M.R. Davey

Transformation of Petunia protoplasts by isolated Agrobacterium plasmids

Abstract Plasmids isolated from Agrobacterium have been used to transform Petunia suspension cell protoplasts. Transformed cells, selected by their hormone independence, possessed features characteristic of crown gall tumours, i.e. continued proliferation on hormone-free media, overgrowth formation when grafted onto host plants, octopine synthesis, and lysopine dehydrogenase activity. These results are discussed in relation to the use of Agrobacterium plasmids in plant genetic manipulation.

Plant regeneration from root protoplasts of Brassica

Abstract Protoplasts isolated enzymatically from roots of Brassica alba (White Mustard), B. campestris (Turnip), B. napus (Rape) and B. oleracea (Cabbage), divided to form callus. Plant regeneration was obtained from protoplast derived tissues of B. napus and B. oleracea , but only rhizogenesis was observed with B. campestris . Tissues of B. alba remained undifferentiated. The suitability of root protoplasts for genetic manipulations in the genus Brassica is discussed.

Origin and development of somatic embryos inMedicago sativa L. (alfalfa)

SummaryEmbryoids which developed from callus obtained from leaves ofMedicago sativa L. (alfalfa) were studied with the light and scanning electron microscopes; embryoids are not common in leguminous plants. Sections showed that in organised callus embryoids apparently originated from groups of embryonic cells, whereas in friable callus, and in the epidermis of cotyledons, hypocotyls and roots of callus-derived plantlets, they often originated from single cells. Many early stages of embryogenesis (1, 2, 3- and 4-celled stages) were observed, particularly in epidermal sites. Stages in somatic embryogenesis, though variable, resembled those described for the normal zygotic embryos of alfalfa. A suspensor-like structure was sometimes present in the embryoids. Numbers of cotyledons, up to six, were observed. The SEM provided confirmation of the many stages of development and the very large number of embryoids present, as well as evidence of the range of form resulting from differential growth of regions of the embryos.

Organogenesis From Root Protoplasts of the Forage Legumes Medicago sativa and Trigonella foenum-graecum

Summary Protoplasts were isolated from seedling roots of Medicago sativa and Trigonella foenumgraecum using a simple enzyme mixture. Medicago root protoplasts entered division after 24 to 48 hours of culture and formed cell colonies with a plating efficiency of 9 to 12 %. Plantlets were recovered via somatic embryogenesis from protoplast derived tissues. Trigonella root protoplasts formed callus at a lower plating efficiency (6.5 to 10 %); such tissues regenerated only roots.

Somatic Embryogenesis in Tissues From Mesophyll and Cell Suspension Protoplasts of Medicago coerulea and M. glutinosa

Summary Mesophyll and cell suspension protoplasts of Medicago coerulea and M. glutinosa divided to form callus. The conditions of plant growth, plant age, protoplast isolation, and protoplast culture were critical for sustained division of mesophyll protoplasts. Those for division of cell suspension protoplasts were less stringent. 2,4-D in combination with a cytokinin induced embryogenesis in both Medicago species. In M. coerulea , BAP was effective as the cytokinin, while M. glutinosa had a requirement for zeatin. The potential of protoplast systems for transfer of characteristics from M. coerulea to M. glutinosa and M. sativa is discussed.

Isolation and Sustained Division of Phaseolus aureus (Mung Bean) Root Protoplasts

Summary A simple enzymatic method is described for the isolation of protoplasts from radicles of 24 hours germinated Phaseolus aureus (Mung Bean) seeds. 8-9 X 10 4 protoplasts were obtained routinely per root explant. Media based on the formulations of Donn (1978) and Kao and Michayluk (1975) supported protoplast division, with liquid over agar cultures being superior to liquid medium alone. Transfer of cell colonies to a modified White’s medium (Wood and Braun, 1961) enabled callus to be recovered from cell colonies. The use of roots for protoplast isolation is discussed in relation to other sources of experimental material such as greenhouse-grown plants and axenic shoots.

Cultural studies of a dividing legume leaf protoplast system

Abstract Cowpea leaf mesophyll protoplasts undergo rapid and sustained division during culture with the eventual production of callus. Regenerated callus will undergo limited differentiation.

Callus formation from root protoplasts of Glycine max (soybean)

Abstract Protoplasts isolated enzymatically from roots of germinating soybean seeds divide to form callus. The ability to culture protoplasts of a more meristematic type is discussed with respect to genetic manipulations in this crop species.

Polyethylene glycol-induced uptake of micro-organisms into higher plant protoplasts: An ultrastructural study

Abstract Protoplasts isolated from suspension-cultured cells of Parthenocissus tricuspidata take up yeast cells, yeast protoplasts, and blue-green algal cells when treated with polyethylene glycol (PEG). Electron microscopy has shown the micro-organisms to become localised in membrane-bounded vesicles in the cytoplasm of the higher plant protoplasts.

Crown gall protoplasts—isolation, culture and ultrastructure☆

Abstract Large quantities of protoplasts were enzymatically isolated from cell suspensions of Parthenocissus tricuspidata crown gall. Their behaviour under fully defined cultural conditions was followed at the light- and electron-microscope level. Some protoplasts regenerated a new cell wall and divided to form small cell colonies; others underwent nuclear division without concomittant cytokinesis to form multinucleate, wall-less structures. In each case there was an absolute requirement for exogenous growth regulators.