CHIMERTSM rr\ Mr PLANTS OF SLTNFLOWER

A]\D ITS SIGI\{IFICANCE

FOR MT]TATION BREEDIITG

T. HEEMELIN I, S. DASKALOV I, A. MICKE 2,

F. STOENESCU 3, MONTCA TUORAS 3

INTRODUCTION

Mutation breeding requires the handling oflarge populations as a mutation in a particulargene is a rare event even with efficient muta-gen treatment. Therefore it is of interest tohandle the mutagen treated plant material ina rather economic way. This refers to plantingdensity in the l/I2 generation but also the har-vest of seeds from M1 plants and the cultiva-tion of the M2 generation for effective mutantscreening.

M1 plants derived from seed irradiation arechimeric for any induced mutation. A parti-cular mutation can be present only in a partof the M1 plant, e.g. spike, branch, sector' groupof flowers. The chimeric pattern differs strong-ly with the ontogenetic system of the plantspecies in question. fn order to decide aboutthe most efficient system of collecting seedsfrom mutagen treated plants for subsequentmutant screening, one should know the chime-ric pattern. The present investigation is partof our ongoing work to clarify the M1 chime-rism in dicotyledonous species (Hermelinet a1,., 1983). \Me hope that the results willhelp sunflo'uver breeders to make efficient useof induced mutations.

MATERIALS AND METHODS

The experimental rvork was carried out atthe Research Institute for Cereals and Indus-trial Crops of Fundu-}ea, Romania, and at theF.A.O./I.A.E.A. Agricultural Biotechnology La-boratory in Seibersdorf, near Vienna, Austria,during the period 1983-1984.

Fl seeds were obtained by crossing the in-bred lines CA-83-f 99 (white seeds) and CG-83-

1 F.A.O.iI.A.E.A. Agricultural Biotechnology Labo-ratory, Seibersdorf, A-1 400 Vienna, Austria.

2 Joint F.A.O./LA.E.A. Division of Isotope and Ra-diation Applications of Atomic Energy for Food andAgricultural Development, A-1 400 Vienna, Austria.

3 Research Institute for Cereals and IndustrialCrops, B 264 Fundulea, România.

2 974 (black seeds). These seeds \/ere irradia-ted with 0, 100 or 200 Gy gamma rays at adose rate of 11 Gy min-l and planted in thefields at Fundulea and Seibersdorf.

1

Fig. 1 - Templâte for simulatedharvesting of mutated seeds in the

sunflower capitulumI-8 .' Angles, for non-random sel.ection ;

l*",l; :s,"1"J, ,1::i*ik":t ::"?i::T:dt

:

Mature capitula of the F1M1 plants were in-vestigated for occurrence of chimeric mutated"sectors" consisting of seeds deviating in co-lour from the intermediate heterozygote of thegrey phenotype. The "mutant sectors" werestudied as to size and shape, described by draw-ing actual patterns in form of a standard sizecapitulum (F'ig. 2). The relqtive areas ,covered bymutant sectors were then calculated (Table 2).

To simulate different harvesting systems ina sunflower mutation breeding project, two rl3-ries of experiments were carried out : (a) si-mulated non-random and (b) simulated randomsampiing of seeds from the F1M1 capitula. Theidea behind was to find out how many seedswould have to be harvested to pick all muta-tions and whether there would be an advan-tage in one or the other harvesting system interms of economic efficiency.

29

o2)

@31

o38

o/*6

ooooeo@

Fis. 2 - chimeric l:atts:l;r,.1"ilirT,

A template was used in the experiments an<lapplied to the capitula showing visible chime-rism. It consisted of eight sectors (I-VIII) andfour circular zones (A-D) as shown in Fi-gqre 1. The central zone, representing about4als of. the total capitulum areà, was nol consi-dered worth harvesting because of pour see.ldevelopment especially in the material grownat Seibersdorf. The seeds to be collected wereidentified by using the template on which theexact positions of selected seeds rvere indica-ted. Grey and "mutant" seeds were recordedindividuaily.

Each capitulum was subjected to eight sr-mulated samplings with 1, 2, 3, 4, 6, B, 12, 16and 24 seeds. Two different selection svstemswere applied :

30

Block seeds x White seeds f1/ nO Ay

oo@@oo@a123*4x56?8

Biock seeds x White seeds f,,/ ZOO Ay

o@oooooo9x rcx t1x nx 13 k 15 16

tlt(Oef lit I'J',\.-/v\-/t \7\/17 18 19 20 21 22

White seeds x Blqck seeds f1! m Ay

30?9

37

o45

oooo@ox Btock seeds/n/e\\-/L-/

t+l l+2to

28n2625

353Il

24

32

6y

o44

c!5249

36

rt? zoo

o43

o51f(50 t(

33

ite seeds

.^

10

flr\7

48

wh

o39

cçl

sunflower capitula. Black areas :areas: grey seeds

a) Non-ranclom sampling. T.he seeds to becollected r','ere all situated in the outer zone(A) and at equal distances, e.g. 1200 apart when3 seeds and 15' apart when 24 seeds were col-lected. The template was turning eight timesand oriented at the angles 1 to B for eachnumber of seeds to be collected to obtain sam-ple replicates.

b) Random sampling. Eight different tem-plates were constructed for each number ofseeds to be colLected. The sampling of seedswas arranged in such a way that probabilitywas êqual to collect seeds in any of the fourzones and in an5r of the eight sectors shown inl'isure 1.

,tla;I

o0tttÉ!t

oçIhodo|:trt

.F!tp

6

€

D

3çp,ooclo'io

1ôF

GIî

Tha frequency of chimeric FlMl sunflowerafter, gamma irradiation. F1 seeds Qerived

crosses between the, lines CA-83-f99(Vtr : whitc seeds) and CG-83-2 974

:.Sql,::r:'

piants were thus probably due to deletions.Considering the fact that seed setting was notaffecteci by the gamma-irradiation, these dele-tions v,iere evidently rather small.

Most iof the 52 chim'eric capitula (Fig. 2)rvere sectorial, but some (e.9. 7, 21 and 35)rvere obviously different. The interpretation ofthe complex configurations of nos. B, 37, 49and 52 is r:ather difficult. One may assume asimultaneous mutation in more than one me-ristem cell, but the type cou.ld also be due ttra rearrangement rn'ithin the meristem andtherefore the four cases are considered each as asingle mutatiqnal event. An irregular borderline betrveen the black and the grey sectors. asin nos. '1 6, 31 and 49 might be the result ofa selective advantage of either the non-mutatedor the mutateC heterozygous tissue which willyieid larger or smaller "mutated sectors" thanexpected frorn the number of meristematiccells normally destined to form the inflores-cence. In ânv case, these capitula confirm thatthere is not a strict predestination of meristemcells and the sector they are responsible for.As seen in Table 2 there is no significant dif-ference as to the relative size of black seeded(mutated) sectors between the two doses. Anincreased sector size after higher mutagen clo-ses, as described by Weiling (1960) for pcas,could not be observed in the material presen-ted here. Assuming that all the 52 cases aredue to a single mutational event, the "Gene-tically Effective Cell Number" as defined byLi and Rédei (1969) is between 2 and 5.

Results of the ttvo systems are shown inTable 3. The recovery of black seeds is pro-portional to the sample size in the range otl-24. A sample size of 24 yielded about 20

times more black seeds than a single seed sam-;, ple. The original orientation of the templatein the non-random series is decisive for thenu-mber of black seeds when only 1 or 2 seeds

were selected as most of the capitula with blaciisectors were oriented towards the top or bot-tom positions (Fig. 2). The heterogeneity in-curred for the random sample sizes of 1 to 4

seeds is likewise due to the non-random orien-tation of the black sectors. Capitula 7, 21 and35 yielded no black seeds in the non-randome;<periment. Out of. 24 seeds harvesl;ed fromcapitula nos. 21, 23, 3B and 51 (ail with smallsectors), only up to 2 seeds were black in therandom selection series.

The number of successful chimeric recove-ries and distribution of black seeds recoveredper capitulum in the, two series of experimentsare summarized in Table 3.

'Table 7

plantsfrom

BxW

WxB

0

100

200

0

100

200

218

487

550

237

619

446

a)

1.159

556

a)

456

qJ+

0

0.5

1.8

0

3,3

2.9

0

4

0

0

2

0

6

10

0

15tc

a Control plants screencd but the nllmber nct reccrdcd

Table 2

Distribution of relative mutated area in 52

chimeric F1M1 sunflower capitula

Number of cepitulaRelative mutatedsector area, o/o

200 Gy

-101r -2021-303l-4041-5051-Tota-t No.

7

6

4

2

6

5

D

4

29

Range

Median4-54

22

RESULTS AND DISCUSSION

A number of 4,727 F1M1 plants from seedstreated with 100 or 200 Gy were examined forchimerism in additjon to a large number ofcontrol plants. Fifty-two capitula showed aportion of seeds deviating in colour from thenormal grey phenotype of the heterozygoticmaterial used. so the incidence of chimericplants was 1.1t1/0. The "seeds" of the "mutantsectors" were in all cases black (Table 1 andFig. 3). No chiimer,ic grlants wer,e observedamong the non-irradiated control. The fre-quency of chimeric plants rvas much higherthan the mutation rate expected for a reces-sive change in a single gene, but in agreemeniwith the probabiiity to induce a break in achrlomosome, being about 10-2 (B r o c k, 1979).The observed black seeded sectors in the F1M1

31J

(B: black seeds)

Expcr.irnental s!te

Table J

Number of chimeric M1 sunflower plants with black seeds, and the number of black seeds horvestetlper capitulum following simulation of different sampling systems (52 eapituta were subjected to

this sirnulated seed samplins)

No. ofseeds

harves-ted percâpi-

tulum

Number of plants identified as havingblâck seeds (a) No. of

seedsharvested

(b)

No. of blâck seeds harvested ExpectedM2

mutantsper capi-

tulum(c)Mean Rânge x2

(l d.f) Total Percapitulum R'ânge

I

2

3

46

I12

16

24

1

2

34

6o

12

16

24

t2.422.931.837.541.8454648

49

4-2812-3726-3534-4134-44

45

45-4748

49

4L6832

r,248t,6642,4963,328

4,9526,6569,984

99191

290

401

582

7841,094

1,543

2,317

122177

303

404621800

t,2521,516

2,605

0.240.460.?00.961.40

1.88

2.633.71

a-D /

o.290.420.?30.9?1.49

1.92

3.01

3.646.26

0-10-20-30-30-40-50-70-100-14

0-10-20-30-40-50-70-90-120-28

0.060.120.18

0.240.350.47

0.660.921.39

48.5 **f33.1 +**

1.62

2.61

0.61

0

0.170

0

75.2

r9.127.429.6

39.641.846.047.4

50.2

3-299-32

16-3515-4034-453B-4?36-5042-514B-52

45.1 *8

23.8 **12.0

19.i1 **2.772.243.70t.520.23

416

832

L,2481,664

2,4963,3284,9926,6569,984

0.070.100.180.240.370.480.750.91

1.56

a) Out of 52 capi.tula with black seeds, mean of I angles or templatesb) 52 capitula, B angles (non-rândom), or B templates (random selection)c) Assuming monogenlc segregation.

Toble 4

Mean number of cases (out of 52) in which black seeds could be recovered after non-random (n) orrandom (r) sampling

Seeds percapitu-

lum

No, of black seeds selected

0 q 5 o B I l0 1l 12-18

I nr

39.636.8

lrL15.2

2nr

29.r32.9

21.916.1

2.03.0

3nr

20.224.6

27.518.1

4.08.0

0.?t.2

4nr

14.522.4

26.513.5

9.611.5

1.54.5

00.1

6nr

10.2t2.4

18.916.6

16.010.9

5.69.8

0.11.9

00.5

00

Bnr

7.010.2

15.013.8

10.0L2,L

2.0l-c

3.0AÀ

02.4

01.2

00.4 0-

12n 6.06.0

11.99.8

9.19.8

7.L7.O

10.15.9

L'4.8

1.05.0

2.52.4

01.1

00.4

00

00

00

16nr

4.04.6

7.07.4

10.08.5

7.08.0

4.06.8

8.04-9

5.53.5

1.53.4

2.O2.5

0.61.0 0.8

00.4

00.4

24nr

3.01.8

1.54.1

6.56.0

10.06.0

t.44.L

6.63.4

2.03.6

6.02.8

3.53.1

5.53.6

1.03.6

2.03.6

3.06.2

32

CONCLUSIONS

Hybrid' seeds heterozygous for a markergene were irradiated with the objective toform of a ,ûriangular s:ector of the M1 capiLu-mutated tissue appeared in several cases infrom of a triangular sector of the M1 capitu-lum. When seeds are treated, the M2 seeds har-vested on the M1 plants are usually derivedfrom nonmutated and mutated M1 embryo cells.Few seeds from the mutant sector have to beharvested to assure a recovery of a mutant.The last column of Table 3 gives the numer ofsuch M2 mutants that can be expected per chi-meric capitulum. About 20 seeds have to becollected to assure harvesting from a mutantsector and to recover at least one mutant plantin the I\{2 generation.

The data indicate that the two approachesnon-random and random sampling yield simi-lar numbers of mutant seeds. From a breedingpoint of view, however', the random seiectionprocedure has sorne advantagcs as it needs lesslabour : any desired number of seeds can besampled easily from the threshed M1 capi-tulum.

Dans l'étude présente, on a utilisé un gène mar-queur de la graine. Les graines F1 obtenues par lecroisement des lignées autofécondées CA-83-199(graines blanches) et C 6-83-2 9?4 (graines noires)ont été irradiées avec 0, 100 et 200 Gy rayonsgamma.

Les capitules mûres des plantes F1M1 ont été exa-minés concernant I'apparition des "secteurs" chimé-riques. Deux séries d'expériences ont été effectuéespour simuler différents systèmes de prélèvement deséchantillons de graines des capitules F1M1 : simulésans-hasard et simulé au hasard. Pour simuler Ieprélèvement des échantillons on a utilisé un modèledivisé en B secteurs et 4 zones circulaires.

On a examiné un nombre de 4727 plantes F1M1,parmi lesquelles 52 capitules ont présentés le chimé-risme. Plusieurs chimères ont eu une forme de sec-teur mais on a observé aussi quelques configurationscomplexes. En ce qui concerne le prélèvement auhasard ou sans-hasard des échantillons, les donnéesont indiquées que les deux procédés permettentI'obtention d'un nornbre similaire de graines mutan-!'s. Du point de vue de i'amélioration, le procédéde prélèvement au hasard des échantillons présenttoutefois I'avantage de nécessiter moins de travailmanuel.

QUIMERISMO EN LAS PLANTAS MI DEGIRASOL Y SU IMPORTANCIA EN LA MEJORA

POR EL METODO DE LA MUTACIÔN

Resû,men

El modelo quimérico de las plantas M1 difiere enfunciôn del sistema ontogenético de la respectiva es-pecie. El conocimiento del modelo permite la co-lecciôn eficiente de las semillas de las plantas M1.

En el presente estudio se ha empleado un genemarcador de la semilla. Las semillas Fr obtenidas porel cruce de las lines cosanguineas CÀ-AS-ISS ise-millas blancas) ;z g 6-83-2 9?4 (semillas negras) seiradiaron con 0, 100 0 200 Gy rayos Gamma.

Los capitulos maduros de las plantas F1M1 fueronexaminados can respecto a la apariciôn de los .,sec-tores" quiméricos. Se han efectuado dos series deexperiencias para esimular dilerentes sistemas detomar las pruebas : de los capitulos F1M1 esimuladocasualmente y esimulado nocasualmente. Un modelodividido en ocho sectores y cuatro zonas circularesse ha empleado pâra esimular la toma de lospruôas.

Se han examinado 4 727 plantas F1M1, de las cua-les 52 capitnlos mostraron quimerismo. Varias qui-meras fueron de tipo sectorial, pero a la vez senotaron algunas configuraciones complejas. En cuantoa la toma casual y no casual de las pruebas, losdatos indicaron que los dos procedimientos permitenobtenere un nûmero similar de semillas maduras.Desde el punto de vista de -la mejora el procedi-miento de tomar casualmente las pruebas presentasin embargo la ventaja de necesitar menos trabajomanual.

REFERENCBS

B r o c k R. D., 7979, Mutation plant breeding f or seeilpl'otein improuement, Seed protein Improve-ment in Cereals and Grain Legumes, IAEA,STI/PUBi496, Vol. I, 43-55.

Hermelin T., Brunner H., Daskalov S.,N a k a i H., 1983, Chimerism in M1 plants ofVicia faba, Capsicum annuurrL and Linum usi-tatissirnum. Chirnerism in lrrailiatecl Dicotgle-donous Plants, IAEA, TECDOC - ZBS. 85-42.

L i S. L., R é d e i c. P., 1969, Estimation ol muta-tion rate in autogannous d.iploid,s, Radiat, Bot.9, 125-131.

Weiling F., 1960, iiber d,ie Hiiufigkeit der nachRôntgenbestrahlung uon Samen iiberlebencledCorpusinitialen, Biometr. Zeitschr. 2, 145-163.

LE CHIMÉRISME CHEZ LES PLANTES MlDE TOURNESOL ET SON IMPORTANCE

POUR L'AMÉLIORATION PAR LA MÉTHODEDES MUTATIONS

Résumé

Le modèle chimérique des plantes M1 diffère enfonction du système ontogénique de I'espèce respec-tive. La connaissance du modèle permet le prélève-ment efficient des graines des plantes Mr.