CN112889667A - Convenient and efficient tomato transgenic method - Google Patents
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Abstract
The invention discloses a convenient and efficient tomato transgenic method, which is mainly improved by the following steps: 1. improving the formula of the culture medium to obtain an explant with stronger activity; 2. the growth speed of the positive seedlings is accelerated by adjusting the hormone proportion, so that the purpose of distinguishing positive buds from non-positive buds is achieved; 3. the treatment time is changed into the growth state of the explant from each operation link as a reference, so that the controllability of the transgene is enhanced; 4. the growth, rooting and seedling hardening of the bud are seamlessly connected, so that the manual operation is reduced, and the risk of bacterial contamination is reduced.
Description
Technical Field
The invention relates to the technical field of transgenosis, in particular to a convenient and efficient tomato transgenic method.
Background
The tomato is originally produced in south America, is a warm-loving and light-loving short-day plant, has a growth cycle of 4-6 months, has no strict requirement on soil, and preferably has a pH value of 6-7. The tomato is a vegetable with rich nutrition, has excellent flavor, is often eaten as a fruit, contains rich trace elements, vitamins and soluble sugar, and has the effects of enhancing the immunity of a human body, relieving summer heat, quenching thirst and promoting digestion. Worldwide, the trade in tomato and its products is a significant position in the vegetable trade. In recent years, the tomato industry in China is rapidly developed and has been the first place in the world, so that the tomato flavor is accurately improved, the adaptability of the tomato is enhanced, the planting range of the tomato is expanded, and the tomato has high economic value. In addition, the genomic sequence information of tomato has been determined, and the growth cycle of tomato is much shorter than that of fruit tree, so tomato has been widely used in scientific research as a model plant for researching fruit quality. Therefore, the efficient tomato transgenic technology is improved and developed, reference can be provided for the research of other fruits, and the method has high scientific research value.
The transgenic technology mediated by agrobacterium usually comprises four links of acquisition of a target gene, construction of an expression vector, agrobacterium transformation and plant tissue culture, wherein the plant tissue culture is the most core part, the most difficult part and the longest period, and is also a key factor for limiting whether a certain plant can realize transgene. The current research results show that the tomato tissue culture theoretically only needs 56 days, but due to the fact that bacterial infection is needed in the transgenic process, operations such as antibiotic addition and the like limit the growth of tomato explants, the transgenic period of the tomatoes is prolonged, even if professional transgenic companies are used, the transgenic materials which are positive need to be obtained for 3-6 months, the transgenic operation is complicated, the influence factors are more, and the stability of the transgenic operation is difficult to control.
The traditional transgenic method has the following defects:
1. the operation is complicated, the growth conditions of the experimental materials in each period are different due to the difference of the materials and the culture conditions, the fluctuation between different batches is large, and the success rate is difficult to control.
2. After infection is finished, the plant is placed in a dark environment for 2 days, and the agrobacterium is excessively propagated, so that the activity of an explant is reduced, the growth of callus and buds is slowed down, and the experimental period is prolonged. At present, professional transgenic companies also need 3-6 months to complete the whole transgenic process.
3. The method utilizes the fact that transgenic buds have resistance genes, and inhibits the growth of normal buds by adding a large amount of antibiotics, so that transgenic buds and non-transgenic buds are distinguished. Although the transgenic buds have resistance, the transgenic buds also need to synthesize and decompose protein of antibiotics, so the antibiotic environment is also a stress to the transgenic seedlings, the growth rate of the transgenic buds is reduced, and the experimental period is prolonged. In addition, tomato is sensitive to hygromycin, so tomato transgenes are not suitable for hygromycin resistance, and the traditional transgenic method also has requirements on a transgenic vector.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a convenient and efficient tomato transgenic method; the traditional tomato transgenic method is improved, the transgenic period is greatly shortened, and the positive rate is improved, and the improvement of the invention is mainly embodied in that: 1. improving the formula of the culture medium to obtain an explant with stronger activity; 2. the growth speed of the positive seedlings is accelerated by adjusting the hormone proportion, so that the purpose of distinguishing positive buds from non-positive buds is achieved; 3. the treatment time is changed into the growth state of the explant from each operation link as a reference, so that the controllability of the transgene is enhanced; 4. the growth, rooting and seedling hardening of the bud are seamlessly connected, so that the manual operation is reduced, and the risk of bacterial contamination is reduced.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a convenient and efficient tomato transgenic method comprises the following steps:
step one, culture of aseptic seedlings
Sterilizing with 10-15% NaClO for 15-20 min, rinsing with sterile water for 5 times, and standing overnight at room temperature in a dark place; then, flatly paving the seeds on 1/2MS culture medium, wherein the seeds can germinate in about 7-10 days and 2 hypertrophic cotyledons are generated;
step two, explant culture
Shearing cotyledons of the tomato seedlings by using sterilized scissors and tweezers, paving the cotyledons on a pre-culture medium, enabling the front faces of the leaves to be downward, preferably 50-60 cotyledons in each plate, and keeping the plate dark and dark for 24-30 hours until the cut of the leaves shows an expansion sign, namely, using the cut in the fourth step;
step three, agrobacterium preparation
Taking out the constructed or frozen agrobacterium, coating the agrobacterium on an LB plate containing corresponding antibiotics, picking a monoclonal, and culturing the monoclonal in an LB liquid culture medium containing the corresponding antibiotics at 28 ℃ and 200rpm overnight until OD600 is 0.5; inoculating 300 mu L of the agrobacterium to a new 30mL LB culture medium, and shaking at 28 ℃ and 200rpm for 5-6 h; the agrobacterium was then centrifuged, the supernatant discarded, and the suspension was incubated with 30mL liquid medium: MS is 4.3g/L, sucrose is 30g/L, pH is 5.5-6.0, and the mixture is re-suspended and uniformly mixed for later use;
step four, explant infection
Soaking the pre-cultured tomato cotyledons in the agrobacterium suspension in the step 3 for 20min, and slightly reversing or rotating every 5min to ensure that the cotyledons are completely contacted with the bacterial liquid as much as possible; then, the bacterial liquid on the surfaces of the cotyledons is sucked dry by using a sterilized paper towel, so that the leaves are prevented from being damaged by compression; placing the leaves on a pre-culture medium, culturing in the dark for 2d, stopping culturing in the dark if the agrobacteria grow around the leaves, and immediately entering the fifth step;
step five, inducing healing and degerming
Transferring the cotyledon in the fourth step to a resistant culture medium, and cutting for about 5-7 d to form a callus mass;
step six, inducing germination
Transferring the cotyledon with the callus onto a hormone plate, and forming a new bud on the cotyledon for about 7-10 days; the thickness of the culture medium is required to be about 0.7-1 cm;
seventhly, rooting culture
And (4) taking the buds in the fifth step off from the stem ends by using sterilized forceps and scissors, inserting the buds into a rooting culture medium, loosening the mouth of a culture bottle every 7-10 days to root for about 15 days, and transplanting after the root system develops strongly.
Further, in the first step, the MS culture medium is MS 2.15g/L, sucrose 30g/L, agar 7g/L and pH 5.5-6.0.
Further, the pre-culture medium in the second step specifically comprises: 4.3g/L of MS, 30g/L of cane sugar, 0.2mg/L of IAA, 2mg/L of 6-BA, 7g/L of agar and 5.5-6.0 of pH.
Further, the anti-resistance culture medium in the fifth step is MS 4.3g/L, sucrose 30g/L, IAA0.1 mg/L, 6-BA 2mg/L, Timentin300mg/L, Kan 200mg/L, agar 7g/L and pH 5.5-6.0.
Further, in the sixth step, the hormone panel comprises: 4.3g/L MS, 30g/L sucrose, 0.1mg/L IAA, 3 mg/L6-BA, 150mg/L Timentin, 50mg/L Kan, 7g/L agar and 5.5-6.0 pH.
Further, in the seventh step, the rooting medium specifically comprises: MS 4.3g/L, sucrose 30g/L, Kan 100mg/L, Timentin300mg/L, agar 7.5g/L, pH 5.5-6.0, and optionally IAA0.1 mg/L.
Compared with the prior art, the invention has the beneficial effects that:
the traditional tomato transgenic method is improved, the transgenic period is greatly shortened, and the positive rate is improved, and the improvement of the invention is mainly embodied in that:
1. improving the formula of the culture medium to obtain an explant with stronger activity;
2. the growth speed of the positive seedlings is accelerated by adjusting the hormone proportion, so that the purpose of distinguishing positive buds from non-positive buds is achieved;
3. the treatment time is changed into the growth state of the explant from each operation link as a reference, so that the controllability of the transgene is enhanced;
4. the growth, rooting and seedling hardening of the bud are seamlessly connected, so that the manual operation is reduced, and the risk of bacterial contamination is reduced.
5. The improved transgenic method has the advantages that the links of bud growth, rooting and seedling hardening are directly and seamlessly connected, the experimental period is greatly shortened, the whole transgenic process can be finished within about 1.5 months at the fastest speed, and the transplanted transgenic material is obtained.
6. The method utilizes the characteristic that agrobacterium-mediated transgenosis can induce plant tissues to form root nodules, enables the transgenic tissues to rapidly grow to form buds by changing the proportion of exogenous hormones, enlarges the time difference between the transgenic buds and non-transgenic buds, and successfully distinguishes the transgenic buds from the non-transgenic buds, and the positive rate of the method is close to 100% (see figure 4).
7. The method uses a lower concentration of antibiotic, only to inhibit bacterial growth, and does not require plant material to have a resistance marker, and therefore the method has no requirement for a transgenic vector. The improved transgenic method comprises the following seven steps, and the flow chart is shown in figure 2.
Drawings
FIG. 1 is a flow chart of a traditional tomato transgene;
FIG. 2 is a flow chart of a modified tomato transgene;
FIG. 3 is an exemplary diagram of the steps of the transgene;
FIG. 4 is a PCR verification of transgenic lines.
Detailed Description
The technical scheme of the invention is further described in detail by combining the drawings and the detailed implementation mode:
as shown in the figures 1-4 of the drawings,
test example: a convenient and efficient tomato transgenic method comprises the following steps:
first, culture of aseptic seedlings
Sterilizing with 10-15% NaClO for 15-20 min, rinsing with sterile water for 5 times, and standing overnight at room temperature in a dark place; then, the seeds are flatly paved on 1/2MS (MS 2.15g/L, sucrose 30g/L, 7g/L agar, pH 5.5-6.0) culture medium, the seeds can germinate in about 7-10 days, and 2 hypertrophic cotyledons are generated (figure 3 a);
second, explant culture
Shearing cotyledons of the tomato seedlings by using sterilized scissors and tweezers, and paving the cotyledons on a pre-culture medium (MS 4.3g/L, sucrose 30g/L, IAA 0.2mg/L, 6-BA 2mg/L, agar 7g/L and pH 5.5-6.0), wherein the front surfaces of the leaves face downwards, 50-60 cotyledons are suitable for each plate, and the leaves are dark and protected from light for 24-30 hours, and when the cuts of the leaves show the signs of expansion, the leaves are used in the fourth step;
third, preparation of Agrobacterium
The constructed or frozen agrobacterium was removed, spread on LB plates containing the corresponding antibiotics, and single colonies were picked and cultured overnight at 28 ℃ and 200rpm in LB liquid medium containing the corresponding antibiotics to an OD600 of 0.5. 300 mu L of the agrobacterium is inoculated in a new 30mL LB culture medium and shaken at the temperature of 28 ℃ and the rpm of 200 for 5 to 6 hours. Then, centrifuging the agrobacterium, removing a supernatant, resuspending the agrobacterium with 30mL of liquid culture medium (MS 4.3g/L, sucrose 30g/L, pH 5.5-6.0), and uniformly mixing the agrobacterium and the supernatant for later use;
fourth, explant infection
And (3) soaking the pre-cultured tomato cotyledons in the agrobacterium suspension in the step 3 for 20min, and slightly reversing or rotating every 5min to ensure that the cotyledons are completely contacted with the bacterial liquid as far as possible. Then, the bacterial solution on the surface of the cotyledon was blotted with a sterilized paper towel to avoid the damage of the cotyledon due to compression (FIG. 3 b). The leaves are placed on a pre-culture medium and cultured in the dark for 2d, during which period the dark culture is terminated if there is evidence of agrobacterium growth around the leaves, and step five is immediately entered.
Fifthly, inducing and healing wounds and sterilizing
Transferring the cotyledons in the fourth step to a resistant culture medium (MS 4.3g/L, sucrose 30g/L, IAA0.1 mg/L, 6-BA 2mg/L, Timentin300mg/L, Kan 200mg/L, agar 7g/L, pH 5.5-6.0), and cutting for about 5-7 d to form callus clusters (figure 3 c).
Sixthly, inducing germination
The cotyledons with callus were transferred to a hormone plate (MS 4.3g/L, sucrose 30g/L, IAA0.1 mg/L, 6-BA 3mg/L, Timentin 150mg/L, Kan 50mg/L, agar 7g/L, pH 5.5-6.0) to form new sprouts on cotyledons about 7-10 days (FIG. 3 d). The thickness of the culture medium is required to be about 0.7 to 1 cm.
Seventhly, rooting culture
And (3) taking the buds in the fifth step off from the stem tips by using sterilized forceps and scissors, inserting the buds into a rooting culture medium (MS 4.3g/L, sucrose 30g/L (optionally adding IAA0.1 mg/L), Kan 100mg/L, Timentin300mg/L, agar 7.5g/L and pH 5.5-6.0), loosening the mouth of the culture bottle every 7-10 days to root for about 15 days (figure 3e), and transplanting after the root system develops strongly (figure 3 f).
Test example: as shown in figure 4 of the drawings,
1. a small piece of leaf was removed from the obtained transgenic seedling, and an appropriate amount of liquid nitrogen was added to extract genomic DNA, and the concentration thereof was diluted to 100 ng/. mu.L. The kit for extracting the genome is purchased from Beijing Tiangen Biochemical technology Co., Ltd (cargo number: DP 350-03).
2. Primers were designed based on the kanamycin resistance gene sequence and the target gene sequence in the vector used for the transgene, and synthesized by the bioscience, kasei, inc.
3. PCR cloning was performed using PrimeSTARMax DNApolymerase (Beijing Baozi physician science and technology Co., Ltd., product No.: R045A). The PCR system composition and reaction procedure are shown in the end of text.
4. To the reaction mixture was added 1/10 volumes of 10 × Loading Buffer (Beijing Baoriri doctor science and technology Co., Ltd., cat # 9157) and the electrophoresis was performed using 1% agarose gel.
5. The results of FIG. 4 were obtained by observing and photographing in a gel imager.
Wherein
The PCR reaction system comprises:
| components | Volume/. mu.L |
| PrimeSTAR Max DNA Polymerase | 12.5 |
| |
1 |
| |
1 |
| Template | 1 |
| H2O | 9.5 |
| Total | 25 |
PCR reaction procedure:
the method for improving the tomato transgenosis has the advantages that:
1. the improved transgenic method has the advantages that the links of bud growth, rooting and seedling hardening are directly and seamlessly connected, the experimental period is greatly shortened, the whole transgenic process can be finished within about 1.5 months at the fastest speed, and the transplanted transgenic material is obtained.
2. The method utilizes the characteristic that agrobacterium-mediated transgenosis can induce plant tissues to form root nodules, enables the transgenic tissues to rapidly grow to form buds by changing the proportion of exogenous hormones, enlarges the time difference between the transgenic buds and non-transgenic buds, and successfully distinguishes the transgenic buds from the non-transgenic buds, and the positive rate of the method is close to 100% (see figure 4).
3. The method uses a lower concentration of antibiotic, only to inhibit bacterial growth, and does not require plant material to have a resistance marker, and therefore the method has no requirement for a transgenic vector. The improved transgenic method comprises the following seven steps, and the flow chart is shown in figure 2.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Claims (6)
1. A convenient and efficient tomato transgenic method is characterized by comprising the following steps:
step one, culture of aseptic seedlings
Sterilizing with 10-15% NaClO for 15-20 min, rinsing with sterile water for 5 times, and standing overnight at room temperature in a dark place; then, flatly paving the seeds on 1/2MS culture medium, wherein the seeds can germinate in about 7-10 days and 2 hypertrophic cotyledons are generated;
step two, explant culture
Shearing cotyledons of the tomato seedlings by using sterilized scissors and tweezers, paving the cotyledons on a pre-culture medium, enabling the front faces of the leaves to be downward, preferably 50-60 cotyledons in each plate, and keeping the plate dark and dark for 24-30 hours until the cut of the leaves shows an expansion sign, namely, using the cut in the fourth step;
step three, agrobacterium preparation
Taking out the constructed or frozen agrobacterium, coating the agrobacterium on an LB plate containing corresponding antibiotics, picking a monoclonal, and culturing the monoclonal in an LB liquid culture medium containing the corresponding antibiotics at 28 ℃ and 200rpm overnight until OD600 is 0.5; inoculating 300 mu L of the agrobacterium to a new 30mL LB culture medium, and shaking at 28 ℃ and 200rpm for 5-6 h; the agrobacterium was then centrifuged, the supernatant discarded, and the suspension was incubated with 30mL liquid medium: MS is 4.3g/L, sucrose is 30g/L, pH is 5.5-6.0, and the mixture is re-suspended and uniformly mixed for later use;
step four, explant infection
Soaking the pre-cultured tomato cotyledons in the agrobacterium suspension in the step 3 for 20min, and slightly reversing or rotating every 5min to ensure that the cotyledons are completely contacted with the bacterial liquid as much as possible; then, the bacterial liquid on the surfaces of the cotyledons is sucked dry by using a sterilized paper towel, so that the leaves are prevented from being damaged by compression; placing the leaves on a pre-culture medium, culturing in the dark for 2d, stopping culturing in the dark if the agrobacteria grow around the leaves, and immediately entering the fifth step;
step five, inducing healing and degerming
Transferring the cotyledon in the fourth step to a resistant culture medium, and cutting for about 5-7 d to form a callus mass;
step six, inducing germination
Transferring the cotyledon with the callus onto a hormone plate, and forming a new bud on the cotyledon for about 7-10 days; the thickness of the culture medium is required to be about 0.7-1 cm;
seventhly, rooting culture
And (4) taking the buds in the fifth step off from the stem ends by using sterilized forceps and scissors, inserting the buds into a rooting culture medium, loosening the mouth of a culture bottle every 7-10 days to root for about 15 days, and transplanting after the root system develops strongly.
2. The method as claimed in claim 1, wherein in the first step, the MS culture medium is MS 2.15g/L, sucrose 30g/L, agar 7g/L, and pH 5.5-6.0.
3. The method according to claim 1, wherein the pre-culture medium in the second step is specifically: 4.3g/L of MS, 30g/L of cane sugar, 0.2mg/L of IAA, 2mg/L of 6-BA, 7g/L of agar and 5.5-6.0 of pH.
4. The method of claim 1, wherein the resistant medium in step five is MS 4.3g/L, sucrose 30g/L, IAA0.1 mg/L, 6-BA 2mg/L, Timentin300mg/L, Kan 200mg/L, agar 7g/L, pH 5.5-6.0.
5. The method of claim 1, wherein in step six, the hormone panel comprises: 4.3g/L MS, 30g/L sucrose, 0.1mg/L IAA, 3 mg/L6-BA, 150mg/L Timentin, 50mg/L Kan, 7g/L agar and 5.5-6.0 pH.
6. The method according to claim 1, wherein in step seven, the rooting medium is specifically: MS 4.3g/L, sucrose 30g/L, Kan 100mg/L, Timentin300mg/L, agar 7.5g/L, pH 5.5-6.0, and optionally IAA0.1 mg/L.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995359A (en) * | 2006-12-29 | 2007-07-11 | 南开大学 | Agrobacterium mediated large size tomato transformation method |
| CN111850036A (en) * | 2020-07-27 | 2020-10-30 | 海南大学 | Agrobacterium-mediated tomato genetic transformation method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995359A (en) * | 2006-12-29 | 2007-07-11 | 南开大学 | Agrobacterium mediated large size tomato transformation method |
| CN111850036A (en) * | 2020-07-27 | 2020-10-30 | 海南大学 | Agrobacterium-mediated tomato genetic transformation method |
Non-Patent Citations (4)
| Title |
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| 张玉等: "农杆菌介导的ICE1基因转化番茄的研究", 《作物杂志》 * |
| 徐小静: "《分子生物学与基因工程技术和实验》", 30 November 2018, 中央民族大学出版社 * |
| 李立芹: "根癌农杆菌介导番茄‘白果强丰’遗传转化体系优化", 《中国农学通报》 * |
| 陈珍等: "农杆菌介导的番茄遗传转化体系优化研究", 《浙江大学学报(农业与生命科学版)》 * |
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