Abstract
A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.
Highlights
There is a great diversity in the genus Citrus, the greatest number of new citrus varieties which are important today originated as bud mutations from existing cultivars (Soost & Cameron, 1975)
Protoplasts can be isolated from different kinds of tissues, such as leaf mesophyll, embryogenic callus, embryogenic suspension cultures and non embryogenic callus (Grosser & Gmitter Junior, 1990)
Protoplast fusion and citrus somatic hybridization have been an important tool in cultivar development schemes in countries such as USA (Gmitter Junior et al, 1992; Grosser, 1994), Israel (Vardi et al, 1975; Vardi & Spiegel-Roy, 1982), Japan (Kobayashi & Ohgawara, 1988; Miranda et al, 1997), and France (Ollitrault & Luro, 1995)
Summary
There is a great diversity in the genus Citrus, the greatest number of new citrus varieties which are important today originated as bud mutations from existing cultivars (Soost & Cameron, 1975). Somatic hybridization through protoplast fusion allows the combination and hybridization of different sexually incompatible genera and species, which is difficult or impossible by conventional breeding (Vardi & Galun, 1989; Grosser & Gmitter Junior, 1990). Protoplast fusion and citrus somatic hybridization have been an important tool in cultivar development schemes in countries such as USA (Gmitter Junior et al, 1992; Grosser, 1994), Israel (Vardi et al, 1975; Vardi & Spiegel-Roy, 1982), Japan (Kobayashi & Ohgawara, 1988; Miranda et al, 1997), and France (Ollitrault & Luro, 1995). This research had the objective to adapt and optimize a protoplast to plant system protocol for Brazilian citrus cultivars
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