INTRODUCTION
The technique of in vitro placental or ovular pollination can be used for
production of interspecific and intergeneric hybrids (Zenkteler 1980). It can help
overcome crossing barriers to interspecific hybridization (Ondřej et al. 2002).
Interspecific hybridization is an important research area in the genus Cucumis
because of the possibility of transferring the genes for resistance to various diseases
from wild Cucumis to the cucumber (Cucumis sativus L.) genome. For successful
transfer of these genes, the use of some in vitro techniques has been suggested
(Lebeda et al. 2007).
The first experiment of direct pollination of ovules was made by Kanta and
Maheswari (1963) with some species of Papaveraceae. The whole process of sexual
reproduction was observed. Well-developed seedlings were obtained after in vitro
intraspecific pollination of Cichorium intybus L. by Castano and De Proft (2000), too.
Zenkteler et al. (2005) used intergeneric in vitro pollination of Melandrium album and
Lychnis coronaria and hybrid plants were observed.
The successful use of in vitro pollination has not yet been reported in the genus
Cucumis. Manipulation with pollen and microspores of C. sativus has been tested
(isolation procedures, viability tests, germination, maturation) (Vižintin and Bohanec
2004). Some attempts at in vitro pollination between C. sativus and C. melo were
made, but no hybrid plants were observed and only calluses were obtained (Ondřej et
al. 2002).
1Cucurbitaceae 2008, Proceedings of the IXth EUCARPIA meeting on genetics and breeding
of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May 21-24th, 2008
360
The main aim of this study was to optimize the isolation of C. sativus and
Cucumis melo pollen grains and use them for in vitro pollination of C. sativus ovules.
MATERIALS AND METHODS
Plant material
Two accessions of Cucumis species were used for testing the viability of pollen grains
and in vitro pollination (C. sativus – CS; C. melo – CM) (C. sativus- ‘Stela F1’, CZ
09H3900744; C. melo – PMR, CZ 09H4000596, CZ 09H4000597, CZ 09H4000599;
donor RICP). The plant material originated from the vegetable germplasm collection
of the Research Institute of Crop Production (RICP, Prague), Department of Gene
Bank, at Olomouc, Czech Republic (Web site: <www.vurv.cz/>, part databases,
EVIGEZ). The plants were cultivated in a glasshouse (25°C/15°C day/night) at the
Department of Botany, Palacký University, Olomouc, Czech Republic.
Methods of isolation pollen grains
Immature male flowers of CS and CM were sterilized (1 min in 70 % ethanol,
10 min in 2.5 % chloramine, three times rinsed in sterile water) and cultivated on ½
MS medium (Murashige and Skoog 1962) at 25oC in the dark for 3 days. Pollen
grains were isolated from anthers directly or by one of three centrifugation techniques
(Tab. 1). Three liquid isolation media used for centrifugation were compared: NLN
(modified NLN 13 solution; Lichter 1981), YST [modified YST solution; Ondřej et
al. (2002)], and VB [modified VB solution; Vižintin and Bohanec (2004)]. The
viability of the isolated pollen grains was evaluated immediately after culture on YS
medium and established using an Olympus BX60 fluorescent microscope, with BW
filter and fluorescein diacetate stain.
Table 1. Methods for isolation of pollen grains.
Designation Description of isolation methods
“a” pollen grains were isolated by squeezing anthers with a glass rod in
isolation media; filtration; three times centrifugation (900 rpm, 10-5-5
min)
“b” pollen grains were isolated by squeezing anthers with a glass rod in
isolation media; centrifugation (500 rpm, 5 min); filtration;
centrifugation (1000 rpm, 3 min)
“c” pollen grains were isolated from anthers chopped with a razor blade in
isolation media; centrifugation (500 rpm, 5 min); filtration;
centrifugation (1000 rpm, 3 min)
“d” pollen grains were isolated directly from anthers (transferred from
anthers directly on or to media )
In vitro culture and in vitro pollination
Immature female flowers of C. sativus (CS) were sterilized and cultured in the
same way the male flowers were. The mature female flowers were then excised in
aseptic conditions and ovules were transferred onto solid media [CP medium: Castano
and De Proft (2000), and YS medium: Ondřej et al. (2002)]. Isolated pollen grains
361
were transferred on and around ovules. In the case of directly isolated pollen grains,
they were cultivated in liquid isolation media in addition (NLN, YST, VB). The Petri
dishes with ovules and pollen grains were cultured for 2 days at 25oC in the dark, and
then fertilized ovules were transferred onto two types of media supporting
embryogenesis (CW medium, GA medium; Skálová et al. 2007). The success of in
vitro pollination was evaluated. Two levels of regeneration were described: green
ovules (the ovules became green and grown) and calluses (max. 2 mm length; Fig. 1).
Figure 1. Developed calluses after in vitro pollination, C. sativus × C. sativus (a, c, d)
and C. sativus × C. melo (b).
RESULTS AND DISCUSSION
The results are summarized in Table 2 and in Figures 2 and 3. No large
differences in pollen grain viability were observed among centrifugation techniques.
The highest viability was observed using C. sativus pollen grains in combination with
isolation medium VB and method “c” (79 %). Direct isolation showed better results
(92 % using C. sativus pollen grains on CP medium). Pollen grains of C. melo had
lower viability (for centrifugation, 68 % in isolation medium NLN and method “a”;
for direct isolation 79 % on CP medium). As ovules of C. sativus became grown and
green after in vitro pollination, this was considered as the first level of regeneration.
The highest level of regeneration was formation of callus. The same results were
obtained by Ondřej et al. (2002). The higher regeneration was observed with in vitro
pollination between C. sativus and C. melo (33 % green ovules and 11 % calluses).
The values for regeneration of C. sativus × C. sativus were lower (20 % green ovules
and 9 % calluses). Direct isolation of pollen grains offered better results again (C.
sativus × C. sativus 36 % green ovules and 3 % calluses; C. sativus × C. melo 39 %
green ovules and 6 % calluses). The positive results with directly extracted pollen
grains were also observed by Castano and De Proft (2000). They obtained seedlings
after in vitro pollination in C. intybus.
Table 2. Summary of number, viability and regeneration success of pollen grains.
Pollen grains of
Cucumis spp./
Type of isolation
C. sativus /
centrifugation
C. sativus /
directly
C. melo /
centrifugation
C. melo /
directly
No. of isolated pollen
grains
540 450 450 450
Average viability (%) 72 86 61 73
Average regeneration
(%)
29 39 44 45
362
The major influence on viability of pollen grains was the condition of donor
plants. No strong influence of different centrifugation procedures was observed on the
viability of pollen grains. On the other hand, direct isolation was more favorable for
pollen grains than using centrifugation. The most positive influence on developing
ovules and embryos was medium CP. The results of Vižintin and Bohanec (2004)
showed that the pollen germination rate is greatly influenced by different factors
(genotypes of accessions, media composition, temperature, etc.). Further
manipulation of pollen grains could bring positive results in in vitro pollination
procedures.
a
0
10
20
30
40
50
60
70
80
90
100
110
120
CS/NLN
CS/YST
CS/VB
CM/NLN
CM/YST
CM/VB
Cucumis spp. / Isolation medium
Viability of pollen grains (%)
Method "a"
Method "b"
Method "c"
b
0
10
20
30
40
50
60
70
80
90
100
110
120
CS/YS
CS/CP
CS/NLN
CS/YST
CS/VB
CM/YS
CM/CP
CM/NLN
CM/YST
CM/VB
Cucumis spp./cultivation medium
Viability of pollen grains (%)
Figure 2. Viability of pollen grains isolated by centrifugation (a) and directly (b).
363
a
0
20
40
60
80
100
120
140
160
180
200
CS/NLN
CS/YST
CS/VB
CM/NLN
CM/YST
CM/VB
CS/NLN
CS/YST
CS/VB
CM/NLN
CM/YST
CM/VB
Cucumis spp./isolation medium
Regeneration (%)
Method "c"
Method "b"
Method "a"
YS medium CP medium
b
0
10
20
30
40
50
60
70
80
90
100
CS/YS
CS/CP
CS/NLN
CS/YST
CS/VB
CM/YS
CM/CP
CM/NLN
CM/YST
CM/VB
Cucumis spp./cultivation medium
Regeneration (%)
Regeneration
(calluses)
Regeneration
(green ovules)
Figure 3. Regeneration (%) after in vitro pollination in Cucumis spp., pollen grains
isolated by centrifugation (a) and pollen grains isolated directly (b).
ACKNOWLEDGEMENTS
This research was supported by NAZV No. QF 4108 Ministry of Agriculture of
Czech Republic (MA CR) and the Ministry of Education of the Czech Republic No.
MSM 6198959215. Comments of Dr. H.S. Paris are acknowledged.
The technique of in vitro placental or ovular pollination can be used for
production of interspecific and intergeneric hybrids (Zenkteler 1980). It can help
overcome crossing barriers to interspecific hybridization (Ondřej et al. 2002).
Interspecific hybridization is an important research area in the genus Cucumis
because of the possibility of transferring the genes for resistance to various diseases
from wild Cucumis to the cucumber (Cucumis sativus L.) genome. For successful
transfer of these genes, the use of some in vitro techniques has been suggested
(Lebeda et al. 2007).
The first experiment of direct pollination of ovules was made by Kanta and
Maheswari (1963) with some species of Papaveraceae. The whole process of sexual
reproduction was observed. Well-developed seedlings were obtained after in vitro
intraspecific pollination of Cichorium intybus L. by Castano and De Proft (2000), too.
Zenkteler et al. (2005) used intergeneric in vitro pollination of Melandrium album and
Lychnis coronaria and hybrid plants were observed.
The successful use of in vitro pollination has not yet been reported in the genus
Cucumis. Manipulation with pollen and microspores of C. sativus has been tested
(isolation procedures, viability tests, germination, maturation) (Vižintin and Bohanec
2004). Some attempts at in vitro pollination between C. sativus and C. melo were
made, but no hybrid plants were observed and only calluses were obtained (Ondřej et
al. 2002).
1Cucurbitaceae 2008, Proceedings of the IXth EUCARPIA meeting on genetics and breeding
of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May 21-24th, 2008
360
The main aim of this study was to optimize the isolation of C. sativus and
Cucumis melo pollen grains and use them for in vitro pollination of C. sativus ovules.
MATERIALS AND METHODS
Plant material
Two accessions of Cucumis species were used for testing the viability of pollen grains
and in vitro pollination (C. sativus – CS; C. melo – CM) (C. sativus- ‘Stela F1’, CZ
09H3900744; C. melo – PMR, CZ 09H4000596, CZ 09H4000597, CZ 09H4000599;
donor RICP). The plant material originated from the vegetable germplasm collection
of the Research Institute of Crop Production (RICP, Prague), Department of Gene
Bank, at Olomouc, Czech Republic (Web site: <www.vurv.cz/>, part databases,
EVIGEZ). The plants were cultivated in a glasshouse (25°C/15°C day/night) at the
Department of Botany, Palacký University, Olomouc, Czech Republic.
Methods of isolation pollen grains
Immature male flowers of CS and CM were sterilized (1 min in 70 % ethanol,
10 min in 2.5 % chloramine, three times rinsed in sterile water) and cultivated on ½
MS medium (Murashige and Skoog 1962) at 25oC in the dark for 3 days. Pollen
grains were isolated from anthers directly or by one of three centrifugation techniques
(Tab. 1). Three liquid isolation media used for centrifugation were compared: NLN
(modified NLN 13 solution; Lichter 1981), YST [modified YST solution; Ondřej et
al. (2002)], and VB [modified VB solution; Vižintin and Bohanec (2004)]. The
viability of the isolated pollen grains was evaluated immediately after culture on YS
medium and established using an Olympus BX60 fluorescent microscope, with BW
filter and fluorescein diacetate stain.
Table 1. Methods for isolation of pollen grains.
Designation Description of isolation methods
“a” pollen grains were isolated by squeezing anthers with a glass rod in
isolation media; filtration; three times centrifugation (900 rpm, 10-5-5
min)
“b” pollen grains were isolated by squeezing anthers with a glass rod in
isolation media; centrifugation (500 rpm, 5 min); filtration;
centrifugation (1000 rpm, 3 min)
“c” pollen grains were isolated from anthers chopped with a razor blade in
isolation media; centrifugation (500 rpm, 5 min); filtration;
centrifugation (1000 rpm, 3 min)
“d” pollen grains were isolated directly from anthers (transferred from
anthers directly on or to media )
In vitro culture and in vitro pollination
Immature female flowers of C. sativus (CS) were sterilized and cultured in the
same way the male flowers were. The mature female flowers were then excised in
aseptic conditions and ovules were transferred onto solid media [CP medium: Castano
and De Proft (2000), and YS medium: Ondřej et al. (2002)]. Isolated pollen grains
361
were transferred on and around ovules. In the case of directly isolated pollen grains,
they were cultivated in liquid isolation media in addition (NLN, YST, VB). The Petri
dishes with ovules and pollen grains were cultured for 2 days at 25oC in the dark, and
then fertilized ovules were transferred onto two types of media supporting
embryogenesis (CW medium, GA medium; Skálová et al. 2007). The success of in
vitro pollination was evaluated. Two levels of regeneration were described: green
ovules (the ovules became green and grown) and calluses (max. 2 mm length; Fig. 1).
Figure 1. Developed calluses after in vitro pollination, C. sativus × C. sativus (a, c, d)
and C. sativus × C. melo (b).
RESULTS AND DISCUSSION
The results are summarized in Table 2 and in Figures 2 and 3. No large
differences in pollen grain viability were observed among centrifugation techniques.
The highest viability was observed using C. sativus pollen grains in combination with
isolation medium VB and method “c” (79 %). Direct isolation showed better results
(92 % using C. sativus pollen grains on CP medium). Pollen grains of C. melo had
lower viability (for centrifugation, 68 % in isolation medium NLN and method “a”;
for direct isolation 79 % on CP medium). As ovules of C. sativus became grown and
green after in vitro pollination, this was considered as the first level of regeneration.
The highest level of regeneration was formation of callus. The same results were
obtained by Ondřej et al. (2002). The higher regeneration was observed with in vitro
pollination between C. sativus and C. melo (33 % green ovules and 11 % calluses).
The values for regeneration of C. sativus × C. sativus were lower (20 % green ovules
and 9 % calluses). Direct isolation of pollen grains offered better results again (C.
sativus × C. sativus 36 % green ovules and 3 % calluses; C. sativus × C. melo 39 %
green ovules and 6 % calluses). The positive results with directly extracted pollen
grains were also observed by Castano and De Proft (2000). They obtained seedlings
after in vitro pollination in C. intybus.
Table 2. Summary of number, viability and regeneration success of pollen grains.
Pollen grains of
Cucumis spp./
Type of isolation
C. sativus /
centrifugation
C. sativus /
directly
C. melo /
centrifugation
C. melo /
directly
No. of isolated pollen
grains
540 450 450 450
Average viability (%) 72 86 61 73
Average regeneration
(%)
29 39 44 45
362
The major influence on viability of pollen grains was the condition of donor
plants. No strong influence of different centrifugation procedures was observed on the
viability of pollen grains. On the other hand, direct isolation was more favorable for
pollen grains than using centrifugation. The most positive influence on developing
ovules and embryos was medium CP. The results of Vižintin and Bohanec (2004)
showed that the pollen germination rate is greatly influenced by different factors
(genotypes of accessions, media composition, temperature, etc.). Further
manipulation of pollen grains could bring positive results in in vitro pollination
procedures.
a
0
10
20
30
40
50
60
70
80
90
100
110
120
CS/NLN
CS/YST
CS/VB
CM/NLN
CM/YST
CM/VB
Cucumis spp. / Isolation medium
Viability of pollen grains (%)
Method "a"
Method "b"
Method "c"
b
0
10
20
30
40
50
60
70
80
90
100
110
120
CS/YS
CS/CP
CS/NLN
CS/YST
CS/VB
CM/YS
CM/CP
CM/NLN
CM/YST
CM/VB
Cucumis spp./cultivation medium
Viability of pollen grains (%)
Figure 2. Viability of pollen grains isolated by centrifugation (a) and directly (b).
363
a
0
20
40
60
80
100
120
140
160
180
200
CS/NLN
CS/YST
CS/VB
CM/NLN
CM/YST
CM/VB
CS/NLN
CS/YST
CS/VB
CM/NLN
CM/YST
CM/VB
Cucumis spp./isolation medium
Regeneration (%)
Method "c"
Method "b"
Method "a"
YS medium CP medium
b
0
10
20
30
40
50
60
70
80
90
100
CS/YS
CS/CP
CS/NLN
CS/YST
CS/VB
CM/YS
CM/CP
CM/NLN
CM/YST
CM/VB
Cucumis spp./cultivation medium
Regeneration (%)
Regeneration
(calluses)
Regeneration
(green ovules)
Figure 3. Regeneration (%) after in vitro pollination in Cucumis spp., pollen grains
isolated by centrifugation (a) and pollen grains isolated directly (b).
ACKNOWLEDGEMENTS
This research was supported by NAZV No. QF 4108 Ministry of Agriculture of
Czech Republic (MA CR) and the Ministry of Education of the Czech Republic No.
MSM 6198959215. Comments of Dr. H.S. Paris are acknowledged.
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