CN119193684B

CN119193684B - A method for genetic transformation of taro

Info

Publication number: CN119193684B
Application number: CN202411731651.8A
Authority: CN
Inventors: 肖遥; 桂艳玲; 黄英金; 张玉凤; 何逸宁; 王延芝; 周庆红; 朱强龙; 罗莎
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2024-11-29
Filing date: 2024-11-29
Publication date: 2025-06-24
Anticipated expiration: 2044-11-29
Also published as: CN119193684A

Abstract

The invention discloses a genetic transformation method of taro, and belongs to the technical field of biological breeding. The method comprises the steps of taking embryogenic callus induced by the stem tip of taro, carrying out propagation, infecting the embryogenic callus induced by the stem tip of taro by using agrobacterium, carrying out co-culture, screening of resistant buds, induction of test tube taro, hardening seedling and transplanting to obtain taro genetic transformation seedlings, wherein the agrobacterium is an agrobacterium strain GV3101 for transforming pRI101-CeSUC3 recombinant plasmids. According to the method, the genetic transformation system of the taro, which is efficient and stable and suitable for taro varieties of different genotypes, is established by screening and comparing the infection mode in the taro genetic transformation process, the culture condition after infection, the hormone coordination and concentration in the bud induction process and whether the test tube taro is induced or not, the problems of difficult genetic transformation of the taro and the like are overcome, and a foundation is laid for biological breeding of the taro.

Description

Genetic transformation method of taro

Technical Field

The invention belongs to the technical field of biological breeding, and particularly relates to a genetic transformation method of taros.

Background

Taro Colocasia esculenta (L) Schott is a traditional medicinal and edible crop, and the bulb of taro is rich in a plurality of physiologically active substances, can relieve various sub-health symptoms of human bodies, and is deeply favored by consumers. Because of the typical asexual propagation characteristics and narrow genetic background, the cultivation of new breakthrough varieties is difficult. Genetic transformation technology is the basis of biological breeding, and can be used for rapid and accurate improvement of crop varieties. However, there is no report on genetic transformation technology of taro.

The agrobacterium-mediated genetic transformation method has important function in plant transgenic breeding due to the advantages of high efficiency, stability, simple operation and the like. The transgenic breeding can provide a rapid, efficient and accurate way for cultivating the new taro breakthrough variety. Therefore, the development of the genetic transformation technology of the taro not only can enrich the breeding method of the taro, but also can change the support taro from traditional breeding to biological breeding.

Disclosure of Invention

The invention aims to at least solve one of the technical problems in the prior art and provides a genetic transformation method for taros.

The technical scheme of the invention is as follows:

in order to achieve the aim of the invention, the invention adopts the following technical scheme:

The invention provides a genetic transformation method of taro, which specifically comprises the following steps:

S1, taking embryogenic callus induced by a stem tip of taro, and carrying out propagation to obtain newly-proliferated embryogenic callus;

S2, transferring the embryogenic callus after co-culture to a differentiation screening culture medium to obtain a resistant bud;

S3, selecting robust differentiation buds, transferring to a seedling culture medium, and obtaining resistant seedlings;

S4, selecting resistant seedlings with the height of 5-7 cm, transferring to a test tube taro induction medium, and obtaining resistant test tube taros;

s5, hardening seedlings of the resistant test-tube taros and planting the test-tube taros in a greenhouse.

Wherein the agrobacterium is agrobacterium strain GV3101 transformed with pRI101-CeSUC3 recombinant plasmid.

Alternatively, the method for obtaining Agrobacterium strain GV3101 transformed with pRI101-CeSUC3 recombinant plasmid comprises the following steps:

The method uses taro cDNA as a template for amplification, wherein the system comprises 2 mu L of DNA template, 8 mu L of DNA polymerase, 0.5 mu L of upstream primer, 0.5 mu L of downstream primer and ddH ₂ O to 20 mu L, the procedures comprise 94 ℃ pre-denaturation for 5min, 94 ℃ denaturation for 30 s,54 ℃ annealing for 20 s,72 ℃ extension for 45 s and 36 cycles, 72 ℃ final extension for 5min and 4 ℃ preservation, and CeSUC3 fragment added with homologous recombination sequences are obtained, wherein the upstream primer F (SEQ ID NO: 1) is 5'-TCTTCACTGTTGATACATATGATGGACGCCATCTCGATCCG-3', and the downstream primer R (SEQ ID NO: 2) is 5'-AGAGTTGTTGATTCAGAATTCTTAGCCAAATCCATGAAGACCCG-3';

AN NdeI and EcoRI restriction endonuclease is adopted to enzyme-cut AN expression vector pRI101-AN annular plasmid to obtain linearized pRI101-AN;

Mixing the linearized pRI101-AN and CeSUC fragments added with the homologous recombination sequences according to a molar ratio of 1:3, and incubating for 30min at 37 ℃ with 1 mu L of the linearized pRI101-AN,3 mu L of CeSUC fragments added with the homologous recombination sequences, 5 mu L of a universal enzyme premix, and ddH ₂ O to 10 mu L;

The recombinant mixed solution is transformed into escherichia coli DH5 alpha, a plate is coated, a monoclonal bacterium is selected, shaking bacteria is carried out, colony PCR is carried out, a monoclonal bacterium solution with a target strip is selected for sequencing, a monoclonal bacterium with the correct sequence is selected for shaking bacteria again, recombinant plasmid pRI101-CeSUC3 is extracted, and agrobacterium GV3101 is transformed by 37 ℃ heat shock.

Optionally, in the above method, the embryogenic callus material induced by the stem tip in step S1 is derived from stem tip induction of different genotype taro varieties such as curved root taro, flower and fruit taro, ganyu taro No. 1, ganyu taro No. 2, etc.;

Optionally, in the method, the proliferation medium in the step S1 is MS+30 g/L of sucrose+TDZ 2 mg/L+NAA 0.1 mg/L+6.5 g/L of agar powder, and the pH is 5.8-6.0, and the proliferation condition is culturing under dark condition, and the temperature is 24-26 ℃;

Alternatively, in the above method, the method of infecting newly proliferated embryogenic callus with agrobacterium in step S1 specifically includes:

pre-treating agrobacterium, re-suspending the thallus by using an agrobacterium infection solution, and adjusting the concentration of the thallus to OD ₆₀₀ = 0.6-0.8 after re-suspending;

Collecting newly proliferated embryogenic callus in a sterile conical flask, pouring a bacterial solution, sealing with a sterile sealing film, infecting for 15min on a 100 rpm shaker at room temperature, and maintaining for 30min under vacuum condition of-0.1 MPa;

wherein the agrobacterium infection liquid comprises the following components of 1/2MS +30 g/L sucrose+20-30 mu mol/L acetosyringone and pH of 5.8-6.0,

Optionally, in the above method, the method for pretreatment of agrobacterium comprises:

Adding corresponding antibiotics into 2mL YEB culture medium, shake culturing 180: 180 rpm at 28deg.C for 12h until thallus is completely recovered;

Adding 500 mu L of recovered bacterial liquid into a 10 mL YEB culture medium, adding corresponding antibiotics, and performing shake cultivation for 12 hours at a temperature of 28 ℃ by 180 rpm until the OD ₆₀₀ of the bacterial liquid reaches 0.9-1.1;

Centrifuging at 5000 rpm for 10min, discarding supernatant, and collecting Agrobacterium.

Optionally, in the method, the co-culture medium in the step S1 comprises the following components of MS+30 g/L of sucrose+20-30 mu mol/L of acetosyringone, and pH is 5.8-6.0, wherein the co-culture condition is that the dark culture is carried out for 3-4 d at 24-26 ℃;

Optionally, in the method, the differentiation screening culture medium in the step S2 comprises the following components of MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+sucrose 30 g/L+acetosyringone 20 mu mol/L-30 mu mol/L+termeitin 250 mg/L+kanamycin 50 mg/L, wherein the pH is 5.8-6.0, the differentiation culture condition is 24-26 ℃, the dark culture is carried out for 7 d-14 d, the culture is carried out under the illumination condition, the illumination intensity is 2000 Lx-3000 Lx, and the light period is 16h illumination and 8h darkness;

Optionally, in the method, the seedling culture medium in the step S3 comprises the following components of MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+sucrose 30 g/L+timetin 250 mg/L+kanamycin 50 mg/L, wherein the pH is 5.8-6.0, the seedling culture condition is that the temperature is 24-26 ℃, the illumination intensity is 2000 Lx-3000 Lx, the photoperiod is 16h illumination and 8h darkness;

optionally, in the method, the test tube taro induction medium in the step S4 comprises the following components of MS+sucrose 80 g/L+timentin 250 mg/L+kanamycin 50 mg/L, pH of 5.8-6.0, and conditions of test tube taro induction culture, namely, temperature of 24-26 ℃, illumination intensity of 2000 Lx-3000 Lx, photoperiod of 16h illumination and darkness of 8 h;

Optionally, in the method, the resistant test tube taro in the step S5 is moved to a normal temperature condition, the seedling is refined for 3-5 d, then the seedling is taken out, a basal medium is cleaned and planted in a matrix, the matrix is turf, the volume ratio of the vermiculite is 2:1, bagging and moisturizing are carried out for 3 d-5 d, the growth condition is that the temperature is 20-30 ℃, the illumination intensity is 2000 Lx-4000 Lx, the light period is 12 h-14 h illumination, 10 h-12 h darkness, and the survival plant is obtained.

The invention has at least one of the following beneficial effects:

1. the genetic transformation method of taro is provided, and agrobacterium is used for infecting embryogenic callus induced by taro stem tip, and co-culturing, screening resistant buds, inducing test tube taro, hardening seedling and transplanting are carried out to obtain the genetic transformation seedling of taro.

2. The invention establishes a high-efficiency stable taro genetic transformation system suitable for taro varieties with different genotypes by screening and comparing infection modes, culture conditions, hormone coordination and concentration and test tube taro induction or not, overcomes the problems of difficult taro genetic transformation and the like, and can be used for biological breeding of taros.

Drawings

FIG. 1 is a physical diagram of the embryogenic callus of taro in example 1;

FIG. 2 is a diagram showing the co-culture of embryogenic callus and Agrobacterium in example 1;

FIG. 3 is a diagram of the transformation vector in example 1;

FIG. 4 is a physical diagram of resistant shoots screened for calli after infection in example 1;

FIG. 5 is a physical view of seedlings grown from resistant buds in example 1;

FIG. 6 is a physical diagram of the resistant seedling-induced test tube taro in example 1;

FIG. 7 shows the PCR electrophoretic detection of pRI101-CeSUC strain of example 1;

FIG. 8 shows the PCR electrophoretic detection of pRI101-CeSUC strain of comparative example 2;

FIG. 9 shows the PCR electrophoretic detection of pRI101-CeSUC strain of comparative example 3.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1 Agrobacterium-mediated genetic transformation of taro

The method specifically comprises the following steps:

(1) Expanding propagation of stem tip embryogenic callus

A. Inoculating embryogenic callus induced by Gantain No. 2 stem tip into callus proliferation culture medium in an ultra-clean workbench, wherein the proliferation culture medium is MS+sucrose 30 g/L+thidiazuron (TDZ) 2 mg/L+naphthalene acetic acid (NAA) 0.1 mg/L+agar powder 6.5 g/L, and the pH is 5.8;

b. Culturing in dark condition at 25deg.C;

(2) Construction of overexpression vector

A. Specific primers were designed based on the sequence of the taro CeSUC3 gene (gene number EVM 0005823.1) and a homologous recombination sequence (primer-streaking portion) was added, the upstream primer F (SEQ ID NO: 1) 5'-TCTTCACTGTTGATACATATGATGGACGCCATCTCGATCCG-3', the downstream primer R (SEQ ID NO: 2) 5'-AGAGTTGTTGATTCAGAATTCTTAGCCAAATCCATGAAGACCCG-3', amplified with taro cDNA as a template, in the system of 2. Mu.L of DNA template, 8. Mu.L of DNA polymerase (PRIMESTAR ^® Max DNA Polymerase, R045A, takara), 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, ddH ₂ O were made up to 20. Mu.L, the procedure was 94℃for 5min, 94℃for 30 s for 20 s for 54 annealing, 72℃for 45 s for 36 cycles, and 72℃for 5min for 4℃for final preservation;

b. Recovering CeSUC target fragment from running gel, connecting to pMD19-T (Takara) sequencing to obtain monoclonal vector with correct sequence, namely CeSUC fragment added with homologous recombination sequence;

c. The overexpression vector pRI101-AN was linearized by digestion with restriction enzymes NdeI and EcoRI (NEW ENGLAND Biolabs) at 25℃for 1h, with pRI101-AN 1. Mu.g, ndeI and EcoRI each 1. Mu.L, buffer 1. Mu.L and ddH ₂ O to 20. Mu.L;

Mixing the recovered linearized vector with CeSUC fragment added with homologous recombination sequence at a molar ratio of 1:3, incubating for 30min at 37℃with 1. Mu.L of linearized pRI101-AN, 3. Mu.L of CeSUC added with homologous recombination sequence, 5. Mu.L of universal enzyme premix (2 x Hieff Clone Universal Enzyme Premix, hirudo Biotech Co., ltd.) and ddH ₂ O up to 10. Mu.L;

d. The recombinant mixed solution is transformed into escherichia coli DH5 alpha (Hua-Vietnam biotechnology Co., ltd.), coated, selected to be monoclonal, shaken, subjected to colony PCR, selected to be a target strip monoclonal bacterial solution for sequencing, selected to be a correct sequence monoclonal and shaken again, and extracted to obtain recombinant plasmid pRI101-CeSUC3, and the recombinant plasmid is subjected to heat shock at 37 ℃ to transform agrobacterium GV3101 (Hua-Vietnam biotechnology Co., ltd.).

(3) Preparation for Agrobacterium infection

A. Picking agrobacterium GV3101 single colony containing pRI101-CeSUC3 plasmid into 2mL YEB culture medium, adding kanamycin and rifampicin, and shake culturing 180 rpm under final concentration of 50 mg/L and 25 mg/L and 28 ℃ temperature condition for 12h until thallus is completely recovered;

b. Adding 500 mu L of resuscitated bacterial liquid into 10 mL YEB culture medium, adding kanamycin and rifampicin, and performing 180 rpm shake cultivation for 12 hours at the temperature of 28 ℃ until the bacterial liquid OD ₆₀₀ reaches about 1.0, wherein the final concentration is 50 mg/L and 25 mg/L;

c. Centrifuging at rotation speed of 5000 rpm for 10 min, collecting thallus, discarding supernatant, and re-suspending thallus with a solution of 1/MS +sucrose 30 g/L+acetosyringone 25 μmol/L, wherein the pH is 5.8, and the concentration of the thallus is adjusted to OD ₆₀₀ =0.8 after re-suspension;

(4) Agrobacterium infects embryogenic callus

A. Collecting embryogenic callus cultured under the dark condition in the step (1) in a 50 mL sterile conical flask, pouring the bacterial liquid in the step (3), sealing by a sterile sealing film, infecting 15 min, and placing the process in a 100 rpm shaking table to shake continuously;

b. Placing in a vacuumizing device again, and keeping for 15min under the vacuum condition of-0.1 MPa to ensure that the callus is fully contacted with the bacterial liquid;

c. removing bacterial liquid in an ultra-clean bench, sucking the bacterial liquid on the surface of the callus by using sterile absorbent paper, uniformly placing the bacterial liquid on a co-culture medium for culture, wherein the co-culture medium comprises the following components of MS+sucrose 30 g/L+acetosyringone 25 mu mol/L, pH of 5.8, and dark culture for 4d at the temperature of 25 ℃;

(5) Screening of resistant shoots

A. Uniformly placing the callus of the co-culture 4d in the step (4) on a differentiation screening culture medium, wherein the differentiation screening culture medium comprises the following components of MS+6-benzylaminoadenine (6-BA) 1.0 mg/L+naphthalene acetic acid (NAA) 0.5 mg/L+sucrose 30 g/L+acetosyringone 25 mu mol/L+timentin 250 mg/L+kanamycin 50 mg/L and pH of 5.8;

b. Culturing at 25deg.C in darkness for 14d, culturing under illumination with illumination intensity of 3000 Lx and photoperiod of 16 hr illumination and darkness for 8 hr;

c. 1 transfer every 15d until the callus differentiated resistant buds;

d. Transferring the resistant buds to a seedling culture medium until the length of the seedling culture medium is 5 cm-7 cm, wherein the seedling culture medium comprises the following components of MS+6-benzylaminoadenine (6-BA) 1.0 mg/L+naphthalene acetic acid (NAA) 0.5 mg/L+sucrose 30 g/L+timentin 250 mg/L+kanamycin 50 mg/L, the pH is 5.8, the seedling culture condition is that the temperature is 25 ℃, the illumination intensity is 3000 Lx, the light period is 16h illumination and 8h darkness.

(6) Test tube taro induction

A. Transferring a seedling with a robust growth height of 5 cm-7 cm into a test tube taro induction culture medium for culture, wherein the test tube taro induction culture medium comprises the following components of MS+sucrose 80 g/L+timentin 250 mg/L+kanamycin 50 mg/L and pH of 5.8;

b. The culture conditions of the test tube taro are that the temperature is 25 ℃, the illumination intensity is 3000 Lx, the light period is 16h illumination and 8h darkness;

c. expanding the test tube taro after 6-8 weeks;

(7) Soil-shifting cultivation of transgenic plants

A. Transferring test tube taro to normal temperature for hardening off 3d, taking out seedling, cleaning basal culture medium, planting in completely wetted matrix (turf: vermiculite volume ratio=2:1), and compacting;

b. And bagging for moisturizing for 3 days, and placing the plants in a greenhouse for growth under the conditions that the temperature is 25 ℃, the illumination intensity is 4000 Lx, the photoperiod is 12 illumination and the darkness is 12 hours, so that the survival plants are obtained.

Comparative example 1 Effect of different differentiation Medium on the budding of Conus embryogenic callus

The genetic transformation of taro was substantially the same as in example 1, except that 6-BA (1.0, 2.0 mg/L) and TDZ (1.0, 2.0 mg/L) were sequentially selected in step (5) and combined with NAA (0.1, 0.3, 0.5 mg/L) respectively to induce buds, and the effects of different concentration ratios on the budding rate of taro were compared.

As shown in Table 1, the differentiation of the embryogenic callus is obviously different under the conditions of different hormone coordination and concentration, when cytokinin is TDZ, the germination rate of the callus is relatively stable and is between 32.2 and 55.6 percent, when cytokinin is 6-BA, the germination rate of the callus is greatly changed, the minimum germination rate is only 15.6 percent, and when NAA content is 0.5 mg/L and 6-BA content is 1.0 mg/L, the differentiation effect is best, and the germination rate can reach more than 70 percent.

Comparative example 2 Effect of vacuuming treatment on genetic transformation Rate of taro

The genetic transformation of taro was essentially the same as in example 1, except that in step (4) the embryogenic callus was not subjected to-0.1 MPa vacuum during the infection, and the effect of both treatments on positive transformation was compared.

Comparative example 3 Effect of darkness treatment after callus infection on genetic transformation Rate of taro

Genetic transformation of taro was substantially the same as in example 1, except that the selection of resistant shoots in step (5) was transferred to a differentiation selection medium without prior dark culture for 14d, and the effect of both treatments on positive transformation was compared.

Table 2 shows comparison of positive transformation ratios of example 1 and comparative examples 2 to 3, extraction of transplanted regenerated plant leaf DNA, PCR detection was performed with untransformed Ganged Gangena 2 plant as negative control, pRI101-SUC3 plasmid as positive control, detected fragment size of 398bp, detection primer F (SEQ ID NO: 3): 5'-CACGGGGGACTCTAGATACA-3', R (SEQ ID NO: 4): 5'-GGCATGTTCAATTCCGAGTGT-3', PCR detection reaction system of 2. Mu.L DNA template, 8. Mu.L universal enzyme premix (2 x Hieff Clone Universal Enzyme Premix, hirudo Co., ltd.) 0.5. Mu.L upstream primer, 0.5. Mu.L downstream primer, ddH ₂ O up to 20. Mu.L, PCR amplification procedure of 94℃pre-denaturation for 4min, 98℃denaturation 10s,54℃annealing 30s,72℃extension 30s,36 cycles, 72℃final extension for 5min, 4℃preservation, and PCR product detection by agarose gel electrophoresis with 1.0% concentration (FIG. 7).

FIG. 7 is a PCR electrophoresis detection chart of pRI101-CeSUC strain in example 1, wherein a total of 23 regenerated plants are obtained, M is a marker (Maker), + is a positive control group, -is a negative control group, -1-23 are pRI101-CeSUC regenerated plants, 100bp, 250bp and 500bp respectively represent the sizes of marker bands, positive plants with fragments of the same size as the positive control group are found, and negative plants without bands are found. That is, of the 23 regenerated plants obtained in example 1, 15 were positive plants and 8 were negative plants.

FIG. 8 is a PCR electrophoretically detected map of pRI101-CeSUC strain of comparative example 2, in which M is marker (Maker) and +positive control group, -negative control group, pRI101-CeSUC3 regenerated plant is 1-29, 100bp, 250bp and 500bp represent marker band sizes, positive plant with fragment of the same size as that of positive control group and negative plant without band. Namely, of the 29 regenerated plants obtained in comparative example 2,2 were positive plants and 27 were negative plants.

FIG. 9 is a PCR electrophoretically detected map of pRI101-CeSUC strain in comparative example 3, in which M is marker (Maker) and +positive control group, -negative control group, pRI101-CeSUC3 regenerated plant is 1-8, and 100bp, 250bp and 500bp represent marker band sizes, respectively, positive plant with fragment of the same size as that of positive control group and negative plant without band. Namely, of the 8 regenerated plants obtained in comparative example 3,3 were positive plants and 5 were negative plants.

FIG. 1 is a physical map of embryogenic callus of taro in example 1, FIG. 2 is a physical map of embryogenic callus of example 1 co-cultured with Agrobacterium, FIG. 3 is a plasmid map of genetic expression vector used in example 1, which shows characteristics of plasmid pRI101-AN, FIG. 4 is a physical map of resistant shoots screened for callus after infection in example 1, FIG. 5 is a physical map of seedlings grown from resistant shoots in example 1, FIG. 6 is a physical map of resistant shoot-induced test tube taro in example 1, and it can be seen from FIGS. 1 to 7 that the method of example 1 can obtain transgenic plants, thereby being applicable to biological breeding of taros.

As can be seen from the comparison of example 1 and comparative example 2, the vacuuming treatment can improve the positive conversion rate, and as can be seen from the comparison of example 1 and comparative example 3, the dark culture 14d can improve the positive conversion rate after the infection of embryogenic callus by agrobacterium.

Comparative example 4 influence of Induction of the survival rate of resistant seedlings by test-tube taro or not

The genetic transformation of taro is basically the same as that of example 1, except that in the step (6), a plant which grows robustly and is 5-7 cm high is transferred into a rooting medium for culture, wherein the rooting medium comprises the following components of MS+6-BA 1.0 mg/L+NAA 1.0 mg/L+sucrose 30 g/L+Temeitin 250 mg/L+kanamycin 50 mg/L, the pH is 5.8, the rooting culture condition is that the temperature is 25 ℃, the illumination intensity is 3000 Lx, the photoperiod is 16h illumination, and 8h darkness is achieved until rooting. The resistant plants obtained in example 1 and comparative example 4 were transplanted, and after 30d, the survival rate was counted and the plant growth was recorded.

As can be seen from Table 3, the survival rate is higher and the seedling growth condition is better after the test tube taro is transplanted.

Example 2 comparison of Positive plant Rate of constructed genetic transformation System in different genotypes of taro variety

Genetic transformation of taro is basically the same as that of example 1, except that embryogenic callus induced by stem tips of a plurality of taro varieties with different genotypes such as curved root taro, flower fruit taro, ganyu 1 and the like is sequentially used to carry out genetic transformation test according to the steps of example 1, and positive plant rates of the taro varieties with different genotypes are compared.

As shown in Table 4, the developed genetic transformation technology of taro can be used for taro varieties with different genotypes, the positive plant rate of the varieties is 48.0% -80.0%, and the positive transformation rate is 2.7% -5.0%.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Claims

1. A method for genetic transformation of taro, characterized in that it comprises the following steps:

S1. Taking the embryonic callus induced from the taro stem tip and multiplying it to obtain newly proliferated embryonic callus; using Agrobacterium to infect the newly proliferated embryonic callus, and then transferring it to a co-culture medium for culture;

S2. transferring the embryonic callus after co-cultivation to a differentiation screening medium to obtain resistant buds;

S3. Select the robust resistant buds and transfer them to the seedling medium to obtain resistant seedlings;

S4. Select the resistant seedlings with a height of 5 to 7 cm, transfer them to the test tube taro induction medium, and obtain resistant test tube taro;

S5. Hardening the resistant test tube taro and planting it in a greenhouse;

Wherein, the embryonic callus induced in the taro stem tip in step S1 is selected from the stem tip induction of any variety of taro, taro with fruit, Ganyu No. 1 and Ganyu No. 2;

The Agrobacterium is Agrobacterium strain GV3101 transformed with pRI101-CeSUC3 recombinant plasmid;

The method of using Agrobacterium to infect newly propagated embryonic callus in step S1 specifically comprises:

Pre-treat Agrobacterium to obtain Agrobacterium cells; then resuspend the Agrobacterium cells with Agrobacterium infection solution, and after resuspension, adjust the concentration of the bacterial solution to OD ₆₀₀ = 0.6-0.8;

The newly proliferated embryonic callus was collected in a sterile conical flask, and the bacterial solution was poured into the flask, which was sealed with a sterile sealing film. At room temperature, the flask was first infected on a shaking table at 100 rpm for 15 minutes, and then kept at a vacuum condition of -0.1 MPa for 15 minutes, with a total infection time of 30 minutes.

The differentiation screening medium described in step S2 includes the following components: MS + 6-BA 1.0 mg/L + NAA 0.5 mg/L + sucrose 30 g/L + acetosyringone 20 µmol/L~30 µmol/L + timentin 250 mg/L + kanamycin 50 mg/L, pH 5.8~6.0, and the conditions for differentiation culture are: 24°C~26°C, first culture in the dark for 7d~14d, and then culture under light conditions, the light intensity is 2000 Lx~3000 Lx, and the photoperiod is 16h light and 8h dark.

2. The method according to claim 1, characterized in that the method for obtaining the Agrobacterium strain GV3101 transformed with the pRI101-CeSUC3 recombinant plasmid comprises the following steps:

Taro cDNA was used as template for amplification. The system was as follows: 2 μL DNA template, 8 μL DNA polymerase, 0.5 μL upstream primer, 0.5 μL downstream primer, and ddH ₂ O was added to 20 μL. The procedure was as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 54°C annealing for 20 s, 72°C extension for 45 s, 36 cycles, 72°C final extension for 5 min, and storage at 4°C to obtain the CeSUC3 fragment with homologous recombination sequence. Among them, the upstream primer F: 5'-TCTTCACTGTTGATACATATGATGGACGCCATCTCGATCCG-3', the downstream primer R: 5'-AGAGTTGTTGATTCAGAATTCTTAGCCAAATCCATGAAGACCCG-3';

The overexpression vector pRI101-AN circular plasmid was digested with NdeⅠ and EcoRⅠ restriction endonucleases to obtain linearized pRI101-AN;

The linearized pRI101-AN and the CeSUC3 fragment with homologous recombination sequence were mixed at a molar ratio of 1:3, and incubated at 37°C for 30 min. The system was: 1 μL linearized pRI101-AN, 3 μL CeSUC3 fragment with homologous recombination sequence, 5 μL universal enzyme premix, and ddH ₂ O was added to 10 μL;

The recombinant mixed solution was transformed into Escherichia coli DH5α, plated, single clones were picked, shaken, colony PCR was performed, and the single clone with the target band was selected for sequencing. The single clone with the correct sequence was selected and shaken again to extract the recombinant plasmid pRI101-CeSUC3 and transformed into Agrobacterium GV3101 with heat shock at 37°C.

3. The method according to claim 1 is characterized in that the proliferation medium used for the propagation in step S1 is MS + sucrose 30 g/L + TDZ 2 mg/L + NAA 0.1 mg/L + agar powder 6.5 g/L, with a pH of 5.8-6.0, and the conditions for the propagation are: culturing in the dark at a temperature of 24°C-26°C.

4. The method according to claim 1, characterized in that the Agrobacterium infection solution comprises the following components: 1/2 MS + sucrose 30 g/L + acetosyringone 20 µmol/L~30 µmol/L, and the pH is 5.8~6.0.

5. The method according to claim 1, wherein the method of pre-treating Agrobacterium comprises:

Place a single colony of Agrobacterium in 2 mL of YEB medium, add the corresponding antibiotics, and culture at 28°C with a shaker at 180 rpm for 12 h until the bacteria are fully revived;

Take 500 μL of the revived bacterial solution and add it to 10 mL of YEB medium, add the corresponding antibiotics, and culture it at 28°C with a shaker at 180 rpm for 12 h until the _OD600 of the bacterial solution reaches 0.9-1.1;

Centrifuge at 5000 rpm for 10 min, discard the supernatant, and collect the Agrobacterium cells.

6. The method according to claim 1, characterized in that the co-culture medium in step S1 comprises the following components: MS + sucrose 30 g/L + acetosyringone 20 µmol/L~30 µmol/L, pH 5.8~6.0, and culture conditions: 24°C~26°C in the dark for 3 d~4 d.

7. The method according to claim 1 is characterized in that the seedling culture medium in step S3 comprises the following components: MS + 6-BA 1.0 mg/L + NAA 0.5 mg/L + sucrose 30 g/L + timentin 250 mg/L + kanamycin 50 mg/L, pH 5.8-6.0, and the conditions for seedling culture are: temperature 24°C-26°C, light intensity 2000 Lx-3000 Lx, and photoperiod of 16h light and 8h dark.

8. The method according to claim 1, characterized in that the test tube taro induction medium described in step S4 comprises the following components: MS + sucrose 80 g/L + timentin 250 mg/L + kanamycin 50 mg/L, pH 5.8-6.0, and the conditions for test tube taro induction culture are: temperature 24°C-26°C, light intensity 2000 Lx-3000 Lx, and photoperiod of 16h light and 8h dark.

9. The method according to claim 1, characterized in that the resistant test tube taro described in step S5 is moved to room temperature conditions, hardened for 3-5 days, then the seedlings are taken out, the base culture medium is cleaned, and the substrate is planted in a matrix, wherein the matrix is peat: vermiculite with a volume ratio of 2:1, bagged for moisture retention for 3-5 days, and grown in a greenhouse, and the growth conditions are: temperature 20°C-30°C, light intensity 2000 Lx-4000 Lx, and photoperiod of 12h-14h light and 10h-12h dark, to obtain surviving plants.