HACKETT ET AL-CYANIDE-RESISTANT RESPIRATION

19. LOUGHMAN, B. C. The uptake and utilization ofphosphate associated with respiration changes inpotato slices. Plant Physiol. 32 suppl.: xxxvii.1957.

20. ROBBIE, W. A. The quantitative control of cyanidein manometric experimentation. Jour. Cell. Comp.Physiol. 27: 181-209. 1946.

21. SHARPENSTEEN, H. Studies on the wound respira-tion of potato, Solanum tuberosum L. PhD thesis,University of Michigan, Ann Arbor 1953.

22. SHARPENSTEEN, H. H. and CONN, E. E. Prepara-tion and properties of potato mitochondria. P. 3Program for the 29th Annual Meeting, Am. Soc.

of Plant Physiologists, Gainesville, Florida Sept.5-8, 1954.

23. STEWARD, F. C., STOUT, P. R. and PRESTON, C. Thebalance sheet of metabolites for potato discs show-ing the effect of salts and dissolved oxygen onmetabolism at 23° C. Plant Physiol. 15: 409-447.1940.

24. THIMANN, K. V. and Loos, G. M. Protein synthesisduring water uptake by tuber tissue. Plant Physiol.32: 274-279. 1957.

25. THIMANN, K. V., YocuM, C. S. and HACKETT, D. P.Terminal oxidases and growth in plant tissues. III.Terminal oxidation in potato tuber tissue. Arch.Biochem. Biophys. 53: 239-257. 1954.

ARGININE-REQUIRING STRAINS OF TISSUEOBTAINED FROM GINKGO POLLEN ' 2

WALTER TULECKEPHYTOCHEMICAL LABORATORY, BIOCHEMICAL RESEARCH DIVIsIoN, CHAS. PFIZER AND CO., INC., BROOKLYN, NEW YORK

The selection of strains of tissue with specificnutritional requirements is one convenient approachto the study of tissue physiology. The recent ad-vances in the manipulation of cells from plant tissuecultures (6, 7, 12, 13) has suggested the possibilityof isolating strains of plant tissue. Techniques simi-lar to those used for detecting biochemical mutantsof microorganisms, such as selective media, singlecell isolation and the plating out of cell suspensions,would be required. Reported here is the use of thisapproach for the isolation and culture of a strain oftissue from the pollen of Ginkgo biloba L. The straindescribed requires arginine for growth.

Tissues may be obtained from the pollen of Ginkgoon media containing yeast extract or coconut milk(15). If the pollen is distributed over the surface ofthe culture medium and allowed to grow, many of thepollen tubes will be abnormal and some of these willform tissues (fig 1). A few tissues proliferate andmay be subcultured; others grow slightly and abort.The tissue masses are made up of small dividing cellssurrounded by larger storage cells; the small cells arebasically haploid in chromosome number. but manyare heteroploid. The cells show no differentiation.

MATERIALS AND METHODSThe pollen used to obtain tissues was stored in

sterile desiccators at 70 C. Earlier work (14) showedthat both fresh and stored pollen forms the tissues, butthe stored pollen forms them more frequently. A uni-

Received February 20, 1959.2 This work was begun while the author was a research

associate at the Brooklyn Botanic Garden, N. Y.

form inoculum was made by suspending the pollen inliquid medium and dispensing aliquots to preparedmedia. After three days, a count was made of thenumber of pollen grains and the percentage viability;later, the number of tissues per number of viablepollen grains was determined.

FIG. 1. A petri dish containing several tissues ori-ginating from Ginkgo pollen. The pollen was dispensedover the surface of a coconut milk medium and incubatedin the dark for eight months at 250 C. The tissues showdistinct differences in their rate of growth; many developinto small masses of cells and others grow rapidly.

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PLANT PHYSIOLOGY'

It is important to point out that the tissues de-scribed here are strains anid not clones. The tissueshave a distinct origin an(d physiology and were picke(dout as cell masses w-hich probably arose from singlepollen grains. However, the pollen grains of Ginkgoare multicellular and "cloning" in the sense of isola-tion from a single cell is virtually impossible. Buta miieasure of success has been achieved in obtainingtissues from single pollen grains. For example, outof 500 germinatedlpollen grains which xxere placedin individual culture bottles on a coconut milk medium,two formed tissues one-half millimeter in size.Growth did not go beyond this stage, but this resultsuggests that cultures may eventually be growni fronmsingle isolate(d pollen grains.

In an effort to (levise nmedlia of known constituentsto replace those containing yeast extract or coconutmilk, v%arious synthetic medlia wNere trie(l. None ofthese were successful. Attempts were tlhen lmade toobtain new strains of tissue fronl pollen pllace(l olme(lia of known compositioni. For this, the basalmedium xvas use(d as noted in table 1.

The growth response of the tissue to various treat-nients was evcaluatedl in the followxing iiiannier. Thetissue inoculum of about 500 mg xvas xweighedl an(ddistributed to five test tubes, each containing 18 to20 ml of medlium. After three (or in some experi-menits four) wxeeks growth in the (lark at 24 to 250 C,the tissue xvas harvested an(d xeiglhedl. The ratio ofthe final fresh weight (lividledl by the inoculum Nxxeightgave an index of groxxwth terml-edI the groNxth Xvalue.The responses to media xxere repeated at least once.Controls xvere use(d for each experimlent acnd( the in-oculum xvas of uniform age.

TABLE I.

RESULLS

'T'lhe l)asal me(liuml xWas supplemented separatelywithi nilne (lifferenlt alinilio acids anl1 the stor-e l p )llelwxas inoculated oIn these me(lia. Table 11 shoxs thalttissuerser fornme(d onv oln mediacontaiin'illg argi-nine, tryptophane, or coconut miillk. Tlhree largetissue masses were formledI on the hasal ineium plisarginine, one smiiall tissue on the me lium with trypto-phane, none on the basal me(lium, l)ut many on themle(liuimi with coconut milk. The large numher oftisstues on the coconut milk me(liulmi is the higLhestfrequency of tissue formation obtained in aln l)ollencultures to (late. This frequency is apparently not (lueto the arginine content of the coconiut mlilk, sinceParis, Duhanmet and( Goris (9) report that coconutnmilk contains only ahout 100 ppm of argginine. ani(lthe number of tissues on the mlediumii wvith argininle at100 ppim was veryr muclh lower than on the coconutmiiilk nle(liulnl.

TAfGLE 11.FORMATION OF TISSUES FROMI POLLEN OF GINKGO ON

MEDIA CONTAINING CgERTAIN AM-\IN-O ACIDS

CONCEN- NUMA\BER OF TISSUESA-MINO ACID TRATION AFTER AFTER

IN PPM 5 MONTHS 11 -MO.NTHS1-Arginine HCI1-Aspartic acid HC11-Glutamic acid HClGlycinedl-Methionine1 -PheniylalaninieI -Proline1.-Tryptophane1-Tryosine(Coconut milk)

706015050305-)0402040

'20 (' volume)

300000010

135

300

()10

CONSTITUENTS

NaH.,PO 4 H.,OKClKNO.

Na.,S4Ca(NO.,)., 4H,0

M-s 7HMIgSO *7H-,0

Na'MoO1 2H.20CuSo4 *5H,0MnSO14 4H,0ZnSO4 * 7H,OH1B0.

Ferric citrate

Thiaminie HCIPyridoxine HCINicotinic acidCalcium pantothenate

GlycineNaphthaleneacetic acidSucroseAgarpH adjusted to 6.0 (NaOH)

MG/L

165.065.080.0

200.0280.0730.0

0.0250.0253.0000.5000.5002.0

0.250.251.251.00

7.500.1020.00g10.OOg

Approximately 10,000 pollen grains x-ere placed onthe basal medium supplemented with each amino acid.The germinlationi was 5%, or 500 per treatmenit. Storedpollen w%Nhich was four years ol l. was used as inoculum.

WVhen large enotuglh, the tissues on arginine andtryptoi)hane xvere transferredl. The trvptophane iso-late xxas smcall an(l soon (lie(l. The three argininestrains grew well an(l one wvas active after mlore than24 transfers (luring 18 nmonths.

Further results xvith argini ne xx-ere obtaine(l x-hensonme 15,000 viable l)ollen grains were place(d on media,respectixvely, xwith 40, 100, 200, 500, 1000, 2000 an(d4000 ppml arginine. Numilerous small tissues werevisible, especially xvith low magnificatiohi, on mediawrith nmore than 500 ppmii. Although only four tissueswvere large enough for subculturing, their occur-rencein(licate(l that such tissues may be obtaine(d repeatellY.

One strain originating on the basal medlium \vitharginine vas dlesignate(d 5857, indicating the molntlh,day and( y-ear when the tissue xvas first isolatedl.Another strain xvhich arose from pollen grown on theblasal medlium pl)us coconutit miiilk xxas (lesignatedl strain81056.

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T'ULECKE-ARGIN INE-REQUIRING STRAINS OF GINKGO TISSUE

I-ARGININE HCI ppm

G:D0

I

crm

10 25 50I-CANAVANINE SO4 ppm

32

28

24

:D 20

16

12

a

4

MEDIUM

BASAL

L-ARGININE-HCI

INH4)S04

L-LYSINE -HCI

L-ORNITHINE-HCI

UREA

PROLINE

L-CITRULLINE

L-ASPARTIC ACIDHCI

L-GLUTAMIC ACIDHCI

GLYCIN E

GROWTH VALUE2 4 6 8 10 12 14 16 Is

1 2 3 4 5 6 7 8

TIME IN WEEKS

FIG. 2 (itpper left). The effect of arginine concentration on the growth of strain 5857. These results are theaverage of two experinments, taken after three weeks growth. Growth value equals the final fresh weight/initialfresh weight.

Fi(. 3 (lower left). The growth response of two strains of pollen tissue: strain 5857 originated from pollengrown on a medium with arginine; 81056 originated on a medium with 20 % coconut milk. Growth value equalsthe final fresh weight/initial fresh weight.

FIG. 4 (ltpper right). Canavanine inhibition of the arginine strain 5857. The growth values were obtained afterthree weeks growth.

FIG. 5 (lower right). The growth of the arginine strain (5857) of pollen tissue oIn basal medium plus compoundsrelated to the ornithine cycle, both in the presence and in the absence of ammonia. The growth values were ob-taine(d after four w%Neeks.

21

10

9

8

C)X0

£ 7

5

4

I-ARGININE HCI ppm _~~~~M 0. 5.MO LAR

D O.5MOLAR +

O.5.M (NH4)2SO4-71 ~ ~ 4

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PLANT PHYSIOLOGY

The 5857 strain of pollen tissue responds to vary-ing concentrations of arginine as is shown in figure2. The growth value at 1,000 ppm is more thandouble that at 100 ppm; concentrations greater than1,000 ppm decrease growth. On the basal mediumwithout arginine, the growth value of 2.0 is probablythe result of the arginine carried over in the inoculum.The tissue dies in the second transfer on media lackingarginine. From these data it is apparent that thebasal medium with arginine at 100 ppm serves to main-tain the tissue, while the optimum concentration is1,000 ppm of arginine. The tissue was maintained atthe lower level because it was considered more respon-sive to additions to the medium, either inhibiting orpromoting, than it would be at higher levels ofarginine.

The growth curves of two strains of tissue, one onarginine (5857) and one on coconut milk (81056)are compared in figure 3. Both strains were placedon two media: the basal medium plus arginine (0.5mM) and the basal medium plus coconut milk (20 %).The growth values, taken at weekly intervals. showthat strain 5857 on the arginine medium grows moreefficiently than either 5857 on coconut milk mediumor 81056 on either the arginine or the coconut milkmedium.

Canavanine, an antimetabolite of arginine, inhibitsthe growth of the arginine-requiring strain. A pre-liminary experiment established the effective levels ofcanavanine inhibition at 10 to 25 ppm. An experi-ment based on this information is shown in figure 4.Canavanine at 25 ppm inhibits growth of the tissuewhen arginine is present in suboptimal amounts (10ppm). Increasing the amount of arginine reversedthis inhibition to a slight extent. In this case, cana-vanine apparently does not function as a competitiveinhibitor; the inhibition is irreversible and argininedoes not relieve it in any direct relation to the amountof canavanine present.

Whether the arginine requirement for strain 5857may be satisfied by compounds related to arginine orby simpler nitrogen sources was tested in the follow-ing manner. Ammonia (as ammonium sulfate), ni-trate (potassium and calcium nitrate), and urea weresterilized by filtering and then added to the basalmedium at several concentrations (0.1, 0.5, and 1.5mM). The growth on these media was compared tothe growth obtained on media containing 0.5 mM argi-nine. Nitrate was found to be a very poor nitrogensource; urea supported fair growth, but ammonia wasthe best substitute for arginine. Since these experi-ments were first transfers from the basal mediumplus 0.5 mM arginine on which the tissue was main-tained, it was assumed that there was a slight carry-over of arginine. Three successive transfers weremade on the nitrate, urea and ammonia media in orderto remove the residual arginine effect. It was con-firmed that nitrate was unavailable; urea was utilizedon the first, but not on successive transfers. Am-monia continued to support relatively high levels ofgrowth. The growth values on basal medium plus 0.5mM ammonium sulfate during three successive trans-

fers (8.5, 10.9, 8.9) compared favorably with thegrowth values obtained on the control basal mediumplus 0.5 mM 1-arginine HCl (11.0, 10.3, 10.2).

The growth of strain 5857 on basal medium pluscompounds related to the urea-ornithine cycle wascompared to the growth obtained on the control argi-nine medium. All the compounds were tested at 0.5mM concentration, both in the presence and in the ab-sence of ammonia nitrogen; the results are shown infigure 5. Without ammonia present, arginine is thebest nitrogen source and ammonia, lysine, ornithineand urea, in that order, partly replace arginine. Pro-line, citrulline, aspartic acid and glutamic acid werepoor nitrogen sources, while glycine was no betterthan the basal medium. Growth of the tissue on thesesame media supplemented with 0.5 mM ammonium sul-fate shows that ammonia increases growth to aboutthe level obtained on basal medium plus 0.5 mMarginine, indicating that ammonia is a good sourceof nitrogen but higher levels of ammonia inhibit thetissue. Even more striking is the growth responseobtained on media with proline and ammonia (growthvalue 18.2) as compared to the control of arginineand ammonia (17.9). Thus, while ammonia alonebecomes toxic at higher levels, it promotes growthof the tissue on media with arginine or proline.

The effect of proline on the growth of the argininestrain of tissue was tested by incorporating prolineor hydroxyproline into the basal medium with argininepresent or absent. The data in figure 6 show thatproline can be utilized by the tissue in the absence ofarginine while hydroxyproline consistently inhibitedgrowth. The inhibition of growth by hydroxyprolinewas directly related to its concentration, but the in-hibition was slight at 6 ppm.

8C)

00"'I

:X6 - 0<~~~~~~~~~~~.

mM

FIG. 6. The growth response of the arginine strain(5857) to media containing proline or hydroxyproline.The growth values were taken after four weeks.

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TULECKE-ARGININE-REQUIRING STRAINS OF GINKGO TISSUE

DISCUSSION

There are several interesting speculations regard-ing the causes of tissue formation from the pollen.Among the factors which might contribute to abnor-mal growth are the method of pollen storage, thegrowth of immature pollen grains and the effect ofconstituents of the culture media. An equally proba-ble and more basic possibility is the abnormal geneticconstitution of some pollen grains. As direct prod-ucts of meiosis and as gamete-forming tissue, thepollen is expected to vary in its genetic composition.The basis for this variability lies in the chromosomalrecombinations which may occur during meiosis. Innatural pollen populations, it can be assumed thatnatural selective mechanisms would operate to preventthe growth and normal function of abnormal pollengrains, including mutants with an unusual biochemicalmake-up. If a particular pollen grain could not syn-thesize or obtain from tissues of the developing ovule,the required level of a factor necessary for growth,it would perish or it would develop abnormally. Butthis aberration in its biochemical apparatus might beovercome by providing a suitable environment. Thesubstrate used for the culture of pollen would thuspermit the survival of imperfect pollen. Althoughthis is not the only explanation possible. the arginine-dependent strains of tissue are considered to arise fromabnormal pollen grains which are biochemical mutantsof this type.

If conditions mentioned above represent a reason-

able evaluation of the circumstances involved in theformation of some tissues from the pollen, then it maybe expected that other strains of tissue with differentspecific nutritional requirements could be isolated.The many tissues originating on coconut milk mediamight represent several kinds of abnormal growth or

combinations of deficencies.A number of papers (2, 8, 10, 16, 5) have in-licated

a significant role for arginine in the metabolism ofboth plant and animal tissues. Fries (3) has shownthat only arginine, of 22 amino acids tested, was ableto replace the activity of an extract of cotyledons andstimulate lateral rooting in decotylized pea seedlings.And Kruse and MIcCoy (5) have demonstrated an

arginine requirement for the growth of cells of theWalker Carcinosarcoma 256. When fed Cl4-labelledarginine. 25 % of the isotopic compoun(d was recovered

from cellular protein; the fate of the remainder was

not determined. Inhibition of these carcinosarcomacells on canavanine was reversed by added arginine.thus indicating a competitive inhibition of arginiineutilization. In the case of the arginine strain ofpollen tissue (5857), the canavanine inhibition is not

reversed by arginine, indicating a non-competitiveblock, perhaps of a more fundamental arginine-requir-ing reaction or possibly a strong binding of canavanineto an arginine-utilizing system.

Two excellent papers by Duranton (1. 2) haveshed light on the dlegracdation of arginine in tissues ofthe Jerusalemii artichoke. With C'4 labelled arginine

(an'idine group) he was able to show that urea was

formed during arginine utilization; with N15 labelledarginine (amidine) he showed the amino nitrogen tobe recycled into the free amino acids of the cells,especially into glutamic acid, but also into prolineand hydroxyproline.

The importance of proline and hydroxyproline inthe protein metabolism of higher plants has been em-phasized by Steward and Pollard (11) in their workon carrot root explants and derived cells. They foundthat C14 proline was incorporated into metabolicallyinactive cellular protein and that part of this prolinewas transformed into hydroxyproline. They alsocalled attention to the fact that hydroxyproline in-hibited growth induction by coconut milk; this in-hibition was reversed by proline. From this andother data they inferred a correlation between rapidgrowth and the proline-hydroxyproline metabolism ofcertain tissues.

Proline stimulates the growth of the arginine strainof pollen tissue, alone, with arginine, and in the pres-ence of ammonia; in the latter case to an extent notequalled by other compounds related to the ornithinecycle. In addition, hydroxyproline inhibits tissuegrowth in the presence of arginine. These results in-dicate a significant role for proline in the metabolismof this tissue, possibly related to arginine and pro-tein synthesis. The findings of Kasting and Delwiche(4) point in a similar direction. They found thatwhen watermelon seedlings were infiltrated with C14arginine, proline was highly labelled compared toglutamic acid. They suggested a close metabolic re-lationship between proline and citrulline.

SUMMARYStrains of tissue may be isolated from Ginkgo

pollen grown on a synthetic medium containing argi-nine. These strains are regarded as biochemical mu-tants which have been selected from large populationsof cultured pollen. In the nutrition of these tissues,arginine is the best nitrogen source, but may be partlyreplaced by ammonia, lysine, ornithine or urea, iinthat order; nitrate and glycine are not utilized.Anmong compounds related to the ornithine cycle, pro-line promotes growth and hydroxyproline is inhibi-tory. Canavanine inhibition of the tissue is onlyslightly reversed by addledl arginine.

ACKNOWNTLEDGEMENTSThe author gratefully acknowledges the excellent

teclhnical assistance of Miss Josephine LaBruna.

LITERATURE CITED1. DURANTON, H. Sort des atomes de la molecule

d'arginine au cours de sa degradation par les tissusde Topinambour. Compt. rend. acad. sci., France246: 3095-3098. 1958.

2. DURANTON, H. Sort des atomes du groupementamidine de la molecule d'arginine, au cours de sadegradation- par les tissus -de Topinambour. Compt.rend. acad. sci., France 247: 502-504. 1958.

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PLANT PHYSIOLOGY

3. FRIES, N. The influenice of amino acids on growthand lateral root formation in cotyledonless peaseedlings. Experientia 7: 378-380. 1951.

4. KASTINIG, R. and DELWICHE, C. C. Ornithine,citrulline, and arginiine metabolisml in watermelonseedlings. Plant Physiol. 33: 350-354. 1958.

5. KRUSE, P. F. and McCoy, T. The competitive effectof canavanine on utilization of arginine in growthof Walker carcinosarcoma 256 cells in vitro.Cancer Research 18: 279-282. 1958.

6. MUIR, W. H., HILDEBRANDT, A. C. anId RIKER, A. J.

The preparation, isolation, and growth in cultureof single cells from higher plants. Anier. Jour.Bot. 45: 589-597. 1958.

7. NICKELL, L. G. The continuous subnmerged cultiva-tioIn of plant tissue as single cells. Proc. Natl.Acad. Sci., U. S. 42: 848-850. 1956.

8. NITSCH, J. P. and NITSCH, C. Auxini dependentgrowth of excised Helianthus tuberosus tissues.II. Organic nitrogenous compounds. Amer. Jour.Bot. 44: 555-564. 1957.

9. PARIS, DENISE, DUHATET. L. anid GORIS, ANDRE.Action des vitamines et des acides amines contenusdans le lait de coco sur la proliferationi d'une souchede tissus de Carotle. Compst. rend. soc. biol. 148:296-299. 1954.

10. RIKER, A. J. anId GUTSCHE, ALICF E. Tlle growtlof sunflower tissue in vitro on synthetic media withvarious organic and inorganiic sources of nitro-en.Amer. Jour. Bot. 35: 227-238. 1948.

11. STEWARD, F. C. and POLLARD, J. K. 14C-prolineaind hydroxvprolioe in the protein metabolismIl ofplants. Nature 182: 828-832. 1958.

12. STEwVARD, F. C., STAPFS, MARION 0. anid SMITH.JOAN. Groxx-tl and organized development (of cul-tured cells. I. Growth and division of freely sus-pended cells. Ancr. Jour. B3ot. 45: 693-703. 1958.

13. TORREY, J. G. Cell division in isolated sinigle plantcells in vitro. Proc. Nat. Acad. Sci., U.S. 43887-891. 1957.

14. TULECKE, \W. Preservation and germilnationi of thepollen of Ginkgo under sterile con(litions. Bull.Torrey Bot. Club. 81: 509-512. 1954.

15. TULECKE, \V. The pollen of Ginkgo biloba: in vitroculture and tissue formationi. Amer. Jour. Bot.44: 602-608. 1957.

16. ZACII.IRIIUS, R. M., CATHFY. H. -M. andCI STE.VARD,F. C. Nitrogenlouis comI1pounds an1d n1itrogen nmetab-olisiim in the Liliaceae. III. Changes in the solublenitrogen compounds of the tulil) anld their relationto flower formationi in the bulb. Anni. Bot. 22193-201. 1957.

STUDIES ON THEI ETHYLENE PRODUCTION OF APPLE TISSUE 1. 2. 8

STkANLEY P. BURG AND KENNETH V. THIMANNBIOILOG.ICA.L IL.AORATORIES, HARVARD UNivERSITT, CA\MARIDElF. ASIA.\ACHt-,FTTS

Ethylene has been identifie(d as a niornmal mietabolicproduct of a wide variety of fruits (1, 15, 24), many

flowvers (6, 10, 29), certain fungi (7, 26, 40) an(d

sone leaves (6, 14, 31). Although studies of the bio-

logical effects of etllvene have revealed thalt in trace

amounts ethylene is able to mo(lify a number of proc-

esses of groNwth anl (levelopnment, the gas is normally

pro(luce(l in such miiinutte quantities thlt physiologicallyactive concentrations seldonm accumiulate. Flowers,for instance, respoln(l to their en(logeniously prodlucedlethylene only when they are confineI without ventila-

tion (10). Ripe fr-uits, however, are an exception in

that their tissues miay contain large internal concen-

trations of ethylene xxvhich are sufficient to accelerate

the ripening of immlnlature fruits. It is uncertain

whether the gas normally initiates ripening, i.e. acts

as a ripening hornmonle, or wlvetlher it is simply a by-

product of the process. There is general agreement,

Received March 16, 1959.2 Costs of this research were defrayed in part bv a

grant to Prof. K. V. Thimann from the Niaria NMoorsCabot Foundation.

This research was carried olnt xvhile S. P. Burg held

a research fellowshil) froml the U'. S. Departmeiit of

Health, Educationi a!ld Welfare.

however, that ethylene l)roduction is restricte(d tcthat stage in the life of the fruit (luring wllich riplen-ing occulrs. In fact, the tiime at which this substancebegins to be prodtuced is verylnear to the timie ofonset of fruiit maturation. A knowvledge of the chleml-istrv of ethflene prodluction thel-efore. shouil(d help to

elucidlate the imietabolic changes whiclh take place justprior to ripening. -Moreover. sloul(d ethylene trulyinitiate natural ripening, it voul(d be all the more im-portanit to know how the gas is svnthesized and underwhat condlitions the synthesis is initiate(l.

Although thel-e are a few reports describinlg thepreparation of enzyme extracts whiclh are capable of

prodlucing ethvlene (7. 12. 13). attemlpts to (luplicatethese results bv the original miiethodls an(l bv othlel-procedlures have so far been unsuccessful. For thi.sreason studies were undlertaken with tissue sections.The apple w,as chosen as an experimental materialbecause of its relatively high rate of ethylenle lrodluc-tion. In the past, research on1 the biosynthesis ofethylene has been hampered by lack of a sufficientlysensitive anDl specific quantitative assay, but the recent

(lev-elopnment of gas chromatography has circumnventedlthis problenm and mnadle possible a new approach.This paper reports some of the results which ha-e beenobtaine(d using gas chromatography to investigate theethylene prodluction of al)ple tissue sections.

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