Plant Cell, Tissue and Organ Culture 25: 161-167, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands. Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle, Lonicera nitida cv. Maigrun S.J. Ochatt INRA, Station d'Amelioration des Espkces Fruiti~res et Ornementales, Route Georges Morel, Beaucouze 49070 Angers, France Received 9 August 1990; accepted in revised form 25 February 1991 Key words: casein enzymatic hydrolysate, group B vitamins, Lonicera nitida, plant regeneration, protoplast culture, woody ornamentals Abstract Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg 1-1 NAA (1-naphthaleneacetic acid), 1.0 mg 1-1 BAP (6-benzylaminopurine) and 150 mg 1-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg 1-1 NAA and 0.2 mg 1-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01mg1-1 NAA, 5.0mg1-1 BAP, 0.5 mg 1-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg 1-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being sub- sequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg 1-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization. Abbreviations: BAP - 6-benzylaminopurine, CPW - Power et al. (1989) medium, 2,4-D - 2,4- dichlorophenoxyacetic acid, FDA - fluorescein diacetate, F.P.E. - final plating efficiency, f.wt. - fresh weight, IAA - 4-indole-3yl-acetic acid, IBA - 4-indole-3yl-butyric acid, I.P.E. - initial plating efficiency, MES - 2-N-morpholinoethane sulfonic acid, M.P.E. - intermediate plating efficiency, MS - Murashige & Skoog (1962) medium, NAA- 1-naphthaleneacetic acid, PVP-10- polyvinylpirrolidone, Av MW 10,000, TIBA - 2,3,5-tri-iodobenzoic acid, Z - zeatin Introduction The ornamental genus Lonicera (honeysuckle) includes several sexually incompatible climbing and shrubby species with fragrant, attractive flowers which are amongst the most widely grown ornamental climbers and shrubs in Europe (Wright 1983). The development of pro- toplast-to-plant systems for honeysuckle may greatly assist in the breeding of novel, ornamen- tally valuable genotypes by both somaclonal (protoclonal) variation and somatic hybridization (through the fusion of protoplasts from climbing and shrubby species). At the onset of this study, though, Lonicera species had rarely been studied in vitro, with only three reports, two concerned micropropagation (Boonnour et al. 1988; Suzuki et al. 1986) while the other used cell cultures for biochemical studies (Tanahashi et al. 1984). Shrubs and trees were typically considered to be