Isolation of an embryogenic line from non-embryogenic Brassica napus cv. Westar through microspore embryogenesis.
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.
Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada.
Meghna R
MR
Malik
Feng
F
Wang
Joan M
JM
Dirpaul
Ning
N
Zhou
Joe
J
Hammerlindl
Wilf
W
Keller
Suzanne R
SR
Abrams
Alison M R
AM
Ferrie
Joan E
JE
Krochko
D029688:Brassica napus / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D001947:Breeding; D035264:Flowers / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D010937:Plant Growth Regulators / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D011058:Pollen / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D010937:Plant Growth Regulators; D010940:Plant Proteins