UNIVERSITY OF CALIFORNIA PUBLICATIONS IN AGRICULTURAL SCIENCES Vol. 2, No. 4, pp. 81-190, plates 22-35 No%'ember 24, 1919 MUTATION IN MATT III OLA' BY HOWARD B. FROST CONTENTS PAGE Introduction 81 Genetic literature relating to Matthiola 84 Methods 85 Experimental data 89 The occurrence of apparent mutants 89 Characteristics and heredity of mutant types 92 1. The early type 92 2. The smooth-leaved type 118 3. The large-leaved type 125 4. The crenate-leaved type 127 5. The slender type 135 6. The narrow-leaved type 141 7. Miscellaneous aberrant types 143 8. Some probabilities of random sampling 145 General discussion 153 Summary 159 Literature cited 161 INTRODUCTION It is hardly safe to use the term mutation without first defining it. In this paper it will be taken to mean a genotypic change, or a change in essential hereditary constitution, due neither to immediate cross fertilization nor to segregation in a heterozygous parent. No attempt will be made to restrict the term to any of the known or supposed types of such genotypic change ; a limitation of this kind, which restricts the generally accepted sense of a widely used term, seems to tend to confusion rather than to clearness. 1 Paper no. 52, University of California Citrus Experiment Station and Graduate School of Tropical Agriculture, Riverside, California. 82 University of California Publications in Agricultural Sciences [Vol. 4 If we use the term factor mutation,2 (Babcock, 1918) where the cytological change occurs within a locus, transforming a factor into a different factor, two analogous terms will apply where the cytological change is external to the locus. When the cytological change consists of a loss, reduplication, or transposition of one or more loci it may be called a locus mutation, and when the change consists in such phenomena affecting a whole chromosome it may be called a chromo- some mutation. If the term mutation is applied to the cytological change itself, the last two types of mutation may be grouped together as extralocus mutations, while the first type consists of intralocus mutations. Examples of factor mutation are white eye in DrosopJiila, and probably the rubrinervis type in Oenothera; an example of locus mutation is (possibly) "deficiency" in DrosopJiila ; and examples of chromosome mutation are Oenothera gigas and 0. lata. It is now evident that the immediate problem with Oenothera relates to the mechanism of heredity in the genus. There are two sharply opposed views. One is that recently emphasized by Atkinson (1917, p. 254), when he says, "The evidence from Oenothera cultures points more and more to the conclusion of Shull that 'a hereditary mechanism must exist in Oenothera fundamentally different from that which dis- tributes the Mendelian unit-characters.' " The opposing view is represented by Muller's (1918) strictly Mendelian explanation for Oenothera, based on "an OenotJi era-like case in Drosophila" ; he says, "The striking parallel between the above behavior and that exhibited in Oenothera makes it practically certain that this, too, is a complicated case of balanced lethal factors." A notable feature of the extensive genetic study of Oenothera is the lack of progress toward any definitely supported explanation of its hereditary mechanism which is not Mendelian. The only definite non-Mendelian hypothesis of chromosome behavior so far proposed, aside from "merogony" and other hypotheses (Goldschmidt, 1916) apparently possible but not proved for Oenothera, which assume loss of chromatin after fertilization, seems to be Swingle's (1911) "zygotaxis, " proposed for the apparently parallel case of Citrus. This suggestion that Fx hybrids may differ, apart from non-uniformity of the Pj gametes, because of the establishment of permanently differ- ent arrangements of the chromosomes in the fertilized egg, still seems to be purely speculative. With more or less "Oenoth era-like" cases in other genera, the only definite progress in analysis seems to have resulted from the assump- 2 Muller (1918) has recently used point mutation in the same sense. L91UJ Frost: Mutation m Uatthiola 83 t ion of Mendelian segregation. With Oenothera itself, the trend of the evidence tends to favor tins form of explanation. This fact is strikingly illustrated by two papers of de Vries i L918, L919) which have appeared since the presenl paper was written, especially as .Midler's (1918) complete report on the beaded-wing case in Drosophila (see especially pp. 471-474, 48!), and 498-499) indicates that de Vries had hardly yet realized the full possibilities of the balanced-factor hypothesis. In the light of Midler's masterly demonstration of these possibilities, we may be confident that "mass mutation" is merely ordinary segregation, and that the "unisexual" crosses of Oenothera are really "Mendelian" in their essential phe- nomena. Some difference of usage respecting the inelusiveness of the term M< mli Han may be involved here, it is true, since apparently de Vries would apply it only to cases where "strictly homologous factors are opposed in homologous chromosomes. Since, however (M idler, 1918), there are good reasons for expecting the occurrence of grada- tions of similarity and of synaptic attraction between opposed loci, and hence of gradations of linkage, the criterion of Mendelian behavior should obviously be the occurrence of segregation between homologous chromosomes, whatever their degree of similarity or amount of cross- ing over. We have no reason to assume that an "unpaired" factor in a parent would so divide as to be included in all gametes; on the other hand, we have learned of a mechanism capable of insuring, in certain particular cases, the inclusion of a certain factor or group of factors either in every functional gamete or in every viable zygote. No doubt, a.s Davis (1917) says, "A great forward step will be taken in Oenothera genetics when types of proven purity have been established ....." Meanwhile, cases of "Oenothera-like" heredity in species known to possess the Mendelian mechanism deserve most thorough investigation. Special interest consequently attaches to the peculiar inheritance of certain apparent mutations of the ten-weeks stock (Matthiola annua Swreet), a species in which various character- istics are typically Mendelian. A remarkable series of aberrant forms in this species3 has been briefly discussed in two preliminary com- munications (Frost, 1912 and 1916), and the present paper gives a fuller account of the same phenomena.4 3 In the variety "Snowflake, " a glabrous, double-producing form with white flowers. * While this paper was in press Blakeslee and Avery (1019), have reported the occurrence of apparent mutations in Datura, which seem to be similar in almost every respect to those here discussed. 84 University of California Publications in Agricultural Sciences [Vol. 4 Apparent mutants were first found in the course of work on another problem, the relation of temperature to variation (Frost, 1911), conducted at Cornell University. Studied incidentally at first, these new forms were later given special attention. About nine thousand plants, of which about two thousand were progeny of mutant-type parents of peculiar heredity (nearly one-fourth of the latter representing crosses with Snowflake), have been examined altogether. Some of these plants have been grown at Riverside, where hybridization studies with mutant types are in progress. The present account considers the origin and characteristics of these types, their inheritance with self pollination, and the rather meager available data relating to their behavior in crossing. In connection with the work at Cornell, special acknowledgment is due to the late Professor John Craig, and to Dr. II. J. Webber and Dr. H. H. Love. Facilities for work were furnished by the depart- ments of Horticulture and Plant Breeding of the New York State College of Agriculture. GENETIC LITERATURE RELATING TO MATTHIOLA The work of Correns (1900) on Matthiola furnished one of the earliest confirmations of Mendel's law, and also pointed to complica- tions not found by Mendel. The earlier literature, according to Correns, gives no indication of the study of Matthiola hybrids beyond the first generation. In his later paper on aberrant hybrid ratios, the same author (1902) discusses complications in maize and in Matthiola. After referring the deviations found in maize to selective pollination, he considers a suggestion of de Vries relating to environmental modi- fication of Mendelian ratios, and himself suggests the possibility of selective elimination of gametes. He says (pp. 171-172), "Solche Einfliisse brauchten nicht alle Sorten Keimzellen des Bastardes gleich- massig zu treffen, sondern sie konnten eine Sorte starker angreifen als die andere." Von Tschermak (1904, 1912) has made extensive studies of Matthiola hybrids, considering mainly, as did Correns, pubescence and flower color. The latter of these papers on hybrids in the genera Matthiola, Pisum, and Phascolus represents a careful analytical test of the "factor hypothesis" of segregating inheritance, leading to the conclusion that the applicability of this hypothesis is strongly con- J 1919] Frost: Mutation in Matthiola 80 firmed by the results secured. This work, with that of Miss Saunders, leaves no possibility of doubt that the typical Mendelian mechanism is present in Matthiola. The most extensive genetic work on Matthiola is evidently thai <>f Miss Saunders, reported by herself (1911, 1911a, 1913, 1913a, 1915, 1916) and by Bateson and Saunders, with others (1902, 1905, 1906, 1908). This also is work on heredity in hybrids, with special emphasis on the factorial interpretation of the various complications relating to pubescence and to "doubleness" of flowers. Goldschmidt (1913) has explained the inheritance of doubleness by sex linkage and lethal action of a femaleness factor in pollen formation, and his interpretation has been criticized by Miss Saunders (1913). I (Frost, 1915) have presented a somewhat different lethal- factor scheme, and Miss Saunders (1916) has since restated her views and criticized mine. Muller (1917) has cited the inheritance of doubleness as a case of "balanced factors," in apparent agreement with my formulation. Apparently no one but the present writer (Frost, 1912, 1916; see also review by Bartlett, 1917) has reported experimental evidence of any notable tendency to apparent mutation in the genus, although de Vries (1906, p. 338) mentions the occasional occurrence of vigorous, rigidly upright individuals (a gigas type?), known at Erfurt as "generals," and refers to the rare mutative occurrence of single flowers on branches of double-flowering plants. Doubleness, and color variations in considerable number, have evidently arisen under culti- vation, probably through mutative changes. METHODS The general cultural methods employed for the first three genera- tions have been very briefly described elsewhere (Frost, 1911). The plants of the first four years were grown in pots in the green- house. The plants of the first generation came from one or both of two packets of commercial seed planted in the fall of 1906. and all plants in the later cultures (possibly excepting series 18) were descendants of these. The cultures will in general be designated by the year in which the seed was sown ; the field and greenhouse cultures of 1911 are indicated by 1911F and 1911 H respectively. Part of the seed planted, especially in 1908, came from unguarded flowers. The seed lots where this occurred will be indicated in the 86 University of California Publications in Agricultural Sciences [Vol.4 tabulation of parental data by italic figures, while protection possibly defective will be indicated by an asterisk. It is not probable that much vicinism occurred in the greenhouse cultures, since this plant is well adapted to self fertilization. In the first year's (1906) cultures the plants in each experimental environment were separately numbered. Each plant was designated by its number preceded by two letters indicative of the environment. For greenhouse temperature these letters were C (cool), M (medium temperature), and W (warm) ; for potting soil"' they were S (sand), L (unfertilized "loam"), and G ("good" soil, fertilized). Thus CS1 CS2, WG9, etc., were pedigree numbers of the first generation, and CG2-M8 and WG9-C10 of the second generation. A few syncotyle- donous plants outside the regular cultures of 1907 were called WG9- synl, etc. For the work at Riverside a new system of numbering was adopted, better suited to ordinary pedigree cultures, and the numbers from this system are used below in the individual treatment of all but one of the mutant types ("early"). This is essentially Webber's (1906, p. 308) system, except that each initial or P, individual of a series is indicated by a letter; a full description has beeu published (Frost, 1917). With Matthiola each type or cross between two types that is tested receives a series number, the apparent mutants themselves always being taken as the initial individuals of their selfed series. The cultures of 1908 included progeny of various parents, one being WG9-C10, an early and few-noded plant suspected of being a mutant. The cultures of 1910 consisted of a second-generation test of WG9- C10, and a first-generation test of other possible mutants, with control lots. The plants were all grown on one bench in one greenhouse (house C), from thirty lots of fifteen seeds each, lots 1-17 relating to WG9-C10. The parents descended from WG9-C10 (see table 7) were selected as those with fewest internodes, a medium number of inter- nodes, and most" internodes in each house of the 1908 cultures, earli- ness of fiowering being considered when parents were alike in number of internodes. The control parents were both few-noded and many- nocled, relatively to their sibs. In 1911 eighty progeny lots were grown in the field at Ithaca. Lots 1 to 28, transplanted from the greenhouse, paralleled the test of ■j Soil experimentally varied only in the 1906 cultures, temperature varied in the two following years also. « For house M, not the highest, which wras exceptionally high, but the next to the highest. 1939 / i-o.i1 • Mutation in Matthiola 87 WG9-C10made in 1910-11 ; all available progeny of WG9 CIO, excepl the erenate-leaved apparenl mutanl WG9-C10-C10, were tested, with check lots between as before. Soil differences and unavoidable differ- ences between lots in time of transplanting combined with hot weather and drought to reduce the value of the results. The remaining fifty- two lots, all field-sown, included a further lest of the heredity of aberrant types other than early. .Most of these lots, however, were progeny of Snowtlake parents, grown to obtain evidence on the relation of temperature to mutation and on the inheritance of douhleness of flowers, and therefore the results are not reported here. The 191111 cultures constituted a coldframe and greenhouse prog- eny test of mutant types, mainly in the second generation, the plants being grown in flats. There was added in 1912-13 a small greenhouse test bearing on the supposed mutative origin of WG9-C10, in view of the apparent possi- bility that AVS1 or "WL10, in the same house with the unbagged WG9. might have been heterozygous for the early type — cross pollination then giving the apparent mutant. Further progeny tests of the mutant types have been made in the field at Riverside, beginning in the fall of 1913. Mainly on account of the unsuitability of the usually hot and dry climate of River- side, the cultures have been largely experimental and always on a small scale, and germination or development has sometimes been un- satisfactory. Cultures have been started in October. November. January, and February, and a trial culture in progress at the time of writing was started in August. Some of the plants of the 1915-16 cultures were kept until the summer of 1917, and many of them flowered for the first time when about a year old. In the cultures of 1913, growth was largely unsatisfactory, and with part of the plants aphid injury interfered more or less with the classification of types. In the cultures of 1914, the seeds were largely lost through toxic effects favored by very shallow planting (as at Ithaca) and strong evaporation from the soil. In subsequent planting, the seeds, planted singly in small paper pots, were dropped into relatively deep holes punched in the soil, and covered with sand. The only field-grown plants closely resembling those grown in the greenhouse at Ithaca, it may be noted, have been those of the 1917 cultures, grown in a lathhouse with added shade from muslin. In the cultures of 1915-16, with partial shade and more frequent irrigation than before, development was in general good ; but even 88 University of California Publications in Agricultural Sciences [Vol. 4 e =D « ^. «! «s e o o 03 oo a* •Si s a j, CO CO CO o o t^ HJ C c oj OJ 3 ° +1 +1 41 s O) Ph ' b- CO rH c •* Ci C5 lO Q. c3 in £1 Tt< CO "# -rjn lO CM "# < 3 3 2 T— 1 8JB[ A\iaA CO 1 a^pil^ds : : : r-4 : >> s\-ei3j +3 n 03 SITU Al3A 111 03 !|[BUIS puB sncua : i— < ; t— i O -uinu saABa^ M c 9}Bf A\l3A luadBtJS : i— I : i— I o -siui sdii.-juai * .£2 >> ■*3 83X5 CO — aapuaIg : »-H : i— i ^h >> a CO M 01 o u £ 0> adAi B 3 paABaj-moja-B^ I H rH (N £ padA} p3AB3] : i— i : rH -aiBuaJO-nuag adA1} u paABaj-ajBuaj^ t— 1 rH t-h : CO i-H CN adAi .3 paABai-mootug CO :: CM O rH rH u >-H CJ 03^ lO GO"tf t~ Tt< GO O o S oo co co rH r- o -t H = rH CM p< 0Q 02 0) M o> o> a > > - b~ oo h 7 v 0_rOrO 1^3,1 5 Ph NHHOejcJ CSC® QO0r-lO_- — Q CjU OrS^rS^^ O r^ o TD > O r*a 4H 0) rQ 0 cci rO o CS Ph a -^ CO c3 60 C3 o c5 c_> ^ - ~ 1 Fh ~ ft . CO rH rH Ph GO P n3 CD OJ 0) 5^ ft CO 60 0 O £ 58 93 P m «« O 4^ r~ T3 CD rH rH o +J as 01 Ph P 3 rH O o C6 T3 o CV OJ CU) P 0> 60 b£+^ H- o o 5j p o - ccj 03 T3 ■!-? 3 CD ^1 rH rH A rH CU 0> +J 0) .9 S > o O o rt rP o p — 0) p o < H < H "5 A B •a n e3 oj L919] Frost: Mutation in Matthiola 89 here the mutant typos, with one exception, often failed to grow satis- factorily or to set seed. Iii feci ion, probably by Fusarium, evidently was the cause of the death of many of these plants in their second season. With all cultures grown after (probably) those of 1906, special care was taken to secure random samples of seed, and after 1908 no plants were rejected. The only exception to this statement is the rejection of one pot out of every fifteen, by number and systematically, in the first twenty progeny lots of 1910. For the earlier cultures, a certain amount of selection must be recorded, as follows. In 1906 the small and the largest plants were omitted at potting, and probably any weak and abnormal seedlings had been omitted at the preceding transplanting. In 1907 all markedly weak, late, or abnormal seedlings, as determined mainly by the appearance of the cotyledons, were omitted at the first transplanting; and the same was done in 1908, except that certain lots from old seed were unselected.7 These last lots were arranged at transplanting in such a way that the weak and abnormal plants came at the end in each lot. EXPERIMENTAL DATA The Occurrence op Apparent Mutants In the cultures of 1906, 88 plants were grown to maturity, none of these being suspected of mutation. In the cultures of 1907, among 170 plants one striking variant appeared; this plant, WG9-C10, was exceptionally small and early in blooming. In the cultures of 1908, 714 plants were available, including ap- parent mutants in several hereditary lines as indicated in table 1. A striking feature of the results is the scarcity of apparent mutants among the seedlings classed as strictly normal at transplanting; prob- ably the scarcity in the preceding years was due mainly to the rejection of abnormal seedlings (see "Methods"). The first, second, and fourth of these forms have been common in later cultures, while the third and fifth have been rarer; the last three, if seen at all elsewhere, have not being recognized as belonging to the same types as these three plants. One tiny plant from WG9, probably not viable, was discarded. 90 University of California Publications in Agricultural Sciences \ Vol. 4 Table 2 shows the numbers and percentages of apparent mutants found in the cultures of 1910 and 1911P. Since the early type seems to differ from Snowflake only in size and earliness, and is probably inherited without special complications, the available progeny of early- type parents are included in the totals. The progeny of all parents recognized as belonging to other aberrant types are omitted. The second column under "Percentage of mutants" omits doubtful types and individuals, but includes some individuals for which some doubt was indicated in the original records. One rare type of 1911, large- Table 2 Aberrant types: occurrence among progeny of Snowflake and early parents. Apparent selective elimination at or after germination in field-sown cultures." Cultures Greenhouse, 1910 Field, 1911, seed house-sown All above Same, Snowflake par- ents only Field,1911,seed field- sown (parents all Snowflake ) Progeny examined'1 338 2072 2410 1304 3927 Percentage of apparent mutants All counted 5.03 ± .82<: 5.31 ± .33 5.27 ± .31 4.3c .41 2.34 ± .24 Doubtful omitted 4.14 4.63 4.56 .77 .31 .29 3.74 ± .38 1.55 .22 0 Germination in greenhouse-sown lots, counting only plants examined for type, 93.2 per cent; in field-sown lots, 45.1 per cent. b Including some plants of uncertain type, indicated for some lots (when apparently not Snowflake) in tables 1 and 3. c For the calculation of these probable errors the percentages on the third line are used as p. leaved, here omitted, has proved to be genetic, but its determination in these cultures was in general uncertain. A stricter criterion for the second column, elimination of all individuals not considered posi- tively determined, was used in the calculations for, the tables for the inheritance of the separate mutant types. Evidently the more rigorous field conditions of 1911 eliminated many of the "mutants" at or soon after germination. The "coefficient of mutability" with good germination, as was the case with the un- selected cultures of 1908, seems to be near 5 per cent, a surprisingly high figure if immediate true mutation is responsible. Before the aberrant types are considered separately, we may examine (table 3) a detailed illustration of their occurrence in larger cultures. It seems probable, from this evidence, that any descendant of WG9 was capable of producing any of the mutant types so far 19191 Frost : Mutation in Mai 91 2 <* >> / - r a - -< ~ a P g ■ tOtOtOtOtOtO— — — — — — — — tO tO IO — — i . — : . i i — z _r y ~i -~ cc to — o -,; o: c> -U co cs oo ^i os in oo ^i to > 3 o to -• — cc -ix «i to — — os oo --i to —. ec — x c" M IOO: :oo i;--|i:io-= * — c *.oo = — -I -1 -I -I -1 -1 — -I -I S - I -I - 1 -I -I -I -1 -1 ii -I ~1 ^J ^1 ^1 ^1 -I -1 osh-1 torfk en to as en ooos^a ~ —. / x ■-: -: y -- u\ y -> ~4 O ~i — — to to — to ic;;ic — — — — — — re: — : : — — : Field lot Generation i Generation 2 Generation 3 Total progi nj ol detei minable I j i" Smooth-leaved Small-smooth- er, ed Crenate-leavcd Semi-crenate- leaved Pointed-crenate- leaved — to i— : p— ': to — — : — : — — : — : CO — : tO .,3 . . .^5 OC04^00CntOOCnOstOOS*»4i-CO^tO- ffl 00 Ol W * K tv Ol !n t5 W OSOCOOOCntOOCOOTt04iCOi*».tO*»tO- Cn-^JOtC04^0it0 0i*' — CO Medium-smooth- leaved Narrow-leaved Narrow-dark- leaved S 1 1 • 1 1 1 1 1 ■ i Small-convex- leaved ( 'otnpar-t Curly-leaved Pointed-light- leaved Large-leaved Medium-large- leaved Large-thick- leaved Small stout- capsuled Jagged-leaved All counted Doubtful omitted Cl B V! J6 k * « ^ "1 ft M. H > ^ ~ — r n> K 92 University of California Publications in Agricultural Sciences [Vol. 4 discovered ; the occurrence of the various types suggests a random distribution among the progeny lots. This conclusion is confirmed, and extended to CG2, by the field-sown lots of 1911. Various parents belonging to mutant types have given other mutant types among their progeny. There is some reason, as table 4 indicates, to suppose that parents of the early type have a more marked tendency to produce these other types than have Snowflake parents.8 Table 4 1910 and 1911F ; sown in greenhouse. Apparent mutants among descendants of WG9-C10 and other ancestors, comparing early parents (pure or heterozygous) with Snowflake parents. Type of parent Progeny Ancestry Total examined Percentage of mutants All counted Doubtful omitted WG9-C10 Pure Snowflake Both Both 1 Early ( Snowflake Snowflake Snowflake Both 1046 558 806 1364 2410 6.50 ± .47" 4 . 30 ± .64 4.34 ± .53 4.33 ± .41 5.27 ± .31 5.64 ± .44 3.41 ± .60 3.97 ± .50 3.74 ± .38 4.56 ± .29 * For the calculation of these probable errors the percentages on the last line are used as p. Characteristics and Heredity op Mutant Types 1. THE EARLY TYPE So far as is known, WG9-C10 (figs. 1, 2) was the only apparent mutant of the early type in the cultures. Since, however, this type visibly differs from Snowflake only or mainly in quantitative characters, it cannot be positively identified without comparative progeny tests, and therefore may have been represented by mutant individuals not used as parents. WG9-C10 was much smaller pro- portionately than were its progeny ; this difference was probably due to an embryonic abnormality, early blind termination of the main axis, which was occasionally observed elsewhere and probably occurred in this case. Plants of this type, as compared with Snowflake, are, in general, fevver-noded, smaller, and earlier in blooming. The principal data from the cultures of 1908 are shown in tables 5 and 6, which also indicate the later conclusions as to the segregation of the early type in the cultures of this year ; figures 3 and 4 illustrate 8 Inspection of the data in detail indicates that this difference is not due to the possible tendency in parents grown in the warm house to more frequent apparent mutation. 1919] Frost: Mutation in Mattliiola 93 Table 5 Cultures of 1908. Time from sowing to emergence of corolla of earliest flower of ■primary cluster. Frequency distributions.' Singles Doubles House C House M House W House C House M House \V Parents: WG9- WG9- WG9- WG9- \\i ,'i WG9- C10 Rest C10 Res ' CIO Res 1 CIO Rest CIO Rest C10 Rest D.I 7- h 110 It 111 U 112 113 111 115 116 1 1 117 "it 118 it 2 119 3 120 "it 4 121 it 2 1 1 122 3 2 123 8 It i 1 2 124 i 4 1 2 125 7 it 5 1 3 126 16 7 7 127 3 it 2 9 2 128 4 3 \ z 8 5 129 7 it 4 8 7 130 1 it 3 12t 4 131 i 2 4 2 12 3 132 "lj 1 2 7 8 133 m 1 i 4 7 134 i 2 2 1 2 7 135 4 4 4 6 6 136 1 i 1 3 1 4 137 7 2 3 3 i 2 138 10 1 9 4 l 1 139 18t 1 i 2 8 l 3 140 i 7 1 r .... 4 13 3 141 10 1 "l 9 i 3 142 4 6 15 l 1 143 4 i 5 "2 10 2 144 4 2 9 2 145 3 6 3 146 i 5 2 147 4 148 2 1 1 149 2 1 150 r 1 2 151 1 1 r 152 2 153 1 i 154 155 1 1 l ■ Daggers (t) indicate the position and number of apparent mutants. Double daggers (t) indicate inheritance of parental type (here, early); all single progeny of WG9-C10 here reported have been tested for inheritance of this type. The conventional statistical constants corresponding to the house totals of tables 5 and 6 have been published (Frost, 1911); the means for flowering given there are too high by one half-day. b To time of observation (upper limit of one-day class). 94 University of California Publications in Agricultural Sciences [Vol. 4 Table 5. Cultures op 1908 (Continued) Singles Doubles House C House M House W House C House M House VV Parents: WG9- C10 Rest WG9- CIO Rest WG9- C10 Rest WG9- C10 Rest WG9- C10 Rest WG9- C10 Rest Days b 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 1 1- 1 it " Daggers (f) indicate the position and number of apparent mutants. Double daggers (t) indicate inheritance of parental type (here, early); all single progeny of WG9-C10 here reported have been tested for inheritance of this type. The conventional statistical constants corresponding to the house totals of tables 5 ami (5 have been published (Frost, 1911); the means for flowering given there are too high by one half-day. b To time of observation (upper limit of one-day class). the difference in earliness between the early and SnowHake types. The parents grouped under "rest" include CGl2 and WG9 themselves, with four progeny of the former and eight of the latter. Of these fourteen parents, not one has produced exceptionally few-noded progeny like those of WG9-C10. Apparently WG9-C10 was heterozygous for a "few-nodedness" factor not carried by any of the other parents tested. Neither in the 1907 cultures nor in the 1908 cultures now under consideration did the data suggest that WG9 itself was similarly heterozygous. Tables 5 and 6 include the first 30 progeny of WG9, for each house, as arranged at the first transplanting," 88 plants altogether; including the remaining plants, mainly weak or abnormal at transplanting, the total is 116. One of the F2 plants (WG9-syn3-M10) was very sug- gestive of the early type, but (tables 12 and 13) it gave only Snow- flake progeny in a small test. * See page 89. Two plants not producing a normal main inflorescence are omitted. L9191 Frost: Mutation in MniilmtUi 95 Tabu 6 ! n-stem internodes below first flower-bearing until . i'n qu< ncy disti b Singles 1 1 lubles House C House M H mse W House ' ' House M House W Parents: WG9 CIO Real WG9 CIO Res 1 Cl( *" Rest WG9 cm Res t WG9 CIO Rest WG9 CIO ' Internodes 16 U 17 18 19 Tit 20 it i 21 2« 22 n "l 1 23 1 24 9 25 i 2f 1 + 1 "2 25 26 29 i" 27 i "i 1 1 22 2t 28 17 6 9 i 29 24 1 14 30 13 1 i 2 22 1 31 8 2 27 32 2 7 5 33 1 15 1 t 3 34 16 4 1 35 2 19 1 t z 5 36 4 1 t .... i 6 37 1 1 t .... 8 38 8 5 39 3 13 40 1 6 41 1 r 8 42 it 1 + + 6 43 It 12 44 9 45 i t z 6 46 4 3 47 3 3 48 4 49 1 3 i' 50 6 51 1 3 2f 52 10 1 53 6 54 2 2 55 1 3 56 7 i 57 8 58 1 59 1 r 60 3 61 62 1 a See note a to table •"">. 96 University of California Publications in Agricultural Sciences [Vol. 4 The differentiation of the early race is very marked ; with the singles, in fact, the later cultures indicate no case of overlapping in the 1908 cultures, in either character, between extracted pure Snow- flake and pure or heterozygous early. The total sterility of the doubles necessarily leaves their constitution somewhat in doubt. The cultures of 1908 so far suggest that "WG9-C10 was a mutant. To be reasonably certain, however, we must have further evidence (1) on the fact and mode of inheritance of the supposed new type, and (2) on the possibility that either WG9 or some other plant of the cultures of 1906 brought the character into the cultures. "We shall now consider somewhat extensive evidence bearing on these points, concluding with a special test of the possibility of vicinism. "When I last saw the warm-house plants of 1906, three were known to be singles, and all but two of the rest were recorded as certainly or probably doubles. Seed was secured from these three singles only, and presumably no other singles occurred in the house. Since this seed was all from unguarded flowers, we must consider the possibility that WTS1 or "WL10, the other warm-house singles, brought the early factor into the cultures. It is also barely possible that pollen was brought to WG9 from some plant not in this greenhouse. These two parents were tested in supplementary cultures, in house C in 1907, and in house W in 1908. The 1907 progeny averaged slightly earlier than those of other parents, but this may have been due to their position, which was much nearer a partition separating the house10 from a warm greenhouse. Unfortunately the internodes were not recorded. In the 1908 cultures these lots were potted two days later than most of the other lots and one day later than the "WG9 lot, and for some unknown reason the "WL10 lot wilted badly for some days. The parents in question gave singles (16 and 11 plants respectively) which when compared with progeny of CG2 and "WG9 (23 and 15) might suggest that the parents were heterozygous for the early type. The results with the similar numbers of doubles decidedly disagree with these, and suggest that cultural accidents produced the differences ; the "WS1 lot was not exceptional, while all the "WL10 progeny were grouped near the lower end of the range of the other lots. In view of all the facts, the data hardly deserve tabular presentation, but they raise a question requiring further study; a later test is reported below. 4) io A temporary substitute for the regular house C. 1919] Frost: Mulnl'mn in Matthiola 97 M 2-to X B o cc 3 £" o o » Si S» N> 3Q d B ^ -r %- so a SB 03 D ^ CL P P O H3 3 ° •a 3 ►a «■ p - •-> cb CHi^03KIMOCOOO^w)Ulil»Ci:Kli-'0*OO^IfflOl*WlOl 3 3 I-1 cc 1— > o , s , 4 TO to a b o o S 32 p p k co 02 /- § TO TO TO £ M g3 21' TO Ka5 TO ?HH 2 b 3 3 hh o o 5 e b> B So£§ O2S"50oo>i«iSj0>i>ioo5^2. 4 i|gaiii^^iiJi«T^M < * 3232= P P s P CL 33 B3 33- tB .0 ?T !T ?r >— ' CB fB (B P P (B (B 32 32! P P ! » 9 ' >->^ TO TO 3 3 O O si ?! 33 33 p p ?r?r ,fe kP. tO ^ to W W CO Ol tO CO rf*. to tO CO tO CO GO !D tOMHOlO M ^^l^to 00 ~J 03 1— to O CO^ICOO2COt^-^JO3^I^JO2»^-^ia2Cn^J0OaiCOCOO3O3^^J^J000O®~-I h- GO^JtOtOO~J^l^I0303>^.CiCnOOOO^Ii*>.C»Oi000000^1^l02030iG2 33 < 9 c 3 o S3j s 98 University of California Publications in Agricultural Sciences [Vol. 4 In the cultures of 1910 and 1911F, all the 1908 progeny of WG9- C10 were tested. On account of the variable nature of the quanti- tative character involved, an elaborate study was necessary. Only small cultures could be grown in the greenhouse ; these were supple- mented by larger lots in the field in 1911, but inhibition of flowering by the hot summer, together with the effects of disease and soil varia- tions, made the field results erratic and necessitated special methods of treatment of the evidence. Singles Doubles • _ _ _^_. _____ ___ _____ __ • :____; ___-_; — ^— _____ _________________ _£__Z__1___- ____* _____ ______ M3 M') I I CI C2 Co Cl M6 M9 M4 M2 M7 C3 C7 W6 Wo W10 Mo MS I I CIO C5 CIO C9 CIO I C9 WG9 Ancestry Chart 1. Cultures of 1910. Internodes: parental values and progeny means (respectively shown by dots and lines) for progeny lots 1 to 17, omitting aberrant progeny. Parental values should be compared only for the same house. Table 7 gives the available data for the parents of the 1910 cultures, and the numbers of progeny available for quantitative data. The order of the pedigree numbers here is the same as that of the progeny lots on the greenhouse bench. For convenience, the 1910 tests of other mutant types, together with tests of several Snowflake parents, are included in the table (lots 18 to 30). 1919] Frost: Muta Matthiola 99 The plants were grown in house C of the previous work. Two or three plants one shown in li'_r. 25) were extremelj vigorous, pre sumahly because of some accidental soil difference; aside from these, a few apparenl mutants, and a few plants otherwise abnormal, the plants were fairly uniform excepl where heterozygosis was to be expected. The data for time of flowering, as with the 1908 cultures, show the same main features as the internode data, and only the latter will be considered in detail. The types were again more widely different in internodes than in earliness, a fad which seems to indicate that the early type grows more slowly than Knowllake. So large and so regular are the differences in internodes that the means of these very small lots seem worthy of present at ion (chart 1 )." Apparently the I'ew-noded character was carried, among the nine parents descended from VYG9 ('10. by all except the three parents having the highest cumbers in their respective houses. Tables 8 and 9 give the internode frequencies for the singles and doubles respectively, by separate progeny lots and by groups of similar ancestry. The range of variation for the cheek lots, omitting the indicated apparent mutants and other apparently abnormal plants, is rather surprisingly small, as is the case with the cool-house cultures of 1908. The three late progeny of WG9-C10 give lots closely corre- sponding in range to the check lots, only one individual falling below the range of the combined check lots. The six early and medium progeny of WG9-C10, on the other hand, give distributions of far greater range than do the check parents, extending to much lower values. Tables 10 and 11 give the ordinary statistical constants for the grouped lots. The mean number of internodes, for both singles and doubles, is about 25 per cent lower in the progeny of the six I'ew- noded parents, the difference being not far from ten times as great as its probable error. The increase in variability with the progeny of the early parents is also striking, and the difference is about five to six times its probable error. With time to flowering, it may be noted, the differences are similar to those with internodes, but somewhat less marked in the case of the mean ; the flowering data are not given here. It is plain that the previous conclusion as to the heterozygous nature of WG9-C10 is sustained. The elimination of the apparent mutants ii Calculated with the apparent mutants and four other apparently abnormal plants eliminated; see tables 8 and 0. 100 University of California Publications in Agricultural Sciences [Vol. 4 w a (0 EH S ft. 1) T3 CC2 a; t-hCOiOO-*-*COCO-*It-h T— 1 C3CO>OCOi-HT«taCNCO •-H CN "# CN i-H JO o .; :-i ■"*0C>t*cNCOCOCO ►h CO ■* Oi 02 t> CD 1— 1 : 1— 1 1—1 5 Is ►J : : ■ ; ;,-, : : : t-h OS : CO t-h Z.2 HCClOO-^rtNNMtH NHHPJHN : o 5 o O : ;^^ : : t-h : : : 00 hNNhh : COCN i-i : o 5 o 5 i i ! I-h ; t-Ht-H f\| H :hw : t— < ! rH : t-h : : : CO : ,-h t-h cM i— i : i— i i— i ; :-h : ; : ; : o ::::::::: :rH :: eo :::: : CO ° ::::::::: :.-,cN **. cm c 5 :(M :i-H : : <5 ; ; ;^ ; :^h ; :^ CM :t-h i* s MNH ,-H 2, i-H N i-H 2 o 8 5 ,: (O : : rH i : »H : t-h i— ( : i— i t-H < 0 "3 0 01 0> DO 3 2^ 6« T3 0? e8 CC •!-! T3 en fl Qj FH 0 t> n 0 rt s 0 -*H 0 on al a 0; tH -= Bi -t- ft ft ti « 0 ^ T3^a — as a a o> o> >h 50 03 © ft fn =4H O en a ts . 0J rj a> be «« w h - S ft * M ft h !>, i>> cS !-H -1^ » 01 sea b 1. « 0 cs > «h """ «8 to ^ o> 3 O T O co a. be p rt ^ a o S S 3 o to .3 ft a 0} -Q 01 0> CO O) (H o ■ ed IS 3 01 to' u b0i> fax)'* cfl O Oi oj H 5 go "I So1 O o ^1 1919] Frost : Mutation in Matthiola 101 Eh On ts. S s is* — T3 51 3-s - 1 •z.2 M _ ,-, _, ^>±> es 2 a > u a rd tn «M =1-1 « h m O so 5 2 - 01 0) 4J &»h -Q.t: r1 "3 S £ .biS1" 3 O h IB'H a * ag o £ o 30, la ate-leav zygous ■*> a +a rj o 'S.S S/S 18 ere leter *-■ ' r— « 2 o £ ■S H -2 *> as T3 r— -rt o 3 J ^ w u M^ .s « .sy.a a rr! « fj.SK a> > 60^ a o bo'H col -CI ins. cs a ™ © a OS .2 r-i a ,q a H t PT? " a ' Sg J2 > & 102 University of California Publications in Agricultural Sciences [Vol.4 OS NO ■0 — o 51 Hi-IHCOCCtH <*HCO C^ 3 O 03 > « !i-l(N irH :MiO»hiOH(OHCM CO i IM o O 03 CO, ►J :rtNHHC.O!«3NH : : :"<< O 5 ►J :::::::: : -i — : : :r*r* : -* i> o : :rH : :h : : ; ; i "S3 £.3 ■5" ; o 5 o 3 :^ :::::::::: : : :::::: :^ ::::::: th to 2 :::::::::::: >> 3 >> ^ a ~~ a ^- h— r. 9 a -~.— N O co 4* 4* ^ *. 4* > 3 H or -i Of- n o' --p o" c i— p c off off to ■< co a> ><_-_ ><; a = § t^ff 2 Mt-'tr1 3 o'f= & fo 5' c p p p C i — r re CO 05 v< en c* c* << 3 •a ■a CO 8 2 ■o c to i3 HtBWMCn 00 -1 *. >-'Cn Mi^OOii^ 4» tO CO en 4* 3 T "- Cn CO 4> OlOCC Cn 00 to 4- o H- -J © o o g If If If If H- If If If If If 3 1-" to 4*- to to to 00 © 4^ CO 00 tO CD © -J © tO i— to to CO CO 4* to to Oi £» en toco CO to tn h-co to © © CO 4>. O.CC h- to Cn P> Or to p— I—© -J K-2 H- If If If B- If If If If If o 3 3 0- MMMtOM l— to 4^ tO tO CO © Cn © © 00 CO CO © to H-> 1— tO P-i t-^ >—L to >-> en to h- 00 h-> 00 CO ©00 t— O tniliHOH -J © to to© *• o |. 35 en CO £. 00 en ©h- i— © co If If If If If If H- If If If 5 3 h— » tO M 3 ■* o 010^1^1^1 © H - >■ p CO 0 - H s w <5 "» I—" © 3 Co ~ 5" ■St. 104 University of California Publications in Agricultural Sciences [Vol. 4 Eh 5SS = .2 oj-- •o at S o -H -H -H -H -H -H ■H H -H 0)1(5 00 rf t-t CO S3[3UIg -H -H 41 ■H -H -H -H -H +1 *> >> « >> as a? --i 0> -r" -u +a tj oj 03 CS hJ^W ►-JhJH O OS O O £ £ sajqnoQ 1 1 1919] Frost: Mutation fa Matthiola 105 ;hh1 the other abnormal plants presumably gives a better comparison as to mean and variability, but the conclusion is the same in either case. The three many-noded (late) parents descended from WG9- ('10 give no definite indication of being genetically different from the "check" lots not descended from W Table 14 1911; field, plants transplanted from greenhouse. Ancestry, seed, and a a mlii is of progt uy." Lot Ancestry Seeds Number of plants alive ■ i'A days after Numbers of plants on mutation and for data lowering sown Gen. 1 Gen. 2 Gen. 3 sowing Total1' Singles Doubles 1} OS /C8 \C10 so 79 77 34 43 71 71 70 36 34 5| rC2 80 80 79 39 39 CIO C5 80 79 78 40 38 1 C8 80 79 78 35 43 6J LCI 80 78 76 36 40 7\ C5 /W18 \ W24 80 79 77 37 40 8/ 80 76 75 41 34 91 10 fM4 80,26 77 76 30 45 M9 80 80 78 36 41 11 12 f CIO . M6 80, 63 77 77 34 42 M2 80,7/ 78 77 36 40 13 14 J M7 80 80 80 37 42 M8 80 74 70 33 37 WG9 ■ 15] C9 /C3 JC7 80 78 78 30 48 16/ 80 76 75 32 43 17] W6 80 74 70 31 39 18 W4 80, £1 70 65 26 38 19 Wll 80 78 76 34 42 20 W9 80, 19 76 72 24 47 21 • CIO • W5 80 79 75 33 42 22 W8 80, //, 76 74 36 36 23 W7 80 75 72 32 40 24 W3 80 73 71 37 34 25 J W10 74 72 66 32 34 26 CIO 80 60 59 27 32 27) 28/ C9 / W10 80 74 73 33 39 \W24 80 80 78 32 45 " For plan of arrangement and parental data, see page 86 and tables 5 and 6. Seeds from unguarded flowers are indicated by italic figures; where two numbers are given the first is the total. b Including twelve plants (all late mutants) with which determination of the form of flower was impossible. 108 University of California Publications in Agricultural Sciences [Vol. 4 and 6, in the order there given, except that the arrangement by inter- nodes reverses the two-day difference in earliness of the parents of lots 19 and 20; for convenient comparison, the parental and parent- lot internode values are included in table 19. Two progeny lots were set in each of the fourteen rows ; probably the soil was less favorable at the east end of the plot, and hence for the even-numbered lots, at least in about the last seven rows out of the fourteen. The plants were beginning to grow very rapidly when moved to the field. On account of deficient soil moisture and excessive heat, the transplanting was slow and in part purposely delayed, covering a period of five days. Lots 21 to 28 were set three days later than lots 80 70 60 00 40 30 20 10 ODD >. fcn o o o 7Z 5 Ph 1 2 3 4 5 6 7 8 9 10 11 12 13 14 (C) (C) (C) Row number (C) Chart 2. 1911, field; lots transplanted from greenhouse. Percentages of progeny lots not flowering by November 3, for singles. Apparent mutants and injured plants eliminated. Odd-numbered lots represented by solid line. (C) indicate check rows. The curves are broken between rows 10 and 11, where a cultural difference enters. 11 to 20, and the later loss of roots resulting seems, in spite of rain coming the next day, to have seriously delayed flowering. Lots 1 and 2 wilted badly after transplanting, and some difference in soil con- ditions in the flats, rather than a genetic difference, was doubtless responsible for the exceptional lateness of these lots. Lot 20 lost an exceptionally large leaf area as a result of transplanting. A fungus disease (a slow stem rot) was more common on lots 20 to 24 than elsewhere ; it doubtless killed some young plants and delayed or pre- vented flowering in some other cases. Possibly the soil was poorer in the later rows. 1919] Frost: Mutation m Miittlimhi 109 Table 15 1911, field; plants transplanted from greenhouse. Plants alive November 8, not having flowered. Singles. Non- Non-flowering, Non-flowering Non-flowering, How Lot Hdwim im- plants Srii.u Sake and early types" Lot plants Snowflake and early types11 1 1 27 26 2 29 28 (27) 2 3 5 2 4 7 5 3 5 9 9 6 11 11 4 7 7 5 8 17 17 5 9 0 0 10 2 2(1) 6 11 1 0 12 9 7 •7 13 19 18(17) 14 20 19 8 15 12 12 16 14 14 9 17 1 1 18 8(7?) 7(6?) 10 19 0 0 20 3 3 11 21 8 8 22b 11 10 12 23 21 20 24 23 23 13 25 16 14 (12) 26 14 12 14 27 23 22 28 24 24 a Omitting non-flowering apparent mutants. For the numbers in parenthesis, "doubtful mutants" are classed as mutants. Two plants accidentally seriously injured, in lots 14 and 25, were counted out with the mutants. b The stem-rot disease (see p. 108) was evidently worst in lot 22; some two or three of the worst infected plants (included above) were nearly or quite dead by November 3. Table 16 Same as table 15, for doubles. Row Lot Non- flowering plants Non-flowering, Snowflake and early types" Lot Non-flowering plants Non-flowering, Snowflake and early types8 1 1 4 2 2 0 0 2 3 3 5 3 3 1 3 4 6 1 0 1(0) 0 4 7 3 2(1) 8 3 1 5 6 7 9 11 13 2 0 4 0 0 2(1) 10 12 14 0 0 1 0 0 1 8 15 2 2 16 6 5 9 10 11 12 13 17 19 21 23 25 1 0 3 5 1 0 0 1 5 1 18 20 22 24 26 3 2 1 4 7 3(2) 2 1 4 5 14 27 1 1 28 10 8 " See notes to table 15. 110 University of California Publications in Agricultural Sciences [Vol.4 Altogether, these cultures are doubtless much less reliable for their size than the greenhouse tests of the early type, but they nevertheless, with due consideration of the points just mentioned, seem to permit of fairly safe conclusions for most of the parents. The plants were examined for flowering every other afternoon from July 4 to November 3, inclusive (73 to 195 days from sowing) . A very large part of the plants flowered in July, some in August, and a few still later. Evidently the high summer temperature largely inhibited flowering; many of the singles and a few of the doubles entirely failed to flower. 100 90 *> 80 o Ctf £ 60 EVE V 1 50 ODD CS c 40 £ 30 — 20 10 1 2 3 4 5 G 7 8 9 10 11 12 13 14 (C) (C) (C) Row number (C) Chart 3. 1911, field; lots transplanted from greenhouse. Percentages of progeny lots with primary cluster flowering or aborted by October 10-16, for singles. Lines as in chart 2. Figures 5 and 6 show the plants in July. Growth was usually vigorous through the season, but the internodes were very short, the branches numerous, and the region of the terminal inflorescence often abortive, so that determination of the number of main-stem internodes was not practicable. The emergence of the earliest corolla on the plant was recorded at the bi-diurnal observations, and at two periods during the season the aborted primary clusters were noted. The data show very definitely the transmission of "earliness" by the fewer-noded progeny of WG9-C10. Tables 15 and 16 show the numbers of plants alive, without having flowered, on November 3 ; the figures are thus a measure of lateness. The two progeny lots in each row are given one line in each of the tables, in order to facilitate separate comparison of the fourteen lots in each end half of the plot. L919] Frost : M utation in Mv July 29 Flowering or :il>oi tcil l>\ Oct. 10-10 1 1 0 0 2 0 0(2) 2 3 3 5 12(13) 2 19 (22) 2 4 6 20 4 24 (26) 8(9) 4 7 2(3) 3(4) 8 1 1(2) 5 6 7 9 11 13 22 25 1 27 29 (30) 2(4) 10 12 14 26 17 2(3) 33 22 2(3) 8 15 4 5(6) 16 0(1) 1(2) 9 10 11 12 13 17 19 21 23 25 19 (20) 25 (27) 4 2 0 20 (23) 29 (31) liiKi :?M, 3(4) 18 20 22 24 26 9 11 7 1 3 12 12 9 1(2) 4(5) 14 27 1 3(4) 28 0 0(1) ° In this table and also in table 18 the numbers in parenthesis include the probable but somewhat doubtful cases. progeny of WG9-C10, is decidedly earlier than the adjacent lots. Lot 25 also appears early, however. Tables 17 and 18 give a direct measure of earliness, relating to the primary inflorescence alone. The clusters visibly aborted were in general relatively far advanced, and those aborted at the earlier date correspond to decidedly early flowering; consequently the flowering and aborted clusters are classed together as early. Chart 3 gives the percentages for singles. Here the data for the doubles show fairly consistent differences in the number aborted at the earlier date, while the October totals are 112 University of California Publications in Agricultural Sciences [Vol. 4 less regular. There are contrasts similar to those of table 15 up to lot 26, which is late, while the check lots 27 and 28 are early. The singles show the type differences very strikingly throughout lots 1 to 20, while lots 21, 22, and 26 give less positive indications of the presence of the early factor. Table 19 gives the numbers of singles flowering, in primary in- florescence or elsewhere, by November 3, when growth had practically stopped. The indications are in general the same as with the data already discussed, with better evidence than usual that lots 21 and 22 Table 18 Same as table 17, for doubles.* Row Lot Aborted by July 29 Flowering or aborted by Oct. 10-16 Lot Aborted by July 29 Flowering or aborted by Oct. 10-16 1 1 15 30 2 6 22 (23) 2 3 3 5 23 12 33 (34) 29 (30) 4 6 21 16 35 30(31) 4 7 17 30 8 8 22 (24) 5 6 7 9 11 13 25 25 (26) 16 41 40 (41) 28 10 12 14 24 23 22 41 40 31 8 15 27 37 (39) 16 11(12) 29 (32) 9 10 11 12 13 17 19 21 23 25 20 21 22 16 (17) 17 35 41 (42) 35 27 (28) 25 18 20 22 24 26 20 (21) 21 18 10 9 32 (33) 42 (44) 27 (28) 16(17) 15 14 27 21 29 (30) 28 20 25 (27) See note to table 17. possessed the early factor. The mean time of flowering is irregular, but shows some effect of the earliness factor. Lot 26 is late as to number flowering, but early as to mean. Table 20, for doubles flowering by August 1, no doubt gives more reliable means ; these means disagree with our scheme only in lot 26 and perhaps lot 22. According to tables 17-20, the fewer-noded check parent of each check row has usually given the earlier progeny. In fact, the agree- ment of parental and progeny differences, throughout the cultures, is decidedly remarkable. It is unfortunate that the later parents were always placed in the east half of the row, especially in view of the fact that there was indication of important differences in soil and L919] Frost : Mutation in Muttliii>l■• n\ 1 1 .,v 3 Number 1 iivs to flowering \ umbei Daj'H to tli.Wrt nil' 1 29.60 1 29 7 147 14 2 32 7 128.57 2 3 21.40 3 5 16 25 34 26 91 94 119.46 4 6 20 27 33 25 105.45 104.08 4 49.57 7 46 30 103.13 8 54 24 105.67 5 6 7 27 . 33 9 11 13 21 22 3 1 30 33 18 91 73 98.85 100.67 10 12 14 21 25 11 34 27 13 91 12 108 30 120.62 8 28 . 50 15 27 17 112.94 16 29 18 118.00 9 10 11 12 13 42 . 56» 17 19 21 23 25 33 36 42 49 55 30 34 25 11 15 100.27 97 . 35 129.36 122.00 136.40 18 20 22 24 26 35 37 45 51 18 21 25 14 13 109 67 117.81 121.76 151.57 121.08 14 47.80 27 46 10 159.40 28 56 8 162.50 1 This parent-lot value does not apply to lot 26, which consists of progeny of WG9-C10 itself. Table 20 Sinnc as table 19, for dotibles flowering by August 7. Row Lot Progeny flowering by Aug. la Lot Progeny flowering by Aug. 1 Number Days to flowering Number Days to flowering 1 1 38 90.26 2 33 91.03 2 3 3 5 36 39 80 . 22 84 . 46 4 6 36 39 80.00 84.10 4 7 36 81.28 8 31 84.32 5 6 7 9 11 13 42 42 37 75.86 77.90 84.32 10 12 14 41 40 35 76.59 80.25 84.80 8 15 46 83.87 16 33 85.21 9 10 11 12 13 17 19 21 23 25 37 42 39 33 32 80.43 78.24 85.95 89.03 88.56 18 20 22 24 26 34 39 30 26 21 84.12 83.85 87.93 88.85 89.05 14 27 35 88.97 28 29 90.28 ■ Only 48 more doubles altogether flowered by November 3, and 25 of these were in the even-numbered lots 20 to 28. 114 University of California Publications in Agricultural Sciences [Vol. 4 probably in the incidence of disease, favoring the plants in the west half. The internode data of 1910, however, show a similar tendency. Small genetic differences are suggested, though it would be remarkable if they were so uniformly present in these plants of a single line of a usually selfed species, descendants of parents and a common grand- parent grown under glass. If such differences exist in the race, conceivably some combination due to crossing might simulate an early mutation. The evidence as a whole, however, does not favor such an origin for our early type ; it is widely divergent from the Snowflake type, and seems to depend on a single main factor difference from Snowflake. Table 21 Cultures of 1912. Ancestry and parental data. Parental data Lot Parent Seeds sown Probable type Days to flowering11 Inter- nodesd 1 WSI-W0I6 Snowflake8 120 . 0 38 15 2 WG9-C10-W6 Early 116.5 33 15 3 WL10-W>2 Snowflake 139 . 5 51 15 4 WL10-W23 Snowflake* 120.5 38 15 5 WSl-W-d Snowflake 141.5 57 15 6 WLIO-W2I4 Snowflake8 126 . 5 38 15 7 WL10-Wo7 Snowflake 145.5 54 15 8 WG9-C10-W8 Early 129 . 5 45 15 9 WS1-W.12 Crenate-leaved '■'' 119.5 34 7r a Suspected before testing of belonging to the early type; first parent also tested in 1910. b A heterozygote between the crenate-leaved and Snowflake types. e Probably open pollinated. d All the parents grew in the same house at the same time. The essential feature of the supplementary cultures of 1912, since no seed of WL10 remained, was a test of two pairs of early and late progeny of WL10 (lots 3 and 4, 6 and 7, table 21), in comparison with two control lots — one (lot 2) from a known early parent, descended from WG9-C10, and one (lot 5) from a late descendent of WS1. Incidentally, WS1-WJ6 and WG9-C10-W8 were retested, and the few available seeds of WS1-W,12 were used to test that phenotypically early parent. The results are given in tables 22 and 23 and chart 4. The very low individual from WS1-W216 came from a very weak embryo, and should be disregarded ; the exceptionally high general range of this lot, which was also visibly behind all others in development, was prob- L919] Frost: Mutaliini m Mullhiola 11.-. Table 22 Cultures of 1912. Number of main-stem internodes below first flower-bearing node. Frequency distributions for singles." f Gen. 1 WSl Wl.'i WL10 WSl WHO WG9 wsi Ann-try -< Gen. 2 Wo 10 CIO \> f22 W23 Wol Wo 14 W27 CIO w2i2 1 Gen. 3 W6 • W8 Internodes 18 it i" 2 1 2t i" i 3 1 1 4 1 1 1 1 3 3 1 2" 1 2 1 It i" 2" i" 19 . 20 21 22 23 24 25 .. 26 27 28 It 29 2 It 30 31 32 3:5 34 35 a See note a to table 5. Table 23 Cultures of 1012. Same as table 22, for doubles.* [ Gen. 1 WSl w 1 ;n \\ 1 III WSl WLIO WG9 WSl Ancestry < Gen. 2 Wo 10 CIO \ V22 W23 Wol Wo, 14 V V27 CIO Wo 12 1 Gen. 3 W6 W8 Internodes 12 1A 1 2" 3t l" 1 1 2 2 1 i" 1 5 1 2" 3 i i" 1 3 1 i" 1' 2 1 2 1 1 2 1 3 2 13 14 15 16 17 .. 18 .. 19 .. 20 .. 21 .. 22 . 23 . It 3 3 2 24 .. 25 .. 1 26 .. 1 27 .. 28 It 29 . 30 .. 31 a See note a to table 5. 116 University of California Publications in Agricultural Sciences [Vol. 4 ably due to some cultural accident, perhaps to an excess of moisture in this row of pots. The lots of plants may seem rather absurdly small for their pur- pose, but the uniformity of development here, with the marked normal divergence in internodes of the types in question, seems to justify a fair degree of confidence. Ten plants here were probably worth fifty in the field. 31 r ingl oubl es ee — -- 30 29 i 1 27 26 25 1 < » ___ 1 "§ 24 c u 22 I — < ► --J 21 1 1 20 19 18 17 16 < 1 W216 I wsi W6 I CIO I WG9 W22 W23 WL10 W2I W2H ' J — WSI WL10 Ancestry W27 W8 W2I2 I I cio wsi I WG9 Chart 4. Cultures of 1912. Internodes: parental values and progeny means, shown as in chart 1. The true parental values are twice those indicated by the ordinate figures, which apply directly to the progeny values. This test, with that of 1910, shows very positively that WS1-W216 was only phenotypically few-noded. Evidently WG9-C10-W8, the parent of field lot 22, really carried the earliness factor, as was some- what doubtfully inferred from the field results; the five progeny of WS1-W212, on the other hand, though from a fewer-noded parent, have values that make the presence of the earliness factor improbable. On the main point at issue the evidence seems satisfactory. Neither of the two very early and few-noded progeny of "WL10 represented 1919] Frost: Mutation in Matthiola 1 17 shows in its progeny any evidence of belonging to the early type; the means are slightlj lower than for the many-noded sibs of these parents, liui far less so than with the parents descended from WG9 CIO. We conclude, thru, thai WG9-CK* was probably a monohybrid, and thai the early bearing gamete entering into its composition was of unknown bill presumably mutative origin. Most of the extracted Late or many-noded parents may now be selected with practical certainty. WG9-C10-C8 and CI i lots 5 and 6 in the 1911F cultures and WG9-C10-M7 and Ms Jots 13 and 14) were genetically very similar to the check parents, as lias already been concluded for two of them from the greenhouse cultures; presumably they wnv pure Snowflake. The data for WG9 CIO itself (lot 26) seem to indicate that the results from the last eight lots are of very doubtful value; still, they show, especially in the original individual records, some evidence of the earliness factor which must be present in part of the individuals. The pour and slow germination of the old seed available may have had an important influence on the result; many of the early embryos may have been non-viable, and the seedlings may have been weaker than those from fresh seed. The 1911 data and observation of the plants in the tield suggest that WG9-C10-AV7, W3, and W10 (lots 23. 24, and 25) are the only remaining extracted late parents, WG9-C10-W5 and W8 (lots 21 and 22) carrying the earliness factor, as the four parents just preceding them in the cultures obviously did. Tables 22 ami 23 con- firm this conclusion for WG9-C10-W8. It is presumably impossible to make a positive separation of the parents homozygous for the presence of the early factor. The green- house data suggest that WG9-C10-M4 was a pure early individual ; the field data (see lot 9) agree, and suggest that WG9-C10-M9 (lot 10) and perhaps WG9-C10-M6 (lot 11) belong in the same class. \V(i!l-C10-C2, C5, and CIO (lots 3, 4. and 40)'- were all evidently heterozygous. Of the parents grown in house W, it would seem that only WG9-C10-W11 (field lot 19) was homozygous early. We have, provisionally, for the available single progeny of WG9-C10: House C House M House W Total Pure early 0 3 14 Hybrid early 3 15 9 Pure late 2 2 3 7 20 12 Statistical data given for the last only for tlie 1910 cultures, not for this field lot. 118 University of California Publications in Agricultural Sciences [Vol. 4 This corresponds well enough with the monohybrid expectation of 5 : 10 : 5 ; in fact, the deviation is just such as would be expected if there was occasional cross pollination of the unprotected flowers of WG9- C10 from Snowflake plants. The large proportion of evidently pure late parents is strong evidence for the monohybrid nature of WG9-C10. The proportions of the two types among the doubles can only be estimated. The 1908 data suggest that 5 of the 10 doubles there reported were early ; this number, with the 13 singles so classed, makes a total of 18 early-type plants out of 30. The ratio is slightly nearer to 1 : 1 than to 3:1, and the former proportion would suggest the peculiar type of inheritance found with the mutant types yet to be described. The evidence of the 1910 distributions, however, shows that the early type largely predominates in the next generation with both singles and doubles, and apparently this is true even when we exclude the progeny of the one parent classed as pure early. The early factor can be positively detected only by progeny tests. No test has shown the presence of this factor elsewhere than in WG9- C10 and part of its descendants. WG9-C10 produced the early and Snowflake types among 20 single progeny nearly in the typical mono- hybrid proportions. Inspection of the double progeny in two genera- tions suggests similar or possibly somewhat lower proportions there. A vicinistic origin for WG9-C10 is improbable. Presumably, then, the early type arose from Snowflake by a single factor mutation, the dominant mutant factor being inherited without special complications. We shall now consider certain apparently mutant types which are characterized by peculiar genetic behavior. 2. THE SMOOTH-LEAVED TYPE This type was first observed in the cultures of 1908 (table 1) and has occurred frequently in later cultures (table 3). It is perhaps the mutant type of most frequent occurrence among progeny of Snow- flake or early parents ; 2410 unselected progeny from house-sown seed of such parents (see table 28) included 28 apparent mutants (14 singles, 11 doubles, and 3 undetermined), a mutation coefficient of 1.16 ± .15 per cent. As grown in the greenhouse at Ithaca, this type (fig. 7, tables 12 and 13) was often many-noded, with correspondingly late flowering. Its most striking peculiarity, shown especially by young seedlings and not evident in the figures, was a lack of buckling between the veins l!M!»j Frost: Mutation in Matthiola 119 pfl H L72 a For a '' In ea d those 0> ■o to to to to £ >« > 35 05 G5 C5 C5 3 CQ ~ f 7 7 7 f 3 -5 - to C5 to 10 ' J> (J-.C~.~~ to C35 to to 35 35 ~P GD a -qooocc^S. 0 T do (isiLi ~ 3 p ^tdo ^ 3-" 03 t/j Ms « B <*d in explar h case rom do > ^OOfDO>- > l—i h— • co o co i— -^ -^ i—i i—i Z-. 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W ^O -.---^.---•H P g" w |_l l-i HOf CO h-1 tO M< o (_■ J W Ct> 13 ; 4£k i-> CO ■*! *; 1-1 ^ O ( i Co B 120 University of California Publications in Agricultural Sciences [Vol. 4 of the leaves, and of general convexity of the upper surface of the leaves. Mature plants developed under favorable conditions in the greenhouse closely resembled Snowflake ; the leaves, however, were noticeably brittle, and the dry capsules so brittle that it was often necessary, as it was not with Snowflake, to shell the seeds individually. Probably the fibrovascular system is in some way defective; Oenothera rubrinervis, which is also brittle (de Vries, 1906, lecture 18), has thin-walled bast fibers. In the field cultures, both at Ithaca (fig. 5) and at Riverside, under conditions less favorable on the whole to the initiation of flowering, this type (fig. 8) differed much more widely from Snowflake. Flower- ing was excessively delayed, and the plants often remained low, with few branches, and rosette-like, with thin, rather narrow leaves. Small brown dead spots, possibly due to excessive transpiration, occurred so frequently on the leaves as to constitute a good diagnostic character for the type. Another peculiarity observed in the field is a reflexed position of the tip of the young leaf when first visible — Snowflake leaves being completely erect from the first. In the 1914 cultures, with better development than in other field cultures, some smooth-leaved plants (figs. 9 and 10) were again more like Snowflake. though later and evidently more leafy. Six smooth-leaved parents have been used in progeny tests, three of these being apparent mutants and three being Ft progeny of two of those mutants. The results are presented in tables 24 and 25 ; these tables require a brief explanation, which will apply also to the similar tables for other types-. For the plan of the new pedigree numbers here used, see "Methods. " The initial plants of a series are designated as the Pl generation in the tables, their progeny as F,, etc. In table 24 the cultures are arranged according to their generations and their pedigree numbers under each generation ; the smooth-leaved parents (P1 or of the Px type) are given first, followed by the extracted Snowflake parents. In table 25 "good germination" indicates that in all lots included (taken as grown, not as summed by parents in table 24) the number of plants determined exceeds 50 per cent of the number of seeds sown, and vice versa; the weighted mean percentages obtained by dividing the total numbers of plants by the respective total numbers of seeds are given for each table in a footnote. All six smooth-leaved parents (tables 24 and 25) gave mixed progeny, part smooth-leaved and part Snowflake. The surprising L919] Frost: Mutation in Miiiihioiu 121 fact is that the parental (smooth-leaved) type appears not in three- fourths of tlic progeny, hut in only about one-fourth. The extracted Snowflake parents tested behave like pure recessives, showing no influence of their smooth-leaved ancestry. Only the aberranl ratio seems inconsistent with the assumption thai the smooth leaved individuals tested were ordinary heterozygous dominants. The relatively weak growth of this type and the apparently poor germination of the seed produced hy il suggest thai normal segregation may be masked by selective elimination. Possibly the smooth-leaved Table 25 Smooth-leaved type: heredity, nummary. Progeny Plants Parents Total examined Smooth-leaved Cultures Seeds Undeter- Deter- mined mined , Number Per cent All smooth- leaved Ithaca 304,0/7 7 156 40 25.6 ± 2.4 All smooth- leaved Riverside 196 I 78 23 (24) 30.8 ±3.4 All smooth- leaved (6) All 500, 277 8 234 63 (64) 27.4 ± 2.0 All Pi smooth- leaved (3) All 244, m 3 115 32 27.8 ± 2.8 All Fi smooth- leaved (3) All 256 5 119 31 (32) 26.9 =*= 2.8 All smooth- Germination leaved good 293, 138 8 187" 55 (56) 29.9 ± 2.2 All smooth- Germinal ion leaved poor 207, 7.9 0 47a 8 17.0 * 4.4 All Snowflake (5, F, and F2) All 208, 50 2 173 • 0 0 ■ Respectively 63.8 and 22.7 per cent of the numbers of seeds planted. factor is lethal when homozygous, as is often the case (Muller, 1918) with dominant mutant factors in DrosopJrila; the data for germi- nation, however, indicate that two-thirds of the mature embryos can hardly belong to the mutant type. We might expect, in view of the weak growth of smooth-leaved plants, that partial elimination of heterozygotes would also occur. That this is the case is suggested, though the numbers are small, by the lower proportion of the mutant type with poor germination (table 25; see also tables 39 and 40) ; it should he noted, however, that transferring the first lot of table 24, the only lot between 50 and 73 per cent, to the "poor" total, makes the percentages practically identical.13 ,:; See also table 2 ami the second paragraph under "Occurrence of Mutants." 122 University of California Publications in Agricultural Sciences [Vol. 4 In connection with the question of lethal action we must consider the inheritance of doubleness of flowers. Snowflake seed regularly gives a mixture of singles and doubles, about 53 per cent being doubles. The doubles, which are totally sterile, are probably (Frost, 1915) pure recessives (dd) for a single-double factor pair. The singles are always heterozygous (Dd) ; crosses with pure single races (Saunders, 1911) show that the approximately 1 : 1 ratio and the failure to produce pure singles, with self pollination, are due to the fact that all the functional pollen is doubleness-carrying (d) . The excess of doubles over 50 per cent has been explained by Miss Saunders (1911) as due to hetero- zygosis of the singles for two linked complementary factors necessary to singleness, and by the present writer (Frost, 1915) as due to lower viability of the "single" gametes or embryos. The absence of func- tional single-carrying pollen is apparently due to a lethal factor acting after separation of the microspore tetrads, since the tetrads themselves appear normal. In any consideration of factors linked with the single-double pair, this semisterility of the pollen must be remembered. For example, any dominant factor completely coupled with D in pollen formation would be totally absent from the functional pollen, and the zygotes produced by selfing would show directly the strength of linkage in the ovules. The available data for the smooth-leaved type (table 24) are far from constituting an adequate test of linkage, but they suggest that the factors are independent. Certainly no high degree of linkage is indicated by the totals, nor do the detailed data suggest that smooth- leavedness is coupled with singleness in some parents and with double- ness in others. We must admit that the peculiar inheritance of this type is not yet positively explained. Evidently larger cultures are needed, and crossing with the Snowflake type and with other commercial varieties; cytological study may also be required. Certain comparisons and speculative possibilities deserve mention, however, especially since the types yet to be discussed furnish additional evidence bearing on them, We may compare the smooth-leaved and double types, as follows : Double Smooth-leaved 1. A rare mutation of pure single 1. Apparently a common mutation ("normal"). of pure Snowflake ("normal"). 2. Eecessive; extracted recessives 2. Apparently dominant ; extracted are sterile mutant-type plants. recessives are fertile normal plants. L9191 Frost: Mutation in MuiiIikiIu 123 BI.E 3. Homozygous dominants nol pro- duced by h\ brids, because func- t ional pollen ca 1 1 issive factor only. i. Recessive (mutant) type the more vigorous. Dominant factor or another fac tor \ i'i\ closely linked with it is incompatible with formation of functional pollen. Recessive type exceeds the ex- pected equality by about .'! per cent among some 7000 indi- vidua Is. Smo i 1 1> 3 I tamo j : a dominants perhaps not produced by hybrids.1 ' i. Recess i\ e I normal i type i he more vigorous; difference much i ea ter t han with single and double. 5. Relation Of dominant factor to \ lability of pollen not yet de- termined. 6. Recessive type exceeds equality by about 215 per cent among 234 individuals. The mosl probable hypothesis for smooth-leavedness, then, would so Ear seem to be essentially the same as for doubleness — complete elimina- tion of the weaker type in pollen formation, and partial elimination in embryo-sac formation. Reciprocal crosses with Snowflake arc obviously necessary; as we shall soon sec, three of the other mutant types have already proved to &< carried bg both eggs and sperms. The case of Oenothera lata (Gates, 1915) suggests the possibility that the smooth-leaved form might arise by reduplication of a chromo- some With ordinary 0. lata the pollen is sterile, but pollination by 0. lamarckiana gives about 15-20 per cent of lata. This deficiency of lata individuals is due, it seems, to a frequent loss of the extra chromosome at meiosis in lata ovules, with a resulting formation of more than 50 per cent of seven-chromosome (lamarckiana) eggs. If the smooth-leaved type originates through duplication of a chromosome, we might suppose that other types of similar heredity involve other pairs of chromosomes. The apparent parallel with O. lata, which Bartletl l 1917) has noted, was long ago suggested by the data, but with at least two or three type's to be described linkage phenomena have seemed to conflict with this interpretation. Possibly different processes have produced different mutant types as with Oenothera; as we have considered types suggestive of O. rubrinervis (early i and of O. lata (smooth-leaved), we may consider next a form which in appearance is remarkably suggestive of O. gigas. it This possibility is only suggested by these cultures, but it becomes highly probable when the data for other types are considered. 124 University of California Publications in Agricultural Sciences [Vol.4 s OS OS .2"° T3.S )NO«'*COtOcC'fM-tNrt tH lO -^f CN CO MOMCqrtiflNONNNH^OiON *t CONO lO^H CDHH31 OD f I lO N N « 'X O CO O CN ffl O N « M 05 , — s >o o — s CO r-- CO i— I G3 CM i— < i-4£M3^CO CO t- T* CO i-l ^NniONHffiNMKH^lfliOOO CM SO CO CM O 00 i-H CO 05 ~f t- CO <— < r— I i— ( »0 'O CM CO > ^J-~^0o ^-O O ©3 cN®J~Jt->Tj<'*) CM Go i-H CM CM "T1 Co CO -*■ -t< co COCOCO'COCOCOCOCOCOCOCOCO CO CO CO CO CO i i i i i I i i i i T i i i i i c3cjcicSojcc!c3drta3c3cScjcJcjrt xooxxcoxxxooxooxooxxoo CMCMCMCMCMCMCMCMCMCMCMC^CMCMCMCM OX X ' CO CO CM (M ft III]." ?C~, CO 00 C5 Cifc S^3 cj 03 c3 ej c3 — 5 00 00 X CO oo — ; s CI CM CM CM CM < -S sc I x 5 CM g L919] Frost: Mutation in Matthiola 125 It should, however, firsl b< aoted that, as will appear Later, phe- nomena of apparenl Linkage in the case of certain other types (crenate, slender, and narrow) suggesl thai these forms commonly arise from Snowflake by segregation rather than 1>.\ immediate mutation. The obvious objection to this hypothesis is the fad thai the apparently mutant t\ pes seem to be dominant to the "normal" or Snowflake t\ pe. Tins objection can be met by assuming the presence o!" dominant in- hibiting fat-tors in the Snowflake parents thai give apparent mutants.15 If the apparenl mutants of the smooth-leaved type arc thus pro- duced by crossing over in a sel of balanced factors, the lethal "balanc- ing" the smooth-leaved factor itself may be distinct from thai winch sterilizes the singleness-carrying pollen. In considering the results here reported, therefore, we must always bear in mind the possible presence of several unidentified lethal factors. If the apparent absence of linkage between the smooth and double factors is not misleading, we must suppose that these factors are carried by different pairs of chromosomes; considerations advanced by Muller (1918, pp. 479-482), however, make it rather probable that the commoner types of apparenl mutants here discussed are all due to factors carried by one pair of chromosomes, the pair containing the factor for doubleness and its normal allelomorph. 3. THE LARGE-LEAVED TYPE A double of this type probably occurred in the 1907 cultures, though its appearance attracted so little attention that no record was made. In the field cultures of 1911 (table 3) several individuals sug- gested a gigas type, though there seemed to be intergradation with Snowflake. In the 1912 cultures a single with leaves "long, rather narrow', thick" developed normally and produced an abundance of good, seed; from this individual (28a) all cultures of this type are descended. This type is stout and coarse throughout, and late to flower. The leaves are strikingly long, thick, and rigid, though as a rule relatively 1S A letter suggesting this explanation was received from Dr. Muller soon after the same idea had been outlined in the "General Discussion" section below. Dr. Muller kindly gave further attention to difficulties at first encountered by the present writer, materially assisting in the formulation of an apparently tenable form of the hypothesis. Since, however, this scheme may seem "far- fetched" and unduly complex, it appears desirable to leave the original discus- sion of the individual types substantially unchanged. When the difficulties encountered by the assumption of frequent true mutation have been more fully presented, the need for some such addition to the scheme will be more evident. 126 University of California Publications in Agricultural Sciences [Vol. 4 narrow ; under unfavorable weather conditions the flowers are often few and defective, while the leaves are resistant and long-lived (fig. 11). Figures 12 and 13 show well the coarse leaves and lateness of well developed large-leaved plants in the 1915-16 cultures, the plants in the latter figure being several weeks the older. The results of the progeny tests are given in tables 26 and 27. All the twenty large-leaved individuals tested have given mixed progeny; the proportion of the mutant type, though much larger than with Table 27 Large-leaved type: heredity. Suihmary. Progeny Plants Parents Total examined Lame-leaved Cultures Seeds8 mined mined Number Per cent 28a 1913,1914, & 1915-16 122 2 73 38 (40) 54.8 ± 3.9 28a-F, (3) 1914 120 2 40 14(19) 47.5 ± 5.3 28a-F, (12) 1915-16 288 2 190 76 (90) 47.4 ± 2.4 28a-F, (4) 1915-16 90 0 54 25 (26) 48.1 =*= 4.6 28a-F, & F2 (19) All 498 4 284 115(135) 47.5 * 2.0 All large-leaved (20) All 620 6 357 153 (175) 49.0 ± 1.8 Large-leaved Germination good 360 3 260b 115(131) 50.4 ± 2.1 Large-leaved Germination poor 260 3 97" 38 (44) 45.4 ± 3.4 Snowflake (1,F,) 1915-16 24 0 15 0 0 a Mainly from unguarded flowers; see table 26. b Respectively 72.2 and 37.3 per cent of the numbers of seeds planted. smooth-leaved, approximates to 50 per cent, not 75 per cent, with little indication of selective elimination with poor germination.10 Here plainly, as with smooth-leaved, no pure mutant-type parent has yet been tested. Since this is also true of the other types, aside from early, that have been somewhat extensively tested, and fifty-three mutant-type parents in all have given Snowflake progeny, it is prob- able that homozygous individuals of these types seldom or never develop. The actual adult ratio with large-leaved is plainly not 2 : 1, but rather 1 : 1, a fact that would suggest absence of the mutant-type factor or factors from the pollen. The small trial cultures started in 1917, however, show that the type is carried by both sperm and eggs. is Since hybrids are of the mutant type in appearance, the possible cross pollination by Snowflake parents could hardly give Snowflake progeny with any pure large-leaved parent. It may, however, have reduced slightly the proportion of large-leaved progeny from heterozygous parents of this type. 1919] Frost: Mutation in Mattliiola 127 If we are dealing here with a type cytologically like Oenothera gigas, or rather the triploid semigigas, abnormal distributions of chromosomes may occur at mciosis, giving unpredictable genetic results. There has been special difficulty, as the numbers of doubtful individuals in table 26 suggest, in separating large-leaved from Snow- flake, though in part of the cases the difference is extreme. Possibly some of the doubtful individuals are genetic intermediates due to irregular meiosis in triploid nuclei; such irregularities in division (Gates, 1915) occur with Oenothera. Both cytological examination and crosses with Snowflake are plainly required. Table 28 Crenate-leaved type: numbers of apparent mutants and association of the type with singleness of flowers. Progeny of Snowflake and early parents Crenate-leaved Culture Total examined" Coefficient of Single Double All mutation 1908 725" 6 1 7 .97 ± .22 1010 338 3 0 3 .89 =*= .32 1911F, seed house- sown 2072 13 3 16 .77 ± .13 All above 3135 22 4 26 .83 ± .11 All unselected 2410 16 3 19 .79 =«= .12 a See note b to table 2. b See note c to table 1. 4. THE CRENATE-LEAVED TYPE This type (tables 1 and 3) is one of the three aberrant types of most frequent occurrence in the cultures here described, having con- st it uted (table 28) about .79 per cent of the progeny of Snowflake and early parents. A large majority of the individuals have been singles, as table 28 shows. If the apparent mutants are produced by some process of segregation of factors, evidently the crenate and single factors were usually coupled in this material; if they are produced by immediate factor mutation, or are individually due to some change in a particular locus, evidently that locus is linked with the single- double locus and the change is more frequent in the single-carrying chromosomes ; and finally, if they are due to reduplication or loss of a chromosome, the apparent linkage remains to be explained. The margins of Snowflake leaves vary from entire or slightly sinuate to coarsely and irregularly dentate or serrate, this character- istic being subject to much environmental modification and varying lL'.S University of California Publications in Agricultural Sciences [Vol. 4 .£ u ^^ o CM CO MH to Ol -c a. 0* -H O O rH CM i-h CM rH CM CM O ■* CO LO ^^ s CM ^-v -— , CD OO rH o o o rf CM CM CO CO — CM, 1^ lo CM •< OS CO rH O 00 OHNOCBHO lO CM 00 i-i CM CM CO OS i— l CO i— 1 rH i-H CD ■o a> CM , , ^ O « lO 5" s to OS oa ^2 CM Tt^ ■s 3 o Q o CM Tt< O h- CO « • — . to Tf »o Is- CM co Q & O-^ O CM CD rH lO CO CM C CO LO 00 CO lO CO m ^ > CO .— . ^^ lO CM OS CD lO CO 5 < — Tf ©S ■* OS CD CO to lo O 10 r}' OS CO CM CO 00 t-H co Ol c' > si CO "* CD CD "* CO ■* r* CO lO O OS 00 a -H CM CO 00 co -a S <1> IOCS lO CM rH O OS CO t- CD 00 OS OS 00 -* a ** c CM CO CM Tj< CO CO CO rH rH rH rH rH O o I- QS rH rH CO 0} U V-T3 d ^ 4) ■g •S.S ©CM CM O -f O O O rH O O O rH CO 00 f-i & E ^ _ * >— i ce -~ 00 T CM >h CM OS io~^ os o »o csToo rHoooooo oo 00 o co CO O Tf CO i-H CM -h t^ lO CM CM CM CM CM CO CO co rH >^, i— I .— < T)H ©* rH rH lO T»H "# g =8 -OS OS ; fa ^fa^fa "8 WEKKrCrClW ^H i: hNWOhOh CO-*COrHrHrHrHrHrHCOrH co" 3 , | r*rir*y-4r*~+r4r*^*r*r* U OS OS OS OS OS OS OS r— QJClOOlfliCjOlOlOlGlCICi ^ r-l rH i— l i— ( i— l i— i en d 0) -tH a a) 03 c3 X o T3 _ crena a-1 a-5 c-1 c-7 c-13 d-8 d-9 d-12 d-15 fa - c s o CM CM CM CM ■-. 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C CN a 00 >-H CN CO CO C I-_ 1 ' i 1 I | (N W lO lO "0 ir - c~ • ," r g cocN >^ £ u II s 'l 1 1 CC c Ph o -3 "3 T3 "0 "0 ~ ;__ O— X3T : * CM CM CM CN b rt o a u 0) « t> «4-l GO o — 0) .- o> en — 0) 0D fee 00 a - en ft c« ,_, — CM is = - 13 — 01 s — o - _ '- +J o — o> +J +3 - o 4^ = r& o O) O) O) a> J3 s 02 fcH o 130 University of California Publications in Agricultural Sciences [Vol. 4 with the position of the leaves on the plant. In the crenate-leaved type this character is much accentuated, as can be seen by comparing figure 14 with figures 1 and 3 ; a warm greenhouse (fig. 14, upper line) gave very marked serration, while a cool greenhouse (lower plant, and also fig. 15) produced leaves much more nearly entire. Under the much more extreme conditions of insolation, temperature, and humidity at Riverside, this type was often much dwarfed in com- parison with Snowflake (figs. 16 and 17; see also fig. 23). In general, growth is weaker than with Snowflake and the stems more slender. Buds and flowers are often produced in great abundance, but the capsules are relatively few, small, and few-seeded. See tables 12 and 13 for internode data. The progeny tests (table 29) show a slightly higher proportion of mutant-type progeny than occurred with smooth-leaved. A striking new feature appears for the first time in these results, the regular presence of linkage, or an association simulating linkage, with the single-double allelomorphs. Further, in all the four apparent mutants tested the crenate factor seems to be coupled with singleness, while among the sixteen Ft and F2 crenate parents there seem to be no crossovers.17 We seem to be justified, for reasons just given, in summing th& progeny as in the tables. Two things appear at once in table 29: (1) there is a great excess of total doubles over the usual 53 pe^r cent; (2) there is a much greater excess of doubles with Snow- flake than of singles with crenate; (3) the supposed double-recessive class (Snowflake double) is about two and one-half times as large as the double-dominant class (crenate single). Table 30 adds two features of special interest. First, there is good evidence of selective elimination with poor germination ; compare the remaining percentages with those for "Ithaca, field," "1915," "P,," and "Germination poor," and see tables 39 and 40; the only excep- tional case is the low percentage for the thirty plants of 1915-16. It would be surprising if the slow and weak growth of the crenate plants did not lead to such a result. Second, there is evidence that the crenate individuals are smaller than Snowflake even before germina- tion. The seeds of crenate parents are less uniform in size than those of Snowflake parents ; small seeds are numerous, and even the larger ones probably weigh decidedly less than normal Snowflake seeds. With five crenate parents included in the cultures of 1913, random if With four of the parents the tests are obviously entirely inadequate; one other, 22d-9, gives no indication of linkage among nineteen progeny. L919] Fids/: Muhilnm in MattMold i::i W 00 - 1 05 — ~4 -. P a i-* 05 oo ►3 n> >>>>>>>>>>>>>> CO o ~ -r"hr---iooonoooo — CD CD fDfflrDrercrc ;. = = ooo = 3333333 ^ 32 r- r*- CD O O <-r-^~-~*^— »~— ^ £jfDO[3330CDCDCD,_^^N._,,^ .-» 3 if ^ "5 4D^i * — -^-^-w to s=» o ESKSB^gggJjJg- 3 3 2 S"^°°o £ o a, gn a, fie a, all 16 side, inati inati o B 3 3 EL e-g S5 T3 7q 3" CO o CO c -ID- Cfl >-> CO ^1 - OeooO«Ooooo»JfflOOWOM^I #*" # * O- " "^ < v 'X ~ ' 00 ts ts *s t* "** .-» ►-.>-, Oo >— — OoOo a a. »_> (-■ p-> I-" a ? Ol-Oi*00*.0JUIOi-'^if'O^ -i | 5" 0 3 '■< 50 a 3 d 5 Oi ^ CO l-1 CT> *■ COtO (-• re lO00**tO^IC0COi->COtOCii~'OiOi P- U^OOS^HWOlOOiHMtOOO 3 5' 2 ST D t— » t— t h-* >— » H- l-1 -J CO to tO 00 tO >— OS C7» 2 C OOt^OlWQiliWi^COOSM-J^ 3 , — 1— •!-» 4iKffl Oi c >— ^J tO 00 tO ^-"l—CO O a> •— '00 00 ®o> oo-^ ■— - i a p rt >->COtOCO^tOCO>-->-'COtOh-CO "' ©COtO-^J**QOOOCOC>tOO>-'CJl -i h-rf-WtOOOfflWWO»]OtO*> H-H-tf-H-H-H-H-H-H-H-H-H-fr o 3 CO^tOi-'tO>-'H-Oi05K-tO*»tO *>-w^J05 to to to > t-H to to to to to tO Hrl to to to ■< ~ 3 -- — — — ■ — a ©©©©© — y. 3"TO W 312.X1 © - • © © © a--1 c to — &. cr c= to Ifs-ss -a -i-i-s^-occrfoo — • — — > 2.-33 .„ 7*© s-jf fc>(6 ft 3 e cn — ™ S' en S' cr h» h-> •> i— > i— > i— > >n. S © — © © © _ H- H- !— ' 1— ' K-» H- 1— * © © © © © © © © © © © o c 4^. CO c» 4*- CO Cn Cn C71 w> Oi wi — CO CO CO CO » - 1 1 1 1 1 1 c © ©©©©©© I to 4* H" i 02 -si -si CO CO o to tO i-> i-1 Cn h- ji tO -14- W C,t rfk H* CO 4- 4- Oi o 2 Q-7 ft a. to to 00 o;hmOhQ Cn 4* H- co ^ — s 5' «_ re" a ■-) © © ©CO CO O © M © to © 0 o o c o> R 4> cr ^r H- CO WM © Cn « 'j. s^ -^ ~-^io 3 /. en -si *■ h- CO to © CO HOM4» i— -si Cn cn ^1 -si CO © H- lO "si Sfi 3' © ~4 4*. H- £>. CO 00 & CO -si to 00 — ' ff C5 l— 1— © *- H- tO 4* CO I— 0 OQ © UO Cn ►— to CO CO © ~I Cn to *- © SO CO tO 4*- o o ^ OS l—1 i— ' i-» Cn 00 tO h- Cn CO jr =0 4*- © Ww © ^- CO O >-^ ^ p ft ?r (6 H- H- tO H- -si CjOmmmWh -si Cn tO CO H-» © H- 4^ -J O ^1 IO © © w © O*-*'!-' > <-• tO h- — • SO • h- h- *> H- 00 cn to to to Cn © *. CO h- tO 4»- to © to -si s—' ^—-o CO 3 ©©O© tO © © © h- H- © ihOMm ■a — 00 CO to _Cn £ ? 134 University of California Publications in Agricultural Sciences [Vol.4 exceed the percentage from selfed parents ; the expected high pro- portion with series 21, however, might well be realized with adequate numbers and good germination. In spite of the small totals, it is very probable that linkage similar to that of the selfed cultures prevails with series 21. Where the crenate type is the pollen parent (series 20) linkage ratios are on our hypothesis impossible, since the eggs are all Snowflake and the sperms all double ; the data, however, though statistically inconclusive, sug- gest that the excess of singles with crenate and of doubles with Snow- flake is greatly reduced but not abolished. Table 33 Hybridization of the Snowflake and crenate-leaved types. Summary. Progeny Cultures Seeds Plants Parents Total examined Crenate Undeter- mined Deter- mined Number Per cent 20aa, bb, & cb 20dc, ed, & ic 20de,ff,gf,gg,&hd All of series 20 21aa, bb, &dd Snowflake par- ents of hybrid* (5) 1913 1914 1915-16 All All All 123 163 120 406 75 271,50 5 0 0 5 1 3 93 14 103 210 25 134 •5(6) 0 6 11 (12) 2(3) (1) 6.5 ± 1.6 0 5.8 =*= 1.5 5.7 * 1.1 12.0 * 4.4 .7 * .5 If we may ignore the doubtful correlation just mentioned a fairly adequate complete hypothesis for the selfing ratio is possible. Assume (1) a gametic ratio19 of 5DC :ldC ADc :5dc, or 16% per cent of crossing over ; (2) non-viability of homozygous crenate (CC) ; (3) low viability of simplex crenate (Cc), eliminating an average of 60 per cent of this type; and (4) coupling of D and C in all parents tested. Evidence has already been presented for assumptions (1), (2), and (3), except as to the intensity of linkage, while (4), as will be seen, is not at all improbable. Random fertilization under these conditions, excepting (3), would give 26DdCc (crenate single) -f- lOddCc (crenate double) -4- 5Ddcc (Snowflake single) -f- 25ddcc (Snowflake double). The other two classes, 5DdCC and lddCC, would be non-viable pure crenate. Adding assumption (3) gives the following comparison: 10 Kepresenting the singleness and doubleness factors by D and d, and the crenate factor and its ''normal" allelomorph by C and c. I '.' in I Frost: Mutation in Matthiola L35 DdCc ddCc Ddco ddcc Theoretical ratio (h = 44.4) 10.4 4 5 25 Calculated for n = 540 L26 4 O CN 1-1 1-1 ^^_ -~. CT !>. CO CO i-" b- IC "* CD co ^# CO ^H 1—1 «s« c CT CT IC t» 00 i- -H T* IC CT CN (^ US IC WH CO CN ■* co CO H '-' ^^ £ .—. CO 03 CN CN t- CO 'O ■* — 3 CD CO CO CO r-< S ^ o Q 00 00 CNt~ 00 i- 00 -* ^t< ■* CN CN c IC iC CO iH iC CN -* co OT 1-1 ^ r3J "m CO CO ic i? c in 1—1 ^ CN O OC CN O^rff O Tfl « B . o 1— 1 T-H CO CT CT ^ IC oS H^ CN CN CN CO H t— CN IC - 0 i— i r-H ^H »— 1 CO >-H CO " IC >> a) X , — ., -~^ in © CN S" £' £ — .£ CT a> -h CN CO M s 3 O Q O V c 00 00 <*tH tH CO -H CN l> CN IC £ '/. 1—1 rt CN " " a; 1 CN CO "* 00 'm 1— 1 T^ CO a ' *—' n n_^ v — ' en CT as o o t(< ** CN IO O CO CO -a S"2 CO ■* CD O Hi- 00 t^ •>* CO 00 IC g -« c 00 00 ICCN CO O ■*># co 0 B as rt CN CN d CJ r_l a>*^ A "£ ^ o ■S.9 o .H OO ^HC i-l O O CN *— ' 1— 1 H 5 s *H © ©3 eg O * c*} ** °o ^*- ^J- O! CO CN -* O OCT CO 9* CO O CN 00 CO *-H t~~ OS CT CN t^ CO CN v(-uo ^n CT T— I CN IO " CO CO rf a Os .« *» CO 5 .=8 ffirt.SW 1 i CO ScxT >-l CO >-H »H CO "* CO IC s „_, 1— t r-H 1-- T— < ^H ^H , , 1—t T— 1 | O 35 CT ^H '"J CT CT CT CT CT CT *4J i-H r-H i-H i-H i— 1 I— 1 '■" ; ct a *"; < o "O ^H CT C CD re, i— 1 l> 'oS ^5 .CO. .O OJ 00 00"o ^1 1 « .- G co co <^ 03 Qu h «j i i ^5 +J cu ^3 "0 Cl, 03 X! _ __ ccX! ^3 — — i ifl CN CN < ic ic ic iC CN CN CN CN •< CN CN< t-- »oio*o * io co -h in c CO * CN CO * IM CN CO CO *CNCO>OCN 0) ■H-H-H-H -H -H -H -H -H -H -H -H -H -H ID P-< * t^ co co inooiooco * co oo cn c/. io io h cc o m ro w io ro cocoococo (M ^h cn * * * co cm co -tramtON CP "C3 a cp 55 CP s 3 z 00 CO* OCOOIHCO 0110**10 i-H (N CO i-HiOI-^CNCO CN lO CN CN lO *00CNO5 00t~O2t~^ * t^ CN O © CO ^H CN CN O ^H * CO * CO CM * CN * LO c 5 T) cp c 1 * * CN CO * * i-H CO * 00 iHiOOOOOO 'c (^ lO CN b- * CN * 00 © U5«NMO V i-H rt CN H ^H CN a a> 1 Q * c M CP ■a cp « c tj o 0 1 Cp cp -a c rHOi-Hi— lOO^CN*— I"— t OHHHr- 1 ©J hcsC T cp CD oo oooo^- vt- s$ co ^J. -|)ly the scheme worked oul for crenate, difficulties appear a1 once. First, the scarcity of Snowflake singles would indicate much closer Linkage than with crenate, while the relative abundance of slender doubles apparentlj contradicts this supposition. Second, the in- adequate results from crossing with Snowflake (table :i(>) surest that the sperms carrj the supposedly crossover slender factor al leasl as often as do the eggs. While crenate as pollen parent gives results agreeing tolerably with the hypothesis, slender gives results differing from these in the w rrong direction. No .Ion I it. however, the disagreements can be over emphasized. Both crenate and slender as seed parent seem to give the expected relations between singles and doubles, and series '-,;! also does this with the Snowflake progeny. Obviously the functional sperms and eggs of these mutant-type parents exhibit different ratios between types, and the peculiar results in other respects with slender may be related to the addeil complication suggested above. The astonishing feature of the data, of course, is the great excess of single slender over double slender in series 2'-] — an excess which suggests an actual significant excess of singles in the totals of all types given by this cross — while with selfed slender there is a great total deficiency of singles. We may at least feel confident that the modifications of the single-double ratio, with this type and with crenate, are due to lethal action which also affects the proportions of viable slender and crenate gametes or zygotes. If differential viability before germination is an important factor with these types, very probably it differs according as Snowflake or the mutant type is the seed parent, and according to the parental environment. Tn other words, partial selective elimination during seed formation may vary with the environment of the embryos, accord- ing as this environment is affected by either the genetic constitution or the external environment of the seed parent. I'ntil such uncer- tainties are eliminated, we are hardly justified in ruling out, for the types discussed, the probability that regular segregation and (in the last two cases* true linkage are concerned in these phenomena. In fact, the definite differences in ratios between reciprocal crosses and between at least one of the crosses and selling encourage further attempts at satisfactory factorial analysis. 140 University of California Publications in Agricultural Sciences [Vol.4 <3 Si hi t* CO 5 G ^?cc OS ONN^H rH a .-v KO "* ^5 CO CO Oi CM < CM CO >0 CO GO O a> ih CO00 i-H CN ^d ^ o3 ^ USO CO 03 3 K3 CO O CM S 3 O Q »-l O CO'* CO 05 CO i-l CO Tjl i-l i-l o "bl MC a CO ** co i-i CO CM OS O i-l co co "d CO OS >o CO -* O •O CM CM f CO *o -H -H -H -H -K c l> CD CO »C -rf 03 s 5 u PH CN CM CN M — cO >. -~ CM CN c c o u s £ O IOC lO CN CM X CM CN Put 3 3 O Q 3S lr-i i-lO O c fefl : rfi O -* CM CM C CM CM CO 1 •"S 13 ( ia CO CO COCN i-H !>• gj < CM OS CM CM CM 0 c is 1-H QJ 03 < it) H ( 9 -H OO i-l O ^H en -a -* lO o OSCO (-- CD i-l CI CM >C "O CO CO i-l rt CM ^H c o co co cc-r- m S co « X! efl CM o< 03 e3 ej O c3. S J. -! : — t; — co co eo-3 Tft cj CM CM CM < CI CC S SO C ej 0J ,Q rg O ■> A 0> rr) > ?H ^ CJ a> oj -5 — 33 ■H SB© C3 — ' »* « ^3 « >-. g ^J g £ z 3 CJ CJ o a <« , t) « 3 .2 EH ' CO .2 ^ • S », es oj M cj O *» ^ S -e » a. 3 Si" a Ch >w i.i: \vi:i» TYPE As table 37 indicates, this type c petes with crenate for second place in frequency of occurrence in the [thaca cultures; in fact, when only the Btrictly unselected cultures are considered the percentage is very close to thai for smooth-leaved. A feature of special interesl is the apparenl association of the mutanl type with doubleness. In ;i cool greenhouse this type (fig. 22) varied from exceptionally late and many-noded to ordinary in both characters. The leaves (see also 6g. L8) were typically narrow, rather strictly entire, often rolled backward or twisted, and typically more ascending than those of Table 37 Narrow leaved type. Numbers of appart nt mutants and association of the type with doublt ness of flout rs. Progeny of Snowflake and early parents Culture Total examined11 Narrow-leaved Single Double All Cocffieient of mutation I'M Is L910 11*11 1". house-sown A.1! above All ini-i'lic. ,| 725b 338 2072 3135 •_• 1 1 ( I 0 1 7 8 8 2 4 12 18 16 2 6 20 28 26 .28 ± .26 1.78 ± .38 .97 * .15 .89 * .12 1.08 ± .14 * See note b to table 2. b See note C to table 1. Snowflake. The apex of the leaf is often more acute than with Snow- flake, and many leaves are mucronate or at least end in a sharp, rigid tip. A striking characteristic is the narrowness of the sepals, resulting in frequent early separation at the edges, partially exposing the petals in immature buds. Under the less favorable field conditions the plants often remain long as dwarf rosettes, and flower late and feebly if at all. Figures 2:5 and 24 show comparatively well developed plants in the field. The type is on the whole very distinct in the field, though there has been some question whether a greenhouse plant such as thai in figure 18 is genetically different from those with short and rigid leaves 'figs. 22 and 24); the very great variability in leaf form due to external conditions makes such a question very difficult without extensive progeny tests. It is now (1918) probable that narrow-dark (p. 143) was not distinguished from narrow in the greenhouse. 142 University of California Publications in Agricultural Sciences [Vol. 4 En u 4) qj iH i-l d o CO ^^ _^ ^ O (M* cn CN c ^3 3 o o a Q oc co ■* r— I CO CO 1-1 " 1—t ^ ^ ^ ^ a> O^-v CO t^H CN "m i-l CO rH c m CC lO CO o cs i— H tH (B_, o3 e^. o TlH T1 a n- : CO *C CI o d ; -h 41 ■H 4) 0C 3 < ai o ** OS s o> Ph (M >> , c*. o-« £ to >. 03 C 1-H CO "* b-^ T C bt c3 lO CI o Ph £ c— £ 3 3 O Q i-H ~ l-_*l OjTJ o OC o o •o H ^■3 « £ a *c *<*■ ■c OS OC 0) rtlO co~ CO CS 02 t* tt< CO CO co I-H ■** OS OS tn T— ( 1-H OS 3 =a =8 i-H 43 i—i co 09 33a 33b All narro leaved "Short s ~E ij CO Ch Ph^ i-H -— 03 > c t~ o C-4 o =1 -t a 4^ - 1 > *- o ai ij w rO a> Ed <\i 4H 4J °H ? O o CD a cS X Z X / oSt : Mnl, ilmn in Malll, ml, i 1 13 The i'i'w singles have produced Few seeds, and these were highly variable in size The capsule often has a defective septum, more or less of the distal portion being absent. Germination was poor in the small cultures secured (table 38, upper pari . with only 10.8 per cenl of the tnutanl t.\ pe among the progeny. This case agrees in mosl respects with those previously discussed, but adds one poinl of interesl in th currence of appareul coupling of mutanl type with doubleness rather than singleness. Seed appears to be less abundant and less well developed than with any of the pre- ceding mutanl types, facts probably significant in relation to the low percentage of narrow progeny from narrow parents, though the large probable error of the percentage must lie considered. 7. MISCELLANEOUS A.BEBEANT TYPES As pari of the aberrant individuals occurring in the greenhouse were cither doubles or singles that produced no seed, while practically no seed was produced by any plants in the field at Ethaca or by even some of the commoner mutant types at Riverside the opportunity for progeny tests lias been almost entirely limited to the types SO far discussed. The narrow-dark-leaved type (table 3) was common and distinct in the field at Ithaca, where it constituted about .48 per cent of the 2072 plants from house-sown seed, and has been readily identified in several cases at Riverside. It was not distinguished in the green house cultures, but was very probably included under narrow-leaved. Possibly a single described as "small-convexdeaved" belonged to this type, though two Geld plants were given this name as distinct from narrow-dark; according to a photograph (fig. 25, second plant from left), another greenhouse plant (a double) may have been similar to narrow-dark-leaved. The narrow-dark-leaved type (figs. 26 and 27) has narrow dark-green leaves, strongly convex upward, and evidently tends to compactness of growth and lateness of flowering; under field conditions it seems decidedly more like Snowflake than like narrow- leaved. The 44 progeny (table 38) secured from the greenhouse single mentionad above included 2 (4) narrow-dark-leaved individuals and '■\ (5) other plants not Snowflake (the last including two smooth, one large, one slender, and one semicrenate), besides five undetermined plants. Plainly flic type of the parent is still in doubt. 144 University of California Publications in Agricultural Sciences [Vol. 4 Another very different greenhouse plant, described as "stout dwarf" (fig. 25, third from left), gave among 29 progeny (table 38) 5 (7) individuals evidently not Snowflake, which may have been narrow-dark or may have belonged to another type that was somewhat similar under the conditions of the tests. The parent resembled Snowflake except in its short internodes and short, stout capsules. Four other plants suspected of mutation apparently entirely failed to repeat their type in their progeny, perhaps because of the smallness of the house cultures. One of these was the plant, much branched for the warm greenhouse, third from the right in figure 18 ; another was a very late plant with a remarkably large number of main-stem leaves; the others were a plant with unusually small flowers and one with some of the leaves somewhat spatulate. Possibly all of these were Snowflake, though the second, which gave poor germination, probably was not. All these four plants have been included as Snowflake parents for tables showing numbers of apparent mutants. The small-smooth-leaved type is well shown in figure 25 (first and fifth from the left) . It is the smallest and weakest of the fairly common and definitely identified types ; it has small, very smooth leaves, and is late in blooming. The two plants shown were both singles, but they set no seed. The* semicrenate-leaved type (table 3) differed slightly but appar- ently definitely from Snowflake, somewhat resembling crenate-leaved in leaf form. The one "pointed-crenate-leaved" plant of table 3 may have been crenate-leaved. The "compact" and "curly-leaved" plants of this table have not been identified with any aberrant types in other cultures. With the remaining six types of table 3 all the individuals have been questioned as possibly Snowflake ; it is now practically cer- tain that some of those in the second, third, and fourth groups belonged to the large-leaved type since studied, but the apparent inter- gradation with Snowflake makes any attempt at a definite reclassi- fication from the records a matter of doubtful value. The second plant from the right in figure 25 was remarkable for its short stem and few but large leaves. Several other more or less exceptional individuals have appeared in the cultures, especially among some plants with abnormal cotyledons, selected from large numbers of greenhouse seedlings in the 1908 cultures, which were examined for syncotyledony. Some of these were very weak plants which finally died without flowering. L919] -' : Mutation in Matthiola I 15 The fluctuations in habit, Leaf Form, etc., within the type are such thai the determination of familiar types is often a matter of some uncertainty, as is shown by data thai have been presented. It maj well be thai among the doubtful types are included several definite bu1 comparative rare mutanl tonus, which occurred too infrequently to afford adequate material for positive classification. v S.iMK 1'Koi: \l:||.|TES OF RANDOM SAMPLING For compactness of presentation and convenience of comparison the materia] in tallies 39 and 40. to winch some incidental references have already been made, is collected heri' rather than scattered through the discussions of the various types concerned. Some statements as to methods are also necessary in connection with each of the topics here treated. First, it should be noted that the percentages previously given have regularly been accompanied by the probable errors of simple sampling. These probable errors have been calculated by the formula E per cent = .6744898 \23l . where p is the percentage of the mutant n type "successes"), q is 1 — p, and n is the size of the sample (the cumber of plants concerned). In the heredity tables for each type, p has uniformly been taken as the percentage of the total of the lots compared, or p0. For the "mutation coefficient" the percentage of the grand total of unselected house sown lots has regularly been used. Evidently the few selected progeny included in tables 1, 28. and 37 should be omitted. All the percentages here an; so low that the probable errors deserve little confidence, even though n is usually fairly large. The rather dose agreement of the percentages of all apparent mutants in the three distinct lots of unselected house-sown cultures suggests that they represent fairly well the population value for the potentialities of the seeds; and even if the mean percentage of the total of the lots for the main comparisons is actually nearer, it is safer to use the larger probable errors resulting from the method here employed. Furthermore strict use of p0 would sometimes require several slightly ditl'ereni probable errors for the same percentage, for use in different comparisons in the same table. 146 University of California Publications in Agricultural Sciences [Vol.4 If the probable error of the difference of any two percentages in the same table is to be obtained, therefore, formulae corresponding to those given by Yule (1911, pp. 264-267) are applicable. Now, it is possible in some of these cases to calculate the actual standard deviation of the percentage in subsamples which make up an aggregate sample. Table 39 gives such actual standard deviations, in comparison with the corresponding theoretical or expected standard deviations given by . v per rent ^> — • Table 39 Standard deviations of percentages of mutant types. Values derived from compared with values expressing the actual variability of subsamples. pq, N / n V Standard deviation of samples of mean size n Type of parent and grouping of progeny Actual Theoretical.J-^-*- n -3 Difference Ea- Smooth-leaved type: All lots by parentage All lots as grown 234 234 6 12 39.0 19.5 27.35 27 . 35 7.5 11.3 7.4 ± 11.0 ± 1.4 1.5 + .1 + .2 Germination good 187 7 26.7 29.95 10.9 / 9.4 ± I 9.2a 1.7 + .9 Germination poor 47 5 9.4 17.02 5.2 114.9 ± \17.6 3.2 - 3.0 Large-leaved type: All lots by parentage All lots as grown 357 357 20 22 17.85 16.2 49.02 49.02 10.7 10.9 13.0 ± 13.7 ± 1.4 1.4 - 1.6 - 2.0 Germination good 260 14 18.6 50.38 11.3 / 12.7 ± I 12.7 1.6 - .9 Germination poor 97 8 12.1 45.36 8.7 f 16.5 ± \16.6 2.8 - 2.8 Crenate-leaved type: All lots by parentage All lots as grown 633 633 20 28 31.65 22.6 29.86 29.86 10.6 12.5 8.6 ± 10.3 ± .9 .9 + 2.2 + 2.4 Germination good 549 20 27 . 45 32.42 10.7 ( 9.5 ± \ 9.3 1.0 + 1.2 Germination poor 84 8 10.5 13.10 10.5 f 12 . 3 ± 116.7 2.1 - .9 Seed-size test, smaller seeds 73 5 14.6 65. ?5 13.2 f 13.9 ± 1 13 . 8 3.0 - .2 Same, larger seeds 193 5 38.6 20.21 9.4 f 6.7 ± \ 7.9 1 4 + 1.9 Same, all seeds, by parentage Same, all seeds, as grown Slender type: All lots by parentage All lots as grown Germination good 266 266 243 243 165 5 10 8 13 7 53.2 26.6 30.4 18.7 23 . 6 32.71 32.71 32.51 32.51 33.33 10.3 22.9 17.5 19.7 14.9 6.6 ± 9.7 ± 9.0 ± 11.8 ± f 10.4 ± 1 10.3 1.4 1.5 1.5 1.6 1.9 + 2.6 + 8.8 + 5.7 + 4.9 + 2.4 Germination poor 78 6 13 0 30.77 27.2 f 14 6 ± \ 14.8 2.8 + 4.5 Parents "extreme" 38 3 12 7 63 16 14.4 J 15 . 5 ± 115.1 4.3 - .3 Parents ' ' ordinary Narrow-leaved type: All lots as grown 205 37 10 3 20.5 12.3 26.83 10.81 14.7 8.1 J 10.6 ± 111. 2 10.2 ± 1.6 2.8 + 2.6 - .75 ■ The second values for some oases in this column are derived from p„ (see text). / ost: Mutation in Matthiola I 17 For example, table -~ gives the percentage of large-leaved plants among the ;!~>7 progeny of the 20 Large leaved parents as 19.0 1 1.8 per cent. This probable error is given by .6744898 \ . where ft p = 49.0 per cent, q 51.0 per cent, and n 357. These ::">7 progeny, as table 39 indicates, came from 20 parents which contributed ;ni average of 17 s". progenj each, and the actual standard deviation of the percentage in these 20 sibships was 10.7 per cent. Obviously the expected standard deviation of simple sampling Eor comparison must represenl samples ool of 357 plants each l>ut of 17.85 plants each. Now a percentage is obviously a mean of values all eitherOor l . Since "Student" (1908) lias shown thai the theoretical standard deviation of the mean in samples is given more exactly by "■ vartat.' .. 1 fvartate than nv (7, y/n — 3 \ a (the value Eor the normal curve conventionally used Eor the probable <']■]■<']■ of the mean) and since n, the mean size of sample, is small enough to make t|lt. direction a matter of considerable importance, V n — 3 is here u^t^l. Since o- variate = VPrJ- Wf> have o-mcan = y_ P^ . where Ti = 17.8"). This gives a theoretical standard devia- // — 3 tion of 13".0 per cent.22 It is true (Yule, L911, p. 260) thai the ordinary method of calcu- lation of the actual standard deviation is not satisfactory for means when the samples vary in size. A method has been used, however, which obviates this difficulty, so thai comparison with the results given by pq .... . . is strictly legitimate. Each squared percentage deviation £ 71 — 3 has been weighted by multiplying it by the number of individual plants which it represents, and the summation of squared deviations has then Keen divided, not by 2/, the number of samples, but by 2/ X 7v. the number of samples multiplied by the mean weight or average size of sample i in other words, by A", the total number of individuals 1 .2S -- In the calculations for table 39 p has been taken as the percentage given in this table, to two decimal places, while with all other numbers employed in calculation, including 77 — 3, three or more decimal places have been used as nee. led. Algebraic proof of the correctness of the method has kindly 1 i furnished by Frank L. Griffin, Professor of Mathematics, Weed College, Portland, Oregon. If it develops that this rather obvious device has not been suggested for the purpose, it is to be presented elsewhere with the mathematical proof. When the \:niates are not grouped in classes the calculation is substantially as easy as without weighting, while the theoretical value is found with much less work than by the method given by Yule (1911, p. -*50), which requires the harmonic mean of the sample sizes. 148 University of California Publications in Agricultural Sciences [Vol.4 e a 5 <*-. tol o 50 a ca s - 8 CC o Oj s ■a (M l>- t-H lO O CO O 1-H •3 « oo cm mo c>r-^^H co OJ t-^ d en V 0 'C -H Ci O: OJ C 3 .Si '5 to 03 ■-H (M n c rt co" cc ■^ 1-1 «.2 w i-H CN 3j= ■a 0. 5 •a °-s O eg L o c S c ^o c -- *-. a> *+j a c u o co 3 a > t^ CM ¥ ^ 13 CO i- *f" Cv ■* oc it; i- c -3 o co -^r iocc ooioo c o b- O oo cm -* co ono c CM »o o V ID ON -"* O OKO C O iC o "a, H CM NO O CO O C o o o £ 03 03 .£ >> s. O 3 03 p C o (5 03 '> 1—1 1—1 1— 1 o •O 03 T-H CM 03 0. 3g 93 T3 a£ T3 Is o S"5 60 J? a a c o 3 S 2 S3 6 > > .2 ■ s so I^cbI^cb r1 0) g 0j •— . £ 3 o cu cu T3 O a - > £ M o a> II 5 II -r >. II bes • 3 t CO ■o II P ood vs. poo With sm With lar Same, /„ With ore Same, /„ With sle With all Same. f„ CO ^ • _c 6 CO parents : With Same, >■►- g O K! - CB co CO O o ^5 II s Ph ^~ o — o> CO 1 ^ 4-» CO ^ ^3 CD 03 t> > c£ CS o c a 01 z V t3 a a > crt 01 T3 cu 01) — - c« CCS ' ' T3 »H fl c al the number of cultural groups or with the first line for each type) the number of parents (/), the average size of the groups of progeny (w), and the mean per- centage of the mutant type (p). This serves as a summary of some of the must important statistical data already presented relating to the inheritance of these types, and also shows the basis of tlie remain- ing pari of this table and of table 40. For comparison of actual and theoretical standard deviations the theoretical value has been calculated from the actual percentage as given in this table. For comparison of means (table 40) the percentage of the corresponding total (p0) has also been used, tins theoretical standard deviation being the second in the table in the cases where the two values are not identical. Since small changes in a percentage have little effect on its theoretical standard deviation, we are fairly well justified in taking the latter, as calculated from the actual percentage in each case, to be the "population" value. Consequently, the difference between the theoretical and actual standard deviations has been expressed in each case as a multiple of the probable error of the theoretical value. Aside from the last line for crenate-leaved, where there is an obvious artificial reason for high variability, there is no very significant difference except with slender. In Ibis case, the deviation of 5.7 times the probable error (line 1) is probably largely due to the genetic differentiation of "extreme" and "ordinary" parents suggested by their appearance and by the wide difference in the heredity per- centages: the differences become moderate when the progeny of the two classes of parents are separated. 150 University of California Publications in Agricultural Sciences [Vol.4 In the two cases (smooth-leaved and crenate-leaved types) where the percentages of mutant types differ greatly with good and poor germination, separation according to germination gives a mean value of the standard deviation decidedly lower than the value for all lots taken together. In the case of the large-leaved type there is little change, while the considerable reduction with the slender type is probably due to unequal separation of lots from parents genetically different. Table 40 shows the simple-sampling probability of the most striking differences of heredity percentages, aside from the characteristic differences between different types. "Student's" (1917) table of probabilities of mean deviations with small samples is used, with interpolation by second differences. "Where the standard deviation of the difference is required it is found from the theoretical values given in table 39 by the formula (Yule. 1911, pp. 264-265) V difference Vcrl ~~H a ■ J Ppgo Po Qo ny — ■ 3 n2 — 3 when one statistical population is assumed (table 40, columns 2 and 3). When two populations are assumed (table 40, columns 4 and 5) the cor- responding formula using p1q1 and p2q2 is employed. In the one case where this is possible (the seed-size test), it is also calculated from the' actual differences of the pairs of percentages in the separate tests, each difference being weighted with the total number of progeny from the parent concerned. Where two values of / (the n of "Student's" table) are involved, the smaller is taken, giving understatements of the probabilities involved ; in the two cases where the difference is more than 2, the values are recalculated, with / as the nearest smaller integer to the geometric mean of the two actual numbers (that is with /0 = V/i/2)- In the case where the probabilities of four devia- tions all in the same direction are combined, the four chances of occurrence are multiplied together; that is, if the J(l + o) of "Student's" table is P, and 1 — P is F, then F1.2.^.^ = F1-F2-F^Fi. "Student" (1908, p. 1) says, "The usual method of determining the probability that the mean of the popidation lies within a given distance of the mean of the sample, is to assume a normal distribution about the mean of the sample . . . ." When this is done with a differ- ence of means, it is at once evident that only half of the chances of deviations as great as the distance of the given difference from zero difference lie below zero difference ; the other half of the chances of 1919] / oat: kfutatton in Matthiolo 15] such deviations Lie in the opposite direction and represenl positive differences still greater than the sample difference. In other words, it' the implications of a sample difference are to be given full weight, this difference musl be considered the most probabh valut of the theoretical "true" difference between two assumed distinct statistical populations. In the presenl case we wish to know the probability thai the "true" or theoretical population means differ in the same srn.sc as the observed sample means. Tliis involves calculation of the proba bility of deviations in one direction (beyond zero difference) from the .sample difference. It' the sample difference of means is considered as positive, then the negative "tail" of the theoretical frequencj curve of sample differe IS I this curve he in.".' centered at thi' observed sample difference) must be compared with the rest of the curve. The positive portion of the curve the \ (1 +a)"4 of the tables, then gives the chances favoring the hypothesis that the sample means truly Pi presenl the population means. The odds in favor of the hypothesis are therefore <_ii\ en bj t he formula 0 =*(! + «> or i±li u* i(i — «) 01 J — K Values calculated from this formula are given in columns 4 and ."> of table 40. When other considerate as than the sample evidence arc to be taken as determinirig the most probable value of the "true" mean, the ease is different. For example, if the probability that our sample per- centages are mere sampling deviations from some theoretical Mendelian value were in question, that theoretical value must be taken as the population mean and only the magnitude of the deviations must be considered. When ,-i difference of means is considered from this latter stand- point, it is assumed that, the two samples come from one statistical population, and hence that zero is the most probable value of the population difference. If we choose to assume that the most probable value of the population difference in our cases is zero, we must calculate the odds against a deviation of the observed amount in either direction from zero difference. The formula for these odds is 2X{(1- a) 1 — a -■> The whole area of the frequency curve is taken as unity, ami a is the area enclosed by any given deviation in both directions from the mean. 152 University of California Publications in Agricultural Sciences \ Vol. 4 Values from this formula are given in columns 2 and 3 of table 40; their magnitude in three cases, however, and the uniform agreement of the direction of difference with the expectation from biological evidence which has been discussed, weigh heavily in each test against the assumption of random sampling from a single statistical population. It does not appear necessary, however, thus to weigh the evidence in detail before deciding which formula is suited to the case. There is no evident theoretical value from which these percentages are reasonably likely to be sampling deviations. This being the case, and granting such general possibilities as that of differential viability, it seems most reasonable to use the former (0X) formula. That is, we should give full weight to the implications of a sample deviation unless there is some definite reason for assuming that some other value better represents the mean of the theoretical statistical population. It must be remembered that the actual probabilities of sampling deviations do not necessarily correspond closely with the probabilities of random, sampling. With the material in table 40, however, aside from the germination comparison in the case of the slender type, table 39 suggests a fair agreement with the conditions of random sampling. The actual standard deviations of the subsamples do not in general differ widely from the corresponding theoretical values, and the differences are negative about as often as positive. The hypothesis of selective elimination with poor germination is strongly sustained (table 40), although only one difference (with the crenate type) has much statistical significance when considered alone. If we may multiply together the members of the four ratios in column 3 of the table, the combined odds (using the /„ values) are 130:1 against occurrence of these four deviations as accidents of simple sampling, when magnitude of deviation alone is considered. If direction of deviation alone is considered the random chance of these four deviations all in the same direction is obviously (-J)4, or the odds favoring the elimination hypothesis are 15:1. Combination of these two chances indicates a high probability for the hypothesis. When the two-population formula is used in calculating the standard devia- tion of the difference (columns 4 and 5) the value of P is consider- ably reduced in some cases, and the combined odds obtained from Fx ■ F., • -F3 ■ F4 are very high. Evidently the best single expression of the simple-sampling odds, though possibly somewhat too high, is the value given last in column 5, or 123,093:1. With the seed-size test of crenate the odds are 499 : 1 with the theoretical standard deviation of the difference, or 1666 : 1 with the 1919J / roat: Mutation vn Matthiola ]•">•'! actual standard deviation. When the relativelj small size and weals growth of erenate seedlings are also taken into account, the relatively small average size of erenate embryos may be considered to be demonstrated beyond reasonable doubt. With "extreme" and "ordinary" slender parents the odds de cide'dly favor the hypothesis of genetic differentiation of parents, in spite of ilif small numbers involved. We must remember thai definite statistical differentiation of lots of progeny grown under uniform con ditions does uo1 necessarily demonstrate gt >tt tie differei s differences in outpul of gametes between the parents; in this case, however, the difference in the appearance of the parents and in the single-double ratio ; ing the progem also surest genetic differentiation. GE X K UAL 1 )ISCUSSIOXL'"' It might be argued with some plausibility that the available evidence hardly justifies conventional factorial analysis, or at least that the data indicate strongly the presence of marked factorial incon- stancy. The aberrant types occur in very small proportions among the progeny of selfed Snowflake parents, in much larger proportions from "mutant-type*' parents, and in intermediate proportions from crosses with Snowflake. It might he supposed that the Snowflake type has a slight tendency to mutate to the other types, and that these have a much mure marked tendency to mutate hack to Snowflake. Various considerations, however, especially the occurrence of apparently regular linkage phenomena, seem to favor the general form of hypothesis which has been presented. As we have seen, it is well known from the behavior of various factors that the typical Mendelian mechanism is present in Matthiola. It cannol be argued here, as sometimes with Oenothera, thai the genetic behavior of the genus or species is fundamentally non- Mendelian. Since the .Mendelian mechanism is demonstrably present, and .Midler's 1918) work on beaded wings in Drosophila seems to establish the adequacy of this mechanism in a closely parallel ease. surely conventional factorial analysis should be carried as far as pos- sible; in fact i .Mu Her. 1018, p. 42:?), a .Mendelian explanation should not be abandoned for anything short of positively contradictory evidence. -'Mutter's (1918) complete report on the beaded-wing case in Drosophila appeared several months after the present paper had gone to the publisher. Certain conclusions given below, very similar to Muller's but not credited to him, were therefore reached independently. 154 University of California Publications in Agricultural Sciences [Vol. 4 In the Drosophila case just mentioned, the "principal" factor for the character in question is "dominant for its visible effect and recessive for a lethal effect," so that no pure beaded individuals appear among the progeny of beaded. The original race regularly gave progeny partly heterozygous beaded and partly homozygous normal, while after a long period of selection a true-breeding beaded race appeared. This latter form, it proved, fails to give normals not because of being duplex for beaded — it is still simplex — but because of its possession of another factor, known only by its lethal effect when homozygous, which is carried by the chromosome bearing the normal allelomorph of the factor for beaded. The locus of this reces- sive lethal factor gives in general about 10 per cent of crossovers with the locus of beaded, but in this case, because of the presence of a factor "which almost entirely prevents crossing over" between the loci of the two lethal factors, viable non-beaded zygotes are very rarely produced. Thus every zygote receiving either two beaded-carrying chromosomes or two non-beaded-carrying chromosomes of the pair concerned fails to develop, and all the insects produced are necessarily heterozygous for both lethal factors. A point of special interest in this case is the fact that by certain crosses individuals can be produced which give certain types among their progeny in very small percentages. Muller suggests that part at least of the supposed mutants of Oenothera may be due to crossing over between chromosomes carrying lethal factors, by which certain recessive factors are permitted to come to expression in viable zygotes. For the inheritance of doubleness of flowers in Matthiola he gives a "balanced-factor" explanation essentially identical with mine (Frost, 1915). There seems to be little reason to doubt that the differential factors for these aberrant Matthiola types have originated by mutation. On the analogy of Drosophila we might expect that the true mutations would be relatively rare, and that most of the apparent mutants, in cases where they appear frequently, would be due to segregation, appearing as the result of crossing over in chromosomes carrying balanced lethal factors. The evidence seems to indicate, however, that the differential factors for the mutant types at all extensively studied are dominant for their visible effects and usually (probably imper- fectly) recessive for a lethal effect, the mutant factors thus being genetically similar to the factor for beaded wings in Drosophila. This would seem to imply the occurrence of certain mutations in pro- 1919] / roat: Mutation in Matthiola L5S portions ;is high as aboul 1 per cent, and a general mutation eoefficienl of perhaps 1.5 per cent, while the onlj Mendelian alternative would seem to be some more complex scheme whose satisfactory formulation mighl require much more extensive hybridization data. To be more specific : (1) these tj pes are do1 single recessives, since they are ool homozygous bu1 split into the mutanl and "normal" types; (2) they ;ire nut simple cases of multiple recessives, as has been proposed by Beriberi Nilsson (1915) for Oenothera mutations. since what is on thai hypothesis the full dominanl t,\ pe reappears with selfing; (3) if these types are single dominants, as they appear to be, they cannol (barring the action of inhibiting factors) arise Erom the pure recessive "normal" or Snowflake type by segregation, hut only by immediate mutation; (4) they are not simple cases of comple- mentary dominant factors, since they occur among the progeny of selfed parents. We mighl assume that a "mutant" type depends on two pairs of factors, one homozygous and the other heterozygous, while both pairs are heterozygous in the "mutating" Snowflake parent. Thus the d a crenate type might have the zygotic formula -= -, where d is the Cv ft factor for double flowers, C a dominant factor for crenate, and / a dominant inhibitor of C, all three loci being situated in the same chromosome, at distances of, say. 16 and 4 units apart, in tl rder indicated. A Snowflake parent producing crenate progeny would then be-: — - or , — •-. and crossover combinations would produce the de% del apparently mutant crenate progeny. The crenate progeny would behave as heterozygous dominants when selfed, and if CC zygotes were non-viable would yield constant Snowflake and inconstant crenate; the extracted Snowflake singles, having the composition Dei — ;— r, could not throw en null iuiliriilinds except bv true mutation of dci c to C. With selfed Snowflake, if we assume 16 per cent and 4 per cent of crossing over in the two positions, and a 60-per-cent selective elimination of crenate zygotes, all CC zygotes being non-viable, sub- stantially the observed percentages of crenate singles and doubles result.'-'" 26 See page 125, footnote. This scheme agrees fairly well with the results from crossing, and gives almost exactly the observed proportion of total doubles (a little over 53 per cent) for selfed Snowflake. Its adequate presentation must be reserved for a later paper. 156 University of California Publications in Agricultural Sciences [Vol.4 Formerly (Frost, 1916) the hypothesis of frequent dominant mutations seemed the more probable, but there is apparently non- conformable evidence. It is true that the peculiar behavior of the slender type might conceivably depend on an occasional mutation in another locus, or an exchange (Shull, 1914) or duplication of loci, giving two similar or identical factors for slender. An apparently fatal objection, however, is the fact that the supposed mutants seem to show linkage with singleness or doubleness at their origin from Snowflake as well as in subsecpient generations — a fact which strongly suggests segregation in the former case. If the apparent mutants are really due to segregation complicated by lethal action, the origin of the complex heterozygosis indicated for Snowflake is doubtful; it may be due to hybridization, but more probably to a gradual accumulation of mutant factors in balanced- lethal combinations. On the analogy of Midler's Drosophila case, especially, it might be expected that the latter would be the true explanation, particularly since self fertilization seems to be the rule in Matthiola. On this basis the term mutant type is used with some confidence in this paper, while the aberrant individuals have been called apparent mutants. We must not forget that some of the mutant types may arise, as with Oenothera gigas and 0. lata, by non-disjunction, or reduplication of chromosomes, and that this fact may determine their heredity. This is not to be expected with the types whose factors show apparent coupling with singleness or doubleness. but it might be true of the apparently unlinked smooth-leaved type. A preliminary study of several types shows that the usual somatic number of chromosomes is probably fourteen, but that positive counts are difficult. AVhile it might be very hard to demonstrate the regular presence of one extra chromosome in an individual or a type, it should be easy to decide between the diploid and triploid numbers. The large-leaved type is so strongly suggestive of 0. gigas that it would not be surprising to find the triploid number in the material now on hand for examination. In a preliminary paper on these types the writer (Frost, 1916) discussed some possible relations of mutation, heterozygosis, and partial sterility, with special reference to Oenothera, mentioning the possibility that special prevalence of heterozygosis in the genus may be, "in large part, a result rather than a cause of mutation." This suggestion is evidently justified even if much of the supposed mutation of Oenothera is really segregation, since it is highly probable that 1919 i / osi : Mutation in Matthiola 1">7 the peculiar phenomena depend on Lethal factors or combinations of factors originally due to tnutal ion. Another possibility there mentioned, advanced by Belling (1914) and since specially discussed by Goodspeed and Clausen (1917), is thai of the occurrence of Lethal combinations of certain factors which in other combinations may be in no way prejudicial to normal develop- ment. As the latter paper shows, it is probable thai in certain crosses between "good species" most of the new combinations brought together in the formation of F, gametes are incompatible with the production of functional gametes. Perhaps in the ease of Oenothera there may exist within a species factors lethal in any combination when homozygous, and other factors lethal only in certain com- binal ions. A balanced-factor explanation for the inheritance of doublcness-7 in Matthiola, a case which Midler 1918) discusses, seems to have been first definitely stated by G-oldschmidl (1913), though he failed to pro- \ ide for one feature of the evidence, the deviation of the heredity ratio from 50 per cent. As has been shown (Frost, 1915), this peculiarity may be due to greater viability of the homozygotes (sterile doubles) during embryonic development, since the doubles are more viable in the mature seeds and more vigorous in later development Saunders, 1915). In this case the "normal" factor is completely eliminated in favor of the mutant (sterile-double) factor in the formation of the sperms, and probably is partially eliminated in the formation of either the eggs or the embryos or both. Here the normal singleness (sporophyll) factor D may act as a lethal in the heterozygous parent, possibly from its general relations of growth vigor in the presence of the more vigorous (/-carrying cells. If the lethal factor is situated in a distinct locus, evidently crossovers are at most extremely rare. Tt is true that Miss Saunders (1911) finds that F, hybrids with pure single forms produce functional single-carrying pollen; with the pure single forms from which the original "double-throwing" mutants arose, however, this might not be true, or a lethal change may have occurred in the singleness factor itself rather than in a factor coupled with it. The Drosophila case would suggest a lethal change in another locus of the single-carrying chromosome. In my paper of 1915 this lethal change in one chromosome ap- parently accompanying the mutation of D to d in the homologous -' For a brief outline of the genetic behavior of doubleness see the discussion of the experimental data for the smooth-leaved type. 158 University of California Publications in Agricultural Sciences [Vol. 4 chromosome was considered puzzling. Evidently, however, it may have occurred in one chromosome before D mutated to d in the other, and even then may have produced its lethal effect. It is evident that if doubleness should arise in the absence of the lethal effect it would tend to be eliminated by the return of one-third of the singles to the homozygous condition in each generation. In fact, it is possible that the lethal change arose later than doubleness, as in the Droso- j)1iila case, or was brought in later by cross pollination, and happened to be preserved as a result of horticultural selection for a high pro- portion of doubles. A parallel-column comparison between the double type and the types especially discussed above has already been given, in connec- tion with the smooth-leaved type. It will now be seen that this com- parison seems to apply to all mutant types, except early, that have been genetically tested, the principal differences between these types relating to the heredity percentage and the apparent presence or absence of linkage with the single-double factors. From the standpoint of its relation to genetic analysis the double- ness factor is remarkably similar to the sex factor in animals. There are two types in each generation, one heterozygous and the other evidently homozygous, and these types are produced by the fertiliza- tion of two kinds of eggs, produced in equal or nearly equal numbers, by a single kind of sperm. Although one of the somatic types is sterile, and the uniformity of the sperms produced by the other is due (evidently) to lethal action, the opportunity for chromosome analysis is similar to that with sex chromosomes. We may say that the doubleness factor and its normal allelomorph (d and D) are carried by chromosome pair I. Already we know several other pairs of factors evidently carried by this pair of chromo- somes. These are, to name only the mutant or possibly mutant member of each pair of factors: P (pale sap color) and W (colorless plastids), both studied by Miss Saunders (1911, 1911a) ; C (crenate- leaved), S (slender; possibly two factors), and N (narrow-leaved). As we have seen, the last three of these are probably lethal when homozygous, and one or more unidentified lethal factors may be con- cerned in the breeding results, while the doubleness factor affects the race much like a recessive lethal, since all dd individuals are completely sterile. Froat: Mutation in Matthiola LS9 SUMMARY This paper describes the occurrence, characteristics, and heredity of certain aberranl planl types which decidedly resemble some of the "mutant" types produced by Oenothera lamarclciana. The parent Eorm is Matthiola annua Sweet, of the horticultural variety "Snow- flake." These aberranl forms may be called mutant types, since it is highly probable that they are originally produced by mutation. The aberranl individuals may be termed apparent mutants, since it may be con- sidered uncertain whether they usually arise by immediate mutation or by segregation. The case acquires special significance because indi- viduals belonging to the mutant types, although the species is known to be typically Mendelian with respeel to various characters, give erratic heredity ratios suggestive of Oenothera. At least eight types have been somewhat carefully studied, and six of these have shown their heritability in progeny tests. Several other types have been named, but for various reasons their distinctness is more or less doubtful. Some of the commoner types have each been produced by many parents, and in several pure lines isolated from the original com- mercial variety. The apparent mutants other than the early type com- pose about four or live per cent of the progeny of Snowflake and early parents, the separate types ranging down from about one per cent. Most of the mutant types are in general inferior to Snowflake in vigor, and the difference in development is greatly increased by certain unfavorable environmental conditions. The proportion of apparent mutants in cultures from Snowflake parents appears to be definitely lower where germination is comparatively poor. The mutant types differ from Snowflake and from each other in various respects. The early type is practically a smaller and earlier Snowflake. The other mutant types, on the other hand, differ markedly from Snowflake in vigor, fertility, and various form and size char- acters. Each type is named from some conspicuous characteristic difference from Snowflake, but usually various other differences can readily be found. Somewhat extensive progeny tests have been made for five of the mutant types, and a little evidence secured for two Or three other types. 160 University of California Publication* in Agricultural Sciences [Vol.4 The early type is probably due to a single dominant mutant factor segregating normally from the corresponding Snowflake factor; the quantitative nature of its differences from Snowflake. however, makes positive determination of this point a matter of great difficulty. At least five other types plainly reproduce themselves, but about 50 to 70 per cent of the progeny are usually Snowflake ; no true- breeding individual of any generation of any of these types has yet been tested. Genetic work with most of these types has been much hampered or even prevented by low vigor and fecundity, and the aggregate data from progeny of parents of four types strongly indi- cate selective viability at germination. It has been determined by crossing that in three of the types the mutant factor (or factors) is carried both by eggs and by sperms. From these facts it seems prob- able that homozygotes of the mutant types are non-viable, and that severe selective elimination occurs during embryonic development; or, in other words, that the mutant factor is imperfectly recessive for a lethal effect. In three types there appears to be linkage with the factor pair for singleness and doubleness of flowers, the mutant factor being coupled with singleness in the tested apparent mutants of two types, and with doubleness in the third type. With two other types these factors seem to be independent. No reversal of coupling has been found in later generations of the former two types, but on the scheme presented crossover singles should be scarce. For one type (crenate-leaved) a hypothesis based on the facts stated gives very closely the ratio obtained from selfed parents. Reciprocal crosses with Snowflake conform less closely to the requirements of the hypothesis, but do not definitely contradict it. The slender type, which shows similar apparent linkage, seems to disagree definitely with the hypothesis ; there is strong evidence, however, that slender individuals may differ genetically among themselves. A more complex scheme providing also for the usual origin of these types from Snowflake by segregation is briefly outlined. The selfing ratios are very suggestive of duplication of a chromo- some (non-disjunction), as in Oenothera lata, but it is hard to reconcile the cases of apparent linkage with this hypothesis. It seems probable that these three linked types have originated and are trans- mitted in the same general way as the double-flowered type, and that all of these four mutant factors (including double) represent changes of some sort within a chromosome of the same pair, which may be Froai : Mutation in tfatth i 161 numbered I Miss Saunder's work shows thai two flower-color factors also belong t<> this linked group. The large leaved type strikingly resembles Oenothera gigas, and il may prove to be triploid in nuclear constitution, In thai case segrega tion maj be irregular and genotypically intermediate individuals maj be more or less frequentlj produa d. It is probable thai further studj of these types will help to explain tin- remarkable genetic behavior of Oenothera and of dints. LTTERATl'HK < ITHI) A i ki son, George P. 1917. Quadruple hybrids in the F, generation from Oenothera nutans and Oenothera ;< i, with 1' generations and back- and inter- crosses. Genetics, vol. 2, pp. 2 1. '5 L'o'o, ]] tables, I diagr., 1 •> t i - ^ - B \i;< oi k. Ernest B. HMs. The role of factor mutations in evolution. Am. Naturalist, vol. 52, pp. 116 128. Barti ett, II. II. L917. Mutation in Mattluola annua, a " Mendelizing" species. [A review of paper of same title by II. B. Frost.] Bot. Gaz., vol. 63, pp. 82-83. Bateson, Wlliam, and Saunders, Edith R. 1902. Experimental studies in the physiology of heredity. I. Experiments with plants. Matthiola. III. Discussion. Roy. Soc. London, Re- ports to the Evolution Committee, vol. I, pp. 32-87, 125-160, 15 tables. Bateson William, Saunders, Edith It., and Punnett, Reginald C. L905. Experimental studies in the physiology of heredity. Matthiola. Roy. Soc. London, Reports to the Evolution Committee, vol. 2, pp. o-44, tables. 1906. Ibid. Stocks. Ibid., vol. 3, pp. 38-53, 4 tables, 2 fi^s. Bateson, William, Saunders, Edith R., Punnett, Reginald C, and Killbt (Miss) II. B. 1908. Experimental studies in the physiology of heredity. Stocks. Roy. Soc. London, Reports to the Evolution Committee, vol. 4, pp. 35-40, I! tables. Belling, John. 1914. The mode of inheritance of semi-sterility in the offspring of certain hybrid plants. Zeitschr. f. indukt. Abstain.- u. Vererbungsl., vol. 12, pp. M03-342, tables, 17 ties. Ill IKESLEE, Al BERT F., AND Avi.RY, B. T. Jr. 1919. Mutations in the jimson weed. .Jour. Heredity, vol. 10, pp. Ill 12i», 11 ties. * An asterisk prefixed to the date indicates that the paper cited has not been seen by the present writer. 162 University of California Publications in Agricultural Sciences [Vol.4 Correns, Carl E. 1900. tiber Levkojenbastarde. Zur Kenntniss der Grenzen der Mendel 'sch en Eegeln. Bot. Centralbl., vol. 84, pp. 97-113. 1902. Scheinbare Ausnahme von der Mendel 'sclien Spaltungsregel f iir Bas- tarde. Deutsch. bot. Ges., Ber., vol. 20, pp. 159-172, 4 tables. Davis, Bradley, M. 1917. A criticism of the evidence for the mutation theory of de Vries from the behavior of species ot Oenothera in crosses and in selfed lines. Nat. Acad. Sei., Proc, vol. 3, pp. 705-710. Frost, Howard B. 1911. Variation as related to the temperature environment. Am. Breeders' Assoc, Ann. Kept., vol. 6, pp. 384-395, 4 tables, 4 charts. 1912. The origin of an early variety of Matthiola by mutation. Ibid., vol. 8, pp. 536-545, 5 tables. 1915. The inheritance of doubleness in Matthiola and Petunia. I. The hypotheses. Am. Naturalist, vol. 49, pp. 623-636, 1 fig., 2 diagr. 1916. Mutation in Matthiola annua, a " Mendelizing " species. Am. Jour. Bot., vol. 7, pp. 377-383, 3 figs. 1917. A method of numbering plants in pedigree cultures. Am. Naturalist, vol. 51, pp. 429-437. Gates, E. Euggles. 1915. The mutation factor in evolution. London, Macmillan, xiv 4- 353 pp., 1 map, 114 figs., bibl. GOLDSCHMIDT, ElCHARD. 1913. Der Vererbungsmodus der gefiillten Levkojenrasson als Fall geschlechts- begrenzter Vererbung? Zeitschr. f. indukt. Abstain.- u. Verer- bungsl., vol. 10, pp. 74-98, diagr. 1916. Nochmals liber die Merogonie der Oenotherabastarde. Genetics, vol. 1, pp. 348-353, 1 pi. Goodspeed, Thomas H., and Clausen, R. E. 1917. Mendelian-f actor differences versus reaction-system contrasts in hered- ity. Am. Naturalist, vol. 51, pp. 31-46, 92-101. IlERIBERT-NlLSSON, N. *1915. Die Spaltungserscheinungen der Oenothera lamarckiana. Lunds Univ. Arsskrift, vol. 12, pp. 4-131. (Review by Ben C. Helmick in Bot. Gaz., vol. 63, 1917, pp. 81-82.) Muller, Hermann J. 1917. An Ocnothcra-like ease in Drosophila. Nat. Acad. Sci., Proc, vol. 3, lip. 619-626. 1918. Genetic variability, twin hybrids and constant hybrids, in a case of balanced lethal factors. Genetics, vol. 3, pp. 422-499', 1 table, 1 fig., 1 diagr. Saunders, Edith R. 1911. Further experiments on the inheritance of doubleness and other char- acters in stocks. Jour. Genetics, vol. 1, pp. 303-376, 8 tables. 1911«. The breeding of double flowers. Fourth Intern. Conf. on Genetics, Proc, pp. 397-405, diagr. *1913. Double flowers. Roy. Hort. Soc, Jour., vol. 38, pt. 3, pp. 469-482. / rost: Mutation wi Uatthiola L63 L913a. < >n the mode of inheritance of certain characters in double-throwing stocks. \ reply. Zeitschr. 1'. indukt. Abstain.- u. Vererbungsl., vol 10, pp. 297 .".I". 1915. A suggested explanation of the abnormallj high records of doubles quoted bj growers of stocks i Vatthiola) . Jour. Genetics, vol. 5, pp. L37 l 13, 3 tallies. 1916. On selective partial steribitj as an explanation of the behavior of the double-throwing stock and the petunia. Am. Naturalist, vol. 50, pp. 186 198. Sin li. (ii ORG] II. 1914. Duplicate genes for capsule form in Bursa bursa-pastoris. Zeitschr. f. indukt. Abstain.- n. Vererbungsl., vol. 12, pp. 97-149, 5 tables, 7 figs. v. " 1908. The probable error of a mean. Biometrika, vol. 6, pp. 1—25, tables, I diagr. 1917. Tables for estimating the probability thai the mean of a unique scries of observations lies between — ~ and any given distance of the mean of the population from which the sample is drawn. fin, I., vol. I 1. ii>. Ill 117. tables. - ingle, Walter T. 1911. Variation in first-generation hybrids (imperfect dominance): its pus sible explanation through zygotaxis. Fourth Intern. Conf. on Genetics, Proc, pp. 381-393, 10 figs. Tschkkmak. Erich vox. *1904. Weitere Kreuzungsstudien an Erbsen, Levkojen u. Bohnen. Zeitschr. f. d. landw. Versuchswesen in Oesterreich, 1904, pp. 533 638. 1912. Bastardierungsversuche an Erbsen, Levkojen, und Bohnen mit Riick- sicht auf die Faktorenlehre. Zeitschr. f. indukt. Abstain.- a. Verer- bun<:sl., vol. 7, ]ip. 81-234, tables. AVeijbf.r, Herbert J. 1906. Pedigree records used in the plant-breeding work of the Department of Agriculture, in L. IT. Bailey, Plant Breeding (New York, Mac- millan), pp. 308-31 9. DE VRIES, HufiO. 1006. Species and varieties: their origin by mutation. Mil. 2, Chicago, Open Court Pub. Co., xviii -1- 847 pages. 1918. Twin hybrids of Oenothera hoolceri T. and G. Genetics, vol. 3, pp. 307- 421, 14 tables. 1019. Oenothera rubrinervis, a half mutant. Pot. Gaz., vol. 67, pp. 1-26, tables. Yule, G. Udnt. 1911. An introduction to the theory of statistics. London, Charles Griffin & Co., xiii -+- 3"*' pa^es, 53 figs. PLATE 22 The Early Type Fig. 1. March 20, 1908. The single progeny of WG9. Plants from house M to the reader's left from stake, from house W to right of stake, from house C below. WG9-C10, the earhr apparent mutant, is the middle plant in the lower row. The stake indicates inches. Fig. 2. About May 1, 1908. WG9-C10 at the left, WG9-C9 (Snowflake) at the right. [164] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 f FROST ) PLATE 22 Fig. 1 Fig. 2 PLATE 23 The Early Type Fig. 3. April 8, 1909. The single progeny of WG9-C9 (Snowflake); arrange- ment as in figure 1 . Fig. 4. April 9, 1909. The single progeny of W69-C10 (heterozygous early). Warm-house plants partly at right of stake in lower row; arrangement other- wise as in figure 3. Compare with figure 3, house by house. [166] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 [ FROST ] PLATE 23 Fie. 3 Pis:, i 4 * s PLATE 24 The Early Type Fig. 5. July 19, 1911. Lots 1 to 10, with lots 11 to 14 mostly in sight at the right. Odd-numbered lot in nearer (west) half of each row. Fig. 6. July 10, 1911. Lots 19 to 28, with lots 15 to 18 mostly in sight at the left. [168] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 ( FROST 1 PLATE 24 Fig. 5 Fig. 6 PLATE 25 The Smooth-leaved Type Fig. 7. April 27, 1909. Smooth-leaved apparent mutants. Compare with figures 3 and 4 as to earliness, noting the difference in date. Fig. 8. May 29, 1914. Progeny of a smooth-leaved parent. Plant at right Snowflake single, the others smooth. [170] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 [ FROST | PLATF. 25 Fig. 7 Pig. 8 PLATE 26 The Smooth-leaved Type Fig. 9. June 28, 1915. Progeny of a smooth-leaved parent. Smooth single at left, Snowflake double at right. Fig. 10. Same date and parent as with figure 9. From left to right: Snow- flake double (also shown in figure 9), Snowflake single, smooth double. [172] UNIV. CALIF. PUBL. AGRI. SCI. VOl | FROST | PLATE 2C Fie. !> Vie. 1(1 PLATE 27 The Large-leaved Type Fig. 11. August 29, 1914. Progeny of a large-leaved parent (28a), near the close of the hot Riverside summer. From left to right: large single, large double, Snowflake single (two, the first injured by aphids). [174] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 [ FROST 1 PLATE 27 Fig. 11 PLATE 28 The Large-leaved Type Fig. 12. July 8, 1916. Progeny of a large-leaved parent. Middle plant Snowflake; the rest large; all single. Fig. 13. July 8, 1916. Progeny of a large-leaved parent, more- than a month older than those shown in figure 12. From left to right: large double, Snow- flake double, large single. [176] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 [ FROST ] PLATE 28 •• .J , b Fie. L3 PLATE 29 The Crenate-leaved Type Fig. 14. April 6, 1909. Crenate-leaved apparent mutants. Note the varia- tion in leaf serration, and especially the slightness of the serration (or crenation) with the one cool-house plant (below). Fig. 15. April 14, 1911. Progeny of a crenate-leaved parent, grown in a cool greenhouse. The first two plants at the right are Snowflake, the rest crenate. [178] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 | FROST | PLATE 29 Fie. 14 knk-l*^ 1 W£ jfl hn 1 ' 1 ' ■ ■ 1 A 1 1r ! 1 I*7| I 1 J 1 rrvvru-A^. ] ^^ ^^m^^i Pie PLATE 30 The Crenate-leaved Type Fig. 16. July 8, 1916. Progeny of a crenate-leaved parent. From left to right: crenate single (two), crenate double, Snowflake double. Fig. 17. July 8, 1916. Snowflake X crenate-leaved, F,. From left to right: smooth, Snowflake single, crenate double (two). [180] UNIV. CALIF. PUBL AGRI. SCI. VOt [ FROST ] PLATE 30 Fie. n; Pie. 1: PLATE 31 The Slendee Type Fig. 18. April 27, 1900. Miscellaneous aberrant individuals, with two typical Snowflake plants (third from the left above, second from the left below). In upper row: second from left, narrow double; second from right, slender double. In lower row at left, slender single (25b). Fig. 19. April 14, 1911. Progeny of a slender parent (25b). Two at the right Snowflake, the rest slender. [182] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 [ FROST | PLATE 31 L L P^^V ^|lj§P^ &* it* yffi 1* *fc^ ^dgg#fr, |, » aigrfr- ij BB^ 3 *■.. *■ ■. > f-A7-0T> Fig. 18 Pie. 1!> * PLATE 32 The Slender Type Fig. 20. June 3, 1914. Progeny of slender parents. From left to right: slender single, slender double, Snowflake double. Fig. 21. July 7, 1916. Snowflake X slender, F,. Middle plant Snowflake; the others slender; all single. [184] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 [ FROST | PLATE 32 hi* ' ■T.-twI r. 7v ■_-.. Kwn^ K. " . wg| • - f-A_'- i 1 v ""^\ ''"— Fig. 21 PLATE 33 The Narrow-leaved Type Tig. 22. April 13, 1911. Narrow-leaved apparent mutants. Fig. 23. June 3, 1914. A narrow-leaved apparent mutant among progeny of a erenate-leaved parent. From left to right: narrow double, erenate single (two). [186] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 I FROST 1 PLATE 33 Fie. 22 Pig. 23 PLATE 34 The Narrow-leaved and Small-smooth-leaved Types Fig. 24. June 28, 1915. A narrow-leaved apparent mutant among F, progeny from Snowflake X slender. Narrow double at left; the rest Snowflake single. Fig. 25. April 14, 1911. Miscellaneous aberrant plants, some being apparent mutants. From the left: first and fifth small-smooth, third stout dwarf, seventh slender. See text. [188] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 [ FROST I PLATE 34 Fig. 24 Fig. 25 PLATE 35 The Narrow-darkxleaved Type Fig. 26. June 3, 1914. A narrow-dark-leaved apparent mutant among progeny of a narrow-leaved parent. Third plant from left narrow-dark single; the other three Snowflake double. Fig. 27. June 28, 1915. Progeny of a "small-convex-leaved(?) " parent (27a). From left to right: narrow-dark single, Snowflake double, smooth single. [190] UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2 [ FROST | PLATE 35 ■ C Pie. 26 Kiir. 127