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 (0 h) and microspores cultured for 3, 5, or 7 d under embryogenesis-inducing conditions. Library comparison tools were used to identify shifts in metabolism across this time course. Detailed expressed sequence tag analyses of 3 and 5 d cultures indicate that most sequences are related to pollen-specific genes. However, semiquantitative and real-time reverse transcription-polymerase chain reaction analyses at the initial stages of embryo induction also reveal expression of embryogenesis-related genes such as BABYBOOM1, LEAFY COTYLEDON1 (LEC1), and LEC2 as early as 2 to 3 d of microspore culture. Sequencing results suggest that embryogenesis is clearly established in a subset of the microspores by 7 d of culture and that this time point is optimal for isolation of embryo-specific expressed sequence tags such as ABSCISIC ACID INSENSITIVE3, ATS1, LEC1, LEC2, and FUSCA3. Following extensive polymerase chain reaction-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 (e

The fatty acid elongase [often designated FAE or b-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium b-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.Very long chain fatty acids (VLCFAs) with 20 carbons or more are widely distributed in nature. In plants, they are mainly found in epicuticular waxes and in the seed oils of a number of plant species, including members of the Brassicaceae, Limnantheceae, Simmondsia, and Tropaeolaceae (Harwood, 1996;Post-Beittenmiller, 1996;Ghanevati and Jaworski, 2001). A strategic goal in oilseed modification is to genetically manipulate high erucic acid germ plasm of the Brassicaceae to increase the content of erucic acid (22:1 D13) and other strategic VLCFAs in the seed oil for industrial niche market needs (Puyaubert et al., 2001;Taylor et al., 2001). Erucic acid and its derivatives are feedstocks in manufacturing slip-promoting agents, surfactants, plasticizers, nylon 1313, and surface coatings; more than 1,000 patents have been issued (Sonntag, 1991(Sonntag, , 1995Leonard, 1994). The current market for high erucate oils exceeds $120 million U.S./annum. Worldwide erucic acid demand is predicted to increase from about 40 million pounds (M pds) in 1990 to about 80 M pds by the year 2010. Similarly, demand for the derivative, behenic acid, is predicted to triple to about 102 M pds by 2010 (Sonntag, 1995). In recent years, production has increased to meet market needs, and high erucic acreage in western Canada is currently at a record high (D. Males, Saskatchewan...

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 embryogenesisrelated 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 seedfocused 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.

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