Zygotic and somatic embryos of cucumber (
Cucumis sati
6
us
L.)
substantially differ in their levels of abscisic acid
Helena Gawronska
a,*, Wojciech Burza
b,1, Elzbieta Bolesta
b,1, Stefan Malepszy
b,1 aDepartment of Plant Physiology,Faculty of Agriculture,Warsaw Agricultural Uni6ersity,Rakowiecka 26-30,02-528 Warsaw, Poland bDepartment of Plant Genetics,Breeding and Biotechnology,Faculty of Horticulture,Warsaw Agricultural Uni6ersity,Nowoursynowska 166a,02-787 Warsaw, Poland
Received 15 November 1999; received in revised form 10 April 2000; accepted 13 April 2000
Abstract
In this work we studied the changes in the level of abscisic acid (ABA) in the somatic embryos (SE) and in the diploid and triploid zygotic embryos (ZE) of the same cucumber line during embryogenesis and seed maturation. Different stages of seed development were selected according to days after pollination (DAP): 21, 24, 28, 35, 42 and 42 plus 14 days of storage for diploid ZE and 35 and 42 plus 14 days of storage for triploid ZE. SE were collected at five growth stages from globular to late cotyledonary. Quantitative analysis of ABA was performed using an enzyme linked immunosorbent assay (ELISA) test. Both types of embryos — somatic and zygotic — essentially differed in their levels of ABA, always being the highest for 2n ZE and lowest for SE. Although the concentration of ABA in ZE of the triploid line was higher when compared with the same DAP, when the comparison was based on embryo development, both the concentration and content of ABA was higher in the diploid line. The pattern of developmental changes in the level of ABA in the diploid ZE was consistent with that known for other species. An increase was observed during embryo development with a peak (51.1mg g−1FW or 0.95 mg per embryo) at the final stage of embryo formation between 21 and 24 DAP. A sharp decrease in the ABA level then took place (more than 3-fold within 4 days) and was followed by a further reduction as the seed matured. The maximal and minimal values for ABA concentration differed about 35-fold. SE differed substantially from their zygotic counterparts not only in that the concentration of ABA was extremely low (0.005 – 0.011mg g−1FW) but also that no significant changes occurred during embryo development and no peak of ABA concentration was observed. Other tissues of the ovule and ovary also contained ABA and could be a source of ABA for the embryo. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Abscisic acid; Cucumber; Somatic; Zygotic; Diploid; Triploid embryos
www.elsevier.com/locate/plantsci
1. Introduction
Artificial seeds are an important part of plant biotechnology and somatic embryos (SE) are the basic component of it [1]. The knowledge of the
physiology of SE development including their hor-monal status is among the major points for the successful application of artificial seed technology. Despite this, comparative studies on SE and ZE are scarce.
Abscisic acid (ABA) plays a key role in embryo and seed development at each stage, both in a stimulatory and inhibitory manner [2,3]. ABA controls the proper embryo formation and matu-ration [4,5], is involved in initiation and progress-ing of tissue desiccation [6,7], as well, as in the acquisition of desiccation tolerance by embryo [8]. Several authors showed that ABA inhibits preco-cious seed germination [7,9] and regulates seed maturation and dormancy [6]. ABA is also
in-Abbre6iations: ABA, abscisic acid; DAP, days after pollination;
ELISA, enzyme linked immunosorbent assay; EtoAc, ethyl acetate; FW, fresh weight; MeOH, methanol; SE, somatic embryos; ZE, zygotic embryos.
* Corresponding author. Tel.: +48-22-8499476; fax: + 48-22-8491946.
E-mail addresses:[email protected] (H. Gawronska)., [email protected] (S. Malepszy1).
1Tel./fax: +48-22-8430982.
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−1as 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 [NH4+][NNO3−] 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 sati6usL.) Borszczagowski cv.
Fig. 2. Somatic embryos of a highly inbred line of cucumber (Cucumis sati6usL.) 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 1×103cells 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 PhillipsTLD36W/33), 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 (v/v) 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 v/v). Aqueous residues were then collected, the pH adjusted to 2.65 – 2.85 followed by a three-fold partitioning against EtoAc (1:1 v/v). 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
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
B0.1% 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
3.1. ABA in zygotic embryos of the diploid line
Endogenous levels of ABA in zygotic embryos differed considerably depending upon the sam-pling date (Fig. 3). The concentration of ABA in diploid ZE increased following pollination with a peak at 24 DAP (51.1 mg g−1 FW) and then
decreased being lowest at 56 DAP (1.5 mg g−1
FW). No data are available for embryos at earlier developmental stages. However, based on data obtained for whole ovules isolated 14 DAP it could be assumed that in the earlier stages the amount of ABA in embryos was very low since the whole ovule contained only 0.014 mg of ABA (Table 1). Essential changes in the concentration of ABA were noted between 21 and 28 DAP when it first increased by nearly double (between 21 and 24 DAP) and then decreased more than 3-fold (within the following 4 days). The maximum ap-peared when the embryo was morphologically completed (torpedo stage), which in the case of this cucumber line is between 18 and 24 DAP as shown by Tarkowska et al. [20]. The maximum and minimum values differed approximately 35-fold. During seed development and maturation a further reduction in concentration was recorded (Fig. 3). The total content of ABA in the embryo
Fig. 3. Concentration of ABA (mg g−1FW) in zygotic embryos of a diploid highly inbred line of cucumber (Cucumis sati6usL.)
Table 1
Level of ABA in the embryos, ovules and in the testa of diploid and triploid highly inbred lines of cucumber (Cucumis sati6usL.)
cv. Borszczagowski at selected days after pollinationa
DAP
Seed part Ploidy level ABA concentration ABA content
FW (mg g−1) 9S.E. LSD0.05 Organ (mg) 9S.E. LSD0.05
Embryo 2n 21 25.41 0.468 8.300 0.398 0.0182 0.309
24 51.06 9.147 0.949 0.1565
35 17.80 3.688 3.970
3n 0.155 0.0202 0.060
56 3.05 0.966 0.024 0.0036
14 0.455 0.012 3.920
Ovule 2n 0.014 0.0001 0.755
21 7.642 0.283 0.536 0.0090
24 25.599 3.636 1.683 0.3020
14 0.376 0.014 0.292 0.014
3n 0.0001 0.032
24 1.532 0.198 0.089 0.0120
35 1.939 0.171 0.079 0.0040
21 3.860 0.137 Ndb Ndb
Testa 2n
24 0.061 0.0001 Ndb Ndb
aData are mean9S.E. (n=3). For each sample ABA was assayed in at least three dilutions that were repeated three times. bNot determined.
depended upon its development and ranged from 0.044 to 0.95mg per embryo (data not shown) with the highest and lowest values observed on the same dates as in the case of ABA concentration. This pattern of change in ABA level in 2n ZE during development is consistent with that re-ported for other species [2 – 4,11]. However, the values obtained in our study of the concentration of ABA in ZE, especially at the peak, (expressed on FW basis) are somewhat higher than those cited in the literature (but are still within a physio-logical range). For example, the respective values for zygotic embryos of bean [21], rapeseed [11] and of soybean [4] are about 1.75, 3 and 17 mg g−1
FW. In some species, however, as, for example, in
Picea glauca, the concentration of ABA in em-bryos when recalculated on a FW basis is almost the same (49.5 mg g−1 FW vs. 51.1 mg g−1 FW)
[22]. It is worth mentioning that the values of ABA concentration in fruit tissues of cucumber recorded by Kim et al. [23] and Kuz’mina [24] for appropriate stages are comparable with those ob-tained in our study.
In order to compare whether other tissues of the ovule, besides the embryo, also contain ABA, its level in the whole ovule, testa, and in the ovary tissues surrounding the seeds, is parallel with the dissected embryos, has been determined on a few dates (Table 1). The content of ABA in the whole ovule increased considerably (by about 40-fold) between 14 and 21 DAP and further by more than
3-fold during the next 3 days (Table 1). Although the concentration of ABA in the ovule both at 21 and 24 DAP was 2 – 3-fold lower than in the embryo, the content of ABA in the ovule was greater (0.54 mg ovule−1 versus 0.40 mg per
em-bryo and 1.68 mg per ovule versus 0.95 mg per embryo, respectively) (Table 1). Also the testa contained ABA (3.86 mg g−1FW and 0.06mg g−1
FW at 21 and 24 DAP, respectively) (Table 1), as well as, the ovary tissues surrounding the seeds (0.382 mg g−1 FW determined on 35 DAP).
The results of this study clearly show that in addition to the embryos, other tissues of the ovules and of the ovary surrounding the seeds also contain ABA. Since, as concluded by Nakamura et al. [25], ABA applied to leaves is translocated to the grains of rice, we may assume that the sur-rounding tissues may also be a source of ABA for the embryos. The large decrease (by about 60-fold) in the concentration of ABA in the testa observed here between 21 and 24 DAP coincides with a substantial increase of this hormone in the em-bryo, also supports this point of view. On the other hand, however, Rajasekaran et al. [26] are not convinced that import of ABA to the embryo from other parts takes place.
3.2. ABA in zygotic embryos of triploid line
were able to collect samples only at 14, 24, and 35 DAP (ovules) and 35 DAP and 56 DAP (em-bryos). The concentration of ABA in 3n ZE was more than double than that in the respective 2n ZE (Fig. 4 vs. Fig. 3). However, it should be noted that ZE of the 2n line were much more advanced in development when compared with 3n and data based on the chronology (DAP) of 2n and of 3n ZE did not relate to the same growth stages. In vivo, 2n zygotic embryos of cucumber were mor-phologically completed 21 DAP (Fig. 1A) while ZE of the 3n line were not fully developed even 35 DAP (Fig. 1B). Moreover, the concentration of ABA in the 2n ZE on 35 DAP was already decreasing (11 days after the peak, Fig. 3). Despite this, if ABA is compared on a per embryo basis its content in 2n ZE was higher than that in 3n ZE (Table 1). In the case of ovules both the concen-tration and content of ABA were always higher in the 2n line than in the 3n (Table 1). On the other hand, however, the concentration of ABA in the ovary tissues of this 3n line at 35 DAP was 170% higher (0.639mg g−1FW) than that of the 2n line.
These data show that the level of ABA both in the ZE and ovules of the triploid line are much lower than in the diploid line. Since this coincides with the period of delay and abnormalities observed during both the ZE and seed development of the 3n line [16], it might partially be due to the low level of ABA. It has been shown for many species that a high level of ABA is required for proper
embryo formation, as well as, for the ‘switch on’ from the middle to late embryogenesis programme that includes acquisition of tissue tolerance to desiccation, and the accumulation of oil and protein bodies and storage compounds [3].
If it is assumed that ovary tissue is a source of ABA for the embryo, and that a high level of ABA is required for proper embryo development the higher concentration of ABA in the ovary tissue of the 3n than that of the 2n (as recorded at 35 DAP) may indirectly suggest that transport of ABA in the 3n line is slower and/or delayed leading to the discrepancies in embryo and seed development observed when compared with the 2n line.
3.3. ABA in somatic embryos
In contrast to the zygotic embryos the level of ABA in the SE in all the growth stages determined was extremely low (Fig. 5). Depending upon the developmental stage of the SE the concentration of ABA ranged between 0.005 and 0.011 mg g−1
FW (Fig. 5) being the highest at the final growth stage (late cotyledonary and beginning of embryo conversion to plantlets). In addition to such ex-treme differences in the concentration of ABA, SE also differed from their zygotic counterparts in that almost no changes and no peak appearance in the concentration of ABA took place along with SE development. Only at the latest stage of SE
Fig. 4. Concentration of ABA (mg g−1FW) in zygotic embryos of a triploid highly inbred line of cucumber (Cucumis sati6usL.)
Fig. 5. Concentration of ABA (mg g−1FW) in somatic embryos of a highly inbred line of cucumber (Cucumis sati6us L.) cv.
Borszczagowski. Details as in Fig. 3.
development was an increase in ABA concentra-tion of about 100% (but still not significant) noted. This study clearly demonstrates that cucumber somatic embryos differ substantially from their zygotic counterparts. The data related to changes in the ABA level in somatic embryos of other species are controversial and not consistent. The lower levels of ABA in SE, with no substantial changes and lack of the peak observed here have been reported for some other species such as larch, white spruce, Vitis 6inifera [27 – 29]. Kamada and
Harada [30] have also reported very low levels of ABA in SE of Daucus carota where embryos cul-tured, as here, on 2,4D free medium while in the presence of synthetic auxin had much higher levels of ABA. In contrast to the above, some other authors have reported relatively high, even com-parable to ZE counterparts, levels of ABA. They have also observed changes during embryogenesis that follow the patterns observed in ZE [31,32]. These discrepancies might, at least to some extent, be explained by different conditions of in vitro culture, as for example the presence of PEG or auxins in the media, both of which may increase the level of ABA [28,33]. Grossmann and Schel-trup [33] showed that auxins stimulated ethylene formation via the induction of ACC synthase ac-tivity, which in turn led to higher levels of ABA. This may explain the higher level of ABA in
Daucus carota SE cultured on media containing
2,4D in the study by Kamada and Harada [30]. In the case of cucumber SE many abnormalities in the morphogenesis may appear [20,34 – 36]. These may be reduced by special treatments with solutions containing various chemicals, including ABA at a concentration of 0.4 mM [34]. Our study indicates that the abnormalities, at least in part, might be due to the extremely low ABA level in the SE. As pointed out by Attree and Fowke [14] and by Nomura and Komamine [15] in the case of somatic embryogenesis a correct level of ABA seems to be essential for synchronised embryo formation and maturation.
Acknowledgements
The authors wish to express their thanks to Professor E.Weiler from Ruhr University, Bochum, Germany for generously providing us with antibody and tracer, to Professor H. Mack-iewicz for plant pollination, to M. Dzieciol for her assistance in ABA assays and to Professor D. Baker from Wye College, University of London, Wye, UK for his kindness in linguistic correction of the manuscript.
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