Plant Physiol. (1987) 84, 1121-11250032-0889/87/84/1 121/05/$0 1.00/0

Gibberellin-Mediated Synergism of Xylogenesis in Lettuce PithCultures

Received for publication December 24, 1986

DAVID PEARCE*, A. RAYMOND MILLER, LORIN W. ROBERTS, AND RICHARD P. PHARISPlant Physiology Research Group, Department ofBiology, The University ofCalgary, Calgary, AlbertaT2N IN4 Canada (D.P., R.P.P.), Department ofHorticulture, Ohio State University, Wooster, Ohio44691-6900 (A.R.M.), and Biological Sciences Department, University ofIdaho, Moscow, Idaho 83843(L.W.R.)

ABSTRACT

Major gibberellins (GAs) in lettuce (Lactuca sativa L. cv Romaine)pith explants have been identified by gas chromatography-mass spec-trometry (GC-MS) or GC-selected ion monitoring (GC-SIM) as GA,, 3-epi-GA,, GA., GA,,, and GA2. Treatment of pith explants with indole-3-acetic acid (IAA) (57 micromolar) plus kinetin (0.5 micromolar) in-duced xylogenesis. In this xylogenic treatment, the concentration of abiologically active, polar GA-like substance(s) increased during the first2 days of culture, although all of the above GAs decresed (as measuredby GC-SIM). In non-xylogenic treatments, where explants were culturedwithout exogenous hormones, or with IAA or kinetin alone, the concen-tration of the biologically active, polar GA-like substance(s) decreasedduring the first two days of culture, as did all of the above GAs (asmeasured by GC-SIM). Treatment of pith explants with exogenous GA,alone did not induce xylogenesis, but GA, at very low concentrations(0.0014 and 0.003 micromolar) synergized xylogenesis in the IAA pluskinetin-treated cultures. These results suggest that changes in the con-centration of certain endogenous GAs may be involved in xylogenesismediated by IAA plus kinetin in lettuce pith cultures.

There are confficting views on the effect of exogenous GA3 onauxin-induced xylogenesis in vitro. Combinations of IAA andGA3 apparently have a synergistic effect on cambial activity andthe differentiation of secondary vascular tissues in woody dicots(29). Some tissue cultures exhibiting xylem differentiation appearto respond favorably to the addition of GA3 to the medium.Several investigators reported that exogenous GA3 stimulatedxylogenesis in tuber explants of Helianthus tuberosus (2, 9, 23,25). Other workers, however, found that GA3 was inhibitory toxylogenesis in bean callus (10), and in cultures of Helianthustuberosus (17, 18, 30).

Aside from tracheary element formation, GA3 has been impli-cated in other cytodifferentiation events. The addition ofGA3 tothe medium stimulated phloem element differentiation in ex-plants ofPinus strobus (5), and primary phloem fiber formationin Coleus stems (1). Also vascular differentiation in needle leavesofPinus was influenced by applied auxin and GA3 (8). Additionalinformation can be found in reviews on the roles of hormonesin vascular differentiation (11, 14, 21, 24, 26, 27).One possible reason for confficting data from studies on cul-

tured tissues may be the presence of endogenous hormoneswithin the explant at the time of excision from the parent plant.A preliminary analysis of freshly excised lettuce pith explants

revealed the presence of endogenous GAs' (22). The presentstudy is devoted to a further examination of the possible role ofthese endogenous GAs in xylogenesis induced by culturing onmedium containing an optimal combination ofIAA and kinetin.

MATERIALS AND METHODS

Plant Material and Tissue Culture. Sterile pith explants wereprepared as previously described (16) from the core of lettuce(Lactuca sativa L. cv Romaine) heads obtained from commercialsources. The explants were rinsed three times with sterile double-distilled H20, blotted on Whatman No. 1 filter paper and trans-ferred to culture media solidified with 1% (w/v) Bacto-agar andprepared according to Murashige and Skoog (20), except that 2%(w/v) glucose was substituted for sucrose. The IAA and kinetinwere added as indicated to the medium before autoclaving; GA,was sterilized by membrane filtration and added as indicated tothe autoclaved medium. Explants used forGA analysis and initialtracheary element determinations were cultured in 125 ml, foil-capped Erlenmeyer flasks (5 explants/flask) containing 50 ml ofculture medium. Each treatment was replicated 4 times (i.e. ineach of 4 culture runs). Explants for GA, feeding experimentswere cultured individually in foil-capped shell vials (25 x 95mm) containing 10 ml of culture medium. All cultures wereincubated in darkness at 250 C.Tracheary Element Counting. After incubation for the speci-

fied period, explants were weighed and tracheary element num-bers determined by microscopic examination of squash prepa-rations of safranin-O-stained explants (3) or explants maceratedin chromic acid maceration fluid (16). Xylem elements inmacerated explants were quantitated according to Dodds andRoberts (4).

Extraction and Purification of GAs. For each treatment in the2-d cultures the explants from all 4 culture runs were combinedbefore extraction. The number and dry weight of explants ineach treatment was: no hormone supplement, 144 (620 mg); 57AM IAA, 152 (750 mg); 0.5 ,AM kinetin, 151 (753 mg); 57 M IAAplus 0.5 ,lM kinetin, 151 (705 mg). The corresponding 4 sets ofuncultured explants (448 explants, 1168 mg dry weight) werealso combined. Each ofthe 7-d cultures ofexplants was extractedseparately; there were 28 to 40 explants in each treatment ineach culture run. The mean dry weights of explants were: nohormone supplement, 7.03 mg; IAA, 7.60 mg; kinetin, 7.54 mg;IAA plus kinetin, 10.05 mg. The extraction procedure was similarto that of Koshioka et al. (13). The freeze-dried pith explants

'Abbreviations: GA, gibberellin; MeOH, methanol; EtOAc, ethylace-tate; SIM, selected ion monitoring; MS medium, Murashige-Skoog me-dium; KRI, Kovats retention index.

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Plant Physiol. Vol. 84, 1987

were ground in a mortar and pestle and extracted with 40 ml of80:20 MeOH:H20. Tritiated GA32 (approximately 1670 Bq, spe-cific activity 8.34 x 10" Bq mmol-') was added to each extractso that workup losses of GAs could be estimated. The extractwas filtered, and the filtrate was forced through a column of C,8reversed-phase material (3 g preparative C,8, particle size 55 to105 gm; Waters Scientific Ltd., Mississauga, Ontario, Canada)which was then eluted with 20 ml of 80% MeOH. The eluatewould have contained free GAs and GA conjugates; nonpolarpigments, kaurene, kaurenoic acid, and probably about 40% ofany GA,2-aldehyde present in the extract would have been re-tained on the column, to be subsequently eluted in 100% MeOH(2 x 30 ml). The 80% MeOH eluate (60 ml total) was diluted to40% MeOH with distilled water, adjusted to pH 6.5 withNH40H, and forced through the column, which was then elutedwith an additional 20 ml of 40% MeOH. This eluate (140 mltotal) will contain most free GAs and GA conjugates (13); someor all of the least polar free GAs (e.g. GA4, GA9, GA,s) shouldremain on the column to be eluted subsequently in 100% MeOH(2 x 30 ml). This 100% MeOH eluate was dried in vacuo at 350C. The MeOH was evaporated from the 40% MeOH eluate andthe aqueous residue was frozen at -70° C, freeze-dried, thendissolved in water (1 ml) and 80% MeOH (1 ml), mixed with0.5 g of Celite, dried in a gentle air stream, and loaded onto aSio2 partition column (prepared from 5 g of deactivated WoelmSiO2 [13] slurried in 95:5 EtOAc:hexane [formate-saturated]).This column was eluted first with 70 to 75 ml of 95:5 EtOAc:n-hexane (to remove free GAs) and then with 100% MeOH (100ml), to remove GA conjugates (13). The EtOAc:hexane eluatewas dried, and the 100% MeOH eluate was neutralized withNH4OH before drying.At each step in the procedure aliquots were taken from solu-

tions and radioactivity was determined by liquid scintillationspectrometry to estimate losses.HPLC. Fractions containing free GAs were chromatographed

using HPLC. Dried residues were dissolved in small volumes ofMeOH:H20:acetic acid, filtered (Millipore HATF filter, 0.45 .mpore size), and injected onto a u-Bondapak C,8 (reversed-phase)column (Waters Associates). Solvent flow, at 2 ml min-' fromtwo M6000A (or M-45) pumps (Waters Associates), was con-trolled by a model 680 Automated Gradient Controller (WatersAssociates). Solvents were MeOH:H20:acetic acid 10:89:1 (pumpA) and 100% MeOH (pump B). The solvent programme forchromatography of the free GA fraction was: 0 to 20 min., 32.5%MeOH (1.5 ml min-' pump A, 0.5 ml min-' pump B); 20 to 45min, linear gradient to 73% MeOH (0.6 ml min-' A, 1.4 mlmin-' B); from 45 min, 100% MeOH. The program for chro-matography of the 'less-polar GA' fraction was: 0 to 20 min,linear gradient from 46% MeOH (1.2 ml min-' A, 0.8 ml min-'B) to 91% MeOH (0.2 ml min-' A, 1.8 ml min-' B); 20 to 30min, 91% MeOH; from 30 min, 100% MeOH. Six-ml fractionswere collected, aliquots were taken for radio-counting, and thefractions were dried (most of the MeOH was evaporated in vacuo,and the aqueous residue frozen and freeze-dried).

Bioassay. Free-GA fractions were assayed for biological activ-ity in a modified (0.5 ,ul drop, 48 h measurement time) Tan-ginbozu dwarf rice micro-drop assay (19). Aliquots of 1/50 and1/100 (7-d cultures), 1/125 and 1,250 (2-d cultures) or 1/200 and1/400 (uncultured explants) were applied to rice plants as 0.5 or1.0 gl drops. The 'less polar GA' fraction was assayed withaliquots of L/2S and 1/50 (7-d cultures) or 1/65 and 1/130 (2-dcultures) or 1/100 and 1/200 (uncultured explants).GC-MS. Samples were methylated with ethereal diazometh-

ane, silylated with 50:50 (v/v) pyridine:BSTFA plus 1% TMCS(Pierce Chemical Co.) for 30 min at 60 to 700 C, and analyzed

2Purchased from IsoCommerz, Dresden, DDR.

by capillary GC-MS. Samples were injected directly onto a DB1-15N or DB5-1SN column (J & W Scientific Inc.) installed in aHewlett Packard GC (5790A Series). The temperature programwas 60° C isothermal for 1 min, 25° C min-' to 2500 C. The gasflow rate was 1.1 ml min-'. The GC was linked by a capillarydirect interface with a Hewlett Packard Mass Selective Detector(5970A Series); the interface temp was 280° C.

Deuterated [ 17,1 7-2HJ (99.2% enrichment) standards of GA,,GA,9, and GA20 were prepared as described in (15), and addedto appropriate fraction residues after bioassay, before methyla-tion for GC-SIM. Endogenous GA,, GA,9, and GA20 were quan-tified from m/e ratios 506/508, 434/436, and 418/420 (respec-tively) using standard curves.

RESULTS

Tracheary Element Formation in Lettuce Pith Explants. Notracheary elements were observed in any treatment after 2 d ofculture. Only after 7 d of culture were tracheary elements ob-served, and only in the IAA (57 FM) plus kinetin (0.5 ,M)-treatedexplants.Measurement and Identification of Endogenous GAs. Major

biologically active GAs in the free GA fraction from unculturedexplants (Fig. 1) were identified by GC-MS (SIM or full scan) asGA,, GA,9, and GA20 (Table I). In addition, the relatively bio-logically inactive GA8 and 3-epi-GA, were identified.

After 2 d of culture, in medium without added hormones orwith IAA or kinetin alone added (all non-xylogenic treatments),the concentration of a biologically active, polar GA-like sub-stance(s) in the explants declined (Table II, fractions 2 and 3).However, in the IAA plus kinetin-treated explants (i.e. the xy-logenic treatment) the concentration of biologically active, polarGA-like substance(s) increased.

Concentrations of other GAs or GA-like substances also ap-peared to be influenced by treatment. The concentration ofGAI,g-

-%

-E

r

tn

aIW

i81

16-

14[

12~

1 5 10 15HPLC fraction no.

FIG. 1. Biological activity in the free GA fraction (after HPLC) froman extract of uncultured lettuce pith explants, measured by the Tan-ginbozu dwarf rice microdrop bioassay. 1,200 (-) or 1/400 (- --- -) ofeach HPLC fraction was applied. GA,, 3-epi-GA,, and GA, were iden-tified by GC-SIM (Table I) in fractions 2 and 3 combined; GA20 infractions 7 and 8; and GA,Ig in fraction 11.

UNCULTURED EXPLANTSGA1 GA19

3-epi GA1GA8

GA20 H

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GIBBERELLINS AND XYLOGENESIS IN LETTUCE PITH CULTURES

Table I. GC-MS ofGAs in Free GA Fraction from Uncultured Lettuce Pith Explants

Compound KRI m/e (Relative Abundance)a Identity

GAi-likeb'c 506 (84) 491 (9) 448 (14) 377 (4) 207 (99) GA,GA, standard 506 (84) 491 (8) 448 (16) 377 (20) 207 (78)3-epi-GAI-likcebc 2802d 506 (30) 491 (2) 459 (3) 448 (8) 377 (7) 3-epi-GA,3-epi-GA, standard 2803 506 (30) 491 (2) 459 (3) 448 (8) 377 (5)GA8rlikeb,c 2824d 594 (52) 579 (4) 535 (3) 504 (2) 448 (10) 379 (8) GA8GA8-standard 2825 594 (52) 579 (3) 535 (3) 504 (1) 448 (10) 379 (7)GAi9-likeb e 462 (4) 434 (42) 402 (11) 374 (33) 345 (8) 315 (8) GA19GA19 standard 462 (3) 434 (42) 402 (14) 374 (32) 345 (16) 315 (13)GA.-likeb,c 2567f 418 (168) 403 (17) 375 (119) 359 (23) 301 (40) GA20GA20 standard 2567 418 (168) 403 (24) 375 (139) 359 (28) 301 (37)' The relative abundances of ions have been corrected for contributions from deuterated internal standards

which co-chromatographed with the endogenous GA, and GA19. The relative abundances of ions in thestandard GAs have been adjusted so that the abundance of M+ (for GA,, 3-epi-GA,, GA8 and GA2o) or thebase peak (for GA19) corresponds with that found in the endogenous GAs. b From HPLC fractions shownin Figure 1: GA,, 3-epi-GA, and GA8 from fractions 2 and 3; GA19 from fr. 11; GA20 from fraction8. c Measurement by GC-SIM. d KRI on DBl-lSN. ' Measurement by GC-MS (full spec-trum). fKRI on DB5-1SN.

Table II. Biological Activity in Free GA Fraction (after HPLC) from Extracts ofLettuce Pith ExplantsBiological activity expressed as ng GA3 equivalent per g dry weight; adjusted for losses based on the recovery

of [3H]GA3 which was added at the beginning of extraction. Only activity which was significantly different (Ps 0.05) from controls is shown.

Treatment

Cultured 2 d Cultured 7 d'Fraction Notcultured No IAA Kinetin AA + No IAA Kinetin Ineti

hormones kinetin hormones knetin2, 3 (GA8, GA1)b 35 7 13 5 62 15 ± 7 46 ± 23 4 ± 2 12 ± 77-9 (GA20)b 39 1611, 12(GA19)b 95 88 20 87 56 44 ± 8 27 ± 19 17 ± 3 20 ± 513, 14(GA4)C 5 8 17± 1 15± 10 4±4 14±53_55d(GA4, GAg)C 35 1 1 2 3 ± 3 12 ± 8a Results for 7 d cultures are the means of 3 (IAA, kinetin) or 4 (no hormones, IAA plus kinetin) replicates,

except as noted in footnote d. One standard error of the mean is shown for each value. b GAs which wereidentified in these fractions. c Examples ofGAs expected to elute from HPLC in these fractions. d FreeGA-like fractions from HPLC of the 'less polar GA' fraction (see "Materials and Methods"). The results for 7d cultures are the means of 2 (IAA, kinetin) or 3 (no hormones, IAA plus kinetin) replicates.

Table III. Relative Concentrations ofGAs in Lettuce Pith Explants, Measured by GC-SIMFor GA, and GA19, amounts were calculated from m/e 506/508 and 434/436, respectively, ([2H2]GA, and

[2H2]GA19 were added to these fractions before GC-SIM). For 3-epi-GA, and GA8, amounts were calculatedfrom m/e 506/508 and 594/508 respectively, based on 506, 508 and 594 abundances in a sample containingequal amounts of [2H2]GA1, 3-epi-GA,, and GA8 standards. Concentrations have been adjusted for losses,based on the recovery of [3H]GA3 after HPLC and subsequent use of part of the fractions in bioassay beforeGC-SIM.

Treatment

HPLC . Cultured 2 dFraction Gibberellin Not

cultured No IAA Kinetin LAA + Kinetinhormones

ng/g dry wt oftissue2, 3 Al 68 ND ND ND ND2, 3 3-epi-A1 112 83 54 47 472, 3 A8 405 42 <8 39 281 1 A1g 48 (95)b 29 (88) 44 (20) 21 (66) 25 (35)

a ND = not detected.b Biological activity in fraction 11, included for comparison with GC-SIM results.

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1EEPlant Physiol. Vol. 84, 1987

like substance(s) (Table II, fractions 11 and 12) remained rela-tively high in explants cultured for 2 d without added hormones,and also in kinetin-treated explants. It declined in IAA-treatedexplants, and in IAA plus kinetin-treated explants. However,after 2 d of culture, the concentration of GA20-like substance(s)(Table II, fractions 7-9) declined in explants from all treatments.Analysis by GC-SIM ofthe polar GA-like substance(s) in residuesremaining after bioassay were consistent with the bioassay resultsin that GA, was readily detected in the fraction from unculturedexplants (35 ng GA3 equivalent by bioassay, 68 ng by GC-SI4)and could not be detected in fractions from the non-xylogenictreatments (Table III). However, no GA, was detected in thexylogenic treatment and the biologically active, polar constituentin this treatment (d 2) remains unknown. The presence of 3-epi-GA, and GA8 in all explants cultured for 2 d was indicated byions of m/e 506 and 594, respectively, at the correct KRI.Amounts of each, estimated by GC-SIM, were relatively high inuncultured explants, and relatively low in all cultured explants(Table III). There was no indication of a significant amount ofGA29 (the biologically inactive C-2 hydroxylated metabolite ofGA2o) in any sample. GA,9 was present in all treatments, but theamounts calculated by GC-SIM (Table III) were lower, except inthe IAA treatment, than those estimated by bioassay. This im-plies that other biologically active, but as yet unidentified, GAswere ptesent in these fractions. Logical possibilities which have

Table IV. Tracheary Element Formation in Lettuce Pith ExplantsCulturedfor 7 d on MS Medium Containing IAA (57 AM), Kinetin (0.5

Mm) ahd Various Concentrations ofGA,Tracheary Elements

GA,Expt. 1 Expt. 2 Expt. 3a

MM number (xIO') per g new tissue (final - initialfresh wt)0 144.02 ± 26.72 (6) 164.27 ± 11.44 (10) 36.28 ± 9.29 (9)0.0003 142.03 ± 12.44 (10) 59.36 ± 12.66 (9)0.0014 257.41 ± 24.14 (10)0.003 237.33 ± 46.82 (9) 244.93 ± 24.14 (10) 76.63 ± 18.96 (9)0.014 136.62 ± 12.80 (10)0.03 132.56 ± 15.23 (9) 139.72 ± 13.23 (10) 34.49 ± 10.21 (9)0.3 111.21 ± 15.65 (9) 36.33 ± 11.27 (9)3 126.64 ± 13.28 (9)'Experiment 3 differed from experiments 1 and 2 in that explants

were trati$ferred to fresh medium after 2 d. b Means ± 1 SE (n).Table V. Tracheary Element Formation in Lettuce Pith ExplantsIn experiments 1 and 2, explants were cultured for 7 d on MS medium

containing LAA (57 MM) and kinetin (0.5 MM), supplemented with variousconcentrations of GA, for either the first 2 d (experiment 1) or last 5 d(experiment 2) of culture.' In experiment 3, explants were cultured for 2d on MS medium containing various concentrations of GA,, thentransferred for 7 d to MS medium containing LAA (57 aM) and kinetin(0.5 gM).

Tracheary ElementsGA,

Expt. ja Expt. 2 Expt. 3MM number (x 104) per g new tissue (final - initialfresh wt)

0 36.28 ± 9.29 (9)b 36.28 ± 9.29 (9) 156.55 ± 22.23 (5)0.0003 38.10± 15.23 (9) 34.59 ± 11.14 (9)0.003 52.27 ± 22.19(9) 38.99 ± 12.75 (9) 93.80 ± 5.77 (5)0.03 4.11 ± 1.01 (9) 23.34 ± 8.15 (9) 107.71 ± 1.48 (6)0.3 10.01 ± 3.45 (9) 33.33 ± 10.30 (9) 56.98 ± 9.30 (6)3 52.15 ± 21.85 (5)a In experiment 1, explants were transferred to fresh culture medium

without added GA, after 2 d; in experiment 2, explants were transferredto fresh culture medium with added GA, after 2 d. b Means ± 1 SE(n)I

similar retention times to GA,9 on HPLC were GA44 and GA36,but neither was detected by GC-SIM in the fractions fromuncultured or kinetin-treated explants. GA20 was identified infractions 7 and 8 from uncultured explants, and measured infraction 7. The amount measured by GC-SIM (27 ng) accountedfor the observed biological activity (12 ng ofGA3 equivalent).

After 7 d of culture, at which stage tracheary elements wereobvious, the concentration of biologically active, polar GA-likesubstance(s) had fallen in the xylogenic explants to a level similarto that found in two of the non-xylogenic treatments (no addedhormones, or kinetin alone) (Table II). The concentration ofGAi9-like substances also fell in the xylogenic explants, as it didin the explants cultured without hormones, or with kinetin alone.However, IAA-treated explants (non-xylogenic) showed an in-crease in the concentration of a biologically active, polar GA-like substance(s) after 7 d of culture (Table II).Tracheary Element Formation in GA,-Treated Explants. GA,

alone, applied at concentrations ranging from 0.003 to 3 uM didnot induce xylogenesis in pith explants during the 7-d cultureperiod (data not shown). GAI treatment for 7 d concurrent withIAA and kinetin treatment synergized tracheary element forma-tion at concentrations of 0.0014 or 0.003 gM, but not at higheror lower concentrations (Table IV). GA, treatment during thefirst 2 d of culture was necessary for synergism of xylogenesis,since treatment for only the final 5 d ofculture was not synergistic(Table V). Treatment with GA, for longer than 2 d was required,since treatment for only the first 2 d ofculture was not synergistic(Table V). Finally, in explants treated with GA, for 2 d beforetreatment with IAA and kinetin, xylogenesis was inhibited (TableV).

DISCUSSIONWe have shown definitively that major biologically active GAs

in lettuce pith explants are GA, and its logical Cl 3-hydroxylatedprecursors (28), GA,9 and GA20. Consistent with these observa-tions is the presence ofbiologically-inactive GA8 (the C2-hydrox-ylated metabolite ofGA, [28]) and 3-epi-GA,.The concentrations of the biologically active GAs (as quanti-

fied by bioassay) changed with time during culture, and werealso modified by exogenous IAA and kinetin. There are few otherreports of the effects of growth regulators on endogenous GAconcentrations (e.g. Refs. in Evans [7]). In this study, the con-centration of biologically active, polar GA-like substance(s) de-clined within 2 d in explants cultured without added growthregulators, or in cultures treated with IAA or kinetin alone. Thexylogenic treatment (IAA plus kinetin) reversed this decline-infact there was an increase in the concentration of biologicallyactive, polar GA-like substance(s) within 2 d, during the periodwhen induction of xylogenesis would be expected (24). Thus,changes in the concentration ofendogenous GAs were associatedwith the induction of xylogenesis in lettuce pith explants, but itis not known if these events were related. However, other evi-dence of a possible role for endogenous GAs in xylogenesis wasthe synergistic effect on xylogenesis of low concentrations ofexogenous GA,. There might be a critical role (rather than simplya synergistic one) for endogenous GAs in the induction ofxylogenesis, since lettuce pith explants cultured for 4 to 5 d onmedium without hormones do not form tracheary elements whentransferred to medium containing IAA plus kinetin, unless thatmedium also contains GA3 (LW Roberts, unpublished results).

Since the increase in polar GA-like biological activity in IAAplus kinetin-treated explants was associated with reduced GA,9-and GA20-like activity, it is possible that its origin was fromexisting pools ofGA,g and GA20, or other GAs ofsimilar polarity.However, the biologically active, polar constituent in these ex-plants was not GA,, the logical dihydroxylated metabolite ofGA20. Its identity remains unknown, although it is under contin-

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GIBBERELLINS AND XYLOGENESIS IN LETTUCE PITH CULTURES

uing investigation. Another explanation for the reduction inGA19 (Table III) and GA20 (Table II) in cultured explants is thatconjugation of GA19, GA20, and/or their metabolites was en-hanced (e.g. as occurs in suspension cell cultures of anise [12]).GA19 and GA20 might have been converted to the biologicallyinactive GA8 or C-3 epi-GA1, but conjugation of these lattermetabolites must have been rapid since their concentrations(measured by GC-SIM) also decreased in all cultured explants(Table III). (A precedent for the conversion of GA20 to 3-epi-GA, exists in Bryophyllum [6]). These hypotheses can be testedby treatment of lettuce pith explants with [3H]- and [2H]GA20and GA19, and such experiments are planned.Our results indicate a positive role for endogenous GAs in

xylogenesis in lettuce pith explants. They also suggest possiblereasons for conflicting results obtained in other studies of theeffect of exogenous GA3 on xylogenesis in vitro. The timing andconcentration of exogenous application appears critical, sinceGA, given before IAA plus kinetin treatment inhibited xylo-genesis (Table V), and GA1 at higher concentrations (i.e. 0.014jM or greater) appeared supra-optimal (Tables IV and V).

Acknowledgments-We thank Dr. William L. Pengelly (Oregon Graduate Cen-ter) for use of his laboratory during the initial phases of this project and Ms. ChristelVelbinger (Ohio State University) and Mrs. Stania Horacek (University of Calgary)for their excellent technical assistance. The help of Dr. N. Murofushi is alsoacknowledged. The authors also acknowledge support from the Natural Sciencesand Engineering Research Council of Canada grant A-2585 (R. P. P.).

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15. LOMBARDO L 1982 Methylenation of carbonyl compounds with Zn-CH2Br2-TiCI4. Tetrahedron Lett 23: 4293-4296

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