Genetic transformation methods for crape myrtle stem segments with buds

Abstract

This invention relates to the field of plant genetic transformation technology, and provides a method for genetic transformation of crape myrtle stem segments with buds. The method includes: infecting crape myrtle explants with Agrobacterium tumefaciens containing the RUBY reporter gene, and co-culturing them in a co-culture medium; then transferring the co-cultured explants to a differentiation medium for differentiation culture; and inoculating the obtained clustered buds into a rooting medium to obtain genetically transformed positive seedlings; wherein the explants are crape myrtle tissue culture seedlings with stem segments containing buds. The crape myrtle stem segments used in this invention have a higher regeneration efficiency than leaves. The adventitious bud induction rate of crape myrtle leaves is 1.0%-4.7%, and the stem segment regeneration and proliferation coefficient is 5-20. A large number of positive plants can be obtained in the later stage, and no browning phenomenon occurs in any of the stem segments after infection.

Description

Technical Field

[0001] This invention relates to the field of plant genetic transformation technology, and more specifically, to a method for genetic transformation of crape myrtle stem segments with buds. Background Technology

[0002] Crape myrtle (Lagerstroemia indica) is a shrub or small tree belonging to the genus Lagerstroemia in the family Lythraceae. It has been cultivated in my country for over 1800 years, and China is also the distribution and origin center of the genus Lagerstroemia. With its beautiful tree shape, vibrant flowers, and a flowering period lasting up to 100 days, crape myrtle is a rare woody flowering plant in summer landscaping and is widely used in public green spaces, residential areas, and roadsides.

[0003] Crape myrtle exhibits strong resistance to various toxic gases and can absorb smoke and dust from the air, making it an excellent pollution-resistant tree species, highly suitable for urban roadside greening. However, its biggest problems are poor cold resistance, poor salt and alkali tolerance, and a lack of yellow-flowered varieties. Traditional breeding techniques are insufficient to improve these traits. The construction of an efficient and stable genetic transformation system is an important prerequisite for cultivating new crape myrtle varieties, and also a key technology that urgently needs to be solved in the molecular biology of crape myrtle.

[0004] Compared to herbaceous plants, woody plants have biological characteristics such as long growth cycles and complex genetic regulation, which slows down breeding progress, limits in-depth research, and makes it difficult to meet rapidly changing social needs. Crape myrtle faces similar challenges as most woody plants, including long growth cycles, browning of recipient materials, difficulties in gene transformation, and a low number of positive seedlings, thus limiting the pace of new variety creation. Existing technologies offer some genetic transformation methods for Crape myrtle, mostly using leaves as explants, which suffer from problems such as leaf browning, low regeneration rates, and low transformation efficiency. Using highly efficient regenerating explants for genetic transformation, and employing modern molecular biology and genetic engineering techniques to integrate the target gene into the Crape myrtle genome to obtain the target trait, is a crucial technical means to solve the problem of breeding new Crape myrtle varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a genetic transformation method for crape myrtle stem segments with buds.

[0006] To achieve the objective of this invention, this invention provides a method for genetic transformation of crape myrtle stem segments with buds. The method includes: infecting crape myrtle explants with Agrobacterium tumefaciens containing the RUBY reporter gene and co-culturing them in a co-culture medium; then transferring the co-cultured explants into a differentiation medium for differentiation culture; and inoculating the obtained clustered buds into a rooting medium to obtain genetically transformed positive seedlings.

[0007] In this invention, the explant is a stem segment with buds from a crape myrtle tissue culture seedling.

[0008] Furthermore, the method includes the following steps:

[0009] Step 1: The expression vector containing the RUBY reporter gene was transferred into competent Agrobacterium tumefaciens cells. After activation culture, positive strains were selected, cultured, and the bacterial cells were extracted. The bacterial cells were resuspended in the infection solution to OD. 600 The value is 0.6-1.2. After being placed in the dark for 1-2 hours, a bacterial suspension is obtained.

[0010] Step 2: Take stem segments with buds from crape myrtle tissue culture seedlings, retaining 1-2 pairs of axillary buds. Place the explants in the bacterial suspension under vacuum conditions for infection. Insert the stem segments with the axillary buds facing upwards into the co-culture medium and co-culture in the dark for 2-3 days.

[0011] Step 3: Transfer the transformed recipient material co-cultured in Step 2 into differentiation medium. After 30-60 days of differentiation culture, a large number of clustered buds are obtained on the stem segments.

[0012] Step four: Inoculate the clustered shoots obtained in step three into the rooting medium to obtain genetically transformed positive seedlings.

[0013] Preferably, the Agrobacterium tumefaciens is GV3101, and the expression vector containing the RUBY reporter gene is a pHDE-35S::RUBY recombinant vector.

[0014] Furthermore, the method for preparing the explant is as follows: using the upper stem segment of a sterile tissue culture seedling of Lagerstroemia indica that has grown for 35 days as the explant, only half to one leaf at the top is retained, containing 1-2 pairs of axillary buds, with a length of 1.0-1.5cm.

[0015] Preferably, the infection solution used in step one is: 10 mmol / L MES (2-(N-morpholino)ethanesulfonic acid), 10 mmol / L MgCl2·6H2O (magnesium chloride hexahydrate) and 200 μmol / L AS (acetylsyleugenol), prepared with sterile water.

[0016] Preferably, the vacuum conditions in step two are as follows: under a vacuum of 0.1 MPa, the stem segment with buds is placed in the bacterial suspension and vacuumed for 10-15 minutes for infection, and the infection temperature is 25-28℃.

[0017] Preferably, the co-culture medium in step two is a WPM medium containing 0.5 mg / L 6-BA, 0.05 mg / L IBA, 30 g / L sucrose and 7 g / L agar, with a pH of 5.8-6.0.

[0018] The co-culture conditions are: 22-24℃, incubation in the dark for 2-3 days.

[0019] Further, in step three, before transferring the co-cultured transformed recipient material into the differentiation medium, the material is washed in sterile water containing 100 mg / L termethin for 3-5 min, then washed in sterile water for 10-30 s, and after absorbing excess water with sterile filter paper, the material is inoculated into the differentiation medium for differentiation culture.

[0020] Preferably, the differentiation medium in step three is a WPM medium containing 0.5 mg / L 6-BA (6-benzylaminopurine), 0.05 mg / L IBA (indolebutyric acid), 200 mg / L termethin, 30 g / L sucrose and 7 g / L agar, with a pH of 5.8-6.0; the differentiation culture conditions are: 22-24℃, light intensity of 1500-2000 lx, 15 h light / day, 9 h dark / day, alternating culture for 30-60 days.

[0021] Preferably, the rooting medium in step four is a 1 / 2 MS medium containing 0.3 mg / L IBA, 200 mg / L termethin, 20 g / L sucrose and 7 g / L agar.

[0022] The conditions for rooting culture are: 22-24℃, light intensity of 1500-2000 lx, 15h light / day, 9h darkness / day, alternating culture for 10-30 days.

[0023] Furthermore, the identification method for genetically transformed positive seedlings in step four includes: extracting DNA from the crape myrtle seedlings, performing PCR amplification using specific primers for detecting the RUBY reporter gene, and detecting the amplification products by electrophoresis.

[0024] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0025] (I) This invention provides a genetic transformation method using crape myrtle stem segments with buds as explants. Compared with the prior art, it effectively solves the problem of easy browning when using crape myrtle leaves as explants. After infection, no browning phenomenon appears in any stem segments.

[0026] (II) The crape myrtle stem segments used in this invention have a higher regeneration efficiency than leaves. The regeneration and proliferation coefficient of crape myrtle leaves is 1-5, while that of stem segments is 5-20, and a large number of positive plants can be obtained in the later stage.

[0027] (III) This invention uses the woody plant Lagerstroemia indica as material, making the transformation and regeneration system with herbaceous plants more difficult. It provides important technical support for subsequent transgenic functional verification and new variety breeding, and also offers a new paradigm for the establishment of genetic transformation systems for other woody plants. Attached Figure Description

[0028] Figure 1This is an example diagram illustrating the entire process of genetic transformation using crape myrtle stem segments with buds as explants in a preferred embodiment of the present invention. A: Stem segments with buds, infected with a solution carrying the target gene, are inserted into a culture medium; B: Induction of shoot clusters from the stem segments with buds; C: Rooting of the regenerated plants.

[0029] Figure 2 The results of PCR electrophoresis detection of positive plants in a preferred embodiment of the present invention are shown. M, Marker; WT (lanes 1-2), wild-type Lagerstroemia indica; RUBY (lanes 3-5), positive plants successfully transformed with the RUBY gene.

[0030] Figure 3 This invention compares the effects of bud induction using crape myrtle leaves and stem segments with buds as explants. A: Adventitious bud regeneration using crape myrtle leaves as explants; B: Adventitious bud regeneration using crape myrtle stem segments with buds as explants. Detailed Implementation

[0031] This invention provides a highly efficient genetic transformation method using crape myrtle stem segments with axillary buds as explants. Compared with other explants, the young stem segments of crape myrtle with axillary buds in the current year can induce a large number of clustered buds with a proliferation coefficient of 20, and the clustered buds grow healthily. Using these as transgenic explants can greatly improve the transformation efficiency and increase the positive plant ratio.

[0032] The present invention adopts the following technical solution:

[0033] This invention provides a highly efficient genetic transformation method using crape myrtle stem segments with buds as explants, comprising the following steps:

[0034] 1. The competent cells of Agrobacterium tumefaciens to be transgenic were introduced, activated, and then positive strains were selected, cultured, and the bacterial cells were extracted. The bacterial cells were resuspended in the infection solution to OD. 600 The value is 0.6-1.2. After placing it in the dark for 1-2 hours, a bacterial suspension is obtained. 2. Take the stem segments with buds from the tissue culture seedlings of Lagerstroemia indica, retain 1-2 pairs of axillary buds, and infect the explants in the bacterial suspension by vacuuming for 10-15 minutes. Insert the stem segments with the axillary buds facing upwards into the co-culture medium and co-culture in the dark for 2-3 days at a temperature of 22-24℃.

[0035] 3. After co-culturing the transformation recipients under light, a large number of clustered buds were obtained on the stem segments after 30-60 days;

[0036] 4. After cutting off the clustered buds (about 1-3cm), inoculate them into the rooting medium for rooting culture. After molecular identification, genetically transformed positive seedlings are obtained.

[0037] The Agrobacterium strain is Agrobacterium tumefaciens GV3101, and the Agrobacterium contains a recombinant vector expressing the target gene pHDE-35S::RUBY.

[0038] The budded stem segments of the crape myrtle tissue culture seedlings are the upper stem segments of sterile seedlings that have grown for about 35 days. Only half to one leaf at the top is retained, with 1-2 pairs of axillary buds, and the length is about 1.0-1.5cm.

[0039] The LB solid medium used for Agrobacterium activation contains the following components: 25 g / L Luria-Bertani (LB) medium, 50 mg / L kanamycin, 25 mg / L rifampin, and 15 g / L agar. The activation temperature is 27-29℃.

[0040] The LB liquid medium used for Agrobacterium culture contains the following components: Luria-Bertani (LB) medium 25 g / L, kanamycin 50 mg / L, rifampin 25 mg / L, and the culture temperature is 27-29℃.

[0041] The infection solution comprises: sterile water, 10 mmol / L MES (2-(N-morpholino)ethanesulfonic acid), 10 mmol / L MgCl2·6H2O (magnesium chloride hexahydrate), and 200 μmol / L AS (acetylsyringone).

[0042] The vacuum conditions are as follows: Under a vacuum of 0.1 MPa, the stem segments with buds are placed in the bacterial suspension and vacuumed for 10-15 minutes for infection. The infection temperature is 25-28℃. After the infection is completed, the vacuum pump slowly releases the pressure.

[0043] The co-culture medium comprises: WPM medium, 0.5 mg / L 6-BA, 0.05 mg / L IBA, 30 g / L sucrose, 7 g / L agar, pH 5.8-6.0.

[0044] Before transferring the explants to the differentiation medium, the stem segments were placed in sterile water containing 100 mg / L termethin and gently shaken for 3-5 minutes. Then, they were immediately placed in sterile water for 10-30 seconds and excess water was absorbed with sterile filter paper before being transferred to the differentiation medium.

[0045] The differentiation medium comprises: WPM medium, 0.5 mg / L 6-BA, 0.05 mg / L IBA, 200 mg / L termethin, 30 g / L sucrose, 7 g / L agar, pH 5.8-6.0.

[0046] The differentiation culture was conducted at a temperature of 22-24℃, with a light intensity of 1500-2000 lx, and was carried out under alternating light and dark conditions for 15h / 9h for 30-60 days.

[0047] After inducing the shoot clusters for approximately 30-60 days, they are inoculated into rooting medium, which consists of: 1 / 2 MS, 0.3 mg / L IBA, 200 mg / L termethin, 20 g / L sucrose, and 7 g / L agar.

[0048] The rooting culture was conducted at a temperature of 22-24℃ and a light intensity of 1500-2000 lx, with alternating light and dark conditions for 15h / 9h for 10-30 days.

[0049] Positive identification of transgenic crape myrtle: DNA was extracted from the obtained crape myrtle seedlings by mixing samples, and PCR electrophoresis was performed using specific primers on the recombinant vector expressing the target gene.

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0051] The WPM culture medium used in the following examples was purchased from CAISSON LABS, model WPP03.

[0052] Example 1: An efficient genetic transformation method using budded stem segments of Lagerstroemia indica

[0053] To observe the transformation process more intuitively, the RUBY visualization vector system was used to construct a Lagerstroemia indica genetic transformation system.

[0054] The plant expression vector (pHDE-35S::RUBY) containing the target gene was transformed into competent Agrobacterium strain GV3101 using a freeze-thaw method, and then cultured in empty LB broth for 2-2.5 h. The starting vector pHDE was provided by the International Bamboo and Rattan Centre, the National Forestry and Grassland Administration, and the Beijing Key Laboratory of Bamboo and Rattan Science and Technology. The full sequence of the vector pHDE-35S::RUBY is formed by tandemly connecting the sequences shown in SEQ ID NO:1-2.

[0055] In a sterile operating table, use a sterile pipette tip to pick up a small amount of bacterial solution and spread it on LB solid medium (containing 50 mg / L kanamycin, 25 mg / L rifampin, and 15 g / L agar). Invert the culture dish and place it in an incubator at 28°C for about 48 hours.

[0056] Single colonies were picked and verified as correct by PCR and sequencing. These colonies were then transferred to LB double-antibody liquid medium (containing 50 mg / L Kan and 25 mg / L Rif) and incubated at 200 rpm and 28°C for approximately 24 hours. After centrifugation at 5000 rpm for 10 minutes, the bacterial culture was collected, the supernatant was discarded, and the extracted bacterial cells were resuspended in an invasion dye solution (containing sterile water, 10 mmol / L MES (2-(N-morpholino)ethanesulfonic acid), 10 mmol / L MgCl2·6H2O (magnesium chloride hexahydrate), and 200 μmol / L AS (acetylsyleugenol)) to adjust the OD and optimize the OD. 600 =0.8, stand in the dark at 28℃ for 1.5h for later use, to obtain bacterial suspension.

[0057] Using aseptic stem segments from sterile 'Binfen Jiaren' crape myrtle seedlings approximately 35 days old as explants, only half to one leaf at the top of the stem segment, containing 1-2 pairs of axillary buds, were retained. Under aseptic conditions, the stem segments and bacterial solution were placed in Erlenmeyer flasks, ensuring full contact between the stem segments and the solution, and then sealed with sealing film. Infection was performed under a 0.1 MPa vacuum, preferably with vacuum for 10 minutes, at a temperature of 28°C. After infection, the vacuum pump was slowly released to prevent damage to the stem segments from sudden depressurization.

[0058] Remove the stem segments and place them on sterile filter paper to absorb the bacterial solution adhering to the stem segments. Then, inoculate the stem segments into a co-culture medium of WPM + 0.5 mg / L 6-BA + 0.05 mg / L IBA + 20 g / L sucrose + 7 g / L agar (preferably pH 5.8) and co-culture at 22-24℃ in the dark for 2 days.

[0059] After co-culturing for 2 days, the stem segments were rinsed in sterile water containing 100 mg / L termethin for 3 minutes, then rinsed in sterile water for 10 seconds, and finally the excess water was absorbed with sterile filter paper. They were then inoculated into differentiation medium containing WPM + 0.5 mg / L 6-BA + 0.05 mg / L IBA + 200 mg / L termethin + 30 g / L sucrose + 7 g / L agar (preferably pH 5.8). The selection culture was carried out under the conditions of 22-24℃, light intensity of 1500-2000 lx, and 15h / 9h light-dark alternation. After 30-60 days, obvious clumps of callus tissue appeared at the bottom of the stem segments, and clustered buds formed.

[0060] Inoculate the newly emerging purplish-red clustered shoots (about 1-3 cm) into rooting medium (containing 1 / 2 MS + 0.3 mg / L IBA + 200 mg / L termethin + 20 g / L sucrose + 7 g / L agar, preferably pH 5.8).

[0061] DNA was extracted from leaves of regenerated plants and analyzed using the Vazyme Plant Genomic DNA Extraction Kit (DC104-01). PCR detection was performed using specific primers for the target gene.

[0062] The specific primer sequences for the pHDE-35S::RUBY vector are as follows:

[0063] F: 5'-ACTGTATGTGCTATGGTATGGACTATGG-3'

[0064] R: 5'-TCCCTTCAATCGTTGCGGTTCTGT-3'

[0065] The results showed that a specific fragment of approximately 512 bp was obtained from PCR of the resistant regenerated plant samples. This fragment was excised, recovered, and sequenced. The sequencing results were compared with the RUBY sequence, and the sequence identity was 100%. A total of 16 regenerated plants were tested in the first batch, with 3 positive plants, resulting in a positive rate of 18.75%. Figure 2 ).

[0066] Depend on Figure 1 The results showed that when stem buds were used as transformation recipients, they could still show red positive buds one month and especially three months after transformation.

[0067] To optimize the formulation of the adventitious bud induction medium for Lagerstroemia indica and to compare the regeneration efficiency of different explant materials, 2-3 pairs of leaves and stem segments containing 1-2 pairs of axillary buds from sterile 'Binfen Jiaren' Lagerstroemia indica seedlings grown for approximately 35 days were used as explants. WPM (Cassion) medium was used as the basal medium, with 6-BA concentrations of 0.25 mg / L, 0.5 mg / L, and 0.75 mg / L, and IBA concentrations of 0.025 mg / L, 0.05 mg / L, and 0.075 mg / L. A two-factor, three-level orthogonal experiment was conducted, resulting in 9 combinations, as shown in Table 1. Each combination was inoculated with 30 leaves and stem segments, replicated 3 times. The callus and adventitious bud development were recorded after 35 days.

[0068] Table 1 shows the callus induction and adventitious bud formation of 'Binfen Jiaren' crape myrtle.

[0069] Table 1. Callus induction and adventitious bud formation of 'Binfen Jiaren' crape myrtle

[0070]

[0071] Note: Different lowercase letters indicate significant differences at the 0.05 level.

[0072] Table 1 shows that differentiation induction using leaves as explants resulted in varying degrees of browning, with the browning rate decreasing with increasing 6-BA concentration. However, no browning was observed with adventitious bud induction using stem segments with buds as explants. Furthermore, the fifth combination, with 6-BA concentrations of 0.5 mg / L and IBA concentrations of 0.05 mg / L, was suitable for inducing adventitious buds from both leaves and stem segments. The highest adventitious bud induction rate was 4.7% for leaves, while the highest adventitious bud proliferation coefficient for stem segments was 20.0, demonstrating a significant effect in inducing clustered buds. Therefore, stem segments can be considered excellent explants for genetic transformation and infection. Figure 3 ).

[0073] It should be noted that transplanting stem segments with buds into the rooting medium requires 25-35 days to achieve good rooting. The upper part of the stem segments and leaves is in the best growth condition and the explants are relatively tender. Therefore, using the upper stem segments of sterile tissue culture seedlings that are 25-35 days old as explant materials results in the highest regeneration rate.

[0074] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A genetic transformation method for crape myrtle stem segments with buds, characterized in that, The method includes: infecting crape myrtle explants with Agrobacterium tumefaciens containing the RUBY reporter gene and co-culturing them in a co-culture medium; then transferring the co-cultured explants into a differentiation medium for differentiation culture; and inoculating the obtained clustered shoots into a rooting medium to obtain genetically transformed positive seedlings. The explant is a stem segment with buds from a crape myrtle tissue culture seedling; Includes the following steps: Step 1: The expression vector containing the RUBY reporter gene was transferred into competent Agrobacterium tumefaciens cells. After activation culture, positive strains were selected, cultured, and the bacterial cells were extracted. The bacterial cells were resuspended in the infection solution to OD. 600 The value is 0.6-1.

2. After being placed in the dark for 1-2 hours, a bacterial suspension is obtained. Step 2: Take stem segments with buds from crape myrtle tissue culture seedlings, retaining 1-2 pairs of axillary buds. Place the explants in the bacterial suspension under vacuum conditions for infection. Insert the stem segments with the axillary buds facing upwards into the co-culture medium and co-culture in the dark for 2-3 days. Step 3: Transfer the transformed recipient material co-cultured in Step 2 into differentiation medium. After 30-60 days of differentiation culture, a large number of clustered buds are obtained on the stem segments. Step four: Inoculate the clustered shoots obtained in step three into the rooting medium to obtain genetically transformed positive seedlings; The method for preparing the explant is as follows: using the upper stem segment of a sterile tissue culture seedling of Lagerstroemia indica that has grown for 35 days as the explant, only half to one leaf at the top is retained, containing 1-2 pairs of axillary buds, with a length of 1.0-1.5cm; The infection solution used in step one was: 10 mmol / L MES, 10 mmol / L MgCl2·6H2O and 200 μmol / L AS, prepared with sterile water; The co-culture medium described in step two is a WPM medium containing 0.5 mg / L 6-BA, 0.05 mg / L IBA, 30 g / L sucrose and 7 g / L agar, with a pH of 5.8-6.

0. The conditions for co-cultivation are: 22-24℃, incubation in the dark for 2-3 days; The differentiation medium described in step three is WPM medium containing 0.5 mg / L 6-BA, 0.05 mg / L IBA, 200 mg / L termethin, 30 g / L sucrose and 7 g / L agar, with a pH of 5.8-6.

0. The conditions for differentiation culture are: 22-24℃, light intensity of 1500-2000 lx, 15h light / day, 9h darkness / day, alternating culture for 30-60 days; The rooting medium described in step four is a 1 / 2 MS medium containing 0.3 mg / L IBA, 200 mg / L termethin, 20 g / L sucrose and 7 g / L agar; The conditions for rooting culture are: 22-24℃, light intensity of 1500-2000 lx, 15h light / day, 9h darkness / day, alternating culture for 10-30 days; Step four, the identification method for genetically transformed positive seedlings, includes: extracting DNA from crape myrtle seedlings, performing PCR amplification using specific primers for detecting the RUBY reporter gene, and detecting the amplification products by electrophoresis; The vacuum conditions described in step two are as follows: Under a vacuum of 0.1 MPa, the stem segments with buds are placed in the bacterial suspension and vacuumed for 10-15 minutes for infection, and the infection temperature is 25-28℃.

2. The method according to claim 1, characterized in that, The Agrobacterium tumefaciens is GV3101, and the expression vector containing the RUBY reporter gene is pHDE-35S::RUBY recombinant vector.

3. The method according to claim 1, characterized in that, Step 3: Before transferring the co-cultured transformed recipient material into the differentiation medium, wash the material in sterile water containing 100 mg / L termethin for 3-5 min, then wash it in sterile water for 10-30 s. After absorbing excess water with sterile filter paper, inoculate the material into the differentiation medium for differentiation culture.

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