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Nov 2019

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Efficient Agrobacterium-mediated Transformation of the Elite–Indica Rice Variety Komboka
农杆菌介导的优良籼稻品种Komboka的高效转化   

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Abstract

Genetic transformation is crucial for both investigating gene functions and for engineering of crops to introduce new traits. Rice (Oryza sativa L.) is an important model in plant research, since it is the staple food for more than half of the world’s population. As a result, numerous transformation methods have been developed for both indica and japonica rice. Since breeders continuously develop new rice varieties, transformation protocols have to be adapted for each new variety. Here we provide an optimized transformation protocol with detailed tips and tricks for a new African variety Komboka using immature embryos. In Komboka, we obtained an apparent transformation rate of up to 48% for GUS/GFP reporter gene constructs using this optimized protocol. This protocol is also applicable for use with other elite indica rice varieties.

Keywords: Agrobacterium-mediated transformation (农杆菌介导转化), Indica rice (籼稻), Oryza sativa (水稻), Elite variety (优良品种), Rice transformation (水稻转化), Plant regeneration (植株再生)

Background

Various methods for genetic transformation of plants have been developed, e.g. PEG-mediated transfection of protoplasts (Shimamoto et al., 1989; Datta et al., 1992), biolistic transformation (Christou et al., 1991) and Agrobacterium-mediated transformation (Slamet-Loedin et al., 2014). Agrobacterium-mediated transformation is one of the most widely used methods to introduce DNA into plants (van Wordragen and Dons, 1992). This method has been used intensively for research and has become a key prerequisite for biotechnology. It has gained in importance since the development of new breeding technologies such as genome editing (Char et al., 2019). For crops, such as rice, genetic transformation could also be used to develop new genetic variations for plant breeding, for example, creating new disease- or insect-resistant lines (Cheng et al., 1998; Helliwell and Yang, 2013; Oliva et al., 2019). Also for rice, Agrobacterium-mediated transformation is the most popular method to transfer T-DNA into plant genomes (Hiei and Komari, 2008). Currently, there are multiple protocols for japonica and indica rice transformation using calli induced from mature seeds or immature embryos (Hiei and Komari, 2006 and 2008; Toki et al., 2006; Nishimura et al., 2006; Sahoo et al., 2011; Sahoo and Tuteja, 2012; Slamet-Loedin et al., 2014; Sundararajan et al., 2017). It is convenient to use mature seeds for transformation because they are available throughout the year and can be stored, although this method is predominantly used for japonica varieties. Methods that involve transformation of rice immature embryos generally yield higher transformation rates compared to mature seeds (Hiei and Komari, 2008; Slamet-Loedin et al., 2014). Overall, japonica varieties such as Kitaake and Nipponbare are apparently easier to transform, compared to indica rice such as IR64 or Ciherang-Sub1 (Oliva et al., 2019). For japonica varieties, highly efficient transformation can be obtained using calli induced from mature seeds with rates of 50-60% (Li et al., 2015). For indica varieties, despite some efforts to increase transformation rate using mature seeds, immature embryos-derived calli are still the tissue of choice for transformation. Notably, transformation efficiency is highly variety-dependent and it is necessary to optimize transformation protocols for each variety.

Komboka (IR 05N 221) is a new elite variety generated by IRRI and released in Tanzania by the National Rice Research Program-KATRIN Research Centre and IRRI-Tanzania (‘Komboka’ = ‘liberated’) (Kitilu et al., 2019). Komboka is high yielding (8.6 t ha-1), semi-aromatic with good grain quality, tolerant to blast and well adapted to upland and lowland areas. In the protocol reported here, we describe detailed steps for the stable transformation of Komboka immature embryos which produce transgenic plants within four months and with a high apparent transformation rate of up to 48%. This protocol was developed by combining and modifying published protocols from Slamet-Loedin et al., 2014 and Hiei and Komari, 2008 which were used to transform different indica rice varieties such as IR64, Ciherang-sub1 (Oliva et al., 2019), therefore, in principle this protocol can likely be adapted for transformation of other indica rice varieties. In this protocol, we highlighted all details, tips and tricks that are essential for setting up transformation protocols for other elite varieties.

Figure 1. Flow chart and timeline for Agrobacterium-mediated transformation of immature embryos of rice var. Komboka (indica) which takes about four months to generate transgenic lines. Briefly, the process starts with growing rice plants to the flowering stage under controlled greenhouse conditions at 30 °C ± 2 °C during the day and 25 °C ± 2 °C during the night, 50-70% relative humidity. Light conditions in the glass greenhouse are determined by natural daylight and additional lamplight (8/16 day/night photoperiod). Daylength varies in the course of the seasons due to the location of our greenhouse along latitude (latitudes and longitudes of Düsseldorf, Germany 51°11'31.2"N 6°47'40.1"E). Under these conditions, Komboka required 13-14 weeks till booting stage and additional 2-3 weeks to flower. 8-12 days post-pollination, rice panicles can be harvested for immature embryos isolation. Selecting the proper stage of seeds for immature embryos isolation is crucial. Immature embryos should be in the late milky stage (Video 1), with a size of 1.3-1.8 mm. After dehusking the seeds and isolating immature embryos (Videos 2 and 3; Figure 2), the isolated immature embryos were moved, as a whole, onto co-cultivation medium. Right after the isolation step immature embryos were co-cultivated with Agrobacteria harboring constructs-of-interest for seven days. Afterwards, germinated immature embryos were cleaned from Agrobacteria with sterile filter papers and shoots were excised. The immature embryos were then transferred onto resting/recovery medium without hygromycin B for five days. After resting, immature embryos were moved to two rounds of selection with 30 mg/L hygromycin B, each for 10 days. After the second selection step, microcalli appeared on brownish maternal immature embryos. Microcalli were carefully separated from the maternal tissues and transferred onto selection medium (30 mg/L hygromycin B) for the third selection round. In cases that the immature embryos did not produce microcalli after the first two rounds of selection, immature embryos were moved to an additional selection round (Supplementary Selection 2) of 10 days each. Resistant and multiplied microcalli after the third selection were transferred onto pre-regeneration medium (30 mg/L hygromycin B) for 1 or 2 rounds of growth for 10 days. Only greening calli were transferred onto regeneration medium (30 mg/L hygromycin B) for 14 days. When small rice plants (plantlets) were regenerated from the calli and reached about 5 cm in height, they were transferred onto rooting medium without hygromycin B for 14 days. Only well-developed plantlets with strong root systems were planted in pots. Pots were submerged in larger buckets (5 L).


Figure 1. Flow chart describing Agrobacterium-mediated transformation using immature embryos of rice variety Komboka


Figure 2. Steps in Agrobacterium-mediated transformation of immature embryos from rice variety Komboka. A. Immature embryo isolation. B. Agrobacterium infection. C. Co-cultivation. D. Resting phase. E. Selection phase. F. Pre-regeneration phase. G. Regeneration phase. H. Rooting phase.

Below, we provide detailed lists of chemicals, equipment, the transformation steps with important notes, as well as all templates for media preparation.

Materials and Reagents

Consumables

  1. Filtropur BT 100 bottle top filter (volume: 1000 ml; membrane: PES; diameter: 90 mm; pore size: 0.2 µm for sterile filtration) (Sarstedt, catalog number: 83.3942.101 ); use to filter stock solutions and for single use only
  2. Filtropur BT 25 bottle top filter (volume: 250 ml; membrane: PES; diameter: 90 mm; pore size: 0.2 µm for sterile filtration) (Sarstedt, catalog number: 83.3940.101 ); use to filter stock solutions and for single use only
  3. Filtropur BT 50 bottle top filter (volume: 500 ml; membrane: PES; diameter: 90 mm; pore size: 0.2 µm for sterile filtration) (Sarstedt, catalog number: 83.3941.101 ); use to filter stock solutions and for single use only
  4. Glass test tubes,14 x 16 mm; 21 ml (DURAN®, catalog number: 26 130 21 )
  5. Gene-Pulse Cuvettes, 0.2 cm gap (Bio-Rad, catalog number: 1652086 ), single use only
  6. Petri dishes, extra deep, disposable, Lab-tekTM (diameter: 100 mm; height: 26 mm; sterile) (Supplier: Thermo Scientific, VWR, catalog number: NUNC4031 ); use to pour Regeneration medium, single use only
  7. Petri dishes, deep, with 3 vents (diameter: 92.3 mm ; height: 20 mm ; sterile ) (Supplier: Greiner Bio One, VWR, catalog number: 391-0493 ); use to pour Pre-regeneration medium, single use only
  8. Petri dishes, with cams (diameter: 92 mm; height: 16 mm; sterile) (Sarstedt, catalog number: 82.1473 ); use to pour YEP, Co-cultivation, Resting and Selection media, single use only
  9. Qualitative filter papers, standard grades, grade 1 and 1 V, Whatman® (diameter: 85 mm) (VWR, catalog number: 516-0593 )
  10. Reaction tubes, 1.5 ml (Sarstedt, catalog number: 72.690.001 )
  11. SafeSeal reaction tube, 2 ml (Sarstedt, catalog number: 72.695.500 )
  12. Scalpel blades (type 11 for scalpel handles using the BAYHA interlocking system; non-sterile) (Behr Labor-Technik, catalog number: 9409911 )
  13. Screw cap tube, 50 ml (Sarstedt, catalog number: 62.547.254 )
  14. Screw cap tube, 15 ml (Sarstedt, catalog number: 62.554.502 )
  15. Serological Pipette, 10 ml (Sarstedt, catalog number: 86.1254.001 ), single use only
  16. Serological Pipette, 25 ml (Sarstedt, catalog number: 86.1685.001 ), single use only
  17. Serological Pipette, 5 ml (Sarstedt, catalog number: 86.1253.001 ), single use only
  18. Stretch foil (Stretchplus; 300.0 m x 40.0 cm; foil thickness: 7 µm)
  19. Surgical tape (3MTM MicroporeTM; Width: 1.25 cm)
  20. Syringe (Injekt®; 20 ml; Luer lock) (Braun, catalog number: 4606205V ), single use only
  21. Syringe filtration unit Filtropur S 0.2 (membrane: PES; filtration surface: 5.3 cm2; pore size: 0.2 µm for sterile filtration) (Sarstedt, catalog number: 83.1826.001 ); use to filter phytohormones and antibiotic solutions, single use only
  22. Tissue culture vessel, MagentaTMGA-7 (Supplier: Sigma-Aldrich; 77 x 77 x 97 mm) (VWR, catalog number: SAFSV8505 ); use to pour ‘Roo’ medium, reusable
  23. Tooth pick made of wood (6.8 cm) (Pap Star, catalog number: 12736 )

Biological material
  1. Komboka seeds (IR05N221, L17WS.06#24)
    Komboka seeds were kindly provided by the International Rice Research Institute (IRRI, Philippine) under a special material transfer agreement (SMTA-MLS).
    Komboka seeds were sown directly into soil in round, 2 L pots (13.2 cm height and 16.7 cm diameter), one plant per pot. A soil mixture of profile porous ceramic (PPC) greens grade and ‘Arabidopsis soil’ with a ratio of 1:1 is used as a standard soil for rice cultivation. See Table S1 for the details of soil composition. As a slowly released fertilizer, 4 g of Osmocote Exact 3-4 M (16% N, 3.9% P, 10% K, 1.2% Mg, 0.45% Fe, 0.06% Mn, 0.02% B, 0.05% Cu, 0.02% Mo, 0.015% Zn) (ICL/SF UK) was added in 1 L soil. In addition, plants were fertilized weekly from the 2nd week and biweekly from the 6th week after germination using Peters Excel (14% N, 6% P, 14% K, 6.5% Ca, 2.5% Mg, 0.12% Fe, 0.06% Mn, 0.02% B, 0.015% Cu, 0.01% Mo, 0.015% Zn) (ICL/SF UK). Fertilization was terminated when plants reached the flowering stage. Rice plants in 2 L pots filled with soil were submerged into a 5 L buckets filled with water, so that the inner 2 L pot is under the waterline. Water can remain in the bucket till rice plants get seeds, no exchange needed. Alternatively, 2 L rice pots can also be placed in 60 x 40 x 6 cm trays and filled with water to the upper edge. In this case, water needs to be exchanged biweekly.
      Rice plants were grown in the glass house with natural daylight and additional lamplight of 8/16 day/night photoperiod, however not strictly required, with day temperature of 30 °C ± 2 °C and night temperature of 25 °C ± 2 °C. The relative humidity was between 50-70% which was manually controlled by spraying water on the greenhouse floor. Plants were grown under a photosynthetic active radiation (PAR) or photosynthetic photon flux density (PPFD) of 200 µmol m-2s-1 with PPFD-blue of 40 µmol m-2s-1, PPFD-green of 70 µmol m-2s-1 and PPFD-red of 80 µmol m-2s-1.


Chemicals
Note: Chemical batches and brands are important factors that may affect transformation success. We found that the indistinctive use of alternative chemical brands often leads to failure when trying to transform Komboka for the first time. Therefore, we highly recommend to use the exact chemicals specified here (brands and catalog numbers), in particular when setting up a new transformation protocol. Make sure to store the chemicals in the required conditions and do not use products after the expiry dates. We declare no competing interest regarding chemicals or instrument choice.

  1. 1-Naphthaleneacetic acid, C12H10O2 (Sigma-Aldrich, catalog number: N0640 ; CAS number: 86-87-3), store at RT
  2. 2,4-Dichlorophenoxyacetic acid , Cl2C6H3OCH2CO2H (Sigma-Aldrich, catalog number: D7299 ; CAS number: 94-75-7), store at RT
  3. 3′,5′-Dimethoxy-4′-hydroxyacetophenone, acetosyringone, HOC6H2(OCH3)2COCH3 (Sigma-Aldrich , catalog number: D134406 ; CAS number: 2478-38-8), store at 4 °C
  4. 6-Benzylaminopurine, C12H11N5 (Sigma-Aldrich, catalog number: B3408 ; CAS number: 1214-39-7), store at RT
  5. Agarose type I, low EEO (Sigma-Aldrich, catalog number: A6013 ; CAS number: 9012-36-6), store at RT
  6. Ammonium nitrate, NH4NO3 ( Sigma-Aldrich, catalog number: A3795 ; CAS number: 6484-52-2), store at RT
  7. Ammonium sulfate, (NH4)2SO4 (Sigma-Aldrich, catalog number: A3920 ; CAS number: 7783-20-2), store at RT
  8. Bacto agar, dehydrated (Fischer Scientific, catalog number: 214050 ), store at RT
  9. Bacto beef extract, desiccated (Fischer Scientific, catalog number: 211520 ), store at RT
  10. Bacto peptone, dehydrated (Fischer Scientific, catalog number: 211677 ), store at RT
  11. Bacto yeast extract (Fischer Scientific, catalog number: 212750 ), store at RT
  12. Boric acid , H3BO3 ( Sigma-Aldrich , catalog number: B6768 ; CAS number: 10043-35-3), store at RT
  13. Calcium chloride dihydrate , CaCl2·2H2O (Sigma-Aldrich, catalog number: C7902 ; CAS number: 10035-04-8), store at RT
  14. Carbenicillin disodium , C17H16N2Na2O6S (Duchefa , catalog number: C0109 ; CAS number: 4800-94-6), store at 4 °C
  15. Cefotaxime sodium, C16H16N5O7S2Na ( Duchefa, catalog number: C0111 ; CAS number: 64485-93-4), store at 4 °C
  16. Chloro cleaner, e.g., DanKlorix® original (2.8 g/100 ml sodium hypochlorite), store at RT
  17. Cobalt (II) chloride hexahydrate , CoCl2·6H2O (Sigma-Aldrich, catalog number: C2911 ; CAS number: 7791-13-1), store at RT
  18. Copper (II) sulfate pentahydrate, CuSO4·5H2O ( Sigma-Aldrich , catalog number: C3036 ; CAS number: 7758-99-8), store at RT
  19. D-(+)-Glucose, C6H12O6 (Sigma-Aldrich, catalog number: G7021 ; CAS number: 50-99-7), store at RT
  20. D-Mannitol, C6H14O6 (Sigma-Aldrich , catalog number: M1902 ; CAS number: 69-65-8), store at RT
  21. D-Sorbitol, C6H14O6 ( Carl Roth, catalog number: 6213.1 ; CAS number: 50-70-4), store at RT
  22. DifcoTM casamino acids, vitamin assay (Thermo Fischer, catalog number: 228820 ), store at RT
  23. Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: D/4121/PB15 ; CAS number: 67-68-5), store at RT 
  24. Ethylenediaminetetraacetic acid disodium salt dihydrate, C10H14N2Na2O8·2H2O (Sigma-Aldrich; catalog number: E6635 ; CAS number: 6381-92-6), store at RT
  25. GELRITETM (Duchefa, catalog number: G1101 ; CAS number: 71010-52-1), store at RT
  26. Glycerol, SOLVAGREEN® ≥ 98 %, anhydrous, Ph.Eur., C3H8O3 (Carl Roth, catalog number: 7530.1 ; CAS number: 56-81-5), store at RT
  27. Glycine , NH2CH2COOH ( Sigma-Aldrich, catalog number: G8790 ; CAS number: 56-40-6), store at RT
  28. Hygromycin B solution, CELLPURE® 50 mg/ml, sterile , C20H37N3O13 (Carl Roth, catalog number: CP12.2 ; CAS number: 31282-04-9), store at 4 °C
  29. Hydrogen chloride, HCl, (Sigma-Aldrich, catalog number: H1758-100ML ; CAS number: 7647-01-0), store at RT
  30. Iron (II) sulfate heptahydrate, FeSO4·7H2O (Sigma-Aldrich , catalog number: F8263 ; CAS number: 7782-63-0), store at RT
  31. Kanamycine sulphate monohydrate, C18H36N4O11·H2SO4·H2O (Duchefa, catalog number: K0126 ; CAS number: 25389-94-0), store at 4 °C
  32. Kinetin, C10H9N5O (Sigma-Aldrich, catalog number: K3378 ; CAS number: 525-79-1), store at 4 °C
  33. L-Arginine, H2NC(=NH)NH(CH2)3CH(NH2)CO2H (Sigma-Aldrich, catalog number: A8094 ; CAS number: 74-79-3), store at RT
  34. L-Aspartic acid , HO2CCH2CH(NH2)CO2H (Sigma-Aldrich, catalog number: A7219 ; CAS number: 56-84-8), store at RT
  35. L-Glutamine, H2NCOCH2CH2CH(NH2)CO2H (Sigma-Aldrich , catalog number: G8540 ; CAS number: 56-85-9), store at RT
  36. Liquid nitrogen
  37. L-Proline , C5H9NO2 (Sigma-Aldrich , catalog number: P5607 ; CAS number: 147-85-3), store at RT
  38. Magnesium chloride, MgCl2 (Sigma-Aldrich; catalog number: M8266 ; CAS number: 7786-30-3), store at RT
  39. Magnesium sulfate heptahydrate, MgSO4·7H2O ( Sigma-Aldrich, catalog number: M2773 ; CAS number: 10034-99-8), store at RT
  40. Maltose monohydrate , C12H22O11·H2O (Duchefa, catalog number: M0811 ; CAS number: 6363-53-7), store at RT
  41. Manganese (II) sulfate monohydrate , MnSO4·H2O (Sigma-Aldrich, catalog number: M7899 ; CAS number: 10034-96-5), store at RT
  42. Myo-Inositol , C6H12O6 ( Sigma-Aldrich , catalog number: I7508 ; CAS number: 87-89-8), store at RT
  43. Nicotinic acid , C6H5NO2 (Sigma-Aldrich, catalog number: N0761 ; CAS number: 59-67-6), store at RT
  44. Potassium chloride , KCl ( Sigma-Aldrich, catalog number: P5405 ; CAS number: 7447-40-7), store at RT
  45. Potassium hydroxide, KOH (Fisher Scientific, catalog number: 10366240 ; CAS number: 1310-58-3), store at RT
  46. Potassium iodide , KI (Sigma-Aldrich, catalog number: P8166 ; CAS number: 7681-11-0), store at RT
  47. Potassium nitrate , KNO3 ( Sigma-Aldrich, catalog number: P8291 ; CAS number: 7757-79-1), store at RT
  48. Potassium phosphate monobasic, KH2PO4 (Sigma-Aldrich, catalog number: P5655 ; CAS number: 7778-77-0), store at RT
  49. Pyridoxine hydrochloride , C8H11NO3·HCl (Sigma-Aldrich, catalog number: P6280 ; CAS number: 58-56-0), store at RT
  50. Rifampicin, C43H58N4O12 (Sigma-Aldrich, catalog number: R3501 ; CAS number: 13292-46-1), store at 4 °C
  51. Sodium chloride, NaCl (Sigma-Aldrich, catalog number: S7653 ; CAS number: 7647-14-5), store at RT
  52. Sodium hydroxide, NaOH (Sigma-Aldrich, catalog number: 30620-1KG-M ; CAS number: 1310-73-2), store at RT
  53. Sodium molybdate dihydrate , Na2MoO4·2H2O (Sigma-Aldrich, catalog number: 331058 ; CAS number: 10102-40-6), store at RT
  54. Sodium phosphate monobasic , NaH2PO4 (Sigma-Aldrich, catalog number: S5011 ; CAS number: 7558-80-7), store at RT
  55. Sucrose , C12H22O11 (Duchefa, catalog number: S0809 ; CAS number: 57-50-1), store at RT
  56. Thiamine hydrochloride, C12H17ClN4OS·HCl ( Sigma-Aldrich, catalog number: T1270 ; CAS number: 67-03-8), store at RT
  57. Zinc sulfate heptahydrate, ZnSO4·7H2O ( Sigma-Aldrich, catalog number: Z1001 ; CAS number: 7446-20-0), store at RT
  58. FastAmp® Plant Direct PCR Kit (Intactgenomics, catalog number: 4612 )
  59. WTWTM TEP 4 Model Buffer Solution pH 4 (WTWTM, catalog number: 108700 )
  60. WTWTM TEP 7 Model Buffer Solution pH 7 (WTWTM, catalog number: 108702 )
  61. WTWTM TPL 10 Trace Model Buffer Solution pH 10.1 (WTWTM, catalog number: 108805 )
  62. Rice soil composition (see Recipes)
  63. Stock solution composition (see Recipes)
  64. Phytohormones and antibiotics (see Recipes)
  65. Cultivation medium composition (see Recipes)
  66. Suspension medium (see Recipes)
  67. Co-cultivation medium (see Recipes)
  68. Resting medium (see Recipes)
  69. Selection medium (see Recipes)
  70. Pre-regeneration medium (see Recipes)
  71. Regeneration medium (see Recipes)
  72. Rooting medium (see Recipes)
  73. Transformation cheat sheet (see Recipes)
  74. GUS staining solution (see Recipes)
  75. X-Gluc solution (see Recipes)

Equipment

Note: Equipment can be adapted to different lab conditions, however, PETRI DISHES, MAGENTA BOXES are VERY IMPORTANT TOOLS which affect transformation efficiency. We highly recommend to use the same brands as we used in this protocol. SCALPELS and TWEEZERS have to be high quality due to the frequent sterilization. Exact type and brand may be adapted to personal choice.
 

  1. -80 °C Ultra low temperature freezer (Panasonic, model: MDF-U76V-PE )
  2. Analytical and Precision Balances (Precisa Gravimetrics AG, Series 321LS, model: LS 2200C and LS 120A )
  3. Autoclave (Systec, model: 3850 EL )
  4. Benchtop pH meter; WTWTM inolabTM 7110 (Supplier: WTWTM 1AA114; Fisher Scientific, catalog number: 11731381 )
  5. Biospectrometer, basic (Eppendorf, catalog number: 6135000009 )
  6. Buckets, 5 L (Auer, catalog number: ER 5,6-226+DK
  7. Erlenmeyer flask, 250 ml (DURAN®, catalog number: 21 216 36 )
  8. Growth chamber 1 [Percival, model: CU-41L5 ; condition: 30 °C, continuous light (24/0 day/night photoperiod) with photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 40 µmol m-2 s-1, PPFD-green of 80 µmol m-2 s-1 and PPFD-red of 70 µmol m-2 s-1]
  9. Growth chamber 2 [Percival, model: CU-41L5 ; condition: 27 °C, 16/8 day/night photoperiod with photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 30 µmol m-2 s-1, PPFD-green of 70 µmol m-2 s-1 and PPFD-red of 60 µmol m-2 s-1]
  10. Glass bead sterilizer (SkinMate Apus Quartz)
  11. High precision tweezers (110 mm; type 5.SA; extra fine tip; stainless steel) (Behr Labor-Technik, catalog number: 6.266 876 )
  12. Electroporator (Bio-Rad Gene PulserTM)
  13. Incubator/shaker 28 °C (Infors HT, Multitron Standard) 
  14. Inoculating loops, PS 10UL PK/25 (Hach, catalog number: 2749125 )
  15. Laboratory bottles with screw cap and pouring ring, 100 ml (Duran®, catalog number: 218012458 )
  16. Laboratory bottles with screw cap and pouring ring, 250 ml (Duran®, catalog number: 218013651 )
  17. Laboratory bottles with screw cap and pouring ring, 500 ml (Duran®, catalog number: 218014459 )
  18. Laboratory bottles with screw cap and pouring ring, 1,000 ml (Duran®, catalog number: 218015455 )
  19. Lightmeter (Quantum Spectrometer, UPRtek, PAR 300)
  20. Magnetic stirring bars, octagonal, with pivot ring, blue, 38 mm (VWR, catalog number: 442-0438 )
  21. Pipette controller, Pipetboy acu 2 (Supplier: Integra (Biosciences); for glass and plastic pipettes from 0.1 to 100 ml) (VWR, catalog number: 613-4437 )
  22. Polycarbonate vacuum desiccator (Sanplatec, catalog number: PC-250KG )
  23. Pots SM-H Container 2.0 L (Meyer-shop, catalog number: 737203 )
  24. Scalpel handles (Supplier: BAYHA GMBH; length: 160 mm) (VWR, catalog number: 233-5202 )
  25. Stereomicroscope (Zeiss, Discovery.v8, brightfield images) 
  26. Stereo zoom and fluorescence microscope (Zeiss, AxioZoom.V16) for GFP imaging
  27. Sterile bench (Thermo ScientificTM HeraguardTM ECO Clean Bench)
  28. Thermometer (Laserliner ThermoSpot) 
  29. Thermal cycler (Bio-Rad, model: T100TM, catalog number: 1861096 )
  30. Vacuum pump (Vacuubrand, catalog number: MZ 2C )

Procedure

  1. Prepare stock solutions
    1. Prepare 17 stock solutions including N6 major 1, N6 major 2, N6 major 3, N6 major 4, B5 minor 1, B5 minor 2, B5 minor 3, B5 minor 4, B5 vitamins, AA macro salts, AA micro salts, Glycine, MS 1, MS 2, MS 3, MS 4 and MS vitamins with composition according to Table S2. Prepare phytohormone and antibiotic stock solutions according to Table S3.
    2. Filter sterilize all stock solutions and keep at 4 °C.
      Notes:
      1. Always filter sterilize the stock solutions (filter pore size: 0.2 µm, Consumables 1, 2, 3, 20 and 21 for filter types), do not autoclave!
      2. Stock solutions can be stored at 4 °C for a maximum of 3 months.
      3. Do not use stock solutions older than 3 months.

  2. Prepare cultivation medium
    1. Prepare cultivation medium according to Table S4-Table S11. Cultivation medium composition.
      Notes:
      1. It is very critical to adjust pH properly: always calibrate the pH meter before use (for pH 5.8, use calibration solutions pH 4 and pH 7).
      2. Always adjust the pH very precisely: for a medium which e.g., pH 5.8 is needed, accepted pH value is 5.80-5.81.
      3. Record the pH values before and after pH adjustment.
      4. Always measure the pH at the same medium temperature and record the medium temperature while measuring pH (Figure 3).


        Figure 3. pH measuring. A. Recording pH and temperature while preparing medium. B. Detail of the right position of the electrode while recording the pH. C. Temperature and pH recording at the display.

      5. pH is measured after all components are added, except for agarose, phytohormones and antibiotics (see Table S4-Table S11 for more details), before autoclaving the medium.
      6. It is critical to use short autoclave cycles for autoclaving media: apply a sterilization time of 5 min (!) at 121 °C (101 kPa) and transfer media to RT as soon as the autoclave has cooled down to 95 °C, do not let media stay longer in the autoclave (Figure 4). Sterilization time varies depending on the amount of medium because the autoclaving process includes additional time for the heating up and cooling down. In this protocol, we prepared medium in 0.5 L bottles, and applied a sterilization time of 5 min at 121 °C (101 kPa), the whole autoclave cycle took 1.5 h.


        Figure 4. Comparison of media autoclaved for 5 min (A) and 20 min (B) at 121 °C (101 kPa). The caramel color is a good indication of over-autoclaved media. Media color should remain almost colorless after autoclaving like show on the left picture.

      7. Add phytohormones and antibiotics after autoclaving only when media have cooled to 40-45 °C (always use the Laserliner thermometer to check temperature!).
      8. Pour plates under sterile bench (biosafety cabinet), see Table S4 for types of Petri dishes and amount to pour for each medium. Types of Petri dishes are different for different media and it is critical to follow our recommendation.
      9. Close the lids only when plates have completely dried and no water condensation is visible on the side of Petri dishes (Figure 5). This is critical because calli prefer to stay dry on the medium, wet calli do not regenerate. But also, do not overdry. Overdried media shrink and form cracks, and due to reduced water content, solute concentrations increase substantially, affecting the sensitive calli and the regeneration process.
      10. Plates are wrapped with stretch foil and stored at room temperature and in the dark (Figure 5). Do not refrigerate media.
      11. Discard unused plates with solid media after 2 weeks.


        Figure 5. Medium drying and storage. A. Completely dried Petri dish. B. Wet Petri dish, with condensed water; the black arrow indicates condense water. C. Plates are wrapped and stored at RT and in the dark.

  3. Competent Agrobacteria preparation and transformation
    1. Prepare Agrobacterium (LBA4404 or EHA105) competent cells.
      1. Streak out Agrobacterium cells from 10% glycerol stock (stored at -80 °C) on a plate with solid YEP medium containing 20 mg/L rifampicin and incubate for 2 days in the dark at 28 °C.
      2. Pick a single colony with a sterile tooth pick or pipette tip and culture in a glass test tube with 5 ml YEP liquid medium containing 20 mg/L rifampicin overnight (12 to 16 h) in the dark and at 28 °C and shake at 200 rpm.
      3. Sub-culture 1 ml overnight culture into 35 ml liquid YEP medium plus 20 mg/L rifampicin in a 250 ml flask, incubate at 28 °C, dark and shake at 200 rpm for about 8 h until OD600 reaches 0.5-0.8.
      4. Chill Agrobacteria on ice for 5 min.
      5. Centrifuge the bacteria in a 50 ml Falcon® tube at 3,000 x g, 4 °C for 5 min.
      6. Resuspend the pellet in 1 ml 20 mM CaCl2 in a 50 ml Falcon® tube (for 50 ml YEP, add 1 ml 1 M CaCl2).
      7. Aliquot (50 μl) in a 2 ml safe lock microcentrifuge tube, flash freeze with liquid nitrogen and store at -80 °C. 
    2. Transformation of competent Agrobacterium cells (LBA4404 or EHA105) with GUS/GFP reporter constructs.
      1. Take an aliquot (50 μl) of competent A. tumefaciens LBA4404 or EHA105 from 10% glycerol stock (stored at -80 °C) and thaw on ice for 10 min.
      2. Mix cells with 1 μl (~100 ng) plasmid DNA in a 1.5 ml microcentrifuge tube.
      3. Fill mixture into a sterile ice-cold electroporation cuvette (0.2 cm gap).
      4. Perform electroporation using an electroporator at 1.8 kV. Pressing until ‘buzzing’ sound can be heard (about 5 ms), leave all other settings as default: 200 Ω, capacitance extender 250 μFD, capacitance 25 μFD.
      5. Add 1 ml YEP medium without antibiotics immediately after pulse, mix cells by pipetting up and down using 1 ml pipet tip.
      6. Transfer suspension into an autoclaved 1.5 ml microcentrifuge tube and incubate for 30 min at 28 °C without shaking.
      7. Spin cells down for 1 min at 210 x g.
      8. Carefully remove supernatant, but leave about 100 μl medium in the 1.5 ml microcentrifuge tube.
      9. Re-suspend the cells in the remaining medium in a 1.5 ml microcentrifuge tube and place 100 μl on a YEP agar plate containing 50 mg/L kanamycin and 20 mg/L rifampicin.
      10. Incubate the plates for 2 days at 28 °C in the dark.
      11. Conduct colony PCR to select for transformed colonies by checking for the presence of the selection marker gene. In our case, to confirm the presence of the Hpt gene (hypoxanthine phosphoribosyl transferase encoding gene), we used the following primers: Hpt_F: 5′-AGCCTGACCTATTGCATCT-3′; Hpt_R: 5′-CATATGAAATCACGCCATGT-3′, amplicon size 200 bp, Tm 55 °C.
      12. Select 1-3 positive colonies and inoculate in a glass test tube with 3 ml liquid YEP medium containing 50 mg/L kanamycin and 20 mg/L rifampicin. Grow bacteria at 28 °C in the dark with shaking speed at 200 rpm overnight.
      13. Prepare glycerol stock of Hpt positive Agrobacterium cultures by mixing 0.6 ml culture with 0.3 ml 10% glycerol (autoclaved) in 2 ml safe lock microcentrifuge tubes, freeze immediately in liquid N2 and store at -80 °C.

      Note: In this study we used a GUS reporter construct (pSWEET13:GUSplus) and a GFP reporter construct (pOsUbi:eGFP) (Figure 6) which were kindly provided by Dr. Joon-Seob Eom (Heinrich Heine University, Düsseldorf, Kyung Hee University, South Korea) and Dr. Bing Yang (University of Missouri, USA) respectively. From other experiments, we know that under pSWEET13 and OsUbi promoters, GUS and GFP are expressed at both calli and seedling stages, therefore we used these two constructs for checking transformation efficiency. One can use any other GUS/GFP reporter construct for the same purpose. Optimal is the use of GUS or GFP intron constructs, since Agrobacteria do not splice the intron and thus can not cause false positive calli or regenerate plants (Vancanneyt et al., 1990).


      Figure 6. Maps of the two plasmids carrying GUS and GFP reporter genes used in this study

  4. Isolation of the immature embryos
    1. Harvest rice immature seeds (8-12 days post-pollination) and place 300-400 seeds in a 50 ml Falcon® tube. It is important to get immature seeds at the right developmental stage. To check for the stage of immature embryos, squeeze immature seeds gently and feel their hardness. Immature seeds should be at the late milky stage at which the endosperm is not completely cellularized yet, see Video 1 for more details. This harvesting step can be done outside of a sterile hood, but all steps below including dehusking, sterilization, isolation of immature embryos are done under sterile condition.

      Video 1. Selection of immature seeds

    2. Under sterile condition, wash seeds with 40 ml ethanol 70% for 30 s with hand shaking in a 50 ml Falcon® tube; discard ethanol. Then wash seeds with 40 ml sterile Milli-Q water three times, discard the water.
    3. Place immature seeds into a Petri dish layered with moistened sterile filter papers and remove the seed coat (lemma and palea) very carefully under a stereo microscope and in sterile conditions (under laminar flow chamber) with scalpel and forceps (Video 2, Figures 7A-7B). Scalpel and forceps should be sterilized by using for example a glass bead sterilizer. Tools need to be cooled down to avoid heat damage to immature embryos.

      Video 2. Dehusking of immature seeds

    4. Place the immature seeds in sterile conditions in a 50 ml Falcon® tube (once lemma and palea have been removed) to start the sterilization procedure (Figure 7C).
    5. Add 40 ml ethanol 70%, under sterile conditions, to the Falcon® tube and shake by hand for 30 s. Discard ethanol. Wash immature seeds with 40 ml sterile Milli-Q water. Discard the water.
    6. In a sterile 50 ml Falcon® tube, prepare a mixture of 5 ml commercial bleach Klorix® with 35 ml sterile Milli-Q water [final concentration of NaClO is 28 g/L (0.376 M)].
    7. Add the mixture to the 50 ml Falcon® tube containing the immature seeds and shake by hand for 5 min.
    8. Discard the Klorix® mixture and rinse the seeds with 40 ml sterile Milli-Q water, repeat the procedure 15 times until all remains of Klorix® are completely washed out.
      Note: It is very important that Klorix® is washed out completely, because Klorix® can affect the germination of immature embryos. We recommend to rinse the immature embryos in a 50 ml Falcon® tube 15 times with 40 ml sterile Milli-Q water each time.
    9. Place immature seeds into a Petri dish with sterile filter paper and carefully isolate immature embryos (IEs) using scalpel and fine forceps under the stereo microscope (Video 3, Figure 7D) in a sterile hood. Be careful and gentle to avoid damaging or wounding the immature embryos!

      Video 3. Immature embryo isolation

    10. Only select undamaged immature embryos with sizes between 1.3-1.8 mm. The immature embryos should be opaque, off-white colored. Transparent immature embryos will not germinate. Place about 50 immature embryos (scutellum face up) on co-cultivation medium (Figure 7E).


      Figure 7. Immature embryo isolation and inoculation with Agrobacteria. A. Removal of palea and lemma under a stereo microscope. B. Dehusked immature seeds in Petri dish with wet Whatman® paper. Scale bar: 1 cm. C. Seed sterilization with ethanol and Klorix®. D. Isolated immature embryos in a plate. E. Immature embryos with scutellum up (white arrows) and immature embryos lying on the side. Scale bar: 1 mm. F. Inoculation with Agrobacteria.

  5. Transformation and co-cultivation
    Notes:
    1. Generally, we recommend to start with 100 immature embryos for one transformation. Depending on the transformation efficiency, the number of immature embryos as starting materials can be reduced or increased.
    2. We recommend to use 12 immature embryos as controls without Agrobacterium inoculation. In the first selection step, 6 control immature embryos will be moved onto selection medium without hygromycin B to check for the regeneration ability. The other 6 control immature embryos will be moved onto selection medium with 30 mg/L hygromycin B to control the effectiveness of hygromycin B selection. These are very important controls, especially when trying to adapt this transformation protocol to other varieties. Hygromycin B concentration, regeneration medium and inoculation time may have to be adapted for other varieties.
    3. We also recommend to record all data for each transformation, e.g., date, number of immature embryo as well as any notes (see Table S12 for the template).

    1. Strike Agrobacteria (LBA4404 or EHA105) from frozen -80 °C stocks on solid YEP plates containing antibiotics (50 mg/L kanamycin and 20 mg/L rifampicin) two days before infection. Then incubate for two days at 28 °C in the dark.
    2. On the day of the transformation, take a 3 mm-size loop of Agrobacterium culture from the YEP plate and suspend in suspension medium (Table S5) in a 50 ml Falcon® tube.
    3. Vortex bacterial suspension and adjust to OD600 0.3. Incubate in dark at 25 °C for 1 h, no shaking needed.
      Notes:
      1. Add acetosyringone just before use and prepare freshly!
      2. Dissolve acetosyringone: for 50 ml suspension culture, dissolve 10 mg acetosyringone in 100 μl DMSO and then take 9.81 μl solution to obtain 0.981 mg acetosyringone; no filter sterilization needed.
      3. Always adjust OD600 very precisely: recommended values, depending on the spectrometer, are between 0.30 and 0.31.
    4. Drop 5 μl Agrobacterium suspension on top of each immature embryo, with scutellum side-up (Figure 7F).
    5. Incubate plates with embryos in the presence of Agrobacteria for 7 days at 25 °C in the dark (Figure 8).


      Figure 8. Immature embryos at the end of co-cultivation phase. A-C. Control calli without Agrobacterium infection. D-F. Agrobacterium-infected calli.

  6. Resting
    1. Check immature embryos for possible contamination (Figure 9). If immature embryos are contaminated, discard them. If possible, uncontaminated immature embryos in the contaminated plate can be rescued and placed on a separate plate. Otherwise discard the whole plate. Contamination can happen if dehusking or immature embryos isolation was not done carefully enough and embryos were damaged with forceps or scalpel during the process, facilitating contamination with other microorganisms (Figure 9).


      Figure 9. Contamination of immature embryos. A. Control immature embryos (no Agrobacterium inoculation) showing bacterial contamination. B. Agrobacterium-inoculated immature embryos showing contamination with other bacteria. C. Control non-infected immature embryos without contamination. D. Agrobacterium-inoculated immature embryos without contamination.

    2. Place immature embryos without contamination on sterile filter paper and remove shoots with scalpel and forceps. Performing this steps under a stereo-microscope to guarantee complete excision of shoots.
      1. No parts of the shoot and rim should be left over, remove everything (Figures 10A and 10B).
      2. Remove the brown tissue from calli if there is excess, but be careful not to damage the immature embryos.


        Figure 10. Immature embryos before (A) and after (B) removing shoots. Arrows indicate parts to be removed: shoot (bottom left), rim (bottom right) and brown tissue (upper). C. Cleaning immature embryo using two layers of sterile filter paper.

    3. Clean the immature embryos by placing them between two layers of sterile filter paper (Figure 10C) and carefully dab off the Agrobacteria. Repeat this procedure at least two times to remove the surplus Agrobacteria.
    4. Transfer immature embryos scutellum side-up onto resting medium (16 immature embryos/plate) and incubate for 5 days in growth chamber 1 [30 °C, continuous light, 24/0 day/night photoperiod, with photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 40 µmol m-2 s-1, PPFD-green of 80 µmol m-2 s-1 and PPFD-red of 70 µmol m-2 s-1] (Figure 11).


      Figure 11. Immature embryos at the end of the resting phase. A-C. Control immature embryos. D-F. Agrobacterium-inoculated immature embryos.

      Notes:
      1. Use the stereo microscope to check for the state of the immature embryos (e.g., if there is any contamination), to clean brown tissues and take immature embryos more gently.
      2. Do not push immature embryos into the medium, let them stay loosely on top of the medium.
      3. Keep immature embryos on the same position when transferring between media (scutellum up-side to the medium).
      4. Seal plates with two layers of Micropore 3M tape.

  7. Selection
    1. After 5 days of resting, remove the brown tissue completely with forceps and a scalpel by scratching or cutting brown tissue off the surface of the immature embryo. Transfer the immature embryos onto the selection medium, containing 30 mg/L hygromycin B (16 calli/plate). Incubate at 30 °C for 10 days in continuous light [24/0 day/night photoperiod, photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 40 µmol m-2 s-1, PPFD-green of 80 µmol m-2 s-1 and PPFD-red of 70 µmol m-2 s-1] (1st selection) (Figure 12).


      Figure 12. Immature embryos at the end of the first selection phase. A, D. Control immature embryos without hygromycin B, immature embryos produced compact and embryogenic calli and started turning green. B, E. Control immature embryos on selection medium with 30 mg/L hygromycin B. Immature embryos do not turn brown completely but are also not growing either. C, F. Agrobacterium-inoculated immature embryos may turn brown partially or completely.

    2. After 10 days of 1st selection, transfer the calli to a freshly prepared plate with selection medium (16 immature embryos/plate). Remove brown tissue as much as possible from the immature embryos and incubate at 30 °C for 10 days in continuous light [24/0 day/night photoperiod, photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 40 µmol m-2 s-1, PPFD-green of 80 µmol m-2 s-1 and PPFD-red of 70 µmol m-2 s-1] (2nd selection) (Figure 13). Some immature embryos turn brown completely after the 1st selection. In this case, do not remove the brown tissue and keep the whole immature embryos.


      Figure 13. Immature embryos after the 2nd selection phase. A, D. Control immature embryos without hygromycin B produced proliferating embryogenic calli. B, E. Control immature embryos with 30 mg/L hygromycin B, immature embryos remain their size (2-4 mm) without producing any callus. C, F. Agrobacterium-inoculated immature embryos on selection medium produced small, compact and light yellow embryogenic microcalli (black arrows).

    3. After 10 days of 2nd selection, take only the embryogenic microcalli from the black immature embryos and place onto fresh selection medium (16 immature embryos/plate), incubate at 30 °C for 10 days in continuous light [24/0 day/night photoperiod, photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 40 µmol m-2 s-1, PPFD-green of 80 µmol m-2 s-1 and PPFD-red of 70 µmol m-2 s-1] (3rd selection) (Figure 14). The embryogenic calli are small, compact clusters of cells, usually light yellow in color (Figure 13 F). Non-embryogenic calli are usually larger, soft, semi-transparent and yellow or gray loosely- held clusters of cells. The “mother immature embryo” can be kept on the selection medium as a back-up in case the freshly-isolated microcalli do not grow, or more microcalli are needed. For this step, microcalli from different immature embryos are kept separately by dividing the Petri dish into small areas (Figure14 C). The separated microcalli will be considered as independent events.


      Figure 14. Microcalli at the end of the 3rd selection phase. A, D. Control calli without hygromycin B are compact, embryogenic and turned green partly. B, E. Control immature embryos with 30 mg/L hygromycin B, immature embryos remain the same size and do not grow. C, F. Agrobacterium-inoculated microcalli proliferating on selection medium.

      Notes:
      1. Use the stereo microscope to check for the state of the immature embryos/calli, to remove the brown tissue and to select the embryogenic calli more precisely.
      2. Don’t push immature embryos/calli into to the medium, let them stay loosely on top of the medium.
      3. Seal plates with two layers of Micropore 3M tape.
      4. If no microcalli is generated after 2nd selection, refresh the selection medium and culture for another 10 days. If after 2-3 additional rounds of selection, no microcalli are produced, discard the immature embryos.

  8. Plant regeneration
    1. Transfer resistant calli to pre-regeneration medium containing 30 mg/L hygromycin B (6-9 calli/plate) and incubate at 30 °C for 10 days in continuous light [24/0 day/night photoperiod, photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 40 µmol m-2 s-1, PPFD-green of 80 µmol m-2 s-1 and PPFD-red of 70 µmol m-2 s-1] (Figure 15).


      Figure 15. Calli at the end of pre-regeneration phase. A, D. Control calli without hygromycin B are mostly green. B, E. Control immature embryos with 30 mg/L hygromycin B; immature embryos remain their sizes and did not grow. C, F. Agrobacterium-inoculated proliferated calli with greening spots.

    2. Select proliferating calli with green spots that covered approximately 2/3 of the calli (Figure 15F) and transfer to regeneration medium containing 30 mg/L hygromycin B (6 calli/plate), for shoot development and incubate at 30 °C for 14 days in continuous light [24/0 day/night photoperiod, photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 40 µmol m-2 s-1, PPFD-green of 80 µmol m-2 s-1 and PPFD-red of 70 µmol m-2 s-1] (Figure 16).


      Figure 16. Calli at the end of the Regeneration phase. A. Regenerated plants from a control calli without hygromycin B. B. Control immature embryos with 30 mg/L hygromycin B, immature embryos did not grow. C. Regenerated plants from Agrobacterium-inoculated calli are putative transformants.

    3. If calli have not produced any shoot or only produce small shoots, transfer the calli to freshly prepared plates of regeneration medium (6 calli/plate) every 14 days and incubate at 30 °C in continuous light [24/0 day/night photoperiod, photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 40 µmol m-2 s-1, PPFD-green of 80 µmol m-2 s-1 and PPFD-red of 70 µmol m-2 s-1], until small plantlets develop (small rice plants regenerated from a callus with at least two leaves and small root system, Figures 16-17).
    4. Select 1-3 plantlets from each callus and transfer into a MagentaTM GA-7 vessel containing rooting medium without hygromycin B. Incubate them for 7 days at 27 °C [16/8 day/night photoperiod with photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 30 µmol m-2 s-1, PPFD-green of 70 µmol m-2 s-1 and PPFD-red of 60 µmol m-2 s-1]. At this stage, plantlets regenerated from different calli are considered independent transformation events, therefore taking more than one plantlet as a backup to ensure that all putative events grow vigorously in the rooting step.
    5. After 7 days, when plantlets reach the top of the MagentaTM box, place another MagentaTM box on top to create more space for the plants to grow (Figure 17A). Place ‘double’ MagentaTM box in the growth chamber with 27 °C [16/8 day/night photoperiod with photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 30 µmol m-2 s-1, PPFD-green of 70 µmol m-2 s-1 and PPFD-red of 60 µmol m-2 s-1] for another 7 days.
    6. Transfer plantlets of approx. 15 cm height into soil by gently washing away excess agarose attached to roots. Grow plantlets in a pot which is submerged in a larger bucket to maintain high humidity condition in the glass house with natural daylight and additional lamplight of 8/16 day/night photoperiod with a day temperature of 30 °C ± 2 °C and a night temperature of 25 °C ± 2 °C. The relative humidity was between 50-70%. Plants were grown under a photosynthetic active radiation (PAR) or photosynthetic photon flux density (PPFD) of 200 µmol m-2 s-1 with PPFD-blue of 40 µmol m-2 s-1, PPFD-green of 70 µmol m-2 s-1 and PPFD-red of 80 µmol m-2 s-1.


      Figure 17. Rooting and planting. A, B. Two-week-old regenerated plants, before transfer to soil. C. Regenerated plant right after planting. D. Regenerated plants, two weeks after planting.

      Notes:
      1. Use the stereo microscope to check for the state of the calli/plantlets, to select the calli and separate plantlets more gently.
      2. Don’t push the calli into to the medium, let them stay loosely on top of the medium.
      3. Seal Petri dishes or MagentaTM GA-7 vessels with two layers of Micropore 3M tape.
      4. Since the plantlets were regenerated under continuous light (24/0 day/night photoperiod), we recommend to grow the plantlet for the rooting step under long-day conditions (16/8 day/night photoperiod) to slowly acclimate them to the upcoming short-day conditions in the glasshouse (8/16 day/night photoperiod).

  9. Screening for transformed plants
    Note: In this protocol, we did not unambiguously determine transformation events. We used plants that had been regenerated in parallel, but without infection by Agrobacteria as controls. In these control plants, we did not observe GUS staining or GFP fluorescence. All transformation events counted here were derived from independent immature embryos (one plant per one transformed immature embryo).

    1. Transformation of 100 Komboka immature embryos with GUS-intron reporter construct using two different Agrobacterium strains (EHA105 and LBA4404) resulted in 14 and 31 putative transformants from independent immature embryos, respectively (Table 1). All regenerated plants were tested for GUS activity. All leaves of regenerated plants showed GUS activity (Figures 18A-18B), except control plants which also underwent the whole protocol but without Agrobacterium infection (Figure 18C). Our results might indicate that the LBA4404 strain is more efficient in transforming Komboka when compared to EHA105, however the relative efficiency of different Agrobacterium strains can not be judged without careful quantitative analyses across many independent transformations.

      Table 1. Apparent transformation efficiency of Komboka using different Agrobacterium strain



      Figure 18. GUS histochemistry of transformed Komboka leaves. A, B. GUS-stained leaves of two plants which are regenerated from two independent immature embryos. C. GUS-stained leaves from one control plant regenerated without Agrobacterium infection, no GUS activity detectable.

    2. Transformation of Komboka with a GFP reporter construct using the Agrobacterium strain LBA4404 resulted in 54 putative independent events (Table 2). Roots of all these 54 plants were GFP positive (Figures 19A-19E). No GFP fluorescence was observed in uninfected plants (Figure 19F). From 54 GFP positive events, 200 bp GFP gene fragment was amplified by PCR from 48 plants (Figure 20). Full T-DNA insertion, inheritance and copy number remain to be validated.

      Table 2. Apparent transformation efficiency of Komboka with GFP reporter construct



      Figure 19. GFP fluorescence of transformed Komboka. A-E. Root of five independent transformants under blue light. F. Root of a non-infected plant regenerated from tissue culture (control). Scale bar: 2000 μm. All photos are taken and displayed at the same setting.


      Figure 20. Representative gel picture for confirmation of a 200 bp GFP gene fragment in transformed plants by PCR. WT: non-infected plant regenerated from tissue culture.

  10. Adjusting the protocol for transformation of other rice varieties
    To adapt this protocol for the transformation of other varieties, we recommend to evaluate the efficiency after each step using GUS or GFP intron reporter constructs (Vancanneyt et al., 1990). For example, after co-cultivation, use five calli for GUS staining or GPF screening to test whether the co-cultivation step was successful or whether co-cultivation time needs to be adjusted.
      We also recommend to use non-infected immature embryos as controls to check for the effectiveness of hygromycin B selection as well as the regeneration ability on media without hygromycin B (see Note 2, Section E), because hygromycin B sensitivity and regeneration ability may differ among varieties. Hygromycin B concentrations can be adjusted between 5-50 mg/L. Criteria for adjustment include: control immature embryos cannot grow or produce microcalli on hygromycin B containing media (Figures 13, 14, 15).
      Another relevant parameter is the stage of immature embryo development, which may differ between varieties; immature seeds should be in the late milky stage (Video 1), typically with a size of 1.3-1.8 mm at about 8-12 days post pollination. Agrobacterium strains (AGL1, LBA4404, EHA105) can be compared. Also, the number of rounds of selection and rounds of pre-generation steps can be adjusted between 1-3 until microcalli or green spots are produced.

Data analysis

  1. GUS staining
    1. Harvest 3 cm leaves and place in 15 ml Falcon® tubes.
    2. Add 5 ml staining solution (Tables S13 and S14).
    3. Vacuum the samples for 10 min at RT.
    4. Close the tubes and incubate at 37 °C overnight in the dark.
    5. Remove staining solution.
    6. Add 70% ethanol for destaining (chlorophyll removal) and incubate samples at 65 °C. The duration of incubation affects destaining, the longer the tissue will be incubated, the higher contrast of blue dye to background can be achieved, usually 2-3 days are fine. During this incubation time, exchange 70% ethanol for 1 or 2 times
    7. As long as the tissue stays in ethanol, it can be stored at RT for long time (at least several months without losing the color). It is recommended to store the samples in a dark place.

  2. GFP imaging
    Roots of GFP-transformed Komboka were observed under blue light with a Zeiss AxioZoom.V16 stereo microscope, filter excitation wavelength: 450-490 nm, filter emission wavelength: 500-550 nm, excitation wavelength: 488 nm, emission wavelength: 509 nm, exposure time 4.59 s, zoom: 0.7, total optical magnification: 7x. All photos are taken and displayed at the same setting: black: 2000, gramma: 1.0, white: 22897.

  3. PCR for GFP encoding gene
    To check for the presence of the GFP gene in regenerated plants, FastAmp® Plant Direct PCR Kit (Intactgenomics) was used to amplify a 200 bp fragment of the GFP coding region using the following primers: VL_GFP_F1 5′-GCAAGCTGACCCTGAAGTTC-3′, VL_GFP_R1 5′-GTCTTGTAGTTGCCGTCGTC-3′. PCR conditions: initial denaturation step at 95 °C for 5 min, followed by 35 cycles each at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 20 s, last extension at 72 °C for 10 min and the reaction was kept at 10 °C.

Recipes

Note: See Supplementary file (Tables S1-S14) for Recipes.

  1. Rice soil composition (Table S1)
  2. Stock solution composition (Table S2)
  3. Phytohormones and antibiotics (Table S3)
  4. Cultivation medium composition (Table S4)
  5. Suspension medium (Table S5)
  6. Co-cultivation medium (Table S6)
  7. Resting medium (Table S7)
  8. Selection medium (Table S8)
  9. Pre-regeneration medium (Table S9)
  10. Regeneration medium (Table S10)
  11. Rooting medium (Table S11)
  12. Transformation cheat sheet (Table S12)
  13. GUS staining solution (Table S13)
  14. X-Gluc solution (Table S14)

Acknowledgments

We gratefully acknowledge funding from the Bill and Melinda Gates Foundation, Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy – EXC-2048/1 – project ID 390686111, and the Alexander von Humboldt Professorship to WBF. We thank IRRI for providing the Komboka seeds, Bing Yang (University of Missouri, USA) and Joon S. Eom (Heinrich Heine University, Düsseldorf, Kyung Hee University, South Korea) for providing the GFP and GUS reporter constructs. We thank Marina Manzanilla for her training effort at IRRI genetic transformation lab. We thank Monika Streubel, Joon S. Eom for their valuable advices in plant transformation.

Competing interests

The Institute for Molecular Physiology, Heinrich Heine University of Düsseldorf (HHU), Germany, The Center for Tropical Agriculture (CIAT), Cali, Colombia and the International Rice Research Institute (IRRI), Philippine contributed equally towards the development of the protocol. The authors declare no competing interests.

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  8. Kitilu, M., Nyomora, A. and Charles, J. (2019). Growth and yield performance of selected upland and lowland rainfed rice varieties grown in farmers and researchers managed fields at Ifakara, Tanzania. Afr J Agricult Res 14: 197-208. 
  9. Li, D., Xu, H., Sun, X., Cui, Z., Zhang, Y., Bai, Y., Wang, X., Chen, W. (2015). Differential transformation efficiency of Japonicarice varieties developed in northern China. Crop Breed Appl Biotechnol 15(3): 162-168.
  10. Nishimura, A., Aichi, I. and Matsuoka, M. (2006). A protocol for Agrobacterium-mediated transformation in rice. Nat Protoc 1(6): 2796-2802.
  11. Oliva, R., Ji, C., Atienza-Grande, G., Huguet-Tapia, J. C., Pérez-Quintero, A., Li, T., Eom, J. S., Li, C., Nguyen, H., Liu, B., Auguy, F., Sciallano, C., Luu, V. T., Dossa, G. S., Cunnac, S., Schmidt, S. M., Slamet-Loedin, I. H., Vera Cruz, C., Szurek, B., Frommer, W. B., White, F. F. and Yang, B. (2019). Broad-spectrum resistance to bacterial blight in rice using genome editing. Nat Biotechnol 37(11): 1344-1350. 
  12. Sahoo, K. K., Tripathi, A. K., Pareek, A., Sopory, S. K. and Singla-Pareek, S. L. (2011). An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods 7(1): 49. 
  13. Sahoo, R. K. and Tuteja, N. (2012). Development of Agrobacterium-mediated transformation technology for mature seed-derived callus tissues of indica rice cultivar IR64. GM Crops Food 3(2): 123-128.
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  16. Sundararajan, S., Sivaraman, B., Rajendran, V. and Ramalingam, S. (2017). Tissue culture and Agrobacterium-mediated genetic transformation studies in four commercially important indica rice cultivars. J Crop Sci Biotechnol 20(3): 175-183.
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简介


[摘要 ] 遗传转化对于研究基因功能和农作物工程引入新性状均至关重要。水稻(Oryza sativa L.)是植物研究中的重要模型,因为它是世界一半以上人口的主食。其结果是,大量的转化方法已经开发了两个籼稻和粳稻米。由于育种者不断开发新的水稻品种,因此必须针对每个新品种适应转化方案。在这里,我们为使用未成熟胚的非洲新品种Komboka 提供了一种优化的转化协议,包括详细的技巧和窍门。在Komboka中,我们使用此优化方案对GUS / GFP报告基因构建体的表观转化率高达48%。该协议也适用于其他优质lite 稻品种。


[背景 ] 为植物的遗传转化的各种方法公顷一直在开发,例如穿孔(Shimamoto PEG介导的原生质体转染。等人,1989;达塔。等人,1992),生物射弹转化(Christou的等人,1991)和农杆菌-介导的转化(Slamet-Loedin 等,2014)。农杆菌介导的转化是将DNA引入植物的最广泛使用的方法之一(van Wordragen and Dons,1992)。该方法已被广泛用于研究,并已成为生物技术的关键先决条件。自从开发新的育种技术(如基因组编辑)以来,它就变得越来越重要(Char 等,2019)。对于水稻等农作物,遗传转化还可以用于开发新的遗传变异以进行植物育种,例如,创建新的抗病或抗虫品系(Cheng 等,1998; Helliwell和Yang,2013; Oliva 等。等,2019)。同样对于水稻来说,农杆菌介导的转化是将T-DNA转移到植物基因组中最流行的方法(Hiei 和Komari ,2008)。当前,存在使用成熟种子或未成熟胚诱导的愈伤组织进行粳稻和in 稻转化的多种方案(Hiei和Komari,2006和2008; Toki 等,2006; Nishimura 等,2006;Sahoo 等,2011)。;Sahoo和Tuteja,2012;Slamet-Loedin 等,2014;Sundararajan 等,2017)。使用成熟种子进行转化很方便,因为它们一年四季都可以使用并且可以存储,尽管这种方法主要用于粳稻品种。与成熟种子相比,涉及水稻未成熟胚转化的方法通常产生更高的转化率(Hiei和Komari,2008; Slamet-Loedin 等,2014)。总体而言,粳稻品种如Kitaake和日丰nbare显然容易变换,相比籼稻大米如IR64或Ciherang-Sub1的(Oliva的等人,2019) 。对于粳稻品种,使用成熟种子诱导的愈伤组织可以达到50-60%的转化率(Li 等,2015)。对于in 品种,尽管有人为提高成熟种子的转化率做出了一些努力,但未成熟的自苔藓来源的愈伤组织仍然是转化的首选组织。值得注意的是,转化效率高度依赖于品种,因此有必要针对每个品种优化转化方案。

Komboka(IR 05N 221)是IRRI产生的一种新的优良品种,由国家水稻研究计划-卡特琳研究中心和IRRI-坦桑尼亚('Komboka'='解放')在坦桑尼亚发行(Kitilu 等人,2019)。Komboka高产(8.6 t ha -1 ),半芳香族,具有良好的谷物品质,耐爆炸,并且非常适合高地和低地地区。在这里报告的协议中,我们描述了Komboka未成熟胚的稳定转化的详细步骤,这些胚可以在四个月内产生转基因植物,并且表观转化率高达48%。这个协议是通过组合和修改从公开的方案开发拉梅希达亚-Loedin 等人。,2014和比睿和Komari ,其用于转化不同,2008 籼稻水稻品种如IR64,Ciherang-SUB1(Oliva的等人,2019),因此,原则上该协议可能可以适于其他转化籼稻水稻品种。在此协议中,我们重点介绍了为其他精英品种建立转化协议必不可少的所有细节,技巧和窍门。

图1. 农杆菌介导的水稻变种未成熟胚转化的流程图和时间表。Komboka(印度)大约需要四个月的时间才能产生转基因品系。简而言之,该过程始于在受控温室条件下,白天将水稻植株生长至开花期,温度为30 °C ±2°C,白天为25 °C ±2 夜间为°C,相对湿度为50-70%。玻璃温室中的光照条件取决于自然日光和其他灯光(8/16天/夜的光照周期)。由于我们的温室沿纬度(德国杜塞尔多夫的纬度和经度,东经51°11'31.2“ N 6°47'40.1” E),白天的时间因季节而异。在这些条件下,Komboka需要1 3 -1 4 周直到启动阶段,另外需要2-3周才能开花。授粉后8-12天,可收获水稻穗以分离未成熟的胚。选择合适的种子阶段以分离未成熟的胚胎至关重要。未成熟胚应在后期阶段乳白色(VID EO 1),尺寸为1.3 - 1.8毫米。将种子去壳并分离未成熟胚(视频2和3;图2)后,将分离出的未成熟胚整体移到共培养培养基上。分离步骤后,将未成熟的胚胎与带有目的构建体的农杆菌共培养7天。然后,用无菌滤纸从农杆菌中清除发芽的未成熟胚,并切下芽。然后将未成熟的胚胎转移到没有潮霉素B的静息/恢复培养基上五天。静息后,将未成熟的胚胎与30 mg / L潮霉素B进行两轮选择,每轮进行10天。在第二选择步骤之后,微愈伤组织出现在褐色的母体未成熟胚上。将微愈伤组织与母体组织小心分离,并转移至选择培养基(30 mg / L潮霉素B)上进行第三轮选择。在该情况下,将未成熟胚没有产生第一两轮选择后microcalli,未成熟胚被转移到一个另外的选择轮(补充选择2)的10天每个。将第三次选择后的抗性和繁殖性微愈伤组织红色转移至再生前培养基(30 mg / L潮霉素B)上,进行1或2轮生长,持续10天。仅将绿化愈伤组织转移至再生培养基(30 mg / L潮霉素B)上14天。当从愈伤组织再生出小的水稻植株(小植株)并达到约5厘米高时,将它们转移到没有潮霉素B的生根培养基上14天。盆中仅种植具有强壮根系的成熟小植株。将盆浸没在较大的水桶(5升)中。



D:\ Reformatting \ 2020-7-1 \ 2003199--1495 Wolf Frommer 843004 \ Figs jpg \ Figure01.jpg

图1.描述利用水稻品种Komboka的未成熟胚进行农杆菌介导转化的流程图



D:\ Reformatting \ 2020-7-1 \ 2003199--1495 Wolf Frommer 843004 \ Figs jpg \ Figure02.jpg

图2 。农杆菌介导的水稻品种Komboka 未成熟胚转化的步骤。A.未成熟的胚胎分离。B. 农杆菌感染。C.共培养。D.休息阶段。E.选择阶段。F.再生前阶段。G.再生阶段。H.生根阶段。



下面,我们提供了详细的化学药品,设备清单,重要注意事项的转化步骤以及用于培养基制备的所有模板。



关键字:农杆菌介导转化, 籼稻, 水稻, 优良品种, 水稻转化, 植株再生

材料和试剂


 


消耗品


Filtropur BT 100瓶顶过滤器(容量:1000 ml;膜:PES;直径:90 mm;孔径:0.2 µm,用于无菌过滤)(Sarstedt ,目录号:83.3942.101);用于过滤原液,仅一次性使用
Filtropur BT 25瓶顶过滤器(容量:250 ml;膜:PES;直径:90 mm;孔径:0.2 µm,用于无菌过滤)(Sarstedt ,目录号:83.3940.101);用于过滤原液,仅一次性使用
Filtropur BT 50瓶顶过滤器(容量:500 ml;膜:PES;直径:90 mm;孔径:0.2 µm,用于无菌过滤)(Sarstedt ,目录号:83.3941.101);用于过滤原液,仅一次性使用
玻璃试管,14 x 16 mm; 21毫升(DURAN ® ,目录号:26 130 21)
Gene-Pulse Cuvettes,0.2厘米间隙(Bio-Rad,目录号:1652086),仅限一次性使用
陪替氏培养皿,超深,一次性的,Lab- TEK TM (直径:100毫米;高度:26毫米;无菌)(供应商:Thermo Scientific的,VWR,目录号:NUNC4031); 用于倒入再生培养基,仅一次性使用
培养皿深,带3个通风口(直径:92.3 mm ;高度:20 mm ;无菌)(供应商:Greiner Bio One,VWR,目录号:391-0493);用于倒入再生前培养基,仅一次性使用                                                                     
培养皿,带凸轮(直径:92 mm;高度:16 mm;无菌)(Sarstedt ,目录号:82.1473);用于倒入YEP,共培养,静止和选择培养基,仅一次性使用
定性滤纸,标准级,1级,1 V,的Whatman ® (直径:85毫米)(VWR,目录号:516-0593)
1.5 ml反应管(Sarstedt ,目录号:72.690.001)
SafeSeal 反应管,2毫升(Sarstedt ,目录号:72.695.500)
手术刀刀片(使用BAYHA互锁系统的手术刀手柄类型11;非无菌)(Behr Labor-Technik,目录号:9409911)
50 ml螺帽管(Sarstedt ,目录号:62.547.254)
15 ml螺帽管(Sarstedt ,目录号:62.554.502)
血清移液管,10毫升(Sarstedt ,目录号:86.1254.001),仅限一次性使用
血清移液管,25 ml(Sarstedt ,目录号:86.1685.001),仅限一次性使用
血清移液管,5毫升(Sarstedt ,目录号:86.1253.001),仅限一次性使用
拉伸箔(Stretchplus ; 300.0 mx 40.0 cm;箔厚度:7 µm)
手术胶带(3M TM Micropore TM ;宽度:1.25 cm)
注射器(Injekt ® ;20毫升; 路厄锁)(布劳恩,目录号:4606205V),单次使用
注射器过滤单元Filtropur S 0.2(膜:PES;过滤表面:5.3 cm 2 ;孔径:0.2 µm,用于无菌过滤)(Sarstedt ,目录号:83.1826.001);用于过滤植物激素和抗生素溶液,仅一次性使用
组织培养容器,Magenta TM GA-7(供应商:Sigma-Aldrich; 77 x 77 x 97 mm)(VWR,目录号:SAFSV8505);用于倒入“ Roo”介质,可重复使用
木质牙签(6.8厘米)(Pap Star,目录号:12736)
 


生物材料


Komboka种子(IR05N221,L17WS.06#24)
国际水稻研究所(菲律宾IRRI)根据特殊材料转让协议(SMTA-MLS)友情提供了Komboka种子。


  将Komboka种子直接播入2升盆(高13.2厘米,直径16.7厘米)的圆形土壤中,每盆一株。异型多孔陶瓷(PPC)蔬菜等级和“拟南芥”比例为1:1的土壤混合物用作水稻种植的标准土壤。小号ee值表S1土壤组合物的详细信息。作为缓释肥料,Osmocote Exact 3-4 M 4克(16%N,3.9%P,10%K,1.2%Mg,0.45%Fe,0.06%Mn,0.02%B,0.05%Cu,0.02%在1 L的土壤中添加Mo,0.015%Zn)(ICL / SF UK)。另外,将植物从每周施肥2 Ñ d 周和每两周从6 个星期使用彼得斯的Excel(14%N,6%的P,14%发芽后K,6.5%的Ca,2.5%的Mg,0.12%的Fe, 0.06%Mn,0.02%B,0.015%Cu,0.01%Mo,0.015%Zn)(ICL / SF UK)。当植物达到开花期时终止施肥。将2升装满土壤的盆中的水稻植株浸入装满水的5升桶中,使内部的2升盆位于水线以下。水可以保留在桶中,直到水稻植株获得种子为止,无需交换。另外,也可以将2 L的饭锅放在60 x 40 x 6 cm的托盘中,并在水的上边缘装满水。在这种情况下,需要每两周更换一次水。


  日光温度为30°C±2°C,夜间温度为25°C±2°C,水稻植物在具有自然日光和8/16日/夜光周期的附加光照的温室中生长,但并非严格要求。相对湿度在50-70%之间,可通过在温室地板上喷水来手动控制。将植物光合有效辐射(PAR)或光合光子通量密度下生长200微摩尔米的(PPFD)-2 小号-1 与PPFD蓝40微摩尔的米-2 小号-1 ,PPFD绿色的70微摩尔米- 2 s -1 和80 µmol m -2 s -1的PPFD-red 。


 


化学制品


注意:化学品批次和品牌是可能影响转化成功的重要因素。我们发现,首次尝试对Komboka进行转型时,对替代化学品牌的不明确使用通常会导致失败。因此,我们强烈建议使用此处指定的确切化学品(品牌和目录编号),尤其是在设置新的转化方案时。确保将化学品储存在规定的条件下,并且在到期后不要使用产品。对于化学品或仪器的选择,我们没有任何竞争利益。


 


1-萘乙酸,C 12 H 10 O 2 (Sigma-Aldrich ,目录号:N0640 ; CAS号:86-87-3),存放于室温
2 ,4-二氯苯氧乙酸,氯2 Ç 6 ħ 3 OCH 2 CO 2 H ^ (Sigma-Aldrich公司,目录号:D7299 ; CAS号:94-75-7),保存于室温                                         
3' ,5'-D 甲氧基-4'-羟基苯乙酮,乙酰丁香酮,HOC 6 H 2 (OCH 3 )2 COCH 3 (Sigma-Aldrich ,目录号:D134406 ; CAS号:2478-38-8),存放于4摄氏                                                       
6-苄基氨基嘌呤,C 12 H 11 N 5 (Sigma-Aldrich ,目录号:B3408 ; CAS号:1214-39-7),在室温下保存                                                       
I型琼脂糖,低EEO(Sigma-Aldrich ,目录号:A6013 ;CAS号:9012-36-6),在室温下存放
硝酸铵NH 4 NO 3 (西格玛奥德里奇(Sigma-Aldrich),目录号:A3795 ; CAS号:6484-52-2),存放在室温下                                         
硫酸铵(NH 4 )2 SO 4 (Sigma-Aldrich ,目录号:A3920 ; CAS号:7783-20-2),在室温下保存                                         
细菌用琼脂,脱水(菲舍尔科学,目录号:214050),保存于室温
干燥的Bacto 牛肉提取物(Fischer Scientific ,目录号:211520),在RT存放
细菌用蛋白胨,脱水(菲舍尔科学,目录号:211677),保存于室温
细菌用酵母提取物(菲舍尔科学,目录号:212750),保存于室温
硼酸H3BO3(Sigma-Aldrich ,目录号:B6768 ; CAS号:10043-35-3),存放在RT                                         
二水氯化钙CaCl 2 ·2H 2 O (Sigma-Aldrich ,目录号:C7902 ;CAS编号:10035-04-8),在室温下存放                                                       
羧苄青霉素二钠,C 17 H 16 N 2 Na 2 O 6 S (Duchefa ,目录号:C0109 ; CAS号:4800-94-6),在4°C下储存                                                       
头孢噻肟钠,C 16 H 16 N 5 O 7 S 2 Na(Duchefa ,目录号:C0111 ; CAS号:64485-93-4),储存在4°C                                                       
氯清洁器,例如,DanKlorix ® 原始(2.8克/ 百毫升次氯酸钠),保存于室温
六水合氯化钴(II),CoCl 2 ·6H 2 O (Sigma-Aldrich ,目录号:C2911 ;CAS号:7791-13-1),在室温下存放                                                       
五水合硫酸铜(II),CuSO 4 ·5H 2 O(Sigma-Aldrich ,目录号:C3036 ; CAS号:7758-99-8),存放于RT                                                       
D-(+)-葡萄糖,C 6 H 12 O 6 (Sigma-Aldrich ,目录号:G7021 ; CAS号:50-99-7),在室温下存放                                         
D-甘露醇,C 6 H 14 O 6 (Sigma-Aldrich ,目录号:M1902 ; CAS号:69-65-8),在RT储存                                         
D-山梨糖醇,C 6 H 14 O 6 (Carl Roth ,目录号:6213.1 ; CAS号:50-70-4),在室温下存放                                                       
的Difco TM 酪蛋白氨基酸,维生素分析(该RMO 费,目录号:228820),STOR È 在室温                           
二甲基亚砜(DMSO)(Fisher Scientific ,目录号:D / 4121 / PB15 ; CAS号:67-68-5),在RT存放
乙二胺四乙酸二钠二水合物,C 10 H 14 N 2 Na 2 O 8 ·2H 2 O(Sigma-Aldrich;目录号:E6635; CAS号:6381-92-6),在室温下存放                           
GELRITE TM (Duchefa ,目录号:G1101 ;CAS号:71010-52-1),在RT存放                                                       
甘油,SOLVAGREEN ® ≥ 98%,无水,欧洲药典。,C 3 ħ 8 ø 3 (卡尔罗斯,目录号:7530.1; CAS号:56-81-5),保存于室温
甘氨酸,NH 2 CH 2 COOH(Sigma-Aldrich,目录号:G8790 ; CAS号:56-40-6),存放在RT                                         
潮霉素B溶液,CELLPURE ® 50毫克/毫升,无菌,C 2 0H 37 Ñ 3 ö 13 (卡尔罗斯,目录号:CP12.2 ; CAS编号:31282-04-9),保存于4℃下                                                       
氯化氢,HC 升,(Sigma-Aldrich公司,目录号:H1758-100ML ; CAS号:7647-01-0),保存于室温
七水合硫酸亚铁(FeSO 4 ·7H 2 O)(Sigma-Aldrich ,目录号:F8263 ;CAS 号:7782-63-0),在室温下存放                                                       
卡那霉素硫酸盐一水合物,C 18 H 36 N 4 O 11 · H 2 SO 4 · H 2 O(Duchefa ,目录号:K0126; CAS号:25389-94-0),储存在4°C
Kinetin,C 10 H 9 N 5 O(Sigma-Aldrich ,目录号:K3378 ; CAS号:525-79-1),在4°C下存储
L-精氨酸,H 2 NC(= NH)NH(CH 2 )3 CH(NH 2 )CO 2 H (Sigma-Aldrich ,目录号:A8094 ; CAS号:74-79-3),存放在RT                                         
L-天冬氨酸,HO 2 CCH 2 CH(NH 2 )CO 2 H (Sigma-Aldrich ,目录号:A7219 ; CAS号:56-84-8),存放在RT                           
L-谷氨酰胺,H 2 NCOCH 2 CH 2 CH(NH 2 )CO 2 H (Sigma-Aldrich ,目录号:G8540 ; CAS号:56-85-9),在室温下保存                                                       
液氮
L-脯氨酸,C 5 H 9 NO 2 (Sigma-Aldrich ,目录号:P5607; CAS号:147-85-3),存放在RT                                         
氯化镁,MgCl 2 (Sigma-Aldrich;目录号:M8266; CAS号:7786-30-3),存放在RT
七水合硫酸镁MgSO 4 ·7H 2 O(Sigma-Aldrich ,目录号:M2773; CAS号:10034-99-8),在室温下存放                                                       
一水合麦芽糖,C 12 H 22 O 11 ·H 2 O (Duchefa ,目录号:M0811; CAS号:6363-53-7),在室温下存放                                                       
一水合硫酸锰(II),MnSO 4 ·H 2 O (Sigma-Aldrich ,目录号:M7899; CAS号:10034-96-5),存放在RT                                                       
肌醇C 6 H 12 O 6 (Sigma-Aldrich ,目录号:I7508; CAS号:87-89-8),在室温下存放                                                       
烟酸C 6 H 5 NO 2 (Sigma-Aldrich ,目录号:N0761; CAS号:59-67-6),存放于室温                                         
氯化钾,氯化钾(Sigma-Aldrich公司,目录号:P5405; CAS号:7447-40-7),保存于室温                           
氢氧化钾,KOH(Fisher Scientific ,目录号:10366240; CAS号:1310-58-3),存放在RT
碘化钾KI (Sigma-Aldrich ,目录号:P8166; CAS号:7681-11-0),存放在RT                                         
硝酸钾,KNO 3 (西格玛奥德里奇,目录号:P8291; CAS号:7757-79-1),存放在室温                                         
磷酸氢二钾,KH 2 PO 4 (Sigma-Aldrich ,目录号:P5655; CAS号:7778-77-0),存放在RT                                                       
盐酸吡rid 醇,C 8 H 11 NO 3 ·HCl (西格玛奥德里奇,目录号:P6280 ;化学文摘社编号:58-56-0),在室温保存                                                       
利福平,C 43 H 58 N 4 O 12 (Sigma-Aldrich,目录号:R3501; CAS号:13292-46-1),在4°C下储存
氯化钠NaCl(Sigma-Aldrich ,目录号:S7653; CAS号:7647-14-5),存放在RT
氢氧化钠,NaOH(Sigma-Aldrich ,目录号:30620-1KG-M; CAS号:1310-73-2),存放在RT
二水合钼酸钠Na 2 MoO 4 ·2H 2 O (Sigma-Aldrich ,目录号:331058; CAS编号:10102-40-6),在室温下保存                                                       
磷酸氢二钠NaH 2 PO 4 (Sigma-Aldrich ,目录号:S5011 ; CAS号:7558-80-7),存放在RT                                                       
蔗糖,C 12 H 22 O 11 (Duchefa ,目录号:S0809 ; CAS号:57-50-1),在室温下存放                                                       
盐酸硫胺素,C 12 H 17 ClN 4 OS·HCl(西格玛奥德里奇,目录号:T1270 ;化学文摘社编号:67-03-8),在室温下保存                                                       
七水合硫酸锌ZnSO 4 ·7H 2 O(Sigma-Aldrich ,目录号:Z1001 ; CAS号:7446-20-0),在室温下存放                                                       
FASTAMP ® 工厂直接P CR试剂盒(Intactgenomics ,目录号:4612)
WTW TM TEP 4模型缓冲溶液pH 4(WTW TM ,目录号:108700)
WTW TM TEP 7模型缓冲溶液pH 7(WTW TM ,目录号:108702)
WTW TM TPL 10微量模型缓冲溶液pH 10.1 (WTW TM ,目录号:108805)
水稻土成分(见食谱)
储备液组成(请参见配方)
植物激素和抗生素(请参阅食谱)
栽培培养基成分(见食谱)
悬浮介质(请参见配方)
共培养培养基(请参见食谱)
静置介质(请参见食谱)
选择介质(请参见食谱)
再生前培养基(请参见食谱)
再生培养基(请参见食谱)
生根培养基(请参见食谱)
转换备忘单(请参阅食谱)
GUS染色液(请参阅配方)
X-Gluc解决方案(请参阅食谱)
 


设备


 


注意:设备可以适应不同的实验室条件,但是PETRI DISHES,MAGENTA BOXES是非常重要的工具,会影响转化效率。我们强烈建议使用与该协议相同的品牌。由于经常消毒,因此小刀和镊子必须是高质量的。确切的类型和品牌可能会根据个人选择进行调整。


 


-80°C 超低温冰箱(Panasonic,型号:MDF-U76V-PE)
分析和精密天平(PRECISA Gravimetrics AG ,系列321LS ,米奥德尔:LS 2200C和LS 120A)
高压釜(SYSTE Ç ,型号:3850 EL)
台式pH计 WTW TM inolab TM 7110(供应商:WTW TM 1AA114; Fis her Scientific ,目录号:11731381)
基本型生物光谱仪(Eppendorf ,目录号:6135000009)
5 L桶(Auer ,目录号:ER 5,6-226 + DK)
三角烧瓶中加入250ml (DURAN ® ,目录号:21 216 36)
生长室1  波斯富街,米Odel等:CU-41L5; 条件:30℃,持续光照的200微摩尔米(24/0白天/夜晚光周期)与光合光子通量密度(PPFD)-2 小号-1 与PPFD蓝色的40微摩尔米-2 小号-1 ,PPFD- 80 µmol m -2 s -1 的绿色和70 µmol m -2 s -1的PPFD-红色
生长室2  波斯富街,米Odel等:CU-41L5; 条件:27°C,16/8日/夜光周期,光子通量密度(PPFD)为200 µmol m -2 s -1 ,PPFD蓝色为30 µmol m -2 s -1 ,PPFD绿为70 µmol m -2 š -1 和PPFD-RED 60微摩尔米的-2 小号-1 
玻璃珠灭菌器(SkinMate Apus石英)
高精度镊子(110毫米; 5.SA型;超细尖端;不锈钢)(Behr Labor-Technik,目录号:6.266 876)
电穿孔仪(Bio-Rad Gene Pulser TM )
孵化器/摇床28°C(Infors HT ,Multitron Standard)
接种环,PS 10UL PK / 25 (Hach ,目录号:2749125)
实验室瓶螺旋盖和浇注环,将100ml (杜兰® ,目录号:218012458)
实验室瓶螺旋盖和浇注环250毫升(杜兰® ,目录号:218013651)
实验室瓶螺旋盖和浇注环,将500ml (杜兰® ,目录号:218014459)
实验室瓶螺旋盖和浇注环,1 ,000个ml的(杜兰® ,目录号:218015455)
照度计(量子光谱仪,UPRtek ,PAR 300)
八角形电磁搅拌棒,带枢轴,蓝色,38 毫米(VWR ,目录号:442-0438)
移液器控制器Pipetboy acu 2(供应商:Integra(Biosciences);适用于0.1至100 ml的玻璃和塑料移液器)(VWR ,目录号:613-4437)
聚碳酸酯真空干燥器(Sanplatec ,目录号:PC-250KG)
Pots SM-H Container 2.0 L(Meyer-shop ,货号:737203)
手术刀手柄(供应商:BAYHA GMBH;长度:160 mm )(VWR,目录号:233-5202)
体视显微镜(Zeiss ,Discovery.v8,明场图像)
用于GFP成像的立体变焦和荧光科学显微镜(Zeiss ,AxioZoom.V16)
无菌工作台(热科技TM Heraguard TM ECO净化工作台)
温度计(Laserliner ThermoSpot )
热循环仪(Bio-Rad ,型号:T100 TM ,目录号:1861096)
真空泵(Vacuubrand ,目录号:MZ 2C)
程序


 


准备库存解决方案
准备17种储备溶液,包括N6主剂1,N6主剂2,N6主剂3,N6主剂4,B5主剂1,B5主剂2,B5主剂3,B5主剂4,B5维生素,AA粗盐,AA微盐,甘氨酸, MS 1,MS 2,MS 3,MS 4和MS维生素与COMPO 小号银行足球比赛根据表S2。根据表S3准备植物激素和抗生素储备溶液。
过滤器将所有储备溶液灭菌,并保持在4 °C下。
                            无德小号:


务必对储备溶液进行过滤灭菌(过滤器孔径:0.2 µm,用于过滤器型号的耗材1、2、3、20 和21 ),请勿高压灭菌!
储备溶液可以在4 °C 下保存最多3个月。
请勿使用3个月以上的储备溶液。
 


准备培养基
按照表S4- 表S 11准备培养基。崇拜ivation介质组合物。
不Ë 小号:


正确调整pH值非常关键:在使用前务必校准pH计(对于pH 5.8,请使用pH 4和pH 7的校准溶液)。
始终非常精确地调节pH:对于需要pH 5.8的介质,可接受的pH值为5.80-5.81。
记录pH调节前后的pH 值。
始终在相同的介质温度下测量pH并在测量pH的同时记录介质温度(图3)。
 


D:\ Reformatting \ 2020-7-1 \ 2003199--1495 Wolf Frommer 843004 \ Figs jpg \ Figure03.jpg


图3. pH测量。A.在制备培养基时记录pH和温度。B.记录pH值时电极正确位置的细节。C.在显示屏上记录温度和pH值。


 


加入所有组分后测量pH,除了琼脂糖,植物激素和抗生素(小号EE 表S4- 表小号11 ˚F 或更多的细节),高压灭菌介质之前。
对于灭菌介质,必须使用短时间的高压灭菌循环:在121°C(101 kPa)下施加5分钟(!)的灭菌时间,并在灭菌器冷却至95°C后立即将介质转移至RT ,不要让介质在高压釜中停留更长的时间(图4)。灭菌时间取决于培养基的量,因为高压灭菌过程包括加热和冷却所需的额外时间。在此方案中,我们将培养基装在0.5 L的瓶子中,并在121°C(101 kPa)下施加5分钟的灭菌时间,整个高压灭菌周期为1.5 h。
 


D:\ Reformatting \ 2020-7-1 \ 2003199--1495 Wolf Frommer 843004 \ Figs jpg \ Figure04.jpg


图4 。在121 °C (101 kPa)下高压灭菌5分钟(A)和20分钟(B)的培养基的比较。焦糖色素是一个很好的迹象的过度灭菌介质。高压灭菌后,介质颜色应几乎保持无色,如左图所示。


 


仅在培养基冷却至40-45°C(始终使用Laserliner 温度计检查温度!)后,高压灭菌后添加植物激素和抗生素。
将灭菌的工作台(生物安全柜)下板,请参阅表S4为类型P ETRI菜肴和量倾为每个平台。类型P ETRI菜迪菲租金不同的媒体和关键是要按照我们的建议。
仅在板完全干燥并且在P etri盘侧面看不到水凝结时才关闭盖(图5)。这是至关重要的,因为愈伤组织更喜欢在培养基上保持干燥,而湿的愈伤组织则不会再生。而且,不要过度干燥。过度干燥的介质会收缩并形成裂纹,由于含水量减少,溶质浓度会大大增加,从而影响敏感的愈伤组织和再生过程。
将板用拉伸箔包裹,并在室温下避光保存(图5)。不要冷藏媒体。
2周后用固体培养基弃去未使用的板。
 


D:\ Reformatting \ 2020-7-1 \ 2003199--1495 Wolf Frommer 843004 \ Figs jpg \ Figure05.jpg


图5.培养基干燥和储存。A.完全干燥P ETRI菜。B.湿法P ETRI菜,冷凝水; 黑色箭头表示冷凝水。C.将板包装并在室温和黑暗中储存。


 


合格的农杆菌制备和转化
准备农杆菌(LBA4404或EHA105)感受态细胞。
              条纹出农杆菌从10%甘油储备细胞与固体YEP培养基含有板(贮存在-80℃)20毫克/升利福平和incubat e计算在2天内的黑暗中28℃。
用无菌的牙签或移液器吸头挑选单个菌落,并在玻璃试管中用含有20 mg / L利福平的5 ml YEP液体培养基在黑暗中和28°C下过夜(12至16 h)培养,并于200°C摇动转速
在250 ml烧瓶中将1 ml过夜培养物加入35 ml液体YEP培养基和20 mg / L利福平中,在28°C下孵育黑暗,并以200 rpm摇动约8 h,直到OD 600 达到0.5-0.8。
在冰上冷却农杆菌5分钟。
离心机在50ml猎鹰细菌® 管以3,000 X 克,4℃ 下进行5分钟。
重悬沉淀在1ml 的20mM的CaCl 2 在50ml 猎鹰® 管(用于50毫升YEP,加入1ml的1M的CaCl 2 )。
等分试样(50 微升)在一个2毫升安全锁微量离心管中,˚F 在-80℃下用液氮和存储间隙冻结。
用GUS / GFP报告基因构建物转化感受态农杆菌细胞(LBA4404或EHA105)。
从10%的甘油储备液(储存在-80°C)中取出等分试样(50μl)合格的根癌农杆菌LBA4404或EHA105,并在冰上融化10分钟。
在1.5 ml微量离心管中将细胞与1μl(〜100 ng)质粒DN A混合。
将混合物填充到无菌的冰冷电穿孔比色皿中(间隙为0.2 cm)。
使用电穿孔器以1.8 kV进行电穿孔。按直到“嗡嗡”的声音可以听到(约5 毫秒),保留所有其他设置为默认值:200 Ω,电容延长250 μFD ,电容25 μFD 。
脉冲后立即添加1 ml 不含抗生素的YEP 培养基,使用1 ml 移液器吸头上下吸液混合细胞。
将悬浮液转移到高压灭菌的1.5 ml微量离心管中,并在28°C下孵育30分钟,不要摇动。
以21 0 x g将细胞旋转1分钟。
小心除去上清液,但在1.5 ml微量离心管中保留约100μl培养基。
重悬在剩余的培养基中的细胞在1.5ml微量离心管中和PLA Ç Ë100微升上含有50mg / L卡那霉素和YEP琼脂平板20毫克/ L利福平。
在黑暗中于28°C孵育平板2天。
进行菌落PCR,通过检查选择标记基因的存在来选择转化菌落。在我们的情况下,确认的存在转移酶基因(次黄嘌呤磷酸核糖转移酶基因编码的),我们使用下面的引物:Hpt_F :5 ' -AGCCTGACCTATTGCATCT-3 ' ; Hpt_R :5 ' -CATATGAAATCACGCCATGT-3 ' ,扩增子大小为200bp,T 米55℃。
选择1-3个阳性菌落,并在玻璃试管中接种含50 mg / L卡那霉素和20 mg / L利福平的3 ml液体YEP培养基。在28℃下生长的细菌中的d 柜以200rpm振摇过夜速度。
通过将0.6 ml培养物与0.3 ml 10%甘油(高压灭菌)在2 ml安全锁微量离心管中混合来制备Hpt 阳性农杆菌培养物的甘油储备,立即在液体N 2中冷冻并储存在-80°C。
 


注意:在这项研究中,我们使用了GUS rep ter构建体(pSWEET13:GUSplus)和GFP报告基因构建体(pOsUbi:eGFP )(图6),它们由Joon- Seob Eom 博士(Heinrich Heine University,杜塞尔多夫,庆州)友情提供。韩国熙大学)和杨冰博士(美国密苏里大学)。从其他实验中,我们知道在pSWEET13和OsUbi 启动子下,GUS和GFP在愈伤组织和幼苗阶段均表达,因此我们使用这两种构建体检查转化效率。出于同一目的,可以使用任何其他GUS / GFP报告基因构建体。最佳的方法是使用GUS或GFP内含子构建体,因为土壤杆菌不会剪接内含子,因此不会引起假阳性愈伤组织或再生植物(Vancanneyt 等,1990)。


 


              D:\ Reformatting \ 2020-7-1 \ 2003199--1495 Wolf Frommer 843004 \ Figs jpg \ Figure06.jpg


图6. 本研究中使用的两个带有GUS和GFP报告基因的质粒的图谱


 


ISO 未成熟胚的特征研
收获稻未成熟种子(8-12天后授粉)和地点300-400种子在50ml猎鹰® 管。在正确的发育阶段获得未成熟的种子非常重要。要检查未成熟胚的阶段,请轻轻挤压未成熟的种子并感觉其硬度。未成熟的种子应处于乳白色晚期,此时胚乳尚未完全细胞化,有关更多详细信息,请参见视频1。该收获步骤可以在无菌罩外进行,但以下所有步骤(包括脱壳,灭菌,未成熟胚的分离)均在无菌条件下进行。
 


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影片o 1 。未成熟种子的选择


 


在无菌条件下,洗涤种子用40毫升70%乙醇30秒与手在50ml猎鹰摇动® 管; d iscard乙醇。然后用40毫升无菌Milli-Q水清洗种子3次,弃去水。
将未成熟的种子穿入培养皿中,该培养皿上铺有湿润的无菌滤纸,并在立体显微镜下和在无菌条件下(在层流室中)用手术刀和镊子小心地除去种皮(内膜和内膜)(视频2,图) 7A-7B)。手术刀和镊子应通过使用例如玻璃珠灭菌器进行灭菌。需要冷却工具,以免热损伤未成熟的胚胎。
 


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                            参见ø2. d 未成熟种子的ehusking


 


放置未成熟在无菌条件下的种子在50毫升猎鹰® 管(一次稃和内稃已被去除)来启动灭菌工序(图7C)。
加40毫升乙醇中的70%,在无菌条件下,向猎鹰® 管,并用手30秒摇动。丢弃乙醇。用40 ml无菌Milli-Q水洗涤未成熟的种子。丢弃水。
在一个50ml无菌猎鹰® 管,准备5毫升商业漂白剂的混合物Klorix ® 用35毫升无菌Milli-Q水[ 终浓度的NaClO 为28克/升(0.376 M)] 。
该混合物添加到第È50毫升猎鹰® 含有未成熟的种子管,并用手5分钟摇动。
丢弃Klori X ® 混合物并用40ml无菌Milli-Q水冲洗种子,重复该过程15次,直到所有残留Klorix ® 被完全洗出。
注:这是非常重要的Klorix ® 完全淘汰,因为Klorix ® 会影响未成熟胚的萌发。我们建议在冲洗一个未成熟胚50毫升猎鹰® 管15倍用40毫升无菌Milli-Q水各添ë 。


地方未成熟种子到培养皿与使用立体显微镜下解剖刀和细镊子无菌滤纸,并仔细分离未成熟胚(IE)的(视频3,连接在无菌罩古尔7D)。请小心轻放,以免损坏或伤害未成熟的胚胎!
 


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录像3 。未成熟的胚胎分离


 


仅选择大小在1.3-1.8毫米之间的未受损未成熟胚。不成熟的胚胎应该是不透明的灰白色。透明的未成熟胚不会发芽。将约50个未成熟的胚胎(盾片朝上)放在共培养培养基上(图7E)。
 


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图7 。农杆菌未成熟胚的分离和接种。A.在立体显微镜下去除内脏和外。B.脱壳未成熟的种子在P ETRI用湿的Whatman菜® PAP ER。比例尺:1 厘米。C.种子消毒用乙醇和Klorix ® 。D.在板上分离出的未成熟胚。E.盾片向上的未成熟胚(白色箭头),侧面未成熟的胚。比例尺:1 毫米。˚F 。农杆菌接种。


 


              转型与共同培养
注意小号:


通常,我们建议使用100个未成熟胚进行一次转化。取决于转化效率,可以减少或增加作为原料的未成熟胚的数量。
我们建议使用未接种农杆菌的12个未成熟胚作为对照。在第一个选择步骤中,将6个未成熟对照胚移至没有潮霉素B的选择培养基中,以检查其再生能力。将其他6个对照未成熟胚移入含有30 mg / L潮霉素B的选择培养基中,以控制潮霉素B选择的有效性。这些是非常重要的控制措施,尤其是在尝试使此转化方案适应其他品种时。潮霉素B的浓度,再生培养基和接种时间可能必须适用于其他品种。
我们还建议记录所有数据,每个转化,例如日期,幼胚的数量以及任何注释(S EE 表S12为模板)。
 


在感染前两天,在含有抗生素(50 mg / L卡那霉素和20 mg / L利福平)的固态YEP平板上,从冷冻的-80°C储存中罢工土壤杆菌(LBA4404或EHA105)。然后在黑暗中于28°C孵育2天。
上转换的天,取3毫米大小的环的农杆菌培养物从YEP板和在悬浮介质中悬浮(表S5)我n中的50毫升猎鹰® 管。
涡旋细菌悬浮液并调节至OD 600 0.3。在黑暗中于25°C孵育1小时,无需摇动。
无德小号:


                     在使用前添加乙酰丁香酮并准备新鲜!
溶解乙酰丁香酮:50 ml的悬浮液培养,溶解10mg的乙酰丁香酮在100 微升DMSO中,然后取9.81微升溶液中,得到0.981毫克乙酰丁香酮; 无需过滤器灭菌。
始终非常精确地调整OD 600 :根据光谱仪,推荐值在0.30和0.31 之间。
将5μl 农杆菌悬液滴在每个未成熟胚的顶部,盾片朝上(图7F)。
在土壤杆菌存在下,在黑暗中于25°C在黑暗中孵育带有胚胎的平板7天(图8)。
 


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图8.共培养阶段结束时的未成熟胚。AC。控制无农杆菌感染的愈伤组织。DF。农杆菌感染的愈伤组织。






                            休息中
检查未成熟的胚胎是否可能受到污染(图9)。如果未成熟的胚胎受到污染,请将其丢弃。如果可能的话,可以拯救受污染平板中未受污染的未成熟胚,并将其放在单独的平板上。否则丢弃整个盘子。Ç 如果dehusking或未成熟胚隔离却没有这样做足够仔细和胚的过程期间用钳子或手术刀损坏,便于与其他微生物(图9)的污染可能发生ontamination。
 


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图9.未成熟胚胎的污染。A.对照显示未成熟胚(无农杆菌接种)显示细菌污染。B. 农杆菌接种的未成熟胚显示出被其他细菌污染。C.对照无感染的未成熟胚而无污染。D. 农杆菌接种的未成熟胚,无污染。


 


将未受污染的胚胎放置在无菌滤纸上,并用手术刀和镊子除去芽。执行此立体声下步骤- 显微镜E要保证完整切除芽。
一个。不应遗留任何枝条和边缘部分,将其全部除去(图10A和10 B)。       


b。如果多余,请从愈伤组织中取出棕色组织,但要注意不要损坏未成熟的胚胎。      


 


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图10.(A)之前和之后(B)去除芽的未成熟胚。箭头指示要去除的部分:芽(左下方),边缘(右下方)和棕色组织(上)。C.使用两层无菌滤纸清洁未成熟的胚胎。


 


通过将清洁的未成熟胚他们的无菌过滤器的两层之间的纸张(图10 C),并仔细DAB 关闭农杆菌。重复此过程至少两次,以去除多余的土壤杆菌。
转印未成熟胚盾片侧向上到停留mediu 米(16个未成熟胚/板)并孵育在生长室5天1  30℃,连续光照,24/0白天/夜晚光周期,光合光子通量密度(PPFD )200 µmol m -2 s -1,其中PPFD蓝为40 µmol m -2 s -1 ,PPFD绿为80 µmol m -2 s -1 ,PPFD红为70 µmol m -2 s -1 ] (图11)。
 


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图11.静止期结束时的未成熟胚胎。AC。控制未成熟的胚胎。DF。农杆菌接种的未成熟胚。


 


无德小号:


使用体视显微镜检查未成熟胚胎的状态(例如,是否有任何污染),清洁棕色组织并更轻柔地取出未成熟胚胎。
不要将未成熟的胚胎推入培养基中,让它们松散地停留在培养基顶部。
在培养基之间转移时(半盾板朝培养基上方),将未成熟的胚胎保持在相同的位置。
用两层Micropore 3M胶带密封板。
 


                            选拔
休息5天后,用镊子和手术刀彻底刮掉棕色组织,从未成熟胚胎的表面刮擦或切下棕色组织。将未成熟的胚胎转移到含有30 mg / L潮霉素B (16愈伤组织/ 平板)的选择培养基上。孵育在30℃下,在连续光照10天[ 200微摩尔米24/0白天/夜晚光周期,光合光子通量密度(PPFD)-2 小号-1 与PPFD蓝色的40微摩尔米-2 小号-1 , PPFD绿色的80微摩尔米-2 小号-1 和PPFD-RED 70微摩尔米的-2 小号-1 ] (1 ST 选择)(图12)。
 


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图12.在第一个选择阶段结束时的未成熟胚胎。A,D。控制没有潮霉素的未成熟胚B,未成熟胚产生致密的胚性愈伤组织并开始变绿。B,E。在含有30 mg / L潮霉素B的选择培养基上控制未成熟胚。未成熟胚不能完全变成褐色,也不能生长。C,F。农杆菌接种的未成熟胚可能部分或完全变成褐色。


 


后1 10天ST 的SelectIO n时,愈伤组织转移到与选择培养基新鲜制备的板(16 immat URE 胚/皿)。在30℃下,在连续光照10天除去棕色组织尽可能从未成熟胚,并培育可能[ 24/0白天/夜晚光周期,光量子通量密度200微摩尔的(PPFD)米-2 小号-1 与PPFD蓝40微摩尔的米-2 小号-1 ,PPFD绿色80微摩尔的米-2 小号-1 和PPFD-RED的70微摩尔米-2 小号-1 ] (2 次选择)(图13)。第一次选择后,一些未成熟的胚胎会完全变成褐色。在这种情况下,请勿去除棕色组织并保留整个未成熟的胚胎。
 


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图13. 第二选择阶段后的未成熟胚胎。A,D。对照没有潮霉素的未成熟胚B产生增殖的胚性愈伤组织。B,E。用30mg / L潮霉素控制未成熟胚。B,未成熟胚保持其大小(2-4mm),而不产生任何愈伤组织。C,F。在选择培养基上接种农杆菌的未成熟胚产生小的,致密的和浅黄色的胚性微愈伤组织(黑色箭头)。


 


后的210天第二选择,只需要在EMB ryogenic microcalli从黑色未成熟胚和地点到新鲜的选择培养基(16个未成熟胚/皿),温育在30℃下在连续LIG10天HT [ 24/0天/夜间光周期,光合通量密度(PPFD)为200 µmol m -2 s -1,其中PPFD蓝为40 µmol m -2 s -1 ,PPFD绿为80 µmol m -2 s -1 ,PPFD-红色的70微摩尔米-2 小号-1 ] (3 次选择)(图14)。胚性愈伤组织是小的,紧密的细胞簇,通常为浅黄色(图13 F)。非胚性愈伤组织通常是较大的,柔软的,半透明的和黄色或灰色的细胞松弛簇。如果新鲜分离的微愈伤组织不生长或需要更多的微愈伤组织,则可以将“未成熟的母亲胚”作为选择保存在选择培养基上。对于此步骤,通过将P etri盘分成小区域,分别保存来自不同未成熟胚的微愈伤组织(图14 C)。分离的微迹将被视为独立事件。
 


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图14. 第三选择阶段结束时的微调。A,D。不含潮霉素的对照愈伤组织B是致密的,胚胎发生的并且部分变为绿色。B,E。用30mg / L潮霉素控制未成熟胚。B,未成熟胚保持相同大小并且不生长。C,F。农杆菌接种的微愈伤组织在选择培养基上增殖。


 


不Ë 小号:


使用体视显微镜检查未成熟胚/愈伤组织的状态,去除棕色组织并更精确地选择胚性愈伤组织。
不要将未成熟的胚/愈伤组织推入培养基中,让它们松散地停留在培养基顶部。
用两层Micropore 3M胶带密封板。
如果第二次选择后未产生微愈伤组织,请刷新选择培养基并再培养10天。如果再经过2-3轮选择后仍未产生微愈伤组织,则丢弃未成熟的胚。
 


植物再生
将抗性愈伤组织转移至含有30 mg / L潮霉素B(6-9愈伤组织/板)的再生前培养基中,并在连续光照下于30°C孵育10天[ 24/0日/夜光周期,光合光子通量密度(PPFD) )200 µmol m -2 s -1,其中PPFD蓝为40 µmol m -2 s -1 ,PPFD绿为80 µmol m -2 s -1 ,PPFD红为70 µmol m -2 s -1 ] (图15)。
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图15。再生前阶段结束时的愈伤组织。A,D。不含潮霉素的对照愈伤组织B大多是绿色的。B,E。用30mg / L潮霉素B控制未成熟胚;未成熟的胚胎保持其大小并且不生长。C,F。农杆菌接种的增殖的愈伤组织带有绿点。


 


选择带有绿色斑点的增殖愈伤组织,其覆盖大约2/3的愈伤组织(图15F),并转移到含有30 mg / L潮霉素B(6愈伤组织/板)的再生培养基中,用于芽发育,并在30°C下孵育14在连续光天[ 24/0白天/夜晚光周期,光合光子通量密度为200微摩尔米(PPFD)-2 小号-1 与PPFD蓝色的40微摩尔米-2 小号-1 ,PPFD绿色的80微摩尔米-2 š -1 和PPFD-RED 70微摩尔米的-2 小号-1 ] (图16)。
 


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图16.再生阶段结束时的愈伤组织。A.从没有潮霉素BB的对照愈伤组织中再生的植物含有30 mg / L潮霉素B的对照未成熟胚,未成熟胚没有生长。C. 农杆菌接种的愈伤组织的再生植物是推定的转化体。


 


如果愈伤组织未产生任何芽或仅产生小芽,则每14天将愈伤组织转移至新鲜制备的再生培养基板(每板6个愈伤组织)中,并在30°C下连续光照[ 24/0日/夜,光合通量密度(PPFD)为200 µmol m -2 s -1,其中PPFD蓝为40 µmol m -2 s -1 ,PPFD绿为80 µmol m -2 s -1 ,PPFD红为70 µmol m -2 s -1 ] ,直到形成小植株(从具有至少两片叶子和小根系的愈伤组织再生的小水稻植株,图16-17)。
从每个愈伤组织中选择1-3株小植株,并转移到装有不含有潮霉素B的生根培养基的Magenta TM GA-7容器中。将它们在27°C下孵育7天[ 具有光子通量密度(PPFD)的16/8天/夜光周期200微摩尔的米-2 小号-1 与PPFD蓝30微摩尔的米-2 小号-1 ,PPFD绿色的70微摩尔米-2 小号-1 和PPFD-RED 60微摩尔米的-2 小号-1 ] 。在此阶段,从不同愈伤组织再生的幼苗被认为是独立的转化事件,因此,以一株以上的幼苗作为后备,以确保所有推定的事件在生根步骤中均能旺盛生长。
7天后,当小植株到达Magenta TM 盒子的顶部时,将另一个Magenta TM 盒子放在顶部,为植物生长腾出更多空间(图17A)。将' double'Magenta TM 盒子放在27° C [ 16/8日/夜的光周期中,光子通量密度(PPFD)为200 µmol m -2 s -1 ,而PPFD蓝色为30 µmol m -2 s -1 ,PPFD绿色的70微摩尔米-2 小号-1 和PPFD-RED 60微摩尔的米-2 小号-1 ] 另外7天。
转移约 轻轻洗掉附着在根部的多余琼脂糖,将其浸入土壤1 5厘米高。在玻璃盆中种植小植株,然后将其种植在较大的水桶中,以保持玻璃屋中的高湿度条件,该玻璃屋具有自然日光和额外的8/16日/夜光周期的日光,白天温度为30°C±2°C,晚上温度为25°C±2°C。相对湿度在50-70%之间。植物在200微摩尔米的光合有效辐射(PAR)或光合光子通量密度(PPFD)下生长-2 小号-1 与PPFD蓝40微摩尔的米-2 小号-1 ,PPFD绿色的70微摩尔米- 2 s - 1 和80 µmol m -2 s -1的PPFD-red 。
 


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图17.生根和种植。A,B。两周龄的再生植物,然后转移到土壤中。C.种植后立即再生植物。D.种植后两周再生的植物。


 


不Ë 小号:


使用体视显微镜检查愈伤组织/小植株的状态,选择愈伤组织并更轻柔地分离小植株。
不要将愈伤组织推入培养基中,让它们松散地放在培养基上。
密封P ETRI菜肴或品红TM GA-7船只用微孔3M胶带的两个层。
由于小植株是在连续光照(24/0日/夜)下再生的,因此我们建议在长日照条件(16/8日/夜)下使小植株长出生根步骤,以使其适应即将到来的短期生长温室的白天条件(8/16天/夜的光照周期)。
筛选转化植物
注意:在此协议中,我们并未明确确定转换事件。我们使用平行再生但未受到土壤杆菌感染的植物作为对照。在这些对照植物中,我们没有观察到GUS染色或GFP荧光。此处计数的所有转化事件均来自独立的未成熟胚(每转化一个未成熟胚中一株植物)。


 


100个Komboka未成熟胚的GUS内含子报道构建使用两个迪菲与转化租金农杆菌菌株(EHA105和LBA4404)导致14 个31 从独立的未成熟胚,分别推定的转化体(表1)。测试所有再生的植物的GUS活性。再生植物的所有叶子均显示出GUS活性(图18A-18B),除了对照植物也经历了整个实验过程,但没有农杆菌感染(图1 8C)。我们的结果可能表明,与EHA105相比,LBA4404菌株在转化Komboka方面更有效,但是如果不对许多独立转化进行仔细的定量分析,就无法判断不同农杆菌菌株的相对效率。
 


表1. 使用不同农杆菌菌株的Komboka的表观转化效率


构建体/ 农杆菌菌株


#未成熟的胚胎


#推定的独立事件


#GUS积极事件


GUS / LBA4404


100


31


31


GUS / EHA105


100


14


14


 


D:\ Reformatting \ 2020-7-1 \ 2003199--1495 Wolf Frommer 843004 \ Figs jpg \ Figure18.jpg


图18.转化的Komboka叶片的GUS组织化学。A,B.从两个独立的未成熟胚再生的两种植物的GUS染色叶。C.来自一株对照植物的GUS染色的叶子在没有农杆菌感染的情况下再生,没有可检测的GUS活性。


 


                            使用农杆菌属菌株LBA4404 用GFP 报告基因构建体转化Komboka 导致54个独立事件(表2)。所有这54株植物的根均为GFP阳性(图19A-19E)。在未感染的植物中未观察到GFP荧光(图19F)。从54个GFP阳性事件中,通过PCR 从48个植物中扩增了200bp GFP基因片段(图20)。完整的T-DNA插入,遗传和拷贝数仍有待验证。
 


              表2 。GFP报告基因构建Komboka的Ap 亲本转化效率


构造/


农杆菌菌株


#未成熟的胚胎


#推定的独立事件


#GFP阳性事件


#PCR阳性事件


GFP / LBA4404


100


54


54


48


 


D:\ Reformatting \ 2020-7-1 \ 2003199--1495 Wolf Frommer 843004 \ Figs jpg \ Figure19.jpg


图19。转化的Komboka的GFP荧光。AE。五个独立转化子的根在蓝光下。F.从组织培养物再生的未感染植物的根(对照)。比例尺:2000m 。所有照片均以相同的设置拍摄和显示。


 


D:\ Reformatting \ 2020-7-1 \ 2003199--1495 Wolf Frommer 843004 \ Figs jpg \ Figure20.jpg


图20.通过PCR确认转化植物中200bp GFP基因片段的代表性凝胶图片。WT:从组织培养中再生的未感染植物。


 


调整其他水稻品种的转化方案
为了使该方案适用于其他品种的转化,我们建议使用GUS或GFP内含子报告子构建体评估每个步骤后的效率(Vancanneyt 等,1990)。例如,在共培养后,使用五个愈伤组织进行GUS染色或GPF筛选,以测试共培养步骤是否成功或是否需要调整共培养时间。


  我们也建议使用非感染的未成熟胚作为对照,以检查潮霉素B选择的有效性以及关于媒体再生能力不含潮霉素B(参见Ñ OTE 2,小号挠度E),因为潮霉素B的敏感性和再生能力品种之间可能有所不同。潮霉素B的浓度可在5至50 mg / L 之间调整。调整的标准包括:控制未成熟的胚不能在含有潮霉素B的培养基上生长或产生微愈伤组织(图s 13、14、15)。


  另一个相关的参数是不成熟胚发育的阶段,不同品种之间可能有所不同。未成熟的种子应处于乳白后期(视频1 ),通常在授粉后约8-12天大小为1.3- 1.8 mm。可以比较农杆菌菌株(AGL 1,LBA4404,EHA105)。同样,选择的回合数和预生成步骤的回合数可在1-3之间调整,直到产生微愈伤组织或绿色斑点。


 


数据分析


 


GUS染色
                                                                                                          收获3厘米叶子和在15ml地方猎鹰® 管。
                                                                                                          加入5 ml染色溶液(表S13和S14)。
                                                                                                          在室温下将样品抽真空10分钟。
                                                                                                          关闭管,在黑暗中于37°C下温育。
                                                                                                          除去染色溶液。
                                                                                                          加入70%的乙醇进行脱色(去除叶绿素)并在65°C下孵育样品。孵育时间影响脱色,组织孵育的时间越长,蓝色染料与背景的对比度越高,通常2-3天即可。在此孵育时间内,将70%的乙醇交换1或2次
                                                                                                          只要组织保留在乙醇中,就可以在室温下保存很长时间(至少几个月不丢失颜色)。建议将样品存放在黑暗的地方。
 


GFP成像
用Zeiss AxioZoom.V16体视显微镜在蓝光下观察到GFP转化的Komboka的根,滤光片激发波长:450-490 nm,滤光片发射波长:500-550 nm,激发波长:488 nm,发射波长:509 nm,曝光时间4.59 s,变焦:0.7,总光学倍率:7倍。所有照片均以相同的设置拍摄和显示:黑色:2000,伽玛:1.0,白色:22897。


 


GFP编码基因的PCR
以检查GFP基因在再生植物的存在,FASTAMP ® 植物直接PCR试剂盒(Intactgenomics )被用于扩增GFP使用下列引物编码区域的200bp的片段:VL_GFP_F1 5 ' -GCAAGCTGACCCTGAAGTTC-3 ' ,VL_GFP_R1 5 ' -GTCTTGTAGTTGCCGTCGTC-3 ' 。PCR条件:最初的变性步骤是在95°C下进行5分钟,然后在95°C下进行35个循环,持续30 s,在55°C下进行30 s,在72°C下进行20 s,最后在72°C下延伸10分钟并将反应保持在10℃。


 


菜谱


 


注意:有关配方,请参见补充文件(表S1-S14)。


水稻土成分(表S1 )
储备液组成(表S2 )
植物激素和抗生素(表S3 )
栽培培养基组成(表S4 )
悬浮介质(表S5 )
C- 培养培养基(表S6 )
静止介质(表S7 )
选择介质(表S8 )
再生前培养基(表S9 )
再生培养基(表S10 )
生根培养基(表S11 )
转换备忘单(表S12 )
GUS染色液(表S13 )
X-Gluc解决方案(表S14 )
 


致谢


 


非常感谢Bill and Melinda Gates基金会,Deutsche Forschungsgemeinschaft(DFG,德国研究基金会)根据德国的卓越战略– EXC-2048 / 1 –项目ID 390686111,以及Alexander von Humboldt教授的资助提供了资金。感谢IRRI提供的Komboka种子,Bing Yang (美国密苏里大学)和Joon S. Eom (美国Heinrich Heine大学,杜塞尔多夫,韩国庆熙大学)提供了GFP和GUS的报告基因。感谢Marina Manzanilla在IRRI基因转化实验室的培训。感谢Monika Streubel 和Joon S. Eom 在植物转化中的宝贵建议。


 


利益争夺


 


德国杜塞尔多夫海因里希海涅大学(HHU)分子生理研究所,哥伦比亚卡利热带农业中心(CIAT)和菲律宾国际水稻研究所(IRRI)对协议的发展做出了同样的贡献。作者宣称没有利益冲突。






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引用:Luu, V. T., Stiebner, M., Maldonado, P. E., Valdés, S., Marín, D., Delgado, G., Laluz, V., Wu, L., Chavarriaga, P., Tohme, J., Slamet-Loedin, I. H. and Frommer, W. B. (2020). Efficient Agrobacterium-mediated Transformation of the Elite–Indica Rice Variety Komboka. Bio-protocol 10(17): e3739. DOI: 10.21769/BioProtoc.3739.
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