NATURE PROTOCOLS | VOL.4 NO.12 | 2009 | 1845 p uo r G gn i h s i lb uP eru t a N 900 2 © natureprotocols / m o c . e r u t a n . w w w / / : pt t h PROTOCOL INTRODUCTION The starchy roots of cassava (Manihot esculenta Crantz) are a vital source of carbohydrate for more than 500 million people living in tropical and subtropical regions. Cassava is ranked among the top five staple crops in global production (FAOSTAT website) and has also recently emerged as a promising biofuel crop given its bioethanol yield per ha 1 . As a consequence of its varied and important uses, cassava production relies on the regular development of cultivars that have enhanced biotic and abiotic stress tolerance, increased nutrient content and improved processing qualities. However, due to heterozygosity, improve- ment of the crop by traditional breeding is a difficult and lengthy task 2 ; the application of transformation technologies to integrate desired traits is therefore viewed as a very useful tool 3 . The ability to readily transform cassava will also enable studies to extend our understanding of gene function and the cassava genome 4 . Protocols for cassava transformation were reported simulta- neously by two different research groups in 1996. Li et al. 5 used Agrobacterium-mediated transformation of somatic cotyledons to then regenerate transgenic shoots by organogenesis. Schöpke et al. 6 , however, performed microparticle bombardment of embryogenic suspension-derived tissues and then regenerated transgenic plant- lets by embryo maturation. In the latter system, transformation relied on the implementation of a protocol to generate totipo- tent cell clusters, known as friable embryogenic callus (FEC) 7 . Agrobacterium-mediated transformation of FEC, a combination of the two original systems, has subsequently emerged as the most efficient and widely used strategy to produce transgenic cassava 8–10 . This combined protocol is superior as, first, by using FEC there is a reduced risk of generating chimeric plants compared to procedures using organized tissues, such as cotyledons 8 . Second, selection (usu- ally antibiotic resistance) of FEC results in fewer nontransformed plantlets being regenerated (i.e., escapes) compared to shoot orga- nogenesis 5,11 . Agrobacterium-mediated transformation of FEC has been used routinely in our laboratory to produce transgenic cassava with enhanced virus resistance 12–14 . It usually takes between 20 and 30 weeks to produce transgenic shoots 11 and despite advancements in the protocol, it remains a tedious and labor-intensive proce- dure. Setbacks and difficulties are largely due to the low regenera- tion frequency of plantlets from somatic embryos 15 , as well as the intrinsic variation (including tissue quality) observed within and between different transformation experiments 9 . To overcome such limitations in transformation systems, it is important to optimize each step of the procedure 16–18 . Our regular use of the protocol has allowed us to assess critically the various stages and introduce several improvements to create a more reliable and robust system. It is our expectation that adoption of this protocol will reduce the workload associated with the production of transgenic cassava, allow implementation of cassava transformation in laboratories where it is needed, as well as provide an opportunity to extend and improve current transformation programs that use a range of farmer-preferred and elite cassava cultivars. Experimental design and overview of the procedure An overview of the protocol is represented in Figure 1 and is divided into phases. Phase I: Production of somatic embryos; phase II: Production of FEC; phase III: Agrobacterium-mediated trans- formation; phase IV: Maturation and development of transformed FEC; phase V: Selection and regeneration of transgenic plantlets; phase VI: Screening and analysis of transgenic plantlets. These phases are discussed below. Phases I and II: Production of somatic embryos and FEC FEC can be generated from leaf explants, shoot apical meri- stems or shoot axillary meristems of cassava cultivar Tropical Manihot Series (TMS) 60444 by primary somatic embryogenesis 11 . The use of axillary meristems (i.e., buds) cultivated on cassava Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava S E Bull 1, 2 , J A Owiti 1 , M Niklaus 1 , J R Beeching 2 , W Gruissem 1 & H Vanderschuren 1 1 Department of Biology, Plant Biotechnology, ETH Zurich, Zurich, Switzerland. 2 Department of Biology & Biochemistry, University of Bath, Claverton Down, Bath, UK. 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 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. 1846 | VOL.4 NO.12 | 2009 | NATURE PROTOCOLS p uo r G gn i h s i lb uP eru t a N 900 2 © natureprotocols / m o c . e r u t a n . w w w / / : pt t h PROTOCOL axillary medium (CAM) in vitro plantlets is preferred as it allows the production of organized embryogenic clusters with reduced accumulation of non-embryogenic friable calli (NEFC; Fig. 1). The production of somatic embryos and FEC is as previously described 11 but with modifications regarding the solidifying agent used and preparation of FEC for transformation. The primary embryogenic clusters are multiplied and purified on Murashige and Skoog medium 19 supplemented with the synthetic auxin, piclo- ram (cassava induction medium, CIM). FEC is then initiated from high-quality embryogenic tissue cultured on a Gresshof and Doy (GD) medium 20 (containing picloram) and is subcultured every 2–3 weeks, resulting in FEC with minimal NEFC. Importantly, because of the possibility of somaclonal variation (chromo- somal rearrangements) an FEC line should not be micropropa- gated for more than ~6 months 21 . The quality of the FEC used for Agrobacterium-mediated transformation is reflected in its ability to regenerate into healthy plantlets. Therefore frequent transfer of nontransformed FEC clusters to Murashige and Skoog medium supplemented with the synthetic auxin, 1-naphthaleneacetic acid (NAA) and carbenicillin (MSN + C250) provides an indication of their transformation viability and ultimately can be regenerated for use as nontransgenic control plantlets. Phase III: Agrobacterium-mediated transformation After ~14 weeks, the FEC are inoculated with Agrobacterium con- taining the pCambia 1305.1 binary vector (AF354045.1). The vector contains the GUSPlus reporter gene to allow easy visuali- zation of transformation success. Previously, FEC were cultured in liquid Schenk and Hildebrandt medium before and following cocultivation with Agrobacterium (as a suspension) with regular sieving to remove NEFC 10,21 . However, we frequently observed morphological changes to the FEC, probably in part due to the sieving process. In addition, FEC produced by this method had poor regeneration efficiency. These observations prompted us to revise the inoculation procedure and to develop a method in which Agrobacterium liquid suspension is delivered directly to FEC clusters on the propagation (GD) solid medium (Fig. 1). A similar method has also been reported recently for the transformation of Brachypodium distachyon, a temperate grass 22 . Phase IV: Maturation and development of transformed FEC The potential stress caused to FEC by Agrobacterium during cocul- tivation necessitates the thorough removal of bacteria followed by a recovery stage for later phases to be successful. For this the FEC are washed in GDS + C500 until the supernatant is clear and trans- ferred to a plastic mesh for culturing/recovery on GD + C250 medium (Fig. 1). The use of mesh allows easy, weekly transfer of tissue and per- mits close regulation of media composition (e.g., pH, salt and vitamin concentrations, antibiotic concentrations) for the developing FEC. After 4 d, the FEC/mesh are transferred to GD media containing a low concentration of hygromycin antibiotic (GD + C250 + H5), which is increased weekly thereafter (GD + C250 + H8 then GD + C250 + H15). This process creates an increasingly stringent environment while ena- bling transformed FEC to express effectively the antibiotic resistance gene and initiate cell division, thus potentially improving successful plant regeneration 6 . pCambia 1305.1 vector contains hptII, which confers hygromycin resistance—a more efficient selection than kan- amycin (nptII) and related aminoglycosides. This is because complete growth inhibition can be achieved at low concentrations of hygromy- cin, resulting in selection of a greater proportion of transformed callus lines 6 . Even though we found that both hygromycin and kanamycin can be used in the described protocol, we prefer the use of hygromycin selection given the advantages mentioned above. Phase V: Selection and regeneration of transgenic plantlets At 3–4 weeks after cocultivation the FEC are cultured on Murashige and Skoog medium supplemented with NAA, carbenicillin and hygromycin (MSN + C250 + H15) to stimulate the maturation and regeneration of hygromycin-resistant embryos (Fig. 1). The maturation and development phase (phase IV), as discussed above, significantly improves regeneration capacity while maintaining a rigorous selection pressure so that the vast majority (>90%) of Grow wild-type, in vitro TMS60444 plantlets ~ 6–8 weeks, 16-h light, 28 °C Transfer stem cuttings to CAM 2–4 days, dark, 28 °C Transfer buds to CIM 2 weeks, dark, 28 °C Transfer embryos to GD 2–3 weeks, dark, 28 °C Grow Agrobacterium LBA4404 (+1305.1 vector) on YEBA+K50/R50/S100, 2 d, dark, 28 °C Grow Agrobacterium LBA4404 (+1305.1 vector) in YEB+K50/R50/S100, 2 d, dark, 28 °C, shake 200 r.p.m. Cocultivate FEC and Agrobacterium 4 d, 16 h light, 24 °C Culture cocultured FEC GD+C250, 4 d, 16-h light, 28 °C Culture cocultured FEC GD+C250+H5, 1 week, 16-h light, 28 °C Culture cocultured FEC GD+C250+H8, 1 week, 16-h light, 28 °C Culture cocultured FEC GD+C250+H15, 1 week, 16-h light, 28 °C Culture cocultured FEC MSN+C250+H15, 1 week, 16-h light, 28 °C Repeat for ~ 6 weeks Isolate developing embryos/cotyledons CEM+C100, 2 weeks, 16-h light, 28 °C Repeat until shoots appear (2–5 weeks) Regenerate transgenic plants CBM+C50, 2 weeks, 16-h light, 28 °C Screen plantlets using rooting test CBM+C50+H10, 2 weeks, 16-h light, 28 °C Advised to also perform Southern blot and PCR tests Wash FEC and Agrobacterium GDS+C500. Repeat until supernatant is clear Isolate FEC and culture on GD 2–3 weeks, 16-h light, 28 °C Repeat purification/subculturing for up to 6 months Propagate and multiply developing embryos on CIM 2 weeks, dark, 28 °C Repeat for total of 6–8 weeks Phase l Production of somatic embryos Phase ll Production of friable embryogenic callus (FEC) FEC viability assay MSN+C250, 10 d, 16-h light replenish every 10 d Phase lll Agrobacterium- mediated transformation Phase lV Maturation and development of transformed FEC GUS assay 1 d, dark GUS assay 1 d, dark Phase V Selection and regeneration of transgenic plantlets Phase Vl Screening and analysis of transgenic plantlets Figure 1 | Schematic diagram for Agrobacterium-mediated transformation of cassava. NATURE PROTOCOLS | VOL.4 NO.12 | 2009 | 1847 p uo r G gn i h s i lb uP eru t a N 900 2 © natureprotocols / m o c . e r u t a n . w w w / / : pt t h PROTOCOL embryos developing in phase V are transgenic. This in turn allows hygromycin to be excluded from further growth medium, reduc- ing the potential negative impact of the antibiotic on plant regen- eration 10 . Developing embryos and cotyledons are transferred to media containing 6-benzylaminopurine (a synthetic cytokinin) and carbenicillin (cassava elongation medium; CEM + C100) to induce shoot growth. It is crucial that carbenicillin is present, otherwise, there is a tendency for Agrobacterium to grow around the embryo/ cotyledon and suppress its development. Fortnightly transfer of the developing green tissue to CEM + C100 will result in a shoot that can be isolated and transferred to a Murashige and Skoog-based medium (cassava basic medium; CBM + C50), lacking hygromycin selection, for establishment of plantlets. Phase VI: Screening and analysis of transgenic plantlets Fully rooted plantlets are screened by transfer of a stem cutting to CBM + C50 + H10 for the rooting test 23 (Fig. 1). After 2 weeks only transgenic plants will have developed new roots (and consequently more leaf tissue) on this hygromycin-containing medium, whereas wild-type control cuttings will remain rootless and eventually become chlorotic. The rooting test is a rapid and reliable screen for transgenic plants clearly identifying the few nontransgenic plant- lets that escaped the initial hygromycin selection phase. Transgenic plantlets should be analyzed by PCR to confirm integration of the gene of interest and also by Southern blot to provide conclusive data pertaining to the copy number and integration pattern of the T-DNA. MATERIALS REAGENTS Acetosyringone (3′,5′-dimethoxy-4′-hydroxyacetophenone; Sigma-Aldrich, cat. no. D134406) ! CAUTION Irritant to eyes, respiratory system and skin. R36/37/38. S26, 36 CRITICAL Risk (R) and Safety (S) codes throughout the materials are based on European Union Commission Directives. A. tumefaciens strain LBA4404 harboring pCambia 1305.1 plasmid, containing GUSPlus reporter gene and hygromycin (hptII) resistance gene. Bacto agar (Difco, cat. no. 0140-07-4) Bacto beef extract (Difco, cat. no. 0115-17-3) Bacto peptone (Difco, cat. no. 0118-17-0) Bacto yeast extract (Difco, cat. no. 0127-07-1) 6-Benzylaminopurine (BAP; Duchefa, cat. no. B0904.0025) ! CAUTION Harmful if swallowed; irritant to eyes, respiratory system and skin. R22, 36/37/38. S24/26, 36. 5-Bromo-4-chloro-3-indoxyl-β-D-glucuronic acid, cyclohexylammonium salt (X-Gluc; Biosynth, cat. no. B-7300) ! CAUTION Harmful if swallowed; irritant to eyes and skin. R 22, 36/38. S26, 36/37, 60. Carbenicillin disodium (Duchefa, cat. no. C0109.0025) ! CAUTION May cause sensitization by inhalation and skin contact. R42/43. S36/37/39. Cassava (M. esculenta Crantz) cultivar TMS60444. Copper(II) sulfate pentahydrate (CuSO 4 ·5H 2 O; Sigma-Aldrich, cat. no. C3036) ! CAUTION Harmful if swallowed; irritant to eyes and skin. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment. R22, 36/38, 50/53. S22, 60, 61. Dimethyl sulfoxide (DMSO; Sigma-Aldrich, cat. no. 41641) N,N -Dimethylformamide (DMF; Sigma-Aldrich, cat. no. 40240) ! CAUTION May cause harm to the unborn child. Harmful by inhalation and in contact with skin; irritant to eyes. R61, 20/21, 36. S45, 53. Gelrite (Duchefa, cat. no. G1101.5000) Gresshof and Doy medium including vitamins (Duchefa, cat. no. G0212.0050) Hydrochloric acid 37% (wt/wt) (HCl; Sigma-Aldrich, cat. no. 84422) ! CAUTION Corrosive, causes burns; irritant to the respiratory system. R34, 37. S26, 45. Hygromycin B (Roth, cat. no. CP12.1) ! CAUTION Very toxic when inhaled, in contact with skin and if swallowed. Risk of serious damage to eyes. May cause sensitization when inhaled and in contact with skin. R23/24/25, 42/43. S26, 28-36/37/39, 45. CRITICAL High-quality product required. Kanamycin monosulphate (Duchefa, cat. no. K0126.0025) ! CAUTION Toxic; may cause harm to the unborn child. R61. S45, 53. Magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O; Sigma-Aldrich, cat. no. 63140) Murashige and Skoog (MS) medium including vitamins (Duchefa, cat. no. M0222.0050) Noble agar (Difco, cat. no. 214230) 1-Naphthaleneacetic acid (NAA; Sigma-Aldrich, cat. no. N0640) ! CAUTION Harmful if swallowed; irritant to the respiratory system and skin; risk of serious damage to eyes. R22, 37/38, 41. S22, 26, 36. Picloram (Duchefa, cat. no. P0914.0010) ! CAUTION Toxic substance; harmful by inhalation, in contact with skin and if swallowed. Irritant to eyes and may cause cancer. R20/21/22, 36, 45. S26, 36/37/39, 45. • • • • • • • • • • • • • • • • • • • • • • • Rifampicin (Duchefa, cat. no. R0146.0005) ! CAUTION Harmful if swallowed. R22. S36. Sterilized deionized water (SDW) Sodium hydroxide (NaOH; Sigma-Aldrich, cat. no. 71690) ! CAUTION Corrosive; causes severe burns. R35. S26, 37/39, 45. Streptomycin sulfate salt (Sigma-Aldrich, cat. no. S6501) ! CAUTION Harmful if swallowed. R22. Sucrose (Roth, cat. no. 4661.3) Tris(hydroxymethyl)aminomethane (TRIS; Chemie Brunschwig, cat. no. 20092391) ! CAUTION Irritant to eyes, respiratory system and skin. R36/37/38. S26, 37/39. Triton X-100 (Sigma-Aldrich, cat. no. 93426) ! CAUTION Harmful if swallowed; risk of serious damage to eyes. Toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment. R22, 41, 51/53. S26, 36/39, 61. Optima compost (G. Optima-Werke) Perlite Wuxal Bio plant fertilizer (Maagoplan, cat. no. 7.610176.068.860) Plastic plant pots EQUIPMENT Autoclave Sterile plastic Petri dishes, 90 mm (Sarstedt, cat. no. 82.1473) Plastic mesh 100 µm, sterile (Lanz-Anliker, cat. no. AH03558) Pipettes 25 ml, sterile (Sarstedt, cat. no. 86.1685.001) Sterile jars (53 mm × 100 mm; Greiner, cat. no. 7.968161) pH meter Balance and precision balance Centrifuge for 50-ml tubes 15-ml sterile, disposable tubes (Huber, cat. no. 7.187262) Microfuge Tabletop shaker Controlled environment chamber (Sanyo MLR, 28 °C, 16-h light/ 8-h dark) Controlled environment room (24 °C, 16-h light/8-h dark) Pipette aid Parafilm (Huber, cat. no. 15.1550.02) Laminar flow hood with Bunsen burner Fridge (4 °C) and freezer ( − 20 °C) Glassware (beakers, Duran bottles and Erlenmeyer flasks) Sterile, disposable syringe filters (0.22 µm; Millipore, cat. no. SLGP033RB) Micropore tape Aluminum foil Scalpel and forceps Binocular microscope Sterile, disposable inoculation loops (Sarstedt, cat. no. 86-1567-010) Static incubator for bacterial cultures (28 °C) Incubator-shaker (28 °C) Spectrophotometer 1 ml disposable cuvettes Microfuge tubes (1.5 ml) Magnetic stirring bars Scissors • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1848 | VOL.4 NO.12 | 2009 | NATURE PROTOCOLS p uo r G gn i h s i lb uP eru t a N 900 2 © natureprotocols / m o c . e r u t a n . w w w / / : pt t h PROTOCOL Disposable Pasteur pipettes Disposable syringes (1 and 5 ml) Glass spreaders for bacteria cultures Vortex Spatula Disposable weighing boats Sterile, disposable 50 ml tube (screw top) (Sarstedt, cat. no. 62.547.254) Sterile, disposable 50 ml tube (flip top) (Nunc, cat. no. NC-362696) REAGENT SETUP Media and stocks Autoclave media and stocks (15 min at 121 °C). All media should be used at room temperature (~22 °C) unless otherwise stated. CRITICAL Solutions stored at 4 °C should be replaced after 3 months; solutions at − 20 °C after 6 months. Media should be prepared weekly. NAA (1 mg ml − 1 stock solution) Dissolve 50 mg in 1 ml 1 M NaOH and adjust volume to 50 ml with SDW. Store at 4 °C. Picloram (12 mg ml − 1 stock solution) Dissolve 0.6 g in ~5 ml of 1 M NaOH and adjust volume to 50 ml with SDW. Filter (0.22 µm)-sterilize, aliquot into 1.5 ml microfuge tubes and store at − 20 °C. BAP (1 mg ml − 1 stock solution) Dissolve 20 mg in ~3 ml of 1 M NaOH and adjust volume to 20 ml with SDW. Filter (0.22 µm)-sterilize, aliquot into 1.5 ml microfuge tubes and store at − 20 °C. CuSO 4 ·5H 2 O (2 mM stock solution) Dissolve 2.49 g in 50 ml SDW (0.2 M stock solution), then dilute 1 ml in 100 ml SDW (final volume) and store at 4 °C. MgSO 4 ·7H 2 O (1 M stock solution) Dissolve 24.6 g in 100 ml SDW. Filter (0.22 µm) sterilize. Store at 4 °C. Rifampicin (25 mg ml − 1 stock solution) Dissolve 625 mg in 25 ml 0.1 N HCl. Filter (0.22 µm)-sterilize and aliquot. Store at − 20 °C. Hydrochloric acid (HCl 0.1 N stock solution) Add 820 µl 37% (wt/wt) HCl to 99.18 ml SDW. Kanamycin monosulphate (50 mg ml − 1 stock solution) Dissolve 2.5 g in 50 ml SDW. Filter (0.22 µm)-sterilize, aliquot into 1.5 ml microfuge tubes and store at − 20 °C. Streptomycin sulfate (100 mg ml − 1 stock solution) Dissolve 5 g in 50 ml SDW. Filter (0.22 µm)-sterilize, aliquot into 1.5 ml microfuge tubes and store at − 20 °C. Carbenicillin disodium (500 mg ml − 1 stock solution) Gradually dissolve 25 g in 50 ml SDW. Filter (0.22 µm)-sterilize, aliquot into 1.5 ml microfuge tubes and store at − 20 °C. Tris/NaCl buffer for GUS assay Add 1.21 g Tris and 2.92 g NaCl to 1 liter SDW (final volume) and adjust pH to 7.2 by adding concentrated HCl. Autoclave and store at room temperature. X-Gluc (10 mg ml − 1 stock solution) for GUS assay Dissolve 100 mg X-Gluc in 10 ml DMF. Store in 1 ml aliquots at − 20 °C. Wrap container in aluminum foil (X-Gluc is light sensitive). Triton X-100 (10% (vol/vol) stock solution) for GUS assay Add 5 ml Triton X-100 to 45 ml SDW and mix gently but continuously until dissolved. Store at room temperature. Acetosyringone (200 mM stock solution) Dissolve 1.962 g in 50 ml DMSO. Aliquot into 1.5 ml microfuge tubes and store at − 20 °C. CAM solid medium for induction of axillary buds Dissolve 4.4 g MS medium including vitamins, 20 g sucrose, 1 ml CuSO 4 (2 mM) and 10 ml BAP (1 mg ml − 1 ) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and autoclave. Allow media to cool before pouring ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. CIM solid medium to induce somatic embryos Dissolve 4.4 g MS medium including vitamins, 20 g sucrose, 1 ml CuSO 4 (2 mM) and 1 ml picloram (12 mg ml − 1 ) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and autoclave. Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. GD solid medium for induction and propagation of FEC Dissolve 2.7 g GD medium including vitamins, 20 g sucrose and 1 ml picloram (12 mg ml − 1 ) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and auto- clave. Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. YEBA + K50/R50/S100 solid medium for Agrobacterium culture Dissolve 1 g Bacto yeast extract, 5 g Bacto beef extract, 5 g Bacto peptone and 5 g sucrose in 1 liter SDW (final volume). Adjust pH to 7.2, add 15 g Bacto agar • • • • • • • • and autoclave. Allow the media to cool and add 1 ml kanamycin (50 mg ml − 1 ), 2 ml rifampicin (25 mg ml − 1 ) and 1 ml streptomycin (100 mg ml − 1 ). Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. YEB + K50/R50/S100 liquid medium for Agrobacterium culture Dissolve 1 g Bacto yeast extract, 5 g Bacto beef extract, 5 g Bacto peptone, 5 g sucrose in 1 liter SDW (final volume). Adjust pH to 7.2 and autoclave. Once the media are at room temperature add 1 ml kanamycin (50 mg ml − 1 ), 2 ml rifampicin (25 mg ml − 1 ), 1 ml streptomycin (100 mg ml − 1 ) and 2 ml (1 M) MgSO 4 . Store at 4 °C. GDS solution for Agrobacterium preparation Dissolve 2.7 g GD medium including vitamins, 20 g sucrose and 1 ml picloram (12 mg ml − 1 ) in 1 liter SDW (final volume). Adjust pH to 5.8 and autoclave. Store at 4 °C. GDS + C500 solution for washing FEC Dissolve 2.7 g GD medium including vitamins, 20 g sucrose and 1 ml picloram (12 mg ml − 1 ) in 1 liter SDW (final volume). Adjust pH to 5.8 and autoclave. Once media are at room tempera- ture add 1 ml carbenicillin (500 mg ml − 1 ) and mix. Store at 4 °C. GD + C250 solid medium for recovery of transgenic FEC Dissolve 2.7 g GD medium including vitamins, 20 g sucrose and 1 ml picloram (12 mg ml − 1 ) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and auto- clave. Once media are cooled add 500 µl carbenicillin (500 mg ml − 1 ). Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. GD + C250 + (H5, H8 or H15) solid medium for maturation of transgenic FEC Dissolve 2.7 g GD medium including vitamins, 20 g sucrose and 1 ml picloram (12 mg ml − 1 ) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and autoclave. Once media are cooled add 500 µl carbenicillin (500 mg ml − 1 ) and the appropriate amount of hygromycin (50 mg ml − 1 ); 100 µl for GD + C250 + H5; 160 µl for GD + C250 + H8 and 300 µl for GD + C250 + H15. Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. MSN + C250 medium for regeneration of wild-type FEC Dissolve 4.4 g MS medium including vitamins, 20 g sucrose and 1 ml NAA (1 mg ml − 1 ) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and auto- clave. Once media are cooled add 500 µl carbenicillin (500 mg ml − 1 ). Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. MSN + C250 + H15 medium for regeneration of transgenic embryos Dissolve 4.4 g MS medium including vitamins, 20 g sucrose and 1 ml NAA (1 mg ml − 1 ) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and autoclave. Once media are cooled add 500 µl carbenicillin (500 mg ml − 1 ) and 300 µl hygromycin (50 mg ml − 1 ). Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. CEM + C100 solid medium for generation of shoots Dissolve 4.4 g MS medium including vitamins, 20 g sucrose, 400 µl BAP (1 mg ml − 1 ) and 1 ml CuSO 4 (2 mM) in 1 liter SDW (final volume). Adjust pH to 5.8, add 8 g Noble agar and autoclave. Once the media are cooled add 200 µl carbenicillin (500 mg ml − 1 ). Pour ~25 ml into sterile 90-mm Petri dishes in a laminar flow hood and allow to solidify (~1 h). Store at 4 °C. CBM for propagation of in vitro plantlets Dissolve 4.4 g MS medium including vitamins, 20 g sucrose and 1 ml CuSO 4 (2 mM) in 1 liter SDW (final volume). Adjust pH to 5.8, add 3 g gelrite and autoclave. Allow media to cool and pour ~35 ml into sterile 53 mm × 100 mm plastic jars. Leave in laminar flow hood for ~1 h for media to solidify. CBM + C50 for propagation of in vitro plantlets Dissolve 4.4 g MS medium including vitamins, 20 g sucrose and 1 ml CuSO 4 (2 mM) in 1 liter SDW (final volume). Adjust pH to 5.8, add 3 g gelrite and autoclave. Allow media to cool before adding 100 µl carbenicillin (500 mg ml − 1 ) and pour ~35 ml into sterile 53 mm × 100 mm plastic jars. Leave in laminar flow hood for ~1 h for media to solidify. Store at 4 °C. CBM + C50 + H10 for screening of transgenic in vitro plantlets Dissolve 4.4 g MS medium including vitamins, 20 g sucrose and 1 ml CuSO 4 (2 mM) in 1 liter SDW (final volume). Adjust pH to 5.8, add 3 g gelrite and autoclave. Allow media to cool before adding 100 µl carbenicillin (500 mg ml − 1 ) and 200 µl hygromycin (50 mg ml − 1 ). Pour ~20 ml into sterile, disposable 50 ml tubes (Nunc; cat. no. NC-362696). Leave in laminar flow hood for ~1 h for media to solidify. Store at 4 °C. NATURE PROTOCOLS | VOL.4 NO.12 | 2009 | 1849 p uo r G gn i h s i lb uP eru t a N 900 2 © natureprotocols / m o c . e r u t a n . w w w / / : pt t h 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. 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 b c d e f g h i j k 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. 1850 | VOL.4 NO.12 | 2009 | NATURE PROTOCOLS p uo r G gn i h s i lb uP eru t a N 900 2 © natureprotocols / m o c . e r u t a n . w w w / / : pt t h 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 ~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 months 21 . 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 OD 600 = 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. NATURE PROTOCOLS | VOL.4 NO.12 | 2009 | 1851 p uo r G gn i h s i lb uP eru t a N 900 2 © natureprotocols / m o c . e r u t a n . w w w / / : pt t h 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. 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). 1852 | VOL.4 NO.12 | 2009 | NATURE PROTOCOLS p uo r G gn i h s i lb uP eru t a N 900 2 © natureprotocols / m o c . e r u t a n . w w w / / : pt t h 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 ~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. NATURE PROTOCOLS | VOL.4 NO.12 | 2009 | 1853 p uo r G gn i h s i lb uP eru t a N 900 2 © natureprotocols / m o c . e r u t a n . w w w / / : pt t h PROTOCOL ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. 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 Gates Foundation (BioCassava Plus program). J.A.O. received a PhD fellowship from the Rockefeller Foundation. We thank Kim Schlegel, Simona Pedrussio and Noemi Peter (ETH Zurich) for valuable technical assistance. We also thank Nigel Taylor (Donald Danforth Plant Science Center) and Peng Zhang (Shanghai Institute for Plant Physiology and Ecology) for discussions on the cassava transformation protocol. Christof Sautter, Samuel C. Zeeman (ETH Zurich) and Ingo Potrykus are acknowledged for their support. AUTHOR CONTRIBUTIONS S.E.B. and H.V. designed the experiments and prepared the paper; S.E.B. and J.A.O. undertook experimental work with technical support from M.N.; and H.V., J.R.B and W.G. supervised the project. Published online at http://www.natureprotocols.com/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/. 1. Balat, M. & Balat, H. Recent trends in global production and utilization of bio-ethanol fuel. Appl. Energ. 86, 2273–2282 (2009). 2. Ceballos, H., Iglesias, C.A., Pérez, J.C. & Dixon, A.G.O. Cassava breeding: opportunities and challenges. Plant Mol. Biol. 56, 503–516 (2004). 3. Taylor, N., Chavarriaga, P., Raemakers, K., Siritunga, D. & Zhang, P. Development and application of transgenic technologies in cassava. Plant Mol. Biol. 56, 671–688 (2004). 4. Raven, P., Fauquet, C., Swaminathan, M.S., Borlaug, N. & Samper, C. Where next for genome sequencing? Science 311, 468 (2006). 5. 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Euphytica 120, 35–42 (2001). TABLE 1 | Troubleshooting table. Step Problem Solution 24 No blue product seen following a GUS assay using FEC material The transformation was unsuccessful. Check the stock of Agrobacterium (PCR for T-DNA 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 accumulation of white/brown tissue The FEC should be spread thinly and evenly over the mesh in Step 22. This is a problem that is exacerbated if the FEC used for transformation are not clean, i.e., presence of 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 assay using developing embryos The transformation was unsuccessful. Check the stock of Agrobacterium (PCR for T-DNA 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 phenotype (thick stems, yellow appearance, poor leaf development) It may be due to poor quality FEC or somaclonal variation. 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A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473 (1962). 20. Gresshof, P. & Doy, C.H. Derivation of a haploid cell line from Vitis vinifera and importance of stage of meiotic development of anthers for haploid culture of this and other genera. Z. Pflanzenphysio. 73, 132–141 (1974). 21. Raemakers, K. et al. Progress made in FEC transformation of cassava. Euphytica 120, 15–24 (2001). 22. Alves, S.C. et al. A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21. Nat. Protoc. 4, 638–649 (2009). 23. Zhang, P., Legris, G., Coulin, P. & Puonti-Kaerlas, J. Production of stably transformed cassava plants via particle bombardment. Plant Cell Rep. 19, 939–945 (2000). . transformation of friable embryogenic calli and regeneration of transgenic cassava S E Bull 1, 2 , J A Owiti 1 , M Niklaus 1 , J R Beeching 2 , W Gruissem 1 & H Vanderschuren 1 1 Department of Biology,. known as friable embryogenic callus (FEC) 7 . Agrobacterium- mediated transformation of FEC, a combination of the two original systems, has subsequently emerged as the most efficient and widely. 2009; doi:10.1038/nprot.2009.208 Agrobacterium- mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach