Transformation system and method based on leaf-induced adventitious root post-transdifferentiation

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a transformation system and method based on transdifferentiation after inducing adventitious roots by leaves, agrobacterium rhizogenes are utilized to infect the leaves, plant hormones with different concentrations are applied, and a suitable system for inducing the adventitious roots by the leaves is screened and obtained; meanwhile, a kanamycin tolerance experiment is carried out, a genetic transformation system based on induction of adventitious roots by leaves is established, and the infection rate, the positive rate and the homozygote rate of the leaves are remarkably increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a transformation system and method based on leaf-induced adventitious root transdifferentiation. Background Art

[0002] The regenerative capacity of plants depends on their ability to regenerate from scratch. De novo organogenesis refers to the process of regeneration through differentiation of meristem centers without going through the somatic embryogenesis stage, which includes two modes: direct and indirect. During indirect organogenesis, cells need to undergo a dedifferentiation process to induce the formation of non-embryonic callus; during direct organogenesis, plant somatic cells can break through the limitations of their original fate under specific conditions and directly form new organs or tissues through transdifferentiation. For example, buds or roots can be induced from existing meristems or damaged organs without going through the callus stage. Studies in Arabidopsis thaliana have shown that the developmental direction of new organs can be regulated by adjusting the ratio of auxin and cytokinin in the culture medium. Taking the two-step method as an example, the root explants are cultured in callus-inducing medium (CIM) with a high auxin / cytokinin ratio, and then transferred to shoot-inducing medium (SIM) with a high cytokinin / auxin ratio. The lateral root primordia can directly transdifferentiate into shoot primordia without going through the dedifferentiation and redifferentiation stages to form callus tissue.

[0003] Transgenic mosaicism is a common phenomenon in the genetic transformation of various species, likely stemming from the multicellular origin of bud organogenesis. This characteristic allows non-transformed cells to be protected by nearby transformed cells, resulting in chimeric transformants. Therefore, while some positive plants may pass various transformation assays, their leaves may appear fully transformed, non-transformed, or in a chimeric state, leading to genetic instability during in vitro micropropagation. Chimeric transgenic plants inevitably produce chimeric gene-edited offspring. For plants capable of sexual reproduction (such as perennial grasses), self-pollination of T0 transformants is typically required to obtain homozygous transgenic plants. However, for asexually reproducing perennial plants (such as poplars), self-pollination of T0 transformants is difficult to obtain homozygous transgenic offspring.

[0004] As an important fast-growing and high-yield forest tree species, poplar has the characteristics of fast growth, long life, tall and straight, good material, strong adaptability, and easy regeneration. It has high economic value and ecological benefits. Due to its small genome and fast reproduction rate, poplar has been widely studied as a model plant among woody plants. Existing studies mostly use the method of Agrobacterium tumefaciens infection to genetically transform poplars, but the phenomenon of a high proportion of chimeras in transgenic offspring has always been common, causing the transgenic phenotype to be gradually lost in generations of in vitro rapid propagation. Therefore, how to improve the homozygous ratio of the poplar genetic transformation system is a difficult problem that needs to be solved urgently. Summary of the Invention

[0005] The present invention provides a transformation system and method based on the transdifferentiation of adventitious roots induced by leaves.

[0006] The technical solutions of the present invention are as follows: The present invention provides a transformation system based on leaf induction and subsequent transdifferentiation of adventitious roots, comprising: Infection suspension containing Agrobacterium rhizogenes; Leaf adventitious root induction medium: contains MS medium, sucrose, indolebutyric acid, naphthaleneacetic acid, and tissue culture agar; Callus induction medium: contains B5 medium, sucrose, naphthaleneacetic acid, and tissue culture agar; Adventitious bud induction differentiation medium I: contains B5 medium, sucrose, isopentenylaminopurine, and tissue culture agar; Adventitious bud induction differentiation medium II: contains MS medium, sucrose, benzyl adenine, and tissue culture agar; Rooting medium: contains MS medium, sucrose, indolebutyric acid, naphthaleneacetic acid, and tissue culture agar.

[0007] Furthermore, the leaf adventitious root induction medium contains: 2.35-2.38 g / L MS medium, 29-31 g / L sucrose, 0.025 mg / L indolebutyric acid, 0.01 mg / L naphthaleneacetic acid, and 7.8-8.2 g / L tissue culture agar; Callus induction medium: contains 3.19-3.23 g / L B5 medium, 29-31 g / L sucrose, 10 μM naphthaleneacetic acid, and 7.8-8.2 g / L tissue culture agar; Adventitious bud induction differentiation medium I: contains 3.19-3.23 g / L B5 medium, 30 g / L sucrose, 8.8 μM isopentenylaminopurine, and 8 g / L tissue culture agar; Adventitious bud induction differentiation medium II: contains 4.74g / L MS medium, 29-31g / L sucrose, 0.5mg / L benzyl adenine, and 7.8-8.2g / L tissue culture agar; Rooting medium: contains 2.35-2.38 g / L MS medium, 29-31 g / L sucrose, 0.1 mg / L indolebutyric acid, 0.01 mg / L naphthaleneacetic acid, and 7.8-8.2 g / L tissue culture agar.

[0008] Furthermore, the leaf adventitious root induction medium contains: 2.37 g / L MS medium, 30 g / L sucrose, 0.025 mg / L indolebutyric acid, 0.01 mg / L naphthaleneacetic acid, 8 g / L tissue culture agar, pH = 5.8; Callus induction medium: containing 3.21 g / L B5 medium, 30 g / L sucrose, 10 μM naphthaleneacetic acid, 8 g / L tissue culture agar, pH = 5.8; Adventitious bud induction differentiation medium I: contains 3.21 g / L B5 medium, 30 g / L sucrose, 8.8 μM isopentenylaminopurine, 8 g / L tissue culture agar, pH = 5.8; Adventitious bud induction differentiation medium II: contains 4.74 g / L MS medium, 30 g / L sucrose, 0.5 mg / L benzyl adenine, 8 g / L tissue culture agar, pH = 5.8; Rooting medium: contains 2.37 g / L MS medium, 30 g / L sucrose, 0.1 mg / L indolebutyric acid, 0.01 mg / L naphthaleneacetic acid, 8 g / L tissue culture agar, pH = 5.8.

[0009] Furthermore, the infection suspension contains water, MS culture medium and sucrose.

[0010] The present invention also provides a transformation method based on inducing adventitious roots from leaves and then transdifferentiating them using the system, comprising: (1) Using Agrobacterium rhizogenes as the material, after activation and culture, centrifuge and discard the supernatant, add the infection suspension to obtain the infection suspension containing Agrobacterium rhizogenes; (2) Select a leaf, remove the leaf tip, and cut it vertically along the main vein to use as an explant; (3) The explants were placed in an infection suspension containing Agrobacterium rhizogenes, incubated, and then placed in a leaf adventitious root induction medium containing acetosyringone and cultured in the dark; (4) After dark culture, the explants were washed with ultrapure water containing Timentin and then ultrapure water, and the leaves were dried; (5) The washed explants were placed in a leaf adventitious root induction medium containing timentin and kanamycin to induce adventitious roots; (6) The induced adventitious roots are placed in callus induction medium, adventitious bud induction differentiation medium I, and adventitious bud induction differentiation medium II in sequence to induce adventitious buds; (7) Use rooting medium containing timentin and kanamycin to culture adventitious buds.

[0011] Furthermore, the Agrobacterium rhizogenes in step (1) is an Agrobacterium rhizogenes carrying a pHDE-RUBY vector.

[0012] Furthermore, in step (5), the concentration of kanamycin is 3.75 mg / L based on the volume of the leaf adventitious root induction culture medium.

[0013] Furthermore, in step (1), the activated culture is carried out until the bacterial solution OD 600 to 0.5-0.7, centrifuged.

[0014] Furthermore, in step (6), the cells are cultured in callus induction medium for 1 day, in adventitious bud induction differentiation medium I for 10 days, and in adventitious bud induction differentiation medium II for 40-75 days.

[0015] Furthermore, in step (6), the amount of timentin added to the entire culture medium for inducing adventitious buds is 100 mg / L.

[0016] The leaves were infected by the method, with an infection rate of 8.47%, a positive rate of 53.63%, and a homozygous rate of 58.13%.

[0017] Beneficial effects of the present invention: 1. The present invention established a tissue culture system based on leaf-induced adventitious roots. After infecting leaves with Agrobacterium rhizogenes and applying different concentrations of plant hormones, a suitable system for leaf-induced adventitious roots was screened and obtained. At the same time, kanamycin tolerance experiments were conducted to establish a genetic transformation system based on leaf-induced adventitious roots. 2. After obtaining positive adventitious roots, the present invention combines the poplar lateral root primordium transdifferentiation system to obtain positive seedlings, which significantly improves the infection rate, positive rate and homozygous rate of poplar compared with the traditional poplar leaf disk method transformation system. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the linear structure diagram of the pHDE-RUBY plant expression vector plasmid.

[0019] Figure 2 Photos of adventitious root growth (AE), induction rate (F) and number of induced roots (G) in adventitious root induction medium from different leaves.

[0020] Figure 3 The effects of different concentrations of kanamycin on adventitious root induction in leaves, where AD are photos of adventitious root growth, E is the adventitious root induction rate, and F is the number of induced adventitious roots.

[0021] Figure 4 Figure 2 shows the phenotypic characteristics of chimeric and homozygous seedlings in the positive transgenic poplar seedlings as a control example, where A is a chimeric leaf, B is a homozygous leaf, C is a wild-type plant, D is a chimeric plant, and E is a homozygous plant.

[0022] Figure 5 The figures are the phenotypic characteristics of different genetic transformation systems of poplar, wherein A is the positive adventitious root induced by the leaves of the embodiment; B is the adventitious bud formed by the transdifferentiation of the positive adventitious root of the embodiment; C and D are the positive adventitious buds induced by the leaves of the control example after culturing for 65 days and 80 days, respectively. DETAILED DESCRIPTION

[0023] The technical solution of this application is described in detail below with reference to specific embodiments.

[0024] The culture medium used in the following examples and control examples is shown in Table 1: Table 1 Culture medium formula Example (1) Using K599 rhizogenes Agrobacterium carrying pHDE-RUBY vector as material (e.g. Figure 1 As shown in the figure), activate the culture with 20 mL of liquid YEP medium containing 50 mg / L Spec and 100 mg / L streptomycin (Str). 600 When the value is around 0.7, centrifuge at 2000 g for 10 minutes at room temperature. Discard the supernatant, add the infection suspension (the formula of the infection suspension is: 0.221 g MS and 3 g sucrose per 100 mL of water), and gently shake to resuspend. This will produce an infection suspension containing Agrobacterium rhizogenes. (2) When the wild-type 84K poplar plants are growing well for 4-6 weeks, select the 3rd to 5th leaves in good condition as explants. After removing the leaf tip, cut the leaves 2 to 3 times vertically along the main vein to use as explants for Agrobacterium infection; (3) The explants were placed in an infection suspension containing Agrobacterium rhizogenes and incubated on a shaker at 220 rpm for 20 min. The explants were then placed on sterile filter paper to remove excess bacterial liquid. The explants were then placed in leaf root induction medium (LRM) containing 80 μM acetosyringone and cultured in the dark for 48 h. (4) After dark culture, the explants were washed five times with 100 mL of sterile ultrapure ddH2O, with 100 mg / L of timentin added for the first three times. The surface moisture of the leaves was then absorbed dry using sterile filter paper. (5) The washed explants were plated on leaf adventitious root induction medium (LRM) containing 100 mg / L timentin and 3.75 mg / L kanamycin for culture. Fresh medium was replaced every two weeks to achieve adventitious root induction. (6) When the induced adventitious roots (positive adventitious roots) extend to 1-3 cm, they are cut and placed on callus induction medium (CIM) for 1 day, then transferred to adventitious bud induction differentiation medium I (SIM-I) for 10 days, and finally transferred to adventitious bud induction differentiation medium II (SIM-II) for 73 days to achieve adventitious bud induction; (7) When the transdifferentiated positive buds on the root segment have grown to 3-5 cm, they are transferred to rooting medium (RM) containing 100 mg / L timentin and 50 mg / L kanamycin to culture into seedlings.

[0025] Control Example (1) Using the GV3101 Agrobacterium tumefaciens strain carrying the pHDE-RUBY vector as the material, activate the culture with 20 mL of liquid YEP medium containing 50 mg / L Spec and 100 mg / L streptomycin (Str). 600 When the value is around 0.7, centrifuge at 2000 g for 10 minutes at room temperature. Discard the supernatant, add the infection suspension (the formula of the infection suspension is: 0.221 g MS and 3 g sucrose per 100 mL of water), and gently shake to resuspend. This will produce an infection suspension containing GV3101 Agrobacterium. (2) When the wild-type 84K poplar plants are growing well for 4-6 weeks, select the 3rd to 5th leaves in good condition as explants. After removing the leaf tip, cut the leaves 2 to 3 times vertically along the main vein to use as explants for Agrobacterium infection; (3) The explants were placed in an infection suspension containing GV3101 Agrobacterium and incubated on a shaker at 220 rpm for 20 min. The explants were then placed on sterile filter paper to remove excess bacterial liquid. The explants were then placed in leaf root induction medium (LRM) containing 80 μM acetosyringone and cultured in the dark for 48 h. (4) After dark culture, the explants were washed five times with 100 mL of sterile ultrapure ddH2O, with 100 mg / L of timentin added for the first three times. The surface moisture of the leaves was then absorbed dry using sterile filter paper. (5) The washed explants were spread on 84K poplar leaf differentiation medium containing 100 mg / L timentin and 50 mg / L kanamycin. Fresh medium was replaced every two weeks for a total of 80 days of adventitious bud induction.

[0026] (6) When the positive buds grow to 3-5 cm, they are transferred to rooting medium (RM) containing 100 mg / L timentin and 50 mg / L kanamycin for culture.

[0027] Experimental analysis 1. To investigate the effects of the components of leaf root induction medium (LRM) on leaf root induction In addition to the example (denoted as Group 2), four other culture medium groups were set up (denoted as control, Group 1, Group 3, and Group 4). The specific settings are shown in Table 2. By changing the culture medium components, the effects of the components (hormone types and concentrations) of the leaf adventitious root induction medium (LRM) on the induction frequency and number of adventitious roots were investigated.

[0028] Specifically, except for the different leaf adventitious root induction medium (LRM) in step (3), in step (5), the operation of the control, group 1, group 2, group 3, and group 4 is as follows: the washed explants are spread on the corresponding LRM medium containing 100 mg / L timentin, and fresh medium is replaced every two weeks.

[0029] After culturing for 5 weeks in step (5), the adventitious root induction rate and number of induced adventitious roots were counted for the control, group 1, group 2, group 3, and group 4.

[0030] The specific statistical formula is as follows: Adventitious root induction rate = number of leaves that eventually induced adventitious roots / total number of infected leaves × 100%; The number of induced adventitious roots = the total number of induced adventitious roots / the total number of infected leaves.

[0031] Table 2 Composition of leaf root induction medium (LRM) The results are as follows Figure 2 As shown, the adventitious root induction rate of Group 2 (i.e., the example of the present invention) reached 100%, with 8 adventitious roots induced per leaf. This shows that LRM medium, which is obtained by adding 0.025 mg / L IBA and 0.01 mg / L NAA to MS medium, is conducive to the induction of adventitious roots in leaves.

[0032] 2. Investigate the effects of different concentrations of kanamycin on adventitious root induction in leaves For comparison, the kanamycin concentration in step (5) of Example 1 was changed to study the effect of different concentrations of kanamycin on adventitious root induction in leaves.

[0033] Specifically, the cleaned explants were plated onto leaf root induction medium (LRM) containing 100 mg / L timentin and varying concentrations of kanamycin (0, 1.25, 2.5, and 3.75 mg / L). Fresh medium was replaced every two weeks. After five weeks of culture, the rate and number of induced adventitious roots were counted.

[0034] The results are as follows Figure 3 As shown, when 3.75 mg / L kanamycin was added to LRM medium, the production of adventitious roots could be completely inhibited.

[0035] 3. Exploring the transformation differences between different genetic transformation systems The example (adventitious root induction followed by transdifferentiation from leaves) was compared with the control example (adventitious bud induction from leaves) to explore the transformation differences between different genetic transformation systems.

[0036] Specifically, after the incubation period, plants exhibiting a red phenotype throughout their tissues were identified as homozygous individuals. Each genetic transformation experiment was performed with three biological replicates, with 30 samples per replicate. All data were analyzed using one-way ANOVA with Graphpad Prism 10 software, and multiple comparisons were evaluated using T-tests (p < 0.05). (Data are presented as percentages and were square-root arcsine transformed before analysis.)

[0037] The specific statistical formula is as follows: Infection rate = number of leaves with positive roots or positive buds / total number of infected leaves × 100%; Homozygous rate = number of homozygous seedlings / total number of positive seedlings × 100%; Positive rate = total number of positive seedlings / total number of induced buds × 100%.

[0038] The specific results are shown in Table 3. Figure 4 、 Figure 5 ,in Figure 4 As a control example, after the GV3101 tumefaciens carrying the pHDE-RUBY vector infected the leaves of 84K poplar, the same batch of adventitious buds induced three phenotypes after growing into seedlings, among which A is a chimeric leaf, B is a homozygous leaf, C is a wild-type plant, D is a chimeric plant, and E is a homozygous plant. Figure 5The figures show the phenotypes during the cultivation process of two genetic transformation systems: leaf-induced adventitious buds (control example) and leaf-induced adventitious roots, followed by root-to-bud regeneration (example). The red roots in A are positive adventitious roots produced on the 24th day during the leaf-induced adventitious rooting process of the example; the red buds in B are positive buds regenerated from the positive adventitious roots of the example through root-to-bud regeneration. The cultivation day at this time is the 84th day after infection, and a large number of positive buds are clearly visible. The red buds in C and D are positive buds produced on the 65th and 80th days, respectively, during the leaf-induced adventitious buding process of the control example. The positive buds on the 65th day are relatively young and tender, not yet ready for transplantation into a rooting medium, while the positive buds on the 80th day are ready for transplantation into a rooting medium, and a small number of positive buds are clearly visible.

[0039] Table 3 Transformation differences in different genetic transformation systems Note: *Lowercase letters in the same column represent significant differences of 5% among three biological replicates.

[0040] It can be seen that compared with the control example, the infection rate of poplar leaves using the method of the present invention was increased from 6.63% to 8.47%, the homozygous rate was increased from 19.1% to 58.13%, and the positive rate was increased from 7.37% to 53.63%.

Claims

1. A transformation system based on leaf induction and subsequent transdifferentiation of adventitious roots, characterized in that: include: Infection suspension containing Agrobacterium rhizogenes; Leaf adventitious root induction medium: contains MS medium, sucrose, indolebutyric acid, naphthaleneacetic acid, and tissue culture agar; Callus induction medium: contains B5 medium, sucrose, naphthaleneacetic acid, and tissue culture agar; Adventitious bud induction differentiation medium I: contains B5 medium, sucrose, isopentenylaminopurine, and tissue culture agar; Adventitious bud induction differentiation medium II: contains MS medium, sucrose, benzyl adenine, and tissue culture agar; Rooting medium: contains MS medium, sucrose, indolebutyric acid, naphthaleneacetic acid, and tissue culture agar.

2. The transformation system based on leaf induction and subsequent transdifferentiation according to claim 1, characterized in that: The leaf adventitious root induction medium contains: 2.35-2.38 g / L MS medium, 29-31 g / L sucrose, 0.025 mg / L indolebutyric acid, 0.01 mg / L naphthaleneacetic acid, and 7.8-8.2 g / L tissue culture agar; Callus induction medium: contains 3.19-3.23 g / L B5 medium, 29-31 g / L sucrose, 10 μM naphthaleneacetic acid, and 7.8-8.2 g / L tissue culture agar; Adventitious bud induction differentiation medium I: contains 3.19-3.23 g / L B5 medium, 29-31 g / L sucrose, 8.8 μM isopentenylaminopurine, and 7.8-8.2 g / L tissue culture agar; Adventitious bud induction differentiation medium II: contains 4.72-4.76 g / L MS medium, 29-31 g / L sucrose, 0.5 mg / L benzyl adenine, and 7.8-8.2 g / L tissue culture agar; Rooting medium: contains 2.35-2.38 g / L MS medium, 29-31 g / L sucrose, 0.1 mg / L indolebutyric acid, 0.01 mg / L naphthaleneacetic acid, and 7.8-8.2 g / L tissue culture agar.

3. The transformation system based on leaf induction and subsequent transdifferentiation according to claim 1, characterized in that: The leaf adventitious root induction medium contains 2.37 g / L MS medium, 30 g / L sucrose, 0.025 mg / L indolebutyric acid, 0.01 mg / L naphthaleneacetic acid, and 8 g / L tissue culture agar, with a pH of 5.

8. Callus induction medium: containing 3.21 g / L B5 medium, 30 g / L sucrose, 10 μM naphthaleneacetic acid, 8 g / L tissue culture agar, pH = 5.8; Adventitious bud induction differentiation medium I: contains 3.21 g / L B5 medium, 30 g / L sucrose, 8.8 μM isopentenylaminopurine, 8 g / L tissue culture agar, pH = 5.8; Adventitious bud induction differentiation medium II: contains 4.74 g / L MS medium, 30 g / L sucrose, 0.5 mg / L benzyl adenine, 8 g / L tissue culture agar, pH = 5.8; Rooting medium: contains 2.37 g / L MS medium, 30 g / L sucrose, 0.1 mg / L indolebutyric acid, 0.01 mg / L naphthaleneacetic acid, 8 g / L tissue culture agar, pH = 5.

8.

4. The transformation system based on leaf induction and subsequent transdifferentiation according to claim 1, characterized in that: The infection suspension contains water, MS medium and sucrose.

5. A transformation method based on leaf induction and subsequent transdifferentiation of adventitious roots using the system of claim 1, characterized in that: include: (1) Using Agrobacterium rhizogenes as the material, after activation and culture, centrifuge and discard the supernatant, add the infection suspension to obtain the infection suspension containing Agrobacterium rhizogenes; (2) Select a leaf, remove the leaf tip, and cut it vertically along the main vein to use as an explant; (3) The explants were placed in an infection suspension containing Agrobacterium rhizogenes, incubated, and then placed in a leaf adventitious root induction medium containing acetosyringone and cultured in the dark; (4) After dark culture, the explants were washed with ultrapure water containing Timentin and then ultrapure water, and the leaves were dried; (5) The washed explants were placed in a leaf adventitious root induction medium containing timentin and kanamycin to induce adventitious roots; (6) The induced adventitious roots are placed in callus induction medium, adventitious bud induction differentiation medium I, and adventitious bud induction differentiation medium II in sequence to induce adventitious buds; (7) Use rooting medium containing timentin and kanamycin to culture adventitious buds.

6. The transformation method based on leaf induction and subsequent transdifferentiation of adventitious roots according to claim 5, characterized in that: The Agrobacterium rhizogenes in step (1) is an Agrobacterium rhizogenes carrying a pHDE-RUBY vector.

7. The transformation method based on leaf induction and subsequent transdifferentiation of adventitious roots according to claim 5, characterized in that: In the step (5), based on the volume of the leaf adventitious root induction culture medium, the concentration of kanamycin is 3.75 mg / L.

8. The transformation method based on leaf induction and subsequent transdifferentiation of adventitious roots according to claim 5, characterized in that: In step (1), the activated culture is cultured to the OD 600 to 0.5-0.7, centrifuged.

9. The transformation method based on leaf induction and subsequent transdifferentiation of adventitious roots according to claim 5, characterized in that: In step (6), the cells are cultured in callus induction medium for 1 day, in adventitious bud induction differentiation medium I for 10 days, and in adventitious bud induction differentiation medium II for 40-75 days.

10. The transformation method based on leaf induction and subsequent transdifferentiation of adventitious roots according to claim 5, characterized in that: In step (6), the amount of timentin added to the entire culture medium for inducing adventitious buds is 100 mg / L.

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