Maataloustieteellinen A ikakauskirja Vol. 60: 201—214, 1988 Adaptation of red clover to the long day environment SEPPO PULLI Agricultural Research Centre Dept, of Plant Breeding SF-31600 Jokioinen Abstract. Five known varieties and five local strains of red clover from the latitudes 60 to 66°N were studied in greenhouse conditions. Photoperiods during the growing period were 12,16and 20 h day, night temperatures 17° and 14°C and light intensity 21.2 klux. The plants were cut three times. Flowering, height developmentand shoot DM yield were assessed at each harvest. The number of shoots and leaves per plant and leaf area per plant were determined at the second harvest and root size at the third harvest. After the second harvest one replicate of each strain under the three light regimes was placed at +4°C under an 8-h light regime at 10 klux for 26 days. For assessment of plant response to hardening conditions, chemical analyses were performed on roots for TNC, long-chain fatty acids and proline. The study showed that the adaptation of red clover to certain daylength conditions in- fluences plant behaviour with respect to its growth, development and hardening. The present study on daylength and growth together with developmental and chemical analysis are useful for breeding. Index words: Red clover, daylength, hardening I. Introduction The genus Trifolium includes approximate- ly 250 annual and perennial species, common- ly called clovers, which are native to the hu- mid, temperate regions of the world. About 25 species are of significance as food for graz- ing animals and of these, about 10are agricul- turally important (Evans 1976). The centre of diversity of clovers is the eastern Mediterranean region where the main limiting factor is summer drought (Cooper 1965). Clovers are either winter annuals sur- viving the dry season as seeds or if perennial, show summer dormancy. Most adapted popu- lations from this region demonstrate the abil- ity to grow at low temperatures and low light intensities. In contrast, local varieties from northern Europe display a different seasonal pattern of growth. These clovers are perenni- al, with considerable frost resistance and winter dormancy. Leaf production at low tem- peratures and low light intensities is poor, but increases considerably during the long day and 201 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=Iyin33Jyb4XRz_3L.YlWHjai1KDET87n01O6nBA.GLmS3mm9jHTsEJmnk5SoK5Ie1EaRZTTnTWbfj9mXXK8lnKxMSjnw-QhrODFo1tPgbFESGtYKBWOWAIa99QHRVNos3RzIGD5V1PVionF9JZ2mDwBalLZgXGw9OWXnkAMtiPTlmnmRtZkqTsNflfsOuiSthXoj2pX_0g abundant radiation of the northern growing season. According to the earliness of flowering (Williams 1927), number of cuttings (Pieters 1928) and number of internodes at the onset of flowering (Hawkins 1952), red clover can be classified into three clones of adaptabili- ty: a) early flowering red clover under a sys- tem of one year leys with two or more cuts per year; b) late flowering single-cut type red clover; and, c) intermediate red clover of the two main types. Valle and Garrison (1959), however, have concluded that the behaviour of late flowering single-cut clover approaches that of early type if late type seed production is carried out in areas south of its origin. The early red clover produces shoots with flowers regularly during the year of seeding (Julen 1959). Plant development is rapid and regrowth after cuttings abundant. As a result of fast stem formation, the leaf rosette of the root crown and the whole root system remain underdeveloped. An open leaf rosette provides inadequate protection to the root crown against frost and also the accumulation of root carbohydrates necessary to winter sur- vival is insufficient. Due to the lack of winter survival cultivation of the early red clover is concentrated in southern Europe. The mid-late red clover is considered to be an intermediate of the early and late clover types. It is grown in the areas between those of the two main types, in central Europe and southern Scandinavia from the latitude 50° to 60°N (Julen 1959). Red clover grown in North America is mainly classified into this group. Despite abundant flowering during the seeding year, mid-latered clover is productive in the stand for two or three years in the areas where it is adapted. Danish and southern Swedish mid-late strains behave mainly as late types do, but are able to flower during the seeding year if growing conditions are favourable enough. Late red clover is characterized by a very slow pace of early development. Flowering of the late type is suppressed during the year of seeding but root development is strong and leaf rosette formation abundant. Flowering of the late type occurs for the first time during the second growing season, but only once, and four weeks later than flowering of the early types. Late red clover is characteristic to Nor- dic growing conditions where the stand is cut once per season and theaftermath is pastured (Julen 1959, 1977). Regrowth is generally modest, at best, but the accumulation of car- bohydrates into the roots is strong and over- wintering ability better than that of early types due to a closed type of rosette around the root crown, and high level of food reserved in the roots. Late red clover has the best adaptation in the central and northern parts of Scandina- via and northern Russia. The late type of red clover grown in Fin- land can be divided into two main groups: the tall, relatively late flowering clover of southern Finland and the low, earlier flower- ing type of northern Finland. The northern type has a very closed leaf rosette around the root crown and excellent adaptation to the long snow cover time (Ravantti 1961). In ad- dition, intermediates of these two main types are also grown in Finland. The red clover var- ieties Tepa (4n), Venla (2n) and Jokioinen(2n) belong to the tall type of southern Finland, and the Swedish local variety Bjursele belongs to the low type of northern areas. The Swed- ish breed Björn can be classified as an inter- mediate of these two. In addition, several lo- cal strains are also grown in Finland. Local strains can be characterized by their adapta- tion to a rather specific environment, but when moved to an another environment their productivity is lowered significantly (Ravant- ti 1980). Julen (1977) conculded that all red clovers are physiologically long day plants but the daylength requirements for flowering vary ac- cording to the geographical adaptation of var- ious strains. Flowering of the early red clover is accelerated and regrowth improved, but overwintering is weakened when it is moved from short day conditions to the long day en- vironment. Correspondingly, the northern late type of clover, when moved to the short day, 202 loses its regrowth abilities. However, overwin- tering is improved which, in turn, influences the yield of the succeeding year. The same trend can be observed among diploid and tetraploid strains. The breeding objective of combining the abundant regrowth of the early type of red clover with the good overwintering of the late type is impossible according to Julen (1977) because both characteristics are completely controlled by daylength. According to this concept, thebreeding of red clover should be carried out separately in each daylength region. True progress in the breeding of red clover will be possible when more detailed in- formation about the physiological behaviour of red clover becomes available. The aim of this study was to survey, for the purposes of breeding, certain adaptation characters of five red clover varieties and five local red clover strains grown in Finland. The experimental parameters of the study were daylength behaviour and the hardening fea- tures of the plants in relation to daylength. The parameters measured characterizing daylength behaviour of the plants were: dry matter accumulation of shoots and roots, plant height and LAI development, leaf and shoot formation, and flowering and harden- ing of the plants. The parameters measured for the hardening process were total nonstruc- tural carbohydrates, long-chain fatty acids and prolin production. 2. Materials and methods Five known varieties and five local strains of red clover from the latitudes 60° to 66°N (Fig. 1) were studied in a greenhouse at the Agricultural Research Centre under different light regimes to determine adaptative be- haviour of the plants. Varieties Jokioinen (2n), Venla (2n) and Tepa (4n) represented breeds of southernFinland. The Swedish var- iety Björn (2n) is an intermediate breed from 63°N latitude, and the Swedish local variety Bjursele (2n) represented the northern most type of red clover from the latitude 66°N. The Finnish local strain Vilo is cultivated in southern Finland, local strains Mustamäki, Jylhä and Airaksinen in central Finland and Hailuoto in northernFinland (Fig. 1). Genetic origins of the local strains were unknown, but they were known to be cultivated > 20 years in the same place. Inoculated seeds were planted in 10-litre pots filled with a fertilized and limed mixture (1 : 1) of sand and peat. Photoperiods were 12, 16 and 20 h, day, night temperatures 17 and 14°C, and light intensity 21.2 klux. First harvest was taken 132 days after seeding (Ta- ble 1), second harvest 47 days later and third harvest 45 days after the second. Flowering and plant height development were assessed at each harvest. The number of shoots and leaves per plant and leaf area per plant were also determined at the second harvest and root size at the third harvest. Dry matter yield of the plants was determined at each harvest. Af- ter the second harvest one replicate of each strain under the three light regimes was placed at +4°C temperature under an 8-h light re- gime of 10 klux for 26 days. For assessment of plant response to the hardening conditions chemical analyses were performed on roots for TNC, long-chain fatty acids and proline. Storage carbohydrates were analyzed accord- ing to Smith (1969), long-chain fatty acids by Fig. I. Origin or location of the studied red clover strains. 203 the method of Hiltunen et ai. (1979) and free proline by the technique of Chinard(1952). 3. Results and Discussion 3.1 Plant height development At the first harvest, average stand height (Fig. 2) was 16 and 67 % higher under the 20-h light regime than under the 16-h and 12-h light regimes, respectively. Plant growth was en- hanced from the beginning under the 20-h light regime. In contrast, under the 16-h and 12-h light regimes height development slowed down after thefirst harvest and the plants be- gan to prepare for winter. At the second har- vest (Fig. 3), average stand height was 49 and 65 % higher in the 20-h group than in the 16-h and 12-h groups, respectively. In each group, strains of northern origin lost their apical dominanceearlier than did strains of southern origin. Height development was lowest in Bjursele, the northernmost variety. From the local strains, the northernmost, Hailuoto, had the earliest cessation in height development for all light regimes. Table 1. Experimental design and timetable. Varieties: Daylengths 1. Venla (2n) 1. 12 h 2. Jokioinen (2n) 2. 16 h 3. Björn (2n) 3. 20 h 4. Bjursele (2n) 5. Tepa (4n) 6. Vilo/Lempäälä (2n) 7. Jylhä/Kaustinen (2n) 8. Mustamäki/Saarijärvi (2n) 9. Airaksinen/Maaninka (2n) 10. Haapaniemi/Hailuoto (2n) Replicates: 4 16.2. Seeding Age of stand: Stand height measurements 26 days Growth analysis 60 » Onset of flowering (20-h) 69 » Ist cut 132 » Onset of flowering after Ist cut (20-h) 158 » 2nd cut 179 » Hardening 189 » Chemical analysis 215 » 3rd cut 224 » The study clearly showed that the long day about equally promoted plant height develop- ment of all strains observed. The results sup- port the observations of Schulze (1957), Umaerus (1963 a) and Tanasch (1979). A cor- responding result was found in the develop- Fig. 2. Height development (cm) of red clover strains at different daylengths from seeding to the first cut 204 ment of the plants after the first harvest. However, height development in 16-h and 12-h light regimes was effectively prevented after the first cut thus showing the good adaptability of the strains studied to long day conditions. In shorter days apical dominance for the generative phase was lost and the plants prepared for winter. The results close- ly followed the observation of Umaerus (1963b). 3.2 Shoot formation and leaf area development Shooting ability and leaf development of the plants were studied at age 60 days, when the initiation of flowering during long day treatment was observed and again at the sec- ond harvest. At age 60 days the number of shoots per plant was equal among the three daylength groups(Table 2). Of the individual strains, the northern types Bjursele and Jylhä showed the best adaptability to long day conditions in- troducing the most shoots during a 12-h day- length. Later development in the shooting ability of the strains measured at the second harvest (Table 3) showed that the longest pho- toperiod promoted the least shoots, suggest- ing the lowest winterhardiness for the plants among the three light regimes. The best shoot- ing development in the long day group was in Bjursele indicating its good overwintering abil- ities. Table 2. Number of shoots per plant of red clover vari- eties (V) grown 60 days at 12, 16 and 20 h day- lengths (D). Daylength (D)Variety (V) 12 h 16 h 20 h Avg. 2,4 1,6 1,5 1,8 2,2 1,4 2,2 2,0 2.1 2,0 2,4 2,2 2.8 1,8 2,4 2,3 1.8 2,2 1,4 1,7 2.2 2,0 2,4 2,2 2.8 2,0 2,4 2,4 2.3 2,1 1,9 2,1 1.9 2,3 2,1 2,1 2.2 2,2 2,4 2,3 2.2 2,0 2,1 2,1 Venla Jokioinen Björn Bjursele Tepa Vilo Jylhä Mustamäki Airaksinen Haapaniemi Fig. 3. Height development (cm) of red clover strains between the first and the second cut (flowering) at different daylengths. 205 Table 3. Number of shoots and leaf area (cm2 ) per plant of red clover strains (V) in the second cut grown at 12, 16 and 20 h daylengths (D). Variety (V) Daylength (D) Number of shoots/plant Leaf area cmVplant 12 h 16 h 20 h 12 h 16 h 20 h Venla 8,1 7,8 6,4 71 146 213 Jokioinen Björn 8,3 9,4 7,3 85 121 223 8,1 8,2 6,6 9,1 6,8 79 130 243 Bjursele Tepa 10,4 8,9 104 132 150 7,8 7,3 135 166 273 Vilo 8,2 9,0 5,2 78 111 265 Jylhä 10,7 10,6 7,7 123 109 180 Mustamäki Airaksinen Haapaniemi 10,2 9,4 6,7 113 116 188 7,7 8,7 4,9 108 148 258 7,1 13,6 6,8 126 142 202 8,68 9,48 6,8 A 102 A 132 A 2198 D.05 D = 1,5 D.05 D =3l V = NS V = 52 D x V = NS D X V = NS Leaf number per plant at age 60 days (Ta- ble 4) was equal among the three groups, but the northernmost strains tended to produce more leaves in both the 12-h and the 20-h groups. Long photoperiods during early de- velopment increased both total leaf areas and the size of individual leaves (Table 5). After further development under the 20-h light re- gime (Table 3), total leaf areas were smaller Table 4. Number of leaves per plant of red clover varie- ties (V) grown 60 days at 12, 16 and 20 h day- lengths (D). Daylength (D)Variety (V) 12 h 16 h 20 h Avg. Venla 9,4 a 7,0 a 7,oab 7,8a Jokioinen 8,6 a 9,1 a 10,7abc 9,5ab Björn 9,4 a 9,2 a B,2abc B,9ab Bjursele 11,6 a 9,6 a 14,7 c 12,0c Tepa 6,4 a 9,5 a 5,6 a 7,2a Vilo 8,9 a 8,9 a 9,4abc 9,lab Jylhä 13,2 a 10,4 a 13,2bc 12,3c Mustamäki 9,3 a 8,2 a 7,Babc 8,4 a Airaksinen 8,4 a 8,5 a B,6abc 8,5 a Haapaniemi 9,5 a 10,1 a 13,6bc 11,1 be 9,5 A 9, IA 9,9 A 9,5 D.05 D = NS V = 2,4 D X V = 7,5 in the northern varieties than in the southern ones and smallest in Bjursele. With regard to breeding, an interesting find- ing was that in the long photoperiod shoot- ing was minimal but leaf area was doubled compared to 12-h and 16-h treatments (Table 3). The strains Bjursele and Jylhä best dis- played the northern behaviour described by Julen (1977). Their shooting dominated over the formation of leaf area. The local strain Vilo and the varieties Venla and Jokioinen fol- lowed thepattern of southern late red clover. The rest of the strains behaved more or less as intermediates. The tetraploid variety Tepa behaved independently from the diploid types but can be classified as an intermediate. 3.3 Flowering behaviour of the strains At first flowering, initiation of blooming in the 20-h photoperiod was latest in Bjursele (Table 6). Local strains from central Finland exhibited a somewhat southern ecotype be- haviour. In a 16-h daylength the onset of flowering of all strains was delayed signifi- cantly and none of the strains flowered in a 12-h daylength. Development in the number of the inflorescences of red clover strains from 206 207 Table 5. Leaf area cmVplant and individual leaflet of red clover varieties (V) grown 60 days at 12, 16 and 20 h daylengths (D). Variety (V) Daylength (D) cmVplant cmVleaflet 12 h 16 h 20 h 12 h 16 h 20 h Venla 72 80 93 7,6 11,4 13,2 Jokioinen 50 86 158 5,8 9,5 14,7 Björn 56 97 110 6,0 10,5 13,4 Bjursele 58 79 150 3,6 8,2 10,2 Tepa 53 129 106 8,2 13,6 18,9 Vilo 61 124 114 6,9 13,9 12,1 Jylhä 89 99 124 8,3 9,5 9,4 Mustamäki 58 81 74 6,2 9,9 9,5 Airaksinen 58 99 118 6,9 11,6 13,7 Haapaniemi 52 95 161 5,5 9,4 11,8 61 A 97A8 1218 6,4 A 10.7A8 12,28 D.05 D =54 D.05 D = 5.7 V = NS V = NS D x V = NS D X V = NS seeding to the first cut at a 20-h daylength (Fig. 4) showed the superiority of the strain Jylhä above the others. The abundance of the inflorescences of red clover strains during the second flowering fol- lowed the pattern observed in the first flower- ing (Table 7). In a 16-h daylength no differ- ences in abundance of flowering among the six flowering strains were observed (Table 8). None of the strains flowered in a 12-h day- length during the study period. Table 6. Beginning of flowering of red clover varieties in days from seeding at 20, 16 and 12 hour day- lengths. Variety (V) Daylength (D) 12 h 16 h 20 h Venla 71 123 Jokioinen 71 Björn 83 Bjursele 88 106 Tepa 78 106 Vilo 78 123 Jylhä 69 123 Mustamäki 69 130 Airaksinen 69 123 Haapaniemi 74 130 75 121 Julen(l977) showed that southern Europe- an strains under constant illumination have the flower-formation ability already at the five-leaf stage, but the flower-bearing stems are weak. Northern types tend to stay in the rosette stage until the development of strong vegetative shoots. In addition to the observa- Table 7. Flowering of red clover varieties (inflores- cences/45 plants) at 20 h daylength (A = in the first, B = in the second and C = in the third cut). Variety (V) Number of inflorescences/45 plants ABC Total (E) Venla 54a 27a 25ab 106 Jokioinen 26a I7a 10a S 3 Björn 30a 24a 10a 64 Bjursele 38a 29a 9a 76 Tepa 28a 25a 12ab 65 Vilo 43a 24a 14ab 81 Jylhä 125 b 60b 33b 218 Mustamäki 43a 36ab 25ab 104 Airaksinen 45a 39ab 23ab 107 Haapaniemi 66ab 29a 29ab 124 47 31 19 D.05 varieties (V) A = 64 » B = 29 » C = 21 tions of Julen, Umaerus (1963b) concluded that the more that daylength exeeds the mini- mum requirements of a plant, the faster its de- velopment will be from the vegetative phase to flowering. If available daylength signifi- cantly exceeds that required, red clover will have rapid flower development and the de- velopment of stems and leaves will remain low. In this study, the only strain of a distinct- ly northern ecotype was Bjursele, as was shown by the abundance and timing of its flowering. A very interesting behaviour was shown by the local strain Jylhä from central Finland. It had an early and abundant flower- ing in all three long day flowerings but flower- ing activity was slight at shorter daylengths. Fig. 4. Development of the number of inflorescences of red clover strains from seeding to the first cut at 20 h daylength. 208 Table 8. Flowering of red clover varieties (V) (inflores- cences/45 plants) at 16 h daylength (A = in the first, B = in the second and C = in the third cut). Variety (V) Number of inflorescences/45 plants B C TotalA (E) Venla 1,0 0,3 1,3 Jokioinen 0,3 0,3 Björn Bjursele 0,8 0,8 Tepa 3,5 3,5 Vilo 0,3 0,3 0,6 Jylhä 0,3 0,3 Mustamäki 0,3 0,3 0,6 Airaksinen 1,0 0,3 1,3 Haapaniemi 1,3 0,3 1,6 Table 10. Dry matter yield g/pot of red clover varieties (V) in the second cut grown 179 days at 12, 16 and 20 h daylengths (D). Daylength (D)Variety (V) 12 h 16 h 20 h Avg. Venla 18,4a 23,5a42,4ab 29,1ab Jokioinen18,6a 23,2a50,2ab 30,7ab Björn 17,9a 25,5a47,5ab 30,3ab Bjursele 18,9a 24,0a 52,7b 31,8ab Tepa 20,0 a 29,3 a 56,9 b 35,4b Vilo 16,1 a 21,9 a 26,9 a 21,6a Jylhä 18,1a 23,9a 60,0b 34,0ab Mustamäki 23,2a 23,7a 53,7b 33,5ab Airaksinen 20,9 a 28,7 a 58,6 b 36,0b Haapaniemi 15,8a 23,9a49,4ab 29,7ab 18,8 A 24,7 A 49,88 31,1 D.05 D = 8,3 V = 12,7 D x V = 24,3 Table 9. Dry matter yield g/pot of red clover varieties in the first cut grown 132 days at 12, 16 and 20 h daylengths (D). Daylength (D)Variety (V) 12 h 16 h 20 h Avg. Table 11. Dry matter yield g/pot of red clover varieties (V) in the third cut grown 226 days at 12, 16 and 20 h daylengths (D). Daylength (D)Variety (V) 12 h 16 h 20 h Avg. Venla 35,9 77,3 142,1 85,1 Venla 13,8 22,6 58,7 31,7ab Jokioinen 31,7 65,5 125,0 74,0 Jokioinen 16,5 22,5 56,0 31,7ab Björn 37,3 69,7 128,7 78,6 Björn 13,8 22,6 53,6 30,0ab Bjursele 32,0 61,6 117,8 70,4 Bjursele 17,3 22,7 52,0 30,6ab Tepa 37,7 79,5 122,3 79,8 Tepa 22,7 28,3 59,9 37,0 b Vilo 32,9 57,3 129,5 73,2 Vilo 9,7 19,7 50,5 26,6 a Jylhä 29,1 69,9 150,0 83,0 Jylhä 12,3 21,9 60,2 31,5ab Mustamäki 41,8 59,3 134,2 78,4 Airaksinen 35,1 73,3 124,9 77,7 Haapaniemi 29,7 74,0 131,0 78,2 34,3 A 68,78 130,5 C 77,9 D.05 D = 5,8 V = Ns D x V = Ns Mustamäki 20,1 25,0 60,2 35,1b Airaksinen 13,8 25,9 62,6 34,1ab Haapaniemi 13,1 22,4 55,4 30,3ab 15,3 A 23,38 56.9C 31,9 D.05 D = 2,7 V = 8,1 D x V = Ns However, the shoot and leaf formation of Jyl- hä followed the pattern of the northern type of red clover. All strains studied showed the growth pattern of one-cut red clover although other northern strains, with the exception of Bjursele, were somewhat less adaptable to long photoperiods and possessed some fea- tures of southern origin. 3.4 Dry matter yields of shoots and roots Dry matter yields of shoots Under the light regimes 12, 16 and 20 h, DM yields were in the ratio of 15:50:55 at the first harvest (Table 9), 20:26:54 at the second harvest (Table 10) and 16:24:60 at the third harvest (Table 11). The different ratios at the 209 second and third harvests clearly show the northern origin of the red clover strains studied. There were no differences among the varieties at the first and third harvests, but at the second harvest the northern varieties and the tetraploid Tepa gave the highest yields as expected in view of their pace of growth and tetraploid character, respectively. In the total yields of the three cuts (Table 12) under the light regimes 12, 16 and 20 h. DM yields were in the ratio of 15:28:27. The ratio shows the importance of daylength to the DM development of red clover. Northern types benefit from the long period of illumi- nation. In the 12 and 16 h daylengths there were no significant differences among varie- ties. The degree of adaptation among strains could be observed at a 20-h daylength, although only the southern strain Vilo and northern strain Jylhä showed the only signifi- cant differences. Root dry matter accumulation Root dry matter accumulation during the entire test period showed that root develop- ment was 59 and 66 % lower in the 12-h groups than in the 16-h and 20-h daylength Table 12. Total DM yieldg/pot in three cuts of red clover varieties (V) grown at 12, 16 and 20 h day- lengths (D). Variety (V) Daylength (D) 12 h 16 h 20 h Avg. Venla 67,3 a 123,4 a 243,2ab 144,6ab Jokioinen 66,8a 111,2 a 231,2ab 136,4ab Björn 69,0 a 117,8 a 229,8ab 138,8ab69,0 a 117,8 a 229,8ab 138,8ab Bjursele 68,2 a 108,3 a 222,5ab 133,0ab Tepa 80,4 a 137,1a 239,1ab 152,2b Vilo 58,7 a 98,9 a 206,9 a 121,5a Jylhä 59,5 a 115,7 a 270,2 b 148,5b Mustamäki 85,1 a 108,0 a 248,1ab 147,1b Airaksinen 69,8 a 127,9 246,1ab 147,9b Haapaniemi 58,6 a 120,3 a 235,8ab 138,2ab 61,4 A 116,98 237,3 C 140,8 D.05 D = 12,5 V = 24,5 D x V = 47,0 groups, respectively (Table 13). Differences in root size among the varieties were smallest in the 12-h group and greatest in the 16-h group showing a varying degree of adaptability. In each daylength group root size was smallest in Bjursele due to genetic development. Bjur- sele has a clearly branched root system instead of the distinct tap root commonly found among clovers. The relationship between the daylength and root size of an invidual root is well described by a second degree regression equation where (x) represents daylength and (y) equals root size (Fig. 5). In the equation R 2 explained 70,1 % of the daylength. y = —1.12 + 0.149x 0.0037x2 (F 31.67***) Although root size accounts only partly for differences in the overwintering of plants, the long day tends to favour root development in the natural conditions of long day and long winter. 3.5 Effects of daylength on red clover hardening Root carbohydrates in relation to hardening Chemical analysis of plants subjected to hardening after the second harvest showed Table 13. Root dry matter weight g/root of red clover varieties (V) in the third cut grown at 12, 16 and 20 h daylengths (D). Variety (V) Daylength (D) 12 h 16 h 20 h Avg. Venla 0,14 0,27 0,38 0,26 Jokioinen 0,10 0,30 0,38 0,26 Björn 0,11 0,35 0,44 0,30 Bjursele 0,09 0,25 0,29 0,21 Tepa 0,15 0,26 0,46 0,29 Vilo 0,20 0,22 0,46 0,29 Jylhä 0,20 0,49 0,41 0,37 Mustamäki 0,08 0,20 0,31 0,20 Airaksinen 0,14 0,38 0,29 0,27 Haapaniemi 0,13 0,43 0,35 0,30 0,13 0,32 0,38 0,28 210 3 that carbohydrate accumulation by the roots, the indicator of hardening, was highest in plants grown under the 20-h light regime (Ta- ble 14). TNC accumulation was highest for Tepa in the 12-h light regime group, Vilo in the 16-h group, and Bjursele in the 20-h group. Great differences in TNC accumula- tion among the clover strains were found in each of the three groups. Umaerus (1963 a) has shown that daylength affects the overwintering of plants. It also in- fluences flowering, onset and end of dorman- cy, as well as the chemical composition of var- ious plant parts. Julen (1977) concluded that the early type of clover has a weak overwin- tering in long day conditions, and the late type adapted to long day conditions tends to have a deep dormancy also in the short day en- vironment. This study agrees very well with that of Julenwith respect to long day environ- ment, but one-cut plants grown under short day behaved differently from the Julen model. The original long day plants hardened in the short day, and at low temperature conditions collected significantly less reserve food than the same plants grown in long day conditions and hardened in short day and low tempera- ture conditions (Table 14). Fatty acid composition and hardening During hardening there are certain changes in the composition of long chain fatty acids. Hardening favours the increase of unsaturat- ed fatty acids at the cost of saturated fatty acids (De La Roche et al. 1972, Kuiper 1970). Cell membranes dominated by unsatu- Table 14. TNC (Vo DM) of the roots of red clover strains (V) grown at 12, 16 and 20 h daylengths and hardened after the second cut for four weeks at +4°C temperature and 8 h daylength. Daylength (D)Variety (V) 12 h 16 h 20 h Avg. Venla 16,2 16,7bc 15,8abc 16,2c Jokioinen ll,oab 18,7de 17,6ed 15,7ab Björn ll,9abc 18,lcde 17,labcd 15,6all,9abc 18,lcde 17,labcd 15,6a Bjursele 10,7 a 13,7 a23,3f15,9ab Tepa 20,4 e 17,2bcd 17,8 d 18,5 d20,4 e 17,2bcd 17,8 d 18,5d Vilo 13,4 c 21,0 f 17,4bcd 17,3c Jylhä 10,0 a 19,8ef 15,4 a 15,1a Mustamäki 15,6d 16,1b 15,6ab 15,8ab Airaksinen 16,1 d 17,5bcd 21,9 e 18,5 d Haapaniemi 12,9bc 17,4bcd 21,6 e 17,3c 13,8 A 17,68 18,38 16,6 D.05 D = 3,4 V = 1,0 D X V = 1,9 Fig. 5. Quadratic relationship between daylength and root size of an individual red clover root. 211 rated fatty acids tend to be less fragile at low temperatures and are thus more easily restored after stress caused by frost. In addition, the abundant availability of unsaturated fatty acids improves the semipermeability of the cell membranes and lowers the freezing point of cell sap. The present study showed that the predominant long chain fatty acid in clover roots was linolic acid (Table 15). The more im- portant fatty acid, linolenic acid, which corre- lates with winter hardiness in cereals, was present in minor quantities. Linolenic acid ac- cumulation was highest in the 12-h daylength group. Under long day conditions linolenic acid content was highest in Bjursele indicat- ing its good overwintering qualities and long day adaptation. Fatty acid analysis clearly showed that all of the red clover strains studied had, in this respect, relatively low adaptability to long day conditions. Although there are great differ- ences of opinion concerning the importance of fatty acid composition in winter hardiness (Willemot 1977, De La Roche 1979), it seems apparent that the low content of lino- lenic acid in the hardening of red clover may be one of the reasons for damage due to low temperatures. Proline production in hardening The amino acid proline has been found to increase significantly under stress conditions caused by high salt concentration of drought, and high and low temperatures (Stewart 1981). Leddet and Schaeverbeke(l97s) con- cluded that in low concentrations proline mainly has an osmotic effect, but in high con- centrations under stress conditions proline has Table 15. Fatty acid content (%) of the roots of red clover strains (V) grown at 12, 16 and 20 h daylengths (D) and hardened after the second cut for four weeks at +4°C temperature and 8 h daylength. Fatty acid Venla Jokioi- Björn Bjur- Tepa Vilo Jylhä Musta- Airak- Haapa- Avg. nen sele mäki sinen niemi Daylength 12 h Palmitic acid (16 :0) 15,9 15,1 15,1 16,0 Stearic acid (18 ; 0) 1,7 2,3 1,8 2,4 Oleic acid (18 : 1) 6,6 5,3 7,1 5,7 Linoleic acid (18 :2) 51,6 51,6 55,4 51,0 Linolenic acid (18 :3) 21,4 22,0 17,4 21,2 Arakidinic acid (20 : 0) 0,9 1,2 1,0 1,1 Eikosadienic acid (20 : 2) 0,5 0,6 0,3 0,8 Behenic acid (22 : 0) 1,2 1,8 1,6 1,6 Daylength 16 h Palmitic acid (16 :0) 19,3 17,7 19,2 20,3 Stearic acid (18 : 0) 2,0 2,1 2,1 2,0 Oleic acid (18 : 1) 10,2 9,3 11,2 7,5 Linoleic acid (18 :2) 46,6 48,6 46,2 49,8 Linolenic acid (18 : 3) 14,7 15,9 14,9 14,7 Arakidinic acid (20 : 0) 1,7 1,4 1,5 1,1 Eikosadienic acid (20 : 2) 0,3 0,3 Behenic acid (22 : 0) 4,8 4,8 4,4 3,8 Daylength 20 h Palmitic acid (16 : 0) 19,0 19,5 18,8 18,6 Stearic acid (18 : 0) 1,6 1,6 1,6 1,6 Oleic acid (18 : 1) 7,3 8,9 7,6 8,5 Linoleic acid (18 :2) 50,1 50,9 51,9 48,7 Linolenic acid (18 :3) 16,7 15,0 14,8 17,5 Arakidinic acid (20 : 0) 1,2 1,0 1,1 1,1 Eikosadienic acid (20 : 2) 0,2 0,2 0,5 0,3 Behenic acid (22 : 0) 3,6 2,9 3,2 3,2 212 13.3 J3,8 22,2 21,2 20,6 19,3 17,3 1,6 1,8 2,6 2,1 2,2 2,0 2,1 8.3 6,3 6,8 7,6 7,1 5,8 6,7 51.6 55,0 45,5 47,9 44,7 49,2 50,9 17.6 20,5 14,5 15,7 18,8 17,7 18,7 0,9 0,8 1,8 1,6 1,6 1,7 1,3 0,3 0,3 0,8 0,4 1.2 1,4 4,5 4,0 4,0 4,3 2,6 18.4 19,4 19,7 20,4 19,9 19,8 19,4 1.9 1,9 1,9 1,9 1,9 1,9 2,0 9.4 7,8 10,2 9,9 9,0 8,3 9,3 48,2 50,8 48,6 47,4 48,2 45,3 48,0 13,8 13,6 15,5 14,3 16,5 18,7 15,3 1.2 1,3 1,0 1,3 1,1 1,2 1,3 0,5 0,1 0,1 0,2 0,1 0,4 0,2 4.9 4,6 2,6 4,5 2,9 3,9 4,1 18.0 20,0 19,9 18,8 18,5 19,0 19,0 1,7 1,5 1,7 1,5 1.7 1,7 1,6 10.1 6,9 9,0 9,5 9,9 9,1 8,7 50.2 52,6 47,8 49,9 50,3 49,7 50,2 14.6 15,6 17,0 14,9 14,5 14,6 15,5 1,0 0,8 1,2 1,2 1,0 1,2 1,1 0,7 0,3 0,1 0,3 0,2 0,3 0,3 3,6 2,3 3,4 3,6 3,8 4,1 3,4 a special mode of action. A significant role of proline is to protect the thylacoid mem- branes of plants against activity losses due to frost. The protection mechanism relies on the production of productive collagens. Proline production is stimulated by abscicic acid through the synthesis of glutamic acid (Stewart 1980). The physiological action of proline classifies it as a cryoprotector. Study of proline showed that red clover has a different protection mechanism against frost depending on the daylengths to which the strains are subjected before the hardening period (Table 16). The protection mechanism is most effectively developed the longer the daylength is. All red clover strains studied reacted to the daylength, but the most distinct proline production was observed in the north- ern variety Bjursele and the local strain Vilo from southern Finland. According to the results, the two red clover strains have the best protection against frost, as observed also in natural conditions. 4. Conclusions The study presented two hypotheses: 1) Adaptation of red clover to a certain day- length condition influences plant behaviour with respect to its growth and development and to hardening; 2) Growth analysis and chemical analysis can be used for characteriz- ing behavioural differences due to daylength. From this study it can be concluded that red clover varieties and local strains originating from different latitudes (60—66°N) all be- haved as late flowering, one-cut types. However, differences could be detected Table 16. Proline content (mg/g DM) of the roots of red clover varieties (V) grown at 12, 16 and 20 h daylengths (D) and hardened after the second cut for four weeks at + 4°C temperature and 8 h daylength. Daylength (D)Variety (V) 12 h 16 h 20 h Avg. Venla .284 c .298bc ,600 c ,394d Jokioinen ,226 a .352de .472 b .350b Björn ,278 c .234 a .516 c ,343b,278 c .234 a .516 c ,343b Bjursele ,318 d .268 b ,758 f ,448e Tepa .374e. .314 c .356 a ,348b Vilo .284 c .294bc .746 f ,441e Jylhä ,270bc .354 e .478 b .367c Mustamäki .324de .474 f .548 d ,449e Airaksinen .240ab ,322cd .370 a .311a Haapaniemi .254abc .454 f ,606 e .438e .285 A .336 A .5458 .389 D.05 D = 0,109 V = 0,015 D X V = 0,030 among the strains. The most distinct north- ern type was Bjursele. The most distinct southernFinnish type was strain Vilo originat- ing from the latitude 61 °N. The rest of the strains possessed some northern and southern features. The most interesting strain was Jyl- hä from the latitude 64°N which displayed some strong northern behaviour together with southern features indicating that adaptation is incomplete. The study showed that chemical methods can be used for the purposes of breeding in determining the adaptation of plants to vari- ous daylength conditions as well as the effects of daylength on the hardening and overwin- tering of plants. Literature Chinako, F.P. 1952. Photometric estimation of proline and ornithine. J. Biol. Chem. 199: 91—95. Cooper, J.P. 1965. The evolution of forage grasses and legumes. In J.P. Hutcnison (ed.)Crop plant evolution, Cambridge, 142—165. Evans, Alica M. 1976. Clovers. In. N.V. Simmons (ed.) Evolution of crops plants. New York, 175 —179. Hawkins, R.P. 1952. Investigations on local strains of herbage plants 2. Types of red clover and their inves- tigations. J. Brit. Grassl. Soc. 8: 213—238. 213 Hiltunen, R., Huhtikangas, A. & Hovinen, S. 1979. Breeding of a zero erucic spring turnip-rape cultivar. Brassica campestris L., adapted toFinnish climaticcon- ditions. I. The use of glass capillary column gas chro- matography in fatty acid analysis. Acta Pharm. 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SELOSTUS Puna-apilan sopeutuminen pitkään päivään Seppo Pulli Maatalouden tutkimuskeskus, kasvinjaloslusosasto 31600 Jokioinen Maatalouden tutkimuskeskuksen kasvinviljelyosaston tutkimuksessa selvitettiin kasvihuoneessa viiden tunnetun puna-apilalajikkeen ja viiden puna-apilan paikalliskan- nan suhtautumista päivänpituuteen. Kasvien alkuperät ja sopeutumisalueet kattoivat leveyspiirit 60—66°N. Päivän- pituudet tutkimusajanjaksona olivat 12, 16ja 20 h, päivä- /yölämpötilat 17°/14° jakasvatusvalojen intensiteetti 21.4 klux. Kasvit niitettiin tutkimusajanjakson aikana kolme ker- taa. Kussakin kasvuperiodissa määritettiin kukkimisen ai- kaisuus ja runsaus, kasvin pituuskasvu jakasvien maan- päällisen osan kuiva-ainesato. Versojen ja lehtien luku- määrä ja lehtiala sekä yksityinen lehdenkoko määritet- tiin toisessa niitossa sekä juuren koko kolmannessa nii- tossa. Toisen niiton jälkeen osa materiaalista vietiin kei- nolliseen karaistumiseen 26 päiväksi. Keinollinen karais- tuminen sisälsi +4°C lämpötilan, 8 h päivänpituuden ja 10 klux valovoimakkuuden. Karaistumisefektin selvittä- miseksi kasvien juurista määritettiin talvehtimisen vara- ravinnot, pitkäketjuiset rasvahapot ja proliini. Tutkimus osoitti, että kasvien sopeutuminen tiettyyn päivänpituuteenheijastuu voimakkaasti sen kasvuun, ke- hitykseen ja talvenkestävyden kehittymiseen. Puna-api- lalla päivänpituus näytteli merkittävää osaa sopeutumis- prosessissa ja tulisi ottaa täysipainoisesti huomioon ja- lostustyössä. 214