Microspores cultured in vitro can be reprogrammed to divide and produce a bipolar embryo. The reaction to stress treatment is a signal for inducing the sporophytic pathway, preventing the development of fertile pollen grain ? the gametophytic pathway. The ultimate goal is to convert each microspore from a heterozygous F1 plant to a doubled haploid plant so that a population of doubled haploids fully represents the genetic variability of the preceding meiosis.
A single microspore cultured in vitro can be reprogrammed from the development of pollen to divisions and production of a bipolar embryo. The final effect of androgenesis in vitro is conversion of each embryo derived from microspore of a heterozygous plant into a doubled haploid plant in such a way that a population of doubled haploids fully represents the genetic variability of the preceding meiosis. DH lines have been already widely used in oilseed rape breeding. Doubled haploids are used by breeders in crossing programs in order to obtain desirable variability in progeny, and also as a part of conventional breeding. Moreover, doubled haploids in conjunction with molecular markers helped to develop the so called 'molecular breeding'.
The fatty acid composition of oil of the zero erucic acid commercial Brassica napus L. is typical for this species. It is rich in oleic acid and contains moderate levels of linoleic and linolenic acid. For human nutrition, it is advantageous primarily to obtain the highest possible content of oleic acid and to maintain the 2:1 ratio of linoleic to linolenic acid, while preserving the average total content of saturated acids. Uni- and multivariate analyses of variance were used for evaluation of doubled haploid lines of winter oilseed rape in respect of five fatty acids: palmitic (C16:0), stearic (C18:0) oleic (C18:1), linoleic (C18:2) and linolenic (C18:3). Some proposals of studying doubled haploid (DH) lines with the use of canonical transformation were also given. In MANOVA, the five original variables (individual fatty acids) were replaced by three 'new' variables (combinations of these acids) and used to evaluate DH lines with respect to the equirements concerning the nutritional role of fatty acids. The first variable was the total content of the saturated acids (C16:0 + C18:0), the second (unchanging) was the content of the monounsaturated acid C18:1, and the third was the difference between polyunsaturated acids, i.e. between linoleic acid, and the doubled content of linolenic acid (C18:2 ? 2 ? C18:3).
Haploid microspore-derived embryos (MDEs) constitute a unique material for the introduction of new traits into winter oilseed rape (Brassica napus). MDEs have been transformed by using Agrobacterium tumefaciens strains EHA105 and LBA4404, both carrying the binary vector pKGIB containing the uidA gene encoding -glucuronidase (GUS) and the bar gene as a marker of resistance to phosphinotricin. Transformed embryos expressed GUS and regenerated plants that were resistant to herbicide Basta, as confirmed by a leaf-painting test. Progeny plants of the transformant T-39 were all transgenic, as they inherited T-DNA from their doubled haploid parental plant. Southern-blot analysis confirmed the integration and transmission of T-DNA into T1 plants. Transformation of MDEs facilitates the obtaining of winter oilseed rape homozygous for the introduced genes.
Microspore culture in conjuction with other technologies such as selection, mutagenesis and transformation has been used for the production of novel genotypes of Brassica napus L. for crop improvement. The example of in vitro selection of microspore - derived embryos includes: a) ploidy level, b) seed oil composition (for example: high level of erucic acid), c) genotypes with restorer gene for CMS-ogura system (by means of isozyme marker PGI-2 ), d) herbicide resistant forms. Efficiency of microspore mutagnesis has been tested by the treatment of freshly isolated microspores with UV and MNU. Direct delivery of foreign gene to the microspores (microprojectile bombardment) combined with the use of Agrobacterium tumefaciens to microspore derived embryos seems to be a promising way of oilseed rape transformation.
Application of doubled-haploid (DH) techniques combined with a method of early selection for erucic acid in cotyledons of microspore-derived embryos (MDEs) from three hybrids of winter oilseed rape (high erucic acid and low glucosinolates) was described. Comparisons of erucic acid content between MDEs and seeds from the same homozygous line DH-Gr85 demonstrated that seed-specific fatty acid composition can be fully stimulated in MDEs after 18 days of culture on the NLN-medium with 0.8% agar and 2% sucrose with or without 10 mM ABA. The erucic acid content (percentage of the total fatty acid content) of cotyledons of embryos from three hybrids and of the seeds derived from plants regenerated from the remaining parts of the embryos were highly correlated (r = 0.78**).
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