Table 1.

Methods for the establishment of the null segregants

Methods	Plant materials	Transformation methods	Cas9 and gRNA delivery	Reference
Mendelian segregation (supporeted by new screening strategies)	Arabidopsis	Agrobacterium-mediated transformation (floral dipping)	Encoding DNA	[35]
	Rice calli	Agrobacterium-mediated transformation	Encoding DNA	[34]
Programmed self-elimination of transgenic plants	Rice calli	Agrobacterium-mediated transformation	Encoding DNA	[36]
Transient expression of CRISPR/Cas9 from DNA or mRNA	Potato protoplasts	PEG-mediated transformation	Encoding DNA	[37]
	B. oldhamii, S. italica, O. sativa, Z. mays, A. thaliana, B. oleracea, B. napus, N. tabacum, S. lycopersicum protoplasts	PEG-mediated transformation	Encoding DNA	[38]
	Tobacco leaf explants	Agrobacterium-mediated transformation	Encoding DNA	[39]
	Wheat calli	Particle bombardment	Encoding DNA or RNA	[40]
RNPs-mediated targeted mutagenesis	Arabidopsis, tobacco, lettuce and rice protoplasts	PEG-mediated transformation	Protein and in vitro-transcribed gRNA	[41]
	Grape, apple protoplasts	PEG-mediated transformation	Protein and in vitro-transcribed gRNA	[42]
	Petunia × hybrida protolasts	PEG-mediated transformation	Protein and in vitro-transcribed gRNA	[43]
	Wheat protoplast, immmature embryo	PEG-mediated transformation, particle bombardment	Protein and in vitro-transcribed gRNA	[44]
	B. oleracea and B. rapa protoplasts	PEG-mediated transformation	Protein and in vitro-transcribed gRNA or synthesized gRNA	[45]
	Maize immmature embryo	Particle bombardment	Protein and in vitro-transcribed gRNA	[46]
	Rice zygote produced by in vitro ferlization of isolated gamates	PEG-mediated transformation	Protein and in vitro-transcribed gRNA	[47]