murashige skoog

7/30/2019 murashige skoog

1/26

PHYSIOLOGIA PLANTARUM, VOL. 15. 1962

A Revised M edium for Rapid G rowth and BioAgsays with Tohaoco Tissue CulturesBy

TosHio MuRAsniGE and FOLKE SKOOGDepnrtmeiil of Botany, University of Wisconsin, Madison, 6, Wisconsin(Received for puhtiealtou .\|irit 1. 1962)

I n t r o d u c t i o nIn experiments with tobacco tissue cultured on White's modified medium1 and 2) supplemenk'd with kiticthi and hidoleacctic

(Fi^'ures 1 an d 2 ). Subse(iueutly it was found Ihiit this jnonio tiou oi'In the isolation of Rrowth factors from plant tissues and other sources

As a point of referetice for this work some of the culture media in mc)steral com positions a re listed in Tables 1 and 2. W hite's nutrient .solution,


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474 TOSHIO MURASHIGE AND FOLKE SKOOG12w0.70.50.2

20 25' ' 30g.Figure 2.

Figure 1.Figure 1. F.ffevt of tobacco lenf extract on the growth of tohacco fnth tissue \'\'-'2. !0uf i iu'd iiim : Exit. stnrtcct Mjir. 7. M158). Cu rve I witii , curve 11 wittiout teat" extadded to the standard hasat nicdiiiiii.Figure 2. Effect of concentration of leaf extract on the yield of tobacco callus tissus (Growiti period , Fe b. 13 to Mar. 6,19 58) . Curve I, fresh we ight: curv e II, dry wei

cul t ivat ion of var ious t i ssues f rotn numerous .species (See Gautheret ' s 1co m pil a t io n) . G au the ret ' s medi tn n (1939j w as devised by comb inl it^ ' a twdi lu ted Kno p ' s n iacro nu t r i en t .solut ion w i th a s l igh t ly mo di f ied Bc r the lm icro nu tr ieu t solut ion. H i ldc bra nd t . Rik er . an d Dufif^ar (1940) em ploy ed tr ianfi ;ulat ion terhnique to i tnprove on Wiii tc 's incdit t tn i 'or the ci i l l ivatiouspecific t i ssues . Th ey rep or te d two new tor t uu lae l incl tided i ti Ta hle s 1 ;uidwhich they desiytuUed as opt imal for cul tures ol the ahove hybr id tobaca lh t s and for sunf lower c rown f all caMus respec t ive ly . These media , accoiu}i to B ur kh old er a nd Nickel l (1949) . w ere s t il l un su i tab le for c i t l livat ionvir t i s - indt iced tumor t i ssues . By employi t i j ; the t r iangulat ion technique ini t ia l t r ia ls and then by tes t ing .ser ia l concentrat ions of each clement inpct ident ly ni the others , these i j tves t igalors devised s t i l l another formliased on the medintn for sut i f l i iwer t i ssue by Hi ldebrandt ct al.. anr l wbtbey repo r ted as m ore s tntable for Rt tmex t issue. Hel ler (19531 ba s tude ta i l ed s tud ies of tbe minera l requi rements in cu l tures of ca r ro t and Vi rg lcre ep er. He first in du ce d defi< iencies of several of the eleuie nls b y rep eatra ns fer s of tbe cu lt t tres to l iquid m ed ia lackinj^ Ihe elem ent in qtiest ion athe n rcintr(Kh i(( 'd the cle m en t in serial co nc cti tra t io ns to .select sat is fac tlevels . For the most par t tbe level of each element was var ied only in cobinat ions in wbich the respect ive levels of a l l other nutr ients were kunchnnscd . In compara t ive t es t s . He l le r found h i s medium to H 've twothree t imes grea te r y ie ld . s than Whi te ' s o r ( i a i i there fs media which wem ploy ed as co ntro ls . T he nu tr ic t it . sol tt tiou devised m ore recet it ly hy Ni t


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MEDIUM FOR TOBACCO TISSUE CULTURES 4 7 5

Mater ia ls and MethodsSource and preparation of tissues. Tobacco tissues, Nicotiana tabacum, var. Wis-have been used exclusively in Ihis study. Fresh pith w as used for the bulktbe work, and continuou sly sutjcullured callus was used for Ihe final con firma tionm. tall fobaccn jjlynts fjrowii in the greenhouse. Ihes were removed , the stems were swabbed witii 95 Vo etban ol and cut inio 5 tocm. long cylinders. Only a 15 cm. region of Ibe stem starting ca. 10 cm . from the

'2 cork Imrer. The cylinders were extruded from the

45 weeks old

n raised to 0.38 mg/1. and inositol had been o m itted. Tbe vigorous m ore com pactexcellent growth and satis facto ry rep rod uc ihility of data (within 10 "y'o) from

Composition of tin- Hasal Medium. Tbe basal medinni whicb has served as control

emicLil Co., Norw icb . N.Y.I. It had the followinji com position , in mg/1. of m ed ium :) Inorg an ic s alt s: NH4NO3, 40 0; KCI, 65 ; KNO3. 80 ; KH2PO4, 12.5; Ca(NO3)2 iH.O,144; MgS0|-7H,,O. 72; NaFe-EDTA, 25; H3BO3, 1.6; MnS04 4H2O, 6.5;"'znS64 7II3O, 2.7: and KI. 0.75.

or 0.04 (for callus); tbiamin IiCI. 0.1: nicotinic acid, 0.5: pyridoxinc MCI, 0.5;


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476 TOSHIO MURASHIGE AND FOLKE SKOOG(c) pH adjustment: The pll ol' :dl media was adjusted lo 5.7-5.8 wilh a tew dropsT 1 N NaOH or 1 N HCl before the agar was added.

Fe was added as agricultural grade sodium-ferric-ethylenediaminetetraacetobtained irom Geif^y Aj^riculUiial Chemicals, New York. N.Y. or Na^Fe KDTA ppared from Nao-EDTA. MW liTl.'M) {Tredcrick Smith tMioni. Corp. Columiius. Ohdissolved 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 .stbe concentrations of tbe otber elements were varied simply by adding them us eithnitrate or ammonium salts and tjy adjusting the amount of NMjNO-j accordinglyattain the specified total inorganic nitrogen level. Tbere was no evident effectgrowtli from [lossible cbanges in pll or in otber conditions arising Irom tliis prcedure.In Tables 1 and 2 the inorganic constituents of the basal medium are listedmilIiinoles/1. {mM) or micromoles/1. (uA/) of eacb element. To facilitate compariso

tbe concentration of each element is also expressed in nig/1 (values in parenthesiThese concentrations will be referred to as tbe 1 x levels, and in many experimemodifications will lc 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 stare both from Nicoliano tabacnm variety Wisconsin 38. T-2 is a water extract fro62.4 kg. of greenhouse grown plants and was used Jiere only in tbe experinierepresented by Figures 1 and 2. T-3 is an extract from field grown plants preparas follow: Healthy green leaves picked from vigorous, nearly mature, ca 1 m. tplants were |)acked into plastic bags, sealed, trucked to a cold storage plant, (piickfrozen and ke|)t indefinitely. The frozen leaves were then groinid in a commei-ctype meat grinder and tluiwed. The juice was expressed witb a liand press, heato near 100 C, cbilled to 5-10C, centrifugcd and concentrated under reduced prsure. The concentrate was stored in a frozen state in 1 1 polyethylene boltles a

I'ahlc t. Macronutrirnt (

White (1943|>Hildchranrtt et o/. (To-Hitdehrandt et a/.' {Sun-Burkhotder & Nickel l' . .Hfltor >Nitsch & Nitsch '

Medium

owposition of lome plant ti.fsue eultiire media.Element (Concentrations itN3.2

8.48.07.1t9.812.05(169)60.0(8401

K1.72.21.73.312.010.039.91.76(68.81

20.0(7821

Ca1.22.11.73.46.0I).510.230.61(24.4)3.0(121)

MR3.00.50.72.92.01.01.80.29(7.10)1.5(36 .8)

1 milt imote.s per l i t e rS4.40.56.43.61.01.01.00.32(10.6)1.6(52 .3)

P0.140.90.241.08.00.91.8{t.O92(2.85)1.25(39.0)

CI0.9

0.91.810.011.00.50.87(30 .9)6.0(212)

if iiieiliuinNa3 0

11.71.78.01,80.1

(2.3)02(4.61

Ve0.00.1-0.10.00.00.00.0f2.90.1l'5.5


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MEDIUM FOR TOBACCO TISSUE CULTURESTable 2, 'Micronutrient comp o.sitlon.s of plant tissue culture media.

All

Medium tilement (Concentrations in tnicromotes per liter of niettium)Cun Zn I .Mo Co Ni Te Be Al

et al.(Tobacco)iIdet>ran


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478 TOS HI O MURAS UI GE AND FOLKE SKOOGTable 3. Effect of increasing the concentration of the nutrients in the basal meiUuw on growth of tobiHio callus tisifue. (Growth iKTiiiil, 11/15-12/18/5S.)

Clianse in totnposition ofm e dill inN o n e ( B a s a l m e d i u m 1 x)All c o n s t i t i i t e n t s 2 x4 XAll o r g a n i c s u h s t a n c e s . . . . 2 x. . . . 4XAll in o r g a n ic s a l t s 2 x4 X

AveniKe final frt-sliweight g ./f las li .5.69.914.96.3(5.49.717.0

Tahle 4. Effect of quadrupling the concentration of the inorganic elements of the bamedium all together find each separatehi on the growth of tobacco callus tissue. (Gropcrio


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MEDIUM FOR TOBACCO TISSUE CULTURES , 4 7 9increased fo 2 x or 4 x, i.e. under Ihe more favorable conditions for

0 mM respeclively.X. The N reqiiiren ient v -ns exa mined in more de tail by lestinj,' the g row thmM in combinations with 14.4 or '28.8 mM K and

mM P. All other inorganic constituents were kept at their 3xThe average final fresh weights of tissue obtained in t)ne experimentinst the N level in Figu re 3. It may he noted that unde r Ibe

h K and low N media. In experim ents with o ther lots of p lanls, P levelsmM were sometimes favorable, but generally P levels of 1.5 mM orThe data show that 50 mM is close lo optimal for N irrespective of the KP concentralions, and tbat tbe N content m ay be increased som ewbat

mM or higber consistently were found to depress tbe yield. Hence, avel of ()0 m;U \ bas been selected as sati sfactory.A'. The K requirem ent was determ ined witb N kepi at the 4 x level (48 mil/);at tbree levels. 8 x. 16 x an d 32 x (0.74, 1.5 and 3.0 mM respectively),ds presenled in curves 1 and 2 increased w ilb Ihe conce ntration of K upmM and fell only sligbtly at 30 mM, tbe highest tested K level. In curve

P. The P requirem eni was determined with N at (he 4x level (48 mM)itb K at tbree levels (7.2 14.4 and 28.8 mM] and with all other inorganicbut with an am ple supply of K il lies in tbe region betw een 0.7 and

mM and as .shown

mM. Other elements were as specified for tbe revised mediumand 2) except that Fe was used at both tbe 0.10 and 0.25 mM levels.tm en ts sucro.se was used al eitber the 2 Vo or 4 Vo level as well as at thepecified 3 Vo level. Data from one experimenf with pi tb , typical of the r esults


8/26

480 TOSHIO MURASHIGE AND FOLKE SKOOG20

10

FIGUR\_

/a'"

- -a1

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 3020

15

10

FiGURf 5

1/ ^7 11

/'\4

""3

f

\2\

P

FIGU

amil

?E 6

r

3/1'2

\ s \ \ \s \ \

CaJ 12 5 30 5 6mM

Figures 3 lo 10 inthisive. Fresh w eight i/ields of e.vcised tohacco pith ealtures in re.f!>to increased concentrations of nutrient elements.Fisiire 3. Effect of ,Y. (Gruwlh pe rio d. M ay 5 tu J un e !*(). 195i)|. All elemenl s a l 3 x levexci'|)l K iind F'.Curve I: K 14.4. P (1.74 m.W. Curvo '2 : K 12S.M. l 0.74 iii .y. C urve 3: K H 4 ln\M. Cu rve 4: K 18.S. P l.r> mM. BC. basal ciinlrnl.4. Effecl of K. (Growlh pfr iod . Miiy 19 lo J un e 16, 1959). N kepi al 4 x (48.8 maufi :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. liaconlrol.5. Effect of P. (Growlh period, C urves I ^ , May 20 to Jun e 17. 1959; Curve1960; Curve .'j. Ju ly lit to .Sept. 1. 1962).N kepi al 4 X and all ulhe r elem ents a t 3 x levels except K. Curves 1, 2. 3 andthe level of K was 7.2, U.4, 28.8, and 20.0 m.V respeclively. Curve 5. see teB(;, hasal control.Figure 0. Effect of Ca. (Growth period. July 9 lo Aug. 10. 1959) N kept at 55 mM, K20 ni:l/. P al 1.0 m.V, and niic ron utri en ts B, Fe, Mn an d Zn at 3 x levels. Mg,and Cl varied . Eaeh kept at ils 1 x, 2 x. and o x levels in Curves 1. 2 and


9/26

MEDIUM FOR TOBACCO TISSUE CULTURES 4H1

20

15

10

\ nGURt\ \ >

-B.C.

Mll

.7- - .?

ss2

y ''\

Mg

FIGUR

o"'

- .ac

E 9

1

i'

2* ;

t

1 .

a

15

TO

I 1.5 2 0 2 dmMFIGURE0

A/

"'ac.

\ 3

s16 2

FIGU

y1. i k.nir?

RE 10

C.1

* - -

2'--?

-

Fe0 0.1 0.2 0.3 0.4 0.5mM

7. /;// et ( o/ My (Grow lh p prio d. .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. E ach kept at t x. 2 x and ox levt'Is in Curves I. 2 arut 3 respec-tivejy. BC, basal control.Effect of S (Growth pe rio d. .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;. an d CI variL'd. Ea


10/26

4 8 2 TOSHIO MURASIIIGE AND FOLKE SKOOG1.0 is on the low side, 1.5 on the hif,'h .side, and 2 or more mM definitely high. For this reason, a 1.25 mM P level has heen selected.

Hfquirements for Ca, M(j, S, and CAWheti IIK- ri'qttirements for Ca, Mg, S., and CI first w ere eva lua ted , theK, and P levels of the media were kept at 55, 20 and 1.0 m.l/ respecliv(".(., close lo the values selected ahove as satisfactory for each of these em en ts. Kach of the iou r e lem ents was tested at four levels (1 x, 2 x, 5 x, 10 x} and in conjunclion wilh three levels (1 x, 2 x, an d 5 x) of th e other telements. The micronulrients B, Fe. Mn and Zn were provided at their levels.Co. By comparison of the cnrves in Figure 6 the reciuirenient for Ca woappear to be relatively higher with the 2x than wilh Ihe other levels of M

S and CI. With these eletnents at the 5x level the Ca concentration cnforms a hroad platean hetween 1.5 and (i n\M. A 3.0 \\\M Ca level has hselected.Mg. Results from one of the experiments with Mg are shown in FigureBoth with the 1 x and 5x levels of Ca. S and CI Icurves 1 and 3) tissue yiincrea.sed as the Mg concentration was increased from 0.3 to 1.5 mM . T3.0 mM level of Mg was witho ut further stim ulating effect when the leof Ca. S and CJ were l x and it was p erh ap s in hibito ry when Ihe levelsIhfsi' eltMiifuts were 5 x. W ith 2x levels of Ca. S. aud CI, the fiual frwcighls (if the pith explants fell as the Mg concentration was raised inmi

to 1.5 m.l/ but was up again when the .Mg concentration was.'i.O m.V. Althoin this case the lowest concentration of Mg resulted in the highest yieldshould be noted that an imbalance existed as tbe cultures in this particutreatment 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 audthe lowest concentration of S (0.3 m.l/) was optim al and higher S concentions caused a steady reduction in tissue yields. However, with tbe 2x orlevels of Ca, Mg aud CI. the 0.3 mM S level was less than acleipiate, ansubstantial increase iu yield was obtained at tbe O.dfi m;V level. S cniicentions higber tban O.(jf) m.U were iiibibitory lo tbe pith explants. In subsequtests witb callus, on tbe otber band, no toxicity was obtained even wilconcentrations as high as 13 mM. On the basis of all results and even thoit may he slightly higber tbau o|)tiuial tor excised pilb cultures, the 1.6 mlevel of S bas been selected.CA. Witb Ca, Mg aud S at tbeir respective 1 x levels, increases in CIcentratiou had no appreciable effect on growtb of pith explants (Figurewbereas with Ca, Mg and S at tbe ir 2 x or 5 x levels, increases iu tbe CI lup to 4.4 mM were stim ula tory . A big he r level of CI (8.7 mM) gave neia further iucrease nor iubibitiou of growtb iu the presence of 2x levels

Ca. Mg and S. hut it was far less stimulatory tbau the lower CI levels in presence of 5 x levels of Ca, Mg and S. Witb callus instead of pitb, CI c


11/26

MEDIUM FOR TOBACCO TISSUE CULTURES 4 8 3Microniitrient requirements

Preliminary studies indicated that 3x to 5x levels of B, Fe, Mn and Zn

Fe. Availahilily of Fe has long been recognized as a critical requirement

1 X. 2 X. and 5x levels of H, Mn and Zn. As .sbowu iu tbe curvesx and 5 x levels of the

mMx levels. A highe r pur ity pre pa ratio n of Na-2-Fe-EDTA was

mM level. Even 0.05 uiiW FemM Fe generally gave cousiderahly lower

mM aremM of

B, Mn, and Zn. (Considering the likely preseuce of impurities, the B, Mn

mM Fe level. There was nomM Mn, and 0.030 m.l/ Zn bave

Other Etements {Cu, Mo, Co, I, and N a). Cu added in tbe range frommM was without effect on the growtb of the tobacco ti.ssue,mM levels, i.e., iu the range used by Heller (1953)et al. (1946), similarly were without effect on yields.


12/26

484 TOSHIO MURASHIGE AND FOLKE SKOOGTable 5 Effect of incrcasefl levels of B, Mn and Z n on the growth of tobacco catlux tis.-(Growlh period 9/15-I0/12/.W.)

Change in comi)osition ofmedium

No ne (Basal meciiuni 1 x)B 5 xB 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 toes.sential elemeuts for planl growtb. Similarly Co is included tor reasons its requirement hi lower plants (Holm-IIausen ct al. 1954) aud tbe possibrole of cobaltous ion in morphogenesis of higber planls (Miller 1954, Salbu ry 1959). A test of CoCU iu eight serial couceiitrafions between 0.000! aO.iri m.l/ and all otber elemeuts at the lx levels was iucluded iu an eaexperim ent. No stim ula tory effect of Co was obtaiu ed at any level but ratha toxic action at tbe two higbest levels. 0.08 aud O.Ki. The selected levels aCu 0.0001. Mo O.OOI aud Co O.OOOt m.U. lod iue arb itra ri ly is reta ined 0.005 mM. Na, o.steusively uonessential but perbaps uot entirely devoid of often claimed slimulatiug effect on plaut growtb. heing a par! of tbe FeSONa^ 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 1 ,V HCl or with NaOH or KOH eitber before or after tbe a gar was addaud had been heated a tew minutes iu tbe autoclave. Tbe value was selectou the experience tbat tbe reaction of eitber more acid or more basic medtends to dritt toward tbis region duriu g tbe beat trea tment for sterilizatiand subsequently witb time even iu uniuoculated fla.sks. Prebeating witb agar pre.sent avoids any appreciable cbauge iu pH during tbe subsequautoclavatiou. Tbe value 5.7 fo 5.8 is suitable for maiulaiuing all the salt soluble forui eveu wilb relatively bigh phosphate levels aud low euough perm it rap id grow tb aud d itfereiitiatiou of the tissue. Th e am on uts of C I. or K iutroduced iu the process are uot inclnded in the stated levels, 6.0, 0or 20 mil/ respectively of tbese ions.

Composition and (jrowth effects of the revised medium. The kinds aamounts of iniueral salts finally adopted for the revised medium are listed Table OA. Quantities bave beeu adjusted upward, beyoud actual needs some cases to provide total concentrations of all elements in round numbas shown iu Tables 1 and 2.Tbe yield of tabacco tis.sue ou tbe revised medium has heen compared wtbose on the original basal m ediuui (1 x) and o tber m edia as sbow u in Tahland iu Figure 11. Not only were the higbest yields obtahied consisten tly


13/26

MEDIUM FOR TOBACCO TISSUE CULTURESComposition of the revised medium. pH adjusted to 5.7-.J.8NaOH (see texlj.

A. Miiu'riil siiUs

willi

4DI. KOli, or

Major elementsSalts mg/l. , m.U

H R O

K I . .CuSOCoCU

Minor ck-tiiSalts

>4- IH2O* ' " ' ' ^OOJ - 2H9O

(iiuo

lUH 1.6.222.38.6O.H'.i0.250.U2:)(I.O'i.-.

|xJf100100305.01.00.10.1

NHjNO, 1650KNO3 1900CaCl2-2H2O 440MgSOj 7H0 370KHaPO^ ; 170N a - E D T . \ 37.327.81

N

NaKe

11.218.83.01.51.250.20O.IO* 5 ml/I oi ' a s to ck sol ut ion contii ininf{ 5.57 g F eS 04 7H2O and 7 .45 g Na j-ED TA per

B . Orf ia i i ic co i is t i luen ts30 g/I.l id a m in ( o p t io n a l ) 1 g /I .GK xin t ' 2 .0 m g/ l .Indo lf iic t ' l i c aci d * 1-30 mg /! .K in e t in = 0.04-10 mg/l .

* See text a nd f iRures 12 :ind 13.

Agar 10 R/ I .nij/o-1 no si to I 100 m g/ l.Nic o t in ic ac id 0 .5 mg / l .Py r id ox in HCl 0 .5 mg/ l ,Ti iin ni in - H( .i O.I nig/l ,

Ta b le 7 . (Comparison of yields nf tissue on different merlinA. Pi th Ti ssu e ( ( ir ow th per i od 10/2-ll /3/ . ')* .H.

M e d i u m

Re v is e dBa s a lHeUermtschHitdebrandt ef ul.

Fresh weight in g/flaskWithout Kdamin

17.34.15.01.95.3

With Ktianiin24.57.117.9y.79.2

Dry w eig ht ID g/FlaskWithout Kdamin

0.470.130.10.100.20

With I'Mamio0.490.260.430.340.37

B. Callus Tissue (Growth period 8/3l-10/I0/.)91.Medium

RevisedBasalHeller

Fresh weight in g/llaskWithout Kdamin

21.55.13.6

With Eciamin22.710.816.2

Dry weight in g.llaskWithouth Kdamin

0.480.22 -0.24

With Kdamin0.460.350.38


14/26

486 TOSHIO MUR.\SH1GE AND FOLKE SKOOG

fj 11. Comparison of yields of tobacco pith tis.Huc ciitliired on various measnl iiu'dumi 1 >. T. rcvistd: H, Heller |19r)3t, N, Nitsch and N{iy.=>6|. nod R, HildchniiuU ct at. 11946).

Organic ConstituentsThe required levels of organic coiLstitutents in the basal medium huve b

le.med only to ;i limited extent.l'Yom Ihe resnlt.s in Table 3. no larf^e increase in yield wonld be exi)ecfrom increased concentralions of the specified organic ingredienls. althouwilh improved mineral supplies some beneficial effect might be oblainedmodifications in these. The list of oilier polentially stinuilatory organic sstances and favorsible combinations of these is of course inexhaustible.Vitamins. The levels of the following vitamins have been rel:iine1. I9(>'2) nn/o-inosilol was tesled and lonnd to mote growlh in cuhiires when


15/26

MEDIUM FOR TOBACCO TISSUE CULTURESKINETtN

4870.04 0.64 2.56 to . 2 0.04 0.64 2.56 10.2

A2

V ^QS^^I^V \S' QP

^ ^ J i k


16/26

4 8 8 TOSHIO MURASHIGE AND FOLKE SKOOGCarhohydrfites. Allhougb tobacco cultures are capahle of utilizing variocarbohydrates and organic acids to some extent as energy sources, sncrois as favorable as any know n m aterial of this type. Concctitrations of 2,an d 4 Vo ar e almost equally .snitable for cu ltu res with low yield.s. How evin several large series of com parative tests 3 Vo was definitely better th

2 Vo, and 4 Vo was often som ew bat less effective tha n 3 "/o in cultu res wmoderate to high yields. For example in one experiment fresh weigbt yielof cnltu res with 2, 3 and 4 Vo sucrose were 14.3, 15.9 and 12.9 g/flask respetively and tbe correspouding dry weights were 0.44, 0.07, and O.fJ2 g/flaDry weights often were somewhat higher per fresb weigbt in cnltures wi4 Vo sucrose tban in the others, bnt on the av erage also the total dry weigper flask was less in Ihese tban in ctiltures w ith 3 Vo suc rose. The level sttcrose in the revised niedititn. therefore, has been set at 3 "/o.Agar ordin arily is kept at 1 Vo to give a suitab ly moist btit rigid nied in

The concentration may need to be varied to some extetit depending on tpreparation and the salt content, pll. etc. of the mediutu. In the case weakly growing tissue cultures agar content may be critical for survivni. bfor vigorously growiug tobacco cultttres it was unimportant. In liifnid ctures, on tbe other band, changes in composition of tlie mcctinm definiteare reqnired for vigorotis bud development to occur (Loewenberg et al. upttl)lished.)Indoteaeetic acid and kiuetin. The opthnal level of IAA or kiiietin wdepend on Ihe type of growth that is desired. It will vary witb the endogenocontents of atixins and kinins and other factors influencing the sensitivity

a partictilar strain or doue of tissue fo the.se substances.Th e curves in figure 13 repre.senting tr esh w eight yields in two exp fiinicwith difterent lAA-kinetin combinations clearly .show that the optimre(itiirenient for either one increased witb the coticentration of the otber. Teffect of a given comhination, e.specially in the intermediate concentratiratige, varied from one experiment to the next as illustrated by tbe twexamples iti figure 13. Note that the response to 3 mg/l. IAA is much greain one experitnent than the response to 4 mg/l, in the otber. Keniarkadevelopmental patterns, differences in form and texture of the callus, exteof organ formation, nnmber and .size of organs, vigor and longevity of cultures are associated witb specific levels aud ratios of IAA and kinetin. Aiiupressir)n of the ran ge in size and form of tbe cu ltu res may be gained frofigure 12. Details of tlie strict quautitative aspects and other characteristiof the growth responses which have emerged as functions of the lAA-kineconcentrations will be presented elsewbere. In general the following appliFor continnotis growth of firm, bealthy tobacco callus in sub cultur0.2 mg/l. of kinetin and 2.0 mg/l. of IAA have been used witb excellent resnfor several years in onr laboratory. However, 0.04 mg/l. of kinetin and mg/l. of I.\.\ has given nuich faster growing, loosely packed ma.sses of cewhich could be transferred and maintained as callus even though vigororoot form ation ordin arily oc curred in these cultures after 2 to 3 weeks. Flong lasting cultures (2 to B montbs) higher kinetin levels were required,


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MEDIUM FOR TOBACCO TISSUE CULTURES25

489

Effects of increasing concen-of IAA and kinetin on freshL'iyht yield of tobacco cn/iiis cultured(Growth period, 4Miii. lo OtI. 1960). Ordinate:

vm h case, tiesiilts are from0.04 0.06 0.16 0.32 0.64 J.28 2.56 5.12 10.24KINETIN mg/l

w, between 1 and 5 mg /l.. depend ing on the kinetinNaplilluileneacctic and 2,4-dichloropbenoxyacetic acids bave been tested as

Re-test of the tobacco leaf extract. In accordance witb the objective to

moted grow th (Figure 15J, but in con trast w ith the earlier tests onon the revised medium a lone wore 13.6 and 0.43 g/flask respectively;ith 3.75 g/1. T-3 extract they rose to 21.7 and 0.58 g/flask respeclively;hereas the ash of this extract gave only 14.6 an d 0.46 g/flask respectively.

The promotion of growth by tbe T-3 extract in this case, therefore, can be


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490 TOSHIO MURASHIGE AND FOLKE SKOOG

a:^6 0

A

-

--- j

n r//26S

/j l ?

V

r1* -O1

2

T

10 20 30 40 0 JO 20 30 40DAYS

H . Efffct.t of theliinic of revised medium fnf the addition of tohaleaf extract on fresh weijield..\. Toba cco pitb sections (pted Oct. 1. 1959I.ti . Tobacco caltusfsulK-ultuSept. 2U. Iil59l!Cmvfs 1 amt 2. It)() mmedium in 250 ml Krnieyer flasks.Curves 3 and -I. 50 ml ofdium in \'2ii mi fta.sks.Curves I and 3 witti T-.'itract. 3.75 g .solids/t ofdium.Curves 2 and 4 wiltiout Textract.

be related not only to extract concentration, but also to tbe vohmie niediuui used. It can be seen in Table 8. that al least at the higher extrlevels, the yield was almost directly proportional to the vohime of medium. More important, yield diflerences between extract levels were pnounced in the 100 ml. volumes, much fess hi the 75 ml. volumes and osligh! in tlie oO ml. voluine.s of uiedium. Yields of tobacco tissue was increasfrom .'to to 00 g/flask by adding tho optimum extract level and tisiug 1instead of 50 ml. of medinni per flask. Figttre 14 reveals, fiirtheriiiore. tIhe tobacco extract enhanced tbe rate of growth of tissne, whereas increin medium volume essentially prolonged tbe logaritbmic growtb period.may be concluded that sto])page of growtb is due to exhaustion of Ibe entmediuui. perhaps uiaiuly the water (see figttre 1ft). Tbe greater final frweigbt.s o! tulture'.s with extract may iu fa(t be due iu part to the earlabsorption of most of tbe available water by the tissue aud consetjueiit lowloss by evaporation from the medium.

Although the fresh and dry weights increased in abont the same mannit is clear from the data in Table 8 as trom other experiments that the dweigbt continues to iucrcasc with the concentration of extract considerabeyond the level which is o|)tinial tor hicreasc in volume of tbe tisstie. Textent to which tliis additiotial increase iti dry weight reflects biosynthesis new cell materials or merely cotitinued absorption of solids from the medihas not been determined..\ttcmi)ts to rcfilacc tlw leaf extract by f;iwwn substances, O[)tinial frweight yields on 50 nil. volume of revised tneditim often have apprtjacbbut rarely exceeded 25 g/flask. wbereas witb leaf extract added tuider oth


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MEDIUM FOR TOBACCO TISSUE CULTURES 491

Effect.t of tobacco leaf extract and its ash content on the growth of tohacco(Growtti period. Sept. 3 to Oct. 2, 1959). From left io right: Basal controt,m utone, with T-3, 3.75 g/J, and with ash from ttie corr esp ond ing am ou nto f T-3.Cultures illustrating grow th an d utilization of niedirt of different compo nilioniGr ow lh pe r io d. .July 2 to .30, lM 'l . i Fl as k 1. ha.sal m edii ini 50 m l: lhi .sk '1 .d mt;dii ii i i .V) m!: fla.sk 3. revis ed m ed iu m lt)O nd ; ami I l i isk 4. revise d m ed iu m w ith

teaf f x t n u t 1(1(1 m l.


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492 TOSHIO MURASHIGE AND FOLKE SKOOGTatjie S. Effects of concentration of extract and iiolumc of medium on tisxue ijirld

revised medium (Grmvlii period. 7/2-7/30/59).

Volume ml/flask

5075100

5075100

T-3 Extract (sotids, g/t)0

25.731 .032.0

0.1550.900.93

0.9 1.9 3.8Fresh weight g/flask

24.730.33


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In pith cultures supplied wilh gibherelHc acid, kinetin and IAA singly andhinatio n of (he tw( ), stim ula ted gro wth. In contra.sf. i.\A alone

With the addition of all the ahove mentioned orgatiic materials to the

DiscuHsionThe use of tissue cultures as a tool iti prohlems of inorganic nulrition of

It is clear, however, that hiological activity of any one substance not only


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4 9 4 TOSHIO MURASHIGE AND FOLKE SEOOGfactory, but some variability was observed in total yields and in morphogenpatterns in response to a given treatment. At least in part this variahility che ascribed to differences in etivironmetital conditions aud treatment of tstock cultures.The reported optimal yields were ohtaitied wilh tbe temperature of tctiltttre room kept between 26 and 2iiC.. hut the temperature was not carftilly controlled nor was ils influence systematically studied.'Hie cttltures w ere kept on tiers of ca 1 meter wide shelves open only one side, so that tbey were exposed to coutinuotts weak and somewbvariable difftise light from fluorescent tttht's and Mazda lamps tnotmted the ceiling. It bas been shown earlier that growth atid organ foruiatioti re.s])oii.se to l\.\ and kiuetin treatments are olitained in darkness as well as the light, hut light is not without uiodifying effects. In general weak diffuartificial light is employed. With iticrease in its intensity tbe tissne becomtuore compact, the fre.sh weigbt tends to decrease aud eveiittially also tdry weigbt is lowertMi. More uniform responses migbt be ohtained hy cloregu lation of tbe light intens ity. It is likely also that tuore rapid and nniforgrowtb might be obtained tbrough stiitahle choices of light quality aespecially of day length.

^'ariahility in resnlts also derives from disuuiforin planting material. Stocultures of difterent age or grown on media wilh dilferent levels of I.\A kinetin. for exatuple, give sub-cnltnres which differ hotb in growtb rate ayield. .\ sla nd ard pretre atuie ut of stock cultures is needed Imt tlifficult specifiy iu advance, as it will depend on the type of growth desired and wvary witb conditions and changes in the stock itself. Requirements of organic nutrirnts probably remain relatively fixed, but tbe need for orgatfactors may fitictnate widely. This is strikingly illustrated by the sporadappearance in control culttn-es of tissue pieces wbicb bave tbe capacity grow in the ab.sence of IAA or kinetin and occassionally witbout cither these in the medium. Tbat increased rates of bio.synthesis is responsible this hebavior is well known iti Ibe case of auxin from the work of (iatither(1959) and coilaboratoi's: and K. J. Fox in our laboratory recently bas .showa relatively bigh kinin contetit in one tissue wbich grew withotit kinetAcqtiired capacity for increa.sed rates of syntbesis of hoth auxin and kininof course also known from Braun's studies of crowti-gall in tohacco.

A significant case of acquired, graded cajiacity of tissue development is treport by (Ihouard auti Agbion (19fit) thai stem segments from the aj)icregion of a flowering tobacco plant produced buds with flowers in rifwhereas segments ftdtn the hasal part of tbe stem prctduced only vegetatihtids. Acqinred differences in accutnnlative or hiosyjithetic capacities muhe responsible for this difference in behavior. It is especially noteworthy thin this case tbe change in Tuorpbogenetic capacity is associated with normontogeny. It is not bnutgbt about by an exogetiotis agent in tbe same sense is tlic case in ttmior indttction: nor is it an act that at present would firespectahle status in the category of so called "biochemical mutations".In the ligbt of Ihe ahove considerations it is to he expected that tissues evfrotn the same j)lant may have difterent quantitative ntttrient reqtiirenien


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Tbe positive response of tbe tobacco tissue to gibberellic acid in the pre-

During the five years this work bas been in progress tbe medium in dif-

The organic addenda in this connection may serve not merely to provide

The mecbanisuis whicb enable tbe tissue to derive nottrishment tor per-2 days,

i).\) and 1.5 g. respectively. Th erefore , about 70 Vu of the w ater furnished"/o was lo.st by evaporation and tra ns pir atio n du riug the four week grow th

rded to utihze about 10 tO 15 Vo of tbe wa ter ava ilable in tbe

itself, and flakes adhere lo the tissue as it "burrows" throughi.e. of liie same order as

S u m m a r yA several fold increa.se in yield of e.xci.sed pith or ca llus c ul tu res of Nico-


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4 9 6 TOSH IO MURASHIGE AND FOLKE SEOOGIn pa rt the iiicreuse was due lo in()rj,'nnif cons lituen ts ui' the ex traespecially N and K, which could be sub.slitiited lor by eitber the ash of textrac t or raised levels of N and K salts in the nied ium .In part the increase was due to or^'anic constituents of the extract wbiincreased 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 addinj* {j[ibberellic acid and Braun's supplements of purines and amino acito a revised medium.A revised m edium ba s been developed (Table 6) w itb eaeb element prvided in sufficient quantity to insure that no increase in yield will result frothe introduction of additional amounts in the ranf,'e ordinarily to l>e expectin plant tissue extracts, etc.Tbe organic constituents bave been retained luichaiif^ed except tbat sucrohas been raised to 3 per cent, nij/o-inosilol has been added as a regular C(nstituent and Edamin has been introduced as an optional ct)nstituent.The revised mediuui is designed for use in bioassays of or^'anic f^jrowfactors. It provides for rapid growth rate, increased response lo orj-angrowtb factors and minimal interference from inorganic and eommon organnutrients.In tbe presence of plant extracts fresb weigbl yields of up to 35 g. on 50 mof medimn bave been obtained. Under these conditions water appears to tbe limiting factor Tor growlh of the tobacco tissues em ployed .Aspects of general application, limitations and behavior of plant lissue ctures are discussed.This work was supported in part by the Research Committee of the GraduSchool. University of Wisconsin with Tniids from tlir Wis. Alumni Rcseart-li Foiinction and in jart by f,'rant G-r)9Hl) fi'oni the Nalionul Science Foiindntion. EdLMiiin wkiiitily riirinshe(t hy tbe Stieftield Ctiemical Co., Norw ieh, N.V. Tho au ltio rs ai-o gralul to Miss Joyce Klenim tor U-chnicat ii.ssi.stance and especiiiUy to Dr. Elt'rieLinsniaier who did the confirimitory experiments on P, Fe, and sucrose levels aon different combinations of IAA and kinetin.Present address of T.M.: Department of Horticulture, Agr. Kxpt. Station, Universof Hawaii, Honolulu, Hawaii.

ReferencesB ra un . A. (',.: A physiolof jical ba sis to r a ti lonoiuou. ' i {growth of t t ie C row n-g all t im ior cell .P r o c . N at i on arA tJ i d . Sr i . IWas t i . l 4 4 :3 44 . lil.'xS.Bi i rk l io h le r . P . H. A Nickc t t , L . ( i . : Alyp ioa l p row ll i o f p h in ls I . Ci i l l iv i il ion of v i ru s lu iof Hiimox on luilriiMil aRar. Bol. Gaz. 110; 42(). 11)41).C h o u a r d , P . & Anh io i i . D. : M odal i les rie In forn ia l io i i dc l id i i rKeons f lo ra i ix s ur des eu t tud e .sofiiiK'iits lif,'i' d e ta lm c. C o ii ip l. r e n d . ACIKI. .S.-i. 1>.'2: 3S (i4 . 1% I.Gaultu-ret , J . H.: Sur Ies | )oss il) i t i t t- de rmdist- r la rullurc imlef inie de.s l issus i ir luI iL-rnde ca ro tt e . C.ompt. re nd . Acad . Sci. Par is *i(): l lK. 1939. Lii cu ll ur e do t is su s vt^g^taiix. ^ta ssl m el Cip. t*ari,s. 19.')9,t le ll t ' r , r t . : t techerclie .s sur la i i i i lr i l ion i i iin


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MEDIUM FO R TOBACCO TISSUE CULTURES 4 9 7Gerloff. G. C. & Skoog, F.; Cobatt as an essential element for hhie-greeiialgae. Physiot. Plant. 7:665. 1954.

lobaeco


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