CN113234754B - Efficient genetic transformation method taking abelmoschus manihot embryo as explant - Google Patents

Abstract

The invention provides a high-efficiency genetic transformation method using goldenflower sunflower seed embryos as explants, and relates to the technical field of plant genetic engineering. The method described in the invention first constructs the GV3101 agrobacterium with the target gene, and through cooperation with Jinhua sunflower Sunflower seed embryos are co-cultivated for transformation, and the transformed seed embryos are degermed, then callus induction, differentiation culture, and rooting culture are performed, and finally transgenic seedlings of sunflower sunflower containing the target gene are efficiently obtained. The invention focuses on constructing a stable genetic transformation system of sunflower sunflower, which has high efficiency, short time and simple operation technology, and the acquisition of transgenic seeds provides important transgenic materials for in-depth research on related molecular mechanisms in the sunflower sunflower.

Description

A high-efficiency genetic transformation method using sunflower seed embryos as explants

technical field

The invention relates to the technical field of plant genetic engineering, in particular to a high-efficiency genetic transformation method using sunflower seed embryos as explants.

Background technique

Since the birth of plant genetic engineering technology, great progress has been made in the study of gene function. At present, widely used plant transgenic methods include particle gun method, Agrobacterium-mediated method, electrode method, etc. However, these methods have disadvantages such as long transformation cycle and poor repeatability. New technologies such as vacuum infiltration method, pollen tube passage method and embryonic tissue infection method have been developed, which are easy to operate and short in the transformation cycle, making the transformation more convenient.

Hibiseu manihot L. belongs to the genus Malvaceae and is an annual herb with medicinal and edible value. Goldenflower sunflower is rich in natural flavonoids, which have anti-inflammatory, analgesic, anti-fatigue, anti-aging, and blood lipid-lowering effects. These flavonoids mainly include hyperin, quercetin, myricetin, quercetin- The abundance of 3-bacoside and quercetin-3-glucoside is dozens of times that of soybean and ginkgo. Although it can be planted in most areas of my country, it is currently an endangered species in my country because its short flowering time (blooming in the morning and decaying in the evening) makes it difficult to pollinate, which leads to low seed setting rate and reduced yield. Therefore, the development of an efficient genetic transformation and regeneration system will create more possibilities for improved breeding, industrial production, and research and development of high-value medicines and health products.

Contents of the invention

The object of the present invention is to provide a method for cultivating a high-efficiency genetic transformation and regeneration system using the sunflower seed embryo as an explant.

In order to realize the above object of the invention, the present invention provides the following technical solutions:

The invention provides a kind of high-efficiency genetic transformation method using sunflower seed embryos as explants, comprising the following steps:

Step 1, transforming the expression vector carrying the target gene into GV3101 Agrobacterium to obtain GV3101 Agrobacterium containing the target gene;

Step 2. Prepare full-grained goldenflower seeds and wash them with clean water for 6-24 hours;

Step 3. Disinfect the sunflower seeds washed with clean water for 6-24 hours in an ultra-clean bench, then cut them along the central axis of the seeds with a sterile scalpel to obtain seed embryos, and immerse the seed embryos into the The GV3101 Agrobacterium liquid was placed on a shaking table for transformation, and after the transformation was completed, the seed embryos were eluted with sterile water;

Step 4, placing the eluted seed embryos in the co-cultivation medium, cultivating in dark for 2-3 days, then cleaning the seed embryos, and then placing them in the degerming medium for degerming;

Step 5, callus induction: place the sterilized seed embryo in the seed embryo callus induction medium for cultivation to obtain callus:

Step 6, callus differentiation culture: the callus obtained in step 5 is placed in the seed embryo callus differentiation medium and cultured so that the callus can complete bud differentiation;

Step 7, rooting culture: the buds of 3-5cm are placed in the rooting medium to induce rooting, and complete colonization;

Step 8, transplanting to the greenhouse.

On the basis of the above scheme, in step 3, the disinfection specifically includes the following steps: placing the seeds in sodium hypochlorite for 5-10 minutes; rinsing with sterile water for 3-5 times; placing them in alcohol for 30s-1min; Rinse 4-5 times.

On the basis of the above scheme, in step 3, the preparation steps of the GV3101 agrobacterium liquid with the gene of interest include:

Step 31, inserting the GV3101 Agrobacterium containing the target gene into the liquid medium, shaking and culturing at 26-30°C for 14-16 hours to obtain a culture solution;

Step 32. Transfer the culture medium into a liquid medium, shake and culture at 26-30°C for 5-6h, until the bacterial concentration OD600=0.4-0.6, centrifuge, resuspend the precipitate with MS culture medium, let stand, and finally obtain GV3101 Agrobacterium broth of the target gene.

The liquid medium is based on YEP medium, and 15-25mg rifampicin and 40-60mg kanamycin are added to each liter of YEP medium.

On the basis of the above scheme, in step 3, the setting parameters of the shaker are: rotation speed 80-100 rpm, time: 0.5-1.5 h.

On the basis of the above scheme, in step 4, the co-cultivation medium is based on MS liquid medium, and 1.5-2mg TDZ, 0.5-1mg NAA, 0.5-1mg NAA are added to each liter of MS liquid medium Folic acid, 20-30g sucrose, 10g glucose, 6-8g agar, adjust the pH value to 5.8-5.9.

On the basis of the above scheme, in step 4, the MS liquid medium is used as the base medium for the degermation medium, and 1.5-2mg TDZ, 0.5-1mg NAA, and 0.5-1mg NAA are added to each liter of MS liquid medium Folic acid, 200-250mg cephalosporin, 10-15mg hygromycin, 20-30g sucrose, 10g glucose, 6-8g agar, adjust the pH value to 5.8-5.9.

On the basis of the above scheme, in step 5, the MS liquid medium is used as the basal medium for the seed embryo callus induction medium, and 4-5mg2.4-D, 0.5- 1 mg zeatin, 300-350 mg hydrolyzed milk protein, 500-600 mg glutamine, 20-30 sucrose and 8-10 g agar, adjust the pH value to 5.8-5.9.

On the basis of the above scheme, in step 6, the seed embryo callus differentiation medium is based on MS liquid medium, and 1-1.5mg6-BA, 1-1.5mg IAA are added to each liter of MS liquid medium , 20-30g sucrose and 8-10g agar, adjust the pH value to 5.8-5.9.

On the basis of the above scheme, the rooting medium described in step 7 is based on MS liquid medium, and 0.1-0.2mgIBA, 0.005-0.01mgNAA, 20-30g sucrose and 8 -10g agar, adjust the pH value to 5.8-5.9.

On the basis of the above scheme, the callus induction described in step 5, the callus differentiation culture described in step 6 and the rooting culture described in step 7 were all carried out at 24-26°C.

Compared with prior art, the beneficial effect of the present invention:

The invention provides a high-efficiency genetic transformation method using sunflower seed embryos as explants, constructing GV3101 Agrobacterium with a target gene and transforming sunflower seeds, and finally obtaining the transgene by inducing callus and differentiation of the sunflower seeds Sunflower plants and seeds. The germination rate of goldenflower seeds is high, the cycle is short, and the stable and efficient genetic transformation system provides more transgenic seeds, which not only provides a large number of transgenic plant materials for in-depth scientific research on the molecular mechanism of goldenflower, but also greatly shortens the experimental cycle , and the equipment required by this method is simple, the operation technique is easy to master, and the conversion efficiency is high.

Description of drawings

The present invention has following accompanying drawing:

Fig. 1 is a schematic diagram of the genetic transformation and regeneration process using the seed embryo as an explant;

Fig. 2 is the flowchart of seed embryo callus regeneration process;

Wherein, figure a is a schematic diagram of callus induction period of sunflower sunflower seed embryo;

The b picture is a schematic diagram of the stage of callus proliferation of sunflower sunflower seed embryos;

The c picture is a schematic diagram of callus differentiation period of goldenflower sunflower seed embryo;

Figure d is a schematic diagram of the callus rooting period of sunflower sunflower seed embryos;

Figure e is a schematic diagram of the root elongation period of the sunflower sunflower;

Figure f is a schematic diagram of the hardening period;

Figure g is a schematic diagram of the solidification period;

The picture h shows the schematic diagram of the goldenflower sunflower transgenic seed

Fig. 3 is the laser confocal image of the eGFP gene in the transgenic seedling;

Fig. 4 is the gel electrophoresis image of the eGFP gene in the transgenic seedlings; wherein #1 and #2 are the seedlings transformed with GFP from goldenflower sunflower.

Figure 5 is a western blot diagram of the eGFP gene in the transgenic seedlings; wherein #1 and #2 are the seedlings of goldenflower sunflower transgenic for GFP.

Detailed ways

The invention provides a kind of high-efficiency genetic transformation method using sunflower seed embryos as explants, comprising the following steps:

Step 1: Transform the expression vector carrying the target gene into GV3101 Agrobacterium to obtain GV3101 Agrobacterium containing the target gene.

Step 2: Prepare full-grained goldenflower seeds and wash them with clean water for 6-24 hours.

Step 3: Disinfect the sunflower seeds rinsed with clean water for 6-24 hours in an ultra-clean bench, then use a sterile scalpel to cut along the central axis of the seeds to obtain seed embryos, and immerse the seed embryos in the The GV3101 Agrobacterium liquid is placed on a shaking table for transformation, and after the transformation is completed, the seed embryos are eluted with sterile water;

Step 4: Place the eluted seed embryos in the co-cultivation medium, cultivate in dark for 2-3 days, then wash the seed embryos, and then place them in the degerming medium for degerming.

Step 5: callus induction: place the sterilized seed embryos in the seed embryo callus induction medium for culture to obtain callus:

Step 6: callus differentiation culture: the callus obtained in step 5 is placed in the seed embryo callus differentiation medium and cultured so that the callus can complete bud differentiation;

Step 7: rooting culture: put the 3-5cm buds in the rooting medium to induce rooting and complete the colonization;

Step 8: Transplant the greenhouse.

In the invention, the expression vector carrying the objective gene is transformed into the GV3101 agrobacterium to obtain the agrobacterium containing the objective gene. There is no special limitation on the specific target gene and suitable expression vector.

In the embodiment of the present invention, with eGFP as the target gene, the eGFP-pROK2 vector was constructed and transformed into GV3101 Agrobacterium. The present invention has no special limitation on the transformation method of the expression vector, and known methods can be used, such as electroporation or heat shock method.

In this study, there is no special order requirement for the use of sodium hypochlorite and alcohol in the process of seed disinfection in step 3.

Sodium hypochlorite disinfection time is preferably 5-10min, more preferably 8min. The alcohol disinfection time is preferably 30s-1min, more preferably 45s. The number of rinses with sterile water is preferably 4-5 times, more preferably 5 times.

In the present invention, the setting parameters of the shaker described in step 3 are preferably 50-100rmp, 0.5-1.5h. More preferably: 80rmp, 1h.

In the present invention, the concentration of the GV3101 Agrobacterium with the target gene in step 3 is preferably OD600 of 0.4-0.6, more preferably 0.5.

In the present invention, the preparation step of the Agrobacterium bacterium liquid with the gene of interest comprises:

Step 1: Inoculate the transformed GV3101 Agrobacterium into a liquid medium, and shake and culture at 26-30° C. for 14-16 hours. The liquid medium is based on YEP medium, and 15-25mg rifampicin and 40-60mg kanamycin are added to each liter of YEP medium.

Step 2: Transfer the culture medium into the liquid medium again, shake and culture at 26-30°C for 5-6h, until the bacterial concentration OD600=0.4-0.6, centrifuge, resuspend the precipitate with MS solution, let stand, and obtain cultured Agrobacterium.

In the present invention, the liquid medium is preferably: YEP medium is used as the base medium, and 20 mg rifampicin and 50 mg kanamycin are added to each liter of YEP medium.

In step 1, the shaking frequency is preferably 150-180 rpm, more preferably 180 rpm, and the incubation time is more preferably 15 h. The temperature is more preferably 28°C.

In step 2, the inoculation amount of the culture solution is 10-12% (v/v), more preferably 10% (v/v). The shaking time is more preferably 5h. The centrifugal speed is preferably 8000-10000rmp, more preferably 10000rmp. The centrifugation time is preferably 8-15 min, more preferably 10 min.

As shown in the specific embodiment of the present invention, the medium for the co-cultivation of seed embryos and Agrobacterium is based on MS liquid medium, and 1.5-2mg TDZ and 0.5-1mg NAA are added to each liter of MS liquid medium. 0.5-1mg folic acid, 20-30g sucrose, 10g glucose, 6-8g agar, adjust the pH value to 5.8-5.9. More preferably: use MS liquid medium as the base medium, and add 2mg TDZ, 0.5mg NAA, 1mg folic acid, 30g sucrose, 10g glucose, and 8g agar to each liter of MS liquid medium to adjust the pH value to 5.8.

In the present invention, the sunflower seed embryos soaked with Agrobacterium are placed again in the co-cultivation medium, and after 2-3 days of dark culture, degerming is carried out in the degerming medium.

The dark cultivation time is preferably 2-3 days, more preferably 3 days.

In the present invention, the seed embryo degerming medium is based on MS liquid medium, and 1.5-2mgTDZ, 0.5-1mg NAA, 0.5-1mg folic acid, 200-250mg Cephalosporium are added to each liter of MS liquid medium 10-15mg hygromycin, 20-30g sucrose, 10g glucose, 6-8g agar, adjust the pH value to 5.8-5.9.

More preferably: use MS liquid medium as the base medium, and add 2mgTDZ, 0.5mg NAA, 1mg folic acid, 250mg cephalosporin, 10mg hygromycin, 30g sucrose, 10g glucose to each liter of MS liquid medium, 8g agar, adjust the pH value to 5.8. The present invention has no special requirements on the number of times of degerming, as long as the aseptic material can be obtained.

In the present invention, the seed embryo callus induction medium is based on MS liquid medium, and 4-5mg 2.4-D, 0.5-1mg zeatin, 300-350mg hydrolyzed milk are added to each liter of MS liquid medium Protein, 500-600mg glutamine, 20-30g sucrose and 8-10g agar, adjust the pH value to 5.8-5.9.

More preferably: use MS liquid medium as the base medium, and add 5mg2.4-D, 0.5mg zeatin, 300mg hydrolyzed milk protein, 500mg glutamine, 30g sucrose and 8g Agar, adjust the pH to 5.8.

In the present invention, the seed embryo callus differentiation medium is based on MS liquid medium, and 1-1.5mg 6-BA, 1-1.5mg IAA, 20-30g sucrose are added to every liter of MS liquid medium And 8-10g agar, adjust the pH value to 5.8-5.9.

More preferably: MS liquid medium is used as the base medium, and 1 mg6-BA, 1 mg IAA, 30 g sucrose and 8 g agar are added to each liter of MS liquid medium to adjust the pH value to 5.8.

In the present invention, when the aseptic seedling grows to 3-5cm, it is transferred to the rooting medium for rooting induction, and the rooting medium is based on MS liquid medium, and 0.1- 0.2mgIBA, 0.005-0.01mgNAA, 20-30g sucrose and 8-10g agar, adjust the pH value to 5.8-5.9.

More preferably: MS liquid medium is used as the base medium, and 0.1 mg IBA, 0.005 mg NAA, 30 g sucrose and 8 g agar are added to each liter of MS liquid medium, and the pH value is 5.8.

In the present invention, the above culture temperature is preferably 24-26°C, more preferably 25°C.

According to the transformation and regeneration method described in the present invention, the gene can be effectively transferred into the sunflower seed embryo, and transgenic seedlings can be obtained efficiently and quickly.

The technical solutions provided by the present invention are described in detail in conjunction with the embodiments.

The induction of embodiment 1 sunflower seed embryo transformation and regenerated seedling

Preparation of transgenic infection solution: The 35s-eGFP-pROK2 vector was constructed using the Seamless Assembly Cloning Kit of Zhongmei Taihe Company. The constructed vector was transformed into GV3101 competent cells by electroporation; a single colony of positive clones was picked, and 5ml YEP+20mg/L Rif+50mg/L Kana liquid medium was shaken overnight at 180rmp on a shaker at 28°C. Transfer to 50ml YEP + 20mg/L Rif + 50mg/L Kana liquid medium for shaking, amplify until the OD600 of the bacterial solution is 0.5, centrifuge the bacterial solution at 10000rmp for 10min at room temperature, discard the supernatant, and use MS medium Resuspended and allowed to stand for 2-3 hours to make a transgenic infection bacterial solution.

Infection and co-cultivation, degerming culture: Disinfect the seeds with sodium hypochlorite for 8 minutes, rinse with sterile water for 5 times, disinfect with alcohol for 45 seconds, rinse with sterile water for 5 times, use a sterilized scalpel to cut along the central axis of the seeds, and place The seed embryos were placed in the bacterial solution, and transformed on a shaker at 80 rpm for 1 hour. After the transformation is completed, the embryos are eluted twice with sterile water, and transferred to MS+2mg/LTDZ+0.5mg/LNAA+1mg/L folic acid+30g/L sucrose+10g/L glucose+8g/L agar culture Based on dark cultivation at 25°C for 3 days. Wash the embryos 4-6 times with sterile water, dry the excess liquid on the surface with sterile filter paper, then transfer to MS+250mg/L cephalosporin+10mg/L hygromycin+2mg/L TDZ+0.5mg/L NAA +1mg/L folic acid+30g/L sucrose+10g/L glucose+8g/L agar medium for degerming.

Callus induction: transfer sterile embryos to MS+5mg/L 2.4-D+0.5mg/L zeatin+300mg/L hydrolyzed milk protein+500mg/L glutamine+30g/L sucrose+8g/L agar , in the medium with a pH value of 5.8, the embryogenic callus was cultured for about 7 days, and the embryogenic callus grew well after 14 days.

Callus differentiation induction: The callus was transferred to the medium of MA+1mg/L 6-BA+1mg/LIAA+30g/L sucrose+8g/L agar for differentiation culture, and the callus completed bud differentiation in about 15 days.

Rooting culture: Induce rooting of 3-5cm shoots in MS+0.1mgIBA+0.005mgNAA+30g sucrose+8g agar, pH value 5.8, and complete colonization in about 10 days.

Transplanting in the greenhouse. Flowering and fruiting are achieved in about 3 months.

Detection of transgenic seedlings: detection at gene and protein levels. The transgenic regenerated seedlings of goldenflower sunflower were taken, and the total RNA was extracted by CTAB method and reverse-transcribed into cDNA.

Primers for eGFP were designed as follows:

eGFP-F: 5'-ATGGTGAGCAAGGGCGAGGAGC-3';

eGFP-R: 5'-TTACTTGTACAGCTCGTCCA-3'.

By means of PCR, the eGFP gene was detected in the seedlings, as shown in FIG. 4 .

Next, the transgenic regenerated seedlings of goldenflower sunflower were taken, the crude protein was extracted, the protein was separated by 12% (w/v) SDA-PAGE, and transferred to PVDF membrane by electrophoresis. The PVDF membrane was blocked in blocking solution for 2h, then the primary antibody (anti-GFP antibody) was mixed with blocking solution and TBST solution at a dilution of 1:20000, incubated with the membrane overnight, and then washed 5 times with TBST solution . Then mix the secondary antibody with blocking solution and TBST solution at a dilution of 1:20000, and incubate with the membrane for 2 hours. After washing with TBST solution for 5 times, add luminescence solution on the membrane to visualize the immune reaction and detect The band of eGFP protein is shown in Figure 5. In addition, green fluorescence containing eGFP was also observed under confocal laser, as shown in Figure 3.

The different concentrations of embodiment 2GV3101 Agrobacterium are on the influence of sunflower seed embryo transformation

Table 1 Effect of different concentrations of GV3101 Agrobacterium on the transformation of goldenflower seed embryos

GV3101 Agrobacterium Concentration (OD600) Number of embryos survival rate 0.4 30 20% 0.5 30 30% 0.6 30 20%

Three copies of GV3101 Agrobacterium were respectively amplified in YEP+20mg/L Rif+50mg/L Kana liquid medium to OD600=0.4, OD600=0.5, OD600=0.6, and the culture conditions were 28°C, 180rmp. The embryos were transformed according to the method described in Example 1, and the survival rate was observed after co-culture and degerm culture.

As shown in Table 1, when the GV3101 Agrobacterium concentration OD600=0.5, the survival rate of the seed embryo is the highest, which is 30%, while the survival rate at OD600=0.4 and OD600=0.6 is only 20%. Therefore, the concentration of OD600=0.5 has the best transformation effect on seed embryos.

Effect of different hormone combinations of embodiment 3 on callus induction

Table 2 Effects of different hormone combinations on callus induction

Put sterile seed embryos in different hormone combinations for callus induction, as shown in Table 2, hormones have a great influence on callus induction, wherein 5mg/L 2.4-D+0.5mg/L zeatin+300mg/L hydrolysis The combination of milk protein+500mg/L glutamine has the best effect of inducing callus, which is 80%. All other combinations were below 80%.

Effect of embodiment 4 different hormone combinations on callus differentiation and budding

Table 3 Effects of different hormone combinations on callus differentiation and budding

The callus is continuously cultured in the medium of MS+5mg/L 2.4-D+0.5mg/L zeatin+300mg/L hydrolyzed milk protein+500mg/L glutamine+30g/L sucrose+8g/L agar to achieve proliferation , the bean-sized calli were respectively placed in the differentiation medium of three different hormone combinations for differentiation culture. The callus differentiation was observed within 2 weeks after subculture.

It can be seen from Table 3 that the combination of 1mg/L 6-BA+1mg/LIAA is the most suitable for inducing callus differentiation, and it only takes 1 week for callus to differentiate and sprout.

The influence of embodiment 5 different hormone combinations on rooting

Table 4 Effects of different hormone combinations on rooting

NAA(mg/L) IBA(mg/L) Rooting rate 0.005 0.1 75% 0.005 0.2 70% 0.01 0.1 60% 0.01 0.2 65%

As shown in Table 4, the combination of 0.1mgIBA+0.005mgNAA can effectively induce rooting of goldenflower sunflower regenerated seedlings, and the rooting rate is 75%, which is the optimal combination for inducing rooting.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

The content not described in detail in this specification belongs to the prior art known to those skilled in the art.

Claims (7)

1. An efficient genetic transformation method taking Abelmoschus manihot embryo as explant is characterized by comprising the following steps:

step 1, transforming an expression vector carrying a target gene into the GV3101 agrobacterium to obtain the GV3101 agrobacterium containing the target gene;

step 2, preparing full Abelmoschus manihot seeds, and washing the seeds for 6 to 24 hours in clean water;

step 3, sterilizing the abelmoschus manihot seeds washed by clean water for 6-24 hours in an ultra-clean bench, cutting the seeds along the central axis of the seeds by using a sterile scalpel to obtain seed embryos, immersing the seed embryos into GV3101 agrobacterium liquid with target genes, placing the seed embryos in a shaking table for transformation, and eluting the seed embryos by using sterile water after the transformation is finished;

step 4, placing the eluted seed embryos in a co-culture medium, performing dark culture for 2-3 days, then cleaning the seed embryos, and placing the seed embryos in a degerming medium for degerming;

step 5, callus induction: placing the sterile seed embryo in a seed embryo callus induction culture medium for culturing to obtain a callus:

step 6, callus differentiation culture: placing the callus obtained in the step 5 in a seed embryo callus differentiation culture medium for culturing to enable the callus to complete bud differentiation;

step 7, rooting culture: placing 3-5cm buds in a rooting culture medium for inducing rooting to complete field planting;

and 8: transplanting in a greenhouse;

in the step 4, the co-culture medium takes MS liquid culture medium as basic culture medium, 1.5-2mg of TDZ,0.5-1mg of NAA,0.5-1mg of folic acid, 20-30g of cane sugar, 10g of glucose and 6-8g of agar are added into each liter of MS liquid culture medium, and the PH value is adjusted to 5.8-5.9;

in the step 4, the degerming culture medium takes an MS liquid culture medium as a basic culture medium, 1.5-2mg of TDZ,0.5-1mg of NAA,0.5-1mg of folic acid, 200-250mg of cefamycin, 10-15mg of hygromycin, 20-30g of cane sugar, 10g of glucose and 6-8g of agar are added into each liter of MS liquid culture medium, and the pH value is adjusted to 5.8-5.9;

in the step 5, the embryo callus induction culture medium takes an MS liquid culture medium as a basic culture medium: wherein each liter of MS liquid culture medium comprises: 5mg/L of 2.4-D, 0.5mg/L of zeatin, 300mg/L of hydrolyzed milk protein, 500mg/L of glutamine, 20-30g of cane sugar and 8-10g of agar, and the pH value is adjusted to 5.8-5.9.

2. The efficient genetic transformation method taking Abelmoschus manihot embryos as explants according to claim 1, wherein in the step 3, the disinfection specifically comprises the following steps: placing the seeds in sodium hypochlorite for 5-10min; washing with sterile water for 3-5 times; placing in alcohol for 30s-1min; washing with sterile water for 4-5 times.

3. The efficient genetic transformation method taking Abelmoschus manihot embryo as explant according to claim 1, wherein in step 3, the preparation of the GV3101 Agrobacterium solution with target gene comprises:

step 31, inoculating the GV3101 agrobacterium containing the target gene into a liquid culture medium, and performing shake culture at 26-30 ℃ for 14-16h to obtain a culture solution;

step 32, transferring the culture solution into a liquid culture medium, carrying out shaking culture at 26-30 ℃ for 5-6h until the concentration OD600 of the bacterial solution is =0.4-0.6, centrifuging, carrying out resuspension on the precipitate by using an MS culture solution, and standing to finally obtain a GV3101 agrobacterium bacterial solution with a target gene;

the liquid culture medium takes YEP culture medium as basic culture medium, and 15-25mg rifampicin and 40-60mg kanamycin are added into each liter of YEP culture medium.

4. The efficient genetic transformation method taking Abelmoschus manihot embryo as explant according to claim 1, wherein in step 3, the setting parameters of the shaking table are as follows: rotation speed 80-100rpm, time: 0.5-1.5h.

5. The efficient genetic transformation method taking Abelmoschus manihot embryo as explant according to claim 1, wherein in step 6, MS liquid culture medium is used as basic culture medium in embryo callus differentiation culture medium, 1-1.5mg of 6-BA, 1-1.5mg of IAA, 20-30g of sucrose and 8-10g of agar are added into each liter of MS liquid culture medium, and pH value is adjusted to 5.8-5.9.

6. The efficient genetic transformation method using Abelmoschus manihot embryo as explant according to claim 1, wherein the rooting medium in step 7 is MS liquid medium as basic medium, and 0.1-0.2mgIBA,0.005-0.01mgNAA,20-30g sucrose and 8-10g agar are added to each liter of MS liquid medium to adjust pH to 5.8-5.9.

7. The efficient genetic transformation method using Abelmoschus manihot embryo as explant according to claim 1, wherein the callus induction in step 5, the callus differentiation culture in step 6 and the rooting culture in step 7 are all performed at 24-26 ℃.

Images