The RootsPlus project is half-way and we are pleased to communicate on the mid-term results each (scientific) partner has obtained.
For one of our private partners, we have further optimised the protocol to obtain Ri-plants from recalcitrant chrysanthemum genotypes. A genotype screen using a fluorescent bacterial strain identified some genotypes for which it is more difficult for the bacteria to enter the plant, but for the majority of the genotypes, obtaining hairy roots is not a real problem but rather the regeneration of shoots from these roots is the biggest bottleneck. Furthermore we are continuously optimising some molecular tools to screen the regenerated plants: a fast qPCR-assay to detect for the presence/absence of Ri-genes was fine-tuned, a dPCR assay to determine the exact copy-numbers of the inserted genes is being validated for this hexaploid plant and Fluorescent In-Situ Hybridisation (FISH) was applied on a selection of Ri chrysanthemum plants. This resulted in the identification of the positions on the chromosomes where the Ri genes were inserted. The amount of locations differed for each transformation event. These tools will now further be implemented on the F1 plants from the company's breeding program with the Ri plants.
Also for apple we collaborate with a private company. Likewise as the colleagues of LUH, we have been able to optimise the protocol to such an extent that we regenerated shoots originating from the same rootstock and scion cultivar. Besides, we established a tissue culture from some of the company's genepool and the first co-cultivation experiments on this material are currently running.
The protocols for hairy root induction in rose and apple and subsequent regeneration of Ri plants were established and are currently optimized also with the help of two BSc students and two MSc students. For rose, whole leaves formed hairy roots at a higher percentage than petioles or leaflets and sonication treatments improved the transformation efficiency in general. This optimized hairy root induction protocol is currently applied to phenotype more than 100 rose genotypes for the subsequent genome-wide association study (GWAS). More than 90 genotypes were characterized already and we will finish the remaining transformations soon and start with the data analysis in the beginning of 2023. The expected output of this GWAS is an insight into genes and genomic regions that are associated with transformability by R. rhizogenes. The integration frequencies of TL-, TR-DNA (60 – 91 %) and of the reporter gene GFP (21 %) were proven. Furthermore, we were able to establish growing rose hairy root cultures samples which were sent to our partners at NCU for phytohormone profiling.
Also, for apple the protocol for induction of hairy roots was established in close collaboration with partners at ILVO. From the 6 genotypes tested so far, a sufficient hairy root induction could only be achieved in one rootstock and one scion cultivar. All bacterial strains tested induced a high number of hairy roots.
Apparently, regeneration of Ri plants was easier in apple than in rose genotypes. By now, we have Ri plants regenerated from hairy roots of two apple genotypes which were proven to contain rol-genes. For rose, regeneration seems quite difficult since we had no success yet despite of testing different approaches for regeneration, i.e. plant growth regulator combinations and concentrations and different regeneration pathways. Nevertheless, some spontaneous regenerants from hairy root cultures were obtained which can now be propagated for being morphologically characterized.
The Toruń Group’s first area of studies is the phytohormone quantification in hairy roots lines differing in their capacity for shoot regeneration. Using a LS MS/MS approach we simultaneously detect and quantify a large set of phytohormones including auxins, cytokinins, gibberelins, abscisic acid, salicylic acid and jasmonic acid, ethylene precursor (ACC) as well as the selected phytohormone conjugates. The hairy root cultures analyzed were developed from tissues of chrysanthemum, rose and sunflower and provided by project partners from EV ILVO, LUH and USVMCN. The cultures analyzed, represented root lines differing in plant source cultivar, kind of transformation event (i.e. presence of left or/and right border sequence from R. rhizogenes) and different shoot regeneration capacity. During preliminary experiments, the effect of the sample preparation method (freezing vs. freeze-drying) on the composition and levels of phytohormones in the rose root cultures was investigated. It was shown that regardless of the sample preparation method, all tested phytohormones were detected in the roots. At the same time, no differences in the analysis efficiency was observed, regardless of the sample preparation method. Then, a comprehensive analysis of a wide spectrum of phytohormones was carried out in the hairy roots of rose representing the rose Gomery variety. IAA and IBA (auxins, the dominant form of auxin was IBA), 2iP (cytokinin), GA3 (gibberellin), abscisic acid, IAMe (methyl derivative of IAA) and salicylic and jasmonic acids (both present in very high concentrations) were detected and quantified. Subsequently, the analysis of the phytohormone content was carried out in samples representing different lines of chrysanthemum hairy root lines. The results require further elaboration and statistical analysis. In addition, several dozen more samples representing the roots of both plants were prepared for analysis. These samples are currently being processed.
The second are of research is focused on physiological traits of Ri plants. The concern of this study is discovering differences and similarities between Ri and wildtype plants in basic physiological processes, manifested under standard cultivation conditions. The hypothesis behind the study assumes that Ri plants, while maintaining their specific traits, will be characterized by a comparable course of basic physiological processes determining the yield and other functional characteristics. For this purpose, Ri plants representing four chrysanthemum genotypes were analyzed during an internship at EV ILVO. For each genotype, the experimental setup consisted of Ri plants containing one or two T-DNA sequences derived from R. rhizogenes and the corresponding wild type (untransformed). In the course of the research, four parameters characterizing the leaf photosynthesis were examined. Furthermore, the root conductivity, the rate of sap secretion by the root systems (root pressure), the fresh and dry mass of roots and the difference in osmotic potential between the sap secreted by the root systems and the soil solution were determined. These parameters were used to calculate root osmotic conductivity. Within the scope of the study, the rate of sap flow through the plant stems was also examined. This parameter was tested in a daily cycle using sensitive sensors recording the flow of sap through the stem of the plant and sending data to the data logger. Subsequent analysis provides daily patterns of sap flow through the stem conducting system. The conclusion issuing from the results of the conducted research indicates that in terms of basic physiological parameters, the Ri plants are comparable to WT plants. This indicates that maintaining their specific and favorable phenotypic traits, the Ri plants would display comparable productivity under standard cultivation conditions.
For sunflower, the aim of the project is to establish a biotechnological method for improving the root system by using the transformation with Rhizobium rhizogenes, in order to obtain plants better adapted to water stress.
The first objective was to establish a method for obtaining transformed roots (Ri roots) and this was achieved by finding a protocol that was applied for several sunflower genotypes and adapted for transformation of H. tuberosus too. Now, the continuous Ri roots production is possible and it is necessary for accomplishing the second objective which is finding a way for regeneration of Ri plants. Although the conversion of the laterally root meristems into shoot meristems was observed, particularly for H. tuberosus Ri roots, the regeneration of Ri plants has not yet been achieved. For establishing the suitable hormonal treatment for plant regeneration we are on the way of finding the endogenous hormonal level of the Ri roots which currently is analyzed by the project partner NCU. In the meantime, since the sunflower Ri plants are not available, we developed as model for analyzing the phenotype of transformed plants, which is the third objective, a composite plant system. For that we developed a hydroponic culture system followed by culture in the solid substrate of sunflower composite plants. Currently the optimization of composite sunflower obtaining is under work. The goal is to increase the rate of Ri roots regeneration. In optimal condition culture the composite plants behaved similar with the controls. Remain to be seen if under hydric stress they will make a difference.