Annelie Ahlman

Transformation of the apple rootstock M.9:29 with the rolB gene and its influence on rooting and growth

To improve the rooting ability, the dwarfing apple rootstock M.9/29 was transformed with the rolB gene by Agrobacterium-mediated gene transfer. The use of sorbitol in the induction medium resulted in a successful transformation, while the use of sucrose failed to give any transformants. Totally 14 putative clones, named ARB1-14, were obtained from ten different leaves. Polymerase chain reaction (PCR) and Southern analyses confirmed that all the clones contained the nptII and rolB genes, while only four of them contained the intact gus gene. The in vitro rooting test showed that all the tested clones rooted to 83-100% on the hormone free rooting medium, while only 1% for the control plants. The root number of the transgenic clones ranged from 3.5 to 9, while the control plants produced only one root. Growth analysis showed that the clone ARB9 and ARB10 had a significant reduced node number and stem length compared with the control plants. However, the relative growth rate (RGR) of the tested clones was similar to that of the control plants, indicating that RGR is not directly related to dwarfism of a plant. The clone ARB10 also showed a significant reduced internode length compared with the control plants. The root length and root morphology did not differ between the transgenic clones and the untransformed control plants.

The rooting ability of the dwarfing pear rootstock bp10030 (pyrus communis) was significantly increased by introduction of the rolb gene

Abstract The pear rootstock BP10030 ( Pyrus communis ) is dwarf, frost hardy and compatible with most pear varieties, but very difficult to propagate due to its poor rooting ability. In order to improve its rooting ability, we transformed this rootstock with the rol B gene. Six transgenic clones were obtained and named 1-6. PCR and Southern hybridisation confirmed that all the transgenic clones contained the rol B and npt II genes, but only two of them contained the gus gene. The in vitro rooting results showed that the transgenic clones rooted from 67 to 100% without auxin, while the untransformed control did not root at all on the hormone free rooting medium. To further confirm the increased rooting ability of these clones, cutting experiments were carried out on four transgenic clones (1-4) and the untransformed control in the greenhouse. The rooting percentage ranged from 71 to 100 for the transgenic clones and only five for the untransformed control. Moreover, the root number was also greatly increased ranging from 11 to 17 for the transgenic clones, but only 3 for the untransformed control. Another important alteration by the insertion of the rol B gene was the root morphology. For the transformed clones, the roots were distributed evenly on the cut surface, but only on one side of the cut surface of the untransformed control. Growth analysis in the greenhouse, conducted on four transgenic clones (1-4) and the untransformed control, showed that three of the transgenic clones had a shortened stem length. The transgenic clones did not show any other visual alteration in shoot phenotype compared with the untransformed control plants.

Origin, timing, and gene expression profile of adventitious rooting in Arabidopsis hypocotyls and stems

PREMISE OF THE STUDY Adventitious root (AR) formation is indispensable for vegetative propagation, but difficult to achieve in many crops. Understanding its molecular mechanisms is thus important for such species. Here we aimed at developing a rooting protocol for direct AR formation in stems, locating cellular AR origins in stems and exploring molecular differences underlying adventitious rooting in hypocotyls and stems. METHODS In-vitro-grown hypocotyls or stems of wild-type and transgenic ecotype Columbia (Col-0) of Arabidopsis thaliana were rooted on rooting media. Anatomy of AR formation, qRT-PCR of some rooting-related genes and in situ GUS expression were carried out during rooting from hypocotyls and stems. KEY RESULTS We developed a rooting protocol for AR formation in stems and traced back root origins in stems by anatomical and in situ expression studies. Unlike rooting in hypocotyls, rooting in stems was slower, and AR origins were mainly from lateral parenchyma of vascular bundles and neighboring starch sheath cells as well as, to a lesser extent, from phloem cap and xylem parenchyma. Transcript levels of GH3-3, LBD16, LBD29, and LRP1 in hypocotyls and stems were similar, but transcript accumulation was delayed in stems. In situ expression signals of DR5::GUS, LBD16::GUS, LBD29::GUS, and rolB::GUS reporters in stems mainly occurred at the root initiation sites, suggesting their involvement in AR formation. CONCLUSIONS We have developed an efficient rooting protocol using half-strength Lepoivre medium for studying AR formation in stems, traced back the cellular AR origins in stems, and correlated expression of rooting-related genes with root initiation sites.

Integration of the rolA gene into the genome of the vigorous apple rootstock A2 reduced plant height and shortened internodes

Summary The aim of this work was to dwarf the vigorous apple rootstock A2 by insertion of the rolA gene. To optimize conditions for a successful transformation, regeneration tests were carried out. The use of sucrose in the regeneration medium gave higher regeneration frequency than sorbitol in some cases and the shoot number per regenerated leaf was higher at 10 |j.M TDZ compared with 2.5 |j.M TDZ on the sucrose medium. Two transgenic clones, verified by PCR and Southern analysis, have been obtained on the sucrose medium together with 2.5 |j.M TDZ and 1.0 or 2.5 |j.M NAA and wounding by forceps. The two clones, named LAI and LA2, contained both the rolA and nptll genes. The results of in vitro rooting showed that LAI had a lower rooting percentage and a reduced root number per rooted shoot than the untransformed control shoots and the clone LA2 on the rooting medium containing 5 |JLM IBA. Growth analysis revealed that both transgenic clones had a reduced plant height and a shortened internode length compared with the control plants. However, the node number and the stem diameter were significantly larger for clone LAI than clone LA2 and the control plants.

Effects of Overexpression of WRI1 and Hemoglobin Genes on the Seed Oil Content of Lepidium campestre

The wild species field cress (Lepidium campestre), belonging to the Brassicaceae family, has potential to be developed into a novel oilseed- and catch crop, however, the species needs to be further improved regarding some important agronomic traits. One of them is its low oil content which needs to be increased. As far as we know there is no study aiming at increasing the oil content that has been reported in this species. In order to investigate the possibility to increase the seed oil content in field cress, we have tried to introduce the Arabidopsis WRINKLED1 (AtWRI1) or hemoglobin (Hb) genes from either Arabidopsis thaliana (AtHb2) or Beta vulgaris (BvHb2) into field cress with the seed specific expression. The hypothesis was that the oil content would be increased by overexpressing these target genes. The results showed that the oil content was indeed increased by up to 29.9, 20.2, and 25.9% in the transgenic lines expressing AtWRI1, AtHb2, and BvHb2, respectively. The seed oil composition of the transgenic lines did not significantly deviate from the seed oil composition of the wild type plants. Our results indicate that genetic modification can be used in this wild species for its fast domestication into a future economically viable oilseed and catch crop.