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Some techniques for the study of roots in place; with special reference to their use in the root development… Frederick, Rexingford Albert 1959

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SOME TECHNIQUES FOR THE STUDY OF ROOTS IN PLACE With s p e c i a l reference to t h e i r use i n the root development of f o u r grasses and two legumes of economic importance. by REXINGFORD ALBERT FREDERICK B. S. A., U n i v e r s i t y of B r i t i s h Columbia, 1958. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n the Department of P lant Science We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1959 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree th a t permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of P l a n t Science.  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8 , Canada. ABSTRACT There are s e v e r a l l i m i t a t i o n s to the use of the ' d i r e c t ' method f o r studying root systems i n t h e i r n a t u r a l h a b i t a t s . Newer techniques are needed i n understanding many c h a r a c t e r i s t i c s of r o o t s , which are s t i l l obsciire. The growing of orchardgrass, c o l o n i a l bentgrass and red c l o v e r i n s p e c i a l c o n t a i n e r s , demonstrated a technique f o r o p t i c a l study of root systems, and i l l u s t r a t e d an apparent l o c a l i z a t i o n of i n d i v i d u a l p l a n t r o o t s i n dense stands. F i e l d t e s t s on square fo o t p l o t s of orchardgrass and Kentucky bluegrass w i t h placements of a dye, a c i d f u c h s i n , at v a r i o u s depths, f a i l e d to r e p l i c a t e the p r e l i m i n a r y l a b o r a t o r y f i n d i n g s , t h a t the absorption by roots of t h i s m a t e r i a l not only s t a i n e d the roo t s red, but was soon evident i n the a e r i a l p o r t i o n s . P l a n t i n j e c t i o n s and s o i l placement of the f l u o r e s c e n t compounds, e o s i n , f l u o r e s c e i n and e s c u l i n f a i l e d to produce fluorescence i n p l a n t s growing i n s p e c i a l c o n t a i n e r s . The s t r a t e g i c placements of small q u a n t i t i e s of r a d i o -a c t i v e phosphorus (p32) a t var i o u s depths i n p l o t s of orchard grass, red fescue, Kentucky bluegrass and c o l o n i a l bentgrass on Alderwood loamy sand, enabled the depth of root penetra-t i o n t o be assessed by measuring r a d i o a c t i v i t y i n the leaves w i t h a Geiger-Muller counter. The movement of P 3 2 i n the s o i l was s t u d i e d on the p r o f i l e s of s i m i l a r p l o t s kept bare of v e g e t a t i o n , and on which s i m i l a r placements were made. i i i A modified technique r e s u l t e d i n the l o c a l i z a t i o n o f the r a d i o a c t i v e phosphorus i n the s o i l . P l a n t s i n e s t a b l i s h e d p l o t s of orchardgrass, red fescue, Kentucky bluegrass and c o l o n i a l bentgrass were i n j e c t e d w i t h P*^ near the crown. The movement t o the roo t s was found to be very slow when r a d i o a c t i v i t y was checked i n the r o o t s from the p r o f i l e s . In a four week o l d a l f a l f a clone, i n j e c t -ed three weeks e a r l i e r , P o c < was recorded to have reached the maximum depth a t t a i n e d by v i s i b l e r o o t s . This was not the case with the grasses. The absorption of l i t h i u m from l i t h i u m c h l o r i d e placed at various depths i n rows of orchardgrass and a l f a l f a was determined by photometric a n a l y s i s of samples from a e r i a l p o r t i o n s . The extent of the root p e n e t r a t i o n was determined by the r e l a t i v e l y higher concentrations of l i t h i u m i n p l a n t s l o c a t e d above these placements. i v ACKNOWLEDGEMENTS I wish t o take t h i s o p portunity to thank the va r i o u s departments and i n d i v i d u a l s , without whose cooperation and as s i s t a n c e t h i s p r e s e n t a t i o n could not have been s u c c e s s f u l . I would l i k e to extend s p e c i a l thanks t o Dr. V.C. Brink, P r o f e s s o r of Agronomy and Chairman of the d i v i s i o n of Pla n t Science, U n i v e r s i t y of B.C., under whose s u p e r v i s i o n t h i s p r o j e c t was undertaken. I s h a l l always be indebted to him not only f o r h i s keen d i r e c t i o n s and a s s i s t a n c e i n conducting and p r e p a r i n g t h i s t h e s i s , but a l s o f o r h i s patience and warm understanding which c o n t r i b u t e d g r e a t l y to a comfortable and pleasant p e r i o d of study. Through him I am a l s o indebted to the D i v i s i o n of P l a n t Science f o r p r o v i d i n g f a c i l i t i e s f o r c a r r y i n g out t h i s programme. I s i n c e r e l y appreciate the cooperation of Mr. Don Pearce, senior t e c h n i c i a n i n the D i v i -s i o n of Pla n t Science, who gave h i s a s s i s t a n c e f r e e l y . Sincere thanks a l s o are accorded Dr. J.J.R. Campbell, P r o f e s s o r of Da i r y i n g , U n i v e r s i t y of B.C., f o r h i s k i n d a s s i s t a n c e i n o b t a i n i n g and"preparing the r a d i o a c t i v e phos-phorus used i n t h i s p r o j e c t . The Department of S o i l Science was very k i n d i n permit-t i n g me t o use the Perkin-Elmer Flame Photometer and have the s e r v i c e s of Mr. Vincent Osborne, Graduate A s s i s t a n t . I am very g r e a t f u l f o r t h i s cooperation. I wish to express s i n c e r e thanks t o the U n i v e r s i t y ' s Department of Mining and Me t a l l u r g y f o r the frequent use of a Geiger-Muller counter, which was necessary i n c a r r y i n g out c e r t a i n phases of t h i s study. My a p p r e c i a t i o n and hearty thanks are accorded Mr. A. Mohammed of the Mathematics department, f o r h i s advice i n the diagrammatic p r e s e n t a t i o n of c e r t a i n r e s u l t s , and to the Extension department of the U n i v e r s i t y f o r c e r t a i n photographs. I wish to thank the Pla n t Products D i v i s i o n of the Department of A g r i c u l t u r e , Vancouver, f o r a l l o w i n g me to use t h e i r vacuum seed counters i n the e a r l i e r phases of my graduate work. L a s t l y , but by no means l e a s t , I would l i k e to tender my sincere thanks to my w i f e , R i t a , not only f o r her moral support and the t y p i n g of t h i s p r e s e n t a t i o n , but a l s o f o r making a v a i l a b l e c e r t a i n l i t e r a t u r e which might have otherwise escaped my a t t e n t i o n . V TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 2 The D i r e c t Method of Root Study 3 ( i ) Simple Washing 4 ( i i ) Trench Washing 4 ( i i i ) S o i l M o n olith 4 ( i v ) ' S o i l Prism' 4 (v) ' S o i l Block' 5 ( v i ) ' N a i l Board' 5 ( v i i ) 'Core Sampling' 5 ( v i i i ) Containers and N u t r i e n t S o l u t i o n s 6 I n d i r e c t Methods of Studying Roots 6 Methods u s i n g dyes or elements not normally present i n q u a n t i t y i n s o i l 6 The use of f l u o r e s c e n t compounds 7 Methods us i n g r a d i o a c t i v e isotopes 8 Some r e l e v a n t comments on the use of P 3 2 f o r root study 11 R e l a t i v e a b s o r p t i o n by p l a n t s of phosphorus from s o i l and f e r t i l i z e r 12 Phosphorus u t i l i z a t i o n by p l a n t r o o t s 14 Phosphorus m o b i l i t y i n p l a n t s 15 Exchange phenomena of phosphorus i n s o i l s 17 Phosphorus m o b i l i t y i n s o i l s 20 R a d i a t i o n e f f e c t s on p l a n t s 22 EXPERIMENTATION 24 1. A technique u s i n g s p e c i a l containers 25 v i 2. A technique u s i n g i n t r a v i t a m dyes and f l u o r e s c e n t compounds 29 3. A technique u s i n g radipphosphorus s o i l placement and pla n t i n j e c t i o n s 32 4. A technique u s i n g l i t h i u m c h l o r i d e placement i n s o i l 44 DISCUSSION AND CONCLUSIONS 47 SUMMARY 51 LITERATURE CITED 55 APPENDIX A. Stand Establishment of Forage Grasses and Legumes 62 APPENDIX B. P l a t e s 80 1. SOME TECHNIQUES FOR THE STUDY OF ROOTS IN PLACE With s p e c i a l reference t o t h e i r use i n the root development of f o u r grasses and two legumes of economic importance. INTRODUCTION Thousands of macro observations are made each year i n crops, but remarkably few of these are made i n crop r o o t s . The g e n e t i c s , morphology and physiology of crops i s l a r g e l y , i t can be s a i d w i t h some t r u t h , the ge n e t i c s , morphology and physiology of the a e r i a l p l a n t p a r t s . Rarely are pla n t r o o t s given much a t t e n t i o n and more r a r e l y i s the p l a n t i n t o t o considered. There are of course good reasons why root observations are r e l a t i v e l y few, f o r i t i s not because of the f a i l u r e to appreciate the s i g n i f i c a n c e of roots t h a t observations are not more commonly made. In the f i r s t p l a c e , r o o t s are u s u a l l y hidden from s i g h t and t h e i r d i r e c t observation i n v o l v e s l a b o r i o u s removal of l a r g e volumes of s o i l . Again, many root systems are made up of t i s s u e s , which when measured i n l i n e a r terms, are hundreds of miles i n le n g t h . Much of t h i s t i s s u e i s f r a g i l e and microscopic. A c c o r d i n g l y , i t i s r a r e l y pos-s i b l e t o obtain by any method of d i r e c t observation more than a small sample of a root system. For these and other reasons d i r e c t study of roots i s d i f f i c u l t . There are, how-ever, i n the l i t e r a t u r e some i n d i c a t i o n s that new knowledge i n science may advance the study of r o o t s . The purpose of 2. t h i s p r e s e n t a t i o n i s to study these newer p o s s i b i l i t i e s . LITERATURE REVIEW There i s abundant experimental evidence which i n d i c a t e s that a root responds q u i c k l y and profoundly to the e n t i r e complex of growth f a c t o r s . Pavlychenko and Harrington (1934, 1935, 1937(a )(b)) have shown t h a t , not only are roots the f i r s t v i s i b l e organs upon germination, but they adapt them-selves to v a r i o u s s o i l c o n d i t i o n s before the stem success-f u l l y emerges. During a p l a n t ' s l i f e , r o ots must continuous-l y adjust themselves to the changing c o n d i t i o n s i n the ground, or otherwise top growth w i l l p e r i s h . Consequently top growth, which depends d i r e c t l y upon the performing c a p a c i t y of the ro o t s , i s i n large measure a r e s u l t a n t of the i n t e r a c t i o n between h a b i t a t f a c t o r s and the root system. There i s con-s i d e r a b l e evidence to show that under a l l c o n d i t i o n s a root p l a y s a l e a d i n g part i n keeping the p l a n t i n the 'best p o s s i b l e balance' w i t h the environment. The d i r e c t method of stud y i n g a root has c e r t a i n de-f i c i e n c i e s . This method, u s u a l l y i n v o l v i n g excavating, washing and/or growing p l a n t s i n s p e c i a l c o n t a i n e r s , i s high-l y a r t i f i c i a l and does not provide much u s e f u l information on p l a n t s growing i n communities. No techniques have yet been devised to enable ' l i v i n g ' r o o t s to be d i s t i n g u i s h e d w i t h c e r t a i n t y from 'dead' r o o t s . The inf o r m a t i o n regarding 3. root s p e c i a l i z a t i o n i s s t i l l v e ry obscure, though i t i s very important to d i f f e r e n t i a t e between a c t i v e and non-active r o o t s . Root dynamics and morphology i s a d i f f i c u l t study on t h i s b a s i s because of the l o s s of the pl a n t a f t e r excavation. The change i n root a c t i v i t i e s w i t h season a l s o i s an impor-ta n t problem, f o r i t i s true t o st a t e that the s u s c e p t i b i l i t y of p l a n t s to damage from root diseases and i n s e c t pests or t o w i n t e r - k i l l i n g i s governed to a considerable extent by t h e i r a b i l i t y to regenerate the damaged roots or t o replac e them by new ones (Simmonds et a l 1935). Excavating and root wash-in g i n v o l v e s a great deal of labour, yet no amount of patience can prevent the l o s s of many microscopic r o o t l e t s as s o i l i s removed, or can f r e e the roo t s completely of s o i l p a r t i c l e s and organisms. F i n a l l y , inasmuch as a root system i s a three dimensional e n t i t y , the exact p o s i t i o n of many ro o t s i s d i f f i c u l t to determine when the s o i l has t o be d i s t u r b e d . The d i r e c t method has y i e l d e d valuable i n f o r m a t i o n i n r o o t s , but as must be evident from the for e g o i n g comments, i t has many serious l i m i t a t i o n s . Although the prime i n t e r e s t was t o review the l i t e r a t u r e and to p r a c t i c e new methods of root study, such as those i n v o l v i n g the use of r a d i o i s o t o p e s , i t seemed appropriate to at l e a s t review the l i t e r a t u r e on the methodology of the d i r e c t method, f o r many of the new methods may make some use of or extend the d i r e c t method. The D i r e c t Method of Root Study 4 . ( i ) Simple Washing. In the a r i d r e g i o n s where subsurface moisture i s a very important item, the e l a b o r a t i o n o f exten-s i v e r o o t systems enhances the s u r v i v a l o f stands of forage grasses and legumes. The d i r e c t i n v e s t i g a t i o n o f the extent of r o o t systems i n p l a n t communities i s a v e r y t e d i o u s and d i f f i c u l t t a s k . Perhaps the f i r s t t o comprehend f u l l y the value of Information on the e x t e n t of r o o t systems o f a g r i c u l -t u r a l p l a n t s from the s t a n d p o i n t o f crop p r o d u c t i o n , was Hales i n 1727, to whom Pavlychenko (1937(b)) r e f e r s . H i s method c o n s i s t e d o f a d i r e c t washing from the ground, an a p p a r e n t l y l o n g and awkward proce e d i n g . ( i i ) Trench Washing. The s i m p l e s t technique d e v i s e d t o c a r r y out s e p a r a t i o n o f the r o o t s from the s o i l was t h a t used by Weaver (1926). T h i s c o n s i s t s of d i g g i n g a t r e n c h some di s t a n c e away from the p l a n t to be s t u d i e d and t r a c i n g the r o o t s back to the p l a n t from the f a c e o f the t r e n c h by c a r e -f u l l y e x c a v a t i n g the s o i l w i t h hand t o o l s . This again i s a very l a b o r i o u s procedure and i s o n l y s u i t a b l e f o r c e r t a i n experiments. ( i i i ) S o i l M o n o l i t h . Perhaps the most popular method used i n r o o t study i s the one used by Weaver and Barland (1949) whereby a m o n o l i t h of s o i l i s taken from the f i e l d and the r o o t s are washed f r e e o f s o i l by means of a j e t of water. In some cases, e s p e c i a l l y under a c l o s e grass sward, i t i s v i r t u a l l y i m p o s s i b l e to separate s i n g l e p l a n t s and t r a c e t h e i r r o o t s down the p r o f i l e . ( i v ) ' S o i l Prism'. A more complicated process c o n s i s t s 5. of h o l d i n g the r o o t s i n the p o s i t i o n they occupy i n the s o i l by means of wire supports w h i l e the washing p r o c e s s take p l a c e . The f i n a l r e s u l t t h e o r e t i c a l l y , i s the q u a s i complete r o o t system w i t h every r o o t i n i t s n a t u r a l p o s i t i o n but without the surrounding s o i l . One of the f i r s t workers t o use t h i s method was King i n 1892 (Troughton 1957) who i s o l a t e d a pri s m of s o i l to the depth of the r o o t system and removed i t i n a g a l v a n i z e d i r o n cage with w i r e - n e t t i n g . A f t e r washing w i t h a f o r c e pump the r o o t s were h e l d i n p o s i t i o n by the cr o s s wires through which photographs were made. (v) ' S o i l Block*. A m o d i f i c a t i o n o r t h i s technique was used by Pavlychenko (1937) whereby a bl o c k of s o i l was t r a n s -p o r t e d to the l a b o r a t o r y f o r washing without the use of the s u p p o r t i n g wires w i t h i n the s o i l . The method i s known as the ' s o i l b l o c k ' method. ( v i ) ' N a i l Board'• Another widely used m o d i f i c a t i o n o f the ' s o i l p r i s m ' method i s the ' n a i l board' method. A board of wood w i t h n a i l s p r o j e c t i n g a t r i g h t a n g l e s t o the major plane on one s i d e i s i n s e r t e d so t h a t the n a i l s p e n e t r a t e i n t o the s o i l from the face o f a t r e n c h . The p r i s m o f s o i l remains a t t a c h e d to the n a i l s when i t i s cut away. This i s removed to the l a b o r a t o r y where the s o i l i s washed away (B l a s e r 1937) ( v i i ) 'Core Sampling'. Other workers have d e v i s e d t e c h -niques f o r s e p a r a t i n g the r o o t s from the s o i l i r r e s p e c t i v e of the p o s i t i o n the r o o t s occupy i n the s o i l . These methods c o n s i s t e s s e n t i a l l y of t a k i n g a s o i l sample from the f i e l d , 6. c o n t a i n i n g both s o i l and r o o t s , soaking i t i n water, and then washing the r o o t s f r e e from s o i l w i t h a j e t or spray o f water a i d e d by hand m a n i p u l a t i o n (Troughton 1957). ( v i i i ) C o ntainers and N u t r i e n t S o l u t i o n s . S e v e r a l methods have been employed where p l a n t s are grown i n n u t r i e n t s o l u t i o n s o r ' i n c o n t a i n e r s which are e a s i l y taken a p a r t . Glass s i d e d Chambers were c o n s t r u c t e d f o r measuring the growth of seminal r o o t s w h i l s t i n c u l t u r e s o l u t i o n by P o l l o c k , Goodwin and Green (1954). T h i s among other t h i n g s p e r m i t t e d m i c r o s c o p i c examination and acc u r a t e measurement of growth. Attempts have been made to study r o o t s growing i n s o i l by means of g l a s s s i d e d p a n e l s i n s e r t e d i n t o the f a c e s o f s p e c i a l l y prepared trenches, and by means of boxes w i t h g l a s s s l i d e s (Troughton 1957). These methods, however, do not pro v i d e much u s e f u l i n f o r m a t i o n when p l a n t communities i n t h e i r n a t u r a l h a b i t a t s are be i n g s t u d i e d . I n d i r e c t Methods of St u d y i n g Roots Methods u s i n g dyes or elements not normally p r e s e n t i n  q u a n t i t y i n s o i l . The a b s o r p t i o n by p l a n t s o f dyes and of elements which are not norm a l l y present i n q u a n t i t y i n the s o i l , such as l i t h i u m and rubidium, has been used to i n d i c a t e the r e g i o n s of r o o t a c t i v i t y . Sayre and M o r r i s (1940) have i d e n t i f i e d l i t h i u m c h l o r i d e s p e c t r o g r a p h i c a l l y at 6708 & i n p l a n t s a f t e r placement i n the s o i l . The s i m i l a r i t y i n che-m i c a l p r o p e r t i e s o f l i t h i u m t o sodium and potassium accounts f o r the ease o f a b s o r p t i o n and accumulation. I t i s noted 7. t h a t l i t h i u m e n t e r s i n t o the base exchange complex o f the s o i l and does not move w i t h the s o i l water. Steward, Prevot and H a r r i s o n (1942) have r e p o r t e d the c a p a b i l i t y o f b a r l e y r o o t s to absorb rubidium and have demonstrated a g r a d a t i o n i n accumulation by the use of s p e c t r o g r a p h i c methods. The use of f l u o r e s c e n t compounds. Based on the f a c t t h a t d i s t i n c t i v e f l u o r e s c e n t substances occur i n grasses and seeds as obtained on f i l t e r paper when the l a t t e r are allowed to germinate, the p o s s i b i l i t y e x i s t s of making r o o t s t u d i e s u s i n g f l u o r e s c e n t compounds. Radley and Grant (1954) i l l u s t r a t e t h a t leguminous seeds, i n p a r t i c u l a r , p r e s e n t many c o l o u r e d f l u o r e s c e n c e e f f e c t s , so t h a t i n a d d i t i o n t o d i f f e r e n t i a t i n g between v a r i o u s s p e c i e s o f each genus, d i s t i n c t i o n may o f t e n be found between d i f f e r e n t s t r a i n s o f the same s p e c i e s . A number of papers have been p u b l i s h e d d e a l i n g w i t h the a p p l i c a t i o n o f the method to the study o f r y e - g r a s s s e e d l i n g s . The d i s t i n c t i o n o f the seeds of p e r e n n i a l r y e - g r a s s (Lolium  perenne L) from those of I t a l i a n r y e - g r a s s (Lolium m u l t i f l o - rium Lam.) i s not at a l l d i f f i c u l t p r o v i d i n g the t e r m i n a l awns of the I t a l i a n s p e c i e s are p r e s e n t . I t i s v e r y n e a r l y im-p o s s i b l e t o make the d i s t i n c t i o n when the seeds have been machine-dressed, owing t o the removal of the awns i n t h i s p r o c e s s . By means of a f i l t e r - p a p e r a n a l y s i s , however, i t has been found t h a t the s e e d l i n g s of I t a l i a n r y e - g r a s s and some of those of f a l s e p e r e n n i a l s give a blue f l u o r e s c e n c e which i s absent i n the case of the t r u e , normal p e r e n n i a l r y e - g r a s s . In f i l t e r e d u l t r a - v i o l e t l i g h t , many p l a n t substances show 8. a more or l e s s c h a r a c t e r i s t i c f l u o r e s c e n c e , i n some cases, even at h i g h d i l u t i o n s , although t h i s may disappear when the pH value f a l l s o u t s i d e a c e r t a i n range. Goodwin and Kavanagh (1948) examined the f l u o r e s c i n g substances i n r o o t s of 135 s p e c i e s of v a s c u l a r p l a n t s r e p r e s e n t i n g 69 f a m i l i e s . A l l were f l u o r e s c e n t , except 6 s p e c i e s i n which the dark pigment i n the r o o t s had a masking e f f e c t . The f l u o r e s c e n t substances are e x t r a c t a b l e w i t h acetone and butanol, and i t was shown t h a t acetone e x t r a c t s of Avena r o o t s c o u l d be separated i n t o t h r e e f r a c t i o n s , each w i t h a c h a r a c t e r i s t i c pH f l u o r e s c e n c e curve. The compound found i n the d i f f e r e n t i a t i n g and mature p o r t i o n s o f the r o o t and almost i f not completely absent from the a p i c a l 3 mm was l a t e r i d e n t i f i e d as s c o p o l e t e n (6-methoxy-7-hydroxy coumarin) (Goodwin and Kavanagh 1949). Goodwin and P o l l o c k (1954) s t u d i e d the p r o p e r t i e s and d i s t r i b u t i o n of f l u o r e s c e n t compounds i n Avena r o o t s and c a l l e d another f r a c t i o n which was found con c e n t r a t e d to a l a r g e extent i n the meristem, the ' r o o t - t i p g l y c o s i d e ' . Subsequently, the p h y s i o l o g i c a l a c t i v i t y o f these and other f l u o r e s c e n t substances were s t u d i e d by P o l l o c k e t a l (1954) i n d i c a t i n g the p o s s i b i l i t i e s o f u s i n g f l u o r e s c e n t compounds as t r a c i n g agents. Methods u s i n g R a d i o a c t i v e I s o t o p e s . Quite r e c e n t l y r a d i o a c t i v e t r a c e r s have been used i n the study of r o o t systems. H a l l et a l (1953) s t u d i e d the r o o t systems o f f o u r s p e c i e s o f crops - c o t t o n , corn, peanuts and tobacco, by growing p l a n t s i n s o i l i n which the 'native phosphate' was l a b e l e d by s t r a t e g i c a l l y l o c a t i n g s m a l l q u a n t i t i e s o f P 3 2 as potassium 9. dihydrogen phosphate (KHgP O4) a t g i v e n d i s t a n c e s from and. below the p l a n t and n o t i n g the uptake by the p l a n t w i t h time. Placement was made by the use o f a 2 cm diameter tube w i t h a s o l i d p o i n t , behind which was l o c a t e d o u t l e t p o r t s . A f t e r d r i v i n g i n t o the s o i l so t h a t the p o r t s were a t the r e q u i r e d depths, the p o r t s were c l e a r e d o f the d e b r i s by pre s s u r e from a hypodermic s y r i n g e which was a l s o used to i n j e c t the phos-phate s o l u t i o n i n t o the tube. In these i n v e s t i g a t i o n s 5 ml KHgP 3 204 of a s p e c i f i c a c t i v i t y o f approximately 50 m i c r o c u r i e s per gram was used. They were a b l e to e s t a b l i s h c o n s i d e r a b l e d i f f e r e n c e s i n r o o t p a t t e r n s and r e g i o n s o f maximum a c t i v i t y by measuring the s p e c i f i c a c t i v i t y i . e . r a t i o o f r a d i o a c t i v e phosphorus to t o t a l phosphorus. Root p e n e t r a t i o n , d i s t r i b u t i o n and a c t i v i t y of grasses under drought c o n d i t i o n s i n deep sand were s t u d i e d by Burton e t a l (1954) i n the southeastern U n i t e d S t a t e s . Turves of Common Bermuda (Cynodon d a c t y l o n ) , C o a s t a l Bermuda (C. d a c t y l o n ) , Swannee Bermuda (C. d a c t y l o n ) , Pensacola Bahia (Pensacola notatum), Pangola ( D i g i t a r i a decumbens) and D a l l i s (Paspalum d i l a t a t u m ) were t r a n s p l a n t e d i n areas i n which r a d i o -a c t i v e phosphorus ( P 3 2 ) i n the form o f superphosphate had been p l a c e d at v a r y i n g depths down t o 8 f e e t and depth o f root p e n e t r a t i o n was asses s e d by measuring the r a d i o a c t i v i t y i n the l e a v e s when a survey meter (Thyac) was p l a c e d near them. P l a c e -ment was accomplished by making a p e r p e n d i c u l a r h o l e t o the d e s i r e d depth with a probe made from 3/4 i n c h g a l v a n i z e d p i p e . A s u i t a b l e l e n g t h of 3/8 i n c h copper t u b i n g was then p l a c e d 10. i n the hole and 5 cc of P 3 2 s o l u t i o n , each placement c o n t a i n -i n g 200 m i c r o c u r i e s of P 3 2 was put i n t o the tube w i t h a s h e i l d e d hypodermic s y r i n g e . Withdrawing the tube 2 inches and washing w i t h 250 cc water, the h o l e was then f i l l e d w i t h screened dry s o i l from the same area. The r e l a t i v e r a t e s of r o o t p e n e t r a t i o n of the s p e c i e s s t u d i e d were l a r g e enough to i n f l u e n c e the s u c c e s s f u l e s t a b l i s h m e n t o f these grasses under unfavourable moisture c o n d i t i o n s . That those grasses capable of r e a c h i n g moisture r e s e r v e s deep down i n the s o i l should (other t h i n g s b e i n g equal) be b e s t a b l e to w i t h s t a n d drought f o l l o w i n g t r a n s p l a n t i n g , was backed up by the r e s u l t s which showed t h a t the drought t o l e r a n t c o a s t a l Bermuda r o o t s reach-ed depths of 8 f e e t while the drought s u s c e p t i b l e carpet grass (Axonopus a f f i n i a ) o n l y reached a depth of 2 f e e t . Boggie et a l (19 58) made s t u d i e s of the r o o t development of c e r t a i n g r asslands i n Aberdeen S c o t l a n d u s i n g r a d i o a c t i v e t r a c e r s . The r o o t i n g h a b i t of 25 s p e c i e s i n c l u d i n g A g r o s t i s , Festuca and T r i f o l i u m were s t u d i e d on m i n e r a l s o i l and peat s o i l w i t h v a r y i n g depth o f P 3 2 and R b 8 6 . Placement was made by d r i v i n g a 2 cm diameter s t e e l spike i n t o the ground w i t h a sledgehammer t o r a t h e r more than the r e q u i r e d depth. Imme-d i a t e l y a f t e r the withdrawal of the s p i k e , a metal tube i n t o which there was a metal ramrod was lowered i n t o the h o l e . The ramrod was withdrawn and 50 ml of a r a d i o a c t i v e phosphate s o l u t i o n c o n t a i n i n g 5 mg K H g P 3 2 0 4 as c a r r i e r was poured down the tube through a f i l t e r f u n n e l (each placement l e v e l con-s i s t i n g t h e r e f o r e about 150 m i c r o c u r i e s P 3 2 ) . The bore h o l e s 11. were f i l l e d w i t h dry s o i l a f t e r washing the tube w i t h 50 ml water and withdrawing the tube. Together w i t h the considerable d i f f e r e n c e s among the r o o t i n g h a b i t , the importance of the top 10 cm f o r root a b s o r p t i o n of n u t r i e n t s was very n o t i c e a b l e i n communities growing on m i n e r a l s o i l s . The l a t t e r was most evident i n A g r o s t i s - Festuca grasslands. Some re l e v a n t comments on the use of P 5 2 f o r root study According to Dr. W i l l a r d F. Libby, Commissioner, United States Atomic Energy Commission (1956), nThe r a d i o i s o t o p e , l i k e the F u l l e r brush man has found i t s way i n t o almost every nook and cranny of our s c i e n t i f i c community. Today there i s h a r d l y a l a r g e u n i v e r s i t y of s t a t e c o l l e g e that does not have a r a d i o i s o t o p e l a b o r a t o r y and a r a d i o i s o t o p e program." Among the many r a d i o i s o t o p e s i n use today, few are more g e n e r a l l y used than P . I t has a number of d e s i r a b l e p r o p e r t i e s from the standpoint of an experimenter; i t i s e a s i l y handled and r e a d i l y adapted to a wide v a r i e t y of uses. The employment of P 3 2 allows the undertaking o f experiments and measurements where o r d i n a r y chemical and i n s t r u m e n t a l measurements are not p r a c t i c a b l e . Two u n d e r l y i n g p r i n c i p l e s are i n v o l v e d . Popu-l a t i o n s o f ions or molecules can be l a b e l e d and r e a d i l y detect-ed, t r a c e d , or a q u a n t i t a t i v e determination made of d i l u t i o n by changes i n s p e c i f i c a c t i v i t y . Secondly, ready i d e n t i f i c a -t i o n and measurement at extremely low concentrations i s poss-i b l e . Radiophosphorus has been used to advantage i n a v a r i e t y of s t u d i e s having a r e l a t i o n to the a v a i l a b i l i t y of s o i l and 12. f e r t i l i z e r phosphorus. These i n c l u d e measurements o f the r e l a t i v e a b s o r p t i o n by p l a n t s of phosphorus from s o i l and f e r t i l i z e r , o f f e r t i l i z e r u t i l i z a t i o n , of phosphorus m o b i l i t y i n s o i l s , of exchange phenomena and o f r o o t growth and d i s -t r i b u t i o n . R e l a t i v e A b s o r p t i o n by P l a n t s o f Phosphorus from s o i l  and f e r t i l i z e r . Spinks and Barber (1947), Dean et a l (1947) and Nelson e t a l (1947) have conducted greenhouse and f i e l d experiments i n which f e r t i l i z e r s l a b e l e d w i t h P have been used to measure the r e l a t i v e amounts o f phosphorus absorbed by crops from s o i l and f e r t i l i z e r . The methods employed amounted e s s e n t i a l l y to the i n c o r p o r a t i o n i n s o i l and crops grown of f e r t i l i z e r s o f known s p e c i f i c a c t i v i t y . Then the r a t i o o f the s p e c i f i c a c t i v i t y o f phosphorus determined t o that of the o r i g i n a l f e r t i l i z e r was assumed to be the f r a c t i o n of the t o t a l phosphorus absorbed by the crop t h a t was d e r i v e d from the f e r t i l i z e r . This was expressed as per cent phos-phorus i n the crop d e r i v e d from the f e r t i l i z e r . An assumption was made t h a t the remainder of the phosphorus was d e r i v e d from the s o i l . The f r a c t i o n r e f e r r e d t o above 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 t e o f f e r t i l i z e r a p p l i c a t i o n and deminished i n s o i l s i n h e r e n t l y h i g h i n a v a i l a b l e phosphorus. Radiophosphorus has a l s o been u t i l i z e d t o e v a l u a t e the a v a i l a b i l i t y o f the phosphorus i n a v a r i e t y o f f e r t i l i z e r s and manures. The p r i n c i p l e s i n v o l v e d are e s s e n t i a l l y a c o u n t e r p a r t o f those d e s c r i b e d f o r e v a l u a t i n g phosphorus y i e l d o f s o i l s . I nstead o f a p p l y i n g the same tagged f e r t i -13. l i z e r to a number of s o i l s , a v a r i e t y of f e r t i l i z e r s are added to the same s o i l . Among such f e r t i l i z e r comparisons are the s t u d i e s w i t h c o t t o n and corn by H a l l et a l (1949), w i t h oats and a l f a l f a by S t a n f o r d and Nelson (1949), orchardgrass and Ladino c l o v e r by B l a z e r and M c A u l i f f e (1949), and wit h a l f a l f a by C a l d w e l l et a l (1954). In a l l i n s t a n c e s the performance of superphosphate and ammoniated superphosphate was used as a b a s i s f o r comparison. In g e n e r a l , c a l c i u m metaphosphate was an e f f e c t i v e m a t e r i a l when a p p l i e d t o a c i d s o i l s . D i c a l c i u m phosphate was l e s s e f f e c t i v e i n band than w i t h mixed placements. In the l a t t e r case i t was n e a r l y as e f f e c t i v e as superphosphate. Olsen et a l (1949)(1950) compared a number o f f e r t i l i z e r m a t e r i a l s on c a l c a r e o u s s o i l ; wheat, b a r l e y , sugar beet and a l f a l f a were the t e s t crops. The phosphorus a v a i l a b i l i t y was g e n e r a l l y i n the o r d e r : super-phosphate, mono-ammonium phosphate, calcium metaphosphate, d i c a l c i u m phosphate, t r i c a l c i u m phosphate. D i c a l c i u m phos-phate was always i n f e r i o r to superphosphate, but r e s u l t s w i t h c a l c i u m metaphosphate v a r i e d w i t h time, s o i l and crop. L i t t l e d i f f e r e n c e was found between the e f f e c t i v e n e s s o f superphosphate, mono-ammonium phosphate and l i q u i d p hosphoric a c i d . Radiophosphorus a l s o has been used f o r s t u d i e s which compared the a v a i l a b i l i t y o f the phosphorus contained i n orga n i c farm r e s i d u e s , and manures. F u l l e r and Dean (1949) and F u l l e r and Rogers (1952) have s t u d i e d leguminous and non-leguminous crop r e s i d u e s and M c A u l i f f e et a l (1949(a)(b)) 14. sheep manure and found t h a t the phosphorus of these m a t e r i a l s compared f a v o u r a b l y w i t h t h a t of superphosphate. Phosphorus U t i l i z a t i o n by P l a n t Roots. In r e c e n t s t u d i e s on m i n e r a l n u t r i t i o n o f crop, P 3 2 has been used to compare the phosphorus a b s o r p t i o n p a t t e r n and the e f f i c i e n c y w i t h which crops u t i l i z e phosphorus from a p p l i c a t i o n s of phosphate f e r t i l i z e r s . S t u d i e s by Bonnet and R i e r a (1953) w i t h sugar cane, Dion e t a l (1949) wi t h wheat, Kapp (1953) w i t h sweet c l o v e r and c o t t o n , Krantz et a l (1949) w i t h p o t a t o e s , corn, c o t t o n and soybeans, Jacob et a l (1949) w i t h potatoes, Spinks et a l (1948(a)(b)) w i t h c e r e a l s , Welch et a l (1949) wi t h soy-beans and Woltz e t a l (1949) w i t h tobacco have shown t h a t when phosphate f e r t i l i z e r i s a p p l i e d a t p l a n t i n g and l o c a l i z e d near the seed, the young p l a n t s , c h a r a c t e r i s t i c a l l y , have a h i g h e r per cent phosphorus i n the crop d e r i v e d from the f e r -t i l i z e r than mature p l a n t s . This p a t t e r n of d e c l i n e i n per cent phosphorus i n the crop d e r i v e d from the f e r t i l i z e r , p r o -b a b l y r e f l e c t s the enlargement o f the root systems, which i n t u r n has i n c r e a s e d the amounts o f s o i l phosphorus a c c e s s i b l e . The d e c l i n e i n the p r o p o r t i o n of f e r t i l i z e r phosphorus w i t h crop m a t u r i t y i s not the same f o r a l l crop s p e c i e s , the d i f -f e r e n c e s b e i n g p r o b a b l y r e l a t e d t o the r o o t systems and t h e i r development. G e n e r a l l y speaking however, the experiments w i t h P 3 2 l a b e l e d f e r t i l i z e r s have shown t h a t , d u r i n g the f i r s t sea-son, crops absorb from 2 to 35 per cent of the phosphorus i n a g i v e n f e r t i l i z e r a p p l i c a t i o n . The t o t a l phosphorus absorbed by a crop u s u a l l y i n c r e a s e s when the r a t e o f f e r t i l i z e r a p p l i c a t i o n 15. i s i n c r e a s e d ; however, the percentage of the phosphorus absorbed decreases. With many s o i l s the a d d i t i o n of f e r t i -l i z e r phosphorus has l i t t l e e f f e c t on the amounts of s o i l phosphorus absorbed by crops, but experiments showing both decreases and i n c r e a s e s i n s o i l phosphorus u t i l i z a t i o n have been r e p o r t e d (U.S.A.E.C. 1956). That the e f f i c i e n c y o f phosphorus u t i l i z a t i o n v a r i e s w i t h the placement and k i n d o f phosphate f e r t i l i z e r was r e p o r t e d by Nelson et a l (1949), S t a n f o r d and Nelson (1949(b)), Olsen and Gardener (1949) and lawton e t a l (1954(a)) u s i n g P 3 2 l a b e l e d f e r t i l i z e r s . With superphosphate, the most common source, the l a r g e s t d i f f e r e n c e s i n f e r t i l i z e r u t i l i z a t i o n were observed d u r i n g the e a r l y stages o f growth and l a r g e l y disappeared as the crop matured. The value o f s u p e r f i c i a l supplemental a p p l i c a t i o n s o f phosphate t o e s t a b l i s h e d stands of p asture and otjier crops has l o n g been d i s c u s s e d . Since i t i s known t h a t phosphate p e n e t r a t e s s o i l s v e r y s l o w l y , broadcast a p p l i c a t i o n s although the most convenient, have not been thought to be worthwhile. N e v e r t h e l e s s , the f e r t i l i z a -t i o n of e s t a b l i s h e d forage stands was s t u d i e d w i t h P 3 2 l a b e l -ed superphosphate by S t a n f o r d et a l (1950), Lawton e t a l (1954(b)) and C a l d w e l l et a l (1954), and the broadcast method was found t o be ve r y e f f e c t i v e . Phosphorus m o b i l i t y i n P l a n t s . The movement of phosphorus i n s i d e the p l a n t i s ve r y e l a b o r a t e and complicated and i t i s on l y p o s s i b l e t o r e l a t e some of the main p o i n t s t h a t have been e s t a b l i s h e d . B r i e f l y , phosphate ions move i n t o the p l a n t sap, 16. and t h i s mobile phosphorus then becomes concentrated at a number of s i t e s where i t i s i n c o r p o r a t e d i n t o the s t r u c t u r e of some f a i r l y complex molecules. In the c e l l s , f o r example, the phosphorus e n t e r s i n t o the composition o f n u c l e i c ac;ids. Whenever i t becomes 'concentrated' i t remains o n l y ' l o o s e l y f i x e d ' . The phosphorus compounds are not compounds of great chemical s t a b i l i t y and can r e a d i l y be b u i l t up and broken down. These u n s t a b l e compounds p l a y an important r o l e i n meristematic t i s s u e s , and a c c o r d i n g l y the phosphorus i s i n g r e a t e s t demands at the growing p o i n t s o f stem and r o o t , i . e . where the c e l l s are v e r y a c t i v e . I f i n s u f f i c i e n t phos-phorus i s r e a c h i n g the p l a n t through i t s r o o t s , then the phos-phorus i s withdrawn from o l d e r l e a v e s and s t a l k s and moved t o the growing p o i n t s . In c e r e a l s , such as wheat, the phosphorus c o n t a i n e d i n the l e a v e s and stem can be m o b i l i z e d and t r a n s -f e r e d to the ears when the g r a i n i s b e g i n n i n g t o form; thus i t i s g e n e r a l l y observed t h a t a l l seeds are r i c h i n phospho-ru s . Rapid c e l l d i v i s i o n , such as occurs i n the germinat-i n g seed, r e q u i r e s t h a t v a r i o u s enzyme systems should be o p e r a t i n g e f f e c t i v e l y ; again there are processes i n which phosphorus p l a y s an i n t i m a t e p a r t . Radiophosphorus i s p r o v i n g a u s e f u l t o o l i n the i n v e s -t i g a t i o n o f v a r i o u s a s p e c t s of these phenomena, i . e . the f a c t t h a t seeds accumulate l a r g e amounts of phosphorus has been demonstrated i m p r e s s i v e l y by autoradiographs of f r u i t s from p l a n t s grown e i t h e r i n r a d i o a c t i v e phosphorus c o n t a i n i n g c u l t u r e s o l u t i o n , or s o i l watered w i t h r a d i o a c t i v e s o l u t i o n 17. (Dick 1957). Stout and Hoagland (1939) i n v e s t i g a t e d the movement of phosphorus i n geranium (Pelargonium zonale) u s i n g r a d i o a c t i v e i s o t o p e s and showed c o n s i s t e n t l y t h a t under c o n d i t i o n s o f a c t i v e growth the xylem i s the path of upward movement o f t h i s s a l t . In t r a c i n g the movement o f radiophosphorus i n the "bean p l a n t , a c o n c e n t r a t i o n g r a d i e n t has been found by Biddulph (1939), which corresponds t o a t r a n s p i r a t i o n d i f f e r e n t i a l . A c c o r d i n g l y , the t r a n s p i r a t i o n stream d e l i v e r s phosphorus to the l e a v e s where i t accumulates as water i s evaporated. This steepens the g r a d i e n t i n the phloem and allows a downward movement through t h a t t i s s u e . He concluded that i f the supply o f phosphorus t o the r o o t i s suddenly removed, a d i f f u s i o n of mobile phosphorus toward the xylem v e s s e l s would take p l a c e , and upon e n t r y i n t o the t r a n s p i r a t i o n stream the ions would be 'swept' upward a g a i n . Exchange Phenomena o f Phosphorus i n S o i l s . A charac-t e r i s t i c f e a t u r e o f s o i l phosphorus i s i t s low s o l u b i l i t y i n water or i n s o i l s o l u t i o n . Phosphorus a p p l i e d t o the s o i l as f e r t i l i z e r i s changed i n t o forms s i m i l a r i n some r e s p e c t s t o the n a t i v e forms a l r e a d y p r e s e n t . A c i d s o i l s c o n t a i n a l a r g e excess of i r o n and aluminum. A l k a l i n e and c a l c a r e o u s s o i l s c o n t a i n calcium. A l l these elements combine w i t h water s o l u b l e phosphates (such as super-phosphate) to convert them i n t o s p a r i n g l y s o l u b l e forms, a process known as f i x a t i o n or r e v e r s i o n . The nature o f f i x a -t i o n may a f f e c t the e f f i c i e n c y o f the phosphorus f e r t i l i z e r s 18. d i f f e r e n t l y on d i f f e r e n t types of s o i l . Hydrated i r o n and aluminum oxides i n a c i d s o i l s are known to absorb s o l u b l e phosphorus from f e r t i l i z e r s to form i r o n and aluminum phosphates. The amounts o f hydrated i r o n and alumi-num oxides i n s o i l s i n c r e a s e In g e n e r a l as weathering processes continue, i . e . h i g h e s t i n r e g i o n s o f h i g h temperature and r a i n -f a l l , and lowest i n regions o f low r a i n f a l l . A c i d s o i l s w i t h t h e i r h i g h e r content o f hydrous oxides w i l l f i x phosphate to a g r e a t e r e x t e n t than a l k a l i n e s o i l s o f s i m i l a r t e x t u r e . Within a group o f a c i d or a l k a l i n e s o i l s , the f i x e d phosphorus i s l e s s a v a i l a b l e on c l a y loams than on sandy loams, because of t h e i r h i g h e r m i n e r a l content. The amount of phosphorus absorbed by a g i v e n s o i l s e r i e s o f v a r y -i n g t e x t u r e s i s r e l a t e d c l o s e l y to the s u r f a c e area of the s o i l p a r t i c l e s . D i f f e r e n c e s i n the s o l u b i l i t y of f i x e d forms o f phos-phorus r e s u l t from d i f f e r e n c e s i n the pH of the s o i l a t the time the f e r t i l i z e r i s a p p l i e d . Iron and aluminum phosphates are l e a s t s o l u b l e a t pH 4, t h e i r s o l u b i l i t y i n c r e a s i n g from 4 t o 8.5. I t has been found t h a t l i m i n g an a c i d s o i l to pH 6.3 i n c r e a s e d t e n f o l d the phosphorus c o n c e n t r a t i o n of the d i s p l a c e d s o i l s o l u t i o n (Olsen and F r i e d 1957). The c a l c i u m phosphates b e g i n t o form around pH 6, then s o l u b i l i t y d e c r e a s i n g as the pH Increases to pH 7.5. In the presence of excess calcium carbonate, the s o l u b i l i t y of the calcium phosphates i n c r e a s e s between pH 7.5 and 9. This e f f e c t o f pH holds true f o r n a t i v e and f i x e d forms of 19. phosphorus. The a v a i l a b i l i t y i s at a maximum i n the pH range 6.5 to 7. As the pH i n c r e a s e s from 7 to 8.5 i n a l k a l i n e s o i l s , the a v a i l a b i l i t y o f phosphorus drops a g a i n due t o the change i n r a t e or c a p a c i t y of the r o o t s to absorb phosphorus or to a decrease i n the c o n c e n t r a t i o n of the H2PO4 i o n . In n e u t r a l , a l k a l i n e and c a l c a r e o u s s o i l s , s o l u b l e phosphorus from f e r t i l i z e r s r e a c t s w i t h exchangeable c a l c i u m i o n s , s o l u b l e c a l c i u m s a l t s , and c a l c i u m carbonate to form s l i g h t l y s o l u b l e c a l c i u m phosphates. The form of c a l c i u m phosphate that appears f i r s t may be d i c a l c i u m phosphate, but t h i s form i s unstable i n the presence of excess calcium ions and changes i n t o h y d r o x y a p a t i t e or s i m i l a r c a l c ium phosphates. The a v a i l a b i l i t y o f the s o i l phosphorus f o r crop produc-t i o n depends p r i m a r i l y on i t s degree o f water s o l u b i l i t y s i n c e the p l a n t o b t a i n s i t s phosphorus from the s o i l s o l u t i o n . The f r a c t i o n o f phosphorus not i n s o l u t i o n i s a s s o c i a t e d w i t h the s o l i d phase. Since the evidence f o r contact o r d i r e c t s o l i d phase f e e d i n g i s l a r g e l y n e g a t i v e , repeated r e s t o r a t i o n o f the water s o l u b l e phosphorus i s necessary to meet the phos-phorus requirements o f the p l a n t . M c A u l i f f e et a l (1947) a p p l i e d P 3 2 to a study of the exchange between s o l u t i o n and s o l i d phase phosphate i n the s o i l system. The amount o f r a p i d l y e q u i l i b r a t i n g s u r f a c e phosphate was measured and found to p a r a l l e l o ther estimates o f phosphorus a v a i l a b i l i t y . Good c o r r e l a t i o n s were shown by Cole et a l (1953) between the phosphate a s s o c i a t e d w i t h the s o l i d phase which r e a d i l y e q u i l i b r a t e d w i t h P 3 2 i n the i o n i c form ( s u r f a c e phosphate) 20. and the p l a n t - a v a i l a b l e phosphorus o f s o i l s . F r i e d and Dean (1952) r e p o r t e d an exchange o f phosphate ions w i t h i n the s o l i d phase phosphorus system. When d i l u t e phosphate s o l u t i o n s r e a c t w i t h i r o n phosphate, phosphate i s absorbed as a mono-layer, but w i t h h i g h e r c o n c e n t r a t i o n s other forms o f absorbed phos-phate appear. Phosphorus M o b i l i t y i n S o i l s . Ionic substances move through s o i l by d i f f u s i o n and as the r e s u l t o f mass flow of the s o i l s o l u t i o n . The r e s u l t s o f these p r o c e s s e s f r e q u e n t l y mean changing a v a i l a b i l i t y o f n u t r i e n t s t o crops. The move-ment o f phosphate ions i n s o i l s i s l i m i t e d by phosphate f i x a t i o n - the p r o c e s s o f changing s o l u b l e phosphates i n t o l e s s s o l u b l e phosphates i n s o i l s and sometimes c a l l e d r e -v e r s i o n . Ordinary chemical methods do not permit the study of phosphorus movement i n s o i l s i n d e t a i l , but through the use of P 3 2 , i t has been p o s s i b l e t o t r a c e the movement of phosphatic f e r t i l i z e r s even under f i e l d c o n d i t i o n s . Heslep and Black (1954) have s t u d i e d the d i f f u s i o n o f f e r t i l i z e r phosphorus i n s o i l s , and have shown t h a t f e r t i l i z e r phosphorus d i f f u s e s a d i s t a n c e o f o n l y 3 t o 4 cm i n f o u r weeks. The d i s t a n c e o f d i f f u s i o n i n c r e a s e d w i t h time and r a t e o f a p p l i -c a t i o n and with the degree o f water s o l u b i l i t y o f the f e r t i l i -z e r . Under otherwise uniform c o n d i t i o n s f e r t i l i z e r phospho-rus d i f f u s e d l e s s i n c a l c a r e o u s than i n a c i d s o i l s . B o u l d i n and Black (1954) have s t u d i e d the v a l i d i t y o f a c t i v i t y measure-ments as estimates o f d i f f u s i o n from tagged phosphate f e r t i -l i z e r s . S u b s t a n t i a l l y the same c o n c l u s i o n s were d e r i v e d 21. from e i t h e r t o t a l phosphorus or P 3 2 a c t i v i t y measurements. Lawton and Vomocil (1954) have e x p e r i m e n t a l l y demonstrated the r a p i d d i s s o l u t i o n o f phosphorus from g r a n u l a r super-phosphate i n con t a c t w i t h moist s o i l and r e p o r t e d t h a t even i n s o i l s as low as 2 to 4 percent moisture, 20 to 50 per cent of the water s o l u b l e phosphorus moved i n one day from f e r t i -l i z e r granules t o s o i l . (At f i e l d c a p a c i t y , 50 to 80 per cent). In t h e i r m i g r a t i o n s t u d i e s Lawton and "Vomocil showed t h a t phosphorus moved v e r y l i t t l e under the c o n d i t i o n s s t u d i e d . Maximum movement of about one i n c h occured at s o i l moistures approximating f i e l d c a p a c i t y on three Michigan s o i l s . Olsen and F r i e d (1957) s t a t e t h a t 'water s o l u b l e phosphorus d i f -f u s e s away from a f e r t i l i z e r p a r t i c l e through the s o i l a d i s t a n c e o f about 1 i n c h ; then i t s movement becomes very slow'. Henderson and Jones (1941) have i n v e s t i g a t e d the i n -f l u e n c e o f water movement on the d i s t r i b u t i o n of phosphate f e r t i l i z e r s i n s o i l s u s i n g mono-calcium phosphate. A p p l y i n g water t o simulate r a i n , they have det e c t e d a phosphorus movement o f from 1.5 to 4 inches depending on the s o i l used. P 3 2 l a b e l e d phosphoric a c i d was a p p l i e d w i t h i r r i g a t i o n water i n f i e l d s t u d i e s made by U l r i c h , Jacobson and O v e r s t r e e t (1947). The phosphorus was r e p o r t e d t o p e n e t r a t e 12 inches when the water reached 20 inches, but 86 per cent of the phos-phorus was r e t a i n e d i n the top 6 inches. That the movement of phosphorus from bandplaced superphosphate was l e s s than f o r phosphoric a c i d a p p l i e d i n i r r i g a t i o n water, was e x e m p l i f i e d by the work of Olsen et a l (1950) i n t h e i r s t u d i e s on the 22. u t i l i z a t i o n of phosphorus by various crops as affected by source of material and placement. Radiation E f f e c t s on Plants. In any tracer experiment, the assumptions are made that the chemical behavior of the radioactive species i s i d e n t i c a l with that of i t s non-radio-active counterpart and that the radio element substitutes at random for the stable forms of the element. This however, has been proved by several investigations to be only approximately true. A l l radioactive isotopes d i f f e r from t h e i r stable forms i n mass, sometimes to a degree which appreciably a l t e r s reaction rates and equilibium conditions. In some cases these isotope effects are large enough to inspire doubt as to the correctness of interpretation of c e r t a i n quantitative measurements. Among the reports describing the b i o l o g i c a l e f f e c t which presumably occurs as a consequence of concentration of the isotope i n question, i s the report of Blume et a l (1950) who showed that nominal tracer l e v e l s of P 3 2 i n the nutrient solu-t i o n have marked e f f e c t s , presumably as a r e s u l t of accumu-l a t i o n by the plants. Figure I demonstrates an appreciable e f f e c t in the 10 m i c r o c u r i e / l i t r e l e v e l , with the e f f e c t d i f f e r i n g i n magnitude for different leaves i n barley. E f f e c t s are v i s i b l e at l e v e l s as low as 8 microcuries over the controls. That incorporation of radioactive phosphorus into the chromosome can bring about chromosomal rearrangements i n plant materials, i s shown by the work of Arnason et a l (1948) 23. 30[-20 I 0 Microcuries P32 per liter of Nutrient Solution FIGURE I THE EFFECT OF P32 CONCENTRATION IN THE NUTRIENT SOLUTION ON THE LENGTH OF BARLEY LEAVES. (BLUME et ol 1950) 2 4 . (1952), w i t h wheat and b a r l e y , thus e x e m p l i f y i n g the c y t o -l o g i c a l e f f e c t s of r a d i a t i o n . These d i f f i c u l t i e s , however, do not preclude the use or P 3 2 , but r a t h e r n e c e s s i t a t e the use of c e r t a i n p r e c a u t i o n s . For i n s t a n c e i n the study o f r o o t systems, the c o n c e n t r a t i o n of P 3 2 used must be such that the maximum r a d i a t i o n exposure of the r o o t w i l l not approach the l e v e l at which damage occurs to the growing t i p (Blume et a l 1950). H a l l et a l (1953) r e p o r t e d t h a t under c o n d i t i o n s or normal phosphorus f e r t i l i t y , a s o l u t i o n r a n g i n g from 10 to 50 m i c r o c u r i e s per ml i s s a t i s -f a c t o r y . Such a c o n c e n t r a t i o n e f f e c t s l i t t l e change i n the l e v e l of s o i l phosphorus by the c a r r i e r of P 3 2 . Blume (1952), i n h i s i n v e s t i g a t i o n s on r a d i a t i o n e f f e c t s on p l a n t s grown i n s o i l t r e a t e d w i t h f e r t i l i z e r c o n t a i n i n g P 3 2 , used s u r f a c e a p p l i c a t i o n s of K H 2 P O 4 c o n t a i n i n g 0-12,500 m i c r o c u r i e s or P 3 2/gram of P 3 ^ and found no e f f e c t or s p e c i f i c a c t i v i t y l e v e l upon phosphorus uptake. EXPERIMENTATION The o b j e c t i v e of t h i s study was s t a t e d e a r l i e r t o be the examination or rorage crop r o o t s by s e v e r a l ' i n d i r e c t ' methods. In other words, i t was hoped t h a t a d d i t i o n a l i n f o r m a t i o n on r o o t morphology, development and f u n c t i o n c o u l d be secured by u s i n g techniques not yet commonly used. L i m i t a t i o n s a s s o c i a t e d with d i r e c t methods of r o o t study, making use of s o i l washing or growth i n c u l t u r e s o l u t i o n s , might i n f a c t , be o b v i a t e d . 25. The s e v e r a l techniques s e l e c t e d f o r t r i a l were as f o l l o w s : -(1) A technique u s i n g s p e c i a l c o n t a i n e r s . (2) A technique u s i n g i n t r a v i t a m dyes and f l u o r e s c e n t compounds. (3) A technique u s i n g radiophosphorus w o i l placements and p l a n t i n j e c t i o n s . (4) A technique u s i n g l i t h i u m c h l o r i d e placements i n s o i l . 1• A Technique U s i n g S p e c i a l C o n t a i n e r s . In the e a r l i e s t phases of t h i s study, i t was thought t h a t c o n t a i n e r s o f s p e c i a l d e s i g n m e r i t e d a t t e n t i o n . This approach to root study pe r m i t t e d simple o b s e r v a t i o n s of growing t i s s u e s and cannot t h e r e f o r e , be s t r i c t l y c l a s s e d as an ' i n d i r e c t ' approach. N e v e r t h e l e s s , i t was f e l t t h a t i t might p r o v i d e a v a l u a b l e compliment f o r l a t e r s t u d i e s u s i n g ' i n d i r e c t ' methods. S p e c i a l c o n t a i n e r s f o r r o o t study have been de v i s e d by a few i n v e s t i g a t o r s . P l a n t p h y s i o l o g i s t s , w i t h a s p e c i a l i n t e r e s t i n a c c e s s o r y growth f a c t o r s , have o f t e n d i r e c t e d r o o t s down g l a s s t u b i n g of narrow bore or between c l o s e l y packed m i c r o s c o p i c s l i d e s . In a l l cases s e e d l i n g r o o t s or e x c i s e d r o o t t i s s u e s were used. Newton and h i s a s s o c i a t e s a t the U n i v e r s i t y o f A l b e r t a ( d i r e c t communication) have e x p l o i t e d the g e o t r o p i c responses of r o o t s by c o n s t r u c t i n g c o n t a i n e r s w i t h s l o p i n g g l a s s f a c e s and by l a t e r o b s e r v i n g the ' p o i n t s ' a t which r o o t s reached the t r a n s p a r e n t f r o n t s . More r e c e n t l y P a t t e r s o n and Law o f the State C o l l e g e of 26. Washington ( d i r e c t communication) have used l o n g l a r g e - b o r e , g l a s s and p l a s t i c tubes c o n t a i n i n g s o i l , and set i n dark chambers, to give i n f o r m a t i o n on the r o o t development of forage and t u r f s p e c i e s . The c o n t a i n e r s used i n t h i s present study, u t i l i z e d some of the f e a t u r e s of those made by other workers, but were e s s e n t i a l l y of new design. M a t e r i a l s and methods. The f i r s t c o n t a i n e r s used were •set up' e a r l y i n the w i n t e r of 1958-59 i n the U.B.U. green-house. They c o n s i s t e d of g l a s s - f a c e d wooden tanks, 121,x4-g-" x l 2 " , f i l l e d w i t h s t e r i l i z e d s o i l . To prevent l i g h t from r e a c h i n g the s o i l below the s u r f a c e , wooden s l i d i n g panels covered the g l a s s f a c e s (see p l a t e s I & I I ) . The tanks were seeded t o orc h a r d g r a s s ( D a c t y l i s glomerata) c o l o n i a l bent-grass ( A g r o s t i s sp.) and red c l o v e r ( T r i f o l i u m pratense) a t the r a t e s o f 100 and 10 pounds per acre ( i . e . 0.39 grams and 0.039 grams/tank). Adequate moisture was p r o v i d e d f o r germi-n a t i o n and growth of the s e e d l i n g s . Observations and r e s u l t s . Roots appeared as f i n e white ' l i n e s ' between g l a s s and s o i l . Sometimes a r o o t would appear near the g l a s s s u r f a c e and then grow 'back' from v i s i o n i n t o the s o i l . In g e n e r a l , the growth was 'downwards' and i n order to p r o v i d e an o b j e c t i v e statement o f the obser-v a t i o n s , measurements, c a p i t a l i z i n g on t h i s f a c t , were made. Once a week the v e r t i c a l d i s t a n c e from the s o i l s u r f a c e to each r o o t 'terminus' a g a i n s t the g l a s s was recorded. F r e -quently, but not always, the terminus was a r o o t t i p . The averages o f the measurements are given i n Table I. This TABLE I ROOT MEASUREMENTS FROM SEEDLINGS GROW IN GLASS-FACED TANKS IN THE GREENHOUSE Species Seeding r a t e Age of s e e d l i n g s (wks.) pounds/acre 1. 2. 3. 4. 5. 6. Red c l o v e r 10 Number of r o o t s Maximum depth Average depth ( i n s ) 3 1.2 1.0 3 3.0 2.5 3 4.7 4.2 3 6.7 6.0 3 9.5 7.5 3 9.0 100 Number of r o o t s Maximum depth Average depth ( i n s ) 5 1.5 1.2 5 3.7 3.5 5 5.7 5.0 5 8.0 7.0 5 10.5 9.2 5 Orchardgrass 10 Number of r o o t s Maximum depth Average depth ( i n s ) 4 1.0 0.8 4 1.7 1.4 4 3.0 2.6 4 4.5 4.0 4 6.2 6.0 4 8.5 8.0 100 Number o f r o o t s Maximum depth Average depth ( i n s ) 12 1.5 1.5 13 2.2 2.0 15 3.5 3.0 15 5.5 5.0 15 7.5 7.2 15 9.7 9.5 C o l o n i a l bentgrass 10 Number of r o o t s Maximum depth Average depth ( i n s ) ; -3 2.0 2.0 3 3.5 3.5 3 5.2 5.2 3 6.8 6.8 100 Number of r o o t s Maximum depth Average depth ( i n s ) - - 10 2.0 1.8 16 3.2 3.0 19 5.5 5.0 19 7.2 7.0 28. t a b l e i s s e l f - e x p l a n a t o r y . A s t r i k i n g o b s e r v a t i o n was t h a t seed under the h e a v i e r r a t e germinated much more r a p i d l y (24 to 48 hours e a r l i e r ) than seeds sown at the l i g h t e r r a t e . The response was not a simple f a c t of numbers, but was p r o b a b l y a s s o c i a t e d w i t h the exudation o f 'germination substances' r e c o r d e d by many w r i t e r s . L o c a l i z a t i o n of i n d i v i d u a l r o o t s i n the dense seeding was very evident, e s p e c i a l l y i n the grasses, where the r o o t s were seen to penetrate almost v e r t i c a l l y , w i t h a minimum of l a t e r a l spread as compared wi t h the more e x t e n s i v e r o o t s of the l i g h t e r seedings. An unexpected o b s e r v a t i o n was t h a t the r o o t s of the s e e d l i n g s i n both l i g h t and heavy r a t e s , reached approximate-l y the same depth i n a 6 weeks o b s e r v a t i o n p e r i o d . I t would seem t h a t p l a n t c o m p e t i t i o n under the c o n d i t i o n s o f t h i s ex-periment d i d not l i m i t the downward extent of the r o o t s . I t would seem too, that the o n l y c o n c l u s i o n to be reached i n t h i s r e g a rd i s that r o o t c o m p e t i t i o n cannot be v e r y e f f e c t i v e u n t i l e x t e n s i v e l a t e r a l r o o t development o c c u r s . In a s i m i l a r man-ner, i t would appear that s e e d l i n g r o o t development i s l a r g e -l y 'downwards', and t h a t l a t e r a l development and f u l l ex-p l o i t a t i o n of the s o i l volume may not occur u n t i l s e e d l i n g s are o l d e r than 6 weeks. L i m i t a t i o n s i n the use of c o n t a i n e r s o f the s o r t used i n t h i s experiment are s e v e r a l . In the f i r s t i n s t a n c e they are f a i r l y c o s t l y and bulky. I t would appear too, t h a t they do not r e s u l t i n a f u l l e x p l o i t a t i o n of the s o i l volume i n a 29. reasonably s h o r t p e r i o d of time even at heavy seeding r a t e s . D espite p r e c a u t i o n s to prevent s p e c i a l c o n d i t i o n s a r i s i n g at the s o i l - g l a s s i n t e r f a c e , i t seems probable t h a t behaviour of r o o t s i n t h i s area i s not e n t i r e l y ' t y p i c a l ' , and t h e r e i s some tendency f o r r o o t s to f o l l o w the i n t e r f a c e . Some of the l i m i t a t i o n s i t was thought, might be overcome by growing s e e d l i n g s i n a l i m i t e d s o i l volume between two g l a s s p l a t e s 1 0 " x l6" bound together with two f " t h i c k wooden s t r i p s between t h e i r l o n g edges. F r i c t i o n tape was used f o r b i n d i n g . The ' s l i d e s ' were then l o o s e l y plugged w i t h g l a s s wool at one end, and f i l l e d w i t h greenhouse s o i l (see p l a t e I I I ) . A f t e r seeding, the ' s l i d e s ' were stacked together i n an u p r i g h t p o s i t i o n , w i t h the two o u t s i d e ' s l i d e s ' unseeded so as to prevent l i g h t from e n t e r i n g the g l a s s f a c e s o f the seeded p l a t e s . The s t a c k was 'set up* i n the l a b o r a t o r y , and kept i n an open tank from which the s l i d e s were sub-i r r i g a t e d . The advantages t h a t t h i s technique has over the former a r e : - (a) i t i s a much cheaper technique. (b) i t f a c i l i t a t e s h a n d l i n g . (c) o b s e r v a t i o n of the r o o t s on both s i d e s of the c o n t a i n e r s i s p o s s i b l e . I t a l s o seems probable t h a t the behaviour o f the r o o t s at the s o i l - g l a s s i n t e r f a c e i s l e s s ' t y p i c a l ' i n t h i s case than b e f o r e . 2. A Technique Using I n t r a v i t a m Dyes and F l u o r e s c e n t Compounds. S p e c i a l c o n t a i n e r s such as those d e s c r i b e d before were 30. used t o study the f l u o r e s c e n c e o f r o o t s , and r o o t s c o l o u r e d w i t h i n t r a v i t a m dyes p l a c e d l o c a l l y i n the solum or i n j e c t e d i n t o the crowns of p l a n t s . Root f l u o r e s c e n c e . I t has been observed t h a t r o o t s o f some p l a n t s f l u o r e s c e b r i l l i a n t l y i n u l t r a - v i o l e t l i g h t and some do not (Radley & Grant 1954) (Goodwin & Kavanagh 1948). I t was hoped t h a t t h i s f a c t might be e x p l o i t e d i n such a way t h a t a s s o c i a t e d r o o t systems, one f l u o r e s c e n t and one n o n - f l u o r e s c e n t might be d i s t i n g u i s h e d . I t i s u s u a l l y very d i f f i c u l t t o d i s t i n -g u i s h r o o t s of d i f f e r e n t s p e c i e s and d i f f e r e n t p l a n t s grow-i n g i n a s s o c i a t i o n . L i t t l e i s known, t h e r e f o r e o f the behaviour of roo t s under such circumstances. I s there r e p u l -s i o n ? I s there mutual e x p l o i t a t i o n o f the solum? A number of questions remain unanswered. Chosen f o r study were a l f a l f a , whose s e e d l i n g r o o t s are known t o f l u o r e s c e b r i l l i a n t l y on f i l t e r paper and other media, and sweet c l o v e r , whose r o o t s do not f l u o r e s c e ( B r i n k 1956). S e e d l i n g r o o t s were observed a g a i n s t the g l a s s w a l l s of the s p e c i a l p l a t e c o n t a i n e r s d e s c r i b e d p r e v i o u s l y . F l u o r e s c e n c e o f r o o t s o f a l f a l f a a g a i n s t a s o i l back-ground was observed, but i t was soon apparent t h a t i t was too weak to be of much use f o r purposes o f d i s t i n c t i o n . Even a f t e r the g l a s s was removed (to permit more s h o r t wave-leng t h s t o reach the r o o t s ) the emission of v i s i b l e l i g h t was ve r y weak. Some attempts were made to r e p l a c e g l a s s 31. w i t h a p l a s t i c ( n o t a b l y p l e x i g l a s s ) which absorbs l e s s s h o r t wave r a d i a t i o n . A number of f l u o r e s c i n g compounds were i n j e c t e d , u s i n g a hypodermic s y r i n g e , i n t o the crowns o f b a r l e y p l a n t s grown i n the s p e c i a l p l a t e c o n t a i n e r s . B a r l e y was chosen because i t s crowns are somewhat l a r g e r a t a y o u t h f u l stage than those of a l f a l f a and sweet c l o v e r . I n j e c t e d were aqueous s o l u t i o n s o f e o s i n , f l u o r e s c e i n and e s c u l i n . Roots were observed f o r s e v e r a l days a f t e r i n j e c t i o n , but no f l u o r e s c e n c e i n v i v o c o u l d be d e t e c t e d . F l u o r e s c e n t substances were a l s o p l a c e d i n s o i l i n the v i c i n i t y of c e r t a i n r o o t s i n the hope t h a t they might be taken up by them and t r a n s p o r t e d g e n e r a l l y through the r o o t system. The compounds d i d not f l u o r e s c e f o r l o n g i n the s o i l , and e v i d e n t l y were not taken up by the p l a n t r o o t s , or were dest r o y e d soon a f t e r e n t r y . Dyeing Roots. Inasmuch as the t r i a l s w i t h r o o t f l u o r e s c e were not p r o m i s i n g , i t was d e c i d e d to see i f i n t r a v i t a m dyes were a p o t e n t i a l a i d i n r o o t study. Again, p l a t e c o n t a i n e r s of the type p r e v i o u s l y d e s c r i b e d , were seeded to b a r l e y . Aqueous s o l u t i o n s and powders of a c i d f u c h s i n , congo r e d and methy-lene blue were both s p e c i a l l y p l a c e d i n the solum and i n j e c t -ed, u s i n g a hypodermic s y r i n g e , i n t o the p l a n t crowns. Root systems from p l a n t s i n s l i d e s c o n t a i n i n g powdered a c i d f u c h s i n , p l a c e d l o c a l l y i n the s o i l , 'took up* the dye, and r o o t l e t s some d i s t a n c e removed from the i n t a k e p o i n t 32. were w e l l s t a i n e d w i t h i n a week, and were r e a d i l y v i s i b l e through the g l a s s . A f t e r two weeks, the crown and l e a f sheath were dyed as were r o o t s t o a depth o f 6 M. Congo r e d was taken up by r o o t s t o a p p a r e n t l y a l i m i t e d extent. Dyeing of t i s s u e s remained f o r a s h o r t d i s t a n c e around the placement r e g i o n and even a f t e r 4 weeks exposure, the a e r i a l p o r t i o n s o f the p l a n t and the nether r e g i o n s of the r o o t systems were not dyed. Methylene blue, not unexpectedly, d i d not dye r o o t or stem. Encouraged by the r e s u l t s of the l a b o r a t o r y t e s t s u s i n g s l i d e c o n t a i n e r s and a c i d f u c h s i n , i t was decided t h a t a f i e l d t e s t would be d e s i r a b l e . Placements o f 10 ml o f an aqueous s o l u t i o n o f a c i d f u c h s i n , were made on e s t a b l i s h e d square f o o t p l o t s of orchard grass and Kentucky b l u e g r a s s at depths of 2", 4" and 12". The method of placement was s i m i l a r to t h a t used f o r the placement of P32 ( d e s c r i b e d l a t e r ) . Even a f t e r 6 weeks, no s t a i n i n g o r the a e r i a l p o r t i o n s was d i s c e r n i b l e . Although f u r t h e r i n v e s t i g a t i o n was not undertaken, i t can be suggested t h a t the f u r t h e r study of the use of dyes i n root study Is warranted. A c i d f u c h s i n i n p a r t i c u l a r i s a w e l l e s t a b l i s h e d s t a i n f o r p l a n t t i s s u e , and i s r e a d i l y absorbed by p l a n t s . 3. A Technique U s i n g Radiophosphorus, S o i l Placement and P l a n t 33. I n j e c t i o n s . F i r s t T r i a l . Many i n v e s t i g a t o r s have used the r a d i o a c t i v e phosphorus i s o t o p e , P 3 2 , as was p o i n t e d out e a r l i e r , f o r the study o f s o i l and f e r t i l i z e r phosphorus. Only two or three workers have used i t as i t i s used i n the pr e s e n t u n d e r t a k i n g , I'or s t u d y i n g r o o t development. Yet P 3 2 has many v a l u a b l e c h a r a c t e r i s t i c s f o r such an undertaking. I t has f o r example, a h a l f - l i r e or reasonable l e n g t h f o r p l a n t work or t h i s k i n d , v i z . 14.3 days; i t i s needed i n a l l a c t i v e c e l l s of the p l a n t , i s q u i t e l a b i l e , and can be s a r e l y used i f o n l y qu a s i o r d i n a r y •clean t e c h n i q u e s ' are employed, v i z . the beta emanations are only moderately e n e r g e t i c , and t h e i r b i o l o g i c a l e f f e c t s are much reduced i n a i r a t d i s t a n c e s of onl y 10". D e t e c t i o n equipment i s r e l a t i v e l y inexpensive and f a i r l y g e n e r a l l y a v a i l a b l e . M a t e r i a l s and Methods. In the s p r i n g and summer of 1959, an enclosure area 45'x45' was d e l i m i t e d on Alderwood sandy loam on the Univer-s i t y farm. I t was so l o c a t e d as t o be reasonably i s o l a t e d from the g e n e r a l p u b l i c ; the area was fenced and s u i t a b l y p osted w i t h r a d i a t i o n hazard s i g n s (see p l a t e IV). In the f i r s t s e r i e s o r experiments c a r r i e d on i n the en c l o s u r e , orchardgrass ( D a c t y l i s glomerata), r e d fescue (Festuca r u b r a ) , Kentucky b l u e g r a s s (Poa p r a t e n s i s ) and c o l o n i a l bentgrass ( A g r o s t i s sp.) were seeded a t the r a t e or 100 l b s / a c r e i n l ' x l ' p l o t s . 34. In order t h a t P co u l d he p l a c e d without confounding e f f e c t s at s e v e r a l depths i n the s o i l , v i z . 0", 2", 4" 12" and 24", f i v e , f o o t square p l o t s of each s p e c i e s were estab-l i s h e d . A l s o t o o b t a i n some measure of e r r o r , the e n t i r e b l o c k was r e p l i c a t e d . The d i s t a n c e between the u l t i m a t e f o o t square p l o t s i n a l l d i r e c t i o n s was 3'. Assignment o f each of the 20 u l t i m a t e p l o t s i n a b l o c k was at random. P r i o r to seeding, a p e r p e n d i c u l a r hole o f r e q u i r e d depth was made w i t h a 3/8" s t e e l rod i n each of those p l o t s where P 3 2 was to be p l a c e d , then 5 mm diameter g l a s s tubes were i n s e r t e d i n the bore, c a r e f u l l y , to prevent blockage by s o i l . A l l tubes had 4" extensions above the s u r f a c e , f i t t e d w i t h 2" o f rubber t u b i n g and s e a l e d o f f w i t h a p i n c h clamp (see p l a t e V ) . A f t e r seeding, the p l o t s were i r r i g a t e d by overhead s p r i n k l e r s once per week throughout the d r y season. The need f o r i r r i g a t i o n was determined by two se t s o f 3 i r r o m e t e r s (see p l a t e VI) i n s t a l l e d a t the h i g h e s t and lowest p o i n t s of the e n c l o s u r e . The i r r o m e t e r s recorded moisture at depths of 6", 12" and 18", and i r r i g a t i o n was commenced when they ap-proached readings o f 75 a t 6" and 40 a t 18". F i e l d c a p a c i t y was e s t i m a t e d at i r r o m e t e r r e a d i n g s between 0 and 10. The f i r s t placements o f P 3 2 were made when the s e e d l i n g s were one month o l d . Due to the l i m i t e d supply a t the time, P 3 2 was a v a i l a b l e o n l y f o r treatment o f orc h a r d grass and Kentucky b l u e g r a s s p l o t s at depths of 2", 4" and 12". Each placement c o n s i s t e d of 100 m i c r o c u r i e s o f P 3 2 i n 10 ml of a 35. 0.5% s o l u t i o n of potassium dihydrogen phosphate (KH2PO4). T h i s was accomplished by f i t t i n g the t i p o f a b u r e t t e con-t a i n i n g the s o l u t i o n i n t o the rubber t u b i n g and r e l e a s i n g the clamp. When s u f f i c i e n t s o l u t i o n was l e t down the tube, the tube was r i n s e d with an equal q u a n t i t y of water (10 ml) from another b u r e t t e i n a s i m i l a r manner. The clamp was then r e p l a c e d . Both b u r e t t e s were mounted on the same p o r t a b l e stand (see p l a t e VI I ) , so as t o f a c i l i t a t e movement and h a n d l i n g of the m a t e r i a l . That the r o o t s o f these s p e c i e s were a b s o r b i n g the r a d i o a c t i v e m a t e r i a l , and hence had p e n e t r a t e d t o these depths, was determined i n t h r e e ways, u s i n g a G e i g e r - M u l l e r counter (see p l a t e s V I I I & IX), (a) p l a c i n g the counter i n d i r e c t c o n t a c t w i t h the f o l i a g e i n s i t u , (b) removing samples of the f o l i a g e o f p l a n t s a t s p e c i f i c d i s t a n c e s from the centre of placement, and t e s t i n g w i t h the counter and (c) p l a c i n g the counter near a ground, oven d r i e d sample of the h a r v e s t e d p l o t . In a l l cases, r a d i a t i o n was r e c o r d e d and compared w i t h the background r a d i a t i o n over p l o t s , and on m a t e r i a l removed from p l o t s on which there were no placements. From a c o n s i d e r a t i o n of the l i t e r a t u r e o u t l i n e d on the m o b i l i t y o f P 3 2 i n s o i l s , i t was r e a l i z e d t h a t any informa-t i o n obtained would be incomplete without some knowledge of the movement o f P 3 2 i n Alderwood loamy sand. Therefore c o n t r o l p l o t s were 'set up' t o study such movement. Two, f o o t square p l o t s were used, and these were kept f r e e o f v e g e t a t i o n . S i m i l a r placements o f P 3 2 were made at 36. the same time as the seeded p l o t s , and to depths o r 4" and 12". The movement or P 3 2 was determined on the same date as was the r o o t a c t i v i t y on seeded p l o t s . This was done by t a k i n g samples of s o i l from s p e c i f i c l o c a t i o n s on the p r o f i l e made through the ce n t r e of each p l o t , and r e c o r d i n g r a d i o -a c t i v i t y by the use o f the Ge i g e r - M u l l e r counter. The sam-p l e s were taken by f o r c i n g g l a s s v i a l s , i - " i n diameter, in i n t o the face of the s o i l p r o f i l e . This then, gave a c y l i n -d r i c a l s o i l sample i n x i n from each l o c a t i o n . Observations and r e s u l t s . Root p e n e t r a t i o n and a c t i v i t y as measured by P 3 2 uptake w i t h the Ge i g e r - M u l l e r counter i s recorded i n t a b l e I I . A l l f o l i a g e l o c a t e d above the s u r f a c e centre o f each p l o t was r a d i o a c t i v e , i e :- 80-4000 c.p.m. were obtained, i n d i c a t i n g t h a t r o o t s were a b s o r b i n g P 3 2 , and hence p e n e t r a t i o n to depths where P 3 2 was l o c a t e d . Readings of 40 c.p.m. and l e s s , were c o n s i d e r e d to be background counts. In f a c t , no counts o u t s i d e the P 3 2 placement areas were found to reach 40 c.p.m. a t any time. F o l i a g e l o c a t e d d i r e c t l y above P 3 2 p l a c e d a t s e v e r a l depths as was s t a t e d , was ve r y r a d i o a c t i v e . In order to determine the area of the 'hot* t u r f , t r a n s e c t s , each p a s s i n g through the su r f a c e centre, were l a i d a c r o s s the p l o t s . In g e n e r a l , i t can be s a i d ' t h a t the r a d i o a c t i v i t y d i d not extend f o r more than 3" from the s u r f a c e c e n t r e . T h i s was true whether the T ^ placement was at 2", 4" or 12". In many i n s t a n c e s the area was even more r e s t r i c t e d . ( s e e t a b l e I I ) . From these r e s u l t s i t may be concluded, t h a t i n t h i s 37. TABLE I I ROOT PENETRATION AND ACTIVITY MEASURED BY P 3 2 UPTAKE WITH A GEIGER-MULLER COUNTER Species Placement depth Distan c e O n from placement 3" centre 6* Kentucky b l u e g r a s s 2" P P N (6 weeks o l d ) 4" P - P N p l a n t e d 22/5/59 12" P N N Orchard grass 2 n P N N (6 weeks old) 4" P N N p l a n t e d 22/5/59 12" P P N P -80-4000 c.p.m. N -0-40 c.p.m. (Background) t u r f o n l y r o o t s of p l a n t s e s t a b l i s h e d immediately above the P 3 2 take i t up. I t i s v e r y probable then, t h a t the r o o t system of a growing pihant i n t u r f i s v e r y l i m i t e d l a t e r a l l y , and t h a t most o f i t s development i s v e r t i c a l . In t h i s experiment, f o l i a g e a t sur f a c e c e n t r e f o r a l l s p e c i e s had a p p a r e n t l y taken up P 3 2 from placements a t a l l depths to 12". This was somewhat unexpected, and as shown below, i s a t t r i b u t a b l e to t e c h n i c a l a b e r r a t i o n s . At the same time f o l i a g e a c t i v i t y was b e i n g measured, sampling s o i l i n and around each P 3 2 placement, i n the p l o t s devoid of v e g e t a t i o n , showed t h a t c o n s i d e r a b l e movement of P 3 2 from i t s o r i g i n a l l o c a t i o n had taken p l a c e . Movements of s e v e r a l inches had occured v e r t i c a l l y i n the v i c i n i t y o f the probe, so that the v e r t i c a l a b s o r b i n g range f o r the 4" placement was 2"-6" and i n the case of the 12" placement i t was 8"-12". L a t e r a l movement was very l i m i t e d . This i s w e l l shown 38. d i a g r a i n m a t i c a l l y i n f i g u r e s 2 & 3. I t was surmised t h a t any c o n s i d e r a b l e movement of P from i t s placement s i t e must be a t t r i b u t a b l e t o di s t u r b a n c e o f the s o i l by p r o b i n g and t w i s t i n g the g l a s s tubes. Even though the sampling procedure was q u i t e coarse, i t was e v i d e n t t h a t the P ^ had, i n f a c t , c r e p t a l o n g the s i d e of the g l a s s 32 tube and t h a t downward movement of the P was p r i m a r i l y i n t o r e c e n t l y d i s t u r b e d s o i l . An i n t e r e s t i n g o b s e r v a t i o n a l s o was t h a t the P 3 2 d i d not move downward i n t o the B h o r i z o n 'hardpan' or 'zone of accumulation' l o c a t e d i n our p r o f i l e s a t 12"-12-|". Given more time than 6 weeks o f course, more movement of the P 3 2 might have occured. Second T r i a l . In an e f f o r t to o b v i a t e P ° & movements l a r g e l y a t t r i b u t a b l e to technique, a second s e r i e s of m o d i f i c a t i o n s o f the t r i a l s a l r e a d y d e s c r i b e d were undertaken. M a t e r i a l s and Methods. The techniques and l o c a t i o n s f o r these t r i a l s are so s i m i l a r to the pre v i o u s ones, that o n l y the m o d i f i c a t i o n s need be d e s c r i b e d . I n s t e a d o f u s i n g a s i n g l e tube, the 5 mm g l a s s tubes of the p r e v i o u s e x p e r i -ments was lowered i n t o the bore, i n s i d e another g l a s s tube, about two inches s h o r t e r , and through which i t c o u l d b a r e l y pass. When i t was time to make placements of P 3 2 , the outer tube alone was withdrawn 1 i n c h . By means of a p i p e t t e , 1 ml of 0.5% KH2PO4 c o n t a i n i n g 100 m i c r o c u r i e s of P 3 2 was poured i n t o the i n n e r tube. This was washed down w i t h 1 ml of water 39. SOIL SURFACE O® ® il O® € o@o 9§P 6 B HORIZON 4 2 6" 2 4 HORIZONTAL DISTANCE FROM CENTRE IN INCHES LE6EN0 OBACK-GROUND 2000 CPM (flgjft 3100 CPM ,22000 ' CPM ,40000 CPM FIGURE 2 THE MOVEMENT OF P32 FROM 4- INCH PLACEMENT IN ALDERW00D LOAMY SAND EXPRESSED DIAGRAMMATIC ALLY ( DRAWN TO SCALE ) 40. SOIL SURFACE 4 2 0 2 4 HORIZONTAL DISTANCE FROM CENTRE IN INCHES LEGEND o BACK-GROUND KLV CPM o , 1 4 0 8 0 0 CPM 1200 CPM 2200 CPM 10000 CPM FIGURE 3 THE MOVEMENT OF P32 FROM 12-INCH PLACEMENT IN ALDERW00D LOAMY SAND EXPRESSED DIAGRAMMATICALLY ( DRAWN TO SCALE ) 41. from another p i p e t t e and then the inner tube was withdrawn. The t h e o r e t i c a l space remaining a t the bottom of the outer tube was s e a l e d by po u r i n g 2 ml o f l i q u i d p a r a f f i n down the tube. This tube was then c l o s e d o f f w i t h a clamp on to a rubber t u b i n g a t the exposed end to reduce abnormal gas ex-changes. Orchardgrass, Kentucky b l u e g r a s s , r e d fescue and c o l o n i a l bentgrass, 8 weeks of age, were used. Placement depths f o r P 3 S were 12" and 24". The movement of P 3 2 i n the s o i l was s t u d i e d c o n c u r r e n t l y at these depths on two p l o t s kept bare of v e g e t a t i o n , u s i n g the same m o d i f i c a t i o n . Observations and r e s u l t s . No r a d i a t i o n g r e a t e r than 40 c.p.m. (background) c o u l d be d e t e c t e d from the f o l i a g e u s i n g the same methods as before, i n d i c a t i n g that none of the s p e c i e s had r o o t s p e n e t r a t i n g to depths of 12" or 24". The p r o f i l e s t u d i e s showed t h a t P 3 2 movement was l o c a l i z e d t o 1 square i n c h on the f a c e of the p r o f i l e i n both p l a c e -ments. These r e s u l t s i l l u s t r a t e that the r o o t s i n p l o t s 32 w i t h 12" placement of P i n the f i r s t technique c o u l d not have reached the maximum of the range shown i n f i g u r e 3. The r e s u l t s from the m o d i f i e d technique are encouraging, f o r they i n d i c a t e t h a t the P 3 2 l o c a l i z a t i o n i s q u i t e s a t i s -f a c t o r y , f o r purposes o f s t u d i e s on t u r f r o o t systems. I t remains o f course, t o see i f P 3 2 moves e x t e n s i v e l y w i t h l o n g e r p e r i o d s of time or under very d i f f e r e n t s o i l and c l i m a t i c c o n d i t i o n s . From t h i s work and the work of P 3 2 i n f e r t i l i z e r s , t h i s seems u n l i k e l y . T h i r d T r i a l . 48:.\ Phosphorus i s a f a i r l y l a b i l e c o n s t i t u e n t of p l a n t s ; not only i s phosphorus q u i t e c o n t i n u o u s l y taken up by r o o t s and t r a n s l o c a t e d to the a e r i a l p o r t i o n s of p l a n t s , but i t i s known t h a t there i s some downward movement from tops t o r o o t s under some circumstances. The c r e a t i o n of an a r t i f i c i a l l y 32 hi g h P c o n c e n t r a t i o n i n the crowns of p l a n t s might l e a d , i f theo r y i s c o r r e c t , to a movement and g r a d i e n t o f P 3 2 i n t o r o o t s which were a c t i v e l y growing and f u n c t i o n i n g . I f t h i s i s the case, then i t should be p o s s i b l e t o d i s t i n g u i s h . : .-between the r o o t systems of two p l a n t s growing i n c l o s e a s s o c i a t i o n , i f one i s i n j e c t e d w i t h P 3 2 . A l s o , the P 3 2 p a t t e r n s e s t a b l i s h e d i n the r o o t systems might give some c l u e s as to times o f r o o t growth, r e g i o n s of g r e a t e s t a c t i v i t y e t c . M a t e r i a l s and Methods. Tagged s i n g l e p l a n t s i n p l o t s o f orchardgrass, r e d fescue, Kentucky b l u e g r a s s , and c o l o n i a l bentgrass were i n j e c t e d w i t h 0.2 ml o f a 0.5% s o l u t i o n of KH2PO4 c o n t a i n i n g 800 m i c r o c u r i e s per ml o f s o l u t i o n . T h i s was accomplished by the use o f a 1 ml hypodermic s y r i n g e . The i n j e c t i o n s were made j u s t above the crown. At one week i n t e r v a l s r o o t samples from cores of s o i l , f n t h i c k , taken at s t r a t e g i c l o c a t i o n s on the p r o f i l e , were checked f o r r a d i o -a c t i v i t y u s i n g the Ge i g e r - M u l l e r counter. These cores were made w i t h -J" diameter v i a l s , and the r o o t s were separated from the s o i l , and p l a c e d on watch-glasses over which the counter was p l a c e d . In many i n s t a n c e s the s o i l came f r e e i n the v i a l s without the r o o t s , i n which cases the r o o t s from 43. the areas were c l i p p e d o f f w i t h a p a i r o f s c i s s o r s . Observations and r e s u l t s . A f t e r one week, P was t r a c e d to a depth of 2" i n orchardgrass, r e d fescue and c o l o -n i a l b entgrass, and to a depth of 6" i n Kentucky b l u e g r a s s . A f t e r 3 weeks P 3 2 was dete c t e d a t the f o l l o w i n g depths. Orchardgrass 7", red fescue 4", Kentucky b l u e g r a s s 7" and c o l o n i a l bentgrass 6". At s i x weeks the depths were 9",6", 9" and 6" r e s p e c t i v e l y . R a d i o a c t i v i t y was recorded o n l y p e r p e n d i c u l a r l y from the i n j e c t e d p l a n t s , i n d i c a t i n g a marked v e r t i c a l development of the r o o t s of i n d i v i d u a l p l a n t s i n a t u r f . The P 3 2 movement downwards o b v i o u s l y i s v e r y slow. I t was ev i d e n t t h a t s o i l samples taken at g r e a t e r depths c o n t a i n e d r o o t s , but a p p a r e n t l y P 3 2 had not reached these r o o t s . Maybe the P 3 2 c o u l d not be d e t e c t e d due to s m a l l volume or masking e f f e c t s by s o i l , or on the other hand these innermost r o o t s may not have been a c t i v e l y func-t i o n i n g r o o t s . That P 3 2 had not moved out i n t o the s o i l , was determined by t e s t i n g the s o i l samples f o r r a d i o a c t i v i t y a f t e r the r o o t s were removed. An i n d i v i d u a l a l f a l f a p l a n t from a row, e s t a b l i s h e d as a c l o n e , one week p r i o r to the above treatments, was a l s o s u b j e c t e d t o the same treatment at the same time. On the t h i r d week a f t e r i n j e c t i o n , P 3 2 was found t o have moved to a depth o f 5^-M. No v i s i b l e r o o t s were obvious below t h i s . The value or i n j e c t i n g P 3 2 i n t o p l a n t crowns f o r the purposes o f r o o t study has s c a r c e l y been e x p l o r e d by these few t r i a l s . The o b s e r v a t i o n s are i n t e r e s t i n g and should be 44. extended. 4. A Technique Using L i t h i u m C h l o r i d e Placement i n S o i l . L i t h i u m , an element i n the same atomic s e r i e s as sodium and potassium, both of which are commonly found i n p l a n t s , i s r a r e l y found i n e a s i l y d e t e c t a b l e q u a n t i t i e s i n s o i l s or p l a n t s , yet l i t h i u m appears to enter p l a n t s i f p r e s e n t i n the s o i l , r e a d i l y and enough t o behave q u i t e p a s s i v e l y . As has been i n d i c a t e d e a r l i e r , l i t h i u m has been used by a t l e a s t one o t h e r group o f workers as a b a s i s f o r r o o t study. On the assumption t h a t l i t h i u m i s p r e s e n t i n o n l y v e r y minute q u a n t i t i e s or not a t a l l i n Alderwood loamy sand, placements of l i t h i u m c h l o r i d e were made at s e v e r a l depths below newly p l a n t e d orchardgrass and a l f a l f a , i n the hope t h a t when the r o o t s of these p l a n t s reached the placements, l i t h i u m could be promptly d e t e c t e d i n the f o l i a g e . M a t e r i a l s and methods. In a manner s i m i l a r to that of P 3 2 placement a l r e a d y d e s c r i b e d , l i t h i u m c h l o r i d e was p l a c e d at depths o f 4", 6" and 10" below o r c h a r d g r a s s and a l f a l f a p l a n t s . The p l a n t s had newly been e s t a b l i s h e d 5 n a p a r t i n rows, and were d e v e l o p i n g a c t i v e l y . One month a f t e r the clones were e s t a b l i s h e d , 100 mg of m i c r o n i z e d l i t h i u m c h l o r i d e was poured down each tube, by f i t t i n g the rubber on the exposed end w i t h a s m a l l f u n n e l . To a s c e r t a i n t h a t a l l the m a t e r i a l was out o f the tube, the tube was withdrawn about •§•", and tapped w i t h an o r d i n a r y p e n c i l . I r r i g a t i o n was done as the need was made apparent by the i r r o m e t e r s mentioned p r e v i o u s l y . 45. One week a f t e r placement, samples were taken of the f o l i a g e of orchardgrass from the p l a n t s a djacent to each placement. At the same time samples were taken from p l a n t s , under which no l i t h i u m c h l o r i d e had been p l a c e d . A l f a l f a sampling had to be delayed, due to i n s u f f i c i e n t top growth. Three weeks l a t e r , however, s i m i l a r samples were taken both of orchardgrass and a l f a l f a . U s i n g 1 gram of oven d r i e d m a t e r i a l , these samples were dry ashed, and s a l t s e x t r a c t e d by a s o l u t i o n 0.2 N i n n i t r i c a c i d . The d e t e r m i n a t i o n o f l i t h i u m was made by the ' d i r e c t i n t e n s i t y method', on a Perkin-Elmer Flame Photometer, u s i n g a top standard of 10 p.p.m. l i t h i u m i n 0.2 N n i t r i c a c i d . Observations and r e s u l t s . The r e s u l t s are shown i n Table I I I . By v i r t u e o f the f a c t t h a t t h e r e might have been some i n t e r f e r e n c e from s a l t s other than l i t h i u m , these r e s u l t s may be regarded as q u a l i t a t i v e r a t h e r than q u a n t i t a t i v e . I t cannot be concluded t h e r e f o r e t h a t the readings o b t a i n e d from no placement of l i t h i u m c h l o r i d e ( i . e . 0.70, 0.75 p.p.m. f o r o r c h a r d g r a s s and 13.5 p.p.m. f o r a l f a l f a ) i s an i n d i c a t i o n of the amounts of l i t h i u m absorbed from Alderwood loamy sand. N e v e r t h e l e s s the r e l a t i v e l y h i g h e r r e a d i n g s obtained f o r v a r i o u s placements might be assumed to i l l u s t r a t e the absorp-t i o n o f l i t h i u m from l i t h i u m c h l o r i d e on a comparative b a s i s . I t can be concluded then t h a t r o o t s of o r c h a r d g r a s s had reach-ed depths of 6" a f t e r 5 weeks, (placement b e i n g made 4 weeks a f t e r e s tablishment) but had not d e c i d e d l y p e n e t r a t e d to 10" even a f t e r 8 weeks. A l f a l f a r o o t s reached a depth of 6" 46. TABLE I I I ' RELATIVE ABSORPTION OF LITHIUM BY PLANTS FROM VARIOUS PLACEMENTS OF LITHIUM CHLORIDE IN ALDERWOOD LOAMY SAND No. Species & placement Readings a t % 10 p.p.m. p.p.m. top standard 1 week a f t e r placement 1 Orchardgrass 0" 7.0 0.70 No placement. 2 tt 4" 9.5 0.95 3 »» 6" 9.0 0.90 4 u 10" 7.0 0.70 3 weeks a f t e r • placement 5 Orchardgrass 0" 7.5 0.75 No placement. 6 4 « 16.0 1.60 7 6" 13.5 1.35 8 10" 8.5" 0.85 9 A l f a l f a 0" 13.5 1.35 No placement. 10 w 4" 17.5 1.75 11 n 6" 20.0 2.00 12 n 10" 15.0 1.50 a f t e r 8 weeks, but a l s o not c o n c l u s i v e l y to a depth of 10" even though the readings were higher (as i n orchardgrass) than no placement. Even though no attempts were made to determine the move-ment o f l i t h i u m i n the s o i l , the p o s s i b i l i t y e x i s t s of making such a study and r e l a t i n g the r e s u l t s to those o b t a i n e d by a n a l y s i s as was done wi t h P 3 2 s t u d i e s . 47. DISCUSSION AND CONCLUSIONS The need f o r newer techniques f o r the study of r o o t systems, cannot be too s t r o n g l y emphasised. There are scores o f phenomena i n r o o t systems not p r o p e r l y understood, due to l a c k of techniques f o r s t u d y i n g them i n p l a c e . There i s no doubt t h a t a l l r o o t s are not a c t i v e l y f u n c t i o n i n g r o o t s , and t h e r e i s a l s o c o n s i d e r a b l e evidence t o show that even a c t i v e -l y f u n c t i o n i n g r o o t s are not f u n c t i o n i n g as such a l l the time. In the F r a s e r V a l l e y f o r example, many areas are l i t e r a l l y under water, or m a i n t a i n a water t a b l e j u s t below the s u r f a c e d u r i n g the w i n t e r months. What happens to the r o o t s o f p l a n t s under these c o n d i t i o n s ? How do the r o o t s keep the p l a n t s i n the 'best p o s s i b l e balance' w i t h the environment at sub-normal temperatures or when the s o i l i s f r o z e n ? Are r o o t s dormant when p l a n t s are o v e r w i n t e r i n g , and 'resume' t h e i r f u n c t i o n when c o n d i t i o n s are f a v o u r a b l e ? What i s the f a t e of r o o t s when changes o f season occur? e t c . These are o n l y a few of the a s p e c t s of r o o t systems, the i n f o r m a t i o n r e g a r d i n g which i s s t i l l v e r y obscure. The d e f i c i e n c i e s i n u s i n g the ' d i r e c t * method expressed, i n c l u d e the i n a b i l i t y to d i s t i n g u i s h between i n d i v i d u a l r o o t s i n a g i v e n sward. That b e i n g a b l e t o do so, c o u l d o f f e r some e x p l a n a t i o n t o the problems of r o o t c o m p e t i t i o n , e s p e c i a l l y i n mixed stands o f forage crops, i s i n d i c a t e d by the work of the few i n v e s t i g a t o r s who used ' i n d i r e c t ' methods. Boggle et a l (1958) f o r example, have shown t h a t the a b i l i t y of c e r t a i n 48. d i f f e r e n t s p e c i e s to grow t o g e t h e r i n a p l a n t community, may be determined by the d i f f e r e n c e s i n t h e i r r o o t i n g h a b i t s . T h e i r work a l s o suggests t h a t d i f f e r e n t p l a n t s growing i n a s s o c i a t i o n may be a b s o r b i n g n u t r i e n t s from d i f f e r e n t depths. Such phenomena of r o o t s cannot be determined by the ' d i r e c t ' method. The main advantage to the ' d i r e c t * method, i s t h a t most of the techniques are q u i t e s u i t a b l e f o r d e s c r i p t i v e and q u a n t i t a t i v e work, and no ' i n d i r e c t ' method can e n t i r e l y p r e c l u d e i t s use i n the comprehensive study of r o o t systems. Growing p l a n t s i n s p e c i a l c o n t a i n e r s and i n n u t r i e n t s o l u t i o n s are h i g h l y a r t i f i c i a l techniques. In s p e c i a l c o n t a i n e r s of the type used i n t h i s study, l a t e r a l spread of some r o o t s was o b v i o u s l y r e s t r i c t e d . In f a c t there was no way of determining whether c e r t a i n r o o t s recorded to be v e r t i c a l at the g l a s s - s o i l i n t e r f a c e , would not have been h o r i z o n g a l under f i e l d c o n d i t i o n s . Growing i n n u t r i e n t s o l u t i o n s , the s i t u a t i o n appears to be even more a r t i f i c i a l . Roots growing i n l i q u i d media c o u l d never conform morpho-l o g i c a l l y t o the same r o o t s growing i n a s o i l . In the s o i l f o r example, r o o t s are a s s o c i a t e d w i t h s o i l a i r , s o i l water and s o i l p a r t i c l e s which e x e r t r e s i s t a n c e s to r o o t p e n e t r a -t i o n and development, which c o n d i t i o n s are never met i n n u t r i e n t s o l u t i o n a l o n e . The r e s u l t s obtained from growing p l a n t s i n g l a s s - f a c e d tanks suggests, however, t h a t these methods may be v a l u a b l e f o r making r e l a t i v e comparisons of r o o t systems, d e v e l o p i n g 4 9 . under c o n d i t i o n s o f dense s e e d i n g versus l i g h t seeding, h i g h f e r t i l i t y v e r s u s low f e r t i l i t y , h i g h moisture versus low moisture, and v a r i o u s c o n b i n a t i o n s of these. That dyes such as a c i d f u c h s i n , congo r e d and methylene blue c o u l d be used i n the study o f root systems, needs much more i n v e s t i g a t i o n . The f a i l u r e t o d u p l i c a t e the p r e l i m i n a r y l a b o r a t o r y t e s t s i n the f i e l d , does not pr e c l u d e the use of those dyes i n root study. For many i n t r a v i t a m dyes not o n l y w i l l s t a i n p l a n t m a t e r i a l , but a l s o are r e a d i l y absorbed by p l a n t s , and are r e l a t i v e l y n o n - t o x i c i n c e r t a i n c o n c e n t r a t i o n s . The s t r a t e g i c placements of r a d i o a c t i v e phosphorus ( P 3 2 ) and of r a r e elements such as l i t h i u m , have proved most promis-i n g i n making r o o t s t u d i e s i n the f i e l d . There i s no doubt t h a t the employment o f P32 allows the u n d e r t a k i n g o f e x p e r i -ments and measurements, where o r d i n a r y chemical and instrumen-t a l measurements are not p r a c t i c a b l e . The p r o p e r t i e s o f P 3 2 d i s c u s s e d p r e v i o u s l y , i n d i c a t e the r e l a t i v e ease w i t h which i t can be handled and adapted t o a wide v a r i e t y of uses. The r e s u l t s o b t a i n e d i n the study of root systems i l l u s t r a t e d c o n c l u s i v e l y , t h a t s e v e r a l r e l a t e d problems c o u l d be s t u d i e d . For example, the d i s t i n g u i s h i n g between ' l i v i n g ' and 'dead' r o o t s , changes i n r o o t a c t i v i t i e s w i t h season, r o o t s p e c i a l -i z a t i o n and root p a t t e r n s can a l l be i n v e s t i g a t e d to a con-s i d e r a b l e e x t e n t . The l o c a l i z a t i o n of r o o t s under a heavy s e e d i n g can be compared wi t h r o o t s under l i g h t seeding, or spaced i n rows. The main disadvantages o f the placement technique used 50. i n t h i s study, was the upward movement of P a l o n g the s i d e s o f the g l a s s tubes. This however, was c o r r e c t e d by the modi-f i c a t i o n i n technique used l a t e r . I t might be argued t h a t t h i s technique has c e r t a i n other disadvantages when c o n s i d e r -i n g the techniques used by Boggie e t a l (1958), Burton et a l (1954) and H a l l e t a l (1953). I t i s the o p i n i o n of the w r i t e r , however, t h a t s i n c e the P 3 2 i n the s o i l i s l o c a l i z e d ; b y the mo d i f i e d procedure, l e a v i n g the tubes i n the s o i l does not cr e a t e c o n d i t i o n s which s e r i o u s l y reduce the value of the technique. The exposed end was c l o s e d o f f w i t h a clamp, and the b u r i e d end t h e o r e t i c a l l y s e a l e d o f f w i t h p a r a f f i n . The e f f e c t then should not be d i f f e r e n t from t h a t of a rock of the same dimensions. The o r i g i n a l idea behind l e a v i n g the tube i n the s o i l , however, was t h a t subsequent placements o f P 3 2 c o u l d be made under i d e n t i c a l c o n d i t i o n s , t o study problems such as changes i n root a c t i v i t y . T h i s o b j e c t i v e seem t o be d e f e a t e d once the m o d i f i e d technique has to be employed, on account of the p a r a f f i n s e a l . One p o i n t which must not be overlooked, i s the f a c t t h a t the volume of phos-phate s o l u t i o n c o n t a i n i n g the P 3 2 was s m a l l e r than i n the o r i g i n a l placement technique ( i . e . 100 mc P 3 2 i n 1 ml KH2PO4 as compared w i t h 100 mc P 3 2 i n 10 ml KH2PO4). This might p a r t l y account f o r the l o c a l i z a t i o n of P 3 2 under the m o d i f i e d technique. Due to l i m i t e d supply of P 3 2 , t h i s could not be i n v e s t i g a t e d f u r t h e r . The o b s e r v a t i o n s and r e s u l t s from p l a n t i n j e c t i o n s were most i n t e r e s t i n g and c e r t a i n l y warrant f u r t h e r i n v e s t i g a t i o n . 51. I f the technique used, c o u l d determine whether P 3 2 had reached depths at which the Geiger counter c o u l d not d e t e c t i t , due e i t h e r to volume or the masking e f f e c t of s o i l , or t h a t the r o o t s a t these depths were not a c t i v e l y f u n c t i o n i n g r o o t s , then i t might be p o s s i b l e t o d i s t i n g u i s h between i n d i v i d u a l r o o t s i n a given sward. As i n d i c a t e d p r e v i o u s l y , t h i s might a l s o give some c l u e s as to the regions of g r e a t e s t a c t i v i t y . The p r o p e r t i e s of l i t h i u m i n the s o i l have been d i s c u s s -ed, and as Sayre and M o r r i s (1940) p o i n t e d out, l i t h i u m enters i n t o the base exchange complex, and does not move w i t h s o i l water. These f a c t s , together w i t h the r e s u l t s obtained, i n -d i c a t e p o s s i b i l i t i e s s i m i l a r t o those of P 3 2 i n the study of r o o t systems. L i t h i u m would have the advantage over P 3 2 , however, a l l other t h i n g s being equal, i n t h a t i t i s s a f e r to handle and does not n e c e s s i t a t e the p r e c a u t i o n s n e c e s s a r y i n the case of r a d i o a c t i v e m a t e r i a l . Furthermore, i t i s l e s s expensive and more e a s i t y a v a i l a b l e than P^ 2. SUMMARY The l i m i t a t i o n s t o the use of the ' d i r e c t 1 method of s t u d y i n g r o o t systems have been expressed, i n d i c a t i n g the d o u b t l e s s need f o r newer techniques i n understanding r o o t problems i n communities of p l a n t s , w i t h s p e c i a l r e f e r e n c e to forage stands. Some ' i n d i r e c t ' techniques were i n v e s t i g a t e d u s i n g o r c h a r d -grass ( D a c t y l i s glomerata), red fescue (Festuca r u b r a ) , 52. Kentucky "bluegrass (Poa - p r a t e n s i s ) . c o l o n i a l bentgrass ( A g r o s t i s sp. ), a l f a l f a (Medicago s a t i v a ) and r e d c l o v e r ( T r i f o l i u m p r a t e n s e ) . Although many of these techniques are merely m o d i f i c a t i o n s o f techniques used p r e v i o u s l y , the r e s u l t s i l l u s t r a t e c o n c l u s i v e l y the scope of t h e i r employment i n understanding b e t t e r t h i s phase of development. The growing o f o r c h a r d g r a s s , c o l o n i a l bentgrass and r e d c l o v e r i n 12"x4-| ,'xl2 , f g l a s s - f a c e d wooden tanks f i l l e d w i t h s o i l i n the greenhouse, has demonstrated a technique f o r o p t i c a l study o f these r o o t systems. This type of s p e c i a l c o n t a i n e r from which d i r e c t measurements were made, has i l l u s t r a t e d an apparent l o c a l i z a t i o n of i n d i v i d u a l p l a n t r o o t s i n dense seedin g as compared w i t h those of l i g h t seeding. Other s p e c i a l c o n t a i n e r s c o n s i s t e d of 1 0 w x l 6 ' n x i " g l a s s ' s l i d e s ' f i l l e d w i t h s o i l . These had the advantage over the former i n t h a t s o i l volume i s reduced, i t i s a much cheaper technique, i t makes h a n d l i n g e a s i e r , and o b s e r v a t i o n o f r o o t s on both s i d e s of the c o n t a i n e r s i s p o s s i b l e . Root f l u o r e s c e n c e was s t u d i e d i n a l f a l f a and sweet c l o v e r s e e d l i n g s growing i n g l a s s ' s l i d e s * . The i n j e c t i o n o f b a r l e y p l a n t s growing i n s i m i l a r s l i d e s , w i t h the f l u o r e s -cent compounds, e o s i n , f l u o r e s c i n , and e s c u l i n f a i l e d to produce f l u o r e s c e n c e i n v i v o . R e s u l t s from the p l a c i n g of the compounds i n the v i c i n i t y of the r o o t s were l a r g e l y n e g a t i v e . L a b o r a t o r y t e s t s were c a r r i e d out w i t h b a r l e y p l a n t s growing i n g l a s s s l i d e s w i t h placements of the dyes, a c i d 53. f u c h s i n , congo r e d and methylene blue at 2" depth. F i e l d t e s t s on square f o o t p l o t s o f orchardgrass and Kentucky b l u e -grass w i t h placements of a c i d f u c h s i n a t depths of 2", 4" and 12", f a i l e d to r e p l i c a t e the l a b o r a t o r y f i n d i n g s t h a t the a b s o r p t i o n by b a r l e y r o o t s of t h i s m a t e r i a l , not o n l y s t a i n e d the r o o t s r e d , but was soon evident i n the a e r i a l p o r t i o n s . The s t r a t e g i c placements of s m a l l q u a n t i t i e s of r a d i o -a c t i v e phosphorus i n a 0.5% s o l u t i o n o f potassium dihydrogen phosphate ( i . e . 100 m i c r o c u r i e s o f P 3 2 / l to 10 ml KH 2P0 4 a t depths of 2", 4", 12" and 24"), was s t u d i e d on square f o o t p l o t s o f o r c h a r d g r a s s , red f e s c u e , Kentucky b l u e g r a s s and c o l o n i a l b entgrass on Alderwood loamy sand. T h i s enabled the depth of r o o t p e n e t r a t i o n t o be a s s e s s e d by measuring r a d i o a c t i v i t y i n the l e a v e s , when a G e i g e r - M u l l e r counter was p l a c e d near them. The movement o f P 3 2 i n Alderwood loamy sand was s t u d i e d on the p r o f i l e s of p l o t s , which were kept bare of v e g e t a t i o n , and on which s i m i l a r placements were made. The r e s u l t s were expressed d i a g r a m m a t i c a l l y and r e l a t e d to those o b t a i n e d on the seeded p l o t s . A m o d i f i e d technique o f p l a c i n g , r e s u l t e d i n the l o c a l i z a t i o n of the r a d i o a c t i v e phosphorus w i t h i n an area of 1 square i n c h on the face of the p r o f i l e . S i n g l e p l a n t s i n e s t a b l i s h e d stands of orchardgrass, r e d f e s c u e , Kentucky b l u e g r a s s and c o l o n i a l bentgrass, were i n -j e c t e d near the crown, each w i t h 0.2 ml of a 0.5% s o l u t i o n of KH2PO4 c o n t a i n i n g 800 m i c r o c u r i e s P 3 2 / m l of s o l u t i o n . The t r a n s l o c a t i o n of P 3 2 from the a e r i a l p o r t i o n s t o the r o o t s , 54. was found to be very slow when r a d i o a c t i v i t y was checked i n the r o o t s from p r o f i l e s . The recorded depths o f P 3 2 move-ment were 7", 4", 7" and 6" r e s p e c t i v e l y a f t e r 3 weeks, and 9'!, 6", 9" and 6" a f t e r 6 weeks. Roots a t g r e a t e r depths were q u i t e e v i d e n t . Movement i n a 4 week o l d a l f a l l ' a c l o n e , i n j e c t e d 3 weeks e a r l i e r , was found t o be 5|-". In t h i s case no v i s i b l e r o o t s were apparent a t g r e a t e r depths. The a b s o r p t i o n of l i t h i u m from l i t h i u m c h l o r i d e p l a c e d a t depths of 4", 6" and 10" i n rows of orchardgrass and a l f a l f a , e s t a b l i s h e d by cl o n e s , was determined by an a l y s e s of the a e r i a l p o r t i o n s by a photometric method. Root p e n e t r a -t i o n t o those depths was determined by the r e l a t i v e l y h igher c o n c e n t r a t i o n s of l i t h i u m found i n p l a n t s l o c a t e d above these placements, when compared w i t h p l a n t s under which there was no placements. 5 5 . LITERATURE CITED. 1. Arnason, T. J". , E. Cumming and J. W. T. Spinks. 1948. Chromosome breakage induced by absorbed radio-active phosphorus, P32. Can. Jour. Res. (c) 26 :109-114. 2. Arnason, T. J., C. 0. Person and J. M. Naylor. 1952. Radiation induced mutations i n wheat and barley. Can. Jour. Bot. 30:743-754. 3. Biddulph, 0. 1939. Absorption and movement of radiophosphorus i n bean seedlings. 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A., V/. L. Nelson, C. D. Welch and N. S. H a l l . 1949. A comparison of phosphorus u t i l i z a t i o n by crops. S o i l Science 68:171-178. 33. Lawton, K., A. E, Eri c k s o n and L. S. Robertson. 1954. ( a ) . U t i l i z a t i o n of phosphorus by sugar beets as a f f e c t e d by f e r t i l i z e r placement. Agron. Jour. 46:262-264. 34. Lawton, K., M. B. Tesar and B. Kawin. 1954 (b). E f f e c t of r a t e and placement of superphos-phate on the y i e l d and absorption of legume hay. S o i l S c i . Soc. Amer. Proc. 18:428-432. 35. Lawton, K., and J . A. Vomocil. 1954. The d i s s o l u t i o n and m i g r a t i o n of phosphorus from granular superphosphate i n some Michigan s o i l s . S o i l S c i . Soc. Amer. Proc. 18:26-32. 36. M c A u l i f f e , C., M. Peach and R. B r a d f i e l d . 1949 (a). U t i l i z a t i o n of phosphorus i n farm manure. I I A v a i l a b i l i t y t o p l a n t s of organic and in o r g a n i c forms of phosphorus i n sheep manure. S o i l Science 68:185-196. 37. M c A u l i f f e . C. and M. Peach. 1949 (b). U t i l i z a t i o n by p l a n t s of phosphorus i n farm manure. I L a b e l i n g of phosphorus i n sheep manure w i t h P32. S o i l Science 68:178-184. 38. M c A u l i f f e , C. D., N. S. H a l l , L. A. Dean and S. B. Hendricks. 1947. Exchange r e a c t i o n s between phosphate and s o i l s . H y droxylic surfaces of s o i l m i n e r a l s . S o i l S c i . Soc. Amer. Proc. 12:119-123. 39. Nelson, W. L., B. A. Krantz, W. E. C o l w e l l , W. G. Woltz, A. Hawkins, L. A. Dean, A. J . MacKenzie and E. J . Rubins. 1947. A p p l i c a t i o n of t r a c e r technique t o st u d i e s of phosphatic f e r t i l i z e r u t i l i z a t i o n by crops. I I F i e l d Experiments. S o i l S c i . Soc. Amer. Proc. 12:113-118. 40. Nelson, W. L., B. A. Krantz, C. D. Welch and N. S. H a l l . 1949. U t i l i z a t i o n of phosphorus as a f f e c t e d by place-ment. I I Cotton and corn i n North C a r o l i n a . S o i l . Science 68:137-144. 59. 41. Olsen, S. R. and R. Gardner. 1949. U t i l i z a t i o n o f phosphorus from v a r i o u s f e r t i -l i z e r m a t e r i a l s 17 Sugar beets, wheat and b a r l e y i n Colorado. S o i l Science 68:163-170. 42. Olsen, S. R., W. R. Schmehl, F. S. Watanabe, C. 0. S c o t t , W. H . F u l l e r , J". V. Jbrdon, and R. Kunkel. 1950. U t i l i z a t i o n o f phosphorus by v a r i o u s crops as a f f e c t e d by source of m a t e r i a l and placement Colorado A g r i c . Expt. Sta. Tech. B u l l . 42. 43. Olsen, S. R. and M. F r i e d . 1957. S o i l phosphorus and f e r t i l i t y . S o i l U.S.D.A. Yearbook pp. 94-100. 44. Pavlychenko, J. K. and J . B. Harrington. 1934. Competitive e f f i c i e n c y o f weeds and c e r e a l crops. Can. Jour. Res. 10:77-94. 45. Pavlychenko, T. K. and J . B. Harrington. 1935. Root development of weeds and crops In competition under dry farming. S c i . A g r i c . 16:151-160. i 46. Pavlychenko, T. K. 1937 ( a ) . Q u a n t i t a t i v e study o f the e n t i r e root systems of weed and crop p l a n t s under f i e l d c o n d i t i o n s . Ecology 18 (1). 47. Pavlychenko, T. K. 1937 (b). The s o i l block washing method i n q u a n t i t a t i v e root study. Can. Jour. Res. (c) 15:33-57. 48. P o l l o c k , B. M, R. H. Goodwin and S. Green. 1954. Studies on r o o t s . I I E f f e c t s of coumarin, s c o p o l e t i n and other substances on growth. Amer. Jour. Bot. 41:521-529. 49. Radley, J . A. and J . Grant. 1954. Fluorescence a n a l y s i s i n u l t r a - v i o l e t l i g h t . 4th E d i t i o n 560 pages. Chapman & H a l l . London, pp. 112-141. 50. Sayre, J . D. and V. H. M o r r i s . 1940. The l i t h i u m method of measuring the extent of corn root systems. P l a n t P h y s i o l o g y . 15: 761-764. 51. Simmonds, P. M., R. C. R u s s e l l and R. J. S a l l a n g . 1935. A comparison o f d i f f e r e n t types o f r o o t r o t of wheat by means of root excavation s t u d i e s . S c i . A g r i c . 15:680-700. 60. 52. Spinks, J . W. T. and S. A. Barber. 1947. Study of f e r t i l i z e r uptake u s i n g r a d i o a c t i v e phosphorus I. S c i . A g r i c . 27:147-156. 53. Spinks, J . W. T. and S. A. Barber. 1948 ( a ) . Study of f e r t i l i z e r uptake u s i n g r a d i o a c t i v e phosphorus I I . S c i . A g r i c . 28:79-87. 54. Spinks, J . W. T. , H. G. Dion, M. Reade and J . E. Behm. 1948 (b). Study of f e r t i l i z e r uptake u s i n g r a d i o a c t i v e phosphorus I I I . S c i . A g r i c . 28:309-314. 55. Stanford, G. and L. B. Nelson. 1949. U t i l i z a t i o n o f phosphorus from v a r i o u s f e r t i l i z e r m a t e r i a l s . I l l Oats and a l f a l f a i n Iowa. S o i l Science 68:157-162. 56. Stanford, G. and L. B. Nelson. 1949. U t i l i z a t i o n o f phosphorus as a f f e c t e d by placement. I Corn and c o t t o n i n Iowa. S o i l Science.68:129-136. 57. S t a n f o r d , G., C. D. M c A u l i f f e and R. B r a d f i e l d . 1950. The e f f e c t i v e n e s s of superphosphate top dressed on e s t a b l i s h e d meadows. Agron. Jour. 42:423-426. 58. Stewart, F. C , P. Prevot and J . A. H a r r i s o n . 1942. A b s o r p t i o n and accumulation o f rubidium bromide by b a r l e y p l a n t s . L o c a l i z a t i o n i n the r o o t s o f c a t i o n accumulation and of t r a n s f e r to the shoot. P l a n t Physcology 17 :411-421. 59. Stout, P. R. and D. R. Hoagland. 1939. Upward and l a t e r a l movement of s a l t i n c e r t a i n p l a n t s as i n d i c a t e d by r a d i o a c t i v e i s o t o p e s of potassium sodium and phosphorus absorbed by r o o t s . Amer. Jour. Bot. 26: 320-324. 60. Troughton, A. 1957. The underground organs or herbage g r a s s e s . Commonwealth Bureau o f P a s t u r e s and F i e l d Crops. B u l l . 44. 61. U l r i c h , A., L. Tacobson and R. O v e r s t r e e t . 1947. Use of r a d i o a c t i v e phosphorus i n a study of the a v a i l a b i l i t y o f phosphorus to grape v i n e s u n d e r f i e l d c o n d i t i o n s . S o i l Science 64:17-28. 61. 62. United States Atomic Energy Commission (U.S.A.E.C.) 1956. A conference on r a d i o a c t i v e isotopes i n a g r i c u l t u r e . TID 7512. 63. Weaver, J . E. 1926. Root development o f f i e l d crops. 291 pp. McGraw-Hill L t d . New York. 64. Weaver, J . E. and R. W. Darland. 1949. Q u a n t i t a t i v e study o f root systems i n d i f f e r e n t s o i l types. Science 110:164-165. 65. Welch, C. D., N. S. H a l l and W. L. Nelson. 1949. U t i l i z a t i o n of f e r t i l i z e r and s o i l phosphorus by soybeans. S o i l S c i . Soc. Amer. Proc. 14:231-235. 66. Woltz, W. G., N. S. H a l l and W. E. C o l w e l l . 1949. U t i l i z a t i o n of phosphorus by tobacco. S o i l Science 68:121-128. 62. , APPENDIX A 63. STAND ESTABLISHMENT OF FORAGE GRASSES AND LEGUMES  INTRODUCTION This s e c t i o n r e p r e s e n t s the work done on a programme, which was o r i g i n a l l y intended to he 'the p r e s e n t a t i o n ' . The l a n d use s i t u a t i o n at the U n i v e r s i t y of B.C. was too u n c e r t a i n at the time, however, to undertake any l o n g term p r o j e c t s , hence the change was made to the f o r e g o i n g . Stand f a i l u r e s of forage legumes and grasses are q u i t e frequent wherever forage crops are grown. That forage seed-ings f a i l because of 'poor seed', 'dry season' or 'winter-k i l l i n g ' , does not n e a r l y b e g i n t o e x p l a i n wtoy some p l a n t s s u r v i v e even when there are ' f a i l u r e s ' ; why we u s u a l l y have to p l a n t 3 to 1000 times as much forage seed as we want p l a n t s . In g e n e r a l , q u i t e a few t h i n g s are known about stand e s t a b l i s h m e n t but there i s v e r y l i t t l e d e t a i l e d work on the problem. There has been much p l o t study a t a macro observa-t i o n l e v e l and i t has l o n g been r e c o g n i s e d that between p l a n t s c o m p e t i t i o n occurs f o r p h y s i c a l f a c t o r s of the environment. Of these, water, n u t r i e n t s , temperature and l i g h t are of p r i n c i p a l s i g n i f i c a n c e both i n n a t u r a l communities and i n sown crops and p a s t u r e s . There i s , however, v e r y l i t t l e d e t a i l e d work r e g a r d i n g such f a c e t s as :-(a) Seed s i z e r e l a t i v e t o germination and v i g o u r . (b) Seed numbers r e l a t i v e t o germination and v i g o u r . (c) Anomolies i n germination. (a) Age of seed r e l a t i v e t o germination and v i g o u r . (e) E f f e c t s o f s c a r i f i c a t i o n . ( f ) E f f e c t s o f soaking seed under d i f f e r e n t c o n d i t i o n s . (g) Seed c o v e r i n g and p l a n t i n g depth. (h) S o i l c o n d i t i o n s - f e r t i l i t y , a e r a t i o n e t c . I t was the purpose o f t h i s p r e s e n t a t i o n , "(therefore, to i n v e s -t i g a t e as many o f these f a c e t s as p o s s i b l e . LITERATURE There are many f a c e t s to t h i s problem, o n l y a few of which can be s t u d i e d . Consider the r e l a t i o n s h i p between s i z e o f seed and depth of seeding. The depth a t which a seed i s sown i n the s o i l , i n f l u e n c e s both i t s germination and i t s s u c c e s s f u l e s t a b l i s h m e n t as a s e e d l i n g . At v a r i o u s d i s -tances below the s u r f a c e , the germination o f a seed may be delayed or dormancy e n f o r c e d by the l o c a l s o i l c o n d i t i o n s -f o r example, high c o n c e n t r a t i o n s o f carbon d i o x i d e i n the deeper l a y e r s of the s o i l may i n h i b i t germination, or the germination o f l i g h t - s e n s i t i v e seeds may be prevented by the absence of l i g h t i n the s o i l . I f germination occurs, the food r e s e r v e s o f deeply sown seeds may be i n s u f f i c i e n t t o permit the shoot of the s e e d l i n g t o r e a c h the s o i l s u r f a c e , or the s e e d l i n g may be so weakened or damaged by the extended growth to the s u r f a c e , t h a t i t s r e s i s t a n c e t o pathogenic a t t a c k or i t s subsequent powers of co m p e t i t i o n are s e r i o u s l y reduced. Seeds which are sown too s h a l l o w l y , may meet d i f f e r e n t hazards which prevent s u c c e s s f u l establishment. In p a r t i c u -l a r , the water r e l a t i o n s o f the s o i l f l u c t u a t e more w i d e l y at the s u r f a c e than i n the deeper l a y e r s , and seeds sown near the s u r f a c e may o f t e n meet c o n d i t i o n s s u i t a b l e f o r germination but which are r a p i d l y f o l l o w e d by p e r i o d s when the s u r f a c e l a y e r s become so dry t h a t many s e e d l i n g s d i e . Moreover, seeds a t the s u r f a c e o f the s o i l are exposed to p r e d a t o r y b i r d s , rodents and s m a l l e r p e s t s w h i l e deep sowing o f f e r s a l a r g e degree of p r o t e c t i o n . In farming and garden-i n g p r a c t i s e i t i s customary to sow l a r g e seeds deeper than s m a l l and t h i s tendency may be c o r r e l a t e d w i t h the g r e a t e r food r e s e r v e s present i n l a r g e seeds which make emergence from a depth more s u c c e s s f u l and a l s o w i t h the f a c t t h a t v e r y few l a r g e seeds demand l i g h t f o r germination. Depth o f seeding i s v e r y s i g n i f i c a n t w i t h forage legumes and grasses which g e n e r a l l y are s m a l l and have l i t t l e food r e s e r v e s . Black (1956) of the Waite A g r i c u l t u r a l Research I n s t i t u t e , t e s t e d three s i z e s of seeds and three depths of sowing, 1-J, and 2 inches, and showed t h a t d e c r e a s i n g seed s i z e and i n c r e a s i n g depth of sowing of subterranean c l o v e r ( T r i f o l i u m subterraneum), one of the l a r g e r seeded legumes, both reduce the weight of the cotyledons a t emergence. Dry weight i n the e a r l y v e g e t a t i v e stage was p r o p o r t i o n a l to seed s i z e , and t o t a l l e a f area and l e a f numbers showed s i m i l a r t r e n d s . P l a n t s o f each o f the three seed s i z e s grew at the same r e l a t i v e r a t e . No e f f e c t of depth of sowing c o u l d be detected , and t h i s was shown to be due to the c o t y l e d o n area at emergence being c o n s t a n t f o r any g i v e n seed s i z e , r e g a r d -66. l e s s of v a r y i n g depth o f sowing and hence of cotyledon weight. Lawrence (1957) conducted a study on the emergence o f 24 l i n e s of i n t e r m e d i a t e wheatgrass (Agropyron intermedium) i n Saskatchewan, r e l a t i n g seed s i z e t o the depth from which emergence would occur. He r e p o r t e d t h a t even though some l i n e s d i d emerge to the extent of 40% a t depths of 2\ to 3 inches, emergence of s e e d l i n g s was delayed w i t h i n c r e a s i n g depth. R o g l e r ( l 9 5 4 ) working i n the Southern Great P l a i n s , r e -p o r t e d h i g h l y s i g n i f i c a n t d i f f e r e n c e s i n the t o t a l s e e d l i n g emergence o f c r e s t e d wheatgrass (Agropyron c r i s t a t u m ) from depths, weights and i n t e r a c t i o n of depths and weights. In most cases, emergence numbers dropped o f f r a p i d l y f o r each seed weight c l a s s as the depth of p l a n t i n g i n c r e a s e d . He a l s o r e p o r t e d t h a t the second and t h i r d l e a v e s developed more r a p i d l y on s e e d l i n g s o f a l l weight c l a s s e s p l a n t e d shallow. Hughes, Heath and M e t c a l f (1953) compiled the r e s u l t s o f s e v e r a l workers, and concluded t h a t on most s o i l s i n humid ar e a s , p r a c t i c a l l y a l l o f the s m a l l seeded forage crops are best sown i M to •§-,t deep or l e s s . They show t h a t a n y t h i n g deeper than l n i s f a t a l to seed the s i z e o f c l o v e r and timothy, u n l e s s the c o v e r i n g s o i l i s l o o s e . Even when seeds are l a r g e enough t o come up from g r e a t e r depths, they emerge so s l o w l y that deep p l a n t i n g i s a handicap. In dry c l i m a t e s and on sandy s o i l s s m a l l seeds must be sown deeper than t 1 ' to i n order to reach moisture. One o f the causes of f a i l u r e i n the e s t a b l i s h m e n t of forage seed i n dry l a n d 67. areas i s the d r y i n g out of seed. Hughes et a l p o i n t out t h a t a l i g h t r a i n a f t e r seeding shallow on l o o s e s o i l i n these areas may germinate the seed but they may die b e f o r e the s e e d l i n g s root enough to become e s t a b l i s h e d . Ludwig and Harper (1958(a)) of the U n i v e r s i t y of Oxford, c a r r i e d out pot and f i e l d experiments on depth o f sowing of maize and have r e p o r t e d t h a t whereas emergence was reduced at depths g r e a t e r than 2.5 cm when s o i l m oisture c o n d i t i o n s were maintained near f i e l d c a p a c i t y , the optimal depth of sowings i n the f i e l d may be 5 or even 10 cm d u r i n g p e r i o d s of drought. They a l s o compared the r e l a t i o n s h i p s between s o i l temperature and depth of sowing on both pot and f i e l d experiments. T h i s l e a d s to a c o n s i d e r a t i o n of temperature and mois-t u r e . Black (1955) has shown f o r subterranean c l o v e r ( T r i f o - l i u m subterraneum), t h a t there i s an optimal temperature (21° C) f o r the t r a n s f e r e n c e of f o o d r e s e r v e s from c o t y l e d o n s i n the e x t e n s i o n o f the h y p o c o t y l , and both depth of sowing and temperature a f f e c t the weight of cotyledons at emergence. Fayemi (1957) of the I l l i n o i s Agr. Expt. Sta., s t a t e s t h a t the r a t e o f germination and seed s w e l l i n g of a l f a l f a , r e d c l o v e r , a l s i k e c l o v e r and Ladino c l o v e r was g r e a t l y i n -f l u e n c e d by temperature. The time from exposure to the i n i t i a l a b s o r p t i o n of water by the seeds became s h o r t e r as the temperature i n c r e a s e d . Laude of the U n i v e r s i t y of C a l i f o r n i a (1956) working on the s e e d l i n g emergence of grasses as a f f e c t e d by low tempera-ture showed that c o l d t o l e r e n c e of the grass s e e d l i n g 68. decreases w i t h advancing stage of the pre-emergence p e r i o d . He found a l s o t h a t t a l l f escue, c r e s t e d wheatgrass and or c h a r d -grass possessed a high e r degree o f t o l e r a n c e than d i d Harding g r a s s , p e r e n n i a l r y e g r a s s and p r a i r i e bromegrass. Gi s t and Mott of Purdue U n i v e r s i t y i n 1957 r e p o r t e d t h a t i n c r e a s i n g temperatures from 60° to 90° P. caused r e d u c t i o n s i n the growth of a l f a l f a , red c l o v e r and b i r d s f o o t t r e f o i l s e e d l i n g s . The growth o f the r o o t s was a f f e c t e d more than was growth of the tops. Among the s o i l f a c t o r s which might i n f l u e n c e s o i l temperature and so determine the success of establishment of s e e d l i n g s i s the c o l o u r of the s o i l . Ludwig and Harper (1958(b)) working w i t h maize and s u r f a c e c o l o u r s r e n g i n g from white to b l a c k through y e l l o w brown and grey, r e p o r t e d t h a t s o i l temperature was markedly changed by such treatments, and i n the darkened s o i l s t h i s was r e f l e c t e d i n i n c r e a s e d emergence. They s t a t e t h a t i n as much as many f a c t o r s other than s o i l c o l o u r determine s o i l temperature, under f i e l d c o n d i t i o n s i n the s p r i n g , the speed and success of e s t a b l i s h -ment are s i g n i f i c a n t l y i n f l u e n c e d by c o l o u r . E f f e c t s a f t e r emergence o f s e e d l i n g s r e q u i r e f u r t h e r study b e f o r e the f u l l agronomic and e c o l o g i c a l s i g n i f i c a n c e of s o i l c o l o u r can be a p p r a i s e d . F a i l u r e to e s t a b l i s h stands of forage s p e c i e s i s f u r t h e r complicated by com p e t i t i o n , not o n l y from weeds and oth e r s p e c i e s , but a l s o between the i n d i v i d u a l s of the stand. P l a n t s d i f f e r i n t h e i r competitive a b i l i t y as p o i n t e d out by 69. B l a s e r et a l (1956). At the V i r g i n i a A g r i c u l t u r a l Experiment S t a t i o n , they have shown that p a s t u r e , meadow and lawn mixtures are a r t i f i c i a l p l a n t a s s o c i a t i o n s i n which the s p e c i e s are not m u t u a l l y b e n e f i c i a l to each other. I t i s d i f f i c u l t to produce and m a i n t a i n a given b o t a n i c a l composition, because the p e r e n n i a l g r a s s e s and legumes used and known to be reason-a b l y w e l l adapted to V i r g i n i a , respond d i f f e r e n t l y to n a t u r a l and imposed environmental f a c t o r s t h a t a f f e c t growth. The s p e c i e s t h a t are b e s t adapted to c u r r e n t environments become ag g r e s s i v e and dominant i n mixtures of any given b o t a n i c a l composition, while those t h a t are l e s s w e l l adapted are sup-pr e s s e d . Since f o r a g e s d i f f e r i n r a t e s o f es t a b l i s h m e n t , they have c l a s s i f i e d the s p e c i e s i n t o groups of ve r y aggres-s i v e , a g g r e s s i v e and non-aggressive s e e d l i n g development, and shown t h a t a g g r e s s i v e n e s s d u r i n g establishment i s a s s o c i a t e d w i t h s e e d l i n g emergence and s u r v i v a l and w i t h growth r a t e of s e e d l i n g p l a n t s . Growth r a t e and s u r v i v a l o f s e e d l i n g s v a r y w i t h the season seeded. Species that are ag g r e s s i v e when seeded i n the s p r i n g are not n e c e s s a r i l y a g g r e s s i v e when seeded i n the summer. The data f o r summer and s p r i n g s e e d l i n g s suggest that a l f a l f a may be very a g g r e s s i v e toward r e d c l o v e r , orchardgrass and other s p e c i e s f o r summer s e e d l i n g s , but w i t h s p r i n g s e e d l i n g s , the r e v e r s e may occur. The v a r i a t i o n s i n s u r v i v a l and growth r a t e among forage s e e d l i n g s under s p r i n g and summer seedings i s a t t r i b u t e d t o d i f f e r e n t i a l responses among the s p e c i e s t o the temperature and moisture c o n d i t i o n s 70. p r e v a i l i n g d u r i n g the season when seeded. The r e s u l t s of B l a s e r et a l (1956) e x e m p l i f i e s those of Troughton (1954). Working w i t h 17 s p e c i e s i n v o l v i n g SE s t r a i n s of grass a t the U n i v e r s i t y C o l l e g e of Wales, Troughton r e p o r t e d t h a t growth, as measured by weight of shoot/ u n i t of r o o t , i n c r e a s e d p r o g r e s s i v e l y w i t h time, and t h a t t h i s was a s s o c i a t e d w i t h the s i z e o f the s p e c i e s . He was able to show t h a t h i s l a r g e s t p l a n t s (ryegrasses and f i e l d brome) may be c l a s s i f i e d as a g g r e s s i v e , while h i s s m a l l e s t (meadow grass and f i n e - l e a v e d f e s c u e s ) as non-aggressive. Under these c o n d i t i o n s , i t would seem t h a t r o o t competi-t i o n i s not a f a c t o r of prime importance i n s e e d l i n g s , but t h a t c o m p e t i t i o n l a r g e l y takes the form of shading. Donald and Black (1958) have r e a l i z e d the s i g n i f i c a n c e of l e a f area i n p a s t u r e growth, and have s t a t e d that an aspect of p a s t u r e p r o d u c t i o n which lends i t s e l f t o a n a l y s i s i n terms o f LAI ( l e a f area index) i s the f a m i l i a r phenomenon of the suppres-s i o n of those s p e c i e s of dwarf s t a t u r e by t h e i r t a l l e r com-p e t i t o r s , e s p e c i a l l y of c l o v e r by grass. This has always been a t t r i b u t e d t o the shading of the dwarf s p e c i e s by the f o l i a g e of the t a l l e r s p e c i e s . Gist and Mott (1958) have shown t h a t r o o t c o m p e t i t i o n as a f a c t o r cannot be d i v o r c e d from f o l i a g e c o m p e t i t i o n . They r e p o r t e d t h a t the l a r g e root growth which i s c h a r a c t e r i s t i c of a l f a l f a , f o r example, does not occur at low l i g h t i n t e n s i -t i e s . Hence the drouth t o l e r e n c e a t t r i b u t e d to a l f a l f a i s the r e s u l t of an e x t e n s i v e r o o t system which develops o n l y 71. when the s e e d l i n g s r e c e i v e adequate l i g h t . Shading of a l f a l f a p l a n t s may thus r e s u l t i n d i r e c t l y i n death of the p l a n t s due t o t h e i r i n a b i l i t y to o b t a i n adequate water d u r i n g drouth p e r i o d s . Donald (1958) a t the U n i v e r s i t y of A d e l a i d e , working w i t h Lolium perenne, an a g g r e s s i v e s p e c i e s , and P h a l a r i s  tuberosa, a non-aggressive s p e c i e s , demonstrates t h a t compe-t i t i o n f o r even a s i n g l e f a c t o r i n v o l v e s i n t e r a c t i o n between d i r e c t and i n d i r e c t e f f e c t s ; when com p e t i t i o n occurs f o r two f a c t o r s , i t l e a d s to m u l t i p l e i n t e r a c t i o n s between two groups of e f f e c t s , and thus g r e a t l y i n t e n s i f i e s the e f f e c t s o f com-p e t i t i o n f o r other f a c t o r s o p e r a t i n g a l o n e . The aggressor s p e c i e s , r y e g r a s s , s u f f e r e d s l i g h t e f f e c t s o f com p e t i t i o n e i t h e r f o r l i g h t or f o r n u t r i e n t s . When both means of com-p e t i t i o n were o p e r a t i v e , the ryegrass was so markedly success-f u l , t h a t i t was able to m a i n t a i n a y i e l d not s i g n i f i c a n t l y d i f f e r e n t from t h a t i n pure c u l t u r e - a negative i n t e r a c t i o n . The P h a l a r i s was depressed by co m p e t i t i o n e i t h e r f o r l i g h t or f o r n u t r i e n t s . When both f a c t o r s were o p e r a t i v e , i t was depressed to a degree exceeding the separate e f f e c t s o f the two f a c t o r s - a p o s i t i v e i n t e r a c t i o n . Donald and Bl a c k (1959), i n t h e i r d i s c u s s i o n on the s i g -n i f i c a n c e o f l e a f a r e a , have s t a t e d t h a t the s i g n i f i c a n c e of l i g h t i s more apparent i n the c o o l temperate r e g i o n s , and that there are many r e g i o n s of the worl d i n which t h i s f a c t o r i s of l i t t l e s i g n i f i c a n c e , more e s p e c i a l l y the a r i d r e g i o n s . Stands o f forage legumes and grasses obtained i n the a r i d 72. r e g i o n s , where moisture i s the c h i e f l i m i t i n g f a c t o r , i s a s s o c i a t e d w i t h depth of seeding, seeding r a t e s and r o o t development. As mentioned be f o r e Hughes e t a l (1953) have p o i n t e d out t h a t , whereas p r a c t i c a l l y a l l the s m a l l seeded forage crops are best seeded at to s " depth or l e s s i n the humid areas, i n the dry c l i m a t e s and on sandy s o i l s s m a l l seeds must be sown deeper i n order to reach m o i s t u r e . Shallow-er seedings under the l a t t e r c o n d i t i o n s may germinate, but may d i e b e f o r e the s e e d l i n g s r o o t enough to become e s t a b l i s h e d . I t has l o n g been e s t a b l i s h e d t h a t l i g h t seeding r a t e s under dry l a n d c o n d i t i o n s enable germinating s e e d l i n g s to compete b e t t e r f o r the l i m i t e d moisture t h a t may be a v a i l a b l e . E x e m p l i f y i n g the r e p o r t s o f p r e v i o u s workers i n c l u d i n g Hughes and h i s a s s o c i a t e s , i s the work of Mueggler and B l a i s d e l l (1955), who demonstrated w i t h c r e s t e d wheatgrass a t Utah F o r e s t S e r v i c e Range S t a t i o n , t h a t i n as much as l i g h t seedings r e q u i r e comparatively l o n g e r p e r i o d s of p r o-t e c t i o n , complete stands were e v e n t u a l l y o b t a i n e d . Subsurface moisture t h e r e f o r e , i s a v e r y important item i n forage stands o f the a r i d r e g i o n s . The e l a b o r a t i o n of e x t e n s i v e r o o t systems i s a c h a r a c t e r i s t i c phenomenon enhanc-i n g t h e i r s u r v i v a l . There seems to be v e r y l i t t l e i n f o r m a t i o n a v a i l a b l e i n connection w i t h r o o t c o m p e t i t i o n o f forage crops, as has been o u t l i n e d i n the main t e x t o f t h i s p r e s e n t a t i o n . EXPERIMENTATION As mentioned b e f o r e , i t was hoped t h a t many f a c e t s of the 73. problem o f stand establishment of f o r a g e crops wou&d be i n -v e s t i g a t e d . There was a change i n programme, however, when on l y a few phases of t h i s study were i n i t i a t e d . None o f these s t u d i e s had yet been pursued to a f i n a l c o n c l u s i o n , but one or two have y i e l d e d c e r t a i n i n t e r e s t i n g o b s e r v a t i o n s , which are r e p o r t e d h e r e i n . S t u d i e d i n t h i s p r e l i m i n a r y manner were (a) l i g h t seeding versus heavy seedi n g of forage grasses and legumes, (b) r o o t development o f c e r t a i n s p e c i e s i n h i g h moisture c o n d i t i o n s and (c) competition of species: growing t o g e t h e r . A. L i g h t s e e d i n g versus heavy seeding of forage grasses and  legumes. M a t e r i a l s and methods. Used i n t h i s t r i a l , were f o u r grasses ( c o l o n i a l b e n tgrass, o r c h a r d g r a s s , r e d fescue, p e r e n n i a l r y e g r a s s and Kentucky b l u e g r a s s ) and two legumes (red c l o v e r and white c l o v e r ) . In the f a l l of 1958 each of these s p e c i e s was seeded i n p o t s 6" i n diameter, f i l l e d w i t h greenhouse s o i l and set up i n the U.B.C. greenhouse. Seeding r a t e s used were 30 pounds and 300 pounds per acre ( i . e . 0.062 grams and 0.62 grams per p o t ) . For each seeding r a t e there were two p o t s of each s p e c i e s . Depth of se e d i n g f o r a l l s p e c i e s were -|". Before seeding, the weighed samples f o r each pot were counted. T h i s was done wi t h the a i d of seed counters a t the P r o v i n c i a l P l a n t Products L a b o r a t o r y i n Vancouver. The average of the d u p l i c a t e samples of each seeding r a t e and s p e c i e s was taken as the amount a c t u a l l y seeded i n each pot. 74. A f t e r seeding, the pots were s u p p l i e d w i t h adequate moisture, and the times of emergence recorded. A f t e r 3 weeks, when emergence was complete (except f o r the b l u e g r a s s and bent-g r a s s ) , the s e e d l i n g s i n each pot were connted once a week. These counts were reco r d e d as percentages of the t h e o r e t i c a l number of seeds p l a n t e d . Observations and r e s u l t s . The r e s u l t s are recorded i n t a b l e IV. Although t h i s t r i a l may be regarded as s u p e r f i c i a l , c e r t a i n o b s e r v a t i o n s were q u i t e apparent. Emergence from the h e a v i e r s e e d i n g r a t e took p l a c e from 24 to 72 hours before the l i g h t e r r a t e . The e a r l i e r emergence was most apparent i n the legumes the time being 48 hours. In the bentgrass, the h e a v i e r seeding emerged 72 hours before the l i g h t e r seeding, but germination on the whole was v e r y slow, and was not completed u n t i l 4 weeks. I t was observed t h a t l a r g e numbers of s e e d l i n g s were l o s t i n the e a r l y days a f t e r emergence i n the h e a v i e r seedings, through heaving of the s o i l a t emergence. This was e s p e c i a l l y t r u e i n the case of r e d c l o v e r and white c l o v e r , and the heaving i s i l l u s t r a t e d c l e a r l y i n p l a t e s X I I I , XIV and XV. I t would have been i n t e r e s t i n g to compare these o b s e r v a t i o n s w i t h a s i m i l a r t r i a l under c o n d i t i o n s o f l i m i t e d moisture, had t h i s programme been continued. B. Root development under h i g h moisture c o n d i t i o n s . The t r i a l s f o r r o o t development are e s s e n t i a l l y the same as d e s c r i b e d f o r the growing of o r c h a r d g r a s s , c o l o n i a l bent-grass and red c l o v e r i n g l a s s - f a c e d tanks (see page 25). I t TABLE IV STANDS OF FORAGE SEEDLINGS FROM HEAVY AND LIGHT SEEDINGS IN 6" DIAMETER POTS Species Seeding r a t e s No. of seeds Emergence Emergence (per cent) pounds/acre per pot time (dys.) 3 ws. 4..ws. 5 ws. 6 ws. Red c l o v e r 300 312 5 100 91 85 80 30 31 7 84 74 74 74 White c l o v e r 300 986 6 85 82 71 68 30 100 8 73 67 64 63 Orchardgrass 300 590 10 89 80 80 80 30 59 11 74 73 73 73 Ryegrass 300 280 9 100 100 100 100 ( P e r e n n i a l ) 30 26 10 96 96 96 96 Red fescue 300 430 11 92 92 92 92 30 43 11 97 86 86 86 Bluegrass 300 3000 13 am 21 20 20 (Kentucky) 30 300 13 - 56 56 56 Bentgrass 300 8000 14 5 4 4 ( C o l o n i a l ) 30 792 17 — 3 3 3 dys - days ws - weeks 76. was hoped, i r t h i s programme was continued, that the t r i a l would be r e p l i c a t e d under c o n d i t i o n s of low moisture, h i g h f e r t i l i t y and low f e r t i l i t y . C. Competition of s p e c i e s growing together. This t r i a l was designed to study the s u r v i v a l of s p e c i e s , growing together i n groups of two, and under three s e t s of c o n d i t i o n s , i . e . when c o n s t a n t l y c l i p p e d to c l i p p e d to l-§-" and u n d i p p e d . I t was thought t h a t a study of t h i s k i n d , might c o n t r i b u t e towards e x p l a i n i n g why some s p e c i e s get 'crowded out' or even disappear from a given sward, as i s the case i n many forage growing ar e a s . The p a i r s of s p e c i e s chosen were ( i ) c o l o n i a l bentgrass and r e d fescue, ( i i ) Kentucky b l u e g r a s s and red fescue, ( i i i ) Kentucky b l u e g r a s s and c o l o n i a l bentgrass, ( i v ) r e d c l o v e r and orch a r d g r a s s and (v) white c l o v e r and Kentucky b l u e g r a s s . Wooden f l a t s , 12"xl8", f i l l e d w i t h greenhouse s o i l were d i v i d e d i n three by t h i n wooden s t r i p s (see p l a t e XVI) so t h a t each s e c t i o n was 12"x6". Each f l a t was seeded to a p a i r of s p e c i e s , a t the r a t e of 100 pounds per acre (50-50 by weight of each s p e c i e s ) . A r e p l i c a t e of each was a l s o set up. Adequate moisture f o r germination and growth was p r o v i d e d . I t was intended t h a t i n each f l a t , one s e c t i o n would be c l i p p e d to •§-", one to l i - " and the other l e f t u n d i p p e d , when the s e e d l i n g s were e s t a b l i s h e d . The t r i a l , however, was not completed before the programme change was made. An i n t e r e s t -i n g o b s e r v a t i o n was made, n e v e r t h e l e s s , and t h i s was apparent 77. at the time of emergence of s e e d l i n g s . As i n a p r e v i o u s t r i a l , there was heaving of the s o i l under t h i s comparatively-heavy seeding. This r e s u l t e d i n the death of most of the white c l o v e r s e e d l i n g s i n the white c l o v e r - Kentucky b l u e -grass mixture. Examples of t h i s phenomenon are i l l u s t r a t e d i n p l a t e s XVI and XVII. SUMMARY In an e f f o r t to o f f e r some e x p l a n a t i o n s f o r the frequent stand f a i l u r e s of forage grasses and legumes, a few t r i a l s were i n i t i a t e d . None of these were pursued t o a f i n a l c o n c l u s i o n due to a change i n programme, but c e r t a i n o b s e r v a t i o n s were made by t h a t time, and are r e p o r t e d h e r e i n . Forage grasses and legumes, grown i n pots a t heavy seed-i n g r a t e s were found t o emerge from 24 to 72 hours e a r l i e r than a t l i g h t seeding r a t e s . Loss of s e e d l i n g s due t o heaving was apparent i n the heavy seedings. Root development under high moisture was d i s c u s s e d p r e v i o u s l y (see page 25). Competition of s p e c i e s growing t o g e t h e r i n f l a t s , was not c a r r i e d out to any e x t e n t . Loss of white c l o v e r s e e d l i n g s from a white c l o v e r - Kentucky b l u e g r a s s mixture, due t o heaving of the s o i l , was n e v e r t h e l e s s evident a t the time of emergence. 78. LITERATURE CITED 1. Black, J. 1956. N. 2. Black, J . N. 1955. The i n f l u e n c e of seed s i z e and depth o f sowing on the pre-emergence and early-v e g e t a t i v e growth of subterranean c l o v e r ( T r i f o l i u m subterraneum) Aus. Jour. A g r i c . Res. 7:98-109. The i n f l u e n c e of depth of sowing and temperature on pre-emergence weight changes i n subterranean c l o v e r ( T r i f o l i u m  subterraneum). Aus. Jour. A g r i c . Res. 6 :203-211. 3. B l a s e r , R, E., Timothy T a y l o r , Walter G r i f f e t h & W i l l i s S k r d l a . 1956. S e e d l i n g c o m p e t i t i o n i n e s t a b l i s h i n g forage p l a n t s . Agron. Jour. 48:1-6. 4. Donald, C. 1958. 5. Donald, C, 1958. 6. Fayemi, A. A. 1957. & J. N. Black. The s i g n i f i c a n c e of l e a f area i n p a s t u r e growth. Herbage A b s t r a c t s 28:Review a r t i c l e . The i n t e r a c t i o n of c o m p e t i t i o n f o r l i g h t and f o r n u t r i e n t s . Aus. Jour. A g r i c . Res. 9 :421-435. E f f e c t of temperature on the r a t e of seed s w e l l i n g and germination of legume seeds. Agron. Jour. 49:75-76. 7. G i s t , G. 1957. R. & G. 0. Mott. Some e f f e c t s of l i g h t i n t e n s i t y , temperature, and s o i l moisture on the growth of a l f a l f a , red c l o v e r and b i r d s f o o t t r e f o i l s e e d l i n g s . Agron. Jour. 49:33-36. 8. Hughes, H. D., M. E. Heath & D. S. M e t c a l f e . 1953. Forages. Rev. Ed. 724 pp. Iowa State C o l . Press pp. 431-447. 9. Laude, H. 19 56. M. The s e e d l i n g emergence of grasses as a f f e c t e d by low temperature. Agron. Jour. 48:558-560. 79. 10. Lawrence, T. 1957. The emergence of Intermediate wheatgrass l i n e s from f i v e depths of seeding. Can. Jour. P l a n t Science 37:215-219. 11. Ludwig, J . W. & J . L. Harper. 1958(a). The i n f l u e n c e of the environment on seed and s e e d l i n g m o r t a l i t y . V I I Depth o f sowing of maize. P l a n t & S o i l X:37-48. 12. Ludwig, J . W. & J. L. Harper. 1958(b). The i n f l u e n c e of the environment of seed and s e e d l i n g m o r t a l i t y . V I I I The i n f l u e n c e o f s o i l c o l o u r . Jour. E c o l o g y 46:381-389. 13. Mueggler, Walter F. and James P. B l a i s d e l l . 1955. E f f e c t of seeding r a t e upon es t a b l i s h m e n t and y i e l d o f c r e s t e d wheatgrass. Jour. Range Management 8:74-76. 14. Rogler, G. A. 1954. Seed s i z e and s e e d l i n g v i g o u r i n c r e s t e d wheatgrass. Agron. Jour. 46:216-220. 15. Troughton, A r t h u r . 1954. The growth of a grass s e e d l i n g . Jour. A g r i c . Soc. - Univ. C o l . of Wales V o l . XXXV. 8 0 . APPENDIX B 81, PLATE I G l a s s - f a c e d tank used f o r r o o t study (panel open) o o PLATE I I G l a s s - f a c e d tank used f o r root study (panel closed) 82. • C A U T I O N ^ SEP • 5 9 PLATE IV E n c l o s u r e f o r P 3 2 s t u d i e s 8 3 . 5 9 PLATE V Foot square p l o t (foreground) PLATE VI rrometers f o r determining i r r i g a t i o n needs 84. SEP • 59 PLATE 711 P o r t a b l e stands h o l d i n g b u r e t t e s f o r p l a c i n g P 3 S SEP • 59 PLATE V I I I The Geiger-Muller counter ( f i e l d u n i t ) 8 5 . PLATE IX The Geiger-Muller counter ( f i e l d u n i t ) SEP • 5 9 PLATE X Some instruments used i n h a n d l i n g ' r a d i o a c t i v e m a t e r i a l ' 86. S E P • 59 PLATE XI Orchardgrass row with l i t h i u m placement PLATE X I I The Perkin-Elmer Flame Photometer 87. Emergence o f white c l o v e r from heavy seeding ( l e f t ) and l i g h t seeding ( r i g h t ) 8 8 . PLATE XV Emergence of p e r e n n i a l r y e g r a s s from heavy seeding ( l e f t ) and l i g h t seeding ( r i g h t ) PLATE XVI S o i l heaving i n seeded f l a t 

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