Materials and methods1. Protoplast

Materials and methods

1. Protoplast donors

Seven carrot accessions were used including one open
pollinated carrot cultivar Dolanka and three pairs of malesterile
(usually marked out as line A) and male-fertile
breeding lines (line B) (Table 1). Protoplasts were isolated
from leaves or hypocotyls of in vitro grown plants or
seedlings, respectively (Fig. 1a, c). Aseptic material was
derived from seeds sterilized using a three-step procedure.
First, seeds were incubated in a water bath at 40C, then
transferred to 0.2% (v/v) solution of fungicide ‘Bravo’
(Syngenta) and placed on a gyratory shaker (160 rpm) and
finally immersed in 20% (w/v) solution of chloramin T
(sodium N-chlorotoluene-4-sulphonamide) (30 min each).
After each step, the seeds were dipped in 70% ethanol for
30–60 s. After three washes with sterile water for 5 min
each, the seeds were air-dried on a sterile filter paper and
germinated in 9 cm Petri dishes on solid Murashige and
Skoog (MS) medium with vitamins (Murashige and Skoog
1962) supplemented with 30 g l-1 sucrose and 6.5 g l-1
plant agar (Biocorp) and maintained at 26 ± 2C in the
dark. In case of protoplast isolation from leaves, about 20
seeds per dish were placed on the germination medium,
and after about 7 days of culture seedlings were transferred
to glass jars containing regeneration medium (R) composed
of MS macro- and micro-elements, 0.1 mg l-1 thiamine
HCl, 0.1 mg l-1 piridoxine HCl, 0.5 mg l-1 nicotinic acid,
3.0 mg l-1 glycine, 100 mg l-1 myo-inozytol, 20 g l-1
sucrose, and 2.5 g l-1 phytagel (Sigma). Cultures were
kept in a climate room at 26 ± 2C under 16 h photoperiod
and light intensity of 55 lmol m-2
s
-1
. In order to obtain
elongated and etiolated hypocotyls, about 30–40 seeds
were placed on the MS medium (the same as for seed
germination) and kept in the dark for about 14 days.
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