PHYSIOLOGIA PLANTARUM, VOL. 15. 1962 A Revised Medium for Rapid Growth and Bio Agsays with Tohaoco Tissue Cultures By TosHio MuRAsniGE and FOLKE SKOOG Depnrtmeiil of Botany, University of Wisconsin, Madison, 6, Wisconsin (Received for puhtiealtou .\|irit 1. 1962) Introduction In experiments with tobacco tissue cultured on White's modified medium (basal meditmi hi Tnhles 1 and 2) supplemenk'd with kiticthi and hidoleacctic acid, a slrikin^' four- lo (ive-told intTease iu yield was ohtaitu-d within a three to Tour week j^rowth period on addition of an aqtteotis exlrarl of tobacco leaves (Fi^'ures 1 and 2). Subse(iueutly it was found Ihiit this jnoniotiou oi' f^rowih was due mainly though nol entirely to inorj^auic rather than organic con.stitttenls in the extract. In the isolation of Rrowth factors from plant tissues and other sources inorj^'anic salts are fre(|uently carried along with fhe organic fraclioits. When tissue cultures are used for bioassays, therefore, il is necessary lo lake into account increases in growth which may result from nutrient elements or other known constituents of the medium which may he present in the te.st materials. To minimize interference trom rontaminaitis of this type, an altempt has heen made to de\eh)p a nieditmi with such adequate supplies of all re(iuired tnineral nutrients and cotntnott orgattic cottslitueitls that no apprecial»le change in growth rate or yield will result from the inlroduclion of additional amounts in the range ordinarily expected to be present in tnate- rials to be assayed. As a point of referetice for this work some of the culture media in mc)st common current use will he cotisidered briefly. For ease of comparis4)n Iheir mineral compositions are listed in Tables 1 and 2. White's nutrient .solution, designed originally for excised root cultures, was based on Uspeuski and Uspetiskaia's medium for algae and Trelease and Trelease's micronutrieni solution. This medium also was employed successfully in the original cttltiva- tion of callus from the tobacco Iiybrid Nicotiana gtauca x A', tanijadorffii, atitl as further modified by White in 194^^ and by others it has been used for the 31 Phu.<t!<)l. Plant.. 15. ifHiJ [473]
Transcript
PHYSIOLOGIA PLANTARUM, VOL. 15. 1962
A Revised Medium for Rapid Growth and BioAgsays with Tohaoco Tissue Cultures
By
TosHio MuRAsniGE and FOLKE SKOOG
Depnrtmeiil of Botany, University of Wisconsin, Madison, 6, Wisconsin(Received for puhtiealtou .\|irit 1. 1962)
Introduction
In experiments with tobacco tissue cultured on White's modified medium(basal meditmi hi Tnhles 1 and 2) supplemenk'd with kiticthi and hidoleaccticacid, a slrikin^' four- lo (ive-told intTease iu yield was ohtaitu-d within athree to Tour week j^rowth period on addition of an aqtteotis exlrarl of tobaccoleaves (Fi^'ures 1 and 2). Subse(iueutly it was found Ihiit this jnoniotiou oi'f^rowih was due mainly though nol entirely to inorj^auic rather than organiccon.stitttenls in the extract.
In the isolation of Rrowth factors from plant tissues and other sourcesinorj 'anic salts are fre(|uently carried along with fhe organic fraclioits. Whentissue cultures are used for bioassays, therefore, il is necessary lo lake intoaccount increases in growth which may result from nutrient elements orother known constituents of the medium which may he present in the te.stmaterials. To minimize interference trom rontaminaitis of this type, analtempt has heen made to de\eh)p a nieditmi with such adequate supplies ofall re(iuired tnineral nutrients and cotntnott orgattic cottslitueitls that noapprecial»le change in growth rate or yield will result from the inlroduclionof additional amounts in the range ordinarily expected to be present in tnate-rials to be assayed.
As a point of referetice for this work some of the culture media in mc)stcommon current use will he cotisidered briefly. For ease of comparis4)n Iheirmineral compositions are listed in Tables 1 and 2. White's nutrient .solution,designed originally for excised root cultures, was based on Uspeuski andUspetiskaia's medium for algae and Trelease and Trelease's micronutrienisolution. This medium also was employed successfully in the original cttltiva-tion of callus from the tobacco Iiybrid Nicotiana gtauca x A', tanijadorffii, atitlas further modified by White in 194 ^ and by others it has been used for the3 1 Phu.<t!<)l. Plant.. 15. ifHiJ
[473]
474 TOSHIO MURASHIGE AND FOLKE SKOOG
125
wog
0.75^
0.50
0.25
» 20 25' ' 30g./i.
Figure 2.
Figure 1.
Figure 1. F.ffevt of tobacco lenf extract on the growth of tohacco fnth tissue \'\'-'2. !0 g/1.uf iiu'diiim: Exi»t. stnrtcct Mjir. 7. M158). Curve I witii, curve 11 wittiout teat" extractadded to the standard hasat nicdiiiiii.
Figure 2. Effect of concentration of leaf extract on the yield of tobacco callus tissus (T-2;Growiti period, Feb. 13 to Mar. 6,1958). Curve I, fresh weight: curve II, dry weight.
cultivation of various tissues frotn numerous .species (See Gautheret's 1959compilation). Gautheret's meditnn (1939j was devised by combinlit^' a twicediluted Knop's niacronutrient .solution with a slightly modified Bcrthelot'smicronutrieut solution. Hildcbrandt. Riker. and Dufif^ar (1940) employed thetrianfi;ulation terhnique to itnprove on Wiiitc's incditttn i'or the ciillivatiou ofspecific tissues. They reported two new tortuulae lincltided iti Tahles 1 ;uid 2)which they desiytuUed as optimal for cultures ol the ahove hybrid tobaccocalhts and for sunflower crown f all caMus respectively. These media, accord-iu}i to Burkholder and Nickell (1949). were still unsuitable for citllivation ofvirtis-indticed tumor tissues. By employitij; the triangulation technique ininitial trials and then by testing .serial concentrations of each clement inde-pctidently ni the others, these ijtvestigalors devised still another formulaliased on the medintn for sutifliiwer tissue by Hildebrandt ct al.. anrl wbichtbey reported as more stntable for Rttmex tissue. Heller (19531 bas tuadedetailed studies of tbe mineral requirements in cultures of carrot and Virgltiiacreeper. He first induced defi< iencies of several of the eleuienls by repeatedtransfers of tbe cultttres to liquid media lackinj^ Ihe element in qtiestion andthen rcintr(Khi(('d the clement in serial concctitrations to .select satisfactorylevels. For the most part tbe level of each element was varied only in com-binations in wbich the respective levels of all other nutrients were keptunchnnscd. In comparative tests. Heller found his medium to H've two tothree times greater yield.s than White's or (iaiitherefs media which wereemployed as controls. The nutrictit .soltttiou devised more recetitly hy Nitschand Nitscb (1956) for Joru.saletn artichoke is based on the hest estimate ofavailable literature for cultures of Ibis species and subsequent modification.s
PhyaSot. Plant.. 15. 1062
MEDIUM FOR TOBACCO TISSUE CULTURES 475
ill accordance with results of testing variations in each element separately,but il lacks iron and other trace elements. It is now clear that none of theabove media provide nearly the prerequisite amounls of some essential ele-ments for the rapid j^rowth rates and large yields Ibat lobacco tissue iscapable of attaining when various organic growth factors are also includedin the medium.
Materials and Methods
Source and preparation of tissues. Tobacco tissues, Nicotiana tabacum, var. Wis-consin 38. have been used exclusively in Ihis study. Fresh pith was used for the bulkof tbe work, and continuously sutjcullured callus was used for Ihe final confirmationtests. .Stems were cut from ca. 1 m. tall fobaccn jjlynts fjrowii in the greenhouse. Iheleaves were removed, the stems were swabbed witii 95 Vo etbanol and cut inio 5 to7 cm. long cylinders. Only a 15 cm. region of Ibe stem starting ca. 10 cm. from thetip was used. Cylinders of pitb parencbynia were bored from near Ibe eenter of theexcised stems with a sterile No. '2 cork Imrer. The cylinders were extruded from theborer with a glass ro<i. and sliced into discs approxiniatoly 2 mm. thick and weighingabout 50 mg. eiicb. Tbe ca. balf cm. end pieces in each cylinder were discarded as aprecaution against contamination. Tbree discs, each placed with one of its ftat snr-faces in coiilact with the niedinm, were planted in each culture flask. As fresb pitbwas readily available, it was a convenient msiterial to use in tbe large scale e.vperi-ments. However, data obtained from one experiment to the next varied to someextent, possibly due to differences in tbe nutrient status of tbe jilants from whicb thepilb was excised. For this reason, improvements in yield at successive stages in tbedevelopment of the nutrient solution and finally the suitability of tbe revised solntionwere confirmed in tests with tobacco callus. Firm, wbite calbis from 4—5 weeks oldstock cultures was cut into roughly rectangular pieces weigbing 40—50 mg. eacb,and tbese were planted in groujis of tliree into eacti culture flask. Tlie stock callus,originally obtained from pitb. h:id lieen subcidtured 10 or more times on mediumidentical witb tbe basal medium described below except tbat the kinetin level badbeen raised to 0.38 mg/1. and inositol had been omitted. Tbe vigorous more compacttissue ohtained by fonr-weekly snhcnlturing on medium modified in this mannergave excellent growth and satisfactory reproducihility of data (within 10 "y'o) fromone exjierimeiit to another.
Composition of tin- Hasal Medium. Tbe basal medinni whicb has served as controlor reference medium throughout this study is a modified White's nutrienl solution,which has been routinely employed in tbis laboratory and witb two addenda. 7?7,i;o-inositol and Edamin (u panereatic digest of lact;ill>umin ftunisbed by .SheffieldCbemicLil Co., Norwicb. N.Y.I. It had the followinji composition, in mg/1. of medium:(All salts were reagent grade unless noted to be otherwise.)
(c) pH adjustment: The pll ol' :dl media was adjusted lo 5.7-5.8 wilh a tew drops ofT 1 N NaOH or 1 N HCl before the agar was added.
Fe was added as agricultural grade sodium-ferric-ethylenediaminetetraacetateobtained irom Geif y Aj^riculUiial Chemicals, New York. N.Y. or Na^Fe KDTA pre-pared from Nao-EDTA. MW liTl.'M) {Tredcrick Smith tMioni. Corp. Columiius. Ohio}dissolved and beated in 1120 with an equimolar amount of FeS04 • THoO.
As the NUjNCXj contenl of the Iiasal medium far exceeded tbat of any other .salt,tbe concentrations of tbe otber elements were varied simply by adding them us eithernitrate or ammonium salts and tjy adjusting the amount of NMjNO-j accordingly toattain the specified total inorganic nitrogen level. Tbere was no evident effect ongrowtli from [lossible cbanges in pll or in otber conditions arising Irom tliis pro-cedure.
In Tables 1 and 2 the inorganic constituents of the basal medium are listed inmilIiinoles/1. {mM) or micromoles/1. (uA/) of eacb element. To facilitate comparisons,tbe concentration of each element is also expressed in nig/1 (values in parenthesis).These concentrations will be referred to as tbe 1 x levels, and in many experimentsmodifications will l»c made in inulti|)les of these, .such as 2 x, 3 x, etc.
Preparation of Uibucco leaf extract. Tbe leaf extracts, T-2 or T-3, used in this studyare both from Nicoliano tabacnm variety Wisconsin 38. T-2 is a water extract from62.4 kg. of greenhouse grown plants and was used Jiere only in tbe experinientsrepresented by Figures 1 and 2. T-3 is an extract from field grown plants preparedas follow: Healthy green leaves picked from vigorous, nearly mature, ca 1 m. tallplants were |)acked into plastic bags, sealed, trucked to a cold storage plant, (piicklyfrozen and ke|)t indefinitely. The frozen leaves were then groinid in a commei-ciaitype meat grinder and tluiwed. The juice was expressed witb a liand press, heatedto near 100 C, cbilled to 5-10°C, centrifugcd and concentrated under reduced pres-sure. The concentrate was stored in a frozen state in 1 1 polyethylene boltles and
* Data from Helter's compilation (1953).in parenthesis are mg/l of medium.Physiol. Plant., 15. J.WS
Data from Nitsch and Nitsch (1956. Data
MEDIUM FOR TOBACCO TISSUE CULTURES
Table 2, 'Micronutrient compo.sitlon.s of plant tissue culture media.
All
Mediumtilement (Concentrations in tnicromotes per liter of niettium)
CuMn Zn I .Mo Co Ni Te Be Al
Whtte (1943)'Gaiittierel 'Hildehmnrtt et al.
(Tobacco)HiIdet>ran<:U et al.
(.Siinftower) . . ,Burtibotitcr &
Nictiell'HeUer ^Nitsch & Nitsch'.Biisiil Medium . . .
1 X LevetRevi-sed
Medium
250.4
6
50
1016
26(0.28)
too0-08)
304.5
20
20
24.5
29(1.60)
100(5.50)
to
0.2
2.2
1.04.63.5
9.4(0.62130(1.92J
4.51.5
8
2.2
0.06
0.1
1.60.12
0.1 1.0 1.0 0.3
• Data from Hetler's compilation (1953)
4.5(0.58)5.0 0.10
(0.64 I (0.0064)
^ Data from
1.0
1.0
0.13
0.10 , —
0.23
0,096) (,0.006)i —Nitsch and Nitsch (1956).
used as needed. T-,'J represents 13.6 kg. of solids in 21.6 1 of concentrate obtainedfrom 465 kg. of tresti leaves.
Cutture conditions and growth measurements. The tissues were grown routinelyin 125 ml. erlenmeyer flasks witli 50 ml. of medium. Fifteen pieces, three in eachof five replicate flasks, were phinted for each treatment. The cultures were kept onshelves inider h)w intensify overiiead florescent lights it] a room at 26-28"r. andahout Hi> "/tl relative humidity.
A growth period of 4 weeks was selected because it extends i)ast the end of thelogarithmic phase of growth (see Figure 1), after which time the fresh and dryweights per flask were determined. Since in most cases the (h'y weights of the tissuewere close to o "/ii of ttie fresh weights, only the latter will he reported. However,certain trends in percentage dry weight of the tissue with change In composition oftlie medium will be considered. Replicate cultures had rather uniform yields, and the
standai'd error, + , of the average final fresh weights in the 5—20 g rangen(n-l)
was around ±0.8g/flask.Testing procedure. In most previous w()rk on quantitative nutrient requirements
either a single element was varied and the others kept constant, or three elementswere varied at a time (the triangulation method). In the present study the require-ment of a given element was estahlished by varying its concentration iu the presenceof several levels of the remaining elements. Tests have l)een limited to 1 x and higherlevels of the elements present in the basal medium. In addition C.IL (.O and Mo havebeen tested.
Results
Prt'Iiniinary teslinj,' of Ihe basal medium showed that douhlint,' and quad-plinfi the levels of inorganic or organic constituents lead to increases in
Physiol. Plant.. 15.1962
478 TOSHIO MURASUIGE AND FOLKE SKOOG
Table 3. Effect of increasing the concentration of the nutrients in the basal meiUuw on thegrowth of tobiHio callus tisifue. (Growth iKTiiiil, 11/15-12/18/5S.)
Clianse in totnposition ofme dill in
None (Basal medium 1 x)All constitiitents 2 x
4 XAll organic suhstances . . . . 2 x
. . . . 4XAll inorganic salts 2 x
4 X
AveniKe final frt-sliweight g./flasli.
5.69.9
14.96.3(5.49.7
17.0
Tahle 4. Effect of quadrupling the concentration of the inorganic elements of the basalmedium all together find each separatehi on the growth of tobacco callus tissue. (Growth
yield of tissne (Table 3) and that Ihe principal, although not always Ihe entireimprovement was from the inorganic salts. When each element was increasedseparately to 4 times its level in tlu' basal medium (4x), each major elementexcept S gave appreciable improvement in yield (Table 4). N and K wereespecially elTective, but increa.shig all the salts to the 4x level definitely hada still greater effect than increasing any one element alone. Of the minorelements, excepting perhaps CI. none was signiticantly better at the 4 x thanat the 1 X level.
The requirements for N, K, and P
In view of Ihe ahove results further tests were carried out with possiblecdmbinations of N. K, and P raised to 2 x, 4 x and 8 x levels. In addition theseC(jmbinations of N, K and P were tested with the remaining elements in-creased to 2x, 4x and 8x levels. The greatest increase in ti.ssue growth wasobtained when N was increased to 4 . K to 8 x, P to 8 x, und all other elementsPhysiot. t'liiHt.. 13. imi
MEDIUM FOR TOBACCO TISSUE CULTURES , 479
were increased fo 2x or 4 x, i.e. under Ihe more favorable conditions forgrowth the optimal levels of N, K and P would appear to be about 50, 15 and1.0 mM respeclively.
X. The N reqiiirenient v -ns examined in more detail by lestinj,' the growthot pith tissue on media containing varying amounts of NILiNOii to give Nlevels of 12, 24, 48 or 96 mM in combinations with 14.4 or '28.8 mM K and0.74 or 1.5 mM P. All other inorganic constituents were kept at their 3xlevels. The average final fresh weights of tissue obtained in t)ne experimentare plotted against the N level in Figure 3. It may he noted that under Ibeconditions of this experiment the increase in K for unknown reasons resultedin a marked increase in yield at the low N levels. In this experiment the highphosphate level (1.5 m.V) depressed growth except perhaps in the case ofhigh K and low N media. In experiments with other lots of planls, P levelsup It) 2.0 mM were sometimes favorable, but generally P levels of 1.5 mM orhigher bave tended to depress the yield of either pith or callus ti.ssue.
The data show that 50 mM is close lo optimal for N irrespective of the Kand P concentralions, and tbat tbe N content may be increased somewbatabove this level witb no appreciable effect on Ihe yield. However, N levelsof 80 mM or higber consistently were found to depress tbe yield. Hence, alevel of ()0 m;U \ bas been selected as satisfactory.
A'. The K requirement was determined witb N kepi at the 4 x level (48 mil/);P kept at tbree levels. 8 x. 16 x and 32 x (0.74, 1.5 and 3.0 mM respectively),and all olber minerals kept at their 3x levels. As shown in Figure 4. tbeyields presenled in curves 1 and 2 increased wilb Ihe concentration of K upio 15 mM and fell only sligbtly at 30 mM, tbe highest tested K level. In curve3 tbe yields are low througboul, probably due to toxicity of the higb P level{see above). A 20 ni.U K level bas been selected.
P. The P requiremeni was determined with N at (he 4x level (48 mM)witb K at tbree levels (7.2 14.4 and 28.8 mM] and with all other inorganicconstituents at tbeir 3x levels. Tbe results are sbown in Figure 5. It may beseen tbat Ihe optimal requirement for P was strikingly dependent on the levelof K. but with an ample supply of K il lies in tbe region between 0.7 and1.5 in.W. liecau.se. as stated, higb P levels were toxic to the pilh explants inthi,s particular lot of i)lnnls. the experiment was repeated in part witb severalotber lots of plants. In the.se cases the K level was set at 20 mM and as .shownhy typical results, curve 4 in Figure 5, the optimum P level was 1.5 mil/.However, sometimes this level and frequently higber levels were growtbiiiliiltilory even though very bealtby looking tis.sue might be produced. Be-cause of these difiicuUies six additional experiments were done, three withpith and three with callus cultures. P levels were adjusted to I.O, 1.25, 1.5,1.0 and 3.0 mM. Other elements were as specified for tbe revised medium(Tables 1 and 2) except that Fe was used at both tbe 0.10 and 0.25 mM levels.Of tbe organic constituents (Table (iB) Edamin was omitted, and for sometreatments sucro.se was used al eitber the 2 Vo or 4 Vo level as well as at the.specified 3 Vo level. Data from one experimenf with pitb, typical of the resultsobtained, are presented in curve 5 in Figure 5. U appeared that the P require-ment may vary witb the levels of Fe and sucrose as well as K. Neverlbeless,the results showed conclusively tbat 1.25 m.U is close to the optimal P level;
Phyaiol. Plant.. 15. 11102
480 TOSHIO MURASHIGE AND FOLKE SKOOG
20
10
FIGUR
\_
/
a'"
- -a
1
E3 .2
"3'--~
N
FIGURE i,
^ 1//
•a.t':-:._
1 1 1 1 1
n
/2
"3
20 40 60 80 960 to 20 mM 30
20
15
10
FiGURf 5
1
/ ^
7 11
/'\4
•
• ""3
f
\2\
P
FIGU
—a
mil
?E 6
r
3/1
'2
\s
\\
\
s\
\
Ca
J 125 30 5 6mM
Figures 3 lo 10 inthisive. Fresh weight i/ields of e.vcised tohacco pith ealtures in re.f!>onseto increased concentrations of nutrient elements.
Fisiire 3. Effect of ,Y. (Gruwlh period. May 5 tu June !*(). 195i)|. All elemenls al 3x levelsexci'|)l K iind F'.Curve I: K 14.4. P (1.74 m.W. Curvo '2: K 12S.M. l» 0.74 iii.y. Curve 3: K H 4 l» 1 5n\M. Curve 4: K 18.S. P l.r> mM. BC. basal ciinlrnl.
4. Effecl of K. (Growlh pfriod. Miiy 19 lo June 16, 1959). N kepi al 4 x (48.8 mM)aufi :il] olher olenienls at 3x levels except P.Curves 1. 2 and 3, the level of P was 0.74. I.f> and H.O iiiM respectievly. BC. liasalconlrol.
5. Effect of P. (Growlh period, Curves I ^ , May 20 to June 17. 1959; Curve 4,1960; Curve .'j. July lit to .Sept. 1. 1962).N kepi al 4 X and all ulher elements at 3 x levels except K. Curves 1, 2. 3 and 4,the level of K was 7.2, U.4, 28.8, and 20.0 m.V respeclively. Curve 5. see text.B(;, hasal control.
Figure 0. Effect of Ca. (Growth period. July 9 lo Aug. 10. 1959) N kept at 55 mM, K at20 ni:l/. P al 1.0 m.V, and niicronutrients B, Fe, Mn and Zn at 3 x levels. Mg, S,and Cl varied. Eaeh kept at ils 1 x, 2 x. and ox levels in Curves 1. 2 and 3respectively. BC, basal control.
P/13/shl. Plant... 15.1963
MEDIUM FOR TOBACCO TISSUE CULTURES 4H1
20
15
10
\nGURt\\
° >
-B.C.
Mill
.7-•- .?
s
s
2
y ''\
Mg
FIGUR
o"'
- .ac
E9
1
i'
2
*•;
t
1 .
—
a
15
TO
I 1.5 2 0 2 dmM
FIGURE0
A
/
"'ac.
\ 3
s
16 2
FIGU
y1.
— i k.
nir? »
RE 10
C.
1
•
— * - -
2
'--••?
— -
Fe
0 0.1 0.2 0.3 0.4 0.5mM
Figure 7. /;//et( o/ My (Growlh ppriod. .7iily 9 lo Aug. 10, 1959). N kept al 55 m.U K at20 mM. P at 1.0 ni.¥, and micronutrients B. Fe. Mn. and Zn at 3x levels. Ca, Sand (.1 viirifd. Each kept at t x. 2 x and ox levt'Is in Curves I. 2 arut 3 respec-tivejy. BC, basal control.
Figure 8. Effect of S (Growth period. .July 10 to Aug. 10, 1959). N kept at 55 m.U K at20 mM. P at 1.0 mA/. and microiiiitdents B. Fe, Mn, and Zn at their H x levelsCa. Mt;. and CI variL'd. Ea<h kt'iit at I x. 2 x and 5 x levpls in curvi-s 1, 2 and 3re.spectively. BC, hasal tontroi.
Figure 9. Effect of CI (Growth period. .July 10 lo Aug. 11, 1959). N ke|)t at iiit m.U. K at20 mM, P at 1.0 m;U. and miironulrients B, Fe. Mn. and Zn at their 3 x h'volsCa. Mg. and S varied. Each kept at its 1 x, 2 x and 3 x levels in curves 1, 2 and 3respectively. BC. hasal control.
Figure 10. Effect of Fe (Growth period, July 15 to Aug. 12, 1959). Nutrients kept at Iliefollowing levels: N 55. K 20, P 1.6. Ca 3.0. Mg 1.5, S l.() and CI (i.O m.U. Mirro-elenients B, Mn, and Zn varied. Each kept at its 1 x, 2 x and 5 x levels in curves1. 2. and 3 respectively. Horizontal arrows represenl yields attained in laterexperiments, see text. BC, basal control.
Physioi. Plant.. IS, IM2
482 TOSHIO MURASIIIGE AND FOLKE SKOOG
1.0 is on the low side, 1.5 on the hif,'h .side, and 2 or more mM definitely toohigh. For this reason, a 1.25 mM P level has heen selected.
Hfquirements for Ca, M(j, S, and CA
Wheti IIK- ri'qttirements for Ca, Mg, S., and CI first were evaluated, the N,K, and P levels of the media were kept at 55, 20 and 1.0 m.l/ respeclively;(".(•., close lo the values selected ahove as satisfactory for each of these ele-ments. Kach of the iour elements was tested at four levels (1 x, 2 x, 5 x, and10 x} and in conjunclion wilh three levels (1 x, 2 x, and 5 x) of the other threeelements. The micronulrients B, Fe. Mn and Zn were provided at their 3xlevels.
Co. By comparison of the cnrves in Figure 6 the reciuirenient for Ca wouldappear to be relatively higher with the 2x than wilh Ihe other levels of Mg,S and CI. With these eletnents at the 5x level the Ca concentration cnrveforms a hroad platean hetween 1.5 and (i n\M. A 3.0 \\\M Ca level has heenselected.
Mg. Results from one of the experiments with Mg are shown in Figure 7.Both with the 1 x and 5x levels of Ca. S and CI Icurves 1 and 3) tissue yieldsincrea.sed as the Mg concentration was increased from 0.3 to 1.5 mM. The3.0 mM level of Mg was without further stimulating effect when the levelsof Ca. S and CJ were l x and it was perhaps inhibitory when Ihe levels ofIhfsi' eltMiifuts were 5 x. With 2x levels of Ca. S. aud CI, the fiual freshwcighls (if the pith explants fell as the Mg concentration was raised inmi 0.3to 1.5 m.l/ but was up again when the .Mg concentration was.'i.O m.V. Althoughin this case the lowest concentration of Mg resulted in the highest yield ilshould be noted that an imbalance existed as tbe cultures in this particulartreatment became very lu'crotic. The 1.5 m.l/ level of Mg wa.s .selected.
S. As is shown by curve 1 in Figure 8. with 1 x levels of Ca, Mg aud CI,the lowest concentration of S (0.3 m.l/) was optimal and higher S concentra-tions caused a steady reduction in tissue yields. However, with tbe 2x or 5xlevels of Ca, Mg aud CI. the 0.3 mM S level was less than acleipiate, and asubstantial increase iu yield was obtained at tbe O.dfi m;V level. S cniicentra-tions higber tban O.(jf) m.U were iiibibitory lo tbe pith explants. In subsequenttests witb callus, on tbe otber band, no toxicity was obtained even wilh Sconcentrations as high as 13 mM. On the basis of all results and even thoughit may he slightly higber tbau o|)tiuial tor excised pilb cultures, the 1.6 m.l/level of S bas been selected.
CA. Witb Ca, Mg aud S at tbeir respective 1 x levels, increases in CI con-centratiou had no appreciable effect on growtb of pith explants (Figure 9),wbereas with Ca, Mg and S at tbeir 2 x or 5 x levels, increases iu tbe CI levelup to 4.4 mM were stimulatory. A bigher level of CI (8.7 mM) gave neithera further iucrease nor iubibitiou of growtb iu the presence of 2x levels ofCa. Mg and S. hut it was far less stimulatory tbau the lower CI levels in thepresence of 5 x levels of Ca, Mg and S. Witb callus instead of pitb, CI con-centrations as high a.s 12 mM were without inhibitory effect. A CI level of(> mil/ has been selected.I'hi/siol. Plant., JS. im2
MEDIUM FOR TOBACCO TISSUE CULTURES 483
Microniitrient requirements
Preliminary studies indicated that 3x to 5x levels of B, Fe, Mn and Znwere not inhibitory, and that in combinations with increased NaFe-EDTAlevels they were .sometimes stimulatory. Iu these experiments the other ele-ments were supplied in the concentrations selected as optimal or very closeto these levels.
Fe. Availahilily of Fe has long been recognized as a critical requirementfor vigorous aud prolonged growth of plaul tissues. Tbe use of chelatingagents, citric or tartaric acid and more recently especially tbe use of EDTAhas greatly improved tbe available snpply of Fe and possibly some othermicronutrients in culture media. In tbe present experiments Fe was suppliedat first as a couuuercial NaFe-EDTA chelate wbicb was tested iu combina-tion -witb 1 X. 2 X. and 5x levels of H, Mn and Zn. As .sbowu iu tbe curvesin Figure 10, increases iu the Fe chelate from 0.05 to 0.25 m.U resulted iniucreased yields of tissue especially in tbe presence of 2 x and 5 x levels of theotber three trace elements. Increa.sing tbe NaFe-EDTA level to 0.5 mMresulted in a definite decrease in yield when the otber trace elements wereraised to their 5x levels. A higher purity preparation of Na-2-Fe-EDTA wasthen u.sed. which in a large numher of tests with both callus aud pith tissuesconsistently gave optimum yields at tbe 0.10 mM level. Even 0.05 uiiW Fepermitted excellent growth, and sometimes close to optimum yields, of botbtypes of tissue, wbereas 0.25 mM Fe generally gave cousiderahly loweryields. Repre.sentative yields at tbe 0.05, 0.10 and 0.25 m,l/ levels obtaiued ina series of three experiments witb pitb ti.ssue are sbowu by horizontal arrowsin figure 10. In view of these results tbe Fe content of t!ie commercial Fe-chelate was verified by chemical analysis. Considering the complex inter-action between the Fe-chelate aud other elements in the nutrient mediumvarious plausible reasons might account for tbe difference in tbe resultsohtained. Tbe curves in figure 10 suggesting an optimal ievel of 0.25 mM areincluded for tbe sake of consistency with tbe data for the (tthor elemeuts. eventhough on tbe basis of all results tbis level is too higb and a 0.10 mM ofNa^Fe-EDTA has been selected as optimal.
B, Mn, and Zn. (Considering the likely preseuce of impurities, the B, Mnaud Zu requireiueuts were fiually tested with callus gnrn-n on media madeup witb Difco purified agar and without addition of Edamiu. Each elemeutand all three in combination were tested in serial concentrations up to tbeir5x levels, aud NaoFe-EDTA was kept at the 0.25 mM Fe level. There was noiubibitiou from their presence, and perbaps a stimulation, at least when alltbree test elements were increased to tbeir 5 x levels (See Table 5). From tbeseand other data the levels 0.10 mil/ B, 0.10 mM Mn, and 0.030 m.l/ Zn bavebeen selected.
Other Etements {Cu, Mo, Co, I, and Na). Cu added in tbe range from0.00003 to 0.03 mM was without effect on the growtb of the tobacco ti.ssue,aud .so was Mo. 0.001 m.V. tbe concentration employed by Torrey (1954)with pea cnlture.s. Iodine, even wben raised to tbe 4x level (0.02 mA/), andNa tested at the 5 and 10 mM levels, i.e., iu the range used by Heller (1953)aud by Hildebraudt et al. (1946), similarly were without effect on yields.
In -spite of the above negative re.sults these elements have heen included inPht/siol. Plant.. 13, JBG2
484 TOSHIO MURASHIGE AND FOLKE SKOOG
Table 5 Effect of incrcasefl levels of B, Mn and Zn on the growth of tobacco catlux tis.-iue.(Growlh period 9/15-I0/12/.W.)
Change in comi)osition ofmedium
None (Basal meciiuni 1 x)B 5xB and Mn 5 xB and Zn 5 xB. Mn and Zn 5 XMn 5 XMn and Zn 5 xZn ax
.•Vverajje final freshwei)fht g. flask.
12.511.612.612.614.313.712.214.0
the revi.sed medium. C.xi and Mo are added because they are kuowu to bees.sential elemeuts for planl growtb. Similarly Co is included tor reasons ofits requirement hi lower plants (Holm-IIausen ct al. 1954) aud tbe possiblerole of cobaltous ion in morphogenesis of higber planls (Miller 1954, Salis-bury 1959). A test of CoCU iu eight serial couceiitrafions between 0.000! andO.iri m.l/ and all otber elemeuts at the lx levels was iucluded iu an earlyexperiment. No stimulatory effect of Co was obtaiued at any level but rathera toxic action at tbe two higbest levels. 0.08 aud O.Ki. The selected levels are:Cu 0.0001. Mo O.OOI aud Co O.OOOt m.U. lodiue arbitrarily is retained at0.005 mM. Na, o.steusively uonessential but perbaps uot entirely devoid of itsoften claimed slimulatiug effect on plaut growtb. heing a par! of tbe FeSOj-Na EDTA solution, tbereiore, is supplied at tbe 0.20 m V level.
pH. Tbe pH of the medium was adjusted to 5.7-5.8 with a few drops of1 ,V HCl or with NaOH or KOH eitber before or after tbe agar was addedaud had been heated a tew minutes iu tbe autoclave. Tbe value was selectedou the experience tbat tbe reaction of eitber more acid or more basic mediatends to dritt toward tbis region duriug tbe beat treatment for sterilizationand subsequently witb time even iu uniuoculated fla.sks. Prebeating witb theagar pre.sent avoids any appreciable cbauge iu pH during tbe subsequentautoclavatiou. Tbe value 5.7 fo 5.8 is suitable for maiulaiuing all the salt insoluble forui eveu wilb relatively bigh phosphate levels aud low euough topermit rapid growtb aud ditfereiitiatiou of the tissue. The amonuts of C I. Naor K iutroduced iu the process are uot inclnded in the stated levels, 6.0, 0.2or 20 mil/ respectively of tbese ions.
Composition and (jrowth effects of the revised medium. The kinds andamounts of iniueral salts finally adopted for the revised medium are listed inTable OA. Quantities bave beeu adjusted upward, beyoud actual needs insome cases to provide total concentrations of all elements in round numbersas shown iu Tables 1 and 2.
Tbe yield of tabacco tis.sue ou tbe revised medium has heen compared withtbose on the original basal mediuui (1 x) and otber media as sbowu in Tahle 7and iu Figure 11. Not only were the higbest yields obtahied consistently outbe revised media hut the pronounced tissue necrosis characteristic of oldcultures on all other media was avoided as is also evident in Figure 11.PhvstoL Plant., 15, 1982
MEDIUM FOR TOBACCO TISSUE CULTURES
Table 6. Composition of the revised medium. pH adjusted to 5.7-.J.8NaOH (see texlj.
Agar 10 R/I.nij/o-1 no si to I 100 mg/l.Nicotinic acid 0.5 mg/l.Pyridoxin • HCl 0.5 mg/l,Tiiinniin- H(.i O.I nig/l,
Table 7. (Comparison of yields nf tissue on different merlinA. Pith Tissue ((irowth period 10/2-ll/3/.')*.H.
Medium
RevisedBasalHeUermtschHitdebrandt ef ul.
Fresh weight in g/flask
Without Kdamin
17.34.15.01.95.3
With Ktianiin
24.57.1
17.9y.79.2
Dry weight ID g/Flask
Without Kdamin
0.470.130.1»0.100.20
With I'Mamio
0.490.260.430.340.37
B. Callus Tissue (Growth period 8/3l-10/I0/.)91.
Medium
RevisedBasalHellerNitscli and NitschHiUtebrandt et at.
Fresh weight in g/llask
Without Kdamin
21.55.13.63.02.S
With Eciamin
22.710.816.217.69.8
Dry weight in g.llask
Withouth Kdamin
0.480.22 -0.240.280.18
With Kdamin
0.460.350.380.480.37
Physio}. Plant.,
486 TOSHIO MUR.\SH1GE AND FOLKE SKOOG
fj 11. Comparison of yields of tobacco pith tis.Huc ciitliired on various me<lia iGrowlhperiod, Oct. 2 to Nov. 3, 1959). Upper row without, lower row witli Kdamin 1.0 g/1. Mediafrom left to riglil: CA. l>asnl iiu'dumi 1 >. T. rcvistd: H, Heller |19r)3t, N, Nitsch and Nitscli
{iy.=>6|. nod R, HildchniiuU ct at. 11946).
Organic Constituents
The required levels of organic coiLstitutents in the basal medium huve beenle.med only to ;i limited extent.
l'Yom Ihe resnlt.s in Table 3. no larf e increase in yield wonld be exi)ectedfrom increased concentralions of the specified organic ingredienls. althoughwilh improved mineral supplies some beneficial effect might be oblained bymodifications in these. The list of oilier polentially stinuilatory organic snh-stances and favorsible combinations of these is of course inexhaustible.
Vitamins. The levels of the following vitamins have been rel:iine<l tin-chunged from the basal medium: thiamin-H(_M 0.1 mg/l. nicotinic acid 0.5mg/L pyridoxine- HCl 0.5 mg/I. On Ihe basis of experience by others (Braim1058, Steinhart ct al. 19(>1. I9(>'2) nn/o-inosilol was tesled and lonnd to pro-mote growlh in cuhiires when <»ther conditions wonld permit high yields.A level of 100 mg/l of nii/o-inositol has been selected.
Nitrogenous eompounds. The combination of amino acids in yeast extractand also various simpler combinations are known to promote gcowlb oftobacco calhis on the ba.sal medinm (Sandstedl and Skoog 1960. La MottelimO). Giycine has beeti retained willumt further testing at '2:0 mg/l. Thecasi-in hydrolysate. Edamin. was tested extensively. In the comparative testswitb diiterent media it snbstantially increased the yield tm all excepi therevi.sed medium (Table 7). The strikingly lower response on the lattermedium prohahly is due mainly to tbe relatively bigh X content. The smallincrease whicb did result from addition of ICdamin perhaps is (hie in part tounknown organic factors. An additional amount of NH4N0:) equivalent to tbeN ct>ntent of the Edamin failed lo replace it and instead lowered the yield.However, NH2-N is not excluded hy these results as being the principal activePhysiol. Plant., 15, 1902
MEDIUM FOR TOBACCO TISSUE CULTURES
KINETtN
487
0.04 0.64 2.56 to. 2 0.04 0.64 2.56 10.2
tAA
2
V ^QS^^I^V \S' QP
^^J i k <HlPfc ''-T^^^^ M H ^ ^
Figure 12. Tohaeco callus ciillures grown on revised medium with increasing levels of IAA(ordinate, mg/i.) and kinetin (abscissa, mg/t.). A without Edamin; growtti period, Oct. 6 to
Dec. 22, 1961. B with 1 mg/t. Edamin; growlti period Oct. 11 lo Dei. 22, 1961.
agent contribtited by the hydrolysate, for it has been shown (Steinhart et ul.1961) that .spruee tls.sne which is ttnable to snrvive on a medinm with NUjNOnas the sole N-sonrce ^vo\\% well on the same meditmi sttpplemented witheitber nrea, arj^inine or other basic atnino acid. The infltience of differentkinds of ca.sein hydrolysnte (kindly snpplied by the Sheffield Chemical Co.)i.s quite variahle. Edamin being among the more active preparations. Theinflnence of Edamin it.self seems to vary to some extent, increasin;? with ageof the ctilttire.s and being even more .striking witb respect to the developinetitof vigorotis sboot.s and root systems than the increase in yield. As may beseen from comparisons of corresponding cnltnres nnder A and H in Fignre 12.tbe presence of Edamin bas resnJted hi a hroader range of IAA and kinetinlevels which permit vigorotis organ development. It has shifted fhe levels andto a slight extent perhaps also the ratio of the two snKstances required for aparticular developmental pattern Io emerge. However, essentially no effectswere ohtained in the presence of Edamin that could not be obtained al.so inits absence by suitable modifications of tbe kinetin and IAA levels.
Physiol. Plant..}a. 1%2
488 TOSHIO MURASHIGE AND FOLKE SKOOG
Carhohydrfites. Allhougb tobacco cultures are capahle of utilizing varionscarbohydrates and organic acids to some extent as energy sources, sncroseis as favorable as any known material of this type. Concctitrations of 2, 3,and 4 Vo are almost equally .snitable for cultures with low yield.s. However,in several large series of comparative tests 3 Vo was definitely better than2 Vo, and 4 Vo was often somewbat less effective than 3 "/o in cultures witbmoderate to high yields. For example in one experiment fresh weigbt yieldsof cnltures with 2, 3 and 4 Vo sucrose were 14.3, 15.9 and 12.9 g/flask respec-tively and tbe correspouding dry weights were 0.44, 0.07, and O.fJ2 g/flask.Dry weights often were somewhat higher per fresb weigbt in cnltures witb4 Vo sucrose tban in the others, bnt on the average also the total dry weightper flask was less in Ihese tban in ctiltures with 3 Vo sucrose. The level ofsttcrose in the revised niedititn. therefore, has been set at 3 "/o.
Agar ordinarily is kept at 1 Vo to give a suitably moist btit rigid niedinm.The concentration may need to be varied to some extetit depending on tbepreparation and the salt content, pll. etc. of the mediutu. In the case ofweakly growing tissue cultures agar content may be critical for survivni. butfor vigorously growiug tobacco cultttres it was unimportant. In liifnid cul-tures, on tbe other band, changes in composition of tlie mcctinm definitelyare reqnired for vigorotis bud development to occur (Loewenberg et al. un-pttl)lished.)
Indoteaeetic acid and kiuetin. The opthnal level of IAA or kiiietin willdepend on Ihe type of growth that is desired. It will vary witb the endogenouscontents of atixins and kinins and other factors influencing the sensitivity ofa partictilar strain or doue of tissue fo the.se substances.
The curves in figure 13 repre.senting tresh weight yields in two expfiinicntswith difterent lAA-kinetin combinations clearly .show that the optimalre(itiirenient for either one increased witb the coticentration of the otber. Theeffect of a given comhination, e.specially in the intermediate concentrationratige, varied from one experiment to the next as illustrated by tbe twoexamples iti figure 13. Note that the response to 3 mg/l. IAA is much greaterin one experitnent than the response to 4 mg/l, in the otber. Keniarkabledevelopmental patterns, differences in form and texture of the callus, extentof organ formation, nnmber and .size of organs, vigor and longevity of tbecultures are associated witb specific levels aud ratios of IAA and kinetin. Aniiupressir)n of the range in size and form of tbe cultures may be gained fromfigure 12. Details of tlie strict quautitative aspects and other characteristic .sof the growth responses which have emerged as functions of the lAA-kinetinconcentrations will be presented elsewbere. In general the following applied.For continnotis growth of firm, bealthy tobacco callus in sub cultures,0.2 mg/l. of kinetin and 2.0 mg/l. of IAA have been used witb excellent resnltsfor several years in onr laboratory. However, 0.04 mg/l. of kinetin and 1.0mg/l. of I.\.\ has given nuich faster growing, loosely packed ma.sses of cellswhich could be transferred and maintained as callus even though vigorousroot formation ordinarily occurred in these cultures after 2 to 3 weeks. Forlong lasting cultures (2 to B montbs) higher kinetin levels were required, andf<»r vigorously growing stich cultures higb levels of both kinethi (3 to i\ mg/I.)and IAA {up to 30 mg/l.) were needed. For induction of bnds the IAA level
PhysluL Plant., 15. ja62
MEDIUM FOR TOBACCO TISSUE CULTURES
25
489
l''if{ure 13. Effects of increasing concen-tration.s of IAA and kinetin on freshit'L'iyht yield of tobacco cn/iiis culturedon revised medium (Growth period, 4weeks. Miii. lo OtI. 1960). Ordinate:averapp frt-sh weight prr flask, at)scissu:ttinctiri concentration. Ttie numbers onttie curves 0. 3, 9. 27 and 1, 2, 4 cor-respond to the concenlralion of IAAadtled in vmh case, tiesiilts are fromtwo experiments represented by sotidand broken Hnes respeclively.
must be kept relatively low, between 1 and 5 mg/l.. depending on the kinetinlevel.
Naplilluileneacctic and 2,4-dichloropbenoxyacetic acids bave been tested assnbstitutes for IAA. In tissues witb high IAA inactivation rates they werepreterable if not essential ftir growth. Levels of 0.05 tt) 0.2 mg/l. bave beensatistactory for tbe growth of callus. These substance.s are so mucb .strongeror longer lasting auxins tban IAA. that all but extremely low concentrationsprevented bud formation.
Re-test of the tobacco leaf extract. In accordance witb the objective toinsure an adequate supply of minerals in tisstie culture bioassays of organicgrowth promoting substances in complex extracts, the stimulating effect oftobacco teaf extract was now re-tesled with the revised medinm. Tbe extractagain promoted growth (Figure 15J, but in contrast with the earlier tests onthe basal medium, on the revised medium the ash of tbe extract was without.significant effect. For example, in one experiment the fresh and dry weightyields on the revised medium alone wore 13.6 and 0.43 g/flask respectively;with 3.75 g/1. T-3 extract they rose to 21.7 and 0.58 g/flask respeclively;whereas the ash of this extract gave only 14.6 and 0.46 g/flask respectively.
The promotion of growth by tbe T-3 extract in this case, therefore, can beattributed fo ils content of organic stibstances rather than inorganic salts.
Effect of volume of medium. Further experiments revealed tissue yield to32 Physiol. Plant., 15, J962
490 TOSHIO MURASHIGE AND FOLKE SKOOG
a:^60
A
-
-
-
- jn
r//2
6
S
/
j l ?
V
r1••* - O — 1
2
T
10 20 30 40 0 JO 20 30 40DAYS
H. Efffct.t of the ixi-liinic of revised medium findnf the addition of tohaccoleaf extract on fresh weightijield.
Cmvfs 1 amt 2. It)() ml ofmedium in 250 ml Krlen-nieyer flasks.
Curves 3 and -I. 50 ml of me-dium in \'2ii mi fta.sks.
Curves I and 3 witti T-.'i ox-tract. 3.75 g .solids/t of me-dium.
Curves 2 and 4 wiltiout T-3extract.
be related not only to extract concentration, but also to tbe vohmie ofniediuui used. It can be seen in Table 8. that al least at the higher extractlevels, the yield was almost directly proportional to the vohime of themedium. More important, yield diflerences between extract levels were pro-nounced in the 100 ml. volumes, much fess hi the 75 ml. volumes and onlysligh! in tlie oO ml. voluine.s of uiedium. Yields of tobacco tissue was increasedfrom .'to to 00 g/flask by adding tho optimum extract level and tisiug 100instead of 50 ml. of medinni per flask. Figttre 14 reveals, fiirtheriiiore. thatIhe tobacco extract enhanced tbe rate of growth of tissne, whereas increasein medium volume essentially prolonged tbe logaritbmic growtb period. Itmay be concluded that sto])page of growtb is due to exhaustion of Ibe entiremediuui. perhaps uiaiuly the water (see figttre 1ft). Tbe greater final freshweigbt.s o! tulture'.s with extract may iu fa(t be due iu part to the earlierabsorption of most of tbe available water by the tissue aud consetjueiit lowerloss by evaporation from the medium.
Although the fresh and dry weights increased in abont the same manner,it is clear from the data in Table 8 as trom other experiments that the dryweigbt continues to iucrcasc with the concentration of extract considerablybeyond the level which is o|)tinial tor hicreasc in volume of tbe tisstie. Tbeextent to which tliis additiotial increase iti dry weight reflects biosynthesis ofnew cell materials or merely cotitinued absorption of solids from the mediumhas not been determined.
.\ttcmi)ts to rcfilacc tlw leaf extract by f;iwwn substances, O[)tinial fresbweight yields on 50 nil. volume of revised tneditim often have apprtjacbedbut rarely exceeded 25 g/flask. wbereas witb leaf extract added tuider other-wise the same conditions yields went over M) g and once reached .'15 g/flask.
Physiol. Plant.. 15,1962
MEDIUM FOR TOBACCO TISSUE CULTURES 491
Figure 15. Effect.t of tobacco leaf extract and its ash content on the growth of tohaccocallus tissue (Growtti period. Sept. 3 to Oct. 2, 1959). From left io right: Basal controt,revised medium utone, with T-3, 3.75 g/J, and with ash from ttie corresponding amount
of T-3.
Figure 16. Cultures illustrating growth and utilization of niedirt of different componilionand volume. iGrowlh period. .July 2 to .30, lM'l.i Flask 1. ha.sal mediiini 50 ml: lhi.sk '1.revised mt;diiiiii .V) m!: fla.sk 3. revised medium lt)O nd; ami Iliisk 4. revised medium with
teaf fx tnu t 1(1(1 ml.
Figure 17, Effect of concentration of gihbercllic aeid (Growth period, Oet. 29 to Nov. 19,t9.59). Numbers denole mg/l.
Ph}/siol. Plant. IS. 19H3
492 TOSHIO MURASHIGE AND FOLKE SKOOG
Tatjie S. Effects of concentration of extract and iiolumc of medium on tisxue ijirld onrevised medium (Grmvlii period. 7/2-7/30/59).
Volume ml/flask
5075
100
5075
100
T-3 Extract (sotids, g/t)
0
25.731.032.0
0.1550.900.93
0.9 1.9 3.8
Fresh weight g/flask
24.730.33<).4
24.332.351.1
30.747.958.5
Dry weight g/flask
0.670.H21.08
0.700.871.31
0.77M71.41
7,5
28.647.9;)4.2
0.861.361.55
Wilh larger volumes of mediitni the differeiice.s in yield in the presence andabsence of leaf extraci were .sHtl more marked (see Table 8). About the samecan be said for dry weight yield.s.
As a preliminary step in diagnosing tbe stimulatory effect of the extractvarious sttbstances have been tested which are known to enhance tbe growtbrate of ti.ssue culttircs.
''Braiin's siif}f>lement,'i'" {cytidylic acid 200 mg/l., guiuiylic acid 200 mg/l..L-asparagine 500 mg/I. and L-gUtlamine 500 mg/l.: private communication)in some cases wbere the controls were relatively low, gave up to nearly 50 %iticrea.se iti fresti weight hut never more than helween 25 and ^ 0 g/flask.Ftirthermore. there wore tio corresponding increases in dry weight, so thatapparently these addenda influenced mainly the rate of water uptake andcell expansion.
Gibberellic acid added to the basal medium {X) resulted in little or nogrowlli stimttlation. In contrast, with the revised meditim cold-sterilized gib-herellic acid markedly increased tlie growth rate. An exauiple of the effectof incMcasiug ctnicentrations is shown iti ligiirc 17. In this case 1 mg/I. appearsto be optitnal, hut generally perhaps bigber levels may be preferable. Asshown hy tbe data hi table 9 tbe gibberellin effect, except perhaps of veryIiigh levels, in these experimetits was entirely on fresh weiglit. It was mo.st
Tahte fl. Influence of yibherellic acid on yield of tohacco callus tissue(Growtti period.
GA mg/l
00.11.0
10100
Yield,
Fresh weight
15..'.26.827.726.325.3
g/nast(
Dry weight
0.550.530.530.560.64
Phl/siol. Plant, 15,19S2
MEDIUM FOR TOBACCO TISSUE CULTURES 493
noticeahle in the early stages of" growth. In .some cases where Ihe tissues didnot ([tiickly exhaust tlie availalile nuHlitim and were kept for 8 weeks orlonger the controls seemed to catch tip witli the treated cultures.
In pith cultures supplied wilh gibherelHc acid, kinetin and IAA singly andin comhinations it was clear that neither gihhereltic acid nor kint'tio alone.or in comhination of (he tw( ), stimulated growth. In contra.sf. i.\A aloneof course gave rise to marked tissue expansion which effect of IAA wasenhanced by gihbereilic acid. IAA together with kinetin permitted conti-Titious growth of the tissue and the rate of this calhis deveiopment wasspeeded greatly hy the further addition of gibberellic acid. Thus, on a suitablemeditnn, the three substances in combiuation exerted growth stimulatoryeffects which readily could he distinguished each one from the others.
With the addition of all the ahove mentioned orgatiic materials to therevised medium, rapid early growth rates comparahle with tho.se on meditmiwith leaf extract added have heen obtained, but so far the final yield.s,especially the dry weights, have heeu lower. In spile of this it is possible thatthe effect of the leaf extract may he accounted for in terms of its contentsof gihherellins. puriue derivatives and otber known constituents. In fact, itnow seems unlikely that its growth protnoting activity is due to an ttnktiowngrowth factor of major importance, although this pos.sihility is not entirelyexcluded by the present results.
DiscuHsion
The use of tissue cultures as a tool iti prohlems of inorganic nulrition ofplants has been much advocated btil so far for several ohvious reasfnis hasproved fruitless in practice. Tbeir use for the detectittn and .study of biologi-cally active organic substances, natttral and synthetic has heen more purpose-ftil. It is clear, however, that hiological activity of any one substance not onlyvaries with the do.sage but depends greatly on tbe milieu in wbich it is placed.Conclusions as to the presence or ftmctioti of a parlicttlar substatice on tbebases of growtli responses must be drawn witb catition. In bioassays forgrowth factors practically an all-or-none response is needed as evidence of anew active agent. Most claims for a new factor hased solely ou quantitativeincreases in yield or growth rate from the administration of ttnknown mix-tures of materials have no validity and liuiited tisefu!ne.ss. Even in cases oftmknown factors supplied in mixttu-es or in tmknown amounts, very largedifferences between treatments and controls may be re<]uired for significantresults. Tbe meditmi specified in table 6 bas beeu designed to minimize theresponse of the tohacco tissue employed to variations in its constituents andto inorganic salts, sugars, etc. commonly pre.sent it tissue extracts. A positivegrowth response, therefore, is likely to reflect the presence iti the medium ofgrowth promoting organic suhstances. This medium bas provided for excel-lent growth of either tobacco calltts or excised pith tissue under ordinaryenvironmental conditions. Replicate cultures within a given treatment of anexperiment were astonishingly alike in size and external appearance. Thereprodtictibility of results from otie experiment to tbe next also has beeti satis-
I'hysiul. I'lORt, J5, J9ti2
494 TOSHIO MURASHIGE AND FOLKE SEOOG
factory, but some variability was observed in total yields and in morphogeniopatterns in response to a given treatment. At least in part this variahility canhe ascribed to differences in etivironmetital conditions aud treatment of tbestock cultures.
The reported optimal yields were ohtaitied wilh tbe temperature of tbectiltttre room kept between 26 and 2ii°C.. hut the temperature was not care-ftilly controlled nor was ils influence systematically studied.
'Hie cttltures were kept on tiers of ca 1 meter wide shelves open only otione side, so that tbey were exposed to coutinuotts weak and somewbatvariable difftise light from fluorescent tttht's and Mazda lamps tnotmted onthe ceiling. It bas been shown earlier that growth atid organ foruiatioti inre.s])oii.se to l\.\ and kiuetin treatments are olitained in darkness as well as inthe light, hut light is not without uiodifying effects. In general weak diffuseartificial light is employed. With iticrease in its intensity tbe tissne becomestuore compact, the fre.sh weigbt tends to decrease aud eveiittially also tbedry weigbt is lowertMi. More uniform responses migbt be ohtained hy closeregulation of tbe light intensity. It is likely also that tuore rapid and nniformgrowtb might be obtained tbrough stiitahle choices of light quality andespecially of day length.
^'ariahility in resnlts also derives from disuuiforin planting material. Stockcultures of difterent age or grown on media wilh dilferent levels of I.\A orkinetin. for exatuple, give sub-cnltnres which differ hotb in growtb rate andyield. .\ slandard pretreatuieut of stock cultures is needed Imt tlifficult tospecifiy iu advance, as it will depend on the type of growth desired and willvary witb conditions and changes in the stock itself. Requirements of in-organic nutrirnts probably remain relatively fixed, but tbe need for orgatiicfactors may fitictnate widely. This is strikingly illustrated by the sporadicappearance in control culttn-es of tissue pieces wbicb bave tbe capacity togrow in the ab.sence of IAA or kinetin and occassionally witbout cither ofthese in the medium. Tbat increased rates of bio.synthesis is responsible iorthis hebavior is well known iti Ibe case of auxin from the work of (iatitheret(1959) and coilaboratoi's: and K. J. Fox in our laboratory recently bas .showna relatively bigh kinin contetit in one tissue wbich grew withotit kinetin.Acqtiired capacity for increa.sed rates of syntbesis of hoth auxin and kinin isof course also known from Braun's studies of crowti-gall in tohacco.
A significant case of acquired, graded cajiacity of tissue development is thereport by (Ihouard auti Agbion (19fit) thai stem segments from the aj)icalregion of a flowering tobacco plant produced buds with flowers in rifrowhereas segments ftdtn the hasal part of tbe stem prctduced only vegetativehtids. Acqinred differences in accutnnlative or hiosyjithetic capacities musthe responsible for this difference in behavior. It is especially noteworthy thatin this case tbe change in Tuorpbogenetic capacity is associated with normalontogeny. It is not bnutgbt about by an exogetiotis agent in tbe same sense asis tlic case in ttmior indttction: nor is it an act that at present would findrespectahle status in the category of so called "biochemical mutations".
In the ligbt of Ihe ahove considerations it is to he expected that tissues evenfrotn the same j)lant may have difterent quantitative ntttrient reqtiirenientsfor optimal growtli i;i vitro, and th;it tbese tuay cbange with time attd condi-tions. In fact it is surprising tbat cultures from all parts of Ihe plant kingdomPhysiol. Plant., 15,1902
MEDIUM FOR TOBACCO TISSUE CULTURES 495
apparently have rather similar requirements, and that often their growth islimited by otie or a few of a small group of common growth factor.s, as forexample by auxin or vitamin Bj.
Tbe positive response of tbe tobacco tissue to gibberellic acid in the pre-.sence of the other growtb factors on the revised but not on tbe hasal mediumis of special interest in showing the futility of attempts to classify tissues ofvarion.s luigin in terms of tbeir requirements for a y>articutar growtli factorwben ctiltured on a medium of uiiknowti general adequacy for tbeir growtb.
During the five years this work bas been in progress tbe medium in dif-ferent stages of its revision has heen tised tor cultivation of various .speciesand strains in our and other laboratories. Tbe results have varied from verygood growth to no growth at all. In no case have yields approached thosefor the ustial tobacco tissue. It seems that tbe mediuui must be modified toaccommodate the needs of any given ti.ssue. Furthermore, several passagesmay be required for the li.s.sue to adapt to high nutrient levels.
The organic addenda in this connection may serve not merely to providespecific growtb factors htit also in part to hufter against excessive electrolyteactivities. Complex formation by amino acids is mentioned as an example oftbis wbicb might be important especially in early .stages. In tbe course of itsgrowth tlie tissue itselt' tuidoubtedly sytithesizes and releases metaboliteswhich not only react with specific constituents but may be said to modifytbe general pliysical and physiological properties of tbe medituu.
The mecbanisuis whicb enable tbe tissue to derive nottrishment tor per-.sistent growth, representing doubling in weigbt on the average every 2 days,for a 3 to 4 weeks period, are unknown and ditlicult to imagine. The com-pleteness of the incorporation of water from the revised meditmi into tissue,especially in the presence of leaf extract, would appear to be a "new" andremarkable phenomenon. In cases of the higbest yields, nearly 35 g. fresbweiglit on 50 mi. and 70 g. on 100 ml. of medium, the dry weights wereca. i).\) and 1.5 g. respectively. Therefore, about 70 Vu of the water furnishedorigitially was incorporated into living material and most of the remaining30 "/o was lo.st by evaporation and transpiration duriug the four week growthperiod. As compared with Ibis, higb yielding dense suspensions of algae havebeen recorded to utihze about 10 tO 15 Vo of tbe water available in tbenutrient .solution for tbe production of cells. It may be seen in fignre HI tbatvery little medium remained in the 50 ml. revised nntrient solution and prac-tically none in the 100 ml. revised medium witb added leaf extract. Tbetissues must make intimate contact with the agar surface. In fact, breaksoccur in the agar itself, and flakes adhere lo the tissue as it "burrows" throughtbe medium. Oxygen availability would appear to be no problem in tbesevigorously growiug cultures. During tbe later stages of growtli the osmoticforces required for transfer of water must he huge, i.e. of liie same order asdevelop in root systems.
Summary
A several fold increa.se in yield of e.xci.sed pith or callus cultures of Nico-tiana tabacum, variety Wiscotisin 38 was ohtaiued by addition of leaf extractto the standard modified White's nutrient medium.
Physinl. Plant. 15,1'.m
496 TOSHIO MURASHIGE AND FOLKE SEOOG
In part the iiicreuse was due lo in()rj,'nnif conslituents ui' the extract,especially N and K, which could be sub.slitiited lor by eitber the ash of theextract or raised levels of N and K salts in the niedium.
In part the increase was due to or^'anic constituents of the extract wbiehincreased both the growth rate :iud linal Tresh weij ht of the cultures.
Similai' increases in growtb rate but not in I'inai yield were oblained byaddinj* {j[ibberellic acid and Braun's supplements of purines and amino acidsto a revised medium.
A revised medium bas been developed (Table 6) witb eaeb element pro-vided in sufficient quantity to insure that no increase in yield will result fromthe introduction of additional amounts in the ranf,'e ordinarily to l>e expectedin plant tissue extracts, etc.
Tbe organic constituents bave been retained luichaiif^ed except tbat sucrosehas been raised to 3 per cent, nij/o-inosilol has been added as a regular C(ni-stituent and Edamin has been introduced as an optional ct)nstituent.
The revised mediuui is designed for use in bioassays of or^'anic f jrowlhfactors. It provides for rapid growth rate, increased response lo orj-anicgrowtb factors and minimal interference from inorganic and eommon organicnutrients.
In tbe presence of plant extracts fresb weigbl yields of up to 35 g. on 50 ml.of medimn bave been obtained. Under these conditions water appears to betbe limiting factor Tor growlh of the tobacco tissues employed.
Aspects of general application, limitations and behavior of plant lissue cul-tures are discussed.
This work was supported in part by the Research Committee of the GraduateSchool. University of Wisconsin with Tniids from tlir Wis. Alumni Rcseart-li Foiinchi-tion and in j»art by f,'rant G-r)9Hl) fi'oni the Nalionul Science Foiindntion. EdLMiiin waskiiitily riirinshe(t hy tbe Stieftield Ctiemical Co., Norwieh, N.V. Tho aultiors ai-o grale-lul to Miss Joyce Klenim tor U-chnicat ii.ssi.stance and especiiiUy to Dr. Elt'riedeLinsniaier who did the confirimitory experiments on P, Fe, and sucrose levels andon different combinations of IAA and kinetin.
Present address of T.M.: Department of Horticulture, Agr. Kxpt. Station, Universityof Hawaii, Honolulu, Hawaii.
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MEDIUM FOR TOBACCO TISSUE CULTURES 497
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