Agrobacterium tumefaciens-mediated transgenic method based on hippeastrum rutilum callus

Abstract

The invention discloses an agrobacterium tumefaciens mediated transgenosis method based on hippeastrum rutilum callus. The method comprises the steps of induction and proliferation of calluses, agrobacterium infection, co-culture and screening of resistant calluses. The invention discloses formulas of a callus induction culture medium, a subculture culture medium, a co-culture culture medium, a screening culture medium and the like, agrobacterium infection time, bacterial liquid concentration and acetosyringone concentration, establishes a hippeastrum striatum genetic transformation system taking the callus as a receptor for the first time, is beneficial to molecular directional breeding, and has a wide application prospect. The transfer of excellent exogenous genes to the hippeastrum hippeastrum is accelerated, the variety of the hippeastrum hippeastrum is improved, a new idea is provided for industrialization development of the hippeastrum hippeastrum, and the method has important application prospects.

Description

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 Art

[0002] Plant transgenic technology has been widely used in plant improvement and genetic breeding. Commonly used methods include Agrobacterium-mediated method, DNA direct insertion method, pollen tube channel method and plant virus-mediated method, among which Agrobacterium-mediated is a widely used transgenic method with the advantages of simple operation, high transformation efficiency, good repeatability, single copy, less gene silencing, and the ability to transform larger fragments. However, the transgenic of monocotyledonous plants is often affected by the difficulty of establishing an effective regeneration system and the restriction of Agrobacterium's affinity to the host, which makes it difficult and has a low probability of success.

[0003] Hippeastrum is a perennial bulbous flower of the genus Amaryllis in the Amaryllis family. It has beautiful leaves, large flowers, high ornamental value, and multiple medicinal values ​​such as detoxification, anti-tumor, and prevention of cardiovascular diseases. It has broad market application prospects and has developed rapidly in my country in recent years. However, there are problems such as few self-cultivated varieties, long growth cycle, short flowering period, and brittle and easy lodging of flower stems in the process of the Hippeastrum industry in my country, which seriously restricts the development of its industry. Hybrid breeding is often used for breeding of Hippeastrum, but there are problems such as long breeding cycle and low controllability of traits. Establishing a genetic transformation system with callus as receptor for molecular directed breeding has the advantages of high transformation efficiency and few chimeras, and has important application prospects. At present, there is no report on the Agrobacterium-mediated transgenic method based on Hippeastrum callus. Summary of the invention

[0004] The object of the present invention is to provide a transgenic method based on Amaryllis callus mediated by Agrobacterium, which comprises the following steps:

[0005] S1. Induction and proliferation of callus: Using leaves of Amaryllis test tube seedlings as explants, callus was induced in callus induction medium, and callus with good growth was selected for subculture on subculture medium. The callus induction medium was: MS + 3.0-3.5mg / L 6-BA + 2.0-3.0mg / L NAA + 0-1.0mg / L KT + 0-0.25mg / LTDZ + 20-30g / L sucrose + 5.0-6.0g / L agar pH 5.4-5.8 or MS + 1.0-1.5mg / L NAA + 1.0-1.5mg / L KT + 0.1-0.25mg / L TDZ + 20-30g / L sucrose + 5.0-6.0g / L agar pH 5.4-5.8, and the subculture medium was MS + 2.0-3.0mg / L 6-BA+0.2-0.3mg / LTDZ+20-30g / L sucrose+5.0-6.0g / L agar pH5.4-5.8;

[0006] S2. Agrobacterium infection: The Agrobacterium to be transformed is cultured 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 in the dark for 2-3 hours, and then add 150-250 μmol·L -1 The callus prepared in step S1 was cut into 0.5-1.0 cm 3 For small pieces, pour the mixture of bacteria and infection solution until they are completely submerged, and infect at 80-120rpm for 10-40min;

[0007] S3, co-cultivation: the infected Amaryllis callus was taken out, air-dried, and inoculated into a co-cultivation medium, and cultured in the dark for 3-7 days. The co-cultivation 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;

[0008] S4. Screening of resistant callus: After co-cultivation, the callus is placed in sterile water containing 400-600 mg / L Cef and shaken to sterilize, then shaken and rinsed with sterile water, dried and inoculated into screening medium for culture, and transgenic plants are detected after the callus grows regenerated plants. The screening medium is 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 + 5.0-6.0 g / L agar, pH 5.4-5.8.

[0009] 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 / LTDZ+500mg / L Cef+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8.

[0010] 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 / LTDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6; 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.

[0011] 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 step of mixing the bacteria with the infection solution is to mix the bacteria with the infection solution until the concentration of the bacterial solution is OD 600=0.6.

[0012] Preferably, the addition of 150-250 μmol·L -1 The AS was added with 200 μmol·L -1 AS.

[0013] Preferably, the dark culture for 3-7 days in step S3 is dark culture for 3 days.

[0014] Preferably, the infection solution is MS+10.0 g / L inositol+1.0 g / L thiamine hydrochloride+30.0 g / L sucrose.

[0015] Preferably, the test tube seedling leaves are base slices of test tube seedling leaves.

[0016] 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.

[0017] Preferably, the sterile water containing 400-600 mg / L Cef is sterile water containing 500 mg / L Cef.

[0018] Advantages of the present invention:

[0019] The invention establishes for the first time a genetic transformation system of amaryllis with callus as a receptor through formulas of callus induction medium, subculture medium, co-culture medium and screening medium, the time of Agrobacterium infection and the concentration of bacterial solution, as well as the concentration of acetosyringone, which 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

[0020] Figure 1 The effect of infection time on callus transformation efficiency.

[0021] Figure 2 The effect of bacterial solution concentration on callus transformation efficiency.

[0022] Figure 3 The effect of different co-cultivation time on callus transformation efficiency.

[0023] Figure 4 The effect of different acetosyringone concentrations (AS) on callus transformation efficiency.

[0024] Figure 5This is the transgenic detection of callus genetic transformation of 'Afre' Hippeastrum mediated by Agrobacterium. AC: GUS transient expression detection, A: CK, B: GUS transient expression negative, C: GUS transient expression positive; DE: GUS stable expression detection, D: young leaves of resistant plants, (left) CK (middle) GUS stable expression negative (right) GUS stable expression positive, E: roots of resistant plants, (left) CK (middle) GUS stable expression negative (right) GUS stable expression positive; F: reporter gene GUS PCR electrophoresis detection, +, positive control, -, negative control, Maker is DL 2000.

[0025] Figure 6 This is the plasmid map of the expression vector pCAMBIA-1304. DETAILED DESCRIPTION

[0026] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0027] Example 1: Agrobacterium-mediated transgenic method based on Amaryllis callus

[0028] 1. Callus induction and proliferation

[0029] This patent uses Hippeastrum Alfresco as the material, selects the lateral small bulbs of the excellent strains that have bloomed as the material, removes the leaves and roots, wipes them clean with 75% alcohol cotton, cuts off the bulb disc, and divides them into 16 pieces with scales of 2-3 cm in length. Then soak the cut bulb disc in 75% alcohol for 20-30 seconds, disinfects it with 0.1% mercuric chloride solution for 8-10 minutes, rinses it with sterile water 4-5 times, and then disinfects it with 0.1% mercuric chloride solution for 4-5 minutes, rinses it with sterile water 4-5 times, cuts off the upper end of the scale, leaves the bulb disc with 1-2 cm scale, and inoculates it on MS+6-benzylpurine (6-BA) 4.0 mg / L+20 g / L sucrose culture medium, with a disinfection success rate of 90-95%. Leaves grew out in about 30 days. A 0.5 cm base slice 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.

[0030] Select callus tissue with good growth and subculture it on MS + 2.0 mg / L 6-BA + 0.25 mg / LTDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6 medium for 30 days, and select callus tissue with good growth as transformation recipient material.

[0031] 2. Agrobacterium infection

[0032] Use a sterile toothpick or pipette tip to pick up the plasmid containing pCAMBIA-1304 to be transformed ( Figure 6 A single colony of Agrobacterium tumefaciens EHA105 (pSoup) Chemically Competent Cell (AC1012) vector was placed in LB solution containing 50 mg / L Kan, placed in a 220 rpm shaker, and cultured overnight at 28°C. 600 When the value reached 0.6, the shaking was stopped and the centrifugation was carried out at 13000 rpm for 1 min. The infection solution was prepared. The bacterial solution was mixed with the infection solution (MS + 10.0 g / L inositol + 1.0 g / L thiamine hydrochloride + 30.0 g / L sucrose). The concentration of the bacterial solution OD 600 =0.6, let stand at room temperature for 2-3h in the dark, then add 200μmol·L -1 Select the well-growing pale yellow callus of Amaryllis prepared in step 1 and cut 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.

[0033] 3. Co-cultivation

[0034] 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.

[0035] 4. Screening of resistant callus

[0036] After the co-cultivation, the callus tissue was placed in sterile water containing 500 mg / L Cef and shaken for sterilization for 1 minute, then washed with sterile water for 1 minute, repeated 3 times, and then dried on sterile filter paper and inoculated into the screening medium. The callus tissue block was inoculated into 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 for screening and culture, and the callus tissue can regenerate plants. The growth of the culture material was observed, and the callus resistance rate was detected after about three weeks.

[0037] 5. Detection of genetically modified plants

[0038] GUS transient expression rate and resistance callus rate detection:

[0039] (1) Pipette 1 mL of X-gluc solution into the X-gluc tube and mix thoroughly until the powder is completely dissolved to prepare X-gluc solution (50×);

[0040] (2) Add 5 mL of GUS staining buffer per 100 μL of X-gluc solution (50×);

[0041] (3) Cut the resistant tissue into small pieces (Agrobacterium tumefaciens containing pCAMBIA-1304 plasmid was transformed as the positive control, callus tissue that was not transformed was used as the negative control, and Agrobacterium tumefaciens without pCAMBIA-1304 plasmid was transformed as the CK control), put 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;

[0042] (4) Elution: Decolorize the material in 70% alcohol 2-3 times until the negative control material turns white;

[0043] (5) Observation: Observe with the naked eye or under a stereomicroscope and calculate the GUS staining rate.

[0044] The results are as follows Figure 5 As shown in Table 1, 60 calli survived and were tested for GUS transient expression. 25 calli showed blue spots, with a GUS transient expression rate of 41.67%. Of the remaining 140 calli for screening, 56 survived and proliferated new calli after 1 month, with a resistant callus rate of 40.00%. After the culture was restored, 36 resistant plants were obtained. Some calli failed to differentiate and gradually withered and died. The reason may be false positive or the original differentiation ability of calli was reduced after infection with Agrobacterium. The resistant plants were tested for GUS stable expression and PCR positive. Two plants were successfully stained and had DNA bands. The final positive conversion rate was 5.56%, and the total positive rate was 1.00%.

[0045] Table 1 Identification of transformation efficiency of Amaryllis callus mediated by Agrobacterium

[0046]

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

[0048] The base sections of leaves of Hippeastrum Alfresco test tube seedlings (prepared by the same method as in Example 1) with a length of about 0.5 cm were used as explants and cultured in different callus induction mediums of 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 for 30-40 days to induce callus tissue. Various indices were statistically analyzed after 40 days of culture.

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

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

[0051] Select the well-growing callus tissue 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 and subculture it on MS + 1.0-6.0 mg / L 6-BA + 0.25 mg / LTDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6 medium.

[0052] The results showed that the proliferation multiples after 30 days were 2.9-4.0, among which MS+2.0mg / L 6-BA+0.25mg / LTDZ+25g / L sucrose+5.0g / L agar pH5.6 had the best effect, which could maintain the callus state, with a proliferation rate of 4.0 and no bud differentiation, making it the most suitable material for transformation receptors (Table 3).

[0053] Table 3 Effects of different 6-BA concentrations added to MS+0.25mg / LTDZ on callus proliferation

[0054]

[0055] Note: Different lowercase letters in the same column in the table indicate significant differences (P≤0.05).

[0056] Example 3: Effects of Agrobacterium infection time and bacterial concentration on callus transformation efficiency

[0057] Steps 1 and 3-5 are the same as in Example 1, and step 2 is as follows (1) or (2):

[0058] (1) Use a sterilized toothpick or pipette 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 an LB solution containing 50 mg / L Kan. Place it in a shaking incubator at 220 rpm and culture it overnight at 28°C. Wait until the OD value of the bacterial solution reaches 1. 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 stand at room temperature for 2-3h in the dark, then add 200μmol·L -1 Select the well-growing pale yellow callus of Amaryllis prepared in step 1 and cut 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 materials. The materials were infected on a shaker at 120 rpm for 10 min, 20 min, 30 min, and 40 min respectively.

[0059] The results showed that different Agrobacterium infection times had different transformation efficiencies ( Figure 1 ).

[0060] (2) Use a sterilized toothpick or pipette 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 an LB solution containing 50 mg / L Kan. Place it in a shaking incubator at 220 rpm and culture it overnight at 28°C. Wait until the OD value of the bacterial solution reaches 1. 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 stand at room temperature for 2-3h in the dark, then add 200μmol·L -1 Select the well-growing pale yellow callus of Amaryllis prepared in step 1 and cut 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 materials, and infect on a shaker at 80° for 40 min.

[0061] The results showed that when the bacterial solution concentration OD600 =0.6, the GUS transient expression rate and resistant callus rate were 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 best infection concentration for callus tissue ( Figure 2 ).

[0062] Example 4: Effects of different co-cultivation times on callus transformation efficiency

[0063] Steps 1-2, 4-5 are the same as in Example 1, and step 3 is as follows:

[0064] The infected Amaryllis callus was removed and placed on sterile filter paper to absorb excess infection fluid. 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.

[0065] Results: The infected callus was cultured on the co-culture medium. After 1-2 days of culture, the surface of the callus did not change significantly and remained bright yellow. The volume did not expand. On the 3rd day, a large amount of purple-brown secretions appeared on the surface of the callus and the volume expanded to a certain extent. Both continued to change in the same direction as the co-culture time increased. Figure 3 As shown in the figure, the GUS transient expression rate and resistant callus rate of callus tissue showed a trend of first increasing and then decreasing with the extension of co-culture time. When co-cultured for 3 days, the GUS transient expression rate and resistant callus rate were the highest, 37% and 34.72%, respectively. When co-cultured for 1 day and 7 days, the resistant callus rate was the lowest, both of which were 2.78. Therefore, co-cultured for 3 days is the optimal co-culture day for genetic transformation of Amaryllis callus ( Figure 3 ).

[0066] Example 5: Acetosyringone concentration screening

[0067] Steps 1 and 3-5 are the same as in Example 1, and step 2 is as follows:

[0068] Use a sterilized toothpick or pipette 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 an LB solution containing 50 mg / L Kan. Place it in a shaking incubator at 220 rpm and culture it overnight at 28°C. Wait until the OD value of the bacterial solution reaches 1. 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, after standing at room temperature in the dark for 2-3 h, 0, 50, 100, 200 or 300 μmol·L -1 Select the well-growing yellow callus tissue of Amaryllis prepared in step 1 and cut 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.

[0069] When performing Agrobacterium-mediated genetic transformation of monocotyledonous plants, some phenolic substances are often added to improve the transformation efficiency. Figure 4 As shown, except AS 0.00 μmol·L -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 AS200.00μmol·L -1 is the optimal acetosyringone concentration for genetic transformation of Amaryllis callus ( Figure 4 )

[0070] Example 6: Screening medium screening

[0071] Steps 1-3 and 5 are the same as in Example 1, and step 4 is as follows:

[0072] After the co-cultivation, the callus tissue was placed in sterile water containing 500 mg / L Cef and shaken for sterilization for 1 minute, then washed with sterile water for 1 minute, repeated 3 times, and then dried on sterile filter paper and inoculated into the screening medium. The callus tissue block was inoculated into MS+1.0 mg / L IAA+0.1 mg / L 6-BA+0.2 mg / L TDZ+0-500 mg / L Car b+0-500 mg / LCef+25 g / L sucrose+5.0 g / L agar pH 5.6 for screening and culture, and the callus tissue can regenerate plants. The growth of the culture material was observed, and the callus resistance rate was detected after about three weeks.

[0073] The callus tissues that were inoculated with Agrobacterium infection solution and washed with co-culture were inoculated on culture media containing different concentrations of Cef, Carb and Cef+Carb for antibacterial experiments. The antibacterial effect of Carb was not as good as that of Cef. On the culture medium without Cef and Carb, the callus tissues were surrounded by Agrobacterium, 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 pollution was the lowest and Agrobacterium could be completely inhibited, followed by Cef500.00 mg·L -1 The contamination rate was 2.22%, but the difference between the two treatments was not obvious, and the addition of multiple antibacterial agents would also reduce the proliferation capacity of callus tissue. -1 It is the optimal antibacterial concentration of Amaryllis callus (Table 4).

[0074] Table 4 Effects of different concentrations of cephalosporin and carbenicillin and their combination on the callus growth of 'Afre' Hippeastrum mediated by Agro-Gan Gong

[0075]

[0076]

[0077] Note: 6-BA (6-Benzylaminopurine) 6-benzylaminopurine, TDZ (Thidiazuron) Thidiazuron, 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: Using leaves of Amaryllis test tube seedlings as explants, callus was induced in callus induction medium, and callus with good growth was selected for subculture on subculture medium. The callus induction medium was: MS + 3.0-3.5mg / L 6-BA + 2.0-3.0mg / L NAA + 0-1.0mg / L KT + 0-0.25mg / LTDZ + 20-30g / L sucrose + 5.0-6.0g / L agar pH 5.4-5.8 or MS + 1.0-1.5mg / L NAA + 1.0-1.5mg / L KT + 0.1-0.25mg / L TDZ + 20-30g / L sucrose + 5.0-6.0g / L agar pH 5.4-5.8, and the subculture medium was MS + 2.0-3.0mg / L 6-BA+0.2-0.3mg / LTDZ+20-30g / L sucrose+5.0-6.0g / L agar pH5.4-5.8; S2. Agrobacterium infection: The Agrobacterium to be transformed is cultured 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 in the dark for 2-3 hours, and then add 150-250 μmol·L -1 The callus prepared in step S1 was cut into 0.5-1.0 cm 3 For small pieces, pour the mixture of bacteria and infection solution until they are completely submerged, and infect at 80-120rpm for 10-40min; S3, co-cultivation: the infected Amaryllis callus was taken out, air-dried, and inoculated into a co-cultivation medium, and cultured in the dark for 3-7 days. The co-cultivation 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; S4. Screening of resistant callus: After co-cultivation, the callus is placed in sterile water containing 400-600 mg / L Cef and shaken to sterilize, then shaken and rinsed with sterile water, dried and inoculated into screening medium for culture, and transgenic plants are detected after the callus grows regenerated plants. The screening medium is 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 + 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+3.0mg / L6-BA+2.0mg / LNAA+0.25mg / L TDZ+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8, MS+3.0mg / L 6-BA+3.0mg / L NAA+1.0mg / L KT+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8, or MS+1.0-1.5mg / LNAA+1.0-1.5mg / L KT+0.1-0.25mg / L TDZ+20-30g / L sucrose+5.0g / L agar, pH 5.4-5.8; the subculture 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 co-cultivation medium is MS+2.0-3.0mg / L 6-BA+0.25mg / LTDZ+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 / LCef+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 / LTDZ+500mg / LCef+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+1.0mg / LNAA+1.0mg / L KT+0.25mg / L TDZ+25g / L sucrose+5.0g / L agar, pH5.6; the subculture medium is MS+2.0mg / L 6-BA+0.25mg / L TDZ+25g / L sucrose+5.0g / L agar, pH5.6; the co-cultivation medium is MS+2.0mg / L 6-BA+0.25mg / L TDZ+25g / L sucrose+5.0g / L agar, pH5.6; the screening medium is MS+1.0mg / L IAA+0.1mg / L 6-BA+0.2mg / L TDZ+500mg / L Cef+25g / L sucrose+5.0g / L agar, pH5.

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4. The method according to claim 1, characterized in that: The Agrobacterium to be transformed is cultured until the 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 step of mixing the bacteria with the infection solution is to mix the bacteria with the infection solution until the concentration of the bacterial solution is OD 600 =0.

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5. The method according to claim 1, characterized in that: The addition of 150-250 μmol·L -1 The AS was added with 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 blade is a base slice of the test tube seedling leaf blade.

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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