A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Summary

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Abstract

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

Introduction

Protoplasts are plant cells that have cell walls removed. As they maintain most of features and activities of plant cells, protoplasts are a good model system to observe and evaluate diverse cellular events, and are valuable tools to study somatic hybridisation¹ and plant regeneration². Protoplasts have been also widely utilized for plant transformation^3,4,5, since cell walls would otherwise block the passage of DNA into the cell. Protoplasts possess some of the physiological responses and cellular processes of intact plants, hence offering fundamental value in basic research to study subcellular protein localization^6,7,8, protein-protein interactions^9,10, and promoter activity^11,12,13 in live cells.

The isolation of plant protoplasts was first reported in 1960¹⁴ and the protocols for both isolation and transformation of protoplasts have been developed and optimized. A standard procedure of protoplast isolation involves the cutting of leaves and enzymatic digestion of cell walls, followed by separation of released protoplasts from non-digested tissue debris. Transformation strategies includes electroporation^15,16, microinjection^17,18, and polyethylene glycol-based (PEG)^4,5,19 methods. A wide range of species have been reported successful for protoplast isolation, including Citrus²⁰, Brassica²¹, Solanaceae²² and other ornamental plant families^23,24. While diverse tissue types are used in various species, a system of transient expression in Arabidopsis mesophyll protoplast (TEAMP) isolated from leaves of the model plant Arabidopsis thaliana has been well established²⁵ and widely adopted to diverse applications.

Soybean (Glycine max (L.) Merr.) is one of the most important protein and oil crops²⁶. Unlike Arabidopsis and rice, obtaining transgenic soybean plants is known to be rather difficult and low efficiency. Agrobacterium tumefaciens-mediated infiltration has been popularly used for transient gene expression studies in the epidermal cells in tobacco²⁷ and seedlings in Arabidopsis^28,29, whereas Agrobacterium rhizogenes has been used for transformation of hairy roots in soybean³⁰. Virus-induced gene silencing approaches have been utilized for downregulation of target genes^31,32 and transient expression³³ in a systemic manner. Protoplasts provide a valuable and versatile alternative to these approaches. Protoplasts can be obtained from soybean's aboveground materials and allow quick and synchronized transgene expression. However, since the initial successful isolation of soybean protoplasts in the 1983³⁴, there have been limited reports on the application of protoplasts in soybean^35,36,37,38, primarily due to relatively low yields of soybean protoplasts.

Here, we describe a simple and efficient protocol for isolation of soybean protoplasts and its application for transient gene expression studies. Using young unifoliate leaves from soybean seedlings, we were able to obtain large quantities of vital protoplasts within a few hours. In addition, we have optimized a PEG-calcium-mediated transformation method that is simple and low cost to deliver DNA into soybean protoplasts with high efficiency.

Protocol

1. Growth of the plants

1. Sow 5 - 10 soybean seeds (Williams 82) in a 13 cm pot in the greenhouse under long-day conditions (16 h light at 1,500 µmol m^-2 s^-1) at 25 °C on the custom soil mix for Soybean (the 1:1:1 ratio of soil, perlite and torpedo sand).

2. Preparation of Plasmid DNA

1. Using a sterile pipette tip or toothpick, pick a single colony or frozen glycerol stock of E. coli carrying the plasmid containing the gene of interest and inoculate it in 20 mL Luria-Bertani (LB) liquid medium with appropriate antibiotics in a 50 mL flask.
2. Incubate the flask at 37 °C overnight on a shaker. Collect the bacteria by centrifuging the suspension at 12,000 x g at room temperature for 5 min and discarding the supernatant. Extract and purify the plasmid following the manufacturer's procedure of a plasmid prep kit.

3. Protoplast isolation

1. Cut newly expanded unifoliate leaves from 10-day old soybean seedlings (Figure 1).
   NOTE: Selection of leaves at an appropriate developmental stage is the key to the success for soybean protoplast preparation. Only use just expanded leaves at early developmental stages. As leaves mature, cell walls become harder to digest.
2. With a fresh razor blade, remove the midrib from the unifoliate leaf and then cut the remains into 0.5-1 mm strips.
   NOTE: Two unifoliate leaves digested in 10 mL enzyme solution will give sufficient protoplasts for more than 10 transformations.
3. Using a pair of forceps, transfer the leaf strips immediately and gently into 10 mL of freshly prepared enzyme solution (Table 1) in a 15 mL tube. Vacuum infiltrate the leaf strips for 15 min at room temperature.
4. Incubate the leaf strips in the enzyme solution with gentle agitation (40 rpm) under low light for 4-6 h at room temperature. Ensure that the enzyme solution turns yellow-green as protoplasts are released. Check the enzyme/protoplasts solution under the microscope (X10).
   NOTE: The released protoplasts are spherical shaped, while the undigested cells have irregular or oval shape.
5. Transfer the 10 mL of enzyme/protoplast solution in a 50 mL tube by gently pouring and add 10 mL of W5 solution (Table 1) at room temperature to stop the digestion. Gently invert the tube a few times. Pour gently the enzyme/protoplasts solution on a clean 75 µm nylon mesh placed on top of a 50 mL tube to remove the undigested leaf tissues.
6. Centrifuge the flow-through enzyme/protoplasts solution at 100 x g in the 50 mL tube for 1-2 min at room temperature. Gently remove the supernatant using a 10 mL serological pipette without disturbing the protoplast pellet.
7. Resuspend the protoplasts and dilute to a concentration of 2 x 10⁵ mL^-1 in chilled W5 solution at 4 °C by counting protoplast number on a hemacytometer under the microscope (x10). Keep the protoplasts on ice for 30 min.
8. Centrifuge the suspension at 100 x g for 1-2 min at room temperature and gently remove the W5 solution using a 1 mL pipette without disturbing the protoplast pellet. Resuspend the protoplasts in MMG solution (Table 1) at a concentration of 2 x 10⁵ mL^-1 at room temperature.

4. Protoplast transformation

1. Make 100 µL aliquots of protoplasts (2 x 10⁴ protoplasts at 2 x 10⁵ mL^-1) in 1.5 mL low adhesion microcentrifuge tubes using uncut 200 µL pipette tips. Put one aliquot aside to serve as negative control. Add 10 µL of plasmid (10-20 µg) into each of the rest aliquot of protoplasts.
2. Slowly add 110 µL of freshly prepared PEG solution (Table 1) on the inner wall of the 1.5 mL microcentrifuge tube, and then gently invert and rotate the tube until the solution becomes homogeneous.
3. Incubate the transformation mixture at room temperature for 15 min.
4. To stop the transformation, slowly add 400 µL of W5 solution to the 1.5 mL tube at room temperature and gently invert the tube until the solution becomes homogeneous. Centrifuge the tube at 100 g for 1-2 min at room temperature and discard the supernatant using a pipette.
5. Add 1 mL of WI solution (Table 1) to the tube and resuspend by gently pipetting 1-2 times. Add 1 mL of 5% (vol/vol) sterile calf serum in each well of a 6-well tissue culture plate to coat the surface and prevent the protoplasts from sticking to the plate.
6. After a few seconds, discard the calf serum using a pipette. Transfer the resuspended protoplasts into a well of the culture plate. Cover the plate with a lid.

5. Protoplast incubation and harvesting

1. Incubate the protoplasts at room temperature for 1-2 days in the dark.
   NOTE: We found that two-days incubation yields stronger fluorescent protein signal in general compared to one-day incubation, and that the fluorescent protein signal may last for up to 3-4 days.
2. Transfer the protoplast solution to a 1.5 mL low adhesion microcentrifuge tube. Centrifuge the tube at 100 x g for 1-2 min at room temperature to harvest protoplasts. Remove the supernatant using a pipette and transfer 10 µL protoplasts onto a glass slide.
3. Observe fluorescent signal under fluorescence or confocal microscopy. Use non-transformed protoplasts as negative control (No signal should be observed).

Results

Different organs of 10-day old soybeans were tested for protoplast preparation (Figure 1) and yields were observed under the microscope (Figure 2). Cell walls from hypocotyl and epicotyl were hardly digested, and some cells stayed attached to each other (Figure 2B, 2C). In cotyledon (Figure 2D) and root (Figure 2A), cell wal...

Discussion

This protocol for the isolation of soybean protoplasts and the application to transient expression studies has been thoroughly tested and works very well in our laboratory. The procedures are simple and easy and require ordinary equipment and minimum cost. Our protocol yields large quantities of uniform, high quality protoplasts compared to previously reported methods^34,35,36,37,

Materials

Name	Company	Catalog Number	Comments
MES	Sigma Aldrich	M8250-100G
Cellulase CELF	Worthington Biological Corporation	LS002611
Pectolyase Y-23	BioWorld	9033-35-6
CELLULASE "ONOZUKA" R-10	yakult	10g
MACEROZYME R-10	yakult	10g
Mannitol	ICN Biomedicals	152540
CaCl2	Fisher	C79-500g
BSA	NEB	R3535S
DTT	Sigma Aldrich	D5545-5G
NaCl	Sigma Aldrich	S7653-1kg
KCl	Fisher	P217-500g
MgCl2	Sigma Aldrich	M8266-100g
PEG4000	Fluka	81240
nylon mesh	carolina	652222N
Tissue Culture Plates	USA Scientific	CC7682-7506
Razor Blades	Fisher	12-640
hemacytometer	hausserscientific	1483
QIAprep Spin Miniprep Kit	Qiagen	27104
EZNA plasmid miniprep kit	Omega	D6942-01
GeneJET Plasmid Miniprep Kit	Thermo Scientific	K0502
Centrifuge 5810	eppendorf	5811000827
Centrifuge 5424	eppendorf	22620401
Jencons Powerpette Plus Pipet Controller	Jencons	14526-202
Zeiss 710 Confocal Microscope	Zeiss	N/A
Nonstick, RNase-free Microfuge Tubes, 1.5 mL	Ambion	AM12450
15 mL Centrifuge Tubes	Denville	C1018-P
50 mL Centrifuge Tubes	Denville	C1060-P
Newborn Calf Serum	Thermo Scientific	16010159
Soil	Ingram's Nursery
perlite	Vigoro	100521091
Torpedo Sand	JKS Ventures
LB Broth, Lennox (Powder)	Fisher	BP1427-500