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 Add 2.15 g of MS powder into 800 ml of water in 1.5 -2.0L autoclavable bottle. Place a stirring bar into the bottle and dissolve MS powder by stirring medium…

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

Material Name	Type	Company	Catalogue Number	Comment
Murashige and Skoog Basal Salt Mixture (MS)		Sigma	M5524
Agar		Sigma	A1296
Petri Dishes (100x15mm)		Kreckeler Scientific	82-4001
Cellulase (Onozuka R‐10)		RPI Corp	C32200
Macerozyme (R10)		RPI Corp	M22010
Cheesecloth wipes		Fisher Scientific	06-665-29
Lab Rotator		Fisher Scientific	1167152Q
Allegra X‐15R Centrifuge		VWR	392932
Axioskop2 Plus Microscope		Zeiss