f zomrfimflmto H3. U” 9. ur‘ Yu‘n..r an, f...“ u ‘ odfiafilf 7V)“. .2)". K .s & o J“... ‘7' 9. ' '15‘.‘ Inr'vrp .trz‘rt. ”L"; 2w- .‘l‘ ‘ ‘I‘ L' ‘W .T ~ (J . v 1 0:1 (ao- . “bunt? CLdJ'LWILHLvu/Yifll. 11 l " l ."kn‘ Lulllfllll 6 . «1 If a: L. ru..v.‘.l,n a. .1 1 . .. . vflsurm LIA, an .va . ‘. A ‘ .. ‘ . . . . ‘. . H r , .. . ‘ . . ‘ JunmwkMMWmuh. 14. ”Ftwofilu‘mflk‘gt, . , ‘ . V ‘ , .. , , ‘ . ‘ ah; .L IE... ‘ \. This is to certify that the thesis entitled A Characterization of Somatic Sectoring in Tredeecantie presented by Michael Lee Christians on has been accepted towards fulfillment of the requirements for LIBRARY Mio’om State Ph. D. degree in The Genetics Program and Department of Botany and Plant Pathology “/2/ hyigga, Major professor Date Juj-Y 27: 1976 0-7639 'r‘mf‘n ‘ n. mom; ‘3" ’ "DAB 6 3"."45' 800K BINDERY INC. LIBRARY BINDERS QIIIIPO‘II, IICIISI; _._.4 v.. / / CD I 0/; =* /... / L7 ABSTRACT A CHARACTERIZATION OF SOMATIC SECTORS IN TRADESCANTIA By Michael Lee Christianson Somatic sectoring in Tradescantia is the most sensitive biological indicator of radiation known, and has been suggested as an admirable test system for the study of chemical mutagens as well. The radio- biology of Tradescantia, as induced chromosome aberrations, is described in an enormous literature, but the investigation of the mechanism(s) of production of somatic "mutations" has just begun. Unless or until the mechanisms of "low—level sectoring (spontaneous and few-fold enhancement)" in Tradescantia are well described and shown to operate in similar relative frequency and at similar efficiency in human beings, the extrapolation of relative biological effectiveness (RBE) or the transfer of "mutation rate" from Tradescantia gp, to Homo sapiens cannot be valid. This dissertation makes a major contribution to the first process, i.e., the characterization of spontaneous and slightly-enhanced somatic sectoring in Tradescantia. Section I of the dissertation describes the chemogenetics of a diploid, purple-flowered I} hirsuticaulis stock and the mutant-colored (red and blue) cells found upon close inspection of the pigmented floral parts. Section II considers the mutant sectors in the stamen hairs, monofilifbrm chains of large, anthocyanin-pigmented cells, in more detail. The red and blue mutant cells are analysed with respect to their 7} pr rel Michael Lee Christianson distribution within and among the hairs on a stamen, as well as with respect to their frequencies and associations with putative chromo- some fragments both spontaneously and after exposure to relatively low amounts of 60Co gamma radiation. These analyses support an argument for somatic crossing-over as the major mode of production of spontaneous sectors as well as an important mode of production of sectors after exposure to as much as 60 R of radiation. Section III considers some conceptual analogies in the genetic behavior of facultative apomicts and somatically sectoring Tradescantia flowers. From this consideration arises a new method of treating somatic sectoring data and the introduction of a previously undescribed somatic genetic parameter, A, the apomictoid fraction. Major findings of this study may be summarized as follows: l) Purple flower color of the I, hirsuticaulis stock is due to the presence of approximately equal amounts of blue (delphinidin) and red (cyanidin) pigments. 2) The total amount of anthocyanin pigment in purple-flowered I. hirsuticaulis is twice that in blue- or red-flowered 1, clone 02 and progeny. 3) Purple-flowered I} hirsuticaulis possesses an allele, E+, which is responsible for both #1 and #é above. Action of the E+ allele may well have a simple molecular explanation. 4) The genotype of the purple-flowered stock is D+E'/D'E+. This designation was arrived at through an analysis of the occasional red Michael Lee Christianson and blue mutant cells produced in the purple floral tissues and is supported by breeding data. 5) Purple-flowered I, hirsuticaulis exhibits three classes of somatic sectors: red-only, red/blue twin spots, and blue-only. The twin spots are shown to be the results of single events. 6) As in 1, clone 02, deletion, as evidenced by the presence of micro- nuclei (chromosome fragments), is indicated to be one mechanism pro- ducing certain kinds of sectors (red-only) in response to ionizing radiation. 7) The predominant, if not exclusive, mechanism of spontaneous sector- ing and an important mechanism even for 60 R-induced sectoring in this I, hirsuticaulis (and by inference, in other Tradescantia) is argued to be mitotic crossing-over. 8) Some conceptual parallels between the genetic behavior of faculta- tive apomicts and sectoring flowers of Tradescantia are described. This results in a description of new somatic genetic parameters, A, the apomictoid fraction, and l-A, the mictoid fraction, those frac- tions of the cell divisions that are typical mitoses, or atypical mitoses where the chromosomes are amenable to exchange, respectively. 9) A genetically accurate method of using somatic sectoring data for the calculation of mitotic map distances and coincidence is described. 10) Mitotic crossing—over through failure of some canalized gene of mitosis is shown to predict a "log response - log dose" plot which simulates that found for somatic sectors in T. clone 02. A CHARACTERIZATION OF SOMATIC SECTORING IN TRADESCANTIA By Michael Lee Christianson. A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program and Department of Botany and Plant Pathology 1975 This Dissertation is dedicated: To the memory of Walt Whitman. To the actuality of Dr. George Landon, To the future of gay women and men, and To Diana Ross. ii Th Robertsr was prin done by Dur: many Sign these are Drs. J. H. R. P. Heri to the Drs respective? nor the ion bility and well, to Dr QIOUp at Sr lad it might have i"tallectue Head! 0. E ACKNOWLEDGEMENTS This bound volume would never have appeared but for Phyllis Robertson who typed the final manuscript. The color plate, Figure 3, was printed by Dr. L. N. Mericle. The layout of Figures 6 - 12 was done by Dr. R. P. Mericle. 1 During my long career at Michigan State University, I have had many significant interactions with faculty and staff. Prominent among these are the guidance and encouragement of my committee members, Drs. J. H. Asher, jr., P. S. Carlson, W. 6. Fields, L. H. Mericle,- R. P. Mericle, and Wm. Tai. A particular expression of gratitude goes ,to the Drs. Mericle, chairman and non-faculty member of my committee, respectively, not just for all the time spent in editorial suggestion, her the long discussions, but for the combination of freedom, responsi- bility and respect that I found in their laboratory. I am grateful, as well, to Dr. A. H. Sparrow for the opportunity to be associated with his group at Brookhaven National Laboratory during the summer of 1973. Had it not been for my friends and members of my living groups, I might have finished sooner, or might not have finished at all. For their intellectual and emotional support, I thank them: L. E. Murry, M. A. Mead, D. E. Clark, K. o. Hoffman,.P. H. deZeeuw, M. w. deZeeuw, J. 5. iii Lee, S. L. White, s. J. Risch, P. Hertz, 5. George, J. N. Enders, s. Itzkowitz, and e. D.-Starks.‘ The financial support came from a National Science Foundation PredoctoralrFellowship. iv LIST OF ' LIST OF 1 INTRODUC' SECTION I Tradescar Gene Pur; Pur; Gene Pred anal Resu SECTION I Purple-fl Gene Red, Red. Red, Sec Her SECTION ’ data fro TABLE OF CONTENTS LIST OF TABLES ................................................ LIST OF FIGURES ............................................... INTRODUCTION .................................................. SECTION I. Chemogenetics of a diploid, purple-flowered, Tradescantia hirsuticaulis .................................... General considerations ................................... Purple pigmentation: qualitative nature .................. Purple pigmentation: quantitative nature ................. Genetics of flower color in Tradescantia ................. Prediction of flower color genotype by somatic cell analyses ................................................. Results of breeding studies .............................. SECTION II. Somatic sectoring behavior in stamen hairs of purple-flowered I, hirsuticaulis .............................. General considerations ................................... Red/blue twin spots: their existence .. ................... Red-only and blue-only sectors: their existence .......... Red/blue twin spots: position and orientation in hairs ... Sector sizes .............. . .............................. Mechanisms of sector production .......................... SECTION III. Towards a theoretically sound method of treating data from mitotic exchange ..... ............................... V Page vii ix 3 3 IS 29 34 38 42 57 57 64 71 72 74 88 104 Parallels between facultative apomicts and somatic. sectoring in Tradescantia .. ............................. 104 Theory of somatic exchange; ............................... ll2 Quantitation of theory .. ................................ 123' Application of quantitated theory ....................... 134 Further implications of the theory ........ .............. lSl SUMMARY ...................................................... l58 APPENDICES A. Particularly interesting somatic sectors in T. hirsuticaulis ............................... T" ......... 160 B. Amounts of nuclear DNA in a mature stamen hairs of 1; clone 02 ............................................. T65 BIBLIOGRAPHY T70 vi LIST OF TABLES TABLE I TO ll 12 13' Petal area, weight, and density of purple I, hirsuticaulis, blue 1. clone 02 and its red segregant, 5-62 ... ........... Progeny expected from inbreeding of purple-flowered I, hirsuticaulis (predicted genotype DTD'E E“) ............... Progeny obtained from self-pollination of purple I, hirsuticaulis ...................... . ........ . ............. Testing linkage of D and E loci in I, hirsuticaulis ....... Seeds per capsule from the cross 5-1 x P .................. . Notation system used for recording somatic sectors in stamen hairs of I. hirsuticaulis .......................... Contribution of multiple sectors to total sectors in I. hirsuticaulis ............................................. Red/blue twin sectors with and without intervening purple cells ..................................................... Red/blue twin sectors: Pair-by-pair analysis of all stamens with red and blue mutant sectors .......................... Red/blue twin sectors: Orientation of red vs. blue component in the hair ..................................... Sizes of non-multiple, non-entire hair sectors in I, hirsuticaulis ...................... . ...................... Numbers and thes of sectors in T. hirsuticaulis after exposures to Co gamma radiation ......................... Distribution of sizes of non-multiple, non-entire red-only and blue-only sectors ................ . ........... .... ..... vii Page 30 47 48 53 55 61 65 66 7O 73 75 79 81 TABLE. Page 14 15 16 T75 18 T9 20 21 22. 23 Sizes of non-multiple, entire hair sectors and non-mutant entire hairs in I, hirsuticaulis .................... ..... 83 Comparison of stamen hair length (number of cells) in 5-42 and 5-62, two red-flowered progeny from 1, clone 02 inbreeding ............................................... 84 Micronuclei association in 60 R-response hairs ........... 91 Micronuclei association in 0 R (Spontaneous) hairs ....... 92 Frequency of somatic sectors in stamen.hairs of I, hirsuticaulis ............................................ 98 Sectors presented as representing crossover events ....... 103 Sectoring data on I. hirsuticaulis and X2 homogeneity test .... ................................................. 135 Numbers of hairs per stamen-and cells per hair in I, hirsuticaulis ........ . ................. . ................. 137 Somatic genetic parameters calculated for I, hirsuticaulis T40 Somatic genetic parameters calculated for Drosophila ..... l48 viii FIGURE 1 2 The ant Visible of flow and pur The col The eff I. clan Co-chro hlr5ut1 \- Absorpt Absorpt Wavelen' red and extract Simulat Spectra- SPeCtre Spectre Spectra hir3uf~ Stocks The st FIGURE 1 2 10 11 12? 13 14 LIST OF FIGURES The anthocyanin chromatophore .......................... Visible absorption spectra of 2% HCl-methanolic extracts of flowers of blue T. clone 02, a red-flowered progeny, and purple I, hirsuticaulis ............................ The color of anthocyanin extracts at various pH ........ The effect of pH on visible absorption spectra of blue 1, clone 02 and a red-flowered progeny ................. Co-chromatograms of anthocyanin extracts of blue 1, clone 02, a red-flowered progeny, and purple I, hirsuticaulis .......................................... Absorption curves of petals and aqueous extracts ....... Absorption curves of stamen hair cells ................. Wavelengths at maximum absorbance of various mixtures of red and blue extracts compared to that of purple extract ................................................ Simulation of purple spectral "fingerprint" ............ Spectral comparison of pigment intensity .. ............. Spectral "fingerprints" of red cells in several stocks . Spectral "fingerprints" of blue cells in several stocks. Spectral "fingerprints" of progeny from purple T. hirsuticaulis compared to those of blue, red,.and purple stocks ................................................. The standard scoring sheet ............................. ix Page 5 14 18 21 23. 25 28 32 41 44 50 63 20 21 22 23 24 25 26 27 28 29 30 31 A sche MJtatT "Dose" Sonati Variat Variat aponix Cytolo- Canali. Chrome! Differe the ac1 Apparer apomict The "Ha A Compa Mitotic SOWatiC VGFTOUS A log-I EXCEpti NUCTQQr FIGURE Page l5 A schematic stamen ............ ......................... 68 T6 Mutation response curves ..' ............................. 78 T7 "Dose"-response curve .................................. 96 TB Somatic exchange and its results ....................... lOl 19 Variation in calculated map distance with apomixis ..... l07 20 Variation in apparent coefficient of coincidence with apomixis ............................................... llO 2T Cytological indications of somatic pairing ............. 114. 22 Canalization of the mitotic gene which keeps chromosomes from pairing ............................... TT7 23 Differences in spontaneous mutation rate explained as the action of modifiers on cellular physiology ......... 122 24 Apparent coefficient of coincidence versus fraction of: apomictoid cells ....................................... l25 25 The "half-tetrad" nature of somatic sectors .. .......... 128 26 A comparison of meiotic and mitotic exchange ........... T30 27 ' Mitotic map of I, hirsuticaulis ..... .... ............... l42 2" 32233232 282§t3§mi§’32§i§{i02"&502352‘s‘tl91“.”.‘Iif“???... 144 29 A log-log plot of the normal curve of error ............ 154 30. Exceptional sectors in I, hirsuticaulis . ............... 162~ 31 Nuclear DNA in a mature stamen hair .................... T67 Strasbur hair cells in sectors, cell enhanced sect (93). and the beginnings of flaking by de alleles, by D‘ Somatic : cal indlcatOr 3" admll‘able (iii), HOW SECtoring int I DElleVe’ DC low-lam Set} Operate in S' hUman DQIHQS (RBE) or the INTRODUCTION Strasburger first described the division of-young, living stamen hair cells in Tradescantia in 1875 (45,134). The existence of somatic sectors, cells of a "mutant“ color, in the stamen hairs of Tradescantia was first reported in 1958 (32). The study of radiation- enhanced sectoring in plants, however, dates back to at least 1935 (93), and the interest in spontaneous somatic sectors, to the-very beginnings of the science of genetics as exemplified in studies of flaking by de Vries (146), patching, by Punnet (112), and unstable alleles, by Demerec (35). Somatic-sectoring in Tradescantia is the most sensitive biologi- cal indicator of radiation known (76,77), and has been suggested as an admirable test system for the study of chemical mutagens as well (141). However, the conversion of these slightly increased rates of sectoring into meaningful estimates of hazards.to human beings is not, I believe, possible at present. Unless or until the mechanisms of low-level sectoring in Tradescantia are well described and shown to operate in similar relative frequency and at similar efficiency in human.beings, the extrapolation of relative biological effectiveness (RBE) or the transfer of "mutation rate" from Tradescantia s2, to 1 ' rem-.11 This tion of s; Tradescant genetics 0 colored (r pigmented ' the stamen crossing-ox as an impor following e III conside tive apomic this conside applicable t the introduc mate“ A, UN Homo sapiens cannot be valid. This dissertation makes a major contribution to the characteriza- tion of spontaneous and slightly-enhanced somatic sectoring in TradeScantia. Section I of the dissertation describes the chemo- genetics of the purple-flowered I, hirsuticaulis stock and the mutant- colored (red and blue) cells found upon close inspection of the pigmented floral parts. Section II considers the mutant sectors in the stamen hairs in more detail and argues for a mechanism, somatic crossing-over, as their major mode of spontaneous production, as well as an important mede of their production at enhanced sectoring rates 6060 gamma radiation. Section following exposure to as much as 60R III considers some analogies between-the genetic behavior of faculta- tive apomicts and somatically sectoring Tradescantia flowers. From this consideration arises a new method of treating sectoring data, applicable to all organisms exhibiting somaticrcrossing-over, and the introduction of a previously undescribed somatic genetic para- meter, A, the apomictoid fraction. Chenogenet General cor Flower of colors c These compo example, al (51.71). M genetic int‘ SUItS 0f crc In Eiéi the Presence the vacuoles CVanin molec mOIeCUIe: SL hydroxy] gr: IS COIOred’ visnne Ilg ConjUOated translthng Section I Chemogenetics of a diploid, purple-flowered, Tradescantia hirsuticaulis General considerations Flowers of the Anthophyta exist in an incredible range and variety of colors caused by the presence of just a few classes of compounds. These compounds can be "diagnostic characters" for the taxonomist: for example, all plants with betalins belong to the order Centrospermae (51.71). More often, variant pigmentation is of horticultural or genetic interest. Indeed, Mendel's first paper (73) records the re- sults of crosses between violetered and white flowered Piggy, In Pjsgm_and Tradescantia, floral pigmentation is due mainly to the presence (or absence) of various anthocyanin pigments contained in the vacuoles of the epidermal cells of the floral parts. The antho- cyanin molecule is more properly a substituted 2-phenyl-ben20pyri1ium molecule: sugar residues attached to the A ring, and 1,2, or 3 hydroxyl groups on the B ring (Figure l). The anthocyanin molecule is colored, of course, because it absorbs Certain wavelengths of visible light. The quanta of energy are absorbed by the electrons in conjugated n molecular orbitals. These electrons make Vpermitted transitions" to excited energy states and subsequently return to the Figure 1. The anthocyanin chrOmatophore.e Substitution with sugar residues at the 3 and 5 positions (arrows) of the anthocyanjgin converts it to theicorresponding,'water-soluble, anthocyanin? Each named anthocyanin or anthocyanidin has.a character- istic hydroxylationpattern for the B-ring; pelargonidin is 3,5,7,4‘- tetrahydroxy—benzopyrilium, cyanidin, 3,5,7,3‘,4i-pentahydroxy- benzapyrilium, and delphinidin, 3,5,7,3I,4',5'-hexahydroxyebenzo- pyrilium.' These pigments are associated with orange, red, and blue flower-color, respectively. «gradnd State"! y reflected 01‘ "3' wavelengths VET ii— The electrc substituent gro'.I the molecular ' on the 8 ring to permitted; the J . terested in f or the papers l cc I perspective (63 116,127,132) ¥ 9 resonance (EP? I linst of ti and coworkers acidified (1-: VAIIOUS Train: \ absm‘PtiOn CUrI "In plarf that is 1' this is Clear dramatically eXpTESsion of E 6 Vground statef, releasing their energy as non-visible quanta. The reflected or transmitted light, then, has differing amounts of various wavelengths removed and thus appears colored to the human eye. The electron-donating or electron-withdrawing properties of the substituent groups on the phenyl ring change the electron density in the n molecular orbital. Molecules with different substituent groups on the B ring then differ in which transitidns to excited states are permitted; they have different colors to the human eye. Readers in- terested in further exploration of this topic are referred to some of the papers I consulted in arriving at the above summary: for historical perspective (69,107,108) for evidence from visible spectrosc0py (115, 116,127,132), from UV spectroscopy (37), and frOm electron paramagnetic resonance (EPR) studies (46,103,126). Most of the English-speaking world follows the method of Harborne and coworkers fbr the stady of anthocyanins, i.e., extraction into acidified (1-2%) alcohol (48,49,50,62,139,l40). Extracts of the various Tradescantia stocks prepared in this way exhibit single peaked absorption curves (Figure 2). While Stevenson (132) says "In plants, the flower pigments usually exist in an environment that is chemically nearly identical to the acidified methanol...", this is clearly not so; the anthocyanin in the petal changes color dramatically upon contact with the acid~methanol.' This is the jn_yjyg_ expression of the long known in 11359 effect of pH on the color of anthocyanin extracts (26,38,110,116,127) (Figure 3). In contrast, Figure 2.." Visible absorption -spectra-of 2% HCT—methanolic extracts of flowers of blue 1} clone o2, a red-flowered progeny, and purple ‘ Lrhirsuticauiis. The three variously colored stocks give smooth single peaks, with rather sharply defined maxima at 532, 548, and 540 nm for red, blue, and purple stocks, respectively, when measured with a Beckman 086 spectrophotometer. *aem_p——‘W_H4A H_—__.__.__-_———_____ Absnrba nce Absorbance Purple Blue 532 - 540 ‘ 548 Wavelength, nm Figure 2 Figure 3; The coloraof-anthocyanin eXtracts at various pH. Shifts in the pH of aqueous extracts.prepared from petals of blue-flowered 1, clone 02 and a red-flowered progenvaere effected throughitheaddition of small amounts of dilute HCl or NaDH; each vial- is labeled as to the.pH of the solution within. Notice that the color of the extract ofred flowers(series labeled "C“) at pH 5.0 and 5.6 is nearly identical to the color of the extract of blue flowers (series labeled “D") at pH 2.8 and 3.1, respectively. _ _ i _ L 95‘.— _—T—-‘ 11 extraction of petals in distilled water.gives a preparation of pH 5.6 - 5.8 (75,135) whose coloraand visible absorption spectra do not. differ from those of intact, living petals or individual-living stamen hairs (82,88). (The routine extraction into acidified water most likely explains.the differences observed by Stewart g§_gl, (133) between spectra of intact tissues and anthocyanin extracts of those tissues.) While anthocyanins are reported to be unstable in non-acidified solution (139), that is not the case with extracts from Tradescantia. Simple aqueous extracts of TradeSCantia stocks have-kept-tbeir original colorrduring more than 6 months of refrigeration. 'While homogenates of certain stocks of Tradescantia are, indeed, very unStable, for reasons not yet known, a nethechnique serendipitously discovered by Mericle and Mericle (in preparation) gives stable, non-acidified, aqueous- extracts of them as well. Initial studies of the visible spectrosc0py of simple aqueous extracts were carried out during 1967 e 1968 by Mericle and Mericle. While these workers had expected to find single- peaked absorption curves (75,87), they observed that both the aqueous extracts and living materials exhibited multi-peaked absorption curves within the visible wavelengths. This type of spectrum, they discover- ed, had just been reported in the botanical literature (119). Multiple-peaked absorption curves, however, were not without pre- cedent, and interpretation, in the physical—chemical literature. Adams and Rosenstein (1) investigated the spectrum of crystal violet, a well known dye. in chanical speci tion curve (a seen at any 91" respective par sequent invest to the same cor While the one colored for 139,140) other anionic form 01 single anionic the QVICOSylate molecule (dEIph ThEOI‘etics extracts of Var spectra] "finge anthocyanin fro (livers, from S cyanin, the bib deiDhinin With cause ibIUEing" While the Dump the Same Coim- 12 known dye, in solutions at various pH. They concluded that each chemical species (ionic form) of the dye had a single-peaked absorp- tion curve (a “fundamental") and that the multimodal absorption curve seen at any given pH was the sum of each molar "fundamental" times the respective partial molar fraction of each chemical species. Sub- sequent investigation by others with different dyes (reviewed, 69) led to the same conclusions. While the English language literature considers equilibria between one colored form and various non-colored species of anthocyanins (e.g., 139,140) other work (38,116) considers a red cationic form and a blue anionicxform of any pure anthocyanin.r Whether a single cationic and a single anionic fbrm actually describes all the ionicSpecies of, say, the gylcosylated 3,5,7,3',4',5‘ -hexahydroxy-2+phenyl-benzopyrilium molecule (delphinidin) is not yet known. Theoretics and physical chemistry aside, the non-acidified aqueous extracts of various Tradescantia stocks give distinctive, multipeaked spectral "fingerprints." Figure 4 shows the spectral curves of the anthocyanin from red-flowered and blue-flowered Tradescantia. The red flowers, from S-62, a red-flowered segregant from 1, clone 02, contain cyanin, the blue flowers of I, clOne 02, a pigment mixture of mostly delphinin with a trace of cyanin (79). Increases in pH are known to cause.?blueing"~of anthocyanins jn_givg_(8,18) and inpyjtrg_(26). While the human eye may see delphinin and cyanin.at different pH as the same color (Figure 3), a spectrophotometer detects differences. 13 Figure 4. The effect of pH on visible absorption spectra of blue '-<:E:Ef 1, clone 02 and a red-flowered progeny. Aqueous extracts of the blue and red stocks, initially pH 5.85 and 5.60—respective1y, had their pH altered by the addition of small amounts of hydrochloric acid. The visible absorption spectra at these new pH were measured with a Beckman 086 spectrophotometer: a) blue stock,b) red stock. The dotted line at 550 nm is to facilitate comparison of the two sets of curves.r e masmmm E: .cuwcoao>m3 1'4 mm.m 1% i% small a itate ......-’ onv omo one P b _ b _ _ _ . _ om m _ w _ mH.m . I . \ _ . _ oo v . _ _ . _ sou cq .msq v Figure 4 Sho extracts. N curve of one pigment. 1” single'Peaka characteri st' The vari general, give tion in a DUI: Dr. A. H. 3133' purple, could routes: throug simultaneous F blue pigments, a red or a blu extracts of pu stamen hair ce maximally tri'.m (compare Figur Hhile Chr clearly show Pigments were 15 Figure 4 shows the results of changing the pH of the blue and red extracts.‘ Note that the spectralcurves change in shape, but the curve of one pigment is never converted into the-curve of the other pigment.i In highly acidic aqueous solution, each pigment~does give a single-peaked absorption curve: these, however, possess a distinct, characteristic wavelength of maximum absorbance.(xmax). Purple pigmentation: ,gualitative nature, The various spectral properties of anthdcyanins, and dyes, in general, give a meansof identifying the nature of the purple pigmenta- tion in;a purple-flowered-Ig hirsuticaulis stock (#2091, obtained from Dr. A; H. Sparrow, Brookhaven National Laboratory).’ The flower color, purple, could be obtained through any one or a combinatidn of several routes: through the presenceof one, purple, pigment, gig the simultaneous presence of approximately equal proportions of red and blue pigments, or 319 a shifting of visual color-throughpr effects on a red or a blue pigment. This latter is ruled out since simple aqueous extracts of purple petals as-well as the petals themselves or individual stamen hair cells show a tetrgmodal absorptibn curve unlike the maximally tgjmodal curves seen for cyanin or delphinin at any pH (campare Figures 6,7 withrFigure 4). . While chromatography of an extract of the purple petals could clearly show whether a single purple pigment, or both red andrblue pigments were the cause of the purple flower color, Tradescantia _ —_ __ pigments do r conditions. gave rise to and unhydroly after the met separation (o solvents). F Tradescantia ‘ strips with d: (drawn from ti l:l:l gave the method preserv red, the delph Pigments from of pigment. The chron, of the purple . Went. but 1 led and blue p Previous TeDOr mnii blue pi Concurren the nature of 16 pigments do not separate well under most standard chromatographic conditions. Progressive hydrolysis and subsequent chromatography gave rise to "extra spots", identified as partial breakdown products and unhydrolyzed pigments (40). My chromatography of anthocyanigins after the methods of several-workers (7,49,94,102) did not give a separation (or, of course, the retention of natural color in the acid solvents). Finally, I attempted chromatography of aqueous extracts of Tradescantia flowers containing anthocyanins: Whatman #1 filter paper stripswith development in mixtures of isopropanol, ethanol, and water (drawn from the methods of Osawa 103, and Alvarez 2). Proportions of 1:1:1 gave the best separation and spot definition, (FigureS). This method preserves the natural color of the pigments; the cyanin spot is red, the delphinin, blue. It proved impossible, however, to elute the pigments from the dried chromatogram in order to quantitate the amounts of pigment. The chromatogram (Figure 5)shows that the anthocyanin pigmentation of the purple I}hirsuticaulis is not the result of a single purple pigment, but instead due to the presence of perhaps equal amounts of red and blue pigments.' Furthermore, the co-chromatogram confirms previous reports (79) that the blue-flowered 1, clone 02 contains mainly blue pigment plus a trace of red pigment, but by chromato- graphy of the anthocyanins, not the anthocyanidins. Concurrent with initial, unsuccessful, attempts at elucidating the-nature of the purplepigmentation gig chromatography, 17 Figure 5. Co-chromatograms of anthocyanin extracts of blue I, glggg_ 92, a red-flowered progeny, and purple I, hirsuticaulis. Ascending chromatography on Whatman #1 filter paper strips with development in the solvent Isopropanol:Ethanoleater::l:l:l of equal amounts of similarly prepared aqueous extracts of blue, red, and purple stocks. The red,.cyanin, pigment has a higher Rf than the blue, delphinin, pigment. I". '- - ‘1:. iii If} {If m oe=m_m RUO o 2...... so»)0.~ UHUflHllHU Aoeaaav +i.co.* .:o>_on spectrophc Non-a I- ma visible re phenomenon and its rec petals, liv tetrahedal trimodal to be easily Se Spectrophotc stocks (Figu When th "Ddal curves defimd maxin 19 spectrophotometric studies were undertaken.toward-the same‘ends.' Non-acidified aqueous extracts of petals from the purple-flowered I,hirsuticaulis have essentially the same absorption spectra in the visible region as the living intact petals (Figure 6);”th15‘ phenomenon was previously reported for the blue flowered 1} clone 02 and its red—flowered progeny (82). The purple pigment, in intact petals,-living stamen hairs, or in aqueous extracts, exhibits a tgtgamodal~absorption spectrum which corresponds to neither of the trimodal curves obtained forblue or red flowers. This difference can be-easily seen in a comparison of absorption curves obtained by micro- spectrophotometry of individual, living, stamen hair cells of the three stocks (Figure 7); When the petals are extracted in acidic methanol (2% HCl), multi- modal curves are not seen. Instead, single peaks with rather sharply defined maxima are obtained; the wavelength at maximum absorbance is specific for each of the variously celored stocks (Figure 2). Mixtures of such acidified methanolicextracts of petals from blue- and red- flowered stocks also show single peaks whose wavelengths at maximal absorbance are intermediate between those of the unmixed extracts, 535 nm and546 nm, respectively, for red-flowered and-blue-flowered- stocks measured with a Beckman 086 spectrophotometer,‘ Indeed, a linear relationship between Amax and proportion of red (or blue) extract isobserved (Figure-8). The Amax of the purple stock is 541 nm, almost exactly midway between those of the unmixed red or blue 20 hi ~ Figure 6. Absorption curves of petals and aqueous extracts. 1. Absorption curves obtained by macrospectrophotometry of living, 0 intact petals and non-acidified aqueous extracts of petals from purple g in flowers of I} hirsuticaulis. The spectrophotometer was a Cary 15. ‘ E 02 p 00 ll ‘2/ / to L__ 430 L if living. from putple ‘ary 15. Absorbance 21 0.4 r ' ",Non-ac1dified aqueous petal extract 0.2 - Intact, living petal 0.0 l l l 1 450 500 550 600 650 700 Wavelength, nm Figure 6 22 Figure 7. Absorption curves of stamen hair cells. Absorption curves obtained by microspectrophotometry of individual, living stamen.hair cells from‘blue-flowered Leia... 02. its red- flowered progeny, and from purple-flowered I, hirsuticaulis. From "plug" measurements uncorrected for cell size or pigment concentration, measured with a Zeiss 01 microspectrophotometer. Absorbance 0.0 Q0 ‘\. armhnmt 5rd- From icentration Absorbance 0.6 0.4 0.2 0.0 0.0 0.0 23 b in Purple, 1. hirsuticaulis r Blue, Clone 02 Red, progeny ’ of Clone 02 J 1 L 1 a 450 500 550 600 650 700 Wavelength, nm Figure 7 24 Figure 8. Wavelengths at maximum absorbance of various mixtures of red and blue extracts compared with that of purple extract. Wavelengths at maximum absorbance for acidified-methanol extracts prepared from equal numbers of petals from blue-flowered 1} clone 02 and its red-flowered progeny, mixtures of the two in various proportions. and comparison with that of an acidified-methanol extract prepared from 9 half the number of petals from purple-flowered I, hirsuticaulis. The spectrophotometer was a Beckman DBG. 25 m mgam_u mHauoo Hauou mo maauoa no» =o«uuoqoom o.H m.o o.o v.o ~.o o.o 111 d d d d d CNm ovm eoueqaosqe mnmtxem ‘mu ‘qafluetanen own extracts, equal amc he Eflill glycoside Tm near of cyanic as strong that the presence There is aqueous l MiX' numbers . similar of the F on the 1 additim extract: curve 'l petals platent the DUI and 511 Chrome 26 extracts, and corresponds exactly to that observed for a mixture of equal amounts of red and blue extract (541 nm). On the other hand, the ugigug, purplish pigments, petunidin and malvinidin or their glycosides, as observed by Stevenson (132), have their respective Amax near but higher than the midpoint of the interval from the Amax of-cyanidin to that of delphinidin. I interpret these experiments as strongly supporting the suggestion from the chromatographic work that the purple color may well be the result of the simultaneous presence of approximately equal amounts of delphinin and cyanin. There is further support for this interpretation in the spectra of aqueous extracts of the red, blue and purple stocks, Mixtures of non-acidified aqueous extracts prepared from equal. numbers of red and blue petals give tetamodal absorptioncurves similar to the tetramodal spectrum of the purple stock; the location of the peaks and shoulders, as well as their relative heights, depend on the proportions of the mixture (88). Indeed, simple mathematical addition of the tgjmodal absorption curves for non-acidified aqueous extracts of equal numbers of red and blue petals gives a tetramodal curve "closely resembling" that from an aqueous extract of purple petals (Figure 9). If the purple color were due to a unique purple pigment, this observed duplication of the “spectral fingerprint“ of the purple-flowered stock by addition of the fingerprints of the red and blue stocks would not be expected. Thus, even without the co- chromatograms of the three stocks (Figure 5), the purple celor of 27 Figure 9. Simulation of purple spectral "fingerprintf. Simulated "purple" absorption curve derived from mathematical addition of the curves obtained by macrospectrophotometry (Beckman DBG) of non-acidified aqueous extracts prepared using equal numbers of petals from blue-flowered 1, clone 02 and its red-flowered progeny. Absorbance mum] kdnn nah“ d WOW Absorbance 28 0.8 P p 0.6 - ",Sum of blue + red curves = simulated purple curve 0.4 _ Ira", Blue, Clone 02 I I I I I ," \ \o"\ I I 0.2 - Red, progeny ;/ of Clone 02 I O o - j L l 450 500 550 600 650 700 Wavelength, nm Figure 9 the _T_. I presence W In the pure informer of T. hi progeny. measure applicah Non are near similar ' he“ as 3 Out trac‘ respectih afGretneni Chromatog amounts 0 only M sufficien. or area m Spectra} c 29 the I, hirsuticaulis stock can be assigned to the simultaneous presence of both red and blue pigments in approximately equal amounts. Purple pigmentation: quantitative nature. In addition to showing the probable anthocyanin composition of the purple-flowered stock, the spectrophotometric data also provide‘ informatiOn on the total amounts of pigment produced by purple flowers of'l} hisuticaulis and blue or red flowers of 1, clone 02 and its progeny.- Maximum absorbance can be used to_givea rough quantitative measure of anthocyanin content (50); this has been demonstrated as applicable to these stocks of Tradescantia (88). Non-acidified aqueous extracts of equal numbers of petals (which are nearly matched for size, Table 1) from blue and red flowers exhibit similar levels of maximum absorbance (see Figure 10 as an example) as well as similar total area under the spectral curve. (Heights of cut- out tracings of spectralcurves, 700-450nm. are 0.56 and 0.55 g, respectively; for red and blue extracts.) These results agree with afbrementioned estimates, based on visual intensity and spot size in chromatograms,.that blue and red flowers contain approximately equal amounts of delphinin and cyanin,.respectively. However. extraction of only half as many (or fewer) petals of the purple I, hirsuticaulis is sufficient to give a comparable maximum absorbance value (Figure 10) or area under the spectral curve. (Height of cut-out tracing of spectral curve,.700-450nm, is 0.66 g, for purple extract ) even Table imag that hheig c Dens 30 Table l. Petal area. weight, and density of purple 13 hirsuticaulis, blue 1} clone 02 and its red segregant,.S-62. Stock Areaa Heightb Densityc purple l.l7‘ 0.0193 0.0164 blue l.36 0.0208 0.0l53 ‘ red l.33 0.02l2 0.0l59 aArea determined by the weights of a cut-out tracing of the projected image of'a water-mounted. flattened petal of each stock relative to' that of-a similarly projected 1 cm2. bHeight 8 average fresh weights of two petals of each stock cDensity # Height/Area 31 Figure l0. Spectral comparison of pigment intensity. Absorption curves obtained by macrospecrophotometry of non- acidified aqueous extracts prepared from equal numbers of petals from blue-flowered 1} clone 02 and its red-flowered progeny, and half as many petals from purple-flowered I, hirsuticaulis. The spectrophoto- meter was a Beckman DBG. Absorbance 32 Purple, 1. hirsuticaulis, 6 petals Absorbance 0.0 Blue, Clone 02, 12 petals 0.0 I Red, progeny of Clone 02, 12 petals 0 0 , ‘* ‘ 1 J 450 500 550 600 650 700 Wavelength, nm Figure l0 A _ ._ _ though t and dens (Table l M of thesr culture the for Th 33 though the petalsof I,-hirsuticaulis are comparable in size, weight, and density to those of red and blue flowered 1, clone 02 and progeny (Table l). The latter implies that theflowers of purple I. hirsuticaulis contain twice as much anthocyanin.as flowers from 1, clone 02 or its progeny, and explains why the red and blue flowers of these latter match the lighter, "/l“, tones of the Royal Horti- cultural Society Colour Chart (RHC) (52) while the purple flowers of" the former match the "full" tone (29,88). The co-chromatograms (Figure 5) show parity in size of the cyanin. spots between the red and purple stocks, as well as parity in size of the delphinin spots between the blue and purple stocks. Equal amounts of extracts from equal numbers of petals of the three stocks:were applied to the chromatogram; the purple stock, then, must have as much blue pigment as the blue stock agg_as much red pigment as the red stock, a total amount twice as much,g§_either stock...Therefore, any proposed genotype for the diploid (75), purpleéflowered. I, hirsuticaulis_ must.take into account a phenotype that differs from both the blue and red phenotypes of diploid I, clone 02 and its pro- geny, not only in regard to the kinds and proportions of anthocyanin. pigment.produced, but also with respect to the total amount of pigment synthesized in each cell. 34 (Erlet'i cs of flower color in Iradgsgggtig . Vihile the inheritance of flower color has been established for many plant species (sunmarized by Paris gill: l06), a: very little is known about flower color in the genus Tradescantia; it is of minor horticultural importance (e.g., the cultivars Iris Pritchard and Purple Dome), despite its extensive use forlcytological studies (e.g., 67*- - 121). Anderson and Diehl (4) suggested that blue flowercolor- "as probably inherited, in diploids, as a simple Mendelian-factor “dominant" to both red and white, or, in tetraploids, as a co- dOminant. Their interpretation stemed from observations of discrete (b1 ue, red, white) classes of flower colOr in populations of diploid mdescantia and a more continuous range of colors including inter- mediate tones observed in a tetraploid population of I. ‘reflexa. (As yet unpublished observations of Mericle and Mericle on Tradescantia lbopulations in Texas and Colorado, however, reveal that diploid populations of atleast five species exhibit "intermediate" colors.) A. H. Sparrow's group at Brookhaven National Laboratory inferred that the diploid, blue-flowered 1, clone 02 was heterozygous for flower color from the observation that red-colored somatic sectors arose in petals and stamen hairs, just as mutant sectors arose in the petals of heterozygous Antirrhinum (32); these red-celled sectors were interpreted as results of mutations from a dominant blue allele to a recessive red allele (l00). Subsequent chemogenetic studies by Mericle and Mericle (79,82); have established that _l_'_. ‘clone 02 is blue flowe factb visua paTET by tl (D'D' radi. segr to S; than to tl stbcl by cl to be C‘C' 93 or t0 be a dun E is:- phinfc ”esiec ’- .3" _J -1 I - 35 flowered due to the presence, in single dose, of a dominant genetic factor, 0+..which gives delphinidin production. Indeed, D+ behaves as a completely dominant Mendelian factor: no visual or spectrophotometric differences are apparent between the parent, 0+0', plant and its 0+0+ progeny. The latter are recognized by the fact that their flowersdo not show the typical, occasional red (D'D') mutant sectors, even following relatively high levels of radiation (79,82). Those progeny from selfing I, clOne 02 which. segregate for-the absence of this D+ factor, D'D', lack the capability to synthesize delphinidin. They are found to be red-flowered, rather than white-flowered, because they can produce cyanidin, presumably due to the presence of another genetic factor, 0+. As both 0+0" and-0+D+ stocks, however, produce similar, small amounts of cyanidin, revealed. by chromatography of floral extracts (79), the C+ factor is indicated to be at a different locus and not simply an allelomorph of 0+. No C'C' (white-flowered) plants have been recovered fromselfing I, glogg_ Qg_or various red x red intercrosses; clone 02 is therefore presumed to be homozygous C+C+ (88). While blue flower color might appear as a dominant.to red flower color, the production of delphinidin is e istatic, not dominant, to the production of cyanidin.- In purplesflowered I, hirsuticaulis, the genes producing del- phinidin and cyanidin are not epistatic and incompletely hypostatic, respectively, but rather, isostatic. It is not that the D+ allele in I, hirsuticaulis has noepistatic capacity. As will be shown later, Perha dbnir glycc CG-dc donir duces by th Wears 36 occasional, mutant, blue cells in the stamen hairs have absorption spectra identical to those of cells in blue-flowered I,'clone'02, i.e., containing mostly delphinin with_traces of cyanin. Perhaps I, hirsuticaulis has a genetic factor that specifically releases the epistatic suppression of C+ in the presence of 0+, or, perhaps the stock carries an allelomorph of C+ insensitive to the action of 0+. As the purple flower color in I, hirsuticaulis is the result of the enhancement of the trace of cyanidin produced in other D+ bearing stocks, or the release of the gpistatic suppression of 6+ in they presence of 0+, the genetic factor responsible is given the tentative designation gf (29.83.88). The action of E+most likely has a simple molecular explanation, perhaps similar to the molecular basis elucidated for the order of dominance in an allelomorphic series at a locus responsible for the glycosylation of flavones in Melandrium (l9,20,2l,23,24) and a variant, co-dominant, allele at that locus (22). In Melandrium, the completely dominant allele, 96, which gives glucosylation of the flavones, pro- duces an enzyme with a Vmax ten-fold greater than the enzyme produced x, which gives xylosylation of the flavone.r In the heterozygote, gng, both enzymes are present in equal amounts; however, by the allele 9 the enzymewith the higher Vmax consumes all the available flavone, the common precursor, before the other enzyme can form detectable amounts of xylosylated product. In one plant, however, the genes G X' appeared to be co-dominant; the heterozygote, g g , produced equal 37 amounts .of‘glycosylated and xylosylated fl'avone. The gXI allele pro- duced an enzyme whose Vmax was similar to that of the enzyme from the 96 allele, and some ten-fold greater than the enzyme from the 9x allele. The "bottom" recessive, the 9 allele, was found to produce an 6 allele. enzyme with 6% of the glucosylating activity of the g The allele E+ in I, hirsuticaulis may well prove to be C+',an allele of C+,which codes for an enzyme molecule which is insensitive to feedback inhibition by delphinidin,.has a higher Km,.or exhibits a higherrvmax (to give-three likely possibilities). If this were so,~ stocks such.as D+D+C+'C+ and olo‘c+'c+' could be obtained through. apprOpriate breeding schemes. "If the C+' enzyme had; for example, a Vmax comparable to therD+ enzyme, while the C+ enzyme did*not, then the' first named stock would have bluedpurple-flowers. While the‘c+ enzyme, with its low vmax’ would produce littleproduct, the enzymes from the two 0+ and one C+' alleles would compete equally well for the common substrate, giving, finally, delphinidin and cyanidin in‘a 2:l ratio, and a flower color intermediate between purple and blue. As will be mentioned later. a blue-purple flowered stock has been obtained; its spectrum and that of a mixture of aqueous extracts of blue 1, clone 02 and of'a red-segregant in proportions of 2:1 are remarkably similar (Figure l3d). The other stock o+o'c+'c+', by a} similarcompetition of enzymes from one 0+ and two C+' alleles would have red-purple flowers. Such a plant has not yet appeared among the very small number of progeny deri Cont desc Eric tron Stuc stuc self Alth his desc base the: the sari 38 derived from breeding experiments with I, hirsuticaulis materials. Continued breeding and biochemical studies will-be needed to completely describe the nature of the E+ factor. Prediction of flower color genotype by somatic cell analyses.- Hhile Anderson and Diehl maintain that Tradescantia "grows (easily from seeds"-(4), that has not been the experience in previous genetiC* studieSon Tradescantia by Mericle and Mericle (79,82). In these studies, however, the plants being utilized, ]}'clone02, were at least self-fertile, whereas the purple Ig‘hirsuticaulis is selfasterile. Althougthrs-A.'H. Sparrow kindly offered a blueéflowered I, hirsulticaulis for my use in making crosses, subsequent examination of- his collection revealed that the stock plant had died (98). Thus, as described below, elucidation of the genotype of the purple stock is based on its capacity to produce somatic sectors and on the pigment' chemistry of those sectors. As will be shown later in this Section, the rare progeny which have been obtained to date from an extensive series of~self-pollinations fully support the postulated genotype. When stamenrhairs and petals of the purple-flowered stock of I, hirSuticaulisare examined under the same conditions used in this laboratory for mutation scoring of 1, clone 02 (76,81), occasional, non—purple cells are seen. As with 1, clone 02, exposure of this purpledflowered~stock to ionizing radiation increases the frequency of these "mutant sectors" -- a subject which will be treated in more 39 deta‘l 1 in Section II of this dissertation. Some of these non-purple mutant cells are red. Microspectrophoto- metry of individual, living, red mutant cells in stamen.hairs of the Purp'l e stock yields absorption curves identical with those of both red mutant cells from the blue ]'_. clone 02 and non-mutant cells of its red- f‘l owered progeny (Figure ll). Since the latter are known from breed- ing experiments to be 0'0' and to have only cyanidin as their antho- cyaninpigment. an assignment of the same cellular pigmentation and genotype is made for the red-mutant cells in the purple flowers, as Was done for their counterparts in the blue Licl-one'oz (79,82). Therefore. as 1111. 'clOne 02, the purple I.-‘hi.rsuticaul is must have a _ genotype of n+0“. The genotype of I. hirsuticaulis differs fromthat of 1. clone 02, however, 'by the presence of E+ in the former.. As was described above, the E+ factor, whatever its molecular nature, affects the final pheno- type by allowing the cells to synthesize, in addition to the "unit" atlhount of delphinidin produced in blue 1. clone 02 cells, an additional "unit" of cyanidin, rather than simply a trace. This results in both an overall purple color and pigmentation that istwice‘as intense. The procedure for determining whether the stock is heterozygous or homozygous for the E+ allele follows that outlined above for the D+ allele; a stock that is E+E' is expected to produce occasional E'E' mutant cells -- these cells being blue in color, as they contain a 0+ allele at the o locus. 40 Figure ll.- Spectral "fingerprints" of red cells in several stocks. Absorption curves obtained by microspectrophotometry of individual, living stamen hair cells consisting of red mutant cells from blue-flowered 1, clone 02 and purple-flowered’Ig'hirsuticaulis,I and non-mutant red cells from 1, clone 02‘s red-flowered progeny.- From "plug" measurements uncorrected for cell size or pigment‘ concentrations, measured with a Zeiss Ol microspectrophotometer. Absorbance 41 0.3 )- Clone 02 0.6 - .I. hirsuticaulis ‘ Red progeny V of Clone 02 0.4 0.2 / \ 0., . . . \ . 450 500 550 600 650 700 Wavelength, nm Figure ll 42 Indeed, others of the aforementioned non-purple mutant cells 33g. blue. Microspectrophotometry of individual, living, blue mutant cells in stamen hairs of the purple I, hirsuticaulis yields absorption curves identical to those found for non-mutant cells of the blue 1, clone 02 (Figure 12). Therefore, the purple stock is assigned a genotype of L E+E'. i “" Additional analyses of the sectoring data in purple I, i hirsuticaulis, presented more appropriately in Section II of this #1 dissertation, leads to the conclusions that the D and E loci are linked, that the D locus is the more distal, and that 0+ and Ef are in a re- pulsed (trans) configuration. Results of breeding;studies Although the purple stock typically sets no seed after self- pollination, persistence in making self-pollinationsihas led to the production of 22 seeds*, of which five have germinated over a period of three years and survived to flowering. One additional seedling was inadvertently lost. Although these data are certainly not "extensive", they do confirm some of the conclusions reached through analyses of somatic sectoring in the purple stock. If the purple stock is indeed D+E'/D'E+, progeny obtained from self—fertilization should form nine * It should be noted that these seeds are assumed to contain embryos which are the results of the sexual process; at present, I have no way of determining whether some -- or all -- of them might have arisen via some apomictic process. 43 Figure l2. Spectral "fingerprints" of blue cells in several stocks. Absorption curves obtained by microspectrophotometry of individual living stamen hair cells consisting of blue mutant cells from purple-flowered I, hirsuticaulis and non-mutant blue cells from blue-flowered 1, clone 02. From "plug" measurements uncorrected for cell size or pigment concentration, measured with a Leitz MPV-l micrOSpectrophotometer. 44 0.8 l 0.6 ~ I. hirsuticaulis Q) 0 fl .8 x... 0.4 '- O m .0 < P Clone 02 0.2 - / \ 0.0 1 1 1 1 450 500 550 600 650 700 Wavelength, nm Figure 12 45 genotypic classes (Table 2). The red-sectoring capability of 0+0" stocks would permit the separate identification of some genotypes having identical phenotypes, e.g., purpleeflowered,’ ‘D+D+E+E’+_ and D+D-E+E', stocks or blue-flowered, D+D+E'E' and 0+0'E'E', stocks. It was originally (88) prOposed that D+D=E+IE+ and 0+0+EfEf progeny might be purple-flowered as well, but identifiableby their ability to sector either red or blue, respectively, but not both types of mutant cells; I am now proposing that these two genotypes producereda—purple and blue-purple flower color, respectively, identifiablewithout recourse to an analysis of their distinctive sectoring capabilities. The phenotypic characteristics of the five progeny plants.are presented in Table .3 with their flower colors recorded as spectral fingerprints in Figure 13. The flowers of those plants listed as red- flowered in Table 3, S-1, S-2, and S-4, have visible absorption spectra very similar to red progeny of 1. clone 02 (Figure 13a). The plant recorded as purple-flowered in Table 3, S-5, exhibits a spectral curve very similar to the purple parent (Figure 13b). In contrast, plant 5'3.‘ scored as blue-purple-flowered* in Table 3,’ exhibits a spectral * This blue-purple plant was originally scored as being blue-flowered, lts flower matching the RHC #41/1. Subsequently, it was recorded as ing "purpl e", no notation of a match'to the Colour Chart being made. there was a possibility that the plant had somatically sectored, Yielding two'distinctly pigmented plants, it was subdivided into 5' Parts. All parts have now bloomed; all produce flowers blue-purple in COlor, matching RHC #39/1- The matchto RHC #38/0 is also very good. In some lights, the flowers of S-3 matchtRl-IC #40/1.’ Indeed, there are my small, (but significant), differences in the spectral fingerprints ”Purple, bl ue-purple and blue pigments. The original .and second (continued page 46) 46 curve-(Figure r313d) intermediate between'that of rpurpleeflowered stocks (Figure 13b) and blue-flowered _T_. clone 02 (Figure 13c). The shoulder c>r1 the S-3 curve at 626 nm is larger than the similar shoulder on purple curves at 630 mi, and smaller than the peak at 614 nm in the b1 ue spectral curve; the main peak of [the S-3 curve at 582 -nm is inter- mediate between the ‘586nmpeak of purple stocks and the 576 nm peak of the blue stock. It is, as noted earlier, very similar to the spectral fingerprint of a mixture of‘delphinidin and cyanidin extracts in propor- tions of 2:1, even thoughxthis latter curve was from a Beckman'DBG spectrophotometer while the otherfingerprints discussed here were from a Cary 15 -spectrophotometer(Figure l3d). That red, .D'D'. Progeny were produced from selfing l. hirsuticaulis confirms the heterozygosity of‘the purple parent at the D locus. It has also been advanced (88) that red progeny might fall into two inten- sity clasSes, "/0" and "ll"; however, RHC notations for S-l, 5-2, and 8-4 are either "/1" or "[3" (Table 3). Quantitative spectrophotometry Suggests this latter difference may well be a result of differences in cell size, not in amount of pigment per cell. Thus, the E locus, in, these 'plantsat least seems to have no effect on cyanidin production, 13 the absence 912:. _‘ notations are both thought to be errors due to the inability at these times to compare the flower color not only to the Colour Chart, but also to flowers of the other, intermittently blooming, I. "hirsuticaulis stocks. «tied U.«~CL1V ..w — Pas-...J v d I 3.0. . .... - F can»... 333 Feb Int 91!. :1 k 0 n»: — 3.9.6.55: . .EQLL Uiefl Unezxw \Aflamvhweural irN riv~ nurse 47 mcouumm 0: p o P -mumiaio nwm 28.8w 2. P o _ PP: as. maouomm o: P o P +m+uiowo emu ems woman P F imiuio+n aspm maopumm o: mums» p p nutm+a+o mapm «Pagan .ospa _ N m._ -m+w+o+o «Paganimapm opacaa .ums N — m.p +m+uio+a mpaazaiumm wapn .ume F P N -u+mio+o mpnaaa meouumm o: F P N +m+meo+o a—agzm ceupccmwncevvcvcnemo.. _ucoemwmpmuoh 288m 233.. . enteewmnwafijtuessofigcemw «mag g ..-m+mio+o mazuoeom umuovumgav mvpamoeuamgwc_mm vesmzopmim—asza co acetamgncv ease emuuoaxe acmmogm .N «Peak u L.‘ 131;” I—l . |.:r H M“: 48‘ Table 3. Progeny obtained from self-pollination of purple I} hirsuticaulis. QQQ§_ QQLQB. Bflgf_ SPECTRUM RED-SECTORING P purple 37/0 Figure 13b 0.15/stamen (430/2780) S-5 purple 37/0 Figure 13b . 0.14/stamen (5/36) S-l red 34/3' Figure 13a. --- S-2. red ' 33/l Figure 13a --- S-4 red 34/3 Figure 13a --- s-3 blue-purple 39/1 figure 13d ‘ 0.00/stamen (1/233)a ,—,‘A_", _- aThe one sector is thought to have arisen via a gene mutation early in floral development, giving a large 0+0" sector, within which a sector-producing mitotic crossover occurred. RHC = Royal Horticultural Society Colour Chart (52). 49 Figure 13. Spectral "fingerprints" of progeny from purple I, hirsuticaulis compared to those of blue, red and purple stocks. All curves are of non-acidified aqueous extracts of petals. a) Spectral curves of a red segregant from 1, clone 02, and the three red-flowered progeny from the parental purple I, hirsuticaulis, S-l, S-2, S-4. b) Spectral curves of the parental purple plant, P, and a purple-flowered offspring, S-5. c) Spectral curve of blue-flowered 1, clone 02. d) Spectral curve of S-3, an offspring of purple I, hirsuticaulis having blue-purple flower color, and of a 2:1 mixture of aqueous extracts of blue-flowered I, clone 02 and one of its red progeny. This latter curve was obtained with a Beckman 086 spectrophotometer, all others, with a Cary 15. 50 ,4 IlLfl‘leleJIvnq . fife? ................................. soon .. OVn ... .os Red. segregant of Clone 03 ' - -.- - - - ' - - - -.-.',-i.o-.'e---.-n o'u .-e-v-e-e-a‘c'nl eoaenuoea< - ' ' ' -.- -.-.-v'd -u-.-0-l-a'4-n-.| 0-. O eonenuoenc wavelength, n- 23 . \\nmwmflww;sit.tono In .lv. .|.I.'.l-.al.|.nv.l.l.l..l.|.I.|.|.- ["8 5-3 .. .I.-. '.'.I.|.I.I.al.|,.l.'.l.'..ls' I‘Vn IIIII .I. .i.I.i.i lilo; oonenuooa< l I.l.l.l.l.l.l.l.|.l.l.l.l.l.l.l.l.L IV—O I I.I.I.l.l.l.l.i.l.l.l.l.|.l.l.l.I.I.I.I.i.l.l.ill1 Gun - al.'.'.'..l.l.'.'.'.4.'.|.'.'.'.-.l.-.a 3N3 OOQIAAOIA< Wavelength, nm Wavelength, on Figure 13 ein' e. 6! 1 Cu. . IFLI All - e I nflu firi- Pub 0 v as me ‘I. Al. I i . 1- y Ar U n V .\4 In r (Inf P e u .1 t I l e. i. PT A&.\ at v f. a id 0 it] A v 7. «AN Fit 5‘ q Fr Al» 4 AU h “2! E An... A u I A . H e. i 1‘ .e Avie I I a! n 9 | rid Ill! 0. Fri L h\v S P N I 51 While no blue, E'E', plants have yet been recovered to directly confirm the E+E' composition of the purple parent, the existence of a blue-purple progeny still confirms the heterozygous E locus in the ' purple parent. If the purple parent were E+E+, then only three geno- types would be expected amongst its progeny: D+D+E+E+, purple, D+D'E+E+, red:purple, and D'D'E+E+, red. A plant with blue-purple flowers is expected only from interaction of two doses of 0+ with one dose of E+. It follows then, that the blue—purple progeny is heter- ozygous at the E locus, and, barring gene mutation, so is the purple parental‘stock.‘ Blue-purple flowers, predicted as having a funitf of delphinidin and half a unit of cyanidin, are expected to be less intensely pig— mented than purple flowers with their two units of pigment, but more intensely pigmented than either red or blue flowers with their one unit of pigment. While 5-3 is recorded as RHC #39/1, it is, however, noticeably more intense than the blue 1, clone 02, recorded as RHC #4111.(79). The Colour Chart was not a perfect match for either flower color. The purple-flowered offspring, S-5, is closely similar to the purple parent. Flowers of both match RHC #37/0. They have identical spectral fingerprints and a red-mutant sectoring rate of 0.14 and 0.15 sectors per stamen, respectively (Table 3). These characters, then, certainly seem to be heritable through the germ line (but see footnote, page 42). rot e. at th Secti' cellu ié'ei 52 Five progeny plants do not permit calculation of an.accurate meiotic map. It is possible to show, however, that,.while these five plants are not at variance with a hypothesis of independent assortment* of the D and E loci, neither are they at variance with the hypothesis of linkage between D and E to the extent (12.1 centimorgans) calculated mitotically in Section III of this dissertation (Table 4). . 0n the basis of either hypothesis, there is an excess of red progeny reCovered, exactly contrary to the case inbreeding results i-‘ from selfing.I, clone 02 (79,82). At present, the breeding data are not extensive-enough to permit interpretation, although some aspects of the haplontic/diplontic-selection questionfi will be discussed in SectionII of this dissertation during a consideration of sizes and cellular genotypes of the various mutant somatic sectors in the parental purple I, hirsuticaulis stock.- Extensive self-pollination of the "self-sterile" purple stock only yielded 22 seeds over a two year period. I hoped that some of the five progeny produced from 19 seeds put to germinate? would prove to cross readily with their parent. In addition, a cross to one of the three red progeny might be a testcross, i.e., D+D-E+E' purple x D'D'E'E' red; in any event, such crosses would reveal the E locus composition of the various red progeny. It was found, however, that the cross S-4 x P gave no seed set. In contrast, S-l x ngave abundant seed set, an average of 3.32 (156/47) seeds per capsule, similar to the value re- ported by-Anderson and Sax (5) for intracrosses with four species of 53 Table 4. Testing linkage of D and E loci in I,'hirsuticaulis. EXPECTED NUMBERS PHENOTYPE GENOTYPE g, D-E'_>_.'0.50a purple,n.s. D+D+E+E+ 0 0.3125 purple D+D'E+E' 1 1.2500 blue-purple D+D+E+E' 1 0.6250 red-purple D+D'E+E+ 0 0.6250 blue,n.s. 0+0+E‘E‘ 0 0.3125 blue 0+0'E'E' 0 0.6250 o'o'c’h:+ red 0'0‘E+E' 3 1.2500 D'D'E'E’ calculated X2, 6 d.f. 4.60 0.25 0.05 aRed/blue twin sectors comprised of’a single red and a single blue sector within the hair were used for the analysis. bThe subtotals comprise sectors from the following experiments A, Spontaneous, 34 R-preresponse and 60 R-preresponse, B. 34 R-response,. C. 60 R-response, D, 34 R-postresponse and 60 R-postresponse. cwith or without non-mutant, purple cells separating the red and the blue sector in the hair. 67 Figure 15. A schematic stamen. A schematic depiction of a stamen with red and blue mutant sectors distributed amongst its hairs to illustrate pair-by-pair analysis. The crosshatched areas represent blue mutant cells, the stipled areas, red mutant cells. All other cells are understood to be purple. This stamen, then, has one red-blue pair in the sgmg_hair, and three red-blue pairs involving sectors in different hairs, a §,g_ratio of 1:3. 68 . -7////7.. .... . eeeoo .... 32:: ..... CC... Figure 15 69 events in stamens containing some 54.5 (30) to 55.4 (Table 21)-hairs would be expected to result in a same:different (3:1) ratio of 1:53.15 - 54.4; instead. the _s_:g_ ratios found were 94:42, 71:83, and 109:193; forspontaneous '(0 R) and responses to 34' R and-1‘60 R 60Co irradiation, respective1y(Table.9).* 5&1; of these, ratios is~very significantly different, (p < 0.001), from that expected on the “basis of random association. It.might be argued that either a red— or blue-sector-producing event maderthe other more likely, (_i_.g_., facilitatedits occurrence)- Sucii would account for the demonstrated non-random distribution ofv sectors without supposing that red and blue sectors in the 'same hair were the result of a single event. The mostextreme casenwould be that of a red or a blue event rgguiring the other to occur at the next-cell division. However, as a consequence of the random segregation of mitotic-chromosomes at that division, at most only 50% of the hairs containing both a red and a blue sector could have those sectors with- out an intervening purple cell. Yet 89% (304/343, Table 8) of those lHStances in which a red and a blue sector occurred in the same hair were without an intervening purple cell: This 89% value is very significantiy different (p < 0.001) from 50%. I interpret this to mean that red and blue sectors occurring in‘a single hair constitute a Mblue twin spot (r/btflg), i.e., each such twin spotydoes indeed constitute a single entity -- the result of gge_initiating event' which is distinct from those events that produce red-only (59) or 70 Table 9. Red/blue twin sectors: Pair-by-pair analysis of all stamens with red and blue mutant sectors. experiment numbers calculated value X2 P Samea Differentb 5/72 Spontaneous 75 36 10/72 Spontaneous 7 3 homogeneity 1 .6037 > 0.500 6/72 33 R-preresponse 3 0 vs. 1:53.5 3411.6 < 0.001 10/72 34 R-preresponse 9 3 vs. l:54.4 3483.8 < 0.001 0 R. TOTAL 94 42 6/ 72 33 R-response 46 60 homogeneity 1.0245 > 0.750 10/72 34'R-response 25 23 vs. 1:53.5 1672.8