£~ENETICAL STUDIES IN APPLES. I I

B~z D, LEWIS _,a~n iVL B. CRANE

JoAn i,J~nes Ho.r~io~da~rcg Institutio'~, Me,~'ton, Lo~do'~:~,

(With Two Text-figures)

]~XmE~SS.VE breeding experiments have been carried out with apples, but little precise knowledge of theh" genetics has been obtained. Thin is mainly due to the apparent continuous vari~tion of man)- characters investigated. Fu~'ther, their heterozygous constitution and the prevalence of self-steI'J.Iity prevents genetical studies from being directly approached.

in a previous report on these experiments (Crane & LawTence, 193.3) an account wan given of genetical studies of fruit oflaraeters, such as colour and russetting of the akin, seaso~ of ripening, shape and size of fa'Nt. Similar studies have been reported upo~ by other investigators, notably Wellington (192"~) and Wilcox& Angeto (19.3.5), and with one doubtful exception simple distrait segregaOion was not found in a~y of the characters studied.

Other characters, such as leaf shape and petiole length (Tydeman, 19.35) and the formation of root burrs appear to be quantitatively inherited. With respect to albinism Crane & Lawrence (19.3g) obtained

results which closely app~:oximated to a .3: i ratio. I-Iowever, such ratio could be expected for either distrait or potysomie inheritance when the genes in a polyploid are in the simplex condition.

In general it was concluded from the above investigations that inheritance Jn apples was polysomic and that a number of genes were involved ia the expression of the characters studied. This to some ex~e~.t. supports the theory edvanced by DarlJ.ngton & Noffett (19.30) that cultivated app]es are secondary polyploids, the so-called diploids (% = .3J-) being in part tetrapldid an.d in part hexaptoJd.

The eh~,racter under investigation to be described in 0his pc,per is the anthocyanin pigmentation of leaves and shoots. The anth.oeyanin coIoration is a convenient character for genetic study, a,s iL develops in the early seedling stage and the p[anSs cazt be scored and the majority discarded, while quite small, thus obviating the labour and expense of growing trees I:o maturity, rl'h_e JavestigatJ.o~ is incomplete, but since to corn.piers a genetical ataalysis ~eould require ag tea, st another general,ion of plants, which would take seven years, ~his preliminary s:eport is

120 (7e,~~e~iccd ,S't.ztd.fe,s i~ Ap 'p les

published. In addidon a brief account is given of a technique need for increasing the proportion of seeds which germbaate. This may be of value when applied[ to o~iter ]?tariffs where under nomnal treatment many seeds fail to germinate.

The experiment was begun h? 1920 by crossing :'Lox:d.Grosvenor'', a variety of Meh~s Mcdv, s, which has normal arena leaves, with M. N4edz~ve~d~;ycoze, in which the leaves ~nd a!I parts of ~he tree are deeply pigmented with anShoeyanin. Some of the I~'~ plants began ~o flower in 1929, and awe of ~hem, with :four other green-leaved varieties,

Shoot.s Ma.h~s N'iedz- Dark purple, wood

wetzlcyay~,~ pm'pb

2Lord Oh'Greener

Cox's Or~nge Pippin

Green Mth red ~inge, wood un- pigmented

TABLE I

Le~f

Tinged red, tuml- ing purpIe Lu au- tumn, pe~iob and midrib bright red

Pale green

Red, wood unpig- Green, petiole mented thlgecl red when

young

Emne~h Early Green with red ])arkgreen,petiole ghlge, woocl ml- wi~h tflage of red pigmented

Goklen Spire Green ])ark green, petioles thtged red

Lord Derby Green Darkgreeu,pe~inle green

Seedlhtg 100l D~rk pttrp[e, wood Ileddish purple Lord C~rosvenor purple x N@dzzc¢~zl~yc~

Seedling 1 . 0 1 7 DarkpurpIe, wood Reddish purple purple (deeper t, han

1001)

Fluwer pe~ais Er ui.g Deep rose purple Skha erhusen put-

[pie, flesh rose purple

Whi~e wi~h a tinge SI<.iaa pMe yellow, of red flesh wMge

Upper side while, Skin yeUowlsh, tower side pale ttushecl and stip- pink pied dull red,

flesh creamy yellow

I_7pper side white, Skin yellow, flesh lower side pale cream pink

Whigs streaked S I ~ goldenyellow w-ith pi~xk flesh creamy

whRe Upper side white Skh~ yellow, flesh

wRh.~inge ofp[~ k, cream lower side pink

]gore purple Sldn pau~plish red, flesh wN~ish red tinged red

Deep rose purple Skha yellowish bro~ua, blotches of ckdI red and crimson, flesh whRe fiinged red

have been used in further breedh~g wo.rk, A description of the seedlings ~md va, rietie, s is .given in Table I.

M. iYfe, dzwetzl<yct,r~c~ readily crosses with cultivated va:d.eties of apple and produces ferdle o:ffsprJng. I t occurs na,~urally together wRh M. v~.cd.~,~s in the Cauo~sus. The principal difference betweett ~hem is the a,nthoeya~in character ]previously described, sad. they are probably only" forms of one polymo <phic group.

D. Lm,vzs AN~ M. B. C~{~u'{t~ 121.

(~ENETI CAL ~ESULTS

The o~!'sprin~ from d~_e original cross (Lord Grosvenor apple × M. ~Viedzv~e~z/~;~c~na) can be grouped provisionally into two main classes: (I) green, in which the cotyledons and leaves ars green, and (9) purl~le, in. which the cotyledons, sSems and leaves produce anShooyanin. The plan.ts in ~he green class are comp~ratively uniform~ bu~ in f;he purple class the m~o~:mt of. a~thocyanin produced varies great, ly, varying from only a slight rings of red to intense pigmentation much deeper tl_mm that of M. Niedzwetz~y~,rm. There is no diffimfl~y, however, in distinguishing between the two groups. In the earlier families no attemp~ was made to classify the purple plants in greater detail, but in later families arbitrary classes were made to ,acfl~uate description.

The seedlings are first recorded h'om 6 go 8 days after germination. At ~his stage the pigment has fulty developed. As the seedlings get older the colour tends to diminish, but again increases at the end of ~ho summer. In 19~9 two small families 34/29 and 35/29 were raised from inter-

Family llO,

a/-~o

:34.f99 29/32 30/32

oI3.5 lt:~s sf3~

Tet,,~l :7,5129

.~la6

TABLE II

iParen~s (1) P u r p l e ) (2) G r e e n x * ~9

L o r d G r o s v e n o r x M~Z'~s 4:0 39 - - - - N iedzwe~z]C~lr~na

S e e d l i n g 1017 x 1001 2 6 - - - - i001 x 1017 26 12 0-$77 0 3 0 - 0 : 3 0 1017 x i001 Z3 18 7"81.3 <0"01 1017 x i001 ,54 12 1-636 0 ' 3 0 - 0 - 2 0 1017 x 1 0 0 i 72 22 0 ' 127 0 , S 0 - 0 7 0 1 0 0 i x 1017 4<~ 17 0-267 0 - 7 0 - 0 5 0 1001 x 1017 ~ n d ree iproea . I 221 87 1 .73 I 0-30-0.10 CJox's O r ~ n g e ]Pippin × i 0 0 1 3 5 - - ,,-- E z m l e t h ]~t~rly x 1017 18 5 ,5-26 0.0;5-0.02

crossing the two purple seedlings 1.01.7 and 1001, and from back-crossing seedling I001 wi%h a greendeaved variety. The resuIts obtai.ned from these F~ seedlings are given in Table II.

Except for the family 30/32 ehey confbrm with a 3 : 1 ratio. The cause of the deviation in this f:~mily is no~ known. These seedlings are, therefore, heterozygous for a single major colour gone. Of ~wo smMI back-cross families, one (:35/29) a,pproxima~es to a I :I rude, bu~ d~e oLher (.3/36), ha.s a deficiency of gree~ plaubs.

The varieties Era.nods Early and Lord G.rosvonor are two of d~o greenest varieties of apples, and were tzssd in these crosses for that ~eason. The leaf petioles of some apple varieties are red; as far as possible these varieties have been avoided. Again some varieties produce seedlings which, have a tinge of red[ pigment in the hypocogyl, whiolz

122 go,noticed Studies in Apples

sabsequently disappears, comparable with the case in R¢.phc,,~v~¢.s sativ'J.~s (Uphof, 192-'1-) and R,~.~&~.s idaeus (Lewis, unpublished), in which this character is due to a single gone. In some families this transitory hypoeotyl eo!ou.r makes the scoring of @~.e pEtrple character a liRle di~euR when the seedlings are very young.

TABLE III r, sumfly Seeds % o12 seeds % of pla~s no. Parents sown germinaf, ed stKvfvh~g

Green x green; untreated seeds Go[del< Spire × Brewnlee's Russet 73 Cox's Oraiage :Dil~pin × Lax~on's Superb 1112 Cox's Orange Pippin × !-Ierring's PippLu 60 Cox's Orange Pippil~ x Emne~h Early 28 Lord Derby x 7gmneth Early 16 Cox's Orange Pipph~ x Charles Ross 15'i Cox's Ora;~ge Pippha x Ellison's Orals.go 126 Ellfsoa's Orange x Cox's Orange Pippin 52 Golden Spire x Beauty of Bath 76

Total 687 Mean

Crosses involving 34. Yisdzwstzfcgana; untreated

1'4/:31 78,0 65.6 11/33. 78,¢ 78-4 16/,32 96.6 96.6 9/33 764: 71-4

10/33 100 100 1/34 64.9 64-9 ~/34 65.8 65.8 6/3".4 98,0 98-0 3/32 92,1 92,1

78.6 77"1

seeds

34/29 1017 x 1001 51 11-7 tt-7 ;~5/29 Cox's Orange Pipph~ × 1{)01 19 42.t 42.1 ~9/32 1001 x 10N 73 .57.5 56.6 30/32 1017 x 1O01 62 72,5 72-5

9/35 1017 x t001 141 46,8 46-8 1/36 I017 x 1001 127 77-I 77-1 2[36 1001 x i017 S~ 72"6 72'6 3/36 Emneth E~r[y x 10[7 59 38.9 38-9

To~at 616 Mean 56-5 56'~

Crosses ipvolving M. zViedz.wetz/cga'nc~; ties.fled seeds

The pe.t'oen¢age of seeds which gerlnin.a.~e, under no:renal condi@ons, in .families d e i v e d from M. Nie.dz'wetzl;yana is usuMly rather low for diploid apples.

tn Table I I I t;he percentage o:/:' seeds germinated and seedI.ings po~ted ~u'e given for the ~hree main classes of seeds, which are as fo].lows: Class I: famJ.l:les derived from two green diploid varieties. Class If:

8/37 Cox's Orange Pippin × 1017 52 100 69.2, 9/37 C',ox's Orange Pippin x 1017 57 98,2 .94.7

1t.0/$'7 ;rinxnef, h Early x 101'7 11-~ 100 82.i 12./37 Golden Spire x 1017 I5 100 I00 i'.4/37 :Lord C4rosvenor x iO01 28 100 75-0 1,5/37 Lord Grosvenor x 1017 20 ],00 9.'5.0 16/37 tO0]. x Golden Spire 81 100 82'7 17/37 I001 ;< i017 74 100 93"2 I8/37 1017 x Golden Sph'e 67 100 92"5 ]_9/37 1017 x Lord Grosven.or 1u)7 100 83-4: 20/37 1017 × 100I 38 100 97"3

Tobal ,671 iKean 99,8 87.'7

123

families derived from crosses involving M. Neid~'wetz/~;yc~'~zr~, the seeds of which were sown in the ordinary manner. Class i I I : families derived from M. Ne,idzwstz/~ya.na, ~he seeds of which were germinated . L.md.er ckifferent condibions, as described[ later. Considerable variation is found in the percentage germinated ia both class I and class t i : however, the d_iJ~erenoe between the means is just on the margin of signgioanee (t = 2-32, io = 0.05 - 0.02).

I~ was thoughe a5 ~rst tha~ the seeds which did no~, germinate contained deeply pigmented embryos; deeply pigmented seedlings are

di~.z"

............. i'":iiS ?,~j,>~.-.

Fig. 1. A~ and A.~ greo,;a, B LJ.nged red, C purplish red, D~ and D: very dark p~rple plants fx'om famiJv 1/36 (1017 x ~,001) photographed 1.5 moa~hs after germinat, ion showilag

" )." the dwarf and sttmged growSh of the d~rk purple pla~J,s.

always small and sgunbed, and have up be now failed to make good trees (Pig, I). I t therefore seemed likely that planfis which, failed Go grow satisfaeeorily would also be weak i.n @sir power of germination. Later resalts showed that ~his was not 0he case, and we mast omlelude that the weakness which aoeompa:nies deep pigmentation is 1lot presel~t in gee early stages of germination. I t is conceivable that She deep pigmen- tation affects chlorophy.Ii Nrmation or photosynthesis, processes which do no~ come im~o play early in germiuatJom

Ss.~n OER~II~A'J;IO~ T~O~IQUE

To overcome ~he d.iI.~iculty o~ poor germination a tee]inJqL~e for brealdl~g the dormancy of seeds described by Flemion (193,3), Tukey

124 (2~eticcr~ ~t'z~di~s i~ A[pp~es

(1934) an.d i%. yon Veh (1986) has been applied. They found tls~t by removing the en.dosl~erm anct n.uceItar tissue the embryos would start to germil_~ate before the seed was mature. The method adopted during th.is ihvestigation was to remove the testa, endosperm and nucellar membrane with a sharp scalpel and to place the embryos in sterHized B.overi dishes of ;vet Spl~c~9,~z~,m moss. Tap water was found to give satisfactory results ; Knop sohition did not improve the growth or development of the embryos. The uncovered dishes were placed ir~ a warm greenhouse,

One hundrsc[ and fifty embryos were excised[ on 6 December 1937 (approximately 7 months after flowering). The resl:~iting seedlings gre)v satisfactorily at first but remained stunted in the spring. The remainder of the embryos were excised 18-22 January 1938. These produced plants which developed normally throughottt. This is in agreement ~ith the work of Tuksy (19.38) who found that if the embryo is immature when excised the resulting seedling is stunted or at.normal.

The development of the embryos after excision is as follows: In twelve hours the cotyledons have divided appreciably; traces of antho- cya~.in and chlorophyll have appeared on the second or third day. The eolour is developed fully in 6-8 days, at which time also the radicIe has begum to grow. ~ t e r 14 days file sesdlings which had developed normally were potted up into a standard compost.

In ~his way 100~/o of t.t~e embryos can be scored for anthocyanin. ~kpproximately 1 3 ~ of the embryos fail to develop into plants. Some of these defective embryos do not develop beyond the stage of producing pigment and expanding the cotyledons. Others start to produce leaves, but the radicle fails to develop (Fig. 2). These embryos probably carry lethal genes, which affect embryonic development. Not only is tee percentage of seorable embryos significantly increased by the treatment from 56.6 t,o 99-8, but t.he percentage of embryos which develop into plants is increased from 56-4 to 87"7 (Table ttI). Therefore only 28 °/o of the seeds which fail to germinate under normal conditions have defec~ive embryos; the remainder (72%) can be ind.nced to grow and form normal plants by removing tiae testes.

Gs~TIcA5 Z~sun~S ~ o ~ sxc~s~D :l~5~-os

AI1 the seeds obtained from crosses made in 1.937 [aa, d their testes and naeellar tissue removed. They were scored. :for anthocyanin (3-8 days lager. The purple group was classifi.ed in t]~e folIowing way: A, very deep purple; B, reddish purple; @, Singe of red or purple. If these rssulSs are compared with those in Table I% it is evident tlt~t the p_roportion of

{ IP~5

purpl.e to green piants has .not been changed significantly by ~he tresb- meet. Therefore the seeds which fail to germinate do not contain an a.bno~'mal p~.'oportion of deeply pigmented embryos, as was su.spected. ]0"nz'~ke±' evidence for ~his is obtained fz'om t.ke twen~y-sb: seeds wl~ich

': k

'< ~2 J"

f , , J • .. .

B

~ C

Pig, 2. S~edlings from fm~lily 19/37 ]?hotogr~phed 20. L 38, 4,5 days afl;¢r exoisiml o:[ the embryos, zl~ no~'ma[ ,seedJb~gs../3, seedlhlgs which fai| ~,o dovelop fit.bet, ~e~ve,s or ~'oo[,s, (5', s{~e(Hings which dl)ve[op leaves btt~ t, be ~'ooLs f-~i[ tic develop. ,4 =i~, H ~uld C = }nat , un~l..size.

did no~ develop beyond opening d~e cotyledons in families 18/37 and 19/37; fwelve ~v'ez'e purple an.el :[btt~'been green, ~.~ close ~p]_)roxim.~t, ion to t.tie expected 1 : 1 ra~io.

lV~mn *>he ~wo F I seedlings (1017 and 1001) a~'e inte~.'crossed, the resnlts approxhnate to a 3 : ] r~i% and back-¢~'ossing either seedli~g to

126 ~e~~et{ccd £%.~cdies ~,~z AppZes

a green, v a r i e t y approx i raa%s ~o a t : 1 rat io. The or ig inal scoring of this

f ami ly was fifey~six purple and twcn.ty-f~re green, b u t as ehe p lan t s

deve loped R was ev ident t ha t e ight plan~s g rouped in the O class were

rea l ly greens. The deficiency of greens in fami ly 16/37 is p r o b a b l y due

to the green p a r e n t carryi114, one or more fac tors for eoloured hypoeo ty l .

This obscures the scoring, so 51~.at some plan~s wh ich are rea l ly green

are p laced i~l class @.'

Family no, Parents 9/37 Cox's Orange Pippin

x 1017 10/37 lgmneth Early :< 1017 12/27 #.,'olden Sph'e x 1017 I5/37 Lord Grosvenor x 10t7 18/37 1017 x Golden Spire 19/37 1017 x Lord Gros~'enor

11/37 Golden Spire x 1001 I-3/37 Lord Derby x 1001 I4/37 Lord C4rosvenor x 1001 16/S7 1001 x @olden Spire

17/.37 1001 x 10t7 20/37 1017 x 1.00l

TABLE IV

Pm'ple

Sum (@sen A B C A[BC ]3 X 2 5o 17 11 14 4~ 48 0.400 0.70-0.50

12 22 !9 58 54 0,009 0-95~0-90 0 0 3 3 12 5-400 0.05-0-02 6 S ;~ 12 8 0.800 0.50-0.30

-- -- -- 48 .'-18 1.162 0.80-0.20 - - -- -- 65 8I 1.753 0.20~0-10

2~3 24I 0.698 0.50-0-30 0%- 0 0 0 g 0 ! 0 1 1 5 3 4 12 15 0-3 3:'~ 0-70-0-;50 9 19 20 48 33 2.776 0-10-0.0.5

14 23 24 61 51 0.892 0-50=0-30 9 19 2.3 51 21 0"666 0"50-0-30

10 S 11 2,9 S 0.22`5 0.70-0.50 19 27 34 80 29 0-149 0.70-0-50

DISCUSSION

The equality of green and purple plants obtained w[hen Lord @rosvenor

(green) was crossed with l]f~h~s NeidzwctWcya'n,c~ (purple) indicates that

~he laRer is heterozygous for a single major colorer gems, The wriation

in the d e p t h of p igmen[a t i on found in the pu rp le classes in atl families

can be explained by ei ther a difference in dosage of t he same geue or by the eKeot of modif iers on the p r inc ipa l colon: gene.

The fac t t h a t two seedlings, 1017 and 1001, beha,ve gene t i ca l ly in the

same way, a l though one is much more deeply p i g m e n t e d t h a n the other,

indica tes t h a t the c[i[~eresce be tween these two p l a n t s is not due to

different dosages of the same gene. Furthermo.re, seedl ings are ob ta ined

f rom s, back-cross of the he%rozygo te to the green which are as deeply

p igmeat .ed as the da rkes t p l an t s p roduced by in te rc ross ing bwo hereto-

zygotes .

F u r t h e r suppor t of tlhe hypo thes i s tha{: modig.ers a re responsible for

v a r i a t i o n wi tk in the purp le class, is (1) t h a t a close a p p r o x i m a t i o n ~o

;3' 1 and 1 :,1 ra t ios is o b t a i n e d f rom in tercrdss ing a n d baek~orossing

D, L E w i s AND M. B. Cr~aNJ~ 127

respectively, and ~he same range of variation occurs in both in tercross and back-cross families; and (2) the inheritance of the pigme:lbted character is the same in seedlings 1017 and 1001, although they are phenotypieally dif~erenL i t is also known that cultivated apples are invariably heterozygous, a condition favoured by ~he prevalence of self-sterility and maintained by elonal reproduction. This would favour ~]ie segrega@on of modifying genes, thus accounting for ~he variation i~ the expression of the colour gone.

A fitrther generation of plants wouid have to be raised, to determine whether, duplex plants are present among the 1~. It is, however, e~fident that even if the major gone is in a polysomic condition modifiers play an important role in the activity of the ptlrple gone.

Hatdane (I93.3) has pointed ou~ ~hat in a polyploid most of the genes will eventually return to the diploid condition and, therefore, disomic segregation of the purple gone would not be in disagreement wRh t h e secondary polyploid 5beery.

SU~A~Y

The inheritance of a : 'purple anthocyanJ.n" character in apples is described. Mc&~s Neidz~ve~,f~yc~.~c~ was found to differ from M. 7]/7a~s in having a single dominan~ gone for "purple pigmentation". Great variation was found within the "purp le" plants, probably due go the segregation of modifiers.

A technique for increasing the percentage of germination by removing the testa is described.. The percentage of scorable plants was increased from 5~ to 99. No differential viability of the "g ree~" and ~'purpte" embryos was found.

~ETFEP~ENCES

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128 Ge%etica,~ -S~%d.i~,s' in AI)I)[~8

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