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

Abstract

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 Kalanchoë accessions belonging to nine species. The highest protoplast yield was obtained from K. blossfeldiana 'Charming Red Meadow' with 10.78 ± 0.51 × 10 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 1.0 mg/l 1-naphthaleneacetic acid (NAA), 0.5 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/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 1 mg/l NAA and 2 mg/l BAP or 2 mg/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 Kalanchoë species in somatic hybridization breeding programs. Key MessageThe study revealed a strong genotype-dependent efficiency of colony and microcallus formation. Plants were regenerated from two Kalanchoë blossfeldiana cultivars on medium supplemented with NAA and BAP.

A More Accessible, Time-Saving, and Efficient Method for In Vitro Plant Regeneration from Potato Protoplasts

“…Protoplast-derived calli are known to induce shoot growth after callus greening [ 50 ]. It has been reported in several papers that light green calli first change to dark green calli, after which the shoots then differentiate; however, when light green calli turn brown, shoots no longer develop [ 47 , 51 ].…”

Kalanchoë Breeding: Past, Present and Future

“…Additionally, protoplast isolation can be applied to create new variation in respect to developing somatic hybrids (individuals that are obtained through the fusion of somatic protoplasts of two different plant species/cultivars). This has been applied in twelve Kalanchoë accessions, belonging to nine species and K. blossfeldiana 'Charming Red Meadow' was found to yield the highest number of protoplasts (Cui et al, 2019). Hereby, there is a potential to facilitate the use of Kalanchoë in somatic hybridizations breeding programs.…”

Tissue Culture in Ornamentals: Cultivation Factors, Propagation Techniques, and Its Application

“…Types of culture medium, culture medium components, strength of the culture medium, carbon sources, pH of the culture medium, supplements of the culture medium, PGRs, and culture conditions have been proven to be vital factors for plantlet generations from protoplasts [ 69 ]. Considering these factors and despite these limitations, plantlets have been generated successfully in several ornamental plant species [ 69 , 73 , 74 , 78 , 94 , 95 ].…”