The culture and differentiation of cereal cells and protoplasts in vitro

Abstract

This thesis describes the development of methodology for: (a) the establishment of somatic and gametic cereal cells in culture; (b) the regeneration of plants from polyploid and polyhaploid callus; (c) the isolation and culture of cereal protoplasts; and finally, also examines the surface properties and metabolic competence of cultured barley mesophyll protoplasts. The cultural requirements for cereal callus initiation and growth are elaborated in Chapter 1. A fully defined nutrient formulation capable of supporting rapid callus proliferation is described. This medium is characterized by its high nitrate to phosphate ratio and a reduced content of ammonium nitrogen. The ability of sibling callus to adapt and survive on medium containing increasing levels of an otherwise toxic nutritional component has permitted the isolation of two cell lines; one of which is capable of growth on nutrient medium with a high content of ammonium and the other, on medium with fructose as the sole carbohydrate source. Methods for retaining the morphogenetic capacity of cereal callus on long-term culture are described in Chapter 2. The procedures are based on the observation that calluses maintained persistently in a de-differentiated state on auxin-containing medium invariably lose their ability to differentiate plants spontaneously on hormone-free medium. These calluses also do not respond de novo to shoot induction medium. In contrast, long-term callus cultures propagated alternately on medium supplemented with and without hormones, retain their morphogenetic potential. Procedures for the production of polyhaploids from wheat anther cultures via the callus to plant route are described in Chapter 3. These procedures were developed by investigating the effects of nutritional, hormonal and cultural conditions on the initiation of callus from somatic and gametic tissues of cultured wheat stamens. Conditions which suppressed diploid callus formation without affecting gametic callus production were then used successfully to elicit a high incidence of callus formation from cultured anthers. Green haploid plants could be readily regenerated from anthers at incipient stages of callus formation following their transfer to shoot regeneration medium. A protoplast isolation technique capable of rapid and efficient protoplast isolation in high yields from a wide range of cereal leaves is described in Chapter 4. Improved methodology for protoplast isolation was necessary because it was found that protoplasts isolated from tissues after long periods of enzymatic treatment (> 12 h) are relatively less stable and metabolically less competent than rapidly (< 3 h) isolated protoplasts. Rapid isolation procedures therefore provide a source of physiologically superior protoplasts suitable for cultural and biochemical experiments. Investigations concerning the stability of barley protoplasts in response to different cultural conditions revealed that lysis occurred rapidly if protoplasts were cultivated directly in nutrients supplemented with auxins and/or cytokinins at concentrations known to support cell division (Chapter 5). This problem was overcome by culturing protoplasts in hormoneless nutrients until cell wall regeneration was completed before nutrients supplemented with hormones were added. Culture of protoplasts in conditioned medium containing no exogenously added hormones during the cell wall regeneration phase also enhanced their capacity to undergo sustained division following the further addition of hormone-supplemented nutrients. These procedures have also been used successfully to cultivate protoplasts isolated from barley callus, maize and sorghum leaves. The use of chemical and biological fusogens as molecular probes to examine the surface properties of barley mesophyll protoplasts in situ is described in Chapter 6. It was found that protoplasts could be: (a) agglutinated electrostatically by charged molecules like polyethyleneglycol, or (b) agglutinated via receptor-ligand interactions by multivalent agents such as Yariv antigen, phytolectins and antiserum. The mechanism of induced and spontaneous agglutination can be explained by mathematical models based on colloidal particle theory, and also by biological models based on the concept of the plasmamembrane being a lipid bilayer with properties conforming to Singer & Nicolson's 'Fluid Mosaic Model of Membrane Structure'. The senescent physiology and biosynthetic competence of cultured barley mesophyll protoplasts are examined in Chapter 7. Results therein show that rapidly isolated protoplasts senesce at a rate comparable to similarly treated detached leaves. Senescence (as determined by the rate of loss of chlorophyll, protein and RNA) in barley leaves and protoplasts during the first three days in culture can be retarded if tissues are incubated in culture medium under low-light illumination (200-500 lx). These incubation conditions also stimulated the incorporation of radioactive precursors into RNA and protein. In contrast, cycloheximide pretreatment of leaves which usually delays senescence, also decreased the biosynthetic capacity of cultured barley protoplasts. Phytolectins, however, stimulated the synthesis of RNA, DNA and protein in freshly isolated protoplasts but not in protoplasts which had regenerated a cell wall or under conditions which prevent lectin binding to the plasmamembrane.