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Generation of Micro-Tom
Based Mutant Lines for Tomato Genomics by
Japanese Solanaceae Genomics Consortium (JSOL)
Hiroshi Ezuraa , Tsuyoshi Mizoguchia, Shin Watanabea, Sayaka Uchiia, Sun Hyon Jina, Yasutaka Kubob, Hitoshi Moric,
Shunsuke Imanishid, Daisuke Shibatae
aGene
Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-shi, Ibaraki-ken, 305-8577, Japan,
bGuraduate School of National Science, Okayama University, 1-1-1 Tsushima-naka, Okayama-shi, Okayama-ken, 700-8530
cGuraduate School of Bioagricultural Sciences, Nagoya University, Furo-cho ChiFull Address,Japan
dNational Institute of Vegetable and Tea Science, NARO, 360 Kusawa, Ano, Mie-ken, 514-2392, Japan
eKazusa DNA Research Institute, Kazusa-Kamatari 2-6-7, Kisarazu-shi, Chiba 292-0818, Japan
Email: [email protected]
2. Micro-Tom Mutant Database
Introduction
The spotlight for most plant scientists must be post-genomic studies such as
transcriptome, proteome, and metabolome. In addition, advance in bioinfomatics
pioneer new category of studies like system biology. Tomato is a model plant that
gives information on fruit storage, product translocation and novel metabolites. It is
highly expected that other solanaceae plant will have constructive feedback from the
genetic and molecular study of tomato. Moreover, the development of L. esculentum
var. Micro-Tom gave us a great advantage to examine tomato plantlet in narrow room
space. Our main objective is to construct an infrastructure for Micro-Tom study.
Mutanegenesis has been highly effective strategy for studying the genetic bases of
traits. One of the mutational approach is a chemical mutagenesis by ehylmetane
sulphate (EMS) treatment which gives rise to a high mutation frequency without
apparent preferences for specific genomic regions. It can also generate many alleles
that enable one to get null phenotypes. At present, we are establishing Micro-Tom
mutant lines by EMS treatment and a construction of the database in silico with aim
to supply the experimental resource and the information for worldwide use. Focusing
on both "Floral and circadian rhythm in neuter plant" and " plant hormone and fruit
development", we have already set up a system for screening mutants. In addition, we
have established an efficient transformation protocol for developing T-DNA tag lines
in Micro-Tom. Here we report the latest progress in our work conducted by Japanese
Solanaceae consortium (JSOL).
Organization: 001 かずさ
To integrate various and
enormous mutant information from
each institutes, Micro-Tom Mutant
Database are under construction in
silico. This is a collaboration work
with National Institute of Genetics in
Japan. final object of the database is
offcorse to supply the experimental
resource and the information for
worldwide use. Start of test operation
is scheduled for during next spring.
Mutant name:
●　phenotype
●　growth stage
□　□　□　□　□　x
□　□　Mutant no.
1. Se ed
1-1.Germ ination
1-2.See dling le thality
1-3.Slow germ ination
2. Plant size
2-1. Extre m e ly sm all
2-2.Sm all plant
2-3. Large plant
3. Plant habit
3-1. Internodele ngth
3-2. Branching
3-3. Aborted grow th
4. Le af m orphology
4-1. Le af w idth
4-2. Le af s ize
4-3. Le af texture
5. Le af color
5-1. Purple leaf
5-2. Ye llow leaf
5-3. Ye llow -gre en le af
5-4. Dull gre en/gray leaf
5-5. Varie gation
6. Flow ering tim ing
7. Inflores ce nce s tructure
8. Flow er m orphology
8-1. Flow e rhom eoticm utation
8-2. Flow e r organ size
8-3. Flow e r organ w idth
0:germination
1:leaf production
2:side shoot
3:inflorescence
4:flowering
5:fruit
6 ripening
Comments
(free text format)
Image upload
IMG
x
9. Flow er color
9-1. White flow er
9-2. Pale yellow flow er
9-3. Strong yellow flow er
10. Fruit s ize
10-1. Sm all fruit
10- 2. Large fruit
11. Fruit m orphology
11-1. Long fruit
11-2. Rounde d fruit
11-3. Othe r fruit m orphology
12. Fruit color
12-1. ye llow fruit
12-2. orange fruit
12-3. dark re d fruit
12-4. Epiderm is
12-5. Gree n fruit
13. Fruit ripe ning
13-1. Early ripe ning
13-2. Late ripening
14. Ste rility
14-1. Partial ste rility
14-2. Full ste rility
15. Dise as e and s tre ss re sponse
15-1. Ne cros is
15-2. Wilting
15-3. othe r dise as e res pons e
Figure 4. Micro-Tom Mutant Database
3. Establish Micro-Tom Transformation System for T-DNA Tag Line
sterized seedling
1 week
seedling
1. Micro-Tom Mutant Line
acet osyringone
After organizing Japanese Solanaceae consortium (JSOL) in 2004, we immediately
decided to establish Micro-Tom mutant line and to set up a system for screening mutants.
We started to cultivate M1 plants at the end of 2004 (Figure 1) and we had harvested 4000
M1 Plants by this spring and obtained 2500 M2 families (Table 1). These M2 Plants have
already cultivated and been ready for screening.
Agrobact erium freezed stock solut ion
incubat ed for 24hr at room t emp.
Agrobact erium
infect ion
remove surplus
Agrobact erium
cocult ivation
acet osyringone
3-5days
callus induct ion
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root induct ion
Figure 1. JSOL Micro-Tom Mutant Line
2 weeks x 3t imes
Tabl e 1. Nu m be r of EMS tre ate d se e ds an d h arve ste d pl an ts
1st
2n d
EMS C on ce n tration
Tre ate d S e e ds Nu m be r
M2 Fam i li e s
0.3 or 0.5%
4000
2500
1.0%
3000
root ing selection
(in t ripricate)
Figure 6. Chimera
both transformed and
t ransformant
か
untransformed cells
After establishing
mutant lin, it is necessary to
develop T-DNA tag lines for
mutant analysis. However,
largest trouble delaying the
study mihgt be absence of highl
efficent transformation
procedure for tomato species.
Therefore, We have established
highly efficient transformation
protocol for developing T-DNA
tag lines in Micro-Tom (Figure
5). We succeeded to achieve
highly efficient transformation
ratio (approximately 40%) by
addition of acetoshringone two
times during cultivation period
and operation of careful rooting
selection to pick up
transformed cells thoroughly
(Figure 7). Especially, it is
effective for highly efficient
transformation to repeat
separate operation at rooting
selection step because organs
undergoing redifferentiation to
form multiple shoots included
both transformed and
untransformed cells (Figure 6).
Figure 5. Micro-Tom Transformation System
n ow h arve sti n g
HOW TO DO ROOTING SELECTION
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Figure 2. JSOL Micro-Tom Mutant Line Team
JSOL Micro-Tom Mutant Line
project is a collaborate work with 5
Japanese institutes. Now M2 Plant
seeds are distributed to each institutes
and cultivated (Figure 2).
At the same time, we are establishing
TILLING SYSTEM for screening
mutants. Some interesting phenotypes
in M2 Plants are discribed in Figure 3.
3 weeks
1st experiment
43 callus from
80 cut leaves
2 weeks
induced shoots
2nd experiment
39 callus from
80 cut leaves
2 weeks
2 weeks
2 weeks
2 weeks
root ing rat io
16/43
root ed shoot s
unrooted shoots
root ed shoot s
unrooted shoots
root ed shoot s
unrooted
shoots
3 weeks
induced shoots
root ing rat io
16/43
root ed shoot s
unrooted shoots
root ed shoot s
unrooted shoots
root ed shoot s
unrooted
shoots
3 weeks
induced shoots
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unrooted shoots
Figure 7. Key Point for high transformation ratio
3. Conclusion
root ing rat io
7/43
root ed shoot s
root ed shoot s
unrooted shoots
root ed shoot s
unrooted
shoots
1st experiment
root ing rat io
39/43callus
39/80 (49%)
2nd experiment
root ing rat io
30/39callus
30/80 (38%)
JSOL Micro-Tom Mutant Line project is a collaborate work with 5 Japanese
institutes. Now M2 Plant seeds are distributed to each institutes and cultivated (Figure 2).
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Figure 3. M2 Plants