Plant Cell Reports (1992) 10: 624- 628 Plant Cell Reports 9 Springer-Verlag 1992 Protoplast fusion-derived Ogura male sterile cauliflower with cold tolerance Thomas W. Waiters 1, Martha A. Mutschler, and Elizabeth D. Earle Department of Plant Breeding and Biometry, Cornell University, Ithaca NY 14853-1902, USA i Present address: Department of Fruit and Vegetable Science, Cornell, USA Received July 24, 1991/Revised version received October 28, 1991 - Communicated by J. M. Widholm Abstract. Cauliflower (Brassica oleracea L. ssp. botrytis) protoplasts with Ogura male sterile and fertile B. oleracea cytoplasms were fused, producing plants with an array of organellar types. Plants with Ogura mitochondria were male sterile; those with B. oleracea chloroplasts were cold tolerant. In some fusions, unfused parental protoplasts were eliminated by double inactivation with iodoacetate and gamma-irradiation; in others, fused protoplasts were physically isolated by micromanipulation or by cell sorting. Double inactivation fusions produced the most plants, including many which were male sterile, female fertile, cold tolerant and diploid. Key words: Brassica oleracea - Cytoplasmic male sterility - Organelle segregation - Selection of fusion products Abbreviations: IA: iodoacetate; FDA: fluorescein diacetate; CMS: cytoplasmic male sterility; mtDNA: mitochondrial DNA Introduction Brassica oleracea contains numerous economically important vegetables including broccoli, cauliflower, cabbage, kohlrabi, brussels sprouts, kale, and collards. Hybrids are produced using self-incompatibility, but breeding and seed production would be simplified if CMS could be used instead (Dickson and Wallace, 1986). The Ogura CMS is particularly attractive for producing B. oleracea hybrids: all tested Brassica accessions act as maintainers of Ogura (Fan et al., 1986; Heyn, 1979; Rousselle, 1979) and Ogura CMS is stable in Brassica (Bartkowiak-Broda et al., 1979). Restorer genes are not required, since B. oleracea is grown for its vegetative tissues rather than for seed production. The Ogura cytoplasm originated in Raphanus sativus (Ogura, 1968) and was subsequently transferred to B. oleracea and B. napus by backcrossing (Bannerot et al., 1974). Brassica plants with this cytoplasm are male sterile, but become chlorotic below 15~ (Bannerot et al., 1977). The cold-temperature chlorosis has been overcome in B. napus by replacing the Raphanus chloroplasts of the Ogura cytoplasm with Brassica ones via protoplast fusion (for example, Pelletier et al., 1983; Yarrow et al., 1986; Jourdan et al., 1989). However, the reduced female fertility of restored Ogura B. napus lines (Pellan-Delourme and Renard, 1987) has limited acceptance of these cytoplasms for hybrid production in this species. In this study, we produced plants with an array of organellar and nuclear types by fusing protoplasts of an Ogura CMS cauliflower line and a nearly-isogenic fertile line with B. oleracea organelles. Some plants were diploid or nearly so, and had Ogura mitochondria with B. oleracea chloroplasts. These plants were male sterile and cold tolerant; their progeny shared these traits and grew vigorously. We also compared recovery of fusion-derived plants using three methods of selection: double inactivation, micromanipulation and cell sorting. Materials and methods Plant materials. Protoplasts were isolated from leaf and hypocotyl explants of cauliflower lines NY 7642A and NY 3317. NY 7642A is an Ogura CMS cauliflower inbred line (Dickson, 1985). NY 3317 was selected from a protoplast regenerant of the fertile maintainer line for NY 7642A (Jourdan et al., 1988) and has normal B. oleracea organelles. Protoplast isolation. Protoplasts were isolated from hypoeotyl explants of 5-7 day old etiolated seedlings (Jourdan et at., 1989) and from plantlet leaves (Jourdan et al., 1990). Hypocotyl protoplasts were treated with FDA for the last 20-30 min of incubation (1 ILl of a 5 mg/ml acetone stock added per ml enzyme solution). Protoplast pretreatments. Protoplasts for double inactivation fusions were treated to prevent division of unfused protoplasts. To pretreat with IA, an appropriate amount of a freshly prepared, filter- sterilized stock of 25 mM IA in W5 solution (Menczel et al., 1981) was added to the protoplasts in enzyme solution for a final concentration of 5 mM IA. Protoplasts were incubated with IA for 20-30 minutes before processing. A 60Co source was used to T- irradiate protoplasts for 180 minutes at 112 rad/min during enzyme incubation; total dosage was 20.2 Krad. No pretreatments were used Offprint requests to. E. D. Earle