Compact shoot architecture of Osteospermum fruticosum transformed with Rhizobium rhizogenes.

KEY MESSAGE: Improved compact shoot architecture of Osteospermum fruticosum Ri lines obtained through Rhizobium rhizogenes transformation reduces the need for chemical growth retardants. Compactness is for many ornamental crops an important commercial trait that is usually obtained through the application of growth retardants. Here, we have adopted a genetic strategy to introduce compactness in the perennial shrub Cape daisy (Osteospermum fruticosum Norl.). To this end, O. fruticosum was transformed using six different wild type Rhizobium rhizogenes strains. The most effective R. rhizogenes strains Arqua1 and ATCC15834 were used to create hairy root cultures from six Cape daisy genotypes. These root cultures were regenerated to produce transgenic Ri lines, which were analyzed for compactness. Ri lines displayed the characteristic Ri phenotype, i.e., reduced plant height, increased branching, shortened internodes, shortened peduncles, and smaller flowers. Evaluation of the Ri lines under commercial production conditions showed that similar compactness was obtained as the original Cape daisy genotypes treated with growth retardant. The results suggest that the use of chemical growth retardants may be omitted or reduced in commercial production systems of Cape daisy through implementation of Ri lines in future breeding programs.

Rhizogenic agrobacteria as an innovative tool for plant breeding: current achievements and limitations.

Compact plant growth is an economically important trait for many crops. In practice, compactness is frequently obtained by applying chemical plant growth regulators. In view of sustainable and environmental-friendly plant production, the search for viable alternatives is a priority for breeders. Co-cultivation and natural transformation using rhizogenic agrobacteria result in morphological alterations which together compose the Ri phenotype. This phenotype is known to exhibit a more compact plant habit, besides other features. In this review, we highlight the use of rhizogenic agrobacteria and the Ri phenotype with regard to sustainable plant production and plant breeding. An overview of described Ri lines and current breeding applications is presented. The potential of Ri lines as pre-breeding material is discussed from both a practical and legal point of view.

Differential efficiency of wild type rhizogenic strains for rol gene transformation of plants.

Rhizogenic agrobacteria induce extensive root proliferation, in several economically valuable, dicotyledonous plant species, a phenomenon referred to as "hairy roots." Besides their pathogenic nature, agrobacteria have proven to be a valuable asset in biotechnology and molecular plant breeding. To assess the potential of frequently used rhizogenic strains, growth in yeast extract glucose broth and antibiotic resistance was analyzed. Growth curves were established for Arqua1, NCPPB2659, LMG150, LMG152, and ATCC15834; and regression analysis of the exponential growth phase resulted in a reliable and standardized method for preparation of a bacterial suspension for inoculation. Cell density did not correlate with the timing of hairy root emergence. The highest number of hairy roots was obtained with an inoculum of 1 × 108 CFU ml-1 for Arqua1, NCPPB2659, and LMG152. Cell density of ATCC15834 did not affect the number of hairy roots formed. The identity of the rhizogenic strains for plant transformation was verified in phylogenetic analysis using average nucleotide identity (ANI), which also provided insight in their genetic diversity within the Rhizobium taxon.

Molecular and cytogenetic characterization of Osteospermum fruticosum lines harboring wild type pRi rol genes.

Transgenic lines engineered through wild type Rhizobium rhizogenes display an altered phenotype known as the Ri phenotype. This phenotype includes a more compact plant habit, which has proved useful to obtain more compact varieties that require less chemical growth regulation. Here, we develop a method for the molecular and cytogenetic characterization of Cape daisy (Osteospermum fruticosum Norl.) Ri lines in order to predict segregation of pRi T-DNA genes. Analysis of copy number variation (CNV) by means of digital PCR indicated large variation in the copy number of the inserted root oncogenic loci (rol) genes, ranging from 1 to more than 15 copies. In addition, up to 9 copies of the auxin biosynthesis genes (aux) were present in a single Ri line. Visualization of pRiA4 and pRi1724 rol and aux insertion in 4 Ri lines was performed through Fluorescence In Situ Hybridization. The number of rol integrated loci varied from 1 to 3 loci. In contrast, the different TR-gene copies were confined to a single locus which consistently co-localized with a TL locus, this was demonstrated for the first time. Based on CNV and FISH a single Ri line, harboring 7 pRi1724 rol gene copies dispersed over 3 integration loci, was selected for breeding. Copy number segregation in R1 progeny of 2, 3, 4 and 5 pRi1724 copies was confirmed, indicating that the evaluation of the breeding value of first generation Ri lines is possible through CNV and FISH.

Segregation of rol Genes in Two Generations of Sinningia speciosa Engineered Through Wild Type Rhizobium rhizogenes.

Rhizobium rhizogenes infects and transforms a wide range of plant species. It thereby introduces new genes located on transfer-DNA of the root inducing plasmid (pRi) into the plant genome and one of its abilities is to alter the host root system. Explants from pRi transformed roots from Sinningia speciosa were regenerated to create naturally transgenic Ri lines. The presence of rol and aux genes in the Ri lines was linked with altered growth characteristics: shorter peduncles, wrinkled leaves, delayed flowering and enhanced root growth. The potential of Ri lines for breeding was evaluated through consecutive backcrossing with the original host genotype. The progeny of reciprocal crosses showed non-Mendelian inheritance suggesting partial transmission of the of the aux and rol genes. The typical Ri phenotype observed in the primary Ri line was partially inherited. These results revealed that the Ri phenotype is a complex trait influenced by the genetic background of the Ri line.