Jos UNIVERSITY OF CALIFORNIA PUBLICATIONS s ‘a ae BOTANY _ Vol. 5, No. 17, pp. 457-582, 2 figures in text, plates 55-85 April 14, 1922 eS A EAT TE INHERITANCE IN NICOTIANA TABACUM A REPORT ON THE RESULTS OF CROSSING ee CERTAIN VARIETIES ea WILLIAM ALBERT SETCHELL ; THOMAS HARPER GOODSPEED : ee AND ROY ELWOOD CLAUSEN By Eee. UNIVERSITY OF CALIFORNIA PRESS Pt Bi 6 3 BERKELEY, CALIFORNIA THE Lifmatny OF cue pat do1 1949 —* ¥ W550 A PeekCEL Bed Al vert Note.—The University of California Publications. are offered in exchange for the pabiy? UNIVERSITY OF CALIFORNIA PUBLICATIONS . =” cations of learned societies and institutions, universities and libraries. Complete lists of all the publications of the University will be sent upon request. Fer sample copies, lists - of publications and other information, address the MANAGER OF THE UNIVERSITY - PRESS, BEREELEY, CALIFORNIA, VU. S.A. All matter sent in exchange should be addressed to THE EXCHANGE DEPARTMENT, UNIVERSITY LIBRARY, BERKELEY, CALIFORNIA, U.S. A. . WILLIAM WESLEY & SONS, LONDON Agent for the series in American Archaeology and Ethnology, ae Geoloey, Physiology, and Zoology. BOTANY.—W. A. Setchell, Editor. Price per volume, $3.50 (vol. 5; $6.00); pacing with pig gia 6, $5.00. . Volumes I; TI, ITI, IV, and IX completed. Volumes VIL VL and in progress. nie wi as Univ. Calif. Publ. Bot. Vol. 5. 1912-1922. Studies in Nicotiana. “I, by William Albert Setchell. Pp. 1-86. Saeniihieg” BSS) Si iets ened i dane a ete a Ee We ERS Peal Me MeeR Ree CN aNee estan Hore dn Us 8 $1.25 1. 2. 3. 18. 16. 17. Quantitative Studies of Inheritance in Nicottana Hybrids. I, by Thomas Harper Goodspeed. Pp. 87-168, plates 1-28. December, 1912... Quantitative Studies of Inheritance in Nicotiana ote aoe by Thomas . Harper Goodspeed. Pp. 169-188, plates 29-34. January, 1913 220... , . On the Partial Sterility of Nicotiana Hybrids made with : enue as Ss Parent, by Thomas Harper Goodspeed. Pp, 189-198. “March, 1913~_ . Notes on the Germination of Tobacco Seed. I, “by Thomas Harper Good- Speed.2 cP ps199-222- May, LORS ose Sa a eee, oath an esata wd Sea ese Ly . Quantitative Studies of Inheritance in Nicotiana Hybrids. 1, by Thomas Harper Goodspéed. ~ Pp; 223-231. “April,/1915 222 Notes on the Germination of ‘Tobacco Seed. IL by ‘Thomas’ Harper Good- speed... Pp233-248-5< dune, A915, ae Se dnatce ed ee eden . Parthenogenesis, Parthenocarpy and Phenospermy in N tokio by Thomas : Harver Goodspeed. Fp. 249-272, plate.35. July, 1915 DNS eee Sas ae . On the Partial Sterility of Nicotiana Hybrids made qrith KN. 84 ylvestris as a Parent. II, by T..H. Goodspeed and A. Hy Ayres. Pp. 273-292, plate 36. OCEOD SH YE DEG Soa ee ha re Geng cabs ARDEA dl Su Ul ce eae ae cise . On the Partial Sterility of Nicotiana Hybrids made with N. sylvestris: as & Parent, IIT, An Account of the Mode of Floral Abscission in the F, Species ~ Hybrids, by: 7. H. Goodspeed and J. N. Kendall. Pp. 293-299. November, ~ BB) BY WILLIAM ALBERT SHETCHELL, THOMAS HARPER. GOODSPEED, AND ROY ELWOOD CLAUSEN April 14, 1922 ¥ : CONTENTS PAGE Introductonyy. wee... Mander: ee ah .. 458 Plan. of themwotkee:.......get.-.--. Peace ee .. 466 Angustifolia-macrophylla series............. .. . 462 1. Parents of the angustifolia-macrophylla series . 463 2. F, of the angustifolia-macrophylla series........ . 467 3. F. of the angustifolia-macrophylla series.. . 469 4. F; and subsequent generations of the Pe aiecmacropls ylla series...... 472 a. STENOPHYLLA derivatives.......... . 475 b. Latrroiia derivatives................ . 476 c. LANCEOLATA derivatives............ . 478 d. Loriro.ta derivatives . 478 e. AURICULATA derivatives........ Ps Tce , ine . 479 Ps. SESSHUUR OMAN G OM VATULV CSMAMMMN LS Pi; 15 20 14 30 20 F 11F.H,P, 13 28 a 32 17 G 11F,H,P;; 12 29 8 35 ) ait 14 H 11FHyPy 13 1 22 32 ae 18 J LRELPs: 8 bear | io 31 17 Kk 11 FH yPas 6 27 15 38 10 Totals 113 240 137 339 » BY 472 Umiversity of California Publications in Botany [ Von. 5 4, F; AND SUBSEQUENT GENERATIONS OF THE ANGUSTIFOLIA-— MACROPHYLLA SERIES From F, of H, and H,, 20 plants were selected for further ex- perimentation and families of 25 were determined upon as the unit. In all except four, the families of 25 each were successfully raised. Of one of the four only 14 plants were obtained, which were all that germinated, while in each of the other three families 24 plants were reared to maturity. Altogether, then, 486 plants were raised of the F, during the season of 1912. It was the intention to grow from each of the selected types, as drawn for illustration. Fifteen of the families from the type parents were successfully reared, but, in some way or other, the seed of type 4 (10F,H,P,P,,) was not to be found, and, unfortunately, no note had been made as to whether or not any seed was produced. No complete sterility, however, was noticed in any members of F, of either H, or H,, and the presumption is that F', seed of type 4 must have been lost in har vesting. The variation within each family was decidedly less than that of the families of F,. Of the 20 families reared wholly or in part, 4 families were rete uniform, varying in minor details only. Five families segrega only in coroll lor, 4 segregated only in leaf base characters, and “fle remaining 7 segregated both in ecoroll color and leaf base characters. In table 2 are summarized the de as to gross behavior of these families of Fr. In ‘those families grown in F, and subsequent generations, a definite attempt was made to fix the original characters of the F, type seleetion in a pure line. The genealogical relation of these selected lines to each other is shown in the chart reproduced herewith. The letters, A, B, C, etc., correspond to the’, family designations noted in table 1, and the sihiboxs refer to type sélection numbers -responding to the type illustrations in plate 63 to 78, or, iw the case of types 17 to 21, 7 to those types as described in the succeeding accounts of the later generations. Ww . 1922)" — Setchell-Goodspeed-Clausen: Nicotiana Tabacum 473 TABLE 2 F; FAMILIES OF THE ANGUSTIFOLIA-MACROPHYLLA SERIES. No. of plants Type Garden in fam- os. Numbers ilies | Results in F3 1 11F,H,P7P. 25 | Segregated both as to leaf and flower color 2 11F.H»P3P3o 25 | Segregated both as to leaf and flower color 3 11F.H2P3Pi4 14 | Segregated only as to leaf 5 11F.H4PaPius 24 | Segregated both as to leaf and flower color Li 6 11F,H4P2Pis 25 | Uniform except as to length and development of wing of petiole 7 11 FH 2PisP.g 25 Uniform both as to leaf and flower color . 8 | 11F.HP:Pa 25 | Segregated as to leaf only 9 11F2HyPaPs 25. | Segregated both as to leaf and flower color 10 11F,H,;PuP:; | 24 | Segregated only as to flower color 11 LLFSHiP aiff 25 7 ae only as to flower color 12 11F,HyPaP i. 25 | Segregated only as to flower color 13 11F,H)P;P x; 25 | Segregated only as to flower color 14 11F,H,P3P3s 25 | Segregated both as to leaf and flower color 15 11F2H2P3Pio 25 | Uniform, slight variation in tint and lobing of corolla 16 11F,H.P3Ps 24 | Segregated only as to leaf 17 11F,.H4P3;P.7 25 | Segregated only as to flower color + ile: ESN 18 11 F,H4P35 P35 25 | Segregated both as to leaf-and. flower color th as to leaf and flower color 19 11 F,H4P35P,s 25 | Segregated 20 | LL Fy HyP P44 25 i niform, close to macrophylla 21 " 11 FL HyP 41 Pos 25 | Uniform, close to angustifolia A714 University of Califorma Publications in Botany [ Vou. 5 In F, primary selection for parents of subsequent generations was based upon the type of leaf borne by the plant, flower color being followed as a secondary matter. In order to systematize the discussion concerning EF’, and subsequent generations, six general types have been selected and named and the discussion of the families has been grouped P, 1909 a MACROPHYLLA Fi 1910 Hs {Ha|_— [| Ha} (Hs} (His) [Hie Fe io! (xe? (BC Ee a) fl] K| Fs i9l2 BA (15 Fa 1913 [6] Gal 20 ~ [|| al Fs 1914 6 | 20) 2) [i2al2t Fe Ios cl) 02 ‘wlealer Fr 1916 7)[6' 6a (20/204 A2)(2al2t| Fig. 1.—Chart showing the relationships ‘of various families of the angusti- folia-macrophylla series. The ditenent FE e connected with their fem - parents; no seed was secured from H,. The F, family a. ions correspon those given in table 1, and the numbers in later generations are the type n under which the populations are described in the text. IF, and F; of type 1 ere grown in 1914 and 1915 respectively. under these headings. The six general types selected and the names given them are as follows: a. STENOPHYLLA derivatives. As a series these approximate very closely in leaf shape to angustifolia. The distinguishing feature of this series is the possession of a distinct, long petiole. b. Warioura derivatives, which are characterized by the possession of a broa with a_petiole shorter than that of angustifolia. The petiole in these #ypés is more or less winged. c. LANCEOLATA derivatives, Which are charaeterized by the posses- sion of a lanceolate leaf like that of typeqs, illustrated in plate 75. This is a non-petiolate form. d. Lortrouis derivatives, characterized by possession of lo: ayes with very narrow blades. The type specimen, type 12, is illu ed in plate 74. This also is a non-petiolate form resembling the LANCEO- LATA derivatives, from which it differs in the extreme narrowing of the blade. 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 475 e. AURICULATA derivatives. The typical form of leaf is that of type 10, illustrated in plate 72. The leaf blade of this form is char- acterized by an abrupt éontraction of the blade at the base, nearly, if not quite, to the midrib. Clasping auricles, from which the name is derived, are usually present in this form. f. SESSILIFOLIA derivatives, of which the leaf of macrophylla may be taken as the type. These derivatives are non-petiolate, as the name indicates. a. STENOPHYLLA derivatives Type 1, as may be seen from the drawing of F,.(cf. pl. 63), seems very close to angustifolia, and had light pink flowers. There were 25 plants of 12F,H,P,P,,, the leaves of which were carefully noted; b t one passed its flowering stage too early to be judged, so that the color of the flowers of 24 only are known. Sixteen plants had srsNoPHYLLA leaves very nearly of type 1, but the extent of the wing structure yaried somewhat. The remaining 9 had LaNcEoLATA leaves of type 14. In flower color the segregation ratio noted was 5 red : 19 pink.” No further generations of this line were grown. Type 17 was not selected for m7 in F,, but was a plant very close to angustifolia. It had, however, somewhat narrower leaves and deep pink flowers. © The 25 plants of 12F,H,P,.P., were uniform and like the F’, parent except as to flower color ; 3 were red and 22 were deeper or lighter pink. This line was not grown through further generations. Type 21, also, was not selected for illustration of the F, plant, but was chosen later for perpetuation because of its extremely close agree- ment with angustifolia. The F, 12F,H,P,P.,, consisted of 25 vigorous plants which seemed to be uniform to the finest detail and agreed in every respect with plants of angustifolia. There were noticed in the plants of this family peculiar fimbriae attached to the corolla, or split corollas, or, in one instance, a split hose-in-hose flower such as occurs at times also in pure angustifolia. This type cat ima a pure recombination of the characters of angustifolia. e Type 21 was continued through tothe sev generation and found to be constant i thegimiformity-6f the individuals in the several families. In 1913, 94 plants of F, e grown; in 1914, 85 plants of F,; in 1915, 10 plants of F,; and in 1916, 8 plants of F,. All these populations closely resembled one another as to individuals as well as those of the F, and the F,, parent. They are all so close to angus- 476 University of California Publications in Botany [ Vou. 5 tifolia as to be practically indistinguishable from it. This line may be regarded, therefore, as a stable derivative very closely approxi- mating angustifolia in all its characters. b. LAvTIFOLIA derivatives Type 2 (ef. pl. 64) approached in F, fairly near to some of the variations of F, (ef. pl. 62). It might be regarded as a STENOPHYLLA derivative possessing an unusually luxuriant development of the wing of the petiole, but it seems more reasonable to classify it as a LATIFOLIA derivative exhibiting marked narrowing at the base of the lamina, such as is shown in LANCEOLATA derivatives. The flower was designated as light pink in the notes taken at the time of flowering. The leaves of I’, showed segregation through a considerable range, 6 being close to the LATIFOLIA type of F,, 6 to the LATmoLIA type of its F, parent (type 2), 8 were SESSILIFOLIA leaves approaching in type those of mac- rophylla, and 4 were AuRICcULATA leaves of type 10. In 13 the flowers were noted as jight pink, in 11 as pink, and in 1 as red. No subsequent generations of this population were grown. Type 3, in F,, had (ef. pl. 65) a distinetly ovate lanceolate leaf with a short fairly br ‘oadly’ mar ‘gined petiole. The flowers were light pink. F,, 12F,H,P,P,,, amoutited to 14 gérminations age" of which ing plants. » The flowers were all the light pink color of the F, parent, agreeing developed, although slowly, into healthy, normal appe with those of angustifolia, but varying somewhat in shape and size. The leaves were of various shapes, 4 were distinctly petiolate, while 10 were sessile. Of the 4 petiolate plants the petiole of 1 was naked and of 3 more or less winged. Of the 10 sEssinirouiA plants, 9 were very similar to type 11, but 1 was rather longer and narrower, although otherwise approaching the same general shape. No further generations from this line were grown. Typed in F, (ef. pl. 67) resembled F, most nearly, but the winged petiole wasson and the corolla tube slightly stouter. The flower color was pink’s,Of 12F,H,P,,P,,, 24 plants were grown. Of these like those of the F, parent, but 1 had Of the 24 d flowers. 23 had leaves almips#exaé This line was not grown in subsequent generations. Type 6 was represented in F, by a plant which resemble having a winged petiole to the leaf and a pink flower. It is well 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 477 sented on plate 68. In the 25 plants of 12F,H,P.P,,, the height, habit, flower shape, and flower color were close to, if not identical with, those of the F, parent. As respects leaf base characters, 14 were LATIFOLIA of type 6, 4 had long, naked petioles, 3 had short, naked petioles, 1 had a long, winged petiole, and 4 were AuRIcULATA plants nearly of type 10. This seems like a considerable segregation, but the leaves are of only two generic types, viz., petiolate and non-petiolate. In subsequent generations selection was made in one line for LATIFOLIA leaves of type 6, and in the other for auricuLATA leaves of type 10, called type 6a to indicate its derivation. Of type 6 as thus established 100 plants of F', were grown in 1914; 100 plants of F, (50 each from 2 different parents) in 1915; 20 plants of F, in 1915; and 20 of F, in 1916. All the individuals thus grown were constant to LATIFOLIA of type 6 as originally selected. 4 Of AuricuLATA of type 6a, similarly, 100 plants of F, were grown in 1913; 94 of F, in 1914; 20 of F, in 1915; and 20 of Fy in 1916. All these plants were uniform and true to AURICULATA of pe 6a, very close to AURICULATA of type 10. In both yor have definitely obtained stable r oii of germi ements exhibiting char- ers different e parents. 13-- 48 F, of type 7 bei ae As (ef. pl. 69), consisted of a family of 25 plants, all ror ous except one (P,,), which was set out in the field later and developed into a ‘‘runt,’’ as often happens with such later plantings. All the plants agreed well with one another in height and habit except the ‘‘filler,’’? and all agreed in inflorescence, flowers, and leaves. There were some variations in size and lobing of the limb of the corolla, indicating possibly minor segregation, but in all general characters there was uniformity to a large degree. The plants agreed well in all characters with the F., parent, and also with the F, parent. The color of the flower was light pink, the petioles of the leaves yaried somewhat in length, were distinctly and more or less roa wee’ and the blade was heart-shaped, at least at the base in the lower leaves In all respects these characters were no more var pee they were found to be in F,. Type 7 continued tobreedtrue in subsggiitttn generations. It was grown in 1913 (100 plants, F,) M1914 (2)families of 50 plants each, F,), 1915 (10 plants, F,), and 1916 (10 plants, F,). All were uni- form as to leaf and flower color. Type 7 is very close to the type of F, and to type 6 described above. It, too, evidently represents a stable recombination of germinal elements derived from both parents. 478 University of California Publications in Botany [ Vou. 5 Type 9 resembles type 5, but had in F, a very short winged petiole and elliptical lanceolate blade. It also had pink flowers. F,, 12F.H,P,,P,, consisted of 25 plants, 18 of which showed LATIFOLIA leaves of type 9, but 7 had sEssmiroura leaves of type 14 (ef. pl. 76). Twenty-one had pink (or hght pink) flowers and 4 had red. No further generations of this line were grown. Type 19 was an F, plant of which no drawing was made, but it resembled F, (ef. pl. 62), having broadly ovate leaves with a long and broadly winged petiole and pink flowers. F;, 12F,H,P,.P,,, con- sisted of 25 plants, 6 of which had sEssmiFoLiA leaves of type 16 (cf. pl. 78) or nearer, perhaps, to those of macrophylla, while 19 had LATIFOLIA leaves of type 19. In 5 plants the flowers were a somewhat darker red than they were in the other 20. This line was not followed further. c. LANCEOLATA derivatives Type 13 is similar to type 12 described below, but the leaves of the F,, plant were more lanceolate and broader.and the flowers were lighter pink. The 25 plants of 12F,H,P,P,, were uniform and like F,, except in flower color. Four were red, 19 ae nd 2 — to light pink. The line was not grown in subsequer ae d. LARIIFOLIA derivatives Type 12, as shown in plate 74, differed very decidedly in leaf shape from either parent. The long linear-lanceolate leaf had the long taper- ing curved tip of angustifolia, but the blade tapered below, making practically a new type. The flowers were like those of angustifolia in shape but were pink. The 25 plants of F,, 12F,H,P,,P,., were exact duplicates of F, as to habit, leaf, and flower shape, but 10 had red and 15 had pink flowers of various shades, mostly dark. None seemed as light pink as angustifolia. Th the most interesting of the types carried through subse- quent generations, representing, apparently, a new combination of leaf the pink flowering F, plants was chosen for seed 12, retained for this and its progeny, while j ne of re ering F’, plants also chosen for seed. Type 12, as thus limited to the pink flowered co scanty germination and few plants for F, in 1913. Apparently it was still varying slightly in color within the pink shades, although fairly uni- characters. and the designation, the designation, 12a, was 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 479 form except for one aberrant (?) plant of a decidedly lighter shade. Two ‘‘normal’’ parents of F, gave 88 and 100 plants of F, in 1914, which were uniform and of a bright pink color. In 1915 F,, showed 10 plants, and in 1916 F’, also showed 10 plants, still uniform and pink. Type 12a on being segregated in the second growing of F, in the season of 1913 yielded 100 plants of F, in 1914, uniform and of deep red flower color. F, of 10 plants in 1915, and_also F,, of 10 plants in 1916, produced uniform individuals of deep red flower color. We find, then, in types 12 and 12a definite fixations of the Lorm- FOLIA type, one with uniformly pink flowers ard one with uniformly deep red flowers. e. AURICULATA derivatives Type 8 is represented in F’, by a plant which had a leaf with an ' extremely constricted base (cf. pl. 70) and deep red flowers. It isy not a typical AURICULATA derivative, but is included under thi hea ing because it resembles the members of this class more closely, tI those of any other. F,, 12F,H,P,P,,, consisted of 25 pla which were uniform in height, habit, and flower color, and in agreement with F, in these respects. The leave a of two distinct types, 16 of hit ang sessnionsh 0 type 16 (ef. pl. 78), the #.. being near to be: i, of macr ophylla. No further generations ‘i this line were grown. Type 10, as shown by the drawing (pl. 72), had a peculiar leaf, near to the macrophylla type, yet deeply constricted at the base into a narrow and extremely abbreviated structure which may resemble a petiole or only a deeply constricted blade. There were, however, auricles partially clasping the stem and slightly decurrent. The leaf form was that characteristic of N. Tabacum var. macrophylla purpurea (ef. Setchell, loc. cit.). All 24 plants of F, had the same type of leaf as F',, but the flowers were of three fairly readily distinguishable shades; 3 were red, 16 pink, and 5 light pink. F, had very dark pink flowers. The line was not grown in further generations. Type 6a is a true AURICULATA derivative which segrega ed in F, from an F, Lavirouia selection. Its occurrence and behavior are de- scribed in connection with the account of type or LATIFOLIA type r five generations it has remained constant for the auricunATA type of leaf. from which it segregated. Grown in the pure line 480 University of California Publications in Botany [ Vou. 5 f. SESSILIFOLIA derivatives Type 11 (ef. pl. 73) im F, gave 25 plantstin 12R9H)P2 P.=all- vigorous except one, but that one showed the,same characters of leaf and flower as the others. All 25 plants possessed a SESSILIFOLIA type of leaf very close to the F, parent and uniform among themselves. There were two distinet shades of color of the flowers, 9 red and 16 pink. No further generations of this line were grown. Type 14 (ef. pl. 76), so far as F, is concerned, was one of those having sessile leaves of a broadly lanceolate type and pink flowers. There were 25 plants in 12F,H,P,P,., 24 had sEssmumrouiaA leaves of type 14, while one (a ‘‘filler’’) had auricuLATa leaves like type 8; 19 had pink (or light pink) flowers, while 6 had red flowers. This line was not followed through subsequent generations. Type 15 (cf. pl. 77) was represented in F,, 12F,H,P,P,,, by 25 vigorous plants which seemed surprisingly uniform and approached macrophylla very closely as to leaf and color of the flower. In the flower, however, the color seemed even darker than that of macrophylla, there were only slight traces of the white tr ‘ee markings on the limb, the limb was mnt Adare deeply lobed, cr tube less stout and with the infundibulum much less abruptly We These dif ences seem to indicate that type 15, which all r F, plants clo: resemble, is not an exact recombination representing macrophylla. Type 15 was represented in 1913 by two families, F, of 10 plants and F, of 100 plants. Both families were uniform as to individuals, and agreed with the F, population grown in 1912 as well as with the F,, ancestor of the season of 1911. As this line seemed to be constant and very close to, although not absolutely identical with, macrophylla, differing in flower shape and leaf shape to some extent, type 15 was considered to be a fixation and no further cultivation of it was made. Type 16 (ef. pl. 78), which in F, approached macrophylla very , by 25 plants. These were all alike and closely resembled in all respeets except in leaf shape. Fifteen had sEssmi- FOLIA leaves of{type 16 while 8 had AuricuLATA leaves approaching those of type . 72). This line was not grown in further generations. Type 18 is the designation given to an F fant, of which no draw- ing was made. It seemed close to macrophylla, but the flower color 1922]. Setchell-Goodspeed-Clausen: Nicotiana Tabacum 481 was pink and the leaves were more slightly attenuate at the base. F,, 12F,H,P,.P,,, gave 25 plants, 13 of which had the gEssILIFOLIA leaf of type 18; 7, AURICULATA of type 10; and 5, auricuLaTa of type 8. In flower color, 17 were some shade of pink and 8 red. The line was not grown in further generations. Type 20 was not selected for illustration in F,, but was a plant chosen because of its very close resemblance to macrophylla, coming even closer than type 16. The F,, 12F,H,P,,P,,, consisted of 25 vig- orous plants of remarkable uniformity. In height, habit, inflorescence, flower, color, shape, fruit, ete., the details follow those of macrophylla so closely as to be indistinguishable unless possibly by careful and labor- ious biometrie study. This type may represent a practically pure recombination equivalent to macrophylla, and is to be compared an contrasted with type 15. \ In 1913 two families of F,, one of 21 plants and the other of 100 — plants, were uniform, as were 3 families of 50 plants each of F, in 1914. In 1914, however, a surprising thing happened. fourth family of F,, consisting of 50 plants, was uniform except one plant which had pink (instead of red) flowers at id an AURICULATA leaf ap- proximating type 8 or 10. It seems ii Mai this plant must have een an intr P, but. its seed was col under bag and grown and is noted below and on the pedigree chart as type 20a. The other 3 plants of F, whose seed was sown in 1915 gave type 20 in F, in families of 10, 9, and 8 respectively, and in turn the seed of 4 individuals of ‘“pure’’ type 20 gave, in 1916, uniformity in families of 10 each. Type 20a, which originated or intruded in 1914, in one plant of F, of type 20 gave in F,, in 1915, 10 plants segregating for flower color and probably also for leaf characters, although the notes taken are inconclusive on the latter point. In 1916 F, of 10 good plants showed uniformly red flowers, but 7 had sEsstniFoLtA leaves, 4 of which were decidedly contracted at the base and 3 had very short winged petioles (AURICULATA of type 8 or type 10). On the whole it seems most likely that the single plant in the F, family was ane intruder, >. A stray xplain it and a matter of inclu- macrophylla as to since all other families of the line have been constant si seed somewhere along the processes of culture wo its appearance is all the more incomprehensible sion in the pedigree of type 20, as it is so close seem practically identical with it. 482 University of California Publications in Botany [ Vou. 5 5. SUMMARY OF FLOWER COLOR OBSERVATIONS IN F, AND SUBSEQUENT GENERATIONS In tables 3 and 4 we have summarized the numerical data with respect to flower color inheritance in F, and in the subsequent popu- lations. In table 3 are assembled data with respect to the behavior of red flowering selections from populations segregating for red and pink. It will be noted that all the five selections which were made bred true for red flower color in the sueceeding generations. In table TABLE 3 INHERITANCE OF RED FLOWER COLOR IN Fs, et seq. Type Numbers Garden Numbers Flower color of population 8 12F3;H2P3Pa 25 red 12a 14F,HyP4Py2P) J 100 red 15 12F3HoP3Pio 25 red 16 12F;H»P3;Ps 24 red 20 12F3H4P4P 44 25 red TABLE 4 POPULATIONS FROM PINK FLOWERING SELECTIONS OF ALL SHADES IN F; et seq. . i " [ na Flower Wor classification Type : Parent NUE Garden Numbers Color ae Pi light pink 3 12F3H.P3Pi4 light pink estes Fonp 14 6 12F3H4P2Pis pink ait P45) S600 v 12F 3H» Pi3P4s light pink ation 5080 25 12 13F,H.PaP2Ps pink socn 88 ooe5 12 13F4H4P4Pi2P 9 pink cand 100 atoll 1 12F3H»P7Pas light pink 5 19 (or light pink) 2. 12F3H.P3P30 light pink 1 24 (13 light pink) 5. 12F;HyPaPis pink 8 16 (or light pink) 9 12F,H.PaPs pink 4 | 21 | (or light pink) 10 | 9 12F.HPaP, pink 3 | 21 | Glight pink) 11 "MOE 3H4P4:Po pink 9 16 (0 light pink) 12 pink 10 15 (0 light pink) 13 pink 4 21 (2 light pink) 14 pink 6 19) (115 eee 7/ pink 3 22) |) ee Meseteares 18 * pink 8 Nie | | RAercees: 19 12F3H4P3sP.3 pink 25 (5 darker red) 187 (Types 2 and 19 excluded) fer) o Totals of segregating populations 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 483 4 are assembled the data from ptnk flowering selections from popu- lations which showed segregation into red and pink. In this table the populations which bred true for pink are assembled in the upper portion of the table, and those which showed further segregation into red and pink are assembled in the lower portion. Of the 18 selections made, 7 bred true for pink (or light pink), and 10 gave segregation in the succeeding generation in about the ratio of 3 pink :1 red. The total figures for the 10 populations—187 pink : 60 red—are in very satisfactory agreement with the simple Mendelian ratio. The family of type 19 behaved in an anomalous fashion, which may indicate mis- classification of the F’, parent; and the family of type 2, which showed only one red plant has been included among those which bred true for pink. Strictly ight pink selections should have given only light pink flowers in subsequent generations; the pink ones should all have give segregating populations. The evidence indicates that this iad ‘ be obtained if segregation-occurred for only one pair tof Mors. The difficulty, in part at least, appears to be the as. adi of modifying factors in the populations. These ae \ ip parently weave an effect on flower color ugh bea th Br cation into -_ hi ig but ni enough \t® Obscure the segregation into .” init % abba hee sults in Nh an approximate agreement .. expectation, but the breeding test clearly is necessary in order to determine the actual distribution of the pink individuals into their genetic classes. 6. LATER SOWINGS OF F, AND F, OF THE ANGUSTIFOLIA-— MACROPHYLLA SERIES In 1916 and 1917 certain families of F, and F,, of H, were grown in order to reéxamine them in the light of data previously collected and to determine whether or not any more definite classifications gould be made than those stated in the preceding pages. The populations grown are described briefly below. & 16F,H.P,, as the population number would indicate, _svaite sowing of seed of 10F,H,P, from the original F, populatio “of me, As in previous cases, the segregation as regards leaf sha as to preclude definite classification. The types previously noted for second generation populations were all in €vi ee and along with them practically every sort of intermediate. The height of plants and general habit likewise agreed with the description previously given. 484 University of California Publications in Botany [ VoL. 5 It was possible as in previous instances to segregate the plants into definite flower color classes. In order to make this segregation as accurate and free from bias as possible a special method of eclassifica- tion was adopted. At the height of the blooming season, single typical flowers were collected from each plant of a population and placed in vials correspondingly numbered. These specimens were then taken into the laboratory, where they could be classified under optimum light conditions. The specimens so collected could then be shifted around into their phenotypic classes and properly compared with each other and with the parent colors. The color classification thus obtained was individually recorded, and later the population was checked over in the field to insure correction of any errors of classification. The surprising feature of this population was a sharp, three-class segre- gation into red, pink, and hght pink; the reds the shade of macro- phylla, the light pinks almost exactly that of angustifolia, and the pinks intermediate between the two. Within the classes there ap- peared to be no significant differences in depth of shade. Two plants bore no flowers. The ratio obtained was 15 red : 23 pink :10 light pink. ; 16F,H,P,, was likewise a sowing of the seed of one % he iin F, plants, in this instance of 10F,H,P,,. As respects habit, height, and leaf shape, there was a strict resemblance throughout of this population to the one described above. Flower color was studied in the same manner and with substantially the same results. However, in this population there was a shading off from pink to light pink, such that it was impossible to draw a sharp line between these two classes as was done in the previous population. The shading off was abrupt, but there were, nevertheless, a few plants on the border line. The observed ratio was 16 red : 34 pink and light pink. YW 1917 six I’, populations, each containing approximately 100 nape. It was impossible, however, to study these plants as as might have been desired on account of conditons ob- - 1917. However, specimens of leaves from each plant preserved and these were studied and classified in the summer of 19 A brief account of each population follows: i WEEP a owing from 16F,H,P,,P,, a STENOPHYLLA selection. With respect tOrleat base characters the segregation was roughly but rather obviously ‘imto two types, a long petioled sTENo- PHYLLA class approximating type 1 in appearance, and an AURICULATA taining duri were pressed 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 485 class approximating type 10. Within the sTmENopHYLLA class there was a variation in the amount of ‘‘wing’’ on the petiole and in the type of blade base, some having the abrupt base of type 1, whereas others had an attenuated type of blade which gradually drew in to the petiole. In the AuRIcULATA class there was also a variation from the strict form of type 10 to a type which lacked the flaring auricle typical for that form, and had a very short naked petiole. In addition to this variation in the amount of ‘‘wing’’ of the constricted class there was also a difference in the presence or absence of attenuation noted for the STENOPHYLLA class, some plants having leaves abruptly drawn in to the midrib, whereas others were very markedly attenu- ated. The difference in this respect appeared to be equivalent in the two distinct classes, i.e., it was independent of any difference in the ‘“netioled’’ or ‘‘constricted’’ condition. With respect to a VS. AURICULATA the segregation was 66 STENOPHYLLA : 32 AURICULATA. © 17F,H,P,,P, was a sowing from 16F,H.,P,,P,, anoth STENO- PHYLLA selection. The leaf classes obtained here were two, STENO- PHYLLA (type 1) and sEssmironiA (type 15). The segregation into the two classes was distinct, but, as in other populations, there was a great deal of yt in each class. \was attenuation of the egperevioustyiady in b fi) ane Som of the petioled individuals had distinct wings, ut hs reer number were naked. Some few individuals had very short petioles. The segregation ratio was 76 STENOPHYLLA : 24 SESSILIFOLIA. 17F,H,P,,P,, was a sowing from 16F,H,P,,P,,, a STENOPHYLLA selection. The population was remarkably uniform in leaf shape, which closely approximated angustifolia with minor differences. The straplike leaves which are a characteristic feature of the upper por- tions of plants of angustifolia were lacking in this population, and the leaf tip and distal portion of the leaf blade did not narrow so gradually in this population as in angustifolia. Otherwise, the char- acters of the plants throughout were closely similar to angustifolia. 17F.,H.P,,P.. was a sowing of seed of 16F,H,P,,P.., a STENOPHYLLA selection. Of the six F, populations studied, this one exhibi greatest diversity in segregation. With respect to leaf base there were two outstanding classes, STENOPHYLLA and SES 15), which could be separated readily. Within the sr however, most of the individuals exhibited a more dition. Within the sessiiroui class, on theother hand, most of the individuals exhibited more or less narrowing of the leaf base, like # 486 University of California Publications in Botany [ Vou. 5 type 14. A few of the sessile individuals, instead of exhibiting gradual and uniform narrowing toward the base of the leaf, were constricted to a degree intermediate between AuRICULATA of type 10 and SESSILI- FOLIA of type 15. With respect to STENOPHYLLA versus SESSILIFOLIA the observed segregation was 67 STENOPHYLLA : 32 SESSILIFOLIA. 17F,H.P,,P, was a sowing of seed. of 16F,H,P,,P;, an F, SESSILI- FOLIA selection. The leaves throughout had the sessile type of leaf base characteristic of macrophylla, but there were many modifications of it in the population. A rough classification with respect to these modifications of the macrophylla type of leaf base gave the following results : On 59 plants, the leaf bases were very nearly the form typical for macrophylla. On 22 plants, the leaf bases were gradually attenuated toward the base, resembling LANCEOLATA of type 13 as a mean. This at- tenuated form of the sessile leaf was a very striking feature of this population. On 10 plants, the leaf bases were intermediate in type between LANCEOLATA of type 13 and the typical macrophylla form. On 2 plants, the base of the leaf immediately above the point of attachment was noticeably constricted, the leaf base thus formed being intermediate between the macrophylla type and AuRICU- LATA of type 10. , On 2 plants, the leaves were intermediate in constriction of the leaf base between the strict macrophylla type and that of the two plants described immediately above. The classification here given is presented only to show that the sessile type of leaf base characteristic of macrophylla is subject to a number of very definite modifications which probably account for some of the complex types of segregation observed in other populations. 17F,H,P,,P,, was a sowing from 16F,H,P,,P,., a SESSILIFOLIA selection, With respect to leaf base segregation there were two dis- tinctclasses, SESSTLIFOLIA (type 15) and aAurIcULATA (type 10). There was also a marked degree of variation within the classes. Within lass the variation was in amount and kind of narrowing le toward the base. A few plants showed a condition AURICULATA type in this respect, whereas others showed a gradu tenuated form of narrowing such as has been noted before in other populations. Within the AuRIcULATA class most of the individuals instead of possessing the slight wing and flaring the sessi of the lea approaching t 1922 | Setchell-Goodspeed-Clausen: Nicotiana Tabacum 487 auricles of type 10 had short naked petioles. A few were strictly of type 10. The following segregation ratio was noted: 61 SESSILIFOLIA : 27 AURICULATA. 7. CROSSES OF DERIVATIVES WITH THE PARENTS In the preceding account we have pointed out that by growing definite hybrid selections in the pure line through a number of gener- ations it has been possible to establish a certain number of stable derivatives which represent more or less obvious recombinations of characters of the original parents. In a Mendelian sense, they repre- sent stable reorganized germinal complexes containing hereditary ele- ments that have been derived from both parents. Obviously such recombinations of Mendelian units must differ in fewer units ff either parental type than did the parental types from each other. test some of these derivatives we have crossed them with the inal parents, usually with the one to which they bore the eloses blance, in order to observe how complex a type of Perceation the hybrids thus obtained would exhibit as co mpoTog with that of the — angust ape ophylla wary In yi far as they have been ‘studied to ‘il description of thes e hybrids and their progenies follows: SESSILIFOLIAxmacrophylla. F\, SESSILIFOLIA of type 20 was crossed with macrophylla giving H., type 209 x macrophyllag and H,, = reciprocal thereof. The derivative parent here very closely resembles macrophylla throughout in flower color and shape, habit, leaf shape, ete. 15F,H,, and 15F,H.,, two families of 50 plants each, were equiy- alent in every respect. The plants were very close indeed to macro- phylla, as is also the SESSILIFOLIA parent. The only difference readily observed was some variation in the amplitude of the corolla. In H,,, a plant with a larger and one with a smaller corolla were selected for pure seed. In F,, grown in 1916, one family of H,, and two families of H,,, of 50 plants each were grown. The flower color i populations was throughout that of macrophylla and the ters also were those of macrophylla. All three families were remark- ably uniform, not only agreeing with one another b indivduals. They all resembled closely the macrophylla type and there The three popula- tions appeared to be replicas of macrophylla throughout except that they were slightly more robust. was no definite segregation of any kind in the 488 University of California Publications in Botany | Vou. 5 LATIFOLIA x angustifolia. KF LATIFOLIA of type 6 was crossed with angustifolia giving H,,—type 69 x angustifoliag and H,,, its recip- rocal. The derivative parent possessed the short winged petiole char- acteristic of LATIFOLIA of type 6. In F, 50 plants of each cross were grown. They exhibited the long naked petiole characteristic of angus- tifolia. In F, two populations of 50 plants each were grown. In color of flowers the two populations were light pink throughout, closely cor- responding in this respect to angustifolia. In leaf shape the segre- gation was sharply into two classes: the STENOPHYLLA type of leaf base (long, naked petiole) and the LATIFoLIA type (shorter, winged petiole). There was some variation in the STENOPHYLLA class sug- gesting intermediacy between angustifolia and LATIFOLIA, but the forms exhibiting it showed a graded series from strict STENOPHYLLA to inter- mediate. The LATIFOLIA class was very uniform and sharply set off from the other class. The segregation ratios observed were as follows: STENOPHYLLA LATIFOLIA 16FH52Pis 42 8 if 6 FH 33Ps35 36 14 Totals 78 22 |! AURICULATA x macrophylla. EF; AuRICULATA of type 6a was crossed with macrophylla giving H., and H,,—=F, type 6a x macrophyllag and H,, and H.,, their reciprocals. It should be observed that type 6a is an early segregant from the LATIFOLIA of type 6 of H,, and H,,. In F, 50 plants were grown of each of the four parents. All four populations were equivalent in every respect. All the plants had pink flowers, although one plant had flowers of a lighter shade than the others, and leaves of a shape somewhat intermediate between the two parents, i.e., they were more contracted at the base than macrophylla, bufgmuch less so than those of type 6a. One plant of H,,, namely 15F,H,,P,,, showed larger corollas than any of the other F, plants of any ‘family, and was selected for further breeding. ur families were raised and they proved to be equivalent except as noted. There was sharp segregation for leaf shape into the§esstuiroi1a and the sharply constricted AURICULATA type. In the sSEssmiro class there were a number of obvious intermedi- ates, as might be eXjected from the characters exhibited by F,, but they formed a continuotissseries with the strict SESSILIFOLIA forms. The AURICULATA class did not intergrade with the dominant class. 1922 | Setchell-Goodspeed-Clausen: Nicotiana Tabacum 489 Segregation for flower color was studied by the method described above. The color distinction between red and pink was sharp and easily drawn. In the pinks, however, there was a continuous series of shades from the deep’ rose pink characteristic of F, to the light pink typical for angustifolia. Numerical data are given in table 5. TABLE 5 H F2 SEGREGATION OF PINK AURICULATA X RED SESSILIFOLIA. Pink Red Pink Red Garden Numbers sessilifolia sessilifolia auriculata auriculata 16F.H54P7 32 6 9 3 16F2H5;Pis 23 11 lat 5 16F2H;; Po 26 13 7 a 16 F2H355Po5 26 11 a 2 16F2H359.P35 26 12 on 2 Totals 133 53 40 16 Expected 136 45 45 15 In 1918 in connection with flower ine yee three more popula- i of F.H,, Mi Ta Bere were collected from each plant and pressed, and leaf shape studies were made on these preserved specimens. The studies were not so satisfactory as those made in the field, where it is possible to examine all the leaves on a given plant; nevertheless, the data derived from the studies agreed substantially with those obtained in 1916 from field studies. It was noted in these studies that there was a distinct class of ‘‘attenuated’’ leaves similar to those which have been described in previous populations. Both attenuation and constriction were observed to occur in the leaves of some individuals, and this gave rise to some difficulty in classification. Numerical data are given in table 6. TABLE 6 ¢ F2 SEGREGATION OF SESSILIFOLIA X AURICULATA. ‘ Garden Numbers SESSILIFOLIA Avmrcpadera 18F.H55P10 67 of bs 18F.H;5P 1 79 18 18F.H5;Pas 79 17 Totals 225 63 490 University of California Publications in Botany [ Vou. 5 STENOPHYLLA x angustifolia. Reeiprocal crosses were made between F’;, STENOPHYLLA of type 21 and angustifolia; H,, =F, type 219 x angustifoliag and H.,,,, its reciprocal. SrENOPHYLLA of type 21 has been deseribed previously as a stable derivative closely approximating angustifolia in all its characters. KF, families of 50 plants of each hybrid were raised in 1915. They were uniform throughout and so close to angustifolia in all characters as to be indistinguishable from it. One plant seemed to be of a slightly darker pink corolla color. 15F,H.,aP,, was the only F, family raised. The flower color of this population was about the shade of angustifolia and uniform through- out the population. The family showed only a slight variation in the base of the blade such as is also seen in populations of angustifolia. 8. DISCUSSION OF RESULTS OF THE ANGUSTIFOLIA—-MACROPHYLLA SERIES OF INVESTIGATIONS Obviously the outstanding result of this series of investigations of hybrids between angustifolia and macrophylla is a demonstration of the complexity of the germinal differences which exist between the two varieties with respect to practically every character contrast which may be made between them. Only in one instance, the contrast be- tween the light pink flower color of angustifolia and the red of macro- phylla, is a simple Mendelian formulation possible. Here evidently the main flower color difference is dependent upon a simple allelo- morphie contrast. Red x light pink gives F, intermediate pink, and F, 1 red : 2 intermediate pink :1 light pink.. The red segregants breed true for red, the light pinks for light pink, and pink continues to segregate in the typical mono-hybrid fashion. Inasmuch as the inter- mediate pinks and light pinks form an intergrading series, it is con- venient to look upon red as the recessive color. Accordingly we give this pair of factors the designation, Rr, following the mnemonic system advocated by Morgan, and shall so refer to it in what follows. The difficulty among the pinks appears to be due not only to phenotypic n but also to the existence of modifying factors which have a slight effeet upon flower color expression. These less strik- ions of flower color we are seeking to analyze further. iape investigations, the complexity of the results is an examination of the data presented in the fore- going pages. Althou the behavior here is complex, in every feature it parallels the Me ndelian expectation for complex factor relations. variat relativels ing modifica In the lea plainly evident fr 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 491 In F, the variety of leaf shapes encountered was nothing short of bewildering and series could be built up from them showing complete intergradation from one type to another. Selection of phenotypes from F,, however, gave F., populations in which the complexity of segregation was usually reduced in a very definite fashion. Most of the populations exhibited fewer classes than F’,, and the selection of F, phenotypes held the expression of F, within very definite limits. Thus selection of SESS™IFOLIA forms gave in F, either all SESSMmIFOLIA or approximately 3 SESSILIFOLIA :1 AURICULATA. In no ease did such selections give F, populations with STENOPHYLLA or LATIFOLIA leaf types. A summary in detail of the type of populations produced is as follows. STENOPHYLLA selections may segregate in a variety of ways. Thus, type 1 showed approximate segregation into 3 STENOPHYLLA : 1 SESSI- 4 LIFOLIA. Type 21 bred true to the STENOPHYLLA characters. Anondll STENOPHYLLA selections grown in 1917, population 17F,H,P,,P,, showed approximate segregation into 3 STENOPHYLLA : 1 ae. 17F,H,P,,P,, 3 STENOPHYLLA:1 sSESstLIroLIA; 17F,H,P,,P, bred true for STENOPHYLLA, and 17F,H,P,,P,, gave a rather indefinite segrega- jon of approximately 3 STENOPHYLLA % \sessmironiA. Larrronta ee vas crossed with angustifolia gave F, srenopryiia and F, approximately 3 STENOPHYLLA : 1 LATIFOLIA. LATIFOLIA selections also segregate in perplexing fashion. The F, population of angustifolia x macrophylla is typically LATIFOLIA in its characters. LariroutA under certain conditions therefore 1s a very complex hybrid expression. Recurrence of complex segregation of a LATIFOLIA selection is shown in F, of type 2. F., of type 3 exhibited a rather anomalous segregation ratio of petioled and sessile forms. Type 5 apparently bred true, although there was one anomalous plant in the population. Type 6 exhibited complex segregation, with an indication of a ratio of 3 LATIFOLIA :1 AURICULATA; with subsequent establishment of both LAaTrrOLIA and AURICULATA In constant races. Type 7 bred true for a type of leaf hke F,; and type 9 gave approxi- mate segregation of 3 LATIFOLIA : 1 SESSILIFOLIA. LORUFOLIA and LANCEOLATA derivatives are really varia SESSILIFOLIA type. They were both produced in constan s of the s. Their ll established y are really quan- genetic relation to the other forms is, however, no by this series of investigations. Although these titative variations from the strict SESSILIFOLIA.type, nevertheless, cer- tain of our-data indicate discontinuous inheritance of these contrasts. 492 University of California Publications in Botany 1 VoL. 5 The same quantitative factors that differentiate the narrow-leaved forms of SESSILIFOLIA from the typical broad-leaved forms may apparently differentiate narrow-leaved STENOPHYLLA, LATIFOLIA, and AURICULATA forms from the more typical broad-leaved ones. It is of interest in this connection to note that LORUFOLIA derivatives have much nazrower leaves than either of the original parents. We have been especially interested in these LORIMFOLIA derivatives beeause they are somewhat like the narrow-leaved forms that Hassel- bring (1912) found among Cuban tobaccos, and which are so well recognized among Cuban growers as to have received the specific desig- nation of lengua de vaca or ‘‘cow’s tongue.’’ Our results indicate that it is possible for such forms to arise by segregation from crosses between broader leaved forms. The lengua de vaca of the Cuban growers is, therefore, probably a segregation product which could easily be eliminated by the adoption of proper pure line methods of breeding. Avricuuata forms appear to breed true whenever segregated. The exception is type 8, which requires further investigation. It may be a leaf type similar to AURICULATA but of different genetic constitution. AURICULATA of type 10 bred true in F,. The AuricunaTa form 6a, which segregated from type 6 bred true thereafter. AURICULATA crossed with macrophylla, H.,, H;;, H;;, and H;, gave SESSILIFOLIA in F, and in F, 3 SEssILIFOLIA : 1 AURICULATA. SEsSILIFOLIA forms have broad sessile leaves, the distinguishing feature being merely their sessile mode of attachment. Of such selec- tions from the original F,, populations, four, with the exception of one anomalous plant, bred true for susstuiroutA. Each of the other three populations segregated into SESSILIFOLIA and AURICULATA In about the ratio of 3 SESSILIFOLIA :1 AURICULATA. Two SESSILIFOLIA selections were grown in 1917. One of these bred true to SESSILIFOLIA ; the other gave 3 SESSILIFOLIA :1 AuRICULATA. The behavior of SESSILIFOLIA in relation to STENOPHYLLA and LATIFOLIA is explained above. On the basis of these results we may distinguish certain definite allelomérphie pairs of factors as follows JOPHYLLA versus SESSILIFOLIA: SS being long petioled like and ss broadly sessile like macrophylla. The heterozygote roach an intermediate condition similar to LATIFOLIA. versus LATIFOLIA: LL being long petioled like angustifolia, and ll Short petioled like LatironiA and with a distinet but not broad wing. The contrast is really one of SSLL, stENOPHYLLA may possibly 4 Ll, stENOPHY 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 493 versus SSI], LatmourA. Both ssLL and ssll are probably typical sEsst- LIFOLIA forms. Here again the heterozygote probably shows an indis- tinet type of intermediacy. Aa, SESSILIFOLIA versus AURICULATA: AA having the broad clasp- ing leaf base characteristic of macrophylla, and aa the deeply con- stricted leaf bases with flaring auricles characteristic of AURICULATA. The contrast here is really one of ssAA, SESSILIFOLIA versus SSaa, AURI- CULATA, for these factors are evidently latent when in combination with SS or Ss. Fig. 2. Leaf base types of the angustifolia-macrophylla series. Left to right: STENOPHYLLA, LATIFOLIA, SESSILIFOLIA, and AURICULATA. Some of the possible genotypes, their phenotypic expression, and genetic behavior are included in table 7. Here only monohybrid segre- gation is considered because it is doubtful, on account of the various types of intermediacy shown by heterozygotes, whether it would be possible to classify dihybrid and trihybrid populations satisfactorily. TABLE 7 GENETIC BEHAVIOR OF VARIOUS LEAF TYPE GENOTYPES. Genotype Phenotype Genetic behavior SSLLAA STENOPHYLLA Breeds true SsLLAA STENOPHYLLA 3 STENOPHYLLA : 1 SESSILIFOLIA SSLIAA STENOPHYLLA 3 STENOPHYLLA : 1 LATIFOLIA SSLLAa STENOPHYLLA Breeds true vd SsLLaa STENOPHYLLA 3 STENOPHYLLA : 1 AURLEULATA SSIIAA LATIFOLIA Breeds true SsIIAA LATIFOLIA 3 LATIFOLIA ESSILIFOLIA Ssllaa LATIFOLIA 3 LATIFOLIA #1 AURICULATA ssLLAA SESSILIFOLIA Breeds true , ssllAA SESSILIFOLIA Breeds trué ssllAa SESSILIFOLIA 3 SESSILMPOLIA : 1 AURICULATA ssllaa AURICULATA Breeds true 494 University of California Publications in Botany [ Vou. 5 Although intermediacy of the heterozygote appears to be the normal thing in these leaf shape contrasts, it is proper to state that this inter- mediacy may depend to some extent upon the effect of modifying factors rather than upon the heterozygous conditon of a pair of allelo- morphs. Thus the intermediate conditon between STENOPHYLLA and AURICULATA is met with in populations which do not contain AURICU- LATA segregation products. There are so many modifying factors in this series of investigations that it is probably impossible for us to declare definitely that in any one instance our observed segregation was wholly the result of segregation of one pair of allelomorphs. Further investigations are in progress, the purpose of which is to isolate and evaluate, if possible, certain of these subsidiary factors. For the present we can only state our certain knowledge of their existence, and our belief as to their various effects. IV. CALYCINA-VIRGINICA SERIES The calycina-virgimea series of hybrids and derivatives has received much less attention than has been given to the previous series; partly because the differences between the parents are less striking and the diversity of segregation products was not so great. Two hybridizations were made: H,, which had calycina for the female and virginica for the male parent and H,, which was the reciprocal cross. 1. PARENTS OF THE CALYCINA-VIRGINICA SERIES Elsewhere Setchell has given descriptions of calycina and virginica (‘‘Maryland’’). Like angustifolia and macrophylla, these two varie- ties possess distinet sets of characters which set them apart from the other Tabacum varieties that have been grown in the University of California Botanical Garden. Calycina is represented in our cultures by a variety, U. C. B. G. 110/05, which was originally received from the Botanical Gardens of Cambridge University. The figure previously published (ef. Setchell, loc. citi pl. 4) well represents the general habit and type of the plant. The particular features of the characteristic teratological flower of yetter shown in plate 79, in which the leaf shape is also ore characteristic fashion. For illustrations of some calycina a illustrated in ich occur in the expression of the split hose-in-hose erred to Goodspeed and Clausen, 1917, plate 45. The legends to the two res of this plate should be reversed as indi- cated in the references to the plate in the text of this earlier article. of the variations flowers the reader is 1922 | Setchell-Goodspeed-Clausen: Nicotiana Tabacum 495 In stature, as previously mentioned, calycina belongs to the low corymbose group of Zabacum forms. In height the central axis usually varies between 60 and 75 em. The laterals, however, which develop later from the base, overtop the central axis and reach a height of 120 to 185 em. Like macrophylla, central axis and laterals bear close panicles of corymbose racemes, the laterals developing successively from the base. The stems and branches are stouter than those of angustifolia and the leaves do not droop so considerably. In these respects calycina occupies an intermediate position between angusti- folia and macrophylla. The leaves of calycina, as plate 79 will show, are sessile, but they are distinetly different from those of either angustifolia or macro- phylla. Curiously enough, however, they do rather closely approxi- mate certain of the derivatives of the angustifolia-macrophylla series, as, for example, the LANCEOLATA and LORIFOLIA leaves of types 13 an 14 respectively, illustrated in plates 75 and 76. The leaves vary from broadly to narrowly lanceolate, tapering toward both base ind apex, and usually with a long curved tip. The broader leaves he borne at the base of the plant, those above them becoming successively narrower in a continuous series until the linear leayes or ‘straplike bracts of the nflorescence a reached. There are no , at the base of the leaf. ~The inflorescence is in the form of a very close panicle of racemes, the secondary axes of which are mostly patent, and more or less re- eurved or bent back. The flower as a whole is of a very characteristic spht hose-in-hose type. The corolla is usually split on one side, some- times twice split, and more or less eurved. The characteristic splt- ting of the corolla is seen even in very young buds and often the pistil protrudes from them. Typically the calyx has an elongated whitish green tube, with 3 to 5 of its tips more or less petaloid. Sometimes strips of petaloid tissue extend down the entire length of the calyx. The pod is ovoid oblong in shape. As it enlarges it splits the calyx, which then withers and drops off like the corolla, leaving a naked, whitish green capsule. The flower color is red fading to bluish purple, apparently the same as that of macrophylla. Virginica is represented by U. C. B. G. 78/05, a strai from the United States Department of Agriculture und fication number ‘‘205-20-7.’’ It is figured by Setch by Goodspeed and Clausen, plate 41, figure 1. Th flower characters are well represented in plate 8 In stature virginica belongs to the moderate pyramidal group of Tabacum varieties. It is conspicuously taller than calycina, the cen- ‘ 496 University of California Publications in Botany [Vou. 5 tral axis reaching a height of 150 to 175 em. While strong laterals develop they do not originate at the base of the plant as in calycina, and they do not overtop the central axis. These characteristics to- gether with the broad spreading basal leaves give the plant its pyra- midal or conical shape. The leaves are very close in general shape to those of calycina, but they taper less abruptly to either end. The apex is prolonged into a fairly long point curved to one side, and the base is expanded into two broad, partially clasping auricles. The inflorescence consists of a more ample panicle than that of calycina. The flowers are light pink in color, identical in this respect with those of angustifolia. The tube and infundibulum are narrow, gradually increasing in diameter from below in a funnel-shaped fashion. The corolla lobes are broad at the base, but have long, slender incurved points. Capsule and calyx present no very charac- teristic features, although the calyx is persistent in contrast to the deciduous ealyx of calycina. It will be seen from the foregoing decor that there are a number of distinct character contrasts between calycina and virginica, a brief note of which may well be made at this point. In flower color, red of calycina is contrasted with light pink of virginica, the same contrast which existed in the angustifolia-macrophylla series. Simi- larly the split hose-in-hose flower of calycina is contrasted with the normal one of virginica; low stature with tall; and a less decided contrast in leaf shape exists, depending upon the presence or absence of auricles at the base of the leaf. On F, OF THE CALYCINA—VIRGINICA SERIES In the season of 1910, 55 plants of 10F,H,, and 58 of 10F,H., were grown. In the season of 1911, 10 plants were grown of each of APH, andthe _ Like other hybrids which have been grown, these populations were uniform and equivalent throughout. It was thought that 10F,H., showéd a more distinctly pronounced trace of calycina characters than did 10% H,,, but the populations of the same seed grown in 1911 showed ppreciable difference in this respect. appearance the F, plants resembled virginica more than The plants were somewhat shorter than virginica, em. In these plants it was noted that some of the laterals overtopped central axis as they do in calycina. The in- florescence was in general of the ample type characteristic of calycina. In gene they did calyc running up to 1 1922 | Setchell-Goodspeed-Clausen: Nicotiana Pabacum 497 The flower color was a deep pink intermediate between the two parents. The flower shape was normal throughout save that on every plant there was a small percentage of calyces with one or more pink and somewhat broadened ealyx tips, or with a streak of white on one side. Sometimes these partially petaloid calyces were partly decid- uous. In shape of flower the hybrid closely resembled virginica except that the corolla lobes were longer and more decidedly mucronate. Calyx and capsule were almost identical with those of virginica, the calyx being typically persistent and acerescent. The leaves were some- what broader proportionately than those of virginica, but they pos- sessed the pronounced auricles of that parent. The usual gradation in leaf shape on each plant from the broad basal leaves to the linear bracts of the inflorescence was in evidence. - The main features of H,, and H.,, are well illustrated in the draw- ings of 10F,H,,P, shown in plate 81. The general habit and charac- ters are illustrated in the photograph of 10F,H,,P.,, which is repro- dueed in plate 83, figure 1. ) 3. F, OF THE CALYCINA-VIRGINICA SERIES In the season of 1911 four F, families of the calycina-virginica series were grown, viz., 11F,H,,P,,, 11F,H,.P,,, 11F,H.,P,, and iP). , As in the angustifolia-macrophylla series, the segregation exhibited in those four families, comprising 97 plants, was nothing short of be- wildering, and in most cases an intergrading series of forms connected one character expression with another. However, an attempt was made to classify the plants into categories suggested by the four pairs of character contrasts existing between the parents. The results of this classification are given in table 8. TABLE 8 NUMERICAL DATA FROM F: POPULATIONS OF THE CALYCINA-VIRGINICA SERIES. Corolla color Corolla shape Stature Leaf width Bl lag) 4 : Garden Numbers 3 al & 2 |e | 6 5 ele te | cel |) Eteach ge q 11F.AisPo5 6 | 11 6 6 8 9 a 11 F.Hy3P 49 7 | 14 4 | 12 4 9 11 11 FHP; 5 9} 11 Ui 8 | 10 4 11 FH Po¢ fy |) Alo) 8 | 10 6 a 8 Totals 23) || 44 || 29) "35" 26) aso 67 | 30 498 University of California Publications in Botany [ Vou. 5 In this cross, corolla color behaved in exactly the same manner as it did in the angustifolia-macrophylla series. The same remarks as to sharpness of segregation apply here as in that series. Red was nearly always readily distinguishable, but pink and light pink formed a more or less completely intergrading series. Taking the results in this way, we obtain 23 red; 73 pink and light pink, which is substan- tially in accord with Mendelian expectations. TABLE 9 F,; SEGREGATION IN CALYCINA-VIRGINICA SERIES. Pink Pink Red Red + Garden numbers normal hose-in-hose normal hose-in-hose 16F2HisPo5 24 14 8 3 16F2HisPa9 25 8 14 3 16F2H.2P7 29 6 10 4 16 F2H2P3s 30 8 11 1 Totals 108 36 43 11 Expected 112 37 37 12 With respect to corolla form some difficulty was encountered be- cause the expression of the hose-in-hose character in the segregants did not seem to be so extreme as it was in the parent, and a large number of the plants showed slight traces of it, but sometimes in a more pronounced form than in the F, hybrids. Accordingly the elassi- fication of corolla form in table 8 is not a wholly satisfactory one. The classification for stature and leaf width is subject to similar remarks as to its definiteness. Here there was also a more or less completely intergrading series of forms and no accurate measurements were taken. However, there is no doubt that there was segregation with respect to these characters, and a range of forms was obtained which completely bridged the gap between the parents. The behavior of ‘these characters is to be considered in the light of their segregation sequent generations. 16 four additional F., populations of the calycina-virginica series grown in order to reéxamine populations for the segrega- tion of nortial versus hose-in-hose flowers, and red versus pink flower 10d of classifying the flowers was that used in studies of 1916 populations previously mentioned. The results of these studies are given in table 9. eolor. The m 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 499 In the segregation the same grading as before of the pinks into two intergrading classes in the proportion of approximately 2 inter- mediate pink : 1 light pink was observed, but it was even more difficult to draw a line between light pink and intermediate pink because of the effect of the hose-in-hose conditon on flower color expression in those plants which bore teratological flowers. In the matter of segre- gation into normal and hose-in-hose flowers, some difficulty was ex- perienced because some otherwise normal flowering plants bore some flowers which showed a tendency for the calyx to become petaloid, and others bore flowers which showed a very slight hose-in-hose ten- dency. A correspondingly slight hose-in-hose tendency is also present in F, plants. These plants were classified as normal. Here again it can be seen that the segregation ratios of 144 pink : 54 red and 151 normal : 47 hose-in-hose are in substantial agreement with Mendelan expectation for contrasts in a single pair of allelomorphs in each ease. Moreover, the dihybrid ratio is substantially in agreement with that expected for independent segregation of the members of these two pairs of allelomorphs. 4, FB, AND SUBSEQUENT GENERATIONS OF THE CALYCINA-VIRGINICA SERIES In 1912 twelve F’, families of H,, and five of H,, were grown. They will be grouped for consideration according to the characters which TABLE 10 F; BEHAVIOR OF RED SEGREGANTS. Garden numbers Red 12F3HysP2sP 1 25 12F3HisPo5Pi9 DNS) 12F3HigP49Pi2 25 12F3AygP49Po4 25 12F 3H P 25 Pos 25 the F, parent exhibited. In table 10 the data with respect t havior of F., populations from red flowering I, plants ar Five such populations gave nothing but red flowering pla clearly that red segregants breed true. In table 11 th flowering plants are similarly collected. The readér will not fail to notice that some pink flowering selections were not heterozygous for 500 University of California Publications in Botany [ Vou. 5 red. This bears out our statements as to the difficulty of classifying pink and light pink. In the seven populations which produced red flowering plants 38 plants had red flowers and 134 pink or pinkish flowers; again in substantial agreement with the behavior of flower color in the angustifolia-macrophylla series. The behavior of segre- gants classified as light pink is shown in table 12. Of the four popu- lations from which data were gathered only one bred true to light TABLE 11 F3; BEHAVIOR OF PINK SEGREGANTS. Garden numbers Red Pink Light pink 12F3Hj3P.;P7 5 16 4 12F3Hy3P.;Po1 ea 21 4 12F3HisP25Po5 6 19 : 12F3HisP49P5 4 20 12F His P4sPio 11 13 ae 12F3HigP49Po5 4 16 5 12F3H2PosPe5 4 20 12F3H PoP 4 21 Totals for segregat- ing populations 38 134 TABLE 12 F; BEHAVIOR OF LIGHT PINK SEGREGANTS. Garden numbers Red Pink Light pink 12F 3 AisP2sPo4 FAs eee 23 12F'3 HigP49P 20 8 12 5 12F3HP7Ps 6 15 4 12F 3H. P2sPs er | 25 pink, one of the others bred true for pink, possibly a slightly darker shade than true light pink, and two segregated for all three colors; they must therefore have been pink heterozygotes. 1 F, two populations each of H,, and of H,, were grown. Popu- lation#13F,H,.P,.P,,P, from an F, population breeding true for red gave i 100 plants all red flowering. Population 13F,H,,P.,P,,P,, from the Same F, population gave 97 plants all red flowering like uation 13F,H,,P.,P.P,, which bred true for pink in F,, gave in F, 96.plants, all pink flowering. These three populations were grown to F, without showing further evident segregation. The calycina. 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 501 pink of the pink flowering derivative was at first considered somewhat darker in shade than the light pink of virginica, but this line also showed the hose-in-hose flower character, which sometimes makes it difficult to determine flower color accurately. In later generations of this line its color was noted as equivalent to the light pink of virginica. TABLE 13 F; BEHAVIOR OF HOSE-IN-HOSE SEGREGANTS. Garden numbers Hose-in-hose 12F3HisP25P.1 25 *12F3H13Po5Po4 23 12F3HisP49P5 24 *12F3AigPa9Pi2 25 12F3AysP19Po4 25 12F;H2P26Ps 24 1 2F 3H Pog P11 25 * Apparently not so extreme as calycina. TABLE 14 F; BEHAVIOR OF NORMAL SEGREGANTS. Garden numbers Hose-in-hose Partial Normal 12F3HjgP2;P7 9 16 12F3Hy3P25Pi1 4 4 17 12F3HigP25Pis 5 20 12F3HisP25P25 3 ae aD, *12F3HisPs9Pi0 Nee 4 20 12F3HigP49P22 ass 25 12F3HigP49 Pos a 21 12F3H.P7Po 5 20 = 12F 3 HPP; Ul 3 15 12F 3H P26 Pos 10 15 Totals 47 153 Taking up corolla form next, we may deal with the different popu- lations in the same manner as was done in the ease of flower color. F, populations from F’, hose-in-hose segregants are recorded in table 13. Seven populations were grown, all of which bred true to the hose-in-hose character, although curiously enough two pop Zhe b>, and 12h,H Pb did not appear to exh treme character expressions as calycina. Only one partially hose-in-hose plant was grown i a) Hor the sake of economy of space it is included in table 14, where it is marked 502 University of California Publications in Botany [ VoL. 5 with an asterisk. Strangely enough, it was one of the two in the table which did not throw hose-in-hose flowers. The other normal selections all threw hose-in-hose flowering plants in the proportion of about 3 normal to 1 hose-in-hose. In subsequent generations only the three families which were pre- viously considered under flower color were grown. Normal flower | selections from 12F,H,,P.,P,, gave two populations, one of 100 and one of 97 plants. The plants all bore normal flowers. In 13F,H,,P..P,,P,, it was noted that some flowers were split, but there was not even a suggestion of approach to the true hose-in-hose condition. The other population 13F,H,,P.,P,;P, was from a hose-in-hose selection in the TABLE 15 F; BEHAVIOR OF TALL SEGREGANTS. Garden numbers Tall Short 12F3HisP25P7 19 6 12F 3H y3Po5Po4 1 24 12F3H_P7P5 6 19 TABLE 16 F; BEHAVIOR OF MEDIUM SEGREGANTS. Garden numbers Tall Short 12F3HisP49P 12 2 23 12F3HygPa9Po2 1 24 corresponding F, population. Ninety-four plants were grown to ma- turity, all of which were strictly hose-in-hose. In subsequent gener- ations these three populations bred true to type save for the sporadic appearance of hose-in-hose flowers on plants which otherwise bore nothing but normal flowers. This, however, is not an unusual phe- nomenon even in pure line cultures of normal flowering varieties of Tabacum, and it is extremely doubtful whether the hybrid derivation of ‘these plants had anything to do with the production of occasional sph owers. espects height of plants the F, data are given in tables 15, 16, and 17, Which give the behavior of tall, medium, and short F,, segre- gants resp@etively. The behavior here is not very convincing. Prob- iffieulty in judging the character and the influence of variation in soil Gondition had something to do with it. 1922 | Setchell-Goodspeed-Clausen: Nicotiana Tabacum 503 In the subsequent generations the behavior was, however, more definite. 13F,H,,P.,P,,P, was grown from a tall F, plant. No defi- nite notes were taken as to height, but the population was noted as varying. In F. and subsequent generations the line bred true to tall. 13F,H,,P.,P,,P,, was grown from a short F, plant. The ninety-seven plants were all of low stature and in subsequent generations the line bred true for low stature. 13F,H,,P.,P.P, was grown from a tall F, plant. Ninety-four plants, although variable in height, all be- longed in the tall class and in subsequent generations the line bred true for tall stature. Nothing but a careful biometrical study under TABLE 17 F; BEHAVIOR OF SHORT SEGREGANTS. . Garden numbers Tall Short 12F3HisPo5Pu 10 15 12F3HisP25Pi9 16 9 12F3Hy3P.;Po1 ri 25 12F3HisP.; Pos 5 20 12F3HigP19P9 iL 24 12F3HigP49Pi0 339 24 B's His P49 Pos 7 18 12F 3 Aig Pap P25 ; 9 16 IF’, op Pos Ps 8 16 12F3H)PosPs 5 19 12F3HopPo5Pi1 mee 25 Totals 68 129 well controlled cultural conditions, however, would yield results capa- ble of strict Mendelian analysis. However, it can be said that none of the results here recorded preclude the possibility of such an analysis, although it evidently can not be done in any simple qualitative manner. As respects leaf width it was found impossible to make even such a rough classification as was attempted in the case of stature. Here again nothing short of a strict biometrical analysis would furnish the basis for a Mendelian formulation. As has been indicated above, three separate lines of this series were carried out to the seventh hybrid generation. Of these a recombination of characters from both parents exhibitin& the tall 504 ° University of California Publications in Botany [ Von. 5 virgimca and the red flower color of calycina. The third had the tall stature of virginica and red flower color, in association with the hose- in-hose flower form of calycina. These three lines apparently bred | true for all their characters. 5. DISCUSSION OF RESULTS OF THE CALYCINA-VIRGINICA SERIES No extended discussion of results is indicated in connection with the calycina-virginica series of hybrids because particular attention was given to so few characters. Just as in the case of angustifolia- macrophylla, so in this series of hybrids the character differences proved to depend upon complex genotypic differences. Apparently the flower color contrast in these two varieties was the same as that in the angustifolia-macrophylla series, and the same relations with respect to dominance and segregation were found to hold for it. Without doubt we are dealing here with the Rr pair of allelomorphs as in the previous instanee. The demonstration of the simple factor relations in the inheritance of the split hose-in-hose form of flower adds to our series another pair of allelomorphs which we may call Ce (calycine). In this case the dominance of normal over split hose-in-hose appears to be nearly, if not quite, complete. The sporadic appearance of split hose-in-hose flowers on otherwise normal plants does not even seem to be clearly associated with the heterozygous genotype, Cc. The data for height are not of sufficient accuracy or extent to warrant an at- tempt at Mendelian formulation. It was again found possible very easily to shuffle and recombine the characters occurring in the parent varieties and to establish recombination derivatives in pure lines. V. ALBA-MACROPHYLLA SERIES 1. PARENTS OF THE ALBA~MACROPHYLLA SERIES Alba, which is one of the parents of the alba-macrophylla series, is the ‘‘White’’ tobacco, U. C. B. G. 30/06, described by Setchell. It is one of the taller forms of Tabacum, ranging in height from 165 to Typically alba is unbranched below; above, it has flowering branches forymbosely arranged in succession from above downward. The leaves are sessile, more ample, more rugose, and more velvety than those of mactophylla. They are narrowed suddenly above the ex- panded, somewhat auricled and partially clasping base. The leaves 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 505 resemble those of macrophylla in shape but differ from them particu- larly in the basal portion. The corollas are white with a yellowish tinge ; but in shape, size, and general proportions they are very similar to those of macrophylla. Line drawings of typical: features of alba are reproduced in plate 82. The reproductions of photographs of the leaf of alba and of the F, hybrid of the alba-macrophylla series are shown in plate 84. Macrophylla, U. C. B. G. 22/07, has been deseribed above. In these two varieties there are definite character contrasts in color of flowers, macrophylla being red and alba white; and in stature, macrophylla being low of stature and alba distinetly taller. Other contrasts also exist, although they are not so definite, in the style of branching and in the shape and texture of the leaves. Like those which have been considered above, this is a hybrid series in which the contrasts between the parent forms are of a distinctly complex character. 2. F, OF THE ALBA-MACROPHYLLA SERIES The crosses between alba and macropyhlla were made in July, 1909. The cross was successful in both directions, and seed was secured from albaQ x macrophyllag, which was given the number H,,, and from the reciprocal which was given the number H.,,. When mature the F, plants were tall, 100 to 200 em., averaging 130 to 160 em. Habit and leaf shape were in general those of alba. The corolla was deep pink of about the same shade as that of the F, of the angustifolia-macrophylla series. The variation in height in these populations possibly indicates a lack of constaney in the alba parent in this respect. In plate 83, figure 2, is shown an F, plant of 10F,H.,. 3. F, OF THE ALBA-~-MACROPHYLLA SERIES In 1911 four F,, populations were grown, viz., 25 plants each of EES ey Pie eerand ll HH and) 23 plantsronellkkysklenby. ». The four populations, although small, proved to be equivalent in every respect. The type of segregation was very complex. That of differ- ences in types of leaves, especially, presented such a series of inter- gradations as to defy any definite classification. Likewise in héight, there was a continuous series of forms from the tallest to the ‘A rough classification was, however, made for purposes ofsreference the segre- hich shows into tall, medium, and short. An excellent illustration gation for this character is shown in plate 85, figure 1, 506 Umversity of Califorma Publications in Botany [ Vou. 5 two adjacent plants of 11F,H,,P.,, one tall and of the general habit of alba, and the other short and of the general habit of macrophylla. The classification for height is given in table 18. Obviously no satis- factory Mendelian formulation can be deduced from these data. As regards flower color, however, the classification is more definite. Four more or less distinct shades were distinguishable, viz., red, pink, light pink, and white. The pink and light pink shades merged into each other, consequently they have not been separately recorded in table 18. Bearing in mind the previous behavior of red and pink, as shown in the angustifolia-macrophylla and calycina-virgimea series, TABLE 18 F, SEGREGATION IN THE ALBA-MACROPHYLLA SERIES. Stature Flower Color Garden numbers tall medium short red pink white 11FHo3Pi3 101 6 8 3 14 8 11 F.Ho3P31 13 6 6 4 12 9 11 F,Ho4Pe a 10 8 3 15 7 11 F,HsP34 6 8 9 6 13 3 Totals 37 39 31 16 54 27 it would appear that we are here dealing with dihybrid populations in which a pair of allelomorphs for color versus white is concerned in addition to that pair upon which the contrast of pink versus red was found to depend. The pair of allelomorphs for the pink versus red contrast has been represented by R and r, respectively. If we repre- sent the contrast of color versus white by W and w, respectively, the two parents in this series would possess the following genotypes: Alba — RRww Macrophylla = rrwWw The light pinks of the previous series would then be RRWW, and the factor R might be regarded as a dominant diluter. According to this ulation, F', of the alba-macrophylla series would be RrWw, pink, » Should segregate in the ratio 3 red : 9 pink : 4 white. The result in the classification of ninety-seven plants in whole numbers is 18 red : 55 pink : 24 white. Agreement is thus fairly close. A check,on the results above noted for the 1911 sowings of the F, as made by growing in 1916, five additional F, popula- population 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 507 tionseet the sameseries, yaz., 16E..P,: 16FH,.P.,;: 16F,H,,P,,; 16F,H,,P.,; and 16F,H,,P,,. The segregation in the resulting popu- lations is recorded in table 19. The method of studying these flowers was the more accurate one previously described in connection with later generations of the angustifolia-macrophylla series. In the classification of flowers it was noted: that reds and whites were sharply distinguishable from pinks. The pinks were of many different shades; some very light, others rela- tively dark, corresponding to the range obtained in the angustifolia- macrophylla series. However, in these populations the range of vari- TABLE 19 F, SEGREGATION IN 1916 SOWINGS OF ALBA-MACROPHYLLA SERIES. Garden numbers Red Pink White Totals 16F.H»;P; 11 18 11 40 16F.H»3P 3. 12 19 11 42 16 F 2H»; P34 8 24 9 41 16F>H.4Po3 5 34 11 50 16F.H4P33 0h 29 14 50 Observed 43 124 56 223 Expected 42 125 56 223 ation of pink appeared to be greater and the intergradations more gradual than in that series. In the whites there was also evidence of differentiation into classes depending upon the amount of yellow or creaminess in the flowers. Some of the whites appeared to belong to a clear white albino class, but most of them had a distinetly creamy tinge. The observed segregation in these populations was in almost exact agreement with the formulation advanced above. 4. F, AND SUBSEQUENT GENERATIONS OF THE ALBA-MACROPHYLLA SERIES In table 20 we have summarized the behavior of the F,, populations as respects color of flowers and height of plant. Of the five red/F, plants from which F,, populations were grown, three proved be homozygous for red and one proved to be a heterozygote of the*genetic constitution rrWw. Of this latter selection two sowings wére made, one in 1912 and another in 1913. The combined results.from these two sowings, 35 red: 14 white, are in fair agreement with Mendelian 508 University of California Publications in Botany | VoL. 5 expectation. The other population exhibited an anomalous type of segregation, and gave 2 red :23 white. It is unfortunate that this line was not investigated further, but the results probably are due to an experimental error. TABLE 20 F; SOWINGS OF THE ALBA-MACROPHYLLA SERIES. F2 Phenotypes Flower Color Stature Garden —EE es Flower Numbers tall (or Color Stature Red pink white short medium) Tall (or M) 12F3H»3Pi3P3 19 sone 6 25 Short 12F3H23P31P1 22 ie Sse 22 boo Red Sh’t (orM), 12F3Ho3P31 P17 25 Bhan shee 4 PAL Tall 12F3H»3P31P 52 24 Ase 60 4 20 Tall 12F3;H23P31P.5 2 Se 23 1 24 Tall 16F3;H.3PisP; |. 16 set 8 Tall 12F 3H»; PisP1; 2 14 6 8 10 Medium 12F3H.3P13P25 6 12 a 22 =e Tall 12F3H»3P31P7 6 14 i) 25 Tall 12F3H»3P31Pi9 10 9 4 ace 23 Pink | Medium 12F3H23P31P20 6 12 5 » 4 19 Tall 12F3;HPsP; 3 22 a ihe 25 Short 12F;HyPssPis 5 13 7 24: i. Tall 12F3H.4P34P20 ro 20 5 en 25 Tall 16F'3Ho4P34P 9 aire 13 4 Tall 12F3Ho3PisPi4 oe wigs 25 a 25 Tall 12F3Ho3Pi3Pi5 ape bee 25 ne 25 Short 12F 3 H.3PisPo4 — mn 25 25 ee White Tall 12F3H4PsP2 arenas S260 24 Sette 24 Short 12F3Ho4P.6P3 Rite oe 25 4 21 Tall 12F3;HoyP5Ps 3 ae 23 = 23 Short 12F3HosP34Po3 Ah 1 23 25 Short 13F3Ho3Pi3P 54 5000 ee 10 10 Short 13F3Ho4P34Po3 fe Ree 10 10 Eight families of F., plants were grown from pink F,,’s. Of these I, plants six proved to belong to the RrWw genotype. The totals from these six populations, viz., 35 red : 74 pink : 34 white, are in fair agreement with the dihybrid ratio 3 red :9 pink :4 white. One of the other populations gave 3 red : 22 pink. It was probably the result ing seed from an F, plant of the genetic constitution RrWW uld give 3 pink :1 red. The observed segregation ratio is not good, but the numbers are small. Two sowings of F,H,,P.,P., gave tot f 33 pink :9 white. The F, plants in this case must have been of the genetic constitution RRWw; in which case expectation would be 3 pink :1 white. No selection was observed to breed true which 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 509 for pink in F,. This, however, is not inexplicable, for only one in nine among the F’, pinks should belong to the RRWW genotype. Sowings were made from seven white F’, plants. Among 190 plants so produced there was one pink flowering individual. It surely rep- resents some kind of experimental error. We may say, therefore, that for flower color the formulation advanced to account for the F, segre- gation ratio, also accounts for the behavior observed in the various F, populations. We have reported the data on height in table 20, largely in order to show that this character, although obviously dependent on factor differences, is so complex as not to permit of a simple qualitative treatment. Thirteen F, sowings from tall F, plants gave ten popu- lations showing only tall plants. Two of the remaining populations showed segregation into 31 tall (and medium) : 13 short. One popu- lation consisted entirely of short plants. The classification of the F, parent of this plant as ‘‘tall’’ was noted as doubtful at the time, the note ‘‘or medium’’ being appended. Two-populations were grown from F’, plants of medium height. One of these populations was uni- formly of low stature; the other showed segregation into 19 tall (and medium) :4 short. Six populations were grown from F,, parents, four of which apparently bred true for low stature, the other two showed segregation into tall (and medium) or short in the ratio 42 : 8. It is interesting to note that at the time of classification the parents of these two later populations were classified as short (or medium), indicating a doubt as to proper classification. More definite data will be necessary before a satisfactory formulation of these height differ- ences can be made, but certain of our results seem to indicate that there is one allelomorphie pair which has a rather marked effect on stature, and that there are other subsidiary pairs of factors which have less marked effects. Only one line in this alba-macrophylla series was carried out to subsequent generations to demonstrate the possibility of fixing char- acter complexes from a hybrid. It was a low stature white flowering line. In F,, 100 plants of 13F,H,,P,,P,,P. bred true to low stature and white flower color. The population was uniform, the plants ex- hibited the general habit of macrophylla rather than that of alba; and the leaves were the same shape as those of macrophylla, but they were slightly rugose, although not so much so as those of alba. In [F'., two populations of 25 plants each were grown, viz., 14F,H.,P4P...P.P,, and 14F.H,,P,,P.,P.P,,. No differences were detectable between these two populations, and the characters exhibited were those we have noted U, OF ILL, LIS. 510 University of California Publications in Botany [ Vou. 5 for F,. In F, 10 plants each of 15F,H,,P,,P,,P,P.,P, amd 15¥',H., P,,P.35P,PssPa2 aud in F’, 10 plants each of 16K. H.,P. eae ee. and 16F,H,,P,,P,,P,.P,,P,;P, were grown. In both cases the parallel populations were equivalent and the characters exhibited and described in F, remained constant. Plate 85, figure 2, is a good illustration of the type of this family as fixed. A photograph of the original F, plant, from which the family descended, is reproduced in plate 85, figure 1. It will be noted that the derivative represents a fixation of the characters of the original F, selection, and that no important seg- regation occurred in it either in F, or in subsequent generations. 5. DISCUSSION OF RESULTS OF THE ALBA-MACROPHYLLA SERIES Here again, as in the calycina-virginica series, no extended discus- sion of results is necessary. Obviously the differences separating the two varieties are of a complex nature genetically as in the two previous eases. The series demonstrates the existence of another pair of allelo- morphs for flower color in this group of 7abacum varieties, viz., Ww, and the part played by it in the production of both red and light pink flower color has been determined. The height contrast again proves to be too complex for qualitative Mendelian formulation. As in the previous eases, the establishment of stable recombination derivatives proved to be a simple task. VI. GENERAL CONCLUSIONS We shall limit the discussion of these results to three main topies upon which these investigations seem to have thrown some light: (1) the origin and interrelationships of varieties of Tabacum; (2) the methodology of Mendelian analysis in Tabacum; and (3) Mendelian heredity in Tabacum. 1, ORIGIN AND INTERRELATIONSHIPS OF VARIETIES OF TABACUM As a result of extensive studies of a considerable assemblage of Tabacum varieties, Comes (1905) came to the conclusion that the species Tabacum could be subdivided into six fundamental varieties: a. var. fructicosa Hook. b. var. lancifolia (W.) Comes. >. var. virgimca (Agdh.) Comes. var. brasiliensis Comes. e r. havanensts (Lag.) Comes. ife var. macrophylla Shrank. 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 511 Inasmuch as practically every Tabacum variety shows combinations of characters of two or more of these fundamental varieties, Comes assumed them to have been derived mostly through hybridization be- tween the fundamental varieties, and he proceeded from purely morph- ological studies to classify the different commercial varieties on the basis of their supposed hybrid derivation. Anastasia (1906), who has criticized this scheme of Comes very severely, reduced the number of fundamental varieties to four, striking out fructicosa and lanctfolia from Comes’ list, and substituting purpurea for macrophylla. Al- though disagreeing as to the fundamental varieties, Comes and Ana- stasia seem to agree in referring existing varieties to derivation, mostly through hybridization, from a relatively small number of fundamental varieties. The Howards (1912) object to the mode of classification of Comes and Anastasia, and point out as a result of their studies of types of Indian tobaccos that no attempt at classification based on derivation can be considered seriously unless supported by actual experimental studies. In her later paper in particular Miss Howard (1913) shows that segregation products may be obtained through hybridization which transcend the limits set by the parents. The Howards propose a scheme of classification based primarily upon leaf and habit char- acters, and they adopted this morphological system purely as a pro- visional means for facilitating identification and reference among the numerous forms of Indian tobaccos. Our results agree with those stated by the Howards, and we raise the same objection to schemes of classification such as Comes and Anastasia have advocated. Any scheme of classification based on morphological considerations alone cannot well meet with the approval of geneticists, for it does not take into account genotypic differences which exist among forms of similar morphological appearance. Thus it is possible, as Miss Howard points out, by crossing different mem- bers of a given group to obtain segregation products which belong in an entirely different morphological group in the scheme of classifica- tion. In particular she points out that ‘‘petiolate’’ forms have been produced as segregation products from two ‘‘sessile’’ parents, yet ‘‘netiolate’’ and ‘‘sessile’’ have been used as primary indexes for classification of tobaccos into groups. : The difficulty from the genetic point of view with any Classieiienien of Tabacum varieties is the same as that which is met with in the classification of varieties of other polymorphic species. ing the species as a whole and viewing the entire assemblage of its varieties, 512 University of California Publications in Botany [ Vou. 5 there is evidently in Tabacum, to those who accept current interpre- tations of heredity, a series of allelomorphie contrasts, the number of which cannot even be guessed, but which need not perhaps be more nu- merous or striking than those which have been discovered in Drosophila. But whereas in Drosophila the factors have been kept in stocks in- volving for the most part single factor differences from a common wild type, in Tabacum, and in other cultivated crop plants such as barley, maize, oats, rice, wheat, ete., these factor differences have been shuftled about through long periods of cultivation until existing varieties are no longer related clearly to a common form or to each other. In some instances in such groups certain factor differences have a more strik- ing visible effect than in others. In such instances we have an obvious mode of classification based not upon number of factor differences so much as upon the striking character differences which arise from certain factor contrasts. Thus in barley we have the classification of varieties advocated by Harlan (1918) based upon recognition of a number of major morphological distinctions, some of which at least have been clearly analyzed in Mendelian fashion; and the same prin- ciple has been recognized in the classification of varieties of maize, where it has led to the absurdity of erection of a heterozygous form, podded maize (vide Collins), as one of the primary group distinctions. In some instances, doubtless, the sorting of factors may give rise to certain recombinations which are more favorable to life processes than others, as Muller has pointed out in another connection, and such genotypes may act as centers around which groups of varieties may be built up, thus giving rise to more or less obvious grouping of varie- ties. The attempt to base a system of classification upon reference to certain fundamental types does not, however, promise much simplhi- fication of the difficulty; moreover, such an attempt rests upon the rather naive assumption that it is unnecessary to account for the fundamental types. From a genetic standpoint, therefore, it would appear that in attacking the problem of classification and interrelations of varieties in a polymorphic species the major premise should be a recognition of the fundamental equivalence of every homozygous genotype. Start- ing from this premise a system of dichotomy beginning with those faetor contrasts which produce the most striking, visible effects and procéeding to those of lesser effect might be set in operation. Such a system. obviously would in certain cases separate some similar varie- ties intO\separate groups, and would lead to recognition of group differences*without obvious morphological distinctions, but the system 1922) Setchell-Goodspeed-Clausen: Nicotiana Tabacum 513 would have a real significance, and the relationships indicated by it would be fundamental ones. It is, however, necessary to have a much more extensive knowledge of Mendelian heredity in Tabacum than we have at present before such a system can be formulated. 2. METHODOLOGY OF MENDELIAN ANALYSIS IN TABACUM From the Mendelian side there are certain obvious facts associated with Tabacum as a species. In the first place, as we have stated before, the species is highly polymorphic. A large and striking assemblage of varieties exists, the most extreme of which hybridize readily and give fully fertile hybrids and full fertility in their derivatives. A few teratological forms are known in which fertility is somewhat re- duced, but the above generalization does not far overstate the facts. The species is, moreover, so highly polymorphic that with respect to any given character a representative collection of varieties may be arranged in a series connecting the most extreme expressions of that character by imperceptible steps. Thus in flower color we have repre- sented in the collection of varieties of the University of California Botanical Garden dark red, red, light pink, pinkish, and white, and descriptions occur in the literature which indicate the existence of further shades of red connecting these. Now flower color is a rather definite character, comparatively speaking, for it appears to be little affected by ordinary environmental conditions. In many polymorphic forms, such for example as maize, there are a large number of such definite characters, and as a consequence studies of inheritance in these forms have resulted in definite Mendelian analysis of many char- acter differences. But in Tabacum unfortunately most of the char- acters involve quantitative elements, and these with few exceptions depend so largely for their particular expression upon environmental conditions that it becomes a difficult matter in a segregating population to distinguish between those differences which are inherent and refer- able to the genotype and those which have come about through the action of extrinsic forces. And yet our assemblage of tobacco varie- ties indicates clearly that there are genotypes which give rise to all possible expressions in these characters. Here we find the reason for the present backward state of knowledge of inheritance in Tabacum, for while there have been numerous investigations which indieate clearly that the Mendelian mode of transmission may be followed in all these character differences, yet there are very few a which have resulted in the precise type of factor analysis characteristic 514 Unversity of California Publications in Botany [Von. 5 of investigations with other forms, specific mention of which is un- necessary. The general features of inheritance in Tabacum varietal crosses are plain enough. The results of our investigations in this connection agree throughout with the conclusions which Miss Howard drew from her studies. When we are dealing with complex differences, the F, is commonly intermediate in character expression between the two parents. Not only is this true as respects the F, plant as a whole but it is also true for individual characters. The F,, commonly consists of a varied assemblage of forms covering the range between the two parents, or even not uncommonly presenting products not ineluded in the range between the two parents. So many and of such variety are the forms obtained that accurate classification is entirely out of the question. But in F, and in subsequent generations segregation, even for characters commonly regarded as quantitative, sometimes occurs in distinet discontinuous classes in marked contrast to the inter- grading series of forms obtained in F,. This is shown particularly well in our analysis of leaf base factors, for in this case we have been able to adopt a qualitative mode of attack on one of the features which contributes to leaf shape. If such an analysis proves successful in one instance, there seems to be little reason why it should not be ex- tended to others. There is, therefore, additional evidence in this suc- cessful application of the mode of qualitative analysis to quantitative characters in support of the oft repeated contentions of East (1913), Hayes (1912), Hayes, East, and Beinhart (1913), Miss Howard (1913), and others that fundamentally the same mode of inheritance holds for quantitative characters in tobacco as for qualitative ones. The distinction between the two classes of characters is purely an artificial one erected for the purpose of convenience in formal treat- ment, and at most depending merely upon an increase in complexity of the factor relations involved and on the greater fluctuation of the characters in response to environmental differences. The question remains to be discussed whether semiquantitative characters admit of a qualitative mode of analysis, and if so, how? Miss Howard (1913) as a result of her extensive studies of inheritance in Indian tobaccos concludes that the easiest way to determine the principles underlying inheritance in these forms is to establish as many extracted homozygous intermediate forms as possible. The estab- lishment of such forms in themselves, however, is only a step in the Mendelian analysis of the differences. Such forms are, as might have been expected on theoretical grounds alone, less different from one 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 515 another and from the parents than the original parents are from each other. Moreover, our experiments show that as a result of simplifi- eation of the factor differences the derivative strains crossed with each other or with the parents give F,, progenies which often exhibit clear- cut segregation in characters which showed intergrading series in the original F, population. In other populations, however, from crosses between derivatives, the populations still exhibit perplexing com- plexities which make classification difficult and uncertain. In such cases we could again resort to the method of establishing intermediate derivatives from them; but if the number of factors concerned in a given character is even moderately large, as is certainly the case with many of these quantitative characters, the number of genotypically different derivatives which may be secured becomes so great as to make the method impracticable. Our experience indicates that the successful factor analysis of these quantitative character differences depends not only upon getting what Castle (1919) has called the residual heredity equivalent throughout the population, but also in establishing the proper kind of residuum which will most emphasize the character differences associated with the pair of factors or pairs of factors under investigation. The prob- lem may be illustrated crudely by considering the pair of flower color factors Rr. If the residuum should contain PP, the effect of which is described below, segregation would give PPRR, PPRr, and PPrr. In character expressions these three different genotypes would doubt- less all be of various shades of dark red, difficult or impossible of ac- curate separation. With such a residuum, therefore, it would be im- possible to investigate satisfactorily inheritance in the factor pair Rr. But if we should substitute pp for PP in the residuum, the segregation products would be ppRR and ppRr, which would be pink, and pprr, which would be red. Here the segregation would be sharp and dis- tinet, and there would be practically no difficulty in classification. How complex such interrelations can be has been shown most clearly by Bridges (1919) in his account of specific modifiers of eosin in Drosophila. As Bridges shows it would easily be possible to obtain populations of Drosophila defying classification, but by keeping the factors separate and studying their character effects with known residual genotypes, it has been possible to determine and locate the factors involved. Doubtless much of the extraordinary success of Mendelian analysis in Drosophila has been due to the fact that factor differences arose under conditions such that the residual otype gave no difficulty ; whereas in crop plants, the geneticist starts with 516 University of California Publications in Botany [ VoL. 5 long established diverse types, evidently related to one another in fundamentally the same manner as are the various Drosophila mutants, but more complexly, and from these complex assemblages he must unravel the tangled skein of heredity. There are, however, other and perhaps quicker ways of establishing a uniform and favorable residual heredity than that of securing and testing homozygous extractives, and these may be employed in certain special cases. Thus, if it be desired to study the relationship of the pair of factors Ss for the petioled versus sessile condition, it should be possible to proceed by crossing back the F, of angustifolia x macro- phylla, for example, to macrophylla, selecting the petioled forms from the back cross for again crossing back to macrophylla, and continuing the process until clear-cut segregation was obtained. Such a mode of procedure should establish a residual genotype equivalent to that of macrophylla itself, and should thereby enable the student event- ually to.study the effect of substituting SS for ss in the macrophylla genotype. In tobaccos technical details make it particularly easy to adopt such a procedure, but it is useless to speculate further upon its results until it shall have been attempted. 3. MENDELIAN HEREDITY IN TABACUM From the standpoint of factor analysis, we have demonstrated clearly in the foregoing pages, the existence of a number of distinct pairs of factors. Two of these affect flower color, one flower form, and three affect the character of the leaf base. The particular effects of the opposing members of these pairs of factors and the interrela- tions which they exhibit so far as these have been investigated have been set forth in the discussions which follow the deseription of each of the three series of hybrids. Although evidently many other factor differences were concerned in these studies, and remain for further investigation, the results which we have described make a beginning toward a more accurate knowledge of Mendelian heredity in Tabacum. So far as our results furnish any data on the question, the six ~ pairs of factors isolated exhibit no linkage relations. The data here are far from complete, but the results are in accordance with theory. According to White (1912), there are twenty-four pairs of chromo- somes in Nicotiana. Assuming for the sake of discussion that each of x pairs of chromosomes bears a set of factors comparable in bérs to any other pair, then the chances of finding linkage when .. of factors are studied is very slight. This large number num only six 1922 | Setchell-Goodspeed-Clausen: Nicotiana Tabacum D17 of pairs of chromosomes may account for the ease with which recom- bination pure lines were established. Even with a large number of factor differences, such as evidently distinguish these Tabacum varie- ties, the chances are slight with so many pairs of chromosomes that linkage will enter in as a factor to cause the continued preservation of a heterozygous condition as a consequence of selection for a certain set of characters. It remains to consider those portions of the Nicotiana literature which deal specifically with the Mendelian inheritance of the charac- ters which we have investigated, and to harmonize our results with those which have been reported previously. Unfortunately there have not been many investigations in Tabacwm which have been prosecuted far enough to arrive at a definite factor analysis of the differences under consideration. The investigations of Miss Howard (1913), promise of the continuation of which has not thus far been fulfilled, in general confirm those which we have presented in this paper. On the strictly analytic side, however, Miss Howard did not carry her work very far. This doubtless was due to the difficulty of making a factor analysis of the characters which she selected for study, viz., (1) time of flowering, (2) height of stem, (3) arrangement of the leaves on the stem, (4) length of the decurrent portion of the lamina, (5) venation of the leaf, (6) leaf shape, and (7) undulation of the surface and margin of the leaf. For most of these characters she demonstrates, by the presentation of numerical data in some cases as far as F,, the probability of the character differences in ques- tion depending upon multiple factor differences. In the case of height certain of her cultures strongly suggest the existence of a pair of allelomorphs, which has a relatively great effect, for in some of her cultures there are definite discontinuous height differences. For the inheritance of length of the decurrent portion of the lamina Miss Howard postulates the existence of at least three or four distinet pairs of factors. As respects leaf base, she records the synthesis of petiolate types from sessile parents, observing in two cases a simple 1:2:1 segre- gation into petiolate : intermediate : sessile. As respects corolla color, she records one F’, population from pink x very pale pink fading into white which consisted of 72 pinks of various shades to 45 whites, but some of the palest pinks were indistinguishable from white. She found evidence of grouping among the pinks, and postulates the existence of two factor differences to account for it. The investigations which we have reported do not throw light upon the factor constitution of the very pale pink varieties with which Miss Howard worked. Our 518 University of Califorma Publications in Botany [ VoL. 5 varieties angustifolia and virginica have lively pink flowers. Of the paler pinks or ‘‘pinkish’’ forms we have a representative in our N. Tabacum var. Cavala, U. C. B. G. 72/05, which has flowers distinctly lighter in color than those of angustifolia or virginica. Our petiolate forms also seem to be of different constitution from those with which Miss Howard worked, for she presents evidence to show that hers are combinations of recessive factors and that they breed true whenever they occur as segregation products, whereas our petiolate forms often gave plants with sessile leaves as segregation products. We have, however, secured evidence that some distinctly short petiolate forms arise from sessile ones, perhaps by modifications of the AURICULATA leaf type in the direction of stripping the auricle and lower portion of the lamina from such leaves, but our results are not yet definite enough to permit of rigid formulation. Further investigation of the relationships of the various petiolate forms is necessary. As respects flower color Allard (1919) has presented some inter- esting data which at first sight appear to contradict those which we have presented. Allard found that carmine x pink gave F, carmine and F, 3 carmine :1 pink. The back crosses gave consistent data. Thus F, carmine x carmine parent gave all carmine, and F, carmine x pink parent gave 1 carmine :1 pink. In F, pink segregants bred true for pink, and carmine either bred true for earmine or gave again 3 carmine :1 pink. The difficulty here is that our red is not genetically identical with Allard’s carmine. Our flowers of macrophylla and calycina at full expansion show a color lying between rose red and pomegranate purple of the Ridgway color scale. This color, which we have called red for the sake of brevity, is very close to carmine, but we have an- other flower color, which we eall dark red, represented by N. Tabacum var. macrophylla purpurea, which is probably identical with the Giant Red flowering tobacco which Allard used in his experiments. We have made some preliminary tests of this dark red, and find that it behaves differently from red. Crossed with our white it gives dark red in F,, instead of pink as was obtained from red x white. Since our white carries the factor R, which is responsible for the production of pink flower color, dark red must differ from pink in a dominant factor. If we call this factor pair Pp, then our various colors of tobacco would have the following genotypes: Darkgredse—. 5 eee WWRRPP Red. 320. a WWrrpp \ Pink? A022 er WWRRpp 1922 | Setchell-Goodspeed-Clausen: Nicotiana Tabacum 519 Obviously this formulation would account for Allard’s results with- out contradicting those which we have presented, but inasmuch as our experimental evidence is not yet complete we refrain from any further discussion of the consequences of this scheme save one. Allard presents certain data for a cross of carmine x white which gave in F, light earmine, and in F’, 3 colored : 1 white, the colored being various shades of carmine and pink. Allard’s discussion of this case is somewhat mixed, but he evidently erroneously expected a simple monohybrid segregation of the 1 :2:1 kind. That more than one factor is con- cerned in the cross is clearly shown by the results of crossing some of the extracted whites with pink varieties. The results of three such crosses gave: 1. Pink (Maryland Mammoth) x Extracted white.................. 36 carmine : 18 pink 2. Extracted white x Pink (Maryland Mammoth)................ 20 carmine : 23 pink 3. Pink (Conn. Broadleaf) x Extracted white.......................- 12 carmine : 39 pink TN Ojb Se ese. sot ssccss2 scaccestessescccoscencnacce ose aueneee Ses laeanees 68 carmine : 70 pink In (1) above we have combined in the carmine class 17 carmine and 19 somewhat lighter than carmine. If we consider a cross of dark red x white according to the genetic formulation given above, the F, should be dark red, and F, should consist of 9 dark red : 3 pink :4 white. Doubtless the pinks and the dark reds would exhibit various shades, but the three classes should be distinct. If we combine ‘‘carmine’’ and ‘‘lighter than carmine’’ to form a carmine class and dark and light pink to form a pink class, Allard’s F,, data reduce to the following form: 149 carmine : 64 pink : 65 white. This ratio compares very favorably with a 9 :3 :4 expectation, viz.: 157 dark red : 52 pink : 69 white. No F, results from sowings from colored F, plants are given, but the single F, white, which gave when crossed with pink approximately equal numbers of carmine and pink flowering plants, is accountable for as of the genotype wwRRPp. Further investigations are in pro- gress for the purpose of determining precisely the relation of dark red and pinkish to the red, light pink, and white colors reported upon in this paper. There are other references in the literature to Mendelian inherit- ance in Tabacum, but inasmuch as these do not bear upon othe characters which we have attempted to analyze it does not ee necessary to discuss them at this point. 520 Unversity of Califorma Publications in Botany [ Vou. 5 VII. SUMMARY Studies of three intervarietal crosses in Tabacum demonstrate that : 1. All the differences between varieties of Tabacum can be analyzed in a Mendelian fashion, if sufficient refinement in methods be intro- dueed. 2. Stable recombinations of parental characters can readily be obtained with three or four generations of self-fertilization. 3. Characters outside the range between the parents are sometimes produced following hybridization, and these may be readily established in stable lines by self-fertilization. 4. The petioled leaf base of angustifolia and the sessile leaf base of macrophylla differ in at least three pairs of factors. 5. A single factor difference exists between normal and spht hose- in-hose flowers. 6. Two pairs of factors account for the relation existing between red, light pink, and white flower color.