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James S. Busse, Monica Figueroa-Cabanas, and Dennis P. Stimart*

Adventitious shoot formation in vitro from Antirrhinum majus L. hypocotyls was investigated using two inbred lines, the most and least regenerative lines selected from screening. Time course analysis indicated cell division in the most regenerative line occurred first in one or a small number of epidermal cells with periclinal and anticlinal divisions. Subsequently, cortical then vascular cells were recruited beneath the dividing epidermal cells. Once shoots formed, their vascular system was continuous with the original hypocotyl explant. The least regenerative line had no cell division directed toward organogenesis. Shoot formation on hypocotyls of A. majus was adventitious in origin, by direct organogenesis and genotype dependent.

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Senay Ozgen, James S. Busse, and Jiwan P. Palta

The important roles of calcium on plant growth and development including cell division and cell elongation is well documented. The purpose of the present study was to determine the impact of root zone calcium on the growth and health of potato apical meristem and on the maintenance of apical dominance. For this purpose, single-node potato cuttings (Solanum tuberosum L. cv. Dark Red Norland) were grown in sterilized modified Murashige and Skoog (MS) media containing varying concentrations of calcium (1 to 3000 μM). After 13 to 30 d of growth, plantlets were harvested and data for height of the main shoot and for the number of axillary shoots produced were recorded. Plantlets were ashed and tissue calcium concentration was determined. Shoot height was retarded with decreasing concentration of calcium in the media. Calcium deficiency induced shoot tip injury and loss of apical dominance. Tip injury was followed by the development of axillary shoots. The number of axillary shoots increased from one to 21 as calcium concentration in the media decreased from 3000 to 1 μM. At calcium concentration of 1500 μM or higher, there was a single main shoot with no axillary shoots. Addition of ethylene glycol tetra acetic acid (EGTA), a calcium chelator, to the media with 2720 μM calcium (sufficient calcium) resulted in the development of shoot injury and in the formation of axillary shoots. Calcium deficiency injury symptoms were prevented by the addition of a calcium analog, strontium, to MS media deficient in calcium. Strontium has been reported to strongly bind to plant cell walls and the inclusion of strontium prevented injury in shoots of plants grown on calcium-deficient media. These results suggest that strontium is able to mimic the role of calcium in the maintenance of cell wall integrity and supports previous studies that showed that calcium deficiency results from cell wall collapse of the subapical cells.

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James S. Busse, Senay Ozgen, and Jiwan P. Palta*

Calcium deficiency in the potato shoot results in sub-apical necrosis. This is a physiological condition whereby necrotic lesions form a few millimeters below the shoot apex ultimately causing shoot tip senescence, loss of apical dominance, and the release of axillary buds. Using a Dark Red Norland tissue culture system, we studied the relationship of root zone calcium levels to shoot tip maintenance. Root zone calcium levels lower than 50 ppm resulted in shoot tip death and prolific branching from axillary buds. Chelator studies with EGTA and tracer studies with 45 Ca, indicated a direct involvement of calcium at the shoot tip for shoot tip maintenance. Interestingly, low root zone calcium deficiency syptoms could be mitigated with 0.001 to 0.01 μM of the auxin analog NAA. Developmental studies of calcium deficiency symptoms indicate no anatomical relationship with shoot tip necrosis as xylem conducting elements were found near the shoot apex regardless of the root zone calcium level. These results have important implications for potato shoot development especially during the early development stage from the seed piece.

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James S. Busse, M. Figueroa-Cabanas, and D.P. Stimart

Developmental anatomy of adventitious shoot formation in vitro from snapdragon (Antirrhinum majus L.) hypocotyls was investigated using two inbred lines, the most (R) and least (NR) regenerative lines selected from screening (Schroeder and Stimart, 1999). Time course analysis indicated cell division in the most regenerative line occurred first in one or a small number of epidermal cells with periclinal and anticlinal divisions within 2 days of placing hypocotyls on induction medium. Subsequently, cortical then vascular cells were recruited beneath the dividing epidermal cells. Once shoots formed, their vascular system was continuous with the original hypocotyl explant. The least regenerative line had no cell division directed towards organogenesis through 6 days. Shoot formation on snapdragon hypocotyls was adventitious in origin, by direct organogenesis and genotype dependent.

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James S. Busse, Senay Ozgen, and Jiwan P. Palta

Shoot tip necrosis has been attributed to calcium deficiency in in vitro cultures, resulting in death of the stem tip, the loss of apical dominance, and axillary branch development. Using an in vitro shoot culture system with Solanum tuberosum L. cv. Dark Red Norland, we studied the development of injury symptoms at the microscopic and tissue levels at a range of media calcium concentrations varying from 6.8 to 3000 μm. Light and electron microscopic studies revealed that the primary injury due to calcium deficiency was the death and collapse of expanding pith cells below the shoot apex. The structure and organization of the shoot apical meristem was the same when plants were cultured on sufficient or suboptimal media calcium concentrations. However, the apical meristem senesced following subapical shoot tissue collapse. Death of the shoot apical meristem was a secondary effect of calcium deficiency, resulting in loss of apical dominance. Studies with 45Ca indicated that calcium was distributed in a gradient along the shoot, with highest concentration at the base and the lowest at the apex. Shoot tip necrosis developed after 20 days of culture on the suboptimal calcium concentration medium. The development of these symptoms and axillary shoot growth was associated with the lack of calcium accumulation in the shoots. Our results provide evidence that a primary injury of calcium deficiency is localized in the expanding pith cells below the shoot apical meristem and this injury results in the collapse of subapical cells. Death of the shoot apical meristem is a secondary injury resulting from calcium deficiency.

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Kevin R. Kosola, Beth Ann A. Workmaster, James S. Busse, and Jeffrey H. Gilman

Air excavation is commonly used to expose structural roots of trees, but its suitability for sampling fine roots for research has not been closely examined. We compared damage and root-diameter class distribution in roots sampled by air excavation with roots sampled by hydropneumatic elutriation of soil cores. We collected samples from six different tree species with a range of fine root diameters: Amur corktree (Phellodendron amurense Rupr.), apple [Malus sylvestris var. domestica (Borkh.) Mansf.], river birch (Betula nigra L.), striped maple (Acer pensylvanicum L.), swamp white oak (Quercus bicolor Willd.), and western redcedar (Thuja plicata D. Don). Root-diameter class distributions for each species were the same for samples collected by either air excavation or by elutriation. Median root diameter was greatest for Amur corktree and western redcedar (≈0.5 mm), intermediate in striped maple and oak, and least in river birch and apple (≈0.2 mm). Root damage was primarily due to loss of root tips. Although species varied in their susceptibility to root damage and whether air excavation caused more damage than elutriation, root diameter was not a good predictor of damage during sampling. Air excavation caused ≈26% greater damage to root samples of river birch and western redcedar than did elutriation. Both sampling methods caused equivalent root damage in all other species. Root anatomy influenced susceptibility to damage during sampling. Epifluorescence microscopy revealed a root hypodermis in all species except Amur corktree and western redcedar. Without the mechanical support of this suberized layer, the cortex of Amur corktree was easily stripped from the stele, leading to extensive damage by both sampling methods. Hydropneumatic root elutriation conferred some protection to roots relative to air excavation (in two of six species); final choice of root sampling method must depend upon the requirements of the individual study and characteristics of the site.