A method for genetic transformation of sugarcane variety liucheng 1541

Abstract

The application provides a genetic transformation method of sugarcane variety Liucheng 1541 and belongs to the technical field of plant genetic engineering. The genetic transformation method is that leaf tissues of the sugarcane variety Liucheng 1541 are induced and cultured on a callus induction culture medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine; the obtained embryogenic callus is infected by agrobacterium; after co-cultivation and recovery culture, the obtained recovered callus is transferred to a screening culture medium containing glufosinate ammonium for screening culture; then the screened resistant embryogenic callus is inoculated on a differentiation culture medium containing glufosinate ammonium for differentiation culture; and after rooting culture, the transgenic plant of Liucheng 1541 is obtained. The method improves the embryogenic callus induction rate, solves the problem of callus browning in the tissue culture process of the variety Liucheng 1541, determines the glufosinate ammonium screening concentration of the embryogenic callus, and establishes the genetic transformation system of Liucheng 1541.

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 sugarcane variety Liucheng 1541. BACKGROUND

[0002] Liucheng 1541, as one of the excellent 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, Liucheng 1541 has been demonstrated in more than 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 sugarcane variety Liucheng 1541.

[0004] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0005] A genetic transformation method of sugarcane variety Liucheng 1541, the genetic transformation method is to take the leaf tissue of sugarcane variety Liucheng 1541 and culture it on a callus induction 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 selection medium containing glufosinate for selection culture, and then the resistant embryogenic callus after selection is inoculated on a differentiation medium containing glufosinate for differentiation culture, and then rooted to obtain transgenic plants of Liucheng 1541.

[0006] Further, 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.

[0007] Further, the callus induction medium is MS medium added with 1.0-2.5 mg / L of 2,4-dichlorophenoxyacetic acid, 0.2-0.8 mg / L of 6-benzylaminopurine, 20-35 g / L of sucrose, 7-8 g / L of agar, and 40-50 mg / L of activated carbon, and the pH is 5.6-5.8.

[0008] Further, the concentration of glufosinate ammonium in the screening medium is 1-3 mg / L.

[0009] The concentration of glufosinate ammonium in the differentiation medium is 1-3 mg / L.

[0010] Further, the screening medium is MS medium added with 1.0-2.5 mg / L of 2,4-dichlorophenoxyacetic acid, 0.2-0.8 mg / L of 6-benzylaminopurine, 200-300 mg / L of thidiazuron, 1-3 mg / L of glufosinate ammonium, 20-35 g / L of sucrose, 7-8 g / L of agar, and 40-50 mg / L of activated carbon, and the pH is 5.6-5.8.

[0011] The differentiation medium is MS medium added with 200-300 mg / L of thidiazuron, 1-3 mg / L of glufosinate ammonium, 0.5-1 mg / L of 6-benzylaminopurine, 20-35 g / L of sucrose, 7-8 g / L of agar, and 40-50 mg / L of activated carbon, and the pH is 5.6-5.8.

[0012] Further, the agrobacterium used for infection is an agrobacterium containing Bar and Cry9Cb genes or an agrobacterium containing Bar , Cry9Cb and Vip3Aa11 genes.

[0013] Among them, Bar the gene is as shown in SEQ ID NO: 1, Cry9Cb the gene is as shown in SEQ ID NO: 2, Vip3Aa11 and the gene is as shown in SEQ ID NO: 3.

[0014] Further, the agrobacterium containing Bar and Cry9Cb genes is constructed by using Bar , Cry9Cb genes to construct a plant expression vector Bar +, Cry9Cb and then using the plant expression vector Bar +, Cry9CbThe Agrobacterium is transformed into Agrobacterium strain EHA105 to obtain;

[0015] The Agrobacterium containing the genes is constructed by using the genes of Bar 、 Cry9Cb and Vip3Aa11 The plant expression vector is constructed by using the genes of Bar 、 Cry9Cb 、 Vip3Aa11 The plant expression vector is transformed into Agrobacterium strain EHA105 to obtain. Bar + Cry9Cb + Vip3Aa11 The plant expression vector is transformed into Agrobacterium strain EHA105 to obtain. Bar + Cry9Cb + Vip3Aa11 The Agrobacterium is transformed into Agrobacterium strain EHA105 to obtain.

[0016] Further, the embryogenic callus is infected by Agrobacterium, that is, the embryogenic callus is picked, and a starting liquid medium is added for incubation; after the starting liquid medium is filtered, the corresponding infection liquid of Agrobacterium is added, and ultrasonic oscillation is performed; after the corresponding infection liquid of Agrobacterium is filtered, the corresponding infection liquid of Agrobacterium is added again, vacuum is extracted, and finally oscillation culture is performed to complete the infection;

[0017] The corresponding infection liquid of Agrobacterium is obtained by the following steps: Agrobacterium is streaked on YEP solid medium containing 50 mg / L kanamycin, and is incubated upside down; a single colony is picked, and is added into YEP liquid medium for oscillation overnight; the bacterial liquid is sucked, and is coated on YEP solid medium containing 5 mg / L rifampicin and 50 mg / L kanamycin, and is incubated upside down; the bacterial body on the medium is resuspended by a starting liquid medium and a pipette blower, and is oscillation cultured to obtain the corresponding infection liquid.

[0018] The starting liquid medium is 1 / 5 MS liquid medium to which 25-35 g / L sucrose, 25-35 g / L glucose and 100-200 μM acetosyringone are added, and the pH is 5.3-5.5.

[0019] Further, the genetic transformation method comprises the following specific steps:

[0020] Embryogenic callus induction: tender leaf tissue of sugarcane variety Liucheng 1541 is selected as callus explants, and is placed on 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 is replaced twice during the induction culture, and the callus induction culture is performed for a total of three times to obtain embryogenic callus.

[0021] Transgenic plant cultivation: the embryogenic callus is picked, and a starting liquid medium is added for incubation; after the starting liquid medium is filtered, the corresponding infection liquid of Agrobacterium is added, and ultrasonic oscillation is performed; after the corresponding infection liquid of Agrobacterium is filtered, the corresponding infection liquid of Agrobacterium is added again, vacuum is extracted, and finally oscillation culture is performed;

[0022] The obtained infected embryogenic callus is transferred into a co-cultivation medium and co-cultivated in dark;

[0023] The co-cultivated callus is transferred onto a solid recovery medium and recovery-cultivated;

[0024] The recovered callus is transferred onto a screening medium containing glufosinate and screened;

[0025] The screened resistant embryogenic callus is inoculated onto a differentiation medium containing glufosinate, dark-cultivated and then light-cultivated, and the callus surface differentiates into seedlings;

[0026] The seedlings are transferred onto a new differentiation medium containing glufosinate and subjected to secondary differentiation cultivation;

[0027] When the seedlings are differentiated into small seedlings, the small seedlings are transferred onto a rooting medium and cultivated until the root system is completely grown, and the transgenic plant of Liucheng 1541 is obtained.

[0028] Further, the co-cultivation medium is a B5 medium added with 1.0-3.0 mg / L 2,4-dichlorophenoxyacetic acid, 100-300 μmol / L acetyl-syringone, 20-30 g / L sucrose and 7-8 g / L agar, and the pH is 5.6-5.8;

[0029] The solid recovery medium is an MS medium added 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, and the pH is 5.6-5.8;

[0030] The rooting medium is an MS medium added 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, and the pH is 5.6-5.8.

[0031] The genetic transformation method of the sugarcane variety Liucheng 1541 has the following beneficial effects:

[0032] The present application explores the induction condition of different concentrations of 2,4-dichlorophenoxyacetic acid on Liucheng 1541, and analyzes the regulation of different medium additives in callus culture to resist browning. The results show that the induction effect of 2.0 mg / L 2,4-dichlorophenoxyacetic acid concentration is the best, and the induction effect of different concentrations shows significant difference; in the medium added with 6-benzylaminopurine, the browning of callus is relieved, and the problem of callus browning in the tissue culture process of Liucheng 1541 is solved; at the same time, through the glufosinate tolerance test, the suitable glufosinate concentration range for maintaining the selection pressure is 1~3 mg / L at the differentiation stage of sugarcane callus, wherein the selection efficiency reaches 93.3% and the transformation efficiency is 15.9% when the glufosinate is 2 mg / L; and the plant expression vector Bar Cry9Cb and the plant expression vector Bar Cry9Cb Vip3Aa11 Liucheng 1541 is genetically transformed by the Agrobacterium-mediated method, and Liucheng 1541 transgenic lines with herbicide resistance and insect resistance characteristics are obtained;

[0033] The Liucheng 1541 transgenic lines obtained by the genetic transformation method of the present application have significant herbicide resistance and insect resistance characteristics through resistance detection; in the glufosinate tolerance experiment, the transgenic lines grow normally when sprayed with 0.2% glufosinate; when the concentration is increased to 0.4%, the growth of plant leaves is slightly inhibited, and the growth of the lines is slow; when sprayed with 0.6% glufosinate, the leaf edges slightly appear yellowing; when sprayed with 0.8% glufosinate herbicide, the leaf edges obviously appear yellowing, and the leaves begin to curl. The non-transgenic lines appear obvious chlorosis and curling phenomenon when sprayed with 0.2% glufosinate; when sprayed with 0.4% glufosinate, the growth of the plants is seriously hindered, the leaves are completely chlorotic and wilted; when the concentration is increased to 0.6%, the plants are completely yellow and dead; the insect resistance experiment shows that the Liucheng 1541 transgenic plants obtained by the genetic transformation method of the present application have insect resistance, and Bar Cry9Cb Vip3Aa11 The insecticidal activity of the positive plants is significantly better than that of Bar Cry9Cb the positive plants, and after feeding for 10d, Bar Cry9Cb the body weight of the borers treated by the positive plants is Bar Cry9Cb Vip3Aa11 3.39 times of that of the positive plants;

[0034] ​​​​​​​​​This invention systematically screened and determined the optimal concentration of 2,4-dichlorophenoxyacetic acid for inducing callus from Liucheng 1541, thereby improving the induction rate of embryogenic callus and laying the foundation for efficient genetic transformation. Through glufosinate-ammonium lethal concentration gradient testing, the concentration range for continuous screening of embryogenic callus was clarified. Combining screening efficiency and transformation efficiency, a genetic transformation screening system for Liucheng 1541 was established.

[0035] This invention is the first to Cry9Cb and Vip3Aa11 The introduction of a dual-gene insect-resistant combination into sugarcane is similar to that of a single gene transfer. Cry9Cb Comparison of insect-resistant genes confirmed that their synergistic effect significantly inhibited the growth of stem borers;

[0036] In this invention, the transgenic sugarcane Liucheng 1541 simultaneously polymerizes Cry9Cb and Vip3Aa11 These two different types of insect-resistant genes, Cry protein, cause osmotic imbalance by disrupting the midgut epithelial cell membrane of insects, while Vip protein induces apoptosis by binding to specific receptors. This dual mechanism of action not only shortens the transgenic breeding cycle but also expands the insecticidal spectrum, especially showing a broader control effect on lepidopteran pests. In addition, the synergistic effect of the multiple toxicity factors of the two proteins can effectively delay the development of resistance in field pests and significantly improve the stability and persistence of insect-resistant traits. Attached Figure Description

[0037] Figure 1 This refers to the callus induction rate of different concentrations of 2,4-dichlorophenoxyacetic acid in Example 1 of the present invention; wherein, 2,4-D is 2,4-dichlorophenoxyacetic acid;

[0038] Figure 2 This is the callus state in Embodiment 2 of the present invention; wherein, Figure A shows browning of the callus, Figure B shows browning of the embryogenic callus, Figure C shows non-browning of the callus, and Figure D shows non-browning of the embryogenic callus.

[0039] Figure 3 This refers to the browning resistance rate of callus tissue in Example 2 of the present invention; wherein, "no additive" represents no additive culture medium, "activated carbon" represents activated carbon culture medium, "proline" represents proline culture medium, "PVP" represents PVP culture medium, and "6BA" represents 6-benzylaminopurine culture medium.

[0040] Figure 4 This is the screening result of the optimal screening concentration of glufosinate in Example 3 of the present invention;

[0041] Figure 5 This is the sugarcane genetic transformation process in Example 4 of the present invention; wherein, Figure A is a slice of young sugarcane tissue, Figure B is callus culture, Figure C is embryogenic callus, Figure D is recovery culture, Figure E is differentiation culture, and Figure F is rooting culture;

[0042] Figure 6 Plant expression vector in embodiment 4 of the present application Bar + Cry9Cb ;

[0043] Figure 7 Plant expression vector in embodiment 4 of the present application Bar + Cry9Cb + Vip3Aa11 ;

[0044] Figure 8 Cultivation of resistant plants in embodiment 4 of the present application; wherein, A figure is the hardening of resistant plants, and B figure is the transplanting of resistant plants into soil

[0045] Figure 9 Part of embodiment 4 of the present application Bar + Cry9Cb + Vip3Aa11 PCR detection results of part of transgenic plants; wherein, A figure is the detection results of screening marker gene Bar , B figure is the detection results of insect-resistant gene Cry9Cb , and C figure is the detection results of insect-resistant gene Vip3Aa11 ; M represents DNA marker ladder, + represents plasmid vector, - represents non-transgenic plants, and 1-21 respectively represent part of Bar + Cry9Cb + Vip3Aa11 resistant plants

[0046] Figure 10 Part of embodiment 4 of the present application Bar + Cry9Cb PCR detection results of part of transgenic plants; wherein, A figure is the detection results of screening marker Bar gene, B figure is the detection results of insect-resistant gene Cry9Cb ; M represents DNA marker ladder, + represents plasmid vector, - represents non-transgenic plants, and 1-21 respectively represent part of Bar + Cry9Cb transgenic plants

[0047] Figure 11 Detection results of screening marker gene of part of positive plants in embodiment 4 of the present application Bar ;

[0048] Figure 12 Test results of herbicide resistance of part of transgenic plants with positive test strips in embodiment 4 of the present application

[0049] Figure 13 Test results of herbicide resistance of non-transgenic plants in embodiment 4 of the present application

[0050] Figure 14 Changes of average daily weight of larvae under feeding treatment of transgenic plant with positive test strip in Example 4 of the present application; wherein, CK represents non-transgenic sugarcane, Bar + Cry9Cb represent Bar + Cry9Cb positive plant, Bar + Cry9Cb + Vip3Aa11 represent Bar + Cry9Cb + Vip3Aa11 positive plant;

[0051] Figure 15 Growth states of larvae at different periods in Example 4 of the present application; wherein, A figure is growth state after feeding non-transgenic sugarcane mixed feed for 3 days, B figure is growth state after feeding Bar + Cry9Cb positive plant mixed feed for 3 days, C figure is growth state after feeding Bar + Cry9Cb + Vip3Aa11 positive plant mixed feed for 3 days, D figure is growth state after feeding non-transgenic sugarcane mixed feed for 10 days, E figure is growth state after feeding Bar + Cry9Cb positive plant mixed feed for 10 days, F figure is growth state after feeding Bar + Cry9Cb + Vip3Aa11 positive plant mixed feed for 10 days, G figure is growth state after feeding non-transgenic sugarcane mixed feed for 25 days, H figure is growth state after feeding Bar + Cry9Cb positive plant mixed feed for 25 days, I figure is growth state after feeding Bar + Cry9Cb + Vip3Aa11 positive plant mixed feed for 25 days. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present application will be described clearly and completely below. In the following description, a lot of specific details are set forth in order to give a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below. The present application will be further described in detail below in combination with specific embodiments, so as to be understood by those skilled in the art.

[0053] Example 1 Screening of callus induction conditions of excellent sugarcane variety Liucheng 1541

[0054] The embodiment is a callus induction condition screening process of a fine sugarcane variety Liucheng 1541, and the process is as follows.

[0055] About 40 cm of a sugarcane tail sheath growing healthily and free of diseases and pests is selected from a field, and tender leaf tissue at a growing point of the tail sheath is used as a callus explant; the callus explant is first washed in sterile water, soaked in 75% ethanol for sterilization for 10 min, washed again in sterile water after sterilization is completed, surface residual water is absorbed using sterile filter paper, the leaf is peeled off again, and then a 0.1 cm thin slice is cut horizontally on callus induction medium containing 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L and 2.5 mg / L of 2,4-dichlorophenoxyacetic acid respectively; 10-13 pieces of the explant thin slice are inoculated in the medium in each 90 mm diameter culture dish, 5 dishes are inoculated for each 2,4-dichlorophenoxyacetic acid concentration, 5 dishes are one repetition, 3 repetitions are set for each concentration, and dark culture is performed in a 28°C constant temperature incubator; callus is obtained after 15 d, the medium is replaced once, and the callus in each dish is inoculated and subcultured under the same culture conditions in the medium with the same 2,4-dichlorophenoxyacetic acid concentration. The subculture is performed three times in total, the total culture period is 45 d, and embryogenic callus is obtained. The callus subcultured three times in succession is photographed and recorded, the number of callus pieces and the number of embryogenic callus pieces are counted, the callus induction rate and the embryogenic callus induction rate are calculated, and the results are shown in Table 1. Figure 1

[0056] The callus induction medium with different 2,4-dichlorophenoxyacetic acid concentrations is specifically MS medium to which 2,4-dichlorophenoxyacetic acid with a corresponding concentration, 0.5 mg / L of 6-benzylaminopurine, 30 g / L of sucrose, 8 g / L of agar and 50 mg / L of activated carbon are added, and the pH is 5.8.

[0057] The callus induction rate (%) = (the number of callus pieces produced / the number of explant pieces inoculated) x 100%;

[0058] The embryogenic callus induction rate (%) = (the number of embryogenic callus pieces / the number of callus pieces) x 100%.

[0059] ​From the above experiments, it can be seen that in the case of inoculating the same number of explants, the larger the value represents the higher the callus induction rate, and the corresponding represents the better the quality of callus, and the concentration of 2,4-dichlorophenoxyacetic acid used is appropriate. For the callus induction rate, at a concentration of 0.5 mg / L, it is 22.94 ± 1.69%; at a concentration of 1.0 mg / L, it is 28.86 ± 1.72%; at a concentration of 1.5 mg / L, it is 35.76 ± 2.08%; at a concentration of 2.0 mg / L, it is 44.38 ± 3.33%; and at a concentration of 2.5 mg / L, it is 35.17 ± 3.06%. For the embryogenic callus induction rate, at a concentration of 0.5 mg / L, it is 33.04 ± 1.69%; at a concentration of 1.0 mg / L, it is 42.67 ± 1.69%; at a concentration of 1.5 mg / L, it is 59.63 ± 1.72%; at a concentration of 2.0 mg / L, it is 68.96 ± 0.98%; and at a concentration of 2.5 mg / L, it is 49.17 ± 3.06%. The above results show that the callus and embryogenic callus under different treatment concentrations all show a clear trend. Among them, the treatment effect of 2 mg / L is the best, which is significantly better than other concentrations, and the treatment effect of 0.5 mg / L is the worst. In summary, in the process of inducing callus of the excellent sugarcane variety Liucheng 1541, the concentration of 2,4-dichlorophenoxyacetic acid can be selected as 1.0-2.5 mg / L, and the induction effect is the best at 2.0 mg / L.

[0060] Example 2 Screening of browning-resistant conditions for the excellent sugarcane variety Liucheng 1541

[0061] This example is a process of screening of browning-resistant conditions for the excellent sugarcane variety Liucheng 1541, specifically, the young leaf slices of sugarcane are inoculated in different culture media (different culture media instead of different 2,4-dichlorophenoxyacetic acid concentrations in the callus induction medium in Example 1), 13 pieces are inoculated in each culture dish, 5 dishes are one repetition, 3 repetitions are set, and embryogenic callus is cultured according to the method in Example 1. The formulations of different culture media are as follows:

[0062] Table 1 Formulations of different culture media in the browning-resistant process

[0063]

[0064] Among them, the browning rate (%) = (the number of browning callus pieces / the number of inoculated explant pieces) x 100%.

[0065] In the process of subculture of sugarcane callus, the browning phenomenon affects the proliferation ability of Liucheng 1541 callus. Under the condition of no addition of culture medium, the callus of this variety is difficult to induce embryogenic callus, and it is prone to death phenomenon in the process of subculture (A figure in Figure 2 In addition, the formed embryogenic callus mostly presents water-stained brown appearanceFigure 2 The transformation efficiency of such calli was generally low (Fig. 2B). In contrast, the Liucheng 1541 calli in the medium with 6-benzylaminopurine appeared healthy and light yellow (Fig. 2C). During the subsequent subculture, these calli gradually developed into granular and high-quality embryogenic calli (Fig. 2D), and the differentiation and transformation efficiency of such embryogenic calli were ideal. Figure 2 Figure 2 The experimental results showed that the browning inhibition effects of the five different browning-resistant media on calli were significantly different (Fig. 3).

[0066] Among them, the browning rate of the medium without addition was the highest (60.92±0.97%), the browning rates of the media with activated carbon, proline and PVP were 56.38±1.07%, 54.78±1.55% and 50.42±1.37%, respectively, all of which showed certain anti-browning effect. The browning rate of the medium with 6-benzylaminopurine was the lowest (36.80±2.04%), and the growth state of the calli in this group was the best. The data showed that during the callus subculture, the addition of 6-benzylaminopurine could effectively inhibit the browning phenomenon and promote the healthy growth of calli. Figure 3

[0067] Example 3 Screening of glufosinate concentration for calli of excellent sugarcane variety Liucheng 1541

[0068] This example is a process for screening of glufosinate concentration for calli of excellent sugarcane variety Liucheng 1541, specifically, high-quality embryogenic calli are inoculated into callus induction medium containing different screening doses of glufosinate (0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L) for subculture at room temperature and in darkness for 15 days; then transferred to differentiation medium containing the same concentration of glufosinate, and cultured at 30°C with 14 h light / 10 h darkness for 20 days; the differentiation state of calli is observed, and the callus differentiation results are as shown in Table 1. Figure 4 .

[0069] Among them, the callus induction medium containing different screening doses of glufosinate is MS medium added with 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L 6-benzylaminopurine, 300 mg / L timentin, corresponding concentration of glufosinate, 30 g / L sucrose, 8 g / L agar and 50 mg / L activated carbon, and the pH is 5.8;

[0070] The differentiation medium with the same concentration of glufosinate is MS medium added with 300 mg / L timentin, corresponding concentration of glufosinate, 0.5 mg / L 6-benzylaminopurine, 30 g / L sucrose, 8 g / L agar and 50 mg / L activated carbon, and the pH is 5.8. ​​

[0071] As can be seen, at 0 mg / L glufosinate concentration, the callus is not inhibited and a large number of sprouts are differentiated (Fig. A in Figure 4 At 1 mg / L glufosinate concentration, the callus is slightly inhibited, but obvious sprouts can still be differentiated (Fig. B in Figure 4 At 2 mg / L glufosinate concentration, the callus differentiation is obviously inhibited, and only a small part of sprouts appear (Fig. C in Figure 4 At 3 mg / L glufosinate concentration, the callus hardly differentiates sprouts (Fig. D in Figure 4 At 4 mg / L and 5 mg / L glufosinate concentration, the callus has no sprouts (Figs. E and F in Figure 4 In summary, the glufosinate concentration is preferably 1-3 mg / L in the genetic transformation process of the excellent sugarcane variety Liucheng 1541.

[0072] Example 4: A genetic transformation method of an excellent sugarcane variety Liucheng 1541

[0073] The present embodiment is a genetic transformation method of an excellent sugarcane variety Liucheng 1541, comprising the following specific steps:

[0074] S1: Embryogenic callus induction

[0075] About 40 cm of the tail sheath of the sugarcane variety Liucheng 1541 growing healthily and free of diseases and pests is selected from the field, and the young leaf tissue at the top growth point is used as the callus explant. The callus explant is first washed in sterile water, soaked in 75% ethanol for sterilization for 10 min, washed thoroughly in sterile water again after sterilization, the surface residual water is absorbed with sterile filter paper, the leaf is peeled off again, and then a 0.1 cm thin slice is transversely cut on the callus induction medium for initial callus induction culture, as shown in Fig. A in Figure 5 The callus induction medium is MS medium added 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, and the pH is 5.6-5.8. In the present embodiment, the callus induction medium is MS medium added 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, and the pH is 5.8.

[0076] After 15 d of induction, the explant slice is twisted and swollen, the core leaf is rapidly elongated to form a tower structure, and the edge is differentiated into uniform distributed light yellow loose cell clusters, i.e. callus, as shown in Fig. B in Figure 5Figure B in the drawings. In the callus induction medium, the explants were subcultured for 15 days, and the original tissues were covered with light yellow calli. The calli were divided into small pieces and subcultured for 15 days in the callus induction medium, and the calli grew into granular embryogenic calli in the edge region, as shown in Figure C in the drawings. Figure 5

[0077] S2, preparation of Agrobacterium infection solution

[0078] Using Bar , Cry9Cb , Vip3Aa11 gene, according to Figure 6 and Figure 7 , a plant expression vector Bar + Cry9Cb was constructed by a conventional method, and a plant expression vector Bar + Cry9Cb + Vip3Aa11 ; wherein, Bar the gene is shown as SEQ ID NO: 1, Cry9Cb the gene is shown as SEQ ID NO: 2, Vip3Aa11 the gene is shown as SEQ ID NO: 3.

[0079] The plant expression vector Bar + Cry9Cb ( Figure 6 ) was introduced into Agrobacterium strain EHA105, and an Agrobacterium strain containing the plant expression vector Bar + Cry9Cb was obtained (i.e., strain Bar + Cry9Cb ).

[0080] The plant expression vector Bar + Cry9Cb + Vip3Aa11 ( Figure 7 ) was introduced into Agrobacterium strain EHA105, and an Agrobacterium strain containing the plant expression vector Bar + Cry9Cb + Vip3Aa11 was obtained (i.e., strain Bar + Cry9Cb + Vip3Aa11 ).

[0081] The strain Bar + Cry9Cb ​Streaked on YEP solid medium containing 50 mg / L kanamycin, 28°C, inverted for 48 h; picked single colony, added 5 mL YEP liquid medium, 28°C, shaken overnight; 1 mL bacterial solution was taken, spread on YEP solid medium containing 5 mg / mL rifampicin and 50 mg / mL kanamycin, 28°C, inverted for 2 d; 100 mL initiation liquid medium was used to resuspend the bacterial body on the medium by a pipette gun, 28°C, 200 r / min, shaken for 2 h to OD 600 = 0.6, i.e. strain Bar Cry9Cb The corresponding infection liquid. The initiation liquid medium is 1 / 5 MS medium with 25-35 g / L sucrose, 25-35 g / L glucose and 100-200 μM acetosyringone, pH 5.3-5.5. In this embodiment, the initiation liquid medium is 1 / 5 MS medium with 30 g / L sucrose, 30 g / L glucose and 100 μM acetosyringone, pH 5.4.

[0082] The same Agrobacterium infection liquid preparation method was used to prepare strain Bar Cry9Cb Vip3Aa11 Strain Bar Cry9Cb Vip3Aa11 The corresponding infection liquid.

[0083] S3, transgenic plant cultivation

[0084] The high-quality embryogenic callus was picked, blown on sterilized filter paper for 1-2 h to make the surface micro-shrink, then 45°C preheated initiation liquid medium was added, 45°C, bathed for 5 min; the initiation liquid medium was filtered out and 45°C preheated initiation liquid medium was added again Bar Cry9Cb Vip3Aa11 The corresponding infection liquid was used, and ultrasonic oscillation was performed for 2 min; the initiation liquid medium was filtered out and 45°C preheated initiation liquid medium was added again Bar Cry9Cb Vip3Aa11 The corresponding infection liquid was used, and ultrasonic oscillation was performed for 2 min; the initiation liquid medium was filtered out and 45°C preheated initiation liquid medium was added again Bar Cry9Cb Vip3Aa11 The corresponding infection liquid was used, and ultrasonic oscillation was performed for 2 min; the initiation liquid medium was filtered out and 45°C preheated initiation liquid medium was added again Figure 5 ​​​​​​​​​​​and transferred to the screening medium, and screened at 28℃ for 15 days. The resistant embryogenic callus after screening was inoculated on the differentiation medium, and transferred to the light incubator after dark culture for 7 days for about 20 days (light time of 14h / d, light condition of 2000lux, and temperature of 30℃; night time of 10h / d, and temperature of 28℃), and the callus surface differentiated into seedlings. The seedlings were transferred to the new differentiation medium for secondary differentiation culture, as shown in FIG. E of Figure 5 When the differentiated seedlings grew to 3-4cm, they were transferred to the rooting medium for culture for 20-30 days until the root system grew completely, and transgenic plants (i.e., transgenic plants of Liucheng 1541) were obtained, as shown in FIG. F of Bar Cry9Cb Vip3Aa11 Figure 5

[0085] Using the same method of Agrobacterium infection of callus, 26 strains of transgenic plants (i.e., transgenic plants of Liucheng 1541) were obtained by using the corresponding infection liquid for infection and culture. Bar Cry9Cb Bar Cry9Cb

[0086] The co-culture medium is B5 medium added with 1.0-3.0mg / L 2,4-dichlorophenoxyacetic acid, 100-300μmol / L acetosyringone, 20-30g / L sucrose, and 7-8g / L agar, and pH is 5.6-5.8; in this embodiment, the co-culture medium is B5 medium added with 2.0mg / L 2,4-dichlorophenoxyacetic acid, 100μmol / L acetosyringone, 30g / L sucrose, and 8g / L agar, and pH is 5.8;

[0087] The solid recovery medium is MS medium added with 1.0-2.0mg / L 2,4-dichlorophenoxyacetic acid, 200-300mg / L timentin, 20-30g / L sucrose, 7-8g / L agar, and 40-50mg / L activated carbon, and pH is 5.6-5.8; in this embodiment, the solid recovery medium is MS medium added with 2.0mg / L 2,4-dichlorophenoxyacetic acid, 300mg / L timentin, 30g / L sucrose, 8g / L agar, and 50mg / L activated carbon, and pH is 5.8;

[0088] ​​​​​​​​The screening medium is MS medium added 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, and the pH is 5.6-5.8; in the embodiment, the screening medium is MS medium added 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, and the pH is 5.8;

[0089] The differentiation medium is MS medium added 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, and the pH is 5.6-5.8; in the embodiment, the differentiation medium is MS medium added 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, and the pH is 5.8;

[0090] The rooting medium is MS medium added with 200-300 mg / L timentin, 1-2 mg / L glufosinate, 1-2 mg / L naphthalene acetic acid, 20-35 g / L sucrose, 7-8 g / L agar and 40-50 mg / L activated carbon, and the pH is 5.6-5.8; in the embodiment, the rooting medium is MS medium added with 300 mg / L timentin, 2 mg / L glufosinate, 2 mg / L naphthalene acetic acid, 30 g / L sucrose, 8 g / L agar and 50 mg / L activated carbon, and the pH is 5.8.

[0091] S4, Cultivation and Transplantation of Resistant Plants

[0092] The plants developed into robust root systems in the rooting medium are taken out and washed with clean water to remove the root residual medium Bar + Cry9Cb + Vip3Aa11 The plants are taken out and washed with clean water to remove the root residual medium Figure 8 , Fig. 2A). The washed plants are placed in a room temperature condition for 14 days of acclimatization, and then transplanted into soil for propagation culture Figure 8 , Fig. 2B). When the new leaves are fully expanded and the plants grow stably, the plants are planted in a field experimental base to obtain Bar + Cry9Cb + Vip3Aa11 resistant plants. Among them, Bar + Cry9Cb +Vip3Aa11 A total of 45 resistant plants were successfully transplanted from the transgenic plants.

[0093] S5. Molecular identification of transgenic plants was performed using the CTAB method. Bar + Cry9Cb + Vip3Aa11 Transgenic plants and Bar + Cry9Cb DNA from the leaves of the transgenic plant was used as a DNA template for PCR reaction using 2×Rapid Tap Master Mix. The total reaction volume was 25µL. The PCR primers are shown in Table 2, and the PCR reaction system is shown in Table 3.

[0094] Table 2 PCR reaction primer sequences

[0095]

[0096] Table 3 PCR amplification system

[0097]

[0098] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, for 34 cycles; final extension at 72℃ for 5 min. The obtained PCR amplification products were detected by agarose gel electrophoresis (1%) at 150V for 20 min. The results are shown below. Figure 9 and Figure 10 As shown.

[0099] like Figure 9 As shown in Figure A, 39 strains amplified to the size of the bands corresponding to the positive control, indicating that positive transformation was obtained. Bar 39 genetically modified plants were identified. Figure 9 As shown in Figure B, 34 strains amplified to the size of the bands corresponding to the positive control, indicating that positive transformation was obtained. Cry9Cb 34 genetically modified plants. (For example...) Figure 9 As shown in Figure C, 28 strains amplified to the size of the positive control, indicating that positive transformation was obtained. Vip3Aa11 28 genetically modified plants were identified. Of the 45 resistant plants, Bar Gene, Cry9Cb Genes and Vip3Aa11 There were 28 plants that were all positive for the gene (i.e.) Bar + Cry9Cb + Vip3Aa11 Positive plants). Bar , Cry9Cb and Vip3Aa11 The negative controls did not amplify any positive bands, indicating that the PCR test was uncontaminated.

[0100] likeFigure 10 As shown in Fig. 6, the use of Bar primers for detection, 26 strains of Bar + Cry9Cb In the transgenic plants, 24 strains amplified bands of the same size as the positive control, proving that 24 strains were transgenic positive plants. Similarly, using the 26 strains of transgenic plants as templates, the use of Bar primers for detection, 23 strains amplified bands of the same size as the target band, proving that 23 strains were transgenic positive plants. The above results showed that a total of 23 strains of transgenic plants were simultaneously transformed with Bar + Cry9Cb gene and Cry9Cb gene (i.e. Cry9Cb + Bar transgenic positive plants). Cry9Cb Bar S6, test paper strip detection of resistant plants Cry9Cb Bar Take well-grown Cry9Cb + transgenic positive plants and

[0101] + +

[0102] + Bar transgenic positive plants, crush them and use Cry9Cb gene detection kit to detect the positive plants, respectively detect Vip3Aa11 + Bar + Bar transgenic positive plants and Cry9Cb + Bar + Cry9Cb + Vip3Aa11 + Figure 11 + Bar transgenic positive plants, a total of 30 plants. In the 30 transgenic positive plants detected, all the detection areas of the plants could appear two red bands, as shown in Fig. 8, indicating that Bar gene was successfully detected. Cry9Cb

[0103] S7, indoor herbicide resistance bioassay of resistant plants

[0104] Select transgenic plants positive for test paper strips (including Bar + Cry9Cb transgenic positive plants and Vip3Aa11 + Figure 12 + Figure 13 transgenic positive plants) for indoor herbicide resistance bioassay.The positive plants and the non-transgenic sugarcane plants with similar growth were transplanted into pots, and 0.2%, 0.4%, 0.6% and 0.8% glufosinate herbicide solutions were sprayed on them, respectively. An additional concentration configuration without herbicide (0%) was set as a negative control. After the spraying treatment, the plants were cultured under the same environmental conditions, and the growth status of the leaves was observed and photographed after 10 days of spraying. Bar The non-transgenic plants showed obvious chlorosis and curling of the leaves when the concentration of glufosinate was 0.2%. When the concentration of glufosinate was 0.4%, the growth of the plants was severely inhibited, the leaves were completely chlorotic and wilted. When the concentration of glufosinate was increased to 0.6%, the plants were completely yellow and died. Cry9Cb The transgenic plants could still grow healthily under the concentration of 0.2% glufosinate, but the growth of the non-transgenic plants was obviously inhibited under the concentration of 0.2% glufosinate.

[0105] S8, In-vitro insect resistance bioassay of positive plants

[0106] 5 g of test paper strips were weighed as transgenic plants (including Bar + Cry9Cb positive plants and Vip3Aa11 + Bar + Cry9Cb non-transgenic sugarcane plants with similar growth were ground into powder with liquid nitrogen, and then 40 g of leaf roller feed was mixed with the leaf powder. After uniform stirring, 10 g of the mixture was placed in a 90 cm plastic dish. Ten 3rd instar larvae with similar sizes were placed in the plastic dish for free feeding, with 5 larvae per dish and 3 replicates per group. The plastic dishes were placed in a 28°C incubator for dark culture, and the feeding experiment lasted for 20 days. Non-transgenic sugarcane leaf materials were used for the same insect resistance bioassay as a negative control. The body weight was weighed every day for 8 consecutive days from the 3rd day of the feeding experiment, and the average body weight change of the larvae fed with each sample was calculated. The development status of the leaf rollers was observed during the feeding experiment, and photographs were taken.

[0107] The experimental results showed that the growth and development of the leaf rollers were inhibited after feeding on the transgenic sugarcane leaves, showing slow growth and significant weight loss, and some larvae died directly without completing pupation. Among them, the dual insect-resistant gene showed more obvious insect resistance effect. After 10 days of feeding, Vip3Aa11 + Bar + Cry9CbThe average weight of the larvae fed with the leaf feed of the positive plant increased from 0.0049g on the 3rd day to 0.0095g, Figure 14 + Figure 15 The average weight of the larvae fed with the leaf feed of the positive plant increased from 0.0062g to 0.0322g, and the average weight of the larvae fed with the non-transgenic leaf feed increased from 0.0053g to 0.1026g Bar ). Due to the slow weight gain of the larvae in the early stage, the weight difference between the control and the transgenic strain was not significant, and the inhibition effect was not obvious. With the passage of time, the weight of the larvae was obviously increased, and the inhibition effect of the transgenic strain on the growth of the larvae was gradually increased, and the effect was more obvious.

[0108] Meanwhile, the larvae in different treatment groups showed significant growth difference after feeding for 5d, 10d and 25d. After feeding for 5d, the control group showed a normal growth and development trend Cry9Cb A graph in the Figure 15 + Bar The growth and development of the positive plant group was slower than that of the control group Cry9Cb B graph in the Vip3Aa11 + Figure 15 + Figure 15 The positive plant group showed a certain growth retardation Bar C graph in the Cry9Cb D graph in the Figure 15 + Bar The positive plant group showed growth inhibition Cry9Cb E graph in the Vip3Aa11 + Figure 15 + Figure 15 The growth retardation of the positive plant group was more obvious Bar F graph in the Cry9Cb G graph in the Figure 15 + Bar The pests of the positive plant group could maintain basic growth and development, but showed obvious growth retardation Cry9Cb H graph in the Vip3Aa11 + Figure 15 + Bar The pests of the positive plant group were smaller in size, and some larvae died directly Cry9Cb I graph in the

[0109] In summary, through the indoor herbicide resistance experiment, the Liucheng 1541 transgenic plant obtained by the genetic transformation method has significant tolerance to glufosinate, and the transgenic plant can still grow healthily under the concentration of 0-0.4% glufosinate; through the indoor pest resistance experiment, Vip3Aa11 + Bar + Cry9CbThe inhibitory effect of the positive plants on the two-point moths is better than that of the negative plants Bar + Cry9Cb The positive plants show weight loss and hindered growth of larvae, indicating that the Liucheng 1541 transgenic plants obtained by the genetic transformation method have insect resistance, and the insecticidal activity of the positive plants is significantly better than that of the negative plants Vip3Aa11 + Bar + Cry9Cb The insecticidal activity of the positive plants is significantly better than that of the negative plants Bar + Cry9Cb The positive plants, after being fed for 10 days, Bar + Cry9Cb The body weight of the moths treated by the positive plants is 3.39 times that of the negative plants Vip3Aa11 + ​ + ​ The body weight of the moths treated by the positive plants is 3.39 times that of the negative plants

[0110] The other parts not specifically described are prior art. Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and the ordinary skilled in the art can also obtain other embodiments according to the present embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A method for genetic transformation of sugarcane variety LiuCheng 1541, characterized in that, The genetic transformation method is that leaf tissues of sugarcane variety Liucheng 1541 are induced and cultured on a callus induction medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine, the obtained embryogenic callus is infected by Agrobacterium, and then co-cultured, recovered and cultured, the obtained recovered callus is transferred to a screening medium containing glufosinate for screening culture, then the screened resistant embryogenic callus is inoculated on a differentiation medium containing glufosinate for differentiation culture, and then rooting culture is carried out to obtain transgenic plants of Liucheng 1541; The Agrobacterium used for infection contains Bar and Cry9Cb Agrobacterium or containing genes Bar , Cry9Cb and Vip3Aa11 Agrobacterium gene; wherein, Bar the gene is as set forth in SEQ ID NO: 1, Cry9Cb the gene is as set forth in SEQ ID NO: 2, Vip3Aa11 the gene is as set forth in SEQ ID NO:

3.

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

8.

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

5. The method of claim 4, wherein the sugarcane variety is Liucheng 1541. The screening medium is MS medium added with 1.0-2.5 mg / L of 2,4-dichlorophenoxyacetic acid, 0.2-0.8 mg / L of 6-benzylaminopurine, 200-300 mg / L of timentin, 1-3 mg / L of glufosinate, 20-35 g / L of sucrose, 7-8 g / L of agar and 40-50 mg / L of activated carbon, and the pH is 5.6-5.

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

8.

6. The method for genetic transformation of the elite sugarcane variety LiuCheng 1541 according to claim 1, characterized in that, containing Bar and Cry9Cb Agrobacterium harboring the gene was used to Bar , Cry9Cb The plant expression vector was constructed using the gene Bar + Cry9Cb The plant expression vector was used to Bar + Cry9Cb Agrobacterium. Agrobacterium containing Bar , Cry9Cb and Vip3Aa11 genes are constructed using Bar , Cry9Cb , Vip3Aa11 genes, plant expression vectors Bar + Cry9Cb + Vip3Aa11 are constructed using the plant expression vectors Bar + Cry9Cb + Vip3Aa11 are introduced into Agrobacterium, and obtained.

7. The method of genetic transformation of the sugarcane variety Liucheng 1541 according to any one of claims 1-3, 5 and 6, wherein, The Agrobacterium infection of the embryogenic callus is that the embryogenic callus is picked up, and a start liquid medium is added for incubation; after the start liquid medium is filtered off, the corresponding Agrobacterium infection liquid is added and ultrasonic oscillation is carried out; the corresponding Agrobacterium infection liquid is filtered off again, the corresponding Agrobacterium infection liquid is added again, vacuum is applied, and finally oscillation culture is carried out to complete the infection. The corresponding infection liquid of the agrobacterium is obtained by the following steps: inoculating the agrobacterium into YEP solid culture medium containing kanamycin, and inverting culture; picking single colony, adding YEP liquid culture medium, and oscillating overnight; absorbing the bacterial liquid, and coating on YEP solid culture medium containing rifampicin and kanamycin, and inverting culture; using the starting liquid culture medium, and resuspending the bacterial body on the culture medium by blowing with a pipette gun, and oscillating culture, so as to obtain the corresponding infection liquid; The starting liquid culture medium is 1 / 5 MS medium added with 25-35 g / L sucrose, 25-35 g / L glucose and 100-200 μM acetosyringone, and the pH is 5.3-5.

5.

8. The method of genetic transformation of the sugarcane variety LiuCheng 1541 according to any one of claims 1-3, 5 and 6, characterized in that, The genetic transformation method comprises the following specific steps: Embryogenic callus induction: young leaf tissue of sugarcane variety Liucheng 1541 is selected as callus explant, and is placed on 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 is replaced twice during the culture, and the callus is subcultured for three times in total, so as to obtain embryogenic callus; Transgenic plant cultivation: the embryogenic callus is picked, and is added with the starting liquid culture medium, and is warmed; after the starting liquid culture medium is filtered, the corresponding infection liquid of the agrobacterium is added, and is ultrasonically oscillated; the corresponding infection liquid of the agrobacterium is filtered again, the corresponding infection liquid of the agrobacterium is added again, vacuum is drawn, and finally oscillation culture is carried out; The obtained infected embryogenic callus is transferred to co-culture medium, and is co-cultured in dark; The co-cultured callus is transferred to solid recovery culture medium, and is recovered; The recovered callus is transferred to screening culture medium containing glufosinate, and is screened; The screened resistant embryogenic callus is inoculated on differentiation culture medium containing glufosinate, and is dark cultured, and then is illuminated and cultured, so that young buds are differentiated on the surface of the callus; The young buds are transferred to new differentiation culture medium containing glufosinate, and are subjected to secondary differentiation cultivation; When the buds are differentiated into seedlings, the seedlings are separated into single plants, and are transferred to rooting culture medium for culture until the root system is completely grown, so as to obtain transgenic plants of Liucheng 1541.

9. The method of claim 8, wherein the sugarcane variety is Liucheng 1541. The co-culture medium is B5 medium added 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, and the pH is 5.6-5.8; The solid recovery culture medium is MS medium added 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, and the pH is 5.6-5.8; The rooting culture medium is MS medium added with 200-300 mg / L timentin, 1-2 mg / L glufosinate, 1-2 mg / L naphthalene acetic acid, 20-35 g / L sucrose, 7-8 g / L agar and 40-50 mg / L activated carbon, and the pH is 5.6-5.8.

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