Efficient tomato living body transgenic method

Abstract

The invention discloses an efficient tomato living body transgenosis method which comprises the following steps: taking a MicroTom seedling, culturing until true leaves grow, cutting the seedling, reserving a hypocotyl, putting the hypocotyl into an illumination incubator, culturing for 1-3 days, sleeving the hypocotyl with a pipette tip, injecting an infection solution into the pipette tip to immerse the section of the hypocotyl, and carrying out transgenosis on the hypocotyl. And infecting the hypocotyl section with the infection liquid. The invention provides a rapid, efficient and stable MicroTom tomato living body genetic transformation method, hypocotyl begins to germinate buds after Agrobacterium tumefaciens infection for 3 weeks, transformed plants bloom after about 3 months, seeds can be collected in 5-6 months, the whole transformation process is faster than that of a tissue culture mode by more than 2 months, the transformation efficiency is 28.57%, and the method is suitable for large-scale popularization and application. The established method is beneficial to the development of gene function research and genetic breeding processes on the MicroTom tomatoes.

Description

Technical Field

[0001] The present invention relates to the technical field of in vivo tomato transgenic technology, and in particular to an efficient method for in vivo tomato transgenic technology. Background Art

[0002] At present, the genetic transformation systems of most plants rely on tissue culture technology to induce cells containing foreign genes to form meristems, and then induce the formation of regenerated seedlings. This genetic transformation method has high requirements for sterile environment and sterile operation technology, and there are problems such as difficulties in the regeneration and identification of resistant plants, low transformation efficiency, and long operation cycle. In addition, high concentrations of exogenous plant hormones are used in the tissue culture process, and the regenerated or transgenic plants are prone to unknown genomic changes. Therefore, tissue culture technology has become an important bottleneck and limiting factor in plant genetic transformation.

[0003] The traditional genetic transformation process of MicroTom tomato, as described in the publication number CN111850036A, mainly includes the disinfection of explants such as seeds or leaves, the induction of seed germination or the regeneration of explants under sterile conditions; the newly emerged cotyledons or explants are pre-cultured, co-cultured with Agrobacterium, screened and cultured (germination), rooted and transplanted, etc. The whole genetic transformation process requires complex and cumbersome preparation of sterile operations, and the transformation cycle is relatively long. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a new and efficient method for in vivo tomato transgenic technology.

[0005] Taking the seedlings germinated from MicroTom tomato seeds as materials, the present invention has established a rapid, efficient and stable in vivo genetic transformation system, in order to lay a foundation for plant gene function research and accelerate the genetic breeding process.

[0006] To solve the above technical problems, the present invention is solved by the following technical solutions: An efficient method for in vivo tomato transgenic technology, comprising the following steps: S1: Cultivate MicroTom seedlings until true leaves grow, cut the seedlings, retain the hypocotyl, and place the hypocotyl in a light incubator for 1 - 3 days.

[0007] S2: Put the pipette tip on the hypocotyl, inject the infection solution into the pipette tip to submerge the cut surface of the hypocotyl, and the infection solution infects the cut surface of the hypocotyl.

[0008] S3: Place the infected hypocotyl in the dark for co-culture for 2 - 4 days.

[0009] S4: Place it in a light incubator for 1 - 3 days.

[0010] S5: Repeat steps S2 - S4 for the cultured hypocotyl 1 - 4 times.

[0011] In the above-mentioned scheme, preferably, in S1, when the seedlings have grown two true leaves, a clean knife is used to make a horizontal cut at a position 0.4 - 0.7 cm below the cotyledons, and the hypocotyl with a length of 1 - 2 cm above the substrate is retained.

[0012] In the above-mentioned scheme, preferably, a clean knife is used to make a horizontal cut at a position 0.5 cm below the cotyledons, and the hypocotyl with a length of 1 - 1.5 cm above the substrate is retained.

[0013] In the above-mentioned scheme, preferably, in S1, the hypocotyl is cultured in a light incubator for 2 days.

[0014] In the above-mentioned scheme, preferably, in S2, the infection solution is used to soak the cut surface of the hypocotyl for 5 - 15 minutes.

[0015] In the above-mentioned scheme, preferably, the infection solution is used to soak the cut surface of the hypocotyl for 8 - 12 minutes.

[0016] In the above-mentioned scheme, preferably, in S1, the culture steps of MicroTom seedlings are as follows: Place MicroTom seeds in a petri dish with moist filter paper and culture them in the dark until the radicles germinate.

[0017] Transfer the seeds after the radicles have germinated to small pots with substrate, place them in a light incubator, and culture them under the conditions of 16 hours of light / 8 hours of darkness, a temperature of 22 ± 1°C, and a humidity of 70%.

[0018] In the above-mentioned scheme, preferably, for the small pots with substrate, the substrate is prepared by mixing peat soil, vermiculite, and perlite in a volume ratio of 3:1:1.

[0019] In the above-mentioned scheme, preferably, in S2, the infection solution is an Agrobacterium tumefaciens bacterial solution carrying a vector with the overexpressed VcRZ-1A-GFP gene.

[0020] In the above-mentioned scheme, preferably, for the preparation process of the infection solution, take Agrobacterium tumefaciens GV3101 carrying a vector with the overexpressed VcRZ-1A-GFP gene and streak-activate it on an LB solid medium containing 25 mg.L -1 rifampicin and 50 mg.L -1 kanamycin, and incubate it upside down at 28°C in the dark for 2 days.

[0021] Pick a single colony and inoculate it into an LB liquid medium containing the corresponding antibiotics. After culturing it at 28°C with shaking at 200 rpm for 12 hours, expand the bacterial solution at a ratio of 1:100 for 12 - 16 hours.

[0022] The collected thalli after centrifugation were resuspended in MS liquid medium, and the OD600 of the bacterial liquid was adjusted to 0.6, and then left to stand in the dark for 2 hours for later use.

[0023] The beneficial effects of the present invention are as follows: The present invention provides a rapid, efficient and stable method for in vivo genetic transformation of MicroTom tomatoes. After 3 weeks of infection with Agrobacterium tumefaciens, hypocotyls begin to germinate young buds, the transformed plants bloom after about 3 months, and seeds can be harvested in the 5th to 6th month. The entire transformation process is at least 2 months faster than the tissue culture method, and the transformation efficiency is 28.57%. The established method helps to carry out gene function research and genetic breeding processes on MicroTom tomatoes. Brief Description of the Drawings

[0024] Figure 1 It is a schematic diagram of tomato seed germination of the present invention.

[0025] Figure 2 It is a schematic diagram of hypocotyl cutting of the present invention.

[0026] Figure 3 It is a schematic diagram of hypocotyl infiltration of the present invention.

[0027] Figure 4 It is a schematic diagram of hypocotyl culture in dark and light environments of the present invention.

[0028] Figure 5 It is a schematic diagram of hypocotyls at 10 days and 14 days after infection of the present invention.

[0029] Figure 6 It is a schematic diagram of hypocotyls at 20 days and 24 days after infection of the present invention.

[0030] Figure 7 It is a schematic diagram of developing seedlings at 35 days and 60 days after infection of the present invention.

[0031] Figure 8 It is a schematic diagram of a seedling developing into a tomato plant of the present invention.

[0032] Figure 9 It is an efficiency evaluation form of hypocotyls of the present invention. Detailed Embodiments

[0033] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments: Refer to Figures 1-9 , Explanation of A-Q in the drawings: A: Germination of tomato seeds; B: Seeds germinated for 3 days, growing radicles; C: Seedlings planted for 14 days; D: Remove the top cotyledons and true leaves of the seedlings, and keep the hypocotyls; E: Hypocotyls are infected with Agrobacterium by using a pipette; F: Infection with Agrobacterium; G: Dark treatment; H: Cover with a transparent cover to keep moisture; I: Hypocotyls infected for 10 days; J: Hypocotyls infected for 14 days, growing callus; K: Hypocotyls infected for 20 days, and callus differentiates into seedlings; L: Hypocotyls infected for 24 days, and callus differentiates into one or more regenerated buds; M: Seedlings infected for 35 days; N: Seedlings infected for 60 days; O: Hypocotyls grow multiple young regenerated buds; P: Hypocotyls regenerate multiple branches; Q: Tomato plants infected for 90 days.

[0034] An efficient method for in vivo genetic modification of tomatoes: 1. MicroTom seedling cultivation: Place MicroTom seeds in a petri dish with filter paper, as shown in the instructions. Figure 1 As shown in A in the figure, add appropriate amount of tap water to moisten the filter paper and culture it in the dark until the radicle germinates. Figure 1 As shown in B. After the radicles germinate, the seeds are transplanted into a small pot with a substrate, wherein the substrate is a mixture of peat soil, vermiculite and perlite in a volume ratio of 3:1:1, and placed in a light incubator, and cultured under the conditions of 16 hours of light / 8 hours of darkness, a temperature of 22±1°C, and 70% humidity.

[0035] 2. Preparation of Agrobacterium tumefaciens infection solution carrying binary expression vector: Agrobacterium tumefaciens GV3101 carrying the overexpression VcRZ-1A-GFP gene vector was added to the culture medium containing 25 mg.L -1 Rifampicin and 50 mg.L -1 The culture was activated by streaking on LB solid medium containing kanamycin and incubated in the dark at 28°C for 2 days.

[0036] Pick a single colony and inoculate it into LB liquid culture medium containing the corresponding antibiotics. After shaking culture at 200 rpm at 28°C for 12 hours, the bacterial solution was propagated again at a ratio of 1:100 for 12-16 hours.

[0037] The bacterial solution was centrifuged using a centrifuge, and the collected bacterial cells were resuspended in MS liquid culture medium containing 30 gL -1 Sucrose, 300 μM acetosyringone, 0.5‰ Silwet L-77, and adjust the bacterial solution OD 600 The concentration of 0.6 was set aside in a dark place for 2 hours.

[0038] 3. Operational procedures for in vivo transformation of MicroTom tomatoes: The MicroTom seedlings were cultured until two true leaves emerged, i.e., at the two-week-old stage as shown in C of the attached instructions. Figure 2 Using a clean blade, a horizontal cut was made at a position approximately 0.5 cm below the cotyledons to remove the apical part, and the hypocotyl with a length of 1.0 - 1.5 cm above the substrate was retained, as shown in D of the attached instructions. Figure 2 It was placed in a light incubator for pre-culturing for 2 days.

[0039] In this example, the sleeve was selected as the cut short 200-μL pipette tip. The cut short 200-μL pipette tip was gently sleeved on the hypocotyl, as shown in E of the attached instructions. Figure 3 Using a 1-mL sterile syringe to aspirate the infection solution, it was slowly injected from the tip of the pipette tip, and the infection solution submerged the cut surface of the hypocotyl. The infection solution infiltrated the cut surface of the hypocotyl for 10 minutes, as shown in F of the attached instructions. Figure 3 The hypocotyl after in-situ infection was placed under dark conditions for co-culturing for 3 days, as shown in G of the attached instructions. Figure 4 as shown in G of the attached instructions.

[0040] After the co-culturing ended, the seedlings were covered with a transparent cover and placed in a light incubator for culturing, as shown in H of the attached instructions. Figure 4 After culturing for 2 days, the infection solution was injected into the epicotyl again, and the infection was repeated once, for a total of 2 infections.

[0041] 4. Detection of transgenic plants: The genomic DNA of wild-type and transformed plant leaves was extracted using the CTAB method. Using the wild-type tomato genomic DNA as a control, PCR identification was performed using the specific primer pair for the exogenous VcRZ-1A-GFP gene (VcRZ-1A-F: 5’-ATGTCAGAAGAACTGGAGTACCGC-3’; VcRZ-1A-R: 5’-AGATCGGAAACCTCCTGAACTCC-3’).

[0042] The PCR reaction system was: 5 μL of 2×Taq Master Mix, 0.5 μL of each primer (10 μM), 200 ng of genomic DNA, and ddH2O was added to make up to 10 μL. The PCR reaction program was: pre-denaturation at 95°C for 4 minutes; 95°C for 30 seconds, 62°C / 60°C for 30 seconds, 72°C for 60 seconds, for 35 cycles; extension at 72°C for 10 minutes. 5 μL of the PCR product was taken for electrophoresis detection in a 1.5% agarose gel, and the conversion rate was statistically analyzed according to the amplification of the PCR product. Conversion rate % = number of regenerated plants with positive PCR detection / total number of regenerated plants * 100%.

[0043] Using a laser scanning confocal microscope, the fluorescence of the green fluorescent protein (GFP) excited in the young roots or leaves of wild-type and T1 generation transgenic tomato plants was observed and photographed.

[0044] When tomato seedlings have grown two true leaves, select seedlings with a hypocotyl diameter greater than 1.5 cm, remove the top, and transform tomato plants using the hypocotyl infection method mediated by Agrobacterium tumefaciens GV3101.

[0045] The transparent cover can maintain humidity, promote callus formation and bud regeneration; after 30 days, remove the transparent cover and culture normally in the incubator.

[0046] Ten days after infection, the top of the hypocotyl slightly swelled, and callus began to appear at the incision. Immediately afterwards, the callus grew rapidly to form a callus mass. One week later, the callus mass differentiated into young buds, and multiple young buds grew from some of the callus. After 45 days of infection, remove the small regenerated buds at the callus site and culture until the plants flower and bear fruit, as shown in I-Q of the attached instructions. Figures 5-8 as shown in

[0047] After infecting the hypocotyls of MicroTom seedlings with Agrobacterium tumefaciens GV3101 carrying the overexpression VcRZ-1A-GFP gene vector, the regeneration rate, multi-bud rate and transformation rate of the T0 generation were respectively counted, the germination rate of the T1 generation seeds under antibiotic screening, and the green fluorescent protein fluorescence of the young roots of the T1 generation plants.

[0048] The results are as shown in Table 1 of the attached instructions. Figure 9 Due to the lack of a suitable growth medium, Agrobacterium tumefaciens could not grow rapidly and inhibited the regeneration of young buds. Using the in vivo method to transform the hypocotyls of MicroTom, the regeneration rate of young buds was as high as 87.58%, and 58.21% of the hypocotyls could regenerate more than two young buds. After 45 days of infection, PCR detection found that the transformation efficiency was as high as 28.57%, significantly higher than the transformation efficiency of MicroTom tomatoes reported previously (3-22%).

[0049] In this example, the harvested T0 generation seeds were directly placed in a contaminated petri dish containing 25 mg.L -1 hygromycin solution for screening, and the germination rate was 66.71%.

[0050] Select the root tissues of the seedlings for GFP fluorescence identification, and the positive rate was 67.02%. It shows that through methods such as antibiotic screening, the transformed plants can be quickly identified, which helps to accelerate the process of gene function research and genetic breeding on MicroTom tomatoes.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An efficient method for in vivo genetic modification of tomatoes, characterized in that: The following steps are involved: S1: Take MicroTom seedlings and culture them until they grow true leaves, cut the seedlings, keep the hypocotyls, and culture the hypocotyls in a light incubator for 1-3 days; S2: placing a sleeve on the hypocotyl, injecting an infection solution into the sleeve to immerse the hypocotyl section, and the infection solution infects the hypocotyl section; S3: The infected hypocotyls were co-cultured in the dark for 2-4 days; S4: Place in a light incubator for 1-3 days; S5: Repeat steps S2-S4 1-4 times for the cultured hypocotyls.

2. The efficient in vivo genetic modification method for tomatoes according to claim 1, characterized in that: In S1, when the seedlings have grown two true leaves, use a clean knife to make a flat cut 0.4-0.7 cm below the cotyledons, leaving 1-2 cm of the hypocotyl above the substrate.

3. The efficient in vivo genetic modification method for tomatoes according to claim 2, characterized in that: Use a clean knife to make a flat cut 0.5 cm below the cotyledons, leaving 1-1.5 cm of the hypocotyl above the substrate.

4. The efficient in vivo genetic modification method for tomatoes according to claim 3, characterized in that: In S1, the hypocotyls were placed in a light incubator and cultured for 2 days.

5. The efficient in vivo genetic modification method for tomatoes according to claim 1, characterized in that: In S2, the infection solution was infiltrated into the hypocotyl sections for 5-15 minutes.

6. The efficient in vivo genetic modification method for tomatoes according to claim 5, characterized in that: The infection solution is infiltrated into the hypocotyl section for 8-12 minutes.

7. An efficient method for in vivo genetic modification of tomatoes according to any one of claims 1 to 6, characterized in that: In S1, the steps for culturing MicroTom seedlings are as follows: MicroTom seeds are placed in a culture dish with moistened filter paper and cultured in the dark until radicles germinate; After the radicle germinates, the seeds are transplanted into small pots with a substrate and placed in a light incubator under the conditions of 16 hours of light / 8 hours of darkness, a temperature of 22±1°C, and 70% humidity.

8. The efficient in vivo genetic modification method for tomatoes according to claim 7, characterized in that: The small pot with a substrate is prepared by mixing peat soil, vermiculite and perlite in a volume ratio of 3:1:

1.

9. The efficient in vivo genetic modification method for tomatoes according to claim 6, characterized in that: In S2, the infection medium is the Agrobacterium tumefaciens bacterial solution carrying the overexpression VcRZ-1A-GFP gene vector.

10. The efficient in vivo genetic modification method for tomatoes according to claim 9, characterized in that: The preparation process of the infection solution, Agrobacterium tumefaciens GV3101 carrying the overexpression VcRZ-1A-GFP gene vector was taken in the presence of 25 mg.L -1 Rifampicin and 50 mg.L -1 Streak out the culture on LB solid medium containing kanamycin and incubate in the dark at 28°C for 2 days; Pick a single colony and inoculate it into LB liquid medium containing the corresponding antibiotics. After shaking and culturing at 200 rpm at 28°C for 12 hours, expand the bacterial solution at a ratio of 1:100 for 12-16 hours. After centrifugation, the collected bacteria were resuspended in MS liquid medium and the OD of the bacterial solution was adjusted. 600 The concentration of 0.6 was allowed to stand in the dark for 2 hours.

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