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JXB Advance Access originally published online on June 13, 2008
Journal of Experimental Botany 2008 59(10):2857-2873; doi:10.1093/jxb/ern149
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Isolation of an embryogenic line from non-embryogenic Brassica napus cv. Westar through microspore embryogenesis

Meghna R. Malik1, Feng Wang1 *, Joan M. Dirpaul1, Ning Zhou1 {dagger}, Joe Hammerlindl1, Wilf Keller1, Suzanne R. Abrams1, Alison M. R. Ferrie1 and Joan E. Krochko1,{ddagger}

1Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9

{ddagger} Corresponding author. E-mail: Joan.Krochko{at}nrc-cnrc.gc.ca

Brassica napus cultivar Westar is non-embryogenic under all standard protocols for induction of microspore embryogenesis; however, the rare embryos produced in Westar microspore cultures, induced with added brassinosteroids, were found to develop into heritably stable embryogenic lines after chromosome doubling. One of the Westar-derived doubled haploid (DH) lines, DH-2, produced up to 30% the number of embryos as the highly embryogenic B. napus line, Topas DH4079. Expression analysis of marker genes for embryogenesis in Westar and the derived DH-2 line, using real-time reverse transcription-PCR, revealed that the timely expression of embryogenesis-related genes such as LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3, and BABY BOOM1, and an accompanying down-regulation of pollen-related transcripts, were associated with commitment to embryo development in Brassica microspores. Microarray comparisons of 7 d cultures of Westar and Westar DH-2, using a B. napus seed-focused cDNA array (10 642 unigenes), identified highly expressed genes related to protein synthesis, translation, and response to stimulus (Gene Ontology) in the embryogenic DH-2 microspore-derived cell cultures. In contrast, transcripts for pollen-expressed genes were predominant in the recalcitrant Westar microspores. Besides being embryogenic, DH-2 plants showed alterations in morphology and architecture as compared with Westar, for example epinastic leaves, non-abscised petals, pale flower colour, and longer lateral branches. Auxin, cytokinin, and abscisic acid (ABA) profiles in young leaves, mature leaves, and inflorescences of Westar and DH-2 revealed no significant differences that could account for the alterations in embryogenic potential or phenotype. Various mechanisms accounting for the increased capacity for embryogenesis in Westar-derived DH lines are considered.

Key words: Brassica napus, embryogenesis, microarray, microspore, transcript profiling

* Present address: National Institute for Nanotechnology, National Research Council of Canada, 11421 Saskatchewan Drive, Edmonton, Alberta, Canada T6G 2M9

{dagger} Present address: Dow AgroSciences, 9330 Zionsville Rd., Indianapolis, IN 46268, USA

Received 13 February 2008; Revised 25 April 2008 Accepted 29 April 2008


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