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The influence of some culture conditions on growth of plant tissues in vitro Florian, Svatopluk Fred 1955

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THE INFLUENCE OF SOME CULTURE CONDITIONS ON GROWTH OF PLANT TISSUES IN VITRO  by SVATOPLUK FRED FLORLAN  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF. THE REQUIREMENTS FOR THE DEGREE OF Master o f ARTS  i n the Department o f Biology end Botany  We accept t h i s t h e s i s as conforming to the standard required from candidates f o r the degree o f MASTER OF ARTS  Members of the Department o f Biology and Botany THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1955  THE INFLUENCE OF SOME CULTUHE CONDITIONS ON GROWTH OF PLANT TISSUES IN VITRO by  S. F. F l o r i a n  ABSTRACT. The response o f various plant tissues to d i f f e r e n t conditions o f culture was compared. The tissues used were cambium-containing carrot roots, undifferentiated carrot c a l l u s , bacteria-free  discs from  sunflower  tumorous (crown-gall) t i s s u e , and segments o f sunflower stems. The culture conditions compared, i n combination, were agar versus l i q u i d medium, shaken versus non-shaken l i q u i d medium, and continuous l i g h t versus continuous dark. The response of the tissues to White's basal nutrient medium with added coconut milk (15 %) and indoleacetic a c i d (0.1 mg/1) and Hildebrandt's improved sunflower medium was also compared under these d i f f e r e n t culture conditions. A g i t a t i o n o f the l i q u i d medium was accomplished through the use o f a newly designed shaker, which consists b a s i c a l l y o f a h o r i z o n t a l l y o s c i l l a t i n g bank of shelves. The tissues rested on the bottom of culture flasks (medicine bottles) on these shelves and were a l t e r n a t e l y exposed to medium and a&r as the l i q u i d medium washed back and f o r t h . Any h o r i z o n t a l l y o s c i l l a t i n g platform could replace t h i s shaker and almost any type and size o f culture f l a s k could be used. Probably any type o f plant tissue could be cultured under these shaking conditions. I t i s not necessary that the tissues adhere to the walls o f the culture vessels as i n other a g i t a t i o n  2  methods so f a r used i n plant tissue culture. Growth (weight increase) of a l l tissues i n shaken l i q u i d medium (in both l i g h t and dark) was markedly superior  (two to s i x times greater  average weight i n 42 days) to that of tissues on agar and i n non-shaken l i q u i d medium. The  s u p e r i o r i t y o f growth i n shaken l i q u i d medium i s probably  due to several factors; nutrients and gasses are supplied to the entire surface of the t i s s u e s , there i s no drying and hardening of the tissue surfaces, r e s u l t i n g i n a greatly increased —'  surface area, harmful  excretions  can not c o l l e c t at the tissue surface, and d i f f u s i o n of nutrients i s not hindered by adsorptive e f f e c t s of agar p a r t i c l e s . To compare the growth o f these cultures with those of other workers using a g i t a t i o n methods i s d i f f i c u l t due to the d i f f e r e n t sources o f plant material, d i f f e r e n t sizes of tissues cultured, and d i f f e r e n t periods of culture used. In general the stimulatory r e s u l t s of shaking obtained appear to be at l e a s t as good as those obtained by Caplin and Steward with the much more elaborate and l i m i t e d *auxophyton*• There was  no sign of eventual  growth stoppage as obtained by White, using r o l l e r tubes. L i g h t consistently stimulated  tissues grown i n l i q u i d medium, p a r t i -  c u l a r l y those i n shaken l i q u i d medium. The e f f e c t was  e s p e c i a l l y marked on  carrot c a l l u s and tumorous sunflower tissues grown i n Hildebrandt's medium. It i s suggested that l i g h t may  play a role i n the synthesis of growth  factors supplied by coconut milk. L i g h t had no s i g n i f i c a n t e f f e c t on  the  growth of tissues on agar medium, i n d i c a t i n g that the primary l i m i t i n g factor i n the growth of such tissues may  be the rate of d i f f u s i o n of  nutrients from the agar. Carrot tissues showed better o v e r a l l growth i n the enriched White's  3  medium while the sunflower tumorous tissue grew better i n Hilda brandt's medium* The  e f f e c t on carrot was  probably primarily through indoleacetic  a c i d and coconut milk* The response of sunflower tissue i s d i f f i c u l t to evaluate at present* A l l carrot t i s s u e s developed chlorophyll throughout a l l of.the experiments i f cultured i n l i g h t , while tumorous sunflower tissue remained white u n t i l placed i n Hildebrandt's medium, when i t turned l i g h t green. The  significance of these differences i s not known. One  experiment shewed that carrot discs derived from d i f f e r e n t  carrots grew at s i g n i f i c a n t l y d i f f e r e n t average rates, i n d i c a t i n g that discs to be compared should be derived from the seme root. The plane i n which the discs were cut did not seem to influence subsequent growth. 'Intra root' v a r i a t i o n i n disc growth necessitates r e p l i c a t i o n .  AGKNOlLEDCTilENTS.  I am grateful to Dr. A.H.Hutchinson,  the former Head of the  Department o f Biology and Botany, and to Dr. T.M.C.Taylor,  the present  Chairman o f the Department, f o r recommending me to Dr. Coleman as h i s assistant and g i v i n g me thus the opportunity to s t a r t the experiments here reported. Dr. Hutchinson and Dr. Taylor took constant interest i n my work and gave me a great deal o f encouragement and help.  The l a t e Dr. L.C.Coleman i s g r a t e f u l l y remembered as a noble man  and an outstanding teacher. Dr. G.A.Setterfield, who took over  the supervision o f my research work a f t e r the t r a g i c death o f Dr. Coleman, did not spare time or trouble to help me with the planning o f experiments, analyzing and i n t e r p r e t i n g o f the r e s u l t s , and preparing of the manuscript; I wish to thank him f o r h i s generous assistance and guidance. I wish also to express my gratitude to the S t a f f o f the Dominion Laboratory o f Plant Pathology i n Saanichton,.B.C., who offered help to promote the success o f these experiments.  The study was carried out under a project supported by a granti n - a i d from the National Cancer Institute of Canada; I appreciate t h i s f i n a n c i a l help which made t h i s study p o s s i b l e .  TABLE 0? CONTENTS  Page I . INTRODUCTION AND REVIEW OP LITERATURE  1  I I . MATERIALS AND METHODS  • • 17  I I I . EXPERIMENTAL RESULTS  • 25  IV. DISCUSSION  • . • 39  V* SUMMARY  VI. LITERATURE CITED  49  . . . . . . . . . . .  52  INTRODUCTION AND REVIEW OF LITERATURE*  I . General background*  The culture o f Isolated plant parts (organs and tissues) In controlled nutrient media has been a major problem i n experimental botany since the turn o f the- century* In the beginning, the establishment o f growth i n v i t r o was simply an end i n i t s e l f but the emphasis has slowly s h i f t e d and today, tissue culture has become p r i m a r i l y a means f o r controlled experiment with l i v i n g processes* Despite, however, notable successes i n c u l t u r i n g plant t i s s u e s and s i g n i f i c a n t advances i n our knowledge which have cone about through the use o f the technique as an experimental t o o l , tissue culture s t i l l has many imperfections and weaknesses which prevent f u l l e x p l o i t a t i o n o f i t s many advantages. To appreciate both the presentday advantages and weaknesses of plant tissue culture i t i s desirable to b r i e f l y | c o n s i d e r i t s development and present status* Plant issue culture was f i r s t attempted by Haberlandt i n 1902* He used r e l a t i v e l y h i g h l y d i f f e r e n t i a t e d c e l l s (e.g. l e a f parenchyma) and very simple mineral nutrients; the experiment was unsuccessful but provided stimulus to other workers who took up the problem* I t was not, however, u n t i l 1922 that p a r t i a l success i n growing plant tissues a r t i f i c i a l l y was obtained* In t h i s year Kotte and Robbins independently  2  c u l t i v a t e d excised tomato roots f o r l i m i t e d periods o f time. Subsequently techniques were slowly improved and i n 1934, White established cultures o f excised tomato roots which were capable of unlimited growth i n culture* White (1943), considers the i n v i t r o c u l t i v a t i o n of excised roots to be organ culture rather than true tissue c u l t u r e . He tissue culture i n the s t r i c t  defines  sense as unlimited a r t i f i c i a l growth o f  undifferentiated t i s s u e . Cultures f u l f i l l i n g t h i s d e f i n i t i o n were obtained i n 1939  when Gautheret, Nobecourt, and White independently  established cultures o f u n d i f f e r e n t i a t e d plant c a l l u s which were apparently  capable of unlimited  sub-culture*  Since thewe o r i g i n a l researches the f i e l d o f plant tissue culture has been extensively and diversely developed. To outline the more recent advances i n the f i e l d i t w i l l be useful to  consider  separately the types of plant t i s s u e s which have been cultured and the general media and methods used.  I I . Types o f Plant Tissue  Cultures*  Plant tissue cultures can be conveniently  divided, according to  the o r i g i n of the plant parts used f o r c u l t i v a t i o n , into the following classes: A* Embryo culture B. Organ culture C. Callus culture D. Culture o f tumorous t i s s u e s E . Cultures o f other o r i g i n  3  While the f i r s t two groups, embryo and organ cultures, do  not  f u l f i l l the conditions o f White's d e f i n i t i o n of true tissue culture ( i . e . the tissue should be u n d i f f e r e n t i a t e d and capable o f unlimited growth), they are included i n t h i s discussion because the techniques used with them are b a s i c a l l y the same as those used i n the culture o f undifferentiated t i s s u e * I t should be further remarked that a l l the c e l l s i n undifferentiated c a l l u s cultures are not the same. Crown g a l l c a l l u s tissue of sunflower and hybrid tobacco c a l l u s tissue contain at l e a s t three d i f f e r e n t kinds of c e l l s : parenchyma c e l l s (large hypertrophic c e l l s ) , meristematic c e l l s (small hyperplastic c e l l s ) , and thick-walled scalariform c e l l s (wound tracheids)(White, 1947;  1939;  Caplin,  Stxuckmeyer et a l * , 1949)* Thus, White's use o f the term  'undifferentiated' i n h i s d e f i n i t i o n of true tissue culture applies at the h i s t o l o g i c a l and morhpological l e v e l s but not at the  cytologies!  level.  A* Embryo Culture.  Attempts to grow plant embryos on a r t i f i c i a l media started  t e a r l y i n the 20th century (e.g. Banning, 1954; i t was not u n t i l 1922  S t i n g l , 1907). However,  that Khudson succeeded i n germinating orchid  seeds on a r t i f i c i a l media under aseptic conditions* The orchid seed i s considered an embryo rather than a seed as i t consists of u n d i f f e r e n t i ated c e l l s and can germinate i n nature only i n symbiosis with f u n g i . • As media Ehudson used mineral s a l t s solutions, glucose and  fructose  as a carbohydrate source, and various plant extracts* He found extracts o f B a c i l l u s r a d i c i c o l a to be p a r t i c u l a r l y b e n e f i c i a l to the development  4  of seedlings. L a t e r Tukey (1933, 1934, 1938, and 1944) successfully cultured embryos o f sweet cherry, peach, and other stone f r u i t s . Considerable improvement i n the technique o f embryo culture was brought about by the introduction o f coconut milk into the nutrient media. T h i s substance was used f o r the f i r s t time by van Overbeek e t a l . (1941) f o r the c u l t i v a t i o n o f Datura embryos. As a r e s u l t i t became possible to grow much younger embryos than had been grown before (van Overbeek e t a l . , 1941, 1942, and 1944), and to obtain hybrids from incompatible crosses o f Datura (Blakealee and Satina, 1944). Following t h i s work embryo culture became widely used as a method o f overcoming embryo abortion i n crosses between incompatible v a r i e t i e s and species. Brink e t a l . (1944) obtained a hybrid between Hordeum Jubatum and Secale cereale; the hybrid embryo does not mature i n the seed but i t was possible to grow i t on an a r t i f i c i a l medium. S i m i l a r l y , Cooper and Brink (1945) were able to produce hybrids between d i p l o i d and t e t r a p l o i d races o f Lycopersicum  pimpinellifolium  through the use o f embryo c u l t u r e . As well as being a means f o r overcoming genetic incompatibility, embryo culture can also speed up breeding programs through the elimination o f dormant period o f seeds. The use o f embryo culture i n plant-breeding was discussed by Skirm (1942) and by Tukey (1944). Weeping cralyapple embryos have been cultured by N i c k e l l  (1951),  and potato embryos by Haynes (1954)» Hybrid embryos of many other plants have been successfully cultured, among them: tobacco, cotton, l i l y , i r i s , v i o l e t , ginkgo, pine, apple, pear, plum, rose, and o l i v e . A general review o f embryo culture has been given by Rappaport (1954).  5  B. Organ Culture* This category includes cultures o f isolated roots, stem apices, leaves, flowers, and f r u i t s * Roots have been most widely cultured o f these organs, p r i m a r i l y because t h e i r i s o l a t i o n , culture, and measurement i s r e l a t i v e l y easy. Tomato roots were used by White (1934) f o r e s t a b l i s h ment of the f i r s t p o t e n t i a l l y unlimited plant tissue c u l t u r e . Since then, tomato roots have been used f o r experimental  studies by many other  authors, among them by Robbins and Bartley (193?), Robbins and Schmidt (1939), Dormer and Street (1949), and Street (1953)* Many other plant species have been used f o r root culture* Bonner and Addicott (1937) cultured pea roots while Bonner and Derivian (1939) cultured, besides pell and tomato roots, roots of radish and f l a x * Bonner (1940) established the minimal n u t r i t i o n a l requirements f o r optimal growth of excised roots o f a l f a l f a , clover, cotton, Datura, carrot, and sunflower. Al mestrand (1949) cultured excised roots of barley and oats. The culture o f stem t i p s o f S t e l l a r i a m e d i a was attempted by White (1933) • Subsequently stem t i p s of Tropaeolum majus and of Lupinus albus were successfully grown by B a l l (1946), shoot t i p s of Psilotum nudum by Marsden and Wetmore (1953), and apices of Adianthum pedatum by Wetmore (1954). Leaves of tobacco were grown on a r t i f i c i a l media by Dawson (1938) and flowers and f r u i t s by Nitsch (1949). Pucher et a l . (1937) have made extensive use o f excised l e a f cultures (e.g. tobacco and rhubarb) i n the study of organic a c i d metabolism i n plants. ,  The l a t t e r cultures, i . e . l e a f , flower, and f r u i t cultures, are  only temporary and end with the f u l l development o f the cultured organ* Cultures o f roots and stem apices, which contain active meristems, can be continued i n d e f i n i t e l y .  C. Callus Culture. The term c a l l u s culture i s generally used to describe the culture of undifferentiated tissue derived from cambium or other meristematic or p o t e n t i - ' a l l y meristematic t i s s u e . Gautheret's (1959) f i r s t tissue culture capable of i n d e f i n i t e growth was derived from the cambium o f carrot roots while White's (1939) came from procembium o f Nicotiana stems. Since these o r i g i n a l experiments, c a l l u s cultures have been obtained from cambium o f many plant species, e.g. Ulmus campestria (Gautheret, 1940), T i t i s v i n i f e r a (Morel, 1944), Rosa sp. (Hobecourt and Koefler, 1945), Helianthus annuus and Vinca rosea (deRopp, 1947), Scorzonera sp. (Gautheret, 1948), S a l i x caprea (Gautheret, 1950), and ferns (Morel and Wetmore, 1951). To the group o f c a l l u s cultures derived from meristems other than cambium belong cultures of undifferentiated tissue p r o l i f e r a t e d from embryos cultured on a r t i f i c i a l media. Such cultures were i s o l a t e d from pro-embryos o f Datura by van Overbeek et a l . (1941, 1948), and from seedlings o f Pinus cultured by Loewenberg and Skoog (1958)• Curtis (1947) was able to produce undifferentiated tissue cultures from embryos of orchids by adding barbiturates (e.g. 10 ppm phenyl ethyl b a r b i t u r i c acid) to the culture media* Though derived from embryos, a l l o f these cultures are true tissue cultures i n the sense o f White's s t r i c t d e f i n i t i o n , as the growth i s undifferentiated and can be continued  7  indefinitely. Several^ c a l l u s cultures have been developed from p o t e n t i a l l y meristematic t i s s u e s . Caplin and Steward (1948, 1949, and 1952) obtained c a l l u s cultures from secondary phloem o f carrot. Cultures have also been derived from storage tissue of potato tuber (Steward and Caplin, 1951), and from similar tissues o f sweet potato and Jerusalem artichoke (Wetmore, 1954). I t should be noted that while cambium produces c a l l u s  'spontane-  ously' i t i s necessary to treat p o t e n t i a l l y meristematic tissues with growth-promoting hormones such as indoleacetic acid (Gautheret, 1946) or coconut milk (Caplin and Steward, 1948) before they w i l l s t a r t  cell  p r o l i f e r a t i o n . However, once removed from contact with organized plant t i s s u e s , a l l c a l l u s cultures normally require an exogenous  source o f  growth-hormone (see below).  C. Cultures of tumorous t i s s u e .  The majority of culture work with tumorous tissue has been c a r r i e d out on tissue derived from tumors o r i g i n a l l y induced by b a c t e r i a o f the genus Agrobactarium (commonly c a l l e d crown g a l l b a c t e r i a ) . Tissue from such tumors i s s i m i l a r to normal undifferentiated c a l l u s i n  general  c e l l u l a r makeup and growth habit but i t requires somewhat d i f f e r e n t techniques of i s o l a t i o n and i s capable of growing on media without growth-hormone. The major problem has been to obtain i n culture,tumorous t i s s u e free of the i n c i t i n g b a c t e r i a . White and Braun (1942) f i r s t i s o l a t e d and cultured bacteria-free crown g a l l tissue from secondary tumors on sunflower. Similar cultures  8  from primary g a l l s on sunflower were l a t e r obtained by deRopp (1947)• Heat treatment was used by Braun (1947) to k i l l the b a c t e r i a i n crown g a l l tissue on Vinca rosea, allowing subsequent culture of bacteria-free t i s s u e . Bacteria-free crown g a l l cultures have been obtained from many other plants, e.g. Scorzonera sp. and Helianthus tuberosus  (Gautheret,  -I 1947, and 1948), and V i t l s v i n i f e r a  and Antirrhinum majus (Morel, 1948).  The problem of crown g a l l and the r e s u l t s achieved i n culturing of tumorous tissue have been reviewed by many authors, among them by Gautheret  (1950), White (1951), and deRopp (1951).  Tumors on plants can be also i n i t i a t e d by otheryigents than Agrobacterium  sp. Tumors on roots of Rumex acetosa induced by wound  v i r u s were cultured by N i c k e l l and Brakke (1954). Genetic incompatibility causes the appearance of spontaneous tumors i n certain crosses o f Nicotiana species (Kostoff, 1930). T h i s Nicotiana hybrid tumor tissue has been cultured very extensively by White (1939), Caplin (1947), Hildebrandt et a l . (1945, and 1946), and Hildebrandt and Riker  (1947).  E. Cultures of other o r i g i n . Tulecke (1953) succeeded i n culturing undifferentiated tissue derived from pollen of Ginfego b i l o b a . The endosperm of maize has been cultured by Strauss and L a Rue  (1954)• Cultured endosporm appears to be  a good source of material f o r observation of mitoses i n vivo (Bajer and Mole-Bajer, 1954). Northcraft (1951) used ammonium oxalate i n a l i q u i d medium to dissolve the middle l a m e l l a of c e l l s i n carrot c a l l u s cultures. In doing so he claims to have obtained cultures o f carrot c a l l u s tissue derived from single c e l l s .  9  Jablonski and Skoog (1954) have cultured p i t h tissue from tobacco. Through the use of indoleacetic a c i d and coconut milk or water extracts o f vascular tissue they obtained continuous c e l l d i v i s i o n and p r o l i f e r a t i o n o f the p i t h c e l l s *  I I I * Tissue Culture Madia* The nutrient media used f o r plant tissue culture consist b a s i c a l l y of mineral s a l t s , carbohydrate, amino acids, vitamins, and growthpromoting hormones (Gautheret, 1942; White, 1943, and 1954). The mineral s a l t s generally used are b a s i c a l l y the same as those used f o r c u l t i v a t i o n of i n t a c t plants i n water cultures. Gautheret (1935) used modified Enop's solution and White (1934, 1943, 1954) used a modification o f the solution o f TTspenski and Uspenskaja. White's o r i g i n a l mineral solution consisted of the following s a l t s : MgS0 , 4  Ca(N0 ) , Na S0 , KNOg, KC1, NaHgPC^, F e ( S 0 ) , MnS0 , ZaS0 , HgBOg, 3  2  2  4  2  4  3  4  4  and K I . T h i s solution supports growth o f most tissues and has been extensively used i n plant tissue culture. More recently some changes which produce increased tissue growth have been proposed. B o l l and Street (1951) suggested the addition o f the trace elements molybdenum and copper. Hildebrandt et a l . (1946) replaced f e r r i c sulphate with the more stable f e r r i c t a r t r a t e . Hildebrandt et a l . (1946) also extensively investigated the optimal concentration requirements f o r the mineral s a l t s and developed improved media f o r sunflower and tobacco t i s s u e s . Different sugars i n varied concentrations have been t r i e d by many workers i n attempts to f i n d the best source o f carbohydrate f o r tissue culture. Thai more important among these studies were those by  10  by Knudson (1984) with cultures of orchid embryos, White (1940) with tomato roots, Bonner (1940) with excised roots of several plant species, Hildebrandt et a l * (1945) with tobacco and sunflower tissue cultures, Dormer and Street (1949), Street and Lowe (1950), Street and McGregor (1952) with culture o f excised tomato roots, and Rappaport (1954) with the culture of plant embryos* In most of these investigations sucrose proved to be superior to any other carbohydrate; dextrose and levulose also gave excellent r e s u l t s * Mannose, maltose, c e l l o b i o s e , galactose, and r a f f i n o s e were s a t i s f a c t o r y f o r some species but poor f o r other species* Generally used now  i s e i t h e r 8 % sucrose (White's and H i l d e -  brandt* s media), or 3-5 % dextrose (Gautheret's medium)* As a source of organic nitrogen White's medium contains glycine and Gautheret's medium cysteine* Addicott and Bonner (1938) used a mixture o f seven d i f f e r e n t amino acids f o r culture of pea roots but these were l a t e r found to be unessential for optimal growth* Various organic and inorganic nitrogenous compounds were tested by Riker  and  Gutsche (1948). These authors found that glycine was not e s s e n t i a l f o r continuous growth o f sunflower g a l l tissue and recommended further experiment with n i t r a t e , urea, alanine, aspartic a c i d , and glutamic  acid.  These substances are claimed to be not only a superior sourte o f nitrogen, but also to have a separte stimulatory e f f e c t on growth o f t i s s u e . The vitamin requirements o f d i f f e r e n t organs and tissues i n culture have been studied widely. The necessity of vitamin B^  (thiamine)  f o r the growth of the majority o f plant t i s s u e s i n v i t r o has been established by the studies of White (1937, and 1940), Robbins and Bartley (1937), Robbins (1939), Bonner (1937, 1938,  and 1940), and Bonner and  11  Devirian (1939). S i m i l a r l y , the tissue culture requirements f o r vitamin B  6  (pyridoxine)  and n i c o t i n i c acid have been studied by Robbins and  Schmidt (1939), White (1940), and Bonner (1938, and 1940); most tissues (and organs) require these vitamins f o r sustained growth* Thiamine, pyridoxine,  and n i c o t i n i c a c i d are now  included i n a l l basic tissue  culture media although c e r t a i n cultures w i l l grow i n the absence o f or the other o f them, e.g. roots o f a l f a l f a do not require  one  pyridoxine  f o r optimal growth (Bonner, 1940)* In addition to these vitamins Gautheret (1950) uses b i o t i n and Ca-pantothenate f o r the culture o f c e r t a i n t i s s u e s , e.g. S a l i x caprea. The  influence o f growth-hormones has been studied extensively i n  cultured normal c a l l u s and i n tumorous t i s s u e s . The work o f Gautheret (1937, and 1939), Duhamet (1939), and others lead to the  conclusion  that indoleacetic a c i d or some other growth-hormone (e.g.naphtalenea c e t i c a c i d or indolebutyric acid) i s indispensable  for.the growth  i n v i t r o of normal plant c a l l u s t i s s u e * An exception to t h i s statement occurs i n the case o f 'habituated' c a l l u s t i s s u e , f i r s t obtained by Gautheret (1948a)* Habituated tissue i s normal c a l l u s tissue which i n the course o f continuous culture has l o s t the need f o r an exogenous supply o f growth-hormone. The mechanism o f t h i s metabolic change i s not understood. Bacteria-free tumorous tissue behaves the same as habituated c a l l u s tissue i n t h i s respect and grows optimally without supplied growth-hormone (Gautheret, 1947)* Tissue cultures have been widely used i n attempts to elucidate the action of growth-hormones i n t i s s u e * The e f f e c t s of indoleacetic a c i d on water absorption by potato tuber tissue have been studied  12 by Commoner et a l . (1942), the h i s t o l o g i c a l e f f e c t s of growth-hormones on crown g a l l tissue by Struckmeyer et a l . (1949), the e f f e c t on growth and r e s p i r a t i o n o f artichoke tissue by Hackett and Thimann (1952), and the influence on meristematic a c t i v i t i e s o f tomato roots by Street (1953). Skoog and Tsui (1948) and Skoog (1951, and 1954)  studied the e f f e c t s o f  growth-honnones on growth, d i f f e r e n t i a t i o n , and organ formation i n c a l l u s culture, and Jablonski and Skoog (1953) examined the e f f e c t o f growth-hormones on c e l l enlargement and c e l l d i v i s i o n i n i s o l a t e d tobacco p i t h t i s s u e . The growth-promoting e f f e c t of coconut milk ( l i q u i d endosperm) on plant tissue cultures was  f i r s t noticed by van Overbeek et a l . (1941) i n  c u l t u r i n g of Datftra embryos. Coconut milk increases the rate o f growth of plant tissue cultures considerably and i s superior to other plant extracts such as tomato j u i c e (Nitsch, 1951). The e f f e c t o f d i f f e r e n t concentrations of coconut milk has been thoroughly studied by Caplin and Steward (1948, 1949,  and 1952), Duhamet (1951a, b, and c ) , and  Cutter and Wilson (1954). The optimal concentration l i e s between 10  and  20 % f o r the majority o f plant t i s s u e s . There i s obviously a d i f f e r e n t content o f the growth-promoting substance i n milk obtained from d i f f e r e n t nuts. The growth-promoting e f f e c t of coconut milk on plant tissue cultures i s s i m i l a r to the e f f e c t of embryo extract on animal tissue cultures. Recently M i l l e r and Skoog ( i n preparation) have i s o l a t e d from coconut milk a factor o f unknown structure which promotes c e l l d i v i s i o n i n i s o l a t e d p i t h tissue and accelerates growth o f tobacco c a l l u s . I t i s to be hoped that a l l active agents i n coconut milk w i l l eventually be  13  chemically characterized as the use o f coconut milk i n plant tissue culture at present introduces unknown factors which hamper c o n t r o l l e d experimentation  and i n t e r p r e t a t i o n of r e s u l t s *  The influence o f environmental conditions on plant tissue cultures has been studied s u f f i c i e n t l y to e s t a b l i s h optimal temperatures, i o n concentration, osmotic pressure, and pH o f media (White, 1932, and 1943; Hildebrandt e t a l . , 1945, and 1946). Optimal values o f these f a c t o r s vary with d i f f e r e n t kinds o f t i s s u e s cultured. For example the optimal temperature f o r growth o f tobacco c a l l u s tissue was found to be 26-32°C and f o r sunflower  c a l l u s tissue 24-28°0. The optimal pH f o r the former  tissue i s 5.0-5.9, and f o r the l a t t e r tissue 5*5-5.9 (Hildebrandt e t a l * , 1945)* While same authors have found that l i g h t has no influence on rate o f growth o f cultured t i s s u e s (Hildebrandt et a l * , 1945), Caplin and Steward (1952) reported a s l i g h t growth-promoting Influence o f l i g h t on the c a l l u s p r o l i f e r a t i o n o f cultured carrot discs* Bunning and Welte (1953) obtained s i g n i f i c a n t differences i n the rate o f growth o f c a r r o t disc cambium under d i f f e r e n t periods o f i l l u m i n a t i o n *  IV* Tissue Culture Technique* For successful c u l t i v a t i o n o f plant tissue i t i s necessary to supply i t with nutrient medium and a i r . The standard technique  i s to  use l i q u i d media f o r root cultures and semi-solid o r s o l i d media (with 0.5-2 % agar) f o r other kinds o f tissue c u l t u r e s . The roots f l o a t on the surface o f the l i q u i d medium and are thus aerated. Tissues on agar media obtain nutrients by d i f f u s i o n from the agar at points o f contact while the greater part o f the tissue i s d i r e c t l y exposed to the a i r .  14 L i q u i d media have not been genrally used f o r cultures other then roots because the tissue does not f l o a t on the medium and consequently s u f f e r s from the l a c k of a i r * White (1939) t r i e d growing c a l l u s tissue i n l i q u i d medium and found that growth was  considerably  slower than on agar and  that h i s t o l o g i c a l d i f f e r e n t i a t i o n occured and roots appeared* He presumed both effedts to be a r e s u l t o f the r e l a t i v e l y anaerobic conditions present beneath the surface of the l i q u i d medium where the^Qasue l a y . I t was  discovered by the workers with animal tissue culture that  a l t e r n a t i n g submersion o f tissue i n l i q u i d medium and exposure to s t e r i l e a i r i n the culture vessel would provide f o r increased rate of growth* Using t h i s p r i n c i p l e Gey and Gey  (1936) developed the r o l l e r tube method*  Cultures are grown on the walls o f tubes f i l l e d with small amount (1-2 ml) o f l i q u i d medium and the tubes are revolved along t h e i r l o n g i tudinal axis on a special drum* The cultures are thus a l t e r n a t e l y washed by the medium and exposed, for longer periods o f time, to the a i r i n the tube* The method was  subsequently modified by Shaw et a l * (1940). These  workers substituted f o r the culture t u b e s , r o l l e r b o t t l e s with a hole i n one  side closed by a cover glass* This cover glass permitted direct  microscopic observations on the tissue culture c e l l s i n vivo* Other modifications, such as introduction o f perforated cellophane to hold the culture, were devised l a t e r . Such techniques are not e a s i l y applied d i r e c t l y to plant tissue cultures as the plant cultures grow as r e l a t i v e l y massive, s o l i d lumps o f tissue and do not r e a d i l y adhere to the walls of the tubes (as do animal cultures) • However, the p o s s i b i l i t y o f accelerating growth of plant tissue cultures through the use of agitated l i q u i d medium 1  15  attracted some workers* deRopp (1946) constructed an apparatus consisting b a s i c a l l y of a U-tube with l i q u i d medium i n one arm and the tissue culture i n the other arm* The tissue r e s t s on some suitable support, such as quartz sand, and the medium i s supplied to i t by t i l t i n g the apparatus on a rack. A f t e r submerging the tissue i n the l i q u i d medium the apparatus i s returned to the o r i g i n a l p o s i t i o n and the tissue exposed to the a i r i n the tube* Another method was devised by Caplin and Steward (1949) and used for extensive studies c a r r i e d out by these authors (Caplin and Steward, 1952; Steward and Caplin, 1952a, b; Steward et a l . , 1952). These workers use a new type o f culture tube which i s closed and rounded at both ends, with a narrower open tube attached, at r i g h t angles, i n the middle of the l a r g e r tube..The tissue s t i c k s to the wall o f one o f the rounded ends o f the main tube. The tube i s attached to a c i r c u l a r disc and revolved at the speed 1 r.p.m., causing the l i q u i d medium to flow slowly from one end of the tube to the other, thereby a l t e r n a t e l y immersing the tissue i n the medium and exposing i t to the a i r i n the tube. The smaller side-neck tube (plugged with cotton) provides f o r the entrance to the tube and a i r exchange. The apparatus was o r i g i n a l l y named revolving k l i n o s t a t and renamed auxophyton i n 1952* White (1953) used the r o l l e r tube method o f Gey and Gey (1936) f o r plant tissue cultures. A l l o f these attempts to u t i l i z e a g i t a t i n g methods i n plant tissue culture have had success i n accelerating growth. However, i n general they are somewhat cumbersome and require c o s t l y glassware and r o t a t i n g devices. In 1952 the l a t e Dr.L.C.Coleman began experiments to develop an a g i t a t i n g method f o r growth acceleration that would be, at the same  16  time, simple, reasonably inexpensive, and r e a d i l y adaptable to l a r g e scale experiments with a l l types o f plant tissue cultures. The present author joined Dr. Coleman e a r l y i n h i s studies along these l i n e s . This report describes the methods eventually developed and the r e s u l t s o f experiments designed t o measure the comparative growth response of several tissues to these and other methods o f tissue culture. The study had, i n e f f e c t , a two-fold aimj} to empirically(develop conditions f o r optimal growth of plant tissue cultures and, using these methods, to gain some insight into the metabolism of various cultured t i s s u e s .  17  MATERIALS AND METHODS. I . Plant M a t e r i a l . In the present experiments these d i f f e r e n t plant materials were used: A. Fresh carrot discs B. Undifferentiated tissue of carrot C. Bacteria-free sunflower tumor tissue D. Stem sections o f sunflower  A. Fresh carrot d i s c s . Carrot discs were cut from carrot roots o f unknown v a r i e t y bought i n l o c a l stores. The roots were washed i n 50 $ alcohol, s t e r i l i z e d f o r 20 min. i n a 0.1 % aqueous solution o f mercuric c h l o r i d e , and subsequently rinsed i n s t e r i l e water. In a s t e r i l e transfer chamber discs f o r culture were cut from the middle t h i r d of the s t e r i l i z e d roots. In preliminary experiments discs with t h e i r diameters p a r a l l e l and at r i g h t angles to the l o n g i t u d i n a l axis o f the root (longitudinal and transverse  discs respectively) were used. To obtqin l o n g i t u d i n a l  discs, r a d i a l cylinders were bored out across the roots with a cannula or a cork borer and then cut into discs with a multibladed cutter s i m i l a r to that used by Caplin and Steward (1949). Only discs  containing  cambium were used f o r c u l t i v a t i o n . Transverse discs were obtained by cutting a transverse  s l i c e , 1 mm thick, across the root with a two-bladed  c u t t e r . (The blades i n t h i s cutter are o f f s e t about 3 mm so that when  18  the upper blade has cut completely across the carrot, and thus cut o f f the unusable portion, the lower blade has not completely severed the s l i c e . Leaving the s l i c e p a r t i a l l y attached to the carrot root i n t h i s manner provides support f o r the s l i c e which makes the cutting of s t e r i l e discs from i t easy)* Discs were cut from the transverse s l i c e with a cannula o r a. borer so that the cambium was running completely across t h e i r diameters. Discs o f various diameters were tested i n preliminary experiments. Discs cut with a 1.8 mm cannula weighed 2 mg, with a 4 mm borer 18 mg, and with a 6 mm borer 42 mg each. Discs 6 mm i n diameter were eventually found to give the most uniform growth and were used i n the main experiments reported here.  B. Undifferentiated tissue o f carrot.  /  Undifferentiated carrot tissue used i n these experiments was derived from c a l l u s p r o l i f e r a t e d by the cambium o f fresh carrot discs i n culture. Fresh transverse carrot d i s c s weighing 18 mg (4 mm i n diameter, see above) were cultured i n agitated l i q u i d White's basal medium with added indoleacetic a c i d and coconut milk: (see below) f o r 14 weeks. They were transferred to fresh media every two weeks during t h i s period. At the end o f t h i s time i r r e g u l a r l y shaped masses o f u n d i f ferentiated t i s s u e , yellowish green with reddish spots, and covered with mamillary outgrowth had formed. These masses of u n d i f f e r e n t i a t e d c a l l u s tissue were cut with a scalpel into small pieces, approximately 80 mg each, and sub-cultured. The culture was maintained and expanded by continuous sub-culture, and the sub-cultured pieces o f c a l l u s were used  19 i n the experiments on tissue culture conditions reported below.  C. Bacteria-free sunflower tumor t i s s u e . Bacteria-free sunflower tumor tissue was i s o l a t e d from a small secondary tumor which developed on the main vein o f a l e a f o f a sunflower plant eight weeks a f t e r the plant was inoculated with Agrobacterium tumefaciens. The secondary tumor was washed i n 50 % alcohol, s t e r i l i z e d for a few minutes i n a 0.1 % aqueous s o l u t i o n o f mercuric chloride, and opened a s e p t i c a l l y . Small pieces of tissue were cut out with a s c a l p e l , cultured i n agitated l i q u i d White's basal medium (see below) f o r 8 weeks, and then sub-cultured. Stock sub-cultures o f the tissue were transferred every two weeks and used .  .. f o r experiments.  D. Stem sections o f sunflower.  In one of the preliminary experiments segments o f sunflower, stem were used. When young sunflower plants were about 5 inches t a l l the leaves were removed and the upper parts o f the stems were cut o f f . The excised stem pieces were washed i n alcohol, s t e r i l i z e d f o r 10 min. i n -the  0.1 % mercuric chloride, and rinsed i n s t e r i l e water. of each stem piece was peeled o f f and the f i r s t  Finally,"epidermis  intemode cut into 4 mm  long segments which were used d i r e c t l y i n the experiment.  (  I I . Handling o f M a t e r i a l . A l l transfers o f tissue cultures were c a r r i e d out i n a transfer  I chamber; the instruments used were dipped i n alcohol and flamed.  20 The amount o f b a c t e r i a l and fungal contamination was 10-15  at f i r s t about  fo but t h i s was lowered to about 5 # when a special small t r a n s f e r  chamber was used* (This part of the experiments was  c a r r i e d out i n the  Dominion Laboratory of Plant Pathology i n Saanichton, B.C.  Because o f  the great amount o f plant pathological work that was being done In the laboratory i t was  d i f f i c u l t to keep the cultures free from contamination  during transfers and weighing) • Afjrer the laboratory was moved to Vancouver a transfer chamber that could be steam-sterilized was used and almost no contamination was  subsequently encountered.  Increase i n wet weight was used as a measure o f growth o f the tissue c u l t u r e s . Tissue pieces from l i q u i d media were surface-dried with s t e r i l e f i l t e r paper i n s t e r i l e P e t r i p l a t e s , transferred into tared s t e r i l e P e t r i p l a t e s , weighed, and returned to fresh media. Tissue  pieces  from agar media were freed of adhering agar and r i n s e d i n s t e r i l e double d i s t i l l e d water before surface-drying and weighing. Contaminated cultures were u s u a l l y discovered within two  dajcs a f t e r t r a n s f e r ; they were immedi-  a t e l y surface-dried, weighed, and discarded. The weight was fr  subtracted  om the o r i g i n a l t o t a l weight o f tissue i n the treatment.  III.  Growth Conditions.  JL. Glassware. Medicine b o t t l e s o f 170 ml i n l i q u i d media and v i a l s 22x94 mm  content  were used f o r cultures grown  f o r cultures grown on agar media.  B o t t l e s end v i a l s were closed with cotton plugs wrapped i n cheese c l o t h which had been previously b o i l e d i n d i s t i l l e d water. A l l glassware used i n the preparation of media and f o r c u l t u r i n g was  a c i d cleaned i n a  21  saturated solution of potassium dichramate  i n concentrated sulphuric  acid, and subsequently rinsed ten times i n hot running water, twice i n d i s t i l l e d water, and twice i n double d i s t i l l e d water*  B. Madia* Two basal media were used i n the experiments: White's standard medium (1943, and 1954*) and Hildebrandt*s Improved sunflower tissue medium (Hildebrandt et a l * , 1946)* Except i n preliminary experiments (see r e s u l t s section) White's medium was supplemented with 15 % coconut milk and 0*1 mg/l 0.01 mg/l  indoleacetic a c i d , and Hildebrandt's medium with  indoleacetic a c i d .  Composition of media i s given on next page. Double d i s t i l l e d water ( f i r s t d i s t i l l a t i o n i n a metal second i n a Pyrex-glass s t i l l ) was used throughout the  still,  experiments.  CP grade reagents were used e x c l u s i v e l y . Stock mineral s a l t  solutions  were kept at room temperature while stock solutions of vitamins and o f indoleacetic a c i d were stored i n a freezer. Fresh stock solutions o f a l l chemicals were prepared every s i x weeks. Coconut milk was obtained from mature coconuts. Each nut  was  opened and the milk poured into a stender and examined f o r q u a l i t y . Disintegrating milk (cloudy and odoured) was discarded. The good q u a l i t y milk was mixed, f i l t e r e d ,  d i s t r i b u t e d i n 150 ml aliquots, and stored i n  a deep-freezer. Preliminary experiments  showed that there was no difference  *The concentration o f vitamins i n White's 1954 book (p.74) i s given, by a mistake, ten times stronger than i t should be.  22  Composition o f Media:  Nutrient ingredient  White's basal medium  NagSg  Hildebrandt's improved sunflower t . medium  200.00 mg  100.00 mg  Ca(N0 ) .4 HgO  200.00 »  800.00 "  MgSO .7 H_0 4 2  360.00 "  720.00  3  2  M  KNO,  80.00 "  160,00 "  KC1  65.00 "  130.00 "  NaH^O^HgO  16.50 "  132.00 •  Fe ( C H O ) F e2( S 4 0 )4 .66 3HgO J  2  4  3  MnSO .4 H O 4 2 ZnS0„.7 H„0 4 S HJ30,,  5.00 " 2.50 « 4.50 "  4.50 "  1.50 "  3.00 "  1.50 «.  3.00 »  KI  0.75 "  0.375"  glycine  3.00 "  12.00 "  nicotinic acid  0.50 "  thiamine  0.10 "  0.10 *  pyridozine  0.10 "  0.80 "  o o  sucrose water  20.00 g 1000.00 ml  •  20.00 g 1000.00 ml  23  i n the growth o f cultures on media with autoclaved coconut milk and those on media with f i l t e r s t e r i l i z e d coconut milk. In experiments reported here coconut milk was added to the media before autoclaving. fhe pH o f the media was always adjusted with 0.1 N NaOH to approximately 5.8 before autoclaving and i n some experiments checked a f t e r autoclaving and also a f t e r a 3-week culture period. No  significant  changes i n pH occurred during autoclaving and only s l i g h t r i s e s (less,than 0.5 pH units) resulted a f t e r 3-week culture of t i s s u e . Both s o l i d and l i q u i d media were used i n the experiments. For s o l i d media 1 % agar (Difco) was added to White*s medium and 0.5 % to Hildebrandt's medium. Madia were aatoclaved f o r 20 min. at 15 l b . per square inch. Ten ml o f medium per culture (both l i q u i d and solid) were used throughout the experiments.  C. A g i t a t i o n .  The major experiments here reported are concerned with measuring the growth response of tissues cultured i n agitated l i q u i d media. A g i t a t i o n o f the l i q u i d media was obtained by p l a c i n g the culture b o t t l e s on a mechanical shaker o r i g i n a l l y conceived by Dr.L.C.Coleman, and constructed by Mr.C.J.Lines of the Dominion Laboratory of Plant Pathology. Saanichton, B.C. This shaker b a s i c a l l y consists of a r i g i d bank o f f i v e wooden shelves, 26x76 inches each, spaced 12 inches above each other. The bank o f shelves i s hung on a movable supporting frame and i s o s c i l l a t e d h o r i z o n t a l l y £ o f an inch, 82 times per minute. O s c i l l a t i o n i s provided by a cam-shaft driven by a £ HP e l e c t r i c motor. Culture b o t t l e s (medicine bottles) were l a i d on the shelves with t h e i r long axis p a r a l l e l  24 to the d i r e c t i o n of o s c i l l a t i o n . The o s c i l l a t i o n caused the medium to wash back and f o r t h on the bottom o f the b o t t l e * thereby a l t e r n a t e l y exposing the tissue inside to medium and a i r .  D. L i g h t conditions. The growth response of tissue cultures to conditions of continuous l i g h t and dark were also studied. Continuous l i g h t i n g of the shaken cultures was accomplished by four 40 W fluorescent l i g h t s suspended on the underside o f each s h e l f o f the shaker. These l i g h t s provided an illumination i n t e n s i t y o f 400 foot-candles to the upper surface of the s h e l f immediately below. To obtain dark conditions on the shaker one s h e l f was covered with thick cardboard and the l i g h t s above extinguished. Cultures which received no shaking were kept on separate stationary shelves with the same l i g h t and dark conditions as those on the shaker.  E . Temperature and Humidity.  Culture was c a r r i e d out i n an insulated room with thermostatically controlled a i r - c o n d i t i o n i n g . The cooling e f f e c t of the air-conditioner and heating action of the l i g h t s were balanced so that the temperature within the culture b o t t l e s remained at 2511° C. L i g h t s beneath the shelves were used to maintain t h i s temperature i n the cultures grown i n dark. The humidity i n the room was maintained at approximately 80 # through / evaporation o f water from large trays.  r  25  EXPERIMENTAL RESULTS.  A preliminary experiment was performed to test the influence o f coconut milk and indoleacetic a c i d (IAA) on the development of c a l l u s tissue from cultured carrot d i s c s . Fresh carrot discs weighing 18 mg were cultured f o r three weeks i n White's basal medium alone and i n combination with IAA (0.1 mg/l) and coconut milk (15 % ) . The cultures were grown i n shaken l i q u i d medium under continuous l i g h t . The r e s u l t s of t h i s experiment are summarized i n Table I . Indoleacetic a c i d had no s i g n i f i c a n t e f f e c t on growth, e i t h e r i n the basal medium alone or i n combination with coconut m i l k . On the other hand, coconut milk gave a h i g h l y s i g n i f i c a n t stimulation of growth with or without IAA. The average weights o f tissues cultured i n media with coconut milk were approximately double those o f tissues i n the media without coconut m i l k . A second preliminary experiment was designed to compare the growth rate o f fresh carrot discs derived from d i f f e r e n t carrot roots and from d i f f e r e n t planes within the same carrot root. Discs i n both transverse and longitudinal planes (see Materials and Methods) were cut from four d i f f e r e n t carrot roots and cultured f o r three weeks, under l i g h t , i n . shaken l i q u i d White's basal medium with added IAA (0.1 mg/l) and coconut milk (15 % ) . The r e s u l t s are summarized graphically i n Figure 1 and are s t a t i s t i c a l l y analyzed i n Table I I . The average growth o f carrot discs derived from d i f f e r e n t carrots showed considerable v a r i a t i o n , the extremes being highly s i g n i f i c a n t (explants from carrot 1 grew approximately twice as fast as those from  26  TABLE I* Results o f experiment designed to test the e f f e c t o f i n d o l e a c e t i c a c i d and coconut milk on the growth o f fresh carrot discs*  A. Mean wet weights o f carrot discs a f t e r three weeks o f culture. O r i g i n a l weight of each disc was 18 mg. Wh - White's basal medium, Wh IAA - Wh plus 0.1 mg/1  indoleacetic acid, Wh CM -  Wh plus 15 % coconut milk, and Wh IAA CM - Wh p l u s 0*1 mg/1 indoleacetic a c i d and 15 % coconut milk* t  Medium  Mean weight, i n mg, o f f i v e carrot discs  Wh  45.0  Wh IAA  58.6  Wh CM  86.8  Wh IAA CM  87.2  B. Analysis o f variance.  Source  D.F.  Total Between media Within media  S.S.  M.S.  19  16,960  3  10,318  3,439  16  6,642  415  L.S.D. - 5 % l e v e l - 27.3 mg 1 % l e v e l - 37.6 mg  F  8.3**  27  carrot 4)* Only s l i g h t mean differences occurred between discs from d i f f e r e n t planes o f the same root. The standard deviations and c o e f f i c i e n t s o f v a r i a b i l i t y show that there i s noticeable v a r i a t i o n i n growth o f d i s c s derived from the same root. The degree o f t h i s 'intra-root' growth v a r i a t i o n seems to change from carrot to carrot (range o f C.V. 14.03 - 30*36) but i s e s s e n t i a l l y independent on the plane i n which discs are taken (mean  C.V*:  transverse discs, 22.94; l o n g i t u d i n a l discs, 20*57)*  TABLE II. Analysis of variance of r e s u l t s (shown i n Figure 1) from experiment on growth o f cultured carrot discs derived t from l o n g i t u d i n a l and transverse planes o f four d i f f e r e n t carrot roots*  Source  D.F.  S.S.  78  57,698,74  Between carrots  7  (Between planes .  Total  Within carrots  M.S.  F  35,136.48  5,019.49  15.79**  1  35.68  35.68  71  22,562.26  317.77  0.11)  Table I I I summarizes the r e s u l t s o f a preliminary experiment designed to compare the e f f e c t s o f shaken l i q u i d and agar based (solid) media on the p r o l i f e r a t i o n of c a l l u s from sunflower stem sections. White's basal medium with added IAA and coconut milk was used i n l i q u i d and s o l i d (1 $ agar) condition. A l l  cultures were kept on the shaker  (which, o f course, has no e f f e c t on the agar medium), under continuous l i g h t , f o r 10 days.  28  Figure 1. Mean wet weight i nrag,Standard Deviation ( S . D . a n d Coefficient o f V a r i a b i l i t y (C.V.) o f cultured l o n g i t u d i n a l and transverse carrot discs derived from four d i f f e r e n t carrot roots. Each disc weighed 18 mg o r i g i n a l l y and was cultured f o r 21 days i n shaken l i q u i d White's basal medium plus O i l mg/1 IAA and 15 % coconut milk* Means are calculated from 5 - 1 6 discs*  0«  M E A N  I  a I I  150  CD  2  S.O.  = L O N G I T U D I N A L  D I S C S  ' ' T R A N S V E R S E  O I S C S  100  ii  X  6  4>  UJ  5  i i  i  ti  P  50  i i  30.4 5  CARROT  260 6  21.5 i  140 i 10  22.7 i  16  279 i  15  174  18.5  _i  10  4  12  C.V. no. o f  discs  29  TABLE I I I * Results of experiment designed to compare the e f f e c t of shaken l i q u i d and agar based media on the p r o l i f e r a t i o n of c a l l u s by sunflower stem segments.  A* Mean wet weights of stem segments a f t e r 10 days culture on White's basal medium plus 0*1 mg/1  IAA and 15 %  coconut milk* Average weight o f the stem segments was o r i g i n a l l y 85 mg*  Type of medium  Mean wet weight, i n mg, of 5 stem segments  Agar (5 %) based  364.2  Shaken l i q u i d  590*0  B. Analysis of variance:  Source  D.F.  S*S«  M.S.  Total  9  171,906  Between media  1  127,464  127,464  Within media  8  44,442  5,555  L.S.D. - 1 # l e v e l - 158.0 mg  F  22.9**  30 The average weight o f the stem sections (4 mm i n length) at the beginning o f the experiment was 85 mg. As can be seen from Table I I I the  shaken l i q u i d media produced «n average growth almost double that  given by the agar media, the difference being s t a t i s t i c a l l y highly significant* Following these preliminary experiments a series o f major experiments designed to compare the growth response o f a number o f tissues to d i f f e r e n t culture conditions was c a r r i e d out. The culture conditions compared were: agar ( i . e . s o l i d medium with an agar base) medium (A) versus l i q u i d medium (Lq); shaken l i q u i d medium (Sh) versus stationary or non-shaken l i q u i d medium (NSh); end continuous l i g h t (L) versus continuous dark (D). These conditions were combined so as to give the following s i x basic culture conditions (treatments) which the tissues were subjected t o :  1. Agar medium, continuous l i g h t (A L) 2. Agar medium, continuous dark (A D) 3* L i q u i d medium, non-shaken, continuous l i g h t (Lq NSh L) 4. L i q u i d medium, non-shaken, continuous dark (Lq NSh D) 5. L i q u i d medium, shaken, continuous l i g h t (Lq Sh L) 6* L i q u i d medium, shaken continuous dark (Lq Sh D) Figure 2 shows, graphically, the r e s u l t s obtained with undifferen t i a t e d carrot c a l l u s tissue grown under these conditions f o r s i x weeks (with weighing a f t e r both three and s i x weeks o f c u l t u r i n g ) . White's basal medium with 0.1 mg/1 IAA and 15 /o coconut m i l k was used throughout c  the  experiment*  31  Figure 2. Bar graph showing mean weights o f undifferentiated carrot c a l l u s tissues., as percentage o f weight at the start o f the experiment, cultured under d i f f e r e n t conditions. A .«* agar medium, Lq ** l i q u i d medium, Sh ** shaken, NSh - non-shaken, L - continuous l i g h t , D - continuous dark. Values at three weeks are means from three r e p l i c a t e s , and at s i x w e e k 3 means from two r e p l i c a t e s . Each r e p l i c a t e s represents s i x i n d i v i d u a l pieces o f c a l l u s tissue cultured separately arid weighed jointly..White's basal medium plus 0.1 mg/l IAA. and 15 $ coconut milk was used i n t h i s experiment.  Figure 3.' Bar graph showing mean weights o f tumorous sunflower t i s s u e s , as percentage o f weight at the s t a r t o f the experiment, cultured under different conditions* Abbreviations used f o r culture conditions and medium are the same as i n Figure 2. A l l values are means o f two r e p l i c a t e s . Each r e p l i c a t e represents s i x individual pieces of tissue cultured separately and weighed j o i n t l y .  FIGURE  2 N X  AL  AO  LqNShL CULTURE  Lq NSh 0 CONDITIONS  Lq ShL  Lq ShO  31  The most s t r i k i n g e f f e c t was that given by the shaken l i q u i d medium. In both l i g h t and dark i t caused a very marked stimulation o f growth over agar and non-shaken l i q u i d media. Agar medium* however, was s l i g h t l y superior to the non-shaken l i q u i d medium* L i g h t appears to stimulate growth i n the l i q u i d medivm ( p a r t i c u l a r l y i n the shaken l i q u i d medium) but to s l i g h t l y retard growth on agar medium.  TATff-'B'-  17. Analysis o f variance on weights o f undifferentiated carrot c a l l u s tissue cultured under d i f f e r e n t conditions for three weeks (Figure 2).  D.F.  Source  Total Replications  S.S.  M.S.  17  468,518.95  2  8,907.11  4,453.55  96,513.77  96,513.77  36.62**  229,633.33  229,633.33  87.14**  74,626.72  74,626.72  28.31**  1.69  Treatments: A. Agar vs l i q u i d m.  1  B. Shaking vs non-sh. 1 C. Light vs dark  1  Interactions: AxC  1  21,805.44  21,805.44  8.27*  BxC  1  10,680.33  10.680.53  4.05  10  26,352.23  2,635.22  Error  A s t a t i s t i c a l analysis o f the r e s u l t s obtained at three weeks i s given i n Table IV. Highly s i g n i f i c a n t differences were obtained between agar and l i q u i d media, shaken and non-ahaken l i q u i d media, and l i g h t and dark. Furthermore, there was a s i g n i f i c a n t i n t e r a c t i o n between the state  32  of the medium ( s o l i d or l i q u i d ) and the light-dark condition'. A l l the differences obtained at three weeks are merely more marked at s i x weeks (Figure 2 ) . At the end o f the six-week growth period the cultures grown i n dark were pale yellowish while those grown i n l i g h t were green and covered with brownish-red spots. The cultures grown i n l i q u i d mediwi i n dark were much more f r i a b l e and usually broke into several pieces before the end o f the culture period ( e s p e c i a l l y those shaken). Occasionally, roots developed on the cultures i n shaken l i q u i d medium, p a r t i c u l a r l y on those i n the dark. No roots developed on cultures grown on agar o r i n non-shaken l i q u i d medivau Cultures grown on agar medium were harder and more compact than those i n l i q u i d medium. There tended to be more v a r i a t i o n i n shape, s i z e , and appearance o f cultures grown on agar or i n l i q u i d non-shaken media than i n those grown i n liquid/shaken medium. A s i m i l a r experiment was c a r r i e d out to test the e f f e c t o f the same culture conditions as i n previous experiment^on the growth o f bacteria-free tumorous tissue of sunflower. Again the medium used throughout was White's basal medium plus 0.1 mg/l IAA and 15 % coconut milk. The r e s u l t s o f t h i s experiment are shown graphically i n Figure 3 and an analysis o f variance i s given i n Table V. The r e s u l t s e s s e n t i a l l y p a r a l l e l those obtained with the carrot c a l l u s tissue although the o v e r a l l growth throughout the experiment was lower. Relative growth was markedly superior i n the shaken l i q u i d medium, p a r t i c u l a r l y under the l i g h t condition. Growth i n the non-shaken l i q u i d and agar media was e s s e n t i a l l y the same i n the l i g h t , but growth i n the non-shaken l i q u i d medium i n the dark was markedly i n f e r i o r to that on  33  agar i n the dark. The e f f e c t o f l i g h t was generally more marked i n t h i s experiment, and i n contrast to the carrot c a l l u s tissue,the tumorous tissue grown on agar was stimulated by l i g h t . The stimulatory e f f e c t o f l i g h t on the shaken l i q u i d medium cultures was p a r t i c u l a r l y noticeable by 6 weeks. The trends i n growth established at three weeks were again only extended and more marked by 6 weeks. .  TABLE V. Analysis of variance on weights o f tumorous sunflower tissue cultured under d i f f e r e n t conditions f o r s i x weeks (Figure 3)•  Source  - D.F.  Total  S.S.  M.S.  F  11  423,259,67  1  14,560.33  14,560.33  2.36  1  32,047.04  32,047.04  5.19  B. Shaking vs Non-sh. 1  224,115.12  224,115.12  36.33**  0. L i g h t vs Dark  1  104,907.00  104,907.00  17.00**  AxC  1  16,695.37  16,695.37  2.70  BxC  1 5  91.12 30,843.67  91.12 6,168.73  0.01  Replications Treatments A. Agar vs L i q u i d m.  Interactions:  Error  The analysis o f variance o f the data obtained at s i x weeks (Table 7) shows that there were highly s i g n i f i c a n t differences between cultures grown i n shaken and non-shaken l i q u i d median, and between cultures grown i n l i g h t and dark. The difference between agar and l i q u i d media only approaches significance and i s somewhat misleading;  non-shaken and shaken l i q u i d  34  media tend to n u l l i f y each other as they are respectively i n f e r i o r superior to agar medium. No  and  s i g n i f i c a n t interactions were obtained.  A series o f major experiments was  c a r r i e d out to t e s t the e f f e c t of  the same growth conditions as before on tissues cultured i n Hildebrandt's improved sunflower medium. The  tissues used were fresh carrot discs,  undifferentiated carrot c a l l u s , and tumorous sunflower t i s s u e . Indoleacetic acid (0.01 mg/l)  was  added to the media f o r carrot discs  carrot c a l l u s , but not for the sunflower t i s s u e . No coconut milk used so that the media were completely chemically  and was  defined.  Figures 4, 5, and 6 summarize graphically the r e s u l t s obtained with carrot discs, carrot c a l l u s , and tumorous sunflower t i s s u e ,  respect-  i v e l y . A l l carrot c a l l u s and sunflower tissues i n the same treatment (culture condition) were weighed together, giving no s t a t i s t i c a l r e p l i cation. With the darrot d i s c s , s i x i n d i v i d u a l discs i n each treatment were weighed separately,  allowing a s t a t i s t i c a l analysis, which i s  given i n Table VI. In the main these experiments p a r a l l e l e d those with White's medium. However, the stimulatory e f f e c t o f l i g h t on growth i n shaken l i q u i d medium was much more marked with the carrot c a l l u s and tumorous sunflower tissues than i n the previous experiments. This e f f e c t was  also much more marked  i n the above mentioned experiments than i n the experiment with the  carrot  d i s c s . Tissues cultured i n non-shaken l i q u i d medium i n the dark showed the poorest growth i n a l l experiments. The  growth on agar media, i n both  l i g h t and dark, and i n non-shaken l i q u i d medium i n the l i g h t was t i a l l y the same f o r a l l t i s s u e s .  essen-  35  TABLE V I . Analysis o f variance  on weights o f fresh carrot discs  cultured under different conditions f o r s i x weeks (Figure 4 ) . S i x discs were cultured i n each treatment and these were weighed  separately.  Source  D.F.  S.S.  M.S.  F  Total  35  306,084.98  A* Agar vs L i q u i d  1  16,866.72  16,866.72  5.77*.  B. Shaking vs Non-Sh.  1  126,150.00  126,150.00  43.21**  C. L i g h t vs Dark  1  25,122.25  25,122.25  8.60**  AxC  1  41,472.00  41,472.00  14.20**  BxC  1  8,893.50  8,893.50  3.04  30  87,580.50  2,919.35  Treatments:  Interactions:  Error  I t should be noted that the t o t a l growth o f the undifferentiated carrot tissues i n Hildebrandt's medium was markedly i n f e r i o r to that i n White's medium with coconut milk. Tumorous sunflower t i s s u e , on the other hand, grew better i n Hildebrandt's medium. I t i s i n t e r e s t i n g that a l l o f the tissues showed a marked decline i n growth during the second three weeks o f culture, except when cultured i n shaken l i q u i d medium i n l i g h t . Such declines were only obtained i n non-shaken l i q u i d medium i n  36  the experiments using White's medium. The r e s u l t s obtained by analysis of variance (Table VI) resemble very c l o s e l y r e s u l t s obtained i n previous experiments. The difference between agar and l i q u i d media i s s i g n i f i c a n t , the differences between shaken and non-shaken l i q u i d media, between l i g h t and dark, and the i n t e r a c t i o n between a g a r - l i q u i d media and l i g h t - d a r k conditions are highly significant. A f t e r 6-week c u l t i v a t i o n the carrot discs grown i n l i g h t  developed  greenish s t r i p s in*cambial region while the regions d i s t a l to cambium were brownish-red. Discs grown i n dark were uniformly pale orange. The appearance of the u n d i f f e r e n t i a t e d carrot c a l l u s t i s s u e s i n t h i s experiment was generally the same as described for the experiments with White's medium (see above). No roots were developed-on any tissue grown i n Hildebrandt's medium. The tumorous sunflower tissue grown i n l i g h t , however, turned green instead o f remaining whitish colourless as i t d i d i n White's medium.  37  Figure 4. Bar graph showing mean weights o f fresh carrot discs i n mg a f t e r three and s i x weeks i n culture. Each treatment consisted o f 6 discs cultured and weighed separately. The o r i g i n a l weight of a disc was 42 mg. iifedium: Hildebrandt* s improved sunflower medium plus O.ol mg/l IAA. Abbreviations used f o r culture conditions are the same as i n Figure 2. x  FIGURE  4  2 L \ N J WEIGHT AFTER 6 WEEKS WEIGHT A F T E R 3 WEEKS  £400 o < u.  O300  ix  <£  233  LU  5  200  < w 100  189  186  ,260 277  LqShL  Lq Sh 0  \\  162  143  229  ,216. 153 83  AL  AD  Lq NSh L CULTURE  Lq NSh D  C0N0ITI0NS  38  Figure 5* Bar graph showing mean weights o f undifferentiated carrot c a l l u s tissues, as percentage of weight at the start o f the experiment, cultured under d i f f e r e n t conditions. Values are means of 1 r e p l i c a t e consisting o f s i x i n d i v i d u a l pieces cultured separately and weighed together. Medium: Hildebrandt's improved sunflower medium plus 0*01 mg£L IAA. Abbreviations used for treatments are the same as i n Figure 2.  Figure 6. Bar graph showing mean weights o f tumorous sunflower tissues, as percentage of weight at the beginning of the experiment, cultured under different conditions. Values are means o f 1 r e p l i c a t e consisting of s i x individual pieces of tissue cultured separately and weighed j o i n t l y . Medium: Hildebrandt's improved sunflower medium* Abbreviations used f o r culture conditions are the same as i n Figure 2.  FIGURE  600  -  5  1 X X 1 WEIGHT AFTER 6 WEEKS WEIGHT AFTER 3 WEEKS  400  X o  U J  297 200  164 154  z  153  \\  - I93v  136  139  FTV1 110  127  UJ  241  K\  152  o cc U J Q .  to <  FIGURE  5 I Li « u.  UJ  5E  o  UJ  tr  *  <  2157  2000 -  x  1200  400  .583 N  426 168 AL  236 AO  5I2, 305  514 140  133 LqNShL CULTURE  Lq NSh D CONDITIONS  Lq Sh L  Lq Sh 0  39  DISCUSSION. The r e s u l t s i n genral corroborate and extend the findings o f other workers. They demonstrate that growth o f plant tissue cultures can be markedly stimulated by c e r t a i n combinations of culture  conditions.  At the same time they raise some i n t e r e s t i n g questions concerning the metabolism o f tissues grown i n v i t r o . e b e n e f i c i a l influence o f coconut milk on growth o f cultured carrot d i s c s , as revealed by the f i r s t preliminary experiment (Table 1 ) , was to be expected. This e f f e c t has been noted by several workers (van Overbeek, 1941; Caplin and Steward, 1948, 1949, and 1952;  Duhamet,  1951a, b, and c) and i s probably at l e a s t p a r t l y due to an as yet uncharacterized  factor, ' k i n e t i n ' , i s o l a t e d from coconut milk recently  by Skoog and M i l l e r ( i n preparation). Since the carrot discs (and the c a l l u s derived from them) w i l l grow i n the absence o f coconut milk i t appears that tissues e i t h e r can .synthesize  the active substance supplied  by the coconut milk, or that t h i s substance can be substituted to some extent by indoleacetic a c i d necessary f o r continuous growth o f normal c a l l u s t i s s u e . Apparently the growth-stimulating action o f coconut milk i s not a r e s u l t o f i t s content o f growth-hormone for i n the same experi-  / ment IAA was completely unable to stimulate growth. Besides, according to a l l studies known to the author coconut milk stimulated growth o f cultured tissues much more than IAA or any other r e l a t e d substance. Because i n the present experiment carrot discs could p r o l i f e r a t e c a l l u s tissue also when grown on medium without IAA or coconut milk (Wh) i t seems probable that the fresh tissue may have an endogenous content o f  40 growth hormone s u f f i c i e n t to support i n i t i a l c a l l u s p r o l i f e r a t i o n . Wiggans (1953) found that 10 mg/l  IAA gave the best growth of carrot  discs while Caplin and Steward (1948) found 0.01  mg/1  to be  optimal.  This suggests that the endogenous supply of growth-hormone may  be  quite v a r i a b l e . The experiment comparing growth o f discs from d i f f e r e n t carrots and d i f f e r e n t planes i n carrots has considerable  p r a c t i c a l significance  i n the design of experiments. Since the average growth rate o f discs r i v e d from d i f f e r e n t carrots was  de-  highly s i g n i f i c a n t l y d i f f e r e n t i t  appears desirable that a l l discs to be compared i n an experiment be derived from the same carrot. This procedure was  followed with the  experiments reported here. The plane from which the disc i s cut seems to have l i t t l e e f f e c t on subsequent growth. Since transverse  discs were  found easier to obtain i n s t e r i l e condition they were used exclusively i n a l l other experiments. There was  also considerable  v a r i a t i o n i n the growth of discs  derived from the same carrot root. Caplin and Steward (1952) found the same to be true f o r discs cut from the secondary phloem of carrot roots. They further noted that discs containing cambium were more variable i n growth than those with only phloem t i s s u e . However, Wiggans (1953) reported that discs containing cambium grew considerably those cut from phloem or xylem. The  f a s t e r than  cambium containing discs are,  there-  fore, excellent material f o r experiments i n which high rates o f growth are desirable while phloem discs w i l l give more uniform r e s u l t s . The v a r i a t i o n i n growth between discs may  be due to v a r i a t i o n s i n the  o r i g i n a l amount of cambium present. I f t h i s i s the case, however, the discs from the l o n g i t u d i n a l plane should grow f a s t e r as they contain  41  more cambium than transverse d i s c s . I t seems that there are minor inherent variations i n the growth capacity o f c e l l s i n d i f f e r e n t parts o f the root. In experiments using discs t h i s ' i n t r a root' v a r i a t i o n can only be controlled by adequate r e p l i c a t i o n and s t a t i s t i c a l treatment. In experiments i n which carrot c a l l u s tissue derived by sub-culture from discs i s used i t would seem advisable to have a l l culture pieces derived o r i g i n a l l y from a single d i s c . This was the case with a l l c a l l u s cultures used i n the experiments described here. The major r e s u l t obtained fobm the experiments with d i f f e r e n t culture conditions was the consistent and s t r i k i n g demonstration that growth i n shaken l i q u i d me dim*, was superior to that on agar mediWi (and. o f course, superior as w e l l to growth i n l i q u i d non-shaken me dim)* Tikis applied to a l l tissues tested i n these experiments, i . e . fresh carrot discs, carrot c a l l u s , tumorous sunflower tissue, and sunflower stem segments, regardlessV^ White*s f  medium with IAA and coconut milk  or Hildebrandt's improved sunflower medium was used. The reason f o r the growth-stimulating action of shaken l i q u i d medium i s undoubtedly complex. Probably the major e f f e c t  i s due to  the action of the medium washing over the t i s s u e . T h i s allows nutrients to enter the tissue at a l l surface points rather than j u s t at the base, as on agar media* The washing over the tissue culture prevents, at the same time, the surface-drying which o c ^ r ^ a ^ i n the parts o f agar-cul tared tissues exposed continuously to a i r . The l a t t e r e f f e c t probably accounts for the loose, f l a c c i d surface of the c a l l u s growing i n shaken l i q u i d medium, as compared with r e l a t i v e l y smooth, harder surgace of c a l l u s grown on agar. The surface area of the cultures grown i n shaken l i q u i d  42 medium i s thus considerably greater, permitting increased exchange o f nutrients and gasses. Secondary reasons f o r the superiority o f the shaken l i q u i d medium may be the prevention o f accumulation of harmful excretions ('staling* products) at the tissue surface of cultures, and greater a v a i l a b i l i t y o f the ions from nutrient solution because the movement o f these substances i s not hindered by adsorption on agar p a r t i c l e s * Another reason f o r greater growth i n shaken l i q u i d medium may be better exchange of (gasses) between the outer atmosphere and the atmosphere o f the culture bottle due to the movement of the b o t t l e . The influence of improved aeration i s corrobated i n d i r e c t l y by r e s u l t s obtained by White  growth of  undisturbed culture with growth o f culture that had been removed from the tube and weighed every three weeks (both cultures were grown on agar medium); the l a t t e r culture grew approximately twice as fast as the former, probably due to better aeration.  Caplin and Steward  (1948, 1949, and 1952), and White (1953),  reported a b e n e f i c i a l e f f e c t o f agitated l i q u i d media on growth o f tissue  / cultures (using the auxophyton and t o l l e r tube techniques, r e s p e c t i v e l y ) • To make a direct comparison between t h e i r r e s u l t s and the r e s u l t s reported here i s very d i f f i c u l t f o r several reasons* F i r s t l y , although a l l these authors used carrot t i s s u e , t h e i r cultures were of quite d i f f e r e n t o r i g i n and d i f f e r e d , most probably, i n the inherent c a p a b i l i t y to grow: Caplin and Steward'used  discs cut from secondary phloem, White used habituated  tissue i s o l a t e d i n 1937 by Gautheret, and the present author used c a l l u s tissue i s o l a t e d some three months before the beginning o f experiments (the experiment with carrot discs cannot be used f o r t h i s  comparison  because they were grown i n medium without coconut milk and the growth  43  was, consequently, much smaller). Secondly, the sizes and weights o f tissue pieces used f o r c u l t u r i n g were d i f f e r e n t * Caplin and Steward used 3 mg  discs,  White rectangulars about 15 mg, and the present author more or l e s s i r r e g u l a r pieces about 70 mg each. T h i r d l y , the culture periods were d i f f e r e n t - Caplin and Steward's 20 days, White's 24 days f o r cultures i n agitated medium and 120 days f o r cultures on agar medium, present author's 42 days. Even i f the f i r s t objection i s disregarded, the d i f f e r e n t weights at the beginning o f experiments and the d i f ferent periods o f c u l t u r i n g make i t Impossible to calculute some ratioi- that could be r e l i a b l y used for comparison o f growth between the c u l t u r e s . Weight increase o f culture pieces i s to some extent geometrical when they are small, but gradually slows down as they enlarge (surface to volume r a t i o diminishes). White (1953) simply assumes a s t r a i g h t - l i n e growth and divides the t o t a l growth by the time of culturing to give an average figure f o r growth per day* Caplin (1947), on the other hand, uses Blackman's (1919) compound i n t e r e s t law to calculate growth as percentage increase per day* Both methods can be extremely misleading and the r e s u l t s may be completely opposite according to which method i s used* The question o f evaluation o f growth i n such a way that the r e s u l t o f one experiment can be compared r e l i a b l y with the r e s u l t o f another experiment i s very important but has been nearly completely neglected by workers i n plant tissue culture. The only serious consideration of the problem was given by Caplin (1947)* In very general terms, the growth response to a g i t a t i o n obtained here may be compared with that obtained by Caplin and Steward i n the following way: Caplin and Steward's cultures grew approximately twice as fast i n agitated medium as on agar medium, i n 20 days, while s i m i l a r cultures used i n t h i s study grew nearly four times faster i n agitated l i q u i d  44  me d i m than on agar medium i n 42 days i f cultures grown i n l i g h t and dark were counted together, and more than f i v e times faster i f only cultures grown i n l i g h t are considered* Since White used d i f f e r e n t periods of culture for agitated and agar media i t i s impossible to make a numerical comparison* I n i t i a l l y his.cultures i n agitated medium grew faster than those on agar but eventually they completely stopped growth and were passed by the  cultures  on agar* No such reversal i n growth trends was found i n the experiments here described, even though the cultures greatly exceeded the size o f White's cultures. The r a t i o s expressing the response of other cultures used i n t h i s study to a g i t a t i o n are generally smaller than i n the case o f carrot c a l l u s tissue but, nevertheless, they are always highly s i g n i f i c a n t (see analyses o f variance)• Certainly, the agitation technique described here i s simpler and more adaptable than those used previously (see Introduction)* Any horizonta l l y o s c i l l a t i n g platform could be s u b s t i t u t e d f o r the shaker used, and 1  almost any. type and size o f bottle or flask could be used as a culture vessel* Furthermore, i t i s not necessary that the tissue adhere to the surface of the culture vessel* This factor l i m i t e d the size o f Caplin and Steward's cultures to about 180 mg and White's to even l e s s * On the other hand, cultures weighing more than 3000 mg have been successfully grown under the shaking conditions described here. Furthermore, organ cultures, which are massive and non-adhesive, can be grown under these conditions but can not be grown i n the special tubes o f the auxophyton or i n r o l l e r tubes. It must be added that one disadvantage o f the here described technique (shared by a l l the techniques which use l i q u i d media and cotton plugs) i s that unless the surrounding humidity i s kept high, rapid evaporation o f the water from the medium can quickly produce detrimental changes i n the concen-  45 t r a t i o n o f nutrients* I t i s recommended, even i f the humidity i s high, that t i s s u e s be transferred to fresh medium every two or three weeks* The finding that growth on agar medium was as good or better than that i n non-shaken l i q u i d medium i s not too s u r p r i s i n g . Although there may have been some unknown growth-promoting  substances contaminating the  agar, the e f f e c t was most probably a r e s u l t o f aeration conditions*  The  t i s s u e s i n the l i q u i d medium sank and were p a r t i a l l y submerged i n the l i q u i d . Such submersion prevents adequate aeration o f the t i s s u e , thereby retarding growth. White (1939) obtained a s i m i l a r e f f e c t although i n h i s case the difference between growth on agar and i n l i q u i d was more.marked than i n these experiments. He used longer times and the tissue i n l i q u i d medium was completely submerged. The presence of continuous l i g h t had s i g n i f i c a n t stimulatory e f f e c t on the growth of a l l tissues cultured i n l i q u i d medium, p a r t i c u l a r l y those in shaken l i q u i d medium* Steward et a l . (1952) obtained increased growth of carrot discs cultured i n rotated l i q u i d medium although the difference due to l i g h t was not so s i g n i f i c a n t as i n the present study. Light and dark conditions had only small, i n s i g n i f i c a n t e f f e c t s on the growth o f tissues on agar medium* This agrees with the f i n d i n g by Hildebrandt et a l . (1945) that l i g h t conditions had l i t t l e e f f e c t on the growth o f tobacco c a l l u s and sunflower tumorous tissues cultured on agar* These r e s u l t s i n general support the interpretation that cultures grown on agar are p r i m a r i l y l i m i t e d by the d i f f u s i o n rate o f nutrients from the agar, whereas i n l i q u i d culture t h i s l i m i t a t i o n i s a l l e v i a t e d and l i g h t can have s i g n i f i c a n t e f f e c t on the u t i l i z a t i o n o f the n u t r i e n t s . Since tissues grown i n shaken l i q u i d medium presumably receive a better supply o f nutrients than those i n nonshaken medium (see above), the e f f e c t of l i g h t would be expected, as was found, to be more marked i n tissues grown i n the former medium.  46  The actual physiological action of the l i g h t i s d i f f i c u l t to ascertain. The  stimulation o f growth exercised by l i g h t i s probably not p r i m a r i l y due  to increased photosynthesis.  F i r s t l y , a l l the culture media contained an  optimal concentration o f sucrose;  secondly, the sunflower tumorous t i s s u e ,  used i n one series o f experiments, was stimulated by l i g h t (Figure 3 and Table V) despite the fact that i t showed no green coloration ( c h l o r o p h y l l ) . It i s i n t e r e s t i n g to note that the most marked stimulation by l i g h t on tissues grown i n shaken l i q u i d medium was given with carrot c a l l u s and sunflower tumorous tissues cultured i n Hildebrandt*s medium without coconut milk (Figures 5 and 6). The stimulation was much l e s s marked when these tissues grown i n White's medium with added coconut milk (Figures 2 and 3 ) . This might suggest that l i g h t plays a r o l e (direct or i n d i r e c t ) i n the synthesis, by the t i s s u e s , o f a growth stimulating factor which i s supplied by coconut milk. Consistent with t h i s hypothesis i s the fact that fresh carrot d i s c s , which might contain an endogenous supply o f such a f a c t o r , were not markedly stimulated by l i g h t when grown i n Hildebrandt's medium. An i n t e r e s t i n g difference i n growth occurred between tissues cultured on White's medium with coconut milk and those on Hildebrandt's medium. A l l tissues on the former medium, except those i n non-shaken l i q u i d medium i n the dark, showed approximately the same growth during the f i r s t and the second three-week culture periods. On the othwr hand, a l l tissues on Hildebrandt's medium, except those i n shaken l i q u i d medium i n l i g h t , showed a marked decline i n growth during the second three-week period. This might suggest that only under the conditions o f shaken l i q u i d medium i n l i g h t can the tissues on a chemically defined medium synthesize an adequate supply o f the growth-promoting substances supplied by coconut milk. Another noticeable difference between the responses to the two  47  nutrient media was that the carrot c a l l u s tissue grew s i g n i f i c a n t l y better on White's medium with added coconut milk (Figures 2 and 5) while the growth of the sunflower tumorous tissue was markedly superior on Hildebrandt's medium (Figures 3 and 6). Fart o f t h i s e f f e c t i s probably d i r e c t l y due to the fact that the concentrations of nutrients i n Hildebrandt's medium are s p e c i f i c a l l y designed f o r sunflower tumorous tissue and may not be optimal for carrot tissue (or at l e a s t not so favourable as the concentrations i n White's medium). However, difference i n growth-hormone, metabolism between the two tissues must also be taken into consideration. Carrot c a l l u s i s a normal tissue and needs an exogenous supply o f growth-hormone (and/or growth factor from coconut milk) f o r i t s growth (Gautheret 1942b, 1946, 1947a, and b) while sunflower crown g a l l tissue generates i t s e l f an excess of growth-hormone and does not respond to growth-hormone i n the medium; (deRopp, 1947). Therefore, enriched White's medium (containing 0.1 IAA. and 15 % coconut milk) was only 0.01 mg/1  mg/l  superior to Hildebrandt's medium (containing  IAA) f o r growth of normal carrot c a l l u s t i s s u e , while  Hildebrandt'3 medium with Improved concentration o f nutrients was superior to White's medium f o r growth o f tumorous sunflower t i s s u e . With the sunflower tissue, i t i s surprising that by merely using more favourable concentrations of nutrients the growth can exceed that with coconut m i l k . The change i n colour o f sunflower tumorous t i s s u e , discussed i n next paragraph, may have been another factor contributing to the r e s u l t . A rather notable phenomenom, which may have been p a r t i a l l y a cause or r e s u l t of the improved growth of the sunflower tumorous tissue i n H i l d e brandt' s medium, was observed. During a l l of the experiments i n which White's enriched medium was used, the sunflower tumorous tissue  remained  whitish and translucent. However, when the tissue was transferred to.  -?  48  H i l d e b r a n d t 3 medium f o r the l a t e r experiments the cultures grown i n l i g h t 1  suddenly turned l i g h t green. The d i f f i c u l t to explain. It may  cause or significance of t h i s change i s  be that the optimal nutrient  concentration  of Hildebrandt's medium overcame a l i m i t i n g factor i n chlorophyll or that the coconut milk contained i n h i b i t o r s of chlorophyll  synthesis  (coconut milk did not, of course, prevent chlorophyll synthesis t i s s u e s ) . The  synthesis  i n other  continuous c u l t i v a t i o n (for some f i v e months) of the sunflower  tissue i n l i g h t might have been also an important f a c t o r influencing the metabolic c a p a b i l i t y of sunflower tumorous tissue to synthesize chlorophyll. Certainly there i s a marked difference between normal carrot tissue  and  tumorous sunflower tissue to synthesize chlorophyll; carrot c a l l u s tissue developed chlorophyll i n both media within about a week. WheT^ther these differences are s p e c i f i c or are due to a more meristematic nature of the tumorous c e l l s cannot be said at present.  49  SUMMARY* The response o f various plant tissues to different culture  conditions  was compared* The tissues used were cambium-containing discs from carrot roots, undifferentiated carrot c a l l u s , bacteria-free sunflower tumorous (crown-gall) t i s s u e , and segments o f sunflower stems* The cubture  conditions  compared, i n combination, were agar versus l i q u i d medium, shaken versus non-shaken l i q u i d medium, and continuous l i g h t versus continuous dark* The response of the t i s s u e s to White's basal nutrient medium with added coconut milk (15 %) and indoleacetic acid (0*1 mg/l) and Hildebrandt's improved sunflower medium was also compared under these d i f f e r e n t culture  conditions*  A g i t a t i o n o f the l i q u i d medium was accomplished through the use o f a newly designed shaker, which consists b a s i c a l l y o f a h o r i z o n t a l l y o s c i l l a t i n g bank o f shelves* The t i s s u e s rested on the bottom of culture f l a s k s (medicine bottles) on these shelves and were a l t e r n a t e l y exposed to medium and a i r as the l i q u i d medium washed back and forth* Amy h o r i z o n t a l l y o s c i l l a t i n g platform  could replace t h i s shaker and almost any type and size o f  culture f l a s k could be used* Probably any type o f plant tissue could be cultured under these shaking conditions* I t i s not necessary that the t i s s u e s adhere t o the walls o f the culture vessels as i n other agitajion methods so f a r used i n plant tissue culture* Growth (weight increase) o f a l l tissues i n shaken l i q u i d medium (in both l i g h t and dark) was markedly superior (two to s i x times greater average weight i n 42 days) to that o f t i s s u e s on agar and i n non-shaken l i q u i d medium. The s u p e r i o r i t y o f growth i n shaken l i q u i d medium i s probably due to several f a c t o r s : nutrients and gasses are supplied to the entire surface o f the t i s s u e , there i s no drying and hardening o f the tissue  50  surfaces, r e s u l t i n g i n a greatly increased  surface  area, harmful  excretions  cannot c o l l e c t at the tissue surface, and d i f f u s i o n of nutrients i s not hindered by adsorption on agar p a r t i c l e s . To compare the growth of these cultures with those of other workers using a g i t a t i o n methods i s d i f f i c u l t due  to the d i f f e r e n t sources o f plant  material, d i f f e r e n t sizes of tissues cultured, and d i f f e r e n t periods o f c u l ture used. In general the stimulatory r e s u l t s of shaking obtained appear to be at l e a s t as good as those obtained by Caplin and Steward with the much more elaborate and l i m i t e d 'auxophyton'. There was  no sign of eventual  growth stoppage as obtained by White, using r o l l e r tubes. There were no s i g n i f i c a n t differences i n the growth o f tissues on agar, i n both l i g h t and dark, and i n non-shaken l i q u i d medium i n l i g h t * Tissues i n non-shaken l i q u i d medium i n the dark always showed the poorest growth. Tissues i n non-shaken l i q u i d medium received i n f e r i o r aeration. L i g h t consistently stimulated  tissues grown i n l i q u i d medium, p a r t i -  c u l a r l y those i n shaken l i q u i d medium. The e f f e c t was  e s p e c i a l l y marked on  carrot c a l l u s and tumorous sunflower tissues grown i n Hildebrandt's medium* I t i s suggested that l i g h t may  play a role i n the synthesis o f growth  factors supplied by coconut milk* Light had no s i g n i f i c a n t e f f e c t on  the  growth o f tissues on agar medium, i n d i c a t i n g that the primary l i m i t i n g factor i n the growth of such tissues may  be the rate of d i f f u s i o n of  nutrients from agar* Carrot t i s s u e s showed better o v e r a l l growth i n the enriched White's medium while the sunflower tumorous tissue did better i n Hildebrandt's medium.. The e f f e c t on carrot was  probably p r i m a r i l y through indoleacetic  a c i d and coconut milk. The response of sunflower tissue i s d i f f i c u l t to evaluate at present.  51  A l l carrot tissues developed chlorophyll throughout a l l of the experiments i f c u l t u r e d i n l i g h t while tumorous sunflower tissue remained .: white u n t i l placed i n Hildebrandt*s medium, when i t turned l i g h t green. The  significance o f these differences i s not known. One  experiment showed that carrot discs derived from d i f f e r e n t  carrots grew at s i g n i f i c a n t l y d i f f e r e n t average rates i n d i c a t i n g that  disds  to be compared should be derived from the same carrot. The plane i n which the discs were cut did not seem to influence subsequent growth. 'Intra root* v a r i a t i o n i n disc; growth necessitates r e p l i c a t i o n .  52  LITERATURE CITED. Almsstrand, A., 1949. Studies on the growth of i s o l a t e d roots of barley and oats. Phys .Plant. 2:372-381.. Bajer, A., and J . Mola-Bajer, 1954. 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