Genetic engineering of black cherry (Prunus serotina) for reproductive sterility and insect pest resistance
Abstract
Black cherry (Prunus serotina Ehrh.) is one of the most valuable hardwoods for high- end cabinetry, furniture, architectural millwork, paneling, and veneer. However, the damage caused by cambial-mining insect pests triggers gummosis in black cherry, a non-specific defense response in which resinous gum is deposited at the site of injury. The gum defects dramatically decrease the yield of high-quality black cherry lumber, and the value can be reduced by as much as 90%. The goal of this project was to optimize the transformation and in vitro rooting system, and to develop transgenic black cherry for reproductive sterility and insect pest resistance. To achieve this, a TERMINAL FLOWER 1 homolog from black cherry was isolated and characterized as one of the strategies for gene containment. The PsTFL1 cDNA contained a 519 bp coding region encoding a putative protein of 172 amino acid residues that have high identity with TFL1 orthologs of other Prunus species. The quantitative polymerase chain reaction (qPCR) detected a single copy of PsTFL1 in the genome of black cherry genotype BC3 with two alleles. The highest mRNA level was detected in shoot tips, and the lowest level in the leaves. Transgenic Arabidopsis thaliana plants overexpressing PsTFL1 showed significantly delayed flowering. Second, an improved Agrobacterium-mediated transformation system for the elite mature genotype of black cherry, BC3, was developed based on an existing protocol developed in our lab. The application of 15 minute vacuum infiltration, Agrobacterium concentration at OD600 of 1.0 or 1.5, and a 3-day co-cultivation were found to be optimal. Rooting of transgenic black cherry shoots was achieved at 30% using half-strength Murashige and Skoog medium supplemented with 2% (w/v) sucrose, 5 &mgr;M napthaleneacetic acid, 0.01 &mgr;M kinetin, and 0.793 mM phloroglucinol, and transgenic plants were successfully acclimatized. Finally, three independent transgenic lines of black cherry overexpressing PsTFL1 were obtained using the improved Agrobacterium-mediated transformation system. The integration of transgenes and the copy number of neomycin phosphotransferase (nptII) were confirmed by PCR and qPCR analysis. The expression levels of PsTFL1 in three 35S::PsTFL1 lines were 3.7 to 5.8 -times higher than that of the wild-type, and these shoots were successfully rooted and acclimatized. To enhance insect pest resistance, the black cherry endogenous genes encoding prunasin hydrolase isoform 3 (PH3) and mandelonitrile lyase isoform 4 (MDL4) were inserted into the black cherry genome respectively, under the control of the phloem-specific promoter rolC or the CaMV 35S promoter. However, in the lines of 35S::mdl4-FLAG, rolC::mdl4-FLAG, and rolC::ph3-FLAG, the mRNA level of mdl4 or ph3 was not increased. Factors that might have caused this in the transgenic shoots were discussed.
Degree
Ph.D.
Advisors
Pijut, Purdue University.
Subject Area
Molecular biology|Botany|Entomology|Forestry
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