protocols-for-obtaining-zygotic-somatic-embryos-for-studying

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

We have cloned a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) gene from Medicago truncatula (MtSERK1) and examined its expression in culture using real time PCR. In the presence of the auxin 1-naphthaleneacetic acid (NAA) alone, root differentiation occurs from the proliferating calli in both the cultured highly embryogenic seed line (2HA) and a low to nonembryogenic seed line (M. truncatula cv Jemalong). Auxin stimulated MtSERK1 expression in both 2HA and M. truncatula cv Jemalong. Embryo induction in proliferating calli requires a cytokinin in M. truncatula and unlike root formation is substantively induced in 2HA, not M. truncatula cv Jemalong. On embryo induction medium containing NAA and the cytokinin 6-benzylaminopurine (BAP), expression of MtSERK1 is elevated within 2 d of initiation of culture in both M. truncatula cv Jemalong and 2HA. However, MtSERK1 expression is much higher when both NAA and BAP are in the medium. BAP potentiates the NAA induction because MtSERK1 expression is not up-regulated by BAP alone. The 2HA genotype is able to increase its embryo formation because of the way it responds to cytokinin, but not because of the cytokinin effect on MtSERK1. Although the studies with M. truncatula indicate that somatic embryogenesis is associated with high SERK expression, auxin alone does not induce somatic embryogenesis as in carrot (Daucus carota) and Arabidopsis. Auxin in M. truncatula induces roots, and there is a clear up-regulation of MtSERK1. Although our analyses suggest that MtSERK1 is orthologous to AtSERK1, which in Arabidopsis is involved in somatic embryogenesis, in legumes, MtSERK1 may have a broader role in morphogenesis in cultured tissue rather than being specific to somatic embryogenesis.

Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures.

The Medicago truncatula line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than wild-type Jemalong. We have compared proteomes of tissue cultures from leaf explants of these two lines. Both 2HA and Jemalong explants were grown on media containing the auxin 1-naphthaleneacetic acid and the cytokinin 6-benzylaminopurine. Proteins were extracted from the cultures at different time points (2, 5, and 8 weeks), separated by two-dimensional gel electrophoresis, and detected by silver staining. More than 2,000 proteins could be reproducibly resolved and detected on each gel. Statistical analysis showed that 54 protein spots were significantly (P < 0.05) changed in expression (accumulation) during the 8 weeks of culture, and most of these spots were extracted from colloidal Coomassie-stained two-dimensional gel electrophoresis gels and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry analysis. Using a publicly available expressed sequence tag database and the Mascot search engine, we were able to identify 16 differentially expressed proteins. More than 60% of the differentially expressed protein spots had very different patterns of gene expression between 2HA and Jemalong during the 8 weeks of culture.

Proteomic analysis of somatic embryogenesis in Medicago truncatula. Explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments.

Medicago truncatula, a model for legume genomics, can be regenerated by somatic embryogensis by the use of a suitable genotype and an auxin plus cytokinin. The stress response induced by explant wounding and culture is increasingly recognized as an important component of somatic embryo induction. We have cloned and investigated the stress kinase gene MtSK1 in relation to somatic embryogenesis in M. truncatula, using the highly embryogenic mutant Jemalong 2HA (2HA) and its progenitor Jemalong. The main features of the MtSK1 protein of 351 amino acids are an N-terminal kinase domain and a C-terminal glutamic acid-rich region, which is predicted to be a coiled-coil. MtSK1 is a member of the SnRK2 subgroup of the SnRK group of plant kinases. Members of the SnRK2 kinases play a role in stress responses of plants. MtSKI expression is induced by wounding in the cultured tissue independent of auxin or cytokinin. However, in both 2HA and Jemalong, as the callus develops in response to auxin plus cytokinin, MtSK1 expression continues to increase. MtSK1 responds to salt stress in vivo, consistent with its role as a stress kinase. The likely role of MtSK1 in stress-induced signaling will facilitate the relating of stress-response pathways to auxin and cytokinin-induced signaling in the understanding of the molecular mechanisms involved in the induction of somatic embryogenesis in M. truncatula.

The stress kinase gene MtSK1 in Medicago truncatula with particular reference to somatic embryogenesis.

Leaf explants of Medicago truncatula were used to investigate the origins of auxin-induced root formation. On the application of auxin there is some callus formation (not the massive amount that occurs in response to auxin plus cytokinin) and roots appear shortly after the first visible callus. Histological examination reveals morphologically distinctive sheets of callus cells that emanate from the veins of the leaf explants and, within this cell type, root primordia are produced as well as some vascular tissue cells. What is suggested is that the vein-derived cells (VDCs) are procambial-like and function as pluripotent stem cells with a propensity to form root meristems or vascular tissues in response to added auxin. The development of root primordia from these pluripotent cells was clearly up-regulated by the use of the sickle (skl) mutant, which is a mutant impaired in ethylene signal transduction while the wild type and the sunn mutant, defective in auxin polar transport, produced similar numbers of roots. The skl mutant in generating many more roots concomitantly formed fewer vascular tissues. The root meristems differentiate similarly to normal roots producing a central cylinder of vascular tissue, which connects with the leaf explant veins. The VDCs appear to be derived from the cells of or near the phloem. The leaf observations suggest that a pool of stem cells exist in vascular tissue that, in combination with auxin and perhaps other factors, drive a diversity of plant development outcomes that is species specific. The way auxin interacts with other hormones is a key factor in determining the stem cell fate. The histological data in this study also assist in the interpretation of the molecular analysis of auxin-induced root formation in cultured leaves of M. truncatula.

Root meristems in Medicago truncatula tissue culture arise from vascular-derived procambial-like cells in a process regulated by ethylene.

SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes have been demonstrated to play a role in somatic embryogenesis in several plant species. As more is learnt about these genes, the view of their role in plant development has broadened. The Medicago truncatula MtSERK1 gene has been associated with somatic embryogenesis and in vitro root formation. In order to study the role of MtSERK1 in development further, the MtSERK1 promoter sequence has been isolated and cloned into a promoter-GUS analysis vector. SERK1 promoter-driven GUS expression was studied in A. tumefaciens-transformed cultures and regenerated plants, in A. rhizogenes-transformed root clones, and in nodulation. In embryogenic cultures, GUS staining is detected after 2 d of culture at the edge of the explant and around vascular tissue. Expression at the explant edge intensifies over subsequent days and then is lost from the edge as callus formation moves inward. MtSERK1 expression appears to be associated with new callus formation. When somatic embryos form, GUS staining occurs throughout embryo development. Zygotic embryos show expression until the heart stage. The in planta studies reveal a number of interesting expression patterns. There appear to be three types. (i) Expression associated with the primary meristems of the root and shoot and the newly formed meristems of the lateral roots and nodule. (ii) Expression at the junction between one type of tissue or organ and another. (iii) Expression associated with the vascular tissue procambial cells. The data led us to conclude that MtSERK1 expression is associated with developmental change, possibly reflecting cellular reprogramming.

Expression of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) gene is associated with developmental change in the life cycle of the model legume Medicago truncatula.

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula.

Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells.

SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are part of the regulation of diverse signalling events in plants. Current evidence shows SERK proteins function both in developmental and defence signalling pathways, which occur in response to both peptide and steroid ligands. SERKs are generally present as small gene families in plants, with five SERK genes in Arabidopsis. Knowledge gained primarily through work on Arabidopsis SERKs indicates that these proteins probably interact with a wide range of other receptor kinases and form a fundamental part of many essential signalling pathways. The SERK1 gene of the model legume, Medicago truncatula functions in somatic and zygotic embryogenesis, and during many phases of plant development, including nodule and lateral root formation. However, other SERK genes in M. truncatula and other legumes are largely unidentified and their functions unknown.

Characterisation of the legume SERK-NIK gene superfamily including splice variants: implications for development and defence.

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.

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.

The Medicago truncatula 2HA seed line is highly embryogenic while the parental line Jemalong rarely produces embryos. The 2HA line was developed from one of the rare Jemalong regenerates and this method for obtaining a highly regenerable genotype in M. truncatula is readily reproducible suggesting an epigenetic mechanism. Microarray transcriptomic analysis showed down regulation of an ETHYLENE INSENSITIVE 3-like gene in 2HA callus which provided an approach to investigating epigenetic regulation of genes related to ethylene signalling and the 2HA phenotype. Ethylene is involved in many developmental processes including somatic embryogenesis (SE) and is associated with stress responses.

Article	Year
Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures. Plant physiology\| PubMed ID: 12970488	2003
Proteomic analysis of somatic embryogenesis in Medicago truncatula. Explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments. Plant physiology\| PubMed ID: 15749990	2005
The stress kinase gene MtSK1 in Medicago truncatula with particular reference to somatic embryogenesis. Plant cell reports\| PubMed ID: 16518633	2006
Root meristems in Medicago truncatula tissue culture arise from vascular-derived procambial-like cells in a process regulated by ethylene. Journal of experimental botany\| PubMed ID: 16714308	2006
Expression of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) gene is associated with developmental change in the life cycle of the model legume Medicago truncatula. Journal of experimental botany\| PubMed ID: 19305022	2009
Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells. Annals of botany\| PubMed ID: 21224270	2011
Characterisation of the legume SERK-NIK gene superfamily including splice variants: implications for development and defence. BMC plant biology\| PubMed ID: 21385462	2011
Stable transformation of Medicago truncatula cv. Jemalong for gene analysis using Agrobacterium tumefaciens. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 23996317	2013
An unusual abscisic acid and gibberellic acid synergism increases somatic embryogenesis, facilitates its genetic analysis and improves transformation in Medicago truncatula. PloS one\| PubMed ID: 24937316	2014
The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive. BMC plant biology\| PubMed ID: 24952658	2014

Kim E. Nolan

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