CN114836462B - Efficient agrobacterium-mediated melon genetic transformation method - Google Patents
Efficient agrobacterium-mediated melon genetic transformation method Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
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- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
A highly efficient agrobacterium-mediated melon genetic transformation method comprising: s1, seed selection and disinfection are carried out, and then the seed selection and disinfection are inoculated on a seed germination culture medium MeM1 for dark culture for standby; s2, using agrobacterium-mediated dyeing medium I M suspension to carry out mediated dyeing on cotyledon explants excised from the melon seedlings which grow robustly in the step S1, and finally placing the impregnated cotyledon explants on agrobacterium co-culture medium MeM2 for co-culture for later use; s3, placing the cotyledon explant subjected to co-culture in the step S2 on an induction bud regeneration culture medium MeM3 for culture, and carrying out secondary culture every two weeks for later use; s4, cutting off the regenerated bud cluster on the culture medium in the step S3, placing the bud cluster in a bud extension culture medium MeM4, continuing to carry out screening culture until the buds are extended to 2cm, and transferring the buds to a rooting culture medium; s5, transferring the buds obtained in the step S4 to a rooting culture medium MeM5 for growth, and then transferring to a nutrition pot for culture; the advantages are that: meanwhile, the method is suitable for thin-skin melons and thick-skin melons, and the yellowing rate of the regenerated buds is reduced to 1.6%.
Description
Technical Field
The invention relates to the technical field of melon biotechnology breeding, in particular to a high-efficiency agrobacterium-mediated melon genetic transformation method.
Background
Melon (cusumis melo L.) is an important melon crop of cucurbitaceae crop melon, is rich in various vitamins, has various colors, fresh and sweet taste, is rich in nutrition and has high economic value, and is a favored fruit for people. The disease resistance of the melon is poor, main diseases include powdery mildew, fusarium wilt, downy mildew, virus diseases and the like, and breeding and utilizing disease resistant variety resources are effective ways for improving the disease resistance property of the melon, but the conventional breeding period of the melon is long, and the variety with improved target properties is difficult to obtain quickly. Along with the rapid development of plant cell engineering and genetic engineering technology, the research and application of genetic engineering and tissue culture technology in crops are more and more extensive, and the development of melon tissue culture technology provides important technical support for the application of transgenic technology. The establishment of a high-efficiency agrobacterium-mediated melon genetic transformation technology system is a key for melon biotechnology breeding.
Although the tissue culture research of muskmelon has been carried out for more than 30 years, there are still many problems to be studied, such as that although the students at home and abroad obtain higher adventitious bud regeneration frequency through cotyledon explant culture, the elongation of cluster buds, rooting of regenerated seedlings and the rate of forming complete plants are not high, and no fixed regeneration system is suitable for all muskmelon germplasm resources so far.
Disclosure of Invention
The invention provides a high-efficiency agrobacterium-mediated melon genetic transformation method, which aims to at least overcome one technical defect, and can simultaneously improve the regeneration rate of thin-skin and thick-skin melon teeth and quickly obtain plants with prolonged buds.
in order to achieve the above object, the present invention provides the following technical solutions:
a high-efficiency agrobacterium-mediated melon genetic transformation method is characterized in that: the method comprises the following steps:
S1, seed selection and disinfection are carried out, and then the seed selection and disinfection are inoculated on a seed germination culture medium MeM1 for dark culture for standby;
S2, using an agrobacterium dip-dyeing culture medium IM suspension to dip-dye the cotyledon explants cut out from the melon seedlings which grow robustly in the step S1, and finally, putting the dipped cotyledon explants into an agrobacterium co-culture medium MeM2 for co-culture for later use;
s3, placing the cotyledon explant subjected to co-culture in the step S2 on an induction bud regeneration culture medium MeM3 for culture, and carrying out secondary culture every two weeks for later use;
S4, cutting off the bud cluster regenerated on the culture medium in the step S3, placing the bud cluster in a bud extension culture medium MeM4, continuing to screen and culture until the buds are extended to 2cm, and transferring the buds to a rooting culture medium;
S5, transferring the buds obtained in the step S4 to a rooting culture medium MeM5 for growth, and then transferring to a nutrient bowl for culture;
Wherein, every 100mL of induction bud regeneration culture medium MeM3 is prepared by the following steps: taking 0.44g of MS finished powder, 3g of sucrose, 0.9g of agar powder and 0.75-1.25 mg/L of CuSO4·5H2The composition of the O and plant growth regulator is 0.5-0.8mg.L-1 6-BA+0.5～0.8mg·L-1Zeatin +0.08 mg.L-1IAA, double distilled water is fixed to 100mL, pH value is adjusted to 5.80-5.85, and 100 microliter of 200mg/mL inhibitor is added after sterilization and temperature reduction to 55 ℃;
The preparation process of the bud extension medium MeM4 per 100mL comprises the following steps: taking 0.44g of finished powder, 3g of sucrose, 0.9g of agar powder and 0.05 to 0.1 mg.L of plant growth regulator-1 6-BA+0.5～1.0mg·L-1GA3, double distilled water with constant volume of 100mL, pH value of 5.80-5.85, sterilizing, adding 100 microliter of 200mg/mL inhibitor and 100 microliter-125 microliter of 20-25 mM silver thiosulfate when the temperature is reduced to 55 ℃.
Preferably, the medium for inducing shoot regeneration, mem3, and the medium for shoot elongation, mem4, both contain a selection agent and are identical to the selection marker on the genetic transformation vector.
Preferably, the screening agent is one of 15-25mg/L kanamycin, 5-10mg/L hygromycin and 8-10mg/L glufosinate.
preferably, the 20mM silver thiosulfate is prepared by the steps of:
(1) The preparation method of 100mM sodium thiosulfate comprises dissolving 158mg sodium thiosulfate in 10ml sterile double distilled water for use;
(2) The preparation method of 100mM silver nitrate comprises dissolving 170mg silver nitrate in 10ml sterile double distilled water;
(3) 100mM sodium thiosulfate and 100mM silver nitrate were mixed in a 4:1 ratio.
Preferably, the preparation process of the agrobacteria infection medium IM per 100mL is as follows: taking 0.44g of MS finished powder, 3g of sucrose, 200 mu l of 1mg/ml 6-BA, 100 mu l of 1mg/ml ABA, 0.27g of MES, adjusting the pH value to 5.45-5.55, adding 200 mu l of 100 mu mol AS after sterilizing and reducing the temperature to 50 ℃, and uniformly mixing.
Preferably, in step S1, the sterilization step is: soaking the pretreated seeds in 70% ethanol for 30-60s, slightly shaking, soaking the seeds in 0.1% mercuric chloride, sterilizing for 8-10min, continuously slightly shaking, washing the seeds with sterile water for 3-4 times, each time for 5-6min, and drying the water with sterile filter paper after the completion of the washing.
Preferably, in step S1, the preparation of the agrobacterium solution: taking out the agrobacterium strain containing the plasmid from the refrigerator at the temperature of minus 80 ℃, dipping the agrobacterium strain into the bacterial liquid, streaking the bacterial liquid on a solid LB culture medium containing the corresponding antibiotics, and culturing in dark at the temperature of 28 ℃; after single colonies with the size of 1mm grow out, selecting the bacteria, placing the bacteria in 5mL of liquid YEB culture medium containing corresponding antibiotics, placing the liquid YEB culture medium in a shaking table at 28 ℃ and at 180rpm for shake culture for 16-18 hours;
100 μl of the culture broth was aspirated, placed in 10mL of liquid YEB containing the corresponding antibiotic, shake-cultured at 28℃and 180rpm until the OD600 was between 0.5 and 0.7, centrifuged at 10mL of the culture broth, centrifuged at 7500rpm for 15min, and the supernatant was decanted.
Preferably, agrobacteria are used to impregnate cotyledon explants when the OD600 is 0.5 in the IM suspension of the medium.
The efficient agrobacterium-mediated melon genetic transformation method has the advantages that:
1. The method designed by the invention can be simultaneously applied to the thin-skin melons and the thick-skin melons, and the yellowing rate of the regenerated buds is reduced to 1.6%;
2. In the process of the method designed by the invention, a specific amount of CuSO is added into the induction bud regeneration culture medium MeM34·5H2the number of explants of regenerated buds can be obviously increased by O, and the regeneration rate of melon buds of thin-skin type and thick-skin type is promoted by combining a specific plant growth regulator, so that the regeneration rate is up to 78.9%;
3. In the process of the method designed by the invention, the specific selection of the plant growth regulator in the bud extension culture medium MeM4 ensures that the bud extension rate after the action of the bud extension culture medium MeM4 is up to 67%; the amount of silver thiosulfate in the method also further reduces the yellowing rate of the regenerated buds.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. In the drawings:
FIG. 1A is a schematic representation of a seed treated in the method of the present invention;
FIG. 1B is a state diagram of cotyledon explants after cultivation in Agrobacterium co-cultivation medium MeM 2;
FIG. 1C is a diagram showing the cotyledon explant after culture with the induction bud regeneration medium MeM 3;
FIG. 1D is a state diagram of regenerated shoot clumps after culture in shoot elongation medium MeM 4;
FIG. 1E is a state diagram of buds after cultivation in rooting medium MeM 5;
FIG. 1F shows the seedlings after the culture of FIG. 1E.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
the following provides details to illustrate the effects of the method of the present invention;
1. Selection of experimental materials
melon seeds: the muskmelon is of the type No. 5, the beech mushroom, the long-lasting crisp and beautiful, the snow honey, the lemon honey, the thin-skin muskmelon is thick, the green honey and the Wu Nong green jade.
2. Basal medium, plant growth regulator and preparation thereof
Basic culture medium components: MS medium (Murashige and Skoog, 1962), sucrose concentration of 30g/L, agar concentration of 8-9g/L, pH of 5.8-5.85.
Growth regulator and preparation thereof: the preparation method of 6-BA comprises dissolving with small amount of 1mol/L HCl, and preparing 1mg/L mother liquor with distilled water.
the preparation of the zeatin and the IAA, IBA, GA3 is to dissolve the zeatin and the IAA, IBA, GA with a proper amount of 95% ethanol, and then add distilled water to prepare 1mg/L mother liquor for later use. The above mother liquor is filtered and sterilized by a microporous filter of 0.22 μm before use, and stored at-20deg.C.
3. preparation of relevant solvents during the experiment:
3.1 seed selection and pretreatment
Selecting a certain amount of filled melon seeds, soaking in water at 50deg.C for 15min, soaking in tap water for 1-2 hr, and washing the seeds with tap water for 2-3 times. The lateral surface of the seed is gently scraped by a knife, and the seed peeled off is placed in a sterilized triangular flask.
Seed disinfection: soaking in 70% ethanol for 30-60s, slightly shaking, soaking seeds in 0.1% mercuric chloride, sterilizing for 8-10min, continuously slightly shaking, washing the seeds with sterile water for 3-4 times, each time for 5-6min, and drying the water with sterile filter paper for later use after finishing;
Preparation of seed germination medium Mem1 (e.g., in an amount of 100 mL): MS finished powder 0.44g, sucrose 3g, agar powder 0.9g, double distilled water to constant volume 100mL, pH value adjustment of 5.80-5.85, high temperature sterilization, temperature reduction to 55 ℃, adding 50 μl of 200mg/mL universal inhibitor (optional termeitin), mixing uniformly, and sub-packaging into sterile culture dish with diameter of 90cm for use.
preparation of Agrobacterium co-culture Medium MeM2 (e.g., in an amount of 100 mL): MS finished powder 0.44g, sucrose 3g, agar powder 0.9g, double distilled water to constant volume of 100mL, pH value adjustment of 5.70-5.75, high temperature sterilization, temperature reduction to 55 ℃ and 100 mu mol AS 100 mu L are added, and the mixture is packaged in a sterile culture dish for standby.
Preparation of induction bud regeneration medium Mem3 (e.g., in an amount of 100 mL): taking 0.44g of MS finished powder, 3g of sucrose, 0.9g of agar powder and 0.75-1.25 mg/L of CuSO4·5H2The composition of the O and plant growth regulator is 0.5-0.8mg.L-1 6-BA+0.5～0.8mg·L-1Zeatin +0.08 mg.L-1IAA, double distilled water is fixed to 100mL, pH value is adjusted to 5.80-5.85, and 100 microliter of 200mg/mL inhibitor is added for standby after sterilization and temperature reduction to 55 ℃;
Screening agents with different types and concentrations such as kanamycin (Kan 15-25 mg/L), hygromycin (Hpt 5-10 mg/L), glufosinate (Basta 8-10 mg/L) and the like are applied according to the screening markers on the genetic transformation vector, and the mixture is packaged into sterile culture dishes after uniform mixing. Preparing 10mg/ml CuSO4.5H2O (mother liquor): 0.1g of CuSO4.5H2O was taken and dissolved in 10ml of sterile water.
The bud extension medium Mem4 was prepared as follows (e.g., in an amount of 100 mL): taking 0.44g of finished powder, 3g of sucrose, 0.9g of agar powder and 0.05 to 0.1 mg.L of plant growth regulator-16-BA+0.5～1.0mg·L-1GA3, double distilled water with constant volume of 100mL, pH value of 5.80-5.85, sterilizing, adding 100 microliter of 200mg/mL inhibitor and 100 microliter-125 microliter of 20-25 mM silver thiosulfate when the temperature is reduced to 55 ℃.
Screening agents with different types and concentrations such as kanamycin (Kan 15-25 mg/L), hygromycin (Hpt 5-10 mg/L), glufosinate (Basta 8-10 mg/L) and the like are applied according to the screening markers on the genetic transformation vector, and the mixture is packaged in sterile culture dishes for standby.
The method for preparing the 20mM silver thiosulfate mother liquor comprises three steps:
(1) The preparation method of 100mM sodium thiosulfate comprises dissolving 158mg sodium thiosulfate in 10ml sterile double distilled water for use;
(2) The preparation method of 100mM silver nitrate comprises dissolving 170mg silver nitrate in 10ml sterile double distilled water;
(3) 100mM sodium thiosulfate and 100mM silver nitrate were mixed in a 4:1 ratio.
The specific operation is that 8ml of 100mM sodium thiosulfate is put into a 50ml sterile centrifuge tube, 2ml of 100mM silver nitrate is added dropwise, the mixture is stirred while being added, the mixture is fully dissolved, filtered and sterilized, and the mixture is stored at 4 ℃ in a dark place.
Preparation of rooting medium Mem5 (e.g., in an amount of 100 mL): MS finished powder 0.44g, sucrose 3g, agar powder 0.9g,100 mu l 1mg/mL I BA, double distilled water 100mL to constant volume, pH value 5.80-5.85, high temperature sterilization, adding 100 micro liter 200mg/mL termeitin when the temperature is reduced to 55 ℃, mixing evenly and split charging into culture bottles for standby.
3.2 preparation of the Agrobacterium-Tight Medium IM suspension
The agrobacterium strains used were GV3101 and EHA105, both of which were rifampicin (Rif) resistant. The transformed vector was pFGC5941, which contains the Bar plant resistance gene. 2 days before inoculating melon cotyledon, taking out agrobacterium strain containing plasmid from refrigerator at-80 deg.c, drawing line with inoculating loop to solid LB medium containing corresponding antibiotic, sealing with sealing film, and culturing at 28 deg.c in dark. After single colonies with the size of 1mm grow out, the selected bacteria are placed in 5mL of liquid YEB culture medium containing corresponding antibiotics, and placed in a shaking table at 28 ℃ and 180rpm for shake culture for 16-18 hours. 100 μl of the culture broth is taken up and placed in 10mL of liquid YEB containing the corresponding antibiotics, shake culture is carried out at 28 ℃ and 180rpm, when the OD600 is between 0.5 and 0.7, 10mL of the culture broth is centrifuged, 7500rpm is used for centrifugation for 15min, the supernatant is poured off, the culture broth is impregnated with the agrobacteria, and the OD600 is measured to be 0.5.
The preparation process of each 100mL of agrobacterium infection culture medium IM comprises the following steps: taking 0.44g of MS finished powder, 3g of sucrose, 200 mu l of 1mg/ml of 6-BA, 100 mu l of 1mg/ml of ABA, 0.27g of MES, adjusting the pH value to 5.45-5.55, adding 200 mu l of 100 mu mol of AS after sterilizing and reducing the temperature to 50 ℃, and uniformly mixing.
provides a high-efficiency agrobacterium-mediated melon genetic transformation method, which comprises the following steps:
S1, seed selection and disinfection are carried out, and then the seed selection and disinfection are inoculated on a seed germination culture medium MeM1 for dark culture for standby (as shown in figure 1A);
S2, using an agrobacterium-mediated dip-dyeing culture medium IM suspension to dip-dye cotyledon explants cut out from the melon seedlings which grow well in the step S1, wherein the method specifically comprises the following steps:
Cotyledon explants (about 50) were transferred to a prepared agro-bacterial dip, wherein a 50mL sterile centrifuge tube contained 20mL agro-bacterial dip, and the centrifuge tube was placed in a vacuum oven and evacuated-0.094 MPa twice for 5min each time. Placing the cotyledon explant subjected to vacuum pumping on sterile filter paper, and adsorbing excessive agrobacterium tumefaciens bacteria liquid;
Then placing the back of the impregnated cotyledon explant on an agrobacterium co-culture medium (MeM 2) upwards, and performing dark culture at 25 ℃ for 3 days in a constant temperature box for later use (as shown in figure 1B);
S3, placing the cotyledon explant subjected to co-culture in the step S2 on an induction bud regeneration culture medium MeM3 for culture, wherein the incision part of the cotyledon explant is fully contacted with the culture medium, and the front surface of the explant faces upwards.
The culture conditions were 25.+ -. 1 ℃ and photoperiod 16h light/8 h darkness. Culturing for 2-6 weeks, and subculturing every 2 weeks (as shown in FIG. 1C);
S4, cutting off the bud cluster regenerated on the culture medium in the step S3, placing the bud cluster in a bud extension culture medium MeM4, continuing to screen and culture until the buds are extended to 2cm, and transferring the buds to a rooting culture medium (as shown in figure 1D);
S5, transferring the buds obtained in the step S4 to a rooting culture medium MeM5 for growth, after the seedlings are 4-5cm high and the root system grows well, opening a bottle cap of the aseptic seedlings before transplanting, hardening the seedlings in an illumination culture room for 1-2 days, transferring the aseptic seedlings from the culture medium to a nutrition pot (shown in figure 1F), covering a heat preservation shed, guaranteeing humidity, setting the temperature of the incubator to be 25 ℃ in daytime/20 ℃ in darkness, and carrying out photoperiod 16h illumination/8 h darkness (shown in figure 1E).
In the method designed by the invention, the invention has relevant experiments and data description about the following problems:
Embodiment one:
1. regeneration efficiency of different types of melon germplasm on MeM3 culture medium
seed selection: melon with thick skin is honey No. 5, beech mushroom, crisp and beautiful, snow honey, lemon honey, melon with thin skin is thick, green honey and Wu Nong green jade;
S1, seed selection and disinfection are carried out, and then the seed selection and disinfection are inoculated on a seed germination culture medium MeM1 for dark culture for standby;
specifically: soaking full melon seeds in water at 50deg.C for 15min, soaking in tap water for 1 hr, and washing with tap water for 2 times. The seed side surface was gently scraped with a knife and the peeled seed was placed in a sterilized triangular flask.
Seed disinfection: soaking in 70% ethanol for 30s, slightly shaking, soaking seeds in 0.1% mercuric chloride, sterilizing for 8min, continuously slightly shaking, washing the seeds with sterile water for 3 times and 5min each time, and drying the water with sterile filter paper for later use;
Preparation of seed germination medium Mem1 (e.g., in an amount of 100 mL): MS finished powder 0.44g, sucrose 3g, agar powder 0.9g, double distilled water to constant volume 100mL, pH value adjustment of 5.80, high temperature sterilization after temperature reduction of 55 ℃, 50 μl 200mg/mL timentin addition, mixing uniformly, split charging into a sterile culture dish with diameter of 90cm for standby.
S2, using an agrobacterium-mediated dip-dyeing culture medium IM suspension to dip-dye cotyledon explants cut out from the melon seedlings which grow well in the step S1, wherein the method specifically comprises the following steps:
Cotyledon explants (about 50) were transferred to a prepared agro-bacterial dip, wherein a 50mL sterile centrifuge tube contained 20mL agro-bacterial dip, and the centrifuge tube was placed in a vacuum oven and evacuated-0.094 MPa twice for 5min each time. Placing the cotyledon explant subjected to vacuum pumping on sterile filter paper, and adsorbing excessive agrobacterium tumefaciens bacteria liquid;
then placing the back of the impregnated cotyledon explant upwards on an agrobacterium co-culture medium MeM2, and carrying out dark culture for 3 days at a constant temperature of 25 ℃ for later use;
Wherein, the preparation of the agrobacterium co-culture medium Mem2 (such as the amount of 100 mL) is: MS finished powder 0.44g, sucrose 3g, agar powder 0.9g, double distilled water to constant volume of 100mL, pH value adjustment of 5.70, high temperature sterilization, temperature reduction of 55 ℃ and 100 mu mol AS 100 mu l are added, and the mixture is packaged in a sterile culture dish for standby.
s3, placing the cotyledon explant subjected to co-culture in the step S2 on an induction bud regeneration culture medium MeM3 for culture, wherein the incision part of the cotyledon explant is fully contacted with the culture medium, and the front surface of the explant faces upwards; the culture conditions were 25.+ -. 1 ℃ and photoperiod 16h light/8 h darkness. Culturing for 2-6 weeks, and subculturing every 2 weeks.
In the steps: preparation of induction bud regeneration medium Mem3 (e.g., in an amount of 100 mL): taking MS finished powder 0.44g, sucrose 3g, agar powder 0.9g and CuSO 0.75mg/L4·5H2the composition of the O and plant growth regulator is 0.5 mg.L-16-BA+0.5mg·L-1Zeatin +0.08 mg.L-1IAA or 0.8 mg.L-16-BA+0.8mg·L-1Zeatin +0.08 mg.L-1IAA, double distilled water is fixed to 100mL, pH value is adjusted to 5.80, and 100 microliter of 200mg/mL inhibitor is added for standby after sterilization and temperature reduction to 55 ℃;
Screening agents with different types and concentrations such as kanamycin (Kan 15-25 mg/L), hygromycin (Hpt 5-10 mg/L), glufosinate (Basta 8-10 mg/L) and the like are applied according to the screening markers on the genetic transformation vector, and the mixture is packaged into sterile culture dishes after uniform mixing. Preparing 10mg/ml CuSO4.5H2O (mother liquor): 0.1g of CuSO4.5H2O was taken and dissolved in 10ml of sterile water.
in this example, melon explants were transferred to a medium that induced buds after agrobacterium-mediated genetic transformation experiments, and after 3 weeks of culture, the number of buds was counted, wherein 2 weeks were subcultured with MeM3 medium, with the following structure in table 1:
TABLE 1 germination Rate of different types of melon on MeM3 induced bud Medium
TABLE 2 germination Rate of different types of melon on MeM3 induced bud Medium
the MeM3 medium of this example was selected to be 0.5mg.L-1 6-BA+0.5mg·L-1zeatin +0.08 mg.L- 1IAA (as in Table 1) or 0.8 mg.L-1 6-BA+0.8mg·L-1Zeatin +0.08 mg.L-1IAA (as shown in Table 2) was used as a plant growth regulator in the induction regeneration medium, and was effective in promoting the regeneration rate of melon buds (length. Gtoreq.1.0 cm,. Gtoreq.1.5 cm) of thin and thick skin type.
Embodiment two:
the steps S1-S3 described in this example are identical to those of the examples, and 0.5 mg.L in MeM3 medium is selected-16-BA+0.5mg·L-1Zeatin +0.08 mg.L-1IAA is used as a plant growth regulator in an induction regeneration culture medium;
S4, cutting off the bud cluster regenerated on the culture medium in the step S3, placing the bud cluster in a bud extension culture medium MeM4, continuing to screen and culture until the buds are extended to 2cm, and transferring the buds to a rooting culture medium (as shown in figure 1D);
1. Bud elongation of different types of melon germplasm on Mem4 medium (statistical value of bud length 1cm-1.5 cm) is shown in tables 3-6 below:
Wherein, the preparation process of the bud extension culture medium MeM4 is as follows (such as the preparation of 100 mL): taking 0.44g of finished powder, 3g of sucrose, 0.9g of agar powder and 0.05 to 0.1 mg.L of plant growth regulator-16-BA+0.5～1.0mg·L-1GA3, double distilled water with constant volume of 100mL, pH value of 5.80-5.85, sterilizing, adding 100 microliter of 200mg/mL inhibitor and 100 microliter-125 microliter of 20-25 mM silver thiosulfate when the temperature is reduced to 55 ℃.
Screening agents with different types and concentrations such as kanamycin (Kan 15-25 mg/L), hygromycin (Hpt 5-10 mg/L), glufosinate (Basta 8-10 mg/L) and the like are applied according to the screening markers on the genetic transformation vector, and the mixture is packaged in sterile culture dishes for standby.
The method for preparing the 20mM silver thiosulfate mother liquor comprises three steps:
(1) The preparation method of 100mM sodium thiosulfate comprises dissolving 158mg sodium thiosulfate in 10ml sterile double distilled water for use;
(2) The preparation method of 100mM silver nitrate comprises dissolving 170mg silver nitrate in 10ml sterile double distilled water;
(3) 100mM sodium thiosulfate and 100mM silver nitrate were mixed in a 4:1 ratio.
The specific operation is that 8ml of 100mM sodium thiosulfate is put into a 50ml sterile centrifuge tube, 2ml of 100mM silver nitrate is added dropwise, the mixture is stirred while being added, the mixture is fully dissolved, filtered and sterilized, and the mixture is stored at 4 ℃ in a dark place.
TABLE 3 bud elongation of different types of melon on MeM4 induced bud Medium (plant growth regulator 0.05 mg.L)-1 6-BA+0.5mg·L-1 GA3)
TABLE 4 bud elongation of different types of melon on MeM4 induced bud Medium (plant growth regulator 0.1 mg.L)-1 6-BA+1.0mg·L-1 GA3)
As shown in tables 3 and 4, buds obtained by inducing different types of melon explants were transferred to a MeM4 medium and grown, and after 2 weeks, the buds were counted and the buds could be prolonged to 1cm-1.5cm, and as shown in tables 3 and 4, specific agents, namely plant growth regulators with corresponding concentrations, were selected in the MeM4 medium, and the bud elongation rate was high.
TABLE 5 yellow/brown index of different types of melon in MeM4 induced bud medium (plant growth regulator 0.05mg.L)-1 6-BA+0.5mg·L-1GA3+20mM/L sodium thiosulfate)
TABLE 6 yellow/brown index of different types of melon in MeM4 induced bud medium (plant growth regulator 0.05mg.L)-1 6-BA+0.5mg·L-1 GA3+25mM/L STS)
TABLE 7 yellow/brown index of different types of melon in MeM4 induced bud medium (plant growth regulator 0.1 mg.L)-1 6-BA+1.0mg·L-1 GA3+20mM/L STS)
TABLE 8 yellow/brown index of different types of melon in MeM4 induced bud medium (plant growth regulator 0.1 mg.L)-1 6-BA+1.0mg·L-1 GA3+25mM/L STS)
As shown in tables 5 to 8, the elongation and browning rate of the regenerated buds were reduced by STS (silver thiosulfate) at different concentrations;
The principle is as follows: in the process of in vitro culture of plants, a large amount of ethylene is generated and accumulated along with the extension of the culture time due to insufficient exchange of gas inside and outside a culture container, and the ethylene has an inhibition effect on cell differentiation and organ formation, wherein the ethylene with high content can cause the browning death of explants and the malformation of plants, and silver ions are a better ethylene activity inhibitor. However, as the concentration of silver ions increases, the silver ions act as heavy metal ions to poison plant organs, for example, the silver ions can competitively enter chlorophyll molecules to change the structure of the chlorophyll molecules, so that the synthesis of the protein with lost functions is correspondingly inhibited. Based on the fact that different concentrations of silver ions in the culture medium have positive and negative effects on plant growth, only when the concentrations are proper, the silver ions can effectively play a role of an ethylene inhibitor, and development of organs is promoted. When the regenerated shoots are elongated in the MeM4 medium, the leaf area is increased
the genetic transformation method designed by the application solves the defect that independent transformation methods are needed to be used for the muskmelon and the muskmelon in the market, has stronger universality, namely, is simultaneously suitable for the genetic transformation of the muskmelon and the muskmelon, and has high regeneration rate and low yellowing rate due to the specific formulas of MeM3, meM4 and the like.
The steps, reagent amounts, etc. not explicitly described in the first and second examples of the present invention are default to the same as those described in the present embodiment, so as to constitute a complete test step.
it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (6)
1. An agrobacterium-mediated melon genetic transformation method is characterized in that: the method comprises the following steps:
S1, seed selection and disinfection are carried out, and then the seed selection and disinfection are inoculated on a seed germination culture medium MeM1 for dark culture for standby;
S2, using an agrobacterium dip-dyeing culture medium IM suspension to dip-dye the cotyledon explants excised from the melon seedlings which grow robustly in the step S1, and finally, putting the dip-dyed cotyledon explants on an agrobacterium co-culture medium MeM2 for co-culture for later use;
S3, placing the cotyledon explant subjected to co-culture in the step S2 on an induction bud regeneration culture medium MeM3 for culture, and carrying out secondary culture every two weeks for later use;
S4, cutting off the regenerated bud cluster on the culture medium in the step S3, placing the bud cluster in a bud extension culture medium MeM4, continuing to carry out screening culture until the buds are extended to 2cm, and transferring the buds to a rooting culture medium;
s5, transferring the buds obtained in the step S4 to a rooting culture medium MeM5 for growth, and then transferring to a nutrition pot for culture;
Wherein,
the preparation process of each 100mL of agrobacterium infection culture medium IM comprises the following steps: taking 0.44g of MS finished powder, 3g of sucrose, 200 mu L of 1mg/ml 6-BA, 100 mu L of 1mg/ml ABA, 0.27gMES, adjusting the pH value to 5.45-5.55, adding 100 mu mol AS200 mu L after sterilization and reducing the temperature to 50 ℃, and uniformly mixing;
Preparation of per 100mL seed germination medium MeM 1: MS finished powder 0.44g, sucrose 3g, agar powder 0.9g, double distilled water constant volume 100mL, pH value 5.80-5.85, high temperature sterilization after temperature reduction to 55 ℃, adding 50 mu L200 mg/mL universal inhibitor, mixing uniformly;
Preparation of MeM2 per 100mL of Agrobacterium co-culture medium: MS finished powder 0.44g, sucrose 3g, agar powder 0.9g, double distilled water constant volume 100mL, pH value 5.70-5.75, high temperature sterilization, after temperature reduction to 55 ℃,100 mu moL AS100 mu L, mixing well;
the preparation process of the induction bud regeneration culture medium MeM3 per 100mL comprises the following steps: taking 0.44g of MS finished powder, 3g of sucrose, 0.9g of agar powder and 0.75-1.25 mg/L of CuSO4·5H2the composition of the O and plant growth regulator is 0.5-0.8mg.L-1 6-BA+0.5～0.8mg·L-1Zeatin +0.08 mg.L-1IAA, double distilled water is fixed to 100mL, pH value is adjusted to 5.80-5.85, 100 microliter of 200mg/mL inhibitor is added after sterilization and temperature is reduced to 55 ℃, and screening agents with different types and concentrations are applied according to screening marks on genetic transformation vectors;
The preparation process of the bud extension medium MeM4 per 100mL comprises the following steps: taking 0.44g of finished powder, 3g of sucrose, 0.9g of agar powder and 0.05 to 0.1 mg.L of plant growth regulator-1 6-BA+0.5～1.0mg·L-1GA3, double distilled water with constant volume of 100mL, pH value of 5.80-5.85, sterilizing, adding 100 microliter of 200mg/mL inhibitor and 100 microliter-125 microliter of 20-25 mM silver thiosulfate when the temperature is reduced to 55 ℃, and applying screening agents with different types and concentrations according to screening marks on genetic transformation carriers;
Preparation of MeM5 per 100mL rooting medium: MS finished powder 0.44g, sucrose 3g, agar powder 0.9g,100 mu L1 mg/mL IBA, double distilled water 100mL, pH value 5.80-5.85, high temperature sterilization, adding 100 microliter 200mg/mL termestin when the temperature is reduced to 55 ℃, mixing uniformly;
The inhibitor is timentin.
2. The agrobacterium-mediated melon genetic transformation method according to claim 1, wherein the method comprises the steps of: the screening agent is one of 15-25mg/L kanamycin, 5-10mg/L hygromycin and 8-10mg/L glufosinate.
3. The agrobacterium-mediated melon genetic transformation method according to claim 1, wherein the method comprises the steps of: configuration of 20mM silver thiosulfate:
(1) The preparation method of 100mM sodium thiosulfate comprises dissolving 158mg sodium thiosulfate in 10ml sterile double distilled water for use;
(2) The preparation method of 100mM silver nitrate comprises dissolving 170mg silver nitrate in 10ml sterile double distilled water;
(3) 100mM sodium thiosulfate and 100mM silver nitrate were mixed in a 4:1 ratio.
4. The agrobacterium-mediated melon genetic transformation method according to claim 1, wherein the method comprises the steps of: in step S1, the disinfection step is: soaking the pretreated seeds in 70% ethanol for 30-60s, slightly shaking, soaking the seeds in 0.1% mercuric chloride, sterilizing for 8-10min, continuously slightly shaking, washing the seeds with sterile water for 3-4 times, each time for 5-6min, and drying the water with sterile filter paper after the completion of the washing.
5. The agrobacterium-mediated melon genetic transformation method according to claim 1, wherein the method comprises the steps of: preparing agrobacterium liquid: taking out the agrobacterium strain containing the plasmid from the refrigerator at the temperature of minus 80 ℃, dipping the agrobacterium strain into the bacterial liquid, streaking the bacterial liquid on a solid LB culture medium containing the corresponding antibiotics, and culturing in dark at the temperature of 28 ℃; after single colonies with the size of 1mm grow out, selecting the bacteria, placing the bacteria in 5mL of liquid YEB culture medium containing corresponding antibiotics, placing the liquid YEB culture medium in a shaking table at 28 ℃ and at 180rpm for shake culture for 16-18 hours;
100 μl of the culture broth was aspirated, placed in 10mL of liquid YEB containing the corresponding antibiotic, shake-cultured at 28℃and 180rpm until the OD600 was between 0.5 and 0.7, centrifuged at 10mL of the culture broth, centrifuged at 7500rpm for 15min, and the supernatant was decanted.
6. The agrobacterium-mediated melon genetic transformation method according to claim 1, wherein the method comprises the steps of: agrobacteria infection medium IM suspension, OD600 of 0.5 was measured and used for the infection of cotyledon explants.
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