A Study of Viviparous Mutants of Maize Citation Robertson, Donald Sage (1951) A Study of Viviparous Mutants of Maize. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/r5q0-4f92. https://resolver.caltech.edu/CaltechTHESIS:07282025-160759217 Abstract The meaning and extent of vivipary among the higher plants are considered. The nature of vivipary in maize as reported by earlier workers is compared with observations made in this study. The use of interchanges among chromosomes of the basic set (A-type chromosomes) and of interchanges between A and B-type chromosomes in genetic studies is outlined. The results of genetic studies with six viviparous mutants are reported. Experiments involving the use of embryo culture techniques to elucidate the cause of vivipary 5 in maize are described. These experiments revealed that viviparous embryos are more tolerant of high carbon dioxide concentrations than normal embryos. Experiments to check the role of the cob in preventing premature germination, as well as experiments to determine if acetaldehyde plays a similar role, yielded negative results. The total auxin contents of viviparous and normal seeds are reported. No obvious differences between the two classes of seeds with regard to this growth substance was round. The significance of the genetic and embryo culture studies as it relates to vivipary in maize is considered. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Genetics and Plant Physiology) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Anderson, Ernest G. Thesis Committee: Unknown, Unknown Defense Date: 1 January 1951 Record Number: CaltechTHESIS:07282025-160759217 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:07282025-160759217 DOI: 10.7907/r5q0-4f92 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17562 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 31 Jul 2025 18:16 Last Modified: 31 Jul 2025 18:40 Thesis Files PDF - Final Version See Usage Policy. 31MB Repository Staff Only: item control page

A STUDY 01"' VIVIPAROUS l lOTAi.liTS JF l\u AI:lE Thesis by Donalds. Robertson In Partial Fulfillment of the Requirem ents For the Degree of Doctor ,: ,f Philosophy California Institute of Technology Pasadena, California 1951 '}' he author- wishes to express his a ppreci a tion to Dr. E.G. Anderson for suggesting this problem and for his advice and encour;_;g(~rnent, to Dr. J. Bonner for his advice with reg ard t o embryo culture ex periments and his permission t0 report the unpublished results of t wo experi ments, to Dr. A. E. Longley for his helpful advice and permission to re port unpublished cytological information, to Dr. H.J. Teas for his helpful suggestions, to Dr. 1 s R. s. Bandurski and H. Bowan for their ;Jermis- sion to r eport unpublished materi a l, to Dr. E. E. Dal e for his advice in pr eparing this manuscript, to Dr.'s G. F. Sprague 1-.rnd C. H. Burnham for su;;p lying needed stocks and to Mr. Ea rl·B. Patterson for caking many crosses for rne in the· su:nmer of 194~) when I un ti,ue ly a ; ,pendectomy. \Y& s hos pi ta l.iZEi d fo r an ABS'fRACT . The .. meaning and extent of vi vipary among the htgher plant_s are. considered. The nature of vivipary 1n maize as reported · by _earlier workers 1s · eompared with observations made in this study. The use of ~rtterchanges amoQ.g ehro~osomes ot* the basic set (A-t,ype chromosomes) • and of 1nterchangea between A and B-type chromosomes in genetic studies is i:,utlined. The results •::>f genetic studies w1tb six viviparous mutants are reported. · Experiments involving the use of embryo culture techniques to ,. elucidate the cause of vivipary in maize are described~ . ' These experiments revealed that v1v1parous 5 embryos are mo-re tolerant of high carbon dioxide conoentr&tions than norm.al embryos. Experiments to ehe.ck the role of the cob in previmting prewature germination, as well as experi• . . .. - • . . ments to determine if' acE:taldehyde pl..ays a ~:!imflar role~ . yielded negative reaul.ts. The tQtal auxin contents of v1v1parousand normal seeds &re reported. ·No obvious differences between the tv,o c1$.iSSes of seeds rl th regard .to . th1a; t?rowth substance was round. Tbe significance °,f-. ~he genetic and embryo culture studies as 1 t relates to ·ViVipaey '1n maize is considered. TABLE Of CGN'I'E.NTS Page 1 1!~1 i•1·0DUCTI Ol~ . ...•....•.....•. • • • • • • • • • • • • • • • • • • • • • • • • • • • THE NATUhE Ofi' VIVI PAHY IN MAI2J:. ..••••••..•••••.•••••.••• A. 5 Vivioarous ~utants ~f Class One ............... . 6 -• B. c. D. Vivi oarou s Mutants of Class Two ............... . ------ a ,_, Viviparous Mutants of Class Three .............. 17 .ft.Summar~ of the Characteristics of the V;!vigarous Mut~ ............................. 19 SPECIAL TOOLS USED T0 LOCATE GE.NES ...................... 19 A. Trans loca ti.Q!l§. . . • . . . . . . • . . . .. • • . • • . • . . . . . . . . . • • 20 B. Translocations Involving &:ItPe Chromosomes .... 24 • ·o·•,· GENET IC s·ruo111s lNVO~VIL~G VIV.lP-i~h: u~ ................... 29 1 Gi:;tH:TIC S'l'UDI E.8 li'iVOI,Vl.NG VI VIPAROUS 0 • • • • • • • • • • • • • • • • • • • 34 ~ G.li.liL'l'IC S'l'UDI£S INVOLvlNG V'I\l•I .DA . .· n·o··Q . u 5• . ...•............• 35 Gl:Nf:..TIC 8TODIES I NVOLVI NG VIVIPAHOUS 7 ••••••••••••••••••• 39 G.LNETIC STUDIES lNV JL VING VIV l :::.,A1i. JU s8 . ....•............. 40 1 ' • OU'-'u . • • • . • . • ........... 43 GENETIC BTODIBS INVOLVING Vlv'IP i-1.J\ 9 E.MBHYO (;0.LTOHE AS A 'I'OOL FOR INVES'TlGATING VI VIPAHY •••.• 45 .A. Tests for a_Qermiqation Inhibitor in the Endosperq.i. . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . 48 B. The Effect of 02 Tension on Ggrmination ........ 51 c. Carbon Dioxide as an Inhibit9r of Germination .. 58 Pa ge i°iiI GCELLA.NE<)US EX.P.Shl ktLNTS B.bAhING ON THE NA'.l.'UHE 0 .F' VIV l PAhY . • • . . • • • • • • • • • • • • • • . . • • . . . • . • • . . . . . . . . . • . . . . . • 6 4 A. The Role of the Cob in Inhibiting Premature .Q...g_rrui B~~ i 9..9. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 6 4 B. li£~:§_1dehyde as a Po s si b l,g__G i:-.! r mi na tion .!.,g.hibitor .. . ..................................... 64 c. Auxin Analyses of Viviparous Mutants ............ 67 DI SCU SSI ON . .................................... •. . . . . . . . . . 70 i-tEP~£HENCES . . . . . • . . • . . . • . . . . . . . . . . • . . . • . . . . . . . . . . . . . . . . . . . 7 5 l INT HODUCTION ¼ost plants are ca;ab le of surviving periods unfc;vorabls for their gro'r-th. In general this ls done by productng spcci r"> lized structures such ns seeds, s JOres or zygotes which arc c~~abl,) of' witbstsndlng •_;1ore cxtre:;1e conditi~ns than the veg8tative body of the pl2nt. Am.-:,ng the a 1.gae the zygote rr2sul ting fr'.Jl:l the fusion of two gametes frequently secretes a thick wall about itself E.~ nd rer:1cdns in a resting st:1tc until condi- tions are favorable for vegetative gro~th. In t he funri, zygotes and spores are employed to bridge periods of adverse gr~wing conditions. Members of the Bryophyt~3 i:,nd Pte r1dophytE:;s rely on sp ores. The sporo::: ::~nd zyGotcs produced by these lower µlDnt s arc relatively undifferontir~ted., frequ ently consis t ing ;)f a sing:..(:> coll wh ich u;·,on germination ;Jroduces thE. vegetative structurE:s. In the higher plants the seed is utilized for this purpose. Unlike spores or zygotes a seed 1 s '°1 co:n - pli c :::. ted structure, being composed of many cells and shows a high degree of differentiation. ~ithin it is found the er::1bryo which up.-Jn germination yields another plant like its parents. The seed is designed to fulfill the needs of thi~ embryo. There are usually found outer coats which serve to protect the embryo from the external environment, and internal storage tissues which sup ply the required nutrients while the embryo is germinating. Such a complica ted structure undoubtedly involves many complex reactions, most of which are essential to the develop~ent of the seed and if interfered with by a mutation of one of the controlling genes will result in aborted seeds. However, reactions ·vrhich can be disrupted without producing lethal changes are to be expected. Changes which are responsible for waxy, sugary, shrunken and brittle endosperm types in maize are examples of such non-lethal Effects. After the egg nucleus is fertilized, the resulting zygote starts its e~bryonlc development. Such develop- ment produces not just a. diminutive copy of the parental plant., but rathe::r a structure specialized for its role in the propaga tion of the species. It ha s been determined experimentally that maize emb ryos are capable of germination soon after pollination (La Hue, 1936; HaagE:n-Smit .§.Sal. 1945), yet in the develo ) ing caryo:)sis this does not take place. This sugg e sts the presence of a mechanism for preventing precocious germination since the young' embryo has an excellent food source in the €ndosperm, together with an adequate water supply. Some suggestions as to what this controlling mechanism is will be made in a later section. At maturity the mechanism f@r inhibiting ger- mination 1s lost but the seed is incapable of germination ctue to its desiccated condition. However, if this control- ling mechanism is lost prematurely, before t he seed is too dry to permit germination, 1~r emature germination· of seeds will result. Such seeds are occasionally observed a.rid 1n some lines of maize the cha racteristic a ppears to be under genie control. Th~se p~emature geroinating seeds ·Mi 11 be ca!.lE.'d viviparous., the term used by Eyster (1931b) to describe these mutants in maize. Ascherson (1875) described a pples which contained premature germinating seeds.- In 1880, _Treichel reported observing thei germina tion of wheat and rye on the stalk. Mc.Kellar (1898) observed germinating seeds within a melon. He also observed seedlings in lemons, oranges, cucumbers, pumpkins, papaws and other fleshy fruits. Oppenheime·r (1922b) indicates that Savelli (1920) had observed seeds germinating in the capsule of Nicgtiana rustica, Citrus limonum and seedlings on e~rs of corn. Bonner (unpublished) obtained a variety of tomatoes which had a tendency to produce germinating seeds within the fruit. One of the earliest refe~ences to vivipa ry in maize is that m.: -: . de by Savelli (1920). Mangelsd6rf (1923) and Lindstrom (1923) briefly describe ears segregating viviparous se~ds. Eyster (1924a) described a viviparous ~utant which was genotypicatly similar to those described by Mangelsdorf and Lindstrom. Also in this paper, he considers briefly the significance of the association of white endosperm color and 'albino seedlings. In a second paper the same year Eyster (1924b) described a second viviparous mutant phenotyp1cally identical to the L! :first one. Mang , lsdorf (1923) termed hi s s ut an ts " ge rmi- na ting seed" (£.§) while Ey st~~r ( l9P4) nr1m fJd his "prirni ti ve Sj)Oro phyte", a te rm hE:· chose becP.use, "th e s:K>rophytes of al l non-s eed--br-ari. nr p lants, whi ch presuma bly A.re ,1 ore 0rimitive than seed-bearing plants a re normally vivinarous" In his later pap er Ey ster (19 71b) us e d ( Fyster, l93lb). the term ''vi v i na rv" (vn) to dE,scr•i b e t hesE· mutants. -.J.::. I ., ti v e in n a tur ::·: n.nd nsidi::: from thE alleLic test rr:portod by Fystcr (19 24b) in his second paper, no genetic studies Ma ng ( lsdorf (19 26) desc1"i bed five diffcren t vivipar~us mut 8n ts , including the mutant of his previous p ,·, per and the 0ne d escr i lie d ·by r... lnds trom ( l9EZi) the same year . He a l so postulated four additional genes t~ 2ccount t'or some rati ,::,s encountered i n segrer. r.t in 0 ears . the gcnH for sugary t>ndoS(H: rm In addi- in the f ourth chrorn .) sGmP . Considered also in t his paper is t he r clatiJnship nf vivtµary to the lack of chlorophyll in th e ~e€d lings and to the endosperm color of the seed. In a later p;-;.per, . Mangel sdorf ( 1960) d 1:1.scri bed · t:": . f: re.sul ts Jf int er cro ss ing the various viviparous mutants considered in hi .s p revious pb"tpers . Fa.rs were ob t a inEd which segregated viviparous seeds in atyJic a l ratios. ThEse are- E:x9l a ine d by a ssuming five addit:ional genes. 5 AlsQ described in this lit,pd' i ::, 1.:; vi vi ps rous mutant which has ~,ink andos)erm color as we l.l as p ink 2;lum1; les. mutant is prJbably the s2 :-ne fas :)W?) This described by Sprague (l J ZC) which 1 s a l.lelic t ,:> :yn 7 .: )f this pi.:iper. Mangel sdorf (19 60) also indicated the possibility of an additional genf wh ich bnd not been tested ag ~in s t his other st0cks. In Lll Msngelsdorf demonstrated six different loci res ponsiblt1 for premature germinati :)n and postulated an addi..., tional nine loci to explain aberrant ratios. (19 31b) questioned the advisability 3f Eyster callinf int~ ser- vice additiona l genes to ex pl3in spurious ratios when dealing with a character such as vivipiry which can be rehdily influenced by environmental CC>nditions. In his paper linkage relations of four viviparous mutants are re ported. These reported linkages will be considered in detail lE<ter. THE NAT □ hE Of VIVI?Ahi IN ~Al~E Jn the basis :)f e ndos perm and se,edl.ing phenotype ·,,;hich accnq Emy t vivi pa rous c~ndition, th~ various vivi parous mutants of corn csn be divided into three classes, as follows: (1) those with yellow endasperm which produce green seedlings, U~) th,:);:e with whi t c or pale yellow endosperm which ,roduce a lbino s eedlings if grown in thE: light, but wt, ich hn.ve a b luish-green tinge if grown in the dark, s.nd (3) those with ·yeLlovl- 6 pink endosperm which produce seedlings that lack chlorophyll but have a f a int pink color whE:n grown in the li ght, but in addition will h&ve a bLu1sh-gr ef;n tinge if grown in thf dark. These three cla sses of vivi parous se eds ha ve previously been re Jorted by various workers. Mutants of cla ss one have been reported by Mangelsdorf (19~6, 1930) and Eyster (1961b) .• Mangelsdorf (1923, l9f:.G, 193-0 ), t.indstrom (192::,), and Eyster (1924a, b, 192,lb) <iescribe uiutants of cla ss two and brief descri p tions of cla ss thrt:e mutants a.re gl ven by l'.iangf lsdor.f (1930) and Sprague J..... Vivinarous M.utan t s of' Cluss One 1:'his group '.Jf mutants i ~ represented by .Y..Ui and. 1.,Q 4 (which a:)peci.r to be allelic) a nd !£8• genes do not development. \t•i si b Ly inf Luence C L'i rotinoid 'These mutant or ch loro ;)hy U. Vivipa rous1, however, ~iff~rs from othsr viviparous types in that seeds homozyg~us for this gene show little or no development of aleurone color. (The geneticaL evidence for this will be considered in the section on genetics). Vivi pa ry in this cute. nt d~es not commence until late in the ontogeny of the caryopsls F. r. t about the time aleurone color is develo ping. This suggests the possibility tha t germination might interfere with development ·J f C·:>lor in thf.: alcurono layer. aleurone co Lor does deve l.09 in ,YB 2 , YJ}. I:hwever, 7 , and .Y.ilg, kernf:ls, 7 Th:!.::-; sup gcsts th~:- t (?erminn.tion :Qer E thD.n ..Y£i · i:! not rf.' :.i Ul. t of This mutant i s phcn?typically nnd r0netically distinct pros- end<)Sfi errn h 1:: d heen digested in this area. r2ti~ ls v0ry closely ~ppr~xim&ted. If thss:.:• ser.·d s In spite of p~ or germination it ls p~ssibl~ to obtain mature hom~2ygous leaves. The mature ~lants are very dwarfed and ~ft 0n :r·:)duce h ,aves Ex tending fr:)Iil the plant in all dire:8i:,i.Jns. Such a .plant la pictur ed in Figure l. Most of thes e ~~~nts praducE functi0n&l t assels , but only a fe~ are str~ng enough to produce an ea r. " ••, :.,'· no.: When F 1 plants of a cr~s~ ·O ~f'. .!..!:!3 seeds with shrunken ger1J1s are ;-:iroduced . seeds. Th :::, tw,) cl;o.sses ThG condl tions responsible for the productL)n 8 FlGU HE 1 Homozy g0us Viv1pn r ous 8 Pla nts . ,. r:-,. .( ·, 1,X 'I!\• '. r;;. la. lb. Fig. la. Dwarfed viv1parous 8 ple.nt illustrating the leaf arrangement tha t fr eq uently occurs. Fig. lb. . Two h.:>mozygous vi \~1:::,,3rous3 plants sh 'J wing the more typical dwarfed condition. • of . thes e cla;s sE,s of seeds are not understood. ical study A histi::tlog- this mutant during the development of the .: )f caryopsis would be helpful in answering this problem. Because these seeds cannot be distinguished until late in ontogeny, such a study has not been undertaken. By utilizing an a;rpropriate technique, it should be possible to select the .!Ra seeds by the early milk stage. This tE:chniquc involves the use ,' Jf a transL')cation between chromosomf-:S 1 and 6 which is closely linked to yg 8 a.nd the gene for yellow endosperm (Y 1). If a plant hetero- zygous for ~uch a tran.slocation, .Yl2e and Y1 ls self-pollinated or backcrossed to a white-endosperm-viviparous 8 ;) lant, endosperm c0 lor will be c 1.,osely linked to .!£a, and U1us it v-ri 11 be poss1 ble to distinguish between 1.2 and n~rmal seeds at an early stage in develo pment. 8 This technique will be discussed in more detail in the section on :!.inkage methods. Viviparous~, viviparous ~ and viviparous , are 0~ 5' representati VEi ~f this class : >f mutants, which are interest- ing because of the apparent lack of carotinoids and chlorophyll development accompanying the e:xpressl.:in of vivipary. Bandurski (unpublished) measured the total car,:>tlnoid content of seedlings of vi vi·-o arous~:) on a Beckman Model D. O. Spectrophotometer at 440 m,,u. He found the total ~ density at th.is wavis, length 1-·ias less than 5% of thP dr-:nsi ty 10 of a simila:r weight uf riorrual material. No suggestion of any compound _f)Of.H.H'' S~ing typical. . earotinoid abnorption was observed. The relationship of vlv1:p~ry to the inhibition of carotinoids 1-1nd chlorophyll :q;fvalop rnent was- interpreted dif ferently i:.y L:;ste.r (l.924u 1 b, 1931b) a.:.~d llangE .: .sd0rf (1£i6). Eyster assumed close linkE.4.ge ·"between the< gene responsible for . vi vipary l',nd unother gene which inhibits chlorophyll and carotinoid formatts>n. lte reported 0btain- ing twenty-two pc.:.le non-viviparous seeds which yitlded :r;,enty...-one gr·esn seedlings nnd one albino seedling upon germint{tion. •rnese twenty-two seeds he cons.i.dered to be Thc:se results can be explained crossovers (il'yster, 19~4a). by hete1·.>-ff;rti 11 za tion and will ·be cons:J.dered telow. In 1931 he repqrted obtaining a few green viviparous see.dlings which, when grown t::> matar1 tJ, p1•,=-duced a c.n1.o- ro phyl t-:b€:ar1ng strain of viviparous uiaize. He did not re,port f:iny tests to deterwine if U1e derived chlo.r<.>phJ tl- . beu.rtng strain was ailelic to the :.>riginal albino mutant. <Since tbis was not done the possibility t~at there wa~ a gen~tlcally distinct chlorophyll-bearing strain seg- regating in his stock h1:1s not be~n excluded . . •M:angel.sdorf (l.926) did not agree \Vi th Eystel't s lnterpretation .of cl9se link~ge. > lie po~nted out . that . . . ·. ,' ,.;;\;_ :.:. ;:>, ~ . -:-' ..' .·: .. . • ••. • . :.·:·:· . . ..-. •.· ' . -. ~-·'. ' • . . . . i;t v1(t'~t·:extrf1:dl~1t ~l{1ke1y · that so' many v1v1parous mutants·· ·.·.•itifc! ~71;uh ; ,' j OA ciOm.W ~nl(i><l :.t1.· ·•.~·•/a?.to~ j~~p~nsibu• _ • for.· .p r.gduct~ ·.jrh1te en4ofpcitl;Jtl · aru{. ' albino ·s~edJ;ings. :.. At. . . . . -~ ·" • . . •' • -· :,. ~· ·;. . ·' :,;· : ~ ' ' ·.- ... •• • ~ .. 11 lenst nine such ::r:.utants from different sources have been described. Mangelsdorf (1926) also observed that some of the \Vhi te seeds on segrEgating • e-ars were donliant, and a:; wac the case with those found by Eyster in 19f:4; tLese only ~)n the sr::-t,dling s t.a.ge, L1ang( -lsdorf grei"'' his plants 0 to maturity and self-pollinated them. SE~d is the result af If this type Df crossing over, the self~pollinated t&r sh~uld produce only ~bite s6eds. This was not ths cc-u,;c . All of thetH- e::.,rs segrEga.ted Yf.~llow and v,hi te seeds. In th.is study pl&nt.s from ·nine such seeds have been grown e.nd self-pollinated. All the, resulting Pars were founc to b€, se·gregating for white endosperm nnd albino viviparous seedlinge. Mangelsdorf (1926) explained such exceptional seeds as a physiological correlation bet~een vivipary, Bndospercr.. color and chlorophyll formation. He sectioned immnture v1 viparous seeds £-md reported that there was . evidence ;J f germlnation even at the early milk · st.,;,gE·, which suggE.,stisd to hi.:n. the possibility that the process of g2rri..in.at1on interfered Y,ith c-=>lor d~posttlon .. O.n this , : interpretation he ex:ilained the two distinct cl!s.sses of vi vip~rous mutants. Th()se which start germlns.ting at an early stage inte:rfE·re with color deposition and, thcrE1fo:re, result in white :Seeq:::, wb.ile those mutants that are cha:ra .e terized by yellow endosperm qo not start to germinate . untll after color deposition has been nccompli2hed. The exce~ti~nal classes are explained as the result of variati~ns in this mechanism. This explanation is inadequate for the following reasons:·: l. Mu tz,,nts used in ~,his study ~v:_v(, ::hown . I r~J obvious signs of gcrminr.tion (1.E:., eloni;ati:,n of · the eoleoptile ::.,ad r.:>ot or E:nl::rgEmcnt ::>f th€ tcutel lum) u.ntll late ln the c!Evelo;:. I.:.cnt of thE seed. ~ t:Lrts Cer:::;.;inr.ti:m in .Y.2o s ,: eds about 28 to 29 da.y s nftE,r pol l1na tion v:h11e ::rnc?dc of .Y.2.g d.o not sh::>w s:ignr. :>f gt~rrntnation until days after pollination. This is consider&bly later thr:n the initiation of color in the endosperm. r.. Occ::sionally ears segregct,ing .Y.25 . have- been found -.·:i t;h a high frer;uency of white SE':eds that be.co:1Je d ,) rmant. Since these seeds did not germin'-'.tc, it \"::)'J.1(3 be expcctr,d, under Mr?.ngclsdorf ts tntr.rpreta tion, th~t they should be yellow. Z. If the- eiceptionnl classes of se~ds c:.ni9m, it woulc. bE:' expected that thE:se ch8.nce: vc.1·:i.ct.inns occurrinc in seeds ~f viviparous wutants chaructorized b:! yellm1 endosperms ,.:md gr~En scr;dlings v:,:,uld occ:isionally produce a seed with a white endos?er~. These are nev~r found. ~prague (1936) explained the existence of •. .?..naV>gc,us cle.sses of se1~ds on ears segregatinf f-,r Y.27,_ as the result of hetera-fertlllzation. This term was first used by Sprague (1929) to de scribe excep tional seeds which have embrjos and endosperms of different genetic constitutions. In a l a ter pa per such seeds were shown to result from the fusion of the egg and polar nuclei with sperm nuclei from two polLen gra ins of' unlike g enotypes ( Spragu~, 1932). Upon self-pollinating a ph:nt heterozygous f ,: ,r one or the albino-pal e -endosperm-vi vi pnrous mutants occ a sional seeds t ha t er e t he product of hetero-fertiliza tion are expected. Only se eds resulting from f emale gameto phy tes 1n which the egg nucleus and ;)olar fusion nucleus both carry the viviparous gene can s how hf'. te ro- f erti liza tion. If th e egg nucleus is fertilized by a sperm from a pol l en grain carrying a non-mutant alte le and t he polar fusion nucleus unites with a s perm from a pollen grain whi~h carri es the mutant al l ele, the resulting seed will have a pale endosperm and ernbry o. c:,. non-vivi ;)arous The t wenty-one non-viviparous-white endos.? E:rm seeds which produced gre e n s e edlings reported by .E.y ster (1924a) a nd similar :mes reported by Mangclsdorf (1926) can be explained as t hs product 6f such hetero-fertili.za,tion. Atl plants resulting from such seeds li-Yhich have been grown to maturity and self-pollinated ha ve produced ea rs which se:gregate for vivi pary. Mang c lsdorf (1926) rea lized that this class of se eds was difficult to explain by his hypothesis since t he etr.bryos remain dormant and 14 therefore color dEc ;)':.>Si tion should proceed unhindered by starch digestion induced by a germinating embry? .. . This class of seed is readily ex J la1nable on the basis of hetero-fertilization. The yellow seeds 'tvhi.ch pr.:,duce albino vivi parous seedlings described by Mnngelsdorf (1926) arc recognized as the :,roduct of the ·. reverse . eombina tion in which the polar nuclei unite with the sperm from a pollen grain carrying the normal allele· rfrd le the egg . nucleus is f .erti.--lized by the sperm from the ;: > ol lf·, n gra1n ca r· rying the mutant allele. Both classe$ of exceptional seeds de$ctibed by hl angelsd:)rf (lSPH) and Eyster (1924a, b, 193lb) are readily expleinable, as the result of hctero-fertilization . .M&ngel~:dorf (191?6) suggest€d that t.he absence of chlorophyll in t he vivi:oarous seedlings of this c!.ass of mutants ,:n ight result fro1ii the inhibition of chlorophyll formation by the act of premature gt,rmina.tion of the viviparous embryos. In sup port of this hypothesis he describe$ some viviparous seeds which occ 1:. sionally a.re found to havi:> chlorophyll. According: to his hypothesis trinse a.re gr12.en because the pren1ature germination started toe> late to interfere v:i th chloroµhy.11 foruiati~m. He also pointed out that h€ had · never found a dormant white se ed which produced an ·~lbino seBdling. Mange-,lsdorft s hypothesis a nd t he tvm pieces of cotroborative evidenc& are inadequate for the 15 follJwing reasons: 1. Fyster (19f4a) rEports finding onr white ., dorm.::.nt :::ccd out of tYrcnty-tvw v;hich .:roc,iced i. \~'r:itE: frHJ.U€n L in E::):r.e of the stocks r-:,f vi viµarous 5 . The fGet tlw.t rm occs.sionn.L chlorophyll- bu1rin,.f; vi vipuroifr ~:;cE.-d ls '.:Jbtuined is not in 1u,.e1f corroborc. ti Vf.: svidence f .Jr his hyp:,thBsis since cart: that ere homozycous f~r the normal clleles of vivipary occ~sionally yield one or more vivipar::>us seeds as the result of tnvir·:mme:ntal ft'\.ctors. t:) Until the plants fr::>m thi .s tLc original vi vi ;rn.r,::ms stock to determine if they are homozygous f~r vivipary snd, if so, 1f ttey are the same mutants &E L).e stock from which they environmenta:. stimulation. Mangelsd~rf's h;pattesls 16 the result of studi22 inv,lv- bryonic growth and ?rocced t~ ger~inate. ~0st of these 16 several did succeed in producing a leaf or two ~hich were normal with reg ' 1ect to chlorophyll formation. If M;_mgels- dorf' s hypothE,sis is c-:>rre.ct these seedlings w::)uld be expected to lack chlorophyll bE'cause they germinated quite prematurely. The preceding three points make it ·very unlikely that the albinism associatE:d with vivipary is the result of thG premalurt germination of the embryo. If th~ so-c~lled crossover classE~ mentioned by Eyster ( l9F4a, b, 19::.Z,lh) t:1n<l exceptional see-d s :re 11orted by M.angc lsdorf (lSf:G) are thtc result of hetf:ro-fertillza- tion, it rould appear that tht·- onc-2 mutant gene ·exprt:sses itself in three vrn.y~- phenotypically (i.e., inhibits the formation of carotinoids in tht ~ndo1;1perm. tmd seedling, inhibits chlorophyll formation and induces prematurE germination). Alth::>ugh thE step affect~d by this gene is n::>t kn-::>vm, it is p'Jssible that some step leading l◊ thr .pr.Jducti::>n ~f curotin•: lids is blocked through its action. This ~ill account for the lack of carotinoids in both the end;::>sp$rlli and embryo of the- mutant sted.s. It is interesting to note that there ar~ no reports of plants_ ~,·i th chl,)ro ,:;; hy 1.1 wi thr.>ut cc..r:>ti-noids _o.Lso bsing pre-sent. 'fhis has l€·d some worke:r,s to sugg~st that the phytyl side -chain of the ch:t:>rophYll !llolt,cule. might be derived fr~m a Cbrotinold or some closely related substance. If this is tiiE case, then it is obvious that a gene inter- 17 ferirtg with the production of carotinoids could also inhibit the formation of chlorophyll. Tatum (1949) · and Da vis (1950) have reported mutant strains of ~E,urospg;ra cr~ssa and .:g. coli, respectively, which have multiple requ1r~,mcnt:3 for· growth sub- stances. These writers postulate a gene block t.:, the formation of a common precursor of all the required growth substances. This suggests a mechanism by which a single gene coul.d _p roduce multiple phenotypic effects as those ·obsex.-ved in viviparous strains of corn. C. Vi Vi:Qar,; .us Mutants of Class Three Vivlp.arous7 is the only representative of this class. The pink color produced by this mut&nt can be ob-· served in •tho developing c,ir,yopsis at the time yellowing of t l"1e,. endosperms of normal s.e eds on the same ear takes plt.ce. The ifilfilature endosperm isan intense pink which fl'ldes a.s the seed tnatu.r es, becoming more of an orange c::>lor apparent'ry· due to the laying dovm of yeLlow pig- ments. At maturity these seeds are sometimes rathc•:r difficu.lt·_ to . separate, by c:)lor of · the_·endosperm al.one, because the plnk pigment becomes masked by the y6llow. . . •• Preliminary studies of the carotinold content · of m~t·u re tieeds .~f this mutant revealed that 1 t 1 s strikingly d.1fferEtrit frora that found in normal seeds segregating _on •tlle:_ .same ear. Separation of the various carot- lnoid COil'Jponents from a petroleum ether extract Of:t.Q7 18 seec;ls by absorbing them on a magnesium oxidE chromatographic colu.mn rev en led that thr~se seeds lacked mos. t, if not all, the color bands f:)Und with H similar analysis of normal seeds. In additi~n, the vivi pa rous seeds showed several carotinoid-like bands that did not appear in t.he normals. These va ri :)us components of normal and vivip- arous seeds have not beE:n identified. Hov:ever, the pre- liminary results indicate that the:.) x.12 7 genE: alters, very markedly, the. normal ·ca.rotinoid dE Velopment of the seed. 1 AdditionY:. evidence that this gene ls i nvolved ln th£ carotinoid llietabolism of the plant is d~monstrated by combining_ 1. t -with tht? white allele of t.he gene for yellow endosp€rm found on chromosome six. BE€ds. that are hoillozygous for the white ·all.elr: ,:~na al so for !J}. 7 have white cndosperms and pink viviparous embryos. WhitE: seeds not carrying the ::!,R 7 a Llele have'. endosperms lacking in carotlnold pigments, but produce seedlings with normal carotinoid and chlorophyll development. In the double recessiv~ seeds no pink color develops in the endosperm, but the viviparous 7 embryo is pink. Thus, in the end:)sperm where no carotinoid p1gnumts ar c produced no pink pigment is formed, and in the e ndos pe rm ·.-.here _normally carotinoids are produced the pink pigment is found. The ln,ck of' chlorophyll in this mutant agnin emphasizes the dependence of this pigment on caiotinoid production. 19 D. A SYti!lmaty of the Characteristics of the Viviparous . 11.utyts • ( The cha.N ~cteristl,cs of all viviparous mutants· considered in this study . are summarized in Table 1. lAB1L . l. . ! Color of' aLeurone 1n presence or A1A2CHPr Color of endos~ierm .vii '.(i ate dough colorless yel.low vp 2 :' ) ate dough purp:te wntte white wtu\:e white bluish- . pink . pink pink+ .bluish- .taiddle dough • . Color of seedling in light Color of, seedling· .·· in dark : · , ... .green green tinge . green • tinge • . } t.P;5· ~xact t1m£: • • .. , green • ' ·:·..'. >•determine • vpJi]~iiddle dough ·: . (dwarf .· plants) · ,· a1ffiault to purple white . .:-_,:: white bluish- green tinge . ' ) i~lies . by~ Obtaining .indications ·of .11~~ge, with knqwn genes, :.-. •, ·, : ·'t•$eli different chromosomes of the basic set and trans-- .· FO locations -involving a chromosome of the basic set and a B-type chromosome. An outline ·of the use of these tools far locating unknown mutants .will show the advantage gained by employing them. A. Translocations In maize, mutant genes affecting the endosperm are some of the best genes for linkage studies, but the number of good endosperm mutants which can be used for t his purpose is limited. Th1s handicap can he overcome by choosing the proper translooations. For . example, if a translocation is selected which involves chromosome nine iri the r egion of the waxy gene and has the other . break in chromosome one it is possible then ·to show linkage between waxy and gEnes in the first chromosome. Translocation l-9a will be used to demonstrate this teclmique. We can repres Ent the configuration formed by thesf: chromosomes in a plant heterozygous for the trans- location diagrammatically a s follows: gl 1 -0- - -------0--, I r--0--- - I I ' I 9 21 The unaltered .cnromo·somes are indicated by the. numbers . . l and 9, while 19 _represents the transloeated chromosdme .: . ·: ,. which has the centromere of l with the trans.located piece of 9 attached and 9l. ··represents th~ translooated chro- . • ' mosome which has the 9 centromere and the translocated piece of chromosome l. attached. f . . . The several different 2-2 combinations which ·result from the _segregat1on of these chromosomes at !tieio· sis are as follows: 1 - 9 resulting from alternate segregation, 19 - 91 91 - l resulting from adjacent-one segregation, 19 - 9 1 19 9 - 9l resulting from adJacent--two. segregation. 'Ac._e ord1ng to Burnham (1949, 1950)- adJaeent-two segregation OQCUrs only when no crossing over takes plac€ in the interstitial segment~ _ Mierospores. produc_ed follQwing alternnte segregati-on have balanced chromosome complements, e.-1 ther l, . • 9 ,or i 9 , 91 .• ·.These· develop_ int=> normal looking functional pollen grains we1i· r111ed with starch. The corresponding ··m1cro$pores:_ p~od~~ed foll,.owi,ng adjacent segregation have unbalanced ehromosome . oomplements and develop into non- fiuictional abort:i:ve poll.,~~ gratns which trmy be empty or partly.,.fill~'. *1th starch~- • Altermt.te and adjacent segre- _gationf1 oc9ur with eq_ual frequency giving rise to pollen which is "se,nisterile." . . Semister.il1tY produced by such . ' a heterozyg ,,us _translocatL:>n behaves like · a dominant · cba rµcter and c an -be used as such. . Since the only rune- . • . tional gametes produced by _a - plant hetero3zygous for the _ translocation are those car~rytng chromosomes oni\ and . nine hnd those · carrying . 19 plu·s 91, the point of trans. . loca tfon ..in th~ t.w.: :., transl;.oca ted chromosoro.e·s must always be included tn the same gamete and thus these p:)lnts •are .. •Similarly tne corresponding poi_nts completely linked. <>n the untransloca.t~d · chr,omosomes . must be -completely linked. Therefore, ·genes in one chromosome closely linked to ·the point of brf.:a:kage wi 1.1 be linked to gen¢s in the other chromosome closet~ the transloriation polnti By se-1€:oting the ap _ p roprlate trans toe-a t1ons 1 t is now possfble t~ USE: th€ few good endosperm genes to test for 11nkage in all chromosomes of the complement. St,J cks · of tra.nslocations which have orie break in chromosome nin~ close to t-hf.:· waxy {~) locus while the other . break~ are in various of the -other chr,:>masomes and similar · stocks of · transloca.tions invt>lving the reee-ssl ve genf.! sugary (.§!!) -have been established. Such translocat1ons enable one · tou.se the excell~nt genes, ·.rus and .§.Y, for linkage . stud-1 es in· all chr,?mosomes of the _c-omplement~ To locate an unknown gene that is not linked to wx or ~u crosses dan b~ made to stocks of tr~n~laca~ -- , ---- -· . · tions .c losely . linked to ttiese genes. The F.-,,::, ratios -from _ --·--- 'Iran-s:_,)c~1.tions Us ed ......... Ln ___ Thi;: ..,...._______ Stud..Y ........,_......, _________ Breal-: pos itions as listed by various 'l-'i Ork8r-s . Anderson Trci.n~- . Burnh&m Anderson And(:' rs0n & Br:i nk .~oc.:i.tion (1934 ) (1938} (192,9}_ (1S40) Anc'if>rson f..,:)ngley (1941} (unoubl:J. l-4a 1 L.49 2-4a • 4; S.66 g L.29 1 L.15 r~ L. 77 4 ~s . 09 2-4c ,-, L1 2 E.19 ~; - ~KlO 4 L.30 4-5d 4 S.21 4-6a 5 I,.19 1 L.33 6 r... 44 4 S.54 4 S.5 4 L.1 8 L.l 8 L.4B 4. L.18 9 L.50 4 S.70 9 L.8 10 L.11 l U.17 1-Sa· .. ... r- 2-9b . C. 9 r.,. 54 f.. E~ .12 ·t f;.,; \..) 0 L. I~ 9 L.12 ~~· L·. 19 9 L.40 3 Z-9a 9 L.l . . L.l '7. () V L.f 4 1.6 9 L.2 4-9b 5-~a 9 L.17 1 I,. 4}: 1 L.6 9 L.5 .... L.l 9 L ,:;,::;. ,..}._ 3 L.15 . 9 E.20 5 L.80 9 S . 21 6 b .79 9 L.40 6 L.17 9 L. f2 5 L.7 9 s.o 6-9x•· 25-78 7-9 a 76 9-l.Ob · l-7c 1-3a 7 L. 27 9 L.20 9 t) .11 l'J S .28 9 L.3 10 nears.a. l L.34 7 L.14 1 8.2 ' Z. '-' T • ,·C).~ .u l S . 25 l fL 19 such <'!r:,s s€·S can ·b e observed f:,r an/ indicat:1.on of link - age wi t;h ~ or ]Y_• Indications of l1nkag,t •w1 th these -genes w'i 11 • then reveal th~it the unplaced gene is on the (lther . chron,,~some involved in 't he trarrnl0ca tlon. The transJ;.oc:a- tion 1::an also be used as a ~aarker in a backo;:.•:Jss test - to d~terrnine tho pcHd tiijn :,f the gene :>n tb.e chrqinosome • • The break ~0sitions of tbe transt~cation can bn established (!ytol:>glcalJ.y and thus give some 1ndtca-- t1ons as to . the physical location of the gene on the chromosome .. .study to locate a chromosome carryi_hg an unkno'nn gene and also in mapping p·::>zitions ,3f genes .. The .transloca ti::>ns used in this study and the cy tolog1 cal posi tlon.s -:)f the breaks are given in Tab le 2. B. mnslocations ,lnvo l v.\ng ~-T;fpe Chromosomes Besides the bt:;.sic set of chromosomes (A..,.type) m,~ize ~ay nlso contain various numbe;rs or supernume-ra..ry eh.romosomes · which have been dlrslgnated as n-typE:~. In l-1. giv~n pQpula.tion the numb/:'?." of B-typt chromosomes may vary from plant to plant. Randolph (1941) indiCcites that pl.ants w1 th from onB to t(;n of th€ se supernumerary chro1 • mosomes are phenotypically fndisttngulsh e.blE• from thc>sc ": lacking .· them. ·.-· . . . . .Roman (194.7 , 1948) has described the behavior • •. ·of ·two A~B · ;interchanges; Be Vtas able to show by lising 25 A-B interchanges that tbe unusuel inheritance pattern of . . . the B-type chromosomes, as reported by Randolph (1041) and Longley (1927), -.;;as the . result of non-di sjuncti0n in the male gametophyte of the D centr,::>t:iere at the division of the generative nucleus to p-rodUCF.1 the two gametic nuclei . . The behavior ~f the translocation b~tween a B-type chr:)mosom.0 nnd the .iihort arm of' chrom.os::>m8 • f::mr in the devel0pmnit, ::>f the- pollen ·grF..ln i s d1.ag r umm_a tica i ly shown in Figure :2: • .• As the result of non-disjunction of the B centro:mere the two gametic nuclei differ in respE:ot to t heir genetic constitut ion. One has the translocated portion • of ch.rom0so.rne four in duz., llc a te (hyperpl<:>id ga UJ et e), while . . tbe other is d~ficient for this section (deficient gamete). Both of thes~ ga~etes aie funct!~nal, thu~, ther e are two · possib1 l,i t·i e s When fertilization takes p.lac E: (1) the byperploid gamE: te, . Cfu"l fertilize th e egg .rnd the deficient fuse With the i?01ar nuclE=:i, and (2) the hy ;>erploid gA.mete Ga..n fuse with polar nuclei .and t ht: defici cnt gami:te ferti 1i,~e th~ egg. It is, t.herE:fore, possible by using L-B inter- of n chr0moaome . be us€,d t:i FQr this rea son A-B irit erchang es can L::>-ctttf: unkn~wn r e ces,!SiVE; genr:s. The rE'CEssive character will be observed in the 1'i progeny if the gene 26 FIGUrl1 2 Pollen Microspore nucleus Su 0-•·•. - 0 • The probable behavior of translocati0n B~4a in the development :of ·, the pollen · grain (Redta:wn from R : : > m a n , 1947, • p. 399). is . located in the porti~n ~f the chromosome that is attttch~d to the B centroawre (Homan 1947). A-B interchanges are 6.lso use-ful in studying embryo-endosperm int ~ractions of some of the viviparous mutants~ If the gene responsible for v1-vipary is located in a section of u chromosome that has been translocated to a D • centromere, then t•,to classes of se\:ds are rf:adily obtainable by pol Lina ting a plant ce.rrying the mutant allele with this A-B interchange p,:>llen. In such a . cross, if tht: defici ent gamete fuses wt tb the egg, and the hyper- ploid gamete fuses \'i.'ith th1~ polar nucl;;:-1, a mutant-embryo, normal-endosperm se ed is -produced. Reciprocall.y, if the hyperploid gamete unites with the egg and the dcfic1ent gamete fuses with t h e polar nuclei, a nor·mal-embry:), mutant- Endosperm seG'. d results. The results obtained from c~mploying A-D interchanges as outlined in the pre;;vious parag:r•aph will be considered in a later section. The A-B tr a.ns Locations used in thesE:, studies are listed in Table 3. TABLE 3 A-B lnterchanrres Used in This SJ:~dy ..--------.------ - - -·~ ' Interchange* Cytological ~0sition of break points** Position of breakpoints relative to marker genes ------B-la ~ ii , -: ..J'. • ,:. ::- . ,_... Proximal t~ hm B-lb S next to centromere B-4a S .25 Pr;:>ximal ·to ..§1! B-7b r.. .3 Between _y 5 ,~ J:Q B-9a L .5 B-Hb S .4 Between wx and c; less thanl'.1& c.o. between breakage point. and 2r! B-lOa * Proximal tog Number rAfers t:> the chromos-:>m€: involved in the inter~ change. ** The position of the break point is given as the dis- tance from the centromere in the long (L) or short (S) arm of the chromosome as a fraction of the total length of the arm. Gl~fi IC STUDI ES INVOLVltG VIVI?ARJUS1 Eyster (1924a) originally described viviparous 1 as a pale endosperm albino se •: dling mutant. In 1931 he r(~ports obtaining a strain of green viviparous maize from th€ ::>riginal stock -. )f .Y.QL. Such green vivi parous plants have n0ver been observed in stocks of .!.'Q.-, , YQ._5 , ~ and .Y.Eg in t he present author's cultures. These mutants are al l characterized by pal e Endo sperm and albino seed- lings. Eyster rE:ports no crosses between the green and ,:slbino strains of .Y.2i _to de:'termine if the two are allelic. Thus, there is a possibi t ity t ha t t Lc ·: ::iriginaL .!Jih mutant ,J,. and Uw derived green one might be different mutants. Th€, _stock of vo _obtained from the Maize Gf.•netics 1 Coope ration for this study was evidently derived from the gree~· ..Y.12 1 strain and this term will henceforth be used to :indicate the ..Y.2i mutant obtained from the Maize Genetics Cooperation. This mutant gene has n::) influence on endo- spE:r m color, except tha t it pr-:>duc-es a pa l e crown on some bf the m~re advanced vivi parous seeds, and does not itite..r fere with the production of chlorophyll. Stoclrn of _y_a 1 and .Y.l¼, also described by Eyster (1931b), obtained from the Maize Genetics Co5peration proved to be allelic when crossed together. A possible oookkeeping error during the tinie these mutnnts were be- ing perpetuated could have be en responsible for the two stoclrn carrying the same gene. However, Eyster (l93lb) 30 does not report an allelic test between the two strains and his evidence for two different mutants seems to be based on different apparent linkage reL:tions. Vi vipar,.:ms 1 was reported as being linked to B with only 0.15% crossing over. As will be sh-, wn later there is a distinct possibility that this is only apparent linkage caused by inhibition of aleurone C'.>lor when the .YQ 1 gene is homozygous. Using data derived from two F",, ears totaling 496 seeds, Eyster (1931b) estimated that •~here was approximately 35% -crossing over between viviparous 1 and the golden locus. The above evidence is not suffi- cient to place .Y.2 on chromosome ten. 1 The evidence · given for the location of .:m 4 in chromosome nine is based on one Fr, ear heterozygous ~ for this mutant and for shrunken (sh). This ear yielded §h .Y2 200, .:ill Y.J?. 14, the f ollow1ng classes of sEH:~cts: f3h .!.12: 22., and .§.h .YI! 87. ~ Using thBse dtita he calcul._ated ll.8 qrossover uni ts between ,Y£ 4 and ..§h (Eyster 1931b). That this . is a poor ear to base F 2 linkage on can refldily ,.) be seen by summing th~· non-viviparous and vi vtparous classes which give values of' 214 and 109, respectively. Similarly, ·. summing the non-shrunken and shrunken classes yields valu~s of 222 and 101. These values deviate considerably from the expected 3:1 ratio. The linkage data reported by Eyster (1931b) are.not sufficient to eliminate possible allelism of the original vivlparous 1 c1nd viviparous 4 mutants. Hereafter, 31 .Y.Qi · will be used to designate yrogeny from the original ..Y.2i ond .!.114 stocks. If Byster's linkage information is correct, this mutant should be either on tb.e short arm of chromosome nine or the long arm of chromosome ten. Crosses of B-10, which includes the loci of 11 and g, t:> three homozygous &nd seven known heterozygous Y2l plants proved conclu!:!iVely that the mutant is not in the long arm of chromosome tEm included in this tr a nsloca tion. Eight crosses of B-9b, v:hich includes the loci of Jill and ~ , and seven crosses of B-Sa , which includes the distal half or the long arm of chromosome nine, with pl a nts known t ,:> be heterozygous for .Y.}21 , gave~ no indica- tions that the mutant w2:1.s ln el ther of the sections of chromo.somE nine carric'd by these B centr()meres. Four F..,~ ears heterozygous for . Yfli ,md sh gave the results in l'able 4. Both classes ,. :,f vivi parous seeds hive been irouped together because of th e difficulty in distinguishing the double re,cessive . . This ratio closely fits ~he expected 9:3:4 ratio, having a chi square value • of lr3262. A deviation as l&rge or Larger than this is expected · by chance about 50;{ of th ~-' time. This indicates ·that there is no linkage between this mutant and shrunken. TAB[& 4 FQ Ratios fr;:>m Plants Heterozygous for Shrunken ~ and Viviparous1 Vp Plant 49-649 -l 49-649-7 49-649-10 -1 9-65.l-5 Vp Sh sh vp 71 79 91 59 174 190 49 ill 8 <)·~ 41 741 231 234 - 68 309 The results of backcrosses of plants heterozygous for Ji.¼ and .lli to .!lli yg1 plants is given in Table 5. The calculated percentages of crossing o.ver arfJ 47 . 6 and 51.0, indicating no linkage between the3e t'NO g e nes. · TABLE f, Backcross Progenies of ...±..Y1H &nd wx vp, wx + vrx vp 1 Pe~r.e nt family Parental combinations + vp wx + Hecombiria\ions ++ wx vp 49-584.3 147 1G4 14~: 58-16:)7 1323 lZ74 l.<'119 Total Percent recoml.Jina tions 140 !:,92, 4,7 .6 1386 5[l)2 ~>l. 0 and wit.ht.he genes~ anci Jill show that ,121 is not in chront:>some nin':, nor in ·the distal. p;:;irtion of the long arm of ten. Tests with B-la, -lb, -4a, and -7b besides those prev-1.ously rep:.>rted have foiled to revea:L any further inf:>rmation as to the locus of this gene. As yet no ind lea tion of its position has b e en obt::-i.ined b>,.1 t further crosses are being pLmned. This mutant is of speci a l inter es t becausE': it inhiLi ts · the ;iroduction of •c:t leur::>ne color in · th E: presence of d~minant A1A~C ,::.. . nnd R. pa rents of the following genotype: i-\A 2 Cd Vp 1 X A 1 Ar;cr vp 1 .., ' Non-vi Vi fP:rous Purple Non-purple lb87 Viviparous Purple . Non-purple 0 ·~ 45.9 dilutely colored .SH: ds A1A2Cr vp 1 A1A2cr vpl 27 -12* ar€ inclucled in this number. If th e hom 1J2</gous vi vipa rous ph pnoty ;K inhi bits color formation ,,.nd is indepc,ndent of any ,'Jf' th e color genes, a ratio of one color ed s eed to three colorless seeds with no colored vivi parous seeds is expected. shows that this is the case. Table 6 The sup i1 ressi011 of C.:}lor form.i4tion in vivipar~us seeds could ex;1lain the .linkage indication with B reported by Eyster (1931.b) and v,hich this study has failed to · confirm. An occa.siop.al viviparous seed 1.s found in the prog6ny of th1:: above cross v;hich is very dilutely colored. 1fo analogou£ clas.:i1 <>f seeds is found .1mong the normal seeds on the same ear. Plants from such sei:dd produce ea.rs which are. hvmr..H1:ygous for vlviµary and have p:redominantl/ color.Less seeds with an occ6.s1onul seed that is faintly colored.. . Such faintly colored seed.:$ could r€sult fr ,.)01 tl1e failure of the viviparous gene to completely suppr-,Jss color formation. A stock of .YQ 2 was obtained . from Maize Gttnetics Cooperation. In this stock -von seeds are smaller than ~. . normal seeds .on the same ear . It is not known if this reduced size is the result of the .!.2 2 allele or of a second closely linked gene such as 1:§l or ~ 2 , as indicated by Eyster (1.9 31a). Eyster (19::Sla , b, 193~5) reported linkage of this gene wi th Pr-, Re , 11,g , Gc and 1 on the basis of F~'. 1 2 1 2 d~ta. The following map dist&nces were calculated: - 40 - ho . .., .:J.§1 • - .Y.12 ~1 2. - 15.5 .§.sl - VP 9.6 12 - ln2 P-:-, ~ 1~ ;:_, . '-,'. YR~) f.'., - 0 "· f ..# 20 0 - 1.2 5.3 35 Burnham reported linkage betwem1 this mutant and .Er of 23% and bE:tween Bt and ll _ of 9;:lb as the result of F...., ~ 2 studies (1935) .. These data indicate that the gene is on -1 chromosome five. To check these values a plsnt hetero¼ygous for vo..., and !:K was out-crossed to a £.r tester. ,__G Plants from 31 of the red seeds and c,O of the purple seeds resulting from this cross were self-pollinated and the resulting ears checked for YQ 2 seeds to determine the constitution of the gametes contributing fil or ?r with regard to .Y.Q 2 • The results of this backcross can be seen 1n T-able 7. TABLE 7 fil.±.. x + V'Q2 pr+ pr + Backcross Progeny of -. Pa1:ental combinations fjecombinatiogs + vp pr + + + • pr vp Total % recombination --------------------------------18 25 12 6 61 GEN.E,'IIC STUDIES INVOLVI.NG VIVIPA.HOUSr::;) Seeds of this mutant were generously furnished by Dr. Burnham. Tests for alLeleism made between this mutant and a phenotypically similar one furnished by Dr. Sprague revealed that the two mutants were alleles. Occasionally seeds of this mutant will fail to ger!linate. The exact conditions responsible for pr.eventing germination are not known. 36 Plants heterozygous for viviparous 5 and homozygous for yellow endosperm pollinated by pollen of L-lb plants produce three classes of se eds: (1) yello w vivip- arous, (2) yellow dormant and ( 3) white dormant. These resultS can be expla ined if the normal a 1.lele of .Y.2 5 is located in the section of the short arm of chromosome one attached to the D centrowere. If the hyperploid sperm fertilizes the polar fusion nucleus and the deficient sperm the egg nucleus of a female garuetophyte which carries .Y.Q 5 , class one seeds wil.l result. If the r everse combination takes place, class t wo seeds wilt I'E' SUl.t. Class three seeds are produced 'py similar nuclear ' fuslons as described for classes one and two, but the female gametes carry the norma l allele for vivipary. Pollen from B-lb plants has been used on fortynine plants of families which are expected to yield fifty percent heteroz.yi;ous vn..:; .... .... plants. ~ ~ Of these forty-nine crosses nineteen have produced the three classes of seeds listed abov,e. White n,: :m-vivi parous seeds from the above pollina- tions should be heterozygous for ----o vo~. Seven such plants were tested and all segregated vivipary. In order to Locate the position -'.)f this mutant on the short arm of chromosome one, crosses were made to translocation l-9a, v:hich is· linked to y. A plant he,t- erozygous for 1.2 5 , w.x and translocation 1-9 8 was crossed to a standard. Plants from' the, seeds of this cross were grown and the pollen checked for sterility. These plants we r e then s e lf-pollina t ed &n d th-:: r·esul t ing ea r s ch1s~,i- fi i.;d • for .Y,B . 5 The results ,o f this bc..ckcross can be seen in Ta ble 8. .I·AEL.L 8 Backcross !?roe:eny, of wx T ,;., X ±±.± + + + vp5 ++ + --'------- To- % Recombifi ecombina tions ta l nation - Hegionl Hegion ~r Hegion 1 , P t{eg.l Peg.2 ++vp wx+vp +T+ wxTvp +++ 'ltx++ +Tvp Parental Fte combina tions wxT+ 32 38 .5 0 17 14 0 l 107 · 5. u E9 . 9 These data indicate t h !::t t thE g ene i s located thirty units from the translocation. Since the tr ans- location break is on.Ly .17 of the cytol:,gice.l distance from the cEntrom-ere on th~,:; short arm of chromosome one, it is probable that .YJ2 5 is distal to this break point. The crosses of this mutant with B-lb reveal some informatiori os to the mechanism involved in producing vivi pe.ry. If only the seeds resulting from f emal e gameto- phytes carrying the viviparous allele are considered two classes of seeds can be r ecognized with regard to the genotype of the erutryo and endosperm. in Tab le 9. These are given 38 IAELE 8 End-:>sperrn and Embryo Gf;:'n:::>types Hesulting from the Pollination of vp.,.) Gametophytes by B-lb Pollen ------------ Gametic Unions Eg g - Hyperploid sperm Polar nuc.-DFfici ent sperm Game tic Onions e fici ent sperm Polar nuc.-Hyperploid sperm Elllbryo vp VP VP vp F-n d0sperm. vp vp vp vp Vp Vp Eg g 7 -----------These two classes of seeds should be useful in determining the se parate r ,::> lE"~s of thE• end·.) Sperm nn.d t:mbryo, if any, in inducing premature germination. If premature germina:ti ,) n is produced by changes only in the endosperm, then seeds of class one should be vivi parous. If inste ad t he vl vi pa rous gene is a ctive -~ nly in the (•mbryo in proruotlng p re: :1 . ature germination, then cla ss t wo see:ds :3hould be Viviparous. Only se eds ::>f c l r1ss t \10 are - :,_bserved to germinate pr ematurely. Spragt1e (.LSZ;6) found a similar situation with viviparous7 mutants. As t he restilt of het e ro-fertiliza- tion h e '.) btained s eeds which had n:,rmnl e!l.ibry os with genetically vi v1p£irous eml osp er rri s and a lso se eds with no r m':d, end:.) sperms which had embryo s tha t wer e ge n e tically viviparous. As in th1~, ca se of vivi pa rous 5 , only the se eds with genetically vivi~arous embryos germina t ed prematur e ly. 39 Ar'S will bE' s!lown below, the si tuati-on is the same in viviparous 00 • ThBse results indicate· that vi vipary is deter- • mined by th€: genotype -::>f the embryo and is independent of the · genotype of thE:· · endosperm. GENE.Tli,; STObI£S INVOLVING VI VIPAii.OUS 7 This mutant is ·characterized by pink endosperm and pink scutelluci.. 'rhe seedlings produced are also pink and lack chlorophyll when germinated in the light, but have~ faint bluish-green color if germinated in darkness. Practically all seeds of this mutant germi- nate prematurely. Nine different mutants of .thi s type, including the .one reported by Sprague (1936), were grown and intercrossed t·o test · for atlE,lism . . These t.e.sts indicated· that all nine are allelic. Dr. Spra.gue (unpublished} had ind tea tc~d the probapJ.e linkage relations of this gene to be !J}. 7 - .QI g1 . • A p+ant _homozygous for the genes responsible for 8 aleui:one color and ·het~rozygous f()r n ·7 , J2! and g1 8 was . crosSed to a plant homozygous (OI' g18 . • The ears resulting • from this · cross .had only purple seeds. These seeds were planted and · 192· of the seedl.i .ngs were classified with regard to_&.!a,~, At' maturity the glassy and normal plants were · sel-f-polliaa.ted aµd t}le · utature ea.rs classlfied for TP.BL.l 10 Backcross Progeny ::::>f Parental Combina1!,on,;J 79 Rccombiqutlons Region l Region£ Region 1,2 9 .1 9 0 To-.. J, Hecombi- tul nation Reg.1 Reg.£ 14.G 0 1.0 Ea nee the p:Jsi tion of g1 8 on chromosome five with respect to .2£ is unknown, it is imp,)ssible to tell • the direction of this linkag e map- with rf:fercnce to the centromere. - Crosses to locate YQ 7 with respect to bm 1... and translocation 5-9 8 , the break point of which ls located .80 of the distance from the centromere on the long arm of chromosome five, have been started &nd should give an answer to this question~ GclH~TlG CTUDI E8 INVOLVING VIVlPjthOUS 8 Vi vi parous 8 origina U.y appfHire<l in stocks derived fr~m seed exposed at the Bikini atom bomb test. :5eed.s of this mutant have yellow endosperms and produce groen seedling~1 \-.hich, if grown to maturity, produce 41 On8 plant heterozygous for .YQ 8 , pollinated by pol Len from a B-la plant, ~duced an ear t:egrf~ga ting viviparous seeds. Such a p0llination produces ears segre- gating 1.a.rge D.nd small - seeds. The lnrge sec-d s are pro- duced when the hypcrplJid ~peru1 fuse::, v-; i th the polf. r nuclei and the deficient sperm fer ti li z.uj t he egg nucleu s , while small . seeds result from .the reverse comb ination. Upon close examination ?f the t~o types of saeds produced by this pollination, it was found that part of t he larg e . se~ds we r e viviparous, ~hile none of the smal l seeds germinated pr ematur ely. Gince '.)Oly large SEE::ds gErruinate prematurely, it appears that the gen8tic constitution of the embryo determine s premature germination. Sixteen kno wn hete1·ozygous plants p-::>ll inated by B-la poilu1 all s egregated for vivip ary. In addition, thirty-six crosses w€ r B made to plants, half of which shoQld be se gregati ng .!as• •Of thE·se, V3 scgrega.tEd for .Y2a• These data r·st.s.b- lish that .Y.Ys ir located in the long i.1rm :)f chrom:)~ome one. To locat~ this gc n 8 more exactly, n cfofs was Plants from seeds of this cross wete check£d for pollen sterility and the se~ist er ile plants self-pollinated to dE=,tcrmin-2 if they wer0 heterozygous for ..YJ2g, a nd al.s:.) outcrosscd to tr, hm 2 or br bm 2 plants. Three ·outcrof!::es were· grovm and the plant s cl ass ified- f o r l.r , bmr and Self~pollinatijns w~ re made to arwth er onl.y bm.~, whil € t he thi.rd inv ,)lVE:d b oth br and . -..:. b~!.l - . . -:,::: The rfJS_ul ts are summariz ed in Tab le LL l8cRtion i.s very cl)se to the locu:-J .12£. The~ trans- 'I'hc 9osi ti.:m - --.: bm.,. TABLE 11 Ba.ckcross Pr~geny Involving T l-7c, br, vp 8 and bm 2 ----------- To- <f, Eecombi type Combin&He combina tions t~:.l na ti on __ti on Region L-7" Hegion 2 Eeg ion 1, }) of F1 Hcg .l Geno- Parental Parent ------'---------~---------·------Tbr+brn ++vp+ 5 9 10 Tbr+ 1 :J l 8 l 6 11 17 8 5 :J l 13 lf} ,) ,r.:., ,., ..., 7 ++vp 08 64.f.: 18.4 1'-t_bm +vp+ . Tat.al hsgi-5n l T:::> tal He- g i.on 2 * 8 , J.. \ ) . "-· 83 1 ?,, ...l.. \.,1 • A total of 175 plants ~er€ cl a ssified far th~ trhnslocation and br without obtaining~ crossover b ct~een them, therefore, in this summary the regions T-vp and Tbr-vp are both considered re g ion l. ,.l t.• Viv1parous 9 originally appeared in stocks derived from seed exposed to an X-ray dos:tge of 20,000 r-units. This mutant is phcnotypicrdly similar to .,Y:g 5 , but is wore st::·onr;ly vlvipr-.ir(;us. Int0rcros :3es havr: shown t.ha t they art not al1.E:11c. Pollination::; wade v;i th A-B int er change stocks g.-;v-e no lndica tions that thE locus 0f th1 £ gene is in any of thE: regions involved in these int erchanges. Table 12 gives Fr data -from ears heterozygous for .Y.Qg and ~· These c:ata do not deviatE signific£. . ntly from a 9:3:3:l ratio expected fo~ two independent genes. The chi s quar e value for tbf': observ(d r :;.t io is 4.677 which is expected about ~O p~rcen t of the time. A stock which was h'JID.J2ygous_ for ~ and heter~nygous for ,YL! 9 was crossed to translocatlon 7-9A 76 . r'b i ob segregated ~ and .Y.Q .... 0 ·w er-e obtai ned. 1 ratios art~ presente-d in Tablr= 13. The re~ul ting Thr obsE'~rved ratio differs very significantly from 9: Z: 3 :l. Thre~ plants from a family in which tw~-thirds ~nct tw.:) plants fr:;;irr, ;_, fr.:.:::1ily in which :)nc-half th8 plants were expected to be heter:>z:;gous from !12g were pollinated by D-7b po lle:n. None of tbesf.~ produced .YE ;_:. seeds. The probabi.lify that only non-viviparous plants were chosen by _chance in making these pollinations is 1/108. There- 44 f0re it is im~r~tuble t hat t~ e . gene is located distal t'.) th2 tran'.3locat1on in th8 long arm of cbr:;mos-:>me seven. Since 1:.§ a n d z.1 1 . loci . :ire in t:hc, long ,n•m :>f chr ·.J!IDsome s0v ~n wlthin the region trans loc ated lo the B cantromere, these genes. Cr<)sses have been made to glossy -i J.. mine the di t; t,:;;n,c C'. b,?:tV,'E:t:•n Yil r &nu thl s t.o d cter- l!:E'nt., . ;'_ .' F<::-PrG~enies :Ji' plants Heter:)?'.;.rgous for wx and v-0 0. ·~ Pl~1.nt Q ------- ·--·----• ;;, Phenoty:oes .· ++ -----------------------.-'769-4 197 769-8 l5f 67 11 4.8 769-14 l f3 100 ---------- ·-----------191 2Jl 1042 • 46 TA.6L1 13 F.,, ProgE:'nies of Pl,ints of the Genotype ' Plant ++ Phenot:t]2t3S vp+ +wx vp V?X -------1659-9 j_t,;4 lH 14 ~1. ,.• , .,., 16t)9-12 101 27 ~~ 1 40 lG59-20 96 50 ,.,'l, ,1_- 68 1699-£ 102 25 20 13 1699-7 76 15 13 17 .· 1699-13 91 12 ~~o 15 1699-16 86 9 ,., l0 14 , · Total 676 ------lfi7 ll5 + II' + wx + VP9 Total - -170 1138 --- Ein8i~to CUL.TU ht'. AS A TOOL. r'Oh INVESTIGATING VIV IPA11'.x'. La Rue (19 36) and flaag en-Smit fil ( 1945) have successfully bultured corn embryos on chemically defined media. La Hue using embryos which had atta1nt~d a scute1lar diameter of 0.6 mm . or greater obtained good growth on a medium of salts and sugar solidified with 1% agar. Haagtm-Smit .§1 fl., with a medium of s tilts and sugar plus some added growth factors, ~btained growth of maize embryos greater than 0.3 mm. . • La Rue (1936) reports thut 0mbryos placed upon thE, culture medium cease a 11 embryoniq growth and proceed 46 to grow c1t once into a seedling. Similar observations have bE::en made wi"th e:.nbryos of other plants, thus pro- viding a practical way to overcome dormancy imposed upon embryos by some constituent of the environr.a.ent within the seed coat. Since maize embryos are potentially capable of germinating at a very early stage if removed from the seed, there must be some mechanism 'l>' i thin th e seed for inhibiting germination and prolonging embryonic growth. It is the alteration of this mechanism by the action of the viviparous gene which is responsible for producing prematurely germinating seeds. Numerous germination inhibitors have been described in the literature. Konis (1940), Evenari (1949) and others have suggested that these substances are responsible . for prev,;nting precocL>us germinati,'.)n of devel- oping embryos. Most of th~ tests for t he presence of germination inhibitors have been made on mature seeds, and thus the effect ,: :,f the inhibitor upon the developing · embryo is not known. On thi::: other hand, r;hen a substance is found which inhibits germinati".Jn of thE: maturing embryo, it is difficult to determine if this substance i s the actual substance utilized by the developing seed for inhibiting premature germination. 4.7 Genetic studiE:s with maiz,e h a ve sh,) wn that sor.w factors responsible for vi vipary a Mendelian wanner. arE;; inherited in Crosses of ---5 vo with E-lb -pollen have revealed that prewatur8 ·germination is dependent upon the genotype of the embryo. If dorma ncy of the develop- ing embryo is caused by th e pr0sence of a germination inhibitoi.·, th L genetic E:VidencE:· would suggest thc,t vivipt.~t·ou s ernbryos gerrui~~ te prematurely because th ey Hre oupabl1:: of ger;;iination in thE;• presence of this inhi Li tor. Pt.:. r a llel test i"or germina tion inhibitlon C Fl n be made wlth vivip,u··ous .s.nd n~)rmal embryos. If an inl1ibitor is respon- si ble for prevE:,nting preiua tur c gerrilinati'.:>n, it s ho~1ld be inhibitory t0 n::>rma l 8llibryos and not to vi vi ;J:'.trous. Embryo culture experiments were used to test f:>r such an inhibitor. In the foll::>wing expeI·imen ts only immature embryos thcit had not startE.od t:, ger:ninatE_; r:ere used. Viviparous 5 embryos were used predominantly in these studies because of the ea se with which potentially viviparous and normal s~eds could be ~e?ar a ted ~t &n early age on th0 basis of ~ndosperm color. Th~ t wo cultur0 ~ediu used .in thbse ex periments were Lt~ Ruii's (19~,6) tnedium number l (1945). 11nd IIaagen-Smit's Twenty cc. of medium \fere added to 1!~:. x 1500 mm. pyrex t es t tubes :_md autoclaved t wenty '.ninutes at fifte e n 0 pounds pressure and 250 F. 48 The seeds we re cut fr0m the cob as aseptically as possi ble hnd t he kernels were th en sterilized by one · of two me thod s as follo ws : (1) placed in 70% ethanol for three minutes and then rins ed in sterile dis t il.Led water or ( 2) placed in a solution of one rar t zephiran chl.oride in 5 ,JOO parts water for t wE;11ty minutes, aft('r which time they we r e rins ed in st er ile distitLed wa tEr. Both ~f these met hods pr0ved ·e ff e ctive in controlling cGntamination. Aftt!r eut ting u window in th e peri car p , f.he embryos were r emoved fr')m the Endosp€rm, measured n.nd placed on thE· S'-lrface r>f ths agar. Thii' cultur e t u bes were kep t in Lh~ li ght at rao~ te~perntur e f~r thG dura- res ; onded the same as those cul t ured in the liEh~. A. 1'.§1st!3 for a Germinati.:Jn Inhibitor in the Endosi:>erm Since embryos removed from the environment of the seed . g e rr:ainated when placed sm the culture mediura, the possi t,i li ty of some inhi hi ting substance being pre sent in the endosp e rm was investigated. To test t hi s , three - experiments were set · uo As follo ws: ; 1 ram. in length, were placed on th e surfface of t h e eul.ture medium ti .long \rd th pieces of frndospcrm, (r~) A hl:)ck 0 f endosperm containing the intact B.wb ryo, 5 mm. in li.::-ngth, ·was removed frcim ten seeds ;:i nd placed on th e surfac e .: , f the culture medium, and (3) A window wrHs cut in thf:: p c rl- co~pared with the c~ntr~l~~ scv~.n days. This diff e rence it r~flected Table ~f thu c~ntr~l J ls roughly t ~ic c th ~t ~f the c~bryos left in ~3ntact with th~ endo2perm . This deluy could be the the different nutri t i :)na.1 tnlfironmE•nt s 0f th<::: G:)ntr;:>l medium, the pLumutes gro?? at. u rate which parul.l.els that of the controls. - ·- . PLu~u l. e Gr-)wth :,.f Emi. r·J JS in 1>}t1tact with________ thr1 End.:,irncrm --------.I-,.;;:;.---=- -- . _______ ._.....__,,_ __ J:.,mb1·y '. J ~,. ex;) ~>~, ed Embry-JS exno~ied in placo ,: ,n a tl•J clt of enct,Jsperm MPan :olumule length --------------------- in crn. -----------~---~---------------To determine if the &b0ve delay ed Eff0ct was Lhc result of an inhibitor of premature germlnation rather U!an an environmental effect, normal and viv1p,:i.r0uf; 1::m- bry0s wer~ exp~sed ~ut left in place in th€ seBd. These were lhen placed under conditions favornbLe f ar gsr~ine- end)s9erm which 12 rapidly los t 0n ~x; J ~ur e ta ths air. quickly fr~zen after r0m0ving th~ embr10s bnd were ~hen froai the materials being .l .yo_phiJ.i2cd would be ~,, L !.cct.0d 51 along with t he water in the ".lyophilizate". Petri dishes (60 x 15 mru.) we re prepared with nutrient medium on which were placed five embryos. · p.. few cc. ~f frozen "lyophilizate" were placed ln a small vessel ~ithin the petri dish, follo wing which th e dishes were i mmediately sealed 1n petroleum jelly and kept at roo m temperature. A control was set u p in which only water was placed in t he enclosed vessel. If pres ent, a v::>latile inhibitor should be liberated when the "lyophilizate" melted, forming an inhibiting atmosphere in the sealed dish. Neither embryos nor endosperm "lyophilizate" produced any inhibitory effect on the germination of the e::nbryos. · b. The Effect of 0£ Tsnsion on Gmlnatton Various workers havs reported that the imper- meab11i t,t of the seed coat to oxygen. will 1nh.ib1 t the . germination of embryos of some species, which, if removed from the confines of the seed coat are capabl e of germination (Atwood, 1914; Harrington, 1923;· f>paeth, 1932; Stier, 1937; Crocker, 1906). Frequently such seeds can be induced to germinate if placed ln an atmosphere of 100% oxygen. This suggests that enough oxygen for germination ·1s capable of ciiffusing through the seed coat if t he oxygen tension is raised high enough. In maize the pericarp and . the underlying layers might act t;:> 52 inhibit the diffusion of sufflcient · oxygen to the develop..ing embryo to perm! t germination.- An experiment was designed to test this. ; . Two 250 mm. desicctLtors were sterilized and u .s ed as culture chambers. In ea.ch were seven ster ile 60 :x 15 mm. • ' \ .::-,:-: : petri dishes containing moist filter })aper on lwhich four seeds wer~ placed. Two classes of seeds were used: (l) whole seeds wfth intHct pericarps, and (2) seeds with a: window of pericar p removed .directly above the embryo without removing any of' the underlying tissues between the pericarp and . th~ embryo. In each desiccator th€· following number and kinds of seeds were placed: ', • 1. Four normal seeds with intact pertcarpi. 2i Eight v1viparous 5 seeds with intact pericarps. 3. Eight norillal seeds with a ,dndow in the .pericarp. 4.. Eight vi viparous 5 seeds with a window in the pericarp. Water was placed in the bottom of each desiccator to ensure a saturated atmosphere. The desiccators wer~ then equip ped as is shown 1n Figure 3to permit the encloe.e d atmosphere to . be changed. Aft.er the seeds were placec:11n the desiccators one desiccator was flushed with · o~ygen to ·produce an 1ooi ·oxygen atmospher~ while the other ret~1ne~ ~ri· atmosphere of ' air. Th.e gas within the des1c- ' ca1:iors ~J~?~ra:nged' once e"!ery twenty-four hours OVeJ- a ,, 53 µer·iod of seven days. At tht:i (;nd of this. time they were opened and the µlumules were measured to detcrmtne the extent to ~hich germination had proce ; ded. The results of this e:xperimE:nt can be seen in TablG 15. Among the intact Seeds germination 0f viviparous ombry<:>s was somewhat more adv&nced tha n norms.ls under condition.$ of utmospheric oxygen. In a pure oxygen atmosphere the amount of germination of the vi vipn.r..:>u.s seeds almost doubled, whereas, t be germination of n,:)rrr.al seecJs remained the s ame. When a window of pericarp was removed above the emhry~, the amount of germinat ion increased in both classes of' seeds. These . results can be explained by making two assumptions: (1) Vi v1p.n.rous e,hbry ,J s &re capablE. of germinating at a lower oxygen tension and !'.~re more sensitive to changes in ~xygen concentration than normals, and (2) the pericarp 1s an efficient barrier to the diffusion of oxygen. Under these conditions the intact seed is ~xpecte,d to have a reduced oxygen tension within the pericarp. This redticed oxygen 6oncentration may be sufficient to permit some germination of the vivi parous t:mbryos, but not •::>f the norma ls. When the amount of ei.xygen availabl E: to the embryos is increased by raising the oxygen concentration, then the viviparous embryo.:;;: may be suffic1£mtly sensitive • to resr;onct to this increase, while the increuse is not suffic.ient to st1mulat€' the normal ~mbryos to germinate. If, however, . ths pericarp is removed, then sufficient 54: .FIGUh.l: 3 A F A. B. C. D. E. Inlet Cotton Filter Clamp Ga s Outlet Cot ton Filter Gas F. PE t ri Di sh G. Wat e r A d es iccator as equip ped for embryo culture studies involving gas mixtures. 55 oxygen under atmos;-lhcric conc!i tions muy reach the E: :bry:>s t ,J stimulate g errdna tlon :>f b ::>th vi vip r: r-:::>us a nd normr: l embry.:>s. TAI;I..L 15 f.ffE:ct of Increasing t hE Op TEnsion and Removing the Peri- carp OpoE..,__1.::_!? c Ge_£gijnation :>t Jb.rma l e nd Vi vJ;12.i!.£.9.!J£_£.Q,\D.T..Y21L- p;;-r.,.ca ... nl" intact ,.~ + ..... -4 ..L vp Air No pcricarp + vp .91 f.69 ±.lF -------------- Ps r1carp intact + No Jericarp vp + vp ¼eo.n plumule length in cm. .07 .6 f +.09 -t.21 Gig. ::>f ciiffcrt::ncE:· between th0 1neans ±.Z7 + + . ,J 7 ±.08 1.4 ±-20 .59 -t.lP + It v10uld ,;ppc.:1r· th, t 'E.xtr·f,::::( 1..y high concentrations o f' :.>xygen ar c LJ.xic 0 embryos. t ,J b'.)th normal Fi.nd vi vipurous Thi~ i nh.1 bi tory effect ;) f high oxygen concfmtra- viviparous c~bry os germinate prematurely because they ar E ca ~a blc of ge rillination at lowe r oxygPn tensi~n, there sb.ould be an :.)J~Yf.E·n c:-: )ncentration which ve"ould pcrmi t vivip~r~us embryos to gPrminate tut not normals. Embry:::,s of ium1c1. ture n ,)rmal c1nd viviparous seeds were exp~sed completely but left in ~Lace on th e endosperm. These seeds we i·e arrangE·d in th( desicca. tor as r:xplained above for the previous experiment. In each desiccator 56 ther€ w~rc foui dishes hol ding a t~tal of sixteen n8rmnl SE:eds t•.n d four ·,·'i th sl :xtec·n vi vipc.rous SeE'·ds ~ After the seeds were .soal.ed in, the desiccators were flushed ,: mt wl th the desired gas mixture. This was repeated once every twenty-four h~urs for six days. The above procedure ~as The gas mixtures were obtained by displacing wnter from five gallon glass bottles. By displacing this gas rzdx ture with water thi.:; ga!; was then forced lnto th<:: d E~-1 cca t:)r s. This method 0f nixing p1.ses w1--1s c()n~idered -3.dcqu ,ite for preliminary tfst:-i for d1fferentia l. effects of reduced oxygen. If such effects are found, m6re refined techni.ques could be used for mixing unc? sup :; ly.ing the gHs. The follo wing concentrations of oxygen were used: ~OJ, The balance of the gas was nitrogen. The results of thesE exp e ri me nts are s u~~arized in Table 16. Effect of Vr~ri:1~1:: Oxyg0n .Tsn :cd.on :3 :)n th r Gc r :uinr-i t i0n of ________ _!ill~YiLfil!dNormaL Embryos :f! /0 ·J 2 t 0 No. of 4.m bryos 'M etln plumule + vp •+ .vp + 14 14 15 O + vp 2 vp + 20 I 14 vp 11 14. .13 .PO .G3 .78 1..15 1.22 ±-06 ±,03 ±-10 ±-18 ±-12 . ±-21 O }~~ii . l .•· ,; ,- ' · •, . :... ________________ ____________ ._.....,.....,_,,.__; . 16 16 0 11 9 0 .64 .10 12 .87 L.19 ±-15±.f2±,Z,}? ±.14 12 l.4 1.4 ±-~;6 ±· 18 1:ti, "g ;':'"i' f 'd;;It:tirenc e ;ll~l!!ns 16 15 • 16 \foan •• plti.mule .• t~,ngth • in cm. 1.39 l.84 t-16 t.16 2.08 1.98 ±-26 ±,20 ·Sig. of + No. or • Embryos differ·e oce .. between .·· :the me;antf 58 At each oxygen concentration tested the growth of nqrmal and viviparous embryos does n~t differ significantly. However, at oxygen concentrati .'.)ns of 2% or lower the viviparous embry ,,s consistently exh1b1 t more germination than normals. This suggests that viviparous ernbryc>s arE' able to germinat e somewhat better than nQrmals at thElSE' lower ,,xygen .tensi t, ns. However, no oxygen co!lcentra- ti :m • tested permitted viviparous e:nbryos to germinate whi 1::-: preventing the germination of normals. Thus a difference in oxygen tensi-0n may have a minor effect, but 1 s not adE,quate to t'1xpluin thi::-: difference between the ff,rmination ::>f normal and v1 viparous embryos. C. _____of ,_,,,,,,_ Germination __.______ Carbon ·01oxide as an Inhibitor , The effect of carbon dioxide was studied, since it might reach high concentrations if the pericarp were impermeable. Normal and viviparous embryos were supplied with mixtures of air and carbon dioxide in desiccators. The f~llowing percentages of carbon dioxide were used: 99% a.nd 100%. are• shown in Table 17. The results of these experiments 59 TAELE 17 1Effect of Variou s co Concen trations on the Germination 2 __________o;;..f;;.._.V"""·1__v__i=p~a... r..;;.o=u=s r:.ind Nor:na_1 Embrzg s :t CO 2 0 No. of !SO 25 +- ·.vp + vp lG 16 9 _ _ __ 100 99 + + ------- vp + vp l ....'?. 16 16 ...l C!. ..., 12 0 0 0 0 vp 13 14 Embryos Mean plumule length in cm. 1.47 2 .17 .18 .39 ±-14 -±-14 ±-04 ±-10 Sig . of differ~nce between the means + + Ge rmination is inhibited by high concentrations of · carbon dioxide. At the lower conc·entra tions .where germination i s not entirely inhibited, viviparous embryos show ·more growth than normals. However, in Bir ( without added C0 0 ) the growth of vivi parous embryos al ~o exceeds t:;, that ::>f n :Jrmals . More precise tests were m!:i de using con- centra tions of 5, 10, 15, 20 , 25, 30 , D5, 45, a nd 50 perc~mt carbon dio.xidt::: in nitrogen with a standard oxygen· tension of l perc eri~. Fresh immature seeds we r e used with th e first five concentrations of carbon dioxi de . For the three high concentrations , mature seeds were used as plants · in the field were killed by frost. The r es·ponse of embryos from these latter seeds seems to be c,:>mparable with those ~btain- ed from the . fresh material so the data have been included 60 Figure 4 Plumulc length cm. 2.0 1.6 1.2 . 0.8. 0.4 10 20 30 40 Plumule length of normal and viviparous embryos as influenced by co~ (1% o used in all experiments) 2 50 here. Ihe results )f ~hes~ ex J erl~cnts ~re pl~tted in figurr:: 4. is lE:;;.i .:'> Lhs.n tn .~11-, ;;:1i10wing a n inhibiting effect af car- ij in~ibited differentially in concentrations of from 10 t) 4:::/ . N--::in.ua l Einbryos arr: inhibited !11,) re str ,_ )ngly th~n are the viviµarou3. This differential effect is For carton d!Jxide to c~ntr~l germination durin~ the i.:arly st.uges of deveLO ;; tnent, it must inhibit the gro ~th process es which Lend to germinati~n, ~ithout Incr ease in scutcllu~ siz~ may be us~d as un indication of effect upon ~h ~ 0mb ry o . Throughout all tests, there 62 TABLE 18 Change in the length of thE •S cutellum as affected by the oxy e=n__ c.2!1£en tr·a tion _ _ _ _ _ _ _ _ _ _ __.._.g__ 0 1 27 28 18 .6 1 .. 05 20 . Expt. 71 • No. of Einnryos i~an c~nge 1,n $.cutellum . 1.34 length. (mm.) it.o . of . Embryos 32 . 22 .61 1.00 20 24 ·aean· ·change in scutellum length (mm.) 1.15 ------------------,.-------------Table 19 contains data from tests in which the embryos were sup lied sufficient oxygen but failed to germinate because of the carbon dioxide conctmtra tions. In all but one case, the scutella enlarged as much as those- grown under atmospheric conditions. ·change in Length of Scutellum as Affected by CO 2 Compared with the Change in Air · Geno- type vp vp ---------------- ----------,..,--10%02 _· . Air 50~ _002 No. of embryos 16 15 10~ 02 Air 50% CO,z 16 16 ' 16 lZ 16 14 1.2 l.4 1.0 l.08 ± ·.09 ±-1 .69 ±-08 +.08 -, --·Mean. · change 1.4 1.8 in scut. ±-14 ±.07 lt1n~th mm. ±.18 ±,09 l.5 ------·-----------·-----------~--------,,.......- >ritg·,. _of dif.f erence bet w€en the means · + The above exp~riments indicate - tl;lat v1v1parous 5 embryos are a"le,, to germinate at hig~er coricell.trations ~:,f carbon . dioxide than are normal embryos) and that . . embryonic growth continues at concentrations of carbon -d1o:xide which inhibit germination of both normal and viviparous embryos. • J\.. lh.g_Hol c of th:.- C'Jb tn lnh'irJi +:i~r, Premature Germination The vz,:,rk of 'J :Jpcnheimer (19E2a., b) with inhibl- tors of germ.ination found in thf! fruits of' tomatoes and other plants suegested to ~angelsdorf (1926) that · the cob 0f ruaize might c-.>ntri bute an inhlbi tor of germination. He observed that if mature seeds are left on the cob, but iE;:it in a moist atmosphere by wrapping the ear tn moist cotton, no germination resulted. Seeds of th~ same age but kept in a moist germinator showed signs of germination after ten days. Attempts to duplicate his experi- went wore not successful. In two such experiments g~r- min~ting seeds were found on the ear and among those ~hich were excised. As an additional test, a corn cob vw : :_; gronnd. in o Waring B11:;ndor, filtered. and the raw filtrate added to the culturr~ medium on which were placed e~cised embryos. All t~n embryos used in this experi- ment germinated after fiv~ days. H. J<ce:te.1 de,hydE: as a Pos:sibl.c Germination Inhibitor - - 1. - fr .:}ill germina. ting. • • - - ,,..II The sAcds u~)on drying lost their ncetaldehyde and were, therefore, able to germinate. '1\:> test the -~ffect of acetaldehyde, n:>rmal crnbryos were cultured on slants of culture medium. Dif- ferent concentrations anc1 different amounts of acetaldehyde 65 solutions wer·e added at the . side of the slant and the tubes turned in a :rmnner which prevented the acetaldehyde solu- tion from touching the embryos. All tub$S were corked ' to prevent the sc·etc1ldehyde from escaping .. Pr e liminary test.s reve.aled that 0.15 cc. of a dilution of ·1 part a.cetalde·hyde in 6 parts 1:)f water was near the thr-eshold for inhibiting germinat11".>n when applied in this manner. One · such experi,ncnt with teri normal embryos produced the results · shown in Table 20. TABLE 20 Effect of .Acetf'tld-ehyde on tre _Germination of Excised • .Maize Embryos ( see text fqr details) Germinatiun after Ger·mination fl ve days after transferring t'.J seven d.a y s with AA. medium minus AA. No. Not Ho. Gertn. 5 4: nl'' all of Mo. not . Germ, Oerw .. 5 th~ tests a giv~t). amount of acetaldehyde ¢ither •did.'. ..nqt prevent germinat'ion or'. had a lethal effect . ·~· .• The embryos classified as showing, gerr.rd~tion in Table 20 ·• did not · germinate in the n..~rntal . and appeared rather . . ~inanneI'. ·.- . ,, ' . sickly. • ln five days after tr:ttri:sfer. to , an ·acetaldehyde •free rriedium'.I · there ·. was very l.tftle' ·growth, •1ild1cating . that acetaldebyd·e is , detrj.meritar to embryos evep at con.. . . c.entrations that will · allow. germihatior:i • • • 66 Viviparous and normal embryos from the same ear were tested for a . difference in response to the ac.etalde- hyde oocn centr~tiori used above.. Ten immature embryos (Z-4 mm. in length) . from .!12 5 seeds and ten normal embryos (3-4.5 mza. in length) were cultured under tbe same conditions as reported in the previous experiment. After thirt-0en days there w~r6 nv indications of germination in any of the viviparous embryos. Five of the embryos fro~ normal seeds showed varying amounts of germination. Similar experiments with .I.Q 4 embryos showed no significant difference in the response of these embryos and their controls t,'.) . acetaldehyde. Normal and vi v1parous embryos do not d1ffer significantly in their response to ucetaldehyde. Plants earring the .!325 allele pollinated by B-lb interchange pol.len pr~ducc two classes of seeds in whi.ch tbe embryo and endosperm differ with respect to the gene for v1vipary. The genotypes of these two classes of seeds a,re given in Table 9, pg. 38. If vivipary ls • the result of the endosperm producing less acetaldehyde, ·, ieeds of Class l (Table 9) should bEf vi Viperous, but this is not the case.' 'l'he Class 2 seeds (Table 9) are vivip- arous, while . th~ Class 1·seeds are dormant. The data obtalnsd· from genetic und embryo-cul- ture experiments indicate that acetaldehyde is not responsible ,forp~eventing premature germination. 67 C. Aux.!!} Analyses of Vivioarous Mutuill Po p?ff (1941) suggested a correlation between the auxir1 content of developing kernels of some of the s mall grains and their alil1ty t~ s prout in the heads. Since it is known that the developing corn seed is a rich source of indole acetic acid (auxin) (Avery, 1935; Stehsel, 1919), a change in the auxin lev e l might be responsi blc for inducing the ~,mbryo t •:> germinate prematurely. Vivi parous and normal seeds from the segrEgating ears of .Y.Q 5 , :!2. 7 , or .Y.2 9 were Rnalyzed. These mutants were used because of the eas P with which the immature viviparous se~ds can be se parated from normal seeds on the ear. The k r, rnels VIere cut from t he ear c:~ nd frozen. The frozen material was lyo;)hiliz,ed and gr-o und. materi a l was analyzed for th 0 This total ether extractable auxin. 'ro extract the auxin the sample was hydrolyzed 0 . at pH 9 at 100 C. for a half-hour. After cooling, the pH was adjusted to pH a and the auxin extracted with five washes of ether. The intensity of the color produced by Salkowski' s reagent was used a s an ind ication •Jf trie amount of au~in present. This intensity was read on a Klett-Summerson colorimeter, using a 540 m_µ filter. The results are summarized in Tabl e 21. 68 '.l:i.B~£ 21 Tot~L Au:xi.n Content of Normal and Mutant Seeds from Ears Segregating for .!J25, !1!1., . and ~9 PhenoF~L:... r1t. :i.utaz:t 5801:..17 Vp5 -17 -9 -9 -13 vp vp vp •,t - 1u -31 vp -:31 5803-28 . ...;f:8 type Vp7 vp -35 25.8 22.or 18.0 18.0 154.9 17.4 79.6 76.7 2.9 68.2 5.8 -Z,9 • Vp9 80.5 74.0 ll.4 10.6 91.0 r;•. ""' o.,:. , 2.8 4.1 3.3 6.9 4.9. -1 -1 80.9 50.4 vp . 43~4 'Vp 60.l 60.2 -:-l.4 -10 • .. ; 10 . . . vp se.o 7.7 6.t 49.5 The auxin content . of n5 do·rman.t dor'Qlant dormant germinating dormant germinating dormant F.6 vp vp :.,.14 to.o 60.5 -26 dormant dormant 3.E3() $}5. 2 -:39 Dormancy 3.70 vp -17 5802-26 92.8 92.8 254.0 242~6 142.4 150.0 163.2 94.2 -17 Condition of Y/seed 4.6 11.4 10.7 vp -2·5 Auxin )1gru • d;ry wt. 01• ·l:Q rl •~ r:nant dorma,nt dormant dormti.nt dormant · dormant germinating dormant dormant dorii:.ant dormant dormant dormtmt dormant germinating 7 seeds does not •diff.~r from that found in norma.i seed·$ from the same ·ear·s ·wh~n·:._o ompared on a per gram or per kernel b~sis. ' Vivip- a,;o~~$) seeds consistently y1€lded .less · auxin tha.n normal '..kernels when ,annly sed. on a . per gram or a ' per kernel basis. :The· dl.t'ferencee in total .auxin between Viviparous and ., normal seeds of t!'ie first two :,;utants do not indicate ' ' that totaL auxin oer ~ is responsiblP- for v1v1paryHowever, the consistently lower va1.i.leS found in .!ng seeds suggest a possible relationship between viv1pary and · auxin content in this mutant. It shou.i.d be pointed out, however, : that the method of extraction does not differontiate between free, physiologically active and bou.nd non-act1 ve auxin since both •Jf these are extructed under the conditions used. A more critical test would be to assay for free auxin in normal and viviparous se0ds. Because the amount of free auxin is relative].y s~all, this analysis would require a bio-assay which has not been done. Since it is the embryo which is responsible for producing vivipary, u more critical test would be an analysis of its auxin content. Preliminary teats of viviparous and normal embryos have failed due to the low level of auxin and the presence of large amounts of oils which interfered wi,th auxin ~xtract1ons. 70 Within a seed th 8 d~velo~1n~ embryo is located in nn environment which is peculiarl? suited for i n~ 1.11 1:;• rt: ,; u i r c,mE>nt s for its gm'mina t.ion. Studies with i Dma t~~e ~~ize embryos have reveRled that they are potenti r:.lly ca ;iable of ge rminatinc s.:)on a fter p::>llina tion (about ten dnys). However, germination doPs not normally occur at this early da te in spite of such an a pparently favorablr •::nvironr·Hmt. Insteacl ,?f' germinating .the embryo continues to grow 1n a mannE?·r which 1. s ty pified by dif- ferenti atlan of leaf primordia, enlargement of these primordia and a pa rellel increa s e in t he size of the scute.Llum. Embryonic growth 'ts furth er mr;.rked by an absence~ '::lf ce Ll clonga tion. E3everal pr ema h .lr e gErmina tion mechrmi sm.s have been postulated by ~orkers in th ~ field of pl ant dormancy. Kidd (1914) suggested t hat c~rbon dioxide, present in the tissue o f deve.lopinp, p ea .: ,nd b1?an erub r y os wa s responsible 0 for p rev\:::nt lng their g eri.uina tion in th € ;,od. However, Ha rriet Bonner (un published), also working with peas, found immature e ,;1bry0s contai ned t, leachab lP- inhibitor of germination which was thought to be r!3spons1bl.e for ma int a ining t he ~eveloping e~bryo in a do~mant state. Pop e (1949), working with barley, was able to induc e vivi pa ry i n this pl ~nt by k ee0ing the germ moist. He proposed t hat t: ,1: i Lm,ature embryo does not germinate because i t is 7 1. a lso f ound asm0tic able to obtain substa~ce s fr~ra th t fruits of thes~ pl~ nts ~a ny o~her workers fw.ve fymc. similar s2r.:1,:tnat ion inhi b itor s in th . fruit s review a f t ht ~0 ~k done i n th i s f i e l t . specu ta ted ::>n tlrn r -. Jl e )f VariJUs workers h ave these, germina tion inhi b:l t:>rs in pr ev enting premature ger~ination of developing seeds. i n di. c at ing th(·~ t th r:i se snbstances ~He a c t ual: y res ;,onsi b le f6r t~is pheno~enon . se ed .,)c e-,jlb ryo t 0 germinate .1.n an ,, nvironmcnt which n::>rmally a more crl t ic~ ~ m2nner. L :Ji.' t.h -: µr~v.:ntLm :,i' germinc.1.ti,)11. The :11utati0ns s tudied in this report ~i ~l ~p p0ar t0 disru J t the njrmal inhibiting .,.. thut. ··: ~) 1,,.,-;....,_4:•._ .i, of t his C<,l:Cbon di.oxide i s due to some IDechanism a s yet unknown. ;-, ;.tW(:h;:nism inv0lving c&rixm C: ioxide ·:; Lich e ✓. pL:dns Vi'lipary can be su;;geste<l. If, : .=;. s the s s ed matures, its enclosing membranes become more p~rmeable to carbon envir onment of ca.rb,::,n dioxide will · decre:as€;;. Sprague (lS Zo ) fLE G. sured the water content -:>f d.ev<2J.oping sE:eds L.nd found it dlso decreased us the: seed matured. This w.s.ter loss teru.inated with :.uaturity at. which time there was not sufficient wat Gt' ~ithin th e seed to support ger- 73 mination and the seed remained dormfint. Vivi ~a rous 1 seeds at the t im e of germina tion u pon the ear have a water content of 36.5±0.7 pe rcent, 1..vhich is comparable to the value of z.s .o t ±O. t -J. percent found by Sprague (l92Ei ) for maturE· seeds a t th€', t ime of germinr~tion. Vi s have, then, t,h5,s pictu:r·e of a maturi.nr: seed with reg a, rd t. :.> its carbon dioxide and water relations. As the seed ;1n t uros, there is a para.1..1.f:.l decreas e :Jf both w::..ter contEmt :J.nd internal carbon di oxide tensi::m. While the water level is high, the carbon di oxide tension of the seed is sufficient to prevent germinatiori; As the seed nw.tures, its lowered ca rbon dioxide tension would permit ger-mina1~ion, but the wat81· c:Jntent is too lov: . On this inte rp retation, the carbon lioxide concentration of the seed should be about 40 -45\ at the time other conditions are favorable for germinati~n, theref..-::re, nor1ml e'.nbry•.)S cann.~t germinr;;.te, but v.i vl ;.)arous one~ ~111 be able to do so because ~r their incr eased carbon dL.'.>x:c. c t .)}_( T &nce . This CJE•ch~.;nism. ct::.n b t: schsttatically represent ed as in Figure 5. There are at leusL six di f fer ent 0mtations that interrupt t he ~ech ~nism f ~r preventin~ ~rematurc ~ennination. TE:st.::: hav0 n::>t bc-t'n :.:i:,.dc, iiith U:c othor viviparous mutants to determine if they have a tolernnce far carbon dioxide similar to th&.t. exhi-r>i tf~d by v J 5 embryos. lJ 20 30 -•-- - - - 40 - - - _5Q_ 6') 70 % co..,,::_. · % CO2, - ' \ \ \ '\ j'...' c::. r- tc· r• ·)(- \ \ 20 30 40 Days from pollination - u( /() ~-··""' ·--··-. ,' • 60 .12.9rrnit the exgressi'Jn :)f vivipary * Fram Sprague , 1936 , pg. 347 10 20 30 40 5•'.) 6'.) 70 80 S·O % Water A schemat ic presentatLm of h::n.• the water a nd CJ;? r e lationsh ips of a maturing seed could K, co~ is not inhibit~ry 10 --- Vi vip&r:Jt.13 cmbr-y'Js can germinate in this runge because water content is sufficient & Insuffici ent water to permit germination below this point ------r- -- ab:JVE this point by C-J2 levels Normal embry..)s are inhibited C00 levels "--· abovP t.hl.s p-Jint embryos o.rf: inhibited by V•i vi par -J11 s .F·lGUL.1:. 5 ,J::,. --..1 75 tU.f Lh.LN G£S Anders:.>n, t: . G., lDZ,8 'rra nolocu tions in r.10.ize: in vol. ving chromosome 9. Genetics 23: 307-313. Anderson, E.G., 1939 Trunolocations in maize involving chromosome 8. Gr netics 24: 385-290. Anderson, E.G., 1941 Trnnslocationa in maize involving th(? short arm of chromosome 1. 4 t\9. Anderson, IL G., clnd Brink, R. A., 1940 in ma ize involving chromosome Z. G".;nctics 26: '152- Translocations G~netics ~ : : f'.99-;:/ )9. Ascherson, P., 1875 Keimung im Inncrn eines Apf0ls. Ver. d. Bot. Ver. d. Prov. Drand enburg, Berlin 17: 79-80. Atwood, W. M., 1914 A physiological study of germination of Avena fatua. Bot. Gaz. 57: 386 -414. Avery, G. s., Jr., Berger, J., and Schulucha, B., 1942 Auxin content of maize kernels during ontogeny, from plants of varying beterotic vigor. Am. Jour. Bot. 29: 765-77f. Burnham, C.R., 1934 Chromosomal interchanges in maize: Heduction of crossing over and th e association of non-homologous ~Jarts (a bstract). Am. Na t. 68: 81-82. Burnham, C. n., 1935 (As cited in) A summary of linkage studies in maize. Memoir 180 Cornell Univ. Agric. Expt. Btation. Burnham, ·c. R.; 1949 Chromosome segregation in maize translocations in relation to crossing over in interstitial segments. Proc. Nat. Acad. Sci. Wash. 35: 049-356. Burnham, C.R., 1950 Chromosome segregation in translocations involving chromosome 6 in mai Z€i . Genetics 35: 447-481. cr·ocker, w., Holf2 of seed coat in delnyed germinaBot. Gaz .. 4.t : 265-::::91. 1906 tion. 76 19 :<) Studies on nutritionally deficient bacterial mutants isolated by means of penicillin. Experientia 6: 41-50. Davis, B. D., Evenari, M., 1949 Germination inhibitors. 15: 153-194. Ey s ter, W. H., 1924a Bot. Hev. A primitive sporophyte in maize. 11: 7-14. Am. Jour. Bot. Eyster, w. H., 1924b A second factor for primitive sporophytein maize. Am. Nat. 58: 436-439. Eyster, W. H. 1931a Heritable characters of maize. XLII Reduced endosperm. Jour. Hered. 22: 251-252. Eyster, W. H., l93lb 574-590. Vivipary in maize. Genetics 16: Eyster, W. H., l9t5 (As cited in) A summary ::,f linkage studies in maize. Memoir 180 Cornell Univ. Agric. Expt. Station. Raagen-Smit, A. J., Siu, R., and Wilson, G., 1945 A method for culturing of excised, immature corn embryos in vitro. Science 101: £34. Harrington, G. T., 1923 Forcing the germination of freshly harvested wheat and other cereals. Jour. Agric. Res. 23: 79-100. Kidd, F., 1914 The controlling influence of carbon dioxide in the maturation, dormancy nnd germination cif seeds. Part II. Proc. Hoy. Soc. (L-~ nd.) B. 87: 609-620. Konis, E., 1940 On germination inhibitors. II On the action of germination inhibiting substances in the tomato fruit. Pulestine Jour. of Bot., Jer. Series. 2: 6-27. La Rue, C. D., 1936 Growth of plant embryos in culture. Bull. Torrey Bot. Club. 6Z: 365-3-82. Lindstrom, E.W., 1923 Heritable characters of maize: XIII Endosperm defects - sweet defective and flint defective. Jour. Hered. 14: 126-135. Longley, A. E., ma.Y__§. 1927 Supernumerary chromosomes in Zea Jour. Agric. Hes. 35: 769-784. 77 ~angelsdorf, P. c., 1923 seeds in ma ize. ThE inheritance of defective Jour. il cred. 1 4: 112-125. '.·;I ang 01. :;d.):cf, P. C., Th r, g<c.mf;t.:i.cs t,nd rnorpholoe:y H-¼SH endosperm cha racters in r1a ize. Ap;ric. 1:xpt. Bull. 279: r,13-€-14. :)f s,::,r!H-:: Conn. j[a ng c Lsdorf, P. C., 1930 Th n !nhed. U 1ncE: of dormuncy ,u1d 1n: r ;WtUr'ft germlnu t ian in :aaize. Gc netics 15: 46:?:-4fM. Maze, P., un o Matura tion :Ol'OV()rjue 1:· dPS g ;:aines. Action antigerminative de-1 1 alcte'hyde fthylique. Com pt. nend. Acad. ~3c1. (Par.t t►) 151: l ?:83-1386. McKellar, A., 1898 Mellon with seed germinating. Chronicle London. 24: 128. o~rd. 19~::2n Keimun6 shemm0nde Bul: stanzen in der r ·'F~· h.t von Solanu,m lycoper 91 cum und. in anderen ?flanzen. Sitzungber. Akad. Wiss. Wien . Oppenheimer, H., .L~~i.1: 59-6 5. Oppenheimer, H., .l9i::Yb Das Unterblei b~n dflr Keimung in den Behaltern der Mutter}lanze. Akad. Wiss. Wien. 161: P78-( l?. Sitzungber Pope,~. N., 1949 Viviparous growth in immature barley kernels. J0ur. Agric. Res. 78: 295-Z09. Popoff, A., B :~) 1941 Ober den AuswRch bei Getreide. Angew. t. 23: 254-285. From E. A. 24: 21592 (1950). Bandolph, L. F., 1941 Genetic characteristics of the B chromosomes in mc.1ize. G'3,netics 26: (~ :) 3-GZcl. fL.ndo.lph, :,. F., r;tnc: Cox, L. G., 1942, Ftictors influt,ncing th e germination of iris seeds 0 n d the rel a ti~n ::>f inhI bi. t ing $Ubst Emces t-,o e,r; bry -? dorw1ncy. Proc. Aw. Soc. Hort. Sci. 43: 284-300. Heinhard, A. W., 1933 Zur Frage dt:r Sumenkeimung bei QQlan4m 1,tgooersicum. Bot. 20: 702-794. Roman, H., Planta Archiv. fur 'Nissen. 1947 Mitotic noncli.sjunction in the case of interchanges involving the B-type. chrot!!osome in maize. Genetics 32: 391-409. ?8 :\ Yrl ::, n, H., l.918 Directs,d fertl .l .i Hi :.ion in maize. Nat . Acad. Sci. Z4: 36-~2. P;·oc. 1920 An~m ~lio delle Jl ~n t ul c e anoru~l i e di germinazlonc in Nic~ti ~nn. Nuavo. Giornnle Bot. Italiano 27: 2-4. s~vclli , ~., 1932 D~rma ncy in seeds of tassvo~d, ame ric a na L. Am. Jour. Bot. 19: 835. Sprague, G. F., 1929 Heter~-f erti lization in maize. Sci 2nce 69 : 526-5[7. Sprag~e, G. F., 1932 The nature and extent of hcterafcrtilizrition in maize .. . Gc.n etics 17: Z:~:B-z·.(3,9. Sprague , G. F., 1936 Tht.• rel D- tion of :no1sture content e.nd ti!!le of h a :::-vct-;t to geroinution of io::wture corn. Am. Soc. Agron::>my. 28: 1.72 -478. Etchsel, M. L., 1840 Unpublished Ph.D. Th~tls, Univ. o f Calif. Berkeley, Calif. .Stier, H. L., 1037 The effect of cert a in seed treatments on th e ger□ inati~n of recent ly harv6~t ed potato ~:et:,ds. Pr::)c. Am. ~;:.,c. Hort. Sci. :.:5: 6'11-605. Tatum,£. ~-, 1919 Amino acid metabolism .i n mutant strains of ri:.icroorganimu::;. fed. Proc. ,:-f.. ' 511-517. Treichel, f.•• , 1880 Uber vorzei tif c Keimtlg . Ver. d. Bot. d. ?rov. Er~ndcnburg , Dc rlin 22 : XI-XIII.