Agrobacterium rhizogenes-mediated fruit tree root gene transformation method and application thereof

Abstract

The invention relates to the field of plant transgenosis, in particular to an agrobacterium rhizogenes-mediated fruit tree root gene transformation method and application thereof. The agrobacterium rhizogenes-mediated fruit tree root gene transformation method comprises the following steps: (a) transforming an expression vector carrying a target gene into an agrobacterium rhizogenes competent cell, scribing a positive monoclonal cell, then carrying out dark culture, selecting a single colony, and carrying out liquid culture and centrifugation to obtain a lower-layer thallus; resuspending the lower-layer thalli by utilizing an infection solution to obtain an infection bacterium solution; (b) putting a pretreated acceptor material into the infection bacterium solution for infection culture; (c) discarding the infection bacterium liquid, and performing co-culture on the acceptor material; and (d) removing non-transgenic lateral roots of the acceptor material, and performing rooting culture. The agrobacterium rhizogenes-mediated fruit tree root gene transformation method has the advantages of short transformation time, high gene transformation efficiency, high plant survival rate, simplicity in operation, no dependence on harsh sterile conditions and the like.

Description

Technical Field

[0001] The invention relates to the field of plant transgenics, and in particular to a method for gene transformation of fruit tree roots mediated by Agrobacterium rhizogenes and an application thereof. Background Art

[0002] Plant molecular design breeding is an important part of modern agricultural technology. Genetic improvement of plants through genetic engineering technology can effectively improve crop yield, quality and stress resistance. However, as a key link in plant molecular design breeding, genetic transformation faces many challenges in its application in fruit trees. In particular, the genetic transformation of perennial fruit trees such as peach and chestnut is particularly difficult, and a large number of fruit tree species lack an effective genetic transformation system.

[0003] At present, published studies on the gene transformation system of fruit trees such as peach and chestnut have shown that existing transformation methods generally have problems such as long transformation cycle, low transformation efficiency and low survival rate. For example, the root transformation of peach requires the preparation of sterile seedlings as explants, and the hairy root transformation method of chestnut also requires the preparation of sterile chestnut seedlings. These methods are not only cumbersome to operate, but also costly, resulting in the long transformation cycle of fruit trees, low efficiency and low survival rate of transgenic seedlings.

[0004] In addition, some transformation methods have the problem of non-reproducibility in practical applications, which further limits the widespread application of these technologies in fruit tree genetic improvement. Therefore, developing a genetic transformation system that is simple to operate, low in cost, high in transformation efficiency and high in survival rate is of great significance for promoting the development of molecular design breeding of fruit trees.

[0005] In summary, the application of existing transgenic system technology in fruit tree woody plants has many limitations, and a new technical solution is urgently needed to solve the above problems in order to improve the efficiency and success rate of genetic transformation of fruit trees, thereby promoting the further development of molecular design breeding of fruit trees.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a method for gene transformation of fruit tree roots mediated by Agrobacterium rhizogenes and its application. The gene transformation method has the advantages of short transformation time, high gene transformation efficiency, high plant survival rate, simple operation, and no reliance on strict aseptic conditions.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] One aspect of the present invention relates to a method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes, comprising the following steps:

[0010] (a) transforming an expression vector carrying a target gene into competent cells of Agrobacterium rhizogenes, streaking a positive single clone and then culturing it in the dark, picking a single colony for liquid culture and centrifugation to obtain a lower layer of bacterial cells; resuspending the lower layer of bacterial cells in an infection solution to obtain an infection solution;

[0011] (b) placing the pretreated receptor material in the infection bacterial solution for infection culture;

[0012] (c) discarding the infecting bacterial solution and co-culturing the receptor material;

[0013] (d) removing the non-transgenic lateral roots of the recipient material and then conducting rooting culture.

[0014] The method for gene transformation of fruit tree roots mediated by Agrobacterium rhizogenes has the advantages of short transformation time, high gene transformation efficiency, high plant survival rate, simple operation, and no reliance on strict aseptic conditions.

[0015] Another aspect of the present invention also relates to a genetic breeding method for fruit trees, including the Agrobacterium rhizogenes-mediated gene transformation method for fruit tree roots.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention provides a method for gene transformation of fruit tree roots mediated by Agrobacterium rhizogenes. The method is simple to operate and eliminates the cumbersome and time-consuming tissue culture process. The method is applicable to a variety of fruit trees that are difficult to transform and significantly shortens the transformation cycle. It only takes 21 days from infection to the emergence of transgenic root plants. The method of infecting the root tips of germinated seeds overcomes the problem of low transformation efficiency and significantly improves the gene transformation efficiency to 27.11%.

[0018] (2) The present invention provides a method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes, which does not rely on strict aseptic conditions and avoids the problem of low survival rate caused by the gradual adaptation of transgenic seedlings to the external environment, thereby significantly improving the survival rate of transgenic root plants, with a survival rate of up to 100%. The morphology of the obtained transgenic plants is the same as that of seedlings, and can be directly used in soil pots and field experiments, laying a foundation for the study of the mechanism of root diseases, salt and alkali resistance, and genetic improvement of important traits.

[0019] (3) The present invention provides a method for fruit tree root gene transformation mediated by Agrobacterium rhizogenes, which is independent of a sterile environment and does not require the preparation of sterile seedlings and tissue culture experiments such as seedling training, thereby greatly saving experimental costs and providing technical support for the future use and promotion of transgenic peach plants with excellent traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The flowchart of the gene transformation of fruit tree roots mediated by Agrobacterium rhizogenes provided by the present invention (taking peach as an example), wherein A is peach seed explants for infection, B is explants infected with bacterial solution, C is co-cultivation, D is explants after 7 days of co-cultivation, and E is peach plants with transgenic roots;

[0022] Figure 2 Bright field (left) and fluorescent field (right) images of the root system of the transgenic peach provided by the present invention;

[0023] Figure 3 The GUS staining results of the DR5::GUS transgenic peach roots provided by the present invention;

[0024] Figure 4 The fluorescence field image of the chestnut transgenic root provided by the present invention;

[0025] Figure 5 This is the GUS staining result of the CmAP2a pro::GUS transgenic chestnut root provided by the present invention. DETAILED DESCRIPTION

[0026] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0027] One aspect of the present invention relates to a method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes, comprising the following steps:

[0028] (a) transforming an expression vector carrying a target gene into competent cells of Agrobacterium rhizogenes, streaking a positive single clone and then culturing it in the dark, picking a single colony for liquid culture and centrifugation to obtain a lower layer of bacterial cells; resuspending the lower layer of bacterial cells in an infection solution to obtain an infection solution;

[0029] (b) placing the pretreated receptor material in the infection bacterial solution for infection culture;

[0030] (c) discarding the infecting solution and co-culturing the receptor material;

[0031] (d) removing the non-transgenic lateral roots of the recipient material and then conducting rooting culture.

[0032] The transformation efficiency of tissue culture is usually genotype-dependent, and the efficiency varies greatly between different transformation schemes. The ideal solution is to develop a transformation system that does not require tissue culture. In the reported CDB delivery system, transformation of multiple species can be achieved without relying on genotype.

[0033] The method for gene transformation of fruit tree roots provided by the present invention is simple to operate and also eliminates the cumbersome and time-consuming tissue culture process. It is applicable to a variety of fruit trees that are difficult to transform and significantly shortens the transformation cycle. It only takes 21 days from infection to the emergence of transgenic root plants. The present invention adopts a method of infecting the root tips of germinated seeds, which overcomes the problem of low transformation efficiency and significantly improves the gene transformation efficiency to 27.11%.

[0034] The transformation method provided by the present invention does not rely on strict aseptic conditions, avoids the problem of low survival rate caused by the gradual adaptation of transgenic seedlings to the external environment, and thus significantly improves the survival rate of transgenic root plants, with a survival rate of up to 100%. The obtained transgenic plant morphology is the same as that of seedlings, and can be directly used in soil pots and field experiments, laying a foundation for the study of root disease mechanism, salt and alkali resistance, and genetic improvement of important traits.

[0035] The transformation method provided by the present invention does not rely on a sterile environment and does not require the preparation of sterile seedlings and tissue culture experiments such as seedling training, which greatly saves experimental costs and provides technical support for the future use and promotion of transgenic peach plants with excellent traits.

[0036] Furthermore, the receptor material includes but is not limited to: seeds with hypocotyls extending 1 to 3 cm (for example, but not limited to any point value of 1 cm, 2 cm or 3 cm or any range value between the two). At this time, the receptor material is relatively young and tender, and lignification is not serious. After being scratched by a scalpel, the infection liquid can more easily enter the cells inside the infection liquid wound.

[0037] Furthermore, the seeds include but are not limited to: chestnut or peach. The transformation method proposed in the present invention is not only applicable to chestnut and peach, but also to other plants with similar physiological characteristics to chestnut and peach.

[0038] The present invention does not specifically limit the type of expression vector, and any conventional expression vector in the art can be used to implement the technical solution of the present invention. In some specific embodiments, the expression vector includes but is not limited to at least one of DR5::GUS, CmAP2a pro::GUS, CmAP2a-RNAi or pK7GWIWG2(II)RR-277.

[0039] The present invention does not specifically limit the target gene, and all target genes that can be carried by the expression vector are applicable to the present invention. In some specific embodiments, the target gene includes but is not limited to: a reporter gene.

[0040] Furthermore, the reporter gene includes but is not limited to: at least one of DsRED, GFP or GUS.

[0041] Furthermore, colony PCR reaction was used to screen positive single clones.

[0042] Furthermore, the temperature of the dark culture is 27-29°C, including but not limited to any one of 27°C, 28°C or 29°C or a range between any two of them.

[0043] Furthermore, the dark culture time is 36 to 48 hours, including but not limited to any point value of 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours or 48 hours, or a range value between any two of them.

[0044] Furthermore, the dark culture is carried out on a solid LB medium containing 95 to 105 mg / L (for example, it may be but is not limited to any point value of 95 mg / L, 97 mg / L, 99 mg / L, 101 mg / L, 103 mg / L or 105 mg / L, or a range value between any two) of Spe.

[0045] Furthermore, the liquid culture is carried out to a bacterial liquid concentration of OD 600 The value is 0.8 to 1.2, including but not limited to any point value of 0.8, 0.9, 1.0, 1.1 or 1.2 or any range value between two of them. 600 When it is between 0.8 and 1.2, the bacterial solution contains a sufficient amount of infectious bacteria, which is beneficial to improving the gene transformation efficiency and shortening the transformation time.

[0046] Further, the liquid culture is carried out in a liquid LB medium containing 95-105 mg / L (for example, it may be but not limited to any one of 95 mg / L, 97 mg / L, 99 mg / L, 101 mg / L, 103 mg / L or 105 mg / L, or a range value between two of them) Spe and 95-105 mM (for example, it may be but not limited to any one of 95 mM, 97 mM, 99 mM, 101 mM, 103 mM or 105 mM, or a range value between two of them) acetosyringone.

[0047] Furthermore, the centrifugal speed is 3400-3600 rpm, including but not limited to any point value of 3400 rpm, 3450 rpm, 3500 rpm, 3550 rpm or 3600 rpm or a range value between any two of them.

[0048] Furthermore, the centrifugation time is 13 to 17 minutes, including but not limited to any point value of 13 minutes, 14 minutes, 15 minutes, 16 minutes or 17 minutes, or a range value between any two of them.

[0049] Further, the infection solution includes: WPM culture medium and additives;

[0050] According to each liter of WPM culture medium, the additives include: 13-17 g sucrose, 0.3-0.7 g 2-morpholineethanesulfonic acid (MES), 0.3-0.7 g CaCl2, 0.6-1.0 mg IBA, 95-105 mM acetosyringone (AS) and 0.03 vol%-0.07 vol% surfactant.

[0051] In some specific embodiments, based on the WPM medium per liter, sucrose can be, for example, but not limited to, any one of 13 g, 14 g, 15 g, 16 g, or 17 g, or a range between any two of them; MES can be, for example, but not limited to, any one of 0.3 g, 0.4 g, 0.5 g, 0.6 g, or 0.7 g, or a range between any two of them; CaCl2 can be, for example, but not limited to, any one of 0.3 g, 0.4 g, 0.5 g, 0.6 g, or 0.7 g, or a range between any two of them; IBA can be, for example, But it is not limited to any point value among 0.6mg, 0.7mg, 0.8mg, 0.9mg or 1.0mg, or a range value between any two of them; acetosyringone, for example, can be but not limited to any point value among 95mM, 97mM, 99mM, 101mM, 103mM or 105mM, or a range value between any two of them; the surfactant, for example, can be but not limited to any point value among 0.03vol%, 0.04vol%, 0.05vol%, 0.06vol% or 0.07vol%, or a range value between any two of them.

[0052] The reason for adding CaCl2 to the infection solution is that calcium ions play an important role in promoting bacterial attachment and stabilizing plant cell structure. In addition, appropriate CaCl concentration can significantly improve the transformation efficiency. IBA is an auxin that can promote rooting of the embryo and promote the rooting rate. The addition of surfactants can increase the deposition, diffusion and retention of the infection solution on the plant surface.

[0053] Furthermore, the surfactant includes but is not limited to: Silwet L-77.

[0054] Furthermore, the preprocessing comprises the following steps:

[0055] Cut off the tender yellow root tip of the receptor material with a length of 1 to 2 mm, and score wounds on the remaining hypocotyl to ensure that all parts of the hypocotyl are covered with dense wounds. In the prior art, a scalpel is used to cut off the swollen part of the hypocotyl of the receptor material to be transformed, while the present invention cuts off the tender yellow root tip part and removes the meristem cells with strong differentiation ability at the root tip. The root tip is the active growth and development area of ​​the root, with high metabolic activity, and wound treatment can enhance the contact efficiency between Agrobacterium and host cells.

[0056] Furthermore, the infection culture temperature is 27-29° C., including but not limited to any point value of 27° C., 28° C. or 29° C. or any range value between the two. When the infection culture is carried out under this temperature condition, the infecting bacteria has a higher activity, can shorten the infection time, and improve the gene conversion rate.

[0057] Furthermore, the infection culture time is 38 to 42 minutes, including but not limited to any one of 38 minutes, 39 minutes, 40 minutes, 41 minutes or 42 minutes, or a range between any two of them.

[0058] Furthermore, the infection culture is carried out in the dark.

[0059] Furthermore, the co-cultivation is carried out in the dark. Co-cultivation in the dark can inhibit plant cell differentiation and reduce light-induced defense reactions, thereby enhancing the efficiency of Agrobacterium T-DNA transfer.

[0060] Furthermore, the co-cultivation is carried out on a moist culture substrate. Co-cultivation is carried out on a moist culture substrate, which can maintain the activity of plant cells and the motility of Agrobacterium, and promote close contact between the two and the transfer of genetic material.

[0061] Furthermore, the culture matrix includes but is not limited to: moistened filter paper and / or 6-8 g / L (for example, it can be but is not limited to any point value of 6 g / L, 7 g / L or 8 g / L or a range value between any two) agar culture medium.

[0062] Furthermore, the co-cultivation temperature is 24-26° C., including but not limited to any point value of 24° C., 25° C. or 26° C. or any range value between the two. If the co-cultivation is not carried out at an appropriate temperature, it will slow down the excessive proliferation of Agrobacterium and maintain the activity of plant cells, balancing the interaction time between the two to optimize T-DNA transfer.

[0063] Furthermore, the co-cultivation time is 6 to 8 days, including but not limited to any point value of 6 days, 7 days or 8 days or any range value between the two. 6 to 8 days has a higher transformation efficiency.

[0064] Furthermore, the rooting culture temperature is 24-26° C., including but not limited to any point value of 24° C., 25° C. or 26° C. or any range value between the two. The rooting culture temperature is adjusted according to the growth habit adaptability of the receptor material.

[0065] Furthermore, the rooting culture time is 13 to 15 days, including but not limited to any point value of 13 days, 14 days or 15 days or a range value between any two of them.

[0066] Furthermore, the photoperiod of the rooting culture includes: 15 to 17 hours of light (for example, but not limited to any one of 15 hours, 16 hours or 17 hours, or a range between the two) and 7 to 9 hours of darkness (for example, but not limited to any one of 7 hours, 8 hours or 9 hours, or a range between the two). The determination of the photoperiod can be adaptively adjusted according to the growth characteristics of the receptor material.

[0067] Furthermore, the culture medium for rooting culture includes: vermiculite and / or matrix.

[0068] Another aspect of the present invention also relates to a genetic breeding method for fruit trees, including the Agrobacterium rhizogenes-mediated gene transformation method for fruit tree roots.

[0069] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0070] Example 1

[0071] This example provides a method for gene transformation of peach roots, and obtains a transgenic peach root line expressing red fluorescent protein. The expressed red fluorescent protein can stimulate a strong red fluorescent signal under a stereo fluorescence microscope. The process is as follows: Figure 1 As shown, the specific steps include:

[0072] 1. Plant the peach seeds stored in sand under low temperature conditions for two months in vermiculite for normal germination, and select the seeds with hypocotyls extending 1 to 2 cm as receptor materials;

[0073] 2. Use the DR5::GUS expression vector with the DsRED reporter gene to transform the expression vector into MSU440 Agrobacterium rhizogenes competent cells;

[0074] 3. Screening was performed using LB solid medium containing 100 mg / L Spe resistance. Positive single clones were screened by colony PCR reaction and streaked onto LB solid medium containing 100 mg / L Spe resistance, and dark cultured in a 28°C incubator for 36 h;

[0075] 4. Pick a single colony and place it in a conical flask containing 100 ml LB liquid medium containing 100 mg / L Spe and 100 mM acetosyringone. Shake and culture at 200 rpm and 28°C until the bacterial concentration reaches OD 600 is 1.0;

[0076] 5. Centrifuge the bacterial solution at 3500 rpm for 15 min. After centrifugation, discard the supernatant and then add an equal volume of sterilized infection solution to resuspend the bacteria to obtain the infection solution. The formula of the infection solution is: 15 g sucrose, 0.5 g MES, 0.5 g CaCl2, 0.8 mg IBA, 100 mM AS and 0.05 vol% Silwet L-77 are added to 1 L WPM medium.

[0077] 6. Use a scalpel to cut off the tender yellow root tips of the peach seeds, and use a scalpel to make dense wounds on the remaining hypocotyls. Then, immediately place the treated explants in the infection solution and place them on a shaker at 28°C in the dark for 40 minutes.

[0078] 7. Discard the infected bacterial solution and place the infected seeds in a culture medium containing 7 g / L agar for co-cultivation in the dark for 7 days. The co-cultivation temperature is 25°C. The culture medium is only used to maintain a moist environment and is not sealed.

[0079] 8. The co-cultivated plants were observed for fluorescence using a stereo fluorescence microscope, and the non-transgenic lateral roots of the receptor material were removed and planted in non-sterilized vermiculite for rooting culture. The photoperiod included 16 h of light and 8 h of darkness, and the culture was carried out at 25°C for 14 days.

[0080] 9. Use a stereo fluorescence microscope to observe the transformed plants after 14 days of rooting. The test results are as follows: Figure 2 and Figure 3 As shown in the figure, the roots of transgenic peach plants contain the DsRed reporter gene, so the entire root and its lateral roots have strong red fluorescence. After GUS staining, blue signals can be seen at the root tip and lateral root primordium of the transgenic roots.

[0081] Example 2

[0082] This embodiment provides a method for genetic transformation of chestnut roots, and obtains chestnut transgenic root plants expressing GFP. The expressed green fluorescent protein can stimulate a strong green fluorescent signal under a stereoscopic fluorescence microscope, and specifically comprises the following steps:

[0083] 1. Select plump chestnut seeds, wash them and plant them in vermiculite for normal germination. Select chestnut seeds with hypocotyls extending 2 to 3 cm as receptor materials;

[0084] 2. Use the CmAP2a pro::GUS expression vector with the GFP reporter gene to transform the expression vector into MSU440 Agrobacterium rhizogenes competent cells;

[0085] 3. Same as Example 1;

[0086] 4. Pick a single colony and place it in a conical flask containing 100 ml LB liquid medium containing 100 mg / L Spe and 100 mM acetosyringone. Shake and culture overnight at 200 rpm and 28 °C. The next day, add 400 ml LB liquid medium containing Spe resistance to the activated bacterial solution and continue to activate until the bacterial solution concentration OD 600 is 0.8;

[0087] 5. Same as Example 1;

[0088] 6. Same as Example 1;

[0089] 7. Discard the infected bacterial solution and place the infected chestnut seeds in a culture box containing moist filter paper for 7 days in the dark at a temperature of 25°C.

[0090] 8. Same as Example 1;

[0091] 9. Fluorescence observation of chestnut transformed plants after 14 days of rooting culture was performed using a stereo fluorescence microscope. The test results are as follows: Figure 4 and Figure 5 As shown in the figure, the transgenic chestnut plant root system contains the GFP reporter gene, so the whole root and its lateral roots have strong green fluorescence. After GUS staining, the transgenic root can be seen with blue signal throughout the root.

[0092] Example 3

[0093] This embodiment provides a method for gene transformation of peach roots, and obtains a transgenic peach root line expressing red fluorescent protein, wherein the expressed red fluorescent protein can stimulate a strong red fluorescent signal under a stereo fluorescence microscope, and specifically comprises the following steps:

[0094] 1. Plant the peach seeds stored in sand under low temperature conditions for two months in vermiculite for normal germination, and select the seeds with hypocotyl extension of 1 to 1.5 cm as the receptor material;

[0095] 2. Use the DR5::GUS expression vector with the DsRED reporter gene to transform the expression vector into MSU440 Agrobacterium rhizogenes competent cells;

[0096] 3. Screening was performed using LB solid medium containing 105 mg / L Spe resistance. Positive single clones were screened by colony PCR reaction and streaked onto LB solid medium containing 105 mg / L Spe resistance, and dark cultured in a 28°C incubator for 40 h;

[0097] 4. Pick a single colony and place it in a conical flask containing 100 ml LB liquid medium containing 105 mg / L Spe and 105 mM acetosyringone. Shake and culture at 200 rpm and 28 °C until the bacterial solution concentration OD 600 is 0.8;

[0098] 5. Centrifuge the bacterial solution at 3600 rpm for 13 min. After centrifugation, discard the supernatant and then add an equal volume of sterilized infection solution to resuspend the bacteria to obtain the infection solution. The formula of the infection solution is: 13 g sucrose, 0.7 g MES, 0.3 g CaCl2, 1.0 mg IBA, 105 mM AS and 0.03 vol% Silwet L-77 are added to 1 L WPM medium.

[0099] 6. Use a scalpel to cut off the tender yellow root tips of the peach seeds, and use a scalpel to make dense wounds on the remaining hypocotyls. Then, immediately place the treated explants in the infection solution and place them on a shaker at 28°C in the dark for 42 minutes.

[0100] 7. Discard the infected bacterial solution and place the infected seeds in a culture medium containing 7 g / L agar for co-cultivation in the dark for 6 days. The co-cultivation temperature is 24°C. The culture medium is only used to maintain a moist environment and is not sealed.

[0101] 8. The co-cultivated plants were observed for fluorescence using a stereo fluorescence microscope, and the non-transgenic lateral roots of the receptor material were removed and planted in non-sterilized vermiculite for rooting culture. The photoperiod included 15 h of light and 9 h of darkness, and the plants were cultured at 24°C for 15 days.

[0102] 9. Fluorescence observation of the transformed plants after rooting culture was performed using a stereo fluorescence microscope. The roots of the transgenic peach plants contained the DsRed reporter gene, so the entire root and its lateral roots had strong red fluorescence. Blue signals were observed at the root tip and lateral root primordium of the transgenic roots after GUS staining.

[0103] Example 4

[0104] This embodiment provides a method for genetic transformation of chestnut roots, and obtains chestnut transgenic root plants expressing GFP. The expressed green fluorescent protein can stimulate a strong green fluorescent signal under a stereoscopic fluorescence microscope, and specifically comprises the following steps:

[0105] 1. Select plump chestnut seeds, wash them and plant them in vermiculite for normal germination. Select chestnut seeds with hypocotyls extending 2.5 to 3 cm as receptor materials;

[0106] 2. Use the CmAP2a pro::GUS expression vector with the GFP reporter gene to transform the expression vector into MSU440 Agrobacterium rhizogenes competent cells;

[0107] 3. Screening was performed using LB solid medium containing 95 mg / L Spe resistance. Positive single clones were screened by colony PCR reaction and streaked onto LB solid medium containing 95 mg / L Spe resistance, and dark cultured in a 28°C incubator for 48 h;

[0108] 4. Pick a single colony and place it in a conical flask containing 100 ml LB liquid medium containing 95 mg / L Spe and 95 mM acetosyringone. Shake and culture at 200 rpm and 28 °C until the bacterial concentration OD 600 is 1.1;

[0109] 5. Centrifuge the bacterial solution at 3400 rpm for 17 min. After centrifugation, discard the supernatant and then add an equal volume of sterilized infection solution to resuspend the bacteria to obtain the infection solution. The formula of the infection solution is: 17 g sucrose, 0.3 g MES, 0.7 g CaCl2, 0.6 mg IBA, 95 mM AS and 0.07 vol% Silwet L-77 are added to 1 L WPM medium.

[0110] 6. Use a scalpel to cut off the tender yellow root tips of the peach seeds, and use a scalpel to make dense wounds on the remaining hypocotyls. Then, immediately place the treated explants in the infection solution and place them on a shaker at 28°C in the dark for 38 minutes.

[0111] 7. Discard the infected bacterial solution and place the infected chestnut seeds in a culture box containing moist filter paper for dark culture for 8 days at a temperature of 26°C.

[0112] 8. The co-cultivated plants were observed for fluorescence using a stereo fluorescence microscope, and the non-transgenic lateral roots of the receptor material were removed and planted in non-sterilized vermiculite for rooting culture. The photoperiod included 17 h of light and 7 h of darkness, and the plants were cultured at 26°C for 13 days.

[0113] 9. Fluorescence observation of chestnut transformed plants after rooting culture was performed using a stereo fluorescence microscope. The root system of the transgenic chestnut plant contained the GFP reporter gene, so the entire root and its lateral roots showed strong green fluorescence. After GUS staining, blue signals were observed throughout the transgenic root.

[0114] Comparative Example 1

[0115] The difference between this comparative example and Example 1 is that the co-cultivation time is 9 days.

[0116] Comparative Example 2

[0117] The difference between this comparative example and Example 1 is that the Agrobacterium rhizogenes bacterial liquid is evenly applied to the surface of the solid culture medium containing Spe resistance, and inverted culture is carried out at 28°C for 2 days until a dense bacterial layer is formed on the surface of the culture medium. The seeds to be infected are placed in distilled water containing 100mM AS and soaked for 20 minutes at room temperature in the dark, and then the seeds are slightly wounded, and the bacteria cultured on the solid culture medium are scraped off with a scalpel and applied to the wounded part of the seeds.

[0118] Comparative Example 3

[0119] The difference between this comparative example and Example 1 is that the seeds are immersed in the suspension of Agrobacterium rhizogenes for infection for 40 minutes as described in Example 1, and then the wounded seeds are smeared with bacteria cultured on a solid culture medium as described in Comparative Example 2.

[0120] Comparative Example 4

[0121] The only difference between this comparative example and Example 1 is that a scalpel is used to remove the tissue below the swollen part of the seed hypocotyl, and dense wounds are scored on the remaining part of the hypocotyl with the scalpel.

[0122] Experimental example

[0123] The gene conversion efficiency and the survival rate of the transgenic plants in each embodiment and comparative example were statistically analyzed, and the results are shown in Table 1.

[0124] Table 1

[0125] Gene transformation efficiency% Survival rate of transgenic plants % Example 1 27.11 100 Comparative Example 1 3.33 100 Comparative Example 2 3.33 100 Comparative Example 3 0.67 100 Comparative Example 4 4.77 100

[0126] As can be seen from Table 1, changing the infection method, infection site and co-cultivation time (Comparative Examples 1-4) all have an impact on the gene transformation efficiency. Therefore, the transformation technology scheme in Example 1 is the optimized version.

[0127] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A method for gene transformation of fruit tree roots mediated by Agrobacterium rhizogenes, characterized in that: The following steps are involved: (a) transforming an expression vector carrying a target gene into competent cells of Agrobacterium rhizogenes, streaking a positive single clone and then culturing it in the dark, picking a single colony for liquid culture and centrifugation to obtain a lower layer of bacterial cells; resuspending the lower layer of bacterial cells in an infection solution to obtain an infection solution; (b) placing the pretreated receptor material in the infection bacterial solution for infection culture; (c) discarding the infecting bacterial solution and co-culturing the receptor material; (d) removing the non-transgenic lateral roots of the recipient material and then conducting rooting culture.

2. The method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The receptor material includes seeds with hypocotyls extending 1 to 3 cm.

3. The method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The infection solution comprises: WPM culture medium and additives; According to each liter of WPM culture medium, the additives include: 13-17 g sucrose, 0.3-0.7 g MES, 0.3-0.7 g CaCl2, 0.6-1.0 mg IBA, 95-105 mM acetosyringone and 0.03 vol%-0.07 vol% surfactant.

4. The method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes according to claim 3, characterized in that: The surfactant includes: Silwet L-77.

5. The method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The pre-processing comprises the following steps: The tender yellow root tip of the receptor material with a length of 1 to 2 mm was cut off, and a wound was scored on the remaining hypocotyl.

6. The method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The expression vector comprises at least one of DR5::GUS, CmAP2a pro::GUS, CmAP2a-RNAi or pK7GWIWG2(II)RR-277.

7. The method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The temperature of the infection culture is 27-29°C; And / or, the infection culture time is 38 to 42 minutes.

8. The method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: Includes at least one of the following technical features: (1) the co-cultivation is carried out in the dark; (2) the co-cultivation is carried out on a moist culture substrate; (3) The co-cultivation temperature is 24-26°C; (4) The co-cultivation period is 6 to 8 days.

9. The method for genetic transformation of fruit tree roots mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: Includes at least one of the following technical features: (1) The temperature of the rooting culture is 24-26° C.; (2) The rooting culture time is 13 to 15 days; (3) The photoperiod of the rooting culture includes: 15 to 17 hours of light and 7 to 9 hours of darkness.

10. A genetic breeding method for fruit trees, characterized in that: The invention comprises the method for gene transformation of fruit tree roots mediated by Agrobacterium rhizogenes as described in any one of claims 1 to 9.

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