Articles
Optimization of protoplast isolation and culture protocol for Mediterranean-adapted broccoli (Brassica oleracea var. italica) cultivars
Article number
1454_54
Pages
387 – 394
Language
English
Abstract
Cytoplasmic male sterility (CMS), achievable through protoplast fusion and regeneration, holds significant potential for enhancing hybrid breeding in broccoli (Brassica oleracea var. italica). However, the commercial application of CMS in B. oleracea has been limited by challenges in efficient protoplast isolation and regeneration.
In this study, we aimed to optimize several steps in the protoplast isolation and regeneration protocol, focusing on the two commercially cultivated B. oleracea cultivars ‘Parthenon F1’ and ‘Naxos F1’, widely grown in the Mediterranean basin.
We first evaluated the impact of different tissue sources (true leaves and hypocotyls) and enzyme concentrations on protoplast yield and viability.
Protoplasts were isolated using two concentrations of Cellulase R-10 and Macerozyme R-10. Comparative analysis showed that true leaves provided the highest protoplast yields using 1.5% cellulase and 0.4% macerozyme concentration, which yielded 2.26×106 protoplasts g‑1 FW in ‘Parthenon F1’ and 2.74×106 protoplasts g‑1 FW in ‘Naxos F1’. In contrast, hypocotyls yielded significantly fewer protoplasts in both cultivars.
Regarding viability, hypocotyl-derived protoplasts isolated with the highest enzyme concentrations exhibited superior viability, whereas mesophyll protoplasts showed higher viability when were digested with 1.5% cellulase and 0.4% macerozyme.
Subsequently, we assessed two different protoplast culture media (PCM) to evaluate plating efficiency and microcolony density in mesophyll-isolated protoplast.
PCM containing 2 mg L‑1 6-Benzylaminopurine (BAP) and 0.5 mg L‑1 naphthaleneacetic acid (NAA) significantly improved plating efficiency and microcolony density in both cultivars, particularly in ‘Naxos F1’. These results suggest that optimized enzymatic conditions and a higher ratio of cytokinin (BAP) to auxin (NAA) is more favorable for protoplast proliferation, thereby contributing to future genetic transformation efforts and enhancing breeding programs, particularly in the context of climate change.
In this study, we aimed to optimize several steps in the protoplast isolation and regeneration protocol, focusing on the two commercially cultivated B. oleracea cultivars ‘Parthenon F1’ and ‘Naxos F1’, widely grown in the Mediterranean basin.
We first evaluated the impact of different tissue sources (true leaves and hypocotyls) and enzyme concentrations on protoplast yield and viability.
Protoplasts were isolated using two concentrations of Cellulase R-10 and Macerozyme R-10. Comparative analysis showed that true leaves provided the highest protoplast yields using 1.5% cellulase and 0.4% macerozyme concentration, which yielded 2.26×106 protoplasts g‑1 FW in ‘Parthenon F1’ and 2.74×106 protoplasts g‑1 FW in ‘Naxos F1’. In contrast, hypocotyls yielded significantly fewer protoplasts in both cultivars.
Regarding viability, hypocotyl-derived protoplasts isolated with the highest enzyme concentrations exhibited superior viability, whereas mesophyll protoplasts showed higher viability when were digested with 1.5% cellulase and 0.4% macerozyme.
Subsequently, we assessed two different protoplast culture media (PCM) to evaluate plating efficiency and microcolony density in mesophyll-isolated protoplast.
PCM containing 2 mg L‑1 6-Benzylaminopurine (BAP) and 0.5 mg L‑1 naphthaleneacetic acid (NAA) significantly improved plating efficiency and microcolony density in both cultivars, particularly in ‘Naxos F1’. These results suggest that optimized enzymatic conditions and a higher ratio of cytokinin (BAP) to auxin (NAA) is more favorable for protoplast proliferation, thereby contributing to future genetic transformation efforts and enhancing breeding programs, particularly in the context of climate change.
Authors
M. Romero-Muñoz, J.M. Gambín, C. López-Sierra, F.J. Vidal-Sánchez, M. Pérez-Jiménez
Keywords
Brassicaceae, micro-propagation, plant biotechnology, protoplast regeneration, biotechnology tools
Groups involved
- Division Plant Genetic Resources, Breeding and Biotechnology
- Working Group Genetic Transformation and Gene Editing
- Working Group Horticultural Biotechnology and Breeding
- Division Ornamental Plants
- Division Vegetables, Roots and Tubers
- Division Temperate Tree Fruits
- Division Vine and Berry Fruits
- Division Horticulture for Development
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