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    · Lupins for Health and Wealth Proceedings of the 12TH INTERNATIONAL LUPIN CONFERENCE Fremantle Western Australia 14 18 September 2008 Jairo A Palta Jens D erger Editors  304 IN J.A. Palta and J.B. Berger (eds). 2008. ‘Lupins for Health and Wealth’ Proceedings of the 12 th  International Lupin Conference, 14-18 Sept. 2008, Fremantle, Western Australia. International Lupin Association, Canterbury, New Zealand. ISBN 0-86476-153-8.   P OTENTIAL AND P ROBLEMS OF  L  UPINUS POLYPHYLLUS    L INDL .   D OMESTICATION   Boguslav S. Kurlovich 1 , Fred L. Stoddard 2  and Peter Earnshaw 3   1 International North Express Co., Leppälaaksontie 2 as 9, 52420, Pellosniemi, Finland 2 Department of Applied Biology, PO Box 27, 00014 University of Helsinki, Finland Email: ABSTRACT We are investigating the agricultural potential of the perennial large-leaved lupin (  Lupinus polyphyllus Lindl.) and its hybrids in Finland and Russia. Naturalised populations of this lupin grow widely in both countries and some workers characterise it as a noxious weed. We, however, recognise its potential as an arable crop. Reduced-alkaloid breeding lines have been isolated. Interspecific hybridisation with  L. mutabilis Sweet has provided hardy perennial materials with high seed oil content, suitable for further development into crops when other limitations are overcome. KEYWORDS alkaloids, oilseed, perennial, biomass, interspecific hybridisation   INTRODUCTION American lupin species of subgenus Platycarpos  (Wats.) Kurl. offer great opportunity for breeding enhancement (Kurlovich and Stankevich, 2002). Large-leaved lupin (  Lupinus polyphyllus  Lindl.) was selected for breeding because it is well adapted to Finnish and North-West Russian conditions. Finnish latitude and climate do not permit the growth of the majority of annual lupins except for the earliest forms of  L. angustifolius . Maturation of perennial large-leaved lupin   is highly reliable in Finland and in the North-West of Russia. Now forms with reduced alkaloid content have been developed and this species has achieved potential as a high-grade fodder crop as a result. Large-leaved lupin is ‘difficult’ in breeding terms, because of its perennial developmental cycle, cross-pollination and indeterminate mode of growth. MATERIALS AND METHODS Research material was obtained from the lupin collection of the N.I. Vavilov Institute, St Petersburg, Russia. Many years (1973-1997) of observing the collection sown in various regions of many countries and the use of methods applicable to lupin (Kurlovich et al.  1990), have enabled us to identify and develop new materials of  L. polyphyllus with economically beneficial characteristics. Mechanisms of pollination, pod set and seed productivity in  L. polyphyllus  were investigated with the purpose of artificial creation of highly productive populations. Four basic pollination methods were used in our germplasm development: 1. Open-pollinated control; 2. Spontaneous self-pollination of plants isolated under gauze; 3. Enhanced self-pollination by shaking of plants isolated under gauze; 4. Open self-pollination of individual plants in isolation plots. Rapid field tests for the presence or absence of alkaloids were carried out by means of paper saturated with alkaloid-sensitive Dragendorff reagent. The quantity of protein, oil, and alkaloids and the composition of the alkaloids (both in seeds and in green mass) were determined in the biochemical laboratory of N.I. Vavilov Institute (St Petersburg, Russia) following standard methods (Mironenko, 1975; Wink, 1992). Two lines of perennial large-leaved  L. polyphyllus , bitter cv. ‘ Pushkinsky ’ and semisweet cv. ‘ Pervenec ’, along with two of annual  L. mutabilis , bitter k-2770 selected in Belarus and sweet cv. ‘  Inti ’ were used as parents for interspecific crosses in 1990-2007. RESULTS AND DISCUSSION Each lupin sample comprises a complex population consisting of several biotypes varying in rates of growth and development, dimensions and quantity of podding branches, and lifespan. Individual plants started flowering and fruiting already in the first year (annual forms), others not until the second, third, fourth or even the fifth year, and some plants died without reproducing. Productivity and quality of a sample population depends on the interrelation of the biotypes. Pod set ranged from 21.5 to 70.5% in open-pollinated control (treatment 1) (Table 1), with an average of 44.3% across five years. Spontaneous self-pollination of plants under gauze (treatment 2) gave only 0.0-5.2% seed set. Enhanced self-pollination following shaking of plants isolated under gauze (treatment 3) provided seed set of 2.5-56.2%. The success of artificial self-pollination depended also on the time of pollination, with maximal podding (50.3-56.2%) obtained after pollination on the third day of anthesis. Pod setting following open self-pollination of individual plants in isolation plots (treatment 4) was only slightly less than in treatment 1, and much greater than in treatments 2 and 3 (17.5-58.3%). The absence in Table 1.  Variability in pod set of  L. polyphyllus  in 5 years following 4 different modes of pollination.  305 IN J.A. Palta and J.B. Berger (eds). 2008 ‘Lupins for Health and Wealth’ Proceedings of the 12 th  International Lupin Conference, 14-18 Sept. 2008, Fremantle, Western Australia. International Lupin Association, Canterbury, New Zealand. ISBN 0-86476-153-8. Pod set, % (mean ± SE) Pollination method 1999 2000 2001 2002 2003 Average Open-pollinated control 21.5 ± 1.3 40.5 ± 2.6 23.8 ± 0.8 70.5 ± 4.2 65.1 ± 4.4 44.3 Spontaneous self-pollination of plants isolated under gauze 0.0 3.8 ± 0.1 0.2 ± 0.03 5,2 ± 0.7 4.6 ± 0.8 2.76 Enhanced self-pollination by shaking plants isolated under gauze 2.5 ± 1.9 26.2 ± 6.1 6.5 ± 2.5 56.2 ± 12.1 51.8 ± 9.2 28.6 Open self-pollination of individual plants in isolation plots 17.5 ± 1.2 38.6 ± 2.3 18.9 ± 0.8 58.3 ± 1.2 55.1 ± 3,1 37.7 separate years (1999) of self-pollination was attributable partly to protandry and partly to herkogamy (Vishnyakova and Kurlovich, 1995). Insects are the primary pollen vectors for cross-pollination. Papillae and hollows in the epidermis on different organs of the flower have been identified as another component of adaptation to cross-pollination (Vishnyakova and Kurlovich, 1995). Furthermore, the stigma of the pistil in large-leaved lupin is set much higher than the anthers and is protected by a collar of hairs (Fig. 1A). Similar hairs are found in  L. mutabilis , whereas in  L. angustifolius (Fig. 1B), they are few and short so they do not significantly obstruct self-pollination .  AB Fig. 1.  The stigmas of (A)  L. polyphyllus  and (B)  L. angustifolius Tripping of individual flowers and shaking of the whole plant has proved a reasonable method for enhancing self-pollination, resulting in a good production of self-pollinated seeds. The level of seed set, however, depends on the level of self-incompatibility of individual plants and on environmental conditions. Pollination treatments 3 and 4 appeared to be the most productive for practical breeding. As a result of over 30 years of self-pollination, numerous presumed recessive mutants have been isolated and fixed. Among these are: reduced alkaloid content; large seeded, suitable for cultivation as a pulse; non-shattering pods; water-permeable seed coat (non-dormant); annual instead of perennial; non-vernalising (spring) instead of vernalising (winter): alternative flower colours; alternative seed colours. The reduced-alkaloid mutant (0.2%) was identified as a single plant in 1990 among self-pollinated inbred lines, by use of the rapid Dragendorff test for the presence of alkaloids. The attribute of reduced alkaloid level was fixed during the next few years of the breeding program. Other medium and low-alkaloid mutants were received from other researchers in Belarus, Russia and Germany. Intercrossing of all of these reduced-alkaloid mutants consistently produced reduced-alkaloid progeny, demonstrating that the mutations were in a common gene. The inheritance of the trait was further studied in reciprocal interspecific crosses between reduced-alkaloid  L. polyphyllus  and high-alkaloid (wild-type)  L. mutabilis plants (Table 2). The alkaloid level in the F 1  generation was generally high while the F 2  segregation ratio was consistent with a single recessive gene. Since the gene conditioning reduced alkaloid content of 0.2 ± 0.02% came to light repeatedly in accessions of different srcin and was sufficiently stable, we gave it the symbol redalkpol  and name reducedalkpolyphyllus . Normal alkaloid content in this material is about 3.2%. Lupanine group compounds accounted for 96.5% of total alkaloid content, the angustifoline group 2.6%, the sparteine 0.6% and gramine 0.1%, and this distribution was similar in both the low-alkaloid and normal-alkaloid material. We aspire to achieve a further decrease in the alkaloid content (to 0.01-0.02%) by carrying out research in the following two directions. First, we continue to investigate whether this new allele is fully recessive. Current investigations indicate that this question is valid, as not only bitter plants but also plants with lower content of alkaloids in vegetative organs (< 0.02%) appear from time to time. We have had three plants showing a consistently low alkaloid content (< 0.02%) in green mass for two years in our experimental field, but they have not yet flowered. It gives us hope to identify more viable sweet plants in the future.  306 PROCEEDINGS 12TH INTERNATIONAL LUPIN CONFERENCE Table 2.  Summary of segregation in F 2  families derived from crossing reduced-alkaloid plants with high alkaloid plants (Classification by Dragendorff reagent). Number of plants F 2  families Year Bitter Sweet χ 23:1    L. polyphyllus  cv. Pervenec x  L. mutabilis line k-2770 2003 19 5 0.22  L. mutabilis line k-2770 x  L. polyphyllus  cv. Pervenec 2004 15 6 0.14  L. polyphyllus  cv. Pervenec x  L. mutabilis line k-2770 2005 88 20 2.41 Table 3.  Characteristics of parents and the best interspecific hybrids from the cross  L. mutabilis x  L. polyphyllus . (Average for 2005-2006). Yield per plant, g Identity Days from plantlets to maturity 1000 seed mass, g Seed protein content, % Seed oil content, % Green mass Seed  L. polyphyllus  cv. Pervenec (1 st  and 2 nd   years) 85-120 21- 25 43.9 ± 2.4 4.2 ± 0.61 2420 ± 79 150 ± 2  L. mutabilis line k-2770 120-140 160-165 46.5 ± 1.8 14.0 ± 1.12 2117 ± 25 296 ± 9 Perennial hybrids 80-120 26- 58 45.8 ± 2.8 7.5 ± 1.36 1820 ± 58 178 ± 10 Annual hybrids 95-125 24-105 46.1 ± 7.6 13.2 ± 3.23 1640 ± 93 201 ± 8 Second, we have crossed sweet  L. mutabilis with our reduced-alkaloid  L. polyphyllus , so we can explore the possibility of combining some of the desirable traits of each species in the presence of stable low alkaloid content. The first results have been encouraging, because the alkaloid level in the F 1  generation appeared very high (up to 3.5%). This suggests that the low-alkaloid mutations in the two species are at different parts of the synthetic pathway. If so, and if the meiosis in these interspecific hybrids behaves in a reasonable manner, we may expect to find transgressive segregants in the F 2  and subsequent generations with even lower alkaloid content. Earlier efforts to produce interspecific hybrids between bitter    L. mutabilis and  L. polyphyllus in Russia (1990-1995) demonstrated the feasibility and efficiency of crossing these species, which have identical chromosome numbers, for investigations of the inheritance of some traits and to derive interesting segregants for further study and practical use. It is not difficult to cross these species and to obtain a wide diversity of hybrids, though the percent of success in fertilisation is lower than in intraspecific crosses. The most typical deflections of development in hybrids are non-uniform allocation of pods on inflorescences flower brushes (rice) and not identical (frequently lowered) quantity of seeds in pods. Cytogenetic analysis of the F 1  interspecific hybrid revealed sometimes abnormal chromosome pairing at metaphase I with a high frequency of univalents in the hybrids in comparison with the parents (Siamasonta, 1996). Because of the widespread naturalised populations of  L. polyphyllus , it is necessary to quantify the frequency of crossing between derivatives of the interspecific hybrids and the natural populations. Significant inflow of bitter genes, among other undesirable traits, may significantly impede the development of this crop. Some 10-15% of the obtained hybrids had individual traits beneficial to the future breeding: larger seed, perenniality, and in annuals a shorter season than  L. mutabilis (Table 3), along with also good seed composition, shattering resistance, frost resistance and high vigour. It was not difficult to isolate each of these characters separately. However, because of the perennial nature of the species and its dependence on cross-pollination, considerable time will be required to recombine some of these attributes into a single genotype. Dominant in Russian and Finnish conditions are: perennial developmental cycle, spring growth habit, shattering of pods, dark blue coloration of flowers, dirty-brown pattern colour of seed coat and bitterness. In addition, by crossing highly inbred lines of  L. polyphyllus (obtained from pollination treatments 3 and 4), we have obtained a wide range of different flower and seed colours, without using  L. arboreus  as was done in the development of the Russell lupins. Our major breeding objectives for large-leaved lupin and its hybrid with  L. mutabilis in Finland since 1996 have been stable low alkaloid content (< 0.02%), different types of pollination (either cross- or self-pollinating), non-dehiscent pods, winter hardiness and frost tolerance. Improved cultivars should also have green mass yield potential in the order of 40-50 Mg fresh matter ha -1 . The first commercial perennial  L. polyphyllus  cultivars with reduced alkaloid content were bred in Russia (cv. Pervenec). Tests and breeding of the best forms of  L. polyphyllus  with reduced alkaloid content for the conditions of Finland is now in progress. They are multiplied by farmers near Tampere. Perennial lupin with reduced content of alkaloids is used as green    PROCEEDINGS 12 TH  INTERNATIONAL LUPIN CONFERENCE  307 mass for preparation of silage. Farmers value this, as the productivity of their animals has increased and acceptability of the silage was good, while no detriment to animal health (cows and horses) was noted. CONCLUSIONS  L. polyphyllus   is not just a weed, and has great potential in the Nordic region as a cultivated plant. Now that reduced-alkaloid forms have been identified, future use of this species for fodder production, green manure, and silage looks possible. Enforced self-pollination for several generations has allowed the isolation of self pollinated forms with valuable attributes. Crossing of these inbred lines, in turn, frequently provides highly heterotic in progeny with new combinations of traits. Enhancing nature’s gift of lupins is possible with effective utilisation of other closely related species with the same number of chromosomes (48), particularly  L. mutabilis , although this process is time-consuming on account of the cross-pollinating nature and perenniality of the species. LITERATURE CITED Kurlovich, B.S., N.S. Nazarova, V.A. Rybnicova, S.I. Pilipenko, L.T. Kartuzova and F.T. Tarba. 1990 . Study of Lupin Accessions of World Collections   (Methods of investigations). VIR, St. Petersburg, Russia, 34p. Kurlovich, B.S. and A.K. Stankevich. 2002. Classification of lupins. pp. 39-88 In: Kurlovich, B.S. (ed.). Lupins. Geography, classification, genetic resources and breeding. Publishing house Intan, St Petersburg, 468p. Mironenko, A.V. 1975.   Biochemistry of Lupins. Minsk, Publishing house Sciences and techniques, 300p. Siamasonta, B.M. 1996. Interspecific hybridisation in the genus  Lupinus.  PhD thesis, Dept of Agricultural Botany, University of Reading, UK. Vishnyakova, M.A. and B.S. Kurlovich. 1995. Mechanisms of pollination and seed productivity in  Lupinus polyphyllus  Lindl. pp. 42-45 In: Berg, G. and Rehhahn, H. (eds.). Yield and Quality in Herbage Seed Production. Proc. International Herbage Seed Group Conference, Martin Luther Universität, Halle-Wittenberg, Germany. Wink, M. 1992. Methoden zum Nachweis von Lupinen-Alkaloiden. pp. 78-90 In: Wink, M. (ed.). Lupinen 1991 – Forschung, Anbau und Verwertund. Universität Heidelberg.
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