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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, 700-8530cGuraduate School of Bioagricultural Sciences, Nagoya University, Furo-cho ChiFull Address,JapandNational Institute of Vegetable and Tea Science, NARO, 360 Kusawa, Ano, Mie-ken, 514-2392, JapaneKazusa DNA Research Institute, Kazusa-Kamatari 2-6-7, Kisarazu-shi, Chiba 292-0818, JapanEmail: ezura@gene.tsukuba.ac.jp

Figure 1. JSOL Micro-Tom Mutant Line

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Figure 3. M2 Plants

IntroductionThe spotlight for most plant scientists must be post-genomic studies suc

h 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).

Figure 4. Micro-Tom Mutant Database

Organization: 001 Ç©Ç∏Ç≥ Mutant no. Å@Å@Å@Å@Å@Å@Å@Mutant name:

1. Seed1-1.Germination1-2.Seedling lethality1-3.Slow germination2. Plant size2-1. Extremely small2-2.Small plant2-3. Large plant3. Plant habit3-1. Internode length3-2. Branching3-3. Aborted growth4. Leaf morphology4-1. Leaf width4-2. Leaf size4-3. Leaf texture5. Leaf color5-1. Purple leaf5-2. Yellow leaf5-3. Yellow-green leaf5-4. Dull green/gray leaf5-5. Variegation6. Flowering timing7. Inflorescence structure8. Flower morphology8-1. Flower homeotic mutation8-2. Flower organ size8-3. Flower organ width

9. Flower color9-1. White flower9-2. Pale yellow flower9-3. Strong yellow flower10. Fruit size10-1. Small fruit10- 2. Large fruit11. Fruit morphology11-1. Long fruit11-2. Rounded fruit11-3. Other fruit morphology12. Fruit color12-1. yellow fruit12-2. orange fruit12-3. dark red fruit12-4. Epidermis12-5. Green fruit13. Fruit ripening13-1. Early ripening13-2. Late ripening14. Sterility14-1. Partial sterility14-2. Full sterility15. Disease and stress response15-1. Necrosis15-2. Wilting15-3. other disease response

ÅúÅ@phenotype

ņÅ@ 0:germination

ņÅ@1:leaf production

ņÅ@2:side shoot

ņÅ@3:inflorescence

ņÅ@4:flowering

ņÅ@5:fruit

ņÅ@6 ripening

ÅúÅ@growth stage

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Generation of Micro-Tom Based Mutant Lines for Tomato Genomics by

Japanese Solanaceae Genomics Consortium (JSOL)

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Figure 2. JSOL Micro-Tom Mutant Line Team

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1. Micro-Tom Mutant LineAfter organizing Japanese Solanaceae consortium (JSOL) in 2004, we immed

iately 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.

2. Micro-Tom Mutant Database

3. Establish Micro-Tom Transformation System for T-DNA Tag Line

3. ConclusionJSOL Micro-Tom Mutant Line project is a collaborate work with 5 Japanese in

stitutes. Now M2 Plant seeds are distributed to each institutes and cultivated (Figure 2).

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.

1st 2nd

EMS Concentration 0.3 or 0.5% 1.0%

Treated Seeds Number 4000 3000

M2 Families 2500 now harvesting

Table 1. Number of EMS treated seeds and harvested plants

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.

Figure 5. Micro-Tom Transformation System

Figure 7. Key Point for high transformation ratio

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 6. Chimera both transformed and unt

ransformed cells