Protoplasting, regeneration and transformation of medicinal mushroom Ganoderma multipileum using succinate dehydrogenase mutation gene as a selection marker

Table 2 Ganoderma multipileum protoplast yields under different protoplasting conditions

Factor^a	Range	Yield of protoplasts (× 10⁵/mL)	Regeneration rate (%)	Viable protoplast (× 10⁵/mL)
pH of lysing enzyme solution^a	5.0	0	0	0
	5.5	33.3	0.78	0.2597
	5.8	17	0.96	0.1632
	6.0	49.5	0.55	0.27225
Mycelia age (days)^b	4	73.8	2.55	1.845
	5	17	0.96	0.1632
	6	115	0.31	0.3565
Lysing enzyme concentration (mg/mL)^c	5.0	0.6	1.09	0.00654
	7.5	17	0.96	0.1632
	10	7.6	0.95	0.0722
^dOsmoticum (OM)	KCl	1.3	0.13	0.00169
	Mannitol	0	0	0
	Sucrose	17	0.96	0.1632
	MgSO₄‧7H₂O	6.5	3.29	0.21385
Enzyme digestion time (h)^e	2	6.1	0.42	0.02562
	3	300	0.41	1.23
	4	17	0.96	0.1632
	5	3.6	1.34	0.04824
All best conditions test 1^f		3	4.83	0.1449
All best conditions test 2^f		3.5	3.53	0.12355
All best conditions Test 3^f		2.5	4.32	0.108
All best conditions Test 4^f		1	4.425	0.0425
OM test 1^g	0.6 M MgSO₄‧7H₂O	11.5	1.37	0.15755
OM test 2^g	0.6 M MgSO₄‧7H₂O	5.5	1.87	0.10285

^aOther parameters: 5-day-old mycelia digested at 30 °C for 4 h in 0.6 M sucrose osmotic stabilizer with lysing enzyme 7.5 mg/mL
^bOther parameters: mycelia digested with lysing enzyme 7.5 mg/mL pH 5.8 at 30 °C for 4 h containing 0.6 M sucrose osmotic stabilizer
^cOther parameters: 5-day-old mycelia digested at 30 °C for 4 h in 0.6 M sucrose as osmotic stabilizer pH 5.8
^dOther parameters: 5-day-old mycelia at 30 °C for 4 h, pH 5.8, lysing enzyme 7.5 mg/mL
^eOther parameters: 5-day-old mycelia digested at 30 °C in 0.6 M sucrose osmotic stabilizer, pH 5.8, lysing enzyme 7.5 mg/mL
^fParameters: 4-day-old mycelia, digested at 30 °C for 3 h in 0.3 M MgSO₄‧7H₂O osmotic stabilizer, lysing enzyme 7.5 mg/mL in pH 6.0
^gParameters: 4-day-old mycelia digested at 30 °C for 3 h in 0.6 M MgSO₄‧7H₂O osmotic stabilizer, lysing enzyme 7.5 mg/mL in pH 6.0

ISSN: 1869-2044