2 BREEDING, GENETICS AND MODELS The classic triangle of U (1935) (Fig. 1.2) shows the inter-relationships of brassicas based on their chromosome numbers and 2n genome descriptors. These species can be inter-crossed using embryo rescue, fusion and other methods. In addition, massive opportunities are emerging from studies of model brassicas such as thale cress (Arabidopsis thaliana) and Wisconsin Fast PlantsTM that identify genes and their products which can be applied in crop species. Further, some breeders are now working with the less well known species in Brassicaceae, such a Brassica carinata, to extract valuable genes for resistance to pathogens, pests and other economic characters. The Brassicaceae is one of the most flexible plant families in terms of interspecifc and intergenomic crosses. Rapid progress is being made in our understanding of their component genes, genomic interactions, protein products and resultant phenotypic characteristics, leading eventually to even wider and more diverse crosses. Concurrently, restriction fragment length polymorphism (RFLP) and linkage maps are being made for most of the major species, and these have shown many common chromosome linkage groups occurring across these species. This is encouraging the research into the potential of single gene transfer into economic crops from the model types. GENOMIC CHARACTERS AND TAXONOMY The following six Brassica species, plus Raphanus sativus, radish 2n = 18, have been inter-crossed, with varying levels of difficulty requiring embryo culture or fusion to obtain hybrids: Brassica nigra Koch, black mustard, 2n = 16; Brassica carinata Braun, Ethiopian mustard, 2n = 34; Brassica juncea L. Coss, brown mustard, 2n = 36; Brassica napus, swede or rutabaga, rape or oilseed rape (canola) 2n = 38; Brassica rapa, turnip and Chinese cabbage, 2n = 20; and Brassica oleracea, cole crops, 2n = 18. Table 2.1 shows the cytoplasmic and genomic descriptors for these species. © G.R. Dixon 2007. Vegetable Brassicas and Related Crucifers (G.R. Dixon) 35 36 Chapter 2 Table 2.1. Designation of cytoplasmic and nuclear genomes of Brassica and Raphanus species. Subspecies or Species variety Cytoplasm 2n genome Common name B. nigra B Bb Black mustard B. oleracea C Cc Cole vegetables B. rapa A Aa Chinensis aa.c Pak Choi Nipposinica aa.n Mizuna Oleifera aa.o Turnip rape Parachinensis aa.pa Caisin (choy sum) Pekinensis aa.p Chinese cabbage, petsai Rapifera aa.r Vegetable turnip Trilocularis aa.t Sarsons B. carinata BC Bbcc Ethiopian mustard B. juncea AB Aabb Vegetable mustard B. napus AC Aacc Fodder rape, oilseed rape (canola), swede R. sativus R Rr Vegetable radish, daikon After Williams and Heyn (1980); these authors suggested using the single upper case letter representing the genome descriptor to designate the cytoplasm in which the nuclear genes are functioning. Hybridization between the seven species in Table 2.1 is easier than to the other cruciferous species. It can still be a difficult and frustrating task, however, to obtain true-breeding new lines with the desired attribute irrespective of whether the species is a diploid or allopolyploid as with B. carinata, B. juncea and B. napus. Using fusion rather than classical hybridization with embryo rescue has improved the chances of success. FLORAL BIOLOGY AS RELATED TO CONTROLLED POLLINATION The flower (Fig. 2.1) differentiates by the successive development of four sepals, six stamens, two carpels and four petals. The carpels form a superior ovary with a ‘false’ septum and two rows of campylotropous ovules. The nucellar tissue is largely displaced by the embryo sac and, when the buds open, the ovules mainly consist of the two integuments and the ripe embryo sac. The buds open under pressure from the rapidly growing petals. Opening starts in the afternoon, and usually the flowers become fully expanding during the following morning. The bright yellow petals grow to 10–25 mm long and 6–10 mm wide. The sepals are erect. Pollination of the flowers is by insects, particularly bees, which collect pollen and nectar. Breeding, Genetics and Models 37 Fig. 2.1. Generalized structure of the flower and half-flower of Brassica (M.H. Dickson). Nectar is secreted by four nectaries situated between bases of the short stamens and the ovary. The flowers are borne in racemes on the main stem and its axillary branches. The inflorescences may attain lengths of 1–2 m. The slender pedicels are 15–20 mm long. After fertilization, the endosperm develops rapidly, while embryo growth does not start for some days. The embryo is generally still small 2 weeks after pollination, and at this stage embryo rescue can be first attempted. The embryo fills most of the seed coat after 3–5 weeks, by which time the endosperm has been almost completely absorbed. Nutrient reserves for germination are stored in the cotyledons, which are folded together with the embryo radicle lying between them. The fruits of cole crops are glabrous siliquae (pods) (Fig. 2.2), 4–5 mm wide and 40–100 mm long, with two rows of seeds lying along the edges of the replus. One silique contains 10–30 seeds. Three to 4 weeks after the opening of a flower, the silique reaches its full length and diameter. When it is ripe, the two valves dehisce. Separation begins at the attached base and works towards the unattached distal end, leaving the seeds attached to the placentas. Physical force ultimately separates the seeds, usually by the pushing of the dehisced siliquae against other plant parts either by wind or in threshing operations. MICROPROPAGATION Microspore-derived embryogenesis was first completed successfully on broccoli by Keller et al. (1975) from isolated anthers. Now embryogenesis of microspores is quite routine in most large-scale crucifer breeding programmes. There are still differences between sources of microspores which affect the number of embryos that are regenerated per anther, but the system is now well developed, and >1000 per bud can be produced. The size of the 38 Chapter 2 Fig. 2.2. Generalized structure of the dry fruit of Brassica (M.H. Dickson). bud and the environment in which the parent plant has been grown make a considerable difference to success in production of embryos. The breeder must ensure that the numbers of regenerates saved does not become larger than can be handled during evaluation. In most cases, a high percentage of the embryos will be haploid and will double spontaneously providing homozygous plants, saving the necessity of working through several generations of inbreeding. Embryogenesis is an expensive procedure, but the time saved in obtaining homozygous lines compared with using four generations of single seed descent for conventional inbreeding can be well worthwhile. GENOMICS AND MAPPING Crucifers enjoy a pivotal role in developing our understanding of plant genomics and mapping, mainly through the extensive study of Arabidopsis thaliana (thale cress). Arabidopsis has a simple five-chromosome genome with minimal levels of duplication, making it an ideal candidate for the first plant genome project, and a model plant for research, as discussed later in this chapter. This work is of special interest to those working with the Brassica crop species, since they share at least 87% coding sequence conservation with Arabidopsis. Breeding, Genetics and Models 39 Molecular markers have been utilized in several Brassica species (particularly B. oleracea, B. napus and B. rapa), and simple maps have been created using isozyme, RFLP and randomly amplified polymorphic DNA (RAPD) markers (Quiros, 1999). The developments in the Arabidopsis Genome Initiative and expressed sequence tag (EST) databases have subsequently displaced these efforts with more powerful tools for the mapping and understanding of Brassica genomes. The uses of comparative genomics have recently accelerated, providing both comparative maps and consensus genetic markers between the species based on ESTs (Brunel et al., 1999; Lan et al., 2000). The research from Arabidopsis is being used to study the functioning of important genes involved in the horticulture and agronomy of Brassica crop species, including the genes responsible for head formation in cauliflower and broccoli (Lan and Paterson, 2000). While current efforts are complicated by loci duplication within the Brassica crop species (as even the diploid Brassica species are derived from smaller ancestral genomes that are thought to be partial amphidiploids), the research should clarify much of the underlying cytogenetics of Brassica genomes (Cavell et al., 1998). Genomic research is currently focused on several Brassica species, which will demonstrate the lateral use of genomics from Arabidopsis in commercially important crop plants such as oilseed rape (canola), mustard and the vegetable crops. These research foci result in the rapid development of molecular tools that can be utilized by breeders in improving germplasm and cultivars. CALLUS CULTURE, FUSION AND TRANSFORMATION Tissue culture techniques have been applied to several B. oleracea vegetables, either for clonal propagation or for development of novel and sometimes improved plant types. Plants have been regenerated from diverse multicellular explants, including: immature embryos; seedling parts such as hypocotyls or cotyledons; stem pieces; leaves; roots; and floral tissues such as flowering stalks or caulilfower curds. Plants can be recovered from single wall-free protoplasts, usually isolated from leaves or hypocotyls of plantlets grown in vitro. Strong genotype specificity is usually noted in studies comparing regeneration from various lines of a given vegetable, with some performing very well and others showing little or no response. This is particularly true in the case of protoplasts. Plant regeneration from somatic tissues makes it possible to maintain populations that fail to produce seeds and are difficult to propagate using standard methods. Embryo culture has been utilized to recover progeny from interspecific hybrids produced either by sexual crosses (e.g. B. napus ϫ B. oleracea) or by protoplast fusion. 40 Chapter 2 Several vegetable lines with new combinations of nuclear and cytoplasmic genomes have been created by protoplast fusion.