A method for inducing the rooting of peppermint transgenics

Abstract

The application discloses a kind of mint transgenic rootlet's induction method, belong to genetic transformation technical field.The mint transgenic rootlet's induction method of the application, steps include:1) preparation rootlet agrobacterium liquid;2) after cutting rootlet, with incision mint seedling is soaked in rootlet agrobacterium liquid 3) mint transgenic plant is transferred to sterile after vermiculite cultivation, and finally cultivate and obtain mint transgenic rootlet.According to the mint transgenic rootlet's induction method of the application, final rootlet conversion efficiency reaches 65%.The application is efficient and fast, short cycle, low cost, wide application range.The method greatly improves the efficiency of mint to obtain transgenic root, and can be applied to root system gene function research and root system and soil microorganism related molecular breeding research.

Description

Technical Field

[0001] This invention belongs to the field of genetic transformation technology, and more specifically, relates to a method for inducing root growth in transgenic peppermint. Background Technology

[0002] Peppermint (Mentha haplocalyx Briq.) is a perennial herb belonging to the genus Mentha L. in the family Lamiaceae. The stems and leaves of peppermint plants are rich in essential oils, which are widely used worldwide in industrial products such as food, cosmetics, fragrances, and tobacco.

[0003] The peppermint genetic transformation system is fundamental to molecular breeding and gene function research using genetic engineering, and is also a key technology in the field of peppermint germplasm innovation. Traditional peppermint genetic transformation is achieved through co-culture with Agrobacterium tumefaciens under tissue culture conditions. The experimental process involves aseptic seedling preparation, explant pre-culture, co-culture with Agrobacterium tumefaciens, screening, rooting, and transplanting, resulting in a lengthy transformation cycle, typically several months. Aseptic seedling preparation is challenging; incomplete explant sterilization leads to high contamination, while over-sterilization causes browning and death of explants. Inappropriate selection medium pressure results in a high false-positive rate in transformed plants. Errors in any step will ultimately lead to transformation failure; therefore, it is always necessary to perform the process by specially trained technicians adhering to aseptic techniques.

[0004] Therefore, in order to better carry out genetic transformation research on mint and improve mint varieties using modern biotechnology, it is necessary to establish an efficient, rapid, and low-cost genetic transformation method. Summary of the Invention

[0005] In view of the above-mentioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a method for inducing root growth in transgenic mint, so as to enable mint to be stably inherited and grow rapidly.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for inducing root growth in genetically modified peppermint, comprising the following steps:

[0008] 1) Preparation of Agrobacterium rhizogenes bacterial culture;

[0009] 2) After the fibrous roots were cut off, the mint seedlings with the cut ends were soaked in Agrobacterium rhizogenes solution to obtain transgenic mint plants;

[0010] 3) The transgenic mint plants were transferred to sterilized vermiculite for cultivation, and the transgenic mint plants with rooting were eventually cultivated.

[0011] The Agrobacterium rhizogenes mentioned is K599.

[0012] The preparation method of the *Agrobacterium rhizogenes* bacterial suspension is as follows: 100 μL of competent *Agrobacterium rhizogenes* cells K599 were thawed on ice using a freeze-thaw method. 5 μL of recombinant plasmid was added, and the suspension was incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and then on ice for 5 min. 1 mL of antibiotic-free LB medium was added to the competent cells containing the plasmid, and the suspension was incubated at 200 rpm in a shaker at 28℃ for 2 h. After centrifugation at 5000g for 2 min, most of the supernatant was discarded in a clean bench, leaving approximately 200 μL. The bacterial pellet was resuspended by pipetting and transferred to a solid culture medium containing Spe antibiotics. The medium was spread evenly and incubated upside down in a 37℃ incubator for 2 days. Single colonies were picked for PCR identification. Positive single colonies were cultured in TY liquid medium and incubated overnight at 28℃ with shaking at 200 rpm. Subsequently, the bacterial suspension was expanded at a 1:100 ratio until the bacterial OD reached the target value. 600 =0.8~1.0, centrifuge at 5000 rpm for 10 min, collect the bacterial culture, discard the supernatant, resuspend in MES buffer, and adjust OD. 600 =0.8~1.0, stand in the dark at 28℃ for 3-4 hours for later use to prepare Agrobacterium rhizogenes bacterial solution.

[0013] The recombinant plasmid is 1305.1-35S:GFP plasmid.

[0014] The 1305.1-35S:GFP plasmid contains the GFP gene.

[0015] The method for constructing the transgenic mint plant is as follows: Select vigorous, current-year-old, and robust mint seedlings with a height of about 10-20 cm, wash off the soil, and cut off their fibrous roots; immerse the seedlings with cuts in Agrobacterium rhizogenes solution for 10-15 minutes to obtain the transgenic mint plant.

[0016] The method for cultivating transgenic mint roots is as follows: Transgenic mint plant cuttings are inserted into sterilized moist vermiculite and transferred to an artificial climate chamber. The specific environment of the artificial climate chamber is a photoperiod of 16 hours of light / 8 hours of darkness, a temperature maintained at 25-27°C, and a humidity maintained at 60%-80%. Water containing 0.1% NAA is applied every 5 days. Once typical capillary roots have formed, transgenic mint roots are obtained.

[0017] The method for inducing root growth in genetically modified peppermint includes the following specific steps:

[0018] 1) Preparation of Agrobacterium rhizogenes bacterial culture: 100 μL of competent Agrobacterium rhizogenes K599 cells were thawed on ice using a freeze-thaw method. 5 μL of recombinant plasmid was added, and the mixture was incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and then on ice for 5 min. 1 mL of antibiotic-free LB medium was added to the competent cells containing the plasmid, and the mixture was incubated at 200 rpm on a shaker at 28°C for 2 h. After centrifugation at 5000g for 2 min, most of the supernatant was discarded in a clean bench, leaving approximately 200 μL. The bacterial pellet was resuspended by pipetting and transferred to a solid medium containing Spe antibiotics. The plate was spread evenly and incubated upside down in a 37°C incubator for 2 days. Single colonies were picked for PCR identification. Positive single colonies were cultured in TY liquid medium and incubated overnight at 28°C with shaking at 200 rpm. Subsequently, the bacterial culture was expanded at a 1:100 ratio until the OD of the culture reached the target value. 600 =0.8~1.0, centrifuge at 5000 rpm for 10 min, collect the bacterial culture, discard the supernatant, resuspend in MES buffer, and adjust OD. 600 =0.8~1.0, stand in the dark at 28℃ for 3-4 hours for later use to prepare Agrobacterium rhizogenes bacterial solution;

[0019] 2) Select vigorous, current-year-old, and robust mint seedlings that are about 10-20 cm tall. Wash them clean of soil and cut off their fibrous roots. Soak the seedlings with the cut ends in Agrobacterium tumefaciens solution for 10-15 minutes to obtain transgenic mint plants.

[0020] 3) Cuttings of the transgenic mint plant material were inserted into sterilized moist vermiculite and transferred to an artificial climate chamber. The specific environment of the artificial climate chamber was a photoperiod of 16 hours of light / 8 hours of darkness, a temperature of 25-27°C, and a humidity of 60%-80%. Water containing 0.1% NAA was applied every 5 days. Once typical capillary roots were formed, the transgenic mint plant was obtained.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention involves activating Agrobacterium rhizogenes K599 containing the 1305.1-35S:GFP plasmid to prepare Agrobacterium rhizogenes bacterial suspension; using the Agrobacterium rhizogenes bacterial suspension to infect mint seedlings with cut ends after fibrous roots have been removed; cultivating and screening to obtain transgenic mint plants; transferring the transgenic mint plants to sterilized vermiculite for culture, and finally cultivating transgenic mint with rooting. The final number of positive rooting results for mint reached 13, with a rooting transformation efficiency of 65%. Attached Figure Description

[0023] Figure 1 Figure 1 shows the establishment of a peppermint rooting transformation system mediated by Agrobacterium rooting to prevent tissue culture (Figure A shows peppermint material with fibrous roots removed; Figure B shows peppermint infected with Agrobacterium rooting; Figure C shows vermiculite tray culture after infection; Figure D shows fibrous roots growing after infection).

[0024] Figure 2 is the diagram of 1305.1-35S:GFP plasmid;

[0025] Figure 3 is the diagram of identifying green fluorescence-positive transgenic hairy roots by a stereoscopic fluorescence microscope (Figure A shows the green fluorescence signal of positive transgenic hairy roots of Mentha '687'; Figure B shows the green fluorescence signal of positive transgenic hairy roots of Mentha cultivar '738'; Figure C shows the green fluorescence signal of positive transgenic hairy roots of Mentha cultivar 'Hu 39'; Figure D shows the result that wild-type roots have no fluorescence signal

[0026] Figure 4 is the diagram of PCR molecular identification results of Mentha transgenic hairy roots (the target fragment of McGFP is 887 bp in length; McWT wild-type and ddH2O are negative controls with no bands). DETAILED DESCRIPTION OF EMBODIMENTS

[0027] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention is further described below with reference to specific embodiments. Unless otherwise specified in the following examples, all technical means used are conventional means well known to those skilled in the art.

[0028] The Mentha materials used in the present invention are collected from the nursery of Institute of Botany, Chinese Academy of Sciences, Nanjing City, Jiangsu Province, and the varieties are the cultivated Mentha variety '687', Mentha variety '738' and Mentha variety 'Hu 39'.

[0029] Example 1

[0030] 1. 100 µL of Agrobacterium rhizogenes competent K599 (TSINGKE, Beijing) is thawed on ice by freeze-thaw method, 5 µL of recombinant plasmid ( Figure 2 ), followed by standing on ice for 5 min, 5 min in liquid nitrogen, 5 min in a 37°C water bath, and standing on ice for 5 min in sequence; 1 mL of antibiotic-free LB medium is added to the competent cells containing the plasmid, and the mixture is cultured at 200 rpm in a 28°C shaker at low speed for 2 h; centrifugation is performed at 5000 g for 2 min, most of the supernatant is poured off in a clean bench, leaving about 200 µL, the bacterial cell pellet is blown and resuspended with a pipette, sucked onto a solid medium containing Spe antibiotic, spread evenly, and the plate is placed upside down in a 37°C incubator to culture for 2 days. Single colonies are picked for PCR identification. Positive single colonies are cultured in TY liquid medium at 28°C and 200 rpm with shaking overnight. Subsequently, the bacterial solution is expanded and cultured at a ratio of 1:100 until the bacterial solution OD 600 = 0.8-1.0, the bacterial solution is collected after centrifugation at 5000 rpm for 10 min, the supernatant is discarded, MES buffer is added for resuspension, and OD is adjusted 600 = 0.8-1.0, the mixture is left to stand at 28°C in the dark for 3-4 h for later use, so that the Agrobacterium rhizogenes bacterial solution is prepared.

[0031] 2. Collect robust, vigorously growing, current-year healthy seedlings with a plant height of about 10-20 cm from the field-grown mint varieties '687', mint variety '738' and mint variety 'Hu 39' respectively, wash off the soil, and cut off their fibrous roots. Soak the cut seedlings in Agrobacterium rhizogenes suspension for 10-15 minutes to obtain infiltrated plant material, that is, transgenic mint plants are obtained.

[0032] The transgenic mint plant material is cut and inserted into sterilized moist vermiculite, then moved to an artificial climate chamber. The specific environment in the artificial climate chamber is a photoperiod of 16h light / 8h darkness, the temperature is maintained at 25-27°C, and the humidity is maintained at 60%-80%. After that, water once every 5 days (with 0.1% NAA contained), and finally transgenic mint hairy roots are obtained after typical fibrous roots are formed.

[0033] 3. Positive identification of transgenic plants

[0034] 1) After typical fibrous roots are formed, wash the material, observe whether the hairy roots have fluorescence signals through a filter with a large-area flashlight-type excitation light source to determine whether the transformation is successful and perform statistics. The transformation efficiency is calculated by the following formula: (number of plants with GFP-positive roots) / (total number of surviving plants) × 100%.

[0035] 2) Cut and mark the hairy roots with fluorescence signals verified by a hand-held excitation light source, take photos under a stereoscopic fluorescence microscope with wild-type roots as a control, with an excitation wavelength of 488nm and an emission wavelength of 505-550nm.

[0036] The results are shown in Figure 3 , the transgenic positive hairy roots of the three varieties all have fluorescence signals under excitation light.

[0037] 4. Extract DNA from hairy roots using the CTAB method. Place approximately 100 mg of root sample (photographed under a stereomicroscope) into a mortar, add liquid nitrogen, and rapidly grind into a uniform powder. Place the powder in a 1.5 mL centrifuge tube, add 600 μL of CTAB separation buffer, and invert the tube to mix thoroughly. Place the centrifuge tube in a 65°C water bath, gently shaking every 30 minutes. After 1–2 hours, remove the tube and cool to room temperature. Add 600 μL of chloroform-isoamyl alcohol (24:1), vortex to mix, and incubate at 4°C for 30 minutes. Centrifuge at 12000 rpm for 10–12 minutes at 4°C. Pipette 400–450 μL of the supernatant into a new 1.5 mL centrifuge tube, add 1 / 10 volume of 3M NaAc (pH 4.8) and an equal volume of isopropanol (pre-treated). Pre-cool at 4℃, immediately invert to mix, which facilitates DNA precipitation; centrifuge at 4℃ and 12000 rpm for 10 min, discard the supernatant; add 600 μL of 70% ethanol (pre-cooled at 4℃ beforehand) to rinse, centrifuge at 4℃ and 12000 rpm for 10 min, discard the supernatant, invert the centrifuge tube on a spread paper towel, and after a few minutes, straighten the centrifuge tube and air dry at room temperature; add 40–100 μL of 1×TE buffer or ddH2O to dissolve and store at -20℃ for later use.

[0038] 5. The specific primer sequences are designed as follows:

[0039] Forward primer F: 5'-CGTAAGGGATGACGCACAATC-3',

[0040] Reverse primer R: 5'-CTTGAAGTCGATGCCCTTCAGC-3'.

[0041] The PCR reaction system was prepared as follows: 10 μL of 2×Hieff PCR Master Max (with Dye), 1 μL of upstream primer F (10 μM), 1 μL of downstream primer R (10 μM), and 8 μL of ddH2O. The extracted DNA was then subjected to PCR amplification. The PCR reaction program was as follows: 94.0℃ pre-denaturation for 4 min; 94.0℃ denaturation for 30 s, 55.0℃ annealing for 30 s, 72.0℃ extension for 2 min, for 32 cycles; 72.0℃ extension for 5 min. The PCR products were analyzed by 1% agarose gel electrophoresis to determine the length of the target gene band. Figure 4 ).

[0042] This method successfully yielded transgenic positive hairy roots for all three peppermint varieties. For peppermint variety '687', 20 transgenic positive hairy roots were obtained, with 20 seedlings infected and 20 rooted, including 9 positive roots, resulting in a rooting conversion efficiency of 45%. For peppermint variety '738', 20 transgenic positive hairy roots were obtained, with 20 seedlings infected and 20 rooted, including 13 positive roots, resulting in a rooting conversion efficiency of 65%. For peppermint variety 'Hu'39', 20 transgenic positive hairy roots were obtained, with 20 seedlings infected and 20 rooted, including 7 positive roots, resulting in a rooting conversion efficiency of 35%.

[0043] Example 2

[0044] 1. Using the freeze-thaw method, 100 μL of Agrobacterium rhizogenes competent cells K599 (TSINGKE, Beijing) were thawed on ice, and 5 μL of recombinant plasmid was added. Figure 2 The bacterial culture was incubated sequentially on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and on ice for 5 minutes. 1 mL of antibiotic-free LB medium was added to the competent cells containing the plasmid, and the cells were incubated at 200 rpm on a shaker at 28°C for 2 hours. After centrifugation at 5000g for 2 minutes, most of the supernatant was discarded in a clean bench, leaving approximately 200 μL. The bacterial pellet was resuspended by pipetting and transferred to a solid culture medium containing Spe antibiotics. The plate was spread evenly and incubated upside down at 37°C for 2 days. Single colonies were picked for PCR identification. Positive single colonies were cultured in TY liquid medium. The culture was then incubated overnight at 28°C with shaking at 200 rpm. The bacterial culture was then expanded at a 1:100 ratio until the OD of the culture reached the target value. 600 =0.8~1.0, centrifuge at 5000 rpm for 10 min, collect the bacterial culture, discard the supernatant, resuspend in MES buffer, and adjust OD. 600 =0.8~1.0, stand in the dark at 28℃ for 3-4 hours for later use to prepare Agrobacterium rhizogenes bacterial solution.

[0045] 2. Collect vigorous, young seedlings of the '687' mint variety from the field. These seedlings should be vigorous, current-year plants, and 3-5 cm tall. Wash them clean of soil and trim their fibrous roots. Soak the seedlings with the cut ends in Agrobacterium rhizogenes solution for 10-15 minutes to obtain the soaked plant material, which is the transgenic mint plant.

[0046] The transgenic mint plants were propagated by cuttings in sterilized, moist vermiculite and then transferred to an artificial climate chamber. The specific environment within the chamber was a 16-hour light / 8-hour dark photoperiod, with a temperature maintained between 25 and 27°C and a humidity level between 60% and 80%. Watering (containing 0.1% NAA) was performed every 5 days until typical fine roots formed, resulting in the transgenic mint plants with rooted growth.

[0047] 3. Positive identification of transgenic plants

[0048] 1) After typical capillary roots have formed, wash the material and observe the capillary roots for fluorescence signals through a filter using a large-area flashlight-type excitation light source to determine whether the transformation was successful and to perform statistical analysis. The transformation efficiency is calculated using the following formula: (Number of plants with GFP-positive roots) / (Total number of surviving plants) × 100%.

[0049] 2) Hair roots exhibiting fluorescence under a handheld excitation light source were cut and labeled, with wild-type roots serving as a control. Images were taken under a stereofluorescence microscope at an excitation wavelength of 488 nm and an emission wavelength of 505-550 nm. The final result showed no fluorescence signal detected in the transgenic hair roots.

[0050] 4. Extract DNA from hairy roots using the CTAB method. Place approximately 100 mg of root sample (photographed under a stereomicroscope) into a mortar, add liquid nitrogen, and rapidly grind into a uniform powder. Place the powder in a 1.5 mL centrifuge tube, add 600 μL of CTAB separation buffer, and invert the tube to mix thoroughly. Place the centrifuge tube in a 65°C water bath, gently shaking every 30 minutes. After 1–2 hours, remove the tube and cool to room temperature. Add 600 μL of chloroform-isoamyl alcohol (24:1), vortex to mix, and incubate at 4°C for 30 minutes. Centrifuge at 12000 rpm for 10–12 minutes at 4°C. Pipette 400–450 μL of the supernatant into a new 1.5 mL centrifuge tube, add 1 / 10 volume of 3M NaAc (pH 4.8) and an equal volume of isopropanol (pre-treated). Pre-cool at 4℃, immediately invert to mix, which facilitates DNA precipitation; centrifuge at 4℃ and 12000 rpm for 10 min, discard the supernatant; add 600 μL of 70% ethanol (pre-cooled at 4℃ beforehand) to rinse, centrifuge at 4℃ and 12000 rpm for 10 min, discard the supernatant, invert the centrifuge tube on a spread paper towel, and after a few minutes, straighten the centrifuge tube and air dry at room temperature; add 40–100 μL of 1×TE buffer or ddH2O to dissolve and store at -20℃ for later use.

[0051] 5. The specific primer sequences are designed as follows:

[0052] Forward primer F: 5'-CGTAAGGGATGACGCACAATC-3',

[0053] Reverse primer R: 5'-CTTGAAGTCGATGCCCTTCAGC-3'.

[0054] The PCR reaction system was prepared as follows: 10 μL of 2×Hieff PCR Master Max (with Dye), 1 μL of upstream primer F (10 μM), 1 μL of downstream primer R (10 μM), and 8 μL of ddH2O. The extracted DNA was then subjected to PCR amplification. The PCR reaction program was as follows: 94.0℃ pre-denaturation for 4 min; 94.0℃ denaturation for 30 s, 55.0℃ annealing for 30 s, 72.0℃ extension for 2 min, for 32 cycles; 72.0℃ final extension for 5 min. The PCR products were analyzed using 1% agarose gel electrophoresis to determine the length of the target gene band.

[0055] Using this method, 20 transgenic hairy roots of the peppermint variety '687' were infected, 10 of which developed roots, and 0 of which developed positive roots.

[0056] In summary, the key to successfully obtaining transgenic positive hairy roots using this method is the growth stage of the infected material.

Claims

1. A method for inducing root growth in transgenic peppermint, characterized by the following steps: include: 1) Preparation of Agrobacterium rhizogenes bacterial culture; the Agrobacterium rhizogenes is K599, K599 contains recombinant plasmid 1305.1-35S:GFP; 2) After the fibrous roots have been removed, the mint seedlings with the cut ends are immersed in Agrobacterium rhizogenes solution to obtain transgenic mint plants. The method for constructing transgenic mint plants is as follows: Select vigorous, current-year-old, robust mint seedlings with a height of about 10-20 cm, wash off the soil, and remove their fibrous roots; immerse the seedlings with the cut ends in Agrobacterium rhizogenes solution for 10-15 minutes to obtain transgenic mint plants. 3) The transgenic mint plants were transferred to sterilized vermiculite for cultivation, and transgenic mint roots were eventually obtained. The cultivation method for transgenic mint roots was as follows: the transgenic mint plant material was inserted into sterilized moist vermiculite and transferred to an artificial climate chamber. The specific environment of the artificial climate chamber was a photoperiod of 16 hours of light / 8 hours of darkness, a temperature maintained at 25-27°C, and a humidity maintained at 60%-80%. Water containing 0.1% NAA was applied every 5 days. Once typical capillary roots were formed, transgenic mint roots were obtained. Among them, the mint variety is mint variety '738'.

2. The method for inducing root development in transgenic peppermint according to claim 1, characterized in that, The preparation method of the *Agrobacterium rhizogenes* bacterial suspension is as follows: 100 μL of competent *Agrobacterium rhizogenes* cells K599 were thawed on ice using a freeze-thaw method. 5 μL of recombinant plasmid 1305.1-35S:GFP was added, and the suspension was incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and then incubated on ice for 5 min. 1 mL of antibiotic-free LB medium was added to the competent cells containing the plasmid, and the suspension was incubated at 200 rpm and 28℃ on a shaker for 2 h. After centrifugation at 5000g for 2 min, most of the supernatant was discarded in a clean bench, leaving approximately 200 μL. The bacterial pellet was resuspended by pipetting and transferred to a solid culture medium containing Spe antibiotics. The plate was spread evenly and incubated upside down in a 37℃ incubator for 2 days. Single colonies were picked for PCR identification. Positive single colonies were placed in TY liquid medium and incubated overnight at 28℃ with shaking at 200 rpm. Subsequently, the bacterial suspension was expanded at a 1:100 ratio until the bacterial OD reached the target value. 600 =0.8~1.0, centrifuge at 5000 rpm for 10 min, collect the bacterial culture, discard the supernatant, resuspend in MES buffer, and adjust OD. 600 =0.8~1.0, stand in the dark at 28℃ for 3-4 hours for later use to prepare Agrobacterium rhizogenes bacterial solution.

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