A method for inducing hairy roots of krameria by agrobacterium rhizogenes mediation

Abstract

The present application relates to plant tissue culture, and in particular to a hairy root agrobacterium-mediated induction method of Kosteletzkya virginica. In the present application, a Kosteletzkya virginica hairy root induction system is established, pre-cultured explants are treated with a hairy root agrobacterium infection solution, and suitable transformation conditions are selected, so that Kosteletzkya virginica hairy roots are successfully induced, thereby providing a reference for further establishing an efficient Kosteletzkya virginica active ingredient production system through hairy root culture.

Description

Technical Field

[0001] This invention relates to plant tissue culture, and more specifically to a method for inducing hairy roots of *Agrobacterium rhizogenes* mediated by *Agrobacterium tumefaciens*. Background Technology

[0002] Malope trifida, a herbaceous plant belonging to the genus Malope in the family Malvaceae, is an important flowering plant with excellent ornamental value. Modern research shows that the flowers and leaves of Malope trifida are rich in flavonoids, which possess significant biological activity. They can help reduce oxidative stress and inflammation, and combat free radical damage to cells. Its potential medicinal value includes antibacterial, diuretic, anti-inflammatory, cholesterol-lowering, and anti-thrombotic effects, thus indicating a broad market prospect. However, due to the low content of medicinally active ingredients in Malope trifida, actual production faces many challenges, including difficulty in ensuring quality and high production costs. Therefore, the production of the effective components of Malope trifida requires the development of new bioengineering technologies.

[0003] Agrobacterium rhizogenes is a group of Gram-negative soil bacteria with a wide host range. The T-DNA fragment on its Ri plasmid can insert and integrate into the plant genome, inducing the production of transgenic hairy roots in plant tissues. Compared with naturally grown plants, hairy roots formed by Agrobacterium rhizogenes infection have advantages such as rapid growth, short culture period, stable genetic traits, no need for exogenous growth substances, and high content of plant secondary metabolites. Therefore, they have become an ideal research subject for studies on plant secondary metabolism regulation, secondary metabolite production, plant trait optimization, and biotransformation.

[0004] As a flowering plant, the holly tree has extremely high ornamental value, but existing research on it is limited, mainly focusing on cultivation, extraction of active ingredients, etc. Currently, there are few reports on the induction of hairy roots in the holly tree in China. Summary of the Invention

[0005] Based on this, the present invention provides a method for inducing hairy roots of *Agrobacterium rhizogenes* mediated by *Agrobacterium tumefaciens*, which at least solves one problem in the prior art.

[0006] This invention provides a method for inducing hairy roots of *Agrobacterium rhizogenes* mediated by *Agrobacterium tumefaciens*, comprising the following steps: Leaves or young stems of *Gnaphalium affine* were obtained, and the leaves or young stems were damaged and inoculated onto a first solid culture medium. They were then cultured in the dark for 2-3 days to obtain pre-cultured explants. The pre-cultured explants were immersed in Agrobacterium rhizogenes infection solution for 5-15 min and then inoculated onto a second solid culture medium. They were then cultured in the dark at 25±2℃ for 2-6 days to obtain co-cultured explants. The co-cultured explants were sterilized and cultured to allow them to grow hairy roots.

[0007] In this invention, a hairy root induction system for *Agrobacterium rhizogenes* was established. Pre-cultured explants were treated with *Agrobacterium rhizogenes* infection solution, and suitable transformation conditions were selected to successfully induce hairy root growth in *Agrobacterium rhizogenes*. This provides a reference for further establishing an efficient production system of effective components from *Agrobacterium rhizogenes* through hairy root culture. The pre-culture process allows the explants to adapt to the in vitro culture environment and induces the secretion of phenolic substances from the wound site, thereby improving the transformation rate.

[0008] In some preferred embodiments, the Agrobacterium rhizogenes infection solution is an infection solution obtained by culturing Agrobacterium rhizogenes C58C1 in MS liquid medium.

[0009] In some preferred embodiments, the pre-cultured explants are immersed in Agrobacterium rhizogenes infection solution for 10 min.

[0010] In some preferred embodiments, the OD600 of the Agrobacterium rhizogenes infection solution is 0.2 to 1.0.

[0011] In some preferred embodiments, the method for preparing the Agrobacterium rhizogenes infection solution includes activation of Agrobacterium rhizogenes and resuspension of Agrobacterium rhizogenes.

[0012] The activation of *Agrobacterium rhizogenes* involved the following steps: *Agrobacterium rhizogenes* strains stored at -80℃ were evenly spread onto YEB or LB solid medium containing antibiotics and incubated upside down at 26-30℃ for 2-6 days; single colonies were picked using an inoculation needle and inoculated into YEB or LB liquid medium containing antibiotics, and cultured in the dark with shaking at 28℃ and 100-400 rpm for 20-26 hours; 10-500 μL of the bacterial culture was added to 10-50 mL of YEB or LB liquid medium containing antibiotics, and cultured in the dark with shaking at 28℃ and 100-400 rpm until the OD value reached 0.2-1.0. The antibiotic could be rifampin.

[0013] Resuspension of Agrobacterium rhizogenes: At room temperature, the activated Agrobacterium rhizogenes was centrifuged at 1000-5000 rpm for 5-20 min, the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in 1 / 2 MS liquid medium and incubated in the dark at 25-30℃ with shaking at 50-200 rpm for half an hour until the OD600 of the bacterial solution was 0.2-1.2, thus obtaining the Agrobacterium rhizogenes infection solution.

[0014] In some preferred embodiments, the method for obtaining leaves or tender stems of *Gnaphalium affine* is as follows: soak *Gnaphalium affine* seeds in water for 30 min to 2 h, disinfect with 75% alcohol for 20 to 30 s, rinse with sterile water 2 to 3 times, disinfect with 4% hypochlorous acid solution for 2 to 4 min, and rinse with sterile water 5 to 6 times; inoculate on solid culture medium (the solid culture medium formula is: 1 / 2 MS solid culture medium, with 30 g / L sucrose, 0 to 0.2 mg / L NAA and 0 to 0.2 mg / L IBA, and 7 to 8 g / L agar powder), and culture in the dark at 28±2℃ for 7 to 14 days, then transfer to a light room with a light intensity of 2000 lx and a light duration of 12 hours / day to obtain sterile *Gnaphalium affine* seedlings, and take leaves or tender stems from the sterile *Gnaphalium affine* seedlings.

[0015] In some preferred embodiments, the leaf or stem is damaged by making 4-5 cuts along the veins of the leaf or by scratching the tender stem. The scratches on the tender stem are made with two smooth cuts.

[0016] In some preferred embodiments, the first solid culture medium is 1 / 2 MS solid culture medium. More preferably, the formulation of the first solid culture medium is: 1 / 2 MS basal medium, with 30 g / L sucrose and 7-8 g / L agar powder added, pH = 6.5.

[0017] In some preferred embodiments, the second solid culture medium is 1 / 2 MS solid culture medium. More preferably, the formulation of the second solid culture medium is: 1 / 2 MS solid culture medium with 30 g / L sucrose and 6-7 g / L agar powder added.

[0018] In some preferred embodiments, the sterilization culture includes the following steps: The co-cultured explants are washed with sterile water and cultured on a first sterile solid medium (formulation: 1 / 2 MS + 400~600 mg / L Cef) for 6~10 days; then transferred to a second sterile solid medium (formulation: 1 / 2 MS + 200~400 mg / L Cef) for 1~2 weeks, during which white hairy roots will gradually appear at the wound site of the explants; then transferred to a third sterile solid medium (formulation: 1 / 2 MS + 50~300 mg / L Cef) for 1~2 weeks; then transferred to a fourth sterile solid medium (formulation: 1 / 2 MS + 50~300 mg / L Cef) for 2~3 weeks, after which sterilization is basically completed; finally, transferred to an antibiotic-free 1 / 2 MS solid medium for culture to obtain the hairy roots of *Abelmoschus esculentus*. The sterilization culture temperature is 25±2℃, and the entire culture is conducted in darkness. Cef represents cefotaxime sodium.

[0019] In some preferred embodiments, the Agrobacterium rhizogenes-mediated induction method for hairy roots of *Aristolochia debilis* further includes a step of screening for high-quality *Aristolochia debilis* hairy root lines. Specifically, this involves selecting thoroughly sterilized *Aristolochia debilis* hairy roots that have undergone PCR identification, transferring them to blank 1 / 2 MS medium, and subculturing them at 110 rpm and 28±2℃ to obtain high-quality *Aristolochia debilis* hairy root lines. PCR identification uses a kit to extract genomic DNA from the *Aristolochia debilis* hairy roots. The positive control is the corresponding engineered *Agrimonia rhizogenes* bacteria, and the negative control is *Aristolochia debilis* leaves. The detection genes are rolB and rolC. PCR amplification is performed, and the products are separated by agarose gel electrophoresis and detected by ultraviolet light to complete the PCR identification of *Aristolochia debilis* hairy roots.

[0020] Due to the adoption of the above technical solutions, the embodiments of the present invention have at least the following beneficial effects: (1) T-DNA of Agrobacterium rhizogenes Ri plasmid was introduced into Malus bacillus to obtain hairy roots. This is the first time that an Agrobacterium rhizogenes-mediated method for obtaining transgenic Malus bacillus hairy roots has been established, laying the foundation for subsequent research on the genetic improvement and functional genes of Malus bacillus. (2) All processes of the hairy root induction system of *Acer buergerianum* are within the range of human control. The hairy roots grow rapidly, have high production efficiency and short production cycle. No hormones need to be added. It has good genetic stability and has good social and economic benefits. Attached Figure Description

[0021] Figure 1 This is a photograph of a sterile *Aeonium alopecuroides* seedling in an embodiment of the present invention.

[0022] Figure 2 This is a photograph of the hairy roots produced by *Agrobacterium rhizogenes* C58C1 induced in *Alopecurus aequalis* in an embodiment of the present invention.

[0023] Figure 3 This is a photograph of the rapid growth of hairy roots on a solid culture medium in an embodiment of the present invention.

[0024] Figure 4 These are PCR detection images of the rolB and rolC genes in hairy roots, as shown in this embodiment of the invention.

[0025] Figure 5 This is a photograph of the hairy roots of *Acer palmatum* cultured in liquid according to an embodiment of the present invention. Detailed Implementation

[0026] The following will provide a clear and complete description of the concept and technical effects of the present invention, so as to fully explain the purpose, solution and effects of the present invention.

[0027] In the following examples, the preparation steps of Agrobacterium rhizogenes infection solution are as follows: (1) Activation of Agrobacterium rhizogenes: Different types of Agrobacterium rhizogenes strains (model: C58C1 / A4 / R1601) stored at -80℃ were evenly spread on YEB solid medium containing rifampicin and cultured at 28℃ for 3 days; single colonies were picked with an inoculation needle and inoculated into YEB liquid medium containing rifampicin (pH=7.0) and cultured in the dark at 200 rpm in a constant temperature shaker at 28℃ for 24 h; 100 μL of the activated bacterial solution was added to 50 mL of YEB liquid medium containing rifampicin and cultured in the dark at 200 rpm in a constant temperature shaker at 28℃ until the OD600 value was 0.8; (2) Resuspension of Agrobacterium rhizogenes: The activated Agrobacterium rhizogenes (model: C58C1 / A4 / R1601) was centrifuged at 3000 rpm for 10 min at room temperature, the supernatant was discarded, the bacterial cells were collected, and 1 / 2 MS liquid medium was added for resuspension. The cells were then cultured in the dark at 200 rpm in a constant temperature shaker at 25℃ until the OD600 value was 0.8, thus obtaining the Agrobacterium rhizogenes infection solution.

[0028] In the following examples, the PCR identification steps are as follows: (1) Extraction of genomic DNA from the hairy roots of *Acer buergerianum*: Take 100 mg of young hairy roots of *Acer buergerianum*, grind them into powder with liquid nitrogen, and transfer them to a 1.5 ml Eppendorf centrifuge tube; immediately add 400 μL Buffer A1 and 4 μL RNase A, mix thoroughly, and incubate at 65 °C for 10 min; add 130 μL Buffer A2 to the mixture, mix thoroughly, incubate on ice for 5 min, and centrifuge at 14000 rpm for 5 min; take 300 μL of the supernatant into a new 1.5 ml Eppendorf centrifuge tube, add 450 μL Buffer A3, and mix thoroughly; transfer the mixture to an adsorption column FastPure gDNA Columns IV, centrifuge at 12000 rpm for 30 s, and discard the filtrate; add 600 μL Buffer AW, centrifuge at 12000 rpm for 30 s, and discard the filtrate; repeat adding 600 μL Buffer AW, centrifuging at 12000 rpm for 30 s. s, discard the filtrate; place the adsorption column back into the collection tube, centrifuge at 12000 rpm for 2 min; place the adsorption column in a new 1.5 ml Eppendorf centrifuge tube, add 100 μL of EB (Elution Buffer) preheated to 70℃ to the center of the membrane of the adsorption column, incubate at room temperature for 4 min, centrifuge at 12000 rpm for 1 min, discard the adsorption column, and store the DNA at -20℃; (2) PCR detection of rolB and rrolC genes in hairy roots of *Pterocarya stenoptera*: The primers for rolB gene amplification were frolb (5'-GCTCTTGCAGTGCTAGATTT-3') and rrolb (5'-GAAGGTGCAAGCTACCTCTC-3'); the primers for rolC gene amplification were frolc (5'-CTCCTGACATCAAACTCGTC-3') and rrolc (5'-TGCTTCGAGTTATGGGTACA-3'); the reaction system (50 μL) consisted of: ddH2O 22 μL, Template DNA 1 μL, Forward Primer (10 μM) 1 μL, Reverse Primer (10 μM) 1 μL, and 2×EasyTaq. ® PCR SuperMix 25 μL; PCR reaction conditions: 94℃ for 5 min, 35 cycles (94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 90 sec), 72℃ for 10 min; positive control was the corresponding Agrobacterium rhizogenes engineered strain, and Acer palmatum leaves were used as negative control; PCR products were separated by agarose gel electrophoresis and detected by UV to determine the presence of rolB and rolC amplification bands; the amplification band size of rolB was 423 bp, and the amplification band size of rolC was 626 bp.

[0029] Example 1: Inducing hairy roots in leaves of *Gnaphalium affine* Pre-culture of explants from *Acer buergerianum*: Leaves of aseptic seedlings of *Acer buergerianum* were cut, and four wounds were made along the veins. The leaves were placed on the first solid medium (1 / 2 MS solid medium) and cultured in the dark at 25±2℃ for 2 days to obtain pre-cultured explants. Co-culture of Agrobacterium rhizogenes and Abelmoschus mandshurica: The pre-cultured explants were immersed in an infection solution containing activated Agrobacterium rhizogenes C58C1 for 10 min, with constant gentle shaking. Then, the residual bacterial solution on the surface of the Abelmoschus mandshurica explants was blotted dry with sterile filter paper, and the explants were transferred to a second solid medium (1 / 2 MS solid medium) and cultured in the dark at 25±2℃ for 2 days to obtain co-cultured explants. Sterilization culture of *Aristolochia debilis*: After rinsing the co-cultured explants several times with sterile water and blotting off the surface moisture with sterile filter paper, the explants were placed on the first sterilization medium (1 / 2 MS + 500 mg / L Cef) for sterilization culture. After 7 days, they were transferred to the second sterilization medium (1 / 2 MS + 300 mg / L Cef) for sterilization culture. During this period, white hairy roots will gradually appear at the wound site of the *Aristolochia debilis* explants. After 2 weeks, the white hairy roots were transferred to the third sterilization medium (1 / 2 MS + 100 mg / L Cef) for sterilization culture. After 2 weeks, they were transferred to the third sterilization medium (1 / 2 MS + 50 mg / L Cef) for sterilization culture. After 3 weeks, when sterilization was basically completed, the explants were transferred to 1 / 2 MS solid medium without antibiotics. The sterilization culture conditions were 25±2℃ and incubation in the dark. Screening of high-quality hairy root lines of *Acer buergerianum*: Hairy roots of *Acer buergerianum* that have been thoroughly sterilized and identified by PCR were selected. Hairy roots with good growth were transferred to blank 1 / 2 MS medium and cultured at 110 rpm and 28±2℃.

[0030] The hairy roots of *Aristolochia debilis* obtained in this embodiment are as follows: Figure 2 As shown, Figure 2 The experimental procedure is recorded in Chinese characters (Malox villosum: plant name, C58C1: type of inducing strain, cultured under sterile conditions on 300 mg / L Cef). The induction rate of hairy roots in Malox villosum leaves was 72.34%.

[0031] The hairy root induction rate (%) = number of explants that induce hairy roots / number of infected explants × 100%.

[0032] Example 2: Inducing hairy roots in young stems of *Gnaphalium affine* This embodiment is basically the same as embodiment 1, except for the pre-culture of the explants of *Acer buergerianum*: tender stems of sterile *Acer buergerianum* seedlings were cut, scratched 3 times and kept smooth at both ends, and placed in 1 / 2 MS solid medium (1 / 2 MS solid medium) and cultured in the dark at 25±2℃ for 2 days.

[0033] In this embodiment, the induction rate of hairy roots from young stems of *Gnaphalium affine* was 63.33%.

[0034] Example 3: Inducing hairy roots in leaves of *Pterocarya stenoptera* This embodiment is basically the same as Example 1, except that Agrobacterium rhizogenes and Aristolochia debilis are co-cultured: the pre-cultured explants are immersed in an infection solution containing activated Agrobacterium rhizogenes A4 for 10 min, and gently shaken continuously. Then, the residual bacterial solution on the surface of the Aristolochia debilis explants is blotted dry with sterile filter paper, and transferred to a second solid medium (1 / 2 MS solid medium). The explants are then cultured in the dark at 25±2℃ for 2 days to obtain co-cultured explants.

[0035] In this embodiment, using Agrobacterium rhizogenes A4 infection solution, the hairy root induction rate of *Agropyron cristatum* leaves was 0%.

[0036] Example 4: Inducing hairy roots in leaves of *Pterocarya stenoptera* This embodiment is basically the same as Example 1, except that Agrobacterium rhizogenes and Aristolochia debilis are co-cultured: the pre-cultured explants are immersed in an infection solution containing activated Agrobacterium rhizogenes R1601 for 10 min, and gently shaken continuously. Then, the residual bacterial solution on the surface of the Aristolochia debilis explants is blotted dry with sterile filter paper, and transferred to a second solid medium (1 / 2 MS solid medium). The explants are then cultured in the dark at 25±2℃ for 2 days to obtain co-cultured explants.

[0037] In this embodiment, using Agrobacterium rhizogenes R1601 infection solution, the hairy root induction rate of Alternanthera philoxeroides leaves was 0%.

[0038] Example 5: Inducing hairy roots from leaves of *Gnaphalium affine* This embodiment is basically the same as Example 1, except that Agrobacterium rhizogenes and Aristolochia debilis are co-cultured: the pre-cultured explants are immersed in an infection solution containing activated Agrobacterium rhizogenes C58C1 for 5 minutes, and gently shaken continuously. Then, the residual bacterial solution on the surface of the Aristolochia debilis explants is blotted dry with sterile filter paper, and transferred to a second solid medium (1 / 2 MS solid medium). The explants are then cultured in the dark at 25±2℃ for 2 days to obtain co-cultured explants.

[0039] In this embodiment, the immersion time was 5 minutes, and the hairy root induction rate of *Acer palmatum* leaves was 43.55%.

[0040] Example 6: Inducing hairy roots from leaves of *Pterocarya stenoptera* This embodiment is basically the same as Example 1, except that Agrobacterium rhizogenes and Aristolochia debilis are co-cultured: the pre-cultured explants are immersed in an infection solution containing activated Agrobacterium rhizogenes C58C1 for 15 minutes, and gently shaken continuously. Then, the residual bacterial solution on the surface of the Aristolochia debilis explants is blotted dry with sterile filter paper, and transferred to a second solid medium (1 / 2 MS solid medium). The explants are then cultured in the dark at 25±2℃ for 2 days to obtain co-cultured explants.

[0041] In this embodiment, the immersion time was 15 minutes, and the explants of *Acer buergerianum* partially browned and produced bacterial solution. The induction rate of hairy roots in *Acer buergerianum* leaves was 24.46%.

[0042] Example 7: Inducing hairy roots in leaves of *Pterocarya stenoptera* Pre-culture of explants from *Acer buergerianum*: Leaves of aseptic seedlings of *Acer buergerianum* were cut, and four wounds were made along the veins. The leaves were placed on the first solid medium (1 / 2 MS solid medium) and cultured in the dark at 25±2℃ for 2 days to obtain pre-cultured explants. Co-culture of Agrobacterium rhizogenes and Acer rubrum: The pre-cultured explants were immersed in an infection solution containing activated Agrobacterium rhizogenes C58C1 for 20 min, with constant gentle shaking. Then, the residual bacterial solution on the surface of the Acer rubrum explants was blotted dry with sterile filter paper, and the explants were transferred to a second solid medium (1 / 2 MS solid medium) and cultured in the dark at 25±2℃ for 2 days. As a result, the Acer rubrum explants turned brown and died.

[0043] In this embodiment, the immersion time was 20 minutes, and the *Aster salsa* explants browned and died.

[0044] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention by the same or equivalent means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. A method for inducing hairy roots of *Agrobacterium rhizogenes* mediated by *Agrobacterium tumefaciens*, characterized in that, Includes the following steps: Leaves or young stems of *Gnaphalium affine* were obtained, and the leaves or young stems were damaged and inoculated onto a first solid culture medium. They were then cultured in the dark for 2-3 days to obtain pre-cultured explants. The pre-cultured explants were immersed in Agrobacterium rhizogenes infection solution for 5-15 min and then inoculated onto a second solid culture medium. They were then cultured in the dark at 25±2℃ for 2-6 days to obtain co-cultured explants. The co-cultured explants were sterilized and cultured to allow them to grow hairy roots. The Agrobacterium rhizogenes infection solution is the infection solution obtained by culturing Agrobacterium rhizogenes C58C1 in MS liquid medium.

2. The method according to claim 1, characterized in that, The pre-cultured explants were immersed in Agrobacterium rhizogenes infection solution for 10 min.

3. The method according to claim 1, characterized in that, The OD600 of the Agrobacterium rhizogenes infection solution is 0.2~1.

0.

4. The method according to claim 1, characterized in that, The method for preparing the Agrobacterium rhizogenes infection solution includes the activation of Agrobacterium rhizogenes and the resuspension of Agrobacterium rhizogenes. The activation of Agrobacterium rhizogenes was carried out as follows: Agrobacterium rhizogenes strains stored at -80℃ were evenly spread on YEB solid medium or LB solid medium containing antibiotics and incubated upside down at 26-30℃ for 2-6 days; single colonies were picked with an inoculation needle and inoculated into YEB liquid medium or LB liquid medium containing antibiotics and cultured in the dark with shaking at 100-400 rpm at 28℃ for 20-26 hours; 10-500 μL of bacterial solution was added to 10-50 mL of YEB liquid medium or LB liquid medium containing antibiotics and cultured in the dark with shaking at 100-400 rpm at 28℃ until the OD value reached 0.2-1.

0. Resuspension of Agrobacterium rhizogenes: At room temperature, the activated Agrobacterium rhizogenes was centrifuged at 1000-5000 rpm for 5-20 min, the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in 1 / 2 MS liquid medium and incubated in the dark at 25-30℃ with shaking at 50-200 rpm for half an hour until the OD600 of the bacterial solution was 0.2-1.2, thus obtaining the Agrobacterium rhizogenes infection solution.

5. The method according to claim 1, characterized in that, Damage is caused to the leaf or the tender stem by making 4-5 cuts along the veins of the leaf or by scratching the tender stem.

6. The method according to claim 1, characterized in that, The first solid culture medium is 1 / 2MS solid culture medium.

7. The method according to claim 1, characterized in that, The second solid culture medium is a 1 / 2 MS solid culture medium.

8. The method according to claim 1, characterized in that, The sterile culture includes the following steps: the co-cultured explants are washed with sterile water and cultured on a first sterile solid medium for 6-10 days; then transferred to a second sterile solid medium for 1-2 weeks; then transferred to a third sterile solid medium for 1-2 weeks; then transferred to a fourth sterile solid medium for 2-3 weeks; and finally transferred to an antibiotic-free 1 / 2 MS solid medium for culture.

9. The method according to claim 1, characterized in that, The Agrobacterium rhizogenes-mediated induction of hairy roots in *Alopecurus aequalis* also includes the step of screening for high-quality hairy root lines.

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