BIOCHEMICAL COMPOSITION

BIOCHEMICAL COMPOSITION

B.S. Kurlovich, L. T. Kartuzova, J. Heinänen, I.I. Benken, Z.V. Chmeleva and M.L. Bernatskaya

Leguminous crops, including lupin, are an important source of protein and other nutritious substances. The major physiological and biochemical feature of lupin is the capability to synthesize plenty of protein. Due to its coexistence with nodule bacteria Bradyrhizobium sp. (Lupinus), lupin possesses high nitrogen-fixing ability, the means of acquiring molecular nitrogen from the atmosphere, which is used to produce protein and other nitrogen substances. Among the rich specific diversity of lupin, there are species, varieties and forms, which accumulate large amounts of protein, oil and other useful substances (Kurlovich, 1998). At the same time, between separate species, varieties and forms there are differences not only in the quantitative content of useful substances, but also in their qualitative structure.

When the lupin collection of the N.I.Vavilov Institute of Plant Industry (VIR) was founded in 1924, it became the object of systematic studies, including biochemical structure of different nutritious and anti-nutritious substances (protein, amino acids, oil, alkaloids, inhibitors and so on). In particular, in 1932 the Institute’s scientists developed an efficient method of rapid determination of alkaloids in seed and plants by means of the assay of Burhard (solution J in KJ), which was for the first time in the world published with the foreword of N.I Vavilov (Иванов et al.,1932), because the methods of alkaloid determination invented in Germany in 1928-29 by von Sengbusch were kept secret. In 1935, the fact of discovery of oil-bearing lupin was published (Ермаков et al. 1935). Features of the diversity of lupins, including biochemical parameters, are also presented in other works (Либкинд, 1931; Шарапов, 1935). Generalization of the accumulated data on lupin’s biochemistry was conducted by M.I.Smirnova in 1938 and published in a slow-paced manner as the serial Biochemistry of Cultivated Plants (Смирнова, 1938).

In 1974, the comprehensive study of N.A Maissurjan and A.I. Atabiekova was published. In this work much attention was paid to the biochemical structure (Maissurjan and Atabiekova, 1974). These works were published in Russian, so the access to them for the worldwide scientific community was limited. The only exception was the book by P.M. Zhukovsky, which was translated in Australia to many languages and is widely quoted (Zhukovsky, 1929).

In the present section, the results of biochemical studying the lupin collection of the N.I.Vavilov Institute of Plant Industry (VIR) in 1980-2000 years are generalized. Biochemical structure was analyzed in the samples of definite species with the same years of reproduction but from different places of growing. Weather conditions in different places of research studies and in different years were diverse, which helped to reveal the substances’ range of variability both within separate accessions and within the limits of a species. Determination of protein was conducted by Kjeldahl’s technique (N X 6,25), lipid by the method of degreased residual. Amino acid structure was analyzed on the automatic analyzer Per Con Inframatic 8620. For calibration of the instrument, the method of Kjeldahl (N x 6,25) was used for protein, and the method of degreased residual was used for oil. In studies of quinolysidine alkaloids (quantitative methods) were conducted by chromatographic techniques described in a number of methodological works (Мироненко, 1975; Wink, 1992, 1995; Allen, 1986; Aniszewski; 1993). Qualitative tests for the presence of alkaloids and searching of low alkaloid (sweet) forms were carried out with the help of sensitive paper impregnated by solution of J in KJ (Иванов et al., 1932). Activity of inhibitors was determined spectrographically with casein as a substratum (Бенкен, 1982,1983). In 10 samples, activity of hemagglutinins was measured by applying the quantitative photometric method (Ярош and Бенкен, 1994). Inhibitors and phytohemagglutinins extracted from the previously degreased flour after infusing it with petrologic ether under room temperature. The activity of hemagglutinins was determined in correlation with rabbit erythrocytes previously treated by trypsin, as they have not produced any effect on agglutination process. Complete lists of the studied species and accessions, as well as their full characteristics are presented in the catalogues issued by the Vavilov Institute in Russian (Каталог мировой коллекции ВИР. Вып.416: Бернацкая, Степанова, 1984; Каталог мировой коллекции ВИР. Вып.496: З.В.Чмелева, И.И.Бенкен, Б.С.Курлович, Л.Т.Картузова, 1989; Каталог мировой коллекции ВИР. Выпуск 511: М.Л.Бернацкая, Б.С.Курлович, З.В. Шошина, 1990; Каталог мировой коллекции ВИР Вып. 568: З.В.Чмелева, И.И.Бенкен, Б.С.Курлович, 1990; Каталог мировой коллекции ВИР. Вып.637: И.И.Бенкен, Б.С.Курлович, Л.Т.Картузова, 1993).

In the present work, an attempt is made to generalize the results obtained.

Results of investigation

Nutrient substances

White lupin – L. albus L. It has been shown by the research accomplished that a significant intraspecific variability of protein content is represented in the collection of VIR: in Kiev Province of the Ukraine from 35.0 up to 53.7%, whereas in humid subtropical environments of Abkhazia up to 55.0%. The largest group (330 samples, or 54.4% of the total number of accessions) in Kiev Province contained 40.1 – 45.0% of protein. The tables with the data and figures on this section are here. 

The highest accumulation of protein (over 50.0%) was marked in 2 samples: k-2202 from the Ukraine, and cv. Herkvell (k-1496) from Germany. High protein content (45.1 – 50.0%) was observed in 27 accessions from Russia, Belarus, Australia, England, France, Palestine, Poland, Sudan and other countries.

As witnessed by the data of the analyses, the amount of methionine in white lupin seed varied within the limits from 177 to 320 mg per 100 g (or 0.4 – 0.7% in protein). Accessions with increased methionine content (280-320 mg/100 g) made only 5.3%, while the content of 201-240 mg/100 g was found in 65.6%. An increased amount of deficient essential amino acid methionine combined with high protein content was observed in k-2209 from Belarus (Mogilev Province), k-495 from Ethiopia, and cv. Astra (k-2375) from England. Accessions with high protein content were analyzed to select among them the accessions with high content of lysine: Herkvell (k-1496), Hadmerslebener Kraftqueill (k-1661) from Germany, k-2094 from Libya, and k-495 from Ethiopia. The latter combined high percent of protein, methionine and lysine with rather high seed productivity. Fig. 29. Distribution of white lupin accessions (L. albus L.) by the level of protein and oil storage

Significant intraspecific variability in oil content (6.2 – 12.0%) was found in Kiev Province among the collection accessions (Fig.29). Breeding cultivars and accessions from Russia, Spain, Poland and Palestine showed increased oil content (9.5%, on the average, and higher), while accessions from Italy and Ethiopia demonstrated reduced amount of oil (less than 9%). On the average, the samples developed in the Ukraine accumulated 9.3 % oil in their seed. A group of accessions with high oil content (over 10.5%) was selected (k-203, 223, 290, 298, 302, 303, 474, 1649, 1784, 1810, 1904, 1984, 2172, 2191, 2273, 2297, 2339, 2587).

Yellow lupin – L. luteus L. The range of protein content variability in the analyzed accessions of yellow lupin was 44.8 – 55.0% in Kiev Province, and 44.6 – 45.2% in Zhitomir Province. For the greatest majority of accessions, typical levels were 40.1 –45.0% in Kiev Province, and 35.1 – 40.0% in Zhitomir Province (Fig. 30).

Narrow-leafed lupin – L. angustifolius L. According to our data (Fig.31 and 32), the amplitude of variability of protein content in the seed of the accessions developed in Moscow Province was 18 - 38%, while oil content ranged from 4.8 to 8.5%. In Kiev Province these parameters were respectively 26.8 – 41.6% and 3.2 – 7.3%. The level of protein content in Moscow Province, if compared with Kiev, was lower, but the level of oil content was higher. Thus, the sources of high protein and oil content selected in different regions were characterized by slightly different values.

Andean pearl lupin – L. mutabilis Sweet. The content of protein in the collection accessions grown in Tambov Province varied from 42.4 to 51.7%, and oil content from 10.5 to 16.3 %. Ecological testing of several samples on different sites showed (Fig.33) that in the area of Sukhumi, Abkhazia, seed had accumulated more protein (up to 54.2%), and in the area of Tashkent, Uzbekistan, more oil (up to 20.8%).

Multifoliate or Washington lupin – L. polyphyllus Lindl. Protein content in green matter (early flowering phase) and in seed of this species was studied. In Leningrad Province (Pushkin Town vicinity), the amount of protein in green matter varied from 13.6 up to 22.2%, in terms of dry matter, with moisture content of 86.4 – 90.5%. Accessions k-285 (Latvia), as well as k-58 and k-190 (Germany) also demonstrated high values of protein amount recalculated on a plant, owing to the high productivity of plants. In the remaining accessions, the gross amount of protein recalculated on a plant was not high because of their low productivity. Samples with high yield were also selected, a part of which ensured high yields of high-quality green matter and seed (k-118, 215, 273, 428, 670, 1422 etc.), while another part consisted mainly of biotypes with high productivity of either green matter (k-84, 116, 119, etc.) or seed (k-112, 155, 213, 317). Seed protein content of the collection accessions of L. polyphyllus Lindl varied within the scope of 42.1 – 48.4%. Table 17 presents the data on the content of protein and its amino acid structure in the alkaloid-containing cultivar Pushkinsky, and the low-alkaloid (sweet) commercial cultivar Pervenets which was bred by us for Russian environments.

Other species of lupin. The data of seed protein content and amino acid structure of protein for 13 lupin species are grouped in Table 17.

Anti-nutritional factors

The study covered alkaloid content in different lupin species, the range of its variability (in alkaloid or bitter forms) as well as the basic structure of alkaloids (Tab. 18). The range of variability in the total content of alkaloids in bitter forms was 0.10 – 4.00%. In low-alkaloid (sweet) forms, residual content of alkaloids was 0.025 – 0.099%. The research resulted in finding new low-alkaloid (sweet) forms of L. polyphyllus Lindl. and other lupin species. We offer two methods of obtaining stable low-alkaloid fodder (sweet) populations of L. polyphyllus Lindl which provide for essential reduction in the number of alkaloid plants in populations (Tab.19). Table 20 presents the data on the activity of proteinase inhibitors (tyrosine and chymotrypsin) in main lupin species.

Discussion

Nutrient substances

White lupin – L. albus L. The research performed has shown that the genetic diversity of white lupin is characterized by a high degree of variability in the content of protein, oil and some essential amino acids. This circumstance opens an opportunity for its broad and effective utilization in future breeding. It is widely known that methionine is one of the most limiting amino acids in leguminous crops.

Unfortunately, our research failed to identify effective sources of rather high content of this essential amino acid. Average values of methionine content are found in varieties from Belarus (280 – 320 mg per 100 g). Identification of an effective source of high methionine content would make it possible to promote the protein of lupin to a qualitatively new stage. In this case, it could rival animal protein by nutritive value. White lupin is also an oil-producing crop. High oil content in the same accessions of VIR’s collection demonstrates stability through different years of cultivation, which testifies to the genetic determination of this character and value of these accessions for breeding cultivars with high oil yield. Worth mentioning is the fact that 5 accessions (k-223, 298, 303, 2339 and 2587) showed high content not only of oil, but also of protein (more than 45%). However, negative correlation between seed oil content and protein content was observed in most cases (Fig.34 and 35); this fact was especially obvious in the process of ecological testing of the accessions in different areas (Fig. 34). Furthermore, it became clear that samples of different origin varied in their ability of protein and oil accumulation. Variations in the degree of protein and oil content in the seed of local forms belonging to different geotypes from western and eastern Mediterranean areas are presented in Fig. 35. Besides, it should be marked that the selected samples had different productivity, and in view of this the output of oil, protein and methionine also was unequal in them. Thus, the study of white lupin accessions has proven that the collection of VIR comprise lupin materials, biochemical performances of which considerably exceed the parameters of quality in commercial cultivars. It opens an opportunity to attain effective breeding results in improving seed quality. For comparison, Table 21 presents the performance of commercial cultivars in the Ukraine and Russia in terms of their efficiency and biochemical structure. Seed yield of these cultivars makes in recalculation on 1 m2 293 – 367 g, content of oil in seed 7.6 – 10.7%, protein 38.0 – 44.3%, methionine 207 – 273 mg/100 g. Per 1 m2, the output of oil is 23.3 – 34.4 g, protein 111.2 – 153.6 g, and methionine 680 – 960 mg/100 g. However, in spite of high biochemical parameters, in most of the identified sources of white lupin’s quality, gross production of protein, oil and essential amino acids per an area unit is rather low due to their poor productivity. For a majority of accessions, negative correlation is marked between high biochemical parameters and efficiency. This fact should be taken into account by plant breeders whose ultimate goal is to create high-yielding cultivars, ensuring combination of high productivity with the highest possible outcome of protein, oil and essential amino acids from an area unit, as it is visually shown in Table 22. The accessions appearing in Table 22 are also recommended for use in breeding practice. It seems expedient to include in hybridization process such accessions that have the maximum content of oil, protein and essential amino acids. In this case, there is a possibility to obtain positive transgressions on seed quality and productivity, combine these properties in the progeny, and thus increase the efficiency of plants.

Yellow lupin – L. luteus L. Yellow or European lupin is one of the most promising fodder crops for Belarus, Russia, Ukraine, Poland and other European countries. It has high protein content in seed and green matter. Most of the high protein content sources listed in Table 23 deserve attention from the viewpoint of breeding owing, first of all, to the year-to-year stability of protein content.

However, when protein accumulation rate is concerned, considerable effect is produced by the amount of rainfall in the areas of their cultivation. (Fig.30, Tab. 23). For instance, in the environments of Zhitomir Province, where the average annual precipitation does not exceed 537 mm, plants were accumulating much less protein than in Kiev Province (about 200 km eastwards), where the rainfall amounts to 800-1000 mm. The dynamics of protein accumulation in ontogenesis has the following regularity: in the flowering phase the content of protein is reduced (if compared with previous phases), and in the phases of beans it slightly increases. Amino acid structure of the accessions, checked by ecological tests in the forest-steppe zone of the Ukraine (Zhitomir Province), testifies to narrow genetic determination of the amino acid pattern in yellow lupin (Бернацкая and Валовненко,1984). Seed proteins are rich in lysine, arginine, hiscidine, leucine, isoleucine, phenylalanine and valine, but show a deficit of methionine. Cultivars resistant to Fusarium wilt — Borluta, Afus, Cyt, Tomik, Refusa Nova —display the tendency to increase the level of methionine in protein in comparison with other accessions. It seems tempting to connect this phenomenon with the presence of resistance to Fusarium wilt.

However, a convincing warning against such assumption is the amino acids structure of widespread but susceptible to Fusarium cv. Akademichesky 1. In lysine and methionine content, it is not inferior to the resistance cultivars. As to the amount of essential amino acids, this cultivar surpasses all investigated accessions and, therefore, can be serve as a source of well-balanced amino acids structure of protein.

Narrow-leafed lupin – L. angustifolius L. Seed of narrow-leafed lupin make an important source of protein for food and feed. As a component in mixed forages for cattle, swine and poultry, they are capable to replace soybean flour. Flour made of lupin seed also has a number of advantages in confectionery (Hill, 1977). By now, the greatest progress in narrow-leafed lupin cultivation has been made in Australia (Gladstones, 1998). This species is distinguished for the wide range of variability in the accumulation of nutrients and other properties If compared with other species (L. albus and L. luteus), seed of L. angustifolius accumulate a little less protein (18.0 – 41.6%). Oil content varies from 3.2 to 8.5%. The degree of accumulation of these substances depends on the plant genotype and the conditions in the places of cultivation. Our research has shown that on the way from north to south (from Moscow to Kiev) protein content in plants was increasing, while oil content was reducing. Besides, negative correlation was found between protein and oil accumulation rates. (Fig. 36). Some accessions from Belarus (Vada-15, Vada-6, Vada-17) were protein-rich in both areas.

Perl lupin – L. mutabilis Sweet. Pearl lupin is a quite unique ancient cultivated species with large seeds, which originated on the American continent. It was domesticated by ancient population of the continent more than 15 thousand years ago. Seed of this species were used for a long as a source of protein after the removal of alkaloids by means of soaking and cooking. Now, low-alkaloid (sweet) forms of this species are available. Andean lupin has a number of valuable traits and properties: large size of seed, high biological value of protein and oil, suitability for food of man, non-dehiscent pods, determined plants (Baer, 1994; Römer, 1994; Gladstones, 1998). Breeding work with L. mutabilis is underway in Chile, Germany, England and other countries. In Russia and other countries of the former USSR, L. mutabilis is not cultivated due to the shortness of vegetation period, though introduction of this crop into cultivation has good prospects. As shown by our research, this species is characterized by extremely high oil content and rather high content of high-quality protein (Tab. 24), the latter depending on the plant genotype and conditions of cultivation. The highest oil content (up to 20.8%) is found in plants grown in arid environments (Tashkent Province, Uzbekistan), while the highest accumulation of protein is observed in humid subtropics of Abkhazia (Sukhumi area). Also revealed was the negative correlation between quantitative accumulation of oil and protein.

Multifoliate or Washington lupin – L. polyphyllus Lindl. Perennial forms of lupins, and multifoliate or Washington lupin (Lupinus polyphyllus Lindl.) in particular, are very interesting for agricultural production in northern countries. Multifoliate Washington lupin can be grown for fodder and seed in the countries with limited agricultural resources and a deficit of vegetation period for other lupin species, for example, in northwest areas of Russia (Kurlovich et al., 1995), Finland, (Aniszewski, 1993), Sweden (Maissurjan and Atabiekova, 1974), and other northern countries. It can be cultivated on low-fertility sandy and acid soils unaccustomed for other crops. Under such conditions, it can be grown and harvested for 4 to 6 years, and provide a yield of green matter up to 40-50 t/ha. Our observations have shown that each sample of Lupinus polyphyllus Lindl. represents a complex population consisting of various biotypes. Their genetic heterogeneity is displayed in variable growth and development rate, size and number of stems, life duration, etc. Efficiency and quality of a population depend on the proportion of various biotypes in it. In view of this, the range of variability in green matter yield at the flowering phase is within the limits of 200 – 2605 g recalculated on one plant, while seed yield is within the limits of 15 – 163 g. Besides, a majority of accessions demonstrate negative connection between high biochemical parameters and yield. This circumstance should be taken into account by plant breeders, whose ultimate goal is to create high-yielding cultivars, ensuring combination of high productivity with the highest possible outcome of protein and other nutrients from an area unit.

Anti-nutritional factors

Alkaloids. The information presented in Table 18 shows that, if not to consider unknowns alkaloids with unidentified toxins which are present in rather small amounts, the studied species of lupin can be grouped in the following manner. With the Mediterranean species, a majority of all most toxic alkaloids are contained in the bitter forms of L. angustifolius and L. luteus . Among these alkaloids lupanine predominates in L. angustifolius and lupinine in L. luteus. More favorable structure of alkaloids is characteristic of one of the oldest domesticated species, L. albus. Its seed from ancient times were used for food after boiling and cooking in Egypt and Italy. It contains in almost equal amounts toxic lupanine and not so toxic 13-hydroxylupanine. Finally, a most interesting alkaloid composition is observed in L. pilosus. Its seed contain not too toxic 13-hydroxylupanine. No wonder, therefore, that even the large seed of bitter forms of this species are sometimes used for food. A different composition of alkaloids is typical for the American species. First of all, their seed are, as the rule, lupinine-free. Only one of the most primitive species, L. subcarnosus, plentifully accumulates this alkaloid. There is a lot of toxic lupanine in bitter forms of L. polyphyllus. L. elegans has rather low total content of alkaloids, but the main share also belongs to lupanine. Medium amount of slightly less toxic sparteine is found in the seed of L. barkeri. Very toxic alkaloids – lupanine and sparteine – are observed in annual sp. L. ornatus. It is interesting that the garden (ornamental) form of this species, known under the name of L. albococcineus, contains little sparteine and traces of 13-hydroxylupanine, but fairly large amount of an unknown alkaloid. Rather low content of lupanine and 13-hydroxylupanine is typical for the unique ancient cultivated American species L. mutabilis. However, it contains much sparteine. L. hybridus, which, according to our data, can be artificially reproduced by crossing L. mutabilis with L. ornatus, is intermediate in terms of its alkaloid structure, and only high content of lupanine draws it closer to L ornatus. Alkaloids of L. hartwegii are mainly represented by not too toxic 13-hydroxylupanine. Toxic lupanine is found in it only in small amounts. A special place in terms of alkaloid content and composition is occupied by L. nanus. Seed of this species contain low quantity of alkaloids, among which 13-hydroxylupanine plays dominating role. The experiments helped to identify great fluctuations in the quantitative content of alkaloids in different lupin species depending on their genetic composition and environmental conditions. The results of our research witness that each of the studied species, in addition to 1 – 2 main alkaloids, contains from one to four accompanying ones. For example, lupinine is always present in yellow lupin, and lupanine in various forms of narrow-leafed and white lupins. Yellow lupin, as a rule, possesses sparteine as the second main alkaloid, and only wild forms show just traces of sparteine. All above-mentioned species are alkaloid-bearing or so-called bitter forms. As to low alkaloid, fodder or sweet forms, the residual content of alkaloids in them does not exceed 0.09%. In low-alkaloid (sweet) forms of L. luteus, for example, sparteine is either completely absent, or present in a very limited amount. While the bitter forms of white lupin are rich in 13-hydroxylupanine, its sweet forms are either completely free of this alkaloid, or possess it as an accompanying one. The first steps of modern sweet lupin breeding were taken in 1928/29 by Reinhold von Sengbush in Germany, who selected the first low-alkaloid forms of L. luteus and L. angustifolius as natural mutants. He was guided by the ideas and lectures of Bauer who cited Vavilov’s law of genetic series in variation and his studies of the problem of the species as a system (Vavilov, 1920, 1931, 1965) as the grounds for the assumption that natural alkaloid-free mutants could be found, if sought hard enough, but they should occur rarely enough, because susceptibility to diseases, insects and animals must under normal circumstances lead to their selective elimination. It is worth mentioning here that, since the methods of alkaloid determination developed in Germany by von Sengbusch were kept secret and German sweet lupin cultivars were sold to a private firm, the Russian scientist N.N. Ivanov from VIR managed to work out, independently from German achievements, an efficient method of rapid alkaloid determination by means of Burhard’s assay (solution of J in KJ). This technique was immediately and for the first time in the world published with N.I Vavilov’s foreword (Иванов et al., 1932).

This publication together with the brainchild of German scientists formed the core of future breeding work with fodder (sweet) low-alkaloid lupin all over the world.

Since that time, lupin has been recognized as valuable fodder crop. Due to its high protein content, sweet lupin is promising as a protein-yielding component in mixed forages where it can successfully replace soybean flour (Hove, 1974). It should be added that it is very important not only just to have low alkaloid (sweet) forms, but also to maintain their genetic integrity. It is known that bitter plants sometimes appear in the plantings of fodder (sweet) cultivars of all species of lupin. It is especially typical for the long-term Lupinus polyphyllus Lindl., because of the difficulties set by its perennial lifestyle and cross pollination (Kurlovich et al.,1995). Low alkaloid content cannot be stabilized in this cross-pollinated species. The genetic code of plants does not enable them to deviate too far from the normal status by means of mutations, and always tends to correct admissible errors. For this purpose, there are such special mechanisms as complementarity, cross-pollination, back mutations, etc.

Due to the complimentary interaction of different genes determining this character, alkaloid content tends to restore (Maissurjan and Atabiekova, 1974; Micolajczyk, 1966, Nowacki, 1963, Турбин and Анохина, 1974). That is why bitter plants appear in crops, preventing wide utilization of grass and seed for fodder. In this connection, one of our problems was to develop methods that would help to avoid or significantly reduce the rates of alkaloid syntheses under cross-pollination in multifoliate Washington lupin.

Fundamental distinction of the developed approach lies in the fact that all sources of low alkaloid content, selected or bred, are not combined to ensure cross-pollination as it was done earlier (Воронов, 1974), but only compatible forms are used, in which low alkaloid content is controlled by the same genetic system. For realization of this approach two methods were designed (Tab.19). The first method of obtaining fodder (low-alkaloid) populations of Lupinus polyphyllus Lindl. was as follows: selected low-alkaloid plants were crossed with each other and the progeny from each cross was grown separately. Populations fully consisting of low-alkaloid plants were selected as initial material for breeding. In the second method, every low-alkaloid plant was crossed with a productive alkaloid form, which had some other valuable characters, and populations with stable low alkaloid content were identified in F2. The latter approach makes it possible to increase the efficiency of plants. Applying these methods and studying the breeding sources led to the development of fodder (sweet) forms of multifoliate Washington lupin with a sufficiently stable level of low alkaloid content. The number of bitter plants in the populations obtained by the above methods was much lower (0.30-0.45 %) than in a population created by a conventional method (Table 12). Besides, the productivity of plants was much higher when the second method had been applied than with the application of the first one.

Utilization of these methods in the Ukraine resulted in releasing a fodder cultivar of multifoliate lupin. This cultivar, Truvor, is currently passing state trials in the Ukraine. In the conditions of northwestern Russia, positive results were attained when we released the commercial cultivar Pervenec (first sweet variety) included in the State Catalogue of Breeding Achievements of Russia.

The proposed techniques exclude any possibility of the appearance of alkaloid plants at the expense of genetic homogeneity in the obtained low-alkaloid populations and the absence of complementary interaction between non-allelic genes that stipulate reconstruction of high alkaloid content. However, in such fodder (sweet) crops as yellow, white and blue lupins, as well as in fodder low-alkaloid multifoliate Washington lupin, partial appearance of bitter plants may happen owing to inverse mutations and also as a result of re-pollination of the low-alkaloid (sweet) forms with the pollen of wild bitter alkaloid ones. In this connection, in all sectors of seed production of fodder low-alkaloid multifoliate Washington lupin forms it is necessary strictly to observe spatial isolation of different samples and undertake continuous check-up over the level of low alkaloid content. Bitter plants, when found, must be removed and discarded prior to the blossoming phase. Low-alkaloid fodder multifoliate Washington lupin is a perennial entomophilous plant. In view of this, to arrange its high-efficiency seed production it seems reasonable to establish a special nursery and to use it for a number of years removing from the vicinity other varieties and wild forms of bitter lupin. In the first year of plant life in such nursery, it is necessary to check up all plants for alkaloids and remove all identified bitter plants before blossoming. Afterwards, such nursery will satisfy the demand for seed for feed purposes during many years and on greater areas.

The content and structure of alkaloids in lupin were described by many authors (Hacbarth and Troll, 1955; Maissurjan and Atabekova, 1974; Мироненко, 1975; Allen, 1986, 1998; Aniszewski, 1993; Wink et al, 1995; Petterson, 1998; and others), but their data differed from our results, because the amount and structure of alkaloids are influenced by the environments.

Inhibitors of proteinase and hemagglutinins. For the promotion of food and feed utilization of lupin, quite interesting is the research on the forms containing protein with anti-physiological effect on an animal organism — inhibitors of proteinase and hemagglutinins. Plants of the leguminous family are known to accumulate them in the greatest amount. However, among legumes there is significant differentiation of species according to the content of inhibitors. It was obvious even from the first report on inhibitors (Borchers et al., 1947) that in the leguminous family, along with the species accumulating greatest amounts of inhibitors (string bean, soybean), there are species with zero activity, among which peas, beans, lentil, vetch and blue lupin were reported.

Information on inhibitors in lupin is limited enough. Some authors (Gallardo et al., 1974) informed on the absence of trypsin inhibitors in the seed of white and yellow lupins. The authors from New Zealand (Hove and King, 1979) investigated the activity of inhibitors in the seed of three species of lupin and did not detect them in white and blue forms. Only L. mutabilis demonstrated minor activity within the limits of 0.3 – 0.4 mg/g. However, there are reports concerning amount of trypsin inhibitors in lupin (Домаш, 1979; Allen, 1986, 1998; Petterson, 1998). Information on lectins (hemagglutinins) in lupin is even more sketchy (Petterson, 1998). There is some evidence that yellow lupin does not demonstrate activity in relation to rabbit’s erythrocytes, and white lupin exerts influence on the procedure of agglutination in cattle (Contreras et al, 1974). As shown our research, the inhibitors were find, but not in all cases and in insignificant amount. Especially low figures characterized the activity of inhibitors in yellow, narrow-leafed and white lupins (Tab.20). With many accessions of yellow lupin, we were unable to detect any activity of trypsin and chymotrypsin at all. Moreover, the highest activity did not exceed 0.1 mg/g. Percentage of enzyme inhibition in the test was only 5 – 10%. The activity of chymotrypsin inhibitors was on the same level or slightly lower. The activity in the samples of L. mutabilis was hardly higher. Even more noticeable amounts of trypsin and chymotrypsin inhibitors were found in some small-seeded species — L. pubescens, L. ornatus and L. douglasii, with the gross activity of about 1.0 mg/g. Comparison with other grain legumes shows that lupin may be recognized as the most low-inhibited crop. In particular, with the use of the same method, the average activity of inhibitors for many soybean accessions was 27 mg/g, for ordinary string bean 10 mg/g, and for peas 2 mg/g.

Therefore, the content of inhibitors in Lupinus spp. much lower than in other leguminous crops. Lectin (hemagglutinin) activity appeared very weak and unstable, 1 – 2 units /mg. It was much lower than in peas, and more than ten times lower than in soybean and string bean. Thus, lupin appeared to contain the least amount of proteins having anti-nutritious properties: inhibitors of proteinase and hemagglutinins. They are practically absent in the main cultivated species and cultivars.

Lupin, like other grain legumes, is cultivated as a source of high-quality protein, essential amino acids, oil and other nutritive substances. In view of the above-mentioned, the major problem of lupin breeding is further improvement of the quality of seed and green matter. As a result of our research on the biochemical structure, accessions of different lupin species from the collection of VIR were identified as initial materials for future breeding. Samples having an increased content of nutrients and low content of anti-nutritional factors were selected. Their variability demonstrated a certain regularity depending on specific and varietal features of plants, their origin and conditions of cultivation. Rather broad polymorphism in the content of different substances was marked in the investigated collection accessions. Thus, it might be expected that breeding efforts toward increasing protein quality and its total amount can be successful. Intravarietal variability in seed protein content from year to year was frequently wider that intervarietal. Among the investigated species, the highest content of protein was found in white and yellow lupins (up to 55 %).

The greatest content of oil was found in the accessions of L. mutabilis Sweet. (in the conditions of Uzbekistan, up to 20.8 %).

Eco-geographic tests of accessions have revealed essential influence of environmental conditions (amount of rainfall, sum of effective temperatures, types of soil) on the level of accumulation of various substances in the plants of all main species of lupin under investigation (Figures 29-34). In humid climate, plants accumulated, as a rule, more protein, while in dry conditions they stored more oil. Narrow-leafed lupin revealed the tendency to increase accumulation of protein and decrease accumulation of oil in the southward direction (from Moscow to Kiev).

Besides, our research has identified certain regularities in the accumulation of protein and oil depending on the origin of accessions. The genotypes from the countries with humid climate (in particular, western Mediterranean area) were usually distinguished for more intensive accumulation of protein, whereas the forms from the countries with droughty climate (eastern Mediterranean area) accumulated more oil. The correlation between protein and oil accumulation was, as a rule, negative, which is visually testified by the data on variation in the degree of protein and oil content in the seed of local forms of Lupinus albus L. belonging to different geotypes. (Fig. 35).

Besides, in spite of high biochemical parameters, for a majority of the identified sources of lupin quality, the gross accumulation rate of protein, oil and essential amino acids per an area unit was rather low, which is connected with their rather low productivity. Most of the accessions of all species displayed negative correlation between high biochemical parameters and yield. This circumstance should to take into account when managing the breeding work with the ultimate goal to create highly productive cultivars and ensure due to high productivity the maximum amount of protein, oil and essential amino acids per an area unit. With these purposes, we also recommend to use high-yielding samples in the breeding process. It seems expedient to engage them in hybridization with the accessions having the highest content of oil, protein and essential amino acids. In this case, there is an obvious possibility to obtain positive transgressions on seed quality and productivity, combine these properties in the progeny, and consequently increase the efficiency of plants. The nutritive value of protein in lupin is reduced because of an unbalanced amino acid composition, as it is characteristic of all fodder crops. There is a deficiency in methionine — an essential sulfur amino acid. Therefore, breeding work toward enhancement of qualitative and quantitative structure of protein acquires primary significance. The content of protein – a composite hereditary character – is influenced by environmental conditions. For grain legume, including lupin, the effect of the genotype upon the level of protein content is more significant than in grain cereals, which instills hope for successful future breeding. Increasing the content of methionine in new lupin cultivars would make it possible to raise lupin protein to a qualitatively new stage – in terms of nutritive value it would rival animal fiber. In order to breed protein-rich forms, it is necessary to deal with complex genetic systems and genomes. In our judgement, there are following possibilities to improve genetically the quality of protein in the part of increasing the amount of essential sulfur amino acids:

· inducement of mutations causing substitution of other amino acids by sulfur amino acids;

· search for regulable genes influencing the ratio of fiber components and enhancing the synthesis of a complex balanced on sulfur amino acids (Бернацкая and Валовненко, 1984);

· identification and creation of transgressive forms with the help of the eco-geographical approach developed by us (Kurlovich et al, 1995, 2000b).

With the purpose of inducing positive transgressions on biochemical parameters, it is expedient to make crosses between protein-rich, amino acid and oil-bearing forms. It is recommended in this case to select pairs for crosses by using the eco-geographical approach; studying valuable accessions in contrast environments and revealing allelic distinctions between them in the control of characters. It appeared (Kurlovich et al, 1995) that when crossing the parents with an identical type of character variability in contrast environments, the hybrid progeny would possess this character within the same limits as the parent forms. But, when the parents had different types of variability, the hybrid progeny could contain transgressive forms, i.e. the forms with the character’s value different from its parents (higher or lower). Distinctions in the variability of characters in future parental forms are revealed by testing them in different environments, however, by similar techniques. By this method it is possible to obtain valuable transgressive forms on any plant traits, including biochemical structure of protein and oil (higher content of them), or lower content of anti-nutritive factors (Kurlovich, 1998; Kurlovich et al, 2000b).

Value of lupin means practical absence of anti-nutritive factors (inhibitors of proteinase and hemagglutinins) in its protein. Seed of lupin (its low-alkaloid sweet forms), unlike other leguminous crops, are suitable for raw use, without heat treatment. It is possible to produce various foods from them, and also to use them as protein-rich components. In view of this, lupin is recognized as an effective alternative of soybean in the world’s agriculture.