Somatic Embryogenesis in the Medicago truncatula Model: Cellular and Molecular Mechanisms

Abstract

Medicago truncatula is now widely regarded as a legume model where there is an increasing range of genomic resources. Highly regenerable lines have been developed from the wild-type Jemalong cultivar, most likely due to epigenetic changes. These lines with high rates of somatic embryogenesis (SE) can be compared with wild-type where SE is rare. Much of the research has been with the high SE genotype Jemalong 2HA (2HA). SE can be induced from leaf tissue explants or isolated mesophyll protoplasts. In 2HA, the exogenous phytohormones 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) are central to SE. However, there are interactions with ethylene, abscisic acid (ABA), and gibberellic acid (GA) which produce maximum SE. In the main, somatic embryos are derived from dedifferentiated cells, undergo organellar changes, and produce stem-like cells. There is evidence that the SE is induced as a result of a stress and hormone interaction and this is discussed. In M. truncatula, there are connections between stress and specific up-regulated genes and specific hormones and up-regulated genes during the SE induction phase. Some of the transcription factors have been knocked down using RNAi to show they are critical for SE induction (MtWUSCHEL, MtSERF1). SE research in M. truncatula has utilized high throughput transcriptomic and proteomic studies and the more detailed investigation of some individual genes. In this review, these studies are integrated to suggest a framework and timeline for some of the key events of SE induction in M. truncatula.

Keywords: Auxin; M. truncatula; cytokinin; ethylene; kinases; somatic embryogenesis; totipotency; transcription factors.

Figure 1

Sequence of steps in somatic embryogenesis. *It has been shown by tracking of a single labeled cell that embryos can develop from single somatic cells (Schmidt et al., 1997). A multicellular origin proposed by Williams and Maheswarin (1986) to occur in some cases has not been unequivocally demonstrated. Haccius (1978) concluded that somatic embryos can derive from a single cell or proembryonal cell complexes which are derived from a single segmenting cell.

Figure 2

Interactions between stress and hormones in somatic embryogenesis. Development of explants from 2HA and wild-type Jemalong. Jemalong leaf explants develop into calli while 2HA explants develop into embryogenic calli capable of producing regenerated plants. Inset shows MtSK1 expression (northern blots) in 2HA explants (E) from leaves (L) cultured for 24 h with and without hormones. Component figures reproduced from Nolan et al. (2006) with permission.

Figure 3

ROS in relation to somatic embryogenesis induction. (A) ROS production in leaf explants. (B) DAB (3,3'-diaminobenzidine) staining for H₂O₂. Bar = 1 mm. (A,B) reproduced with permission from Wang et al., 2011.

Figure 4

Effects of different hormones on somatic embryo induction. (A) MtWUSCHEL expression; (B) MtSERK1 expression; (C) MtSERF1 expression. AVG (10 μM aminoethoxyvinylglycine) and AgNO₃ (10 μM) are ethylene biosynthesis and ethylene perception inhibitors, respectively. Aux = NAA = 10 μM 1-naphthalene acetic acid, Cyt = BAP = 4 μM 6-benzylaminopurine. (A) From Chen et al. (2009), authors’ copyright. (B) From Nolan et al. (2003), www.plantphysiol.org, Copyright American Society of Plant Biologists. (C) From Mantiri et al. (2008a,, www.plantphysiol.org, Copyright American Society of Plant Biologists.

Figure 5

Model for sequence of events in somatic embryogenesis from M. trunctula leaf explants. Excision and plating of the explant produce ROS that probably in interaction with hormones cause chromatin remodeling involving PKL, PRC complexes, and TRITHORAX genes, and a stress kinase that maybe linked to transcript degradation via RNA processing bodies. Cell divisions follow and callus is produced. Cytokinin-dependent WUS expression is essential for SE and there is an initial expression analogous to that occurring in the ovule followed by stem cell development linked to PEMs in patches on the callus, correlating with CLV3 and STM expression. AGL15, SERK, and SERF are part of the correct hormone milieu leading to expression of BBM and LEC genes and the embryogenic program. The changes diagramed go hand in hand with hormonal changes. Auxin is initially high then lowers as differentiation starts, followed by auxin regulation associated with embryo patterning in the formation of the bipolar embryo. Cytokinin is important for specific genes and the cell cycle. Endogenous ethylene is produced and endogenous ABA/GA ratios specific to M. truncatula influence SE. Ethylene, ABA, and GA can be involved in regulating auxin responses. Abbreviations are in the text.