Soybean protoplast culture and direct gene uptake and expression by cultured soybean protoplasts

• Published in: Plant physiology (Bethesda) Vol. 84; no. 3; pp. 856 - 861
• Main Authors: Lin, W, Odell, J.T, Schreiner, R.M
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01.07.1987
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMAT; Agronomy. Soil science and plant productions; ANTIBIOTICOS; ANTIBIOTICS; ANTIBIOTIQUE; Biological and medical sciences; Biotechnology; Callus; CHIMAERAS; CHIMERE; COTILEDONES; COTYLEDON; COTYLEDONS; CULTIVO DE TEJIDOS; CULTURE DE TISSUS; DNA; Electroporation; ENZYMIC ACTIVITY; Fundamental and applied biological sciences. Psychology; G0640LYCINE MAX; GENE; GENES; Genetic engineering; Genetic engineering applications; Genetic technics; Genetics and breeding of economic plants; GLYCINE MAX; Methods. Procedures. Technologies; Plant breeding: fundamental aspects and methodology; Plants; Plasmids; Promoter regions; Protoplast culture; PROTOPLASTE; PROTOPLASTO; PROTOPLASTS; QUIMERA; Soybeans; TISSUE CULTURE; TRANSFERASAS; TRANSFERASE; TRANSFERASES; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Chloramphenicol acetyltransferase; Cell culture; Callus; Vegetals; Electroporation; Gene expression; Glycine max; Oil plant(vegetal); Genetic transfer; Leguminosae; Dicotyledones; Protoplast; Angiospermae; Spermatophyta; Fodder crop; Vector; Hybrid gene
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.84.3.856

A method was developed for culturing protoplasts freshly isolated from developing soybean (Glycine max L.) cotyledons. First cell divisions were observed within 5 days after protoplast isolation and microcalli, consisting of about 20 cells, were formed within 10 days. Thirty days after protoplast isolation, callus tissues were observed without the aid of a microscope. A 30 to 50% plating efficiency was consistently obtained. Using a polyethylene glycol-electroporation technique, DNA was introduced into these protoplasts. The protoplasts were then cultured to form callus. Chloramphenicol acetyltransferase (CAT) activity was detected in protoplast cultures 6 hours after introduction of a 35S-CAT-nopaline synthase 3′ chimeric gene. The highest CAT activity was detected in 3-day-old electroporated protoplast cultures, indicating transient expression of the introduced gene. Some CAT activity was detected in 40-day-old callus cultures and in geneticin (G418) selected callus tissues which also received a chimeric neomycin phosphotransferase II gene, indicating the presence of stable transformants. A control chimeric gene with an inverted 35S promoter failed to produce any CAT activity in this system.