B. W. W. Grout

Reconsideration of the term ‘vitrification’ as used in micropropagation

The term vitrification is currently used to describe two types of processes related to tissue-cultured plant material. The first is used to describe organs and tissues having an abnormal morphological appearance and physiological function. The second is used to describe the transition from liquid to solid state, i.e. the formation of ice during low temperature storage of in vitro cultured cells, tissues and organs. Use of the same term to define two greatly different processes in the same research area can only lead to confusion, especially for key words. Thus it is appropriate to reconsider the usage of vitrification in the first sense mentioned above. It is recommended that the term vitrification should no longer be used to indicate plant material with an abnormal morphological appearance and physiological function, and should be substituted by the term ‘hyperhydricity’.

Contaminated liquid nitrogen vapour as a risk factor in pathogen transfer

Liquid nitrogen in storage containers will gather particulate matter from the atmosphere, or the surfaces of containers placed into it, with time. Some of these accumulating particles may be pathogenic organisms and it can be demonstrated that their viability may be conserved by immersion in the liquid nitrogen. This contamination constitutes a risk to the status of stored samples that can, largely, be avoided by the use of appropriate techniques for sealing sample containers and sterilizing their outer surfaces. The present study uses fungal spores and organic crystals to demonstrate that such particles contained in liquid nitrogen are released back into the environment when nitrogen vapour cools programmable freezers or dry shippers. This demonstrates that storage in the vapour phase above liquid nitrogen still carries a real risk of sample, or facility, contamination. Regardless of the safety of the stored sample, this is a potential source of cross-contamination between repositories or experimental sites, both locally and internationally, that could have serious consequences in clinical and agricultural situations. This study provides evidence to suggest that this possibility, as yet unquantified, should be included in risk analysis of storage protocols for reproductive materials. The risk becomes diverse when, for example, semen and embryos are frozen at an agricultural site and the dry shipper can co-transport spores of contaminating crop plant pathogens to the destination site.

Wax development on leaf surfaces of Brassica oleracea var. Currawong regenerated from meristem culture

Abstract The surface wax of seedlings and plantlets regenerated from tissue culture of Brassica oleracea var. Currawong was investigated. Preparations examined by scanning electron microscopy showed differences in the quantity of wax on the leaf surfaces of plants from tissue culture when compared with seedlings. These leaf surfaces also exhibited different contact angles to small water droplets. These differences could be eliminated if the regenerated plants were hardened. The excessive wilting and eventual death of the regerated plants consequent on their removal from culture conditions may be explained by these observations.

Cryopreservation and the maintenance of cell lines

Techniques for cell-line preservation which enable reliable and reproducible recovery of material with unchanged, defined characteristics are essential in many biotechnology industries. Cryopreservation is one such technique, and in this review we explore some of its problems, successes and potential future applications.

Survival of shoot meristems of tomato seedlings frozen in liquid nitrogen

Abstract An explant containing the primary shoot meristem was dissected from intact tomato seedlings after thawing from liquid nitrogen. Surviving explants produced shoots directly by normal meristem growth when cultured in the presence of gibberellic acid. Without gibberellic acid all surviving explants produced callus tissue and subsequently adventitious shoots, with no direct outgrowth of the primary meristem. Dimethyl sulphoxide (15%) in culture medium and a cooling rate changing continuously from 20 to 55 °C min−1 between 0 and −120 °C were required for optimal survival. Nonfrozen material produced shoots directly without the requirement for gibberellic acid indicating that hormonal regulation of organised growth by the shoot meristem had been altered by the freeze/ thaw process.

Introduction to the in Vitro Preservation of Plant Cells, Tissues and Organs

As a consequence of the developing technology for sustaining growth and development of plant materials in vitro, tissues from a great diversity of species are cultured in this way for an equally diverse number of reasons (Bhojwani 1990; Nijkamp et al. 1990; Pollard and Walker 1990). These will include transient structures such as isolated protoplasts and excised shoot and root meristems, that must proceed with development as a necessary adjunct to their survival, and somewhat more stable structures such as single cells, cell clusters, somatic embryos and fully differentiated plantlets. These may be maintained in liquid growth medium or on the surface of, or embedded in, medium made semi-solid with various gelling agents. Such in vitro cultures are employed in all aspects of basic and applied research in the plant sciences, encompassing molecular biology, biotechnology, crop improvement, plant production and basic metabolic and anatomical studies.

Cellulose microfibril deposition at the plasmalemma surface of regenerating tobacco mesophyll protoplasts: A deep-etch study.

SummaryThe reappearance of cellulose microfibrils at the naked surface of protoplasts enzymatically isolated from tobacco (Nicotiana tabacum L. var. Xanthi) mesophyll tissue has been closely studied using the techniques of thin-sectoining and the deep-etch modification of the freeze fracture procedure.A 16 h lag period was recorded between the time of isolation and the sudden appearance of considerable lengths of cellulose microfibril at the outer protoplast surface. The microfibrils were not associated with any structured particles or apparently differentiated regions of the plasmalemma. Terminal regions of the microfibrils appeared to have tapering ends, or else be sinking into the membrane substance. There was no evidence to suggest transport of intact microfibrils in vesicles through the cytoplasm to the plasmalemma.The reported observations have been discussed with respect to the various working hypotheses which have been proposed for the ‘in vivo’ construction of cellulose microfibrils.

BIOCHEMICAL AND ULTRASTRUCTURAL EXAMINATION OF CRYOPRESERVED HEPATOCYTES IN RAT

Abstract We investigated the ultrastructure and metabolic capabilities of isolated rat hepatocytes after cryopreservation using 1.5 M Me 2 SO as protectant and a slow cool/fast thaw regime. Ultrastructural assessment of the cryopreserved population revealed only approximately 10% of cells with normal morphology. Conjugation of bilirubin by the cryopreserved cells was reduced to 20% of that seen in unfrozen hepatocytes and there was a net loss of glycogen measured in cryopreserved cells incubated in conditions which stimulated glycogen synthesis by unfrozen cells. These results are in contrast to other reports in which cryopreserved hepatocytes have been successfully used for transplantation to reverse hepatic insufficiency.

Further observations on cell-wall formation around isolated protoplasts of tobacco and tomato.

Freeze-etch observations of protoplasts isolated from tobacco (Nicotiana tabacum L.) mesophyll tissue and tomato (Lycopersicum esculentum Mill.) fruit locule tissue are described which clarify earlier observations (Burgess, J., Fleming, E.N., Planta 131, 173–178, 1976; Planta 133, 267–273, 1977), obtained using scanning electron microscopy. of “fibres” associated with “projections” from these cell surfaces. It is demonstrated (1) that the “fibres” consist of bundles of small numbers of microfibrils which have become artifactually thickened by the deposition of coating materials, and (2) that the apparent association between “fibres” and “projections” results from microfibrils being lifted preferentially from protoplast surfaces in regions rich in “projections” (plasmalemmasomes). With the higher resolution available using freeze-etching it can be demonstrated that microfibril deposition does not occur in discontinuous zones on these protoplast surfaces. Globules associated with microfibril termini in radish (Raphanus sativus L.) roots are illustrated and it is proposed that turgor pressure differences between isolated protoplasts and intact tissue may account for the absence of similar globules from isolated protoplast surfaces.

Additives for the enhancement of fusion and endocytosis in higher plant protoplasts: An electrophoretic study

Abstract A range of additives has been employed to enhance both endocytosis and fusion in isolated higher plant protoplast systems. The effects of these compounds, at various concentrations, on the electrical properties of the plasmalemma have been investigated for a range of protoplast types using methods of whole cell electrophoresis. Enhancement of endocytosis and fusion is considered in terms of electrical repulsion and attraction and the presented data are considered as a guide-line for the design of similar experimental systems.