PNL Volume 17 1985
CROSS OF WL 3527 (orc) x Wt 11145 (Orc)
Ludwicki J., . Agricultural University/SGGW, Warsaw, Poland
and W. K. Swiecicki Plant Experiment Station, Wiatrowo, Poland
The composition and content of carotenoids was investigated in dry
and fresh seeds of lines Wt 3527 (orc I- yellow cotyledons), Wt 11145
Orc 1 - orange cotyledons), and in dry F2 seeds of the hybrid Wt 3527 x
Wt 11145. F2 seeds were divided into five groups: yellow, dark yellow,
light orange, orange, brick (see [1]).
The seeds were ground and hydrolyzed (KOH in alcohol) prior to ex-
traction with hexane. Chromatographic separation of carotenoids was
done on an MgO column using hexane as the mobile phase followed by 10
and 20% solutions of acetone in hexane.
Moreover, the separation of pigments was investigated by TLC on
silica gel using solvent mixture of hexane-ethylacetate-methanol
(75:20:7) as the mobile phase.
The content of a given carotenoid was estimated on the basis of a
spectrophotometric assay using tabular values of E1%1cm, viz: beta-carotene
2592; beta-cryptoxanthin 2386; lutein 2550.
For the determination of the provitamin A value, the biological
activity of beta-carotene was taken as 100% beta-cryptoxanthin as 57%, and
lutein as 0%.
PNL Volume 17
Column chromatography of pigment extracts from dry seeds of line
Wt 3527 showed the presence of two fractions migrating in 10% and 20%
solution of acetone in hexane. But carotenoids from dry seeds of line
Wt 11145, fresh seeds of line Wt 3527, and all F2 recombinants had three
fractions, eluted in turn by hexane, 10% and 20% solution of acetone in
hexane. Fresh seeds of line Wt 11145 did not contain the carotenoid
eluted from the column by 20% solution of acetone.
The TLC method showed that the fraction eluted from the column by
20% solution of acetone contained not only lutein but also two addi-
tional carotenoid pigments (Fig. 1). The following substances were iden-
tified in solutions obtained by separation of carotenoids by column
Further examination of the pigment in the 20% fraction which migra-
ted below lutein on the TLC chromatogram (Fig. Id) had absorption maxima
in the following wave lengths (nm): 420, 445, 472, corresponding to
taraxanthin. The lowest migrating carotenoid was not identified (Fig.
Wt 11145 had two to four times more total carotenoids than the
other lines, but 90% of this was lutein (Table 1). Lutein was the pre-
dominant carotenoid in dry seeds. Fresh seeds contained greater amounts
of 6-carotene and beta-cryptoxanthin. The amount of beta-cryptoxanthin in dry
seeds was the least variable constituent: 94-110 mkg/lOOg.
The highest provitamin value was found in dry seeds of an F2 from
cross Wt 3527 x Wt 11145 having dark yellow cotyledons (Table 2). Other
segregants had less than half the provitamin A.
1. Blixt, S., and W. K. Swiecicki. 1983. PNL 15:9-10.
Fig. 1. Carotenoid separation
extracted from seeds of
hybrids WT 3527 and WT 11145.
a) beta-carotene;
b) beta-cryptoxanthin;
c) lutein;
d) taraxanthin;
e) unidentified;
s) standard all-trans-
PNL Volume 17 1985
Table 1. Carotenoid content isolated from pea seeds by column chromatography.
Fresh and dry seeds of parents and dry seeds of selected F2's.
Table 2. Provitamin A value of pea seed (in g of -carotene/lOOg)