Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol82.Issue3-4.Dec1999:
Scientia Horticulturae 82 (1999) 299±307
Effect of silver nitrate on anther culture embryo
production of different Brassica
oleracea morphotypes
J.S. Dias*, M.G. Martins
Instituto Superior de Agronomia, Technical University of Lisbon,
Tapada da Ajuda, 1300 Lisbon, Portugal
Accepted 17 March 1999
Abstract
The effect of three concentrations of silver nitrate on anther culture embryo production was
studied in 27 morphotypes of Brassica oleracea.
Embryo yields were significantly increased in the majority of the morphotypes by the addition of
silver nitrate to the media. Embryo yields were not increased in only a few of the almost nonembryogenic accessions. The magnitude of the response to silver nitrate varied with the different
morphotypes. In general, the best results were obtained with the addition of 10 mg lÿ1 AgNO3.
Without the addition of silver nitrate no embryos would have been obtained in some of the nonembryogenic morphotypes. Data suggests that silver nitrate addition to anther culture media
promoted embryo production in different B. oleracea morphotypes. # 1999 Elsevier Science B.V.
All rights reserved.
Keywords: Embryogenesis; Coles; Variability; Ethylene
1. Introduction
Anther culture provides a tool for the rapid production of homozygous lines for
hybrid seed production which could be valuable in self-incompatible, outcrossing
crops such as vegetable brassicas. Although anther culture has been applied to
* Corresponding author. Fax: +351-13635031
E-mail address: joaosilvadias@mail.telepac.pt (J.S. Dias)
0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 5 2 - 7
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J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
most morphotypes of Brassica oleracea (Keller and Armstrong, 1981, 1983;
Chiang et al., 1985; Ockendon, 1985; Arnison and Keller, 1990; Arnison et al.,
1990; Martins and Dias, 1993), the low percentage of response and the major
genotype effect on the ability to culture anthers limit the practical application of
the technique in breeding programmes. The problems may be overcome either by
integration of the genetic capacity for anther culture response into the breeding
material by conventional or molecular breeding as suggested by Ockendon and
Sutherland (1987) and Touraev et al. (1997) respectively, or by general
improvement of the anther culture method.
Biddington et al. (1988) described the increase of embryo production in anther
culture of different cultivars of Brussels sprouts (`Hal', `Valiant', and `Oliver') by
the inclusion of the ethylene antagonist silver nitrate (AgNO3) in the medium.
Despite this, little work has been done on the applicability of silver nitrate in the
medium to the different horticultural morphotypes of B. oleracea.
The objective of the present paper is to analyse the variability and embryogenic
ability of 27 B. oleracea morphotypes to the inclusion of three different
concentrations of silver nitrate in the medium.
2. Material and methods
2.1. Plant morphotypes
The names of the 27 morphotypes of B. oleracea used in the present study, their
accession codes and origin are listed in Table 1. Donor plants were openpollinated cultivars, F1 hybrids and breeding material with 2±3 years of selection
(kale Galega, kailan, and tronchudas) or five generations of self-pollination (kale
Demi-moellier, kale Cavalier Vert and cauliflowers).
2.2. Plant growth conditions
Plants were grown in plastic pots of 16 cm diameter filled with (1) sand and (2)
Levington M21 compost, fertilised with 20 g of the slow-release fertiliser
Osmocote Plus1 (15N : 11P : 13K 3MgO), and further fertilised as necessary
with Bayfolan1 6N : 4P : 6K, in the field or in the greenhouse depending in their
juvenile period and requirement for vernalisation in a cold room at 48C. After
floral differentiation and the start of generative development all the plants were
transferred to a climate room with a day/night temperature of 22/168C, 16 h
photoperiod and a photosynthetic photon-flux density of 150 mE mÿ2 sÿ1 given
by `warm-white' tubular fluorescent lamps. Flower buds were harvested from
plants kept at least two weeks under these conditions, after the first flowers had
opened. Six-to-eight plants per accession were used for anther culture. Before bud
collecting, an assessment was made of pollen developmental stage in an attempt
301
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
Table 1
Names, codes and origins of the B. oleracea morphotypes used to study the effect of silver nitrate
on anther culture embryo production
Code
Origina
Brassica oleracea var. acephala:
1
Kale Demi-moellier
2
Kale Demi-moellier F1
3
Kale Demi-moellier F1
4
Kale Cavalier F1
5
Kale Cavalier Vert
6
Kale Galega
INRA P2.30
INRA 429xP2.30
INRA 429X918
INRA C51XF155
INRA CB171
ISA 703
FRA
FRA
FRA
FRA
FRA
PRT
Brassica oleracea var. alboglabra:
7
Kailan
ISA 702
CHN
Brassica oleracea var. botrytis:
8
Cauliflower
9
Cauliflower
10
Cauliflower
11
Cauliflower
12
Cauliflower
13
Cauliflower Siria F1
INRA 702
INRA 923
INRA 924
INRA 7714
INRA 8181
CLAUSE
FRA
FRA
FRA
FRA
FRA
FRA
Brassica oleracea var. capitata:
14
Cabbage Hawke
15
Cabbage Corac,aÄo de Boi
ISA 704
ISA 705
ENG
PRT
Brassica oleracea var. costata:
16
Tronchuda cabbage Grelo
17
Tronchuda cabbage Grelo
18
Tronchuda cabbage Grelo
19
Tronchuda cabbage Grelo
20
Tronchuda cabbage Algarvia
21
Tronchuda cabbage Penca da PoÂvoa
22
Tronchuda cabbage Portuguesa
23
Tronchuda cabbage Portuguesa
24
Tronchuda cabbage Murciana
ISA
ISA
ISA
ISA
ISA
ISA
ISA
ISA
ISA
PRT
PRT
PRT
PRT
PRT
PRT
PRT
PRT
PRT
Brassica oleracea var. italica:
25
Broccoli Arcadia
26
Broccoli Green Duke
27
Broccoli Marathon
ISA 706
SAKATA
SAKATA
Morphotypes
number
name
55/1
55/2
55/3
61
207
454
455
CT 32/92
CT 62/92
USA
JAP
JAP
a
INRA Institut National de la Recherche Agronomique, Rennes, France; ISA Instituto
Superior de Agronomia, Technical University of Lisbon, Portugal; SAKATA Sakata Seeds Co.,
Japan; CLAUSE Clause Seeds Co., France.
302
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
to plate only potential embryogenic anthers with late-uninucleated microspores
and binucleated pollen.
2.3. Anther culture
Buds were surface sterilised by a 15 min dip in a 3% calcium hypoclorite
solution followed by three rinses in pure sterile water. The anthers were removed
from the buds without their filaments. They were excised under a stereomicroscope and plated in 94 mm Petri dishes containing 12 ml of Gamborg's B5
medium (Gamborg et al., 1968) modified by Keller et al. (1975) and by Yang
(1989) containing 14% sucrose, 0.1 mg lÿ1 NAA and 0.1 mg lÿ1 2,4-D,
100 mg lÿ1 L-serine, solidified with 7 g lÿ1 of agarose, and pH adjusted to 5.8
before sterilisation in a autoclave at 1108C for 15 min. Different media with the
three concentrations of silver nitrate (0, 5 and 10 mg lÿ1 AgNO3) were made. For
every six anthers taken from each bud always two of them were placed in the
different Petri dishes with the different concentrations of silver nitrate. Three
flower buds, with potential embryogenic anthers, were harvested from single
plants each time. A Petri dish with six anthers (two per each treatment) was
considered as a replication unit. Depending on genotype and flower ability
between 216 and 612 anthers were plated by morphotype. Petri dishes were
sealed with Parafilm1 and placed in a 358C incubator for 24 h. The dishes were
then incubated in the dark at 258C. After 15 days in culture, the Petri dishes were
observed weekly. After the appearance of the first embryos, the dishes were
exposed to a 16 h photoperiod and a photon-flux density of 40 mE mÿ2 sÿ1 in a
climate room at 248C.
2.4. Data analysis
The number of embryogenic anthers and the number of embryos produced by
each anther were counted and the results expressed as a percentage of
embryogenic anthers, number of embryos per embryogenic anther and number
of embryos produced per 100 cultured anthers. For the number of embryos
produced a statistical analysis was performed using a log-linear model
(McCullagh and Nelder, 1989) since data follows the Poisson distribution. The
means were compared according to pairwise t-tests.
3. Results
3.1. Embryogenic capacity of the morphotypes
There was considerable variation in the response of the different morphotypes
to anther culture (Table 2). Almost all the morphotypes produced few or no
Table 2
Effect of the concentration of silver nitrate (0, 5 and 10 mg lÿ1 AgNO3) on anther culture embryo yield of different morphotypes of B. oleracea
Morphotypes
Kale Demi-moellier
Kale Demi-moellier F1
Kale Demi-moellier F1
Kale Cavalier F1
Kale Cavalier Vert
Kale Galega
Kailan
Cauliflower INRA702
Cauliflower INRA923
Cauliflower INRA924
Cauliflower INRA7714
Cauliflower INRA8181
Cauliflower Siria F1
Cabbage Kawke
Cabbage Corac,aÄo de Boi
Tronchuda Grelo 55/1
Tronchuda Grelo 55/2
Tronchuda Grelo 55/3
Tronchuda Grelo 61
Tronchuda Algarvia
Tronchuda Penca PoÂvoa
Tronchuda Portuguesa
Tronchuda Portuguesa
Tronchuda Murciana
Broccoli Arcadia
Broccoli Green Duke
Broccoli Marathon
b
No. of embryos per
embryogenic anthers
Embryos per 100 anthers
culturedb
0
5
10
0
5
10
0
5
10
4.5
3.2
0.2
0.4
0.0
3.0
0.0
0.2
0.3
1.4
0.0
0.0
2.3
0.4
0.4
2.1
4.7
1.6
0.8
1.4
0.0
0.4
0.0
0.0
0.9
4.6
1.1
8.0
4.2
0.5
0.7
0.4
3.8
0.8
0.0
1.0
2.8
0.3
0.5
2.7
0.6
0.8
3.6
7.5
1.4
1.3
2.1
1.4
0.0
1.8
0.4
1.3
4.2
3.6
7.6
5.1
0.5
1.1
0.0
3.4
0.4
0.0
1.7
3.5
0.7
0.7
3.8
0.6
0.8
3.8
8.3
1.2
1.3
2.4
1.4
0.0
0.7
0.4
1.7
4.6
1.4
1.7
1.1
1.0
1.0
0.0
1.8
0.0
1.0
1.0
1.0
0.0
0.0
1.3
1.0
1.0
1.1
2.2
1.1
1.0
1.5
0.0
1.0
0.0
0.0
1.0
2.2
1.0
1.8
1.2
1.0
1.3
1.0
1.9
1.0
0.0
1.0
1.0
1.0
1.0
1.3
1.0
1.0
1.5
2.4
1.1
1.2
1.5
1.0
0.0
1.3
1.0
1.0
2.6
1.2
2.2
1.3
1.0
1.3
0.0
1.9
1.0
0.0
1.2
1.1
1.0
1.3
1.4
1.0
1.0
1.6
2.6
1.0
1.4
1.7
1.0
0.0
1.0
1.0
1.0
2.5
1.0
7.6b
3.7b
0.2a
0.4b
0.0a
5.3a
0.0a
0.2a
0.3b
1.4b
0.0a
0.0b
3.0b
0.4a
0.4a
2.3b
10.6b
1.9a
0.8b
2.1b
0.0b
0.4a
0.0b
0.0a
0.9b
10.2a
1.1b
14.6a
5.1ab
0.5a
0.9ab
0.4a
7.1a
0.8a
0.0a
1.0ab
2.8ab
0.3a
0.5ab
3.4ab
0.6a
0.8a
5.6a
17.8ab
1.6a
1.5ab
3.1ab
1.4a
0.0a
2.2a
0.4a
1.3ab
10.6a
4.3a
16.7a
6.5a
0.5a
1.5a
0.0a
6.4a
0.4a
0.0a
2.1a
3.8a
0.7a
0.8a
5.3a
0.6a
0.8a
6.0a
21.4a
1.2a
1.8a
4.2a
1.4a
0.0a
0.7b
0.4a
1.7a
11.6a
1.4ab
Replications of six anthers per Petri dish. Some few Petri dishes had only five anthers due to accidents.
The means followed by the same letter, in the same row, do not significantly differ at p 0.05 according to pairwise t-tests.
303
a
288
216
432
540
252
264
252
504
288
288
288
612
264
488
264
468
360
486
396
286
216
232
452
516
234
216
280
Percentage of
embryogenic anthers
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
No. of anthers
cultureda
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J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
embryos without silver nitrate treatments. Kale `Demi-moellier' INRA P2.30 (1),
tronchuda cabbage `Grelo' ISA 55/2 (17) and broccoli `Green Duke' (26) were
the only good embryogenic accessions.
3.2. Influence of silver nitrate
Embryogenic anthers and embryo yields were significantly increased in the
majority of the morphotypes by the addition of silver nitrate to the media (cf.
Table 2). Embryo yield was not increased only in some of the almost nonembryogenic accessions such as cauliflower INRA 702 (8), and tronchuda
cabbage `Portuguesa' ISA 455 (22), and in the poor embryogenic tronchuda
cabbage `Grelo' ISA 55/3 (18). In these accessions, the total number of embryos
produced with the addition of silver nitrate was less than in the control (0 mg lÿ1
AgNO3) which may suggest errors in the culture of the anthers. Also, in the
almost non-embryogenic accessions (8, and 22), only one embryo from one
anther was produced which is not significant for analysis purposes. Excluding
these situations, silver nitrate addition was not found to inhibit embryo
production when compared to the untreated control in any other morphotype
(cf. Table 2). Therefore, silver nitrate seems to act as a promoter of
embryogenesis in anther culture of different B. oleracea morphotypes above
concentrations of 5±10 mg lÿ1 AgNO3.
The magnitude of the response to silver nitrate varied with different
morphotypes (cf. Table 2). In general, the best results were obtained with the
addition of 10 mg lÿ1 AgNO3. The exceptions to this rule were accessions kale
`Cavalier Vert'(5), `kale Galega' (6), kailan (7), tronchuda cabbage `Portuguesa'
(23), and broccoli `Marathon' (27), that have a higher embryo yield with the
concentration of 5 mg lÿ1 AgNO3. Although only in the two last accessions (23
and 27) did treatment with 5 mg lÿ1 AgNO3 induce embryo yields that were
significantly greater.
There was also variation comparing the influence of the addition of silver
nitrate in the non or poor embryogenic and in the good embryogenic anther
culture morphotypes. In the good embryogenic, there was a significant effect with
increase of silver nitrate concentration from 0 to 10 mg lÿ1 AgNO3, in accessions
kale Demi-moellier INRA P2.30 (1), and tronchuda cabbage Grelo ISA 55/2 (17),
but with broccoli `Green Duke' (26) the effect was not significant (cf. Table 2).
Biddington et al. (1988) and Ockendon (1985) also found that highly embryogenic Brussels sprouts (e.g. `Gower') may respond to silver nitrate indifferently. In the non or poor embryogenic morphotypes, the increase of silver
nitrate concentration from 0 to 10 mg lÿ1 AgNO3 was significant in accessions
kale `Cavalier' (4), in cauliflowers (9, 10, and 12), in tronchuda cabbages `Grelo'
(16 and 19), `Algarvia'(20), and `Penca da PoÂvoa' (21), and in broccoli `Arcadia'
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
305
(25). Without the addition of silver nitrate, no embryos would have been obtained
in the non-embryogenic morphotypes, kale `Cavalier Vert' (5), cauliflowers
INRA 7714 (11) and INRA 8181 (12), and tronchuda cabbages `Penca da PoÂvoa'
(21), `Portuguesa' ISA CT32/92 (23) and `Murciana' (24). Biddington et al.
(1988) also found in Brussels sprouts cultivars that silver nitrate is a promoter of
anther culture in very poorly embryogenic genotypes.
In the fair embryogenic morphotypes, the increase of silver nitrate
concentration from 0 to 10 mg lÿ1 AgNO3 was significant in accessions kale
`Demi-moellier' (2) and cauliflower `Siria' (13).
4. Discussion
The results confirmed the poor or even no embryogenic response of the
majority of the morphotypes of B. oleracea to anther culture without silver nitrate
treatment, and the variability in both cultivar-to-cultivar and plant-to-plant
response.
Additionally, the effect of silver nitrate concentration also varied greatly from
morphotype to morphotype. Overall three different categories of response to the
addition of silver nitrate were detected: (i) a small number of the morphotypes
tested showed little or no response to the addition of silver nitrate at any
concentration tested; (ii) a few morphotypes were stimulated to form embryos by
the addition of 5 mg lÿ1 AgNO3; and (iii) the great majority of the morphotypes
were stimulated to form embryos by the addition of 10 mg lÿ1 AgNO3. The
magnitude of the response to silver nitrate was variable with the different
morphotypes. The significant increase in embryo yield of some accessions with
the increase of silver nitrate concentrations from 0 to 10 mg lÿ1 AgNO3 contrasts
with the results of Dunwell (1979) who showed in Nicotiana tabacum anther
culture that silver nitrate increased embryo induction only slightly. Since Ag
inhibits ethylene action in plant tissue (Beyer, 1976a; Beyer, 1976b, b), this
suggests that AgNO3 promotes embryogenesis in B. oleracea morphotypes by
blocking the inhibitory effect of endogenous ethylene on embryo production. The
tendency of the high concentration of AgNO3 to improve the embryogenic
response was not evident in some morphotypes maybe due to the inhibition of
embryo formation at high levels of silver nitrate. Biddington et al. (1988) also
made similar observations and suggested that it was possible that these genotypes
either produced lower levels of endogenous ethylene or were less sensitive to
ethylene for embryo induction. The experiments of Dunwell (1979) with
Nicotiana tabacum anther culture also suggest that there may be an optimum
level of ethylene for embryo induction above which induction is inhibited or
reduced. The response to ethylene seems to be dependent also on the growth stage
of the anther.
306
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
5. Conclusions
From a practical point of view, the present results, even in spite of the variation
between morphotypes and plants, seem promising because in some morphotypes
silver nitrate treatment produced embryos in what would otherwise have been
unresponsive morphotypes, and in most of the accessions studied, the
embryogenic capacity was effectively enhanced.
Acknowledgements
Funding of this research was provided by the National Board of Scientific and
Technological Research (Junta Nacional de Investigac,aÄo CientõÂfica e TecnoloÂgica), Lisbon, Portugal, under project AGR/1483/92 ``Aplicac,aÄo das teÂcnicas de
haploidizac,aÄo androgeÂnica ao melhoramento das couves tronchudas''.
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Effect of silver nitrate on anther culture embryo
production of different Brassica
oleracea morphotypes
J.S. Dias*, M.G. Martins
Instituto Superior de Agronomia, Technical University of Lisbon,
Tapada da Ajuda, 1300 Lisbon, Portugal
Accepted 17 March 1999
Abstract
The effect of three concentrations of silver nitrate on anther culture embryo production was
studied in 27 morphotypes of Brassica oleracea.
Embryo yields were significantly increased in the majority of the morphotypes by the addition of
silver nitrate to the media. Embryo yields were not increased in only a few of the almost nonembryogenic accessions. The magnitude of the response to silver nitrate varied with the different
morphotypes. In general, the best results were obtained with the addition of 10 mg lÿ1 AgNO3.
Without the addition of silver nitrate no embryos would have been obtained in some of the nonembryogenic morphotypes. Data suggests that silver nitrate addition to anther culture media
promoted embryo production in different B. oleracea morphotypes. # 1999 Elsevier Science B.V.
All rights reserved.
Keywords: Embryogenesis; Coles; Variability; Ethylene
1. Introduction
Anther culture provides a tool for the rapid production of homozygous lines for
hybrid seed production which could be valuable in self-incompatible, outcrossing
crops such as vegetable brassicas. Although anther culture has been applied to
* Corresponding author. Fax: +351-13635031
E-mail address: joaosilvadias@mail.telepac.pt (J.S. Dias)
0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 5 2 - 7
300
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
most morphotypes of Brassica oleracea (Keller and Armstrong, 1981, 1983;
Chiang et al., 1985; Ockendon, 1985; Arnison and Keller, 1990; Arnison et al.,
1990; Martins and Dias, 1993), the low percentage of response and the major
genotype effect on the ability to culture anthers limit the practical application of
the technique in breeding programmes. The problems may be overcome either by
integration of the genetic capacity for anther culture response into the breeding
material by conventional or molecular breeding as suggested by Ockendon and
Sutherland (1987) and Touraev et al. (1997) respectively, or by general
improvement of the anther culture method.
Biddington et al. (1988) described the increase of embryo production in anther
culture of different cultivars of Brussels sprouts (`Hal', `Valiant', and `Oliver') by
the inclusion of the ethylene antagonist silver nitrate (AgNO3) in the medium.
Despite this, little work has been done on the applicability of silver nitrate in the
medium to the different horticultural morphotypes of B. oleracea.
The objective of the present paper is to analyse the variability and embryogenic
ability of 27 B. oleracea morphotypes to the inclusion of three different
concentrations of silver nitrate in the medium.
2. Material and methods
2.1. Plant morphotypes
The names of the 27 morphotypes of B. oleracea used in the present study, their
accession codes and origin are listed in Table 1. Donor plants were openpollinated cultivars, F1 hybrids and breeding material with 2±3 years of selection
(kale Galega, kailan, and tronchudas) or five generations of self-pollination (kale
Demi-moellier, kale Cavalier Vert and cauliflowers).
2.2. Plant growth conditions
Plants were grown in plastic pots of 16 cm diameter filled with (1) sand and (2)
Levington M21 compost, fertilised with 20 g of the slow-release fertiliser
Osmocote Plus1 (15N : 11P : 13K 3MgO), and further fertilised as necessary
with Bayfolan1 6N : 4P : 6K, in the field or in the greenhouse depending in their
juvenile period and requirement for vernalisation in a cold room at 48C. After
floral differentiation and the start of generative development all the plants were
transferred to a climate room with a day/night temperature of 22/168C, 16 h
photoperiod and a photosynthetic photon-flux density of 150 mE mÿ2 sÿ1 given
by `warm-white' tubular fluorescent lamps. Flower buds were harvested from
plants kept at least two weeks under these conditions, after the first flowers had
opened. Six-to-eight plants per accession were used for anther culture. Before bud
collecting, an assessment was made of pollen developmental stage in an attempt
301
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
Table 1
Names, codes and origins of the B. oleracea morphotypes used to study the effect of silver nitrate
on anther culture embryo production
Code
Origina
Brassica oleracea var. acephala:
1
Kale Demi-moellier
2
Kale Demi-moellier F1
3
Kale Demi-moellier F1
4
Kale Cavalier F1
5
Kale Cavalier Vert
6
Kale Galega
INRA P2.30
INRA 429xP2.30
INRA 429X918
INRA C51XF155
INRA CB171
ISA 703
FRA
FRA
FRA
FRA
FRA
PRT
Brassica oleracea var. alboglabra:
7
Kailan
ISA 702
CHN
Brassica oleracea var. botrytis:
8
Cauliflower
9
Cauliflower
10
Cauliflower
11
Cauliflower
12
Cauliflower
13
Cauliflower Siria F1
INRA 702
INRA 923
INRA 924
INRA 7714
INRA 8181
CLAUSE
FRA
FRA
FRA
FRA
FRA
FRA
Brassica oleracea var. capitata:
14
Cabbage Hawke
15
Cabbage Corac,aÄo de Boi
ISA 704
ISA 705
ENG
PRT
Brassica oleracea var. costata:
16
Tronchuda cabbage Grelo
17
Tronchuda cabbage Grelo
18
Tronchuda cabbage Grelo
19
Tronchuda cabbage Grelo
20
Tronchuda cabbage Algarvia
21
Tronchuda cabbage Penca da PoÂvoa
22
Tronchuda cabbage Portuguesa
23
Tronchuda cabbage Portuguesa
24
Tronchuda cabbage Murciana
ISA
ISA
ISA
ISA
ISA
ISA
ISA
ISA
ISA
PRT
PRT
PRT
PRT
PRT
PRT
PRT
PRT
PRT
Brassica oleracea var. italica:
25
Broccoli Arcadia
26
Broccoli Green Duke
27
Broccoli Marathon
ISA 706
SAKATA
SAKATA
Morphotypes
number
name
55/1
55/2
55/3
61
207
454
455
CT 32/92
CT 62/92
USA
JAP
JAP
a
INRA Institut National de la Recherche Agronomique, Rennes, France; ISA Instituto
Superior de Agronomia, Technical University of Lisbon, Portugal; SAKATA Sakata Seeds Co.,
Japan; CLAUSE Clause Seeds Co., France.
302
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
to plate only potential embryogenic anthers with late-uninucleated microspores
and binucleated pollen.
2.3. Anther culture
Buds were surface sterilised by a 15 min dip in a 3% calcium hypoclorite
solution followed by three rinses in pure sterile water. The anthers were removed
from the buds without their filaments. They were excised under a stereomicroscope and plated in 94 mm Petri dishes containing 12 ml of Gamborg's B5
medium (Gamborg et al., 1968) modified by Keller et al. (1975) and by Yang
(1989) containing 14% sucrose, 0.1 mg lÿ1 NAA and 0.1 mg lÿ1 2,4-D,
100 mg lÿ1 L-serine, solidified with 7 g lÿ1 of agarose, and pH adjusted to 5.8
before sterilisation in a autoclave at 1108C for 15 min. Different media with the
three concentrations of silver nitrate (0, 5 and 10 mg lÿ1 AgNO3) were made. For
every six anthers taken from each bud always two of them were placed in the
different Petri dishes with the different concentrations of silver nitrate. Three
flower buds, with potential embryogenic anthers, were harvested from single
plants each time. A Petri dish with six anthers (two per each treatment) was
considered as a replication unit. Depending on genotype and flower ability
between 216 and 612 anthers were plated by morphotype. Petri dishes were
sealed with Parafilm1 and placed in a 358C incubator for 24 h. The dishes were
then incubated in the dark at 258C. After 15 days in culture, the Petri dishes were
observed weekly. After the appearance of the first embryos, the dishes were
exposed to a 16 h photoperiod and a photon-flux density of 40 mE mÿ2 sÿ1 in a
climate room at 248C.
2.4. Data analysis
The number of embryogenic anthers and the number of embryos produced by
each anther were counted and the results expressed as a percentage of
embryogenic anthers, number of embryos per embryogenic anther and number
of embryos produced per 100 cultured anthers. For the number of embryos
produced a statistical analysis was performed using a log-linear model
(McCullagh and Nelder, 1989) since data follows the Poisson distribution. The
means were compared according to pairwise t-tests.
3. Results
3.1. Embryogenic capacity of the morphotypes
There was considerable variation in the response of the different morphotypes
to anther culture (Table 2). Almost all the morphotypes produced few or no
Table 2
Effect of the concentration of silver nitrate (0, 5 and 10 mg lÿ1 AgNO3) on anther culture embryo yield of different morphotypes of B. oleracea
Morphotypes
Kale Demi-moellier
Kale Demi-moellier F1
Kale Demi-moellier F1
Kale Cavalier F1
Kale Cavalier Vert
Kale Galega
Kailan
Cauliflower INRA702
Cauliflower INRA923
Cauliflower INRA924
Cauliflower INRA7714
Cauliflower INRA8181
Cauliflower Siria F1
Cabbage Kawke
Cabbage Corac,aÄo de Boi
Tronchuda Grelo 55/1
Tronchuda Grelo 55/2
Tronchuda Grelo 55/3
Tronchuda Grelo 61
Tronchuda Algarvia
Tronchuda Penca PoÂvoa
Tronchuda Portuguesa
Tronchuda Portuguesa
Tronchuda Murciana
Broccoli Arcadia
Broccoli Green Duke
Broccoli Marathon
b
No. of embryos per
embryogenic anthers
Embryos per 100 anthers
culturedb
0
5
10
0
5
10
0
5
10
4.5
3.2
0.2
0.4
0.0
3.0
0.0
0.2
0.3
1.4
0.0
0.0
2.3
0.4
0.4
2.1
4.7
1.6
0.8
1.4
0.0
0.4
0.0
0.0
0.9
4.6
1.1
8.0
4.2
0.5
0.7
0.4
3.8
0.8
0.0
1.0
2.8
0.3
0.5
2.7
0.6
0.8
3.6
7.5
1.4
1.3
2.1
1.4
0.0
1.8
0.4
1.3
4.2
3.6
7.6
5.1
0.5
1.1
0.0
3.4
0.4
0.0
1.7
3.5
0.7
0.7
3.8
0.6
0.8
3.8
8.3
1.2
1.3
2.4
1.4
0.0
0.7
0.4
1.7
4.6
1.4
1.7
1.1
1.0
1.0
0.0
1.8
0.0
1.0
1.0
1.0
0.0
0.0
1.3
1.0
1.0
1.1
2.2
1.1
1.0
1.5
0.0
1.0
0.0
0.0
1.0
2.2
1.0
1.8
1.2
1.0
1.3
1.0
1.9
1.0
0.0
1.0
1.0
1.0
1.0
1.3
1.0
1.0
1.5
2.4
1.1
1.2
1.5
1.0
0.0
1.3
1.0
1.0
2.6
1.2
2.2
1.3
1.0
1.3
0.0
1.9
1.0
0.0
1.2
1.1
1.0
1.3
1.4
1.0
1.0
1.6
2.6
1.0
1.4
1.7
1.0
0.0
1.0
1.0
1.0
2.5
1.0
7.6b
3.7b
0.2a
0.4b
0.0a
5.3a
0.0a
0.2a
0.3b
1.4b
0.0a
0.0b
3.0b
0.4a
0.4a
2.3b
10.6b
1.9a
0.8b
2.1b
0.0b
0.4a
0.0b
0.0a
0.9b
10.2a
1.1b
14.6a
5.1ab
0.5a
0.9ab
0.4a
7.1a
0.8a
0.0a
1.0ab
2.8ab
0.3a
0.5ab
3.4ab
0.6a
0.8a
5.6a
17.8ab
1.6a
1.5ab
3.1ab
1.4a
0.0a
2.2a
0.4a
1.3ab
10.6a
4.3a
16.7a
6.5a
0.5a
1.5a
0.0a
6.4a
0.4a
0.0a
2.1a
3.8a
0.7a
0.8a
5.3a
0.6a
0.8a
6.0a
21.4a
1.2a
1.8a
4.2a
1.4a
0.0a
0.7b
0.4a
1.7a
11.6a
1.4ab
Replications of six anthers per Petri dish. Some few Petri dishes had only five anthers due to accidents.
The means followed by the same letter, in the same row, do not significantly differ at p 0.05 according to pairwise t-tests.
303
a
288
216
432
540
252
264
252
504
288
288
288
612
264
488
264
468
360
486
396
286
216
232
452
516
234
216
280
Percentage of
embryogenic anthers
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
No. of anthers
cultureda
304
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
embryos without silver nitrate treatments. Kale `Demi-moellier' INRA P2.30 (1),
tronchuda cabbage `Grelo' ISA 55/2 (17) and broccoli `Green Duke' (26) were
the only good embryogenic accessions.
3.2. Influence of silver nitrate
Embryogenic anthers and embryo yields were significantly increased in the
majority of the morphotypes by the addition of silver nitrate to the media (cf.
Table 2). Embryo yield was not increased only in some of the almost nonembryogenic accessions such as cauliflower INRA 702 (8), and tronchuda
cabbage `Portuguesa' ISA 455 (22), and in the poor embryogenic tronchuda
cabbage `Grelo' ISA 55/3 (18). In these accessions, the total number of embryos
produced with the addition of silver nitrate was less than in the control (0 mg lÿ1
AgNO3) which may suggest errors in the culture of the anthers. Also, in the
almost non-embryogenic accessions (8, and 22), only one embryo from one
anther was produced which is not significant for analysis purposes. Excluding
these situations, silver nitrate addition was not found to inhibit embryo
production when compared to the untreated control in any other morphotype
(cf. Table 2). Therefore, silver nitrate seems to act as a promoter of
embryogenesis in anther culture of different B. oleracea morphotypes above
concentrations of 5±10 mg lÿ1 AgNO3.
The magnitude of the response to silver nitrate varied with different
morphotypes (cf. Table 2). In general, the best results were obtained with the
addition of 10 mg lÿ1 AgNO3. The exceptions to this rule were accessions kale
`Cavalier Vert'(5), `kale Galega' (6), kailan (7), tronchuda cabbage `Portuguesa'
(23), and broccoli `Marathon' (27), that have a higher embryo yield with the
concentration of 5 mg lÿ1 AgNO3. Although only in the two last accessions (23
and 27) did treatment with 5 mg lÿ1 AgNO3 induce embryo yields that were
significantly greater.
There was also variation comparing the influence of the addition of silver
nitrate in the non or poor embryogenic and in the good embryogenic anther
culture morphotypes. In the good embryogenic, there was a significant effect with
increase of silver nitrate concentration from 0 to 10 mg lÿ1 AgNO3, in accessions
kale Demi-moellier INRA P2.30 (1), and tronchuda cabbage Grelo ISA 55/2 (17),
but with broccoli `Green Duke' (26) the effect was not significant (cf. Table 2).
Biddington et al. (1988) and Ockendon (1985) also found that highly embryogenic Brussels sprouts (e.g. `Gower') may respond to silver nitrate indifferently. In the non or poor embryogenic morphotypes, the increase of silver
nitrate concentration from 0 to 10 mg lÿ1 AgNO3 was significant in accessions
kale `Cavalier' (4), in cauliflowers (9, 10, and 12), in tronchuda cabbages `Grelo'
(16 and 19), `Algarvia'(20), and `Penca da PoÂvoa' (21), and in broccoli `Arcadia'
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
305
(25). Without the addition of silver nitrate, no embryos would have been obtained
in the non-embryogenic morphotypes, kale `Cavalier Vert' (5), cauliflowers
INRA 7714 (11) and INRA 8181 (12), and tronchuda cabbages `Penca da PoÂvoa'
(21), `Portuguesa' ISA CT32/92 (23) and `Murciana' (24). Biddington et al.
(1988) also found in Brussels sprouts cultivars that silver nitrate is a promoter of
anther culture in very poorly embryogenic genotypes.
In the fair embryogenic morphotypes, the increase of silver nitrate
concentration from 0 to 10 mg lÿ1 AgNO3 was significant in accessions kale
`Demi-moellier' (2) and cauliflower `Siria' (13).
4. Discussion
The results confirmed the poor or even no embryogenic response of the
majority of the morphotypes of B. oleracea to anther culture without silver nitrate
treatment, and the variability in both cultivar-to-cultivar and plant-to-plant
response.
Additionally, the effect of silver nitrate concentration also varied greatly from
morphotype to morphotype. Overall three different categories of response to the
addition of silver nitrate were detected: (i) a small number of the morphotypes
tested showed little or no response to the addition of silver nitrate at any
concentration tested; (ii) a few morphotypes were stimulated to form embryos by
the addition of 5 mg lÿ1 AgNO3; and (iii) the great majority of the morphotypes
were stimulated to form embryos by the addition of 10 mg lÿ1 AgNO3. The
magnitude of the response to silver nitrate was variable with the different
morphotypes. The significant increase in embryo yield of some accessions with
the increase of silver nitrate concentrations from 0 to 10 mg lÿ1 AgNO3 contrasts
with the results of Dunwell (1979) who showed in Nicotiana tabacum anther
culture that silver nitrate increased embryo induction only slightly. Since Ag
inhibits ethylene action in plant tissue (Beyer, 1976a; Beyer, 1976b, b), this
suggests that AgNO3 promotes embryogenesis in B. oleracea morphotypes by
blocking the inhibitory effect of endogenous ethylene on embryo production. The
tendency of the high concentration of AgNO3 to improve the embryogenic
response was not evident in some morphotypes maybe due to the inhibition of
embryo formation at high levels of silver nitrate. Biddington et al. (1988) also
made similar observations and suggested that it was possible that these genotypes
either produced lower levels of endogenous ethylene or were less sensitive to
ethylene for embryo induction. The experiments of Dunwell (1979) with
Nicotiana tabacum anther culture also suggest that there may be an optimum
level of ethylene for embryo induction above which induction is inhibited or
reduced. The response to ethylene seems to be dependent also on the growth stage
of the anther.
306
J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307
5. Conclusions
From a practical point of view, the present results, even in spite of the variation
between morphotypes and plants, seem promising because in some morphotypes
silver nitrate treatment produced embryos in what would otherwise have been
unresponsive morphotypes, and in most of the accessions studied, the
embryogenic capacity was effectively enhanced.
Acknowledgements
Funding of this research was provided by the National Board of Scientific and
Technological Research (Junta Nacional de Investigac,aÄo CientõÂfica e TecnoloÂgica), Lisbon, Portugal, under project AGR/1483/92 ``Aplicac,aÄo das teÂcnicas de
haploidizac,aÄo androgeÂnica ao melhoramento das couves tronchudas''.
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