Euphytica 111: 153–160, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

153

Introgressive hexaploid oats from theAvena abyssinica(♀) × A. sativahybrid: performance, grain lipids and proteins

Hannu Ahokas1,2 & Marja-Leena Manninen11Crops and Soil, ARC, Myllytie 10, FIN-31600 Jokioinen, Finland;2Division of Genetics, P.O. Box 56, FIN-00014University of Helsinki, Finland

Received 18 September 1998; accepted 5 July 1999

Key words: Avenahybrids, fatty acids, grain protein fractionation, interspecific recombinant, lipid extraction, oatcrosses

Summary

A fertile hexaploid oat plant was obtained after several generations of selection for seed set and plant type froma colchicine-produced decaploid hybrid,Avena abyssinica(2n = 4x = 28, AABB)× A. sativa(2n = 6x = 42,AACCDD). The selected line proved to be stably fertile and in many characteristics equal or superior to thehexaploid parent. The grain protein fractions showed two qualitative differences from those of the pollen parentand several differences from the maternal parent. The fractionating extraction used was new for oats. The fatty acidcomposition of grains of the hybrid derivative was similar to that of the pollen parent, but different from that of thematernal parent. The maternal parent (A. abyssinica) had a relatively high 16:0 fatty acid content (ca. 20.5 mol%)compared with the level of the hexaploid parent and the hybrid derivation (ca. 17.5 mol% each) in field-grown grain.However, in grain produced in the greenhouse, the hexaploids had ca. 20.5 mol% of 16:0 fatty acid and a decreasein 18:1 fatty acid, whereas seed of theA. abyssinicaparent showed only a slight increase (ca. 21.5 mol%) in 16:0fatty acid. These and other responses statistically significant may be due to adaptation to temperature conditionsbeing wider in the hexaploids than in the East-AfricanA. abyssinica. A new method of grain lipid extraction wasintroduced and showed good reproducibility. The derived hexaploid oat can be crossed withA. sativafor breedingpurposes and due to its early maturity might also have direct use in northern or high-altitude cultivation.

Introduction

With the aim of transferring the hexaploid nuclear gen-ome of common oats (Avena sativaL. cv ‘Hannes’) asthe pollen parent to the cytoplasm of the tetraploid (A.abyssinicaHochst. CI 2108), crosses were made in1973 by one of us (H.A.). The hybrids proved highlysterile, and some of the F1 seeds were treated with col-chicine. An amphiploid F1 plant from the colchicinetreatment was partially fertile and produced someseeds. The material was grown for several subsequentgenerations isolated among barley trials and resultedfinally in stable, fertile oats different from the ori-ginal amphiploid but resembling mostly the commonoats. This proved to be hexaploid (unpublished), hencebeing a carrier ofA. abyssinicacytoplasmic factorspossibly different from those of the pollen parent. Both

species,A. abyssinicaandA. sativa, form chloroplas-tidic stromacentres (Steer et al., 1970), which containa glycoside and saponins related to fungal resistance(Nisius, 1988; Bowyer et al., 1995). Also, bothA.abyssinicaandA. sativacontain the fast electromorphof the chloroplastidic enzyme ribulose bisphosphatecarboxylase compared to the slow electromorph of CgenomeAvenaspecies (Steer, 1975). Possibly, mito-chondrial DNA endonuclease restriction site diversityexists between crossed parents (cf. Rines et al., 1988)and was maternally transmitted fromA. abyssinicaspecies to the hexaploid derivative, but such DNAvariation does not necessarily exert any phenotypicdifference.

Hybrids betweenA. abyssinicaandA. sativahavebeen reported by Emme (1932), Kihara & Nishiyama(1932), Lesik (1948), Zillinsky (1956), Marshall &

154

Myers (1961), Nishiyama (1962), Thomas & Lawes(1968), Arias & Frey (1973), Sharma (1975) and Fritz& Sorrells (1990). Amphidecaploids of Nishiyama(1962) resulted in some hexaploid and octoploid spon-taneous recombinants. Likewise, the current selectionwith normal fertility was found to be hexaploid whosegrain protein and lipid properties and some agronomicvalues are described and compared with the parentaloats. Grain lipids of oats vary between cultivars orgenotypes (Hutchinson & Martin, 1955; Baker &McKenzie, 1972; Brown et al., 1974; Sahasrabudhe,1979), hence apparently being dependent on genotype.We also report a note that enviromnent, temperature,may regulate the grain lipid composition in hexaploidbut less in tetraploid oats. Temperature difference hasbeen found to change oat grain lipids in field andcontrolled chambers (review by Welch, 1995).

Materials and methods

Plants and statistics

Plants were grown in the field in single-row plots onsandy soil in 1996 and in three different soil types:heavy clay, sand and mould (classification accordingto Aaltonen et al., 1949) without replications on eachsoil type. Fields were fertilized with 400 or 550 kgha−1 of N-P-K (20–4–8) on 10 m2 plots in 1997 inJokioinen, SW Finland. Other material was grown insingle row plots on heavy clay soil in Elimäki, SEFinland during different seasons before 1995. Plantswere grown in the 1996 season in a greenhouse inpotting soil (Kekkilä Oy, Finland) with automatic soilirrigation.

Yield data are based on cleaned grains retained ona 1.8-mm sieve (cv Hannes and the hybrid derivativeHA 94-1113/1117) or cleaned by hand and retained ona 1-mm sieve (A. abyssinica). Grain weight was basedon 5× 100 grains, excluding the 2× 100 extremes.Yield was calculated at 15% moisture content (ovenmethod at 130◦C). Husk percentage was determinedfrom combine-harvested grains after husking by handfrom 3× 100 grains.

When possible, significances were determinedwith Kruskal-Wallis one-way analysis of variance (H)or variance ratio (F) as shown in Tables and probabil-ities indicated as follows:∗∗∗ p <0.001,∗∗ p <0.01,∗ p <0.05,◦ p <0.10.

Lipid extraction and derivation

Lipids were extracted from meal samples (1.3 g)ground to pass a 0.5-mm sieve (Cyclotec, Tecator)collecting the adhering meal from the mill. Grindingof the hand-husked caryopses was performed on theday of starting extraction in 50-ml Teflon-FEP tubes(Nalge). The samples were suspended in the solventsand the tubes were rolled during the extraction in aMixer 820 (Swelab), ensuring a top-to-bottom mo-tion of the suspension at +20◦C. The solvent to mealratio used was in 5:1 (ml/g). An 1-h extraction wasended with centrifugation in a swing-out rotor (JS13.1) at 7500 rpm (average 5730 RCF) and +20◦Cfor 10 minutes. This was followed by another 1-hextraction with the same solvent, and a third one forabout 11.5 h. The supernatants were pooled and storedin stoppered glass flasks at –20◦C for less than threedays. The second and third solvents were used withthe same schedule. The order of the three solventswas 2-propanol, H2O (Milli-Q) saturated 1-butanoland absolute methanol. The solvents were of analyt-ical grade (Merck). The extracts pooled were taken todryness using a rotary evaporator (Heidolph) and dis-solved with about 2 ml of petroleum benzine (boilingrange 40–60◦C, Merck) also applying a cup sonicator(Bransonic 5). The lipids (200µl) were methanolyzed(Kates, 1964; Appelqvist, 1968) in petroleum ben-zine + 10 mM Na-methylate (2 + 3 ml) at 50◦C for 15min, shaken with an aqueous solution of NaCl (1370mM) + NaHSO4 (22 mM) at room temperature. Theupper phase was taken to dryness and dissolved with1 ml of petroleum benzine. The samples were ana-lyzed within two hours using a 1-µl injection volume.An Auto System (Perkin-Elmer) gas-liquid chromato-graph was used with hydrogen as the carrier gas andwith a Supelcowax-10 glass capillary 30 m× 0.75 mmcolumn (Supelco). The quantities are given in molarpercentages of the total fatty acids.

Grain protein contents

Grain protein was extracted from 25 to 30 mg mealsamples including the husks and determined in duplic-ate samples by the UV method as described by Ahokas(1978). They were calculated as protein using a set of10 standard samples from the same season and fieldplot samples determined with Kjeldahl and obtainedfrom Boreal Plant Breeding Co., Jokioinen. The pro-tein contents in the 10 standards varied from 12.2 to17.4% (N× 6.25).

155

Table 1. Field trial results of the introgressive (Ha 94-1113/1117) oats and its parents in 1997

Oats Soil type Days from planting to Height Sraw Grain Yield Husks Protein

(fertilizer Heading Maturity, (cm) stiffness mass (kg/ha) (%) % kg/ha

kg ha−1) combine- (1–10; (mg)

harvestable 1 = weak)

Mould

(400)

Hannes 50 98 123 3 32.9 6290 22.4 15.2 959

A. abyssinica

CI 2108 65 98 100 4 14.2 4150 19.3 15.3 634

HA 94-1113/1117 45 98 116 3 31.8 5200 24.6 16.4 852

Heavy clay

(550)

Hannes 47 93 104 9 31.1 3930 25.2 11.4 447

A. abyssinica

CI 2108 57 93 95 7 14.4 2420 17.5 11.3 273

HA 94-1113/1117 43 93 95 8 33.2 3620 24.5 13.1 473

Sand

(550)

Hannes 47 97 128 5 30.1 5490 24.6 14.3 785

A. abyssinica

CI 2108 61 97 110 3 14.6 3290 17.9 15.7 516

HA 94-1113/1117 45 97 117 5 33.6 5990 23.6 15.9 956

Means (Significance against Hannes)

Hannes 48 96 118 5.7 31.4 5235 24.1 13.6 730

A. abyssinica

CI 2108 61∗∗ 96 102 4.7 14.4∗∗∗ 3288 18.2∗∗∗ 14.1 474

HA 94-1113/1117 44∗∗ 96 109 5.3 32.9∗∗ 4937 24.4 15.1 745

Statistic H – H – F H F H H

Grain protein fractionation

Grinding of embryoless single grains and samplepreparations for SDS-PAGE (sodium dodecyl sulph-ate – polyacrylamide gel electrophoresis) from singlegrains were performed as described elsewhere but us-ing 5% β-mercaptoethanol in the extraction buffer(Ahokas, 1994). A de-embryoed single-grain samplewas weighed in a 2-ml Eppendorf tube, and an acid-resistant steel ball (SKF, RB 4, 762 RJ) was droppedinto the tube before milling in a Retsch MM2 Mixerwith vibration at position 50 for 5 or more minutes ifneeded. The meal was extracted twice (450 and 400µl) with Tris-buffered (40 mM, pH 8.0) 2-propanol(50%, v/v) andβ-mercaptoethanol (0.5%, v/v) at 25◦Cfor 1 h, and the supernatants (400µl) were precipit-ated one after the other with 2.5 volumes (1 ml) ofmethanol in the same tube at room temperature (Aho-

kas, 1994). After the addition of methanol, the tubewas shaken and allowed to stand for 15 min. The pre-cipitate was collected by spinning at 15.000 RCF for15 min. This is the methanol precipitated fraction. Acentrifugal force of 5.000 RCF was applied to the tubecarrying a steel ball. The dried proteins left after thebuffered 2-propanol form the residual proteins. Thedry samples were dissolved in Laemmli’s sample buf-fer (Laemmli, 1970), heated at 93–95◦C for 2 min,made about 5% withβ-mercaptoethanol and carboxy-amidated by mixing 2µl of 500 mM freshly madeiodoacetamide per tube and incubating at +50◦C for15 min. The extraction-precipitation procedure wasdescribed for wheat (Ahokas, 1994), also used with ly-megrass (Greipsson et al., 1997) and barley (Ahokas &Poukkula, 1999). This is the first published applicationto oat. The samples were fractioned on 10% SDS-PA

156

Figure 1. SDS-PAGE fractionated proteins of husked and embryoless single-grain samples of oats. Tris-buffered 2-propanol extracted andmethanol-precipitated (tracks2 through7) and residual proteins (tracks8 through13). Molecular marker bandsA throughH (track 1), 170,116.4, 85.2, 55.6, 39.2, 26.6, 20.1 and 14.3 kg mol−1, respectively.2, 3, 8and9, Avena abyssinicaCI 2108; 4, 5, 10and11, interspecificrecombinant HA 94-1113/1117; the protein of intermediary size is indicated with black dots;6, 7, 12and13, A. sativacv Hannes. The proteinunique to cv Hannes is indicated with the arrow. The residual proteins of unique sizes inA. abyssinicaare indicated with squares (8 and9).The loaded samples correspond to 4.1, 4.7, 5.3, 5.4, 4.8 and 5.3 mg of meal (tracks2 through7) and 2.1, 2.4, 2.6, 2.7, 2.4 and 2.8 mg (tracks8through13), respectively.

gels (Shapiro et al., 1967; Laemmli, 1970) stainedwith Serva Blue R.

Results

The epiblast of HA 94-1113/1117 is of the fatuoid-type (cf. Baum, 1977: Figure 292) differing from thetypes of cv Hannes andA. abyssinica(cf. Baum, 1977:Figure 290 and Figure 211, respectively).

The hybrid derivative HA 94-1113/1117 headsthree to four days earlier than cv Hannes and up to 20days earlier than theA. abyssinicaparent (Table 1).In earlier trials, the stabilized hybrid derivative hadgenerally shown a two to four days’ earlier maturity

than cv Hannes, though weather conditions in the 1997season forced all the material to mature in a similartime. The mean 1997 yield on widely different soiltypes was slightly but not significantly lower in HA94-1113/1117 than in Hannes. HA 94-1113/1117 maybe valuable at northern and high-altitude margins ofoats cultivation due to its earliness, and may be betteradapted to sandy soils than cv Hannes.

The grains of HA 94-1113/1117 are light colouredand slightly heavier than those of cv Hannes (Table 1).The protein yield of the derivative HA 94-1113/1117is probably higher than that of the parents (Table 1).The grain protein patterns as revealed by the frac-tionation and SDS-PAGE are not identical to either

157

of the parents. The methanol-precipitable proteins ofHA 94-1113/1117 show quantitative changes in theabundance of the polypeptides, one of the proteins be-ing a putative intergenomic recombinant, and one cvHannes protein missing in HA 94-1113/1117hence re-sembling the pattern ofA. abyssinica(Figure 1). Theseproteins belong to the fraction soluble in buffered 2-propanol. The protein pattern ofA. abyssinicadifferedfrom the derivative and the hexaploid parent in mostof its proteins as revealed by SDS-PAGE (Figure 1).In the less variable residual protein fraction, the majorfractions of the hexaploids appear similar and theA.abyssinicapattern differs from that of the hexaploidapparently at five sizes of proteins, the rest of thevariation appearing rather quantitative (Figure 1).

The lipid extraction method introduced in thisstudy gives a smooth baseline and avoids pluggingthe capillary column used. Plugging has been a prob-lem with some oat extracts. Starting from separategrindings and extractions, the method shows excellentreproducibility (Table 2) as also found with other oatsamples from different seasons (to be published). Thequantitative distribution of fatty acid types is fairlysimilar between the two hexaploid oat lines in thefield-grown material whereas theA. abyssinicaacces-sion produced relatively more 16:0 and 18:1 and relat-ively less 18:2 fatty acids (Table 2). In the greenhouse,theA. abyssinicaaccession had a lower 18:1 fatty acidcontent and the hexaploids appeared likewise to havea lower 18:1 content but they had an increase in their16:0, 18:2, 18:3 and 20:1 proportions as comparedwith the field samples. HA 94-1113/1117 shows pro-portions of 18:0 (greenhouse) and 18:1 (both field andgreenhouse) intermediary between cv Hannes andA.abyssinica. All genotypes slightly increased their 20:2proportion in the greenhouse. The molar ratio of totalC18:C16 fatty acids was higher in the field samplesthan in the greenhouse samples, theA. abyssinicaac-cession showing the lowest ratio in both environments(Table 2).

Gigantic mutants have appeared in HA 94-1113/1117, hence resembling the relatively high mut-ability of this or a similar locus in common oats(Ahokas, 1996). Also a variant with black lemma hasbeen found in HA 94-1113/1117. This is caused by atentative spontaneous mutant.

Discussion

In general, the transfer of genes from di- and tetraploidAvenaspecies to hexaploid oats was considered diffi-cult (Frey, 1994). It has been achieved by chromosomesubstitution (Thomas et al., 1975; Sharma, 1978) andtranslocation (Aung & Thomas, 1978). Reductionsboth to octoploid and hexaploid levels were detected inA. abyssinica× A. sativaamphiploids by Nishiyama(1962). The twoA. abyssinica× A. sativaamphip-loid populations studied by Fritz & Sorrells (1990)produced euploids only at the decaploid level, theinstabilities leading to aneuploids and showing gen-otypic effects. The chromosome pairing efficiencyis likely controlled also by the temperature, whichis variable and moderate during the season of oatmeiosis in Finland. The temperature was probablyhigher in their environments (Nishiyama, 1962; Fritz& Sorrells, 1990). The new protein types in HA 94-1113/1117 suggest intragenic recombination, thoughalso other, more complicated reasons are possible fora protein of a new size. The two genomes A and B ofthe tetraploidA. abyssinicahave a close relationshipto one another (Katsiotis et al., 1997). Hence, both Aand B may tentatively recombine with the A genomeof the hexaploidA. sativa.

Among the environmental factors controlling lipidcontents in plants (Harwood, 1994), the temperatureeffect is the most probable in the present pair of en-vironments sampled, field vs greenhouse. The overalltemperature in the greenhouse was about 8◦C higherthan in the field. In the spring and early summer, thegreenhouse temperature hardly sinks below 10◦C atnights, while subzero temperatures are not uncom-mon in the field. Cv Hannes and HA 94-1113/1117appeared to have an ability to adapt to high temperat-ures by variations of their lipid deposition. InA. sativagrains (Beringer, 1971a), inBrassica napusL. leaves(Williams et al., 1996) and in developingHelianthusannuusL. seeds (Sarmiento et al., 1998), such lipidadaptation induced by temperature change may occurin a period of hours, hence apparently being vital forthe plant. In wheat grain lipids, the acyl composi-tion of the individual lipids 18:1 and 18:2 respondedsignificantly to temperature (Williams et al., 1995).Among the present field grown vs greenhouse grownoats, the accession ofAvena abyssinica, an originalAfrican oat, was the least affected, while both thehexaploid oat cultivar parent and the selected hybridresponded with increased 16:0, 18:2, 18:3 and 20:1and decreased 18:0 and 18:1 proportions in the warmer

158

Table 2. Fatty acid composition of lipids (molar %) extracted from meal of husked caryopses

Fatty acid and Name Field grown in 19961 Greenhouse grown in 19961 Field vs

Hannes HA 94- A. abys- Signi- Hannes HA 94- A. abys- Signi- greenhouse

1113/1117 sinica ficance 1113/1117 sinica ficance (Significance,

CI 2108 (H) CI 2108 (H) H)

16:0 Hexadecanoic 17.5 17.1 20.1 ◦ 20.7 20.7 21.6 ∗∗or palmitic 17.4 17.3 20.4 20.1 21.0 21.1

17.9 17.7 20.6

16:1 9-Hexadecenoic 0.4 0.4 0.3 0.5 0.5 0.3

or palmitoleic 0.3 0.3 0.4 0.4 0.4 0.3

0.4 0.4 0.3

18:0 Octadecanoic 1.4 1.4 1.2 0.9 1.1 1.2 ◦ ∗∗or stearic 1.4 1.2 1.2 0.9 1.1 1.2

1.5 1.3 1.2

18:1 9-Octadecenoic 37.3 38.1 39.5 ∗∗ 30.8 31.7 35.9 ◦ ∗∗or oleic 37.6 38.2 39.8 31.3 31.5 36.5

37.6 37.9 39.9

18:2 9,12-Octa- 40.2 40.0 35.0 ∗ 42.9 42.3 35.8 ◦ ◦decadienoic 40.3 39.7 34.6 43.3 41.7 35.4

or linoleic 39.8 40.0 34.4

18:3 9,12,15-Octa- 1.4 1.4 1.5 2.2 2.0 1.9 ∗∗decatrienoic 1.4 1.4 1.4 2.2 2.0 2.1

or linolenic 1.3 1.3 1.4

20:0 Eicosanoic 0.1 0.1 0.1 0.1 0.1 0.1

or arachidic 0.1 0.1 0.1 0.1 0.1 0.1

0.1 0.1 0.1

20:1 11-Eicosenoic 0.8 0.7 1.2 ◦ 0.9 0.9 1.2 ◦ ◦0.7 0.7 1.2 0.9 0.8 1.2

0.7 0.7 1.1

20:2 11,14-Eicosa- 0.2 0.1 0.2 0.2 0.2 0.3

dienoic 0.1 0.1 0.1 0.2 0.2 0.2

0.1 0.1 0.1

22:0 Docosanoic 0.1 0.0 0.1 0.1 0.1 0.2 ∗or behenic 0.0 0.1 0.1 0.1 0.6 0.2

0.0 0.0 0.1

22:1 13-Docosenoic 0.6 0.6 0.7 0.7 0.6 1.5

or erucic 0.7 0.8 0.8 0.6 0.6 0.8

0.6 0.6 0.7

Molar ratio C18:C16 4.47 4.55 3.72 ◦ 3.71 3.60 3.46 ∗∗

1 Results of independent extractions of different meals are listed.

greenhouse environment (Table 2). Fatty acid compar-isons of mutants, transgenics, somaclones and specieswith chilling sensitivity, or tolerance or the chillingacclimation in some plant species have been published(Lindberg et al., 1964; Beringer, 1971a,b; Murata etal., 1982; Hugly & Somerville, 1992; Kodama et al.,1994; Bertin et al., 1998; Martínez-Force et al., 1998;

Sarmiento et al., 1998). They indicate that lipids ofleaf or seed tissues under chilling conditions morefrequently have double bond desaturation and oftenhigher proportions of C18 over C16 fatty acids. Achange of the molar C18:C16 ratio in the fatty acidswas also found here as a response to cool (field) andwarm (greenhouse) environment, and a significant in-

159

crease in the molar proportion of oleic (18:1) acid as aresponse to cool environment (Table 2).

HA 94-1113/1117can be crossed withA. sativaforfurther recombination.A. abyssinicaCI 2108 is notknown to be any special source of disease resistance.The route described, however, may provide a way totransfer genes from tetraploids to hexaploids.

Acknowledgement

We are obliged to Ms Tuula Louhi (Boreal PlantBreeding Co) for advices on gas chromatography.

References

Aaltonen, V.T., B. Aarnio, E. Hyyppä, P. Kaitera, L. Keso, E. Kiv-inen, P. Kokkonen, M.J. Kotilainen, M. Sauramo, P. Tuorila &J. Vuorinen, 1949. A critical review of soil terminology and soilclassification in Finland in the year 1949. J Sci Agric Soc Finl21: 55–66.

Ahokas, H., 1978. A simple and rapid screening method for the de-termination of protein and tryptophan in kernel halves and smallsamples of barley meal. J Sci Food Agric 29: 47–52.

Ahokas, H., 1994. Searching for DNA introgressed from wheat andfor wheat-like grain proteins in a rice× wheat hybridizationderivative. Euphytica 72: 177–182.

Ahokas, H., 1996. Unfecund, gigantic mutant of oats (Avenasativa) shows fecundity overdominance and difference in DNAmethylation properties. Euphytica 92: 21–26.

Ahokas, H. & M. Poukkula, 1999. Malting enzyme activities, grainprotein variation and yield potentials in the displaced genetic re-sources of barley landraces of Finland. Genet Resour Crop Evol46: 251–260.

Appelqvist, L.-Å., 1968. Rapid methods of lipid extraction and fattyacid methyl ester preparation for seed and leaf tissue with specialremarks on preventing the accumulation of lipid contaminants.Arkiv för Kemi 28: 551–570.

Arias, J. & Frey, K.J., 1973. Selection for seed set in crosses ofAvena sativaL. × A. abyssinicaHochst. Euphytica 22: 413–422.

Aung, T. & H. Thomas, 1978. The structure and breeding behaviourof a translocation involving the transfer of mildew resistancefrom Avena barbataPott. into the cultivated oat. Euphytica 27:731–739.

Baker, R.J. & R.I.H. McKenzie, 1972. Heritability of oil content inoats,Avena sativaL. Crop Sci 12: 201–202.

Baum, B.R., 1977. Oats: wild and cultivated. A monograph of thegenusAvenaL. (Poaceae). Agriculture Canada, Monograph No.14.

Beringer, H., 1971a. Influence of temperature and seed ripening onthe in vivo incorporation of14CO2 into the lipids of oat grains(Avena sativaL.). Plant Physiol 48: 433–436.

Beringer, H., 1971b. An approach to the interpretation of the effectof temperature on fatty acid biosynthesis in developing seeds. ZPflanzenernähr Bodenk 128: 115–122.

Bertin, P., P. Bullens, J. Bouharmont & J.-M. Kinet, 1998.Somaclonal variation and chilling tolerance improvement inrice: changes in fatty acid composition. Plant Growth Regul 24:31–41.

Bowyer, P., B.R. Clarke, P. Lunness, M.J. Daniels & A.E. Osbourn,1995. Host range of a plant pathogenic fungus determined by asaponin detoxifying enzyme. Science 267: 371–374.

Brown, C.M., A.N. Aryeetey & S.N. Dubey, 1974. Inheritance andcombining ability for oil content in oats (Avena sativaL.). CropSci 14: 67–69.

Emme, E.K., 1932. K izuchenijo tsitologii pentaploidnyh gibridovovsov. Summary in German: Beitrag zur Zytologie derpentaploiden Haferbastarde. Trud Prikl Bot Genet Sel Ser 2(1):169–176.

Frey, K.J., 1994. Remaking a crop gene pool: the case history ofAvena. Proc Natl Sci Counc ROC, Part B, Life Sci 18(2): 85–93.

Fritz, S.E. & M.E. Sorrells, 1990. Effect of mass selection for seeddensity in populations derived fromAvena abyssinica×A. sativaamphidecaploids. Euphytica 46: 85–93.

Greipsson, S., H. Ahokas & S. Vähämiko, 1997. A rapid adaptationto low salinity of inland-colonizing populations of the littoralgrassLeymus arenarius. Int J Plant Sci 158: 73–78.

Harwood, J.L., 1994. Environmental factors which can alter lipidmetabolism. Prog Lipid Res 33: 193–202.

Hugly, S. & C. Somerville, 1992. A role for membrane lipid poly-unsaturation in chloroplast biogenesis at low temperature. PlantPhysiol 99: 197–202.

Hutchinson, J.B. & H.F. Martin, 1955. The chemical compositionof oats. I. The oil and free fatty acid content of oats and groats. JAgric Sci 45: 411–418.

Kates, M., 1964. Simplified procedures for hydrolysis or methan-olysis of lipids. J Lipid Res 5: 132–135.

Katsiotis, A., M. Hagidimitriou & J.S. Heslop-Harrison, 1997. Theclose relationship between the A and B genomes inAvenaL.(Poaceae) determined by molecular cytogenetic analysis of totalgenomic, tandemly and dispersed repetitive DNA sequences.Ann Bot 79: 103–109.

Kihara, H. & I. Nishiyama, 1932. The genetics and cytology ofcertain cereals. III. Different compatibility in reciprocal crossesof Avena, with special reference to tetraploid hybrids betweenhexaploid and diploid species. Jap J Bot 6: 245–305.

Kodama, H., T. Hamada, G. Horiguchi, M. Nishimura & K. Iba,1994. Genetic enhancement of cold tolerance by expression ofa gene for chloroplastω-3 fatty acid desaturase in transgenictobacco. Plant Physiol 105: 601–605.

Laemmli, U.K., 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227: 680–685.

Lesik, F.L., 1948. Eksperimental’nyi sintez amfidiploidov ovsa.DokI Akad Nauk SSSR 60: 299–300.

Lindberg, P., E. Tanhuanpää, G. Nilsson & L. Wass, 1964. The fatty-acid composition of ripening grain. Acta Agric Scand 14: 297–306.

Marshall, H.G. & W.M. Myers, 1961. A cytogenetic study of certaininterspecificAvenahybrids and the inheritance of resistance indiploid and tetraploid varieties to races of crown rust. Crop Sci1: 29–34.

Martínez-Force, E., R. Álvarez-Ortega, S. Cantisán & R. Gar-cés, 1998. Fatty acid composition in developing high saturatedsunflower (Helinathus annuus) seeds: maturation changes andtemperature effect. J Agric Food Chem 46: 3577–3582.

Murata, N., N. Sato, N. Takahashi & Y. Hamazaki, 1982. Compos-itions and positional distributions of fatty acids in phospholipidsfrom leaves of chilling-sensitive and chilling-resistant plants.Plant Cell Physiol 23: 1071–1079.

Nishiyama, I., 1962. Cytogenetic studies inAvena. IX. New syn-thetic oats in the progenies of induced decaploid interspecifichybrids. Jap J Genet 37: 118–130.

160

Nisius, A., 1988. The stromacentre inAvenaplastids: an aggreg-ation ofβ-glucosidase responsible for the activation of oat-leafsaponins. Planta 173: 474–481.

Rines, H.W., B.G. Gengenbach, K.L. Boylan & K.K. Storey, 1988.Mitochondrial DNA diversity in oat cultivars and species. CropSci 28: 171–176.

Sahasrabudhe, M.R., 1979. Lipid composition of oats (Avena sativaL.). J Amer Oil Chem Soc 56: 80–84.

Sarmiento, C., R. Garcés & M. Mancha, 1998. Oleate desaturationand acyl turnover in sunflower (Helianthus annuusL.) seed lipidsduring rapid temperature adaptation. Planta 205: 595–600.

Shapiro, A.L., E. Viñuela & J.V. Maizel, Jr., 1967. Molecularweight estimation of polypeptide chains by electrophoresis inSDS-polyacrylamide gels. Biochem Biophys Res Commun 28:815–820.

Sharma, D.C., 1975. Chromosome pairing problems in interploidytransfer of leaf rust resistance in oats. Euphytica 24: 503–510.

Sharma, D.C., 1978. Disomic alien chromosome substitution andaddition in hexaploid oat. Euphytica 27: 581–586.

Steer, M.W., 1975. Evolution in the genusAvena: inheritance ofdifferent forms of ribulose diphosphate carboxylase. Can J GenetCytol 17: 337–344.

Steer, M.W., J.H.W. Holden & B.E.S. Gunning, 1970.Avenachloro-plasts: species relationships and the occurrence of stromacentres.Can J Genet Cytol 12: 21–27.

Thomas, H. & D.A. Lawes, 1968. Evaluation of the use of specieshybrids and synthetic amphiploids in the improvement of the oatcrop. Euphytica 17: 404–413.

Thomas, H., J.M. Leggett & I.T. Jones, 1975. The addition of a pairof chromosomes of the wild oatAvena barbata(2n = 28) to thecultivated oatA. sativaL. (2n = 42). Euphytica 24: 717–724.

Welch, R.W., 1995. The chemical composition of oats. In: R.W.Welch (Ed), The Oat Crop, pp. 279–320, Chapman & Hall,London etc.

Williams, J.P., M.U. Khan & D. Wong, 1996. Fatty acid desaturationin monogalactosyldiacylglycerol ofBrassica napusleaves duringlow temperature acclimation. Physiol Plant 96: 258–262.

Williams, M., P.R. Shewry, D.W. Lawlor & J.L. Harwood, 1995.The effects of elevated temperature and atmospheric carbondioxide concentration on the quality of grain lipids in wheat (Trit-icum aestivumL.) grown at two levels of nitrogen application.Plant Cell Environ 18: 999–1009.

Zillinsky, F.J., 1956. Cross compatibility relationships among someAvenaspecies and polyploids. Can J Agric Sci 36: 107–113.