volved in induction and regulation of a number of genes coding for storage and LEA proteins [4,10 –
12], as well as, enzymes in fatty acids and lipids metabolism in seeds [13]. It is also suggested that
ABA plays an essential role in somatic embryoge- nesis and somatic embryos maturation [5,14,15].
Moreover, it has been shown for many, but not all the plant species, that exogenously applied ABA
to the growth medium increases the number of properly developed SE and improves their quality
[14,15].
Triploids, when
compared with
respective diploid lines, differ from them in several aspects of
development and morphology including delayed and somewhat abnormal embryo and seed devel-
opment [16].
Not much data are available in the literature, related to differences in ABA levels between so-
matic and zygotic embryos and not between diploid and triploid lines having the same genetic
background. To our knowledge, such data are not available for cucumber. Moreover, the limited
data on the role of ABA in somatic embryogenesis of other plant species, as pointed out by Nomura
and Komamine [15], are still controversial and not conclusive.
The aim of this study was to examine changes in the level of ABA in SE and in ZE of highly inbred
diploid and triploid lines of cucumber Cucumis sati6us
L. during
embryogenesis and
seed maturation.
2. Material and methods
2
.
1
. Plant material All types of embryos used in the study were of
the same origin, i.e. a highly inbred line of cv. Borszczagowski. Zygotic embryos were collected
from ethanol sterilised fruits from greenhouse grown, hand pollinated plants. ZE were collected
on the following days after pollination DAP: 21 Fig. 1A, 24, 28, 35, and 42. Samples of ZE
dissected at 42 DAP and stored for further 14 days for simplicity designated as a 56 DAP were also
included in the study. Due to the delay and abnor- malities in the development the ZE of 3n line were
collected only at 35 DAP Fig. 1B and 56 DAP. Whole ovules, testa and ovary tissues surrounding
the seeds were also collected at certain time points.
Somatic embryos of the same diploid inbred line were collected from established, embryogenic cell
suspension cultures.
2
.
2
. Initiation of the suspension culture Liquid cultures were initiated directly from pri-
mary explants of sterile seedlings by isolating vege- tative shoot apices about 1.5 mm long. Each ten
shoot apices were placed in 100 ml of liquid medium in a 350 ml Erlenmeyer flask. A modified
liquid Murashige and Skoog [17] was used, to which macroelements and iron were added at half
concentration, and microelements and vitamins at full concentration. The medium was also supple-
mented with 250 mg l
− 1
edamine, 40 g l
− 1
su- crose, 5 g l
− 1
glucose. 2,4D was added at a concentration of 1 mg l
− 1
as a sole source of growth regulators. The pH of the medium was
adjusted to 5.6 before autoclaving 17 min, 121°C. The nitrogen content [NH
4 +
][NNO
3 −
] was approximately 10:19.5. The explants developing in
liquid media were transferred to fresh media every 2 – 3 weeks, each time about 300 mg of tissue as
the inoculum per 100 ml of medium. The cell
Fig. 1. Zygotic embryos of diploid A and triploid B highly inbred lines of cucumber Cucumis sati6us L. Borszczagowski cv. at 21 and 35 days after pollination, respectively.
Fig. 2. Somatic embryos of a highly inbred line of cucumber Cucumis sati6us L. Borszczagowski cv. at four developmental stages: A globular, B heart, C early cotyledons and D late cotyledons bar is 1 mm.
suspension was formed by the separation of cells from the explant and their division. At 8 – 10
months a stable suspension culture counting from the moment of initiation was used for the
experiment.
2
.
3
. Inoculation into a hormone-free medium The suspension was ready for inoculation into a
hormone-free medium 8 – 12 days after the initia- tion of a fresh culture. Eighty milliliters of the
culture were filtered through a nylon sieve with a mesh diameter 150 mm, and then centrifuged for 5
min at 100 × g. The pellet was resuspended in a medium of the same composition but without
2,4D. Cells were washed in this manner three times. After resuspension in a liquid medium at a
density of 1 × 10
3
cells per ml cells and the aggre- gates were placed in a Petri dish 10 cm in diame-
ter, 4 ml liquid culture each. The culture was then placed in a growth chamber with a light intensity
of about 200 Lx LF PhillipsTLD36W33, tem- perature 26°C and a 16 h photoperiod. SE were
hand harvested at the following growth stages: globular Fig. 2A, early heart, late heart Fig.
2B, early cotyledons Fig. 2C and late cotyledons Fig. 2D. Plant material was kept frozen −
76°C until ABA extraction and analysis.
2
.
4
. ABA extraction and purification Plant tissues were homogenised in 80 aqueous
MeOH vv in the presence of 0.001 of BHT 2,6-ditert-butyl-4-methylphenol, Merck and 1
Polyclar AT Sigma, shaken twice 1.5 h each at 4°C in the dark and centrifuged 2 × 15 min, 7000
rpm. The supernatants were then combined and MeOH dried out. Aqueous phases were adjusted
to pH 7.65 – 7.85 and partitioned against EtoAc 1:1 vv. Aqueous residues were then collected,
the pH adjusted to 2.65 – 2.85 followed by a three- fold partitioning against EtoAc 1:1 vv. The
EtoAc fractions were combined and the EtoAc evaporated to dryness. The extracts were dissolved
in TBS buffer and stored at − 30°C until ABA analysis.
2
.
5
. Quantitati6e analysis of ABA Quantitative analysis of ABA was performed by
ELISA with rabbit polyclonal antibody raised
against mouse IGg RAMIG and anti-ABA mouse, monoclonal antibody MAB. ABA la-
belled alkaline phosphatase and Sigma 104 phos- phatase
substrate p-Nitrophenyl
phosphate, disodium, hexahydrate were used as a tracer and
substrate, respectively. The assay was run as de- scribed by Weiler [18] and Mertens et al. [19] using
96 wells ELISA plates Maxisorb, NUNC and ODs were read by an ELISA reader Dynatech
MR 5000 under Revelation 2.0 control. For each plate a standard curve was obtained. According to
Mertens et al. [19] the anti-ABA MAB shows 100 reactivity with 2-cis-s-ABA and very low
B
0.1 cross reactivity with compounds struc- turally similar to ABA. Professor E.W. Weiler
Ruhr University, Bochum, Germany generously provided the antibody and tracer as a gift. In
order to check whether samples contained im- munoreactive compounds other than ABA, several
dilutions of the standard curve were spiked with three dilutions of samples with the highest and
lowest concentration of ABA. The spiked standard curves were parallel to the original one in its linear
range data not presented. In most cases approxi- mately 1 g of fresh weight FW of tissue in at
least three replications was used for extraction. For each sample ABA was assayed in at least
three dilutions each repeated three times. Data were analysed by ANOVA 1 and the differences
were estimated using Student’s t-test.
3. Results and discussion
