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nolic compounds and seed reserves. The hydrolyt- seeds and ripe brown seeds. The clectrophoresis
ic enzymes considered in the present study take gels (12%) in denaturing conditions (SDS-PAGE)
part in the norma) metabolism of the embryo sac were prepared according to Laemli ( 1970). Protein
and their activities show severa! changes during samples were diluted l :3 (v/v) with a solution con-
embryogenesis (Georgieva, 1977). Enzymes of the taining 62.5 mM Tris-HCI (pH 8.3). IO% glycerol,
group of esterases, which hydrolyze esters, pep- 2% SDS and 5% 2-mercaptoethanol and the solu-
tides, amides and halides (Walker and Mackness, tion was heated to l 00°C for 5 min. Molecular
1983) have been localized in the proembryo and weight standards from 14 to 205 KD were used as
nuclear endosperm of Lilium regale, whereas acid markers. Electrophoresis was carried out at 20Co in
phosphatase activity was only detected in the cells 25 mM Tris-glycine buffer (pH 8.3) containing
of embryo and embryo sac (Georgieva, 1991 ). 0.1 o/o SDS at 25 mA. The gels were photographed
Phenolic compounds have been demonstrated in after staining with l o/o Coomassie blue.
the covering tissues of the embryo during embryo-
genesis and seed maturation of Brassica napus RESULTS
(Twanowska et al., 1994). They have also been
reported in mature seeds where they play a role of Three morphological stages were identified during
defense (Wronka et al., 1994). the development of Brassica macrocarpa seeds. A
fust stage consisted of formation of the embryonic
MATERIALS AND METHODS axis and cotyledons. A second stage was character-
ized by celi expansion, during which the embryo
In the Botanica! Garden of the University of Paler- increased in size and weight as storage substances
mo, Brassica macroccupa Guss. seeds from Favig- were accumulated. A third stage, when the embryo
nana (Egadi islands) were sown in Aprii in pots in a ceased growing and the seed ripened, was charac-
greenhouse and transplanted outside in May. Plants terized by lipid and protein deposition and water
stru1ed to flower in October and dormant seeds were loss. Changes in the cytochemistry of the seed tis-
released from the siliquas in May-June of the fol- sues were identified in each stage. In the first stage,
lowing year. Maximum germination percentage was esterase and acid phosphatase activity was detected
achieved 3-4 months after harvesting. in the endosperm. integument and suspensor.
Developing siliquas were harvested at various Esterase activity was strongest in the embryo (Fig.
stages of maturation and seeds were frozen and sec- 1), micropyle and suspensor cells. During the sec-
tioned with an Amesh Labtek cryostat. Unfixed, ond stage, the cells of the ovule integuments filled
frozen sections were examined for esterase activity with starch grains and those destined to form the
using naphthol AS-D acetate and for acid phos- palisade layer became richer in esterases. Esterase
phatase using naphthol AS-81 as substrates. The dia- activity and chloroplasts were detected in the
zonium salt fast blue 88 was used for both reactions. embryo; phenols were localized in the epidermis.
Sections incubated in media without the substrates During embryo growth, esterase and phosphatase
were used as controls. Reactions were performed at activities decreased in the endosperm, while phenols
37°C (Gahan, 1984). Phenols were detected in increased in the cells of the extemal and internai
frozen sections by ferric chloride or by fast blue 88. ovule integuments. The embryo was surrounded by
Mucilage was detected with ruthenium red, and endosperm rich in phenols. The deposition of phe-
starch with Lugol's (IKJ) solution. Carbohydrates nols occurred centrifugal ly, from the internai
were detected by the PAS reaction, and lipids by the endosperm layer (Fig. 2) to the integuments (Fig. 3).
Sudan 8lack 8 test (Gahan, 1984). Observations Early in the third stage, the endosperm was reab-
were made by light microscope. Description of the sorbed and seed integuments were rich in esterases
microscopic features of the seed integuments is and phosphatases (Fig. 4). The epidermal cells of
according to Vaughan et al. ( 197 1). the outer integument contained, in the space
Protein reserves were studied using ripening seeds between the celi wali and the protoplast, severallay-
at three levels of maturation: green seeds with a ers of mucilaginous substances which at maturity,
completely developed embryo; hard green-brown upon imbibition, absorb water and swell (Fig. 5).
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