===================================================================================== *Agrobacterium*-mediated transformation of *Arabidopsis thaliana* T87 cultured cells ===================================================================================== Materials ========= Biological materials -------------------- - :doc:`../cell_lines/rpc00008` - *Agrobacterium tumefaciens* strain GV3101, harboring a vector with an appropriate transformation construct, growing on LB agar plates Equipment --------- - Glassware and equipment used for culturing T87 cells (:doc:`../cell_lines/rpc00008`) - Cell culture dish, plastic, 9 cm - Cell culture dish, plastic, 15 cm - Multi-well cell culture plate, 6-well - Conical centrifuge tube, plastic, 15 mL - Nylon membrane: Product. Cut into a circle with a diameter of about 14 cm. Sterilize by autoclave at 121°C for 20 min. - Forceps: Sterilize before use Chemicals and stock solutions ----------------------------- - Gamborg's B5 Medium Salt Mixture: 399-00621, FUJIFILM Wako Pure Chemical Corporation - MES hydrate: 2-(N-Morpholino)ethanesulfonic acid hydrate, M8250, Sigma-Aldrich - Gamborg's B5 vitamin solution, 1000×: G1019, Sigma-Aldrich - 1-Naphthaleneacetic acid (NAA): Potassium 1-naphthylacetate, 161-04021, FUJIFILM Wako Pure Chemical Corporation - Agar, powder: BD BACTO Agar, 214010, Becton Dickinson - Claforan: Dissolve in distilled water, sterilize by filtration, and store at −30°C - Kanamycin: Dissolve in distilled water, sterilize by filtration, and store at −30°C Culture media ------------- - :doc:`Jouanneau and Péaud-Lenoël (JPL) medium, 1 µM NAA, liquid (medium no. 5) <../media/medium_5>` - Callus-Inducing medium (CIM), 1 µM NAA, 0.6% (w/v) agar, pH 5.7 .. csv-table:: :header: "Chemical/Stock solution", "For 1 L" "Gamborg's B5 Medium Salt Mixture", "1 bag" "Sucrose", "30 g" "MES hydrate", "0.5 g" "Gamborg's B5 vitamin solution, 1000×", "1 mL" "NAA (1 mM)", "1 mL" "H\ :sub:`2`\ O", "---" * Adjust pH to 5.7 with KOH (1 N) .. csv-table:: "CIM", "1 L" "Agar, powder", "6 g" - Luria-Bertani (LB) medium, liquid (Wise 2006 [3]_). - JPL medium, liquid, supplemented with 200 g/mL Claforan - CIM, supplemented with 200 g/ml of Claforan, 15-cm dish - CIM, supplemented with 30 g/mL Kanamycin and 200 g/mL Claforan, 15-cm dish - CIM, supplemented with 30 g/mL Kanamycin and 200 g/mL Claforan, 9-cm dish - JPL medium, liquid, supplemented with 5 g/mL Kanamycin and 200 g/mL Claforan Methods ======= Preparation of T87 cell culture (2--3 days) ------------------------------------------- #. Pass 7-day-old T87 suspension cell culture through a 1-mm Stainless test sieve (:ref:`rpc00008: Arabidopsis thaliana T87 Figure 3 `). #. Subculture the T87 cell suspension into JPL medium in an Erlenmeyer flask (10-fold dilution). #. Incubate the T87 cell culture under continuous light at 22 with rotary shaking at 120 rpm for 2--3 days. Preparation of *Agrobacterium* culture (3 days) ----------------------------------------------- 4. Inoculate a colony of *Agrobacterium* in LB liquid medium containing appropriate antibiotics. #. Incubate the *Agrobacterium* culture at 30 for two days. #. Transfer the *Agrobacterium* culture to a larger volume of LB medium containing appropriate antibiotics. #. Incubate the *Agrobacterium* culture at 30 overnight. #. Transfer 500 µL of the *Agrobacterium* culture to a 1.5-mL microcentrifuge tube. #. Centrifuge the tube. #. Wash the *Agrobacterium* cells with JPL medium three times. #. Resuspend the *Agrobacterium* cells in 500 µL of JPL medium. Co-cultivation of T87 cells with *Agrobacterium* (2 days) --------------------------------------------------------- 12. Transfer 5 mL of the T87 suspension cell culture into a well of a 6-well cell culture plate. #. Add 50 µL of *Agrobacterium* culture to the T87 cell culture. #. Incubate the co-cultivation culture under continuous light with rotary shaking at 120 rpm at 22 for two days (`Notes 1 `__). Removal of *Agrobacterium* (3--4 days) -------------------------------------- 15. Transfer the co-cultivation culture into a 15-mL conical centrifuge tube. #. Centrifuge the tube at 600 rpm for 30 sec. #. Wash the T87 cells with 3 mL of JPL medium three times. #. Resuspend the T87 cells in 5 ml of JPL medium supplemented with 300 g/mL Claforan. #. Transfer the T87 cell suspension into a well of a 6-well cell culture plate. #. Incubate the T87 cell culture under continuous light with rotary shaking for 3--4 days. Antibiotic selection of transgenic cells (17 days~) --------------------------------------------------- 21. Transfer the T87 suspension cell culture into a 15-mL conical centrifuge tube. #. Wash the T87 cells with JPL medium one time. #. Resuspend the T87 cells in JPL medium (total volume 2 mL). #. Spread the T87 cells over a nylon membrane placed on CIM (15-cm dish) supplemented with 200 g/mL Claforan. #. Incubate the dish under continuous light for 3 days. #. Transfer the nylon membrane to CIM (15-cm dish) supplemented with 30 g/mL Kanamycin and 200 g/mL Claforan. #. Incubate the dish for further 2 weeks. #. Transfer an individual green callus onto CIM (9-cm dish) supplemented with 30 g/mL Kanamycin and 200 g/mL Claforan. #. Incubate the callus cultures. Induction of suspension cell cultures ------------------------------------- 30. Transfer a piece of green callus into JPL liquid medium supplemented with 5 g/mL Kanamycin and 200 g/mL Claforan. #. Maintain the transgenic culture according to :doc:`../cell_lines/rpc00008`. Notes ===== .. _note-1: #. *Agrobacterium* grow in the co-cultivation culture. Overgrowth of *Agrobacterium* damages T87 cells. #. Some research papers also report transformation methods of the Arabidopsis T87 cell line. [1]_ [2]_ References ========== .. [1] Hata T, Mukae K, Satoh S, Matsuo M, Obokata J (2021) Preculture in an enriched nutrient medium greatly enhances the *Agrobacterium*-mediated transformation efficiency in *Arabidopsis* T87 cultured cells. Plant Biotechnology 38: 179--182. PMID: `34177340 `_. DOI: `10.5511/plantbiotechnology.20.1211b `_ .. [2] Ogawa Y, Dansako T, Yano K, Sakurai N, Suzuki H, Aoki K, Noji M, Saito K, Shibata D (2008) Efficient and high-throughput vector construction and *Agrobacterium*-mediated transformation of *Arabidopsis thaliana* suspension-cultured cells for functional genomics. Plant & Cell Physiology 49: 242--250. PMID: `18178967 `__. DOI: `10.1093/pcp/pcm181 `__ .. [3] Wise AA, Liu Z, Binns AN (2006) Culture and maintenance of *Agrobacterium* strains. In: Wang K (ed) Methods in molecular biology, volume 343: *Agrobacterium* protocols, second edition, volume 1. Humana Press, Totowa, New Jersey, pp 3--13. PMID: `16988330 `__. DOI: `10.1385/1-59745-130-4:3 `__