Köves, Erzsébet

Varga, Magdolna

English

Varga Magdolna

Köves Erzsébet

University of Szeged

M E T H O D O L O G I C A L E X A M I N A T I O N S C O N C E R N I N G T H E G R O W I N G O F O A T S E E D L I N G S F O R A U X I N - A S S A Y By M A G D A L É N A V A R G A a n d E L I S A B E T H K Ö V E S I n s t i t u t e f o r P l a n t P h y s i o l o g y o f U n i v e r s i t y , S z e g e d , H u n g a r y I n t r o d u c t i o n F o r d e t e r m i n a t i o n of g rowth - subs t ances , especial ly f o r aux in -as say , t he most w ide - sp read ing m e t h o d is the use of t he Avena s t r a i g h t - g r o w t h coleopt i le test. Acco rd ing to o u r obse rva t ions , t he mos t cr i t ical f a c t o r in such e x a m i n a -t ions is t o ob t a in oa t co leopt i les of su i table size, i. e. 2 0 m m , a t t h e t ime requi red . T h e r e f o r e it is v e r y i m p o r t a n t t h a t t he t i m e needed f o r t he g r o w i n g of t he oa t seedlings should be s t a n d a r d i z e d by keeping precisely all the ex te rna l cond i t ions . T h e most f a v o u r a b l e c i r cums tances of the g rowing of o a t co leopt i les were s tudied t h o r o u g h l y b y several a u t h o r s (1, 3, 6, 7). H o w e v e r , no pub l i ca t i on is k n o w n dea l ing w i t h t he role a n d e f f ec t of t he inh ib i t ing substances , occu r r ing in t he husks of t he corns, on t he g r o w t h a n d aux in-sens i t iv i ty of t he co leop-tiles. T h e presence of g r o w t h - a n d ge rmina t i on inh ib i t ing substances in t he husks is k n o w n f o r a long t ime (2), h o w e v e r , t he wa te r - so lub le inh ib i to r s in husks A-ere s tudied in deta i l on ly r ecen t ly ( 4 ) . W i t h p a p e r c h r o m a t o g r a p h y , K Ö V E S ( 4 ) sepa ra t ed six inh ib i t ing subs tances f r o m the w a t e r - e x t r a c t of o a t - h u s k s a n d in the course of t he i den t i f i c a t i on they p r o v e d to be phenol ic ac ids a n d the i r depsides a n d po lydeps ides , respec t ive ly . Whi le g r o w i n g the test seedlings, of course , w e must reckon w i t h the i r presence, the i r leaching i n t o the m e d i u m , a n d the re fo re , w i th the i r b io logical e f f ec t on t he ge rmina t i ng co rns a n d on t he deve lop ing seedlings. T h e a im of th i s p a p e r is to ana lyse the role th i s f a c t o r . R e s u l t s a n d c o n c l u s i o n s Avena sativa L. of „FLEISCHMANN" va r i e ty , ha rves ted in 1959 w a s used f o r e x p e r i m e n t a l ma te r i a l . T o c lcar u p the c f f cc t of the husk , expe r imen t s in P L T R I d ishes w e r e m a d e as p r e l i m i n a r y w o r k . 1 0 0 — 1 0 0 oa t - co rns were ge rmina t ed on f i l t e r p a p e r w e t -40 M A G D A L E N A V A R G A A N D E L I S A B E T H K Ö V E S ted wi th w a t e r of equal quan t i t y . The 1st sample con ta ined oa t s wi th husk , t h e 2d o n e oa t s w i thou t husk and the 3d the same bu t the husks were pu t bes ide t h e naked corns. T h e potency and the percen tage of the ge rmina t ion o b t a i n e d in these three samples are shown in Fig. 1. F r o m the d a t a w e can d r a w the conclusion tha t the husks re ta rd the ge rmina t ion on ly in p a r t owing t o t h e i r inh ib i to r con ten t , since the potency of the germina t ion observed in t h e 3d sample is g rea te r than tha t of the intact oa t s (1st sample) but fal l beh ind the ge rmina t ion of the dehusked corns (2d sample) . In add i t ion to the i nh ib i t o r con ten t the husks delay the germinat ion because they a re a mechanical obs tac le , f u r t h e r because they retard the swelling of the corns; t ha t was d e m o n s t r a t e d by measur ing the swell ing of oats with and wi thou t husk. Fig . 1. P o t e n c y of g e r m i n a t i o n o f o a t s w i t h a n d w i t h o u t h u s k a t 2 3 ° C . ( A v e r a g e of 3 p a r a l l e l s a n d 2 r e p e t i t i o n s ) The results concerning the intensity of g r o w t h of the coleopti les a re ind i -ca ted in Fig. 2. Accord ing to the da t a to a t t a in the length of 20 m m , 102 h o u r s a re required fo r the seedlings grown f r o m huskless corns and 139 hours fo r tha t of wi th husk; in the la t ter case the lag is one day and a ha l f . Thus , t h e d i f f e r ence observed in the germinat ion at the beginning is no t equa l ized w h e n the 20 m m length is a t ta ined . T h e exper iments were cont inued wi th oa t s sown into sand. As a m e d i u m , r iver -sand was used a f t e r wash ing tho rough ly and annea led at 7 0 0 ° C . O a t s w i th and wi thou t husk were swelled fo r 8 hours in t a p - w a t e r and sown i n t o sand in glass dishes wet ted wi th water co r respond ing to 8 0 % w a t e r - c a p a c i t y . T h e germina t ion took place in d a r k , at 2 3 ° C and 95°/o re la t ive h u m i d i t y . In the course of the germina t ion , repeated three times, it was seen tha t t h e coleopt i les of the seedlings f r o m dehusked corns genera l ly a t t a ined the length of 20 m m a f t e r 84 hours while the seedlings f rom in tac t corns required 93 hours fo r the same cond i t ion . Hence, the d i f fe rence in d e v e l o p m e n t is no t so much as in P E T R I -dishes. E X A M I N A T I O N S FOR A U X I N - A S S A Y 41 F u r t h e r on we wan ted to k n o w h o w we can reckon wi th the wash ing ou t of the inh ib i to rs f r o m the husks in to the sand and therein, w i th the e f f e c t on the d e v e l o p m e n t of the seedlings. So, the sand used earl ier was dr ied a t 105 C , sifted out , wet ted again and used repea tedly as a med ium. In one series husked corns were employed , whi le in the o ther one a lways unhusked ones, 9 t imes repeated, and the t ime needed to reach the 20 m m coleopti le length , I. e. the coleoptile length stated dur ing the s t a n d a r d t ime in the basic exper iment (84 hours for dehusked and 93 hours fo r husked corns) was recorded. Especial ca re has been t aken cf the p r o p e r iden t i ty of the exper imenta l f ac to r s in o rde r to on / be able to ascribe the di f ferences , occur r ing in the g rowth , a lone to the biologi-cal e f fec t of the substances accumula ted in the sand. Results are shown in Fig. 3. T h e average length of the coleoptiles, a t t a ined du r ing the s t a n d a r d t ime, g rown f i rs t in pure sand, is identical wi th tha t of the cont ro l . Sown the 2d and 3d t ime, in the case of both husked and dehusked corns, the coleopti les grew longer du r ing the same time, tha t is to say some sort of s t imu la to ry e f f ec t was shown; later , however , the length of the coleoptiles g radua l ly decreased. In the case of the husked corns, sown the 5th, 6 th and 7th t ime, a de f i n i t e inhibi t ing e f fec t appea red which was in full agreement with our expec ta t ions on the basis of our previous exper imen ta l results (4). Accordingly , the s t imu-lat ion observed in the 2d and 3d sowing is cer ta in ly caused fo r the most pa r t by the s t imula to ry e f fec t of the ac t ive substances being washed ou t f r o m the husks and present in the sand still in low concen t ra t ion ; which, however , if accumula ted in larger quan t i t y , soon result a r emarkab le inhibi t ion. I t was f a i r l y surpr is ing the excessive e longat ion of the coleopti les noted in the 8th and 9th sowing of husked corns. As these seedlings were th inner and curved , even in most cases d e f o r m e d , the phenomenon may be a t t r i bu t ed to the toxic e f f ec t of the inhibi t ing substances and o ther decomposi t ion p roduc t s CORNS WITH HUSK CORNS WITHOUT HUSK CORNS+ UUSK STRIPPED OFF t— ¿ g 50 72 84 Q6 108 120 132 « 1 " 0 U Q S Fig. 2 . G r o w t h of c o l e o p t i l c s f r o m h u s k e d a n d d e h u s k e d o a t s a t 2 3 ° C . ( A v e r a g e of 3 p a r a l l e l s a n d 2 r e p e t i t i o n s ) 1 s t 2 n a 3 r d * t h 5 m 6 № 7 t n 8 m 9 m PLANTING f j f , 3 ' T h c ¡cngth o f co l cop t i l e s a t t a i n e d in t h e r e p e a t e d s o w i n g d u r i n g t h e s t a n d a r d t i m e (93 h o u r s in husked a n d 84 h o u r s in d e h u s k e d c o r n s ) . A v e r a g e o f 3 p a r a l l e l s a n d 2 r e p e t i t i o n s M A C D A L E N A VARGA A N D E L I S A 3 E T H KOVES C O R N S WITH 14USK CORNS WITHOUT HUSK (secreted by the roots or microorganisms) in the sand. I t is n o t e w o r t h y t h a t even the ge rmina t ion itself a p p e a r e d also uneven wi th repea ted sowing of hus-ked corns. O n the o ther hand , the mild s t imula tory e f f ec t could be not iced up to the 5 th sowing of the unhusked corns, however , no inhibi t ion fo l lowed . In this case the biological e f fec t of the substances washed f r o m the grains o r p e r h a p s secreted by the roots m a y be assumed. T o suppor t the a b o v e assumpt ions e f f o r t s have been m a d e to d e m o n s t r a t e p a p e r - c h r o m a t o g r a p h i c a l l y these substances f r o m the sand. For this pu rpose the water-soluble substances of 10 g husks were ex t rac ted in 1 % N a H C O s a n d acidif ied to p H 3 shaken in to ether, then in o rde r to separa te the c o m p o u n d s c h r o m a t o g r a p h e d in i s o p r o p a n o l - a m m o n i a - w a t e r 1 0 : 1 : 1 solvent on SCH & SCH 2043 b paper . In the same way the substances accumula ted in t h e sand —used 5 to 7 t imes — were also extracted and l ikewise c h r o m a t o g r a p h e d . T h e spots developed on the ch roma tog rams were m a r k e d in U V light and to iden-t i f y them sprayed with reagents for acids and phenol ic c o m p o u n d s (4, 5, 8). C o m p a r i n g the c h r o m a t o g r a m s obtained f r o m the ex t rac t s of the husks a n d sand could be stated as fo l lows : In sowing husked corns p r imar i ly the presence of inhibi tors released f r o m the husks is considerable in the sand, namely , large molecular t ann ic acids, p -oxybenzoic acid, ferul ic acid, p -coumar ic acid and salicylic acid were found there in ; besides, in smal ler a m o u n t , seme o t h e r sub-stances — not or ig inat ing f r o m the husks — could be also demons t r a t ed f r o m the sand samples. These substances a re p re sumab ly secreted by the roots or t hey may be the metabol ic p roduc t s of the ge rmina t ing corns and of the mic ro -organisms, respectively. T h e results of the above exper iments suggest t h a t the most f a v o u r a b l e con-di t ions fo r oat-seedl ing cu l t iva t ion would be to sow dehusked corns a l w a y s in E X A M I N A T I O N S FOR A U X I N - A S S A Y 4 3 pure sand. As the dehusk ing p rac t i ca l ly can not be carr ied ou t in serial expe-riments, good test-seedlings can be ob ta ined even if the oa t is sown husked but wi th the v iew to the s t a n d a r d i z a t i o n of the t ime fo r cu l t iva t ion the sand is to be used but once. An o the r i m p o r t a n t p rob lem concerning the g rowing of the oat-seedl ings is to ob ta in the max ima l auxin-sens i t iv i ty of the coleopti les which d e p e n d s main ly on the va r i e ty and the c i rcumstances of the g rowing of the seedlings. T h e ques-t ion is, w h e t h e r the inh ib i t ing substances leached into the med ium or secreted, have or not any in jur ious e f f ec t a lso on the auxin-sens i t iv i ty which m a y be expected on the basis of prev ious exper imenta l results. For this pu rpose the husked and dehusked corns were repea ted ly sown in the same medium in the identical w a y as described above, and fo l l owing the 1st, 3d , 5th and 7th sowing, the g rowth of the 5 m m sections cut subapical ly f r o m the coleoptiles of 20 m m long, were de te rmined in I A A concen t ra t ion -series. N o def in i te cor re la t ion could be found between the auxin-sens i t iv i ty of the coleopti les of the dehusked corns and the q u a n t i t y of the biological ly ac t ive substances leached into the med ium or secreted; on the o the r hand , in the case of the husked corns the corre la t ion could posi t ively be recognized. These results a re in Fig. 4. T h u s our supposi t ion p roved to be r ight , t h a t is, the sensitivity of the coleopti les to I A A is p ropo r t i ona t e ly decreasing wi th the increasing concen t ra t ion of the substances washed ou t f r o m the husks and accumula ted in the med ium, which is mani fes ted in the g radua l decrease of IAA CONCENTRATION Fig . 4. G r o w t h of c o l e o p u l e s ec t ions in I A A obse rved in 1st ( ), 3 d ( ) , 5 th ( — — ) a n d 7 t h ( ) sowing in t h e same m e d i u m 44 MAGDALENA VARGA A N D ELISABETH KÖVES the g r o w t h react ions observed in the consecut ive sowings. C o n s e q u e n t l y t o use the same med ium repea ted ly is to be avo ided , even f o r the sake of secur ing the m a x i m a l sensi t ivi ty of the test. S u m m a r y In c o n n e c t i o n w i t h t h e g r o w i n g of o a t - s e e d l i n g s f o r a u x i n - a s s a y , t h e p o t e n c y a n d p e r c e n t a g e o f t h e g e r m i n a t i o n , t h e r o l e a n d e f f e c t of t h e s u b s t a n c e s w a s h e d o u t i n t o t h e s a n d , e s p e c i a l l y of t h e i n h i b i t i n g s u b s t a n c e s of t h e h u s k s o n t h e g r o w t h a n d a u x i n - s e n s i t i v i t y o t t h e s e e d l i n g s , h a v e b e e n d e m o n s t r a t e d . T h e p r e s e n c e of t h e h u s k — b e s i d e o t h e r f a c t o r s — d e l a y s t h e g e r m i n a t i o n o f t h e c o r n s a s w e l l as t h e t i m e r e q u i r e d t o a t t a i n t h e l e n g t h of 2 0 m m o f t h e c o l c o p t i l e s , c h i e f l y d u e t o t h e i n h i b i t i n g c o n t e n t . T h i s d e l a y is c o n s i d e r a b l y g r e a t e r in P E T R I - d i s h e s t h a n in s a n d . I h e i n h i b i t i n g s u b s t a n c e s o f t h e h u s k s a r c f o r t h e m o s t p a r t w a s h e d o u t b y w a t e r a n d a r e a c c u m u l a t e d in t h e m e d i u m , w h e n c e t h e y c a n b e d e m o n s t r a t e d w i t h p a p e r c h r o m a t o g r a p h i c m e t h o d ( l a r g e m o l e c u l a r t a n n i c a c i d s , p - o x y b e n z o i c a c i d , p - c o u m a r i c a c i d , f e r u l i c a c i d , s a l i -c y l i c a c i d e t c . ) . U s i n g r e p e a t e d l y t h e m e d i u m , t h e w a s h e d o u t s u b s t a n c e s f i r s t s o m e w h a t s t i m u l a t e , l a t e r — in h i g h e r c o n c e n t r a t i o n - r e m a r k a b l y i n h i b i t t h e g e r m i n a t i o n o f t h e c o r n s , t h e g r o w t h a n d d e v e l o p m e n t o f t h e s e e d l i n g s , f u r t h e r t h e y d e c r e a s e a l s o t h e a u x i n -s e n s i t i v i t y o t t h e c o l c o p t i l e s . T h u s t o s t a n d a r d i z e t h e t i m e r e q u i r e d f o r t h e g r o w i n g o f t h e Avena t e s t s e e d l i n g s a n d t o s e c u r e t h e m a x i m a l a u x i n - s e n s i t i v i t y t h e m e d i u m m u s t n o t b e used m o r e rh . in n n r p R e f e r e n c e s (1) BENTLEV J A : N a t u r a l l y - o c c u r r i n g a u x i n s a n d i n h i b i t o r s . A n n . R e v . P l a n t P h y s i o l . A 4 7 — a u ( 1 9 3 8 ) . (2) EVENARI, M . : G e r m i n a t i o n i n h i b i t o r s . Bot . R e v . 15, 1 5 3 — 1 9 4 ( 1 9 4 9 ) . ( 3 ) H U L L , H . M „ W E N T , F M „ AND Y A M A D A , N . : F l u c t u a t i o n s i n s e n s i t i v i t y o f t h e A v e n a t es t d u e t o a i r p o l l u t a n t s . P l a n t P h y s i o l . 2 9 , 1 8 2 — 1 8 7 ( 1 9 5 4 ) . (4) KÖVES, E : P a p i e r c h r o m a t o g r a p h i s c h e U n t e r s u c h u n g e n d e r ä t h e r l ö s l i c h e n k e i m u n g s - u n d w a c h s t u m s h e m m e n d e n S t o f l e d e r H a f e r s p e l z e . A c t a B io l . S z e g e d . 3 , 1 7 9 — 1 8 7 ( 1 9 5 7 ) (5) ^OVES, E . , A N D VARGA, M . : G r o w t h - i n h i b i t i n g s u b s t a n c e s in r i c e - s t r a w . A c t a B io l . S z e g e d ! *•» 1 J 16 (1 7 J o ) . (6) NITSCH, J P . , AND NITSCH, C . : S t u d i e s on t h e g r o w t h o f c o l e o p t i l e a n d f i r s t i n t e r n o d e s e a i o n s . A n e w , s e n s i t i v e , s t r a i g h t - g r o w t h t e s t f o r a u x i n s . P l a n t P h y s i o l . 3 1 , 9 4 — 1 1 1 (7) TVCE, G M : T h e g r o w t h o f s e g m e n t s of A v e n a c o l c o p t i l e s in d i s t i l l e d w a t e r a n d in i n d o l v l a c e t i c a c i d d u r i n g d i f f e r e n t m o n t h s of t h e y e a r . A n n . B o t . 2 1 , 4 6 1 — 4 6 4 ( 1 9 5 7 ) ( } «AIRUAL- M " a n d , K ö v e . s > E " V o r k o m m e n , V e r t e i l u n g u n d q u a n t i t a t i v e V e r ä n d e r u n g d e s f . o « ! S = l n " l n e n O r g a n e n d e r B o h n e n p f l a n z e . N a t u r w i s s . 4 5 , 4 6 8 - 4 6 9 ( 1 9 3 8 ) . 

Methodological examinations concerning the growing of oat seedlings for auxin-assay

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Methodological examinations concerning the growing of oat seedlings for auxin-assay

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