Protoplast

INTRODUCTION

Introduction

Solution to sexual incompatibility barriers – distant species;

Fusion of cells – full hybrids – crop improvement programmes;

EC Cocking (1960) – enzymatic degradation of plant cell wall - viable naked cells

Freshly isolated mesophyll protoplasts

JB Power, UK

Introduction

ISOLATION OF PROTOPLASTS

ISOLATION OF PROTOPLASTS FROM PLANTLETS OF Solanum phureja

Freshly isolated

First mitotic division

Multicellular colonies (7-d-old)

Macroscopic p-calli (several wks)

Factors that influence yield and viability of protoplasts

Factors that influence yield and viability of protoplasts

Factors that influence yield and viability of protoplasts

Factors that influence yield and viability of protoplasts

Commercial Enzymes

Factors that influence yield and viability of protoplasts

Factors that influence yield and viability of protoplasts

Factors that influence yield and viability of protoplasts

Viability of the Protoplasts

Detection methods:

1)Observation of cyclosis or cytoplasmic streaming;

2)Oxygen uptake

3)Photosynthetic activity;

4)Staining with fluroescein diacetate - common

Nutritional Requirements

Plant regeneration

- Plant regeneration from hypocotyl protoplasts of Brassica napus

Stage

Event

AFreshly isolated

protoplasts

B 1-d-old protoplasts

C Callus (14-d culture)

D Shoot buds differentiated

ECalli with regenerated shoots (Zeatin + IAA)

F Plant regenerated