A wide array of new molecular biology and genetic manipulation techniques are available that give plant breeders new opportunities to create and manipulate genetic variation to produce improved plants more easily. This chapter discusses the role of biotechnology in Lotus improvement. It considers what the objectives in a breeding program are and how they may be achieved using traditional breeding methods and if not, whether biotechnology may assist the breeder. The primary requirements of any breeding program are two fold: a source of variation and the means to manipulate that variation. The chapter also considers the current situation for Lotus tissue culture and transformation and then discusses possible applications for crop improvement. Regeneration systems have been developed in Lotus species for a variety of reasons, including maintenance of selected genotypes, induction of somaclonal variation, and production of transformed plants or somatic hybrids.

Currently, significant academic and industrial activity is focused on sourcing feed stocks from non-food biomass crops for the sustainable production of energy, power and chemical products. Crops identified as suitable for Northern Europe include Miscanthus, switchgrass (Panicum virgatum), reed canary grass (Phalaris arundinacea) and short rotation coppice willow and poplar (Salix and Populus spp.). All of these crops provide biomass that is amenable for conversion by thermochemical processes i.e. those based on heat and pressure. There are concerns that for some processes the conversion efficiency of biomass is poor compared with coal and oil due to comparatively low energy density, high moisture content, and poor storage and handling properties. Many of these parameters can be improved by pre-processing feed stock materials prior to their conversion. We examine the energy crop species that are suitable for Northern Europe; discuss the processes of combustion, gasification and pyrolysis, and explore how differences in chemical composition influence conversion efficiency. Finally, we review biomass upgrading (pelletisation, torrefaction and treatment with sub-critical (hydrothermal upgrading) and with supercritical water).

Proanthocyanidins (PAs) are agronomically important biopolymers in higher plants composed primarily of catechin and epicatechin units. The biosynthesis of these natural products is regulated by transcription factors including proteins of the R2R3MYB class. To gain insight into the genetic control of the catechin and epicatechin branches of the PA pathway in forage legumes, here the effects of the expression of FaMYB1, a flavonoid R2R3MYB repressor from strawberry, in Lotus corniculatus (birdsfoot trefoil), were tested. It was found that in leaves of T-0 transgenic lines the degree of PA inhibition correlated with the level of FaMYB1 expression. These effects were heritable in the transgene-positive plant T-1 generation and were tissue specific as the suppression of proanthocyanidin biosynthesis was most pronounced in mesophyll cells within the leaf, whereas other flavonoid and phenolic compounds were substantially unaltered. The data suggest that FaMYB1 may counter-balance the activity of the endogenous transcriptional MYB-bHLH-WD40 (MBW) complex promoting proanthocyanidin biosynthesis via the catechin and epicatechin branches and that FaMYB1 does not interfere with the expression levels of a resident R2R3MYB activator of PAs. It is proposed that in forage legumes leaf cell commitment to synthesize proanthocyanidins relies on the balance between the activity of activator and repressor MYBs operating within the MBW complex.

Recently, there has been tremendous world-wide interest in dedicated energy crops as a source of renewable carbon neutral feed-stocks for the production of energy and heat by combustion processes. However, in order for the potential benefits of decreased greenhouse gas emissions and improved fuel security to be delivered, it will be essential for the cultivation of these crops to be achieved in a sustainable manner. In this chapter we identify those species with greatest potential for cultivation in the UK, explore the effect of biomass chemical composition on combustion efficiency and, with particular focus on Miscanthus species, discuss how these crops may be best improved by strategies including genetic engineering, gene discovery and breeding strategies.

Proanthocyanidins (condensed tannins) can play an important part in ruminant nutrition both by increasing ruminal efficiency and preventing pasture bloat. In this chapter we discuss the control of this pathway by transcription factors and focus particularly upon genes of the bHLH and R2R3MYB classes. Results from studies using transgenic approaches, tilling and similar techniques are discussed here.

Lotus corniculatus and Lotus uliginosus are agronomically important forage crops used in ruminant livestock production. The condensed tannin (CT) content, dry matter (DM) production, and persistence of these species are key characteristics of interest for future exploitation of these crops. Here we present field data on 19 varieties of L. corniculatus, 2 varieties of L. uliginosus and, additionally, a glasshouse experiment using 6 varieties of L. corniculatus and 2 varieties of L. uliginosus. Current methods for the quantification of condensed tannins in crop species are slow and labor intensive and are generally based upon polymer hydrolysis following the extraction of chlorophyll in a liquid phase. Presented here is a high-throughput protocol for condensed tannin quantification suitable for microtiter plates based upon the precipitation of condensed tannin polymers in complex with bovine serum albumin (BSA) with subsequent hydrolysis of precipates using butan 1-ol/ hydrochloric acid.

Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

Bryant, D. N., Bailey, P., Morris, P., Robbins, M. P., Wang, T., Martin, C. (2005). Identification of putative AtTT2 R2R3-MYB transcription factor orthologues in tanniferous tissues of L. corniculatus var. japonicus cv Gifu. Molecular Breeding for the Genetic Improvement of Forage Crops and Turf. Proceedings of the 4th International Symposium on the Molecular Breeding of Forage and Turf. XXth International Grassland Congress, July 2005, Aberystwyth, Wales, Humphreys, M. O. (ed), p. 166

Julier, B., Lila, M., Huyghe, C., Morris, P., Allison, G. G., Robbins, M. P. (2002). Effect of condensed tannin content on protein solubility in legume forages. Pages 134-135 in: Durand, J-L., Emile, J-C., Huyghe, C., Lemaire, G. (Eds). Multifunctional Grassland: Quality Forages, Animal Products and Landscapes. Grassland science in Europe 7. European Grassland Federation. ISBN:2950411037.

Robbins, M. P., Allison, G. G., Bettany, A. J. E., Dalton, S. J., Davies, T. E., Hauck, B., Hughes, J-W., Timms, E. J., Morris, P. (2002). Biochemical and molecular basis of plant composition determining the degradability of forage for ruminant nutrition. Pages 37-43 in: Durand, J-L., Emile, J-C., Huyghe, C., Lemaire, G. (Eds). Multifunctional Grassland: Quality Forages, Animal Products and Landscapes. Grassland Science in Europe, 7, ISBN:2950411037. Proceedings of the 19th General Meeting of the European Grassland Federation, La Rochelle, France, 27-30 May 2002.

SUMMARY - The efficiency of proteins of legume forages in ruminant nutrition is poor because of their extensive degradation in the rumen. The objective of this study was to evaluate the variation available for protein degradation in tannin-free (alfalfa and white clover) and tannin-rich (Lotus sp.) legume species. Protein and dry matter degradation were measured by in sacco incubation of forage samples in the rumen of fistulated cows, for 2, 8 or 48 h. Condensed tannin content was measured on Lotus samples. Variation in protein degradation was low among alfalfa and white clover cultivars, but the degradation was more important for alfalfa than for clover cultivars. Protein degradation was less important in Lotus sp. than in alfalfa or clover, and the variation among Lotus cultivars was mainly related to condensed tannin content.