Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

Francine Hiromi Ishikawa,Eustáquio Souza Dias,Elaine Aparecida De Souza,Quélen De Lima Barcelos

doi:10.1590/s1413-70542010000100009

Francine Hiromi Ishikawa, Eustáquio Souza Dias + Show 2 more

Open Access

https://doi.org/10.1590/s1413-70542010000100009

Copy DOI

Abstract

The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.

Highlights

Anthracnose is one of the most important diseases of the common bean (Phaseolus vulgaris L.)
The efficiency of the provided osmotic support to the protoplasts following the removal of the cell wall was evaluated for 6 different osmotic stabilizers
The best results were obtained with 0.6 mol l-1 NH Cl and 0.37 mol l-1 NaCl salts, which produced an average of 9.6 x 105 and 8 x 105 protoplasts ml-1, respectively (Fig. 1)

Summary

Introduction

Anthracnose is one of the most important diseases of the common bean (Phaseolus vulgaris L.). Protoplast release is an important biological tool for experiments addressing genetic transformation and other molecular approaches such as pulsed-field gel electrophoresis (PFGE) and flow cytometry. Althhoouugghh pprootoopplaasstt foormmaattioonn inn C. lindemuthianum has been reported before, any existing protocol should be used for each strain under study. Most of these studies used Novozym 234, this enzymatic complex is no commercially available. Factors such as lytic enzymes, osmotic stabilizers, mycelium age and the type of microorganism can affect the maximum release of protoplasts (Peberdy, 1995)

Objectives

Methods

Results