Transgene insertion

Plant cell lines

• rpc00014: Catharanthus roseus CRA cell suspension culture

• rpc00015: Catharanthus roseus V208 cell suspension culture

• rpc00039: Nicotiana tabacum GV7 transgenic cell suspension culture

• rpc00040: Nicotiana tabacum GF11 transgenic cell suspension culture

• rpc00041: Nicotiana tabacum GT16 transgenic cell suspension culture

• rpc00042: Nicotiana tabacum TBY2-AtRER1B transgenic callus culture

• rpc00062: Nicotiana tabacum BY-TIPG transgenic cell suspension culture

• rpc00069: Coptis japonica 156-S transgenic cell suspension culture

• rpc00091: Nicotiana tabacum TBY2-31/ST transgenic cell suspension culture

• rpc00093: Nicotiana tabacum TBY2-41/ST transgenic cell suspension culture

• rpc00095: Nicotiana tabacum TBY2-R31 transgenic cell suspension culture

Genotypes

Cell line	Transgene insertion
CRA	Not detected (tmr)
V208	tmr
GV7	35Spro-GFP-AtVam3p
GF11	35Spro-GFP-AtFim1
GT16	35Spro-GFP-tubulin
TBY2-AtRER1B	35Spro-GFP-AtRer1B
BY-TIPG	35Spro-NtTIP1;1-GFP
156-S	35Spro-CjSMT
TBY2-31/ST	35Spro-GFP-AtSYP31/35Spro-ST-mRFP
TBY2-41/ST	35Spro-GFP-AtSYP41/35Spro-ST-mRFP
TBY2-R31	35Spro-mRFP-AtSYP31

Protocol

1. PCR

   • Primer sets 《Primer sequences》

     Cell line	Target	Forward	Reverse	Product size
     CRA	tmr	35Spro_F3	GV7_R	Not detected (370 bp)
     V208	tmr	35Spro_F3	GV7_R	370 bp
     GV7	35Spro-GFP-AtVam3p	35Spro_F3	GV7_R	1.5 kbp
     GV7	35Spro-GFP-AtVam3p	GFP_F	GV7_R	1.3 kbp
     GF11	35Spro-GFP-AtFim1	35Spro_F3	GF11_R	2.0 kbp
     GF11	35Spro-GFP-AtFim1	GFP_F	GF11_R	1.7 kbp
     GT16	35Spro-GFP-tubulin	35Spro_F3	GT16_R	2.1 kbp
     GT16	35Spro-GFP-tubulin	GFP_F	GT16_R	1.9 kbp
     TBY2-AtRER1B	35Spro-GFP-AtRer1B	35Spro_F3	RER1B_R	1.4 kbp
     TBY2-AtRER1B	35Spro-GFP-AtRer1B	GFP_494_F	RER1B_R	800 bp
     TBY2-AtRER1B	35Spro-GFP-AtRer1B	GFP_F	RER1B_R	1.2 kbp
     TBY2-AtRER1B	35Spro-GFP-AtRer1B	GFPS65T_F	RER1B_R1	565 bp
     BY-TIPG	35Spro-NtTIP1;1-GFP	35Spro_F3	GFP_R	1.3 kbp
     BY-TIPG	35Spro-NtTIP1;1-GFP	NtTIP1_F	GFP_R	1.0 kbp
     156-S	35Spro-CjSMT	35Spro_F2	SMT_R	1.5 kbp
     156-S	35Spro-CjSMT	35Spro_F2	NOSterR1	2.0 kbp
     TBY2-31/ST	35Spro-GFP-AtSYP31	35Spro_F5	SYP31stop_R	1.8 kbp
     TBY2-31/ST	35Spro-ST-mRFP	35Spro_F2	mRFPstop_R	1.1 kbp
     TBY2-41/ST	35Spro-GFP-AtSYP41	35Spro_F5	SYP41stop_R	1.8 kbp
     TBY2-41/ST	35Spro-ST-mRFP	35Spro_F2	mRFPstop_R	1.1 kbp
     TBY2-R31	35Spro-mRFP-AtSYP31	35Spro_F5	SYP31stop_R	1.8 kbp

   • PCR reaction mixture

     Component	10 µL reaction
     TaKaRa Ex Taq™ Premix	5 µL
     Forward primer (10 µM)	0.5 µL
     Reverse primer (10 µM)	0.5 µL
     Template DNA	1 µL
     Sterile distilled water	3 µL

   • PCR reaction

     Temperature	Time	Cycles
     94°C	1 min
     94°C	30 sec	35 cycles
     55°C	30 sec
     72°C	1 min/1 kbp
     72°C	10 min

2. Check the size of the PCR product.

3. If necessary, check the DNA sequence of the PCR product.