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ARS Home » Midwest Area » Madison, Wisconsin » Vegetable Crops Research » Research » Research Project #442904

Research Project: Agrobacterium-mediated Transformation in Cranberry: Establishment and Optimization

Location: Vegetable Crops Research

Project Number: 5090-21220-004-029-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 1, 2022
End Date: Oct 31, 2024

Objective:
1) Establishing an efficient generation system for Cranberry by optimizing tissue culture protocols. 2) Developing Agrobacterium mediated genetic transformation system for cranberry using different varieties.

Approach:
Objective 1: Using leaf discs, we will establish an efficient adventitious regeneration system for Cranberry (Vaccinium macrocarpon Ait.). The approach involves: Selection of plant material from various cultivars of Cranberry. Initiation and maintenance of in vitro shoot utilizing various tissue culture media. The optimization of regeneration media by employing various combinations of plant growth regulators. Investigation of their effect on the shoot and root development. Using this optimized regeneration protocol, we will explore genetic variability in cranberry cultivars (>6). Objective 2: We will produce callus and adventitious buds using optimized tissue culture protocols from objective one from young leaves. The ploidy levels will be measured using a flow cytometer. The green fluorescent protein (GFP) and beta-glucuronidase (GUS) will be transformed into Agrobacterium tumefaciens by constructing the plasmid. Different concentrations of Agrobacteria with different incubation time periods will be tried to find the best infection and transformation rate. The green fluorescence of leaf discs after the transformation will be examined using a fluorescence microscope. The portions of the petri dish that showed green fluorescence will be monitored. The regenerated shoots will be cultured for root growth and transplanted to potting media. The transformation of the GFP and GUS gene expression will be verified by isolating DNA and amplifying genes with PCR. Southern blotting will be performed to confirm the transformation and determine the copy numbers. The reproducibility of the protocol will be evaluated using various cultivars of Cranberry, and the optimal cultivar for transformation will be selected.