A genetic transformation method for callus of apricot plum leaf

Abstract

The present invention relates to the field of plant genetic engineering technology, and in particular to a method for genetic transformation of plum blossom leaf callus of the apricot plum family. The method first prepares a plum blossom genetic transformation receptor, and then RUBY The gene is a reporter gene to transform Agrobacterium to prepare an infection solution, and then the callus is infected and co-cultured to finally screen and obtain resistant callus. The method provided by the present invention can identify resistant callus by observation or PCR detection. The present invention successfully infects plum callus and obtains RUBY The red callus expressing the reporter gene has a high induction rate, which provides a good foundation for the establishment of the plum blossom genetic system and genetic improvement.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a genetic transformation method for callus of apricot plum leaf. Background Art

[0002] plum bossom( Prunus mume Plum blossoms are a kind of woody plant of the genus Prunus in the family Rosaceae. As one of the traditional famous flowers in China, it has a history of introduction and cultivation of more than 3,000 years in my country. It has an important position and cultural connotation in the ecological application of Chinese gardens. Plum blossoms can be divided into true plum series, apricot plum series and cherry plum series according to their provenance composition. Among them, the apricot plum series is an interspecific hybrid of plum blossoms and apricots. Plum blossoms have a wide range of applications and are important early spring flowering plants. They can also be used as food and medicine, etc. They have high ornamental and economic value.

[0003] In order to cultivate plum varieties with strong resistance and high ornamental value for use in landscaping, it is necessary to carry out the selection and improvement of plum varieties. However, in the process of variety selection, conventional hybrid breeding technology is generally adopted, which has a long breeding cycle and low efficiency. The development of plant genetic engineering has provided a new way for plum breeding, but the efficient genetic transformation system of plum is still in the preliminary research stage, and it is difficult to establish an efficient genetic transformation system for complex woody plants. At present, there is still a lack of effective genetic transformation methods for leaf callus tissue of apricot plum series.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a genetic transformation method of callus tissue of plum blossom leaves of the apricot plum series.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for genetic transformation of plum blossoms, which uses leaves as explants, and obtains successfully transformed callus tissue through callus induction, infection culture and screening; wherein the culture medium formula used for callus induction includes: 1.0±0.2 mg / L 6-BA, 1.0±0.2 mg / L TDZ and 0.5±0.1 mg / L 2,4-D.

[0008] Preferably, the culture medium formula used for callus induction is based on WPM as the basic culture medium, and further includes 30±5 g / L of sucrose, 7±1 g / L of agar, and a pH value of 5.8-6.0.

[0009] Preferably, the OD600 value of the bacterial solution used for infection is 0.7±0.1.

[0010] Preferably, the bacterial solution used for infection contains RUBY The reporter gene Agrobacterium GV3101 resuspension solution comprises: 100±20 μmol / L acetosyringone, pH 5.8-6.0.

[0011] Preferably, containing RUBY The method for preparing the resuspension of Agrobacterium GV3101 with reporter gene comprises: firstly RUBY Agrobacterium GV3101 carrying the reporter gene plasmid was activated in LB solid medium supplemented with 75±5 mg / L spectinomycin and 50±5 mg / L rifampicin, and a single clone colony was picked; then the colony was propagated in LB liquid medium supplemented with 75±5 mg / L spectinomycin and 50±5 mg / L rifampicin; finally, the cells were collected by centrifugation and resuspended.

[0012] Preferably, the culture medium used for infection culture is the same as the culture medium used for callus induction.

[0013] Preferably, the callus induction and / or infection culture is carried out at a temperature of 22-26° C. in the dark.

[0014] Preferably, the formula of the screening culture medium includes: 15±2 mg / L spectinomycin and 100±5 mg / L timentin.

[0015] Preferably, the explants are selected from fresh leaves of the branches of the apricot-plum series plum tree 'Xiangruibai', which are newly grown for 1-2 months.

[0016] Preferably, the explant is disinfected, and the disinfection includes: soaking and disinfecting with 75±5% alcohol for 30±5s, rinsing with sterile water for 30±5s, soaking and disinfecting with 2-5% sodium hypochlorite solution for 10-15min, and rinsing with sterile water for 5±2 times.

[0017] In a second aspect, the present invention provides the application of the genetic transformation method of plum blossoms in the preparation of transgenic plum blossom callus or the study of plum blossom gene function and the improvement of plum blossom varieties.

[0018] Preferably, the application includes at least one of the following methods for determining whether Agrobacterium has been successfully transformed:

[0019] (1) Observe with the naked eye. If the callus tissue is red, the transformation is successful.

[0020] (2) Perform PCR identification using primer pairs with nucleic acid sequences such as SEQ ID NO.01~02.

[0021] Beneficial effects:

[0022] The invention provides a method for genetic transformation of plum blossom leaf callus of apricot plum series, firstly preparing a plum blossom genetic transformation receptor, and then RUBY The gene is a reporter gene to transform Agrobacterium to prepare an infection solution, and then the callus is infected and co-cultured to finally screen and obtain resistant callus. The method provided by the present invention can identify resistant callus by observation or PCR detection. The present invention successfully infects plum callus and obtains RUBY The red callus expressing the reporter gene had a high induction rate, which provided a good foundation for the establishment of the genetic transformation system and genetic improvement of plum blossom. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0024] Figure 1 This is a photo of the plum blossom leaves inoculated into callus induction medium in the present invention.

[0025] Figure 2 This is the state of the plum blossom callus after proliferation in the present invention.

[0026] Figure 3 The present invention is a plum blossom callus tissue that is successfully infected and has obvious red color.

[0027] Figure 4 The figure is a comparison between the uninfected plum blossom callus and the transgenic plum blossom callus in the present invention (A: uninfected, B: transgenic).

[0028] Figure 5 In the embodiment of the present invention, RUBY PCR verification results of transgenic plum callus DNA induced by Agrobacterium tumefaciens GV3101 reporter gene; where M is a marker, WT is wild-type plum callus as a negative control, and 1-2 is transgenic plum callus. DETAILED DESCRIPTION

[0029] The present invention provides a method for genetic transformation of callus tissue of plum blossom leaves of apricot plum series. RUBY The reporter gene was used to obtain positive callus tissue through Agrobacterium transformation, providing a technical basis for the research on plum genetic breeding and gene function.

[0030] It is difficult to establish an efficient genetic transformation system for complex woody plants. The present invention first prepares callus receptors for genetic transformation of plum blossoms, which has high efficiency, short cycle and strong stability. Then transgenic callus is obtained through callus infection, and transgenic plum blossoms are obtained through induced regeneration, thereby improving the characteristics of plum blossoms and obtaining new and superior varieties.

[0031] RUBYThe principle of the reporter system is to use tyrosine as a substrate and three enzymes: CYP76AD1, DODA, and GT catalyze the synthesis of betalain as a reporter molecule. These three enzymes are integrated into an open reading frame. RUBY The reporter system can avoid adding exogenous substrates and judge the expression of genes by producing red. RUBY The reporter gene provides a method for efficiently screening the successfully transformed plum callus receptor materials by directly observing and screening the positive transformed materials.

[0032] The invention establishes an Agrobacterium-mediated callus genetic transformation system, which is of great significance for plum variety improvement.

[0033] In a more specific embodiment, the present invention provides a method for genetic transformation of callus tissue of plum blossom leaves of the apricot plum series, comprising the following steps:

[0034] (1) Preparation of plum blossom genetic transformation receptors: Fresh leaves of plum blossoms of the current year were used as explants. After disinfection, they were inoculated on callus induction medium to induce plum blossom callus, which was then used as genetic transformation receptors after subculture.

[0035] (2) Preparation of infection medium: RUBY The reporter gene Agrobacterium GV3101 was streaked and activated in LB solid medium supplemented with 75 mg / L spectinomycin and 50 mg / L rifampicin, and a single clone colony was picked and cultured in LB liquid medium supplemented with 75 mg / L spectinomycin and 50 mg / L rifampicin to obtain a bacterial solution, which was collected by centrifugation and resuspended to obtain a bacterial solution for callus infection.

[0036] (3) Infection and co-cultivation: The plum blossom callus and the infection bacterial solution are shaken and cultured to obtain co-cultured callus, the callus is taken out from the infection bacterial solution, excess water is absorbed on filter paper, and then transferred to the co-cultivation medium.

[0037] (4) Screening: After a period of co-culture, the callus tissue on the co-culture medium is transferred to a screening medium supplemented with antibiotics. The screening medium is replaced every 20 days. The surviving callus tissue is the resistant callus tissue.

[0038] (5) Identification of resistant callus: ① RUBY Gene expression observation: Obvious red color was observed on the resistant callus, indicating RUBY The gene was successfully expressed; ②PCR identification: further detect and identify the transgenic callus at the molecular level, and use PCR technology to detect the callus with obvious red expression.

[0039] In the present invention, the plum variety is preferably 'Xiangruibai' of the apricot plum series.

[0040] Preferably, in step (1), fresh leaves of the branch of the apricot plum tree 'Xiangruibai', which are newly grown for 1-2 months, are selected as explants, and the leaf surfaces are free of black spots, insect pests or damage.

[0041] Preferably, in step (1), the disinfection method is: soak the plum blossom leaves in 75% alcohol for 30 seconds, rinse with sterile water for 30 seconds, soak and disinfect in 2-5% sodium hypochlorite for 10-15 minutes, rinse with sterile water for 5 times, and absorb the surface moisture with filter paper; cut the sterile leaves into 1 cm×1 cm square pieces, and inoculate them on the callus induction medium with the back of the leaves facing up.

[0042] Preferably, in step (1), the formula of the callus induction medium for plum blossom leaves is: WPM + 1.0 mg / L 6-BA + 1.0 mg / L TDZ + 0.5 mg / L 2,4-D + 30g / L sucrose + 7g / L agar, pH 5.8-6.0; the growth conditions for inducing callus tissue are: culture temperature of 22-26°C, dark culture.

[0043] Preferably, in step (2), the resuspended RUBY The reporter gene Agrobacterium tumefaciens GV3101 was cultured in LB medium, the cells were collected by centrifugation, and resuspended in a resuspension solution, which included 1 / 2MS + 30g / L sucrose + 100μmol / L acetosyringone (AS); the OD600 value of the bacterial solution used for infection after resuspension was 0.7.

[0044] Preferably, in step (2), the LB solid culture medium formula is: 10 g / L tryptone + 5 g / L yeast extract + 10 g / L sodium chloride + 15 g / L agar; the LB liquid culture medium formula is: 10 g / L tryptone + 5 g / L yeast extract + 10 g / L sodium chloride.

[0045] Preferably, in step (3), the plum blossom callus is shaken and infected in the bacterial solution for 15 minutes, the culture medium used for co-cultivation is a callus induction medium with a pH of 5.8-6.0, the co-cultivation time is 5 days, the culture temperature is 22-26° C., and the culture is carried out in the dark.

[0046] Preferably, in step (4), the type and concentration of antibiotics added are: 15 mg / L spectinomycin + 100 mg / L timentin.

[0047] Preferably, the present invention further comprises the following steps: observing the callus after co-cultivation, counting the number of red callus, and performing PCR identification on the red callus.

[0048] Furthermore, the present invention also provides the application of the above-mentioned method in the preparation of transgenic plum callus or the study of plum gene function and the improvement of plum varieties.

[0049] The present invention uses the Agrobacterium transformation method and plum blossom callus as the receptor material to successfully introduce the target gene into the plum blossom callus, and establishes a genetic transformation method for plum blossom callus, which is of great significance for the verification and research of plum blossom gene function. RUBY The reporter system is a plant genetic transformation and gene expression reporter system. It can obtain red plum callus tissue visible to the naked eye, directly determine whether Agrobacterium has been successfully transformed, and efficiently screen and non-destructively observe gene expression.

[0050] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0052] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0053] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0054] Example 1

[0055] This embodiment provides a method for genetic transformation of callus tissue of plum blossom leaves of the apricot plum series.

[0056] Here are the steps:

[0057] (1) Preparation of Plum Blossom Genetic Transformation Receptor:

[0058] Cut the fresh leaves of plum blossoms of the current year and disinfect them: rinse the leaves under running water for 15 minutes, then soak the leaves in 75% alcohol for 30 seconds in a clean bench, rinse with sterile water for 30 seconds, and then soak them in 2-5% sodium hypochlorite for 10-15 minutes (in this embodiment, 2% sodium hypochlorite is used for soaking for 15 minutes), rinse with sterile water 5 times, each time for 30 seconds, and then soak them in sterile water after washing; after the disinfection, dry the leaves on filter paper, cut them into 1 cm × 1 cm size, with the back of the leaves facing up, and inoculate them on callus induction medium ( Figure 1 ).

[0059] The formula of callus induction medium is based on WPM as the basic medium, with three plant hormones of 6-BA, 2,4-D and TDZ added, 30 g / L sucrose, 7 g / L agar, and pH 5.8-6.0. In this embodiment, the concentration of 6-BA is 1.0 mg / L, the concentration of 2,4-D is 1.0 mg / L, and the concentration of TDZ is 0.5 mg / L.

[0060] During the induction process, the callus state was observed and the contaminated leaves were cleaned in time; the culture temperature was 22-26°C, cultured in the dark, and subcultured every 20 days after the callus grew ( Figure 2 ), the induction rate was 66.7%.

[0061] (2) Preparation of infection medium:

[0062] will contain RUBYPreparation of Agrobacterium infection solution for Agrobacterium GV3101 with reporter gene: pick a small amount of Agrobacterium strains with a pipette tip, streak on LB solid medium containing 50mg / L spectinomycin and 25mg / L rifampicin, and place the streaked LB solid medium in a 28℃ incubator for 48h until a single colony is produced. Among them, the specific components of LB solid medium are: 10g / L tryptone + 5g / L yeast extract + 10g / L sodium chloride + 15g / L agar. Pick a monoclonal colony with a sterile pipette tip, add it to LB liquid medium containing 75mg / L spectinomycin and 50mg / L rifampicin, shake and culture overnight at 200r / min and 28℃; detect the OD600 value of the culture solution after culture, collect the bacteria after centrifugation, resuspend the culture solution to OD600=0.7, and obtain the infection solution used to infect callus tissue. The specific composition of the resuspension is: 1 / 2MS + 30g / L sucrose + 100μmol / L acetosyringone (AS), pH 5.8-6.0.

[0063] (3) Infection and co-cultivation: The plum blossom callus induced in step (1) was immersed in the infection solution with OD600 = 0.7 in step (2) for 15 min. During this period, the solution needs to be shaken continuously. After immersion, the infected callus was taken out and placed on filter paper to absorb the excess bacterial solution. After the callus was completely dried, it was inoculated on the co-culture medium and co-cultivated for 5 days.

[0064] Among them, the co-culture medium formula is callus induction medium: WPM + 1.0 mg / L 6-BA +1.0 mg / LTDZ + 0.5 mg / L 2,4-D; the culture environment is: culture temperature is 22-26℃, and culture is carried out in the dark.

[0065] (4) Screening: After 5 days of co-culture, the callus on the co-culture medium is transferred to a screening medium supplemented with antibiotics. The screening medium is replaced every 20 days. The surviving callus is the resistant callus, which has a distinct red color.

[0066] (5) Identification of resistant callus:

[0067] RUBY Gene expression observation: Obvious red color can be observed on the resistant callus ( Figure 3 ), while uninfected plum callus does not show red ( Figure 4 ),show RUBY The gene was successfully transformed and expressed, and positive callus tissue was obtained.

[0068] PCR identification: In order to further detect and identify transgenic callus at the molecular level, genomic DNA of callus with obvious red expression was selected as template for PCR amplification. The following upstream primers and downstream primers were selected to obtain amplification products, and uninfected plum callus was used as negative control.

[0069] Upstream primer: cgacactctcgtctactccaa.

[0070] Downstream primer: aacttccgcaagattaccgc.

[0071] PCR reaction conditions: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 30 s; 35 cycles; extension at 72°C for 2 min.

[0072] PCR amplification obtained a band of about 800 bp in size, in which all positive callus tissues could amplify the band, while no band could be obtained from uninfected plum callus tissues ( Figure 5 ).

[0073] In this example, 17 transgenic plum calli were finally identified, and the number of infected calli was 100, so the transformation efficiency was calculated to be 17%, indicating that the infection effect of this example was relatively high.

[0074] Comparative Examples 1-8

[0075] The difference between Comparative Examples 1-8 and Example 1 is that the formula of the callus induction medium in step (1) is different, see Table 1 for details.

[0076]

[0077] Compared with Comparative Examples 1-8, the callus induction rate (the data in Table 1 are the results of three repeated treatments) of the culture medium formula used in Example 1 of the present invention is the highest, and the callus state is the best.

[0078] Comparative Examples 9-10

[0079] The difference between Comparative Examples 1-8 and Example 1 is that the concentration of the infection solution used to infect the callus tissue in step (2) is different, see Table 2 for details.

[0080]

[0081] Compared with Comparative Examples 9-10, the infection effect of the infection solution concentration used in Example 1 of the present invention was the best, with obvious red callus and an induction rate of up to 17.0%. However, Comparative Examples 9 and 10 failed to successfully obtain transgenic callus.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A genetic transformation method of the apricot plum family plum blossom 'Xiangruibai', characterized in that: The fresh leaves of the branches of the plum tree 'Xiangruibai', which are newly grown for 1-2 months, are used as explants, and callus tissue is induced, infected and cultured, and screened to obtain successfully transformed callus tissue; wherein the culture medium formula used for callus tissue induction includes: 1.0±0.2 mg / L 6-BA, 1.0±0.2 mg / L TDZ and 0.5±0.1 mg / L 2,4-D; The medium formula used for callus induction is based on WPM as the basic medium, and also includes 30±5g / L sucrose, 7±1g / L agar, and pH value is 5.8-6.0; The OD600 value of the bacterial solution used for infection was 0.7 ± 0.1; The bacterial solution used for infection contains RUBY A resuspension of Agrobacterium tumefaciens GV3101 containing a reporter gene, the resuspension comprising: 100±20 μmol / L acetosyringone, pH 5.8-6.0; The culture medium used for infection culture is the same as the culture medium used for callus induction; contain RUBY The method for preparing the resuspension of Agrobacterium GV3101 with reporter gene comprises: firstly RUBY Agrobacterium GV3101 carrying the reporter gene plasmid was activated in LB solid medium supplemented with 75±5 mg / L spectinomycin and 50±5 mg / L rifampicin, and a single clone colony was picked; then the colony was propagated in LB liquid medium supplemented with 75±5 mg / L spectinomycin and 50±5 mg / L rifampicin; finally, the bacterial cells were collected by centrifugation and resuspended; The temperature for callus induction and / or infection culture is 22-26°C, in dark; The formulation of the screening medium included: 15 ± 2 mg / L spectinomycin, 100 ± 5 mg / L timentin; The explants were disinfected, and the disinfection sequentially included: immersion in 75±5% alcohol for 30±5s, rinsing with sterile water for 30±5s, immersion in 2-5% sodium hypochlorite solution for 10-15min, and rinsing with sterile water for 5±2 times.

2. Application of the method for genetic transformation of plum blossoms according to claim 1 in preparation of transgenic plum blossom callus or study of plum blossom gene function and improvement of plum blossom varieties; The application includes at least one of the following methods for determining whether Agrobacterium has been successfully transformed: (1) Observe with the naked eye. If the callus tissue is red, the transformation is successful. (2) Perform PCR identification using primer pairs with nucleic acid sequences such as SEQ ID NO.01~02.

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