An agrobacterium-mediated transgenic method based on callus of rhododendron simsii

Abstract

The application discloses a kind of Agrobacterium-mediated transgenic methods based on Anthracene callus.The method includes callus induction and proliferation, Agrobacterium infection, co-culture, and resistant callus screening.The application discloses callus induction medium, subculture medium, co-culture medium and screening medium and other formulations, Agrobacterium infection time and bacterial liquid concentration, and acetyl-syringone concentration, first establish Anthracene genetic transformation system with callus as receptor, which is conducive to molecular directed breeding, accelerates excellent foreign gene transfer to Anthracene, improves Anthracene variety, provides new ideas for Anthracene industrialization development, and has important application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a Agrobacterium-mediated transgenic method based on Hippeastrum callus. BACKGROUND

[0002] Plant transgenic technology has been widely applied in plant improvement and genetic breeding. Common methods include Agrobacterium-mediated method, DNA direct insertion method, pollen tube channel method and plant virus-mediated method, etc. Among them, Agrobacterium-mediated method is a widely used transgenic method, which has the advantages of simple operation, high transformation efficiency, good repeatability, single copy, less gene silencing phenomenon, and can perform large fragment transformation. However, the transgenic of monocotyledonous plants is often affected by the difficulty in establishing an effective regeneration system and the limitation of Agrobacterium on host affinity, which is difficult and has a low success probability.

[0003] Hippeastrum is a perennial bulbous flower of Amaryllidaceae Hippeastrum, which has beautiful leaf posture, large flowers, high ornamental value, and various medicinal values such as detoxification, anti-tumor and prevention of cardiovascular diseases, and has a broad market application prospect. In recent years, it has developed rapidly in China. However, there are problems in the process of Hippeastrum industry in China, such as few self-cultivated varieties, long growth cycle, short flowering period, and fragile flower stems prone to lodging, which seriously restrict the development of the industry. The breeding of Hippeastrum often uses hybrid breeding, but there are problems such as long breeding cycle and low controllability of traits. Establishing a genetic transformation system with callus as the receptor for molecular directional breeding has the advantages of high transformation efficiency and few chimeras, and has important application prospects. At present, there is no report on Agrobacterium-mediated transgenic method based on Hippeastrum callus. SUMMARY

[0004] The purpose of the present application is to provide an Agrobacterium-mediated transgenic method based on Hippeastrum callus, which comprises the following steps:

[0005] S1, callus induction and proliferation: the leaves of the in vitro seedlings of Azalea indica are used as explants to induce callus in callus induction medium, and the callus with good growth is subcultured in subculture medium, the callus induction medium is MS+3.0-3.5mg / L 6-BA+2.0-3.0mg / L NAA+0-1.0mg / L KT+0-0.25mg / L TDZ+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 is MS+2.0-3.0mg / L 6-BA+0.2-0.3mg / L TDZ+20-30g / L sucrose+5.0-6.0g / L agar pH 5.4-5.8;

[0006] S2, Agrobacterium infection: the Agrobacterium to be transformed is cultured to an OD 600 value of 0.6-0.8, the bacterial cells are collected by centrifugation, the bacterial cells are mixed with the infection solution, and the mixture is placed at room temperature in the dark for 2-3h, then 150-250μmol·L -1 of AS is added and reserved; the callus prepared in step S1 is cut into 0.5-1.0cm 3 pieces, and the pieces are poured into the bacterial cell and infection solution mixture until completely immersed, and the mixture is infected at 80-120rpm for 10-40min;

[0007] S3, co-culture: the infected callus of Azalea indica is taken out, air-dried, and inoculated in a co-culture medium, and dark culture is performed for 3-7d, and the co-culture medium is MS+2.0-3.0mg / L 6-BA+0.2-0.3mg / L TDZ+20-30g / L sucrose+5.0-6.0g / L agar pH 5.4-5.8;

[0008] S4, selection of resistant callus: after co-culture, the callus is placed in sterile water containing 400-600mg / L Cef, shaken to remove bacteria, washed with sterile water, dried, and inoculated in a selection medium for culture, and after the callus grows into a regenerated plant, the transgenic plant is detected, and the selection medium is MS+1.0-1.5mg / L IAA+0.1-0.2mg / L 6-BA+0.2-0.3mg / L TDZ+0-500mg / L Carb+400-600mg / L Cef+20-30g / L sucrose+5.0-6.0g / 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+20-30 g / L sucrose+5.0 g / L agar pH 5.4-5.8; the co-culture medium is MS+2.0-3.0 mg / L 6-BA+0.25 mg / L TDZ+20-30 g / L sucrose+5.0 g / L agar pH 5.4-5.8; 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 Carb+500 mg / L Cef+20-30 g / L sucrose+5.0 g / L agar pH 5.4-5.8 or MS+1.0 mg / L IAA+0.1 mg / L 6-BA+0.2 mg / L TDZ+500 mg / L Cef+20-30 g / L sucrose+5.0 g / 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-culture 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.

[0011] Preferably, the culturing of Agrobacterium to be transformed to an OD 600 value of 0.6-0.8; the mixing of the bacterial cells with the infection liquid is mixing of the bacterial cells with the infection liquid to an OD 600 value of 0.6; and the mixing of the bacterial cells with the infection liquid is mixing of the bacterial cells with the infection liquid to an OD 600= 0.6.

[0012] Preferably, the adding 150-250 μmol·L -1 of AS is adding 200 μmol·L -1 of 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 myo-inositol+1.0 g / L thiamine hydrochloride+30.0 g / L sucrose.

[0015] Preferably, the leaf blade of the test tube seedling is a basal section of a leaf blade of the test tube seedling.

[0016] Preferably, the cutting the callus into 0.5-1.0 cm 3 pieces is picking well-grown Euphorbia lathyris L. yellow callus in a clean bench and cutting the callus into 0.5-1.0 cm 3 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 application:

[0019] The present application first establishes a genetic transformation system of Euphorbia lathyris L. with callus as a receptor by formulating a callus induction medium, a subculture medium, a co-culture medium and a screening medium, and by adjusting the time and concentration of Agrobacterium infection and the concentration of acetosyringone, which is conducive to molecular directed breeding, accelerates the transfer of excellent foreign genes to Euphorbia lathyris L., improves Euphorbia lathyris L. varieties, provides a new idea for the industrial development of Euphorbia lathyris L., and has important application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the influence of infection time on the transformation efficiency of callus.

[0021] Figure 2 is the influence of the concentration of bacterial solution on the transformation efficiency of callus.

[0022] Figure 3 is the influence of different co-culture times on the transformation efficiency of callus.

[0023] Figure 4 is the influence of different concentrations of acetosyringone (AS) on the transformation efficiency of callus.

[0024] Figure 5are the transgenic detection of Agrobacterium-mediated genetic transformation of 'Alfresco' Hippeastrum callus. A-C: GUS transient expression detection, A: CK, B: GUS transient expression negative, C: GUS transient expression positive; D-E: GUS stable expression detection, D: tender 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: report gene GUS PCR electrophoresis detection, +, positive control, -, negative control, Maker is DL 2000.

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

[0026] The following examples are further illustrations of the application and are not intended to limit the application.

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

[0028] 1. Callus induction and proliferation

[0029] This patent takes 'Alfresco' Hippeastrum as the material, selects the lateral small bulbs of the excellent strain that has already opened flowers, removes the leaves and root system, and then cuts the bulb disc, divides it into 16 pieces, and takes the scales with a length of 2-3 cm. Then the cut bulb disc is soaked in 75% alcohol for 20-30 seconds, disinfected with 0.1% mercury solution for 8-10 minutes, washed with sterile water for 4-5 times, disinfected with 0.1% mercury solution for 4-5 minutes, washed with sterile water for 4-5 times, cut off the upper end of the scale, leave the bulb disc with 1-2 cm scale, inoculate on MS+6-BA 4.0 mg / L+20 g / L sucrose medium, and the disinfection success rate is 90-95%. Leaves grow out after about 30 days, take the aseptic test-tube seedling leaf 0.5 cm or so base section as the explant in the 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, and the callus induction rate is counted after 40 days of culture.

[0030] Select the callus with good growth for subculture 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, and select the callus with good growth as the transformation receptor material.

[0031] 2. Agrobacterium infection

[0032] Use a sterilized 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 shaking incubator at 220 rpm, and cultured overnight at 28°C. 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.

[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 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.

[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 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;

[0042] (4) Elution: Decolorize the material by transferring it to 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 surviving calli were tested for GUS transient expression. 25 showed blue spots, with a GUS transient expression rate of 41.67%. Of the remaining 140 calli for screening, 56 survived and proliferated into new calli after one month, resulting in 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. This may be due to false positives or reduced differentiation capacity after Agrobacterium infection. The resistant plants were tested for GUS stable expression and PCR positivity. Two plants were successfully stained and showed DNA bands, resulting in a final positive conversion rate of 5.56% and an overall positive rate of 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 0.5 cm or so base sections of the leaves of the test-tube seedlings of 'Alfresco' Hippeastrum (prepared in the same way as in Example 1) were used as explants and cultured 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 for 30-40 days to induce callus, and the various indicators were counted after 40 days of culture.

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

[0050] Table 2 Effect of different plant growth regulator combinations on induction of aseptic seedling leaves Note: In the same column of the table, different lowercase letters indicate significant differences (P≤0.05).

[0051] The well-growing callus induced in 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 subcultured in 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.

[0052] The results show that the proliferation rate was 2.9-4.0 in 30 days, and the best effect was in MS + 2.0 mg / L 6-BA + 0.25 mg / L TDZ + 25 g / L sucrose + 5.0 g / L agar pH 5.6, which could maintain the callus state, the proliferation rate was 4.0, and no bud differentiation occurred, which was most suitable for use as a transformation receptor material (Table 3).

[0053] Table 3 Effect of different 6-BA concentrations in MS + 0.25 mg / L TDZ on callus proliferation

[0054]

[0055] Note: In the same column of the table, different lowercase letters indicate significant differences (P≤0.05).

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

[0057] Step 1, 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 gun head to pick up the single colony of Agrobacterium tumefaciens EHA105 (pSoup) Chemically Competent Cell (AC1012) containing pCAMBIA-1304 plasmid to be transformed in LB solution containing Kan 50mg / L, and put it in a 220rpm shaker for overnight culture at 28℃. When the OD value of the bacterial solution reaches 0.6, stop shaking and centrifuge. Prepare the infection solution, mix the bacterial solution with the infection solution (MS+10.0g / L myo-inositol+1.0g / L thiamine hydrochloride+30.0g / L sucrose), and then the OD value of the bacterial solution is 0.6. After 2-3h standing at room temperature in the dark, add 200μmol / L AS standby. In the clean bench, cut the well-grown pale yellow callus of K. coccinea prepared in step 1 into 0.5-1.0cm small pieces, put them into sterile 50mL centrifuge tubes, and then pour the infection solution mixture into the tubes to fully immerse the materials. Shake at 120rpm for 10min, 20min, 30min, and 40min, respectively. 600 600 -1 3

[0059] The results show that different Agrobacterium infection times have different transformation efficiencies. Figure 1

[0060] (2) Use a sterilized toothpick or gun head to pick up the single colony of Agrobacterium tumefaciens EHA105 (pSoup) Chemically Competent Cell (AC1012) containing pCAMBIA-1304 plasmid to be transformed in LB solution containing Kan 50mg / L, and put it in a 220rpm shaker for overnight culture at 28℃. When the OD value of the bacterial solution reaches 0.4, 0.6, 0.8, or 1.0, stop shaking and centrifuge. Prepare the infection solution, mix the bacterial solution with the infection solution (MS+10.0g / L myo-inositol+1.0g / L thiamine hydrochloride+30.0g / L sucrose), and then the OD value of the bacterial solution is 0.6. After 2-3h standing at room temperature in the dark, add 200μmol / L AS standby. In the clean bench, cut the well-grown pale yellow callus of K. coccinea prepared in step 1 into 0.5-1.0cm small pieces, put them into sterile 50mL centrifuge tubes, and then pour the infection solution mixture into the tubes to fully immerse the materials. Shake at 120rpm for 10min, 20min, 30min, and 40min, respectively. 600 600 -1 3

[0061] The results show that when the bacterial solution is shaken to an OD value of 0.6, the transformation efficiency is the highest.​​​​​​​​​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 ).

[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 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.

[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 slightly compared to the beginning. Both of these changes 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, which were 37% and 34.72%, respectively. When co-cultured for 1 day and 7 days, the resistant callus rate was the lowest, which was 2.78 for both. Therefore, co-culture for 3 days is the optimal co-culture day for genetic transformation of Amaryllis callus tissue ( 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 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. 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, 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.

[0069] When performing Agrobacterium-mediated genetic transformation of monocotyledonous plants, 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 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 is ( 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 co-cultivation, the callus was placed in sterile water containing 500 mg / L Cef for 1 min, then washed with sterile water for 1 min, repeated 3 times, then dried on sterile filter paper, and then inoculated into the screening medium. The callus was inoculated into MS + 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 + 5.0 g / L agar pH 5.6 for screening culture, and the callus could regenerate plants. The growth of the culture material was observed, and the resistance rate of the callus was detected after about three weeks.

[0073] The callus after Agrobacterium infection and co-cultivation was inoculated into the medium containing different concentrations of Cef, Carb and Cef+Carb for antibacterial experiment. Carb had less antibacterial effect than Cef. The callus was contaminated by Agrobacterium at a rate of 100% without adding Cef and Carb, and the callus proliferation rate was very low, only 4.44. When Cef 500.00 mg / L -1 +Carb 500.00mg·L -1 , the contamination was the lowest, and Agrobacterium could be completely inhibited, followed by Cef 500.00 mg / L -1 , the contamination rate was 2.22%, but there was no significant difference between the two treatments, and adding multiple antibacterial agents would reduce the proliferation ability of callus. Therefore, Cef 500.00 mg / L -1 was recommended as the best antibacterial concentration of callus of Euphorbia pulcherrima (Table 4).

[0074] Table 4 Effect of different concentrations of cefotaxime and carbenicillin and their combination on Agrobacterium-mediated growth of 'Afre' Euphorbia pulcherrima callus

[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 B, AS (Acetosyringone) acetosyringone, Cef (Cefotaxime sodium claforan ctx) cefotaxime, 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 + 1.0 mg / L KT + 20-30 g / L sucrose + 5.0-6.0 g / L agar (pH 5.4-5.8), MS + 3.0-3.5 mg / L 6-BA + 2.0-3.0 mg / L NAA + 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 is MS + 2.0-3.0 mg / L 6-BA + 0.2-0.3 mg / LTDZ + 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 AS 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 immersed, and infect at 80-120 rpm for 10-40 minutes; 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 at pH 5.4-5.8 for 3-7 days in the dark. S4. Screening of resistant callus: After co-cultivation, the callus was placed in sterile water containing 400-600 mg / L Cef and degermed by shaking. The callus was then rinsed with sterile water by shaking. After drying, it was inoculated into a screening medium for culture. After the callus regenerated plants, transgenic plants were detected. The screening medium consisted of 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 + 3.0 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 + 20-30 g / L sucrose + 5.0 g / L agar pH5.4-5.8; the co-cultivation medium is MS + 2.0-3.0 mg / L 6-BA + 0.25 mg / L TDZ + 20-30 g / L sucrose + 5.0 g / L agar pH5.4-5.8; the screening medium is MS + 1.0 mg / L IAA + 0.1 mg / L 6-BA + 0.2 mg / L TDZ + 500 mg / L Carb + 500 mg / L Cef + 20-30 g / L sucrose + 5.0 g / L agar pH5.4-5.8 or MS + 1.0 mg / L IAA + 0.1 mg / L 6-BA + 0.2 mg / L TDZ + 500 mg / L Cef + 20-30 g / L sucrose + 5.0 g / 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, 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.

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4. The method according to claim 1, wherein In step 2, the Agrobacterium to be transformed is cultured to a bacterial solution OD 600 When the value reaches 0.6, the bacteria are collected by centrifugation and mixed with the infection solution until the concentration of the bacterial solution 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 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 + 10.0 g / L inositol + 1.0 g / L thiamine hydrochloride + 30.0 g / 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 In step S2, the callus prepared in step S1 is selected in a clean bench and the light yellow callus of Amaryllis with good growth is 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 120 rpm for 40 min.

10. The method according to claim 1, characterized in that After co-cultivation in step S4, the callus tissue was placed in sterile water containing 500 mg / L Cef and shaken to sterilize.

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