Broccoli transgenic plant cultivation method based on RUBY visual marker

Abstract

The invention relates to the field of cruciferous crop breeding, and discloses a broccoli transgenic plant cultivation method based on an RUBY visual marker, which comprises the following steps: infecting an explant by adopting agrobacterium rhizogenes K599 carrying an RUBY expression vector, and optimizing a culture medium: a culture medium containing 0.9 mg.L <-1 > TDZ for bud induction and a culture medium containing 0.1 mg.L <-1 > NAA and 0.2 mg.L <-1 > IBA for root induction; and culturing to form a complete plant, and transplanting after acclimatization and domestication. The invention provides a positive material screening method based on an RUBY visual marker. According to the method, in the differentiation culture stage, non-destructive visual judgment can be achieved through phenotypic colors, the screening efficiency is improved by 60% or above, and the detection cost is greatly reduced; by means of an optimized culture medium formula, the bud induction rate reaches 75% or above, the root induction rate reaches 90% or above, and the regeneration period is shortened to 2.5-3 months and is shortened by one month compared with a traditional method. The method is suitable for broccoli transgenic breeding.

Description

Technical Field

[0001] This invention belongs to the field of cruciferous crop breeding, specifically relating to a method for cultivating transgenic broccoli plants based on RUBY visual markers. Background Technology

[0002] Broccoli, an important cruciferous vegetable, is rich in vitamins, flavonoids, and glucosinolates, among other functional components, possessing both nutritional and health benefits, and is widely cultivated globally. With the development of genetic engineering technology, improving the stress resistance, quality, and yield of broccoli through transgenic methods has become an important direction in breeding research.

[0003] Currently, the transgenic cultivation of broccoli mainly relies on the Agrobacterium-mediated transformation system, but the existing technology has the following key problems: (1) Traditional methods mostly use antibiotic resistance markers (such as kanamycin resistance) or reporter genes (such as GFP green fluorescent protein). The former requires positive verification by molecular detection such as PCR after resistance screening, and the latter requires observation with a fluorescence microscope. Both have the defects of being cumbersome, time-consuming, and costly, making it difficult to achieve rapid batch screening; (2) The regeneration of broccoli explants (such as cotyledons and hypocotyls) is sensitive to the composition of the culture medium (especially the hormone ratio). The existing culture medium formula is prone to causing abnormal callus differentiation, low shoot induction rate, or poor root development, and the regeneration cycle often exceeds 3 months; (3) After Agrobacterium infection, explants are easily affected by excessive growth of Agrobacterium. It is necessary to accurately control the antibiotic concentration to inhibit Agrobacterium while protecting the explant activity. The existing methods are difficult to balance the relationship between the two, resulting in large fluctuations in transformation efficiency. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for cultivating transgenic broccoli plants based on RUBY visual markers.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for cultivating transgenic broccoli plants based on RUBY visual markers includes the following steps:

[0007] (1) Material preparation: Select 7-8 day old broccoli seedlings, and use seedlings with roots removed as explants; prepare Agrobacterium rhizogenes K599 bacterial culture (OD) carrying the RUBY marker gene vector. 600 =0.4-0.6);

[0008] (2) Agrobacterium infection and co-culture: The explants were immersed in the bacterial solution for 10 min, the bacterial solution was dried and then transferred to the co-culture medium, and cultured in the dark at 25°C for 2 days.

[0009] (3) Differentiation culture: The explants after co-culture were transferred to the differentiation medium and cultured for about 20 days at 23-25℃ and 16h / d light (2000-3000 lux). Explants with red transgenic roots marked by RUBY at the wound site were screened out.

[0010] (4) Bud induction culture: Cut the red transgenic root system into 0.5-1cm root segments, transfer them to the bud induction culture medium, and culture them under the same culture conditions as in step (3) for 25-30 days to form seedlings 2-3cm high;

[0011] (5) Root induction culture: The seedlings are cut off and transferred to the root induction culture medium, and cultured for 10-15 days under the same culture conditions as in step (3) to form complete plants;

[0012] (6) Acclimatization and transplanting: After the complete plants have been acclimatized by closing the bottle for 3-5 days and opening the bottle for 2-3 days, they are transplanted into nutrient soil and kept moist until they survive.

[0013] Further, the Agrobacterium rhizogenes K599 bacterial culture in step (1) is cultured in LB liquid medium (containing 50 mg·L⁻¹) -1 Kana, 50 mg / L -1 Streptomycin was prepared by shaking and culturing at 28°C and 180 rpm for 16 h.

[0014] Further, in step (2), the co-culture medium is MS medium + 30 g·L⁻¹. -1 sucrose + 7g·L -1 Agar.

[0015] Further, in step (3), the differentiation medium is MS medium + 30 g·L⁻¹. -1 sucrose + 7g·L -1 Agar + 200 mg / L -1 Carboxybenzyl + 200 mg·L -1 Termethin.

[0016] Further, in step (4), the bud induction medium is the differentiation medium + 0.9 mg·L⁻¹ -1 TDZ.

[0017] Further, in step (5), the root induction medium is the differentiation medium + 0.1 mg·L⁻¹ -1 NAA + 0.2 mg·L -1 IBA.

[0018] Furthermore, the matrix formulation in step (6) is peat moss:vermiculite:perlite in a weight ratio of 3:1:1, and is sterilized.

[0019] Beneficial effects: Compared with existing technologies, the advantages of this invention are: combining the RUBY visual marker with Agrobacterium rhizogenes K599 optimizes the culture medium formulation and culture process, which is of great significance for solving existing problems in the transgenic breeding of broccoli. Specifically, the advantages are as follows:

[0020] 1. Using the red root system marked by RUBY, positive materials can be screened by the naked eye during the differentiation culture stage (20 days) without the need for PCR, fluorescence microscopes and other equipment, improving the screening efficiency by more than 60% and significantly reducing the detection cost;

[0021] 2. Through optimized culture medium formulation, especially specific concentrations of TDZ, NAA and IBA, the shoot induction rate can reach over 75%, the root induction rate can reach over 90%, and the regeneration cycle is shortened to 2.5-3 months, which is 1 month shorter than the traditional method;

[0022] 3. Through phased acclimatization treatment, plant wilting caused by environmental stress after transplanting is avoided, and the transplant survival rate is stabilized at over 85%, meeting the needs of subsequent breeding and functional research. Attached Figure Description

[0023] Figure 1 This is a growth diagram of each stage during the cultivation process using the method of the present invention. Detailed Implementation

[0024] This invention uses broccoli seedling explants as recipients and transforms them into a vector containing the RUBY marker gene via Agrobacterium rhizogenes K599. Using a specific culture medium, transgenic root differentiation (visual screening) → shoot induction → root induction are sequentially achieved, ultimately obtaining complete transgenic plants that are then domesticated and transplanted. The invention is described in detail below with specific examples.

[0025] Example 1

[0026] A method for cultivating transgenic broccoli plants based on RUBY visual markers includes the following steps:

[0027] (1) Material preparation:

[0028] 7-8 day old broccoli inbred line (number ZC2060) seedlings were selected. Under aseptic conditions, the seedlings with their roots removed were used as explants, and fresh wounds were retained to facilitate Agrobacterium infection. The hypocotyl length was 0.8-1.2 cm.

[0029] Preparation of Agrobacterium rhizogenes K599 bacterial suspension carrying the RUBY marker gene vector: Agrobacterium rhizogenes K599 was transformed with a vector carrying WUS, IPT, PLT5 growth factors and the RUBY marker gene. After plating, single colonies of Agrobacterium rhizogenes K599 carrying the RUBY marker gene vector were picked, and after PCR positive detection, they were inoculated into a solution containing 50 mg·L⁻¹ of...-1 Kana, 50 mg / L -1 Streptomycin was cultured in LB broth at 28°C with shaking at 180 rpm until the bacterial culture reached OD. 600 A value of 0.4-0.6 indicates the bacterial solution is suitable for infection.

[0030] (2) Agrobacterium infection and co-culture: The explants from step (1) were immersed in the bacterial solution and allowed to stand at 18-22℃ for 10 min for infection; after infection, excess bacterial solution on the surface of the explants was blotted dry with sterile filter paper and transferred to co-culture medium, and incubated at 25℃.

[0031] Dark culture for 2 days; the co-culture medium was MS medium + 30 g·L⁻¹. -1 sucrose + 7g·L -1 Agar, pH 5.8.

[0032] (3) Differentiation culture: The co-cultured explants were transferred to differentiation medium and cultured in an incubator with the following conditions: temperature 23-25℃, light intensity 16h / d, and light intensity 2000-3000 lux for about 20 days until visible RUBY-marked red transgenic roots appeared at the explant wound. Explants with visible RUBY-marked red transgenic roots at the wound were selected; those without red roots were negative and directly discarded. The differentiation medium was MS medium + 30 g·L⁻¹. -1 sucrose + 7g·L -1 Agar + 200 mg / L -1 Carboxybenzyl + 200 mg·L -1 Termetitin, pH 5.8.

[0033] (4) Bud induction culture: The red transgenic roots obtained in step (3) were cut into 0.5-1cm root segments using a sterile knife and transferred to the bud induction medium. They were cultured under the same conditions as in step (3) for 25-30 days. The root segments first swelled to form callus tissue, and the callus tissue further differentiated to form buds, eventually developing into seedlings with a height of 2-3cm. The bud induction medium was the differentiation medium + 0.9 mg·L⁻¹. -1 TDZ. TDZ is a cytokinin-like hormone that can efficiently induce callus tissue to differentiate into buds, significantly improving the bud induction rate.

[0034] (5) Root induction culture: Using a sterile knife, cut off the seedlings obtained in step (4) and transfer them to the root induction medium. Culture them under the same conditions as in step (3) for 10-15 days until robust adventitious roots form at the base of the seedlings, thus obtaining complete transgenic broccoli plants. The root induction medium is the differentiation medium + 0.1 mg·L⁻¹ -1 NAA + 0.2 mg·L -1IBA; NAA works synergistically with IBA to promote root development and avoid root thinning caused by single hormones.

[0035] (6) Acclimatization and transplanting: Transfer the complete plant obtained in step (5) along with the culture bottle to the greenhouse (temperature 22-25℃, humidity 60%-70%). First, close the bottle to harden the seedlings for 3-5 days, then open the bottle cap to harden the seedlings for 2-3 days, so that the plant can gradually adapt to the external environment and obtain the acclimatized plant.

[0036] After acclimatization, remove the plants from the culture medium, gently rinse the roots with clean water to remove any remaining culture medium, and transplant them into a sterilized substrate (by weight, peat moss: vermiculite: perlite = 3:1:1). After transplanting, cover with a shade net to retain moisture for 3-5 days, and then manage water and fertilizer normally. The survival rate can reach over 85%.

[0037] Example 2

[0038] The cultivation method is basically the same as in Example 1, except that the concentration of Agrobacterium rhizogenes is changed.

[0039] The following experiment further illustrates the effects of different concentrations of Agrobacterium rhizogenes on explant induction.

[0040] Table 1 Effects of different concentrations of Agrobacterium rhizogenes on explant induction

[0041]

[0042] As can be seen from the table above, OD 600 =0.4-0.5 Explant viability is high, with certain regeneration potential; OD 600 When OD = 0.6, the bacterial cells are in the middle of the logarithmic growth phase, and the balance between activity and density is optimal; as OD... 600 The value increased from 0.7 to 0.9, and the increase in contamination rate and explant necrosis rate was much greater than the decrease in positive rate, indicating that transformation efficiency decreased rather than increased at high concentrations, the risk of uncontrolled contamination was high, and the explant necrosis rate was high. Therefore, OD 600 Agrobacterium rhizogenes with a concentration of 0.6 is the optimal choice for inducing broccoli explants, followed by OD. 600 =0.4-0.5 Agrobacterium rhizogenes.

[0043] Example 3

[0044] The following experiment was used to verify the optimized culture medium (bud induction medium containing 0.9 mg·L⁻¹) for a specific hormone concentration in this invention. -1 TDZ, root induction medium containing 0.1 mg·L -1 NAA + 0.2 mg·L -1Compared to traditional culture media, IBA (Induced Basalt-Based Acid) demonstrates the significant advantages of optimized formulation in improving shoot induction rate, root induction rate, and regeneration cycle during the transgenic regeneration of broccoli explants.

[0045] 1. Test materials

[0046] Explants: Cotyledons of sterile broccoli seedlings (7 days old, 0.8cm × 0.8cm in size, with fresh wounds on each leaf to ensure uniformity);

[0047] Agrobacterium: Agrobacterium rhizogenes K599 bacterial culture carrying the RUBY marker gene vector (OD) 600 =0.6, preparation method is the same as in Example 1, to ensure consistent batch activity;

[0048] Basic components of the culture medium: MS basal medium, 30 g·L -1 Sucrose, 7g·L -1 Agar, 200 mg·L -1 Carboxybenzyl, 200 mg·L -1 Termetidine (pH 5.8, all groups share the same basic components, only the hormone concentration differs);

[0049] Hormone reagents: TDZ (thiafenuron), NAA (naphthaleneacetic acid), IBA (indolebutyric acid, all analytical grade).

[0050] 2. Experimental group design

[0051] The experiment consisted of one experimental group (Example 1 of the present invention) and six control groups, with each group being repeated three times, and each repetition using 50 explants to ensure data reliability.

[0052] Table 2 Group Design

[0053]

[0054] 3. Experimental Procedure

[0055] The experimental procedures for each group were basically the same as those in Example 1, except that the culture medium hormones were different, and the hormone data used were from Table 2.

[0056] 4. Statistical methods:

[0057] The mean ± standard deviation of the three replicates for each group was calculated using Excel (e.g., bud induction rate of experimental group = (replication 1 + replication 2 + replication 3) / 3, with the standard deviation noted).

[0058] One-way ANOVA was performed using SPSS 26.0 software, and the differences between groups were tested using Duncan's multiple comparison method (P<0.05 was considered significant, and P<0.01 was considered highly significant). The results were labeled with letters (e.g., a, b, c, where different letters indicate significant differences).

[0059] Positive rate = (Number of red root explants / Total number of explants) × 100%;

[0060] Bud induction rate = (Number of positive explants that formed bud points / Total number of positive explants) × 100%;

[0061] Root induction rate = (Number of shoots forming robust root systems / Total number of shoots) × 100%;

[0062] Regeneration cycle = the total number of days from Agrobacterium infection to the formation of a complete rooted plant;

[0063] Transplant survival rate = (Number of surviving plants / Total number of transplanted plants) × 100%.

[0064] 5. Test Results

[0065] Table 3 Effects of different culture hormones

[0066] Group Positive rate (%) Bud induction rate (%) Root induction rate (%) Regeneration cycle (d) Transplant survival rate (%) experimental group 68±2.5a 78±3.1a 93±2.3a 78±3.5a 88±2.7a Control group 1 65±2.8a 58±3.4c 68±3.7d 105±4.2d 68±3.9d Control group 2 66±2.6a 62±3.2b 72±3.5c 98±3.8c 72±3.6c Control group 3 67±2.7a 77±2.9a 69±3.6d 80±3.7a 70±3.8d Control group 4 67±2.6a 76±3.0a 71±3.4c 81±3.6a 73±3.5c Control group 5 66±2.8a 57±3.3c 92±2.5a 82±3.9a 86±2.9a Control group 6 67±2.5a 63±3.1b 91±2.6a 83±4.0a 85±3.0a

[0067] (Note: Different letters in the same column indicate significant differences, P<0.05; data are mean ± standard deviation)

[0068] It can be seen from the above table:

[0069] (1) The bud induction rate (78%) of the experimental group was significantly higher than that of control group 1 (58%) and control group 2 (62%), the root induction rate (93%) was significantly higher than that of control group 1 (68%) and control group 2 (72%), the regeneration cycle (78 days) was shortened by 27 days compared with control group 1 (105 days), and the transplant survival rate (88%) was significantly higher than that of the traditional group; proving that the overall optimized scheme of the experimental group surpassed the control group in terms of efficiency, cycle and survival rate.

[0070] (2) The 5-bud induction rate of the control group (57%) was significantly lower than that of the experimental group (78%), and the 6-bud induction rate of the control group (63%) was also significantly lower than that of the experimental group, indicating that the TDZ concentration increased from 0.5 to 0.9 mg·L⁻¹. -1 The core reason for the increased shoot induction rate is (i.e., low TDZ hormone deficiency and high TDZ inhibition of callus differentiation); the root induction rates of control groups 5 and 6 were close to those of the experimental group (91%-92% vs 93%), proving that after root hormone fixation optimization, TDZ only affects shoot induction and does not interfere with root induction, demonstrating effective variable control. Therefore, 0.9 mg·L - 1TDZ is the optimal concentration for bud induction from broccoli explants, below 0.5 mg·L⁻¹. -1 Hormone deficiency leads to slow bud differentiation, with levels exceeding 1.2 mg / L. -1 It inhibits callus activity at a rate of only 0.9 mg / L. -1 It can balance differentiation efficiency and cell viability.

[0071] (3) The root induction rates of control group 3 (69%) and control group 4 (71%) were significantly lower than those of the experimental group (93%), demonstrating that the NAA+IBA synergy in the experimental group was superior to the traditional single NAA scheme in control group 3 and the traditional single IBA scheme in control group 4. Moreover, the synergistic effect (balanced development of primary root and lateral root) was the key to improving the root induction rate (single NAA results in weak primary root, and single IBA results in fewer lateral roots). The bud induction rates of control groups 3 and 4 were close to those of the experimental group (76%-77% vs 78%), demonstrating that after TDZ fixation optimization, root hormones only affected root induction and did not interfere with bud induction, indicating that variable control was effective.

[0072] Conclusion: The present invention "0.9 mg·L -1 TDZ bud induction +0.1 mg·L -1 NAA + 0.2 mg·L -1 The optimized culture medium scheme for "IBA root induction" can achieve a shoot induction rate of over 75% and a root induction rate of over 90% for broccoli explants, shortening the regeneration cycle to 2.5-3 months (1 month shorter than the traditional method), and maintaining a stable transplant survival rate of over 85%. The overall performance is significantly better than the control group. This invention solves the problems of low shoot-root induction rate and long cycle in traditional methods by precisely optimizing the TDZ concentration and root hormone ratio. The technical solution is scientifically reliable and can be efficiently applied to the transgenic broccoli cultivation practice.

[0073] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for cultivating transgenic broccoli plants based on RUBY visual markers, characterized in that, Includes the following steps: (1) Material preparation: Select 7-8 day old broccoli seedlings, and use seedlings with roots removed as explants; prepare Agrobacterium rhizogenes K599 bacterial culture (OD) carrying the RUBY marker gene vector. 600 =0.4-0.6); (2) Agrobacterium infection and co-culture: The explants were immersed in the bacterial solution for 10 min, the bacterial solution was dried and then transferred to the co-culture medium, and cultured in the dark at 25°C for 2 days. (3) Differentiation culture: The explants after co-culture were transferred to the differentiation medium and cultured for about 20 days at 23-25℃ and 16h / d light (2000-3000 lux). Explants with red transgenic roots marked by RUBY at the wound site were screened out. (4) Bud induction culture: Cut the red transgenic root system into 0.5-1cm root segments, transfer them to the bud induction culture medium, and culture them under the same culture conditions as in step (3) for 25-30 days to form seedlings 2-3cm high; (5) Root induction culture: The seedlings are cut off and transferred to the root induction culture medium, and cultured for 10-15 days under the same culture conditions as in step (3) to form complete plants; (6) Acclimatization and transplanting: After the complete plants have been acclimatized by closing the bottle for 3-5 days and opening the bottle for 2-3 days, they are transplanted into the substrate and kept moist until they survive.

2. The method for cultivating transgenic broccoli plants based on RUBY visual markers according to claim 1, characterized in that, The Agrobacterium rhizogenes K599 bacterial culture in step (1) was cultured in LB liquid medium (containing 50 mg·L⁻¹) -1 Kana, 50 mg / L -1 Streptomycin was prepared by shaking and culturing at 28°C and 180 rpm for 16 h.

3. The method for cultivating transgenic broccoli plants based on RUBY visual markers according to claim 1, characterized in that, In step (2), the co-culture medium is MS medium + 30 g·L⁻¹. -1 sucrose + 7g·L -1 Agar.

4. The method for cultivating transgenic broccoli plants based on RUBY visual markers according to claim 1, characterized in that, In step (3), the differentiation medium is MS medium + 30 g·L⁻¹. -1 sucrose + 7g·L -1 Agar + 200 mg / L -1 Carboxybenzyl + 200 mg·L -1 Termethin.

5. The method for cultivating transgenic broccoli plants based on RUBY visual markers according to claim 1, characterized in that, In step (4), the bud induction medium is the differentiation medium + 0.9 mg·L⁻¹ -1 TDZ.

6. The method for cultivating transgenic broccoli plants based on RUBY visual markers according to claim 1, characterized in that, In step (5), the root induction medium is the differentiation medium + 0.1 mg·L⁻¹ -1 NAA + 0.2 mg·L -1 IBA.

7. The method for cultivating transgenic broccoli plants based on RUBY visual markers according to claim 1, characterized in that, The matrix formula described in step (6) is peat soil: vermiculite: perlite in a weight ratio of 3:1:1, and is sterilized.

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