In vitro propagation of the rose rootstock 'Moneyway' was investigated on the following media: Murashige and Skoog (MS), Quoirin and Lepoivre (QL) and Woody Plant (WP). Growth, which was measured as length of shoots after a 6-week period, was faster on MS and QL than on WP. In spite of the better growth, chlorosis of newly formed leaves occurred from the third week on and was correlated with a lower chlorophyll content of shoots.
The effect of indole-3-butyric acid (IBA) on the formation of non-transformed and rol gene transformed roots on stem slices of in vitro cultured shoots of Rosa hybridu L. 'Moneyway' was examined. Formation of adventitious roots on this rootstock was dependent on the IBA dose; it was not affected by the presence of other root primordia on the same explant. Application of 0.32 to 1 PM IBA during 5 days, followed by transfer to medium without hormones resulted in maximum root formation (90%) after three weeks. The formation of such untransformed roots was completely inhibited by transfer to medium with 5 mg 1-l kanamycin two days after excision. Ri roots were formed upon inoculation with A. rhizogenes LBA9402 harbouring two plasmids: pRil855, comprising the rol genes and the binary plasmid p 35Sgusintron with the nptll gene for kanamycin resistance and the CaMV 35Sgusintron gene. The formation of these Ri roots on kanamycin-containing medium was independent of the presence of IBA. Stem slices inoculated with a disarmed A. tumefuciens GV3 101, harbouring only the nptll gene, formed callus and subsequently roots in the presence of kanamycin exclusively on medium with high IBA concentrations (10 or 100 pw. Root formation at 100 PM IBA was considerably improved by transformation with the rolB gene under the influence of the strong CaMV 35s promoter. In addition, low IBA (0.1 and 1 ,uM) stimulated the formation of roots only on stem slices transformed with A. tumefuciens harbouring the rolA+rolB+rolC genes; the rooting response at 10 PM IBA was much improved. It was concluded that the 35SrolB gene and especially a combination of rolA, B and C genes promote the rooting response.Abbreviations: BAP = 6-benzylaminopurine; FeEDDHA = ferric ethylenediamine di(o-hydroxyphenylacetate); IBA = indole-3-butyric acid; LSD = least significant difference; RIM = root induction medium; SE = standard error; X-glut = 5-bromo-4-chloro-3-indolyl glucuronide
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 cultivare are discussed.
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