Abstract
Despite the increasing use of protoplasts in plant biotechnology research, shoot regeneration from protoplasts remains challenging. In this study, we investigated the factors involved in protoplast isolation, callus induction, and shoot regeneration in Petunia hybrida cv. Mirage Rose. The following conditions were found to be most optimal for protoplast yield and viability: 0.6 M mannitol, 2.0% cellulase, and 6 h digestion time. A plating density of 10 × 104 protoplasts/mL under osmoticum condition (0.58 M mannitol) showed high microcolony viability in liquid culture. The Kao and Michayluk medium was found to be appropriate for callus proliferation from microcalli under a 16-h light photoperiod. Calli cultured in Murashige and Skoog medium containing 1.0 mg/L 6-benzylaminopurine and 0.2 mg/L 3-indole butyric acid showed the highest shoot regeneration frequency and number of shoots obtained per explant. Random amplification of polymorphic DNA analysis showed that the protoplast-derived shoots exhibited the same banding patterns as those of donor plants. Collectively, these findings can contribute to solving problems encountered in protoplast isolation and shoot regeneration in other petunia cultivars and related species. As the protocol developed by us is highly reproducible, it can be applied in biotechnology research on P. hybrida cv. Mirage Rose.
Highlights
Plant protoplasts are totipotent and can regenerate into various organs
Target gene editing via ribonucleoprotein (RNP) complex delivery using protoplast-based technology is much less likely to produce off-target mutants compared with Agrobacterium-meditated transformation [8,9,10]
0.6 M mannitol and 2.0% cellulase were found to be most optimal for protoplast isolation in P. hybrida cv
Summary
Plant protoplasts are totipotent and can regenerate into various organs. In addition, they can take up foreign genetic material such as DNA, chromosomes, organelles, and viral particles [1,2].Plant protoplasts have garnered interest as experimental single cells in various fields of plant biotechnology, such as genetic manipulation, protoplast transient gene expression, plant gene functional characterization, and genome editing [3,4,5,6,7]. Plant protoplasts are totipotent and can regenerate into various organs. They can take up foreign genetic material such as DNA, chromosomes, organelles, and viral particles [1,2]. Plant protoplasts have garnered interest as experimental single cells in various fields of plant biotechnology, such as genetic manipulation, protoplast transient gene expression, plant gene functional characterization, and genome editing [3,4,5,6,7]. Despite the success of clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated genome editing in several plant species using Agrobacterium-mediated transformation, off-target effects cause unwanted results. Shoot regeneration from a protoplast-derived callus has far remained challenging, a few studies have reported genotype-dependent shoot regeneration from protoplast-derived calli [16], with most genotypes being minor or of no commercial importance
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