Nanoparticle-mediated hippeastrum rutilum transformation method

Abstract

The invention discloses a nanoparticle-mediated hippeastrum hippeastrum transformation method which comprises the following steps: (1) uniformly mixing and combining a PEI (polyetherimide) modified carbon quantum dot solution and a pSQ5 plasmid solution to obtain a CDQs-PEI / pSQ5 plasmid vector mixture; (2) uniformly mixing hippeastrum hippeastrum pollen, a CDQs-PEI / pSQ5 plasmid vector mixture and a germination solution, and standing for transformation; (3) drying the converted pollen mixture, and collecting the dried pollen; (4) removing stamens; (5) pollinating, bagging, and collecting after the seeds are mature. According to the nanoparticle-mediated hippeastrum rutilum transformation method, CDQs-PEI and a pollen tube channel transformation method are combined, and an efficient hippeastrum rutilum transgenic method independent of tissue culture and a callus regeneration system is established.

Description

Technical Field

[0001] This invention relates to the field of molecular biology of garden plants, and in particular to a nanoparticle-mediated transformation method for amaryllis. Background Technology

[0002] Amaryllis (Hippeastrum hybrida) is a perennial bulbous flowering plant belonging to the genus Hippeastrum in the family Amaryllidaceae. It has upright flower stalks, large flowers, vibrant colors, and a variety of flower shapes, making it highly valued for its ornamental qualities. As a newly emerging bulbous flower in recent years, amaryllis has gained widespread popularity and is used for potted plants, cut flowers, indoor decoration, and landscaping, showing great market potential.

[0003] Transgenic technology is the fastest-growing and most influential modern biotechnology, offering advantages over traditional breeding methods such as accuracy and efficiency, significantly improving breeding efficiency. Currently, there are no reports on transgenic technology for amaryllis in existing technologies. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a low-cost, easy-to-operate, and effective nanoparticle-mediated transformation method for amaryllis.

[0005] The nanoparticle-mediated transformation method for amaryllis of this invention specifically includes the following steps:

[0006] (1) Mix the PEI-modified carbon quantum dot solution and the pSQ5 plasmid solution to obtain a CDQs-PEI / pSQ5 plasmid carrier mixture;

[0007] (2) Mix amaryllis pollen, CDQs-PEI / pSQ5 plasmid vector mixture and germination broth, and let stand for transformation;

[0008] (3) Dry the transformed pollen mixture and collect the dried pollen;

[0009] (4) Remove the stamens;

[0010] (5) Pollinate, bag the seeds, and harvest them after they mature.

[0011] The nanoparticle-mediated transformation method for amaryllis according to the present invention wherein the germination broth solvent is pure water, which contains 61 mg / L boric acid, 91.5 mg / L CaCl2 and 60 g / L sucrose.

[0012] The nanoparticle-mediated amaryllis transformation method of the present invention includes step (1) as follows: 1.6 ml of PEI-modified carbon quantum dots of 100 μg / mL and 2 μl of pSQ5 plasmid of 1 μg / mL are mixed and bound at room temperature for 30 min.

[0013] The nanoparticle-mediated transformation method of amaryllis described in this invention includes step (2) as follows: 15 mg of amaryllis pollen, 0.8 ml of CDQs-PEI / pSQ5 plasmid vector mixture and 0.8 ml of germination broth are mixed and transformed at room temperature for 30 min.

[0014] The nanoparticle-mediated transformation method of amaryllis described in this invention includes step (3) as follows: 1 ml of the transformed pollen mixture is dropped onto a 500-mesh nylon cloth, with absorbent paper underneath, and dried at room temperature for 30 min before being gently scraped off and collected.

[0015] In the nanoparticle-mediated transformation method of amaryllis described in this invention, in step (5), mature seeds are harvested two months after pollination when the capsules are about to split open.

[0016] The nanoparticle-mediated transformation method for amaryllis described in this invention, wherein the specific variety of amaryllis is 'Lion King'.

[0017] The nanoparticle-mediated transformation method for amaryllis in this invention differs from existing technologies in that it combines CDQs-PEI with pollen tube pathway transformation, establishing a highly efficient amaryllis transgenic method independent of tissue culture and callus regeneration systems, achieving a transformation efficiency of 13.9%. This method provides a new approach for plant transgenics. Currently, there are no reports of amaryllis transgenics; this study is the first to establish an amaryllis transgenic system and obtain transgenic amaryllis plants.

[0018] The nanoparticle-mediated amaryllis conversion method of the present invention will be further described below with reference to the accompanying drawings. Attached Figure Description

[0019] Figure 1 The pSQ5 carrier used in the nanoparticle-mediated amaryllis transformation method of this invention is shown in the image.

[0020] Figure 2 The images show the transgenic detection results of the nanoparticle-mediated transformation method for amaryllis according to the present invention; where: A1 is a root tip slice of control seedlings under white light; A2 is a root tip slice of control seedlings under red fluorescence; A3 is a root tip slice of control seedlings under green fluorescence; B1 is a root tip slice of transgenic seedlings under white light; B2 is a root tip slice of transgenic seedlings under red fluorescence; B3 is a root tip slice of transgenic seedlings under green fluorescence.

[0021] Figure 3 The image shows a transgenic plant 1 obtained by the nanoparticle-mediated transformation method of amaryllis according to the present invention; wherein: A is a control; B is a front view of the transgenic plant; C is a top view of the transgenic plant;

[0022] Figure 4 Transgenic plant 2 and transgenic plant 3 were obtained by the nanoparticle-mediated transformation method of amaryllis according to the present invention. Detailed Implementation

[0023] I. Materials and Reagents

[0024] Hippeastrum 'Lion King'

[0025] plasmid vector pSQ5 (vector map as shown) Figure 1 (as shown);

[0026] CDQs-PEI (synthesized by Xi'an Ruixi Biotechnology Co., Ltd.);

[0027] Germination solution: H3BO4, 61 mg / L; CaCl2, 91.5 mg / L; sucrose, 60 g / L; the remainder was water.

[0028] II. Conversion Methods:

[0029] (1) Take 1.6 ml of PEI-modified carbon quantum dots (CDQs-PEI) at 100 μg / mL and 2 μl of pSQ5 plasmid at 1 μg / mL. Mix well and bind at room temperature for 30 min.

[0030] (2) 15mg pollen + 0.8ml CDQs-PEI / pSQ5 plasmid vector mixture + 0.8ml germination broth (61mg / L boric acid + 91.5mg / L CaCl2 + 100g / L sucrose), mix well, and let stand at room temperature for 30min for transformation;

[0031] (3) Take 1 ml of the transformed pollen mixture and drop it onto a 500-mesh nylon cloth, place absorbent paper underneath, dry at room temperature for 30 minutes, and then gently scrape it off and collect it.

[0032] (4) Remove the stamens;

[0033] (5) Pollination and bagging; about 2 months after pollination, when the capsule is about to split open, harvest the mature seeds.

[0034] III. Detection of transgenic plants

[0035] (1) Sow the seeds in seedling trays, transplant them into flower pots after half a year and observe them under a fluorescence microscope.

[0036] (2) Observation under a fluorescence microscope.

[0037] The pSQ5 vector contains two reporter genes, GFP and RFP. Under a fluorescence microscope, pSQ5 transgenic plants can be observed to exhibit both green and red fluorescence simultaneously.

[0038] Root tips were taken, longitudinally sectioned, and prepared into sections for observation under a fluorescence microscope, with non-transgenic plants used as controls.

[0039] One year after transplanting into flowerpots, the phenotypic traits of the transgenic plants were statistically analyzed.

[0040] IV. Results

[0041] 1. Genetically modified organism (GMO) testing

[0042] like Figure 2 As shown, under the same excitation light intensity, the control showed no fluorescence, while the transgenic plants exhibited both red and green fluorescence. A total of 36 transformed plants were obtained, of which 5 were positive for fluorescence detection. The transformation efficiency was 13.9%.

[0043] 2. Phenotypic traits of transgenic plants

[0044] A total of 5 transgenic plants were obtained. Among them, 2 plants showed no significant morphological differences compared with the control, while 3 plants showed significant differences compared with the control.

[0045] like Figure 3 As shown, the number of leaves in the transgenic plant is significantly increased. The control (A) has 4-5 leaves, while this plant (B) has 14 leaves, an increase of about 200% compared to the control. The leaf opposite characteristics have changed. The control leaves are axially symmetrical, while the leaves of this plant are basically centrally symmetrical (C).

[0046] like Figure 4 As shown, transgenic plant 2 (left) and transgenic plant 3 (right) showed slow growth. The remaining plants showed no significant phenotypic differences compared to the control.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A nanoparticle-mediated transformation method for amaryllis, characterized in that: Includes the following steps: (1) Mix the PEI-modified carbon quantum dot solution and the pSQ5 plasmid solution to obtain a CDQs-PEI / pSQ5 plasmid carrier mixture; (2) Mix amaryllis pollen, CDQs-PEI / pSQ5 plasmid vector mixture and germination broth, and let stand for transformation; (3) Dry the transformed pollen mixture and collect the dried pollen; (4) Remove the stamens; (5) Pollinate, bag the seeds, and harvest them after they mature.

2. The nanoparticle-mediated amaryllis transformation method according to claim 1, characterized in that: The germination solution is a pure water solvent containing 61 mg / L boric acid, 91.5 mg / L CaCl2, and 60 g / L sucrose.

3. The nanoparticle-mediated amaryllis transformation method according to claim 2, characterized in that: Step (1) is as follows: Take 1.6 ml of PEI-modified carbon quantum dots with a concentration of 100 μg / mL and 2 μl of pSQ5 plasmid with a concentration of 1 μg / mL. Mix them well and bind at room temperature for 30 min.

4. The nanoparticle-mediated amaryllis transformation method according to claim 3, characterized in that: Step (2) is as follows: Mix 15mg of amaryllis pollen, 0.8ml of CDQs-PEI / pSQ5 plasmid vector mixture and 0.8ml of germination broth, and let it stand at room temperature for 30min for transformation.

5. The nanoparticle-mediated amaryllis transformation method according to claim 4, characterized in that: Step (3) is as follows: Take 1 ml of the transformed pollen mixture and drop it onto a 500-mesh nylon cloth, place absorbent paper underneath, dry at room temperature for 30 minutes, and then gently scrape it off and collect it.

6. The nanoparticle-mediated amaryllis transformation method according to claim 5, characterized in that: In step (5), the mature seeds are harvested two months after pollination, when the capsules are about to split open.

7. The nanoparticle-mediated amaryllis transformation method according to claim 1, characterized in that: The specific variety of amaryllis mentioned is 'Lion King'.

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