Garlic tuberous root-like body induction generation method and tuberous root-like body mediated genetic transformation method

Abstract

The invention belongs to the field of biotechnology and tissue culture, and particularly relates to a garlic tuberous root-like body induced generation method and a tuberous root-like body mediated genetic transformation method. The method comprises the following steps: massively culturing garlic aseptic seedlings; under a dark condition, inducing the garlic explant to generate a root-like body by RIM; under an illumination condition, inducing the garlic root-like body to generate a tuberous root-like body by using the RTB induction culture medium; inducing the RTB embryos to germinate into regenerated seedlings; inducing the regenerated seedlings to root and develop into seedlings; infecting a garlic explant, and incubating the infected garlic explant in a dark place; inducing the co-cultured explant to generate RTB and screening positive RTB; inducing the positive RTB embryos to germinate into positive regenerated seedlings; the rooting culture medium RM induces rooting seedlings of the positive regenerated seedlings. The garlic RTB can be efficiently induced, exogenous genes are successfully transformed through an RTB-mediated method, and the established garlic RTB-mediated regeneration system and genetic transformation system are efficient and stable.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and tissue culture, specifically relating to a method for inducing the formation of garlic tuberous roots and a method for genetic transformation mediated by tuberous roots. Background Technology

[0002] Garlic is a perennial herbaceous plant belonging to the lily family. Garlic cannot reproduce sexually through pollination and can only reproduce asexually through bulbils, resulting in a low reproduction rate and susceptibility to viral diseases. During cultivation, garlic is more susceptible to various diseases. Furthermore, various insect pests have a significant impact on garlic yield and quality. Therefore, resistance molecular design breeding and variety improvement in garlic have become urgent problems to be solved in garlic agricultural production.

[0003] Current research on the genetic transformation of garlic includes: establishing a genetic transformation system for garlic by infecting garlic root callus with Agrobacterium; mediating the regeneration and genetic transformation of immature garlic leaves; establishing an Agrobacterium-mediated genetic transformation system for garlic embryos; studying the efficient regeneration of garlic by protoplasts isolated from garlic bud primordium callus; reporting that apical bud meristem callus can mediate the genetic transformation of garlic; and establishing traditional somatic embryogenesis and organogenesis systems for garlic.

[0004] Existing technologies disclose methods for inducing rhizomatous tubers (RTBs) and RTB-mediated genetic transformation. However, the following technical challenges remain in RTB induction and RTB-mediated genetic transformation: screening of culture medium types, seed germination, screening of plant growth regulator types and concentrations, identification and determination of RTBs, screening of optimal explants, screening of culture media for RTB embryo differentiation and germination, and selection of the Agrobacterium infection time.

[0005] However, plants from different evolutionary branches and families exhibit varying sensitivities and responsiveness to plant growth regulators (types and combinations), as well as inducing factors such as light and darkness. In garlic, root induction was achieved using root segments as explants; shoot tips and garlic scape segments were unsuccessful. In contrast, groundcherry used leaves as inducing explants, with roots and stems also failing. The fundamental difference in explant selection between these two plant species lies in the different natural characteristics of leaf and root segment tissue structures in monocotyledonous and dicotyledonous plants.

[0006] The induction of root formation in garlic is a prerequisite for the generation of regenerative stem cells (RTBs), and an efficient RTB-mediated regeneration system is a prerequisite for the construction of its genetic transformation system. Currently, domestic and international research on garlic largely focuses on Agrobacterium infection of garlic root callus, immature leaves, garlic embryos, apical meristem callus, shoot tips, and bulb leaf discs, as well as establishing genetic transformation systems for garlic using particle bombardment. However, the transformation efficiency is generally low. While efficient garlic regeneration has been achieved through inducing garlic shoot primordia callus and constructing traditional somatic embryogenesis and organogenesis systems, the limitations of explants are significant. Research on the efficient RTB-mediated regeneration and genetic transformation of novel somatic embryo structures in garlic has not yet been systematically reported, greatly limiting the advancement of basic research and the expansion of production applications for garlic and other Liliaceae plants. Summary of the Invention

[0007] The purpose of this invention is to provide a method for inducing the formation of garlic tubers.

[0008] Another object of the present invention is to provide a method for genetic transformation mediated by garlic tubers.

[0009] The method for inducing the formation of garlic tubers according to the present invention includes the following steps: (1) Cultivate aseptic garlic seedlings on aseptic culture medium; (2) Under room temperature and dark culture conditions, garlic explants were placed on rhizogenes induction medium RIM to induce rhizogenes. The formulation of the rhizogenes induction medium RIM was: MS salt, MS organic, 15.0 mg / L NAA, 15.0 g / L sucrose, 3.45 g / L gellan gum, pH 5.8. (3) Under room temperature and light conditions, the obtained rhizomorphs together with the parent tissue were placed on RTB induction medium to induce RTB. The RTB induction medium was formulated as follows: MS salt, MS organic, 20.0 mg / L LTDZ, 30 g / L sucrose, 3.45 g / L gellan gum, pH 5.8.

[0010] According to the method for inducing the formation of garlic tubers according to the present invention, the garlic explant is a root segment, bulbil, bulbil disc, stem tip segment, leaf segment, or garlic scape segment of garlic.

[0011] According to the method for inducing the formation of garlic tubers according to the present invention, in step (3), the light intensity is 180 µmol·m⁻¹. -2 s -1 .

[0012] According to the method for inducing the formation of garlic tubers according to the present invention, in step (1), the aseptic culture medium is formulated as follows: 1 / 2MS salt, 1 / 2MS organic, 10 g / L sucrose, 6.5 g / L agar powder, pH 5.8.

[0013] The method for garlic tuber-mediated genetic transformation according to the present invention includes the following steps: (1) Cultivating aseptic garlic seedlings; (2) Select sterile garlic seedlings as explants, infect them with Agrobacterium in a transformation and permeation medium, and then incubate them in a co-culture medium. The formulation of the transformation and permeation medium is: 1 / 2 MS salt, 1 / 2 B5 organic, 0.03% silwet L-77, 0.01 mg / L 6-BA + 50 g / L sucrose, pH 5.8; (3) Under dark conditions, the co-cultured garlic explants were transferred to RTB induction-selection medium for RTB induction and screening of RTB resistance. The RTB induction-selection medium was formulated as follows: MS salt, MS organic, 2.50 mg / L NAA, 2.50 mg / L 2,4-D, 25 mg / L hygromycin B, 500 mg / L carbenicillin sodium + 30 g / L sucrose, 3.45 g / L gellan gum, pH 5.8; (4) Under light conditions, positive garlic explants were induced to germinate in embryo germination medium to obtain positive regenerated seedlings. The embryo germination medium was formulated as follows: MS salt, MS organic, 2.5 mg / L 6-BA, 30 g / L sucrose, 6.5 g / L agar powder, pH 5.8. (5) Place the regenerated seedlings in rooting medium RM and keep them under light conditions (25℃, 16h light / 8h dark, illuminance of 180 µmol·m⁻¹). -2 s -1 Inducing the embryo to take root and develop into a seedling.

[0014] According to the garlic tuber-mediated genetic transformation method of the present invention, in step (4), the light intensity is 180 µmol·m⁻¹. -2 s -1 .

[0015] Beneficial technical effects: 1. According to the technical solution of this application, the dual starvation treatment (MS component and carbon source) designed in the garlic sterile seedling culture medium can significantly increase the number of roots to 25-30, while the number of roots induced by conventional MS culture medium (no element starvation and carbon source starvation) is 15-20.

[0016] 2. According to the technical solution of this application, in the induction step of garlic roots, the carbon source is hemisugar starvation (15.0 g / L), which is completed by high concentration (10 mg / L) NAA in synergistic dark induction; high concentration (20.0 mg / L) TDZ and strong light (180 µmol / L) are used. -2 s -1 The induction of RTB structure was achieved through a combination of factors. The induction medium for garlic roots and RTB did not involve any missing components, while the carbon source was supplied using hemisugar starvation (15.0 g / L), synergistically combined with a high concentration (20.0 mg / L) of TDZ and strong light intensity (180 µmol / m²). -2 s -1 The treatment of this method, compared with existing methods such as the induction of root tubers in physalis, is equivalent to inventing a new starvation method. Attached Figure Description

[0017] Figure 1 This diagram illustrates the induction and development of RTB in garlic root explants. Figure A shows early-stage rhizomes produced 10 days after inoculation; Figure B shows early-stage rhizomes produced 15 days after inoculation; Figure C shows early-stage rhizomes produced 20 days after inoculation; Figure D shows early-stage rhizomes produced 25 days after inoculation; Figure E shows early-stage rhizomes produced 30 days after inoculation; Figure F shows mid-stage rhizomes produced 35 days after inoculation; Figure G shows mid-stage rhizomes produced 40 days after inoculation; Figure H shows mid-stage rhizomes produced 45 days after inoculation; Figure I shows mid-stage rhizomes produced 50 days after inoculation; Figure J shows mid-stage rhizomes produced 55 days after inoculation; Figure K shows mid-stage rhizomes produced 60 days after inoculation; Figures L and L1 show rhizomes produced 70 days after inoculation. The images show the front and back of mature rhizomes produced 80 days after inoculation, M and M1 respectively, the front and back of mature rhizomes produced 90 days after inoculation, O image is a longitudinal section of early-stage rhizomes produced 10 days after inoculation, O1 image is a longitudinal section of early-stage rhizomes produced 20 days after inoculation, O2 image is a longitudinal section of early-stage rhizomes produced 30 days after inoculation, O3 and O4 images are longitudinal sections of early-stage rhizomes produced 60 days after inoculation, O4 image is an enlarged version of O3 image showing the root cap, O5 image is a longitudinal section of early-stage rhizomes produced 80 days after inoculation showing the Fast-cell-division zone (FCDZ) of the epidermis, and O6 image is a longitudinal section of early-stage rhizomes produced 90 days after inoculation showing the epidermis and root cap structure of inflatable rhizomes. Figure 2This image shows frozen sections illustrating the process of RTB formation induced by garlic rhizomes and their development. Figure A shows the early-stage RTB structure 20 days after rhizome induction; Figure A1 shows the mid-stage RTB structure 35 days after rhizome induction; Figure A2 shows the mature RTB structure 50 days after rhizome induction; Figures B and B1 are longitudinal frozen sections of RTB structures developed for 15 days, showing the cortex and potential cell division pools; Figure B2 is a longitudinal frozen section of RTB structures developed for 20 days, showing residual rhizomes; Figures B3 and B4 are longitudinal frozen sections of RTB structures developed for 20 days, showing numerous proembryos; Figures B5 and B6 are longitudinal frozen sections of RTB structures developed for 30 days, showing swollen and irregular morphology; Figures B7 and B8 are longitudinal frozen sections of RTB structures developed for 35 days, showing the early globular embryo stage; Figures B9 and B11 are longitudinal frozen sections of RTB structures developed for 50 days, showing torpedo-shaped transitional embryos; Figures C through C6 illustrate the process of RTB development into a clump-like regenerated plant. Figure 3 Display pCAMBIA2300- TkTCS -OE interaction analysis of garlic bulbils with *Penicillium chrysogenum* and *Phytophthora alli*. Figure A shows the phenotype of wild-type garlic bulbils interacting with *Penicillium chrysogenum* for 17 days; Figure A1 is a magnified version of Figure A; Figure A2 shows the mycelial growth of *Penicillium chrysogenum* after 17 days of interaction; Figure A3 is a magnified version of Figure A2; Figure B shows pCAMBIA2300- TkTCS - Phenotypic interaction between OE buds and Penicillium chrysogenum over 17 days. Figure B1 is a magnified version of Figure B, and Figure B2 is a pCAMBIA2300-. TkTCS - OE bulbils interacted with *Penicillium chrysogenum* for 17 days, showing *Penicillium chrysogenum* mycelial growth. Figure B3 is a magnified version of Figure B2. Figure C shows the phenotype of wild-type garlic bulbils interacting with *Phytophthora indicum* for 28 days, with Figure C1 being a magnified version of Figure C. Figure C2 shows the mycelial growth of *Penicillium chrysogenum* after 28 days of interaction between wild-type garlic bulbils and *Phytophthora indicum*, with Figure C3 being a magnified version of Figure C2. Figure D shows pCAMBIA2300- TkTCS - The phenotypes of garlic bulbils and Phytophthora alligatoris after 28 days of interaction are shown in Figure D1, Figure D2 shows the mycelial growth of Phytophthora alligatoris after 28 days of interaction between pCAMBIA2300-TkTCS-OE garlic bulbils and Phytophthora alligatoris, and Figure D3 is a magnification of Figure D2. Detailed Implementation

[0018] The invention will now be described in detail with reference to garlic.

[0019] Table 1 Abbreviations, Names and Components of Culture Media Used .

[0020] According to the technical solution of this application, the following improvements have been made to the induction of garlic tuberous root formation: 1) All components (inorganic salts, organic matter) of the sterile seedling MS medium were halved, along with a low concentration (10.0 g / L) of carbon source. A dual starvation treatment (MS component, carbon source) was designed to trigger a "survival stress response" in the plant through "nutrient signal regulation," while simultaneously reducing nutrient competition between the upper parts and roots. This creates physiological conditions conducive to root differentiation and elongation, which is more beneficial for the rapid development of the garlic sterile seedling root system, thereby increasing the number of rhizosomes and RTB-induced primitive explants. The dual starvation treatment (MS component, carbon source) designed in the garlic sterile seedling medium significantly increased the number of roots to 25-30, while the number of roots induced by normal MS medium (no elemental starvation and no carbon source starvation) was only 15-20.

[0021] 2) A hemisugar starvation (15.0 g / L) induction method was designed in the rhizomorph induction medium to achieve efficient rhizomorph generation; 3) High concentration (20.0 mg / L) TDZ with strong light (180 µmol·m⁻¹) -2 s -1 This enabled the reprogramming of garlic cell fate, regulation of hormone signaling networks, and remodeling of key physiological metabolism, thereby initiating the efficient induction of RTB structure.

[0022] The garlic tuber-mediated genetic transformation according to this application involves the following improvements: 1) The Agrobacterium transformation osmotic medium used was 1 / 2 MS salt + 1 / 2 B5 organic components. This balanced the "osmotic pressure requirement" and "material physiological tolerance," avoiding hyperosmotic damage, reducing ion toxicity, ensuring osmotic efficiency, and preventing "nutrient overload" from inhibiting cell activity. If MS medium were used as the basic osmotic medium and co-culture medium for Agrobacterium transformation, positive rhizogenes and RTBs were not obtained, and transformation was unsuccessful. B5 was chosen instead of MS as the organic component because, under in vitro explant culture conditions, exogenous vitamin supplementation can significantly maintain cell activity and regulate metabolic efficiency. Transgenic operations cause a certain degree of mechanical damage, leading to the production of reactive oxygen species (ROS). Excessive ROS can damage cell membranes, nucleic acids, and proteins. B5 has a high vitamin content, which increases the vitamin content and helps to scavenge ROS through "antioxidant" action, maintaining cell homeostasis. Silwet L-77 works by reducing liquid surface tension and enhancing cell hydrophilicity, promoting contact and penetration between gene-carrying Agrobacterium and plant tissues, thereby significantly improving transformation efficiency. A low concentration of 0.01 mg / L 6-BA can regulate cell division, differentiation and growth status of explants, providing a "physiological basis" for the integration of exogenous genes, the survival of transformed cells and subsequent plant regeneration.

[0023] 2) Hygromycin B is used as the screening agent in the RTB induction screening medium because it can efficiently penetrate the cell walls and cell membranes of monocotyledonous plants such as garlic. Furthermore, garlic itself lacks the enzyme system to degrade hygromycin B (no natural resistance), and untransformed cells cannot resist its inhibitory effect, thus achieving the experimental effect of "untransformed cells die, transformed cells survive" during screening. If kanamycin is used as the screening agent for garlic transformation, positive rhizogenes and RTB, as well as positive plants, cannot be screened out. Example 1: Induction and genetic transformation of garlic tubers

[0024] (1) Aseptic seedling culture: Select healthy and plump garlic bulblets, first disinfect them with 75% (v / v) ethanol for 30s-1min, then rinse 3-5 times with sterile water; then disinfect them with 0.1% mercuric chloride for 8-10min, and rinse 3-5 times with sterile water; sow the disinfected garlic bulblets according to their growth polarity into tissue culture bottles containing 1 / 2 MS (10 g / L sucrose) aseptic seedling medium. Then, at 25 ± 1°C, 16 h light / 8 h dark, with a light intensity of 180 µmol·m⁻¹ -2 s -1 Under tissue culture conditions, once the garlic aseptic seedlings have grown to 2-3 true leaves, four types of explants can be obtained: root segments, bulbils, bulbil discs, and stem tips. In addition, by directly treating potted seedlings at different growth stages using the above disinfection methods, three types of explants can be obtained: stem segments, leaf segments, and garlic scape segments.

[0025] (2) Seven types of explants obtained, namely garlic root segments (about 1-2 cm long), bulbils (without root primordia and dormant buds), bulbils (about 1-2 mm thick), shoot tips (with one leaf primordia; about 1 cm long), stem segments (about 1-2 cm long), leaf segments (about 1-2 cm long), and garlic scape segments (about 1-2 cm long), were placed in Rhizoid induction medium (RIM) to induce the production of rhizoids.

[0026] (3) The induced rhizoids were transferred to Rhizoid tuber induction medium (RTBIM) to induce RTB formation.

[0027] (4) The induced RTB was transferred to RTBGM (RTB germination medium) to induce embryo germination and obtain regenerated seedlings; (5) Transfer the induced regenerated seedlings to the rooting medium (RM) to induce rooting and develop into seedlings.

[0028] like Figure 1The figures show the induction of rootoids from garlic root explants. Figure A shows early-stage rhizomes produced 10 days after inoculation (scale bar = 1 cm); Figure B shows early-stage rhizomes produced 15 days after inoculation (scale bar = 1 cm); Figure C shows early-stage rhizomes produced 20 days after inoculation (scale bar = 1 cm); Figure D shows early-stage rhizomes produced 25 days after inoculation (scale bar = 1 cm); Figure E shows early-stage rhizomes produced 30 days after inoculation (scale bar = 1 cm); Figure F shows mid-stage rhizomes produced 35 days after inoculation (scale bar = 1 cm); Figure G shows mid-stage rhizomes produced 40 days after inoculation (scale bar = 1 cm); Figure H shows mid-stage rhizomes produced 45 days after inoculation (scale bar = 1 cm); Figure I shows mid-stage rhizomes produced 50 days after inoculation (scale bar = 1 cm); Figure J shows mid-stage rhizomes produced 55 days after inoculation (scale bar = 1 cm). Image K shows the mid-stage rhizomes produced 60 days after inoculation, with a scale bar of 1 cm. Images L and L1 show the front and back views of mature stage rhizomes produced 70 days after inoculation, with scale bars (L and L1) of 1 cm. Images M and M1 show the front and back views of mature stage rhizomes produced 80 days after inoculation, with scale bars (M and M1) of 1 cm. Images N and N1 show the front and back views of mature stage rhizomes produced 90 days after inoculation, with scale bars (N and N1) of 1 cm. Image O shows a longitudinal section of early stage rhizomes produced 10 days after inoculation, with a scale bar of 2 mm. Image O1 shows a longitudinal section of early stage rhizomes produced 20 days after inoculation, with a scale bar of 2 mm. Image O2 shows a longitudinal section of early stage rhizomes produced 30 days after inoculation, with a scale bar of 2 mm. mm; Figures O3 and O4 are longitudinal sections of early-stage rhizomes produced 60 days after inoculation. Figure O4 is an enlarged version of Figure O3, showing the root cap. Scale bar (O3) = 2 mm, scale bar (O4) = 1 mm; Figure O5 is a longitudinal section of early-stage rhizomes produced 80 days after inoculation, showing the epidermis and FCDZ. Scale bar = 1 mm; Figure O6 is a longitudinal section of early-stage rhizomes produced 90 days after inoculation, showing the epidermis and the root cap structure of the inflatable root. Scale bar = 1 mm.

[0029] like Figure 2As shown, Figure A shows the early-stage RTB structure 20 days after rhizobium induction, with scale bars = 2 mm; Figure A1 shows the mid-stage RTB structure 35 days after rhizobium induction, with scale bars = 2 mm; Figure A2 shows the mature RTB structure 50 days after rhizobium induction, with scale bars = 2 mm; Figures B and B1 are longitudinal frozen sections of the RTB structure at 15 days of development, showing the cortex and potential cell division pools, with scale bars (B and B1) = 300 μm; Figure B2 is a longitudinal frozen section of the RTB structure at 20 days of development, showing residual rhizobium, with scale bars = 300 μm; Figures B3 and B4 are longitudinal frozen sections of the RTB structure at 20 days of development, showing numerous protoembryos, with scale bars (B3 and B4) = 300 μm; Figures B5 and B6 are longitudinal frozen sections of the RTB structure at 30 days of development, showing an swollen and irregular morphology, with scale bars (B5 and B6) = 300 μm. μm; Figures B7 and B8 are longitudinal frozen sections of the RTB structure at 35 days of development, showing the early globular embryo stage, with scale bars (B7 and B8) = 300 μm; Figures B9-B11 are longitudinal frozen sections of the RTB structure at 50 days of development, showing the torpedo transitional embryo, with scale bars (B9-B11) = 300 μm; Figures C-C6 show the process of RTB development into a clump-like regenerated plant, with scale bars (C and C1-C3) = 1 cm and scale bars (C4-C6) = 2 cm. Example 2: Study on the effect of root-like induction on garlic explants 2.1 Effects of 2,4-D and NAA on the number of rootoids induced in garlic explants under darkness, semi-sugar starvation, and full-sugar treatments

[0030] Explants were obtained in the same manner as in Example 1. Garlic root segments, shoot tips, and garlic scape segments were cultured on rhizogene induction medium (RIM), which was supplemented with MS salts, MS organics, 3.45 g / L gellan gum, different concentrations (0, 5.0, 10.0, and 15.0 mg / L) of NAA, different concentrations (0, 5.0, 10.0, and 15.0 mg / L) of NAA+2,4-D, and different concentrations (0, 7.5, 15.0, 22.5, and 30.0 g / L) of sucrose, at pH 5.8. Rhizogene formation was induced in complete darkness at 25 ± 1°C.

[0031] Table 2. Effects of 2,4-D and NAA on the number of rootoids induced in garlic explants under darkness, semi-sugar starvation, and full-sugar treatments.

[0032] Note: The average number of rhizomorphs produced per explant is the ratio of the total number of rhizomorphs induced by each treatment to the total number of explants inoculated. The mean and standard error for each treatment were calculated from 3000 explants in 300 culture dishes (30 replicates, 100 explants per replicate). Uppercase and lowercase letters indicate significant differences at the 1% and 5% confidence levels, respectively. Significant differences were analyzed using the Duncan test in SPSS 19.0.

[0033] As shown in Table 2, among all garlic explants—root segments, bulbils (without root primordia and dormant buds), bulbil discs, shoot tips (with one leaf primordia), stem segments, leaf segments, and garlic scape segments—except for root segments, none showed the ability to form rhizomorphs. The results indicate that NAA is crucial for the induction of garlic rhizomorphs; in media without NAA, rhizomorphs failed to form. Among all tested concentrations, 10.0 mg / L NAA induced the highest number of rhizomorphs, reaching 63.50, demonstrating the best effect. The synergistic effect of the combined treatment of 2,4-D and NAA on rhizomorph induction was significantly weaker than that of NAA alone, with a maximum of 13.53 rhizomorphs per explant, only about one-fifth of the number induced by NAA alone. Furthermore, hemisugar starvation was more effective than standard sucrose concentration in increasing the number of rhizomorphs induced in a single root segment explant. Therefore, the optimal induction combination was determined to be the synergistic treatment of 10.0 mg / L NAA and hemisugar starvation. 2.2 Effects of TDZ on RTB induction in garlic explants under dark conditions

[0034] During RTB induction, the rhizomorphs, along with their original explants, need to be transplanted onto RTB induction medium (RTBIM). This medium contains MS salts, MS organics, 3.45 g / L gellan gum, and different concentrations (0, 10.0, 20.0, and 30.0 mg / L) of TDZ, at pH 5.8. The induction process is carried out under strong light (180 µmol·m⁻¹). -2 s -1 (This will be carried out under)

[0035] Table 3. Effects of TDZ on RTB induction in garlic explants under dark conditions.

[0036] Note: The mean number of RTBs induced by a single explant is the ratio of the total number of RTBs induced by each treatment to the total number of explants inoculated. The mean and standard error for each treatment were calculated from 3000 explants in 300 culture dishes (30 replicates, 100 explants per replicate). Uppercase and lowercase letters indicate significant differences at the 1% and 5% confidence levels, respectively. Significant differences were analyzed using the Duncan test in SPSS 19.0.

[0037] As shown in Table 3, the 20.0 mg / L TDZ treatment induced a significantly greater number of RTBs compared to the 10.0 and 30.0 mg / L TDZ treatments. In this treatment, 90.12% of the rhizomorphs developed into RTB structures, a slightly higher percentage than the 87.47% and 87.78% in the 10.0 and 30.0 mg / L TDZ treatments, respectively. Furthermore, no RTBs were induced in the medium without TDZ. Example 3: Genetic transformation mediated by garlic tubers

[0038] Build includes TkTCS Recombinant plasmid pCAMBIA2300 -TkTCS -OE, TkTCS This gene encodes trichosanthin (TCS) pollen protein from the Cucurbitaceae plant *Trichosanthes kirilowii*. Clinical studies have shown that its encoded product can inhibit tumor growth and exhibits broad-spectrum resistance to various human and animal viruses, including HIV and hepatitis B. The following steps were used to transform and integrate this gene into the garlic genome, enabling its expression. The resistance of transgenic garlic (bulbs) to *Penicillium chrysogenum* and *Phytophthora alli* was then analyzed.

[0039] (1) The collected Agrobacterium cell precipitate was resuspended in the Agrobacterium transformation and infiltration medium (TIM) to ultimately reduce its OD. 600 The concentration should reach 0.5-0.6. During the infection of garlic explants, shake continuously for 8-10 minutes to ensure full contact between the explants and the transformation medium. After infection, remove the explants from the transformation medium, first absorb excess transformation medium with sterile filter paper, then spread them evenly on co-culture medium (C-CM) for incubation. Then place them in a constant temperature incubator at 28℃ in the dark for 72 hours for co-culture treatment.

[0040] (2) Under dark conditions, garlic explants after co-culture were transferred to RTB induction-selective medium RTBISM (RTBinduction-selective medium) to induce RTB and screen for RTB resistance; (3) Under illumination conditions (illuminance of 180 µmol·m⁻¹) -2 s -1 Positive RTB was used to induce embryo germination in RTBGM embryo germination medium to obtain positive regenerated seedlings; (4) Place the regenerated seedlings in rooting medium RM and keep them under light conditions (25℃, 16h light / 8h dark, illuminance of 180 µmol·m⁻¹). -2 s -1 Inducing the embryo to take root and develop into a seedling; (5) After the regenerated seedlings induced by RTB were opened and hardened off for 7 days, the roots were rinsed with a small stream of clean water to remove any remaining agar blocks. The seedlings were then transplanted into a culture medium (vermiculite:potting soil = 1:1) and kept under low light (90 µmol·m⁻¹). -2 s -1 The plants were kept moist under plastic wrap. After 15 days, the plastic wrap was removed, and the plants were transplanted to the field for further cultivation.

[0041] Pathogenicity testing was performed using a fungal spore suspension inoculation method. Spore suspensions of *Penicillium chrysogenum* (ACCC31569) and *Phytophthora indicum* (ACCC30382) were incubated at 28°C and 180 rpm for 8 h. Spores were then collected by centrifugation at 5000×g for 10 min. The spore precipitate was resuspended in a 0.05% Tween-20 aqueous solution to achieve a concentration of 10. 4 -10 6 CFU / mL was used for subsequent disease detection. Garlic bulbs infected with *Penicillium chrysogenum* for 17 h and with *Phytophthora alligatorii* for 28 h were harvested. They were then soaked in DAB solution (1 mg / mL, pH 3.8) for 8–12 h until visible DAB staining appeared on the bulb surface. The stained bulbs were then fixed and stained with 0.6% (w / v) Coomassie Brilliant Blue R250 in methanol. The epidermis of the bulbs was observed under a differential interference contrast microscope (BX61, Olympus), and images were captured using a color camera with integrated image analysis software (Image-Pro Plus 4.1, Media Cybernetics). TkTCS Microscopic samples of five biological replicates were selected from both OE garlic plants and wild-type plants.

[0042] like Figure 3As shown, Figure A shows the phenotype of wild-type garlic bulbils interacting with *Penicillium chrysogenum* for 17 days, with a scale bar of 1 cm; Figure A1 is a magnified version of Figure A, with a scale bar of 0.5 cm; Figure A2 shows the mycelial growth of *Penicillium chrysogenum* after 17 days of interaction between wild-type garlic bulbils and *Penicillium chrysogenum*, with a scale bar of 25 μm; Figure A3 is a magnified version of Figure A2, with a scale bar of 150 μm; Figure B shows pCAMBIA2300- TkTCS - Phenotypic analysis of OE buds and Penicillium chrysogenum over 17 days, scale bar = 1 cm; Figure B1 is a magnified version of Figure B, scale bar = 0.5 cm; Figure B2 is a pCAMBIA2300- TkTCS - OE bulbils interacted with *Penicillium chrysogenum* for 17 days, showing *Penicillium chrysogenum* mycelial growth (scale bar = 25 μm); Figure B3 is a magnified version of Figure B2 (scale bar = 150 μm); Figure C shows the phenotype of wild-type garlic bulbils interacting with *Phytophthora alligatorii* for 28 days (scale bar = 1 cm); Figure C1 is a magnified version of Figure C (scale bar = 0.5 cm); Figure C2 shows the *Penicillium chrysogenum* mycelial growth after 28 days of interaction between wild-type garlic bulbils and *Phytophthora alligatorii* (scale bar = 25 μm); Figure C3 is a magnified version of Figure C2 (scale bar = 150 μm); Figure D shows pCAMBIA2300- TkTCS -OE garlic bulbils and Phytophthora allium 'OE' were compared phenotypes over 28 days, scale bar = 1 cm; Figure D1 is a magnified version of Figure D, scale bar = 0.5 cm; Figure D2 is a pCAMBIA2300- TkTCS -OE garlic bulbs were interacted with Phytophthora alligatoris for 28 days. Phytophthora alligatoris mycelial growth, scale bar = 25 μm; D3 is a magnified version of D2, scale bar = 150 μm.

[0043] Wild-type garlic bulbs rapidly exhibited hypersensitive cellular responses when inoculated with suspensions of *Penicillium chrysogenum* and *Phytophthora alli* spores, respectively, with fungal growth and conidiophore formation completed within a short period (Figures 3A-A3 and C-C3). In contrast, in... TkTCS In -OE plants, most cells invaded by Penicillium chrysogenum and Phytophthora alligatorii hyphae rapidly accumulate H2O2 and undergo cell death, thereby inhibiting the further growth of Penicillium chrysogenum. Figure 3 Figures B2 and B3 in the diagram) and the growth of Phytophthora indicum ( Figure 3 (D2 and D3 diagrams in the diagram). TkTCS-OE garlic bulbs had a lower proportion of dead cells than wild-type. Statistical analysis of over 500 hyphae-invaded cells in each microscopic sample indicated that wild-type bulbs and... TkTCS -OE bulbs showed an average proportion of approximately 49% and 12% of HR-positive cells in Penicillium chrysogenum infection, respectively, compared to 75% and 29% in Phytophthora alligatorii infection. Compared to the susceptible wild type, TkTCS -OE bulbs remained viable in most of the cells invaded by hyphae, with the hyphae retaining their normal morphology; this allowed the fungus to continue growing and forming conidiophores. These results indicate that, compared to susceptible wild-type plants, TkTCS -OE bulbs exhibit a later and weaker cellular hypersensitivity response induced by Penicillium chrysogenum and Phytophthora indicum.

[0044] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method for inducing the formation of garlic tubers, characterized in that, The method includes the following steps: (1) Cultivate aseptic garlic seedlings on aseptic culture medium; (2) Under room temperature and dark culture conditions, garlic explants were placed on rhizogenes induction medium RIM to induce rhizogenes. The formulation of the rhizogenes induction medium RIM was: MS salt, MS organic, 15.0 mg / L NAA, 15.0 g / L sucrose, 3.45 g / L gellan gum, pH 5.

8. (3) Under room temperature and light conditions, the obtained rhizomorphs together with the parent tissue were placed on RTB induction medium to induce RTB. The RTB induction medium was formulated as follows: MS salt, MS organic, 20.0 mg / L LTDZ, 30 g / L sucrose, 3.45 g / L gellan gum, pH 5.

8.

2. The method for inducing the formation of garlic tubers according to claim 1, characterized in that, The garlic explants are garlic root segments, bulbils, bulbils, stem tips, leaf segments, or garlic scape segments.

3. The method for inducing the formation of garlic tubers according to claim 1, characterized in that, In step (3), the light intensity is 180 µmol·m⁻¹. -2 s -1 .

4. The method for inducing the formation of garlic tubers according to claim 1, characterized in that, In step (1), the sterile culture medium is formulated as follows: 1 / 2MS salt, 1 / 2MS organic, 10 g / L sucrose, 6.5 g / L agar powder, pH 5.

8.

5. A method for genetic transformation mediated by garlic tubers, characterized in that, The method includes the following steps: (1) Cultivating aseptic garlic seedlings; (2) Select sterile garlic seedlings as explants, infect them with Agrobacterium in a transformation and permeation medium, and then incubate them in a co-culture medium. The formulation of the transformation and permeation medium is: 1 / 2 MS salt, 1 / 2 B5 organic, 0.03% silwet L-77, 0.01 mg / L 6-BA + 50 g / L sucrose, pH 5.8; (3) Under dark conditions, the co-cultured garlic explants were transferred to RTB induction-selection medium for RTB induction and screening of RTB resistance. The RTB induction-selection medium was formulated as follows: MS salt, MS organic, 2.50 mg / L NAA, 2.50 mg / L 2,4-D, 25 mg / L hygromycin B, 500 mg / L carbenicillin sodium + 30 g / L sucrose, 3.45 g / L gellan gum, pH 5.8; (4) Under light conditions, positive garlic explants were induced to germinate in embryo germination medium to obtain positive regenerated seedlings. The embryo germination medium was formulated as follows: MS salt, MS organic, 2.5 mg / L 6-BA, 30 g / L sucrose, 6.5 g / L agar powder, pH 5.

8. (5) Place the regenerated seedlings in rooting medium RM and keep them under light conditions (25℃, 16h light / 8h dark, illuminance of 180µmol·m⁻¹). -2 s -1 Inducing the embryo to take root and develop into a seedling.

6. The method for garlic tuber-mediated genetic transformation according to claim 5, characterized in that, In step (4), the illuminance is 180 µmol·m⁻¹. -2 s -1 .

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