Protoplast isolation and culture from Kalanchoe species: optimization of plant growth regulator concentration for efficient callus production

A high yield of isolated protoplasts and efficient regeneration protocols are prerequisites for successful development of somatic hybrids. In the present study, protoplast isolation and regeneration were evaluated in 12 Kalanchoe accessions belonging to nine species. The highest protoplast yield was obtained from K. blossfeldiana Charming Red Meadow' with 10.780.51x10(5) protoplasts per gram fresh weight. We observed significant differences of protoplast yield while there was no distinct difference in viability among the accessions. Seven accessions reached the microcolony stage and four developed microcalli in medium supplemented with 1.0mg/l 1-naphthaleneacetic acid (NAA), 0.5mg/l 6-benzylaminopurine (BAP) and 0.5mg/l 2,4-dichlorophenoxy acetic acid (2,4-D). Using five selected accessions we optimized the PGR (plant growth regulators) concentrations using combinations of NAA, BAP and 2,4-D. K. blossfeldiana cultivars Charming Red Meadow' and Paris' produced significantly different numbers of calli depending on the PGR concentrations. For plant regeneration, the medium was supplemented with 1mg/l NAA and 2mg/l BAP or 2mg/l zeatin. Shoots were regenerated on medium supplemented with NAA and BAP for K. blossfeldiana Charming Red Meadow' and K. blossfeldiana Paris'. The plants successfully developed roots on the medium supplemented with IAA. The medium containing zeatin induced root formation directly from callus in K. blossfeldiana Charming Red Meadow'. Our findings have the potential to facilitate the use of Kalanchoe species in somatic hybridization breeding programs. Key Message The study revealed a strong genotype-dependent efficiency of colony and microcallus formation. Plants were regenerated from two Kalanchoe blossfeldiana cultivars on medium supplemented with NAA and BAP.