UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

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

Item Metadata

Download

Media
831-UBC_1955_A8 F6 I6.pdf [ 3.02MB ]
Metadata
JSON: 831-1.0106344.json
JSON-LD: 831-1.0106344-ld.json
RDF/XML (Pretty): 831-1.0106344-rdf.xml
RDF/JSON: 831-1.0106344-rdf.json
Turtle: 831-1.0106344-turtle.txt
N-Triples: 831-1.0106344-rdf-ntriples.txt
Original Record: 831-1.0106344-source.json
Full Text
831-1.0106344-fulltext.txt
Citation
831-1.0106344.ris

Full Text

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 of ARTS in the Department of Biology end Botany We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS Members of the Department of 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. Florian ABSTRACT. The response of various plant tissues to different conditions of culture was compared. The tissues used were cambium-containing discs from carrot roots, undifferentiated carrot callus, bacteria-free sunflower tumorous (crown-gall) tissue, and segments of sunflower stems. The culture conditions compared, in combination, were agar versus l i q u i d medium, shaken versus non-shaken l i q u i d medium, and continuous ligh t versus continuous dark. The response of the tissues to White's basal nutrient medium with added coconut milk (15 %) and indoleacetic acid (0.1 mg/1) and Hildebrandt's improved sunflower medium was also compared under these different culture conditions. Agitation of the l i q u i d medium was accomplished through the use of a newly designed shaker, which consists basically of a horizontally o s c i l -lating bank of shelves. The tissues rested on the bottom of culture flasks (medicine bottles) on these shelves and were alternately exposed to medium and a&r as the li q u i d medium washed back and forth. Any horizontally o s c i l -lating platform could replace this shaker and almost any type and size of culture flask could be used. Probably any type of plant tissue could be cultured under these shaking conditions. It i s not necessary that the tissues adhere to the walls of the culture vessels as in other agitation 2 methods so far used in plant tissue culture. Growth (weight increase) of a l l tissues in shaken l i q u i d medium (in both l i g h t and dark) was markedly superior (two to six times greater average weight in 42 days) to that of tissues on agar and in non-shaken l i q u i d medium. The superiority of growth in 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 tissues, there i s no drying and hardening of the tissue surfaces, resulting in a greatly increased surface area, harmful excretions —' can not collect at the tissue surface, and diffusion of nutrients i s not hindered by adsorptive effects of agar particles. To compare the growth of these cultures with those of other workers using agitation methods i s d i f f i c u l t due to the different sources of plant material, different sizes of tissues cultured, and different periods of culture used. In general the stimulatory results of shaking obtained appear to be at least as good as those obtained by Caplin and Steward with the much more elaborate and limited *auxophyton*• There was no sign of eventual growth stoppage as obtained by White, using r o l l e r tubes. Light consistently stimulated tissues grown in l i q u i d medium, p a r t i -cularly those in shaken l i q u i d medium. The effect was especially marked on carrot callus and tumorous sunflower tissues grown in Hildebrandt's medium. It i s suggested that lig h t may play a role in the synthesis of growth factors supplied by coconut milk. Light had no significant effect on the growth of tissues on agar medium, indicating that the primary limiting factor i n the growth of such tissues may be the rate of diffusion of nutrients from the agar. Carrot tissues showed better overall growth in the enriched White's 3 medium while the sunflower tumorous tissue grew better in Hilda brandt's medium* The effect on carrot was probably primarily through indoleacetic acid 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 tissues developed chlorophyll throughout a l l of.the experiments i f cultured in l i g h t , while tumorous sunflower tissue remained white unt i l placed in 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 different carrots grew at significantly different average rates, indicating that discs to be compared should be derived from the seme root. The plane in which the discs were cut did not seem to influence subsequent growth. 'Intra root' variation in disc growth necessitates replication. AGKNOlLEDCTilENTS. I am grateful to Dr. A.H.Hutchinson, the former Head of the Department of Biology and Botany, and to Dr. T.M.C.Taylor, the present Chairman of the Department, for recommending me to Dr. Coleman as his assistant and giving me thus the opportunity to start the experiments here reported. Dr. Hutchinson and Dr. Taylor took constant interest in my work and gave me a great deal of encouragement and help. The late Dr. L.C.Coleman i s gratefully remembered as a noble man and an outstanding teacher. Dr. G.A.Setterfield, who took over the supervision of my research work after the tragic death of Dr. Coleman, did not spare time or trouble to help me with the planning of experiments, analyzing and interpreting of the results, and preparing of the manuscript; I wish to thank him for his generous assistance and guidance. I wish also to express my gratitude to the Staff of the Dominion Laboratory of Plant Pathology in Saanichton,.B.C., who offered help to promote the success of these experiments. The study was carried out under a project supported by a grant-in-aid from the National Cancer Institute of Canada; I appreciate this financial help which made this study possible. TABLE 0? CONTENTS Page I. INTRODUCTION AND REVIEW OP LITERATURE 1 II. MATERIALS AND METHODS • • 17 III. EXPERIMENTAL RESULTS • 25 IV. DISCUSSION • . • 39 V* SUMMARY 49 VI. LITERATURE CITED . . . . . . . . . . . 52 INTRODUCTION AND REVIEW OF LITERATURE* I. General background* The culture of Isolated plant parts (organs and tissues) In controlled nutrient media has been a major problem in experimental botany since the turn of the- century* In the beginning, the establishment of growth in vi t r o was simply an end in i t s e l f but the emphasis has slowly shifted and today, tissue culture has become Despite, however, notable successes i n culturing plant tissues and significant advances in our knowledge which have cone about through the use of the technique as an experimental tool, tissue culture s t i l l has many imperfections and weaknesses which prevent f u l l exploitation of i t s many advantages. To appreciate both the present-day advantages and weaknesses of plant tissue culture i t i s desirable to briefly|consider i t s development and present status* Plant issue culture was f i r s t attempted by Haberlandt in 1902* He used relatively highly differentiated c e l l s (e.g. leaf parenchyma) and very simple mineral nutrients; the experiment was unsuccessful but provided stimulus to other workers who took up the problem* It was not, however, u n t i l 1922 that partial success i n growing plant tissues a r t i f i c i a l l y was obtained* In this year Kotte and Robbins independently primarily a means for controlled experiment with l i v i n g processes* 2 cultivated excised tomato roots for limited periods of time. Subsequently techniques were slowly improved and in 1934, White established cultures of excised tomato roots which were capable of unlimited growth in culture* White (1943), considers the i n vitro cultivation of excised roots to be organ culture rather than true tissue culture. He defines tissue culture in the s t r i c t sense as unlimited a r t i f i c i a l growth of undifferentiated tissue. Cultures f u l f i l l i n g this definition were obtained in 1939 when Gautheret, Nobecourt, and White independently established cultures of undifferentiated plant callus which were apparently capable of unlimited sub-culture* Since thewe original researches the f i e l d of plant tissue culture has been extensively and diversely developed. To outline the more recent advances in the f i e l d i t w i l l be useful to consider separately the types of plant tissues which have been cultured and the general media and methods used. I I . Types of Plant Tissue Cultures* Plant tissue cultures can be conveniently divided, according to the origin of the plant parts used for cultivation, into the following classes: A* Embryo culture B. Organ culture C. Callus culture D. Culture of tumorous tissues E. Cultures of other origin 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 of White's definition of true tissue culture ( i . e . the tissue should be undifferentiated and capable of unlimited growth), they are included i n this discussion because the techniques used with them are basically the same as those used in the culture of undifferentiated tissue* It should be further remarked that a l l the c e l l s in undifferentiated callus cultures are not the same. Crown gall callus tissue of sunflower and hybrid tobacco callus tissue contain at least three different kinds of c e l l s : parenchyma cells (large hyper-trophic c e l l s ) , meristematic c e l l s (small hyperplastic c e l l s ) , and thick-walled scalariform cells (wound tracheids)(White, 1939; Caplin, 1947; Stxuckmeyer et a l * , 1949)* Thus, White's use of the term 'undifferentiated' in his definition of true tissue culture applies at the histological and morhpological levels but not at the cytologies! l e v e l . A* Embryo Culture. Attempts to grow plant embryos on a r t i f i c i a l media started t early i n the 20th century (e.g. Banning, 1954; Stingl, 1907). However, i t was not u n t i l 1922 that Khudson succeeded in 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 undifferenti-ated c e l l s and can germinate in nature only in symbiosis with fungi. • As media Ehudson used mineral salts solutions, glucose and fructose as a carbohydrate source, and various plant extracts* He found extracts of Bacillus radicicola to be particularly beneficial to the development 4 of seedlings. Later Tukey (1933, 1934, 1938, and 1944) successfully cultured embryos of sweet cherry, peach, and other stone f r u i t s . Considerable improvement in the technique of embryo culture was brought about by the introduction of coconut milk into the nutrient media. This substance was used for the f i r s t time by van Overbeek et a l . (1941) for the cultivation of Datura embryos. As a result i t became possible to grow much younger embryos than had been grown before (van Overbeek et a l . , 1941, 1942, and 1944), and to obtain hybrids from incompatible crosses of Datura (Blakealee and Satina, 1944). Following this work embryo culture became widely used as a method of overcoming embryo abortion i n crosses between incompatible varieties and species. Brink et a l . (1944) obtained a hybrid between Hordeum Jubatum and Secale cereale; the hybrid embryo does not mature in the seed but i t was possible to grow i t on an a r t i f i c i a l medium. Similarly, Cooper and Brink (1945) were able to produce hybrids between diploid and tetraploid races of Lycopersicum pimpinellifolium through the use of embryo culture. As well as being a means for over-coming genetic incompatibility, embryo culture can also speed up breeding programs through the elimination of dormant period of seeds. The use of embryo culture in plant-breeding was discussed by Skirm (1942) and by Tukey (1944). Weeping cralyapple embryos have been cultured by Nickell (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 , violet, ginkgo, pine, apple, pear, plum, rose, and olive. A general review of embryo culture has been given by Rappaport (1954). 5 B. Organ Culture* This category includes cultures of isolated roots, stem apices, leaves, flowers, and f r u i t s * Roots have been most widely cultured of these organs, primarily because their isolation, culture, and measurement i s relatively easy. Tomato roots were used by White (1934) for establish-ment of the f i r s t potentially unlimited plant tissue culture. Since then, tomato roots have been used for 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 for root culture* Bonner and Addicott (1937) cultured pea roots while Bonner and Derivian (1939) cultured, besides pell and tomato roots, roots of radish and flax* Bonner (1940) established the minimal nutritional requirements for optimal growth of excised roots of 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 of stem tips of Stellariamedia was attempted by White (1933) • Subsequently stem tips of Tropaeolum majus and of Lupinus  albus were successfully grown by B a l l (1946), shoot tips 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 of excised leaf cultures (e.g. tobacco and rhubarb) in the study of organic acid metabolism in plants. , The lat t e r cultures, i.e. leaf, flower, and f r u i t cultures, are only temporary and end with the f u l l development of the cultured organ* Cultures of roots and stem apices, which contain active meristems, can be continued indefinitely. C. Callus Culture. The term callus culture i s generally used to describe the culture of undifferentiated tissue derived from cambium or other meristematic or potenti-'ally meristematic tissue. Gautheret's (1959) f i r s t tissue culture capable of indefinite growth was derived from the cambium of carrot roots while White's (1939) came from procembium of Nicotiana stems. Since these original experiments, callus cultures have been obtained from cambium of many plant species, e.g. Ulmus campestria (Gautheret, 1940), T i t i s vinifera (Morel, 1944), Rosa sp. (Hobecourt and Koefler, 1945), Helianthus annuus and Vinca rosea (deRopp, 1947), Scorzonera sp. (Gautheret, 1948), Salix caprea (Gautheret, 1950), and ferns (Morel and Wetmore, 1951). To the group of callus cultures derived from meristems other than cambium belong cultures of undifferentiated tissue proliferated from embryos cultured on a r t i f i c i a l media. Such cultures were isolated from pro-embryos of Datura by van Overbeek et a l . (1941, 1948), and from seedlings of 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 barbituric acid) to the culture media* Though derived from embryos, a l l of these cultures are true tissue cultures in the sense of White's s t r i c t definition, as the growth i s undifferentiated and can be continued 7 indefinitely. Several^ callus cultures have been developed from potentially meristematic tissues. Caplin and Steward (1948, 1949, and 1952) obtained callus cultures from secondary phloem of carrot. Cultures have also been derived from storage tissue of potato tuber (Steward and Caplin, 1951), and from similar tissues of sweet potato and Jerusalem artichoke (Wetmore, 1954). It should be noted that while cambium produces callus 'spontane-ously' i t i s necessary to treat potentially 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 start c e l l proliferation. However, once removed from contact with organized plant tissues, a l l callus cultures normally require an exogenous source of growth-hormone (see below). C. Cultures of tumorous tissue. The majority of culture work with tumorous tissue has been carried out on tissue derived from tumors originally induced by bacteria of the genus Agrobactarium (commonly called crown g a l l bacteria). Tissue from such tumors i s similar to normal undifferentiated callus i n general cellular makeup and growth habit but i t requires somewhat different techniques of isolation and i s capable of growing on media without growth-hormone. The major problem has been to obtain in culture,tumorous tissue free of the inciting bacteria. White and Braun (1942) f i r s t isolated and cultured bacteria-free crown ga l l tissue from secondary tumors on sunflower. Similar cultures 8 from primary galls on sunflower were later obtained by deRopp (1947)• Heat treatment was used by Braun (1947) to k i l l the bacteria in crown gall tissue on Vinca rosea, allowing subsequent culture of bacteria-free tissue. Bacteria-free crown gall 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 vinifera and Antirrhinum majus (Morel, 1948). The problem of crown ga l l and the results 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 in i t i a t e d by otheryigents than Agrobacterium sp. Tumors on roots of Rumex acetosa induced by wound virus were cultured by Nickell and Brakke (1954). Genetic incompatibility causes the appearance of spontaneous tumors in certain crosses of Nicotiana species (Kostoff, 1930). This 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 origin. Tulecke (1953) succeeded i n culturing undifferentiated tissue derived from pollen of Ginfego biloba. The endosperm of maize has been cultured by Strauss and La Rue (1954)• Cultured endosporm appears to be a good source of material for observation of mitoses in 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 lamella of c e l l s i n carrot callus cultures. In doing so he claims to have obtained cultures of carrot callus tissue derived from single c e l l s . 9 Jablonski and Skoog (1954) have cultured pith tissue from tobacco. Through the use of indoleacetic acid and coconut milk or water extracts of vascular tissue they obtained continuous c e l l division and p r o l i f e r a -tion of the pith c e l l s * III* Tissue Culture Madia* The nutrient media used for plant tissue culture consist basic-a l l y of mineral salts, carbohydrate, amino acids, vitamins, and growth-promoting hormones (Gautheret, 1942; White, 1943, and 1954). The mineral salts generally used are basically the same as those used for cultivation of intact plants i n water cultures. Gautheret (1935) used modified Enop's solution and White (1934, 1943, 1954) used a modification of the solution of TTspenski and Uspenskaja. White's original mineral solution consisted of the following salts: MgS04, Ca(N0 3) 2, Na 2S0 4, KNOg, KC1, NaHgPC^, Fe 2(S0 4) 3, MnS04, ZaS04, HgBOg, and KI. This solution supports growth of most tissues and has been extensively used i n plant tissue culture. More recently some changes which produce increased tissue growth have been proposed. Boll and Street (1951) suggested the addition of the trace elements molybdenum and copper. Hildebrandt et a l . (1946) replaced fe r r i c sulphate with the more stable f e r r i c tartrate. Hildebrandt et a l . (1946) also extensively investigated the optimal concentration requirements for the mineral salts and developed improved media for sunflower and tobacco tissues. Different sugars i n varied concentrations have been tr i e d by many workers i n attempts to find the best source of carbohydrate for 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 of 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 results* Mannose, maltose, cellobiose, galactose, and raffinose were satisfactory for some species but poor for other species* Generally used now i s either 8 % sucrose (White's and Hilde-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 of seven different amino acids for culture of pea roots but these were later 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 essential for continuous growth of sunflower gall tissue and recommended further experiment with nitrate, urea, alanine, aspartic acid, and glutamic acid. These substances are claimed to be not only a superior sourte of nitrogen, but also to have a separte stimulatory effect on growth of tissue. The vitamin requirements of different organs and tissues i n culture have been studied widely. The necessity of vitamin B^ (thiamine) for the growth of the majority of plant tissues in vitro 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). Similarly, the tissue culture requirements for vitamin B (pyridoxine) and nicotinic acid have been studied by Robbins and 6 Schmidt (1939), White (1940), and Bonner (1938, and 1940); most tissues (and organs) require these vitamins for sustained growth* Thiamine, pyridoxine, and nicotinic acid are now included in a l l basic tissue culture media although certain cultures w i l l grow in the absence of one or the other of them, e.g. roots of a l f a l f a do not require pyridoxine for optimal growth (Bonner, 1940)* In addition to these vitamins Gautheret (1950) uses biotin and Ca-pantothenate for the culture of certain tissues, e.g. Salix caprea. The influence of growth-hormones has been studied extensively i n cultured normal callus and in tumorous tissues. The work of Gautheret (1937, and 1939), Duhamet (1939), and others lead to the conclusion that indoleacetic acid or some other growth-hormone (e.g.naphtalene-acetic acid or indolebutyric acid) i s indispensable for.the growth in vitro of normal plant callus tissue* An exception to this statement occurs in the case of 'habituated' callus tissue, f i r s t obtained by Gautheret (1948a)* Habituated tissue i s normal callus tissue which in the course of continuous culture has lost the need for an exogenous supply of growth-hormone. The mechanism of this metabolic change i s not understood. Bacteria-free tumorous tissue behaves the same as habituated callus tissue in this respect and grows optimally without supplied growth-hormone (Gautheret, 1947)* Tissue cultures have been widely used in attempts to elucidate the action of growth-hormones in tissue* The effects of indoleacetic acid on water absorption by potato tuber tissue have been studied 12 by Commoner et a l . (1942), the histological effects of growth-hormones on crown ga l l tissue by Struckmeyer et a l . (1949), the effect on growth and respiration of artichoke tissue by Hackett and Thimann (1952), and the influence on meristematic a c t i v i t i e s of tomato roots by Street (1953). Skoog and Tsui (1948) and Skoog (1951, and 1954) studied the effects of growth-honnones on growth, differentiation, and organ formation i n callus culture, and Jablonski and Skoog (1953) examined the effect of growth-hormones on c e l l enlargement and c e l l division in isolated tobacco pith tissue. The growth-promoting effect of coconut milk (liquid endosperm) on plant tissue cultures was f i r s t noticed by van Overbeek et a l . (1941) in culturing of Datftra embryos. Coconut milk increases the rate of growth of plant tissue cultures considerably and i s superior to other plant extracts such as tomato juice (Nitsch, 1951). The effect of different 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 % for the majority of plant tissues. There i s obviously a different content of the growth-promoting substance in milk obtained from different nuts. The growth-promoting effect of coconut milk on plant tissue cultures i s similar to the effect of embryo extract on animal tissue cultures. Recently M i l l e r and Skoog (in preparation) have isolated from coconut milk a factor of unknown structure which promotes c e l l division in isolated pith tissue and accelerates growth of tobacco callus. It i s to be hoped that a l l active agents in coconut milk w i l l eventually be 13 chemically characterized as the use of coconut milk in plant tissue culture at present introduces unknown factors which hamper controlled experimentation and interpretation of results* The influence of environmental conditions on plant tissue cultures has been studied sufficiently to establish optimal temperatures, ion concentration, osmotic pressure, and pH of media (White, 1932, and 1943; Hildebrandt et a l . , 1945, and 1946). Optimal values of these factors vary with different kinds of tissues cultured. For example the optimal temperature for growth of tobacco callus tissue was found to be 26-32°C and for sunflower callus tissue 24-28°0. The optimal pH for the former tissue i s 5.0-5.9, and for the l a t t e r tissue 5*5-5.9 (Hildebrandt et a l * , 1945)* While same authors have found that l i g h t has no influence on rate of growth of cultured tissues (Hildebrandt et a l * , 1945), Caplin and Steward (1952) reported a slight growth-promoting Influence of l i g h t on the callus proliferation of cultured carrot discs* Bunning and Welte (1953) obtained significant differences in the rate of growth of carrot-disc cambium under different periods of illumination* IV* Tissue Culture Technique* For successful cultivation of 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 for root cultures and semi-solid or solid media (with 0.5-2 % agar) for other kinds of tissue cultures. The roots float on the surface of the l i q u i d medium and are thus aerated. Tissues on agar media obtain nutrients by diffusion from the agar at points of contact while the greater part of the tissue i s directly exposed to the a i r . 14 Liquid media have not been genrally used for cultures other then roots because the tissue does not float on the medium and consequently suffers from the lack of air* White (1939) tried growing callus tissue in l i q u i d medium and found that growth was considerably slower than on agar and that histological differentiation occured and roots appeared* He presumed both effedts to be a result of the relatively anaerobic conditions present beneath the surface of the l i q u i d medium where the^Qasue lay. It was discovered by the workers with animal tissue culture that alternating submersion of tissue i n l i q u i d medium and exposure to sterile a i r in the culture vessel would provide for increased rate of growth* Using this principle Gey and Gey (1936) developed the r o l l e r tube method* Cultures are grown on the walls of tubes f i l l e d with small amount (1-2 ml) of l i q u i d medium and the tubes are revolved along their longi-tudinal axis on a special drum* The cultures are thus alternately washed by the medium and exposed, for longer periods of time, to the a i r in the tube* The method was subsequently modified by Shaw et a l * (1940). These workers substituted for the culture tubes,roller bottles with a hole in 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 of perforated cellophane to hold the culture, were devised later. Such techniques are not easily applied directly to plant tissue cultures as the plant cultures grow as relatively massive, solid lumps of tissue and do not readily adhere to the walls of the tubes (as do animal cultures) • However, the pos s i b i l i t y of accelerating growth of plant tissue cultures through the use of agitated l i q u i d 1 medium 15 attracted some workers* deRopp (1946) constructed an apparatus consisting basically of a U-tube with l i q u i d medium in one arm and the tissue culture in the other arm* The tissue rests 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. After submerging the tissue i n the l i q u i d medium the apparatus i s returned to the original position and the tissue exposed to the a i r in the tube* Another method was devised by Caplin and Steward (1949) and used for extensive studies carried out by these authors (Caplin and Steward, 1952; Steward and Caplin, 1952a, b; Steward et a l . , 1952). These workers use a new type of culture tube which i s closed and rounded at both ends, with a narrower open tube attached, at right angles, i n the middle of the larger tube..The tissue sticks to the wall of one of the rounded ends of the main tube. The tube i s attached to a circular 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 alternately immersing the tissue in the medium and exposing i t to the a i r in the tube. The smaller side-neck tube (plugged with cotton) provides for the entrance to the tube and a i r exchange. The apparatus was originally named revolving klinostat and renamed auxophyton in 1952* White (1953) used the r o l l e r tube method of Gey and Gey (1936) for plant tissue cultures. A l l of these attempts to u t i l i z e agitating methods in plant tissue culture have had success in accelerating growth. However, in general they are somewhat cumbersome and require costly glassware and rotating devices. In 1952 the late Dr.L.C.Coleman began experiments to develop an agitating method for growth acceleration that would be, at the same 16 time, simple, reasonably inexpensive, and readily adaptable to large-scale experiments with a l l types of plant tissue cultures. The present author joined Dr. Coleman early in his studies along these l i n e s . This report describes the methods eventually developed and the results of experiments designed to measure the comparative growth response of several tissues to these and other methods of tissue culture. The study had, in effect, a two-fold aimj} to empirically(develop conditions for optimal growth of plant tissue cultures and, using these methods, to gain some insight into the metabolism of various cultured tissues. 17 MATERIALS AND METHODS. I. Plant Material. In the present experiments these different plant materials were used: A. Fresh carrot discs B. Undifferentiated tissue of carrot C. Bacteria-free sunflower tumor tissue D. Stem sections of sunflower A. Fresh carrot discs. Carrot discs were cut from carrot roots of unknown variety bought in local stores. The roots were washed in 50 $ alcohol, s t e r i l i z e d for 20 min. i n a 0.1 % aqueous solution of mercuric chloride, and subsequently rinsed in sterile water. In a sterile transfer chamber discs for culture were cut from the middle third of the s t e r i l i z e d roots. In preliminary experiments discs with their diameters parallel and at right angles to the longitudinal axis of the root (longitudinal and transverse discs respectively) were used. To obtqin longitudinal discs, radial cylinders were bored out across the roots with a cannula or a cork borer and then cut into discs with a multibladed cutter similar to that used by Caplin and Steward (1949). Only discs containing cambium were used for cultivation. Transverse discs were obtained by cutting a transverse s l i c e , 1 mm thick, across the root with a two-bladed cutter. (The blades in this cutter are offset 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 slice p a r t i a l l y attached to the carrot root i n this manner provides support for the slice which makes the cutting of sterile discs from i t easy)* Discs were cut from the transverse slice with a cannula or a. borer so that the cambium was running completely across their diameters. Discs of various diameters were tested in 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 in diameter were eventually found to give the most uniform growth and were used i n the main experi-ments reported here. B. Undifferentiated tissue of carrot. / Undifferentiated carrot tissue used in these experiments was derived from callus proliferated by the cambium of fresh carrot discs i n culture. Fresh transverse carrot discs 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 acid and coconut milk: (see below) for 14 weeks. They were transferred to fresh media every two weeks during this period. At the end of this time irregularly shaped masses of undif-ferentiated tissue, yellowish green with reddish spots, and covered with mamillary outgrowth had formed. These masses of undifferentiated callus 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 of callus were used 19 i n the experiments on tissue culture conditions reported below. C. Bacteria-free sunflower tumor tissue. Bacteria-free sunflower tumor tissue was isolated from a small secondary tumor which developed on the main vein of a leaf of a sunflower plant eight weeks after the plant was inoculated with Agrobacterium  tumefaciens. The secondary tumor was washed in 50 % alcohol, s t e r i l i z e d for a few minutes in a 0.1 % aqueous solution of mercuric chloride, and opened aseptically. Small pieces of tissue were cut out with a scalpel, cultured i n agitated l i q u i d White's basal medium (see below) for 8 weeks, and then sub-cultured. Stock sub-cultures of the tissue were transferred every two weeks and used . .. for experiments. D. Stem sections of sunflower. In one of the preliminary experiments segments of sunflower, stem were used. When young sunflower plants were about 5 inches t a l l the leaves were removed and the upper parts of the stems were cut o f f . The excised stem pieces were washed in alcohol, s t e r i l i z e d for 10 min. i n - t h e 0.1 % mercuric chloride, and rinsed in sterile water. Finally,"epidermis of each stem piece was peeled o f f and the f i r s t intemode cut into 4 mm long segments which were used directly in the experiment. ( I I . Handling of Material. A l l transfers of tissue cultures were carried out in a transfer I chamber; the instruments used were dipped in alcohol and flamed. 20 The amount of bacterial and fungal contamination was at f i r s t about 10-15 fo but this was lowered to about 5 # when a special small transfer chamber was used* (This part of the experiments was carried out i n the Dominion Laboratory of Plant Pathology in Saanichton, B.C. Because of the great amount of 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 in wet weight was used as a measure of growth of the tissue cultures. Tissue pieces from l i q u i d media were surface-dried with sterile f i l t e r paper in sterile P e t r i plates, transferred into tared sterile Petri plates, weighed, and returned to fresh media. Tissue pieces from agar media were freed of adhering agar and rinsed in sterile double d i s t i l l e d water before surface-drying and weighing. Contaminated cultures were usually discovered within two dajcs after transfer; they were immedi-ately surface-dried, weighed, and discarded. The weight was subtracted f rom the original total weight of tissue in the treatment. III. Growth Conditions. JL. Glassware. Medicine bottles of 170 ml c o n t e n t were used for cultures grown in l i q u i d media and v i a l s 22x94 mm for cultures grown on agar media. Bottles end v i a l s were closed with cotton plugs wrapped in cheese cloth which had been previously boiled in d i s t i l l e d water. A l l glassware used in the preparation of media and for culturing was acid cleaned in a 21 saturated solution of potassium dichramate in concentrated sulphuric acid, and subsequently rinsed ten times in hot running water, twice in d i s t i l l e d water, and twice in double d i s t i l l e d water* B. Madia* Two basal media were used in the experiments: White's standard medium (1943, and 1954*) and Hildebrandt*s Improved sunflower tissue medium (Hildebrandt et a l * , 1946)* Except in preliminary experiments (see results section) White's medium was supplemented with 15 % coconut milk and 0*1 mg/l indoleacetic acid, and Hildebrandt's medium with 0.01 mg/l indoleacetic acid. 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 s t i l l , second i n a Pyrex-glass s t i l l ) was used throughout the experiments. CP grade reagents were used exclusively. Stock mineral salt solutions were kept at room temperature while stock solutions of vitamins and of indoleacetic acid were stored in a freezer. Fresh stock solutions of a l l chemicals were prepared every six weeks. Coconut milk was obtained from mature coconuts. Each nut was opened and the milk poured into a stender and examined for quality. Disintegrating milk (cloudy and odoured) was discarded. The good quality milk was mixed, fi l t e r e d , distributed in 150 ml aliquots, and stored i n a deep-freezer. Preliminary experiments showed that there was no difference *The concentration of vitamins in White's 1954 book (p.74) i s given, by a mistake, ten times stronger than i t should be. 22 Composition of Media: Nutrient White's basal Hildebrandt's improved ingredient medium sunflower t . medium NagSg 200.00 mg 100.00 mg Ca(N0 3) 2.4 HgO 200.00 » 800.00 " MgSO .7 H_0 360.00 " 720.00 M 4 2 KNO, 80.00 " 160,00 " KC1 65.00 " 130.00 " NaH^O^HgO 16.50 " 132.00 • Fe ( C H O ) 5.00 " 2 4 4 6 J3 Fe 2(S0 4) 3.6 HgO 2.50 « MnSO .4 HO 4.50 " 4.50 " 4 2 ZnS0„.7 H„0 1.50 " 3.00 " 4 S HJ30,, 1.50 «. 3.00 » o o KI 0.75 " 0.375" glycine 3.00 " 12.00 " nicotinic acid 0.50 " thiamine 0.10 " 0.10 * pyridozine 0.10 " 0.80 " sucrose 20.00 g • 20.00 g water 1000.00 ml 1000.00 ml 23 in the growth of 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 of the media was always adjusted with 0.1 N NaOH to approximately 5.8 before autoclaving and in some experiments checked after autoclaving and also after a 3-week culture period. No significant changes in pH occurred during autoclaving and only slight rises (less,than 0.5 pH units) resulted after 3-week culture of tissue. Both solid and l i q u i d media were used in the experiments. For solid media 1 % agar (Difco) was added to White*s medium and 0.5 % to Hildebrandt's medium. Madia were aatoclaved for 20 min. at 15 l b . per square inch. Ten ml of medium per culture (both l i q u i d and solid) were used throughout the experiments. C. Agitation. The major experiments here reported are concerned with measuring the growth response of tissues cultured in agitated l i q u i d media. Agitation of the l i q u i d media was obtained by placing the culture bottles on a mechanical shaker originally conceived by Dr.L.C.Coleman, and constructed by Mr.C.J.Lines of the Dominion Laboratory of Plant Patho-logy. Saanichton, B.C. This shaker basically consists of a r i g i d bank of five wooden shelves, 26x76 inches each, spaced 12 inches above each other. The bank of shelves i s hung on a movable supporting frame and i s o s c i l -lated horizontally £ of an inch, 82 times per minute. Oscillation i s provided by a cam-shaft driven by a £ HP ele c t r i c motor. Culture bottles (medicine bottles) were l a i d on the shelves with their long axis par a l l e l 24 to the direction of os c i l l a t i o n . The osc i l l a t i o n caused the medium to wash back and forth on the bottom of the bottle* thereby alternately exposing the tissue inside to medium and a i r . D. Light conditions. The growth response of tissue cultures to conditions of continuous l i g h t and dark were also studied. Continuous lighting of the shaken cultures was accomplished by four 40 W fluorescent li g h t s suspended on the underside of each shelf of the shaker. These ligh t s provided an illumination intensity of 400 foot-candles to the upper surface of the shelf immediately below. To obtain dark conditions on the shaker one shelf was covered with thick cardboard and the lights above extinguished. Cultures which received no shaking were kept on separate stationary shelves with the same li g h t and dark conditions as those on the shaker. E. Temperature and Humidity. Culture was carried out in an insulated room with thermostatically controlled air-conditioning. The cooling effect of the air-conditioner and heating action of the lights were balanced so that the temperature within the culture bottles remained at 2511° C. Lights beneath the shelves were used to maintain this temperature in the cultures grown in dark. -The humidity i n the room was maintained at approximately 80 # through / r evaporation of water from large trays. 25 EXPERIMENTAL RESULTS. A preliminary experiment was performed to test the influence of coconut milk and indoleacetic acid (IAA) on the development of callus tissue from cultured carrot discs. Fresh carrot discs weighing 18 mg were cultured for three weeks in White's basal medium alone and in combination with IAA (0.1 mg/l) and coconut milk (15 % ) . The cultures were grown in shaken l i q u i d medium under continuous l i g h t . The results of this experiment are summarized in Table I. Indoleacetic acid had no significant effect on growth, either in the basal medium alone or in combination with coconut milk. On the other hand, coconut milk gave a highly significant stimulation of growth with or without IAA. The average weights of tissues cultured in media with coconut milk were approximately double those of tissues in the media -without coconut milk. A second preliminary experiment was designed to compare the growth rate of fresh carrot discs derived from different carrot roots and from different planes within the same carrot root. Discs in both transverse and longitudinal planes (see Materials and Methods) were cut from four different carrot roots and cultured for three weeks, under l i g h t , in. shaken l i q u i d White's basal medium with added IAA (0.1 mg/l) and coconut milk (15 % ) . The results are summarized graphically in 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 of carrot discs derived from different carrots showed considerable variation, the extremes being highly significant (explants from carrot 1 grew approximately twice as fast as those from 26 TABLE I* Results of experiment designed to test the effect of indole-acetic acid and coconut milk on the growth of fresh carrot discs* A. Mean wet weights of carrot discs after three weeks of culture. Original 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 plus 0*1 mg/1 indoleacetic acid and 15 % coconut milk* t Medium Mean weight, in mg, of five carrot discs Wh 45.0 Wh IAA 58.6 Wh CM 86.8 Wh IAA CM 87.2 B. Analysis of variance. Source D.F. S.S. M.S. F Total 19 16,960 Between media 3 10,318 3,439 8.3** Within media 16 6,642 415 L.S.D. - 5 % level - 27.3 mg 1 % level - 37.6 mg 27 carrot 4)* Only slight mean differences occurred between discs from different planes of the same root. The standard deviations and coefficients of v a r i a b i l i t y show that there i s noticeable variation i n growth of discs derived from the same root. The degree of this 'intra-root' growth variation seems to change from carrot to carrot (range of C.V. 14.03 - 30*36) but i s essentially independent on the plane in which discs are taken (mean C.V*: transverse discs, 22.94; longitudinal discs, 20*57)* TABLE II. Analysis of variance of results (shown in Figure 1) from experiment on growth of cultured carrot discs derived t from longitudinal and transverse planes of four different carrot roots* Source D.F. S.S. M.S. F Total 78 57,698,74 Between carrots 7 35,136.48 5,019.49 15.79** (Between planes . 1 35.68 35.68 0.11) Within carrots 71 22,562.26 317.77 Table III summarizes the results of a preliminary experiment designed to compare the effects of shaken l i q u i d and agar based (solid) media on the proliferation of callus 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 solid (1 $ agar) condition. A l l cultures were kept on the shaker (which, of course, has no effect on the agar medium), under continuous l i g h t , for 10 days. 28 Figure 1. Mean wet weight in rag, Standard Deviation ( S . D . a n d Coefficient of Variability (C.V.) of cultured longitudinal and transverse carrot discs derived from four different carrot roots. Each disc weighed 18 mg originally and was cultured for 21 days in shaken l i q u i d White's basal medium plus Oil mg/1 IAA and 15 % coconut milk* Means are calculated from 5 - 1 6 discs* 150 CD 2 100 X UJ 5 50 i i 6 0 « M E A N a I I S . O . I = 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 i P t i i i 30.4 260 21.5 1 4 0 i i 22.7 279 i i 5 CARROT 6 1 0 16 15 4> i i 174 18.5 _ i C.V. 1 0 12 4 n o . o f d i s c s 29 TABLE III* Results of experiment designed to compare the effect of shaken l i q u i d and agar based media on the proliferation of callus by sunflower stem segments. A* Mean wet weights of stem segments after 10 days culture on White's basal medium plus 0*1 mg/1 IAA and 15 % coconut milk* Average weight of the stem segments was originally 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. F Total 9 171,906 Between media 1 127,464 127,464 22.9** Within media 8 44,442 5,555 L.S.D. - 1 # level - 158.0 mg 30 The average weight of the stem sections (4 mm in length) at the beginning of the experiment was 85 mg. As can be seen from Table III 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 of major experi-ments designed to compare the growth response of a number of tissues to different culture conditions was carried out. The culture conditions compared were: agar ( i . e . solid 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 six basic culture conditions (treatments) which the tissues were subjected to: 1. Agar medium, continuous l i g h t (A L) 2. Agar medium, continuous dark (A D) 3* Liquid medium, non-shaken, continuous l i g h t (Lq NSh L) 4. Liquid medium, non-shaken, continuous dark (Lq NSh D) 5. Liquid medium, shaken, continuous l i g h t (Lq Sh L) 6* Liquid medium, shaken continuous dark (Lq Sh D) Figure 2 shows, graphically, the results obtained with undifferen tiated carrot callus tissue grown under these conditions for six weeks (with weighing after both three and six weeks of culturing). White's basal medium with 0.1 mg/1 IAA and 15 c/o coconut milk was used throughout the experiment* 31 Figure 2. Bar graph showing mean weights of undifferentiated carrot callus tissues., as percentage of weight at the start of the experiment, cultured under different 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 replicates, and at six w e e k 3 means from two replicates. Each replicates represents six individual pieces of callus tissue cultured separately arid weighed jointly..White's basal medium plus 0.1 mg/l IAA. and 15 $ coconut milk was used in this experiment. Figure 3.' Bar graph showing mean weights of tumorous sunflower tissues, as percentage of weight at the start of the experiment, cultured under different conditions* Abbreviations used for culture conditions and medium are the same as in Figure 2. A l l values are means of two replicates. Each replicate represents six individual pieces of tissue cultured separately and weighed jointly . FIGURE 2 N X AL AO LqNShL Lq NSh 0 Lq ShL Lq ShO CULTURE CONDITIONS 31 The most striking effect was that given by the shaken l i q u i d medium. In both ligh t and dark i t caused a very marked stimulation of growth over agar and non-shaken l i q u i d media. Agar medium* however, was slightly superior to the non-shaken l i q u i d medium* Light appears to stimulate growth in the l i q u i d medivm (particularly in the shaken l i q u i d medium) but to slightly retard growth on agar medium. TATff-'B'- 17. Analysis of variance on weights of undifferentiated carrot callus tissue cultured under different conditions for three weeks (Figure 2). Source D.F. S.S. M.S. Total Replications 17 2 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 BxC 1 1 468,518.95 8,907.11 96,513.77 229,633.33 74,626.72 21,805.44 10,680.33 4,453.55 1.69 96,513.77 36.62** 229,633.33 87.14** 74,626.72 28.31** 21,805.44 8.27* 10.680.53 4.05 Error 10 26,352.23 2,635.22 A s t a t i s t i c a l analysis of the results obtained at three weeks i s given in Table IV. Highly significant 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 li g h t and dark. Furthermore, there was a significant interaction between the state 32 of the medium (solid or liquid) and the light-dark condition'. A l l the differences obtained at three weeks are merely more marked at six weeks (Figure 2). At the end of the six-week growth period the cultures grown in dark were pale yellowish while those grown in li g h t were green and covered with brownish-red spots. The cultures grown in l i q u i d mediwi in dark were much more friable and usually broke into several pieces before the end of the culture period (especially those shaken). Occasionally, roots developed on the cultures i n shaken l i q u i d medium, particularly on those in the dark. No roots developed on cultures grown on agar or in non-shaken l i q u i d medivau Cultures grown on agar medium were harder and more compact than those in l i q u i d medium. There tended to be more variation in shape, size, and appearance of cultures grown on agar or in l i q u i d non-shaken media than in those grown in liquid/shaken medium. A similar experiment was carried out to test the effect of the same culture conditions as i n previous experiment^on the growth of 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 results of this experiment are shown graphically in Figure 3 and an analysis of variance i s given in Table V. The results essentially parallel those obtained with the carrot callus tissue although the overall growth throughout the experiment was lower. Relative growth was markedly superior in the shaken l i q u i d medium, particularly under the light condition. Growth in the non-shaken l i q u i d and agar media was essentially the same i n the l i g h t , but growth in the non-shaken l i q u i d medium in the dark was markedly inferior to that on 33 agar in the dark. The effect of light was generally more marked i n this experiment, and in contrast to the carrot callus tissue,the tumorous tissue grown on agar was stimulated by l i g h t . The stimulatory effect of li g h t on the shaken l i q u i d medium cultures was particularly noticeable by 6 weeks. The trends in growth established at three weeks were again only extended and more marked by 6 weeks. . TABLE V. Analysis of variance on weights of tumorous sunflower tissue cultured under different conditions for six weeks (Figure 3)• Source - D.F. S.S. M.S. F Total 11 423,259,67 Replications 1 14,560.33 14,560.33 2.36 Treatments A. Agar vs Liquid m. 1 32,047.04 32,047.04 5.19 B. Shaking vs Non-sh. 1 224,115.12 224,115.12 36.33** 0. Light vs Dark 1 104,907.00 104,907.00 17.00** Interactions: AxC 1 16,695.37 16,695.37 2.70 BxC 1 91.12 91.12 0.01 Error 5 30,843.67 6,168.73 The analysis of variance of the data obtained at six weeks (Table 7) shows that there were highly significant differences between cultures grown in shaken and non-shaken l i q u i d median, and between cultures grown in li 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 inferior and superior to agar medium. No significant interactions were obtained. A series of major experiments was carried out to test the effect of the same growth conditions as before on tissues cultured in Hildebrandt's improved sunflower medium. The tissues used were fresh carrot discs, undifferentiated carrot callus, and tumorous sunflower tissue. Indole-acetic acid (0.01 mg/l) was added to the media for carrot discs and carrot callus, but not for the sunflower tissue. No coconut milk was used so that the media were completely chemically defined. Figures 4, 5, and 6 summarize graphically the results obtained with carrot discs, carrot callus, and tumorous sunflower tissue, respect-ively. A l l carrot callus and sunflower tissues in 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 discs, six individual discs in each treatment were weighed separately, allowing a s t a t i s t i c a l analysis, which i s given in Table VI. In the main these experiments paralleled those with White's medium. However, the stimulatory effect of l i g h t on growth in shaken l i q u i d medium was much more marked with the carrot callus and tumorous sunflower tissues than in the previous experiments. This effect was also much more marked in the above mentioned experiments than in the experiment with the carrot discs. Tissues cultured in non-shaken l i q u i d medium in the dark showed the poorest growth in a l l experiments. The growth on agar media, in both light and dark, and in non-shaken l i q u i d medium in the l i g h t was essen-t i a l l y the same for a l l tissues. 35 TABLE VI. Analysis of variance on weights of fresh carrot discs cultured under different conditions for six weeks (Figure 4). Six discs were cultured in each treatment and these were weighed separately. Source D.F. S.S. M.S. F Total 35 306,084.98 Treatments: A* Agar vs Liquid 1 16,866.72 16,866.72 5.77*. B. Shaking vs Non-Sh. 1 126,150.00 126,150.00 43.21** C. Light vs Dark 1 25,122.25 25,122.25 8.60** Interactions: AxC 1 41,472.00 41,472.00 14.20** BxC 1 8,893.50 8,893.50 3.04 Error 30 87,580.50 2,919.35 It should be noted that the total growth of the undifferentiated carrot tissues in Hildebrandt's medium was markedly inferior to that i n White's medium with coconut milk. Tumorous sunflower tissue, on the other hand, grew better in Hildebrandt's medium. It i s interesting that a l l of the tissues showed a marked decline i n growth during the second three weeks of culture, except when cultured in shaken l i q u i d medium i n l i g h t . Such declines were only obtained in non-shaken l i q u i d medium in 36 the experiments using White's medium. The results obtained by analysis of variance (Table VI) resemble very closely results obtained in previous experiments. The difference between agar and l i q u i d media i s significant, the differences between shaken and non-shaken l i q u i d media, between light and dark, and the interaction between agar-liquid media and light-dark conditions are highly significant. After 6-week cultivation the carrot discs grown in l i g h t developed greenish strips in*cambial region while the regions distal to cambium were brownish-red. Discs grown in dark were uniformly pale orange. The appearance of the undifferentiated carrot callus tissues i n this experiment was generally the same as described for the experiments with White's medium (see above). No roots were developed-on any tissue grown in Hildebrandt's medium. The tumorous sunflower tissue grown in l i g h t , however, turned green instead of remaining whitish colourless as i t did in White's medium. 37 Figure 4. Bar graph showing mean weights of fresh carrot discs in mg after three and six weeks in culture. Each treatment consisted of 6 discs cultured and weighed separately. The original weight of a disc was 42 mg. iifedium: Hildebrandt* s improved sunflower medium plus O.ol mg/l IAA. Abbreviations used for culture conditions are thexsame as in Figure 2. FIGURE 4 2 £ 4 0 0 o < u. O300 i -x <£ LU 5 2 0 0 < w 1 0 0 L \ N J WEIGHT AFTER 6 WEEKS WEIGHT AFTER3 WEEKS 1 8 9 143 AL 233 ,216. 186 \ \ 162 153 83 229 AD Lq NSh L Lq NSh D LqShL CULTURE C0N0ITI0NS ,260 277 Lq Sh 0 38 Figure 5* Bar graph showing mean weights of undifferentiated carrot callus tissues, as percentage of weight at the start of the experiment, cultured under different conditions. Values are means of 1 replicate consisting of six individual 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 in Figure 2. Figure 6. Bar graph showing mean weights of tumorous sunflower tissues, as percentage of weight at the beginning of the experiment, cultured under different conditions. Values are means of 1 replicate consisting of six individual pieces of tissue cultured separately and weighed jointly . Medium: Hildebrandt's improved sunflower medium* Abbreviations used for culture con-ditions are the same as in Figure 2. FIGURE 5 600 -400 1XX1 WEIGHT AFTER 6 WEEKS WEIGHT AFTER 3 WEEKS 2 0 0 X o U J z U J o cc U J Q . to < 5 I Li « u. x U J 5E o U J tr * < 1200 2 0 0 0 -400 426 168 AL 164 153 - I93v 154 \ \ 127 139 136 FTV1 110 297 FIGURE .583 236 N5I2, 305 1 4 0 133 2157 514 AO LqNShL Lq NSh D Lq Sh L CULTURE CONDITIONS 241 K \ 152 Lq Sh 0 39 DISCUSSION. The results i n genral corroborate and extend the findings of other workers. They demonstrate that growth of plant tissue cultures can be markedly stimulated by certain combinations of culture conditions. At the same time they raise some interesting questions concerning the metabolism of tissues grown in v i t r o . e beneficial influence of coconut milk on growth of cultured carrot discs, as revealed by the f i r s t preliminary experiment (Table 1), was to be expected. This effect 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 least partly due to an as yet uncharacterized factor, 'kinetin', isolated from coconut milk recently by Skoog and Mil l e r (in preparation). Since the carrot discs (and the callus derived from them) w i l l grow i n the absence of coconut milk i t appears that tissues either can .synthesize the active substance supplied by the coconut milk, or that this substance can be substituted to some extent by indoleacetic acid necessary for continuous growth of normal callus tissue. Apparently the growth-stimulating action of coconut milk i s not a result of i t s content of growth-hormone for in 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 of cultured tissues much more than IAA or any other related substance. Because in the present experiment carrot discs could proliferate callus 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 of 40 growth hormone sufficient to support i n i t i a l callus proliferation. 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 variable. The experiment comparing growth of discs from different carrots and different planes in carrots has considerable practical significance in the design of experiments. Since the average growth rate of discs de-rived from different carrots was highly significantly different i t appears desirable that a l l discs to be compared in 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 effect on subsequent growth. Since transverse discs were found easier to obtain in sterile condition they were used exclusively in a l l other experiments. There was also considerable variation in the growth of discs derived from the same carrot root. Caplin and Steward (1952) found the same to be true for discs cut from the secondary phloem of carrot roots. They further noted that discs containing cambium were more variable in growth than those with only phloem tissue. However, Wiggans (1953) reported that discs containing cambium grew considerably faster than those cut from phloem or xylem. The cambium containing discs are, there-fore, excellent material for experiments in which high rates of growth are desirable while phloem discs w i l l give more uniform results. The variation in growth between discs may be due to variations in the original amount of cambium present. If this i s the case, however, the discs from the longitudinal plane should grow faster as they contain 41 more cambium than transverse discs. It seems that there are minor inherent variations in the growth capacity of c e l l s in different parts of the root. In experiments using discs this 'intra root' variation can only be controlled by adequate replication and s t a t i s t i c a l treatment. In experiments in which carrot callus tissue derived by sub-culture from discs i s used i t would seem advisable to have a l l culture pieces derived originally from a single disc. This was the case with a l l callus cultures used in the experiments described here. The major result obtained fobm the experiments with different culture conditions was the consistent and striking demonstration that growth in shaken l i q u i d me dim*, was superior to that on agar mediWi (and. of course, superior as well to growth in l i q u i d non-shaken me dim)* Tikis applied to a l l tissues tested in these experiments, i . e . fresh carrot discs, carrot callus, tumorous sunflower tissue, and sunflower stem segments, regardlessV^ fWhite*s medium with IAA and coconut milk or Hildebrandt's improved sunflower medium was used. The reason for the growth-stimulating action of shaken l i q u i d medium i s undoubtedly complex. Probably the major effect i s due to the action of the medium washing over the tissue. This allows nutrients to enter the tissue at a l l surface points rather than just 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 of agar-cul tared tissues exposed continuously to a i r . The l a t t e r effect probably accounts for the loose, fl a c c i d surface of the callus growing in shaken l i q u i d medium, as compared with relatively smooth, harder surgace of callus grown on agar. The surface area of the cultures grown in shaken l i q u i d 42 medium is thus considerably greater, permitting increased exchange of nutrients and gasses. Secondary reasons for the superiority of the shaken l i q u i d medium may be the prevention of accumulation of harmful excretions ('staling* products) at the tissue surface of cultures, and greater avai l a b i l i t y of the ions from nutrient solution because the movement of these substances i s not hindered by adsorption on agar particles* Another reason for greater growth in shaken l i q u i d medium may be better exchange of (gasses) between the outer atmosphere and the atmosphere of the culture bottle due to the movement of the bottle. The influence of improved aeration i s corrobated undisturbed culture with growth of culture that had been removed from the tube and weighed every three weeks (both cultures were grown on agar medium); the latt 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 beneficial effect of agitated l i q u i d media on growth of tissue / cultures (using the auxophyton and t o l l e r tube techniques, respectively)• To make a direct comparison between their results and the results reported here i s very d i f f i c u l t for several reasons* F i r s t l y , although a l l these authors used carrot tissue, their cultures were of quite different origin and differed, most probably, in the inherent capability to grow: Caplin and Steward'used discs cut from secondary phloem, White used habituated tissue isolated in 1937 by Gautheret, and the present author used callus tissue isolated some three months before the beginning of experiments (the experiment with carrot discs cannot be used for this comparison because they were grown in medium without coconut milk and the growth indirectly by results obtained by White growth of 43 was, consequently, much smaller). Secondly, the sizes and weights of tissue pieces used for culturing were different* Caplin and Steward used 3 mg discs, White rectangulars about 15 mg, and the present author more or less irregular pieces about 70 mg each. Thirdly, the culture periods were different - Caplin and Steward's 20 days, White's 24 days for cultures in agitated medium and 120 days for cultures on agar medium, present author's 42 days. Even i f the f i r s t objection i s disregarded, the different weights at the beginning of experiments and the dif ferent periods of culturing make i t Impossible to calculute some ratioi- that could be reliably used for comparison of growth between the cultures. Weight increase of culture pieces i s to some extent geometrical when they are small, but gradually slows down as they enlarge (surface to volume ratio diminishes). White (1953) simply assumes a straight-line growth and divides the total growth by the time of culturing to give an average figure for growth per day* Caplin (1947), on the other hand, uses Blackman's (1919) compound interest law to calculate growth as percentage increase per day* Both methods can be extremely misleading and the results may be completely opposite according to which method i s used* The question of evaluation of growth in such a way that the result of one experiment can be compared reliably with the result of another experiment is very important but has been nearly completely neglected by workers in plant tissue culture. The only serious consideration of the problem was given by Caplin (1947)* In very general terms, the growth response to agitation obtained here may be compared with that obtained by Caplin and Steward in the following way: Caplin and Steward's cultures grew approximately twice as fast in agitated medium as on agar medium, in 20 days, while similar cultures used in this study grew nearly four times faster in agitated l i q u i d 44 me dim than on agar medium in 42 days i f cultures grown in l i g h t and dark were counted together, and more than five times faster i f only cultures grown in l i g h t are considered* Since White used different 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 in 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 in growth trends was found in the experiments here described, even though the cultures greatly exceeded the size of White's cultures. The ratios expressing the response of other cultures used in this study to agitation are generally smaller than in the case of carrot callus tissue but, nevertheless, they are always highly significant (see analyses of variance)• Certainly, the agitation technique described here i s simpler and more adaptable than those used previously (see Introduction)* Any horizont-a l l y o s c i l l a t i n g platform could be substituted 1for the shaker used, and almost any. type and size of 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 limited the size of Caplin and Steward's cultures to about 180 mg and White's to even less* 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 in the special tubes of the auxophyton or in r o l l e r tubes. It must be added that one disadvantage of 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 of the water from the medium can quickly produce detrimental changes in the concen-45 tration of nutrients* It i s recommended, even i f the humidity i s high, that tissues be transferred to fresh medium every two or three weeks* The finding that growth on agar medium was as good or better than that in non-shaken l i q u i d medium i s not too surprising. Although there may have been some unknown growth-promoting substances contaminating the agar, the effect was most probably a result of aeration conditions* The tissues in the l i q u i d medium sank and were par t i a l l y submerged in the l i q u i d . Such submersion prevents adequate aeration of the tissue, thereby retarding growth. White (1939) obtained a similar effect although in his case the difference between growth on agar and in l i q u i d was more.marked than in these experiments. He used longer times and the tissue in l i q u i d medium was completely submerged. The presence of continuous l i g h t had significant stimulatory effect on the growth of a l l tissues cultured in l i q u i d medium, particularly those in shaken l i q u i d medium* Steward et a l . (1952) obtained increased growth of carrot discs cultured in rotated l i q u i d medium although the difference due to ligh t was not so significant as in the present study. Light and dark conditions had only small, insignificant effects on the growth of tissues on agar medium* This agrees with the finding by Hildebrandt et a l . (1945) that l i g h t conditions had l i t t l e effect on the growth of tobacco callus and sunflower tumorous tissues cultured on agar* These results in general support the interpretation that cultures grown on agar are primarily limited by the diffusion rate of nutrients from the agar, whereas in l i q u i d culture this limitation i s alleviated and ligh t can have significant effect on the u t i l i z a t i o n of the nutrients. Since tissues grown in shaken l i q u i d medium presumably receive a better supply of nutrients than those in non-shaken medium (see above), the effect of ligh t would be expected, as was found, to be more marked in tissues grown in the former medium. 46 The actual physiological action of the li g h t i s d i f f i c u l t to ascertain. The stimulation of growth exercised by light i s probably not primarily due to increased photosynthesis. F i r s t l y , a l l the culture media contained an optimal concentration of sucrose; secondly, the sunflower tumorous tissue, used in one series of experiments, was stimulated by li g h t (Figure 3 and Table V) despite the fact that i t showed no green coloration (chlorophyll). It i s interesting to note that the most marked stimulation by li g h t on tissues grown in shaken l i q u i d medium was given with carrot callus and sunflower tumorous tissues cultured in Hildebrandt*s medium without coconut milk (Figures 5 and 6). The stimulation was much less marked when these tissues grown in White's medium with added coconut milk (Figures 2 and 3). This might suggest that l i g h t plays a role (direct or indirect) i n the synthesis, by the tissues, of a growth stimulating factor which i s supplied by coconut milk. Consistent with this hypothesis i s the fact that fresh carrot discs, which might contain an endogenous supply of such a factor, were not markedly stimulated by l i g h t when grown in Hildebrandt's medium. An interesting difference in 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 in 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 in shaken l i q u i d medium in li g h t , showed a marked decline in growth during the second three-week period. This might suggest that only under the conditions of shaken l i q u i d medium in li g h t can the tissues on a chemically defined medium synthesize an adequate supply of the growth-promoting substances supplied by coconut milk. Another noticeable difference between the responses to the two 47 nutrient media was that the carrot callus tissue grew significantly 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 of this effect i s probably directly due to the fact that the concentrations of nutrients in Hildebrandt's medium are specifically designed for sunflower tumorous tissue and may not be optimal for carrot tissue (or at least not so favourable as the concentrations in White's medium). However, difference in growth-hormone, metabolism between the two tissues must also be taken into consideration. Carrot callus i s a normal tissue and needs an exogenous supply of growth-hormone (and/or growth factor from coconut milk) for i t s growth (Gautheret 1942b, 1946, 1947a, and b) while sunflower crown gall tissue generates i t s e l f an excess of growth-hormone and does not respond to growth-hormone in the medium; (deRopp, 1947). Therefore, enriched White's medium (containing 0.1 mg/l IAA. and 15 % coconut milk) was superior to Hildebrandt's medium (containing only 0.01 mg/1 IAA) for growth of normal carrot callus tissue, while Hildebrandt'3 medium with Improved concentration of nutrients was superior to White's medium for growth of tumorous sunflower tissue. With the sunflower tissue, i t i s surprising that by merely using more favourable con-centrations of nutrients the growth can exceed that with coconut milk. The change in colour of sunflower tumorous tissue, discussed in next para-graph, may have been another factor contributing to the result. A rather notable phenomenom, which may have been pa r t i a l l y a cause or result of the improved growth of the sunflower tumorous tissue in Hilde-brandt' s medium, was observed. During a l l of the experiments in which White's enriched medium was used, the sunflower tumorous tissue remained whitish and translucent. However, when the tissue was transferred to. -? 48 Hildebrandt 1 3 medium for the later experiments the cultures grown in l i g h t suddenly turned light green. The cause or significance of this change i s d i f f i c u l t to explain. It may be that the optimal nutrient concentration of Hildebrandt's medium overcame a limiting factor in chlorophyll synthesis or that the coconut milk contained inhibitors of chlorophyll synthesis (coconut milk did not, of course, prevent chlorophyll synthesis in other tissues). The continuous cultivation (for some five months) of the sunflower tissue in lig h t might have been also an important factor influencing the metabolic capability 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 callus tissue developed chlorophyll in both media within about a week. WheT^ther these differences are specific or are due to a more meristematic nature of the tumorous cells cannot be said at present. 49 SUMMARY* The response of various plant tissues to different culture conditions was compared* The tissues used were cambium-containing discs from carrot roots, undifferentiated carrot callus, bacteria-free sunflower tumorous (crown-gall) tissue, and segments of sunflower stems* The cubture conditions compared, in 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 acid (0*1 mg/l) and Hildebrandt's improved sunflower medium was also compared under these different culture conditions* Agitation of the l i q u i d medium was accomplished through the use of a newly designed shaker, which consists basically of a horizontally o s c i l l a -ting bank of shelves* The tissues rested on the bottom of culture flasks (medicine bottles) on these shelves and were alternately exposed to medium and a i r as the l i q u i d medium washed back and forth* Amy horizontally o s c i l -l a t i n g platform could replace this shaker and almost any type and size of culture flask could be used* Probably any type of plant tissue could be cultured under these shaking conditions* It i s not necessary that the tissues adhere to the walls of the culture vessels as i n other agitajion methods so far used in plant tissue culture* Growth (weight increase) of a l l tissues in shaken l i q u i d medium (in both ligh t and dark) was markedly superior (two to six times greater average weight in 42 days) to that of tissues on agar and in non-shaken l i q u i d medium. The superiority of growth in 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 tissue, there i s no drying and hardening of the tissue 50 surfaces, resulting in a greatly increased surface area, harmful excretions cannot collect at the tissue surface, and diffusion of nutrients i s not hindered by adsorption on agar particles. To compare the growth of these cultures with those of other workers using agitation methods i s d i f f i c u l t due to the different sources of plant material, different sizes of tissues cultured, and different periods of cul-ture used. In general the stimulatory results of shaking obtained appear to be at least as good as those obtained by Caplin and Steward with the much more elaborate and limited 'auxophyton'. There was no sign of eventual growth stoppage as obtained by White, using r o l l e r tubes. There were no significant differences in the growth of tissues on agar, in both ligh t and dark, and in non-shaken l i q u i d medium in light* Tissues in non-shaken l i q u i d medium in the dark always showed the poorest growth. Tissues in non-shaken l i q u i d medium received inferior aeration. Light consistently stimulated tissues grown in l i q u i d medium, p a r t i -cularly those in shaken l i q u i d medium. The effect was especially marked on carrot callus and tumorous sunflower tissues grown in Hildebrandt's medium* It i s suggested that l i g h t may play a role in the synthesis of growth factors supplied by coconut milk* Light had no significant effect on the growth of tissues on agar medium, indicating that the primary limiting factor in the growth of such tissues may be the rate of diffusion of nutrients from agar* Carrot tissues showed better overall growth in the enriched White's medium while the sunflower tumorous tissue did better in Hildebrandt's medium.. The effect on carrot was probably primarily through indoleacetic acid 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 cultured in light while tumorous sunflower tissue remained .: white u n t i l placed in Hildebrandt*s medium, when i t turned ligh t green. The significance of these differences i s not known. One experiment showed that carrot discs derived from different carrots grew at significantly different average rates indicating that disds to be compared should be derived from the same carrot. The plane in which the discs were cut did not seem to influence subsequent growth. 'Intra root* variation in disc; growth necessitates replication. 52 LITERATURE CITED. Almsstrand, A., 1949. Studies on the growth of isolated roots of barley and oats. Phys .Plant. 2:372-381.. Bajer, A., and J . Mola-Bajer, 1954. Endosperm, material for study of the physiology of c e l l division. Acta Soc.Bot.Poloniae 23:69-98. B a l l , E«, 1946. Development in sterile culture of stem tips and subjacent regions of Tropaeolum majus L. and of Lupinus albus L. Am.J.Bot.33: 3ol-318. Blakeslee, A.F., and S.Satina, 1944. New hybrids from incompatible crosses in Datura through culture of excised embryos on malt media. Sci.99: 331-334. Bo l l , W.G., and H.E.Street, 1951. Studies on the growth of excised roots. I, The stimulatory effect of molybdenum and copper on the growth of excised tomato roots. New Phytol.50:52-75. Bonner, J., 1937. Vitamin B-, a growth factor for higher plants. Sci 85: 183-184. ,1938. Thiamin (vitamin B ) and the growth of roots: the relation of chemical structure to physiological ac t i v i t y . Am.J.Bot.25:543-549. 1940. On the growth factor requirements of isolated roots. Am.J.Bot.27:692-701. • and F.Addicott, 1937. Cultivation in vitro of excised pea roots. Bot.Gaz.99:144-170. and P.S.Devirian, 1939. Growth factor requirements of four species of isolated roots. Am.J.Bot.26:661-665. Braun, A.C., 1947. Thermal studies on the factors responsible for tumor i n i t i a t i o n in crown g a l l . Am.J.Bot.34:214-240. Brink, R.A., D.C.Cooper, and L.E.Ausherman, 1944. A hybrid between Hordeum  jubatum and Secale cereale reared form an a r t i f i c i a l l y cultivated embryo. J.Hered.35:67-75. Bunning, E., and H.Vfelte, 1953. Photoperiodische Reaktionen an pflanzlichen Gewebekulturen. Phys.Plant.7:197-203. Caplin, S.M., 1947. Growth and morphology of tobacco tissue cultures in v i t r o . Bot.Gaz.108:379-393. and F.C.Steward, 1948. Effect of coconut milk on the growth of explants from carrot root. Sci.108:655-657. 53 Caplin, S.M., and F.C.Steward, 1949. A technique for the controlled growth of excised plant tissue in l i q u i d media under aseptic conditions. Nature 163:920-924. — 1952. Investigations on the growth and meta-bolism of plant c e l l s . II. Variables affecting the growth of tissue explants and the development of a quantitative method using carrot root. Ann.Bot.n.s.16:219-234. Commoner, B., S.Fogel, and W.H.Muller, 1942. The' mechanism of auxin action. The effect of auxin on water absorption by potato tuber tissue. Jim. J.Bot.30:23-28. Cooper, D.C., and R.A.Brink, 1945. Seed collapse following matings between diploid and tetraploid races of Lycopersicon pimpinellifolium. Genetics 30:376-382. Curtis, J.T., 1947. Undifferentiated growth of orchid embryos on media containing barbiturates. Sci*105:128. Cutter, W.M., and K.S.Wilson, 1954. Effect of coconut endosperm and other growth stimulants upon the development in vitro of Cocos nucifera. Bot.Gaz.115:234-240. Dawson, R.F., 1938. A method for culture of excised plant parts. Am.J.Bot. 25:522-524. Dormer., K.J., and H.E.Street, 1949. The carbohydrate nutrition of tomato roots. Ann.Bot.n.3.13:199-217. Duhamet, L., 1939. Action de l'hetero-auxine sur l a croissance de racines isolees de Lupinus albus. C.r.acad.sci., Paris, 208:1838-1839. > 1951a. Action du l a i t de coco sur l a croissance des cultures de tissus de crown-gall de vigne, de tabac, de topinambur et de scorsonere. C.r.Soc.Biol.145:1781-1783. 1951b. Action du l a i t de coco sur les tissus accoutumes aux hetero-auxines de scorsonere, de tabac et de vigne. C.r.Soc.Biol. 145:1783-1785. 1951c. Action comparee du l a i t di coco sur une souche de tissue de carotte et sur des fragments preleves sur des racines. C.r.Soc.Biol. 145:1841-1843. * Gautheret, R.J., 1935. Recherches sur l a culture des tissus vegetaux: Essais de culture de quelques tissus meristematiques. These, Univ. de Paris. 229 pp. — — — 1937. Nouveilss recherches sur l a culture du tissu cambial. C.r .Acad.Sci., Paris, 205:572-574. — 1939. Sur l a possibilite de realiser l a culture indefinie des tissus de tubercules de carotte. C.r.Acad.Sci., Paris, 208:118-120. 54 Gautheret, R.J., 1940. Recherches sur le bourgeonnement du tissu camblal d'Ulmua campeBtria eultive in v i t r o . C.r.Acad.Sci., Paris, 210:632-632. —,1942a. Manuel technique de culture des tissus vegetaux. Preface de Alexis Carrel. Paris: Masson et Cie, 172 pp., 95 fi g s . 1942b. Hetero-auxin.es et cultures de tissus vegetaux. Bull.Soc.Chim.Biol.24:13-47. - — 1946. Comparaison entre 1'action de l'acide indole-acetique et celle du Phytomonas tumefaciens sur l a croissance des tissus vegetaux. C.r.Soc.Biol., Paris, 140:169-171. 1947a. Sur les besoins en hetero-auxine des cultures de tissus de quelques vegetaux. C.r.Soc.Biol., Paris, 141:627-629. 1947b. Action de l'acide indole-acetique sur l e developpement des tissus normaux et des tissus de crown-gall de topinambour cultives to v i t r o . C.r.Acad.Sci., Paris, 224:1728-1730. — — — 1948a. Sur l a culture de t r o i s types de tissus de Scorsonere: tissus normaux, tissus de Crown-Gall et tissus accoutumes a 1'hetero-auxine. C.r.Acad.Sci., Paris, 226:270-271. 1948b. Plant tissue culture. Endeavour 7, 26:75-79. —'< 1950. Remarques sur les besoins n u t r i t i f s des cultures de tissus de Salix caprea. C.r.Soc.Biol., Paris, 144:173-174. Gey, G.O., and M.K.Gey, 1936. The maintenance of human normal cells and tumor ce l l s in continuous culture. I, Preliminary report: Cultivation of mesoblastic tumors and normal tissue and notes on methods of cultivation. AM.J.Cancer 27:45-76. " * Haberlandt, G., 1902. Eulturversuche mit isolierten Pflanzenzellen. Sitzungs-ber.Akad.Wiss.Wien, Math.-naturw.Kl.111:69-92. Hackett, D.P., and E.V.Thimann, 1952. The effect of auxin on growth and respiration of artichoke tissue. Proc.N.A.S.(USAt #8:770-775. Hanning, E., 19o4. Zur Physiologie pflanzlichen Embryonen. I. Ueber die Cultur von Cruciferen-Embryonen ausserhalb des Embryosacks. Bot.Ztng. 62:45-80. Haynes, F.L., 1954. Potato embryo culture. Am.Pot.Jour.31:282-288. 1947. Hildebrandt, A.C., and A.J.Riker. Influence of some growth regulating substances on sunflower and tobacco tissue in v i t r o . Am.J.Bot.34:421-427. and B.M.Duggar, 1945. Growth in vitro of excised tobacco and sunflower tissue with different temperatures, hydrogen-ion oeneentrations, and amounts of sugar. Am.J.Bot.32:357-361. 1946. The influence of the composition of the medium on growth in vdtro of excised tobacco and sunflower tissue cultures. Am.J.Bot.33:591-597. 55 Jablonski, J.R., and F.Skoog, 1953. Cell enlargement and c e l l division in excised tobacco pith tissue. Phys.Plant.7:16-24. Knudson, L«, 1922. Non-symbiotic germination of orchid seeds. Bot.Gaz.73:1-25. Kostoff, D., 1930. Tumors and other malformations on certain Nicotiana hybrids. Sbl.Bakt.Parasit.Infect., Abt.2, 81:244-260. Kotte, W., 1922. Kulturversuche mit isolierten Wurzelspitzen. Bei t r . a l l g . Bot.2:413-434. Loewenberg, J.R., and F.Skog, 1952. Pine tissue cultures. Phys.Plant. 5:33-36. Marsden, M.P.F., and R.H.Wetmore, 1953. In vitro cultures of the 3hoot tips of Psilotum nudum. An.J.Bot.41:641-645. Morel, G., 1944. Sur l a posibilite de realiser l a culture indefinie des tissus de vigne. C.r.Acad.Sci., Paris, 219:36-37. — — — 1948. Recherches sur l a culture associee de parasites obligatoires et de tissus vegetaux. Ann.Epiphyties 14:123-234. Morel, G«, and R.H.Wetmore, 1951. Fern callus tissue cultures. Am.J.Bot. 38:141-143. Nickell, L.G., 1951. Embryo culture of weeping crabapple. Proc.Am.Soc.Hort. Sci.57:401-405. — and M.K.Brakke, 1954. Secretion of alfa-amylase by Rumex virus tumors in vi t r o . Biological studies. Am.J.Bot.41:390-394. Nitsch, J., 1949. Culture of fr u i t s in v i t r o . Sci.ll0:499. Nitsch, J.P., 1951. Action du jus de Tomate sur l a croissance des tissus de crown-gall.cultives in vitr o . C.r.Acad.Sci., Paris, 223:1676-1677. Nobecourt, P., 1939. Sur l a perennite et 1'augmentation de volume des cultures de tissus vegetaux. C.r.Soc.Biol., Paris, 130:1270. — - — - and L.Kofler, 1945. Culture de tissus de tige de rosier. C.r.Acad.Sci., Paris, 221:53-54. Northcraft, R.D., 1951. The use of oxalate to produce free-living cells from carrot tissue cultures. Sci.113:407-408. van Overbeek, J., M.E.Conklin, and JLF.Blakeslee, 1941. Factors in coconut milk essential for growth and development of very young Datura embryos. Sci.94:350-351. 1942. Cultivation in vitro of small Datura embryos. Am.J.Bot.29:472-477. 1944. Factors effecting the growth of Datura embryos in vi t r o . Am.J.Bot.31:219-224. 56 Rappaport, J., 1954. In vitro cultures of plant embryes and factors control-l i n g their growth. Bot.Rev.20:201-225. Riker, A.J., and A.E.Gutsche, 1948. The growth of sunflower tissue in vitro on synthetic media with various organic and inorganic sources of nitrogen. Am.J.Bot.35:227-838. Robbins. W.J., 1922. Cultivation of excised root tips and stem tips under sterile conditions. Bot.Gaz.73:376«390. — 1939. Thiamin and plant growth. Sci.89:303-307. • and M.A.Bartlpy, 1937. Vitamin B]_ and the growth of excised tomato roots. Sci.85:246-247. and M.B.Schmidt, 1939. Growth of excised tomato roots i n a synthetic medium. Bull.Torrey Bot.Club 66:193-200. deRopp, R.S., 1946. Apparatus for the prolonged sterile culture in vitro of whole plants or excised plant tissues. Sci.104:371-373. — 1947. The response of normal plant tissues and of crown-gall tumor tissues to synthetic growth-hormones. Am.J.Bot.34:53-62. 1951. The crown-gall problem. Bot.Rev.17:629-670. Shaw, D.T., L.C.Kingsland, and A.M.Brues, 1940. A r o l l e r bottle tissue culture system. Sci.91:148-149. Skirm, G.W., 1942. Embryo culturing as an aid to plant breeding. Jour.of Hered.33:211-215. Skoog, F., 1954. Substances involved in normal growth and differentiation of plants. Abnormal and Pathological Plant Growth, Brook* Symp.in B i o l . No 6, 1-21. - — 1951. Chemical control of growth and organ formation i n plant tissues*. Annee Biol.26:545-562. - — r and C.Tsui, 1948. Chemical control of growth and bud formation in tobacco stem segments and callus cultured in v i t r o . Am.J.Bot. 35:782-787. Steward, F.C., and CM.Caplin, 1951. A tissue culture from potato tuber: the synergistic action of 2,4-D and of coconut milk. Sci.113:518-520. ; 1952a. Investigations on growth and metabolism of plant c e l l s . III. Evidence for growth inhibitors in certain mature tissues. Ann.Bot.n.s.l6:477-489. 1952b. Investigations on growth and metabolism of plant c e l l s . IV. Evidence on the role of the coconut-milk factor in development. Ann.Bot.n.s.l6:491-504. 57 Steward, F.C., S.M.Caplin, and F.K.Millar, 195S. Investigations on growth and metabolism of plant c e l l s . I. New techniques for the investigation of metabolism, nutrition, and growth in undifferentiated c e l l s . Ann.Bot.n.S.16:57-77. Stingl, G., 19o7. Experimentelle Studie uber die Ernahrung von pflanzlichen Embryonen. Flora 97:308-331. Strauss, J., and C.D.LaRue, 1954. Maize endosperm tissue grown in v i t r o . I. Culture requirements. Am.J.Bot.41:687-694. Street, H.E., 1953. Factors controlling meristematic activity in excised roots. V. Effects of beta-indolylacetic acid, beta-indolylacetonitrite, and alfa-(l-naphtylmethylsulphide)-propionic acid on the growth and survival of roots of Lycopersicum esculentum, M i l l . Phys.Plant.7:212-23o, — • — — and J.S.Lowe, 1950. The carbohydrate nutrition of tomato roots. II. The mechanism of sucrose absorption by excised roots. Ann.Bot.n.s. 14:307-329. and S.M.McGregor, 1952. The carbohydrate nutrition of tomato roots. III. The effects of external sucrose concentration on the growth and anatomy of excised roots. Ann.Bot.n.s.16:185-205. Struckmeyer, B.E., A.C.Hildebrandt, and J.A.Riker, 1949. Histological effects of growth-regulating substances on sunflower tissue of crown-gall origin grown in v i t r o . Am.J.Bot.36:491-495. Tukey, H.B., 1933. A r t i f i c i a l culture of sweet cherry embryos. J.Herdd. 24:7-12. — 1934. A r t i f i c i a l culture methods for isolated embryos of deciduous f r u i t s . Am.Soc.Hort.Sci.32:313-322. . 1938. Growth patterns of plants developed from immature embryos in a r t i f i c i a l culture. Bot.Gaz.99:631. 1944. Plant breeding by incubator methods. Sci.Monthly 58: 321-322. Tulecke, W.R., 1953. A tissue derived form the poilen of Ginkgo biloba. Sci.117:599-600. Vickery, H.B., G.W.Pucher, A.J.Wakeman, and C.L.LEavenworthm 1937. The metabolism of amides in green plants. I. The amides of tobacco leaf. Jour.Biol.Chem.119:369-382. Wetmore, R.H., 1954. The use 'in vitro' cultures in the investigation of growth and differentiation in vascular plants. Abnormal and Pathological Plant Growth, Brookhaven Symp. in B i o l . , No 6, 22-40. White, P.R., 1932. Influence of some environmental conditions on the growth of excised root tips of wheat seedlings in l i q u i d media. Plant Phys. 7:613-628. 58 White, P.R., 1933. Plant tissue cultures. Results of preliminary experiments on the culturing of isolated stem-tips of S t e l l a r i a media. Protoplasma 19:97-116. - — - — 1934. Potentially unlimited growth of excised tomato rootstips in a l i q u i d medium. Plant Physiol.9:585-600. 1937. Vitamin in the nutrition of excised tomato roots. Plant Physiol.12:803-811. — 1- 1939a. Potentially unlimited growth of excised plant callus in an a r t i f i c i a l nutrient. Am.J.Bot.26:59-64. — 1939b. Controlled differentiation in a plant tissue culture. Bull.Torrey Bot.Club 86:507-513. 1940a. Vitamin Bfi, nicotinic acid, pyridine, glycine and thiamin in the nutrition of excised tomato roots. Am.Jour.Bot.27:811-821. 1940b.Sucrose vs. dextrose as carbohydrate source for excised tomato roots. Plant Physiol.15:355-358. > 1943. A handbook of plant tissue culture. Lancaster, Pa.: Jaques Cattell. 277 pp. — — 1951. Neoplastic growth in plants. Quat.Retf.Biol.26:1-16. 1953. A comparison of certain procedures for the maintenance of plant tissue cultures. Am .J.Bot.40:517-524. 1954. The cultivation of animal and plant c e l l s . The Ronald Press Company, New York. 239 pp. — and A.C.Braun, 1942. A cancerous neoplasm of plants. Autonomous bacteria-free" crown-gall tissue. Cancer Res.2:597-617. 

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.831.1-0106344/manifest

Comment

Related Items