A method for inducing hairy roots in a wye-shaped aseptic system

Abstract

The application provides a hairy root induction method of Prunus mume sterile system, and constructs a Prunus mume hairy root genetic transformation system based on a RUBY reporter gene as a screening marker. The application screens conditions such as explant type, growth environment, infection method and infection concentration which affect the induction efficiency of Prunus mume hairy roots, successfully induces the growth of Prunus mume hairy roots and obtains red hairy roots started by the RUBY reporter gene, and provides technical support for the introduction of exogenous genes and the exploration of the function of genes in Prunus mume body by using genetic engineering technology by using RUBY as a screening marker.

Description

Technical Field

[0001] This invention belongs to the field of plant culture technology, specifically, it relates to a method for inducing hairy roots in a sterile plum blossom system. Background Technology

[0002] Plum blossom (Prunus mume), a deciduous tree belonging to the genus Prunus in the family Rosaceae, is the first of my country's ten traditional famous flowers. With a cultivation history of over 3000 years, it is an important early spring woody ornamental flower with extremely high ornamental, economic, and cultural symbolic value. To expand the planting range of plum blossoms in my country and promote their international application, it is of great significance to promote variety selection and the improvement of superior traits. Conventional breeding methods, such as bud mutation breeding and introduction and domestication, have certain limitations: long cycles, high consumption of human and material resources, susceptibility to natural conditions, and low predictability of target traits. Plant genetic engineering technology can overcome the limitations of traditional breeding, utilizing exogenous genes for targeted improvement, achieving targeted breeding of plum blossoms, and innovating plum blossom germplasm resources. However, current research mainly focuses on establishing efficient and stable regeneration systems for plum blossoms, and the construction of a plum blossom intrinsic genetic transformation system has not yet been reported. Transgenic plum root systems can be rapidly obtained through Agrobacterium rhizogenes-mediated hairy root transformation technology, which can be used for research on plum root development, secondary metabolism, and stress resistance mechanisms.

[0003] In plant genetic engineering, Agrobacterium-mediated transformation is a mature and widely used method for introducing exogenous genes. Although *Agrobacterium tumefaciens* is the preferred engineered strain for genetic engineering, *Agrobacterium rhizogenes*-mediated transformation of plant hairy roots also has many advantages. For example, differentiated hairy roots grow rapidly, originate from the same cell, are easy to genetically transform, have a high differentiation rate, and are stable in ploidy. *Agrobacterium rhizogenes* is a Gram-negative bacterium belonging to the genus *Agrobacterium* of the family Rhizobiumceae. It has broad infectivity. After infecting the host, it stably integrates the *rol* gene into the host plant genome through the T-DNA of the Ri plasmid, inducing the plant to produce a large number of adventitious roots, forming chimeric plants with transgenic hairy roots and non-transgenic aboveground parts. At the same time, it can also integrate exogenous genes carried in the Ti plasmid into the plant genome, making it a good material for studying plant-specific gene expression and elucidating the function of regulatory genes.

[0004] T-DNA integration is known to be random, thus requiring efficient screening methods to identify positive hairy roots. RUBY, a visually perceptible reporter gene system, has been successfully applied to positive screening in rice and Arabidopsis thaliana. This system couples three betaine biosynthesis genes (CYP76AD1, DODA, and GT) to an open reading frame, forming the RUBY reporter system, which converts tyrosine into red betaine. This reporter gene system can efficiently screen positive transformed tissues and organs with the naked eye, thus possessing great application potential in the genetic transformation of woody plants, enabling morphological marker screening in positive hairy roots of plum blossoms.

[0005] Because woody plants are perennial and highly heterozygous, constructing a stable genetic transformation system based on Agrobacterium tumefaciens is difficult. However, Agrobacterium tumefaciens-mediated transformation of plant hairy roots has significant application value. Currently, there are no reports on using Agrobacterium tumefaciens to induce the culture of plum blossom hairy roots using tissue culture techniques to obtain tissue chimeras. Summary of the Invention

[0006] The purpose of this invention is to provide a method for inducing hairy roots in a sterile plum blossom system based on the RUBY reporter gene as a screening marker, in order to establish a plum blossom transformation system mediated by Agrobacterium rhizogenes, and lay the foundation for future identification of specific gene functions in plum blossoms and genetic improvement of plum blossoms.

[0007] To achieve the objective of this invention, in a first aspect, this invention provides a method for inducing hairy roots in plum blossoms using a sterile system, which involves infecting plum blossom explants with Agrobacterium rhizogenes K599 containing the RUBY reporter gene to induce the explants to generate hairy roots.

[0008] The explants are leaves, stem segments, and petioles of sterile tissue culture seedlings and substrate seedlings.

[0009] Furthermore, the Agrobacterium rhizogenes K599 containing the RUBY reporter gene is obtained by transforming Agrobacterium rhizogenes K599 competent cells with a plasmid containing the RUBY reporter gene, and the RUBY reporter gene is preferably driven by a 35S promoter.

[0010] Furthermore, the methods for preparing aseptic tissue culture seedlings include:

[0011] (1) Remove the pulp from the plum fruit, wash and dry the seeds, soak them in sterile water, and then soak the kernels obtained by mechanically removing the pits in sterile water overnight. Select the dried and plump kernels after soaking, rinse them with running water for 30 minutes, and then transfer them to a sterile tissue culture table. First, soak them in 2% NaClO for 15 minutes, then disinfect them with 75% alcohol for 30 seconds, and finally rinse them with sterile water 3 to 5 times. Peel out the plum seed embryo with 1 / 4 of the cotyledon using a sterile scalpel, place the embryo with the plum bud facing down in the embryo induction medium, and culture it in an artificial climate chamber at 24℃ with 16 hours of light and 8 hours of darkness until it germinates to obtain sterile tissue culture seedlings of plum.

[0012] (2) Remove the primary roots of the tissue culture seedlings and retain the upper part of the roots as explant material, or take the stem and root segments of the tissue culture seedlings as explant material; place the explants in MS pre-culture medium and pre-culture them in the dark at 24℃ for 2-3 days before using them for infection.

[0013] The embryo induction medium consisted of 4.43 g / L QL medium powder + 30 g / L sucrose + 0.5 mg / L 6-BA + 1.0 mg / L TDZ + 7 g / L agar, pH 5.8-6.0. The QL (Quoirin & Lepoivre) medium powder, suitable for tissue regeneration in Prunus plants, was purchased from Beijing Coollife Technology Co., Ltd.; TDZ was a phenylthiazolyl urea hormone.

[0014] The MS pre-medium consisted of 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar, pH 5.8-6.0.

[0015] Furthermore, the methods for preparing substrate seedling leaves, stem segments, and petioles include:

[0016] (1) Remove the pulp from the plum fruit, wash and dry the seeds, and store them in a 4℃ cold storage in sand. Regularly check and spray water to prevent the seeds from getting moldy until they show white. Sow the white seeds in substrate trays, and plum seedlings will be obtained after 20-30 days.

[0017] (2) Take leaves with petioles and young stem segments from the substrate seedlings as explant materials; after sterilization and drying, the explant materials are pre-cultured in MS pre-medium at 24℃ in the dark for 2-3 days before being used for infection.

[0018] The matrix is ​​composed of peat moss and perlite mixed in a 2:1 volume ratio;

[0019] The MS pre-medium consisted of 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar, pH 5.8-6.0.

[0020] The aforementioned method, in step (2), includes the following steps for disinfecting the explant material: disinfecting with 75% alcohol for 30 seconds, then disinfecting with 5% NaClO for 15 minutes, and finally rinsing with sterile water 6 to 8 times.

[0021] Furthermore, Agrobacterium rhizogenes K599 containing the RUBY reporter gene was cultured in LB selective medium, and the bacterial cells were collected by centrifugation. The bacterial cells were then resuspended in MS resuspending buffer to OD. 600 =0.6~1.0 (preferably 0.6), used as an infection solution for infecting plum blossom explants;

[0022] The LB selective medium is composed of 10 g / L tryptone + 5 g / L yeast extract + 10 g / L sodium chloride + 15 g / L agar, with spectinomycin at 75 mg / L and streptomycin at 50 mg / L.

[0023] The MS resuspension was prepared as follows: 4.43 g / L MS medium powder + 30 g / L sucrose + 200 μM AS, pH 5.8-6.0.

[0024] Furthermore, the plum blossom explants were soaked in the infection solution for 10 minutes, blotted dry with sterile filter paper, and then co-cultured in MS co-culture medium at 24°C in the dark for 2–3 days.

[0025] The MS co-culture medium is: 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar + 200 μMAS, pH 5.8-6.0.

[0026] Further, the co-cultured explants were transferred to MS rooting medium and cultured at 24°C under light for 20–30 days, with subculture every 7 days until callus tissue and hairy roots differentiated at the explant wound site. After PCR detection and positive identification, the main root was removed, leaving only the hairy roots, and then the seeds were sown in seedling trays for substrate culture.

[0027] The MS rooting medium consists of: 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar + 0.05 mg / L NAA + 0.5 mg / L 6-BA; the MS rooting medium contains 7.5 mg / L spectinomycin and 200 mg / L cephalosporin, with a pH of 5.8-6.0.

[0028] Preferably, the primers used for PCR detection are:

[0029] 35S:RUBY-F:5'-CGACACTCTCGTCTACTCCAA-3';

[0030] 35S:RUBY-R:5'-GCGGTAATCTTGCGGAAGTT-3'.

[0031] In this invention, the preferred plum variety is the 'Early Green Calyx' of the True Plum series.

[0032] Secondly, the present invention provides the application of plum blossom hairy roots prepared according to the method in plum blossom gene function research and plum blossom genetic improvement breeding.

[0033] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0034] (I) This invention is the first to induce the formation of hairy roots in plum blossoms using Agrobacterium rhizogenes K599 under tissue culture conditions. In existing technologies, the tissue culture system for Prunus species is immature, and plum callus culture is difficult and time-consuming. Currently, there are no reports on Agrobacterium rhizogenes genetic transformation systems for plum blossoms, which is a pressing issue that needs to be addressed. Hairy roots are special plant mutant tissues produced after Agrobacterium rhizogenes infects plant cells, possessing advantages such as rapid growth, hormone autotrophy, stable transformation, and strong secondary metabolism. As a transgenic organ, the functional verification of subsequent exogenous gene introduction can be explored through the induction of plum blossom hairy roots.

[0035] (II) This invention, through Agrobacterium rhizogenes-mediated transformation, screened the explant types, growth environments, infection methods, and infection concentrations for transforming plum blossom hairy roots, ultimately determining the optimal method using OD... 600 A bacterial solution with a concentration of 0.6% was used to immerse sterile tissue culture seedlings, stem segments of substrate seedlings, and petioles, resulting in the highest efficiency in inducing hairy roots. The establishment of this system has accelerated the research process of plum blossom molecular biology and also provides materials for subsequent use of hairy roots to induce complete transformed plants.

[0036] (III) This invention uses the RUBY gene carrying betalain biosynthesis as a reporter system to obtain naked-eye visible red hairy roots and red callus tissue of plum blossoms, thereby determining whether the transformation is successful. It can be used as a non-destructive screening system in the process of plant genetic transformation. Attached Figure Description

[0037] Figure 1 The plum seedlings in the preferred embodiment of the present invention; wherein (A) are aseptic tissue culture seedlings of plum blossoms; and (B) are 20-30 day old plum blossom substrate seedlings.

[0038] Figure 2 This is a pre-culture of plum blossom explants in a preferred embodiment of the present invention; wherein (A) is a sterile tissue culture seedling with primary roots removed as an explant; (B) is a stem segment as an explant; (C) is a leaf as an explant; and (D) is a petiole as an explant.

[0039] Figure 3Different methods for infecting explants with Agrobacterium rhizogenes in a preferred embodiment of the present invention are described; wherein (A) is the immersion method; (B) is the dipping method; and (C) is the injection method.

[0040] Figure 4 The image shows the growth of callus induced by explants in a preferred embodiment of the present invention; where (A) and (B) are callus formation from aseptic tissue culture seedlings; (C) is callus formation from stem segments; (D) and (E) are callus formation from leaves; and (F) is callus formation from petioles.

[0041] Figure 5 The growth status of differentiated hairy roots is shown in the preferred embodiment of the present invention; wherein (A) and (B) are hairy roots induced by aseptic tissue culture seedlings of plum blossom; (C) and (D) are hairy roots induced by stem segments; (E) are hairy roots induced by leaves; (F) and (G) are hairy roots induced by petioles;

[0042] Figure 6 In a preferred embodiment of the present invention, PCR verification of plum blossom hairy root DNA induced by K599 Agrobacterium rhizogenes carrying the RUBY reporter gene was performed; wherein (A) is the verification using the RUBY reporter gene system-specific primer 35S:RUBY-F / R; wherein M is the marker, 1 is a wild-type root as a negative control, 2 is K599 Agrobacterium transformed with the 35S:RUBY plasmid as a positive control, 3-6 are red hairy roots, and 7-10 are white hairy roots; B) Verification using beet pigment synthesis gene-specific primers CYP76AD1-F1 / R1, DODA-F1 / R1, and GT-F1 / R1; where M is the marker, 1 is a wild-type root as a negative control, 2, 7, and 12 are K599 Agrobacterium transformed with the 35S:RUBY plasmid as positive controls, and 3-6, 8-11, and 13-16 are amplification bands of the CYP76AD1, GT, and DODA genes using hairy roots as templates, respectively.

[0043] Figure 7 The following are the effects of different conditions on the induction of hairy roots in plum blossoms in a preferred embodiment of the present invention; wherein (A) is the effect of infection method on the induction of hairy roots in explants from aseptic tissue culture seedlings; (B) is the effect of growth environment on the induction of hairy roots in stem segment explants; (C) and (D) are the effects of explant type on hairy root induction under aseptic tissue culture and substrate environments, respectively; (E) is the effect of infection concentration on the induction of hairy roots in stem segment explants; different lowercase letters indicate significant differences;

[0044] Figure 8 The substrate culture for inducing hairy root seedlings is shown in the preferred embodiment of the present invention; wherein (A) represents the growth status of the seedlings; and (B) represents the growth status of the hairy roots in the seedling substrate. Detailed Implementation

[0045] This invention provides a method for inducing hairy roots in a sterile system for plum blossoms. Under tissue culture conditions, K599 Agrobacterium containing the RUBY reporter gene is used to infect plum blossom explants, inducing the growth of hairy roots in the explants.

[0046] The present invention adopts the following technical solution:

[0047] This invention provides a method for inducing hairy roots in a sterile plum blossom system, comprising the following steps:

[0048] (1) Cultivating aseptic tissue culture seedlings of plum blossoms;

[0049] The pulp of the collected plum cultivar 'Zao Lü E' was removed, and the seeds were washed and dried. The seeds were then soaked in sterile water. The kernels were then removed by mechanical treatment and soaked in sterile water overnight. After disinfection, the kernels were placed in embryo induction medium and cultured in an artificial climate chamber at 24°C with 16 hours of light and 8 hours of darkness until embryo induction of sterile tissue culture seedlings of plum blossoms was achieved.

[0050] (2) Cultivating plum blossom substrate seedlings;

[0051] The seeds obtained in (1) were placed in a 4℃ cold storage sand storage, and were regularly turned over and sprayed with water to prevent the seeds from getting moldy until they turned white. The white seeds were then sown in substrate (peat moss: perlite = 2:1, volume ratio) seedlings and cultured for 20-30 days to obtain plum blossom substrate seedlings.

[0052] (3) Pre-culture of explants;

[0053] This invention uses different growth parts of plum blossom under two different growth environments as explant materials: sterile tissue culture seedlings, stem segments, root segments, and substrate seedling stem segments, leaves, and petioles. The study investigates the effects of different growth environments and explant types on the induction of plum blossom hairy roots.

[0054] (4) Preparation of Agrobacterium rhizogenes infection solution, specifically including:

[0055] a. Preparation of Agrobacterium rhizogenes: The preserved Agrobacterium rhizogenes transformed with the 35S:RUBY plasmid was streaked on LB selective medium and cultured at 28°C in the dark for 2-3 days until the strain recovered.

[0056] b. Preparation of infecting bacterial culture: Pick a single colony revived from step a and incubate it in LB liquid selective medium for 12-16 hours until OD500 reaches zero. 600 =1.0, centrifuge at 5000 rpm for 10 min to collect the bacterial pellet, and resuspend the bacterial cells in MS resuspending buffer to OD. 600 =0.6, 0.8, 1.0, incubate in the dark for 2-3 hours, and resuspend the bacterial solution as the infection solution of Agrobacterium rhizogenes for infecting explants;

[0057] (5) Infection of plum blossom hairy roots:

[0058] a. Immersion method: The explants pre-cultured in (3) were immersed in the Agrobacterium rhizogenes infection solution in (4) for 10 min, dried with sterile filter paper, placed in MS co-culture medium, and cultured in the dark at 24℃ for 2-3 days;

[0059] b. Dipping method: Incubate the revived bacterial culture from (4) to OD200. 600 =2.0, after collecting the bacteria, spread them onto LB selective medium and culture until the bacterial solution on the plate is pasty; take the tissue culture seedlings whose primary roots have been removed in (3) and scrape the bacterial solution on the plate 2-3 times with the cut surface so that the pasty Agrobacterium rhizogenes wraps the cut surface of the hypocotyl of the seedling, place it in MS co-medium, and culture in the dark at 24℃ for 2-3 days;

[0060] c. Injection method: Using a 1ml syringe, inject 0.1ml of Agrobacterium rhizogenes infection solution into the tissue culture seedlings at a position 1cm away from the root hypocotyl. Use sterile filter paper to absorb the surface bacterial solution, place in MS co-culture medium, and incubate in the dark at 24℃ for 2-3 days.

[0061] (6) Induction of hairy roots of plum blossom: The explants after co-culture in (5) were transferred to MS rooting medium and cultured at 24℃ for 20-30 days. Subculture was carried out every 7 days until callus tissue was generated at the wound site of the explant and hairy roots were differentiated. The red hairy roots visible to the naked eye were screened, and samples were taken and PCR was performed.

[0062] (7) Substrate culture of plum blossom hairy roots:

[0063] After removing the main root and keeping only the hairy roots, the tissue culture seedlings that were successfully identified as having hairy roots in (6) were planted in a seedling tray for substrate culture.

[0064] Furthermore, (3) the specific methods for explant pre-culture include:

[0065] a. Preparation of explants: Take the primary roots of the sterile tissue culture seedlings of plum blossom in (1) and remove them, leaving the upper part of the roots; take the stem and root segments of the tissue culture seedlings in (1); take the leaves with petioles and the young stem segments of the substrate seedlings in (2), disinfect them in the tissue culture table, and cut wounds with a sterile blade; the above plant tissues are used as explants for Agrobacterium rhizogenes-mediated transformation;

[0066] b. Pre-culture of explants: The explants obtained in step a were placed in MS pre-culture medium and pre-cultured at 24°C in the dark for 2–3 days.

[0067] In some embodiments of the present invention, (1) the method for cultivating plum blossom embryo-induced aseptic tissue culture seedlings is as follows:

[0068] Select plump, dried kernels after soaking, rinse with running water for 30 minutes, and in a tissue culture environment, first soak them in 2% NaClO for 15 minutes, then disinfect them with 75% alcohol for 30 seconds, and finally rinse them with sterile water 3 to 5 times. Use a sterile scalpel to peel out the plum seed embryo along with 1 / 4 of the cotyledon, place the embryo downwards in the embryo induction medium and wait for it to germinate to obtain sterile plum tissue culture seedlings.

[0069] Preferably, the plum blossom embryo induction medium is: 4.43 g / L QL medium powder + 30 g / L sucrose + 0.5 mg / L 6-BA + 1.0 mg / L TDZ + 7 g / L agar, pH 5.8-6.0.

[0070] In some embodiments of the present invention, (3) the explants of the sterile tissue culture seedlings are robust sterile seedlings that have grown in tissue culture bottles for 20 to 30 days, and after the primary roots are removed, i.e. the hypocotyl is removed, they are placed in a pre-culture medium for culture.

[0071] Preferably, the stem internodes are selected, the axillary buds at both ends are removed, and the stem and root explants are cut into 1.0-1.5 cm segments.

[0072] Preferably, the plum blossom leaves and petioles are the 3rd to 5th fully unfolded tender green leaves with petioles from the top of the seedling in the plug tray substrate.

[0073] Preferably, the tender stems of the plum blossom substrate seedlings are cut into 1.0-1.5cm stem segments.

[0074] Preferably, the specific method for disinfecting the explants, including the stem segments, leaves, and petioles of the substrate seedlings, is as follows:

[0075] Inside the tissue culture station, disinfect with 75% alcohol for 30 seconds, then disinfect with 5% NaClO for 15 minutes, and finally rinse with sterile water 6-8 times; the MS pre-medium is: 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar, pH 5.8-6.0.

[0076] In some embodiments of the present invention, the 35S:RUBY plasmid described in (4)a carries the naked-eye visible red protein reporter gene RUBY, and is transformed into Agrobacterium rhizogenes K599 competent cells using the freeze-thaw method, and positive selection is performed on LB solid selective medium containing antibiotics.

[0077] Preferably, the LB selective medium contains 75 mg / L spectinomycin and 50 mg / L streptomycin.

[0078] Preferably, single colonies are selected for PCR verification using the following primers: 35S:RUBY-F:5'-CGACACTCTCGTCTACTCCAA-3'; 35S:RUBY-R:5'-GCGGTAATCTTGCGGAAGTT-3'.

[0079] In some embodiments of the present invention, the MS resuspension in (4)b is: 4.43 g / L MS culture medium powder + 30 g / L sucrose + 200 μM AS, pH 5.8-6.0.

[0080] In some embodiments of the present invention, (5)a describes the slow infection of explants for 10 minutes at 28°C and 60 rpm in a shaker.

[0081] Preferably, the MS co-culture medium is: 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar + 200 μMAS, pH 5.8-6.0.

[0082] In some embodiments of the present invention, (5)b describes taking 2ml of post-resuscitation OD 600 =2.0 bacterial suspension, centrifuged at 6000 rpm for 1 min, discarded 1.8 ml of supernatant, and the remaining 0.2 ml of bacterial suspension was pipetted and mixed well, then spread onto LB selective medium and incubated at 28℃ for 24 h until the bacterial suspension on the plate became a paste.

[0083] Preferably, the explant is obliquely cut to remove the primary roots, scraped and dipped in a plate of bacterial paste, wiped clean with sterile filter paper, and then placed in LB co-culture medium for culture.

[0084] In some embodiments of the present invention, (6) the MS rooting medium is: 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar, 0.05 mg / L NAA, 0.5 mg / L 6-BA, pH 5.8-6.0, spectinomycin content of 7.5 mg / L, and cephalosporin content of 200 mg / L.

[0085] Preferably, DNA is extracted from the screened hairy roots and subjected to PCR detection to identify genes including the RUBY reporter gene, CYP76AD1, DODA, and GT (GenBank accession No.: OL411875.1).

[0086] In some embodiments of the present invention, (7) the tissue culture seedlings with successfully identified hairy roots are cut off from the main root and only the hairy roots are retained and planted in a substrate (peat moss: perlite = 2:1, volume ratio) for cultivation.

[0087] This invention also provides a method for screening positive hairy roots based on the RUBY reporter gene, which allows for naked-eye visual screening of induced transformed hairy roots.

[0088] The Agrobacterium rhizogenes used in this invention is K599 Agrobacterium rhizogenes carrying the RUBY reporter gene, or it can be Agrobacterium rhizogenes transformed with the target gene. Agrobacterium rhizogenes carrying the RUBY reporter gene can achieve morphological marker screening of positively transformed roots and efficiently explore the plum blossom hairy root induction system, while the hairy roots induced by Agrobacterium rhizogenes carrying the target gene can be used for functional studies of specific genes or promoters and other components.

[0089] 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.

[0090] The sterile plum seedlings and substrate seedlings in the following examples were cultivated in the Advanced Innovation Center for Molecular Design Breeding of Forest Trees, Beijing Forestry University; Agrobacterium rhizogenes strain K599 was purchased from Shanghai Weidi Biotechnology Co., Ltd.; plasmid 35S:RUBY was purchased from Miaoling Plasmid Platform; and the plant DNA extraction kit was purchased from Tiangen Biotech Co., Ltd.

[0091] The following examples provide a method for inducing hairy roots in a sterile plum blossom system. The method for establishing this induction and transformation system includes the following steps: (1) Using sterile tissue culture seedlings, sterile seedling stem segments, root segments, and substrate seedling leaves, stem segments, and petioles as experimental materials, pre-culture is carried out to obtain plum blossom explants; (2) Using Agrobacterium rhizogenes K599 containing the RUBY reporter gene, plum blossom explants are infected and co-cultured by immersion, dipping, and injection methods to obtain hairy roots with the red protein reporter gene RUBY visible to the naked eye under natural light; (3) The hairy roots growing on the plum blossom explants are cut off, positive identification is performed, and then the main root is cut off for substrate culture.

[0092] Example 1: Obtaining plum seedlings and pre-culturing explants

[0093] 1. Experimental Methods

[0094] 1.1 Obtaining plum seedlings

[0095] (1) Obtaining sterile tissue culture seedlings of plum blossoms: The pulp of the collected true plum variety 'Zao Lü'e' was removed, and the seeds were washed and dried. The seeds were then soaked in sterile water. After mechanical processing, the kernels were soaked in sterile water overnight. After sterilization under sterile conditions, the kernels were placed in embryo induction medium and cultured in an artificial climate chamber at 24℃ with 16h light / 8h darkness until embryo induction of sterile tissue culture seedlings was achieved. Figure 1 A);

[0096] The embryo induction medium was: 4.43 g / L QL medium powder + 30 g / L sucrose + 0.5 mg / L 6-BA + 1.0 mg / L LTDZ + 7 g / L agar, pH 5.8-6.0;

[0097] The disinfection method is as follows: Select plump kernels after soaking, rinse with running water for 30 minutes, soak in 2% NaClO for 15 minutes in a sterile environment, then disinfect with 75% alcohol for 30 seconds, and finally rinse with sterile water 3 to 5 times. Peel out the embryo with 1 / 4 of the cotyledon using a sterile scalpel, and place the embryo downwards on the embryo induction medium for growth.

[0098] (2) Obtaining plum blossom substrate seedlings: The seeds obtained in (1) were placed in a 4℃ cold storage sand storage, and were regularly turned over and sprayed with water to prevent the seeds from getting moldy, until they showed white. The white seeds were then sown in substrate (peat moss: perlite = 2:1, volume ratio) seedling trays. After 20-30 days, plum blossom substrate seedlings were obtained. Figure 1 B);

[0099] 1.2 Culture of explants

[0100] (1) Preparation of explants: Take healthy aseptic tissue culture seedlings from 1.1 that are 20-30 days old, remove the primary roots, and retain the upper part of the roots; take the internodes of the stems of tissue culture seedlings from 1.1, remove the axillary buds at both ends, and cut them into stem segments of 1.0-1.5 cm; take the roots of tissue culture seedlings from 1.1 and cut them into root segments of 1.0-1.5 cm; take the 3rd to 5th fully unfolded tender green leaves with petioles from the top of the substrate seedlings from 1.1 and cut them into 1 cm × 1 cm segments and separate the petioles; take the tender upper part of the stems of the substrate seedlings from 1.1 and cut them into stem segments of 1.0-1.5 cm; the above plant tissues are used as explants for Agrobacterium rhizogenes-mediated transformation;

[0101] (2) Pre-culture of explants: Explants obtained from substrate seedlings were sterilized in a tissue culture station and wounds were made with a sterile scalpel; all explants from (1) were placed in MS pre-culture medium and pre-cultured at 24°C in the dark for 2-3 days. Figure 2 );

[0102] The specific method for disinfecting the explants of the substrate seedlings is as follows: disinfect with 75% alcohol for 30 seconds, then disinfect with 5% NaClO for 15 minutes, and finally rinse with sterile water 6 to 8 times.

[0103] The MS pre-medium consisted of 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar, pH 5.8-6.0.

[0104] 2. Experimental Results

[0105] 2.1 Growth status of plum seedlings

[0106] Seeds of the 'Early Green Calyx' variety of *Prunus mume* showed the lowest contamination rate in embryo induction medium and exhibited robust, upright growth. The sterile seedlings that developed from the plumules after 20–30 days could be used as explants for Agrobacterium rhizogenes-mediated infection. Figure 1 A); Seeds that have been stratified in sand and then planted in a substrate can germinate normally. Seedlings that have germinated for 30-40 days can be used as explants for Agrobacterium rhizogenes-mediated infection. Figure 1 B); The above explants can be used to explore the effects of plum seedlings under different growth environments on hairy root induction.

[0107] 2.2 Pre-culture of explants

[0108] A total of six explants were obtained from four growth sites under two different growth environments: aseptic tissue culture seedlings with primary roots removed, aseptic tissue culture seedling stem segments, aseptic tissue culture seedling root segments, substrate seedling stem segments, substrate seedling leaves, and substrate seedling petioles. Figure 2 ).

[0109] Example 2: Induction and System Screening of Hairy Roots in Plum Blossom

[0110] 1. Experimental Methods

[0111] 1.1 Preparation of Agrobacterium rhizogenes infection solution

[0112] The preserved Agrobacterium rhizogenes K599 bacterial culture transformed with the 35S:RUBY plasmid was streaked onto LB selective medium and cultured at 28°C in the dark for 2 days until the bacterial culture recovered. Single colonies of the recovered culture were picked and cultured in LB liquid selective medium for 12–16 hours until OD500 was reached. 600 =1.0, centrifuge at 5000 rpm for 10 min to collect the bacterial pellet, and resuspend the bacterial cells in MS resuspending buffer to OD. 600 =0.6, 0.8, 1.0, incubate in the dark for 2-3 hours, and resuspend the bacterial solution as the Agrobacterium rhizogenes infection solution for the explants;

[0113] The 35S:RUBY plasmid carries the naked-eye visible red protein reporter gene RUBY. It was transformed into Agrobacterium rhizogenes K599 competent cells using the freeze-thaw method and positive selection was performed on LB solid selective medium containing antibiotics.

[0114] The LB selective medium contained spectinomycin at a concentration of 75 mg / L and streptomycin at a concentration of 50 mg / L.

[0115] The primers used for PCR verification of single-clone colonies were: 35S:RUBY-F:5'-CGACACTCTCGTCTACTCCAA-3'; 35S:RUBY-R:5'-GCGGTAATCTTGCGGAAGTT-3';

[0116] The MS resuspension was prepared as follows: 4.43 g / L MS medium powder + 30 g / L sucrose + 200 μM AS, pH 5.8-6.0;

[0117] 1.2 Infection of the hairy roots of plum blossom

[0118] (1) Immersion method: The pre-cultured explants were immersed in Agrobacterium rhizogenes infection solution at 28°C and 60 rpm for 10 min. The surface bacterial solution was then blotted dry with sterile filter paper, and the explants were placed in MS co-culture medium and cultured in the dark at 24°C for 2–3 days. Figure 3 A);

[0119] (2) Dipping method: Cultivate the revived bacterial culture to OD. 600 =2.0, after collecting the bacterial cells, spread them onto LB selective medium and culture until the bacterial solution on the plate becomes a paste; scrape the bacterial solution from the explant cut surface of the primary root of the tissue culture seedling 2-3 times to make the paste-like Agrobacterium rhizogenes cover the hypocotyl cut surface, and place it in MS co-medium, incubate at 24℃ in the dark for 2-3 days. Figure 3 B);

[0120] (3) Injection method: Using a 1ml syringe, inject 0.1ml of Agrobacterium rhizogenes infection solution into the tissue culture seedlings 1cm from the root hypocotyl. Blot the surface bacterial solution with sterile filter paper, place in MS co-culture medium, and incubate in the dark at 24℃ for 2–3 days. Figure 3 C);

[0121] The MS co-culture medium was: 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar + 200 μM AS, pH 5.8-6.0;

[0122] The method for culturing bacterial culture using the dip-plate method is as follows: Take 2 ml of OD... 600 =2.0 bacterial suspension, centrifuged at 6000 rpm for 1 min, discarded 1.8 ml of supernatant, resuspended the bacterial precipitate in the remaining 0.2 ml and spread it onto LB selective medium, and incubated at 28℃ for 24 h until the bacterial suspension became paste-like;

[0123] 1.3 Effects of different infection conditions on the induction of hairy roots in plum blossom

[0124] The stem explants from the substrate seedlings in Examples 1 and 2.2 were infected using immersion, dipping, and injection methods, respectively. Different types of explants from Example 2.2 were infected using the immersion method. The stem explants from the substrate seedlings in Example 2.2 were infected using infection solutions of different concentrations. Each group underwent three replicate experiments under the above conditions. All infected explants were then inoculated into MS rooting medium for induction. After 30–40 days, the number of hairy roots and the induction rate were recorded to screen for the optimal explant type, infection method, and infection concentration for growth.

[0125] The MS rooting medium consisted of: 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar + 0.05 mg / L NAA + 0.5 mg / L 6BA, pH 5.8-6.0, spectinomycin content of 7.5 mg / L, and cephalosporin content of 200 mg / L.

[0126] The different growth environments are: sterile tissue culture environment and substrate environment;

[0127] The different explant types are: aseptic tissue culture seedlings, aseptic tissue culture seedling stem segments, aseptic tissue culture seedling root segments, substrate seedling leaves, substrate seedling stem segments, and substrate seedling petioles.

[0128] The different infection methods are: immersion method, dipping method, and injection method;

[0129] The different infection concentrations are: OD 600 =0.6, 0.8, 1.0.

[0130] 2. Experimental Results

[0131] 2.1 Effects of explants from different growth environments and different infection methods on the induction of hairy roots in plum blossom

[0132] After obtaining sterile tissue culture seedlings and substrate seedlings of plum blossom, explants from sterile seedlings, stem segments of sterile seedlings, and stem segments of substrate seedlings were infected using the immersion method; the hypocotyls of tissue culture seedlings were infected using the dipping and injection methods. Experiments showed that after 7 days of culture, the *Agrobacterium rhizogenes* immersion method resulted in callus formation at both ends of the wounds of 30 explants under different growth conditions. After 14 days of culture, callus tissue with visible red deposits grew at both ends of the stem segments. Figure 4 C), after 25–30 days of cultivation, hairy roots could be successfully induced from the callus tissue of both aseptic tissue culture seedlings and substrate seedlings. Figure 5 (C and D), the induction rates were 13.33% and 16.67%, respectively; Agrobacterium tumefaciens was exuded from the wounds after 2-3 days of culture using both dipping and injection methods, and this explants subsequently turned black, wilted, and lost viability. The experimental results are shown in Table 1. Figure 7 (A and B).

[0133] Table 1. Effects of explants from different growth environments and different infection methods on the induction of hairy roots in plum blossom.

[0134]

[0135] Experimental results show that during the induction of hairy roots in plum blossoms, the immersion method can successfully induce hairy roots by infecting aseptic tissue culture seedlings; hairy roots can be successfully induced in explants from both aseptic tissue culture seedlings and substrate seedlings, and the hairy root induction rate is higher when the stem segments of substrate seedlings are disinfected and used as explants.

[0136] 2.2 Effects of different explant types on the induction of hairy roots in plum blossom

[0137] Different types of explants, including pre-cultured aseptic tissue culture seedlings, stem segments, root segments, and substrate seedling stem segments, leaves, and petioles, were infected using the immersion method with Agrobacterium rhizogenes K599 infection solution. Experiments showed that after 7–14 days of culture, 12 out of 30 aseptic tissue culture seedling explants with primary roots removed were infected with Agrobacterium rhizogenes, and callus tissue with red dot-like deposits formed at the hypocotyl incision site. Figure 4 After 20-30 days of culture, the callus tissue of 6 seedlings (A and B) differentiated into a total of 7 independent hairy roots, including 4 white hairy roots and 3 red hairy roots. Figure 5 (A and B), the induction rate of hairy roots in tissue culture explants was 20%; Agrobacterium rhizogenes infected stem and root segments of 30 sterile tissue culture explants respectively. After 14 days of culture, callus tissue with red deposits grew from the wounds at both ends of 10 stem segment explants. Figure 4 C), after 25–30 days of culture, hairy roots successfully differentiated from the callus tissue of the four stem segments. Figure 5 (C and D), the induction rate was 13.33%; among them, after infection with Agrobacterium tumefaciens, the root explants oozed out, turned black and wilted after 7 days, and failed to induce hairy roots, with a hairy root induction rate of 0%; Agrobacterium rhizogenes infected 30 stem segments, 30 leaves and 30 petioles of seedlings after substrate disinfection, and after 7-14 days of culture, 80% of the explant wounds could generate callus tissue, and the proportion of callus tissue with red deposits was low ( Figure 4 (D, E, and F) After 20–30 days of culture, callus tissue from five stem segment explants differentiated into hairy roots, including four white hairy roots and one red hairy root, with a stem segment explant induction rate of 16.67%. Hairy roots were successfully induced from four leaf explants, and gradually turned red as the hairy roots grew. Figure 5 E), the induction rate of leaf explants was 13.33%; five petiole explants successfully induced hairy roots ( Figure 5 The induction rate of petiole explants (F and G) was 13.33%. The experimental results are shown in Table 2. Figure 7 (C and D)

[0138] Table 2. Effects of different explant types on root induction in plum blossom.

[0139]

[0140]

[0141] Experimental results show that using stem segments and petioles of tissue-cultured seedlings and substrate seedlings with primary roots removed as explants for Agrobacterium rhizogenes induction of hairy roots in plum blossoms can achieve better induction results. Therefore, the optimal explants for inducing hairy roots in plum blossoms are stem segments and petioles of sterile tissue-cultured seedlings and substrate seedlings.

[0142] 2.3 Effects of different infection concentrations on the induction of hairy roots in plum blossom

[0143] Using OD respectively 600 Stem explants of substrate seedlings were infected with Agrobacterium rhizogenes infection solutions of 0.6, 0.8, and 1.0. After culturing for 2–3 days, OD... 600 Agrobacterium moniliforme oozed around stem explants infected with 0.8 and 1.0 oz., and could not be inhibited even after multiple changes to the inhibitory culture medium; OD 600 =0.6% of the stem segment explants infected with the virus showed callus formation at both ends of the wound in about 80% of cases after 2-3 days. After 20-30 days, 5 callus tissues differentiated into hairy roots. OD 600 =0.6% hairy root induction rate was 16.67%. Experimental results are shown in Table 3. Figure 7 E.

[0144] Table 3. Effects of different infection concentrations on the induction of hairy roots in plum blossoms.

[0145]

[0146] Experimental results show that, during the induction of hairy roots in plum blossoms, the use of OD... 600 A concentration of 0.6% Agrobacterium rhizogenes infection solution can achieve a good induction effect on stem segment explants. Therefore, the optimal infection concentration for inducing infection from plum blossom hairy roots is OD0.6. 600 =0.6.

[0147] In summary, the optimal induction conditions for the hairy roots of plum blossom are: using OD... 600 A 0.6% Agrobacterium rhizogenes K599 infection solution was used to immerse tissue culture seedlings, stem segments, and petioles explants in a sterile environment and a substrate environment, which showed a good induction effect on the hairy roots of plum blossoms. Figure 8 ).

[0148] Example 3: Molecular detection of plum blossom hairy roots induced by the present invention

[0149] 1. Experimental Methods

[0150] T-DNA integration is known to be random, thus requiring efficient screening methods to identify positive hairy roots. A naked-eye visible reporter gene system, RUBY, has been successfully developed and applied to positive screening in rice and Arabidopsis thaliana. The system fuses three betaine biosynthesis genes (CYP76AD1, DODA, and GT) into an open reading frame, converting tyrosine into red betaine. This reporter gene system can efficiently screen positive transformed tissues and organs with the naked eye, thus possessing great application potential in the genetic transformation of woody plants.

[0151] Using Agrobacterium rhizogenes strain K599 containing 35S:RUBY as a positive control and wild-type roots as a negative control, three red and three white hairy roots were randomly selected for PCR identification. Genomic DNA was extracted and used as a template for PCR amplification using the 35S:RUBY vector-specific primer 35S:RUBY-F / R and three primers for betaine biosynthesis genes: CYP76AD1-F1 / R1, DODA-F1 / R1, and GT-F1 / R1.

[0152] Specific primers were designed based on the RUBY reporter gene sequence as follows:

[0153] 35S:RUBY-F:5'-CGACACTCTCGTCTACTCCAA-3',

[0154] 35S:RUBY-R:5'-GCGGTAATCTTGCGGAAGTT-3';

[0155] The PCR product size is 815 bp.

[0156] Specific primers were designed based on the sequences of the three betaine biosynthesis genes as follows:

[0157] CYP76AD1-F: 5'-CGATACCACATCCTCCACATTC-3',

[0158] CYP76AD1-R: 5'-GGACCTGCTTGATTTCCTTCT-3';

[0159] The PCR product size is 95 bp.

[0160] DODA-F: 5'-TTGCGAGAAAGGTGGAAGAG-3',

[0161] DODA-R: 5'-GGATACATGAGCATGAGTGGAA-3';

[0162] The PCR product size is 112 bp.

[0163] GT-F: 5'-GGCATCGAGAACACCGATAAG-3',

[0164] GT-R: 5'-GCTGAAATGCCTGGAGATGTA-3';

[0165] The PCR product size is 108 bp.

[0166] PCR amplification reaction system: A 25 μl reaction system was used, consisting of 1 μl DNA template, 1 μl each of forward and reverse primers, 12.5 μl Taq enzyme, and 9.5 μl double-distilled water.

[0167] PCR amplification conditions: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s; 35 cycles; 72℃ extension for 5 min; store at -20℃.

[0168] Take 2 μl of amplification product and add 2 μl of DNA loading buffer, place it on a 1% agarose gel for electrophoresis detection, and observe the results after 15 min using a gel imaging system. The hairy roots with the target band in the correct position are positive.

[0169] 2. Experimental Results

[0170] PCR detection results of RUBY reporter gene, CYP76AD1, DODA, and GT genes in the hairy roots of *Prunus mume* are as follows: Figure 6 As shown, white and red hairy roots randomly selected from Example 2 were able to amplify the target fragment, indicating the presence of the RUBY reporter gene system, which shows that the induced roots are plum blossom hairy roots.

[0171] Example 4: Startup efficiency of the RUBY reporter gene system with hairy roots in plum blossom

[0172] In Example 3, PCR detection confirmed the presence of the RUBY reporter gene in all three white and three red hairy roots randomly selected from Example 2. Furthermore, with prolonged culture time (40–50 days), the leaf-induced hairy roots gradually changed from white to red, and red dotted deposits were observed in the callus tissue. In Example 2, of the seven independent hairy roots induced from six tissue culture seedlings, four were white and three were red, indicating a RUBY reporter gene activation rate of 42.86%.

[0173] Experimental results showed that during the induction of plum blossom hairy roots, the 35S promoter initiated the expression of the RUBY reporter gene and obtained positive hairy roots with red selection markers visible to the naked eye at an efficiency of 42.86%, which can realize the morphological selection marker of positive transformed hairy roots; the remaining white hairy roots did not express the RUBY reporter gene system, but were also positive hairy roots.

[0174] 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 method for inducing hairy roots in a sterile plum blossom system, characterized in that, Using Agrobacterium rhizogenes K599 containing the RUBY reporter gene to infect plum blossom explants, hairy roots were induced to form in the explants; The explants are the root portion of sterile tissue culture seedlings after primary roots have been removed, or the leaves with petioles or young stem segments of substrate seedlings, which are pre-cultured in MS pre-medium at 24°C in the dark for 2-3 days before being used for infection. The MS pre-medium consisted of 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar, pH 5.8-6.

0. Agrobacterium rhizogenes K599 containing the RUBY reporter gene was cultured in LB selective medium, and the bacterial cells were collected by centrifugation. The bacterial cells were then resuspended in MS resuspending buffer to OD. 600 =0.6, used as the infection solution for infecting plum blossom explants; soak the plum blossom explants in the infection solution for 10 min, blot dry with sterile filter paper, and then place them in MS co-culture medium for co-culture at 24℃ in the dark for 2~3 days; After co-culture, the explants were transferred to MS rooting medium and cultured at 24°C under light for 20-30 days. Subculture was performed every 7 days until callus tissue and hairy roots were generated at the wound site of the explants. After PCR detection and positive identification, the main root was removed and only the hairy roots were retained. Then, the seeds were sown in seedling trays for substrate culture. The MS rooting medium is: 4.43 g / L MS medium powder + 30 g / L sucrose + 7 g / L agar + 0.05 mg / L NAA + 0.5 mg / L 6-BA, with spectinomycin at 7.5 mg / L, cephalosporin at 200 mg / L, and pH 5.8-6.

0. The plum blossom variety is 'Early Green Calyx' of the True Plum series.

2. The method according to claim 1, characterized in that, The *Agrobacterium rhizogenes* K599 containing the RUBY reporter gene was obtained by transforming *Agrobacterium rhizogenes* K599 competent cells with a plasmid containing the RUBY reporter gene, and the RUBY reporter gene is driven by a 35S promoter.

3. The method according to claim 1, characterized in that, Methods for preparing aseptic tissue culture seedlings include: Remove the pulp from the plum fruit, wash and dry the seeds, then soak them in sterile water. Next, mechanically remove the pits from the seeds and soak the kernels in sterile water overnight. Select the soaked and dried plump kernels, rinse them with running water for 20-30 minutes, and then transfer them to a sterile tissue culture table. First, soak them in 2% NaClO for 15 minutes, then disinfect them with 75% alcohol for 30 seconds, and finally rinse them with sterile water 3-5 times. Use a sterile scalpel to peel out the plum seed embryo with 1 / 4 of the cotyledon attached. Place the embryo with the plum bud facing down in the embryo induction medium and culture it in an artificial climate chamber at 24℃ with 16 hours of light and 8 hours of darkness until germination, thus obtaining sterile plum tissue culture seedlings. The embryo induction medium is: 4.43 g / L QL medium powder + 30 g / L sucrose + 0.5 mg / L 6-BA + 1.0 mg / L TDZ + 7 g / L agar, pH 5.8-6.

0.

4. The method according to claim 1, characterized in that, Methods for preparing substrate seedlings include: (1) Remove the pulp from the plum fruit, wash and dry the seeds, and store them in a 4℃ cold storage in sand. Regularly check and spray water to prevent the seeds from getting moldy until they turn white. Sow the white seeds in substrate trays, and plum seedlings will be obtained after 20-30 days. (2) Take leaves with petioles and young stem segments from substrate seedlings as explant materials; after sterilization and drying, the explant materials are pre-cultured in MS pre-medium at 24℃ in the dark for 2-3 days before being used for infection; The matrix is ​​composed of peat moss and perlite mixed in a 2:1 volume ratio.

5. The method according to claim 4, characterized in that, The method for disinfecting explant materials in step (2) includes: disinfecting with 75% alcohol for 30 seconds, then disinfecting with 5% NaClO for 15 minutes, and finally rinsing with sterile water 6 to 8 times.

6. The method according to claim 1, characterized in that, The LB selective medium consisted of 10 g / L tryptone + 5 g / L yeast extract + 10 g / L sodium chloride + 15 g / L agar, with spectinomycin at 75 mg / L and streptomycin at 50 mg / L. The MS resuspension was prepared as follows: 4.43 g / L MS medium powder + 30 g / L sucrose + 200 μM AS, pH 5.8-6.

0.

7. The method according to claim 1, characterized in that, The primers used for PCR detection are: 35S: RUBY-F: 5'-CGACACTCTCGTCTACTCCAA-3'; 35S: RUBY-R: 5'-GCGGTAATCTTGCGGAAGTT-3'.

8. The application of plum blossom hairy roots prepared according to any one of claims 1-7 in plum blossom gene function research and plum blossom genetic improvement breeding.

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