonic callus was removed and the infection solution on its surface was dried with sterile filter paper. It was blown dry on a clean bench. The resulting callus was transferred to the co-culture medium and co-cultured in the dark at 21°C for 3 days. The co-cultured callus was transferred to the solid recovery medium and recovered at 28°C for 7 days. Figure 5 As shown in Figure D; the recovered callus tissue was transferred to the screening medium and screened and cultured at 28℃ for 15 days; the screened resistant embryonic callus tissue was inoculated on the differentiation medium, cultured in the dark for 7 days, and then transferred to the light incubator for about 20 days (the daytime light time is 14h / d, the light conditions are 2000lux, and the temperature is 30℃; the night time is 10h / d, and the temperature is 28℃), and young shoots differentiated on the surface of the callus tissue; the young shoots were transferred to a new differentiation medium for secondary differentiation cultivation, as shown in Figure D; the recovered callus tissue was transferred to the screening medium and screened for 15 days; the screened resistant embryonic callus tissue was inoculated on the differentiation medium, cultured in the dark for 7 days, and then transferred to the light incubator for about 20 days (the daytime light time is 14h / d, the light conditions are 2000lux, and the temperature is 30℃; the night time is 10h / d, and the temperature is 28℃), and the young shoots differentiated on the surface of the callus tissue; the young shoots were transferred to the new differentiation medium for secondary differentiation cultivation, as shown in Figure D; the recovered callus tissue was transferred to the screening medium and screened for 15 days; the recovered embryonic callus tissue was transferred to the differentiation medium, and ... Figure 5 When the differentiated seedlings grow to 3-4 cm, they are transferred to the rooting medium and cultured for 20-30 days until the root system is fully grown. Bar + Cry9Cb + Vip3Aa11Transgenic plants (i.e., transgenic plants of Liucheng 1541), such as Figure 5 As shown in Figure F.

[0036] The same method of infecting callus tissue with Agrobacterium was used, and the strain Bar + Cry9Cb Corresponding infection fluid was used for infection and culture, and 26 strains were obtained. Bar + Cry9Cb Transgenic plants (i.e., transgenic plants of Liucheng 1541).

[0037] The co-cultivation medium is a B5 medium supplemented with 1.0-3.0 mg / L 2,4-dichlorophenoxyacetic acid, 100-300 μmol / L acetosyringone, 20-30 g / L sucrose, and 7-8 g / L agar, with a pH of 5.6-5.8. In this embodiment, the co-cultivation medium is a B5 medium supplemented with 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 100 μmol / L acetosyringone, 30 g / L sucrose, and 8 g / L agar, with a pH of 5.8. The solid recovery medium is MS medium supplemented with 1.0-2.0 mg / L 2,4-dichlorophenoxyacetic acid, 200-300 mg / L timentin, 20-30 g / L sucrose, 7-8 g / L agar, and 40-50 mg / L activated carbon, with a pH of 5.6-5.8. In this embodiment, the solid recovery medium is MS medium supplemented with 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 300 mg / L timentin, 30 g / L sucrose, 8 g / L agar, and 50 mg / L activated carbon, with a pH of 5.8. The screening medium is MS medium supplemented with 1.0-2.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.8 mg / L 6-benzylaminopurine, 200-300 mg / L timentin, 1-3 mg / L glufosinate, 20-35 g / L sucrose, 7-8 g / L agar, and 40-50 mg / L activated carbon, with a pH of 5.6-5.8. In this embodiment, the screening medium is specifically MS medium supplemented with 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L 6-benzylaminopurine, 300 mg / L timentin, 2 mg / L glufosinate, 30 g / L sucrose, 8 g / L agar, and 50 mg / L activated carbon, with a pH of 5.8. The differentiation medium is MS medium supplemented with 200-300 mg / L timentin, 1-3 mg / L glufosinate, 0.5-1 mg / L 6-benzylaminopurine, 20-35 g / L sucrose, 7-8 g / L agar, and 40-50 mg / L activated carbon, with a pH of 5.6-5.8. In this embodiment, the differentiation medium is specifically MS medium supplemented with 300 mg / L timentin, 2 mg / L glufosinate, 0.5 mg / L 6-benzylaminopurine, 30 g / L sucrose, 8 g / L agar, and 50 mg / L activated carbon, with a pH of 5.8. The rooting medium is an MS medium supplemented with 200-300 mg / L timentin, 1-2 mg / L glufosinate, 1-2 mg / L naphthaleneacetic acid, 20-35 g / L sucrose, 7-8 g / L agar, and 40-50 mg / L activated carbon, with a pH of 5.6-5.8. In this embodiment, the rooting medium is an MS medium supplemented with 300 mg / L timentin, 2 mg / L glufosinate, 2 mg / L naphthaleneacetic acid, 30 g / L sucrose, 8 g / L agar, and 50 mg / L activated carbon, with a pH of 5.8.

[0038] S4. Cultivation and transplanting of resistant plants The roots that have developed into a strong root system in the rooting medium Bar + Cry9Cb + Vip3Aa11 The transgenic plants were taken out and rinsed thoroughly with clean water to remove the residual culture medium on the roots ( Figure 8 Wash the tissue culture seedlings and place them at room temperature for 14 days for adaptation. After they gradually adapt to the natural environment, they are transplanted into the soil for propagation and cultivation ( Figure 8 When the new leaves were fully expanded and the plants were growing stably, they were planted in the field experimental base. Bar + Cry9Cb + Vip3Aa11 Resistant plants. Among them, Bar + Cry9Cb + Vip3Aa11 A total of 45 transgenic plants were successfully transplanted to obtain resistant plants.

[0039] S5. Molecular identification of transgenic plants Extraction by CTAB method Bar + Cry9Cb + Vip3Aa11 Transgenic plants and Bar + Cry9Cb The DNA of the leaves of the transgenic plants was used as the DNA template for PCR reaction using 2× Rapid Tap Master Mix. The total reaction volume was 25 μL. The PCR primers were shown in Table 2 , and the PCR reaction system was shown in Table 3 .

[0040] Table 2 PCR reaction primer sequences

[0041] Table 3 PCR amplification system

[0042] The PCR reaction program was 34 cycles of pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 15 seconds; the final extension was at 72°C for 5 minutes. The PCR amplification products were detected by electrophoresis on agarose gel (1%) at 150V for 20 minutes. Figure 9 and Figure 10 shown.

[0043] like Figure 9 As shown in Figure A, 39 strains were amplified to bands that matched the size of the positive control, indicating that positive transfection was achieved. Bar There are 39 genetic plants. Figure 9 As shown in Figure B, 34 strains were amplified to bands that matched the size of the positive control, indicating that positive transfection was achieved. Cry9Cb There are 34 genetic plants. Figure 9 As shown in Figure C, 28 strains were amplified to bands that matched the size of the positive control, indicating that positive transfection was achieved. Vip3Aa11 There are 28 gene plants. Among the 45 resistant plants, Bar Gene, Cry9Cb Genes and Vip3Aa11 There were 28 plants with positive results for all genes (i.e. Bar + Cry9Cb + Vip3Aa11 positive plants). Bar 、 Cry9Cb and Vip3Aa11 No positive bands were amplified in the negative controls, indicating that the PCR detection was free of contamination.

[0044] like Figure 10 As shown, using Bar When primers were tested, 26 strains Bar + Cry9Cb Among the transgenic plants, 24 amplified bands with the same size as the positive control, proving that the 24 plants were transgenic. Bar Gene-positive plants. Bar + Cry9Cb Transgenic plants were used as templates. Cry9Cb Primer detection showed that 23 strains amplified bands with the same size as the target band, proving that the 23 strains were transgenic. Cry9Cb Gene positive plants. The above results show that there are 23 plants in total. Bar + Cry9Cb Transgenic plants were simultaneously transferred Bar Genes andCry9Cb Gene (i.e. Bar + Cry9Cb positive plants).

[0045] S6. Resistance plant test strip detection Take the ones in good growth condition Bar + Cry9Cb Positive plants and Vip3Aa11 + Bar + Bar Part of the leaves of the positive plants, crushed and used Cry9Cb Gene detection kits are used to detect positive plants and detect Bar + Cry9Cb Positive plants and Vip3Aa11 + Figure 11 + Bar There are 15 positive plants each, a total of 30 plants. Among the 30 positive plants tested, two red stripes can be seen in the test area of ​​all plants, such as Bar As shown, it indicates that the detection is successful Cry9Cb Gene.

[0046] S7. Indoor herbicide resistance bioassay of resistant plants Select transgenic plants with positive test strips (including Bar + Cry9Cb Positive plants and Vip3Aa11 + Figure 12 + Figure 13 Positive plants) and non-transgenic sugarcane plants with relatively consistent growth were transplanted into pots and sprayed with 0.2%, 0.4%, 0.6% and 0.8% glufosinate herbicide solutions, respectively. At the same time, a herbicide-free (0%) concentration was added as a negative control. After spraying, they were cultured under the same environmental conditions. The growth of the leaves was observed and photographed 10 days after spraying. The transgenic lines grew normally when sprayed with 0.2% glufosinate; when the concentration was increased to 0.4%, the growth of the plant leaves was slightly inhibited, and compared with the control, the line grew slowly; when sprayed with 0.6% glufosinate, the leaf edges showed slight yellowing; after spraying with 0.8% glufosinate, the leaf edges showed obvious yellowing, and the leaves began to curl ( Bar When the non-transgenic strains were sprayed with 0.2% glufosinate, their leaves showed obvious chlorosis and curling. When sprayed with 0.4% glufosinate, the growth of the plants was severely stunted, and the leaves completely lost their green color and wilted. When the concentration was increased to 0.6%, the plants completely withered and died. Cry9Cb ). The transgenic plants continued to grow healthily at a concentration of 0.2% glufosinate ammonium, but the growth of non-transgenic plants was significantly hindered at a concentration of 0.2% glufosinate ammonium.

[0047] S8. Indoor insect resistance bioassay of positive plants Weigh 5g of transgenic plants with positive test strips (including Bar + Cry9Cb Positive plants and Vip3Aa11 + Bar + Cry9Cb Leaves from positive plants and non-transgenic sugarcane plants with similar growth patterns were ground into a powder using liquid nitrogen. 40g of stem borer feed was then weighed and mixed with the leaf powder. After thorough mixing, 10g of the mixture was weighed and placed in 90cm plastic dishes. Ten third-instar larvae of uniform size were placed in each plastic dish and allowed to feed freely, with five larvae per dish, and three replicates per group. The plastic dishes were incubated in a dark incubator at 28°C for 20 days. Non-transgenic sugarcane leaves were used as negative controls for the same insect resistance tests. Starting on the third day of the feeding experiment, larvae were weighed daily for 8 consecutive days, and the average weight change of larvae fed each sample was calculated. Throughout the feeding experiment, the development of the stem borers was observed and photographed.

[0048] The experimental results showed that the growth and development of the two-spotted borer was inhibited after feeding on transgenic sugarcane leaves, showing slow growth and significant weight loss. Some larvae died directly before completing pupation. Among them, the insect-resistant effect of the bivalent insect-resistant gene was more obvious. After 10 days of feeding, Vip3Aa11 + Bar + Cry9Cb The average weight of larvae fed with positive plant leaves increased from 0.0049g on the third day to 0.0095g. Figure 14 + Figure 15 The average weight of larvae fed with positive plant leaf feed increased from 0.0062g to 0.0322g, while the average weight of larvae fed with non-GMO leaf feed increased from 0.0053g to 0.1026g ( Bar Because the insects' weight gain was slow in the early stages, there was no significant difference in weight between the control and transgenic lines, and the inhibitory effect was not obvious. Over time, the insects' weight increased significantly, and the transgenic lines' inhibitory effect on insect growth gradually increased, becoming more significant.

[0049] At the same time, the larvae in different treatment groups showed significant growth differences after 5, 10 and 25 days of feeding. After 5 days of feeding, the control group showed a normal growth and development trend ( Cry9Cb Figure A in the figure), Figure 15 + Bar The positive plant group grew and developed more slowly than the control group ( Cry9Cb Figure B in the figure). Vip3Aa11 + Figure 15 + Figure 15 The positive plant group showed some growth retardation ( Bar Figure C in the figure); after 10 days of feeding, the control group grew further ( Cry9CbD in FIG. 1, Figure 15 + Bar The positive plant group showed growth inhibition Cry9Cb E in FIG. 1, Vip3Aa11 + Figure 15 + Figure 15 The positive plant group showed more obvious growth inhibition Bar F in FIG. 1, Cry9Cb G in FIG. 1, Figure 15 + Bar The pests in the positive plant group could maintain basic growth and development, but showed obvious growth retardation Cry9Cb H in FIG. 1, Vip3Aa11 + Figure 15 + Bar The positive plant group was smaller in size, and some larvae died directly Cry9Cb I in FIG. 1.

[0050] In summary, through the indoor herbicide resistance experiment, the Liucheng 1541 transgenic plants obtained by the genetic transformation method of the application have significant tolerance to glufosinate ammonium, and can still grow healthily under the concentration of 0-0.4% glufosinate ammonium; through the indoor pest resistance experiment, Vip3Aa11 + Bar + Cry9Cb The positive plants have better inhibition effect on the two-point moths than Bar + Cry9Cb The positive plants, showing weight loss and growth inhibition of larvae, indicate that the Liucheng 1541 transgenic plants obtained by the genetic transformation method of the application have pest resistance, and Vip3Aa11 + Bar + Cry9Cb The positive plants have better inhibition effect on the two-point moths than Bar + Cry9Cb After 10 days of feeding, Bar + Cry9Cb The weight of the moths treated by the positive plants is Vip3Aa11 + ​ + ​ 3.39 times of that of the moths treated by the positive plants.

[0051] The other parts not specifically described are prior art. Although the above examples make a detailed description of the application, it is only a part of the examples of the application, not all the examples, and the ordinary skilled in the art can obtain other examples according to the examples without creativity, which are within the protection scope of the application.

Claims

1. A genetic transformation method for the superior sugarcane variety Liucheng 1541, characterized in that: The genetic transformation method comprises the following steps: taking leaf tissue of the sugarcane variety Liucheng 1541 and inducing it on a callus induction medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine; infecting the obtained embryonic callus with Agrobacterium; co-culturing and recovery culture; transferring the recovered callus to a screening medium containing glufosinate ammonium for screening culture; inoculating the screened resistant embryonic callus on a differentiation medium containing glufosinate ammonium for differentiation culture; and then performing rooting culture to obtain transgenic plants of Liucheng 1541.

2. The genetic transformation method of the superior sugarcane variety Liucheng 1541 according to claim 1, characterized in that: The concentration of 2,4-dichlorophenoxyacetic acid in the callus induction medium is 1.0~2.5 mg / L, and the concentration of 6-benzylaminopurine is 0.2~0.8 mg / L.

3. The genetic transformation method of the superior sugarcane variety Liucheng 1541 according to claim 2, characterized in that: The callus induction medium is prepared by adding 1.0-2.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.8 mg / L 6-benzylaminopurine, 20-35 g / L sucrose, 7-8 g / L agar and 40-50 mg / L activated carbon to MS medium, with a pH of 5.6-5.

8.

4. The genetic transformation method of the superior sugarcane variety Liucheng 1541 according to any one of claims 1 to 3, characterized in that: The concentration of glufosinate in the screening medium was 1-3 mg / L; The concentration of glufosinate in the differentiation medium is 1~3 mg / L.

5. The genetic transformation method of the superior sugarcane variety Liucheng 1541 according to claim 4, characterized in that: The screening medium is MS medium supplemented with 1.0-2.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.8 mg / L 6-benzylaminopurine, 200-300 mg / L timentin, 1-3 mg / L glufosinate, 20-35 g / L sucrose, 7-8 g / L agar, and 40-50 mg / L activated carbon, with a pH of 5.6-5.

8. The differentiation medium is MS medium supplemented with 200~300 mg / L timentin, 1~3 mg / L glufosinate, 0.5~1.5 mg / L 6-benzylaminopurine, 20~35 g / L sucrose, 7~8 g / L agar and 40~50 mg / L activated carbon, with a pH of 5.6~5.

8.

6. The genetic transformation method of the superior sugarcane variety Liucheng 1541 according to any one of claims 1 to 3 and 5, characterized in that: The Agrobacterium used for infection is Bar and Cry9Cb Agrobacterium containing genes or Bar 、 Cry9Cb and Vip3Aa11 Agrobacterium of genes; in, Bar The gene is shown in SEQ ID NO: 1, Cry9Cb The gene is shown in SEQ ID NO: 2, Vip3Aa11 The gene is shown in SEQ ID NO:

3.

7. The genetic transformation method of the superior sugarcane variety Liucheng 1541 according to claim 6, characterized in that: Contains Bar and Cry9Cb Agrobacterium tumefaciens is a gene-producing Bar 、 Cry9Cb Gene, construction of plant expression vector Bar + Cry9Cb , and then use plant expression vector Bar + Cry9Cb Transformed into Agrobacterium to obtain; Contains Bar 、 Cry9Cb and Vip3Aa11 Agrobacterium tumefaciens is a gene-producing Bar 、 Cry9Cb 、 Vip3Aa11 Gene, construction of plant expression vector Bar + Cry9Cb + Vip3Aa11 , and then use plant expression vector Bar + Cry9Cb + Vip3Aa11 Transformed into Agrobacterium to obtain.

8. The genetic transformation method of the superior sugarcane variety Liucheng 1541 according to any one of claims 1 to 3, 5 and 7, characterized in that: Embryogenic callus is infected with Agrobacterium by picking up embryogenic callus, adding starting liquid culture medium, and warming; filtering out the starting liquid culture medium and then adding the corresponding infection liquid of Agrobacterium, and ultrasonically shaking; filtering out the corresponding infection liquid of Agrobacterium, and then adding the corresponding infection liquid of Agrobacterium again, vacuuming, and finally shaking culture to complete the infection; The corresponding infection solution of Agrobacterium is obtained by streaking Agrobacterium onto a YEP solid medium containing kanamycin and inverting the medium; picking a single colony and adding it to a YEP liquid medium and shaking it overnight; aspirating the bacterial solution and applying it onto a YEP solid medium containing rifampicin and kanamycin and inverting the medium to culture; using the starting liquid medium, pipetting the resuspended bacteria on the medium and shaking the medium to obtain the corresponding infection solution; The starting liquid culture medium is 1 / 5 macroelement MS medium supplemented with 25-35 g / L sucrose, 25-35 g / L glucose, and 100-200 μM acetosyringone, with a pH of 5.3-5.

5.

9. The genetic transformation method of the superior sugarcane variety Liucheng 1541 according to any one of claims 1 to 3, 5 and 7, characterized in that: The genetic transformation method comprises the following specific steps: Embryogenic callus induction: Young leaf tissue of the sugarcane variety Liucheng 1541 was selected as callus explants and placed on a callus induction medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine for induction culture. The callus induction medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine was replaced twice during the induction process. The callus was subcultured three times to obtain embryonic callus. Transgenic plant cultivation: Pick embryonic callus, add starter liquid culture medium, and incubate; filter out the starter liquid culture medium and then add the corresponding infection liquid of Agrobacterium, and shake with ultrasound; filter out the corresponding infection liquid of Agrobacterium, add the corresponding infection liquid of Agrobacterium again, vacuumize, and finally shake and culture; The infected embryogenic callus was transferred to co-cultivation medium and co-cultivated in the dark; The co-cultured callus tissue was transferred to a solid recovery medium and the culture was resumed; The recovered callus tissue was transferred to a screening medium containing glufosinate-ammonium and cultured for screening; The selected resistant embryogenic callus was inoculated on a differentiation medium containing glufosinate ammonium, cultured in the dark and then cultured in the light, and young shoots were differentiated on the surface of the callus; The young shoots were transferred to a new differentiation medium containing glufosinate ammonium for secondary differentiation cultivation; When the cells differentiate into seedlings, they are individually transferred to a rooting medium and cultured until the root system is fully grown, thereby obtaining transgenic plants of Liucheng 1541.

10. The genetic transformation method of the superior sugarcane variety Liucheng 1541 according to claim 9, characterized in that: The co-culture medium is B5 medium supplemented with 1.0-3.0 mg / L 2,4-dichlorophenoxyacetic acid, 100-300 μmol / L acetosyringone, 20-30 g / L sucrose, and 7-8 g / L agar, with a pH of 5.6-5.8; Solid recovery medium is MS medium supplemented with 1.0-2.0 mg / L 2,4-dichlorophenoxyacetic acid, 200-300 mg / L timentin, 20-30 g / L sucrose, 7-8 g / L agar, and 40-50 mg / L activated carbon, with a pH of 5.6-5.8; The rooting medium is MS medium supplemented with 200~300 mg / L timentin, 1~2 mg / L glufosinate, 1~2 mg / L naphthaleneacetic acid, 20~35 g / L sucrose, 7~8 g / L agar and 40~50 mg / L activated carbon, with a pH of 5.6~5.8.

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