Transgenic Kalanchoë blossfeldiana, Containing Individual rol Genes and Open Reading Frames Under 35S Promoter, Exhibit Compact Habit, Reduced Plant Growth, and Altered Ethylene Tolerance in Flowers

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.

Rhizobium rhizogenes rolC gene promotes leaf senescence and enhances osmotic stress resistance in Arabidopsis: the positive role of abscisic acid

“…The dwarfing and well-branched phenotypes observed in the present study indicate reduced apical dominance of rolC-OX plants (Figure 1A-C), an effect that was also seen in carnation and tobacco plants overexpressing rolC (Eva Casanovaa 2003). It has been previously speculated that over-expression of rolC may lead to increased CTK levels (Favero et al 2021), a feature that might contribute to the phenotypes observed here. In general, high ratios of CTKto-auxins promote plant shoot differentiation (Naseem et al 2012); however, here, the gene expression data did not support the idea based on the CTK transcript levels.…”

“…The rol C sequence was amplified from the total DNA of R. rhizogenes A4 strain (ATCC43057) (Favero et al 2021) on a MyCycler (Biorad) using TaKaRa LA Taq polymerase (Mountain View). The amplification product was BP‐cloned into the entry vector pDONR221 and subsequently recombined with the destination vector pK2GW7 to generate 35S:rol C fusion constructs according to Favero et al (2021).…”

“…The rol C sequence was amplified from the total DNA of R. rhizogenes A4 strain (ATCC43057) (Favero et al 2021) on a MyCycler (Biorad) using TaKaRa LA Taq polymerase (Mountain View). The amplification product was BP‐cloned into the entry vector pDONR221 and subsequently recombined with the destination vector pK2GW7 to generate 35S:rol C fusion constructs according to Favero et al (2021). The recombinant plasmids containing rol C clones were transferred into electrocompetent Agrobacterium tumefaciens C58C1 (pGV3850) by electroporation, then rol C gene was transformed to Arabidopsis plants by the floral dip method (Chen et al 2022).…”

“…In addition, plants transformed solely with rolC gene display a series of morphological abnormalities, including reduced height, increased number of branches, and better rooting ability, etc. (Favero et al 2021). The morphological alterations and abiotic stress resistance induced by rolC indicate its mechanism of action interferes with common factors that modulate plant development and stress responses.…”

Kalanchoë Breeding: Past, Present and Future

“…Later, F1 plants were inbred and it was found that the same correlation between the presence of rol-genes and compactness continued in the obtained F2 plants (Lütken et al, 2012b). Moreover, the study of individual rol genes and less characterized open reading frames from R. rhizogenes have very recently been shown to present higher ethylene tolerance in transgenic lines containing 35S::ORF14 (Favero et al, 2021).…”