Minisymposium 28: Biotechnology

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Abs # M2802: Phage site-specific recombinases for marker gene excision in tobacco chloroplasts

Presenter:	Lutz, Kerry A       Contact Presenter
Authors	Lutz, Kerry A (A)   Bosacchi, Massimo H (A)   Kittiwongwattana, Chokchai  (A)   Clark, Mark  (A)   Mailga, Pal  (A)
Affiliations:	(A): Rutgers University

Plastid transformation requires uniform alteration of the 1,000-to 10,000-plastid genome copies present in a cell by selection for antibiotic resistance encoded in a marker gene. Once all plastid DNA copies are transformed the marker gene is no longer needed to maintain the transformed state. We describe here excision of plastid marker genes using two different site-specific recombinases, the CRE-loxP and the phiC31 integrase. The Cre-loxP site-specific recombinase was previously described for efficient removal of plastid marker genes but required transformation of the nuclear genome with a nuclear-encoded plastid targeted Cre gene (1, 2). We report here a novel protocol for CRE-mediated marker gene excision without transformation of the plant nucleus (3). The approach involved leaf agroinfiltration for CRE expression, followed by plant regeneration in the absence of selection and yielded ~10% plastid marker-free plants without an integrated nuclear Cre. Unfortunately, marker excision sometimes involved deletions between loxP sites and fortuitous plastid DNA sequences (4). We are testing if such undesirable deletions can be avoided by using an alternate site-specific recombinase, the phiC31 integrase (INT). At the time of the meeting we will report if INT can efficiently excise plastid marker genes while avoiding undesirable deletions in the plastid genome. 1. Hajdukiewicz, et. al. Plant J 27, 161 (2001) 2. Corneille, et. al. Plant J 72, 171 (2001) 3. Lutz, et. al. Plant J 45, 447 (2006) 4. Corneille, et. al. Plant J 35, 753 (2003)