Nicotiana (tobacco) cell lines

Plant cell lines

• rpc00001: Nicotiana tabacum BY-2 cell suspension culture

• rpc00009: Nicotiana tabacum T-13 cell suspension culture

• rpc00035: Nicotiana tabacum Xan-1 callus culture

• rpc00036: Nicotiana tabacum BY-2H callus 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

• rpc00043: Nicotiana tabacum 3n-3 callus culture

• rpc00044: Nicotiana glauca G89 callus culture

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

• rpc00084: Nicotiana tabacum NI cell suspension culture

• rpc00085: Nicotiana tabacum ATR-r cell suspension culture

• rpc00086: Nicotiana tabacum NaCl-r 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	rbcL	Transgene insertion	psbA	SSR
BY-2	Nicotiana tabacum	—	—	BY-2
T-13	Nicotiana tabacum	—	—	T-13
Xan-1	Nicotiana tabacum	—	—	Xan-1
BY-2H	Nicotiana tabacum	—	—	BY-2H
GV7	Nicotiana tabacum	35Spro-GFP-AtVam3p	—	—
GF11	Nicotiana tabacum	35Spro-GFP-AtFim1	—	—
GT16	Nicotiana tabacum	35Spro-GFP-tubulin	—	—
TBY2-AtRER1B	Nicotiana tabacum	35Spro-GFP-AtRer1B	—	—
3n-3	Nicotiana tabacum	—	—	3n-3
G89	Nicotiana glauca	—	—	G89
BY-TIPG	Nicotiana tabacum	35Spro-NtTIP1;1-GFP	—	—
NI	Nicotiana tabacum	—	—	NI
ATR-r	Nicotiana tabacum	—	p.Ser264Thr	ATR-r
NaCl-r	Nicotiana tabacum	—	—	NaCl-r
TBY2-31/ST	Nicotiana tabacum	35Spro-GFP-AtSYP31 35Spro-ST-mRFP	—	—
TBY2-41/ST	Nicotiana tabacum	35Spro-GFP-AtSYP41 35Spro-ST-mRFP	—	—
TBY2-R31	Nicotiana tabacum	35Spro-mRFP-AtSYP31	—	—

Protocols

DNA barcoding — rbcL

DNA barcoding — rbcL

Transgene insertion

Transgene insertion

DNA sequencing — psbA gene

1. PCR

   • Primer set 《Primer sequences》

     Target	Forward primer	Reverse primer	Product size
     psbA	PCRpsbALEFT	PCRpsbARIGHT

   • PCR reaction mixture

     Component	10 µL reaction
     TaKaRa Ex Taq™ Premix	5 µL
     Forward primer (100 µM)	0.125 µL
     Reverse primer (100 µM)	0.125 µL
     Template DNA	1 µL
     Sterile distilled water	3.75 µL

   • PCR reaction

     Temperature	Time	Cycles
     94°C	1 min
     94°C	30 sec	35 cycles
     55°C	30 sec
     72°C	1 min
     72°C	7 min

2. Check the DNA sequence of the PCR product.

   • psbA: p.Ser264Thr (c.791G>C)

Fragment analysis — SSR (3 loci)

1. PCR with each of the primer mix

   • Primer sets 《Primer sequences》

     Primer mix	Primer name	Concentration
     3493/3494	3493	1 µM
     	3494	0.1 µM
     3495/3496	3495	1 µM
     	3496	0.1 µM
     3497/3498	3497	1 µM
     	3498	0.1 µM
     	uni-FAM [1]	10 µM

     [1]

     Fluorescent labeled primer at 5′ end with 6-FAM™

   • PCR reaction mixture

     Component	10 µL reaction
     AmpliTaq Gold™ 360 Master Mix	5 µL
     uni-FAM	0.2 µL
     Primer mix	2 µL
     Template DNA	1 µL
     Sterile distilled water	1.8 µL

   • PCR reaction

     Temperature	Time	Cycles
     95°C	10 min
     95°C	30 sec	40 cycles
     55°C	30 sec
     72°C	30 sec
     72°C	10 min

2. Capillary electrophoresis of each of the PCR products with the Applied Biosystems™ 3130xl Genetic Analyzer

3. Data analysis with the GeneMapper^® Software