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Agrobacterium-mediated transformation of friable   embryogenic calli and regeneration of transgenic   cassava
 

Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava

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Author: Bull 2009

Author: Bull 2009

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    Agrobacterium-mediated transformation of friable   embryogenic calli and regeneration of transgenic   cassava Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava Document Transcript

    • protocol Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava S E Bull1, 2, J A Owiti1, M Niklaus1, J R Beeching2, W Gruissem1 & H Vanderschuren1 Department of Biology, Plant Biotechnology, ETH Zurich, Zurich, Switzerland. 2Department of Biology & Biochemistry, University of Bath, Claverton Down, Bath, UK. 1 Correspondence should be addressed to H.V. (hvanderschuren@ethz.ch) or S.E.B. (s.e.bull@bath.ac.uk). Published online 3 December 2009; doi:10.1038/nprot.2009.208© 2009 Nature Publishing Group http://www.nature.com/natureprotocols Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking ~6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop. INTRODUCTION The starchy roots of cassava (Manihot esculenta Crantz) are Agrobacterium-mediated transformation of FEC has been used a vital source of carbohydrate for more than 500 million routinely in our laboratory to produce transgenic cassava with people living in tropical and subtropical regions. Cassava is ranked enhanced virus resistance12–14. It usually takes between 20 and 30 among the top five staple crops in global production (FAOSTAT weeks to produce transgenic shoots11 and despite advancements website) and has also recently emerged as a promising biofuel in the protocol, it remains a tedious and labor-intensive proce- crop given its bioethanol yield per ha1. As a conse­quence of dure. Setbacks and difficulties are largely due to the low regenera- its varied and important uses, cassava production relies on the tion frequency of plantlets from somatic embryos15, as well as the regular development of cultivars that have enhanced biotic and intrinsic variation (including tissue quality) observed within and abiotic stress tolerance, increased nutrient content and improved between different transformation experiments9. To overcome such processing qualities. However, due to heterozygosity, improve- limitations in transformation systems, it is important to optimize ment of the crop by traditional breeding is a difficult and each step of the procedure16–18. Our regular use of the protocol lengthy task2; the application of transformation technologies to has allowed us to assess critically the various stages and introduce integrate desired traits is therefore viewed as a very useful tool3. several improvements to create a more reliable and robust system. The ability to readily transform cassava will also enable studies It is our expectation that adoption of this protocol will reduce the to extend our understanding of gene function and the cassava workload associated with the production of transgenic cassava, genome4. allow implementation of cassava transformation in laboratories Protocols for cassava transformation were reported simulta- where it is needed, as well as provide an opportunity to extend neously by two different research groups in 1996. Li et al.5 used and improve current transformation programs that use a range of Agrobacterium-mediated transformation of somatic cotyledons to farmer-preferred and elite cassava cultivars. then regenerate transgenic shoots by organogenesis. Schöpke et al.6, however, performed microparticle bombardment of embryogenic Experimental design and overview of the procedure suspension-derived tissues and then regenerated transgenic plant- An overview of the protocol is represented in Figure 1 and is lets by embryo maturation. In the latter system, transformation divided into phases. Phase I: Production of somatic embryos; phase relied on the implementation of a protocol to generate totipo- II: Production of FEC; phase III: Agrobacterium-mediated trans- tent cell clusters, known as friable embryogenic callus (FEC)7. formation; phase IV: Maturation and development of transformed Agrobacterium-mediated transformation of FEC, a combination FEC; phase V: Selection and regeneration of transgenic plantlets; of the two original systems, has subsequently emerged as the most phase VI: Screening and analysis of transgenic plantlets. These efficient and widely used strategy to produce transgenic cassava8–10. phases are discussed below. This combined protocol is superior as, first, by using FEC there is a reduced risk of generating chimeric plants compared to procedures Phases I and II: Production of somatic embryos and FEC using organized tissues, such as cotyledons8. Second, selection (usu- FEC can be generated from leaf explants, shoot apical meri­ ally antibiotic resistance) of FEC results in fewer nontransformed stems or shoot axillary meristems of cassava cultivar Tropical plantlets being regenerated (i.e., escapes) compared to shoot orga- Manihot Series (TMS) 60444 by primary somatic embryogenesis11. nogenesis5,11. The use of axillary meristems (i.e., buds) cultivated on cassava nature protocols | VOL.4 NO.12 | 2009 | 1845
    • protocol because of the possibility of somaclonal variation (chromo- Grow wild-type, in vitro TMS60444 plantlets ~ 6–8 weeks, 16-h light, 28 °C somal rearrangements) an FEC line should not be micropropa- gated for more than ~6 months21. The quality of the FEC used for Transfer stem cuttings to CAM Agrobacterium-mediated transformation is reflected in its ability 2–4 days, dark, 28 °C Phase l to regenerate into healthy plantlets. Therefore frequent transfer of Production of Transfer buds to CIM somatic nontransformed FEC clusters to Murashige and Skoog medium 2 weeks, dark, 28 °C embryos supplemented with the synthetic auxin, 1-naphthaleneacetic acid Propagate and multiply developing embryos on CIM (NAA) and carbenicillin (MSN + C250) provides an indication of 2 weeks, dark, 28 °C Repeat for total of 6–8 weeks their transformation viability and ultimately can be regenerated for use as nontransgenic control plantlets. Transfer embryos to GD Phase ll 2–3 weeks, dark, 28 °C Production of friable Phase III: Agrobacterium-mediated transformation© 2009 Nature Publishing Group http://www.nature.com/natureprotocols embryogenic callus (FEC) After ~14 weeks, the FEC are inoculated with Agrobacterium con- Isolate FEC and culture on GD 2–3 weeks, 16-h light, 28 °C taining the pCambia 1305.1 binary vector (AF354045.1). The Repeat purification/subculturing for up to 6 months FEC viability assay MSN+C250, 10 d, 16-h light vector contains the GUSPlus reporter gene to allow easy visuali- replenish every 10 d zation of transformation success. Previously, FEC were cultured Grow Agrobacterium LBA4404 (+1305.1 vector) on YEBA+K50/R50/S100, 2 d, dark, 28 °C in liquid Schenk and Hildebrandt medium before and following Phase lll cocultivation with Agrobacterium (as a suspension) with regular Agrobacterium- Grow Agrobacterium LBA4404 (+1305.1 vector) in YEB+K50/R50/S100, 2 d, dark, 28 °C, shake 200 r.p.m. mediated sieving to remove NEFC10,21. However, we frequently observed transformation morpho­logical changes to the FEC, probably in part due to the Cocultivate FEC and Agrobacterium sieving process. In addition, FEC produced by this method had 4 d, 16 h light, 24 °C poor regeneration efficiency. These observations prompted us Wash FEC and Agrobacterium to revise the inoculation procedure and to develop a method in GDS+C500. Repeat until supernatant is clear which Agrobacterium liquid suspension is delivered directly to FEC Phase lV clusters on the propagation (GD) solid medium (Fig. 1). A similar Culture cocultured FEC Maturation and GD+C250, 4 d, 16-h light, 28 °C development of method has also been reported recently for the transformation transformed FEC of Brachypodium distachyon, a temperate grass22. Culture cocultured FEC GD+C250+H5, 1 week, 16-h light, 28 °C GUS assay 1 d, dark Phase IV: Maturation and development of transformed FEC Culture cocultured FEC GD+C250+H8, 1 week, 16-h light, 28 °C The potential stress caused to FEC by Agrobacterium during cocul­ tivation necessitates the thorough removal of bacteria followed by Culture cocultured FEC a recovery stage for later phases to be successful. For this the FEC GD+C250+H15, 1 week, 16-h light, 28 °C are washed in GDS + C500 until the supernatant is clear and trans- ferred to a plastic mesh for culturing/recovery on GD + C250 medium Culture cocultured FEC MSN+C250+H15, 1 week, 16-h light, 28 °C Phase V (Fig. 1). The use of mesh allows easy, weekly transfer of tissue and per- Repeat for ~ 6 weeks Selection and regeneration of mits close regulation of media composition (e.g., pH, salt and vitamin transgenic plantlets concentrations, antibiotic concentrations) for the developing FEC. Isolate developing embryos/cotyledons CEM+C100, 2 weeks, 16-h light, 28 °C After 4 d, the FEC/mesh are transferred to GD media containing a low Repeat until shoots appear (2–5 weeks) GUS assay concentration of hygromycin antibiotic (GD + C250 + H5), which is 1 d, dark Regenerate transgenic plants increased weekly thereafter (GD + C250 + H8 then GD + C250 + H15). CBM+C50, 2 weeks, 16-h light, 28 °C This process creates an increasingly stringent environment while ena- bling transformed FEC to express effectively the antibiotic resistance Screen plantlets using rooting test CBM+C50+H10, 2 weeks, 16-h light, 28 °C Phase Vl gene and initiate cell division, thus potentially improving successful Screening and analysis Advised to also perform Southern blot and PCR tests of transgenic plantlets plant regeneration6. pCambia 1305.1 vector contains hptII, which confers hygromycin resistance—a more efficient selection than kan- Figure 1 | Schematic diagram for Agrobacterium-mediated transformation of amycin (nptII) and related aminoglycosides. This is because complete cassava. growth inhibition can be achieved at low concentrations of hygromy- cin, resulting in selection of a greater proportion of transformed callus axillary medium (CAM) in vitro plantlets is preferred as it allows lines6. Even though we found that both hygromycin and kanamycin the production of organized embryogenic clusters with reduced can be used in the described protocol, we prefer the use of hygromycin accumulation of non-embryogenic friable calli (NEFC; Fig. 1). selection given the advantages mentioned above. The production of somatic embryos and FEC is as previously described11 but with modifications regarding the solidifying agent Phase V: Selection and regeneration of transgenic plantlets used and preparation of FEC for transformation. The primary At 3–4 weeks after cocultivation the FEC are cultured on Murashige embryogenic clusters are multiplied and purified on Murashige and Skoog medium supplemented with NAA, carbenicillin and and Skoog medium19 supplemented with the synthetic auxin, piclo- hygromycin (MSN + C250 + H15) to stimulate the maturation ram (cassava induction medium, CIM). FEC is then initiated from and regeneration of hygromycin-resistant embryos (Fig. 1). The high-quality embryogenic tissue cultured on a Gresshof and Doy maturation and development phase (phase IV), as discussed above, (GD) medium20 (containing picloram) and is subcultured every significantly improves regeneration capacity while maintaining a 2–3 weeks, resulting in FEC with minimal NEFC. Importantly, rigorous selection pressure so that the vast majority (>90%) of 1846 | VOL.4 NO.12 | 2009 | nature protocols
    • protocol embryos developing in phase V are transgenic. This in turn allows Phase VI: Screening and analysis of transgenic plantlets hygromycin to be excluded from further growth medium, reduc- Fully rooted plantlets are screened by transfer of a stem cutting to ing the potential negative impact of the antibiotic on plant regen- CBM + C50 + H10 for the rooting test23 (Fig. 1). After 2 weeks only eration10. Developing embryos and cotyledons are transferred to transgenic plants will have developed new roots (and consequently media containing 6-benzylaminopurine (a synthetic cytokinin) and more leaf tissue) on this hygromycin-containing medium, whereas carbenicillin (cassava elongation medium; CEM + C100) to induce wild-type control cuttings will remain rootless and eventually shoot growth. It is crucial that carbenicillin is present, otherwise, become chlorotic. The rooting test is a rapid and reliable screen for there is a tendency for Agrobacterium to grow around the embryo/ transgenic plants clearly identifying the few nontransgenic plant- cotyledon and suppress its development. Fortnightly transfer of the lets that escaped the initial hygromycin selection phase. Transgenic developing green tissue to CEM + C100 will result in a shoot that plantlets should be analyzed by PCR to confirm integration of the can be isolated and transferred to a Murashige and Skoog-based gene of interest and also by Southern blot to provide conclusive medium (cassava basic medium; CBM + C50), lacking hygromycin data pertaining to the copy number and integration pattern of the© 2009 Nature Publishing Group http://www.nature.com/natureprotocols selection, for establishment of plantlets. T-DNA. MATERIALS REAGENTS • Rifampicin (Duchefa, cat. no. R0146.0005) ! CAUTION Harmful if • Acetosyringone (3′,5′-dimethoxy-4′-hydroxyacetophenone; Sigma-Aldrich, swallowed. R22. S36. cat. no. D134406) ! CAUTION Irritant to eyes, respiratory system and skin. • Sterilized deionized water (SDW) R36/37/38. S26, 36  CRITICAL Risk (R) and Safety (S) codes throughout • Sodium hydroxide (NaOH; Sigma-Aldrich, cat. no. 71690) ! CAUTION the materials are based on European Union Commission Directives. Corrosive; causes severe burns. R35. S26, 37/39, 45. • A. tumefaciens strain LBA4404 harboring pCambia 1305.1 plasmid, • Streptomycin sulfate salt (Sigma-Aldrich, cat. no. S6501) ! CAUTION containing GUSPlus reporter gene and hygromycin (hptII) resistance gene. Harmful if swallowed. R22. • Bacto agar (Difco, cat. no. 0140-07-4) • Sucrose (Roth, cat. no. 4661.3) • Bacto beef extract (Difco, cat. no. 0115-17-3) • Tris(hydroxymethyl)aminomethane (TRIS; Chemie Brunschwig, • Bacto peptone (Difco, cat. no. 0118-17-0) cat. no. 20092391) ! CAUTION Irritant to eyes, respiratory system and skin. • Bacto yeast extract (Difco, cat. no. 0127-07-1) R36/37/38. S26, 37/39. • 6-Benzylaminopurine (BAP; Duchefa, cat. no. B0904.0025) ! CAUTION • Triton X-100 (Sigma-Aldrich, cat. no. 93426) ! CAUTION Harmful if Harmful if swallowed; irritant to eyes, respiratory system and skin. swallowed; risk of serious damage to eyes. Toxic to aquatic organisms, R22, 36/37/38. S24/26, 36. may cause long-term adverse effects in the aquatic environment. R22, 41, • 5-Bromo-4-chloro-3-indoxyl-β-D-glucuronic acid, cyclohexylammonium 51/53. S26, 36/39, 61. salt (X-Gluc; Biosynth, cat. no. B-7300) ! CAUTION Harmful if swallowed; • Optima compost (G. Optima-Werke) irritant to eyes and skin. R 22, 36/38. S26, 36/37, 60. • Perlite • Carbenicillin disodium (Duchefa, cat. no. C0109.0025) ! CAUTION May • Wuxal Bio plant fertilizer (Maagoplan, cat. no. 7.610176.068.860) cause sensitization by inhalation and skin contact. R42/43. S36/37/39. • Plastic plant pots • Cassava (M. esculenta Crantz) cultivar TMS60444. EQUIPMENT • Copper(II) sulfate pentahydrate (CuSO4·5H2O; Sigma-Aldrich, cat. no. • Autoclave C3036) ! CAUTION Harmful if swallowed; irritant to eyes and skin. Very • Sterile plastic Petri dishes, 90 mm (Sarstedt, cat. no. 82.1473) toxic to aquatic organisms, may cause long-term adverse effects in the • Plastic mesh 100 µm, sterile (Lanz-Anliker, cat. no. AH03558) aquatic environment. R22, 36/38, 50/53. S22, 60, 61. • Pipettes 25 ml, sterile (Sarstedt, cat. no. 86.1685.001) • Dimethyl sulfoxide (DMSO; Sigma-Aldrich, cat. no. 41641) • Sterile jars (53 mm × 100 mm; Greiner, cat. no. 7.968161) • N,N -Dimethylformamide (DMF; Sigma-Aldrich, cat. no. 40240) • pH meter ! CAUTION May cause harm to the unborn child. Harmful by inhalation • Balance and precision balance and in contact with skin; irritant to eyes. R61, 20/21, 36. S45, 53. • Centrifuge for 50-ml tubes • Gelrite (Duchefa, cat. no. G1101.5000) • 15-ml sterile, disposable tubes (Huber, cat. no. 7.187262) • Gresshof and Doy medium including vitamins (Duchefa, cat. no. • Microfuge G0212.0050) • Tabletop shaker • Hydrochloric acid 37% (wt/wt) (HCl; Sigma-Aldrich, cat. no. 84422) • Controlled environment chamber (Sanyo MLR, 28 °C, 16-h light/ ! CAUTION Corrosive, causes burns; irritant to the respiratory system. 8-h dark) R34, 37. S26, 45. • Controlled environment room (24 °C, 16-h light/8-h dark) • Hygromycin B (Roth, cat. no. CP12.1) ! CAUTION Very toxic when • Pipette aid inhaled, in contact with skin and if swallowed. Risk of serious damage • Parafilm (Huber, cat. no. 15.1550.02) to eyes. May cause sensitization when inhaled and in contact with skin. • Laminar flow hood with Bunsen burner R23/24/25, 42/43. S26, 28-36/37/39, 45.  CRITICAL High-quality product • Fridge (4 °C) and freezer ( − 20 °C) required. • Glassware (beakers, Duran bottles and Erlenmeyer flasks) • Kanamycin monosulphate (Duchefa, cat. no. K0126.0025) ! CAUTION Toxic; • Sterile, disposable syringe filters (0.22 µm; Millipore, cat. no. SLGP033RB) may cause harm to the unborn child. R61. S45, 53. • Micropore tape • Magnesium sulfate heptahydrate (MgSO4·7H2O; Sigma-Aldrich, • Aluminum foil cat. no. 63140) • Scalpel and forceps • Murashige and Skoog (MS) medium including vitamins (Duchefa, • Binocular microscope cat. no. M0222.0050) • Sterile, disposable inoculation loops (Sarstedt, cat. no. 86-1567-010) • Noble agar (Difco, cat. no. 214230) • Static incubator for bacterial cultures (28 °C) • 1-Naphthaleneacetic acid (NAA; Sigma-Aldrich, cat. no. N0640) ! CAUTION • Incubator-shaker (28 °C) Harmful if swallowed; irritant to the respiratory system and skin; risk of • Spectrophotometer serious damage to eyes. R22, 37/38, 41. S22, 26, 36. • 1 ml disposable cuvettes • Picloram (Duchefa, cat. no. P0914.0010) ! CAUTION Toxic substance; • Microfuge tubes (1.5 ml) harmful by inhalation, in contact with skin and if swallowed. Irritant to • Magnetic stirring bars eyes and may cause cancer. R20/21/22, 36, 45. S26, 36/37/39, 45. • Scissors nature protocols | VOL.4 NO.12 | 2009 | 1847
    • protocol • Disposable Pasteur pipettes and autoclave. Allow the media to cool and add 1 ml kanamycin (50 mg • Disposable syringes (1 and 5 ml) ml − 1), 2 ml rifampicin (25 mg ml − 1) and 1 ml streptomycin (100 mg ml − 1). • Glass spreaders for bacteria cultures Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and • Vortex allow to solidify (~1 h). Store at 4 °C. • Spatula YEB + K50/R50/S100 liquid medium for Agrobacterium culture  Dissolve • Disposable weighing boats 1 g Bacto yeast extract, 5 g Bacto beef extract, 5 g Bacto peptone, 5 g sucrose • Sterile, disposable 50 ml tube (screw top) (Sarstedt, cat. no. 62.547.254) in 1 liter SDW (final volume). Adjust pH to 7.2 and autoclave. Once the • Sterile, disposable 50 ml tube (flip top) (Nunc, cat. no. NC-362696) media are at room temperature add 1 ml kanamycin (50 mg ml − 1), 2 ml REAGENT SETUP rifampicin (25 mg ml − 1), 1 ml streptomycin (100 mg ml − 1) and 2 ml (1 M) Media and stocks  Autoclave media and stocks (15 min at 121 °C). All media MgSO4. Store at 4 °C. should be used at room temperature (~22 °C) unless otherwise stated. GDS solution for Agrobacterium preparation  Dissolve 2.7 g GD medium  CRITICAL Solutions stored at 4 °C should be replaced after 3 months; including vitamins, 20 g sucrose and 1 ml picloram (12 mg ml − 1) in 1 liter solutions at  − 20 °C after 6 months. Media should be prepared weekly. SDW (final volume). Adjust pH to 5.8 and autoclave. Store at 4 °C. NAA (1 mg ml − 1 stock solution)  Dissolve 50 mg in 1 ml 1 M NaOH and GDS + C500 solution for washing FEC  Dissolve 2.7 g GD medium including© 2009 Nature Publishing Group http://www.nature.com/natureprotocols adjust volume to 50 ml with SDW. Store at 4 °C. vitamins, 20 g sucrose and 1 ml picloram (12 mg ml − 1) in 1 liter SDW (final Picloram (12 mg ml − 1 stock solution)  Dissolve 0.6 g in ~5 ml of 1 M NaOH volume). Adjust pH to 5.8 and autoclave. Once media are at room tempera- and adjust volume to 50 ml with SDW. Filter (0.22 µm)-sterilize, aliquot into ture add 1 ml carbenicillin (500 mg ml − 1) and mix. Store at 4 °C. 1.5 ml microfuge tubes and store at  − 20 °C. GD + C250 solid medium for recovery of transgenic FEC  Dissolve 2.7 g GD BAP (1 mg ml − 1 stock solution)  Dissolve 20 mg in ~3 ml of 1 M NaOH and medium including vitamins, 20 g sucrose and 1 ml picloram (12 mg ml − 1) in adjust volume to 20 ml with SDW. Filter (0.22 µm)-sterilize, aliquot into 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and auto- 1.5 ml microfuge tubes and store at  − 20 °C. clave. Once media are cooled add 500 µl carbenicillin (500 mg ml − 1). Pour CuSO4·5H2O (2 mM stock solution)  Dissolve 2.49 g in 50 ml SDW (0.2 M stock ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solution), then dilute 1 ml in 100 ml SDW (final volume) and store at 4 °C. solidify (~1 h). Store at 4 °C. MgSO4·7H2O (1 M stock solution)  Dissolve 24.6 g in 100 ml SDW. GD + C250 + (H5, H8 or H15) solid medium for maturation of transgenic Filter (0.22 µm) sterilize. Store at 4 °C. FEC  Dissolve 2.7 g GD medium including vitamins, 20 g sucrose and Rifampicin (25 mg ml − 1 stock solution)  Dissolve 625 mg in 25 ml 0.1 N 1 ml picloram (12 mg ml − 1) in 1 liter SDW (final volume). Adjust pH to HCl. Filter (0.22 µm)-sterilize and aliquot. Store at  − 20 °C. 5.8, add 8 g Noble agar and autoclave. Once media are cooled add 500 µl Hydrochloric acid (HCl 0.1 N stock solution)  Add 820 µl 37% (wt/wt) carbenicillin (500 mg ml − 1) and the appropriate amount of hygromycin HCl to 99.18 ml SDW. (50 mg ml − 1); 100 µl for GD + C250 + H5; 160 µl for GD + C250 + H8 and Kanamycin monosulphate (50 mg ml − 1 stock solution)  Dissolve 2.5 g in 300 µl for GD + C250 + H15. Pour ~25 ml into sterile 90-mm Petri dishes 50 ml SDW. Filter (0.22 µm)-sterilize, aliquot into 1.5 ml microfuge tubes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. and store at  − 20 °C. MSN + C250 medium for regeneration of wild-type FEC  Dissolve 4.4 g MS Streptomycin sulfate (100 mg ml − 1 stock solution)  Dissolve 5 g in 50 ml medium including vitamins, 20 g sucrose and 1 ml NAA (1 mg ml − 1) in SDW. Filter (0.22 µm)-sterilize, aliquot into 1.5 ml microfuge tubes and 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and auto- store at  − 20 °C. clave. Once media are cooled add 500 µl carbenicillin (500 mg ml − 1). Pour Carbenicillin disodium (500 mg ml − 1 stock solution)  Gradually dissolve ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to 25 g in 50 ml SDW. Filter (0.22 µm)-sterilize, aliquot into 1.5 ml microfuge solidify (~1 h). Store at 4 °C. tubes and store at  − 20 °C. MSN + C250 + H15 medium for regeneration of transgenic embryos  Tris/NaCl buffer for GUS assay  Add 1.21 g Tris and 2.92 g NaCl to 1 liter Dissolve 4.4 g MS medium including vitamins, 20 g sucrose and 1 ml NAA SDW (final volume) and adjust pH to 7.2 by adding concentrated HCl. (1 mg ml − 1) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble Autoclave and store at room temperature. agar and autoclave. Once media are cooled add 500 µl carbenicillin X-Gluc (10 mg ml − 1 stock solution) for GUS assay  Dissolve 100 mg (500 mg ml − 1) and 300 µl hygromycin (50 mg ml − 1). Pour ~25 ml into X-Gluc in 10 ml DMF. Store in 1 ml aliquots at  − 20 °C. Wrap container in sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify aluminum foil (X-Gluc is light sensitive). (~1 h). Store at 4 °C. Triton X-100 (10% (vol/vol) stock solution) for GUS assay  Add 5 ml Triton CEM + C100 solid medium for generation of shoots  Dissolve 4.4 g MS X-100 to 45 ml SDW and mix gently but continuously until dissolved. Store medium including vitamins, 20 g sucrose, 400 µl BAP (1 mg ml − 1) and at room temperature. 1 ml CuSO4 (2 mM) in 1 liter SDW (final volume). Adjust pH to 5.8, add Acetosyringone (200 mM stock solution)  Dissolve 1.962 g in 50 ml DMSO. 8 g Noble agar and autoclave. Once the media are cooled add 200 µl Aliquot into 1.5 ml microfuge tubes and store at  − 20 °C. carbenicillin (500 mg ml − 1). Pour ~25 ml into sterile 90-mm Petri dishes CAM solid medium for induction of axillary buds  Dissolve 4.4 g MS in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. medium including vitamins, 20 g sucrose, 1 ml CuSO4 (2 mM) and 10 ml CBM for propagation of in vitro plantlets  Dissolve 4.4 g MS medium BAP (1 mg ml − 1) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g inclu­ding vitamins, 20 g sucrose and 1 ml CuSO4 (2 mM) in 1 liter SDW Noble agar and autoclave. Allow media to cool before pouring ~25 ml into (final volume). Adjust pH to 5.8, add 3 g gelrite and autoclave. Allow media sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify to cool and pour ~35 ml into sterile 53 mm × 100 mm plastic jars. Leave in (~1 h). Store at 4 °C. laminar flow hood for ~1 h for media to solidify. CIM solid medium to induce somatic embryos  Dissolve 4.4 g MS medium CBM + C50 for propagation of in vitro plantlets  Dissolve 4.4 g MS medium including vitamins, 20 g sucrose, 1 ml CuSO4 (2 mM) and 1 ml picloram including vitamins, 20 g sucrose and 1 ml CuSO4 (2 mM) in 1 liter SDW (12 mg ml − 1) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble (final volume). Adjust pH to 5.8, add 3 g gelrite and autoclave. Allow media agar and autoclave. Pour ~25 ml into sterile 90-mm Petri dishes in a laminar to cool before adding 100 µl carbenicillin (500 mg ml − 1) and pour ~35 ml flow hood and allow to solidify (~1 h). Store at 4 °C. into sterile 53 mm × 100 mm plastic jars. Leave in laminar flow hood for GD solid medium for induction and propagation of FEC  Dissolve 2.7 g GD ~1 h for media to solidify. Store at 4 °C. medium including vitamins, 20 g sucrose and 1 ml picloram (12 mg ml − 1) in CBM + C50 + H10 for screening of transgenic in vitro plantlets  Dissolve 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and auto- 4.4 g MS medium including vitamins, 20 g sucrose and 1 ml CuSO4 (2 mM) clave. Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood in 1 liter SDW (final volume). Adjust pH to 5.8, add 3 g gelrite and autoclave. and allow to solidify (~1 h). Store at 4 °C. Allow media to cool before adding 100 µl carbenicillin (500 mg ml − 1) and YEBA + K50/R50/S100 solid medium for Agrobacterium culture  Dissolve 200 µl hygromycin (50 mg ml − 1). Pour ~20 ml into sterile, disposable 50 ml 1 g Bacto yeast extract, 5 g Bacto beef extract, 5 g Bacto peptone and 5 g tubes (Nunc; cat. no. NC-362696). Leave in laminar flow hood for ~1 h for sucrose in 1 liter SDW (final volume). Adjust pH to 7.2, add 15 g Bacto agar media to solidify. Store at 4 °C. 1848 | VOL.4 NO.12 | 2009 | nature protocols
    • protocol PROCEDURE Induction of somatic embryos from TMS60444 ● TIMING 6–8 weeks 1| Make stem cuttings of in vitro TMS60444 plantlets, removing leaves and shoots, and place horizontally on CAM. Seal plates with parafilm, wrap in aluminum foil and incubate at 28 °C for 2–4 d.  CRITICAL STEP For easier handling in Step 2, leave a sufficient length of stem (~5 to 10 mm) either side of the axillary shoot (Fig. 2a). The apical shoots should be used for planting in CBM to establish a new population of in vitro plantlets (28 °C, 16-h light/8-h dark).  CRITICAL STEP To minimize tissue multiplication in subsequent steps, it is advised to use material from a minimum of ~75 plantlets.  CRITICAL STEP Work in this and subsequent steps is conducted under sterile conditions in a laminar flow hood using sterile tools and media.© 2009 Nature Publishing Group http://www.nature.com/natureprotocols 2| Remove the axillary buds (Fig. 2a) with sterile syringe needles using a binocular microscope and place them on CIM. If the bud has already started to shoot, simply remove the emerging leaves and transfer the growing tip to media. Seal plates with parafilm, wrap in aluminum foil and incubate at 28 °C for 2 weeks. 3| Transfer the developing embryos, gently removing any NEFC with sterile syringe needles, to CIM (Fig. 2b). Seal plates with parafilm, wrap in aluminum foil and incubate at 28 °C for 2 weeks. 4| Repeat Step 3. At this stage the embryos will be developing and will need to be divided (Fig. 2c). a d g j b e h k c f i l Figure 2 | Procedure for producing transgenic cassava plants. (a) Swollen axillary bud on CAM. (b) Immature somatic embryos (indicated by arrows) developing on a bed of NEFC on CIM. (c) Maturing somatic embryos on CIM. Dotted line indicates approximate suggested division for further propagation. (d) Cluster of FEC on GD appropriate for Agrobacterium inoculation. (e) FEC following cocultivation spread onto mesh on GD + C250. (f) Developing embryo/ cotyledon (indicated by arrow) on MSN + C250 + H15. Transformed FEC seen as swollen, yellowish structures. Nontransformed are smaller, white clusters. (g) Developing embryo/cotyledon transferred to CEM + C100. (h) Developing embryos/cotyledons from MSN + C250 + H15 used for GUS assay. Blue precipitate clearly visible throughout all tissue. (i) Appearance of immature shoots following several weeks on CEM + C100. (j) In vitro transgenic cassava plantlet. (k) GUS-stained leaves. (l) Rooting assay of transgenic plantlets (left and center) and wild-type TMS60444 (right) on CBM + C50 + H10. Scale bars represent 5 mm. nature protocols | VOL.4 NO.12 | 2009 | 1849
    • protocol 5| The embryos should now be developing finger-like structures, although some will still appear more compact, coral-like (Fig. 2c). If the latter is more abundant then cycle again as in Step 3.  CRITICAL STEP After 6–8 weeks one should aim to have in excess of 20 plates of CIM/somatic embryos (with ~10 embryos per plate) to generate sufficient amounts of FEC. Generation of FEC ● TIMING 6 weeks 6| Divide the embryos and transfer to GD. Seal plates with parafilm, wrap in aluminum foil and incubate at 28 °C. After 2 weeks use a binocular microscope to check for the presence of FEC.  CRITICAL STEP FEC are small clusters of off-white/yellowish small ball-like structures. FEC development is variable both in terms of timing and efficiency; retaining the embryos on GD for a further 1–2 weeks should make identification easier. However, due to nutrient depletion and thus stress, FEC should not be retained on a plate for more than© 2009 Nature Publishing Group http://www.nature.com/natureprotocols ~4 weeks.  CRITICAL STEP The limitation of space in incubators may become a consideration as tissue is multiplied, but stacking Petri dishes 2 or 3 high has had no discernible affect on tissue quality or growth.  CRITICAL STEP Various experiments were performed to establish the effect of different incubators on tissue development (data not shown). From this it was apparent that tissue development was improved in chambers with closely regulated conditions (e.g., Sanyo MLR Plant Growth Chamber) rather than generic plant growth chambers. In the latter it was common to see a considerable quantity of moisture on the lids of the Petri dishes, which presumably affected both light distribution and media/culture conditions. 7| Isolate clusters of FEC using sterile syringe needles and place on GD (Fig. 2d), ~10 clusters per plate. Seal plates with parafilm and incubate at 28 °C, 16-h light/8-h dark for 2 weeks.  CRITICAL STEP Do not disrupt the clusters of FEC as this will attenuate their growth. If there is any doubt about the FEC, it is advised to transfer to GD and then after 2–3 weeks the FEC should have approximately doubled in size; any cluster that does not should be discarded. 8| Every 2 weeks multiply the FEC tissue on GD.  CRITICAL STEP The multiplication and maintenance of FEC not only relies on the transfer of material but also on the continuing isolation of healthy, young FEC from NEFC. This process can continue for ~6 months.  CRITICAL STEP The extent to which FEC can be multiplied and maintained has not been determined in this paper, but there have been reports of an increased likelihood of somaclonal variation in tissue maintained for more than 6 months21. The experience of the worker to consistently maintain and isolate good quality material may affect the life span of the FEC. It is therefore recommended that a new induction (Step 1 onwards) is started every 1–2 months to maintain a regular supply of young, healthy FEC. Preparation of Agrobacterium inoculum ● TIMING 4–5 d 9| Streak A. tumefaciens (LBA4404 strain), carrying the pCambia 1305.1 binary vector, from a glycerol stock onto YEBA + K50/R50/S100 plates, invert and incubate at 28 °C for 2 d in the dark. ! CAUTION Handle genetically modified organisms according to good laboratory practice. 10| Remove a colony using a sterile inoculation loop and inoculate 5 ml of YEB + K50/R50/S100 in a 15-ml sterile disposable tube. Grow overnight in an incubator-shaker at 28 °C and 200 r.p.m. 11| Measure the optical density (OD), allowing cultures to grow until λ = 600 nm is 0.7–1. Allow any solids to settle and remove ~0.5 ml to inoculate 25 ml of YEB + K50/R50/S100 in sterile 250 ml flasks. Grow overnight in an incubator-shaker at 28 °C and 200 r.p.m. 12| Measure the OD, which needs to be 0.7–1 at λ = 600 nm. 13| Transfer bacterial suspension to a sterile 50 ml disposable tube and centrifuge at 4,000g for 10 min at room temperature. Pour off medium and using a 25-ml sterile pipette resuspend the pellet in 25 ml of GDS. Centrifuge again as above and discard supernatant. Invert the tube on tissue for ~1 min to remove excess liquid. 14| Resuspend pellet in GDS and dilute to OD600 = 0.5 using GDS. Add acetosyringone to a final concentration of 200 µM. 15| Place cultures on a horizontal shaker (~50 r.p.m.) for 45 min at room temperature. 1850 | VOL.4 NO.12 | 2009 | nature protocols
    • protocol Agrobacterium-mediated transformation of FEC ● TIMING 4 d 16| Pipette the Agrobacterium suspension onto the FEC while gently disrupting the clusters.  CRITICAL STEP The clusters need to be soaked but do not flood the plate. Leave for ~5 min in the laminar flow hood before covering and sealing with parafilm. 17| Coculture the FEC + Agrobacterium plates at 24 °C for 4 d, with 16-h light/8-h dark. 18| Gently scrape the FEC + Agrobacterium from the plate using sterile forceps and place in 25 ml GDS + C500 in a sterile 50 ml disposable tube.  CRITICAL STEP Do not put too much material in the tubes (i.e., a maximum of about six plates worth of material) otherwise washing is less effective. Vortex the suspension for 5–10 s and then allow the FEC to settle.© 2009 Nature Publishing Group http://www.nature.com/natureprotocols 19| Using a 25-ml pipette remove the supernatant and pour in ~25 ml GDS + C500 to the FEC. Mix gently by inverting tube. 20| Repeat Step 19.  CRITICAL STEP The supernatant must be clear and depending on the extent of bacterial growth during cocultivation this usually requires 3–5 washes. Gently resuspend FEC in 25 ml GDS + C500 using a 25-ml pipette. 21| Place a sterile section of plastic mesh in the base of a sterile 90-mm Petri dish and three pieces of sterile filter paper in the upturned lid. 22| Gently pipette some FEC suspension onto the mesh and spread the FEC evenly in a thin layer (Fig. 2e). Remove remaining liquid using the pipette.  CRITICAL STEP Ensure a thin layer is spread otherwise regeneration is hindered by overgrowth of material. This is especially likely if NEFC were remaining during FEC propagation/cocultivation. Using forceps transfer the mesh to the filter papers and leave for ~10 s to remove any excess liquid. Recovery and maturation of transformed FEC ● TIMING 3–4 weeks 23| Transfer the mesh/FEC to GD + C250 medium. Seal with parafilm and incubate at 28 °C, 16-h light/8-h dark for 4 d.  CRITICAL STEP This stage is a recovery phase for the FEC (i.e., no antibiotic selection) and suppresses Agrobacterium growth. 24| Transfer the mesh/FEC to GD + C250 + H5. Incubate at 28 °C, 16-h light/8-h dark for 1 week. GUS assay (Box 1) should be performed at this stage to check for transformation success. ? TROUBLESHOOTING 25| Transfer the mesh/FEC to GD + C250 + H8. Incubate at 28 °C, 16-h light/8-h dark for 1 week. ? TROUBLESHOOTING 26| Transfer the mesh/FEC to GD + C250 + H15. Incubate at 28 °C, 16-h light/8-h dark for 1 week.  CRITICAL STEP The stepwise increase of hygromycin selection on GD plates allows transformed FEC to mature and thus improve their regeneration efficiency. ? TROUBLESHOOTING Regeneration of transgenic plants ● TIMING 7–11 weeks 27| Transfer the mesh/FEC to MSN + C250 + H15. Seal with parafilm and incubate at 28 °C, 16-h light/8-h dark for 1 week. 28| Repeat Step 27 for several weeks.  CRITICAL STEP After ~3 weeks, small, green/white tube-like structures will appear, which should be retained on the mesh. Continue transferring mesh/FEC to MSN + C250 + H15 for as long as embryos/cotyledons are appearing. Box 1 | EXPRESSION OF -GLUCURONIDASE TO DETERMINE TRANSFORMATION   SUCCESS A GUS-based assay can be used as a visual representation of transformation success, using FEC following cocultivation (Step 24), embryos/cotyledons (Step 29) or developed plantlets (Step 32). 1. Immerse the selected material in GUS assay solution (890 µl Tris/NaCl buffer, 100 µl X-Gluc (10 mg ml − 1), 10 µl 10% Triton X-100, vol/vol). 2. Incubate at 37 °C for 12 h. 3. Remove the GUS buffer and destain tissue in 70% ethanol (vol/vol). nature protocols | VOL.4 NO.12 | 2009 | 1851
    • protocol 29| When green cotyledons have developed (Fig. 2f) use sterile syringe needles with binocular microscope to transfer to CEM + C100 (Fig. 2g). Gently remove any FEC sticking to the structures. Place ~8 embryos/cotyledons per plate.  CRITICAL STEP The inclusion of C100 in this media is crucial to prevent growth of Agrobacterium around the embryo/ cotyledon, which significantly hinders regeneration. GUS assays (Box 1) can be performed on isolated tissue to ensure developing embryos/cotyledons are transgenic (Fig. 2h). ? TROUBLESHOOTING 30| Transfer developing tissue to CEM + C100 every 10–14 d, gently removing any callus tissue with a sterile scalpel. As the tissue develops it will be necessary to reduce the number of embryos per plate (i.e., to four or six depending on size). 31| Repeat Step 30 until juvenile leaves and shoots appear (Fig. 2i). This can be as early as 2 weeks but normally requires© 2009 Nature Publishing Group http://www.nature.com/natureprotocols ~5 weeks. ? TROUBLESHOOTING 32| Remove the shoot and place in CBM + C50, seal with parafilm or micropore tape and incubate at 28 °C, 16-h light/8-h dark for ~3 weeks.  CRITICAL STEP Ensure that there are no leaves larger than ~1 cm in diameter on the transferred apical shoot; too large a leaf will die before the plantlet is established leading to accumulation of dead material in the jar. After 1–2 weeks roots will be visible (Fig. 2j). A GUS assay (Box 1) can be performed on the plantlet leaf material (Fig. 2k). ? TROUBLESHOOTING Preliminary screen using the rooting experiment ● TIMING 2 weeks 33| Perform a rooting experiment to screen for hygromycin-resistant transgenic lines by isolating the apical shoot and trans- planting in CBM + C50 + H10. Puncture the lid twice using a sterile syringe and cover the holes with a piece of micropore tape. To ensure the media are effective, also plant wild-type TMS60444 as a negative control (Fig. 2l). Transfer of in vitro plantlets to soil ● TIMING 5 +  weeks 34| Prepare CBM but with only 2.5 g liter − 1 of gelrite.  CRITICAL STEP This softer medium will enable easy transfer of plantlets to soil, minimizing risk of damage and loss. 35| Remove apical shoots of established in vitro plantlets, transplant into CBM, seal pots with parafilm and incubate at 28 °C, 16-h light/8-h dark for ~2–3 weeks. ! CAUTION Ensure that the shoot is ~3 or 4 cm in length otherwise the plant may be too small to survive transfer to soil. The aim is to develop the plantlets so that new leaf material appears and the roots are a few centimeters in length. 36| Using forceps, gently pull the plantlet from the medium. Gently knocking the culture pot to loosen/break up the medium will assist removal of the plantlets. 37| Wash the roots under lukewarm tap water for ~1 min to remove all the media. ! CAUTION Ensure the water is lukewarm to prevent stressing of the plantlet, which will occur if water is too cold. Roots can be trimmed to ~3 cm in length using a pair of scissors, stimulating their growth. 38| Remove leaves so that only the uppermost two or three leaves remain. Lay the plantlets on damp paper towels in a tray and spray frequently to keep moist. Cover with a lid.  CRITICAL STEP Removal of the lower, larger leaves prevents the possibility that they wilt and die before the plant is established, leading to the accumulation of decaying material in the pot. 39| Fill a small plastic pot (with holes) ¾ with a mix of ½ compost (Optima) and ½ perlite. Using a 1-cm wide stick, make a hole in the soil and carefully deposit the plant. Compress soil slightly and fill the pot with the compost/perlite mix. Place the pots in a tray (without drainage holes) containing enough water to reach ~1/3 up the pot. Spray to keep foliage moist and cover with a transparent lid. After 1–2 h pour off the excess water from the tray and cover with a transparent lid. 40| Retain the pots in the covered tray in a climate-controlled room or glasshouse at 28 °C, high humidity (>50%) and with 16-h day length. After 1 week, open the cover, leaving a 1–2 cm gap to minimize/prevent fungal growth. After about 2 weeks the cover can be removed completely. Every 2 weeks, water with 0.2% Wuxal Bio plant fertilizer.  CRITICAL STEP Optimizing fertilizer usage, light and temperature will enable rapid growth of the plants. Systemic insecticides and biological control agents may also be used to prevent pests and diseases. 1852 | VOL.4 NO.12 | 2009 | nature protocols
    • protocol ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. Table 1 | Troubleshooting table. Step Problem Solution 24 No blue product seen following a GUS The transformation was unsuccessful. Check the stock of Agrobacterium (PCR for T-DNA assay using FEC material and bacterial genes, e.g., antibiotic resistance gene). In addition, FEC material may have been of poor quality. Perform viability assay to assess (Fig. 1) 24–26 Overgrowth of material leading to The FEC should be spread thinly and evenly over the mesh in Step 22. This is a problem accumulation of white/brown tissue that is exacerbated if the FEC used for transformation are not clean, i.e., presence of© 2009 Nature Publishing Group http://www.nature.com/natureprotocols NEFC. Therefore take care to remove NEFC during subculturing in Steps 7 and 8 Overgrowth of Agrobacterium The washes following cocultivation (Steps 18–20) are important to remove as much Agrobacterium as possible. Thus only transfer the FEC to mesh once the supernatant is clear. This problem is pronounced if material is not spread thinly and evenly on the mesh (see above) 29 No blue product seen following a GUS The transformation was unsuccessful. Check the stock of Agrobacterium (PCR for T-DNA assay using developing embryos and bacterial genes, e.g., antibiotic resistance gene). In addition, FEC material may have been of poor quality, therefore perform viability assay (Fig. 1). In addition, ensure that the correct concentration of antibiotic is being used for selection 31 Poor regeneration efficiency Generally 40–70% of embryos/cotyledons transferred to CEM will generate a shoot. Continued cycling of material on CEM should improve regeneration. Avoid transferring embryos/cotyledons that have an unusual phenotype or that are immature 32 Shoot growth is attenuated or unusual It may be due to poor quality FEC or somaclonal variation. Perform FEC viability assay phenotype (thick stems, yellow prior to transformation (Fig. 1) appearance, poor leaf development) CEM, cassava elongation medium; FEC, friable embryogenic callus; NEFC, non-embryogenic friable callus. ANTICIPATED RESULTS In our laboratory we customarily use 10 plates of FEC (each containing ~10 clusters of FEC) for transformation with a single expression construct. On the basis of this, one should yield ~5–15 embryos/cotyledons per GD plate that are suitable for regeneration. Thus, in total, this protocol reliably produces in excess of 50 plantlets from ~100 clusters of FEC. However, the efficiency will vary depending on the quality of the FEC and their regeneration capacity. It is important to screen the plantlets (PCR and Southern blot analyses) to show the presence of transgene and to determine individual lines and T-DNA insertion frequency. Approximately 90% of in vitro plantlets transferred to soil survived and developed healthy phenotypes. Acknowledgments This work was partially funded by the Bill & Melinda 3. Taylor, N., Chavarriaga, P., Raemakers, K., Siritunga, D. & Zhang, P. Gates Foundation (BioCassava Plus program). J.A.O. received a PhD fellowship Development and application of transgenic technologies in cassava. from the Rockefeller Foundation. We thank Kim Schlegel, Simona Pedrussio Plant Mol. Biol. 56, 671–688 (2004). and Noemi Peter (ETH Zurich) for valuable technical assistance. We also 4. Raven, P., Fauquet, C., Swaminathan, M.S., Borlaug, N. & Samper, C. thank Nigel Taylor (Donald Danforth Plant Science Center) and Peng Zhang Where next for genome sequencing? Science 311, 468 (2006). (Shanghai Institute for Plant Physiology and Ecology) for discussions on 5. Li, H.Q., Sautter, C., Potrykus, I. & Puonti-Kaerlas, J. Genetic the cassava transformation protocol. Christof Sautter, Samuel C. Zeeman transformation of cassava (Manihot esculenta Crantz). Nat. 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