Agrobacterium-mediated transgenic method on basis of hippeastrum calli

Abstract

An agrobacterium-mediated transgenic method on the basis of Hippeastrum calli, which method comprises the steps of the induction and proliferation of calli, agrobacterium infection, co-culture and the screening of resistant calli. Provided are formulas such as a callus induction medium, a subculture medium, a co-culture medium, and a screening medium, the time and bacterial liquid concentration of Agrobacterium infection, and the concentration of acetosyringone. A Hippeastrum genetic transformation system using a callus as a receptor is established for the first time, which facilitates molecular directed breeding, accelerates the transfer of excellent exogenous genes into Hippeastrum, and improves the variety of Hippeastrum. The method provides a new idea for the industrial development of Hippeastrum and has important application prospects.

Description

An Agrobacterium-mediated transgenic method based on amaryllis callus Technical field:

[0001] The invention belongs to the field of biotechnology, and in particular relates to an Agrobacterium-mediated transgenic method based on amaryllis callus. Background technology:

[0002] Plant genetic modification has been widely used in plant improvement and genetic breeding. Common methods include Agrobacterium-mediated transgenesis, direct DNA insertion, pollen tube transfection, and plant virus-mediated transgenesis. Agrobacterium-mediated transgenesis is a widely used transgenesis method, offering advantages such as ease of operation, high transformation efficiency, good reproducibility, single-copy transgenesis, minimal gene silencing, and the ability to transform larger fragments. However, transgenic monocots are often hampered by difficulties in establishing an effective regeneration system and limited host affinity of Agrobacterium, resulting in significant challenges and a low success rate.

[0003] Hippeastrum (Hippeastrum) is a perennial bulbous flower of the genus Amaryllis in the Amaryllis family. It has beautiful leaves and large flowers, high ornamental value, and multiple medicinal uses, including detoxification and swelling reduction, anti-tumor and cardiovascular disease prevention. It has broad market application prospects and has developed rapidly in my country in recent years. However, the Chinese Hippeastrum industry faces problems such as a small number of independently cultivated varieties, a long growth cycle, a short flowering period, and fragile and easily collapsed flower stems, which have seriously restricted its development. Hybrid breeding is commonly used in Hippeastrum breeding, but it suffers from long breeding cycles and low trait controllability. Establishing a genetic transformation system using callus tissue as a receptor for molecular directed breeding has the advantages of high transformation efficiency and low chimera production, and has important application prospects. Currently, there are no reports of Agrobacterium-mediated transgenic methods based on Hippeastrum callus tissue.

[0004] Summary of the invention:

[0005] The object of the present invention is to provide an Agrobacterium-mediated transgenic method based on Hippeastrum callus, which comprises the following steps:

[0006] S1. Induction and proliferation of callus: Callus was induced by culturing leaves of Hippeastrum test tube seedlings as explants in callus induction medium. Callus with good growth was selected and subcultured on subculture medium. The callus induction medium was: MS + 3.0-3.5 mg / L 6-BA + 2.0-3.0 mg / L NAA + 0-1.0 mg / L KT + 0-0.25 mg / L TDZ + 20-30 g / L sucrose + 5.0-6.0 g / L agar pH 5.4-5.8 or MS + 1.0-1.5 mg / L NAA + 1.0-1.5 mg / L KT + 0.1-0.25 mg / L TDZ + 20-30 g / L sucrose + 5.0-6.0 g / L agar pH 5.4-5.8. The subculture medium was MS + 2.0-3.0 mg / L 6-BA + 0.2-0.3 mg / L TDZ + 20-30 g / L sucrose + 5.0-6.0 g / L agar pH 5.4-5.8;

[0007] S2, Agrobacterium infection: Cultivate the Agrobacterium to be transformed until the bacterial solution OD 600 When the value reaches 0.6-0.8, collect the bacteria by centrifugation, mix the bacteria with the infection solution, let it stand at room temperature for 2-3 hours in the dark, and then add 150-250 μmol·L -1 The callus prepared in step S1 was cut into 0.5-1.0 cm 3 Pour the mixture of bacteria and infection solution into small pieces until they are completely immersed, and infect at 80-120rpm for 10-40min;

[0008] S3. Co-cultivation: The infected Amaryllis callus was removed, air-dried, and inoculated into a co-cultivation medium containing MS, 2.0-3.0 mg / L 6-BA, 0.2-0.3 mg / L TDZ, 20-30 g / L sucrose, and 5.0-6.0 g / L agar, pH 5.4-5.8, for incubation in the dark for 3-7 days.

[0009] S4. Screening for resistant callus: After co-cultivation, the callus was placed in sterile water containing 400-600 mg / L Cef and shaken to sterilize, then rinsed with sterile water and dried. After inoculation, it was cultured on a screening medium containing MS, 1.0-1.5 mg / L IAA, 0.1-0.2 mg / L 6-BA, 0.2-0.3 mg / L TDZ, 0-500 mg / L Carb, 400-600 mg / L Cef, 20-30 g / L sucrose, and 5.0-6.0 g / L agar (pH 5.4-5.8).

[0010] Preferably, the callus induction medium is MS + 3.0 mg / L 6-BA + 2.0 mg / L NAA + 0.25 mg / L TDZ + 20-30 g / L sucrose + 5.0 g / L agar pH 5.4-5.8, MS + 3.0 mg / L 6-BA + 3.0 mg / L NAA + 1.0 mg / L KT + 20-30 g / L sucrose + 5.0 g / L agar pH 5.4-5.8 or MS + 1.0-1.5 mg / L NAA + 1.0-1.5 mg / L KT + 0.1-0.25 mg / L TDZ + 20-30 g / L sucrose + 5.0 g / L agar pH 5.4-5.8; the subculture medium is MS + 2.0-3.0 mg / L 6-BA + 0.25 mg / L TDZ+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8; the co-cultivation medium is MS+2.0-3.0mg / L 6-BA+0.25mg / L TDZ+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8; the screening medium is MS+1.0mg / L IAA+0.1mg / L 6-BA+0.2mg / L TDZ+500mg / L Carb+500mg / L Cef+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8 or MS+1.0mg / L IAA+0.1mg / L 6-BA+0.2mg / L TDZ+500mg / L Cef+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8.

[0011] Preferably, the callus induction medium is MS + 1.0 mg / L NAA + 1.0 mg / L KT + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar, pH 5.6; the subculture medium is MS + 2.0 mg / L 6-BA + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar, pH 5.6; the co-cultivation medium is MS + 2.0 mg / L 6-BA + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar, pH 5.6; and the screening medium is MS + 1.0 mg / L IAA + 0.1 mg / L 6-BA + 0.2 mg / L TDZ + 500 mg / L Cef + 25 g / L sucrose + 5.0 g / L agar, pH 5.6.

[0012] Preferably, the Agrobacterium to be transformed is cultured to a bacterial solution OD 600 When the value reaches 0.6-0.8, the Agrobacterium to be transformed is cultured to the bacterial solution OD 600 When the value reaches 0.6, the bacteria are mixed with the infection solution until the bacteria solution concentration OD600 =0.6.

[0013] Preferably, the addition of 150-250 μmol·L -1 The AS is added 200 μmol·L -1 AS.

[0014] Preferably, the dark culture of 3-7 days in step S3 is dark culture of 3 days.

[0015] Preferably, the infection solution is MS+10.0 g / L inositol+1.0 g / L thiamine hydrochloride+30.0 g / L sucrose.

[0016] Preferably, the test tube seedling leaves are base slices of test tube seedling leaves.

[0017] Preferably, the callus is cut into 0.5-1.0 cm 3 Small pieces are selected in the clean bench to cut the light yellow callus of Amaryllis with good growth into 0.5-1.0cm 3 For small pieces, the 80-120 rpm infection for 10-40 min is 120 rpm infection for 40 min.

[0018] Preferably, the sterile water containing 400-600 mg / L Cef is sterile water containing 500 mg / L Cef.

[0019] Advantages of the present invention:

[0020] The present invention establishes for the first time a genetic transformation system for amaryllis using callus as a receptor by adjusting the formulations of a callus induction medium, a subculture medium, a co-cultivation medium, and a screening medium, the time and concentration of Agrobacterium infection, and the concentration of acetosyringone. This system is conducive to molecular directed breeding, accelerates the transfer of excellent exogenous genes to amaryllis, improves amaryllis varieties, provides new ideas for the industrial development of amaryllis, and has important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the effect of infection time on callus transformation efficiency.

[0022] FIG2 shows the effect of bacterial solution concentration on callus transformation efficiency.

[0023] FIG3 shows the effect of different co-cultivation times on callus transformation efficiency.

[0024] FIG4 shows the effects of different acetosyringone concentrations (AS) on callus transformation efficiency.

[0025] Figure 5 shows transgenic detection in callus of 'Afre' Hippeastrum mediated genetic transformation. AC: GUS transient expression assay, A: CK, B: GUS transient expression negative, C: GUS transient expression positive; D: GUS stable expression assay, D: Young leaves of resistant plants, (left) CK (center) GUS stable expression negative, (right) GUS stable expression positive; E: Roots of resistant plants, (left) CK (center) GUS stable expression negative, (right) GUS stable expression positive; F: PCR electrophoresis assay of the reporter gene GUS, +, positive control, -, negative control. Maker: DL 2000.

[0026] FIG6 is a plasmid map of the expression vector pCAMBIA-1304. Specific implementation method:

[0027] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0028] Example 1: Agrobacterium-mediated transgenic method based on Hippeastrum callus

[0029] Callus induction and proliferation

[0030] This patent uses 'Afresco' Hippeastrum as the material, selecting lateral bulblets from high-quality, already-flowered strains. After removing leaves and roots, clean the bulb with 75% alcohol cotton pads, then cut the bulb disc into 16 pieces with scales 2-3 cm in length. The cut bulb discs are then soaked in 75% alcohol for 20-30 seconds, disinfected with 0.1% mercuric chloride solution for 8-10 minutes, rinsed 4-5 times with sterile water, and then disinfected again with 0.1% mercuric chloride solution for 4-5 minutes. After rinsing 4-5 times with sterile water, the top of the scales are cut off, leaving a 1-2 cm disc with scales. The discs are then inoculated onto a medium containing 4.0 mg / L MS, 6-benzylpurine (6-BA), and 20 g / L sucrose. The disinfection success rate is 90-95%. Leaves grew out after about 30 days. A 0.5 cm base section of the sterile test tube seedling leaf was taken as an explant and cultured in callus induction medium MS + 1.0 mg / L NAA + 1.0 mg / L KT + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6 for 30-40 days to induce callus tissue. The callus induction rate was calculated after 40 days of culture.

[0031] Calli with good growth were selected and subcultured on MS+2.0 mg / L 6-BA+0.25 mg / L TDZ+25 g / L sucrose+5.0 g / L agar pH 5.6 medium for 30 days. Calli with good growth were selected as transformation recipient materials.

[0032] 2. Agrobacterium infection

[0033] Use a sterile toothpick or pipette tip to pick up a single colony of the Agrobacterium tumefaciens EHA105 (pSoup) Chemically Competent Cell (AC1012) vector containing the pCAMBIA-1304 plasmid (Figure 6) to be transformed and place it in LB solution containing 50 mg / L Kan. Place it in a shaking incubator at 220 rpm and culture overnight at 28°C. Wait until the OD value of the bacterial solution reaches 0. 600 When the value reaches 0.6, stop shaking the bacteria and centrifuge at 13000 rpm for 1 min. Prepare the infection solution, mix the bacterial solution with the infection solution (MS + 10.0 g / L inositol + 1.0 g / L thiamine hydrochloride + 30.0 g / L sucrose), and the bacterial solution concentration OD 600 =0.6, let it stand at room temperature for 2-3 hours in the dark, then add 200 μmol·L -1 In the clean bench, select the well-growing pale yellow callus of Amaryllis prepared in step 1 and cut it into 0.5-1.0 cm 3 Place the small pieces into a sterile 50 mL centrifuge tube, then pour the infecting solution mixture to fully immerse the material, and infect on a shaker at 120 rpm for 40 min.

[0034] 3. Co-cultivation

[0035] The infected amaryllis callus was removed and placed on sterile filter paper to absorb excess infection liquid. After 15-30 minutes, the bacterial liquid on the surface of the material was air-dried, and then inoculated into MS + 2.0 mg / L 6-BA + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6 co-culture medium and cultured in the dark for 3 days.

[0036] 4. Screening of resistant callus

[0037] After co-cultivation, the callus was placed in sterile water containing 500 mg / L Cef and decontaminated by shaking for 1 minute. The callus was then rinsed with sterile water for 1 minute, repeated three times, blotted dry on sterile filter paper, and inoculated onto screening medium. Callus fragments were then inoculated onto MS medium supplemented with 1.0 mg / L IAA, 0.1 mg / L 6-BA, 0.2 mg / L TDZ, 500 mg / L Cef, 25 g / L sucrose, and 5.0 g / L agar, pH 5.6, for screening. Plantlets were regenerated from the calli. The growth of the culture was observed, and the callus resistance rate was measured approximately three weeks later.

[0038] 5. Detection of genetically modified plants

[0039] GUS transient expression rate and resistance callus rate detection:

[0040] (1) Pipette 1 mL of X-gluc solution into the X-gluc tube and mix thoroughly until the powder is completely dissolved to prepare the X-gluc solution (50×);

[0041] (2) Add 5 mL of GUS staining buffer per 100 μL of X-gluc solution (50×);

[0042] (3) Cut the resistant tissue into small pieces (Agrobacterium tumefaciens containing the pCAMBIA-1304 plasmid was transformed as a positive control, untransformed callus tissue was used as a negative control, and Agrobacterium tumefaciens without the pCAMBIA-1304 plasmid was transformed as a CK control), place them in a 50 mL centrifuge tube, add an appropriate amount of prepared GUS staining working solution to completely cover the material, wrap it with tin foil, and place it at room temperature for 3 hours to overnight;

[0043] (4) Elution: Decolorize the material by transferring it to 70% alcohol 2-3 times until the negative control material turns white;

[0044] (5) Observation: Observe with the naked eye or under a stereomicroscope, and calculate the GUS staining rate.

[0045] The results are shown in Figure 5 and Table 1. Of the 60 surviving calli, 25 showed blue spots, resulting in a GUS transient expression rate of 41.67%. Of the remaining 140 calli screened, 56 survived and proliferated into new calli after one month, yielding a resistant callus rate of 40.00%. After subsequent culture recovery, 36 resistant plants were obtained. Some calli failed to differentiate and gradually withered and died, likely due to false positives or reduced differentiation capacity after Agrobacterium infection. The resistant plants were tested for stable GUS expression and PCR positivity, with two successfully stained and exhibiting DNA bands. The final positive conversion rate was 5.56%, and the overall positive rate was 1.00%.

[0046] Table 1 Identification of transformation efficiency of Amaryllis callus mediated by Agrobacterium

[0047] Example 2: Effects of different hormone combinations on callus induction and proliferation

[0048] Using 0.5 cm basal sections of leaves of 'Alfrey' Hippeastrum (Hippeastrum Alfresco) test tube seedlings (prepared as in Example 1) as explants, calli were induced after culturing for 30-40 days in different callus induction media (MS + 0-4.0 mg / L 6-BA + 0-3.0 mg / L NAA + 0-3.0 mg / L KT + 0-1.0 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar, pH 5.6). Various indices were measured after 40 days of culture.

[0049] The results showed that the callus rate was 0 on the culture medium without plant growth regulators, but after adding plant growth regulators, the induction rate was 4.4-97.8% (Table 2).

[0050] Table 2 Effects of different plant growth regulator combinations on leaf induction in sterile seedlings Note: Different lowercase letters in the same column in the table indicate significant differences (P≤0.05).

[0051] Well-growing callus induced by MS + 1.0 mg / L NAA + 1.0 mg / L KT + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6 was selected and subcultured on MS + 1.0-6.0 mg / L 6-BA + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6 medium.

[0052] The results showed that the proliferation rate after 30 days was 2.9-4.0, among which MS + 2.0 mg / L 6-BA + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6 was the best, maintaining the callus state with a proliferation rate of 4.0 and no bud differentiation, which was the most suitable as a transformation recipient material (Table 3).

[0053] Table 3 Effects of different 6-BA concentrations added to MS+0.25mg / LTDZ on callus proliferation Note: Different lowercase letters in the same column in the table indicate significant differences (P≤0.05).

[0054] Example 3: Effects of Agrobacterium infection time and bacterial concentration on callus transformation efficiency

[0055] Steps 1 and 3-5 are the same as in Example 1, and step 2 is as follows (1) or (2):

[0056] (1) Use a sterilized toothpick or pipette tip to pick up a single colony of Agrobacterium tumefaciens EHA105 (pSoup) Chemically Competent Cell (AC1012) containing the pCAMBIA-1304 plasmid to be transformed and place it in LB solution containing 50 mg / L Kan. Place it in a shaking incubator at 220 rpm and culture it at 28°C overnight. Wait until the OD value of the bacterial solution reaches 0. 600 When the value reaches 0.6, stop shaking the bacteria and centrifuge. Prepare the infection solution, mix the bacterial solution with the infection solution (MS + 10.0g / L inositol + 1.0g / L thiamine hydrochloride + 30.0g / L sucrose), and the bacterial solution concentration OD 600 =0.6, let it stand at room temperature for 2-3 hours in the dark, then add 200 μmol·L -1 In the clean bench, select the well-growing pale yellow callus of Amaryllis prepared in step 1 and cut it into 0.5-1.0 cm 3 Small pieces were placed in a sterile 50 mL centrifuge tube, and the infecting solution mixture was poured in to fully immerse the material. The materials were infected on a shaker at 120 rpm for 10 min, 20 min, 30 min, and 40 min respectively.

[0057] The results showed that different Agrobacterium infection times resulted in different transformation efficiencies (Figure 1).

[0058] (2) Use a sterilized toothpick or pipette tip to pick up a single colony of Agrobacterium tumefaciens EHA105 (pSoup) Chemically Competent Cell (AC1012) containing the pCAMBIA-1304 plasmid to be transformed and place it in LB solution containing 50 mg / L Kan. Place it in a shaking incubator at 220 rpm and culture it at 28°C overnight. Wait until the OD value of the bacterial solution reaches 0. 600 When the value reaches 0.4, 0.6, 0.8 or 1.0, stop shaking the bacteria and centrifuge. Prepare the infection solution, mix the bacterial solution with the infection solution (MS + 10.0g / L inositol + 1.0g / L thiamine hydrochloride + 30.0g / L sucrose), and the bacterial solution concentration OD 600 =0.6, let it stand at room temperature for 2-3 hours in the dark, then add 200 μmol·L -1 In the clean bench, select the well-growing pale yellow callus tissue of Amaryllis prepared in step 1 and cut it into 0.5-1.0 cm 3 Place the small pieces into a sterile 50 mL centrifuge tube, then pour the infecting solution mixture to fully immerse the material, and infect on a shaker at 80° for 40 minutes.

[0059] The results showed that when the bacteria were shaken to a concentration of OD 600= 0.6, the GUS transient expression rate and resistant callus rate were the highest at 53.33% and 38.38%, respectively. 600 =0.4, the lowest GUS transient expression rate was 23.33%, and the bacterial concentration OD 600 =1.0, the lowest resistance callus rate was 10.61%. 600 =0.6 is the optimal infection concentration for callus tissue (Figure 2).

[0060] Example 4: Effects of different co-cultivation times on callus transformation efficiency

[0061] Steps 1-2, 4-5 are the same as in Example 1, and step 3 is as follows:

[0062] The infected amaryllis callus was removed and placed on sterile filter paper to absorb excess infection liquid. After 15-30 minutes, the bacterial liquid on the surface of the material was air-dried, and then inoculated into MS + 2.0 mg / L 6-BA + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6 co-culture medium and cultured in the dark for 1-7 days.

[0063] Results: After infection, callus tissue was cultured on co-culture medium. After 1-2 days of culture, the callus surface showed no noticeable changes, remaining bright yellow and showing no initial expansion. On the third day, a large amount of purple-brown secretions appeared on the callus surface, and the callus volume expanded slightly compared to the initial stage. Both changes continued in the same direction with increasing co-culture time. As shown in Figure 3, the GUS transient expression rate and the resistant callus rate in the callus tissue initially increased and then decreased with increasing co-culture time. The GUS transient expression rate and resistant callus rate reached their highest levels at 37% and 34.72%, respectively, after 3 days of co-culture. The resistant callus rate was lowest at 2.78% after 1 and 7 days of co-culture. Therefore, 3 days of co-culture is the optimal number of days for genetic transformation of Amaryllis callus (Figure 3).

[0064] Example 5: Acetosyringone concentration screening

[0065] Steps 1 and 3-5 are the same as in Example 1, and step 2 is as follows:

[0066] Use a sterile toothpick or pipette tip to pick up a single colony of Agrobacterium tumefaciens EHA105 (pSoup) Chemically Competent Cell (AC1012) containing the pCAMBIA-1304 plasmid to be transformed and place it in LB solution containing 50 mg / L Kan. Place it in a shaking incubator at 220 rpm and culture overnight at 28°C. Wait until the OD value of the bacterial solution reaches 0. 600When the value reaches 0.6, stop shaking the bacteria and centrifuge. Prepare the infection solution, mix the bacterial solution with the infection solution (MS + 10.0g / L inositol + 1.0g / L thiamine hydrochloride + 30.0g / L sucrose), and the bacterial solution concentration OD 600 =0.6, and then stand at room temperature in the dark for 2-3 h, and then add 0, 50, 100, 200 or 300 μmol·L -1 In the clean bench, select the well-growing pale yellow callus tissue of Amaryllis prepared in step 1 and cut it into 0.5-1.0 cm 3 Place the small pieces into a sterile 50 mL centrifuge tube, then pour the infecting solution mixture to fully immerse the material, and infect in a shaker at 120 rpm for 40 min.

[0067] When performing Agrobacterium-mediated genetic transformation of monocotyledonous plants, some phenolic substances are often added to improve the transformation efficiency. The results are shown in Figure 4. -1 Except for the GUS transient expression rate at 0.00 μmol·L -1 and 50.00 μmol·L -1 When the concentration increased to 100.0 μmol·L -1 When AS was 200.00 μmol·L -1 When the concentration of AS200.00 μmol·L was 0.177 μmol·L, the transformation efficiency was significantly improved, and the GUS transient expression rate and resistant callus rate were the highest, which were 63.337% and 33.33% respectively. -1 The optimal acetosyringone concentration for genetic transformation of Amaryllis callus (Figure 4)

[0068] Example 6: Screening medium screening

[0069] Steps 1-3 and 5 are the same as in Example 1, and step 4 is as follows:

[0070] After co-cultivation, the callus was placed in sterile water containing 500 mg / L Cef and shaken for 1 minute to sterilize. The callus was then rinsed with sterile water for 1 minute, and this process was repeated three times. The callus was then blotted dry on sterile filter paper and inoculated onto screening medium. Callus fragments were then inoculated onto MS medium supplemented with 1.0 mg / L IAA, 0.1 mg / L 6-BA, 0.2 mg / L TDZ, 0-500 mg / L Carb, 0-500 mg / L Cef, 25 g / L sucrose, and 5.0 g / L agar, pH 5.6, for screening. Plantlets were regenerated from the calli. The growth of the culture was observed, and the callus resistance rate was measured approximately three weeks later.

[0071] The callus tissues that had been infected with Agrobacterium infection solution and washed with co-culture were inoculated onto culture media containing different concentrations of Cef, Carb, and Cef+Carb for antibacterial experiments. Carb had a lower antibacterial effect than Cef. When the callus tissues were grown on culture media without Cef and Carb, Agrobacterium grew all around the callus tissues, with a contamination rate of 100%. The callus proliferation rate was extremely low, only 4.44. When Cef was 500.00 mg·L -1 +Carb 500.00mg·L -1 When the concentration was 500.00 mg·L, the contamination was the lowest and the Agrobacterium could be completely inhibited, followed by Cef 500.00 mg·L -1 The contamination rate was 2.22%, but the difference between the two treatments was not significant, and the addition of multiple antibacterial agents would also reduce the proliferation capacity of callus tissue. Therefore, it is recommended to use Cef 500.00mg·L -1 It is the optimal antibacterial concentration of Amaryllis callus (Table 4).

[0072] Table 4 Effects of different concentrations of cephalosporin and carbenicillin and their combination on the growth of callus tissue of Hippeastrum 'Afre' mediated by Agro-Gan Gong

[0073] Note: 6-BA (6-Benzylaminopurine) 6-benzylaminopurine, TDZ (Thidiazuron) thiadiazole, KT (Kinetin) kinetin, NAA (α-napthaleneacetic acid) naphthaleneacetic acid, HygB (Hygromycin B) hygromycin, AS (Acetosyringone) acetosyringone, Cef (Cefotaxime sodium claforan ctx) cephalosporin, Carb carbenicillin.

Claims

1. A transgenic method based on Amaryllis callus mediated by Agrobacterium, characterized in that: The following steps are involved: S1. Induction and proliferation of callus: Callus was induced by culturing leaves of Hippeastrum test tube seedlings as explants in callus induction medium. Callus with good growth was selected and subcultured on subculture medium. The callus induction medium was: MS + 3.0-3.5 mg / L 6-BA + 2.0-3.0 mg / L NAA + 0-1.0 mg / L KT + 0-0.25 mg / L TDZ + 20-30 g / L sucrose + 5.0-6.0 g / L agar pH 5.4-5.8 or MS + 1.0-1.5 mg / L NAA + 1.0-1.5 mg / L KT + 0.1-0.25 mg / L TDZ + 20-30 g / L sucrose + 5.0-6.0 g / L agar pH 5.4-5.

8. The subculture medium was MS + 2.0-3.0 mg / L 6-BA + 0.2-0.3 mg / L TDZ + 20-30 g / L sucrose + 5.0-6.0 g / L agar pH 5.4-5.8; S2, Agrobacterium infection: Cultivate the Agrobacterium to be transformed until the bacterial solution OD 600 When the value reaches 0.6-0.8, collect the bacteria by centrifugation, mix the bacteria with the infection solution, let it stand at room temperature for 2-3 hours in the dark, and then add 150-250 μmol·L -1 The callus prepared in step S1 was cut into 0.5-1.0 cm 3 Pour the mixture of bacteria and infection solution into small pieces until they are completely immersed, and infect at 80-120rpm for 10-40min; S3. Co-cultivation: The infected Amaryllis callus was removed, air-dried, and inoculated into a co-cultivation medium containing MS, 2.0-3.0 mg / L 6-BA, 0.2-0.3 mg / L TDZ, 20-30 g / L sucrose, and 5.0-6.0 g / L agar, pH 5.4-5.8, for incubation in the dark for 3-7 days. S4. Screening for resistant callus: After co-cultivation, the callus was placed in sterile water containing 400-600 mg / L Cef and shaken to sterilize, then rinsed with sterile water and dried. After inoculation, it was cultured on a screening medium containing MS, 1.0-1.5 mg / L IAA, 0.1-0.2 mg / L 6-BA, 0.2-0.3 mg / L TDZ, 0-500 mg / L Carb, 400-600 mg / L Cef, 20-30 g / L sucrose, and 5.0-6.0 g / L agar (pH 5.4-5.8).

2. The method according to claim 1, characterized in that The callus induction medium is MS + 3.0 mg / L 6-BA + 2.0 mg / L NAA + 0.25 mg / L TDZ + 20-30 g / L sucrose + 5.0 g / L agar, pH 5.4-5.8, MS + 3.0 mg / L 6-BA + 3.0 mg / L NAA + 1.0 mg / L KT + 20-30 g / L sucrose + 5.0 g / L agar, pH 5.4-5.8, or MS + 1.0-1.5 mg / L NAA + 1.0-1.5 mg / L KT + 0.1-0.25 mg / L TDZ + 20-30 g / L sucrose + 5.0 g / L agar, pH 5.4-5.8; the subculture medium is MS + 2.0-3.0 mg / L 6-BA + 0.25 mg / L TDZ+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8; the co-cultivation medium is MS+2.0-3.0mg / L 6-BA+0.25mg / L TDZ+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8; the screening medium is MS+1.0mg / L IAA+0.1mg / L 6-BA+0.2mg / L TDZ+500mg / L Carb+500mg / L Cef+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8 or MS+1.0mg / L IAA+0.1mg / L 6-BA+0.2mg / L TDZ+500mg / L Cef+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.

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3. The method according to claim 2, characterized in that The callus induction medium is MS + 1.0 mg / L NAA + 1.0 mg / L KT + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar at pH 5.6; the subculture medium is MS + 2.0 mg / L 6-BA + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar at pH 5.6; the co-cultivation medium is MS + 2.0 mg / L 6-BA + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar at pH 5.6; and the screening medium is MS + 1.0 mg / L IAA + 0.1 mg / L 6-BA + 0.2 mg / L TDZ + 500 mg / L Cef + 25 g / L sucrose + 5.0 g / L agar at pH 5.

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4. The method according to claim 1, wherein The Agrobacterium to be transformed is cultured to a bacterial solution OD 600 When the value reaches 0.6-0.8, the Agrobacterium to be transformed is cultured to the bacterial solution OD 600 When the value reaches 0.6, the bacteria are mixed with the infection solution until the bacteria solution concentration OD 600 =0.

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5. The method according to claim 1, wherein The addition of 150-250 μmol·L -1 The AS is added 200 μmol·L -1 AS.

6. The method according to claim 1, characterized in that The dark culture of 3-7 days in step S3 is dark culture of 3 days.

7. The method according to claim 1, characterized in that The infection solution is MS+10.0g / L inositol+1.0g / L thiamine hydrochloride+30.0g / L sucrose.

8. The method according to claim 1, characterized in that The test tube seedling leaf is a base slice of the test tube seedling leaf.

9. The method according to claim 1, characterized in that The callus tissue was cut into 0.5-1.0 cm 3 Small pieces are selected in the clean bench to cut the light yellow callus of Amaryllis with good growth into 0.5-1.0cm 3 For small pieces, the 80-120 rpm infection for 10-40 min is 120 rpm infection for 40 min.

10. The method according to claim 1, characterized in that The sterile water containing 400-600 mg / L Cef is sterile water containing 500 mg / L Cef.

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