A method for inducing transgenic hairy roots from walnut stems

Abstract

The present invention discloses a method for inducing transgenic hairy roots in the stems of Carya serrata, belonging to the field of forest genetic engineering. The method utilizes Agrobacterium rhizogenes to infect semi-lignified or lignified stems of Carya serrata to obtain stable transgenic hairy roots. These roots can be induced to develop within one to two months, with a rooting rate as high as 94.43% and a transgene-positive rate of 98.38%. This method provides a cost-effective, tissue culture-independent transgenic method, and clearly demonstrates that transgenic roots can be used to verify gene function. This method overcomes the technical barriers to stable genetic transformation in Carya serrata, providing technical support for the research of functional genes.

Description

Technical Field

[0001] The invention relates to the field of forestry genetic engineering, in particular to a method for inducing walnut stems to produce transgenic hairy roots. Background Art

[0002] Currently, the main plant genetic transformation systems include Agrobacterium-mediated transformation, biolistic bombardment, pollen tube pathway, and protoplast transformation. Among them, biolistic bombardment has relatively low transformation efficiency and high equipment costs; pollen tube pathway transformation is seasonally restricted and has inconsistent efficiency; and protoplast transformation involves a complex protoplast culture and regeneration process, requiring high technical skills and adaptability to only some plant species. Agrobacterium-mediated transformation utilizes T-DNA from the Ti or Ri plasmids in Agrobacterium tumefaciens or Agrobacterium rhizogenes cells to integrate the target gene into the plant genome during infection. This method offers the advantages of high transformation efficiency, simplicity, and low cost. However, plant genetic transformation mediated by Agrobacterium rhizogenes typically uses sterile seedlings, leaves, or stem segments of sterile seedlings as explants, co-cultivated with bacterial solution to obtain transgenic roots, resulting in demanding experimental conditions. Transgenic plant production using Agrobacterium tumefaciens requires extensive callus induction, differentiation, and subculture, resulting in a long, cumbersome, and inefficient process.

[0003] Carya serrata is a commercial tree in the Juglandaceae family, rich in phenolic compounds. Its explants are prone to browning and are difficult to induce differentiation. Traditional tissue culture methods using stem segments as explants have reportedly been successful in inducing plant regeneration, but rooting rates are less than 50% and are limited to seedlings that are only two to three months old. Although regeneration systems using stem segments as explants have been established, no genetic transformation systems have been reported. This may be due to the cumbersome tissue culture procedures and the poor receptivity of non-embryonic callus to exogenous DNA. Because embryonic cells have a strong receptivity to exogenous DNA, reported genetic transformation systems for Carya serrata have mostly used somatic embryos as explants, but transformation rates remain below 60%, with some reports as low as 6.10%. Due to the cumbersome, time-consuming, and demanding experimental conditions of somatic embryo induction, the use of somatic embryos as transformation material remains limited. Currently, girdling and transverse incision methods have been used to induce hairy roots in the hypocotyl or stem of thin-shelled pecan seedlings. However, the induction rate remains low, at 56.5%, and the transgenic positive rate is only 45.20%. Typically, the hypocotyl or stem tends to differentiate into wound-healing cortical tissue. However, girdling and transverse incision create smaller wounds, making it difficult to induce root formation in the stem. Furthermore, the hypocotyl tissue is relatively tender, and the stem tissue above the incision tends to wither and die. This may be the main reason for the low induction rate of transgenic hairy roots in hypocotyl or stem tissue. Given the shortcomings of existing genetic transformation systems for thin-shelled pecan, such as low transformation rates, cumbersome tissue culture procedures, high costs, and low positive rates, it is crucial to develop an efficient and reliable genetic transformation system for thin-shelled pecan. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for inducing thin-shelled pecan stems to produce transgenic hairy roots, so as to solve the problems existing in the above-mentioned prior art. The method can simply and efficiently induce thin-shelled pecan stems to produce transgenic hairy roots.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for inducing slender-shelled pecan stems to produce transgenic hairy roots, comprising the following steps:

[0007] The binary vector plasmid containing the target gene is transferred into Agrobacterium rhizogenes to prepare recombinant Agrobacterium rhizogenes;

[0008] The stems of Carya sylvestris were infected with the infection solution of recombinant Agrobacterium rhizogenes and co-cultured, and then rooting culture was carried out.

[0009] Optionally, the target gene includes the CiGATA12a gene.

[0010] Optionally, the recombinant Agrobacterium rhizogenes infection solution is prepared by the following steps: a positive single colony of the recombinant Agrobacterium rhizogenes is cultured in LB medium containing antibiotics and acetosyringone until the OD 600 The concentration of the recombinant Agrobacterium rhizogenes was 0.5-0.7, and the cells were collected by centrifugation. The cells were resuspended with a resuspension solution to obtain the recombinant Agrobacterium rhizogenes infection solution.

[0011] Optionally, the concentration of antibiotics in the LB medium is 50 mM, and the concentration of acetosyringone is 15 μM; and / or the components of the resuspension are: an aqueous solution containing 10 mM MgCl 2 , 150 μM acetosyringone and 10 mM MES.

[0012] Optionally, the OD of the recombinant Agrobacterium rhizogenes infection solution is 600 It is 1.0-1.4, and the infection time is 24h.

[0013] Optionally, the infection and co-cultivation methods include the following steps:

[0014] Ring peel the bark of the thin-shelled 0.5-1 cm pecan stem and scrape off the cambium tissue remaining in the xylem as the infection site;

[0015] The infected part is impregnated with the impregnation liquid and sealed and wrapped, and then placed in a dark environment at 20-28° C. for co-culture for 24-48 hours, the sealed and wrapped materials are removed, and the infected part is covered with a culture medium.

[0016] A more specific option is to dip the infection liquid with a medium such as paper towels or cotton, wrap the infected area, and wrap the infection medium with plastic wrap to prevent the evaporation of the bacterial liquid; finally, place the stem in a dark environment at 20-28℃, culture for 24-48 hours, remove the plastic wrap and infection medium, and cover the infected area with the culture medium.

[0017] Optionally, the stem is a semi-lignified or lignified stem of the current year of the thin-shelled pecan seedling or variety seedling.

[0018] Optionally, the temperature of the rooting culture is 25-28°C.

[0019] Optionally, the Agrobacterium rhizogenes includes C58C1, K599 and MSU440.

[0020] The present invention discloses the following technical effects:

[0021] (1) High infection success rate: The percentage of plants that produce hairy roots after infection, i.e., the rooting rate, can reach as high as 94.43%, far higher than the 15.0-56.5% reported previously. In addition, among the hairy roots produced, the proportion of transgenic-positive roots is high, reaching as high as 98.38%.

[0022] (2) Short cycle: Depending on the age of the mother tree of the stem, the rooting time is between 1 and 2 months. Compared with traditional tissue culture transformation, the time to obtain transgenic tissue is greatly shortened.

[0023] (3) Simple operation: the materials used do not need to be sterile seedlings, and tissue culture is not required. It is only necessary to select seed seedlings or variety seedlings that are not separated from the current year's semi-lignified or lignified stems for operation; the ring-barking and bacterial liquid wrapping method is easy to operate and does not require high experimental equipment. The present invention provides an economical and efficient non-tissue culture-dependent transgenic method, and it is clear that the transgenic roots can fully exert the gene function.

[0024] (4) Can be used to improve the traits of cultivated varieties: Given the ability of the present invention to induce transgenic hairy roots in the seedling stems of varieties, specific genes can be introduced to improve the resistance, quality, yield and other traits of existing thin-shelled pecan varieties. Traditional methods of improving variety traits through hybridization are uncertain because parental traits will segregate in the offspring, often making it difficult to screen for offspring with excellent comprehensive traits, and the screening cycle can take decades. The transgenic method provided by the present invention can not only retain the original traits of the cultivated variety, but also improve existing undesirable traits, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is a flowchart of the infection method; (a) girdling; (b) wrapping the girdled area with a paper towel soaked in infection solution; (c) wrapping the paper towel with plastic wrap; (d) covering the girdled area with soil; scale: 5 mm.

[0027] Figure 2 Dynamic changes of the infected site; (a) callus formation one week after infection; (b) rooting two weeks after infection; (c) rooting three weeks after infection; (d) rooting four weeks after infection; (e) rooting five weeks after infection; (f) rooting six weeks after infection; scale bar: 5 mm;

[0028] Figure 3 Results of gene conversion verification: (a) GUS staining results; (b) electrophoresis diagram of gene conversion verification using intron-spanning primers;

[0029] Figure 4 The H2O2 and O2 levels in Carya serrata overexpressing CiGATA12a before and after treatment with methyl viologen (MV) 2- Content changes; (a) H2O2 content; (b) O 2- Content; Un-treatment refers to samples without MV treatment, MV treatment refers to samples treated with MV, different capital letters indicate that the content of the wild type is significantly different after MV treatment, different lowercase letters indicate that the content of 35S::CiGATA12a is significantly different after MV treatment, and asterisks indicate that the content of the wild type and 35S::CiGATA12a is significantly different. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The present invention provides a method for inducing transgenic hairy roots in the stems of Carya serrata, comprising the following steps: 1. preparing intact stems; 2. constructing a binary vector plasmid containing the target gene and introducing it into Agrobacterium rhizogenes; 3. preparing an infection solution; 4. infection, transformation, and co-cultivation; 5. rooting culture and detection of positive hairy roots. This method is further described below with reference to specific examples.

[0036] Example 1

[0037] In this example, the stems of Carya serrata were used to induce hairy roots that overexpress CiGATA12a and verify its function. The specific steps are as follows:

[0038] 1. Prepare semi-lignified stems

[0039] Soak fresh pecan seeds for 5-6 days, changing the water daily. Then, bury the seeds in perlite and place them in an incubator at 24-35°C for germination. Water them regularly to maintain humidity. Once germination is complete, the seeds have cracked, and roots and buds have developed, transplant them into a container containing a sterilized substrate (a mixture of garden soil, perlite, and vermiculite in a 3:1:1 ratio by volume) and water thoroughly. Cultivate at 25-28°C under light conditions until the stem base becomes semi-lignified.

[0040] 2. Prepare 35S::CiGATA12a vector to transform Agrobacterium rhizogenes

[0041] RNA was extracted from leaves of Carya 'Boni' and, after passing quality control, first-strand cDNA synthesis was performed. Primers for the CDS amplification of the CiGATA12a gene were designed based on the reference genome: the upstream primer is: 5'-ATGGAGGCACCTGAGTTTT-3' (SEQ ID No. 1); the downstream primer is: 5'-GATCAGCTGCCTGAAATC-3' (SEQ ID No. 2). PCR amplification was performed using the synthesized cDNA as a template, resulting in a product size of 1050 bp, the sequence of which is shown in SEQ ID No. 3. The CDS of CiGATA12a was ligated into the pCAMBIA2301 vector using homologous recombination, and Escherichia coli was transformed. Positive single colonies were selected, cultured, and plasmids were extracted. Heat-stimulated plasmids were then introduced into Agrobacterium rhizogenes C58C1.

[0042] The sequence shown in SEQ ID No.3 is:

[0043]

[0044] 3. Prepare infection solution

[0045] Pick a single colony of 35S::CiGATA12a Agrobacterium rhizogenes and place it in 1 mL of LB liquid medium containing 50 mM kanamycin (Kan) and shake it at 28°C and 200 rpm for 24 h. Transfer 1 mL of the Agrobacterium culture to 20 mL of LB medium containing 50 mM Kan antibiotic and 15 μM acetosyringone (AS) and shake it at 28°C and 200 rpm until the OD 600 The cells were then centrifuged at 6000 rpm for 10 min to collect the cells, and the Agrobacterium cells were resuspended in a resuspension solution (containing 10 mM MgCl2, 150 μM AS and 10 mM MES) to an OD of 600 = 1. Let it stand at room temperature for 2-3 hours (2 hours was selected in this experiment) and set aside.

[0046] 4. Infection, transformation and co-cultivation

[0047] Use a blade to ring the semi-lignified stems of the thin-shelled pecan seedlings with a ring width of 0.5-1 cm (0.5 cm was used in this experiment), and scrape off the remaining cambium in the xylem ( Figure 1 (a)); Use a paper towel to dip the bacterial solution and wrap the ring-barking part ( Figure 1 (b)), and then wrap with plastic wrap to prevent the bacterial solution from evaporating ( Figure 1 After culturing in the dark for 24 h, the plastic wrap and paper towel were removed and the infected area was covered with a substrate (peat: perlite: vermiculite = 3:1:1 (volume ratio)). Figure 1 (d)).

[0048] 5. Rooting culture and detection of positive hairy roots

[0049] Seedlings were cultured at 25-28℃ and callus appeared one week after infection ( Figure 2 Middle (a)), root tissue appeared two weeks after infection ( Figure 2 Middle (b)), more hairy roots differentiated three weeks after infection ( Figure 2 Middle (c)), four weeks after infection, the roots elongated rapidly ( Figure 2 Middle (d)), capillary roots appeared five weeks after infection ( Figure 2 Middle (e)), six weeks after infection, the base of some roots began to lignify ( Figure 2 Middle (f). GUS staining was used to detect positive roots, and the results showed that all hairy roots were stained, while the wild type WT without vector transformation did not show blue ( Figure 3(a)). Intron amplification primers were designed for positive detection. The upstream primer was 5'-ACCTTTGTGAGCCGTATGACG-3' (SEQ ID No. 4); the downstream primer was 5'-CTAGATCAGCTGCCTGAAATCG-3' (SEQ ID No. 5). The PCR amplification results showed that the brightness of the transgenic band was much higher than that of the WT ( Figure 3 Middle (b)) further proved that the hairy roots were positive.

[0050] 6. Identification of CiGATA12a function using hairy roots

[0051] Transgenic hairy roots were soaked in 5 mM methyl viologen (MV) and the H2O2 and O 2- The results showed that MV treatment caused the H2O2 and O 2- Compared with the control, the H2O2 and O 2- The contents were significantly lower than those in the wild type ( Figure 4 The results indicate that transgenic hairy roots can be used to identify gene functions and that CiGATA12a can scavenge reactive oxygen species induced by stress.

[0052] Example 2 Effects of different factors on the induction of hairy roots

[0053] As shown in Table 1, different factors were evaluated, including the Agrobacterium rhizogenes strains (K599, C58C1, MSU440), the concentration of the infection solution (OD 600 =0.6, 1.0, 1.4) and infection time (12h, 24h, 36h) on the rooting rate and positive rate of stem induction. A three-factor three-level orthogonal design L9 (3 3 ) were tested (Table 2). Agrobacterium was transformed with the pCAMBIA2301 vector carrying 35S::GUS. After preparing the infection solution, the semi-lignified stems of the seedlings (about 2 months old) were ring-barked and infected. After 1.5 months of culture, the rooting rate and positive rate were statistically analyzed. The results showed that the rooting rate of different factors and levels was 85.01-94.43%, and the positive rate was 94.37-98.38%. The order of factors affecting the induction of transgenic hairy roots was bacterial solution concentration > bacterial type > infection time, and the best combination was C58C1, OD 600 =1.0, infection time 24h, under this condition, the rooting rate of plants was 94.43%, and the positive roots of all rooting plants were 98.38% (Table 2).

[0054] Table 1 Three factors and three levels

[0055]

[0056] Table 2 Three-factor three-level orthogonal experimental design and results

[0057]

[0058] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for inducing transgenic hairy roots in walnut stems, characterized in that: The following steps are involved: The binary vector plasmid containing the target gene is transferred into Agrobacterium rhizogenes to prepare recombinant Agrobacterium rhizogenes; The stems of Carya sylvestris were infected with the infection solution of recombinant Agrobacterium rhizogenes and co-cultured, and then rooted. The target gene is CiGATA12a gene, and its nucleotide sequence is as shown in SEQ ID NO.3; The OD of the recombinant Agrobacterium rhizogenes infection solution 600 is 1.0, the infection time is 24h; The method of infection and co-cultivation comprises the following steps: Ring peel the bark of the thin-shelled 0.5-1 cm thick pecan stem and scrape off the remaining cambium tissue in the xylem as the infection site; Impregnate the infected site with the impregnation solution and seal the infected site, then place it in a dark environment at 20-28°C for 24-48 hours, remove the sealing material, and cover the infected site with a culture medium; The stem is a semi-lignified stem of the current year of the thin-shelled pecan seedling or variety seedling; The Agrobacterium rhizogenes is C58C1; The recombinant Agrobacterium rhizogenes infection solution was prepared by the following steps: a positive single colony of the recombinant Agrobacterium rhizogenes was cultured in LB medium containing antibiotics and acetosyringone until the OD 600 The concentration of the recombinant Agrobacterium rhizogenes was 0.5-0.7, and the cells were collected by centrifugation. The cells were resuspended with a resuspension solution to obtain the recombinant Agrobacterium rhizogenes infection solution.

2. The method according to claim 1, wherein The concentration of antibiotics in the LB medium is 50 mM, and the concentration of acetosyringone is 15 μM; and / or the components of the resuspension are: an aqueous solution containing 10 mM MgCl 2 , 150 μM acetosyringone and 10 mM MES.

3. The method according to claim 1, wherein The temperature of the rooting culture is 25-28°C.

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