THE CHROMOSOMAL CONSTITUTION OF CERTAIN CULTIVATED VARIETIES.

BY MUI~IEL V. ROSCOE. (With Eight Text-figures.)

INTRODUCTION. TEE cytology of many varieties of the cultivated apple has already been studied by several workers including tlybin (1926, 1927), Shoemaker (1926), Kobel (1926(~, ]926b, 1931), Heilborn (1928), Nebel (1929, 1930a., 1930b), ])arlington and Moffett (].930) and Moffett (].931). Through these studies there has developed an interest in the relationship of cln'omosomal constitutiou to some practical problems of the grower and breeder. Some of these aspects have been discussed by Crane and Lawrence (1929, 1930), and of particular interest is their treatment of polyploidy and its bearing on fertility and on seedling vigour. Correlations between diploidy and triploidy and pollination may be made fl;om the pollen-germination ex- periments of Florin (1926).and Kobel (1931). Most recent among the investigations on are those of Brittain and Eidt, who have been worldng with pollination and genetical aspects of diploid v. triploid varieties. These experiments have been carried on chiefly at the Dominion Experimental Station at Kcntville, a station located in the Annapolis-Cornwallis Valley, the apple-raising section of Nova Scotia. Their data show correlations between ploidy and effective pollination, set of fruit, seed count, seed germination and growth rate of seedlings. The present cytological work was carried on during the summer and fall of 1932 and developed out of an interest in the experiments of Brittain and Eidt. Some eighteen varieties were chosen for study and included in these were certain varieties extensively grown in the - polis Valley region 1. Among such are Wagner, Golden gusset, (I~oxbury ~usset), Stark and Fallawater.

MATERIALS AND 1V[ETHODS. The material for the study was obtained from tile orchards of tl~e Dominion Experimental Station at Kentville, with auxiliary material 1 The pollination and genetical aspects of these varieties as ~ and ~ parents are presented by ~Brittain and Eidt. 158 Constitution of Ce~'tain Cull/ivated Al)~)le Vc~,~'ie~ies from cuttings in the case of C4olden l~usset, Nonpareil and Stark. The cuttings were developed in the greenhouse and the pollen mother cell divisions from these gave appearances very like those of buds developing out of doors. Carnoy's fluid was used as a fixing agent and the buds embedded in celloidin. The sections were stained with Heidenhain's iron-alton haema- toxylin and studied with ghe aid of 1.5 ram. and 2 ram. Zeiss apochrd- magic objectives used[ with ]0"< and 15× oculars. Observations have been concerned chiefly with the reduction divisions of the pollen mother cell, but in several instances [,hess have been accompanied by study of somatic divisions. Petal, stamen and ovary tissue afforded good[ opportunities for making somatic counts.

OBSEI~VATIONS.

Ba~z/cs' 65'imson Grase~tstei~. 2~ = 51 (somatic and reduction divisions). The triploid condition of this variety places it with the clonal varieties of reported by Nebel (1930 b). Petal tissue has provided somatic divisions showing 51 chromosomes, l~eduetion divisions reveal M. I plates with 22 and 23 units comparable with those in Seark. Irre- gular chromosomal distribution in both the heterotypie and homotypic divisions followed by polyspory indicates the similarity of division pheno- mena in this and varieties such as Stark, Nonpareil and Fallawater.

65"imson Bea~ty of New B~'~,~zswic/c (Ea,~'ly Red Bi'rd). 2~ = 3~; ~, = 17 (M. I). The reduction divisions axe featm'ed by regularib~ with normal inter- kinesis and tetracary.

Deaco~ Jones. 2n -- ca. 36 (tapetal cells); q~ = 17 (i~{. i and A. I). Most M. I divisions show 17 bivaleng chromosomes at the plate, but in a few instances these are 16 and 15. This means that probably one and two of these were quadrivdents, although such a morphological nature was not recognisable. With this condition occurring even rarely, it is considered possible that the form is similm: to the diploid species with complex cin'omosomes examined by Darlington and Noffett. The rarity of such occurrences in nay material should[ be stressed. The divi- sions are regular and only a few A. I cases show slight lagging. All the chromosomes arc eventually inchded in the interkinetic nuclei. MIUI~IEL V. ~osooE 159 Delicio~ts. ~ = 17 (M. I, M. II, A. II). The regular divisions lead ~o normal interkinesis and[ getraeary. Shoemaker has reported Delicious as having ~a = 14 chromosomes, but it; is appa,rent; from bhe counts in bo~h hegerogypic and homotypic divi- sions tha~ the number is 17 and ~hat ~he behaviour is that of an ordinary diploid, lrig. 3 shows 17 chromosomes in each of the me~,aphase plates of the homotypic dixdsion. D~tehess. ~z = 17 (H. I, A. I.) Nobel lists bhis as a diploid form, COharlamowsky (Duchess of) Olden- burg. The eorm~ and ~he chromosomal action confirm Nebel's reporf,. O

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\ Figs. I-4. Diploid variebies. l, Golden 12,ussog : 1)i. I, polar view, showing 17 chromosomes. 2, Wellhlgbon: M. I, polar view showing 17 chromosonms. 3, Delicious: M. II, 17 chromosomes ill each ptal, o. 4, Wolf I/,iver: A. I [cat, ured by regularit,y of chromo- scram behaviour. Fcdlc~vc~te~'. 2~ = 51 (somagic divisions). Somatic metaphasc plates observed in cells of petal tissue indicate a t,riploid condition for ~his variety. Only a limited nmnber of pollen mo~her cells were available for a study of reduction divisions. M. I pla~es observed showed 21 chromosomal units. The conspicuous lagging of chromosomes in hegerogypic figures indical;es a similarigy of behaviour for Ieallawa~er, Shark and Nonpareil. 160 Constitutio~a of Certain Cultivated Apple Varieties . n = 17 (5I. I, M. II, A. II). Fig. 1 is a drawing of a polar view of an M. I plate, and emphasises tile count of ]7. The divisions are typical of diploid forms and do not show lagging or irregularity of action. . n = 17 (M. I, T. I). The variety has regular divisions. . n = 17 (M. II). While no phases of the heterotypic division have been seen for this variety, the regularity of the interkinetic figures, coupled with the con- stancy of 17 in the M. II places, leaves no doubt as to its diploid character. NonpareiZ (). 2n = 51 (reduction divisions). The difficulty of making counts for triploid forms is especially great in working with reduction divisions only. As Darlington and Moffett point out, the tendency toward multiple association reaches greater limits in triploid than in diploid species. However; the units which have been seen in Nonpareil do not range higher than quadrivalents and in this respect differ from the triploids reported upon by these English workers. Bivalents are represented in greater numbers than the quadri- valents, trivalents or univalents. Diakinesis reveals varying counts, but always in excess of 19. M. I exhibits units ranging from 20 to 29. Fig. 6 illusCrates a plate with 29 such units. A determination of the number of qnadrivalents, trivalm~ts, bivalents and univalents in this and other cases shows them to aggregate 51. Diakinetic figures are thus supported by iVI. I and A. I findings. Interkinesis shows chromosomes unincluded in the nuclei, and the homo~ypic divisions very often result in polyspory. The lagging of the chromosomes in the first division is pronounced and late anaphases show univalents scattered all along the spindle. Lateral views all show the tardy action of the chromosomes. M. II places show 2~ to 25 chromosomes it~ one plate with many fewer in the second plate. A. II may reveal a group entirely free from the spindle and lying in the cytoplasm. While the larger number of observations were made on greenhouse material, these were confirmed by the figures from buds collected out of doors. Red Spyl n = 17 (M. I). Lateral views of M. I, T. I and in~erkinesis show regular chromo- somal distribution which leads to the formation of orthoploid tetraspores. ~{Ur~X:¢L V. I%OSCOE 161

Reinette Rouge d'Hiver. ~ = 17 (M. I, T. I, M. II). Lateral views of M. I, T. I and T. II exhibit meiotic regularity.

8ta,rlc. 2n = 51 (reduction divisions). In M. I of this variety 22-25 chromosomes are usually present and these are represented by varying numbers of quadrivalents, trivalents, bivalents and univalen~s. Fig. 5 shows a metaphase plate with 23 ehro- lIlo8omes. The table below suggests the variability of the chromosomal associa- tion observed at metaphase, but at the same time emphasises the ~riploid nature of the variety.

QuadN- valonl~s Trivaleni)s ]3ivalenl~s Univalents Units Tot)al 5 3 8 6 22 51 1 7 12 2 22 51 1 5 . 13 6 25 51 0 7 14 2 23 51 It is seen that the ~otal count is always 51 in these ~. I plates and the triploid character is vouched for by the N. II figm'es. M. II plates differ greatly in counts, but cells showing plates with 28 and 23 chr'omo- seines (Fig. 8) or with plates of 28 and 19 chromosomes and 4 separate chromosomes forming a thh'd small plate on a separate spindle confirm the he~erotypie counts. All figures of b6th divisions are distinguished by lagging of the chromosomes. Fig. 7 is a drawing of an anaphase illustrating such lagging. Mos~ homoVpic divisions show chromosomes ]ying off ~he spindles and frequently also supernumerary spindles are present. Polycary, polyspory and even diad forma~sions resN~. Such figures are similar to those described by P~ybin in the triploid t~einette du Canada.

Wagner. ~ = 17 (I~. I, M. II, A. II, T. II). M. I shows 17 bivalent chromosomes constantly and the regularity of the division stages in the form is impressive.

Wellington. 2n = ca. 34; n = 17 (IV[. I, A. I, T. I, 13{. II). Lateral views of M. I, A. I, and T. I all show great regularity.

Winter Banana. 2~ = 34; n = 17 (diakinesis and 1~{. I). Division figures show complete absence of lagging chr'omosomes. Chance observation of a somatic division in a tapetal cell gave 68 chromo- Journ. of Genetics xxvnI 11 162 Constitution of Ce~'tain Cultivated Apple Va~'ieties seines as the ~o~al for the metaphase pla~es of the two nuclei, leading to a coufirmagion of ~he reduced number, 17, in the pollen mogher cell.

l'Volf River. ,~ = 17 (M. I and M. II). The regularity of division figures is marked.

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:Figs. 5-8. THploid variet, ies. 5, S~rk: i~[. I pl~Le wi~h 23 chromosomes (6 briwlen~s, 16 biva.lon~s, 1 uniw~lent). 6, Nonpareil (l%oxbmT l%usseS) : 1~{.I pl~e wi~h 29 chromo- somes, 7, S~rk: A. I, fe~bm'cd byirregul~rif,y of ohromosom~d beh~viour. 8, S~a.rk: ~'[. II pl~es of 28 ~nd 23 chromosomes show ineclu~li~y of distribution in ~he he~ero- ~ypic division.

York Imperial. 2~ = 34; q~ = 17 (M. I, M. II). The he~ero~ypie divisions show more lethargy on the part of the chromosomes than in the preceding diploids and in some cells exhibit alight lagging. The results are hOg serious since, as shown by ghe M. II counts, all the chromosomes are included in the interkine~ic nuclei. The homo~ypie division is regular in ~he distribution of ~he chromosomes. MUgIEL V. ]~oscoE 163

DISCUSSION. Dil~loi&,l , tr@loidy c~ct~ a,~eul~loidy. With 34 and 51 as somatic counts ~he varieties of this investigation are all slmwn to be euploid. Since I~ybin's publication of t/.einet~e du Canada as a triploid in 1927, many varieties have been admitted to this category. The re-examination by Nobel and by Darlington and Moffett of some of Kobd's aneuploid varieties has shown these to be triploid *. The revised[ counts have been accepted by Kobel and indeed supported by his recent findings in Bohnapfel (1931). Nobel (1929) noted the strong tendency toward euploidy in apples and the data now a,t hand cover a large number of varieties and[ strongly support his earlier conclusion. Darlington and Moffett along with Crane and Lawrence find that aneuploid seedlings which wore derived from triploid parents (selfed or crossed with diploids) are not vigorous, and from the work already done, it seems safe to predict that rarely will aneuploid forms appear in the cultivated apple. Kobel earlier (1926 b) thought that the poor germination of pollen in certain varieties of apples and pears was due to the presence of abnormal chromosome numbers and supported this for the apple by his counts for Wamer's King, Bohnapfel, Gravenstein and Sehihner yon Boskoop. In view of the revised counts for Gravenstein and Sch51~er yon Boskoop referred to above, it is suggested that triploidy and no~ aneuploidy is associated with low germination percentages. Ub'o~nosomcd behc~vio~o'. A study of the species described reveals that the meiotic divisions of the triploid species vary considerably fl'om those of the diploid species. In diploids, ~he reguIarigy with which the chromosomes separate and pass to the poles in the heterotypie division is in most cases pronounced. This regularity is maintained in the homotypie division. Such a normal course of behaviour has been mentioned by Darlington and lVIoffett for the diploid varieties of their investigation. Of these they say: "Abnor- malities-such as the occasional lagging of univalents--are of sporadic occurrence." I-Ieilborn studied divisions in buds of diploids which had been developed in a warm greenhouse and believed ~he irregularities had been largely induced by increased temperagm'es. I~[ethus attributed the eause of irregular meiosis to outside factors. Nobel fonnd that 1 The v~rieties ex*~ttthled include ll,ibst, on Pippin, (4r~vonsgein, /~Mdwin and Belte de .Boskoop (Nobel, 1929) and Brtmfley's Seedling and Crimson Bramley (Darlinggon and Moffob'., 1930). 164 Uonstitv, tion of Certain Cul, tivated Apple Varieties material of a given variety showed grea~er irregularities in his 1927 than in his 1928 collections, and. in some dil)loids quite an amount of irregularity is recorded ( bacca,ta,, M. seheidecl~eri, Mahts var. _Yellow Newtown), Nebel thinks ~he explanation is to be found in un- favourable enviromnen~al conditions rather than in any inherent factors. The findings of the present investigation are such as ~o indicate a pro- hemmed regularity of division for most diploids, zhly irregularity of meiosis is so slight that the effect upon spore formation may be considered negligible. For triploid varieties, chromosomal behaviour is, as may be noted in foregoing descriptions of S~ark and Nonpareil, quite different from that in the diploid species. Varying numbers of chromosomes are directed toward different poles, and varying numbers of laggards are seen on the spindle. This means in most cases that the nucM of interkinesis contain different numbers of chromosomes, wNle frequently some of the laggards are not included in these daughter nuclei. I-Iomotypie divisions reveal still further instances of lagging chromosomes and abnormal figttres which lead to the conditions of polycary and polyspory. The effec~ upon the formation and functional ability of the pollen is obviously dele- terious. It is apparent from ills foregoing account of meiosis that distri- bution of ch'omosomes in triploids is such as to lead to morphologically and physiologically poor pollen. It is to be noted that the present investigation deals with diploid and triploid varieties subjected to the same environmental influences. The contrast in ch'omosomal action in the two cases is significant.

Causal factors in meiosis. The study of chromosomal behaviour in triploids has already received much attention, and the meiotic phenomena in such are now well known. Although griploid species and varieties doubtless originate in different ways, it is generally conceded that hybridisation is a frequent cause. Woodworth (1929) has described meiosis for Betulajaclcii, undoubtedly a hybrid between B. lenta, (diploid) and B. pumila, (tetraploid). Of the meiotic irregularities he says: "The apparent cause...is the lack of homology between the chromosomes during gemini formation." Here the irregularities are attributed to the hybrid ancestry, and are con- cerned not only with numerical constitution but also with the non- homology of the ehronmsomes entering into thafi constitution. While ~he origin of triploid varieties in the apple is not necessarily analogous with that of the ~riploid species of Betula referred to, the MU~IEL V. Rosco~ ]65 meiotic behaviour of the triploid apple varieties under consideration is of the nature of meiotic behaviour in tripleids with a hybrid origin. For the triploid apple the non-homology of the units is' indicated by the great variety of their associations as represented by the formation of quadrivalents, trivalents, etc. Darlington and Moffett have pointed out that various sorts of multi valcnt associations occur in Mahts varieties, and that meiotic irregularities lead to dissimilar numbers in the homo- typic plates. Nebel found two modes of conjugation in M. sl)ectabilis, with the forming of 17 tri va,lent, groups in the one case, and of 25 bivalent groups in the other ease. The present work gives further instances of such associat~ions for triploid varieties of the cultivated apple. Table I shows that :for Stark~ various countsbetween 22 and 25 may occur. A greater range of counts was fmmd for Nonpareil and although the majority of these were between 22 and 25, yet variable numbers ranging as high as 29 (Fig. 6) have been found. Apparently, in the case of Stark and perhaps for the majority of the eases in Nonpareil, the "third supposed haploid set," to borrow Darlington and[ Moffett's term, pail' among themselves. In the remaining eases in Nonpareil, where more than 25 units are present, it seems that there has not been complete pairing within the "third haploid set." Darlington and l~loffett state that the meiotic abnormalities in tri- ploids lie in the "multivalent association of chromosomes." If this in itself were an explanation of the cause of the irregularities observed in triploids, the same logic would lead us to expect more prominent irregu- larities in the reduction divisions in diploids, since in the diploid species which these workers have described quadrivalent and sexivalent chromo- somes are commonly present. The balanced or nnbalaneed relationship seems to bear on meiotic behaviour, and from the results of the present investigation , it is be- lieved that for diploids the association of homologous units with a balance of their somatic number, 2,n = 34, accounts for the general normality of the division figures. Similarly, it is believed[ that the triploids show the irregularities manifest in triploids of hybrid origin, and that the unbalanced nmnber and the non-homology of the uNts concern ed can be considered the causative agencies of su oh abnormalities.

~UNI~IAItY. 1. Of the eighteen varieties of cultivated apples studied, fore'teen have been found to be diploid aud tour triploid. 2. Euploidy is characteristic of cultivated apples. 166 Constitution of Ce~'lain d'qdtivated Apple Va~'ielies

3. The diploid varieties considered are distinguished by regularity of meiosis with normal chromosome distribution. 4. The triploid varieties are distinguished by irregularity of meiosis with unequal distribntiou of ~he chromosomes. 5. Certain similaritie~ between the division figures o f triploid varieties and hybrid forms are pointed out. 6. It~ is considered that constitutional ra~her than environmental factors cletermine chromosomal behaviour. 7. Homology and s, bs,lanced rel,~t,ionship of the chromosomes ac- count for the normal reduction figures in diploid varieties.

I am indebted to Dr W. H. Bri~tain of Macdonald College for assis- tance rendered dm:ing the course of the investigation. I wish also to thank Mr (J. ~. Eidt of the Dominion Experimental Station at Kentville for his kind co-operation and especially for his services in securing material from the Experimental Station orchards.

REFERENCES.

]3RITT~IN, W. H. and EIDT, C. (J. "The relation between seed and seedling production and fruibfnhmss in apple crosses." Journ. Ag.r. Research (in the press). Cmt~E, M. ]3. and L~tW~ENOE, W. J. C. (1929). "Genetical and eyto[ogical aspects of incompatibility ~nd sterility in cultivated fruits." Jou~'n. Pore. and Herr. Sci. 7, 4, 284--301. (1930). "Fertility and vigour of apples in relation to elu'omosome nmnber." Jour~. ae~,. 22, 153-63. DAXaLINGTON,(I. D. and MOF]rET% A. A. (1930). "Primary and secondary chromosome balance in Pyrus." Jou~'n. Gen. 22, 129-51, i pl., 4I figs. FLORI~, I~UDOL~" (1926). "Pollen production and incompatibilities in apples and pears." Mere. Heft. Sos. New Yorlc, 3, 87-118, 3 pls. HEm~ORN, 0. (1928). "Zytologische Studien fiber PollensteriligSt yon Apfelsorten." Nve~zs]¢. Bet. Tidslc,r. 22, 1-2, 185-199, 3 figs. KerneL, F. (1926 a). "Zytologisehe Abnormit/i~en bet Apfel- und Birnsorten trod ihre I~olgen." Actes Soc. Helvdtiquc Sci. Nat. 107, 205-6. I~eview in Biol. Abst. 2, 15578, 1928. --(1926b). "Untersuchungen tiber die Keimf~higkeit des PollEns unserer wiehtigsten Stein- und t~ernobstsorten, mif, einem Ueberbliek tiber die ]3efrueh- tungsverh/tlgnisse derselben." La,ndw. Jahrb. Sch'weiz, 40, 550-89, 3 figs. Review in Biol. Absl. 2, 18~H5, 1928. -- (1931). Lehrbuch des Obslbaus r~f 'physiologischer ar~ndla, ge. Berlin. ~OFFET~', A. A. (1931). "A preliminary aeeotmg el chromosome behaviour in the Pomoideae." Jou,rn. Pore. and Herr. Sci. 9, 100-10, 11 figs. NEBEL, ]3. (1929). "Zur Cytologic yon Malus und ViCis." Die Gt~rtenbauwisse'nsch~ft, :1, 549-92, 55 figs. MUIClEL V. I~OSCOE 167 (1930 a). "Chromosome counts in Vitis and Pyrus." Amer. Nat. 63, 188-9. (1,930 b). "J~eeent findings in cytology of frait~ (Cytology of Pya'us, III)." Proe. Ame'r. Soo. IIort. Nci. 27, 406-10, 2 pls. I~Y~i~, V. A. (1926). "Cytologiea,1 investigations of ~he genus A~fal,tts (prelimhlary accotmt)." Bull. Appl. Bet. Genetics and Plant Breeding, t6, 3, 187-200, 2 pls. (English summa.ry.) -. (1927). "On ~h~ number o£ chromosomes observed ffl ~he som~Sie gnd reduction division of the Clfltiwt.ted apple in eonneclffon wi~h pollen Sgel'ility of some of its varieties." Bull. Al)l)l. 13ot. Genetics and Plant .Breed#~g, t7, 191-20, 5 pls. (English smnmary.) SImJs~IAK~I, J. S. (1926). "Pollen developmeng in ~he a.pple, with special reference to chromosome behaviour." Bog. Gaz. 8t., 148-72, 3 pls. WooDwo~'rtr, I~. H. (1929). "Cygologieal studies in the Betul~ceae. I." Bet. Gaz. 87, 331-63, 2 pls.