Erica E. Benson

Cryopreservation and post freeze molecular and biosynthetic stability in transformed roots of Beta vulgaris and Nicotiana rustica

Crypopreservation methods were firstly developed for root-tips from hairy root cultures of Beta vulgaris, established after transformation by Agrobacterium rhizogenes. The effects of culture age, pre-growth, cryoprotection, freezing rate and post-freeze culture conditions were determined. The resulting freezing protocol was then used to cryopreserve transformed root cultures of Nicotiana rustica. Both species were viable after freezing (ca. 80%), according to fluorescein diacetate vital staining. However, on average the regeneration of proliferating roots from surviving root-tips was low (<20%). Growth rates, secondary metabolite production and T-DNA structure of a number of hairy root lines were examined and found to be unchanged after cryopreservation.

The detection of lipid peroxidation products in cryoprotected and frozen rice cells: consequences for post-thaw survival

Abstract Lipid peroxidation was measured in frozen/thawed and unfrozen rice cells using the thiobarbituric acid (TBA) assay for malondialdehyde (MDA), a breakdown product of lipid peroxides. MDA was detected in increased amounts in frozen/thawed rice cells compared to unfrozen controls, suggesting that freezing injury can promote lipid peroxidation. The MDA content of rice cells was influenced by the carbon source (sucrose or fructose) in the recovery medium. Levels of peroxidation were greater in the presence of fructose.

Rice cell cryopreservation: the influence of culture methods and the embryogenic potential of cell suspensions on post-thaw recovery

Protoplast-derived plants were regenerated from cryopreserved embryogenic suspension cultures of the Japonica rice cultivar Taipei 309 at frequencies comparable to those of the original non-frozen cultures. The duration of cryogenic storage did not affect the embryogenic potential of suspension cultures re-established from thawed material. Cells from embryogenic cultures regrew after thawing significantly faster than those from non-embryogenic cultures. The method and frequency of subculture of cells after thawing and the nitrogen status of the recovery medium significantly influenced the rate of cell regrowth. The most rapid post-thaw cell regrowth occurred in cell samples maintained on filter paper overlying semi-solid medium lacking inorganic nitrogen and subcultured 3 days after thawing.

Variation in free-radical damage in rice cell suspensions with different embryogenic potentials

Levels of free-radical-mediated lipid peroxidation were monitored in cell-suspension cultures of Oryza sativa L. possessing different embryogenic potentials. Oxidative stress was evaluated using assays which sequentially assessed the stages of lipid peroxidation (diene conjugation, peroxidation, and the formation of secondary lipid-peroxidation products). Lipid peroxidation was significantly higher in a cell line which had lost embryogenic ability compared with lines which still retained this capacity. Superoxide dismutase (EC 1.15.1.1) activity did not vary significantly between the embryogenic and previously embryogenic lines; however, catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) activities were significantly lower in the line which had lost embryogenic ability. Metabolic activity as estimated by reduction of triphenyl tetrazolium chloride decreased with diminishing embryogenic potential and was especially low in cell lines which never exhibited embryogenic capabilities. The possible involvement of free radicals in the loss of embryogenic potential of rice cells is discussed.

Nodulation of non-legumes by rhizobia

In discussing required inputs from basic studies to applied nitrogen fixation research, Gibson (1) has highlighted three major areas which must be investigated if much of the basic research is to become of practical benefit. These include (i) the development of improved nitrogen fixing strains of rhizobia, cyanobacteria, Azospirillum and Frankia species by molecular biology techniques, coupled with physiological studies relating to the ability of such engineered microorganisms to survive in the soil, (ii) the production, storage, transport and application of inoculants and (iii) the control by the host plant of the development of various symbiotic associations and subsequent nitrogen fixation. His belief was that the objective of nitrogen self-sufficiency for agriculturally important plants was most likely to be achieved through symbiotic associations similar to those occurring in legume or actinorhizal nodules.

Free radicals in stressed and aging plant tissue cultures.

Free radicals are highly reactive molecular species which possess an unpaired electron. Oxy free radicals are especially important in biological tissues, since metabolism is dependent on the transfer of electrons, oxidation/reduction reactions and molecular oxygen. Free radical activity is therefore a normal feature in both plant and animal cells (e.g. in electron transport, lipid metabolism, detoxification and phagocytosis). However, free radicals can also initiate harmful reactions in the cell and their activity is tightly controlled. Cells are equipped with antioxidants to ensure that any free radicals that “leak” from normal metabolic processes are removed. Unfortunately such control is challenged if tissues undergo pathological disease, severe stress and physical injury. Under these circumstances, reduced antioxidant status and metabolic impairment can soon lead to free radical attack of macro molecules (lipids, proteins and DNA). In the long term these events can lead to further metabolic disorder, necrosis and cell and tissue death.

The Application of Germplasm Storage in Biotechnology

Biotechnology has precipitated a vast change in the way in which plants are utilised. Plant tissue culture techniques and genetic engineering provide a new approach to plant breeding. Furthermore large scale in vitroculture is a medium through which plant metabolism can be exploited to provide products of potential benefit to industry. These developments have also affected the applications and needs of plant germplasm conservation, these can be categorised into several main areas: 1. The conservation of important genotypes (including cultures used in genetic engineering and industrial processes). 2. The commercial exploitation of cultures which need conserving for patenting purposes. 3. The control of time-related change (e.g. somaclonal and culture variation). 4. Reduction of handling risks (contamination hazards of long-term routine culture work). 5. Reduction of costs (maintenance costs of keeping plants in long-term culture at normal growth rates).

The embryogenic potential of rice cell suspensions affects their recovery following cryogenic storage

The rates of recovery from cryogenic storage of suspension cultures of the Japonica rice cultivar Taipei 309, as determined by the reduction of triphenyl tetrazolium chloride and cell regrowth, were significantly influenced by the embryogenic potential of the non-frozen cultures.

The Control by Cryopreservation of Age-Related Changes in Plant Tissue Cultures

Cryopreservation is the process by which viable tissues are stored at ultra-low temperatures (-196°C) in liquid nitrogen. Metabolism is suspended and evidence suggests that genetic stability is maintained. The following provides a brief account of cryopreservation methodology (see also Withers, 1987; Benson and Withers, 1988).