A . C A R R U T H E R S A N D J . F . T . O I . D F I E L D1
Received for publication February 6, 1964
F o r m a l d e h y d e s o l u t i o n is o n e of t h e m o s t effective a n d con- venient r e a g e n t s for c o n t r o l l i n g m i c r o b i o l o g i c a l activity in t h e beet factory process. T h e total f e r m e n t a t i o n i n c o n t i n u o u s diffu- sion is f r e q u e n t l y less t h a n in b a t t e r y o p e r a t i o n b u t t h e c o r r o - sive effects of acid p r o d u c t i o n a r e m o r e a p p a r e n t in c o n t i n u o u s diffusers a s t h e f e r m e n t a t i o n t e n d s t o b e localized t o w a r d s t h e juice e n d of t h e dilTuser. W h e r e a s heavy f o r m a l d e h y d e d o s i n g was c o m m o n in t h e r e t u r n w a t e r systems of b a t t e r y diffusers, with t h e a d o p t i o n of c o n t i n u o u s diffusers t h e use of f o r m a l d e - hyde at t h e juice e n d has g r e a t l y i n c r e a s e d a n d m a y e x c e e d I lb of formalin ( 4 0 % f o r m a l d e h y d e ) p e r ton of b e e t . In t e r m s of a n h y d r o u s f o r m a l d e h y d e , t h i s is e q u i v a l e n t to a b o u t 0.1 p e r 100 sugar, o r a b o u t 1 % o f t h e n o n - s u g a r s i n j u i c e a n d . since m u c h of the f o r m a l d e h y d e is c a r r i e d f o r w a r d in t h e r a w j u i c e to process, the fate of f o r m a l d e h y d e in t h e p u r i f i c a t i o n stages is of c o n s i d e r - able i m p o r t a n c e .
D e t e r m i n a t i o n o f f o r m a l d e h y d e
F o r m a l d e h y d e was d e t e r m i n e d c o l o r i m e t r i c a l l v b y t h e m e t h o d of Xash (1)- w h i c h d e p e n d s on t h e synthesis of d i a c e t v l d i h y r o l u - tidine b y t h e H a n t z s c h r e a c t i o n b e t w e e n f o r m a l d e h y d e a n d ace- tvlacetone in t h e p r e s e n c e of excess a m m o n i u m salt. Each test solution was d i l u t e d to a f o r m a l d e h y d e c o n c e n t r a t i o n in t h e range 1 to 8 p p m a n d t h e a b s o r p t i o n was m e a s u r e d at 410 mµ in a 1 cm cell.
F r e e f o r m a l d e h y d e i n r a w j u i c e
A l t h o u g h f o r m a l d e h y d e is a very r e a c t i v e s u b s t a n c e , t h e bac- terial effect is n o t sensibly d i m i n s h e d e v e n after several h o u r s contact w i t h r a w j u i c e . C o n s e q u e n t l y t h e i n h i b i t i v e effect of f o r m a l d e h y d e c a n b e t r a n s m i t t e d b y t h e j u i c e f l o w t o r e l a t i v e l y inaccessible p a r t s o f t h e c o u n t e r - c u r r e n t diffusion svstetn t o a n extent n o t possible w i t h , for e x a m p l e , c h l o r i n e w h i c h i s a n ex- tremely efficient b a c t e r i c i d e w h e n a d d e d t o r a w j u i c e b u t r e a c t s so r a p i d l y w i t h j u i c e c o n s t i t u e n t s t h a t after a few m i n u t e s con- tact no i n h i h i t i v e effect r e m a i n s .
I t i s t h e r e f o r e a p p a r e n t t h a t t h e m a j o r i t y o f t h e f o r m a l d e - hyde i n t h e j u i c e s t r e a m i s n o t d e c o m p o s e d b y o r i r r e v e r s i b l y with juice c o n s t i t u e n t s .
1 Research Laboratories. British Sugar Corporation Limited, T h e Grange, Brameote, Nottingham. England.
2 Numbers in parentheses refer to literature cited.
106 J O U R N A L OF T H E A. S. S. B. T.
T h e v a l i d i t y o f t h i s d e d u c t i o n w a s i n v e s t i g a t e d b y a d d i t i o n o f k n o w n a m o u n t s o f f o r m a l d e h y d e t o r a w j u i c e ; t h e s a m p l e s w e r e h e a t e d a t 8 0oC f o r 3 0 m i n u t e s a n d t h e f o r m a l d e h y d e c o n - t e n t w a s t h e n d e t e r m i n e d b y t h e N a s h p r o c e d u r e . A f o r m a l i n u s a g e o f 1 l b p e r t o n o f b e e t i s e q u i v a l e n t t o a n a v e r a g e f o r m a l d e - h y d e a d d i t i o n o f a b o u t - 0 2 % o n j u i c e b u t , s i n c e f o r m a l i n i s g e n e r a l l y a p p l i e d b y i n t e r m i t t e n t s h o c k d o s i n g , t h e e x p e r i m e n t w a s c o n d u c t e d w i t h f o r m a l d e h y d e a d d i t i o n s r a n g i n g f r o m . 0 0 1 % t o 0 . 1 5 % o n j u i c e . T h e r e c o v e r y o f f o r m a l d e h y d e a s a p e r c e n t a g e o f t h e a p p l i e d d o s a g e i s r e c o r d e d i n F i g u r e 1 .
F i g u r e I.—Recover)' of f o r m a l d e h y d e from r a w j u i c e .
E v e n a t t h e lowest dosage, 5 0 % o f t h e a p p l i e d f o r m a l d e h y d e was r e c o v e r e d a n d t h e r e c o v e r y rose r a p i d l y t o a l m o s t 9 0 % w i t h a f o r m a l d e h y d e a d d i t i o n o f . 0 2 % o n juice, a n d t o a b o u t 9 8 % a t h i g h e r dose r a t e s . F o r m a l d e h y d e a p p l i e d d u r i n g diffusion i s in c o n t a c t w i t h a m i x t u r e of j u i c e a n d p a r t i a l l y e x h a u s t e d cos- settes s o t h a t t h e possibility arises o f c o m b i n a t i o n w i t h p r o t e i n o r o t h e r p u l p c o n s t i t u e n t s . O n t r e a t i n g m i x t u r e s o f j u i c e a n d p u l p w i t h f o r m a l d e h y d e a t a level o f 0 . 0 1 % o n t o t a l l i q u i d s how- ever, t h e recoveries r a n g e d from 7 8 t o 8 3 % w h i c h a r e very simi- l a r t o those o b t a i n e d w i t h j u i c e a l o n e .
I t i s t h e r e b y c o n f i r m e d t h a t t h e m a j o r i t y o f t h e f o r m a l d e - h y d e i n t r o d u c e d i n t o t h e diffuser r e m a i n s in a free or very lightly b o u n d f o r m .
T h i s c o n c l u s i o n i s p e r h a p s r a t h e r u n e x p e c t e d i n v i e w o f the c o m m o n s u p p o s i t i o n t h a t t h e b a c t e r i c i d a l effect o f f o r m a l d e h y d e m a y b e d u e t o c o m b i n a t i o n w i t h b a c t e r i a l p r o t e i n . D e s p i t e the large excess o f p r o t e i n n i t r o g e n o v e r a d d e d f o r m a l d e h y d e , very l i t t l e i f a n y o f t h e f o r m a l d e h y d e r e a c t e d w i t h p u l p c o n s t i t u e n t s .
VOL. 13, No. 2, J I I Y 1964 107 T h e s l i g h t m e a s u r e d loss of- f o r m a l d e h y d e in j u i c e a p p e a r s t o h e real a n d was n o t d u e t o i n t e r f e r e n c e b y t h e j u i c e a m i n o acids i n t h e H a n t z s c h r e a c t i o n since 0 . 0 2 % f o r m a l d e h y d e c o u l d b e r e c o v e r e d q u a n t i t a t i v e l y from s y n t h e t i c s o l u t i o n s c o n t a i n i n g 20 moles of e i t h e r g l u t a m i n e or g l y c i n e p e r m o l e of f o r m a l d e h y d e . P r o b a b l y a s m a l l a m o u n t of t h e f o r m a l d e h y d e c o m b i n e s w i t h some j u i c e c o n s t i t u e n t s since t h e form o f t h e . c u r v e a p p r o x i m a t e s to t h a t of a s e c o n d o r d e r r e a c t i o n a n d t h e u n a c c o u n t e d f o r m a l d e - hyde a t t h e h i g h dose levels i n c r e a s e d w i t h i n c r e a s i n g r a w j u i c e brix. T h i s loss w o u l d h a v e o n l y a slight effect in r e d u c i n g t h e a m o u n t o f f o r m a l d e h y d e c a r r i e d t h r o u g h w i t h t h e j u i c e t o process.
Since a m i n o acids can b e d e t e r m i n e d b y t h e S o r e n s e n f o r m o l titration, based o n t h e r e a c t i o n b e t w e e n excess f o r m a l d e h y d e a n d a m i n o a c i d t o f o r m a n N - m e t h y l e n e d e r i v a t i v e , t h e e x t e n t o f t h i s reaction i n j u i c e was i n v e s t i g a t e d . A s t h e r e a c t i o n w i t h a m i n o acids is r e v e r s i b l e , t h e H a n t z s c h r e a c t i o n c a n n o t be u s e d to esti- mate free f o r m a l d e h y d e , since t h e progressive r e m o v a l of formal- dehyde leads t o c o m p l e t e recovery. T h e r e a c t i o n was t h e r e f o r e investigated b y p o t e n t i o m e t r i c t i t r a t i o n t o p H 8.05 o f s o l u t i o n s c o n t a i n i n g 3 3 m - m o l e s o f g l y c i n e p e r l i t r e w i t h f o r m a l d e h y d e addition i n t h e r a n g e 0.06 t o 2 . 5 % . T h e c o n c e n t r a t i o n o f m e t h - ylene d e r i v a t i v e was e s t i m a t e d from t h e t i t r a t i o n a n d t h e c o n c e n - trations o f free a m i n o acid a n d f o r m a l d e h y d e w e r e c a l c u l a t e d b y difference. T h e s e v a l u e s t o g e t h e r w i t h t h e c a l c u l a t e d c o n c e n - tration e q u i l i b r i u m c o n s t a n t s a r e r e c o r d e d i n T a b l e 1 .
Table I.—Interaction of formaldehyde and glycine. (Concentrations in m-moles per litre).
Formaldehyde N-methylene Residual Residual K X 103
added derivative formaldehyde glycine m-moles-1 .litres 20.7 1.8 18.9
41.2 5.1 36.1 78.8 H . 2 67.6 158.6 17.9 140.7 241 21.4 220 320 23.7 296 485 26.5 459 6S0 28.1 602 777 29.0 748
T o o b t a i n m e a s u r a b l e yields o f t h e m e t h y l e n e d e r i v a t i v e , t h e e x p e r i m e n t a l c o n c e n t r a t i o n s o f a m i n o acid a n d f o r m a l d e h y d e were h i g h e r t h a n o c c u r i n factory p r a c t i c e a n d e v e n so. t h e a m o u n t o f m e t h y l e n e d e r i v a t i v e f o r m e d was very low a t t h e lowest f o r m a l d e h y d e a d d i t i o n w h e r e a s very l i t t l e free a m i n o acid r e m a i n e d a t t h e h i g h e s t f o r m a l d e h y d e level. C o n s e q u e n t l y
32.6 29.1 21.4 18.1 11.8 9.3 6.9 4.5 3.1
2.9 4.8 7.7 7.1 8.2 8.6 8.4 10.4 12.5
108 JOURNAL OF THE A. S. S. B. T.
t h e e q u i l i b r i u m c o n s t a n t s c a n n o t b e c a l c u l a t e d very precisely a t these t w o f o r m a l d e h y d e c o n c e n t r a t i o n s b u t n e v e r t h e l e s s i t a p p e a r s t h a t t h e e q u i l i b r i u m c o n s t a n t b e c o m e s s m a l l e r a s t h e for- m a l d e h y d e c o n t e n t i s r e d u c e d . F o r a n a v e r a g e a m i n o a c i d con- c e n t r a t i o n in j u i c e of 15 m m o l e s p e r l i t r e a n d f o r m a l d e h y d e c o n c e n t r a t i o n s of e i t h e r . 0 3 % or . 0 0 3 % a K v a l u e of 0.0029 w o u l d c o r r e s p o n d t o c o m b i n a t i o n o f o n l y a b o u t 3 % o f t h e i o r m a k l e h y d e a n d even if t h e e q u i l i b r i u m c o n s t a n t was as h i g h a s 0.008, o n l y 1 0 % o f t h e f o r m a l d e h y d e w o u l d b e c o m b i n e d w i t h a m i n o acids.
It is t h e r e f o r e c o n c l u d e d t h a t t h e g r e a t e r p a r t of t h e f o r m a l d e - h y d e i n t r o d u c e d i n t o t h e j u i c e e n d o f t h e diffuser will b e c a r r i e d t h r o u g h a s free f o r m a l d e h y d e i n t o t h e c a r b o n a t a t i o n system.
D e g r a d a t i o n o f f o r m a l d e h y d e i n c a r b o n a t a t i o n
T h e effect o f l i m i n g a n d c a r b o n a t a t i o n o n f o r m a l d e h y d e i n r a w juice was e x a m i n e d by c l a r i f i c a t i o n of juice c o n t a i n i n g for- m a l d e h y d e - C 1 4 .
P a r a f o r m a l d e h y d e - C 1 4 (1.16 mg = 100 µ.C) p l u s inactive p a r a f o r m a l d e h y d e (4.8 mg) was s u s p e n d e d in 1.1 ml w a t e r a n d c o n v e r t e d to f o r m a l d e h y d e - C 1 4 by h e a t i n g at 1 0 0 ° C in a sealed t u b e for 5 h o u r s . A test e x p e r i m e n t w i t h i n a c t i v e p a r a f o r m a l d e - h y d e s h o w e d t h a t m o r e t h a n 90%, c o n v e r s i o n was o b t a i n e d i n 3 0 m i n u t e s u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s .
1 ml (91 µc) of t h e r a d i o a c t i v e f o r m e l d e h y d e s o l u t i o n was a d d e d to 30 ml of r a w j u i c e from t h e l a b o r a t o r y m i c r o - b a t t e r y to give a t o t a l f o r m a l d e h y d e c o n c e n t r a t i o n of a b o u t 0 . 0 2 % a n d samples o f t h e u n t r e a t e d m i x t u r e w e r e r e m o v e d for c o u n t i n g , Since f o r m a l d e h y d e is v o l a t i l e , a p p r e c i a b l e losses o c c u r r e d if the r a d i o a c t i v e j u i c e was d r i e d d i r e c t l y for c o u n t i n g b u t this loss was p r e v e n t e d b y c o n v e r t i n g t h e f o r m a l d e h y d e i n t o t h e non- v o l a t i l e d i m e d o n e d e r i v a t i v e o n t h e s a m p l e c o u n t i n g p a n . 2 5 µl a l i q u o t s of t h e r a d i o a c t i v e j u i c e w e r e t r a n s f e r r e d to a tared s a m p l e p a n , 40 µg of i n a c t i v e f o r m a l d e h y d e was a d d e d t o g e t h e r w i t h 100 µl of a 1% s o l u t i o n of d i m e d o n e in m e t h a n o l . W a t e r was a d d e d to give a total v o l u m e of 600 µl to fill t h e source area o f t h e s a m p l e p a n a n d t h e s o l u t i o n was e v a p o r a t e d t o dry- ness u n d e r a n infra r e d l a m p . T h e s a m p l e was r e w e i g h e d and c o u n t e d u s i n g a P h i l i p s e n d w i n d o w G . M . t u b e t y p e 18505 un- der c o n d i t i o n s of s t a n d a r d g e o m e t r y as r e p o r t e d p r e v i o u s l y (2) • T h e c o u n t r a t e was c o r r e c t e d for self a b s o r p t i o n to g i v e a cal- c u l a t e d z e r o a b s o r p t i o n r a t e of 7580 c o u n t s m i n p e r 25 µl of juice.
Vol. 13, No. 2, July 1964 109 A s a m p l e o f t h e r a d i o a c t i v e j u i c e (15 m l ) was h e a t e d t o 8 0 ° C in a c e n t r i f u g e t u b e in a w a t e r b a t h a n d s t i r r e d w i t h a fine jet of air. T h e juice was defecated u s i n g d r y l i m e ( 1 . 6 % ) for 5 m i n - utes a n d gassed w i t h c a r b o n d i o x i d e from a low p r e s s u r e r e s e r v o i r t o t h e first a n d t h e n t o t h e s e c o n d c a r b o n a t a t i o i i end p o i n t s . T h e c a r b o n a t a t i o i i p r e c i p i t a t e s w e r e r e m o v e d bv c e n t r i f u g a t i o n . A sample o f t h e second c a r b o n a t a t i o i i juice was d r i e d a n d c o u n t e d as before a n d it was f o u n d t h a t 7 5 % of t h e i n i t i a l r a d i o a c t i v i t y had passed t h r o u g h i n t o t h e second c a r b o n a t a t i o i i juice.
A l i q u o t s o f t h e s e c o n d c a r b o n a t a t i o n j u i c e w e r e a p p l i e d to a s t r i p of W h a t m a n X o . 1 filter p a p e r , 50 cm in l e n g t h , a n d subjected to h i g h v o l t a g e e l e c t r o p h o r e s i s at 100 v / c m for 15 m i n - utes i n 1 % a m m o n i u m c a r b o n a t e . T h e filter p a p e r was t h e n ex- posed for 24 h o u r s for a u t o r a d i o g r a p h y w i t h Ilford X-ray I n d u s - trial G film. T h e p o s i t i o n s o f t h e b a n d s o f r a d i o a c t i v e p r o d u c t s revealed o n t h e film w e r e r e m a r k e d l y s i m i l a r t o those r e p o r t e d previously (2) for t h e acidic p r o d u c t s p r o d u c e d b y a l k a l i n e de- g r a d a t i o n of fructose or g l u c o s e : t h e e x p e r i m e n t was t h e r e f o r e r e p e a t e d w i t h o u t f o r m a l d e h y d e b u t s u b s t i t u t i n g u n i f o r m l y la- belled fructose-C14 (4.9 mg == 100 µc) p l u s i n a c t i v e fructose
(30 mg) . T h e t w o second c a r b o n a t a t i o i i juices w e r e a p p l i e d side b y side t o t h e s a m e p a p e r s t r i p for e l e c t r o p h o r e s i s , a n d a u t o r a d i o - g r a p h s o f t h e p r o d u c t s a r e r e p r o d u c e d i n F i g u r e 2 a n d 3 . T o o b t a i n c l e a r s e p a r a t i o n of t h e p r o d u c t b a n d s , t h e a m o u n t of:
r a d i o a c t i v e m a t e r i a l was r e d u c e d t o 1 5 mµc for t h e s e p a r a t i o n shown in F i g u r e 2 w i t h an a u t o r a d i o g r a p h i c e x p o s u r e of 7 days, while t h e a p p l i c a t i o n was d o u b l e d for F i g u r e 3 to show t h e less intense p r o d u c t s a t t h e e x p e n s e o f s o m e o v e r l o a d i n g o f t h e m o r e active b a n d s .
D u r i n g l i m i n g a n d c a r b o n a t a t i o i i b o t h t h e f o r m a l d e h y d e a n d the fructose w e r e c o n v e r t e d a l m o s t e x c l u s i v e l y i n t o a n i o n i c p r o - ducts. X o c a t o n i c c o m p o n e n t s c o u l d b e d e t e c t e d i n e i t h e r system and t h e r e was n o d e t e c t a b l e loss o f activity f r o m e i t h e r s e c o n d c a r b o n a t a t i o i i j u i c e o n t r e a t m e n t w i t h a s t r o n g c a t i o n e x c h a n g e resin ( Z e o K a r b 225) w h e r e a s 9 2 % o f t h e a c t i v i t y a r i s i n g f r o m f o r m a l d e h y d e d e g r a d a t i o n a n d 9 5 % o f t h e a c t i v i t y a r i s i n g f r o m fructose d e g r a d a t i o n c o u l d be a b s o r b e d on a s t r o n g anion ex- c h a n g e r ( D e - A c i d i t e F F ) . ^
T h e p r i n c i p a l p r o d u c t b a n d s h a v e b e e n l a b e l l e d 1 t o 8 , t h e h i g h e r n u m b e r s r e p r e s e n t i n g t h e m o s t m o b i l e o f t h e e l e c t r o p h o r e - tic b a n d s . T h e e l e c t r o p h c r o g r a m s h o w n in Figure 3 was c u t i n t o strips t o isolate t h e p r o d u c t b a n d s , i n c l u d i n g s o m e faint i n t e r m e - diate b a n d s w h i c h w e r e visible o n t h e o r i g i n a l a u t o r a d i o g r a p h .
T h e strips w e r e e l u t e d and counted to determine the r e l a t i v e yields a n d these v a l u e s a r e also r e c o r d e d o n F i g u r e 3 .
• J O U R N A L OF T H E A. S. S. B. T.
F i g u r e s 2 a n d 3 . — A u t o r a d i o g r a p h s following electrophoresis of de- g r a d a t i o n p r o d u c t s of f o r m a l d e h y d e C14 a n d fructose C14.
A t t h e p r e s e n t stage o f t h e i n v e s t i g a t i o n , t h e fructose degra- d a t i o n p r o d u c t s h a v e b e e n e x a m i n e d i n m o r e d e t a i l t h a n t h e p r o d u c t s from f o r m a l d e h y d e a n d o n l y a few of t h e l a t t e r h a v e b e e n positively i d e n t i f i e d , b u t from e l e c t r o p h o r e t i c a n d c h r o m a - t o g r a p h i c s e p a r a t i o n s i t a p p e a r s t h a t t h e m a j o r i t y o f t h e b a n d s consist of t h e s a m e acids, or closely r e l a t e d i s o m e r i c acids, regard- less o f w h e t h e r t h e s o u r c e was f o r m a l d e h y d e o r fructose. T h e r e l a t i v e p r o p o r t i o n s o f t h e p r o d u c t s a r e clearly d i f f e r e n t w i t h t h e d i f f e r e n t s o u r c e s a n d it is p r o b a b l e t h a t , as w i t h fructose, t h e p r o p o r t i o n s o f t h e f o r m a l d e h y d e p r o d u c t s will v a r y a c c o r d i n g t o t h e i n i t i a l c o n c e n t r a t i o n a n d t h e d e f e c a t i o n c o n d i t i o n s .
B a n d 8 consists of f o r m i c acid a n d was o b t a i n e d in h i g h e r yield from f o r m a l d e h y d e t h a n f r o m fructose. M o s t o f t h e re- m a i n i n g b a n d s d o n o t consist o f a s i n g l e s u b s t a n c e a n d a f t e r elu-
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VOL. 13, No. 2, JULY 1964 111 tion from t h e e l e c t r o p h e r o g r a m the b a n d s can be f u r t h e r re
solved b y c h r o m a t o g r a p h y . T h e c a r b o n c h a i n l e n g t h increases with decreasing e l e c t r o p h o r e t i c m o b i l i t y a n d , a l t h o u g h s i m p l e carboxylic acidvS c o n t r i b u t e to t h e m o r e m o b i l e b a n d s , t h e princi
pal c o n s t i t u e n t s a r e t h e hydroxy-acids f o r m i n g t h e lower h o m o logues of t h e saccharinic acid series.
T h e acids f o r m i n g b a n d 7 c o n t a i n two c a r b o n a t o m s a n d can he resolved i n t o separate b a n d s of glycollic a n d acetic acid by c h r o m a t o g r a p h y in n - p r o p a n o l : .880 a m m o n i a 70:30.
B a n d G consists p r e d o m i n a n t l y of lactic acid w h i c h r e p r e s e n t s the major single p r o d u c t of a l k a l i n e d e g r a d a t i o n of fructose a n d is also o n e of t h e p r i n c i p a l p r o d u c t s of t h e d e g r a d a t i o n of formal- dehyde.
T h e faint b a n d 5 in t h e fructose s e p a r a t i o n is p r o b a b l y gly
ceric acid. T h i s b a n d was less clearly resolved from lactic acid in the f o r m a l d e h y d e s e p a r a t i o n .
B a n d 4 c o n t a i n s 2:4 d i h v d r o x v b u t y r i c acid a n d this b a n d rep
resents a h i g h e r p r o p o r t i o n of t h e p r o d u c t s from f o r m a l d e h y d e d e g r a d a t i o n t h a n from fructose d e g r a d a t i o n . T h e b a n d i s m o r e diffuse from t h e f o r m a l d e h y d e t r e a t m e n t a n d m a y c o n t a i n o t h e r 4 carbon a t o m acids.
Band 3 is again t h e m o r e p r o m i n e n t in t h e f o r m a l d e h y d e d e g r a d a t i o n . T h i s b a n d p r o b a b l v c o n t a i n s p e n t o s a c c h a r i n i c acids.
B a n d 2 is a p r o m i n e n t p r o d u c t of t h e fructose d e g r a d a t i o n and in t h e b a n d from this system several of t h e isomeric gluco- saccharinic acids have b e e n identified. T h i s b a n d is n o t a m a j o r product of t h e f o r m a l d e h y d e d e g r a d a t i o n b u t a series of b a n d s of low i n t e n s i t y o c c u r in this r e g i o n .
B a n d 1 is a l m o s t i m m o b i l e a n d may be d u e e i t h e r to lactones o r u n r e a c t e d c a r b o h y d r a t e s . T h i s b a n d d o e s n o t c o n t a i n any u n - reacted f o r m a l d e h y d e a n d n o n e o f t h e o t h e r b a n d s c o u l d b e de
fected by e l e c t r o p h o r e s i s of t h e r a d i o a c t i v e r a w j u i c e before liming.
C a r b o h y d r a t e s c a n be p r o d u c e d by aldol c o n d e n s a t i o n s from formaldehyde, a n d even i n t h e n i n e t e e n t h c e n t u r y syrups con
taining hexoses h a d b e e n p r e p a r e d from p a r a f o r m a l d e h y d e b v Butleroff. a n d from f o r m a l d e h y d e by L o e w ; a l t h o u g h t h e overall yields were low, these syrups c o n t a i n e d α-acrose w h i c h was shown by Fischer to be DL-fructose.
I t was t h e r e f o r e t o b e e x p e c t e d t h a t s o m e h v d r o x v acids w o u l d be formed by a l k a l i n e d e g r a d a t i o n of f o r m a l d e h y d e , p a r t i c u l a r l v as it is n o t essential for t h e c o n d e n s a t i o n to p r o c e e d as far as hex- oses before d e g r a d a t i o n is i n i t i a t e d . It is h o w e v e r r a t h e r sur
prising t h a t t h e v a r i o u s acids s h o u l d b e o b t a i n e d s o q u i c k l y a n d
112 JOURNAL OF THE A. S. S. B. T.
i n such g o o d yield, a n d t h a t t h e p r o d u c t s s h o u l d b e s o s i m i l a r to those f o r m e d by a l k a l i n e d e g r a d a t i o n of fructose.
R a t e o f d e c o m p o s i t i o n o f f o r m a l d e h y d e d u r i n g l i m i n g The r a t e of d e c o m p o s i t i o n of f o r m a l d e h y d e was e x a m i n e d initially b y l i m i n g a n a q u e o u s s o l u t i o n o f f o r m a l d e h y d e a t 8 0 ° C a n d r e s i d u a l f o r m a l d e h y d e was m e a s u r e d b y t h e N a s h p r o c e d u r e . X o facilitate analysis t h e c o n c e n t r a t i o n o f f o r m a l d e h y d e was h i g h e r t h a n i n factory p r a c t i c e a n d , a s t h e t i m e r e q u i r e d for car- b o n a t a t i o n was e x p e c t e d t o b e l o n g r e l a t i v e t o t h e r a t e o f decom- p o s i t i o n , t h e r e a c t i o n was s t o p p e d a t t h e r e q u i r e d t i m e i n t e r v a l s by n e u t r a l i z a t i o n w i t h h y d r o c h l o r i c acid. Because of t h e analogy w i t h fructose d e g r a d a t i o n , r e d u c i n g m a t e r i a l s w e r e also m e a s u r e d w i t h t r i p h e n y l t e t r a z o l i u m (3) d u r i n g t h e d e g r a d a t i o n .
O n e m l o f f o r m a l i n ( a p p r o x i m a t e l y 3 3 % f o r m a l d e h y d e ) was d i l u t e d t o 2 5 m l w i t h w a t e r a n d t h e s o l u t i o n was h e a t e d i n a w a t e r b a t h t o 8 0 ° C . D r y l i m e (0.4 g ) was a d d e d t o t h e s o l u t i o n at 8 0 ° C a n d at 1 m i n u t e i n t e r v a l s a 1 ml a l i q u o t was w i t h d r a w n , n e u t r a l i z e d i m m e d i a t e l y w i t h 0.7 ml of N h y d r o c h l o r i c acid, a n d d i l u t e d t o 2 5 m l for d e t e r m i n a t i o n o f t h e c o n c e n t r a t i o n s o f f o r m a l d e h y d e a n d r e d u c i n g m a t e r i a l . X h e c o n c e n t r a t i o n s o f t h e l a t t e r w e r e e x p r e s s e d r e l a t i v e t o fructose s t a n d a r d s .
The e x p e r i m e n t a l r e s u l t s a r e r e c o r d e d in F i g u r e 4. It was o b s e r v e d t h a t t h e s o l u t i o n s u d d e n l y d e v e l o p e d a yellow c o l o r a t i o n 5 m i n u t e s 10 seconds after l i m i n g .
A slow d e c o m p o s i t i o n of f o r m a l d e h y d e o c c u r r e d d u r i n g the first 4 m i n u t e s after l i m i n g b u t t h e r e a f t e r t h e f o r m a l d e h y d e was r a p i d l y d e c o m p o s e d , t h e c o n c e n t r a t i o n f a l l i n g t o z e r o w i t h a n
TIME (MINUTES)
Figure 4.—Degradation of formaldehyde during liming ( n o additives).
VOL. 13, N o . 2, J U L Y 196-1 1 1 3
almost e q u i v a l e n t p r o d u c t i o n o f r e d u c i n g m a t e r i a l . T h e r e d u c i n g material was n o t s t a b l e a n d c o l o r was p r o d u c e d soon after t h e c o n c e n t r a t i o n o f r e d u c i n g m a t e r i a l b e c a m e significant. T h e s u b - sequent d e c o m p o s i t i o n of t h e r e d u c i n g m a t e r i a l followed a c u r v e which was a p r o l o n g a t i o n of t h e i n i t i a l f o r m a l d e h y d e d e c o m p o s i - tion.
T h e slope o f t h e f o r m a l d e h y d e d e c o m p o s i t i o n c u r v e does n o t follow any s i m p l e 1st o r 2 n d o r d e r r e a c t i o n a n d s o m e o t h e r species must be i n v o l v e d to i n i t i a t e t h e r a p i d stage of d e c o m p o s i t i o n . Possibilities for such a " s t a r t e r " c o u l d i n c l u d e m a n y of t h e acidic products d e t e c t e d i n t h e r a d i o c h e m i c a l e x p e r i m e n t s b u t s o m e o f the m o r e r e a c t i v e n o n - a c i d i c p r o d u c t s o f r e d u c i n g s u g a r de- gradation s e e m e d m o r e p r o b a b l e . T h e e x p e r i m e n t was t h e r e f o r e repeated i n t h e p r e s e n c e o f a d d e d g l y c e r a l d e h y d e a t a n i n i t i a l c o n c e n t r a t i o n of 0 . 0 0 S °o. G l y c e r a l d e h y d e is n o t s t a b l e u n d e r alka- line c o n d i t i o n s a n d is in e q u i l i b r i u m w i t h d i h y r o x y a c e t o n e w h i c h has b e e n t e n t a t i v e l y i d e n t i f i e d in low yield as a p r o d u c t of h e x o s e d e g r a d a t i o n . T h e d e c o m p o s i t i o n o f t h e f o r m a l d e h y d e , t h e p r o - d u c t i o n a n d s u b s e q u e n t d e g r a d a t i o n o f r e d u c i n g m a t e r i a l a n d the color p r o d u c t i o n followed a s i m i l a r p a t t e r n w i t h glyceralde- hyde a d d i t i o n , a s s h o w n i n F i g u r e 5 , b u t t h e r e a c t i o n rates w e r e greatly i n c r e a s e d ; t h e f o r m a l d e h y d e c o n c e n t r a t i o n fell t o zero in a b o u t \l/2 m i n u t e s a n d t h e r e d u c i n g m a t e r i a l was d e c o m p o s e d more r a p i d l y .
T h e c o n c e n t r a t i o n o f r e d u c i n g m a t e r i a l d o e s n o t fall e x p o n - entially t o zero s h o w i n g t h a t e i t h e r t h e i n i t i a l r e d u c i n g m a t e r i a l is a m i x t u r e of d i f f e r e n t s u b s t a n c e s , or t h a t s o m e of t h e p r o d u c t s
Figure 5.—Degradation of formaldehyde during liming (effect of yccraldchyde starter 0,008%.
• -
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o f t h e i n t i a l d e g r a d a t i o n o f r e d u c i n g m a t e r i a l a r e also reducing.
A s i m i l a r t y p e of c u r v e is o b t a i n e d on m e a s u r i n g t h e r a t e of c h a n g e o f r e d u c i n g m a t e r i a l d u r i n g a l k a l i n e d e g r a d a t i o n o f fruc- tose o r g l u c o s e .
P r o b a b l y m a n y o t h e r u n s t a b l e a l d e h y d e s a n d k e t o n e s will a c c e l e r a t e t h e a l k a l i n e d e g r a d a t i o n of f o r m a l d e h y d e a n d it was f o u n d t h a t a d d i t i o n of e v e n a m i n u t e a m o u n t of fructose
( 0 . 0 0 4 % ) was sufficient to r e d u c e t h e t i m e of c o m p l e t e formalde- h y d e d e c o m p o s i t i o n t o a b o u t 2 1 / 2 m i n u t e s .
T h e d e g r a d a t i o n o f f o r m a l d e h y d e a t t h e l o w e r c o n c e n t r a t i o n s realized in factory p r a c t i c e was also e x a m i n e d in s y n t h e t i c solu- t i o n s . All t h e s o l u t i o n s w e r e t r e a t e d w i t h 1.6% l i m e a t 80°C a n d t h e d e g r a d a t i o n r a t e s for 0 . 0 2 1 % f o r m a l d e h y d e w i t h o u t addi- tives, w i t h 1 0 % sucrose o r w i t h 0 . 0 0 7 % fructose a n d w i t h 0.12%
fructose a r e r e c o r d e d in F i g u r e 6. At this l o w c o n c e n t r a t i o n it was n o t possible t o d e t e c t c o l o r p r o d u c t i o n w h i c h c o u l d b e attri- b u t e d t o t h e f o r m a l d e h y d e d e g r a d a t i o n .
Figure 6.—Degradation of formaldehyde at low concentration (effect of fructose and sucrose).
At c o n c e n t r a t i o n s s i m i l a r to t h o s e in r a w j u i c e , t h e rate of a l k a l i n e d e g r a d a t i o n o f f o r m a l d e h y d e i n a q u e o u s s o l u t i o n without a d d i t i v e s was very slow; less t h a n 5% b e i n g d e c o m p o s e d in 10 m i n u t e s . I n 1 0 % sucrose s o l u t i o n h o w e v e r t h e r e a c t i o n rate was greatly i n c r e a s e d : t h i s i n c r e a s e m a y b e d u e t o t h e g r e a t e r solu- bility of l i m e in sucrose s o l u t i o n , or to t h e p r e s e n c e of trace im- p u r i t i e s i n t h e sucrose w h i c h i n i t i a t e t h e c h a i n r e a c t i o n o r perhaps to a l k a l i n e d e g r a d a t i o n of t h e s u c r o s e itself to y i e l d s u c h starters.
T h e i n c r e a s e d r e a c t i o n r a t e was n o t d u e t o t r a c e s o f fructose i n t h e s u g a r s i n c e t h e c o n c e n t r a t i o n o f r e d u c i n g s u g a r s i n solution was- less t h a n 0 . 0 0 0 3 % a n d w i t h o u t t h e s u c r o s e t h i s a m o u n t of
VOL. 13, No, 2, JULY 1964 115
fructose d i d n o t m e a s u r a b l y i n c r e a s e t h e r e a c t i o n r a t e . E v e n w i t h - out sucrose h o w e v e r . 0 . 0 0 7 % fructose b r o u g h t a b o u t a very r a p i d d e g r a d a t i o n o f t h e f o r m a l d e h y d e a n d w h e n t h e fructose c o n c e n - t r a t i o n was i n c r e a s e d t o a b o u t t h a t o f total r e d u c i n g sugars i n raw j u i c e , a l m o s t c o m p l e t e d e s t r u c t i o n o f t h e f o r m a l d e h y d e oc- c u r r e d w i t h i n 1 m i n u t e of l i m i n g .
A l t h o u g h t h e r e m a y well b e m a n y c o m p o u n d s i n raw j u i c e c a p a b l e of i n i t i a t i n g t h e c h a i n r e a c t i o n , it is n o t nccessarv to in- voke s u b s t a n c e s o t h e r t h a n sucrose a n d r e d u c i n g sugars i n juice i n o r d e r t o e x p l a i n t h e c o m p l e t e d e g r a d a t i o n o f f o r m a l d e h y d e d u r i n g l i m i n g a n d c a r b o n a t a t i o n .
C o n c l u s i o n s
1 . W h e n f o r m a l d e h y d e i s i n t r o d u c e d t o w a r d s t h e h e a d e n d of the diffuser. very l i t t l e if a n y of t h e f o r m a l d e h y d e reacts with p u l p c o n s t i t u e n t s . O n l y small a m o u n t s c o m b i n e i r r e v e r s i b l y w i t h juice c o n s t i t u e n t s o r c o m b i n e i n t h e r e v e r s i b l e c o n d e n s a t i o n w i t h a m i n o acids a n d t h e g r e a t e r p a r t is c a r r i e d f o r w a r d as free for- m a l d e h y d e i n t o t h e r a w j u i c e t o process.
2 . T h e f o r m a l d e h y d e i s r a p i d l y a n d c o m p l e t e l y d e c o m p o s e d d u r i n g l i m i n g . A b o u t 7 0 % o f t h e f o r m a l d e h y d e a d d e d t o raw juice c o u l d b e a c c o u n t e d for a s a c i d i c p r o d u c t s i n s e c o n d c a r b o n - a t a t i o n j u i c e .
3 . T h e a c i d i c p r o d u c t s c a n b e s e p a r a t e d b y e l e c t r o p h o r e s i s into a series of p r o d u c t s i n c r e a s i n g in c a r b o n c h a i n l e n g t h w i t h decreasing m o b i l i t y . T h e p r o d u c t s a r e v e r y s i m i l a r t o t h e p r o d u c t s of a l k a l i n e d e g r a d a t i o n of f r u c t o s e , c o n s i s t i n g of s i m p l e c a r b o x - ylic acids a n d t h e l o w e r h o m o l o g u e s o f t h e s a c c h a i i n i c acid series.
4 . F o r m a l d e h y d e a l o n e i s c o m p a r a t i v e l y s t a b l e i n a l k a l i n e solution. I n t h e p r e s e n c e o f d e g r a d a t i o n p r o d u c t s o f f o r m a l d e h y d e itself, o r o f f r u c t o s e o r o f s i m p l e u n s t a b l e a l d e h y d e s , t h e f o r m a l d e - hyde i s r a p i d l y d e c o m p o s e d t o g i v e a n a l m o s t e q u i v a l e n t yield o f r e d u c i n g m a t e r i a l . T h i s r e d u c i n g m a t e r i a l i s u n s t a b l e i n alka- line s o l u t i o n a n d d e c o m p o s e s t o y i e l d t h e a c i d i c m a t e r i a l t o g e t h e r with small a m o u n t o f y e l l o w p r o d u c t s .
Influence of Size of Fruit and Seed on Germination of A Monogerm Sugar Beet Variety1
G . J . HOGABOAM- AND F . W . S.NYDER Received for publication November 29, 1963
T h e a d v e n t of t h e m o n o g e r m s u g a r b e e t has a c c e l e r a t e d the t r e n d t o w a r d c o m p l e t e l y m e c h a n i z e d p r o d u c t i o n . H o w e v e r , mech- anized c u l t u r a l o p e r a t i o n s in t h e s p r i n g d e p e n d on t h e successful s o l u t i o n of such p r o b l e m s as precision space-planting, adequate e m e r g e n c e , weed c o n t r o l , a n d disease c o n t r o l . A sugar b e e t variety that p r o d u c e d u n i f o r m l y sized a n d s h a p e d fruits3, each containing a single seed t h a t g e r m i n a t e s r a p i d l y , w o u l d seem to be t h e most d e s i r a b l e for c o m p l e t e m e c h a n i z a t i o n .
In a study of b u l k e d fruits (seedballs) of m u i t i g e r m varieties P r i c e a n d C a r s n e r (4)' o b s e r v e d t h a t seeds g e r m i n a t e d m o r e rapid- ly a n d c o m p l e t e l y a n d grew m o r e vigorously from seedballs re- m a i n i n g on 4- a n d 3.5-mm screens t h a n those from seedballs pass- i n g t h r o u g h a 3 m m . b u t r e m a i n i n g on a 2.5 mm screen. In con- trast. U s t i m e n k o (9) , in s t u d y i n g b u l k e d fruits of monogerm strains, o b s e r v e d t h a t seeds in fruits w i t h a high w e i g h t p e r 1,000 fruits g e r m i n a t e d slower a n d less c o m p l e t e l y t h a n those in fruits h a v i n g less weight. I n o n e m u i t i g e r m a n d t w o m o n o g e r m vari- eties h e f o u n d t h a t t h e large fruits c o n t a i n e d , b o t h a b s o l u t e l y a n d relatively, a larger mass of p e r i c a r p t h a n t h e s m a l l e r fruits.
H e ascribed t h e slower g e r m i n a t i o n t o t h e relatively greater a m o u n t of p e r i c a r p . D e c o r t i c a t i n g t h e fruits h a s t e n e d emergence.
W a t e r a b s o r p t i o n by d e c o r t i c a t e d fruits was c o m p l e t e after 40 to 50 h o u r s , w h i l e n o n - d e c o r t i c a t e d fruits r e q u i r e d 80 to 90 hours O n l y one-half to o n e - t h i r d as m u c h w a t e r was i m b i b e d by decorti- cated fruits.
Savitsky (5) o b s e r v e d t h a t t h e weight of seeds increased in p r o p o r t i o n to t h e size of t h e m o n o g e r m fruits. Savitsky et al
(6) r e p o r t e d t h a t seeds in large fruits of i n b r e d m o n o g e r m lines g e r m i n a t e d m o r e slowly t h a n those in small fruits.
S e d l m a y r (7) , u s i n g seedballs of a u n i f o r m size-class, d e m o n - s t i a t e d that speed of g e r m i n a t i o n of t h e m u i t i g e r m s u g a r beet variety US 401 is h e r i t a b l e . Differences in speed of germination
1 Cooperative investigations of the Crops Research Division. Agricultural Research Service. U. S. Department of Agriculture, and the Michigan Agricultural Experiment Sta- tion. Approved for publication as Journal article 3264 .Michigan Agricultural Experiment Station.
2 Research Agonomist and Plant Physiologist, respectively. Crops Research Division, Agricultural Research Service, U. S. Department of Agriculture. East Lansing. Michigan
3 A fruit is defined as an individual structure which contains one or more true seeds. In this paper, the term seed refers only to the true seed contained within the fruit.
4 Numbers in parentheses refer to literature cited.
VoL. 13, N o . 2, J U L Y 1964 117
were d u e m a i n l y t o t h e physical a n d c h e m i c a l n a t u r e o f t h e ma- ternal tissues of t h e fruit w h i c h s u r r o u n d t h e t r u e seed (7, 8).
P o x t a t o r a n d H e l m e r i c k (1) o b s e r v e d t h a t large-fruited p a r e n t s produce p r o g e n i e s w i t h l a r g e fruits.
Before b r e e d i n g for u n i f o r m fruits t h e b r e e d e r s h o u l d k n o w whether t h e r e is a most d e s i r a b l e size a n d w h a t o t h e r c h a r a c t e r i s - tics of fruit a n d seed m a y i n f l u e n c e his choice of size. Since pre- vious w o r k suggested t h a t t h e size of fruit i n f l u e n c e s g e r m i n a t i o n performance, t h i s research was u n d e r t a k e n to s e p a r a t e t h e effect of fruit size from t h e effect of seed size on g e r m i n a t i o n w h e n the seed is g e r m i n a t e d w i t h i n t h e i n t a c t fruit.
M e t h o d s and Materials
T h e d a t a w e r e c o l l e c t e d o n a n i n d i v i d u a l p l a n t basis a s a control o n v a r i a b i l i t y t h a t exists b e t w e e n p l a n t s for q u a l i t y a n d c o n c e n t r a t i o n of i n h i b i t o r s in t h e i r f r u t s as well as t h e m e c h a n i - cal p r o p e r t i e s of t h e fruit affecting g e r m i n a t i o n . S i m p l e a n d partial c o r r e l a t i o n s w e r e t h e n used t o e v a l u a t e t h e d a t a for w i t h i n plants, for b e t w e e n p l a n t s , a n d for t h e variety.
In t h e e x p e r i m e n t a l p r o c e d u r e s (3) e m p l o y e d to i m p r o v e the m o n o g e r m v a r i e t y SP 5832-0. each r o o t of 141 selections was planted in a soil b e d in t h e g r e e n h o u s e at East L a n s i n g . M i c h i - gan, i n D e c e m b e r 1958. M a t u r e fruits w e r e h a r v e s t e d s e p a r a t e l y from each of 131 p l a n t s . All fruits w e r e t r e a t e d w i t h f u n g i c i d e b u t received no o t h e r t r e a t m e n t . E l e v e n to 22 g r a m s of fruits from each of 19 p l a n t s w e r e sized for d i a m e t e r a n d t h e n for t h i c k n e s s . T h e p e r c e n t a g e s by w e i g h t of fruits w i t h a single o v a r i a n cavity ( m u l t i p l e o v a r i a n cavities d i s c a r d e d ) in each of t h e size classes were listed by p l a n t s o u r c e . In this study, t h e 1 2 / 6 4 - i n c h dia- meter class refers to fruits t h a t passed t h r o u g h a 13 (54-inch r o u n d - hole screen a n d r e m a i n e d o n a n l l / 6 4 - i n c h screen. T h e 9 / 6 4 - i n c h thickness class refers to fruits t h a t passed t h r o u g h a 1 0 / 6 4 - i n c h slotted screen a n d r e m a i n e d on an 8 64-inch screen.
A n i n d e x o f u n i f o r m i t y was c a l c u l a t e d b o t h for d i a m e t e r a n d thickness o f fruit. T h e i n d e x e s w e r e c a l c u l a t e d from t h e fruit- size d i s t r i b u t i o n d a t a as follows: T h e m o d a l class was assigned a value of zero, t h e class on e i t h e r side of t h e m o d e was assigned a value of 0 . 1 , t h e second classes from t h e m o d e w e r e assigned a value of 0.2, t h e t h i r d a' v a l u e of 0.4, a n d t h e f o u r t h a v a l u e o f 0.8. T h e assigned class v a l u e was m u l t i p l i e d b y t h e p e r c e n t a g e by w e i g h t of fruits in t h a t class a n d t h e s u m of these p r o d u c t s i s the i n d e x o f u n i f o r m i t y . T h u s , t h e s m a l l e r t h e v a l u e s o f t h e index, t h e m o r e u n i f o r m t h e size of fruits.
V o l u m e of fruit was c a l c u l a t e d since it offered t h e possibility o f b e t t e r r e l a t i n g f r u i t size t o g e r m i n a t i o n r e s p o n s e t h a n e i t h e r
118 J O U R N A L OF T H E A. S. S. B. T.
d i a m e t e r o r thickness. T h e f o r m u l a for t h e v o l u m e o f a n oblate s p h e r o i d (V — 4/3 a2b) a p p e a r e d to be t h e best o n e for an initial e s t i m a t i o n of t h e v o l u m e p e r fruit w h e r e " a " is t h e d i a m e t e r and
"b" is t h e thickness. T h e a c t u a l v o l u m e s were d e t e r m i n e d for each size class by v o l u m e t r i c d i s p l a c e m e n t in S t o d d a r d ' s solvent5. T h e fruits used in this d e t e r m i n a t i o n w e r e from t h e same variety b u t n o t necessarily from t h e same 19 p l a n t s as used for radio- g r a p h i n g .
D a t a on t h e r e l a t i o n of fruit d i a m e t e r a n d fruit thickness t o g e r m i n a t i o n t i m e w e r e o b t a i n e d for each p l a n t .
An X-ray t e c h n i q u e (2) was used to d e t e r m i n e t h e diameter of t h e seed w h i l e i n t a c t w i t h i n its fruit. Samples of fruits from each of 19 p l a n t s , sized for d i a m e t e r a n d thickness, w e r e placed on sticky c e l l o p h a n e t a p e by p l a n t source a n d by size class for X-ray. T h u s it was possible to o b t a i n d a t a on an i n d i v i d u a l basis.
A total of 6,782 single-cavity fruits, r a n g i n g from a m i n i m u m of 228 to a m a x i m u m of 480 fruits p e r p l a n t , was r a d i o g r a p h e d . In a d d i t i o n to the d i a m e t e r of t h e seed, t h e n u m b e r of fruits c o n t a i n i n g no d e v e l o p e d seed a n d those c o n t a i n i n g 1, 2, 3, or 4 seeds i n t h e single o v a r i a n cavity w e r e r e c o r d e d . T h e identity of each fruit was m a i n t a i n e d on t h e g e r m i n a t i o n b l o t t e r a n d the t i m e r e q u i r e d to g e r m i n a t e was r e c o r d e d for each seed. Weighted averages for seed size w e r e used to c a l c u l a t e t h e a v e r a g e seed size for a p l a n t , since fruit size classes t h a t w e r e r a d i o g r a p h e d were n o t always t a k e n i n p r o p o r t i o n a l a m o u n t s .
T h e data o b t a i n e d by i n d i v i d u a l fruits w e r e e v a l u a t e d for i n t e r - r e l a t i o n s h i p s b y s i m p l e a n d p a r t i a l c o r r e l a t i o n s . T h e data consisted of: (A) g e r m i n a t i o n t i m e ; ( B ) fruit d i a m e t e r ; (C) fruit thickness, (D) seed d i a m e t e r ; (E) n u m b e r of seeds in ovar- ian cavity; a n d (F) v o l u m e of t h e fruit. C o r r e l a t i o n coefficients were c a l c u l a t e d for w i t h i n - p l a n t samples, for b e t w e e n - p l a n t sam- ples, a n d tor all s a m p l e s c o m b i n e d t o r e p r e s e n t t h e variety.
R e s u l t s Fruit size
T h e fruit d i a m e t e r a n d thickness d a t a ( T a b l e 1) for this mon- o g e r m variety revealed d i a m e t e r s r a n g i n g b e t w e e n 6 / 6 4 a n d 17/64 a n d thicknesses from 4 / 6 4 to 11/'64 of an i n c h . Each p l a n t had at least a few fruits in t h e smallest d i a m e t e r a n d t h i c k n e s s class w h i l e only a b o u t half of t h e p l a n t s h a d fruits in t h e largest dia- m e t e r a n d thickness class. P l a n t s differed in u n i f o r m i t y of fruit size.
5 Trade names are mentioned for identification only and do not constitute recommen-' dation by United States Department of Agriculture.
VoL 13, N o . 2, J U L Y 1964 121
T h e c a l c u l a t e d v o l u m e e x c e e d e d t h e actual v o l u m e p e r fruit, however, w h e n t h e c a l c u l a t e d v o l u m e was s u b s t i t u t e d i n t h e formula Y = 0.598x + 0.0013. this c o r r e c t e d v o l u m e closely ap- p r o x i m a t e d t h e a c t u a l v o l u m e p e r fruit a s m e a s u r e d b y volu- metric d i s p l a c e m e n t .
T h e s h a p e o f fruits v a r i e d b y p l a n t s from o n e i n w h i c h t h e fruit, t h i c k n e s s was 56% of t h e d i a m e t e r to o n e in w h i c h it was 72% ( T a b l e 2).
Seed size ayid numbers of seeds per cavity
T h e d i a m e t e r o f t h e seed g e n e r a l l y v a r i e d d i r e c t l y w i t h t h e d i a m e t e r of t h e fruit a n d also fruit thickness. T h i s t r e n d a n d its exceptions may be n o t e d in t h e s u m m a r y of fruit a n d seed char- acteristics i n T a b l e 2 . T h e s m a l l e r fruits t e n d t o c o n t a i n r e l a t i v e l v larger seeds as s h o w n by t h e "seed d i a m e t e r as % fruit d i a m e t e r "
column.
] 2 2 JOURNAL OF THE A. S. S. B. T.
T h e d a t a o n t h e c o n t e n t o f t h e o v a r i a n cavities, o b t a i n e d b y m e a n s of t h e X-ray t e c h n i q u e , are s u m m a r i z e d in Table 3. Indi- v i d u a l p l a n t s of this m o n o g e r m variety v a r i e d greatly in t h e per- c e n t a g e of fruits c o n t a i n i n g a single seed. Six of t h e p l a n t s had m o r e t h a n 10 p e r c e n t seedless fruits ( a b o r t e d seeds) . F o u r of the p l a n t s h a d 10 p e r c e n t or m o r e of t h e i r fruits c o n t a i n i n g t w o or m e r e seeds. M o r e t h a n 23 p e r c e n t of t h e fruits of p l a n t 41 con- t a i n e d m u l t i p l e seeds. Of t h e fruits in t h e 16.. 24 of an inch dia- m e t e r si/e-class, m o r e t h a n 8 % w e r e seedless.
Speed of germination
T h e g e r m i n a t i o n d a t a w e r e used t o calculate t h e a m o u n t o f delay in g e r m i n a t i o n t h a t can be e x p e c t e d from an increase in fruit d i a m e t e r o r a n increase i n fruit thickness. W h e n fruit t h i c k - ncss was m a i n t a i n e d b e t w e e n (S\A to 8 64th of an inch, t h e time t o a t t a i n 8 0 % g e r m i n a t i o n was a p p r o x i m a t e l y a day l o n g e r f o r each increase of 2 64th of an inch in fruit d i a m e t e r . W h e n the fruit d i a m e t e r was m a i n t a i n e d at e i t h e r 9 to 11 64th or 11 to
13 64th of an i n c h , an e x t r a clav was r e q u i r e d to a t t a i n 80%
t e r m i n a t i o n by increasing t h e thickness class from 5 to 6 1/2 to 6i <, to 8 G4th. Except for t h e smallest seeds a n d fruits, g e r m i n a - tion t i m e c o r r e l a t e d positively with fruit v o l u m e ( T a b l e 4).
Of t h e 6,782 single-cavity fruits t h a t w e r e r a d i o g r a p h e d , 2.5%
n e v ? sacrificed to era in e x p e r i e n c e in r e a d i n g t h e r a d i o g r a p h s and 1 0 . 2 % c o n t a i n e d n o d e v e l o p e d seeds. O f t h e r e m a i n i n g f r u i t s , 9 7 . 6 % g e r m i n a t e d w i t h i r t h e 25-day l i m i t o f t h e e x p e r i m e n t , 1.9% h a d seeds t h a t w e r e fully d e v e l o p e d a n d a p p e a r e d as though
1 Expressed as cubic centimeters x 10,000.
VoL 13, No. 2, JULY 1964 123
t h e y should h a v e g e r m i n a t e d , a n d 0 . 5 % h a d seeds that a p p e a r e d incompletely d e v e l o p e d a n d w e r e n o n - v i a b l e .
Correlation studies
T h e w i t h i n - p l a n t c o r r e l a t i o n s w e r e c a l c u l a t e d for each p l a n t , but only t h e e x t r e m e s a n d t h e m e a n s of these d a t a a r e g i v e n in.
T a b l e 5 . T h e b e t w e e n - p l a n t a n d w i t h i n - v a r i e t y c o r r e l a t i o n s a r e also listed.
Table 5.—Correlation coefficients between various fruit dimensions, seed diameter, and germination time for samples from 19 plants of monogerm s«S.ir beet variety SP 5832-0.
* Significant at 5% level and * * at 1% level.
W i t h i n - p l a n t c o r r e l a t i o n s for g e r m i n a t i o n t i m e v e r s u s fruit size [ d i a m e t e r ( A B ) , t h i c k n e s s ( A C ) , o r v o l u m e (AF)] a n d also seed d i a m e t e r ( A D ) r e v e a l e d 10 p l a n t s w i t h significant positive c o r r e l a t i o n s , t w o w i t h n o c o r r e l a t i o n ( n u m b e r s 2 a n d 60), a n d o n e w i t h a n e g a t i v e c o r r e l a t i o n . T h e e x t r e m e n e g a t i v e w i t h i n - p l a n t c o r r e l a t i o n s o f c h a r a c t e r s w i t h g e r m i n a t i o n t i m e
( F a b l e 5 ) w e r e o b t a i n e d from p l a n t 4 8 w h i l e t h e e x t r e m e posi- tive ones w e r e from p l a n t 110.
In all cases, t h e r e was a s i g n i f i c a n t positive c o r r e l a t i o n be- tween fruit d i a m e t e r a n d fruit t h i c k n e s s ( B C ) , fruit v o l u m e
( B F ) , a n d seed d i a m e t e r ( B D ) . F r u i t t h i c k n e s s was positively correlated, w i t h seed d i a m e t e r ( C D ) a n d fruit v o l u m e was posi- tively c o r r e l a t e d w i t h seed d i a m e t e r ( F D ) . S i m p l e c o r r e l a t i o n s between seed d i a m e t e r a n d g e r m i n a t i o n t i m e ( A D ) a r e m i s -
124 J O U R N A L OF T H E A. S. S. B. T.
l e n d i n g d u e t o t h e h i g h c o r r e l a t i o n o f s e e d d i a m e t e r ( D ) with f r u i t size c h a r a c t e r i s t i c s ( B , C , & F ) . P a r t i a l c o r r e l a t i o n s o f seed d i a m e t e r w i t h g e r m i n a t i o n t i m e , b y r e m o v i n g t h e e f f e c t o f fruit v o l u m e ( A D . F ) , r e v e a l e d f o u r s i g n i f i c a n t c o r r e l a t i o n s w i t h i n p l a n t s ( t h r e e w e r e n e g a t i v e a n d o n e p o s i t i v e ) . I n a l l c a s e s their v a l u e s w e r e l e s s t h a n + 0 . 2 9 . T h e b e t w e e n - p l a n t a n d w i t h i n - v a r i e t y p a r t i a l c o r r e l a t i o n s f o r t h e s e t h r e e c h a r a c t e r s w e r e n o t s i g n i f i c a n t . T h e w i t h i n - p l a n t c o r r e l a t i o n s f o r f r u i t d i a m e t e r v e r s u s seeds- p e r - c a v i t y ( B E ) w e r e n o t o b t a i n e d f r o m t h e e l e c t r o n i c c o m p u t e r , b u t f o r f r u i t t h i c k n e s s v e r s u s s e e d s - p e r - c a v i t y ( C E ) t h e values r a n g e d f r o m 0 . 0 0 t o + O . 3 6 a n d f o r f r u i t v o l u m e v e r s u s seeds- p e r - c a v i t y ( F E ) f r o m + 0 . 0 1 t o + 0 . 4 9 . N o n e o f t h e b e t w e e n - p l a n t c o r r e l a t i o n s o f s e e d s - p e r - c a v i t y w i t h f r u i t d i a m e t e r ( B E ) , f r u i t t h i c k n e s s ( C E ) , o r f r u i t v o l u m e ( F E ) w e r e s i g n i f i c a n t . T h e w i t h i n - v a r i e t v c o r r e l a t i o n o f s e e d s - p e r - c a v i t y w i t h f r u i t vol- u m e ( F E ) w a s h i g h l y s i g n i f i c a n t a t + 0 . 2 2 .
D i s c u s s i o n
T h e d a t a c l e a r l v r e v e a l a s i g n i f i c a n t p o s i t i v e r e l a t i o n s h i p be- t w e e n f r u i t size ( d i a m e t e r , t h i c k n e s s , o r v o l u m e ) a n d t h e time r e q u i r e d f o r g e r m i n a t i o n . O f t h e f r u i t size c h a r a c t e r s , v o l u m e a p p e a r s r o b e t h e b e s t f o r c o r r e l a t i o n s t u d i e s b e c a u s e i t involves b o t h d i a m e t e r a n d t h i c k n e s s . I n g e n e r a l , fruit, v o l u m e g a v e the h i g h e s t c o r r e l a t i o n s w i t h t i m e r e q u i r e d f o r g e r m i n a t i o n . The s i m p l e c o r r e l a t i o n s b e t w e e n g e r m i n a t i o n t i m e a n d s e e d d i a m e t e r g a v e v a l u e s v e r v s i m i l a r t o t h o s e b e t w e e n g e r m i n a t i o n t i m e and f r u i t s i z e d u e t o t h e h i g h p o s i t i v e c o r r e l a t i o n s b e t w e e n s e e d size a n d t h e v a r i o u s f r u i t s i z e a t t r i b u t e s .
W h e n t h e e f f e c t s o f f r u i t v o l u m e w e r e r e m o v e d b v a partial c o r r e l a t i o n t h e n p l a n t s 4 8 . 5 9 , 6 0 . a n d 1 1 0 h a d rA D . F v a l u e s o f
— 0 . 2 3 , — 0 . 1 8 , — 0 . 1 9 a n d + 0 . 2 8 r e s p e c t i v e l y . A l l o t h e r p l a n t had n o n s i g n i f i c a n t v a l u e s . W h e n c o e f f i c i e n t s o f d e t e r m i n a t i o n were c a l c u l a t e d , t h e m i n o r r o l e o f s e e d d i a m e t e r per s e i n g e r m i n a t i o n t i m e w a s a p p a r e n t .
F r o m t h i s s t u d y i t i s e v i d e n t t h a t f r u i t s i z e , w h e t h e r i t b e d i a m e t e r , t h i c k n e s s , o r v o l u m e , i s a p o o r i n d i c a t o r o f t h e content o f t h e o v a r i a n c a v i r y . M a n y f r u i t s 9 6 4 i n c h i n d i a m e t e r o r larger w e r e f o u n d t o c o n t a i n a b o r t e d s e e d s . M u l t i p l e s e e d s w e r e found i n b o t h s m a l l a n d l a r g e f r u i t s . C o r r e l a t i o n s b e t w e e n f r u i t volume a n d s e e d s - p e r - c a v i t y w e r e p o s s i b l e w i t h t h e d a t a f r o m a few o f t h e p l a n t s . T h e r v a l u e s r a n g e d f r o m + 0 . 0 1 t o + 0 . 4 9 which w o u l d m e a n t h a t f o r s o m e p l a n t s t h e r e i s a t e n d e n c y f o r t h e l a r g e r f r u i t s t o h a v e m o r e s e e d s p e r c a v i t y b u t f o r o t h e r plants t h i s t e n d e n c y d o e s n o t e x i s t . T h u s , i f a b r e e d e r i s g o i n g t o s e -
Vol. 13, N o . 2, J U L Y 1961 125
lect against m u l t i p l e seeds p e r cavity, h e m u s t e x a m i n e a d e q u a t e - ly all fruit sizes on a p l a n t r a t h e r t h a n just t h e l a r g e r fruits.
T h e s h a p e of t h e w h o l e m o n o g e r m fruit was e x a m i n e d by dividing t h e fruit t h i c k n e s s by t h e fruit d i a m e t e r ( T a b l e 2) a n d by t h e c o r r e l a t i o n s b e t w e e n fruit d i a m e t e r a n d fruit thickness
( T a b l e 5). Since t h e e x t r e m e s in t h e thickness by d i a m e t e r r a t i o only differed from t h e m e a n by 12.5% a n d since t h e b e t w e e n - p l a n t c o r r e l a t i o n for fruit d i a m e t e r versus fruit t h i c k n e s s was +0.77, t h e r e a p p e a r s to be o n l y a small c h a n c e to c h a n g e t h e shape of f r u i t of this variety by b r e e d i n g .
If t h e c o m p o s i t e d a t a a r e c o n s i d e r e d as r e p r e s e n t i n g t h e var- iety, t h e n g e n e r a l r e l a t i o n s b e t w e e n fruit size a n d seed d i a m e t e r may b e i n d i c a t e d . T h e l a r g e r t h e fruit, t h e slower t h e seed w i t h i n it g e r m i n a t e d . Seed d i a m e t e r h a d no significant effect on speed of g e r m i n a t i o n . F o r a n y g i v e n p l a n t in w h i c h v i a b l e seeds of different d i a m e t e r s w e r e c o n t a i n e d in fruits of fixed v o l u m e , differences in speed of g e r m i n a t i o n v a r i e d by as m u c h as 24 h o u r s , b u t g e n e r a l l y t h e v a r i a t i o n a v e r a g e d 12 h o u r s or less. In c o m p a r i s o n , seeds of t h e s a m e d i a m e t e r in fruits of d i f f e r e n t sizes, from t h e s a m e p l a n t a n d p a r t i c u l a r l y from d i f f e r e n t p l a n t s ( p r e s u m a b l y differing i n k i n d s a n d a m o u n t s o f i n h i b i t o r s ) , dif- fered in speed of g e r m i n a t i o n by as m u c h as 10 days. E x c e p t for the m a r k e d l y slower g e r m i n a t i o n of t h e seeds in t h e t w o smallest fruit v o l u m e classes ( T a b l e 4) w h i c h a p p e a r s to be c o n t r o l l e d in- t e r n a l l y by t h e seeds, s p e e d of g e r m i n a t i o n of seeds in l a r g e r fruits is r e g u l a t e d m a i n l y by t h e m a t e r n a l tissues of t h e fruit. G e n e r a l l y because of c h e m i c a l i n h i b i t o r s in t h e fruit, seeds in situ in fruits g e r m i n a t e m o r e slowly t h a n seeds r e m o v e d from t h e m . H o w e v e r , a s t i m u l a t o r y effect of t h e fruit on speed of g e r m i n a t i o n has b e e n observed (7, 8 ) . If s a m p l e s of fruits h a v i n g o n l y a s t i m u l a t i n g a c t i o n c o u l d b e isolated, t h e n l a r g e r fruits p r o b a b l y w o u l d b e desired.
S u m m a r y
S a m p l e s of w h o l e fruits from 19 p l a n t s of t h e m o n o g e r m s u g a r beet v a r i e t y S P 5832-0 w e r e sized for d i a m e t e r a n d t h i c k n e s s i n t o a n u m b e r of size classes. An X-ray t e c h n i q u e was used to e x a m i n e the c o n t e n t s a n d t o d e t e r m i n e t h e d i a m e t e r o f t h e seed w i t h i n each of 6,782 fruits. Seedless o v a r i a n cavities w e r e f o u n d in 10.2%
o f t h e fruits. T w o o r m o r e seeds p e r cavity w e r e f o u n d w i t h i n some of t h e fruits from 1.5 of t h e p l a n t s . T h e fruits w e r e p l a c e d o n b l o t t e r s a n d t h e t i m e r e q u i r e d for g e r m i n a t i o n was r e c o r d e d (in 1/2-day i n c r e m e n t s ) for each seed t h a t g e r m i n a t e d w i t h i n 25 days.
126 J O U R N A L OF T H E A. S. S. B. T.
F r u i t size, as m e a s u r e d by d i a m e t e r , thickness, or v o l u m e , sig- nificantly i n f l u e n c e d t h e t i m e r e q u i r e d for g e r m i n a t i o n . Gen- erally seeds in t h e l a r g e r fruits g e r m i n a t e d m o r e slowly. F r u i t size was a p o o r i n d i c a t o r of e i t h e r seedless fruits or m u l t i p l e - seeded fruits.
Since fruit v o l u m e was h i g h l y c o r r e l a t e d w i t h seed d i a m e t e r , a p a r t i a l c o r r e l a t i o n w h i c h r e m o v e d t h e effects of fruit v o l u m e was used t o d e t e r m i n e t h e r e l a t i o n s h i p b e t w e e n seed d i a m e t e r a n d g e r m i n a t i o n t i m e . T h e rA D . F v a l u e for t h e variety o f —0.03 i n d i c a t e d t h a t seed d i a m e t e r per se h a d l i t t l e effect on t h e ger- m i n a t i o n r a t e . W i t h i n i n d i v i d u a l p l a n t s , h o w e v e r , t h e rA D . F
values r a n g e d from —0.23 to +0.28 w h i c h i n d i c a t e d t h a t certain p o p u l a t i o n s c o u l d be isolated in w h i c h seed d i a m e t e r per se could i n f l u e n c e t h e g e r m i n a t i o n r a t e .
If g e r m i n a t i o n t i m e (speed of g e r m i n a t i o n ) is to be t h e cri- t e r i o n for selection of m o n o g e r m p l a n t s , t h e n , in m o s t cases, large seeds w o u l d n o t be d e s i r a b l e b e c a u s e of t h e h i g h c o r r e l a t i o n be- t w e e n large seeds a n d large fruits.
Literature Cited
(1) . DOXTATOR, C. W. a n d R. H. H E L M E R I C K . 1962. Selection for seed size in m o n o g e r m varieties. Am. Soc. Sugar Beet T e c h n o l . J. 12 (3):
268-272.
(2) H O G A B O A M , G. J. 1961. R a d i o g r a p h i n g as a m e t h o d of observing some seed characters in m o n o g e r m sugar beet fruits. Am. Soc. Sugar Beet T e c h n o l . J. 11: 605-609.
(3) H O G A B O A M , G. J., F. W. SNYDER a n d H. W. BOCKSTAHLER. 1959. Se- lecting for yield a n d sucrose i m p r o v e m e n t of a m o n o g e r m sugar beet variety. A m . Soc. Sugar Beet T e c h n o l . Eastern R e g . Proc.
10: 21-28.
(4) P R I C E . C. a n d E. CARSNER. 1916. Seed size in relation to d e v e l o p m e n t and yield of sugar beets. Am. Soc. Sugar Beet T e c h n o l . Proc. 4: 263-269.
(5) SAVITSKY, V. F. 1954. R e l a t i o n between the weight of fruit a n d weight of germ in mono- a n d m u l t i g e r m beets. Am. Soc. Sugar Beet Tech- nol. Proc. 8 (2) : 16-22.
(6) SAVITSKY. V. F., G. K. RYSER. G. E. R U S H a n d C. P. PARRISH. 1954.
Inter-relation between weight of seed a n d fruit a n d u t i l i t a r i a n char- acters in i n b r e d lines a n d hybrids of m o n o g e r m sugar beets. Am. Soc.
Sugar Beet T e c h n o l . Proc. 8 (2) : 399-403.
(7) SEDLMAYR, T. E. 1960. I n h e r i t a n c e of speed of g e r m i n a t i o n in sugar beets (Beta vulgaris L.). Doctoral dissertation. Michigan State Uni- versity.
(8) SNYDER. F. W. 1959. Influence of the seedball on speed of germination of sugar beet seeds. Am. Soc. Sugar Beet T e c h n o l . J. 10: 513-520.
(9) U S T I M E N K O . S. P. 1957. Effect of p e r i c a r p on the s p r o u t i n g energy of seeds of m o n o g e r m sugar beet. (In Russian) Sakh. Svekla 2 (12):
24-27.
Levels of T o t a l Nitrogen, Potassium and Sodium in Petioles and in Thin Juice of Sugar Beets
1,4 M E R L E O . P A Y N E2, L E R O Y P O W E R S3 AND G R A C E W . M A A G2Received for publication January 16. 1964
In 1961 s t u d i e s w e r e c o n d u c t e d to d e t e r m i n e levels of total nitrogen, p o t a s s i u m a n d s o d i u m i n t h e p e t i o l e s a s c o m p a r e d w i t h levels of those c h e m i c a l s in t h e t h i n j u i c e of s u g a r beets (Beta vulgaris L.). T h e p e t i o l e s a r e a s t r u c t u r e of t h e tops of t h e s u g a r beet a n d t h e t h i n j u i c e i s p r e p a r e d from t h e r o o t s o f t h e s a m e plant. T h e p r i m a r y p u r p o s e o f the s t u d y was t o d e t e r m i n e w h e t h e r genotypes differ as to levels of these c h e m i c a l c o n s t i t u e n t s in t h e petioles a n d i n t h e t h i n juice a n d , i f so, w h e t h e r s o m e g e n o t y p e s tend t o h a v e h i g h e r levels i n t h e p e t i o l e s a n d l o w e r levels i n t h e thin j u i c e , w h e r e a s for o t h e r g e n o t y p e s t h e r e v e r s e i s t r u e . I n other Avoids is t h e r e an i n t e r a c t i o n of g e n o t y p e s a n d m a t e r i a l analysed ( p e t i o l e s or t h i n j u i c e ) as r e g a r d s levels of total n i t r o g e n , potassium a n d s o d i u m ? Such i n f o r m a t i o n is of g r e a t f u n d a m e n t a l and p r a c t i c a l i m p o r t a n c e t o t h e b e e t s u g a r i n d u s t r y a s t h e s e chemicals h a v e b e e n f o u n d t o b e associated w i t h yield a n d q u a l i t y .
Literature R e v i e w
E m m e r t ( 3 , 4 , 5,)5 w o r k i n g w i t h t o m a t o e s , l e t t u c e a n d cu- c u m b e r s d e v e l o p e d r a p i d m e t h o d s for e s t i m a t i n g n i t r a t e n i t r o g e n , phosphate, a n d p o t a s s i u m i n p l a n t s . H e b e l i e v e d t h a t i n a s m u c h as t h e n u t r i e n t s d e r i v e d by a g r o w i n g p l a n t from t h e soil m u s t enter in s o l u t i o n t h r o u g h t h e s t e m t h a t t h e c o n c e n t r a t i o n of a given n u t r i e n t i n t h i s m a t u r e c o n d u c t i v e tissue s h o u l d b e d i r e c t l y p r o p o r t i o n a l to t h e a v a i l a b l e s u p p l y of t h e n u t r i e n t in t h e soil.
Hence, a m e a s u r e of t h e c o n c e n t r a t i o n of n u t r i e n t s in this con- ductive tissue m a y be a b e t t e r m e a s u r e of t h e a b i l i t y of t h e soil t o s u p p l y n u t r i e n t s t o g l o w i n g p l a n t s t h a n c h e m i c a l tests o f t h e soil itself. Also, he felt t h a t an o p t i m u m c o n t e n t of n u t r i e n t s
1Cooperative investigations of the Colorado Agricultural Experiment Station, the Crops Research Division, Agricultural Research Service. U. S. Department of Agri- culture, and the Beet Sugar Development Foundation. T h e Colorado State University gratefully acknowledges financial support from the fames G. Boswell Foundation admin- istered by the Agricultural Research Center of Stanford Research Institute, the National Institutes of Health, the National Plant Food Institute and contract-research funds [12- 14-100-4549 ( 3 4 ) ] from the Agricultural Research Service of the U. S. Department
of Agriculture. Approved bv the Colorado Agricultural Experiment Station for publi- cation as Scientific Series Article No. 884.
2Professor of Chemistry and Research Assistant Colorado State University, respectively, Colorado State University.
3Geneticist, Crops Research Division, Agricultural Research Service. U. S. Depart- ment of Agriculture.
4T h e writers arc indebted to R. Ralph Wood of the C.reat Western Sugar Company for obtaining thin juice samples bv an oxalate method standard with his company and to the Western Data Processing Center at the University of California at Los Angeles
or use of the computing facilities for analysing data. J o b No. 398.
5Numbers in parentheses refer to literature cited.
128 J O U R N A L OF THE A. S. S. B. T.
in this k i n d of tissue exists for the v a r i o u s stages of g r o w t h of each k i n d of c r o p , regardless of t h e k i n d of soil in w h i c h the c r o p is g r o w i n g . H i s researches s u p p o r t e d these d e d u c t i o n s and h e n c e seemed to justify t h e p r a c t i c e of a n a l y s i n g t h e m a t u r e con- d u c t i n g tissues of t h e p l a n t to d e t e r m i n e t h e a b i l i t y of t h e soil t o p r o v i d e t h e n u t r i e n t r e q u i r e m e n t s . G a r d n e r a n d Robertson (6) anaylsed petioles of sugar b e e t s a n d f o u n d it useful in deter- m i n i n g fertilizer n e e d s as r e g a r d s n i t r a t e , p h o s p h a t e a n d po- tassium.
U l r i c h (16, 17, 18) a n d T o l m a n a n d J o h n s o n (15) conducted e x t e n s i v e e x p e r i m e n t s w i t h s u g a r beets, s t u d y i n g yield a n d quality as r e g a r d s fertilizer practices. He f o u n d a n e g a t i v e r e l a t i o n be- tween levels of n i t r a t e n i t r o g e n in t h e petioles a n d percentage sucrose of t h e roots. He also established t h e o p t i m u m level of n i t r a t e n i t r o g e n in t h e petioles as r e g a r d s p e r c e n t a g e sucrose to b e a p p r o x i m a t e l y 1000 p p m . H e f o u n d t h a t once t h e critical n u t r i e n t level for an e l e m e n t has b e e n established for a crop t h r o u g h m a n y field e x p e r i m e n t s , p l a n t analysis has t h e following a p p l i c a t i o n s : (A) d e t e r m i n a t i o n of t h e k i n d of n u t r i e n t that m i g h t be deficient in t h e field; (TV) e s t i m a t i o n of t h e t i m e of a p p l i c a t i o n a n d t h e a m o u n t of fertilizer to a p p l y : (C) aid in selecting the location of fertilizer e x p e r i m e n t s : a n d (D) aid in m a i n t e n a n c e of t h e p r o p e r level of soil fertilitv. Also, some beet sugar c o m p a n i e s have used t h e t e c h n i q u e s he d e v e l o p e d to deter- m i n e fertilizer practices and established dates of harvest. Prob- ably o n e of t h e m o r e i m p o r t a n t c o n t r i b u t i o n s of t h e researches by U l r i c h is the s t i m u l a t i o n of r a t h e r e x t e n s i v e researches on the effects of fertilizer practices on t h e yield, q u a l i t y a n d processing of s u g a r beets.
R o r a b a u g h a n d N o r m a n (12) f o u n d t h e o r d e r i n which some of t h e c o m m o n b e e t - s i r u p i m p u r i t i e s adversely affect crystal- lization of sugar, d u r i n g factorv processing, to be as follows: (A) c a r b o n a t e a n d c h l o r i d e salts, (B) a m i n o acids, (C) b e t a i n e and n o n - n i t r o g e n o u s o r g a n i c acids, a n d (D) sulfate salts. T h e y found t h e c a r b o n a t e s a n d c h l o r i d e s to be strongly m t l a s s i g e n i c and to be p r e s e n t in relatively large c o n c e n t r a t i o n s in t h e b e e t syrups.
H e n c e , t h e c a r b o n a t e s a n d c h l o r i d e s a c c o u n t e d for a large frac- t i o n of t h e total sugar lost in molasses. T h e v c o n c l u d e that the most fertile g r o u n d for i m p r o v e m e n t of t h e crystallization char- acteristic lies in t h e e l i m i n a t i o n of c a r b o n a t e a n d c h l o r i d e salts w i t h l o w e r i n g of p y r o l i d o n e c a r b o x y l i c acid a n d t h a t glutamic acid is t h e second most likely p o i n t of attack. T h e v f u r t h e r point o u t t h a t t h e case against p y r o l i d o n e c a r b o x y l i c acid a n d glutamic acid is a two-edged o n e , since n o t o n l y a r e they m a j o r contribu-
Vol. 13, No. 2, JULY 1961 129
tors to sugar loss in molasses, but also d e c o m p o s i t i o n of g l u t a m i n e . the a m i d e from which they o r i g i n a t e , causes processing difficulties through l o w e r i n g of: buffering capacity of j u i c e a n d l o w e r i n g alkalinity. T h e r a t h e r extensive s t u d i e s o f C a r r u t h e r s a n d O l d - field (2) in general agree w i t h those of R o r a b a u g h a n d N o r m a n
(12). In a d d i t i o n C a r r u t h e r s a n d Oldfield p r e s e n t m e t h o d s of assessing q u a l i t y that a p p e a r t o h a v e c o n s i d e r a b l e m e r i t a n d h e n c e extensive a p p l i c a t i o n .
H a d d o c k , L i n t o n a n d H u r s t (7) f o u n d that n i t r o g e n fertili- zation a n d n i t r o g e n plant c o m p o s i t i o n a r e closely associated with sucrose storage in beet r o o t s a n d sugar recoveries from ex- tract j u i c e . Also, they f o u n d t h a t t h e s o l u b l e n i t r o g e n c o n s t i t u e n t s of the s u g a r beet roots a r e highly associated w i t h , if n o t responsi- ble for, v a r i a t i o n s in p u r i t y a n d sucrose p e r c e n t a g e as well as in dry m a t t e r p e r c e n t a g e . F r o m t h e i r studies t h e p a r t i c u l a r com- ponents w h i c h a p p e a r to be most h i g h l y associated with c h a n g e s i n q u a l i t y a r e t h e g l u t a m i n e a n d a m m o n i a fractions. T h e y believe the g l u t a m i n e n i t r o g e n to be of greatest signiiicance in q u a l i t y variation, because of its high association, w i t h q u a l i t y factors, a n d because t h e c o n c e n t r a t i o n of this form of n i t r o g e n is ten t i m e s that of a m o m n i a n i t o g e n .
R o u n d s et al. (13) f o u n d t h a t t h e n i t r o g e n levels caused greater v a r i a t i o n s in t h e a m o u n t s of n o n s u g a r s p r e s e n t in the beet roots t h a n did t h e varieties tested. Significant i n t e r a c t i o n s of varieties X n i t r o g e n fertility levels w e r e f o u n d for s o d i u m con-
t e n t . T h e d a t a p r e s e n t e d i n d i c a t e t h a t b o t h varieties a n d n i t r o g e n fertility levels can a p p r e c i a b l y influence t h e a m o u n t of n o n - sugars in beets. T h e association of total n i t r o g e n a n d n i t r o g e n c o m p o u n d s w i t h r e d u c e d p u r i t y a n d e x t r a c t i o n i n t h e roots was p r o n o u n c e d . R y s e r et al. (14) in a c o m p a r i s o n of harvest d a t e s found t h a t t h e sugars from t h e late harvest w e r e h i g h e r t h a n from t h e early harvest w h i l e t h e p u r i t i e s w e r e lower. T h e un- expected decrease in p u r i t y was n o t a c c o u n t e d for by an increase in the level of a m i n o n i t r o g e n . Levels of n i t r a t e n i t r o g e n in t h e petioles, a m i n o N i n t h e roots, a n d X a c o n t e n t i n t h e roots w e r e greatly influenced by h i g h n i t r o g e n fertilization, b u t varietal dif- ferences w e r e j u s t as s t r i k i n g . In Xa c o n t e n t , a f o u r or fivefold difference b e t w e e n low X a types a n d h i g h X a types was n o t u n c o m m o n .
O w e n et al. (8) f o u n d t h a t highest p u r i t y was n o t always associated w i t h highest p e r c e n t a g e sucrose. They a t t r i b u t e d t h e i r resuits t o b e d u e t o a n i n t e r a c t i o n b e t w e e n g e n o t y p e s (as r e p r e -
s e n t e d b y h y b r i d s ) a n d locations. P o w e r e t al. (9) f o u n d t h a t