Introduction of ROL genes in Rosa hybrida L. for improved rootstock performance

In contrast to many other cut flowers, which are harvested completely at the time of flowering, rose plants ( Rosahybrida L.) continuously produce flowering shoots, which are harvested on a regular basis. For the release of axillary buds and development of flowering shoots high energy inputs are required in rose culture. However, for enviromnental reasons the use of fossil fuels should be limited i.e. the energy efficiency of rose production needs to be improved. The energy efficiency in horticulture was already remarkably improved by technical and cultural measures, which has been evolved since the energy crisis in the seventies. Breeding of roses, which produce more efficiently per unit energy input, is another powerful tool to save energy. The aim of the research described in this thesis is to genetically change roses in such a way that they can be cultured at low energy input.The production of flowering stems of roses is dependent on the number of axillary shoots or basal shoots. Axillary bud release is a plant developmental process, which is controlled by plant hormones, such as auxin and cytokinin. Auxin is produced in the apex and leaves of growing plants and is transported to the roots, while cytokinin is produced in the root tips and transported to the aerial parts. This means that the hormonal status of the roots influences the development of the whole plant. Many cut roses are therefore grafted on a rose rootstock; not only to enhance axillary bud release and flower production, but also to overcome deficiencies in adventitious root formation. The separation of rose plants in a scion and a rootstock part gives opportunity to specific breeding programmes for flower and root characteristics. In contrast to a high breeding activity in cut roses, breeding efforts for rose rootstocks have been relatively poor. This opens perspectives for the development of new rose rootstocks, either via cross breeding or more recently also via genetic modification. The latter approach has the advantage that gene transfer is no longer determined by crossing potential, but principally unlimited from one species to another species. In this thesis we aimed at the improvement of rootstock performance by the introduction of the ROL genes from the bacterium Agrobacterium rhizogenes. These genes influence plant development by interference in hormone metabolism or hormone sensitivity.A prerequisite for genetic modification is the availability of reliable transformation procedures. At the start of this research such a transformation protocol was not available for rose. Since rose is a recalcitrant species in this respect, much attention has been paid to the development of methodological principles of genetic modification, such as micropropagation, transformation and regeneration.First, the micropropagation of the rose rootstock 'Moneyway' was examined. On standard media either shoot growth was inadequate or leaves became chlorotic, suggesting an iron deficiency. Replacement of FeEDTA by the more stable iron chelate FeEDDHA in the medium resulted in the development of green shoots for more than three months.Adventitious root formation of rose was studied on stem slices of micropropagated shoots. First it was shown that the formation of adventitious roots was dependent on the auxin dose and was not affected by the presence of other root primordia on the same stem slice. Secondly, to study the effects of ROL genes, a method was developed for the production of transformed roots, using 5 mg/L kanamycin for selection. Kanamycin resistant roots were formed on stem slices, which were inoculated with A.rhizogenes, harbouring the complete Ri plasmid encompassing the ROL genes and a binary plasmid with the neomycin phosphotransferase ( NPTII ) gene for kanamycin resistance. In contrast to adventitious root formation, the formation of these Ri transformed roots was independent of the presence of auxin. This autonomous formation of Ri roots might be due to the transfer of AUX genes, which offer an alternative synthesis route for auxin. Stem slices, inoculated with A . tumefaciens harbouring only the NPTII gene, formed on kanamycin containing medium with high auxin concentrations first callus and subsequently roots. The formation of such kanamycin resistant roots was significantly improved upon transformation with the ROLB gene under the strong CaMV 35S promoter and even more by transformation with a combination of ROLA, B, C genes. These experiments demonstrated that the ROLB gene and especially the ROLA, B, C genes are in principle suitable genes for improvement of the rooting ability of rose rootstocks.For the production of transformed rose plants a study was undertaken to establish a regeneration procedure. Plants were regenerated from excised adventitious roots via somatic embryogenesis. First, the roots were incubated on callus induction medium containing a high concentration of auxin (50 μ M 2,4-D). For embryo induction calluses were transferred to hormone-free medium. The use of Gelrite instead of agar during callus induction andlor embryo induction stimulated embryogenesis; up to 16% of the calluses formed organized structures. Approximately 40% of these structures further developed into shoots. Despite the long lasting callus phase during regeneration, the majority of the regenerant plants did not show any signs of somaclonal variation, indicating that we developed a suitable regeneration procedure.This regeneration procedure was applied for the regeneration of plants from excised kanamycin resistant roots. For this purpose first hundreds of kanamycin resistant roots were produced, with an efficiency of up to two roots per stem slice. Regeneration of plants from these roots lasted up to 12 months, but finally nine independent transformants were produced: four transformants with the reporter gene 35SGUSINT , one transformant with the ROLB gene behind its own promoter (B1) and four transformants with a combination of ROLA, B, C genes also driven by their own promoters (ABC1 to ABC4). Rooting experiments in vitro showed that adventitious root formation on shoots and leaves was enhanced by the presence of these ROL genes. Even in the absence of exogenous auxin roots were induced, probably due to an increased sensitivity for endogenous auxin. The presence of the ROLA, B, C genes also enhanced adventitious root formation on stem slices of micropropagated plants, whereas the ROLB gene surprisingly decreased it. We suggested that this might be due to overexpression of the ROLB gene. The rooting experiments in vitro were confirmed by rooting experiments with cuttings from greenhouse grown plants; adventitious root formation was improved threefold upon introduction of the ROLB gene or a combination of ROLA, B, C genes.Next to an increased rooting ability of ROL gene transformed plants, many pleiotropic effects were observed in the greenhouse: increased (ROLB) or decreased ( ROLA,B, C) apical dominance, reduced plant weight and altered leaf morphology. Northern analysis showed that altered leaf shapes were correlated with the presence of specific ROL transcripts; wrinkled leaves with a ROLA messenger of 650 nt, round-edged leaves with a ROLB messenger of 1050 nt and small and lanceolate leaves with a ROLC messenger of 850 nt.The ROLB transformant (B1) and one ROLA, B, C transformant (ABC1) were used as rootstocks in combination plants with the cut rose cultivar Madelon as a scion. Growth and development were followed on hydroculture at two temperatures during three months. Although the formation of adventitious roots was initially enhanced by the ROLB gene, further root development was inhibited. Despite the smaller root system, the performance of the untransformed scion was as in the control. In contrast, root development was enhanced by the presence of ROLA, B, C genes, while the development of the untransformed aerial part was also affected. Regarding the aim of this research, the most important observation was that the release of axillary buds was increased from 0.1 to 0.6 and from 0.3 to 1.3 basal shoot per plants at 15 and 20°C, respectively. This enhanced axillary bud release might reflect either an increased production of cytokinin in such well developed root systems or a cytokinin-like action as mentioned for ROLC transformed plants. In this respect it should be mentioned that the ROLC gene was indeed active in ROLA, B, C transformed roots. Since the formation of these basal shoots is correlated with flower production, we expect that application of ROLA, B, C transformed rootstocks will lead to a more energy efficient flower production both at 20°C and 15°C.In the final part of this thesis, prospects for the applications of ROL genes for crop improvement are discussed. As suggested in many reports, the ROL genes are principally suitable tools to modify plant developmental processes such as adventitious root formation and axillary bud release. However, practical application of ROL gene transformed plants might be hampered by the many pleiotropic side effects, as observed in completely transformed roses. Based on the research described in this thesis, we offer a novel approach for application, which is clearly different from earlier strategies. It was demonstrated for the first time that expression of ROL genes in the rootstock led to a beneficial stimulation of axillary bud release of the untransformed scion, without the transmissibility of many undesired pleiotropic effects

Somatic embryogenesis and shoot regeneration from excised adventitious roots of the rootstock Rosa hybrida L. 'Moneyway'

Plants were regenerated from excised adventitious roots of the rose rootstock 'Moneyway' via a three step procedure: callus induction, induction of somatic embryos and shoot development. Callus was induced on excised roots after incubation for 4 weeks in the dark on SH-medium (Schenk and Hildebrandt) containing 50 μM 2,4-dichlorophenoxyacetic acid. For embryo induction, calluses were transferred to hormone-free SH-medium and incubated for 8 weeks. The use of Gelrite instead of agar during callus induction stimulated somatic embryogenesis (up to 16% of the explants formed organized structures), whereas the presence of 6-benzylaminopurine in this phase inhibited subsequent regeneration. Yellow solid calluses with embryo-like cotyledons or primordia and friable calluses with embryos were selected, and upon incubation in the light shoots developed. Shoot development was faster and more frequent on solid callus than on friable callus (64% and 21 % of the calluses finally formed one or more shoots, respectively). Eleven out of thirteen regenerants developed similarly to control shoots. Finally this regeneration method is compared with other systems for somatic embryogenesis and opportunities for the production of transgenic rose rootstocks and rose cultivars are discussed

Production of ROL gene transformed plants of Rosa hybrida L. and characterization of their rooting ability

Transgenic plants of the rootstock Rosa hybrida L. cv. Moneyway were produced via a two-step procedure. First, kanamycin-resistant roots were generated on stem slices from micropropagated shoots, which were cocultivated with Agrobacterium tumefaciens containing the neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, together with individual ROL genes from A. rhizogenes. Root formation was quite efficient and up to two kanamycin-resistant roots per stem slice were produced. In the second step, these roots were used to regenerate transgenic plants via somatic embryogenesis. Although regeneration lasted up to 12 months, production of several transformants was successfully accomplished. Untransformed escapes were not found, indicating that the initial selection on kanamycin resistance was reliable. The presence of a combination of ROLA, B and C genes enhanced adventitious root formation on micropropagated shoots and explants of stems and leaves. It appears that the auxin sensitivity was increased to such a degree that cells were able to respond even to endogenous auxins present in shoots and leaves. Rooting experiments in greenhouse demonstrated that adventitious root formation on cuttings was improved threefold upon introduction of these ROL genes. It is concluded that a method was developed for the production of ROL gene transformed roses with improved rooting characteristics