A new protoplast culture system in Daucus carota L. and its applications for mutant selection and transformation

Abstract

Petiole protoplasts from in vitro-grown carrot plants are a very good alternative to traditionally obtained protoplasts from suspension cultures. High plating and regeneration efficiencies were obtained in most of the breeding lines that were tested. The embedding of the protoplasts in alginate was crucial for initiating cell division and further development. Several streptomycin resistant and chlorophyll-deficient plant lines were selected for using the petiole protoplast system. Maternally inherited streptomycin resistance was demonstrated by sexual crosses. Protoplast fusion of several chlorophyll-deficient lines did not result in complementation, indicating the cytoplasmic nature of the mutations. Petiole protoplasts were used for direct transformation with plasmid DNA pNUNV containing NPTII as a selectable marker. High transformation frequencies (up to 1%) were obtained after PEG treatment of the protoplasts. Kanamycin resistance was shown to be inherited as a single dominant nuclear trait.

Plant regeneration from leaf-derived protoplasts within the Daucus genus: effect of different conditions in alginate embedding and phytosulfokine application

“…Then, the released tissue was washed twice by centrifugation at 100g for 5 min. After a second wash in CPPD medium containing 0.1 mg l -1 a-naphthaleneacetic acid (NAA), 0.2 mg l -1 zeatin, pH = 5.6 (Dirks et al 1996), 1 ml aliquots of protoplast-derived tissue and somatic embryos were spread on filter paper placed on solidified R medium in glass jars. The procedure for regeneration from ETAFs was performed without the alginate depolymerization step.…”

“…Therefore, before applying this procedure to plant breeding programs, successful protocols for plant regeneration from protoplasts of both partners should be elaborated. For carrot, well known as a model species for plant tissue culture systems, efficient procedures for protoplast isolation and plant regeneration from different types of source tissue, such as suspension cultures, petioles, leaves, and hypocotyls have been presented (Grambow et al 1972;Dirks et al 1996;Grzebelus et al 2012a). However, to our knowledge, similar studies with regard to wild Daucus species do not exist.…”

Plastid transformants of tomato selected using mutations affecting ribosome structure

“…For the isolation of protoplasts, leaves from cultured shoots were cut into small strips and pre-plasmolysed for 10 min. in CCP pre-plasmolysis solution [CCP medium (Dirks et al 1996) with 75 g/l mannitol]. For enzymatic digestion, the pre-plasmolysis solution was replaced by enzyme solution EK-1 (0.6% cellulase R10 Onozuka, 0.6% macerozyme, 0.15% driselase, 0.2% cellulysin, 0.75% glycine, 0.1% CaCl 2 ·2H 2 O, 6% mannitol; pH 5.6; all w/v).…”

“…The protoplast suspension was transferred to centrifuge tubes, pelleted (10 min, 60g) and washed twice with W5. The pellet was finally resuspended in 9 ml CCP medium (Dirks et al 1996) with 110 g/l sucrose, overlaid with 1 ml W5 and centrifuged (5 min, 100g). The protoplasts were recovered from the interface, diluted with W5 and counted.…”

“…Ten droplets of PEG solution [40% (w/v) PEG 6000 in 0.4 M mannitol, 0.1 mM Ca(NO 3 ) 2 , 21 mM HEPES; pH 7.1] were added and mixed gently and incubated for 10 min on ice. The protoplasts were then washed twice in W5, resuspended in 1.6 ml CPP medium (Dirks et al (1996), supplemented with 60 g/l glucose, 0.1 mg/l 2,4-D and 0.2 mg/l zeatin riboside) and kept at 4 • C. The following day the protoplasts were embedded in alginate according to Dirks et al (1996) and cultured in 3 ml CPP medium (as above) in the dark at 27 • C. At days 9 and 16, 1 ml CPP medium with 0.2 mg/l zeatin riboside was added (at day 16, CPP medium with only 6 g/l glucose).…”