Sclereid Distribution in the Leaves of Pseudotsuga Under Natural and Experimental Conditions Author(S): Khalil H

Home , Cortex (botany), Sclereid

Sclereid Distribution in the Leaves of Pseudotsuga Under Natural and Experimental Conditions Author(s): Khalil H. Al-Talib and John G. Torrey Source: American Journal of Botany, Vol. 48, No. 1 (Jan., 1961), pp. 71-79 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2439597 . Accessed: 19/08/2011 13:16

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected].

Botanical Society of America is collaborating with JSTOR to digitize, preserve and extend access to American Journal of Botany.

http://www.jstor.org January, 1961] AL-TALIB AND TORREY-SCLEREID DISTRIBUTION 71

SMITH, G. H. 1926. Vascular anatomyof Ranalian flowers. Aquilegia formosav. truncata and Ranunculus repens. I. Ranunculaceae. Bot. Gaz. 82: 1-29. Univ. California Publ. Bot. 25: 513-648. . 1928. Vascular anatomy of Ranalian flowers. II. TUCKER, SHIRLEY C. 1959. Ontogeny of the inflorescence Ranunculaceae (continued), Menispermaceae,Calycan- and the flowerin Drimys winteri v. chilensis. Univ. thaceae, Annonaceae. Bot. Gaz. 85: 152-177. California Publ. Bot. 30: 257-335. SNOW, MARY, AND R. SNOW. 1947. On the determination . 1960. Ontogeny of the floral apex of Micheiat of leaves. New Phytol. 46: 5-19. fuscata. Amer. Jour. Bot. 47: 266-277. SNOW, R. 1942. Further experimentson whorled phyllo- WARDLAW, C. W. 1949. Experimentaland analytical stud- taxis. New Phytol. 41: 108-124. ies of pteridophytes.XIV. Leaf formationand phyllo- STERLING, C. 1945. Growth and vascular developmentin taxis in Dryopterisaristata. Ann. Bot. (N.S.) 13: 163- the shoot apex of Sequoia sempervirens.II. Vascular 198. developmentin relation to phyllotaxis.Amer. Jour.Bot. . 1957. On the organization and reactivityof the 32: 118-126. shoot apex in vascular plants. Amer. Jour. Bot. 44: TEPFER, S. S. 1953. Floral anatomy and ontogeny in 176-185.

SCLEREID DISTRIBUTION IN THE LEAVES OF PSEUDOTSUGA UNDER NATURALAND EXPERIMENTALCONDITIONS' KHALIL H. AL-TALIB AND JOHN G. TORREY

ABSTRA CT

AL-TALIB, KHALIL H., and JOHN G. TORREY. (U. California,Berkeley.) Sclereid distributionin the leaves of Pseudotsuga under natural and experimentalconditions. Amer. Jour. Bot. 48(1): 71-79. Illus. 1961.-A studyof the distributionof sclereids in cleared leaves taken from1-, 2-, and 4-year-oldshoots of an adult tree of Pseudotsuga menziesii (Mirb.) Franco showed a repeated patternof sclereid distributionalong the shoot axis with many sclereids in the basal leaves grading into few or no sclereids in the terminal leaves of each year's growth. Attemptswere made to influencesclereid distributionby bud defoliationof attached branches with and without auxin treatmentand by testing the effectsof growth-regulatingsubstances on sclereid formationin leaves of excised buds of Pseudotsuga cultured in vitro. Whereas removal of the basal 84 of the leaves at the time of bud unfoldinghad no effecton bud, leaf or sclereid development,removal of the leaves of the upper half or complete defoliationled to prematureexpansion of next year's terminalbud with leaves developingin part from presumptivebud-scale primordia. Indoleacetic acid at 0.5% in lanolin paste applied to the defoliated region preventedthis prematurebud expansion. Defoliation of the basal half did not affectsclereid formationin the terminal leaves. Sclereid developmentin leaves of prematurelyexpanded buds on defoliated branches was normal except in the few cases where bud expansion occurredin the presence of low-auxinconcentrations. Then, sclereid developmentwas inhibited. Sclereid formationin leaves of excised buds grown in nutrientculture was generally much less frequentthan in intact branches, and auxin treatment still furtherreduced the frequencyof sclereids. It was concluded that sclereid initiationand differ- entiationin the intact plant may well be under the control of hormonalfactors in the plant, one of which may be auxin.

IN recentyears, sclerenchymatousidioblasts or tissues of various membersof the Coniferales. Allen sclereidshave been the subject of muchinvestiga- (1945) noted the occurrence of sclereids in the tionby plantanatomists. Their occurrence has been leaves of Pseudotsuga taxifolia. Sterling (1947) noted in leaves, stems,roots, fruitsand seeds of studied the ontogeny and morphology of the scle- angiosperms.Further, their morphologyand on- reids in the shoot of P. taxifolia and observed dif- togenyhave been studiedand theirtaxonomic im- ferences in the number of sclereids between indi- portanceevaluated (Foster, 1944, 1945, 1946, 1947, vidual branches. He also observed that sclereids in 1955a, 1955b,1956; Bloch,1946; Bailey and Nast, the cortex were less abundant but were larger than 1948; Rao, 1951). those in the pith. Griffith(1950) reported the In gymnosperms,sclereids have been less in- presence of numerous crystalliferous idioblastic tensivelystudied; theyhave been shownto occur, sclereids in the leaves of certain species of Arau- however,in both the vegetativeand reproductive caria. Sacher (1954) found that sclereids occur in the cortex of shoots of Pinus ponderosa, extending 1 Received for publication April 25, 1960. up to within 2 mm. of the shoot apex. Kitamura This work was supported in part by Research Grant (1956) studied the distributionof foliar sclereids Health Serv- RG 2861, National Institutesof Health, Public verticillata and found that various ice. The authors express their appreciation to Dr. A. S. of Sciadopitys Foster and Dr. L. Machlis for helpful advice. zones of the leaf differin the number of sclereids 72 AMERICAN JOURNAL OF BOTANY [VO1. v

l j S &0 ' f: ? t

. , , ..

,.,...... - . :.x : . . j::: fi .;,-: . ' '' ...... :.; . ., .-. . . : .... r . , .. . .: . .. !o- :...... - :

...... 1w

. ... . I . r

.. I ' : . . :. ,., ......

' 1 .::

' . . : . ': j : : . . ! .l ;> ,' '.'.'.',.'' . .. ,<.

.: . . , ' :Ri < . .. . : .: : :

',:.,s,. 1.: ' . . . . t . .:: . u .: .: . ', S .. d C 00000X ff SE ;j {0000nDS00; 0

" 0 aV0000- 0,0f'0,j,'"'00000 M\\s'S} wi} . _ !; ' - W.t, ^;7 t j ,' t50'; y< t X1i; 011'f' f ' 'Ds

y \ . \ 111|0f'leg

* . ,. .i . : . : S1: 42 i3-;itv

... . *

_ .r :

. . I S ...... _l | i| S I __ i l l I l * - 1@ ...... | l|. I!, i1l-I1 | ,_-- ._ : January, 1961 1 AL-TALIB AND TORREY-SCLEREID DISTRIBUTION 73

TABLE 1. Sclereid distributionin leaves along the shoot axis ziesii in natureand an accountof certainexperi- of 1- and 2-year-oldbranches of Pseudotsuga menziesii ments designed to affectthe regular patternof sclereidformation. A. 1-year-oldbranch (1954) MATERIALS AND METHODS.--Freshleaves and buds Segmentnumber Averagenumber of were obtained froma single old tree of P. menziesii, (15.leaves per Numberof sclereids sclereids per approximately50 ft. tall and 1 ft. in diameterat segment) per segment leaf and S.E. the base, growingin the BotanicalGarden, Univer- 1 (Terminal) 32 2.13 ? 0.41 sity of California,Berkeley. Preservedspecimens 2 128 8.53 ? 0.72 of P. menziesii, P. macrocarpa, P. glauca and P. 3 228 15.20 ? 0.82 sinensis were obtained from the Herbarium of the 4 338 22.53 0.79 ? of 5 (Basal) 286 19.07 ? 2.95 University California. The freshmaterial was collectedat monthlyin- B. 2-year-oldbranch tervalsfrom October 1953 to June1957, was killed 1954 and fixedin formalin-aceticacid-alcohol (FAA) Averagenum- withaspiration and was thenstored in freshFAA Number of ber of sclereids untilneeded. For thestudy of sclereiddistribution, Segment leaves in Total number per leaf and number segment of sclereids S.E. large numbersof leaves frombranches at various levelson the treewere collected, marked according 1 (Terminal) 20 31 1.55 ? 0.27 2 19 158 8.33 ? 0.62 to theirlocation along a branchand thencleared 3 15 245 16.33 ? 0.65 as wholeleaves in sodiumhydroxide. 4 16 428 26.75 ? 0.95 For clearing,the leaves werefirst boiled in 70% 5 (Basal) 18 450 25.0 ? 2.28 ethylalcohol to removepigments (several hours forold leaves fromthe intact plant; 30-60 min.for 1953 1 (Terminal) 15 34 2.27 ? 0.35 leaves fromcultured buds). Afterwashing in water, 2 17 99 5.82 ? 0.38 the leaves were placed in sodium hydroxide solu- 3 18 228 12.67 ? 0.72 tion (5% for old leaves; 2.5/o for young leaves 4 19 356 18.74 ? 0.59 from cultured buds) and placed in an oven at 35- 5 15 258 17.20 ? 1.87 40?C. Several changes of sodium hydroxide solu- tion were needed to complete clearing in some cases. Afterwashing with water again, the leaves were de- presentwith the highest number of sclereidsoccur- hydrated through an ethyl alcohol series to 70%, ringtoward the tip of the leaf. Withthe exception at which stage they were stained with safranin. De- of the studyby Kitamura,there appears to be no hydration was then completed through to absolute quantitativestudy of sclereiddistribution in plants. ethylalcohol, followedby 2 washes of absolute ethyl Duringa preliminarystudy of the occurrenceof alcohol-xylene (1:1) and finallywith pure xylene, sclereids in the leaves of Pseudotsuga menziesii then mountingin piccolyte. In cleared preparations (Mirb.) Franco2 (Douglas fir), therewas noteda of this type, cellular detail is quite evident and the ratherstriking and distinctivepattern of sclereid large ramifyingsclereids are readily discernible un- distributionin theleaves along theshoot axis. The der relativelylow magnification (fig. 1). regularityof thepattern suggested that sclereid formationin leaves of P. menziesiiwas rigidlycon- Defoliation experiments on the intact tree were trolledby internalphysiological factors and that performedin order to determinethe effect,if any, experimentalmethods might be used to upset the of leaf removal on the distribution of sclereids in regularityof sclereidformation in an attemptto the remaining leaves. Complete or partial defolia- discoverthe physiologicalfactors affecting sclereid tion of the newly formedbranches was made slight- distribution.In this paper are presentedthe data ly before or at the time of expansion of the bud, at concerningsclereid formation in leaves of P. men- which occurs between February and April Berke- ley. The unopened buds were descaled with forceps 2 Recent nomenclaturalchanges require the use of this and then the leaves removed individually with scis- name in place of the more familiar one, P. taxifolia sors and sharp-pointedforceps. Lanolin paste was (Lamb.) Britt. applied to the defoliated areas of the branches to

Fig. 1-3; 5-8.-Fig. 1-3. Cleared leaves of Pseudotsuga menziesii.-Fig. 1. Portion of cleared leaf of P. mnenziesii, showingmature ramifying selereids in spongyparenchyma on either side of the midrib. The elliptical structuresare tylo- soids in the resin canal cavity. X45.-Fig. 2. Cleared whole leaf of P. menziesii taken fromthe basal part of a 1-yr.-old branch. Note the abundance of stellate-shapedsclereids. X5.-Fig. 3. Cleared whole leaf of P. mnenziesiitaken fromthe uppermostpart of a 1-yr.-oldbranch. Note the completelack of sclereids. X 5.-Fig. 5. One-yr.-oldbranches of P. menziesii: left,untreated intact branch with next year's unexpanded terminalbud; center,completely defoliated branch treated withlanolin paste, showingnext year's prematurelyexpanded terminalbud and lateral buds; right,completely defoliated branch treatedwith 0.5%oIAA in lanolin, showingnext year's unexpanded terminalbud. X1.-Fig. 6. Cleared whole leaf of P. sinensis,showing numerous filiform sclereids. X7.5.-Fig. 7-8. Cleared leaves of P. menziesii taken from excised buds culturedin vitrofor 2 mo. X1O.-Fig. 7. Medium contained 10-6 M indolebutyricacid.-Fig. 8. Medium contained 10-4 M indolebutyricacid. Note complete absence of scleieids. 74 AMERICAN JOURNAL OF BOTANY [Vol. 48

TABLE 2. Parallel samples of leaves of P. menziesii with Mouriria and Boronia where they are associated and withoutlateral buds in their axilsa withvein endings(Foster, 1947, 1955b). A wide range of variation in the numberof Number of leaves Total numberof sclereids in sample sclereidsin the leaves of an individualbranch was in sample Bud in axil No bud in axil noted. The differencein sclereid numberin the 1 19 18 basal leaves of a 1-yr.-oldbranchlet (fig. 2) and in 1 10 11 the terminalleaves of the same branchlet(fig. 3) 1 14 14 was verystriking. A detailedanalysis was made of 1 23 22 the averagenumber of sclereidsin theleaves in re- 4 91 (22.7) 90 (22.5) lation to the distanceof the leaves fromthe ter- 4 147 (36.8) 149 (37.2) minal bud. A 1-yr.-oldbranch was dividedalong 6 158 (26.3) 139 (23.1) the axis into segmentsof 15 leaves each and the 23 329 (14.3) 336 (14.5) totalnumber of sclereidsin the leaves of each seg-

a mentwas counted(table IA). The average number of sclereids per leaf is given in It was at firstsupposed that the observedvaria- parentheses. tionin sclereidnumber was associatedwith the age of the leaves, with the youngerleaves containing preventdesiccation. In certainexperiments, growth- fewersclereids than the olderleaves. A similaran- regulatingcompounds were added to the lanolin alysiswas made of 2-yr.-oldand 4-yr.-oldbranches. pasteto determinetheir effect on sclereidformation Fromtable lB it becomesapparent that the pattern on thepremise that the leaves whenattached might of sclereiddistribution is repeatedeach year in the influencebud expansionby virtueof theirproduc- 2-yr.-oldbranch and the selereidformation is not tion of auxin. Plain lanolinwas applied to control relatedto age of leaf per se but to positionof the branches. All applicationswere renewedevery 2 leaf along the shootaxis foreach year of shootde- days. velopment.In fig.4 is shownthe sclereiddistribution in successiveyears of shootgrowth in a 4-yr.- OBSERVATIONS AND EXPERIMENTAL RESULTS.-The old branch expressedas average numberof scle- distributionof foliar sclereidsin intactshoots.- reids per 20 leaves. For each year's new shoot Sclereidsare found as scatteredidioblasts in the growth,regardless of the age of the branch,the spongy parenchymaof the leaf of Pseudotsuga averagenumber of sclereidsper leaf is at a mini- menziesiiand are absentfrom the palisade paren- mumin theapical leaves,rises to a maximumin the chyma.They are distributedin approximatelyequal leavestoward the base untilat theextreme base the numberson both flanksof the midrib(fig. 2), but average numberdecreases. There is variationin are neverfound in thespongy parenchyma adaxial the differentmaxima; this variationis evidentin to the vein. The prevailingdiffuse distribution of table lB as wellas in fig.4. The consistentdecrease sclereidsthroughout the spongy mesophyllof P. in numberof sclereids at thebase was relatedto the menziesiicontrasts strikingly with some of the spe- factthat several of the mostbasal leaves have few cies of Monsterawhere they are restrictedto a spe- or no sclereids,so thatthe average number per seg- cificseries of mothercells (Bloch, 1946) or as in mentis reduced.

THE SCLEREID DISTRIBUTION IN A FOUR YEAR OLD BRANCH

600- w -J 500- 0

Cr w 400- a. z 3003 0 0 200- w 100 -j 0

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 I 2 3 4 5 956 1955 1954 1953 SEGMENT Fig. 4. Histogram slhowingtlhe patternof sclereid distributionalong the axis of a 4-yr.-oldbranch of P. rnenziesii.Each segmentcontained approximately20 leaves. January, 1961] AL-TALIB AND TORREY-SCLEREID DISTRIBUTION 75

TABLE 3. The distributionof sclereids in the terminaland to form. In the fully formedbut unexpanded basal samples of leaves of preservedshoot specimensof leaves, sclereid initiationhas already been com- Pseudotsugaa pleted,while sclereid initiation in primordialleaves has notyet occurred. Defoliation of theunexpanded Numberof sclereids per sample buds at the time of bud expansionwas performed Terminal Basal in orderto studythe effectof leaf removalon tife P. glauca 542 (108) 828 (165) developmentof the remainingleaves and to ascer- P. macrocarpa 204 (41) 533 (106) tain the effectof defoliationon selereid develop- P. sinensis 1006 (201) 1713 (342) ment. P. menziesii 15 (3) 93 (18) Removalof up to 3,4of the lower leaves of the branchletdid not affectthe normaldevelopment of a 5 leaves per sample. (The average numberof sclereids the remainingterminal leaves. It was found,how- per leaf is given in parentheses.) ever,that completedefoliation or removalof the leaves of the upper half of the elongatingbranch In searching' furtherfor morphological relation- caused a prematureexpansion of the next year's ships which might influence sclereid distribution, terminalbud. Since at the timeof defoliationthese leaves which bore axillary buds were sampled. A leaves were primordialand sclereidinitiation had number of leaves with lateral buds in their axils not yet been determined,it was of special interest were taken from various branches, together with to studythe effectof defoliationon leaf develop- leaves at the same level containingno axillary buds. mentand sclereidformation in these prematurely In table 2 the data presented show that the pres- expandingstructures. ence of a lateral bud does not affectthe number of Followingremoval of the leaves along the upper sclereids formedin the leaf subtendingit. Although half of the bud or all of the leaves of the bud, the number of sclereids formed in each leaf varies thereoccurred within a period of 4-8 wk. a pre- from one level of the axis to another, the data fur- matureexpansion of nextyear's bud (fig. 5). The ther indicate that the distributionof sclereids in the leaves of this newlyexpanded bud, as well as the leaves is very similar on all sides of the axis at a shoot,are considerablysmaller than normal. These given level. leaves develop from the oldest basal primordia The distributionof sclereids in the leaves of pre- which, under ordinary conditions,would have served specimens of Pseudotsuga glauca, P. macro- formedbud scales. Thus the defoliationalters the carpa, P. sinensis and P. menziesii was also studied. These specimens had been collected fromgeograph- TABLE 4. Theeffect of defoliation and ofauxin treatment ical regions outside the San Francisco Bay area. As on thepremature expansion of terminalbuds in P. is seen in the data in table 3, strikingvariations in inenziesii the average number of sclereids fromspecies to species was observed. In P. sinensis, the sclereids were Concentra- long and filiform(fig. 6) in contrastto the stellate tionof in- shape in the other species studied. In general, how- doleacetic Numberof Numberof Numberof ever, the pattern of sclereid formation in leaves acid in branches Numberof unexpand-expanded along the shoot axis was consistent,with the basal lanolin(%) tested deadbuds ed buds buds leaves containing more sclereids than the upper or Lowerhalf defoliated Be- terminal leaves within any one year's growth. 0.0 10 0 10 0 cause of the regularityof patternof sclereid forma- 0.1 10 0 10 0 tion, Pseudotsuga seemed to be particularly suited 0.5 10 0 10 0 for studies of the physiological mechanisms control- 1.0 10 4 6 0 ling the process. Experimental procedures were de- Lowerthree-fourths defoliated vised which might be expected to upset or modify 0.0 25 0 25 0 interest normal sclereid occurrence. Of particular 0.1 25 0 25 0 were experiments with defoliated shoot branches 0.5 25 0 25 0 growing on the intact tree and experiments with 1.0 25 16 9 0 isolated buds of P. menziesii cultured in vitro. Upperhalf defoliated The effectsof defoliation and auxin treatmenton development.- Within the dormant bud of 0.0 10 1 2 7 leaf 10 0 7 3 Pseudotsuga immediately prior to bud expansion 0.1 0.5 10 1 8 1 found there are the fully formed, unexpanded 1.0 10 9 1 0 leaves of the currentyear's growth. These are sur- rounded and enclosed by thin, brown, outer bud Completelydefoliated scales and inner transparent bud scales which 0.0 20 1 2 17 loosen and unfold as the bud expands. Within the 0.1 20 0 15 5 bud at the shoot apex itself,the bud scale and leaf 0.5 20 2 16 2 18 16 2 0 primordia for next year's bud are already beginning 1.0 76 AMERICAN JOURNAL OF BOTANY [Vol. 48 normaldevelopmental pattern of the foliarpriimor- expandedbuds followingdefoliation with and with. dia, causingcertain primordia to developinto leaves out auxin treatment. which otherwisewere destined to become bud In table 5 are presenteddata fromcounts made scales. of leaves fromprematurely expanded buds follow- The timeof defoliationis critical,there being a ing completedefoliation. In one treatment,lanolin limitedperiod only duringwhich defoliation will only was applied to the defoliatedarea; in other producepremature bud expansion.In Berkeley,only treatmentslanolin containing0.1% and 0.5%c in- duringApril and May, whenthe meristem is form- doleaceticacid had been applied. The leaves were ing primordiawhich normally form scales, was it killed and cleared afterthey had completedtheir possibleto cause prematurebud expansionthrough expansion.Since auxintreatment usually suppresses defoliation. bud expansion,only in those cases wherebud ex- Experimentswere set up to determinethe rela- pansion did occur,such as at low IAA concentra- tiveeffectiveness of partialdefoliation of the upper tion, were determinationsof sclereid distribution and lowerportions of the branchand completede- possible. foliationwith and withoutauxin treatmenton bud The numberof sclereidsin the leaves of newly expansion. The resultsof these experimentsare expandedbuds followingdefoliation and treatment summarizedin table 4. withplain lanolinwas foundto vary widely,from is It apparentthat the presenceof the leaves pre- as fewas 3 or 4 to as manyas 30 or 35 per leaf. vents prematureexpansion of the terminalbud. In general,however, the average numberof scle- However,it is clear that only the terminalleaves, reids per leaf approachedthat of normalleaves. i.e., onlythe upper1/4 of theleaves along the axis, Thus,primordia which would normally develop into contributesignificantly to the suppressionof bud bud scales lacking sclereidshave developed into expansion,since no bud expansionoccurred when essentiallynormal leaves withtypical sclereid dis- up to 3/iof thelower leaves were removed. One can tribution. also concludefrom this experiment that indoleacetic Auxin treatment,which was shown to suppress acid appliedto the stemsurface in place of the up- leaf expansionin the terminalbud, appears also to per leaves afterdefoliation effectively prevents bud inhibitsclereid formation in thoseleaves whichdo expansion at relatively low concentrationfor developfollowing defoliation. this methodof treatment.At high concentration, Sclereid formationin leaves of isolated buds the auxin becomestoxic. It is interestingto note grownin vitro.-In experimentswith isolated buds that the inhibitoryeffect must be transmittedin of P. menziesiigrown in nutrientmedia (Al-Talib nonpolarfashion toward the apex. Completede- and Torrey,1959), leaf developmentwas foundto foliationdiffers in its effectfrom defoliation of the be markedlyinfluenced by the constituentsof the upper half in that,besides inducinga higherpro- medium.Of particularinterest was the effectof portionof prematurelyexpanded terminal buds, it auxins oII leaf developmentand of sclereidinitia- causes earlierterminal bud expansionand also ex- tionin thesebuds. Leaf expansionoccurred in the of pansion lateralbuds along the axis (fig. 5). absenceof added auxin in the mediumand, in the The effectof defoliationand auxin treatmenton case of mostauxins, when they were added at con- sclereid of the lower of formation.-Defoliation %/ centrationsof 10-6 M. However,a-naphthalene- the branchdid not affectthe numberof sclereids acetic acid (NAA) and 2,4-dichlorophenoxyacetic in the remainingterminal leaves of the bud. The acid (2,4-D) at 10- M stimulatedcallus develop- lack of effecton sclereiddistribution in theseter- minal leaves of the bud was not unexpectedsince theterminal leaves at the timeof bud openinghad TABLE 5. Sclereid formationin leaves of prematurelyex- alreadyformed their sclereid initials. The earlyde- panded buds of P. menziesiifollowing defoliation, with terminationof sclereid initials in all the fully and withoutIAA treatment formedleaves of the expandingbud precludedthe study of factors in Numberof Total number Averagenum- influencingsclereid initiation Sample newlyformed of sclereids ber of scle- theseleaves. However,in thecase of thepremature number leaves in sample reids per leaf expansion of the bud of next year's growth following defoliation,sclereid initiation actually occurs as Lanolin o01l) a part of the developmentof the leaves from their 1 23 191 8.3 primordial condition at the meristem. Here the ef- 2 11 210 19.] fects of defoliation and auxin treatmenton sclereid 3 15 163 11.0 formationcan be observed. Lanolin plus 0.1% IAA During the course of these studies on Pseudotsu- 1 7 6 0.9 ga, no sclereid formationin the bud scales of vege- 2 12 23 1.9 tative buds was ever observed. On occasion, scle- 3 8 5 0.6 reids were found in the scales and bracts of the Lanolin plus 0.5% IAA female cones. It was interesting,therefore, to ob- 1 14 6 0.4 serve sclereid formation in leaves of prematurely January, 1961] AL-TALIB AND TORREY-SCLEREID DISTRIBUTION 77 mentat the expenseof leaf growth.At highercon- (1926) observedthat the presenceof youngleaves centrations (10-5 and 10-4 M), all auxins tested retardedthe growth of axillarybuds. Thimannand retarded leaf expansion. Two reputed auxin an- Skoog (1934) studiedthe inhibitorysubstance in tagonists, 2,6-dichlorophenoxyaceticacid (2,6-D) Vicia faba and were able to reproducethe inhibi- and 2,3,5-triiodobenzoic acid (TIBA) at 10- M tory effectwith externally supplied auxin. Avery had no effecton leaf development. At higher con- (1935) showedthat the amountof auxin in leaves centrations,they also produced a retardationof leaf tendsto vary inverselywith their age, an observa- expansion. tion which has been confirmedby more recent In table 6 are presented data showing the effect workers(e.g., Wetmoreand Jacobs,1953). Delisle of various growth-regulatingsubstances related to (1937) and Goodwin (1937) were both able to auxins on sclereid formation in leaves of excised demonstratethat inhibitory influences from young buds culturedin vitro. The leaves examined in this leaves affectingassociated lateral bud or leaf devel- experiment were taken at random from the basal opmentcould be replaced by the applicationof part of 10 buds cultured for 2 mo. beginning in auxin in lanolin on the petiolarstumps of the ex- November under conditions described by Al-Talib cised leaves. and Torrey (1959). The basic medium containing All of thesestudies suggested that in Pseudotsuga 2% sucrose and added auxin was used throughout. auxin production,especially by the terminalleaves It is evident that growth regulators at a concen- in the expandingshoot, may act to preventthe pre- tration of 10- M, where they did not cause actual matureexpansion of the terminalbud. The experi- death of the bud, induced a very marked reduction mentsreported above, in which defoliatedshoots in the number of sclereids formed as compared to were treatedwith auxin, tend to confirmthe idea the controls without auxin (fig. 8). At lower con- thatauxin productionin theyoung leaves is impor- centrationsof auxins, there was no significantdif- tantin controlling,the developmentof leaf primor- ference in the leaves between the treated and the dia as well as influencingleaf expansionin the ter- untreated buds (fig. 7). In general, the average minalbud. That foliageleaves could developfrom number of sclereids formed per leaf was relatively presumptivescale primordiaunder experimental low. In other experimentsin which the isolation of conditionswas earlyobserved by Goebel (1880) in the buds from the tree was made in April, late in experimentson Prunus. Such changeshave been bud development,the average number of sclereids discussedfrom the pointof view of bud-scalemor- per leaf was consistentlyhigher. These observations phologyby Foster (1928). The evidencefrom the sug,gestthat early isolation of the bud in some way presentexperiments clearly suggests that the deter- reduced the capacity of the leaves to initiate scle- minationof foliage-leafprimordia might be use- reids. Since completely normal bud development fullyexamined with respect to the auxin relations was not achieved in vitro (Al-Talib and Torrey, of the structuresconcerned. Clearly, in theabsence 1959), it is possible that nutrients essential for of determinationsof theauxin productionby leaves normal cellular differentiationwere lacking in the of Pseudotsuga,no firmconclusions concerning the medium used in these experiments.Auxin treatment role of auxinin the determinationand development at high concentration furthersuppressed the ini- of foliarprimordia can be made fromthese studies. tiation process. The determinationof sclereid initials in foliar DISCUSSION.-The relation between auxin and the leaves of Pseudotsugamenziesii is clearly under behavior of buds has been studied in the past by someprecise control of changingphysiological con- those interestedin the problem of apical dominance ditionsactive duringleaf and shoot development. and by others studying the effectof defoliation on Insofaras thereis evidenceon the matter,it would lateral branching and leaf development. Dostal seem probable that hormonal factors,especially

TABLE 6. The efect o1 various p/anltgrowth-regulators onl the occurrence of sclereids in leaves of isolated buds of P. menziesii culturedin vitro fortwvo mo.

Growth Regulator 10- MNI 10-5 M 10-4 M Averageno. of Averageno. of Averageno. of Numberof sclereidsper Numberof sclereidsper Numberof sclereidsper leaves leaf and S.E. leaves leaf and S.E. leaves leaf and S.E. IAA 24 3.7 + 0.8 18 2.8 + 0.6 25 0.5 ? 0.1 IBA 25 2.5 ? 0.5 21 3.2 ? 0.5 29 0 NAA 19 1.7 ? 0.4 poorlydeveloped leaves poorlydeveloped leaves 2,4-D 21 2.5 ? 0.4 poorlydeveloped leaves poorlydeveloped leaves 2,6-D 24 3.4 ? 0.6 25 2.2 ? 0.5 25 0.2 + 0.1 TIBA 20 2.5 ? 0.5 21 1.9 ? 0.4 None 24 2.3 + 0.4 78 AMERICAN JOURNAL OF BOTANY [Vol. 48 auxinm,may play a role in determining,when and It is uncertainwhether the inhibitoryeffects of wheresclereid initials develop. Taken together,the auxin are specificin nature or resultfrom non- experimentson defoliationwith and withoutauxin specificinhibition of leaf developmentin general. treatmentand thoseon auxin treatmentof isolated It is a strikingfact that in the leaves of excised buds all suggestthat auxin levels in the leaf in- buds grownin culture,whether in the presenceor fluencethe developmentof sclereids,with high absence of added auxin, the average number of auxin levels tendingto suppresssclereid develop- sclereids per leaf is quite low, comparingvery ment. The observed distributionof sclereids in closelyto the averagenumber produced in the ter- leaves bornealong the intactshoot axis of Pseudo- minal leaves of each year's growth in the intact tsugafits in wellwith the idea thathigh auxin levels plant. There are at least 2 possible interpretations in the youngestterminal leaves are associatedwith of this result: (1) the auxin level in the isolated low sclereidinitiation, and low auxinlevels in older, buds grown in vitro is high, leading to suppression more basal leaves occur wheresclereids are found of sclereid development; or (2) isolation of the morefrequently. The lack of sclereidsin thebasal- buds deprives them of some unidentifiedsubstance mostleaves remainsunexplained. normally provided by the plant which is essential In partiallydefoliated buds, removal of thebasal to sclereid differentiation.In the absence of ex- leaves does not affectsclereid development in the perimental observations which bear directly on remainingleaves. This resultwould be expectedif either of these interpretations,conclusions as to the thebasal leaves in themselvescontribute little auxin controllingfactors in sclereid development in these to the developingshoot system.In completelyde- leaves must be deferred,although the possible role foliatedbuds which showed prematureexpansion of auxins in determiningsclereid distributioncan- of next year's buds, sclereids do develop in the not be disregarded. newlyformed leaves derivedfrom primordia which In recent experimentalstudies on sclereid forma- normallywould have developed into bud scales tion in leaves of Camellia,Foard (1958) produced lacking sclereids. In these buds the numberof evidence that sclereid initials develop to maturity sclereidsformed per leaf variedwidely, some leaves in detached immature leaves cultured in a liquid containing3 or 4 while othershad 30 to 35, but, nutrientmedium with agitation and artificial illu- in general,the averagenumber of sclereidsper leaf mination. High sugar concentrationsin the medium approachedthat found in normalleaves (compare inhibited sclereid differentiation; this inhibition tables 5 and lA). Unfortunately,no record was was attributedto an osmotic effect. In surgical ex- made of the gradientof sclereid distributionin periments with leaves attached to the plant, Foard leaves of plantswhich had been defoliated. (1959) showed that sclereid formation was deter- When completelydefoliated buds were treated mined by the position along the margin of the leaf. with auxin in lanolin, the developmentof foliar No evidence for hormonal control was forthcoming leaves frompresumptive bud-scale primordia was from these experiments which do show, however, markedlyreduced, but in certain cases, foliage that determinationof sclereid initiation in leaves is leavesdid develop. In such leaves,the incidenceof subject to experimentalmanipulation and thus sub- inhibited sclereidswas verylow. Here auxinclearly ject to study by experimentalmeans. the developmentof sclereids. From the experimentswith excised buds grown DEPARTMENT OF BOTANY in culture,it is also evidentthat high auxin con- UNIVERSITY OF CALIFORNIA centrationstend to suppresssclereid development. BERKELEY 4, CALIFORNIA

LITERATURE CITED

ALLEN, G. S. 1945. Einbryogenyand the developmentof branching in two species of Aster. Amer. Jour. Bot. apical meristems of Pseudotsuga taxifolia (Lamb.) 24: 159-167. Britt. Ph.D. dissertation,Univ. Calif., Berkeley. DOSTXL, R. 1926. fIber die Wachstumsregulierendede des Laubblattes. Acta Sci. Nat. Morav. 3: AL-TALIB, K. H., AND J. G. TORREY. 1959. The aseptic cul- Wirkung ture of isolated buds of Pseudotsuga taxifolia. Plant 83-209. de- Physiol. 34: 630-637. FOARD, D. E. 1958. An experimentalstudy of sclereid velopment in the leaf of Camellia japonica. Plant AVERY, of phytohor- G. S. 1935. Differentialdistribution Physiol. 33 (Suppl.): xli. mones in the developingleaf of Nicotiana, and its rela- . 1959. Pattern and controlof sclereid formationin tion to polarized growth. Bull. Torrey Bot. Club 62: the leaf of Camellia japonica. Nature 184: 1663-1664. 313-324. FOSTER, A. S. 1928. Salient featuresof the problemof bud- BAILEY, I. W., AND CHARLOTTE G. NAST. 1948. Morphology scale morphology.Biol Rev. 3: 123-164. and relationshipsof Illicium, Schizandra and Kadsura. . 1944. Structure and developmentof sclereids in I. Stem and leaf. Jour. Arnold Arboretum29: 77-89. the petiole of Camellia japonica L. Bull. Torrey Bot. BLOCH, R. 1946. Differentiationand pattern in Monstera Club 71: 302-326. deliciosa. The idioblastic developmentof the tricho- -. 1945. Origin and developmentof sclereids in the sclereids in the air roots.Amer. Jour. Bot. 33: 544-551. foliage leaf of Trochodendronaralioides Sieb. and Zucc. DELISLE, A. L. 1937. The influenceof auxin in secondary Amer.Jour. Bot. 32: 456-468. January,1961] GRUN-BARRIERS IN SOLANUM 79

. 1946. Comiparativemorphology of the foliar scle- histogenesisin certain species of Araucaria. Ph.D. Dis- reids in the genus Mouriria Aubl. Jour. Arnold Ar- sertation,Univ. Calif., Berkeley. boretum27: 253-271. KITAMURA, R. 1956. Development of foliar sclereids in . 1947. Structureand ontogenyof the terminalscle- Sciadopitys verticilata Sieb. and Zucc. Bot. Magazine reids in the leaf of Mouriria huberia Cogn. Amer. (Tokyo) 69: 519-523. Jour. Bot. 34: 501-514. RAO, T. A. 1951. Studies on foliar sclereids. A prelimi- - . 1955a. Comparative morphology of foliar scle- nary gurvey. Jour. Ind. Bot. Soc. 30: 28-39. reids in Boronella Bail. Jour. Arnold Arboretum36: SACHER, J. A. 1954. Structureand seasonal activityof the 189-198. shootapices of Pinus lambertianaand Pinus ponderosa. . 1955b. Structure and ontogeny of terminal scle- Amer. Jour. Bot. 41: 749-759. reids in Boronia serrulata. Amer. Jour. Bot. 42: 551- STERLING, C. 1947. Sclereid formationin the shoot of 560. Pseudotsuga taxifolia. Amer. Jour. Bot. 34: 45-52. . 1956. Plant idioblasts. Remarkable examples of THIMANN, K. V., AND F. SKOOG. 1934. On the inhibitioniof cell specialization. Protoplasma 46: 184-193. bud developmentand other functionsof growthsub- GOEBEL,K. 1880. Beitrag zur Morphologieund Physiologie stances in Vicia faba. Proc. Roy. Soc. London 114: des Blattes. Bot. Zeit. 38: 800-816. 317-339. GOODWIN,R. H. 1937. The role of auxin in leaf develop- WETMORE, R. H., AND W. P. JACOBS. 1953. Studies on ab- ment in Solidago species. Amer. Jour. Bot. 24: 43-51. scission: the inhibiting effectof auxin. Amer. Jour. GRIFFITH-I,M. 1950. A study of the shoot apex and leaf Bot. 40: 272-276.

EARLY STAGES IN THE FORMATION OF INTERNAL BARRIERS TO GENE EXCHANGE BETWEEN DIPLOID SPECIES OF SOLANUM' PAUL GRUN

A B S T R A C 1 GRUN, PAUL. (Pennsylvania State U., UniversityPark.) Early stages in the formationof internalbarriers to gene exchange between diploid species of Solanum. Amer. Jour. Bot. 48(1): 79-89. Illus. 1961.-Internal barriersrestricting development of F1 hybridsin crosses among closely related diploid species of Solanum belonging to the series Tuberosa and Commersonianawere studied to determinethe early stages in developmentof barriers to gene exchange. Both of the parental series (species complexes) are native to South America, the Tuberosa occurring along the length of the Andes, while the Commersoniaoccur at lower altitudes in Argentina,Paraguay, and Bolivia. They are distinguishedby a fair number of morphologicalcharacters. Following crossingsbetween the species of each series and between species of the differentseries comparisons were made of the number of berries formedper attemptedcross, seed per berry,percentage seed g,ermination,and F1 vigor and pollen fertility.Although the species used were closely enough related that all could readily be hybridized,berry and seed set followingcrosses between species of differentseries were on the average lower than they were followingcrosses between species of the same series. These barriers are just formingand are expressed in a varyingmanner, so that there were significantdifferences between species of the same series, and even between clones of the same species in barrier expression. The barriers of some of the species were expressed only when they were tested as female or as male parent. The inter-seriesF1 seeds germinatedas well as did those of the parent species and the hybridshad a normal vigor and pollen fertility.

WHILE extensive informationhas been gained of series (species complexes)Tuberosa and Commer- internal barriers to crossing of plant species that soniana werechosen for intensivestudy because it are distantlyrelated, our knowledge of early stages is knownthat they can easilybe hybridized(Choud- in the formation of barriers, those operating be- huri,1944; Koopmans.1931; Swaminathan,1953; tween closely related taxa, is somewhat less satis- Hawkes,1956b; Wangenheim,1957a; and Magoon factory.The object of the study reported here was et al., 1958) and that,if any earlybarriers leading to analyze barriers to exchange of genes between to partialisolation have evolved,they are notas yet closely related species in the belief that such early masked by the accumulationof complexbarriers barrier formationis a very critical part of the proc- leadingto completegenetic isolation. Analysisof ess of speciation. Diploid species of Solanum of the theircrossing relationships was undertakento ob- tain informationon the first-expressedbarriers, 1 Received for publication April 28, 1960. those that limitthe numberof fertileF1 hybrids Authorizedfor publication Sept. 21, 1959, as paper No. formed. The object of the study was to answer the 2403 in the JournalSeries of the PennsylvaniaAgricultural followingquestions: (1) How does the degree of in- ExperimentStation and ContributionNo. 250 fromthe De- partmentof Botany and Plant Pathology. The Pennsylvania tercrossabilityof species that are members of the State University. same series compare with that of species that belong