UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on the morphology and chemical composition of the roots of several annual and perennial grasses Pongskool, Virote 1962

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1962_A4 P6 S7.pdf [ 4.46MB ]
Metadata
JSON: 831-1.0105718.json
JSON-LD: 831-1.0105718-ld.json
RDF/XML (Pretty): 831-1.0105718-rdf.xml
RDF/JSON: 831-1.0105718-rdf.json
Turtle: 831-1.0105718-turtle.txt
N-Triples: 831-1.0105718-rdf-ntriples.txt
Original Record: 831-1.0105718-source.json
Full Text
831-1.0105718-fulltext.txt
Citation
831-1.0105718.ris

Full Text

STUDIES ON THE MORPHOLOGY AND CHEMICAL COMPOSITION OF THE ROOTS OF SEVERAL ANNUAL AND PERENNIAL GRASSES by VIROTE PONGSKOOL B. S. A., Kasetsart U n i v e r s i t y , Bangkok, Thailand, 1957. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n the Department of P l a n t Science We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1962 In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia, Vancouver 8 , Canada. ABSTRACT Root and top development of an annual grass, b a r l e y (Hordeum sativum), and of three v a r i e t i e s of p e r e n n i a l grass, orchard grass ( D a c t y l i s glomerata) i s f o l l o w e d during the f i r s t year from seeding. Reserve carbohydrate and l i g n i n l e v e l s f o r both root and top at s e v e r a l dates are given. A t t e n t i o n i s a l s o d i r e c t e d towards the development of techniques f o r root study, and to root i n t e r a c t i o n s of grass and legume seedlings i n pure and mixed stands. ACKNOWLEDGMENTS I wish to take t h i s opportunity to express my indebted-ness t o those who introduced me t o Canada and who guided me i n my p e r i o d of study. I would l i k e to extend s p e c i a l thanks to Dr. Vernon C. Br i n k , Professor of Agronomy and Chairman of the D i v i s i o n of Plant Science, U n i v e r s i t y of B r i t i s h Columbia, under whose su 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 d i r e c t i o n and as s i s t a n c e i n conducting and preparing 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 u s e f u l and pleasant p e r i o d of study. I a l s o thank f o r t h e i r co-operation, Mr. Don Pearce, senior t e c h n i c i a n , Mr. Jan B r u s s e l , former l a b o r a t o r y t e c h n i c i a n i n the D i v i s i o n of Plant Science, and Mrs. Agnes B e l l , Miss Lyn J o l l y , and Mrs. Suzanne L o r e n c z i , S e c r e t a r i e s . I would l i k e t o express my deepest personal g r a t i t u d e to Mr. Roderick C. B a i l e y , of the 4-H Club Branch, the Department of A g r i c u l t u r e , B r i t i s h Columbia, and my former colleague at the Thailand-UNESCO Fundamental Education Centre, Ubon, Thailand, and t o h i s f a m i l y whose r e a l i n t e r e s t made i t p o s s i b l e f o r me to come to study i n Canada under the E x t e r n a l A i d Programme (Colombo P l a n ) . G r a t i t u d e i s a l s o expressed t o my Government f o r gr a n t i n g me leave of absence. G r a t e f u l acknowledgment i s a l s o made to the Government of Canada f o r the f i n a n c i a l a i d . TABLE OF CONTENTS INTRODUCTION 1 REVIEW OP LITERATURE 2 Root Study Techniques 2 Seasonal r o o t development and l e n g t h of l i f e of r o o t s 4 Root i n t e r a c t i o n of crops growing i n a s s o c i a t i o n 7 Chemical composition of r o o t s 8 EXPERIMENTATION P r o j e c t 1 - D i r e c t o b s e r v a t i o n i n s o i l of growing r o o t s 12 P r o j e c t 2 - D i r e c t o b s e r v a t i o n of p o s s i b l e i n t e r -a c t i o n s of grass and legume r o o t s i n s i t u 16 P r o j e c t 3 - The development of a r o o t washing machine 20 P r o j e c t 4 - Comparative r o o t development of an annual grass ( b a r l e y Hordeum sativum) and a p e r e n n i a l grass (orchard grass D a c t y l i s glomerata) from seed 24 P r o j e c t 5 - Time of onset of s e e d l i n g competition i n forage grass stands 33 P r o j e c t 6 - Seasonal trends In l i g n i n and r e s e r v e carbohydrate i n the r o o t s and tops of b a r l e y 4l P r o j e c t 7 - Seasonal trends i n l i g n i n and r e s e r v e carbohydrate i n the r o o t s and tops of a f i r s t year stands of three orchard grass v a r i e t i e s 4-9 GENERAL DISCUSSION AND SUMMARY 59 APPENDIX 63 LITERATURE CITED 69 STUDIES ON THE MORPHOLOGY AND CHEMICAL COMPOSITION OP THE ROOTS OP SEVERAL ANNUAL AND PERENNIAL GRASSES INTRODUCTION Much i s known about the a e r i a l p a r t s of crops, and t h e i r morphology and physiology has been the subject of innumerable s t u d i e s . By con t r a s t the studi e s of the roots of crops are few. The reason f o r the p a u c i t y of work on roots appears to stem l a r g e l y from the labouriousness of current root study techniques and from the f a c t that i n t a c t root systems are obtained only under very s p e c i a l and a t y p i c a l c o n d i t i o n s . I t i s apparent, however, that a greater knowledge of the whole p l a n t , tops and r o o t , would be h i g h l y d e s i r a b l e i n the s o l u t i o n of many kinds of problems i n crop production g e n e r a l l y and, more s p e c i f i c a l l y , i n forage and t u r f production. Good studi e s on root-top i n t e r a c t i o n s , on root decomposition i n s o i l w i t h age, on root chemistry, on root competition and even on root morphology are comparatively few. In t h i s study, work on roots i n i t i a t e d by a former student, Mr. Rex F r e d e r i c k ( 1 9 5 9 ) , i s continued. Examination and development of techniques f o r root study are continued and a beginning i s made i n the study of root chemistry and root/top i n t e r a c t i o n s . A d d i t i o n a l l y , a comparison i s made of r o o t development i n an annual and p e r e n n i a l grass. 2 REVIEW OF LITERATURE The l i t e r a t u r e on the r o o t s of grasses has been the subject of a recent, e x c e l l e n t review by Troughton (1957), of the Welsh Plant Breeding S t a t i o n , Aberystwyth, Wales. Inasmuch as t h i s covers much of the l i t e r a t u r e r e l a t i v e t o the f i e l d , l a b o r a t o r y , and greenhouse stud i e s l a t e r described, the l i t e r a t u r e surveyed i n t h i s t h e s i s i s l i m i t e d . No t r u l y comprehensive review on the e v o l u t i o n , morphology and physiology of root s f o r the p l a n t kingdom appears t o have been made. A p i c t o r i a l p r e s e n t a t i o n of root morphology In higher crop p l a n t s was developed by Weaver (1926), a f t e r p a i n s t a k i n g excavation s t u d i e s ; these stu d i e s were extended by Canadian, German and Russian workers (e.g. Pavlychenko, 1937, 1942). Evans (1958) has stud i e d , i n considerable d e t a i l , the development of the timothy root system and Cormack (194-9) has reviewed knowledge of the root h a i r and i s c u r r e n t l y engaged i n a more up-to-date p r e s e n t a t i o n . Root study techniques Since t h i s study grew out of an e a r l i e r study by Fr e d e r i c k (1959) and, inasmuch as he reviewed i n some d e t a i l techniques used i n root study to 1959, there i s l i t t l e p o i n t i n r e p e a t i n g h i s review. The most s i g n i f i c a n t a d d i t i o n to root study during the l a s t three years appears to be that 3 of Burton (1962) who has used an "exhaustion of root reserves" i n darkness or " e t i o l a t i o n " technique to determine the " e f f e c t i v e " root development i n t u r f . Turf plugs are cut, a e r i a l growth i s trimmed to the s o i l surface, and the plugs are placed i n darkness under c o n t r o l l e d temperature. A e r i a l growth produced i s p e r i o d i c a l l y c l i p p e d , d r i e d and weighed. Dry matter produced during the dark p e r i o d i s an index of the f u n c t i o n i n g root and food reserves. The method has much to recommend i t from the viewpoint of convenience, and from the f a c t that i n older t u r f i t i s w e l l - n i g h impossible to d i s t i n g u i s h between l i v i n g and recently-dead r o o t s . Obviously, the method can co n t r i b u t e l i t t l e to the study of the physio-logy and morphology of root s i n s i t u . There appears to be a heightened i n t e r e s t i n root study techniques i n the l a s t two years, but most progress seems to be concerned w i t h m o d i f i c a t i o n s of the gl a s s or p l a s t i c window techniques described by F r e d e r i c k (1959)• Also new developments i n root-washing machines ( W i l l i a m and Baker, 1957), ( F r i b o u r g 1953) have r e s u l t e d i n great savings of labour i n root study where s o i l c o n d i t i o n s favour t h e i r use and seems to make l a r g e - s c a l e examination of root s of t u r f p o s s i b l e . I t would appear that a d d i t i o n a l methods f o r root washing by machine may yet be developed but a l l are l i k e l y to s u f f e r from the f a c t that very f i n e r o o t s are not recovered and from the f a c t that dead r o o t s , or i n a c t i v e r o o t s , are not r e a d i l y 4 d i s t i n g u i s h e d from l i v i n g and f u n c t i o n i n g r o o t s . Root washing, too, i s of l i m i t e d value i n s o i l s where there i s much coarse organic matter i n t o which f i n e roots are p e n e t r a t i n g . Seasonal root development and le n g t h of l i f e of root s According t o Troughton (1957) during the f i r s t few months of a p l a n t ' s l i f e , i f i t i s i n a s u i t a b l e environment, the root system grows very r a p i d l y , the growth r a t e i n c r e a s i n g from zero j u s t before germination to a maximum some weeks l a t e r , a f t e r which I t decreases. The growth of the roots c o n s i s t s of an increase i n t h e i r number, weight, and len g t h , and v a r i e s considerably between species. Remarkably few root s t u d i e s begin w i t h the s e e d l i n g development, and even fewer are comparative between species, or consider root and top development co n c u r r e n t l y . Plummer (1943) and others have concluded that root development during the f i r s t month or two l a r g e l y determined the i n d i v i d u a l grasses' success or f a i l u r e . Species which developed r o o t s r a p i d l y e s t a b l i s h e d stands much more r e a d i l y than those whose root systems developed slowly. There seems to be general agreement w i t h Keim and Beadle (1927) and wi t h Simpson and Moore (1955), and Robocker, C u r t i s and Ahlgren (1953) that root depth and weight i n young grasses i s simply r e l a t e d to co l d and drought t o l e r a n c e . Although Kauter (1933) a ^ d Sprague (1933) found no co n s i s t e n t trends 5 i n the d i s t r i b u t i o n of dry matter between tops and roots during the e a r l y months of a grass p l a n t ' s l i f e , Troughton (1956) s t a t e d that the r e l a t i o n s h i p between roots and shoots 1 weight i n a steady sta t e environment can be expressed by the k a l l o m e t r i c formula. The formula could be w r i t t e n y = bx when y represents the weight of r o o t s and x the weight of the shoots, b a constant equal to the value of y when the value of x was u n i t y and to a constant equal to the r a t i o : l o g a r i t h m i c growth r a t e of y (roots) l o g a r i t h m i c growth r a t e of x (shoots). The values of x and y are obtained by weighing p l a n t s at i n t e r v a l s over a p e r i o d of time. The values of b and k were then obtained by c a l c u l a t i o n or g r a p h i c a l means. When the r e l a t i o n s h i p between the two s e r i e s of values can be expressed by t h i s formula, then the p o i n t s produced on a graph by p l o t t i n g t h e i r l o g a r i t h m i c values against each other w i l l be along a s t r a i g h t l i n e . The formula can be used only w i t h p l a n t s measured on at l e a s t three occasions i n a season. T i l l e r production i n young grass no doubt would complicate measurements f o r s t o l o n s and rhizomes although often non-photosynthetic and often below ground would have to be c l a s s e d as stem. The subject of many studi e s has been the time of root and shoot i n i t i a t i o n and growth. In temperate l a t i t u d e s w i t h p e r e n n i a l grasses most root growth seems to take place i n s p r i n g (Stuckey 19^1) , but some roots are i n i t i a t e d almost every month of the year i f the s o i l i s not frozen or droughty. I t i s apparent that a good deal of root growth occurs during the w i n t e r months i n forage species i n unfrozen s o i l when no a e r i a l growth occurs (Jacques and Schwass 1956)• Commonly, however, root growth diminishes as a e r i a l growth i s produced i n s p r i n g and may cease a l t o g e t h e r by f l o w e r i n g time and during seed maturation. Again, under c o o l , temperate c o n d i t i o n s , Goedewaagen and Schuurman (1950) and Troughton (1951) concluded that the annual death and decay of roots of forage p l a n t s s t a r t e d i n May and continued i n t o mid-winter. D e f o l i a t i o n of the r o o t s of forage grasses, e s p e c i a l l y when more than k0% of the photosynthetic surface i s removed, r e s u l t s , almost immediately, i n the c e s s a t i o n of root growth as C r i d e r (1955) has observed. This i s the case w i t h n e a r l y a l l common temperate forage grasses except orchard grass where i t i s a second d e f o l i a t i o n , soon a f t e r the f i r s t , which seems to r e s u l t i n root growth stoppage and, a d d i t i o n a l l y , death. Although t h i s simple r e l a t i o n s h i p between top removal and root growth stoppage e x i s t s , the broader i n t e r - r e l a t i o n s h i p between tops and root development seems complex: no worker has reported, f o r example, that the maximum growth of root s and shoots i s ever c o i n c i d e n t a l . 7 Root i n t e r a c t i o n of crops growing i n a s s o c i a t i o n Competition occurs between p l a n t s f o r "elements" i n the environment, and of these, most s t r i k i n g l y f o r water, l i g h t and c e r t a i n mineral elements. I t i s often stated that competition i s more intense between species of the same growth form than between those which are d i s s i m i l a r (Weaver and Clements, 1938): t h i s competition between smooth brome grass, a rhizomatous grass and a l f a l f a , a tap-rooted legume, i s not as intense as between Kentucky bluegrass, another sod grass, and smooth brome. Within c e r t a i n l i m i t s the greater the d e n s i t y of a p l a n t p o p u l a t i o n the smaller should be the i n d i v i d u a l p l a n t and i n general the weights of r o o t s per p l a n t should vary i n v e r s e l y w i t h the r a t e of seeding and stand d e n s i t y ; (de P e r a l t a 1935, Nedrow 1937,and o t h e r s ) . The l i t e r a t u r e on root competition, however, i s q u i t e l i m i t e d and conclusions l a c k d e f i n i t i o n . Very few s t u d i e s , f o r example, consider root development i n r e l a t i o n to s o i l volume, s o i l area, or pore space. Nor have many st u d i e s been attempted to c l a s s i f y the major element i n the competition system such as l i g h t , or water or n u t r i e n t s . Ahlgren and Aamodt (1939) i n a study of root systems of four common cool temperate forage grasses grown i n mixed and pure stands could not f i n d any c o n s i s t e n t trends i n r e l a t i o n s h i p s between ro o t s and shoots, but d i d a t t r i b u t e some v a r i a t i o n s to harmful root i n t e r a c t i o n s . Aberg, Johnson and W i l s i e (194-3) i n very s i m i l a r experiments found no evidence of e i t h e r mutually b e n e f i c i a l or a n t a g o n i s t i c a s s o c i a t i o n s ; responses were merely of the "compensating" type. Troughton (1956) d i d , however, i n a c a r e f u l study f i n d root i n t e r a c t i o n s between p e r e n n i a l ryegrass and timothy. The root i n t e r a c t i o n s of grass and legume growing i n a s s o c i a t i o n i s undoubtedly more complex than those of grass and grass because of the added involvement of nit r o g e n f i x a t i o n i n the root nodules of the legume. Again a modest l i t e r a t u r e y i e l d s i n c o n c l u s i v e statements: Russian workers (Duhanin 1940), Ivanov (1950), Schwendiman, H a f e n r i c h t e r and Law (1953), and Roberts and Olson (194-2) found an increase i n root production per u n i t area when grass and legume were grown i n a s s o c i a t i o n . Troughton (1956) found that one p l a n t of white c l o v e r grown w i t h two to s i x p l a n t s of timothy or p e r e n n i a l ryegrass d i d not a f f e c t the growth of the grass r o o t s i n twenty-two weeks' growth from seed. On the other hand, Jacques (1943) and a few others have recorded smaller root weights f o r grasses grown i n the presence of a legume. Chemical composition of root s P l a n t p h y s i o l o g i s t s and p l a n t chemists have long been i n t e r e s t e d i n "reserve" m a t e r i a l s as opposed to " s t r u c t u r a l " m a t e r i a l s . There has been, moreover, a long-standing view that reserves are depleted during periods of r a p i d forage growth, during w i n t er as root s are extended, and f o l l o w i n g d e f o l i a t i o n . Seasonal v a r i a t i o n has been noted i n the l e v e l s of nitrogenous exudates, and mineral c o n s t i t u e n t s i n roots but, as Weinman (19U8) and Troughton (1957) p o i n t out, much c o r r e l a t i v e work i s needed before g e n e r a l i z a t i o n s can be made. C u r i o u s l y l i t t l e work on the " s t r u c t u r a l " c o n s t i t u e n t s of r o o t s e x i s t s i n the formal l i t e r a t u r e of p l a n t physiology but, inasmuch as the m i c r o b i o l o g i c a l degradation of these c o n s t i t u e n t s i n s o i l i s of considerable s c i e n t i f i c and t e c h n i c a l i n t e r e s t , the r e l e v a n t l i t e r a t u r e , viewed i n a broad sense i s l a r g e . L i g n i n , as one of the i n t e r e s t i n g s t r u c t u r a l components of r o o t s , i s the subject of exhaustive reviews of G o t t l i e b and Hendricks (19U5), and by Brauns and Brauns (i960). B o l l e n and Lee (1957) are of the view that the s t r u c t u r a l l i g n i n - c e l l u l o s e complex i s metabolized slowly by s o i l micro-organisms. Kholodnyi (1951) b e l i e v e s that s o i l l i g n i n , as i t decomposes, re l e a s e s root growth s t i m u l a t i n g substances but apparently he does not s p e c i f y whether or not these are deriv e d from root or shoot l i g n i n . ' Although Weinman (19^6) does not b e l i e v e that h e m i c e l l u l o s e normally should be considered as 'reserve 1 i n e t i o l a t e d Bromus c a r i n a t u s i t i s converted to simpler substances. A great deal of v a r i a t i o n a c t u a l l y e x i s t s i n the views of workers as t o the r e l a t i v e importance of the var i o u s carbohydrate f r a c t i o n s as "reserves" a c c o r d i n g l y there i s a good deal of v a r i a t i o n i n the chemical procedures used i n the determination of reserve carbohydrates. Weinman (19U6) and Dale Smith (1962) have commented b r i e f l y 10 on the procedures i n use. Because of t h e i r very probable importance i n the recovery of forage crops f o l l o w i n g g r a z i n g or mowing, i n competition w i t h weeds, i n winter and summer hardiness, seasonal accumulation of reserve carbohydrates has a t t r a c t e d a good deal of a t t e n t i o n . Despite a good deal of i n t e r e s t , however, the number of studie s are few, (McCarty 1938, Klapp 1937, Weinman 1948, Brown 1943). Most of the s t u d i e s r e l a t e to e s t a b l i s h e d p e r e n n i a l forage crops and, In general, i t seems that carbohydrate reserves are stored i n r o o t s during periods of slow herbage growth i n s p r i n g , summer, and, esp-e c i a l l y , e a r l y autumn, that the s t r u c t u r a l m a t e r i a l s are p o s s i b l y l e s s important and l e s s abundant i n r o o t s than i n tops, and tha t r o o t s are important storage organs f o r reserve substances. A c t u a l l y the chemistry of root s i s not very w e l l known, although doubtless these g e n e r a l i z a t i o n s are v a l i d . However, i t i s i n t e r e s t i n g to note that r e c e n t l y (Baker 1955, 16l) at the Grassland Research S t a t i o n , at Hurley, England, bas a l stems, leaves, and p l a n t crowns were found to be qu i t e as important f o r storage as root s i n c e r t a i n grasses, and that r o o t s functioned p r i m a r i l y i n the uptake of mineral n u t r i e n t s and water. Reserve substances i n grass roots are considered to be mainly carbohydrates, e.g. sugars, f r u c t o s o n s , d e x t r i n s , s t a r c h pentosans, h e m i c e l l u l o s e , and true c e l l u l o s e are considered to be l a r g e l y s t r u c t u r a l . (Weinman 1948). C e r t a i n l y p l a n t s l o s e , i n the production of tops i n darkness, carbohydrates of the former category and r e l a t i v e l y l i t t l e carbohydrate i n the l a t t e r category. Some workers (McCarty 1938) b e l i e v e t h a t , f o r a given species, root s t r u c t u r a l m a t e r i a l s d i f f e r g r e a t l y from those of the a e r i a l p a r t s , but s i n g u l a r l y l i t t l e chemistry has been undertaken which would e s t a b l i s h t h i s p o i n t . 12 EXPERIMENTATION P r o j e c t 1 - D i r e c t observation i n s o i l of growing r o o t s . a) Object To observe roots i n s o i l d i r e c t l y has been the goal of many s t u d i e s . Thus f a r a l l methods developed can be s e r i o u s l y c r i t i c i z e d , f o r a l l d i s t u r b the s o i l , or i n other ways create u n d e s i r a b l e changes i n the root environment or s a c r i f i c e the root or p a r t s of r o o t s . Glass or p l a s t i c windows have been t r i e d by a number of workers f o r the study of r o o t s i n p l a c e , ( C r i d e r 1955) (Lavin 196l) and others, and despite d e f i c i e n c i e s i n method, are u s e f u l f o r r a t e of root growth s t u d i e s , species i n t e r a c t i o n s t u d i e s , and so f o r t h . In our s t u d i e s g l a s s "wafers" were used which c a l l e d f o r a minimal s o i l volume, the p o i n t being that root responses would be more r a p i d l y perceived and that emanations from small q u a n t i t i e s of r a d i o t r a c e r s , dyes, e t c . would not be " l o s t " i n a l a r g e s o i l mass. b) Method 92" x 16 i n c h window-glass r e c t a n g l e s were held 1/4 i n c h apart i n twin-grooved wooden frames (see P i g . l ) . F i b e r g l a s s wool was placed to a depth of \ i n c h at the bottom of the lacuna so that s o i l could be held and f r e e movement of water permitted. Nicholson loam s o i l was a i r d r i e d , screened through an 8-mesh screen, and c a r e f u l l y fed i n t o the lacuna between the p l a t e s . The p l a t e s were tapped l i g h t l y to be assured that the 13 s o i l was compacted f a i r l y u n i f o r m l y . Seeds were plante d 3 per i n c h . The "wafers" were placed v e r t i c a l l y on a t h i n l a y e r of moistened v e r m i c u l i t e and covered each side w i t h black p l a s t i c sheets to present l i g h t from reaching the s o i l - g l a s s i n t e r f a c e . The s o i l was kept moist by watering every other day. c) Observations and d i s c u s s i o n The b l a c k p l a s t i c sheets were opened p e r i o d i c a l l y to examine the root systems of grass and c l o v e r . This technique allowed the worker to observe the root system from both sides of the gl a s s p l a t e s . Although the roo t s grown between the narrow p l a t e s probably act somewhat d i f f e r e n t l y from those i n nature there i s some value to t h i s method ( F r e d e r i c k 1958). Some feat u r e s of general morphology of the root systems can be seen and Lav i n (1961) has observed and measured the r a t e of root growth by a s i m i l a r technique. The root systems of p l a n t s s t u d i e d , v i z . , p e r e n n i a l ryegrass, by t h i s method were photographed ( F i g . l ) . The photos give a general idea of the roots-system-arrangement under the s o i l c o n d i t i o n s but p l a i n p o l a r i z e d l i g h t would be necessary t o obtain good records (Haas and Rogler 1953), P a r t r i d g e (l9ho), Schultz (1956), and Parmeijer (1956) photographed the root systems of various p l a n t s i n s i t u and 14 showed that the p i c t u r e s from multidimensional p r o f i l e study and those from in_ s i t u study were s i m i l a r . No attempt was made to measure the d a i l y r a t e of root growth. The roo t s of both red c l o v e r and ryegrass reached the bottom-most part of the "wafers" only three weeks a f t e r emergence. Fig. 1. - Roots of 10-day old perennial ryegrass plant showing at the between glass-soil interface. Note the roots do not necessarily follow the interface. 16 P r o j e c t 2. - D i r e c t observation of p o s s i b l e i n t e r a c t i o n s of grass and legume ro o t s in. s i t u . a) Object; 32 P as a r a d i o t r a c e r substance i n r o o t s was used f a i r l y s u c c e s s f u l l y by F r e d e r i c k (1959) and others ( H a l l e_t aJL 1953), (Burton e_b al_ 1954, 1957), (Boggle et_ al_ 1958, i960) i n the study of root d i s t r i b u t i o n of grasses i n the f i e l d . I t was hoped th a t the use of X-ray photos or r a d i o t r a c e r s w i t h more 32 energetic emanations than P could be used w i t h the " t h i n -wafer technique" described above. The p o s s i b i l i t y of usi n g r a d i o t r a c e r s such as r a d i o - i o d i n e could not be undertaken because of d i f f i c u l t i e s i n o b t a i n i n g land tenure guarantees, and hence the d i r e c t i o n of the work was a l t e r e d somewhat. Although the b e n e f i c i a l e f f e c t s of as s o c i a t e d growth of grass and legumes have been on record f o r a very long time and on a_ p r i o r i grounds, one must assume that important i n t e r -a c t i o n s between t h e i r r o o t s occur, the nature of the i n t e r a c t i o n s has never been c l e a r l y e s t a b l i s h e d . Undoubtedly i n death and decay of ro o t s of grass and legume, and in_ v i t r o , m a t e r i a l s such as amino a c i d s , C0 2 e t c . are r e l e a s e d which are important, but r o o t - i n i t i a t e d i n t e r a c t i o n s between grass and legume occur long before observable death and decay of r o o t s . In the hope that morphological rapprochements of grass and legume roots could be observed by the t h i n wafer technique, i n t e r p l a n t i n g s of grass and legume were undertaken. The technique and observ-a t i o n s are described below. b) Method The method was s i m i l a r to the "wafer" technique des-c r i b e d i n P r o j e c t 1. Red c l o v e r and p e r e n n i a l ryegrass seeds were placed a l t e r n a t e l y i n the wafers. c) Observations and d i s c u s s i o n The mixed root systems of c l o v e r and ryegrass were observed d a i l y . Roots of both p l a n t s reached the bottom of the wafer p l a t e s i n about three weeks and s t a r t e d to send the roots o u t s i d e . This means tha t the root system extended at about 1" per day or more. Weaver (1926) notes that root e l o n g a t i o n i n grass seedlings of common grasses of -§•" a day i s a good r a t e . The root system of both ryegrass and c l o v e r moved down side by side i n d i f f e r e n t l y . Though the seeds of c l o v e r germinated n e a r l y two days ahead they d i d not show much advantage i n elongation or i n occupying the s o i l volume. The observation p e r i o d was r e l a t i v e l y short but i t seemed that both root systems may grow side by s i d e . Because roots of grass could not r e a d i l y be d i s t i n g u i s h e d from r o o t s of legumes, observation was of n e c e s s i t y c a s u a l , but i t seems that no s p e c i f i c a t t r a c t i o n or r e p u l s i o n i s i n v o l v e d . However, t h i s does not n e c e s s a r i l y b e l i e the f i e l d observation by Haynes, et a l . (1956), that the root growth of corn tended to be i n the d i r e c t i o n of no root occupancy. The root system of grass d i d not appear to be more su p e r i o r i n "penetrating a b i l i t y " to legume. ( F i g . 2 ) . This opinion i s supported by the observations of Taylor (i960) (a and b ) , who a l s o f i n d s the p e n e t r a t i n g a b i l i t y of legume roo t s to be not s i g n i f i c a n t l y greater than those of non-legumes. Nevertheless, i n t e r a c t i o n may take place i n the v i c i n i t y of root h a i r s , i . e . i n the rhizosphere zones. Rosene and W a l t h a l l (1949) reported the v e l o c i t y of water absorption by i n d i v i d u a l root h a i r was not at the same r a t e ; legume r a t e s were higher than grass r a t e s . When the r a t e of absorption i s higher, n a t u r a l l y the amount of n u t r i e n t intake would be higher p r o v i d i n g that the amount .<3f) root h a i r was constant. Dittmer (1937, 1938, 1949, 1959, a and b) reported the t o t a l surface area of 4321.30 square f e e t of 14,335,568,288 root h a i r s of a matured s i n g l e winter rye p l a n t . However, there i s no rep o r t f o r a legume p l a n t on a s i m i l a r b a s i s . Fig. 2 - Root systems of red clover and perennial ryegrass, two weeks old, growing side by side. The primary roots of red clover are thicker than those of the perennial ryegrass at the same age. 20 P r o j e c t 3 - The development of a root washing machine. When i t was r e a l i z e d that work w i t h r a d i o t r a c e r s could not be continued, the l i t e r a t u r e r e l a t i v e to techniques used i n studying the roo t s of forage crops and sports t u r f by washing and/or excavation techniques was examined. The main techniques are simple enough, b a s i c a l l y , and c o n s i s t of (a) washing away s o i l from ro o t s or (b) p l o t t i n g roots as they appear i n successive p r o f i l e s , but dozens of m o d i f i c a t i o n s and combination of these occur and many v a r i a t i o n s In equip-ment used have been proposed. I t appeared, from the l i t e r a t u r e survey, that a root washing machine developed and used at the Grassland Research I n s t i t u t e , at Hurley, England, worked w e l l and would be g e n e r a l l y u s e f u l . A c c o r d i n g l y , a machine s i m i l a r to that used at the I n s t i t u t e was constructed. I t i s described below and comments on i t s usefulness are made. a) Method A machine f o r the root washing s i m i l a r to the one used by W i l l i a m s and Baker (1957), only d i f f e r e n t i n the d e t a i l s , was constructed. ( F i g . 3). The machine was mounted on the angle i r o n frame, I t co n s i s t e d of four washing t r a y s . A 1/4 H.P. e l e c t r i c motor was used to d r i v e the t r a y s by means of rubber b e l t s . The t r a y s turned at 60 r.p.m. Each t r a y was movable from the f u n n e l . The diameter of the t r a y was 12 Inches, the depth 6 inches, and 3-mesh galvanized screen Fig. 3 - General view of the root washing machine. 2 2 f o r the supports. 31-mesh brass screen was used on the bottom. Two faucets were f i x e d above each t r a y ; the i n t e n s i t y and flow of the streams of water could be regulated by the f a u c e t s . ( P i g . 4). b) Observations and d i s c u s s i o n The machine worked w e l l w i t h samples l i m i t e d i n s i z e . The s o i l - r o o t samples from the f i r s t harvest b a r l e y (to be r e f e r r e d to l a t e r ) were washed w i t h the machine a f t e r s e c t i o n s of s o i l had been crumbled. The machine could not handle the l a r g e r sample s i z e s e f f e c t i v e l y , i . e . samples which were l a r g e r than the t r a y i t s e l f . However, the machine i s g e n e r a l l y very e f f e c t i v e f o r root washing and one can support P r i b o u r g (1953) who found that a root washing machine was 18 times f a s t e r i n washing 100 root samples of Ladino c l o v e r than manual washing. I t was found that i f dead ro o t s or b i t s of wood, e t c . were mixed i n the s o i l - r o o t sample, the machine-washed samples were not clean and time had to be spent i n c u l l i n g . I t i s suggested that an "up and down" motion, (Pribourg 1953) seen i n old-type washing machines might be u t i l i z e d i n f u t u r e c o n s t r u c t i o n , i . e . a l t e r n a t e immersion and removal of the sample from a w e l l of water might wash more u n i f o r m l y and g e n t l y . Fig. k - View from top showing rotating containers and adjustable water jets. 24 P r o j e c t 4 - Comparative root development of an annual grass ( b a r l e y Hordeum sativum) and a p e r e n n i a l grass (orchard grass D a c t y l i s glomerata) from seed. a) Object Although i t i s g e n e r a l l y acknowledged that the root system produced by an annual i s much smaller than that produced by a p e r e n n i a l grass i n a given season, a search of the l i t e r a t u r e d i d not r e v e a l any t r i a l s where the two root systems had been produced under comparable c o n d i t i o n s . B a r l e y and orchard grass were seeded i n s p e c i a l boxes set i n the f i e l d at about the same time i n e a r l y summer. P e r i o d i c a l l y the root production was assessed by washing c e r t a i n r e p l i c a t e s f r e e of s o i l , d r y i n g , and weighing the product. b) Method Boxes were constructed of plywood 12" x 12" x 12" and 19-mesh nylon screen f i x e d on the bottoms. Nicholson s o i l was screened to remove f i n e r o o t s , o l d r o o t s , e t c . In a l l , 40 boxes were e s t a b l i s h e d to b a r l e y ; one-half were f e r t i l i z e d at the r a t e of 1000# per acre (15 g. per box) w i t h 10-20-10 mineral f e r t i l i z e r , and one-half were not f e r t i l i z e d . The f e r t i l i z e r was thoroughly mixed w i t h the s o i l p r i o r to adding the s o i l to the boxes. The boxes were c a r e f u l l y placed so that the rims were at general s o i l l e v e l and c a r e f u l l y f i l l e d w i t h f e r t i l i z e d or u n f e r t i l i z e d s o i l as the f i e l d design r e q u i r e d . ( F i g s . 5 and 6). 25 B a r l e y seed was sown on May 19, 1 9 6 1 , and when the p l a n t s were 1-|" high they were thinned to 4 per box. Water was l i g h t l y and u n i f o r m l y a p p l i e d on a l t e r n a t e days through-out the growing season. Three v a r i e t i e s of orchard grass, i . e . L a t a r , Danish, and S-143 v a r i e t i e s , were seeded i n 48 boxes f i l l e d w i t h s o i l f e r t i l i z e d at 1000# per acre, i n the same fa s h i o n as f o r b a r l e y . Because of time i n p r e p a r a t i o n and the l a t e a r r i v a l of the seed of the L a t a r v a r i e t y , seeding d i d not take place u n t i l June 14. At that time the seed was broadcast at the r a t e of 170 l b s . per acre (1.8 grams per box). Seeds were covered w i t h 1/4"- s o i l a f t e r seeding. B a r l e y roots and tops from four boxes were washed, d r i e d and weighed i n each of f i v e occasions during the grow-i n g season. Orchard grass roots and tops from four boxes were washed, d r i e d and weighed on each of four occasions during the growing season. c) Observations and r e s u l t s Both b a r l e y and orchard grass developed normally through the growing season and disease and i n s e c t damage were minimal. I t i s notable t h a t the top/root r a t i o s i n the annual grass, (barley) increase s t e a d i l y throughout the growing season but those of the p e r e n n i a l grass (orchard grass) remain remarkably constant through the growing season Fig. 5 - Barley plants in 12M x 12" x 12" boxes as seen in the field. Fig. 6 - Removing a barley container prior to harvesting tops and washing roots. 28 ( F i g s . 7 and 8 ) . T h i s o b s e r v a t i o n i s g e n e r a l l y i n t e r p r e t e d t o mean t h a t , although the r o o t s of annual grasses do grow through the growing season, minimal amounts of r e s e r v e •arbohydrates are t r a n s l o c a t e d to the r o o t s and most of these m a t e r i a l s tend to remain i n the a e r i a l p a r t s of p l a n t s c l o s e t o the r e g i o n s of t h e i r e l a b o r a t i o n . Annuals, i t has l o n g been r e c o g n i s e d , are poor c o n t r i b u t o r s to the organic matter l e v e l s of s o i l s . On the other hand, i n i t s f i r s t year of development, the orchard grass p l a n t c o n t r i b u t e s about one-half of t h e i r e l a b o r a t e d organic matter to the s o i l as r o o t . I t i s a l s o notable' t h a t the b a r l e y r o o t was dying and d i m i n i s h i n g i n weight by l a t e dough stage. By l a t e f a l l h a r v e s t , the y i e l d of orchard g r a s s , both i n top and r o o t , had w e l l exceeded those of the annual c e r e a l . ( F i g s . 9 and 10). 29 ^ F i g . 7 • - Top/root r a t i o s at s e v e r a l growth stages f o r f e r t i l i z e d and u n f e r t i l i z e d barley-p l a n t s . i LATAR DANISH 6 10 GRAND EARLY FALL 0 I 1 1 1 1 SAMPLING DATES AND STAGES OF GROWTH Fig, 8~ - Top/root ratios at several growth stages for Latar, Danish and S-143 orchard grass varieties. o o cc s CL O 0 5 S-143 ORCHARDGRASS (FERTILIZED) TOP l ROOT BARLEY (FERTILIZED) MAX WT. TOP MAX WT ROOT i i i i i i 0 30 60 90 120 150 180 Gms Fig. 9 - Comparison of root and top development in mid-season of an annual and perennial grass in the year of seeding. Aug 8 25 days Aug 5 46 days 32 Sept. 26 72 days S - 1 4 3 ORCHARDGRASS (FERT. ) JVIAX WT. TOP 1 I MAX. WT ROOT Sept. I 8 119 days BARLEY ( FERT. ) ROOT TOP 30 60 90 Gms 120 150 180 Fig, 10 - Comparison of root and top development in late season of an annual and perennial grass in the year of seeding. 33 P r o j e c t 5. - Time of onset of s e e d l i n g competition i n forage grass stands. a) Object There seems to be some u n c e r t a i n t y i n the l i t e r a t u r e as to the time or stage at which competition i n forage seedings might be expected when water and n u t r i e n t s are not l i m i t i n g . I t i s apparent t h a t " c o m p e t i t i o n " w i l l never be e a s i l y d e f i n e d but i n g e n e r a l i t may be assumed t h a t i n standard forage seedings r e s e r v e foods i n the seed " c a r r y " the s e e d l i n g i n the i n i t i a l stages of i t s development. As the s e e d l i n g develops, and as n u t r i e n t s and water are drawn from the s o i l , i n t e r a c t i o n s between s e e d l i n g s must soon occur. I t would seem t h a t the f i r s t c o m p e t i t i v e r e a c t i o n s occur i n the s o i l , not i n the atmosphere, although B l a c k (1956) and others have shown t h a t l i g h t and shading may become important f a c t o r s at a s u r p r i s i n g l y e a r l y stage i n dense s e e d l i n g stands. Root competition, i . e . competition f o r s o i l s o l u t e s and water, In view of the tremendous s u r f a c e areas and pore space areas i n a common s o i l , would, on a_ p r i o r i grounds, not be expected to develop a t an e a r l y s e e d l i n g stage. The l a r g e range i n seeding r a t e s which g i v e s a t i s f a c t o r y stands i n many " r a t e s of seeding" experiments would seem to support t h i s c o n t e n t i o n . Nonetheless, c r i t i c a l o b s e r v a t i o n s i n grass s e e d l i n g r e a c t i o n s are remarkably few, and I t was decided t h e r e f o r e , to examine the time of onset 34 of s e e d l i n g r o o t competition i n a p r e l i m i n a r y way. b) Method Seeds of p e r e n n i a l r y e g r a s s ( L o l i u m perenne) were sown i n boxes l i k e those p r e v i o u s l y d e s c r i b e d , and set out i n the f i e l d . Seeding r a t e s were e s t a b l i s h e d at 25 l b s . per acre (a common forage seeding r a t e i n the Lower F r a s e r V a l l e y ) , at 100 l b s . per acre (a r a t e commonly used f o r lawns) and at 400 l b s . per acre (a r a t e known to be used o c c a s i o n a l l y by greens-keepers f o r s p e c i a l t u r f s e e d i n g s ) . F i f t e e n boxes were f e r t i l i z e d a t 1000 l b s . per acre w i t h 10-20-10 m i n e r a l f e r t i l i z e r , and f i f t e e n were not f e r t i l i z e d . Seeding took p l a c e on August 15, 1961. The boxes were p l a c e d i n the f i e l d and s p r i n k l e r i r r i g a t e d every other day. T h i r t y - s i x days a f t e r p l a n t i n g and counting, the a e r i a l p o r t i o n s of the s e e d l i n g s (about 3" i n height) were cut w i t h s c i s s o r s at ground l e v e l , d r i e d and weighed. Immediately afterwards, the s e e d l i n g r o o t s were washed f r e e of s o i l , d r i e d , weighed and counted. c) Observations and d i s c u s s i o n The s e e d l i n g counts per box, the weights of tops and r o o t s , the t o p / r o o t r a t i o , and the average weights of s i n g l e s e e d l i n g s 1 top and r o o t are given i n Table 1 and F i g u r e 11. Counting the s e e d l i n g p o p u l a t i o n s per box was done from the r o o t stubs r a t h e r than before h a r v e s t s i n c e i t was much e a s i e r , p a r t i c u l a r l y i n the dense stands. The 35 number of s e e d l i n g s i n a p o p u l a t i o n was checked by counting the r o o t s ; t h i s was done a f t e r the r o o t mass of a box was d r i e d and weighed. The r o o t mass was soaked i n hot water, squeezed dry, and each stub and r o o t p u l l e d f r e e . Soaking made the r o o t s e l a s t i c and easy to p u l l out. S e v e r a l s t r i k i n g items emerge from the t a b l e d data. F i r s t i t i s to be noted t h a t the to p / r o o t r a t i o i s v e r y s i m i l a r i n n e a r l y a l l boxes and suggests t h a t the percentage of r o o t recovered was much the same by the method used r e g a r d l e s s of treatment. Next, the most s t r i k i n g o b s e r v a t i o n i s t h a t the s e e d l i n g s i z e at the low r a t e of seeding (25 l b s . p/a) was very much s m a l l e r than at the h e a v i e r r a t e s . F e r t i l i z e r , as would be expected, made l i t t l e d i f f e r e n c e i n s e e d l i n g emergence, i . e . the numbers of s e e d l i n g s i n the comparable f e r t i l i z e d and u n f e r t i l i z e d boxes were about the same. At a l l r a t e s f e r t i l i z e r a d d i t i o n r e s u l t e d i n the p r o d u c t i o n of l a r g e r tops but not i n very much l a r g e r r o o t s . The response t o f e r t i l i z e r was r e l a t i v e l y g r e a t e s t at the lowest r a t e of seeding. The l a r g e s t s e e d l i n g s were obtained at the i n t e r -mediate seeding r a t e of 100#per acre i n both f e r t i l i z e d and u n f e r t i l i z e d boxes. In both f e r t i l i z e d and u n f e r t i l i z e d s o i l s at the 400 l b s . per acre r a t e the s e e d l i n g s , though numerous, were about the same average s i z e as those at the lowest seeding r a t e ; i t may be s i g n i f i c a n t , nonetheless, TABLE I TIME OF ONSET OF SEEDLING- COMPETITION IN FORAGE GRASS STANDS FERTILIZED Seeding Box Plant Weight Weight Top/Root Av. Wt. Av. Wt. Rates No. No. of of Ratio Single Single Tops Roots Top Root (gm.) (gm.) (gm.) ' (gm.) 1 112 3.2573 0.8774 3.7124 0.0290 .00783 25 pounds 2 109 1.8189 1.4796 1.2293 0.0166 .01357 per 3 117 1.4620 0.6442 2.2694 0.0125 .00550 acre 4 109 1.3970 0.6652 2.1001 0.0128 .00610 5 119 2.2926 1.5483 1.4807 0.0193 .01301 X - S.E.- 113.4 2.0455 1.0429 0.0180 .00920 X ±2.062 ±0.3422 ±0.1949 1 499 17.020 6.7522 2.5206 0.0341 .01353 100 pounds 2 486 18.320 6.7382 2.7188 0.0377 .01386 per 3 458 15.920 5.4126 2.9412 0.0347 .01181 acre 4 487 13.960 5.1217 2.7256 0.0287 .01051 5 464 15.785 5.4896 2.8754 0.0340 .01183 X - S.E.- 478.8 16.201 5.9028 0.0338 .01231 X ±7.678 ±0.7301 ±0.3528 1 1638 21.180 8.805 2.4054 0.0129 .00537 400 pounds 2 1644 17.965 7.290 2.4643 0.0109 .00443 per 3 1657 17.560 7.235 2.4270- 0.0106 .00436 acre 4 1705 15.420 6.930 2.2251 0.0090 .00406 5 1708 17.195 6.595 2.6072 0.0100 .00386 X ± S.E.- 1670.4 17.864 7.371 X ±15.05 ±0.9359 ±0.3795 0.0107 .00442 TABLE I - cont 1d NON - FERTILIZED Seeding Rates Box No. Plant No. Weight of Tops (gm.) Weight of Roots (gm.) Top/Root Ratio Av. Wt. Single Top (gm.) Av. Wt. Single Root (gm.) 25 pounds per acre 1 2 3 4 110 120 118 101 0.5199 0.6354 0.6216 0.4215 0.3823 0.4795 0.4692 0.3738 1.3599 1.3251 1.3248 1.1276 .00472 .00529 .00526 .00417 .00347 .00399 .00397 .00370 X ± S.E.-A 112.5 ±4.330 0.5496 -0.1575 0.4264 ±0.02646 0.0049 0.0038 100 pounds per acre 1 2 3 4 5 383 421 494 476 446 10.375 12.432 13.500 13.140 14.000 4.3752 4.6470 5.5690 6.2103 5.8345 2.3713 2.6759 2.4241 2.1158 2'. 3995 .02708 .02953 .02732 .02760 .03139 .01142 .01103 .01127 .01304 .01308 X - S.E.-X . 444 ±19.69 12.690 ±6.325 5.3272 ±0.3510 0.0286 0.0120 400 pounds per acre 1 2 3 4 5 1480 1605 1778 1660 1662 9.858 12.535 12.160 14.858 15.830 6.1247 6.3484 6.8406 7.7200 7.8694 1.6095 1.9745 1.7776 1.9246 2.0115 .00666 .00780 .00683 .00895 .00952 .00413 .00395 .00384 .00465 .00473 X - S.E.^ 1637 ±48.32 13.0482 ±1.055 6.98062 ±0.3527 0.0079 0.0043 38 t h a t , i n the u n f e r t i l i z e d s o i l s , the s e e d l i n g s a t the h i g h seeding r a t e were somewhat l a r g e r than those at the low s e e d l i n g r a t e . The f a c t t h a t the l a r g e s t s e e d l i n g s should be produced at the h e a v i e s t seeding seems at f i r s t to be anomalous. I t has f r e q u e n t l y been assumed t h a t at the low seeding r a t e s c o m p e t i t i o n f o r n u t r i e n t s and water would be minimal and t h e r e f o r e more vig o r o u s s e e d l i n g s develop at low seeding r a t e s . However, the occurrence o f • l a r g e r s e e d l i n g s at h i g h seeding r a t e s has been observed both i n the f i e l d and the greenhouse on s e v e r a l occasions ( F r e d e r i c k , B r i n k ) and cannot be d i s m i s s e d as e r r o r . Furthermore, K i t t o c k and P a t t e r s o n (1959) a l s o r e p o r t t h a t the i n d i v i d u a l grass s e e d l i n g weight, r o o t p e n e t r a t i o n , height and l e a f area were g r e a t e r at h e a v i e r seeding r a t e s than at low. However, there must be l i m i t s as the o b s e r v a t i o n s from the 400 l b s . per acre seeding r a t e a t t e s t . An e x p l a n a t i o n f o r the o b s e r v a t i o n i s not easy. Why should the s e e d l i n g s i n t h i n stands a c t u a l l y be s m a l l e r than i n t h i c k stands? The f a c t t h a t r o o t competition has not occurred, In these experiments at 36 days, may account f o r the comparative v i g o r of the s e e d l i n g s a t the 100 l b . per acre r a t e ; t h a t r o o t competition has occurred may account f o r the s m a l l e r s i z e of the s e e d l i n g s at the 4-00 l b . per acre r a t e . But the s m a l l s i z e of the t h i n stand s e e d l i n g s must be a t t r i b u t e d t o other f a c t o r s . Perhaps there i s a dosage response and heavy seedings modify the s o i l 3 9 environment to t h e i r advantages through e x c r e t i o n s . Perhaps the s u r f a c e environment i n t h i n stands i s s u b j e c t to g r e a t e r extremes of temperature and s e e d l i n g s are a d v e r s e l y a f f e c t e d . C e r t a i n l y i t i s known t h a t small d i f f e r e n c e s i n s e e d l i n g d e n s i t y p r o f o u n d l y a l t e r the su r f a c e temperature regime. Maybe h e r e i n l i e s one of the c h i e f v a l u e s of the companion crop as has been so o f t e n suggested but not e x p e r i m e n t a l l y e s t a b l i s h e d . I t i s r e c o g n i s e d t h a t i n these experiments s o i l moisture was not l i m i t i n g and, as has been suggested, q u i t e d i f f e r e n t responses might o b t a i n i f i t were. e> 3 5 3 3 0 UJ 20 * 15 £ 10 UJ £ 5 TOP R 0 O T | | 11 TOIL I K$j FERTILIZED iH UNFERTILIZED ROOT TOP f ROOT 25 PER ACRE r l l ROOT TOP ROOT | TOP ROOT 100 PER ACRE 4 0 0 PER ACRE R A T E S OF SEED ING Fig,11 - Average dry weights of 36 day seedlings of perennial ryegrass from stands established at rates of 25 , 100, and 400 pounds per acre. 41 P r o j e c t 6 - Seasonal trends i n l i g n i n and r e s e r v e carbo-hydrate i n the r o o t s and tops of b a r l e y . a) Object I t may be s a i d t h a t the knowledge of the chemistry of r o o t s i s l i m i t e d . Good r o o t samples from f i e l d grown p l a n t s are obtained w i t h d i f f i c u l t y and the u s u a l p r e c i s i o n achieved i n chemical a n a l y s i s i s t h e r e f o r e o f t e n without much meaning. Attendant on sampling i s always a l o s s of f i n e branch r o o t s of unknown magnitude. Moreover, contamination of a r o o t sample, w i t h s o i l m i n e r a l and organic matter and dead r o o t s i s l i k e l y . S t u d i e s of r o o t chemical composition tend t h e r e f o r e to be d i r e c t e d towards the a n a l y s i s of gross m a t e r i a l s , such as " r e s e r v e " carbohydrates, which emphasize the r o o t as a storage organ. As has p r e v i o u s l y been d i s c u s s e d i n the l i t e r a t u r e review there are a number of such s t u d i e s . Without doubt the importance of r e s e r v e carbohydrates i n r o o t s has been d e l i n e a t e d , d e s p i t e d i f f i c u l t i e s i n d e f i n i n g t h e i r chemical nature and i n d e c i d i n g on s u i t a b l e methods f o r a n a l y s i s . I t has sometimes been assumed t h a t i n r o o t s s t r u c t u r a l carbohydrates and a s s o c i a t e d m a t e r i a l s such as l i g n i n are not as f r e e l y e l a b o r a t e d as i n a e r i a l p l a n t p a r t s . However, as Troughton (1957) p o i n t s out, i n grasses there i s l i t t l e evidence f o r t h i s p o i n t of view and comparatively l i t t l e chemical study i n any event has been undertaken. 42 I t appeared t o be worthwhile to extend knowledge of the gross chemistry of r o o t s of annual grasses and to r e l a t e t h i s to t h e , a n a l y s i s of the s e r i a l p a r t s and to the gross chemistry of the r o o t s and tops of p e r e n n i a l g r a s s e s . I t was decided to d i r e c t e f f o r t towards the a n a l y s i s of r e s e r v e carbohydrates which have been accorded a good d e a l of a t t e n t i o n i n r o o t s and to l i g n i n , r e a l l y a complex of substances, which are presumed to decay s l o w l y i n s o i l . b) Methods The b a r l e y r o o t s and tops were from the p l a n t s s t u d i e d i n P r o j e c t 4. Reserve carbohydrates were de t e r -mined by a method developed by Weinman (1946) and modi-f i e d by L i n d a h l et_ aJL. (1948); sugars, however, were determined by the method proposed by Somogyi (1945) and not by the procedure recommended by L i n d a h l et_ al_. ( i b i d . ) . L i g n i n was determined by the A.O.A.C. (i960) method. The a n a l y t i c a l procedures are given i n some d e t a i l i n the appendix. c) Observations and d i s c u s s i o n The method f o r r e s e r v e carbohydrate d e t e r m i n a t i o n , d e s p i t e a good d e a l of e f f o r t spent i n t r y i n g t o make i t s a t i s f a c t o r y , l e f t much to be d e s i r e d ; the causes of d i f f i c u l t y remain u n c e r t a i n . The method f o r l i g n i n gave r e s p e c t a b l e r e s u l t s and i s to be p r e f e r r e d to s e v e r a l " s h o r t " methods (e.g. the " A r i z o n a " method) which were 43 a l s o t r i e d . The r e s u l t s of a n a l y s i s are given i n Table 2, and i n F i g u r e s 12, 13, and 14. The r e s e r v e carbohydrates f o l l o w a p a t t e r n which might be expected i n an annual grass such as b a r l e y , v i z . a marked d e c l i n e i n carbohydrate r e s e r v e s , as a percentage of the t o t a l r o o t dry matter, occurs as the p l a n t matures and, c o n v e r s e l y , as the p l a n t matures, r e l a t i v e l y more r e s e r v e carbohydrate i s found i n the a e r i a l p o r t i o n s of the p l a n t . S l i g h t l y more res e r v e carbohydrate was e l a b o r a t e d i n both r o o t and top In f e r t i l i z e d p l a n t s than i n n o n - f e r t i l i z e d p l a n t s . Very l i t t l e r e s e r v e carbohydrate remains i n the r o o t s of b a r l e y by season's end. L i g n i n content, as one might expect, i n the r o o t s of b a r l e y , i n c r e a s e s percentagewise, as the " r e s e r v e " carbohydrate content drops. On the other hand, as r e s e r v e carbohydrate percentage r i s e s i n the a e r i a l p a r t of the p l a n t the l i g n i n percentage remains about even or drops s l i g h t l y . TABLE 2 BASIC GRAVIMETRIC DATA AND CHEMICAL ANALYSES FOR BARLEY 44 Stage Wt. Gm. Ratio % L i g n i n Average % avai l a b l e carbohydrate s Top Root T/R Top Root Top Root Non- • F e r t . 1.028 0.844 1.216 2.968 12.60 6" 1.069 1.067 1.001 2.742 no June 1.223 1.036 1.180 2.544 more 16/61 1.204 1.018 1.182 2.544 sample Av. 1.131+0.048 0.991+0.050 1.140 2.699+0.100 12.60 3.40 5.62 F e r t . 2.128 1.361 1.563 1.629 12.22 6" 2.483 1.384 1.794 1.820 12.18 2.020 1.050 1.923 1.821 13.90 2.280 1.111 2.052 1.665 10.02 2.228+0.100 1.226+0.085 1.816 1.734+0.050 12.080+0.795 2.25 4.58 Non-F e r t . 4.304 2.089 2.060 3.007 12.68 10" 3.473 2.080 1.669 2.654 12.72 June 3.679 2.054 1.791 2.565 13.05 23/61 3.668 1.710 2.144 2.530 13.12 Av. 3.781+0.181 1.983+0.091 1.906 2.689+0.109 12.892+0.112 1.87 4.18 F e r t . 11.468 3.774 3.038 2.032 11.80 10" 9.684 2.759 3.510 2.253 11.62 10.294 3.002 3.429 2.141 11.71 12.697 4.307 2.948 2.167 11.19 Av. 11.036+0.666 3,460+0.355 3.189 2.148+0.044 11.58+0.135 1.47 4.17 Non-F e r t . 30" Bloom- 31.001 6.890 4.499 6.330 15.27 i n g 44.552 10.309 4.321 6.723 13.65 J u l y 41.724 8.380 4.979 4.323 13.95 14 45.592 9.214 4.947 5.406 10.94 Av. 40.717+3.340 8.705+0.721 4.677 5.695+0.534 13.452+0.908 8.56 1.36 6% TABLE 2 cont'd. 45 Stage Wt. Gm. Ratio % Lignin Average % available carbohydrates Non-Fert. 30" Bloom- 31.001 6.890 4.499 6.330 15.27 ing 44.552 10.309 4.321 6.723 13.65 July 41.724 8.380 4.979 4.323 13.95 14/61 45.592 9.214 4.947 5.406 10.94 Av. 40.717+3.340 8.705+0.721 4.677 5.695+0.908 13.452+0.908 8.56 1.36 Fert. 115.175 21.275 5.413 6.861 11.14 30" 125.339 21.344 5.872 6.226 11.24 Bloom- 104.648 17.609 5.942 6.981 12.57 ing 120.604 14.462 8.339 5.323 13.68 Av. 116.442+4.445 18.673+1.652 6.236 6.438+0.380 12.157+0.603 9.60 1.57 Mon-Fert. Matur- 57.390 7.120 8.060 — 13.43 ing 74.445 8.950 8.317 6.674 13.39 Aug. 77.575 11.405 6.301 6.403 11.45 5/61 77.068 11.730 6.570 8.664 12.40 Av. 71.619+4.792 9.801+1.037 7.307 6.538+0.713 12.667+0.470 10.56 2.25 Fert. 187.515 23.135 8.087 8.590 10.84 Matur- 191.085 24.030 7.951 10.480 10.88 ing 142.515 13.090 7.378 8.370 7.62 194.725 20.560 9.471 6.845 12.23 Av. 178.960+12.24 21.466+1.365 8.337 8.571+0.745 10.392+0.979 12.64 4.76 Non-Fert. 49.500 4.740 10.443 7.780 17.72 Late 54.94 5.710 9.621 8.307 17.02 Sept. 59.38 4.730 12.553 8.201 18.25 18 Av. 54.606+2.357 5.060+0.325 10.791 8.196+0.159 18.133+0.356 11.55 1.34 Fert. 143.120 11.480 12.466 8.904 17.29 Late 159.510 11.380 14.016 6.381 17.95 140.210 13.420 10.477 8.377 14.04 Av. 141.580+6.007 12.093+0.664 11.707 8.054+0.606 16.36+1.208 14.73 1.98 S A M P L I N G DATES • 12 - The seasonal change in available carbohydrate percentage of fertilized and unfertilized barley plants. Fig. 13 - The seasonal change in lignin percentage of fertilized and unfertilized barley plants. 48 SAMPLING DATES . . i Fig. 14 - Logarithm plus one of dry matter in grams, of top and root per box of fertilized and unfertilized barley plants. 49 P r o j e c t 7 - Seasonal trends i n l i g n i n and r e s e r v e carbohydrate i n the r o o t s and tops of a f i r s t year stands of three orchard grass v a r i e t i e s . c) Object and Methods Comparative g r a v i m e t r i c data f o r dry matter e l a b o r a t e d i n top and r o o t of an annual grass, b a r l e y , and a p e r e n n i a l gr a s s , orchard grass, were given under P r o j e c t 4. E x t e n s i o n of the comparative study t o i n c l u d e r e s e r v e carbohydrate and l i g n i n a n a l y s i s of the orchard grass tops and r o o t s seemed to be d e s i r a b l e though i t i s r e a l i z e d t h a t the p l a n t i n g dates of the b a r l e y and orchard grass d i f f e r e d by three weeks. E s s e n t i a l l y the methods f i n a l l y adopted were those a l r e a d y given under P r o j e c t 6 f o r b a r l e y . Although the r e s u l t s r e l a t e t o a s i n g l e season and to the year of seeding, only, some advantage may be claimed over some e a r l i e r a n a l y t i c a l s t u d i e s of a s i m i l a r nature w i t h p e r e n n i a l grasses i n t h a t dead r o o t s do not complicate the assays. P o i n t was added to t h i s study i n t h a t " L a t a r " orchard grass developed at Washington State U n i v e r s i t y , at Pullman, Wash., was r e l e a s e d as a v a r i e t y i n h e r e n t l y low i n l i g n i n . I t was claimed t h a t the low l i g n i n forage of t h i s v a r i e t y improved d i g e s t i b i l i t y by 18 to 20$ ( S o s u l s k i i960). I t was f e l t t h a t i t would be i n order t o determine l i g n i n l e v e l s i n the forage of t h i s v a r i e t y produced under the maritime environment of the U n i v e r s i t y of B r i t i s h Columbia f i e l d s and a l s o t o determine the l i g n i n l e v e l s i n r o o t s as W e l l a s f o r a g e . 50 b) Observations (please see Table 3 and F i g u r e s ) . The trends i n the percentages of r e s e r v e carbohydrate and of l i g n i n i n tops and r o o t s of the p e r e n n i a l orchard grass v a r i e t i e s d i f f e r markedly from those of the annual b a r l e y . In the f i r s t p l a c e r e s e r v e carbohydrates tend t o accumulate percentage-wise r a t h e r s l o w l y at f i r s t i n the p e r e n n i a l grass r o o t s , but as the season advances t h i s accumulation a c c e l e r a t e s ; by c o n t r a s t as the season advanced i n the annual grass r e s e r v e s disappeared from the r o o t . I t i s p r o b a b l y unwise t o p l a c e much emphasis on the i n t e r - v a r i e t a l d i f f e r e n c e s shown i n F i g s . 15, 16, and 17, because of the s i n g u l a r i n s t a b i -l i t y of the c o l o u r development i n the chemical d e t e r m i n a t i o n . The l i g n i n d e t e r m i n a t i o n s by the A.O.A.C. method were q u i t e s a t i s f a c t o r y and show c o n s i s t e n t i n t e r - v a r i e t a l p a t t e r n s ; r o o t l i g n i n , i t i s to be noted, i s c o n s i s t e n t l y h i g h e r than "tops" l i g n i n . I f there i s a tendency f o r the l i g n i n p e r c e n t -age t o f a l l or r i s e i n the tops or r o o t s of orchard grass i t i s not ve r y marked. Furthermore, there i s l i t t l e evidence t o support the c o n t e n t i o n t h a t " L a t a r " orchard grass i s i n h e r e n t l y lower i n l i g n i n than other v a r i e t i e s . TABLE 3 51 GRAVIMETRIC DATA AND CHEMICAL ANALYSIS FOR THE ORCHARD GRASS VARIETIES (Grasses Planted July 14, 1961) Wt. Gm. Ratio % Lignin Average % available carbohydrates Top Root T/R Top Root Top Root July 22/61 LATAR 16.460 21.642 25.762 18.432 12.434 12.819 14.027 10.977 1.324 1.688 1.837 1.680 11.000 8.713 8.662 9.523 14.47 14.00 14.23 15.48 Av. 20.574+2.036 12.564+0.628 1.632 9.476+0.545 14.545+0.326 2.80 1.30 DANISH 15.883 24.811 30.742 20.750 13.297 16.007 15.416 13.185 1.504 1.551 1.993 1.347 10.980 7.058 8.224 8.063 15.10 14.28 14.17 14.68 Av. 23.046+3.148 14.477+0.724 1.591 8.581+0.840 14.557+0.211 3.94 4.43 S-143 13.064 12.715 12.046 15.277 11.220 10.599 9.169 11.619 1.163 1.199 1.313 1.314 11.690 7.643 7.612 9.482 15.94 14.14 13.82 14.49 Av. 13.276+6.999 10.652+0.537 1.247 9.107+0.969 14.597+0.468 1.25 1.42 Aug. 5/61 2nd Harv. 10" 27.310 LATAR 28.680 30.015 28.530 16.200 18.365 18.385 20.350 1.717 1.571 1.632 1.402 9.103 7.916 8.250 9.610 13.16 12.64 12.79 12.96 Av. 28.759+0.460 18.325+0.847 1.570 8.721+0.389 12.837+0.112 3.54 1.87 DANISH 39.390 38.735 38.330 36.450 25.500 24.935 26.450 25.980 1.545 1.553 1.449 1.403 9.371 10.100 9.714 10.450 14.09 15.19 14.55 14.86 Av. 35.726+0.631 25.716+0.322 1.389 9.909+0.234 14.682+0.234 1.75 1.06 S-143 43.065 49.198 34.592 30.095 21.470 22.210 22.580 23.900 2.005 2.215 1.532 1.259 9.226 9.734 8.602 9.307 14.57 13.03 14.24 14.34 Av. 39.237+4.271 22.540+0.508 1.740 9.232+0.233 14.045+0.345 3.85 1.69 TABLE 3 - cont'd. 52 Wt. Gm. Ratio % Lignin Average % available carbohydrates Top Root T/R Top Root Top Root Aug. 3/61 3rd Har. LATAR 53.890 54.850 51.800 5 2 . 2 7 0 45.150 4 2 . 9 2 0 40.880 43.120 1.193 1.277 1.267 1.213 9.995 11.82 9.237 9.406 15.74 15.30 12.33 12.30 Av. 53.202+0.708 43.017+0.872 1.237 10.114+0.594 13.917+1.056 3.08 1 . 9 2 DANISH 76.900 8 1 . 2 5 0 71.570 59.800 58.800 62.000 59.370 57.000 1.308 1.311 1.205 1.049 8.194 10.02 10.28 8.764 13.49 12.44 12.37 1 2 . 7 2 Av. 72.380+4.637 59.292+1.034 1.220 9.314+0.499 12.755+0.259 8.19 5.33 S-143 75.090 75.250 68.800 69.430 49.50 54.00 64.00 50.00 1.517 1.393 1.075 1.240 3.303 7.567 9.430 9.258 14.08 13.55 14.03 13.37 Av. 72.142+1.753 58.75+3.362 1.227 8.639+0.435 13.757+0.176 7.32 3.22 Sept. 26/61 4th Har. Late in 85.80 the 9 2 . 7 0 season 94.90 LATAR 86.90 73.45 83.05 59.5 78.80 1.168 1.116 1.594 1.102 4.768 4 . 5 2 4 5.052 13.15 12.91 13.59 13.68 Av. 90.075+2.208 73.70+5.125 1.222 4.781+0.152 13 . 332+0.182 14.89 1 3 . 2 5 DANISH 126.64 134.58 107.78 91.90 110.06 82.6 96.15 62.10 1.151 1.629 1.120 1.479 4.837 4.956 5.599 1 3 . 2 4 12.96 13.89 13.87 Av. 115.225+9.594 37.728+10.210 1.313 5.127+0.236 13.485+0.232 14.86 10.00 S-L43 1 2 6 . 1 6 132.31 1 2 6 . 2 4 129.51 72.30 91.10 56.60 86.70 1.744 1.452 2.230 1.493 5.563 4.926 5.313 6.151 14.04 14.29 14.58 15.28 Av. 128.555+1.475 76.675+7.803 1.676 5.513+0.256 14.547+0.268 14.80 7.24 53 Fig, 15 - The seasonal change in carbohydrate percentage of Latar orchard grass, top and root. Eig. 16 - The seasonal change in carbohydrate percentage of S - 1 4 3 orchard grass, top and root. 55 Fig, 17 - The seasonal change in carbohydrate percentage of Danish orchard g rass, top and root. Fig # IB - The seasonal change in lignin percentage of Latar orchard grass, top and root. C5 15 . 10 5 . S-143 ORCHARD GRASS — ROOT  TOP SAMPLING DATES F i g . 19 - Seasonal change in lignin percentage of S-143 orchard grass, top and root DANISH ORCHARD GRASS SAMPLING DATES . 20 - Seasonal change in lignin percentage of Danish orchard grass, top and root. 59 General D i s c u s s i o n and Summary That r o o t s , t h e i r morphology, physiology and chemistry, are d i f f i c u l t to study i s g e n e r a l l y conceded. In s i t u often they cannot be seen and hence many root phenomena must be studi e d by i n d i r e c t means. Roots die and decay slowly, and f r e q u e n t l y i t i s d i f f i c u l t to d i s t i n g u i s h l i v i n g from dead r o o t s . Very often too roo t s are separated from t h e i r s o i l environment only through p a i n s t a k i n g e f f o r t and these only incompletely. Nonetheless i t s u r e l y can be s a i d that p l a n t ^ s t u d y without root study i s incomplete and t h a t , hard though i t may be to gain, knowledge of roo t s should be v i g o r o u s l y sought a f t e r . I t may be added t h a t i n no f i e l d i s root study more important than In forage crops, where i t i s r e a l l y the p e r e n n i a l root and crown system which confers i n the crop any gain i n dry matter production i t may make over annual arable crops such as c e r e a l s and r o o t s . The p r o j e c t s aforementioned provided an opportunity to examine techniques which are claimed t o be or promise to be u s e f u l i n root study. I t i s g r e a t l y to be r e g r e t t e d that land tenure p o l i c y d i d not permit the use of r a d i o -t r a c e r s w i t h emanations more energetic than those given by P^gj p o s s i b l y the t h i n wafer technique could be so used that X-ray photos of growing ro o t s could be taken and so avoid the hazards attendant i n the use of such t r a c e r s as r a d i o -i o d i n e . In any event the " t h i n wafer technique" appears to produce f a i r l y normal roots and the c o n d i t i o n s at the s o i l -g l a s s i n t e r f a c e do not appear to he h i g h l y abnormal f o r root growth. Using t h i n wafers i t seems p o s s i b l e that a c a r e f u l l y planned s t a t i s t i c a l approach could be made to the i n t e r -a c t i o n s which may e x i s t between r o o t s of grasses and legumes when grown i n a s s o c i a t i o n . One handicap remains, v i z . no easy method f o r the d i s t i n c t i o n of grass and legume roo t s has been developed. Sprague and h i s ^ a s s o c i a t e s (1962) have r e f e r r e d very r e c e n t l y to some of the p u z z l i n g features i n forage crop competition. The observations i n the p r o j e c t s w i t h seedlings o u t l i n e d e a r l i e r tend to support t h e i r contention. I t i s somewhat s u r p r i s i n g , f o r example, to f i n d t h i n l y e s t a b l i s h e d seedlings a c t u a l l y doing l e s s w e l l than seedlings i n t h i c k e r stands. Also i t would appear that s o i l volume and r e l a t e d f a c t o r s must be more c a r e f u l l y taken i n t o account i n s e e d l i n g s t u d i e s , f o r w i t h l i g h t , n u t r i e n t s and water not l i m i t i n g , other f a c t o r s of s o i l might w e l l become important f a c t o r s i n competition. I t seems, f o r example, to be g e n e r a l l y conceded that seedlings competition i s e a r l y manifested long before the t o t a l solum i s e x p l o i t e d and before l i g h t and other f a c t o r s become l i m i t i n g but the p r e c i s e nature of the elements of competition are f a r from c l e a r . The root and top i n t e r r e l a t i o n s h i p s seen w i t h an annual grass, b a r l e y and a p e r e n n i a l grass, orchard grass, are much as might he expected. As Foth (1962) has r e c e n t l y observed w i t h another annual crop, corn, pronounced changes i n r a t e of growth of both top and root occurred i n b a r l e y ; the weight of top occurred more r a p i d l y r e l a t i v e l y than root to produce a marked increase on the top/root r a t i o and g r a i n development occurred l a r g e l y a f t e r root growth ceased. The p e r e n n i a l grass i n i t s f i r s t year from seeding however, maintained a f a i r l y uniform top to root r a t i o and by the end of the growing season had elaborated much more dry matter than had the annual grass. Carbohydrate reserves have been stud i e d now f o r a number of forage crops and despite d i f f i c u l t i e s i n the development of s u i t a b l e assays f o r "reserve" substances the general f e a t u r e s of t h e i r accumulation w i t h season and w i t h management systems i s f a i r l y c l e a r . The pa t t e r n s f o r orchard grass conform r a t h e r w e l l w i t h those to be expected from other work'(e.g. Smith 1962) i n that accumulation a c c e l e r a t e d w i t h the advance of the season. I t would appear that the p a t t e r n f o r b a r l e y i s that expected f o r an annual grass but i t i s apparently the f i r s t r e c o rd of such. The l i g n i n trends as expressive of the s t r u c t u r a l elements of grasses are not so easy to understand. The a n a l y s i s i s c a r r i e d out, a c t u a l l y , w i t h more p r e c i s i o n than that of "reserve" carbohydrates but the r o l e of l i g n i n i s probably not so e a s i l y d e f i n e d . I t i s , probably, s i g n i f i c a n t 6 2 t h a t the l i g n i n i n the r o o t s of the annual grass tended to i n c r e a s e w i t h m a t u r i t y a n d ' i s , probably, i n d i c a t i v e of the degradation of components of dead or dying r o o t s more e a s i l y " a t t a c k e d " by the s o i l f l o r a and fauna. The r e l a t i v e constancy of the l i g n i n component r e l a t i v e t o i n c r e a s i n g carbohydrate r e s e r v e s i s somewhat unexpected on a_ p r i o r i grounds. I t i s c l e a r from the f o r e g o i n g s t u d i e s t h a t d e s p i t e inadequacies i n technique t h a t worthwhile c o n t r i b u t i o n s can be made to the knowledge of the b i o l o g y and chemistry of the r o o t s of forage crops. 63 APPENDIX 64 A. METHOD USED FOR THE DETERMINATION OF *RESERVE» CARBO-HYDRATES (AFTER LINDAHL (194871 Procedure Weigh out a 0 .1- to 1- gram sample of m a t e r i a l and t r a n s f e r t o 125-ml. erlenmeyer f l a s k which has p r e v i o u s l y been weighed a c c u r a t e l y to the nearest 0.01 gram. Add 10 ml. of d i s t i l l e d water and heat f o r one-half hour on a b o i l i n g water - bat h to g e l a t i n i z e s t a r c h , I n s e r t i n g a small g l a s s f u n n e l i n t o the neck of the f l a s k t o minimize the l o s s of water. Cool t o room temperature, wipe the moisture from the outs i d e of the f l a s k and the i n s i d e of i t s neck w i t h a f i l t e r paper. Place the f l a s k on the balance, and by means of a dropper add as many drops of d i s t i l l e d water as necessary to r e p l a c e the water l o s t by e v a p o r a t i o n . The counterpoise to be used must equal the weight of the f l a s k p l u s the weight of the sample p l u s 10 grams. P i p e t t e 10 ml. of b u f f e r s o l u t i o n (pH 4.45J prepared by mixing three volume p a r t s of 0 . 2 N a c e t i c a c i d w i t h two volume p a r t s of 0 . 2 N sodium a c e t a t e s o l u t i o n ) and 10 ml. of c l a r a s e (0.5$) s o l u t i o n i n t o the f l a s k thus i n c r e a s i n g the l i q u i d volume of the d i g e s t t o 30 ml. Stopper the f l a s k t i g h t l y w i t h a w e l l - f i t t i n g rubber stopper, and incubate f o r 44 hours at 37° C., shaking the f l a s k o c c a s i o n a l l y . Cool t o room temperature, add 50 to 100 mg. of powdered n e u t r a l l e a d a c e t a t e , shake, and al l o w p r e c i p i t a t e and r e s i d u e 65 to s e t t l e . Test f o r completeness of the r e a c t i o n w i t h a s i n g l e drop of d i l u t e potassium oxalate s o l u t i o n and f i l t e r , without washing, through a dry h i g h l y r e t e n t i v e f i l t e r paper (Whatman No. 42) i n t o a dry f l a s k c o n t a i n i n g 100 to 200 mg. of powdered potassium o x a l a t e . Shake, t e s t f o r completeness of deleading w i t h a drop of d i l u t e lead acetate s o l u t i o n , stopper, and l e t stand from three to four hours, or overnight i n a r e f r i g e r a t o r . F i l t e r and hydrolyze a 15 ml. a l i q u o t w i t h 0.75 ml. of 25$ h y d r o c h l o r i c a c i d f o r one h a l f hour on the b o i l i n g water bath, a t t a c h i n g the f l a s k to a r e f l u x condenser. Cool, t r a n s f e r q u a n t i t a t i v e l y to a 50-ml. standard f l a s k , n e a r l y n e u t r a l i z e w i t h 25$ sodium hydroxide- s o l u t i o n (using a few drops of methyl - red s o l u t i o n as i n d i c a t o r ) , and make to volume. The f i n a l e x t r a c t volume (50 ml.) contains 15/30 (one-half) the t o t a l a v a i l a b l e carbohydrate of the o r i g i n a l d i g e s t . Sugar determination Reagent: 1 l i t e r of the s o l u t i o n contains 28 gm. of anhydrous disodium phosphate, 100 ml. g. normal sodium hydroxide, 40 gm. g. sodium potassium t a r t r a t e s a l t , 8 gm. of c u p r i c s u l f a t e ( c r y s t a l l i n e ) , and 180 gm. of anhydrous sodium s u l f a t e . The s o l u t i o n i s prepared i n t h i s way. The phosphate and t a r t r a t e are d i s s o l v e d i n 'about 700 ml. of water, the sodium hydroxide i s added, and then, w i t h s t i r r i n g , 80 ml. of a 10$ copper s u l f a t e s o l u t i o n are introduced. F i n a l l y the 66 sodium s u l f a t e i s added and, when d i s s o l v e d , the s o l u t i o n i s d i l u t e d t o 1 l i t e r and allow t o stand f o r a day or two, d u r i n g which time i m p u r i t i e s separate out. The c l e a r top p a r t of the s o l u t i o n i s decanted and the remainder f i l t e r e d through a good grade of f i l t e r paper. T h i s reagent keeps i n d e f i n i t e l y w i t h no s i g n of d e t e r i o r a t i o n . Technique: The i o d e m e t r i c technique i s used i n the de t e r m i n a t i o n . 5 c.c. of the reagent and 5 c.c. of the e x t r a c t s o l u t i o n are mixed i n a 30 x 200 mm. Pyrex t e s t - t u b e , covered w i t h a g l a s s f u n n e l , and heated by immersion i n a vigo r o u s b o i l i n g water bath f o r ten minutes. A f t e r c o o l i n g , 6 ml. potassium i o d i d e (2|-$ s o l u t i o n ) i s added by running i t from a p i p e t t e down the w a l l of the t e s t - t u b e , without s t i r r i n g or a g i t a t i o n . F o l l o w i n g t h i s , about 1.5 ml. of approximately 2.ON s u l f u r i c a c i d are added; the a c i d i s r a p i d l y dropped, r a t h e r than p e r m i t t e d t o flow i n t o the t e s t - t u b e , w i t h simultaneous a g i t a t i o n , so t h a t the e n t i r e contents of the tube are mixed and a c i d i f i e d at once. For t i t r a t i o n 0.05N t h i o s u l f a t e d i s used. T h i s i s prepared from time t o time by d i l u t i o n from 0.1N s t o c k s o l u t i o n . S u b t r a c t from the blank t i t r a t i o n value the t i t r a t i o n v a lue f o r the p l a n t d i g e s t , c a l c u l a t e as glucose, and r e p o r t as " t o t a l a v a i l a b l e carbohydrate". Blank determinations should be c a r r i e d out whenever the new c l a r a s e (trade name of t a k a d i a s t a s e ) s o l u t i o n i s used. 67 For t h i s purpose, p i p e t t e 10 ml. of d i s t i l l e d water, 10 ml. of b u f f e r s o l u t i o n , and 10 ml. of the c l a r a s e s o l u t i o n i n t o a 100-ml. erlenmeyer f l a s k . Incubate, and subsequently t r e a t the blank d i g e s t i n e x a c t l y the same way as the other d i g e s t s (except f o r the g e l a t i n i z a t i o n treatment which i s omitted i n the case of the b l a n k s ) . For the standard, 40 mg. of glucose i s used. Procedure and treatment f o r the standard are e x a c t l y the same as i n the case of blank d e t e r m i n a t i o n . B. METHOD USED FOR THE DETERMINATION OF LIGNIN Weigh 1 gm. of sample i n the thimble l i n e d w i t h f i l t e r paper, p l u g w i t h c o t t o n wool and p l a c e i n the Soxhl e t . E x t r a c t w i t h alcohol-benzene ( l + 2) f o r f o u r hours. Replace the e x t r a c t o r w i t h a l c o h o l , a l l o w i n g the Soxhlet t o f i l l twice and r e p l a c e w i t h ether (the h o t - p l a t e must be cooled down bef o r e p l a c i n g the ether on) f o r one-half hour. Dry the thimble a t 130° C. i n the oven f o r one hour, and t r a n s f e r sample to a 100 ml. wide-mouth erlenmeyer. Add 40 ml. ifo s o l u t i o n of pe p s i n i n 0.1N H c l , w e t t i n g sample w e l l by adding a smal l p o r t i o n of the s o l u t i o n , s t i r r i n g or shaking thoroughly and f i n a l l y washing down the s i d e s of f l a s k s w i t h remainder of s o l u t i o n . Incubate at 40°C. o v e r n i g h t . Add 20 - 30 ml. hot water and f i l t e r through gooch c r u c i b l e w i t h a t h i n asbestos l a y e r , wash w i t h water. T r a n s f e r to a 600 ml. beaker w i t h 150 ml. 5$ HgSO^. Re f l u x 68 v i g o r o u s l y on hot p l a t e f o r 1 hour, adding E^O o c c a s i o n a l l y to m aintain o r i g i n a l volume. F i l t e r through gooch c r u c i b l e w i t h a t h i n asbestos l a y e r . Wash r e s i d u e w i t h three 20 - 30 ml. p o r t i o n s e t h e r . Dry i n oven at 130°C. (+ 15 minutes). T r a n s f e r r e s i d u e t o 200 ml. beaker. Add 20 ml. 72$ HgSO^ at 20°C. to r e s i d u e and h o l d two hours at 20°C, s t i r r i n g o c c a s i o n a l l y . Add 125 ml. HgO, f i l t e r , wash w i t h water. Wash r e s i d u e from f i l t e r paper and r e f l u x as before f o r two hours, u s i n g 150 ml. 3$ RgSO^. F i l t e r r e s i d u e onto gooch w i t h asbestos pad and wash w i t h hot water u n t i l f r e e of a c i d . Dry at 105 - 110° C. and determine l i g n i n by l o s s i n weight on i g n i t i o n at 600° C. P r e p a r a t i o n of Sample Samples are oven-dried and ground i n a Wiley m i l l through a 40 - mesh screen. Samples are kept i n a i r - t i g h t c o n t a i n e r s u n t i l ready f o r use. 69 LITERATURE CITED 1.' A. 0. A. C. O f f i c i a l methods of a n a l y s i s of the A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists. 9th Ed. (i960). 91.92. 2. Aberg, E., I . J . Johnson and C. P. W i l s i e (1943) . A s s o c i a t i o n s between s p e c i e s of grasses and legumes. J . Amer. Soc. Agron. 35* 357-69. 3. Ahlgren, H. L. and 0. S. Aamodt (1939) . Harmful r o o t i n t e r a c t i o n s as a p o s s i b l e e x p l a n a t i o n f o r e f f e c t s noted between v a r i o u s s p e c i e s of grasses and legumes. J . Amer. Soc. Agron. 31 : 982-5. 4. Baker, H. K. 1955. The e f f e c t of c u t t i n g on the r o o t develop-ment and herbage p r o d u c t i o n of Dolium  perenne. Ph.D. T h e s i s , Univ. Durham, Durham, England. 5. Boggie, R., Hunter, R. P., and Knight, A. H. 1958. S t u d i e s of the r o o t development of p l a n t s i n the f i e l d u s i n g r a d i o a c t i v e t r a c e r s . J . E c o l . 4 6 : 621-39. 6. Boggie, R. & Knight, A. H. i960. S t u d i e s of r o o t development i n a grass sward growing on deep peat u s i n g r a d i o -a c t i v e t r a c e r s . B r i t . Grassland Soc. J . 15 (2) : 133-136. 7. B o l l e n , W.B. & Lu, K. C. 1957. E f f e c t of D o u g l a s - f i r sawdust mulches and i n c o r p o r a t i o n s on s o i l m i c r o b i o l a c t i v i -t i e s and p l a n t growth. S o i l S c i . Soc. Am. Proc. 21: 35-41. 8. Brauns, P. E. & Brauns, D. A. I960. "The Chemistry of L i g n i n 1949-1958". Academic Press, London. 9. Brown, E. M. 1943. Seasonal v a r i a t i o n s i n the growth and chemical composition of Kentucky b l u e g r a s s . Res. B u i . 360. Mo. A g r i c . Exp. S t a . Columbia, Mo. 70 10. Burton, 1954. 11. Burton, 1957. G. W., De Vane, E. H. and C a r t e r , R. L. 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 i n southern grasses measured by y i e l d s , drought symptoms and p32 uptake. Agron. J . 46 : 229-33. G. ¥., Gordon, M. P. and Jackson, J . E. S t u d i e s of drought t o l e r a n c e and water use of s e v e r a l southern g r a s s e s . Agron. J . 49 : 498-503. 12. Burton, 1962. G. W. and Jackson, J . E. A method f o r measuring sod r e s e r v e s . Agron. J . 54: 53-55. 13. Cormack, R.G.H. 1949. 14. C r i d e r , F. J, 1955. The development of r o o t h a i r s i n angiosperms. Bot. Rev. 15: 583-612. Root-growth stoppage r e s u l t i n g from d e f o l i a t i o n of g r a s s . U.S. Ag. Techn. B u i . 1102: 1-23. 15. Dittmer, H. J, 1937. 16. Dittmer, H. J . 1938. Q u a n t i t a t i v e s t u d y i n g of the r o o t s and r o o t h a i r s of a wi n t e r rye p l a n t (Secale c e r e a l e ) Am..J. Bot. 24: 417-20. Q u a n t i t a t i v e study of the subterranean members of three f i e l d g r a s s e s . Am. J . Bot. 25: 654-7. 17. Dittmer, 1949. H. J . 18. Dittmer, H. J . 1959. Root h a i r v a r i a t i o n s i n p l a n t s p e c i e s . Am. J . Bot. 36: 152-7. Method to determine the l e n g t h of i n d i v i d u a l r o o t s . T o r r e y Bot. Club, 86^ 59-61. B u i 19. Dittmer, H. J . 1959. Study of the r o o t systems of c e r t a i n sand dune p l a n t s i n New Mexico. E c o l . 40: 265-73. 20. Duhanin, K.S. 1940. (Decomposition of r o o t and stubble r e s i d u e s of grasses and grass mixtures on s o i l s of the a r i d south-west). Pocvovedenie No. 11: 49-54. 71 21. Foth, H. D. 1962. Root and top growth of corn. Agron. J. 54: 49-52. 22. F r e d e r i c k , R. A. 1959. 23. F r i b o u r g , H. A. 1953. 24. Goedewaagen, 1950. M. "Some techniques f o r the study of r o o t s i n p l a c e " . M.S.A. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B. C. Rapid method of washing r o o t s . Agron. J . 45: 334-5. A. J . and Schurman, J . J . (Root p r o d u c t i o n under f i e l d crops and grass as a source of org a n i c matter i n the s o i l ) . Land. T i j d . , Wageningen 62: 469-82. 25. G o t t l i e b , S. and Hendricks, S. B. 1945. S o i l o r g a n i c matter as r e l a t e d t o newer concepts of l i g n i n chemistry. S o i l S c i , Soc. Am. Proc. 10: 117-125. 26. Haas, H. J . and Rogler, G. A. 1953. Technique f o r photographing grass r o o t s :"- '; . i n s i t u . Agron. J . 45: 173. 27. H a l l , N.S., 1953. Chandler, W.F., van B a v e l , C.H.M., Reid, P.H. and Anderson, J . H. A t r a c e r technique t o measure growth and a c t i v i t y of p l a n t and r o o t systems. B u i . No. 101, North Car. Expt. S t a . Tech. pp. 1-40. 28. Haynes, J . L., S t r i n g f i e l d , G. H. and Johnson, M. J . 1959. 29. Ivanov, P.K. 1950. 30. Jacques, W.A. 1943. E f f e c t of corn p l a n t i n g p a t t e r n on y i e l d , r o o t e x t e n s i o n and i n t e r s e e d e d cover crops. Agron. J . 51.: 454-456. (The i n f l u e n c e of p e r e n n i a l herbage on the s t r u c t u r e and water regime of the s o i l ) . Pocvovedenia 3-11. Root development i n some common New Zealand p a s t u r e p l a n t s . N. Z. J . S c i . Tech. 25: 97-117. 31. Jacques, W.A. and Schwass, H.A. 1956. Root development i n some common New Zealand pas t u r e p l a n t s . N. Z. J . S c i . Tech. 37: 569-83. ~ 72 32. Kauter, A. 1933. ( C o n t r i b u t i o n to the knowledge of r o o t growth i n grasses.) Ber. Schweiz. bot: Ges. 42: 37-109 ( D i s s e r t a t i o n No. 731, Z u r i c h ) . 33. Keim, P.D. and Beadle, G. W. 1927. R e l a t i o n of time of seeding to r o o t develop-ment and w i n t e r s u r v i v a l of f a l l - s e e d e d grasses and legumes. E c o l . 8_: 251-64. 34. Kholodnyi, N. G. 1951. Doklady Akad. Nauk. S.S.S.R. 81: 673. 35. K i t t o c k , D. L. and P a t t e r s o n , J . K. 1959. Measurement of r e l a t i v e r o o t p e n e t r a t i o n of grass s e e d l i n g s . Agron. J . 51: 512. 36. Klapp, E. 1937. ( P r i n c i p l e s governing the value of herbage p l a n t s f o r hay and p a s t u r e u s e ) . Proc. 4th I n t . Grass Congress. Aberystwyth, Wales, 108-115. 37. L a v i n , P. 196l. G l a s s - f a c e d p l a n t e r box f o r f i e l d observ-a t i o n s on r o o t s . Agron. J . 53: 265-8. 38. L i n d a h l , J . , Davis, R.P., and Shepherd, W. 0. 1948. A p p l i c a t i o n of the t o t a l a v a i l a b l e carbo-hydrate method to the study of carbohydrate r e s e r v e s of switch cane (Arundinara t e c t a ) . P l a n t P h y s i o l . 24: 285-290. 39. McCarty, E. C. 1938. The r e l a t i o n of growth to v a r y i n g carbo-hydrate content i n meredian brome. Tech. B u i . 598. U.S.D.A. 40. Morgen, E. W. 1958. Growth and development i n c e r t a i n economic g r a s s e s . Ohio A g r i c . Exp. Sta., Agron. S e r i e s No. 147. 41 . Nedrow, W. W. 1937. S t u d i e s on the ecology of r o o t s . E c o l . 1 8 : 27-52. 42. Parmeijer, W. L. 1956. P a i n t i n g exposed r o o t s In s i t u f o r photographing. Queensland, J . Agr. S c i . 13: 66-7. 73 43. P a r t r i d g e , N. L. 1940. A c o n t a i n e r f o r growing p l a n t s f o r r o o t s t u d i e s i Am. Soc. Agron. J. 32_: 407-8. 44. Pavlychenko, T. K. 1942. Root system of c e r t a i n forage crops i n r r e l a t i o n t o the management of a g r i c u l t u r a l s o i l s . N a t i o n a l Research C o u n c i l of Canada Pub. No. 1088. 45. Pavlychenko, T. K. 1937. Q u a n t i t a t i v e study of the e n t i r e r o o t 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 . E c o l . 18: 62-79. 46. P e r a l t a , P. de 1935. Some p r i n c i p l e s of competition as i l l u s t r a t e d by Sudan g r a s s . E c o l . 5_: 335-404. 47. Plummer, A. P. 1943. The germination and e a r l y s e e d l i n g develop-ment of twelve range g r a s s e s . Am. Soc. Agron. J . 35: 17-34. 48. Roberts, J . L. and Olsen, P. R. 1942. I n t e r r e l a t i o n s h i p s of legumes and grasses i n a s s o c i a t i o n . J . Am. Soc. Agron. J . 34: 695-701. 49. Robocker, ¥. C , C u r t i s , J . T. and Ahlgren, H. L. 1953- Some f a c t o r s a f f e c t i n g emergence and establishment of n a t i v e grass s e e d l i n g s i n Wisconsin. E c o l . 34: 134-9. 50. Rosene, H. P. and W a l t h a l l , A. M. J . 1949. V e l o c i t i e s of water a b s o r p t i o n by i n d i v i d u a l r o o t h a i r s of d i f f e r e n t s p e c i e s . Bot. Gas. I l l : 11-21. 51. S c h u l t z , A. M. and B i s w e l l , H. H. 1956. Method f o r photographing r o o t s . J . For. 53: 138. ~~ 52. S o s u l s k i , P. W., P a t t e r s o n , J . K., and Law, A. G. i960. The l i g n i n content of grass s t r a i n s . Agron. J . 52: 130-134. 53- Schwendiman, J . L., H a f e n r i c h t e r , A. L., and Law, A. G. 1953- The p r o d u c t i o n of tops and r o o t s by grass and sweet c l o v e r when grown i n mixtures. Agron. J . 45: 110-114. 74 54. Simpson, M.J.A., and Moore, L. B. 1955- S e e d l i n g s t u d i e s i n f e s c u e - t u s s o c k grassland, I. Some e f f e c t s of shading, c u l t i v a t i o n , and ' f r o s t . N.Z. J . S c i . Tech. Sec. A. 37: 93-99. 55. Smith, Dale. 1962. 56. Somogyi, 1945. M. Carbohydrate r o o t r e s e r v e s i n a l f a l f a , r e d c l o v e r , and b i r d s f o o t t r e f o i l under s e v e r a l management schedules. Crops. S c i . 2: 75-78. A new reagent f o r the de t e r m i n a t i o n of sugars, J . B i o l . Chem. 160: 6l-68. 57. Sprague, M. A., I l n i c k i , R. D., Chase, R.W. and Kates, A. H. Growth of forage s e e d l i n g s i n competition w i t h p a r t i a l l y k i l l e d grass sods. Crop S c i . 2: 52-55; 58. Sprague, H. 1933. M. Root development of p e r e n n i a l grasses and i t s r e l a t i o n to s o i l c o n d i t i o n . S o i l S c i , 36: 189-209. 59. Stuckey, I. 1941. H. Seasonal growth of grass r o o t s . Bot. 28: 486-91. Am. J . 60. T a y l o r , H. I960. M. and Gardner, H. R. Use of wax s u b s t r a t e s i n r o o t p e n e t r a t i o n s t u d i e s . S o i l S c i . Soc. Am. Proc. 24: 79-81 61. T a y l o r , H. I 9 6 0 . 62. Troughton, 1951. 63. Troughton, 1956. M. and Gardner, H. R. R e l a t i v e p e n e t r a t i n g a b i l i t y of d i f f e r e n t p l a n t r o o t s . Agron. J . 52_: 579-81. A. Stud i e s on the r o o t s and storage organs of herbage p l a n t s . B r i t . Grass. Soc. J . 6: 197-206. A. St u d i e s on the growth of young grass 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 the r e l a t i o n s h i p between the shoot and r o o t systems. B r i t . Grass. Soc. J . 11: 56-65. 75 64. Troughton, A. 1957- The underground organs of herbage g r a s s e s . B u i . No. 44. Commonwealth A g r i c u l t u r a l Bureau, Farnham Royal, England. 65. Weaver, J . E. 1926. Root development of f i e l d crops. McGraw-H i l l Book Co., L t d . pp. 291. 66. Weaver, J . E. and Clements, P. E. 1938. P l a n t Ecology. McGraw-Hill Book Co., L t d . ed. 2nd. 67. Weinman, H. 1946. Determination of t o t a l a v a i l a b l e carbohydrates i n p l a n t s . P l a n t Physio. 22: 279-290. 68. Weinman, H. 1948. Underground development and r e s e r v e s of gr a s s e s . B r i t . Grass. Soc. J . 3_s 115-140. 69. W i l l i a m s , T. E. and Baker, H. K. 1957. S t u d i e s on the r o o t development of herbage p l a n t s . I . Techniques of herbage r o o t i n v e s t i g a t i o n s . B r i t . Grass. Soc. J . 12: 49-55. ADDENDUM Blac k , J . N. 1956. "The i n f l u e n c e of seed s i z e and depth of sowing on pre-emergence and e a r l y v e g e t a t i v e growth of subterranean c l o v e r . " Aust. J . A g r i c . Res. 7: 98-109. 76 ADDENDA Black, J . M. 1956. "The i n f l u e n c e of seed s i z e and depth of sowing on pre-emergence and e a r l y vegetative growth of subterranean c l o v e r " . Aust. J . A g r i c . Res. 7: 98-109. Evans, M. ¥. 1958. Growth and development i n c e r t a i n economic grasses. Ohio Agr. Exp. Sta. Agron. Serie s No. 147. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0105718/manifest

Comment

Related Items