Genetic transformation method based on adventitious root generation of betula alnoides leaf

Abstract

The present application relates to a kind of genetic transformation methods based on southwest birch leaf adventitious root generation, comprising the following steps: with the leaf of southwest birch petiole as transformation receptor, the pre-culture is carried out to the transformation receptor;The transformation receptor is pre-cultured again and then is infected with the transformation fluid containing Agrobacterium tumefaciens;The leaf after infection is co-cultured;The leaf after washing is co-cultured;The leaf after washing is induced to culture adventitious root;Wherein the Agrobacterium tumefaciens includes the expression vector carrying enhanced green fluorescent protein gene.The method of the present application, without experiencing callus induction and differentiation process can directly realize rooting, greatly shorten the transformation cycle;Efficient washing method is used, and the sterilization strength is increased, and the transgenic efficiency of southwest birch is improved, so that the genetic transformation based on the leaf adventitious root generation of southwest birch mediated by Agrobacterium tumefaciens is successfully realized, and the genetic transformation rate can reach more than 10%, with the advantages of short cycle, high transformation rate and simple operation.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a genetic transformation method and system based on the adventitious root formation of Southwest birch leaves. Background Technology

[0002] Southwest birch (Betula alnoides Buch.-Ham. ex D.Don) is a tall tree belonging to the genus Betula in the family Betulaceae. It is naturally distributed in Yunnan and Guizhou provinces of my country, and has also been successfully introduced and cultivated in Guangdong and Fujian provinces. It grows rapidly, has a straight trunk, and produces excellent timber, making it a superior species for cultivating large-diameter timber. Southwest birch is a typical mycorrhizal plant, establishing a symbiotic relationship with mycorrhizal fungi through its roots, thereby efficiently absorbing limiting nutrients.

[0003] Currently, researchers are conducting genetic transformation studies on betulaceous plants. Patent CN105861542B provides a method for birch transgenic transformation based on zygotic embryo regeneration of adventitious buds. However, due to the extremely small size of birch seeds, the operation is difficult, and each seed has a different genetic background. Patent CN114836468A provides a highly efficient method for birch root transgenic transformation. This technology requires a traditional "explant-callus-adventitious bud-rooting" process, which is cumbersome and has a long rooting cycle, making it unsuitable for studying root gene function. For example, Liu Xueyu et al. (2021) reported a genetic transformation system based on Agrobacterium rhizogenes-mediated hairy roots of Betulia sclerotium. Because the hairy roots are induced by the Ti plasmid of Agrobacterium rhizogenes, their root structure, growth characteristics, and effective components differ significantly from normal plant roots, thus affecting the functional identification of root trait-related genes. Studies have shown that the symbiotic efficiency between the roots and mycorrhizal fungi of *Betula spp.* varies considerably, severely impacting the species' growth efficiency. Therefore, the demand for improved varieties with highly efficient root symbiosis is extremely urgent in practical production. Given the long traditional breeding cycle of *Betula spp.*, isolating key root genes through genetic engineering and genetically improving their traits has become an important approach to *Betula spp.* However, the lack of a genetic transformation system for *Betula spp.* severely restricts the progress of molecular breeding. Summary of the Invention

[0004] Therefore, it is necessary to provide a genetic transformation method based on the adventitious root development of leaves of *Betula spp.* to address the current lack of a genetic transformation system for *Betula spp.*. This method has enabled the successful application of genetic transformation technology in *Betula spp.* and achieved a high transformation rate.

[0005] One aspect of the present invention provides a genetic transformation method based on the adventitious root formation of *Betula spp.* leaves, comprising the following steps:

[0006] Using petiolate leaves of *Betula septemlobus* as transformation recipients, the transformation recipients were pre-cultured; after pre-culture, the transformation recipients were infected with a transformation solution containing *Agrobacterium tumefaciens*; the infected leaves were co-cultured; the co-cultured leaves were washed; and the washed leaves were subjected to adventitious root induction culture.

[0007] In one embodiment, the Agrobacterium tumefaciens contains an expression vector carrying an enhanced green fluorescent protein gene.

[0008] In one embodiment, the strain of Agrobacterium tumefaciens is GV3101.

[0009] In one embodiment, the expression vector is pSAK277::eGFP.

[0010] In one embodiment, the pre-culture medium is a WPM medium containing 0.1 mg / L to 0.3 mg / L NAA, 15 g / L to 25 g / L sucrose, and 5 g / L to 7 g / L agar.

[0011] In one embodiment, the OD of the conversion solution 600 The value is 0.3 to 0.5.

[0012] In one embodiment, the infection time is 10 min to 15 min.

[0013] In one embodiment, the co-cultivation conditions include:

[0014] (1) The culture was carried out in the dark; (2) The culture medium for co-culture was MS medium; (3) The co-culture time was 2d to 4d; and (4) The co-culture temperature was 23℃ to 25℃.

[0015] In one embodiment, the cleaning method after co-cultivation is as follows:

[0016] (1) Rinse once with sterile water; (2) Soak once with a cefotaxime sodium aqueous solution with a concentration of 500mg / L to 1000mg / L for 1 minute; (3) Soak once again with a cefotaxime sodium aqueous solution with a concentration of 400mg / L to 600mg / L for 1 minute; (4) Rinse twice with sterile water.

[0017] In one embodiment, the culture medium for adventitious root induction culture is WPM medium containing 4 mg / L to 6 mg / L kanamycin, 190 mg / L to 210 mg / L cefotaxime sodium, 15 g / L to 25 g / L sucrose, 5 g / L to 7 g / L agar, 0.05 mg / L to 0.15 mg / L NAA, and 0.01 mg / L to 0.03 mg / L 6-BA.

[0018] In one embodiment, the conditions for the adventitious root induction culture include:

[0019] (1) Temperature is 23℃~25℃; (2) Light intensity is 2000lx~2500lx, and the daily light duration is 14h~16h; and (3) Culture time is 15d~30d, and the culture medium is replaced every 5d~8d.

[0020] In one embodiment, the steps for preparing the conversion solution include:

[0021] Agrobacterium tumefaciens containing an expression vector carrying enhanced green fluorescent protein, stored at -80℃, was inoculated into YEP culture medium containing antibiotics for the first culture to obtain culture 1; culture 1 was taken and YEP culture medium without antibiotics was added for the second culture to obtain culture 2; culture 2 was centrifuged and enriched and then added to resuspension to prepare the transformation solution.

[0022] In one embodiment, the resuspension contains 8 mmol / L to 12 mmol / L MES, 8 mmol / L to 12 mmol / L MgCl2, and 90 μmol / L to 110 μmol / L acetosyringone.

[0023] In one embodiment, after adventitious root induction culture, the step further includes identifying the product obtained after adventitious root induction culture; the identification method is fluorescence microscopy detection.

[0024] In another aspect, the present invention provides a genetic transformation system based on the adventitious root formation of Betula alba leaves, constructed using the aforementioned genetic transformation method.

[0025] Compared with traditional technologies, the present invention also has the following beneficial effects:

[0026] (1) This invention uses asexualized whole leaves of Southwest Birch with petioles as transformation recipients, which unifies the genetic background of transgenic progeny lines, improves material utilization, and saves material collection time.

[0027] (2) This invention uses the leaf adventitious root system for genetic transformation, which does not require the traditional process of explant-callus-adventitious bud-rooting, and directly achieves the transformation from explant to root. The rooting rate can reach 100%, which greatly shortens the transformation cycle and has obvious advantages in the identification of genes related to plant root traits.

[0028] (3) In the cleaning process after co-cultivation, the present invention adopts the method of rinsing-soaking-rinsing, which improves the sterilization effect, reduces the difficulty of sterilization in the later stage, and improves the transgenic efficiency of Southwest Birch, with a genetic transformation rate of more than 10%. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 Figure 1 shows the growth and identification of the adventitious root transformation system of *Betula septemlobus* leaves at each stage of its establishment. Figure A: Growth status of *Betula septemlobus* clonal tissue culture seedlings used for leaf sampling. Figure B: Growth status of infected *Betula septemlobus* petiolate leaves after 15 days of culture on adventitious root induction solid plate medium containing selectants and antibacterial agents. Figure C: Scanning electron micrograph of the transformed root system. Figure D: Fluorescence micrograph of the transformed root system. Figure E: Overall view of the transgenic line. Figure F: Fluorescence micrograph of the overall view of the transgenic line. Detailed Implementation

[0031] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0033] the term

[0034] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0035] In this invention, the selection scope of "and / or", "or / and", and "and / or" includes any one of two or more related listed items, as well as any and all combinations of the related listed items.

[0036] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the above-mentioned numerical intervals are considered to be continuous.

[0037] Unless otherwise specified, MES in this invention refers to 2-(N-morpholino)ethanesulfonic acid-water.

[0038] Unless otherwise specified, Cef in this invention refers to cefotaxime sodium.

[0039] Unless otherwise specified, Kan in this invention refers to kanamycin.

[0040] The first aspect of this invention provides a genetic transformation method based on the adventitious root formation of *Betula spp.* leaves, comprising the following steps:

[0041] Using petiolate leaves of *Betula septemlobus* as transformation recipients, the transformation recipients were pre-cultured; after pre-culture, the transformation recipients were infected with a transformation solution containing *Agrobacterium tumefaciens*; the infected leaves were co-cultured; the co-cultured leaves were washed; and the washed leaves were subjected to adventitious root induction culture.

[0042] Optionally, the Agrobacterium tumefaciens contains an expression vector carrying an enhanced green fluorescent protein gene.

[0043] Optionally, the strain of Agrobacterium tumefaciens is GV3101.

[0044] Optionally, the expression vector is pSAK277::eGFP.

[0045] Optionally, the pre-culture medium is a WPM medium containing 0.1 mg / L to 0.3 mg / L NAA, 15 g / L to 25 g / L sucrose, and 5 g / L to 7 g / L agar.

[0046] Optionally, the OD of the conversion solution 600 The value is 0.3 to 0.5.

[0047] Optionally, the infection time is 10 min to 15 min.

[0048] Optionally, co-cultivation conditions include:

[0049] (1) The culture was carried out in the dark; (2) The culture medium for co-culture was MS medium; (3) The co-culture time was 2d to 4d; and (4) The co-culture temperature was 23℃ to 25℃.

[0050] Alternatively, the washing method after co-cultivation is as follows:

[0051] (1) Rinse once with sterile water; (2) Soak once with a cefotaxime sodium aqueous solution with a concentration of 500mg / L to 1000mg / L for 1 minute; (3) Soak once again with a cefotaxime sodium aqueous solution with a concentration of 400mg / L to 600mg / L for 1 minute; (4) Rinse twice with sterile water.

[0052] Optionally, the culture medium for adventitious root induction is WPM medium containing 4 mg / L to 6 mg / L kanamycin, 190 mg / L to 210 mg / L cefotaxime sodium, 15 g / L to 25 g / L sucrose, 5 g / L to 7 g / L agar, 0.05 mg / L to 0.15 mg / L NAA, and 0.01 mg / L to 0.03 mg / L 6-BA.

[0053] Optionally, the pH of the culture medium for adventitious root induction culture is 5.5 to 6.5.

[0054] Optionally, the conditions for adventitious root induction culture include:

[0055] (1) Temperature is 23℃~25℃; (2) Light intensity is 2000lx~2500lx, and the daily light duration is 14h~16h; and (3) Culture time is 15d~30d, and the culture medium is replaced every 5d~8d.

[0056] Optionally, the step of preparing the conversion solution includes:

[0057] Agrobacterium tumefaciens containing an expression vector carrying an enhanced green fluorescent protein gene, stored at -80°C, was inoculated into YEP culture medium containing antibiotics for a first culture. The culture from the first culture was taken and YEP culture medium without antibiotics was added for a second culture. The culture from the second culture was centrifuged and enriched, and then resuspended to prepare the transformation solution.

[0058] Optionally, the resuspension contains 8 mmol / L to 12 mmol / L MES, 8 mmol / L to 12 mmol / L MgCl2, and 90 μmol / L to 110 μmol / L acetosyringone.

[0059] Optionally, the antibiotic-containing culture medium is YEP culture medium containing 50 mg / L Rif and 100 mg / L Spe.

[0060] Optionally, the culture conditions for the first culture include:

[0061] (1) The culture time is 10h to 14h; (2) The culture temperature is 27℃ to 29℃; and (3) The culture speed is 200rpm / min to 240rpm / min.

[0062] Optionally, the OD of the second culture... 600 The value is 0.5 to 0.7.

[0063] Optionally, after adventitious root induction culture, the method further includes a step of identifying the product obtained after genetically transformed adventitious root induction culture; the identification method is fluorescence microscopy detection.

[0064] In a second aspect, the present invention provides a genetic transformation system based on the adventitious root formation of Betula alba leaves, constructed using the genetic transformation method described above.

[0065] The following detailed description is provided in conjunction with specific embodiments. Unless otherwise specified, all raw materials and instruments used in the following embodiments are commercially available. Unless otherwise specified, all processes involved are conventionally selected by those skilled in the art.

[0066] Example 1

[0067] This embodiment 1 provides a genetic transformation method based on the adventitious root formation of Southwest Betula leaves, the method comprising the following steps:

[0068] (1) Select young petioles from healthy, fully developed Southwest Birch tissue culture seedlings with a height of more than 2 cm as transformation recipient materials and inoculate them into a pre-culture medium for pre-culture. The pre-culture medium is formulated as WPM basic medium with the addition of 20 g / L sucrose, 6.0 g / L agar, 0.2 mg / L NAA, and pH 5.8.

[0069] (2) Preparation of transformation medium: Take 100 μL of Agrobacterium (GV3101 strain carrying pSAK277::eGFP expression vector) stored at -80℃, and inoculate it into 5 ml of YEP culture medium supplemented with 50 mg / L Rif and 100 mg / L Spe. Incubate for the first time at 28℃ and 220 rpm / min for 12 h. The next day, take the above overnight culture and transfer it into fresh antibiotic-free YEP culture medium at a ratio of 2% for the second culture until OD. 600 The value was 0.7. 100 μmol / L acetylsyleugenone was added, and after 2 h, the bacterial culture obtained from the second culture was enriched by centrifugation. Then, the concentration was adjusted to OD using a resuspension containing MES. 600 =0.5, to obtain a conversion solution for infection of Southwest birch.

[0070] The resuspension contained MES and MgCl2·6H2O at a concentration of 10 mmol / L, acetylsylgenone at a concentration of 100 μmol / L, and a pH of 6.

[0071] (3) In a clean bench, the Southwest Birch leaf recipients that were pre-cultured for 1 day in step (1) were transferred into a sterile tissue culture bottle, and then 100 ml of the conversion solution prepared in step (2) was added and soaked for 10 min to obtain infected petiolate leaves.

[0072] (4) The infected petioles were transferred to culture dishes containing MS medium, with 10 petioles inoculated per dish, and co-cultured in the dark at 23℃~25℃ for 2 days.

[0073] (5) The leaves of (4) after co-culturing for 2 days were cleaned. The cleaning process was as follows: rinsed once with sterile water, soaked for 1 min each with 1000 mg / L and 500 mg / L cefotaxime sodium (Cef) aqueous solution, and then rinsed twice with sterile water.

[0074] (6) After cleaning, the leaves were transferred to a solid plate medium containing a selector and an antibacterial agent for adventitious root induction. Adventitious root induction culture was carried out at 23℃–25℃, 2000 lx–2500 lx, and a light / dark cycle of 16 h / 8 h. The medium was changed every 7 days. After 15 days of culture under the above conditions, white resistant adventitious roots were observed and detected at the petiole using scanning electron microscopy and fluorescence microscopy, thus obtaining transgenic lines. The rooting rate and the average number of roots per leaf (average number of roots / leaf) were recorded at 20 and 30 days of culture. The solid medium for adventitious root induction culture was WPM basal medium supplemented with 5 mg / L kanamycin (Kan), 200 mg / L cefotaxime sodium (Cef), 20 g / L sucrose, 6.0 g / L agar, 0.1 mg / L NAA, 0.02 mg / L 6-BA, and a pH of 5.8.

[0075] Example 2

[0076] The genetic transformation method in this embodiment is the same as that in Example 1. The difference from Example 1 is that the cleaning method is as follows: rinse once with sterile water, soak twice with 500mg / L Cef for 1 minute each time, and then rinse twice more with sterile water.

[0077] Example 3

[0078] The genetic transformation method in this embodiment is the same as that in Example 1, except that the infection conditions are as follows: the concentration of the transformation solution (OD) is... 600 The concentration was 0.3, the infection time was 15 min, and the co-culture time was 3 days.

[0079] Example 4

[0080] The genetic transformation method in this embodiment is the same as that in Example 1, except that the infection conditions are as follows: the concentration of the transformation solution (OD) is... 600 The concentration was 0.3, the infection time was 10 min, and the co-culture time was 4 days.

[0081] Example 5

[0082] The genetic transformation method in this embodiment is the same as that in Example 1. The difference is that 6-BA was not added to the solid culture medium for adventitious root induction culture.

[0083] Comparative Example 1

[0084] The genetic transformation method in this comparative example is the same as that in Example 1. The difference from Example 1 is that the washing conditions are: rinse once with sterile water, soak in 1000 mg / L Cef for 2 min, and rinse twice more with sterile water.

[0085] Comparative Example 2

[0086] The genetic transformation method used in this comparative example is the same as that in Example 1, except that the concentration of Kan in the solid culture medium for adventitious root induction culture is 10 mg / L.

[0087] Comparative Example 3

[0088] The genetic transformation method in this comparative example is the same as that in Example 1, except that the infection time is 7 min and the co-culture time is 4 d.

[0089] Comparative Example 4

[0090] The genetic transformation method used in this comparative example is the same as that in Example 1, except that the Cef concentration in the solid culture medium for adventitious root induction culture is 100 mg / L.

[0091] Comparative Example 5

[0092] The genetic transformation method in this comparative example is the same as that in Example 1, except that the infection time is 15 min and the co-culture time is 3 days.

[0093] Comparative Example 6

[0094] The genetic transformation method in this comparative example is the same as that in Example 1, except that the washing method is to wash with sterile water three times.

[0095] Comparative Example 7

[0096] The genetic transformation method used in this comparative example is the same as that in Example 1, except that the concentration of Kan in the solid medium for adventitious root induction culture is 15 mg / L.

[0097] Comparative Example 8

[0098] The genetic transformation method used in this comparative example is the same as that in Example 1, except that the concentration of the transformation solution (OD) is different. 600 The concentration was 0.3, and the infection time was 7 minutes.

[0099] Comparative Example 9

[0100] The genetic transformation method in this comparative example is the same as that in Example 1, except that the washing condition is rinsing with sterile water 5 times.

[0101] Comparative Example 10

[0102] The genetic transformation method used in this comparative example is the same as that in Example 1, except that the concentration of the transformation solution (OD) is different. 600 The concentration was 0.7, the infection time was 7 min, and the co-culture time was 4 d.

[0103] Comparative Example 11

[0104] The genetic transformation method used in this comparative example is the same as that in Example 1, except that the Cef concentration in the solid culture medium for adventitious root induction culture is 300 mg / L.

[0105] Comparative Example 12

[0106] The genetic transformation method used in this comparative example is the same as that in Example 1, except that the concentration of the transformation solution (OD) is different. 600 The value was 0.7, and the co-culture time was 3 days.

[0107] Comparative Example 13

[0108] The genetic transformation method used in this comparative example is the same as that in Example 1, except that the concentration of Kan in the solid medium for adventitious root induction culture is 20 mg / L.

[0109] Comparative Example 14

[0110] The genetic transformation method used in this comparative example is the same as that in Example 1, except that the concentration of the transformation solution (OD) is different. 600 The value was 0.7, and the infection time was 15 minutes.

[0111] Comparative Example 15

[0112] The genetic transformation methods used in this comparative example are the same as those in Example 1, except that the concentration of Kan in the solid medium for adventitious root induction culture is 25 mg / L.

[0113] Comparative Example 16

[0114] The comparative example is the same as Example 1, except that the concentration of NAA in the adventitious root induction culture medium is 0.3 mg / L and no 6-BA is added.

[0115] Comparative Example 17

[0116] The genetic transformation method in this comparative example is the same as that in Example 1, except that the concentration of NAA in the solid culture medium for adventitious root induction culture is 0.3 mg / L.

[0117] Comparative Example 18

[0118] The genetic transformation method in this comparative example is the same as that in Example 1. The difference is that the basic medium in the solid medium for adventitious root induction culture is 1 / 2 MS and no 6-BA is added.

[0119] Comparative Example 19

[0120] The genetic transformation method used in this comparative example is the same as that in Example 1. The difference is that the basic medium in the solid medium for adventitious root induction culture is 1 / 2 MS.

[0121] Comparative Example 20

[0122] The genetic transformation method in this comparative example is the same as that in Example 1. The difference is that the basic medium in the solid medium for adventitious root induction culture is 1 / 2 MS, the concentration of NAA is 0.3 mg / L, and 6-BA is not added.

[0123] Comparative Example 21

[0124] The genetic transformation method in this comparative example is the same as that in Example 1. The difference is that the basic medium in the solid medium for adventitious root induction culture is 1 / 2 MS, and the concentration of NAA is 0.3 mg / L.

[0125] The effects of different genetic transformation treatments on the transformation efficiency of *Betula spp.* in Examples 1-4 and Comparative Examples 1-15 are shown in Table 1. The effects of different genetic transformation treatments on adventitious root induction in Examples 1 and 5 and Comparative Examples 16-21 are shown in Table 2.

[0126] The cleaning methods in Table 1 are as follows: A. Rinse with sterile water 3 times; B. Rinse with sterile water 5 times; C. Rinse with sterile water once, soak in 500mg / L Cef twice for 1 minute each time, and then rinse with sterile water twice more; D. Rinse with sterile water once, soak in 1000mg / L and 500mg / L Cef for 1 minute each, and then rinse with sterile water twice more; E. Rinse with sterile water once, soak in 1000mg / L Cef for 2 minutes, and then rinse with sterile water twice more.

[0127] Table 1. Effects of different treatments on adventitious root transformation of leaves from *Betula septemlobus*

[0128]

[0129]

[0130] Table 2 Effects of different culture media on adventitious root induction of *Betula septemlobus* leaves

[0131]

[0132]

[0133] Because tissue culture of *Betula spp.* started relatively late, and the plant itself has pubescence and a high content of phenolic substances, technical challenges such as difficult explant disinfection and easy browning occur during in vitro tissue culture, seriously affecting the successful construction of *Betula spp.* regeneration and genetic transformation systems. This embodiment uses *Agrobacterium tumefaciens* to transform asexually derived *Betula spp.* leaves. Infection is achieved through a transformation solution containing *Agrobacterium tumefaciens*, combined with a highly efficient washing method after co-culture and a high-rooting-rate culture medium. This allows for the direct generation of a large number of adventitious roots without callus induction, thus successfully achieving *Agrobacterium tumefaciens*-mediated genetic transformation of *Betula spp.*. This embodiment successfully constructs a highly efficient genetic transformation system for *Betula spp.* based on the efficient genetic transformation method of adventitious root formation from *Betula spp.* leaves. Rooting can be achieved directly without callus induction and differentiation, with a rooting rate of 100%, significantly shortening the transformation cycle. The efficient washing method enhances sterilization and reduces the difficulty of sterilization, thereby improving the transgenic efficiency of *Betula spp.*, achieving a genetic transformation rate of over 10%. It has advantages such as short cycle, high transformation rate, and simple operation.

[0134] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0135] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A genetic transformation method based on the adventitious root development of *Betula spp.* leaves, characterized in that, Includes the following steps: Using petiolate leaves of Betula stenoptera as transformation recipients, the transformation recipients were pre-cultured; after pre-culture, the transformation recipients were infected with a transformation solution containing Agrobacterium tumefaciens; the infected leaves were co-cultured; the co-cultured leaves were washed; and the washed leaves were subjected to adventitious root induction culture. The Agrobacterium tumefaciens contains an expression vector carrying an enhanced green fluorescent protein gene; The culture medium for inducing adventitious roots was WPM medium containing 4 mg / L~6 mg / L kanamycin, 190 mg / L~210 mg / L cefotaxime sodium, 15 g / L~25 g / L sucrose, 5 g / L~7 g / L agar, 0.05 mg / L~0.15 mg / L NAA, and 0.01 mg / L~0.03 mg / L 6-BA. The conditions for adventitious root induction culture include: (1) temperature of 23 ℃~25 ℃; (2) light intensity of 2000 lx~2500 lx, and light duration of 14 h~16 h per day; and (3) culture time of 15 d~30 d, and culture medium replacement frequency of once every 5 d~8 d.

2. The genetic transformation method according to claim 1, characterized in that, The strain of Agrobacterium tumefaciens is GV3101, and the expression vector is pSAK277::eGFP; the pre-culture medium is WPM medium containing 0.1 mg / L~0.3 mg / L NAA, 15 g / L~25 g / L sucrose, and 5 g / L~7 g / L agar.

3. The genetic transformation method according to claim 1, characterized in that, The conversion solution's OD 600 The value is 0.3~0.5, or / and, and the infection time is 10 min~15 min.

4. The genetic transformation method according to claim 1, characterized in that, The co-culture conditions include: (1) culture in the dark; (2) the culture medium for co-culture is MS medium; (3) the co-culture time is 2 d to 4 d; and (4) the co-culture temperature is 23 ℃ to 25 ℃.

5. The genetic transformation method according to claim 1, characterized in that, The cleaning method after co-cultivation is as follows: (1) Rinse once with sterile water; (2) Soak once with a cefotaxime sodium aqueous solution with a concentration of 500 mg / L to 1000 mg / L for 1 min; (3) Soak once again with a cefotaxime sodium aqueous solution with a concentration of 400 mg / L to 600 mg / L for 1 min; (4) Rinse twice with sterile water.

6. The genetic transformation method according to claim 1, characterized in that, The steps for preparing the conversion solution include: Agrobacterium tumefaciens containing an expression vector carrying an enhanced green fluorescent protein gene, stored at -80℃, was inoculated into YEP culture medium containing antibiotics for the first culture to obtain culture 1; culture 1 was taken and YEP culture medium without antibiotics was added for the second culture to obtain culture 2; culture 2 was centrifuged and enriched and then added to resuspension to prepare the transformation solution; The resuspension contains 8 mmol / L to 12 mmol / L MES, 8 mmol / L to 12 mmol / L MgCl2, and 90 μmol / L to 110 μmol / L acetosyringone.

7. The genetic transformation method according to claim 1, characterized in that, Following the adventitious root induction culture, the process also includes identifying the products obtained after the adventitious root induction culture; the identification method is fluorescence microscopy detection.

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