Progress and Challenges:

Progress and Challenges: Forage Breeding in Temperate Australia K.F.M. Reed\ R. Culvenor', Z. Jahufer', P. Nichols', K. Smith' and R. Williams 4 I Agriculture Victoria, Pastoral and Veterinary Institute, Cooperative Research Centre Molecular Plant Breeding, PB 105, Hamilton, Victoria; 2Plant Industry Division, CSIRO, Canberra, ACT; 3Agriculture Western Australia, South Perth, Western Australia ; 4New South Wales Agriculture, Tamworth Centrefor Crop Improvement, Tamworth, New South Wales

Key words:

Phalaris aquatica, Lolium perenne, Trifolium repens, Trifolium subterraneum, Medicago sativa, plant toxins, pests, diseases, edaphic !imitations, c1imatic adaptation

Abstract: Austra!ia has approximately 2.5 M ha of high quality perennial pasture. Agriculture has however, also developed in many relatively dry regions characterised by a short growing season, and infertile, acid soi!. Australia's improved pasture includes introduced species not previously cultivated elsewhere (e.g. phalaris (Phalaris aquatica), tall wheatgrass (Thinopyron ponticum), barrel medic (Medicago truncatula), strand medic (M. littoralis), subterranean/sub clover (Trifolium subterraneum), michel'slbalansa clover (T. michelianum) and yellow serradella (Ornithopus compressus). Common varieties selected from naturalised species, or developed from accessions collected from the Mediterranean basin, have gradually been replaced by cultivars representing a range of maturity times, bred in the target environment using phenotypic selection and multi-site progeny testing. Over the last forty years the zone of adaptation of pasture species has greatly expanded. Achievements include expanding the zone of adaptation by providing a range of maturity - e.g. some subterranean clover cultivars flower up to two months later than the earliest cultivar; barrel medic cultivars vary by 3 weeks. Further achievements include seed!ing vigour, cool season vigour, drought tolerance and yicld. In addition, the domestication of annual legurnes has involved selection for hard seededness for regions with short growing seasons where cropping phases rotate with pasture. The successful domestication of phalaris involved a mutation for complete seed retention and reduction of alkaloid concentrations. Selection for low oestrogenicity has been important in sub and

303 G. Spangenberg (ed.), Molecular Breeding ofForage Crops , 303-316. © 2001 Kluwer Academic Publishers.

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K.F.M. Reed et al. red clover (Trifolium pratense). Disease screening has made legurnes resistant to root pathogens and foliar diseases including viruses. Modem luceme and medic cultivars are resistant to several aphid species; some resist stern nematode. Edaphic limitations have been overcome by selecting for waterlogging resistance and tolerance to high levels of exchangeable aluminium in acid soils. Challenges yet to be overcome include resistance to many grassland pests including molluscs, mites, Collembola and other insects including Coleoptera, Orthoptera and Lepidoptera. Root rot and virus diseases remain serious problems with legumes. With several important forage species, elimination of toxins, persistence on acid and infertile soils - e.g. low Molhigh Mn; ability to absorb Zn at high pH - are required. We need to slow the rate ofbreakdown of hardseededness in some annual legumes. Susceptibility to herbicide residues and leaching of nutrients from mature herbage represent serious economic losses. Molecular markers are needed for seed retention, root architecture and Al tolerance. Possibilities for molecular breeding include the introduction of genes for drought tolerance, disease resistance, Al tolerance, increased acquisition of P from less soluble sources, increased soluble carbohydrate content and bioat-safe legumes.

1 Introduction

Australia has approximately 2,000,000 hectares of reliable high rainfall pasture and 500,000 ha of irrigated pasture . These pastures produce over 80% of our dairy produce. Most of the high quality veal and lamb results from the high rates of liveweight gain and early turn-off achieved on these quality pastures which however represent less than 10% of the land used for grazing in Australia. Most of the high quality pasture is in the wet coast temperate agro-ecological zone; some in the temperate highlands. Despite the excellent environment of the wet coast temperate zone, Australia is by far the driest continent - excluding Antarctica. With a mean rainfall of 460 mm per year it receives slightly more than half the rainfall of the other continents and has a much lower river discharge. The rainfall in many regions (e.g. most of New South Wales) has an even distribution over the year but is so erratic that vast areas are best regarded as having a modified Mediterranean climate . Thus reliance on annual legurnes for Australian pasture is especially significant. Soils over vast areas are subject to seasonal desiccation that can be quite severe. The humid climate of the late Tertiary period impoverished many soils by leaching and most soils are naturally infertile by international agricultural standards. The acidic soil types podzols and lateritic sand plains - dominate; black earths, rendzinas and red

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brown earths make up the other major soil groups in the medium to high rainfall areas. The native grassland evolved under a less humid climate than the current one and in the presence of soft-footed marsupials. Many native species have proved vulnerable to the heavy stocking of cloven-hooved livestock so characteristic of the high rainfall zone. In a comprehensive review of limitations of pasture species in southem Australia , Reed and Cocks (1982) emphasised the success of the annual legurne development program. They drew attention to the limited use of perennial grasses that had been developed for the short growing seasonlwheat sheep zone, and the pressing need for perennial legumes . Attention was also given to the competition that subterranean clover was encountering in the high rainfall zone where decades of good clover production and increasing soil fertility had made competition from grasses and nitrophilous weeds a significant challenge for the annuallegume. 2 Early Selection Programs Phalaris, a summer-donnant perennial grass of Mediterranean ongm, proved to be a productive and drought-hardy speeies for sown pasture in temperate SE Australia where it currently oceupies 2 M ha. The biology and use of phalaris was deseribed by Carlson et al. (1996). Phalaris has been eultivated since the early 1900's. The first cultivar, Australian, was derived without seleetion from an eeotype - probably ofItalian origin. Cultivars have since been developed by seleetion and breeding in Australia, USA, South America and New Zealand . Breeding of phalaris in Australia initially involved limited seleetion within aceessions exhibiting particular attributes such as high summer donnancy for use in areas with shorter growing seasons. The main breeding effort aimed to overeome defieiencies in Australian through programs of reeurrent seleetion in populations based on Mediterranean accessions and Australian. Selection criteria included higher seedling vigour , autumn and winter growth rates and reduced levels of toxie alkaloids . Perennial ryegrass (Lolium perenne) is the most popular grass and has been sown over 6 M heetares, mainly with the Australian eeotype, Vietorian - released in 1936. Ecotypes from New South Wales and Tasmania were released in the 1960s. Sinee the introduetion ofNui perennial ryegrass in the late 1970's a number of New Zealand eultivars have been marketed in Australia . The majority have useful rust resistanee and have been promoted for the higher rainfall dairy producing environments. Imports of European cocksfoot persisted only in the high rainfall areas until Currie (ex Algeria)

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and Porto (ex Portugal) were released in the 1960s and 1970s respectively. Demeter tall fescue (ex Morocco) was released in the 1960s. Early in the zo" century, the use of superphosphate and subterranean c1over, revolutionised the carrying capacity of pastoral holdings. It has been sown on as much as 80% of the present 20 M ha it occupies (Donald 1970). Early selection of subterranean clover was directed at providing varieties with different flowering times, to enable the species to be grown in lower rainfall environments to that suited to the original cultivar - Mt Barker. Breeding commenced in the early 1960' s to counter the high levels of formononetin (Francis and Millington, 1965) that were causing infertility problems in ewes. Later breeders focussed on resistance to clover scorch (Kabatiella caulivora), a leaf disease that devastated large areas of high rainfall pasture in the early 1970's (Chatel and Francis 1974). Running in parallel to the crossing and selection programs has been a large effort to collect germplasm from throughout the Mediterranean basin and surrounding areas, particularly in the 1970's and 1980's . Such new germplasm has been evaluated alongside crossbred varieties and been used as new parents for crossmg. Luceme is a perennial legurne used to produce high quality hay and pasture, and to increase the grain yield and protein of following crops. Although a cross-pollinated autotetraploid, where cultivars are a heterogeneous mix of highly heterozygous plants, breeders have successfully increased the pest and disease resistance of luceme in locally adapted material since the 1950's . Rapid breeding of aphid-resistant cultivars followed the unwelcome introduction of three aphid pests (Therioaphis trifolii f. maculata, Acyrthosiphon pisum and Acyrthosiphon kondoi) in the late 1970's. The introduction of perennial ryegrass and white clover into Australia dates from early European settlement. White clover presently occupies an estimated 6 M ha in southem Australia. This zone encompasses a wide range of environments contrasting in c1imatic, biotic and edaphic factors, and diverse grazing enterprises (Ayres et al. 1992). The white clover zone extcnds from Queensland along the coast and adjacent table1ands of New South Wales and Victoria to Mt Gambier in South Australia. Under irrigation, white clover use extends beyond the boundary of the temperate perennial pasture zone to the perennial grass/annual legurne zone, Mediterranean zone and coastal subtropics.


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The white clover cultivars Irrigation, Haifa and Siral were the first to be developed in Australia . Cultivar Irrigation was selected from an ecotype found in northem Victoria . Cultivars Haifa and Siral were developed from ecotypes found in Israel and Algeria respectively. Noted for cool season vigour, the accessions underwent some evaluation and phenotypic selection for persistence in a limited number of environments in Australia (Oram 1990). 3 Recent Progress

The history of phalaris improvement in Australia (Oram and Culvenor 1994) iIlustrates the polygenie control of many traits of economic importance. More recently plant breeders have developed a range of perennial ryegrass cultivars based on the old Australian ecotypes, Victorian (GelIert et al. 1999, Croft et al. 2000) and Kangaroo VaIley. Selections from the ecotypes have been based on phenotypic selection for seasonal production, crown rust (Puccinia coronata) resistance (Kimbeng 1999) and virus tolerance (viz. barley yeIlow dwarf virus and ryegrass mosaic virus). These new cultivars are attracting keen interest from producers as they combine improved productivity with the renowned adaptation/persistence of the local ecotypes. Germplasm from the Mediterranean has lead to the development of cultivars weIl adapted to Australian conditions in several forage species (Donald 1970, Reed 1996). This approach has recently been used successfuIly in the development of summer dormant perennial ryegrass and tall fescue cultivars that exhibit tolerance to drought (Anderson et al. 1999). Much work is now required to utilise early, winter active, Mediterranean genotypes in improving fine leaved perennial turf grasses for persistence and seed production potential in the temperate Australian environment. Breeders of perennial ryegrass are now addressing nutritive value. They have recognised the relative importance of various nutritive value traits (Smith et al. 1997) and large benefits in milk production have been predicted from the use of perennial ryegrass cultivars with increased digestibility (Smith et al. 1998). Tetraploid cultivars are now being developed in recognition oftheir potential to raise nutritive value (Nair et al. 1999). Since 1983 there has been anational program of subterranean clover improvement. Crossing and early generation selection has been conducted in Perth and advanced breeding lines satisfying selection criteria have been evaluated nationaIly. Breeding efforts have largely concentrated on improved adaptation to each of the major agro-ecological environments with

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selection criteria largely devoted to improving persistence. A breeding method to enable selection for adaptation to target agro-ecological environments in the segregating generations is now employed (Nichols 1993). Specific characters of importance to the differing environments are also incorporated (Nichols et al. 1994). For the low rainfall environment, early flowering for adequate seed setting, coupled with high hardseededness to improve persistence through the cropping phase, are of major importance. The challenge to identify more productive late maturing sub clover to better compete with companion species and weeds was weIl met - with material introduced from Sardinia (Clark and Hirth 1987). For high rainfall areas, particularly those with semi-permanent and permanent pasture systems, emphasis has been towards improved disease resistance, particularly to clover scorch and root rot caused by Phytophthora clandestina. Low formononetin level has continued to be a major selection criterion for all material, while in recent years breeding has concentrated on incorporating redlegged earth mite resistance into adapted genotypes (Ridsdill-Smith and Nichols 1998). To date 33 subterranean clover cultivars have been registered in Australia (Nichols et al. 1996). Increasingly, luceme has been recognised for its potential to improve the sustainability of farming systems. In particular, luceme has been shown to arrest land degradation associated with fertility decline, dryland salinity, soil acidification and the development of herbicide-resistant weeds. Luceme cultivars have been developed using recurrent cycles of phenotypic selection, often among and within half-sib families, to maximise the frequency of individuals within breeding populations that express desirable traits. Robust and precise screening and evaluation techniques have been developed by all breeding programs for most major pests and diseases to better identify superior plants and maximise potential advances . Selected clones within populations are usually caged, then crossed in a greenhouse, often with honeybees. Current aims are to both improve luceme and to improve its utilisation in sustainable systems of mixed farming. This will ensure that lucerne-based pastures play an increasing role in the development of sustainable farming systems. Since 1980 major luceme breeding programs based in three states have released a suite of successful cultivars. The area sown to luceme - excluding luceme use in seed mixes - has increased considerably over the past 15 years and now exceeds 800,000 ha. The New South Wales program re1eased Nova as the first Australian-bred 1uceme resistant to aphids (Williams 1999). Aurora followed in 1986 and rose to dominate the market in that state. The NSW program was first to exploit a dominant major-gene resistance to


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Phytophthora megasperma f sp. medicageninis root rot in the cultivar Aquarius. Aquarius has the highest resistance to this disease of any lucerne in the world (JA Irwin, pers. comm.). Aquarius has extended the adaptation of lucerne into heavier soils and temporarily waterlogged areas. Cultivars Genesis and Venus have extended lucerne usage into dry margins. The South Australian program has released cultivars with multiple-pest and disease resistance for all of the key lucerne growing zones. This is the broadest testing program (Auricht 1999). Cultivars such as Sceptre, Eureka and Super 7 have all demonstrated broad adaptation, high productivity and persistence . The variety Jindera, an exceptionally prostrate cultivar, has found an important niche as a ground cover. The Queensland program bred the successful cultivars Trifecta and Sequel (Clements et al. 1984). Trifecta was re-selected for resistance to Stemphylium botryosum leaf spot to produce cultivar Quadrella. Sequel HR has high resistance to the damaging Colletotrichum trifolii crown rot fungus. Later releases include Hallmark and UQLl. As with other species, development of cultivars with adaptation to the Australian environment is expected to increase the use of white clover pasture. A number of imported cultivars have been widely used but show poor adaptation to drier environments. Environmental constraints such as summer moisture stress, viruses (alfalfa mosaic virus - AMV, clover yellow vein virus - CYVV, and white clover mosaic virus - WCMV), nematodes (clover cyst nematode Heterodera trifolii and root-knot nematode Meloidogyne spp.), redlegged earth mite (Halotydeus destructor), lucerne flea (Sminthurus viridus), blue green aphid (Acyrthosiphon kondoi) and salinity, limit white clover performance in Australia. Summer moisture stress has been identified as a key constraint to white clover's vegetative persistence (Archer and Robinson 1989). White clover breeding was initiated in 1989. The National program based in Victoria with cooperators in NSW and Queensland, focused on developing cultivars for the high rainfall/irrigated dairy pasture. Another program is focussed on the sheeplbeef pasture of the northern tableland in NSW (Ayres et al. 1996). The National program has developed broadly adapted cultivars with improved seasonal growth. A large-leaved, spring/early summer active white clover cultivar, Mink, was released in 2000. The breeding program for Mink began with a collection of 5000 plants from some of Victoria's oldest white clover pasture, in the Northern Irrigation Region - pasture sown up to 50 years aga with the cultivar Irrigation (Lee et al. 1993). The NSW program is developing cultivars with broad adaptation and drought tolerance.

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Grasslands NuSiral, was developed from Siral by selecting for flowering intensity and seed yield. 4 Opportunities for Biotechnology

A major step in the domestication of phalaris occurred with the discovery of a mutant in cultivar Australian that completely retained its seed (Oram et al. 1985). This economically important character requires homozygous recessive alleles at 4 loci with other modifying genes also possibly involved. Achieving a high proportion of retainers still presents a difficulty when breeding new cultivars. The development of molecular markers for seed retention would be valuable in reducing the time taken to backcross the character into non-retaining populations or to increase the frequency of retention in advanced populations . Other challenges currently facing phalaris breeders in Australia include improving persistence of the winter-active cultivars under heavy grazing pressure, improving performance and survival on acid and low fertility soils widespread in SE Australia and the elimination of toxins. Winter-active populations are currently under selection for higher ground cover and persistence under heavy grazing (Culvenor et al. 1999) but the long periods of evaluation required make this slow and costly. Markers associated with persistence and spreading ability would reduce the time and cost of breeding programs. Large gains in the acid soil tolerance of phalaris should be possible by the introgression of genes for high Al tolerance from P. arundinacea. Early backcross generations displayed superior tolerance of acid soils (Oram et al. 1990) but retaining this character while subsequently selecting for a P. aquatica plant type has been less successful. Molecular markers for the Al tolerance genes specific to P. arundinacea could facilitate their transfer to P. aquatica. Recent discoveries on the possible role of organic acid secretion by roots in Al tolerance raise the possibility of transforming species that are sensitive to Al (De la Fuente-Martinez and Herrera-Estrella 1999). This approach is currently being used in the development of transgenic white clover and lucerne plants expressing a chimeric bacterial citrate synthase gene for enhanced phosphorus use efficiency and Al tolerance (G Spangenberg, pers. comm.; Lyons et al. 2000). Acquisition of P by plants is also a frequent limitation in Australian soils. Plants able to exploit less available sources of soil P may be developed if genes from soil microorganisms that can exploit these sources can be transferred (Richardson 1998).


3I I

Selection for improved nutritive quality has received littIe attention in phalaris apart from selection for reduced alkaloid content. Work in more widely-studied species such as perennial ryegrass should eventually be relevant to phalaris since homology between species is likely to exist for the control of processes that influence nutritive quality. It may then become possible to introduce into phalaris attributes such as the high water soluble carbohydrate trait found in perennial ryegrass (Humphreys et al. 1989). The diverse range of sowing environments and grazing industries, along with perennial ryegrass-endophyte interactions (Reed et al. 2000), make symbio-genomics and the breeding of perennial ryegrass for Australia a complex challenge. However the use of marker assisted selection and transgenesis will greatly increase the range of traits that can be targeted in a breeding program for perennial ryegrass with functional endophyte characteristics . To this end a comprehensive program exists within the Cooperative Research Centre for Molecular Plant Breeding, employing modem breeding methods to develop cultivars with a range of novel attributes (Spangenberg et al. 2000a). A comprehensive molecular map of perennial ryegrass is being developed based on DNA simple sequence repeats. The initial target traits to map include drought tolerance, virus resistance , resistance to crown rust and forage quality (Forster et al. 2000) Concurrently, transgenie plants with reduced expression of pollen allergens, down-regulated lignin biosynthesis and increased fructan biosynthesis are being developed (G Spangenberg, pers. comm .; Spangenberg et al. 1998, 2000b; Lidgett et al. 2000; Donato et al. 2000). Genetic variation for minerals related to grass tetany has been observed in half-sib families of perennial ryegrass adapted to Australian conditions (Smith et al. 1999). However large environmental and family x environment effects would make screening families difficult in a conventional breeding program. Traits that are needed in subterranean clover include: higher digestibility of mature foliage, reduced leaching of nutrients following summer rain; deeper roots; greater resistance to soil fungal pathogens including Pythium, Rhizoctonia, Fusarium and Phytophthora; delayed hardseed breakdown, tolerance to herbicide residues from the crop phase and greater seedling vigour. Research in Agriculture Victoria has led to the production of transgenic subterranean clover plants expressing different antifungal protein genes for enhanced resistance to the above soil pathogens (G Spangenberg, pers. comm.; Aldao et al. 2000). Other important annual legumes such as Persian clover, T resupinatum ssp majus, lack resistance to rust (Uromyces

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trifolii-repentis Liro) . In subterranean clover we need to improve tolerance to low molybdenum and high manganese levels in soil, In T. subterranean spp brachy calycinum , luceme and in the annual medics, inability to absorb zinc at high pR can often be a serious limitation to production. Some of these challenges may best be addressed through use ofbiotechnology. Conventional breeding has been unable to address several key limitations in the use and adaptation of luceme. The most important of these are the propensity for luceme to cause bloat, and its inability to grow on acid soils particularly those where soil aluminium levels are high. Gene transfer protocols for Australian-bred luceme have been developed and programs in Agriculture Victoria and CSIRO are focussed on engineering bloat-safe luceme using tannin genes , luceme with sulphur-rich/rumen-resistant proteins, luceme for acid soils and high soil aluminium and low lignin luceme with improved forage quality. Other areas of potential importance for molecular approaches include the incorporation of virus resistance and resistance to several insect species notably white-fringed weevil (Graphognathus leucoloma). The generation of molecular markers for disease resistance and adaptation traits such as tolerance to salt and acid soils also promise major advances in both the efficiency of the breeding process and in the quality of their future outcomes. The National white clover program is currently evaluating transgenic white clover plants developed in Agricu1ture Victoria that are immune to alfal fa mosaic virus (AM V) (Kalla et al. 2000). These have been field tested at Hamilton under a strict protocol approved by the Australian Genetic Manipulation Advisory Committee. Plants expressing the AMV coatprotein coding gene may be released for breeding in the near future . Novel transgenic germplasm with tripIe virus (AMV, CYVV and WCMV) resistance is now being developed (Spangenberg and Chu 2000). It is estimated that infection ofwhite clover by AMV, CYVV and WCMV, causes annual losses to the Victorian dairy industry of $20 m, $3.5m, and $7.0 m respectively (Garrett et al. 1998) . Delayed senesence would greatly enhance the value of forages, particularly in short growing season regions where nutriti ve value can decline rapidly at the end of the season. Research in Agriculture Victoria has developed transgenic white clover plants with delayed leaf senescence by the regulated expression of chimeric isopentenyltransferase genes (G Spangenberg, pers. comm.; Ludlow et al. 2000). Research to develop transgenic white clover with resistance to clover cyst nematode Heterodera trifolii, and root-knot nematode Meloidogyne spp.,


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commenced recently in CSIRO after the significance of these pests was highlighted in economic terms: Berg and Hinch (1996) estimated that the dairy industry suffers a loss in excess of $20 M per annum due to nematode damage of white clover . The economic losses due to redlegged earth mite, luceme flea and blue green aphid damage to sub clover and white clover is also most significant. Introgression of resistance into clover would greatly enhance pasture quality, persistence and production. Berg (1993) estimated that annual losses due to these pests exceed $200 M per annum. In addition to transgenic development, the identification of molecular markers for complex attributes will improve the efficiency of conventional breeding and accelerate progress in the improvement of white clover for our livestock feeding systems. Traits to be targeted would include root architecture, leaf size, stomatal aperture, stolon branching frequency, stolon thickness, water use efficiency and seasonal yield potential (Forster et al. 2000). Measurement of such attributes is often confounded by genotype x environment interaction and experimental error (Jahufer et al. 1999). This lowers the accuracy in predicting the performance of genotypes. Initial studies on the use of AFLP's in white clover germplasm characterisation have delivered promising results (Kölliker et al. 2000). References Anderson MW, Cunningham PJ, Reed KFM, Byron A (1999) Perennial grasses of Mediterranean origin offer advantages for Central Western Victorian sheep pasture. Australian Journal ofExperimental Agriculture 39: 275-284. Areher KA, Robinson GG (1989) The role of stolons and seedlings in the persistence of white clover (Trifolium repens L. cv. Huia) in temperate pastures on the Northem Tablelands of New South Wales. Aust J Agric Res. 40: 605-16. Aldao G, Drayton M, Kalla R, Cammue B, Spangenberg G (2000) Development oftransgenic subterranean clover expres sing different chimeric AFP genes for enhanced resistance to fungal disease . In: Abstr acts Second International Symposium Molecular Breeding Forage Crops 2000 , Lorne and Hamilton, 19-24 November 2000, p 110. Auricht GC (1999) Lucerne: an Australian breeding success story . In: Proceedings 11th Australian Plant Breeding Conference, Langridge P, Barr A, Auricht G, Collins G, Granger A, Handford D, J Paull (eds.). I: 71-76. Ayres JF, FitzGerald RD, Jahufer MZZ, Norton MR (1992). White clover improvement for the Australian sheep industry. Wool Technology and Sheep Breeding 4: 162-67. Ayres JF, Caradus JR., Lane LA, Murison R (1996) White clover breeding for dryland sheep and cattle pastures in Australia. Proc. White Clover Symposium, Lincoln, New Zealand, pp. 155158. Berg G ( 1993) Invertebrate pests ofwhite clover. In: White Clover: A key to increasing milk yields. Mason W (ed) 91-94. Dairy Research and Development Corporation, Melbourne.

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Berg G, Hinch J (1996). Investigating the impact ofnematodes on white c1over. Final Report to the Dairy Research and Development Corporation on project DAV 254. Carlson IT, Oram RN, Suprenant J (1996) . Reed canarygrass and other phalaris species. In ' Cool-season forage grasses .' Moser LE, Buxton DR, Casler MD (eds) pp. 569-604 . Chatel DL, Francis CM (1974) Susceptibility of subterranean clover to clover scorch (Kabatiella caulivora ). Journal of the Australian Institute of Agricultural Science 40: 8082. Clark SG, Hirth J (1987) Growth and persistence of Mediterranean genotypes of midseasonlaIe maturing subterranean clover cultivars in Victoria . Australian Journal of Experimental Agriculture 27: 551-557 . Clements RJ, Turner JW, Irwin JAG, Langdon PW, Bray RA (1984) Breeding diseaseresistant, est-resistant lucerne for sub-tropical Queensland. Australian Journal of Experimental Agriculture and AnimaJ Husbandry 24: 178-88. Croft VM, Cunningham PJ, Anderson MW, Smith KF, Reed KFM (2000) Lolium perenne L. (perennial ryegrass) cv. Avalon . Australian Journal 01 Experimental Agriculture (in press). Culvenor RA, Dobbie MJ, Wood JT (1999) Breeding for improved persistence in winteractive phalaris . In: Proceedings of the I1 th Australian Plant Breeding Conference, Langridge P, Barr A, Auricht G, Collins G, Granger A, Handford D, Paull J (eds) 2: 252253. De Ja Fuente-Martinez JM, Herrera-Estrella L (1999) Advances in the understanding of aluminum toxicity and the development of aluminum-tolerant transgenic plants. Advances in Agronomy 66: 103-120. Donald CM (1970) Temperate pasture species . In: Australian Grasslands Milton Moore R (ed) 303-320. ANU Press, Canberra. Donato R, Liu B, Wu XL, Spangenberg G (2000) Down-regulation of major pollen allergens in lransgenic ryegrasses. In: Abstracts Second International Symposium Molecular Breeding Forage Crops 2000, Lorne and Hamilton , 19-24 November 2000, p 15. Forster JW, Jones ES, Kölliker R, Drayton MC, Dumsday JL, Dupal MP, Guthridge KM, Mahoney NL, van Zijll de Jong E, Smith KF (2000) Development and implementation of molecular markers for forage crop improvement. In: Spangenberg G (ed), Molecular breeding of forage crops, Kluwer Academic Publishers , Dordrecht, chapter 6 (this volume). Francis CM, Millington AJ (1965) Varietal variation in the isoflavone content ofsubterranean clover: its estimation by a microtechn ique. Australian Journal of Agricultural Research 16: 557-654. Garrett R, Zhang L, Chu P (1998) White clover virus resistance. Final report October 1994-June 1998, to the Dairy Research and Development Corporation Project CSP 133 Phase I. Geliert VM, Smith KF, Reed KFM (1999) Capturing the yield potential of the Victorian perenn ial ryegrass ecotype . In: Proceedings 1I th Australian Plant Breeding Conference Langridge P, Barr A, Auricht G, Collins G, Granger A, Handford D, Paull J (eds) 2: 255256. Humphreys MO (1989) Warer-soluble carbohydrates in perennial ryegrass breeding . I. Genetic differences among cult ivars and hybrid progeny grown as spaced pJants. Grass and Forage Science 44: 231-236. Jahufer MZZ, Cooper M, Bray RA, Ayres JF (1999) Evaluation of white clover populations for summer moisture stress and adaptation in Australia. Australian Journal of Agricultural Research 50: 561-574 .

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