Hollingshead, Lillian,      1900-

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Lillian Hollingshead

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Chromosomal chimeras in Crepis,

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Chromosomal chimeras in Crepis

CHROMOSOMAL CHIMERAS IN CREPISLILLIAN HOLLINGSHEADty--^University of California Publications in Agricultural SciencesVolume 2, No. 12, pp. 343-354, plates 54, 55, 2 figures in textIssued March 26, 1928University of California PressBerkeley, CaliforniaCambridge University PressLondon, EnglandCHROMOSOMAL CHIMERAS IN CREPISBYLILLIAN HOLLINGSHEADGates (1924) stated that "it is unknown at the present time howwidespread polyploidy in somatic tissues may be." Several writershave since that time reported cases of somatic polyploidy and it wasthought worth while to put on record instances of this conditionrecently discovered in the Genetics Laboratory of the University ofCalifoi'nia.In the course of an examination of root tips of various Crepisspecies and species hybrids, two plants which were partly tetraploidhave been found. ^ These are not the first cases of chromosomal chi-meras reported in Crepis, Lesley (1925) and Nawaschin (1926) havingpreviously recorded the phenomenon. Lesley 's report is a note statingthat in an P^ between C. biennis («= 20) and C. foetida (w^5)a few neighboring cells were found having about twice 25 chromo-somes, whereas most of the cells contained the expected 25. Nawaschinreported the occurrence of a tetraploid area in the form of a narrowsector in a diploid root of C. Bioscoridis (w^4).The first of the two cases to be described was that of a chimeralroot of a derivative from a cross between Crepis biennis {n= 20) andC. setosa (^= 4) which had 24 chromosomes in most of the somaticcells. In this root, however, a large number of cells was found whichobviously had many more than the noi*mal 24 chromosomes, severalapproximated 48, and two cells gave clear accounts of 48 chromosomes.The normal and tetraploid chromosome complexes are shown infigure A. By an examination of successive sections it was determinedthat the tetraploid cells were confined to a definite region whichextended from at least vei-y close to the root cap to a point where nodivision figures could be found. Apparently the longitudinal outlinesof the tetraploid area were fairly regular, since only one case occurredin which diploid and tetraploid cells were found in different sectionsoccupying the same position relative to the circumference, and thiswas on the line of demarcation between the two areas.1 Since writing the above a root of C. Hakelei (/i^8) containing severalneighboring cells with about twice the normal chromosome number and one ofC. mothtana (w^6) with one tetraploid cell have been found.344 University of California Publications in Agricultural Sciences [Vol. 2Figure B was made up from an examination of the whole root andshows that the tetraploid area occupied the major portion of the rootand that its cross-section was very irregular in shape. The dottedlines indicate the portions of the boundary between the 2n and 4«areas which could not be accurately determined. It is uncertainwhether the tetraploid area extended into the central cylinder or not.The tetraploid area at a is two cell layers deep, at h it is only one, butopposite a at c it occupies most or all of the cortex. While there isconsiderable variation in cell size \vithin both areas the average sizeof the tetraploid cells is larger than that of the diploid.^8S>»^•^Fig. A. 1, Diploid chromosome complex of biennis-setosa liybrid derivative(2n= 24). 2, Tetraploid complex (2w^48) from chimeral root of the sameplant.The shape of the tetraploid area is of some interest from the pointof view of development as presumably the doubling of chromosomestook place in one of the initial cells from which the root developed.The area does not show the comparative regularity in shape exhibitedby Nawaschin's tetraploid sector. On the other hand, tetraploidy hereis confined to one definite area, which was not the case with Lesley's(1925) tomato chimeras, where isolated areas of tetraploid cells wereobserved. The condition is similar to that which Langlet (1927)found in two roots of Thalictrum but differs from that reported byWinge (1927) in Tragopogon hybrids where the tetraploid parts oftwo roots by reason of their larger cells rendered the cross-sectionsof the roots eccentric. The plant bearing this chimeral root was ofnormal appearance in the rosette stage but unfortunately died beforeflowering.The second case showing a chimeral condition was a plant ofCrepis Burcniana (?^= 4). Of the thirty roots of this plant whichwere examined two were tetraploid, having 16 chromosomes in all the1928] Hollingsliead : Chromosomal Chimeras in Crcpis 345plates observed. Plate 54, figures a and b, shows the chromosome com-plexes with surrounding areas from the outer cortex in comparableregions of the diploid and tetraploid roots. Each of the chromosomesof the diploid complex could be recognized in the tetraploid and thelongest one of the set could be identified four times in the best tetra-ploid plate. Undoubtedly there has been a doubling of the diploidset. An examination of plate 54 shows that the average size of cellsand nuclei is larger in the tetraploid root.Fig. B. A cross-section of root of hiennis-setosa hybrid derivative showingthe extent of the tetraploid area. The outer ring is the extension of the root capand contains no dividing cells.The occurrence of two tetraploid roots in the same plant may betaken as evidence that part of the central cylinder from whichbranches arise had become tetraploid. It was thought possible thatthe plant above ground might have been affected similarly, and if so,the pollen produced on a tetraploid branch would be larger than thaton a diploid. Examination of pollen fi*om various branches, however,showed no noticeable size differences, so it was concluded that tetra-ploidy was probably confined to the roots.In this same plant a root was examined which contained a numberof large cells. Most of them Avere clearly multinucleate, from two tofour nuclei having been counted in single cells (pi. 55). Plate 55 hshows one of the smallest of these with 2 nuclei, and 2 nuclei may beobserved in one of the cells in a. In the larger cells nuclei were to befound in successive sections. These cells were scattered throughout346 University of California Puhlicaticns in Agricultural Sciences [Vol. 2the cortex, mostly near the i^eriphery, and in one region a group ofthem seems to be responsible for the somewhat misshapen appearanceof the root in cross-section (pi. 55 a,c). The cells vary in size, some-times extending through three or four sections 7/x thick. They aremore or less vacuolated, depending on their size, but even the smallestones could be distinguished almost at a glance by their less densely,staining cytoplasm.The normal chromosome complex of 8 was to be seen in severalplates of normal cells. In one very large vacuolated cell a largenumber of chromosomes was observed, apparently in metaphase(pi. 55 c?). Note the V-shaped arrangement of the metaphase plate.The chromosomes could be seen in 3 successive sections of 7/i, thickness.Nawaschin (1926) has reported a giant cell with over 500 chi'omo-somes in a root tip of Crcpis tectorum and takes up favorably thetheory that it has arisen by successive chromosome divisions withoutaccompanying cell divisions. The cell he shows is not greatly unlike• some of tliose seen in this material. Here, however, there seems to besome evidence that the large cells are the result of fusion of severalsmaller ones. The V-shape of the one plate observed in a giant cellindicates that it may be a combination of two plates and that a nuclearfusion has taken place. Plate 55 c gives the impression that two largecells are in the process of fusion and indeed the cell wall has practic-ally disappeared. It seems likely that such cell fusion might be asso-ciated with an abnormal or pathological condition of the root, furtherevidence of which was to be seen in small black areas probably repre-senting degenerated cells (pi. 55 b).The origin of tetraploidy in diploid tissue has been discussed byvarious investigators. In some cases it has been associated with specificoutside influences. The effect of narcotics in inducing tetraploidy hasbeen investigated by Nemec (1903) and Sakamura (1920). Blakesleeand Belling (1924) found tetraploid shoots in Datura plants subjectedto cold. Lesley (1926) found tetraploid areas in tomato plantsaffected by mosaic and thought it might be jiossible that local changesdue to this disease might affect mitotic processes. Cases of polyploidcells have been attributed to abnormal processes related to degenera-tion as in the investing cells of the ovaries of Anasa tristis (Wilson,1906). The doubling of chromosome numbers in Winkler's (1916)Avell-kno^Mi chimeras has been attributed to the effect of wounding.Jorgensen and Crane (1927) have recently secured tetraploidy inSolaitum by the use of Winkler's method. Winge (1927) finds that3928] Hnllingshead : Chromosomal Chimeras in Crepis 347most of the cells of the "crown galls" on sugar beets which can beinduced by inoculation with Bacterium tumefaciens have the tetra-ploid chromosome number.Nawaschin did not venture any suggestion as to causal agencies inconnection with his tetraploid sector in Crepis Dioscoridis. Lesleybelieved that it was unlikely that cold played any part as a causalfactor in tomato chimeras, as only the roots seemed to be affected. Ithas been suggested by Mr. C. W. Haney that watering greenhouseplants with cold water would pro\'ide the necessary conditions if sud-den lowering of temperature has anything to do with the productionof tetraploid root cells. The plants described here were entirelynormal as far as could be observed and tetraploidy could not beascribed to any special factor in the environment.Winkler had suggested that certain tissues may regularly becomepolyploid and Breslawetz (1926) has reported tetraploidy as theuniversal condition in the dermatogen of the root tips of Cannabissativa.- De Litardiere (1923) found tetraploid and octoploid cellsin the deraiatogen of Spinacia oleraeea. In both these cases it wouldseem that the transforming of diploid into tetraploid cells must haveoceui-red many times in the same root.The possibility of fragmentation giving rise to these increasednumbers is easily excluded in most cases. The two most favorablyreceived theories to account for doubling are: (1) the fusion of nucleifrom two cells; (2) the division of the chromosome complex withoutcytoplasmic division. Breslawetz has brought forward evidence thatnuclear fusion gave rise to the tetraploid cells which made up thedermatogen of the roots in Cannabis sativa. As no diploid complexeswere to be seen in that region of the root one would conclude thatfusion of diploid to form tetraploid nuclei had taken place before anynormal diploid divisions occurred, or at least at an early stage in thedevelopment of the root. If so, one wonders why evidences of nuclearfusion were still to be found in well developed roots. On the otherhand, the paired condition of the chromosomes in some of the tetra-ploid cells in Spinacia oleraeea convinced de Litardiere that thesecells had just completed a chromosome division without separation ofthe resulting daughter chromosomes.The occurrence of multinucleate cells in a root of the CrepisBureniana plant which was partly tetraploid has been noted above.The significance of this phenomenon in the origin of the tetraploid2 De Litardiere (1924) found rare diploid cells in the peribleni and in oneease a tetraploid cell in the plerome of roots of this species.348 University of California Publications in A gricttltwal Sciences [Vol. 2roots is questionable. The presence of several nuclei in one cell andthe abnormal appearance of these large cells would incline one tobelittle the possibility that tetraploidy here had originated by cell andnuclear fusion. However, it has been pointed out that the one chromo-some complex observed in a giant cell indicated that nuclear fusionhad taken place. We cannot, therefore, dismiss the possibility that afusion of nuclei from two cells gave rise to a cell which was tetraploidand thence to tetraploid roots. It seems more likely, however, that theoccurrence of tetraploidy and of a root with giant multinucleate cellsin the same plant was merely a coincidence.Whatever the method of origin, the frequent occurrence of tetra-ploidy in somatic tissues throws some light on two much discussedquestions. First, there is that of the mode of origin of diploid gametes.Rosenberg (1926-27), Karpechenko (1927), and others have describedprocesses in the reduction divisions of apogamous species and inter-specific hybrids bj' which diploid gametes are formed. The increasingfrequency with which tetraploidy has been recorded in root tips makesit seem likely that it would be found in other tissues were theyexamined as consistently. Its occurrence in the cells of the germinalline would lead to the formation of gametes with twice the normalchromosome number. This lias been shown to occur in Datura wheretetraploid shoots were found. Presumably a smaller area might beaffected and only a portion of the gametes formed on one shoot mightbe diploid.In the second place, the frequent occurrence of somatic tetraploidyhas a bearing on the origin of tetraploids and tetraploid hybrids.Prirmda kewensis arose as a bud sport probably from an Fi hybrid ofP. verticillata and P. florihunda. It has the sum of the diploid chro-mosome numbers of the parent species and Clausen and Goodspeed(1925) have suggested that it is a true tetraploid hybrid, the bud sporthaving arisen by a doubling of somatic chromosomes. A similarexplanation with the doubling occurring immediately subsequent tofertilization was suggested by these investigators to explain the originof a tetraploid hybrid between Nicotiana tabacum and N. glutinosa.Rosenberg (1926) has recently proposed an explanation for the originof the tetraploid AcgUops—Tritic^im hybrid of Tschermak and Bleier(1926) which depends on the chance meeting of diploid gametesformed by a " semi-heterotypic " division. In the light of the fore-going facts it seems much simpler to suppose that a doubling of thechromosomes took place in the fertilized egg, or in some cell of the1928] Hollingshead : Chromosomal Chimeras in Crepis 349young embryo which gave rise to the growing point of tlie stem.Nawaschin was led to favor this theory of the origin of tetraploids bythe frequency of 4j( plants in Crepis tecforum. He calculated the fre-quency of diploid gametes from the number of triploid plants obtainedin over 4,000 individuals, and on this basis determined the number oftetraploids which should occur by chance meeting of those gametes.He found the expected number of tetraploids to be much less than thatactually occurring. He concluded, therefore, that tetraploids arosethrough the doubling of chromosomes in the fertilized egg cells.In view of the increasing number of cases in which tetraploidy hasarisen in normal diploid tissue, one is justified in concluding that itmay play a part in the origin of polyploid species and interspecifichybrids.I acknowledge with pleasure my indebtedness to Dr. J. L. Collinsand Professor E. Bi Babcock for the material used in this study.SUMMARYTetraploidy was observed in the roots of two different plants. One,a C. biennis x C. setosa hybrid derivative, had one root partly tetra-ploid. The other, a plant of C. Bureniana, had two roots whollytetraploid.No external factors could be associated with the tetraploidy.Giant multinucleate vacuolated cells occurred in another root ofthe same C. Bureniana plant. Evidence of cell and nuclear fusion wasobserved. It is doubtful whether this phenomenon has any significancein relation to the origin of the tetrajjloid roots.Tetraploidy arising in somatic tissue probably plays a part in theorigin of polyploid species and intei-specific hybrids.350 University of California Puhlications in Agricultural Sciences [Vol.2LITERATURE CITEDBlakeslee, a. F., and Belling, J.1924. Chromosomal chimeras in the Jimson weed. Science, vol. 60, pp. 19-20.Breslawetz, L.1926. Polyploide Mitosen bei Cannabis sativa L. Berichte der deut. Bot.Gesell., vol. 44, pp. 498-502.Clausen, E. E., and Goodspeed, T. H.1925. Interspecific hybridization in Nicotiana. II. A tetraploid glutinosa-tahacum hybrid, an experimental verification of Wiiige's hypothesis.Genetics, vol. 10, pp. 278-84.Gates, E. E.1924. Polyploidy. Brit. Jour. Exper. Biol., vol. 1, pp. 153-82.JORGENSEN, C. A., AND CrANE, M. B.1927. Formation and morphology of Solnnum chimeras. Jour. Gen., vol. 18,pp. 247-73.Kaepechenko, G. D.1927. The production of polyploid gametes in hybrids. Hereditas, vol. 9,pp. 349-68.Langlet, O. F.1927. Beitriige zur Zytologie der Eanunculazeen. Svensk. Bot. Tidskr.,vol. 21, pp. 1-17.Lesley, M. M.1925. Chromosome chimeras in the tomato. Am. Nat., vol. 59, pp. 570-74.LiTAKDIEEE, M. E. DE1923. Les anomalies de la caryocinese somatique chez le Spinacia olcracea L.Eevue Gen. Bot., vol. 35, pp. 369-81.1924. Sur 1 'existence de figures didiploides dans le meristeme radiculairedu Cannabis sativa L. La Cellule, vol. 35, pp. 21-25.Nawaschin, M.1926. Variabilitat des Zellkems bei Crepis Arten in Bezug auf die Artbildung.Zeitschr. f. Zellf. u. Mikr. Anat., vol. 4, pp. 171-215.Nemec, B.1903. Tiber die Einwirkung des Chloral-hydrates auf die Kern und Zell-teilung. Jahrb. Wiss. Bot., vol. 39, pp. 645-730.EOSENBERG, O.1926. tJber die Verdoppelung der Chromosomenzahl nach Bastierdung.Berichte der deut. Bot. Gesell., vol. 44, pp. 455-60.1926-27. Die semiheterotypische Teilung und ihre Bedeutung fiir die Entste-hung verdoppelter Chromosomenzahlen. Hereditas, vol. 8, pp. 305-38.1928] nollingsliead: Cliromosomal Chimeras in Crrpis 351Sakamura, T.1920. Experimcntelle Studien iibei" die Zell und Kernteihing mit besondererRiieksiclit auf Form, Grosse, mid Zalil der Chromosomoi. Jour.Coll. Sci. Imp. Univ. Tokyo, vol. 39, i)p. 1-221.TSCHEBMAK, E., UND BLEIEH, H.1926. Tiber fruehtbare Aegilops Weizenbastarde. (Beispiele fiir die Eutste-hung neuer Arten durch Bastardierung.) Berichte der deut. Bot.Gesell., vol. 44, pp. 110-32.Wilson, E. B.1906. Studies on chromosomes III. Jour. Exper. Zool., vol. 3, pp. 1-40.WlNGE, O.1927. Zytologisfhe Untersuehungen iiber die Natur maligner Tumoren.I. "Crown gall" der Zuckerriibe. Zeitsclir. f. Zellf. u. Mikr.Anat., vol. 6, pp. 397-423.Winkler, H.1916. tJber die experimentelle Erzeugung von Pflanzen mit abweichendenChromosomenzahlen. Zeitsclir. f. Bot., vol. 8, pp. 417-.531.PLATE 54Comparable areas of a, diploid, b, tetrnploid roots of a Crepis Burenkina plant.[352]UNIV CALIF PUBL AGR. SCI . VOL 2 HOLLINGSHEAD I PLATE 54-^-rPLATE 55Photomicrographs of root of the Crepis Burcniana plant showing giant multi-nucleate cells.a. A cross-section showing a group of giant cells.h. One of the smaller giant cells with two nuclei.c. Two giant cells apparently fusing.d. A large cell containing a V-shaped metaphase plate with many chromosomes.[354]UNIV. CALIF. PUBL. AGR. SCI . VOL 2 HOLLINGSHEAD I PLATE 55. fr "'.'tlRi*-^!§^B'^^'^"^t^'^m%*M^>'f.V-*>.,.^ m^ .*V

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Chromosomal chimeras in Crepis,

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