Effect of silver nitrate on anther culture embryo production of different Brassica oleracea morphotypes

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 l 1 AgNO 3. 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 0304-4238(99)00052-7

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 (AgNO 3 ) 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 M2 1 compost, fertilised with 20 g of the slow-release fertiliser Osmocote Plus 1 (15N : 11P : 13K 3MgO), and further fertilised as necessary with Bayfolan 1 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 mem 2 s 1 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

Table 1 Names, codes and origins of the B. oleracea morphotypes used to study the effect of silver nitrate on anther culture embryo production Morphotypes Code Origin a number J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307 301 name Brassica oleracea var. acephala: 1 Kale Demi-moellier INRA P2.30 FRA 2 Kale Demi-moellier F1 INRA 429xP2.30 FRA 3 Kale Demi-moellier F1 INRA 429X918 FRA 4 Kale Cavalier F1 INRA C51XF155 FRA 5 Kale Cavalier Vert INRA CB171 FRA 6 Kale Galega ISA 703 PRT Brassica oleracea var. alboglabra: 7 Kailan ISA 702 CHN Brassica oleracea var. botrytis: 8 Cauliflower INRA 702 FRA 9 Cauliflower INRA 923 FRA 10 Cauliflower INRA 924 FRA 11 Cauliflower INRA 7714 FRA 12 Cauliflower INRA 8181 FRA 13 Cauliflower Siria F1 CLAUSE FRA Brassica oleracea var. capitata: 14 Cabbage Hawke ISA 704 ENG 15 Cabbage Corac,aÄo de Boi ISA 705 PRT Brassica oleracea var. costata: 16 Tronchuda cabbage Grelo ISA 55/1 PRT 17 Tronchuda cabbage Grelo ISA 55/2 PRT 18 Tronchuda cabbage Grelo ISA 55/3 PRT 19 Tronchuda cabbage Grelo ISA 61 PRT 20 Tronchuda cabbage Algarvia ISA 207 PRT 21 Tronchuda cabbage Penca da PoÂvoa ISA 454 PRT 22 Tronchuda cabbage Portuguesa ISA 455 PRT 23 Tronchuda cabbage Portuguesa ISA CT 32/92 PRT 24 Tronchuda cabbage Murciana ISA CT 62/92 PRT Brassica oleracea var. italica: 25 Broccoli Arcadia ISA 706 USA 26 Broccoli Green Duke SAKATA JAP 27 Broccoli Marathon SAKATA JAP a INRA ˆ Institut National de la Recherche Agronomique, Rennes, France; ISA ˆ Instituto Superior de Agronomia, Technical University of Lisbon, Portugal; SAKATA ˆ Sakata Seeds Co., Japan; CLAUSE ˆ 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 l 1 NAA and 0.1 mg l 1 2,4-D, 100 mg l 1 L-serine, solidified with 7 g l 1 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 l 1 AgNO 3 ) 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 Parafilm 1 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 mem 2 s 1 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 l 1 AgNO 3 ) on anther culture embryo yield of different morphotypes of B. oleracea Morphotypes No. of anthers cultured a Percentage of embryogenic anthers No. of embryos per embryogenic anthers Embryos per 100 anthers cultured b 0 5 10 0 5 10 0 5 10 1 Kale Demi-moellier 288 4.5 8.0 7.6 1.7 1.8 2.2 7.6b 14.6a 16.7a 2 Kale Demi-moellier F1 216 3.2 4.2 5.1 1.1 1.2 1.3 3.7b 5.1ab 6.5a 3 Kale Demi-moellier F1 432 0.2 0.5 0.5 1.0 1.0 1.0 0.2a 0.5a 0.5a 4 Kale Cavalier F1 540 0.4 0.7 1.1 1.0 1.3 1.3 0.4b 0.9ab 1.5a 5 Kale Cavalier Vert 252 0.0 0.4 0.0 0.0 1.0 0.0 0.0a 0.4a 0.0a 6 Kale Galega 264 3.0 3.8 3.4 1.8 1.9 1.9 5.3a 7.1a 6.4a 7 Kailan 252 0.0 0.8 0.4 0.0 1.0 1.0 0.0a 0.8a 0.4a 8 Cauliflower INRA702 504 0.2 0.0 0.0 1.0 0.0 0.0 0.2a 0.0a 0.0a 9 Cauliflower INRA923 288 0.3 1.0 1.7 1.0 1.0 1.2 0.3b 1.0ab 2.1a 10 Cauliflower INRA924 288 1.4 2.8 3.5 1.0 1.0 1.1 1.4b 2.8ab 3.8a 11 Cauliflower INRA7714 288 0.0 0.3 0.7 0.0 1.0 1.0 0.0a 0.3a 0.7a 12 Cauliflower INRA8181 612 0.0 0.5 0.7 0.0 1.0 1.3 0.0b 0.5ab 0.8a 13 Cauliflower Siria F1 264 2.3 2.7 3.8 1.3 1.3 1.4 3.0b 3.4ab 5.3a 14 Cabbage Kawke 488 0.4 0.6 0.6 1.0 1.0 1.0 0.4a 0.6a 0.6a 15 Cabbage Corac,aÄo de Boi 264 0.4 0.8 0.8 1.0 1.0 1.0 0.4a 0.8a 0.8a 16 Tronchuda Grelo 55/1 468 2.1 3.6 3.8 1.1 1.5 1.6 2.3b 5.6a 6.0a 17 Tronchuda Grelo 55/2 360 4.7 7.5 8.3 2.2 2.4 2.6 10.6b 17.8ab 21.4a 18 Tronchuda Grelo 55/3 486 1.6 1.4 1.2 1.1 1.1 1.0 1.9a 1.6a 1.2a 19 Tronchuda Grelo 61 396 0.8 1.3 1.3 1.0 1.2 1.4 0.8b 1.5ab 1.8a 20 Tronchuda Algarvia 286 1.4 2.1 2.4 1.5 1.5 1.7 2.1b 3.1ab 4.2a 21 Tronchuda Penca PoÂvoa 216 0.0 1.4 1.4 0.0 1.0 1.0 0.0b 1.4a 1.4a 22 Tronchuda Portuguesa 232 0.4 0.0 0.0 1.0 0.0 0.0 0.4a 0.0a 0.0a 23 Tronchuda Portuguesa 452 0.0 1.8 0.7 0.0 1.3 1.0 0.0b 2.2a 0.7b 24 Tronchuda Murciana 516 0.0 0.4 0.4 0.0 1.0 1.0 0.0a 0.4a 0.4a 25 Broccoli Arcadia 234 0.9 1.3 1.7 1.0 1.0 1.0 0.9b 1.3ab 1.7a 26 Broccoli Green Duke 216 4.6 4.2 4.6 2.2 2.6 2.5 10.2a 10.6a 11.6a 27 Broccoli Marathon 280 1.1 3.6 1.4 1.0 1.2 1.0 1.1b 4.3a 1.4ab a Replications of six anthers per Petri dish. Some few Petri dishes had only five anthers due to accidents. b The means followed by the same letter, in the same row, do not significantly differ at p ˆ 0.05 according to pairwise t-tests. J.S. Dias, M.G. Martins / Scientia Horticulturae 82 (1999) 299±307 303

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 l 1 AgNO 3 ) 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 l 1 AgNO 3. 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 l 1 AgNO 3. 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 l 1 AgNO 3. Although only in the two last accessions (23 and 27) did treatment with 5 mg l 1 AgNO 3 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 l 1 AgNO 3, 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 l 1 AgNO 3 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 l 1 AgNO 3 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 l 1 AgNO 3 ; and (iii) the great majority of the morphotypes were stimulated to form embryos by the addition of 10 mg l 1 AgNO 3. 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 l 1 AgNO 3 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 AgNO 3 promotes embryogenesis in B. oleracea morphotypes by blocking the inhibitory effect of endogenous ethylene on embryo production. The tendency of the high concentration of AgNO 3 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''. References Arnison, P.G., Keller, W.A., 1990. A survey of anther culture response of B. oleracea L. cultivars grown under field conditions. Plant Breeding 104, 125±133. Arnison, P.G., Donaldson, P., Jackson, A., Semple, C., Keller, W.A., 1990. Genotype- specific response of cultured broccoli (Brassica oleracea var. italica) anthers to cytokinins. Plant Cell Tiss. Org. Cult. 20, 217±222. Beyer, E., Jr., 1976a. Silver ion: a potent antiethylene agent in cucumber and tomato. HortScience, 11, pp. 195±196. Beyer, E., Jr., 1976b. A potent inhibitor of ethylene action in plants. Plant Physiol., 58, pp. 268±271. Biddington, N.L., Sutherland, R.A., Robinson, H.T., 1988. Silver nitrate increases embryo production in anther culture of Brussels sprouts. Ann. Botany 62, 181±185. Chiang, M.S., Frechette, S., Kuo, C.G., Chong, C., Delafield, S.J., 1985. Embryogenesis and haploid production from anther culture of cabbage (Brassica oleracea var. capitata L.). Can. J. Plant Sci. 65, 1033±1037. Dunwell, J.M., 1979. Anther culture in Nicotiana tabacum: the role of the culture vessel atmosphere in pollen embryo induction and growth. J. Exp. Bot. 30, 419±428. Gamborg, O.L., Miller, R.A., Ojima, L., 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell. Res. 50, 151±158. Keller, W.A., Armstrong, K.C., 1981. Production of anther-derived dihaploid plants in autotetraploid marrowstemkale (Brassica oleracea var. acephala). Can. J. Genet. Cytol. 23, 259±265. Keller, W.A., Armstrong, K.C., 1983. Production of haploids via anther culture in B. oleracea var. italica. Euphytica 32, 151±159. Keller, W.A., Rajhathy, T., Lacapra, J., 1975. In vitro production of plants from pollen in Brassica campestris. Can. J. Genet. Cytol. 17, 655±666. Martins, M.G., Dias, J.S., 1993. Comparac,aÄo de dois tipos de gelificante, agarose e gelrite, na aptidaäo para a androgeânese e no rendimento em embrioäes em Brassica oleracea L.. SECH, Actas de Horticultura 10, 966±971.

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