Agrobacterium rhizogenes-mediated arrowhead genetic transformation method
By using Agrobacterium rhizogenes-mediated transformation, the expression vector of the reporter gene was transferred into the shoot tip of Sagittaria salsa bulbs, which solved the problems of difficult callus induction and hairy root transformation in Sagittaria salsa genetic transformation, and achieved rapid rooting and positive root screening, thus improving transformation efficiency.
Patent Information
- Application Number
- CN202610245327.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-02
- Publication Date
- 2026-04-24
AI Technical Summary
Genetic transformation methods for arrowhead are difficult to induce callus formation and differentiation through conventional tissue culture, and hairy root transformation technology that does not require tissue culture is relatively difficult in monocotyledonous plants, making it difficult to induce seedlings from transgenic positive roots.
Using Agrobacterium rhizogenes-mediated transformation, the expression vector of the reporter gene was transferred into Agrobacterium rhizogenes. Genetic transformation was carried out using the shoot tips of Sagittaria sagittifolia bulbils. Positive underground roots were selected by screening for the reporter gene, and transgenic positive bulbs were then regenerated into positive plants.
This method achieves convenient and effective genetic transformation of arrowhead, enabling rapid root induction and easy screening of transgenic positive roots, avoiding the risk of damaging roots by uprooting, and improving transformation efficiency.
Smart Images
Figure CN121915094A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to plant transgenic biotechnology, specifically to a method for genetic transformation of arrowhead mediated by Agrobacterium rhizogenes. Background Technology
[0002] arrowhead( Sagittaria trifolia L . Arrowhead (Sagittaria sagittifolia) is a perennial herbaceous aquatic plant belonging to the genus Sagittaria in the family Alismataceae. It is a distinctive aquatic vegetable widely cultivated in Jiangsu, Guangxi, and Yunnan provinces of my country, and is considered one of the "Eight Aquatic Treasures." Compared to other horticultural crops, research efforts focused on arrowhead production, practice, and bioengineering breeding are relatively weak. Currently, there are numerous arrowhead varieties in China, but research on arrowhead is mostly concentrated in cultivation. Research on molecular genetic breeding and functional gene identification has progressed slowly due to a lack of whole-genome information, stable genetic transformation systems, and gene silencing and editing systems. Stable genetic transformation systems are essential for gene function verification in crop genetic breeding and molecular improvement. Suitable genetic transformation systems can efficiently and conveniently obtain transgenic plants, greatly facilitating plant breeding. Several horticultural plants have already achieved high-quality, highly resistant, and other new germplasm through stable genetic transformation, completing efficient targeted breeding of horticultural plants.
[0003] However, different plant genetic transformation methods vary considerably, requiring researchers in each species' research field to explore suitable genetic transformation pathways for that species. Exploring a stable genetic transformation system for Sagittaria sagittifolia is of significant practical importance for serving molecular biology research related to the plant. Previous experimental studies revealed difficulties in inducing callus formation from Sagittaria sagittifolia explants, and even greater challenges in differentiating and regenerating plants. Specifically, most plant genetic transformations utilize leaf or petiole sections or segments, aseptically cultured to induce callus formation, and then induced callus differentiation to obtain transgenic seedlings. However, our team's previous research found that using Sagittaria sagittifolia leaf and petiole tissue culture failed. The waxy surface of Sagittaria sagittifolia leaves and the well-developed aerenchyma of the petioles, along with their strong water resistance, make it difficult to induce callus formation from the contact surface cells on a culture medium supplemented with plant hormones. Therefore, it is difficult to induce callus formation and differentiation through conventional tissue culture for Sagittaria sagittifolia genetic transformation. Tissue culture offers the advantage of a nutrient-rich medium and the ability to add plant hormones that promote growth and rooting to induce rapid plant growth, but it is difficult to induce callus formation and differentiation. Arrowhead is a monocotyledonous plant, and only its bulb tissue has the ability to root, unlike plants such as water celery and kalanchoe, where stems, petioles, and leaves easily regenerate roots after being cut. Currently reported tissue-free hairy root transformation techniques are mostly used to infect plant stems, leaves, and petioles, or to inject plant buds to produce new branches for rooting. These techniques are suitable for plants with strong root regeneration capabilities, as the stems and leaves of monocotyledonous arrowhead do not easily regenerate roots. The advantage of tissue-free transformation is that it does not require callus induction, and the non-sterile environment eliminates the need to consider tissue sterilization. However, while Agrobacterium rhizogenes-induced tissue-free genetic transformation can produce transgenic positive roots, inducing seedlings (transgenic seedlings) from positive roots is difficult in most species. Summary of the Invention
[0004] Purpose of the invention: The purpose of this invention is to provide a method for genetic transformation of arrowhead mediated by Agrobacterium rhizogenes.
[0005] Technical solution: The method for genetic transformation of arrowhead mediated by Agrobacterium rhizogenes according to the present invention includes using the shoot tip of arrowhead bulb as the recipient material, transferring an expression vector containing a reporter gene into Agrobacterium rhizogenes, using Agrobacterium rhizogenes to mediate genetic transformation, screening plants with positive underground roots through the reporter gene, obtaining transgenic positive bulbs, and then regenerating positive plants.
[0006] Preferably, the Agrobacterium rhizogenes includes strain K599.
[0007] Preferably, the reporter gene includes GUS Reporter genes, GFP Reporter genes, EGFP Reporter genes.
[0008] Preferably, the expression vector includes pCAMBIA2300 and pCAMBIA1300-35S-EGFP plasmids.
[0009] Preferably, it includes the following steps: (1) Disinfection of arrowhead stem tips and preparation of sterile stem tips: Cut off fresh arrowhead stem tips, take fresh arrowhead bulbs and cut off the stem tips of healthy and intact bulbs, peel off the outermost 1-2 layers of scales and soak them in dish soap, rinse with running water and peel off 1-4 layers of scales again to obtain stem tips 2.5-3.5 cm long, disinfect with sodium hypochlorite solution and rinse with sterile water more than 3 times to obtain sterile stem tips for use; (2) Culture and activation of Agrobacterium rhizogenes: The expression vector containing the reporter gene was transferred into Agrobacterium rhizogenes, and the obtained positive Agrobacterium was cultured and activated using a culture medium; (3) Agrobacterium rhizogenes infection of shoot tips: Soak sterile shoot tips in activated Agrobacterium bacterial solution and incubate in the dark for 2-3 days; (4) Induction of rooting by shoot tip tissue culture: After washing the shoot tips infected with Agrobacterium rhizogenes, the shoot tips of Sagittaria sagittifolia were transferred to MS medium, with the shoot tips facing upwards, and cultured in a light incubator at 23~27℃ to induce rooting; (5) Positive identification of new roots: After the shoot tip tissue culture of arrowhead has rooted and grown to a height of 8-12 cm, multiple new roots at different positions are cut off, and positive roots are identified according to the reporter gene characteristics (if the reporter gene is GUS, staining is used for identification; if the reporter gene is GFP or EGFP, fluorescence observation is used for identification). (6) Transfer the plants with positive roots to pot substrate and harden them in a constant temperature incubator. When the plants grow to have a lush root system, transplant them into field pot soil for cultivation until harvest to obtain bulbs. After identifying positive bulbs based on reporter gene characteristics, promote germination and regeneration to obtain transgenic positive plants.
[0010] Preferably, in step (1), the stem tip is first soaked in 74-76% alcohol for 0.5-1 min, washed with sterile water 3-4 times, and then disinfected with sodium hypochlorite for 7.5-8.5 min. The concentration of the sodium hypochlorite solution used is 3.8-4.2%.
[0011] Preferably, in step (2), Agrobacterium culture is carried out using LB liquid medium, wherein a final concentration of 45-55 mg / L Kanamycin and 45-55 mg / L Streptomycin is added.
[0012] Preferably, the MS liquid culture medium for Agrobacterium activation in step (2) contains 0.55~0.65 mg / L NAA, 0.14~0.16 mg / L 6-BA and 40~50 mg / L acetylsuccinone.
[0013] Preferably, in step (3), the bottom of the sterile stem tip needs to be cut off by 0.4~0.6cm before being infected with Agrobacterium tumefaciens solution, and the stem tip needs to be inverted and shaken 2~3 times every 8~10 hours during the dark culture process.
[0014] Preferably, in step (4), the bacteria are first washed 2-4 times with sterile water, and then carbenicillin is added to the sterile water and washed 1-3 times until the sterile water is clear and transparent; after washing, the bacteria are transferred to MS solid medium (containing 45-55 mg / L treptomycin, 0.55-0.65 mg / L NAA, 0.14-0.16 mg / L 6-BA, 180-220 mg / L carbenicillin and 0.01-0.03% activated carbon).
[0015] Further optimization specifically includes the following steps: (1) Disinfection of arrowhead stem tips and preparation of sterile stem tips: Cut off fresh arrowhead stem tips and rinse with running water; Take fresh arrowhead bulbs and cut off the stem tips of healthy and intact bulbs. Peel off the outermost 1-2 layers of scales and soak in dish soap. After rinsing with running water, place in a clean bench and peel off 1-4 layers of scales again to obtain stem tips about 3 cm long. Disinfect with sodium hypochlorite solution and rinse with sterile water 3-5 times to obtain sterile stem tips for later use. (2) Culture and activation of Agrobacterium rhizogenes: ... GUS The reporter gene pCAMBIA2300 plasmid was transformed into Agrobacterium rhizogenes strain K599, and the obtained positive Agrobacterium was cultured and activated using LB liquid medium; (3) Agrobacterium rhizogenes infection of shoot tips: Soak sterile shoot tips in activated Agrobacterium bacterial solution and incubate in the dark for 2-3 days; (4) Induction of rooting by stem tip tissue culture: The stem tips of Sagittaria salsa infected with Agrobacterium rhizogenes were taken out in a clean bench, washed, and transferred to MS medium. The stem tips were placed with the stem tips facing upwards and cultured in a 25°C light incubator to induce rooting. (5) Positive identification of new roots: After the arrowhead shoot tip tissue culture has rooted and grown to about 10 cm in height, multiple new roots from different positions are cut off, and the root tips are stained with GUS staining solution and the color change of the root tips is observed. If they turn blue, they are positive roots. (6) The plants with positive roots were transferred to pot substrate and hardened off in a constant temperature incubator. When the plants grew to have a lush root system, they were transplanted into field pot soil for cultivation until harvest. The positive bulbs were identified by staining with GUS dye solution and then germinated to obtain transgenic positive plants.
[0016] In a further preferred embodiment, in step (1), the stem tip is first soaked in 75% alcohol for 1 min, washed with sterile water 3-4 times, and then disinfected with sodium hypochlorite for 8 min. The concentration of the sodium hypochlorite solution used is 4%.
[0017] More preferably, in step (2), the LB liquid medium used for Agrobacterium culture should be supplemented with a final concentration of 50 mg / L Kanamycin (hereinafter abbreviated as Km) and 50 mg / L Streptomycin (hereinafter abbreviated as Strep), and the liquid medium used for activating Agrobacterium is MS liquid medium (containing 0.6 mg / L 6-BA, 0.15 mg / L NAA and 40~50 mg / L acetosyringone (hereinafter abbreviated as AS)).
[0018] In a further preferred embodiment, in step (3), the bottom of the sterile stem tip needs to be cut off by 0.5 cm before being infected with Agrobacterium tumefaciens solution, and the stem tip needs to be inverted and shaken 2-3 times every 8-10 hours during the dark culture process.
[0019] In a further preferred embodiment, in step (4), the bacteria need to be washed three times with sterile water, and then carbenicillin is added to the sterile water for two more washes until the sterile water is clear and transparent. After washing, the bacteria are transferred to MS solid medium (containing 50 mg / L trep, 0.6 mg / L NAA, 0.15 mg / L 6-BA, 200 mg / L Carbenicillin (hereinafter abbreviated as Cb) and 0.01% activated carbon).
[0020] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The method described in this invention is more convenient and effective than methods that use explants to induce callus differentiation and regeneration; compared with genetic transformation methods that do not require tissue culture, it can rapidly induce rooting and facilitate the screening of transgenic positive roots, such as when the genetic transformation vector carries a visual reporter gene (e.g., GFP , EGFP (etc.) will be able to quickly identify positive roots growing on the culture medium by fluorescent light detection, without having to pull the arrowhead seedlings out of the soil by the roots for identification (avoiding root damage from pulling them out). Attached Figure Description
[0021] Figure 1 Image showing the germination process of arrowhead seeds (comparison of direct germination and germination after sodium hypochlorite disinfection).
[0022] Figure 2 This is a comparison of arrowhead stem tips treated with different disinfectant concentrations (2% and 0.5%) for 15 minutes in Example 1.
[0023] Figure 3 This is a vector diagram illustrating the operation flow of the method of the present invention.
[0024] Figure 4 The arrowhead stem tip is inoculated onto the culture medium after infection, as described in this invention example.
[0025] Figure 5 The results of GUS staining of transgenic positive roots identified in the examples of this invention are shown.
[0026] Figure 6 In this example of the invention, transgenic positive tissue culture seedlings are hardened off in small pots.
[0027] Figure 7 The image shows the GUS staining results of the underground bulbs of the transgenic positive tissue culture seedlings in this invention example. Detailed Implementation
[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0029] Example 1
[0030] In the early stages of the study, tissue culture was conducted on arrowhead leaves and petioles, but it was found that callus formation was difficult to induce. The original plan was to use arrowhead seeds, sterilized and germinated, to induce callus formation from the tender leaf and petiole tissues produced by seed germination. However, it was found that arrowhead seeds themselves have a low germination rate, and because the seeds are so small, sterilization with too low a concentration of disinfectant is ineffective, while too high a concentration results in extremely low germination rates. It was impossible to achieve both sterilization and germination assurance simultaneously. Arrowhead shoot tip tissue culture is feasible, but some shoot tips are prone to inadequate sterilization. While individual contaminated shoot tips can be discarded after sterilization and placement in separate culture media, centralized use for Agrobacterium infection after sterilization easily leads to overall microbial contamination. Arrowhead shoot tips are delicate tissues, and sterilization requires careful control of disinfectant concentration and time. Too high a concentration and too long a time result in softening and severe damage to the shoot tip, hindering differentiation, rooting, and shoot formation. Incomplete sterilization leads to a high contamination rate. Previous reports on shoot tip sterilization methods often use mercuric chloride and mercuric chloride, but these hazardous chemicals are currently difficult to procure in experiments. In the early stages of this invention, the following disinfection methods were used to determine the method for obtaining sterile stem tips for transformation. The specific steps are as follows: The 'Suzhou Yellow' variety of arrowhead, widely cultivated in production, was used as the experimental material. Fresh arrowhead tubers were cleaned, and healthy tuber tips of 5-6 cm were cut off. The outermost 1-2 layers of scales were removed, and the tubers were soaked in detergent for 5 minutes, rinsed under running water for about 15 minutes, dried with filter paper, and then placed in a clean bench to remove another 1-4 layers of scales, yielding tuber tips of about 3 cm. The obtained tuber tips were then soaked in 75% alcohol for 0.5 or 1 minute, rinsed 3-4 times with sterile water, and then soaked in 0.5%, 2%, 4%, or 17% NaClO for 8 minutes or 15 minutes, rinsed 3-4 times with sterile water. The tuber tips were then cultured on MS solid medium supplemented with 6-BA and NAA. The contamination rate was calculated after one month of culture. The results (Table 1) showed that disinfection with 75% alcohol for 0.5 or 1 minute, followed by disinfection with 4% sodium hypochlorite for 8 minutes, resulted in the highest efficiency in obtaining sterile arrowhead tuber tips, which can be used for further experiments.
[0031] Table 1. Arrowhead stem tip disinfection test
[0032] Example 2 This embodiment uses the pCAMBIA2300 vector carrying the GUS reporter gene as an example to describe the technical solution of the present invention in detail. However, those skilled in the art will understand that any other reporter gene (such as GFP / EGFP) and expression vector suitable for plant transformation can be used in the method of the present invention. This embodiment provides a method for genetic transformation of Sagittaria sagittifolia mediated by Agrobacterium rhizogenes, the specific method including the following steps performed in sequence (such as... Figure 3 (As shown): 1) Disinfection of arrowhead stem tips and preparation of sterile stem tips The tubers of the 'Suzhou Yellow' variety of arrowhead were selected as the experimental material. Fresh arrowhead tubers were cleaned, and healthy tuber tips of 5-6 cm were cut off. The outermost 1-2 layers of scales were peeled off, and the tubers were soaked in detergent for 5 minutes, rinsed under running water for about 15 minutes, dried with filter paper, and then placed on a clean bench to peel off another 1-4 layers of scales, yielding tuber tips of about 3 cm. The obtained tuber tips were then soaked in 75% alcohol for 1 minute, rinsed 3-4 times with sterile water, and then soaked in 4% NaClO for 8 minutes (this sterilization condition was determined based on the optimal method in Example 1). After rinsing 3-4 times with sterile water, the tuber tips were placed in petri dishes for later use.
[0033] 2) Culture and activation of Agrobacterium rhizogenes Use pCAMBIA2300 (carry) GUSActivated Agrobacterium tumefaciens bacterial culture was prepared using the reporter gene-containing Agrobacterium tumefaciens K599. Specifically, 50 µL of Agrobacterium tumefaciens bacterial culture carrying the transformation plasmid was cultured overnight in 10 mL of LB broth (with Km and Strep added to a final concentration of 50 mg / L) for activation. The next day, 2 mL of the activated bacterial culture was transferred to 50 mL of fresh LB broth (with antibiotics added as above) and cultured vigorously until the Agrobacterium tumefaciens bacterial culture reached OD. 600 After centrifuging at 0.4~0.5 for 8 min, the supernatant was discarded. The obtained bacterial cells were resuspended in MS liquid medium (with a final concentration of 0.6 mg / L NAA, 0.15 mg / L 6-BA and 50 mg / L AS) and shaken on a shaker at 28°C for 1 h to further activate and infect the Agrobacterium rhizogenes used.
[0034] 3) Agrobacterium rhizogenes infects the shoot tip Immerse the sterile stem tips in activated Agrobacterium bacterial solution and incubate in the dark for 2-3 days.
[0035] 4) Shoot tip tissue culture to induce rooting After soaking and washing the stem tips, transfer them to a culture medium containing antibiotics that inhibit Agrobacterium and incubate them in a 25°C light incubator. Specifically, wash the stem tips 3-5 times in sterile water until the washing solution is clear and transparent. Finally, insert the washed stem tips into a tissue culture flask containing MS solid medium (containing 50 mg / L Strep, 0.6 mg / L NAA, 0.15 mg / L 6-BA, 200 mg / L Cb, and 0.1 g activated carbon) (stem tip facing up) and place it in a 25°C light incubator to cultivate roots (e.g., ...). Figure 4 ).
[0036] 5) Positive identification of newly formed roots After the arrowhead tissue culture has rooted, cut off the arrowhead root system (three roots per plant, each root divided into three sections, approximately 0.5 cm in length), and completely immerse them in GUS staining solution; incubate at 37℃ for 1-24 hours, then observe the color change of the root tissue (if the color is not clear to the naked eye, it can be destained with alcohol for 1-3 hours before observation). Figure 5 As shown, roots that turn blue when stained are positive roots.
[0037] 6) Hardening off seedlings Plants with positive roots were transferred to potting mix and hardened off in a constant temperature incubator (e.g.) Figure 6 Once the plants have developed a robust root system, they are transplanted into pots in the field for cultivation until harvest to obtain bulbs. These bulbs are then stained with GUS dye (e.g., ...). Figure 7(As shown) After identifying positive bulbs, transgenic positive plants were obtained through germination and regeneration. Transgenic positive arrowhead plants can be obtained by germination after confirmation. This embodiment successfully obtained transgenic arrowhead positive roots and plants, confirming the feasibility and effectiveness of this method.
Claims
1. A method for genetic transformation of *Sagittaria sagittifolia* mediated by *Agrobacterium rhizogenes*, characterized in that, Using the shoot tip of arrowhead bulb as the recipient material, an expression vector containing a reporter gene was transferred into Agrobacterium rhizogenes. Genetic transformation was carried out using Agrobacterium rhizogenes-mediated transformation. Plants with positive underground roots were screened by reporter gene, and transgenic positive bulbs were obtained and then regenerated into positive plants.
2. The method for Agrobacterium rhizogenes-mediated genetic transformation of Sagittaria sagittifolia according to claim 1, characterized in that, The Agrobacterium rhizogenes includes strain K599.
3. The method for Agrobacterium rhizogenes-mediated genetic transformation of Sagittaria sagittifolia according to claim 1, characterized in that, The reporter genes include GUS Reporter genes, GFP Reporter genes, EGFP Reporter genes.
4. The method for Agrobacterium rhizogenes-mediated genetic transformation of Sagittaria sagittifolia according to claim 1, characterized in that, The expression vectors include pCAMBIA2300 and pCAMBIA1300-35S-EGFP plasmids.
5. The method for Agrobacterium rhizogenes-mediated genetic transformation of Sagittaria sagittifolia according to claim 1, characterized in that, Specifically, the following steps are included: (1) Disinfection of arrowhead stem tips and preparation of sterile stem tips: Cut off fresh arrowhead stem tips, take fresh arrowhead bulbs and cut off the stem tips of healthy and intact bulbs, peel off the outermost 1-2 layers of scales and soak them in dish soap, rinse with running water and peel off 1-4 layers of scales again to obtain stem tips 2.5-3.5 cm long, disinfect with sodium hypochlorite solution and rinse with sterile water more than 3 times to obtain sterile stem tips for use; (2) Culture and activation of Agrobacterium rhizogenes: The expression vector containing the reporter gene was transferred into Agrobacterium rhizogenes, and the obtained positive Agrobacterium was cultured and activated using a culture medium; (3) Agrobacterium rhizogenes infection of shoot tips: Soak sterile shoot tips in activated Agrobacterium bacillus solution and incubate in the dark for 2-3 days; (4) Induction of rooting by shoot tip tissue culture: After washing the shoot tips infected with Agrobacterium rhizogenes, the shoot tips of Sagittaria sagittifolia were transferred to MS medium, with the shoot tips facing upwards, and cultured in a light incubator at 23~27℃ to induce rooting; (5) Positive identification of new roots: After the arrowhead shoot tip tissue culture has rooted and grown to a height of 8-12 cm, multiple new roots at different positions are cut off, and positive roots are identified according to the reporter gene characteristics; (6) Transfer the plants with positive roots to pot substrate and harden them in a constant temperature incubator. When the plants grow to have a lush root system, transplant them into field pot soil for cultivation until harvest to obtain bulbs. After identifying positive bulbs based on reporter gene characteristics, promote germination and regeneration to obtain transgenic positive plants.
6. The method for Agrobacterium rhizogenes-mediated genetic transformation of Sagittaria sagittifolia according to claim 5, characterized in that, In step (1), the stem tip is first soaked in 74-76% alcohol for 0.5-1 min, washed with sterile water 3-4 times, and then disinfected with sodium hypochlorite for 7.5-8.5 min. The concentration of the sodium hypochlorite solution used is 3.8-4.2%.
7. The method for Agrobacterium rhizogenes-mediated genetic transformation of Sagittaria sagittifolia according to claim 5, characterized in that, In step (2), Agrobacterium is cultured using LB liquid medium, with a final concentration of 45-55 mg / L Kanamycin and 45-55 mg / L treptomycin added.
8. The method for Agrobacterium rhizogenes-mediated genetic transformation of Sagittaria sagittifolia according to claim 5, characterized in that, In step (2), the MS liquid culture medium used for Agrobacterium activation contains 0.55~0.65 mg / L NAA, 0.14~0.16 mg / L 6-BA and 40~50 mg / L acetylsuccinone.
9. The method for Agrobacterium rhizogenes-mediated genetic transformation of Sagittaria sagittifolia according to claim 5, characterized in that, In step (3), the bottom of the sterile stem tip needs to be cut off by 0.4-0.6 cm before being infected with Agrobacterium tumefaciens. During the dark culture of the stem tip, it needs to be inverted and shaken 2-3 times every 8-10 hours.
10. The method for Agrobacterium rhizogenes-mediated genetic transformation of Sagittaria sagittifolia according to claim 5, characterized in that, In step (4), the bacteria are first washed with sterile water 2 to 4 times, and then carbenicillin is added to the sterile water and washed 1 to 3 times until the sterile water used for washing is clear and transparent. After washing, the bacteria were transferred to MS solid medium; the MS solid medium contained 45-55 mg / L treptomycin, 0.55-0.65 mg / L NAA, 0.14-0.16 mg / L 6-BA, 180-220 mg / L arcenicillin and activated charcoal.