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.

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.

Medicago truncatula is used as a pasture legume and a source of nitrogen for grain crops in southern Australia. Alfalfa mosaic virus (AMV) infection reduces herbage production and nodulation. The coat protein gene of a South Australian strain of AMV (AMV N20) has been transferred to Medicago truncatula cv. Jemalong 2HA using Agrobacterium-mediated transformation. The most detailed investigations were carried out with the coat protein gene in the sense orientation (CP+). Progeny (T1, T2, T3) raised from self-pollinated primary transformants (T0) containing the coat protein CP+ gene were resistant to AMV. Based on Southern analysis and segregation, the transformants contained a single gene copy. In the T3 generation, one line was immune and one line showed resistance to AMV N20. The immune line contained no detectable virus when plant sap from either inoculated or systemic leaves was bioassayed on Phaseolus vulgaris. This line was also immune to the heterologous AMV S40 isolate. A line with the coat protein gene in antisense orientation (CP–) showed delayed systemic infection but was not immune. We conclude that coat protein mediated protection (CPMP) is an effective strategy for controlling AMV infection and should be further evaluated in the field.

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.

Medicago truncatula (Jemalong 2HA) can be regenerated by somatic embryogenesis utilising 1-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP). There is a requirement for both NAA and BAP for callus induction and embryo formation. There is no requirement for a drop in auxin concentration to induce embryos. Abscisic acid (ABA) when present with NAA and BAP during embryo formation at a concentration of 1 µM, increases the number of embryos per callus. The ABA treatment stimulates embryo numbers in both light and darkness. The conversion efficiency of embryo to plant is unchanged irrespective of the presence of ABA during embryo formation, indicating that ABA does not improve the regeneration of the embryos once formed. Importantly, the presence of light in the embryo formation period causes a marked inhibition of embryo conversion.

Fertile 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.

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.