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Transient Transformation of Maize BMS Suspension Cells via Particle Bombardment

LZ Liang-Zi Zhou
Thomas Dresselhaus Thomas Dresselhaus

Published: Dec 5, 2019 DOI: 10.21769/BioProtoc.3451 Views: 3821

Edited by: Marisa Rosa

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Abstract

Maize is one of the most important crop species and serves also as a model plant for grass research. A major bottleneck in maize research is stable transformation, which is both time and cost consuming, but also a technical challenge for most labs due to limited access to sufficient and optimal plant growth facilities. However, many studies in maize cell biology, physiology and biochemistry don’t require stable transformed plants and can be accomplished by using transiently transformed suspension cultures. Here, we report a detailed protocol to establish Black Mexican Sweet (BMS) maize suspension cell cultures and transiently transform it via particle bombardment. We demonstrate how reliable subcellular protein localization data can be obtained within a very short time period and analyzed using open access software.

Keywords: Particle bombardment search Transient transformation search BMS suspension cells search Co-localization analysis search Maize search

Background

Maize is indispensable for producing food, animal feed, industrial products and biofuels. Thus, continuous research to improve its germplasm will be fundamental to meet increasing global demands under challenging environmental conditions (Shiferaw et al., 2011). Moreover, maize has also served as a genetic system and model species for grass research for over a century (e.g., Dresselhaus et al., 2011). The availability of the first genome sequence of the maize inbred line B73 in 2009 (Schnable et al., 2009) and reference genomes of additional inbred lines in successive years (e.g., Jiao et al., 2017; Sun et al., 2018) further fueled its usage as a model and crop plant. Unfortunately, functional studies of maize genes are still difficult and only a few academic labs have established efficient maize transformation systems. Limitations include access to state-of-the-art growth facilities required to achieve high and efficient transformation rates. Although highly efficient transformation methods have been reported recently (e.g., Anand et al., 2019), its routine use is restricted to labs experienced in maize transformation that possess also access to excellent plant growth facilities.

However, stable maize transformation is not always necessary as a number of analyses including primary studies using large sets of candidate genes can also be done using cell cultures. Subcellular localization analysis of candidate proteins, for example, is essential to understand gene functions. The majority of studies investigating the subcellular localization of maize proteins used heterologous systems including onion epidermal cells as well as leaves and suspension cells of tobacco (e.g., Amien et al., 2010; Juranić et al., 2012). These systems might lack important components for proper protein localization and often form large aggregates likely due to mis-folding of heterologous proteins (e.g., Srilunchang et al., 2010). Here, we describe a transient transformation system that uses a non-embryogenic maize BMS (Black Mexican Sweet) suspension cell culture (Spencer et al., 1990). Since its development in 1979, various studies have used this approach to investigate the subcellular (co-) localization of multiple candidate proteins (e.g., Uebler et al., 2015), among other applications. We detail the procedures to culture BMS cells on solid and in liquid medium, to prepare plasmids coated with gold particles, to bombard cells, and to image and analyze them using open-access software packages. These methods can be used to establish stable BMS cultures comparable to Arabidopsis PSB-D suspension cultures, which have been shown to be valuable, for example, for co-localization studies and biochemical assays to identify protein, RNA and DNA interaction partners of stable transformed candidate proteins (e.g., Antosz et al., 2017).

Materials and Reagents

  1. Aluminum foil (Roth, catalog number: 2596.1)
  2. Microscope Slides (VWR, catalog number: 631-1552)
  3. Coverslips (VWR, catalog number: MENZBBAD024004SC13)
  4. 25 ml glass flask (VWR, catalog number: 214-0248)
  5. 1.5 ml microcentrifuge tubes (Sarstedt, catalog number: 72.706.400)
  6. Petri dish 92 x 16 mm with cams (Sarstedt, catalog number: 82.1473)
  7. Petri dish 60 x 15 mm with cams (Sarstedt, catalog number: 82.1194.500)
  8. Parafilm (VWR, catalog number: 291-0057)
  9. Corning cell strainer 40 μm (Sigma-Aldrich, catalog number: CLS431750-50EA)
  10. Sterile syringe filter, pore size 0.22 μm (Berrytec, catalog number: 1302601)
  11. 12 ml Syringe (Norm-Ject, catalog number: 4100-000V0)
  12. 1100 psi Rupture disks (Bio-Rad, catalog number 1652329)
  13. 1.0 μm Gold microcarriers (Bio-Rad, catalog number: 1652263)
  14. Macrocarriers (Bio-Rad, catalog number: 1652335)
  15. Macrocarrier holders (Bio-Rad, catalog number: 1652322)
  16. Stopping screens (Bio-Rad, catalog number: 1652336)
  17. BMS cell line (Arabidopsis Biological Resource Center ABRC, stock: CCL84842; see also Spencer et al., 1990) 
  18. proZmUbi:ZmEA1-GFP plasmid (from Uebler et al., 2015)
  19. pro2x35S:ER-mCherry (ABRC, stock: CD3-959; see also Nelson et al., 2007)
  20. Ethanol (Sigma-Aldrich, catalog number: 32205-1L-M)
  21. Glycerol (Sigma-Aldrich, catalog number: G9012-2L)
  22. Double distilled water (ddH2O), produced by MilliporeSigma Milli-Q Advantage A10 water purification system)
  23. 2,4-Dichlorophenoxyacetic acid (Sigma-Aldrich, catalog number: D70724-5G)
  24. Potassium hydroxide (KOH) (Merck, catalog number: 1.05033.1000)
  25. Gelrite (Duchefa Biochemie, catalog number: G1101)
  26. PureLink HiPure Plasmid Midiprep Kit (Thermo Fisher Scientific, catalog number: K210004)
  27. Calcium chloride (CaCl2) (Roth, catalog number: 5239.1)
  28. 0.1 M Spermidine solution (Sigma-Aldrich, catalog number: 05292-1ML-F)
  29. MS salts (based on Murashige and Skoog, 1962) including vitamins (Duchefa Biochemie, catalog number: M0222)
  30. Sucrose (Roth, catalog number: 4661.4)
  31. Liquid MS medium (see Recipes)
  32. MS plates (see Recipes)

Equipment

  1. PDS-1000/He TM System (Bio-Rad, catalog number: 1652257)
  2. Sterile laminar flow bench (KR Biowizard Golden Line Safety Cabinet, KR-130 GL or similar)
  3. Vortex (Scientific Industries, model: Vortex-Genie 2)
  4. Table centrifuge (Eppendorf, model 5424)
  5. Pipette (Eppendorf, model: Research® plus, series in 2.5-1,000 μl volume)
  6. pH meter (Mettler Toledo SevenEasy S20 pH Meter)
  7. Steel sieve (size around 1.5-2 mm, purchased in a supermarket)
  8. Standard refrigerator (4 °C and -20 °C)
  9. Laboratory autoclave machine
  10. Standard microwave oven
  11. Spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 1000, catalog number: ND-1000)
  12. Tissue culture incubator (for example from Binder allowing cultivation at 26 °C in the dark)
  13. Compact incubation shaker (Minitron from Infors HT, 130 rpm at 26 °C in the dark)
  14. Plant growth chamber (walk-in growth chamber or greenhouse; long-day growth conditions with 16 h of light at 28 °C and 8 h of dark at 22 °C each at 65% relative humidity)
  15. Confocal microscope (Zeiss LSM880, Leica SP8 or similar)

Software

  1. ImageJ (National Institute of Health and the Laboratory for Optical Computational Instrumentation, http://imagej.nih.gov/ij, Schneider et al., 2012)
  2. ZEN Lite software package (Zeiss, http://zeiss.com/microscopy/int/products/microscope-software/zen-lite.html)

Procedure

Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant transformation > Bombardment

Cell Biology > Cell isolation and culture > Transformation

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