STUDEES ON THE DEVELOPMENT OF BARLEY EMBRYOS EN CULTURE Thesis fear the Degree of Ph. D. MECHIGAN STATE UNIVERSITY Aian W. Coackerfiine 1961 This is to certify that the thesis entitled STUDIES ON THE DEVELOPMENT OF BARLEY EMBRYOS IN CULTURE presented by Alan W. Cockerline has been accepted towards fulfillment of the requirements for Ph. D-degree in Botany %M/z/}_. V Major professor DateS‘S‘ é/ 0-169 LIBRARY Michigan State University ABSTRACT STUDIES ON THE DEVELOPMENT OF BARLEY EMBRYOS IN CULTURE By Alan W. Cockerline The purpose of this investigation was to make a study in zitgg of the morphological development of the embryo and F? young seedling of barley, Hordeum distichon L. (Hannchen, C.I. g 531, a two-rowed variety). The embryos selected for study ; were excised from caryopses in various levels of development. L!‘ The culture media were entirely synthetic, with special emphasis being placed on the carbohydrate source, and in particular the sugars. The cultured samples ranged in level of development from the relatively undifferentiated late proembryos on up through the level of late differentiation to ”mature" embryos. For purposes of the investigation the cultured embryos were. grouped into four levels of morphological differentiation. These were: level I - late proembryo; level II - early differentiation; level III - middle differentiation; and level IV - late differentiation. Under the experimental conditions of this investigation the morphological responses of the cultured embryos appeared to be directly related to the level of embryonic differentia- tion at the time of inoculation into culture. The cultured I Alan W. Cockerline nbryos did not follow the normal sequence of i§_§itu_and in iyg_deve10pment. Though no responses were observed for ultured late proembryos, embryos excised and placed in ulture at early and middle differentiation germinated recociously to form aberrant plantlets, while embryos ultured at late differentiation germinated readily to roduce "normal" seedlings. It would seem that the development of callus, under the xperimental conditions, is characteristic for embryos ultured at early and middle differentiation. In the cul- ures of early differentiating embryos the callus formation 5 extensive and apparently a forerunner of subsequent evelopment. Thirteen sugars and seven intermediate (glycolytic) espiratory products were used as primary organic carbon ources in the media. In general, the pentoses and the ntermediate respiratory products were poor carbon sources. ucrose was the most satisfactory sugar for the culture of arley embryos under the experimental conditions. The esponses exhibited by the cultured embryos to the various arbon sources appear to be characteristic of the level of ifferentiation at the time of inoculation and the variation P ‘9-“‘1. h-u‘.‘.- .o.. 14 'Ifi-L‘ .. ”j d 3 Alan W. Cockerline between carbon sources is a matter of degree rather than type of response. Level IV responds most favorably to a sugar concentration of 3%; level III to 2%; and, level II to 4%. It would seem that the responses of the various levels of development are somewhat related to the concentration of agar in the media. Over a range of from 0.65 to 0.8% agar, the most satisfactory concentration for the culturable levels, was 0.8%. Eimhm‘j . an A. _- STUDIES ON THE DEVELOPMENT OF BARLEY EMBRYOS IN CULTURE BY Alan W? Cockerline A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1961 ii ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. L. W. Mericle and Dr. R. P. Mericle for their constructive criticism and encouragement throughout the course of the investigation, and to Dr. G. B. Wilson, Dr. W. B. Drew, Dr. E. S. Beneke and Dr. C. A. Hoppert for their valuable suggestions. Thanks are also due to Mr. P.G. Coleman for his un- reserved help in the preparation of the photographic material. Acknowledgment is made to the Atomic Energy Commission for financial assistance, equipment and supplies. TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . GENERAL CONCEPTS . . . . . . . . . . . . . . . . . HISTORICAL REVIEW . . . . . . . . . . . . . . . . Media Mineral Nutrition Carbon Nutrition - Carbohydrates Other Organic Supplements Physical Factors Summary MATERIALS AND METHODS . . . . . . . . . . . . . . Levels of Development Growth of Plant Materials in the Greenhouse Determination of Embryonic Level of Development (prior to harvesting) Sterilization of Living Material Contamination Culture "Chambers" Media Cultural Conditions EXPERIMENTAL RESULTS . . . . . . . . . . . . . . . General Morphological Development Responses to Organic Carbon Sources 11 12 14 23 34 37 39 39 46 48 49 50 51 54 54 56 68 81 Preliminary Experiments Intermediate Experiments Final Experiments Agar DISCUSSION . . . . . . . . . SUMMARY AND CONCLUSIONS . . LITERATURE CITED . . . . . . APPENDIX . . . . . . . . . iv Page 81 82 83 91 92 102 104 116 I4 Table 2. LIST OF TABLES Page Summary of the responses in_vitro, relative to the level of differentiation of the embryo at the time of inoculation . . . . . . . . . . . . 59 In vitro responses of barley embryos to various organic carbon sources . . . . . . . . . . . . 84 vi LIST OF PLATES Plate Page I. Seedlings germinated from caryopses rolled in a moist paper towel . . . . . . . . . . . . ix II. Morphological relationships between the various levels of differentiation . . . . . . 41 III. Culture chamber formed by placing plastic cups in a petri dish . . . . . . . . . . . . 53 IV. Aberrant plantlet on the 17th day of culture . 61 V. Aberrant plantlet on the 15th day of culture . 63 VI. Aberrant plantlet on the 20th day of culture . 65 VII. Aberrant plantlet on the 15th day of culture . 67 VIII. Characteristic callus formation of level II (Early Differentiation) on the 12th day of culture . . . . . . . . . . . . . . . . . . . 71 IX. Seedlings produced by a "mature" embryo, excised from an ig_vivo ripened caryopsis . . 74 X—XIV. Embryos at level IV (Late Differentiation) are cultured (in the dark) on media in which the concentrations of sucrose are: 1, 2, 3, 4, 5, and 6% . . . . . . . . . . . . . . 76 XV. Seedlings on the 10th day of culture exhibiting a "plateau" . . . . . . . . . . . . 88 XVI. Late differentiating (level IV) embryos cultured on a medium with an agar concen— tration of 0.65% . . . . . . . . . . . . . . 90 LIST OF APPENDIX TABLES Table Page 1. Media used in the investigation . . . . . . . 116 2. Components of three media for plant embryo cultures ... . . ..... . . ... . . . . . . . 118 3. LaRue's modification of White's Basic Medium . . . . . . . . . . . . . . . . . . . 119 4. Percent sugar (on a dry weight basis) at the various levels of development . . . . . . 122 viii PLATE I. Seedlings germinated from caryopses rolled in a moist paper towel; two days after ger— mination, and four days of incubation. O INTRODUCTION The purpose of this investigation was to make a study ig_vitro of the morphological development of the embryo and young seedling of barley, Hordeum distichon L. (Hannchen, C.I. 531, a two-rowed variety). The embryos selected for study were excised from caryopses in various levels of development. The culture media were entirely synthetic, with special emphasis being placed on the carbohydrate source, and in particular the sugars. The research problem.was planned to give answers to the following questions: 1. Can barley embryos be cultured ig_yi££g, when using well-defined media devoid of biological extracts such as coconut milk, etc.? 2. When cultured in_yitrg, does the embryo follow its "normal" in situ, in vivo sequence of embryological development? 3. Is precocious germination characteristic of the embryo in purely synthetic media? 4. How does root and shoot development in_vitro compare with "normal" germination? 5. Eight arbitrary stages of embryological development can be readily recognized and excised for culture. Do these levels of differentiation exhibit characteristic responses in vitro? 6. Do qualitative and quantitative variations in the primary organic carbon source have a pronounced effect on the development of the embryo, and seedling in vitro? 7. Will variations in the concentrationof agar have any effect on growth responses? 8. Are there quantitative and qualitative differences in sugars extractable by means of 80% ethanol reflux which might be useful in attempting to further characterize the various stages of embryological development? GENERAL CONCEPTS When reduced to its simplest form, the technique broadly classified as “plant tissue culture," may be defined as follows: "the cultivation of isolated plant tissues and organs in_yi§£g" (White, 1951). In addition to the study of "normal" and tumor tissues, the in_yit£g studies of plants have included among others, considerations of the following: root culture; endosperm culture; shoot apex culture; and, the culture of flower buds. Gustaf Haberlandt (1902) was the first to formulate specifically the concept of plant tissue culture, p§£_§g (White, 1943). His work is generally considered as being basic to the establishment of this field. In addition, he was the first investigator to express clearly the idea of cultivating isolated plant cells ifl.!l££2: as well as being the first to make well-organized studies of the cultivation of cells. The crux of Haberlandt's classic paper, ”Kulturversuche mit isolierten Pflanzenzellen," as translated by White (1954), is contained in the following excerpt: There has been, so far as I know, up to the present, no planned attempt to cultivate vegetative cells of higher plants in suitable nutrients. Yet the results of such attempts should cast many interesting sidelights on the peculiarities and capacities which the cell, as an elementary organism, possesses: they should make possible O conclusions as to the interrelations and reciprocal influences to which the cell is subjected within the multicellular organism. The term "embryo culture" has frequently been used in a rather vague sense. It is most commonly employed to describe the growth and development of plant embryos (as well as ensuant seedlings) in various culture media; with however, a seeming indifference towards age, size, and level of differentiation of the embryos at the time of excision and inoculation. The in vitro study of plant embryos has been approached in three general ways: (a) seedling culture; (b) culture of parts of embryos and seedlings; and (c) embryo culture in toto. Two fundamental problems in the development of successful techniques for the cultivation of excised plant tissues and organs have been: the choice of tissues which were responsive to such an approach; and, the development of satisfactory media. The technique of tissue culture, as such, has now advanced well beyond the point wherein it is an end in itself. Early investigators in the field of plant embryo culture were primarily concerned with such objectives as: overcoming certain doemancy phenomena in some seeds; and, obtaining hybrids which were previously considered to be unobtainable. Currently interest is centered on such aspects as: processes -1;volV€d J27 ar A ful :ure nece following c;ltures: Dietei tiated eml abserved t shryonic 2115 he re early germ iescribed Rijven between "p Zion accor :issues (i beaome Sep 3911 elong, irks off a TituI-e 0f ‘. filture im “lture, de mar”Shines involved in the course of germination; morphogenesis in vitro; and, specific nutritive requirements. A full appreciation of the subject of plant embryo cul- ture necessitates familiarity with, and comprehension of the following concepts: precocious germination; pregerminal cultures; and, postgerminal cultures. Dieterich (1924) in his work with relatively undifferen- tiated embryos of the Cruciferae, and Gramineae, frequently observed that the embryos instead of continuing their embryonic development, produced a premature germination. This he referred to as "kfinstliche Frfihgeburt," or artificial early germination. Such a form of development is often described in the literature as precocious germination. Rijven (1952) pointed out, that one shouki distinguish between "pregerminal" and "postgerminal" cultures. Germina- tion according to Rijven, begins "at the moment the embryonic tissues (in a state of cell division and plasmotic growth) become separated by an intercalated section of incipient cell elongation. The appearance of the cell elongation marks off a new phase of the life cycle, as it affects the nature of growth and structure." He thus divides embryo culture into two distinct phases: (a) pregerminal embryo culture, dealing with embryos excised from the ovule during embryogenesis, and with the attempt to prolong the embryonic gm‘tl‘. postgel at simi valved ling de J I growth until a fully differentiated embryo results; and (b) postgerminal embryo culture, which includes embryos excised at similar or later stages, and dealing with processes in- volved in the embryo's germination, and its subsequent seed- ling development. HISTORICAL REVIEW An historical review of plant embryo culture must of necessity include numerous citations from the broader field of plant tissue culture. As one might expect, many of the fundamental techniques and basic media utilized for the cultivation of plant embryos have evolved naturally from the extensive investigations of isolated plant tissues and organs in_yi§;2, The history and much of the literature pertaining to plant tissue culture has been described and discussed in various reviews and manuals, such as those of: Gautheret (1942, 1945, 1947, 1955); Guilliermond (1942); Nobecourt (1943); Rappaport (1954); Riker and Hildebrandt (1958); Street (1957); and, White (1931, 1936, 1941, 1943, 1946, 1951, 1954). In addition, the work with plant tissue cultures has in itself derived many ideas and techniques from that done with animal tissue cultures. The amount of research carried on with animal tissue cultures has been far more extensive than that with plants (Riker and Hildebrandt, 1958). Reviews on this subject, amongst others, have been published by Parker (1950) and Murray (1953). Following Haberlandt's paper, "Kulturversuche mit iso- lierten Pflanzenzellen" (1902), many believed that each * I o , individual cell had the capacity to divide and proliferate indefinitely. Early attempts to maintain cells in a proli- ferating state were unsuccessful. Positive results in the area of cultures produced from single plant cells were not published until 52 years later. Muir et_§1, (1954) reported on cultures produced from single cells of marigold (crown gall Origin) and tobacco (from "normal" stem). Cultures of animal tissues grown from single isolated cells were reported as early as 1948 by Sandford, Earle, and Likely. Subsequent papers relative to the cultivation of plant tissues from single cells, were published by: Nickell (1956); Muir §;_§1, (1958); Steward §§_§l, (1958a, b); and, Jones §t__l, (1960). Two early works might well be ranked as classics in the field of plant embryo culture. They are those of Brown and Morris (1890) and Hannig (1904). Brown and Morris's paper, "Research on the germination of some Gramineae. I," published in the Journal of the Chemical Society (London), is one of the earliest publications on the subject of plant embryo culture.~ Brown and Morris were primarily concerned with the post- germinal culture of mature grass embryos. Hannig, who made studies on various representatives of the Cruciferae and in particular of Raphanus sativus, should be given credit as the first investigator to report on the pregerminal culture of relatively immature plant embryos in_vitro. sul‘se Kastl< (1924) gation descril as cocc embryon Studie mst be endeavc closel) cereal Ant two Sin resulti either ( undoubte Ever. 3501 it P055112, of H791! 0v ”Carrence. five siDCe 5 l I \ I 4 A l / The works of Hannig, Brown and Morris, together with the subsequent investigations of Stingl (1907), Buckner and Kastle (1917), Andronescu (1919), Molliard (1921) and Dieterich (1924), established a foundation for future in yitrg investi- gations of plant embryos. Throughout the literature are to be found numerous papers describing the effects of various biological additives, such as coconut milk and tomato extract, on pre- and postgerminal embryonic development. Stingl's paper, "Experimentelle Studie fiber die Ernahrung von pflanzlichen Embryonen" (1907) must be ranked as one of the forerunners in this field of endeavor. His media p§£_§§.consisted of the endosperm of closely related grass species, into which mature, excised cereal embryos were implanted. Andronescu's contributions to plant embryo culture were two simple but very fundamental observations: seedlings resulting from cultured Zea Mays embryos were frequently either dwarfed, or weak and etiolated. Earlier investigators undoubtedly had made similar observations. It remained, how- ever, for Andronescu to publish these findings thereby making it possible for others to realize that when viewed in the light of their own research, such wasrurtunusual but rather a common occurrence. Numerous reports on a wide variety of materials have since substantiated his results. I l t - O 10 After removing the cotyledons, Buckner and Kastle cul- tured embryos of lima bean on nutrient agar and obtained relatively normal seedlings. Molliard's paper, "Sur 1e development des plantules fragmentees" (1921) was one of the earliest indications that there are some regions of the plant embryo, particularly the cotyledons, which have a greater proliferation capacity than others. Many papers dealing with the cultivation of embryos which have had organs removed and/or the culture of isolated organs and organ- fragments have since been published. The principal contri— bution of the forementioned authors was, I believe, to be found in their then unique approach to the subject of plant embryo culture. In almost any in_vitro study of growth and development, a familiarity with some of the abnormal and unusual manifestations which may be attributed to technique (such as wounding, etc.), rather than to the treatment pg; §§J permits a more meaningful interpretation of the results. Dieterich's pregerminal cultures of relatively undiffer- entiated embryos of the Cruciferae and the Gramineae were in essence an extension of Hannig's investigations. The basic idea of precocious germination, as described in detail under the section entitled GENERAL CONCEPTS, can be traced directly to his publication, "Uber Kultur von Embryonen ausserhalb des Samens" (1924), in which he described the condition known 11 ukunstlitflae Fiuhgeburt." Unfortunately to this date, the coblem Of Eunecocious germination is the rule, rather than he excepticui, in pregerminal embryonic studies. Extensiv a‘use has been made of postgerminal or seedling mlture as a means of obtaining viable plants from hybrid :rosses. This technique has frequently permitted the develop— ment of a young plant, which was otherwise impractical or impossible when a fully ripened seed was used. Excision and culture of the embryo has been one method of circumventing the problem of abortion in_situ, and l vivo. The initial impetus for this line of research was provided by Laibach's work with flax in 1925. Amongst others, subsequent reports with somewhat similar objectives, were those of: Jorgensen (1928); Laibach (1929); Werckmeister (1934); Starstead (1935); Skirm (1936); Lammerts (1942); Smith (1944); Blakeslee and Satina (1944); Cummings (1945); MacLean (1946); Sanders (1950); bnzak §§_§1, (1951); and, Weaver (1957, 1958). Media Media used for the culture of plant embryos are either iQuid or partially solidified with agar. Liquid media are epOrted onlyr rarely. In addition to distilled water the arious medial contain: mineral salts; carbohydrates; nitro- 3Km$ COmpOtunds; vitamins; auxins; and frequently biological dmacts sucll as coconut milk and tomato extract. t_? I V :2 l...“‘ 12 Media IISEHi for the culture of plant embryos are almost as numerous 133 the papers on this subject. Understandably the requirements of the material to be cultured will vary, and so accordingly will that portion of the environment formed by the culture base. Once a given medium has been published, its subsequent citations usually appear in a modified form. Media frequently used for the culture of plant embryos, are those of: Tukey (1934); Randolf and Cox (1943); White (1943); and Rijven (1952). An additional VEZIIEMI’ medium satisfactory for this type of investigation is that of LaRue (1955, unpublished). The composition of the afore- mentioned media will be found in Tables 1 and 2 of the APPENDIX. Mineral Nutrition Certain solutions appear with a high degree of regularity. These are as follows: Berthelot's Trace.Elements (1934); Randolf and Cox's Mineral Solution (1943); White's Mineral $01ution (1943); and, Nitch's Trace Elements (1951). The composition of these various solutions is to be found in Appendix Tables 1 and 2. The early investigators made use of very simple media. Over the years the direction has been towards that of in- creasing COHQPlexity. The more recent solutions have attempted to take intC> consideration the nutritional requirements of “KT 13 the materiaJ. to be cultured, in so far as they are known. Iron salJ:s used in the various media have had a tendency to precipitateu This accordingly renders the medium iron deficient. Street §§_al. (1952) believe that the precipi— tation of the iron salts may result in an increased pH of the medium. A number of investigators have replaced the .-..r— A relatively unstable iron sulfate by complexes such as ferric citrate. It would seem that the use of chelates permits one 1 to maintain embryos in culture over a much longer period of L! time without having to renew the media or stock solutions. Unfortunately very little is known of the role of the micro—elements pg; fig, in the nutrition of plant embryos in yitgg. Investigations on this subject have been primarily limited to the role of certain trace elements in plant tissue cultures. According to White (1951), excised tomato roots require iron, copper and molybdenum. White also believes that zinc, manganese, boron, and iron are probably necessary. Nobecourt (1938) and Gautheret (1939) introduced into their media a complex of accessory salts as suggested by Berthelot (1934) including beryllium, titanium, cobalt, nickel, and boron. An absolute ruecessity for such elements in tissue culture, has yet to be «demonstrated. Various investigators have made use of Berthelot's complex additives, and were unable to detect any significant influence. 14 Boll arui Street (1951) growing tomato roots in White's medium: “SifKJ highly purified chemicals, found that additions of copper (0.01.ppm) and molybdenum (0.0001 ppm) were necessary for successful growth. It should be noted however, that in normal reagent grade salts, these chemicals seem to be generally present as impurities in concentrations closely paralleling those added by B011 and Street. Possibly the failure to grow very young stages of some embryos in culture may in part be due to the lack of some micro element or group of trace elements in the medium. I doubt this. Rather I believe that the situation is a great deal more complex, and is related to the environmental organization as a whole. Carbon Nutrition - Carbohydrates Carbohydrates in the medium supply a source of energy for the maintenance of the various processes in the cultured embryo. Some investigators place great credence in their value as osmotic agents. Early workers such as Kotte (1922) and Robbins (1918, 1922) assumed that monosaccharides, especially glucose, would be a most suitable: carbon source. Tukey (1933) grew peach embryos On media contuaining 0.5 to 2% glucose. In 1934 he reported that althouQTI 2% glucose was beneficial for embryos removed 15 from the Seed at an early stage of development, it was inhibitory for later stages. White (1934, 1940b), Thielman (1938) and LaRue (1936a), showed that in the culture of tissue fragments, and embryos, sucrose was a more satisfactory energy source. Van Overbeek _£__l. (1944) noted that in the culture of the "heart shaped" relatively immature Datura embryos, sucrose was a much better carbon source than glucose. Doerpinghaus (1952) cultured "heart shaped" Datura embryos (10 species) in media containing sucrose, glucose, fructose, mannose, and glycerol. He found sucrose to be a superior carbon source for all species tested. The othersigars showed a species dependence. Three species of the "stramonium" type grew well on 4% sucrose. 2. meteloides grew well on all sugars except fructose. Q. discolor grew only on sucrose and glucose. LaRue (1936b) succeeded in culturing smaller embryos than had previously been reported. He observed that younger embryos require a higher sugar concentration than do the older ones. Tukey (1938), reported that the concentration of sugars for excised peach embryos, varied with the time lapse between fer1zilization and excision. In 1942, Lammerts found that for Peach, tangerine, and apricot embryos, 2% sucrose is better f°r Chaltures of relatively undifferentiated embryos, 16 while a CODLHentration of 0.5% is more satisfactory for the mature ones. Sanders (1950) found that growth in Datura §E£§Egggflg embryos, with 4% sucrose was 42 times that obtained with 0.9%. Over the same range, comparable embryos of three other Datura species increased only 1.2 to 2.7 times. Sanders' cultures were restricted to relatively well differentiated embryos. In 1953 Rietsma §t_§1, published a report on the culture of Datura stramonium embryos over an incubation period of 8 days. They observed that while the "pre—heart" stages grew well on 8-12% sucrose, the "late heart," and "torpedo" stages (mature embryos) developed more satisfactor- ily at 4%. The authors suggest that "culturing of plant embryos in very early stages can be made possible by studying the relations between the phase of development and nutritive requirements." Ziebur §t_§1, (1950) and Ziebur and Brink (1951) reported limited success in the culture of young Hordeum embryos on a medium containing 12.5% sucrose. They noted that a concen- tration of 2% was quite satisfactory for the well differen— tiated embryos. PUIViS (1944) in excised and vernalized rye embryos, foundl flowering is; increased with rising sucrose concentrations, reaChing a nuaximum at 2%. In 1947 ESteinberg reported that sucrose was superior to F ’-a.:, W_-T_—Ffig ~9— .\ .. 17 11 other Slngars tested for the culture of tobacco seedlings. fie noted, Tumwever, that substantial growth was obtained with both glucose and fructose. Knudsonl 01916) and Knudson and Lindstrom (1919) cultured intact albino corn seedlings in both light and dark. They found little difference between sucrose, glucose, and fructose ?j as carbon sources. The sugars failed to sustain growth, although those plants cultured with sugar in the media lived t'*‘_._ ‘ longer. ‘1” .1:- -M-... M.m.-__ .. . In 1923 Brannon reported that fructose was superior to glucose in intact seedling cultures of timothy. However it was not found to be superior for pea, alfalfa, radish or Bryophvllum. Juhren and Went (1949) cultured squash seedlings in the dark. They reported that fructose was a more effective carbon source than was sucrose. They believed that ”fructose “my be more effectively utilized than sucrose in the synthesis of certain growth promoting substances." Burstrom (1941, 1948) cultured excised wheat roots in media containing sucrose, glucose, and fructose. The most satis- factory growth responses were obtained from glucose. In additional cniltures of excised wheat, flax, and sunflower roots using {galactose in addition to the above mentioned sugars; galeuctose was found to be "toxic" to wheat roots, but not to flaXI<3r sunflower. Burstrom concluded that "galactose 18 was used fox- respiration instead of for synthetic purposes." Rijven (1952) found that 8% sucrose was the most suitable concentraticni, and carbon source, for Capsella bursa—pastoris. A mixture Of equal parts of isotonic solutions of fructose and glucose did not yield as good or better results. In White's (1940a) cultures of excised tomato roots ?2 sucrose was found to be superior to glucose, as well as to f equi-molecular concentrations of maltose, raffinose, and i arabinose. Dormer and Street (1949) reported that excised Li , tomato roots utilized sucrose at a greater rate than glucose or fructose, or equal molecular quantities of the two. They concluded that the absorption of sugar was closely related to phosphorylation. Smith (1932) and Hill and Patton (1947) reported that reducing sugars undergo partial hydrolysis with autoclaving. In their cultures of excised tomato roots, Street and Lowe (1950) assumed that there was no significant hydrolysis when sucrose was autoclaved. Rijven (1952) reported that he did not find any difference in the activity of un—autoclaved and autoclaved monosaccharides and disaccharides in his Capsella cultures. After autoclaving sucrose, Ball (1953) found from 0'7'0~8% glucnose or fructose present. He notes that callus cultures Of 53eguoia sempervirens when grown on two differently SteriliZed HHSdia.(heat sterilized and filter sterilized), 19 :Xhibit Cluite different growth patterns. Undoubtedly there are differenmces in media which have been either heat or filter SterdJlized. Ball made analyses of his media relative to hydrohytic products before and after sterilization. Those investigators who did not find any difference between the two based their conclusions solely on the manner in which the cultured tissues or embryos reacted to this kind of treatment. Nickell and Burkholder (1950) reported that in their studies of Rumex tumors in_vitro, sucrose, glucose, and fructose were the best carbon sources. Their's was one of the first publications listing soluble starch as a good organic carbon supplement. All organic acids were, with the exception of aspartic, rated as being poor carbon sources. Pyruvic acid at all levels (0.001-0.032 M or 88 ppm to 3817 ppm) proved to be inhibitory. The carbon sources and their ratings are summarized as follows: (1) Excellent - sucrose, Glucose, fructose, and soluble starch; (2) Good — raffinose, melobiose, cellobiose, glycerol, and aspartic acid; (3) POOr - galactose, xylose, arabinose, lactose, sorbose, rhamnose, snarbitol, inositol, mannitol, sodium acetate, oxalic acid, maleic: acid, and tartaric acid. Lee (19EHDa, 1950b) cultured seedlings of Lyc0persicum g513111911131131;Mill. He substituted equimolecular concentrations 1 ..‘l- ‘4‘ ”I". a. 'u 4‘... WL_o . ‘ . ||Iil|ll: ‘ H.1I nl. n I. 20 of ma1t°S&3, glucose, fructose, raffinose, and arabinose for 2% sucrose. The seedlings exhibited a greater growth response with sucrose than with any of the other sugars. In general no significant responses were observed with either raffinose or arabinose. One of the most exhaustive series of publications dealing with the effects of various carbon compounds in tissue culture, are those of Hildebrandt and Riker (1946, 1947, 1948, 1949, and 1953). These investigators studied the effects of various sugars, polysaccharides, organic acids, and alcohols on callus cultures of marigold, paris-daisy, periwinkle, sunflower, and tobacco tissues. Glucose, fructose, and sucrose were reported as excellent carbon sources for all five species. However, as would be expected, with certain species specific effects were observed. Significant increases in weight were noted for marigold on mannose, maltose, and cellobiose. Significant increases were also reported for periwinkle on galactose, maltose, lactose, and cellobiose. Only slight increases were listed for: marigold on starch, dextrin, pectin; paris-daisy on maltose, lactose, cellobiose, raffinose; starch, inulin, dextrin, pectin; sunflower on maltose, lactose, raffinose, starch, inulin, dextrin, pectin; and tobacco on xylose, mannose, lactose, raffinose, starch, dextrin, and pectin. | 1 k mm Q a... L. I ma M] 3:11 1 ..lltl‘- (IVAaI. r Iv (I’ll'i‘u‘nll‘ll‘ h, l ..l _ , a; :b, I--‘Ii¢-l‘1fl: ... , {In a: . uldd. 1.. I , 21 All Organic acids as a substitute for sucrose were un— favorable for growth although slight increases were observed. The organic acids were incorporated into the media at a con- centration of 0.5% or 5000 ppm. They included such acids as: succinic, stearic, fumaric, glutaric, acetic, malic, formic, tartaric, and glycolic. Alcohols at a concentration of 0.5% as the sole carbon source proved to be unfavorable for all tissues. The study included such alcohols as : methanol, ethanol, butanol, mannitol, dulcitol, and erythritol. The sugar concentrations ranged from 0.015 to 8.0%. Glucose, sucrose, and fructose were best in a range of from 0.5 to 2.0%, with an optimum of 1.0 to 2.0%. Almost as good for sunflower, as glucose, fructose, and sucrose, were: cellobiose and maltose. For periwinkle, raffinose, galactose, and lactose were almost equal in response to glucose, sucrose, and fructose. Concentrations of sugars below 0.5% and above 4.0% gave little or no growth. Van Overbeek gt_§l. (1944) based their explanation for the superiority of sucrose upon the findings of Duodoroff QE 1;. (1943), who reported that dry preparations of gseuggmas saccharophila will readily form glucose-l-phosphate with sucrose, but not with glucose or fructose, or a mixture of both. Van Overbeek g; 1;. reason there should be in these i? «4 substancefis a difference in availability for phosphorylation. A Similar type of explanation was that of White (1951) who concluded that ”sucrose utilization probably involves an enzymatic phosphorylation at the surface of the absorbing cells so that the material actually absorbed is freshly formed (nascent) hexose—phosphate which cannot be replaced by hexose and phosphate in uncombined form nor by the addi— tion of preformed hexose—phosphates to the medium." Plantefol (1938) and Plantefol and Gautheret (1939) found that carrot tissues can be adapted to glycerol as a carbon source, if first grown in a mixture of glucose and glycerol. Embryos and tissues of various plant species react in different ways to the various sugars, both quantitatively and qualitatively. In general sucrose would seem to be the most satisfactory organic carbon source for plant embryos. However, as is quite apparent from the variety of citations on this subject, one must be very careful when making a broad generalization from very specific data collected under equally restrictive conditions. Consideration should be given to the following factors when evaluating the various sugars: availability of the different sugars; paths of translocation; changes during translocation; specific roles of these sugars in growth and development; differences in 22 23 requirement for sugar; and, differences in ability to utilize sugar. Other Organic Supplements Numerous investigators using varied tissues, organ— fragments and embryos have reported that the cultured material can assimilate inorganic nitrogen. Nitrates have proven to be the most suitable nitrogen source p§;_§g. In a few instances ammonium salts have been reported to be as good a source of nitrogen. Generally organic nitrogenous compounds have been assumed to be inadequate nitrogen sources, and even sometimes toxic in culture (Burgeff, 1936; and Knudson, 1932). Brown (1906) reported an increase in dry weight of excised barley embryos grown in media containing asparagine, aspartic acid and glutamic acid. Spoerl in 1948 noted that in orchid embryos, under certain conditions, arginine and aspartic acid were as effective as ammonium nitrate, if used as a substitute for the latter. However, when used in addition to ammonium nitrate they did not show any stimulation to growth. Spoerl also reported that the effect of amino acids on the growth of orchid embryos depended on such factors, as: concentrations of acids used; light conditions; and age and species of orchid embryos. In "unripe" orchid seeds only arginine supported growth. Eighteen other amino acids tested inhibited growth. In mature embryos, 24 aspartic acid was a good nitrogen source; glutamine a neutral one; and other amino acids appeared to inhibit growth. Ziebur §t_§l. (1950) observed that casein hydrolysate when supplied to a medium in combination with phosphate ions and sodium chloride, had an inhibitory effect on the post- germinal development of immature Hordeum embryos, and a F? stimulating effect on the pregerminal growth of these ‘2 1 embryos. They also reported that the excised immature embryos i continued their "embryonic growth" but did not germinate. Ea ‘ Casein hydrolysate has been categorized by some investigators as a "so-called" embryo factor. Similar effects have been noted with water extracts of dates, bananas, wheat gluten hydrolysate, and tomato juice (Kent and Brink, 1947). The inhibition of germination is attributed by these authors to the high osmotic pressure (a somewhat controversial point) created by the addition of these extracts and especially by casein hydrolysate in con— junction with sodium chloride. The same authors also assumed that the amino acids present in casein hydrolysate not only act as suppressors of precocious germination of the immature embryos, but also as nutrients helping to increase the rate of embryonic growth. Sanders and Burkholder (1948) attempted to analyze the growth prOmoting action of the amino acid complex present 25 in casein hydrolysate. They made use of postgerminal growth of young Datura embryos as a bioassay. Addition of casein hydrolysate (100—800 ppm) and equal parts of cysteine and tryptophane (6.67 mg. per 100 mg. of cas. hyd.) to the basic medium, resulted in significant growth of the embryos. Similar results were obtained by substituting in the medium a mixture of twenty amino acids, approximating the composition of casein hydrolysate. It was noted that single amino acids or incomplete mixtures of the twenty acids did not promote embryonic growth at rates equal to those obtained by the complex mixture. This led Sanders and Burkholder to conclude, "that the growth stimulus of the combination of the combination of the twenty amino acids, results from a physiological inter- action rather than from the summation of effects of the individual acids." Rijven's (1952) results would seem to contradict those of Sanders and Burkholder. Using Capsella embryos, glutamine proved superior to the amino acid mixture. Two sets of con- ditions must be taken into consideration when evaluating this apparent contradiction. Rijven cultured immature embryos of Capsella, while Sanders and Burkholder studied Qgtgga. Also, Rijven incorporated glutamine rather than glutamic acid into his medium. w. 1‘. ... .53. , .... a...“ 26 Rijven (1952) suggests that glutamine is significant in the nitrogen metabolism of Capsella. In 1949 Street reported that glutamine was the prevailing amide in crucifer seedlings. Rijven also observed that while the young Capsella embryos apparently can use glutamine as a nitrogen source, asparagine would seem to be of limited value. In addition he noted that the small embryos when grown in a medium with asparagine exhibited significant starch formation, while those grown in a glutamine-containing medium showed negligible starch V1.2“ “’— ":3 formation. The role of compounds with the amino group, in early embryonic development, is somewhat vague. Rijven (1952) reported no detectable improvement when purine derivatives were added to his medium for Capsella embryos. Rappaport _t_§l. (1950) observed that nucleic acids proved inhibitory to postgerminal cultures of Datura. Curtis (1947) and Curtis and Nichol (1948) reported that barbituates in the medium, resulted in unorganized tumor outgrowths from orchid embryos. It has been noted by numerous investigators that tissue cultures grown on a medium containing only mineral salts and carbohydrates showed only very limited growth. Some authors have referred to this as "residual growth." It has been demonstrated that the promotion of growth in excised materials requires incorporation into the media of varied accessory substances and factors. Lil-3‘ " 11‘4“ 27 Kotte (1922) added "Liebig Fleisch" extract (tomato) to his medium. Robbins (1922) made use of peptone and autolyzed yeast as additives. White (1932a) cultured 0.2 mm. Portulaca embryos on a medium which had in addition to minerals and 2% sucrose, yeast extract. White (1934), Fiedler (1936) and Gautheret (1939) reported, that a "pasteurized" extract of yeast is a useful source of required additional substances. Attempts to identify clearly the nature of these substances has as yet had limited success. Kogl and Haagen-Smit (1936) considered thiamin and biotin as essential growth factors. They believed ascorbic acid to be a non—essential substance on the basis of lack of stimulation in their experiments. However Bonner and Bonner (1938) reported that ascorbic acid promoted growth in excised pea embryos. Virtanen and Hausen (1950) gave evidence to suggest that the role of ascorbic acid, in the culture of pea and wheat embryos, is that of a reducing agent of nitrates in the medium. Bonner (1938) found nicotinic acid to be a growth factor for excised pea embryos. Bonner and Axtman (1937) reported that pantothenic acid favored growth in peas. Van Overbeek gt 3;. (1942) observed that young Datura embryos failed to develop cotyledons on media devoid of supplementary factors. They used an arbitrary mixture of 28 the "so—called” growth factors, which was composed of: glycine, 3.0 ppm; Thiamine, 0.15 ppm; ascorbic acid, 20.0 ppm; pantothenic acid, 0.5 ppm; nicotinic acid, 1.0 ppm; pyridoxine hydrochloride, 0.2 ppm; adenine, 0.2 ppm; and succinic\acid, 25.0 ppm. This mixture was effective in stimulating the growth of Datura embryos, but not of those r] less than 0.5 mm. in length. l' Rijven (1952) reported negative results when the following a.- - 1g-“ “3;. mixture (in ppm) was added to his medium for Capsella embryos: thiamine, 0.15; nicotinic acid, 1.0; pyridoxine-HCl, 0.2; calcium pantothenate, 0.2; inositol, 0.5; p-amino benzoic acid, 0.5; riboflavine, 0.1; folic acid, 0.01; and biotin 0.0004. 0 Rytz (1939) found that various varieties of pea embryos did not react in the same way to thiamine treatment. Coconut milk has been used to stimulate the growth of immature plant embryos. It has been observed frequently that, even when vitamins and other growth factors were added to the media, the embryos failed to grow without the addition of a biological extract. Van Overbeek §£_§1, (1941, 1942) used coconut milk in their media for immature Datura embryos. When the coconut milk was autoclaved the embryos grew as "an unorganized mass." When the coconut milk was filter-sterilized, "normal" growth of the very young embryos resulted. These embryos developed .||||IIHI. .. ‘ _ I. _ 29 from an initial length of 0.15 mm. to 8.0 mm. after seven days. The authors believed that two substances or complexes were to be found in coconut milk. A heat stable one producing cell proliferation, and a heat labile one for "normal" differentiation. Norstog (1956) and Chang (1957) succeeded, in varying degrees, in culturing relatively undifferentiated barley embryos. Norstog used a medium in which coconut milk was incorporated 90% by total volume. On this medium embryos as small as 0.15 - 0.20 mm. (late proembryos) were grown. Only four such immature embryos were cultured to seedlings, following a precocious form of development. As did other investigators, Norstog believed that coconut milk contains a factor or factors essential for root and shoot development. Following the technique of Norstog, Chang also incorpor- ated 90% by volume coconut milk into his medium. Thirty—eight embryos with an initial size of 0.5 x 0.30 mm., were grown to an average size of 1.20 mm. x 0.90 mm., after two weeks in culture. He noted that the ig_y;££g embryos differed signi- ficantly from those developing ig_y;gg, in that the cultured embryos were larger at all morphological stages of differen- tiation, and slower in passing through the various levels of development, than those in situ, and ig_yiyg. The cultured embryos never reached a stage comparable to the final level v.2: ““‘j‘ _ . l .. 30 of differentiation, although seedlings were obtained. Van Overbeek _§_al. (1942) observed that Datura embryos growing on autoclaved coconut milk, did not show any root formation. They assumed that a heat stable substance in coconut milk brings about suppression of root development. Cook and Doyle (1916) found that when germinating coconut embryos, in_vivo, the developing roots remained in the fibrous outer shell and did not penetrate into the milk. Other investigators who have made use of coconut milk for various plant parts and embryos, are: Caplin and Steward (1948); Ball (1948); Nickell(l950); Steward and Caplin (1951); Morel and Wetmore (1951); and Steward §t_§1. (1952). Numerous attempts have been made to identify the factor or factors present in coconut milk and responsible for growth promoting activity. Van Overbeek g; 31. (1944) found that with partial purification of the original ”sap," they obtained 170 times more activity. Steward and Caplin (1951) and Steward gt 11. (1952) reported the activity in coconut milk was due to a heat stable, water soluble, organic com- pound. Mauney (1952) with data somewhat contradictory to that of Steward, reported that most of the activity was in the "meat," and not in the milk. After purification he found the factor to be a heat stable, acid and alkali labile, non- volatile, and water soluble organic compound. Mauney ":::”“‘"”*‘::? 31 prepared a concentrate 4350 times as active on a fresh weight basis as the raw material. In many cases coconut milk has been a success in pro- moting growth which was not deemed otherwise possible. How— ever, in an equally large number of instances it has also been a failure. Van Overbeek et a1. (1942) observed that the addition of coconut milk to media for very small (radially sym.) Datura embryos, did not promote growth. Haagen-Smit _3 a1. (1945) reported that coconut milk did not promote any significant growth of immature corn embryos (0.3 mm. in length). Ziebur and Brink (1951) found that the addition of coconut milk to the media did not have a favorable effect on the growth of immature barley embryos (0.3—1.1 mm. in length). The data of Ziebur and Brink do not contradict that of Norstog (1956), nor Chang (1957), in that the latter two investigators used the biological additive at 90% by total volume, while in contrast Ziebur and Brink used 20% by volume. Van Overbeek gt a1, (1944) reported that the following exhibited an embryo factor, although not equal to that of coconut milk: yeast extract; wheat germ; almond meal extract; and extracts of Datura ovules. The value of endosperm as a biological additive to the culture media was noted as early as 1907, by Stingl. Excising embryos of wheat and oats, and then implanting them into 32 endosperm of their own species, as well as making reciprocal implants into the endosperm of each other, Stingl observed that: wheat embryos grew better in oat endosperm than they did in wheat endosperm; and oat embryos grew better in wheat endosperm than they did in oat endosperm. Blakeslee and Satina (1944) reported that a powdered malt extract solution was an effective substitute for the culture of immature Datura embryos. However, as with coconut milk, it caused an inhibition of growth after autoclaving. Solomon (1950) reported evidence to the contrary. He found that the growth factor in malt was not destroyed by autoclaving, but rather was masked by the presence of a newly formed growth inhibitor. This inhibitor was water soluble, non-volatile, and heat stable. Dickson and Burkhart (1942) reported that the growth promoting action of malt extract was due to soluble nitrogenous substances presentin the extract in the form of amino acids. Sanders (1950) observed that Seitz-filtered malt interfered with the germination of Datura embryos, decreased root formation, and inhibited shoot development. Van Overbeek (1942) suspected an auxin present in the coconut milk as being responsible for the inhibition of root growth. After testing various extracts of coconut milk he concluded that, "it is the auxin in the medium which keeps ‘ “:1, 21.40-1- ‘ifl ‘V “LL... Il 33 the embryo in the embryonic stage. Lowering of the auxin level causes the embryo to go into the seedling stage." As one might expect, studies relative to auxins, and their effects on the growth and development of plant tissues and embryos, have yielded a wide variety of results. Went's (1926) publication, "On growth accelerating substances in the coleoptile of Avena sativa,“ was the forerunner in this field of endeavor. Sanders (1950) was unable to detect any favorable effect of IAA on Datura embryos. He reported slight improvement in growth of Datura stramonium embryos, with the addition of alpha napthalene acetic acid (0.05 and 0.1 ppm). At a higher concentration of 0.5 to 10.0 ppm an "irregular twisting and bending" of the embryo was observed. Rijven (1952) reported that the concentrations of IAA 0.001 ppm stimulated the growth of Capsella embryos, Concen- trations above 1.0 ppm were inhibitory. The influence of auxins on the cultures of mature plant embryos is somewhat different. Solacolu and Constantinesco (1936), and Gautheret (1937), working with bean embryos used IAA in concentrations of from 1.0 to 100.0 ppm. They reported, amongst other observations, a swelling of the hypocotyl eventually producing an unorganized mass of cells. Kraus E; E1. (1936) and Hamner and Kraus (1937), found similar '02:" ““7277 34 results with bean seedlings. Lachaux (1944) found that IAA increased respiration of Jerusalem artichoke tissue cultures but was ineffective in carrot tissue. Physical Factors Very few investigations have been carried out to clarify the significance of pH. Van Overbeek gt gt. (1944) observed that for "heart shaped" Datura embryos, two to four days after excision, a pH of 7 was optimal, and that for later cultures a pH of 5.5 was optimum. Rijven in 1952 was unable to determine a definite optimum for the culture of immature Capsella embryos. He felt that "other factors of nutritional nature interfered with the growth of the embryos." Rijven noted that shifts of pH may upset ionic balances of the medium and in this way interfere with normal growth. Street _t_gt. (1952) reported that a shift towards alkalinity in White's medium may occur during experimentation, with the resulting precipitation of iron salts, thus rendering the iron unavailable. Street refers to this as a “stalling factor" in growth. The current literature reports media with a pH range of from 5.7 to 6.0. Some researchers add phosphate buffers, while others apparently rely on phosphates in the biological additives to act as stabilizers. 35 Van Overbeek et a1. (1944) grew Datura embryos at various temperatures. Relative growth was measured at regular intervals for the first five days of the experiment. An optimum of 32°C. was reported. Rijven (1952) found an opti- mum temperature of 30°C.during the first twenty—four hours of his experiment with Capsella embryos. He points out, however, that over a period of 96 hours, other factors ("probably nutritional") appeared, and rendered difficult an exact determination of non-nutritional factors. Rijven observed a slight inhibiting effect of light on pregerminal embryonic growth, not significant however until after the fourth day of culture. As previously noted some investigators place great emphasis on the effects of osmotic pressure. Dieterich (1924) reported significant differences in the developmental pattern of young embryos in culture, when: submerged in the agar medium, embryonic growth was continued; and, when placed on the surface of the agar, precocious germination resulted. Rappaport (1954) observed that in the culture of Qgtgtg embryos, the optimal sugar concentration of the medium varies with the age at the moment of excision. He states that "the younger the embryos the higher must be the sugar concentra— tion." The range of concentrations was from 8.0 to 0.5%. 36 Ziebur gt gt. (1950) attributed the inhibition and prolonging of embryonic growth, resulting from the addition of 1% casein hydrolysate to the medium, to the high osmotic pressure created by the addition of amino acids and sodium chloride. They replaced sucrose with mannitol (believed to be nutritionally inactive with barley embryos), maintained a high osmotic pressure, and prevented germination. Other authors have reported that osmotic pressure to some degree controls behavior of the embryos. Uhvitis (1946) observed that germination of mature alfalfa seed can be inhibited by changing the osmotic pressure of the environ- ment, using sodium chloride or mannitol. Duym gt gt. (1947) reported that the inhibition of germination caused by extracts of sugar beet balls, can be partly attributed to the osmotic pressure caused by inorganic salts present in the balls. Pope (1944, 1949) induced barley embryos to germinate while still on the plant, by exposing them tg gttg, and lowering the osmotic pressure of the environment by applying wads of cotton soaked in water. Street and McGregor (1952) in their report on the culture of excised roots, relative to sucrose concentration, concluded that "the effect of varying sucrose concentration on the growth of roots is not caused by the resulting changes in the osmotic pressure of the medium." '1 37 In 1952 Went suggests, in his paper, "Physical factors affecting growth in plants," that: Specific diffusible growth factors, needed for differen— tiation, are produced within the embryo and have a tendency to diffuse into the surrounding medium. In larger embryos, enough of these substances are accumu- lated to initiate differentiation, but in the smaller ones these growth factors would diffuse away, and would fail to reach a critical concentration which would cause differentiation. Possibly this may in part explain why well differentiated embryos can be grown in nutrient media without the addition of growth factors. Relatively undifferentiated embryos it would seem develop satisfactorily only if a complex such as coconut milk, etc. is introduced into the media. Went concludes that: It would appear there is a dependence on the balance between the rates of production of these growth factors not only whether an organism can grow at all in a given medium, but also what its growth rate will be. Summary It would seem that there are no real general principles which can be applied per gg to the culture of plant embryos, except for the fact that both minerals and an organic carbon source are necessary constituents of the media. The require- ments for "immature" embryos frequently differ from those of "mature" embryos. Additives, in addition to the above- mentioned, have been demonstrated as being necessary for the 38 culture of very young embryos. Such additives frequently include biological extracts, which unfortunately, even to the present date, have been poorly delimited. 39 MATERIALS AND METHODS As noted in the Introduction, the material used for this study was Hordeum distichon L. (Hannchen, C.I. 531). The cultured samples ranged in level of development from the relatively undifferentiated late proembryos on up through the level of late differentiation to "mature" embryos. The “mature" embryos were excised from in situ, and i vivo ripened caryopses. One thousand, nine hundred and seventy-eight embryos were used for the investigation. Of this number, 800 were set aside for the exploratory chromatographic studies, as reported in the Appendix. The tg vitro observations pgt_gg_were based on 1178 embryos. Levels of Development As stated in the Introduction: eight arbitrary stages of embryological development can be recognized readily and excised for culture. Extensive studies on the embryogeny of barley have been reported by Merry (1941, 1942), Eunus (1954), and Mericle and Mericle (1957). Mericle and Mericle "arbitrarily divided" barley embryogeny into 13 stages. Their study was based on: 7 proembryo stages (a-g) and 6 stages of differentiating embryo (1—6). The authors describe stage 6 as "being the fully differentiated embryo as found in the ‘55 1*. .- :‘qufl WEI; \ I 40 PLATE II. Morphological relationships between the various levels of differentiation. Each representative figure is properly pro- portioned to the smallest member of the series (x 100). Fig. 1. Level I Late Proembryo 0.12 x 0.09 mm. 2. Level I Late Proembryo 0.30 x 0.15 mm. 3. Level II Early Differentiation 0.40 x 0.20 mm. 4. Level II Early Differentiation 0.60 x 0.30 mm. 5. Level III Middle Differentiation 0.80 x 0.40 mm. 6. Level IV Late Differentiation 1.30 x 0.85 mm. I I E T A L P 42 seed." These stages were based on characteristic histological and morphological features of the embryo. Barley embryogeny may be divided into two broad categories: proembryos; and differentiating embryos. As reported by Chang (1957) and used in this investigation "the former may in turn be arbi- trarily divided into three sub—groups: early, middle, and late proembryos." Additionally the differentiating embryos may be categorized, as: early, middle, and late differentiation. The youngest embryos successfully excised for culture work were those referred to as late proembryos. For purposes of this investigation the cultured embryos have been grouped into four levels of morphological differentiation. These are: Level I - late proembryo; Level II - early differentiation; Level III — middle differentiation; and, Level IV — late differentiation. This study has been based on the foremen- tioned levels of development. Therefore, for the sake of brevity, morphological manifestations of the cultured embryos are frequently correlated with the various levels: i.e., level I, level II, etc. Plate II has been designated to show the morphological relationships between the various levels of development. As noted in the descriptive text for this plate, all figures are at a magnification of approximately 100x. Each represen— tative figure is properly proportioned to the smallest member of the series. 43 The various levels of development were categorized on the basis of two sets of factors: (a) length and width dimensions of the embryo; and (b) characteristic morphological features. (a) length and width dimensions of the embryos tgygt Reference Stages I Late Proembryo LP f-g 11 Early Differentiation ED 1-2 III Middle Differentiation MD 3-4 IV Late Differentiation LD 5-6 Level Dimensions in mm. I Late Proembryo .12 x .09 to .30 x .15 II Early Differentiation .37 x .18 to .60 x .30 III Middle Differentiation .67 x .35 to .82 x .40 IV Late Differentiation .90 x .45 and beyond (b) characteristic morphological features Level I (Late Proembryo - P1. II, Fig. 1-2): Morphologically there are no readily apparent signs of differentiation. Immediately after excision the "youngest" of the late pro- embryos will be characterized in form by a shape closely approximating that of a prolate spheroid. Towards the end of level I this orbicular shape becomes quite pronounced, tending in some instances to appear slightly obovate. 44 Level II (Egrlv Differentiation - P1. II, figs. 3-4): That part of the embryo which is away from the axis of the seed (abaxial), can be observed early in level II as a slight swelling near the distal end of the embryo. The swelling becomes more pronounced towards the end of level II, at which time, this convexity assumes the shape of a semi-circular ridge. This ridge of tissue represents the morphological manifestation of the developing coleoptile (the sheath which surrounds the shoot apex and the developing leaves in the Gramineae). The obovate form of the developing embryo is quite pronounced by the end of level II. Level III (Middle Differentiation — Pl. II, fig. 5): In this level of development the circular ridge of tissue representing the developing coleoptile is completed. Early in this level the shoot apex can be observed as a pronounced protuberence within the ring of tissue formed by the developing coleoptile. The swelling, denoting the shoot apex, does not completely fill the circle of tissue formed by the coleoptile, rather it takes on the appearance of a "nodule," somewhat centrally located. Towards the end of this level the primordium of the first true leaf can be observed as a delicate fissure "cutting across" the swelling of the coleoptile, and bordering "on the edge" of the forementioned "nodule." The developing radicle (primary root) can be observed histologically, but is not obvious morphologically. M»: 45 It is within this level that the scutellum (single coty- ledon characteristic of Gramineae) first becomes readily apparent. It forms a fan-shaped shield surrounding the coleoptile, and extending out in a distal orientation from the mesocotyl. When compared with the scutellar development in level IV the "fan" is quite thick and "fleshy” in appearance. Level IV (Late Differentiation - Pl. II, fig. 6): The primordia of two true leaves can be observed within the encircling ridge of the developing coleoptile early in this level. As differentiation proceeds the coleoptile shows a marked extension and takes on the form of a "very stubby tube" at the apex of which can usually be Observed a small slit, through which the first true leaf emerges when ger- minated 1 vivo, and which is generally torn and ruptured by germination tg.vitro. The scutellum is one of the most pronounced features of this level of development. In some instances it has a truly "fan-shaped" appearance. In con- trast to level III it becomes almost "paper thin” at its edges. However, in other cases it will be more "shield- shaped" and give the appearance of extending almost to the top of the coleorhiza (sheath encasing the radicle of a monocotyledenous embryo). The coleorhiza is very prominent in level IV and frequently shows a marked swelling at the tip. W‘TE k.- 46 Histologically a primary root primordiam and 4—5 seminal root primordia are well developed. Three to four leaf primordia can be observed histologically at this level, although only two are usually discernable morphologically. Growth of Plant Materials in the Greenhouse The source material (excluding field-grown crops) was collected from plants prepared for, and grown under the following conditions. The basic soil mixture consisted of the following: three parts loam; one part sand; and one part peat moss. The seeds were sown in ten inch pots; five plants to a pot. Available stock was maintained by weekly plantings, in a series of five, with a theoretical total of twenty-five plants at harvest time. In order to obtain relatively uniform embryos, only the main stalks were allowed to develop and the tillers (side "branches") were removed as they appeared. The pots in each linear series were rotated one quarter of a turn daily and changed in position once a week. Such procedure was standard and necessary in order to compensate for varying environ- mental conditions within the greenhouse, such as: influence of heating pipes; variation in natural light exposure; and position of the pots relative to window vents. 47 The Plants were watered by "flooding" the pots once each week supplemented by intermediate sprinkling when necessary. A regular fertilization schedule was carried out every ten days using the commercial preparation, Vigoro. Disease preventative measures such as spraying with Malathione, dusting with sulfur, and fumigating with "Nicofume," were carried out at regular intervals. A supplementary light source was supplied daily from 0-1800 hours. Use was made of several one thousand watt incadescent lamps, placed at three foot intervals, and set. so as to be a minimum of two feet from the top of the heading plants. During the fall, winter, and spring the temperature was controlled as closely as the physical set-up and environmental conditions permitted. Daytime temperatures ranged from 75-850F., and nighttime temperatures from 60-650. A tempera- ture drop of from 15-200 was necessary for the production of well-developed viable heads. Lacking this change, or having temperature ranges in excess of the forementioned, invariably resulted in the development of basically sterile heads. Under the above-mentioned conditions one could, in general, expect to have the relatively undifferentiated late proembryos, on the average, 55 days after sowing. Embryos at the level of late differentiation were usually available on or after the 6lst day from the time of planting. . 48 'Determination of Embryonic Level of Development (prior to harvesting) In the culture of plant embryos time is a very critical factor. One must always allow for the fact that a certain num- ber of ovaries will fail to develop, with resultant sterility. The pure mechanics of excision is susceptible to human error. A number of embryos will be injured or destroyed, even with very careful technique. In general the terminal and proximal four caryopses of any given head of Hordeum distichon L., var. Hannchen, are frequently somewhat out of phase with the re- mainder. These eight caryopses are therefore lost for experi- mental purposes. The longer the elapsed time between har- vesting and the actual excision and inoculation of the embryos, the greater is the chance for deterioration of the material, and a change in the level of development which would render it useless for a given series of treatments. A procedure for determining the level of differentiation of the embryo, while tg_situ, in vivo was worked out. On the whole this has proven to be most satisfactory for the variety of material studied, particularly for plants cultivated in the greenhouse during the late fall, winter and early spring,when environmental conditions can be reasonably controlled. The technique used to ascertain the level of differentia- tion of the embryos, before microsc0pic examination, and the occu We] the C0 49 practicaJ- for rapid analysis in the greenhouse was based upon the time of emergence of the awns (bristle-like appendage, occurring on the lemma* of each floret) from the "boot" (vernacular description of the terminal leaf sheath enveloping the immature head). It was observed that there was an apparent correlation between the level of differentiation of the embryos and the number of days elapsed after the awns first emerged from the boot. Elapsed Time Level of Development 6 - 7 days I Late Proembryo 8 - 10 days II Early Differentiation ll - 12 days III Middle Differentiation l3 and beyond IV Late Differentiation Sterilization of Living Material The procedure which produced the most satisfactory results (relative to effective sterilization with an absence of readily recognizable injury) was as follows: A. l. **Pa1ea, lemma and glumes intact. Place material in a vial containing a 1% solution of Kromet*** *Lemma - the lower of the two bracts inclosing the flower in the grasses, formerly called the flowering glume. **Palea - tiny upper bract which with the lemma incloses the flower in the grasses. ***Kromet - a commercial complex of sodium hypochlorite, with wetting agent, supplied by the Wyandotte Chemical Corporation, Detroit, Michigan. 50 (2 1/2 min.), with intermittent agitation: shake 1/2 min.; rest 1 min.; shake 1/2 min.; and rest 1/2 min. 2. Decant Kromet solution. Rinse rapidly in three changes of sterile basic salt solution (in triple distilled water). B. 1. Remove: glumes; palea, lemma, and awn. 2. Place material ("naked" fruits) in a 1% solution of Kromet and agitate as above. 3. Decant Kromet solution, and rinse rapidly three times in basic salt solution, as above. C. Place sterilized material (preparatory to excision) in a sterilized petri dish containing filter paper moistened with basic salt solution. Spread the material evenly, permitting no layering. Note: total elapsed time in the disinfectant = 5 min. Contamination The degree of contamination was relatively low. The cultures were regularly 95-100% free of contaminants. Organisms most frequently encountered in the few samples which had to be discarded, were: Aspergillus sp.; Penicillium sp.; and Mucor sp. Fungal infection almost invariably pre- ceded bacterial contamination. Material cultured during the late fall, winter and early spring exhibited only negligible traces of contamination. Embryos excised and cultured from samples collected in the greenhouse during the summer months, were likewise low in their degree of infection. However, since conditions in the green- house during the summer months were frequently unfavorable to 51 good heading, it was often necessary to make use of samples colleCtECl from field-grown crops. It was from such material that the greatest amount of contamination developed. The techniques of sterilization and excision were standard through- out. The fungus growth was characteristically initiated at the point of inoculation of the embryo, usually on the surface of the embryo pg; gg, One might therefore conclude that the contaminant was already within the embryonic tissue, and accordingly not affected by ordinary means of sterilization. Undoubtedly more drastic techniques would have eliminated this variable. Unfortunately such methods probably would also have been detrimental to the inoculum. Culture "Chambers" Three different types of culture chambers were used in this investigation. The type of chamber used is noted separately for each experiment, and their descriptions are as follows. I. Plastic cups (Plate III): in a set of four, with depressions 15 mm. in diam., and a depth of 10 mm. II. Round—bottom vials (Plates X-XIV): 25 mm. in diam. x 100 mm. in length; cotton stoppered, and "sealed" with Sargent, grade M, Parafilm. III. Test tubes (Plate IX): 30 mm. in diam. x 195 mm. in length; cotton stoppered, and "sealed" with Parafilm. 52 PLATE III. Culture chamber formed by placing plastic cups in a petri dish , ("sealed" with masking tape) in the bottom of which are two moistened filter papers. .' 54 Media Three media were used during the course of the investi- gation, and are noted separately for each experiment. These were: White's (1943), Gautheret's (1950), and LaRue's (1955, unpublished). These media are described in detail in Table l of the APPENDIX. In addition, Table 3 of the APPENDIX gives further information relative to LaRue's modification of White's Basic Medium. The media pgt_gg.were heat—sterilized by autoclaving at 250°C. (or 1210C., Heat of Condensation), 15 to 17 pounds pressure for an exposure of 15 minutes. The vitamin solutions were sterilized by filtration through a bacterio-sinter filter, and incorporated into the final medium. Separate stock solutions were maintained for the: basic salts, trace elements, and vitamins. Such additives as auxins (i.e., IAA) were prepared freshly and heat sterilized along with the basic medium. The pH for all media was adjusted to 5.7 by means of 0.1 N KOH. Cultural Conditions With the exception of the first Preliminary Experiment, all cultures were maintained in the dark at from 75-800F. One experiment was carried out in a "growth control laboratory." Embryos excised at level IV (Late Differentiation) and cultured 55 on Whit—9' S unmodified basic medium in test tubes (Plate IX) , were grown under the following conditions, as per Mericle and Mericle (1957): day temperature, 72°F.; night temperature, 60°F.; and light, 1200 ft.-candles during an 18-hour day. 56 EXPERIMENTAL RESULTS The investigation pgt_gg_was based on eight experiments. Studies 1-7 were concerned with the tg_vitro activity of various levels of embryonic differentiation. Experiment #8 (Chromatographic Studies), although certainly purposeful, was exploratory in nature and is reported on and discussed in the APPENDIX. The seven cultural studies of excised barley embryos were grouped into the following three categories: preliminary; intermediate; and final experiments. As noted earlier in MATERIALS AND METHODS a total of 1,978 embryos were used for the investigation. Of this num- ber 800 embryos were used for the chromatographic studies. The following is a numerical breakdown of the number of embryos upon which the results for the remaining seven experi- ments were based. 1. Preliminary Experiments (Culture medium: White, 1943) #1. 10 embryos 2. 60 embryos 3. 60 embryos 130 II. Intermediate Experiments (Culture medium: Gautheret, 1950) #4. 40 embryos 5. 48 embryos §§. . \ ‘K ‘ x ,. i i ! 57 III. Final Experiments (Culture medium: LaRue, 1955) #6. 384 embryos 7. 576 embryos 960 No observations were reported for contaminated specimens. Where an individual within a set (with reference to embryos cultured in plastic cups as a set of four) became contaminated, the whole set was discarded and that portion of the experiment repeated. The embryos cultured in plastic cups were grown in very close proximity to one another. Under such circum- stances it was felt that the presence of an infected sample in the immediate vicinity of "healthy" embryos would introduce a variable which might be significant in an interpretation of the results. The mechanics of the experimentation was a limiting fac- tor in the number of samples used at any one time. This involved such considerations as: availability of samples at the pr0per level of differentiation; degree of sterility within the heads; and the time factor (elapsed time) between harvesting, sterilization, excision and inoculation. The most satisfac- tory results can be obtained only when a small number of fruits are processed at any one given time. Permitting the sterilized material to "lie around" in a moist chamber for an "excessive" period of time introduces, at the very least, two new variables: deterioration of the material; and significant changes in the level of development. 58 The entire investigation must be classified as "explora- tory." It was therefore considered that the studies, at this stage, would be most useful if a wide range of treatments were carried out using a limited number of samples per treat- ment; and in particular a small number of "replicates," rather than attempting to concentrate on any one particular area. 1. Summary of the responses in vitro, 59 relative to the level of differentiation of the embryo at the time of inocuhtion TABLE Level I. Late Proembryo Level II. Early Differentiation Level III. Middle Differentiation Level IV. Late Differentiation Reference LP Stage f—g ED 1-2 MD 3—4 LD 5-6 No responses were observed with embryos cultured at level I. Level II highly aberrant Level III less aberrant Level IV seedling rather than nlantlet plantlet than II plantlet partial germination "complete" germin- generally "complete" common ation more frequent germination than II, but not as cqmmpn as IV shoot usually shoot not restric- "normal" shoot restricted to an extension of the (~01 ponti 1e lst true leaf occasionally emerges from coleoptile ted to an extension of the coleoptile lst true leaf frequently emerges from coleoptile development common lst true leaf regularly emerges from coleoptile; up to three true leaves (;E_Vitr0) primary root rarely emerges from coleorhiza; no seminal roots observed callus a regular occurrence extensive callus development; embryo per se often "obscured" formation of callus apparently a "pre- requisite" for sub- seguent developmgpt responses character— istic; relatively predictable under gxpt'l conditions primary root frequently emerges from coleorhiza, but not as common as IV; seminal roots infrequent callus prevalent, but not as common as in II callus not as extensive as in II. rarely "obscures"the embryo; frequently concentrated in mesocotyl and cole- optile regions callus does not seem to be as sig- nificant as in II responses more variable than in II primary root regularly emerges from coleorhiza. seminal roots abundant no callus observed at any time responses character- istic and predictable under most conditions 60 PLATE IV. Aberrant plantlet on the 17th day of culture. Produced by an embryo inoculated at level II (Early Differentiation). Note: coleoptile protruding from a substantially callused and swollen mesocotyl region; the outline of the first true leaf can be seen vaguely within the coleoptile (x130). PLATE IV 62 PLATE V. Aberrant plantlet on the 15th day of culture. Produced by an embryo inoculated at level III (Middle Differentiation). Note: callus develop- ment in the mesocotyl region, though not as sub- stantial as in Plate IV; a deterioration at the tip of the coleoptile; the first true leaf clearly visible within the sheath; and the second leaf visible vaguely at the edge of the callus for- mation (x75). PLATE V 64 PLATE VI . Aberrant plantlet on the 20th day of culture. Produced by an embryo inoculated at level II (Early Differentiation). Note: the pronounced collar formed by the coleoptile at the base of first and second true leaves; the darkened shield-like formation below the coleoptile is the scutellum bending upward; and the loose and shaggy callus development (x70). PLATE VI 66 PLATE VII. Aberrant plantlet on the 15th day of culture. Produced by an embryo inoculated at level III. Note: the coleoptile is represented by a vestigial "ring" at the base of the first true leaf; characteristic callus; and the vague outlines of the recurved second leaf approxi— mately 3.5 cm. from the tip of the coleoptile (x85). PLATE VI I 68 The EXPERIMENTAL RESULTS of the investigation are summarized in Tables 1 and 2. General Morphological Development Table 1 summarizes in detail the morphological char- acteristics of the developing embryo and subsequent seedling (or plantlet) tg_ytttg, relative to the level of differen— tiation at the time of excision and inoculation. Both the shoot and the root arising precociously from embryos cultured at levels II and III generally have a somewhat abortive appearance. To this aberrant type of development I have applied the term "plantlet," rather than seedling. Only the latter can properly be applied to the young plant which has evolved from embryos placed in culture at level IV (Late Differentiation). Plates IV, V, VI, and VII are examples of aberrant plantlets. A detailed description of these is included in the legend for each plate. Attention should be drawn to the four, somewhat different, nuinifestations of the coleoptile, as shown in the fore- nRentioned plates. Plate IV shows a coleoptile protruding ZEJTCHn a substantially callused and sWollen mesocotyl region. 1355 in Plate IV, in Plate V the coleoptile also has emerged :ElTCun an extensively callused mesocotyl region, though it is rullcfli more pronounced in its development and the first true 69 leaf can be observed readily within the coleoptile. As noted in MATERIALS AND METHODS, the first true leaf as it emerges from the coleoptile during precocious germination does not pass through the "emergent" pore at the tip of the sheath. In precocious germination the first true leaf generally emerges by ripping and tearing the tip of the coleoptile as it comes out. Sometimes there is a shredding and partial deterioration of the tissue at the extremity of the coleoptile even before the first true leaf has reached this point (Plate V). In Plate VI the coleoptile forms a pronounced "collar" at the base of the first and second leaves. In contrast, in Plate VII the coleoptile is represented only by a vestigial "ring" at the base of the first true leaf. When placed in a moistened paper towel, germination from in situ, in vivo ripened caryopses usually occurs within 48 ihours (Plate I). Under favorable conditions, embryos excised aand.cultured at level IV (Late Differentiation) generally germinate in from 48 to 72 hours. Germination of embryos Chaltured at earlier levels of differentiation is not as clear <211t. Precocious germination of early differentiating embryos (Juevel II) is usually 10-15 days from the time of inoculation. EIn'bryos inoculated into culture at level III will germinate, if at all, by the 10th day. 7O PLATE VIII. Characteristic callus formation of level II (Early Differentiation), 12th day of culture. Note: the more or less spherical shape; mask- ing of the morphological features; pronounced swelling of the coleoptile; and the shoot apex represented as a slight, lateral protrusion. (x180) PLATE VIII 72 RO017-8 produced at levels II and III frequently are poor in root hair development. Root hairs derived from embryos inoculated at level IV will vary from sparse to dense pubes- cence. In some cases epidermal hairs can be observed on the surface of the "tips” of the coleorhizae of embryos inoculated at level IV. The callus formation so very characteristic of level II (Early Differentiation) is shown in Plate VIII. The callus development may be of two distinct types or a combination of both. It may be highly compact in appearance, as in Plate VIII, or it may be very loose and irregular, as in Plate VI. The compact type of growth tends to assume a more or less spherical shape. As noted in Table 1, during the process of callus development, the characteristic morphological features of the embryo are frequently all but obliterated. Table 1, level III (Middle Differentiation), shows Inesponses which are more variable than level II. All of the Eunbryos used in the study have been very carefully selected ‘tCD fit one of the four levels of development. However in level IEJII, even with these precautions, the samples often act in the fashion of either level II or level IV. 73 PLATE IX. Seedling produced by a "mature" embryo, excised from an tg_vivo ripened caryopsis. Culture chambers exposed to 18 hours of illumination daily. Note: the three leaves in Figure 8, and the abundant root hair development throughout. Using Figure l as the standard (with an arbitrary value of 1), the scales of the remaining seven figures are shown in the form of a ratio. K Fig. l 2 days 2 4 " 1:2 3 6 " 1:2 4 8 " 2:5 5 10 " 3'10 6 12 " 2:5 7 l4 " 2:5 8 20 " 1:5 PLATE IX .1 I Iciclut ll 75 PLATES X - XIV. Embryos at level IV (Late Differentiation) are cultured (in the dark) on media in which the concentrations of sucrose are: l, 2, 3, 4, 5, and 6%. The figures can be read directly in concentration of sugar (i.e., Fig. l = 1%). This series of plates emphasizes the re- sponse of the late differentiating embryos to a sugar concentration of 3% (xl.7). Plate X 5 days XI 7 days XII 11 days XIII 14 days XIV 20 days respi sour: pent phos int 11 m1 81 Responses to Organic Carbon Sources IQiirteen sugars and seven intermediate (glycolytic) respijratory products were used as primary organic carbon sourceas. These compounds included the following: four pentoses; five hexoses; four di—hexoses; four hexose-, phosphates; a triose; a triose phosphate; and pyruvic acid. The thirteen sugars were used as the primary organic carbon source for all four levels of development. The intermediate respiratory products were used only for level II (Early Differentiation) in Intermediate Experiments 4-5. Preliminary Experiments Experiment 1: Embryos excised at level IV (Late Dif- ferentiation) were cultured on White's unmodified basic medium in test tubes (Plate IX). The culture chambers for experiments 2-3 were round- bottom vials. Experiment 2: Late differentiating embryos were cul- tured on a modified version of White's medium, in which the organic carbon source, sucrose, was incorporated into the media at l, 2, 3, 4, 5, and 6% (Plates X-XIV). Experiment 3: The purpose of this experiment was to compare the responses tg vitro of embryos excised and inoculated at level IV into media (White's) which contained SUCIOS W353 Amanda) pt 82 sucrose as the organic carbon source, or media in which there wasea combination of fructose and glucose. 1&1e following treatments were used: a. sucrose 3% 'b. fructose 3 c. glucose 3 d. fructose 1.5 e. glucose 1.5 1.5 f. fructose + glucose ( each, for a total of 3% sugar) Intermediate Experiments The purpose of experiments 4—5 was to study the effect on embryos cultured at level II (Early Differentiation) when various intermediate (glycolytic) respiratory products were used as the sole carbon source. In a secondary series of treatments these compounds were used in combination with sucrose (3%). The embryos used for experiments 4-5 were cultured in plastic cups on Gautheret's medium, modified by the incor- poration of the various intermediate products. Experiment 4: Pyruvic acid as the primary organic carbon source was incorporated into the media at: 2000, 1000, 500, 100, and 50 ppm. A secondary series of treatments made use of the forementioned (in the same series of concentrations) in combination with sucrose. Experiment 5: The following intermediate products were incorporated into the media at a concentration of 500 ppm 83 (0.03%): glucose—l-phosphate; glucose—6-phosphate; fructose- 6-phosphate; fructose-l,6-diphosphate; glyceraldehyde; and phosphoglyceric acid. As above, a secondary series of treat- ments made use of the intermediate products in combination with 3%.sucrose. Final Experiments The final series of experiments (6-7) had three purposes: to study the tg.vitro responses of all four levels of develOp- ment on media containing sucrose as the primary organic carbon source; to see if a variation in the concentration of agar would affect the cultured embryos; and to study the responses of all four levels to sugars, other than sucrose. The embryos used for experiments 6-7 were cultured in plastic cups on LaRue's medium, modified by: various sugar concentrations; varied sugars; and various concentrations of agar. Experiment 6: Sucrose was incorporated into the media at the following concentrations: 4, 3, 2, l, 0.5, and 0.25%. The following concentrations of agar were used: 0.8, 0.75, 0.70, and 0.65%. Experiment 7: Using an agar concentration of 0.8%, twelve sugars, other than sucrose (as listed in Table 2), were in- corporated into the media as the primary organic carbon source. mCOflumWN'qx‘JAn F N m-HnHlIH. N0 COiUQCIHnanW II a m + UHUM UHHmohHmonmmofim II g m + oomnmpHmumumHm N+ « m + «omHtIo.HImmouosum N+ « m + «omIoImmouosum m+ g m + vomlwlmmoosHm II w m + womIHlmwousHm NImoo. + ON Ham @ mwouusmc mmOHosm + GHUM UH>5H>m II g pHom UHHmomHUOHQmonm II g momgmonqu>Hm + v muggmmoanUIo.Hlmmou05Hm II g oumnmmonmlolwmouosum + g mumnmmoamImemousHm II v opusmmozmlHIwwoosHm NImoo. mo. H ON oHom 0H>NEE N+ 0H Asumw Xm.H ©V meHUSHm + wmousHm NIm. II NH MIm. II NH ¢IN II NH ¢IN II NH 0mOQH0m H NIm. II NH mIm. II NH «IN II NH «IN II NH omoHNx A+cg mIm. II NH mIm. II NH «IN II NH «IN II NH mmoxNH AIVn MIm. II NH mIm. II NH vIN II NH «IN II NH omOQHH o mIm. m H NH mIm. N H NH «IN « H NH «IN II NH mmocams 1+cn mIm. m + NH mIm. N + NH VIN v + NH ¢IN II NH 0w0u00H mIm. m + NH NIm. N + NH «IN « + NH «IN II NH omopumHmm H+vn mIm. m N+ NH MIm. N N+ NH «IN g N+ NH ¢IN II NH mmosHQmum H+VH MIm. m m+ NH MIm. N m+ NH «IN a m+ NH ¢IN II NH mmouHmE MIm. m v+ NH mum. N ¢+ NH ¢IN g v+ NH gIN II NH mmOHQOHHoo MIm. m m+ Nm MIm. N v+ NH ¢IN a v+ NH ¢IN II NH mmouosum HIVQ mIm. m m+ Nm mIm.. N m+ NH «IN « m+ NH «IN II NH mmoosHm H+co mImN. m 5+ 05H glmN. N 5+ om ¢ImN. g 5+ om gImN. II om 0m0HUSm mam.umo.mmm. # mom.umo.mmmu # mcmuumonmmmm" # mnmuumoummmmn # GUHDOm GOQHMU UHGmmHO >H H0>¢H HHH H0>0H HH H0>0H H Hm>mq . 84 mmoHSOm sonumo UHcmmHo mSOHHm> ou mOMHnEw hmHHmn mo mmmcommwu OHHH> EH .N mHm0H £000 How U0umsH0>0 0903 m0mcomm0n 0gp H0>0H £000 How 0500 0gp 0H 080300 mcHumn 0gp QmsonuH¢ .UGHumu 0HQ0H0>0H 9005 0a? .:i on 098900“ 0Hnm>u0mno o: AIIV Scum 0005: 0Hmom 038 .0H000 mcHumu =MH0HHHQH0: c0 mo EHOM 0£u CH U0©H000H mH NMMMWM.mW A000H500 Gonnmo UHsmmuov muc0fium0uu mDOHH0> 0£u ou momunfi0 >0HH0Q mo m0mgomm0u 03p mo GOHumsH0>0 0A9 E0pmhm mGHHMm muusoonm mucumuHmm0H 0u0HU0EH0usH monHm> 0A9 HHH3 GOHHMGHAEOU SH @005 G053 .0mouosm How GOHu0H>0HQ90 I m .0 $.GH p0mm0nmx0 .p0um0u 0:0Hp0HHG0ocoo mo 0mc0H I mom .0 o0um0u 003 0mcmu 0 c0£3_:* :H U0000HQX0 .00Hsom gonumo mo COHumuug0ogoo ESEHHQO I poo .0 000H500 gonumo UHgmmHo msoHH0> 0gp cu momuna0 >0Humn mo m0msomm0u 03¢ mo mmcHHMH =o0pmsomum= I mm0m .Q U0u05H0>0 can o0HsuHso mOthE0 mo H0AESG I # .0 mGoHu0H>0HQQ¢ N 0HQ09 mo GOHHMGMmem The suga: II(Eark III (Mid the cone Tabl of barle As? hon sou: regardl incorpo quantit is unsa the 0t] is to 1 II - 4 In Althot inten sourcI they into glue, su9a 86 The SUgar concentrations for levels I (Late Proembryo) and II (Early Differentiation), were: 4, 3, and 2%. For levels III (Middle Differentiation) and IV (Late Differentiation), the concentrations were: 3, 2, and 0.5%. Table 2 gives a tabulation of the tg_vitro responses of barley embryos to various organic carbon sources. As Table 2 indicates, the responses to the various car- bon sources are characteristic of the level of differentiation, regardless of the sugar or intermediate respiratory compound incorporated into the medium. The response is primarily quantitative rather than qualitative. A carbon source that is unsatisfactory for one level is also unsatisfactory for the other two. The significant difference between the levels is to be found in the optimum concentration of sugar: level II - 4%: level III - 2%; and level IV - 3%. In general, pentoses are unsatisfactory carbon sources. Although responses in varying degrees were observed for the intermediate respiratory products, they too were poor carbon sources. Even though responses of +2 and +3 were observed, they were for treatments in which sucrose was incorporated into the medium along with the intermediate compound. It should be noted that the combination of fructose and glucose was not as effective as sucrose or even as either sugar when used alone as the sole organic carbon source. 87 PLATE XV. Embryos cultured during late differentiation often exhibit two trends. During the first ten days following inoculation the most satisfactory responses may be in media with exceedingly low concentrations of sucrose. A "plateau" is frequently attained by the 10th day, after which greater responses can be observed on media with sugar concentrations of 2-3%; and in the final stages of incuba- tion (21 days) 3% is superior. This plate shows seedlings, on the 10th day of culture, exhibiting the forementioned "plateau." Fig. l 4% sucrose (Scale: each unit equals 2 3% " 1 mm.) 3 2% II 4 1% II 5 0.5% " 6 0.25% " _:_:__::__::____: 89 PLATE XVI. Late differentiating (level IV) embryos cultured on a medium with an agar con- centration of 0.65%; fourteen days after inoculation, germination having occurred on the tenth day in culture. Note: abor- ted shoot development and the absence of roots (xl.6). _‘__._ I PLATE XVI w’ u* - O . : ‘_-- j .. -é 91 (At level IV (Late Differentiation) glucose and fructose were rated as being equally effective, although in levels II and III glucose was considered to be slightly superior. Embryos cultured during late differentiation often showed two trends. When using a series of treatments in which the concentrations of sucrose ranged from 4.0 - 0.25%, the lower concentrations of 0.25, 0.5, and 1.0% produced the greatest response of the seedlings during the first ten days after inoculation (Plate XV). After this period, those embryos cultured at 2 and 3% not only equalled but surpassed the development of cultures at the lower concentrations. In the final stages (21 days) sucrose at 3% was definitely superior. Agar The responses of the various levels of development would appear to be somewhat related to the concentration of agar in the media. As the agar becomes less solid (lowered con- centration) the growth responses of the three levels were decreased. At a concentration of 0.7%, levels II and III exhibited very erratic growth, and showed no response at 0.65%. Level IV responded over the entire range (0.8 - 0.65%); how- ever as can be seen in Plate XVI, at a concentration of 0.65% the shoot development was aborted and root development rare. .n 92 DISCUSSION To deve10p a meaningful modification of previously reported culture techniques, three sets of facts are required: well-defined components of the media; the concentrations used; and the level of differentiation of the embryos cultured. In order to study embryological development tg_vitro, one must be able to culture an embryo from its relatively undifferentiated state. Few investigators have been able to do this. Those who have succeeded have had only limited success. When they did succeed in getting very young embryos to grow, they did so by incorporating biological extracts, such as coconut milk, into the medium (Van Overbeek gt_gt,, 1941, 1942; NorstOg, 1956; and Chang, 1957). The use of such an additive presents two problems: the components of the extract are not delimited;_and no two "batches" of the coco- nut milk will be precisely the same. The environmental conditions must be such that they can be "duplicated" through- out a series of treatments. If one desires to study the effects of a given treatment on cultured material there is no room for unknown variables in the medium, whiCh of course eliminated the use of coconut milk in this investigation. A superficial examination would lead one to conclude that the concentrationsfiused in the various media are rather well I 93 established. The basic minerals can be incorporated into the media over a rather wide range of concentrations with seem- ingly no deleterious effects; for other components this is not true. Concentrations used for the various carbon sources, and in particular the sugars have been reported as being relatively "critical" (LaRue, 1936b). A number of investi- gators have indicated that the satisfactory development of the very young embryos in culture required a high sugar con- centration, while in contrast, the older embryos did better on media containing a low concentration (LaRue, 1936b; Tukey, 1938; Lammertz, 1942). There would seem to be a variance of opinion as to what is high and what is low. For example: a concentration of 2% sucrose will be considered as being low by one individual (when compared with 6%); and yet another will consider it as being high (when contrasted with 0.5%). There has been a tendency to describe the cultured embryos as being either mature or immature with a paucity of parameters, morphological or otherwise, to delimit one from the other. It would seem that what is not mature (a term at best, very difficult to define) is immature. The detailed embryological development of barley has been extensively studied by such investigators as Merry (1941, 1942), Eunus (1954), and Mericle and Mericle (1957). This study has been based on the work of Mericle and Mericle. 94 Since no dependable culture technique was available, one had to be developed, and therefore the technique became the problem. From the problem evolved certain fundamental obser- vations which mayin the future permit one to use the technique of plant embryo culture more effectively as a means of studying the responses of cultured barley embryos to various treatments. Although late proembryos (level I) showed no response in culture, barley embryos at the level of early (II), middle (III), and late (IV) differentiation can be cultured on well- defined media devoid of biological extracts. The morphological responses of the cultured embryos are characteristic for the level of differentiation at the time of inoculation. Embryos cultured at level IV readily germinate to produce "normal" seedlings, while embryos cultured at levels II and III ger- minate precociously to form aberrant plantlets. The development of a callus is also characteristic of levels II and III. Sucrose is by far the most satisfactory carbon source for the culture of barley embryos. The responses to the various sugars are characteristic of the level of differen- tiation at the time of inoculation and the variation between carbon sources is a matter of degree rather than type of response. The well differentiated embryos of level IV respond most favorably to a concentration of 3%, and the relatively 95 undifferentiated embryos of level II develop best at a con- centration of 4%. The responses