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James F. Harbage

Micropropagation of three Echinacea species, E. angustifolia, E. pallida, and E. purpurea, was investigated as a potential means of germplasm preservation of species faced with overcollection in the wild and rapid clonal propagation of elite individuals with unique medicinal or ornamental properties. Comparison of explant sources indicated vegetative explants resulted in high contamination rates when collected from shoot-tips (100%),but not when collected from nodal explants (11% to 39). Seed coat removal reduced contamination from 100% in intact seeds to near 0% in excised embryos. Removal of seed coats (pericarp and integument layers) also eliminated dormancy requirement for germination. All species responded with shoot multiplication and loss of rooting when BA or thidiazuron was added to culture medium. Medium with thidiazuron resulted in excessive adventitious shoot formation. Shoot multiplication rates were low (one to three shoots/explant) on medium with BA levels low enough to avoid adventitious shoot formation. Medium containing half-strength MS minerals resulted in more shoots with smaller leaves than full-strength MS minerals. Cultures did not perform well on Woody Plant Medium. Increasing subculture frequency from every 4 weeks to every 2 weeks increased shoot multiplication rates from 1.4 to 1.8 shoots per subculture and total shoots produced after 12 weeks of culturing (per initial explant) from 2.8 to 23.9. Rooting occurred readily on shoots isolated from E. purpurea without addition of IBA. Rooting was low or non-existent on shoots from E. angustifolia and E. pallida, respectively, regardless of IBA level, light conditions, or temperature. Methods described in this study allow rapid multiplication of three Echinacea species and subsequent rooting of E. angustifolia and E. purpurea. Future improvements in root induction treatments will allow more effective use of micropropagation for Echinacea germplasm preservation and multiplication. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA), 1H-indole-3-butyric acid (IBA).

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James F. Harbage

Micropropagation of three Echinacea species, E. angustifolia DC., E. pallida Nutt., and E. purpurea Moench., was investigated as a potential means of germplasm preservation of species faced with overcollection in the wild and rapid clonal propagation of elite individuals with unique medicinal or ornamental properties. Very high contamination rates occurred with shoot-tip explants but not with nodal segments. Contamination rates for seed explants were inversely related to the number of seedcoat layers removed, ranging from 100% contamination from intact seeds to near 0% contamination from excised embryos. Dormancy of seed explants was also eliminated when the pericarp and integument were removed. Addition of benzyladenine (BA) to the culture medium induced shoot multiplication and inhibited root formation in all three species. Shoot multiplication rates were low (1-3 shoots per culture) when seed explants were placed on a medium with BA levels low enough to avoid adventitious shoot formation (0.45 μm). Shoot count was higher on half-strength Murashige and Skoog (MS) minerals, while leaf size was greater on full-strength MS minerals. Cultures did not perform well in Woody Plant Medium. Reducing subculture frequency from 4 to 2 weeks increased shoot multiplication from 1.4 to 1.8 shoots per subculture and total shoots produced per subculture after 12 weeks from 2.8 to 23.9. Rooting occurred readily on shoots isolated from E. purpurea cultures and was not promoted by addition of IBA to the rooting medium. Rooting was low and nil on shoots from cultures of E. angustifolia and E. pallida, respectively. Methods described in this study allow rapid multiplication of three Echinacea species and subsequent rooting of E. purpurea. Future improvements in root induction treatments will allow these methods to be used effectively for micropropagation and maintenance of disease-free germplasm of Echinacea species. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); 1H-indole-3-butyric acid (IBA).

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Dennis P. Stimart and James F. Harbage

The role of the number of adventitious roots of Malus domestics Borkh. `Gala' microcuttings in vitro on ex vitro root and shoot growth was investigated. Root initiation treatments consisted of IBA at 0, 0.15, 1.5, 15, and 150 μm in factorial combination with media at pH 5.5, 6.3, and 7.0. IBA concentrations significantly influenced final root count and shoot fresh and dry weights, but not plant height, leaf count, or root fresh and dry weights at 116 days. Between 0 and 0.15 μm IBA, final root counts were similar, but at 1.5, 15, and 150 μm IBA, root counts increased by 45%, 141%, and 159%, respectively, over the control. The pH levels did not affect observed characteristics significantly. There was no significant interaction between main effects. A significant positive linear relationship was found between initial and final root count. The results suggest a limited association between high initial adventitious root count and subsequent growth. Chemical name used: 1 H -indole-3-butyric acid (IBA).

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James F. Harbage and Dennis P. Stimart

Many physiological responses in plants are influenced by pH. The present chemiosmotic hypothesis suggests that auxin uptake into plant cells is governed by pH. Since auxin is used widely to enhance rooting, the influence of pH on 1H-indole-3-butyric acid (IBA) induced adventitious root formation was examined. Roots were initiated aseptically in 5 node apical shoot cuttings of micropropagated Malus domestica 'Gala'. Initiation was induced using a four day pulse in IBA and 15 g/L sucrose at pH 5.6 and 30C in the dark. Observations showed pH rose to 7.0 or greater within 1 to 2 days from microcutting placement in unbuffered initiation medium. Root numbers from shoots in media containing 1.5 μM IBA buffered with 10 mM 2[N-morpholino] ethanesulfonic acid (MES) to pH 5.5, 6.0, 6.5 or 7.0 with KOH resulted in average root numbers of 14.2, 10.9, 8.7, and 7.1, respectively, while unbuffered medium yielded 7,6 roots per shoot. Comparison of MES buffered medium at pH 5.5, 6.25 or 7.0 in factorial combination with IBA at 0, 0.15, 1.5, 15.0, and 150.0 μM resulted in a significant pH by IBA interaction for root number. At 0, 0.15 and 1.5 μM IBA root numbers were greatest at pH 5.5. At 15.0 μM IBA, pH 6.25 was optimal and at 150.0 μM IBA all three pH levels produced equivalent root numbers. A calorimetric assay to measure IBA removal from the initiation medium by microcuttings of `Gala' and `Triple Red Delicious' showed more IBA removal at pH 5.5 than at pH 7.0. Possible reasons for the effect of pH on adventitious root formation will be discussed.

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James F. Harbage and Dennis P. Stimart

We investigated the role of ethylene on adventitious rooting of `Gala' (easy-to-root) and `Triple Red Delicious' (difficult-to-root) apple (Malus domestica Borkh.) microcuttings. Root count increased significantly as IBA level increased, with highest root counts on `Gala'. Ethylene evolution increased significantly with IBA level without significant differences between cultivars. Basal section removal of microcuttings in the area of root origin reduced root count without changing ethylene evolution. Ethylene treatment of proliferated shoots before microcutting excision failed to enhance rooting. IBA-induced ethylene evolution was eliminated nearly by AVG, but root count remained IBA dependent. ACC reversed IBA plus AVG rooting inhibition, but ACC alone failed to influence root count. Polar auxin transport inhibitors NPA and TIBA stimulated ethylene evolution without increasing root count. Adventitious rooting of apple microcuttings was not associated with ethylene. Chemical names used: 1-H-indole-3-butyric acid (IBA); aminoethoxyvinylglycine (AVG); 1-aminocyclopropane-1-carboxylic acid (ACC); 2,3,5-triiodobenzoic acid (TIBA); N-1-naphthylphthalamic acid (NPA).

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James F. Harbage and Dennis P. Stimart

Involvement of pH and IBA on adventitious root initiation was investigated with Malus domestica Borkh. microcuttings. The pH of unbuffered root initiation medium (RIM) increased from 5.6 to 7 within 2 days. Buffering with 2[N-morpholino] ethanesulfonic acid (MES) adjusted to specific pHs with potassium hydroxide prevented pH changes and resulted in a 2-fold higher root count at pH 5.5 compared to pH 7 or unbuffered medium. As pH decreased, lower concentrations of IBA were required to increase root counts. Colorimetric measurement of IBA in buffered RIM showed greater IBA loss and higher root count were associated with lower pH levels in all cultivars. This suggests that IBA loss from RIM depends on medium pH, which affects root count. Root count differences between easy-to-root through difficult-to-root cultivars were not consistent with amount of IBA loss from RIM. Cultivar differences in root count could not be explained solely by IBA loss from RIM.

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James F. Harbage, Dennis P. Stimart, and Ray F. Evert

Anatomical events of adventitious root formation in response to root induction medium, observing changes during induction and post-induction stages, were made with microcuttings of `Gala' apples. Shoot explants on root induction medium containing water, 1.5 μm IBA, 44 mm sucrose, or 1.5 μm IBA + 44 mm sucrose after 4 days of treatment averaged 0, 0.2, 2.2, and 11.9 meristemoids per microcutting, respectively. Meristemoids formed in response to sucrose were confined to leaf gaps and traces. Time-course analysis of root induction with 1.5 μm IBA + 44 mm sucrose over 4 days revealed that some phloem parenchyma cells became densely cytoplasmic, having nuclei with prominent nucleoli within 1 day; meristematic activity in the phloem was widespread by 2 days; continued division of phloem parenchyma cells advanced into the cortex by 3 days; and that identifiable root primordia were present by 4 days. Cell division of pith, vascular cambium, and cortex did not lead to primordia formation. Meristematic activity was confined to the basal 1 mm of microcuttings. Time-course analysis of post-induction treatment revealed differentiation of distinct cell layers at the distal end of primordia by 1 day; primordia with a conical shape and several cell layers at the distal end by 2 to 3 days; roots with organized tissue systems emerging from the stem by 4 days; and numerous emerged roots by 6 days. Root initiation was detectable within 24 hours and completed by day 4 of the root induction treatment and involved only phloem parenchyma cells. Chemical names used: 1 H -indole3-butryic acid (IBA).

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James F. Harbage, Dennis P. Stimart, and Carol Auer

The influence of root initiation medium pH on root formation was investigated in relation to uptake and metabolism of applied IBA in microcuttings of Malus ×domestica Borkh. `Gala' and `Triple Red Delicious'. Root formation and uptake of H 3-IBA were related inversely to root initiation medium pH. Maximum root count (10.3 roots) and IBA uptake were observed at pH 4.0. Regardless of pH, overall root count of `Gala' was higher (13.5 roots) than `Triple Red Delicious' (4 roots). Uptake of IBA was highest at pH 4.0 for `Gala' (1.7% uptake) and at pH 4 and 5 for `Triple Red Delicious' (0.75% uptake). Metabolism of IBA was the same regardless of root initiation medium pH or cultivar examined. One-half of the IBA taken up was converted to a compound that coeluted with IBAsp during high-performance liquid chromatography. Apparently, pH regulates root formation by affecting IBA uptake but not metabolism. The level of auxin in tissue appeared unrelated to root formation between genotypes. Chemical names used: 1H-indole-3-butyric acid (IBA); 5-H 3-indole-3-butyric acid (H 3-IBA); indole-3-butrylaspartic acid (IBAsp).

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Bjorn H. Karlsson, Jiwan P. Palta, Laurie S. Weiss, James F. Harbage, and John B. Bamber

Cold-induced changes in gene expression have been demonstrated in a number of species that vary in freezing tolerance and acclimation capacity. Relative freezing tolerance was measured based on ion leakage for both nonacclimated and acclimated S. commersonii and S. cardiophyllum parents, F1 and backcross progeny segregating for cold tolerance and acclimation capacity. Western blot analyses showed increase in a dehydrin band (47 kD)(antisera courtesy of T. Close) following cold acclimation of cold tolerant S. commersonii, and a slight increase in cold sensitive S. cardiophyllum. Expression of 47 kD cosegregated with non acclimated freezing tolerance but not with acclimated freezing tolerance. Our results show that (i) expression of dehydrins is a heritable trait in the Solanum diploid population, (ii) there is no direct relationship between relative freezing tolerance and the presence or absence of dehydrm protein following cold acclimation and (iii) based on assays measuring the residual activity of the lactate dehydrogenase (LDH) enzyme following freezing, the cryoprotective influence of `boiling stable' proteins was species dependent and is related to the freezing tolerance of the species. Supported by USDA/NRI grant 91-3700-6636 to J.P.P. and J.B.B..