Introduction

As a perennial plant, willow is known to recycle resources during the winter from leaves to stems, stools and roots to provide reserves for re-growth in the spring (Bollmark, Sennerby-Forsse et al. 1999; Karp and Shield 2008). An understanding of the changing nitrogen source/sink relationships during this recycling and of global nutrient budgets will help improve our understanding of the key physiological characteristics important for growth and thus biomass yield.

Nitrogen plays a limiting and defining role in willow growth with some willow varieties showing a strong correlation between relative growth rates and total tree nitrogen content (Ericsson 1981). Two reviews have addressed work performed to date on defining and calculating nitrogen-use-efficiency (NUE) in plants other than willow (Good, Shrawat et al. 2004; Brauer and Shelp 2010) and more recently a review by Weih (2010) has gone some way towards transferring the concepts of NUE assessment to willow. In this work, the arguments made by Moll et al. (1982) and advocated by Weih are accepted, that there is a need to separate nitrogen utilisation

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efficiency (UtE) and nitrogen uptake efficiency (UpE) factors. We use UpE here, however, in place of presenting UtE, we have accepted the arguments of Siddiqi et al. (1981), that nitrogen utilisation index (NUI) is more appropriate to use. NUI defines nitrogen utilisation as biomass per unit of tissue specific (stem) nitrogen

concentration. This takes into account that variation in net nitrogen losses, such as through leaf litter, or nitrogen partitioning can mislead the estimation of utilisation efficiency.

If large amounts of nitrogen are lost through either leaf abscission in the winter (Bollmark, Sennerby-Forsse et al. 1999) or (when the willow is harvested) through removal of the stems (Sennerby-Forsse 1995) then this may affect re-growth in spring. Although leaf abscission is the primary natural source of nitrogen loss from the tree it must also be considered that nitrogen reserves stored in the stem, lost from the system through harvesting, have a deleterious effect on re-growth in spring without further nitrogen inputs. Nitrogen is available via the leaf litter (which is why it is important to harvest after leaf fall), through soil mineralisation (depending upon the soil type) and through aerial N deposition, yet in commercial (and most

experimental) SRC plantations, 30-80 kg of nitrogen fertiliser is added after harvest in order to compensate for removal of nitrogen by harvesting the stems (Sennerby- Forsse 1995). Hence a reduction in stem nitrogen levels, or increase the stool as a nitrogen sink during dormancy, would allow for decreased nitrogen fertilisation.

Willow field trials with varying nitrogen fertilization (and irrigation) regimes have highlighted the importance and degree of response variation in Nitrogen-Use Efficiency (NUE) to biomass yield (Christersson 1987; Bowman and Conant 1994; Hodson, Slater et al. 1994; Nielsen 1994; Labrecque, Teodorescu et al. 1998; Weih

2001). The poor understanding of the regulatory mechanisms for controlling nitrogen economy in willows and other crops are highlighted in two recent reviews addressing NUE (Hirel, Le Gouis et al. 2007; Weih, Asplund et al. 2010). However, what is evident is that there is a large amount of variability of NUE in willows (Weih and Nordh 2002; Weih and Nordh 2005)

3.2.1 Aims and Strategy

Nitrogen 15 ratios have been used in the past to study nitrogen partitioning and utilisation in woody species (Cote and Camire 1985; Gebauer and Schulze 1991; Millard 1996; Nasholm, Ekblad et al. 1998). In this study, fertilizer enrichment with the stable nitrogen isotope N15 was used to follow finite amounts of nitrogen between different growth stages in a selection of willows from the K8 mapping population known to differ in biomass yield potential. A pot experiment was devised to assess aspects of development and NUE during the first 6 months growth of the cuttings. The experiment consisted of 252 willows derived from cuttings of the two parents of the K8 mapping population and 12 of their progeny. The progeny were specifically chosen because of the consistency and variance of their biomass yield (Hanley 2003) (See Chapter 4). The cuttings were planted in April 2008, and a third of the population was harvested at each of 3 harvest points spanning what was estimated to be three key developmental events and shifts in physiological

stratagems. These three points represent the initial growth/canopy establishment phase (June), the height of the growth phase (August) and the retardation of growth and remobilisation of resources for winter phase (October) (Bollmark, Sennerby- Forsse et al. 1999; von Fircks, Ericsson et al. 2001) (Table 3-2).

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Half the population was treated with the stable isotope N15 (10% atom for atom) which allowed for the surveillance of any nitrogen reallocation (after assimilation from the soil and initial allocation) between August and October through Isotope Ratio Mass Spectrometry (IR-MS). This approach has been successfully used in species such as Norway Spruce (Picea abies) (Gebauer and Schulze 1991) and Poplar (Dluzniewska, Gessler et al. 2007).

Although biomass yields are of large importance when considering biofuels, increasing final sugar yields will also be of importance when considering

lignocellulosic ethanol optimisation.

The specific objectives of this study were to establish: i) whether biomass yields of specific willow genotypes in a pot experimental system correlated with performance in the field ii) what the growth priorities of the trees are at different stages of growth from establishment of the cutting iii) how nitrogen is allocated at different stages of development iv) whether there is any secondary allocation of nitrogen between organs at different stages of development and v) whether higher biomass yielding genotypes of willow have increased NUE.