Abstract
Reduced growth habit is a desirable trait for ornamental potted plants and can successfully be obtained through Rhizobium rhizogenes transformation in a stable and heritable manner. Additionally, it can also be obtained by transformation with Agrobacterium tumefaciens harboring specific genes from R. rhizogenes. The bacterial T-DNA harbors four root oncogenic loci (rol) genes and 14 less known open reading frames (ORFs). The four rol genes, i.e., rolA, rolB, rolC, and rolD, are conceived as the common denominator for the compact phenotype and the other less characterized ORFs seem auxiliary but present a potential breeding target for less aberrant and/or more tailored phenotypes. In this study, Kalanchoë blossfeldiana ‘Molly’ was transformed with individual rol genes and selected ORFs in 35S overexpressing cassettes to comprehensively characterize growth traits, gene copy and expression, and ethylene tolerance of the flowers. An association of reduced growth habit, e.g. height and diameter, was observed for rolB2 and ORF14-2 when a transgene single copy and high gene expression were detected. Chlorophyll content was reduced in overexpressing lines compared to wild type (WT), except for one ΔORF13a (a truncated ORF13a, where SPXX DNA-binding motif is absent). The flower number severely decreased in the overexpressing lines compared to WT. The anthesis timing showed that WT opened the first flower at 68.9 ± 0.9 days and the overexpressing lines showed similar or up to 24 days delay in flowering. In general, a single or low relative gene copy insertion was correlated to higher gene expression, ca. 3 to 5-fold, in rolB and ΔORF13a lines, while in ORF14 such relation was not directly linked. The increased gene expression observed in rolB2 and ΔORF13a-2 contributed to reducing plant growth and a more compact habit. Tolerance of detached flowers to 0.5 μl L−1 ethylene was markedly higher for ORF14 with 66% less flower closure at day 3 compared to WT. The subcellular localization of rolC and ΔORF13a was investigated by transient expression in Nicotiana benthamiana and confocal images showed that rolC and ΔORF13a are soluble and localize in the cytoplasm being able to enter the nucleus.
Highlights
Rhizobium rhizogenes-mediated plant transformation has for the last 4 decades been intensively applied in many studies with the pursuit of introducing compact habit and reduced plant growth traits in plants, primarily ornamentals (Christensen et al, 2008; Pérez de la Torre et al, 2018)
The open reading frames (ORFs) predicted by FgenesH with the highest similarity was ORF10 resulting from the algorithm and was 228 nucleotides long from position 15,012–14,785 in the reverse strand of the pRiA4 and consisted of one exon
Albeit sequences shared 68% identity and 76% similarity at amino acid level, the ORF13a sequence from pRiA4 is 34 amino acids shorter than that of pRi8196; the pRiA4 ORF13a sequence starts at the amino acid 36 of the ORF13a of pRi8196 and the alignment continues to the end of both sequences
Summary
Rhizobium rhizogenes-mediated plant transformation has for the last 4 decades been intensively applied in many studies with the pursuit of introducing compact habit and reduced plant growth traits in plants, primarily ornamentals (Christensen et al, 2008; Pérez de la Torre et al, 2018). These approaches have been pursued in several ornamental plants, e.g., Calibrachoa (Gennarelli et al, 2009), Kalanchoë (Christensen et al, 2008), Mecardonia (Pérez de la Torre et al, 2018), Petunia (Mishiba et al, 2006), and poinsettia (Islam et al, 2013) Some of these strategies target alteration of gibberellic acid (GA) metabolism; GA20 oxidases have silenced (Topp et al, 2008), GA2 oxidases have been over-expressed (Gargul et al, 2013), the Arabidopsis thaliana Short Internodes gene has been overexpressed (Lütken et al, 2010; Islam et al, 2013), and knotted homeobox genes have been modulated (Lütken et al, 2011). The terminology of “natural transformation” has been used as a label for these strategies and it presents a very promising beacon for generating compact habit and reduced plant growth (as reviewed by Lütken et al, 2012a; Desmet et al, 2019)
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