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AIR2-CT94-1617
Genetic Improvement of Willow (Salix) as A Source of Bioenergy for the EC |
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Proposal No: | AIR2-CT94-1617 |
| Date Prepared: | September 1999 | |
| Source: | Final report November 1998 |
Introduction
Willow (family Salicaceae) is not a new crop in agriculture. Salix viminalis L. (osier) and some other Salix species have been cultivated for hundreds of years to produce raw material for wicker baskets and furniture. Willow has been selected earlier to produce slender and straight rods, not to maximise the above ground biomass in a highly mechanised cultivation system. Willow is thus a novel crop when it comes to plant breeding aimed to produce biomass on agricultural land. Rapid improvements in these aspects can therefore be expected. Plant material used earlier in willow coppice plantations consisted of clones propagated from wild or naturalised stands, of the species S. viminalis in particular. Even though earlier cultivated clones have been found superior to others of similar origin, they do have weaknesses as far as extensive commercial cultivation is concerned. Through plant breeding, crop traits such as biomass yield, plant shape and resistance to pests, diseases and frost can be significantly improved. thereby increasing the economic gain from cultivation.
Objectives
The objectives of the project have been to improve willow (Salix) as an energy crop by (a) applying new technologies (molecular fingerprinting and marker identifications) to sharpen breeding efforts, (b) improving market competitiveness by producing clones with improved yield, frost, pest and disease resistance (c) maximising productivity over a wide range of soil and climatic conditions (d) assessing economic and technical aspects of market potential.
Activities
For European conditions it is important to produce varieties which are resistant to leaf rust, as the natural infection pressure of rust is higher in central and southern Europe compared to the northern parts where the first marketed varieties of willows have been produced. Great attention has therefore been paid in the project to leaf rust resistance and improvement of techniques to breed for rust resistance. Other adaptive traits in the plant material were also studied in field trials, that were planted in five European countries. Though these field trials are of a limited size for yield estimations in practice, they can be used to assess some technical and economical aspects of importance for willow growing. These aspects dealt with traits that may be changed by plant breeding. These include rust resistance, frost tolerance, biomass yield and wood density for example. The field trials; containing 16 clones of different origin with varying degree of rust resistance, have been established and evaluated using molecular fingerprinting and marker techniques. The genetic variation of the rust in relation to willow hosts has been investigated and a number of crosses have been made to produce material suitable for all parts of Europe. Crosses have also been made especially for the study of molecular markers for rust resistance and frost tolerance.
Results
The results from the specific tasks can be summarised as follow:
Fingerprinting Molecular screening techniques and statistical analysis methods have been developed for the identification and characterisation of willow species, hybrids and cultivars. Three different techniques were tested, RAPD (Randomly Amplified Polymorphic DNAS), AFLP (Amplified Fragment Length Polymorphism) and SSR (Simple Sequence Repeats). All of these methods are based on PCR (Polymerase Chain Reaction), which is faster than hybridisation-based techniques such as RFLPS. Compared to RAPD, AFLP fingerprints are more reproducible because of longer primers with more specific annealing in the PCR reaction. Another advantage over RAPD is that many more loci are screened in each AFLP- reaction. The advantages of SSRs are that they are often multi-allelic and heterozygotes can be distinguished from homozygotes.
The two screening methods of choice, AFLP analyses for multi-locus DNA fingerprinting and SSR markers for the use as single-locus probes, have been used to identify the most important biomass cultivars, including commercial cultivars. The following types of willow material have been screened:
Groupings of the clones were well in accordance with previous knowledge about relatedness between clones. One case of a clone duplicated in willow collections was detected by these marker studies. The methods can now be used to identify identical or closely related cultivars and, thus, prevent the duplication of crosses in future/on-going breeding programmes.
Molecular markers for leaf rust resistance Bulk segregant analysis aimed at identification of AFLP markers for resistance to leaf rust was performed in two families of biomass willow. Several AFLP bands differed between the bulked rust resistant and the bulked susceptible progenies, but when individuals were screened rather than bulks, these putative markers did not show any linkage to resistance or susceptibility. This result might be due to the resistance being polygenic. In order to identify markers for that type of resistance it is necessary to have a genetic map in which the molecular markers span the genome at even intervals. To construct such a map a total of 470 AFLP and 13 SSR markers were analysed for linkage. However, numbers of linkage groups did not fit with the chromosome number of Salix viminalis, the willow species analysed, indicating that more markers are needed to produce an authentic map.
Molecular markers for different virulence factors of the leaf rust The rust populations of Melampsora spp. on Salix used for short rotation forestry are very diverse with a large variation in virulence and aggressiveness, i.e. variation both regarding host spectrum and disease intensity after successful rust infections. Molecular tools such as isozymes, RFLP, RAPD and AFLP have been evaluated as markers for reliable identification of rust isolates of use when screening willow breeding materials for resistance to rust and for recordings of temporal and regional variations in rust populations. AFLP turned out to be the best method for characterisation of the rust in that many loci were screened in each reaction and that the reproducibility was high.
Breeding of willow The breeding part of the project concerns breeding of willow clones for European conditions, as an extension of the current research program on Salix at Svalöf Weibull AB. Special attention has been focused on resistance to leaf rust and there has been much improvement in this, and other characters, selected for in the breeding material. During the project period twelve willow varieties from the Svalöf Weibull Salix breeding programme have certified for plant variety protection within the EU. Results from the clonal trials will influence the current willow breeding program. Since these trials have been located in places with very different climatic conditions, the effect of biotic and abiotic factors on biomass production have been varying a lot. Especially the effect of drought is of importance for future breeding efforts. New collections have therefore been made of material from Greece to improve the drought resistance characteristics in the breeding material.
From the clonal trials, on the other hand, there was shown a consistency in leaf rust reaction between varieties in the various locations. The rust resistance in the varieties 'Bjorn' and 'Tora' was very strong and it is tempting to continue with crosses containing this Siberian type of rust resistance. That has been an important information for the breeding program. However, there is still a need for studies of variation in rust resistance genes in this kind of material, in order to ensure durable resistance.
Clonal trials The plant survival was high in almost all clones, ranging from 91 to 100 %. However, in two of the clones, due to poor cutting quality, the survival was less good. Stem wood production, as expected, varied a lot between the sites and the highest overall production was found in the English trial where the standing biomass after three years of growth was 29.4 tonnes dry matter per hectare. Within each site the ranking between clones differed. One clone, 'Tora', ranked among the three best yielding clones in four of the five trials. In the French trial however, 'Tora' was the lowest yielding clone due to poor drought tolerance. Weed occurrence and water deficiency were the most important factors limiting willow growth. The levels of insect damage in the trials were in general low during the project period. However, in England, the trial was twice heavily attacked, by chrysomelid beetles and sawfly larvae. Also the leaf rust infection levels were low during the period in all trials and all clones. Two clones, 'Björn' and 'Tora', were almost free of rust. Frost damage of any importance was only recorded in the Swedish trial where the clone 'Jorunn' lost almost a full years production due to frost.
A willow stand is expected to have a life span of 20 to 30 years. If harvest takes place every third year, at least 6 to 7 cutting cycles will be included in that time. This project studied only the first cutting cycle, the establishment phase of the willow plants. If the establishment is successful (good survival, no weed) the following cutting cycles are expected to have an increased yield. Plant survival is a very important factor influencing yield and stand development. It is especially important for production during the first cutting cycle. In that stage the plants have not yet become fully established and are therefore not able to occupy the available space. In the following cutting cycles, stand closure is reached more rapidly and the established plants next to a gap are able to fill up the available space. Reduced production due to low plant survival, in this case due to poor cutting quality, has been observed in the clones 'Christina' and 'Marie'. However, in the English trial, the growth was already during this cutting cycle large enough to compensate for the gaps to some extent.
The overall development and performance of each of the five field trials have been very much influenced by the local climatic conditions and the management. As stated above, good water availability is of major importance for successful willow growing. Because of low water availability in the Danish and the French trial during the project period, some of the clones have been observed as being more sensitive to drought than others, an observation that has not been made before. During the project period, pests like insects and fungi have not had any significant effect on willow growth. However, if the trial in England had not been sprayed with insecticides in the establishment phase 1994 and 1995 the development of that trial could have taken another turn. In the Swedish trial, the impact of frost was clearly demonstrated as one clone lost almost a whole years production of biomass because of autumn/winter frost damage.
Economical assessment and information A comparison is made relating to the economic competitiveness of various energy crops between, on the one hand, the individual energy crops and, on the other, energy crops versus conventional crops. The energy crops investigated are Salix, reed canary grass, elephant grass and solid fuel energy grain. The comparison relates to conditions in Denmark, that are in many respects similar to those in most European countries with regard to the introduction of biofuels. Energy crops clearly show the best profitability on good arable land, but is by far the most competitive versus grain on inferior arable land. The difference in profitability is relatively small between the most profitable energy crops and grain. The profitability rating order between conventional crops and energy crops can be reverted by factors such as whether straw is sold off or not, the price of grain, yield levels, the arrangement of the area subsidy systems etc. The energy crop showing the highest profitability is Salix followed by elephant grass and reed canary grass. Energy grain are the energy crops with the lowest profitability. The most important factors for the profitability of Salix cultivation are the price of fuels and yield levels, both of which can be improved by plant breeding. Salix cultivation costs, such as harvesting and transportation, can also be influenced indirectly by plant breeding.
Handbook for Willow Growers
A handbook (Short-Rotation willow Coppice - Growers Manual for willow growers) was translated and modified to English from a Swedish version, as part of the project activities. To cover the cost of this work an EC Sponsorship Grant has been received (FAIR-CT97-4822). The interest in this handbook, that was ready for distribution at the beginning of 1998, has been very great. About 3000 copies out of the total of 4000 had been distributed at the time the project ended.
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by CPL Press:
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