Method for pollen transformation in cucurbitaceous crops

Abstract

The present invention relates to a method for pollen transformation in cucurbitaceous crops and, more particularly, to transformation of cucumber pollen. Transformed pollen can be obtained by forming a MNP-DNA complex in which plasmid DNA for transformation is combined with magnetic nanoparticles, and transforming cucumber pollen on the magnetic plate, followed by pollination.

Description

Method for transforming pollen in cucurbit crops

[0001] The present invention provides a method for transforming pollen of a cucurbit crop.

[0002] Cucurbit crops, including cucumbers, are widely cultivated and consumed worldwide. While cultivars being developed domestically are being bred using traditional methods, this requires considerable time. Cucurbit crops are particularly vulnerable to viral diseases. Rapid climate change due to global warming is leading to a growing diversity of viral outbreaks. Furthermore, traditional breeding methods are challenging to develop and cultivate cultivars that can quickly adapt to changing environments. Consequently, the concept of "speed breeding," which aims to shorten breeding time, is being adopted globally, and "gene editing" technology is being proposed as a potential method to shorten this time.

[0003] Recently, a system for exogenous DNA delivery using DNA-coated magnetic nanoparticles was developed, successfully producing transgenic cotton. This technology, called "pollen magnetofection," utilizes positively charged Fe3O4MNPs as exogenous DNA carriers to deliver them into the highly permeable, thin-walled pollen pores prior to pollination. When the magnetofected pollen is fertilized onto the stigma of the flower, exogenous DNA-transformed seeds can be obtained without the regeneration process typically encountered in tissue culture for Agrobacterium-mediated transformation. However, other research groups have struggled with this technique in monocotyledonous plants. Despite these previous results, another group discovered that pretreatment of pollen with a transfection buffer induces pore opening, and this pretreatment was essential for the production of transgenic maize. These results highlight the challenges and limitations of applying pollen magnetofection to diverse plant species and suggest the need for further research and optimization.

[0004] Therefore, there is a need for technologies to develop and breed varieties that can shorten the breeding time for cucurbits and quickly adapt to the environment.

[0005] [Prior Art Literature]

[0006] [Patent Document]

[0007] 1. Republic of Korea Publication Patent No. 10-2014-0137048 (published on December 2, 2014)

[0008] The purpose of the present invention is to provide a method for transforming pollen of a cucurbit crop, comprising the steps of (1) forming a MNP-DNA complex by combining a transformation plasmid DNA and magnetic nanoparticles; (2) collecting pollen from male flowers of a cucurbit crop; (3) adding the MNP-DNA complex of step (1) and the pollen of step (2) to a transformation buffer and then transforming the pollen on a magnetic plate for 30 to 40 minutes; and (4) obtaining transformed pollen by filtering and drying the transformed pollen after the transformation of step (3).

[0009] Another object of the present invention is to provide a cucurbit pollen transformant produced by the method.

[0010] In order to achieve the above object, the present invention provides a method for transforming pollen of a cucurbit crop, comprising the steps of (1) forming a MNP-DNA complex by combining a transformation plasmid DNA and a magnetic nanoparticle; (2) collecting pollen from male flowers of a cucurbit crop; (3) adding the MNP-DNA complex of step (1) and the pollen of step (2) to a transformation buffer and then transforming the pollen on a magnetic plate for 30 to 40 minutes; and (4) obtaining transformed pollen by filtering and drying the transformed pollen after the transformation of step (3).

[0011] In addition, the present invention provides a cucurbit pollen transformant produced by the method.

[0012] The present invention relates to a method for transforming pollen of cucurbit crops, and more particularly, to transforming cucumber pollen. By forming an MNP-DNA complex by combining transformation plasmid DNA and magnetic nanoparticles, cucumber pollen is transformed on a magnetic plate, and then the resulting product is pollinated again to obtain transformed pollen.

[0013] Figure 1 is a schematic diagram showing the results of the application of pollen magnetofection using pollen and nanoparticles in cucumber.

[0014] Figure 2 shows the results of confirming the efficiency of introducing the GUS reporter gene into cucumber using pollen magnetofection technology.

[0015] Figure 3 shows the results of confirming the efficiency of seeds produced by artificial fertilization using wild-type cucumber seeds and pollen to which the GUS reporter gene was transferred using pollen magnetofection technology, after germination, through GUS staining.

[0016] Figure 4 shows the results of confirming the efficiency through GUS staining in male flowers and pollen of plants grown after germination of seeds produced by artificial fertilization using pollen to which the GUS reporter gene was transferred using pollen magnetofection technology.

[0017] Hereinafter, the present invention will be described in more detail.

[0018] The present invention provides a method for transforming pollen of a cucurbit crop, comprising the steps of (1) forming a MNP-DNA complex by combining a transformation plasmid DNA and magnetic nanoparticles; (2) collecting pollen from male flowers of a cucurbit crop; (3) adding the MNP-DNA complex of step (1) and the pollen of step (2) to a transformation buffer and then transforming the pollen on a magnetic plate for 30 to 40 minutes; and (4) obtaining transformed pollen by filtering and drying the transformed pollen after the transformation of step (3).

[0019] An additional step of pollinating the transformed pollen of step (4) above into the stigma of a female flower of a cucurbit crop may be included.

[0020] The above cucurbit crop may be at least one selected from the group consisting of cucumber, melon, pumpkin, watermelon, melon, watermelon, and yam, but is not limited thereto.

[0021] The combination of the above step (1) can be carried out by mixing the plasmid DNA for transformation and the magnetic nanoparticles in a weight ratio of 3:1 to 5:1 and reacting them at room temperature for 15 to 20 minutes.

[0022] The above transformation plasmid DNA may include, but is not limited to, the Cauliflower mosaic virus 35S promoter or the OsMTD2 promoter.

[0023] The pollen of the above step (2) can be collected through a step of squeezing the anther of the male flower of a cucurbit crop into a transformation buffer and then removing the anther residue.

[0024] The transformation buffer of step (3) above may contain, but is not limited to, 10% to 20% (w / v) sucrose, 0.01% (w / v) boric acid, and 0.03% (w / v) calcium nitrate.

[0025] The above step (4) may additionally include a step of storing the transformed pollen at 1°C to 10°C after filtering and drying.

[0026] An additional step of sealing the flower petals after the moisture in step (4) above may be included.

[0027] In addition, the present invention provides a cucurbit pollen transformant produced by the above method.

[0028] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the following examples and other embodiments merely illustrate the content of the present invention and are not intended to limit the scope of the present invention. The examples and other embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0029]

[0030] <Experimental Example 1> Materials

[0031] Cucumbers (Cucumis sativus L.) provided by Nongwoo Bio Southern Breeding Research Institute (Miryang, Gyeongsangnam-do, Republic of Korea) were grown in a living modified organism (LMO)-regulated field at Pusan ​​National University in Miryang, South Korea. MagnetoFACTOR-24 plates and magnetic nanoparticles (PolyMAG-200) were purchased from Chemicell (Berlin, Germany).

[0032]

[0033] <Experimental Example 2> Self-transformation of cucumber pollen

[0034] 0.5 μg of MNP (PolyMag200) and 2 μg of plasmid DNA were placed in a PCR tube at a 1:4 ratio and left at room temperature for 15 minutes to form MNP-DNA complexes. After that, anthers of male flowers were collected and placed in a 24-well plate. To extract pollen, the anthers were squeezed in 500 μl of magnetic transformation buffer (15% (w / v) sucrose, 0.01% (w / v) boric acid, 1 mM Ca(NO3)2). Then, all anther residues were removed from the well. The MNP-DNA complexes were added to the magnetic transformation buffer together with the pollen particles. To proceed with the reaction, the 24-well plate was placed in a magnetic field for 30 minutes using a MagnetoFACTOR-24 plate. Then, the magnetically transformed pollen grains were carefully spread on filter paper, the buffer was removed, and the pollen grains were dried. Dried pollen grains were transferred to 2 ml Effendorf tubes and stored overnight at 4°C. One day later, the stigmas of female flowers were manually pollinated with self-transformed pollen (female flowers had to be sealed 1 day before hand-pollination), and the petals of the hand-pollinated flowers were sealed with a twist-string to prevent pollination by pollinators. Pollen grains were pollinated by two different methods: directly transferring the pollen grains to the stigmas of female flowers using forceps (approximately 310 pollen grains were pollinated to the stigmas of female flowers), or carefully pipetting them onto germination medium.

[0035] To determine whether pollen pretreatment is essential for the efficiency of cucumber pollen self-transformation, collected cucumber pollen grains were pre-treated in self-transformation buffer in a refrigerator at 4°C for 10 min before reacting with the MNP-DNA complex. Pollen pre-treatment was performed only with the GUS gene construct.

[0036]

[0037] <Experimental Example 3> Confirming the expression of exogenous genes

[0038] To confirm the expression of exogenous genes in pollen through self-transformation, we examined transgenic seeds obtained by artificial pollination with pollen transformed with the GUS gene fused to the cauliflower mosaic virus 35S promoter (p35S) (hereinafter referred to as p35S:GUS) and transgenic seeds obtained by artificial pollination with pollen transformed with the GUS gene fused to the OsMTD2 promoter, a pollen-specific promoter of rice (Oryza sativa) (hereinafter referred to as pMTD2:GUS). The self-transformed pollen was dried and incubated overnight at 4°C. Subsequently, it was stained with a GUS solution containing 50 mM sodium phosphate buffer and 1 g / L X-Gluc for 6 h. The self-transformed pollen was germinated on pollen germination medium at 25°C for 24 h and then subjected to the same staining method. Additionally, pollen grains transformed with plasmids containing fluorescent proteins were observed for GFP and YFP channels using a laser scanning confocal laser scanning microscope K1-Fluo (Nanscope Systems, Korea).

[0039] To assess exogenous gene expression in germinated seeds, seeds from fully mature fruits artificially pollinated with the p35S:GUS plasmid containing self-transformed pollen grains were harvested. These seeds were germinated in square dishes (SPL Life Sciences) with an appropriate amount of water at 25°C for 40 h. The germinated seeds were collected in 15 ml conical tubes (SPL Life Sciences) and stained with GUS solution using the same staining method.

[0040] To confirm the expression of exogenous genes in transgenic plants, various tissues of T1 cucumbers were collected into wells of a 24-well cell culture plate (SPL Life Sciences) and stained with GUS solution using the same staining method.

[0041]

[0042] <Experimental Example 4> Pollen Germination and Viability Test

[0043] Cucumber pollen collected from 20 female flowers was germinated in pollen germination medium containing the same components as 500 μl of transformation buffer in a well of a 24-well cell culture plate (SPL Life Sciences). To test pollen germination and viability in vitro, pollen grains were incubated in the pollen germination medium at 25°C. After incubation for 10–60 min, pollen was observed using a BX53 microscope (Olympus, Tokyo, Japan).

[0044]

[0045] <Experimental Example 5> Screening and PCR analysis of transgenic plants

[0046] Genomic DNA was extracted from cotyledons of self-transgenic cucumber seedlings, confirming successful insertion of the corresponding gene. DNA extraction was performed using the conventional CTAB method. Subsequently, PCR was performed using a Mastercycler nexus gradient (Eppendorf) with specific primers for the Cas9 and GUS reporter genes (Table 1).

[0047] Primer sequence (5'->3')PurposeHyg_FGTGCTTGACATTGGGGAGTTDNA certificationHyg_RGATGTTGGCGACCTCGTATTDNA certificationCas1_FTTCATCCAGCTCGTGCADNA certificationCas1_RGGCTTGATGAACTTGTAGAACTDNA certificationCas2_FTTCATCCAGCTCGTGCADADNA certificationCas2_RGGCTTGATGAACTTGTAGAACTDNA certification

[0048]

[0049] <Example 1> Checking pollen viability

[0050] Transgenic seeds were produced by manually transferring the self-transformed pollen to the stigma of cucumber female flowers. However, it is important to confirm that the pollen grains maintain their viability before artificial pollination. Therefore, after the pollen self-transformation process, the self-transformation buffer containing the same composition as the pollen germination medium was removed from the pollen and dried to prevent induction of pollen tube germination. It was also checked whether this process damaged the pollen grains, which could affect viability.

[0051] To investigate whether the viability of pollen was affected by the process of removing the self-transformation buffer from the pollen and drying it, cucumber pollen grains were observed immediately after the drying step of the pollen self-transformation process. The dried pollen grains were observed to be intact and undamaged (Fig. 2A).

[0052] To test the viability of stored pollen before artificial pollination, dried pollen was stored overnight at 4°C in 2 ml Effendorf tubes. The stored dried pollen grains successfully germinated in pollen germination medium (Fig. 2B).

[0053] We also investigated the temporal dynamics of exogenous gene expression within self-transformed pollen grains. The activity of the GUS reporter gene was observed to increase over time (Figures 2C to 2E). The observed increase in GUS activity confirmed that the exogenous gene was successfully transferred to the pollen and was actively transcribed and translated over time, demonstrating that the exogenous gene can be transiently expressed in pollen.

[0054] These results indicate that the pollen drying and storage processes do not significantly affect pollen viability and germination.

[0055]

[0056] <Example 2> Expression of exogenous genes in pollen

[0057] To test the transient expression of exogenous genes, two GUS plasmids (Fig. 2F) were autoinfected into cucumber pollen. Each plasmid contained a different promoter for the GUS gene. The goal was to obtain transgenic seeds by artificial pollination of pollen transformed with the GUS gene fused with the cauliflower mosaic virus 35S promoter (p35S). However, because the expression level of the p35S:GUS plasmid in cucumber pollen is low, the OsMTD2 promoter, a strong pollen-specific promoter from rice (Oryza sativa), was also utilized.

[0058] GUS expression efficiency was found to be higher with the pMTD2:GUS plasmid (56%) than with the p35S:GUS plasmid (25%) (Figs. 2C to 2E). These observations suggest that the OsMTD2 promoter used in the pMTD2:GUS plasmid is not only more effective in driving gene expression in cucumber pollen than the cauliflower mosaic virus 35S promoter, but also a suitable promoter for driving gene expression in pollen to improve the efficiency of transgenic plant production.

[0059]

[0060] <Example 3> Integration and expression of GUS reporter in transgenic plants

[0061] After artificial pollination using self-infected pollen transformed with the p35S:GUS plasmid, female flowers were immediately sealed with twine. Cucumbers were then grown in a genetically modified organism (LMO) field until fully ripe. Seeds were then harvested and germinated for GUS staining. As a result, GUS activity was successfully detected in T1 cucumber seeds (Fig. 3).

[0062] To determine whether pretreatment of cucumber pollen is essential for the transformation of cucumber plants, we pretreated collected cucumber pollen grains in a magnetic transformation buffer at 4°C for 10 min before reacting them with MNP-DNA complexes. While pretreatment affected pore size, increasing pore size, it did not significantly impact the transformation efficiency of cucumber plants (Fig. 3E).

[0063] Furthermore, GUS expression was detected not only in transgenic seeds but also in pollen from T1 cucumber plants (Fig. 4). However, not all pollen showed GUS expression, indicating that the transgenic cucumber plants were heterozygous. Furthermore, strong GUS expression was detected in T2 embryos following selfing of T1 plants. Furthermore, to analyze the next-generation inheritance of the exogenous gene, PCR analysis was performed to amplify the GUS gene integrated into the T1 cucumber genome, confirming its expression (Fig. 4C).

[0064]

[0065] The foregoing description of the present invention is for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0066] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. (1) A step of forming a MNP-DNA complex by combining plasmid DNA for transformation and magnetic nanoparticles; (2) Step of collecting pollen from male flowers of cucurbit crops; (3) A step of adding the MNP-DNA complex of step (1) and the pollen of step (2) to the transformation buffer and then transforming on a magnetic plate for 30 to 40 minutes; and (4) A method for transforming pollen of a cucurbit crop, comprising a step of obtaining transformed pollen by filtering and drying after transformation in step (3).

2. In claim 1, A method for transforming pollen of a cucurbit crop, characterized in that it further comprises a step of pollinating the transformed pollen of step (4) above into the stigma of a female flower of a cucurbit crop.

3. In claim 1, A method for transforming pollen of a cucurbit crop, characterized in that the cucurbit crop is at least one selected from the group consisting of cucumber, melon, pumpkin, watermelon, melon, watermelon, and bitter melon.

4. In claim 1, The method for transforming pollen of a cucurbit crop is characterized in that the combination of step (1) above comprises mixing the plasmid DNA for transformation and the magnetic nanoparticles in a weight ratio of 3:1 to 5:1 and reacting them at room temperature for 15 to 20 minutes.

5. In claim 1, A method for transforming pollen of a cucurbit crop, characterized in that the above transformation plasmid DNA comprises a cauliflower mosaic virus 35S promoter or an OsMTD2 promoter.

6. In claim 1, A method for transforming pollen of a cucurbit crop, characterized in that the pollen of the above step (2) is collected through a step of squeezing the anther of a male flower of a cucurbit crop into a transformation buffer and then removing the anther residue.

7. In claim 1, A method for transforming pollen of a cucurbit crop, characterized in that the transformation buffer of step (3) above contains 10% to 20% (w / v) sucrose, 0.01% (w / v) boric acid, and 0.03% (w / v) calcium nitrate.

8. In claim 1, A method for transforming pollen of a cucurbit crop, characterized in that the step (4) above additionally includes a step of storing the transformed pollen at 1°C to 10°C after filtering and drying.

9. In claim 1, A method for transforming pollen of a cucurbit crop, characterized in that it further comprises a step of sealing the flower petals after the moisture in step (4) above.

10. A cucurbit pollen transformant produced by any one of the methods of claims 1 to 8.

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