Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol86.Issue4.Dec2000:
Scientia Horticulturae 86 (2000) 267±278
Gamma-irradiated pollen induces the formation of
2n endosperm and abnormal embryo development
in European plum (Prunus domestica
L., cv. ``Rainha ClaÂudia Verde'')
A. Peixea,*, M.D. Camposa, C. Cavaleiroa, J. Barrosoa, M.S. Paisb
a
Inst. CieÃncias Agr. MediterraÃnicas, Universidade de EÂvora, 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 2n endosperm, 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; Prunus sp.
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
Abbreviations: Gy, grays; DAP, days after pollination; cv., cultivar; LSD, least signi®cant
difference
*
Corresponding author. Tel./fax: 351-266760821.
E-mail address: [email protected] (A. Peixe).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 5 6 - 4
268
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
Rosaceae botanical 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
domestica L. 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 a 60Co source providing 10 Gy minÿ1.
2.3. In vitro and in situ conditions for pollen germination and pollen tube growth
For the in vitro tests, the pollen irradiated under 0, 100, 200, 500 and 1000 Gy
was cultured on Brewbaker and Kwack (1963) medium at 258C and 100%
relative humidity. The germination rates were recorded 24 h later using 300
pollen grains per treatment. The pollen tube growth was measured at 3, 6, 24 and
48 h after inoculation using 24 pollen tubes per treatment. An Olympus light
microscope Mod. CK40 was used for both observations.
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
269
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 7 mm 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 of L-glutamine, 250 mg lÿ1 of hydrolysate
caseine, 10 mM of adenine, 0.1 mM of a-naphthaleneacetic acid (NAA) and 1 mM
of benzylaminopurine (BAP). The medium was geli®ed with 0.7% of difco bactoagar. All cultures were maintained at 258C, with a 16 h photoperiod for embryo
germination and plant recovery.
2.7. Ploidy level determination
Embryos and/or endosperm were removed from seeds obtained from ®eld
controlled pollinations with 100 and 200 Gy irradiated pollen, under a stereo
binocular microscope with magni®cation capacity up to 70. The ploidy level of
the embryos and endosperm was evaluated by ¯ow cytometry at the Plant
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A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
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 icecold 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 40 mm 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,
Fig. 1. Effect of the irradiation levels on pollen germination in vitro. Observations made after 24 h
in culture. LSD95%.
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
271
Fig. 2. Effect of the irradiation levels, on in vitro pollen tube growth.
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.
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A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
Table 1
In situ observations of pollen tube growtha
Irradiation level (Gy)
Average length of the biggest pollen tube,
expressed in % of the style reached
a
0
100
200
500
1000
100a
100a
100a
70b
50b
Numbers followed by the same letters are not signi®cantly different at 5% level for ANOVA
test.
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.
Fig. 3. Interaction plot for the effect of irradiation on the percentage of fruit set at 30 and 70 DAP.
LSD95%.
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
273
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.
Fig. 4. Embryo and endosperm development at 30, 40 and 50 DAP for pollination: (A) with untreated
pollen (magni®cation 150); (B) with 200 Gy irradiated pollen (magni®cation 150); (C) with
untreated pollen (magni®cation 75); (D) with 200 Gy irradiated pollen (magni®cation 75); (E) with
untreated pollen (magni®cation 75); (F) with 200 Gy irradiated pollen (magni®cation 120).
274
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
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 (%)
Empty seeds (%)
Seeds only with endosperm (%)
Seeds with abnormal development
of endosperm and embryo (%)
100
0
0
0
100
0
0
0
100
0
0
0
95
0
0
5
92
0
8
0
69
6
0
25
0
30
53
17
0
40
48
12
0
67
0
33
After 50 DAP, the cellular endosperm of the normally developing seeds from
control, continues its normal development, and the embryo reached the heartshape 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.
Fig. 5. Heart-shape embryo at 70 DAP, from pollination with 200 Gy irradiated pollen;
ampli®cation: 40.
275
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
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 (%)
Abnormal plants (%)
Non-germinated embryos (%)
100
0
0
100
0
0
100
0
0
27
55
18
50
33
17
80
5
15
0
0
100
0
0
100
0
0
100
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).
Fig. 6. DNA histograms from ¯ow cytometry analysis: (A) control from young leaves; (B) 2n
endosperm from a seed pollinated with 200 Gy irradiated pollen; (C) normal 3n endosperm from
0 Gy seeds. All samples were collected at 70 DAP.
276
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
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 VassilevaDryanovska (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.
In apple, James et al. (1985) and Nicoll et al. (1987) also obtained the
formation of seeds just with endosperm or with endosperm and embryos, after
pollination with irradiated pollen. According to these, at high levels of radiation,
only a single sperm nuclei was present per tube, allowing the fertilization either
of the egg cell or of the fused polar nuclei.
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
277
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 to Prunus 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 acknowledgement 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.
References
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mentor pollen to induce self-fertilization of two self-incompatible Upper Amazon Cacao clones.
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Barroso, J.M., 1990. Estudo da biologia ¯oral numa populacËaÄo regional de ameixeiras `Rainha
ClaÂudia' (Prunus domestica L.). Ph.D. Thesis. Universidade de EÂvora, EÂvora, Portugal.
Bouvier, L., Zhang, Y.X., Lespinasse, Y., 1993. Two methods of haploidization in pear, Pyrus
communis L.: greenhouse seedling selection and in situ parthenogenesis induced by irradiated
pollen. Theor. Appl. Genet. 87, 229±232.
Brewbaker, J.L., Kwack, B.H., 1963. The essential role of calcium ion in pollen germination and
pollen tube growth. Am. J. Bot. 50, 859±865.
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Chapman, G.P.C., et al. (Eds.), Experimental Manipulation of Ovule Tissues. Longman, London,
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irradiated and non-irradiated apple pollen. Agronomie 11, 483±489.
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Sestili, S., Ficcadenti, N., 1996. Irradiated pollen for haploid production. In: Jain, S., et al. (Eds.), In
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Vassileva-Dryanovska, O.A., 1966a. Development of embryo and endosperm produced after
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Gamma-irradiated pollen induces the formation of
2n endosperm and abnormal embryo development
in European plum (Prunus domestica
L., cv. ``Rainha ClaÂudia Verde'')
A. Peixea,*, M.D. Camposa, C. Cavaleiroa, J. Barrosoa, M.S. Paisb
a
Inst. CieÃncias Agr. MediterraÃnicas, Universidade de EÂvora, 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 2n endosperm, 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; Prunus sp.
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
Abbreviations: Gy, grays; DAP, days after pollination; cv., cultivar; LSD, least signi®cant
difference
*
Corresponding author. Tel./fax: 351-266760821.
E-mail address: [email protected] (A. Peixe).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 5 6 - 4
268
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
Rosaceae botanical 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
domestica L. 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 a 60Co source providing 10 Gy minÿ1.
2.3. In vitro and in situ conditions for pollen germination and pollen tube growth
For the in vitro tests, the pollen irradiated under 0, 100, 200, 500 and 1000 Gy
was cultured on Brewbaker and Kwack (1963) medium at 258C and 100%
relative humidity. The germination rates were recorded 24 h later using 300
pollen grains per treatment. The pollen tube growth was measured at 3, 6, 24 and
48 h after inoculation using 24 pollen tubes per treatment. An Olympus light
microscope Mod. CK40 was used for both observations.
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
269
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 7 mm 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 of L-glutamine, 250 mg lÿ1 of hydrolysate
caseine, 10 mM of adenine, 0.1 mM of a-naphthaleneacetic acid (NAA) and 1 mM
of benzylaminopurine (BAP). The medium was geli®ed with 0.7% of difco bactoagar. All cultures were maintained at 258C, with a 16 h photoperiod for embryo
germination and plant recovery.
2.7. Ploidy level determination
Embryos and/or endosperm were removed from seeds obtained from ®eld
controlled pollinations with 100 and 200 Gy irradiated pollen, under a stereo
binocular microscope with magni®cation capacity up to 70. The ploidy level of
the embryos and endosperm was evaluated by ¯ow cytometry at the Plant
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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 icecold 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 40 mm 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,
Fig. 1. Effect of the irradiation levels on pollen germination in vitro. Observations made after 24 h
in culture. LSD95%.
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271
Fig. 2. Effect of the irradiation levels, on in vitro pollen tube growth.
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.
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Table 1
In situ observations of pollen tube growtha
Irradiation level (Gy)
Average length of the biggest pollen tube,
expressed in % of the style reached
a
0
100
200
500
1000
100a
100a
100a
70b
50b
Numbers followed by the same letters are not signi®cantly different at 5% level for ANOVA
test.
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.
Fig. 3. Interaction plot for the effect of irradiation on the percentage of fruit set at 30 and 70 DAP.
LSD95%.
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
273
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.
Fig. 4. Embryo and endosperm development at 30, 40 and 50 DAP for pollination: (A) with untreated
pollen (magni®cation 150); (B) with 200 Gy irradiated pollen (magni®cation 150); (C) with
untreated pollen (magni®cation 75); (D) with 200 Gy irradiated pollen (magni®cation 75); (E) with
untreated pollen (magni®cation 75); (F) with 200 Gy irradiated pollen (magni®cation 120).
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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 (%)
Empty seeds (%)
Seeds only with endosperm (%)
Seeds with abnormal development
of endosperm and embryo (%)
100
0
0
0
100
0
0
0
100
0
0
0
95
0
0
5
92
0
8
0
69
6
0
25
0
30
53
17
0
40
48
12
0
67
0
33
After 50 DAP, the cellular endosperm of the normally developing seeds from
control, continues its normal development, and the embryo reached the heartshape 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.
Fig. 5. Heart-shape embryo at 70 DAP, from pollination with 200 Gy irradiated pollen;
ampli®cation: 40.
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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 (%)
Abnormal plants (%)
Non-germinated embryos (%)
100
0
0
100
0
0
100
0
0
27
55
18
50
33
17
80
5
15
0
0
100
0
0
100
0
0
100
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).
Fig. 6. DNA histograms from ¯ow cytometry analysis: (A) control from young leaves; (B) 2n
endosperm from a seed pollinated with 200 Gy irradiated pollen; (C) normal 3n endosperm from
0 Gy seeds. All samples were collected at 70 DAP.
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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 VassilevaDryanovska (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.
In apple, James et al. (1985) and Nicoll et al. (1987) also obtained the
formation of seeds just with endosperm or with endosperm and embryos, after
pollination with irradiated pollen. According to these, at high levels of radiation,
only a single sperm nuclei was present per tube, allowing the fertilization either
of the egg cell or of the fused polar nuclei.
A. Peixe et al. / Scientia Horticulturae 86 (2000) 267±278
277
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 to Prunus 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 acknowledgement 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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