Adventitious shoot regeneration and genetic transformation of Prunus serotina for reproductive sterility
Black cherry (Prunus serotina Ehrh.) is a valuable hardwood in the eastern United States and Canada. Demand for high-quality black cherry wood is increasing and there is a need to establish plantations with improved black cherry trees. Genetically improved trees containing foreign genes are subject to government regulatory guidelines for field planting because of the potential for dispersal of transgenic pollen and seeds, and the environmental impact could be difficult to predict and control. To reduce the dispersion of some genes, engineering reproductive sterility will help simplify the impact, and thus facilitate regulatory and public approval. In this dissertation, a regeneration and transformation system for mature black cherry explants was developed, and transgenic black cherry plantlets with the AGAMOUS (AG) gene cloned from black cherry or the apple AG gene were produced. ^ A regeneration system for black cherry from a juvenile (F) and two mature genotypes (#3 and #4) was developed. Adventitious shoots regenerated from leaves of in vitro cultures on woody plant medium with thidiazuron (TDZ) and naphthaleneacetic acid (NAA). The best regeneration for genotype F (91.4%) with a mean number of 8.2 shoots was observed on medium with 9.08 μM TDZ and 1.07 μM NAA. Genotype #3 had the highest regeneration (41.7%) with a mean number of 4.8 shoots per explant on 9.08 μM TDZ and 1.07 μM NAA, whereas genotype #4 had a 38.8% regeneration with a mean of 3.3 shoots per explant. Silver thiosulphate at 60 or 80 μM increased the percent regeneration of the mature genotypes #3 (75%) and #4 (58.1%). Adventious shoots were rooted (70 to 76%) and rooted plantlets survived acclimatization in the greenhouse. ^ PsAG (a putative flower homeotic identity gene) was cloned and characterized from black cherry and its MIKC-type structure shown to be a homolog of the Arabidopsis thaliana AG. It is a single-copy gene in black cherry. A phylogenetic analysis indicated that PsAG is a C-class flower homeotic gene, and it has high similarity to other AG homologs such as those from Prunus persica and P. mune. PsAG meets the criteria for AG subfamily gene structure with a typical MIKC structure. In situ hybridization showed that PsAG was expressed mainly in the floral meristem, such as stamen and carpel primordia when the flowers are in the early stages of development, and PsAG accumulated in the tissues of the ovary, stigma, style, and stamens. When the flowers matured, PsAG had enhanced expression in the ovary, style, and stigma with decreased expression in the stamen. PsAG remains to be expressed in the ovules. The expression pattern was consistent with those of AG and homologs from other species, such as apple and peach. ^ A protocol for Agrobacterium-mediated transformation was developed for leaf explants of black cherry. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the apple AG gene or black cherry AG gene was used. Regenerating shoots were selected 27 days or 48 days after co-culture. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial number of leaf explants tested ranged from 0.9% for construct MdAGRNAi (apple) and 1.2% for PsAGRNAi (black cherry). Southern blot analysis on five randomly chosen PCR-positive shoots confirmed the presence of the nptII and AG transgene, and late selection protocols used in this study have greatly improved the transformation efficiency in black cherry. ^
Paula M. Pijut, Purdue University.
Biology, Molecular|Biology, Genetics