Kim E. Nolan

Auxin Up-Regulates MtSERK1 Expression in Both Medicago truncatula Root-Forming and Embryogenic Cultures

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.

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

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.

Regeneration of Medicago truncatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants

Plant regeneration has been achieved by somatic embryogenesis in Medicago truncatula Gaertn. (barrel medic) c.v. Jemalong, an annual legume species. Regenerated plants were obtained from cultured leaf tissue explants on a four-step modified B5 basal medium. Induction of embryo formation occurred on a medium containing 10 μM NAA and 10 μM BAP, and embryo maturation was promoted after transfer to a medium containing 1 μM NAA and 10 μM BAP. Shoot development, secondary somatic embryogenesis and occasional plantlet development occurred on a subsequent transfer to 0.1 μM NAA and 1 μM BAP. Plantlet formation could also be completed by transfer of well developed shoots to 0.05 μM NAA. A high frequency of primary somatic embryos could only be obtained by using the same culture protocol with tissue from regenerated plants. Explants from regenerated plants showed a large increase in the number of primary embryos per callus and the number of calli producing embryos. Explants from plants derived from the seed of one regenerated plant also showed increased embryo formation. Although high embryo formation rates can be reproducibly obtained from this seed, embryo conversion rates to plants are currently low.

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

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.

Genetic transformation of Medicago truncatula using Agrobacterium with genetically modified Ri and disarmed Ti plasmids

SummaryFertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R1 progeny.

The Development of the Highly Regenerable Seed Line Jemalong 2HA for Transformation of Medicago truncatula — Implications for Regenerability via Somatic Embryogenesis

Summary Medicago truncatula is an annual legume that can be regenerated from tissue explants via somatic embryogenesis at low frequency using the cultivar Jemalong. Regenerated plants from Jemalong show very large increases in somatic embryogenesis. This large increase in somatic embryogenesis is inherited with progeny segregating into classes we categorised as high, moderate and low regenerators. The low regenerators have a similar frequency of somatic embryogenesis to Jemalong. Continued selection from the high regenerators allowed the development of the highly regenerable seed line Jemalong 2HA in which the progeny are all high regenerators. Jemalong 2HA is isogenic with Jemalong except for the enhanced somatic embryogenesis in Jemalong 2HA. A cycle of tissue culture resulting in an increase in somatic embryogenesis, may be a tool that can increase somatic embryogenesis in some recalcitrant species. Jemalong 2HA can be described as a super-embryogenic mutant and is valuable not only for its ease of transformation but also can provide insights into the nature of totipotency.

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

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.

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

BACKGROUND AND AIMS 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. METHODS Callus development was examined from cultured leaf explants of the highly regenerable genotype Jemalong 2HA (2HA) and from mesophyll protoplasts of 2HA and wild-type Jemalong. Callus development was studied by histology, manipulation of the culture system, detection of early production of reactive oxygen species and visualization of SERK1 (SOMATIC EMBRYO RECEPTOR KINASE1) gene expression. KEY RESULTS Callus formation in leaf explants initiates at the cut surface and within veins of the explant. The ontogeny of callus development is dominated by the division and differentiation of cells derived from pluripotent procambial cells and from dedifferentiated mesophyll cells. Procambium-derived cells differentiated into vascular tissue and rarely formed somatic embryos, whereas dedifferentiated mesophyll cells were competent to form somatic embryos. Interestingly, explants incubated adaxial-side down had substantially less cell proliferation associated with veins yet produced similar numbers of somatic embryos to explants incubated abaxial-side down. Somatic embryos mostly formed on the explant surface originally in contact with the medium, while in protoplast microcalli, somatic embryos only fully developed once at the surface of the callus. Mesophyll protoplasts of 2HA formed embryogenic callus while Jemalong mesophyll protoplasts produced callus rich in vasculature. CONCLUSIONS The ontogeny of embryogenic callus in M. truncatula relates to explant orientation and is driven by the dynamics of pluripotent procambial cells, which proliferate and differentiate into vasculature. The ontogeny is also related to de-differentiated mesophyll cells that acquire totipotency and form the majority of embryos. This contrasts with other species where totipotent embryo-forming initials mostly originate from procambial cells.

Developmental Biology of Somatic Embryogenesis

Somatic embryogenesis (SE) is a remarkable developmental process enabling nonzygotic plant cells to form embryos and, ultimately, fertile plants. It is an expression of totipotency. This chapter initially considers the genotypic component and the progenitor stem cells where SE is induced to form the initial asymmetric division of the somatic embryogenesis program. These cells are part of a stem cell niche dependent on the surrounding cells. Recent evidence is discussed that, before the SE pathway can be initiated, a GA-modulated pathway that represses inappropriate embryogenesis needs to be derepressed. The current understanding of how stress and hormones induce the activation of specific SE genes is examined. Important stress components are reactive oxygen species and the signalling of stress-related hormones. The action of the key developmental hormones auxin and cytokinin in relation to developmental genes is considered and, based on current understanding, a model is presented for the mechanism of SE. While there are many SE applications in contemporary biotechnology, understanding the reprogramming process associated with SE remains an important question for developmental biology.

INVITED REVIEW: GENETIC REGULATION OF SOMATIC EMBRYOGENESIS WITH PARTICULAR REFERENCE TO ARABIDOPSIS THALIANA AND MEDICAGO TRUNCATULA

SummaryInvestigations into the mechanisms of somatic embryogenesis (SE) have largely focused on the hormonal regulation of the process and a repertoire of strategies has been developed to regenerate many species via SE. However, the genes that regulate the induction and development of somatic embryos have not been defined. In the recent times, regeneration via overexpression of genes, such as WUSCHEL or LEAFY COTYLEDON, in Arabidopsis has started to provide a basis for understanding the genes involved in SE. This has gone hand in hand with the availability of genome sequence information and the availability of mutants in model plants such as Arabidopsis and Medicago. An improved understanding of zygotic embryogenesis and the maintenance and differentiation of stem cells in the shoot meristem also helps to provide novel insights into the mechanisms of SE. This review examines the current understanding of the genetic regulation of SE in the context of current molecular understanding of plant development.