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Chapter4 Protoplast Isolation and Culture1

Julie Russell Kikkert

ate into whole . 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 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 is usually impermeable to DNA. Several protoplast-based genetic engineering techniques Key Factors emerged, including protoplast fusion, DNA microinjection, , and polyethylene glycol (PEG)-mediated Numerous factors affect the isolation and culture of pro­ gene transfer. Today, other methods, such as ­ 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 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 and treatment times (Sasamoto et al. 1989) and the growth medium (Russell and that have been used for poplars. 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. 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). Agar or more uniformly exposed to the enzymes. The results were ctgarose plating generally produces few or no protoplast­ higher protoplast yields and better viability. Leaves pro­ derived calli (Chupeau et al. 1993; Park and Son 1988, 1992; cessed with an Omni-mixer required only a 4-h digestion, Russell and McCown 1986b ). One reason for using liquid whereas other labs used a 10- to 16-h digestion (table 2). Park plating is to avoid buildup of toxic exudates around the

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 27 ~ Section I In Vitro Culture '\;;jjjj/

VJiil

V/Jiiil Table 3. Conditions for the culture of Populus protoplasts that led to sustained cell division and eventual plant recovery. V!iiil Culture Plating Basal NH/ Growth Species method density1 medium2 (ammonium nitrate) Osmotic urn regulators ~ ...:# P. alba Liquid 2.5x104 MS No 0.08 M sucrose 1.0 J.LM 2,4-D (Sasamoto et al. 1989) 0.6 M mannitol 0.1 J.1M BA ~ P. alba x P. glandulosa Agar/ 2.4x105 MS No 0.2 M mannitol 9.0 J.LM 2,4-D .) (Park and Son 1988) Gauze 0.4 M glucose 22.2 J.1M BA 4 P. alba x P. grandidentata Liquid 5.0x10 WPM No 0.366 M sucrose 1.0 J.1M NAA ~ (Russell and McCown 1988) Floating 0.046 M mannitol 0.1 J.1M BA Disc 0.046 M sorbitol 'o:!Jfl 0.046 M xylitol ~ 0.046 M inositol P. glandulosa Liquid 1.0x105 KM8P Yes 0.6 M sucrose 5.0 J.1M 2,4-D -.a/ (Park et al. 1990) 0.5 J.1M BA ~ P. koreana x P. nigra Liquid un- KM8P Yes 0.6 M sucrose 0.4 J.1M 2,4-D somatic hybrid with available 0.5 J.LM BA ~ P. x euramericana (Park et al. 1992) ~ 4 P. nigra Liquid 3.7x10 WPM No 0.366 M sucrose 1.0 J.1M NAA ~ (Lee et al. 1987) 0.046 M mannitol 0.1 J.lM BA 0.046 M sorbitol viii~ 0.046 M xylitol ~ 0.046 M inositol P. nigra x P. maximowiczii Agar/ 2.4x105 MS No 0.2 M mannitol 9.0 J.LM 2,4-D \.:J!i) (Park and Son 1992) Gauze 0.4 M glucose 0.4 J.1M BA -..9 P. nigra x P. trichocarpa Liquid 0.5x104 WPM No 0.366 M sucrose 1.0 J.1M NAA (Russell and McCown 1988) Floating 0.046 M mannitol 0.1 J.LM BA ~ Disc 0.046 M sorbitol ...;6) 0.046 M xylitol 0.046 M inositol ...;6) P. simonii Liquid 3.0x106 KM8P Yes 0.4 M glucose 13.6 J.tM 2,4-D (Wang et al. 1995) 1.1 J.1M NAA -w) 0.9 J.1M KT 'Q!ii) P. tomentosa Liquid 2.5x105 KM8P Yes glucose 2.2 J.lM 2,4-D (Wang et al. 1991) 2.5 J.1M NAA .v) 2.2J.1M BA ~ P. tremu/a Liquid 1.0x105 WPM No 0.366 M sucrose 1.0 JlM NAA (Russell and McCown 1988) Floating 0.046 M mannitol 0.1 J.LM BA 'Qii) Disc 0.046 M sorbitol ...a) 0.046 M xylitol 0.046 M inositol ~ P. tremula x P. alba Liquid 5-8x104 Chupeau Yes 0.55 M glucose 14.0 J.tM 2,4-D (Chupeau et al. 1993) 0.05 J.1MTDZ \8) 1 Protoplasts per mi. ~ 2 Three basal media formulations have been used for protoplast culture of poplars in an equal number of cases. WPM is a lower ~ salt and lower chloride medium than MS. KM8P is a complex medium containing numerous vitamins, sugars and sugar alcohols, organic acids, casamino acids, and coconut water. WPM=Lioyd and McCown (1980); MS=Murashige and Skoog (1962); ~ KM8P=Kao and Michayluk (1975). ~

~

~ cells (Chupeau et al.1993; Russell and McCown 1986b). plating ~ethod, in which the protoplasts were cultured In liquid culture, the medium can be refreshed periodi- in contact with a polyester screen at the surface of the ~ cally, thus reducing the concentration of toxic exudates. medium (figure 1). The protoplasts adhered to the screen, ~ Russell and McCown (1986b, 1988) used a floating disc which aided in medium replenishment and observation. -c)

28 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. ~ \$I

lou# Protoplast Isolation and Culture

Table 4. Plant growth regulators used for shoot regeneration from protoplast-derived calli of Populus.

Concentration in Regeneration Medium (J.lM)

Species I(T2 TDZ3 ZT4 2,4-05 P. alba (Sasamoto et al. 1989) 0.1 1.0

P. alba x P. glandulosa (Park and Son 1988) 4.6

P. alba x P. grandidentata (Russell and McCown 1988) 0.1

P. glandu/osa (Park et al. 1990) 7.5

P. koreans x P. nigra somatic hybrid with P. x euramericana (Park et al. 1992) 5.0

P. nigra (Lee et al. 1987) 0.1 1.0

P. nigra x P. maximowiczii (Park and Son 1992) 6.8

P. nigra x P. trichocarpa (Russell and McCown 1988) 0.1

P. simonii (Wang et al. 1995) 4.44 2.32 2.28 0.54

P. tomentosa (Wang et al. 199·1) 2.2 1.0

P. tremula (Russell and McCown 1988) 0.4 0.01

P. tremula x P. alba (Chupeau et al. 1993) 0.1 1 benzyladenine 2 kinetin 3 thidiazuron 4 zeatin 5 2,4-dichlorophenoxyacetic acid 6 a-naphthaleneacetic acid

Protoplasts will float only when sucrose is used as the ditions until colonies have formed. Poplar protoplasts pro­ osmoticum; however, sucrose is toxic (Chupeau et al. 1993) duced anthocyanins and did not divide even when cul­ or not optimal for some genotypes (table 3). Sucrose, glu­ tured in dim light (Chupeau et al. 1993; Russell and cose, and mannitol were the most commonly used osmotica. McCown 1986b). Plating density also affects protoplast Another apparent requirement for poplar protoplasts, development. With poplars, the optimal densities range though not listed on table 3, is culturing under dark con- from 0.5 x 104 to 3.0 x 106 protoplasts per ml of culture

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 29 Section I In Vitro Culture

medium (table 3). Optimal plating densities vary with the systems have only recently been accomplished for many· genotype, culture medium (Russell and McCown 1988), genotypes. Somatic hybridization, which requires proto­ and culture method. plasts, has been reported in 3 cases. Saito {1980b) fused Protoplasts can be sensitive to the types and concentra­ protoplasts of P. x euramericana with those of either the tions of nitrogen in the culture medium. The usual concen­ same species or of Paulownia taiwaniana. Heterokaryons trations of ammonium nitrate in the basal formulations (4.94 were identified by differential staining, but were not cul­ mM in WPM; 20.6 mM in MS; 7.4 mM in KM8P) are some­ tured. In a second report, leaf protoplasts of P. nigra var. times toxic and thus, are eliminated or reduced in the proto­ charkowiensis x P. nigra var. caudina were fused with callus­ plast culture medium (Russell and McCown 1988; Russell derived protoplasts of Hibiscus sabdariffa (Ito et al. ·1986; 1993). In poplars, 7 of the 10 reports of successful culture Oji-Paper 1989). Plants were regenerated that were poplar­ were without ammonium ions (NH4·)in the medium (table like. Thirdly, protoplasts of P. koreana x P. nigra were fused 3). Similarly, the response to organic nitrogen sources is geno­ with protoplasts of P. x euramericana (Park et al. 1992; Park type dependent. When Russell and McCown (1988) added and Son 1994). Two of the regenerated plants showed inter­ casein hydrolysate and coconut water to their WPM-based mediate protein band patterns in SDS-PAGE analysis. protoplast medium, the supplements were required for 1 Chupeau et al. (1994) recovered plants after electroporation genotype, toxic to another, and neither required nor toxic to of protoplasts with genes encoding resistance to 3 selective yet another. Organics in the medium lowered the optimal agents; paromomycin, chlorsulfuron, and phosphinothricin. plating density. Wang et al. (1995) found that either ammo­ Standard electroporation techniques developed for herba­ nium nitrate or glutamine plus aspartic acid supported low ceous crops were used. Transgenic plants that expressed the rates of colony formation, but that a combination doubled genes were obtained with relatively high frequency. The the rates of colony formation. With P. tremula x P. alba, or­ patterns of gene integration were single and clear. ganic nitrogen produced no apparent benefits. Rather, opti­ mal protoplast culture occurred when a low initial concentration of mineral nitrogen was successively increased over time (Chupeau et al. 1993). Plant growth regulators are another important compo­ Conclusions nent of the protoplast culture medium. However, the opti­ mal type and concentration varies (table 3). The most Several poplar species and hybrids have been cultured commonly used auxin and cytokinin are 2,4-D {2,4-dichlo­ from protoplasts to whole plants. Common requirements rophenoxyacetic acid) and BA (benzyladenine), respec­ for protoplast culture are the use of shoot cultures as a tively. Chupeau et al. {1993) were the only group to test source tissue, liquid plating medium, and culture in the thidiazuron (TDZ) for poplar protoplast culture. TDZ is a dark. Other important factors for each genotype include substituted phenylurea with cytokinin-like activity, and the enzyme concentration and incubation time, the isola­ in combination with 2,4-D, was required for sustained tion medium, and the protoplast culture medium, includ­ development of protoplast-derived cells of P. tremula x P. ing osmoticum, nitrogen, and plant growth regulators. alba. TDZ also reduced the release of exudates from the Growth regulators for plant regeneration from protoplast­ cells. In another study, Sasamoto et al. {1989) found that derived calli are also genotype dependent. Though proto­ the optimal growth regulator combination of 1 J.1.M 2,4-D plast isolation and culture must be optimized for each new with 0.1 J1M BA could overcome inhibitory conditions of genotype, the parameter ranges for poplars are better de­ osmoticum or ammonium ions to promote cell division fined than in the past. The ability to successfully isolate and colony formation. and culture poplar protoplasts allows for their use in physi­ Plant growth regulators are also important in the me­ ological and genetic engineering studies. dium for organogenesis from the protoplast-derived calli. The requirements appear to be highly genotype depen­ dent (table 4). Zeatin and BA were commonly used, with TDZ sometimes required. Standard micropropagation techniques were used to propagate and move regenerated Literature Cited plants to the greenhouse and field.

Protoplast-Based Genetic Engineering in Ahuja, M.R. 1983a. Developmental potential of mega and normal protoplasts in Populus. In: Potrykus, 1.; Harms, Poplars C.T.; Hinnen, A.; Hutter, R.; King, P.J.; Shillito, R.D., eds. There are few reports of protoplast-based genetic engi­ Proceedings of the 6th International Protoplast Sympo­ neering in poplars. This is because of the availability of sium; Basel, Switzerland: Experientia Supplementum. other gene transfer techniques and because regeneration 45:28-29.

30 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. Protoplast Isolation and Culture

Ahuja, M.R. 1983b. Short note: Isolation and culture of ed. Plant protoplasts and genetic engineering I: Biotech­ mega and normal protoplasts in aspen. Silvae Genetica. nology in agriculture and forestry, Vol. 8. Berlin: 32: S-6. Springer-Verlag: 262-274. Butt, A.D.1985. A general method for the high-yield isola­ Lee, J.S.; Lee, S.K.; Jang, S.S.; Lee, J.J. 1987. Plantlet regen­ tion of mesophyll protoplasts from deciduous tree spe­ eration from callus protoplasts. of Populus nigra. Re­ cies. Plant Science. 42: 55-59. search Report of the Institute of Forest Genetics. Korea. Cheema, G.S. 1988. Isolation and culture of protoplasts 23: 143-148. from totipotent cell cultures of Populus ciliata. In: Puite, Lloyd, G.; McCown, B. 1980. Commercially-feasible K.]. et al., eds. Progress in plant protoplast research: Pro­ micropropagation of mountain laurel, Kalmia latifolia, ceedings of the 7th international protoplast symposium; by use of shoot-tip culture. Proceedings of the Interna­ Wageningen, The Netherlands; 1987 December 6-11. tional Plant Propagators Society. 30: 421-427. Current plant science and biotechnology in agriculture. McCown, B.H. 1988. Recent advances in protoplast cul­ Dordrecht, The Netherlands: Kluwer Academic Publish­ ture of horticultural crops: Ornamental trees and shrubs. ers: Issue 7: 107-108. Scientia Horticulturae. 37: 257-265. Chun, Y.W. 1985. Isolation and culture of in vitro cultured McCown, B.H.; Russell, J.A. 1987. Protoplast culture of Populus alba x P. grandidentata protoplasts. Journal of Ko­ hardwoods. In: Bonja, J.M.; Durzan, D.J., eds. Cell and rean Forestry Society. 71: 45-59. tissue culture in forestry, Vol. 2: Specific principles and Chupeau, M.C.; Lemoine, M.; Chupeau, Y. 1993. Require­ methods: Growth and development. ·Dordrecht, The ment of thidiazuron for healthy protoplast development Netherlands: Martinus Nijhoff Publishers: 16-30. to efficient tree regeneration of a hybrid poplar (Populus Mgaloblishvili, M.P.; Sanadze, G.A.; Dakakishvili, K.G.; tremula x P. alba). Journal of Plant Physiology. 141:601- Badridze, G.S.H. 1988. The effect of dithiothreitol on the 609. isoprene action and photosynthetic assimilation of car­ Chupeau, M.C.; Pautot, V.; Chupeau, Y. 1994. Recovery of bon dioxide in protoplasts. Izvestiya Akademii Nauk transgenic trees after electroporation of poplar proto­ Gruzinskoi Ssr Seriya Biologicheskaya. 14: 103-109. plasts. Transgenic Research. 3: 13-19. Murashige, T.; Skoog, F. 1962. A revised medium for rapid Dalakishvili, K.G.; Mgaloblishvili, M.P.; Sanadze, G.A. growth and bioassays with tobacco tissue cultures. 1989. The relationship between photosynthetic assimi­ Physiologia ·Plantarum. 15: 473-497. lation of carbon dioxide and isoprene biosynthesis and Oji-Paper. 1989. Regeneration of arboraceous plant from reamination reactions. Soobshcheniy AAkademii Nauk protoplast culture - Populus charkowiensis x Populu·s Gruzinskoi Ssr. 136: 129-t"32. caudina, Hibiscus sabdariffa hybrid propagation. Patent Douglas, G. 1982. Protoplast isolation from totipotent cell­ Number JP 1043138. cultures of Populus hybrid TT32. In: Fujiwara, A., ed. Park, Y.G.; Choi, M.S.; Kim, J.H. 1990. Plant regeneration 1982. Tokyo: Proceedings of the 5th of Populus glandulosa from mesophyll protoplast. Ko­ International Congress of Plant Tissue and Cell Culture: rean Journal of Plant Tissue Culture. 17: 189-199. 605-606. . Park, Y.G.; Han, K.H. 1986. Isolation and culture of mesophyll Ito, K.; Tatemichi, Y.; Shibata, M. 1986. Regeneration of protoplasts from in vitro cultured Populus alba x glandulosa. poplar-like plants from fusion between Populus (hybrid) Journal of the Korean Forestry Society. 73:33-42. and Hibiscus. In: Abstracts of the International Congress Park, Y.G.; Kim, J.H.; Son, S.H. 1992. Induction of somatic of Plant Tissue and Cell Culture; 1986 August 3-8; Min­ hybrid by protoplast fusion between Populus koreana x neapolis, MN, U.S.A.: University of Minnesota: 389. Ab­ Populus nigra var. italica and Populus euramericana cv. stract #441. Guardi. Journal of the Korean Forestry Society. 81:273- Jang, S.S.; Lee, J.J.; Lee, J.S.; Lee, S.K. 1987. Isolation, cul­ 279. ture and fusion of protoplasts of Populus glandulosa Park, Y.G.; Shin, 0.1.; Woo, J.H.; Sui, I.W.; Son, S.H. 1987. Uyeki. Research Report of the Institute of Forest Genet­ Protoplast isolation from mesophyll of Populus alba. Ko­ ics. Korea. 23: 137-142. rean Journal of Plant Tissue Culture. 14: 49-54. Kao, K.N .; Michayluk, M. 1975. Nutritional requirements for Park, Y.G.; Son, S.H. 1986. Factors affecting the isolation of growth of Vicia hajastana cells and protoplasts at very low mesophyll protoplasts from Populus euramericana cv. 1- population density in liquid media. Planta. 126: 105-110. 214. Journal of the Korean Forestry Society. 74: 29-36. Kim, C.S.; Kim, Y.W.; Kim, Y.S. 1988. Breeding for Park, Y.G.; Son, S.H. 1987. Protoplast isolation from callus allotriploids with quick growing hybrid aspen (Populus and suspension cultured cells of Populus alba. Korean alba x P. glandulosa), 2: Factors affecting protoplasts from Journal of Genetics. 9: 133-140. mesophyll cells and microcolony formation. Korean Park, Y.G.; Son, S.H. 1988. Culture and regeneration of Journal of Breeding. 20: 314-323. Populus alba x P. glandulosa leaf protoplasts isolated from Kirby, E.G.; Campbell, M.A.; Penchel, R.M. 1989. Isolation in vitro cultured explant. Journal of the Korean Forestry and culture of protoplasts of forest trees. 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USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 31 Section I In Vitro Culture

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