A mutant of Brassica napus with increased palmitic acid content

Plant Breeding 008\ 030*033 "1999# Þ 1999 Blackwell Wissenschafts!Verlag\ Berlin ISSN 9068!8430

A mutant of Brassica napus with increased palmitic acid content 0 0\2 T[ SCHNURBUSCH0\1\ C[ MO Ý LLERS and H[ C[ BECKER 0

Institut fur P~anzenbau und P~anzenzuchtung\ Georg!August!Universitat\ Von!Siebold!Strasse 7\ DÐ26964 Gottingen\ Germany^ 1 Present address] Institute of Plant Biology\ University of Zurich\ Zollikerstr[ 096\ CH!7997 Zurich\ Switzerland^ 2 Corresponding author With 0 _gure and 0 table Received May 2\ 0888:Accepted October 01\ 0888 Communicated by W[ Friedt

Abstract

Materials and Methods

An induced mutant from European winter oilseed rape with increased palmitic acid content was phenotypically characterized and genetically analysed[ The mutant showed a palmitic acid content of 8[1) compared with 3[4) in the parental cultivar[ The oleic acid content decreased from 50[5) to 33[1)\ whereas the linoleic and linolenic acid contents increased[ The mutant plants grew poorly and their seed oil content was only 20[1) compared with 31[7) in the parental cultivar[ The inheritance of the mutant was oligogenic and determined by at least four genes[ In the F1 generation\ palmitic acid content was negatively correlated with oil content[ This mutant may be useful to improve understanding of the genetic regulation of storage lipid synthesis\ but has no immediate value for oilseed rape improvement[

The mutant M3825 was isolated after ethylmethane sulphonate treat! ment of winter rapeseed\ Brassica napus L[\ cv[ {Wotan| "Rucker and Robbelen 0884#[ The mutant plants were self!pollinated for several generations and controlled for stable expression of a high palmitic acid content[ The M4 line 41:0933:85 was reciprocally crossed with a line from {Wotan| "47:0949:85# and the cross was propagated until the F1[ The parents of the cross\ the F0 and the F1 generation were grown in 0887 at the experimental _eld station Reinshof near Gottingen\ Germ! any[ Randomly chosen plants were bagged before ~owering[ Fatty acid composition of the seeds was analysed by gas chromatography according to Rucker and Robbelen "0885# from 20 "{Wotan|#\ 02 "mutant#\ 64 "F0# and 146 "F1# plants\ respectively[ Oil and protein content were determined in 649 mg seed samples by near!infrared re~ectance spectroscopy "Reinhardt 0881#[ The fatty acid composition of leaves and roots of the parent lines was determined from 19 2!week! old plants\ as described by Rucker and Robbelen "0886#[ The minimum number of genes\ k\ was estimated by the approach originally proposed by Castle and elaborated by Wright "0857#]

Key words] Brassica sp[ * fatty acids * mutants * oil con! tent * palmitic acid The optimal composition of vegetable oils for human con! sumption is a compromise between the demands of nutrition physiology and the requirements of food technology[ From a nutritional aspect\ the content of saturated fatty acids should be as low as possible\ and in this respect\ oilseed rape has the most valuable oil of all major oil crops "Scarth and McVetty 0888#[ It contains only about 3) palmitic acid "C05 ] 9# and about 1) stearic acid "C07 ] 9# "Luhs and Friedt 0882#[ However\ this low level of saturated fatty acids is the reason for the low melting point of rapeseed oil[ To produce margarine\ rapeseed oil has to be blended with other vegetable oils rich in saturated fatty acids\ such as palm or coconut oil\ or it has to be arti_cially hydrogenated[ The industrial hydrogenation of vegetable oils requires a high energy input and results in the formation of trans fatty acids\ which are undesirable for human nutrition "Fritsche and Steinhart 0886#[ Therefore\ an increased content of saturated fatty acids is of interest for the production of a rapeseed oil suitable for mar! garine production[ In Swedish breeding programmes "Jonsson and Uppstrom 0875# palmitic acid contents up to 00) in oilseed rape and up to 08) in turnip rapeseed "Brassica rapa# were observed[ In Finnish turnip rapeseed\ Tanhuanpaa et al[ "0884# identi_ed one quantitative trait locus which increased palmitic acid content by nearly 3) and which explained 74) of the variation observed[ The objective of this study was to investigate a recently detected mutant with increased palmitic acid content "Rucker and Robbelen 0886#[ This mutant was phenotypically char! acterized under _eld conditions and the inheritance of palmitic acid content was analysed[ U[ S[ Copyright Clearance Center Code Statement]

k  "P0 Ð P1#1:7"s1F1 Ð VE# where P0 and P1 are the mean values of the two parents\ s1F1 is the variance of the F1 plants and VE is the environmental variance among individual plants[ VE was estimated by pooling the variances within the genetically uniform generations P0\ P1 and F0[ The standard error of k was calculated according to Lande "0870#[

Results The high palmitic acid mutant had a drastically changed fatty acid pattern in the seed oil "Table 0#] the content of palmitic acid was about doubled\ and the content of oleic acid "C07 ] 0# was reduced whereas the contents of linoleic acid "C07 ] 1# and linolenic acid "C07 ] 2# were increased[ The mutant was pheno! typically very di}erent from the original cultivar[ It was earlier! ~owering and much less vigorous[ The dry matter yield was only about 19) of the yield of the control plants[ This cannot be easily explained\ because the changes in the fatty acid pattern of the mutant were seed speci_c[ The palmitic acid content was not increased in leaves and roots "Table 0# and the content of other fatty acids was unchanged in leaves and roots "data not shown#[ This is in agreement with the results of Rucker and Robbelen "0886#[ Figure 0a shows the frequency distribution of the palmitic acid content in the seeds of the parents and the F0[ The two reciprocal F0s did not di}er signi_cantly and were pooled[ The three genotypes are clearly separated\ without any overlap[ If the increased palmitic acid content is due to a mutation in one

9068Ð8430:1999:0891Ð9030 , 04[99:9

SCHNURBUSCH\ MOÝLLERS and BECKER

031 Table 0] Comparison of the cv[ {Wotan| and the high palmitic acid mutant "HP!mutant# in the _eld in 0886:87\ the palmitic acid content "C05 ] 9# of leaves and roots under controlled conditions in a growth chamber\ and phenotypic cor! relation coe.cients between pal! mitic acid and other seed traits in F1!plants of the cross {Wot! an| × HP!mutant

Trait Seed Palmitic acid ")# Palmitoleic acid ")# Stearic acid ")# Oleic acid ")# Linoleic acid ")#0 Linolenic acid ")# Oil ")#1 Protein ")#1 Leaf Palmitic acid2 ")# Root Palmitic acid2 ")# Plant Beginning of ~owering "days from 0 April# Plant height3 "cm# Dry matter yield3 "g#

{Wotan|

HP!mutant

Correlation coe.cient with C05 ] 9 content

3[4 2 9[1 9[3 2 9[0 0[4 2 9[0 50[5 2 0[6 08[7 2 0[1 09[0 2 9[5 31[7 2 0[7 08[6 2 0[2

8[1 2 9[2 0[0 2 9[1 0[6 2 9[1 33[1 2 1[9 15[8 2 0[0 03[7 2 9[8 20[1 2 1[7 13[9 2 0[1

9[73 Ð9[97 Ð9[46 9[04 9[29 Ð9[24 9[10

03[7

03[7

06[3

06[0

12 048[1 2 3[8 33[7 2 10[5

07 78[7 2 09[8 8[2 2 5[2

0

Per cent of total fatty acids 2 SD[ Per cent of dry matter 2 SD[ Values are means from a bulk of 19 plants grown in a growth chamber[ 3 Values are means of _ve plants 2 SD[ 1 2

Fig[ 0] Frequency distribution of palmitic acid content in the cv[ {Wot! an|\ the high palmitic acid mutant "HP!Mutant#\ and their F0 "A# and of the segregating F1 population "B#

gene\ the F1 should show a clear 0 ] 1 ] 0 segregation[ However\ the F1 showed a skewed normal distribution\ and hardly any F1 plant reached the high palmitic acid values of the mutant "Fig[ 0b#[ The minimum number of genes\ k\ was estimated from the F1 variance "9[6547# and the environmental variance within parents and F0 "pooled estimate 9[0460#[ This resulted in an estimate for k of 3[4 2 0[9\ suggesting that at least four genes are involved in the di}erence in palmitic acid content between the mutant and the original cv[ {Wotan|[ The correlations between the palmitic acid content of the F1 plants and other seed traits are given in Table 0[ Increased palmitic acid content is correlated with decreased content in oleic acid\ and increased content in linoleic and linolenic acid[ The most important result is the signi_cant negative correlation between palmitic acid content and oil content[ The inheritance of the mutant was found to be oligogenic or polygenic[ Several methods have been proposed to estimate gene numbers for quantitative traits] all su}er from certain disadvantages "for a brief review see Mayo 0876#[ We used the CastleÐWright index which is based on the following four assumptions "Hill et al[ 0887#] "0# that one parent contains only the increasing and the other parent the decreasing alleles for the trait in question^ "1# that all genes a}ect the trait equally^ "2# that the loci controlling the trait are unlinked^ "3# that dominance and epistasis are absent[ Violation of these assump! tions leads to an underestimation of the true number of genes[ Therefore\ the estimated number of four genes is a minimum number^ the actual number may be larger[ The results from this quantitative analysis are in agreement with the observed F1 distribution "Fig[ 0#[ All parental plants of the mutant had a palmitic acid content above 7)\ but only seven out of 146 F1 plants reached this value[ This result cannot be explained by monogenic or digenic segregation[ A complex inheritance of palmitic acid content was also observed in soybean\ with at least four genes involved "Schnebly et al[ 0883# and in sun~ower with three genes involved "Perez!Vich et al[ 0888#[ However\ it seems unlikely\ that the mutagenic treatment

Brassica napus mutant with increased palmitic acid content

032

resulted in the change of at least four genes for palmitic acid content at the same time[ One explanation might be that the original material used for mutagenesis was not completely uniform[ The M0 generation was very large "19 999 plants# and the initial selection pressure very severe "Rucker and Robbelen 0884#\ so that selection for minor genetic variation existing before mutagenic treatment might have been e}ective[ The changed palmitic acid content of the mutant was associ! ated with changes in other fatty acids\ namely a decrease in oleic acid and an increase in polyunsaturated fatty acids[ A decrease in oleic acid content was also observed in several other crops such as soybean "Rebetzke et al[ 0885\ Hartmann et al[ 0885#\ sun~ower "Fernandez!Mart(nez et al[ 0886#\ turnip rape! seed "Jonsson and Uppstrom 0875\ Tanhuanpaa et al[ 0884# and linseed "Ntiamoah et al[ 0884\ Rowland et al[ 0884#[ The changes in other fatty acids were not consistent in the various crops[

mitic acid content with high yield by selection in further gen! erations will require a large breeding programme because of the complex inheritance of palmitic acid content[ An even more severe problem may be the negative correlation between pal! mitic acid content and oil content[ A better knowledge of fatty acid metabolism in the future will help us to understand whether this correlation is due to pleiotropic e}ects of the genes involved\ or whether high palmitic acid content can be com! bined with high oil content[ Therefore\ the mutant described in the present study will be valuable for further studies in pin! pointing the genes involved in the complex biosynthesis of stor! age lipids in rapeseed[

Discussion

References

The observed results give some indications as to which of the genes involved in the fatty acid biosynthesis might be a}ected in the mutant[ The ratio of C05 to C07 products was doubled in the present mutant "9[007 vs[ 9[942#\ indicating that the activity of the b!ketoacyl!ACP synthase II "KAS II# is reduced "for review see Harwood 0885\ Ohlrogge and Jaworski 0886#[ Furthermore\ the reduced oil content observed in the present mutant could well be a consequence of a reduced KAS II activity[ However\ an Arabidopsis mutant with a reduced KAS II activity "James and Dooner 0889\ 0880\ Wu et al[ 0883# did not show elevated linoleic acid and linolenic acid contents as found in the present experiments[ The increased contents of linoleic acid and linolenic acid together with a reduced oil con! tent resembles more the phenotype of an Arabidopsis mutant with a reduced diacylglycerol acyltransferase activity "DAGAT# described by Katavic et al[ "0884#[ DAGAT is the _nal enzyme in the Kennedy pathway and thus may regulate triacylglycerol biosynthesis[ If the function of this enzyme is drastically impaired\ diacylgylcerols "DAGs# should accumulate "Katavic et al[ 0884#[ However\ the analysis of the mature seed lipids of the mutant by thin!layer chromatography did not reveal an accumulation of DAGs "data not shown#[ Further genetic and biochemical analyses are required to clarify whether the activity of the KAS II and:or the DAGAT are impaired\ and to identify the other genes involved in the mutant phenotype[ An alternative approach to increase palmitic acid content is the transformation of rapeseed with thioesterase genes[ After transformation with acyl!ACP thioesterase from soybean\ Hitz et al[ "0884# observed a palmitic acid content of 8[1) compared with 2[8) in the untransformed control[ Jones et al[ "0884# and Martini et al[ "0884# reported the production of a C05 ] 9!rich oil in rapeseed after transformation with medium!chain acyl! ACP thioesterases from Cuphea species[ These transgenic lines contained myristic acid "C03 ] 9# and a high amount of palmitic acid\ up to 15) "Rudlo} and Wehling 0887#[ The increase in palmitic acid content in the transgenic plants was correlated with a decrease in C07 ] 0\ whereas yield and oil content were not signi_cantly changed "Rudlo} et al[ 0888#[ In conclusion\ the mutant has several disadvantages for use in a breeding programme[ The poor vegetative growth and the low yield may be a side!e}ect of the mutagenesis and not a physiological relation\ because the changes in palmitic acid content in the mutant are seed speci_c[ To combine high pal!

Fernandez!Mart(nez\ J[ M[\ M[ Mancha\ J[ Osorio\ and R[ Garces\ 0886] Sun~ower mutant containing high levels of palmitic acid in high oleic background[ Euphytica 86\ 002*005[ Fritsche\ J[\ and H[ Steinhart\ 0886] Trans fatty acid content in German margarines[ Fett:Lipid 88\ 103*106[ Hartmann\ R[ B[\ W[ R[ Fehr\ G[ A[ Welke\ E[ G[ Hammond\ D[ N[ Duvick\ and S[ R[ Cianzio\ 0885] Association of elevated palmitate content with agronomic and seed traits of soybean[ Crop Sci[ 25\ 0355*0369[ Harwood\ J[ L[\ 0885] Recent advances in the biosynthesis of plant fatty acids[ Biochim[ Biophy[ Acta 0920\ 6*45[ Hill\ J[\ H[ C[ Becker\ and P[ M[ A[ Tigerstedt\ 0887] Quantitative and Ecological Aspects of Plant Breeding[ Chapman and Hall\ London[ Hitz\ W[ D[\ C[ J[ Mauvis\ K[ G[ Ripp\ R[ J[ Reiter\ L[ Debonte\ and Z[ Chen\ 0884] The use of cloned rapeseed genes for the cytoplasmic fatty acid desaturases and the plastid acyl!ACP thioesterases to alter relative levels of polyunsaturated and saturated fatty acids in rap! eseed oil[ Proc[ 8th Int[ Rapeseed Congr[ "GCIRC#\ Cambridge\ 1\ 369*361[ James\ D[ W[\ and H[ K[ Dooner\ 0889] Isolation of EMS!induced mutants in Arabidopsis altered in seed fatty acid composition[ Theor[ Appl[ Genet[ 79\ 130*134[ James\ D[ W[\ and H[ K[ Dooner\ 0880] Novel seed lipid phenotypes in combinations of mutants altered in fatty acid biosynthesis in Ara! bidopsis[ Theor[ Appl[ Genet[ 71\ 398*301[ Jonsson\ R[\ and B[ Uppstrom\ 0875] Quality breeding in rapeseed[ In] G[ Olsson "ed[#\ Svalof 0775Ð0875 Research and Results in Plant Breeding\ 062*072[ TLs Forlag\ Stockholm[ Jones\ A[\ H[ Maelor Davies\ and T[ A[ Voelker\ 0884] Palmitoyl!acyl carrier protein "ACP# thioesterase and the evolutionary origin of plant acyl!ACP thioesterases[ Plant Cell 6\ 248*260[ Katavic\ V[\ D[ W[ Reed\ D[ C[ Taylor\ E[ M[ Giblin\ D[ L[ Barton\ J[ Zou\ S[ L[ MacKenzie\ P[ S[ Covello\ and L[ Kunst\ 0884] Alteration of seed fatty acid composition by an ethyl methansulfonate!induced mutation in Arabidopsis thaliana a}ecting diacylglycerol acyl! transferase activity[ Plant Physiol[ 097\ 288*398[ Lande\ R[\ 0870] The minimum number of genes contributing to quan! titative variation between and within populations[ Genetics 88\ 430* 442[ Luhs\ W[\ and W[ Friedt\ 0882] The major oil crops[ In] D[ J[ Murphy "ed[#\ Designer Oil Crops\ 4*60[ VCH\ Weinheim[ Martini\ N[\ J[ Schell\ and R[ Topfer\ 0884] Expression of medium! chain acyl!"ACP# thioesterases in transgenic rapeseed[ Proc[ 8th Int[ Rapeseed Congr[ "GCIRC#\ Cambridge\ 1\ 350*352[ Mayo\ O[\ 0876] The Theory of Plant Breeding\ 1nd edn[ Clarendon Press\ Oxford[ Ntiamoah\ C[\ G[ G[ Rowland\ and D[ C[ Taylor\ 0884] Inheritance of

Acknowledgements The authors thank Prof[ Dr G[ Robbelen and Dr B[ Rucker for pro! viding the original mutant\ Dr D[ Stelling for providing the parental lines for the crosses\ A[ Schierholt for data on fatty acids in leaves and roots and C[ Reuter for excellent technical assistance[

033 elevated palmitic acid in ~ax and its relationship to the low linolenic acid[ Crop Sci[ 24\ 037*041[ Ohlrogge\ J[ B[\ and J[ G[ Jaworski\ 0886] Regulation of fatty acid synthesis[ Annu[ Rev[ Plant Physiol[ Plant Mol[ Biol[ 137\ 098*025[ Perez!Vich\ B[\ J[ Fernandez\ R[ Garces\ and J[ M[ Fernandez!Mart! inez\ 0888] Inheritance of high palmitic acid content in the seed oil of sun~ower mutant CAS!4[ Theor[ Appl[ Genet[ 87\ 385*490[ Rebetzke\ G[ J[\ V[ R[ Pantalone\ J[ W[ Burton\ B[ F[ Carver\ and R[ F[ Wilson\ 0885] Phenotypic variation for saturated fatty acid content in soybean[ Euphytica 80\ 178*184[ Reinhardt\ T[!C[\ 0881] Entwicklung und Anwendung von Nah!Infra! rot!Spektroskopischen Methoden fur die Bestimmung von O Ý l!\ Protein!\ Glucosinolat!\ Feuchte! und Fettsaure!Gehalten in intakter Rapssaat[ Cuvillier!Verlag\ Gottingen[ Rowland\ G[ G[\ A[ McHughen\ L[ V[ Gusta\ R[ S[ Bhatty\ S[ L[ MacKenzie\ and D[ C[ Taylor\ 0884] The application of chemical mutagenesis and biotechnology to the modi_cation of linseed "Linum usitatissimum L[#[ Euphytica 74\ 206*210[ Rucker\ B[\ and G[ Robbelen\ 0884] Development of high oleic acid rapeseed[ Proc[ 8th Int[ Rapeseed Congress "GCIRC#\ Cambridge\ 1\ 278*280[ Rucker\ B[\ and G[ Robbelen\ 0885] Impact of low linolenic acid content on seed yield of winter oilseed rape "Brassica napus L[#[ Plant Breed! ing 004\ 115*129[ Rucker\ B[\ and G[ Robbelen\ 0886] Mutants of Brassica napus with altered seed lipid fatty acid composition[ In] J[ P[ Williams\ M[ U[

SCHNURBUSCH\ MOÝLLERS and BECKER Khan\ N[ Wan Lem "eds#\ Physiology\ Biochemistry and Molecular Biology of Plant Lipids\ 205*207[ Kluwer Academic Publ[\ Dord! recht[ Rudlo}\ E[\ and P[ Wehling\ 0887] Release of transgenic oilseed rape "Brassica napus L[# with altered fatty acids[ Acta Hort[ 348\ 268* 274[ Rudlo}\ E[\ H[ U[ Jurgens\ B[ Ruge\ and P[ Wehling\ 0888] Selection in transgenic lines of oilseed rape "Brassica napus L[# with modi_ed seed oil composition[ Proc[ 09th Int[ Rapeseed Congress "GCIRC#\ Canberra[ Compact Disk[ Scarth\ R[\ and P[ B[ E[ McVetty\ 0888] Designer oil canola * a review of new food!grade Brassica oils with focus on high oleic\ low linolenic types[ Proc[ 09th Int[ Rapeseed Congress "GCIRC#\ Canberra[ Com! pact Disk[ Schnebly\ S[ R[\ W[ R[ Fehr\ G[ A[ Welke\ E[ G[ Hammond\ and D[ N[ Duvick\ 0883] Inheritance of reduced and elevated palmitate in mutant lines or soybean[ Crop Sci[ 23\ 718*722[ Tanhuanpaa\ P[ K[\ J[ P[ Vilkki\ and H[ J[ Vilkki\ 0884] Identi_cation of a RAPD marker for palmitic!acid concentration in the seed oil of spring turnip rape "Brassica rapa ssp[ oleifera#[ Theor[ Appl[ Genet[ 80\ 366*379[ Wright\ S[\ 0857] Evolution and the Genetics of Populations\ Vol[ 0[ Univ[ of Chicago Press\ Chicago[ Wu\ J[\ D[ W[ James\ H[ K[ Dooner\ and J[ Browse\ 0883] A mutant of Arabidopsis de_cient in the elongation of palmitic acid[ Plant Physiol[ 095\ 032*049[