Agrobacterium-mediated transformation of lettuce for enhancement of disease resistance

Abstract

Lettuce is affected by a number of bacterial and fungal pathogens, which can result in a dramatic decrease in yield. Traditional breeding approaches for disease resistance are normally reliant on resistant germplasm being available in sexually compatible plant species. Plant genetic engineering may be used to supplement more conventional breeding methodologies, where introgression of disease resistance into elite cultivars is problematic using traditional methods. The objective of this thesis was to use Agrobacterium-mediated transformation to transfer four synthetic antimicrobial genes, based on magainin 2, an antimicrobial peptide from the African clawed toad, into lettuce in an attempt to enhance disease resistance.

Magainin 2 was found to be non-toxic to lettuce seedlings in vitro at 50 µg/ml. In vitro experiments indicated that magainin was toxic to three important pathogens of lettuce, Erwinia carotovora subsp. carotovora (causal agent of soft rot), Pseudomonas marginalis (causal agent of soft rot) and Xanthomonas campestris pv. vitians (responsible for leaf spot of lettuce). Preliminary studies suggested that three fungal lettuce pathogens, Sclerotinia minor (causal agent of lettuce drop), S. sclerotiorum (causal agent of lettuce drop) and Botrytis cinerea (responsible for Botrytis and grey mold), were insensitive to magainin at 50 µg/ml.

Transformation of lettuce was attempted using Agrobacterium strain AGLI containing the binary vector pBINMgA, and Agrobacterium strain EHA105 carrying either binary vector pBINMgB, pBINMgC or pBINMgD. These vectors each encoded one of four different synthetic genes encoding peptides based on magainin 2. Regenerated transformed shoots of Cobham Green (butterhead type) were successfully obtained. However, attempts to transform lettuce cultivars Bambino, Prizehead and Summer Gem (crisphead types) did not meet with any success, confirming the previously reported recalcitrance of crisphead lettuce cultivars to Agrobacterium-mediated transformation. Identification of transformed shoots was confirmed by their ability to root on kanamycin-supplemented medium, and polymerase chain reaction (PCR) analysis to detect the selectable marker gene (nptII). During this phase of the study, it was discovered that the antibiotic Timentin was unable to control the growth and/or eliminate Agrobacterium after the initial co cultivation period. It was mainly for this reason that the final number of independently-derived transgenic Cobham Green plants was restricted to 39.

Further PCR analysis revealed that 35 of the 39 primary transformants contained, in addition to the presence of the nptII gene, the expected chimeric magainin gene. X campestris growth was inhibited in the presence of crude leaf extracts from two of the primary transformant lettuce lines, and from leaf extracts from one line in the T1 generation. Extracts from untransfonned control lines had no effect on the growth of X campestris. There was evidence that magainin 2 activity was inhibited by some component of the lettuce extract, which may be due to proteolytic degradation by plant proteases. Segregation analysis on the selfed progeny of transformed lines revealed that most lines conformed to either a 3:1 or 15:1 ratio for transmission of the nptII gene, indicating a single T-DNA insertion or two unlinked TDNA insertions, respectively.