Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Summary

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Abstract

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago ¹ and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) ^2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) ^2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 10⁶-10⁷ protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Protocol

Part 1. Preparation of solid medium for growing plants.

We typically culture plants on concentrated Murasige and Skoog (MS) medium supplemented with 1% sucrose and 0.7% agar. It contains all necessary ingredients to keep plants healthy.

For preparing 1L of MS, media pH 5.7

1. Add 2.15 g of MS powder into 800 ml of water in 1.5 -2.0L autoclavable bottle.
2. Place a stirring bar into the bottle and dissolve MS powder by stirring medium on a stirring plate.
3. While stirring, add, drop by drop, 1N KOH to adjust pH of MS medium to 5.7.
4. Add 10 g of sucrose, keep stirri....

Discussion

To ensure the high yield of intact protoplast it is very important to start-up with healthy plants. Use filter-sterilize solutions for isolating protoplasts. Remember that protoplasts are fragile. Therefore, when you are handling protoplasts, do not mix, pipet or vortex them vigorously since it will brake them. Instead, mix protoplasts by slowly rotating or taping the centrifuge tube. The described procedure yield intact protoplasts that are viable for at least 96 h. Therefore, protoplasts can be used to study a var.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MurashigeandSkoogBasalSaltMixture(MS)	Sigma-Aldrich	M5524
Agar	Sigma-Aldrich	A1296
PetriDishes(100x15mm)	Krackeler Scientific, Inc.	82-4001
Cellulase(OnozukaR‐10)	Research Products International Corp.	C32200
Macerozyme(R10)	Research Products International Corp.	M22010
Cheeseclothwipes	Fisher Scientific	06-665-29
LabRotator	Fisher Scientific	1167152Q
AllegraX‐15RCentrifuge	VWR international	392932
Axioskop2PlusMicroscope	Carl Zeiss, Inc.