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, he....

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....

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

List of materials used in this article

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