After 1 hor, exogenous deoxyribonlei acid was degraded within a culture medim at 25 C (pH 6) co ti protots of Daucus careots L. var. sativa Low te eature nc ton (1 C) or the additi of 45 m1-molar sodm citrate to the medim eliminated DNase activity for at least 4.5 hours. Tis DNase actiity was not reduced at pH 7 or 9, nor by addition of 200 mimlar adenosine 5T-triphosphate.Techniques were deveIoped to ensure hb protoplast platag efficiencies and high regenerative capabies after low temperature treatment and the addition of sodim citrate to the medium. Results ndicated citrate concentratons to 45 mm and 1 C te rtures revealed lttle or no effect on protoplast regeneration capaces Protoplast viabilty was 90 to 95% at the time of platig as deteried by phenosafranin stainig and an estimated 50 to 60% of these undergo cell division in the sold agar meum.Although genetic transformation with bacteria is a well documented phenomenon (4), demonstration ofgenetic transformation in eucaryotic organisms has been limited to fungi (12), algae (17), and cultured animal cells (10). There have been reports of DNA uptake and subsequent genetic transformation in higher plants (6,7). However, due to the irreproducibility of the results or the methods used to ascertain DNA uptake and expression, reported DNA transformation in higher plants or plant cell cultures now appears controversial (8).The action of exogenous nuclease on foreign DNA added to cultured plant cells or protoplasts presents a major problem in the uptake of undegraded transforming DNA. This problem was first delineated by Bendich and Filner (3) were grown in liquid suspension in the medium described previously (20) were treated with I mg/ml lysozyme for 1 hr at 37 C. SDS was added to a final concentration of 1% and the mixture was incubated at 60 C for 10 min. Nuclease-free pronase (Sigma) (2 mg/ml) was added and the incubation continued for an additional hr at 37 C. CsCl (Harshaw Chemical) was added to a refractive index of 1.400 and then centrifuged to equilibrium at 218,000g, 21 hr, in a Spinco type 65 rotor. The viscous DNA fraction was collected, diluted 1:5 with 1 x SSC (0.15 M NaCl, 0.015 M Na-citrate, pH 7) and centrifuged at 300,000g, 12 hr, 5 C in a Spinco SW 50.1 rotor.The DNA pellet was resuspended in 0.1 x SSC, and the A260/A590 was between 1.8 and 2.Protoplast Isolation. One g fresh weight of mid-log phase suspension cultured D. carota cells was harvested by centrifugation (75g, 4 min) and resuspended in 20 ml of a solution consisting of 4% Cellulysin (Calbiochem), 2% Macerase (Calbiochem), and 0.4 M sorbitol (pH 6). The cells were incubated at 30 C with gentle shaking in a 250-ml flask for 3.5 to 4 hr. Complete cell wall removal was indicated by complete cell lysis upon addition of SDS to a representative sample (5