Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume <em>Medicago truncatula</em>

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

University of Newcastle, Callaghan, New South Wales, Australia

Leymus chinensis is a dominant species in the Inner Mongolia steppe, northern China. Plant growth in northern China grassland is often limited by low soil nitrogen availability. The objective of this study is to investigate whether rhizomes of Leymus chinensis are involved in the contribution of N uptake. The N concentration, (15)N concentration and (15)N proportion in roots, rhizomes and shoots after 48 h exposure of roots (L(root)) and rhizomes (L(rhizo)) separately and roots and rhizomes together (L(r+r)) to 0.1 mM (15)NH (4)(15)NO(3) solution were measured using root-splitting equipment and stable isotope ((15)N) techniques, respectively. The N content and dry mass were not affected by the labeling treatment. In contrast, the (15)N concentration in shoots, rhizomes and roots was significantly increased by the labeling in rhizomes, indicating that the inorganic nitrogen was absorbed via rhizomes from the solution and can be transported to other tissues, with preference to shoots rather than roots. Meanwhile, the absolute N absorption and translocation among compartments were also calculated. The N absorption via rhizomes was much smaller than via roots; however, the uptake efficiency per surface unit via rhizomes was greater than via roots. The capacity and high efficiency to absorb N nutrient via rhizomes enable plants to use transient nutrient supplies in the top soil surface.

Absorption and translocation of nitrogen in rhizomes of Leymus chinensis.

• The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.

From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula.

In wheat, tillering and water-soluble carbohydrates (WSCs) in the stem are potential traits for adaptation to different environments and are of interest as targets for selective breeding. This study investigated the observation that a high stem WSC concentration (WSCc) is often related to low tillering. The proposition tested was that stem WSC accumulation is plant density dependent and could be an emergent property of tillering, whether driven by genotype or by environment. A small subset of recombinant inbred lines (RILs) contrasting for tillering was grown at different plant densities or on different sowing dates in multiple field experiments. Both tillering and WSCc were highly influenced by the environment, with a smaller, distinct genotypic component; the genotype × environment range covered 350-750 stems m(-2) and 25-210 mg g(-1) WSCc. Stem WSCc was inversely related to stem number m(-2), but genotypic rankings for stem WSCc persisted when RILs were compared at similar stem density. Low tillering-high WSCc RILs had similar leaf area index, larger individual leaves, and stems with larger internode cross-section and wall area when compared with high tillering-low WSCc RILs. The maximum number of stems per plant was positively associated with growth and relative growth rate per plant, tillering rate and duration, and also, in some treatments, with leaf appearance rate and final leaf number. A common threshold of the red:far red ratio (0.39-0.44; standard error of the difference=0.055) coincided with the maximum stem number per plant across genotypes and plant densities, and could be effectively used in crop simulation modelling as a 'cut-off' rule for tillering. The relationship between tillering, WSCc, and their component traits, as well as the possible implications for crop simulation and breeding, is discussed.

Developmental and growth controls of tillering and water-soluble carbohydrate accumulation in contrasting wheat (Triticum aestivum L.) genotypes: can we dissect them?

Medicago truncatula is a model legume that has all the genomic resources to be suitable as a model for functional genomics. Transformation to produce transgenic plants is part of this toolkit, enabling a spectrum of approaches to study gene function: including knockdown, overexpression, reporter genes for gene expression, and proteins tagged with fluorescent proteins such as GFP. A special genetic line is necessary for transformation and Jemalong 2HA derived from cv. Jemalong is used in the methods described. Leaf explants can be used for the transformation of the embryonic stem cells to produce the transgenic somatic embryos for regeneration. An auxin and a cytokinin are the key hormone requirements for regeneration by somatic embryogenesis but other hormones such as abscisic acid can be used to augment the system. As the explants used in this system are from leaves, rather than immature embryos or meristematic tissues often used in other species, it is a quite straightforward system. Agrobacterium tumefaciens containing a binary vector suitable for the particular objectives is used to deliver the transgene of interest.

Stable transformation of Medicago truncatula cv. Jemalong for gene analysis using Agrobacterium tumefaciens.

Cultivated legumes account for more than a quarter of primary crop production worldwide. The protein- and oil-rich seed of cultivated legumes provides around one-third of the protein in the average human diet, with soybeans (Glycine max (L.) Merr) being the single largest source of vegetable oil. Despite their critical importance to human and animal nutrition, we lack an understanding of how early seed development in legumes is orchestrated at the transcriptional level. We developed a method to isolate ovules from the model legume, Medicago truncatula Gaertn, at specific stages of embryogenesis, on the basis of flower and pod morphology. Using these isolated ovules we profiled the expression of candidate homeobox, AP2 domain and B3 domain-containing transcription factors. These genes were identified by available information and sequence homology, and five distinctive patterns of transcription were found that correlated with specific stages of early seed growth and development. Co-expression of some genes could be related to common regulatory sequences in the promoter or 3'-UTR regions. These expression patterns were also related to the expression of B3-domain transcription factors important in seed filling (MtFUS3-like and MtABI3-like). Localisation of gene expression by promoter-GUS fusions or in situ hybridisation aided understanding of the role of the transcription factors. This study provides a framework to enhance the understanding of the integrated transcriptional regulation of legume embryo growth and development and seed filling.

Transcriptional regulation of early embryo development in the model legume Medicago truncatula.

Somatic embryogenesis (SE) can be readily induced in leaf explants of the Jemalong 2HA genotype of the model legume Medicago truncatula by auxin and cytokinin, but rarely in wild-type Jemalong. Gibberellic acid (GA), a hormone not included in the medium, appears to act in Arabidopsis as a repressor of the embryonic state such that low ABA (abscisic acid): GA ratios will inhibit SE. It was important to evaluate the GA effect in M. truncatula in order to formulate generic SE mechanisms, given the Arabidopsis information. It was surprising to find that low ABA:GA ratios in M. truncatula acted synergistically to stimulate SE. The unusual synergism between GA and ABA in inducing SE has utility in improving SE for regeneration and transformation in M. truncatula. Expression of genes previously shown to be important in M. truncatula SE was not increased. In investigating genes previously studied in GA investigations of Arabidopsis SE, there was increased expression of GA2ox and decreased expression of PICKLE, a negative regulator of SE in Arabidopsis. We suggest that in M. truncatula there are different ABA:GA ratios required for down-regulating the PICKLE gene, a repressor of the embryonic state. In M. truncatula it is a low ABA:GA ratio while in Arabidopsis it is a high ABA:GA ratio. In different species the expression of key genes is probably related to differences in how the hormone networks optimise their expression.

An unusual abscisic acid and gibberellic acid synergism increases somatic embryogenesis, facilitates its genetic analysis and improves transformation in Medicago truncatula.

Youhong Song

School of Environmental and Life Sciences

University of Newcastle, Callaghan, New South Wales, Australia

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Youhong Song

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University of Newcastle, Callaghan, New South Wales, Australia

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

School of Environmental and Life Sciences