Somatic embryogenesis protocol: cotyledons
were cultured in culture jars with 20 ml of the somatic induction medium (SIM; Collado et al., 2010) in darkness at 25.0[degrees]C [+ or -] 2.0.
Count averages on the cotyledons
were low and there were no significant differences between treatments in the number of immatures (F = 0.98, df = 1, 57, P = 0.3233) or adults (F = 0.0, df = 1, 57, P = 0.9600) observed on d 7, or the eggs (F = 0.04, df = 1, 57, P = 0.8454), immatures (F = 1.55, df = 1, 57, P = 0.2182), or adults (F = 0.66, df = 1, 57, P = 0.4207) observed on d 14.
remains same throughout the gestation.
Elongation was also observed in shoots induced from cotyledons
in the presence of 2 g [L.sup.-1] activated charcoal in Pinus pinaster (ALVARLZ; MAJADA; ORDAS, 2009), Pinus peuce (STOJICIC et al., 2012) and Pinus heldreichii (STOJICIC et al., 1999; STOJICIC; BUDIMIR, 2004).
indica callus cultures such as the embryogenic, organogenic, rhizogenic and non-embryogenic callus lines were obtained successfully, especially in cotyledons
. This study principally characterized the primary and secondary somatic embryos.
The seed coat was removed, and 100 g of the obtained cotyledons
were separated and placed flat side up on a moist tissue paper in glass trays (Figure 2).
Chlorophylls and carotenoids contents: The cotyledons
of 10 old radish plantlets were placed in acetone (80%) to extract photosynthetic pigments.
The optimal infection time for obtaining the highest regeneration frequency (99.31%) and number of shoots (1.90) for the cotyledon
node explants were 15 min (Table 4).
The material (root, hypocotyl, cotyledons
, epicotyl and eophyll) were fixed in glutaraldehyde (1% in 0.1 M phosphate buffer, pH 7.2) (Karnovsky, 1965) and later transferred to 70% ethyl alcohol following the protocol of Johansen (1940).
However, the cultivation of explants in flasks with lower gas exchange was more effective in callus induction than regeneration, and hypocotyls were more effective than cotyledons
in morphogenesis induction.
Lesions that appear on the cotyledons
are small, water-soaked and pale in colour, chlorotic (yellow) or necrotic (brown).
Dave and Batra  conducted the first study of somatic embryogenesis in cumin using different explants including root, hypocotyls, and cotyledons
. They stated that hypocotyl explants in MS medium complemented with 8 mg/L BA showed the best response to embryogenesis and emphasized the necessity of cytokinins hormone for production and development of cumin somatic embryos .
The model developed could explain about 77% of the variations in the pH of the cotyledons
, suggesting that 23% of the variations were due to other factors not investigated in this work (Table 1).