mom m Iggfpuggg wamugyw Thesis for flu Dam of M. S, MICHIGAN STATE UNEVERSITY Lynéon W. Kannanberg $959 .. , m,- ' LIBRARY " II." b 3" ~-'~ "v- - w w-‘wr ~ -- .‘ "“”‘,1" "1f LVLUY 1...} “-1.” \-L.Lx .4- .14- -"L3L»T\ l" ‘ L37KATU .4. rutnzxfl;uv: v. ,'.yv -.~v‘—7~ n A‘i J.....JJ.LJ 1 a \ o *“01 of G“1*uate Studiex of L13n1;;n ‘_ .W ‘ lie QCIJ‘.‘ \ J-( Mk 1‘. . a -'. ,— _‘ ._-', -. , xiglieq an-.Lce Submitted to ' o? A;rj-ulture and . c"- Fj c+ 1 (f- State b.1ve :; y - . in par iaL fulfillment of the reguirements ;LJ.:$ Cb JUIL.LL ""~ J ,, ,‘L ." ‘. . ‘1 . . DCJ‘JCLIGLJU 3'21ng (JUL : 4.1) L) I. x);IS \f' \C) *3 11‘ _. .L, a -- ,‘.__. “ -. _ . - ‘ ' 4118 Jubuor IV-LsJ.AD-J t) 81.: IC.-~S “1.; ...-..-Ct"‘rb d. F”ClrJL_LtJN tnls research :rouect and for n15 heszul aav1ce 1n tne .. M-.. 3.- r' #«4-« ...-, .... .' - gregarat.gn o; Un-o mahuocxl,t. 1'9 "V*""O '2. L'V'f't P .. pd " *‘MJ "u‘J‘ICJ‘C."- :3 :V': .31" “1 .- 11.; autul. r 1.1 {L‘i CAUJ— .LLI Lftw C..J»JJ_VD£~L.C‘.4 LA..1‘-I. La :CJ‘AI‘ -. ‘ r‘ ‘ " " ~ I "'\'~" ' ’~ -I 1 ‘1 a ‘. r. I 2 V7 "’.-' I r- f. 'w -".‘ J—L‘ g9;..92;t OJ. nib Q17 n-1,... l .111. 6.9;..ICCL‘4JJV ..:.L:.1TJCE.LI¢L .Lul‘ bile g. ‘v\- ‘L r r ‘ . ‘rx r‘vfi ~ ‘ "4 - ~."- I. ~.--r .‘l "a r 5‘ .‘ .r‘ aua;”oance, BFCuhrJ;GUCJE, an: uevwv.on o. h. hlLe, JGIUQIQ, during the caurse of LL15 study. TLLIJY I. IaIFCLl.K 3.7flfifil A w Lvngon u. nannenberc a 5, a - 1 fi‘ . "T lai‘fi 21;. 3-5.). .nbl. - 3-,12 ‘ ¢, ‘,.,.. .-~ ‘1...” z‘ ,. 3...; -. .' .:W ‘ -' ~.i .1 .. -. DLLUn-l_-te(.1 LA) tut: JLLVO]. 0-; ‘..I<1.L.L.:Lue 0LLA=-.LKJu O-L r-J.L,.1_L_<1fL jzate halvers;ty cl agriculture and hylllsa DClEHCB 1n gartia; fu¢;1..ment or fine TUHMJTG.CntS - . .: I , . ‘ ‘1' .-. ‘ ‘ L‘JIJLLLL U: QUA— .Ji.LJ.‘-‘J Derartucnt of Farm C1033 Year $L9 Approved QM E. E W (\l ‘1 ‘JJ.-a-‘-¢\/L 1 . . . \ x w r? ’: \u"~fi« 5" ' v__- a... x. L‘ .A. \J t J A L k) ‘— L4 w 1., K... o.) kl A. I .L -- L— --1- L ' u i h... l . 1.: . . A. v k) . 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J 4‘ ~‘ ~, 5 ' r - i‘ ' ‘~ ‘. -’ .fi .- ‘ I *‘p l I I‘.':.'.-;.;'_L0lu :_'.-‘U;‘C€‘.:>. ELEV; um, L;LI‘C€‘J 1.9.0., TEJ‘. ,CcivvJV, 3. iew Bdtl’fl plaid aid pcnta:loid plants. difice no aneugioi‘s Mere I-uni it was ‘ * ', r 'l I“: 1‘ r \nfi ‘ ." - r: . ~ - w. a. . "vr . . - O. Lui'qiv 91113-48 10.1. .Iaa'..+v0 1128148 ; l'D 121.61“ 111181 12.1-0131“) HLdtllJUD (.0 I- N m hy; otke involvin. di_ierent sourcec. Ti 51; \ own in the field and greenhouse were examined for several HorthIO'icai and yh;sioloLicel characteristics known to De ‘ieide. ;be1es its indicated tuet under fielr -' / .-,..,-.' 3-..“ . ,. :-.. .. ‘. .. -. -r 131’};th W/ Jrea Lyn .L “La-LIL )u- ’ if1CI$10CL1 .LCiJ. Lire‘-L, .L. 91-81 IJlAt .Ll-lI :91. :3de bud, . < '- ~ ' -‘ ‘ r ' 1" ‘ ‘0 , ,.r 'L r“ r ‘I .4. \fi. '. ‘ .-~ .1 r -4 .—~ increased out 11-8? llOMeILh,, lor:;er .1J.ec aux Igviic, in; er 'A w . J- .. . 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VI 4-....‘bx v v A x 54 UV A L1-.’.:.»J _-A ..... .— J. .L J L V r, . U 9 I. ‘ fi‘ ‘ - I: 1 1‘ y- ‘ ‘\ ‘ ‘ i . " '3 ' " ' 3 . ‘ - -_‘ .4 - _, A .. ~ _ , — —. .- r -. ‘ ._ s, Kata. ‘ O *.-u‘ A J. J—J L I" A. ‘4.- /_.LL - VL ' 3‘- V k — ’ n - \4 ~/ ’ Ljv‘ JC—_Lk 7“ K] 5.7... ‘ L ~‘r m '\ _~ '3 3 _ " It 1 u I : f. 1 ‘ ' -~‘ 1 ‘ I: .4 ‘ ‘l. e. )7 \ J- - " -. —‘ 1 ~ I 1‘. \JJ‘. CLU.,..4V..,. I v.1 d. C- ’JHI _ VIC/-4 11 sJ./+.L_i L ’3 JAULLJ 4U. .2U - u .L I kay.’ A 1‘3 ”l I ’ C” ‘ W ‘ “" - .- r " ‘ 1 - ' ~. 4- - -' ' I—a _ ' . - ‘ .. ‘ . ~VV . ILL k1 Ak‘ V' .1) J 3.1 J -jsl.C‘D dUJ Ui—I-L V“ . JL..(~‘. L~l 3 LA: VJCvA t: A UV} q- ‘h/L‘I -. .* ‘ . . . .1 1. . . «r L m . 1’" q tx- w. v: .- .. ”.5 ~ ‘ a, ,1 "AL.“ 1. (if... L‘K/uv‘r,‘eul; .)\.~LJICK.,._) ui .i... - p: : - ., .,..... .1 I, '. .. " ,5, r ,- y- _ .. It .13 -144” ".".,L,.3(.;. '..£1..:.U the btl‘Ql'rL (31.;(34‘A 21.1. CagnchVd' V‘l. iii/1.1. “1,- -. .t .- L ,. .. - : -. . .t. .. , .- . .: , - .- - ”'1 ,_ - .pl- , , ,. Cid-"3'81“ i-‘EI‘. __L L, .2 Ln- 3 1*. 323:2: u -I 3.- (31' H-131“. L1 LIE/11 ‘JaLLI‘LCLL .1 .1) I J-u;i.1'-.l_.h‘uutl ,4 - 4. 'A 1.- .. ., ...-.:. - .. .-._.' :fi.,-,.»t-,~ . . ‘ - -.—' .‘ .... -9 ’ adaeta.1.sn c.) d. I‘ ~31". '3f;.-il“c‘f...r-.Z-L. - di‘euerhfz. ‘3 tilt: CuEJ-..LQTJJ21C8 Ui 4A ‘ -—- J ‘ ‘ a “L- '- ‘ r " ' u L t- ' v \'~l 9 I \'Y -: r '1 (M, (3111‘:4 "xx v” 7’30 (1:) 1 3 h L (1;) 9118 Ci. £431 GAIL lJC-x VJ; 1.1. 1.8 i LKDLC /, l‘J‘f .1 D . rN' . a" ' . ‘ -'< ‘ “" - ’Vr' V'\ I ‘ ~r-“" 1 '7' .1“ —' "' .’ . u r ' \ '. . - j;‘ I ' ”‘- 'V 1 ~ . ’ "v' . 1"" lrlulCCLLe Erie oi “5C ‘3 3 LBJ LII, e.'ae.1'u_.'-.j '5 LL81 :_|»).LIA.I_ .L.L.Lv\ll b’LIlL/ BVOIutlQ.;dl J, Chen e5. Djfterepces in re:;onee o; certiin sources to environaentel cha:.c* is dLscucsed fro“ the Viewgoint of the severit; of their previcus area of adaptatien. . 1"" 7 _,.._. I “-511. -ICJL/‘L C]: I U‘l‘i o O O o o y Y ‘ , . — -~‘ '*“-.f"" "."‘“I ‘1'.~ 31'1 __ royvi'ga U: LL- 4. ..—LLL-~.1‘J . ',- . , ' 4.: ,. CnafaCtCrleLCo o of polgpl \ - ' x-v-l—-‘ i u ‘ r u , ‘ "\"1 -‘ -‘ ‘ - 2 :~ ~ - . I ,-, v. 1213.4...1114LIJXI‘L3 nuD JL)1\.ILJ..) DeterminetLon of lbflfihiflaLiQfl Exxxination Examination gorphological and .-' " fuh-DLLJLJ O O o o o o o -‘ v‘~‘ .- -5”'.5DI l‘.‘ o o o o o 0 Di bL L' r‘v >3 ‘ ' Int." 1“ rvfi --.~~- _‘.T\‘ DL.’r :ion CL A reps-At intzoquction ecei ved L re. 1 have not been Able to Let seed from this Aotnanited n_'ter:1i gTOkh in isol;tion conditions in Cenoerr21snd two sites in sou nern victorie. j ployin: hird [oilinetion Me n ' mcinrtion union uroduced as”e ed. Homeve., ceit in combination I (T) C -- J.’ _ A p ,‘ - O}. on" LL'DtIi-37,:.L:Lct in. AU . _u..-.;l EMU!) ”Izmir .‘ "2- - '1‘]? .t (‘5‘ (.1 -“ .~\ _' p. ‘3‘ - ‘ ‘ I 4— ~ 1‘ ,r. . - ‘~ " 'I _ ' \ J ‘- ". ‘ Fol. .Lvu,b: I'D-3 liAve LlV'e‘. (BALL/(3 1.720» Seed HIOCACclnu." A .1 ~ '.‘- . r. ,.'t , “"vvw— ' ~~ .i'\ _u, Ar ' .'~ ~ -..' ‘ ~‘.. .~v- - w . . ‘ be A also comhenced AAAt muCL oi tLe i. AmletnA mctdrlwl in circulition at present is orvo: l; unees lIAul e in:red An. eu'.ests y.- Qistingulsnlhg Datween the ploleil Arosps and “Aminb §LlJCTQQS€3 irom elected from a nquer of lHtTJUHCLlONS. Rely observed tnAt A high percentage of 6x plants Lave ineffective hedulation with br. Er‘man's Turkish isolates of finizobium trifolii as well as uncertain flowering and very poor seed production under the Australian environ- ment. On the whole, massive inoculations were necessary to estaslish T. ambiguum bacteria in competition with other strains of ahizobium trifolii. In later correspondence (ll), it was noted that in Canberra the diploids (226A and 2771) were better in seed production and had a number of effectively nodulated plants. These plants were Small but denser and could easily out yield the polyploids. The tetraploid CPI 9949 was described as being strong-growing, leafy, shy flowering, and subject to early and effective modulation. The other tetraploid, CPI 683A, was described as a poor agronomic type but a good seed producer. This also modulated early and effectively with the Turkish isolates. In addition to the Australian material the folloainb sources were also studied. ho description accompanied these sources. t” C 33109. deceived from 'r. E. A. Hollowell, head of Clover section Forage and Range Research Branch, Agricultural nesearch Service, U. S. D. A., Beltsville, karyland. Pellett Clover. Irradiated material received from Dr. n. J. Letzger Farm Crops Dept., Corvallis, Orebon. PI 22966 , PI 228370, PI 22962 6. Received from hortheast Regional Plant Introduction Station, Geneva, R. Y. Iran is listed as the country of ori;in. 10 Early in the vinter of 1957-53 part of the seed from each source, with the exception of the Iranian materials, was planted in flats in the greenhouse. The Iranian naterials, PI 229o25, fl 225270, and PI 22962Ao, were not received until the spring of I953. deeds not alanted were held for later use since one of the techniaues tried for determinin; chromosome number reguired gerninating seed. This will be described later. At the completion of the aforeuentioned experinent (I) , the remainini seeds were sown in flats along with \R in the spring of ' the newly arrived Iranian seeds. The same technique was used in the sowing of all seeds. Seeds were planted ; inch deep in regularly spaced rows in sand-filled flats. Full width artitions were used to prevent >\ C) mixtures when two or more sources were planted in the same flat. Prom seedling emergence until the termination of the study nutrient solutions were applied to greenhouse plants at 10 day intervals. Khan the seedlings reached a suitable size, the more viporous ones from each source were transplanted to four inch pots. Discard of the less vigorous types in each source may have eliminated some slower ironing or slower germinating types. Aneuploids, triploids, pentaploids, etc., may fit into this category.’ A loam-peat potting mixture was used. Labeled 6-inch stakes were used to identify plants as to source and number. Each pot was then placed in another four-inch pot. The void between the pots allowed root tips emerging from the drainhole of the upper pot to be uncontaminated by foreign materials. Clean root tips were desirable for cytological investigations. Ho control was exerted over light and temperature until Cetober, 1953. By June, 1958 many plants from th earlier seedinps had produced I ' ‘ ‘ .‘ ‘_'\_ _‘,.I. , I...” ‘ ’. ‘v--'- 1 I “‘ ;r" ' rfllbLj—HLBS . 5. 1711413138 V3.3 .u Veer J l < .._._ Conan Ol L-£.?:.‘..)C -_'J_.:.1.L.J mills. Qa-‘uLlJ c:— H. I.“ . ‘ -\ I ~‘ - - ‘4- -4~, L- r‘ 1“ ~ - g g.', .‘ - -' v -- “i" - ‘3‘. -‘ . r~- . ' ‘ .‘ 1 pottei in ,-ur lnCH :Ooa as described above. inc ldmgthCl oi eaca - = -J‘ ..c, L._ _‘ .- ...-«“,.-... «‘3 4— “‘ ~ ° '3 ‘ -..'~--. r .- - :3 .—-' .~ — «3. " plug-la"; 1Nils v...~r}.'. tr: fl-;;_'-L:-11U€atv 'v.-’ the .LlCLU Ilka belJ at UJDL LCL;;D.L.I'L .. in t 1.“ . ‘r‘ ,_‘1* I". #n ‘r- H‘ “.1, , _ _ . .. li‘l' .l. a \ .‘-‘., flu ust all plants lrum the earl er seedinés were transplanted to the *- field thether or not rhizomes had seen produced. however, CULtlh35 were made from plants whicn had initiated rhizomes after the earlie I field transplantini. fhese cuttings were also nept in the greenhouse in labeled double pots. With the enception of a few seedlinps from the Iranian sources, all of the later seedlings were maintained in the reennouse. These comprised the bulk of the greenhouse plants. a Lew plants from each of the Iranian sources were transplanted ts the field in order to have replicctioh of each source in both the c,remhouse and the field. Plants in the field were ssace planted at two foot interVals ' art. lj-lj-l; fertilizer‘was a plied in a rin, around E. “i C; U‘ \H F H (D (l) ('1‘ w of approximately lOCO lbs/acre. Identification of (D Q. ( x-) }_J 5.? :3 C _ l (f- H J C.) plants was accomplished by stamping the source and plant number on a I metal tar. The ta_ wa< then Stapled to a S/A” x 2" i 8” w oden stage ' l1 my ‘ i, :L .n.). x ‘.1-,.., a ., ‘ W :, ,‘,_,. ‘ ,....'.,;1 ltluuea my the plant. only those plants whicn nae tutcin_s Sl H. 0 CT (.4 D T U) in the greenhouse were individually identified in the field. ine owners were idegti;ied only o3 source. Since plants initially were placed in the field acre or less accordin; to the time of rhizome proauction the plants 01 host 3 urces were spresented in varying numbers at the or 12 i. Deterninltion of nloigy level for each source, 2. JLQJlta o' the e:;ect of ploid; and source on variocs morgholo icel ard physioloLicel Characteristics inciucin: lee; size, stom ta lentth and nunoer, size of floral carts, pollen size and Vliullitj, fertility, seed size a.d weight, vivor, winter hareiness, Crowth haoit, and flowering, Fertility sttdies. \0 Comparisons could not be made oetween all tlents represented in both the field and the greenhouse anone all the sources oeCause: l. The Iranian source materials were not vegetatively .‘nce rhizome initiation Jas not yet a_garent pit) a:< teCl e at the time of transglentin . These seedlings were apyroximntel; six months y‘un er than the transplants fron the other sources. 2. Some sources had onl, a few olents producing rhizomes at ‘ tr» . ': I‘ ‘ygj -“ \;' 'o ‘ ~ - ,~ ‘ I t V ‘ ~ ' I ~-r- r‘ ‘0 ‘ tne th3 Cl uranbrldhulng. LCUSBUMGHULJ, the numoei 01 plants represented ootn 1r tne lieli an; LFBSHHOLoe was Losses occurred 'n cuttinvs from QOCL the Lreenhcuse \J‘ I F]. Fr- and cld for a v riety of reasons. 4. In the ¢reenhouse erratic ilouering further re uced the . ‘ p l‘eeluwub‘e data. J. I‘Luu L L oo;crmeo;i;s to castle llietiflkh ‘m r 7". - 1 r\ F.-;- the Same Llchbs. Determination of nloidy level A H , - D- (o Elo.oy levels or the various sources were net nnoun hith ,.— J \.Q the exce;tion of the naogrial received fron Eel; Which had been tentative identified. In most sources, hongver, v.riation oetneen lants within a source was sucn that it was suSyected a ranbe of ploidy levels hi.ht exist. Generally Trifolium chromosomes are small and not amenable to ordinary Shearing technicues. Three methods of cytolQAical examination were investit ted in an attempt to find a fast and accurate orocedire to determine chromosome numoer: l. Examination of root tips from germinated seeds, 4. Exahihation of pollen nether cells, b. Exanination of root tits from greenhouse slants. A Examination of root ties from berninated seeds Seeds were scerified with sandpaper and treated with nrasan. Petri dishes labeled accordinb to source here used as Cerninators. Each Petri dish vas hold at room temperature and under A; hour illumination. The seeds were terminated on blotters seturated hitfl 0.2, KLCB solution. Identification of individual seeds has facilitated by the following steps. holes 2” in diameter were punched in a Slotter and numbered consecutively with indelible inn. dtaplint a nonoerlorated I , blotter to the slotter helped to nold the seeds in place. the seed was placed in each hole. I V“ -‘ Crl- " ’ Tilt: Univ “ch. \— "13 e o germination in thich the first flush of mitotic division in the root tio occurred was not Anntn. Therefore, ea .les of root tips were taker from the time the Seed coats ruptured until the roots were apyroximately 2-h km long. The root tip Iron a girticular plant vas placed in a labeled l0 nl shell vial containing .eolzu 14 3- hydroxyquinoline and held at approximately b.5OC. After 5 hours the hydroxyquinoline was replaced with hewcomer's solution. The root tip was kept at A.50C. in Newcomer's solution until used. Standard procedures were used to prepare slides for study. Fropionic carmine was used as the stain. No divisions were found in root tips saupled as outlined above. To deto mine if mitotic divisions were under diurnal control, root tips were taken every half hour over a 24 hour period. In addition the maximum length of the root before sampling was increased to approx- imately 6 millimeters. Several stages of divisions were found among the larger root tips at varying times. Therefore diurnal control was not apparent. This method of detennination of chromosome number was discontinued for these reasons: 1. Plant recovery was too slow after the root tip had been removed, 2. The number of dividing cells per slide were too few for rapid determination of chromosome number, 3. Space limitations in the field and éreenhouse were such hat it was desirable to save only those plants which (+- showed good vigor. This would have been quite difficult to determine in germinated seedlings. Examination of pollen mother cells (PRC) During meiosis chromosome associations can be determined and counted at late Diakinesis and hetaphase 1. In T. ambiguum, dividing PIC'S were found when the heads had just becun to emerge from their protective sheaths in the axils of the flowering shoots. In this study one or two sucn heads were removed from each of several plants. heads from a particular olant were placed in labeled l0 ml. shell vial con- taining Newcomer's solution and held at approximately 4.50C. for at less 12 hours or until used. Since the florets of a clove; head mature acrOpetally the area of the head in which the PLC'S are UiVlanL also moves from the oottom to the tap of the head. This region must oe determined by trial and error procedure although floret size is often .' an indication of the approximate region oi the head in which divisions are taking place. Anthers here renoved from florets and Sguasned in a drop of m. ine oreuaration was then briefly ooserved propionic carmine stain. , 5 under lEUX to determine if the ELC's were underpoinp meiosis. If meiotic stages were observed, a cover slip was put in place and the ared. Once the area of the head was deternined in (D preparation was on f). :4 L (4' which divisions were occurring all florets at this level were isoli and slides mere prepared as needed. Cfteh the anthers from two or three florets were combined in one slide. Althou h the ploidy level of several plants was determined using the PLO method, it was discon- tinued for the following reasons: 1. The process of finding the exact level of the head in which meiosis was taking place was too slow. a. Nultivalent configurations, especially in the tetraploid and hexaploid levels, made accurate counting verg difficult. Cytological xauine ion of root tiLs of greenhouse ;lm ts Th eaost successful of the three net} [eds 101 determinin:; mitotic divisions in the root sins slants. Eacn plant in the Creenhouse was douole- potted in four inch gots. The void oeuw, n the oottoms of the tvo pots allowed root tips to area unconte azzinated b, foreign materials after emergin¢ from the drainr isle of the firs' pot. In this study root tips were taaen at various times between 9 a.,. and 5 P.n. With no apparent differences in the nunoer of dividing cells found. tpon rezoval, root tips were placed in a labeled 10 ml shell vial containing £CQ m s-hydroxyquinoline at aporoximately 4.50C. After approximately 2 . 3 hours, the hy'roxyquinoline was replaced by Farmer's solution (3 parts Ethyl alcohol: 1 part glacial acetic acid). Several dross of aceto carnine were added to the fixative (9). After 12-24 hours the 1 In fixative vas poured 011 and 1N HCL added for l5 minutes to facilitate maceration. Finally, the root tins were transferred to 709 ethgl alcohol until used. Best results were obtained when slides were pregared from root ti ps within four days after removal. The aceto carmine added to the fixative served two rur>os s: 1. Since onLv the flier istematic 1e ion of the root tip took up stain readily, the remainder of the root tip could oe discarded v:hen a Si ide vas or wated L 4. Since materials tree ted with LCL do not stain readily with ‘5 v aceto carmine, pre-treatment with stain intensified th staining of the final product. r‘ In each slide preparation, 2-5 root tips were souasned in l7 aceto carmine t: which iron citrate had been added. The hummer of root tips used was dependent on their size. Best results were obtained when a hard even pressure was exerted on the cover slip durinb the smearing process. Lhile pressure was maintained halted tax Was a plied to the perineter of the cover slip. This helped to prevent bubbles due to the intake of air under the cover slip when pressure was released. Leaf length/breadth ratios Artificially doubled plants tend to have a larger leaf length/ breadth ratios than their undoubled pro;enitors (29). Trifolium ambiguum was examined for this characteristic. Ei;ht plants from each source were sampled in the field and eleven plants from each source were sampled in the greenhouse. In either case seaple size has based on the source hith the smallest number of plants. Length and width measurements were made on each leaflet of the largest eaf of the plant. Generally the three leaflets from the largest leai appeared more fully develoged than those from snaller leaves. Length measure ents were made in millimeters along the midrib of each leaflet from the base of the leaflet to its aoex: width measurements were made in millimeters at the widest portion of the leaflet. To obtain the length/breacth ratio for the leaf, the sum of the lengths of the three leaflets was divided by the sum of the widths of the three leaflets. The ratios so obtained were used in calculating the mean length/breadth ratio for source and ploidy level in the field and greenhouse respectively. Since some plants in the fielv had oeen ve statively propagated 18 from duplicate plants in the greenhouse, correlation coefficients were calculated to determine environmental effects on length/breadth ratios. Leaf length x breadth data One of the characteristics often found in artificially induced polyploids is larger leaves (23). This characteristic was studied in T. ambigpum by using the same data conpiled for eacn leaf in the deter— mination of length/breadth ratios. In this study, however, the sum of the length of the three leaflets was multiplied, rather than divided, by the sum of the widths of the three leaflets. The lenéth-breadth product so obtained indicates the relative size of the leaf. Average size was then calculated for each source and ploidy level in both the greenhouse and field. Stomata length and number In artificial polyploids the size of stonata increase and the number of stomata per unit area decrease as the ploidy level increases (23). To determine if such a trend existed in T. ambipuum, stonatal measurements and counts were made on fourteen plants from each of the three ploidy levels. Seven of the plants from each ploidp level were selected from those in the field and seven from those in the greenhouse. In each place at least one plant from each source was included. one of the largest leaflets was removed from each plant and transferred to ice water until used. Since it has been reported that the numb r and size of stomata vary according to the portion of the eaf studied, all studies were made at one region of the leaflet (6). The area examined was the lower epidermis at a point adjacent to the mid-rib and approximately 19 half-way up the long-axis of the leaflet. lne leaflet was taped to a glass Slide b; lashing tape in 'which a diamond shaped hole approximately 3/15" long had been cut. The masking tape was placed so as to position the hole over the area to be studied. A drOp of water placed under the leaflet prevented immediate wilting and emphasized the outline of the stomata. By placin; a drOp of immersion oil directly on the leaflet and using a 15 x 50 (oil immersion) magnification, the stomata could be accurately measured on a micrometer scale. Measurements were made in divisions. At this magni- fication each division was equal to 2 microns. The avera e stomata lentth was determined from the measurements of ten stomata selected at random from those in the aforementioned area. Under the conditions of this experiment the stomata were closed when measured. Th0 repl'cations of ten stom ta each were measured for each leaflet removed from plants in the greenhouse. The small (0-1.2) microns deviation between r plication averages indicated that one replication was adeeuate. These averages were then used in calculating the ploidy avers e for the field and preenhouse respectively. The average numoer of stomata per unit area of each leaflet was determined by counting the numoer of stomata in the field of view at 750x using an American Optical AT hicrostar. The numbers found at two locations was averaged. From these averabes the averawe number of stomata per unit area was calculated for each ploidj level in the field and greenhouse, respectively. In addition, an overall mean for number of stomata per unit area was determined by combining the data for field and greenhouse. A spot check on the stomata of the ujjer epidermis indicated that although these were 5 aller and acre numerous, the same trend of size and nuroer occurred between ploidy levels. Althou,h the sa ple size in this investigation has suite 3 all, the ranges of the results indicated that further salgliné would have been of little value. Floral initiation in the greenhouse Difficulties in flower induction were reported between septemoer P1 and Karch in l. ambiguum under greenhouse conditions (l?) (13). In early Octooer 1959, plants in the greenhouse were subgected to a 17 hour day with a temperature range of ADO-50°F at night and 700-750E uurinb the day. Normal day lentth was extended by the use of 150 Matt reflector flood lamps spaced at A ft intervals over eacn bencn. The liphts were adjusted according to plant ;rowth so as to give maximum libht intensitg. Temperatures adjustments were automatically controlled by outdoor liJht conditions. nutrient solutions were applied at 10 day intervals. under these conditions flowering was initiated. The numoer of plants showing their first flower was noted bimonthly. This study was terminated on Key 15, 1959. Average number of heads per plant in the field Fewer flowers are reported on artificiallj‘induced polyploids ‘7‘ than in their diploid oarents ll3 . l. a bi uum was studied for tnis J. I. x_i characteristic. The number of flower heads on Several (6-9) slants from each source were counted at the seen of initial flower production 3 A a -‘ D ' “ ". — a J‘ ' ,H ‘1 f n ‘ ,‘ . , " ‘ 1‘.. ‘ll ‘:. ‘3’ _ '- -2 :V‘ .5. a ,L‘. : -"- ' in the ilelQ. LrOl tale tha the averate ndmOCi oi neiis per fling has calculated for each source and for each ploidy level. he allowances were made for rhizomes producing flowers since at the time of this study florierin w on rhizomes was insignificant. Time was not sufficient to note any differences in the length or intensity of the flowering period. because the nunoer 0; heads was correl;teo with the size and vigor of the plant, some oias prooaoly occurs in favor of those plants trln planted in June L 58 which at the time of the study were generally more viiorous than those trans pie WI ted in Auéust 1955. however, Some of ts may have flowered the sources represented only in the Au ust tr .nsplan aoout the same even if transplanted earlier. Average number of heads per plant in the greenhouse Flowering was first noted in the reenhouse on hov euoer 15, 1953. Subs eqLen tly, mature heads were removed and their numbers recorded at two week intervals until June 1, 1959. 3y the latter date only a few plants were still flowerinp and the study was terminated. Data were compiled only on the basis of flowers produced per plant irrespective of rhizome developitent or length and intensity of the flowering period. The mean number of heads per plant was calculated for eacn source and plaid; level. Some bias is introduced in that one of the tetraploids and four of the hexaploids initiated flowering on comparatively few (4-7) plants. AveraQe number of florets per head Artificially induced polyploids of clover reportedly have larger heads than diploiis (23). Conseouently, the number of florets per head would be expected to increase as the ploidy level increased. \ ~ «4 ~.' 1‘ ' «. 4‘9 .. r " d a"); DUI 1.:1; 111', 04.4. (Lila ‘ -\.7,»‘ . n ('H F‘ V.‘ — ‘n “Is ,‘ V‘ y «w i ‘- ; ', : . The nunoer c1 llorets per head was determln: \ counting the florets from fully matured heads. "he mean hunter of florets per head was calculated for each plant. This avera_e was then used in tabulating the average numoer of florets per head for 00th source and ploidy level. stunted or d&fldfied heads were not used as well as >-.-: those which had florets removed in the course of fertility studies. Tie numoer of heads examined per plant was also depencent on the intensity of the flowering period and the time of flower initiation relative to the termination of the study. At the conclusion of this stud; several sources were still inadequately sampled because of erratic flowering. Four hexaploid sources, for example, had 5 or less plants ea pled. Tine did not allow adeguate field samplin; in the spriné of '5‘. however, florets were counted on 6 field plants which, by virtue of vegetative cuttings, were also represented in the greenhouse. The numoer of florets per head in each case was within the range found for the greenhouse. Floret and pistil length in the field and in the greenhouse Induced tetraploids of clover have been reported to have larger floral parts than their undouoled progenitors (23). To examine the effect of polyploidy on the size of florets and length of pistils in T. anoipuum, samples for measureients were taken at the field and the greenhouse. At both places the nuxoer sampled in each source was dependent upon the number of plants flowering and/or the numoer of plants in that source. When possible at least eight plants per source were studied. Lehith measurements of floret were made on three fully opened flormms. Pistil length was also determined from these same florets. In the field the florets were _en°rally tine” from nilferett heads. however, time of scutlin; and by h ("L >.« ,__1 (u flowerinb in the greenhouse was erratic at 'enerally removed from the same head. necessity the three ilorets were 9 Before the pistil was extracted, each iloret was measured in millimeters from the top of th: receptacle to the tip of the standard. Variation in floret length was never more than one millimeter between and within heads from the seme plant. The plant averages for floret length were used in calculating mean floret lenith for source and for ploidy level in the greenhouse and field, respectively. In ad ltion an overall avers e was obtained by combining the plant averages of floret length from both the greenhouse and the field. In the field the mean floret lenbth for both source and ploioy level was calculated using data Obtained from each source. In the greenhouse the mean was determined only for those sources that had five or more plants flowerin; at the time of sampling. since only one of the seven hexaploid suurces in the treenhouse was adeguately sanpled, the 6x mean floret lenLth for plcidy level was not calculeted. The pistil was extracted from each floret and measured in millineters fromthe base of the ovary to the uppergost part of the style. he allowance was made for the amount of curvature in the style. Variation in :istil length was never more than 0.5 mm between and within heads from the same floret; generally, the deviation was in the range of 0.0 mm to 0.3 km. The number of plants sampled and the methods of calculation to determine the mean length of pistil coincide with those for length of floret measurements. Generally the pistil was either shorter than or about the same A} r- length of the lon est stauen. however, some instances were noted in which the pistil was longer than any of the stamens. The eifects on fertility, if any, were not noted. In the Iranian materials, the pistils were easier to extract than in the other sources. This indicated a weak vascular connection between the bistil and the recebtacle. If this connection is comparatively weak, the nourishment required for complete seed development may be laching. In addition, shattering would prouably occur more readily. ‘ Pollen diameter and staina"ility Artificially induced polfploids tend to huve larger pollen grains than their undouoled pro suitors (s) (23 . In addition a higher frequency of non-stainable pollen is regerted. hon-stainaule pollen grains are assumed to be non—viable. Pollen diameter and stainaoility was studied for the 2x, Ax, and 6x ploidy levels in Trifolium anhiLuum. Bangles were initially taaen from plants flowering in the greenhouse. ‘eecause of erratic flowering however several sources were not aceguately samgled. Therefore in order to make a more complete survey, a minimum of four sanples from each source were taken in the field. Enouwn plants were ex sled from each source so that a minimum of eight plants had been examined in the field and greenhouse combined. Each sample consisted of one flore q removed from a plant. The floret was then placed on a moist olotter in a labeled Petri dish and refrigerated until used. Unless refrigerated, germination of pollen grains occurred. terminated pollen grains were '.L not only more difficult to measure but were clso di-‘icult to sistinwuish V A) \h from the no n-sta ned pollen Lrains When the ,erninated ¢rain did not re sin intact rlOF' ts Iron plants in the greenhouse were renoved then fully opyned; in the tielo, however, it was necessar; to use florets at a state of envelojmsnt just prior to openin; because of the prcolem of pollen eatin inseCLs and hes visitations. ,. _ L _ v 4 '1:,.‘ . .. . . o a ‘ ., o n ,. o v r. 7 “-7 slides were pre_arel ior collen cianoter moaburerfiloa and wullen >,etl oac;;‘to V \ A were str i (11 s tainaoilitv counts as ollows. Eloret part eiipo ethe stanens. If dehiscence had not occurred, the anthers were cut ooen. In eitn r case the anther‘ were brushed on a clean ;lass slide A (D in an area approximating the of a 22r n.m scuare cover slip. lnis area was then ringed cy a oand of aceto carmine stain in order to prevent hollow pollen grains from floatin; off when the COVer slip was dro; ed in place. Ien well-iilleo yollen grains (s) were randomly selected from different poroions of the slide and their die eters measured on a mlCLO~ 'goi. The roasure;erts were recordei as divisions with each division e tiValent to 2.5 ticrcns at the aiorelentioned ma niLic tion. An a"era;e was ootained for tns ten counts ircm eacn floret. These alanc aVora es were then used to calculate the average :loidy group in the field and greenhouse, T83j€CElVBlJ. In the field the Lean goll n diaaeoe: for )oth source and gloic, level was calculated rein, data oit: ine ire; each source. n the t_1'~eo:_'ncuioe the mean pollen di, eter was det O b. (\1 (\J l ti \4 4-56 1-24 “13 O \1 ) \A/ }._J O \C) H \I‘c \1 O\ o \O r— o 0 Lu ,.— l [s \ J.‘ O o \I . Average number 0" The averag same between sources Floret len;th in the field and in the Tables 15 and 1 r‘\ 40 ‘ a \J O I',’ . ». K ,— ,. ‘. J. L Nil—(‘4‘: C1; +vA: In the A (4’ ufper 1i its of floter of tne ‘fi .3 the 1.1 e . 1": I) C) v. hous L) .1 .‘-rs 7-0L , :df‘ehb ,1 16 indicate that: field average floret (w pIOidy level increase . ,.‘ us ,‘ ‘v- -\ L ‘- 'l ‘.\ 4 . aud [we flOISL; lJilLLuL l althou;h the 6x florets ten At the AK and 6x 1 th evels, 3 p1311. such trsnd we r‘ a L.‘ A \A 2) v greeuction increased. lorets per head tendenca r head may haVe been I . O 2'. are a;:?0kl¢at€ly ange remained approximately :3 a,;arent in the :Jreen- 11’; 1,178 treenn "use is agngleatelg the of some sources touard 0 due to sanglinb greenhouse (Q U 'th tend to increase as L) V ever, in the ;r enho 3, .J L113 to be lonher. ran a L ’- l'VCl‘; s -e‘ in a source ‘w ' ’l 'M“. 1A 6 - 'Lv ‘1 i 7 l‘sv H . I , - ‘-. ..!_.—.«-,.-‘ SUGM :lmllar lloret lehbtn lOI glauto 1n tn; ireehnluse and plants in the lielu. ment, however, 4x plants than when under field T" a " r- v- 1‘ 0.1. r3111; ‘aL\J i (D overlaigin; ‘ V Juld Eenerally not 0e an .3 U (U: ‘31 ! - ,.J. been that ‘ the r‘ 2' I .' ’xvu " '1’ ‘3‘. l.V-t-IJfL..C‘Ilb o d Gtérmlnlh¢ 1 H. - . _ .3. ,1 4. .‘ n _ , v ploi: leVel. “Onever, a :Luflu “it” Co.;e1atixel 1d“ “‘ «J— N 7 .—' ‘ ,N .\ — " - \u ‘1 .‘ '- ,.‘ —~ . v. ‘ 1 WI“; '; Ilurbufi hlhnt be «tPJI J easiect 01 DElLQ a ne-a)ltlo. Table 1A. avers; ’3 IlLBIEOE‘I‘ -- -1 '2 LA- , , t2 1, grenn in tle greenhouse, ,—. v" 01 01-... '- llUlEtb ,,‘, 1;? El lirter ly;e-5y. “ 31/ vx " I. .7 x‘ 1 ~ r. ‘m. .. r q , ,.' -\ :18? n sell .1. I On; hhl‘a clov-:1 3.1.. “to Ploidy to. of Ploidy Ploidy rloioy level source plants X SD mange X JD range 2x - CPI 226a 60 87 21.7 35—145 6x - CPI 99A9 PI 229625 PI 228370 PI 229624b CPI 6161 CPI 23A08 CPI 13115 CPI 23158 CPI 10803 1-4 (\J \A) \0 \J‘! Sh-lB? 52—127 92—172 A7-ll7 43—120 59-101 113-153 84-131 43-103 90 79 21.1 (\‘I (\4) o \J‘Y 35-lh5 42—172 AB—lh \; \Jo " ' - ... . I, .J ,- .. ; .. ,, 1. j -.... e 1). AVHF3LB leh;tn in ml-limete‘ 11*“ hula clo er T .~ -v «‘4 , / Ploidy ho. of Ploii' Ploidy Ploidy r' 1 J 4 level Source plants 1 JD hangs h o‘ in'e CPI 277 3 11.1 0.55 10.0-12.0:10.b 0.7; 9.7-12.0 I» CPI 9949 ll.h 3.53 10.5-12.0 P I _/ "fl H I\ J C! O\ A J \fl U4 12.011. 6.79 11-00-1503 "U H A) I \ g5.) \ 0 ‘Q Q U1 r“ I ~ (I) ”U H M \ ’- ’7 A) 1:“ OJ 0 C o \ If H oi..~ 1007—1144 5 O H1 H on 1-4 C“ F4 CI; H .7\ O \7 \ 0 O \I‘ U1 6x - 12... 5.3.5300 O ”U H K.) ‘E‘- C1. -U 00 ._l \L‘ C Q \1) 3 :q C") 'r H H I!) ’.._J F.) \_,"v A!) H U F.) O O C». I\ H (U 0 ‘ '7 l L.) p 0 IV C11 23153 8 12.5 0.77 12.0-14.3 11. 01-121. 0 5 O "C' H t4 L) i» (1‘ \o (O H \A ‘ C t O C i if) C D k.) \0 FJ (. \L m H K 0 .L‘ O O \r' (\J H h x O.» l L4 1: I b H k, D (\\ H O |,._.. k e h“ (,.' O l 1’ \r C l“' .1 r, 7“ :‘V ..-' '-. ""r "I TdOle LU. .1." 0.1. AU. '3". biUJCVl plants grOTL unge per plant). ‘1 (j ("J 51 w (W) A (D ,.J ‘J t " ‘ -.-' 1 7 ’3 V ..3. 1:) ‘C‘ 1' 1' 1 t“."- _;+ -" £111:— IA 111 llli—L.L.Ll.t‘~l VI L) O- .A.... .. CUO *reeuhsuse cuncltions, April 1999 (j florets x a "3 Plaidy KO. of Plvidy Ploidy Plaidy level Source plants X JD Range A 3D ran;e H A) . C) O o O \D 3...: F“ o \O I 1.1 I , . H 2X - CPI 2L64 9 l .q H O\ H K) b3 CFI 2/ E I C) "T‘ H O‘ rt :— H p,_: 1—4 . .Q W H [\ 3 J \O O\ (\ \ 1 \C H H \J’Y H 0 K») C; \O o {\3 I H \k‘ 0 PI 229624b 5 11.6 0.91 10.7—13.0 ;1.3 0.99 9.2-13.0 6x - CPI 6161 1 11.7 15.1 14.3-16.0 0 "L f—’ {\D \Q .1" (_ P, \I‘ CPI 23158 CPI 10333 PC 1 15.3 K] 0\ FC 3310 35 13.9 A. \n (I, Pistil length "5 The following inlormation was obtained from stuiies on leneths of pistil (feeles l7 and 18). l. Average pistil length was asgroximetely the same for - D - I“ a given source ooth in the field and greenhouse. This environgent. It should C“ O" ' l' . ‘ 7 '4—3 , “D- J inaiCetea liotle eilec I "| -" -.- '.' v r 1 r‘ I -/~ r. « o \ A 1 t ‘ 3" . - "z ‘ ‘ t 1 »’ r ~ ‘. I . 2. my plOlQJ leel lnCJBdSCL, arerawe gistll ldNLLh also teneeo to increase. f‘ m“ _‘ f“ T w o ‘ I ‘ 'q J. . r ".‘ .V P ‘ 1 . u .‘1 v,- u - ‘- "‘ -‘ ). lne loner llmltb oi Vhe ox dveldtcu oi pistil len_tn were transcended in manr instances 5; the 4x avere e". however, the lower linits of the LX and 6x averages were exceeded in only one instance by a 2x giant. This may be one of the more effect've criteria Ior nelping to esteollen ploidy level, at least at the 2X level. Pollen dinmeter r q 1 .7. (r ' rm; Lg (3 ad 5" ' "a g ', *‘ '\- " ' "hr 13' 1-“ 4 w _ 8 90.1.4 v1'1 cal/JV on.“ L40 lIlCI'v'iSC, do £J.LU1L.‘: o Vol .Lfl crea nowever, eespite tne S¢flli semple Size to (D Q. A *3 m U H (D on F.) \C go '3 Q A (J V O the same. relien oiemeter apparently won a net as an GlferlVS means it of indicating ploidy level except ior those planes Mhicn were at the ‘1 I u .- -‘ q - ”,.‘ ,«- / ‘ ‘ ~x upper liiits 01 the ox rence. Some tendency ior snaller lelcn in the Lreenneuse can oe I :‘ ,.‘ fl ‘ ‘V‘ 'f ‘* . ,w ‘r " ' '.'~m; - K c'\ ' V >- I '.,) »‘ L l' " \v“ i. noted (Ta”le 4o). however tula may hell have Oeen e iqMCulUn 0; mDHflD WW : Qvn' lav '7 I“ We‘AVl-V*qn‘ ‘év—LJ .{ o ."1.V.x_..c...~ e 1..L_ LU 1.1 .1..L..._L_L.'..:;‘u\3l;; O ‘ l Kara clover giants grgwn in the field, plant). Ploidy R0. of Ploiug Ploidy Ploidy v «H i . level 3 urce plants A ou flange A JD range 2x - 031 2264 8 A.O 0.14 3.7-4.1 03- CEI 277 4.3 0.33 A.l—5.0 A.l 0.29 fi.7-5.0 4x - CPI 5554 9 5.1 0.3l h.7—5.h C3 *1 H \O \1W 1-" \O O . b O \O O I \ w \‘x b O ‘T \r. O O\ 'U H m A \O O\ I\ \h CU \ft 0 kg 0 O I“ ‘ 0 . \J‘! O C]; I U1 0 OJ AOL‘-60l *‘C H A) I\ \f‘ O (x It 0 \7 \fl 0 b C I‘ \C \J‘I O (\T- I O\\ O H \n O I\ C O \ L \IU 503‘603 O .74 H (\J \_D «9‘ Q1 0.1 O» KIT 0 .Q C‘ o \ \A— CPI 10303 8 5.7 O.A5 5.3—6.3 O 0.1 C \ p U? r. \f‘ i N.) O (D Table 13. Average 1 n¢th in millimeters of pist' Kura clover plants firozn unier greennsuse ' (3 florets per plant). Ploidy Ho. of Floidy Ploidy floidy level Source plants X 5D nenge X 3D ran‘e 2x - CPI 226k 9 4.0 C CPI 2771 6 h.1 0.1L 4.0-4.4 A.O 0.11 A.O-4.4 AX - CPI 6334 1 4.5 CPI 99L9 l 5.1 PI 229625 9 5.4 0.40 L.9-é.0 PI 228370 7 5.5 0.34 5.0-6.0 \T" \u C) O \A W p 0 \IO Ox 0 6X - CPI 6161 l 6.0 8 5.;-6ol \ .3 KT! o 011 23408 CPI 19115 CPI 23158 Table 19. Average diameter in microns of pollen grains frOn Aura clover plants grown in the fiela, Lay 1&59 (10 measurements per plant). Pl°idy “30° 01‘ Ploidy Ploidy Ploidy level source Plents X 3D Range X SD range 2x - CPI 2264 9 32.2 2.33 30.0-36.0 \ t +4 0 4}. CPI 2771 8 30.7 1.A9 23.8-33.1 1 1.39 31.A-35.3 l C kw H 0\ CK} {XL :- CZ L m K» o 32.9 1.22 31-7-3ho3 3104-330]. "U H I\) h ‘ u) \o -Q Q P P P- \7 \JQ (\J O (\J C/ O \7 Jr— 3A.1 2.57 31.9-37.A 32.9 1.94 25.3-37.4 0 w: H A) \ A) 4:" C) 0.» .J.‘ be Px‘ o ('1 ”J H F" L.‘ A 1“ r...» \4h 0.1 K) C) o P b \p O \n K.) K C, O U I \J O O .Q CPI 2315 8 3a.l 1.2) 3;.l-,o. CPI lCQD, 3 3o.o 2.23 ;3.o—;9.7 '13 O O) K o \l o O \13 o I\, 1 ,1“ \p I l o p l i] o -\'1 -1 —\ . I‘\ ' I", ;‘\’ . \J-LPA'. . 7 r11 0 K») \0 F; O \O P KL) I...) 0 :2. ..—J O m fl \ n (.1 O O I \x [\‘I 0 \FA‘; 5 P. ?\ O C' H C‘\ N r’} 1 - ,~.\‘ \ ,. -.,- ‘A' . . -4. ' 1“ ' m ~ w '7' ‘ ’1‘ ‘_. ‘ " I" I“ I ‘ ' 18.0.1.8 Aw}. {1v gifiaE'd CLCI.--;eLzer in “110 .011.) O- 904'. 311 LTC._LILQ .L lighu [Kurcl 1 r‘ ’- 1 '-t. ‘ : '1 ' ' I ‘. R r- V 'r‘fi- ‘f f‘ *3 ‘ _-‘ r~ ' ~ ‘ P " .I ;’- clover plints C..rot~.; uncer yreennonse conoitlons, nag 1997 (1 measurejents per plant). Ploidy ho. of Ploioy Ploioy . Ploidy level Source plants A 3D Range A 5D ranLe 4 ~ GQL)_/-UQO C o \J\ KO 2x - CPI 2264 6 2?.5 .v-s ZrZIO-jlorz 29.3 19K); A.’/.O-,;...7 O "U H f‘ \ ’\. P Q K)‘ C o O I—’ . K1 I‘d CPI 9949 1 29.5 ’T‘ H 1‘ V b) ~11 E: \ Kl} O \J) b O 4: 5...: H \J’. \fi r.) o p \ I Kt ‘ 1‘ O (7\ }1 221370 3 32.4 3.19 49.v—37.4 .‘ ,‘\ x .’ a, - v r I“ k‘ - A r r: h ’41,.“ 3 r~~ 5 , - ..\ ’1' I: "I ZQL/OLLLD 0 221.9 ‘1.)9 RO.’_/:).) JK..I+ 4.1—4.5 QID./-/’J.3 6x - CPI 6161 1 34.8 I 1‘ O W r (K \L. C 0.1 (\3 o \ 0 K23 IVY O\ o {J I K .1. h o \ CPI 23 CPI 13115 CPI 23158 (S \L‘ \1) H‘ ) \O Ko 0 \ K .3 4:“. O O\ A O (x L) K»- C: o C I K ( 0 ”v 1‘ (I ‘ K I \u‘; C The CPl-EBLOB pentaploid slants have average pollen diaieters within the range of the CPI-ZjLOB hexaploid plants. Pollen diameter apparently is not radically different b tween these two ploidy levels. Pollen stainaoility As shown in Table 21, a tendency exists for increased pollen non-stainaoility as ploidy level increases. This is indicated by the upper limits of the ploidy ranges. however, pollen infertility (as measured by non—stainaole pollen) does not necessarily become greater with an increase in ploidj level. For example, two of the hexaploid sources and one of the tetraploid ssurces had less non- stainaole pollen on the average than either of the diploid sources. Examination of the aforementioned table snows extreme variaoility in the ranges of most sources. rollen stainaoilit, is apparently more a function of the genetics of an individual plan than of ssurce or ploidy level. Pollen non-stainaoility of the CPI-23AOS-Sx plant is well vdthin the range of the CPI-ZBAOa-ox plants. Thus, pollen stain- ability is not an adequate method to distinguish 5X types in this Source. Fertility studies The following results are shown in Taoles 22-25 and Appendix 1. Seed set tends to decrease as the ploidy level increases (Table 22) o 2. ho self fertility was Observed in hand selfed plants Table 21. LA Averaie percent of non-stained pollen from florets of hura clover plants groan in the greenhouse and field respectively, Lay 1959 (Average of 2 counts of 100 each per plant). Ploidy N0. of Ploidy Ploidy Ploidy level Source plants X SD Range X SD range 2x - CPI 226A 11 6.9 4.02 2.5-13.5 CPI 2771 15 5.3 7.48 0—23.5 6.1 6.21 0-23.5 AX - CPI 6884 9 1.2 0.83 0.5—3.0 CPI 09A9 8 27.1 14.04 A.O-49.0 PI 229625 8 9.5 5.89 1.0-20.5 PI 228370 10 6.8 8.83 0.5-27.5 PI 22962Ab 9 17.2 1A.49 4.0-27.0 11.2 13.34 0.9-A9.0 5x - CPI 23A08 A 17.9 11.87 6.5-34.5 6x - CPI 6161 12 A.7 A.h7 1.0-15.0 CPI 23108 8 25.4 19.6? 10.0-68.5 CPI 18115 8 18.1 18.52 6.5-61.0 CPI 23158 8 22.0 16.6A 3.0-51.0 CPI 10803 8 2.6 2.81 0-?.5 PC 8 15.9 11.18 3.5-36.0 PO 33109 30 8.2 7.04 1.0-35.0 13.; 13.34 0—68.5 Table 22. within and between ploidy levels. Average seed yield per floret from crosses of T. a bi_tum No. of No. of no. of Seeds/ Type of Cross plants florets seeds florets 2x selfed 15 1020 O 0 2x K 2x multiplant intersource CPI 2264 15 3279 3318 1.01 011 2771 11 2158 2339 1.08 Combined 26 5437 5657 1.04 2x X 2x multiplant intrasource CPI 2264 17 1418 635 0.45 CPI 2771 8 1061 762 0.72 2x,X 4x reciprocal 14 3794 20 0.005 2X.X 6x reciprocal 20 3049 55 0.02 4x selfed 10 617 0 0 4x X 4x multiplant intersource CPI 6884 1 173 134 0.77 PI 229625 2 566 568 1.00 PI 228370 6 1957 1715 0.88 PI 2296211 1 405 36 5 0.90 Combined 10 3101 2782 0.90 4x X 6X reciprocal 14 '1620 458 0.28 Approx. ' 5x X 6x reciprocal 2 50 19 0.38 6x selfed 16 935 0 . 0 6x X 6x multiplant intersource FC 33109 7 314 150 0.48 CPI 23408 1 60 27 0.45 4. 5. 65 a 5 v regardless of ploidy level (Table 22). In addition, no seeds were found in non—pollinated heads examined . during other phases of this study. Lewis (22) has shown that induced autotetraploiay often resulted in the reestablishment of self compati”i1ity at the 4x level. IT. ambipuum, however, showed no self compat- ibility at the 2x, 4x, 5x, and 6x ploidy level. Intraploidy crosses involving two plants either from the same or different sources, generally had one of three levels of fertility. These levers were: (Tables 23-24 and Appendix B) 1. High fertility in ooth parents, 2. High fertility in one parent out low fertility in the other, 3. Low fertility in both parents. 2X 1 4x and 2x X 6x crosses, generally had very low fer- tility Table 22 and Afpendix 8, Tables 41 and 42). however in both cases, one cross was noted in which one parent set a number of apparently viable seeds. 4x X 6x crosses gave an appreciable seed set in the majority of crosses (Table 22 and Appendix 8, Table 46). host crosses indicated relatively high or low fertility in both parents, out one cross had very high fertility in one parent and none in the other. One cross was made between a 5n and a 6n plant (Table 1 22). 19 apparently viable seeds were set on the 5n Table 22--Continued 40. of ho. of no. of deeds/ Type of cross plants florets seeds florets CPI 23158 226 l 89 31 0.35 PC 411 1 55 35 0.64 Combined 10 518 243 0.47 6x X 6x multiplant intrasource FC 33109 9 1558 1045 0.67 +-"1'4 .'. . ' . blllvy‘ k..“.t.1 Jr? 71, 1-, ‘4. \ &ULUQ JU’4U)0 c1.._‘111_ - n L ‘Jarent {1:}. riant 7. . ‘1 1211.8 parent jource llant NO. ho. of florets .‘ , ,- 4 _. Je'j‘aL-J flouots I ‘ - e’ r r: ‘ Cfl £4,314, (.3; 7:31 II II A 1 hultiplant intersource multiplant intersource 011 2264 apt in: u; .L (MR-C- multip an; intersource I1111;;i;;1;;31t intrassurce LII I . .- '11 .. r 1 11.1.1.1. Lleplduqb intrasource ('1 ‘T i , "- :. , -1 nultiplaht intrascurce ~ - 7,. e 5; '01 .1 £2,011 n‘.“ ’7: fl: I I 22.31; r“; I‘ {4+7 r“ f r‘ I /H7 (‘7 ‘3. ‘ I r“; . ; Cu 11 7 C7 ‘- .- I / 4L+O O». \}I ,,1 ‘..| {H H \L' Cl‘ \Jk I‘d \J‘ U K; \D (‘\_‘. x)‘ u} 114.; \l (I) \f, I" :47? V- .I f. \-‘ .5.” Table 23--Continued c a Female parent hale parent 71% Source Plant Source Plant Tyfi No. of No. of Seeds/ no. no. ,EZI florets seeds florets 1: CPI 2264 749 X CPI 2264 773 H 26 o o " " X CPI 2264 781 h 18 C 0 CPI 2264 786 X multiplant 8 50 21 0.42 intrasource ” " X CPI 2264 764 h 50 0 0 " ” A CPI 2264 749 H 43 0 0 CPI 2264 747 X multiplant B 196 2 0.01 intrasource " " X CPI 2264 93 H 25 0 0 " " X CPI 2264 760 H 22 0 0 CPI 2264 761 X multiplant 8 60 47 0.78 intrasource " " X CPI 2264 749 H 47 0 0 CPI 2264 {60 X multiplant 5 58 22 0.38 intersource " " X CPI 2264 747 u 23 0 0 CPI 2771 813 X multiplant 8 193 193 1.00 intersource ’ ’ " " X multiplant 3 108 65 0.60 intrasource CPI 2771 807 K multiplant 8 234 273 1.17 intersource " " X CPI 2771 817 H 82 0 0 CPI 2771 799 X multiplant o 134 141 1.05 intersource " " X multiplant 8 82 49 0.60 intrasource " 2 X CPI 2771 794 h 24 0 0 CPI 2771 811 X multiplant 8 176 158 0.90 intersource Table 23-—Continued c — 3 Female parent hale parent ‘8 a 4 . - ~ 8 .. .. bource Plant source Plant '3 5 no. of (—1 no. no. iSCj florets m o .2 Q. CPI 2771 811 X multiplant 3 122 intrasource CPI 277 810 X multiplant o 43 intrasource CPI 2771 810 X CPI 2771 798 H 48 P .) Lv 8* - pollination by honey bees h** — pollination by nand Table 24. Fertility data from 4x X 4x crosses of T. ambiéuum (See ‘ppendin 8, Table 45). o - 3 Female parent hale parent ‘8.8 a Source Plan Source Plant '8.S ho. of ho. of deeds/ ,cra _ no. no. -$r§* florets seeds florets CPI 228370 662 X CPI 228370 680 8* 18 4 0.22 n n X n 599 H 1,9 20 0.41 n n X H (328 H 3 5 38 1.09 CPI 298370 628 X " 662 H 40 25 0.63 n u ' u ’ P so - , w k 0A5 1’1 44 l) 0.0% H* — pollination by hand (‘7 1 Table 25. Fertility dd a from 6x X 6x crosses of T. awoiguum (see Apfendix.5, Tables L7-50). c «+94 Female parent hale parent Oi; Source Plant Source Plant :Efij No. of ho. of Seeds/ no. no. arg‘ florets seeds florets CPI 6161 7 X PC 411 h* 28 27 0.96 " 7 X CFI 23608 335 H 22 7 0.32 " 7 X FC 33109 851 H 32 26 0.81 PO 33109 897 X multiplant B** 51 31 0.61 intersource " " X EC 33109 962 H 59 15 0.25 PO 33109 851 X CPI 6161 7 H 27 23 0.85 " " X PO 33109 993 h 36 21 0.58 PC 411 X multiplant 3 55 35 0.64 intrasource " " X CFI 6161 7 H 29 12 0.41 PO 33109 860 X EC 33109. 982 H 32 11 0.34 " " X PO 33109 869 s 179 A7 0.26 H* - pollination by hand 3** - pollination by bees O) O The l plant. Lone were set on the 6n plant. approximately 50 florets were involved in each plant but the exact number was lost. ‘The 2x multiplaht intersource cross produced a Higher average seed set per floret than either of the 2x multiplant intraseurce crosses (Table 22 and Appendix 3, Table 36, 37, and 33). This was true not only for tne crosses as a WflOle but also (or those individual plants represented in both the intersource and intra- source crosses. The 6x multiplant intersource cross produced a lower average seed set per floret than the intrasource cross (-able 22 and Appendix 3, Table 47 and 46). Seed size following results were noted: Seed weight increased signiiicantly as the ploidy level became hipher (Table 26). Screens which reloved the Lost tetraploid seed also removed some diploid and hexaploid seed (see Tawle 27). For example, although 613 of the Ax seed was retained on the 1/16-1/17 screens, these screens also retained 135 of the 2x .nd 10; of the ox seed. Therejore, separation of mixtures of 2x, Ax, and 6x seeds on tnis basis would be impractical. A l/lu hand screen removed 40; of the 6x seed but none of Table 26. Ana ysis of variance for avers e wt. in grams per lud . q .— _ .‘_ r‘ . x.” . A.“ _- __ ._ ' _‘ -I‘ {.5 ' ”A“ 4 1'. SBBGS Ol 49., 4.x, 8.113 GA 131010.; .LUVC-Zla 'vJ. Lara (,.LuVJ‘I‘ I‘iogCCthelJ. Source df SS 1 S F iotal 14 .0630 Reps. A .0001 .000C3 0.5 \ N Ploidy . 06-24 . 03120 5.40. we Error 8 .0005 .00006 A): - . 1761 6x - .2936 Standard deviation of ploidy average, .0035 Ens. Duncan's multiple range test indicates 2x 4x, Ax 6x, and 2x 6X at 15 level. 7i. Table 27. Percent of hura clover seed passing through hand screens. Screens 2X 4X 6X 1/12 100 110 99.7 1/13 100 100 93.0 1/14 100 100 60.3 1/15 100 99.1 26.0 1/16 98.5 71.3 5.3 1/17 87.1 38.9 1.3 1/13 56.9 9.8 0.3 1/19 L3.8 6.1 0.05 1/20 19.4 0.9 0.04 1/21 0.1 0 0 the 2x and Ax Seed. A 1/15 screen TJhOVGd 7a” of the 6x seed, 1g of the 4x, and none of the 2x seed. Inerefcre, hexaploid seed night be effeCLively renoved from hixtures of 2x, Ax, and 6x seeds 0, screening. The size of seeds fro» interploidy crosses, however, would de end on the ) '71 ‘ -‘ .1 4" 4“ ‘ .1..L :12. U ‘- ‘3 A.“ _. l o: the lenale uaICubo Thus, a seed CEVleJed '1 ' 3-“ l .10le 19V: U L (L on the 6x parent of a Ax X 6X cross would approniiate ox seed size. Since these studies were carried out on ;green- house materials extrapolations to field harvested seed may be unwarranted. Vigor rating The follOWing results were noted: (Tables 23 and 29) l. The range in vigor ratin;s is about the same over all ploidy level but there is an increase in average vi;or as ploidy level increases. 2. CPI 10303, a hexaploid source, contained a larie propor- tion of plants receiving a high vipor rating. The diploid source, CPI-2771, has rated second in vi or \A) . nhile the other diploid, CfI-226h, had the lovest ratinL. This indicated thrt vi;or is prooaoly pore stronply correlated with source than vith plaid; level. dhiZOme development Average rhizome production increased at the higher ploidy levels (see Table 30). This tendency was not found in all sources. For ex- ample, one hexaploid and two tetraploid sources produced fever rhizomes *0 0\ Table 26. Average of 0-10 ratings for vigor of hura clover plants grown in the field, Lav 1959 (0 = dead, 10 = most viiorous). U .— Ploidy Ave. n0. of Ploidy iloidy level Source hating Plants hangs X range 2x - CPI 2264 6.3 30 l-7 CEI 2771 7.6 9 1-10 5.0 1—10 4x - CPI 0684 h.8 27 1-9 Crl 9949 6.7 15 3-9 11 229625 5.5 lo 1-8 PI 225570 5.1 7 1-7 PI 229624b 5.6 16 1-8 5.5 1-9 6x - CPI 6161 6.9 13 4—10 LEI 23108 7.1 73 1-10 611 13115 6.9 32 2-9 CrI 23155 6.0 32 1—9 CPI 10803 8.5 12 7—10 PC 7.1 76 1-10 EC 33109 6.4 55 1-10 6.9 1-10 77 average of 1—10 ratings for vigor oi hura clover plants . 'I -' -. ' 7' - 1’ .- I / ‘g — .— \ l, ‘ . r‘~ -\ .' v ‘ x . n the lielo “av 1733 l — least vi ozous lo - most ’ a C , Ave. 10. of Ploidy lloidy Source hating Plants Range X range 011 2264 3.0 47 0-7 CPI 2771 7.6 9 1-10 3.3 0-10 CPI 6886 3.1 44 0-9 031 h; 9 5.6 13 0—9 rI 229625 5.4 17 0-8 PI 2‘5370 1.1 10 0—7 PI 22962hb 4.7 18 0-8 6.2 0—9 CPI 6161 5.6 16 0-10 CrI 23603 6.9 81 0-10 CPI 18115 5.5 39 0-9 CPI 23158 6.9 39 0-9 CPI 10803 8.5 12 7-10 PC 6.6 87 0-10 PO 33109 5.8 62 0-10 6.2 0-10 78 Table 30' Average number of rhizomes produced by hura clover plants EFOWH in the field, Lay 1959. Ploidy no. of Ploidy Ploidy level Source X plants Range X rah;e 2x - CPI 2264 0.36 30 0-4 CPI 2771 1.44 9 0—5 0.6 0—5 4x - CPI 6884 1.52 27 0—10 CPI 9949 1.85 15 0-8 PI 229625 0.63 16 0-3 PI 228370 0.29 7 0-2 PI 229624b 1.81 16 0-11 1.5 0—11 6x - CPI 6161 3.46 13 0-10 CPI 23408 3.01 78 0-15 CPI 18115 2.34 32 0-12 CPI 23158 2.97 32 0-25 CPI 10803 7.91 12 0-24 PC 2.34 78 0—12 PO 33109 1.39 55 0-15 2.7 0-25 on the average than did one of the diploid sources. as the ploidy level increased, there was a tendency for a higher level of rhizome production by some plants; yet some plants of each ploidy level produced no rhizomes. Rhizome production is apparently a function of the genetics of a given plant under a certain environment as well as a function of source and ploidy level. It was noted that rhizome development was not necessarily correlated with the overall vigor of the plant. For example, some plants which produced several rhizomes had soarse vegetation while others with several rhizomes had dense veretation. Growth habit Despite the small sample size in some instances, each source had a wide range of ratings with the exception of two of the tetra- ploids, PI 228370 and CPI 9949 (see Taole 31). This indicates that growth habit is apparently more a funCLion of the genetics of an individual plant rather than a function of source or ploidy level. However, definite differences do occur between source averates. This preponderance of one or another type night indicate better surviVal of that tvoe under the environment in which the source arose. II Winter hardiness All plants transplanted from the 5rsenhouse to the field nursery prior to August survived the unusually severe winter of 1958— 59. 0f the later transplants, 88; survived (Table 32). all but the three Iranian tetraploid sources were represented in both trans— plantings; the Iranian materials were only among the second transplants. Table 31. Average of 1-5 rating for degree of erectness of hura clover plants grown in the field, hay 1739 (l = prostrate, 5 = erect). Ploidy ho. of Ploidy Ploidy level Source K plants mange K ran;e 2x - CPI 2264 3.22, 27 1-5 CPI 2771 2.89 3 1-4 3.14 1—5 Ax - CPI 633A A.SO 2h 1-5 CPI 9949 b.08 12 3-5 PI 229625 3.14 14 1—5 rI 228370 3.71 7 3-4 PI 22962Lb 2.69 13 1-5 3.74 1-5 6x — CEI 6161 1.80 . 10 1—4 CPI 23h08 2.85 77 1—5 CPI 13115 3.93 29 1-5 CPI 23158 3.57 12 105 CPI 10803 3.25 23 1-5 PC 3.19 51 1—5 FC 33109 2.h9 67 1-5 3.05 1-5 1 Table 32. Survival of plants of aura clover during the winter of 1953-59. No. of ho. of _ Ploidy plants plants A Minter level Source Oct.1958 May 1959 survival 2x - CPI 226A 39 28 72 C31 2771 9 9 100 AX - CPI 638A 21 17 81 CPI 99h9 10 S 80 PI 229625 13 19 83 PI 223370 10 7 70 PI 22962Ab 20 16 80 6x - CPI 6161 9 8 89 CPI 23AC8 55 53 96 CPI 18115 31 28 9O CPI 23158 13 8 62 CPI 10803 2 2 100 PC 79 78 99 FC_33109 38 35 82 82 Under the conditions at hast Lansing, there was no apparent corre- lation between ploidy level and winter hardiness. For eXanple, the three sources suffering the host Minter loss included a diploid, tetraploid, and hexaploid source. The results did indicate, however, that some sources had less winter hardiness than others. This would be expected if the sources had arisen under varying environments or had been probagated for several years under less extreme conditions than in their place of origin. Source ratinas As a :roup the hexaploid sources are rated highest in ooth the field and the greenhouse with the exception of CPI 23408 which is rated lower in the greenhouse (see Tables 33, 34 and 35). matings in the field and greenhouse both rank the Iranian tetraploids and the diploid, CPI 226A, low. The direct use of these last—mentioned sources is questionable. The field ratings were the most useful from a practical VIEWpOint but the general a5 eement between ratings indi- cates that greenhouse data are important (Figures 11-18). Diseases and insects In the greenhouse two abnormal situations involving flowering were noted. In one, certain florets developed a condition in whicn the anthers turned black. Usually only one or two florets per head 16‘ 9—... were infected but occasionally a few more showed this condition. T pistil was apparently unafjected. Overall seed set would not be lowered appreciably unless the condition became more intense. cc \o Table 33. Rankinys for various Characteristics of sources of hura clover brown under field conxitions. Ave.9;_ Ave. o Ave. Ave. Ave. pollen Ploidy heads ;e floret pistil pollen stain- level source plar length length size ability 2x — LII 2264 8 l 1 2.5 9 CPI 2771 4 2 2 l 11 4X - CPI 6834 11 5 4 8 14 CPI 9949 10 4 3 A 1 PI 229625 3 7.5 6.5 6.5 7 PI 228,70 1 3 6.5 5 10 l I 229624b 2 6 5 c3. 5 5 6X C11 61r1 9 ll 10 l2 l2 CPI 23408 12 13 10 2.5 2 CPI 18115 7 10 13., 9 4 CPI 23153 5 7.5 8 10.5 3 CPI 10303 14 9 10 l3 13 PC 6 14 l2 l4 6 PC 33109 13 12 13.5 10.5 8 gable 33—-Continu:d Ave. Ave. Ave. length vigor vigor Ave. Ave. x rating rating no. of erectness breadth 0—10 1—10 rhiZOmeS rating Total 3 1 1 2 8 36.5 5 13 13 4 5 00- 7 2 2 o 14 75.0 6 8.5 8 3 13 65.5 1+ C 5 3 0 31.1.5 1 3 3 1 11 44.5 8 6.5 9.5 l, 13 2 11.5 12 9 7 9.5 9.5 10 5 6 11 11 14 14 14 12 11 11.5 9.5 14 10 7 5 1 94.o 4 92.0 12 79.5 10 76.0 9 121. 7 103. 2 95.0 Table 34. Rankinss for various characteristics of sources of Kura g, clover grown under greenhouse conditions. : : :5ve.no.: : : Ave. % : ave. : : . Ave. no.:florets: Ive. : Ave. : pollen : length : Ploidy : :heads per: for :pistil : pollen: stain- : .x : level : Source : plant : head :length : size : ability: breadth: Total 2x - CPI 2264 14 6.5 1 2.5 9 2 35.0 CPI 2771 13 10 2 1 ll 6 43.0 CPI 992.9 2 14 3 4 l 9 32.0 PI 22x625 4 4.5 6.5 6.5 7 l 49.; ”I H A) (s. 1 \O O\ I\ p U H 10 p o \b \J" o O O\ o \J‘7 \D 1‘- K. \ \l o 0 6X - CPI 6161 3.5 13 10.0 12 12 5 60.5 CPl 23405 3 12 10.0 2.5 2 12 41.5 Table 35. Overall ranking of sources of hura clover based on totals ire" Table 33 and Table 3A. Field Greenhouse Ploidy Total Flo i dy Total source level points Source lev vel points CPI 10803 6x 121 EC 33109 6x 03.5 PC 6X 103 CPI 6161 ox 00.5 FC 33109 6X 95 CPI 10803 0X 00.0 CPI 6161 6x 94 PC ox 53.0 CPI 23403 6x 92 CPI 18115 6x 47.5 CPI 18115 6X 79.5 CPI 23158 6x A6.0 “I 15“ OX 70 O C‘rI 2771 23c 43.0 CPI 63’1 x 73.0 CPI 0004 hi A2 CHI 9949 AK 05.5 CPI 23403 ox 41.5 CPI 2771 2x 60 0 II 4~9olto 4X 57.0 PI 229625 in: 54., CHI 226:2. a 35.0 PI 228,70 4X #4 5 PI 223370 4k 33.5 PI 229624b #1 A4 5 CPI 9v49 4A 35.0 CII 226A 2x 3o.5 PI 229625 #x 29.5 87 Fig. 11. 2x plants of Kara. Fig.. 12.. 2x plant of Kara clover during winter of clover on right. '58-'59. 4x plants can 6x plants on left.. be seen on extreme right. Winter '58-'59. Bee cage used in this study is in background. --'-.". :‘:.o ‘4. . ..l£.I‘ Fig. 15.. 6: plant of‘!; Fig. 14.. Vigorous 2n plant ambiggums Winter of Kura clover grown '58-'59 in the field. .June, 1959 {'V Fig. 'F'.. H?" 17. 3 we on- Vigorous 4n plan ’. Same 6n plant as in Fige 16o t Fige of Kura clover grown in the field. June, 1959 18. Vigorous 6n plant of Knra clover grown in the field. June, 1959 Leaves from 2n, #1:, and 621 plants of . Knra clover grown in the field. June, 1959 The other aonornal condition resulted in the failure of an entire head or part of a head to develop and nature normally. The number of heads affected as well as the extent of the abnor ality on an individual head appeared to vary from plant to plant. In its most severe manifestation the number of florets lost to this malady would lower potential seed set aporeciaoly. THIS condition was not apparent under field environment. Under field and greenhouse conditions, most plants appeared to be free of vir‘s infection. However, two plants in the field apparently succumbed to virus while a few appeared to be infected to varying degrees. In the field, there was serious infestation of an insect wnich destroyed the upper part of the pistil. Although some plants appeared to be relatively unaffected, it Was difficult in others to find an 'undamaged floret. If this sane condition persisted prOportionately under normal field Operations, tnis factor would prooably be extrenely imywrtant in total seed set. DIJCLBSICN Ploidy levels Although cytological examination revealed only one ploidy level in most sources, two predominantly hexaploid sources contained a few tetraploids and pentaploids, respectively. In one source, CPI 6161, 4x plants were found while in the other, CPI 23h08 5X plants were revealed. In CPI 6161, a Ax X 6x cross might be eXpected, but no 5x plants were found in this source. In CPI 23A08, the 5x plants presumably arose from 4x I 6x crosses but no ax types were noted in this source. In addition, aneuploids would be expected in CPI 23L08 because of 5x X 6x crosses; no definite aneuploids were As previously suggested, irregular types mi:ht have existed in larger numbers and in other sources and night have been discarded because of low viior or slower germination. Another possibility is that these plants resulted from seed mixtures between sources of different ploidy levels. But from the information at hand, the most plausible explanation would seem to be that these irrepular typ s arouse from intersource crossings between different ploidy levels. Thus the LX types of CPI 6161 might arise from a cross betteen a 2x source and hexaploid CPI 6161 plants. The Ex type might have resulted '1 from a cross between a Ax source and hexaploid CPI 23408 plants. The \(j 0 fact that aneuploids were not found sugpests that these irreLular types were rl's. Even at relatively low freouencies, interploidy miXCures would have a marked depressing effect on fertility. Because of its strong asexual capacity, hura clover is not subject to selection solely on the basis of a strong sexual mechanism. This permits experimentations with ploidy in extending the area of adaptation of the species. However, no polyploids greater than 6x have been found in this study. In addition none of the lite‘ature reviewed mentions higher ploidy levels in I. ambippum. In other crops there is often maximum plant response tithin a certain ploidy range. The amount of response decreases according to how far a diven ploidy level is above or below this ptimum range. On this basis, even if the hexaploids of hura clover represent an Optimum ploidy level, at least an octaploid might be expected, but as noted above, none have yet been reported. The species mrr be presently under bin; critical evolutionary stages from which a more stable polyploid level may emerge. At these higher levels plants might show even greater vigor than the hexaploids. For several characteristics examined in this study, rances increased directly with ploidy level. This increased variability in polyploids might be an effect of the high degree of recombination possible in polyploids over a long period of time. Leaf length/breadth ratios In two hexaploid sources, CPI 10803 and PC, there were high CXDrrelation coefficients for length/breadth ratios in the field and the greenhouse. In addition, each had similar averapes anu Standard deviations in both places. Average ratios and standard deviations of the other hexaploid sources varied between field and greenhouse. n the field, plants of 011 10503 and PC showed higher variability than all but one of the other hexaploid sources. In the g‘eenhouse, however, the variability of these two hexaploid sources was about average for all iexaploids. The standard deviations of the length— breadth product indicated a similar pattern of variability. If subjected to stress in a given environment over long periods surviving genotypes may produce a similar phenotype. when tanen from this critical environment, genes whicn had been previously maSKed might then express themselves and new phenotypes would arise (3) (h). pressed in the new environment would vary between plants >< The change“ e and would depend on such factors as the effect of environmental Change renotype, the number of renes controlling a given k.) on the expression of trait, the number of chromosomes on snich these genes are located and the amount of recomoination between these genes. Organisms wnich have not been exposed to critical environments may lack the genetic plasticity necessary to produce different pheno- types under comparatively minor environmental Changes. 0n the other hand, such plasticity may be present to a hi,h degree in organisms from areas of environnental stress. Accordinp to this hypothesis CPI 10803 and PC mibht have been grown in a favorable environment for hexaploid T. amoiiuum while the other hexaploid sources niiht have cane from more critical areas. The difference in magnitude of standard deviations ootained from the same plants of a Jiven source grown in di: fetent environments are, at oest, indicative of phenothic plasticity. The elasticity maJ oe Lore or less directly related to envzron er al stresses of the area of adaptation. According to this hJoothesis, s_urce Crl 23153 would have arisen under the host critical environment. :34 Leaf lengt x breadth product Leaf size a ear 5 to es a function of source as Well a; ploidy level. Apcarentlv the environment under union a particular clone arose stronglJ inil uences size oi is M haturel selection under some environients has prooaolJ been instru enoal in reloval 01' ceitain of the large leaf thes expected in higher ploidJ levels. For example, plants with large leaf areas and, consequently, large transpiration 5,), surfaces would presunaoly be sele cte against unter drougnty conditions. Ki Pollen non~stainaoility sin“ non-s taina ole pollen it is assuned that the ;er- C) (J) In discu cent of non~stainaole pollen is directly related to the percent non- fertile pollen. Ii the above thothesi s is at least reasonaoly accurate, the low percer t non-st ainaoili t;' round in ianJ slants at the .L 2x, Ax, and 6x ploidg l vale create ale ndicates reéular Leiosis in these plants. A tenioncy t0ward more re: ular meiosis has baen reported in induced polJploids after a few generations (23 . ihis agpareht tendency toward natural selection of fertile types is trooaoly Caniic ited in T. anoiguum DJ its stron;LJ rhizonato us haoit MfilCh .ould favor vi orous \0 clones re any neictic difficulties. ’) CPI 10303, knich hypothetically ma, have come iron a non- critical en=ironment, had a very lox percenta e o: non-stainaole pollen. however, CEI ZElSB, Whicn hypothetically was adapted to a .4 critical environment, had a high percentage of non—stainaole pollen. ”1“ - ,.‘ a ‘- »_ v fir‘\~‘ ‘~ I . ‘- ’.' " luls ui;nu oe erectel i: natuzal as F. ection in igin;e areas of adap- tation was such that these perennial plants were selected more on their ability to survive as individual tlants rather than their aoility to function as a strongly sexual organism. ln a non-critical environment, ed V (T; on the other hand, the ability to produce large quantities of 5 mi ht be advantageous. The more recomoination types produced by an individual the greater are the Opportunities for types more adapted than their parents. The heavy flowerin; and strongly rhizoaatous haoit of CPI 10503 lends support to this hypothesis. Conversely, orl 23153 had fewer flowers on the averace then the other heaaploid sources, but average rhizome production in this source was aoout the same as that of the other hexaploid sources with the exception of Dbl 10303. However, all plants in the field vere included in the Study on nunOer of rhizomes. Thus while Cfl lJSOB was represented primarily in the vigorous first transplants, the other sources were more or less repre— sented in both the earlier and the less vigorous later transglants. In addition, the rayieity and extensiveness of rhizome devcloymcnt might vary according to environment. Therefore in its area of adaptation a plant might react entirely different than in the enviro TUUtS in which this study was carried out. 95 Conclusions drawn from the results of fertility studies are based on the assunption that pollinations were eguslly effective in all crosses. If this assumption is generally valid, definite incom- patibility mechanisms must exist in T, angisuum. Three levels of fertility were noted in the various crosses: 1. High fertility in both parents, 4. hi_h fertility in one parent, but low fertility in the other, 3. Low fertility in ooth parents. The hicotiana type of incompatibility mechanism might help explain the above phenomena. Tuis type of incompataoility has been reported in many Species of the Trifolium and does produce the effects noted above. Other incompatibility mechanisms would also be a plicable to the above phenomenI. The complexity of the mechanism would most probably increase with an increase in ploid; level. The matinés in this Stud; hcve shomn that fertility exists within and between all ploidy levels. Hagoerg (8) noted a marhed reduction in seed set when l—Zz diploid clover was grown with tetra- pleid clover. 45 diploid clover halved the seed yield of the tetra— ploid. If such a similar reaction occurred in interploidy hixtures in T..ambi;uum, it would be especially important to maintain isolation of the PlOidY EPOHPJ- If the 2X, AK, and ox ploidy levels are not iso- lated from one another, it is possible that 2x, 3x, Ax, tx, and ex seeds would be harvested in the same lot. The 3x and 5x would not breed true to ploidy level. The 4x plants produced from a 2x X ex mating would also fail to bread true. hertilitv v.uld be guise low in any one of the above three cases. duosecuent generations would have a hiiher deiree of aneuploiuy due to such natin,s as 3x A ax, 5x X bx, etc. T is again would decrease overall fertility. .‘ .,-_. W .. LL.— ‘ .. ‘--.~ “ ,-‘ ‘ r ._ .- v‘ :p ‘ w -. ._ ~. ‘1‘." _. 1 .l - _: H In sun aiy, curse problems apaeal 'J aiiect lBlbllluV in ‘-. as Dl;iuuh. 1. Different pl:id; levels, ’- . . o -‘ 4. Chron some aberrations, espeCially in the hither plaidy level. This includes chromosome aberrations arising from fragmentation of individual chromosomes as hell lflr‘" 0 $118 (I) as eiiierences in wnole cnronosome number theie chromosome type 0; aberration 10? ex; pie, 1"" 2.1 ‘1‘ 'v. "it "w"- n‘ TI"\."I‘~Q .Y [2' ‘ x . " - ",3 11‘1-3 ‘30 _1\;h‘[_h IeDLA ilolll CA Us): VJ 1.1K Oi Jlnt' \illuh-1J--’J.{AQ. r) . .-. ~r -: “.3". - '- f" n ., \ J. IRCJmfatldlllbq D start). o.+ -3- w -" o . -- A , -'-l. . - , ~ —-\ . 7‘ ., ' .-~ -: ~ - ...-1 -‘. I The lascer tau are trooaoid rsasons lo; 1L3 ioker seed set Fertility at the bx level The fix nultiplant intersource cross produced a loner aVJraQe seed set per floret than the intraaource cross. This was contrary h _',A_ ...,l. .1, 'C‘ '3 V . I~ _. ‘ ‘r,-'-" . to tie results iron the 4x nultiplant CLQS-€S. sole oi this deviation l in trend Li;hC be exolained oy the iollorinL: 1) see activity mi_ht have O€,L less durin” the OK inter- SC'LLI‘CG CI‘C’SS; 1" 2) The recigrucsi crosses involvin_ so ,‘lQ? inuicate that . 9““. 4'4': . 'r - ”\n".'."7'1q “ ‘--f.‘ " l"l""l (‘ 7-‘ T. “' lnCoiialelllC' ROChlhloma Q3 Bhlst between some Oi one plants from tUiS source. It is possible that the plants of EC 35l09 in the intrassurce cross were lore mutually conpatiole than the plants of this source in cne inter— source cross. TLis may ac ;ant for tne low seed set on sons of the r‘ 3;lQ$'s in the latter cro . 5i1ce dif— ferent plants were in soon cross, it we s not ,tssioie to correlate seed set and type of cross in the some plant; 5) Seven 13lsnts 0: EC 33109 were in the intersource 'ross but only one plant from eacn of to Ie- other sources zas involved. Cne or these, CPI z31os—521, has later found to have aptroximately 7G; non-:ertile pollen (based on pollen stainaoility). Therefore, even if these plants were mutually corratiole the 03'“*tu1i “ for tne intro- duction of CDnrdol le tyoes from otner sources is elatively lot. however, reci groc :l intcis3crce cr :sses at the ox level have indic;ted tnat inc1up toullltv mechanisms exist between sour es as wle l as witnin sources. This could have further complicated the on inte sou1ce cross. Seed set, in the aioremem“ ionad ml 1:.t rich 705 non— st ained oollen grains, however, was aogut averaie for bat particular multi- plant cross. Les ice the mist percentage of a1,are tl“ iniertile pollen, a large trogrrtion or e,;s muss have seen :ertile. In most slants whici have n3r;al meiosis, three of the iour nuclei formed from the moraJIOIe tether cell degenerate. ihe relaininL ne,ssgore, 1e gicropglar end, tnen ur er ocs usually tne one iurtnest iron t1 w ._ .,. a _ - , l 4. CCCUF, l l": .K.’ v lL‘a‘ 1:“ 4L 5 O m f s \", LU ..."1- 1”,... .. uim usually hub tho ‘J’ '1“ ([3 chance in developed in eacn of induced polypleid, and the effects of golv' However, SOme discrepancies did exist. to have are r ported production in the Lield increased amoi dun. In addition, per head in Aura clover are regorted in induced result of recomeination of time. that a 1101' floret. ~30 J\— .. there has 3 p ‘— “Q‘RVV- r1 I ‘l 'r,‘ ._-, .— ’V -. ."-~ ’ "'\ i Brier liq hero r1110. L filb'vlll‘do, rILlQrJiLC :‘uiu there has and natural Selection OVer _ 7. ., - ‘ ' 2. . ' - . -"JJ-U‘ it'lCJi-L-.J-’ in l. '11 w_1_ . r. \J y‘u ’_ _, u n v - ~,- v- a r; w J. d—LlL—kJ—U-Lbb 3 ‘. 4. ,. 4 ' — -. re I 'llbo‘i (.L ; 1,.'..).1_l;_..'.,11, ouiI'Vl‘u' Cb. A ' 1 .. ..‘ ,- I-31 ;._1_ id. 3143 .1- ,L:3 -hiu‘ QC gal or near LQTgfil f" 1 .' ,1. L ,. vulcidj in general 0 eheral acreurent OGLWeen . | .. . . {“ U r , .- ‘~ ~.— c.1-_- I]. u L311. ‘1 ‘ L—L unile incuced golgploids flover directly with ploidg'level in T. - no decrease in the numoer of florets level increased altuough decreases Tnese cnanhes are prose" eXtenced Leriod v c A 8.3". -uw » » -~ 2' . . «-.. 1 J JL - ,.l .A'XLL‘E. L FL-\J.J \1b-- . as“, ~).L\11“.J r1“. ' f', '1, . .' .. 1' . ,4..r1 - V ., -_ ., 4 .: , _' ,1 ,._’ .; lrl-l‘../:l..llllh c..‘1--‘l,._:.zlzl-. 1'1. .‘3. (xx—:3) CCJLsILOQlJ eMobb .Ln ‘llelu, H! r‘ O L: C, ( I W O (D '1) d (D H: (D tetraploid, and heiaplolu lOrmS. Plants :rem :ou““ examined for ploidy level. host sources were asparently gure lOF a given cloiiy level, but try predominantly heiaplold sources has, retrectivaly, a few tetrag’ id and sentaploid plants. since no aneuploids were found in these sources, it has nygothesized that these variants were Fl's from interploidg matinhs involving differ- ent sources. Several morphological and physiolorical cnaracteristics or plants in the greeihouse and field were examined in an atte at to _ - so; , v.0: ' (qt-“,4 1. 1,11. 13: .‘ - D r.,‘ :.;. .. d8t€f1hlne lt'ODfoliJle l‘dlctlona.1l;xs DELl'Jden ;1'.L1-'.Lklu.' level.) cilia b.1808 characteristics. Generally, the following changes were noted in 1, field plants as ploidv level increased: larger leaf length/breadth 9 ratios (no chanée «as n ted between 4x and 6x averaues), increased \ but later flowerin;, longer florets and pistils, laréer pollen, larger and heavier seeds, increased rhizome production, and treater vigor. Althea h non-stainaoility of pollen tended to increase with ploidy level, some polyploid sources had low percentuges of non- stainaole polle1. winter losses were smal and a;garently not related to ploidy level. Disease and insect probleis generally a ,eared to be of little consequence excegt that nany :lorets in the lield were noted which had the up er ;ortion of the distil" destroyed or severely 1 .L 100 U} daaaged of insect . If this situation prevailed in conhercial tro- duction, a nigh seed loss might occur. In the freenhouse, floret lEEEth, number of heads per plant, and nuloer and size of stometa did not reflect the above trends at I) all PIGiGY levels. Overall vi;“r was al;o less in the fireennouse. L High intrasource correlation coefficients for leaf length/ breadth ratios of plants froth in the field and greenhouse were found for only two sources. These two sources also had similar intra- source averages and standard deviations in the field and :reenhouse. An hypothes‘s for the above phenomena was formulated and discussed. CytolOEical examination was the only efficient criterion found for identification of ploidy level of any Liven plant. however, under the environmental conditions prevalent in this study, plants which exceeded one or more of the followin; limits could tentatively be regarded as iexaploids: length X width :ro uct treeter than ltOQO sq. mm., floret length greater than 14 mm., pistil length in excess of 6.0 mm., and pollen diameter greater than 38 microns. Plants having a pistil length of less than A.5 mm. would probably be diploids. This included the bulk of the diploids examined in this Study and appeared to be en important criterion. hexaploid seed can apnarently be efficiently removed from mixtures with 2x and/or 4x seeds by oer screen size. Crosses were made within and between plaid; levels. Intra- ploidy fertility appeared to decrease as the floidy level increased. Some effective crosses were ootained from all interploidy crosses; however, the ex l ox crosses arouuced seed rather consistently. no selfcompatioility was noted. in adtition, self—incompatibility mechanisms also appeared to exist Ivithin an between sources of all ploiiv levels. Fertility data ep«na ized the imoortance of maintain- ing effective isolation of ploidy levels if regular t;pes are to be maintained. It is sugLested that Kura clover's strong asexual capacity enables the species to experiment with ploidy for maximum adaptation cause of coexiSLence of Zn, Ax, and 6x IJ I (D to a given envirornent. oparent lack of niLher ploidy levels, it is L» types as well as the a further hgoozhesized tzit th soecies may presentl; be undergoing important evolutionary Cr nges. In lichiLan, Kura clover has a hiLh octential as a more or less termanent l011L_e 0103 on soils hit h drainabe oroble.s, out in any given area only one ploidg level should be Crotn. bnder the . environment at East Lansin; the neXlJlOld scuxces snoued the hOSt promise. However, ertain plants at the digloid and tetraplsid levels had good vi Lor. At all nloicL levels, ruthless se lectlon would be necessary before a breeding proLra; was initiated. Seed yield would be related not only to ploid; level out also to the diver— sity of g rm plasm within a ploidy level. Mus, a vari W3 involving materials from several sources of the sane ploiug level would yrobably outyield a variety involving only one or two sources becazuse of the concentration oi incompatible tyres in the latter. This is an im— portant consideration at the diploid and tetraploid level since only a few plants were i‘ound which red to es dc siraole agronouic 5C r04 tJ 995 o 132 r5 sexual reo-ssuction in Aura clover csdlu be (U L7 ? L) C‘. F1 5: O L— .. ,, .. " ,.L , L -‘.,~~ --‘.’ ,...i ‘1‘; L‘ .. N benual prorauasion. aeoliaJle Lloheo f“ 1‘ new 05 r" ,“1 r». a ,- \" -: ‘1 “I ”I“ *- v yr. J; :30”: .;- 6U. J)! L. a _L.L_Lu;'~ o i011 Ol ‘ ‘ f. ‘ ' ,‘(~‘« . "f: *3. fi.“' " ',‘*“."~ "‘ ..""“ IN"‘ . ‘3"! r I . ‘h‘k‘ '. could be :rop- .~: :1qu linieilhlm—Jluf vaicnout the liJiCI LJHL, leldallltd in ILL)" "~71 333115, progeny resulting from sexual orogagatiin. \r‘J‘ F‘ I 3.? .r I;* . n v '\+‘ "*V' "V l V“'\ \l' ‘: (‘- 'LV‘fI‘, C‘ V| ‘1'; ‘, O ’_ r“-_) u -‘ - l ‘r . «._"2 ‘1"! l 8.1le en-JlVJJ—J I £31“OjibL“-U"u‘j leJLL—zll 3‘21 lva‘ £Jek.) " #14. \4 JV ediltvClCl—LJ—vl QeBJLI «AL-J e in even a profrzm. Asexual pro;a5ation has been successfuli, utilized L in the propagation of sermuda grass (Cyrodon dact;lon). \0 o A. 9. lo. LlTjnAltfil CLTLD Anonymous, "That Lek Clover.” A erican me WJo11'ne.l ,65:394-39L, YCV. 1945- bingefors, 3. "The Svaloi Tetraploid Led Clover Ulva. experiences from Trials and Cultivation in C ntral Sweden. Plant LreeninLLaos tracts, 4~.L3,e, 175.5 UriLinal not seen. slrusen J. ”The Function and Evolution of Ecotgoes, Ecospecies, otner natural Entities.” {Lg;ac1 Lr iversisets Arssmritt, Clausen, J. and Milliam L. niesey. "‘enOTruic dxor ssion of Geno otvpes in Contra stin- bnvironments.” scottish Plant Ereeding Station aejort, 1p. Al-bl, 1993. Erdman, L. U. and U. L. heans. ”Strains of Ihizooia Effiective on Trilolium amsiguum." AfironongJournal,A 3 :3nl-3L3, 1950. Evans, A. K. "The Production and Id 3nt1iicetion of roi'bloihs '1 ' .l. in Fed Clover, Hfiite Glover and L‘ucernz.” Lew 1LLtoloList, Curavich, D. A. "Intersge ci :i'ic Co jzatsuility v‘thin the Genus Trifolium and the vature oi seed Deva loga;1t i1 the gross l. a_oinum 1.5. by T. hLoridti L.” Lnoubii_ned fh.U. thesis, University or misconsin, l949. '\ ;~- - J» Hagberg, A. ”The Stability of Tetraploids and tne nisn Oi Cross Pollination and Contahination in Field Gondi tions Pla.rt freedinL Abstracts, M‘: x, 195 . privinal not see.. r—r Haney, h. J. COFIBCFHuchC ,1997. rim 1‘- Helv F. N. ”3vmbiotic Variation in irii .2 u Witn speCial Relerelce to the Latuie 01 assistinc,. Australisn Journal 0: ;iolovical science, lo(l):l—l~, Feb. \L 5.! ‘lnlstyanbi‘infifii.. Li: I! . Correspondence, UCL. lb, l9t5. ' c.,.,.. .‘I - ~_-- .I.r -' “‘1' -‘_--- “m: .‘_.. . «nil '. ,123 . henA-dln, L. 0 LI 0 I'Il MICK/CiIAlCCAJ. L.) 'IlO:-'Q.L Ale k;u..&.L/..L\ILL.UI~4L1 b LOVQ} 8 V 5 L - 14 t‘ ~ w , z a. - a ‘ “V is (T.rlfO+LlAi11)o [Logo-L303. dflr. ”fiat/Q.) *"O. ("'5 J"; “In. la. 4 .. ._J . H ‘C o (\3 C) o hf“. 10A 1 Hollovzll, E. A. ”Aura Clover.“ Tim-t;5reph renngiet, Elsie I"-’£—:5 3.88 3:2. “CH .31 GIlClfll, (1.51.030, Ludofloi‘LO’ 19 \II ‘ \w 0 sets 0: _u..uc:w11&utopolQfiiloiov‘111.h.ite Ihe Journal Hutton, B. K. ”Sons 311 Clover, Barrel redic, ans ”liters WJeLrass." of the austlilLHM Institute of A_:icult r;_ 22(-23 l, SBLt. 175 v '-~-~ 22. DC“,..IIC\'J. “J. ._._l Kashirina, l. E. ”Aura Clover as a Pasture Plant.” (in mussian) Bot. Zhur. (1» 03 kva) 41:31 5. June, 1958. L \1 I 0‘. LN Keim, hayne F. "Inte1-1cc11ic H; oriciza tion in lr11oi1ua Utilizing Embryo Culture Techniques." n:ron013 Journgl, 45:61Cl-1’3L1‘t, D‘SC. 1953. :I‘J- I ‘. ‘. . "deths o: 1 s .« -n H P s nqAJW ~ 1 +1. ” , am now" . W —..A. e"— [LL.~:. r‘v'C::,’;\~_LA.c’—C 0'— all: 13" .1 .‘Va‘\_k'u..-l/ CA. QCLJAAIVL’ ‘1 r‘ "7‘ " -';‘ I l:12~~lJ/, 3,1- .f... ' , ‘r‘ 1“ - r; ”A . 'erL' ‘l , v ', ' ' - f -n g j _‘ - - ‘_ . . hulLHt, n. u. an- 3. A. HollOWeil. "I1el .1lu-nce 01 lCMKcrabhle ans Pnotope: iod on Crowtu ans Elowering oi Crimson Clover 1:1 ' y. .- - - r .' p. .. V .. ., .. '1 '1 . if L; , 1:. :- (Lo idcar.‘ atlUL 1.)." figf9'3». JouIle, DU.~7,-~,&, l9, . homarov, V. L. "flora F355," vol. ll, LeLu4inoeae b? 3. h. Schischkin. 432 p, 1945. f. c L... O :3 O h ._J k. (D C" y. Q H O L.) ,- a ( T Q. -~r 1 ',_‘—~. -- -'vr 1" ,w' -: L1chns -a - l. MththZQnd. ”invest. Clover.” Plant jresdinL Austrac CriLinal no; seen. V3, ‘.~\1..LZ‘A—, Lei-Qt. Lewis D. "Comfietition ani Jom11auce of Incom 1tioiliLJ milel r ' I“ Y .. ’ u I"W ‘ ' :1, l . 1J1.‘101..d P334811," “‘63: C’- _' 4..- ) :1-) )‘l\/ , .1. 21+] 0 M — -- 4' v- ‘ ‘w " -‘ vnv ‘ ,r ‘ "rx vr T" Buta, big {1 o CAAJJ .1". o . J‘NLLAJLLIA‘L Chin. "SUUCLLeJ OI] .Llib .L'CQ .40. IUlJ- —! n .: '1 ,m .- . , ..,'.:. .. r .H, _-.‘. A. ... ,. , 3,11 plolds in 10.1_ e Cr e.” in-iqm uiufhul o. . Luth- 1-. Plant jreedLDL ,l7:27-37, l;S/. ‘ 1“" ' . 3"- v "3 v f“ 1. v i. ‘ A ~\-' ‘1'“117 Earle , you 1. and u. 1. Allen. ”Mogula ti on J: tus o1 1‘ < na-«nv» n “v-11 ‘ca "wmc l;~vzr _ r- ct i-‘*’ .. 0 (L x -1. 2.-\.11].. 1»... .1.-- .3 .1. o .. ~. 0 K' .J 3.; ’1 (.1 , U Q ., ’)l... a" 1 4' -1 ‘1' 1‘) " "\-~ ' ”L‘2r. 3 v "fi. ' "0 .. _‘ "r. - «r - ‘. '«-« 13‘ reliete, :. ”uo_e “suit bfiat hem cloveL." A e11c11 3:9 a; r 1-, #1.; r ”1" ; " '- ' - 11.1’ 0 -.~ :L}. L) 7-.+t)v', .20 \r o l ," xi“). ‘V‘l‘ - " h ‘) ‘, ~’. ‘ '- “i ‘ -.-‘1" "‘ "‘1 " ‘ ‘.\’~‘ 0 "TALL-t new glove: 11;").--11'.” :L...€I"..C.Ll- .1737} L1, Azir '11., .- ~. : ’ 1" y\ .‘ ._- 4J-44, u-u. 12.3. ,‘1 a J. J- . . .. a a, . .1' v.” ~ 1| _ ;" ,“1' ‘ 'rr~ -.. {‘1'} (A. .' ‘ 'v‘ a, 5‘ 13774.45- L211) 1‘ . “I '121’1418 OJ. E 33-1...‘35J v 4.»)VV to); o ” ‘£_._':/.J:1._L:1.‘ :. , {'C/ :‘Jv U—u‘ v'l’ ———-——h— r‘ ' '1 \“ ' 1 VOL. —L; L/J... 1»,~:-.L ’ , a ,.x,., .- v“.wufi . “roilett ulm~1er .11ott11.-1.” n s11c-1 .23 »...H.-, 195 ":1‘,‘ ‘ . l-. 4‘ V r‘ . “ 'iv‘w'n I A a ~‘- -, - ~,-. c . 4‘ z w ‘- é- .\ U ’;1 v‘ ‘9‘} o 31.3.15», Jo 1-. , o J. J».‘Oll«.lha‘vii¢‘~u, Ftl’lu m. :L. Le‘lquo “.LAUAJCGJ. . -A -4“ ' 1" ‘\ 0 .‘ 1" .nxn 1"“ -‘ . 1h ‘ |-_‘ ' ‘ -J“ ‘ "I ~ ‘ ' ‘ .1 ”I 1.2.: mg”! t 1a-; and. 4.3415311 (,JUVGI o .I udulgl 9, LONG . .L 7.4.: ‘ ————-— ./‘~ )2 3 1:3; 0 (3 ~‘ V W-f‘ (‘ f l_ L_ 11 ‘1 .V+ “ 1.: F)“ _ "41‘_./ “"t : --‘/: - _ " _‘ r- n “ 1A 3 ‘i: n .3 r) 3" i T' ./v'o JUJQV'VA.’ :1. Jun; gvu on ; -‘JL: ”3.1 24-111330 ll; U.;_;‘J-Ol(l uIA stud/O- ... . , ',-' _ ,‘ ‘1 .. .. m ' In. ‘3 4 ‘ V -., . -' - A, :1 ‘ ,A. .‘ ' .A : tebrdf I‘lJl-J med (44-0 (81 (4. Fl; ..’_L.'_ 2.1:- Lit—3.5 21-..”..I) c “'4. "1.11., ST : - r _ .. ,— ‘x r ,. .3 '_ r A A’, ’ ". :1 L16 ( ecqle cercglaj." fierflfliudo, AJ:9{7-ou4, i;,7. ” *T I, ' v- ' .. r‘. . '. : ..: .‘ "34'1““ )1. uacen, J. ELL-.1 c.1021. hwy. 1;. .-/.LBLJ . H I 4.01.1.t and J. Dad "Tr1Qol " a '“ ”3:1777, L7} . Or;;;aaL not seen. APPENDIX A LEAF SHAPES OF GREENHOUSE PLANTS OF KURA CLOVER Figures Figures Figures Figures 19 and 20 - 2: plants 21 to 25 inclusive and number 15 of Fig. 26 to 52 inclusivo - 6x plants 55 to 56 inclusive - abnormal leaf types 26 - 4x plants 106 M: nub ma .uun re- 9).. 108 +2.... .93 2 noun no... \om mm .uaa hmwoww ‘ ‘L“I‘III!|.IIII|III‘ ‘1 109 mm .u«& ‘2» HL0 #05 “sh mm 63 ¢>~.¢~m ooh nub 110 28 Fig. 27 Fig. 111 ‘1... on .ug momo\ H05 m: om 6:” 112 AH0.\ “hes mm .uam WORM UK 050‘ t>o~ 5 .....n ... .. wimp“ C an .ndn- woamm um 114 wm .uah mmzm Eu n- mm .uan r¢- HmU .ugwh V l v b-..— APPENDIX B FERTILITY DATA Tables 56 to 50 inclusive B - pollination by honeybees H - pollination by hand TABLE 56 Fertility data from.a 2x X 2: multiplant intersource croee Plant Nb. Of No. of Iource No. Florete} Seeds Seede/Floret CPI 2264 765 B" 246 261 1.06 " 778 152 175 1.14 9 788 146 142 0.97 ' 785 61 50 0.82 ' 780 52 72 1.58 9 771 112 97 0.87 9 762 15k 169 1.10 l 750 555 455 1-25 ' 92 559 527 0-96 ' 749 566 #02 1.10 ' 775 80 58 0-73 I 86 81+ 108 1.29 ' 95 527 510 0-95 ' 97 51k 468 0.91 9 - 85 295 2&6 0.84 CPI 27 819 189 172 0.91 ' 808 254 291 1.15 ' 815 195 195 1.00 ' 795 167 198 1.19 ' 792 174 219 1.26 ' 802 550 505 0.92 ' 809 189 260 1.58 ' 807 25k 275 1.17 ' 799 154 141 1.05 F 795 118 151 1.11 ' 811 176 158 0.90 Total Total Overall Florete leede Average 021 226“ 5279 5518 1.01 CPI 2771 2158 2559 1.08 116 TABLE 57 Fertility data from a 2): X 2x multiplant cross within source CPI 2264 Plant No. of N0. of N0 . Florete Seeds Seede/P‘loret 765 B 65 49 0.75 96 111 65 0.57 747 196 2 0.01 748 51 7 0.25 749 145 46 0.52 790 74 55 0.45 104 52 14 0.27 764 54 55 0-65 761 60 47 0.78 91 54 58 0.70 773 165 55 0.21 782 58 46 0.79 760 58 22 0.58 776 85 78 0.94 788 101 71 0.70 79 65 28 0.44 786 50 21 0.42 Total Total Overall Florets Seeds 0 Average 141a 1655 0.45 TABLE 55 Fertility data from a 2x X 2x multiplant cross within source CPI 2771. Plant No. of No. of N0. Florete Seeds Seeds/Floret 814 520 195 0.61 799 82 49 0.60 821 46 45 0.95 810 45 51 0.72 804 191 127 0.66 815 108 65 0.60 811 122 92 0.75 815 149 160 1.07 Total Total Overall Florets Seeds Average 1061 762 0.72 118 1181: 59 Fertility data from 2x X 2x reciprocal crosses within source CPI 2264 Pluto NOe Of No. Of No. Florets Seeds Sheds/Floret 747 H5“ 14 0 0 95 25 0 0 749 H 22 0 0 760 25 0 0 786 H 50 0 0 764 48 0 0 786 H 45 0 0 749 46 29 0-65 749 H 49 22 0.45 761 47 0 0 749 H 26 0 0 775 51 o o 749 ' H 18 0 0 788 16 9 0.56 775 H - -' 764 58 55 0.92 775 H 55 49 1.40 788 55 58 788 H 44 2 0.05 765 44 25 0.52 765 H 67 0 0 764 68 0 0' 119 TABLE 40 Fertility data from 21: X 21: reciprocal crosses within source CPI 2771 Plant N0. of N0. of N0 . Florets Seeds Ieeds/Floret 817 H ' 76 0 O 807 . 82 0 0 799 H 24 O 0 794 -- -- -- 805 H 29 12 0.41 821 50 12 0.40 798 H 57 1 5 O. 55 810 58 28 0.74 120 TABLI 41 Fertility data from 2x X 4x reciprocal crosses Plant N0. of N0. of flource N0. Floretsw Seeds Seeds/Floret CPI 2264 95 H 24 0 0 021 6161 1 25 0 0 CPI 2771 1817 H 74 0 0 PI 229625 557 75 14 0.19 CPI 2771 806 H 61 0 0 or: 6884 29 65 0 0 CPI 2264 766 B 580 0 0 r1 228570 654 150 0 0 UPI 2264. 775 B 451 0 0 PI 2296240 722 275 0 0 UPI 2771 814 s 609 2 0.005 PI 228570 652 448 1 0.002 or: 2264 747 B 555 5 0.005 21 228570 626 428 0 0 121 TABLE 42 Fertility data from 2x X 6x reciprocal crosses Plant N0. of N0. of 80urce No. Florete Seeds Seeds/Floret 0r1 2264 770 H 50 0 0 or: 25158 244 50 0 0 CPI 2264 75 H 60 0 0 no 55109 875 60 2 0.05 or: 2771 792 n 44 28 0.64 to 55109 890 44 0 0 brI 2264 759 H 26 0 0 CPI 6161 5 ,.. -- .. CPI 2771 827 B 528 0 0 CPI 6161 10 175 2 0.01 or: 2771 795 B 565 0 0 to 55109 859 202 0 0 CPI 2771 851 B - -- -- CPI 25158 258 195 0 0 CPI 2264 775 B 296 10 0.05 so 55109 1091 79 0 0 or: 2264 74 B 555 0 0 ’0 55109 851 259 9 0.04 or: 2264 747 B 582 2 0.005 no 55109 855 107 2‘ 0.02 Total Total Overall Florets Seeds Average 5049 55 0.02 1225 TABLE 45 Fertility data from a 4x X 4x multiplant intersource cross Plant N0. of N0. of Iource No. Plorets Seeds leeds/Ploret PI 229625 516 B 575 567 0.98 ' 557 191 201 1.05 PI_228570 685 559 296 0.82 ' 687 412 548 0.84 ' 658 157 171 1.09 ' 616 278 525 1.17 ' 654 554 271 0.81 1 679 417 504 0°73 PI.229624b 711 405 565 0.90 CPI 6884 25 175 154 0.77 Total Total Overall Florete Sieds Average 5101 2782 0.90 125 - TABLE 44 Fertility data from 41 1.4x intersource reciprocal crosses Plant No. of No. of Source N0. Florets. Seeds Seeds/Floret PI 229625 552 B 428 0 ‘ f 0 CPI 9949 129 164, 0 0 PI 229625 505 B 551 215 0.65 r1 2296241: 697 559 250 0.64 PI 229625 490 H 55: 9 0.17 H 228570 672 g. 55 50 0.94 124 TABLE 45 Fertility data from 4x Xu4x intrssource reciprocal crosses Plant N0. of N0. of Source N0. Florete leeds Seeds/Floret or: 6884 29 a 49 59 0.80 ‘ 60 49 51 0.65 P: 229625 571 a 45 28 0.65 ‘ 490 46 5 0.07 21 229625 540 H 52 20 0.65 9 575 40 57 0095 PI 229625 565 H 49 55 0.71 ' 507 #8 56 0275 PI 228570 662 a 18 4 0.22 ' 680 18 14 0.78 P: 228570 662 H 49 20 0.41 ' 599 50 56 0.72 PI 228570 662 n 55 58 1.09 ' 628 40 25 0.65 PI 228570 628 n 22 15 0.68 ' 625 - -- -- PI 228570 615 H 28 0 0 ' 657 27 0 0 r1 229624b 755 H 21 18 0.86 ' 708 52 22 0.69 r1 229624b 722 n 57 17 0.46 ' 724 44 25 0.57 125 TABLE 46 Fertility data from 4x x 6x reciprocal crosses Plant N0. of No. of lource N0. Florets Seeds Seeds/Floret CPI 6884 60 55 26 0.74 PO 55109 1085 42 o o CPI 6884 65 65 16 0.25 CPI 18115 276 58 6 0.10 CPI 6161 1 75 58 0.51 '0 55109 1055 75 44 0059 PI 228570 672 47 18 0.58 PO 55109 1081 50 5 0.10 P1 229625 507 101 4 0.04 CPI 25408 555 105 4 0.04 PI 229625 579 550 57 0.16 CPI 18115 275 414 156 0.55 PI 229624b 724 205 104 0.51 F0 55109 860 -- -- .. Total Total Overage Florets Seeds Average 1620 458 0.28 126 TABLI 47 Fertility data from a 6: X 6x multiplant intrasource cross involving source FC 55109 Plant No. of No. of N0. Florets. Seeds Seeds/Floret 956 B 197 170 0.86 1050 122 108 0.89 965 175 58 0-55 856 179 128 0.72 880 200 107 0.54 862 140 96 0.69 1006 164 96 0.59 1079 122 97 0.80 870 259 185 0.71 Total Total Overall Florets Seeds Average 1558 1045 0.67 127 TABLE 48 Fertility data.from a 6x X 6: multiplant intersource cross A Plant No. of No. of Source No. Florete Seede Siode/Floret EC 55109 855 B #1 20 0.49 " 172 42 27 0.64 9 1055 48 29 0.60 9 897 51 51 0.61 ' 1059 47 12 0.26 9 1067 57 . 27 0.75 9 1004 48 4 0.08 CPI 25408 521 60 27 0.#5 CPI 25158 226 89 51 0.55 PO 411 55 55 0.64 Totnl Total Ovorall Plorete Seede Lvnrege 518 245 0.47 128 TABLE 49 Fertility data from 6x X 6x reciprocal crosses within PO 55109 Plant No. of No. of No. Florete Seeds Seeds/Floret 897 u 59 15 0.25 962 58 52 0-55 1050 H 57 - 1055 57 0 0 851 H 56 21 0.58 995 54 29 0.85 1022 H 19 5 0.16 1087 20 7 0.55 982 H 52 12 0.58 860 52 11 0.58 860 B 179 1+7 0 . 26 869 268 17 0.06 129 TABLE 50 Fertility data from 6x X 6x intersource reciprocal crosses Plant . 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