Control of plant regeneration in vitro: Manipulation of environmental parameters, polyamine metabolism, and auxin biosynthesis
Abstract
Genetic engineering of indica rice (Oryza sativa L.) requires an in vitro culture system from which plants can be regenerated. To optimize regeneration of plants from callus, several cultural parameters first were tested. Callus induction from immature zygotic embryos and plant regeneration were dependent on basal medium and amendments, explant plating density, growth regulator concentration, and plant cultivar. Maximum regeneration was achieved by plating immature embryos of IR54 on N6 callus induction medium supplemented with 1 g/L casein hydrolysate, 0.2% (w/v) Gelrite, and 0.5 mg/L 2,4-D, then subculturing one callus per plate to regeneration medium lacking 2,4-D. Second, to ascertain whether in vitro culture of rice necessitates the biosynthesis of polyamines, we measured polyamine pools using high performance liquid chromatography and used polyamine biosynthesis inhibitors during callus and cell suspension culture of IR54. Regeneration of plants from callus and growth of cell suspension cultures were correlated with polyamines. Although dedifferentiation was unaffected by polyamine biosynthesis inhibitors, 10-25 mM difluoromethylarginine (DFMA, a specific inhibitor of arginine decarboxylase) significantly inhibited somatic embryogenesis, suspension culture growth, and plant regeneration. Thus, polyamine metabolism via arginine decarboxylase is involved in somatic embryo development in rice. Finally, to determine whether endogenous auxin levels could be manipulated to obtain selective growth of transgenic cells, carrot (Daucus carota L.) protoplasts were treated with a plasmid encoding constitutive expression of tms2. Dividing protoplasts were subcultured to selection medium containing 50 $\mu$M kinetin and 0 to 100 $\mu$M auxin amide (indole- or napthalene-acetamide). At low protoplast plating density putatively transformed cell clusters were larger and whiter than controls; however, that this phenotype was due to the expression of tms2 could not be confirmed in amidohydrolase assays or by Southern analysis. Prospects for the use of amidohydrolase activity as a selectable marker are discussed.
Degree
Ph.D.
Advisors
Hodges, Purdue University.
Subject Area
Botany
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