Euphytica 60 : 105-113, 1992 .©1992 Kluwer Academic Publishers . Printed in the Netherlands .

The importance of Endosperm Balance Number in potato breeding andthe evolution of tuber-bearing Solanum species

Rodomiro Ortiz' & Mark K . Ehlenfeldt2' Rutgers University, Cook College ; 2 USDA-ARS, Blueberry & Cranberry Research Center, Chatsworth,NJ 08019, USA

Received 31 October 1991 ; accepted 6 March 1992

Key words: bridging crosses, chromosome addition lines, Endosperm Balance Number, evolution,2n gametes, imprinting, interspecific crosses, ploidy manipulations, tuber-bearing Solanum species

Summary

Endosperm failure is considered the primary reason for the lack of success in intra- and interspecific crosses .The Endosperm Balance Number (EBN) hypothesis is a unifying concept for predicting endosperm functionin intraspecific, interploidy, and interspecific crosses . In the EBN system, every species has an `effectiveploidy' (EBN), which must be in a 2 :1 maternal to paternal ratio in the endosperm for crosses to succeed . Theknowledge of EBN is very useful in the transfer of genes from exotic germplasm, and in the development ofnew breeding schemes in potato . The paper describes the strategies for introducing 2x(lEBN), 2x(2EBN),4x(2EBN) and 6x(4EBN) germplasm into the cultivated 4x(4EBN) potato gene pool . A new methodologyfor producing 4x(4EBN) and 2x(2EBN) chromosome addition lines is also discussed . EBN has evolutionaryimportance in the origin of tuber-bearing Solanums . The role of the EBN in the origin of diploid andpolyploid potato species, and as a barrier for hybridization and speciation of sympatric species within thesame ploidy level is demonstrated . The origin of 3x and 5x cultivated tuber-bearing Solanums may also beexplained using the EBN concept . EBN has been reported to exist in other plant species : alfalfa, beans,blueberries, rice, soybeans, squashes, tomato, forage legumes, grasses, ornamentals and Datura stramoni-um. This indicates that EBN may have broad application and could be useful for germplasm transfer andbreeding in other crop species .

Introduction

The endosperm is a unique and very importantplant tissue of the angiosperms. Like the embryo, itis the result of double fertilization . In normal fertil-ization within 2x species, one sperm nucleus (lx)fertilizes the central cell (2x) and the other the eggcell (lx) . This results in the formation of a 2x em-bryo associated with a 3x endosperm (Fig . la) . Theendosperm, however, has a transitory life becauseit does not form germ cells .

Brink & Cooper (1947) were the first to establish

that endosperm failure was the primary reason forthe lack of success in intra- and interspecific crossesand ultimately, embryo death . Different hypothes-es had been postulated earlier to explain seed fail-ure. The most widely accepted suggested that thefailure was related to the relative ploidy levels ofmaternal : endosperm : embryo tissue (Muntzing,1930). These tissues are in a ratio 2 :3 :2, respec-tively, in intra- and interspecific diploid (2x) cross-es . The theory suggested that any deviation fromthis ratio resulted in the failure of the cross . Thehypothesis fell from favor after the success of sever-

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A

Normal fertilization2x x 2x

centralcell2x

egg1x

00

'CD = colchicine doubled .

B

Triploid block4x x 2x

Unknown EBN

Standard EBN

4x `A'

x 2x (2EBN)x 4x (4EBN)

4x `B'

x 2x (2EBN)x 4x (4EBN)

2x `C'

x 2x (2EBN)x 4x (4EBN)

4x `C' (CD)'

x 2x (2EBN)x 4x (4EBN)

00

Fig . 1 . a . Normal double fertilization in 2x x 2x crosses .b . Triploid block after 4x x 2x crosses .

al crosses which did not follow the postulated rule .An alternative hypothesis was postulated by Lin(1984). He demonstrated in diploid maize, thatnormal endosperm development required a 2 :1 ma-ternal to paternal genome ratio in the endosperm .Deviations from this 2 :1 ratio resulted in a failure ofendosperm development and ultimately, in seedabortion. This work clearly indicated that not onlythe ratio and total dosage, but also the paternalsource were critical in determining endospermfunction .

EBN concept

Lin's work satisfactorily explained the results ob-tained in intraspecific interploidy crosses such asdiploid x autotetraploid or extracted diploid xtetraploid crosses ; however, the 2 :1 maternal to

paternal genomic requirement in the endospermwas violated in some interspecific interploidy cross-es between tuber-bearing Solanum species (Irik-ura, 1968; von Wangenheim, 1955), Lycopersiconspp. (Cooper & Brink, 1945), Avena spp. (Nishiya-ma & Yabuno, 1978), Medicago spp. (Clement,1963 ; Lesins, 1961 ; McCoy & Smith, 1983), Gossy-pium spp . (Stephens, 1942), and Dactylis spp .(Carrol & Borrill, 1965 ; Jones & Borrill, 1962) .These results led to the development of a newconcept termed Endosperm Balance Number(EBN) (Johnston et al ., 1980) . In this theory, nor-mal seed development depends on the balance ofgenetic factors in the endosperm which are contrib-uted by both the female and male gametes . In theEBN system, every species has an `effective ploidy'(EBN), not necessarily equivalent to its real ploi-dy, which must be in a 2:1 maternal to paternalratio in the endosperm for crosses to succeed .Endosperm development fails when there is a de-viation from the 2:1 maternal to paternal EBNratio .

EBN assignment

The EBNs are themselves arbitrary values, as-signed to species based on their crossing behaviorin matings with known EBN standards (Table 1) .Solanum chacoense, a South American diploid spe-cies, is the 2EBN standard used in assigning EBNvalues to tuber-bearing Solanum species . In the

Table 1 . Assignment of EBNs to unknown species by crosses with known EBN standards

Result

Conclusion

failed4x

3xfailed

failedfailed

3xfailed

`A' is 4x (4EBN)

`B' is 4x (2EBN)

inconclusive

`C' is 2x (1EBN)

course of extensive testing, 2x(1EBN), 2x(2EBN),4x(2EBN), 4x(4EBN) and 6x(4EBN) species havebeen identified (Johnston & Hanneman, 1980,1982). In tuber-bearing Solanum species, EBNsare whole numbers; however, EBN need not al-ways be an integer. The genetic model of Ehlen-feldt & Hanneman (1988b) suggests that EBN is anadditive, three-gene trait, and that single genechanges may occur .

Predictive value

The knowledge of the EBN values has been usefulin predicting the success or failure of interspecificcrosses as well as the ploidy of the progeny . Thefailure of triploid production (triploid block) after4x x 2x crosses (Marks, 1966) can be explained interms of EBN (Fig . 1b) . Triploid block is the conse-quence of an imbalance between the genetic factorscontributed by female and male gametes . In 4x x2x crosses the maternal to paternal genome EBNcontributions are in a 4:1 ratio in the endosperm .This imbalance results in the failure of endospermdevelopment and seed abortion .

The importance of EBN is also illustrated bycrosses between the two sympatric 4x species S.acaule (2EBN) x S. tuberosum Gp . Andigena(4EBN) which do not succeed (von Wangenheim,1955) in accord with the EBN hypothesis . Thiscross fails due to endosperm breakdown, yet if theploidy of the S. acaule parent is doubled, an altered8x(4EBN) S. acaule x 4x(4EBN) S . tuberosumcross can be readily made yielding 6x progeny . Inthis case, the cross which succeeds has a 10x (8 :2ploidy ratio) endosperm, but a 4 maternal : 2 pater-nal EBN ratio. The cross which fails has a hex-aploid endosperm but only a 2 maternal : 2 paternalEBN ratio .

Utilization of EBN knowledge for potato breeding

The EBN concept has been very useful in exoticgermplasm transfer and the development of newbreeding schemes . The knowledge of EBN can be

107

used for directed ploidy manipulations and the de-sign of bridging crosses in potato breeding .

Utilization of 2x(1EBN) species and theirintroduction into the cultivated 4x gene pool

S. etuberosum and S. brevidens are two 2x(1EBN)species from Chile. They possess many valuableattributes, e .g. PVY resistance, PLRV immunity,frost tolerance ; however, they cannot be easilycrossed with most other 2x species . The differencesin EBN between the 2x species is the primary causeof incompatibility, which prevents their utilizationin a breeding program . However, an understand-ing of the EBN concept allows the manipulation ofthis germplasm in potato improvement . For exam-ple, Johnston & Hanneman (1982) applied theknowledge of EBN to the production of interspec-ific hybrids between the non-tuber-bearing 2x(1EBN) species S . brevidens and the tuber-bearing2x(2EBN) species S . chacoense . By colchicine dou-bling the 2x(1EBN) species and crossing the resul-tant 4x(2EBN) individual with the 2x(2EBN) spe-cies, triploid (3x, 2EBN) hybrids were produced .Alternative production of similar 3x hybrids maybe accomplished through the use of 2n gametes(gametes with the sporophytic chromosome num-ber) in the 2x(1EBN) species . The resultant 3x(2EBN) hybrids can then be crossed, via 2n gameteproduction, with cultivated 4x(4EBN) Gp. Tuber-osum to produce 5x hybrids (Ehlenfeldt and Han-neman, 1984) . The scheme for the production of 5xhybrids is illustrated in Fig . 2 . Similar methods canbe used for the incorporation of 4x(2EBN) germ-plasm .

Endosperm Balance Number if also useful inunderstanding the first successful crosses of 2x(1EBN) S. etuberosum with the 2x(1EBN) tuber-bearing species S. pinnatisectum (Hermsen & Tay-lor, 1979) . The sterile hybrids produced were suc-cessfully doubled to produce 4x(2EBN) plantswhich were crossed as males with 2x(2EBN) S .verrucosum (Ramanna & Hermsen, 1982) . The 3x(2EBN) hybrids from this cross and their colchi-cine-doubled hexaploids were subsequently usedto incorporate PLRV resistance into 4x materials .

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Species parent

2x(1)

2x(2)

4x(2) 4x(2) 4x(4)I

parent

colchicineor 2n gamete

Icross

3x(2)I

2n gamete productionI

I4x(4) -, cross cross cross

W4) 4x(4)

6x(4) 5x(4) 4x(4)

Crossing

2x(2) ----j..

Fig. 2. Ploidy and EBN manipulations in the production of 4x(EBN) compatible lines and in the production of 4x(4EBN) and 2x(2EBN)chromosome addition lines . EBN in brackets . EC = extra-chromosome(s) .

Utilization of 2x(2EBN) species and 2n gametes insexual polyploidization

The use of 2x(2EBN) Solanum species and 2n ga-metes in potato breeding has been extensively doc-umented (Peloquin & Ortiz, 1991) . Two breedingschemes have been primarily utilized . The firstmethod involves the production of 4x(4EBN) off-spring via unilateral sexual polyploidization in

which one parent is a 4x(4EBN) species with ngametes, and the other is a 2x(2EBN) species with

2n gametes. The second method, bilateral sexualpolyploidization, involves the use of 2n gametesfrom both 2x(2EBN) parents to obtain 4x(4EBN)offspring . Both of these methods rely on the trip-loid block imposed by EBN requirements to elim-inate triploids and produce balanced 6x endospermand 4x embryos .

Production of 4x and 2x chromosome addition lines

The manipulation of EBN may also be used in abreeding scheme to generate 4x(4EBN) chromo-some addition lines through 5x(4EBN) x 4x(4EBN) crosses . The methodology is illustrated inFig. 2 . Utilizing this scheme, specific chromosomescarrying the allele of interest can be introduced intothe 4x ploidy level for further genetic manipula-tion .

Another potential application of EBN in plant

crossl

3x(2)I

or colchicine

cross cross

Hybrid parent

6x(4)

3x(2)

5x(4)

cross

cross

crossi'

45x(4)

2x+EC(2) 4x+EC(4)

chromosome addition lines

breeding is the production of 2x chromosome addi-tion lines using 2x species with 1 and 2 EBNs (Fig .2). The production of a 3x(2EBN) from intermat-ing species and subsequent use of the interspecific3x hybrids in crosses with the 2x(2EBN) speciesparent will lead to the production of 2x(2EBN)chromosome addition lines (Fig . 2). This materialcan then have further utilization in a programwhich emphasizes breeding at the 2x level . Haploidextraction from 5x(4EBN) clones is an alternativemethod for producing materials at the 2x level (Eh-lenfeldt & Hanneman, 1988a) . Haploid extractionappears to screen for correct balance in the endo-sperm and results in 2EBN progeny with traitsderived from the 1EBN parent .

The evolutionary importance of EBN

The control of endosperm development by EBNsuggests that endosperm plays an important role inspeciation. Plant evolutionists (Grant, 1971 ;Hawkes, 1979 ; Regal, 1977 ; Stebbins, 1976) havegiven only minor consideration to this point, how-ever, as Brink (1952) pointed out, the fact that seeddevelopment depends largely on endosperm devel-opment clearly dictates that endosperm dysfunc-tion could act as an isolating mechanism .

A hypothetical example can serve to clarify therole of the EBN in evolution . A mutation in EBNin a self-pollinated species would lead to the pro-

duction of three types of individuals (a/a . a/a', a'/a') . Fertilizations between homozygous types willbe unsuccesful because the EBNs will no longer bein a 2 :1 ratio . In this way, each one will be isolatedfrom the other and they will develop as sympatricspecies. In this regard, EBN can be considered as ascreen for diploid fidelity . Hence, EBN could act asboth a species initiator, and a mechanism for themaintenance of the species identity . This doublerole of the EBN is especially important amongsexually reproducing, non-clonal 2x species .

Diploid species evolution

Two types of diploid species occur with respect toEBN, 2x(1EBN) and 2x(2EBN) . The 2x(1EBN)types found in North America, Central Americaand scattered locations in South America, appearto correspond well to Hawkes (1990) `B' genomespecies, which suggests they are evolutionally 'ol-der' species. The 2x(2EBN) types found mainly inSouth America, with only one species (S. verruco-sum) found in limited Mexican locations (Correll,1962), appear to correspond generally to `A' ge-nome species of more recent origin .

The following example illustrates the role ofEBN as a genetic isolating mechanism for sym-patric species of the same ploidy level, and its uti-lization as a taxonomic descriptor .

Solanum chacoense (2x, 2EBN) and S . commer-sonii (2x, 1EBN) are two sympatric Argentine spe-cies in the series Commersoniana (Correll, 1962)and appear to have undergone the kind of specia-tion described above, with S. chacoense havingdiverged from S. commersonii. Within their range,rare triploid hybrids occur . Since S. chacoense pos-sesses stylar barriers (Chujoy, 1985) which preventfertilization by S. commersonii, these hybrids un-doubtedly are the product of 2n eggs from S. com-mersonii and 1n gametes from S. chacoense. Thestylar barrier in S. chacoense would not be un-expected in such a successfully diverged speciessince initially it would be needed to avoid having itseggs from being swamped by its progenitor's pol-len .

The triploid hybrids produced are potentially

interfertile with either of the two parent species,yet owing to the triploid nature of these hybrids,the segregation ratios of EBN genes, and stylarbarriers, this cross-fertility exists only on a verylimited scale . Hence, these triploids, no matterhow vigorous, are limited in their sexual reproduc-tion (Ehlenfeldt, 1984) and are unlikely to supplanteither of the parental species. Within their locales,however, these hybrids may act as both a geneticbridge and a genetic buffer between the two spe-cies .

Polyploid species evolution

The role of 2n gametes in polyploid evolution ofplants has been discussed by Harlan & de Wet(1975) . The origins of 4x and 6x tuber-bearing Sola-num species are a rich source for such examples(Mendiburu & Peloquin, 1977 ; den Nijs & Pelo-quin, 1977; Camadro & Peloquin, 1980 ; Iwanaga &Peloquin, 1982 ; Watanabe & Peloquin, 1989) .EBN complements, and is an integral factor in thefunction of 2n gametes in the evolution of pol-yploids (Table 2) .

The cultivated 4x potato (4EBN) with tetrasom-ic inheritance was presumably the result of crossesbetween two 2x(2EBN) species producing 2n ga-metes. Thus, a viable plant was obtained becausethe EBN ratio in the endosperm was 4 maternal : 2paternal after the fusion of one 2n gamete withanother 2n gamete . In such tetrasomic polyploidSolanum species, EBN provides a selective screenfor gene flow from the 2x to 4x level: 2n gametesproduced by 2x species can successfully fertilize neggs (2x) of the 4x allowing unilateral introgres-sion. Interestingly, 2n pollen is more vigorous than1x pollen (Simon & Peloquin, 1976) and shouldtherefore fertilize the 4x preferentially . Addition-ally, the highly heterozygous first division restitu-tion type of 2n pollen (as might be expected to beproduced by the 2x) outcompetes the 2x pollenwhich function during the self-pollination of the 4x,thus allowing a continuous introgression into the4x .

An example of the role of EBN in the origin ofdisomic polyploid species in the tuber-bearing So-

109

1 10

lanums is given by S. acaule, a 4x(2EBN) species .This species could have originated after the fusionof 2n gametes from two different 2x(1EBN) species(Table 2) or alternatively by the somatic doublingof a sterile 2x(1EBN) hybrid .

An alternative hypothesis is that this species re-sulted from bilateral sexual polyplodization be-tween two 2x(2EBN) species, followed by a changeof EBN as part of the diploidization process . Thiscould result in the reduction of the EBN, and theoccurrence of bivalent pairing instead of quadriv-alent formation during meiosis .

Watanabe (1988) found a low frequency of 2npollen in this species, which he suggested could bethe result of the suppression of the meiotic muta-tion by modifiers during the process of diploidiza-tion to maintian high fitness by autogamy of ngametes. In this regard, MacKey (1970) pointedout that disomic polyploids are self-pollinated spe-cies which possess the following advantages : a)genotypic fixation : the heterosis can be maintainedby heterozygosity between homeologous loci, b)specialization for a particular environment, c) ad-aptation to dispersal, d) guaranteed fertilization, e)

Table 2 . Origin of 3x, 4x, 5x and 6x tuber-bearing Solanum species via sexual polyploidization and the role of the EBN

1. 3x(2EBN)2x(1EBN, 2n gametes) X 2x(2EBN, n gametes) -* 3x(2EBN)Example: natural 3x interspecific between S. commersonii and S . chacoense .or4x (2EBN, n gametes) X 2x(2EBN, n gametes) -* 3x(2EBN)Example: cultivated 3x S . X juzepczukii.

2. disomic 4x(2EBN)2x(lEBN, 2n eggs) x 2x(1EBN, 2n pollen) -* 4x(2EBN)or2x(2EBN, 2n eggs) x 2x(2EBN, 2n pollen)yielding a 4x(4EBN), then diploidization - 4x(2EBN)Examples : S. acaule, S. stoloniferum.

3. tetrasomic 4x(4EBN)2x(2EBN, 2n eggs) x 2x(2EBN, 2n pollen) -* 4x(4EBN)Example: cultivated 4x potato S. tuberosum .

4. 5x(4EBN)3x(2EBN, 2n eggs) x 4x(4EBN, n pollen) - 5x(4EBN)Example : S . X curtilobum .

5 . disomic 6x(4EBN)4x(2EBN, 2n eggs) x 2x(2EBN, 2n pollen) -* 6x(4EBN)Examples : S. demissum and other 6x Mexican species .

pollen economy and f) ability to develop pene-tration resistance against seed borne diseases . In-deed, immunity against potato spindle tuber viroid(PSTV), one of the few diseases transmitted bytrue seed, has been found in S . acaule (Internation-al Potato Center, 1988) .

Similar origins may be postulated for 6x(4EBN)species . The disomic 6x Mexican species S. demis-sum could have originated from a cross betweentwo sympatric species : S. verrucosum, the only 2x(2EBN) Mexican species, and S . stoloniferum, adisomic 4x species (Table 2) . The structure of theknown ploidy/EBN combinations suggests that nopolysomic hexaploids exist among the tuber-bear-ing Solanum species .

Origin of odd-ploids

As mentioned earlier, triploid hybrids may be theresult of 2x(1EBN) x 2x(2EBN) crosses, when the2x(1EBN) species produces 2n gametes (Table 2) .Other odd-ploid and unexpected hybrids can occa-sionally occur, however, through exceptional

events (see Ehlenfeldt & Hanneman, 1988b for alimited discussion) . The expected method via 2x(1EBN) x 2x(2EBN) crosses has been demon-strated experimentally (Chujoy, 1985 ; Ehlenfeldt,1984) by producing 3x(2EBN) hybrids in crossesbetween S. commersonnii (2x(lEBN) with 2neggs) x S. chacoense (2x(2EBN), n pollen) . John-son & Hanneman (1980) have also produced other3x(2EBN) hybrids in a similar manner . An alterna-tive method for obtaining a 3x(2EBN) hybrid isillustrated by the origin of the cultivated 3x S . xjuzepczukii (Table 2) . This hybrid species has beenconsidered the product of natural hybridization be-tween the sympatric species 4x(2EBN) S . acauleand 2x(2EBN) Gp . Stenotomum (Hawkes, 1962) .S. X curtilobum, the cultivated 5x species in the

Andes, was probably the result of a cross betweenS . X juzepczukii (3x, 2EBN, 2n eggs) x Gp . Andi-gena (4x, 4EBN, n pollen) . The 5x hybrid speciesshould be 4EBN (Table 2) . In fact, it was foundthat hybrids could be easily produced by matingS . X curtilobum with Gp. Tuberosum, a 4x(4EBN)species (Hawkes, 1962) .

Conclusion

EBN is a unifying concept for predicting endo-sperm function in intraspecific, interploidy and in-terspecific crosses . EBN, is characteristic of a spe-cies and is more important than the chromosomenumber in both predicting the success of a cross anddetermining the ploidy of the offspring . EBN maybe considered a simplification to predict the successof the endosperm development without fully eluci-dating all of the other interactions which are impor-tant in determining endosperm development, e.g .,imprinting of gene action in maize endosperm(Kermicle, 1978) .

EBN also provides an opportunity to understandthe role of the endosperm in evolution and its prop-er utilization in the development of breeding meth-ods for gene transfer . Through the correct ploidy/EBN manipulations, virtually all of the divergentpotato species may be utilized and their genes in-trogressed into the cultivated germplasm pool .

Finally, the endosperm dosage concept, as em-

111

bodied in the EBN system, is not restricted to thetuber-bearing Solanum spp. This concept has beenused to explain the results of interspecific/interploi-dy crosses in Cucumis spp . (Dane et al ., 1980),Dactylis spp . (Van Santen, 1988), Datura stramoni-um (Johnston, 1980), Glycine spp . (Zhang & Palm-er, 1990), Impatiens spp. (Arisumi, 1982), Lotusspp. (Negri & Veronesi, 1989), Lycopersicon spp .(Ehlenfeldt & Hanneman, 1992), Medicago spp .(Veronesi et al ., 1986), Oryza spp. (Khush, per-sonal communication), Petunia spp . (Maizonnier,1976), Phaseolus spp. (Shii et al ., 1982), Trifoliumspp. (Parrott & Smith, 1984), Triticum spp . (Dhali-wahl,1977), and Vaccinium spp. (Dweikat & Lyre-ne, 1988) . This indicates that the EBN hypothesismay have a broad application and could be usefulfor germplasm transfer and breeding other cropspecies .

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