The effects of anther culture and plant genetic background on Agrobacterium rhizogenes-mediated transformation of commercial cultivars and derived doubled-haploid Brassica oleracea

Abstract

The production of transgenic roots was scored for eight Brassica oleracea cultivars from broccoli, cabbage, cauliflower and kale following inoculation with an Agrobacterium rhizogenes cell line carrying a binary plasmid bearing the green fluorescence protein (gfp) gene in the T-DNA. Significant differences in the numbers of explants producing transgenic roots were observed between cultivars, ranging from 1.4% for Marathon F1 to 57.8% for the Green Duke F1. Three F1 cultivars were subjected to anther culture, and doubled-haploid (DH) lines were used for transformation. The DH lines produced showed considerable variation for transgenic root production with some lines showing increased efficiency compared to the parental F1 cultivar. Grouping of the DH lines into response classes with respect to transgenic root production allowed the development of potential genetic models to explain the variation in performance released from each F1 cultivar. No apparent segregation distortion for transgenic root production was observed in the DH lines following anther culture.

Identification of genetic factors controlling the efficiency of Agrobacterium rhizogenes-mediated transformation in Brassica oleracea by QTL analysis

“…This observation is consistent with our earlier findings that the process of anther or microspore culture to generate homozygous lines in B. oleracea does not distort segregation for transgenic root production in the resulting population (Cogan et al 2001). Regions of the genome that contain enhancing QTL associated with transgenic root production originate from both parents, with the enhancing alleles coming from A12DHd for the QTL on linkage groups O1 and O3, while GDDH33 has an enhancing QTL allele on linkage group O7.…”

“…An optimised transformation system for B. oleracea has been developed utilising A. rhizogenes . We have already shown that the effects of plant genes regulating A. rhizogenes-mediated transformation of B. oleracea can be identified and established that plant genes controlling transformation segregate in a quantitative manner (Cogan et al 2001). In addition, we have shown that there was no segregation distortion associated with transformation efficiency as a consequence of producing anther culture-derived DH lines from a range of B. oleracea germplasm.…”

Double haploids, markers and QTL analysis in vegetable brassicas

“…The timescale for producing inbred lines for use as parents of hybrid cultivars is reduced from 6+ years to approximately 2 years (Dias 2001). In addition, where strong incompatibility alleles are present in breeding material, DH production only requires one round of bud self-pollination to produce seed of homozygous lines, compared to Wve rounds of self-pollination to produce an F6 inbred where there will still be residual heterozygocity (»98% homozygous).…”

Production of Transgenic Vegetable Brassicas

“…It is likely that future research optimizing transformation conditions will use the green fluorescent protein (GFP) reporter gene that enables transformation to be monitored non-destructively, as shown by Cogan et al (2001) with broccoli. Due to the low efficiency of transformation, several authors have used transient GUS expression as a simple assay for establishing the optimal conditions for transformation.…”