Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum)

Botanical Studies

Table 2 Transient transformation rates of A. rhizogenes A4 or A8196 strain harboring the pCAMBIA1303 plasmid in ice plant seedlings and distributions (%) of GUS staining in different tissues of 3- and 5-day-old ice plant seedlings

Ages of seedlings	A. rhizogenes strains	Days after 2-day coincubation periods	Transient transformation rates^a	% of tissues showing GUS activities^b
3-day-old with root tip removed	A4	1	100.0 ± 0.0*	Cotyledon	100.0 ± 0.0^#
				Hypocotyl	0.0 ± 0.0
				Root	95.0 ± 0.9^#
	A8196	1	100.0 ± 0.0*	Cotyledon	100.0 ± 0.0^#
				Hypocotyl	0.0 ± 0.0
				Root	100.0 ± 0.0^#
	A4	2	96.7 ± 1.1*	Cotyledon	96.7 ± 0.7^#
				Hypocotyl	6.3 ± 0.1
				Root	95.3 ± 0.1^#
	A8196	2	100.0 ± 0.0*	Cotyledon	100.0 ± 0.0^#
				Hypocotyl	5.6 ± 0.0
				Root	100.0 ± 0.0^#
	A4	5	92.9 ± 2.3*	Cotyledon	83.3 ± 0.5^#
				Hypocotyl	1.9 ± 0.6
				Root	85.2 ± 0.5^#
	A8196	5	100.0 ± 0.0*	Cotyledon	100.0 ± 0.0^#
				Hypocotyl	0.0 ± 0.0
				Root	100.0 ± 0.0^#
	A4	7	33.3 ± 1.1	Cotyledon	33.3 ± 1.1^#
				Hypocotyl	0.0 ± 0.0
				Root	31.4 ± 1.0^#
	A8196	7	0.0 ± 0.0	Cotyledon	0.0 ± 0.0
				Hypocotyl	0.0 ± 0.0
				Root	0.0 ± 0.0
5-day-old with root tip removed	A4	1	100.0 ± 0.0*	Cotyledon	66.7 ± 1.1^#
				Hypocotyl	0.0 ± 0.0
				Root	55.0 ± 0.9^#
	A8196	1	100.0 ± 0.0*	Cotyledon	45.0 ± 1.2^#
				Hypocotyl	0.0 ± 0.0
				Root	47.5 ± 1.2^#
	A4	2	97.5 ± 0.6*	Cotyledon	65.0 ± 1.0^#
				Hypocotyl	0.0 ± 0.0
				Root	55.0 ± 0.9^#
	A8196	2	100.0 ± 0.0*	Cotyledon	50.0 ± 1.3^#
				Hypocotyl	2.4 ± 0.1
				Root	47.6 ± 1.2^#
	A4	5	47.2 ± 1.2	Cotyledon	31.5 ± 1.0^#
				Hypocotyl	0.0 ± 0.0
				Root	29.6 ± 0.9^#
	A8196	5	0.0 ± 0.0	Cotyledon	0.0 ± 0.0
				Hypocotyl	0.0 ± 0.0
				Root	0.0 ± 0.0
	A4	7	45.0 ± 0.0	Cotyledon	28.3 ± 0.9^#
				Hypocotyl	0.0 ± 0.0
				Root	30.0 ± 0.9^#
	A8196	7	0.0 ± 0.0	Cotyledon	0.0 ± 0.0
				Hypocotyl	0.0 ± 0.0
				Root	0.0 ± 0.0

^aNumber of transiently transformed seedlings/total number of seedlings × 100%. The 60 to 80 ice plant seedlings were infected with A. rhizogenes strain for each independent transformation assay. Data are mean ± SD (standard deviation) from at least 3 independent experiments
^bNumber of GUS stained tissues/number of transiently transformed seedlings × 100%
* P < 0.05 comparing 1-, 2-, or 5-day after infection and 7-day after infection of the same ice plant type under the same bacteria strain infection condition based on pairwise Student t-test
^#P < 0.05 comparing cotyledon, or root tissues and hypocotyl tissues of the same type ice plants under the same bacteria infection condition based on pairwise Student t-test