Table 3.

Systematic comparison of transformation methods.

Method	Recipient cells	Principles	Key points	Advantages	Disadvantages	Source
PEG mediated transformation (PMT)	Protoplasts	The receptive cells (protoplasts) absorb foreign DNA precipitated with divalent calcium ions on the membrane surface under the action of PEG, and the cell membrane will release lipids to reveal pores and facilitate the entry of foreign nucleic acids after short heat shocks	Preparation and regeneration of protoplasts; Appropriate selection of PEG, temperature and osmotic buffer; Cell permeability inducers can be added	Not necessary special vectors and bacteria intermediary	The preparation of protoplasts is cumbersome and there are no uniform standards due to variable proportion and specific components of cell wall for different species and even within the same individual at different stages of development; Not all fungi are suitable for the preparation of protoplasts, limited to specific species and types	Case et al. (1979)
Agrobacterium Mediated Transformation (AMT)	Conidia, mycelia, fruiting bodies, etc.	A. tumefaciens integrates foreign genes between the transfer DNA (T-DNA) left and right border repeats of the binary vector into the host through the virulence region of the T-vector, DNA transfer is achieved during co-cultivation of A. tumefaciens with the fungus	Integration of foreign genes by A. tumefaciens, pay attention to Appropriate concentration of inducer AS, temperature and time of co-culture, ratio of recipient cells to A. tumefaciema, etc.	The types of receptor cells is not limited; Efficient, more single copy integration and easier to produce stable genetic transformants	Time-consuming and requires tedious optimization of various factors during co-cultivation; The T-DNA inserted into genome lacks integrity and thus difficult to identify the insertion sites; It is hard to obtain enough mutants for high-throughput screening	Bundock et al. (1995)
Electroporation (EP)	Protoplasts or spores	Electric pulses induce membrane permeabilization providing a local driving force for ionic and molecular transport through the pores	Selection and control of electric field condition parameters depend on types of cells, such as the capacitance, the pulse duration and frequency; and Pay attention to low temperature heat dissipation	Can be applied to any fungi in vivo or frozen; Efficient and easily optimized protocols; High repeatability; and Multiple copy integration is possible.	Depends on the electrophysiological characteristics of the fungus; Requires additional expensive instrumentation; and Raises the rate of cellular death.	Alagui et al. (1989)
Biolistic Transformation (BT), also known as particle bombardment and microprojectile bombardment	Any cell types or species	DNA are coated with small tungsten or gold particles and accelerated to penetrate the cell wall at high velocity	Metal particles packaging for exogenous nucleic acids and the instrumental settings	Not limited to cell types or species and independent of the physiological properties of the fungi; Transformation with multiple transgenes is possible; Foreign genes can be transformed instantaneously in the cytoplasm without being integrated into the genome	DNA can be damaged; Produces multiple copies of introduced genes; The projectile preparation is complex; Low efficiency; and Expensive.	Klein et al. (1987)
Agitation with glass beads	Yeast, and few species with thin cell walls	Agitation of the fungal cells with glass beads in the presence of carrier and plasmid DNA allows DNA absorption	The nature of recipient cells, and the effect of agitation amplitude on cell activity	Simple, fast, cheap, and safe protocol	Cells require osmotic support and may cause cell disruption; Genome randomly assigned; Low transformation efficiency	Costanzo and Fox (1988)
Vacuum infiltration	Certain type of fungi with low sporulation rate	Vacuum generates a negative atmospheric pressure that causes the air spaces between the cells to decrease allowing the infiltration of Agrobacterium	Mediating the incorporation of Agrobacterium for fungi transformation	Can be used for fungi that produce little or no spores	Requires the use of bacteria which may have unwanted consequences	Bechtold et al. (1993)
Shock wave mediated transformation (SWMT)	Conidia	Acoustic cavitation changes the permeability of the membrane facilitating the absorption of DNA	Determination of Shock wave parameters (frequency, energy, voltage, and number of shock waves)	Simple, safe, cheap, and good reproducibility; High efficiency and may be applicable to species that have never been transformed; Uniform parameters can be used to transform diverse species of fungi	Relatively high cost of the shock wave source; Expertise in shock wave physics required; Will destroy a certain amount of DNA, but the tandem shock wave will enhance the transformation and make up for the loss of DNA to some extent	Magana-Ortiz et al. (2013)