Margareta Welander

Plant regeneration from leaf and stem segments of shoots raised in vitro from mature apple trees

Summary Plants were regenerated from leaf segments of shoots raised in vitro from mature apple trees of the cultivars Akero, McIntosh, McIntosh Wijcik, Gravenstein and the apple rootstock M26. The regeneration capacity was influenced by wounding, macroelements, light conditions, polarity and maturity of the leaves. The highest number of shoots per leaf segment was formed using young expanding leaves on a medium based on N 6 macroelements plus 22 µM BAP and 1 µM NAA. Dark treatment during the first 3 weeks of culture and placing the leaf segments on the medium with the abaxial surface uppermost enhanced shoot formation. The morphogenetic response for stem segments was very low since only McIntosh Wijcik formed shoots and at a low frequency. The histological studies revealed both embryoidlike structures and adventitious shoots. Shoots regenerated from both leaves and meristems were rooted and placed in the greenhouse for comparison. Trees of the cultivars McIntosh, McIntosh Wijcik and M26 were planted in the field for further evaluation of growth habit and fruit setting.

Transformation of Apple Rootstock M26 with Agrobacterium tumefaciens

Summary Different tissues of the apple ( Malus X domestica Borkh.) rootstock M26 were inoculated with various wild-type strains of Agrobacterium tumefaciens . Galls formed on leaf but not on stem or petiole explants. Wild-type strain A281 was more effective than C58 and A348. Disarmed A. tumefaciens strains with the binary vectors pCGP257 and pBI121, which contain the npt II and gus genes, were used to transform leaf explants. The appropriate transformation protocol included an initial dark incubation period and the use of Gelrite-gelled medium. Transformation was confirmed by histochemical localization and fluorometric assays for GUS, by NPT II dot blot assay, and by Southern blot analysis for both gus and npt II . The extent to which transformed shoots initiated roots in the presence of kanamycin was quantified and related to the expression of the incorporated npt II gene.

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.

Transformation of the apple rootstock M26 with the rolA gene and its influence on growth

Abstract In vitro produced leaves of the apple rootstock M26 were infected with Agrobacterium tumefaciens strain GV3 101 containing a binary vector carrying the nptII gene and the rolA gene on the T-DNA. Four transformed clones were obtained from 433 infected leaves. Two of the transformed clones had a single copy and two had double copies of the T-DNA integrated. In all the four transformed clones the T-DNA was integrated at different positions in the plant chromosomal DNA. All transformed plants exhibited reduced stem growth compared to untransformed control plants. Three of the clones also showed reduced internode length and two of these exhibited reduced leaf area, shoot dry weight, root dry weight and plant dry weight compared to the control plants. No correlation between copy number and plant phenotype was shown.

Effects of Light–Temperature Regimes on Plant Growth and Essential Oil Yield of Selected Aromatic Plants

The effects of different light–temperature conditions on leaf growth and essential oil yield and oil composition of Mentha×piperita L, M spicata L, M longifolia L, M rubra L and Chamomilla recutita (L) Rausch were studied in a biotron experiment. A majority of the Mentha L species exposed to a 21–3 h photoperiod, simulating conditions typical of the July environment in northern Sweden, produced significantly higher concentrations of menthol than treatments with shorter photoperiods. Night temperatures had little effects on the menthol levels under the 21 h photoperiod regime. The total yields of menthol and menthone in the Mentha L species were significantly higher in the third to fifth leaf pairs compared to the topmost leaf pair position. The different environmental treatments had no evident effects on leaf growth (measurements based on the five topmost leaf pairs). The combination of 21–3 h photoperiod and 25–18°C thermoperiod resulted in the highest levels of α-bisabolol in C recutita (L) Rausch. Also, bud or flower formation was only induced in treatments with photoperiods ⩾17 h.

Genetic transformation of the apple rootstock M26 with the RolB gene and its influence on rooting

Summary The apple rootstock M26 ( Malus × domestica Borkh.) was transformed using the binary vector pCMBB: GUS in the Agrobacterium tumefaciens strain C58C1. The vector contained the npt II gene under the nos promotor and a tandem gene construct of the gus and the rol B genes both under the rolB promotor. Two transformed clones were verified with PCR and Southern blot analyses. One clone contained all three genes, whereas in the other clone only rol B and npt II genes were integrated in the plant genome. Rooting experiments using both intact shoots and stem segments showed that integration of the rol B gene increased auxin sensitivity and rooting ability compared with untransformed control shoots. GUS activity driven by the rol B promotor was higly expressed in the root meristem but not in the extended root. In the shoot, GUS activity was confined to the procambial strands. In the bud, the leaf primordia showed rather intense uniform staining. During growth of the leaf the provascular cells of the midrib showed weak GUS expression and developed leaves were almost unstained.

Regulation of in vitro shoot multiplication in Syringa, Alnus and Malus by different carbon sources

SummaryThe influence of different carbon sources on shoot multiplication was studied in the apple rootstock M.9. (Malus pumila Mill.), Syringa chinensis Willd. cv. Saugeana and Alnus glutinosa (L.) Gaertn. Both nodal and shoot tip explants were used. The sugars sucrose, glucose, fructose and the sugar alcohols mannitol and sorbitol at 88 and 175 mM were investigated. The carbon source influenced the percentage explants forming shoots, number of shoots per explant, number of separable nodes per shoot as well as shoot length. In Syringa, the carbohydrates influenced mainly the survival percentage of the explants, with the best carbon source being mannitol at both concentrations and sucrose at 88 mM. Overall the most effective carbon source in Alnus was glucose at 88 mM. Malus explants grew best on sorbitol, and fructose at 175 mM. The capability to metabolize a certain type of carbon source was reflected by the sugars and sugar alcohols reported in the sieve-tube exudate within a given genus (Zimmermann and...

Overexpression of the Arabidopsis gai gene in apple significantly reduces plant size

Genetic engineering is an attractive method to obtain dwarf plants in order to eliminate the extensive use of growth retardants in horticultural crop production. In this study, we evaluated the potential of using the Arabidopsisgai (gibberellic acid insensitive) gene to dwarf apple trees. The gai gene under 35S promoter was introduced in the apple rootstock A2 and the cultivars Gravenstein and McIntosh through Agrobacterium-mediated transformation. One transgenic clone was recovered for Gravenstein and McIntosh, and several transgenic clones for A2, confirmed by Southern blot analysis. Two weak bands were detected by Southern blot analysis in all the untransformed controls, possibly indicating the existence of the internal GAI gene in apple. Most of the transgenic plants showed reduced growth in vitro. Growth analyses in the greenhouse showed a clear reduction in stem length, internode length and node number for the dwarf clones. The normal phenotype of some transgenic clones appears to be associated with silencing of the introduced gai gene, confirmed by RT–PCR analysis. In general, transgenic clones showed reduced rooting ability, especially for the extremely compact ones.

Changes of leaf water potential and endogenous cytokinins in young apple trees treated with or without paclobutrazol under drought conditions

Abstract Leaf water potential and endogenous cytokinins in xylem sap of young apple trees treated with or without paclobutrazol (PBZ) under drought stress conditions were investigated. Three drought and rewatering cycles were used in the experiments and leaf water potential was measured every 2–4 days during the experimental period (39 days). Zeatin (Z) and zeatin riboside (ZR) from the xylem sap in the trunks were measured during the drought periods. The results showed that leaf water potential of the plants decreased significantly when drought was applied, while for the PBZ treatment the reduction of leaf water potential was significantly less in all the three drought cycles, particularly in the first and second cycles. The concentrations of Z and ZR in xylem sap of the drought treated plants decreased significantly in the first drought cycle. In the second cycle, Z continued to decrease but ZR did not change significantly by day 4 but increased at day 8 for both treatments compared with the control. In the third cycle, there was no difference in Z concentration between the drought treatment alone and the control, but Z increased significantly for the PBZ treatment. ZR increased significantly for the drought and drought plus PBZ treatments compared with the control in the third cycle.

Optimisation of growing conditions for the apple rootstock M26 grown in RITA containers using temporary immersion principle

The use of bioreactors may provide an efficient and economic tool for mass clonal propagation of plants if technical problems can be solved. In this paper, we report the results of experiments aimed at optimising conditions for apple rootstock M26 grown in RITA containers using the temporary immersion principle. We tested different types and sizes of explants, different concentrations of plant growth regulators (BAP, kinetin and IBA) in the multiplication and elongation phases, and medium exchange during the shoot elongation period. The results show that the higher concentrations of cytokinins were required during the shoot multiplication phase, while the lower concentrations were better during the shoot elongation phase. Hyperhydricity was increased with increasing concentration in of cytokinins during both shoot multiplication and shoot elongation phases. The best shoot production in terms of shoot number and shoot quality was obtained using 4.4 μmol BAP and 0.5 μmol IBA during the shoot multiplication phase and 1.1 μmol BAP and 0.25 μmol IBA during the shoot elongation phase. Medium exchange twice during the shoot elongation phase resulted in higher shoot production compared with no exchange of the medium. However, it also resulted in increased hyperhydricity. Immersion frequency of 16 times per day gave a higher multiplication rate and longer shoots than 8 times per day. The explant size of 0.5 cm or 1 cm resulted in a significantly higher shoot production rate compared with that of 1.5 cm, but shoot length and hyperhydricity were not affected by the explant size. Shoot cultures from the liquid media rooted normally in the RITA containers with more than 90% rooting and the rooted plantlets acclimatised well in the greenhouse.