Protoplast isolation and regeneration of fertile plants from arabidopsis Trp mutant, trp1-100

• Published in: Korean journal of biological sciences Vol. 2; no. 2; pp. 239 - 242
• Main Authors: Lim, Seonhee, Kim, Youngsoon, Lee, Euiseung, Rose, Alan, Last, Robert, Cheong, Hyeonsook
• Format: Journal Article
• Language: English
• Published: Taylor & Francis Group 01.01.1998
• Subjects: Arabidopsis thaliana; PAT; Plant regeneration; Protoplast; Trp1-100
• ISSN: 1226-5071
• DOI: 10.1080/12265071.1998.9647414

Arabidopsis trp1 mutant plants, deficient in phosphoribosyl anthranilate transferase (PAT) activity, accumulate anthranilate compounds, which render them blue fluorescence. The visible phenotype of trp1 makes the PAT gene an excellent reporter gene in the mutant. In order to develop a system for the homologous recombination using the phenotypic characteristic of trp1-100, we established optimum conditions for the isolation and regeneration of protoplast from auxin-conditioned, trp1-100 root cultures. Tryptophan had to be supplemented in the germination medium for the efficient cell division and subsequent plant regeneration. When 10 μM tryptophan was added to the germination medium, we obtained the highest yield of protoplasts (3×10 6 cells/g) and the best viability (92%). Thirty percent of root protoplast derived from meristematic cells underwent cell division within 5 days in callus-induction medium. Regenerated rosette leaves (2-3 mm) were transferred to rooting medium and finally acclimated to the soil for flowering