Induction and mechanism of Brassica napus cv. Topas microspore embryogenesis

Brassica napus cv. Topas microspores develop into mature pollen in vivo, or in vitro at 25{dollar}{bsol}sp{bsol}circ{dollar}C. However, when cultured at 32.5{dollar}{bsol}sp{bsol}circ{dollar}C for 24 h prior to 25{dollar}{bsol}sp{bsol}circ{dollar}C this normal developmental pathway is blocked and replaced by haploid embryo formation. Haploid embryos have great potential in plant breeding programs and other biotechnological applications.; Embryogenesis of B. napus microspores is conventionally induced with heat shock. The mechanism that triggers the induction of embryogenesis is not clear although heat shock proteins (HSPs) have been proposed to play a role during the induction process. A novel approach of inducing embryogenesis using an anti-microtubule agent, colchicine, without heat has been developed. Embryogenic frequencies of more than 15% were obtained from microspores cultured in 25 {dollar}{bsol}mu{dollar}M colchicine for 42 h at 25{dollar}{bsol}sp{bsol}circ{dollar}C. The microspore developmental stages most responsive to colchicine are unicellular vacuolate and late unicellular, somewhat earlier stages than the population responsive to heat induction. This approach offers an ideal system to discriminate factors specific to the heat-shock response and those associated with induction of embryogenesis thereby to evaluate the role of HSPs. The ability of colchicine to induce embryogenesis suggests that disruption and reorganization of microtubules is sufficient to alter the developmental fate of microspores. Therefore the importance of HSPs during induction of microspore embryogenesis is in question.; Colchicine is known to be a microtubule depolymerizer and a chromosome doubling agent. An additional advantage of the colchicine induction is that 90% of the plants recovered from colchicine-induced embryos were doubled haploids, compared to 6% from heat-induced embryos. This one-step process to simultaneously induce embryogenesis and chromosome doubling offers a simple, time, labour and cost saving approach to produce fertile plants which is very advantageous for plant breeding programs, genetic analysis and other biotechnological applications.; Prior to this study, HSPs were believed to control the developmental switch from gametogenesis to embryogenesis in model systems of B. napus and Nicotiana tabacum. The colchicine-induced embryogenesis allowed us to reexamine this hypothesis. The present study demonstrates that the HSPs investigated are not required for embryogenesis from B. napus cv. Topas microspores. HSP19 is not expressed nor is expression of HSP70 elevated during embryo induction with colchicine, indicating that neither HSP is required for colchicine induction of embryogenesis and the presence of HSPs in the heat-induced cells may be the cell's natural response to heat shock. Protein banding patterns observed on SDS-PAGE appear to be similar at different unicellular stages in vivo. Extra bands appear in early and late bicellular pollen and a different banding pattern is evident in mature pollen. Similar patterns are seen from in vitro pollen cultured at 25{dollar}{bsol}sp{bsol}circ{dollar}C after 42 h and 96 h of incubation. This change in protein expression does not occur in samples induced to undergo embryogenesis with either heat or colchicine treatments. Thus, the extra protein bands shown at later stages of microspore development in vivo or in vitro at 25{dollar}{bsol}sp{bsol}circ{dollar}C represent the pollen-specific proteins and the expression of these proteins are inhibited under embryogenic conditions.; The data present evidence to support the hypotheses proposed for this project: the HSPs investigated are not required for the induction of microspore embryogenesis and inhibition of pollen-specific gene expression and cytoskeletal reorganizations are most likely the true mechanisms that regulate the induction of microspore embryogenesis.