The Application of Ooubled Haploid Plants to Population ... - MSpace

2.12
maturation and plant regeneration (Tayfor et al., 1993). Crop impmvements through
gene transfer (Huang, 1992) and mutagenesis of haploid cells have also been shown
(Swanson et. aL.1989; Huang, 1992).
In Brassica. DH plants are usually produced through tissue culture of male
gametes or microspores (Feme and Keller, 1995). Microspore culture in Bmsic6 was
first demonstrated by Lichter (1982). The procedure used to produce OH plants in
canola involves the seledion of buds containing late uninucleate to early binucieate
microspores which have not undergone first pollen mitosis (Keller et al.. 1975; Pechan
and Keller. 1988). At this stage, microspores have the ability to switch fiom
gametophytic to sporophytic control by undergoing symmetric instead of asymmetric
division which occurs in vivo to promote gametogenesis (Zaki and Dickinson. 1990;
1991 ). Microspores can be characterized cytologically (Kott et al., 1988a) or using flow
cytometry (Fuchs and Pauls, 1992). Buds are macerated to release microspores which
are then cultured in a liquid medium to recover haploid embiyos. Haploid plants are
then regenerated frorn these embryos and chromosomes are doubled by submersing
the plant roots in a diluted solution containing an antimicrotubule agent, such as
colchicine. Colchicine has been applied in the microspore stage of culture to double
chromosomes and even enhance embryogenesis in B. napus (Iqbal et al., 1994; Zaki
and Dickinson, 1995; Zhao et al., 1996) and in anther culture of wheat (Barnabas et al..
1991 ; Navarro-Alvarez et al.. 1994). When applied prior to the first pollen mitosis
colchicine ptomotes symmetric cell division. Application of colchicine at the single cell
stage avoids chimenc expression of different ploidy levels and associated poor seed