Nicotiana (tobacco) cell lines

Plant cell lines

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

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

    • 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