138 M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 the cell-alginate mixture into strontium chloride solution containing the cell wall-digesting enzymes (Aoyagi and Tanaka,1999).In this way,protoplast isolation and gel solidification proceeded simultaneously.These authors claimed that the viability of the immobilised protoplasts was higher using this procedure than with more conventional methods.It will be important to determine the general applicability of this procedure to other higher plant systems.Interestingly,the authors also claimed comparable success with yeast protoplasts by employing this procedure. 4.3.Plating density and protoplast growth in culture The final (overall)density of protoplasts in the culture medium (plating density)is crucial for maximising wall regeneration and concomitant daughter cell formation. Generally,the optimum plating density is in the range 5x10-1x10 protoplasts ml- An excessively high plating density rapidly depletes nutrients,and protoplast-derived cells can fail to undergo sustained division.Cells stimulate mitotic division of adjacent cells by releasing growth factors,including amino acids,into the surrounding medium,a process commonly known as 'medium conditioning'or 'nurse'culture.Consequently, protoplasts fail to undergo sustained division when cultured below a minimum inoculum density threshold.Medium preconditioned by supporting the growth of actively dividing cells for a limited period is valuable in stimulating growth of isolated protoplasts. Similarly,actively dividing cells can promote or 'nurse'the growth of recently isolated protoplasts.Nurse cells can be from the same or different species.For example,division of protoplasts from embryogenic rice cell suspensions was most effectively stimulated by nurse cells of Italian ryegrass (Lolium multiflorum;Jain et al.,1995).Protoplasts/ spheroplasts or cells that have been X-irradiated to inhibit division can also exert a similar nurse effect. Isolated protoplasts may be cultured,using the procedures already described,in liquid medium over semi-solid medium containing the nurse cells.Dispensing protoplasts in a limited volume of medium on a membrane overlaying the semi-solid nurse cell layer promotes division.Chen et al.(2004)described a procedure for regenerating shoots from hypocotyl-derived protoplasts of red cabbage (Brassica oleracea)in which the target protoplasts were mixed in a 1:1 ratio with viable protoplasts of tuber mustard (Brassica juncea),the latter being essential for sustained division and colony formation from red cabbage protoplasts.Light microscopy revealed that regenerated plants had the expected somatic chromosome complement of red cabbage (2n=2x=18),confirming that sponta- neous fusion did not occur between protoplasts of the two species during culture.The applicability of this approach of mixing viable test and nurse protoplasts warrants more extensive evaluation in other systems,especially where protoplast-derived cells fail to undergo sustained mitosis as a monoculture. 5.Totipotent protoplast systems Many of the morphological changes that occur during the development of protoplasts to cells were described in detail in early publications.In contrast,anatomical investigationsthe cell–alginate mixture into strontium chloride solution containing the cell wall-digesting enzymes (Aoyagi and Tanaka, 1999). In this way, protoplast isolation and gel solidification proceeded simultaneously. These authors claimed that the viability of the immobilised protoplasts was higher using this procedure than with more conventional methods. It will be important to determine the general applicability of this procedure to other higher plant systems. Interestingly, the authors also claimed comparable success with yeast protoplasts by employing this procedure. 4.3. Plating density and protoplast growth in culture The final (overall) density of protoplasts in the culture medium (plating density) is crucial for maximising wall regeneration and concomitant daughter cell formation. Generally, the optimum plating density is in the range 5
104 –1
106 protoplasts ml1 . An excessively high plating density rapidly depletes nutrients, and protoplast-derived cells can fail to undergo sustained division. Cells stimulate mitotic division of adjacent cells by releasing growth factors, including amino acids, into the surrounding medium, a process commonly known as dmedium conditioningT or dnurseT culture. Consequently, protoplasts fail to undergo sustained division when cultured below a minimum inoculum density threshold. Medium preconditioned by supporting the growth of actively dividing cells for a limited period is valuable in stimulating growth of isolated protoplasts. Similarly, actively dividing cells can promote or dnurseT the growth of recently isolated protoplasts. Nurse cells can be from the same or different species. For example, division of protoplasts from embryogenic rice cell suspensions was most effectively stimulated by nurse cells of Italian ryegrass (Lolium multiflorum; Jain et al., 1995). Protoplasts/ spheroplasts or cells that have been X-irradiated to inhibit division can also exert a similar nurse effect. Isolated protoplasts may be cultured, using the procedures already described, in liquid medium over semi-solid medium containing the nurse cells. Dispensing protoplasts in a limited volume of medium on a membrane overlaying the semi-solid nurse cell layer promotes division. Chen et al. (2004) described a procedure for regenerating shoots from hypocotyl-derived protoplasts of red cabbage (Brassica oleracea) in which the target protoplasts were mixed in a 1:1 ratio with viable protoplasts of tuber mustard (Brassica juncea), the latter being essential for sustained division and colony formation from red cabbage protoplasts. Light microscopy revealed that regenerated plants had the expected somatic chromosome complement of red cabbage (2n=2x=18), confirming that sponta￾neous fusion did not occur between protoplasts of the two species during culture. The applicability of this approach of mixing viable test and nurse protoplasts warrants more extensive evaluation in other systems, especially where protoplast-derived cells fail to undergo sustained mitosis as a monoculture. 5. Totipotent protoplast systems Many of the morphological changes that occur during the development of protoplasts to cells were described in detail in early publications. In contrast, anatomical investigations 138 M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171