Euphytica 18 (1969) : 71-78

GENETIC NECROSIS IN TRITICUM xAEGILOPS PENTAPLOID HYBRIDS

K. A. SIDDIQUI' and J . K . JONES

Department of Agricultural Botany, The University, Reading, England

Received 22 June 1968

SUMMARY

Ten varieties of Triticum aestivum and three synthetic T. durum + Aegilops squar-rosa2 hexaploids were crossed with an Ae. bicornis -}- Ae. squarrosa amphiploid . 55hybrid plants involving 9 different combinations developed necrosis and died beforematurity . The two plants which survived were both from the F i with a synthetichexaploid, T. durum var. Carleton + Ae. squarrosa .

The genetic necrosis developing in these hybrids differed from hybrid necrosis andred hybrid chlorosis both in symptoms and in the genetic basis . The symptoms weresimilar to those reported for a number of Triticum x Aegilops hybrids in which Ae .bicornis and Ae . squarrosa were involved but the genetic basis was not determined .The survival of 2 of the 13 progenies from one cross is discussed .

INTRODUCTION

Pentaploid hybrids are the recurrent parents in the series of backcrosses that isrequired for the extraction or reconstitution of tetraploid components from a naturalhexaploid species (KERBER, 1964 ; SIDDIQUI, 1964). The Fi pentaploid hybrids betweenTriticum aestivum (2n = 6x = 42, AABBDD) and natural or synthetic tetraploidshaving the genomes AABB are usually vigorous, although HERMSEN (1963a) hasreported inviability and necrosis in some combinations . The present paper reports anattempt to produce a pentaploid having the genomes BBDDA for the subsequentextraction of theBBDD component from natural and synthetic AABBDD hexaploids,and the necrosis that occurred . Necrosis is used to indicate death of tissue withoutimplying any reference to or association with other terms that have been used todescribe specific types of partially or completely defective growth .

EXPERIMENTAL RESULTS

Ten varieties of T. aestivum, including some European and Pakistani varieties, andthree synthetic T. durum Ae. squarrosa hexaploids, obtained from Dr E . R . SEARS

(Missouri), were crossed with the amphiploid Ae. bicornis + Ae . squarrosa (2n = 4x= 28, SbSbDD), obtained from Dr R . Riley (Cambridge) (Table 1) . Since manyPresent address : Atomic Energy Agricultural Centre, Tando Jam, West Pakistan .

2 The + sign indicates an amphiploid .

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K. A . SIDDIQUI AND J. K . JONES

anthers in the amphiploid contained less than 5 % stainable pollen and the most fertileanthers contained less than 50 %, the hexaploids were used as pollen parent in most ofthe the crosses . All the 192 reciprocal crosses were unsuccessful .

During 1962 and 1963, 1,992 florets of the Ae. bicornis + Ae . squarrosa amphiploidwere pollinated and 88 hybrid seeds were obtained (Table 1). More varieties of T.aestivum and all 3 synthetic hexaploids were used in 1963, but the crosses were lesssuccessful. The total seed set was 17 % in 1962 but was only 3 % in 1963. This reduc-tion in seed set occurred for all combinations that were crossed extensively in bothyears .

Seed set in the Ae. bicornis + Ae. squarrosa amphiploid was lower in 1963 also .Self-pollination of 50 florets in 1962 produced 27 seeds (54 %) but in the followingyear 16 seeds were obtained from the 100 selfed florets . It is probable, therefore, thatthe reduced success of the crosses with the amphiploid in 1963 was due to the lowerfertility of this parent . It seems likely that this difference between the fertility in twoyears was due to variation in some environmental factors .

Table 1 . Results from crosses of the amphiploid Ae. bicornis + Ae. squarrosa (2n = 28, SbSbDD)with varieties of T. aestivum and synthetic T. durum + Ae. squarrosa hexaploids (2n = 42, AABBDD) .

' Other varieties (1963) : C591 (30), H-23-42 (30), Peko (112), Cappelle Desprez (48) = 220 .

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Pollen parent Summer 1962 Summer 1963

No. floretspollinated

No. seedsobtained

(%) No. floretspollinated

No. seedsobtained

(%)

T. aestivumChinese Spring

disomic 46 4 254 6monosomic 5B 38 12 - -monosomic 6D 40 3 - -monosomic 7D - 70 0

Koga 20 0 412 6Svenno 10 1 72 2C518 22 8 22 0April Bearded - 280 22A.T. 38 - 46 1Other varieties' - 220 0

subtotals 176 28 (16) 1376 37 (3)

T. durum + Ae. squarrosaamphiploids

Carleton + Ae. squarrosa 44 10 192 12Iumillo + Ae. squarrosa - - 130 1PI 94587 + Ae. squarrosa 74 0

Subtotals 44 10 (23) 396 13 (3)

Totals 220 38 (17) 1772 50 (3)

NECROSIS IN TRITICUM X AEGILOPS HYBRIDS

The results are too variable to indicate genetic differences in crossability . T. aestivumvar. Chinese Spring and the synthetic hexaploid T. durum var. Carleton - Ae. squar-rosa were successful parents in both years, but reciprocal crosses between the F Ihybrids of these two hexaploids and the amphiploid were unsuccessful . This failuremight have been caused by the partial sterility of the F i hybrids or could indicate thepresence of non-allelic recessive crossability genes in the hexaploid parents .

All of the 88 hybrid seeds were intermediate in size between those of the Ae. bicor-nis d- Ae. squarrosa amphiploid and the respective hexaploid parents . The seeds werenot wrinkled and 57 of the 68 that were sown germinated after 3 to 5 days whereas theselfed amphiploid seed germinated after 8 to 10 days . There was therefore no indi-cation of irregularity in seed development .

Forty-three of the seeds were germinated in Petri dishes and the seedlings were thengrown in John Innes compost No . 2. All of seedlings appeared to be normal imme-diately after germination but 34 died at the seedling stage within 1 to 5 weeks beforeany secondary tillers had developed (Table 2) . Most of these seedlings produced 2 or 3leaves only, and the maximum number of leaves on the primary tiller was 6 . The firstsigns of necrosis occurred on the youngest leaf, which died and turned brown . Theolder, fully expanded leaves subsequently developed a similar necrosis .

Table 2 . Survival of pentaploid SbBDDA hybrids, (Ae. bicornis = Ae. squarrosa amphiploid ;K

hexaploid wheat) .

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Pollen parent No. seedssown

No. plants No . dead No. survived

seedlingstage

aftertillering

Grown in compostChinese Spring

disomic 4 2 0 2 0monosomic 5B 12 9 8 I 0monosomic 6D 3 3 3 0 0

April Bearded 12 11 11 0 0C518 7 6 3 3 0Svenno 3 3 3 0 0A.T . 38 1 1 1 0 0Carleton -r Ae. squarrosa 9 7 5 l 1lumillo + Ae. squarrosa 1 I 1 0 0

Subtotals 52 43 35 7 1

Grown in sand cultureApril Bearded 8 6 5 1 0Svenno 2 2 2 0 0Carleton + Ae. squarrosa 6 6 4 1 1

Subtotals 16 14 1 1 1 1

Totals 68 57 46 8 2

K . A. SIDDIQUI AND J . K . JONES

Some plants of three different pentaploid hybrids involving the hexaploids ChineseSpring, C518, and T . durum var. Carleton + Ae. squarrosa produced from 5 to 19tillers and lived for 2 to 4 months . Other plants from each of these progenies died at theseedling stage. Necrosis in these tillering plants started on the youngest leaf of theoldest, primary tiller . This leaf and then successively older leaves and ultimately theentire tiller turned brown . Similar necrotic symptoms appeared in all other tillers andthe plants died .

Since HERMSEN (1963b) reported that environmental conditions could influence theexpression of hybrid necrosis in some intervarietal wheat hybrids, some of the penta-ploid hybrids were grown in different cultural conditions . Sixteen of the hybrid seed-lings from three of the hexaploid parents were germinated in Petri dishes and thentransferred to pots of sterilized sand, which was supplied with a Long Ashton nutrientsolution (HEwrrT, 1952) . Four plants of each parent were grown in the same sandculture, and all grew vigorously and produced ears . All except one of the pentaploidhybrids died either at the seedling stage or after producing one or more secondarytillers . Eight of these plants were sprayed with 10 ppm aqueous solution of gibberellicacid either before or after the first appearance of necrosis . This did not promote anynew growth but delayed the onset of necrosis in the older leaves for up to 4 weeks . Allof the plants which were sprayed eventually died after showing the usual necroticsymptoms .

Two of the hybrids from the cross (Ae. bicornis + Ae . squarrosa) x (T. durum var .Carleton + Ae. squarrosa) did not develop necrosis except at the tips of some of theyoungest leaves at the time of ear emergence . One was grown in compost and theother in sand culture but not sprayed with gibberellic acid . Both plants grew vigorous-ly and produced many tillers and ears, but were completely sterile .

The chromosome number of 14 necrotic seedlings was determined . Eleven had 35chromosomes and three, all from crosses with Chinese Spring monosomics, had 34 .There was no difference between the morphology and necrosis in the 34 and 35 chro-mosome seedlings. There is therefore no evidence that aneuploidy was a cause ofnecrosis in the hybrids with the Ae. bicornis + Ae. squarrosa amphiploid .

Of the 57 hybrid pentaploids that were obtained from crosses between the Ae.bicornis + Ae. squarrosa amphiploid and a number of natural and synthetic hexa-ploids, all but two developed necrosis and died (Table 2) . The two plants which surviv-ed were from the same combination (Ae. bicornis + Ae . squarrosa) x (T. durum var .Carleton + Ae. squarrosa) but other plants from the same progeny developednecrosis and died . Both of the viable hybrids were pentaploid and completely sterile .

Some necrosis occurred in the Ae. bicornis + Ae. squarrosa amphiploid also . All50 plants grew vigorously for about two months and produced an average of 21 tillersper plant. Necrosis then began to appear on the youngest leaf of one of one of thesecondary tillers, not on the oldest tiller as in the hybrids. Subsequently this tiller andall other tillers died . Fifteen plants developed this necrosis and died before earemergence, 5 of 25 in 1962 and 10 of 25 in 1963 . Two other plants developed necrosisafter ears had begun to emerge and all the vegetative tillers died . The flowering tillersdid not show any necrosis and other normal tillers developed later on these plants .

The chromosome numbers of 25 of the plants were determined . All but one had28 chromosomes . The exception, an aneuploid having 27 chromosomes, was one of

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the 15 plants that died before ear emergence, but 4 other necrotic plants had a tetra-ploid complement. Although it was not possible to check that this was a normal com-plement by studying chromosome pairing, it seems unlikely that aneuploidy was themain cause of necrosis in these tetraploids. The occurrence of necrosis and the re-duction of fertility in 1963 may have been different symptoms of the instability ofgenetic expression in the amphiploid .

The amphiploid Ae. bicornis -±-- Ae. squarrosa was crossed with Secale cereale andS. montanum also (SIDDIQUI, 1964) . No necrosis was observed in the 7 hybrids thatwere grown . The viability of these is of no significance except in indicating that thenecrosis genes from the amphiploid are not expressed in all interspecific hybrids .

DISCUSSION

Partially or completely defective growth and development has been reported formany intervarietal and interspecific hybrids in Triticum and Aegilops . The nature andthe sequence of the characteristic symptoms have been described by a number ofterms including firing (MCMILLAN, 1936), progressive lethal necrosis (CALDWELL andCOMPTON, 1943), inviability (SEARS, 1944), lethality (HERBERT and MIDDLETON, 1955),semi-lethality (Roy, 1955), hybrid necrosis (HERMSEN, 1962), red hybrid chlorosis(HERMSEN, 1966), and necrosis types 1 and 2 (NISHIKAWA, 1962) . In several cases thegenetic basis of the symptoms has been determined . The symptoms for particularparental genotypes are usually fairly uniform, and the defective hybrids can bearranged into groups having similar symptoms . It is therefore useful to compare thegenetic necrosis reported in this paper with these groups .

HERMSEN (1966) made an extensive study of two types of genetic necrosis, hybridnecrosis and red hybrid chlorosis, that occur in intervarietal hybrids of wheat . Redhybrid chlorosis, determined by complementary genes Chi and Ch2 , is characterizedby a reddening of the foliage in addition to chlorotic and yellowish discolouration .Since red colouration did not develop in any of the hybrids with the Ae. bicornisAe. squarrosa amphiploid, there is no evidence that genes Ch, and Ch2 were involved .

Hybrid necrosis, determined by complementary genes Ne, and Nee, is characterizedby a particular development of necrosis without any red colouration . The necrosisstarts at the tip of the oldest leaf, usually but not always on the oldest or primarytillers . A change in colour either to dark or to yellow green precedes the completedegradation of chlorophyll and the development of brown necrotic leaves . This pre-mature gradual death of leaves and leaf sheaths may start at any stage from the seed-ling, when it is usually complete and lethal, until after ears have emerged . Variation inthe nature of necrosis is attributed to multiple allelomorphs of Net and Nee differing inrespect of the intensity of the necrosis and the stage at which it develops . HERMSEN(1966) has reported that plants having the genotype for weak necrosis may be pheno-typically normal . Many varieties of T. aestivum and T. durum have been classifiedaccording to the alleles of Nei and Nee that they carry (HERMSEN, 1963c ; ZEVEN, 1965,1967) .The lethal necrosis reported by CALDWELL and COMPTON (1943) and by HERBERT

and MIDDLETON (1955) had symptoms very similar to those of hybrid necrosis and theformer was similarly caused by two complementary genes . The symptoms of firing

NECROSIS IN TRITICUM X AEGILOPS HYBRIDS

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K. A . SIDDIQUI AND J. K . JONES

(MCMILLAN, 1936) differed in that the leaves died as a whole rather than from the tip .This could be a consequence of the late development of necrosis after the ears hademerged . HERMSEN (1966) considered firing to be a weak form of hybrid necrosis andreported a basis of two complementary genes .

These four reports of necrosis are characterized by symptoms developing first onthe oldest leaf generally of the oldest tiller, and proceeding gradually to youngertissues. There is no red colouration and growth is not retarded either before or duringthe first development of necrosis . This necrosis appears to be a premature ageing ofdifferentiated tissue, the apical meristem remaining normal and new leaves beingproduced at least until the condition has spread throughout the plant .

The development of the necrosis reported in the T. aestivum x (Ae. bicornis + Ae .squarrosa) hybrids was different. Necrosis started at the tip of the youngest leaf of theoldest primary tiller and subsequently developed in older leaves . Further, the differ-ence between varieties carrying or not carrying particular alleles of Nel and Nee wasnot expressed in hybrids with the (Ae. bicornis + Ae . squarrosa) amphiploid . ChineseSpring carries an allele of Nel which gives weak hybrid necrosis on some leaves afterear emergence in intervarietal hybrids . lumillo and Carleton both carry alleles of Nelwhich cause severe necrosis in hybrids (HERMSEN, 1963b ; NISHIKAWA, 1967). Svennois reported to be a non-carrier of Nel and Nee . Crosses between the (Ae. bicornis + Ae .squarrosa) amphiploid and lumillo, Chinese Spring or Svenno all produced hybridswith severe necrosis which died before ears emerged, whereas 2 of the hybrids withCarleton did not develop necrosis . Of the other varieties which were parents of ne-crotic hybrids, Koga I carries Nel (HERMSEN, 1963b), the Pakistani varieties mostprobably are Nei or non-carriers (TSUNEWAKI and NAKAI, 1967a, 1967b ; ZEVEN, 1966and pers. comm. 1968) and April Bearded has not been tested . Hybrids of the amphi-ploid with varieties known to carry Nee were not obtained, but the results, includingthe sequence of necrosis, suggest that it is unlikely that alleles at the loci of Net andNee caused the necrosis in these hybrids . It could however have been caused by asimilar complementary action of other genes in the species of Triticum and Aegilops .

Complementary inviability in hybrids with various combinations of the fourgenomes A, B, D and Sb has been reported by SEARS (1944), Roy (1955) and NISHI-KAWA (1962). SEARS (1944) also reported inviability in hybrids between einkorn wheat(AA) and Ae. umbellulata (CuCu). In all cases in which necrosis leading to lethalityoccurred in seedlings or young plants, the symptoms followed the same pattern as thatfound in the hybrids with the Ae. bicornis + Ae. squarrosa amphiploid. Growthbecame retarded and necrosis developed on the youngest leaf of the oldest tiller, oftenthe only tiller. The older differentiated leaves remained green for a longer time beforebecoming necrotic . Such genetic necrosis developing either in or near to the oldestapical meristem is distinct from hybrid necrosis which appears to be the prematuredegeneration of older tissues, and from red hybrid chlorosis which produces a charac-teristic colouration and does not develop initially in the apical meristem. The degene-ration of the first shoot apex is followed by the spreading of the condition, possible,of a toxic chemical, to other apices and to differentiated tissue .

Some genetic analysis of this apical necrosis has been reported . SEARS (1944)reported monofactorial segregation in the A genome of Triticum of independentfactors which gave inviability with Ae. umbellulata and Ae. bicornis respectively. Roy

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NECROSIS IN TRITICUM X AEGILOPS HYBRIDS

(1955) reported evidence that similar necrosis was caused by complementary inter-action between factors in the Sn and D genomes . NISHUCAWA (1962) suggested afactor in either the A or B genomes giving complementary necrosis with a factor inthe D genomes. Any of these complementary interactions could be the cause of thenecrosis reported for the hybrids with the Ae. bicornis + Ae. squarrosa amphiploid .Further, since SEARS (1944) showed that similar necrosis in different interspecifichybrids could be caused by different factors in the common parent, the geneticnecrosis reported in this paper could be determined by other factors in two or more ofthe genomes .

The occurrence of necrotic and normal plants in the same F l progeny of (T. durumvar. Carleton + Ae. squarrosa) x (Ae. bicornis Ae. squarrosa) and the develop-ment of necrosis at different stages in the same progeny is an additional problem .Both of these parents had been induced from triploid and diploid hybrids, respect-ively, and would initially have been completely homozygous . Most plants in varietiesof T. aestivum are homozygous also. It was expected therefore that each F l progenywould be uniformly heterozygous, and consequently have similar phenotypes .

Genetic variation in the parents or in the gametes from them could have beenproduced by gene mutation, by aneuploidy or by irregular recombination . Occasionaloutcrossing also occurs in T. aestivum but is unlikely to have occurred unnoticed inthe induced polyploids . Since both Ae. bicornis + Ae. squarrosa and T. durum var .Carleton + Ae. squarrosa were produced several generations earlier, gene mutationsproducing heterogeneity in interspecific progeny could have occurred . Such mutationswould not be detected except in the hybrids, and both polyploids had been maintainedas a population and not as isolated single plant progenies . Occasional aneuploidy wasobserved in the Ae. bicornis + Ae. squarrosa amphiploid, but it was not associatedwith the occurrence of necrosis and the symptoms were different from those in thehybrids . The occurrence of aneuploids suggests that meiosis may be irregular in somecells of the amphiploid although there is no direct evidence of this. Occasional re-combination in multivalents or homoeologous bivalents could produce geneticheterogeneity in gametes and consequently in progeny, but it seems unlikely that thiswas the main cause of the variation in the development of necrosis and of the absenceof necrosis in two plants form the same progeny .

The alternative hypothesis is that the variation was not caused by genetic variationbut was due to instability in phenotypic expression . The occasional absence of ex-pression of weak necrosis in T. aestivum hybrids (HERMSEN, 1966) and the survival of1 or 2 plants to produce a single spike when most of the progeny died at the seedlingstage (Roy, 1955) may be comparable examples of phenotypic instability (STEBBINS,

1958), which seems to be the most likely cause of the variation that occurred. Theoccurrence of such phenotypic instability in progenies that are known to be geneticallyuniform could provide useful material for the study of the physiology and biochemis-try of the several complementary necroses that have been reported .

The occurrence of necrosis and other defective growth in interspecific hybrids is apotential barrier to the transference of factors in the breeding of wheat, although inmost cases the complementary necrosis factors appears to be characteristic of parti-cular genotypes rather than of species . The necrosis factors may have some evolution-ary significance also, but since post-fertilisation barriers are wasteful and therefore

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inefficient isolating mechanisms, the several complementary interactions which pro-duce genetic necrosis are probably the consequences of changes that have occurredat related loci since the species became isolated .

REFERENCES

CALDWELL, R. M . and COMPTON, L . E ., 1943. Complementary lethal genes in wheat causing a pro-gressive lethal necrosis of seedlings . J . Heredity 34 : 66-70.

HERMSEN, J. G. TH ., 1962. Bastaard-necrose bij tarwe (Hybrid necrosis in wheat) - Verslagen vanLandbouwk . Onderz . no . 68 .5 : pp. 129 (Summary in English) .

HERMSEN, J . G. TH., 1963a . The genetic basis of hybrid necrosis in wheat . Genetica 33 : 245-287 .HERMSEN, J . G . TH ., 1963b. Hybrid necrosis as a problem for the wheat breeder . Euphytica 12 : 1-16 .HERMSEN, J . G . TH ., 1963c . Sources and distribution of the complementary genes for hybrid necrosis

in wheat . Euphytica 12 : 147-160 .HERMSEN, J. G . TH ., 1966 . Hybrid necrosis and red hybrid chlorosis in wheat . Proc . 2nd Int. Wheat

Genetics Symp ., Lund 1963, Hereditas, Suppl . 2 146-166 .HEwirr, E . J., 1952 . Sand and water culture methods used in the study of plant nutrition . Tech .

Comm. 22, Comm. Bureau, Hort . Plant Crops .KERBER, E. R., 1964. Wheat : Reconstitution of the tetraploid component of hexaploids . Science 143 :

253-255 .MCMILLAN, J . R. A ., 1936 . `Firing' - A heritable character of wheat . J. Coun . Sci . Industr . Res . 9 :

283-249 .NISNIKAWA, K., 1962 . Hybrid lethality in crosses between emmer wheats and Aegilops squarrosa. III.

Gene analysis of type 2 necrosis . Seiken Ziho 14 : 45-50 .NISHIKAWA, K., 1967 . Identification and distribution of necrosis and chlorosis genes in tetraploid

wheat . Seiken Ziho 19 : 37-42 .Roy, R. P ., 1955 . Semi-lethal hybrids in crosses of species and synthetic amphidiploids of Triticum

and Aegilops . Indian J . Genet. Plant Breed . 15 : 88-98 .SEARS, E . R ., 1944 . Inviability of intergeneric hybrids involving Triticum monococcum and T. aegilo-

poides.SIDDIQUI, K . A ., 1964 . Cytogenetic studies in the tribe Triticeae . Ph . D . Thesis . University of Reading,

England .STEBBINS, G . L., 1958 . Hybrid inviability, weakness and sterility . Adv . Genet . 9 : 147-215 .TSUNEWAKI, K. and NAKAT, Y . 1967a . Necrosis genes in KUSE wheat . Wheat Inf. Serv. Kyoto Univ .

Nos. 23/24 : 39-46.TSUNEWAKI, K. and NAKAI, Y. 1967b . Distribution of necrosis genes in wheat . 1 . Common wheat

from Central Asia. Can . J . Genet . Cytol . 9 : 69-74 .ZEVEN, A. C., 1965. First supplementary list of genotypes of hybrid necrosis of wheat varieties .

Euphytica 14 : 239-243 .ZEVEN, A. C., 1966 . Geographical distribution of genes causing hybrid necrosis in wheat . Euphytica

15 : 281-284 .ZEVEN, A . C., 1967. Second supplementary list of genotypes of hybrid necrosis of wheat varieties .

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