This file was created by scanning the printed publication. Errors identified by the software have been corrected; however, some errors may remain. Chapter4 Protoplast Isolation and Culture1 Julie Russell Kikkert ate into whole plants. Figure 1 shows the steps involved and table 1lists reports of poplar protoplast isolation and Introduction culture. Woody plants were once considered difficult to regenerate from protoplasts, but breakthroughs occurred Protoplasts are cells without cell walls. In plants proto­ in the mid-1980s with Populus spp. protoplasts as one of plasts are usually liberated by tissue digestion with cellu­ the first succesful genera (McCown 1988; McCown and lases and pectinases purified from wood-rotting fungi. To Russell1987; Russell and McCown 1986a,1986b). Critical keep the plasma membrane from bursting during proto­ to this success was the use of juvenile or juvenile-like source plast isolation, an isotonic or slightly hypertonic medium tissues, such as in vitro grown plants (shoot cultures) or is used. Because of the lack of external constraints on the embryogenic cell cultures (McCown 1988), and optimum membrane, protoplasts are spherical (figure 1B). One use protoplast isolation and culture conditions (Russell1993). of plant protoplasts is to isolate single cells for physiologi­ Currently, whole plants have been regenerated from a wide cal studies. In Populus (poplar), protoplasts have been used range of poplar species and hybrids (table 1, section D). to study reactions of the photosynthetic pathway ~chapter compares reports of plant regeneration from (Dalakishvili et al. 1989; Mgaloblishvili et al. 1988; Sanadze poplar protoplasts and highlights the important common et al. 1986, 1990). success factors. For a more thorough description of gen­ Mter the demonstration of totipotency of tobacco pro­ eral protoplast culture, refer to reviews by Kirby et al. toplasts by Takebe et al. (1971), many laboratories began (1989); Rashid (1988); and Russell (1993). developing protocols to regenerate plants from herbaceous and woody plant protoplasts to use in genetic engineer­ ing. Protoplasts were considered essential for gene trans­ fer because the cell wall is usually impermeable to DNA. Several protoplast-based genetic engineering techniques Key Factors emerged, including protoplast fusion, DNA microinjection, electroporation, and polyethylene glycol (PEG)-mediated Numerous factors affect the isolation and culture of pro­ gene transfer. Today, other methods, such as Agrobacterium­ toplasts. A variety of poplar genotypes have been success­ mediated gene transfer and biolistics, are available to trans­ fully cultured by several laboratories. When the procedures form cells within intact tissues. Still, protoplasts are 1 are compared, the following common features emerge. method that has proven valuable for genetic engineering research. To obtain transgenic plants, the genetically altered pro­ Protoplast Isolation toplasts must regrow their cell walls, divide, and regener- The source tissue is a common and important factor in successful poplar protoplast culture. In 10 of the 12 reports listed in table 1, section D, the protoplast source was leaves from shoot cultures. Further, most of the shoot cultures were from nonseedling trees, which allowed researchers to work with already proven clone varieties. In woody species, shoot cultures yielded higher numbers of proto­ 1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. plasts with better viability than did leaves from green­ Micropropagation, genetic engineering, and molecular biology house- or field-grown plants (Smith and McCown 1982). of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. In addition to being juvenile, shoot cultures are aseptic, Department of Agriculture, Forest Service, Rocky Mountain Re­ adapted to tissue culture conditions, and maintain genepc search Station. 326 p. and physiological tissue uniformity (McCown 1988). To 24 Protoplast Isolation and Culture Figure 1. Stages in the culture of protoplasts through plant regeneration for Populus alba x P grandidentata. A) Leaves from shoot cultures are the source tissue. B) Newly isolated protoplasts plated in liquid medium with a polyester screen disc for support (openings in the screen are 150 11m). C) Cell divisions approximately 2 weeks after protoplast isolation. D) Protoplast-derived calli developing on the screen disc (ruler markings represent 1 mm). E) Shoot regeneration. F) Shoot multiplication. G) Harvest and rooting of micro cuttings that are ready for transfer to the greenhouse. For detailed procedures, see Russell and McCown (1988). USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 25 Section I In Vitro Culture Table 1. Reports of protoplast isolation and culture in Populus species, which are categorized by the most advanced protoplast development for a given report. Species Source1 tissue Reference Protoplast isolation P. alba C,CS Park and Son 1987 SH Park et al. 1987 P. nigra var. charkowiensis L Ito et al. 1986 x P. nigra var. caudina P. deltoides L Sanadze et al. 1986 Xin et al. 1991 P. x euramericana L-SDL Saito, 1976, 1980a SH Park and Son 1986 P. glandulosa Jang et al. 1987 P. nigra x P. laurifolia SH Russell and McCown 1986a P. tacamahaca x P. trichocarpa L Butt 1985 P. tremuloides SOL Verma and Wann 1983 P. trichocarpa x P. tacamahaca cs Douglas 1982 Protoplast division P. alba x P. glandu/osa cs Youn et al. 1985 L Kim et al. 1988 SH Park and Han 1986 P. alba x P. grandidentata SH Chun 1985 P. ciliata c Cheema 1988 P. davidiana SH Park et al. 1988 P. glandulosa SH Park et al. 1988 P. sieboldii SH Saito et al. 1987 P. tremula L Ahuja 1983a,b P. tremuloides L Ahuja 1983b Organogenesis from protoplast-derived calli P. alba SH Sasamoto et al. 1989 Recovery of whole plants from protoplasts P. alba x P. glandulosa SH Park and Son 1988 P. alba x P. grandidentata SH Russell and McCown 1986b, 1988 P. nigra var. charkowiensis SH Ito et al. 1986; x P. nigra var. caudina Oji-Paper 1989 somatic hybrid with Hibiscus sabdariffa P. glandulosa SH Park et al. 1990 P. koreana x P. nigra SH Park et al. 1992 somatic hybrid with P. x euramericana P. nigra c Lee et al. 1987 P. nigra x P. maximowiczii SH Park and Son 1992 P. nigra x P. trichocarpa SH Russell and McCown 1988 P. simonii cs Wang et al. 1995 P. tomentosa SH Wang et al. 1991 P. tremula SH Russell and McCown 1988 P. tremula x P. alba SH Chupeau et al. 1993 1 C=callus; CS=cell suspensions; L=leaf; SDL=seedlings; SH=shoot cultures ensure high yields of viable protoplasts, growth conditions A second component of protoplast isolation is tissue of shoot cultures must be optimum. Lighting conditions digestion. Table 2 lists the enzymes and treatment times (Sasamoto et al. 1989) and the growth medium (Russell and that have been used for poplars. Cellulase RIO and McCown I988) are especially important. Macerozyme RIO were the most commonly used, often in 26 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. Protoplast Isolation and Culture Table 2. Enzyme treatments used to liberate protoplasts from Populus shoot culture-derived leaves. Only reports in which whole plants were regenerated from protoplasts are listed. Enzyme concentration (percent w/v) Cellulase Cellulase Cellulase Macerase Macerozyme Hemi- Driselase Pecto- Species (Cooper) R1 0 RS R10 cellulase lyase Time P. alba (Sasamoto et al. 1989) 1.0 0.25 1.5 h P. alba x P. glandulosa (Park and Son 1988) P. nigra x P. maximowiczii (Park and Son 1992) 2.0 0.8 1.2 2.0 0.05 1.8 h P. alba x P. grandidentata P. nigra x P. trichocarpa P. tremula (Russell and McCown 1988) 0.5 0.1 4h P. x euramericana (Park et al. 1992) 1.0 0.4 1.2 2.0 0.05 10 h P. g/andulosa (Park et al. 1990) 1.5 0.5 0.5 0.5 0.05 12 h P. koreana x P. nigra (Park et al. 1992) 2.0 0.4 1.2 2.0 0.05 10 h P. tomentosa (Wang et al. 1991) 2.0 0.5 14 h P. tremula x P. alba (Chupeau et al. 1993) 0.1 0.02 0.05 16 h combination with Hemicellulase, Driselase, or Pectolyase. and Son (1988, 1992) digested poplar leaf tissue for only 1.8 h, However, the exact concentrations and time of treatment even though the leaves were sliced with a scalpel. The shorter vary with the genotype and the researcher preferences. The digestion may have been possible because the leaves were digestion environment may contain many toxic com­ placed in fresh enzyme solution 4 times. Thus, toxic compo­ pounds, such as enzyme impurities and components re­ nents were removed along with the old enzyme solution. leased after tissue wounding. Thus, digestive time is a Osmoticum, salts, growth regula tors, and buffers in the critical factor for good protoplast viability (table 2). Be­ isolation medium are also important for long-term viability cause enzymes are relatively large molecules, they pen­ of poplar protoplasts {Park and Son 1992; Chupeau et al. etrate tissues slowly. To increase penetration, leaf tissue is 1993 ). However, no general recommendations are made here. usually sliced or chopped before enzyme treatment. How­ ever, cell exposure to the enzymes is still not uniform, Protoplast Culture and Regeneration which results in over digested cells near the edges of the cuts and under digested cells in the interior of the leaves. Key factors for poplar protoplast culture through sus­ Russell and McCown (1986a) demonstrated that when tained division and plant regeneration are listed on tables leaves of poplar shoot cultures were processed in an Omni­ 3 and 4. One of the most common factors is plating in mixer, the epidermis was stripped away and the cells were liquid medium (10 of the 12 reports) (table 3).