Key words : alkaloid spectra', gas chromatog raphy, Datura stramonium. Abstract: Alkaloid spectra of diploid (Zn) and tetraploid (4n) plants and transgenic root ...
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ANNUAIRE DE
L'UNIVERSITE DE SOFIA
"ST. KLIMENT OHRIDSKI"
FACULTE DE FORMATION
D'EDUCATEURS ET D'INSTITUTEURS
LIVRE 4 - loeme SESSION SCIENTIFIQUE, SOFIA'03
Partie II
Tome 96
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SOFIA· 2005
Annuaire de l'Uuiversite de Sofia "St. Kliment Ohridski" 2004, volume 96, livre 4, pp 95-98 10 erne session scientifique, Sofia ' 03
ALKALOID SPECTRA OF DIPLOID AND TETRAPLOID PLANTS AND
TRANSGENIC ROOT CULTURES OF DATURA STRAMONIUM
STRAHIL BERKOy1 ,A . PA VLOy 2 , Y. GEORGIEy2 , P. STANIMIROYA 1,
R KOYACHEYA 2 , S. PHILIPOy 3
of Botany, Bulgarian Academy ofSciences, 23 Acad. G. BoncltevStr., 1113 Sofia, Bulgaria. 211lstitute of Microbiology, Bulgarian Academy of Sciences, 26 Maritza Blvd, 4002 Plovdiv, Bulgaria 3lnstitute of Or ganic Chemistry with Centre ofPhytochemistry, Bulgarian A cademy 0/ Sciences, 9 Acad. G. Bonchev Str. , 1113 Sofia, Bulgaria
1 Institute
Key words : alkaloid spectra', gas chromatog ra phy, Datura stramonium . Abstract: Alkaloid spectra of di ploid (Zn) and tetrap loid (4 n) plant s and tran sgeni c root cultures (hair): roots) orDa /llra stra mo nium we re investigated by gas-chro rnatog raphy,'mass-s pectro me try. Thirty-nine alk aloids /twc nty -nine alkaloids for intact plants and twenty alka loids [or hai rv roots/ were found. Differences betwee n the alkaloid spectr a of intac t and hai r, roots were estab lished. Ploidv level JOG S not influence signifi cantly the alkaloi d pattern.
Polyploidy is a w ides pread phenomenon in nature 11 j. As compared to dip loids , natural polyp loids have altered morph ological. physiological and biochemica l cha racte ristics 12j. Such alternati ons and especially variati ons in accumulation o f bioactivc co mpo unds are of interest fo r induction of polypl oid forms (geno m e mut ant s) in medicinal plants and their in vitro cultures f3-7]. Dat ura st ramonium L. (Solanaceae ) is one of the most inten si vely investigated J)((/ U I'O species bCCJ USC it is alm ost an ideal plant for research . It j " easy to grow e ither in g ree nho use or fie ld , sclfi ng and cro ssing is si mple and ~I large number o f see ds is produced. Furt h.. .. n n orc . l ) . stramo ntu m is an importan t medicinal plant containing spasmodic a:id hypn otic tropane alkaloids . Rece ntly: trangcnic root c ultures. so called 'hairy roots' arc intensively investigated JS J possible alternat ive for industrial production of bioactive molecules l8]. D stram om um hairy roots arc stud ied for their hyoscyamine production . This alkalo id is w idely used in the modem medic ine [9J. In thi s paper we summarize our results on the a lka loid s in genome dou bled (polyploid) Da tura stramonium plants and their transgenic root cultures .
MATERIALS AND METHODS
Seeds from the initial diploid form of Datura stramonium L. (2n = 24) was collected near vicinities of Lovech, Bulgaria. Tetraploid form of this species (4n = 48) was induced as described in ref. 6 . Trasgenic root cultures from diploid and tetraploid plants were obtained and maintained in liquid culture after the method described in ref. 7 Alkaloid identification was performed with GC, GC-MS as described in ref. 6, 7. Chromosome counting was performed after ref. 7. RESULTS AND DISCUSSION Thirty-nine alkaloids were detected in the plant samples and root cultures of D. stramonium (Table 1). Thirty-tree of them were identified, They can be classified in five subgroups of the tropane alkaloids according to ref. 10: 3 monosubstituted, 2,3-disubstituted, 3,6- disubstituted, 3,6,7- trisubstituted and 3-substituted-6,7-epoxytropanes , One pyrrolidine type alkaloid (hygrine) was detected . Alkaloid spectrum of D. stramonium is organ dependent. There was no significant difference between diploid and tetraploid plants in respect to synthesized alkaloids. The differences between the alkaloid spectra of diploid and tetraploid plans are mainly in alkaloids, which are less than 0.1 % of the alkaloid fractions. Thus , the presence or absence of such alkaloids will depend on the quantity of the injected sample , and their absenc e can not be unambiguously proved by a single GC-MS analysis, 11K ploidy influences the ratio between the main alkaloids [6], The difference between alkal oid spectra of diploid and tetrapl oid hair. roots C:l.J.1 be explained with the formation of sell aggregates on tetraploid level 17 J, Hyoscyamine was found to be major alkaloid in all plant organs and root cultures. HO\\'eVCL when the plants arc grown under artificial conditions , scopolamine is accumulated as major alkaloid in the aerial parts l6. 7 j Intact root s, wh ich arc the place of alkaloid synthesis: have the most complex alkaloid spectrum . It contain s 3-monosubstitutcd. 2,3-d isub stitutcd , ~U1d 3-substitutcd-6 ,7 3J1-dlSllbstituted, 3,6,7-trisubstll Uk d alkaloids cpoxyt ropancs. In contrast to them. alkaloid spectrum of hairy root s has mainly 3-monosubstituted and 3,6-disubsrituted alkaloids, Other significant difference between the intact and hairv. roots is that intact roots contain a rclativclv. hizh v perce ntage of different alkaloi d subg roups 3-m onosubstitutcd (30 -35°;;) of hyoscyamine) , 3-substitutcd- 6,7 -epoxy-rropJlle (10% scopolamine) and 3,6.7 trisub stitutcd ( 19% 3.6-ditigloyloxy-7 -bydroxv tropane ) in the ir alkal oid fractions ' 6 J, 9G
Table!. Alkaloids found in diploid and tetraploid plants transgenic root cultures of D. stramonium.
was (4n
A lkaloid
M+I
2n
l ed In
es be ...,
,)
ld lS
Leaves
Roo ts
b as e io n
4n
2n
Se ed s
4n
2n
Ha iry ro ot s
4n
2n
4n
141 / 84
+
+
139/82 183/124 2231124 259/124 259/ 124 271 /124 271/124 289/124
+ +
+
+ + + +
+
+
-t-
+
+
+
+
+
Py rrolizidine a 7lkaloids H ygrin e ( 1)
3-Alo noslibstitllted trop anes T ro pi no ne (2 ) 3-Acc toxytropa ne (3 ) 3 -Ti gl o yl o !l.y 1ro pane (4 ) 3a- Phe ny lac eto xytropa ne (5 ) 3 p- Phe ny lac e to xyt ro pa ne (6) 3a- A po tro poy lox ytro pane (7) JP- Ap otro po yl o !l.y trop an e (8) Hyos c yami ne (9 ) 3-(3 ' -Acetox ytr opoyloxy jtropanc ( 10) 3-( 2 ' Hyd ro xyt ro po y lo x y jtro pan e ( 11) A lka lo id A ( 12 ) A lkaloid C ( 13)
+
+
+
+
+
+
+
331 /1 2 4
-t
+
30 51124 -/124 -/1 2 4
+ +
+
+ + + + +
-t
2, S-Dt substitiued tropan es 0
Methylecgon ine ( 14)
0
3,6-Di.wbstllllted tropanes
d
3 -Acc:t oxy -6 -h yd rox y1rop an e ( 15) 3- Hyd roxy- S-acc toxy t ropane ( 16) 3.6-D iacdo xy1ropa ne ( 17) 3-H yd rox y-6-t igl o vl oxyt ropa ue ( 18) 3-Tig lo yloxy-6-hyJroX\"l10 pa nc: ( 19) 3-T igloyloxy-c-iso buryloxvtro pa nc ( 20) 3- T rop ovl oxy-o-hvdroxvtr op uuc ( 2 1) 3- T ig lo do:-.:y-G m eth yl bury loxvt rop.uic ( 22 ) 3 -T rop o y I0 xy -6 - t i g 10 y'lo xyt ro pJJIC ( 23) 3- Tro po vlo xv -G-ncc to xyt ro pnnc (24) ] .G-D itig lo \ '\') :-'::1 ropal1c l 2:;)
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d " ,
