Turning point article plant protoplasts

摘要: What led me in 1960 to begin work develop an enzymatic procedure for the isolation of plant protoplasts? The truth is that I initially had no idea wanted isolate protoplasts! arrived 1959 at University Nottingham as a lecturer physiology. already completed 3 yr Civil Service Commission postdoctoral fellow bacterial chemistry, after receiving my Ph.D. biochemistry from Bristol 1956 studies on synthesis amino acids and proteins barley. Two viewpoints impacted these 6 research. first was whole too complex detailed biochemical analysis experimentally it would be better reduce cell level then reassemble relate plant. second appreciation with bacteria single cells small groups much more amenable biological studies. During this time, also been interested development new analytical procedures experienced way which their introduction opened up research areas sciences. From results year published supervisor improved determination ninhydrin (Yemm Cocking, 1955), play key role quantitative acid composition proteins. Also, by researching bacteria, produce quickly than plants, two significant papers keen isolated plants aspiration developing ‘bacterial’ type culture system divide, separate cells. approach adopted investigate use chelating agents rapidly elongating region roots tomato seedlings. This established separation largely dependent Ca middle lamella. However, although extensive achieved, division root observed became evident were negatively impacting physiology biochemistry. Chelating therefore little use; thoughts turned possibility breaking down not just lamella but wall itself order release protoplast within wall. read about fungal protoplasts enzymes degrading walls and, probably importantly, discussions workers Microbiological Research Establishment, Porton when fellow. My training told have cellulases cellulose rather lysozyme. knew possible physically wall, provided plasmolyzed away In undergraduate practicals cut through pieces plasmolized beetroot ends cut-through As recounted, illustrated picture May seedling tips using Myrothecium verrucaria cellulase (Cocking, 1983), survey wide range commercially available preparations ability protoplasts. Seedling chosen because material could thereby readily obtained different known stages differentiation, minimal problems penetration enzyme. Many enzyme tested, all without success. While progress, D. R. Whitaker (National Laboratories, Ottawa) purification M. verrucaria. He generously few grams his preparation covering letter, dated 25 November 1959, said ‘In tends highly crystalline, should think its degradation slow process—quite apart accessibility factors due other components acting physical barrier.’ Thinking might ruled out such physiochemical factors, put sample bottom deep freeze. Only everything else failed did test preparation; released 1960). Microscopically observing being meristematic exciting, examine inside extra clarity there looked appreciated indispensability fundamental importance plasma membrane protoplast. respect interesting recall whether inevitably bounded question arose solid characterized certainty identified animal recalled Henry Harris recent perceptive meticulous historiography doctrine (Harris, 1999), distinction between cytoplasmic only finally classical plasmolysis