1. Is My Gene Important for Seed Development in Plants?? Gene: AT3G53370 Jonathan Milgrom Spring 2004

2. What Does My Gene Do? Transcription Factor* Repressor of Spinach gene rps1 Active in roots Highly conserved in plant kingdom Transcription factor *Dao-Xiu Zhou, Cordelia Bisanz Seyer

3. Is This Function Integral to Seed Development ? Knockout the gene and find out! Engineer agrobacteria to insert tDNA Grow mutant seeds Genotype Can an Arabidopsis plant develop without a functioning form of my gene? Agrobacteria naturally performs this task tDNA AT3G53370

4. Madison & Salk Provide the Knockouts 5’3’ SP #1 SP #4 Madison Salk AT3G53370 Exon

5. Knockout Analysis Madison project began with… Superpool Salk project began with… Mutant seeds Superpool tDNA line/ Genotype Mutant Seed Plant Sp #4 PCR products amplified with RV (gene specific) and tdna primers 4/15/04 (small length suggests insertion)

6. Madison Results Identified Madison DNA Pool SP #4 DNA pool #6 Madison PCR products amplified with RV (gene specific) & tdna primers 5/18/04 3 SP#4SP#4 AT3G53370 3’ 5’ tDNA RV

7. SALK Results Homozygous tDNA exist Knockout is not embryo lethal genotype ** Wild Type ** Heterozygous *** Homozygous tDNA 9876541 Plant # Wild type PCR products of various SALK lines amplified with FW & RV (gene specific) primers 6/4/04 PCR products of various SALK lines amplified with RV (gene specific) and tDNA primers 6/1/04 1 4 5 6 7 8 9 WtWt C WtWt C Tubulin 7 Plants Genotyped…

8. Does My Gene Play Some Role in Seed Development? Scarlet Runner Bean equivalent Gene activity/mRNA accumulation patterns Lec1 mutant Arabidopsis 14 day old embryo RT-PCR of 6 organs

9. Next…? Study rsp1 spinach gene equivalent in Arabidopsis Study lec1 gene Identify Madison tDNA line and Genotype