For Cereal Grain Protein Imrovement

IAEA-TECDOC-259

INDUCED MUTANTS FOR CEREAL GRAIN PROTEIN IMROVEMENT

PROCEEDING RESEARCA F SO H CO-ORDINATION MEETING ORGANIZED BY THE JOINT FAO/IAEA DIVISION OF ISOTOP RADIATIOD EAN N APPLICATION ATOMIF SO C ENERGY FOR FOO AGRICULTURAD DAN L DEVELOPMENT WITH SUPPORT FROE MTH GESELLSCHAF STRAHLENR TFÜ UMWELTFORSCHUND UN - H GMB SWEDISE TH D HAN INTERNATIONAL DEVELOPMENT AUTHORITY AND HELD IN NICOSIA, CYPRUS, 21-25 APRIL 1980

A TECHNICAL DOCUMENT ISSUED BY THE INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1982 INDUCED MUTANTS FOR CEREAL GRAIN PROTEIN IMPROVEMENT IAEA, VIENNA, 1982

Printe IAEe th AustriAn y i d b a January 1982 PLEASE BE AWARE THAT ALL OF THE MISSING PAGES IN THIS DOCUMENT WERE ORIGINALLY BLANK The IAEA does not maintain stocks of reports in this series. However, microfiche copies of these reports can be obtained from

INIS Microfiche Clearinghouse International Atomic Energy Agency Wagramerstrasse 5 P.O. Box 100 A-1400 Vienna, Austria prepaymenn o f Austriao t n Schillings 40.0 r agains0o IAEe on t A microfiche service coupon. FOREWORD

programmA e directed toward genetie sth c improvemen seef o t d protein bot qualitn i h quantitd an y s initiate ywa Join e th ty db FAO/IAE A Division of Atomic Energy in Food and Agriculture in 1969. The problems related to protein improvemen foon i t d were reviewe FAO/IAEn a y b d A Symposiu Plann mo t Protein Resources : Their Improvement through the Application of Nuclear Techniques held in Vienna, 8-12 June 1970 (STI/PUB/258). From 1971, this programme received financial support from the Federal Republic of Germany throug Gesellschafe th h Strahlenr fu t Umweltforschund -un g (GSF programme Th ). e was terminated in 1978 and its achievements were summarized and reported by B. Sigurbjornsson, R.D. Brock and T. Hermelin at the FAO/IAEA International Symposium on Seed Protein Improvement in Cereals and Grain Legumes, 4-8 September 1978 (STI/PUB/496).

A follow-up programme, the'Coordinated Researcf o he ProgrammUs e th n o e Nuclear Technique Improvemene th r fo s Cereaf o t l Grain Protei s initiatenwa d in 1980 with Financial supporSwedisd an F thGS froe Internationath m l Development Authority (SIDA). The programme has the aim of advancing towards practical utilizatio obtainee th f o nd mutants achievee whicb n hca d through : Testin. I g mutant line grair sfo n yield, protein yield, protein qualit d generayan l agronomic performance, witobjective th h e of releasing the best ones as varieties ; II. Using protein mutants in cross breeding to obtain advanced breeding materials having the potential of being released as varieties; II. Nutritional evaluation of advanced breeding materials.

e firsTh t research coordination meetin thif go s programm s helet wa a d the Agricultural Research Institute, Nicosia, Cyprus, 21-25 April 1980. Participants presented their results obtained after the termination of the previous programme and research plans in the future. On this occasion, a closer cooperatio s establishenwa d with another international programme having similar objectives, the FAO/SIDA/SAREC Project on Improvement in Nutritional Quality of Barley and Spring Wheat.

The results and discussions at the meeting indicated that genetic improvemen graif to n protei qualitn ni d quantityan y combination ,i n with high grain productivity, is more difficult than was originally assumed. Some advanced protein mutation breeding projects are disturbed by problems wit pathogenw hne biotyper so pesf so t insects weakeo T . e nth negative association between protein content and grain yield will be a common targe researcf nexe o t th t r yearsh fo papere Th . s include thin i d s publication present result breedinf so g effort alsd san o various approaches to overcome the difficulties in genetic improvement of protein quality and quantity. CONTENTS

Evaluation of barley mutant lines in dry areas of Cyprus ...... 7 A Hadjichristodoulou, Athena Delia High protein mutant ricn si e ...... 9 1 . M. Ismachin, K. Mugiono Testing, cross-breeding, inductio nutritionad nan l evaluatio wheaf no d an t triticale mutants ...... 1 3 . P.C. Parodi, NebredaIM. Achievements in seed protein improvement programme ...... 53 M. TahirNadeem Improved quantitative protein mutants, selected from a barley mutation breeding programme using advanced selection procedures ...... 5 5 . H. Walther Problems and possibilities in genetic improvement of protein yields in cereals ...... 71 F. Scholz Utilization of high grain protein wheat mutants ...... 81 CR. Bhatia, R. Mitra incorporatioe Th f mutanno t gene widy sb e crosse theid san r consequencee th r sfo quality complex in hexaploid wheats ...... 93 K. Nagl Breeding varieties with increased protein content in rice ...... 109 C. Gangadharan, S.N. Ratho, PandeK. Research work on high-lysine barley at Svalöv ...... 137 Tallberg,A. K:E. Karlsson, StayV. Protein improvement in cereals I. Wheat ...... 143 . Konzak,CF G.L. Rubenthaler Protein improvement in cereals II. Barley ...... 153 S.E. Ullrich, T.K. Blake, A. Kleinhofs, C.N. Coon, R.A. Nilan higf o he approacproteius w Ae ne th r n hfo varietie mutantd san breedinn si g for high grain conten Triticumn i t aestivum ...... 1 16 . Brunori,A. Figueroa,A. MickeA. Studies of barley high-lysine mutants and seed proteins at Risci ...... 165 H. Doll, J. Jensen, B. K

A. HADJICHRISTODOULOU, ATHENA DELLA Agricultural Research Institute, Nicosia, Cyprus

ABSTRACT

e higTh h yielding barley lines, Athenais, Attikd ian Beecher, were treated with/^-rays, fast neutrons and S1!S at different doses. Screening was carried out for both agronomic performance and quality. Lines having yield lower than the control were not selected as, at present, there is no premium price for grain with improved quality. Some lines were selected from trials conducted at four locations with up to ll?o higher grain yield, 1% higher DBG and Kjeldahl protein content and 20fo higher protein yield. These lines will be tested at more locations and for 2-3 more years in order to evaluate their adaptatio areay dr d thei o an st n r suitability for commercial cultivation»

INTRODUCTION

Cyprun I s barle s considereywa mose th dt productive crop under rainfed conditions (Hadjichri stodoulou 1S7Ud )an only tecently the new durum wheat variety Arenas and a few triticale lines gave equa r highelo r yields thabese th nt barley variety Athenais (Agricultural Research Institute, 1980). Several methods including introduction, hybridization and mutation, were use n ordei d improvo t r e barley varieties. l thesal ey B methods promising varieties were selected, some of which were recommended for release to growers (Hadjichristo- doulou, Josephide Delid an s a 1980). Mutant lines of barley have been used in many countries s varietiea s parenta r o sn crossini s g programmes (Sigurbjorn- sso Micked nan , 197U; Sigurbjjornsson, 1975Î Micke,' 1979a and 1979b) .e mutanth Mos f o t varieties were released late, since 1970.and their most common attribute e shorar s t and strong stem, high yield, lodging resistance, early maturity, disease and malting and milling quality.

Mutation breedin r improvefo g d protei s beenha n given very little attentio paste th n .ni However, since 197e 0th attention of breeders has increased and PAO, IAEA and other International Agencies have intensified their efforto t s improve protein content and quality by induced mutations and hybridization. Until to-day there is very little information on commercial varieties (having high agrondmic performance) released from direct multiplication of mutants. There is one report from India (Sharma and Sutar, 1977) in which it is mentioned that the R-16 barley mutant outyielded the mother variet grain yi n yiel proteid an d n yield. Several protein mutants have been induce n barlei d t theiybu r yiel lowes dwa r than the mother variety, and for this reason they are being used in crossing programmes. The most widely used induced mutant is^ perhaps, the high lysine mutant 1508, developed t Risoa , Denmark,

The aim of the mutation breeding in Cyprus is to develop new varieties having higher protein content and producing at least similar yield as their mother variety under dryland conditions. Varieties with lower yields cannot be released because there is no premium price for protein content or quality achievo T . e thi e followinth s g procedures were followed:

(a) Selectio exercises nwa treaten i d d populations for the direct release of mutants and (b) Crosses were made between introduced high prqtein and lysine spontaneou induced an s d mutants with high yielding varieties. There are plans to cross also induced protein mutants selected from our mutation breeding programme.

The available results of induced mutants for direct release wil discussee b l n thii d s report»

MATERIALS AND MCTHOES

As the aim was to induce mutants for commercial cultivation, e highesth t yielding varietie Cyprun i s s were treated with ionizing radiatio r EMSo n . Selectio n treateni â populations continuously aimed at both high agronomic performance and protein improvement (high DBG and N content).

In 1969 the barley varieties Athenaia (main variety in Cyprus Beeched an ) r (new promising variety) were treatee th t da IAEA Laborator n Seibersdorfi y , Austria, with )^-rays (10, 20 , 30 rad fasd )an t neutrons (200, UOO rad)0 60 , .

In 1971 Athenais was treated with 'K-rays (15 and 25 krad) and fast neutrons (500, 700 and 1000 rad). It was also treated in our laboratory with E.lvI.S. (0.5/4 and 0.9$).

commerciaw n 197ne I e 3th l variety Attik treates wa i d wit kra5 h2 d V<-ray 100d an s 0 rads fast neutrons.

n 197 I materiaw 5ne f Athenaio l s barle treates e wa yth t da IAEA Laboratory at Seibersdorf using higher doses of /^-rays (25» krad 5 fasd U an d )t an neutron 5 3 s (700, 100 130d 0an 0 rad).

In general, the screening and evaluation procedure was the following: In the first generations, Mg-M-,, visual single plant selection was carried out for good agronomic performance. Som . linee M unifor sl al , linewer mM d ean s screenen i d nurserie r improvefo s d protei alsd r agronominan ofo c performance.

Screenin f proteio g s don t nwa earla e y stagee DBth Gy b s method, as mg of dye Acid Orange 12, bound by the protein f flour o mose g Th m t presen.0 advance80 n i t d materias wa l also screened for N content by the Kjeldahl method.

9 Selected varieties were evaluated in Randomized Complete Blocks preliminare Th . y screenin 3 replications 2- dons n i ewa g , o small plots (l.2m ) and at one location. The most advanced materia screenes lwa replicationsx trialn i dsi f o s , large n plot size, lUm , and at 2-3 locations. Seed rate was 100 kg/ha. At sowing 2k kg Vha and 30kg FgO^/ha were applied. At tillering stag additionan a e N/hg k a1 2 lwer e applied,

The selection proces n treatei s d materia similas wa l n i r l populationsal . e Detailselectioth r fo s n procesn i s population f Attikio s , treated wit kra5 2 h d V^-ray 100d an s 0 rads fast neutrons at the IAEA Laboratory at Seibersdorf, Austria, are given below:

In 197*4- 600 single M^ plants were harvested. Prom each M, plant 5«5ne spaces on ,> wa lon w d- sow ro g n Mgi n , securing one plant every 10 cm. Single plants were selected on the , basis of agronomic characters (disease reaction and plant type). Four hundred and ninety seven plants were selected, 192 from e 100th d populatiokra5 02 ra frod5 e population«30 mth d nan ,

These plants were sown in a nursery, each Mg plant in one M, row, »2 J long, with one check every 10 lines. They were harvested in bulk and screened by the DEC method. The most promising 75 M, lines, on the basis of agronomic performance and DBG, were teste n Randomisei d d Complete Blocks with four replication . (seveM s a ns trials wit line0 1 h e son eacd an h n with five, in addition to the c hecks) . Plot size was l4.«8m . The best eight lines, selected from these trials for high DBC and grain yield were include trialn i d s wit replicationx hsi s and plot size Il4fli2. (The results for 1977-79 are given in Table. 5) d s an 1,2 k , ,3

RESULTS

A general observatio e materianth fro l mal l screenes dwa that ther significans wa e t variation among lines selected from treated population content r N DB fo d s Can . However, verw yfe lines combined high agronomic performanc improved an e d protein,,

10 The lines with the highest protein improvement had lower agronomic performance than their mother variety.

One of the first promising mutant lines was M71-Ath.-l4.95-3» selected from Athenais treated with 1000 red in 1971. This line, teste replicaten di d yield trial t eigha s t environments over the years 1975-79, produced grains having 10# higher protein content than Athenais (ranging with environmen to 13^)t fro% 5 n. However, its grain yield was on average 5?S lower, so the line was not promoted.

The most promising lines were selected frovariete mth y Attüci treated wit kra5 2 h d v^-ray 100d an s0 rads fast neutrons (Tables 1, 2, 3» k and 5). These mutants were tested in Randomized Complete Blocks wit replicationh6 ll^d man s large plots bese tTh .line , M-Att-73-335-3 gave significantly higher grain yield (ll#), DBG (7$ Kjeldahd )an l protein content (7#).

Line M-Att-73-122-2 had the highest DBG and K&eldahl protein content but the lowest grain yield, lower than the mother variety. There were also significant differences between mutant line Attikd an sr plan fo i t heigh 1000-graid an t n weight but differences in earliness and volume weight were small.

Kjeldahl protein yiel availabls dwa e locationfroo tw n s (Table 5). With the exception of M-Att-73-122-2 and M-Att-73- 360-1 all other l^es gave higher protein yield than the mother variety .e eighth Fou f to r mutant lines produced 10-20/ß more protein per ha than the mother variety.

Three Athenais mutants were selected from populations treate 1975n di . They were teste replicaten i d d yield trials locatioe on t a 1978/7n ni outyielded 9an d their mother variety by 6-16 grain $i n yiel 1U-21?d dan protein Si n yield.

11 DISCUSSION

The encouraging finding of this work is that a few mutant lines could be selected from thousands of lines screened, which combine high agronomic performanc alsd an eo improved protein. The superiority of these lines in grain yield, DBG and Kjeldahl protein content were moderate, up to 7-11$, but protei o 2C$>.t np u yiel s Othedwa r studies reported higher protein and lysine improvements but with reduced grain yieid. Doll (1972) found a large number of induced variation in dry matter yiel f mutanto d f Garlsbero s I gbarleyI t non f bu thes,o e e line highed ha s r grai r proteino n yield tha controle nth n I . another study Koie and Doll (1979) reported that the protein yield of the high lysine mutants was 73-79^ of that of e parentth starcd an s h e mutant yielth 38-88s f e do th wa s f $o

yield of the parents0 Persson (1975) also reported that high lysine barleys yielded 95-100/ e standarth f So d variety under favourable condition t thebu s y suffered more under unfavourable conditions.

Barle s growi y Cyprun i n s under rainfed conditione th d an s amount of annual rainfall and its distribution during the growing season vary significantly from year to year. Two seasons very seldom are similar. Therefore, lines must be tested over a period of at least four years and at 2-3 locations in order to assess e adaptatioth qualitd nan f mutanyo t lines. Testin n mangi y environments is necessary also to identify mutants with small difference protein i s n content earlien shows a A n .i n r study (Hadjichristodoulo Deliad an u , 1978e Leasth ) t Significant values were smaller when several trials were combined together, than when each tria analyses wa l d separately„

ACKNOWLEDGEMENTS The authors wis o acknolwedgt h e assistancth e DanielL f o e , G. Alexandrou, Chr. Theodorides, M. Mouzouris, G. Papageorghiou, A. Demetrio . PharmakideA d an u n fiel i sd laborator an d y work.

12 REPSESND3S

Agricultural Research Institute, Cyprus. 1980. Annual Report for 1979, 16-17.

Doll . 1972H , . Variatio n proteini n quantit qualitd yan y induced in barley by EMS treatment. Induced Mutations and Plant Improvement (Proc. Meeting Buenos Aires, 1970). IAEA, Vienna (1972) 331-3U1.

Had jichristodoulou . 197UA , . Comparative Stud f Yielo y d dan Quality of Grain Cereals under Rainfed Conditions. Technical Bulleti , Agricultura15 . nNo l Research Institute, Cyprus, pp. 1-12.

Hadjichristodoulou, A«, and Athena Delia« 1978. Induced micro mu iont ta n barlei s r proteifo y n conten qualitd an t y rainfalw lo n i l areas. Seed Protein Improvemen Nucleay b t r Techniques (Proc. Meeting Baden J. IAEA , Vienna (1978)

Hadjichristcdoulou . JosephideC , A. , Athend an s a Delia. 1980. Improvement of grain barley varieties in Cyprus - "Kantara" a new variety. Technical Bulletin, 32, Cyprus Agricultural Research Institute, Nicosia, 14 p.

Koie B. and H. Doll. 1979» Protein and carbohydrate components Rise th o n i high-lysine barley mutants. Seed Protein Improvemen Cerealn i t Graid an s n Legumes I (Proc l Vo , . Meeting Neuherberg, 1978) IAEA, Vienna (1979) 205-21U.

Micke f mutatio . o 1979a A e , Us . n inductio o altent e th r ontogenetic patter f crono p plants. Crop Improvement

by Induced Mutation, 1979. Gamma-Field Symposia, No0 18, 1-23. Micke, A» 1979b. Mutant varieties of crop plants. Induced Mutations for Crop Improvement in Africa (Proc. Seminar, Ibadan, 1978) 257-265.

13 Persson barlee Th y1975. ,G .protei n projec Svaloft ta . Breeding for Seed Protein Improvement Using Nuclear Techniques (Proc. Meeting Ibadan, 1973). IAEA, Vienna (1975) 91-97.

SigurbjornBson, B. 1975. The improvement of "barley through induced mutation. Proceeding of the Third International Barley Genetics Symposium, 81^-95,

Sigurbjornsson Mick. A d ean 197U. ., B Philosoph Accomplishmend an y t of mutation breeding. Polyploidy and Induced Mutations in Plant Breeding IAEA, Vienna (197U) 303-343.

Sharma, R„P» and R.S. Sutar 1977. J. Nucl. Agric,, Biol, 6 11U.-118 (cite Mutation i d n Breeding NewsletterU 1 . ,No (1979) P.2, IAEA, Vienna.

14 Table 1. Grain yield of Attiki mutants during 1977-9 tested at several locations.

Grain Yield kg/ha 1977/8 1978/9 Mean Dromol . Laxia Ayii Mean Dromol . Laxia Akhera Mean 1977-9

M-Att-73-122-2 2580 e 3038 a 2783 a 2800 d 2220 b 3270 a 2U145 c 26U5 c 2723 M-Att-73-335-2 3375 be 311+2 a 3128 a c 321ab 5 2798 a 3712 a 267e 8b 3063 ab 3139 M-Att-73-335-3- 3832 a 3113 a 3270 a 3UD9 a 27U5 a 3787 a 276e 8b 310b a 0 3253 M-Att-73-337-1 3135 cd 3128 a 3030 a 3098 c 2760 a U095 a 306b 0a 3305 a 3202- M-Att-73-360-1- 3270 bo 3173 a 3000 a 31U8 be 2692 a 33U5 a 2835 abc 2957 b 3052 M-Att-73-396-2 c 351äb 8 3000 a 3225 a 32U8 abc 2752 a 3832a 32i|7 a 3277 a 3262 M-Att-73-Ull-2 337e 5b 32UO a 3128 a 32Uc ab 8 2632 a 3623 a 2558 c 2938 "b 3093 M-Att-73-l4l5-l 3585 ab 31i43 a 3308 a 33*4b a 5 2655 a 3592 a 2550 c 2932 b 3139 Attiki 2880 de 2910 a 2895 a 2895 a 2738 a 3173 a 2888 abc 2933 b 291U. Athenais - - - - 2U60 ab 314.72 a 2715 be 2882 be 2882

Mean 3233 3098 3085 3155 26U5 3590 277U 3003 3075 SX 123.7 92. h 151.5 71.7 219.7 113.0 11U.14- 9U.1 C.V. % 9.2 7.3 12.0 9.7 15.0 10.3 12.7 13. k Precipitation (mm2 U0 ) 258 230 307 ON 1 VO VO CM CM CO CO K"\ KN co CO Is- o * H ON o ON ON CTl ON O ON ON ON CM H (M H H

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16 Tabl » Crude3 e Protein Conten Attikf to i mutantn si 1977/ severat 8a l locations (Kjeldahl Method).

Location Line Dromolaxia Laxa l Ayii Mean

M-Att-73-122-2 16.2 a 11.5 a 13.9 a 13,9 a M-Att-73-335-2 14.9 ab 11,6 a 13.1 a 13.2 abc M-Att-73-335-3 15.0 b a 11.14b a - 14.1 a 13.b 1a M-Att-73-337-1 14.0 be 11 „2 ab 12.9 a 12.7 bed M-Att-73-360-1 e . Ub 10 13.3 c 12.9 a 12.2 d M-Att-73-396-2 15.2 ab 11.3b a 13.5 a 13.c 3ab M-Att-73-411-2 14.1 be 10.2 c 13.6 a 12.6d c M-Att-73-415-1 15.6 a 11.3 ac b . Uab 13 13.2 a Attiki 114.. 1 be 11.0 abc 12.8 a 12.6d c

Mean 14.7 11.1 13.3 13.0 SX 0.409 0.294 0.513 0.241 C.V. 4.8 4.6 6.7 5.5

Table 4. Performance of Attiki mutants (means of several locati on. s)

Grain DBC Kjjeldahl Head. Plant 1000 Volume Line Yield 6 loc. Protein(?0 date Height grain wt.(kg/ ? kg/ha) 3 loc. l=lst (cm) wt(gms)hlj 6 locc March U loc. 5 loc loc.U . 1977-9 1977-9 1977-8 3 loc. 1977-9 1977-9 1977-9 1977-9 M-Att-73-122-2 2723 21.5 13. 9a 20 87 142.9 56.3 M-Att-73-335-2 3139 19.6 13.2abc 20 85 144.7 58.2 K-Att-73-335-3 3253 20.6 9 1 13. b 5a 90 W-.U 57.6 M-Att-73-337-1 3202 19.2 12 .7 bed 21 90 45.2 57.3 M-Att -73-360-1 3052 19.2 12.2 d 19 85 42.6 58.1 M-Att -73-3 96-2 3262 19.8 13.3 abc 19 91 44.5 58.7 M-Att-73-l4ll-2 3093 19.8 12.6 cd 19 93 B. 58 43.0 M-Att-73-415-1 3139 20.3 13. k abc 19 90 40.8 57.6 Attiki (mother 2914 19.3 12.6 cd 20 92 45.5 57.9 var.) Athenais (check)* (2882)3 (18.6)3 (19)1 (89)* (42 3 (57.2 2) .8)

* Superscripts indicate number of locations in which Athenais was included in the trials. Table 5. Kjeldahl Protein Yield (kg/ha) in 1977/8 t severaa l locations.

Line Bromolaxia Laxia Ayii Mean

M-Att-73-122-2 U18 3U9 387 385 M-Att-73-335-2 503 36U mo U26 M-Att-73-335-3 575 355 1461 U614- M-Att-73-337-1 «9 350 391 393 M-Att-73-360-1 U35 330 387 38U M-Att-73-396-2 535 339 U35 U36 M-Att-73-Ull-2 U76 330 U25 mo M-Att-73-U15-l 559 355 ^37 U50 Attiki i^9 329 382 387

Mean [4.88

18 HIGH PROTEIN MUTANTS IN RICE*

M. ISMACHIN KARTOPRAWIRO . MUGIONK , O National Atomic Energy Agency, Jakarta, Indonesia

Abstract

HIGH P3CTEIN IUÏ7ANT N RIGSI S .

This protein programr s beeha en carrie t ^incou d e 1973t lo .^ of high protein mutants were obtiired. Their superio n proteii r n leve s maintainei l d through;:out tested generations. Their yield potentials have been tested sinc generation. eM, . Seventeen linec which have shown a nore stable in their protein contents were continually tested. Result f theso s e teste wer- re e ported. One mutant line which see™s to be resistant to brown planthopper is also mentioned.

INTBODUCTION

Under the IAEA research contract "o„ 1303/GG >Thich uas carried out since 1973 a- lot of high protein mutants vere obtained. The preliminary tests on their agronomic traits and yield potential have been reporte Neuherbert a d g meetin n 197i g 8 (1).

Further test have yielded unsatisfie o browdt date ndu aplan-t -

hopper attack. Since 197 rice 7th e f aliosproducino l al t g area were infecte y browb d n planthopper r protei.Ou n mutant e suscepar s - tibl o browt e n planthopper,therefor s veri yt i e difficul o carrt t y

out the yield test. However, it was found that mutant line no.

2238/2/1/1 e resistanseenb o t s o thit s insect.

e presencTh browf o e n plant hopper cause e criterioth d r fo n

releasin w varietne a g y enlarge. Thi hige sth h neanf o e s on tha f i t protein mutant will be released it nust first have a high potential grain yiel d seconan d d resistan browo t n planthopper t wil.I e lb more aceptable by the farners if the taste is good.

* A co-operative research project supported by IAEA Research Contract No. 2561/JP. Papepresentee b o rt Cyprut da s meeting 21-25 April 1980.

19 Among 85 selected lines, seventeen have shown more stable in their protein level compared with the others. This paper reports

the results of yield potential tests, agronomic traits and the sta-

bility in protein content of those mutants.

MATERIALS AND METHODS

The materials, mutagenic treatments and other experimental

details have been described previousl Badee th nt a ymeetin g (2).

Selected mutant lines were tested their yield potentials

together with the original variety, Pelita 1/1,. at Bogor during

two successive generations, viz. M,- and M,_. Seventeen lines which

showed more stabl n theii e r protein level were continued their

yield potential test n furthesi r generations Plo. M_ t ,d vizan g .K

size was 5 ni with spacing of 25cm x 25cm. Fertilizers were applied e phosphorouTh . ha rate r d 120ka50^f th tpe o an es T ggI a h ^p^ r e cP

was given at transplanting time and the application of nitrogen was split into three equa transplantint la e doseon : s g tine ,a secon d

at 21 days' after transplanting and the final one at 50 days after

transplanting. The plots were continually flooded during the

growing period except when nitrogen

was being applied. The Kg, !', Kg and I-!- -.VTP j,l;\i.ted during the

dry season 1977 t ceaso,ve n 1977/197? y f-aro,dr n t 19?8we d ,an

season 19n8/1979, respectively. All of these experiments were

carried out at Bogor because the experiments at the other locations

were attacked by brown planthopper. The folloving experiments at

Bogor, during dry season 1979 and wet season 1979/1980 w-re also

damage by this insect.

Therefore, this report is only involving data observed up to

Mq generation. e proteiTh n conten s detsrninewa t C e methodDB th y d b d an ,

stabilit f proteio y n conten s analysewa t y usin b de stabilit th g y

par=ir;eters proposed by Eberhart ar, ü Russell (3). The regression of

each variet n experimena n i yn environmenta a n o t l a inde d an x

function of the squared deviations from this regression would

20 provide.estimates of the desired stability parameters. It is accepted that lines with regression coefficient less tha0 (b<1.01. n ) usually adapt to low fertile environment. .On the contrary, lines

with regression coefficient more than 1.0 ( b^1.0 ) will ^usually

adapt to high fertile environments. The closer b to 1 and the closer more squareth e s stabli dline0 e deviatioo th et . } , (s n

RESULTS AÏID DISCUSSION

The possible change in protein and starch yield observed in M,

and M is shown in TABLE I and II. From the grain yield data it

seems thae environmentath t l condition tes . s notadurinM/ wa t e sth g

goo s durin a M d teste e graith g .Th ninitiae yielth f o dl variety

in the Mg test was about a half of its yield in the H„ test.

Three possible changes in protein and starch yield found in

the mutant lines were decreas protein i e d starcan n t differena h t

ratio, subtitution of starch by protein and increase in protein

and starch at different ratio.

Amon 1 mutan6 g t lines g werwhicM n ei hgroupe havins a d g

decrease in protein and st&rch, only 32 lines were included into the yiel togethed_ M, tes n i t r with mutants froe otheth mo tw r

groups. Result of this test is presented in TABLE II. The changes

in the number of line members of each group are considered due to

the difference n genetisi c environmental interactio f eaco n h line. Walther and Seibold observed similar c^se in the yield of barley

mutant lines (#).

Seventeen mutant lines selected from group - increase in protein and starch - which maintained their protein level above the initial variety, Pelita 1/1, throughout generations v:ere tested

their yield potentials during two following generations. The results

were sunmerize' n TABLîi E III e grain. Th proteid -an n e yielth f o d mutant st significantl mostlno e ar y y difference compare graie th no t d and protein yield of Pelita 1/1. There are many factors influence the yield which sometimes difficult to be measured. It seems that

the protein mutants are more attractive to birds than the initial variety. In fact, the mutants were more damage than Pelita 1/1, but 21 the degre measurablet f danagno o e s wa e n TABL.I E III, therefore,

the potential protein yield was calculated by manipulating its yield

component and its protein content. The mutant lines no. 713/V3/1|

8oV5/3/^i 190V/2/3 and 2581/V2/3 have certain numerically a higher

protein yield both in the mean protein yield and potential protein

yield than Pelita 1/1, but are not statistically significant.

According to Gomes (5) this may give indication of a real geno-

typ ee experimen effecth d an t t might therefor e wortb e h repeating

with more effort to reduce the error.

e yielTh d componen f theso t 7 mutan1 e t line presentee sar d

in TABLE IV together with data of plant height, amylose and protein

content. Some mutant e significantlsar y lowe n plani r t heighd an t

numbe f fertilo r e seed paniclr spe e compare Pelito t d a 1/1. Onl5 y

mutant lines have significantly higher in panicle sterility than

d almosPelitan o significan1 n t 1/ a t chang n numbei e productivf o r e

tillers, thousand seeds weight and amylose content fron the initial

variety. However, all of the mutants protein contents are highly

significantly higher'than their original variety, Pelita 1/1.

e proteiTh n conten s negativha t e correlation with plant

height, number of fertile seeds per panicle, thousand seeds weight d amylosan e conten possitivd an ; t e correlation with nunbef o r

productive tiller panicld san e sterilit However. TABL( y) V EI ,

it seems that enougno t h informatio o provt n a esignifican t corre-

lation between protein content and the those traits, except for

its correlation to number of productive tillers and to plant

height.

The protein content is largely enfluenced by the environmen- l conditionsta . When varietie e comparesar d ove seriea r f o s

environments rel&tive ,th e rankings usually differ. This causes

difficult n demonstratini y e significanth g t superiority an f o y variety. By using stability parameters proposed by Eberhart and Russelestimates wa t i lstabilite th d proteif o y n content ove7 r

successive générations. T.'i3LI3 V shows the recuits of this estima-

tion lineae Th . r regression coefficien d squarean ) d(b t deviation

(G,'" n e&c)i h mutant revea e wid th variatiof lo e n proteii n n

22 perforrance across generations. The average of protein content

in s eachighl t-ii u n ht y significantly higher thae initiath n l

variety ( see TA.1LE .IV as average of protein content over two

generation d TABL s averagan sa EV e over sev»n generation . Mutan) s t

line . 1164/3/3/2sno , 2039/3/4/ 2644/2/3/d 5an characterisee 2ar d

by having b •=* 1.0 and especially nutant line no 1164/3/3/2 which

also has s t=z 0. This indicates that these linec are widely

stable ir their protein content over generations. The other lines

although their protein leve e maintainear l d higher than Pelit1 1/ a

in every generation, their protein, per cent differ depend upon

the environmental conditio f eaco n h generation e proteiTh . n content e originath f o l variet s considerei y e unstablb o t d e with b<^1.0

and s,2 = 0.156. d The yield potential test carried out at Bogor in wet season

1979/1980 were attacke browy b d n planthopper l mutan.Al t lines

were seriously damage except line no. 2238/2/1/1. Thie line seems

to be résistant to brown planthopper. Its protein content, grain

and protein yield are shown in TABLE VI. This mutant is now being

largely gro\ produco t m e enough seed r includinfo s g inte th o

amltilocation test, becaus t alsi e o e seemresistanb o t s o t t

grassy/rugged stunt disease besides its taste is acceptible for

the people in this country C atnylose c.ontent is about 22 /o ) .

CONCLUSION

This research programme starte n 197i df o s yielde3 t ha lo a d

protein mutants which som f the o ee abl mmaintaio ar t e hige th nh

yield potential of the original variety.

The protein content of some of these high yielding mutants

are concidered to be stable in generation to generation while

the othersfiiffer according to the environmental condition during

the generation they grew. The proteir levels of these mutants are

always significantly higher thaoriginae th n l variet n everi y y

generation.

23 e presenTh t statu browf o e n planthoppe e firsth ts ri pes t

of rice in Indonesia. However, among the protein mutants only

mutent 1'in . 2238/2/1/eno s show1ha possibla n e resistanco t e

this pest , thereforeIt . , become a sgrea t proble utilizo t r e those

mutant lines into practical efforts.

ACKNOWLEDGEMENTS

,£, The authors gratefully acknowledge the financial assitance

provided by FAO/IAEA/GSF for the project.

Thanks are also due to V.r. Oiman Sujor.o, Mr. Tatang and

Suticn. Mr r theifo a r valuable technical assistance.

REFERENCES

(1) KARTOPBAWÏRO, M.I. , MUGIONO, RIY.ANTI, A.M., HUTABAHAT, D., "Evaluation of high protein mutant lines in rice " Seed Protein Improvement in Cereals and Grain Legumes, IAEA (1979) ^2. (2) I;AR::OPRA'.':::?O, ::.i., rr/oiorro, RIYA:;TI, ;>.:•:.,'induced nutations

r higfo h protein conten n rici t e ;"Seed Protein Irrprovcneny b t Nuclear Techniques, IAEA (19?8) 15?.

) EBE3ÏÏART(3 , S.A., RUSSELL, W.A., "Stability parameterr fo s

comparing varieties'^' Crop Science, 6(196G) 36.

('4) '.'ALTirjR H., SEIBCLD, K.H. , "Induced variation in protein mu-

tnr.ts after multiple E!;S and X-ray treatments", Seed Protein

Improvemen Nucleay b t r Techniques, IAEA (197^) 131.

(5) GO.f.'So, K.A., Statistical procedures for ric« experinents,

IRRI (1968).

24 TABL . I VARIATIOE F CHANGEO N PROÏBMNN I S , STARC GRAID AN H N YIELD HIGg M HF O PROTEIN MUTANT LIMES BOGORT ,A .

No Changn i e Châflge in Yield of of protein starch grain Protein Starch f£ protein Ratio lines yield yield ( g/m2) (S/m2) (g/tn2) it d.n. (g/m2) (g/m2)

Initial variety 1 - - - 193.3 14.7 17.8. 7 7.58 to 1/1 Dicreas n proteii e 1 6 n - 13.5 - 169.7 1 ; 12 10.1 1.2 8.9 8.22 and starc- at h to to to to to to to different ratios - 1.7 - ^.1 1 : 29 135.9 12.9 129.5 11.90 2 1 Subtitutio f o n + 0.1 - 37.7 : 1 1 153-9 14.8 138.9 8.69 starc y proteib h n to to to to to to to + 5.6 - 3.2 1 :337 193-3 I8.it 175.5 10.26 Increase in protein 12 + 3-3 + 0.2 : 1 0 199.0 17.9 178.8 8.25 and starc- t a h to to to to to to to different ratios + 11.6 + 96.6 1 : 9 300.3 26.2 ?75.2 10.15 TABLE II. VARIATION OF CHANGES IN PR07DIN STARCH AND G3AIN YIELD OF M HIGH P3OTEIN MUTANT LINES AT BOGOR.

No Change in Changn e"i Yielf o d of protein starch Ratio grain protein starch % protsin lines yield yield (g/m2) i. d.m. (g/m2) (g/m2) (g/m2)

Initiel variety 1 359 24.4 334.6 6.81 Dicreace in protein 13 - 16.7 - 257.3 1 15 85 7.7 77.3 7.87 and starch at - to to to to to to to different ratios - 0.9 - 665 1 113 291 23.5 268.1 9.45 Substitution of 16 + 0.2 - 77.3 1 1 283 24.6 257.3 7.51 starch by protein to to to to to to to + 11.2 - 4.2 1 376 413 35.6 378.4 9.09 Increase in protein 27 + 7.6 + 15.2 1 2 383 32.0 349.8 7.26 and starch at - to to to to to to to different ratios + 28.0 + 232.0 1 11 619 52.4 566.6 9.20 TABLE III. TIÏS GRAIN YIELD ATID PliOTEIN YIELD OF TIIE PROTEIN MUTANTS TESTED OVEH J> SUCCESSIVE GE;fE2A?IONS ( M - M ).

Kean grain Mean protein Potential yield protein yield Genotype yield ( g/ha ) ( kg/ha ) ( kg/ha)

1. 38/1/3 41.66 3^8.86 ^693 .2 2. 713/4/3/1 ItO. 't2 3V5.90 513.64 3. 804/5/3/4 U0.28 3^2.90 517.49 . 1164/3/2/k 4 30.82 270.91 566.71 . 1164/3/3/5 2 29.^7 260.15 490.68 6. 161 4/1/2 38.79 337.72 460.25 . 190VV2/7 3 37.66 351.82 542.17 . 2039/3/4/8 5 to.23 337.83 441.59 9. 2299/2/2/3 28.57 250.99 463.22 10. 2394/3/3/2 3^.8^ 301.07 520.96 11. 2394/3/3/5 27.53 233.29 497.89 12. 2575/2/1/1 33.28 300. k9 438.69 13. 2581/4/2/3 39.86 337.99 484.73 14. 258 V 1/V3 32.09 291.73 588.06 15. 2590/3/1/2 2^.2** 208.22 415.19 . 2644/2/3/16 2 31.32 270.140 450.34 17. 2692/5/2/4 33-30 286.9^ 477.07 1&\ Pelits 1/1 »fO.96 311.56 461.55

L.S.D. (0.05) 11.22 9^.79 'L.S.D. (0.01) 15.03 127.01 ) C.V# ( . 19. W 19.11

PoteniiaL protein yield was calculated as

A x 3 x C x D x P 1000

. pToductivKo = A e tillers fertil. No = e3. seeds/panicle C = Thousand Grain Weight D = Tic. plants per ha P = Protein content ( % )

27 *> O -p -p • IACO O ß

(H O OOOOCOOOCOOO CTSCDOOOOOO OSOOOOOOOOOO O O

O -4-> tN w e^ IN O D O N I O K O CNA Ot [N CO H -P "if, VJD NAOMD E O^ O W K EH l PH O •d <-x G E P - M o i O X O « M f N I - r - > . < !- 3 b d - 3 COvjDV£>OO*-vQC\J[--lAr\JCO*-lAr?\ON O ON IN K\ CJ O t H - > « O P--CO [NINONIAC^-VD [NCOCO IAINOO N O ^ S 0 E1 H O

?•» EH -P C ^cc t) -H rH rH CO S ~" ^ H O- PIAQIAQÇÇDOIAQQIAIAIAIALAIAO -*VOCO ON p ^ C 2: w a -P P< W-»-' (M C\S f\J •^ § H M W EH u fc H o , p v •w rH rH S § ü W H O- +> -Ö -ri ^ cr>vKM D r\j CM c^- ir\ c^- H °C > t H ( • K K O 4> ü C d O vOSt . . tA . O MO . . _ _ . . . -p J5 P, co CO CO CO C-^Cl O C O- M t < O O OO INCN I ON I OO V C H o R W ü t; M <ï M VD ü vo •H W O V EH !=> - g O b Ö CM IACO t •P M L? d -rl O O rH «> INIAOJ ONCO tNVD OOV f\i IN c\jco o O 1 o pt t-* v^ ~ ON ONO EH l •P EH •^5. C O •H M K O O O K KN. •p ft O •H K O •P W n -P O «H O {-, VD'VJD- J o "r o ß K W Opt) •H t) O EH •Ö rH <\J •P ü H r O t ce l -r M O H h. W EH g; (ii -P 4) 4) (-> ft O O IA PH S O O M 11 « £ U -P S O •H O K ü U W P* •rH T) H) Ö KA 1>-CO P3 t) W COÜ O il IT\VD tNCO N OO (M tAVO C^-00 TABLE V. STABILITY PARAMETERS OF P1ÎOTEIK 1ÎUTANTS TESTED OVER 7 GENERATIONS ( M - M ) AT MUAS - ABOGOR .

Mean protein content over Genotype b all generations Ed ( % )

1. 38/1/3 8.07 0.415 0.013 2. 713/4/3/1 8.73 0.640 0.214 3. 8o4/5/3/^ 8.99 1.670 0.113 4. 1164/3/2/4 9.12 1.444 0.055 . 1164/3/3/5 2 9.01 1.150 0.008 6. l6lVl/2 8.28 0.219 0.320 7. 1904/1/2/3 8.95 0.449 0.076 8. 2039/3/4/5 8.72 1.103 0.117 9. 2299/2/2/3 9.12 0.786 0.068 10. 2394/3/3/2 9.36 1.671 0.768 11. 2394/3/3/5 9.16 1.496 0.122 12. 2575/2/1/1 8.84 0.65 0.003 13. 2581/4/2/3 8.87 0.138 0.443 14. 2584/1/1/3 8.90 0.004 0.172 15'. 2590/3/1/2 8.81 0.398 0.015 16. 2644/2/3/2 8.88 1.064 0.147 . 2692/^/2/17 4 8.80 0.710 0.125 18. Pelit1 1/ a 7.38 0.171 0.156

L.S.D. ( 0.05 ) 0.57 L.S.D. ( 0.01 ) 0.76 c.v. (#) 6.19

29 TABLE VI. THE PROTEIN CONTENT, GRAIN YIELD AMD PROTEIN YIELD OF A PROTEIN MUTANT UNE (2238/2/1/1) WHICH ALSO RESISTAN O BROV/TT N PLANT HOPPER.

Generation

M M M M 7 6 5 8 »9

1. Protein conten: ) (% t 2238/2/1/1 8.73 8.57 - 8.69S.kk 8.65 Pelita 3/1 7.2k 7.25 - 6.817.3^ 7-92

2. Grain yield (g/ha) : 2238/2/1/1 20.30 30.67 3^-67 Pelit1 3/ a 35.85 52.65 3^.38

3. Protein yield (kg/h^t 2238/2/1/ - 1 176.^1 258.85 299.90 Pelita 1/1 - 2'^.1't 386.^5 272.29

30 TESTING, CROSS-BREEDING, INDUCTION AND NUTRITIONAL EVALUATION OF WHEAT AND TRITICALE MUTANTS

PATRICIO C. PARODI, ISABEL M. NEBREDA Departmen f Plano t t Science, Schoo f Agricultureo l , Catholic Universit f Chileyo , Santiago, Chile

ABSTRACT

Through eight years of research our Project was able to identify several outstanding mutants, derived e froth m gamma irradiatio wheax si tf no genotypes teste prcr fo d_ tein content, yield, adaptability and disease resistance ove wida r e rang environmentaf eo l conditions. Short^ ly before releasing some of these mutants for commercial cultivation, race 15 B of stem rust (Puccinia graminis f.sp. tritici), absent from Chile for over 20 years, was identified, causing devastating effects on the mutants and other material. Only low levels of resistance were found among Chilean spring wheat cultivar advanced an s d breeding lines availabl .1979n ei .

This paper presents the results of our research from 1971 until 1979, and a series of procedures which have been programmed to solve the problem, and rapidly induce resis_ tance to the material, without losing its characters, specially high yiel improved an d d protein content.

INTRODUCTION

Quisenberr Reitd hav) an y (2 ze stated that whea mans i t y things. To a botanist wheat is grass. To a chemist, it is organic compounds, and to a geneticist a challenging

31 organism. To a farmer, it means a cash crop, and to a hau 1er, freight laborera o T . meant ,i s employementa o ;t merchant, it is produce. To a miller, it is grist, and to a baker, flour. The banker sees it as chattel and the po litician as a problem. Animals brouse and feed on it and it sustains parasites. The conservationist uses it as ground cover. In religion it is used as a symbol. The ar tist and photographer see it as a model. To millions it provides a livelihood, and to millions more a lifegiving food.

For the Chilean population, and specially the less privileg_ ed segment ,maie wheath n s ti sourc calorief eo proteind an s , obtaining approximately 44 and 50%, respectively, from wheat- derived products. For the country's economy, wheat is the main component of the agricultural sector, with a trade volu $ 450.000.000US f o e m whicf ,o h nea e hal rspens on i f t import; ing wheat to satisfy demand.

To Chilean plant breeders, wheat is indeed a challenge and a responsibility. Ther nees ei improvo t d e national production, providin farmere th g s with better cultivars, which must meet a large numbe requirementsf o r , among which disease resistan_ present a s c i eparamounf to t importance. These cultivars mus adaptee tb wida o et d rang f environmentaeo l conditions, dictated by Chile's narrow and long North to South geography. These cultivars must be plastic and easy to manage, so that the small farmer, who uses limited inputs, can obtain a pro- fit from his modest investment, and also capable of reacting positivel modere th o nt y procedure bettee th f rso technically equipped farmer. They must produce yields that allow for a sale price competitive with the world market, to which the countr wids i y e open. They must provid millere eth , baker and pasta-producer wit acceptabln ha e produc turo t n into flour, bread and pasta. Finally, they must reach the consu-

32 realtivela e for f th o m n i r yme inexpensive, nutritious pro_ duct.

e advenTh f triticalto TriticosecalX ( e e Wittmackm co a s )a mercial crop, with its high yield potential, wide adaptability, better disease resistance, higher protein content and improved amino acid balance, may relieve some of the pressu re from wheat, partially replacin increasin, it g g national grain production, and providing more nutritious food products. Triticale, however, has problems of its own, which require intensive effort solveo st .

Both species, therefore, considered together, compound the problems, but simultaneously, both working in association, may offer very important answers to the national agricultural, economic and nutritional situation.

In orde o acceleratt r productioe eth superiof o n r material, we initiated a Program of Mutation Induction, jointly finan£ ed by the International Atomic Energy Agency and the Catholic University of Chile.

This progra e fac bases th i mt n mutationo d mose th m ti e ar s portant sourc variabilitf eo organismsl al n i y . They yma spontaneoun occua s a r s process during evolutiony ma r ,o be induced through chemical or physical agents. Basically, induced variability doet diffeno s r from variability natur_ rally occurring.

Mutation induction is a fairly recent supplementary metho dology, very valuable in plant breeding, particularly when the objective is to improve one or two easily identifiable character cultivaa n i s r whic otherwiss hi e acceptablee .Th main advantages of this method are, a) that the basic geno_ type of the cultivar to be improved is only slightly alter compares a , ed d with hibridizatio o differentw f no t genoty_ pes, as the desired character or characters are added, and

33 2) that the length of time required to develop the improved cultiva considerabls i r y shorter than that needed whe- nhy bridization is used for the some purpose.

In spite of this, it is necessary to consider possible pleio tropic effects of the mutated gene or genes, as well as other undesirable mutations that may take place. When some of these effect observede ar s advisabls i t ,i croso et e sth mutant with the parent line, and select from the progeny in dividuals carrying the desired mutations, but free of other undesirable changes.

A favorable mutation may be recovered in an homozygous con

4 generationM r o j M- g e ditioF compares ,th a e n th i n o t d or F 7 generation when hybridization is used. The.satisfac tory performanc mutana f eo t line, however onlt ,no y depends on the new favorable mutation that has been induced. The genotyp whico t e belongt hi s determine agrs it rese o th sf to nomic characteristics, suc adaptabilitys a h , disease resistance, quality and yield. It is thus necessary to extensive_ ly evaluat mutant'e eth s behavior under field conditions C3).

Our Projec s initiatetwa 1971n i d , havin basie th g c objecti ve of increasing the protein content of spring wheat genoty_ pes, adapted and high yielding in Chiles North Central Region.

MATERIAL AND METHODS

sprine th See f go d wheat genotypes Huelquen, Collafen, Yafen, Bluebird an 3 (.Triticu1 PL ° 77 AdN m aestivuQuilafed an ) . mL n . duru(T m Desf. s irradiate)wa d with gamma ray dosen i s f so 10 and 25 krad. The M]_ generation was planted in the field 30 days after irradiation harvested ,an bulkn i d , preserving treatment identity. The M2 generation was spaced-planted in 5 m long 50-row plots, with an average population of approxi^ mately 15,000 individuals per treatment; every treatment was followe y fivdb parentae th row f so l genotyp controls ea . 34 The materia s fertilizelwa d with optimum level nitrogef so n

and phosphorus. Starting with M2 the populations were inocu lated wit racha e composit Puccittif eo a graminis f.sp. triti- cvi; only natural infestatio . reconditaP f no striiformi. ;P_ s and other pathogen provideds swa ) (1 .

Plant selectio generation2 s initiateM nwa e th n i d , basen o d four selection criteria plant) (a : s that showed phenotypic differences from the parent genotype; (2) plants that showed an improved leve diseasf lo e resistance plant) (3 ; s thad tha superior agronomic characters plant) (4 d s ;an identica o t l the parent genotype. Theee selection criteria were applied separately or jointly on the population, depending on its cha racteristics. A total of approximately 25,000 plants were selected, wit averagn ha abouf eo t 2,000 plant r treatmentspe . materiae th rese Th f to s harveste lwa bulkn di , preserving treatment identity. Two hundred plants of each parental geno type were selecte randot a d harvested man controlss a d .

The protein content of each selection was estimated by the dye-binding capacity CDBC) method, usin e sampla g th f eo seed of all the spikes of each plant selected. All those plant sprotei a thad tha mornr o contene percentaon f to - ge points abov respective eth e control were spaced-planted in 2 m long 5-10 row plots, depending on seed availability. The rest of the selections, except the very poor ones, were planted in 2 m long 2-row plots.

Selectio s generationU continueM nwa e th n i d _ , sa usin e th g criterie m a described. There were three different populations in JMßr Cl) high—protein selections; T2) average or low-protein selections; and C3) non-selected, bulk—harvest, ed populations proteiC DE . n determinations were continued on all selected individuals. In the M4 generation each se_ lected mutan plantes m lon twa 2 gn i d5-ro w plots, witha uniform planting density orden ,i obtaio rt preliminarna y

35 yield estimate. Control plots were so spaced as to provide a reliable comparison of mutants and controls. Selection s conductewa plotsn o d , whe mutane nth reached tha d adequate homozygosis individuan o r ,o l plants when they were stile ls gregating. Analyses and selection for protein content were continue describeds a d , adding Kjeldahl analyse o checst k unusually high or low values.

The procedure was continued in the M5 generation. However, the best homozygous mutants were studied in replicated yield experiment Experimene th t a s t Statiothren i d e nan location s within the potencial area of adaptation. The rest of the ma terial was grown in a screening nursery, consisting of 2 m long 5-row plots, with an adequate number of controls. More mutant d locationg genereM an s d an a ^ sM , werMg ee addeth n i d tions yield experiments numbee Th . selection f ro s madn i e generationg M o t j M shows si Tabln no . e1

In 197 generationg 6(M mutan)a t designate UC-s a ds inclu3wa d ed in the National Cooperative Yield Experiment (NCYE) run by the Ministry of Agriculture. In 1977 mutant UC-4 was added to the NCYE; two more mutants, UC-5 and UC—6 were added in 1978 197n I e include. 9w NCY e mutantsx th Esi n i d , UC-3, UC-4, UC-5, UC-6, UC-7 and UC-8.

yeare Ith n s 197 197o 6t 9 several mutants were selecter dfo inclusio varioun ni s multiline composites, wit objective hth e of studying their behavior by comparing the performance of

Tabl . eNumbe1 selectionf ro ^ M se madth n ei

to M5 generations

Generation M3 M4

Number of selections 8420 4520 2687

36 each composite wit constituens hit t line witd san h high- yielding controls.

The number and type of yield experiments conducted during these numbee yearsth mutantf d ro ,an s studie showe ar d n on Table 2.

Tabl . Numbe2 e typd ran yielf e o d experiment numbed an s mutantf o r s studie n generationi d g M o t r sM

Type of experiment Experiment Multiline Number of station Regional Cooperative composites mutants Generation Number of experiments studied

M5 7 3 0 0 156

M6 18 8 16 3 400

M7 21 6 8 1 463

M8 14 9 9 1 318 Mg 14 8 6 8 430

yiele th dr experimentFo basicalle sw y use randomizea d d complete block design with four replications e signjTh . L ficance of the differences among treatments was estimated through Duncan's new multiple range test at the 0.05 level. Industrial quality analyses were contracted wit inden ha - pendent laboratory.

RESULTS AND DISCUSSION

The first treatment effect was expressed in seed viability and survival. The 10 krad treatment showed an average su£ vival of 76.8%; the 25 krad treatment produced a significantly higher lethality, average survival being estimated as 58.3%. No significant differences were observed within

37 each genotype in their ability to survive the irradiation treatments. Part of the lethality was expressed as albino plants, which died 18-35 days after emergence (il.

Tabl . Yiele3 selectef o d d mutant controld san s during four years

Yield, k/ha Mutants equal or Mean of better than the Yead ran Mutants ' Mutant srang' e the best best control generation mean Low High control Number Percentage

Experiment station

1975 (M5) 3604 2142 4805 3630 76 54 .3

1976 CM6) 5107 4211 6138 5969 207 57 .5

1977 (M7) 5766 4409 6919 6578 92 21 .9

1978

1975 (M5) 5193 3909 6563 5101 26 54 .2

1976 (M6) 4146 2764 5403 5175 86 56 .6

1977 (M?) 4870 3630 6723 6308 34 28 .3

1978 (Mg) 4847 4071 5471 5310 13 8.1

Tabl show3 e summara syielde groua th f f f selectso o ypo ^ ed mutants, studie yieln i d d experiment Experimene th t sa t Station and in regional locations during four years. The mutants' yield comparee ar s d year'e wityiele th th hf so d best control. An important number of mutants, fluctuating per year 57.5%d betweean shows 1 ,ha 8. ngraia n n production capacity statistically equa better lo r tharespective nth e best control, which suggests thayiele tth d potentiae th f lo original germ plas s oftemwa n improve irradiatioe th y db n treatments. Additionally, the data show that, from a grain yield stand point, severamutante th f lo s havpotentiae eth l growte ob n commercially, favorably competing wit bese hth t genotypes available in the area in which they were analyzed.

38 Table 4. Protein content of selected mutants and controls during four years

Protein conten% t Mutants equal or Mean of better than the Year and Mutants ' Mutants ' range the best best control generation mean Low High control Number Percentage

Experiment station

1975 (M5) 11.7 9.8 13.8 11.4 71 50.7

1976 (M6) 12.1 11.0 13.3 11.8 201 56.0

1977 (M7) 12.5 11.1 14.3 13.0 143 34.0 1978 (Mg) 11.5 9.9 13.3 12.4 61 21.8 Regional experiments

1975 (M5) 13.8 10.8 15.4 12.3 35 72.9

1976 (M6) 13.4 12.0 15.3 12.7 112 73.7

1977 (M7) 12.2 11.1 13.5 13.0 24 20.0 1978 (Mg) 11.5 10.1 13.2 11.5 79 49.4

proteie Th n conten f thito mutantssf o sam t ese , also compar_ ed wit year'e th h s best control shows ,i e TablTh n no . 4 e protei materiae th nl al meal f no analyze Experimene th t a d t Statiogeneran i s nwa l simila bese th thao t rf to control . An important numbe mutantf o r s statisticallswa yt equabe r o l ter than the best control, with protein values as high as 14.3% (.1977), that is, 1.3 percentage points, or 10%, high er thabese th nt control mose Th .t outstanding grouf po mutants, include regionan i d l experiments e aveth _ n o d ,ha rage, excep 1977n i t , protein contents abov e beseth t res_ pective control proteie Th . n e bescontenth t mutantf to s in each year, were above the best control by 0.5 to 3.1 percentage points, equivalent to 3.8 to 25.1% higher protein content. The percentage of mutants that had protein contents statistically equal or better than the best control, ranged between 20.0 and 73.7%. The data show that the irra_ diation treatments used, followed by strict selection proce

39 dures, were effective in improving the original material's protein content, and allowed the identification of superior genotypes.

This information allows to conclude first that the Project's basic objective, improving the material's protein content was fullfilled, and second, that improved protein content was reached without deterioratin yiele th g d potentiae th f lo parental genotypes ofted ,an n improvin. it g

From a practical point of view, that is, considering the pos_ sibilit releasinf o y g th.e best mutant r commercialsfo , grow advisabls i ing t i , examino et e their individual performance.

This information is shown on Tables 5 to 10 for mutants UC-3 to UC-8.

Mutant UC-3 ,s studie Tablwa , 5 e d durine yearo th tw gt a s Experiment Station, and since 1976 included in regional and cooperative experiments yiels It s highl.dwa y satisfactory, until 1977 consistently above the controls' mean and the best control. During that period its test-weight ranged between 79.92 and 82.01 k/hl, and its protein content between 11.9 and 12.4%. Until candidata 197 s 7wa registratior efo n i n the National Cultivars Registry. However, starting in 1978, UC-3 starte severele b o t d y attacke Pucciniy db a gramini. f s sp. tritic^, disease that deteriorated its yield and test- weight. At present, we are trying to add stem rust resistan UC-co et 3 throug backcrosha s program usin single gth e seed descent method, and it has been withdrawn from the NCYE.

Mutant UC-4, Tabl afte, 6 eyeae replicateron f ro d testing at the Experiment Station entered regional experiments in 1975yiels It s satisfactor.dwa y throughou perioe th t n i d which it was analyzed, in spite of its susceptibility to Puc- cinia graminis,that was evident for the first time in 1978, under conditions of artificial inoculation, and become more

40 Table 5. Yield, test-weight and protein content of mutant UC-3 in several years and locations

Yield, k/ha Test Protein Number of Controls' Best weight content Year Experiments Locations Mutant mean control k/hl %

1974 1 1 5968 4935 5113 82.01 12.3 1975 1 1 4132 3748 3965 81.50 12.4 1976 18 17 5514 4328 4671 79.93 11.9 1977 18 14 4939 4517 4649 80.27 12.4

1978 21 17 5131 4126 4563 77.76 11.8 1979 15 11 7141 7297 8106 77.75 12.0

Tabl . Yield6 e , test-weigh proteid an t n conten mutanf to t UC- n severa4i l year d locationan s s

Yield, k/ha Test Protein Number of Controls '1 Best weight content Year Experiments Locations Mutant mean control k/hl %

1974 1 1 5632 4935 5113 79.21 12 .7

1975 3 3 5070 3961 4075 77.86 12 .8 1976 8 8 5176 4471 4705 78.86 12 .8 1977 18 14 4999 4517 4649 81.85 12 .6 1978 19 18 4499 3549 3928 78.50 12 .1 1979 3 5 7068 7133 7742 78.15 12 .0

sever 1979n ei . Mutant test-weigha UC- d 4ha t between 77.86 and 81.85 k/hl, and a protein content between 12.0 and 12.8%. Presently it is undergoing the some process described for UC-3.

Mutant UC-5, Table 7, behaved quite similarly to UC-3 and UC-4, decreasing its yield since 1978 due to a stem rust at tack. Stem rust also affecte s test-weightit d , which decreas_ ed from 80.2 78.3o t 0 1 k/hl proteis .It n content, however, remained stable, ranging from 12. 13.0%o 6t . UC- alss 5i o being t backcrossestei mo t rus efforn d a ad tn i dresiso t _ tance. 41 Table 7. Yield, test-weight and protein content of routant UC-5 in several years and locations

Yield, k/ha Test Protein Number of Controls ' Best weight content Year Experiments Locations Mutant mean control k/hl

1975 1 1 4667 4012 4207 80.20 13 .0 1976 1 1 6396 4500 4780 80.12 12.7 1977 5 5 6427 4813 5115 80.13 12.9 1978 20 17 4539 4098 4529 79.44 12.7 1979 1 1 6537 7525 7796 78.31 12.6

Table 8. Yield, test-weight and protein content of mutant UC-6 in several year d locationan s s

Yield, k/ha Test Protein Numbef o r Controls ' Best weight content Year Experiments Locations Mutant mean control k/hl %

1974 1 1 5812 4235 4416 81.35 12.9 1975 1 1 5229 4473 4680 81.28 13.2 1976 1 1 7090 4905 5187 81.47 12.8 1977 5 5 6294 4888 5010 82.42 13.0

1978 18 17 4625 3303 3695 80.46 12.5 1979 14 11 7998 7207 7822 80.25 12.6

Performance of mutant UC-6 is shown on Table 8. This mutant has consistently kept its high yield potential through the six years of testing, yielding better than the mean of the controlbese th t d controan s eacf lo h test-year s showha nt I . to posses higsa h test-weight, whic ranges hha d from 80.2o 5t 82.42 k/hl, and also a comparatively high protein content that has fluctuated between 12.6 and 13.2%. As the previously dis_ cussed mutants, shows UC-ha 6 degrena susceptibilitf eo o yt Puccinia graminis, however, at a comparatively lower level. Be_ cause of this information, we have reached the decision to

42 Table 9. Yield, test-weight and protein content of mutant UC-7 in several years and locations

Yield, k/ha Test Protein Number of Controls ' Best weight content Year Experiments Locations Mutant mean control k/hl

1975 1 1 5877 4511 4631 78.95 11.9

1976 1 1 6341 5016 5245 78.48 12.4 1977 5 3 6615 6235 7142 78.70 12.6

1978 5 3 9492 8158 8906 77.55 11.3

1979 9 7 5945 7145 7300 60.38 11.8

initiate seed multiplication of UC-6, and to register it Nationae ith n l Variety Registry additionn I . , UC-s 6i also being backcrossed to sources of stem rust resistance.

Table 9 summarizes the performance of mutant UC-7. This genotype has been most severely affected in its yield by stem rust s drastis showit ,a y nb c yield reduction i n 1979. Damag alss eha o occurre test-weights it n i d , that decreased in 1979 to 60.38 k/hl. Mutant UC-7 is also being backcrosse steo t d m rust resistance sources.

Performanc mutanf eo t UC- shows 8i n Tablno . Thie10 _ sge notyp maintaines eha higa d h yield level, surpassine th g mean of the controls throughout the entire period of tes^t ing, including 1979, when it was severely attacked by Puc- cinia graminis, speciall area e norte th th .f ht o a y UC-8' s test-weigh bees tha n adequate althoug s showha tena nt hi - dency towards decreasing, from 82.60 k/h 1976n li ,79.3o t 7 k/h 1979n i lproteis It . n conten ranges tha thesn i d e four years between 12. 13.0%d 2an . UC- alss 8i o being backcross ed to sources of stem rust resistance.

The yield performance of these six mutants, in percentage bese meath e t oth f no contro eacf lo h yea f studyro s ,i

43 Table 10. Yield, test-weight and protein content of mutant UC-8 in several year d locationan s s

Yield, k/ha Test Protein Number of Controls ' Best weight content Year Experiments Locations Mutant mean control k/hl %

1976 1 1 6532 4417 4786 82.60 12.2 1977 1 1 6354 5823 6708 81.60 12.3 1978 1 1 8547 8158 8906 80.00 13.0 1979 10 7 8730 7315 8135 79.37 12.5

observee b y showma dt Figurn I no that . e1 , wit excee hth £ tio UC-8f no tendence ,th towards i y decreasa s yieldn ei , probably caused by stem rust attack, which justifies the measures resistanc d takead o nt e through backcrossing.

The specific reaction to Puccinia graminis of the six mutants is presented on Table 11, showing their reaction at the north regioe oth f n wher attace eth bees kha n most intensee th t ,a Pirque Experiment Station under conditions of artificial inoculation, and in locations south of Talca. The first signs importann oa f t attac f thiko s pathogen were notice 1978n i d , as shown by a susceptible reaction of UC—3 at Ovalle in the north end of the region, under conditions of natural infesta- tion d als ,an Pirquet oa moderateld ,an y resistan moderao t _ tely, susceptible reactions of mutants UC-4, UC-5 and UC-6 at Pirque.

Stem rust attack became more severe at Ovalle in 1979, and the_ re was also a significant increase in intensity at the Experi^ ment Station. Presently, no stem rust attack has been recorded south of Pirque, except UC-7 in 1979, which had a 10% of s foliait r area affecte ruste th ,y db wit moderatelha y resist^ ant reaction.

44 1974/75 1975/76 1976/77 '977/78 ^978/79 1979/8O YEAH rGuRE 1 YIELD or MUTANTS uc-3 TO uc-8 IN PERCENTAGE E CONTROLE MEA0«TH TH 7F O N ? Table 11. Reaction of six mutants to Puccinia graminis f.sp. tritici

Reaction to Puccinia gxaminis Mutant UC-3 UC-4 UC-5 UC-6 UC-7 UC-8 3 Yea 2 r 1 I 1 2 312 3 123 1 2 3 1 2 3

1974; 0 0 0 0 0 0 1975 0 0 0 0 0 0

1976 000 0 0 000 0 0 0 0 0 0

1977 000 0 0 000 0 000 0 ts o 0

1978 30S2 30S 0 S 05M S 5Mt S 0 0 0 5MR 0 0 0 0 0

1979 80S 80S 0 S S 3050 50 S S 40 0 0 2 QMS 5MR 0 50MR 80S 10MR 80S 30S 0

North= 1 Pirqu= 2 ; e with artificial inoculation Sout= ;3 h Resistant= R 2 moderatel= R ;M y resistant moderatel= S ;M y susceptible susceptibl= ;S e The incidence of other pathogens, such as Puccinia striiformis, Puccinia recondita Septoria sp. and BYDV has been recorded , but will not be reported here because of its compara^ tively minor importance.

Tabl 2 e1 shows resulte th f industriaso l quality analyses mutante oth n s UC-3, UC-4, ÜC- UC-6d 5an , using Marianela,a wheat cultivar of high industrial quality, as a control. In general mutante ,th s showe posseso t d s adequate qualit- le y vels; UC-6 appeare superioe b rest e o th t d .o rt

These results suggest that mutation induction through gamma rays irradiation selectioe th d ,an n process that followe, it d were successfu producinn li identifyind an g g mutant genotypes of superior performance. Selected materiapotentiae th d lha l to produce high yields, associated with protein levels higher than paren e thosth f teo genotypes highed ,an r thameae nth n of the best cultivars of the area in which the experiments we_ re conducted.

Unfortunately Puccinie ,th a graminis attack that starten i d e area th northere d tha th f ,an o tt 197a d coul8nen d even_ tually progress towards the south, deteriorated the yields of the best mutants, which had been selected in the absen racee th ,f co e race biotyper o s s that cause probleme th d . To recover their yield potential it will be necessary to ado thet d m genetic resistanc o steet m rust, process alrea_ dy initiated throug backcrosha s progra me sin baseth -n o d gle seed descent method.

The t onlprobleno y s affecteha m populatione th d s derived through mutation induction alst ,bu o those originated through other methods. Therefore, it is correct to conclude that mutation induction through radiatio viabla s ni e plant breed_ ing method that allows, whe e correcnth t dose e appliedar s , to induce genetic variability o identifT . e besth yt genoty_

47 Table 12. Industrial quality characteristics of mutants UC-3, UC-4, UC-5 and controd UC-an 6 l Marianela1

Mutant Control Variable UC-3 UC-4 UC-5 UC-"5~ Marianela

Chemical analyses Ashes 0,637 0,697 0,735 0,798 0,549 Sedimentation 23 49 40 40 48 Maltose 1,607 3,904 2,710 4,100 1,454 Farinogram Absorptio) (% n 64,5 69,4 69,5 74,2 61,9 Dough development (time) 4'15" 5'25" 4 '10" 4'10" 7'45" Stability (time) 25" 20" 30" 30" 35" Resistance (time) 4'25" 5'45" 4 '10" 4-40" 5 '55" W value 44 55 42 50 50 Bread characters Loaf volume (cm ) 675 685 700 730 655 Loaf weight (g) 152 155 157 159 157 Crumb color (1-100) 81 84 82 84 84 Crumb texture (1-100) 78 84 82 84 87

Industrial Quality LaboratorA EG y

pes it is required to work with large enough populations, usin e appropriatth g e analytical method deteco t s t thosn i e dividuals that posses desiree th s d genotypic characters.

FUTURE OUTLOOK

1. Mutants UC-3, UC-4, UC-5 and UC-7 are presently lost for all practical purposes commercias ,a l cultivars.

2. It is still feasible to use mutant UC-6 as a commercial cultivar because: s susceptibilitIt ) a equas i y r leslo s thane th tha f to other commercial cultivars that are available. yiels It d) b potentia beet no n s drasticallha l y impaired, even under the worst conditions of artificial stem rust inoculation.

48 At presen have tw e approximatel f UC-o g k 6 see 0 d16 y which will be multiplied during 1980.

3. Mutant UC-8 wil yiele lb d teste more on de year befor- ede ciding its eventual multiplication.

4. Mutants UC-3, UC-4, UC-5, UC-6, UC- UC-d 7an 8n i wil e lb eluded in a backcross program with four sources of resis_ tance to Puccinia graminis which were identified in 1979. This program, already initiated, will be conducted in an accelerated manner, using the single seed descent method, under greenhouse conditions, aimin producint a gd an o tw g hala f generation recoveo e t g r years e a spe th ro ,s notypes with added resistanc o steo t t e o m tw rus n i t three years.

This program will be supplemented with artificial stem rust inoculation ensuro ,t e appropriate identification of the resistant individuals.

As soon as the adequate level of homozygosis is reached, the resistant lines wil e transferelb fiele th o t do t d increase seed, still under conditions of artificial stem rust inoculation. When seed becomes availabl adequan i e _ e amountst mutante ,th s wil e testelb yielr prod fo an d_ tein conten replicaten i t d experiment Experimene th t a s t Statioregionat a d an n l locations. Artificial stem rust inoculation will be maintained throughout the testing procesExperimene th t a s t Station.

5. These mutants addition , i wheae th to nt cultivar s Maria- nela, Aurifen and Sonka, all high yielding and well adapjt ed, but susceptible to stem rust, will be gamma irradiât; ed in dosis of 10 and 25 krad in May of 1980; the Mj_ gen£ ration will be planted in the field in June of the same year, in populations of approximately 100000 individuals per treatment.

49 The main objective of this program is to obtain stem rust resistance. Therefore, the first selection cri. terium wil resistance lb diseasee th o et , startinn i g

the M2 generation, working under conditions of artificial stem rust inoculation. Resistant individuals will be analyze proteir fo d n content firsa n O t. attempt, those that show levels of protein equal or better to their parental types will be advanced to the next gene_ ration thosf I . e individual scare t exisar no _ r o to sd ce, selection will be expanded to those that show accep_ table protein levels.

When appropriate levels of homozygous are reached, po£ generation^ M e siblth t a y , these genotypes wile b l analyzed for yield and protein content in replicated experiment e Experimenth t a s t Statio d regionanan _ lo l cations.

6. During 1980, selection will be initiated on the M, ge- neratioo tritical wheao tw tw d tf an no e genotypem sga ma irradiated in 1978. The same criteria of selection and procedures described in point 5 will be used. becomet i f I s . necessary7 , more whea d triticaltan _ ema terial will be irradiated in 1981 and 1982.

8. Three e initiateyearw o ag s prograa d wheaf o m d tr;tan L ticale multiline composites, which at present, due to the crisis derived from the stem rust situation, has re_ ceived further financial support from the University's Directio Researchr fo n Projecs ,a 95/79° tN .

Under the terms of that Contract we have grouped wheat and/or triticale genotypes produced by gamma irradiation and conventional breeding methods f simila,o r phenotype, carryint bu g different gene resistancr sfo Puccinio et a graminis, and if possible different levels of resistance

50 and/or toleranc barleo et y yellow dwarf virus ford ,an m ed multiline composites of up to nine constituent lines.

During 1979 we worked with, a total of eight wheat and four triticale composites. That year we selected 125 new wheat lines and 49 new triticale lines that can be grouped into composites in 1980. This process will be continue intensifiedd an d , testin multilinee gth t sa the Experiment Station under artificial stem rus£ tin culation sees a dd ,availabilitan y permit regionat sa l locations.

9. In an effort to search for additional sources of resis_ tanc steo t e m rus wile w t , le fieldracB th gro 5 e1 n i w, in 1980, with artificial stem rust inoculationm re e ,th mant seed from our reserve stock of mutants produced durin duratioe th g Researcf no h Contrac 1033/RG/G° tN S whic beed hha n discarde variour fo d s reasons.

This material will be grown in non-replicated plots, in the forscreenina f mo g nursery, wit primare hth y objec_ tiv identifyinf eo s stegit m rust reaction.

Any promising material will be further tested with, the same procedures describe poinn di . t5 materiae Th . l10 derived fro varioue mth s approaches describ_ whichd ean d , statisticalle showb o t s y better thae nth controls with which it is compared, will be analyzed for milling and baking quality by contract with an independ_ ent laboratory.

. Dependin11 n fundgo s availability ,numbea f selectero d mutants wil analyzee lb determino dt e their amino acid composition.

. Outstandin12 g material wil subjectee lb agronomio t d c studies mainly planting date, planting rate resd ,an - ponse to fertilizers. 51 13. All protein determinations will be made through, the dye- binding capacity method; some selected lines will be fur ther analyzed by micro-kjeldahl.

LITERATURE CITED

. 1 Parodi, P.C Isabed .an Nebreda. lM . 1979. Proteid nan yield respons wheax si tf eo (.Triticum spp.) genotypes to gamma radiation. In Seed Protein Improvemen Cerealn ti Graid an s n Legumes, Vol. II: 201-209. IAEA, Vienna 1979. STI/PUB/496. ISBN-92-0-010179-8. 2. Quisenberry, K.S. and L.P. Reitz (Editors). 1967. Wheat and Wheat Improvement. ASA Agronomy Series 13. American Societ Agronomyf yo , Inc., Madison, Wisconsin, USA. 560.pp. 3. Sigurbjornsson . ,1970B . Introduction: Mutationn i s plant breeding programs Manuan I . Mutation lo n Breeding. Technical Report Serie 119° sN . IAEA, Vienna, Austria p:l-7.

52 ACHIEVEMENT SEEN SI D PROTEIN IMPROVEMENT PROGRAMME

M. TAHIR NADEEM NIAB, P.O. Box 1208, Faisalabad, Pakistan

ABSTRACT

In wheat, a mutant M-536 with higher protein content and the sam eparentas yielit s a d l variet rusa d t yan resistan t mutant M-546 were selected triticalA . e selection, NIAB T.157-4, with good performanc botn i e h yiel d proteian d n conten s foundtwa . Unfortunately it was inferior to the local standard wheat variety with regards to the Chapati making quality, so it will hve to be used mixed with wheat.

Results Obtained

During this study, some promising mutants of wheat and tsiticale were selected. Wheat;

Mutant line M-536, a derivative of local wheat variety C-273 having good Chapati-making quality (a form of unleavened bread eaten Pakistann i isolates wa )higa s hda protein line t I gave sam. th e e grain e parentyielth t s comparativelda bu , s comparea w lo y o t d prevalent high yielding wheat varieties. Attempts are being made to improv s graiit e n yield potential without affectin s proteiit g n content.

Mutant line M-546 which does not have a high protein content, but is quite resistant to various rusts, could be used as a source of disease resistant germplasm. The mutant was also evaluated for its bread, milling and Chapati-making qualities. The results showed that e mutan th comparabls wa ts breadit r fo e, millin Chapati-makind gan g qualities with leading wheat varieties of this region i.e. ZA-75 and. LU-26» Attempt e beanar s g mad o improvt e s proteiit e n contente Th . mutant could prove very useful in adverse weather conditions.

Triticale:

A strai f triticalno e NIAB T.157-4 which performed well botn i h irrigated and rainfed conditions regarding its yield and protein content was evaluate r Chapati-makinfo d g quality resulte Th . s indicated that e straith n coul t competno d e with prevailing standard variet f rainfeyo d area in Pakistan (variety-Pathwar) for all the attributes, such as colour, texture, chewabilit tastd an y e which ultimately adversely affected the overall acceptability. This might be due to its dark flour colour which biased the opinion of the judges. However, experiments arn progresi e improvo t s s Chapati-makinit e g qualit mixiny yb t gi wit h bread whea n appropriati t e proportions.

53 Programme Status

i. Mutant line M-536 is being used as a germplasm of good quality wheat variety. Attempts are also being made to improve its grain yield.

ii. Mutant line M-546 may be used as a source of rust resistant germplasm e tryinar incorporato e t W g. e this character inthige on oh yielding wheat variet thao graie ys affectee b tth nt yielno n i dy dma adverse climatic (weather) conditions.

iii. Experiment beine sar g conducte establiso t d h appropriate proportions of bread wheat to promising triticale mutants (good grain character and yielding ability) to improve their Chapati-making quality. e otheTh r programm botr fo eh whea d triticalan t e will remain similar to that of previous years:

January - June: Technological and nutritional qualities' tests and harvesting of crop.

Jul Decembery— : Protein analysis, crop growing seasod nan compilatio resultsf o n .

a) The promising (high protein) homozygons wheat strains will be grown to test their stability for protein content.

b) Generations obtained after mutagenic treatment will be raised and screened for various characters.

c) The resultant advanced lines will be evaluated for protein, amino acids, and other technological parameters.

54 IMPROVED QUANTITATIVE PROTEIN MUTANTS, SELECTED FRO MBARLEA Y MUTATION BREEDING PROGRAM USING ADVANCED SELECTION PROCEDURES

H. WALTHER Abteilung für Pflanzengenetik, Gesellschaft für Strahlen- und Umweltforschung, Grünbach, Federal Republic of Germany

Abstract

Withi mutatioa n n breeding progra selectioa m n experimens ti describe sprine th r g fo dbarle y varieties Asse (6-rowedd )an Edelmut (2-rowed advanced mutant line) after applicatiof no EMS-treatments (1-, 2-, 3 x replicated) and X-ray irradiations (replicated)x 3 . Selection occurre proteir fo d n yields ,a major selection criteria, using a partial bulk breeding method during segregating generations.

As an example for this research, experiment CM3 - M6) , results are demonstrate e successfuth r fo d l selectio f improveno d protein lines from variety Edelmut, including descriptiof no the breeding program n improve,a d selection criterir fo a quantitative protein selection, and a successful selection procedure.

1. INTRODUCTION

Selection for quantitatively inherited characters like yield, protein content, lysine yield and lysine content, needs a defined selection procedure durin generatioe th g n where segregation occurs. This includes the estimation of gain through selection (GS) in early generations, which depends from population size (n), heritability of the selection criteria (h ), the induced genotypic variance (a ) and most off all from an appropriate selection intensity (i), which varies under practical breeding condition eacn i s h consecutive generation differeno t e ,du t selection rates (

From protein-mutation breeding programs, no selection procedure has been described so far, including the sequence of selection intensities in a quantitative genetic approach. This experiment was started to determine a practicable selection procedure with optimized selection intensities.

55 MATERIA. 2 METHODD LAN S

2.1 Breeding method

mutatioe Th n progra startes mwa d with 10.00 singl2 OM e plants for the whole experiment, or with 125O single plants per treatment and variety. All M2 plants were transferred to M3 and planted in single rows as base population (B). All M3 lines were teste proteir fo d n characters wer% 0 e5 selected ,an d an d orthogonan a n i 5 M ld replicatean teste4 M n i d d 2-years (generations) experiment (see Fig. 1).

5 % of the best lines were transferred into a two-years replicated yield performance trial in M6 + M7. At the same time the separatio withie th f no line variation (partial bulk lines) starte. M6 n i d

Except for M3 the primary and empirical selection procedure was basegeneraa n o d l selectio r eacfo hn% selectio5 rat f eo n step.

Starting with M4 + M5, always two successive generations were grown orthogonal with same numbers of replications and lines, allowing for more detailed testing of the genotypic base of variation.

Durin verificatioe th g e breedinth f no g program ,sligha t reductio numbee th f linen ro i n s occurre usin, M3 d go t fro 2 mM only 8.OOO lines instead of 1O.OOO for selection in further generations. This was caused through the fact that only lines without drastic morphological segregation coul usee r b d fo d quantitative selection purposes base .Th e populatio eacr fo nh variet d treatmenan y t consisted therefor f 1.OOeo O M3-liner so M2-plants (see Tab.1). From the total base population of 8.000 _2 M3-lines we expected a usable mutation rate of 10 depending on at least 1OO genes, quantitatively contributing to protein productio d leadin an n expectatioa o t g t leasa 0 f t2 no definable and significantly improved mutants after segregation.

All parameters were estimated from randomized complete block designs including a correction for within-block-error components.

2.2 Selection criteria

We used as selection criteria the protein yield measure in 2 g protein/ d suggeste whic, mha e hw previoun i d s experiments bivariata e whicr anus fo de hw e mode r estimatiofo l e th f o n values [1]. 56 According to the negative relation between % protein i.d.m and grain yield the criteria protein yield is the only one measur deteco et t quantitative-genetic improvementss i t I . estimated as product of the two components, % protein i.d.m and grain yield in g grain/m (see Fig. 2.).

In analogy to a one dimensional variation, we estimated also for this bivariate selection criteria at every selection step an expected relative gain through selection as

, 3L - H GS + , x. = S RG e M. i P.

followine th ann i d g generatio realizea n d relative gain through selectios na

RGS = P * ~ M * rr i+l i+i

This enable decido t s u de already during early generations over suffucient genetic variation, whicnecessara s i h y requirement for a successful selection. All values were estimate evern i d y generation relativ e originath o et l parent from whic populatioe hth deriveds nwa .

e use w generan i d r S estimatio Fo RG s selectio a le th f no n rate tha te populatio th par f to n which deviated significantly

(LSD) froparene th m t mean. This selectioe nth ratd ean 5& corresponding selection intensity decides at a given heritabilit e selectioth f o y n givea criteri t r a no d an a induced genetic variatio e basth en i npopulatio n ovee th r maximum gain and progress which can be achieved through selection.

The estimation of the selection parameters occurred through an analysi variancf so e give Tabn responsn i ni 2 . o et generations M3, M4, M4 + M5, allowing in this order an increasing precision in the prediction of an expected gain through selection resultf .I s fro generation2 m e sar r also r mor , fo oavailabl eM4 tha+ 3 locatio1 n (M e d nan year withi generatione non able o estimatar t e e )w d ean interpret also the genetic basis of the induced variation 2 throug. r hc

The correlation between the expected and the realized RGS- parameters was found to be very high with r = O.84*t~.O.89**~ , indicatin googa d reliability between both parameters.

57 2.3 Selection procedure

This empirical selection procedure, usin t eaca g h selection step only those line selectios sa n intensity which deviate significantly from their parents, is however just one of a number of possible selection procedures. The search for an optimal selection procedure, applicabl mutatioa r fo e n breeding program, and thus the maximization of the selection progress s stil,i open la n problem.

To optimize selection progress and procedure, we have tested a serie differenf so t selection procedures, varying onln i y different selection intensities at the first, second and third selection steps and having the same given variance and heritability in the base population. The selection intensity only decides therefor n thii e s experiment ovenumbee th r f o r selectable improved lines within .this program.

In a number of different earlier and related experiments we found for protein yield heritability coefficients ranging 2 betwee 2 n h 2 = O.25 and h = O.75, in average h = O.S. In a proposed linear relation between heritabilit d selectioan y n intensite yth value f o r selectio% (o s5 7 - n5 2 rate, ) s

Within this selection program 18 different selection procedures were tested varying in their sequence of selection intensities at the subsequent selection steps (linear or progressive selection usind )an ode- 1 g r 2-years result r eacfo sh selection step (see Tab. 3 ). For some procedures, progressive selection rates were determined through tests for significance. maximue Th m numbe f significantlro y improved line achieves swa d with selection procedure No. 15 = 1OO/50-5O/*-. However procedures 12, 17 and 18 proved also quite valuable.

If the value of a selection procedure is judged by its achievable RGe foun w e selectioS th d n intensity appliee firsth t a d selection mos e b ste to t p decidin r furthefo g r achievable progress. With strong selection intensities applied at the first selection stechance pth detectinf eo g best lines drops rapidly.

58 From Fig. 4 it is obvious that strong selection in M3 (1O...O.1 %)

leads inevitably to low RGSr~values in M6. This is caused by the fact that lines selecte characterr fo d s witheritabilityw lo h s ,a is know proteir nfo n yield, have lower chance foune b n o i dt s early generations when high selection intensitie appliede sar . With the lowest selection intensity (i = O.798,ore* = 50 %) the highest RGS was found in M6.

e fundamentaTh l questio optiman a r fo nl selection procedure might be answere relatiny db r eacfo gh procedur S -valuee RG th e o est th total input needed up to the stage of comparison.

As measure for the total labour input we used the number of field plots up to M6, since the amount of labour needed in planting, harvesting and analytical screening is thought to be the same for each field plot, independen varyinf to g plot sizes ranging between O.8 and 5.0 m2.

Thd labouean relatio rS RG inpu f no t reveale o importantw d t results whic demonstratee har constana Fign t i da .5 t selection

intensit2.89= 6 bes5 M ( i 2 tf yo line M6)n i s .

1. All selection procedures, based on 2-years selection steps are superior to those based on 1-year selection steps.

2. The optimum in selection progress measured at a heritability 2 5 mighO. e achieve= tb leveh onlt f y procedurno o dlb y , 15 . eNo usin maximua g f inputmo alst procedurey ,bu ob s defined under e samusin, th 18 e f g, o input 17 onl 3 , 2/ y12 . Thi. No s indicates 3 populatioM e e leas e expecteth th b e tf n b o nca % o tha t d5 2 t sufficient selectio e selectionTh rat. optimizo S t e RG n e th e optimum in this experiment was found highest between 200O...300O input units (field plots).

In summarizing the results from the 6 best selection procedures we could demonstrate that the best selection results were obtained with an initial selection rate of 25...50 % at the first selection selectioa sted an p n rat 0.3...3. f o e secon th t da 0% selection e seconstepth t d.A selection stee applicatioth p f no significance testf advantago s si e (see Tab. .4)

The maximum output of improved lines was found with procedure e e optimuth th , , S S witwit15 15 RG mRG . h. h yieldinNo No , procedureprocedure18 d an g 7 ' 1 s1 s improve6 1 d an d8 1 line , 50 s respectively.

59 3. RESULTS

In Figresulte th .presente6 e sar d from selection procedure No. 15, showing the maximum average output of 3O significantly- improved protein-yield line treatmenr spe wit , n M6 ha n i t average progress of 12 % and, for the 4 different mutagenic

treatments, a range from 8.9... 17.1 % RGSr, respectively.

From replicated treatments (X3, EMS3) more improved protein lines coul selectee b d fros a d m single treatments (EMS1)t .A the same timmutagenie th e c treatments also differee th n i d way in which improvement was achieved. X3-treatments, for example, resulted lines with high grain yield contributions to protein yield. From EMS3-treatments more protein-improved lines were found with raised level proteif so n content ta constant level graif so n yield.

In average howeve e proteith r n yield improvemens wa % 2 1 f to achieved approximatel graiy b % n 0 yiel5 o t yd increaso t d ean 5O % by increased protein content.

e bivariatTh e distribution indicate snumbea alsr f fo oro mutants improved levels with + 20 % protein content at constant grain yield levels or with simultaneous increase in grain yield and protein content of + 10 to 15 % above the parental control lines.

Since these values were derived from partial bulk, plotn i s generation som, M5 e+ additiona 4 sM l segregatioe b n nca expecte furthey b d r selection within families starting from generation M6 with single plant selections.

4. DISCUSSION

In addition to previous results [1] the problem of an optimized selection procedure, applicabl quantitativo et e mutant selection, s investigatewa d durin generatione th g s M3-M6.

A significant increas n selectioi e n progress coul demonstratee b d d by using 2-years (generations) selection steps instean a f o d annual selection scheme. It would be recommendable, to include r durin phase gth f earleo y selection als) (M4M6 o, ,morM5 e than 1 location in order to increase the precision of all selection parameters. This might raise the level of selection progress (RGS ) by an additional increment and might lead to a second dimensio optimizatiof no n with appropriate allocatio adaptef no d environmental combination during early generations. 60 The application of different schedules for selection intensities conclusioe th leo t d o intensift n e selectioyth n pressure only n latee seconi i th nd r an dselectio n step o ordet n i s t rno sacrific considerablea e advance through selectio losy nb f so improved mutants under strong selectio M.3.n i ne selectio Th n intensity during the first selection step should range between i = 0.798...1.271 (25-5O % ) according to heritability values expected betwee O.5O...0.75= h n secona r .Fo d selection step an increased selection intensity in the range of i = 2.231... 3.050 (3.0...0.3 % ) is recommended when 2 generations (years) e include e ar seconth n di d selection step.

To optimize a selection procedure, the induced genetic variation knowne oughb o t .A rathe r valuable information about significant genetic components of variation can be used from a two-generation analysi variancef so / e samgivinth et a gtim e o information abou operative th t the eoperativ heritabilite foun) her h ( yd: within the population under consideration.

All results described are relevant so far for the first phase of the breeding program, using the between family variation. An additional increas protein i e n yiel expectes i d d froe mth second progra e phasth f o em wher withie eth n family variation is utilized.

REFERENCES

[1] WALTHER , SEIBOLD,H. , K.H., 1979, Improved protein mutants selected from barley after multipl X-rad an yS eEM treatments , Proc. Sympos. FAO/IAEA/GSF, Neuherberg, 4.-8.9.78, . SeeIn d Protein Improvemen Cerealn i t Graid san n Legumes, 327-343, IAEA, Vienna

[2] SATTERTHWAITE, E.F., 1946, An approximate distribution of estimate variancf so e components, Biometric0 11 , 2 s

61 TABL . I EXPERIMENE R SELECTIOTFO F IMPROVENO D PROTEIN STRAIN N SPRINI S G BARLEY SELECTION CRITERIA: PROTEIN YIELPROTEINg N I D /

EMS1 EMS 2 EMS3 X3

Parent Edelmut M2 1250 1250 1250 1250 Asse M2 1250 1250 1250 1250

Edelmut M3 1OOO+ 1000 1000 1000

Asse M3 1OOO 1OOO 1OOO 10OO

+ = Base population

62 TABLE II ANALYSIS OF VARIANCE FOR ESTIMATION OF SELECTION PARAMETERS IN A SINGLE ENVIRONMENT MICROENVIRONMENTS= (M3 4 ,M DIFFERENN I D )AN T COMBINATION ENVIRONMENTF SO S (M4 + M5 = GENERATIONS, LOCATIONS, YEARS = MACROENVIRONMENTS).

Generation Sourc f variatioo e n „> MS Expectations of MS Components of Selection variance0 parameter

M3 Strains G-l M3 s = VM3 = Vs

h2 =(S2_S2)/S2

xM+1.96sph

2 2 M4 Replications R-l Ml 0" +GCT e r Strains G-l M3 a2 +Ra2 o-2 = (M3-M5)/R e g g bV OP^p - ! h Error (G-l) (R-l) MS ^ ex; , i ^ x +LSD5%

M4+M5 Replications U(R-l) Ml CTe r Sph = y/M3/UR 2 Environments U-l M2 o- = (M2-M4-Ml+M5)/GR hO = ag/CT|h' bOP Strains G-l M3 a2 +RO-2 +URa2 er2 = (M3-M4)/UR CX,i>x LSD e gu g g p+ 5% M4 Strain x Environms . (U-l) (G-lu ) °g + e ° OgU= (M4-M5)/R Error M5

determinatior Fo parenta% = O 1 a , i l f lineno s were included into analysi variancef so . For determination of o~2(only mutants were included into analysis of variance. b = R = replications, U = environments, G = genotypes, P = parents, O = offspring, ph = phenotypic c = F-Test according to [2] 2 h • i • h Sp = S GaiG n: through selection Relative gain through selection, expected: RGSe = XM. + GS^ - x"p. Relative gain through selection, realized . :-x . RGSM pX . = r TABL SELECTIOI EII N PROCEDURES TESTED WITHI NPARTIAA L BULK BREEDING PROGRAM WITH SPRING BARLEN YI GENERATIONS M3-M6. VALUES GIVEN AS AVERAGE OVER MUTAGENIC TREATMENTS EMS1, EMS2, EMS3, X3

Selection procedure Selected strains from Selection steps Selection rates Selection rates Selection intensities No. base population per ste p= %o

1-year linear 100/50 /50 /50 100/5 5 /12./2 0 5 0 /O. 798/1. 271/1 .647 1 1OOO 500 250 125 5 100/2/2 5 5/2 100/2 6.25/ 56 .5 /1 0 /1. 271/1. 968/2. 516 1 1 3 26 0 10O25 O 100/10 /10 /10 100/1 I / 0/O. I /I0 . 755/2. 665/37 .36 1 0 31 0 1OO10 O 100/ 5/5/5 0.25/ 100 1 /5 / 0. 0 /2. 063/3. 110/3. 367 4 1000 50 3 1 100/ 1/1/1 10O/ 1 / 0.1 / 0. 665/3. 1 /2 .0 367/37 .36 1 51 lOOO 1 O 100/ !+/ !+/ 1+ ' 100/ O.I/ 0.1 / 0.1 0 /3. 367/3. 367/3. 367 6 1OOO 1 1 1 progressive 100/50 /25 /10 1.2/ 5 100/55. 2 I / 0 798/1. /O .0 271/29 .58 7 lOOO 50O 125 13 100/25 /10 / 5 100/25 / 2.5 / 0.1 0 /I. 271/2. 338/3. 367 8 1000 250 25 1 100/10 / 5 / 1 100/10 / 0.5 / 0.1 /10 . 755/2. 892/37 .36 9 lOOO 1OO 5 1 2-year progressive 100/50 -50 / 5 100/50 -50 / 2. 798-0. 5 /O .0 798/2. 338 1000 500 500 25 1 100/5/ O 0-5 5 0. / 100/5 0 -5 0 798-0. /O .0 798/22 .89 5 O 5O lOO0 50 O 1 100/2/ 5 5-2 0.2/ 100/2 55 -2 5 0 /I. 271-1. 271/3. 1103 0 25 1000 025 100/10 -10 / 1 1OO/1O -1O / O. 755-1. I /I .0 755/37 .36 13 lOOO 1OO 1OO 1 100/ 5-5/1 5 100/O./5- I 063-2. /2 .O 063/37 .36 14 lOOO 50 50 1 progressive, 100/50 -50 /*!0 3. / 100/5 0 -5 0 798-0. /O .0 798/28 .26 lOOO 500 5OO 30* + test of 2 100/*1* 1 1-* 1004 /0. 3.2/ 2 -3. 249-2. /2 .0 249/22 .96 16 1000 32 32 4* significance IOO/>XE->XE/*I 100/248 .1. 6-24 / .6 281-1. /I .0 281/29 .45 1000 246 246 18* ! 100/2/* 5 5-2 100/25 -25 / 1.6 O /I .271-1 .271/2.562 1000 250 250 16*- selectio= S 1= ) n step bas= B 2 e)= population witlOO= hn O strain varietr spe d treatmenyan t 3} = favorable selection procedures significan= ) 4 0.0= p 5 t ta 5bes= } t strain TABLE IV. SUMMARY OF RESULTS FROM BEST SELECTION PROCEDURES. MEAN VALUES FROM EMS1, EMS2, VARIET R EMS3FO 3 ,X Y EDELMUT

Selection proceduro eN 8 1 7 1 2 1 5 1 1 1 0 1 %0

Generation M3 1000+ 1000 1OOO 1000 1000 1000100 0.0

M4 50O O 25 5OO 6 5024 0 0 25 J50-25 798-. 0 1 .271 M5 500 500 500 250 246 250 M6 25 5* 16 30 * 18 3 3.0-0.3 2.231-3.050

+ 5 RGSr 23.2 18.6 23.7 26.4 21.5 19.8 1 + 38.5 34.5 39.1 31.9 38.1 35.1

+ = Number of strains from base population B in M3 per treatment Strains selected through test of significance

PT' 'PERFORMANCE TEST OOOOOOOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO0 200000 SEEDS TREATED R ;• REPLICA TIONS P, MO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOQOOO.O L - •• LOCATIONS Y '< YEARS M1 OOOOOOOOOOO-OOOOOOOOOQOOOOOOOOOOOOOOOOOO SEEDSO 000 200 M1-BULK OOOOOOOOOOOOOOO OOOOOOOOOOO OOOO OOOOOOOOOOO O 200000 SEEDS M2-BULK M2 10000 M2-SINCLE PLANT.

M3 10000 M3-ROWS

SELECTION 5000 PT,2R BETWEEN FAMILIES

M5 5000 M5 PT, 2R

M6 250 M6 PT, 2R ,2 L 250* 10 025000= SINÖLE PLANTS

M7 250 M7 PT,2R , 2L 250x100 = 25000 ROWS

2R , PT 1000M8 Ma SELECTION BETWEEN AND WITHIN FAMILIES MS 2R PT, 1000M9

R,L,YPT, 50M10 M 10

Fig Partia1 . l bulk breeding method user selectiofo d n of mutants with improved protein yield. 65 FREQUENCY P = PARENT- POPULATION MMUTA= POPULANT- TION

30 --

20 --

10 -

ISO-PRO TEIN YIELD-LEVEL PROTEINImg :

500 600 glrrT GRAIN YIELD

Fig. 2 The use of relative gain through selection (RGS) as quantitative parameter for uni- and bivariate selection characters, including selection limits, determined by tests of significance.

66 o\ -J

WcxsSEIfO% C TtONPA TE

Fig Expecte3 . d relation between heritability (hd Q)an selection rate (

120 -

U 110 -

p, 10 ———- 0- — o oo

0.0 0.798 1.271 7.755 2.065 2.665 3367 BASE-POPULATIONi IB)*M3 h- -H— —)—— 700 50 25 01 <*fl -*— H—————————————— 7000 500 250 700 50 70 / NUMBER STRAINSOF FROMB. IN M3-100Q

Fig. 4 Realized relative gain through selection (RGS ) aftei6 nM r applicatio differenf no t selection O.798= i C intensitie .3 .3.367). M n i ) .(i s RCSr in % PROTEIN YIELD : g PRO T EIN l m to VARIETY ; EDELMUT PARENTfP) GENERATIONS: M3-M6 MEAN TREATMENTSOF EMS1. EMS2, EMS3. X3 SELECTIONlNTENSlfY: 'MS- 2.392 BEST5 = STRAINS

130

120 --

110 -

0\ 6o 0-04

YEARS2- = SELECTIONSTEPS• o= 1-YEAR SELECTIONSTEPS

1000 1500 2000 2500 3000 NUMBER OF PLOTS PER TREATMENT M6 TO FROM M3

Fig Realize5 . d relative gain through selection (RGS) in relation to total input from M3 to M6 (number of plots per treatment), according to different selection procedure .15. . ) . 1 ( s % PROTEIN i. dm. S ELECT/ON PROCEDURE NO. 15 to in% VARIETY : EDELMUT. SIGNIFICANT STRAINS FROM ML* MS. P A RENTIP) 120 - EM . 51 • = EMS2 o = EMS3 O = X3 115 - ® = TREATMENT MEAN (5)= TOTAL MEAN

110 --

705 -

-l o P=11.8%- - 0 10

1171% X3 15 STRAINS 1U.5% EM S3 3t, STRAINS 112.0% EMS2 STRAINS39 108.9% EMS1 STRAINS32

90 -- = 52.5g PROTEIN/m

90 95 100 105 110 115 120 125 GRAINlmg PARENTto W in% 2 P*t,901 glm2

Fig. 6 Results from selection after M4 + M5, obtained under selectio ndifferenr procedurfo 5 1 . teNo mutagenic treatments (EMS1, EMS2, EMS3, X3). PROBLEMS AND POSSIBILITIES IN GENETIC IMPROVEMEN PROTEIF TO N YIELD CEREALN SI S

F. SCHOLZ Zentralinstitut für Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, 4325 Gatersleben, German Democratic Republic

Abstract Decisive succes breedinn si r highgfo , protein yiel cerealn i d s has not yet been achieved anywhere. Notable increases in protein content were always compensated by at least nearly corresponding grain yield reduction. This proved also true with many induced high-protein barley mutants obtaine long-tern i d m experiments which we had begun more than two decades ago. Also use of the mutants in cross-breeding gave likewise inadequate results. Interrelating problems as to the potentials of genetic improvemen proteif o t n yiel cerealn i d e beinar s g discussed. Consider- ing nitrogen balance and requirements of the crop, one should be aware that more grain protei unir npe t aren onl producee aca yb d if more nitroge grair nfo n protei availables ni . Hence, protein yield can only be increased by improving the ability for H uptake frosoie th ml and/o mobilizatiorN d translocationan n froe mth vegetative e organgrainth o t s. This, however, appeare b o t s possible on a comparatively limited scale only, unless additional nitrogen fertilizing is provided.

Introduction

In considering grain protein improvement, we mean two different aims: (1) increase of protein content, more exactly protein yield per ur.it area, and (2) improving protein quality, i.e. amino acid composition. Here I would like to concentrate upon the first problem. The las tdecaden eighte r o ts have see nmosa t considerable increas yieldf o e n cerealsi s , especiall industrialle th n yi y developed countries. The increase has related and is relating not onl graio t y n yield alst proteio sbu t o n yields. This became possible mainl meany yb f increasino s g fertilization, especially by nitrogen. Also plant breeding, of course, has substantial shar thin i e s developmen mora n ei t, indirec t way, however. 71 Plant breeders have provided varieties which were able to utilize the higher amounts of fertilizers, and that by higher yield potentials, higher harvest indices, improved lodgin diseasd gan e resistance, etc. 7/e, however, wan havo t e more plane Th : s beinti g expected to produce even more protein same placn th ,i e f eamouno carbof o t - hydrates, and that, if ever possible, without additional nitrogen fertilizing difficulto to e loob t .no o kt Thi, d ssincdi e cereal forms with high protein contents could comparatively easily be foun collectionn di d latesan als n ro mutatioy ob n induction. Therefore, attempt breeo t s d high-protein (and high-yielding) varieties were repeatedly made: already before the first world war (e.g. USA — maize), then in the twenties and thirties (e.g. German Canadd yan abarle— wheat)d yan finalld ,an ye nowth r ,fo fifteer o lasn te tn years, very intensel mann yi y countries. Decisive success in breeding for high protein yield has not yet been achieved, however. This is true at least for humid and semiarid regions where nitrogen mobilizatio d translocationan n in the plant is apparently better than under arid conditions. Notable increase protein si n content were alv/ays compensateds a , show reliabln ni e field trials least a y tb , nearly corresponding grain yield reduction. In some cases, maximum gains in protein yield in the order of about 5 percent or so are possible. Such gains, however, eve possible varietiew nar ne n ei s with increased grain yields and unchanged protein contents. The initial optimis regards ma possibilitiee sth genetif so c improvemen proteif to moro nt yieldgivo et y realismewa d sha . The rather easy increase proteif so n contenteasilt no e n yb ca s converted into increase proteif so n yield unir spe t area. Improv- ing protein yields appears to be just as difficult as improving grain yields.

Studies on induced high-protein mutants in barley

This situation proved also true witinducer ou h d high-protein barley mutants obtained in long-term, special experiments which 12 begud weha n more tha decadeo ntw s ago sooe .W n succeede findn i d - 'mutantg in s containin fourta o t hp g u more protei graie th nn ni than the original variety, e.g. 15 % as compared to 12 % /ï,2,2/. In the course of time we were able to isolate and to test in field trial differen1 s11 t mutant line sprinf so winted gan r barley, 58 of them over three or more years. In no case a high-protein mutant line reached the grain yields of its original variety; there were deficits of about 10 to 20 percent. The protein yields per unit area of some of the best mutant lines were in the order of abou percent5 t above thos theif eo r original varietiest bu , cose areducef tth to d grai Thi- n« syield 5/ tendenc , s/4 y proved unchanged in field trials with different, moderate or high, nitrogen levels. Analyse yielf so d component mutane th mann si f tyo line- sin dicated tha mosn i t t case reductiosa numbern ni grainf o s r spe 2 spike and/o tillerinf ro decisivs gi ) (spike reductior m fo er spe n of grain yield. Decreased grain size (thousand grain weight) was found in some cases only. We, t thahowevera t giv tno p u e tim d d expecte,di an e e b o t d abl "breako t e negative "th e correlation between protein content and grain yield. We attempted this by crossing the mutants with several high-yielding varieties or strains, in order to get by recombination more "harmonic" genotypes. Starting in 1967, we used the eight best mutants of spring barley for several cross combinations fij• After re-slectio higr nfo h protein conten made tw e field trials with 62 different strains in about 130 individual tests. The yields were again inadequate, just as with the primary mutants: Grain yield 85-9f so 0 percen maximud tan m protein yields of abou 5 percent10 compares ta high-yieldine th o t d g crossing parent. 6/ > s/5 We have used a large number of induced mutants being highly

isogenic to their original varieties and which are therefore, s very suitabl drawinr efo g critical conclusions resultr .Ou s give evidence thaexpectee th t d declinnegative th f eo e correlation

13 between protein conten d graitan takt n no yiele d placedi d . Thus, this correlation cannot be due to linkage of certain genes or to other genetic causes which could be "removed" by breeding. The increas proteif o e n yields, wi.tnout additional N-fertilizing, appears possible tb o limitea n o e d scale only.

Interrelating pJEqblems_as__tp_ increasing protein yields

i.e, so .f gainf i I protein si n easilcontent no cone n b y ca t - verted into gains in protein yield, we should look for other reasons. e coulOn d suggest that bioenergetic implications would cause unfavourabln a e balanc proteif i e n conten beins i t g increased. Bhati Rabsod aan J gavn$ e clear bioenergetic considerationn si cereal breedin proteir gfo n improvement. They have verified that eac percenh1 t increas protein i e n content same placn th ,i e f o e amoun f carbohydratesto , must caus energetin ea c los abouf o s t 1 percent and therefore a corresponding loss in grain yield of likewise nearly 1 percent, because energy requirements for protein biosynthesis is about twice as high as for carbohydrates. These inevitable costs for protein increases, however, are of an order which is not too high and may be accepted. In any case, decisive th e b e t situatione reasono th the n r ca ynfo . y opinionm n I come ,w e close reao rt l implication cone w f -si side e nitrogerth n balanc requirementd ean e th crope l th .Al f so protein produced derives from inorganic nitrogen taken up from the soil (protein has about 16 % N). More grain protein per unit areonln producee aca yb morf i d e nitroge r grainfo n proteis ni available. Providing unchanged manuring conditions proteie th , n yield can be increased on two ways: (1) by improved nitrogen uptake frosoie th ml improvey and/ob ) r(2 d nitrogen translocation from vegetative graine organth o .t s f nitrogeI n uptake frosoie th beins mli g improved, this must be compensated by higher N-fertilizing, at least for the subsequent crop. Furthermore, variatio uptakN f no e appear vere b yo t ssmal l

74 d recen an n both i d t,ol barley varieties thae reasoe b .Th t y nma genotypes with high N uptake give a higher yield and had always an advantage in natural evolution as well as in selection by man /§, ^/. Thus, improving nitrogen uptake appears to offer limited reserves only. Better nitrogen mobilizatio vegetative th n ni e organd san translocation to the grain may offer more possibilities, because apparentle oth f y considerable variatio thif no s trai e.gs ta . whean i t /ÏQ/. Some gai breediny nb indeey gma realizee b d n di increasing the proportion of plant nitrogen in grain at harvest harvesr (o t nitrogen index). This inde larga o xt e exten- in s ti fluenced by the harvest index or grain/straw ratio. The high yield potential of the current semidv/arf varieties is due to their higher harvest index sucd ,an h varieties already hav vera e y high proportion of plant protein in grain at harvest ffj: About 70 % or eveuptakW e n beins th morei f o eg usegrair fo d n protein formation. Further improvements, therefore ,difficulte b wiD1 . These implications considere above-mentionede th als n oi d pape Bhatiy rb Rabsod aan furthea nn i /7_/^le d ron 7 an / have stimulated a calculation, naturally a simplificated one, on the nitrogen balanc cerealn i e s (see table). There I proceed from an initial, "normal" situation (first column): 5000 kg/ha grai protei % grain e n2 1 th yiel d n ,ni an d under given manurin othed gan r environmental conditionn o d san the assumption that 70 % of the 1\T uptake can be used for grain protein. Under these conditions graie th , n yield necessarily must decrease from 5000 to 4000 kg/ha, if protein content increases % (secon 5 1 o dfrot 2 column)m1 . Higher protein yield onle sar y possible if a higher percentage of N uptake (e.g. 80 %") can be user graifo d n protein (third uptakcolumnN f i er )o itsel s i f improved (fourth column) f bot.I h source additionaf o s r fo lN grain protein are effective in appropriate amount, then even 5000 kg/ha grain yield with 15 % protein may be achieved, at least theoretically (fifth column). Calculatio nitrogen no n balanc yieldd ean cerealn si s

Initial situation; 5000 kg/ha grai protei% n2 1 yiel d ndan (further explanation in text) (1) (2) (3) (4) (5) (6) 12 % 15 % 15 % 12 %

Grain yield 5000 4000 4567 4375 5000 6250 Protein yield 600 600 685 656 750 750 N requirement 96 96 110 105 120 120 of grain protein Necessary N uptake 137 137 137 150 150 150 (70 % or 80 % of it into tEe" grain) (70) (70) (80) (70) (80) (80)

All figuren si kg/ha (except last line in #)

The last columtable th ef no show calculatiosa n with ,graia n yiel 625f o d 0 kg/ha instea 500f o d 0 kg/ha. This would theoretically be possibl highee th highee f uptakrN i eth d rean percentag y % 0 (8 e grair fo nt ofi protei utilizes ni varieta y db y insteawit% 2 h1 d of 15 % protein content. Then we would have gains in protein yield as well as in carbohaydrates, i.e. total grain yield. Hence, several specialists have considered it to be probably of more advan- tag breeo t e d primaril r graiyfo n yield, tryin keeo proe t g pth - tein content unchanged thao proteie ,s tth n yield- woulim e b d proved together with grain yield. Perhaps we should briefly return to the harvest index for both grain and protein yield. Recently, Kramer /Î2/ gave a survey of this problem in wheat. The considerable grain yield increases in the past seem primarily to be due to improved harvest indices (grain/straw ratio). As the biomass production seems to be rather constant there will be a strong positive correlation between harvest index and grain yield. This leads to a likewise strong negative correlation between grain yiel proteid an d n content, since th redistributee b availablo t s ha large a N eo t d r biomasf so grain. This implies also, in my opinion, that by means of a higher

76 harvest index the protein yield will increase though the protein content is decreasing. In agreement with this is the fact that high protein yields per unit area are usually obtained with varieties having high grain yield potentials (and high harvest indices rathet ) bu graiw rlo n protein contents. According to Kramer, the genetic variation for grain protein content is largely an "improper" one, because the observed genetic resule variatioth f "proteit to no s ni n derivea genes" s i t d,bu genee th sl effec involveal distributioe f o tth n di mattey dr f no r (and thus protein). "Protein genes" really are "plant-type genes" which influenc harvese eth t inde harvese welth s x a s la t nitrogen index.

Some aspect possibilitief o s breedinr sfo g

The increas proteif eo n yield cerealsn si , without additional nitrogen fertilizing, appears to be possible on a limited scale only possibilitiee .Th s are leastt ,a , much more limited than formerly supposed. However, we should be aware that we still do have some real possibilities. Much more emphasi futurn si e work shoul placee b d genotypn o d e waydifferenceo tw s e allowinth r sfo least ga t small protein yield improvements without additional nitrogen fertilizing: ) Geneti(1 c variatio duratioe amoune th th uptakN d n f tan ni no e from the soil may be very important, though variability for this trait limitede seemb o t s . Long leaf area duratio d increasenan d greee levelN th n si vegetativ e tissueusefue b y lsma indications. ) Improve(2 mobilizatiodW translocatiod nan n frovegetative mth e organs to the grain may offer better possibilities, especially as nitrogen conten ripn ti e stra comparativels wi y high, % abou 5 t0. d evean n higher with high fertilizinglevelT I f so broadea n .I r sense, also high harvest indices produce an effect comparable to improved K translocation. However e shoul,w fulle b d ylimitee awarth f o ed effectf so these possibilities. If vie want to increase the protein yields essentially, the e shoulnw expect o no d d ablplan e e b o tth t e o t

77 so without additional nitrogen muse .demant W tno muca d h higher output without increasing input , e.g..If , protein conteno t s ti be increased from 12 to 15 % and correspondingly protein yield 0 kg/ha75 o t ,fro thefac0 y e regar m60 pa havth e n t w o o t et dtha t nitrogen requirement for the grain protein increases from 96 to 120 kg/ha. Only part of these additional requirements can probably be provide plane th ty b ditself , varieti.ea y .b y with improved uptakN e and/or translocation rese .Th t must come from additional N fertilizing. Breeding can support this in an indirect way, by developing varieties which are better able to utilize high levels of manuring, especially nitrogen r thes.Fo e aims, induced mutants may be valuable tools and can perhaps serve as pace-makers.

References

/"I/ Scholz Versuch, ,F. züchterischer zu e n Steigerun Eiweisss gde - gehalt Gerstr sde Hilft experimenteller mi e ede n Muta- tionsauslösung, Qual. Plant. Mater. Veget (1960.6 ) 276-92. / Scholz/2 Qualitätsproblem, ,F. Futtergerstenzüchtungr de n i e , dargestellt an Ergebnissen von Mutationsversuchen, Z. Pflanzenzucht. 44 (1960) 105-28.

/3,/ Scholz , "Utilizatio,F. inducef no d mutation barley"n i s , Barley Genetic I (ProcsI d Internat.2n . Barley Genet. Symp. Pullman, 1969), Washington State Univ. Press, Pullman (1971) 94-105.

Scholz, F., "Induced high-protein mutants of barley - problems breedinn i proteir gfo n content", Breedin Producd gan - tivit Barlef yo y (Proc. Symp. Kromeriz", 1972), Institute of Cereal Crops, Kromeriz (1973) 255-65. Scholz, F., "Problems of breeding for high protein yield in barley", Barley Genetics III (Proc. 3rd Internat. Barley Genet. Symp. Garching, 1975)? Verlag Karl Thiemig, München (1976) 548-56.

/5/ Scholz, F., "Experience and opinions on using induced mutants cross-breeding"n i , Induced Mutation Cross-Breedinn si g (Proc. Advisory Group Vienna, 1975), IAEA, Vienna (1976) 5-16.

78 /7_/ Bhatia, O.E., Rabson, A., Bioenergetic considerations in cereal breedin proteir gfo n improvement, Scienc^ e19 (1976) 1418-21.

/8_/ Sandfaer, J., Haahr, V., Barley stripe mosaic virus and the w barlene d yan varietiesd yielol f O d Pflanzenzucht. ,Z . 74 (1975) 211-22.

7 Doll/9 , "Storag,H. e protein cereals"n si , Genetic Diversity Muhamiaed. A Plantsn i y b . Akse. R ,ed R.Cd lan n .vo Borstel, Plenum Press Yorw ,Ne k (1977) 337-47.

/10/ Johnson, V.A., Mattern, P.J., Whited, D.A., Schmidt, J.W., "Breedin higr gfo h protein conten qualitd tan wheat"n i y , New Approaches to Breeding for Improved Plant Protein (Proc. Panel Meeting Röstanga, 1968), IAEA, Vienna (1969) 29-40.

/11_7 Rabson, R. , Bhatia, C.R., Mitra, R.K. , "Crop productivity, grain protei energyd nan : Inputs, subsidie limitad san - tions", Seed Protein Improvemen Nucleay tb r Techniques (Proc. Meetings Baden and Vienna, 1977), IAEA, Vienna (1978• - )3

/Î2/ Kramer, T., Environmental and genetic variation for protein content in winter wheat (Triticum aestiyum L.), Euphytic (19798 a2 ) 209-18.

79 UTILIZATIO HIGF NO H GRAIN PROTEIN WHEAT MUTANTS

C.R. BHATIA, R. MITRA Biology and Agriculture Division, Bhabha Atomic Research Centre, Bomba 0850 y40 , India

ABSTRACT;

The progress of work since the Neuherberg meeting in 1978 is reported. The aspects covered are: 1. Agronomic evaluation of a new selection (TW-3)« 2. Yield evaluation of fw-i (induced high protein mutant) derivatives in ÏV . 3 Transfe . anFy d f higo r h lysine o sprint gen ) s e( g wheat ss derivativesit fro d m an Mahrattal Ha P . Na , 4- Performance of other sources of high grain protein spring * wheatPla5 d g an slea f senescenc graid an e n protein.

INTRODUCTION:

This paper reports the progress in our research since e fielth 197f proteio dn i 8 n improvemen n wheati e t Th . objectives of our programme are genetic, biochemical and agronomic evaluation of high protein and lysine mutants or stock f o understanwheatt o s d an , e physiologicath d l basis e higth h r graifo n protein character differene Th . t aspects investigate e resultth d dan s obtaine e reportedar d .

1. Agronomic evaluation of Tff-g;

A selection from Rageni-15 (PI-383308) which is a high protein mutant reporte Khay Kasand b d an nfouns wa 'o yiel t d d equal or better than the popular cultivar Kalyan Sona, at Trombay. It 6 waday5- s s earlie n maturityi r , higheg ove 0 1 rn TKi rd Wan havin r cenpe t1 g point more grain protein than Kalyan Sona. During 1978-79 this cultur givens wa es TW-3 a , r testinfo , n gi

81 Vidarbha Region of the Maharashtra State and at the Indian Agricultural Research Institute (IARI), New Delhi. In Maharashtra, grain yield of TW-3 was -the same as that of 5 location f Kalyao t e locationou son Son4 t (Tabl a t a an, 1) e it was inferior. Mean yield of 5 locations were 2760, 3014 and 3219 kg/ha respectively, for TW-3, Kalyan Sona and HD 2251 which was the top yielding entry in these trials-. Grain protein could only be estimated at one location where the values were 11.6 and r 13.cenPe 3 t respectivel r Kalyafo y n l SonTW-3d al t an aA . locations, TW-3 was early in flowering as well as maturity and had higher TK n Wcomparisoi o Kalyat n n Sona (Tabl. 2) e

Grai nt satisfactoryyielno Delh w s f TW-3o dNe wa i t a , , thoug e graith h n produce highed dha r protein concentration (Table 3). Another culture, TV/-2, included in this trial is a. population selected for more protein per grain (larger seed 2 size) from Kalyan Sona whic describes hwa d previousls ha t I . y no advantage in grain protein though its grain yield, is at par with Kalyan Sona. In this experiment Kalyan Sona was not included. TW-3 is included In late sown Initial evaluation

trial other! at s locations,

2« Yield evaluation of TW-1 derivatives ; 3 4 Ab- reported previousl , TW-higa y s hi 1 protein mutant isolated from Kalyan Sona. In grain yield, it was 10-15e foun"b o $t d lower than TCalyan s Sonhenced wa an a t i , crossee paren th othed o t dan t r "high yielding cultivars. ^Results of a replicated, yield trial conducted at Trombay Which included 1Ü, TW-1 derivatives are given in Table 4. All "the cultures have given better yield and had higher TGW and grain pxotein than Kalyane . th Sona n I . previous generation f thes o highed l eha al ,r grain protein than Xalyan Sona. This year, the crop was heavily infected with leaf lust and only entry nunrber 12 showed resistance.

82 Som f thé«*o e * cultures wil givee b lr testinnfo t gothea r location im the next cropping season.

3. Transfer of higfr lysine gene ('s) to spring wheats:

In order to utilize genes for high lysine concentration in grain protein, Nap Hal (PI 176217) its derivatives (75Y 41226, 75Y 51254) and Mahratta (CI 8500) were used. Bue two Nap Kal derivatives were received from Dr. V. Johnson,

University of Nebraska and their pedigree is given in Table 5- AH the four high lysine parents are extremely late and do not produc seey an ed when grow n fieli n d unde r environmentou r .

2 plantAmonP e th gs that flowere d producean d d about five gram seed, 50 plants from each cross were analyzed for their

grain protein and lysine (g/l6gîT). In the ?2 generation, there were several plants having range lysin th f 2.9 o en i e 5 - 3-35 (Table 5). The progeny of these plants were grown in field and selections were mad r desirablefo e , plant typyieldd an e . •Thes , selectionF e s were analyze r lysinefo d generaln I . , the lysine values were considerably lower, even in the control Kalyan Sona and TW-1 . This could be either due to the analytical error in lysine estimations or the sudden increase in temperature during grain filling. Most of these lines are rather late in flowering and maturity as they are derived from crosses having the high lysine parent of winter growth habit, or extremely late maturity. These were grown in P, and promising selections will be analyzed for lysine.

4• Other sources for high grain protein In spring wheats:

With the long range objective to investigate the genetic and physiological basis of high grain protein in different stocks, we are collecting and evaluating diverse high protein spring wheat stocks that can grow well under our environment. Eleven high grain protein spring wheat selections obtained from Dr. V.A. Johnson have been1 evaluate r graifo d n proteid nan yield. These stock e reportear s havo t d e 'minor1 gener fo s 83 5 hig he differenproteiar d an j t froe well-knowmth n high protein gene (s ) in Atlas 66 (Pro 1 and Pro 2 ) which are reporte e linkeb o t dd witn gen r vernalizatioVr hfo e n requirement located on the short arm of chromosome 5D • In observation nurseries during 1978-7 îd 979-80an 9 , mosf o t

these Hues had higher grain protein (Table 6). Some of these cultures can be useful donors for high protein genes in spring wheats, and have been crossed to TVM, TW-3 and other lines.

. 5 Flag leaf senescenc graid an en protein:

Increase in grain protein enhances the nitrogen requirements (milligrams of IS per gram of photosynthate) for cereal •7 estimates wa grain t I d . s tha poin$ 1 t t increas grain i e n 7 protein in wheat increases the nitrogen requirement by 6% . High protein wheat and rice genotypes are also reported to show high leaf proteinase activity and increased mobilization of N from foliage ' ' . Possibly, greater demand of N for seed growth hasten e breakdowth s f leao n f proteins, thereby initiating early observesenescencs wa t i soybean i d s a e n Early senescence of the leaves would reduce the supply of photosynthate r graifo s n developmen henced an t , reduce grain yield11.

Under our growing conditions, we have repeatedly observed early flag leaf senescence starting fromn i leap ti f high protein stocks, though we have not been able to collect quantitative data because of the enormous amount of -work involved in obtaining such information. Genetic approach to avoid photosynthate losses due to early senescence would be to select genotypes which either already have excessive 'source' compared TO the^'r 'sink size or have a greater capacity to 1 compensate for the loss of green area. If part of the leaves e shadear r removedo d , others increase their photosynthetic

84 rates and when the grains are removed the leaves reduce their photosynthesis-12' 13>14.

This phenomenon of compensation was studied in six different wheat genotypes, Shera, Moti, ÏÏ.P. 310, Sonalika, Mexi-Pa N.Pd an k . 876, grow n poti n s wit replications5 h t A . the time of anthesis, either all or the upper three leaf blades were cut in one or two tillers. The other tillers, with full complement of leaf blades served as controls-. Grain weight, grain numbe r spikepe r , IKW, grain nitroge r cend pe nan t straw nitrogen % were estimated. The amount of N per grain. was calculated.

Defoliation decreased grain weigh r spikepe e t th , reduction varying from 5 (Moti, Mexi-Pak and Sonalika) to 25 (Shera N.Pd , an Ü.P 0 . 31 876. r cen)pe . t 8) (Table d an 7 s

This decrease was due to reduced TKW, grain number per spike remainin e samegth s expecteda , . Grain JUfa increasel al n i d the cultivars, increase being more in those cultivars where grain weigh r spik pe TKd t an eW were reduce a greate o t d r extent. Nitrogen per grain basis also decreased. Tne data shows that removal of leaf blades, led to reduced supply of e grainsth o t s .N wel a C s Marginaa l l reductio n graii n n was weight per spike and ÎVT per grain^observed in three cultivars (Moti, Mexi-Pa Sonalika)d kan . f considerablo Thi s i s e interes suggestd an t s that these cultivars either hava e better capacity to compensate by increased assimilation in other plant parts for the loss of green, photosynthetic area of the leaf blades or they already have excessive 'source1 compared to the 'sink'. We feel that such experiments can help in the identification of yield limiting parameter n specifii s c high protein genotypes/mutants. Further, cultivars showing marginal reductio grain i n n yield following defoliation could provide better parent for crossing high protein mutants. 85 . 6 Programm f woro e k till 1982;

a) Test of advanced breeding materials derived from cross-treading, using high protein mutants and spontaneous stock as parents for their grain yield, protein quantity qualitd an , othed yan r agronomic characters (including disease resistance.'-.

b) Investigation of nitrogen incorporation into seed by using 15N fertilizer of high protein genotypes.

) c Investigatio f proteino n compositio structurd an n e of high protein lines.

ACKNOV/IEDGEMENTS: e gratefuWar e o Proft l . V-.A. Johnso r seedsnfo , staff of the IAEA laboratory for lysine analyses, Drs. S.B. Atal V.Ld an e . Chopr r varietafo a l testind gan Mr. K.N. Suseela r technicanfo l assistance.

REFERENCES

(1) Khan, A.H., Mahmud-ul Hasan, Productio f proteino n riéh wheat by mutation breeding. J. Agri. Res. (Punjab), .10 (1972), 230-232.

(2) Ifarahari , BhatiaP. , , O.E., Gopalakrishna, T. , Mitra, R., Mutation induction of protein variability n wheai riced . "Evaluatioan tIn , f Seeo n d Protein Alterations by Mutation Breeding" (STI-PUB-426), IAEA, Vienna (1976) 119-127.

(5) Bhatia, O.E., Desai, R.M., Suseelan, K.N., Attempts to -combine high yiel increased an d d grain protei n wheati n , In, "Seed Protein Improvement by Nuclear Techniques" (STI-PUB-479), IAEA, Vienna (1978) 51-57.

86 (4) Bhagwat, S.G., Bhatia, C.R., Gopalakrishna , JoshuaT. , , B.C., Mitra, R.K , .Narahari , PawarP. , , S.E, Thakare, R.G. Increasing protein production in cereals and grain legumes n "SeeI , d Protein Improvemen n Cereali t d an s Grain Legumes", Vol. II (STI-PUB-496), IAEA, Vienna (1979) 225-236.

(5) Johnson, V.A., Mattern, P.J., Kuhr, S.l., Genetic improvemen f wheao t t protein, ibid, 165-179.

(6) law, C.N., Young, O.P., Brown, J.W.S., Snape, J.W., Worland, A.J., The study of grain protein control in wheat using whole chromosome substitution lines, In "Seed Protein Improvement by Nuclear Techniques" (STI-PUB-479), IAEA, Vienna (1978), 483-502.

(7) Bhatia, C.R., Rabson, R., Bioenergetic considerations in cereal breeding for protein improvement, Science 194 (1976) 1418-1421.

(8) Hailing, M.J., Boland , WilsonG. , , J.H., Relation between acid proteinase activit redistributiod yan f nitrogeno n during grain developmen wheatn i t ,. HiysiolAusPI . J . . 3 (1976) 721-730.

(9) Perez, C.M., Cagampang, G.B., Esmana, B.V., Monserrate, R.U., Juliano, B.O., Protein metabolism in leavers and developing grains of rices differing in grain protein content, PI. Pnysiol. 5J. 0973) 537-542.

00) Mitra, R., Narahari, P., Gopalakrishna T., Bhatia, C.R., Intervarietal differences in some metabolic functions associated with protein accumulation in rice grains, Theor Appl& . . Genet 8 09764 . ) 145-151.

(11) Sinclair, T.R., de Wit, C.T., Analysis of the carbon and nitrogen limitations to soybean yield, Agron. J. 68 (1976) 319-324.

87 (12) Lupton, P.G.Ti.,, Ali, M.A.M., Studie n photosynthesio s s f wheato r ea , Anne th . n i appl. Biol (19667 _5 . ) 281-286.

(13) Evans, L.T., Wardlaw, I.P., Pischer, H.A., Wheat, n "CroI p ïhysiology, Some Case Histories" (ed. Evans, L.T.), Cambridge Univ. Press' (1975) 101-149.

(H) Lupton, P.G.H., Oliver, H.H., Murty, K.S., Euwali, K.N., The importance of sink/source balance in determining yielding capacit wheatn i y n "PiftI , h Intern. Wheat Genetics Symp. Vol 2 (ed . . Ramanujam)S . Delhw Ne i, (1979) 891-898,

TABL . 1 E GRAIN YIEL TW-P DO FIV- 3AT E LOCATIONS (kg/ha) 1978-79

Cultivars/Lecation Akela Washim Amravati Yectmal Tharsa General Mean

HD-2251 3134 2521 375& 4050 2638 3219

Kalyan Scna-C 3186 2854 2963 37C6 2359 3014

TW-3 2802 2448 2963 3239 2344 27fO

AKWI-13 1723 1741 1609 1«32 1797 1701

LSD/Vi « kg a 473.9 691.3 347.8 443-5 346.7

* Calculated from 16 varieties.

TABLE S. ANCILLARY DATA OP THE CUIfflVARS.

•— ——— ——————————_—_————————____^—_——

Cultivar «M Threshability Grain colour

HD-2251 57-64 99-109 35-42 Amber

Kalyan Sona 52-60 92-107 32-37 Araber

TW-3 47-59 07-101 46-49 Ey Amber

AKWI-13 64-82 110-115 40-49 M Amber

88 TABLE 3. CHAIN YIELD AND GRAIN PROTEIN AT IARI, NEW DEMI (1978-79)*

Rust reaction** Strain Yield in Grain qAa protein % Brown Yellew

W 2 34.65 13-1 VHS F TW 3 24.75 14.4 VMS F HD 2009 43.20 14.1 TR F ffL 711 32.40 13.1 MS F HD 2204 36.45 14. C TR F Sonalika 31.50 13-3 IS F leve$ 5 IS t Dla 8.22 CV 5.7B?&

* Medium fertilit d irrigatedyan . Plot size 3.6 . Rando8m m block design. ** Rust react ions are from the artificially innoculated plots with mixtur l racesal f o e .

lABIE 4. YIELD AND 1000 KERNEL WEIGHT OP PROMISING SELECTIONS.

Entry Pedigree Grain yield 1000 kernel wt. Kernel protein% 6/m2 __

1 7 Ï S TW-K x 1 327 26.6 15.0 2 KS (Check) 263 21.9 14.6

3 1*17™'i M "1 TA TTrvO^ SV rj 350 28.6 15.4

4 Xu * l Z KS en 370 26.0 15.9

5 TW-1 x KS ?7 321 27.2 16.3

6 TW-1 x KS F? 362 29.7 15-2 7 Tif-1 x Motl J 325 26.4 15.6

8 TW-1 x KS F7 341 27.4 15.8

9 TW-1 x KS F7 330 26.4 16.0 10 TW- Motx 1 1 l 346 25.9 15.5 11 TW-3 (Oieck) 263 29.2 14.1 12 CT-1 x HP 876 453 33.2 15.2

1SD 55« 29.5 1.7 C7 2.7 1.9

89 TABLE 5. GRAIN NITROGEN AND LYSINE IN SELECTED Fg PLANTS.

Frequency Cross/Parent Percent nitrogen g lysine/16 g N 1.75-1.90 2.2-2.5 2.75-3.1 1.8-2.0 2.15-2.35 2.55-2.85 2.95-3-35

TV/ 1 x Mahratta ) 3(0 4 1 3 7) (13)0 (0 (131 4 3 « ) 9 9 3 0 1 Mahratt S K x a 1 0 (10) 0(22) 24 (0) 21 (0) 6 3 5 NapHa S K x l 9 0 10 29 11 51234 x KS 5 39 6 0 (10) 2 (14) 39 9 0 2 4 1 4122S K X 6 3 C (10) 7 (2) 38 5

PercentN g lysine/16 g K 9 2. 1 W T 2.6 (1.8) Kalyan3 2. Sona (KS) 2.3 (1.8) 2 2. 75Y 51234 (NapHal/CI 13449) 3-7 2 2. 75Y 41226 (IlapHal/CI 13449) 3.7 Mahratta (CI 8500) 2.9 2.7 NapHal (PI 176217) 3-2 2.8

* Lysine value n parenthesii s s refeF o t r

TABLE 6. HIGH PROTEIH SPRING WHEAT FEOM HEBHASKA.

Grain protsin 1978 Yuma Ppdii?rp« Grain n Trombai protei % yn originan i Sourc% l e No. grown cropea^ree 1978-79 1979-80

40406 No. 66/Gallo ., 17.1 15.3 15.9 10421 Tob-CNO"S"/Tob-8156/1BB(l8M) 16.8 14.3 13.7 CM 5403 - 8RT - 1 PB r 4043n 0 m 17.7 15.9 15.8

10439 N II 17.7 15.5 15-4 10443 (CalAc-8156/CUO"S") Cal-Sar 16.8 CM-5756-7H-1PB 18.3 14.1

1045H 7 H 17.2 16.3 19.0

2 10484 B_JJ-CHO//CNO/IK64 -SB64 )EN- 16.6 15.1 C«' 5437-19H-8PB 17.7

40504 {CaVCC-815 x 6CNO»S " )CHO"S"-8156 18.9 3 1B - C M K 5533 - 4 19.1 17.2 n n 40515 17.0 4 16.2 15.6 4053 5534-3H-11P2M C " B " 18.0 16.9 16.6 40542 M H ff 17.7 17.3 17.3 KS 14.2 14.6 TW-3 15.3 14.1

90 TABLE 7. CUWIVARAL RESPONSE TO DEFOLIATION AT ANTHESIS.

Varieties/ Grain wt/spike (g) Grain number/ _„., " TB spike Percent „ defoliation 50 100 0 50 100 0 50 100

Shera 1.41.149 1.11 34.7 33.7 33.8 36.3 31.730.6 (77) (74) (87) (84) Moti 1.433 1.36 40.2 41.7 41.5 37.7 36.134.1 (io o) (95) (96) (90) I'exk Pa i 1.49 1.42 1-47 44.2 44.2 45^5 34.5 32.8 31.2 (95) (99) (95) (90) Sonalika 1.21.207 1.21 31.3 31.7 32-0 39.9 35.36.67 (94) (95) (89) (92) 0 U31 P 1.34 1,02 1.03 38.5 41.5 39.2 35-3 24.24.4 4 (76) (77) (69) (69)

6 N87 P 1.54 1.13 1.17 r . 36.35 3 7 43.2 34.6 35.1 (73) (76 || (81)

LSD at ^% level Varietal mean (V) 0.123 2.68 3.3Y Treatment mean (T) 0.31C NS 4.7£ VXT interaction 0.071 NS 1,94

Values in pay nthesis are relative to defoliation" control

TABL . 8 E CUI/TIVARAL RESPONS DEFOLIATIOO T E ANTHESIST A N .

Varieties/ Grai $ N n Stra5 wN? mg N/grain

« I 0 defoliation 50 100 C 5C 10« 56 10C

Shera 3.11 3-36 0.77 0.89 0.81 1• 13 1.04 1.C3 (106) (108) (116) (105) (92) (91) Moti 2.46 2.57 2.44 0.88 0.83 0.83 0 • 93 0.93 0.83 (104) (99) (94) (94) (100) (89) Hexk Pa i 2.77 2.81 2.86 0.72 0.94 0.98 0.96 0.92 0.89 (101) (1C3) (131) (136) (96) (93) Sonalika 2.87 3.03 3.01 1.03 1.06 1.12 1.15 1.08 1.10 (106) (105) (103) (109) (94) (96) UP-310 2.92 3.05 3-21 0. 77 1.03 1.05 1.03 0.74 0.78 (104) (110) (134) (136) (72) (76) NP 876 2.82 2.99 2.96 0. 85 0.89 0.82 1 .22 1.03 1.04 (106) (105) (105) (96) (84) (85) LSD at 5% level Varietal mean (V) 0.17 0.14 Treatment mea) (T n 0.24 0.19 VXT interaction NS 0.08

Values in parenthesis are relative to defoliation" control.

91 THE INCORPORATION OF MUTANT GENES BY WIDE CROSSES AND THEIR CONSEQUENCES FOR THE QUALITY COMPLEX IN HEXAPLOID WHEATS

K. NAGL Bundesanstal r Pflanzenbafü t Samenprüfungd uun , Vienna, Austria

Abstract

The incorporation of mutant genes by wide crosses and their consequences for the quality complex in hexaploid wheats.

A hexaploid spring wheat with high baking quality and two high yielding winter wheat (6n) varieties with rather low quality levels were used in crosses wit n inducea h d high protein sphaerococcoid mutant (^n) .line1 1 s deriving from the spring wheat cross have been tested over two years. Apart from a general transgressive enlargement of grain weight protein content was enhanced by 12,5% in average. Protein yields were improved in the range of 0 - 33^. The majority of lines exceed the limits for high baking quality. Different ratios betwee nn glute gluteno d nan nprotein s indicate additional nutritive improvement.

F,-progenie f winteo s r wheat crosses exhibit different segregation patterns with 50^ durum and 25% aestivum types in one cross but only 1% durum and 70% aestivum types in the other. The distribution of positive protein recombinants vary wite differenceth h f segregantso s . Genetic variability and even population means were increased for different protein traits. These wide crosses offer the possibility to increase the diversity of segregants in combination with protein improvement and thus it is a promissirig too n plantbreedini l r morfo e d efficienan g t selection.

Objectives for quantitative protein improvement

e breedinTh r nutritionafo g l qualit n wheai y n contrasi t barleo t t y is complicated by the fact that wheat is primarly used for a wide variety of human food product n for i sf whol o m e wheat products (for example breakfast cereals, chappaties and whole wheat bread) or milled to more refined product n whici se bra d gerth e hseperate an nar m d froe endospermth m . Therefore difference n nutritionai s l quality f mighsignificanco e b t e already by including or excluding by-products of the milling process so that wheat qualit s largeli y a yrelativ e conceps considerei d an t n i d relation to the particular use of the product. In addition, nutritional improvement had to be integrated into the requirements of cereal chemists for technological quality with the necessary processing characteristics

93 w materialora f n theio d rsan suitabilit d functionalitan y n producini y g n acceptabla e food wit e requirementan d th h f farmero s millerd an s r fo s economical yields. Best usage woul made b df hig o e h protein grain that possesses traditional food processing properties. n animaWheaa s a tl fee f increasino d g importance especiall r countriefo y s with wheat over- production should be defined to make clear if the term fodder wheat as a grain product means high yielding wheats, usefu s energla y source and/or wheat unsuitable for food processing and mainly low in protein and nutritional qualit r wheao y t with high nutritional quality standart bu d lacking some quality criteria for technological use. Economically successful breeding for this purpose will largely be dependent on the stimulation given by a premium for high nutritional quality equal to premium payin r higfo g h baking qualit n differeni y t countries.

Nutritional improvement of wheat for breadmaking in the sense of yeast leavened products had to be concentrated on endosperm proteins and should balance both nutritiona d technologicalan l requirement- to s gether with yield and quality interactions. Negative examples exist for several very high yielding winter wheat varieties being extensively used l Europeaial n n wheat breeding programme t acceptablno t bu s breadr fo e - making since their dough e stickmachineth o t s s when mixe t higa d h speed (l). Gluten proteins includin e gliadith g d gluteninan n fractions impart dough forming properties that differ from those of doughs made from any other cereal grains glutei t I .n formation rather thay distinctivan n e nutritive property that gives bread wheat its prominence in the diet. In hige th h f almoso lysin l al te source prolaminee sth s presen n highesi t t proportion but of lowest nutritional value are proportionally reduced rather sharply, but these protein components are evidently important to e propertiethus e f wheato s . Moreover, lysine conten f wheao t t prolamines is remarkable higher than in maize so reduction of this protein fraction would not have the same consequences for lysine enhancement in wheat. The n endosperno m protein e ric ar sn lysin i h e (about 't/E) while endosperm proteins are rather poor in lysine with about 2%. This enlarges difficulties to improve the nutritive value of wheat flour. Therefore location in the kerne f improveo l d protein-lysine leveln importana s i s t consideration since it will determine the degree to which the effects are transmissable to milled floure endosperth s .A m normally constitutes morf eo tha$ 8C n the wheat kerne y weightlb s unlikeli t i , y that high protein froy an m source as measured by whole kernel analysis would be confined only to the bran milling fraction knows i t n.I thaproteie th t n los n millino s g low-protein wheat s generalli s y greater thahigr fo nh protein wheat) (2 s d thae absencan th t f negativo e e correlation between protein contend an t lysine content in the protein was found at higher levels of protein in wheat (3)« Therefore the total enhancement of protein in the wheat endosperm or the whole grain if used in whole wheat products or for animal feedin s stili g n acceptablla e genetic rout n nutritionai e l improvement.

94 In addition to positive changes in the pathways of protein synthesis and protein composition, grain sizgraid an e n shap s wel a ekernes e l l confi- guratio e furthear n r attribute progressiva f o s e selection programme whic affecy ma h t protei d lysinan n e conten certaia o t n extent.

Improvement of N-efficiency

Increasing the protein concentration in the grain is not only of interest in the context of breeding for nutritional improvement alone but also from the point of view of a higher efficiency in nitrogen utilization with rising costs of nitrogen fertilizers. The breeding goal of high protein varietie s realizesi d whe e nitrogenth n concentration i n the grain s improve si absence th a reduction i f deo n graii n n yield. Thi s similai s a maximu o t r m yiel f graio d n protei unir pe nt aereat bu , thi s usualli s y obtained with varietie o havinsto a higg h yield potential and a low grain protein content (GPC %). But a low grain protein content is undersirable for nutritional purposes and the baking quality. Therefore breeder o improv t % whil y C tr seGP e maintainin yielde th g level and overcoming the negative genetic correlation between grain yield and GPC %. This negative correlation can partly be explained by bioenergetic reasons (compare o carbohydratet d s fro a certaim n amount f glucoso e only about hale quantitth f f proteio y n coul producee b d d by photosynthesis) e otheth n rO .han d yield increase f modero s n wheat varieties are mainly a result of breeding for increased harvest index and thereby the protein reservoir present in the vegetative tissue is reduced. A reduced amount of straw means at the same time that a smaller amount of N has to be redistributed to a larger biomass of grain thus it surprisint ino s g tha e tnegatival ar grai % nC GP yiely d correlatedan d . When merele differencear % y C baseGP harvesn n o dsi t index differences, variatio n proteii n a mor o t en e efficiencontendu t f graino o t ts i n utilization of nitrogen. Any breeding program in which the yield improvemen s realizei t y mattea chang dr y b dn i re distribution will thue sb confronte unles% C GP a dro s n y i additionapb d l fertilize applieds i r . Only whee efficiencth n underlyine th f o y g physiological phenomens ai improved n expecca increaso e t t on ,% withou t C i GP r e tfo haviny pa o gt in grain yield (higher absorption capacity of N from the soil, differences in activit e rooth t f o systey d translocatioan m n efficienc r nitroyfo - genous substances).

A very strong correlatio 0,85- = s bee r )ha ( nn reported betwee% C GP n d harvesan t index (HI) which support e ideth sa together witphysioe th h - logical considerations mentioned before that thes variableo tw t e no e sar merely statisticall t functionallbu y y related. This means tha larga t e part of the variation in grain protein is due to variation of the harvest index. The same may be true for grain yield which shows similar high negative correlations with grain protein content. If these parts appear to be large in comparison to the total genetic variation for the protein 95 trait, little real variatio resula s a nproteif o t n lefe geneb ty sma which can be used to increase grain protein content without yield reduction. To improve precision in comparing different genotypes on basis of GPC %, s suggestecorrectioa i wa te us o Kramey t b d n regressioe O C valurth y b e n coefficient b to a common harvest index of ^J>% to remove the improper genetic variatio a derive s a n d l geneeffecal f so t involvee th n i d distributio y mattedr f o rn (and thus protein) between grai d strawan n . On this basis, after correction the high protein variety Atlas 66 does not seem to be justified for its frequent use in breeding programs for high protein compared with other parental lines.

In this contex e suggestioth tn adjustea e s us madi ndo t e value based regressioe oth n n coefficien meaa o nt grai ncomparable yielth f o d e varietie r straino s n questioni s . Grain yield result e easiear s r available and oftern more exact than harvest index values and might give similar information e .justifieb Thi e strony th ma s n go d functional relationships between GPC % and grain yield on the one hand and grain yield and harvest e otherindeth n o .x Such adjusted values have bee comparisoe nth user fo d n f lysino e value n basio a commos f o s n protein contenNebraske th n i t a wheat breeding programm removo t e e lysine variability resulting from protein differences.

This complexity indicates that clear cut definitions are necessary in single case e tersth m eve r wheafo n t protein mutant before discussine th g breeding goa o avoilt d misunderstanding.

Breeding strategies and quality complex

e genetiTh c potentia r quantitativfo l e protein improvemen whean i t t might be exploited in different ways by mutation breeding methods. The duplicity resulting from the polyploid nature of wheat may in fact endow wheats with greater rather than less genetic potential for desired changes als n qualiti o y factors e rang.Th f possibilitieo e s whic provides i h y b d applying induced mutation n breedini s s reflectei g y differenb d t kindf o s breeding progress. Thes e reachinar e g from minor adjustment come th -n i s position of quality factors by immediate utilization of micro mutants to drastical changes of a wheat genotype possibly useful as a cross parent in hybridization programmes for protein improvement. Confirmation of positive breeding value of mutant characteristics in such macro mutants is required by incorporation of mutant genes into different genetic backgrounds. Great attention should possibilite als th paie ob o t d f o y obtaining transgressive effect n quantitativeli s y inherited characters.

Suc hradicaa l mutatio f socalleo n d major gene s induceswa a y b d chemical mutagen e (DEScommerciath n )i l spring durum variety Adud an r restored the primitive phenotype of a sphaerococcum wheat. Even if such changes cannot be regarded as progressive as a whole mutant, they represent a multiple gene source for different positive breeding or mutation

96 characters probably useful in cross breeding which were already summarized at the Baden-Meeting (5)- The most important ones are short stiff straw, high protein content and spherical grain. On search of new protein genes a hybridization program has been initiated several years ago involving cross parent wheao tw tf o sspecie s (tetra d hexaploidan - o )t hybridize genetically diverse germ plas d producan m wida e e rangf o e physica d chemicalan l propertie kernee th f lo s constituent o improvt s e nutritional qualito stud t e interaction d th y an y f differeno s t yield and quality components. Gene transfer between durum and aestivum wheats have taken place mainl y incorporatiob y f duruo n r emmeo m r genes into aestivum wheats to improve disease and insect resistance and vice versa to incorporate dwarfing genes and genes to improve fertility and yield in durum wheats. Little is known about gene transfer for direct changes of quality components between both species perhaps mainly because they are traditionally used for different food purposes. Durum wheats are generally distinguished by certain quality characteristics from aestivum wheats which have superiority in paste production but nevertheless cheaper indigenous wheats especially strong types are often blended with durum. s alsIha to reported tha a durut m type wheat with high breadmaking quality s producewa y crossinb d a durug m wheat variety wit a commoh n bread wheat variety, backcrossin breae th do t gwhea t variet r threfo y e generations (6). Mediterraneae th n I n area Near EasU.S.S.R.d an t , durum wheats have been grow manr fo ny year r breadmakinfo s g purposes n commo.I n wheat flous i r superio l othe al o tha t rf r o tproductioe specieth r fo s f leaveneo n d bread.

A short characterization of the requirements for technological quality in Austri s givei a o elucidatt n e experimental dat n tablo a . Whea2 e t baking qualit s officialli y y evaluate e "Wertzahlth y b d Z (wort"W h figure) introduced by H. Fuchs 1953« This figure is based on the experience that gluten amoun d glutean t n qualit n compensatca y r eacfo e h other (within limits) in contributing to baking quality. The formula for the WZ is (2 x wet gluten content -f 3 x Qo). Qo being the "structural swelling volume a certai f "o n amoun glutef ) accordino tg 1 ( n Berlineo t g d an r Koopman. To estimate the proteolytic destruction of gluten Qo/Q-,.., the >>u proteolytic swelling volume (Q-zO) is added. Wheat reaching or exceeding a WZ of 118 is accepted as high quality wheat in the case that its gluten content is at least 28$ and its Qo at least 12* ml (e.g. 32# gluten content Z norW o givmQ e l th e118)m an8 1 d. Proteolytic destructio f gluteo n n should not exceed 35^- The sedimentation test developed t.y Zeleny (?) is another useful method for estimating the strength of wheat. The sedimentation value is also influenced both by the quantity and the quality of the gluten and hence can be used as an index of bread baking strength. This tess show ha s tusefulnes it n n earlsi y generation wheat breeding a microtechni word an k c have been developed saving enough whear fo t planting. Dividing the sedimentation value by the percentage of protein gives a "specific sedimentation value" that is an estimation of gluten 97 quality alone e rati.Th o gluten content/protein content give rouga s h indication about gluten or dough forming and non gluten protein components interesting from technologica wels la s nutritionala l standpoints wa t I . pointed out by Hansel (8) that the intervariety correlation between grain yiel proteid an d n conten0,78**- = r s highe( t) wa r than between grain yiel d glutean d n conten = •* r 0,59*( t ) which suggests tha shoult i t e b d soraewheateasier to breed for higher gluten content than protein content without decreasing yield .A furthe r negative correlation between gluten/ protein ratio (G/P) and Qo (r = - 0,^2*) indicates that with increasing G/P the gluten quality tends to decrease.

Volume weigh d kernean t l guida usee weigh b s o floua dn t e ca t r yield. Kernel shap d uniformitan e kernef o y mose lth t siz e importanar e t factors influencing test weight e othe.Th r important e densitfactoe th th s f i ro y grain. Abov certaia e n leve 3 kg/hi(7 l tese )th t weigh relativels ha t y little influence on flour milling yield. In several cases kernel weight was superior to test weight in predicting milling yield. Kernel weight ia sfunctio kernef o n l sizkerned ean l density. Large dense wheat kernels normally have a higher ratio of endosperm to non-endosperm than do smaller less dense kernel d thusan s coul more db e reliable th r fo e test weight.

e generaTh l characteristic f duruo s m wheats shoul summerizee b d d because one of the cross parents in this experiment derived from this specie d despitan s s differenit e t sphaerococcoid mutant habit carries typical durum genes whic e manifestear h y purb d e durum segregante th n i s offspring. Protein d e durumstarcsan th e somewhaf ar o sh t different from those of common wheat. The physical dough characteristics of durum wheats range from medium strong to very weak; even at very high protein levels they never approac e characteristicth h f strono s g aestivurr. wheats. Durum doughs do not exhibit the degree of elasticity found in the stronger bread wheats whicw swellinlo he refeth g o t rvolum d sedimentatioan e n value and the high degree of proteolytic gluten destruction. The majority of durums are amber in grain colour and contain a high concentration of yellow carotenoid pigments which are about twice as much in the endosperm of durums than in bread wheat. This feature has long been an attribute of paste products made from durum s opposea s d those from other wheats. Durum wheats generally have large kernels which are longer than common wheat; thousand-kernel weight is higher on average and so is hectoliter weight. e sphaerococcoiTh d mutant use n thii d s experimen a drasti s i t c exception with regar o kernet d l shape (spherical) kernel weigh d voluman t e weight due to the small somerwheat shrivelled grains.

Discussio f experimentao n l data

Spring wheat

The tetraploid sphaerococcoid mutan s primarilwa t y progenitoa use s a d r r higfo h protein conten n crossei t s with aestivum wheats firse th tn I . 98 breeding cycle, a spring wheat was used as the recipient variety distinguished frol otheal m r Austrian aestivum spring wheats release y lighb d t grain colour and awned spikes to have certain markers for this breeding material. The tetraploid cross parent high in protein content but poor in grain yield, grain weight and with negative baking characteristics was combined wit e higth h h baking qualit d mediuan y m protei d graian n n yield level of the hexaploid spring wheat. Selection pressure was directed only to aestivum types with improved protein content without deleterious effect n bakino s g quality maintainin r improvino g g grain e yielth t a d same time. Earlier results of this hybridization programme are published proceedinge ith n e researcth f o s h co-ordination meetin t Badea g n (5)- Selected experimental lines were tested over two years on a dry climate location favourabl o exprest e e qualitth s y criteria. Thes line1 1 e s have been compared with the cross parents and the two most distributed spring wheats in Austria. The Swedish variety Weibulls Svenno with high baking Germae qualitth d n an y variety Mephisto ,a hig h yielding one t les,bu s suitabl comparison r i baking fo e2 tabed e e date givean .th Th ar an1 ln o n with the aestivum parent (100/e). Grain weights of all lines except one show transgressive improvement of 8% in average in the range of 4 - 173>. Grain e firsyieldth tf o s five line e improvesar r maintainedo d , yields othee oth f r line e slightlar s y reduced. Protein conten s generalli t y improved in all lines with an anverage of 12,5% in the range of 8 - 18% and protei r graipe n n show transgressive improvemen o l t case al e n du si t grain weight and protein enhancement. In technological quality, only 3 lines (2351, 2006, 2279) do not reach the limits for high baking qualitqualite th f o yy evaluation system mentioned before. Transgressive positive changes in worth figure (WZ) and sedimentation values have taken place with few exceptions despite the low quality values of the sphaerococcoid cross parent. Volume weight positive th e als n ar si o e range of the aestivum varieties. But there are greater differences between lines with lowest and highest wet gluten content (34,1% - 45,1% = J>2% rel.) than between lines with lowest and highest protein content (14,15e - 15,5515 = 1056 rel.). This range is also expressed in the differences of gluten/protein (G/P) ratio which amounts about 26%. Due to the selection pressur n boto e h yiel d proteinan d o negativ,n e correlation was calculated between these traits for the experimental lines. A high significant negative correlatio 0,97*- = r )( n howeve s founwa r d between these trait r crosfo s s parent d varietiesan s t thera tendenc .Bu s i e o t y a negative correlation between wet gluten content and grain yield. A negative correlation was confirmed between G/P-ratio and Qo (r = - 0,31) which indicates that a low G/P-ratio resulting from protein enhancement without proportional increas t glutewe f no e would affect both gluten qualit d nutritionaan y l quality positively f interest migho I . e b t o t t select sucw G/P-genotypelo h n whici s h changes have been obtainen i d limits of the requirements for high baking quality such as line N^ 2353 and 2321 for more detailed investigation of the nutritive quality complex.

99 Even if final conclusions could not be drawn at the moment because of the restricted material (about 50 other lines from this cross are still investigates)an e shorth d t testing time, ther e indicationar e s thae th t drastic sphaerococcoid mutant could successfull usee b yprotein i d n improvement of hexaploid spring wheats.

Winter wheats

a secon n I d breeding cycl o Austriatw e n winter aestivurr, wheats with high yield potentia t rathebu l w levello r n technologicai s l quality have been use n widi d e crosses wite sphaerococcoith h d mutan o improvt t e grain protein. Wintef higheo r rfa wheaty economicab e ar s l importance than spring wheats in our country like in other countries in and outside of Europe.

Neuho n awnea 1 f d variety especially adaptey climatdr o t d e conditions is early ripening with good winterhardiness but insufficient lodging resistance. Probstdorfer Gigant awnlesB and suitable for growing over wider climatic diffentiated region,medium late ripenin s gooha gd lodging resistance and winterhardiness. Both variétés are low in gluten content t reachencd no e limitan o th h d e r hig sfo h baking quality.

e selectioTh f segreganto n t onlno y s restricteswa aestivuo t d m types like in the spring wheat cross because beside intermediate types pure durum segregants are of interest to develop winter durum wheats with sufficient winterhardiness. Winter durum e highlwheatb y yma s evaluated in countries with durum growing but with incinsistent yields of the spring crop. Temperature stress could be the main limiting environmental facto r growinfo r wintea g r cro n thesi p e countries. Winter durum wheats will also be of certain significance in triticale breeding to improve winterhardines f hexaploio s d winter triticale. This breeding material grown under rather low temperatures during the winter 1978/79 which caused frost damag f differeno e t wintee degreth n i re crops have been the precondition o select s better fo t r winterhardiness.

The frequency of useful segregants belonging to different classes of wheat types (durum, intermediate aestivum s quit)i e differene th n i t progenies of aestivum wheat crosses. About half of the total F,-plants deriving from the cross with cv. Probstd. Gigant and grown as lines in F are durum types preliminary classified by grain and plant characteristics. About 25& belong to each class of intermediate and aestivum types. A family of 1^ F -plants (N- 653) the most promissing for selection shows a segregation % dururatik2 f mo J>7% intermediat d 2~\%an e aestivum types. The remaining F,-plants are distributed in the ratio of 60% durum 10S& intermediate and ~5Q% aestivum. In all cases the durums belong to the prevailing class. This will affec totae th t l selection progres somn i s e y witwa h regar o specifit d c characteristic e differenth f o s t wheat types suc s larga h e kernel siz f duruo ed evean m n intermediate typer o s differences in yield structure.

100 In protei e totanth contenlf o F.-plant % k$ t s excee proteie th d n range of the aestivum cross parent Probstd. Gigant. The highest percentages of positively changed segregants in protein content exceeding the range of cv. Probstd. Gigant belong to intermediate types with 6?/° followd by k^% of aestivum and J>k% of durum types. If the progenies of F family 653 and the remaining plants of other progenies are classified in the some manner it turns out that durum and intermediate types of family 653 with &k resp. 75^ segregants and aestivum types of the remaining progenies mose th t e promissinar wit% 65 h g classe r proteifo s n selectio n thii n s generation and cross.

e traiIth f t protein/grai n thii st pu classificatio s i n n system only protein/100g Giganf &o %0 9, t - plant 1 range 7, e foun0 th sf ar o en i d grain compared with 36/6 of total F,-plants. Best improvement is reached for durum and intermediate types in the progenies of family 653 with &2% arri 52# in the range of 7,1 - 10,0 g protein/1000 grain. Other classes do not look very promissing for selection progress on this trait. Improve- ment in these both classes results from considerable enlargement of grain weight together with the best response to protein enhancement. Protein amount per spike taken as indicator for protein yield might not be very reliabl earln i e y generation widf o s e crosses. Therefore t resultno e sar include e tablesth n i d. Preliminary data indicate only little improvement in this trait at that stage (Table 3).

Selection progress could be predicted or achieved not only by increasing the genetic variability alone but also by maintaining or elevating the population mean at the same time. This is the case for different n differentraiti d an s t classe f segregantso r wheao s t types respectively. The general population mean of protein content and protein/1000 grain in the cross with cv. Probstd. Gigant is increased by 15% and 10Ï« respectively. Highest value r thesfo s e trait e reachear s d with duru d interan m - mediate types. There are indications for improvement in protein per spike especially in aestivum plants of the socalled remaining progenies together with a rather low mean for the plant height (Table 5)-

e profilTh f segreganteo F,-generatioe th n i s secone th f do n cross with cv. Neuhof 1 is quite different from that with cv. Probstd. Gigant. Aestivum types are the largest fraction with 702>, durum types the smallest one with 1% the intermediate types take the position between both classes with 2J>%. The differences in the composition of both cross progenies probabely result froe differenth m t genetic constitutions of the recipient varieties. Cv. Probstd. Gigant has a rather complicated pedigree including four varieties compared with cv. Neuhof 1 deriving from a single cross.

The distribution of high protein recombinants in positive direction is not as pronounced as in the cross with cv. Probstd. Gigant because the cross parent Neuho originall1 f a highe s ha yr protein level. This wile lb evident comparing the population means of protein content and protein per 101 e aestivugraith f o n m cros sprogeniee parenth d an t s that shows exace th t Same protein level for the progenies of both crosses but only slight increase compare Neuhoo t d . Highes1 f t value e reachee intermear sth n i d - diate types that hav a largee r portio f segreganto n n highei s r protein classes (Table 4 and 5)- In general the values for protein per spike are highe thesn i r e cross progenies.

e utilizatioTh f thio n s drastic protein mutan n widi t e crosses with high yielding winter aestivum wheats offers the possibility to increase the diversity of segregants in combination with the improvement of the protein complex. Thi sbasimora e mighth r e fo se b tefficien t selection i n different direction r nutritionafo s l improvemen d producan t e propertieus t s of wheat. Thersouna s ei d hop confiro et positie th m . vG aspec. S f o t Stephens statement that interspecific crosses are one of the most promissing (but also most frustrating) tool of plant breeding.

Research plan for wheat protein improvement

Spring wheats

Developed experimental lines of aestivum spring wheats improved in protein by incorporation of mutant genes from a sphaerococcoid (A-n) cross parent shoul e testeb d n ploi d t trial t severaa s l locationo t s confirm selection progress in yield and protein under different environmental conditions. Material could als e distributeob adaptibilite th r fo d y test in other regions. Younger breeding material will be included in this test series.

Interactions between different genotype d environmentan s s should clarify the expression and stability of protein genes in new lines.

Analytical analyses will be carried out for grain protein and lysine together with some investigation f technologicao s l qualit r specififo y c use purposes. This seems important to select genotypes which are well balanced in nutritional and technological quality. Analyses of protein and eventually of lysine in whole grain and flour would be necessary to estimat protein/lysine th f i e e comple s totalli x r partlo y y transmitted e endospermtth o e bes.Th t selected lines coul usee b dn crosse i d s with other high protei r higo n h yielding gene source o accumulatt s e useful genes.

Winter wheats

The selection of improved single plants or homogenous lines of progenies deriving from crosses with two high yielding winter wheats (6n) hige anth dh protein sphaerococcoid mutant Ctn sprinf )o g habit wile lb continued. This involves durum, intermediat d aestivuan e m types which should be analyzed with respect to positive changes in nutritional and technological quality n singl.I e cases breeding material changer o d 102 unchange n harvesi d t index coul e analyzeb d r N-conten fo de stra th wn i t to study the N-distribution pattern in different genotypes. Yield tests of breeding material will be started as soon as possible and the comparison with foreign high protein varieties will give possibilitth e o t y estimate protein producitivity e optima.Th l utilizatio f differeno n t wheat types (durum, intermediate aestivum) segregating in the progenies of such wide crosses is intended to develop new and improved recombinants.

References

( 1 ) Rimpau, J., Niemayer, U., Röbbelen, G., Protein analysis of "non-baking" high yielding varieties of wheat. Seed protein improvement by nuclear techniques (Proc. Meeting Baden 1977) IAEA, Vienna (1978) 533-5^2.

(2) Farrand, E.A., Hivton, J.J.C., Study of relationship between wheat protein contents of two U.K. varieties and derived flour protein contents of varying extraction rates. I. Studies on an experimental commercia la laboratormil d an l y Bubler mill 56-66 . Studies.II y -b hand dissectio f individuao n l grains, Cereal Chem 1 (197*.5 0 66-7^.

) Johnson(3 , V.A., Mattern, P.J., "Protein improvemen wheaf o t t breeding", Improvin Nutriene th g t Qualit Cerealf o y , AID, II s Washington D.C. (1976)

(4) Kramer, Th., Environmental and genetic variation for protein content in winter wheat (Triticum aestivum L.). Euphytic , 197a928 Vol. 209-218.

) Nagl(5 ,n . induce-A K , d tetraploid sphaerococcoid semidwarf mutans a t a useful cross-parent for stem height reduction and protein improvement in hexaploid wheat. "Seed protein improvement by nuclear techniques (Proc. Meeting Baden 1977) IAEA, Vienna (19?8) 69-78.

(6) Pomeranz, Y., Composition and funcionality of wheat-flour components. Wheat chemistry and technology 2 nd. ed. 1971 Y. Pomeranz 585.

) Zeleny(7 T Criteri. L , wheaf o a t quality. Wheat chemistr technologd an y y Pomeran. Y 197. 1 ed . . 19 z nd 2

) Hansel(8 , Intercultivara,H. l correlations between yiel d qualitan d y components in winter wheat. Annual Wheat Newsletter Vol. 18, 1972, 18-19.

103 Table 1 Protein and yield data of selected spring wheat lines tested in plot trials (one location, 2 years average 1978/79)

Grain yield Protein Protein Protein 1000 grain Nr. dt/ha rel.# content rel.Se 1000 grain rel.Sé yield rel.2> weight rel.% 6 kg/ha (g)

Sphaerococcoid mutant (4n) 16,7 60 •18,1 •138 4,78 108 302,3 55 26,4 77 Aestivum strain (spring whea 6n.- t ) 41,7 100 13,1 100 4,43 100 544,9 100 34,2 100

Weibulls Svenno 48,7 117 13,1 100 5,16 116 661,0 121 39,2 115 Mephisto 51,4 123 12,7 97 4,98 112 652,6 120 40,0 117

2351 47,3 113 15,3 -117 '6,08 137 722,4 133 40,0 117 2353 46,0 110 14,9 114 5,30 120 684,3 126 35,6 104 2280 41,6 100 15,0 115 5,^3 123 619,6 114 36,1 106 2261 42,5 102 14,1 108 5,30 120 598,5 110 37,5 110 2007 43,0 103 14,1 108 5,00 113 603,7 111 35,5 104

2210 39,0 94 15,5 118 5,62 127 604,5 111 36,7 107 2006 40,4 97 14,5 111 5,53 125 585,0 107 38,0 111 2278 39,3 96 15,1 115 5,48 124 592,3 109 36,2 106 2279 37,9 91 14,7 112 5,38 121 555,8 102 36,4 106 2277 38,8 93 14,1 108 5,10 115 543,8 100 36,2 106 2321 38,5 92 14,7 112 4,94 112 563,7 103 33,7 99 Table 2 Technological quality v-alues of selected spring wheat lines (1979)

Protein Wet Wet gluten Swelling Proteolytic Worth Sediment . Yellow hi content gluten Protei% n volume ml destruction figure Test pigment weight Nr. i.dry subst. % Qo/Q30 t gluteowe f n (2 x wet gluten ml m* • « i p • p kg % % + 3 x Qo) y substdr , »

Sphaerococcoid mutant (4n) 21,0 52,8 2,51 3/1 67 115 33 5,38 69,9 Aestivum strain (spring wheat - 6n) 15,8 36,0 2,25 16/12 25 120 40 3,29 77,6

Weibulls Svenno 17,0 39,8 2,34 20/15 25 140 40 3,11 78,1 Mephisto 15,7 36,0 2,29 13/7 46 111 65 3,71 77,2

2351 18,1 41,2 2,28 11/6 45 115 54 3,44 76,8 2353 17,4 35,7 2,05 20/15 25 131 48 3,40 78,1 2280 16,2 37,5 2,31 20/14 30 135 49 3,62 77,2 2261 16,7 40,1 2,40 18/13 28 134 55 3,26 78,4 2007 16,0 38,1 2,38 18/1 4 22 130 52 4,28 75,7

2210 18,6 45,1 2,42 15/11 27 135 60 3,78 77,7 2006 16, 't 41,6 2,54 13/10 23 122 4o 4,23 74,9 2278 17,3 42,1 2,43 21/15 29 147 59 3,78 77,8 2279 16,5 40,4 2,45 16/10 38 129 36 3,45 78,4 2277 16,2 38,8 2,40 20/14 30 138 53 3,80 77,6 2321 16,9 34,1 2,02 19/13 32 125 52 3,66 78,4 Table 3 Distributio d classificatioan n f segreganto n n Fi-generatioi s a cros f o ns cv. Probstd. Gigant (winter wheatSpaerococcoix ) 6n , d mutant (spring whea) 4n t

. Probstdcv . Total Progenie e besth tf 3 o sfamil 65 - N° y Remaining progenies Trait Gigant Fi-plants Durum Intermediate Aestivum Durum Intermediate Aestivum _/ M- -/ -/ _/ _/

Protein conten% t 12,1 - 13,0 11 ------13,1 - 14,0 67 16 6 7 29 40 -- - 14,1 - 15,0 18 25 12 9 29 46 33 29 15,1 - 16,0 4 14 18 9 14 11 50 6 o 10,1 - 17,0 - 25 23 33 23 3 17 59 o\ 17,1 - 18,0 - 14 23 30 5 - - 6 18,1 - 19,0 - 5 15 9 - - - - 19,1 - 20,0 - 1 3 3 - - - -

Protein/1000 grainsg 3,1 - 4,0 - - - - - 3 - 6 0 5, 4, - 1 7 12 3 - 29 23 17 50 0 6, 5, - 1 56 34 9 30 56 40 33 38 6,1 - 7,0 29 18 6 18 10 23 33 6 7.1 - 8,0 4 21 29 43 5 11 17 - 0 9, 8, - 1 k 9 29 9 - - - - 9,1 -10,0 _ 6 24 - - - - - Table Distributio d classificatioan n f segreganto n s in F,-generatio a cros f o ns cv. Neuhof 1 (winter wheat 6n) x Sphaerococcoid mutant (spring wheat

cv. Total Trait Neuhof 1 F,-plants Durum Intermed. Aestivum Aest.sphaeroc.

Protein conten% t 12, 13,- 1 0 5 - - 10 - 0 1 13, 14,- 1 0 8 - - 16 5 3 4 14, 15,- 1 0 24 25 4 37 21 3 2 15, 16,- 1 0 11 25 11 6 21 1 2 16, 17,- 1 0 30 38 48 20 32 17,1 - 18,0 3 17 12 22 10 18 18,1 - 19,0 5 - 15 - 3 19, 20,- 1 0 - - - - -

Protein/1000 grainsg 0 4, 3, - 1 - - - - - 0 5, 4, - 1 3 - - 8 - 5,1 - 6,0 31 31 25 4 4o 39 6 4 0 7, 6, - 1 33 13 30 25 50 3 2 0 8, 7, - 1 28 62 51 23 11 0 9, 8, - 1 5 - 15 4 - 9,1 -10,0 - - - - -

107 fable 5 Population means of different traits in F, generations derived from crosses winter aestivum wheat x tetraploid sphaerococcoid mutant

Protein Protein Protein Plant content % 1000 graig n spike g height cm

cv. Probstd. Gigant 13,6 5,88 0,18 77,6

Family 653

Durum 16,5 7,89 0,17 84,5 Intermediate 16, ^ 6,76 0,17 85,0

Aestivum 14,9 5,45 0,14 75, H

Remaining progenies

Durum 14,3 5,60 0,15 82,3 Intermediate 15,6 6,16 0,16 77,9 Aestivum 15,8 5,82 0,20 77,0

Total 15,6 6,48 0,17

. Neuhocv 1 f 15,0 6,39 0,23 81,7

Durum 16,4 6,78 0,17 83,2 Intermediate 16,7 7,29 0,20 76,0 Aestivum 14,8 6,42 0,20 78,1* Aestivum (sphaeroc.) 15,8 6,12 0,19 69,2 Total 15,6 6,47 0,20

108 BREEDING VARIETIES WITH INCREASED PROTEIN CONTEN RICETN I 1

C. GANGADHARAN, S.N. RATHO . PANDE,K 2

ABSTRACT

Efforts mad o identifet y high protein cultivars from germplasm collections in India and abroad and their utilization to evolve productive varieties with higher protein majoe contenth r fo tric e ecosystem f sIndio e ar a and summarised and discussed. Genetical/anatomical studies related to protein content are considered. The relevance projece th f breeo o t d varieties with increased protein conten s stressedti .

A project for breeding high yielding rice varieties with increased protein content, sanctioned and financed by the Indian Council of Agricultural Research, New Delhi, has been in operation from mid-July 1973 to end of February 1976 Centrat a , l Rice Research Institute, Cuttacks it n O . expiry, the project was incorporated as a regular item of the breeding programme of the Institute.

e earlierth n I - years e efforth , s directetwa o t d identify breeding lines/mutants with increased protein content in agronomically well managed and under controlled irrigation in transplanted medium duration rice varieties for the monsoon season. As rice ecology is very diverse and as rice is

cultivated under uplands, medium lands and low lands, it was felt necessar wideo t ye scop th no encompas t e s these important rice growing conditions projece mako th y impact f an e,s i i t t

Contribution from Quality Breeding Laboratory, Central Rice Research Institute, 1 Cuttack-753006, Orissa, India. 2 Respectively Scientist S-2Genetice th , n S-li l s, S- Divisio f CRRIno , Cuttack-753006, Orissa, India. 109 l alla . Hence, screenin studd an gf cultivars/hybri o y r o d mutant lines suited to these conditions have been initiated. Salient results achieved in this effort are given below.

wore Th k don C.R.R.t a e I comes under four categories: ) Screening(1 Breeding) (2 ; ) Genetical(3 ;) Anatomical(4 d an , . Results under each heae summarisear d discussedd an d , with the accent,-, on breeding.Micro-Kjeldahl method was used for nitrogen estimatiorsand protein content was computed with a facto basist f 5.9o rwe 5n o .

1. Screening

worle Th d rice germplas bees ha m n screene proteir fo d n content Internationae mainlth y yb l Rice Research Institute, Manila, Philippines. Central Ric^ Re-cearch Institute also has done screenin ricf o g e varieties fros nationait m l germplasm collection and has identified accessions/cultivars with a crude protein content of over 10/i in brown rice. Table 1 lists such high protein varieties. This work has indicated the gcnetical variability presently available in rice cultivars and ready for utilisation. All the same, there is need to intensify and complete screening of available cultivars in the genetic stocks.

Perhap earliese th s t repor protein to n contenf o t brown rice is by Sen (1916) from India and Fraps (1916) in the U.S thed .an y reported ove % proteir8 brown i n n rice. A compilation by Juliano (1966) shows that the variability protein i n conten reportes a t y workerb d s aroun worle th d d till 1966 was 5.5 to 14.3% for brown rice and 4.5 to 14.3% for milled rice.

110 Table 1: List of high protein cultivars

Variety Protein % Variety Protein%

1. Pirurutong 13.90 Ptb.2. 18 2 11.40

Gaise. 2 n Mochi 13.50 19. Ptb.30 11.2 0 3. Ptb.23 12.60 . Shall20 a 11.20 4. Ptb.29 12.50 21. Ptb.25 11.10

5. ARC.6638 12.45 22. ARC.5995 10.88 Chaliat. 6 a 12.40 R.57. 23 5 10.80 . Ghunghu7 n Jhigan 12.10 Ch.6. 24 3 10.75

Omri. 8 9 t.3 12.00 Earl. 26 y Kolipi.70 10.70 Mnp.6. 9 4 12.00 Rdr.. 26 2 10.70 10. Crythroceros 11.95 27. R2 Nungi 10.60 11. Chow Sung 11.90 28. Ptb.10 10.60 12. Hr.22 11.85 29. Bcp.6 10.60 13. Mnp.99 11.80 30. Dular 10.50 14. Hr.19 11.60 31. Adt.27 10.27 Gare. 15 m 11.60 32. R.10 Chatri 10.20

AC.80. 6 1 6 11.50 33. Zinia 63 10.10 17. ARß.6600 11.48 Mtu.. 34 9 10.10

Juliano, Ign^cio, Pangeniban and Perez ( 1968) had reported prote-in content above 14% in five varieties (Table 2) from an evaluation of the International Rice Research Institute

germplasm.

Tabl : 2 Hige h protein rices after Juliano et.al (1968).

Varieties Protei browf o n% n rice Rikuto Norin.20 14.5 Omrit.39 14.7 Chok-Jye-Bi-Chal 15.1 Gros a- 2 15.1 Sénto 16.7

111 Recently Coffman and Juliano (1979) had said that from the extensive screening of 7760 varieties they had identified six varieties with oveprotei% 14 r n severai n l yield tests and these have been utilised in the breeding programme for protein enhancement. They also observed that those varieties were j aponicas which matured early, with low dry matter productio .werd an ne susceptibl o severat e l important diseases and insects, presumably under Philippines condition. This is a relevant poin practican ti l breeding» Whee temperatth n e aponicaj s have bee ntropica e grownh n ni l Philippinese th , varieties have unaergon a drastic change in their entire physiology and thereby in the different growth processes and biological reactions and this environmental disturbance or shock (like reouction in duration, susceptibility to new species or races or biotypes of diseases and pests) can contribut proteio -c e n enrichment. Such drastic changes have been know o exisnt t with referenc productivito t e y also (Gangadharan, 1978).

There have been very encouraging reports fron a m evaluatio e newlth f yo n acquired germplasm froe Northth m - Eastern state f Indiaso . Fro preliminara m y screeninf o g 118 varieties/ 71 were found to have a protein content range of 11—16 % which is remarkably high (Anonymous 1973). A subsequent study of morphological variants from their collections has also reported 11-13 %e culti,/ar proteies i t n i ns (Srivastava and Nanda, 1977).

Sampat Seshd an h u (1957) aft^r comparing protein content of a few genotypes suggested that the morphological character of "long glume" might be correlated with high protein content of grain. But subsequent work has failed to confirm this association. In fact, Coffman and Juliano (197g) have stressed the absence of a marker either in the seedling or vegetative stage for high protein genotypes. This is reasonable as they quoting

Ferez et»al.1973 thae differenceth t protein i s n contens twa

112 mainly due to difference in the efficiency of translocation e grai th f foliao o nt afterN r anthesis rather thao nt differences in total plant N. But unlike in maize, with opaque or floury endosperm, there is no identifying feature in the rice grain for high levels of protein. Thus laborious laboratory screening still remains the only method available.

So there seems to be natural variability available for this important character of profound biological significance in rice, awaiting exploitatio rice-staple th n ni e developing countries, including India.

2. Breeding

Time and adjoin (Rao/ 1970, Rao et.al/ 1911, Gangadharan et. al, 1974, 1975, Gangadharan and Misra, 1977, Rao, 1978) it s beeha n stressed thae uplandth t s pron droughw o lyint elo d g an t water stagnant lands subjec o submersiot n from flash floodr o s constitute the major heavy precipitations/hurdles for a breakthrough in kharif (May-December) or monsoon dependent rice culture in India. Efforts have been mad breeo t e d varieties with increased protein for these twin vulnerable situations as well as medium

lands under controlled irrigation. Both recombination

d mutatioan n breeding1 have been» of < e madus e Significant results obtaine thin i d s regar summarisee ar d d below:

Uplands;

projece Ith n o evolvt e varieties with drought tolerance, mutation improvo breedint f o mad s ee gwa productiviteus n yi known drought tolerant varieties/hybrid lines as an alternate approach. Known drought tolerant varieties like talnakanda 41,' 'ch.45,"Mtu.l7,' fa. 113-322,. bK ' 2 'had been treated with either physica chemicar o l l mutagen screened an s productivitr fo d y under moisture stress conditions. Productive drought tolerant mutants had been identified in the variety Vltu. 17* and the

hybrid line'CR.113-32, proteie Th ' n conten mutante th f to s and the parents is given in the Table-3.

113 Table 3: Crude protein content in mutants and parents of varieties for uplands. Designation of Protein Designation of Protein mutent/parent mutant/parent»

CRM. 4-1 7.4 CRM7 .4- 8.4

CRM. 4-2 8.3 CRM. 4 -8 7.6

CRM3 4- . 8.1 CRM9 .4- 7.6

CRM4 - 4 . 8.8 CRM. 4-10 7.1

CRM5 .4- 7.7 CRM. 4-11 6.9

CRM. 4-6 9.4 CRM4-12 8.5

Mtu.17 (Parent) 8.1

CRM. 13-3037 6.3 CRM. 13-3511 6.6

CRM. 13-3052 7.5 CRMJ3-3512 7.0

CRM. 13-3055 8.3 CRM. 13-3532 8.9

CRM. 13 -30 58 5.7 CRM. 13-3535 8.5

CRM. 13-3061 6.3 CRM. 13-3 53 7 6.6

CRM. 13 -30 62 6.1 CRM. 13-3538 9.3

CRM. 13-3241 7.9 CRM. 13-3558 8.6

CRM. 113-32 (Parent) 9.6

From the latter/ a very esrly mutant/ induced by gamma irradiation (30 Kr)/ designated CRM.13-3241 maturing in seventy days when direct seeded had been identified which was found suitable as a catch crop after the drought or flash flood during kharif or as a regular crop in rabi with limited water resource n relai r yo s cropping. This mutan s testetwa d under x nitrogesi n level replicatea n i s d randomised block desigo t n evaluat s responsit e n yiel i proteid e an d n conten o incremeatt s

of nitroge e nresult th dore e d presentear an ss n Tabli d , 4 e

114 Table 4: Yield, protein content ana protein yield (kg/ha) of CRM. 13-3241 undex si r nitrogen levels.

. leveN l Mean grain Mean protein Protein yield yield (kg/h a) conten) (% t (kq/h a)

0 N 616 5.98 41.0 20 N 920 6.20 57.0 40 N 1325 6.44 85.4 60 N 2078 7.36 152.9

80 N 2308 7.32 180.5 100 N 2226 9.60 214.2 C.D. at 5% 164 0.92 C.D. at 1% 226 1.86

might I e seeb t n froabove th m e Table, that bote hth grain yiel proteid an d n content were significan unde% 1 t ta r different nitrogen levels. The mean grain yield and protein content increased steadily with increasing nitrogen doses. The yield declined after 80 kg/ha level whereas the protein content increased. In fact protein content was significantly superio leveN g k l 0 rove immediats 10 onl rit t a y e lower dose s A Krame . N rg k (197.5 0 o8 f ) explaine wheatn i d , even when added nitrogen faile o increast d e yield contributet i , e th o t d enhancement of protein content in this very early rice mutant.

Medium lands;

o higtw h e proteiTh n varieties Piruruton Gaised an g n Mochi had been hybridize lina d e an ddesignate d CRHP.8 with stable protein conten s selectedtwa . Despit consistens eit t high protein content, the culture yielw glutinoud lo an dd sha s endosperm. Hence back cross programm o improvs taket wa ep u n e these characters using semi dwarf hybrid Padma as the recurrent parent and CRHP.8 as the other. Seven cultures (bRHP.lr?) were selected frosegregatine th m g generation thif so s back cross CCRHP.' x 8' 'Padma. f thesO ' e selections, 'CRHP.l'recorded higher yield than

115 'Padma. e averagTh 1 e protein content, yielproteid an d n yield/hectare is give Tabln i n (Ghos. 5 e Sampath& h , 1975).

Table 5: Grain yield/ average protein and protein yield/ha f CRHo P cultures.

Culture P.arentage Average grain Average protein Protein yield (kg/ha) content (%) yield (kq/ha)

CRHP.l (CRHP x Padma .8 x ) 3807 10.07 383 Padma«

2 -00- 3327 8.36 278

3 -do- 3162 8.97 283

4 -do- 3316 8.78 292

5 -do- 3316 8.20 272

6 -do- 2771 9.90 274

7 -do- 3299 9.90 326

8 Gaisen Kochi x Pirurutong 3170 11.55 366

Padma T.141 x T(N)1 2926 7.27 205

IR.8 Peta x DGWG 4000 7.37 294

'CRHP .1 'consistentlI t yieldeou yÙ 2/ dd ha Paöm d an a more protein. Grain yiel 'CRHP.l*whef o d n compared with'lR.8 was lesproducet sbu d more protein (kg/ha) than IR.8. This was also 7-10 dayd 20-2an s 5 days earlie duration i r n than 'Padma'and 'IR.8* respectivel e dat presentee th ar ad an y n i d the Table 6. raoi: o e for Drotei proteid an n n yield. Culture Flowering Grain yield Protein Protein yield duration (days) (kq/ha) conten) t(% (kg/ha.

CRHP.l 85 4850 10.2 494 Padma 92 4250 7.8 351 IR.8 108 5575 7.1 396

Besides, a higher protein, medium duration variety Mtu.9 (Anonymous, 1969) was irradiated with gamma rays from a

116 Csourc^ O selectiond an e s were mad improvo t e e yiel pland an d t habit. Viable semi—dwarf mutafcts with yields comparable to that of the parent were obtained and their protein content evaluated. Results are presented in Table 7.

Table 7: Protein content of Mtu.9 and its mutants.

Mutant/Parent Protein% CRM.18-2 9.6 " -4 8.5 6 8. 5 - "

8 8. -6

-8 9.0

" -9 8.0 -16:: 9.4 4 9. Mtu.9 (Parent)

t mighI e notetb d tha mi-c'.v.'^r- s t f rnutcmts with

comparable and even higher protein content tl>an the parent could be identified and most of these mutants also possessed higher yield potential because of the nonlodging habit and pleiotropic effect conferred by semi-dwarfism on them through induced mutation (Gangadharan et.al, 1975).

Possibly there is no urgency to bre- d for varieties with increased protei mediun ni m lands thae amenablar t o goot e d agronomy, especially with control of irrigation. It is well established that increased leve f nitrogeo l n enhances protein content in most varieties and there is an obvious improvement of protein leve n varietiei l s cultivate weln i d l drained an d well fertilized medium lands.

It has been shown above with the early mutant that protein content coul e enhancedb y 100mucb s a ds a %h ti.rough nitrogen application stude Th y Gangadhara.yb n et.al (1978) al'so confirm enhancemene th s proteif to n content through fertilizer management. Thusincreasint a /m ai whe e nw g rice

117 production through the application of higher fertilizer inputs in lands with controlled irrigatio kharie th f nfo f o crod an p the entire rabi (December-April) generally, n thera s i e automatic elevation in protein yield of crops harvested from such lands.

Low lands;

In the project to evolve varieties suited to low lands with long duration (ove days0 grai15 rd an ) n quality, attempt

was also made to evaluate and identify promising lines in F. generatio x crossesi f o ns having higher protein content. This s importani t because submerge t lenw landno lo d o d sthemselve s o efficient t agronom genetid an y c superiorit desirably an r yfo e trait under such conditions will be an advantage. Hence, 309 cultures belongin x crossesi o t gs have been analyser fo d protein content (4. 4o 13.6%t werd )an e see havo nt e considerable variation in protein content. As the level of protein content in these cultures were generall w (4.lo y 5s 7%)o i t % ,8 arbitrarily considered as high in varieties for submerged low a land numbed an s f selectionro s with more tha protei% 9 n n are listed in Table 8.

Table 8s Protein content of some of the low land progenies and parents.

Name of Cross/ e th No f .o No. of high Range of Parent. progenies protein protein analysed. progenies content screened) (% .

. 1 CR.15 x 1Haldiamagur a 141 32 5.3-13.6 2. Haladiamagura x Jagannath 97 12 6.0-10.4

. Panka3 x JTagannatj h 13 1 6.3- 9„0

. CR.154 Mahsurx 1 i 4 6.67 8. -

5. CR.151 x CR.1014 38 4.89 7. -

6. CR.140 x Jagannath 16 4.4-8.1

7. Haldiamagura - 5.8

8. CR.10£U4 - 7.3 . Panka9 j - 6.4 10«, Jagannath - 6.9 11. CR.151 — 6.2

118 In a breeding trial, eighteen fixed lines from P_ generation of five crosses with their five parents were grown in a completely randomised block with two replications in low land theid an s r yield, protein percent, protein yield, protein per seekerned an d l weight were studied e analysiTh . f so varianc r graifo e n yield/plot, protein yield/unit area, protein/seed value, protein percent and kernel weight is given in Table 9. Both parents and selections differed significantly.

Table 9: Analysis of variance for different characters»

Mean scruares Source Grain yield Protein Protein/ Prot- Kernel yield n / ei weightseed , unit value. % arâa.

Entries 22 1.67** 4342.31** 0.0009** 1.44** 3850.8** ** Cultivars 4 0.29* 1810.96** 0.001038 0.99** 3601.45** ** Fixed cultures 17 1.78** 4669.96** 0.001178 0.61** 3447.39** Varieties vs fixed cultures. 1 5.40** 8889.19** 0.000251 17.10**11707.32** Error 23 0.053 31.3 0.000052 0.0621 17.76

CV 6.96 3.01 5.82 4.46 0.3232 CD 0.48 11.57 0.01207 0.423 1.206

SEM 0.007 3.95 0.00412 0.1438 0.4122 Heritability (broad sense) ß. S3 0.93 0.98 0.89 0.917 0.99 leve% 1 significan* probabilitf d * lo an d % an 5 * t a t y respectively.

The values of correlation coefficient for yield, protein per se^.d, proteito/ n percentage and 10 kernel weight are given in Table./ It is seen that the negative correlation coefficient was not significant for the protein content vith the grain yield/plot, protein yield/plot, protein/seed value and 10 kernel weight. This shows the nonsignificant reduction in grain yield with increase in protein content which is advantageous,

119 labi : Correlatio10 é n among different characters.

Protein j Protein/ Protein 10 kernel yield/plot. seed value. % weight.

Yield/plot 0.1831 -0.1086 -0.170 -0.0423 Protein yield/plot - -0.0711 -0.0597 -0.1340 Protein/ seed value - - -0.0414 -0.7558

Protein percentage - - - -0.5670

A study of 25 medium duration hybrid lines and 5 popular high yielding varietie r responsfo s n yiel i eproteid an d n content to increasing do.^es of nitrogen (0, 50, 100, 150 and 200 kg N/ha) under transplanted condition showed negative correlation between flowering duration and protein content as well es grain yield and protein content (Gangadharan et.al, 1978). The analysis of

variance is given in Table 11. Govindaswamy et.al (1973) had also reporteu significant negative correlation between these characters.

Tabl : Analysi11 e f varianco s r proteifo e n conrenl and grain yield.

Sourcef o s df Protein content Grain yield variation. MS F MS F

Block 1 1.4 4.77 9139961.7 7.478 Fertilize) r(F 4 97.5 32.828**16157145.5 13.022*

Erro) r(a 4 0.3 1222234.8 - Varietie) s(v 29 13.2 0.080 66024750.8 2.628**

Interaction (VxF) 116 1.4 O.OC8 7543116.2 0.300 Erro) r(b 145 164.6 25117083.8 -

Total : 299

% leveSignifican1 - probabilitf d * o l* an d % 5 an t * a t y respectively.

120 In the above experiment it was seen that protein content wahybrio stw hig r dN/hg fo hk CR.115-44-7 r line0 a fo t a s 2 (10,95%) and CR.129-11 (9.92%). CR.115-44-72 was remarkable in tha t registereti proteia d n conten f 13.4to 15.2d 8an 8 at 50 and 100 kg N/ha respectively.

Besides through induced mutation/ effor mads twa o t e improv introducee th e d Malayasian variety 'Mahsuri' which was very popula kharia s a r f variet mosn i y t rice growing state Inain i smand an ay productive mutants were isolated. Some of these had been evaluated for protein content as well e resultanth d presentee ar s d below (Table 12).

Table 12: Crude protein content (/.) o£ Mahsuri and its mutants.

Culture Protein Culture Protein content. content

Mahsuri (Parent) 6.9 CRM. 10-4616- 7.0

CRM. 10-3619 6.3 CRM. 10-4622 6.3

CRM. 10-3 622 6o4 CRM. 10-4623 7.4

CRM.l 0-3629 7.6 CRM. 10-4626 6.0

CRM. 10-3630 6.3 CRM. 10-4660 8.3

CRM. 10-4611 7.3 CRh. 10-4 671 6.6

CRM. 10-4615 8.3 CRM. 10-4676 7.9

Results given above show that there is variability for this biologically important character generated in the hybrids and it is upto breeders to properly exploit it. Results from mutation breeding indicate that variability towards negative and positive sides in comparison with the parent variety is induced for protein content in rice and through careful screening/ mutants v/ith more protein than the parents can be selected. One added advantage of mutation breedin s thaproteii e g th t n enhancemene b n ca t achieved without disturbing the desirable group of traits

121 parene th f o ts achieve i variet t i yielr s fo da y d (Gangadharan et.al, 1974, 1975).

From the foregone, it would be clear that despite protein conten mose th t t f variablo bein e on g characterf eo s governed by many physical, chemical ana biochemical factors,

there is possibility to genetically upgrade the varieties that are to be grown in different rice ecosystems of monsoon India. As the ecosystems drastically differ the aim of a uniform leve proteif o l e feasiblnb content thesno r fo ey tema situations s suggestei t i o S d. thauplandr fo t s (wita h fertility statu f 30-4so e b sbovd N/hag 0k y an ema % ) 9 considere higs proteir a d fo h n content correspondine Th . g figur mediur fo e m lands(wit fertilita h y leve g N/haovef k o l 0 r5 ) may be 10% and above and for low lends (with the fertility status betweerm aboved 20-3an N/hag % 0.k 8 e )b

Secondly it mgist be recognised that protein content per se will remain a matter of little concern for the cultivator, till price policies make production of high protein rices economically attractive. Thus, yield remains the primary concer e sacrificeb wild t an nno l e sakdth e mucr fo h of protein. Also it is known that more energy is required for productioe th proteinf no s thar sugarfo n starcr so therd an h e will be proportion-ate reduction in yield with increase in protein (Bhatia and Rabson, 1976). A workable compromise is to emphasise protei basie nth yield/hacf o criteri e on s ,a a for selection in rice breeding programmes so that the varieties evolved will hove a desirable minimum of protein with yield. Available information shows that selection for higher protein is possible in later generations after stabilizing yiel earlien i d r generations. (Ghosh et.al, 1976, Anonymous 1976)

122 It is seen that protein content shows a linear response to added nitrogen upto 200 kg N/ha, where as the yield declines after 80-100 kg N/ha level. This association is really beneficia e havw e optio s th ea l o manipulatnt e fertility level economir fo s c yield returns combined with higher protein. Through fertilizer application alone protein content was raised by 2-4.5% in rice. (Anonymous,1972).

Another significant findin s tharesponse wa gtth o t e protein conten higd t an undeh w fertilitrlo y conditions swa genetically distinct (Gangadharan et.al, 1978 selectiond an ) s te viablob e shoul made db e unde e appropriatrth e levelf o s fertility.

Perhaps this linear response of protein content to increasing dose nitrogef so beneficias uniqus it ni r fo e l effects. Increased level f fertility,thougo s h helpfuo t l boost production knowe ,ar o aggravatnt e pes diseasd an t e and incidences/are reported to have broken out in epidemic form in intensive cultivation areas in India/ Indonesia, Philippines etc. or say the application of fertilisers has a negative or balancing effec yieln o t d through these pesdiseasd an t e damages. But in the case of protein content no such deleterious side effec seens i t . Besides, reports show that varieties cultivated under high fertility have not only higher yield and protein content but also higher hulling percent (Ghosh et.al, 1971). Increase in protein content is seen to improve milling and reduce breakage in rice varieties ( Juliano, 1972).

Interestingly enough, higher levels of nitrogen are also seen to facilitate a deeper dispersion of protein bodies in the.endosperm helpin reduco t g e losse proteif o s n froe th m kernel during millin polishingd an g . Thu coult si e saidb d

123 that the technology of high yielding varieties with the stress on fertilizers consumption also serves as the technology for boosting protein yiel f ric o dn India i e »

Consumer premiua y finr spa fo me qualit r scenteo y d rices, because of their limited production and demand. Such G laissez-fair practicae b e t "policno r higy fo lma y h protein f theio m rai productio e ricesth o s react a , wids s a hi n a e segment of the population to ensure better nutrition, especially weanine oth f g children rice-stapln i , e societies. Hence, the succes f productioo s popularisatiod an n f higo n h protein rices will depend on the price support extended by the concerned Governments, State or Central, in India.

. Genetica3 l

The interest in genetics of protein content of rice develope de sixtie onlth f yo n Indi i s towardd d en an a e th s there were reports by early seventies. The available information is summarised below:

A study of the segregating population from several crosses during the last few years showeu that protein content t havdino de significant association either with plant height r earo - bearing negativtillerd ha t sbu e association with single plant yield. (Anonymus, 1972, Chutima et.al, 1979).

recena Ther s ewa t repor f positivto e nonsignificant correlation between yield and protein content among the induced mutants of

o rictw e varieties, whic d negativha h e correlation between these characters (Monyo et.al, 1977) which madauthore th e s claim that these mutants were breakers of negative correlations. (Monyo, et.al, 1979).

Govindaswamy et.al (1974) from a study of the cultures otripla f e cross 'Padma 'x ('Geisenmochi' x 'Pirurutong') obtained low heritability for protein content but higher genetic

advance than for plant height, possibly because of the

124 inheritanc f thio e s character being governe y additivdb e genes as reported by Ghosh et.al, 1971, Mohanty and Reddy, 1972 and Swaminathan, et.al, 1971.

Hi1lerisi ambers et.al (1973) found significant genetic var-iation for protein content in a study of £ S1X cross-es involving four parents. It was also found that high protein content was significantly correlated with light kernels; early headin shord Chamuran d g tan staturo aSe (1979 . e ) have a-lso reported associatio higf no h protein content with earlines ricn i s e varieties.

The three essential amino acids viz. Js lysine, threonine and methionine were estimate e higth hn i proteid n culture, 'CRHP.81. Ther negativs ewa e correlation between protein conten lysind an t e contentpercene on r tFo . increase th n i e grain protein, there was a reduction of about 0.3 to 0.4% of lysine content. But the availability of lysine increased markedly with the incre-ase of protein. (Anonymous,1973).

Inheritanc f proteio e n conten s studiee crostwa th sn i d 'CRHP.8' x 'Ratna'o Inoiviaual seeds were used for the estimation of protein conten n hybridsti , back-croc„ F sf o sc-ed d an s population. Reciprocal differences were observed for protein content in the F.. crossed seed, but the mean value in F„ generation was more or less the same. A dose effect was recognised in prote.in values of hybrids in a study of F' reciprocal crosse bacd an sk crosses e patterTh _ .F f o n segregation suggested that protein conten possibls i t y governed

by a few major genes with minor genes influencing its level.

(-.Anonymous, 1974).

IRRI used a modified diallele breeding scheme to concentrate genetic factors for protein content along with resistances to major disease pestsd an s . They found that higher protein content is less associated with lower grain yield in the progeny lines

125 tha n theii n r parentsuc4 1 hd linean s s with higher yield an d protein had been identified (Anonymous, 1975)«

All evaluation of the varieties for protein contents showed that environmental factors greatly influenced the protein content. Genetic studies showed that low values for brown rice protein (BRP)percentage, protei seer npe d 0 (P/S10 d )an kernel weight were dominant ovehighe rth s valuese Th . heritability estimates for P/S were relatively higher than percentageP BR thos r efo effective b . y ma Selectio P eBR r nfo frogeneratio- F, me donb n earlie y i et selectioma nbu s rP/ f no generations. Both ERP and P/S were negatively correlated with grain yield and resistance to bacterial blight (Anonymous, 1976).

Genetic studie n proteio s n conten £,ponicaJ f to s betwee w proteilo higa nd an nh variety' showed thae broath t d generation, F d an sens sF , e 0.46 s F„ heritabilit wa n I .~ F e th f yo ther negativs wa e e correlation betwee proteie th n n content and other agronomic traits. Early, short stature pood an dr yielding segregants tendeproteie b o t d n rich (Higashd an i Kushibuchi, 1976).

From a study of 23 rice varieties with protein content varying from 10-14%, it was founa that low protein exhibited partial dominance over high inheritance proteith d an n s wa e controlle majow fe a r geney db s besides minor ones. Though maternal influence was noted in the first generation, the F„

mean values were mor r leseo s similar s suggestei t I . d thet selectio proteir fo n n content shoule th e carrien b di t ou o advanced generation (Ghosh, et al, 1976).

o ët.aGu l (1978) from inheritance studie pooteif o s n content, heading date and other characters found in F~, the dominance of low protein over high protein and it was negatively correlated v/ith plant height, panicle weight and number, grain yield and 1000 grain weight but was positively correlated 126 with early maturity. Heritàbilit proteir fo y n content ranged from 0.3 0.56o 1t .

Recently Ratho, et.al 6.97pressn i 9 ) mad fula e l 7 dialle7x l studo t crosnature t th y se genf so e e action governing protein content. The varieties were 'Ambemohar' « Nira- ) Z ('1 ,'Wase-aikokuLx 9,'Tkm.6', 'Ratna1, 'Padmad an ' 'Tainan. Mut.587- 3 . Variou-4' s genetic componentd an s

combining ability effects were estimc.ted. It was seen that bote additivth h dominancd an e e component f genetio s c variance were significant. There was asymmetry in the distribution of dominant and recessi /e alleles and the dominant alleles were in excess showing over dominance. The number of effective factor pair x wits estimatee si wa sth h e b o t d sominanc higf o e h protein oveproteiw lo r n contente Th . parents ('L x Z1)- Nira1/ 'Tkm.6' and 'Wase-aikoku1 had more numbers of dominant genes and 'Tainan.3 Mut.587 -4 ' and 'Ratna1 had mor f recessivo e e gene proteir fo s n content e GCATh ., SCA and Reciprocal mean squares were significant. Parents like 1 Wase-aikoku d 'Padmaan 1 1 were gooc general co/nbinerse Th . crosses ('LxZ)- Nira x Tainan.3 Mut.587-4* and 'Ambemohar x (LxZ)- Nira1 were the good specific combiners. Most of the crosses involving the best general combiner/ 'Wase-aikoku1 had intermediate positiv effectsA SC e .

In a trec-ent study of eighteen fixed lines from F,- 1 - x si e th f o generations of five/crosses (listed in Table 8 ) along with their parents growrandomizea n i n d block wito replicationtw h s 'PanJ Gangadharad an e n (under publication) observed significant variatio proteir fo n n content amon e fixeth g d line parentsd an s . The pattern of transgressive segregation was towards the negative siae as there was no line superior in protein content to the parents, whereas a few of them had lower protein. This coul because existence db th f o e negativf o e e correlation between the protein content end yield. Correlation

127 (r = 0.90) between protein per seed value and kernel size was found positive and significant. The magnitude of heritsbility for both protein percent and protein per seed were al most of the same order. The broad sense heritability was above 90% charactere th l al r sfo studied.

It may thus be seen that knowledge on this important character of nutrition of rice grain is scrappy and needs to be intensified to thoroughly understand the mechanism of gene actio interactiod an n o facilitatt n e faster incorporatiof no

this character in desired varieties. Such intensive study requires faster methods of protein estimation than the traditional micro- kjeldahl techinique. Gangaaharan et.al. (1979) have foun Near-Infra-ree th d d reflectance system

fasteproteir fo r n estimatio millef no d wheat fractions and so could be of use in rice also. Fifteen or more different nuclear or related methods are available for N estimation which are faster, nondestractive and requiring

only a minimum of sample (Niemann , 1979-A) .According to him, kjeldahl metho estimatioN f o d n gives slightly lower value doet t i accoun no s a sr nitrattfo nitritd A an e . N e review of these techniques is under publication (Neimann, 1979-B)

4. Anatomical

Anatomical studies to understand the distribution of protein e bodieendosperth n i s m were undertaken:

Histochemical studies have shown thae t th mos f o t waxy endosperm types have thicker aleurone layer/ deeper distributio protief o n n bodies insid e endosperth e a d an m higher percentage of protein. Among the hybrid derivatives, 'CRHP.81 had the thickest aleurone layer (66.8 « on the side and 35 p. on the ventral side) and deeper distribution of protein bodies (Anonymous, 197l).

Disttrbution of protein bodies was found to be restricte peripherao t dp l portio sub-aleuronf no e layen i r 128 the varieties, 'IR.81, 'Jaya't 'Ratna1 anu'Vijaya1 (Anonymous/ 1972}. But the seeds raised under high rates of nitrogen (above 100 kg N/ha) present uniform distribution of protein bodies inside the endosperm. The reticulum of protein was observed to spread deep towards the centre of the endosperm in the samples obtained from heavily fertilised plots.

comparativA polished an e w studra d f o yric f theso e e high yielding varieties showed that the loss of protein during millin less samplegn wa si s grown under high fertility. This wa sdeepee possiblth o rt distributioe ydu proteif no n bodies insic- endospere th e m after heavy nitrogenous fertilizer application. Hence, higher rateN applicatio f o s onlt no y o ensurnd e more protein but also give a better distribution pattern, makin graie th g n nutritionally superior even milleds aftei t ri .

The increase in the protein content of rice results in an increase in the number of protein bodies in the endosperm. e distributioTh f thesno e protein bodie mors i s e unifor deeped an m r in high protein than low protein samples. The difference in protein conten browf t o mille d an n d rice tend decreaso st s a e the protein content increases. The high protein samples had a thicker aleurone layer. The protein content and the thickness of aleuronc laye e qivear r n belo 'Tabln wi 3 (Govindaswam1 e a an y Ghoch, 1974).

Saral d Reddan a y (1979) found fro 0 studa m9 f o y 4 grei12 loca nd an lshup e mutants thaproteie th t n bodien i s many mutants were distributed deep inside the endosperm while e locaith n l varietie e distributioth s s rathenwa r uniform throughou endosperme th t . s see wa Als milling% nt i 12 otna t a t , protein retention varied from 79-96%, irrespectiv proteie th f o en percentage.

Thus , it may be seen that the pattern of protein distribution is a varietal character that can be considerably

129 Table 13: Protein content, aleurone layer thickness etc f somo e 'CRHP1 lines and varieties.

Protein Aleurone layer thicknes) (u s Distribution Variety content (%) Dorsal Ventral Mean of protein bodies in endosperm.

CRHP.l 10.05 38.0 17.2 27.60 Peripheral/ scattered»

" .2 7.78 32.0 I5e7 23.85 -do— " 3 7.97 38.5 18.5 28.50 -do-

4 7.97 33.2 20.5 26.85 -do-

5 8.05 38.2 22.5 30.35 -do-

6 9.45 40.5 24.6 32.55 -do-

7 10.94 48.0 29.5 43.75 Deeper, Uniform

8 11.72 66.8 34.5 50.65 -do-

Chow Sung 9.98 41.8 24.7 33.25 -do-

Santo 10.78 45.0 22.7 33.85 -do- Mn p6. 9 11.05 45.0 25.0 35.00 -do-

Omri9 t.3 10.87 43.7 23.8 33.75 -do-

Krishna 9.95 30.5 16,4 23.45 -do-

GEB4 2 . 8.28 25.0 15.4 20.20 Peripheral, Scattered. T. 141 9.05 30.5 18.5 24.50 -do-

Ratna 7.78 26.5 13.8 20.15 -do- Padma 8.05 27.2 14.0 20.60 -do- Jay? 7.41 25.0 15.1 20U60 -do-

IR.8 7.45 27.5 16.2 21J85 -do-

influenced through manipulation of nitrogenous fertilizer application. In the programme for breeding varieties with increased protein in rice , deeper distribution of protein bodieendospere th importann n a i s s i m t featur e reckoneb o t e d with as it provides a genetic guarantee against heavy loss of available protein through milling ana polishing.

130 Relevanc f increasino e g protei n ricn i Indian i e ;

Though therrecora s i ef ric o d e protein from 197n i 6 India s significancit , charactea s a e profounf ro d biological value, both for the present anc future generations (Gangadharan et.al, 1978) was not realised till the late sixties; in fact till IRRI starter projec'a breedinr fo t g rices with increased protein in the Philippines (Beachell £t.al, 1972). The screenin f germplasgo s confirmeha m e availabilitdth f higo y h protein cultivars, breeding has established the possibility of evolving protein- rich varieties with the support of genetics» High protein rices have higher hulling and milling percentages

with less grcin breakage ancn havca .e excellent cooking quality Juliano, attributes (Anonymous, 1969,/1972). vera Rics y ha efavourabl e and balanced amino acid profile for its protein and this balance is not altérée, by increasing the protein percentage o pointstw r o w prolaini ,e lo bbecaus on y s hig d it an nh f eo *• glutelin moitiés, when compared to other cereal proteins (Nelson, 1979) maie Th . n increaso t tas s i k quantite eth y whic s fairli h y easier than improving cjualit f proteio y n in rice s alreadA . y pointed out, proteie n on contene th s i t niLJor character that has shown a linear response to increasing level f fertiliseo s r withou deleteriouy tan s side effecr to efficien n nitrogei t n utilization. Nutritional studien o s weaning children have shown that the increased protein was

retained anci used by the children (Anonymous, 1973&)» So, all that is required is a resolve at appropriate centres to e thar futurse ou t e varieties hav minimua e f 8-9o m r/a protei n themi n , when they reac e consumerth h .

It was felt that any increase in protein content in rice would be of benefit to millions (Anonymous, 197^a)< According to Bollich (1976 y improvemenan ) proteie th n i tn conten f ricto e through breeding would be valuable in view of the low nutritional status of peoples rece-iving 10% or more of their calories from rice. Br€ssan Eliad an i s (1979) have estimated majo 5 2 tha n ri t 131 rice consuming area r 1970fo s , particularl mose th t n i ypopulated , rice provided more thacaloriee o thirdth tw n f mord so an se than hal proteinf o f . e accepEvew f ni t tha r capittpe a incomes alone cannot truly reflect the extent of hunger and malnutrition depending on complex interaction of various factors of production,

distribution, socio-political system e footh sds a etc d an . procurement styles vory in rural and urban ar>..as, there is nothing losd everythinan t f higo e h us proteig e gaineth n i d rices ,ths A e. spiralling global inflation continue o shrint s k the purchasing power of common man every year, there is a short of nutritional insurance that high protein rices provido t e e consumeth r free:.

e 'chilOth n n Indiai d 1,Gopalan (1979) observed "Children ...... constitut totar ou e lf nearl o populatio % 40 y n ...... children below 5 years of age number nearly 100 million ...... It is the present state of health and

nutrition of this crucial age group that will largely determin qualite th e calibrd r natioan you 200n f i no e 0 A.D. an o higdo pricIndir n to beyond" fo hs , o produc i et aso f I .e and. popularise protein- rich ric..-s as expeditiously as possible,

ACKNOWLEDGES SNT

. H.KDr .o t Pandee Thankdu e , ar sDirector , Central Rice Research Institute, Cuttack and Mr. M.J.Balakrishna Rao, Head, Genetics Division, CRRI, Cuttac havinr fo k g accorded facilities.

RESEARCH PLAN

1. Evaluate the available breeding lines/mutants of rice for protein content for upland, medium

land and low lands typical in the monsoon tract f Indiao s .

132 . 2 Generate genetic variability through recombination breeding and induced mutation.

3« Evolve rice varieties monsooe suiteth o t dn rice ecologies with high protein yield per hectare,

. 4 Evaluatio qualite th f proteif no o y n with special reference to lysine, tryptophan and methionine.

5. Find out the relationship of common breakfast ric'e preparation (beaten rice, puffed rice, popped rice etc.) with the bio-chemical qualitie e ricth e f endospermo s .

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133 18. GANGADHARAN, c, MISRA, R.N. Breeding for monsoon rices In India, (Proc SABRAd .3r O Cong. Feb.11, Canberra, Australia) (1977). .5 1 9.GANGADHARAN KONZAK, ,C , C.F BRUINSMAD .AN , B.L. Possible genetic differences in the wheat kernel protein gradient (Proc. Seed Protein Improvement in cereals and grain legumes, Neuherberg, Sept, 1978) IAEA, Vienna (1979), 432. 20. GANGADHARAN, C., MiSRA, R.N. AND SREEDHARAN, P.N. Use of radiation and chemical mutagen ricn si e breeding Nuclea. ,J r Agricd .an Biology (±1975) 4(l)»8. 21. GANGADHARAN SREEDHARAN, . ,C , P.N. MISRD ,AN A R.N. Inductiof no useful mutation ricen si , (Proc radiationf o . e Sympus n d .o san radio isotope studien si planf so t productivity, Pantnagar, India) (1974), 13.

22. GANGADHARANAT, C., RATHO, S.N, PATNAIK, A, AND KHAN, S.K. Towards more protei ricesn ni RISL ,I O (1978), XXVI181i ) (3 I. 23. GHOSH, A.K., ANQ SAMPATH, S. Breeding rice varieties with more protein. Indi agric. aJ . Sei. (1977) (4)5 ,4 i 156. 24. GHOSH, A ,K., MITRA, G.N., AND SAMPATH, S. Genetic improvement of protein content in rice, Indian J. agric. Sei. (1976), 46(1) :26. 25. GHOSH, A.K. NANDA,B.B, GOVINDASWAMY, S AND NAYAK, B.B. Influence of nitrogen on the physico-chemical characteristics of rice grain. Oryzas(197l), 8(l):87. 2b. GOPALAN, C. The child in India, XXII Jawaharlal Nehru Memorial Lecture, Teen Murti House, New Delhi, November 13 (1979). 27. GOVINDASWAMY, S, AND GHOSH, A.K. Breeding for high protein content in rice. Indian J. Genet.(1974), 34(A):628. 28. GOVINÜASWAMY, S, GHOSH, A .K., MAHANA, N.K., AND DASH, A.B. Genetic variability and correlation studies on protein content and some quantitative characteristic ricf so e (Oryza sativ ) Oryza(l973)aL. . 10(1): 1. 29. GUO, Y.c.c. (Kuo, I.C.), XIE, S.J. (HSIEH, S.C .). Breeding studies on high protein rice. II, Inheritance of grain protein content, heading date, and other characters in later generations of a cross between earl latd an ye Japonica rice varieties Agricf o . .J . Res» China (1978), 27(3)s 198. HIGASHI. 30 KUSHIBUCHID AN , ,T . ,K Studie breedinn so higr fo gh protein rice . Inheritanc,II hige th h f proteio e n propert Japonica n i y a rice cross. JapBree. J . d (1976), 26(1). :17 31. HILLERISLAMBERS , RUTGER,D , J.N., QUALSET, C.O., WISER,W.J. Geneti 'environmentad an c l variatio protein ni n conten ricf to e (Oryza sativa L.), Euphytica (1973), 27: 264. 32. JULIANO, B.O. Physico-chemical data on the rice grain. Int.Rice Res.Inst. Tech. Bull (1966), 6, 159. 33. JULIANO, B.O. Physico-chemical properties of starch and protein in relatio griio nt n qualit nutritionad an y l valu ricef eo . Rice Breeding, IRRI, Los Banos, Laguna, Philippines (1972), 389. JULIANO. 34 , B.O., IGNACIO, C.C: PANGANIBAN, V.M. PEREZ,C.MD ,AN . Screenin higr gfo h protein rice varieties. Cereal Sei. Today(1968), 13: 299. 35. KRAMER, T» Environmental and genetic variation for protein content in winter wheat, Euphytica (1979), 28, 209. 36. MOHANTY, H.K, AND REDDY, C.S. Inheritance of protein content in rice, J.Res. (OUAT), Bhubaneswar(1972) 1(2): 106. 37. MONYO, J.H., AND SUGIYAMA, T. Breeding for high protein and balanced amino acids in rice. Proc.3rd SABRAO Cong. Canberra(1977) . . 52 - 5A , 2

134 38. MONYO, J.H., SUGIYAMA, T., AND KIHUPI, A.N. Potentially high yielding and high protein rice in induced mutation breeding (Proc, Symp. Seed protein improvement in cereals and grain legumes, Neuherber Septemberg, , (1978(1979)A IE ), , 293. NIEMANN. 39 , E.G. Screenin proteir fo g n quantit qualitd r an fo y d an y other nutritional factor breedinn si g programme. Gamma Field Symposia on crop improvement by induced mutation. Inst. Rad. Biol. Japan. (19783 ) a 9 40. NIEMANN, E.G. Nuclear technique for the determination of protein content in plant material. Atomic Energy Review (1979 b) (in print) . NELSON. 41 , O.E. Inheritanc aminf eo o acid conten cerealsn ti . (Proc. Symp. Seed Protein Improvemen cerealn ti graid an s n legumes, Neuherberg, Sept. 1978, IAEA, Vienna (1979), 79. 42. PEREZ,C.M., CAGAMPANG,G.B., ESMAMA, E.V., MONSERRATE, R.VD .AN JULIANO, B.O. Protein metabolis leaven i m developind san g grains of rices differing in grain protein content, Plant Physiol. (1973) , 51: 537. RAO,M.J.B.K. 43 . Problem ricf so e varietal improvemen Indian ti . Inter Rice Comm. Newslet. (1973), 22(2), 21. . RAO,M.J.B.K44 . Recent developmen breedinn ti g approache varietar sfo l improvement in rice, Proc. National Symp. on increasing rice yields in kharif, Cuttack (Feb. 1978). ,67 45. RAO,M.J.B.K., SAMPATH,S, AND MURTY,K.S. Breeding varieties for special situations, Indian Farming (1971), 21 (7) j 45, 46. RATHO, S.N., NANDA, B.B., SAHOO,K.M. Genetics of protein content in rice. IL RISO (1979) (in press;. 47. SARALA, A .K., AND REDDY, G.M.,. Role of local germplasm and induced mutation improvemene th n proteie si th f to n contenn ti rice. Theoritica Applied lan d Genetics (1979), 54(2) :75, «8. SAMPATH, S , AND SESHU, D.V. Variability of protein content in rice. Current Sei. (1957), 26 (5fr: 139. SEN. 59 , J.N., PUSAfc BUL SOHSIMPSOND n LAN (I ,CHOU, , A C.CY . . ,I ,A . BULL Inst. Med. Res., Federation Malaya (1951 7)(1916, 5 ). , 62 ), 50. SEO, B.W. AND CHAMURA, S. Occurrence of varietal differences in protein content, Jap Cro. .J p Sei. (1979), 48(1). ,34 51. SRIVASTAVA, NANDAD.PD AN ., ? B.B. Variatio grain ni n protein ni some groups of rice varieties from the collection of North-East India. Oryza (1979), 14(1): 45. 52. S WAMI NATHAN, M.S. Genetic and Agronomic enrichment of the quantity and quality of protein in cereals and pulses. In new approaches to breeding for improvement plant protein, (1969), IAEA Vienna, 71.

135 RESEARCH WORK ON HIGH-LYSINE BARLEY AT SVALÖV

A. TALLBERG, K.-E. KARLSSON . STOV , Y Svalö , SvalôvAB f , Sweden

Abstract

High-lysine barley breeding with Hiproly as gene source has resulted iyiela n dmose leveth tn i advancel f thado materia% t 9 obtaine9 f o l d in corresponding normal cultivars e averagTh . e lysine conten n thii t s advanced materia e see4.0s i th l dn 5i whic a significanN g/1g s i h6 t improvement but still not as high as desired. By combining different high-lysine factors a further substantial increase in lysine content s beeha n obtained n thesI . e high-lysine double-récessive e glutelith s n fractio s increasewa n d wherea e hordeith s n fractio s decreasedwa n e Th . grain weight of the double-récessives was, however, reduced compared wit e hparen th tha f o t lines. DBC/N analyse f commerciao s l varieties showe differencesg bi d , which were consistent for the varieties at two localities and when compared for different years. This indicates that in a favourable genetic background the lysine content in the seed might be improved. Possible relations between phytohormone activitie r contento s d graian s n growth in both normal and high-lysine barleys were investigated. The normal types exhibited high levels of auxins and low levels of gibberellin- like substances wherea e reverse high-lysinth sth s trur ewa fo e e types. It is suggested that auxins in particular may play an important role in grain growth. Further research wor n phytohormono k d othean e r effects on grain growth and development in normal and high-lysine barley genotypes is in progress.

Practical breeding with Hiproly as gene-source Effort o product s a ehigh-lysin e barley variety, e.ga barle. y line wit a substantiah l improved nutritiona t givelno valuw n no eo t hav p u e any practical results. This means not that the work has failed, on the contrary, the progress has been highly remarkable. The difficulties have been much greater than anticipated and it seems reasonable to assume that it will take longer than originally planned. Inview of tne results produced so far there are, however, good reasons to believe that we will be successful in production of high-lysine barley. e difficultieTh o product s a high-lysine e barley variety with good agronomic characters are due to the fact that the high-lysine genes influence several other properties in the plant sinfultanously, especially the grain weight. We have, however, now succeeded in elevating the yield level of the high-lysine material by intense crossing and selection and y broadeninb e genetith g c back-ground e graiTh . ne mos yielth tf o advanced d high-lysine material with % compareHiprol 9 9 s i yd wite commerciath h l variety Tellu s standarda s e proteiTh . n yiel f thio d s materia s increasei l d with 10 % with a lysine content of approximately 4.05 g/16 gN.

1508/Hiproly double-récessives. Although the lysine yield is increased with 20 % compared with a standard variety e lysinth , e conten e see s th insufficienti dn i t o enhancT . e th e lysine conten e barleth n i ty seed different high-lysine factors have been combined s 3a-genly e jnutann i eTh . ts combine150wa 8 d wite lys-genth h e from a high-lysine back-cross line, Hiproly x Mona^. The lysine content in the double-récessives was raised to an optimal level, 5.6 g/16 gN, whic abovs i h e that recommende y FAOb d , 5.44 . g/1gN 6 137 Sequential extractio f protein o n) show 2 , s(1 stha e hordeith t n fraction is drastically reducee double-récessivesth n i d , displaying % onl 0 1 y hordein proteins (Tabl , whil 1) e gluteli th e n fractio s increasei n d % comparewit 0 4 h d with mutan 3a-gens ly t e 1508effece eth Th .besidef o t s the suppressed hordein synthesis is a highly increased content of salt- soluble proteins, whic s alsi h o reflecte e double-récessivesth n i d n I . Mona, Bomi and Hiproly x Mona^ 7 % of total N is attributed to non-protein nitrogen (NPN) e greate.Th r proportio f freo n e amino acid mutann i s t 1508 (3) is reflected in a higher amount of NPN in the mutant itself e double-récessiveth n i d an . Thi) %) % s6 3 increas(1 (1 s f non-proteio e n nitroge mutann i n e double-récessiveth t d 150an 8 a consequenc s i s e th f o e suppressed hordein synthesis, which also has been reported by K0ie and Kreis (4). The increased content of non-protein nitrogen and thus the free amino acids e double-récessiveith n s cause e insufficienth s t separation between mutant 1508-segregant d double-récessivean s e DBC-analysith n i s s employer fo d selection (Fig. 1). As amino acids and peptides will not precipitate, although dye will adhere to the basic amino groups, a lower DBC-value is . recorde ) 6 , (5 d These high-lysine barley double-récessives (lys 3a/lys) are characterized by extremely shrunken seeds due to major changes in seed metabolism affecting both proteins and carbohydrates. It might still be possible, however, that by transferring these high-lysine barley genes into new genetic back-grounds the agronomic characters are improved.

Mutant 7/Hiproly double-récessives. Up till now the only high-lysine barley mutant described without reduced seed siz mutans i ee increas Th 7 inducet. f 8) lysino e Bomn , i ds (7 i e % compareonl 0 1 y d with Bomi, most a singlprobabl o t e du recessivy e gene inheritance (8). In an attempt to achieve an increased lysine content in the seed without a drastic reduction in grain weight mutant 7 was crossed with the high-lysine back-cross line, Hiproly x Mona^. e double-récessiveTh s displa o lysintw y e level d representativean s s from each grou e referre ar p s DR- d DR-Ba an Ao t .d Lysine conten f proteito s i n increased with 10 % and 17.5 %, respectively, compared with the high-lysine parents. Sequential extractio f protein o n) show 2 , s(1 s tha e contenth t t f hordeio n protein mutann i ss decrease i 7 t d whereas salt-soluble proteins s wel a s glutelina l e slightlar s y increased (Tabl . DR-A2) e , wite loweth h r lysine content, has almost the same protein composition as mutant 7, while in DR-B with the higher lysine content, the glutelin synthesis is substantially enhanced in addition to an impaired hordein synthesis. In Fig. 2 the lysine content of the protein fractions in % of total lysine is presented. The glutelin fraction contributes with most of the lysine compared with the other fractions. The high lysine content of the glutelins in Hiproly x Mona^ explains the higher content of lysine in the double- récessives compared with mutant 7. The effect of the lys-gene on an enhanced glutelin synthesis in combination with the hordein suppressing mutant 7 s resulteha n improvea n i d d lysine content withou e deficiencth t f o y storage protein, characteristi f hordein-deficieno c t mutants e graiTh . n e weighdouble-récessiveth f o t % e loweonl 5 ar s1 y r tha a norman l lysine standar y puttinb d e double-récessivean dth g s into favourable genetic backgrounds there are good reasons to believe in increased grain weights.

Differences in lysine content between commercial varieties. Besides the substantial increase of lysine content in Hiproly and the other high-lysine mutants, smaller difference e lysinth n i se level have been found among commercial varieties. Varietie e sbreedin th use n i d g program for high-lysine barley have been checked with DBC/N analyses. 0 plant2 s from each variety were analysee regressioth d an d n liner fo s different varieties compared.Difference e lysinth n i se level between

138 commercial varieties were as big as half the difference between high- lysine lines includin e jys-genth g d normaan e l varieties s alswa ot I . found that the relative differences between varieties were the same for material grown at two localities. For some varieties the differences were consistent even when different years were compared. I ne positivlighth f o t e transgressio n lysini n e contente founth n i d earlier mentioned double-récessives it will be interesting to see if the lysine level in high lysine barley still can be increased by choosing the "right" parents e effecTh . f sucto h modifiers wil e furtheb l r studied e high-lysinth t a e normae th leve t la s wel a ls lysin a l e level.

Phytohormone n high-lysini s e barleys Grain growth in cereals is presumably controlled by the activity of various phytohormone d differencean s n graii s n weigh y thuma t s possibly o differencet e du e b n hormoni s e activity s thereforha t I . e been considered worthwhil o investigatt e e changeth e n activiti s r o y content of several phytohormones during grain development in both normal and high-lysine genotypes in order to find out if these changes in somewa e relatear y o observet d d difference n graii s n growth. Gibberellin-like activities and auxin contents were determined at various stages of grain development in the normal cultivars Mona and e high-lysinth Bomd an i e type v 7360S s 8 (Jys^-gene d Risan ) 0 1508 (lys 3a-gene). Very great differences were found betwee e normath n l e high-lysinanth d e types with respec o bott h gibberellin-like activit d auxian y n content. Thue pea n th gibberellin-liksi k e activity found abou 3 weekt s after anthesi5 time2- s wa shighe n i r e high-lysinth e type se norma th tha n i ln cultivars (9). In contrast to these findings auxin content was much higher in the normal types than in the high-lysine ones. Ris0 1508 exhibited extremely low levels of auxin throughout the entire grain filling period whereas Sv 73608 first developed an auxin peak similar to that foun n Mont i lated bu a r rapidly approache n auxia d n level which s onlwa y hal f thao f t obtaine e normath n i ld cultivar s therefori t I . e suggested that auxin play n importana s e tdevelopmen th rol n i e f o t barlee precisth t y bu egrai actio) (9 nd interactio an n e variouth f o ns phytohormone n thii s s proces s stili s e lpossibl th unknow s i s ea n e high-lysineffecth f o t ee hormon geneth n o se system. Further work n thio s proble n progresi s i m t Svalöva s .

References :

K0IE, B. and NIELSEN, G. 1977. Extraction and separation of hordeins.- In Techniques for the separation of barley and maize seed protein (Eds. MIFLIN, B.J. and SHEWRY,'P.), EUR 5687, p.25-35. SHEWRY, P.R., PRATT, H.M., LEGGATT, M.M. and MIFLIN, B.J. 1979. Protein metabolism in developing endosperms of high-lysine and normal barley.- Cereal Chem. 56: 110-117. BRANDT, A. 1976. Endosperm protein formation during kernel development f wilo d a high-lysin typd an e e Larley mutant Cerea- . l Chem : 89053 . - 901. K0IE, B. and KREIS, M. 1978. Hordein and starch synthesis in developing high-lysin d normaan e l barley seed t differena s t N-fertilizer levels. - In Carbohydrate and protein synthesis (Eds. MIFLIN, B.J. and ZOSCHKE, 6043R M.)EU ,. p.137-150. MUNCK . 1976L , . Aspecte selectionth f o s f higo ,e h desigus lysin d an n e cereals. - In Evaluation of seed protein alterations by mutation breeding, IAEA/FAO/GSF, STI/PUB/426, Vienna, p.3-17.

139 TALLBERG, A. 1980. Comparison between screening methods for lysine with the use of a barley material with a varying amino acid composition. - Aeta Agric. Scand. 30: 26-32.

DOLL, H., K0IE, B. and EGGUM, B.O. 1974. Induced high lysine mutants in barley. - Rad. Bot. 14: 73-80.

DOLL . 1976H , . Genetic studie f high-lysino s e barley mutant ;s. - In Barley Genetics III (Ed. GAUL, H.), Verlag Karl Thiemig, München, p. 542-546.

MOUNLA, M.A.Kh.,. BANGERTH, F. and STOY, V. 1980. Gibberellin-like substances and indole type auxins in developing grains of normal- and high-lysine genotype f barleyo s - Physio. . !Plant n press)(i , .

TABL, 1 E PROTEIN FRACTIONS IN % OF TOTAL M X 6,25

NPN SALT-SOLUBLE HÜRDEINS GLUTELINS RESIDUE PROTEINS

4 4 MONA 1 3 7 18 7

HIPROL X MONAY 5 8 31 43 20 6 9 4 BOM I 9 2 7 17 5 MUTANT 1508 13 43 17 22 18 DOUBLE-RECESSIVE 16 47 10 31 13

TABL, 2 E PROTEIN FRACTION F TOTAO % N L N I S X 6,25

NPN SALT-SOLUBLE HORDEINS GLUTELINS RESIDUE PROTEINS

2 DR-3 A 1 3 8 24 13

DR-B 8 32 26 31 11

1 MUTAN3 T 7 8 2 9 22 19

9 3 HIPROLY X MONA 3 3 5 8 31

MONA 7 23 47 20 10

140 65-

O " ^) a i O

60- O

0 «^ Q « 01 a U

13 I/I 0l 55- \ro Um Q

50-

45-

r l l 11 12 13 14

crude protein IN x6.25)

© Double-récessives 9 15OS- segregants A Hiproly- segregants Fig. 1. Relation between dye-binding (g bound dye/kg sample d crud)an e protein percentag n segreganti e s from x (Hiprol1508 x y

141 LYSIN FRACTION EI TOTAF NO L LYSINE

-40

-30

-20

-10

SAL T - S IGLUTE L u 0 in \ u U) m G ü D m c j un-A 0 z m U) r Z U) Cfl ) DR- ( B 0 C r ta ^ MUTANT-(f 7 C r m m r ( J) HIPnOLY x MOMA5 m MCJMA

Fig. 2. Lysine content in protein fractions in per cent of total lysine in double-récessives from Mutan x (Hiprol 1 t Mona^x y )

142 PROTEIN IMPROVEMENT IN CEREALS I. WHEAT*

CF. KONZAK Washington State University G.L. RUBENTHALER Western Wheat Quality Laboratory, United States Department of Agriculture, Scienc Educatiod ean n Administration-Agricultural Research, Pullman, Washington, United States of America

Abstract

BREEDIN HIGR FO GH PROTEIN CONTEN CEREALN I T WHEAT. I S . Preliminary studies were made of progenies selected from crosses between standard hard red spring wheats and a high protein germ plasm sourc . 1 Resulte t Magnier s1 4 f indicate thagenetie th t c capacito t y produce grain of higher protein content at equal or higher grain yield level realistia s i s c goale principaTh . l obstacle thio t s s goal appear to be similar to those encountered in any breeding effort to transfer a few desired traits from unadapted, diverse germ plasm sources to locally adapted wheats. Thus, it is not unexpected that, compared with the locally adapted wheats ,progenw onlfe a y y selections from simple crosses show promise for competitive or higher yield than local cultivars, as wel s adequatla e disease resistanc d acceptablean e agronomic characteristics (straw stiffness, adaptation d satisfactor)an y processing qualities along with the desired higher protein production potential. Especially encouragin indicatione ar g s that progeny with processing qualities superio parene th d chec o an tt r k varieties have been recovered in the early stages of research. Additional germ plasm resources for further increasing wheat protein content and nutritional value appear to be available for exploitation. Induced mutations offer promis complemeno et t natural germ plasm sources for genetically increasing wheat protein content. New infrared spectroscopy methods for rapid screening selections for protein and lysine content offer promis enhanco t e e protein improvement research.

1. INTRODUCTION

Several wheat breeding research program Unitee th n di s- Statein w no s clude improved protein content and/or nutritional valumajoa s a er goar fo l future wheat cultivars e StatTh .e Universit d Experimenan y t Statio- re n search programs in Nebraska, Kansas, South Dakota, Montana, California, and Washington all have significant high protein wheat breeding research in progress. The Nebraska-USAID wheat protein improvement program especially s madha e exceptional progres breedinn i s g high protein harwinted re d r

^Scientific Paper No.5715 . College of Agriculture Research Center, Washington State University, Project Nos. 1568 and 1570. Supported in par IAEy b t A Contracts RB-159 RB-1690d 0an .

Mentio f tradno e names doet implno s y endorsemen e authorsth y tb , Washington Stat eDepartmen. UniversityS . U e Agriculturef th o t r ,o , Agricultural Research Service, Division of Science and Education.

143 wheats and in identifying germ plasm sources [1]. Also, several high protein selections from Kansas and South Dakota have been widely used by breeders. In some other states, such as North Dakota, Minnesota, Oklahoma, and Texas, wheat protein improvement also has long been an overall objective. private Oth f e breeding firms, Seed Research Associates, Scott City, Kansas, was perhaps first in the U.S. to market high protein hard red winter wheats under private contracts, with the production used for blending with lower protein standard wheats. (Ken Gertzen, S.R.I., personal communication, 1980). researce goae th th f increasinf lo o s Centra i hl al e genetio t lth g c potential for higher grain protein content while retainin traditionae th g l industrial processing properties; and, of course the necessary agronomic and disease resistances for competitive yielding. Improving the nutritional value of wheat protei exploratore s generallth i n n i t yye y stages, although significant progres s beesha n mad n identifyini e g germ plasm sources. Only recently have there been indications that genetic recombination among high lysine germ plasm sources might permit achievement of even higher lysine/protein content levels in wheat [2, 3; Allan et al., personal communication, 1980]. A major impetus for breeding higher protein wheats has been prompted by millin. S . d U bakin conceran ge th g f no industr breao t ye tren th kf lowe o d r protein wheat productio . Insufficien4] , [1 n t supplie higf o s h protein wheat for blending has made it difficult to meet requirements of U. S. national bread labeling laws [4]. The price paid to growers for low protein (below 10.5 percent) bread wheats severele ofteb y nma y discounted premiumt ,ye s are pair proteifo d n levels over 14. 16.o 5t 5 percent. However achievo t , e the higher protein levels, growers must often apply mor eN fertilizer , which is becoming increasingly more costly. Consequently, there is an increased incentive for breeders to develop high protein wheats. This paper describes some preliminary resultauthor'e th f o s s program at Washington State Universit increaso t y e proteith e n content potentiaf lo hard sprinre d g (bread) wheats selectioe th , d evaluationan n methods employed. Some related aspect wheaf o s t protein improvement wor n progresi k Pullmat a s n and at some other institutions are briefly reviewed.

2. MATERIALS AND METHODS

Several source higf o s h protein genes have been introducee th n i d Washington State University spring wheat breeding program A (Tabl . I) e semidwarf mutant, Magnif 41 ert^ l (CI17689) induced by the senior author Argentine ith n e high protein cultiva rbees Magniha n , use41 f d most extensively and some progenies rapidly advanced to preliminary trials from increases of selected F 3 lines. This was done in order to obtain preliminary indication probleme th e encountered b f o so t s , especially since Magni1 4 f is poorly adapte o locat d l conditions becausd weakee ,an th f ro e mixing properties associated wits proteinit h . Also, unlike most other sources of high protein genes mutane th , , lik1 toriginae eth Magnit er 1 4 fl Magnif 41, carries resistance to leaf rust (Puccinia recondita f. sp. tritici) n increasingl,a y important diseasareae th .n i e In the studies described, the local high-quality bread wheats, Wared and Borah were included as single cross parents. The selections discussed here were evaluate Washingtoo tw t a d n State station test sites: Lindn ,o fallow, alternate year cropping, rainfall 220 mm during the fallow year, durinm m croe 0 th g20 p year (September 1—August 31) d Roya;an l Slope, rill irrigated, following a two-year alfalfa hay crop. The evaluation trials were replicated three times with fourteen selections plus the two check varieties (Wared, Wampum) in each trial. For the results reported here, the e checth datkr varietiefo a s were averaged across locations l valueal d s,an are expressed in percent of the Wared check variety, which was the normal protein parent of cross K74102. The protein and lysine composition data reported here were obtained using a Technicon Infralyzer-Plus near infrared spectrometer employin e standarth g d calibration proteinr fo s , with lysine calibrations base samplen o d s evaluate yearo tw d s earlier against samples checke n amina y ob d acid analyzer. The lines selected for the tests and included in this report were evaluated visually in the field for uniformity and for potential produc- tivitlineß F 1976n s i sa y . Only those lines with superior seed quality were teste proteir fo d n content. Thus, approximatel ybettee 180th f 0ro quality lines, representin crosse3 2 g involvinl al s 1 g t Magnier 1 4 f (CI17689), were sown as single seed increase plots at Pullman in 1978.

144 Samples from the 1978 harvest were screened visually for seed quality (plump- ness, smoothness), and 143 of those considered satisfactory were submitted for a preliminary processing quality evaluation. Seventy lines with apparently high protein, most with mixing properties in the satisfactory range, were then entered into the replicated agronomic trials discussed above. A sample of the experimental lines were recently evaluated in preliminary tests for loaf quality and mixing properties. The 13 lines discussed here represent the residue from about 4,00 singl^ 0F e lines availabl initiae th r lfo e evaluations and are the best-performing part of the seventy lines that were yield tested in 1979. FoEty-one of the seventy lines have been replanted in four replicate agronomic trials at three test locations in 1980.

. 3 RESULT DISCUSSIOD SAN N

Preliminary data (Table II) on the thirteen experimental lines selected from yield trials at Lind (low rainfall, dryland) and Royal Slope (irrigated) indicate that a few of the seventy lines tested may prove superior to their locally adapted paren grain ti n yield, protein content, and/or protein yield (others were lower yielding or equal to the Wared check). It is particularly interesting that superior performance occurs most often at the irrigated test site, indicating perhaps that their potentia superioritr fo l s leasywa t restricte moistury b d e availabilit that a y t site. However, ther s alsi e a o suggestion frodate th ma thaapparene th t t yield and/or protein production superiority may occur at either the low potential location (K74127-422), at the irrigated high potential location (K74102-46, K74127-292, 334, 337, 339), or at both (K74102-56, K74102-134). Perhaps the adaptation of developmental patterns, including the maturation period of the selections to the test environments strona s ,ha g influenc observee th n eo d agronomic performance.

TABLE I. SOURCES OF HIGH PROTEIN AND/OR HIGH LYSINE GERM PLASM USED IN SPRING WHEAT BREEDIN WASHINGTOT GA N STATE UNIVERSITY, PULLMAN, WASHINGTON

Entry r o SelI P , Sourcr eo Vernalization Useful numbeI C r origin response traits

April Bearded CI7337 England S Protein, Lysine Atlas 66/Comanche NB68-24709 Nebraska I Protein Magni1 4 f PI344466 Argentina S Protein l t Magnier 1 4 f CI17689 Washington S Protein Nal pHa PI176217 India S-W Protein, Lysine Nap Hal PI176221 India S Protein, Lysine l NapHa PI176223 India S Protein, Lysine Mahratta CI8500 Australia S Lysine Marfed Mutant MJD72175 Washington Lysine SD69103/Atlas 50 KS745720 Kansas W Protein lines KS745820 Hybrid English CI6225 England W Protein Pearl CI3285 Sweden S Lysine 22A CI5484 USSR S Protein Rageni 15 PI383308 Pakistan S Protein T. aest. albidum CI15002 Nepal S Protein, Lysine T. aest. caesium CI15011 Nepal S Lysine T. aest. nigricolor CI15007 Nepal S Protein, Lysine T. aest. pseuc CI15005 Nepal S Protein, Lysine ingrediens

145 TABL . PRELIMINAREII Y AGRONOMIC PERFORMANC HAR F EO SPRIND DRE G WHEAT DERIVATIVE HIGF SO H PROTEIN MAGNIF 41 ert l (CI17689).

LIND (DRY) ROYAL SLOPE (IRRIG) Yiel Waref do K % C d Yield % of Wared CK . SelNo . Grain Plant Tes. tWt Grain Protein Lysine Plant Tes. tWt Grain Protein Lysine Size^. 1cm ). Ht Kg/hl Ht . cm . Kg/hl

K74102-23 L 45.0 76.5 99 94.7 105 85.0 78.5 111.8 112.9 114 K74102-46 M 40.0 77.2 79 76.8 85 85.0 79.1 113.0 126.7 118 K741p2-56 L 55.0 75.9 113 108.9 120 82.5 77.2 110.9 115.7 120 K74102-118 M 47.5 77. 2 105 96.7 106 77.5 81.7 120.0 117.0 112 K74102-134 M 55.0 11.1 105 104.7 111 90.0 81.1 112.8 116.2 107 K74102-206 M 52.5 11.1 102 98.9 107 90.0 81.1 118.6 116.4 108 K74110-256 M 40.0 11.1 96 93.4 97 80.0 79.8 106.6 106.8 97 ON K74123-341 M 52.5 77.9 107 98.2 104 72.5 77.9 99.4 102.3 98 K74127-292 M 55.0 77.2 76 77.1 76 70.0 78.5 109.3 117.8 104 K74127-334 M 42.5 75.9 67 76.6 68 75.0 79.8 100.0 109.7 100 K74127-337 M 50.0 74.6 91 92.3 87 77.5 79.8 111.3 117.7 103 K74127-339 M 42.5 81.1 84 83.6 81 77.5 81.1 107.3 110.4 106 K74127-422 S 52.5 77.9 109 105.0 '110 82.5 78.5 91.5 94.8 85 3) (2, Wared (CK) M 55.0 78.5 1431® 223. 2(2' 5 16 3) 85.0 79.2 4849.0«706.4e'3)17.24(2'3) Wampum (CK) M 55.0 78.5 99 88.5 100 90.0 79.8 117.7 113.0 114

— L = Large, M = Medium, S = Small 2/ — Actual values Kg/Ha 3/ — Protein 15.6-Lind, 14.6-Royal Slope; Lysine 2.31-Lind, 2.55-Royal Slope e resultTh f theso s e tests show thaprime th t e componen proteie th f o tn yield for a variety is grain yield. Even fairly large (1-2%) differences in protein content r linfo e s ,a K74127-33 4 (17.9 Royat Linda t %a % l ,16 Slope ) are insufficient to make up for lower grain yield performance. Moreover, any premium price paid to the grower for higher protein grain is likely to be temporar d rarelan y y wil proteie lth n premiu adequate mb - in coveo t ee th r creased N fertilizerscost r fo s . Thu wilt i se importan lb achievo t t e superiority in both grain yield and grain protein content. Protein yield may busefua e l calculatio estimato t n e genotyp e efficiencus eN t thibu ys measure valuo n f io se until grain yield e equa ar scomparablo t l e standardsr ou n I . research, progress in breeding during the development of the high protein germ plasm lines has led to the identification and release of Wampum which shows equal yield performance wit hw potentia Warelo t a d l environmentst ,bu distinctly superior performanc higt a e h potential environmentss i t I . encouraging to note from Table II that line K74102-56 shows yield and protein superiority to both the Wared parent and the competing Wampum variety, indicating that it should be possible not only to increase grain protein conten alst tbu o grain yield ovecurrene th r t cultivars. Because the high protein germ plasm source Magnif 41 ert l was poorly adapted to Washington conditions, it yielded well below local standards, yet always produced high protein grain [3]. It cannot be assumed that the high protein production of Magnif 41 or mutant ert l is associated with thei w yieldinlo r g ability since these wheats transmit their high protein trai higheo t r yielding progeny. It is not yet known if any of the higher yielding lines carry the mutant semidwarfing trait of Magnif 41 ert 1. This trait probably is inherited independentl f proteiyo n content. However, sinc1 e t Magnier 1 4 f is slightly shorter in height than Wared, it is possible that lines K74102-118, K74110-256, K74123-341 K74127-29d ,an 2 carry this gene instead of Rht?. Because Rht2 is closely associated with gibberellin insensitivity gene Gai2, a simple test for seedling response to GAß will identify the gene involved gent MagniRh f o e e s inherite i 1 ejrTh 4 f.1 t d independently from the Rht? GaJ2 association. e "doublth Non f eo e short", 2-gene recombinants survive rigoroue th d s screening tests applied to this set of materials. However, intensive pedigree selection among F/ and F r populations of the "very short" segregates from crosses K7410 Waredx 21 K7412d (Magnit )an er 71 4 f (Borahx Magnif 41 ert 1) may yet produce some high yielding, high protein shorter height lines better adapted for irrigated culture. The most notable weaknesses introduced into the crosses from the Magnif 41 ert 1 parent include the tendency to produce seed with a shrunken endosperm, often associated with late maturity itsel1 t Magnit s er Ye i f . 1 4 f comparatively earlier thalocae th n l cultivar othee s th use rs a dparent n i s yielw crosses als1 y hav lo a t Magnif oma o de o eer t Th bee 1 .e 4 f ndu lower number of flowers formed per spikelet and possibly some floret sterility. Surprisingly largee th , r hard Magnie grainth f fo s paren to readils wer t eno y recovered as might have been expected if its high protein gene(s) are associated with large grain size. In addition, the relatively weaker mixing properties of the Magnif 41 parent was severely limiting, such that perhaps only the few lines currently under agronomic evaluation survived screening thu s hopei s t fardI .thaback-u e th t p reselections made frovarioue th m s sub-line f theso s e crosses wil t leasa l t include some combination higf o s h protei betted an n r baking qualit higr o y h protei d hignan h yiel n linei d s exploitabl y furtheeb r breeding. However, more recent preliminary baking and processing data t compiled(noye t ) indicate that lines K74102-23, K74J02- 56, K74102-206 produced bread superior to the check varieties and had acceptable mixing properties, while lines K74127-334 was distinctly superior to the checks d line,an s K74127-33 K74127-33d 7an 9 wer echeckse equath o t l, Wared and Wampum. These results and especially those for K74102-56 suggest there may be no significant obstacle to achieving satisfactory processing properties at higher than usual protein levels. Other high protein sources, including mutants investigated in Washington State University breeding research: A brief mention of our experiences using other sources of genes for higher protein or lysine content seems appropriate. We have used as parents CI8500, CI7337, CI3285, several Nebraska Atlas 66/Commanche/Lancer lines, Nap Hal lines PI176217, PI176221, and PI176223, as well as mutants Rageni 15 from Pakistan, and Marfed mutant MJD7217 severan 5i l crosses with local wheats. Selected lines from this group of single crosse locao t s l cultivar r selectiono s onle w enterinar sno y g preliminary replicated yield trials. 147 wits Magnie A th h 1 ert4 f ^1 crosse s discussed earliere th f ,o verw fe y lines screene qualitr fo d y have shown acceptable mixing properties. However, early indications are that advances may have been made toward adaptation to local conditions combined with increased protein and/or lysine contentf O . 0 lineth78 e s submitte preliminarr fo d y quality screening (milling, mixing properties, protein content and water absorption) only 20 lines appear to have satisfactory mixing properties combined with high p proteinNa e Th . materiall Ha d notoriouslsha y poor milling propertie d introducean s d poor agronomic traits into the crosses. Crosses with Mahratta (CI8500), a high lysine source from Australia, have produced a large number of promising lines currently under yield performance evaluation. However the weak mixing propertie mosf so t lines will limit thei s parenta r e further valuus fo s o t e r crosses .linee th Man sf o y fro Mahrattx l m t Magnier a 1 sho4 f w increased lysine or increased protein and some may combine both [5], The Rageni 15 genotype introduced a number of undesirable and unadapted traits reducing the frequency of promising segregates. Marfed mutant MJD72175, selected as a possible higher lysine source, also has proved difficult to use as a parent. MJD72175 may carry some chromosome abnormalities, since sterility and defective recombinants have beecrosS F nd s an encountere 2 ? e th n i d progeny. However, a number of the problems encountered with the cross combinations mad w eger ne usin me plasth g m resources were unpredictable when the crosses were made. Most seriou appearance f thesth so w s racei ene f seo of stripe rust (Puccinia striiformis West d lea)an f rust (Puccinia recondita Rob. ex Desm. f. sp. tritici) to which many of the locally adapted parents proved susceptible. Thus w crosse,ne s including more disease resistant parent madee b somn casesw sI o t .fe ehav d ,eha however, 3-wa d 4-waan y y crosses were mad includo t e e more germ plasm from locally adapted lines with disease resistanc betted ean r processing properties. Selections from those crosses are only now reaching the stage of preliminary laboratory and field screening prior to entry into yield trials. Because of the strong focus on practical objectives of the research described, and because of the encounter with new disease problems, it has not been possibl r prograou n identifo ei t m a y'parent 1 source suitablr fo e induction of mutations for high protein content. Consequently, this type e futureth o fn i researc. t ye s i h w resultNe s from other research program Washingtot a s n State University: Allan. E Peterson. Rubenthale. J L . R . . ,C G d ,an r (personal communication, 1979), USDA-SE Pullmant a A , have recently determined thalocae th t l cultivar, Luke (CI14586), is possibly a better germ plasm source for high lysine than CI13447 or CI13449. The cultivars Hyslop (CI14564) , Gaines (CI13448), and Nugaines (CI13968) also appear to have high lysine composition. Analyses of 165 Fj lines selected from ten crosses among the high protein or high lysine sources SD69103 witl ,Ha h CI7337 p locallNa d ,an y adapted winter wheats Hyslop, Luke, Nugaines, Wanser d Bur,an t showed that heritabilitf o y protein content was relatively high (60%) but that for lysine was lower (47%). Numerous line d eithesha r high protei higr o n h5 lysine16 e th ,f o wit 1 1 h lines having higher lysine than Nap Hal and with a protein content equal to CI7337. Most significantly, however, 16 lines had lysine values significantly above Luke (3.39 g lys/100 g protein) indicating transgressive segregation for lysine content, and suggesting that genes for lysine in Luke may differ from l (PI176217)Ha p thosNa n i e. Another interesting cultiva Yamhils i r l whic s provee hb ha o t d nutritionally superio poultrn i r y feeding, possibly becaus highea f o e r threonine and lower carbohydrate gum content [6-8; J. McGinnis, personal communication, 1980]. As a further aspect of our effort to increase the genetic potentia proteir fo l n productio wheatn i n e hav,w e recently initiated cooperative research witVolcane th h i Institute, Rehovot, Israel to further develo d exploian p t som dicoccoide. eT_ s Körn germ plasm resources r proteifo n improvement. Duru d commoan m n dicoccoidewhea. T_ tx s dérivâtes percen1 2 o t wit t8 1 hprotei n already have been recovere Volcane th t a di Institute by Adriana Grama (personal communication, 1980). At a nearby laboratory in Israel, Avivi [9 ] recently found that some selections of wildicoccoide. T_ d s produced plump grain wit proteia h n conten per7 2 -f o t cent in the field and that can produce plump grain with up to 43 percent protein when grown under greenhouse conditions. These results surely suggest there is far greater genetic potential for increasing the protein content of wheat than was ever imagined previously. The achievement of 43 percent protein in any wheat grain, no matter the yield level, suggests that the genetic regulatio f proteio n sucn i n h genotype bees ha sn relieved, allowing protein to accumulate in the endosperm as long as a nitrogen supply is available to 148 construce discoverTh . sucf it to y h high protein accumulation capacitn i y strains of wild T_. dicoccoides (the presumed progenitor of both durum and common wheats) suggest least a s t thawheae th t tcapacite genomth s r eha fo y mutation toward protein production levels far beyond those yet realized. It should thu e possiblb s o induct e e mutants affectin regulatore th g y processes of protein accumulation. New results at Kansas State University: Also of interest here, are result mutatiof so n experiment Heyne. G s . conducteE , . KansaDr y b ds State University, Manhattan, Kansas harn winted ,o re d r wheats. Ethylmethanesul- fonate (EMS) treatments were applie fouo t d r wheats (about 16,00 plant^ 0M s were grown). Selections were ß madM proteir d fo e an 2 nM contene th n i t generations, followin pedigrea g e syste selectionf o m . Sub-lines fro3 2 m families are still under evaluation in agronomic trials (Table III). In- dications are that some variations (both i) in protein content are present amon linese th g . There appeasome b o et rdifference s als mixogran i o m characteristics. Only a few of the lines selected are agronomically equal in performanc Heyne. G chece . th ,(E ko t epersona l communication, 1980). Genetic necessar e b test y sma establiso t y h whether protein content improvements have in fact been induced by the treatments.

4. CONCLUSION AND SUMMARY

Research aimed at genetically increasing the capacity of wheats to accumulate endosperm protein has begun in earnest at several research organizations in the United States. Industries already appear willing to pay extra for extra protein, but this is not the only factor affecting progres protein i s n enhancement breeding research. Neithe growee th rr rno industry wants to bear the cost of the added N needed to achieve the desired protein levels, since the cost of the added N may exceed the protein pre- miums offereopee th n n marketso d . This situatio y resulnma n increasei t d risk to wheat producers, and fluctuating supply and demand. The preferred alternative is to develop wheats genetically capable of more efficiently converting soil N into both yield and grain protein. Some millers alread e contractinar y g with producer groo hige t s th wh protein strains already released (Seed Research Associates, Incorporated, personal communication, 1980).

TABLE III. PROTEIN CONTENT VARIATIO HAR N WINTED I N DRE R WHEATS FOLLOWING EMS TREATMENTS. DATA OF E. G. HEYNE (1980, PERSONAL COMMUNICATION).

Protei/ n%- Cultivar Families Lines Avg. Range

Kaw Lines 5 20 14.7 13.7-16.7 Raw Check - 33 13.6 13.1-14.5 KS 644 Lines 8 32 13.8 12.7-16.0 KS 644 Check - 55 12.7 12.1-13.5 Parker Lines 4 16 14.9 13.8-16.2 Parker Check - 9 13.9 13.8-14.1 Shawnee Lines 6 24 15.9 14.6-17.3 Shawnee Check — 31 13.5 13.2-15.5

-Wheat grain moistur e0.3t ( abou )% 10 t

Major stimuli to protein improvement research in wheat have been new knowledg availablf eo e protein gene resource developmene th d an s f to efficient protein screening methods. Of the protein screening methods currently available w neane re infrare,th d analyzers offer rapid, non- destructive means for screening, applicable also to mutation experiments. Indication successfuf so l inductio higf no h protein mutants from mutation research should increasingly stimulate researc othet ha r laboratories. Further evidence of progress made in advancing both protein content and grain yiel severay b d l research laboratories will also encourage

149 competitive effort y otherssb s alread,a y e takinseemb o t sg placen I . l casesal , however majo,a r proble s beeretentioe mha th n f traditionano l processing properties. For the most part, this problem now seems resolvabl proteir efo n improvements. However, research to increase protein nutritional value in typical bread-type wheat y encountesma r more serious obstacle sufficientlt ye t sno y researched probabls i t I . e that increased lysine wil t affeclno t soft wheat quality since protein properties are of less importance. Neverthe- less wilt i , e importanlb determino t t influence th e lysinf eo e improvement on the processing properties of wheats, since it has been suggested on the basis of the barley, sorghum, and maize research that to increase lysine in wheat it will be necessary to reduce gliadins important to processing n contrasI . o thest e other cereals valuee th , d food product characteristics of polyploid wheats are uniquely dependent on the content of prolamines (gluten and glutenin fractions). It is already known that the water soluble proteins have little influence on processing properties. e possiblb y Thus ma achiev o t t e,i e some nutritional improvemena vi t increasing soluble proteins. highls Alsoi t i ,y probable that genetic recombination made b n e ca samon varioue th g s protein compensato t s r fo e "weakening" effects of added lysine residues in proteins introduced to achieve increased protein nutritional value [3]. Unlike most of the high lysine sources in other cereals, the higher lysine germ plasm sources in wheat produce plump grains, and recent results suggest that some of the genetic variatio r lysin fo ne independenb s additiv i ey ma d f proteian eo t n content. Indications from these studie d otheran s s conducte Pullmat a d n [10] show that near-infrared reflectance spectroscop y als ma ye usefu ob r aminfo l o acid content screening merit further investigatio d testinan n g unde widea r r range of conditions n thesI . e studies, relationshi f lysinpo y infrareeb - re d flectance spectroscopy (IR amind )an o acid analyzer (AAA) then obtaines wa d slightly better than between the AAA and the U-D dye binding protein vs. Kjeldahl predictior fo , f lysinno e content [10] e requirement.Th r fo s calibratiof similao n undesirablt a se rt a ye sample o t s na s ei scomplication . promise Buth t e that even whole unground e analyzeb grai y nma d with somf eo the newer commercially available instruments may be of such significance to nutritional improvement that the method be afforded more consideration by other laboratories. The high equipment costs for the near infrared reflectance spectrometer y bes sma e offse tb y shareb t d uses o screeT . r fo n amino acid content (know r lysine nfo s essentia i t )i havo t l e accesn a o t s amino acid analyzer for the analysis of current calibration samples; and when ground sample usede ar s , fine, uniform sample grindin e importanb y ma g t with some equipment. Results from some preliminary analyse f sprino s g wheats brehigher fo d r protein content indicate tha t shouli t e possiblb d simultaneouslo t e - in y crease both yiel d proteian d n content potentials while also retaining traditional flour processing properties. That some difficulties have been encountered using certain germ plasm sources, and that the frequency of desirable recombinants is low, is not at all unusual when introducing divergent germ plasm sources r resultOu . thin i s s regar e consistenar d t with those from other laboratories. Induced mutation d shoulan n d ca s have an increasingly important role in complementing other germ plasm sources for protein content and nutritional value improvement. The impact of induced protein mutants can be greatly advanced via making known mutants more accessible to breeders and via greater knowledge of their value and potential through biochemica d genetian l c analyses.

REFERENCES

[1] JOHNSON, V. A., Protein in hard red winter wheats, Baker's Digest 52^ 2 (1978. )22 [2] KUHR, S. L., WILHELMI, K. D., JOHNSON, V. A., MATTERN, P. J., "Results secone oth f d high protein-high lysine wheat observation nursery grow 1976"n i n , Agricultural Experiment Station Research Bulletin 291, Lincoln, Nebraska (1980). ] KONZAK [3 , GenetiF. . ,C c controe contentth f lo , amino acid composition, and processing properties of proteins in wheat, Adv. Genet. 19 (1977) 407.

150 DEPARTMEN. S . U ] AGRICULTUREF TO [4 , SEA, Wheat Protein Conference (Proc. Conf. Manhattan, Kansas, 1978) Departmen. S . ,U Agrif o t - culture-Scienc Educatiod ean n Administratio , Peoria#9 M nAR , Illinois (1979). ] KONZAK WARNER, MUNG[5 , RUBENTHALER, V. F. L. . . ,. N ,C ,R , L. . ,G FINNEY, P. L., "Advances in technology and in genetics information for breeding improvements in wheat protein potentials", Seed Protein Improvement by Nuclear Techniques (Proc. Res. Coord. Meet. Vienna, 1977), IAEA, Vienna (1978) 519. [6] KIRSTEIN, D. D., Effect of a Pectinase Enzyme Preparation on the Feeding Value of Wheat for Broiler Chicks, Master of Science Thesis, Washington State University, Pullman (1979). [7] KIRSTEIN, D. D., McGINNIS, J., Effect of ergozyme 100 on the feeding valu wheaf eo broiler fo t r chicks, Poultry (1979ô Seijr . ) 1073, Abs. ] VOHRA[8 KRATZER, ,P. Growt, H. . ,hF inhibitory effec certaif o t n polysaccharides for chickens, Poultry Sei. 43 (1964) 1164. ] AVIVI[9 , "Hig,L. h grain protein conten wiln i t d tetraploid wheat Triticum dicoccoides Körn.", Proc. 5th Int. Wheat Genetics Sym- posium New Deli, 1978. (RAMANUJAM, S., Ed), India Society of Genetics and Plant Breeding (1979) 372. [10] RUBENTHALER BRUINSMA, L. Lysin, . ,G L. . ,eB estimatio cerealn i n y sb near-infrared reflectance, Crop (1978 _ Sei18 .) 1039.

151 PROTEIN IMPROVEMEN CEREALN TI . BARLEYSII 1

S.E. ULLRICH, T.K. BLAKE . KLEINHOFSA , , C.N. COON, R.A. NILAN2

ABSTRACT Stud t Washingtoa y n State Universit e inheritancth f o y f higo e h lysine genef theso barlen e i se us e y th (Hordeu o t d le m s vulgärha ) L. e mutant a pedigre n i s e breeding progra r improvefo m d protein. Crosses between several high lysine mutants and locally adapted, high yielding lines has resulted in the isolation of several lines with good protein quality and promising yield. Some of these lines have been evaluated in chick feeding trials with an indication of improved weight gain over normal barley. Althoug o linen he read ar r srelease fo y , parent building is progressing satisfactorily.

INTRODUCTION Protein improvement work with barley has been ongoing at Washington State Universit r manfo yy years untit e earl,bu th l y 1970' e emphasith s s s solelwa n maltino y g quality. Although malting quality improvemens ha t been and is still an integral part of the overall barley program, nutritional quality improvement has received considerable attention since the discover f 'Hiprolyo y 1 (Munc t al.e k ,e inductio 1970th e d th )an f o n Ris0 mutants (Ingversen et al., 1973 and Doll et al., 1974) with their increased grain lysine content. e overalTh l objectiv e proteith f o en improvement projec o prot s -i t duce nutritionally superior (protei d energyan n ) high yielding varieties. o accomplisT h this objective, background genetic studie d nutritionaan s l evaluations have been conducted. Basic researc e inheritancth n o h f o e several high lysine genes was conducted by Muench (1975) and Somers (1977)

Scientific Paper No. 5690. College of Agriculture Research Center, Washington State University, Project Nos. 1006, 0430, 0277 and 0233. 2 Assistant Professor of Agronomy and Assistant Agronomist, Depart- ment of Agronomy and Soils; Graduate Research Assistant and Professor of Genetics and Agronomist, Department of Agronomy and Soils and Program in Genetics; Associate Professor of Animal Sciences, Department of Ani- mal Sciences and Professor of Genetics and Agronomist, Department of Agronomy and Soils and Program in Genetics, respectively.

153 e relationshiTh p betwee hige th nh lysine gene f Hiprolo s d 'Bomian y ' Ris0 1508 was determined by Muench et al. (1976), which resulted in the apparent "double mutant", D-129. Screening techniques useful for breeding high lysine barley were also developed, as well as the building of an improved protein germ plasm base utilizing high lysine genes (Muench, 1975 and Somers, 1977). Initial nutritional evaluations of this germ plasm using chicks was conducted by Coon et al. (1979). Coincident with the genetic research a pedigree breeding program was developed utilizing several high lysine mutant d locaan s l materials adapted to the Pacific Northwest's semi-arid conditions. Interest in high lysine barley stems from the fact that lysine is e mosth t limiting amino aci barlen i d y when considering monogastric nutrition. The implications are obvious in areas where protein supple- mentatio n huma i nd livestoc an n k diet s lackini s g and/or expensive. Barley, primarily a carbohydrate energy source, has the potential to supply significant amounts of critical amino acids. e objectiveTh f thio s s o describreport e prograte ar th e o datt m e at Washington State University in breeding barley with nutritionally improved protein and to relate the plans for future work.

MATERIALS AND METHODS The basic approach to improving protein quality in barley at Wash- ington State Universit bees o increasha yt n e effectiveth e lysine content e grainith n . Thi s beeha sn attempte e introductioth y b d f higo n h protein and/or high lysine mutants. The basic sources used were Hiproly, Bomi Risfi 1508, D-129 (double mutant with the Hiproly plus Ris0 1508 genes) and Bomi Ris0 7. Crosses were made between these mutants and a number of

locally adapted varieties and advanced breeding lines including 'Steptoe', 'Blazer,1 'Boyer', 'Advance', 'Vale 70', 'Piroline', 'Vanguard', 'Klages', WA 7228-67, WA3564, WA 6468-67 ('Mari1 x 'Luther2' x 'Trail!1), and WA 6194-63 [ 'Forma1 x ( 'Triple-bearded Mariot1 x 'White Winter1) ]. The resultant lines were advance a pedigre y b d e selection system. Although lysine and protein contents were followed, selection criteria were primarily agronomic characters. Double as well as single crossed material have been utilized. e originaTha th s somi tf eo l advanced high lysine

154 breeding lines were crossed again to either another high lysine mutant r adapteo d variety/line. Thi s don wa so eithe t e r increas e lysineth e / protein contene adaptationth r o t . Advanced lines from the CIMMYT and ICARDA breeding programs have also been evaluated. Dats collectewa a d reportean d d herein from replicated yield trials d singlan w selectionro e s grow t Pullmana n . Yiel d percenan d t plump m sievem m sieve 9 m d thi1 9 )an 1 x n )x 4 (thrwer 2 2. e2. un measured(o . Protein was determined by the Kjeldahl method and lysine by the Udy dye binding method described by Mossberg (1969). Lysine content results were verifie y aminb d o acid analysis. Nutritional quality of some of the original advanced high lysine selections were evaluated with day-old male broiler chicks in replicated trials. These trials included barley test diets containing .3% supplemental lysin d .08%an e supplemental threonine, barley test diets containing % supplementa.3 l lysin d barlean e y test diets containin o supplementan g l lysin r threonineo e . Weight gaifeed an nd consumption were measured over weeo tw k a period.

RESULTS AND DISCUSSION e resultTh e preliminarysar thur fa s A hig. h protein and/or lysine variety release is not in the immediate offing, but parent building is progressing well. The lines and data presented in Tables 1 and 2 indicated the identification of the most promising material selected and e 197tested 197th an 8 9 n i dcro p years, respectively. The lines represented in Table 1, and other lines not shown, were used as parents or subjected to further selection in 1979. One line, Ris0 150 8x 7228-67 s advance,wa a majo o t dr yield tria n 1979i l n tha.I t trial its performance was similar to that of 1978 in terms of grain, protein and lysine yield, which placed it 10th among 24 entries. This line appears to show more promise as a parent, than a potential variety. o otheN r lins survive ha ee place b a majo o n t di d r yield nursery. However, there were severa * lineF l s selecte n 197i d 9 that appea o havt r e good potentia d severaan l l linea preliminar n i s y yield trial performed wel yieln i l d (Tabl . Thes2) e e lines, P.O.N , Mex47 . . 188 d Klage,an s t hav S 7211xno ed particularl4di y high lysine contents, however. 155 Tabl . l Improvee d protein selections with greatest promise froe 197th m 8 crop yea t Pullmana r , Washington.

Line/ Grain Protein Protein Lysinn i e Lysinn i e Lysine Cultivai Plump Thin yield in grain yield protein grain yield

°/ -Kg/ha- % -Kg/ha- V -Kg/ha- A67-2 8x Blazer 4 F , 76 7 5200 11.2 582 3.82 .428 22.2 70/2230 1x Piro l ine5 F , 26 14 2500 18.8 469 4.03 .757 18.9

Vanguar 70/22089x d 5 ,F 67 8 3600 15.2 548 4.10 .624 22.4

Hiprol6 F , WAx y 64 S5 86 3 3000 17.2 516 3.93 .676 20.3 A13 x Steptoe, FS 84 4 3200 14.8 473 4.22 .625 20.0

Ristf 150 87223-67x 6 ,F 81 6 4410 11.0 485 4.10 .451 19.9 Risrf 1508 55 10 2780 14.3 398 4.61 .659 18.3 Hiproly 36 17 2040 18.6 379 3.53 .656 13.4 Steptoe 90 4 4250 12.4 527 3.00 .372 15.8

+Numbered e crosselinear 0 s7 spreceeder o involvin A y b d g Hiproly. Data for the first five entries are from single rows, while data for the second four entries are from replicated preliminary yield trial. The single rows and yield trial were adjacent in the field. Table 2. Improved protein selections with greatest promise from the 1979 crop year at Pullman, Washington.

Line/ Grain Protein Protein Lysinn i e Lysinn i e Lysine Cultivar"1" Plump Thin yield in grain yield protein grain yield

01 -Kg/ha- V -Kg/ha- v -Kg/ha-

A412-75x Steptoe4 F , 81 5 5000 13.5 675 4.21 .568 28.4

A412-75 x 11304-73, F4 22 30 4700 14.8 696 4.14 .613 28.8 (A) A412-75 x 10500-74, F4 80 6 5700 13.5 770 3.66 .494 28.2 B A412-75 x 10500 74^ ^F4 68 9 5400 13.6 734 4.54 .617 33.3 C A412-75x 10500 74,^ ^F4 48 15 4500 14.2 639 4.17 .592 26.6 7 P.O4 . .N (ICARDA) 89 2 4430 14.9 660 3.39 .505 22.4 Mex8 18 . (CIMMYT) 94 1 5650 12.6 712 3.45 .435 24.6 Klages x 7211S 4 95 1 4970 13.1 651 3.48 .456 22.7 Risul 1508 4 38 3440 15.0 516 4.62 .693 23.8 Hiproly 3 32 2040 20.6 420 4.12 .828 16.9 Steptoe 95 2 5650 12.0 678 2.90 .348 19.6

+Numbered lines preceeded A or S are crosses involving Hiproly. e firsth Dattr fivfo a e entrie e frosar m single rows, whil e secon eth entriex datr si d fo e afro ar s replicatea m d preliminary yield trial. The single rows and yield trial were adjacent in the field. A close scrutin e dat2 reveal Table n th d i af an o y 1 s that protein and lysine yield/ha relies both upon grain yield and protein and lysine content, respectively. The dilema of combining high yield with high protein or lysine is seen in these data, as it has been in data from other breeding programs around the world. The apparent pleiotropic relationships between high lysine and shrunken endosperm has not satisfactorily been broken. The morphology of the kernels of the lines depicted in Tables 1 and 2 is such that the exceptional yielders have plump kernels with médiocre lysine e contenhighesth d an t lysine lines have shrivelled kernels. Ther s somi e e definit e source th gai n eo n mutant f Hiprolo s d an y Ris0 1508, however s evidence,a e percenth y b dt plum d thian pn data. The protein and/or lysine production per hectare is most critical and this mus e reconcileb t d wit a high h energy (primarily carbohydrate) yield. It is clear then, that high yielding cultivars must be developed wit n elevatea h d protein and/or lysine content. Ultimately e proteith , n and lysine produced in the kernels must be available to the people and livestock consumin e barleyth g o evaluatT . e this aspect, several high lysine parental lines were fed to chicks (Table 3). These lines were the progen f Hiprolo y x adaptey d variety crosses. These line turn i s n have been crossed with other adapted varietie o product s e e th som f o e material depicted in Tables 1 and 2. Weight gain and feed efficiency (feed/gain) were measured and comparisons were made among the high lysine parents and Ris0 1508 and Steptoe, with Soybean oil meal used as a control. (Table 3). Weight gain se highe b ten r o higt dfo r h lysine lines than either the Ris0 1508 or Steptoe checks for both amino acid supplemented and non- supplemented diets. However, statistical differences wer t demoneno - strate mosn i d t cases. Feed efficiencie e oppositesth tendee e b Th o .t d feed/gain ratios tendee higheb o t dr (less efficient e higth h r lysin)fo e lines than for the checks. The overall indication is that the high lysine barley produced higher gain, but in some cases at the expense of greater consumption. A problem with the availability of the lysine or the overall energy balanc e higr valuth o eh f lysino e e e indicatedbarleb y ma y . Progress in combining high yield with high protein quality in barley is apparent in the material developed at Washington State University and

158 in other programs in the U.S., Europe and the international crop improvement centers, namely CIMHYT and ICARDA. However, we have not seen a commercial cultivar released to date. The work at Washington State University will continue to emphasize w mutanne d introducean t d breeding line evaluations. High quality material will be incorporated into the existing pedigree high lysine breeding program. High lysine parent building and advanced testing of lines will continue along with nutritional evaluation of both parents and advanced lines.

A summarTabl . 3 ef chic o y k performance durin o weetw gk feeding periods with barley test djets supplemented with lysine and threonine and not supplemented."''

Weight gain Feed/gain ratio5 Line/ , .3% lys No .3% lys No CultivarT .08% th rs ly Suppl% .3 . r th .08% s ly % .3 Suppl.

g/chick A 13 97.5 b 2.17ab -- -_ A 13-10 94.9 b 99.2 b 2.16ab 2.02a — A 67-28 90.9 b c b 85.0 2 . 22 2.14a — B 134-27 89.4 b 80.G bc 2.23ab 2.35a -- C 195-31 86.8 b c b 82.c b 4 76.7 2.47a 2.21a 2.39ab Ristf 1508 84.7 b 78.1 bc 2.11ab 2.21a — Steptoe 89.1 b 69.1 C 56.2 c 2.07ab 2.25a 2.48ab Soybean oil meal a 2 125. 124. 3a 120. 3a 1.93 b 2.03a 2.10 b tData from Coo al.t e n, 1979. ^Numbered lines are crosses involving Hiproly. G feed consumed/g weight gained. a'b'cMeans within columns containin a commog n t significantlletteno e ar r y differen > .05) .P ( t

159 References

Coon, C. N., R. Shepler, D. McFarland, and J. Nordheim. 1979. The nutritional evaluation of barley selections and cultivars from Washington State. Poultry Sei. 58:913-918. . Egqum0 . B . K0i.DollB d an e, 1974 H. , . Induced high lysine mutants in barley. Radiation Bot. 14:73-80. Ingversen . DollH . K0iB d .an , e ,J. 1973 . Induced seed orotein mutants f barleyo . Experientia 29:1151-1152. Mossberg, R. 1969. Evaluation of protein quality and quantity by dye binding capacity a too :n plan i l t breeding . 151-160p , : IN . New Approaches to Breeding for Improved Plant Protein: Proceedings a Pane f o l Meetinw ApproacheNe n o g o Breedint s r Planfo g t Protein Improvement (Rostanga). Int. Atomic Energy Agency, Vienna 519/PUB/212. Muench . R 1975 . ,S . Protei d lysinan n e gene n barlei sn i d thei an ye us r quality improvement. Ph.D. Dissertation, Washington State University. Muench, S. R., A. J. Lejeune, R. A. Nilan and A. Kleinhofs. 1976. Evi- dence for two independent high lysine genes in barley. Crop Sei. 16:283-285. Munck, L., K. E. Karlsson, A. Haqberg and B. 0. Egqum. 1970. Gene for improved nutritional valu barlen i e y seed protein. Science 168: 985-98. 7 Somers, D. A. 1977. Inheritance of the high lysine genes in Hordeum vulgäre L. M.S. dissertation, Washignton State University.

160 A NEW APPROACH FOR THE USE OF HIGH PROTEIN VARIETIE MUTANTD SAN BREEDINN SI R GFO HIGH GRAIN PROTEIN CONTEN TRITICUMTN I AESTIVUM

A. BRUNORI . FIGUEROAA , . MICKA , E Lab. VACOIND, CSN Casaccia, Roma, Italy; Joint FAO/IAEA Divisio Atomif no c Energy in Food and Agriculture, Vienna, Austria

Abstract

Analysi kinetice th f so nitrogef so n accumulatio developinn i n g grai reveales nha d large difference rate duratiod th an e n si nitrogef no n deposition among genotypes of bread wheat. This has suggested the existence of particular genes governing the process of nitrogen accumulation»

Some experimental lines derived from the high lysine variety Nap Hal have apparently inherite pattere dth nitrogef no n accumulatiof no Nap Hal, indicating that kinetics of nitrogen deposition can be trans- ferre crossingy db .

One could assume that by transferring kinetics, genotypes could contribute the amount of nitrogen per grain which they are able to accumulate. Furthermore combininy ,b g high rate d lonsan g duratiof no nitrogen accumulation, it was hoped that transgressive types could be obtained which might accumulate amounts of nitrogen per grain superior tbese o th that f parento t verifo T . y these point o set stw f crosse so s were made. Analysis of the nitrogen content per grain of individual F plants supporte above th d e hypothesis numbe A . plantP f ro s were selected and the nitrogen amount per grain of individual plants of F, families will be determined to confirm the results obtained in P„.

Introduction Programme improvemene th r proteie sfo th f to n conten whean i t t usually varietief o involve us liner e so eth s with high protein percentage th n i e grai charactee donors nth a r sfo r 'high protein*. Although several genotypes are available which present high protein grain (Konzak 1977)» the variety playes •Atla ha majo e ' dth s66 r rol donos e a hig r rfo h protein genes (Johnson et al. 1969, 1978, 1979)« Following its use in crossbreeding, very often hige ,th h protein percentag founs ei taln i d l plants with small grains, and thi reducy s ma yieldin e eth w linesg ne abilit e .th f yo To avoid a reduction of yield, Jain has proposed to select first for seed weigh d successiveltan proteir yfo n percentag alwayt no e sear negativel y correlated and combinations of large grain and high protein percentage are possible (Jain et al. 1976). So far the search for high protein genes has been based on the protein percentage of mature grain and for this purpose, the grain protein percentage of the wheat world collection has been scored. Unfortunately, knowledge of how proteins are accumulated in developing wheat grains is very poor. We anticipate y proteind wa thae e accumulatetth sar reveay dma l differences among genotypes indicating the existence of a different kind of 'protein genes* which cannot be identified any more by analyzing the mature grain.

161 Consequently have ,w e investigate consecutivo tw n i d e year kinetice sth s of nitrogen accumulation in a number of varieties and mutant lines, possessing different percentage proteif mature so th n eni grain determinationl Al . f so seed weight and nitrogen content were based on those grains developing from the two lateral florets of each spikelet with the exclusion of the apical and basal spikelets. Commencing at mid flowering, 5-10 spikes per genotype were harvested twice a week. Depending on the genotype 9-12 samples were collected betteo T . r asses potentiae sth proteir lfo n accumulation, part plante ofth s were give supplementarna y dos nitrogef eo n fertilizer between heading and flowering, to secure nitrogen availability to the developing grain.

The stud nitrogef yo n accumulatio developine th n ni g grais nha indicate existence dth sourcef eo higf so h protein genes alternativo et widwela s 'Atla a s ela ' rangs66 ratef eo durationd san nitrogef so n accumulation (Brunor . 1979al »t ie 1980) orden I . tak o rt e advantagf eo the genetic variability reveale thin i d seto s tw crossef wayso , s were mad answeo et followine rth g questions:

there Ar e ) potentiaa l source higf so h protein genes besides those from 'Atlaconvenientle b ' than s66 ca t y use breedinn i d g programmes? possiblt i s I genetiw builo et ) ne b a d c potentia higr lfo h protein contents by combining high rates add long duration of nitrogen deposition?

Results to date Nitrogen accumulation studies. Data relativ firse th to t eyea r study (1976/7? nitrogen )o n accumulation have already been published (Brunori et al. 1979t 1980). Relevant observation yearo tw se th investigatio r sfo summarizee b n nca followss a d : Among the genotypes investigated a large variation was observed for seed weight, nitrogen amount per grain and nitrogen percentage of grain.

Few genotypes showed higher amounts of nitrogen per grain than that of the high protein variety 'Atla ' whics66 h nevertheles highese th d tsha percentag nitrogef eo grain e th n ni . Nitrogen amount per grain appeared to be a stable characteristic whilst a considerable variation was observed for seed weight and nitrogen percentage.

In general, late nitrogen fertilization affected the amount of nitrogen per grain in a positive way, though a large variation in response was evident among genotypes. High protein lines, however, did not respond to late nitrogen fertilization. Rate duratiod san nitrogef no n accumulation varied widely depending upogenotypee nth , suggestin existence gth differenf eo t genes governing the process of protein accumulation.

In 1977/7 weathee 8th r conditions were drastically different from those experienced in 1976/77« Compared to the first year, the second was cold and rainy, and this may explain the "low^r rates and longer duration of nitrogen accumulation observed in general. However, genotypes which had shown high rates and/ar long duration of nitrogen deposition maintained these characteristics.

Lines derived frohige th mh lysine variet Halp ,yNa which have been selected for the high lysine percentage in the protein, appeared to have inherite kinetice th d nitrogef so n accumulatiod an l nHa frop mHa consequentl same yth e amoun nitrogef o t grainr npe . This finding indicated thakinetice th t nitrogef so n depositio relates amoune i n th f o to t d nitrogen per grain and that both characteristics can be transferred together by crossing.

162 The observation nitrogen so n amount grair spe maturf no e th seed d san results of kinetics studies led us to conclude the following: - There is a level of nitrogen per grain, characteristic for each genotype, which canno exceedee tb d even when large amount nitrogef so n are available during the process of seed development. - Several gene higr sfo h protein contenpresene b y tma t which regulate the accumulation of nitrogen according to different kinetics. - Kinetics of nitrogen accumulation can obviously be transferred by crossing. Therefore, a breeding programme for the improvement of the protein content of the grain was initiated to test the following hypotheses : 1. Genotypes which accumulate in the grain larger amounts of nitrogen than "Atlas 66" and which present kinetics of nitrogen accumulation clearly different may represent alternative sources of high protein genes. To verify this point the high yielding, low protein variety "Kalyansona" was crossed as mother with "Atlas 66" (USA), "F 26-70" (Romania) and "5-3" (India). large Th e . variatio2 ratef no duratiod san proteif no n accumulation observed among genotypes might permit the creation of a new genetic potential for protein accumulation. By combining genotypes with supplementary kinetic nitrogef so n deposition larger amountf so nitroge grair npe n tha bese n th that f o parent croste th use sn i d coul expectede db r thiFo . s fouf o purpos rt se crosse ea mades swa : "5 - 3" x "F 26-70" "TW-l" x "Lancota"

(high rate (very high (late high (high initial and long rates) rates and long rates) duration) duration)

up 307-65" "51092" "TW-l" "Atlas 66" (long duration) (high rates) (late high rates (long duration) and long duration)

Potential donor higr sfo h protein genes

Analysi nitrogee th f so n conten grair graid pe t nan n yielr pe d spike of individual F_ and parental plants of crosses involving the varieties "Kalyansona", "Atlas 66", "5-3" and "F 26-70" has shown that high protein parents can actually transfer to the recipient variety the full potential of nitrogen accumulation per grain. "5-3" and "P 26-70" were more effective than "Atlas 66", confirming the expectation of the potential value of these two lines as donors for high protein genes.

Combination of the grain yield per spike of "Kalyansona" and of the nitrogen conten hig e grair th h pe t f nproteio n parent s observeswa d in all the crosses indicating that yield per spike and nitrogen accumulatio necessarilt no grair e npe nar y competing processes. Quite unexpected, transgressive types with amounts of nitrogen per grain higher than that of "F 26-70" were observed in the cross "Kalyansona 26-70"F " "crossee x Th . s have been plannebasie th sn do resulte kinetice th th f f o so nitrogef so n accumulation obtainen i d 1976/77. In that year the kinetics of nitrogen accumulation of "F 26-70" was at any time superior to that of "Kalyansona" and no complementation between the kinetics of the two genotypes could be expected. However, in the season 1977/78 the kinetics of nitrogen depositio "Kalyansonaf no 26-70F " d ""an presente possibilita d r yfo complementation. The climatic conditions in 1977/78 were quite similar to thos 1978/7f eo 9 under werg whicF e th hgrow thi d explaiy nan s ma n the transgression observed in this cross. 163 Building new genetic potential for protein accumulation

In fouf threo rF t population eou secone th f dso grou crossesf po , plants were observed with amount nitrogef so grair pe n n higher than thosf eo the best parent supporting the hypothesis that it may be possible to achiev genetiw ne ea c potentia nitroger fo l n accumulation three th er Fo . crosses whos „ planteF s showed transgressive amount nitrogef so grainr npe , the study of the kinetics of nitrogen deposition has indicated for both years considerable area possiblf so e interaction betwee kinetice e th n th f so parent genotypes F frocrose e th mth 307-65F s" n I . ""51092x " transgression for nitrogen content per grain was not so evident. However, at varia e with the results of 1976/77» the kinetics of nitrogen accumulation of 1977/78 did not indicate the possibility for any complementation. Summe n 197ri s simila9wa o tw thao re t 197f o tth d therefore - 8an F e th n ,i pools of genes may have expressed themselves according to the performance of 197'8 with little, if any, chance for complementation.

Though negative, the results of the cross "F 307-65" x "51092" add support to the hypothesis that complementation of kinetics may occur in F provided that the parents used in the cross show complementing kinetics or nitrogen accumulation under the climatic conditions under which the F_ is grown.

Although it is premature to draw firm conclusions, the knowledge of the kinetic nitrogef so n accumulatio developinn ni g grains appeare b o st very usefu planninn i l g breeding programme improvemene th r e sfo th f o t protei creatioe th w genetin ne r conten f fo no cd potentiaan t r higfo lh protein grain.

References

BRUNORI, A., H. AXMANN, A. FIGUEROA, and A. MICKE, 1980: Kinetics of nitrogen and dry matter accumulation in the develop!jg seed of some varietie d varietiesan d mutansan t line Triticuf o s m aestivum. Pflanzenzüchtun. Z 201-218, g84 .

KONZAK, C.F., 1977î Genetic control of the content, amino acid composition, and processing properties of protein in wheat. Advances in Genetics 19, 407-582.

JAIN, H.K., N.C. SINGHAL, and A. AUSTIN, 1976: Breeding for higher protein yields in bread wheat: Experimental approach and phenotype marker. Z. Pflanzenzüchtung 77, 100-111.

JOHNSON, V.A., P.J. MATTERN S.Ld ,an . KUHR, 1979: Genetic improvemenf o t wheat protein : SeeIn .d protein improvemen cerealn i t d graian s n legumes, IAEA, Vienna, 165-181.

JOHNSON, V.A., P.J. MATTERN, D.A. WHITBD, and J.W. SCHMIDT, 1969: Breeding for high protein content and quality in wheat. In: New approaches to breedin r improvegfo d plant protein, IAEA, Vienna, 29-40.

JOHNSON, V.A., P.J. MATTERN, K.i). WILHELM!, and S.L. KUHR, 1978: Seed protein improvement in common wheat (Triticum aestivum). In : Seed protein improvement by nuclear techniques, IAEA, Vienna, 23-32.

164 STUDIES OF BARLEY HIGH-LYSINE MUTANTS AND SEED PROTEINS AT RIS0

H. DOLL, J. JENSEN, B. K0IE, M. KREIS, J. TORP Agricultural Research Department Ris$ National Laboratory DK-4000 Roskilde, Denmark

ABSTRACT

Studie e beinar s e effecgth f carriehigh-lysinn o o t t ou d e genes on yield, the relation between starch and protein production, e starcth h synthesi n high-lysini s e genotypes possibilite th , r yfo lysine rich storage protein, the induction of low-hordein mutants e inheritancanth d f hordeino e resulte e studieth Th .f e o s ar s reviewed for possible approaches to surmount the main obstacle of of utilizing high-lysine mutants in barley breeding, i.e., the negative association between lysine conten graid an t n yield.

1. Effect of high-lysine genes on grain yield

Studies of high-lysine mutants have shown that such mutants n generai l hav a reducee d grai imparen a n o t yiel de starcddu h accumulatio e endospermth n i n . This yield depression represents the main obstacle for utilizing high-lysine types in agriculture. An estimatio e yielth f dno depression associated wita h particular high-lysine gen s veri e y time consuming, because th e e geneffece separate th b n questio i e f o o t t s dha n froe mth effecte genetith f o s c backgroun othed an d r mutationsr ou n I . studies of mutant Ris0 1508 we have compared the segregated normal high-lysind an - e lines after crossin e mutanth g t with a standard variety and producing chromosome doubled haploids from the F. [1]. This method is not suitable for studies of many mutants, and we have therefore been looking for a more simple test procedure. e mutantth l have Al w s e found hav a distince t effecn o t seed size already in the F_ generation, where a segregation of shrunken see s i easild y detectable. Figur show1 e e weighth s t

165 80

O) E 60

Z 0 LU 40

O UJ UJ 20 O) o Iys3a homozygotes + normal seeds

0L 10 2 5 5 1 10 25 30 bottom FLOWER POSITION top Fig. 1. Relation between seed weight and flower position of indi- vidua- spikF n _ seeda eF l n froi e scros th m s Ris0 150 x 8Sultan . The seeds are segregating for the high-lysine gene lysZa.

of the individual F„ seeds in a spike from the cross Ris0 1508 x Sultan. The genotype of the seeds was detected by electrophoresis (see below) o thing.Tw s appear from figur First. 1 e e ,th seeds being homozygous for the recessive mutant gene lysSa are considerably lighter than the normal seeds. Second, seed weight is strongly dependent on the position of the flower in the spike. However, apart fropositioe th m n effect variatioe ,th n seei n d weight among e.g e norma.th l seed s smali s l comparee th o t d difference between normal d high-lysinan - e seeds. w evaluatinno e ar possibilite e th gW utilizinf o y g segre-

gating F2 seeds for the estimation of the effect of a high-lysine calculatioa gene d ,an n procedure, which eliminate positioe th s n effect on seed weight within spikes, is developed. Sixteen high- -lysine mutants and lines were crossed with 'Sultan', and F_ seeds have been harvested. The seeds are weighed and analysed for starch and hordein content. The method requires that it is possibl o detect e e segregateth t d mutant homozygotes amon„ F g r thiseedsbees fa ha s n o .S , possibl 56 , 16 mutantr fo , e8 , 7 s 527, and 1508 by means of SDS-polyacrylamid gel electrophoresis of the hordein. Studies of the lysSa gene in Ris0 1508 have shown that the

seed weight reduction estimate basie th f segregatin so n o d 2 F g

166 seed vers i s y similagraie th no r t yiel d depression observen i d field tests of advanced lines.

2. Relations between starch proteinand production

The relations betwee chemicae th n l compositioe th d nan grain yield have been studie high-yieldingn i d , well-adapted varietie morn i wels a ses a lexoti c varietie d linean s s [2]. A procedure was developed for rapid routine-determination of the predominant nitrogeneous constituents as well as the major classe carbohydratef so s [3], Variatio grain i n n yiels dwa primaril e resulth y variatiof to amoune th n f starcti no h produced e yiel .Th proteif o d s positivelnwa y correlated with starch percentage yieldth t ,bu proteif graie o th s n wa i n negative correlated with starch yield becaus increase th e n i e starch yiel s highewa d r thae increasth n protein i e n yielr fo d a certain increase in grain yield. The compositio proteif no n depended upo e starcnth h yield level. While the amount of hordein produced per unit area was relatively constant amoune ,th non-hordeif to n proteins produced was positively correlated with the starch yield. These relations between protein and starch gave rise to a higher content of proteie lysinth n i f higheeo n r yielding thaw yieldinlo n - va g rieties variatioe .Th e qualite proteith th n i f no s cony i n - sidered to reflect an increasing need for metabolic proteins in high yielding varieties.

3. Starch synthesis in high-lysine barley

The shrunken endosperm and smaller kernel weight of the high-lysine mutant almoss i s t entirel consequenca y - re a f eo duced starch synthesis throughout the development of the endosperm. A much higher than normal sucrose content in the high- lysine mutant endosperms indicates biochemicae blockth n i s l pathways leading from sucrose to starch. A concomittant reduction in prolami d starcan n h synthesi s observe smutantl wa al n i ds tested even thoug e gene th e hlocate ar s n differeno d t chromosomes or in different loci. The combination of two high-lysine genes in one single barley genotype, for example mutant gene 1508 with mutant gene 527 or 29, further reduced prolamin and starch synthesis [4]. The double mutant 527/1508 almost completely prevented prolamin synthesis and reduced starch synthesis much more than each mutant gene alonetherefors i t .I e probable thagenee th t s 1508, 7 affec52 d t2 an 9starc h synthesi differenn i s t ways.

167 In an effort to get more detailed information about starch synthesi e endosperth n i s high-lysinf mo e mutants, investigations were performed on starch phosphorylase, ß-amylase, starch synthetase and ADP-glucose pyrophosphorylase from developing endosperm mutanf so s tparen it 150 d t8an variet y Bomi [5], Although mutant 1508 endosperms contained much less soluble ß-amylase than Bomi, this difference was not likely to be responsible for the reduced starch synthesis of mutant 1508. Un- primed starch synthetase activity was 40% lower in mutant 1508 than in Bomi [6]. So far this is the only enzymatic difference observed in mutant 1508 as compared to Bomi likely to be re- e reducelateth o t dd rat f starco e h synthesi mutante th n i s.

4. Lysine rich proteins in high-lysine barley

The possibilities of finding genotypes with an improved abilit o synthesizt y e lysine rich proteins with storage propertie e elucidatedar s n somI . e high-lysine mutants, e.g. Hiproly and e reducemutanth , 56 td hordein conten partls i t y counter- balance concomitana y b d t increas n ß-amylasei e , z-proteid an n o proteastw e inhibitors e increas.Th n thesi e e proteint no y sma f significanco onl e b y e lysinth r efo e content coult i t d,bu f importanco als e ob overcominr fo e e graith g n yield reduction relate moso t d t high-lysine mutants. A variety wit vera hw conten lo y f z-proteito bees nha n found. Genetic studies have shown that this reduction in z-protein is due to a single recessive gene. This gene will be utilized e significanc ia studnth f o y f z-protei o ee lysinth r efo n conten e grai th wels a tns a l yiel f high-lysino d e barley. Screening of barley varieties from world collections has shown large variation in the content of ß-amylase, which is another lysine rich seed protein with storage properties. Se- lected varieties are being regrown for repeated analysis in an attempt to find lines with increased or decreased ß-amylase content.

5. Screening for low-hordein mutants

Seeds of a chromosome doubled line of Sultan have been treated with sodium azide, ethyl methanesulphonate, or gamma rays e treate.Th d seeds gav totaa e f abouo l t 15000 fertile

1 plantse M spik on s harveste d ewa ,an d from eac . plantM h e Th . material has been screened for low-hordein mutants by analysing two seeds from each M. spike by the turbidity test [7]. If one or both of the two seeds had a clearly reduced turbidity, and thereb w hordeilo a y n content e embryo,th e growar s n agair fo n

168 repeated turbidity test. The hordein composition, lysine content, genetics, and grain yield of the selected mutants will be studied during the contract period. The above mentioned mutagenic treatment and screening wil e repeateb l n 1980i d .

6. Genetic studies of hordein

e storagTh e proteibarlee th f yno endosperm, hordein, contain majoo tw s r fraction f polypeptidesso , hordein- d hordein-2an 1 , which differ somewha aminn ti o acid compositio d othenan r chemical properties [83. The polypeptide composition of hordein is studie y SDS-polyacrylamib d l electrophoresisge d , which also gives a separation of the two hordein fractions. We have shown tha e compositiotth f hordein-no d hordein-an 1 s controllei 2 d by two corresponding loci, tiorl and HorS [9], which are linked with 7.4 - 0.9% recombination and located on the short arm of chromosome 5 [10]. HOT! is nearest to the centromere, and the hordein loce locatear i d e locclosth i o t eMl-ad Ml-k an containin knowge th mos nf to milde w resistance gene barleyn i s . All available data indicate that Horl and HorS contain the structural genes codin r hordeinfo g . Both locf io appeae b o t r compound nature because each one codes for several polypeptides. The high-lysine mutant Ris0 56 is characterized by a very w contenlo hordein-2f o t a somewha d an , t increased hordein-1 content [11]. It has now been shown that this change is due to a mutation at or near the locus HorS coding for hordein-2 polypeptides. The mutant gene is recessive in its qualitative effect e electrophoretith n o c banding patter f hordein-2no - co t ,bu dominant with respect to the relative amount of the hordein fractions [11], Homozygous mutant seeds were abou smalle% 10 t r than normal seeds.

7. References

[1] DOLL K0IE, ,H. , Influenc ,B. high-lysine th f eo e gene from barley mutant 150 n grain,carbohydrat8o proteid an e n yield, Seed Protein Improvement by Nuclear Techniques (Proc. Meeting Baden, 1977), IAEA, Vie'nna (1978). 107. TORP] [2 Relation, ,J. s between productio f starcno d an h percentage, qualit yield an yf protei o d barleyn i n . ,Z Acker- u. Pflanzenbau 148 (1979) 367.

169 [3] TORP, J., Patterns of variation in major chemical consti- barlee tuentth f yo s grain, Thesis, Departmen f Geneticsto , Royal Veterinary and Agricultural University, Copenhagen, and Agricultural Research Department, Ris0 National Labo- ratory (1979)pp.89. [4] KREIS, M., Starch and prolamin level in single and double high-lysine barley mutants, Physiologia Plantaru 8 (1980)1394 m . KREIS] [5 , Starc,M. h synthesi barlen i s y mutants deficienn i t storage proteins, Thesis, Université Catholique de Louvain- La-Neuve, Belgium (1979)pp.127. [6] KREIS, M., Primer dependent and independent forms of soluble starch synthetase from developing barley endosperms, Planta (in press). ] K0IE [7 NIELSEN, ,B. "Extractio, ,G. separatiod nan hordeins"f no , Techniques for the Separation of Barley and Maize Proteins, Rep. EUR-5687e (1977. 25 ) K0IE.] [8 INGVERSEN, ,B. ANDERSEN, ,J. , A.J., DOLL EGGUM, ,H. , B.O., Compositio nutritionad nan l qualit barlef o y y protein, "Evaluatio f Seeno d Protein Alteration Mutatioy b s n Breeding, IAEA, Vienna (1976) 55. DOLL.] [9 BROWN, ,H. , A.H.D., Hordein variatio wiln i n d (Horaeum spontaneum) and cultivated (H. vulgäre) barley, Can. J. Genet. Cytol. 21 (1979) 391. [10] JENSEN, J., J0RGENSEN, J.H., JENSEN, H.P., GIESE, H., DOLL, , Linkaghordeie H. th f eo n loci Eorld Hor2an witpowe th h - dery mildew resistance loci Ml-k and Ml-a on barley chromosome 5, Theor. Appl. Genet.(in press). [11] DOLLnearlA , ,H. y non-functional mutant e storagallelth f eo e protein locus Hor2 barleyn i , Heredita press)n (i s .

170 STUDIES OF WHEAT AND MAIZE MUTANTS WITH RESPECT TO PROTEIN CONTENT AND QUALITY

M. DENIC, KOSANA KONSTANTINOV . DUMANOVIJ , C Maize Research Institute, Belgrad - Zemun, Yugoslavia

Abstract

STUDIE WHEAF S O MAIZD TAN E MUTANTS WITH RESPEC PROTEIO T N CONTENT AND QUALITY. n introductorI papee y th resultpare previour f rth o tou f so s work on the general picture of genetic variation in protein content and composition in hexaploid winter wheat and maize were summarized. papee Parth f ro t deals wit e comparisohth e changeth f no proteif o s n complex in M -19 mutant line of wheat with those of opaque-2 mutant of maize r thi.Fo s purpose distributio nitrogef no varioun i n s Osborne protein fraction theid san r amino acid compositios nwa investigated case maizf th o e n .I e within each Osborne protein fraction poly.peptid.es were determined. Distribution of lysine in various polypeptide d aminsan o acid compositio majof o n r polypeptide f zeino s s were analyzed date .Th a obtained show that changes occure n amouni d d aminan t o acid compositio Osbornf o n e protein whea9 fraction1 - t M mutan n i s t line were less pronouncen i d comparison with those in opaque-2 mutant of maize. Distribution of lysine in various Osborne protein fractions and their polypeptides in whea s differenwa t n comparisoi t n wit e sam maizehn th i e . Wheat mutant lines were analyzed with respec graio t n yiel d proteian d n yiel wells a d .

INTRODUCTION

Since cereal grains are a major sorce of vegetable proteins, increasin quantite gth graif yo d improvinnan g cereal grain protein have become increasingly important mutatio.A n breeding project provide goosa d opportunit o investigatt y possibilite eth y of approaching this goal. r result possibilitiee e studOu th th f o yn so f inducino s g heritable variatio mutatioy nb proteif o n n conten d compositioan t n i n hexaploid winter wheat (Bezostaj hav) 1 ae demonstrated significantly brodened variation of these traits (3 - 7)- Compared with controls, an increased variation in protein content was found in Mp and M, generations. Multiple regression analysis of T-L data strongly indicated that the observed variations contain a heritable component. At low and high doses of the inutagenic agents studied

171 correlation coefficients were relatively low, whereae th t a s medium doses higd significanan h t inter-year correlations were obtained. With respect to protein composition of the three essencial amino acid primarf o s y importanc whean i e t (14) lysine ,th d ean methionine content proteie th f o sn were negatively correlated with the protein content doet i prevent sno t ,bu n increasa t f theio e r contents in per cent of the kernel weight with increasing protein content.The threonine contenproteine th f o t , however s abou,wa t the same at all protein contents. Protein content and looo-grain weight were often negatively correlate mutann i d t lines. Detailed analysis have showen that different rautagenic agent gamma-rays( s var) , neutronsEl y d an S ,EH n "mutatioi n spectra" with relative regarth o t d e contributiot na the other variable observee th o st d protein yield (5)« Special

attentio mutan e gives th nwa o tnt line advancen i s d generations (Hq) with elevated protein content (1). This type of mutation material (deriving from variety Bezostaja 1) have showen negative correlation between grain yield per plant and protein content. Usual negative correlation between protein content and lysine content in protein fount no thin i ds swa case. Large variatio contenn ni Osbornf o t e protein fractions was found. Prom the densitoraetric tracings of the polypeptides from each Osborne protein fraction in selected mutant lines there seem to be more quantitative than qualitative differences between the control and the mutant lines. From the data on the distribution of lysine in various polypeptides it seems that there were no very lysine-rich polypeptides. An induced variation was found in some quantitative characters (plant hight and grain weight) in other varieties , Bankut 12o5 and hybri j (9,lo)ol d effece . Th mor s wa te clearcu Bankun i t t 12o5 because of the higher stability of the control material of this variety. These results indicated tha tgamma-radiatiow mediulo d an m n dose e preferablar s n breedini e g work e isolatioaiminth t a g f o n high yielding mutant lines free from deleterious mutations. In the case of maize the contents of protein and sixteen amino acids were determine endospere th n i d m par f singlo t e kernels belongin e grou th differenf o po t g t sublines inbre r fivfo de generations following gamma-irradiatio kernel f o ne lin th e f o sV-51 2 and the group of genetically different maize lines (2, 4-, 11). A considerable variation in both protein content and composition s observedwa . After mutagenic treatment with gamma-irradiation variatio n lysinni e conten alss wa to observed .regressioA n analysis of values from the H^ and Iv generations after irradiation permitted the conclusio ntotae thath lf t o variatio abou% 5 6 t n linean(i r scale- s heritable)wa e correlation.Th s found suggested groupinf o g the amino acids. Grou : lys1 p , his, arg, asx, thrt me ,d an gly l ,va

172 were clearly Intel-correlated, very strongl e cas f th lys-arg-glyo e n yi , somewhat paire weaketh sr rfo asx-hi d asx-metsan thesl .Al e amino acids were negatively correlated with the protein content. Group 2: the remaining amino acids were, with certain exceptions (e.g. ile-val) more or less negatively correlated with group 1 amino acids, and they showed mostly positive correlation with protein content. Corre- latio f lysino n totao et l protein content showed that line mediuf o s m protein content exhibit the highest lysine produc'cion. Mathematical analysis of the number of principal causes of variation of this material demonstrated that nine dimension e needesar o explait d n 96 % of the total variation in mutant group, but only six dimensions e grou th e cas geneticallf o pf th o e n i y different lines e induce.Th d variation had certain similarities with the variation occuring in genetically different lines with the exception that the total variation in the case of mutant lines was considerably smaller as compared wit groue hth f geneticall o p y different lines. One of the most promissing developments for the improvement of the quality of the storage proteins came after the discovery of Mertz et al.(15) thae opaque-th t 2 mutandeastia o t maizf o td cle e change in the amino acid pattern of the endosperm protein. Further studies (13) showed that those change overale th n i sl amino acid composition in opaque-2 endosperm could be accounted for by changes in the relative proportion of the Osborne protein fractions. Concerning the polypeptides separated by electrophoresis on polyacrylamide gel there was an additional polypeptide around 2o,ooo MW in the normal genotype (16). Amino acid analysis showed that polypeptide highef o s contributW rM e increaseth o et d lysine contenn i t the mutant s alswa o t .founI d that opaque-2 gene reduces protein conten d l,ooo-graian t n weigh different a t t exten differenn i t t inbred lines. Variation of endosperm texture and some biochemical traits, resulting from the action of modifier genes within a number of homozygous opaque-2 converted materials of different genetic backgrounds was studied (12) n genera .I founs wa dt i l thaamoune th t proteif o t n was slightly increased, lysine content as pre cent of protein (and also his, arg, asx and gly) somewhat decreased, but content of glx, leu and phe (and perhaps pro and ala) slightly increased in modified endosperm compared to standard opaque-2 soft floury endosperm. A tendency was indicated for RNase activity to be decreased with increa- sine proportioth g e modifieth f o n d endosperm. Further studies (17) showed tha day2 2 t s after pollination there highea s wa r accumulatio zein-f normae no th n 2li genotype than i n the mutant. However, the absolute amount of glutelin in the mutant endosperm was higher than in the normal genotype. Analysis of alpha- amino nitrogen showed a higher amount of all 19 free amino acids studie mutanr pe d t endosper me endosper th tha n ni normaf o m l maize.

173 However, there wer o sucen h differenc relativn i e e concentrations for most of the free amino -acids of both genotypes. e papeTh r deals wit e comparisohth e changeth f o nf proteis o n complex in M - 19 mutant line of hexaploid winter wheat with those occure n opaque-i d 2 mutan maizef o t . Selected wheat mutant lines were analyzed with respec graio t n yiel d proteian d n yield wells ,a .

RECENT RESULT DISCUSSIOD SAN N

In orde o compart r e typ th ef changeo e protein i s n composition of M-19 mutan f wheao t t with thos f opaque-o e 2 mutan maizef to , fractionation of proteins was performed using the method of Osborne. From the data presented in Table 1 and 2 it is evident tha whean i t t mutant albumins were reduced, whereas globulins and glutelins increased knows i n opaque-t i ni s .A 2 mutanf o t maize glutelin d albuminan s s were increased, whil alcochoe th e l fraction (zeins) was reduced.

Table 1.- DISTRIBUTION OF NITROGEN IN PROTEIN FRACTIONS OF y weightMAIZdr D WHEAg AN E r )Tpe MUTANN g (m T

n : te o Pr Maize Wheat % of Mother rncf s on ti Normal Mutant normal Mutant % of variety mother

Albumins 0.57 1.95 342 5.43 d.68 86

Globulins 0.54 0.5B 107 2.40 3.00 125

Zeins , . Gliadins ; 5.14 2.45 48 5.82 6.30 108

Glutelins 2.08 3.93 189 1.93 2.93 152

Extraction was performed in the presence of 2-mercojpto- ethanol

The amino acid compositio f variouno s Osborne protein fractions' in maize and wheat is shown in Tables 3 - 6. In the case of water soluble fraction in wheat mutant (Table 3) a remarkable increase (more mothef o tha % 5 n r variety s foun )wa lysn i d , his and asx, whereas the amounts of pro,val,met,ile and tyr were decreased. These change e lesar ss pronunce comparin i d - son with the same fraction in opaque-2 of maize, where the s morwa eamounx thaas f nto twic mucs a ecomparison i h n with normal genotype. Therefore, the relative amounts of other ami- o acidsn , except tyr, were reduced.

174 Table 2.- RELATIVE DISTRIBUTION OF NITROGEN IN PROTEIN FRACTION MAIZF SO WHEA D EAN T MUTANT (in % of total N)

Protein Maize Wheat % of fractions Normal Mutant normal Mother Mutant % of variety mother

Albumins 6.83 21.91 321 34.21 27.67 81

Globulins 6.48 , 6.47 100 15.40 17.74 115

Zeins ion Gliadins1' 61.70 27.47 35 37.37 37.25

Glutelijis 24.98 44.15 177 12.39 17.33 140

1)Extraction was performed in the presence of 2-mercopto- ethanol Table 3.- AMINO ACID COMPOSITION OF WATER SOLUBLE' FRACTION (ALBUMINS) FROM WHEA MAIZD AN T E (MOLE PERCENT)

Maize Wheat Amino acids % of Mother % of Normal Mutant normal variety Mutant mother

Lys 4.17 3.37 80.81 2.97 3.53 118.85 His 1.99 1.36 68.34 1.90 2.10 110.53 Arg 5.14 3.25 63.23 4.20 4.10 97.62 Asp 18.79 43.27 230.28 7.85 8.38 106.75 Thr 4.45 2.83 63.59 3.35 3.33 99.40 Ser 4.76 3.02 63.44 6.06 5.78 95.38 Glu 14.15 11.81 83.46 25.16 25.96 103.18 Pro 8.41 6.45 76.69 11.04 10.21 92.48 Gly 9.28 5.97 64.33 7.44 7.59 JL02.02 Ala 9.17 5.94 64.78 6.77 7.07 104.43

Cys1' ------

VoJ 5.69 3.24 56.94 5.46 5.10 93.41 Met0 0.40 0.19 47.50 0.93 0..81 87.09 lie 3.54 2.09 59.04 3.35 3.13 93.43

LPU 5.72 3.43 59.96 7.17 6.83 95.26

Tyr 1.95 2.21 113.33 2.57 2.31 89.88 Phe 2.40 1.57 65.42 3.78 3.78 100.00 1) Cye and Met are partially decomposed or oxydized. 175 Tabl AMIN- 4. e O ACID COMPOSITIO d SOLUBLNa F O N E PROTEINS (GLOBULINS) FROM MAI'/ D WHEAAM E T (MOLE PERCENT)

Maize Wheat Amino acids % of Mother % of Normal Mutant normal variety Mutant mother

Lys 3.63 3.50 96.42 , 3.06 2.99 97.71 His 4.22 3.84 90.99 2.44 2.38 97.54 Arg 5.08 6.55 128.94 5.84 5. 04 100.00

Asp 6.98 6.52 93.41 5.97 6.13 102.68 Thr 5.24 4.85 92.56 3.53 3.46 98.02 Ser 7.86 6.27 11 IB. 6.65 6.20 93.23 Glu 16.21 15.69 96.79 25.64 26.03 101.52 Pro 12.27 10.60 86.39 8.61 8.60 99.88 Gly 9.87 10.22 104.07 8.32 8.15 97.96 Ala 8.55 8.83 103.27 6.41 6.48 101.09 Cys1' 0.66 1.11 (168.18) 0.47 0.75 (159.57) Vol 6.09 5.97 98.03 6.12 5.63 91.99

Met1) 0.55 0.48 87.27 1.06 1.09 102.83 He 3.17 3.21 101.26 3.27 3.38 103.36 Leu 7.71 7.52 97.54 6.96 7.11 102.15 Tyr 2.18 2.27 104.13 1.99 2.08 104.52 Phe 2.35 2.59 110.21 3.65 3.69 101.09

1) Cys and Mat are partially decomposed or oxydized.

Data presente n Tabli d sho4 e w that change n amini s o acid compositio f wheao n t mutant globulins were withi% n5 _ rang+ f o e e motheth f o r variety. Exception d valan c n 1-honlI r .ar s se ey cas opaque-f o e 2 mutan% 10 werc ty ph eb proportion d an y ar f so higher than in normal genotype. At this level the amounts of ser, pro and met were reduced in opaque-2 mutant.

I'll, nujo.'j in i uni no ncid t'oni| ion i L I on of ;i 1 rr>c;ho I rouiihli.1 prol^Jnii in wliivU. iiiuL.iiiL woiro los.", prouoimcod in c-oni|>ii r i ;;on with I ho:',<> Jn opaquo-2 mut,ml (Tnhl.e 5). The nnunnii'-1 of ,"

Maize W h e a t Amino acids % of Mother % of Normal Mutant normal variety Mutant mother

Lys 0.14 0.29 207.14 0.71 0.73 102.82 His 1.20 1.47 122.50 1.51 1.53 101.32 Arg 1.13 1.67 147.79 1.94 1.90 97.94

Asp 4.57 4.15 90.81 2.61 2.66 101.91

Thr 2.83 3.08 108.83 2.51 2.51 100.00

Ser 5.64 6.47 114.72 6.10 6.48 106.23

Glu 22.93 22.05 96.16 35.38 35.64 100.73 Pro 11.16 12.22 109.50 16.03 15.40 96.07

Gly 2.43 4.58 183.93 4.93 5.18 105.07 Ala 12.85 12.43 96.73 3.76 3.77 100.26

2) Cys 0.29 0.38 (131.03) 0.56 0.66 (117.86)

Vol 3.59 3.35 93.31 3.86 3.89 10U.78 2) Met 1.18 1.02 H6 . 44 1.08 1.12 103.70 lie 8.96 2.78 93.92 3.41 3.44 100.88

Leu 18.96 15.53 80.85 8.79 7.99 90.90

Tyr 3 44 3.63 105.52 2.32 2.47 106.47

Phe 4.44 4.89 110.13 4.50 4.65 103.33 1) UN I raclion w;u; performed in the. prnsouco of 2-mort-qpto- eLhnnol 2) e partiallCys'anar t Me d y decompose oxydizer o d d acid composition of glutelin fraction (Table 6). Most changes in the wheat mutant were within +_ 5% except for ser and val wich were othee lowerth rn .O hand opaque-e th , 2 mutant showed a higher amoun f lyso t , arg a lowe d ,an r asxu le ,d an gly e il , amoun f glxto . The separation of polypeptides within each Osborne protein fraction was performed using SDS polyacrylamide gel electrophoresis (PAGE) e dat.Th a obtaine whean o d t mutant lines were reported earlier. Here are presented data on maize. With respece patterth o f polypeptidet o n s thers wa e distinct difference in zeins between normal genotype and opaque-2 mutant (Fig.1). 177 Table 6. AMINO ACID COMPOSITION OF NaOH SOLUBLE PROTEINS (GLUTELINS) FROM MAIZE AND WHEAT (MOLE PERCENT)

Maize Wheat Amino acids Norntal % of Mother Mutant % of Mutant normal variety mother

Lys 4.02 5.16 128.36 3.06 2.99 97.71

His 3.61 3.61 100.00 2.44 2.38 97.54 Arg 3.77 4.66 123.61 5.84 5.84 100.00 Asp 6.88 8.01 116.42 5.97 6.13 102.68 Thr 3.87 4.16 107.49 3.52 3.46 98.29

Ser 5.23 5.43 103.82 6.55 6.20 93.23 Glu 20.13 13.96 69.35 25.64 26-03 101.52 Pro 9.28 8.50 91.59 8.61 8.60 99.88 Gly 5.83 6.51 110.71 8.32 8.15 S 7. 96 Ala 9. 33 9.19 98.50 6.41 6.48 101.05 Cys1' — - - 0.47 0.75 (155. 57) vol 6.73 7.12 105.79 6.12 5.63 91.99 Met" 1.4« 1.68 113.51 1.06 1.09 102,83 lie 3.46 4.14 118.96 3.27 3.33 103.36 Leu 10.20 10.80 105.88 6.95 7.11 102.30 Tyr 2.92 3.21 109.93 1.99 2. Go 1042 .5 i Phe 3.20 3.85120.31 3.55 3-69 10] .09 j i

The group of small adjacent polypeptides or larger well separated polypeptides on the gel were cut and hydrolized in .6 N HC1 at 110°C for 20 hours. After hydrolyses the samples were purified usin ion-exchange th g e rezin Dowex-50. Lysine content in each section was determined by amino acid analyzer. The l represenge number e picture th th e section n e th tf so o e th . of s gels that were analyzed for lysine content or amino acid composition. The results obtained (see Table 7) show that the highest amoun f totao t l lysin protein i e s foun nwa glutelinen i d s (40.4% d lowe) an globuli n i r n (31.7% albumind )an s (23.8%)e .Th lowest amount of lysine was found in zeins (2.1% in normal and n opaquo-i 6.0% 2 rnutnnt )Distributio. f lysino n e .in various polypeptides within one Osborne protein fraction of maize is differen n comparisoi t n wite sam f th wheaheo t proteinse th n .O basis of these data it seems that there are no very lysine rich polypeptides. 178 CI

Fig.l. Electropf oregraniB of Osbornc protein fractions from maize endosperm: a - albumins; b - globulins; c - p;]utclins; - dzein s from normal genotype - zeinr e ; , from opnqnp-J? mutant

Data presented in Table 8 show content of amino acids of some poly- peptdde f zeino s s from endosper normaf o m d opaque-lan 2 mutanf o t maize. These data show differences between genotype n amini s o acid composition of polypeptides of the same molecular weight. Differences were also found polypeptidebetweeo tw e e nsamth th e f genotypeso . For estimation of grain yield and protein yield wheat mutant lines were planted in plots. The plants were harvested, grain yield and protein content was measured. Data obtained are presented in Table 9. It shoul pointee b d t thaou dt these n advancei line e sar d generation (H,, generation mosd )thef an o t m were preselecte proteir fo d n content. Thus, this is not random variation of the traits above mentioned. Selected mutants deriving from EMS treated material showed decreased grain yield with increased protein content as per cent of dry matter. With respec proteio t n yiel line9 d s showed 5 lowehighe1 d r ran protei n yield in comparison with the mother variety Bezostaja 1. This means that increased protein content per dry matter will not increase protein yield if the grain yield is reduced at higher extent. This suggests that in these mutant lines there was reduction in carbohydrates but not real increase in protein content. As expected some of mutant lines deriving from El and gamma-rays treatment showed higher protein yield in the case when protein content was slightly reduced. Futher work is require n ordei d o explairt n this kin f relationshipo d .

179 Table ?• Lysine content in various subtractions (polypoptido^) within each Osborne protein fraction in m a i 7. o ^

2") Fraction Section number of each rçel -' „n|.a1 123 4 5 fi

ALBUMINES : nmole lys per 1.14 2.13 1.6? 2.2? 1.33 - 8."« section cenr totaf Pe o t l 13.4 25.1 19-7 26.3 15-* - 2?.8

GLOBULINES: nmole lys per 2.33 1.65 3-35 3-95 - - 11.28 section Per cent of total 20.7 14.6 29-7 35.0 - -31.7

ZEINS: r pe nmol 0.1s ly e 8 0.11 0.14 0.05 0.15 O.OQ Q.7? section5^ 0.55 - 0.71 - O.HJ - 2.19 r cen total^25.f Pe o t 0 15-3 19. 9 6. 420. 8 12.1 52. 25.1 - 32.4 - /)2.5 - A.n

GLUTEIINS: nmole lys per 2.32 4,55 3.57 4.15 - - 14.30 section Per cent of total 16.1 30.2 24.8 28.8 - - 4O.4

" ^Mean valu f fouo e r .«sectionn.

2)'For corresponding noet numbern ao fi~.e se s l

3"o fir.'Dat)th sn t rov i a i correspon e normath o lt d penotype, wherea e seconth w corresponn ro i sd e th o t d opaque -2 mutant of maize.

180 Tabl . e8 AMDT O ACID COMPOSITIO SOMF NO E POLYPEPTIDEF SO ZEINS FROM ENDOSPERM OF NORMAL AND OPAQUE - 2 MUTANT 1)

n molr pe e sampl e mol r cenpe e t

AMnro ACID NOR MAL MUTANT NOR MAL MUTANT

sec. 5 sec6 . sec. 3 sec. 5 sec. 6 sec. 3

LYS 11.8 12.9 4.1 8.0 6.7 1.9

HIS 2.3 3-3 1.4 1.6 1.7 0.7

ASP 13-7 90 11.8 9.3 4.8 5-3

THR 4.6 13.1 6.9 3.1 6.8 3.1 SER 25.0 23.6 20.7 16.8 12.3 9-4 GLU 16.5 34-2 42.0 11.1 17.8 19.0 PRO 15.3 18.0 31.2 10.3 9-4 14.1 GLY 17-7 11.8 11.8 12.0 6.1 5-3 ALA 18.5 26.6 32.6 12.4 13.8 14.8

VAL 3-4 2.0 10.8 2.3 1.1 4-9 ILE 2-5 4-4 3-3 1.7 2.3 1.5 LEU 14.0 27.8 34.2 9-5 14.4 15.5 PHE 2.8 5-4 9-9 1.9 2.8 4.5

TOTAL 148.1 192.4 220.7 100.0 100.0 100.0

1) Trp, argwerr ty e , d degradecysan t ,me d during hydrolyses.

In the future work with wheat main attention will be given to the studies of selected mutants and their derivatives segregating from the crosses with local varieties with respec graio t n yield, protein yield and yield of lysine. In the case of maize attention will be given e studietth o higf o s h protein opaque- 2e studie th version o t s d san of modified opaque-2 versions with respece improvementh o t f o t the protein quantit d qualityan y .

181 Table 9« Grain yield, protein yield and protein content of wheat mutant lines ' deriving from the three experiments (treatment with EMS, El and gamma-rays)

Protein content Grain Yield Protein Yield Line

f~t\ 0 % of % of 2 of mother kg/pi ot""' mothef '6o r D.M. M.V. kg/plot ^ variety variety

1 2 3 4 5 6 7

Experiment 1: Treatment with EMS

M.V. 14.3 100.0 3.01 100.0 0.43 100.0 1. 16.7 116.8 2.83 94-0 0.47 109.3 2. 16.9 118.2 2.83 94.0 0.48 111.6 3- 16.7 116.8 2.75 91.4 0.46 107.0 4. 16.4 114.7 2.22 73.8 0.36 83.7 5. 17.1 119.6 2.81 93.4 0.48 111.6 6. 17.3 121.0 2.57 85.4 0.44 102.3 7. 18.0 125.9 2.48 82.4 0.45 104.7 8. 16.7 116.8 2.82 93-7 0.47 109.3 9- 14.9 104.2 2.99 99-3 0.45 104.7 10. 17.6 123.1 2.75 91.4 0.49 114.0 11. 14.9 104-2 2.57 85-4 0.38 88.4 12. 15.7 109.8 2.70 89.7 0.42 97-7 13- 16.2 113.3 2.08 69.1 0.34 79-1 14. 16.8 117.5 2.23 74-1 0.38 88.4 15- 15.2 106.3 2.29 76.7 0.35 81.4 16. 15-4 107-7 2.28 76.1 0.35 81.4 IT- 16.0 118.9 2.18 72.4 0.35 81.4 18. 15.1 105.6 2.44 81.1 0.37 86.0 19. 15.2 106.3 2-39 79.4 0.36 83.7 20. 14.8 103-5 2.33 77.4 0.34 79.1 21. 14.1 98.6 2.45 81.4 0.35 81.4 22. 15-7 109.8 1.56 51.8 0.24 55.8 continued

182 1 2 3 4 5 6 7

Experiment 2: Treatment with EI

M. V. 14.6 100.0 2.54 100.0 0.37 100.0 24. 13.9 95.2 2.47 97.2 0.34 91.9 25- 14.9 102.1 3.02 118.9 0.45 121.7 26. 14.7 100.7 2.82 111.0 0.41 110.8 27. 15.1 103.4 2.82 111.0 0.43 116.2 28. 14.3 97-9 2.89 113.8 0.41 110.8 29. 14.0 95.9 3.02 118.9 0.42 113.5 30. 14.6 100.0 2.12 83.5 0.31 83.8 31. 14.7 100.7 2.36 92.9 0.34 91.9 32. 13.9 95.2 2.50 98.4 0.35 94.6 33- 14.4 98.6 2.29 90.2 0.33 89.2 34. 15.2 104.1 2.62 103.1 0.40 108.1 35- 14.9 102.1 2.72 107.1 0.41 110.8 36. 15.4 105.5 2.55 100.4 0.39 105-4 37. 16.2 111.0 2.06 81.1 0.33 89.2 38. 14-7 100.7 2.42 95.3 0.36 97-3 39. 15.3 104.8 2.13 83-9 0.33 89.2 40. 14.7 100.7 2.49 98.0 0.37 100.0 41. H.7 100.7 2.89 113.8 0.42 113-5

Experiment 3: Treatment with Gamma-rays

M. V. 15.2 100.0 2.81 100.0 0.43 100.0 43- H-7 95-5 3.12 111.0 0.46 107.0 44- 15.4 101.3 3-01 107.1 0.45 104.7 45- 15.4 101.3 3.18 113.2 0.49 114.0 46. 15.0 98.7 2.89 102.8 0.43 100.0 47. 14.2 93-4 3.16 112.5 0.45 104.7 48. 15.9 104.6 2.59 92.2 0.41 95.3 49- 16.2 106.6 2.58 91.8 0.42 97.7 50. 15-1 99-3 2.65 94.3 0.40 93.0 51. 15-1 99-3 2.84 101.1 0.43 100.0

' Wheat mutant lines in M,. generation.

. m 0 1 siz e plos f o eTh twa 2 }' 2

•" M.V. denotes mother variety.

183 ACKNOWLEDGEMENTS This stud s supporteywa granty db s froFune Republif th mo d c Internationae ofth Serbiy b d aan l Atomic Energy Agenc Viennan i y ,

REFERENCES

/!/ DENl6,M., " Some charasteristics of proteins in mutant lines of hexaploid wheat",Seed Protein Improvement by Nuclear Techniques,IAEA,Vienna (1978)365. /2/ DENIC,M.,DUMMOVIC,J.,BERGSTRAND,K.G.,EHRENBERG,L., On principal components of variation in amino acid composition of maize endosperm proteins after gamma-irradiation, Genetik (1969)25L aJ . / DENIC,M.,DUMANOVIC,J.,EHRENBERG,L./3 , Indukovano variranje sadrzaja protein aminokiselini a au zrn u meke psenice, Savremena Poljopr. 11-12(1969)85. /V DENICjM.,DUMANOVIC,J., "Improvement of cereal grain protein quantity and quality through induced mutation", Peaceful Use Atomif so c Energy (Proc Conf. Geneva,1971) 12, UN, New York, and IAEA, Vienna (I972)2ol. /5/ DENIG,M.,DUMANOVIC,J.,EHRENBERG,L.,EKMAN,G.,SIMIC,R., Heritable variations in protein yield and its components, induced by radiation and mutagenic chemicals, Genetika 8 (1976)163. /6/ DUMANOVIC,J.,DENIC,M., "Variation and heritability of lysine conten maize"n i t Approachew ,Ne Breedino st r fo g Improved Plant Protein (Proc.Panel Röstanga,1968),IAEA, Vienna (I969)lo9. / DUMANOVIC,J.,EHRENBERG,L.,DENIC,M./? , "Induced variatiof o n protein content and composition in hexaploid wheat", Improving Plant Protei Nucleay nb r Techniques (Proc.Symp. Vienna,197o),IAEA, Vienna (I97o)lo?. /8/ DUMANOVIC,J.,DENIC,M.,JOVANOVIC,C.,EHRENBERG,L.,"Variâtion in content and composition of protein in wheat induced by mutation",Nuclear Techniques for Seed Protein Improvement (Proc.Meeting Neuherberg,1972),IAEA,Vienna (1973)153. / DUMANOVIG,J.,DENIG,M./9 , Variatio somf o n e quantitative character whean i s t induce y gamma-rab d y irradiation,J,Sei. Agr.Res (1967)98o .2 . /lo/ DUMANOVIU,J.,DENIC,M., Radiation induced heritable variation of quantitative characters in wheat, Hereditas 6_2 (1969)221.

184 /Il/ DUMANOVIC,J.,DENIC,M., "Variatio heritabilitd nan f o y lysine conten maize"n i t Approachew ,Ne Breedino st g for Improved Plant Protein,IAEA,Vienna (1969)lo9. /12/ DUMMOVIC,J.,DENIC,M.,KONSTMTINOV,K., Variation i n kernel phenotyp d soman ee biochemical propertien si opaque-2 from different genetic backgrounds, Genetika 6 (1974)211. /13/ JIMENEZ,J.B.,"Protein fractionation studie higf o s h lysine corn", Proc.High Lysine Conf.Corn Industries Res Fundation, Washington D.O. (1966)74. /14/ JOHNSON,V.A.,MATTERN,P.J., "Protein Improvemen Wheaf o t y b t Breeding", Improving the Nutrient Quality of Cereals II,AID Wahington,D.C.(1976)134. /15/ MERTZ,E.T.,BATES,L.S.,NELSON,O.E., Mutant gene that changes protein compositio d increasenan s lysine content, Science 14-5 (1964)279. /16/ POPOVIO,V.,DENIO,n.,DUîlANOVl6,J., Influenc opaque-e th f o e 2 gen proteinn o e maizn i s differenf o e t genetic backgrounds, Genetik (1974)7a6 - /17/ SIMIO,R.,DENl6,M., Relationship between protein composition and the content of free amino acids in the endosperm of normal genotyp n opaque-a d an e maize,f 2o . Genetik (1975)25_ a7 -

PROJECT: "STUDIES Or WHEAT AND MAIZE PROTEIN MUTANTS ;/ITH RESPECT TO GRAIN YIELD, AND PROTEIN YIELD AND QUALITY"

Principal investigator: Dr. M. Denic Maize Research Institute, Belgrade-Zemun, Yugoslavia

Programme

e cas th f wheao en I t main attentio e studies giveth i n o t n s of protein mutants in advanced generations and their derivatives. This work will include: - Déterminacio graif o n n yiel mutantf o d d segregatesan s frocrossee th m s with local varieties; - Determinati proteif o n n conten d yiel an tmutanti o d d an s tehir derivatives; - Detrmination of lysine content and lysine yield of mutants and their derivatives. f thio sm o increas t worai s e i k Th e nutritional valuf o e wheat varieties.

185 e cas th maizf o en I e attentio e studies givei nth o nt f o s high protein opaque-2 versions and to the studies of modified opaque-2 version with respece improvementh o t proteif o t n quantity and quality. This work will include determination of protein content and lysine content in: modifie- d endosper differenn i m t stages (back-crosses) vith material with modifier genesd ,an - Endosperm vith incorporated opaque-2 gene in the genotypes with high protein (295 lines).

186 THE IDENTIFICATION AND STUDY OF GENETIC VARIATION USEFU IMPROVINLN I G GRAIN PROTEIN ATTRIBUTE WHEAN SI T

C.N. LAW, J.W. SNAPE, P.I. PAYNE, A.J. WORLAND AND P.A. HARRIS Plant Breeding Institute, Maris Lane, Trumpington, Cambridge, U.K.

ABSTRACT

Followin gshora t revie previouf wo s studie protein so n genef so whea related tan d species resulte ,th f studieso proteif so n genes located on chromosomes of the homoeologous group 5 of Triticum aestivum and Triticum spelta, and chromosome 2M from Aegilops comosa, particularly their effect on grain yield by using substitution lines of common wheat, are reported. In a similar way, protein genes located on the homoeologous chromosome group 1 of Aegilops umbellulata, Ae. sharonensis and Ae. columnaris, producing high molecular weight protein subunits assumed to affect bread-making quality, were investigated.

INTRODUCTION

In previous work carried out under contracts 1667/RB, 1667/R1/RB and 1667/R2/RB it has been shown that intervarietal chromosome substitutions for chromosomes of homoeologous group 5 have effects on total grain protein production (Law et al., 1978). In particular, two genes, Pro! and Prog, were tentatively located on chromosome 5D. One of these genes, Pro!, was possibly identical to e vernalisatioth n gene Vrn3 o tha,s t this gen s probabli e y less important in breeding for increased protein because of correlated effects on maturity e seconTh . d gene, Pro2, however t appeano d o havy t r,di an e adverse correlated effects. Following the earlier reports by R. Morris (Morri al.t e s , 1978) that chromosom s responsibl Atlaf wa o 6 D 6 s5 e r fo e the high protein value of this variety, it was thought that the gene Pro2 located in studies at this institute, could be the site of the

187 gene responsibl Atlae th 6 effect 6 sr fo e .wore th Park f sincto e 1978 has therefore been concerned with establishing whether the Atlas 66 5D effect can be observed in European wheat backgrounds and also, if this effece identifiedb n tca , whethe allelis i t ri o Pro2 t c . A major part of the earlier work was also concerned with investigatin potentiae th g l contributio f "alieno n " chromosomes from related specie o increasint s g grain protein levels. Because numerous alien chromosome substitution lines were available for homoeologous grou , initia2 p l studies were confine o thit d s grou f chromosomeo p s (Law et al., 1978). Using particular experimental designs, it was possibl o rant e k several alien chromosomes against their wheat homoeologues in terms of their ability to produce grain protein. In this way it was shown that chromosome 2M from Aegilops comosa was more potent in producing grain protein tha U fro2C n m Aegilops umbellulata thad m fro,an t2R m Secale montanue leasth ts potenmwa t protein producerwheae th tr Fo . chromosomes mose th t s effectivwa A ,2 e produce proteinf ro , followed by 2B and then 2D. In all the experiments 2D was shown to be less effective than 2M, so that several lines carrying 2M substituted for 0 consistentl2 y produced higher protein amounts than their recipient varieties. Further experiments investigatin effece M substitute2 th g f to d in the variety Maris Widgeon have been carried out. Apart from the effects that genes from alien sources may have on grain protein production s evideni t ,i t that grain proteine b typn ca e changed appreciably by introducing genes from alien sources. In the earlier work several different protein subunit t presene no sth n ti variety Chinese Spring were controllee founb o t d y chromosomb d U 1C e of Aegilops umbel!ulata. One of these different subunits is of high molecular weight and is probably a glutenin, which could be of value in improving bread-making quality. Work to introduce the genes responsible r som fo f theso e e subunit underways i s B technique, "5 usin e th g o t " induce homoeologous recombination betwee e alieth n n chromosoms it d an e related wheat homoeologues.

188 RESULTS OBTAINED SINCE 1978 1. Stud f intervarietao y l chromosome substitution lines e substitutioTh n programme involvin s carriewa g D t Atla5 ou d 6 6 s f Hobbito b usinsi a ,semi-dwara g f variety s growa K U n e widelth n i y the recipient monosomic series. Because the substitution of a chromosome into a recipient variety requires a lengthy backcross programme, the substitutio D intf Atla5 o n o6 6 s stoppeHobbi e seconwa th b t si ta dd backcross, and the selected monosomic substitution lines were crossed reciprocally with Hobbi monosomib si t . Monosomic5D c s were then selected d disomian d fe c l anlinese d s extracted. This procedure permit a direcs t compariso e madb eo t nbetwee e effectD chromosomAtlae 5 th n th 6 6 sf o s e s homologuit d an Hobbin n equivaleni o e b si t t segregatinbu t g genetic backgrounds. A total of 40 different extractions, 20 for the Atlas 66 5D chromosome and 20 for the 5D homologue in Hobbit sib, was obtained from these reciprocal crosses and the performance of these was observed e fieldith n . The results of this experiment are summarised in Table 1 and show D chromosom5 thae th t en effec a agai s n percentagha to n e protein, which canno e accounteb t termn i yielf r o s fo d d variation, sin^. meane th ef o s the two 5D chromosome populations are not significantly different in yield, whereas they are for percentage protein. However, rather surprisingly, the subsamplc- carrying chromosome 5D of Atlas 66 gave a lower percentage protein than the subsample carrying its homologue from Hobbit sib n thiI . s particular cross, therefore e increaseth , d protein of Atlas 66 over Hobbit sib is not due to chromosome 5D. Evidently, n thio s test, chromosom f boto D h 5 d AtlaeHobbi an 6 shoul6 b s si t e b d more poten t increasina t g grain protei nf Chineso tha D 5 n e Sprin- g the recipient variety used by R. Morris in detecting the Atlas 66 5D effect.

In other trials involving substitutiod an B 5 , n 5A line r fo s 5D from Atlas 66, Cappelle-Desprez and Cheyenne into Chinese Spring, all three chromosomes were show o havt nn effec a e n totato l protein production majoe Th .r effects were associated wite substitutioth h n f chromosomo D but5 e , significantly e totath , l protein productior fo n

189 CS(Cappelle-Despre slightls wa ) y5D z greater than CS(Atla ) althoug5D 6 6 s h

both lines were much higher than Chinese Spring. Cappelle-Desprez feature parentage th n i s f Hobbio e t sib s possibli thao , s t i t e thae th t

Cappelle-Desprez 5D chromosome is present in this variety. If this

were the case, then the results of this investigation involving Chinese

Spring substitution lines would agree with the reciprocal monosomic

results described above.

n anotheI r investigations homozygous recombinant lines derived from

crossing Hobbit sib with a substitution line of this variety in which

chromosome 5A has been replaced by its homologue from Triticum s pel ta

were studied. Triticum spelta 5A differs from its homologue in Hobbit

sib by the presence of 3 marker genes. These are Vrnl for spring-winter

habit, ^ for speltoid ear and Bj[ for awn inhibition. A fourth factor

s alsi o possibly discernible r grai fo thid s ni san , hardness. This i s

f locatethio e shor th m s n ar tchromosomdo s i probabl d an e y homoeoallelic

with Ha, the more potent gene for hardness on 5D. Because of these

markers t i wa, s possibl o identift e y recombinan non-recombinand an t t

chromosome thao e s precisio , th t 5A f f o sgeneticano l analysis wa s

enhanced considerably.

Using this material, amounting to 74 lines, in randomised block

experiments sown in the winter and in the spring of 1978/79, it has

been possibl o shot e association a w n betwee e marketh n r gened an s

percentage protein. This informatio s ni show indicated n Tabli nan , 2 e s

tha gena t r geneo e s locateproximae th n i d l region f eitheo s e th r

lon r shoro g s i responsibl A t 5 armeffecte f th o s grair n o sfo e n protein

levels. It is possible that some of these effects may be due to a

gene homoeoalleli o Prot c n chromosomo 2 . 5D e

effece Th f chromosomo t. 2 grain o M n2 e protein productioe th n ni

variety Man's widgeon

A further experiment was carried out in 1979 using Maris Widgeon

and the Man's Widgeon trans 2D/2M line in which a translocated chromosome

involvin chromosommajoga M 2 e r th par f eo t combined wit e shorth hm ar t

of 2D has been substituted. This experiment consisted of a drilled trial

in which each plot was 1.5 m x 4.5 m and each genotype was replicated

190 six times. Again percentage protei e Mari s higheth wa n s r Widgeofo r n trans 2D/2M (13.28) tharecipiene th n t variety (12.88 )however- , with this degre f replicatioo e n this differenc t significantno s ewa A . combined analysis has therefore been made on the performance of these o linetw s ove yeare th r s 1976 throug cleas i o 1979t t hI r .fro m these results (Table 3) that the translocated line is consistently higher throughout all the trials. Analysis of this combined data in terms of seasons and line/season interactions, shows that there is a significant linear relationship between the environmental component and its inter-

action with genotype. This relationship can be described in terms of the following equation: line response = 13.96 ± 0.32 + (1 ± 0.08)ie where £ stands for the mean environmental effect. This indicates that the 2M effect is greater as the mean percentage protein increases with the environment othen I . r words, under poor protein producing conditions thM effec2 e t becomes negligible, whereas under good protein producing conditions, percentage protein differences arising froe introductioth m n M int2 f o o Maris Widgeoe appreciablb n ca n % 2 s d muccoula an s e a e hb d different under very high protein producing conditions. Unfortunately, since UK conditions, and the genetical backgrounds of UK varieties, ten o product d e lower protein amounts ,s likeli the t i n yM 2 thae th t effect will only be small in terms of UK plant breeding prospects. Further work, in which the causes of this percentage protein difference associated with this chromosom e identifiedb y ema , should indicate whethe e extrapolationth r s predicte e abovth ey b dequatio e valiar n d or not.

3. Alien chromosome introductions and protein type In work carriet thia st ou Institutd e (Payne, Corfield an d Blackman, 1979 a clos) e correlatio s founwa n d between certain high molecular weight (HMW) subunits of glutenin and bread-making quality. Studies using wheat aneuploid lines have shown that thes W subuniteHM s e determinear y geneb d n homoeologouo s s grou 1 chromosomesp . However, e numbeth f differeno r W subunitHM t s doe t appeae extensivb no s o t r e within the hexaploid wheat. This possible limitation has therefore prompted screening of some of the related species of wheat. Rather 19] surprisingly, this showha s n tha Aegilope t th man f o y s accessions carry HMW subunits not present in hexaploid wheat and in some cases these units are much more than those presen whean ti t itself. Wors ha k therefore been initiate o introduct d chromosomee th e s carryin genese th g , genee th sr o themselves, responsibl r controllinfo e g som f theso e W eHM subunits. From earlier work mentioned above, it had already been established that chromosome 1C of Aegilops umbel!ulata carries genes controlling U at least 2 HMW subunits. One of these subunits has an approximate molecular weight of 120,000. Crosses have been made between substitution lines of 1C in Chinese Spring with Chinese Spring nullisomic 5B U tetrasomic 5D. The resulting hybrids have been further backcrossed e nullisomitth o B tetrasomi5 c D stoc5 c d plantan k s deficienB 5 r fo t and carrying a single dose of chromosome 1C have been selected. In U these plants homoeologous chromosome pairin s beeha gn observed an d recombinant lines involving chromosome 1C with chromosome IB have been identifie y endosperb d m protein screenin f halo g f grains. These recombinant lines are now being grown for further study. The reason for carrying out this recombinant exercise involving chromosom evidens s i thai t U i t 1C et that this chromosome carries genes which adversely affect yield. It is also probably responsible for the production of different^ -gliadin patterns, presumably arising from allelic difference e locth i t a responsibls r thesfo e e proteins located e shoroth n e grout th arm chromosomes1 pf o s y selectinB . g recombinant products involving this chromosome it is hoped to recombine out the genes responsibl e adversth r efo e yield effect d als o an tesst o t whether the variatio gliadin i n n patterny consequencan s ha s n qualityeo . In other work W subunit, HM gene r fo ss have been transferred into adapted wheat background e "shot-gun y meanth b s f o s " approac whicn i h h the amphiploid between wheat and related species has been used in crosses with nullisomic 5B tetrasomic 5D rather than single chromosome addition lines. In two instances involving Ae. sharonensis and Ae. columnar!s, backcross derivatives, genetically very close to the recipient wheat parent, have been obtained which carry genes for HMW subunits present in the alien species. These lines are being investigated further in order

192 to determin e consequencth e f suco e h gene substitution n flouo s r quality and other grain attributes.

FUTURE WORK

1. s Chromosominfluencit d an n graio M e2 e n protein production Further work is necessary to describe the effect of this chromosome from Ae. comosa on grain protein production. At the moment, the effects f thio s chromosome have only been describe n threi d e different genetic backgrounds s therefori t I . e necessar o explort y e consequencth e f o e 2M substitutions for chromosomes of homoeologous group 2 in other varieties, particularly thos presenn i e t commercial use. In addition M effec2 e e causen proteith th ,to f o s n production e varietith n y Man's Widgeon e understoodneedb o t salreads i t I .y known that this substitutio o effecn s n proteitha o n n typt that bu ei t influences some of the components of yield appreciably. Thus, increased grain size has consistently been observed in all the substitution lines involving 2M over a number of seasons. Whether this increase is associated with reduced grain numbers per ear is not known at the moment, but such a compensating effect is quite likely and could have a major influence on the final amounts of protein in the grain. Studies of these yield component d othean s r associated characters will therefor e carrieb e t ou d in the near future. e introductioTh . 2 f geneo n s from alien species affecting protein type This promising area of research should be expanded and more extensive survey f Aegilopo s d relatean s d species carrie e samdth eoutt A tim. e available chromosome additio d substitutioan n n lines involving alien species should also be screened. Work to introduce genes controlling HMW subunit y meanb s f homoeologouo s s recombination shoul e encourageb d d an d the consequenc f suco e h gene introduction n qualityo s , amino acid levels and total grain protein examined.

. 3 Effect f homoeologouo s s grou chromosome5 p othed an s r chromosomes n proteio n levels e grouTh 5 chromosomep s have been pinpointe s carryina d g genes important in the control of grain protein levels. Further investigations 193 f theso e effects shoul continuede b d , using aneuploi inter-vanetad dan l chromosome substitution lines currently available, with a view to

establishin e componength t grain protein characters being controlley db

these chromosomes. However, variation amongst other group f chromosomeo s s

needs to be considered also, as is evidenced by the different grain

protein levels obtained fro e studmth f alieo y n chromosomes homoeologous

o thost f grou o ef wheat o 2 p .

REFERENCES

Payne, P.I., Corfield ,Blackmand K.Gan . , J.A. (1979).

Identification of a high-molecular-weight subunit of glutenin

whose presence correlates with breadmaking qualit n wheati y f o s related pedigree. Theoretical Applied Genetics _55, 153-159.

Law, C.N., Young, C.F., Brown, J.W.S., Snape, J.W. and Uorland, A.J.

(1978). The study of grain protein control in wheat using whole

chromosome substitution lines. In Seed Protein Improvement by

Nuclear Techniques, IAEA, Vienna, 483-502.

Morris, R., Mattern, P.J., Schmidt, J.W. and Johnson, V.A. (1978).

Studies on protein, lysine, and leaf-rust reactions in the wheat

cultivar Atlas 66 using chromosome substitutions. Proc. 5th Int.

Wheat Genetics Symp., New Delhi, 447-454.

Table 1. Percentage protein and yield for reciprocally derived disomic

substitutions for chromosomes 5D of Hobbit sib and Atlas 66.

Percentage Yield protein

Hobbit sib 5D/Atlas 66 x Hobbit sib 5D (20 lines) 12.83±0.08| ***161± 9.6] s n v s I {carries } Hobbi50 b si t

Hobbit sib 5D x Hobbit sib 5D/Atlas 66 (.20 lines) 12.31±0.1lJ 171±11.5, {carries Atlas 66 50}

Atlas 66 (2 lines) 13.85 194

Hobbit sib (2 lines) 12.08 193

*** P < 0.001

194 Tabl . 2 e Mean percentage protein associated with each marker class amongst homozygous lines obtained from recombining chromosome 5A of T. spel ta with its homologue Hobbit-siD in a Hobbit-sib background.

Marker genotypes No. of lines Winter Sowing Spring Sowing Vrnl (T.sp.) 33 11.70 13.40 vrnl 40 11.87 13.43

43 n.59* * | 13.57)* bl (T.sp.) 30 11.92J 13.32)

Q 44 11.58)* 13.607* £ (T.sp.) 29 11.92) 13.303

ha 40 11.66-1* Ha (T.sp.) 33 11.94J 14.163

0.05-0.0P * 1 ** P 0.01-0.001

Tabl . 3 e Percentage protei f Mario n s Widgeo d Marian n s Widgeon yeare th 2D/2 sr 1976fo M , 197 1979d 8an .

1976 1978(1) 1978(2) 1979 Mean Maris Widgeon 15.7 12.3 13.7 12.9 13.6

Maris Widgeon 2D/2M 16.7 12.8 14.4 13.3 14.3

Mean 16.2 12.5 14.4 13.1 Difference 1.0 0.5 0.7 0.4

195 CEREAL PROTEIN QUALITY

BJ^DR . EGGUNO M National Institute of Animal Science Departmen f Animao t l Physiolog Chemistryd yan , Rolighedsvej 25, DK-1958, Copenhagen, Denmark

Abstract Discoveries of strains of naize, barley and other crops having higher levels of essential amino acids,, have shown the differences in nutritional quality that can occur among strains of crop varieties. Comparisons are made between the total lysine conten f commoo t n cereal d selectean s d high-lysine mutants t appearI . s from these comparisons thae totath t l lysine content expresse n perceni d f proteio t s veri n y hig n somi h e varieties. However e digestibilitth f i , e individuath f o y l amino acid component s takei s n into consideratio e picturth n s somewhai e t different. Experi- mental data show that lysine especially, has a loui availability in several of the cereal grains s assumei t I . d that thi s becausi s e I/sin s mainli e y depositee th n i d protein fraction f loweso s t digestibility. Base n theso d e observations e validitth , y concepe oth f f additivito t f groso y s e questionedvalueb n ca ss alsi t oI . documented that when barley, rye, wheat, maize and sorghum are fertilized with increasing amounts of nitrogen more protein will be deposited in the prolamins. As the prolamin frac- a pootiot highl s bu ri n y digestible sourc f lysineo e , more digestible proteit bu n of lower biological valu s synthesisedi e r oat d ricFo e situatio.an s th e s diffei n - rent as glutelin (relatively rich in lysine) is the main storage protein in these grains. Tannin e presenar s a numbe n i tf plan o r t materials. Present work shows that barley also contains significant amounts of tannins. Experiments with rats showed tha a highlt y significant regative correlation exists betwee e tannith n n contenf o t barley and protein digestibility. By adding increasing amounts of tannin to rat diets it was found that tannin has a specific affinity for proline, glycine and glutamic acid.

INTRODUCTION

Recent years have brought a greater awarness of the need for more plentiful, as wel s mora l e nutritious foods. Discoverie f straino s f maizeo s , barle d othean y r crops with higher levels of essential amino acids have shown the differences in nutritional quality than occuca t r among strain f croo s p varieties. Discoverie f growtho s - inhibiting substances in grain and other plant parts have indicated the need to consider both positive and negative factors when working with nutritionalv quality. Increasing the proportion of essential nutrients is not a sufficient answer, these nutrients mus e readilb t y availabl e biologicath o t e l system. Therefore factor- af s fecting the availability of the nutrients must be taken into consideration when judgin e nutritionath g l valua foodstuff f o e .

197 ANIMO ACID COMPOSITION AND ANIMO ACID DIGESTIBILITY OF CEREAL GRAINS

It is well established that the normal cereal grains are all low in lysine. There- fore international agencies, plant breeders, biochemist o forths d an s, havmuct pu eh effort into the task of raising the lysine level in cereal protein. Plertz (1) content/ compared the lysine of common cereals and selected high-lysine mutants with the

FAO 1973 pattern of 5.2^ lysine in the protein being ideal for the infant. This comparison showed that the best of the cereal grains, oats, has about 73/Ü of the ideal level, and the poorest of the grains, sorghum and millet, have 35$ and 36f, respectively e RisTh . a barley mutant contains 5.6$ lysine, whic s veri h y high. Nor- l maizma e contains 52/5, whereas opaque-2 maize s currentla , y produce n Mexicoi d , contains 87-90e ideath lf $ o level. High-lysine sorghum contains nearly twic s muca e h s normaa ) l (63% sorghum n als ca e see ob t I .n from this comparison that wheat contains about 57% and current varieties of rice 71$ of the ideal level of lysine. However, if we take digestibility into consideration, the picture will be somewhat different. Eggum (2), in experiments with rats measured true protein digestibility (TO f protei)o n eighi n t different cereal grain d comparean s d witgrose th h s values e resultTh . e give ar sn Tabl i n . I e

TABL - I E TOTA D TRUAN LE DIGESTIBLE PROTEI N EIGHI N T CEREAL GRAINS Total protein TO protein a Cereal grain in DM3 ($) in DM ($) Difference (%}

Barley 10.13 8.31 17.96 Oats 10.75 9.04 15.90 Wheat 12.63 11.32 10.37 Rye 9.13 7.03 23.00 Maize 10.06 8.81 12.42 Sorghum 12.54 10.63 15.23 Rica 8.96 8.90 0.66 Triticale 13.07 12.12 7.30

Soya bean meal 53.11 50.35 5.19 mattey Dr , r DN a

It appears from Tabl thaI ee difference th t s between tota d digestiblan l e protein in the cereal grains vary considerably. For rice there is almost no difference between total and digestible protein, whereas in rye this difference is more than 20$. This indicates that crude protein values of rice and rye are not additive from a nutritional point of view irrespective of their amino acid composition. As for the other cereal grains the differences between total and digestible protein are smaller but/ e neverthelesry thar fo n s considerable r purposeFo . f comparisonso e value,th r fo s soya bean meal are aLso given. The results show that besides the high-protein content in soya bean meaproteie th l n digestibilit s alsi y a generao s highi t I l. experien- e thac e digestibilitth t f protein-uico y h feed foodstuffan d s highei s r than i n most cereal grains (3).,

198 proteie Ath s n digestibilit n differeni y t protein sources varies considerably, e assumeb y ima td thae digestibilitth t e individuath f o y l amino acids also mora var r lesn o ei y s similar manner.

Tablen gives values for lysine in several cereal grains. Digestible amino acids uere measured accordin methoe th f Kuikeo a dt g d Lymaan n n (4). Although this method is criticized because of microbial activity in the alimentary tract, valuable informatio e obtaineb n ca n d with this method (.5).

TABL. II E TOTA D TRUAM LE DIGESTIBLE LYSIN M CEREAI E L GRAINS

Total lysine Digestible lysine Lysine source in DM in On Difference

(S/l6g N) (g/169 N) (*)

Barley 3.69 2.80 24.11 Oats 4.03 3.21 20.34 Wheat 2.55 2.02 20.80 Rye 3.67 2.40 26.60 Maize 2.73 2.31 15.38 Sorghum 1.83 1.33 23.30 Rice 3.54 3.51 0.85

Soya bean meal 5.98 5.48 8.30

The differences between values of total and digestible.lysine are much more pronounced than for protein. As discussed by Eggui7) (6) this is probably due to the fact that lysine is mainly deposited in the protein fractions of lowest digestibility. In the highly digestible prolamin fraction almost no lysine is found, whereas the glutamic acid content is very high, which explains the high digestibility o~ glutamic acid, shown in Table III.

TABLE III. TOTAL AND TRUE DIGESTIBLE GLUTAHIC ACID IM CEREAL GRAINS Total glutamic Digestible glutamic Glutamic acid acid in DM acid in DM Difference (g/16g N) (g/16g N) (50

Barley 25.06 22.90 8.62 Oats 22.10 19.71 10.81 Wheat 35.77 35.41 1.01 Rye 23.62 21.52 8.89 Maize 17.46 16.05 8.08 Sorghum 21.24 19.12 9.98 Rice 17.16 17.18 0.00

Soya bean meal 17.82 17.04 4.38

199 As expected e differenceth , s between tota d digestiblan l e glutamic acie mucar dh smaller than for lysine and total protein. These differences are also discussed by Hunck (7), Thomke(8), Schille- va D T d Eggur e an (9)th m ,r (5)fo s A . lues for the other amino acids, they are all intermediate between lysine and glutamic acit shoulI d e stresseb (5)d . d that aspartic acid, glycine d alaan , - nine haue TD values in the louer part of the range, whereas the histidine, arginine,, and serine values are in the upper part. This is in agreement with correlation coefficients found by Pomeranz et al. (10) between lysine and the three amino acids, aspartic acid, glycine, and alanine, whereas the correlation coefficient between lysine and glutamic acid was negative in barley protein. It is documented that when barley, rye, wheat, maize, and sorghum are fertilized with increasing amounts of nitrogen more protein will be deposited in their prolamin fraction. As this fraction is a poor source of lysine (10) but highly digestible (5), more digestible proteif loweo t rbu n biological value r Fo s obtainei d , 12)(119 , .8 , oat d rice situatioan s th e s differeni n s glutelia t n (relatively ric n lysinei h s i ) the main storage protein in these cereal grains (10). Juliano et . (13al ) observe a negativd e correlation between lysine conten f proteio t : proan n - tein content of browrr rice only in samples with protein below 10$. In further uork with rice, Eggum and Juliano (14) observed that TD and net protein utilisation (PJPU) were positively correlated with lysine content of rice protein (3.17-4.06 g/'5gM). Utilisable protein ranged from 5.1 s mainlo 11.19wa t d yan 2 determine N con-an y b d - of milled rice (r=0.988). Digestibilit f amino y o acid f milleo s d rice protei, t n fecal analysis ranged from 94.1 to 100.0/J in samples with 1.38 and 2.74/a M, dry :=sis. According to Eggum and Juliano (15), an increase in milled rice protein conte" (N x 6.25) from 9.12 to 11.09 in IR 8 and from 11.09 to 14.56 in IR 480-5-9 oui-- tM fertilizeo r applicatio o significann d ha n t e lysineffecth n o et contene -~ f o t d proteilittlha d ean n effec n truo t e digestibility, biological t -rovaluene -d an , tein utilisatio e proteith f n o growini n g rats. Maruyam . (16al )t e a however, foun n rati d s that increase n proteii s n content were accompaine n increasa y b d n i e indispensable amino acids. Patter. (17al )t e n found thae negativth t e correlation between proteic an n lysine appear o becomt s e nonsignifican t highea t r level f proteio s n wheati n , however, genetically high-protein ehea s beeha t n e equafounb r o higheo t ld n lysini r e content as a percentage of protein than are normal wheats grown in the same environment e aminTh . o acid compositio f oato ns remarkabli s y constant ove a widr e ran^f o e protein content (18,_12 , 19)10 , .

e relationshiTh p between protein conten d proteian t ns i qualit e ry n oati d y an s illustrate y Eppendorfeb d r (12) (se. e2) Figd an . 1

200 g/16gN TD,B\/,NPU 5.00 100 r /o

— 90 4.00

- 80 3.00 - 70

2.00 - 60 ± 3 0 3. 0 2. W inN DM(%) Fig Relationship. 1 . s between true digestibility (TD), biological value (BV), net protein utilisation (IMPU), lysine, threonine, methionine, and cystine, and the concentration of nitrogen in grain of oats (var. Selma). (From Eppendorfer, 1975, with permission). g/16gN TD,BV,NPU S.OOr %

200 -

200-

10 20 3.0 N in DM(%) Fig Relationship. 2 . s between true digestibility (TD), biological value (BV), net protein utilisation (NPU), lysine, threonine, methionine d cystine e conan , th -d ,an centration of nitrogen in grain of spring rye (uar. Petkus), 201 From Fig.1 it is evident that the concentrations of lysine in particular, but thos f threonineo e , cystine d methioninan , s wela e onl ar l y slightly decreased with increasing nitrogen content. Consequently, B\l almost remains unchanged. TO, however, increases linearly with protein content and consequently NPU. In Fig. 2 concentrations of lysine, threonine, methionine, and cystine are also shown as a function of M content of grain. This graphic presentation clearly shows that the decrease of the BV is very closely paralleled by decreasing concentrations of those amino acids. Figure 2 is probably representative of similar relationships between protein content and protein quality in barley, wheat, maize, and sorghum. N applications normally increase yield or protein content, or both, of grain and, therefore, alse productioth o r are f pe proteino a d individuaan n l amino acid- in s cluding lysine. A decrease in the nutritional value of protein, whether expressed as a reductio n lysini n r NPUo e , will probably alway more b s e than balance a large y b d r protein and amino acid production (11 ).

E INFLUENCTH F TANNIO E M PROTEIO N N UTILISATION

Tannins are present in a number of plant materials at very high levels, often 1 C;1 or more of dry weight (20). They may be of significance in some common feed d d foodstuffsan an - , suc s sorghua h m grain ) 1 (2 s rape seed meal (22). Mili t al.(23e c ) suggest thae tanninth t s affec e rat f th methylatioto e d an n the formatio f complexeo n s wite constituentth h e feedth .f o sGalli c acid galloan d - tannins posses a sdistinctiv e inhibitory e digestiveffecth n o t e enzymes, forming complexes wite proteith h n par f enzymeo t r bindino s e simplth g e constituents th f o s digested feed into complexes. However e mod f th actio,o e f enzymo n e inhibitioy b n tannin is still uncertain. Furthermore, types of protein and tannin influence the reaction. Axtell et al. (24) put forward the hypothesis that seed proteins become complexed or bound with tannin compounds of the whole grain, and that the complexée protein e substantiallar s y less availabl r utilisatiofo e y monogastrib n c animals. Experiments with chick d tannife s c acid othean d r tannins have shown that about 0.5% tanni e diec th acit n i resultd n depressiosi , hig5% h t f growta moro n d -an h tality occurs (25). n agreemeni Thi s i s t with wor f Baeluo k d an m Petersen (26) and Peterser. (27) who found that tannin had a negative effect on feed conversion. Furthermore, chicken fed on diets with added tannin had a significant- y lowel r flavor quality thad thos d di dietn fe e s without tannin. As mentioned above, tannin e presen ar sa numbe n i tf plan o r t materialst a t bu , different levels. Barley, the most widespread cereal grain in Denmark, also contains significant amounts of tannins, which night partly explain the relative lo'j digestibilit f barleo y y protein o evaluatT (28) e influenc.th e f boto e n protein and tannin content on protein digestibility in barley, 29 samples were tested (29). e concentrationth s A f nitrogeo s d tannian n n hav a edetrimenta l influencn o e protein digestibility e relationshith , p between protein digestibilit d contenan y f o t nitroge d tanni multipla an n s determinef o wa n e eus regressioy b d n equations ,a follows: = 82.6 O T 0 4JJ3.8 x IMjO9 j -JJ6.2 7x tanni n (fYj

sb1=0.81; sb2=2.11; R=0.72; r^O.60; r2=0.60; t=3.G 202 The regression coefficien s significantli t y different from zero t tes a ,t showing P<0.001. Hence, the nitrogen content has a positive influence on protein digestibility, whereas tanninegativa s ha n e influence. To evaluate the effect of tannin on protein utilisation, Eggum and Christen- sen-(2;0 gave increasing amounts of tannin in diets to rats. Soya bean meal was used as dietary protein. True digestibility (TD), biological value (BU), and net protein utilisation (NPU) were employed as biological criteria (see Table iv.).

TABL . IV E INFLUENC F TANNIO E N PROTEIO N N UTILISATIO N SOYI N A BEAN PROTEIN3

Tannin supplement ($) D (JÉT ) BV ($) NPU ($)

0 92.9 77.9 72.4 0.5 81.1 77.9 63.2 1.0 78.4 81.3 63.6 1.5 73.2 77.9 57.0

aSource: (29)

It would appear from these results that tannin exerts a severe negative effect on protein digestibility. A supplement of 0.5$ resulted in a decrease in TD from 92.9 to,81.1$, whereas 1.5$ tannin in the diet reduced TD to 73.2£. The biological uias/ value almost unaffected by tannin. The results do not indicate that methionl.ne is more sensitive to tannin than other amino acids - as has been proposed by other tannif a specifiI d ha n c workereffec n thio ts s(30) particula. r amino acid e biologicath , l valu f soyo e a bean protein would have been reducey b d a tannin supplement. This was not the case. To investigate this aspect of the problem, the availability of the individual amino acids was measured. The group without tannin was compared with the group supplemented with 1.5$ tannin in terms of the TD of the individual amino acids. Tabl showV e s thal aminal t o acid n soyi s a bean protein have high availabilities. With e diettannith n ,i n however availabilite th , l aminal of o y acids decreaset bu s to a different degree. As discussed earlier, methionine is not more severely affectee othedth thare ar aminn o acids n facti ; , this aci s leasi d t affectee th y b d addition of tannins. The availability of some of the other amino acids such as proline, glycine, and glutamic acid is severely reduced. Proline is not absorbed at all , whereas about 60/; of the glycine and more than 3Q/^ of the glutamic acid are recovered in the feces. As none of these amino acids is essential for rats, this decreas n availabiliti e y will hav o negativn e e n agreemeninfluenci , BU n o et with the results in Table V. The theory of the detoxification effect of methionine - and thereb a highey r requiremen r thifo ts amino acid whe - doent appl d tannino sfe y s i n to the present data. Whether or not proline, glycine, and possibly glutamic acid have a detoxifying effect is unclear. However, these three amino acids in particular are present in very high concentrations in gelatin. As discussed by Van Buren and Robin- n (31)so , bonding between gelati d tannian n n molecule s strongi s . This could indicate that tannia specifi s ha n c affinit r prolinefo y , glycine d glutamian , c acid.

203 TAELE M. AF1INO ACID AVAILABILITY IN SOYA BEAN PROTEIN WITHOUT AMD WITH 1.5# E TANNIDIETH TN I N

Ofa Tannin 1 .5% Tannin 0$ Tannin 5$1. £ Tannin TD (JE) TD (%) TO (58) D (56T )

Lys 93.3 81 .1 Val 90.0 76.9 riet 91.9 80.6 Iso 90.7 78.1 Cys 94.0 84.7 Leu 90.1 77.6 Asp 93.4 78.7 Tyr 90.6 77.3 Thr 90.4 78.5 Phe 93.3 78.4 Ser 96.6 83.1 His 97.4 75.6 Glu 94.9 68.1 Arg 97.4 75.3 Pro 93.5 0.9 Try 94.2 83.9 Gly 89.3 41.5 NH, 93.4 64.9 O Ala 88.3 75.1

REFERENCES

(1) MERTZ, E.T. Breeding for improved nutritional value in cereals. In "Protein Nutri- tional Qualit f Foodo y d Feedssan . (Friedman, Wendel, ed.), . Parpp 2 t Dekker. York1-12 w Ne , . (1975). ) EGGUM(2 , B.Orelationshie Th . p between tota d digestiblan l e protein (amino acids) and total and digestible energy in cereal grains as determined in experiments with rats. Xth Int. Congress of Nutrition, Kyoto, Japan, Aug. 3-9, (1975). (3) EGGUPl, B.O. Aminosyrekoncentration og Proteinkvalitet. Stougaards Forlag, Copen- hagen (1968) 90pp. ) KUIKEN(4 , K.A., LYFIAN, C.M. Availabilit f amino y o acid n somi s e foods . NutrJ . 6 3_ . (1948) 359. ) EGGUM(5 , B.O A stud. f Certaio y n Factors Influencing Protein Utilizatio n Rati n s and Pigs. Thesis, Royal Veterinary and Agricultural University, Copen- hagen (1973) 173pp. (6) EGGUM, B.O. Biological availability of amino acid constituents in grain protein, pp. 391-408 n "NucleaI . r Technique r Seefo sd Protein Improvement", International Atomic Energy Agency, Vienna. (1973) 422pp. (7) PIU\'C:<, L. The variation of nutritional value in barley. I. Variety and nitrogen fertilizer effects on chemical composition and laboratory feeding experiments. Hereditas 52_ (1964) 1. (8) THOMKE, S. Über die Veränderung des Aminosäuregehaltes der Gerste mit steigendem Stickstoffgehalt. Zeit.. Tierphysiol., Tierernähr., Futtermittelk_ 2T . (1970. )23 ) SCHILLER(9 . UntersuchungeK , n übee Variabititädi r Futtergerstenproteinn vo t . Mit2 . - teilung: Über den Einfluss ökologischer Faktoren auf die Verteilung der Eiueissarten im Protein von Gerstekaryopsen. Landuirtsch. Forsch. XXIV (1971. )15

204 (10) POWIERANZ, Y., ROBBINS, G.S., UESENBERG, D.M., HOCKETT, E.A., GILBERTSON, 3.T. Amino acid composition of two-rowed and six-rowed barleys. 3. Agr. Food Chem. 21 (1973) 218. (11) EGGUM, B.O. Über die Abhängigkeit der Proteinqualität von Stickstoffgehalt der Gerste. Zeit. Tierphysiol., Tierernähr., Futtermittelk. 2£ (1970) 65. (12) EPPENDORFER, U. Effects of fertilizers on quality and nutritional value of grain protein. 11th Kolloquiu f Internationao m l Potash Institute, Ranne, Denmark, 3un5 (1975)2- e . (13) 3ULIAMQ, B.O., ANTONIO, A.A., ESMANA, B.V. Effect f proteiso n contene disth -n o t tributio propertied an n f ricso e protein . Sei.3 . Food Agr 4 (19732 . ) 295. (14) EGGUM, B.O., 3ULIANO, B.O. Nitrogen balance in rats fed rices differing in protein content . Sei3 . . Food Agr (1973. 24 . ) 921. (15) EGGUM, B.O., 3ULIANO, 8.0. Higher protein content from nitrogen fertilizer applicatio d nutritivan n e valu milled-ricf o e e protein . Sei.3 . Food Agr. 26 (1975) 425. (16) MARUYAHA, K., SHANDS, H.L., HARPER, A.E., SUNDE, PI.L. An evaluation of the nutritive value of neu high protein oat varieties (cultivars). 3. Nutr. 105 (1975) 1048. (17) PATTERN, P.3., 30HNSON, U.A., STROIKE, 3.E., SCHMIDT, 3.U., KLEPPER ULNER, L. , , R.L. Status of protein quality improvement in wheat, pp. 287-297. In "High- Quality Protein Maize", Halsted Press, Stroudsburg, Pennsylvania, (1975) 524pp. (18) BENGTSSON, A., EGGUFI, B.O. Virkningen af stigende N-gadskning pâ havre og bygpro- teinets kvalitet. Tidskr. Plant. 73 (1969) 105. (19) SCHRICKEL.D.3., CLARK, W.L. Status of protein quality improvement in oats. pp.398-411. In 'IHigh-Qualityi. Protein Maize". Halsted Press, Stroudsburg, Pennsylvania. (1975) 524pp. (20) SINGLETON, V.L., KRATZER, F.H. Toxicity and related physiological activity of phenol substance planf o s t origin . Agri3 . . Food Chem 7 (1969V . ) 497. (21) CHAIMG, S.I., FULLER, H.L. Effec f tannio t n conten f graio t n sorghum n theio s r feeding value for growing chicks. Poultry Sei. 43 (1964) 30. (22) CLANDININ, D.R., HEARD . Tannin3 , n press-solveni s d solvenan t t processed rapeseed meal. Poultry Sei 7 .(1968£ ) 688. (23) rilLlC, B.L., ST03ANOVIC" , VUCREVICS. , . LucernN , e tannins . IsolatioII . f tannino n s from lucerne, their nature and influence on the digestive enzymes in vitro. 3. Sei. Food Agri. 23. (1972) 1157. (24) AXTELL, 3.D., OSWALT, D.L., ClERTZ, E.T., PICKETT, R.C., 3AMBUNATHAN , SRINIVASANR. , , G. Component f nutritionao s l qualit n graii y n sorghum . 374-386pp , n .I "High-Quality protein Maize". Halsted Press,S'troudsburg> Pennsylvania. 524pp. (25) VOHRA, P., KRATZER, F.H., 30SLYN, M.A. The growth-depressing and toxic effects of tannins to chicks. Poultry Sei. _45 (1966) 135. (26) BAELUM, 3., PETERSEN, V.E. II. Forsag med slagtekyllinger. pp. 311-315. In "Bilag Landokonomisk Forsagslab. efterarsmade" Copenhagen. (1964). (27) PETERSEN, U.E. A comparison of the feeding value for broilers of corn, grain, sorghum, barley, e influencwhea th d oatse variou an d tth an ,f o es grainn o s the composition and taste of broiler meat. Poultry Sei. 48^ (1969) 2006.

205 (28) EGGUM, B.O. Current method f nutritionao s l protein evaluation . 289-302pp , n .I "Improving Plant Protei Nucleay b n r Techniques". International Atomic Energy Agency", Vienna (1970). (29) EGGUFl, B.O., CHRISTENSEN, K.D. Influenc f tannio e protein o n n utilizatio feedn i n - stuffs with special referenc barleyo t e 135-143. ,pp "Breedinn .I r fo g Seed Protein Improvement using Nuclear Techniques". International Atomic Energy Agency", Vienna (1975). (30) NELSON, R.F., FINKEL, B.J. Enzyme reactions with phenolic compounds: Effectf o s 0-methyltransferase and high pH on the polyphenol oxidase substrates in apple. Phytochemistr Z (1954y ) 321, (31) BUREN, VAN, J.P., ROBINSON, U/.B. Formation of complexes between protein and tannic acid . Agri3 . . Food Chem. _1_7 (1969) 772.

206 CONCLUSIONS AND RECOMMENDATIONS OF THE RESEARCH CO-ORDINATION MEETING OF FAO/IAEA/GSF/SIDA CO-ORDINATED RESEARCH NUCLEAF O PROGRAMM E US RE TH N EO TECHNIQUE CEREAR SFO L GRAIN PROTEIN IMPROVEMENT

The meeting considered the recommendations on plant breeding for seed protein improvement and analysis for improved protein content and quality mad previout a e s meeting FAO/IAEA/SIDe th f so A Coordinate- dBe searc Nucleaf o h e ProgrammUs r e Techniqueth n o e Seer fo sd Protein Improvement (l, 2, 3, 4). These recommendation acknowledgede sar . Howevere vieth n , i f wo fact that some new findings have been obtained since the previous meeting, and that the Coordinated Research is in ±he final phase of breeding, the participants consider that plant breeders would benefit from the recommendations regarding mutation breeding, use of mutants in cross-breeding for seed protein improvement, and nutritional evaluation of high-protein breeding material.

1. MUTATION BREEDING FOR SEED PROTEIN IMPROVEMENT 1.1 Objectives When breedin higher gfo r quantit improved yan d qualit proteif yo n in existing high-yielding varieties of cereals, the plant breeder should conside nutritionae rth l locae needth lf s o huma n populatio livestocr o n k d seean k those improvements which will provid greatese eth t benefio t consumerse th feedine th * o t dieanimalsf g o r to . Good agronomic performance (high grain yield) should also be empha- sized when seeking improved seed protein. Increasing protein production per hectare by increasing grain yield, without reducing protein content, is generally very important. However, improving protein content or qual- itythereford ,an e improvin nutritionae gth l valu unir foof pe ed to dan feed, must be the primary objective of a mutation breeding programme for protein improvement. proteie Th n mutant must fulfi agronomie lth c requirements, disease and pest resistance, and other desirable characteristics (e.g., baking quality in wheat) normally associated with high yielding commercial varieties. A premiu paie higher b m fo d o t pric rs graieha n protein content and/or improved protein quality appropriatn a d ,an e marketing system has to be established to compensate the farmer for his extra efforts and to assure the expected quality to the consumer. 2 1. Protein mutant proteiA n definee mutanwhice b on y characterizes hi s tma da y db higher grain protein quantit seedr yflourpe r ,pe , and/o plantr rpe , and/o improven a y rb d grain amino acid composition.

A breeder may like to select one or the other of these different protein mutants depending upon his specific needs. 3 1. Strategy breedee Th r should have extensive collection gerf so m plasr mfo his breeding programmes. The collections should be screened for protein quantity and quality.

207 Two kinds of genetic improvements through induced mutations in respect of protein should be aimed at: ) Improvement(a s resulting from genes with major effectso T . detect such a mutant, a population of at least 2000 to 10,000 progenies mus screenede th . This estimate bases th ei n o d assumptio mutatioa f no n rat 10~3f eo d an ; ) Improvement(b s resulting from genes with minor effectA . population size resulting from at least 200 to 1,000 treated seeds must be screened on the assumption of a mutation rate of 10~2. It should als notee ob d thalargee tth populatioe rth n screened, morane dth e accurat fiele analyticad eth dan l conditions greatee ,th r chance wilth findine f leb o g mutants with improvement protein si n con- tenqualityd tan . 4 1. Selectio mutantf no s In the Ü2 generation, selection against plants having undesirable agronomic characters shoul madee db . Under circumstances where improvements resulting from mutations with major effect materiad an aimee s, ar grows dat li n under uniform conditions and can be screened by accurate analytical procedures, selectio proteif no effectivee b ny mutantma U M .n si case Also th e n ,i where improvement in protein quality is the major objective, the M-j seed produce analyseplante g b M y n do proteir sma dfo n quality. Under most conditions, however d late,an rselectin^ M e th n gi generation thir sfo s characte generalls ri y considere more b eo dt worthwhile, and therefore, selection at this stage is recommended. Confirmatio furthed an n r evaluatio yielf no proteid dan n contens i t necessar subsequenn yi t generations. Although morphological character associatee b y sma d with changen si protein qualit quantityr yo shoult ,i noticee db d thagena tt - thino s si eral phenomenon. 1.5 Growth of mutant lines in the field wels Ii t l known that environmental conditions influence seed protein quantity and quality. Differences in plant protein due to environmental non-uniformit muce b hy ygreatema r than difference genetio t e sdu c factors. Thus, in order to detect moderate changes (say 10$) in total protein content or of a particular amino acid, all environmental factors that influence seed protein shoul kepe db unifors a t s possiblml a plant al e b r sefo o whict e har screened. Important agronomical traits such as grain yield, protein content and protein qualit mutann yi t lines shoul checkee db y standardizedb d performance trials carried out under various environmental conditions.

The meeting acknowledged the guidelines which had been prepared at the previous meeting to assist co-operators in achieving greatest possible environmental uniformity in their experimental fields (Annex recommendes wa referencf o t I I . de2) that these guideline followee sb d participantl al y b programmee th n si . 6 1. Samplin mutanf go t materia proteir lfo n analysis There should also be standardized procedures for taking grain sample preparind san g thechemicar fo m l analysis. Such procedures permit reliable analytical result obtainee b o st d frofiele mth d material. Clear identification of samples is essential to both analyst and plant breeder so long as non-destructive techniques are not available. The meeting acknowledged the guidelines which had been prepared at the previous meeting for outlining sampling and identification procedures (Annex II of reference 2) and recommended that they be followed by all partici- programmee pantth n si . 208 1.7 International protein mutants preliminary evaluation, test (IPMPET) Preliminary evaluatioproteif o e us n r mutantno crosn si s breeding are of great interest to the participants in the programme. Exchanges of breeding materia result d preliminare lan th f so y evaluations amone th g participants should be made available through IAEA.

2. USE OF MUTANTS IN CROSS BREEDING AND GENETIC ANALYSES Many protein mutants have been established throug programme hth e and are being incorporated into hybridization breeding for further improvemen proteinf to combininr fo r ,o g improved protein attributes with agronomically important characters, suc higs ha h grain yiel resisd dan - tance to pests or diseases. The meeting considered that plant breeders would benefit from the recommendations regarding the methodology in cross breedin usiny gb g protein mutant parents geneticasn a i d san l analysis of mutants.

2.1 Cross breeding recommendes i t I 2.1.1d that protein mutant ranga usee sb n f di eo crosses with adapted varieties. A number of cross procedures may be considered but it is suggested that backcross, three- way or double crosses should be used to introduce the high protein germplasm int olocalla y adapted genetic background. It is strongly recommended that selection be carried out on 'lines* rather than single plant thao s t selectio- im r nfo proved protein attributes shoul confinee db or'late, F o dt r generations somn I .e circumstances appropriate b y ma t ,i e to intermate lines in order to break repulsion linkages. 2.1.2 Consideration shoul givee programma db o nt accumulatinf eo g high protein gene 'eliten i s ' breeding line meany sb f so selection from appropriate multiple crosses involving mutant lines, including use where possible of male sterile facilitated crossing systems. 2.1.3 It is recommended that preliminary characterization of the phenotype proteif so n mutant lines shoul carriee db sinct dou e such characterization could be of value in selection. Thus, particular protein changes resulting from mutation idens ,a - tified by using electrophoretic amino—acid analyses could be use markers selectioe da th n si n programme following hybridization. 2.2 Inter-specific hybridization It should be appreciated that useful sources of protein improving genes exis relaten ti d specie wheatf so , barle riced yan . Such sources should not be ignored and efforts should be made to use lines carrying 'alien1 genes established as conferring desirable protein attributes. 3 2. Researc geneticae th n h o d phenotypioalan l characterizatiof no mutant high protein lines The efficient exploitatio higf no h protein mutant lines depends upo understandinn na numbere th f genegf o so s involved, their linkage relationship with other genes whethed thet ,an no y r rhavo e adverse pleiotropic effects on other characters of agronomic interest. The efficienc whicy yb h mutant selectee b gene n sca d would also benefit if pleiotropic effects on more readily identifiable characters can be established. Both these sources of information are not available at the moment and in most instances the required information retires extensive research programmes which will take some time to complete. It is recommended, however, that such work should be initiated and should consider: ) Geneti(a c analysi determino st estimatr eo numbee eth f ro genes involved and the nature of their interaction. These 209 determinee b y ma d using appropriate biometrical investigations and in the case of wheat, aneuploid analytical techniques shoul considerede db . urges i t dI tha ) genetican i t(b l analysi thif so s type random lines at least in PC resulting from the cross of the high protein mutant line wit recipienha t variety shoule db studied in order to establish whether adverse correlated effects are associated with high protein levels. (c) The selected F^ lines should also be used in physiological and biochemical studies to define the role of the mutant genes more exactly. Such studies would conside identifd ran y whether the mutant effects arise from alterations in grain yield components, alteration vegetativn si e amounts, leaf senescense, increased ability to translocate Nf more efficient uptake of N, or disturbed starch synthesis in the grain. (d) It is also recommended that in association with genetic analyses, mutant lines should be studied using protein electrophoretic techniques to establish whether they carry alterations in structural genes of endosperm proteins, or in genes affecting the regulation of sunn genes.

. 3 NUTRITIONAL VALUE EVALUATION

participante Manth f yo s have materia potentiaf lo e eithelus r rfo commercial utilizatio germplasms a r no nutritionaA . l evaluatio sucf no h material is recommended.

3.1 Estimation of protein content and quality The, protein content of cereal grains is estimated as the Kjeldahl nitrogen content times 6.25. A conversion factor of 5»? has been used for wheat and rice, but experimental evidence for using different factors in those welt cropno ls si established . Protein conten alsn e ca tob measured by DBC, provided samples with known protein content are used for calibration. The protein qualit measuree b n yca severan i d l ways. relatioe Th . n betweeKjeldahd an C nDB l nitrogen conten bees ha tn used extensiveln yi barley, where it gives an accurate estimate of the protein quality. We believe the DBC - Kjeldahl N method can also be used to measure quality in other cereals, provided that samples with known protein quality are usecalibrationr fo d . Single seed methods have been develope detectioe th r fo dspef o n - cific high-lysine genes, e.g. fros ,ly m Hiprol crosn yi s breeding programmes. These methods include the determination of specific enzymes prolamie th r o n compositio determines na electrophoresisy b d . Screening for large changes in barley hordein composition is carried out in several laboratories by electrophoresis or the turbidity test. The final evaluation of the protein quality should primarily be base estimationn do contene limitine th th f f so o t g essential amino acids. e participantTh s expresse neee r standarth dfo d d sample distri-e b o st r s decidebutedwa t requesI o .t d e IAEtth A Laborator administeo yt dise rth - tribution of such samples. Subject to available resources, the laboratory will assist participant renderiny sb g analytical services (NinhydrinN

lysine, Kjeldahl-N, amino acid analyses). t

3.2 Lysine loss in processing Los availablf so e processinlysino t e ef du o y g wa depend e th n so processing (5) mose .Th t important factors involve temperature ar d e level, time and reducing substances.

210 Heat processing has both beneficial and deteriorative effects on the nutritional valu proteinsf eo . Moderate heat treatment, especially with high moisture, not only enhances palatability and acceptance but also im- proves protein quality. Severe heat treatments such as toasting, puffing and gun explosion techniques have been reporte causo dt drastiea c reductio nutritive th n ni e value of wheat, oat, and rice. Amino acid analysis of acid and enzyme hydrolysate heat-processed an w ra f so d proteins revealed that lysins ewa *he amino acid affected to a large extent. Increasing the degree of heat treatment cause progressivda e decreas ease th e n witei h which lysine could be released from the protein by enzymatic action. Similarly, the most important loss during bread making is that of lysine (6). This loss occurs primaril cruse th tn yi (15-20$ d )an to a much lesser extent in the crumb (approx. 5$)« The loss of lysine bees ha n muce founb ho dt highe brean i r whico dt h reducing sugard sha been added. Consequently, the risk is more pronounced in yeast fermented bread compared to unleavened bread (7).

3.3 Baking quality When applicable, baking quality assessments should be done in advanced breeding materials. Local procedures for processing should be applied. yi.trn I o 4 digestibilit3. y

completr Fo e nutritional utilizatio proteina f no ingestee ,th d protein source mus digestee tb constituens it o dt t aminoe th acid d san amino acids must be absorbed in a chemical form which can be utilized physiologically. Data about amino acid composition provide indispensable information on the levels and distribution of the amino acids contained in a protein source. The composition data alone, however, do not pro- vidinformatioy an e n abou digestibilite th t proteia f yo n sourcr eno abou utilizatioe tth amine th of no acid vitrn i s f ocontainedo e us e Th . enzymatic methods provides an approach for obtaining information which may be related to the relative degree to which different proteins are digested and utilized in vivo. A number of methods have been used for estimating the digestibility of various materials. However, the most promising one seems to be the method developed by Hsu et. al \B). The sample is ground to a fine powder. adjustes i H p Approximatel d d an proteif o O g Hg m suspendes l ni 0 m y30 0 5 n di to 8.0. The multienzyme solution (trypsin, chymotrypsin, peptidase) is added and the subsequent fall in pH recorded after 10 minutes is used to estimate the digestibility. The method is further developed (one more enzyme Satterley )b (lu)l a remarkabld t e ,an y good agreement- ob e sar tained between in vivo and in vitro data for digestibility. 3«5 Animal feeding test

Methods for the evaluation of nutritional value of protein in food and feedstuffs have been reviewe many db y workers. From these works, strict standardizatio experimentae th f no l condition recommendeds si . The standardization is especially important in breeding work where small improvements of protein have to be detected. In agreement with the recommendations of PAO Guideline No. 16 (lO) IAEe andth A meeting 197recommendes i 7t ra (3) e bioassar ,th dfo y procedures in plant breeding. Furthermore, the nitrogen balance method is recommended balance Th . e technique also include measursa proteir efo n digestibility whic consideres i h vera e yb valuablo dt e criterionn I . the same procedur digestibilite eth individuae th f yo l amino acidn sca also be measured. Published results by Eggum (ll) show that the individual amino acid component absorbet same no th e e o sar degreet d , e.g., lysine locatealeurone th n i d e laye cerealf ro s show significantlsa y lower digestibility value compared to e.g., glutamic acid located pri- endospermmarile th n yi . However biologicas ,a l value (BV measura s )i e

211 of retained nitroge percenn ni digestef o t d nitrogen, this criterion will indirectly give a close estimate of the availability of the most limiting amino acid. However must , i kep e tb minn i t d tha cerean i t l foo feer do d other amino acids than lysine (e.g., threonine) might become a limiting factor for protein utilization. . 4 COOPERATION BETWEEN FAO/IAEA/GSF/SIDA PROGRAMM PAO/SIDA/SARED EAN G PROGRAMME It is recommended to develop closer cooperation between the programmes havin gcommoa n objectiv improvinf eo g grain protein content.

4.1 Exchange of documentation and literature should be made available routinel participanto yt otherd san requesn so t througi IAEAan .O hPA 4.2 PAO and IAEA should facilitate exchange of breeding material between the scientists participating in both of the programmes. 4.3 Countries from the PAO/3IDA/SAREC programme should be invited to participate in the International Preliminary Evaluation Test. Responsibility for assembling and distribution of breeding material resultd an s wil wite Joine lb hth t PAO/IAEA Division. 4 4. Representatives from FAO/SIDA/SAREC programme should participate Researce inth h Coordination Meeting finad san l Symposiue th f mo FAO/IAEA/GSF/SIDA programme wels , a representative s la e th f so FAO/IAEA/GSF/SIDA programme should participate at the Seminars organized by the PAO/SIDA/SAREC programme. Each project should funparticipatioe dth representativess it f no . 5 4. Cooperation amon institutee gth s withi same nth e country supported eithey b r cooperatioe projecth d tan n amon countriee gth s included in both programme IAEAe sth .should an encourageO e db PA e th y db Bilateral contacts between the participating institutes should also be promoted. 4.6 The PAO and the IAEA should try to make funds available through thei resourcen row througr so h fund trusn si continuo t e follow-up Technical Meetings after the termination of formal Research Contracts. Thi essentias si maintaio lt n momentu nationae th . mof l programmen so protein improvement by exchanging technical information, breeding material and by personal contacts. The funds will be required only for travel expenses of selected participants to the meetings. representativA 7 4. e from SID SAREd Aan C shoul invitee db particio dt - Steerine th pat n ei g Committee Meeting FAO/IAEA/GSF/SIDe th f so A programm evaluatior efo plannind nan furthef go r activities.

212 REFERENCE

1. Nuclear Techniques for Seed Protein Improvement (Proc. Meeting, Neuherberg, 1972), IAEA, Vienna (1973), Recommendation, 411-415; Apendi 416xI ; Apendi7 41 I xI

. 2 Breedin Seer gfo d Protein Improvement Using Nuclear Technique (Proc. Meeting, Ibadan, 1973), IAEA, Vienna (1975), Recommendation 181-186; Annex I 189-190; Annex II 191-192; Annex III 193-209

. 3 Nutritional Evaluatio Cereaf no l Mutants (Proc. Meeting, Munich and Vienna, 1976), IAEA, Vienna (1977), Conclusions and Recommendations 151-183 4. Seed Protein Improvement by Nuclear Techniques (Proc. Meeting, Baden, 1977) IAEA, Vienna (1978), Anne 393-402I x ; AnneI xI 403-410; Anne 411-42I xII 0

. 5 Eggum, B.O. (1979)> Protein Qualit Cerealf yo s Processen i d Various Ways, IAEA p. 383

6. Eggum, B.O. (1968), Amino-syrekoncentration og Protein-kvalitet, Stougaards Forlag, 99 pp

. 7 Eggum, B.O Duggald .an , S.K. (1977) Proteie ,Th n Qualit somf yo e Indian Dishes Prepared from Wheat, J. Sei. Fid. Agric. 28:1052 . 8 Hsu, H.W., Vavak, D.L., Satterlee, L.D Millerd .an , G.A.,(1977), A multienzyme technique for estimating protein digestibility, Jour. Food Science 42:1269-1273

9. Satterles, L.D., Marshall, H.F. Tennypond ,an , J.M., (1979), Measuring Protein Quality Chem,l JourOi .. .SocAm . 56:103 10. PAG Guideline on Protein Methods for Cereal Breeders as Related to Human Nutritional Requirements (PAG Guideline . 16)sNo G ,PA Bulletin 52 (1975) 22 11. Eggum, B.O. (1973)» A Study of Certain Factors Influencing Protein Utilization in Rats and Pigs, Beretn. 406, fra Fors^gslaboratoriet, P P 3 17

213 LIS PARTICIPANTF TO S

Dr. Miloje Denic" Maize Research Institute "Zemun Polje" 9 8 x Bo . 0 . P 11081 Belgra Zemund- , Yugoslavia Hadjichristodoulo. A . Dr u Agricultural Research Institute Agronomy Section Nicosia, Cyprus Dr. Ismachin M. Kartoprawiro National Atomic Energy Agency Pasar Jumat Atomic Energy Research Centre P. 0. Box 2, Kebayoran Lama Jakarta Selatan, -Indonesia Mitr. R . a Dr Biology and Agricultural Division Bhàbha Atomic Research Centre Trorabay, Bombay 400 085 India

Dr. Patrici . ParodoC i School of Agriculture Catholic Universit f Chilyo e Cacilla 114-D Santiago, Chile

. Solari. Dr M . R , Institute Naciona Tecnologie d l a Agropecuaria Rivadavia 1439 Buenos Aires, Argentina

. TahiM . r Dr Nadeem Nuclear Institute for Agriculture and Biology Jhang Road, P. 0. Box 128 Paisalabad, Pakistan

Dr. A. Brunori CHEN, Centro de Studi Nucleari della Casaccia S.P. Anguillarese Km. 1 + 300 Rome, Italy

Dr. Hans Doll Risrf National Laboratory Agricultural Research Department DK-4000 Roskilde, Denmark

Björ. Dr Eggu. n0 m National Institut Animaf eo l Scienqes Rolighedsve5 j2 DK-1958 Copenhagen, Denmark Dr. A. Tallberg Swedish Seed Association S 26800 Svalov, Svjeden

Dr. C. P. Konzak Washington State University Pullman, Washingto. nA 99164. S . U ,

215 Dr. C. N. Law Plant Breeding Institute Maris Lane, Trumpington Cambridge CB2 2LQ, England . KarDr l Nagi Bundesanstal r Pflanzenbafü t Saraenprüfund un u g A-1201 Vienna, Austria S-.ho. P . lz Dr Zentralinstitut fUr Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR DDR-4325 Gatersleben, German Democratic Republic

GSF Representatives Pendric. I . Dr k Institut für Ökologische Physik der Gesellschaft Strahlenr fü Umweltforschund -un MüncheH gmb n Herrenhäuserstr. 2 3000 Hannover, Federal Republic of Germany Dr. H. Walther Gesellschaft für Strahlen- und Umweltforschung mbH München Institu r Strahlenbotenifü t k Abteilun r PflanzengenetigfU k Graf Seinsheira Stras se D-8059 Grünbach, Federal Republi f Germano c y

PAO/SIDA/SARBC Project Members

. BansaC . H l. Dr Plant Breeder Indian Agricultural Research Institute New Delhi 110012, India Dr. Abdel-Salam Gomaa, Director Wheat Research Section Field Crops Research Institute Agricultural Research Centre Giza, Egypt

Tahi. M . rW . Dr Plant Productio Protectiod nan n Division (AGP), FAO délia Vi e Term Caracalli ed a 00100 Rome, Italy

Host Institute Mrs. Athena Delia Agronom. 0 . R . yA Sectio. A n Agricultural Research Institute Nicosia, Cyprus

IAEA Hermeli. E . T . n Dr Division of Research and Laboratories Vienna, Austria Kawa. T . i Dr Joint FAO/IAEA Divisiof no Atomic Energ Foon Agriculturyd i dan e Vienna, Austria » CO CO o ° ° 6 21 CN