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Published on March 23, 2007; 10.1104/pp.106.092932

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Received November 16, 2006
Accepted March 10, 2007

Transcript Profiling and Identification of Molecular Markers for Early Microspore Embryogenesis in Brassica napus

Meghna R Malik , Feng Wang , Joan M Dirpaul , Ning Zhou , Patricia L Polowick , Alison MR Ferrie , and Joan E Krochko *

Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9

* Corresponding author; email: Joan.Krochko{at}nrc-cnrc.gc.ca.

Isolated microspores of Brassica napus are developmentally programmed to form gametes; however, microspores can be reprogrammed through stress treatments to undergo appropriate divisions and form embryos. We are interested in the identification and isolation of factors and genes associated with the induction and establishment of embryogenesis in isolated microspores. Standard and normalized cDNA libraries, as well as subtractive cDNA libraries, were constructed from freshly isolated microspores (0h) and microspores cultured for 3d, 5d or 7d under embryogenesis-inducing conditions. Library comparison tools were used to identify shifts in metabolism across this time-course. Detailed EST analyses of 3d and 5d cultures indicate that most sequences are related to pollen-specific genes. However, semi-quantitative and real-time RT-PCR analyses at the initial stages of embryo induction also reveal expression of embryogenesis-related genes such as BBM1, LEC1 and LEC2 as early as 2d to 3d of microspore culture. Sequencing results suggest that embryogenesis is clearly established in a subset of the microspores by 7d of culture and that this time point is optimal for isolation of embryo-specific ESTs such as ABI3, ATS1, LEC1, LEC2 and FUS3. Following extensive PCR-based expression profiling, 16 genes were identified as unequivocal molecular markers for microspore embryogenesis in B. napus. These molecular marker genes also show expression during zygotic embryogenesis, underscoring the common developmental pathways that function in zygotic and gametic embryogenesis. The quantitative expression values of several of these molecular marker genes are shown to be predictive of embryogenic potential in B. napus cultivars (eg. Topas DH4079, Allons, Westar, Garrison).




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