Abstract
Chromosome doubling of microspore-derived plants is an important factor in the practical application of microspore culture technology because breeding programs require a large number of genetically stable, homozygous doubled haploid plants with a high level of fertility. In the present paper, 29 populations of microspore-derived plantlets from cabbage (Brassica oleracea var. capitata) and broccoli (Brassica oleracea var. italica) were used to study the ploidy level and spontaneous chromosome doubling of these populations, the artificial chromosome doubling induced by colchicine, and the influence of tissue culture duration on the chromosomal ploidy of the microspore-derived regenerants. Spontaneous chromosome doubling occurred randomly and was genotype dependent. In the plant populations derived from microspores, there were haploids, diploids, and even a low frequency of polyploids and mixed-ploidy plantlets. The total spontaneous doubling in the 14 cabbage populations ranged from 0 to 76.9%, compared with 52.2 to 100% in the 15 broccoli populations. To improve the rate of chromosome doubling, an efficient and reliable artificial chromosome doubling protocol (i.e., the immersion of haploid plantlet roots in a colchicine solution) was developed for cabbage and broccoli microspore-derived haploids. The optimal chromosome doubling of the haploids was obtained with a solution of 0.2% colchicine for 9–12 h or 0.4% colchicine for 3–9 h for cabbage and 0.05% colchicine for 6–12 h for broccoli. This protocol produced chromosome doubling in over 50% of the haploid genotypes for most of the populations derived from cabbage and broccoli. Notably, after 1 or more years in tissue culture, the chromosomes of the haploids were doubled, and most of the haploids turned into doubled haploid or mixed-ploidy plants. This is the first report indicating that tissue culture duration can change the chromosomal ploidy of microspore-derived regenerants.
Highlights
Microspore culture is an effective alternative technique for the production of doubled haploid (DH) parental lines to generate F1 hybrids (Abercrombie et al, 2005)
The chromosome doubling of haploids derived from microspores is an important step in the practical application of microspore culture technology
Our objectives were to ascertain the spontaneous doubling of the populations, to assess the impact of tissue culture duration on chromosome doubling in the populations and to develop an efficient and reliable artificial chromosome doubling protocol for microspore-derived haploids of cabbage and broccoli
Summary
Microspore culture is an effective alternative technique for the production of doubled haploid (DH) parental lines to generate F1 hybrids (Abercrombie et al, 2005). DH lines can be used for marker identification, gene mapping and various genetic manipulations (Forster et al, 2007; Ferrie and Möllers, 2011; Ferrie and Caswell, 2011). This technique has been successfully used in cabbage and broccoli, and large-scale DH lines have been developed (Cao et al, 1990; Takahata and Keller, 1991; Duijs et al, 1992; Hansen, 1994; Pink, 1999; da Silva Dias, 2003; Yuan et al, 2009, 2011, 2012). Microspore-derived haploids can spontaneously double their chromosomes during the very early stages of embryogenesis or can be induced to become DHs in the later stages of development (Palmer et al, 1996)
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