INCOMPATIBILITY IN FLOWERING PLANTS

472

INCOMPATIBILITY I N FLOWERING PLANTS

BY D. LEWIS John Innes Horticultural Institution, Bayfordbury, Hertford

(Received 26 ApiL 1949)

CONTENTS

I. 11.

111. IV. V.

VI. VII.

VIII. IX. X.

Introduction . . . . Heteromorphic incompatibility Homomorphic incompatibility Polyploidy and incompatibility Physiology of incompatibility . Interspecific hybrids . . Number of alleles . . . Mutation of the S gene . . Summary . . . . References . . . .

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PAGE 472 473 477 -180 48-1 489 490 40 I 49 3 494

I . INTRODUCTION

Many species of flowering plants fail to produce seed when they are self-pollinated. Cross-pollination within certain groups of individuals of these species is also in- effective, This is not due to the female or male organs being sterile, but simply to their being brought together in wrong combinations. This hindrance to fertilization, which is caused by the failure of the pollen to penetrate the female stylar tissue, has in the past been referred to as self- and cross-sterility. But it is now more appro- priately called incompatibility.

In a survey of the distribution of incompatibility throughout the families of flowering plants, based upon the data available before 1937, East (1940) estimated that self-incompatibility was to be found in more than three thousand species, distributed among twenty families. Thus incompatibility is not confined to a few specialized families but is widespread, and is, therefore, an important breeding mechanism in plants. It is, in fact, one of the several mechanisms in flowering plants which encourage outbreeding. For this reason it is rarely, if ever, to be found in forms which have some other efficient out-breeding system, although it is sometimes present together with less efficient means such as the non-synchronous development of the male and female organs.

T o observe incompatibility a special test must be made, hence our knowledge of its distribution is extremely fragmentary and much less complete than what is known of the occurrence of other breeding mechanisms such as the separation of the sexes; these are observed in the course of taxonomical description. Nevertheless, more is known about the gentics of self-incompatibility than about the other outbreeding mechanisms; it is this aspect which shows some remarkable facts, some of which still lack a satisfactory explanation.

Incompatibility in flowering plants 473 But apart from the studies on the genetics of incompatibility for its own sake, the

genetical system is such that it can be used as a tool for experiments on other aspects of genetics which are often beyond the reach of more usual genetical techniques.

Another aspect of incompatibility is its importance in commercial crop production. Many cultivated plants are self-incompatible, but only with a plant that is propagated vegetatively and cultivated as a clone for the fruit or seed is incompatibility an important factor in the crop produced. A single variety of a sexually raised crop such as clover, despite the self-incompatibility of this species, can produce a full set of seed because there is adequate provision for compatible pollination between the genetically different plants within the variety. Vegetatively propagated fruit trees, on the other hand, will only be compatibly pollinated if two or more of the right varieties are planted together: thus a detailed knowledge of incompatibility in fruit trees has been essential for modern commercial fruit-growing.

Incompatibility in plants is of two main types, namely: (I) heteromorphic-this is associated with differences in floral structure such as the length of the style and the level of the anthers; ( 2 ) homomorphic-this type is not associated with differences in floral structure.

The earlier work on incompatibility will not be described in detail, as good re- views have already been published (East, 1929; Brieger, 1930).

11. HETEROMORPHIC INCOMPATIBILITY

The main floral differences associated with heteromorphic incompatibility, which were first described by Darwin in 1877, are in the lengths of the styles and the levels of the anthers. The most common and simplest heterostyled system is that in which there are two kinds of flowers-borne on different plants-one kind having long styles and low anthers,pin, and the other having short styles and high anthers, thrum. Darwin also showed that these differences are accompanied by incompatibility, such that only pollinations between pin and thrum plants, that is between anthers and styles at the same level, are fertile. Pollinations between flowers of the same kind, that is between anthers and styles at different levels, are either completely or partially sterile.

One of the most interesting features of heterostyled incompatibility is the association of several very different characters, for apart from the differences in style length and anther height there are differences in pollen size and size of stigmatic cells. Thrum pollen is larger than pin while the stigmatic cells of the thrum stigmas are smaller than those of pin. In all, there are six contrasting pairs of characters concerned in the difference between pin and thrum; these are given in Table I. The remarkable feature about this complex of characters is that many of them are not obviously related physiologically. It is conceivable, however, that the size of stigmatic cells and the length of the style are merely two ways of measuring the same effect, and similarly, pollen size and anther height may be one physiological character. This view is supported by observations made on cell size and style and anther

BR XXlV 31

474 D. LEWIS lengths in Psychotn’a malayana. The lengths of the style and of the anther filaments were found to be proportional to the size of the component cells (Ernst, 1932). This would appear at first sight to simplify the complex of characters by reducing the number to four.

The same author (1936), however, found in Primula viscosa exceptional plants in which the usual association of the characters had been broken down. Homostyle plants were found which had the stigma and anthers at the same level-the reverse of the normal-and these plants were self-compatible, as would be expected if the incompatibility reaction of the pollen and style were conditioned by their positions. This dissociation, therefore, separates anther and style characters. But a further separation was found in some rare plants; some had large pollen grains in low anthers and others had small pollen in high anthers. Altogether, the characters which have so far not been separated by Ernst are: on the male side, the incompatibility reaction and the size of the pollen and, on the female side, the size of the cells and the length and incompatibility of the style.

Table I. Characters found in pin and thrum plants ~ ~~

Character

Incompatibility reaction of pollen Incompatibility reaction of style Length of style Size of stigmatic cells Height of anthers 1 Size of pollen

Pin

‘ Pin ’ ‘ Pin ’ Long Large Low Small

Thrum

‘Thrum’ ‘ Thrum ’ Short Small High Large

The genetic control of the pin and thrum complex of characters is by two alleles of a single gene S; the thrum plants being heterozygous Ss and the pin plants homozygous ss. The incompatibility reaction of the pollen is determined solely by the genotype of the somatic tissue and not by the genotype of the individual pollen grain: for example, the pollen of thrum plants, although it segregates into half S and half s, all behaves like thrum pollen. This has been found in all the distylic species which have been analysed; these are: P. sinensis (Bateson & Gregory, I~OS), P. acaulis (Gregory, I~IS), P. oficinalis (Dahlgren, 1916), P. viscosa, P . hortensik (Ernst, 1936), P. obconica (Lewis, unpubl.), Pulmonaria angustifolia (Darwin, 1876), Fagopyrum esculentum (Dahlgren, 1922 ; Garber & Quisenberry, 1927). Homozygous thrum plants SS derived from self-pollinating a normal heterozygous thrum plant are known in Primula sinensik.

The simple breeding behaviour of the system is given in Table 2. It will be seen that, when only compatible pollinations occur, the two types are maintained in approximately equal numbers. This mechanism is similar to the X Y sex determination (in animals and plants) and has similar consequences on outbreeding.

Breeding results obtained by Ernst, with the exceptional homostyle plants referred to earlier, have extended our knowledge of the genetic control. As a result of these experiments it appears that the S gene is composed of at least three subunits, one

Incompatibility in jlowering plants 475 unit controlling the stylar characteristics, a second the anther height and the third controls the size and incompatibility of the pollen.

These units are almost completely linked, the exceptional plants being the result of a rare cross-over between the units. Thus the complex of characters has been gradually built up through the evolution of a major gene by the co-ordination of smaller units.

Type of pollination

Table 2 . Breeding behaaiour of pin and thrum plants

Parents Progeny Thrum Pin

Phenotype 1 Genotype ss ss ss

Compatible

Incompatible

Pin x thrum ss x ss 0 : 1 : 1 Thrum x pin ss x ss 0 : 1 : 1 Thrum x thrum ssxss 1 : 2 : 1 Pin x Pin ss x ss o : o : all

In Primula sinensis, a species which has been much studied genetically, no such break-up of the character complex due to a rearrangement of the units of the S gene has been found, but two independent genes are known modifying the S effect. The mutant Primrose eye (a) shortens the style and the mutant Fertile Double (m) raises the level of the anthers (de Winton & Haldane, 1933). These genes also affect the incompatibility of the style and pollen (Beale, 1939; Mather, 1948). Thus there is in this case a breakdown of the character complex but in a different genetic way.

Homostyle plants, that is, plants with styles and anthers at the same level, have been found in wild populations of P. vulgaris by Crosby (1940), and in Limonium vulgare (Baker, 1948). These are examples of the break-up of an incompatibility mechanism in nature.

An interesting variant of the distylic system is found in Linum grandifirum. In this species there are long- and short-styled flowers, the anthers of each, however, differ only very slightly in level and the pollen grains do not differ in size. But the pollen grains show differences in turgor pressure ; the pollen from the short-styled flower has a higher turgor pressure than that from the long-styled flower. Similar differences in turgor pressure are present between the styles of different lengths. These differences in turgor pressure are related to the incompatibility (Lewis, 1943). Only when there is a 4: I ratio between the turgor pressures of the pollen and style is the pollination compatible; when the ratio is too high (in thrum x thrum) the pollen bursts, when the ratio is too low (in pin x pin) the pollen does not germinate.

A more complex type of heterostyly is found in Lythrum salicaria and Oxalis species in which there are long-, mid- and short-styled flowers. Each flower has anthers at two different levels, the stigma being at a level different from both sets of anthers. Compatible pollinations, as in distyly, are between stigmas and pollen of the same level ; incompatible pollinations are those between stigmas and pollen of different levels (Darwin, 1877).

31-2

476 D. LEWIS As there are two levels of anthers in each flower, it follows that pollen from dif-

ferent sets of anthers on the same plant behave differently. For example, a mid- styled plant has pollen from long and short anthers; pollen from these two types of anthers, although they have an identical distribution of genotypes, behave differently on long and short styles. This suggests a close physiological connexion between anther height and the growth reaction of the pollen.

Work on the inheritance of tristyly in Lythrurn by Barlow (1923) and East (1932) has established a genetical control based on two independent genes. The long-styed plant has the recessive alleles of both genes, the mid carries the dominant allele M of the mid gene but carries the recessive of the short gene s, while the short-styled plant carries the S allele which is dominant to s and epistatic to M.

The three types and their genotypes are the following: Long, ss mm; Mid, ss M; Short, S mm or S M.

There were certain irregularities in the breeding experiments that were difficult to explain, and East introduced the idea of two linked mid genes, each lethal when homozygous. Recent work by Fisher & Mather (1943), designed to clear up these abnormalities, has shown that the gene for Mid is polysomic, but a decision between tetrasomic and hexasomic could not be made. Thus we have the situation in a plant with a high chromosome number-probably an old polyploid-in which the incompatibility system is controlled by one disomic and one polysomic gene.

The evolution of heterostyled incompatibility. There are certain features common to all heterostyled systems which are not found in other types of incompatibility. As these features are important in understanding the differences between incompatibility systems and also essential to any attempt to formulate the evolution of heterostyled incompatibility, they are summarized below :

( I ) The genetic control is by a small number of alleles, that is, by one gene with two alleles in distyly and by two genes each with two alleles in tristyly. (2) One allele is dominant to another. (3) The incompatibility reaction of the pollen is controlled by the genotype of

the zygote from which it came or by the anther producing it and not by its own gametic genotype.

(4) Short style is always dominant to long. I t has been pointed out (Lewis, 1 9 4 ) that these aspects of the genetic control

have an internal restriction on the number of different incompatibility genes-and hence reactions-that can be maintained in a heterostyled species. For example, the balanced dominance of the alleles and the zygotic determination of the pollen reaction, which are essential features of the system, could not be obtained with many alleles. There is also an external restriction on the number of alleles by the morphological differences, for the number of different style lengths showing discontinuous variation is clearly limited.

Both the incompatibility reactions and the morphological differences are means of encouraging outbreeding. The question arises as to which developed first. Were

Incompatibility in jlowering plants 477 the morphological differences present first, in which case they would determine the type of genic control of incompatibility? Or was the incompatibility present first, and has it by its internal restriction to two or three mating types made possible the development of a morphological differentiation?

The incompatibility system found in Capsella grandzjlora is of special interest in this connexion. Riley (1936) found that there were only three types of incompatibility reaction in Capsella and that these were controlled by two genes each with two alleles as in tristylic species. One allele is dominant to its partner, and there is zygotic determination of the pollen reaction but no heterostyly. This is therefore an example of the internal genetical restriction limiting the number of incompatibility reactions. The genetical control typical of heterostyly has arisen without the morphological differentiation. This supports the view that the incompatibility came first and the heterostyly followed as a reinforcement.

On the other hand, Mather & de Winton (1941) have shown in Primula sinensis that, although thrum is dominant to pin, the homozygous thrum plants are less fit than pins. The loss of fitness of homozygous thrums, it is argued, came from the heterozygosis enforced by incompatibility sheltering deleterious recessives from selection. To explain the dominance of thrum these authors suggest that pin was once a deleterious mutant. As outbreeding became advantageous the pin mutant became the starting-point for a more or less parallel development of heterostyly and incompatibility.

111. HOMOMORPHIC INCOMPATIBILITY

The pre-Mendelian workers, including Kolreuter, Muller, Hildebrandt and Darwin, made several observations on self-incompatible homomorphic plants. As they found no case of cross-incompatibility between different individuals they believed every plant to be unique in respect of incompatibility. I t was not difficult for Darwin to accommodate this concept into his somewhat elastic theory of heredity. But to the post-Mendelian geneticist it was very difficult to explain, and this undoubtedly stimulated work on the problem.

Correns (1913) with Cardamine pratensis soon found, however, several cases of cross-incompatibility by testing the progeny of controlled pollination. Correns explained his results by assuming that there were certain ‘line stuffs’ or factors with which, when both the pollen and stylar parents carried the same factor, the pollination was incompatible. It was assumed that incompatibility of the pollen was determined by the factors carried by the sporophytic tissue and not by the factor carried by the individual pollen grain. We now know that this last assumption is wrong, but this mistake is not surprising when we consider that Correns was un- wittingly dealing with difficult material-a polyploid.

The true genetical control of homomorphic incompatibility was discovered independently by East & Mangelsdorf (1925), working with Nicotiana sanderae, and by Filzer (1926) with Veronica syriaca: the results of this early work merit not

478 D. LEWIS only a description but a note on the problems confronting the earlier workers, because the genetical control in these species has since been found to apply to many diverse flowering plants (see Table 3).

The control is by a single gene S, which has a large number of different alleles Sl,z,3,...,n. As in heterostyled incompatibility these alleles operate both in the pollen and the styles; the pollen, being a haploid spore, has one allele; the style, being diploid sporophytic tissue, has two. These alleles act so that pollen grains are unable to grow and effect fertilization in styles that have the same allele as the pollen. But, unlike the action in heterostyly, the pollen grain incompatibility is determined by its own S allele and not by the genotype of the sporophyte from which it came. It is also unlike heterostyly in that the alleles have an independent action without dominance in the style.

Basically there are three different kinds of pollination with this system; these and the progeny to be expected are given in Fig. I .

S l i , s;s, Fig. I . Pollen-tube growth and types of progeny obtained from the three basic

types of pollination. (By courtesy of M. B. Crane.)

The pollination shown on the left is incompatible because the male and female parents have both S alleles in common; all self- pollinations and all cross-pollinations within a group of individuals with both S alleles in common are of this kind. The two other pollinations are compatible because they both have at least one S allele which is not common to the two parents. In the pollination (centre of figure) which has only one allele common to both parents, only half the pollen is inhibited. In general, this will not affect the production of seed as half the pollen on the stigma is often more than enough to fertilize all the eggs. Thus these two kinds of compatible crosses cannot be distinguished by the fertility of the cross. But a distinction can be made by the behaviour of their progeny; the pollination with one allele in common gives progeny which fall into two intra-incompatible, inter-compatible groups, while the other pollination gives four different groups.

Incompatibility in jkwering plants 479 Nicotiana sanderae, the plant used in this early work, was known by East to be

a hybrid between N. alata and N . forgetiana; he therefore inbred his material for twelve generations before attempting a detailed analysis. As the inbreeding continued the results showed more and more order. He was, however, trying for a long time to explain his data, as did Correns, on the assumption that the pollen reaction was sporophytically controlled. It was not until the idea of gametophytic pollen control occurred to him that the solution of the problem was found. Perhaps his adherence to the other view was due to the fact that no gametically controlled pollen characters were known, for it was not until 1921 that the first was described in rice by Parnell.

Shortly after the discovery of the S gene, Brieger & Mangelsdorf (1926) found in N . sanderae an example of linkage between this gene and a flower colour gene, thus giving confirmation to the whole scheme. Subsequently, similar linkages have been found by Sterling Emerson (1941) in Oenothera organensis and Riley (1944) in Nemesia strumosa.

Table 3. Homomorphic species in which an incompatibility system has been .found to be controlled by a single multiple-allele series

Species

Antirrhinum glutinosum Nemesia strumosa Nicotiana sanderae Oenothera organensis 0 . rhombapetala Papaver nudicaule Petunia violacea Prunus avium Solanum (3 species) Tradescantia virginiana Trifolium pratense T. repens Veronica syriaca

- Authority

Gruber, 1932 Riley, 1934- East ~ & Mangelsdorf, I 9 2 5 Emerson, 1938 Hecht, 1944 Fabergk, 1942 Harland & Atteck, 1933 Crane & Brown, 1937 Pushkar Nath, 1942 Anderson & Sax, 1934 Williams & Silow, 1933 Atwood, 1940 Filzer, 1926

There are several self-incompatible species in which the simple S scheme applies but in a modified form. In addition to these are a few exceptions which are not readily fitted into the scheme at all.

Results obtained in Verbascum phoeniceum by Sirks (1926) were explained on a modified S scheme. It was assumed that the S, as well as the S, allele in the style has the power to inhibit pollen carrying S although S, in the style does not inhibit

1’. pollen carrying S,. This clearly conflicts with the specific one-to-one action fundamental to the whole S scheme. Lawrence (1930), however, pointed out that V . phoeniceum has a tetraploid chromosome number, and may therefore be an allopolyploid. He found a reasonable fit with the data by assuming duplicate S genes which would be expected if both ancestral species had an incompatibility mechanism.

Complicated results were obtained with Brassica oleracea in which there was segregation of different incompatible groups, one-way incompatibility where re-

480 D. LEWIS ciprocal pollinations gave different results, and the ,segregation of self-compatible plants (Kakizaki, 1930). A complicated scheme with an inhibiting S gene and a stimulating T gene, each with many alleles, was invoked. Unfortunately the data on the critical tests were not sufficiently extensive to accept it with complete con- fidence.

In Beta vulgaris, Owen (1942) explained incompatibility with two sets of duplicate

Since the work of Correns (1928)~ Tolmiea mensiesii has continually been quoted as a self-incompatible species in which cross-incompatibility had not been found. This species is a tetraploid (2n = 28) and many examples of cross-incompatibility have been found, however, by Wigan (1940). But the data obtained were so complicated, that even by the adoption of a polysomic condition they could not be reduced to a reasonable scheme.

At the opposite extreme to Tolmiea, in which cross-incompatibility is rare, is the supposed case of incompatibility in Cacao. Certain strains of Cacao contain plants which will not set seeds with their own pollen; other strains are fully self-fertile (for review see Posnette, 1945). The self-sterile trees are inter-sterile and can only be effectively pollinated by a self-fertile tree, although the pollen of the self-steriles is effective on the self-fertiles. (The cross-fertility between two self-steriles reported by Muntzing (1947) is an exception.) This raises doubts as to whether true incompatibility is involved and suggests some form of sterility.

Mather (1944) has pointed out that the Cacao case is at variance in another respect. I n other species, incompatibility is due to the inhibition of pollen germination or pollen-tube growth by stylar tissue. Thus the eggs are not wasted by incompatible pollination. In the self-sterile strains of Cacao, however, pollen-tube growth is normal, the eggs are fertilized, but subsequent development is arrested. This violates one of the chief features of incompatibility, the conservation of eggs. A similar case has been described in Gasteria (Sears, 1937), in which fertilization occurs after self- pollination but no seeds are formed. Here again there is no evidence that true incompatibility is involved.

genes $,2,3 ,..., 11 and '1,2,3 ,..., 11'

IV. POLYPLOIDY AND INCOMPATIBILITY

When the chromosome number of an organism is increased there is a drastic upset in the genic balance, and thus in the physiological processes which the genes control. This upset in balance by polyploidy has been a useful tool in experiments on the genetics of sex, first in animals (Bridges, 1922) and later in plants (Westergaard, 1940). Similarly, with incompatibility it has been useful for obtaining knowledge on the genetic control.

Two kinds of data are available : (I) the experimental results with artificial tetraploids; and (2) the circumstantial evidence from natural polyploid species. As there is little known about their origin, this has the least precision, but it adds to the more precise data obtained from the artificial polyploids.

Incompatibility in flowering plants 481 The effects of polyploidy on incompatibility in autotetraploids arising under con-

trolled conditions are summarized in Table 4. It will be seen that in more than half the species the incompatibility system has been completely broken down-the tetraploids are self-compatible. In the remaining species there has been no effect detected or, at most, only a tendency towards self-compatibility. In the majority of these species the analysis has not been carried any further than to record the state of self-incompatibility. I n Oenothera organensis, Trifolium repens, Petunia (axillaris ?) and Pyrus communis the analysis has been carried further. This work has been described and discussed in some detail (Lewis, 1947) and only a brief summary will be given here. The key to the variable behaviour of the tetraploids is to be found in Oenothera organensis.

Table 4

Species

Antirrhinum molle Campanula persicifoliu Petunia (axillaris?)*

Pryus communis

P . m l u s

Solanum ( 3 species)

Trtfolium repens Ananas sativus

Brassica campestris B. rapa Oenothera organensis

0. rhombipetala Raphanus sativus Taraxacum kok-saghya

Origin of tetraploid

Colchicine Spontaneous Colchicine

Spontaneous

Progeny of triploids

Colchicine

Colchicine Colchicine

Colchicine Colchicine Colchicine

Colchicine Colchicine Colchicine

Self-compatible Self-compatible Self-compatible

Self-compatible

Self-compatible

Self-compatible

Self-compatible No effect

No effect No effect Slight increase in

No effect No effect No effect

self-compatibility

Author

Straub, 1941 Gairdner, 1926 stout & Chandler, 1941

Crane & Lewis, I942

Johansson, 1945

Livermore & Johnstone, 1939

Atwood, 1944 Kerns & Collins,

Howard, 1942 Howard, 1942 Lewis, 1943

Hecht, 1944 Howard, 1942 Bannan, 1946

I947

* The species used was probably the garden form, P. violacea.

In a tetraploid, the styles will have four S alleles and the pollen grains will have two. In the styles of tetraploid 0. organensis the four S alleles, even when they are four different ones, S,, S,, S,, S,, all operate efficiently, without interaction between one another, to inhibit haploid pollen carrying any one of these alleles. This is to be expected, because we know that the two different alleles operate in the diploid style without interactions and there is no reason why this independence should not be extended to four alleles.

In the diploid pollen grains of a tetraploid, however, new and complex interactions occur. If we consider the types of pollen produced from a tetraploid plant with S,S,S,S, some of the pollen will be homogenic S,S, and S,S, and the re- mainder will be heterogenic S,S,. Diploid grains with the two like alleles, S,S, and S,S,, behave like haploid grains carrying S, and S, respectively, in other words, there has been no upset in the genic balance controlling incompatibility, The

482 D. LEWIS diploid grains with S,S,, however, may show either of two different kinds of genic interactions according to the particular pair of alleles involved.

First, with the pairs, S,s,, s3s6, and S3S4, neither of the two alleles is able to function efficiently due to competitive interaction between each allele of the pair. In these cases the pollen grains have a much reduced incompatibility reaction and thus are not fully inhibited in any style even in one carrying both of the alleles. Plants producing such pollen grains are either weakly self-incompatible or completely self-compatible according to the strength of the original reaction (see Fig. 2 ) .

s1 I1 SI k SlSl

Diploid selfed, incompatible

Tetraploid selfed, Diploid x tetraploid, Tetraploid x diploid, compatible compatible incompatible

Fig. 2. The incompatibility behaviour of diploid and tetraploid plants. The compatibility of the tetraploid selfed and the ‘diploid x tetraploid’ is due to the S alleles in heterogenic pollen (S,,*) failing to operate owing to competitive interaction.

Secondly, with other pairs of alleles, s4s6 and S,S,, there is no competitive interaction between the alleles, but one allele is dominant to the other. In consequence, such pollen grains function as if they were homogenic for the dominant allele. Plants producing only this type of heterogenic pollen grain are as self-incompatible as the diploids because there is a full reaction between the dominant allele in the diploid pollen and the tetraploid style.

These two effects, competition and dominance in the diploid pollen grain, can explain most of the effects of polyploidy found in the other species. Species which show no change could be explained by dominance or by the absence of interaction, either competitive or dominant. Species which show a change to self-compatibility can be explained only by competitiwe interaction.

In Petunia (axillaris?) Stout & Chandler (1942) showed that when using the diploid and tetraploid forms of the same genotype the p was self-compatible, the

Incompatibility in jlowering plants 483 2 x x p cross was compatible while the p x 2 x cross was incompatible, Thus showing that the breakdown is in the pollen and not in the style. Furthermore, they found that the offspring from the selfed tetraploid were all self-compatible, all inter-compatible, all were incompatible when crossed as females with their diploid parent and all were compatible when crossed as males with their diploid parent. All this can be explained on the assumption that all the offspring are heterozygous for S; this would clearly be the case if only heterogenic S,S, pollen functions as in Oenotheru.

In the tetraploid form of Pyrus communis, which arose spontaneously and of which the diploid form is known, Crane & Lewis (1942) found also that the 2x x p cross was compatible while the p x zx cross was incompatible, again indicating the pollen as the site of breakdown. In the styles of this tetraploid after self-pollination, Lewis & Modlibowska (1942) found both compatible and incompatible tubes; these were interpreted as arising from heterogenic S,S, and homogenic S,S, and S,S, pollen respectively.

As early as (1929) Crane & Lawrence interpreted their incompatibility results with the hexaploid Prunus domesticu, in which reciprocal differences occurred, by an effect of dominance in the pollen grain. A somewhat similar effect of dominance was also invoked by Lawrence (1930) in his interpretation of Sirk’s Verbascum data. He considered it to be caused by the unlike allele positively promoting pollen-tube growth.

Recent studies with artificially produced tetraploids of Trifolium repens by Atwood (1944) have shown that among the progeny from the cross S,S,S,S, x S3S,S4S, three plants were self-incompatible and twenty-six were self-compatible. This can readily be explained on the two effects of competition and dominance; the self- incompatible plants would segregate types of heterogenic pollen which only showed dominance, the self-compatibles would segregate at least one pollen type which shows competitive interaction. Atwood, however, favoured the view that self- compatibility was caused only when a plant produced more than one type of heterogenic pollen, e.g. an S,S2S3S4 plant.

Among natural polyploids there are some examples known of genera in which both the diploid and tetraploid species are self-incompatible. The best known is T. prutense (2x) and T. repens (p), which are both self-incompatible, with a disomic S control (Williams & Silow, 1933 ; Atwood, 1940). The p species may be an old auto-tetraploid which has become functionally a diploid. The disomic S control could have arisen after the species had become polyploid. Alternatively, the qc species might be an allotetraploid between self-fertile and self-sterile species.

Reference has already been made to Verbuscum phoeniceum with its tetrasomic S control, but in this case there are no true diploids for comparison. There are no genera known to me, however, in which diploid species are self-compatible and tetraploids are self-incompatible. In fact, the reverse condition, self-incompatible diploids and self-compatible tetraploids, is the rule (cf. Lewis, 1947). This suggests that the tetraploids have flourished without an outbreeding mechanism and that the

484 D. LEWIS incompatibility system has been broken down by genic unbalance in the tetraploid as in the artificial tetraploids.

In concluding the effect of polyploidy there are two points of interest in connexion with gene action. First, those effects peculiar to the diploid pollen grain are primary properties of the S gene; there can have been no selection for such an action because these genes have not been subjected to such an environment in the past. Thus we have dominance which has not been produced by selection.

Secondly, it is of interest that the action of the S gene in the style is quite independent while the action in the pollen shows these interactions. We shall see later that not only are the pollen and stylar effects of the S gene different in their interaction but also that they can be separated by mutation.

V. PHYSIOLOGY OF INCOMPATIBILITY Several theories have been advanced on the physiological and chemical nature of incompatibility, but there are few conclusive results at present to support any of them. One of the main difficulties has been the failure to demonstrate the reaction in vitro. Pollen can be germinated in sugar solutions to which have been added chemicals, stylar extracts or crushed sty!ar tissue. But, apart from a claim by Yasuda (1934) working with Petunia, all the attempts to demonstrate in vitro a differential effect of compatible and incompatible stylar tissues have failed.

First let us consider the positive evidence. I t has been known for a long time that incompatibility is due either to failure of the pollen to pentrate the stigma or, if it does penetrate, to the early cessation of its growth. Sears (1937) has summarized the previous work and also compared pollen-tube growth after compatible and incompatible pollination in many species. This work may be summarized as follows.

In some species, such as Brassica oleracea, Raphanus sativa, Secale cereale, the incompatible tubes rarely penetrate the stigmatic surface; the removal of this surface removes the inhibition of the pollen in Brassicu. Thus in these species there is evidence for a localization of the reaction in the stigmatic surface. In other plants, Nicotiana sanderae, Petunia violacea, Nemesia strumosa and many others, the pollen germinates readily and the tubes penetrate the style to a distance which varies not only with the species but also with the particular S allelomorph involved. In some species the tubes stop after z mm. growth while in others they may reach the base of the style.

That incompatibility is due solely to a reaction between diploid stylar tissue and haploid pollen tubes has been proved in several species. In Nicotiana sanderae East (1934) was able to get some seeds after selfing by pollinating flowers which had not opened, that is, before the incompatibility had developed fully. In Trifoliiriri pratense a low percentage of seed setting was obtained by Williams & Silow (1933) after selfing certain individual plants. This was due to a rare pollen tube being able to traverse the whole length of the style.

Incompatibility ilt jowering plants 485 The plants obtained from such pollinations segregated into heterozygotes, S,S,,

and homozygotes, S,S, and S,S,. Thus, if the pollen tube can manage to reach the ovary, there is nothing to stop the fusion of pollen and egg nuclei with the same S allele. Furthermore, the S alleles have little effect on the viability and vigour of the plants. Homozygosity for the S alleles in Nicotiana, with one exception (S, S,), does not in itself impair the vigour. But even the exception is probably spurious, for a rare S, S, plant with normal vigour was found, indicating that in this case the loss of vigour was due to a closely linked gene.

In finding a physiological explanation of incompatibility the basic choice is between a positive inhibition and an absence of stimulation by the style on pollen containing a common allele.

East (1929) put forward the theory that incompatibility is due to a reaction between pollen and style similar to an immunity reaction in animals. ‘The pollen-tube passes down the stylar tissue intercellularly, as a parasitic growth. If we assume that the secretions of the pollen-tube bearing a given gene, say S, , act as antigens against the stylar tissue bearing the gene S,; if we further assume that the stylar tissue in which the S , is present forms antibodies against such a pollen-tube and thus inhibits its growth; then all requirements are satisfied.’

Two main lines of argument lead to this view: ( I ) Pollen tubes will grow in an artificial medium and in styles of different species; therefore they do not appear to require specific stimulating substances. Thus incompatibility is due to a positive inhibitory reaction rather than a lack of some stimulant. (2) The reaction is highly specific such that only pollen and styles with like alleles react together.

Sears (1937) supports East’s view pointing out that, in the species in which the incompatible pollen fails to germinate, it would be difficult to explain incompatibility on the lack of a stimulant because pollen will germinate in moist air.

Further evidence that incompatibility is due to a positive inhibition rather than a lack of a stimulant was obtained from the differential effects of temperature on compatible and incompatible tube growth (Lewis, 1942). Pollen-tube growth was examined in temperatures ranging from 10 to 35’ C. in Prunus avium and Oenothera organensis and in the heterostyled species Primula sinensis and P. obconica. In all cases it was found that, whereas compatible tube growth was faster at a high temperature than at a low temperature as would be expected, incompatible growth was very much slower and stopped earlier at a high temperature. A similar effect was found in Pyrus species by Modlibowska (1945). These results could not easily be interpreted on the lack of a stimulant view but are precisely what would be expected on the positive inhibition view. The most satisfactory explanation of the growth- temperature curves obtained was that, as the pollen tubes grow in the style the effects of the inhibition reaction accumulate until a threshold is reached and the tubes stop growing. This inhibition reaction, like all reactions, proceeds faster at higher temperatures, thus causing the tubes to stop growing earlier.

These temperature experiments also show that the incompatibility is not confined to particular zones of the style in these species. Style-grafting experiments by

486 D. LEWIS Emerson (1940) also showed this. The amount of growth of incompatible tubes was not affected by the part of the style in which the growth occurred and Emerson concluded that his experiments support East’s view that ‘ incompatible tubes are inhibited in some way upon coming in contact with the tissues of the stigma or style, rather than that compatible pollen tubes receive some necessary growth stimulus from compatible tissues’. Emerson also found that there was no perceptible diffusion of incompatibility substances in the style of Oenothera.

Ingenious experiments with Petunia violacea by Straub (1946, 1947) have led him to postulate a theory of incompatibility which, he considers, differs radically from East’s immunity theory. After a careful study of the papers of East (1929) and Sears (1937) it appears that the two theories do not differ fundamentally and that the difference considered by Straub is due to a misunderstanding of some of East’s rather ambiguous statements.

Straub’s theory is that there is a substance, which is formed in the pollen grain in a definite amount and with a specific quality determined by the S allele present. This substance is necessary for the growth of the pollen tube in the style, probably by its enzyme-like ability to decompose the stylar tissue. When all the substance is used up in growth the tubes stop growing. In a compatible style most of the substance is available for growth, but in an incompatible style the majority of it becomes inactivated by another specific substance, which is formed in the style by the same allele.

The main evidence for this theory rests on the following experiments.

( I ) Incompatible pollen tubes in P. violacea will only grow 15 mm. down the style. Styles were pollinated and afterwards cut off at certain distances from the stigma at a time before the tubes had reached the part to be cut. The cut ends of these styles were then placed in a sugar medium, the tubes grew out of the cut end into the sugar, and the total tube length obtained in the medium was measured. When the length of the style cut off was 6 mm. or less the amount of growth of the emerging tubes in the medium was the same from an incompatible as from a compatible style. For lengths greater than this, the longer the style the shorter were the tubes in the medium, but those from an incompatible style were much shorter than those from a compatible style.

(2) Style grafts were made in which the pollen tubes first grew through a compatible style and then into an incompatible style below the graft. The final amount of growth in the incompatible tissue depended upon the length of compatible tissue the tubes had traversed. With qmm. of compatible style above the graft the tubes grew 15 mm. in the incompatible tissue, but with 20 mm. the tubes grew only 6.5 mm.

(3) By pollinating with incompatible pollen and later applying more of the same pollen, it was found that the first group of pollen tubes caused no stronger inhibition of the second group.

There are certain criticisms which can be made against the interpretation of these

Incompatibility in jiowering plants 487 results. First, the results of the critical experiments are, as Straub admits, only on the verge of significance. Secondly, the results can be interpreted equally well in another way. For example, assuming a theory based on inhibition, it is possible that in Exp. I there has been some .inhibition while the tubes were traversing the incompatible style; this would have an effect on the subsequent tube growth in the medium Exp. I would then be interpreted as follows:

The pollen tubes grow some 6 mm. in the style before the active inhibiting substances in the pollen become available. Thus the total growth in the medium of tubes emerging from pieces of style which do not exceed 6 mm. in length is independent of whether the style is incompatible or compatible. After 6 mm. growth the inhibition reaction starts, culminating in complete stoppage at 15 mm. ; thus with a 12 mm. piece of style there should be, as found, a difference of growth in the medium between tubes emerging from incompatible and compatible styles.

Again, the results of the second experiment are better explained by the inhibition theory, as the results of this experiment are not in good agreement with Straub’s growth-substance theory. For in another experiment Straub found that the absolute maximum growth of tubes in a compatible style was 140 mm. This, he interpreted, was due to the limited amount of substance in the pollen grain being just sufficient for 140 mm. growth.

Returning to Exp. 2 above, the difference of length between the two pieces of compatible style used is 16 mm. ; thus the tubes in growing through this piece would require only of their total substance. This slight depletion could hardly account for the subsequent growth in the incompatible tissue beyond the graft being reduced to more than a half, i.e. from 15 to 6.5 mm.

On the inhibition explanation of Exp. I that the active substance is not available in the tube until after 4-6 mm. growth there is no difficulty in explaining the difference found in Exp. 2.

The results of Exp. 3 are given as decisively disproving East’s immunity theory; and they certainly do disprove an immunity theory which relies on the formation of the inhibitory substance in the style being stimulated by the pollen tube. It does not exclude an inhibition reaction which is brought about by a preformed stylar substance and this is undoubtedly what East meant in his original theory. Clearly Straub’s and East’s theories only differ in that the pollen component of the reacting system has in Straub’s a vital function in tube growth as well as its specific incompatibility reaction with the stylar component. In East’s theory no such growth function was involved. .

For Straub’s theory to be fully acceptable even for Petunia more critical evidence is necessary. There are, however, certain lines of evidence which make such a theory untenable as a general explanation for other species.

First, it could not apply to the species in which pollen germination is inhibited. Secondly, in Oenotheru organensis, it is difficult to account for the stoppage of incompatible pollen tubes before more than a minute fraction of the contents (including presumably the active substance) have passed from the pollen grain into the tube.

488 D. LEWIS Thirdly, except on the positive inhibition hypothesis, it is difficult to explain the previously mentioned results with temperature and the following results (Lewis, unpubl.). Incompatible tubes at 30' C. stop growing after 45 min., that is after I mm. growth. If pollinated styles treated in this way are then transferred to 15" C. there is no resumption of growth, despite the fact that when a pollinated style is kept from the beginning at 15' C. the tubes grow to at least 120 mm. This result strongly indicates an irreversible inhibition, and makes it difficult to visualize how, on the limiting growth substance of Straub, all of this substance should be lost in I mm. growth in 45 min. at 30' C., while at 15" C. some substance is still available after 60 hr. and 120 mm. growth. There is still one other line of evidence in Oeno- them from X-ray mutations. This will be briefly mentioned here but will be treated in more detail in a later section. Mutations of the S gene produced by X-rays are to alleles which have lost their ability to initiate.the incompatibility reaction in the pollen grain (Lewis, 1946). If incompatibility is due to the inactivation of a vital growth substance, then we must assume that its incompatibility specificity has been destroyed without damaging its growth-promoting property. No doubt this is possible, but the results are more simply explained on the inhibition theory.

Several attempts have been made to overcome incompatibility by the application of growth substances to the style. There is as yet no good evidence that this has been successfully achieved by a primary action on pollen-tube growth. There is, however, definite evidence of secondary actions which in some cases can increase the seed set after incompatible pollination.

The abscission of the flower and style was delayed by the application of a-naphthalene acetamide in Prunus aviuin and Oenothera organensis, but incompatible' and compatible tube growths were unaffected except by high concentrations, in which case all tubes were completely inhibited (Lewis, 1946). With certain plants it is possible to stimulate the swelling of the ovary and development of empty seeds, with or without pollination (Overbeck, Conklin & Blakeslee, 1.941 ; Lewis, 1946). It is probably this last effect which Eyster (1941) observed before he made the somewhat premature statement that a-naphthalene acetamide treatment had overcome incompatibility in Petunia.

It is possible for this kind of ovary stimulation to have a slight indirect effect on the number of seeds produced after incompatible pollination. For example, in Oenotheru organensis it is known that after self-pollination there is about one compatible tube, due to mutation, in IOO flowers incompatibly pollinated; this one tube will fertilize a single ovule. But one fertilized ovule does not give sufficient stimulus to make the ovary swell. Application of growth substance stimulates the ovary and the single seeds can develop (Lewis, 1948).

Interesting and suggestive results with growth substances have been obtained in Lilium by Emsweller & Stuart (1948). By treating the ovaries after incompatible pollination they have been able to get a very substantial number of seeds. It is not clear whether this is due to the longer life of the style-which was observed- allowing more time for the tubes to reach the ovary, to a stimulus of the ovary, or

Incompatibility in flowering plants 489 to a real primary effect on incompatible tube growth. Only a detailed study of pollen tube growth can decide this.

If a successful chemical treatment is found to overcome incompatibility by a primary action it will be a great step in elucidating the physiological basis. But until evidence is available to the contrary the most acceptable theory is that a substance in the pollen reacts with one in the style to stop pollen-tube growth by an irreversible blocking mechanism.

VI. INTERSPECIFIC HYBRIDS

The study of incompatibility relationships in interspecific hybrids has given infor- mation on the genetic mechanism which would be difficult to obtain by other means.

When two self-incompatible species are crossed and the Fl and F, progenies are analysed it is found that in a few cases the results are orderly. Hybrids between two diploid Solanum species, S. caldasii and S. subtilius, give four incompatibility groups in the Fl. This shows that the S alleles in the two species are homologous and that each series can work efficiently in a hybrid genetic background (Pal & Pushkar Nath, 1942). A similar relationship has been found in the hybrid between S. rybinii and S. simplidfolium by Black (unpubl.).

This indicates that these species are closely related, their common ancestor already had the incompatibility system, and that the modifiers affecting incompatibility have remained much the same in the two species.

The most instructive analyses come from hybrids between a self-incompatible and a self-compatible species, for in these cases the relevant modifiers will be balanced differently in the two species.

The hybrid between Nicotiana alata which is self-incompatible (S, S,) and N . langsdorfli which is self-compatible (S, S,) is self-compatible (East, 1929; Anderson & de Winton, 1931). The interpretation of this is simply that the self-compatible species has a gene $4, which is allelomorphic with the S series in the other species. Pollen carrying S, is compatible on any style; thus any plant with S, is self-compatible.

An unusual relationship was found with one strain of N . alata by Anderson & de Winton (1931). This strain when used as a female inhibited the pollen of the self- compatible N . langsdorfli. The results were satisfactorily explained on the assumption that the alata plant carried an S allele which, apart from having the usual property of repelling its own pollen, also repelled the pollen carrying the self- compatibility allele from the N . Zungsdorfli.

This very interesting result was also found in the hybrid between Petunia axillaris (self-compatible S,S,) and P. violacea (self-incompatible S,S,). The S, allele in the style inhibits S, pollen as well as S, (Mather, 1943 ; Bateman, 1943). These examples indicate that the S allele can have two properties: the property of inhibiting (i) its own type pollen, and (ii) the pollen of another species with an S, allele. This will be referred to later when further evidence for a dual nature of the S gene has been described.

g R X X l V 32

490 D. LEWIS Apart from the direct effects of the S alleles there are effects due to modifying

genes. An example of a single gene, non-allelomorphic with the S gene, which had the effect of making plants self-compatible was found in Nicotiana by Brieger (1930).

Mather (1943) found in the hybrid between Petunia violacea and P. axillaris strong evidence for the presence of polygenic modifiers, that is, a number of genes each with small effects. These were present in the self-compatible species and had the effect of weakening the action of the S alleles. In view of his findings he discusses the evolution of incompatibility systems and stresses the importance of these polygenes in making the gradual transition from inbreeding to outbreeding which would be required.

VII. NUMBER OF ALLELES The incompatibility gene is different from most other genes in that a large number of different alleles is an advantage to the population. With most genes two alleles, the normal or wild type and the more or less rare mutant type, are the usual limits of variation tolerated. There are well-known cases of multiple alleles controlling such characters as blood groups in man, coat colour in rodents and petal colour in plants, but these are the exception rather than the rule. It is also true that, where a search with special methods has been made, slight quantitative differences have been found in the effects of alleles that had previously been thought to be identical (‘rimof&eff, 1932; Stern & Schaeffer, 1943). From this evidence it seems highly probable that most genes have the potentiality for existing in a large number of alleles, but only those controlling incompatibility are brought out by natural selection. The incompatibility gene therefore offers rare opportunities for studying the mutational potentialities of genes.

The evidence for a large number of alleles comes from every species which has been adequately tested. In Nicotiuna sanderue, East & Yarnell (1929) found fifteen different alleles. In Antirrhinum glutinosum eight were found by Gruber (1932). In Prunus aoium Crane & Brown (1937) found eight different alleles. In Oenothera organensis Emerson (1939) reports forty-five alleles in about five hundred plants. From four plants of Nemesia strumosa picked at random, Riley (1934) found at least six and possibly seven different alleles. In Trijolium repens Atwood (1944) found 80% of the alleles to be different, and in T. pratense Williams (1947) found in one population 86% and in another 93 yo to be different.

The determination of the number of alleles is a laborious task. ‘l’o obtain the minimum number of eight in Prunus, Crane Sz Brown had identified twenty-six incompatible groups. With Oenothera the labour was greatly reduced by the excellence of this material for pollen-tube examination, for it was possible to identify a pollination between plants which had one allele in common.

The work in Trifolium was made practicable by the use of homozygotes, S,S,, obtained as a rarity from self-pollination, All the plants to be tested were first crossed to the homozygote; thus all the progeny of the crosses had only one unknown allele, the other allele from the homozygote being common to all. Hence the number

Incompatibility in flowering plants 49 f of different groups among these progeny is equal to the number of alleles present in the sample.

Using a formula of Sewall-Wright, Bateman (1947) has calculated from the data in Trifolium that the number of different alleles in the populations sampled is 212 with 5 yo limits of 442 and I 15. This estimate has been criticized by Fisher (1947) on the grounds that the upper limit may be too low; if this is so the number of alleles in these populations may be even higher than the estimate. It is impossible to estimate the total number of alleles for the whole of such a large species as T. prateme of which the two populations sampled are but a small part. I t is safe to guess, however, that the number is immense.

In a species with a few individuals it is possible to make a direct observation of the total number of alleles. The total population of Oenotheru orgunensis was found to be approximately 500 plants and the number of alleles 45. An attempt to calculate the mutation rate of the S gene necessary to maintain this number has been made by Sewall Wright (1939). The calculation required a higher mutation rate than has actually been found (cf. Lewis, 1948), and before a satisfactory solution can be found to this interesting problem there are several unknown quantities to be estimated.

VIII. MUTATION OF THE S GENE

The advantages of the S gene for mutation experiments are as follows: (I) The large number of observable alleles offers great scope for the detection of different mutations. (2) The pleiotropic action of the gene, that is, the action in the pollen and style, allows a study of mutation affecting either or both of the actions. (3) Incompatibility is a sieve through which only pollen grains with mutant S alleles can pass; thus it is itself a practical means of securing rare mutants.

Mutations affecting incompatibility genes, which have appeared in the course of studies made for other purposes, have been recorded in Antirrhinum glutinosum (Gruber, 1932) ; Trifolium pratense (Williams & Silow, 1933); Beta vulgaris (Owen, 1942) and Pupuver nudicuule (FabergC, 1942). All these four mutations were to alleles giving self-compatibility.

A deliberate attempt to estimate the spontaneous mutation rate of the S gene by Lewis (1948) gave rates per million pollen grains varying from 4'3 & 0.3 to 1.7 & 0.2

in different clones of Oenotheru organensis and 2.3 ? 0.5 to 0.2 & 0.2 in Prunus uvium. These rates include all types of changes, many of which have since been found not to be mutations to new incompatibility alleles which are fully operative. In fact no mutation from one S allele to another fully operative but different allele has been found in more than seventy million pollen grains. Some spontaneous mutations result in complete inactivation to a self-compatibility allele. Others appear to be slightly less efficient types of the original allele. It is already clear from these results that if a change from one S allele to another does ever occur in one mutational step-and only future work can show this-then it must be a very rare event. This conclusion clearly makes the origin of the many alleles found in nature a problem which, if

492 D. LEWIS solved, may tell us something about the fundamental problem of the origin of new genetic material.

Mutations of the S gene induced by treating pollen mother cells with X-radiation have revealed new facts about the structure of this gene.

Before the results of these experiments can be fully assessed it is necessary to describe the technique. The pollen resulting from the treated mother cells is applied to incompatible stigmas. The few grains which contain a mutant S allele with a new reaction will grow down the style and give seeds. Thus there is a rigorous selection for mutations of the S allele which affect the pollen side of the reaction; there is, however, no selection for any mutation which affects the stylar side of the reaction.

All the eight X-radiation mutants which have been analysed show the same change. They all have a changed pollen reaction but an unchanged stylar reaction. For example, a mutant S, allele gave full compatibility to the pollen grain on styles carrying either the old or the new S, allele. In the style, however, the mutant allele retained its original power of repelling S, pollen. Clearly this shows that the S gene behaves towards X-rays as two independent units, one controlling the pollen reaction and the other the style reaction. That no mutations of the style-controlling unit were found does not imply that they are rare, for the selective force of the method used readily accounts for their absence.

Evidence has already been given from interspecific hybrids in Nicotiana and Petunia that the S gene has two properties. The S, pollen from a self-compatible species is repelled by S, in the style of the self-incompatible species. Thus these cases are the reverse of the X-ray mutants, for the S, allele confers an incompatibility reaction on the pollen grain but not on the style.

Further evidence for the dual nature of the S gene comes from the interactions obtained with polyploidy. Two different S alleles interact in the pollen grain, resulting in competition or dominance, but in the style they are fully independent.

The general evidence from X-ray mutations with other genes is that they are loss or inactivation changes. This appears to be the simplest explanation for the loss of incompatibility by the pollen grain carrying the X-ray mutated allele. In view of the dual nature of the S gene, however, it is possible to explain the present results by a non-loss change. The pollen-controlling part of the S gene could have mutated to a new type of incompatibility allele, the style-controlling part remaining unchanged. I n this case a self-pollination would be compatible not because the pollen fails to develop any reaction but because it develops a reaction different from that in the style.

It 'is also evident that if the two gene parts governing the two functions behave towards the drastic action of X-rays as independent units it is probable that they will be independent to the milder agents causing spontaneous mutation. From this it follows that to obtain a mutation from one fully operative allele to another there must be two simultaneous mutations of the same kind in each part of the gene. This would be so rare that it would appear to solve the difficulty of the observed absence of spontaneous mutations to incompatibility alleles. However, it aggravates rather than resolves the problem of the origin of the many alleles in natural populations.

Incompatibility in flowering plants 493

IX. SUMMARY I . Incompatibility is a physiological mechanism which enforces outbreeding.

I t is widespread throughout the families of flowering plants. There are two main types: (I) Heteromorphic. This is associated with differences in floral structure; distylic with two types of flower, thrum having a short style and high anthers, pin having a long style and low anthers; tristylic with three types of flowers, with long, mid and short styles. ( 2 ) Homomorphic, in which there are no floral differences.

2. Heteromorphic incompatibility is associated with six contrasting pairs of characters; these are normally inherited as a single unit. The genetic control is by one gene, S, with two alleles in distyly, and by two genes each with two alleles in tristyly. The incompatibility reaction of the pollen is sporophytically and not gametophytically determined. Short style is always dominant to long. Major modifying genes and polygenes which affect the expression of the S gene are present. In Primulu hortensis abnormal plants exist in which the complex of characters has been broken down, presumably by abnormal crossing-over between subunits of which the S gene must be composed. Both the morphological and the genetic determination restrict the number of different types, and hence of alleles.

3. Homomorphic incompatibility in many species is controlled by a single gene with a large number of alleles, Sl,S2,S3,...,S,t. Pollen is unable to grow in a style which has the same allele as the pollen. Unlike heteromorphic incompatibility the pollen reaction is gametophytically determined and the two alleles in the style are independent. In some species, most of which are polyploids, the simple S type of control does not work without modifications.

4. In a new tetraploid incompatibility breaks down and frequently the tetraploids are fully self-compatible. This breakdown is due to interactions between two different alleles in diploid pollen grains. With some alleles the interaction is competitive so that neither allele functions. This results in self-compatibility. With other alleles, one may be dominant to the other, thus retaining the self-incompatibility of the diploid plant.

5 . Incompatibility is due in some species to the failure of pollen germination, in others to the pollen tubes failing to penetrate the full length of the style. Style- grafting and temperature experiments indicate that the stoppage of pollen-tube growth is due to a reaction between a substance in the pollen tube and a complemen- tary substance in the style, the pollen and style substances being reactively different for each allele. The reaction blocks some process necessary for tube growth and is irreversible.

6. Incompatibility in species crosses and their derivatives shows that species without an incompatibility system have polygenic modifiers which weaken the action of the S gene. From Nicotiana and Petunia hybrids there is evidence that the S gene can have an additional effect of inhibiting pollen from a self-compatible species.

7. The spontaneous mutation rate of the S gene in Oenothera and Prunus, including all changes, is of the order of one in a million pollen grains, but no mutations

494 D. LEWIS to a fully operative incompatibility allele have been found in seventy riiillion tested grains.

Mutant alleles produced by X-rays are all of a type which have their pollen- controlling activity destroyed but their stylar activity unimpaired. The X-ray results and the evidence from polyploidy and interspecific hybrids all point to a dual structure of the S gene. The solution of the problem of the extremely low mutation rate with the high number of alleles in natural populations may throw light on the origin of new genic material.

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INCOMPATIBILITY IN FLOWERING PLANTS