CA2069122A1 - Methods and compositions for the production of taxol, taxol precursors and derivatives of taxol from plant tissue culture - Google Patents

Abstract

ABSTRACT

Sucessful tissue culture methods have been developed which results in stable, long-term cell lines derived from Taxus explants. Some of the cells lines are habituated, that is, do not require plants hormones as supplements to the nutrient culture medium, for long-term maintenance.
The cell lines established by use of the novel methods of the present invention are capable of producing taxol, and tacol-related compounds. These cell lines offer a rapidly repriducible, continuously-available sourse for the production of taxol and taxol-related compounds. This production may be scaled to commercial levels by use of the cell lines in bioreactors. The cell lines provide screening assays for species and cultures of Taxus that are sources of taxol and taxol-related compounds. In addition to obtaining the same compositions as presently directly extracted from

Description

A_~.~ A ~ L ~ ~/ ' L .~r 1-1 ~ I VL~ I ~AnV1~ 1nh~ U ~

~I~DS ~1) CO~O~ oN8 P'~ P~bU~N oF ~AtlOI"
TAXOL ~R~CCUR~R8 AND ~ IVAT~ O~
~ROM Y~NT TI 8 ~O~ 'rP.~ SN' ~
~h~ inv0n~ion deAl~ Wlth th~ p~odu~ n ~na uae t~r c~nd a6~ u~pen~ion culture~ b~ ~IXU~ gro~n on ~3mbran~ , ln 6haker a~llt~ a, ~nd ln ~ior~aatoXq ~or th~ l?rodu~ion ~t t~xo~, ~r~o~s~r~ ~n~ lnt~ iAt~
o t)l~ bloo~s~ioal pathway r~l~ultlng ln t~ol ~ro~uc~lon, ~n~
~a~lv~ d~rlvAtiv~ o~ taxol, includln~ o~p~alo~nnino an~
~c~atin. T~xol and ~axol-x~l~.t~d ~o~our~ haw ~c~ait:lv~
~t~vlty ln aana~r mod~ y~ ~nd ~l~rotu~
~t~112ing aa~Ay~, ~h~ pound~ pot~
power~ul ah~mot~erapeutla ag~nt~.
I~xol tN~C-125g73) la a oyta~oxio di'cQ~p~n~ wlsh a oom~ x ch~mis~ry, initi~lly i~ol~ta~ froPI th~ ~t~ rX
o~ t~a woat~rn y~, q'~U3 brovl~olls Nut~. ~ by t~Ani ~nd }o Go-wortcerB ~n ~g71. ~ub~guen~ly, ~t nas be~n ~2~own~co ~ pr~nt in t~o ~ a~ ~ll a~ 'ch~ roo~ o~ tb~
p}~nt ~Wltl7~ru~ 90; ~7ld~aok, 1~90~. It i~ the ~nly pl~nt ~ro~ur~t ~cn3wn to ~r~m~t~ a~ly t~
microtubul~ and inhl~lt ~ho tubulln d~u~ ly ~roo~u~, ~nd a~aa~s to ~Q th~ prototy~o o~ ~ new ola~ o~ ~na~r ah~mothera~ tlc ag~n~ u~n~a~ an~ Cord~ll, 19~5~
~n addltion to t~xol "nany othOr clo~qly r~lat~d cyto'coX$c taxan~ dQrivat~vB~ h~ s~n ~aol~ted rGm th~
Uonu~ Nc~auqhlln ~t ~l., 1981; ~ t al., lg8~J
5~in~oton ~t ~ lq92; ~IUAng ~ al . ~ 1 9B6) ~ ~ut ~r rotubul~ ~t~b~ g capn~ ky ~n~ ch~eth~ra~tic 4~ ar~ not xnoun.
T~xcl ~xhl~lt~ aot~lrlty ~ln~ o K~ ~n~r cell l~no u~e~ to scra~n ~o~ ~ot~n~ial ch~m~r~pou'cia ~b~erlto ln till!ll~4 ou~ture. Durlng ~x~alinlea~ ~v~lu~t~on ~tu~
lt h~ boon ~hown to h~tQ a~lvity ~galn~t b~'ch ~ n~
~lld t~mor lin~s un~ oQll lln~ ~W~n~
19?1; suffnQ~ And cord~ll, 19~$J D~n~how~r ot al., 19~7~, P~aentl~ t~xol ha~ b~/an ~hown ~o ~ act~v~ ln t~e o 3 o human Ovari~J~ ca~cinom~ x¢n~ t mod~l and ~:~ hav~

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~ ~Y~: I ~Y~ U 2 1 . ~ . .1 . J _ I ~ ,, ~,, ~ ~, ", , J I I t ~ 't '' -t ' t C~

- 2 - 20~9122 ~roml~e ln ~h~ troa~Qnt o~ a~A~c6~ ragY~iV~, an~
drug-r~actory ~vArlan d~ aeuiro ~ ., lgi~g;
Thl~n, ~t ~1., 1990~ .
~xol 1~ ~ potent lnh~b~t~ btr ~Q~ 11cat~on th~t act~ aa an ~n~1mltot1~ ~gent, blockln~ ~ol1~ ~n th~ oa~M
pha~ o~ tha o~l~ (SCbl~ ~nd t~rowit~, 19~0J ~orowlt4: ot ~l., 1986~. ~t 1~ uni~a ln it~ ab~ y to b~n~
gtoiahi~mQ~r~ y to ~olymeriz~d ~bulin ~oth in ~rltro ~T~d ~3~ VlYo, an~ lndua~- ths a~bly o~ ~l~lln ~0 orYD
lolum-~table ~ç~grog~ as~o~cu~ul~ ~ruatur~ h~
~tru~ur~ rl~si~t ~ol~erlz~tlon L~y d~lutlon, ~alc~um ion~, cold ~nd a s~u~bor o~ m~ero~ulo-d1cru~tln~ ~ruç~
M~crotubul~s a~am~l~ tubulln ~n th~ ~b~sfloQ ot sxo~no~l~
~uano~ine 5 ~ phs~h~ or ~ crotubul~-a~ooiat~d protl~lnB A~ lo~t t8mper2tur~ ch~P~ ~nd 130rowitz, 1Ç~O~
~nrn4~ nd ~rowlts, 198~ ~ow~ nd q'l~dR~ , 198~) and ~3B~ ~I alln~ o~l ~tudiso l~ 3n conduoted wlth ~aXC~. llo~v~r, ~ 0~ ~upply o~
taxol h~- pr~l~lud~d ~xt~e~ llnlq~l ~'CUdl~. 13acau~
~0 there is a ~tron~ po~ ility th~t t~#:C~l w~ ho~
8isni~1aant aCt~Vity ~ln~t ~ d~ r~ng~ o~ aan~ar~, lt ~ ~ cr~a ~ al to snaka ~ bl~ t~ aup~ nt t~u~ ~ultur~ t~ohnlguo~ haY~ thu ~t~n~i~l to pr~dua~
an abund~nt su~}~ly o~ nd ~ iolog~ ~ally ~ctive 2 5 ~o~pound~ and ~ ur~or~ . In ~d~lltlon t~ ~rovi~ing a r~p~dly avai~ R and ~r~n~abl~ ~oura~ t~xol, ~not~r b~n~lt a~ t~6~ue cultura a~ urcD i~ tho ~n~ ti~r~
o~ y~w tre~, a ~rg~ r oP whi~h r.eed ~o b~ h~rvn~4fl ~n or~er to l~o~at~ the kllo~ t~ ti~ pr*~nt~y n~dod ~or cl~n~e~l u~.
~xol haJ ~n rou~in~ly ~r~ct~ ~ro~ ~ch~ drl~d ~t~D~
b~ o~ T. b~-v~ n ~ tlon ~XQ~ al~o ~
i~olatod rrcm ~ov~r~l othor ~pac~o~ ~f y~ ~g~nU~ T~uo, ~a~lly ~2xaceae), ~r~alu~ing ~. b~cGzl~a and ~ p1dati}
(Southton ~md Bucxlnqh~ lg8~1 and hlor~ r~cently ~
c~aaens~a c~r. Capitata Qnd ~. m0d~ cJY~ naltor~ Lnd k~ll twit:l~eru3? 1~ A~ 90) . 5q~ d~

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h?~v~ n ~hown t~ h~v~ hlgh~r ~e~ o~ t~xol tl~n r~xuR
bre~-~lfsY~a ~blQ ~).
Although t~xol h~ n ~xtraa'co~l t~ tu~al ~oura~, ex~r~t~ on ha~ n ~ ~ely p~ ta~lng, And pr~duo~ ~ro~ly lnc~decluat~ or b4t~ pro~ and ant~ t~v~ naod~ U~8 ~h~ae ~.mpo~nd~ ~r~ ~o~
~x~ ely low cona~nt~ n~ in ~lant~ tO.Ol~ dry welg~t ba~lo) . Although tn~ ~a~onal C~ncor $n~t~t~ h~ ba~n ~b~ to ~?~c~C oVor g~,004 ~o~nd~ d ~. b~c~oll~
ba~k, only l-a k~ o~ t~ol h~Y~ ba~r ~xtra~t~d durlng tha ~a~ ~lv~ y~ar~ ~u~n~ 9D) . ~a6ed on an ~Y~
at~d yield of O . Oâ9~ col on ~ ~!lry ~ig~t ~oa~ it 1~ 0~1m~ted thA~ ~ to~l o~ 720,000 pouna4 ~ bark or 3~, 000 tr~ woul~l n~od ~ b4 h~rv~t~ ~0 ob~aln ~u~ol~nt ~xol ~or ~ dlath ph~r~ utl~al UB~D, if~ tho nati~ 'r. ~rD~l~oll~ i~ t~ lo c~ur~ n~d~r, 1~0) .
in g~n~rnl hav~ ~ory ~14w ~rowth rAtoq ~n~ lony prop~tlon ~rio~l~;. Th~ lt dl~flc~lt t~ i~l~0nt br~adinq S;rogram~ aim3d at ~cra~nlns~ ~r ~p~ale~ pr~duoin~
;3n 2~bundant ~upply o~ thcoo ao~po~ u~ to ~h~ V~y ~low ~o~rth o~ 1l11 y~w~ d the lo~r y191d~ 0~ e~X~l obt~in~d, th~r~ la wid~r~ad ~on~a~n th4t a~t~nat~
~ut~ ~rQ n~d~d to ntainta~n an aS~ t~ and c~ntl~u~u~
~u~ply o~` thi~ d~ug. ~he~i~t~ al~ O~ t` the w~rl~ h~
bo-n lnvolv~d in ~n ~tt~mpt eO whl~ ot~ o~
t~xol t~u~rl~tn-vo~g~le~ ~9t ~ 8t), ~ut tha~s ~oxt~
hav~ onl~ ~e~ ~lith llm;t~ . EvQn ~ ~o~o o~ th~
V~rioUE~ ~trat~ empl~y~d p~u~e ~exol I thoy naqre llttl~ pr~ a~l v~lu~ oau~0 ~h~y ara ~lilc~y to ~urnlP~ t~xol ~nd t~xo~ t~ co~po~ ln ~or~ ~hsn aa flmoun~cs (D~ni~ ~t al., 1988~ .
Con~1ictf~ BXl~t ~tw~on th~ nb~ ~or l~rq~ un~
o~ t~xol, ~n ~Ytrar~ly ~ hamoth~r~p~utla ~n~
3~ an~ tho cona~rn o~ ~nviyon~Qnt~ t~ to pr~a~r~e th~ y~w tr~s (g~rAU~ T~xu~ a~lly ~!~Yaa~a~3 whlch at ~r~e-t Ar~
t~lO only 60urc~ o~ t~x~ r~ov~r, A ll~r~OU4 llmltat~on ln t~xt~A~tlng t~xol ~lr~c~ly ~ral~l y~w tree~ th~ thl~

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2~122 hod ha~ only vexy low yl~la, ir!~hf~ u~lc~ to ~at~ ~y n~od~ of ~n~or ra~aroh~r~ hQ~a con~ t~r~g allni~l tri~l~ on ~h~ a o~ ~xol. ~v~n nl~ro ~v~x~ ~ho~t~g~
o~ taxo~ would on~ p~ re~t~nt or oAn~s wi~h t~ol wA~; init~t~t~a, Bu~h ~hc~rtR~ wo~
corr6~1~ta~ wlth ~roh~ itl~f#ly hlç~h oo~t~ o~ the flru~.
q~hRrla i8 ~n lm~d~ ate e~ aruclal n~od to ~wlo~
rapidly r~prod~aiblo, contlnuou~ly ~vn~ lAbls~, r~ w~Sl4 ~ouros~ o~ t~s~nl ~nd t~ol-xoL~t~ c~ und~D, ~ h~ or rg~ ~a~ produatl~n. ~saaU~R ~bW ~o g~a~ y ~lowly, plnntlng ~or~ ysw tr~ not ~ ~rb~i~ing ~ol~tion. Ch~mic~ h~ a~ no~ b~n ~oc~QB~ul.
T~x~nc rlng-~ontalnlng ~lX~lo~d co~aundl~ hav~ b00n obta~n~d trom coll ~ulturR~ o~ T. ~v~ol~, bu~ ~on~-term, ~ablR gro~h ~ ~uch Cu~tur~, or o~ ~Bt~ d a~ll llne~ of ;~rly ~ hav~ not ~n r~ rt~ ~Chr~tQn ~t ~ 19~1) .
~h~ mathod~- an~ s:ompo~ltlbn~ o~ pr~n~ i~v-Dn~ n ar~ ~ no~utlon ~o th~ psobl~ of tax~ up~ly b~a~u~ y ra~& to ~ UC~ cult~sl o~ ~AXU~ tt~ ~v~l~p Htabl~ lon~-t~ ~æll t ln~ wt~lc~ prc~duc~, ~v~n 8ve~prndl~o~, tAxol, l~no~ to u~a t~i~ tho~ coll lin~ r~ o~ to ~?rodu~
tA~ nd ta~col-ralat~d a~m~oUn~; in la~g~ t~ o th~n ar~ ~08~ With curxon~ ~axol-5~rodu6;tl0n mb~

~5 ~AaY 0~
~uoc~s~ul tissue cul~u~ h~lv~ b~ O.ovul~p6d whlch r~ault ~n long~ t~l~ ce~l lln~ d~ri~ from ~plAn~ omo o~ t~e a~ lnl~ ar~ hflbitu~
o no~ r~ plarlt hor~non~ ~g ~s~pplQm~n~ ~o ~h~ nu~ri~ u~.tu~- mcd~ ox lonq-t~ n~en~nG~.
Th~ ~.ll lin~ s~Abli~h-d ~ w~ o~ ~h~ D~¢tho~ th~
~r~s~nt lnver tion aro ~p~bl ~ o~ ~ro~lu~ taxol, ~n~
t~xol-r~l~t4t~ aola~oundc. ~h~uq~ ~ell lln~ 02~ rapi41y ~proauo~blR, cOr~lnuou~ly~ All~bl~ ~bura~ ~or productlon o~ taxol ans!l t~Axol-r~lat~d ~C~mpoun~
~roduct~n ~n~y ~e ~cal11d tO aoyD~ltaroiA~ loy U~ 0 th8 CQll l~n-~- ln l~io2~ tor~. ~n ~ddltlon to ~a~nln~

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~ om~?o~ on~ ntly ~r~ ly @~ d ~r~m Y~ew ca~po~l~lon~ ~xhl~i~ln~ t~ llk~
vl~y, I~-Y~ b~ d ~ro~ ~xtr~ o~ th~ ~a c~ n~o, or~lnç7 n~w h~ ono ~!or C~ 0~ p3t~ C
t ~v~ n~.
cy~otoxlc dlk~n~ vl~h A~onot~t~
~nt~ln~o~la~ti~ aatlvity ag~ln~ ol~ tu~ d 1aU~C~D~C c~ll llnQs, 4nd ac'ctvlty a~ a ~h~moth~r~la ag~ ln ollnlc~l t~ial~. Taxol i~ ~ only ~l~me ~rodu¢t 10 ~on~ly XnOWI ~co prQ~o~ a~ y o~ ~aro~ubul~
a~ ~o ln~bl~ th4 t~bul ~ n ~ ~ly ~oc~ . Thl~
~ec~anl~m ~u~g~t~ ~h~t ~A~ol iu ~ pro~oty~ n~w ola~ of oh~m~tha~ap~aela aq~rlt~ e~at ~tt~ek o~n~ ro~th hle ~a~i lon. ~or ~U~pO~I; o~ ln~n$10n ~ d h~r~ln, ~t~Xbl~ n~ral~y ~ nt to l~olud~ aompound-h~h a~- ~tructurally rol~t~ to t~xol aT~ do~lnad to ln~u~ r~ uro~r~ o~ taxol 1R ~ioah~ aal ~t~w~yo wh~ah ~r~ G~bl~ o~ ~r~ducln~ ~xol, to lnt~ t~ o~
tho~ yo, ~nd ~o ~ lYn~ d ~ S~y 2~ ~nt~olito~ e~r t~lxol ~a~halo~annlr~ aa~ln). "~xol~
llX~ act~vlty" ~ r~ to ~o~o~t~on~ whl~h ~h~w ~o~tlY~
r~-ult~ in ~ on~ t~l~t ~ dlctlva of ~
~hQr~othsrapy: a ~ otubul~ stabil~lny ~a~y, aan~r mcd~ y, or 411nlaal ~rl~
By co~lnin~ ~a~o not ~vlcu~ly ~o~ln~ t:u p~o~u~0 ~uo ~ulturo~ and ~y u~lng nev ~ n~ cQ~ ltion~
ob~ul lo~ term ~rowth h~ n nchlclv~ld, an~
inc~ aell lin~ ~rodua~d. A IA~thod for ~t~ hlng a c~lltur~l oS` ~all~ d~rl~d ~x~ ~e q~ml~ ~xu~ h~ n u~ao~ul in p~o~ualng ~oll lin~a ~ eor~t~ tax~l and t~xol-r~u~o~ co~ound~ into t~n ~lt~r~ n~v~on~en~
c~ o~ th~ ultur~ hoa aro n~ ~llow-:
1. 8~1ect 6n ~pproprlAt~ t~u~ lourc~r ~n 4~nt ~hlc:h Wlll ~ u~ed to initlat~ ~h~ a~ ur~.
.
3 5~h4 g~n4r~ u~ u~uro~ 1~ pl~th~ g~nu~
T~xu8, Yar~ou~ ~2cl~ ~xolg whlah thXol T~Ay b~ l~ol~t~d lnalud~ . Ps~ol~ t~ b~¢oAt~ . au~tdat~
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2~6~122 6 ~

~:~n~donsl~ ~, C~pltata, T. m~O~ . D~n~ rm~ nA 5~.
m~d~ cv. Rlak~li. Pr~ r~o~ re~ ~o~ ~xpl~nts u~ed - 'co p~oduce tPXol-- ~xpI:~o~ln9~ CUltl~ A~O t~ i plAntE~
~r~ whlah t~ unt ~ t~x~l thAt m~ty b~ ~xtr~ot4d d~r~ctly ila hi~h uhon comp~r~d ~ u~ xtrac:~d 4r~n oth~ 0Ci6~1 by co~}lpar~ t~ da gro~ oo~pa~g~ble a~our~ 0~ 6t~rti~ m~r~Ql~ By th~t arlti~r~on, T.
~di~ or~ rtlcul~rly ~ d~ C~. H1Q~ I fflOr~
pr~t~!rr~d ~oura~ n ~. ~ovl~oI ~ta .
~arX ha~ boan th~ u~u~l ~ou~c~ scol, an~ nu~ s and roots al~ Ar~ o~ ~x~r~6~1ng ~Xo~ w~x, 1~ W~ tn~ t~t ~ p~ u~ 4~urc~ o~ n~
1~ a 6t~, roQr~ p~rtloul~rly a youn~, ~rQ~h~ ~ccul~nt 4t~3. Y~ at~Y ~ tho~ tn~ ~p~r 8~ tho b~nn~ny n~ a new q~owth se~on ~o~ ths pl~n~ tur~ ~t~
~ctlon~ o~ ~xu~ m~dl~ cv. Hiok4ii ur~ p~rticul~rl y ~r~rr~d b~&a~a ~t~ r)P ~ d~a pl~nt~ con~sln h~hor lev~l- o~ ~oxal ~han rax~o ~7r~oll~ ~nd ot~r p~ o. Explarlt~ D~ ~out ô.~-0.5 hUR ~n dla~tor ~o ar~ it~bl~ .

a. C~lLtur~ t~ nt i~ cAll~lo-~ndual~ du~ to ~or~ calll~6.

Ono o~ th~ con~n~t~on~ o~ ~o~r~ d lng to ~UoOI~Sl~U,!l ~AXW ouït~ Y lnolu~ U~8b 0~ Sl ~sallul~-2~ induain5~ dlu~ in wnl~h to ~r~w ~ho ~lant.
~ o Ior~ Qn s~l~rlt, ~ ti~u~ ob~a~n~d ~ro3n th~ 3plant prca~ss~ y sur~c~ ~t~ 2al:i~n nnd ~atl~no~ und~
~sap'ci~ c~ndlt10n3. ~eotloning 1~ aaoo~pll~h~d by au~n~
a s~ lnto plea~ of o.~proxl~at~ly 8-d ~ ln lsng~h, ~r~m im~aturo 6t~ th~ ~rR elpproxi~ately 3-5 ~m in diA~Dt~r, and trans~err1ns~ the ~ctl~n~ to ~ u~ oult~
~ns~lly, ~t~ di~h~-, w~n c~llu~-~nduo~n~ ~flUt~lant ~Q~liQ. 'rhl4 1~ dlU~ ~hloh ~tl es~le o~ lrtlt~s~-~n~ AnCI
E~romotinU the ~o~mation o~ a~ gro~th Pr~a~ ~ ox3~l~n~.
3~ ~ p~e~erre~d me~ ~bor51'~ B5 ~s~ tGnn~or~ ot ~l., 1~68) ~u~ onteo~ wlth pl~nt ~o~n~ uch Al~ ~,4^D ~n~

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L~ YL). I ~y~ , J _ 1 J_ I IJ U U IJ~J ~ 0_ U~ / ~ J
~ L I ~ L ~ U r ~ V L C I O~ I. A rl V l ~ C r~ r ~l A n ~ y ~1 4 1 .S J ~

klnetin ~lth~r ~lo~ r ln ~ ina~ion. Ils~lum o~
Mur~hlg~ ~nd SXoog (~ura~hi~, T. ~nd F. 8X~o~, 19~2~ la ~1JO ~ bl~ r t;~ pU~p~ hn~l ~Ay ~ u~ menl:odl.
n~ a~Qnt~ ~uch a~ G~l~it~ ~gcott Il~b~r~ ri~
WArW1Ck, R.I.~ 0~ r ~Y~ ~lx~d ~rlth th~ r,ut~l~r,t m~
to for~ a ~olid ~upport ~n ~h~ aulturo V08l6~ or malnt~n~n~ ~n~ ~rowth o~ ~hs 4x~1~n~.

3 . ~ubcul~r1 hq ~ol~tod ~e~or~ llu~ a~
lnt~Al~ whi~h ~romOt~ ~ub~lon~ g~o~h.

Callu~ ~or~tl~n io d~3~0~1r~d wh~ obulilr ~ggr2g~t~ of undl~Qr~n~lated ~ re~ rvAd, Tni~
g~norally o~Cl1~8 on go~ o~ th~ oxpl~nts within ~ou~ ~-4 w~ok~. ~uboulturl~g ini~l~lly 1~ ~r~ fl at ~ w~
lntQrvala, but msy bQ ~ rod a~ a fu~tlon ~ cult~r4 l 5 gr~wth .

sl~cting a ~ubQlon~ to ~V~ ll lln~.
!

A~ lh;uort~ t Ptq~ ln ~r~einq o{~ll 11n~ r~
c:2lyabla of ~rod~inç~ ~axol 1~ a~ o~ln~ ~h~ ~llua ~plo ts ~olcot ~o~ subc~lturin~ to ~vslop ~ olon* o~ th~
ao callu~ Physical ~lection ~ltaria hav~ n d,~v~lop~d as ~n n~peat ot th~ pro~nt lnv~ntlon. C~llu~ aue wlth red ~ Drin~ snoul~ not ~a oRleot~d ~aau~a th4 r~d c~lor ln~i~Atea tnQ pr~tnao o~ p~noll~ ~xuAat~o whiu~ inhlbi~
a~llu~ ~rowth. ~11U# ~oction~ ~xbibi~in~ ~ y~llow calor 2s ~ r~era~ly ~lact~ ~or ~uboultur~n~ ure~
~rowing vell ~n th~ d~rk w~ll b3 gr~n. Fri~ ty 119 ~notn~ al~tlon ~rle.rlor. ~ le'Larm~n~d by o~sr~ng ~ cru~bly, ~r~glle~ t~ turs2 o~ the c~.llue.
Ano~er ~Actor ~1ch indic~ts~ o~m~lea 1:~A1; ~ llX~ly to r~ t ln ~uc~G~ul c~3ll lln~ r ~U:IepO4~ o praa~nt lnv~ntlon l~ r~lst~ ~h rat~ a~ the uallu~
aell~. Ini~ially, al~ c~llu~ lo~ q~w~ t~king ab~ut 2~4 mc~nth~ to ~oubl0~ ~r~ ~r~Able ~9~u~1~n4~ ti~o~ ~r~
.~ f r~ abaut S-16 do.ys . 8ubsu~urin~ wa~ p~ri~ mD~

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v ~ r ,-l ~ v L L l ~ L~~ Lr~ r~nr~A- ~ ~ 1 4 ~ I S U ~
2~S9122 ln~:~rval~ out a-6 or~ s ~ ~lthou~h ~ y b~ l~h~rter o~ lonç~r d~n~lln5J Dn ~ch~ ~alth o~ th~
CAllU~ lnCI~ orlt~d by ~lor, ~d by th~s ~12d~ of th oall~a ~t~ growth, s ~y ~rLa~ ~ub~ul~cu~lng n~ c~llu~ ollos~llng the~o crlt~rl~, r~ln~ ly hom~n~ou~ n~ w1~h rA~rd to th~a~ crits~ ,, d~v~ chough ~ tlm~
bRtor~on ~n ~x~l~,nt bolng ~ d ln cul~ur~ ~nd th:3 appo.a~anca o~ h~lonQ~ varl~ ome o~ th~ lnt~rval~ ~r~
O m~s~ d i~l yQ~

. ~ulturin~ ~ne c~ll lina on a ~ ld ~ rt ln ~a~nten~na~ ~edi~D~

~ so ~tl~r ~np~r~ant fAc:tora P~ ~ucc~ xu~s cultur~ ~re a ~ol~d ~uppor~ ~n th~ ~or~ oP a ~3Qbr~n~
lB r~t, and ~naintonanc~ ~dla ~or l~n~ m aulture ~rawth~
2h~ opti~ ul~u~a clondl~lDn~ ~or t~ aot~ b~lon~
Ar~ than determln~d by a ~ultiY4s:1ats ~n~ly~1~ o~ ~otors Whlch n~ likDly to or~wt cultllrn c~ro~thl ~r~ nao a~
~b~eno~ 11gb~, t~r~ur~, p~ d~ up~ t~
2 0 ~ ncludln~ plant hor~on~ nns~ ~gllro . f3~a~ h~ n us~l by thoc~ o~ ~k~ll ln th~ ~rt a,e ~ gdr ln nutr1~n~
dlum for ~ AB CUltUr~l~. Unexp~ a~ly, ~ruato~3 An~
gluao~8 ~re u3~d ~or th~ pr~nt lnYontit~n t~ ~nhanco . oultur~ ~obrth, W1th or wi~ho~ U~ro~a. ~1~3o ~ur~ri~lngly, high~r conc~ntr~tion~ t~An pr~viou~ly roco~ndQd ~r auaro~ ~f u~d a~ a ~nql~ ~u~ Y~re ~ound ~o ~nhan6e cultu~c~ h conc~ntrst1ona o~ ~ther gl~co~s or ~uc'co~s ~ d ~ln~ o gavo oxo~ nt ro~ul~o. Nona o~ th~o U~Ar pro~a~lB h~d b~n x~or~o~
ror !r~xt~
ORtl~u~ cUl~urs ~int~n~n~ or3 lnalud~sd uP~ o~
~rn~ rt to s~Upport ~ ae11~ ~ Thl~ ctruc~
11itate~d ~ub~ultur1n~, ~oll~otion o~ r~aat~, ~nA
~or~atlon o~ C~ll ou~pon~1o~. A~ t~ Ult~
growt21 1B ~C~ ev~d, the C~11 llnelg are ]C~ in ~aln~n~m ~di~m, Par ~c~me o~ tho C~ll lln~ bl~ t~on oe~:ur~, ~ Y L.~ ~ U -~ I . I ' J -- ~ V V I ~ I I J I * ~ 0 4 ~ J I ~ t ( J
2~69~22 o g ~hat 1~, th~ oult~ r~ msin'c~1ne~ wltho~ hq ne~d ~or h~ nal 4uppl~n~6, ln addi~:lon to ~llu~ c~ll lln~, c~ u~n~i~n O~l'cure3 ~r~ ln~ond ~rom we~ ttlbli,~hlad Dt~le c~llu~
cul llr~a ~ d~orib~d ~bc~v~ br tr~ rring ;hn ~noculu~
of C~110 ~2-3 grAm~) o~ ~rl~blH o~llu~ alu~ into ~ d ~dlu~ cont~in~d ln ~ ~ey~r ~ X~ n~r~lly 250 ~1 ~1R~ ao~ta~nln~ 140 T~1 o~ e~1~) . F~k~ ar~ pla~d on ~ ro~y shaker ~cnown to tho~e ~ ~X~ll in the ~rt an~
opo~ d ~ out 125 rp~.
An impo2~tant u~ o~ thb ~w ~ell l~n~ to prodw@
tnxol ~nd t~xol-r~la~ed compound~. C~l~u~ ~nd ~u~p~ion b~ n~ Of ~axuB d~ lop~ ~y t~e m~ 3 o~ th~
~rR~nt iJ~Y~ntion pro~u~o t~xol, ~s~s~rl~r~ ~nd int~nnadl~te~ o~ a bl~h~mla~ thway rs~ in~ ~n tax~l pr~dUCt~on, ~nc1udlnsJ O~!;phalon)~nino and ~acca'cln~ ~n~
a~clva d~riv~ r~3 oi~ ~xol. J~o~h l~lotic ancl y~blot~c~
ltors, proour~or ffi~ nd oth~r ~l~c~thtior~
~ochn~q~ r-a us~ o anh~naa c~c~o3ldary ~otsbollt - 2~ ~rodu~t~n. ~?~x~l, and taxol-r~latQd aompaund~, ar~
oxtra~tad ~ro~n ~llu~ u~n~i~n ~u~ r~ ana oul~
mediu~, An ~ot~V~ o~n~o~t1on ia on~ ~t ~8 c~abla o~ lnq ~O~ Y~ U~ ln a ~l~r~tubul~ ~tabill~in~
a~ay, a ~Pncer ~od~l a~y, a cll~o~ x ~ny ~5 com~n~tlon o~ t2~ t~st~ TheK~ aoll lin~ ~r- ~
continuous ~o~rc~ b~au~ thoy ~a~ :be ~ntnined lnds~n~t~ly, ~3 hav~ noW ~Qn m~intaln~d for S~ y~ar~, und~r ~pti~ ~on~ltlon~ and rl~t~in ~ lr ab~ y ~o produ~ t~xo~, ~ot~ro ~r~ n~ p~a~o~s~ o~
~h~y ~row well in l~rg~ wal~ b~or~tor~ th~ a~
oan ~ ~ryopr430rv~ ol prC~duo~on l.bill~g r~tain~ t~ crlfopr~r~ation or ~to~ 10-15'~
e~ ln~ ro ~ult~bl~ oUXGO~ to ~roW ln biortl~oto~D to p~oduco la~o quan~lti~o ol~ ~hXOl ~ ~nd 3~ t~xol-x~la~s:d oc~m~ound~. ~o~ h~ ~po~tl~n~ ~r~
rOund ln th~ aultu~ m an~ y b~ obt~n~a by ~olloatln~`th~ ~dlum. Ut~ in~ dl~r~nt ~io~aa~:
~lgna, th~ ~4~0n~ry zn~tclboli~ n~ ln ~lutlan 2~ny .

~ .
.

~ YLl I ~Y~ U -1 1 . ~ . J _ I ~J_ ' I L~ JV-~ 'J()._ 'J.~
J ~ IL I ~ .lrlll ~ L~ n~ Ln ~ n- ~14~4~
2~63~ ~2 b~ ~R~ly ~mo~d iro~ th~ Isolu~103~ tlll~lng ~2xc;,h~n~
r~cl~ whi~h QYt~l~Ct only the ~ eoondAry olit~ ~rcm th~ ~Dlution ~nd no~ tho ~d.la 8 r~ ?b~ c~u~ b~:~ . Ot~r ~o~po~tl~ne~ ~r~ produo~d ~r~d r~tained lntr~c~llulnrly ~nd mu~t b~ axtr~otod from tt~
~hQ ~owth o~ T~xu~-d~ c~ in~ ln blor~otor~
on~blR~ tho production o~ ~uch l~r~ ~Rnti~ n ar~
~r~n~y ~as~bla. ~hll~ 2;roC~ nt)t only ~ o aE a 0 ret~w~bl~ ra~U~ce o~ chsurlo~h~ra~¢ut~ g~nt~ bue ~ o saY~A ~be larg~ n~er oP ~X~ 'rhat ~r~ ntly bolng n~v30ta~ to le~olat~ th~ ~o~a~o~an~.
~ha ~n~t~od~ an~ co~po~ n~ o~ thi- px~nt inventlon ~acill~ht~ new ~ro~n~ng ts~t~ to ~l~t~mln~ whl~h 13~p61C3.~D
Rnd ~rlst~ of ~9XU~ ~ra am~ l4 ~o lc~ng-term, ~tablQ
~ult~lr~ nd whlch Raur~:g~ ~r~ lik~ly to y~ ~ld ~hxtsl an~
- ~xol-r~l~t~d c~m~und~l. No~ onl~ ~ra r~ ly ra~rO~u~ , oont~nuc~u#ly Q~ , hec3~lblo llnd r~n~w~ble ~ourc~ o~ th~ OO~O~ h0 ~rov~d, but ~yu o to ~ac~ thd UnXn~ r~tlon~ in taxol~r~la~o~
compoundsl, ~omo v~iatio~ not obtA~n~bl~ rrDm th~ urQ.l ~ourt~, 9~0 al~o provla~ o tr~ tlt~ Dy ~ ~v~n ~ore pot~nt ch~motha~ pout~a P~ge~llt3 th~ln I~Bve b~4n l~orted.
Pra~louely u~nown a~pon~nt~ o ~ au~tus~
coll extra~t~ w~th taxol-llk~ ~ot~ity h~ve b~on ~lUtQd a~ lcz by HPLC ~h~ gh pro~ uid chro~toqra~hs~
.~ The~o p~aX~ co~t~n rA~t~ dhi~h ar~ poslt~ ln mlcrotubuls~ 2ins~ y~ ~oa~ct~on ~0 o~ o~ltlon~ wlt~ t~xol~ ctlvity ro~ ~ulturot~, c:ompoa~t~On~ tha~ m~y not b~ lpr~n~ ln n~t~ ourc~, #Ixpan~s~ th~ horl zonr~ ~or d.e~relo~mont of otlomoth~ra~
~q~nt~. Al~a, b~u~- call lln~ ~lY~er in their }~C!
~ ~ ~Y~ r~l~t~ to tho~ co~7o~ltlona, ~sll c~lltur~
o~r~ to ~A A u~e~ul ~oyr~ o~ n~w ~r~msnt ag~t~, ~no oel~ lino~ di~ar ~n ~ h~unt and natur~ of taxol ~nd ~axol~ tod Compour~d~ oy o~,ue~
oul~ure undqr a d~inoi ~t oP 0nvlronm~en'c~1 aond~lon~.

., , .;-:
. : ~. . ,.,: , :., ..................... :

.

2~9122 ~ 11 --Ir~hla V lllu~tr~ d~i f f~r~c~ n~ ~o~ o~ tb4 ooll l~hg~ witn r~g~rd éo tl~ r~n~ ~f ~r~nt OI ~xol ~ro~u~. C4m~p~nDr~t~ ot ~ ars t~x~ ika ~i~ thc~y ar~
e~bl~ OL` ~ n~ cr~tubul~3 ~nd/or o~ giv~
po~ lve ~ull:~ ln o~ncer ~o~ y~tom~, ~or sax~mpl~, oxlQ~ty OI ~ toat ~g~3nt: ~o ~ ncther a~t o~ th~ ~r~nt ln-J~n~lon 1~ to ~odua~
nQw T~xU~ cell line~i ~y lndu~lng ~iu~ on~ ta~ion~ o~
iQl~ct~d c011 llns~ ~o lnd.uc~d by irr~ lon ~rQAtm~nt and ~hcmical mutAg~ins~ ~rh~ ~llu~ an~ oiub~lone ~loation crltori~ and mQ'chodEi ~o inClu~:~ eRllu~ ~nd ~ t~n cQ
lln~ a~i d~scribQ~l herRln, ~Lr~: UFil!~ to ~o~u~ o~ll lin~
l~rom the ~nu~e~ ~bU~C~. Ul~x~vlost li~ht, ln par~ioul~r ~-A~ ~n~ e~l ~uta~a~ u~h Q~ ~hy~
lS ma~h~n~ ~ul~on~, ~re ~llt~b~ ~u~qQn~.
Anoth~ a~ t o~ p~ant inv~nt~on 1~ ~o r~ n~ate T~ plant~ ~rom ~n ~ rv ~ultur~s. F~r ths rc~n~r~tion o~ t~xol-~o~ ing plantl~, o~ a~llt~r~d ~n a higll a~l~lnsoytokinin xati~ undsr a 16 h~ur r~gl~ was ln~u~d ~o ur~or~o o~g~n~n~ y ~o~ially d~r~a~lng eh~ o,uxln: ~ytolc~nin ra~lo ~to ~P ~a-~ sop~nt~nyl~nln~
2 - 1~ mg~l in com~n~t~on wi~ ~A (ln~ol~-3-bu~rlo ~cld) 0.1 ~ 2 . 0 ~ o~ ~X~pl~ . 5hoo~ odu~od ~y tbe~ llus wbr~ th~n ex~ d 4~t~ a~11y ~pproxim~ly at ~ho ~t~a ~ 1 om ~hoot~) ~n~ pl~a~d on ~org~ B~5 ba~Al medium ~uppl~ tnd ~ 20 qlfl suara~ to lnduc~
r~o~ ~orlution. W~ll root~d ~noQt~a Ar-3 ~h~n tran~rr~d on~o ~Qrll~ tro-mix 200 ~o~lng ~odlu~, ~a~ t~d An~
tr~n~ rr~d t~ th~ qrQ~nho~ to ~v~lc>p lnto ~ a.
~2uenar~ r. of plAn~o tro~ ~u~tu~ wlth ino~ae~d produo~ion o~ tAxol pro~ lan~ souroe~ th~t ~-r~
~n~ o~ p~od~ qre~ r ~4~n~ tl-xol And t~xol-r~ o~poun~ th~n natural plu~ An pro~u~.
gC~'iVl~lD of ~h~ inv~ntl~n ~nalud~ ~rowing O~lU~:
3~ ~nd ~u~p~tn~lon ~ltur~ to ~roduce ~t~blo ~
lin~ ich ~ro aa~ oS proau~ng ~axol ~n~ ~'ch~ ~xol-relatod ~om~oun~ olo-~d h~in. ~ aell lln~6 ~r~
u~aiul a~ mater3Al ~or ~o p~oductl~n o~ taxol And t~xo~-: ~ ;

J _ I ~ . J ~J ~ J - I I t l_~
m ~ L ~L ~ rlr, I VL~I~Lt~nul~cn ~,nw~n-~ ~14~41~

2~69122 r~la~od co~pound~ in ~ r~o~r~, r~ ~x~dplæ, l~ orat~y ec~l~ bior~ct~r~. ~orn~h~ro~ CR10 ~or~aaot~r~ aro ~l~o aon~w~pl~t~d ~n OY~br ~ rcldu~s4 com31l~rc~a~ n~l~io~ o~
t~xol, aotlv~ der~vn~ o~ ~axol, c~h~ nnln~, S ~Aoo~ ln, ~nd okh~r t~:~ol ~ r~ r~ ~nd d~rl~rativ~, guantiti~e ~hat are re~uiræ~ ~or ~n~;30r ~e~a~ah an~
tb~rapy. ~w co~o~ltions w~th t~xol llk~ ~ativi'cy m~y b~ ob~ n~d ~ro~ o~ tr~ct~ or ~l~uo oultu~ .
~ultur~s Are u~e~l to ~cr~n ~clXU4 ~ Oi6~5;1 Qnd ~ell lln~s 1~ ~o~ ~ro~luc~lon o~ taxol. N~w t~xol-llk~ c~ ound~ ~ay b4 roun~ ln ~ultur~-.

D~Oll~
t,ho ~ro~ gr~du~l tr~n~sr o~ In Ylt~o ~en~rat~ ~lant~ ~ thA ot~ lo ~nvlron~nt ln s~rdDr to lS ~ d ~udden d~sloc~lon du~ ~o ox~s~v~2 wat4r 1~
~Q~S ap~ara~wY iEo~ gr~wln5~ ne6 ~n ~ lar~e ~cal~
A.~rll~t: Ronte~ ~cty~ irlit blo~otor~
~raun Bl ch (~ 'ca~ ~ir 11~ blor~aator~
~Q LH ~ ntation ~00 ~erl~) ~tlrr~: N~S~ Bxun~wlok (C~llla~n & B~o~lo~
F~ nt~t ~ on ~orf`u~lon: N~w B~un~wlck ~embrAnG-~low~ nut~nt ~oduD~ ~low~ ov~r a ~mbrAt~
. 25 Wn~h ~upp~rt~ t~e c611~, ~h~ u~ 1~ r~r~ulat~d.
OthQ~ no~ l de~l~n~s ~r~ ~u1~4bl~
~aJ,.lla~ an ag~req~t~ o~ d~i~r~ntiat~d cell~.
n ~t~o or lr~ v~,vo ~yfa~o~ wh4~rolr~
a r~ult 1~ produa~d ~y tout ~n~ ld r~ult dat~r3~1n~ 'co be pr~dlctlv~ o~ ~llnlo~ ec~o ~ the ' Ag~n~.
aOmPO~ttiOn ~r livln~ asll~ ~nalnta~r~eCI Yn ~1 ~o .
~L~: g~oup of 4~ ln YltX`o ~or whiah Jro~
charact~gtice ~Yo ~cor~ ~t~blo un~r ~ ~efineA ~o~ o~
env~onmant~l condl~i~n~, and ~hl~ o di~r~rontl~t~a ~o~
o~r ~ n~ h~ rlo~o~, - , ; . ~.
.... . . .

- 13 .v 2 ~ ~91 22 ~o~1~vlon o~ livlnq ~Q~l~ thRt nræ not ~tt~d tc ~ ~ol~d ~uh~tr~t~ but r~th0r sr~ ~u~p~ ln a llgul~.
ClU~ S qlo~ul~r or no~ul~r 4~r~t~ o~ a~llN
ob~ ra~ n th~ s~urfaco o~ ~ a~
~n~ d~um ln whl~h oultur~d oell~
~qr~t~ into t~ m~lurd ~JArlou3 m~t~ollt~ an~ other aompon~n~ c~ ro~lrth and, dlvl~lbn.
h~r~n ~o ~ ch~ r~to ~t ~ h o cell~ ln cultvura doubl4 by ml~o~ doubllng rat~.
111 ~ t~chnigu~ appll~Dd to ~ o~ll cul~u~ wt~ch ~nar~o th~ y~ld o~ ~xol Ox ~xol-r~ te~
co~poun~o th~ ~r~ produ~d by th3 cultur~ ~h~ h~
o~ t}~ iria ~ at m4y bo ~mnunolo~ 1, t2~t 1~, th~
lS coll~ m~y ~ount ~ dQ~n~ aga~n~'c forelqn ~ant~- by produclng taxol .
BlotlQ: ~x~mpl~: lnalud~ aut~cl~ d gunu~l o~ll w~ll ~rllgmen~a ~nd s~or~.
c~tl~ t ~x~plo~ ls~e: lud~ ~alt~ orh h~
m~t~ ,vanadat~ d ~nd m~rcury.
F~J~ty: ~ra~ ru~bly t~ a oonsi~t~noy, }~b~u~ a~ c~ n vlt~ wnlel~ e.r~ m~lnt~ ~n~à
~y ~u~ul~r~ ~nd do n~t r~ n~trlont modlum s~ppl em~n~ed wlth honnon~ for ~int~nan~; th~ ltur~
~rQ usually char~ct~le~d by rolatiYoly rapld ~rowtl~
rat~-~s condltlon wher~lr a ~ n~ np~bl~ o~
balng ~ intaln~ by ~r~al ~u~:ul~r~ v~thout th~ u~4 of honllcna~ upplem~nt~ to th~ nutri~nt In~dlu~ in o~hl~h tt~ cell~ aro ~in~ln~d.
~: llt~r~lly l)ln S111~8E-~7, an ~ la.l on-~lr~n~hDnt wh~ah ~l~ia~ th~ ln ~vo aonq~tlon~ tanarlc~ ~nCI~
~nnrally, ~rowth and r~o~ual:lon o~ ~lolo~ie~l Illl~tC~ B .
3~ a nutri~nt ~om~o~ibn ~or tn~ ~rowth o~ l~vin~
~nat~ u~h aa cell~, til~U~ fflbry4~ d th~
R~ lon t~ t~mo ~t wh~ch. ~ giY~n ~onlpounfl will elu~
~ B cr~romatogr~p~ lu~n ooun~ln~ fro~a th~ s ~t ~ YIJ~ I t I . I . .~ J ' U~' ' ~IU'I ~,J~ 1 't ' '' I '_' t-J

_14 20~9122 .rhioh ~ ~mpl~ w~ in~r~uoa~l o~o tho aolu~n.
_: ~ m~buli~ thht L~ no~ p~
~r~ry ~a~c~b~ n~twork o~ th~ c~ cond~y ~aht~oll'e~ ~r~ u~ua~ rQ~nt u~ly in c~ t ~p~al~e ~n ~om~ w~y (~.~. d~n~
t any ~t~ln~ th~t c~ul~ b~ u~ to ~ulture ~ell~ on a ro~ry ~AX~ O.g. ~rl~ 0y~r r1a~
~r5[lDs~: ~'orcln~ ~bubblin~) alr throuyh ~ gu1d m~dlu~, :~: cor~dll:lon o~' c6~11 aul~ S~owth wher~ln dou~l~ng ~m~ n~ ~h~ ~ul~l~r~ ~V~ r~aahad an Q~aU~ librium und~ a de~ln~ t o~ cull:ure ~on~tlon~ .
5u~ cultura ~r1~ ro~ lo o~ c~ r~
a par~nt oultur~.
~ n a~ll aul~ur~ c~o~ltlon ~rlY~d ~rom a t 5 ~ampl~ ~2' cultur~d cell~ 1 a~m~ b~lng tr~ rr~d eo a dlfr~rent tl~u~ cultur~ v~n~l ~han thl~ aont~lnin~
~b-. aulture o~ o~llA ~rom ~hloh th~ o~ lnea.
Tl~uo c~l~r~: compn~lon o~ l~vln~ u~ ~lnt~ln~d ~ Y~er~, th~ t~ o~ll cUltu~ nd ~s~u~ cultur~ ar~
o~tan u~ad ~n~*r~h~ngo~bl5~ y ulll ~ hor~ln ~o r~er t~ ~ny a~o~ition wh~r~ ~ pl~r~llty o~ r~
~ln~ain~l In vltro.

~R~F n~A~
'~ oth~ o~i~ct~ Imd ~dv~n~ n o~ tha ~nv~ntlon wll~
~5 b~ne ~ r~nt upon raa~n~ th~ ~ollowln~ dot~tl~d d~crl~tlon ~nd u~on re~r~n~ to ~h~ ~a~ ln wh~t:h:

FIG. 1. 8t~m ~ an~ ~hc~ln~ callu~ ~orm~lt~c~n A~t~r 3 w61ok~ in cu1turs~.

FIC. a. Coll ~u~*~ on~ rat1ng pr6l~r~ yellow 3~ c~ ko t2~ le~t, ~n~ r~d c~ whioh ar3 to b~ ~voided, to th~ rigl~.
F~G. 3. ~ad br~wn o~llu~ ~re ~ ~ at th~ top, pr~ d y~llow cal1u- at thr, bc~ttor~.

:

. . . .
,~

V U 7 ._ V_. .J _ ~
L ~ - J ~ J ~ r ~ Lhnu~ ,n~An~ ~ l D,Y~4 1~

~069122 -- 15 ~

FI~. 4, A ~ul~ure o~ oallu~ lin~ C~1 b~ brlln~ r~rt~;
two raPte ~r~ ~hown, ons ~ 4 w~ek growth ~orl~
t) an~ th~ oth~r ~t~ ~ 7 w~ rowt2~ ~srlod fri~ht), E~ah or ~h~ rn~ W~ lnl~lallY ~ rith 3 ~ 0 oallu~.

PIG. 5. ~n vltro ~lcrotubule ~nbly and ~t~hillzin~
~atl~lty ~!leDIonatrntDd ~y t~x~ ol~od ~rom ooll li-~o CR~ ur~d by dar)t-~lel~ mloro~eopy (to~ ~ot T~Q cont~ol t~ot~om ~ ~tur~ y~ ln ~Danner wit~out ~he ad~ltion o~ taxol, `:
FI~. 6 . ~E>I,C ~ro~ f coll ~xtr~ how~ng sc~l~
~ak~ at 18 ~nd a5 ~lnu~ 81~ltlon. 1~ pro~ile to th~
le~t l~how~ au~hon~lc taxol, ~2~ mann~n~ nd ~Aa~at~n, ~h~ pro~le to thQ 5~1ght ~l~o lncl~ r~ultli o~ ~n ~xtrElct o~ &~11 lin~ c~-~. PG~X~ 4 and 5, at 1~ ~nd a~
minut~ ~lu~:lon, ~rer~ po~lt~v~ ~e ~i~rotubul~-~t~billslnq ~q~y (~o ~l~t~rl~ nd r~

~2TA~D D~CR~ 0~1 Ualng thc ~n~th~ o~ t~e p~ nt ~n~r~ntlon, succc~
~û in pro~uolnq st~5bl~, long-t~ n~l8 ha~ bo~n aahl~v~. The ~ollowlny ~c~inati~n~ o~ st~p~ ~re n,~
~o~ ~reA~lon o~ xU~ C;Gll lin~s.
Tll~ cal~ Rnd ~oll 6u~lonq o~ th4 pr~nt ~ nvont I on arQ indua~d ~ro~ th~ a ~p~cle3 lnolu~inq 2~ T. br6v~011~, T. b~cc~t~ pAnd~n~ p~d~t~
~dl~ cY. D~n~i~P~i0, ~nd ~ dl~ ov. I~io~
Young ~t~ xplant~ ~r~ pr~ red a~ a tlu~u~ ~urc~
to lnduc~ callu3. Youn~ ~t~ ro thc~ ~h~t ~pp~Ar n~v qro1lrthe on a ~ pl~t th~ ~t~ gen~rGlly ~r~
a~out 0.~-o.~ d~a~ r. Youn~ Or ~auNJ~ 4lfl av. Illok~ o p~e~errc~ b~u~4 ~cn~ ,t~ ound to p~cdua~ hlgher lc~vol~- o~ tax~ n ~th~r ~axu3 o~ole~
lnoludlng th~a ~ts~ oP ~ r~vi~ol~ Al~hough ot~er plant p~rto ha~ h~ l$ty ~o produc~ callu~ youn~
~5 13tQ1tl~ ro~ id~d t~l¢ b~t ~ ltil on A ~or~ nt ~ ia `:

J ~ 1 1 , .J . ., ~ o ~ o 2~6~122 ~ ung ~o~ ~xpl~nts Pro~ x~oc~ ~ca~ çad and ~actlor~d und@r ~uo~tlo aondlt~ on~ atlon~ ~r~s gxo~n on c~lluai-lndu~in,~ dlum. 6ult~hl~ ~dlu~ inc~lu~
O~nb~rg l ~ B~ ~dt UTP ~Gam~r~ ~t ~ 8) ~l~a~ls ~
fo~ti~i~d witl~ ~mbor~ 5 Ylt~mln~ cDR~ln hyd~ly~te (1.0 ~Jl), And a ~ain~ln~t~on or ~exuc~o~a~, g1U~3B~ and t~uc~roe~ ~ot~lllng ~s-40 ~ c)r ~ala~ oe~ n~, hlgh concs~rltratlon~ of gluao~e~ l~rl~ot~ 3t~ or ~uaro~ 40-~0 i~ pr~ rQ~ to th~ ~tarldl~rd 20-30 ~/1 oP ~uoro~ao ir~
nu~r~n~ a~ ur~rl~ingl~, yl~co6a ~nd/or ~ru~q~
~I~!Iy ~pl~!lGU I~UCrO8e. PlAI~t h~on~ r~ o ~uppl~n~ ths m~dlum a4 pl~nt growt~ r~sgulDto~
;; ~allu~ lncluot~on. A-larin~ y bo us~d ln oomblnAl:10r~ h 1~ kin~tln, th~ latter ~ A r~ngR o U.1-1. 0 ~g/1. In dQtormlning ratios of ho~morles t~ U~, a hlg~ ~uxlz~:
~ytDklnin r~tio ~ r~fe~red l~o~ a~llu~ lndu~tlo~, Ps~r ~xa~31~, 1 mg~l ~, 4-D to o. 2 r~g~l kls~tln~ 3 ~Jl ~P
n~p~t~al~n~ ~catic~ Mc1d (N~) ~o 1 ms klnotln. With ~eg~ l to Auxlns~ ~ t ~ r~n~ .0~ m~ 2,4-D
~t a ran~ o~ o~l-a~ gJl) o~ IBA (ind~lo bu'c7ri~ AalCl~
a'c ~ re~nS~ of l.~-~.Q ~q/l) a3~ ~uit4bl~ an illu~tratl~r~ bodlmenk, 1~ 2,4-~ an~ 0.~ p~m 3~ ti~
~o ~d~d to th- culture~ ne or ~n co~ination wlth ~5 Gol~ito or qg~r a~ ~elllng agQnta.
~x~lant~ ar~ plao~ ln tl8~u~ ~ul~ura v~e~, ~an~r~lly p~trl dlsh~ o~nt~lnin~ d1u~ lo~ w~h B~lr~t~, at ~bou~ ~ 2 q~l oono~ntr~tlon. Prlor to k~in~
~l-pen~d ~n~co ~terll~ pe~rl dld~4ll, th~ :nQdiu~ ~:oy l:~o ~t~rlllzQd, ~or ~cam~ uto~l~vQd at ~a~ o~ about lS
~' nut~
Explant~ are ~n~ub~t~d ~ s~ c~o~apl~t~ d~rlc ~t t~p~rAtur~ o~ 2bout 23-25 ~ hcll p~l o~ ~he~ ~Qa.it~
pr~ ly ~alnt~ln~d ln ~ r~n~o o~ , 40l~
3~ cultura~3 ~ay pra~er to qrow ln tha ll~htt ~t ox~pl~l, ~o~ hle~ rl~l~ra~con,t li~h~ ~or 1~ hours ln ~ a4 hour ayc~lQ, Indu~Q~ c~llu~ ~B ~ g~ ell ~ul~ 1~ lnltl~lly VQry ;, . ~ .. .

': ~ ' ~ ', " ' ',: .

~ 17 ~ 2l~ 691 22 BlOW grr~w~n~ ~n~ prodUoBO ~hsholln QXUdltOO ~OhU01nS~ th~
ca11~o to t:urn s!~fl) whl~h ~r~ dLItrln~Q~n~al to th~ growth 0~ th~ c~l~U~, avsn wn~n ~a ~llU~ lo r~ontly ~u~ul~ur~d. A~ d~arlb~d in m~ro d~t~ h~ t~r~al~
3 s.nd ~l~thod6- ~aa~lon h~1n~ ~b~l~n~4 ~y b~ t~d ~
th~ ~uboultur~ o~ ~ndu~ cnllu~ 5 on ~h~ c:al ~ crlt~ lnoludlnsa comp~r~tiv~a ~rowth ra~, s~s6~n~0 o~
r~ ~olQrlng, ~rQ~enc~ o~ yellow col~in~, and Pri~hlli~y FIG. ~ I'or c~ u~n~on ~olo~ crlt~xl~; and P'IG.
3 f~r c~llu~ ~olor ~rit~r~
8u~aultureç! s~ d ~r t~ t~ di~lo~d h~r~$~l, ~r~ tran~4~r~ to lol~d 8u~0rt~ erzlbly msr~r~m~
r~ts (~G. 4~ (Si~ h~mic~l ~4.) ~n ~edia ~Uc~
ohowrl in ~I!able I~ ~r~n~ r~f~ w~r~ prsf~rr-~d Par tha 1~ aultu~ o~ c~llu~3 b~cr~u~ thi~ syst~ lar to a ~mam~an~-flow`' typ~ ~or~a~ox. ~y u~ o~ A r~t, ~
aultur~ diu~ han~d ~td ~an~ lat~d ~lly, and t~UB
a~n aao~l~ b~ ad4~t~d to l~rg~ ~c~l~ oultu~a, Oth1~r r~ on~ ~or u~lng~ raftc arD th~t callu~ t~nd to ~row ao ~tt~r on ~ne~r~ne r~t~ ~han on agur o~ oth~r 0~11ing ag~nt~, ~n~ al~a lC 1~ r to h~r~t callu~ both ~or ~naly~ an~ ~ubculturln~y purpo~e~.
Taxol, ~tiY~ d~iv-~tlv~ o~ e~xol, c~phal~nnln~, l~a~tln and oth~r pl:o~ur~ora A~d l~to~dt~ t~xol 23 ~ x~ od u~lng t~~ Arlllllysll~ by ~o~p~rln~ ~e r~ on tlm~¢ o~ ~uth~ntlc ~nd~xd~ ~n~ plant ~x~ra~t~.
Thls An~ly~ acco~l~ ~h2~1 by ~raat$ng cnllul-, ~r~ nCI ~IPT,C an~ly~ n tl~ ~xtract~, And ooll~ctin~
40m~n~ntl- of mA~or ~ak~ nl~ ~o~pon~n~ of ~ AIU~
ar~ t4~t~d r~r ~otivity ln m~crotubul~ ~t~bll~21ng ~ ye (~XG. B, ~atori~llo ~nd ~h~dB) ~and in ~n Y~tro u~na~2 ~odsl ~y~ter~ d ~ ~o1De~ he~ n . c~nCer t: Q
su~t~ or ~ar~enlr~ lncludl0 Bl~ M~l~no~na; MX-l ~nia~
x~nogra~t; P3R8 l~uk~ml~; K~7 I,12~0 lou1c3m~.
Tslx~1 D¢tiYlty l~ o aon~ n a~11 lln~ ~y ~o~lng ~a'c ~X~:r~c~ hav- tho ~bilaty tD ~t~blll2 miar~tu~ul~ ~tlY~ty in ~h~ ~m~ r a~ t~xol ~P~a. ~.
Anot~r m~ns o~ idon'c~ t1on o~ t~ acc~mpli~ d ~ ~ U '~ ' J ~ ' U ~ ~./ U -t '_/(- _. U . . ~ t . . '! ~ (j ---'' ' -- ' ' '----'`' "`--'`''`' '` ' `' ~ '~L ~ rlll I V~C I ~ ~ vl~L~ ~vr~hn~

~69~22 . by ex~r~c~tln~ O~llutl ~1311 lin~ pur~yln~ thl~ ~xtrrt~t by HP~ And ~d~nt$t~n~ t~xol b~,~ util~n~ H~ ~nd a-~ high ~olutl~n nu~le~r m~s~n0tie ~ort~noD (~) ~na~y~i~
(P~lZor~ 1., 1992) ~lr~re~t bloreac~or d~lgno ~ av~ to ~axi~lz~
gro~rtb r~t~8, ~alx~l, aC~1Yd~ d~rlvatlva~ of ~a~ol, ~eph~lom~nnine, bacc~tin ~nd o~ pr~cur~or~ or ~f~xol mad~ by ~ah o~ th~ o~ll lin~ nd o~or~t~d ~nto aolu~os~
(W~tb~r~ ~nd G11~ 48 J P~ t al., lg~7) . Pl~nt ~0 C~ mo~ z~d in Y8~1~US gul~ Ana ~mbran6l r~n~
ha~o ~n U. ~d in bl~r~to~ IBrode~11u~, 19~5~ .
Alg~n~t~, ~gar, aqaro~4, kh~p~ o~rr~ n~n, ~olyacrylA~id~, polyllr~t~Ano And vAriou~ ran~
~on~i~rat~on~ haLv~ ~B~n us~d to ~antra~ ~lla t~rad~u~, 1~ lg~a ~ ~m~d6rod and sk~k~r~ l9gO) ~ By o~in~iglng Qn~lr~ nt~l b~d1~ on~ ~unh a~ v~
co~po~ on~/dln~ a~ ~xyg~n, tom~r~tur~, pl~nt 2~ox~onoa, ~H of th~ ~r~wth ~dit~, n~trl~nt~, vl~amin~ an~
r~moval o~ t~x~l fro~ ~h~ dlia, d~ir~d com~ur O oxtr~:tion ~ilay be n~xlmi2~d .
~hn 1~VQ1~ 0~ t~xol ~ound ln tn~ C~Q11 lin~ o~ th~
pr~nt inv~ntion ara ~ a~t ~ to 2 ~o~d ht~har thæ.n in ~t~ ollV~5 and b~rk og ~axu~ modl3 ~. ~lck~ Ta~ls I) . ~ha~a l~v~ nay b~ lncr~æed ~th~r~ In ~n~ar~l ~5 s~ varl~ty of tis~u~ ~ulture~ h~ bn~n ~Ihown th~t c~ltur~o p~nduclng low or ~det~ot~blo lsY~l~ o~ on~
~rodu~t6 Are ~l~vata~ varl-ty b~ ~an~ d~ng ~p~ tl~n o~ culturo ~on~t~onc-, pr~r~or ~a~ln~
of biotlc ~H~ln~ln, lg85t ~F~Icul et al., 199~), an~
~biotlc slioito~ lnclud~nq ~)~e l~o ~ ultr~-v~ olot lrra~lo.ticn ~X~tu~ch ~n~ tl~n~orf~r, 19~0) ~nd h~
~etal lons (~h~ All ~nd ~hi~ah~ , 19~ l~ nddltlon or ~r~our~on3 'CO ~1~11 cultu~ OI ~pt~g~um hr~lîor~
h~ beor~ r~3~or~d to ln~A~ pro~uation o~ th~
~n~it~nor co~p~und tri~iollda IMlslawa~ oe ~ 19~5).
Proaursor reedlng~ bio~ici ~n~ tlc 811ai~or~ a~ ~ofin~ld h~r~ln, Rre sul~b1~ ~or Ul~e with !~tt2n2~ cultl~r~.
The lndu~tion o~ 2lut~tlon~ in oall 1~ by .

' ' ~ ,` ' - . ~ ~ . ' . ' . .~!
.~.

~ ' '' .

I~ \ U ~ J ~ ) U~ J t '.~0_. IJ._ I ~ . I tO
' n~ ; v ~ r ~ J L ~ L~ '~ UI~ r w~ 14 U S 413 9 ~ 3 ~0~912~
_ ~9 _ lrradl~tlon ~nd ch~ hl ~llt~g~n~ ro~ult ~n a~ e~
wh~ v~r~rodU~ e~xol, ~lV~ tv~t~vo~ ~ t~ol, c~phalom~nnln~ b~ao~tin an~ ot~L~r pr~u~ r~ 0~ tAX~
ln ~d~itior~ cttic~ ~nd ~bl~tlu ~ trra, ~e30di~g Or S ~ uraorf~, ~nd o'chsr cl ~ citE~tlc~n tochnlq~ ln ah~k~
cultur~ and larg4 ~cale ~lor~Aotoa~ c~upl~d ~ith lar~
wala ~uri~lc:h~lon ~ay $n~r~a~ rodllct yl~ld.
~lant~ ~ay ~ n~ra~ Prom ~ u~ure~ to b~
yet P~noth~ ~oUrG~ o~ 'c4xo~ a~nd, ~or~ in~v~r~lngly, perh~ o~ ~h~ naw ~und~ id~n~ d ~n ~ltur~ t}~at have t~xol-like 3~t~vity.

Tne 2~ t.r1n~ ar~ x~tlv~ ~x~mplfa~ o~ ~ p~ent ln~an~lon.

p~:avAration ~1~ A ~Lll~i 0~1~ I.in~ m ~xu~
~eW15,t ap~enring, uc~oul~3n~ lcsy) ~tMI~ grow~h Pr~
T~1~8 2~l~u'1a ~v. ~ k~ll w~ harvQ~t~ rQaa loo~t~
~n ~rhe P~nnsylvan~ 6t~to un~r~$ty, ~l~ ity Park C~mpu3 ln th~ ~r~ng and ~ ly ~ro~ d. Tn~
n~dl~ wcr~ r~move~ 'chQ ~t~ na tho ~tosn~ a-lt ~n~o 3 C5~ ~ctlon~. Th~ youn~ ~uc~ul~nt ~ts~ tl~e~u~ w~o cte~ b~c~u~ ~t conta~n~ th~ hl~h~t 1~VQ1~ 0~ taxol ln ~ p~ 8 ~ o~CI. 'rh~ o~lantn w~r4 eur~ao~
~t~rlll2~ fo~ ~0 ~eaon~ nnl an~ ln~edlht~ly pl~ced ln lot ~ ac~ ~olut~on (~lv) plu~ n ~o for 20 ~nuto~, rin~d 3X wlt~ ~taril~ ~At~r and ~otlon~d un~r n~ptlc ~ndl'clon4. O~ar 0~n~A~rCI ~ur~a~ ~t~rl~ lon proa~flur~ ~re alllo ~ultnl)l~ aoo ~t~rili~t ~D ~te~ wer~ cut ~nto sl~atlqrl3 undar ~p~l~ conditio~e ~nd pl~d on GAmborg~ ~5 m~ lu~ an~ ~lnu~ 2,4~, Xinetin or ~ plu~ Xlnt~eln ln th~ a~ ~rk ~t ~3'a.
Un~iff~r~n~iA~ed o~llu~ e s~ ln~tlally ~lo,r gr~ng, how~v~dr~ thr~h lnany ~u~culture~ a ~r~a~le y~llowish 3~ a~lor~ ol~ill lln~- d~ n~t~ ~1 with ~t~lbl~ slrow'ch cha~a~tari~uu w~3 obtaln~ hl4 c~ r~w- on ¢a~bor~'a B5 ~dia (~blo ~:E) ~n tl~o dar~ At a~Dc wlth~ut v v r ,~l ~ VL C l DL~ c r~ r 7~ J 14 ~ '~4 1 ~ ~ v - ~ 4 ~.
2069~ 22 ~ 20 --.:
~h~ ~upply ci~ ~ny ~xo~nou~ l~ox~nonoo .
- q!ha c~ nA Cl ~ int~lin~ ~n ~adlA æu~ nt~d wlth ~eln h~droly~nt~ oo;~ ~nd ~rUctoD~, wh~
~ound to Dlg~ icant~y ~n~r~ o ~* g~ow~h r~ o~
s c~ol~ lin~ b~L~ IY~ ~ .
~r q~;ll lln~ we~ rl~ r~ h~ c~llu~
aoll llne Gl w~ ~ubclos~ n modl~ g's ~5 ~dl~
~bown in T~ h~ oallu~ o~ll lina os~ theh t~an~f~rr~d to mentbr~n~ ~a~t~ . 43 cont~intng li~ld ~dl~ ~hown ln T~:Le II at 25'C ln th~ d~rk. ~hi~
l~no W~ ubolon~d ~nd 1l ~11 llne Witll ~t~ble gr~w~
c)~Rr~t~ri~tlca w~ d~ rod (cR13 ~o ~x~1~ 2) .

12X~E.~ ~
9~_ ~
pr~ In~r~2n A ~allu~-d~riv~d o~ll lin~ d~ k~d CR-l, d~ri~d ~om ~ oallu~ .gnate~ ~1/hss ~ho ~ollowlng ~hJIr4~eeria~io~l gro~ ir a~l~Ure~ ,n ~h~ nutxl~n~
ao m~ is ~nl~r~ 5 ~e~dium~ q~o~ in th~ X ~t ~0-26'C) do~ n~t r~lr~ æ~ nou~ pl~nt ho~non~ a h~b~t~at~ lin~); RhDw2l ln~rat~ oo~ d t~
lt~ bq~Qlin~ ~y 3ddltion t~ um o~ c~ln hydroly~t~, glu~e and ~mCto~ to th~ Alu~ e Ta~l~ IIl);
26 ssthi~lt~ ~ d~u~lln~ r~om ~ 1~ d~ysl ~nd ~ w~
~t~ row~h in ~ul~u~ a~ll lln~ wa~ OApi~ a of - ~ar~t~n~ taxol, ~ctlv~ ~ar~ tiv~ o~ taxol, ~h~lo~arm~n~, bacc~tin ~nd othsr preaur~or~ <~f t~:~cl into th~a growth m~
~n~ c~ sriYed 0411 11~ dQ~ nAt~d ~Rl p~wdu~od r~pid ~rowth o~ c~llU~ aft~r ~uboulturlng: ~h~ ~h~
c~llU~ uHe~Qâ on a r~mbrnno ~t ~pro~uc~ 50-70 gr~ o~
ca~ a wl~h~n 7-lo w~oX~ o ~Gulturln~, ~u~ thD
add~tion or ~ro-h melllum. tF~ 4) 3~
" ~_~

: . .
.," : :;

- . : . .. .
. - - , ~ : , .::
..

V~ n~r~ 4 ~ ~r~ l ~ y v l u ~

2~$9122 ~.

rwO ~uo~ ion cul~ur~ l~ne~ w~jr~ ~loo ~dtl~d ~y ~:n- m~hs~ oi? ~ ~r~ n~ ln~Qnt~on ~ro~ llu~
llno ~:1. A ~uap~n~lon ~u~ n~ ~3 w~ d~riv~d ~i~h s~abl~ gr~ ohz~r~c~eri~t~c~ W~n ~oW3~ at as~c ln [email protected] wltn modifi~ a~mborg~ ~5 ~la ohown in ~ lo XY.
suopen~on oQll l~n~ ~rodu~ in t~ mann~ w~ra typically ~low ~r~w~nSI, now~v~r, ~ou~h ~u~olor~ln~
lQ c:~ll llne ~3 wa~ a~t~in~d And h~4 ~ 3~od~ra~ ~r~ r~t~.
~hin ~ell l~no ~ odu~o taxol, ~t~ ~r~u~r~ ~nd ~otlv~ derlvat~rQ~ o~ ol 4nd ~ocr~t~ th~ h~la~l~
nto tho sol~ orl and aan ~A~i1Y b~ r~ov~d ~ro~ ~S~
~luti~n U~1 nq ~x~8,n~ r~ln~ .
Anotho~ ~u~p~n~lt)n ~ult~ no d~gn~
W~9 o~taino~ ln a ~i~R W~'y ~ro~ CRl oall~ lin6~ rown on m~mbrAno ra~t~. A tin~ o~ 3u~n~1On ~ oun~ ~n th~ nu~rl~nt ~olutlon in which t~ ca~lu~ ~Rl Whf!l gr~w~-g on mQr~r~n~ r~rt~ . Th~ ~u~ au~.~U~
od~ced u6ing ~tho~ o~ tb~ ~r~ht invant~on wherg~ln o~lls sl~ughQd o~f a211u~ c~ n~ C~l lh~o ~u~ n ln th~ nutr~nt ~n~d~, w~r~ qrown ~n 1 ~ slhh~r i~
with a~ltat~on in th~ d~rX ~ ~bout ~4-a~'c ln C~m~or~
~ ~edium ~ 4hown ln ~bl~ ~v. ~h~ ~u~pan~ion oul~ure wa~; do~ign~t~d SR-l. eu~n~n oultur~ ~R~l w~ ~Ap~bls ~t ~acrRtlng taxDl, ~ctlve d~rlva'ci~ o~ t~xol, acphalom~nnls~o, b~a~tin ~n~ r proo~roor~ o~ ~Xt~l lnto ~ ~rowth ~d~.

A ~a~pl~ o~ ~ell~ ~rom call lln~ Gl (EX~L~ hle~
g~ow~ ~n th~ dllrk W/~18 ~ ~rad tc~ ~ llq~t aycle o~ la h~ur~ ht, 12 h~r~ do.rX, ~t ~'c. Tr~r~H~-~ to tho llght ln~ tla~ly c~us~d ~ ~lo~ ~o~n in g~owth ~t~
b~own dl~ao~or~tlo", ~o ~v~ld a~ ath ~hl~ cllllu~ lin~
plaa~d on ~ov~r~l hormon~ r~ eont~ln~ng ~,4-D ~nd ; ~ ~
.

, "
,.", ~ t~yL~ t ~ J ~ I () V ~ ~ IJ~J ~ J.~

2~122 - 2a klnQt~n. I`hl3sc- aoll l:Ln~ cl~ha~ and ~ ahllu~
c~ 1n~ des1qnat~d L~3 which g~ow~ on Ga~b~rg'a ~ ~edi~
w~ ~el~q~ ITI) ~n ~ ltgl~ 3 r~ul nou~ plAnt hor~onQs (1 ~m ~,4-D ~nd o.a ~p~ kinu~1n) in ord~r to ~rosr. ~hl~ ~allu~ lln6~ h~ P~on RcC~ t~d to ~no~bx~r.o rn~ nd ~u~n~ion oul~ h~v~ b~on initi~ted. LG3 wa~ ~ubault~a on di~r~nt hor~
~gl~ con~tin~ v~iou~ bln~tlom3 9~' 2~i~0~ntyla~nin~ and in~ ~bu~yric ~ 0.l, ~ .o ~
0 5.0 pp~ h~ ~11 lin~ p~blo o~ ~oer~ting taxol, V~ ~erlvatlv~3 o5~ taxol, c~l~h~l~mann~ne, ~ c~1n ~nd oth~r p~c~lr~o~s of t~xol lnto *ho ~ lu~, ~h~ c~llu~ coll lin~ L~3 w~ ~CUl~ d on ~o dl~ferer~t ho~#Qn~ r~gim~ ytokinl~s~ and ~uxlno lr~ the ~i~ht at ~s ~. ~y u~lng ~h~ thod ~h~t ~o~atlQn w~
irlducsd. rhl~ ~ohn~qu~ w111 b~ lnv~luabla ln r~Stsneration o~ pl~n~ w~th ~up~lar Itaxol proau~ln~
oapa~ ti~3 ~nd Afl. ~ 1/ oP ~ atin~ taxol l~v~ n ~l~nt~ ~rown und~r A~ptlo cond~tlon~ S~ a 2~ ou~pon~ion cu~eu~e d~3rSv~A ~ro~

~La2~ 5 xol, oo~halo~Q~nnlna ~n~ ~aoc~tlr, ~ra ld~nt1~1~d ~5 in ea~h ~ axw w~dl~ ~v. ~l~k~ t CP.l, ~3, ~Rl, And hG3 u~ln~ HPLC ~rlaly~la ~ t~lri~ d Mat~oda). 'rhi6 wa~ ac¢o~p~h~d by making~ ~om~arls~ o~
~tent~on ~m~s b~tw~n ~lAnt n~ ~u~h~nt~ o ~t~n~ard~. ~Axol ~c~-.rity~ on~ d ~y o~o~1n~
th~t oxtrac~ v~ llty t4 ~tabllls~ ~iarotubul~s :~ ln th~ 0~4 ~annor ~a ~ ntl~ taxol ~FIC. ~). rh~ thlr~
l~ne Or ~Yidsnae t~a~ tAxo~ on~Aln~ in ~ o~ll wa~ 1~y puri~yln~ ~xol on NP~ ~nd lden~leyln~ tAxol ~1~ purl~l~d ~Ampl~ b~ nucl~r mA~nnt~ o ra~nan~
3S ~n~ly~Lo. 4erlYa~v~ o t-~xol hnd ~ddit1~n~1 pr~ r ~rs alBO ~nt~ied by ~PI,C ~n ~ach 1~ c~ll lin~. ThiE~
wa~ aoaampllohed hy ex~ract1ng ~ o~ ~h~ a~ n~, 2~9.~22 a3 -runnln~ ~e ~xtrac~e on ~ 1~, e411~ctlng ~or ~Q~k~ ~nd t~ting the~ aa~ ln ln vitro ~l~o~llbul~ ~tA~ in~
a~ya~ a~rlal~ and M~thod~).
Th~ lo~ o~ tax~l pr~du~ltd ln un~lial~d ~ul~ur~l-S o~ m~dlfa av. ~lck~1 C~ll lin~ CStl, ~3, ~, nn~
~3 Taxol l~v018 w~re ~-3 t~Q0~ h~ U~t~r ~h~n t~ la Po~nd ln ~x.t~ x ~r~ yoUr~ ~te~ ,o ~ sr~lnod by ~P~C
an~ly~1~. Ev~n ~nor~ intG~r~t~ng wer~ th~ IIPLC pro~ 3 roYeal~ng ~reiv~ou~ly uni~nl~i~ a~ with 'c~ ke ~ct~vi~:y, and ~ro~ dl~r'~renGo~ ~ong o~ n~. For 6~xa~bpl~, dif~rQn~a in ~ N~C ~hromta~r~ pro~ o~
oRll li~ SF~ nd 9-3 ~ ~aren~ wh~n ~cm~r~a wlth thel ~IPI,C pro~ Or c:~ll llne~ CR~ hown ln ~. C, und~x condl~ion~ AID d~in~d fDr ~IC. 6. P~k 4, ~
whl~ t~ taxol-lilco actl~ y sn~ wh~n 1~ ~ra~nt ~n C~ dl3: lni~h~d o~ ~,b~t ln 3-3 . P~c 5 1 p~e0~n~ in CR-1 but dl~lnluh~a in ~-1. Inter~tingly, ~lth~x p~kB ~ nor S ~e~unt!l ~n C~-l w3~ d~t~C~d ~n dlrQc~
~xt~a~to 6~ d~ lok~ undar th~ ot)n~ on~ ~od ~o d~ t th~ ~3~ o~ o~llu~ ~d ~ul~ur~ XtPaclt~.
It ~ tpest~d tllat oths~r pr~lt ~ wlll r~ult i~
dl~r~nt ~row~h p~raD~et~r~ o~ aul~ arl~ uGad ~r d$rrer~nt HPLC m~thO~!IB ~ ~11wa. The ~th~
illu3tr~t~d ~or CR'~, ln FIG. 6 ~r~ de~cxlb~d 3~ex~sln.
O~h~ mothod~ ed ~n hydroph~bialty or o~h~
~l~tlr,oul~hin~ m~ d~ for ring~contQ~nlnq ~tru~ture~ ~r~
~Ult~

~ho BU~pen~lon cult~r~ l~no~ ~3 ~n~ 61~1 ~r~ grown ~n lL ~la~Xs ln Ga~obor~ ' o ~B ~e~a 1~ V), ~lot~ c~
uto~ Yud tunçl~l G~ll wall ~a~p~nl~ nd 13p~X~4) ~nd ~bl~lc ~ltls 02 h~ n~t~ nd ~5 m~roury) ~llcitatlon ~r~ ~ult~bl~ llcl~c,r~. Doth a~l 11na~ a1Loltation ~ may ~ult ln ~1Dtt~t~d 1~Y~1~ c t~xol, 1~ prQ~ or~ ~nd t~xo1 dloriv~lv0~.

, , ...... -- . .. .. _ ., v ~ v ~ V ~ v l .7 '`: ~ ' J I S v ~/ 4 ~ O
2~69~22 D~L~ 7 Indua~on ,~ tu~
a~ ~ald In or~e~ ~o lnd~uoo mut8~tl~na whioh ~oducs high 1~
o~ t~xol, 1~ oo~xn~r~ an~ ao~lv~ tivo~ o~ t~xol 1n ~ l linQ~ CR1, s~, ~Rl an.d ~:3 Ar~ o~d ~o UV
~xr~dlRt10n ~r~a~nt ~nd ~a~1 mu~qa~n# 5uo~ ao ~S.
Bsl~ction ar~ t~rlA and ~e~o~ o2 ~uleur1ny tax~l~
l?ro~ucin~ c~ll line~ cordlng to ~o~e ~ o~l7 Wlf.
hQ~in. In th~ shlon, ~.Ut~t~Oh~ hAt ~sor~ h~
outpUl~ ~ the ~xol~p~hâuclhg ~ , m~y b~ t~d.

~e~. ~n A n~vQl ~D~thod o~ c~crag~ pr~rred ~or ~ua cu~ r~-~
1~ ~a~ tc~ r~fluw lnot~at~on ~ p~rAturo ~o 1~-15'C!. ~:row~:~
Wl~ ~low~ to a hlb~rnat~on-l~k~ ~t~to. To re~urr, c~all~
tO norm~ t~mpQr~tursl WA~ ~lowly ral~-d to tho opti~ ~or g~o~th.
llu~ a~ n~ CRl ~nd ~G3 arc m~int~ln~ on ~ r~n~ ~ar~6 plac~d ~nsid~ NAg~n~a ~A-7 v~sssl~, while shaX~r aultus~a o~ ~3 ~nd S~ ar~ Aint~ln~d ~n lL ~ha~r cul~ur~ k~ and Bu~cu~ 3d ~ery 2 W~
Cry~r~rV~lon ~or lons! ta~ ht~r~g~ o~ ~11 cult~e~
CRl, ~:3 ~ nd ~ urlng the ~ ?h~ of ~r~ Wa~
a~com~llshod by ~lng ~t~nda~d ~yopro~0r~a~ion ~thod~
U8 ~ ng ~orbitol ~nd D~80 ~ th& ~ry~prot~t~nt Cr~opr~ v~tlbt~ wa0 ~ho~n ~o hav~ ~r~ t~ntia~ ~o~
lon~ tor~ pros-~rV~ Qo of coll D~llt~rss Yho~ b~o~yn~h~tlc cP~olty tor 3ynthQ~i~ an~ ~ooumu~ n of ~ond~
mot~bolit~ romn~2~ inthct ~o~lowing r~l~o~ r~m ~stc~r80e, 1'h~ growth o~ ~UB~n~lOn C:9~ 1 lin~ ~1 in ~irllt, 35 ~t~rr~e an9 p~rIusion bl~r~ot~r6 ~ult~ ln ~xol ~roduot$~n, Alt~rnat~Y~l~, naw r~4C~3r~s mAy 1~ d~sLqn~.

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- , .

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~ ' ~Y~.: I ~Y~ V ~ I . ~ 1 . ' J _ I V_ ~ V J V V J~ <.) ~0~91 22 ~hQ p2:a~0r ~d~ t~n~ o~ ~nt~lr~n~ont4~1 condltlc~ s~oh ~
~a~ po~ltlon, ~l~eolv~ o~s~n, rat~ par~inq ~nd a~ee~ gita~lon, t~m~er~tllr~ o~ tho grbWt~ ~dlu~, ~utri-nt~ o~7an~c Elup~l o~nt~, r~ov~1 o~ orld~ry ~ro~ua~ ~rom th~ diu~ ~rbugh ~h~ UB~ of rQ~ln~ ~nà
~licltation ~r~ r~ul~ t~ ~ptlmiaQ t:he ~y~t~m0.
~h~ growth ~ the c~llu~ lln~ uit~blo ln a "mem~r~n~ flo~ lor~ o~. ~hi~ ~y~to~ h~a nlltriQr~t ~olu~lon rlowln~ o~r tn~ br~ ~hi~h 6~p~rt;~ tho c~llu~ ~nd 1~ ~P~x~ulated. Nutri~ntf~ aan b6~ d. ni-ne~dsd ~y ~eR~u~in~ conductl~fity nnd th8 pr~genc~
t~xol, ~axol der~ tiv~ an~ pr~aur~or0 ln thQ meA~u~ ~an ba a~ y~d b~ ~a~pl~n~ ~ra~ ~ho 301~tl~ In llna. ~hl~
~t~ Up By~t9m 18 analogou~ t~ thR g~o~rth o~ thR oallu~
~5 cnll 11n¢ CRl on m~ r~n~p x~.~t~. '~$8 ~yG~ an alç~o b-~ 12~ ~y hav~ng th~ nut~l~nt ~ ray~a o~to th~
o~llu4 Int~r~ltt~ntly th~ou~ a ~yat~ oon~$.t~r~g ~ ~
n~ , a p~rls~Altic ~u~p ~nd ~ r, thor~by ~u~plying ~h~ nut~i~n~ ~o~ unl~Dr~ly ~o th~ en~ nllu~ nd 2~ o by lncrua~ing nutr~nk u~ak~, ef~ial~ncy, ~n~
mlnl~nls5,ng nutrl4nt U8~-~lle th~ in~t~ntlon i~ ~uu~;~p~ to v~iou~
r~odi~14ati3~s ~ alt~ tl~ ~orm~ o~lc ~dim~n~
thor~ v~ boan hown ~y w~y o~ oxnm~ n 'ch~ draw~ng~
~n~ her~in ~Ar~ da-cr1~a ln d4~11. It ~holl1d ~D
undor~toa~, how~or, th~t lt 1~ not 1nk~nded to l~mlt ~h~
1n~ ~ntion ~o tha ~artia~ r ~orm~ dlac1c~d, ~u~ an th~
aon~rhry, t~ tan~1on i~ to c~o~0~ ~11 nodi~aJ~io~
~o ~e~ulv~l~h~, as~ al~rn~t~v~ llln~J within ~ lt ~nd ~COPB of the inv~ntion ~ ~et~1n~ by ~h~ ap~ndod ~llaim3 .

.
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- : , , . " , 206g:122 -- 26 ~

~I~I Co~p~rl~n ~f ~x~ v~ und ln dl~r0nt E~larlt tl~u~ of th~ g~n~ TavrU~ nr~ ~xpr~ed I~D
p~roont~g~ o~ e ~ry w~i~h~.
~--,_ =~_ ~ e~ ~_ ~ b~ ~ ~ ~ ~
~ep~udon~ ~ h~h~ ~
~ . _.= ,. ~__ ___ _ ... ,._ .. .. ...
5 ~_ ~ , ~.01~ __ _ --_ tnnn~ 0.001' 0.0~1~ 0.~ ~ ~ 3~ û.~
O.ODl`O.~OIr 0.û0S3' i~hlro O.OW D.~33' O.~ID7~ 0.~117 ~ __ ___ ___ .._ _~, __ ..... . ~,~., __ 0.003' 0,~61 ~ O,QD~ 0~002' 0.00~-.003" O.~ 0.01~4' 0.0141 0.00~
l),0~15' ~.0(~' O.OlS6' L ~_.. ~ ~ __ _~ _ _ I
~Wl~erup~al., 1~9U~Withuup,K.M.,S.A.Lo~k,M.W. S~s~D,T.
nd ~.M. ~lu~chil~ pp. n~dle~ ~ount~ of t~l comp~blc t~
tho ba~k of 1~ ~ lia; Andy~is and l3~lnd~nl J. Natu~l Pr~dllct~ ~3:1~4~-l~S (1~90).
Vidense~ C~ Rl., l9~0 ~Yide~ t, N., Y. Lim, A. C~np~li and C. ~ nn.
~5 T~xol eon~4nt ;n b~rk, wood, xoot, Icaf, twiSs ~d ~dline fr~ everal tsxus Ip~c~. J. Nat. I'rod. ~3~ 1610 p99~.
Wlc~nl~sinhe ~nd Art~

.. ..

.' ! , !, '. . . ' ~ '' ', 2~69122 -- 2~ ~

T~ II Com,~)o~itiDn ~ Gambo~ B5 n~odiuDt.
~al ~al~
rn~lL
Ammon~um ~ulfat~ 134.~0 B~rlc acid 3.000 Calcium chl~d~ ~nhydrou~ ~13.2 Cobalt chlo2ide h~Ahydr~te 0~
Cupnc 9ulf~ ~n~hyd~t~ O.OQS
D~s~iu~ ~TA dihydra~ 37.250 1~ Polrous slltf~te hcp~ydr~t~ 27.8~0 M~n~u~ ~f~ anhydsous l22.0~0 ~nego sulf~te monohyd~to 10.0Pot~sa~uln iodid~
Pot~si~n nit~ ~500.0aD
~odtum molybdate dlhydr~t~ 0.2~0 ~odlum phosph~te monobasi~ ~nhydrou~ 13V.gOO
Zino sulf~ heptahyd V~
My~lno~ilol lW.~Thiam~ C1 10.0 Nicotlnlc ~ 1.0Sug~r~
a5 SuC~n~e 20,0~.0 , - . ' .. ' . .

2~69122 - ~B -M~ifl~ nborg~ B~ m~un) I~Dr g~owth of c~llU
n~al S~}~
~L
Ammor~um ~ulf~te 134.00D
s sario ~d 3-000 Cdciurr chlori~e anhy~ .24 Cobd1 chlo~ide h~hydra~ ~.025 Cupna s~lfalcpent~hyds~tc ~.OQ5 Pl~di~ TA dlhydr~t4 37.2~0 ~o~rou~ ~ul~t~ h~ydrate 2?.~50 b~n~alum sul~ate an}syd~ou~ 122.0gO
M~galte3~ sulfat~ mon~hydrata 1~.~0 Pot~u~ iodids 0,7SO
Potas~um n~atg 2S~.O~
~ium molybdah dihydr~te O.~Q
Sodium pho~ R~e rns~ob~sie a~hydrous 130~5 - ~inc ~ulhto h~p~hytlr~to ~.(~OO
Vit l~y~inosltol 1~.~
2 0 ~amina HCI lû.O
Pyrldoxine l.O
NiGo~nle ~cid 1.O
Sug~ls Suc~ose 2O,~.O
a~ Gluco~e 2,~
P~ucoD~e 2,S~O.O
C:~ln hyds~ly~atc 1,O~O.~

.-', , `, .,: ~ !

~, ' ' ~ '': . ,:

..

2g --~A~I~ IV M~dified ~ r~ BS modil~ fo~ rth of c~ll suspens~ons.
B~al S~lt~

Arnmonium sulfat~ 134Q~O
Bo~ aGsd 3.aOD
C~ob~lt chlonde hexahydrat~ o.ms C~o Wlgdte pe~ yd~t~ O~i dium ~T~ ~3;hyd~ata 37~2SO
P~rmus s~ oh~ptahydrAte 27,~54 1~ ~nesium sulfate allhy~nut 1~2 Mitn~nc9e g~llhte n~nohyd~dte 10.~
Pota~um ~ e 0.~50 Pot~s~ium ni~t~
Sod~um molybd~ta dlhy~ate O~Q
Sodiuln photphatc ~nonoba~ic dnhy~ous 13U:500 ~:inc ~ te heptahydrato ~tr~
~y~ino~ltol 200.0 llùan~ino EICI ~o,o 2 0 Pyridoxln~
NicoU~lc acld ~.
~u~a~
Saoroso 20,~0.0 2,500.0 ~uc~s~ 2,50~.0 ein hydtoly~s~e 200~0 TAll~ V
_____ ~ _ (~JI lu~ ublin~ (d4ys)Ta~ol ~ _~
CR~l 1~ O.OOl~o ~. . ~_ _ 3~) ~R-l 5 g S~.001~.016 - .. ... _ ,,, _ . . ~ ~
3 3-~ 0~1-0~08 ~ , . _ I
LG-3 15-1~ O~Qol^O.OQ0 ...... ~ _ .. _ ~
LCS-3 1~-16 0.001~0,~1~
~,~,,, . ,,", ~_ __ .

~0~22 - ~o -~ha ~iere~tubul~ 4t~hll~1n~ bio~ y i~ 6ed ~o ucr~n ~or ~ b~ 1~ ty of ~ lti~ to ~tubllle~ tho ~or~tlon c,~ ~nlcrotubu~4~. ~h~ra 1~ a dllr~t, ~rs~aloti~
~oxrelAt~on b~we~n ro~ul~3 11~ th~ m~c~ubu~e lDI~ ay and th~ ch~ rapa-lt~ ~Ct~on o~ ~ ~om~?o~ n in o~na~r l a~ay~ ~h~r~ror~; ~ho ~ro~ubul~ n~lliz~ng Oa~ Ay i3 xeoognl~ed ~y tho~ o~ ~Cill in ~;h6~ ~r~ ~ a OC~ n~nl;~ tel~t ~02' ~ aCtiVQ ~he~c~th~r~aUtlC ~g~nto ~or r~ ical ~rl~ln.
~o ~ndu~'t th~ ~lo~ y, ~uxon~ rottlbul~
~rot,~in~ are pr~ od Qooord~ ng ~o ~o m~od~ 0 lS ah~l~nski ~t al . ~ lg73 ~ u~ r~h cal~ br~in~ .
Endo~nou~ m~rotu~ule-a~iso~at~d ~rot:eln~ r~
ramo~r2d ~ro~ prot~in~ ~h~h h~ ba~n ~yclod on~ ti~e, ~y oyclln~ ~ tlme~ lr3 et-~ ~>r~a~o~ o~ luts.~o (~l~m~l l~nd Lln, l~a~). A~t3r co~ tlon o~ tl~ a~ , th~ t 2~ t~bul~n pcll~ obt~n~d i~ ~olubil~ ln o~ld ~ bu~r ~nd 1~ ~uxth~r ~ur~fl~d byphq~ho~ellulosG ahro~*og~n~hy acaordin~ ~ W~ngart~n ~ al. tl~75).
~o~ay u~;in~ ~a~ iol~ ~lcro~aopy 1~ p~onn~ d~
~olloot~:
~1~ tho t~t ~x~ract~ ar~ tho~ ~olub111~d ln P~
burP~r a~ crl~e~ ~b~
uffor: 50 m~ 4-plp~r~ 4dl~hAne~ul~ania a~
~Pi~) a~ l?lI 61 9; 1 ~M ~n~no~ium ~ul~ a ~thylene glyaol-hi~ ~b-a~no~thyl 6~hçlr~-N,N,N~ ,N~-t~tr~acetlc h~ld ~ECTA] tu~ulln and ~ P ~toak.

2 . ~ ~
. a ~nd 3) ~pp~a~i~nntely 90S ~ l'a2~us at~m ~zpl~nt~ ahow~d 3S cnllu~ ~n~u~tlon ~t~r ~ouæ 2-4 ~/~aknl o~ irll~int~o~.
w col~red CA~ we~fd BE110Ct:~d ~ A~IIt~d erOl~l th~
~xpl~nt, nnd ~ubaulturad. ~ UB oh~ctori~ticall~ ~Ow - . .
- , '- : ~ ' ' .. :

L. ~YUI I ~Y~ U ~ 1 . J
7L ~ vrlr I V~L l~ Jl~n ~vr~ n~ 4 i ~n~tlal ~lov growtn, ~nly doubl~nq ln ~1~out 3-~ ~onth~ o~
lon~er. ~th~r dl~ ulty to be ov~room~ 'ch~t ~o~t~
sallw~ turn 2~ ~row~ aolor w~in 1-3 w-~ko ~f~ex ~uhoul~uru!, ~nd ev~n~u~lly d~. R~d-~Olor~A ~xudat~ w~r~
l!lno~Qr ~lqn of lm~ n~ ~11 d~
Cl~mp~ o~ y~ aolo~Qd~ ~ri~bl~ a~llu~ w~r~ ~3al~ed f~r ~u~c~llturln9~ d ~nd ~rown colar~d c1~p3 w~ro ~O~
at~d. S~ tabl~ olum~ were o~n ~urro~d~d wlth brown-aol~ d ~dsad) c~ .u~ c~ , tho llv~ c~ t~o~
ap~r~ng ~ nodul~e ~ro~ud~nç~ rrom a br~ colc~d ~s ol;' callus.
3 ~ 1 ~ ~Y~
~rled ff~thyl~nQ chl~r~ xtr~ usr~ ~isctolYod in ~th~nol ~or HPL~ ~n~ly~ n~n~Atl~ oon~ t~d ~
~ w~Pr~ 6K ~ver~uid ~hrb~Q~o~r~h ln~eator, Nat~o ~DE n~ultl~lv~nt d~liv~y ~y~t~ nd s~a~Qr~: 4~4 tun~bl~a Ab~Or~n~ d~ta~tor. A ~yna~x-~o n pheny~ a~ 4.~ mm x ~0 m~) wlt21 ~ g~r~ m~dl~le w~ UDoCI~ ~ha ~ob~la ~nahe~ aon~ko~ Or ~ ~thanol, ~moni~m ~q~ta~ bu~r ~n~ ~c~tonli:rll~ ~un ls~or~tic~lly ~4t 2~:41:39, ~beor~no~ w~ da~cted ~t th~ ~b~orp~l~n m2~xlmum 0~ t~XC~l ~hl~ 227 ~. Auth~ntiC ~tAn~rd~ o~
t~xol (1~, c~ nnin~ ~a) ~ ba~fl~n ~3) w~ro u~à
to ld~t~y the p~kd ~a- 6) ~ t~ndoxd~, 2S ~qht: M6thyl~ne chl4~ xtraot ~ro~ C~-l. Poo)~ nu~r 4 ~nd 5 ar~ us~ld~nti~ chibltln~ ivl~y in th~
3ior~tub~ bl~ y.

,: , , ~ , , .~ ' , .

. . .- . . . : , ~
~ ~ , V ~ I V r ~ I f ~ J ~ ~. J ~ G . ~ ~ V
2~69122 - 3~ ~
., `:
x,~g~r~oo,~
Irh~ r~f~PArg~n~ t,~d bslow ~r0 in~or~ r~ln by ,r~rbn~ ~ ~o ths ext~n~ t~A~ th~y ~uppl~ nt, ~ n~
pP ov i a~A a ba ~ ,und i o~, v A~ ~A~ch ~hod 4 ~ y, 3 t~,ohnlq~lss,, nd/or c~m~osltlon,3 ~pl~yad. heLAeln, ~rodollus, P. -~ B ~ot~nti~ loe of l~noblll~sR~ion ln pl~nt a~ll bi~t~hnol~ frr~nd~ 10~4uhnolagy, 3s~o-~5 (lg~S) ,~odoliu~, P. I~mo~ Ad P1~ t; coll~ oura~
1~ bioch~lod~ n: ~ior~aactor l~obl~ d ~n,s~ ~nd c~ d: M. Mo~-Youn~ vi~r A~ d ~ci~n~ ndon ~nd N~w York, ~Ba, ~p. 167-lg6.
chrls~n ~t ~1., Prndt~tl~n o- ~x~l or T~xol-liko Co~po~lnd~ ln asll CUltllre~. U.~, P4~ant No. ~,0~,304 ~g91).
~ni~ N., A.~. Gr~n, D. ~u~n4~d, F.
Gueritto-Vo-sloluin, ~. NAnqat~l and P. Potl~r. A hlg~
e~loi~nt, practical ~pxcach ~ n~urnl t~xol~ ~0 e~
Amu~. Che~. ~oc~ 110; 5~17-5~
Dor~ohowar, ~2~C~ E.X. Rc~n~ky~ roo~ow, ~.~.
L~nqn~atce~ ~nd D . ~ ~t~ qer, ~h~ I tr ~ al ~ t~xc~l ln patlsnt~ Wl~n ~dvanc~ ~nna~r. ~ano~r ~rt. R~ort~
7~ ) tll71~1i77 ~1987) .
F~lzone, C,P., B~n~i, A.J. ~ Le~mt~ J.T.J. ~l~g2).
2S ~h~r~ct~ri~ on o~ eaxol ln m~tnyl~n~ ehlori~ by N~
~ec~ro~oopy, '~otrahsdron ~4~t~ro 3~ 2.
~ul, H., M. ~ nd ~. T~batU. ~nduation o~ ~h$konln by ~ndcg~nou~ poly~acah~r~ d~ ln ~ryt~orhl~n o~ u~n~lon oult~rea . PlAnt C~ cpoFt~
73-76 ~1990).
Gamb~rg, O~h., 11.~ r ~nd ~t. O~l~a. Nutr~nt requlr~m~nt~ o~ ~u~p~n~on cultur~ oyb2~n root c~
E~p . C~ ., 50 ~ g0 1 1~68~ ~
~u--~e~-vb~gbl~n~ c~ Lhl41~ ~n~ ~. Po~r. TAXO1 And d~ri~Ativ~s: ~ b~oq4n~t~ th~ . Jourr~al o~
N~tu~nl P~oducts, 50 ~ 9-18 (19a7) O
H~al, E. ~n~ C.M, I.ln. aluth~kat~lhd~d ~ol~k~rl~tian o~ tu~ulin: oh~r~tQrl~tlo~ o~ th~ r~a~t~on an~
~pplic:~ti~n to t~a lnr~o~aal~ pur~tl~t~on ~ ~ub~llln.
A~ch. ~l~c~e~. Bio~hys., 20~:29-~0 (1981).
~a~n~in, P . F . E~t~lrl~ appxoa~ to th~ l!on~tlon o~
Qcond~lry nstu~l prod~t~ In pl~nt ~oll ~u~p~r~aion J'. Na~, ~ro~. 4~ 19~$).

, :
..

L ~ Irl~ 11 VLL IC~L~ L~ n JVr~ n' ~14~ C~

~069122 Hor~ t~, S.~., L. Lot~t~i~, J.~. Nanfr~dl, W. I~loll~do, J, ~i~rneB~ ~.N. Roy, ~.~. 6ahi~ L. ~orb~ro. ~n~ R.
~h~. Irax~ls MeQhanl~m ~ ~c~lor~ ~nd r~ t~t-o~ Annal~
N~ York A~do~ o~ ~oi~ e~, N~w Yo~c; Th~ d~y, v~l.
466, Pp. 7~3-743 ~19 Ha~ , N~ d 6 . N . ~1~aDh~ 1nk4s~ b~t~
e f~t~ o~ t~xo:l, oc~lohlcin~ ~nd ~TP on tubulin ~olym~ri~t~on. J. 0~ bi~lo~1chl ~haml~ery, 2~ ~3) S 1~2-134~ g) .
1~ Huan~, C.~Ø, ~.G.I. ~nq~t~n, N.~. ~aqri ~nd a.
r~n~ya~e. ~w t~xanes :ero~ vlfoll~, 2. J.
o~ Natural Pr~d.uctt3 49 t~ 19~S~, Xar~oh, R. arld ~. Mitton~or~er. Ultravlol~t 3:ad~tlon ~ ncr~ nl~ot~ne pro~uc~lon ln ~1~ ~allus o~lturo~. J. Plant Physlol, 3~ 0~ 1990).
Klnq~on, ~ . N~wkln~ ~n~ Ov~ngton. Now ~x~n~ rrom T~u~ br~ . J. Na~ur~l Product~, 4~ 66-470 ~lcGu~r~, W,P" E.K. P~win~Xy, ~.~. Ro~n~h~in, F.a.
cr~tm~ln~, D.A. Et~ingor, D.~ t~os 0, P~.cù ~c)nohQw~r.
TAXO1: A un~ antln~ls~tlo ag4n~ wl'eh ~lgnlf t ~ct~vity ln ~d~ncad ov~rlan epl~ al n~o~la~. Annal~
o~ In~rnal ~S~dlcln~ s~?3-~7g tl95~-McI.au~3hl$n, J.~ . Hill~r R.C. Pow~ll and C.R. a~lth Jr. ~9-Xy~lroxy~c7a~tln I~ 0-~oac~tylo~ph~l~nnin~, and l:O-d~na~tyl~axQl ~ N~w ~n'clt~2~0r tax~ne~ ~r~m T4 ~lllch~an~. J ~ Na~ur~l P~adut~ 44t~ 12-31~
Mlller, R.W., ~.G. Pow~ll an~ C.~. 8~ h ~r~ Antile~omlc a~knl~d~ fro~ ~U~L~o w~ chl~n~ Zuao. g. Or~niQ
Ch~n~try 46:146g-14~ 81) .
w~, }S., M. Il~y~-hl an~ ak~y~ cou~u~ation o3!
antlnea~la~tio aC~Qnts by ~ nt ti~l-U~ ~ulturo~, n~
Prlm~ry ~n~ ~eoond~y M~tabol~m o~ Pl~t ~11 Cul~ur~;
~!d . K . N . Neum~nn, w, ~ xd . 8pr ~ n~
3s ~erlln ~ lb~rq 19~S, ~ol 3~, pp ~3~-a~6.
,~lqo, T. and F. ~o~. A r~vi~ me~ or rapl~
growt~ dn~ ~iOAI~S~ wlth tob~ccc~ t~Aua ~ultur~s.
Phy~lol Plan~. 15:473-497 ~1g6~).
P~rnOo~ n~ . Horow~tz. TeXal binq- to poly~ lz~d tu~ulin ~n vltro. ~ ~tll sis~ t. l) ~47s~4a~
.~ ~19Bl).
Payne, ~.~., Shuler 2~.~. and P.~ llu~ lg~73 . L~rge 90~1~ Pl~n~ ~ell Cultur~. ~n~ L~r~ Baale c~ll Cu~tur~
T~chnolo~y, Ed: ~orn R. ~der~on. l~ne~r Pu~ h~
4~ ~unlch, ~ snno., ~4w Yorlc. p~. 194-~9, : . .

. n~ n,~ n ~ .~ L I J~ r~ uLc ~ nl~1~Cr~ 4 ~ 1 0 2~6912~

sohl~I', P~B And ~. H~rbW~, T~xol ~ bll~
nllcrotu~ul~a in ~ou~ ~lb~obl~t a~ . Proo. ~ln~, Ac~d.
8~1. 77 ~3) ~ 15~l-15~s ~1~89~ .
8h~1~n~Xl, M., ~ !cln dnd ~ n~r. M~o~tu~?Ul~
~e~bly ln ~ sl~en~e o~ add~d nuol~c~tlde~. Pr~c. PJa~.
Ac~d. 8ai, U~A, 7~ 5-768 ~1973~
~lCl~rod, O. A~ kj~X-~r~eX, Rlgln~te ~o lmmoblllzation m~trix 1~or a~ . Tr~n~ ln ~lo~ohnolo~y~
~71-78.
1~) SnAd~r, R. I:tv~rvlQt1 o~ ~up~ly ~u~o. ~o~k~hop on ~xol ~nd r~ Cu~ror~t ~nd ~-~tu~ tl~ p~n~or~ad byl D~vel~ nt~ 4r~eutl~ gr~, bt~ n o~ nc~r trhstr~Qn~, N~tl~n~ nc~r in~tl~ut~ 90). ~traat.
80u~htan, ~.W. and ~. Bu~lngh~s. D~ctto~ry o~
1~ A1~A101d~ Ch~PmAn ~ H~11, No~ Y~rX ~193~), pp ~o~, 1024.
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~u~Pne~ 19gO). l~ o~ nd ~arly dbv~l~p~ent o~
~ol. ~tork~hop On T~xol And T~XUSDS ~rront and ~u~u~
2~ Pars~o~t~v~. Nd~iOn41 C~na~ Instltut~ 0.
~b~tract.
Etu~n~so, ~. ar~d C.~, Co~d~ r A~ old~
~k~ id~/ od. A. Bro~i, Acad~ Pr~, Ino. ~BS), Yol . ~nr. ~p 6~ . 0 at~d S7 ~g .
~u~no~ . Di~aov~ry ~n~ y ~ lopm~nt c~ ~xol.
Nor~coAop ~n t~xo~ an~ ~xw: Currunt ~n~ future p~r-~-ati~a~. Etpan-~n~e~ b~ ev~ ntal 'ch~rAp~ c pro~rA~, 3i~ on ~ n~r t~ nt, N~t ion~l ~a~lo~r ln~t~uto ~1990). ~ o~.
~hlg~n~ n~n~ t Al. ~ Ph3~a I~ trl~
~r taxol ~n ~aoond-l~n~ th~ea~y ~ o~arl~ rcln~ A
gynscoloqlo on~logy gro~p l-tudy~ P~oo. 1~. ~oo. Clin.
Onaol~ 9: 156 ~l~P0~.
Thr~ nd I.PI. ~hlt~h~ bts~ n~
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~t:rua'cur~ o~ T~XOl~ a noYDl ~ntll0uk~mlG an~ titwllor .

, . . ~ J ' .~ ~ r ,11 . V L ~ V 1~ 1 4 ~ j 4 ~ 5 ~ D ~ d 2 ~ 2 2 ag~n~ ~rO~ T~XUB b~vlr~ . J. ~herlo~h ~h~lCQl ~oc~e~ty 93 ~ 23~5-~327 ~1~71) .
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i~ 1On. J. N~tUra1 PrOdU~tB ~3:1~4~-1a~S t1~~-,: .
. ~ ,. . .

- ,,

Claims (29)

1. A method for establishing a call line culture of Taxus, said method comprising:
(a) selecting a young stem as a tissue source for an explant;
(b) culturing the explant in callus-inducing medium to form a callus;
(c) subculturing selected sections of the callus to promote subclone growth;
(d) selecting a subclone to develop a cell line; and (e) culturing the cell line on a solid support in maintenance medium.

2. The method of claim 1 wherein the Taxus is further defined as Taxus media cv. Hicksii.

3. The method of claim 1 wherein the callus-inducing medium comprises Gamborg's B5 medium supplement with plant hormones.

4. The method of claim 3 wherein the plant hormones are selected from a group consisting of 2,4-D, kinetin and a combination of 2,4-D and kinetin.

5. The method of claim 1 wherein the selected sections of callus are selected based on the following criteria:high growth rate compared to growth rate of other callus, lack of red coloring, said coloring indicating the presence of exudates which inhibit growth, presence of yellow coloring, and friability.

6. The method of claim 1 wherein the solid support for maintenance of the cell line comprises a membrane raft and the maintenance medium comprises modified Gamborg's B5 medium according to Table IV.

7. The method of claim 1 wherein the maintenance medium comprises casain hydrolysate, glucose and fructose.

8. The method of claim 7 wherein the concentration of glucose and fructose totals about 10-40 g/l.

9. The method of claim 1 wherein the maintenance medium comprises a sugar selected from a group consisting of sucrose, glucose and fructose, and wherein the concentration of said sugar is about 40-80 g/l.

10. The method of claim 7 wherein the concentration of casain hydrolysate is about 0.2-1.0 g/l.

11. A method of producing a cell suspension culture from Taxus comprising:
(a) preparing a cell line according to claim 1;
(b) obtaining medium conditioned by growth of the cell line supported on membrane rafts, said medium containing cells from the cell line in suspension' and (c) culturing the cell suspension in shaker flasks in the dark at a temperature of about 24-26°C in Gamborg's medium according to Table IV.

12. A method of producing a compound selected from the group consisting of taxol, a precursor or intermediate of the biochemical pathway resulting in taxol production, and a derivative of taxol, said method comprising :
(a) culturing a cell culture according to claim 1 under conditions which allow production of the compound; and (b) collecting the compound from the cells in culture or from the conditioned medium of the culture.

13. The method of claim 12 further comprising enhancing production of secondary metabolites of the compounds by applying alicitation techniques.

14. The method of claim 13 wherein alicitation techniques comprise feeding taxol precursors to the cell cultures.

15. The method of claim 12 further comprising the isolation and purification of taxol from non-taxol-containing compounds and from contaminants.

16. The method of claim 12 wherein the cell compositions are cultured in large scale bioreactors.

17. A cell line having the following characteristics:

(a) stable growth in culture; and (b) capability of expressing a compound selected from the group consisting of taxol, a precursor or intermediate of the biochemical pathway resulting in the production of taxol, and a derivative of taxol.

18. The cell line of claim 17 further defined as comprising maintaining the capability of expressing a composition of step b, after cold storage and reconstitution of the cell culture.

19. The cell line of claim 17 wherein Taxus comprises Taxus media cv. Hicksii.

20. The cell line of claim 17 wherein the compound is capable of inhibiting cell growth in cancer model systems or of producing positive results in a microtubule-stabilizing bioassay.

21. The cell line of claim 20 wherein the cancer model system comprises a cell line selected from a group consisting of ?16 melanoma, MX-1 mammary xenograft, P388 leukemia, KB, and L1210 leukemia.

22. The cell culture of claim 17 further defined as producing an amount of taxol that is at least about 1-2 fold increased above that produced by direct extraction from stems, leaves and bark of Taxus media cv. Hicksii.

23. The cell line of claim 17 further comprising at least one mutation which confers on the cell a phenotype wherein the compound is overproduced compared to the non-mutated cell culture.

24. A method of regenerating plants from cultures of Taxus, said method comprising:
(a) producing a cell line according to claim 1;
(b) subculturing the cell line in the light at a temperature of about 25°C in decreasing concentrations of hormone-containing media to induce sheet formation; and (c) regenerating plants from the sheet-containing cultures.

25. A method of producing taxol, said method comprising:
(a) placing a cell culture derived from Taxus in a bioreactor;
(b) operating the bioreactor so that taxol is produced by the cell line and is secreted into the nutrient medium of the bioreactor; and (c) collecting the taxol from the medium,

26. The method of claim 26 wherein the cell culture is produced according to claim 1.

27. A compound exhibiting microtubule stabilizing activity, said compound selected from the group consisting of peaks eluting on an HPLC profile of a cell extract from a Taxus cell line, said HPLC profiles being produced from methylene chloride extracts of a phenyl column.

28. The compound of claim 27 wherein the Taxus cell line comprises CR-1 and the peak selected elutes at 18 minutes.

29. The compound of claim 27 wherein the Taxus cell line comprises CR-1 and the peak selected elutes at 25 minutes.