Electroporation and Agrobacterium-Mediated Spore Transformation

Abstract

Genetic transformation is a key technology in modern fungal research. Most commonly, protoplasts are transformed using the polyethylene glycol-mediated transformation protocols. Because protoplasts are generated by treatment of mycelia with a crude enzyme preparation, the results tend to be inconsistent. Furthermore, some species cannot be transformed by this method. Electroporation (EP) and Agrobacterium tumefaciens-mediated transformation (AMT) are two alternative methods. These methods allow the transformation of spores or mycelia, they are simple to perform and provide consistent results. In this chapter, we describe EP and AMT protocols for the fungus Colletotrichum gloeosporioides f. sp. aeschynomene (C. gloeosporioides). These protocols can be used as baseline for the calibration of similar transformation protocols in other species.

Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora

“…Then the culture was spread on a new LB agar plate with antibiotics and grew overnight. Before transformation, the bacterial cells were scraped from the LB plate, suspended and further diluted to various concentrations in Agrobacterium induction medium (IM) , which consisted of liquid minimal medium (MM) [34] supplemented with 200 μM acetosyringone in 50 ml sterile conical centrifuge tubes. The centrifuge tube with Agrobacterium suspension was wrapped in foil and incubated at room temperature for 2 h on a platform shaker with gentle agitation to induce vir gene expression.…”

Genetic Transformation Systems in Fungi, Volume 1

“…Here, we describe protocols for the F. oxysporum transformation by AMT and GBT methods with some modifi cations which can also be applied for transformation of other fi lamentous fungi Singh & Rajam 2013 ;Minz & Sharon 2010 ). …”

The use of T‐DNAtagging to isolate mutants ofColletotrichum gloeosporioidesandColletotrichum acutatumwith reduced virulence againstHevea brasiliensis

“…Arg.) Many investigations showed differences in the biology, epidemiology, pathogenicity and fungicide sensitivity of C. acutatum and C. gloeosporioides (Jayasinghe et al 1997;de Groot et al 1998;Zhang et al 2008b;Thambugala and Deshappriya 2009;Minz and Sharon 2010); however, little is known about the genetic basis of these differences. C. gloeosporioides has been described as the causative agent of CLD of rubber tree in many parts of the world (Jayasinghe et al 1997).…”

“…C. acutatum was first recorded as a causative agent of CLD in rubber tree in Yunnan province, and since then, it has been found in many plantations in China (Zhang et al 2008a). Many investigations showed differences in the biology, epidemiology, pathogenicity and fungicide sensitivity of C. acutatum and C. gloeosporioides (Jayasinghe et al 1997;de Groot et al 1998;Zhang et al 2008b;Thambugala and Deshappriya 2009;Minz and Sharon 2010); however, little is known about the genetic basis of these differences.…”