第63回⽇本植物⽣理学会年会『ゲノムと新技術が磨くバイオリソース』2022年3⽉22⽇

Diverse Resourcesof Plant Cell Cultures
and Quality Control
for Their Advanced Utilization
多様な植物培養細胞リソースとその⾼度な利活⽤のための品質管理

Toshihiro Kobayashi
RIKEN BioResource Research Center

1

RIKEN BRC plant resources
NBRP Arabidopsis / Cultured plant cells, genes

Plant seeds

Plant DNA

Plant cell lines

130,889 lines

711,240 clones

72 cell linesnArabidopsisnArabidopsisnBrachypodiumnPhyscomitrium patens

nPoplarn

Cassavan

Striga hermonthicanBrassica rapanThellungiella halophilanTobacco BY-2nVectors

n39 plant species

2Plant Cell Resources
in RIKEN BRC

3

Plant cell cultures– Rapid growth– Homogenous population ofundifferentiated cells– Easy to control culture
environment– Used for
Cell biology
Cell engineering
4

RIKEN BRC plant cell lines
Model cell line — Tobacco BY-2
including transgenic cell lines

Cell lines of model plant species
Arabidopsis
Lotus / Glycine

Rice
TomatoSecondary metabolite-producing
cell lines
Plant cell lines that lost differentiation potential
except for Carrot kurodagosun
Brachypodium embryogenic callus5Mission– Maintain living cells
by subculturing– Keep original culture
conditions– Risk hedge
Loss of plant cell lines✕ Contamination of microbes✕ Accident / Mistake✕ Growth failure✕ Changes in characteristics

Multiple strains
Multiple locations

6Collection / Preservation

Number of cell lines

100
Preparing

80
60
40

Released

200

2003

2006

2009

2012

2015

2018

2021

RIKEN Gene Bank (Plant Cell Bank)
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Distribution
140

Number of orders

120
100
80
60

Domestic

40
200Overseas
2003

2006

2009

2012

2015

2018

2021

25 countries

Overseas distribution
8Plant cell collectionsDSMZ

VTTRIKENBRC

https://www.dsmz.de/

http://culturecollection.vtt.fi/

https://web.brc.riken.jp/ja/

https://abrc.osu.edu/

GermanyFinland

JapanUSA——23 species
58 cell lines

39 species
72 cell lines

2 species
4 cell lines

ABRC

only within USA9

Featured Plant Cell Lines
and Resource Information

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GFP bioresources
Transgenic BY-2 cell lines

GF11 Actin filament

GV7 Vacuolar membrane
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GFP bioresources
Transgenic BY-2 cell lines
TargetConstruct

Cell line

Actin filaments

GFP-AtFim1

GF11

Golgi bodies

GFP-AtRer1B

TBY2-AtRER1B

MicrotubulesGFP-Tubulin αGT16

Vacuolar membrane

GFP-AtVAM3

GV7

NtTIP1;1-GFP

BY-TIPG

Preparing other 10 transgenic BY-2 cell lines
including BY-2 cell lines expressing other fluorescent proteins
GFPリソースのご利⽤に関して
https://web.brc.riken.jp/ja/method/link/gfp_conclude

12Resource information
Exp-Plant Catalog — Plant Cell Lines
List
Browsable
Searchable
Sortable

Details
Links to– Medium preparation– Maintenance history
Photographs
https://plant.rtc.riken.jp/resource/cell_line/cell_line_list.html

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Resource information
Exp-Plant Catalog — Plant Cell Lines
ListBrowsable
Searchable
Sortable

Details
Links to– Medium preparation– Maintenance history
Photographs
https://plant.rtc.riken.jp/resource/cell_line/cell_line_list.html

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Resource information
Exp-Plant Catalog — Plant Cell Lines
List
Browsable
Searchable
Sortable

Details
Links to– Medium preparation– Maintenance history
Photographs
https://plant.rtc.riken.jp/resource/cell_line/cell_line_list.html

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Resource information
Exp-Plant Catalog — Plant Cell Lines
List
Browsable
Searchable
Sortable

Details
Links to– Medium preparation– Maintenance history
Photographs
https://plant.rtc.riken.jp/resource/cell_line/cell_line_list.html

13Technical support
RIKEN BRC plant cell line documentation
Medium preparation
Subculture method
Culture conditions

https://plant.rtc.riken.jp/resource/cell_line/web_documents/index.html

14Technical support
RIKEN BRC plant cell line documentation
Medium preparation
Subculture method
Culture conditions

https://plant.rtc.riken.jp/resource/cell_line/web_documents/index.html

14Technical support
RIKEN BRC plant cell line documentation
Medium preparation
Subculture method
Culture conditions

https://plant.rtc.riken.jp/resource/cell_line/web_documents/index.html

14Technical support
RIKEN BRC plant cell line documentation
Medium preparation
Subculture method
Culture conditions

https://plant.rtc.riken.jp/resource/cell_line/web_documents/index.html

14Technical support
RIKEN BRC plant cell line documentation
Medium preparation
Subculture method
Culture conditions

https://plant.rtc.riken.jp/resource/cell_line/web_documents/index.html

14Technical support
RIKEN BRC plant cell line documentation
Medium preparation
Subculture method
Culture conditions

https://plant.rtc.riken.jp/resource/cell_line/web_documents/index.html

14Technical support
Other documents
Technical notes
Protocols
Brochureshttps://epd.brc.riken.jp/ja/manual/cell15

Technical support
Other documents
Technical notes
Protocols
Brochureshttps://epd.brc.riken.jp/ja/manual/cell

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Quality Control
for Confirming Authenticityof Plant Cell Lines

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The need for quality control
Plant cell lines with similar appearance

Pokeweed PAR

Pokeweed PAW

Vinca major Vma-1

Tomato Sly-1

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Genotyping check
1. Identification of plant species 【First year】
DNA barcoding● rbcL● psbK-psbI

2. Identification of plant cell lines
DNA barcoding● rbcLNCBI
GenBank【Annual】● psbK-psbI● matK ● atpF-atpH

Fragment analysis● SSR● In/Del

DNA sequencing● SNP● Mutation

Previousyears

Transgene insertion
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Genotyping
Tomato cell lines
DNA barcoding

DNA barcoding

DNA sequencing

rbcL

matK

Tm-1

Sly-1

Solanum
lycopersicum

Sly-1

Not tested

GCR26

Solanum
lycopersicum

GCR26/GCR237+/+

GCR237

Solanum
lycopersicum

GCR26/GCR237

Tm-1/Tm-1

Cell line

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Identification of species
DNA barcoding

Cell lines

Identification

Mistake6130 species

0psbK-psbI

11 species

0rbcL

96 genus *0rbcL* Cell lines with only genus nameSpecies not registered in NCBI Genbank
Matched only to genus

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Identification of cell lines
Plant species

Cell lines/species
MistakeDNADNA
Fragment Transgene
barcodingsequencing
analysis
insertion(Indistinguishable)(Total)23 species

1 (23)◯05 species

2–3 (11)◯0

Nicotiana tabacum

13◯

Arabidopsis thaliana

4

◯

◯

Solanum lycopersicum

3

◯

◯

0Spinacia oleracea

3◯◯0Catharanthus roseus

2◯◯0Coptis japonica

2◯◯0Lotus japonicus

3◯

Sesamum indicum

3◯

Glehnia littoralis

2

◯

0 (2)

Luffa aegyptiaca2◯

0 (2)

◯◯0◯

◯

00 (2) Culturetype◯

0 (2)Color21

Summaryn Plant cell resources in RIKEN BRC– Released 72 cell lines (39 plant species)– GFP-expressing cell lines– Secondary metabolite-producing cell linesn Resource information– Exp-Plant Catalog– RIKEN BRC plant cell line documentationn Quality control for confirming authenticity– Genotyping
Plant species (First year)
Plant cell lines (Annual)
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