photosynthesis and cannabinoid content

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Biochemical S ystematicsand Ecology 1975, Vol. 3, pp. 15 to 18. PergamonPress, Printed n England.

Photosynthesis and Can nab inoid Co nten t of Tem perate andTropical Pop ulations of Cannabis sativa

F . A . B AZZ AZ, D . D USEK * , D . S . SE IGLER and A . W. HA NE Y

D e p a r tm e n t o f B o ta n y , U n i v e rs it y o f I l li n o i s , U rb a n a , I L 6 1 8 0 1 , U S A

K e y W o r d I n d e x - - -Ca n n a b i s s a t i v a ; C a n n a b i n a c e a e ; p h o to s y n th e s i s ; c a n n a b i n o i d s ; p o p u l a t i o n s t u d ie s .

A b s t r a c t - - F o u r p o p u l a t io n s o f Cannabis sat iva L . g rown f rom seeds co l lec ted in Panama, Jamaica , Nepa l , ande a s t c e n t ra l I l l in o i s w e re g ro w n u n d e r c o n t ro l l e d c o n d i t i o n s i n g ro w th c h a m b e rs , O n e s e t w a s g ro w n u n d e r w a rmcon d i t i ons (32 ° day and 23 ° n igh t ) and the o ther se t wa s g r ow n under lo we r temp era tu res (23 ° day and 16 ° n igh t ) .C O 2 e x c h a n g e a n d t r a n s p i r a t i o n w e re e x a m i n e d u n d e r v a r i o u s t e m p e ra tu re s a n d l i g h t i n t e n s it ie s . O b s e rv a t i o n s ong ro w th , a n d a n aly se s f o r c h l o ro p h y l l a n d / k 1 TH C ( t e t r a h y d ro c a n n a b i n o l ) c o n te n t w e re m a d e . U n d e r w a rm g ro w thc o n d i t i o n s , t h e c e n t ra l I l l i n o is p o p u l a t i o n h a d t h e h i g h e s t p h o to s y n th e t i c r a te a t a l l t e m p e ra tu re s in v e s t i g a te d .Th e N e p a l p o p u l a t i o n h a d i n t e rm e d i a te r at es , w h i l e t h e J a m a i c a a n d t h e Pa na m a p o p u l a t i o n s h a d t h e l o w e s t r a te .T h e J a m a i c a a n d Pa n a m a p o p u l a t i o n s h a d i n s i g n i f i c a n t ch a n g e s i n p h o to s y n th e t i c r e s p o n s e t o c h a n g e s i nt e m p e ra tu re s b e tw e e n 1 5 ° a n d 3 0 °. U n d e r c o o l g ro w i n g c o n d i t i o n s t h e c e n t ra l I l l i n o is p o p u l a t i o n h a d t h e h i g h e s tra te o f pho to syn the s is w i th a de f in i te peak a t 25 °. Nep a l p lan ts had in te rme d ia te ra tes o f p ho tosyn thes is , w h i let h e Pa n am a a n d J a m a i c a p o p u l a t i o n s h a d t h e l o w e s t ra te . D i ff e re n c e s in c h l o ro p h y l l a n d d ru g c o n te n t w e re a l sos i g n i f i c a n t b e tw e e n th e s e p o p u l a t io n s . F ro m th e s e d a ta i t is s u g g e s te d t h a t t h e f o u r p o p u l a t i o n s c a n b e g ro u p e di n to d i f fe re n t e c o t y p e s g e n e t i c a l l y a d a p te d t o t h e i r r e s p e c ti v e e n v ir o n m e n ts .

I n t r o d u c t i o nH e m p (Cannabis sat iva L.) is a widelyd is t r ibu ted p lant in var ious par ts o f the wor ld .Or ig ina l ly ind igenous to temperate par ts o f

Asia, probably to the desert region near theCasp ian Sea [1 ] , i t no w g rows as a wee d in avar iety of habi tats ranging f rom sea level intropical areas [2] to 7000 f t in the HimalayaM oun ta ins [3 ] . The spec ies was f i rs t i n tr oducedin to the Un i ted S ta tes f rom England in 1632 bythe pi lgr ims and later f rom China [4 ] . Present ly,i t i s a common weed in the eastern and mid-western Un i ted S ta tes [5 ] .

Cannabis has had a long history of associa-t ion wi th man. Schu l tes est imates that i t hasbeen used for over 6000 years, and regards i tas one o f the o ldest cu l t iva ted p lants [6 ] . The

plant is a source of f iber , o i l , and a narcot icdrug. The i l l ic i t use of Cannabis as a sourceof mar ihuana (p lant shoots) and hash ish( res in) is widesprea d thro ugh out the wo r lddesp i te severe legal pen al t ies in some countr ies.The act ive pr incip le in the resin, which isfound on f lowers, leaves and stems, is most lyZ~ , - te t rahydrocannab ino l (C2,H3oO 2) . Th iscompound is b iosynthet ica l ly der ived f romcannabid io l (C2,H3oO2) and may be b io -

* Present address: Univers i t y o f I l l i no is , Co l lege o fM e d i c i n e , C h ic a g o , I L 6 0 6 8 0 , U S A .

synthet ica l ly ox id ized in to cannab ino l(C2,H 2 ,O6) [7 ] .

I t has been reported that p lants f romdi f fe rent geograph ic locat ions d i f fe r in the

amount of resin they produce. For example,p lants f rom warm c l imates produce 10 t imesas much o f the act ive pr inc ip le , te t rahydro-cannabinol , as plants f rom central Europegrown under ident ica l cond i t ions [8 ] . Fur ther -more, i t has been suggested that temperatureand humid i ty a f fec t the res in content ; thusthere is ev idence that the drug content isboth genet ica l ly and env i ronme nta l ly cont ro l led .

In th is paper we examine the d i f fe rences inpho tosyn the t i c capac i t y and the cannab ino idcon ten t o f popu la t i ons o f Cannabis sat lvaf rom four d i f fe rent geograph ic locat ions grown

under two d is t inc t env i ronmenta l reg imes.On the bas is o f these d i f fe rences, we suggestthat the popu la t ions f rom these areas may begrouped in to temperate and t rop ica l ecotypes.

R e s u l t sLight sa tura t ion curves for the net photo-synthes is o f four popu la t ions were s imi la r fo ra l l popu la t ions under both growing cond i t ions(F ig . 1 ) . Satura t ion was not reached even a t12 00 00 lux i nd i ca t ing tha t a ll popu la t i onsbe long to the sun p lant group. Photosynthet icresponse to temperature o f p lants grown under

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F I G . 1 . L I G H T S A T U R A T I O N C U R V E S F O R 4 P O P U L A T I O N S O F :CAN NAB IS SATIVA (z~ I L L I N O I S . • N E P A L , O P A N A M A ,[ ] J A M A I C A ) G R O W N U N D E R C O OL C O N D I TI O N S .

warm t em pera t u re cond i t i ons sepa ra t es t hef o u r p o p u l a t i o n s i n t o t w o d i s t i n c t g r o u p s(F ig . 2a ) . P ho t osy n t hes i s o f Nepa l and I l l i no i spop u la t i on s i nc reased w i t h a ir t em pera t u rer ise f rom 20 ° to 25 ° then dec l ined sharp ly .P ho t osyn t hes i s o f t he Jam a i ca and P anam ap lan t s i nc reased w i t h t em pera t u re r i se f rom20 ° t o 30 ° (F i g . 2a ) . M ax im um ne t pho t o -

syn t hes i s was 15 . 4 , 14 . 5 , 14 . 5 and 12 . 5 m gCO2 dm -~ h r - , r espec t i ve l y f o r Nepa l , I l l i no i s ,P anam a and Jam a ica p l an t s . The ra t e o fp h o t o s y n t h e s i s o f t h e t w o g r o u p s w e r esta t i s t i ca l l y d i f fe ren t (5% leve l ) a t 25 ° on ly .

P h o t o s y n t h e t i c r a t e s o f p l a n t s g r o w n u n d e rc o o l c o n d i t i o n s ( F i g . 2 b ) w e r e s i g n i f i c a n t l yl o w e r t h a n t h e r a t e s o f t h o s e g r o w n u n d e rwa rm con d i t i ons , espec ia l l y f o r p l an t s o f t het rop i ca l popu la t i ons . Ra t e o f pho t osyn t hes i so f t he I l l i no i s p l an t s i nc reased sha rp l y a t a i rtemp era ture s be twe en 15 ° and 25 ° thendec reased s l i gh t l y a t 30 ° . P ho t os yn t hes i s o f

t he N epa l p l an t s was l ess sens i t i ve t o chan gein a i r t em pera t u re t han t ha t o f t he I l l ino i s p l an t sand t he pho t osyn t he t i c ra t es o f t hese t wopopu la t i ons were s t a t i s t i ca l l y d i f f e ren t f r omeach o ther a t 15 ° on ly . Ph oto sy nth et i c ra teso f t h e P a n a m a a n d J a m a i c a p o p u l a t i o n si nc reased on l y s l i gh t l y w i t h t em pera t u re r i sef rom 15 ° to 30 ° and we re not s ig n i f i ca nt l yd i f f e ren t f r om each o t he r a t a l l t em pera t u resi nves t i ga t ed . A s a g roup , howeve r , t he i rp h o t o s y n t h e t i c r at es w e r e s i g n i f ic a n t l y d i f fe r e n t(5% l eve l ) f r om t he I l l i no i s and Nepa l g roupa t a l l t em pera t u res excep t t ha t o f t he I l l i no i sp lan ts a t 15 ° (F ig . 2b) . On the bas is o f these

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d a t a t h e p l a n t s g r o w n u n d e r c o o l c o n d i t i o n sa n d t h e p la n t s g r o w n u n d e r w a r m c o n d i t i o n sc a n b e g r o u p e d i n to t w o d i s t i n c t g r o u p s :t em pera t e ( I l l i no i s and N epa l ) a nd t rop i ca l( Jam a i ca and P anam a) .

The ch lo rophy l l con t en t o f l eaves f romp lan t s o f d i f f e ren t o r i g i n g rown unde r coo lc o n d i t i o n s w e r e m a r k e d l y d i v e r s e b u t n od i f f e rences were obse rved be t ween t he p l an t s

g r o w n u n d e r w a r m c o n d i t i o n s . T h e c h l o r o -p h y l l d a t a o f p l a n t s g r o w n u n d e r c o o l c o n d i -t i ons aga in show t ha t t he p l an t s can beg r o u p e d i n t o t e m p e r a t e ( I l l i n o i s - N e p a l ; 8 . 9m g and 8 . 5 m g o f ch l o rophy l l s a and b pe r gmd r y w t r e s p e c ti v e ly ) a n d tr o p i c a l ( J a m a i c a -P anam a; 2 . 2 m g and 1 . 2 m g pe r gm d ry w t )w i t h t h e f o r m e r g r o u p h a v i n g s i g n i f i c a n t l ym ore ch lo rophy l l pe r l ea f d ry w t t han t hela t t e r . The re l a t i ve l y l ow ch lo rophy l l con t en to f t he t rop i ca l p l an t s m ay be a cause o f t he i rl o w p h o t o s y n t h e t i c r a te s.

A l t h o u g h t h e r e w e r e s i g n i f i c a n t d i f f e r e n c e si n c a n n a b i n o i d c o n t e n t b e t w e e n t h e p o p u l a -



TEMPERATEAND TROPICAL POPULATIONS OF CANNABIS 17

t ions unde r the two gro wth cond i t ions, t i~esedi fferences we re general ly of l e ss use inseparat ing the populat ions into temperate andtropical group s (Table 1 ) . C ontrary to avai lable

TABLE 1. CAN NAB INOID CON TENT OF LEAVES OF PLAN TSF R O M F O U R L O C A L IT IE S G R O W N U N D E R W A R M 3 2 0/ 23°AN D CO OL (23°/16 ° CON DITIONS

mg g-1 dry wtOrigin

o f Con d i t i on To ta lplant CBD THC CBN cannabinoid

Panama warm 0.64 4.80 0.80 6.24Panama cool 0.65 6.18 0.43 7.26Jamaica warm 0.88 4.07 1.11 6.06Jamaica cool 1.42 13.20 0.30 14'92Nepal warm 0.40 2.97 1.14 4.51Nepal cool 0.88 12.94 1 57 15"39Ill inois warm 1-23 0.46 0.05 1.74Ill inois cool 2.89 2.99 0.00 5.88

Cannabinoids were extracted with l ight petroleum and ana lyzedbyGL C. Cetyl alcohol was used as internal standard,

i n format ion, the to ta l cannabino id content aswe l l as t he euphor ic A , - t e t rahydrocan nab ino lwere much h igher in p lants grown under coo lcondi t ions than in p lants grown under warmcond i t i ons . However , under warm g row thcondi t ions Nepal and Jamaica plants di f fermo re f rom I l li no is and Pan am a p lants thanbetween each o ther . A l though the photo-synthet ic ra te and cannabino id content gener-

al ly separate the populat ions into two groups,the data f rom these two param eters do notcompletely correspond. This suggests thatm ore than one parameter m ust be used inassessing ecotypic di f ferent iat ion in Cannabisand probably in other species.

D i s c u s s io n a n d C o n c l u s i o n sI t has long b e e n argued tha t the genusCannabis may co nsist of more than one species.Certain authors have considered C. indica Lam.to be the p lant which grows w i ld in t rop ica lAsia and is used as a source of Marihuanaand hashish ( :1,6] . Cannabis sadva is assumedto be the species which for many years hasbeen under cu l t i va t ion in var ious par ts o f theworld as a source of rope. However, currentfeel ing among most botanists is that Cannabisi s a monotyp ic genus w i th many ecotypesand cu l ti va ted races [ 1 ,6 ] .

Since the plant has been us ed as a sourceof drug, f iber, and oi l for thousands of yearsone can assume a large amount of breedingand selection for va r ious qua l it ies has occurred,making the taxonomy and evo lu t ion o f

Cannabis very compl icated. In the U.S. thes i tuat ion is aggravated by the in t roduct ion,

over long periods of t ime, of Cannabis f romvar ious sources inc lud ing Eu rope and Chinaand the m ore recent smal l-scale int rod uct io nof t rop ica l Cannabis for i l l ici t use as a sourceof drug.

A l t hough the p las t i c i t y o f Cannabis haslong be en recognized, i t has not b ee n com -pletely understood [6 ] . I t is kn ow n, forexample, that the spec ies exh ib i ts morpho-log ica l var ia t ion wh en grow n under var iousenv i ronmenta l cond i t ions and that the p lantgrows under a var ie ty o f cond i t ions and hasbeen col lected in various types o f habi tats inthe U ni ted Sta tes r5 ] . Eve n sex in the spec iesis in fluenced by var ious env i ronmenta l fac torssuch as UV l ight [9 ] , day- length [101,temperature [1 1] and n i t rogen co nce ntrat ion

in the soi l solut ions [121. However, ourresul ts c learly show that physiological andbiochemical ecotypes are present in Cannabisand that these ecotypes can be dist inguishedf rom each o ther by the i r ra te o f photosyn-thes is , ch lorophy l l and cannabino id content .Cool growth condi t ions d is t ingu ish theseecotypes m o re c lear ly than w arm gro wthcondi t ions.

E x p e r i m e n t a lCannab is sa t i re s e e d s w e r e c o l l e c t e d fr o m m a t u r e

p l a n t s i n e a s t c e n t ra l I l l i n o i s , n e a r t h e c o a s t i n P a n a m a ,

a t a b o u t 2 0 0 0 f t e l e v a t i o n i n c e n t r a l J a m a i c a , a n d a ta b o u t 6 0 0 0 f t n e a r K a t m a n d u , N e p a l .

S e e d s f r o m t h e s e l o c a l i ti e s w e r e s o w n i n l o a m - f i l l e d

8 - i n , c l a y - p o t s i n c u b a t e d a t 2 2 ° f o r 1 1 d a y s a n d t h e n

t ra n s f e rr e d t o t w o g r o w t h c h a m b e r s w h e n t h e s e e d l i n g s

w e r e a b o u t 4 c m t a l l . T h e g r o w t h c h a m b e r s w e r e

p r o g r a m m e d t o a p p r o x i m a t e t r o p i c a l a n d te m p e r a t e

t e m p e r a t u r e s . T h e t e m p e r a t u r e o f t h e " w a r m c h a m b e r ' "

w a s 3 2 ° d u r i n g t h e d a y a n d 2 3 ° a t n i g h t , w h i l e t h a t o f

t h e c o o l c h a m b e r w a s 2 3 ° a n d 1 6 °; d a y l e n g t h w a s 1 5 h r .

L i g h t i n t e n s it y , g e n e r a t e d b y 1 6 c o o l w h i t e f l u o r e s c e n t

a n d e i g h t 2 5 W i n c a n d e s c e n t l a m p s w a s a p p r o x i m a t e l y

4 0 0 0 0 l u x n ea r th e t o p o f t h e p l a n ts . S i n c e Cannab is is a

d i o e c i o u s s p e c i e s a n d t h e r e m a y b e d i f f e r e n c e s

b e t w e e n m a l e a n d f e m a l e p l a n t s , o n l y m a t u r e y e ts e x u a l l y u n d i f f e r e n t i a t e d p l a n t s w e r e u s e d f o r a l l

m e a s u r e m e n t s .

M e a s u re m e n t o f p h o to s y n t h e s is . N e t p h o t o s y n t h e s i s ,

h e r e a f t e r r e f e r r e d t o a s " p h o t o s y n t h e s i s " . a n d t r a n s p i r a -

t i o n o f p l a n t s w e r e m e a s u r e d u s i n g a s e m i - c l o s e d

i n f r a - r e d g a s a n a l y s i s s y s t e m . P l a n t s h o o t s w e r e

e n c l o s e d in a n a s s i m i la t i o n c h a m b e r i n w h i c h l i g h ti n te n s i t y, a i r t e m p e r a t u r e , w i n d s p e e d , r e l a ti v e h u m i d i t y

a n d C O 2 c o n c e n t r a t i o n w e r e p r e c i s e l y c o n t r o l l e d , A i rw a s c i r c u l a t e d b e t w e e n t h e a s s i m i la t i o n c h a m b e r a n d a

G r u b b P a r so n i n f r a - re d g a s a n a l y z e r w h i c h d e t e c t e dc h a n g e s i n C O 2 c o n c e n t r a t i o n s i n th e a i r s t r e a m . F o r a

d e t a i l e d d e s c r i p t i o n o f t h e g a s a n a l y s i s s y s t e m a n d

m e a s u r e m e n t t e c h n i q u e s s e e B a z z a z a n d 1 3 o y e r [ 1 3 ] .L ig h t sa tu ra t i on cu rv es a t 25 °, 70 ~= 2% re la t i ve



18 F .A . BAZZAZ, D . DUSEK, D . S . SE IGLER AN D A. W. HANEY

h u m i d i t y , 5 m s ec - ~ w i n d s p e e d a n d 3 0 0 - 3 1 0 p p m C 0 2w e re e s ta b l i s h e d f o r p l a n t s f r o m t h e f o u r p o p u l a t i o n s .A f te r a s teady ra te o f CO2 re lease in the dark wasa c h i e v e d , l i g h t i n t e n s i t y w a s i n c re a s e d s t e p w i s e b yre m o v i n g l a y e rs o f c h e e s e c l o t h p l a c e d b e tw e e n t h ea s s i m i l a t io n c h a m b e r a n d t h e l i g h t s o u rc e a b o v e i t . A teach o f severa l i n tens i t i es , repeated pho tosyn the t i cm e a s u re m e n ts w e re m a d e a f t e r a s t e a d y rat e o f C O 2e x c h a n g e h a d b e e n a c h i e v e d b e tw e e n t h e e n c l o s e dp l a n t s h o o t a n d t h e a i r c i r c u l a t in g i n th e s y s te m . U n d e r8 0 0 0 0 l u x l i g h t i n t e n s i t y a n d t h e c o n d i t i o n s s p e c i f i e da b o v e f o r r e l a t i v e h u m i d i t y , w i n d s p e e d , a n d C O 2c o n c e n t ra t i o n , p h o to s y n th e s i s o f p l a n t s f r o m t h e f o u rp o p u l a t io n s fr o m b o t h g r o w t h c h a m b e r s w a s m e a s u re dat 20 °. 25 ° and 30 ° amb ien t tem pera tu re . A dd i t i o na lp h o to s y n th e t i c m e a s u re m e n ts w e re m a d e a t 1 5 ° o np l a n ts g r o w n in t h e " c o o l c h a m b e r " . A t e a c h se t ofe n v i r o n m e n ta l c o n d i t i o n s a b o u t 1 0 re a d i n g s o n e a c hfo 4 d i f f e re n t i n d i v i d u a l p l a n t s f r o m e a c h p o p u l a t i o n

w e r e m a d e .De t e rm i n a t i o n o f c h l o ro p h y l l a n d c a n n a b m o i d s .C h l o ro p h y l l f r o m m a tu re l ea v es w a s e x t ra c te d w i t ha c e to n e , a n d o p t i c a l d e n s i t y m e a s u re m e n ts w e re m a d ea t 6 4 5 a n d 6 6 3 n m w i t h a B e c km a n D U s p e c t r o p h o t o -m e te r . To ta l c h l o ro p h y l l w a s c a l c u l a t e d a c c o rd i n g t ot h e m e t h o d o f A r n o n [ 1 4 ] . C a n n a b i n o id s [ c a n n a b i d i o l( C B D ) , t e t r a h y d r o c a n n a b i n o l ( T H C ) a n d c a n n a b in o l( C B N ) ] w e r e e x t r a ct e d w i t h p e t r o le u m e t h e r f r o mw h o l e s h o o t s o f s e x u a l l y u n d i f f e re n t i a t e d p l a n t s . Th ee x t ra c ts w e re a n a l y z e d b y G L C o n a 3 m x 3 m m s ta in l e sss te e l c o l u m n p a c k e d w i t h 5 % S E-3 0 s i l i c o n e li qu i dp ha se , T h e c h r o m a t o g r a p h w a s p r o g r a m m e d t oinc rease te mp era tu re f rom 150 ° to 250 ° a t 4 deg per

m i n a f t e r a n 8 - ra i n p o s t - i n j e c t i o n p e r i o d . U n d e r t h e s ec o n d i t i o n s t h e a v e ra g e re t e n t i o n t i m e f o r t h e t h re ee a n n a b i n o i d s w a s a b o u t 1 7 m i n . C e t yl a lc o h o l w a sfo u n d t o b e a s u i t a b l e i n t e rn a l s t a n d a rd a n d w a s u s e dto qu a n t i t a t e t h e re s u l ts . Th e c a n n a b i n o i d s t a n d a rd s

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photosynthesis and cannabinoid content

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