Gamma-irradiated pollen induces the formation of
2
n
endosperm and abnormal embryo development
in European plum (
Prunus domestica
L., cv. ``Rainha ClaÂudia Verde'')
A. Peixe
a,*, M.D. Campos
a, C. Cavaleiro
a, J. Barroso
a, M.S. Pais
b aInst. CieÃncias Agr. MediterraÃnicas, Universidade de EÂ vora, Ap. 94, 7000 EÂvora, Portugal
b
Faculdade de CieÃncias de Lisboa, Centro De Biotecnologia Vegetal, Campo Grande, Bloco C-2, 1700 Lisboa, Portugal
Accepted 14 March 2000
Abstract
The effect of gamma radiation on pollen germination capacity and pollen tube growth was evaluated in vitro and in situ conditions. In vitro experiments, revealed that irradiation signi®cantly affects pollen viability, mainly for levels higher than 200 Grays (Gy). Also, for levels higher than 200 Gy, in situ observations showed that no pollen tube reached the ovule. Fruit set results con®rmed that for irradiation levels higher than 200 Gy, all fruits dropped before 90 days after pollination (DAP). Most of the seeds obtained from 200 Gy pollination treatments were empty. Other seeds contained only endosperm or endosperm and embryos with abnormal development. For those seeds, ¯ow cytometry analysis revealed sometimes the presence of a 2nendosperm, indicating that double fertilization did not occur and leading to the possibility of haploid embryo formation.#2000 Elsevier Science B.V. All rights reserved.
Keywords: Fruit set; Gamma rays; Parthenogenesis; Pollen viability;Prunussp.
1. Introduction
In situ parthenogenesis, induced by irradiated pollen, is like anther or microspore culture, one of the techniques used for haploid production. In the
*
Corresponding author. Tel./fax:351-266760821.
E-mail address: [email protected] (A. Peixe).
Abbreviations: Gy, grays; DAP, days after pollination; cv., cultivar; LSD, least signi®cant difference
Rosaceaebotanical family, this technique has been successfully used in apple and pear. In apple, the ®rst haploid plants were successfully regenerated in the cv. ``Erovan'', after pollination with irradiated pollen, at levels from 500 to 1000 Gy, followed by in vitro culture of immature embryos on Murashige and Skoog medium (Zhang et al., 1988). The same technique was also successfully applied to other genotypes by Zhang and Lespinasse (1991). These authors used irradiated pollen for controlled pollination at levels from 200 to 500 Gy. Haploid plants were obtained by in vitro germination of immature embryos, isolated from seeds collected 2 and 3 months after pollination. The embryos were subjected to 2 months of cold treatment (38C) before in vitro culture.
The ef®ciency of haploid embryo production is strongly affected by the irradiation level (250 Gy was more ef®cient than 500 Gy), picking time and also by the quality of the irradiated pollen (De Witte and Keulemans, 1994). Bouvier et al. (1993) applied this technique in pear cultivars and obtained one haploid (at 500 Gy) and two mixoploid plants (at 250 Gy). However, those plants did not survive more than 3 months under in vitro culture.
The present paper, describes the effect of pollination with gamma-irradiated pollen on pollen viability, fruit setting and seed development, in Prunus domesticaL. cv. ``Rainha ClaÂudia Verde''.
2. Materials and methods
2.1. Plant materials
For this study, the Prunus domestica L. cv. ``Rainha ClaÂudia Verde'' was chosen as the female parent and the cv. ``Stanley'' was used as the male parent. Both genotypes are hexaploid with 2n6x48 chromosomes.
2.2. Pollen irradiation
Pollen of the cv. ``Stanley'' was subjected to gamma irradiation, in petri dishes (80 mm), under a60Co source providing 10 Gy minÿ1
.
2.3. In vitro and in situ conditions for pollen germination and pollen tube growth
For in situ observations of the pollen tube growth, 5 days after pollination, four pistils for each radiation level were ®xed in FAA and stained with 0.1% aniline blue in a 0.1 N K3PO4 solution according to the procedure described by Martin (1959). These observations were made under a Leitz-Biomed microscope, equipped with a mercury lamp of 100 W and an excitation UV ®lter Leitz BP 340±380 nm.
2.4. Pollination procedure
Flowers of cv. ``Rainha ClaÂudia Verde'', reaching the balloon stage were emasculated and bagged. After hand pollination with non-treated and irradiated pollen, the branches were rebagged and the bags were left on until the styles withered, which happens 3±4 days after pollination.
2.5. Histological observations
Four samples of embryo sacs and immature seeds from control, 100 and 200 Gy treated ¯owers, were collected at 7, 12, 24, 48 and 72 h after pollination from branches placed in the phytotron and at 4, 6, 8, 15, 25, 30 and 50 days after pollination from trees in the orchard. The fruitlets were ®xed in FAA and paraf®n embedded according to the procedure of Johansen (1940). In those samples, embryo and endosperm development were observed after sectioning at 7mm with a rotary microtome, and saffranine and fast green staining.
2.6. Fruit collection for embryo rescue
Fruit samples were collected from the orchard at 60, 70 and 90 days after pollination. The embryo sacs were removed under sterile conditions from seeds super®cially disinfected with Ca(ClO)2 (3% available Cl) and cultured in Gamborg B5 medium (Gamborg et al., 1968), supplied with 4% sucrose, 100 mg lÿ1
of myoinositol, 500 mg lÿ1
ofL-glutamine, 250 mg lÿ1of hydrolysate
caseine, 10mM of adenine, 0.1mM ofa-naphthaleneacetic acid (NAA) and 1mM of benzylaminopurine (BAP). The medium was geli®ed with 0.7% of difco bacto-agar. All cultures were maintained at 258C, with a 16 h photoperiod for embryo germination and plant recovery.
2.7. Ploidy level determination
Cytometry Services, Schijndel (Netherlands), using a PAS II cytometer (Partec GmbH), equipped with a high pressure mercury lamp (OSRAM HBO 100 W/2), and using the excitation ®lters UG-1, BG-31, KG-1 and TK-420 and emission ®lters TK560 and GG435.
The plant material (a few cm2) was chopped with a sharp razor blade in an ice-cold neutral buffer, and placed in plastic petri discs. Neutral DNA buffer (pH 7) modi®ed by De Laat and Blaas (1984) with 15 mM hepes, 1 mM EDTA, 80 mM KCl, 20 mM NaCl, 0.5 mM spermine, 300 mM sucrose, 0.2% triton X-100, 15 mM DTE (dithiothreitol) and 2 mg lÿ1
DAPI was used.
After chopping, the buffer (ca. 2 ml), containing cell components and large tissue remnants was passed through a nylon ®lter of 40mm mesh size.
Young leaves from trees of ``Rainha ClaÂudia Verde'' were used as control, in ploidy determinations by ¯ow cytometry.
3. Results
3.1. Effect of irradiation on pollen viability, in vitro and in situ conditions
Irradiation had a signi®cant effect on pollen germination rates (Fig. 1). When compared with the control (0 Gy), the germination capacity signi®cantly decreased, even at the lowest irradiation levels tested.
The values observed for in vitro germination rate in control pollen were 40% on average. These values are in agreement with those presented by Barroso (1990), on trials performed with the same variety. The average rate for germination,
observed in treatments with an irradiation dose of 200 Gy is about half of the control. With these results, we can estimate that the LD50 dose for this European plum variety is nearly 200 Gy. Our results also show that pollen tube growth is affected by irradiation. Both the maximum pollen tube length, and the growth rate are signi®cantly affected (Fig. 2).
With 200 Gy irradiation level, the pollen tube growth shows an intermediate adjustment between control and 500 Gy treatments. Data collected 24 h after inoculation reveal that signi®cant differences from this treatment, the control, and the 100 Gy treatments. However, pollen tube growth still continues between 24 and 48 h after inoculation and the observations made at 48 h after inoculation reveal that only treatments signi®cantly different from control are those subjected to 500 and 1000 Gy irradiation levels.
Most of the work available about the effect of ionising radiation on pollen viability is performed under arti®cial conditions. Since the results obtained in these conditions are sometimes different from those obtained in situ, we also decided to perform in situ experiments to con®rm our in vitro results.
The results obtained from in situ pollination (Table 1), show that for treatments up to 200 Gy, at least one pollen tube is able to grow through the style and reach the ovule.
These results are in agreement with those observed in in vitro tests. Thus we can conclude that for irradiation levels up to 200 Gy, if the pollen tube is able to reach the ovule in its viability period, fertilization is possible.
3.2. Effect of irradiation on fruit set
Data about the effect of radiation on fruit set is presented in Fig. 3. We can see that fruit drop until 30 days is signi®cantly higher for all the treatments performed with irradiated pollen, when compared with control. For treatments with 100 and 200 Gy irradiated pollen, this fruit drop is probably due to the pollen tube reaching sometimes the ovule after its receptivity period. For higher doses, the fruit drop happens because the pollen tube is not even able to reach the ovule. These results are in agreement with those reported for pollen tube growth in in vitro and in situ conditions.
A continuous fruit drop is observed from 30 to 70 DAP, which is signi®cant for all treatments except for control. This indicates that an abnormal seed development occurred, due to irradiation, inducing a precocious embryo abortion and consequently the fruit drop.
Table 1
In situ observations of pollen tube growtha
Irradiation level (Gy)
0 100 200 500 1000
Average length of the biggest pollen tube, expressed in % of the style reached
100a 100a 100a 70b 50b
aNumbers followed by the same letters are not signi®cantly different at 5% level for ANOVA
test.
3.3. Effect of irradiation on seed development
At 4 DAP, mature embryo sacs containing the egg apparatus (the egg cell and two synergids), the central cell with two polar nuclei, and three antipodes were observed for all treatments studied (0, 100 and 200 Gy).
Until 30 DAP, whenever the development of the embryo and the endosperm was initiated, it was similar for the control and for the ¯owers pollinated with irradiated pollen (Fig. 4A and B).
The ®rst differences in embryo and endosperm development can be found only after 40 DAP (Fig. 4C and D). By that time, the embryos of control and 100 Gy pollination continue their normal growth, while those obtained with 200 Gy irradiated pollen, had practically the same size as at 30 days. Concerning endosperm development, in control seeds, the endosperm had already turned to cellular near the borders, while in 200 Gy irradiated seeds it still reveals a nuclear condition.
After 50 DAP, the cellular endosperm of the normally developing seeds from control, continues its normal development, and the embryo reached the heart-shape stage (Fig. 4E). No signi®cant differences were found by that time, between the development of the control and 100 Gy seeds. In seeds issued from 200 Gy irradiated pollen, the embryo sac development was by that time in a very early stage, showing still nuclear endosperm and globular embryos (Fig. 4F).
After reaching the heart-shape stage, the development of the embryos issued from pollination with untreated and 100 Gy irradiated pollen became very fast. They reached the cotyledonar stage 60 days after pollination, and most of the seeds collected from those treatments were fully developed (Table 2).
At the same time, most of the seeds observed from 200 Gy treatments were empty or formed only endosperm (Table 2). Whenever embryos were produced, they usually reached only the heart-shape stage (Fig. 5) and no further development was observed in collections made at 60, 70 and 90 DAP.
Table 2
Effect of the irradiation level on seed quality at 60, 70 and 90 DAP
Irradiation level 0 100 200
Days after pollination 60 70 90 60 70 90 60 70 90
Well formed seeds (%) 100 100 100 95 92 69 0 0 0
Empty seeds (%) 0 0 0 0 0 6 30 40 67
Seeds only with endosperm (%) 0 0 0 0 8 0 53 48 0 Seeds with abnormal development
of endosperm and embryo (%)
0 0 0 5 0 25 17 12 33
The embryo rescue techniques used were not successful for those embryos produced after pollination with 200 Gy irradiated pollen. Germination was achieved with embryos derived from control and from 100 Gy pollination treatments. However, many of the plants produced from the pollination with 100 Gy irradiated pollen, showed abnormal phenotypes (Table 3). Albinoism and leaves or stems malformations were the most frequent abnormalities.
The ploidy level of the plants produced from the 100 Gy treatments was estimated by ¯ow cytometry analysis and revealed that they were all hexaploid. Flow cytometry analysis was also performed on seed endosperm resulting from the 200 Gy treatments. Data analysis revealed the presence of a 2n endosperm (Fig. 6).
Table 3
Effect of irradiation level on seed germination and plant quality
Irradiation level (Gy) 0 100 200
Days after pollination 60 70 90 60 70 90 60 70 90
Normal plants (%) 100 100 100 27 50 80 0 0 0
Abnormal plants (%) 0 0 0 55 33 5 0 0 0
Non-germinated embryos (%) 0 0 0 18 17 15 100 100 100
Fig. 6. DNA histograms from ¯ow cytometry analysis: (A) control from young leaves; (B) 2n
4. Discussion
It appears that the European plum, in particular cv. ``Stanley'', is more sensitive to ionizing radiation than other species from the same family, like apple and pear. In apple, gamma irradiation levels up to 1000 Gy, had no signi®cant effect on pollen germination (Zhang and Lespinasse, 1991). In pear, Bouvier et al. (1993), observed only small differences on pollen germination capacity between the irradiated pollen up to 500 Gy and unirradiated control pollen. Our results are very similar to those presented by Adu-Amphomah et al. (1991) in cacao and Rode and Vaulx (1987) in carrot, where all the irradiation levels tested also had a signi®cant effect in pollen viability.
The fruit set rates of 27±32%, that we observed at 30 DAP, using untreated pollen, agree with those presented by Barroso (1990) for the same cultivar.
The full fruits drop observed for pollen irradiation levels over 200 Gy, could be explained by the absence of pollen tube reaching the ovule at 5 DAP. The signi®cant fruit drop observed after 30 DAP for treatments with irradiated pollen at levels of 100 and 200 Gy could be explained by the absence or occurrence of fertilization, followed by a rapid rejection of the male genome or by a rapid collapse of the entire zygote, due to problems related to mitosis in irradiated pollen as previously referred by Lecuyer et al. (1991).
The plants obtained from seeds resulting from pollination with 100 Gy irradiated pollen may have resulted from an uncompleted transmission of the male genome. According to Sestili and Ficcadenti (1996), low levels of radiation may damage only part of the generative nucleus while maintaining its capacity to fertilize the egg cell, and lead to hybridisation.
The ¯ow cytometry results, that revealed the presence of a 2n endosperm obtained after pollination with 200 Gy irradiated pollen are of great importance. This endosperm may have a potential utility as well as in the regeneration of maternal homozygous diploid plants as for genetic studies and breeding programs (James et al., 1985; Nicoll et al., 1987). On the other hand, it may con®rm the parthenogenic development of the embryos obtained. According to Vassileva-Dryanovska (1966a,b), these haploid embryos could be obtained following two different ways. The ®rst one concerns the stimulation of the female nucleus to divide by a pycnotic male chromatin while the second concerns the fertilization of the egg nucleus by damaged sperm, the chromatin of which would be subsequently eliminated in the cytoplasm.
Although the ploidy level of the embryos obtained from 200 Gy pollination treatments may be of great interest, their small dimension, as well as the unsuccessfully evolution in vitro, did not allow us to determine it by ¯ow cytometry. Advances in embryo rescue techniques are essential to improve the possibilities of application of in situ parthenogenesis toPrunus species.
Our present results show that haploidization approach, by the use of irradiated pollen, could open new possibilities for breeding of these species, until now recalcitrant to any other technique of haploid production.
Acknowledgements
The authors wish to thank Ph. Druart and M. Mota for reviewing the paper and for all the suggestions made for its improvement. Also a special acknowl-edgement to Lia AscencËaÄo for helping with the histological procedures, to the Instituto TecnoloÂgico e Nuclear for pollen irradiation and to Eng. Marino Martins to allow the installation of ®eld trials in his orchard.
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