THE INCOMPATIBILITY SIEVE FOIl PP~O]gUOING POLYPLOIDS

The possibilities of improving our cultivated fruit ~rees by inerea.sing the chromosome number are not yet fully known, but t]ae good qualities of the existing triploid varieties of apples and pears justify the hope that improvements may be obtained by polyploidy. These good qualities of the triploids havs been fully disoussed by Nilsson-Ehle (1938}, consequently only a brief reference to them will be made. The average keeping qnality of the present triploid, varieties of apples is be~ter than that of the diploids. Crane & Zilva (1931) have shown that the highest ~dtamin C content in apples is fonnd in the triploid group. Further, 9he f~et that one-third of our best modern pear varieties and one-six-bh of the apple varieties are triploids shows that they have ~ahable qualities. This is more striking when ig is remembered tha$ the numb@.} of triploids arising natm'ally is very limited[ in comparison with the number of dJploids available for selection, t~rom our knowledge of the tetraploid l%~tility pear, tetraploids%honld have the advantage of self-compatibility, thus obxdating the necessity of ml,xed'orohards. Larger f r e t of better flavonr than the diploid is another advantage of the tetraploid pear (Crane, I9~8).

Because of the promising qualities of polyploid fruits many attempts to produce them have been made. Nilsson~Ehle (1988) produced tetraploid a.pples by growing a large number of natural seed from trip!old varieties. Nebel & l~uttle (1938) have stated ths,~ a gripIoid form of the apple NcIntosh would be of great importance in the U.S.A., bni; that a tetraploid would hays to be produced before triploids could be obtained. With the method to be described here, triploids can be obta,ined in one stage, thus sa~dng many yea~s.

Of the two most importan.6 methods of producing polyp]aids, colchieJne l;re~.tment ~nd heat-shed,s, eolchieins has been so successful in plants with sof~ tisst\es and rapid growth that i~, bar superseded ~he heat~shoek method. This success of co].ohieinc is dus t~o jt~s relatively prolonged action on a vegetative growing point. ]{lent-shocks necessarily being of shori, duration, are not sui~a.ble for naultieelhJar tJ.ssues. [But: in woody slow growing plant;s coleNoine has not me~ with t:he same gueec'ss, and consequendy in fruit trees I ha,re made use of the hen.t-shuck meldxod.

I-Isag-shocks, to be @cti.ve, must be applied at a time in the life cycle of a plane when one cell dixdsion a-ff'eets the whole of the organism, i.e. the first zygotic, the pollen mother cell, and pollen grs~in, divisions. Treat, recur of the firs~ zygotic di~dsion has })sen used effectively in maize by l~audolph (1932), but it mnsl; be earei\d].y timed and h~s been :fmmd unsatisfa,eto~:y :in many o~her p]ant~s. The value of hea,~ treatme~ae on the pol].e~ mother cell or pollen gra, in ¢|ivisions is reduced by th.e fact ~bat celt. divisions in an aa~il.~er are not synch.rm~ized,, so that the resulting pollen is a mixtm:e of diploid and[ haploid grains. When this }?o!leni~ used .the hapl'oid generally has a dJs~ince advantage over the diploid, so thaf the requj.red diploid pollen grab.s do not effect fertilization. Therefore a sieve is required which allows only the diploid pollen grains tlhrough to the ovary, Such. a, sie~,e is present in .m~y self-ineomj?s,tible plants, e.g. 1)e~.~.~i~, species (Sicat & Chandler, 19~1), ~ola..~,u,~ species (Johnstone, 19.39) and Py.r~.s com,~m.,~lis (Crane & Lewis, 19~12}; for diploid pollen

262 Ir~co,m.2)c~tibibity sieve for pro&~.ci.ng pol;ql)loida

grains are compatible and fully efl!ective on their own style, while haploid, grains axe in- compatible on tff/eir own styles (of. Lewis & l~'[odlibowska, 1942). Hence +she produetip[~ of triploids should be assured in seIf-ineompatible pla,trts by heat-treating the pollei mother cells, followed by self'- or other inoo:mpatible eross~polliaation. Tlisre is one con> plioation in the use of the method, viz. only tlie grains '>vliieh are heterozygotls for gw~ different S gene~ are eompa~ibIe (Lewis, 1943). Tlierefore a heat,shook at tlie second meiotic divis[~on would no[ neeessa,rily produce self-eompat:ib!e diploid pollen grMns, and R must l)e given at a time before ~he segregation of ~he incompatibility gen.es.

]~ETIIODS OF GIVINg TffE H]~AT-St[OOX8

~{ea~-shocks were given in 194:0 to sweet cherries (2x), a seedling plum (4x) and pears (2~), by placing a thermostatica,lly controlled electric heating cylinder over tff~.e ends .of ~he branches. The treatment temperatuse was varied between ¢0 arid 46 ° C., and the duration of treatment tk'om 20 rain. to 2 hr. Tlle letlial temperaments was a])ou.l~ -[6 ° C. i±" applied fez more than 15 min.,"ba[ a Sicher teinperattu'e of 50 ° was not [etlial for 2 3 rain. Since the metl,od proved. to be successfal, it was tried on a larger some in 19-'tt by placing whole tl'eos in healed chambers; unfor~lmately this method wa.s g failm'e becmise t;reat- ments which were drastic enomgli to cause polyploidy were semi-lethal when applied to the whole p]ant. Trees were eitlier killed[ outright or the buds were killed. In 19-1.9, these- fore, the original method of treating indbddu~l branches was again used.

The timing of the treatment in relation to tl~e stage of development of the polle~ mother eel]. could not be accurately estimated, because there is such gre~t variation both wRhin an anther and between flowers on the same brailch, i"~evertheless, treatments were made to coincide roughly with (I) the pre-meiotlo mitosis, (2) early prophase and (3) second metaphase of meiosis. The pollen was examined at frequent intervals a res treatment, and found to be abnormal in most cases. In the early prophase ~reatments fa ihre of pairing w~s evident but it was never complete; in the'second metaphase tree% merit, failure of cell-wall formation could be seen. In some cases, no effect of the "0reatment could be detected; while in others, the pollen was completely killed. Before pollination pollen was selected with a high proportion of abnormally large viable grains, which presumably were diploid.

~ESULTS

Pgt~.ms. Since %he domestic plums are already hsx~pioid there w..a..s no reason to increase the number to nonaploid by heat treatment. Kowever,.a very good tetraploid seedling p l a n (No. 1193), raised by Mr ~ . B. Crane from a cross between P,r'~,,~us <~os~estica vat. Jeffe~:son (Oz) and P. ceriseS'era (2x), was treated, in the hope of getting a hexaploid. This seedling is completely selfqnoompatibIe, havi~Jg produced no fmfits after self-pollination of 1709 flowers. A hexaploid from this seedling should have larger fruits an[[ be self- compatible.

~ollen mother cells which had been treated at the secon.d metaphase at '~4 ° C. for 1 hr. on 27 February produced many giant grains, This pol].en was applied to eighty-six untrea,ed flowers and[ to ninety-JJ.vs treated flowers, Rom the total of whic[h nine fruits we're obta.h~ed (Table 1). Four se,ed[ings were el)tabled from the seed; three of them were normal in vigom' and one was rather w e a l They all proved to be ~,etsa]?loids and no(

D. L ~ w : s 963

hexsploids as w~s expected. This f~mctionJng of hs,,ploid pollen grains m a y be due to gene meats.Siena at the S locus, caused by the heat-shocks.

C/mr~',fes. T h e production of a, triploid sweet cherry would be of little value, because in -a frofft with only two ovules, gametic st, eri]Ry is ~ seriohs ca,:as of A~rai t futness . A teh'aploid world not suffer i?om @.is defect and might show improve.men~s in othe_~ chsraeters. The chance of lX'O&:oing a tet: 'aploid by hsat t rea tment qsnatural!y smaller.

<"Ibuo the seIec, t i o : of the diploid grains by self-ineompatibili:.y sho~]cl, bri%g this within prs, otioal limits. Ten clJJ~'erent, varieties were treated in the spmings of 19@-2 at the early p:'op~ase and second metaphase of meiosis. The pollen after treatmen~ was h'equently abnormal; dyads, and :tetra, de: eonta.i~i~g five, six s~d seven cells were produced. ~{ol's than 800 1%were were sel£pollinated with the ires.ted pollen, and onlytwo fruits were o%t£ned, these being bn the vaiety @o?ernor Wood self-polE~atsd with pollen treated at second mete,phase. These ~'uRs contained very small seeds which failed to germinate.

.~ o'- 2, . af tsr @m~p:Um.~.~:bZe p<?ZZi~:at.io:: l#'ith hs:~,t-t're&tecl ~9ol~,sn Table 1. Y r u { t ~ c~:~ .se5 .. #? o..z~.o~d

Plmms. 2~:.=39: 8eedl iug 110S

Oherxies, 2~ = ] 6 : ten variet%a :Pears, 2~1. = B~:

~'ertilRy

Co~ference

]3. dL4ms~sfis x Confe;:en{'.~

Trsaf.ment

~lean ~iax. Time p obliw rue.sofas ° O. ~ C, rain., ato£ Fmi~s Seeds P]an~s nnmhcr

66 60 95 a 5 $ 8 8S ~6 ~6 60 S§* 6 4 I Sff 44 ~8 50-~0 S0O ~ - £ 0 --

45 ~7 50 186 3 6 6 51 .4-5 50 88 38 5 15 15 51 ~4 45 120 23 I 5 5 51 66 46 120 i16 0 . . . . 40 ~1 90 g~ 1 3 S 51 ~O ~1 90 $2 ] :- - -

2oHen;a~o~]:e:'s ~ a v e both trea~ed, t ]~mp~y. "* Untreate£ ovules fertilized ])y tregted

&cc~..r~ c,a~#, ~7<~Ze.~. The results; gives in Table t, show tYaa.t the method is more successful wJ.t.h }?ears thaw vdt]~ che.ries. T.rJploids have been obtained irom the varieties Ife:'@]ib~ and C,onferenoe. These variet.ies are ftttiy self-h.]compa.tible., in ~hs 0ooi g:reenhous%

a~te.: se]:fi]_w.: or wRho@:, pc!End,ilion. alth.oug]a t hey e~n ].)rod.uoe part]ie_~toea.rp~c f]:uJts ~ 1 ~. wt~ en g:'ow£ in the open afl, e.r frost inj ury to the styles. [['h6 different tempe.ra,tu:cs a.]?p]ie8 i~,o the buds did_ ~ot have a. oou.s£sl.e:~t e,a'e~o on t]ie success . . . . . of ~he treatment. In Fertility.,. a .high tempers,ture fo~ a short time gave ]oettox results than a, ]owe: temperature for a longer J2e:'.]od. P, at with (bnfe:'enee the reverse was true. Three treated branc]:ss of the var ie ty 1)oye.s.s6 dn q_gmice failed t:o set, a~y frui[,. Since the o~mles were treated at the .same %line ss the pollen ]nether cells % was expected: i]_: ~dew o:[ the varia.@on of tb_ue of flower developmen~-, on a b~a::cl:~ tha t seine of the. e~gs might ha,ve beef di]?loid. ])toweve:'~ no tet: 'aploids were prodl:ced.

The aot.iOl], o~" selec@on fo:r l i e diploid, s.i: tl.le ex:t:emse of i}he haploid poIlezL l]] all: inoompatl]ole style is reversed in a compati]de, style. For exa.~]p]e: w]~e.]~ hea t treated pollen, of I¢oyenne-au~ . . " -~ "~.@omloe' was applied to t].e styles of I:qp,roved [[~e:'tj:Lty (~:x)~ Q913, "haploid pollen effecbed fertilization producing h'J.p]oids bu~ no tetra,]ploids. A cross which ..might l?e expected .to ]?:{ocRme a small p:roportio~ of tetra.pickle is ff3cnrr~ d~Am.anlis (3,z:) ].~ eat treabed _,o.n{ere::ee, si~ ce t]~..{,~ i.s incompal;l]2e when :~iorma.l po]le:n is ~:sed (Orane :: Lewis; J.942). ]~ut with treated poJ.]e.n one fre{t from eig].~[,y-two flowers wa,: obtsiued a,nd this cent<lined :.i o:,hm a hu~ el:apt): seed,s,

Join're. of (Jenet :cs <i5 i8

2 , 6 4 I~zoo'~~,pcr~ib{f@/ sieve fo)" l)~'odzcci'~zg pofyi)Ioid~ ]?our varieties of apples were t~ea~sd, of which Cox's Orange Pippiu and I-le~ring's

Pippin produced fruit after seH'-pollination with heat treated pollen: but only two se~_ds were obtained aad these failed to germinate.

The ddscrimina'5ing acgion of' the style varies according to whether the grottp is self- compatible or not. In self-compatible groups it hinders or px~events the funcbo~ing of- abnormal eameues.~ ~ This abno~,.~.ah~y" ' '~ may be d.u.s to ~aenic segregation or chromosomal change within the no~'mal breeding g~:onp, m' to the.presence of gametes from outside this group. In these cases the style has a stabilizing influence, reducing the aolugl and poten- tial variability and particularly eliminat:ing unreduced pollen from. the breeding group. In self-incompatible groups, the sieve action of the style is ~ormally to eliminate self'- and cross-incompatible pelion. But, after selflng, it is the abnormal gametes which are favoxtrsd by a secondary selection in the style. Thus, haploid gametes fail in self-pollina- tion, while diptoid gametes succeed.

The 5y_,por~nce of this action in ~he occurrence of nat~.~al polyl?J,oids i~ ~he self- incompatible ~II~',~av,. ,%/~os'uopres.z~v~ atfd yl. ~a~,~zs was pointed out by Levan (1936). i t is also s~ factor which has encottraged triploidy in the selfdneompatfble species of flhd.ij)a,, tt~/c~c.i~t/~,~x, L i ~ { ~ , and Trad¢}c~,#ia. . [['he triploid v~ieties of Py.m~s co~~z~,~z.fs and. ~P. ~.uel~s can be accounted for i5. tk~ same way, sines in a large tree nearly all the flowers must be self-pollinated whether they are cross pollinated or not.

~eafi-sliooks applied to the pollen mo~her cells, followed by sslLpo~nation is effective in producing triploids in fruit trees. The method depends upon the sieve-like action of incompatibility on ~he pollen, allowing only diploid pollen tubes to reach the ovary, although much of the pollen is haploid. Since only pollen grains which carry two different S genes are compatible the heat-shock must be given b e f o r e th.ese genes segregate.

By this met, hod triploids have been produced from diploid pears. In diploid cherries, ~uits have been obtained, but the seeds were small and in~dable. In a self-incompatible plum (2'~=39), fruits and seeds were formed aRsr self-pollination with treated pollen, but fibs seedlings had the s~me chromosome number as the parent.

I am indebted to Dr P. T. Thomas and Mr S. K. Revel for determini~_g the c!xromosome numbers of zdany seedling pears.

P,.'ElPERE~f@ES

CP~-c-wz, Bi. B. & LEwis, D. (194~fi). Y~ ~e~a~. 43, 31-43. Cs~_w~, IN[. B. & Zlzv-a, S. S. (19.31). J. Yw~o/,. 9,228 91- Jo~sTO~, ?T. E. (1939). Avast. Pot. J. t6, 2.88-30~. L~VA~, A. (1936). Hemgitt,~.% L~.~cf., 22; 278-80. L~wIs, D. (19¢3). J. g,~z¢~., (in the Press). LEWIS, D. c~. ]iODL[[BOVCSI;A: I. (t942). ,r, g~Tts~. 43, 21].-22, N ~ L , ]3. R. & R.UTXr~Z, 1K. L. (I938). d;'{~'~, iRr.] -. ,% Agile. E.¢p, ~t~t. no. 183. Nwsso~,'-E:l~L~, t{. (l, o8)- fl~.~'sdftas, L~.~(f, 24 ]95-209. ~,~r:ooLr~, L- I~. (]922). P~'o¢. zVc~.t. ,dc,.uf. 5~ci., I I"a.~)~., IS: ff2"-2~-9. STO~T, &. B. ~c Cm{mDz~g, C. (1961). Sc./¢uc~, 94., 118.