Plant regeneration from stem cortex protoplasts ofBrassica napus

✍ Scribed by K. Klimaszewska; W. A. Keller

• Publisher: Springer
• Year: 1987
• Tongue: English
• Weight: 979 KB
• Volume: 8
• Category: Article
• ISSN: 0167-6857
• DOI: 10.1007/bf00040949

✦ Synopsis

Cell layer strips composed of the epidermis and 7-9 layers of subepidermal cells were isolated from the 3-4 terminal internodes of Brassica napus cv Westar plants at the early flowering stage. The strips were precultured for one day in modified liquid MS [1 l] medium and subsequently incubated for 17-18h in a 0.4M mannitol solution containing 1% Macerozyme and 1% Cellulase 'Onozuka' R-10. Protoplast yield was 2-2.8 x 10 6 per 1.0 g of tissue. Protoplasts were cultured at 1 x 105/ml in three different media: S I [13], B [12] and L [8l. The first cell divisions occurred after 2-8 days of culture at frequencies of 20-54%. The highest growth rate of colonies was obtained in L medium containing 0.4 M sucrose and 2% Ficoll. After 4 weeks, green calli, 1-2 mm in diameter were transferred onto B 5 12] medium with 3 mgl-~ zeatin, 1% sucrose, 0. ! M mannitol and 0.5 % agarose for shoot regeneration. Up to 20% of the calli regenerated shoots which subsequently were rooted and established in soil in the greenhouse.