Optimum age of siliques for rescue of hybrid embryos from crosses between Brassica oleracea, B. rapa and B. carinata

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1 Optimum age of siliques for rescue of hybrid embryos from crosses between Brassica oleracea, B. rapa and B. carinata M. H. Rahman 1 Danisco Seed, DK-4960 Holeby, Denmark. Received 6 January 2004, accepted 11 May Rahman, M. H Optimum age of siliques for rescue of hybrid embryos from crosses between Brassica oleracea, B. rapa and B. carinata. Can. J. Plant Sci. 84: Interspecific hybrids were produced from crosses involving Brassica rapa var. Yellow Sarson (AA), Canadian B. rapa (AA) cultivars, B. oleracea var. alboglabra (CC), B. oleracea var. italica (CC), rapidcycling B. oleracea (CC) and B. carinata (BBCC) to introgress the yellow seed color or self-incompatibility genes into B. napus. Embryo rescue technique was employed for this purpose. In interspecific crosses where a CC genome species was used as the female parent, a significantly lower number of ovules were fertilized compared to the crosses where the BBCC genome species was female. Embryo growth and development were slower in the crosses where the CC genome species was female than in the crosses where BBCC genome species was female. The efficiency of the embryo rescue technique from 16 to 40 d after pollination (DAP) with 4-d intervals was investigated in the crosses using CC genome species as female. The highest numbers of embryos were rescued between 20 and 28 DAP depending upon the specific cross undertaken. However, the survival rate of the embryos rescued at 20 DAP was very low compared to the embryos rescued at 24 and 28 DAP. The survival rate of the embryos rescued at 32 DAP was generally high, but the number of embryos rescued at this stage was significantly lower than at 24 and 28 DAP. No embryos were obtained at 16 DAP or at 40 DAP. Thus, using the CC genome species as female parent, the maximum efficiency of the embryo rescue technique was achieved when embryos were rescued between 24 and 28 DAP. In the case of crosses using the BBCC genome species as female, rescue of hybrid embryos was successful at 18 and 22 DAP. Key words: Brassica, interspecific cross, embryo rescue, embryo development Rahman, M. H Âge optimal des siliques pour la récupération des embryons issus de croisements entre Brassica oleracea, B. rapa et B. carinata. Can. J. Plant Sci. 84: L auteur a produit des hybrides interspécifiques en croisant Brassica rapa var. «yellow sarson» (AA), des cultivars canadiens de B. rapa (AA), B. olearacea var. alboglabra (CC), B. olearacea var. italica (CC), B. olearacea à cycle court (CC) et B carinata (BBCC). L objectif était d introgresser les gènes codant la couleur jaune des semences ou l autostérilité dans B. napus en recourant à la technique de récupération des embryons. Le nombre d ovules fécondés est sensiblement plus faible quand le parent femelle porte le génome CC plutôt que le génome BBCC dans les croisements interspécifiques. L embryon croît et se développe aussi plus lentement. L auteur a étudié l efficacité de la technique de récupération des embryons de 16 à 40 jours après la pollinisation (JAP) à intervalles de quatre jours pour les croisements où le parent femelle portait le génome CC. Il a recueilli le plus grand nombre d embryons entre 20 et 28 JAP, selon le croisement. Le taux de survie des embryons récupérés à 20 JAP était néanmoins très faible comparativement à celui des embryons récupérés entre 34 et 28 JAP. Ceux récupérés 32 JAP se caractérisaient généralement par un taux de survie élevé, mais leur nombre était sensiblement plus faible que celui des embryons récupérés 24 et 28 JAP. Aucun embryon n a été récupéré 16 JAP ni 40 JAP. En utilisant une espèce du génome CC comme parent femelle, la technique de récupération des embryons atteint donc son efficacité maximale quand les embryons ont entre 24 et 28 jours. Lorsque le parent femelle a le génome BBCC, cette technique donne de bons résultats quand les embryons ont 18 ou 22 jours. Mots clés: Brassica, croisement interspécifique, récupération des embryons, développement de l embryon In Brassica interspecific crosses, hybrid seed can easily be obtained in some crosses, whereas in other crosses either it cannot be obtained or a large number of cross-pollinations are needed (Downey et al. 1980). Various types of barriers to interspecific crossability have been reported. These are broadly classified into pre-fertilization and post-fertilization barriers. An example of the pre-fertilization barrier is when 1 Present address: Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5 ( 965 fertilization is prevented due to failure of the pollen tube to penetrate the stylar tissue. Post-fertilization embryo growth and development can be arrested due to lack of endosperm development and/or imbalance between the developing embryo and the endosperm. This ultimately results in abortion of the hybrid embryo (Hakansson 1956; Hadley and Openshaw 1980; Singh et al. 1990). Different techniques of plant cell and tissue culture, such as ovary, ovule and embryo culture as well as protoplast fusion, have proved useful for production of interspecific Abbreviations: DAP, days after pollination

2 966 CANADIAN JOURNAL OF PLANT SCIENCE hybrids (Khush and Brar 1991; Sharma et al. 1996). Rescue of hybrid embryos and their culture in vitro helps to overcome post-fertilization barriers in interspecific crosses. The successful application of this technique depends on the stage of the embryo being rescued and cultured in vitro. According to Raghavan (1976) and Raghavan and Srivastava (1982), two basic stages of embryo growth exist with regard to nutritional independence, viz. heterotrophic and autotrophic. The heterotrophic stage extends from fertilization up to the heart stage, while the autotrophic stage begins at the late heart stage. In the heterotrophic stage, growth of the embryo depends on the endosperm for its nutrients. In the autotrophic stage, the embryo becomes sufficiently independent of the endosperm for subsequent growth. Therefore, the requirements for culture of the embryos become less complex with the increasing maturity of the embryos. A high possibility of damage during rescue and sensitivity to osmotic shock under in vitro culture are added difficulties in the culture of young embryos compared with older ones (Maheshwari and Rangaswamy 1965; Raghavan 1977). Inomata (1993) reviewed embryo rescue techniques, namely ovary, ovule and embryo culture, for the production of wide cross hybrids in Brassica. Rescue of embryos from developing siliques followed by in vitro culture has been reported (Takeshita et al. 1980; Bajaj et al. 1986; Chen et al. 1988; Quazi 1988). This technique has been applied as early as 10 d after pollination (DAP) of silique age and as late as 30 DAP. The objective of this paper was to determine the optimum time of application of the embryo rescue technique in interspecific crosses where Brassica CC and BBCC genome species are used as female. These interspecific crosses were done to develop yellow-seeded B. napus germplasm (Rahman 2001) and to introgress self-incompatibility genes into B. napus. MATERIALS AND METHODS Plant Materials Two zero erucic acid yellow-seeded Brassica rapa (AA) var. Yellow Sarson breeding lines of Danisco Seed s (DS) accessions YS49 and YS50, two self-incompatible Canadian B. rapa (AA) cvs. Horizon and AC Parkland, one black-seeded B. oleracea var. alboglabra (CC) line (DS accession ), one self-incompatible B. oleracea var. italica (CC) broccoli cv. Green Duke, one black-seeded rapid-cycling B. oleracea (CC) line (DS accession ) and one yellow-seeded B. carinata (BBCC) line (DS accession ) were used in this study. The parents Horizon and AC Parkland are open-pollinated varieties, Green Duke is an F 1 hybrid, and all other parents are inbred materials. Parental plants were grown in a growth chamber, 20 /15 C day/night temperature and 16 h diurnal periods. The nine crosses that were made are listed in Table 1. The two crosses of B. oleracea var. italica Canadian B. rapa were made to introgress self-incompatibility genes, while the other seven crosses were made to introgress yellow seed color genes into B. napus. In crosses using B. oleracea as the female parent, maternal plants were used per cross, Table 1. Average number (±SE) of fertilized ovules per silique in nine interspecific crosses of Brassica Number of fertilized Cross ovules per silique B. oleracea var. alboglabra B. rapa var. Yellow Sarson YS ± 0.36 B. oleracea var. alboglabra B. rapa var. Yellow Sarson YS ± 0.18 B. oleracea var. italica cv. Green Duke B. rapa cv. Horizon 4.2 ± 0.29 B. oleracea var. italica cv. Green Duke B. rapa cv. AC Parkland 3.5 ± 0.17 B. oleracea var. alboglabra B. carinata 7.2 ± 0.62 Rapid-cycling B. oleracea B. carinata 8.2 ± 0.68 Mean of six crosses 4.6 ± 0.17 B. carinata B. rapa var. Yellow Sarson YS ± 0.52 B. carinata B. rapa var. Yellow Sarson YS ± 0.53 B. carinata B. oleracea var. alboglabra 7.9 ± 0.79 Mean of three crosses 10.1 ± 0.36 and five maternal plants were used when B. carinata used as the female parent. Randomly chosen pollen from 5 10 male parental plants were used in crossing. Embryo Rescue and in vitro Culture In crosses where the female parent was B. oleracea, a total of 1535 immature siliques, harvested at 16, 20, 24, 28, 32, 36 and 40 d after pollination (DAP) were used for the rescue of hybrid embryos. In crosses where B. carinata was the female parent, the rescue of hybrid embryos was done at 18 and 22 DAP, where 10 siliques per cross for each of the two dates were used. Excised siliques were surface sterilized with 70% ethanol for 3 min followed by 5% calcium hypochloride solution for 20 min. Siliques were washed with sterile water three times and were longitudinally dissected under a stereo microscope, and the number of fertilized ovules (larger in size compared to the unpollinated ones) were counted and excised. Recognizable embryos were extracted with a needle from the ovules and cultured (1 3 embryos per plate) on the solid embryo culture medium and were incubated at 24 C with continuous illumination of 12 µmol m 2 s 1 (approx. 4.6W m 2 ). Embryo culture medium was Murashige and Skoog s (1962) medium supplemented with 0.1 mg NAA and 0.1 mg Kinetin L 1, and sucrose concentration was 3%. The medium was filter-sterilized and 6 g agar L 1 medium was added aseptically to solidify the medium. Subculturing of the embryos, if required, was done on the same medium. Germinated embryos were transferred to Murashige and Skoog medium with the following modifications: 2 mg Benzylaminopurine L 1, 2% sucrose, no caseinhydrolysate, and solidified with agar (6 g L 1 ); and plantlets were regenerated. For root induction, plantlets were grown on modified Kao and Michayluk (1974) media (without myo-insitol, vitamins, glucose, ribose, sorbitol and manitol, and CaCl mg L 1, H 3 BO 3 10mg L 1 and half the amount of iron compounds) and solidified with 10g agar L 1. The interspecific hybrid nature of the embryos was confirmed by isozyme electrophoresis on the regenerated plants.

3 RAHMAN EMBYRO RESCUE IN BRASSICA 967 Fig. 1. Number of rescued embryos per pollination at different days after pollination (DAP) in six interspecific crosses using Brassica oleracea as the female parent. Solid bar inside the main bar shows the proportion of rescued embryos that survived under in vitro culture. The numbers of siliques excised for each date and cross is given at the bottom of the bar. (Note: at 16 DAP, siliques per cross and at 40 DAP, siliques per cross were excised but no embryos could be rescued. Statistical Analysis Comparisons of mean fertilization rates of ovules of the CC and BBCC genome species following interspecific crosspollination and yield of embryos at different dates after pollination were done by two sample t-test with unequal variances (non-pooled t-test). RESULTS In vivo Fertilized Ovules In the crosses of B. oleracea var. alboglabra Yellow Sarson (CC AA) ovules per silique (mean 1.7 ± 0.20) and in B. oleracea var. italica B. rapa (CC AA) crosses ovules per silique (mean 3.9 ± 0.18) were fertilized; while in B. oleracea var. alboglabra/rapid-cycling B. oleracea B. carinata (CC BBCC) crosses ovules per silique (mean 7.7 ± 0.46) were fertilized. In contrast, in two crosses of B. carinata Yellow Sarson (BBCC AA) ovules per silique (mean 11.2 ± 0.37), and in B. carinata B. oleracea var. alboglabra (BBCC CC) cross 7.9 ± 0.79 ovules per silique were fertilized (Table 1). Thus, the number of fertilized ovules was significantly lower in six crosses where the CC genome species was the female parent compared with the three crosses where BBCC genome species as the female parent (4.6 ± 0.17 vs ± 0.36; t = 13.79, P = 0.001). In vivo Embryo Growth and Development Embryo growth and development were slower in the crosses where B. oleracea var. alboglabra was the female parent than in the crosses where B. carinata was the female parent. In B. alboglabra B. rapa var. Yellow Sarson crosses, the first detectable globular to early heart stage embryos were observed at 20 DAP. Embryos were heart to early torpedo stage at 24 DAP, late heart to torpedo stage at 28 DAP, and at 32 DAP all embryos had reached the torpedo stage. In B. carinata B. rapa var. Yellow Sarson crosses embryos reached the globular to heart stage at 14 DAP, torpedo stage at 18 DAP and walking stick to mature stage at 22 DAP. In the B. carinata B. alboglabra cross, embryo development was different from that in B. carinata B. rapa var. Yellow Sarson crosses. In this cross, embryo growth and development were faster and at 18 DAP the embryos turned to a round seed-like structure. Embryo Rescue: B. oleracea as the Female Parent In all six crosses where B. oleracea var. alboglabra/b. oleracea var. italica/rapid-cycling B. oleracea were the female parents,

4 968 CANADIAN JOURNAL OF PLANT SCIENCE no recognizable embryo was found at 16 DAP. At 20 DAP, depending on the crosses, embryos per pollination (mean 0.17 ± 0.06) were rescued (Fig. 1). Generally rescued embryos did not survive except in the B. oleracea var. italica B. rapa crosses (14.3% survival). The cross B. oleracea var. italica B. rapa cv. AC Parkland yielded the highest number of embryos at 20 DAP. For the other five crosses, the highest number of embryos were rescued either at 24 DAP or at 28 DAP. The survival rate of these embryos was generally high, %, except the B. oleracea var. alboglabra B. carinata cross where a low survival (16.7%) of the rescued embryos was recorded at 24 DAP. At 32 DAP, a significantly lower (t = 2.50, P = 0.05) number of embryos was obtained than at 24 and 28 DAP. However, the survival rate of these embryos was high, %. At 36 DAP, all hybrid embryos had degenerated in five of the six crosses, and at 40 DAP all ovules turned dark brown and no live embryo was found. Thus, in this study, using the CC genome species as the female parent, the maximum efficiency of the embryo rescue technique was achieved when embryos were rescued between 24 and 28 DAP. The specific type of the cross had a significant effect on the number of embryos rescued per silique. The highest numbers of embryos were rescued in B. oleracea var. alboglabra Yellow Sarson (B. rapa) crosses; and the lowest number in rapid-cycling B. oleracea B. carinata cross. Variation in number of embryos rescued per pollination was found even in crosses of similar genome combination, e.g., in CC AA cross combination, B. oleracea var. alboglabra Yellow Sarson crosses yielded a higher number of embryos than B. oleracea var. italica B. rapa crosses. Embryo Rescue: B. carinata as the Female Parent In the three crosses where B. carinata was the female parent, embryos per pollination (mean 6.6 ± 0.32) were rescued at 18 DAP and embryos per pollination (mean 7.3 ± 0.35) were rescued at 22 DAP (Table 2). The survival rate of the embryos rescued at 18 DAP was 57 83% (mean 72%), while it was 93 96% (mean 94%) for the embryos rescued at 22 DAP. The interspecific hybrids were confirmed by isozyme electrophoresis of the following five enzyme systems: glucosephosphate isomerase, leucine aminopeptidase, phosphoglucomutase, 6-phosphogluconate dehydrogenase and malate dehydrogenase. DISCUSSION The optimum stage for the rescue of hybrid embryos depends on the time at which endosperm abortion occurs and the time at which subsequent embryo degeneration starts. In crosses where B. oleracea was the female parent, silique of the ages between 24 and 28 DAP were found to be most suitable for rescue of hybrid embryos. At this stage, a significant fraction of the rescued embryos were autotrophic (late heart stage or older), which accounted for a higher survival rate compared to the embryos rescued at 20 DAP (globular to early heart embryos, i.e., heterotrophic). At 32 DAP, a significantly lower number of embryos were rescued, which suggests that degeneration of the hybrid embryos starts after 24 to 28 DAP. From the observation that none of the rescued embryos corresponded to the walking stick stage, it is assumed that degeneration of hybrid embryos occurs before they reach this stage. However, in these interspecific crosses, only a small fraction of the fertilized ovules yielded rescueable embryos. Over all crosses, the average number of fertilized ovules per silique was 4.6. At 24 DAP, embryos per silique were rescued, i.e., only 4% of the fertilized ovules yielded rescueable embryos. This indicates that in the majority of the fertilized ovules, embryo growth was arrested at an early stage of development. Chen et al. (1989) applied similar embryo rescue technique at DAP on B. oleracea var. alboglabra B. rapa (Yellow Sarson and self-incompatible types) crosses using B. oleracea as the female parent, where the average number of rescued embryos per pollination was 0.24 for all crosses and 0.18 specifically for B. oleracea var. alboglabra Yellow Sarson crosses. They did not report specific data on the effectiveness of this technique at DAP. In the present study, significantly higher numbers of embryos were rescued from B. oleracea var. alboglabra Yellow Sarson crosses at 24 and 28 DAP than to at 20 DAP. For production of interspecific hybrids in Brassica, application of ovary or ovule culture, as another approach to embryo rescue, could also be effective. Inomata (1993) obtained, on an average, 0.83 hybrid/cultured ovary through culture of ovaries from interspecific crosses between different types of B. rapa (female) and B. oleracea (male). Through culture of fertilized ovules from B. oleracea B. rapa crosses, Lu et al. (2001) obtained 0.07 hybrids per pollination using B. oleracea as the female parent and 0.25 hybrids per pollination using B. rapa as the female parent; Heath and Earle (1996) obtained 0.28 hybrids per pollination using B. oleracea as the female parent; and Diederichsen and Sacristan (1994) obtained 0.75 hybrids per pollination using B. oleracea as the female parent and 0.89 hybrids per pollination using B. rapa as the female parent. In crosses where B. carinata was the female parent, most of the embryos rescued at 18 and 22 DAP were autotrophic, which would account for the high survival rate of these embryos. However, application of embryo rescue technique will probably not be required for production of hybrids of these three crosses, as a good number of hybrid seeds per pollination can be harvested in vivo (Rahman 2001). Data presented in this paper show that interspecific hybrid embryo growth and development are highly dependent on the genome of the maternal species. They also suggest that the genotype of the female parent may influence the success rate of embryo rescue. The results of this study can be applied to efficient production of Brassica interspecific hybrids, by application of embryo rescue technique, while using the CC or BBCC genome species as female in the cross. In interspecific crosses using other species, e.g., B. rapa (AA), B. nigra (BB), B. napus (AACC), etc., as the female parent, the optimum age of the silique for embryo rescue may be different from that in the present study. Investigation would therefore be needed to determine the optimum time for embryo rescue for a given specific interspecific cross.

5 RAHMAN EMBYRO RESCUE IN BRASSICA 969 Table 2. Number of rescued embryos/pollination at 18 and 22 d after pollination (DAP) and percent survival of the rescued embryos under in vitro culture in three interspecific crosses using Brassica carinata as the female parent 18 DAP 22 DAP Cross No. rescued embryo % survived embryo No. rescued embryo % survived embryo B. carinata B. rapa var. Yellow Sarson YS B. carinata B. rapa var. Yellow Sarson YS B. carinata B. oleracea var. alboglabra Mean SE (±) ACKNOWLEDGEMENTS I thank Dr. Morten Helt Poulsen (former Director of Breeding, Danisco Seed, Denmark) for his support during the course of this study, and Dr. Anna Haldrup (present address: Royal Veterinary and Agricultural University, Denmark) for assistance in embryo rescue. Bajaj, Y. P. S., Mahajan, S. K. and Labana, K. S Interspecific hybridization of Brassica napus and B. juncea through ovary, ovule and embryo culture. Euphytica 35: Chen, B. Y., Heneen, W. K. and Jönsson, R Resynthesis of Brassica napus L. through interspecific hybridization between B. alboglabra Bailey and B. campestris L. with special emphasis on seed colour. Plant Breed. 101: Diederichsen, E. and Sacristan, M. D The use of ovule culture in reciprocal hybridization between B. campestris L. and B. oleracea L. Plant Breed. 113: Downey, R. K., Klassen, A. J. and Stringam, G. R Rapeseed and mustard. Pages in W. R. Fehr and H. H. Hadley eds. Hybridization of crop plants. ASA, CSSA, Madison, WI. Hadley, H. H. and Openshaw, S. J Interspecific and intergeneric hybridization. Pages in W.R. Fehr and H.H. Hadley eds. Hybridization of crop plants. ASA, CSSA, Madison, WI. Hakansson, A Seed development of Brassica oleracea and B. rapa after certain reciprocal pollinations. Hereditas42: Heath, D. W. and Earle, E. D Resynthesis of rapeseed (Brassica napus L.): a comparison of sexual versus somatic hybridization. Plant Breed. 115: Inomata, N Embryo rescue techniques for wide hybridization. Pages in K. S. Labana, S. S. Banga, and S. K. Banga, eds. Breeding oilseed brassicas. Springer-Verlag, Berlin, Germany. Kao, K. N. and Michayluk, M. R A method for high frequency intergeneric fusion of plant protoplasts. Planta 115: Khush, G. S. and Brar, D. S Overcoming the barriers in hybridization. Pages in G. Kalloo and J. B. Chowdhury, eds. Distant hybridization of crop plants. Monographs on Theoretical and Applied Genetics. Springer-Verlag, Berlin, Germany. Lu, C. M., Zhang B., Kakihara, F. and Kato, M Introgression of genes into cultivated Brassica napus through resynthesis of B. napus via ovule culture and the accompanying change in fatty acid composition. Plant Breed. 120: Maheshwari, P. and Rangaswamy, N. S Embryology in relation to physiology and genetics. Pages in R. D. Preston, ed. Advances in botanical research 2. Academic Press, New York, NY. Murashige, I. and Skoog, F A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15: Quazi, M. H Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture. Theor. Appl. Genet. 75: Raghavan, V Experimental embryogenesis in vascular plants. Academic Press, New York, NY. Raghavan, V Applied aspects of embryo culture. Pages in J. Reinert and Y. P. S. Bajaj, eds. Applied and fundamental aspects of plant cell, tissue and organ culture. Springer- Verlag, Berlin, Germany. Raghavan, V. and Srivastava, P. S Embryo culture. Pages in B. M. Johri, ed. Experimental embryology of vascular plants. Springer-Verlag, Berlin, Germany. Rahman, M. H Production of yellow-seeded Brassica napus from interspecific crosses. Plant Breed. 120: Sharma, D. R., Kaur, R. and Kumar, K Embryo rescue in plants: a review. Euphytica 89: Singh, A. K., Moss, J. P. and Smartt, J Ploidy manipulations for interspecific gene transfer. Adv. Agron. 43: Takeshita, M., Kato, M. and Tokumasu, S Application of ovule culture to the production of intergeneric or interspecific hybrids in Brassica and Raphanus. Jpn. J. Genet. 55:

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