-- .._ ' _‘:,<-a l L.- ms mucus: as ISOLATED ‘ mm SEGMLNLS or SEQUDIA SLMPELLVLREN: E ff”. " (LAMA) LNNL CULTURED LN \m‘ ' I - m VALLLNLLs CHEMICAL ANN omen WWM mmam MICHIGAN sun UNIVERSITY Donald Francis Rusted 1956 1‘ HLBIS This is to certify that the thesis entitled The response of isolated stem segments of Sequoia senpervirens (Lamb.) Endl. cultured in vitro to various chemical and other environmental treatments. presented by Donald Francis Restool has been accepted towards fulfillment of the requirements for H]. D. degree infinitely— Major professor Date April 2, 1956 0-169 THE RESPONSE OF ISOLATED STEM SEGEENTS OF B-‘TUOIA snmavxaaws (LANBJ ENDL. CULTURED IE VITPO TO VAPIOUS CHEKICAL AND OTHER ENVIRONHENTAL TREATMENTS By DONALD FRANCIS RESTOOL Submitted to the School of Graduate Studies of Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY repartment of Botany and Plant Pathology 1956 57:91-- r7/ 7 g A; A 3 ACKNOELEDGEEENTS The writer wishes to express his sincere appreciation to Dr. G. P. Steinbauer, Chairman of his Guidance Committee, under whose leadership, supervision, and guidance this study was undertaken. To the remaining members of the committee, Dr. H. J. Stafseth, Dr. W. B. DreW, Dr. L. W. Mericle, Dr. G. W. Prescott, and Dr. S. H. Wittwer, the writer extends his gratitude for their interest and suggestions in carrying on this investigation. To Er. Ian W. Tervet, special thanks are due for encouragement and for procurement of certain facilities at Ihgway Proving Ground, Utah. To the Chemical Corps, United States Army, for permission to use government facilities. For unfailing patience and continuous encouragement, the writer Wishes to express his gratitude to his wife, wary E. Restool. Donald Francis Restool candidate for the degree of Doctor of Philosophy Final examination: April 3, 1956, 10:00 A. M., Botany Seminar Room. Dissertation: The Response of Isolated Stem Segments of Sequoia sempervirens (Lamb. Endl. Cultured in Vitro to Various Chemical and Other Environmental Treatments. Outline of Studies Hajor subject: Plant physiology Minor subjects: Horticulture, General Botany Biographical Items Born: April 2h, 1915, Petoskey, Michigan Undergraduate Studies, Michigan State College 193 -l9hO. Graduate Studies, Michigan State College 19t6-19So. continued 1953-1956 in absentia. Experience: Member Army of the United States l9hO-l9h6. Graduate Assistant, Michigan State College 19t6—19h9. Plant Physiologist, Crops Division, Dugway Proving Grounds, Dugway, Utah 1951 to date. Member of: Phi Kappa Phi, Tau Sigma, Sem. Bot., Botanical Society of America. THE RESPOWSE OF ISOLATED STEH SEGTEfiTS OF sacrcu s:atrer‘-;*Irsx J (D the segment was placed in th equal number were inverted in the agar so that the growth regulator Iould have the normal path of translocation. The segments were weighed before and after the experiment and the dry weight and percentage dry weight for each individual culture was determined. Experiment VI. The effect of combinations of adenine sulfate and -20- naphthaleneacetic acid upon the production of buds by excised segments was studied. The procedures outlined in the three previous experiments, both for the preparation of segments and for the integration of the medium, were not changed except to accomodate several concentrations of the two chemicals (Table l). Adenine sulfate was added at the rates of 0, ho, 80 and 120 milligrams per liter to three sets of cultures containing naphthaleneacetic acid at the rates of 0, SO and 100 gammas per liter. Each individual culture was weighed before and after the experi- ment. Observations were made upon the increment of growth, the per- centage dry weight and the number of buds and shoots produced. -21.. EXPERIMENTAL RESULTS Experiment I Light, Growth Supplements, Polarity Bud and Callus Growth. Two weeks after the implantation of the stem segments in the nutrient medium, small nodule-like masses of cells appeared on the sides of the segments. The difference between bud and callus development could not be determined With accuracy until the fifth week; so, initially, the percentages of cultures that produced any type of proliferation were computed and recorded for the weekly intervals (Table 2). Once started, the computation of these percentages was con- tinued biweekly beyond the fifth week until the end of the experiment at the tenth week. Such very small differences existed between treatments of the nor- mal and inverted placement of the segment in agar (Figures 1 and 2), that the data for these two treatments were combined and reproduced graphically in Figure 3. The curves of Figures 1, 2, and 3 all indi- cate that the segments cultured in the light without growth supplements began to display external growth at an early and rapid rate. The other treatments of light and growth supplements show nothing conclusive ex- cept that those segments in darkness, without growth supplements, were less successful, at the end, in producing external proliferations. It is apparent that continuous light in the absence of growth supplements -22.. exerted a great influence upon the early ability of the cells to in- crease. After the fifth week, the growth pattern of the cultures, in regard to the formation of buds, began to appear. Buds could be distinguished from the nodules of callus with certainty. An attempt then was made to determine which treatments had the greatest tendency to produce buds and, conversely, callus as expressed by percentages of the total number of cultures over a period of time. Accordingly, at intervals of two weeks until the end of the experiment, these calculations were made (Table 3). Analysis of the figures shown in Table 3 indicates significant dif- ferences between the supplement treatments in darkness. The percentage of cultures producing buds over a five week period of time was much less in darkness with supplements than without supplements. Other differences between treatments, such as between normal and inverted segments, appear to be present but, since the observations are expressed as percentages, and not of number of buds, the differences may not actually be significant. By plotting the data on Figures h, S, and 6, an inhibition of the ability of cultures to produce buds, as expressed by percentages of the total, can be observed in cultures maintained in darkness and furnished with the certain growth supplements. Conversely, this treatment enhanced the ability of the cultures to form callus. No treatment completely prevented the initiation of buds. The results are, therefore, a matter of degree of control, rather than of absolute control. According to the limits set up in this experiment, about fifteen percent of the cultures grown in the dark'with growth -23- supplements produced buds; whereas, about eightybfive percent of those grown in the dark without growth supplements produced buds (Figure 6). Thus, some tendency toward the inhibition of buds appears to be possible in the light depending upon whether or not the growth supplements were added to the medium. Data regarding the number of buds per segment offer a better basis for conclusions regarding the ability of the segments to initiate buds than the previous percentage data. Records of the number of buds per culture were taken on the eighth and tenth weeks of the experiment. The data for the two periods showed no differences, therefore, only the data for the tenth week are shown in Table h. These data show significant differences between light and darkness in the number of buds produced per segment. A greater number of buds per segment is produced in the light than in the dark. This condition was always obvious When the periodic observations were made. The cultures in the light that possessed shoots almost invariably had clusters of short shoots. Nearly all cultures in the dark had long, etiolated shoots, very few in the dark with supplements and a few more in the dark without supplements. The influence of developing shoots upon the growth of other shoots or upon the callus itself was not studied. Fresh and Dry Weights. Eighty-eight days after the start of the experiment, the cultures were opened and the tissues were weighed individually in the fresh condition and collectively.for each treatment in the dry condition (Tables 6 and 7). The rather large probable error -gh- associated with each mean average weight for each treatment indicates considerable variability among segments. But the figures show clearly that much more fresh weight was produced in the light than in the dark. There were no significant differences between supplement treatments in the light nor between inverted or normal segments. In darkness, however, a significant difference existed between the supplement treatments. A greater amount of tissue was produced in the dark as a result of the addition of the supplement. The same general conclusions can be drawn for the final dry weight. In regard to the figures for the percentage dry weight, (Table 8) no significant difference can be found. The conclusion must be reached, therefore, that within the limits of the experiment, no differences exist in regard to the relative production of dry matter among the treatments. Although no significant differences are apparent in the fresh weight data in regard to those cultures in the light with and without growth supplements (Table 6), the actual appearance of these cultures, during the latter stages of the experiment showed great differences. The cul- tures maintained in the light with supplements formed vigorous, dark green, thick growths of shoots and callus; whereas, the segments in the light without supplements appeared to lack vigor and color. Had the experiment progressed longer, the segments given growth supplements would have surpassed, no doubt, the others in wet weight. Evidence to this effect is brought out in Table 5. The segments which had been set aside for another investigation were weighed one year after the end of -25... this experiment. The differences in weight are significant and the conclusion can be reached that the supplementary substances greatly enhanced growth over a long period of time. Dead Cultures. Some of the segments receiving the supplementary substances began to die early in the experiment. At first it appeared that the supplements, probably yeast extract, possessed an inhibitory action upon growth, but, When surviving segments showed excellent growth, the early deaths of these segments became of interest. The percentage of cultures that failed to grow (disregarding contamination) are tabulated in Table 9. Those in the light with supplement show a high percentage of deaths. No other relationships are apparent. Thus, throughout the whole experiment, fewer segments survived in the light with supplements than in any other treatment. These results indicate an early toxic effect of the growth supplements. The toxic property gradu- ally disappeared until at the end of the experiment the growth supple- ments actually enhanced growth among surviving segments. Epptg. Toward the end of the experiment roots began to appear, although the initiation of roots was not expected at so low a level of auxin. The roots did not show any specific inclination to grow into the agar. A count of the total number of roots showed 31 grOWing into the agar and h? into the air above the agar. Another observation re- garding roots revealed that roots were initiated follOWing the produc- tion of shoots in twentyhfive cultures and appeared before shoots in only seven. On the other hand, shoots grew in the complete absence of roots in 139 cultures. -26- Downward GrOWingiShoots. In fifty-three cultures, the buds gave rise to shoots Which grew downward into the agar (PLATE 3). There was a slight indication that most of these shoots occurred in the cultures not furnished with growth supplements but they were so few in number that no conclusion can be drawn. As a matter of interest, a few shoot tips normally discarded were inverted in nutrient agar with the result shown in PLATE h. These tips showed excellent growth into the agar without the formation of roots at any time. The growth of these excised shoots can be observed by the amount of penetration into the agar. Table 10 represents a balance sheet concerning the dispositions of the four hundred cultures at the end of the experiment. Observations on grOWth were made with the totals shown at the top of the table; whereas, observations on final weight were made on somewhat fewer samples because some cultures were saved for future experimentation. Experiment II Maleic Hydrazide, Initial Weight, Position In previous work, all shoots and segments were removed from the parent material without consideration as to position, relative age, or initial weight. The segments were excised as uniformly as possible. The variability of final wet weight within treatments in Experiment I led to the suggestion that the original location of the segment on the shoot might have an influence upon subsequent growth, and that variations in original weight might be responsible for the observed variations in -27- the amount of callus produced. An experiment accordingly was set up to test this hypothesis Within the usual limits of the technique utilized for the cultures. The experimental design is shown diagrammatically in Figure 7. A study of the action of maleic hydrazide as a growth inhibi- tor was superimposed upon the study of the effects of position and ori- ginal weight. Examination of Table 11 shows that the original weights of segments given the several treatments of maleic hydrazide were remarkably uniform in the various distances from the apex of the segment. Maleic Hydrazide. The action of maleic hydrazide as a growth in- hibitor is very clearly shown in Figure 8 in Which maleic hydrazide at 10, 100, and 1000 parts per million gradually killed cultures until the twelfth week at which time all of these cultures, except a few at ten parts per million, were dead. The cultures receiving no maleic hydra- zide and those receiving one part per million progressed equally well until the twelfth week at which time these cultures began to die. The deaths of these cultures, after the twelfth week, was caused by the development of an unknown deficiency in the medium as shown by the fact that the rate was approximately equal for the controls receiving no maleic hydrazide and for the one part per million treatment. In spite of the toxicity of lO, 100, and 1000 parts per million of maleic hydrazide to the Sequoia segments, several cultures in each series became contaminated with bacteria and fungi of undetermined iden- tity. Apparently the toxicity of maleic hydrazide at this range did not affect the growth of these microorganisms. ~28- Shoot and Callus Growth. As in Experiment I, the time course of the treatments upon the cultures, as expressed by the production of external proliferations, was followed. The 100 and 1000 parts per million treat- ments of maleic hydrazide produced no external grOWth and the 10 parts per million treatments produced a negligible amount of growth. These treatments, therefore, receive no further consideration. In regard to the controls and the 1 part per million series (Figure 9), no difference in the production of external proliferations can be observed. After the twelfth week, a necrosis of tissues is evident. The effect of maleic hydrazide as a growth inhibitor was followed in the controls and in the cultures receiving 1 part per million of maleic hydrazide. Figure 10 shows the nearly equal amount of growth by shoots in both series. Thus, maleic hydrazide, at 1 part per million, did not inhibit the elongation of shoots. It must, however, be remem- bered that no shoots were formed at the 10, 100 and 1000 parts per mil- lion maleic hydrazide treatments. The number of shoots and buds produced by each treatment is shown in Table 12. Again no significant differences occur between the con- trols and the 1 part per million treatment. Table 13 also shows the parallel ability of both treatments to pro- duce callus. The reduction in percentage of cultures after the twelfth ‘week is included to show that an unknown factor other than maleic hydra- zide had become effective in killing the tissues. Pbsition and Initial Weight. In spite of the fact that 10, 100 and 1000 parts per million of maleic hydrazide had completely killed all -29- tissues by the twelfth week, the cha acteristics of the exper: imenta I design permitted a consideration of the effects of position and initial weight upon the ability of the segments to proliferate. The data shown in Figure ll portrays clearly the fact that the proliferation of callus and shoots was not Confined to any position or weight group within the limits of the experiment. At the twelfth Jeek , almost all the cultures receiving the two treatments aere "rOsin: satisfactorily. Four percent the losses which occurred among the total number of segments was caused by death of the segment and fifteen percent was ca we d by conta- mination of the culture. The growth of the surviving cultures of this experiment was inferior to t? at of the first experiment. Terminal wet weights of the cultures were not taken. Althou3h roots occurred in Experiment I without naphthaleneacetic acid, no roots appeared in this experiment with naphthaleneacetic acid at a concentration of 100 2:3ammas per rliter. Fxperimcnt III Temperature Shoot and Callus Growt.. This experiment consisted of two trials as nearly alike as possible. However, some minor differences in the treatments existed. The first, be~un in April, con ained cultures with 3 segments weighing initially about 160 milli3r ams; the second, begun in September from another burl, contained segments weighing about 80 milli- A grams (Table lb). The original se3nents all measured one centimeter -3o- in length. Furthermore, in the interpretation of the effects of tem- perature in this experiment, three different time lengths must be considered. In regard to the production of shoots and buds, the principal difference between the two trials lies in the fact that, although some buds occurred in the first trial, none occurred in the second trial (PLATE 6). Naphthaleneacetic acid at 100 gammas per liter completely in- hibited the formation of buds in one case and not in the other although the number of buds produced was small (Table 15). The shorter time length of the second trial does not account for the absence of buds which always appeared within thirty days in other experiments. The presence of a few buds and shoots in the first trial, however, made possible a comparison of the effects of temperature upon their re- lative number and subsequent growth. According to the data of Table 1h, the maximum production and growth occurred at ZhOC. and at room temper- ature both in light and in darkness. A 10W, constant temperature of 6°C. permitted the slow growth of both shoots and callus (Table lb and 15). Although growth was seriously retarded at 600., no damage to the tissue was apparent. The shoots were long and etiolated because of the darkness. A completely different condition existed in the 37°C. treatment. These cultures also showed retardation of grOWth but considerable nec- rosis and browning of the tissues was evident. In the surviving cul- tures, seven buds had appeared early in the experiment. The development of these organs was immediately diverted from the organized growth of -31- normal buds to the undifferentiated growth of callus. At the end of the experiment, these buds were reduced to a mass of callus-like cells. Fresh and Dry'Weights. Table 1h includes all the data on initial and final weights per segment for the controlled temperatures and for the room temperature series. Each individual segment was weighed be- fore and after the experiment. The low production of fresh weight in the 6°C. and 37°C. cultures obviously resulted from temperature ex- tremes. The best growth occurred at ZhOC. and at room temperature both in light and in darkness. The experimental differences between treat- ments do not allow any close comparisons except between the ZhOC. and the 30°C. components of the second trial. In this trial, a temperature of 30°C. resulted in the formation of significantly less tissue than the 2hOC. temperature (Figure 12). Thus, it can be concluded that room temperature is a suitable temperature,but not necessarily the optimum temperature, for the production of cells of Sequoia in culture. Experiment IV Aeration Shoot and Callus Growth. The effects of reduced aeration in sub- merged cultures (PLATES 8 and 10) gave wholly unexpected results in view of the findings of White (1939a). The submerged cultures, instead of initiating buds, produced only callus; whereas, the controls produced numerous buds although some of these produced callus only. The data of Table 16 clearly indicate that a decided difference existed in the two treatments in regard to the ability of the cultures to produce buds. The actual production of buds by the controls indicates the inherent ability of all the segments to form buds. The submerged cultures, therefore, completely inhibited the initiation of buds. PLATES 1, §, 2, and IQ represent a photographic record of this ex- periment. PLATE 7 illustrates the method of submersion of the culture by insertion of the segment in agar followed by the addition of five milliliters of distilled water. PLATE 8 shows the type of callus masses produced in the submerged cultures. The water had been removed for photographic purposes. PLATE 9 presents an example of the type of callus produced by the aerated cultures. The excellent shoot growth of many of the aerated cultures is shown in PLATE 10. Wet and Dry Weight. Although a reduction in aeration destroyed the ability of the segments to form'buds, it did not actually enhance the production of callus cells as measured by final wet weight (Table 16). The aerated cultures produced six times the callus produced by the sub- merged cultures. The aerated segments showed a higher percentage of dry matter. , Expe riment V Polarity of Segments in Medium Shoot and Callus Growth. Too few shoots were produced by either treatment to reach any conclusions about the relative production by in- verted and normal segments. The callus formed by each treatment appeared at the base of the segment and on the sides at the surface of the agar. Both series of cultures exhibited remarkable similarities in rate and appearance of growth. Fresh and Dry Weight. The two treatments produced similar amounts of fresh and dry material (Table 17). No significant difference could be found in the increments of fresh weight formed during the experiment. Experiment VI Adenine Sulfate - Naphthaleneacetic Acid Bud and Callus GrOWth. The level of naphthaleneacetic acid that inhibited bud formation according to Experiment IV consisted of about 100 gammas per liter. This amount of the growth regulator was used in the controls of this experiment with the idea that adenine sulfate, at LG, 80, and 120 milligrams per liter, would overcome the bud inhibition according to its concentration. Examination of Table 18 shows that 100 gammas of naphthaleneacetic acid permitted the formation of an average of only 1.3 buds per culture when no adenine sulfate was present in the medium. The presence of 50 gammas per liter of naphthaleneacetic acid permitted the formation of 0.3 buds per culture. These figures are not sufficiently different to allow conclusive estimates of variability; nevertheless, they are low enough to act as controls for measurement of the bud-inducing effects of adenine sulfate. The table shows that ade- nine sulfate at the rate of h0, 80, and 120 milligrams per liter did not, in any case, cause an increase in the number of buds per culture. In fact, a decrease seems to be in effect. The total length of the shoots produced by the various treatments (Table 18) offers no insight into the effect of adenine sulfate upon the growth of shoots inasmuch -314- as the growth for all treatments averaged 2.3 to h.h centimeters per shoot and no individual record was kept of the time of the initiation of each bud. Fresh and Dry Weight. The data for the initial weight, the final fresh weight, and the final dry weight are included in Table 19. The growth of the cultures is expressed as the average amount of fresh matter produced per culture in each treatment during the course of the experi- ment. A gradual decrease in the amount of fresh matter produced is apparent with increases in the concentration of adenine sulfate in the medium. (Figure 1h). A decrease occurs also in the treatments receiving 50 gammas per liter of naphthaleneacetic acid in Which case the fresh weight dropped from 60 milligrams to 19 as a result of the addition of 120 milligrams of adenine sulfate to the medium. That no growth occurred in the series receiving no naphthaleneacetic acid seems to indicate that, in this experiment, the growth regulator was necessary for the proliferation of callus and of shoots. No roots occurred in any treatment. The results obtained at the end of this experiment in regard to the production of buds (Table 18), and more specifically to the production of fresh matter (Table 19), all indicate that the adenine sulfate exerted an inhibitory effect which was partially overcome by increasing amounts of naphthaleneacetic acid. The effect of shoots, When present in the culture, upon the amount of callus produced was not studied. However, since some of the growth -3 5.. increment consisted of shoots, this amount was subtracted from the means shown in Figure 13 and the result shown in Figure 1h. This procedure is justified on the grounds that the shoots after initiation acted more under their own organismal influence than under the influence of the medium. Admittedly, the effects of shoots upon callus growth can not be resolved by this experiment; nevertheless, the growth of callus shows a progressive drop with increased concentration of adenine sulfate. General Observations Cultures Maintained at Room Temperature The results obtained from preliminary investigation as well as the results from the six previously described experiments indicated consider— able variation in response to environment among the segments from differ- ent burls. For instance, lOO gammas per liter naphthaleneacetic acid did not always completely inhibit the initiation of buds. In most experi- ments, a few buds always occurred. Furthermore, the rate of grOWth between experiments generally varied in amounts not always attributable to the environment. These variations in response could be attributed to differences existing in the responses of different burls, since each experiment was prepared from a freshly sprouted burl at different sea— sons of the year. In order to exercise some sort of control over the interpretation of data between experiments, a group of cultures from each experiment were subjected to the conditions of room temperature in the type of light available. -35- The data obtained from the cultures maintained at room temperature was subjected to light of several intensities according to the experi- ment. The light ranged from continuous at high intensity to the weak, diffused daylight entering the laboratory during the Winter. In addition the room temperature varied somewhat from artificial heat of Winter to the air conditioning of summer. These data are shown graphically in Figure 15. A group is shown for each experiment except Experiment II which utilized a medium too different to be compared to the others (Table 1). Experiment I, cultivated at a high continuous light intensity, produced rapid and abundant growth. Groups from Experiments III, IV, and VI although of different time lengths, were all maintained in simi- lar environments of room temperature and daylight. Two groups from Experiment V stored for 192 days in very weak daylight produced little, but similar, amounts of tissues. Some general considerations are possible in regard to the inhibi- tion of buds by several treatments. Table 20 portrays, in diagrammatic form, the treatments utilized throughout the investigation and their comparative effect upon the inhibition of shoot growth and the produc- tion of callus by the segments. As indicated, a certain degree of bud inhibition was manifest in Experiment I in a treatment utilizing dark- ness and growth supplements. In the temperature series, the buds formed were inhibited from further development in darkness at 3700. Buds were completely inhibited at all temperatures in another trial of the same series by 100 gammas of naphthaleneacetic acid from a fresh supply. _3 7.. In this treatment, growth of callus was somewhat less than in other ex- periments. In Experiment IV, reduced aeration completely inhibited the initiation of buds although the controls produced them. In Experiment V, the production of buds was inhibited but not completely suppressed by 100 gammas of naphthaleneacetic acid, again from the fresh supply. The matter of the breakdown of the older naphthaleneacetic acid into a chemical of less activity received attention. Chemical analysis by two methods showed the older crystals to be 99.6 percent pure and the fresh supply to be 98.8 percent pure. These differences are hardly significant and the conclusion must be drawn that individual differences in burls caused differences in response to higher levels of the growth regulator. -38- DISCUSSION EEEEEE' The establishment of the proper nutrient medium for any type of tissue culture involves a large number of trials with combina- tions of nutrients, vitamins, amino acids, and growth regulators at a great expense of time and effort. However, the importance of the estab- lishment of the proper medium cannot be minimized. Once the optimum nu- trient balance has been determined, experiments can be devised regarding the response of the tissues to various chemicals simply by maintaining the tissue in the optimum basic medium at the same time varying the con- centration of the substance in question. The problem is complicated somewhat by the fact that tissue cultures fluctuate greatly in response to environmental conditions (de Capite, 1955) because of their delicate nature. At any rate, tissue culture, at best, is a technique capable of eliminating some, but not all, variables inherent to the work with whole plants. Fortunately, Ball has worked with Sequoia tissue and has determined that a basic medium of Knop's Solution, three percent sucrose and lxlO"’6 grams per liter of indoleacetic acid gave excellent results in terms of growth in his studies on differentiation of tissues and movement of ra- dioactive chemicals (Ball, 1950, 1953a, b, c, d, e). In many preliminary tests, Ball's medium proved to be entirely satisfactory substituting alpha naphthaleneacetic acid for indoleacetic acid because of its greater stability in solution. In addition, the B vitamins, cysteine, yeast -39- extract, and coconut milk often enhanced the results of other investiga- tors. Early in the study of roots in culture (Bonner, 1937; Robbins and Bartley, 19373 White 1937), the effective agent in yeast extract for the indefinite growth of most excised roots was found to be thiamin. Gau- theret (1937) also included thiamin in the media for the growth of tissue cultures of cambial origin. However, Nobecourt (l9h0) did not include thiamin in his original formula for carrot tissues Which synthesized as much thiamin as did normal tissue. This vitamin was found necessary for tomato roots by Rabideau and Whaley (1950). The reports of beneficial activities for cultures other than roots is not so clear. It was deemed unnecessary for the culture of excised asparagus tips (Loo, l9h5) and for the growth of the first leaf of isolated stem tips of rye (deRopp, l9h6). The beneficial results in stem cultures of normal tissues (Gau- theret, 1937; Henderson, Durrell, and Bonner, 1952) and of atypical tis- sues (Burkholder and Nickel, l9h9; Skoog and Tsui, l9h8) probably indi- cate that thiamin is synthesized in most tissues of stem origin but sometimes not at optimum amounts. Pyridoxine also gave favorable results for root cultures (Robbins and Schmidt, 1939; Day, l9hl), for sunflower tissue (Hildebrandt, Riker, and Bugger, l9h6), and for virus tumors of Bumgx (Burkholder and Nickel, 19h9). Pantothenic acid was found to be occasionally beneficial for the growth of excised corn root tips (Robbins and White, 1936) and indis- pensable for cambial cultures of hawthorne (Morel, l9h6a). Pantothenic -ho- acid reduced the inhibiting effects of tannins in the formation of callus by cambial cultures of Quercus (Jacquoit, 19h?) which, up to that time had not been cultured. Cysteine was used for carrot root cultures by Nobecourt (1937) and for cambial cultures by Gautheret (1937). Nob6hourt considered cysteine unnecessary for carrot root cultures (l9h2) but later included it in the medium for rose stem cultures (Nobecourt and Kgfler, l9b5; Nobecourt, 19h6). The effects of the various growth supplements upon other tissues were evaluated and the decision was made to include them in the basic medium, inasmuch as no harmful effects, at the low concentrations used, were found in any case. The results of Experiment I showed that grOWth was definitely enhanced by the addition of these supplements. The deci- sion to include biotin and thiamin received further justification from the fact that traces of these substances have been found in Difco agar (Robbins, 1939; Day, l9h2). White (1953), in a criticism of the tech- niques currently in use, objected to the use of agar in critical studies because of its "unknown nutritive properties". The inclusion of ascorbic acid received attention, not especially for its metabolic effects, but for the fact that its presence in the medium reduced the necrosis and browning of cut surfaces. Wetmore and Morel (l9h9) made tissue cultures of horsetails and ferns in Which growth ceased as soon as the medium became dark brown. They suggested that as- corbic acid reduced the action of the polyphenol oxidase system at the injured edges of the fragments. This treatment eliminated the browning .1, 1.. and diffusion of the substance into the agar with the result that growth progressed. Another beneficial effect of ascorbic acid was demonstrated by Waygood (l9h9) in Which hydrogen was transferred from the malic dehy- drogenase - Coenzyme I system to molecular oxygen by way of an ascorbic acid oxidase. At any rate, ascorbic acid always exerted a decidedly beneficial effect upon the early growth of Sequoia stem segments espe- cially in regard to the control of the browning and necrosis of cut surfaces. Several sources of carbon received attention. Sucrose invariably gave the best results and subsequently was used in all experiments except the second in which glucose was used. Examination of Figure 8 reveals that after the twelfth week all cultures, regardless of treatment, began to die at an increasing rate. These cultures seemed unable to derive some substance from the agar. On sucrose, however, the cultures of the other experiments always progressed considerably beyond this point with no decrease in growth or vigor. Sucrose, no doubt, remains the best carbon source for Sequoia, a result also found for this tissue by Ball (1953a). White (19h0) obtained ten times the growth of excised tomato roots in two percent sucrose than in two percent glucose. Each tissue according to species has its particular optimum carbon source as well as a certain optimum concentration. A comprehensive work on carbohydrates as sources of energy for tissues is that of Hildebrandt and Hiker (l9h9, 1953). They concluded that many basic differences in carbohydrate utilization exist among species and among types of tissue. By way of explanation they offered the theory that these tissues may have missing or incomplete .bg- enzyme systems, may be impermeable to certain carbohydrates, or may be suppressed by the formation of inhibiting metabolites. The problem of the adjustment of the p H to the optimum was met by the addition of appropriate amounts of 0.1 normal sodium hydroxide be- fore autoclaving. The tissue grew well at a p H of 5.6. Gautheret (19h7b, c, d) found that for carrot tissues, the tissue had a tendency to change the p H of the substratum to a value slightly higher than the proper p H of 5.5, but that growth proceeded normally between p H 6 and 9. He found a buffer action by the tissue which, if not immediately killed, neutralized a high or low p H and destroyed its toxicity. de Capite (1955) influenced by these findings of Gautheret found a p H of 6 to be entirely satisfactory for the growth i2 vitro of three species of plants. In the case of Sequoia tissue, the final p H did not differ from the initial by more than one log. Experiment I consisted partly of a test as to the enhancement of growth by the addition of certain vitamins, cysteine, and yeast extract. Several points are obvious. In the first place, the addition of these growth supplements initially retarded growth and killed a large number of segments (Table 9). Secondly, after a certain length of time, the surviving cultures in the treated series became greener and more compact until, at the end of the experiment, they had completely surpassed the controls in general vigor. Thus, the time length of an experiment may have some effect upon the interpretation of the results, especially in regard to tissue cul- tures which, unlike intact plants, do not possess normal physiological .443- periods of vegetative or reproductive growth. A tissue culture depend- ent upon a medium for minerals and carbohydrates without the presence of roots and leaves either becomes adjusted to its environment or expires. The balance of enzyme systems within the original segment origin- ally may not be able to cope with the demands of a certain environment. Later, the adjustment of already present systems, or of new systems, may enable the segment to shift its biological functions to a more favorable balance. This phenomenon has been observed many times but especially in regard to the "habituation" of certain tissues to indoleacetic acid Gautheret, l9h6, 19h7e) and to naphthaleneacetic acid (Morel, 19h6b). These cultures adjusted themselves to the absence of growth substances by the gradual assumption of the ability to synthesize them while grOWing. hhite (19h9) refers to cellular adaptation as the boosting of a subcritical physiological system or of the shifting in the relative amounts of pre-existent systems. Plant Material. Statistics regarding the Redwood tree in its native habitat are well known. Generally the tree is considered to be capable of living between 500 and 1300 years and to grow to a height of 360 feet With a circumference of forty-seven feet (Hollister, 1953). Very little information, however, is available regarding the origin and development of the burls. According to Betts (19h5) burls are irreg- ular swellings or growths near the base of the trees consisting of closely clustered groups of twisted fibers through Which adventitious buds are scattered. -1411- All of the burls received from a commercial source in California sprouted when placed in water. Those burls received during the winter sprouted at a slower rate than those received during summer. Although originally the burls were considered to be uniform in growth and con- dition of the shoots, a remarkable dissimilarity existed between them in the number, color, texture, and rate of growth of the sprouts produced. These differences between burls are probably reflected in some of the variability within experiments in regard to subsequent development and final wet weights. The effect of seasons of the year upon the sprouting of burls pro- bably changes the response of the shoot segments to treatments because of the differences in auxin content among burls from different positions on the tree. In addition the shoots may have variable abilities to pro- duce auxin especially after excision. Burls resemble most the tumor growths of plants. According to Kulescha and Gautheret (l9h8) differ- ences in the normal tissue, gall tissue, and habituated tissue of black salsify contain different amounts of indoleacetic acid, a fact which accounts for their relative differences in grOWth and response to added growth regulators. They also determined (19h?) that tissue cultures of Jerusalem artichoke taken at several seasons grew in proportion to the amount of growth substance normally present for that time of year. Kulescha (l9h9) found the proliferating capacity of Jerusalem artichoke to be related to the quantities of contained auxin depending upon the season of the year. Differences in the auxin content of the excised segments of Sequoia burl shoots may account for the fact that 100 gammas -h 5.. of naphthaleneacetic acid did not always inhibit the initiation of buds. Another alternative is that this concentration of naphthaleneacetic acid represents a threshold value at which buds may or may not be entirely inhibited depending upon the amount of natural auxin in the plant mate- rial. Another disturbing difference between the growths from different burls existed in the texture of the sprouts or shoots. Some, depending upon the burl, were thick, soft, and fleshy; whereas, occasionally, a burl gave rise to hard, brittle shoots. Ball (1950), using the same species, makes no mention of the texture of the shoots other than to state: "Thick green shoots from a burl were used". £E§EE° In Experiment I, the use of continuous light of rather high intensity resulted in the production of greater amounts of tissue than in darkness. The results of the other experiments in diffuse daylight of variable intensities are not strictly comparable but much less tissue was produced than in continuous light. These results, of course, were not unexpected. de Capite (1955) studied the effects of light intensity upon three types of tissue in culture and determined that all three grew best at 350 foot candles, with less grOWth at 150 and 750 foot candles. His data consistently shows a greater wet weight in continuous artifi- cial light than in either natural light or continuous darkness. The presence of sucrose in the medium eliminated the necessity for photosynthesis and any benefit from light probably resulted from the higher temperature caused by the light (de Capite, 1955) or from the formation of substances dependent upon photochemical reactions. -h6- Since shoots appeared in some cultures and not in others, a method is suggested Whereby the effect of shoots upon the growth of callus might be studied by means of the final wet weights obtained from a great number of cultures. The data (Table h) revealed that more shoots are formed in light than in darkness. Again an unanswered question arises as to the effect of shoots upon the initiation of buds as expressed by apical dominance. Refinements in the present technique may offer information on this subject. Temperature. The growth of cells in culture depends greatly upon an optimum temperature according to the findings of de Capite (1955). He suspected that tissues in culture, because of the delicate structure and of the lack of an epidermis, might be much more sensitive than the intact plant to external factors. Likewise, in the growth of Sequoia tissue the question arose whether or not room temperature, With its lack of control, represented a suitable temperature for growth as opposed to a constant optimum temperature. The results of Experiment III show that the room temperature of about 2100. provides almost as good an environment as that of a con- trolled temperature of ZhOC. Lack of facilities prevented a more exhaus- tive study of temperature optimums but the findings indicate that as long as the entire experiment is conducted under the same conditions of temper- ature, consistent results within the experiment may be expected. However, as de Capite (1955) demonstrated that changes of a few de- grees between experiments cause differences in wet weights that would -L; 7.. completely invalidate the results of experiments compared at greatly differing room temperatures. de Capite using three different species of plants found that the growth of tissues 12.11352 was strongly influ- enced by temperature with a rapid drop in final wet weight on each side of the optimum temperature. In addition, he found that high light inten- sities have a tendency to raise the temperatures of the tissue. Optimum temperatures for the three species ranged between 23 and 26°C. and he concluded that optimum temperatures for the growth of tissue cultures are higher than those observed for intact plants of the same species. The principal difference between the segments used in these experi- ments and the cuttings used for propagation of plants lies in the compar- ative sizes and sources of carbohydrates. In cuttings, the carbohydrates are stored in the tissues; the segments derive carbohydrates from the medium by diffusion. Both, however, form callus to varying degrees. Shippy (1930), studying the callusing of apple cuttings, found that the complete range for the formation of callus occurred between 0° and hOOC. with injury to the tissues above 32°C. He found that variable tempera- tures did not change the relationship in which callusing was accelerated or retarded according to the degree and duration of the temperature. Skoog (l9hh) placed tobacco tissue cultures in temperatures ranging from 5°C. to 33°C. The amount of growth increased with the temperature but the formation of buds was greatly suppressed at 33°C. This result is in agreement with the suppression of bud growth found at 37°C. in Experiment III. Skoog further found that at 5°C. and 12°C. the growth rate was so slow that very little differentiation occurred. This condi- .1, 8- tion also existed at 6°C. in Experiment III. Hildebrandt, Hiker, and Duggar (19h5) found an optimum temperature of 26°—32°C. for tobacco and _ 2h°-28°C. for sunflower cultures. In using final wet weight as the meas- ure of grOWth, they found considerable variation between individual pieces. The culture of root tips presents a different problem from the cul- ture of stem tissues. However, a parallel condition may exist in a sup- posed seasonal fluctuation in growth rates. White (1937), while investi- gating the seasonal fluctuation in growth rates of excised tomato root tips, utilized controlled temperatures in the range from 5°C. to h0°C. Growth was practically nil below 10°C. and at hOOC. and slow at 15°C. and 35°C. Optimum grOWth was best, and very sharply so, at about 30°C. He concluded that the temperature range of a laboratory probably explained the seasonal variations and that, for root tips at least, the temperature conditions must be rigidly controlled. Aeration. The inhibition of buds by the submerged cultures in Expe- riment IV is directly opposite to the results obtained by White (1939a). White proposed the theory that low oxygen gradients in the tissue may be important factors in the differentiation of organs. He found that, in the case of tobacco tissue cultures, the mere submersion of cultures re- sulted in the production of stems and roots even with cultures which had long produced only callus. Several important differences exist between the tobacco and redwood tissues, however. White's tobacco tissue was derived directly from neo- plastic growths on the stem; whereas, the redwood segments, although originally from a burl, were derived from normally appearing stems. A -t9- difference between the media of the two tissues lies in the fact that tobacco tumor tissues can grow indefinitely without the addition of growth substances and in the case of the redwood tissues, 100 gammas per liter were added. In the case of the redwood tissue, this amount was utilized to control or greatly inhibit the production of buds. Thus, the relationship between aeration and control of bud formation requires further investigation especially in regard to the interaction of oxygen tension and amount of growth regulator furnished. Skoog and Tsui (19h8) confirmed the work of White (1939a) on tobacco tissue but also found that the addition of indoleacetic acid or naphthaleneacetic acid prevented bud formation in submerged cultures. Other evidence from their experi- ments also led to the conclusion that oxygen tension is not the only fac- tor active in organ formation. They noted that shoots removed from the liquid continued to produce shoots and leaves rather than callus, and that a severed shoot placed on agar generally reverted to callus at any point on its surface. The amount of growth as expressed by the increase fresh weight at the end of Experiment IV revealed that the submersion of the cultures greatly inhibited growth. Gautheret (1950) also found a suppression of growth in submerged cambial explants of Salix caprea. Polarity. In order to take advantage of the polar movement of auxins, the shoot segments were normally inverted in the agar. This procedure necessitated an accurate and time consuming marking of the apical or basal end of the segment so that it might be inverted in the agar without error. Any differences between normal and inverted segments -50- in regard to growth were tested in Experiments I and V. In both experiments, the production of fresh material was uniform between the two treatments. Apparently, within the limits of these experiments, the polarity of the segment had little effect upon its ability to absorb naphthaleneacetic acid from the medium. However, this result cannot be conclusive for a number of reasons. The possibility exists that certain amounts of naphthaleneacetic acid could have moved up the outside of the segment by diffusion on the moist surface. The concentration may have been high enough for the growth regulator to move upward against the gradient in the vascular bundles as found by Snow (1936). The polarity may have been reversed by the formation of new tissues of opposite polarity as found in inverted Tagetes cuttings by Went (19hl). Thus, the sensitivity of the experiments was not conducive to con- clusive results in regard to the polar movement except that, within the limits of the experiment, no effect of reversal of polarity upon growth was evident. Several other results, closely related to the movement of auxins, have a certain amount of interest. Went (1937) proposed a number of hormone-like factors called "calines" which are formed in various organs, transported to other organs where they produce growth effects. For in- stance, caulocaline, produced in roots specifically causes the elongation of stem and buds. Fhizocaline must be furnished by cotyledons and leaves before roots can be formed. LikeWise phyllocaline must be present and necessary for leaf growth. Normal growth of the plant thus depends upon ~51- the relative distribution of the calines in conjunction with auxin. Some evidence against this theory is discernable in the work of Loo in which he cultivated excised stem tips of asparagus (l9h5, l9h6b) and of dodder (l9h6a). In both cultures of stem tips, growth progressed satisfactorily in the complete absence of roots although in the case of asparagus the growth was slower among non-rooted tips than among rooted tips. The appearance of cladophylls also enhanced the growth. These results, however, do not exclude a partial need by the stem-tips for caulocalene in the growth of shoots. de Ropp (l9h6), on the other hand, working on the influence of roots upon the growth of rye showed that, in stems that failed to rege- nerate roots, grOWth was confined to the first leaf of the stem. The amount of growth increased With increased root system. Galston (l9h8) found that roots developed in the dark on asparagus stem tips when exposed to one to ten gammas per cubic centimeter of indoleacetic acid in the medium, but that no roots developed in the light in the presence of the same concentrations. He assumed the presence of a material formed in the light other than auxin essential for root initia- tion. The stem-tips shown in PLATE 3 were excised from the tips of Sequoia shoots and allowed to grow for eighty days. They grew well, as demon- strated by the amounts of penetration into the agar, Without the formation of roots. In fact, most of the cultures of these experiments produced shoots and callus without the formation of a single root. Furthermore, by actual count of the cultures, the formation of what roots occurred nearly always was preceded by the initiation and growth of shoots. In Sggggia cultures, therefore, there seems to be no specific need for a substance formed in roots in order to allow the formation and growth of shoots. Maleic Hydrazide. In order to investigate a condition which might lead to the production of callus cells only, the growth inhibiting chemi- cal, maleic hydrazide (Schoene and Hoffman, l9h9), was incorporated into the nutrient medium at several concentrations with the idea that the ma- leic hydrazide might act as a selective inhibitor permitting the growth of unorganized callus at the same time suppressing the initiation of buds and shoots. The quick deaths of the segments in the 10, 00, and 1000 parts per million maleic hydrazide concentrations indicates that this chemical moves rapidly in all directions in the shoot, a condition found by Linder (1951) in stems. The possibility exists, however, that the maleic hydra- zide could have killed the tissues of the segment at the surface, thus blocking the absorption of nutrients from the agar. At any rate, the two treatments remaining gave several comparisons between cultures growing With and without one part per million of maleic hydrazide. A complete parallelism existed in the growth responses of Sequoia tissue to media with and without one part per million of maleic hydrazide but some instances of the stimulatory effect of small dosages have been reported. Greulach and Atchison (1950) claim a stimulation of the pro- duction of onion roots at the one part per million level. In their expe- riment ten parts per million showed extreme growth inhibiting properties. Another example cf a stimulatin" c- 00 is found in the work of Erocn r1 " " 1 -‘ " In '. -‘ x r A ~ V -: . 's '1. ' A . ‘r‘ u ' fl (1932) tho cet‘xminou IesL ual tox1cit lbs in the 8011. In tro Case of that maleic hydrazic inhihitzd toe growth of humex tissue directly in rd to Sequoia tis 919, the fact that one part per million did not inhibit growth in eighty days indicates that this amount can be tolerated. Tl: range of concentration between one and ten pafl sper million could possibly contain a concentration at which stim Lia ion of groxth might occur. . ~ Some anti-auxin (antagonistic) effects of maleic hydraaice also have been reported. According to Fitohcock and Zimme rmann (1951), combinations of 2, h - dichlorophenoxyacetic acid and maleic hydrazide produced addi- agonistic effects on several plants. Johnson and Euchholz (1951) report that proliferation and rooting 'nduced by 2, h - dichloro- phenoxyacetic acid could be eliminated in combination with maleic hydra— - 1 2108. Leopold and s 4 r’x lein (1951) concluded that the inlmi tion of growth by low concentrations of maleic hydrazide is completely relieved by the addition of auxin and that maleic hydrazide is not a growth regulator because it cannot remote growth in the absence of auxin. The results of i he treatment of Sequoia tissue with one part per million of maleic hydra- L C,— 'ide do not eliminate the possibility of an antagonistic effect of the two chemicals inasmuch as the naphthaleneacetic acid at 100 gammas per liter may have been sufficient to overcome the inhibition exerted by one part per million of maleic hydrazide but not of tour alts per million. -Sh- Evidence that some organisms can withstand large concentrations of maleic hydrazide was obtained from the fact that cultures became conta- minated even at the concentration of 1000 parts per million. Since all of the Sequoia tissue died in concentrations above ten parts per million, the survival of microorganisms seemed contradictory to conclusions drawn concerning the toxicity of the chemical. taste (1952) observed that, although maleic hydrazide was strongly inhibitory to Cytophaga, it had little effect upon Azotobacter in the soil. Nickel and English (1953) studied the effect of maleic hydrazide on soil bacteria and other micro- organisms. They found that 100 parts per million had no activity against many of them and that 280 parts per million had no effect against Rhizo- bium. Some withstood concentrations up to 1000 parts per million, the same concentration used in this experiment. Thus, in this experiment, maleic hydrazide failed to act as a selective inhibitor in the suppression of organized growth. Toxicity was rapid and general at ten parts per million and above. Segments grew equally well whether or not one part per million had been added to the medium. Adenine Sulfate. The review of the literature indicated that the formation of buds in tissue culture could be inhibited by high auxin concentrations and enhanced by high adenine concentrations (Skoog and Tsui l9h8). In regard to tobacco tissue the optimum concentrations for bud formation appear to be in the vicinity of 100 milligrams per liter for adenine sulfate (Skoog, 1953) and for complete inhibition of buds, 10 gammas per liter of indoleacetic acid (Killer and Skoog 1953). -55.. Carrot callus responded well in increased callus production up to 10 milligrams per liter of naphthaleneacetic acid (Wiggans 195h). The above investigators established the amount of adenine sulfate which would overcome the bud inhibiting properties of auxin as somewhat near 100 milligrams per liter. higgans (195h) pointed out that the ratio probably differed among species, organs and tissues. In regard to Sequoia tissue, a concentration of 100 milligrams per liter of naphthaleneacetic acid was found capable, in most cases, to inhibit bud formation. If this were true, then a concentration of ade- nine sulfate should exist that would counteract the inhibition caused by the naphthaleneacetic acid. As in the experiment of wiggans (l95h) With carrot tissue, no apparent increase in bud formation resulted from hO, 80, 100, and 120 milligrams of adenine sulfate used to counteract 50 and 100 gammas per liter of naphthaleneacetic acid (Table 18). As a matter of fact, any amount of adenine sulfate above forty milligrams appears to be toxic to growth of the segments. Since forty milligrams is the lowest amount used, it follows that the inhibition of buds by 50 and 100 gammas per liter of naphthaleneacetic acid was not overcome within the toxic range of adenine sulfate, a condition similar to that noted by higgans (l95b) in the case of carrot tissue. The inhibition of buds by the naphthaleneacetic acid is somewhat overshadowed by the general toxicity of the adenine sulfate. Examination of Table 18 reveals from little data, to be sure, that the higher concen- tration of naphthaleneacetic acid actually overcame some of the toxicity of the adenine sulfate and allowed a few more buds to be produced. -55.. Howell and Skoog (1955) point out a stimulating effect of l milligram per liter of adenine sulfate in the grOWth of excised pea epicotyls. The inhibiting action of adenine sulfate upon the production of fresh weight is shown in Table 19. Fresh weight rapidly increases with concen- tration of naphthaleneacetic acid in the presence of all concentrations of adenine sulfate and again the toxicity of the adenine sulfate appears to be lessened by the increased amount of naphthaleneacetic acid. The same results are apparent in regard to final dry weight (Table 19). The differences are not so obvious in regard to percentage dry weight but the same results apply. Thus the evidence of this experiment points toward a toxicity to the tissues exerted by the adenine sulfate. This toxicity is reflected by the decreased production of buds, and of fresh and dry weight. Very little data exists in the literature on the effect of adenine upon fresh and dry weights, since the emphasis is usually directed toward organo- genesis. However, Skoog and Tsui (l9h8) using adenosine show progressive decreases in the mean fresh weight of callus tissues from h8.b milligrams at 0.5 milligrams per liter of adenosine to 28.5 milligrams at 50 milli- grams per liter. The dry weight dropped from h.5 to 2.8 milligrams. The effect of adenine sulfate upon the initiation of buds and upon the production of callus cells apparently varies Widely among species and tissues. The findings of higgans (l95h) that the concentration of auxin Within the tissue may be high enough to prevent the expression of the adenine sulfate in the range in which it can be added without toxicity also can be applied to Sequoia stem segments in culture. _S 7.. SUMMARY AND CONCLUSIONS Stem segments, excised from shoots of Redwood burls (Sequoia semper- virens),were cultured in vitro under various conditions of chemical (maleic hydrazide and adenine sulfate) and other environmental treatments (growth supplements, light, polarity, temperature, reduced aeration). The effect of the original position of the segment on the stem and of original weight were studied. The effects of the above environmental treatments were expressed mainly in the relative control over the ini- tiation of buds and shoots and in the relative amounts of fresh weights produced by each treatment. The basic medium consisted of one adapted from Gautheret containing the mineral nutrients of Knop's Solution, Berthelot's oligodynamic ele- ments, three percent sucrose, two percent agar, naphthaleneacetic acid, and certain growth supplements: cysteine, thiamin, pantothenic acid, biotin, inositol, pyridoxine,and ascorbic acid. The segments responded in the following manner to the various treatments: 1. The ability of the segments in culture to form cell masses was initially greater and more rapid in the light in the absence of growth supplements (and yeast extract). In darkness, however, the presence of growth supplements (and yeast extract) enhanced the power of the seg~ ments cultured in darkness Without supplementary substances. (FIGURE 3). ~58- 2. The percentage of proliferating cultures that produced buds was highest in darkness Without the growth supplements (and yeast extract) and lowest in darkness with growth supplements. The percentages of cul~ tures that produced buds in the light With and without growth supplements (and yeast extract) gradually approached the high percentage shown for darkness without supplements (FIGURE 6). 3. A greater number of buds per culture occurred in the light than in the dark (TABLE h). h. Cultures maintained in light produced a greater final fresh weight than those in darkness. Among segments in darkness the presence of growth supplements (and yeast extract) enhanced the production of fresh matter over that produced in the absence of the supplements (TABLE 6). Although the presence of supplements initially inhibited growth (FIGURE 3) or killed segments (TABLE 9), the surviving cultures overtook and surpassed the cultures without supplements in the production of fresh weight (TABLES S and 6) and in general appearance and vigor. 5. The initiation and growth of roots occurred in the absence of naphthaleneacetic acid (EXPERIMENT I). Roots were completely inhibited in the presence of SO and 100 gammas per liter of naphthaleneacetic acid (EXPERIMENTS II, III, IV, V and VI). 6. Shoots grew vigorously without the presence of roots in most cultures of all experiments. 7. Maleic hydrazide, at concentrations of 10, 100, and 1000 parts jper million, killed all segments (FIGURE 8). No differences in the Ielative production of shoots or callus were observed between cultures Wflith and without one part per million of maleic hydrazide (FIGURE 9). -E9— Certain microorganisms maintained growth and contaminated cultures containing 100 parts per million of maleic hydrazide in the medium. 8. The effect of position on the shoot and of the original weight of the segments upon subsequent growth gave no significant differences Within the limits of the experiment (FIGURE 11). 9. Naphthaleneacetic acid at a concentration of 100 gammas per liter reduced and sometimes completely inhibited the initiation of buds (EXPERIE’ENT III). 10. The optimum temperature for the production of fresh weight occurred in a controlled temperature of 2hOC. and less than 3000. Cul~ tures maintained at room temperature grew almost as well as those at the controlled temperature of 2hOC. Buds developed slowly, but normally, at 6°C. At 37°C. buds were initiated but failed to develop (TABLE 15). ll. Submerged cultures failed to produce buds. However control cultures receiving the normal aeration produced numerous buds. The submerged cultures produced less callus than the controls (TABLE 16). 12. Adenine sulfate, at concentrations of to, 80, and 120 milli- grams per liter, progressively inhibited, rather than enhanced, bud ini- tiation and fresh weight production by the cultures in treatments of naphthaleneacetic acid at 0, SO, and 100 gammas per liter. The inhibi- tion of buds and fresh weight by adenine sulfate was partially alle- viated by the increasing concentrations of naphthaleneacetic acid (TABLE 13. Within the limits of the technique, no differences between the normal or inverted polarity of the segments in agar could be determined with respect to the production of buds or fresh weight. -60— LIT”RATUHE CITE Ball, Ernest. 1950. Idfferentiation in a callus culture of Sequoia sempervirens. Growth 1hz295-325. Ball, Ernest. 1953a. Histological effects of absorbed radioisotopes upon the callus of Sequoia sempervirens. Botan. Gaz. 11h:353‘363. Ball, Ernest. 1953b. Hydrolysis of sucrose by autoclaving media, a neglected aspect in the technique of culture of plant tissues. Bull. Torre Bot. Club 89:509-hll. Ball, Ernest. 1953c. Modifications in a ca lus culture of Sequoia sempervirens after growth on P32 and 83). Growth 17:215-a28. Ball, Ernest. l953d. Persistence of Clb in a callus culture of Sequoia sempervirens. Growth 11:169—182. Ball, Ernest. l953e. Studies of the accumulation of certain radioiso- topes by a callus culture. Amer. Jour. Bot. £9:306-316. Berthelot, A. 193h. 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Compte rend. soc. biol. 1h1:627-o29. Gautheret, L. J. 1950. Action du contact d'un milieu aqueux sur la morphorenese des cultures de tissu cambial de Salix caorea. Compte rend. soc. biol. lthz626- 629. Greulach, V. A. and E. Atchison. 1950. Inhibition of growth and cell division in onion roots by maleic hydrazide. Bull Torrey Hot. Club Haberlandt, G. 190%18Ku1turversuche mit isolierten pflanzenzellen. Sitzber. Aka d. " .Uien., Hath., natur - w. Klasse. Abt. I, 111: 69-92. Henderson, James H. M., Mary E. Iurrell, and James Bonner. 19 culture of normal sunflower tissue. Amer. Jour. Bot. 39: Hildebrandt, A. C. and A. J. Biker. 19h9. The influence of various carbon compounds on the growth of marygold, Paris daisy, periwinkle, sunflower, and tobacco tissues in vitro. Amer. Jour. Bot. 36:7h-85. Hildebrandt, A. C. and A. J. Hiker. 1953. Influence of concentrations of sugars and polysaccharides on callus tissue growth in vitro. Amer. Jour. Bot. h0:66-76. Hildebrandt, A. C., A. J. 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Uber ein neues auxin (heteroauxin) aus hain. XI Hitteilung. Zeitschr. Physiol. Chem. 228:90-103. Cited in: Leopold, A. C. 1955. Auxins and Plant GrowthT_—U. of Calif. Press, Berkeley and Los Angeles, Calif. Koritz, H. G. and F. Skoog. 1952. Colorimetric determination of adenine in plant tissues. Arch. Biochem. Biophys. 38:15-21. Kotte, halter. 1922. Kulturversuche mit isolierten wurzelspitzen. Eeitr. allg. Botan. 2:h13-h3h. Cited in: Kotte, halter. 1922. Jurzelmeristem in gewebekultur. Ber. deut. botan. Ges. £9:269-272. Leopold, A. C. and U. H. Klein. 1951. Naleic hydrazide as an anti- auxin. Science llh:9-10. Linder, Paul J. 1951. Absorption of some new herbicides by plants. Proc. N. E. heed Control Conf. 7-12. Lee, S. A. 19b5. Cultivation of excised stem tips of asparagus in vitro. Amer. Jour. Bot. 32:13-17. Lee, S. W. l9h6a. Cultivation of excised stem tips of dodder in vitro. Amer. Jour. Bot. 32:295-300. Lee, S. w. 19h6b. 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Compte rend. 205:521-523. Nobecourt, P. 1939. Sur la perennite et 1'augmentation de volume des cultures de tissus vegetaux. Compte rend. soc. biol. 130:1270-1271. Nobecourt, P. l9hO. SynthESe de la vitamine B dans des cultures de tissus végétaux. Compte rend. soc. biol. LLyons) 133:530-532. Nobécourt, P. l9h2. Sur les facteurs de croissance necessaires aux cultures de tissus de carotte. Compte rend. 215:376-378. ’ 1 ' I ’ 1 Nobecourt, P. 19h6. Culture prolonqee oe tissus vegetaux en l'acsence de facteurs de croissance. Compte rend. 222:817-818. NobEcourt, P. and L. Kofler. 19h5. Culture de tissus de tize de rosier. Compte rend. 221:53-5h. Peal, Arpad. l9lh. Uber phototropische “eizleitungen. Ber. d. bot. Gcs. gg:h99-502. Peal, Arpad. 1919. Uber phototrooische reizleitung. Jahrb. Wiss. Bot. 583h06‘1580 Rabideau,. G. S. and W. C. Uhaley. 1950. The growth and metabolism of excised roots in culture. II The respiratory rates of excised tomato roots. Plant Physiol. 25:33h‘339. Robbins, h. 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Changes induced by indoleacetic acid in nucleic acid contents and growth of tobacco pith tissue. Science 118:hh3—hth. Skoog, F. 1939. Experiments on bud inhibition with indole-3-acetic acid. Amer. Jour. Bot. g§3702-707. Skoog, F. 19hh. Growth and organ formation in tobacco tissue cultures. Amer. Jour. Bot. 31:19-2h. Skoog, F. 1953. Induction of bud formation in plant tissues. U. 8. Patent Number 2,653,087 to uisc. Alumni Research Foundation. Sept. 22, 1953. Skoog, F. and C. Tsui. 19h8. Chemical control of growth and bud forma- tion in tobacco stem segments and callus cultured in vitro. Amer. Jour. Bot. 35:782-787. Skoog, F. and C. Tsui. 19h9. Chemical induction of bud formation in plant tissues. Biol. Bull. 22:268-269. SnOW, P. 1936. Upward effects of auxin in coleoptiles and stems. New Phytol.‘25:292-30h. Thimann, K. and F. Skoog. 1933. The inhibiting action of the growth substance on bud development. Proc. Nat. Acad. Sci. 19:71h—716. taygood, E. R. l9h9. The respiratory carrier function of ascorbic acid in the wheat plant. Amer. Jour. Bot. 36 Suppl:930. ~66— Went, F. W. 1928. Wuchstoff und wachstum. Rec. Trav. Pot. Neerland. 25:1—116. Cited in: Meyer, B. S. and D. B. Anderson. 1952. Plant Physiology, D. Van Norstrand and Co., New York. tent, F. h. 1937. Specific factors other than auxin affecting growth and root formation. Plant Physiol. 13:55-80. hent, F. W. l9h1. Polarity of auxin transport in inverted Tagetes cuttings. Botan. Caz. 103:386-390. wetmore, R. H. and G. Morel. 19U9. Polyphenoloxidase as a problem in organ culture and auxin diffusion studies in horsetails and ferns. Amer. Jour. Bot. 3Q Supp13830. thite, Philip R. l93h. Potentially unlimited growth of excised tomato root tips in a liquid medium. Plant Physiol. 9:585-600. White, Philip R. 1937. Seasonal fluctuations in growth rates of excised tomato root tips. Plant Physiol. 12:183-190. White, Philip R. 1939a. Controlled differentiation in a plant tissue culture. Pull. Torrey Bot. Club éé:507-513. hhite, Philip R. 1939b. Potentially unlimited growth of excised plant callus in an artificial nutrient. Amer. Jour. Bot. 29:59-6h. 'Khite, Philip R. l9hO. Sucrose vs dextrose as carbohydrate source for excised tomato roots. Plant Physiol. 25:355-358. White, Philip R. 19h6. Plant tissue culture 11. Botan. Rev. 12:521-529. White, Philip R. 19h9. Growth hormones and tissue growth in plants. Biolog. Progress 1:267-280. white, Philip R. 1953. A comparison of certain procedures for the . maintenance of plant tissue cultures. Amer. Jour. Bot. £93517-52h. 'hhite, Philip R. 195k. The cultivation of animal and plant cells. The Ronald Press Co., New York. hlggans, Samuel C. 195a. Growth and organ formation in callus tissues derived from Daucus carota. ’ er. Jour. Bot. £13321-326. -57- TABLE 1. COMPONENTS OF NUTRIENT SOLUTIONS USED IN EACH EXPERIHENT. INGREDIENTS USED IN SEVERAL CONCENTRATIONS WITHIN AN EXPELIUENT ARE SHOWN IN THE AMOUNTS USEL SEPARATEL BY DASHES. TYPE OF EXPERIMENT I II III IV V VI Maleic- CORPONENT Light, Hydrazide, Adenine- (Amount Vitamins, Position. Temper- Aera- Polar- Auxin per liter) Polarity Init. ht. ature tion ity Ratio Knop's Solution* 500 m1 100 500 500 500 500 Berthelot's 0.5 ml 1 0.5 0.5 0.5 0.5 Solution** l(+ Cysteine-RC1 0-10 mg 10 lo lo 10 10 Thiamin RC1 0-1 mg l 1 1 l 1 Ca-d-panto- 0-1 mg l l 1 1 1 thenate Biotin 0 mg 0 l l 1 1 i‘InOSitol O-IOO mg 100 100 100 100 100 Pyridoxine-HCl 0—1 mg 1 l l l 1 Ascorbic Acid hOO mg 0 h30 hOO hOO h00 Yeast Extract O-h gm 0 O 0 0 0 Sucrose 30 gm 0 30 30 30 30 Glucose 0 gm 30 0 0 0 0 Agar 10 gm 6 10 10 10 10 Naphthaleneacetic 0 100 100 100 100 0-50— Acid 100 Adenine Sulfate 0 mg 0 0 0 0 O-UO- 2H2O 80-120 Kaleic Hydrazide 0 mg 0—1-10 0 0 0 0 Liethanolamine 100—1000 Salt * Knop's Solution: Components per liter of stock (twice strength) 1.0 .25 .25 .25 gm Ca(N03) . 2H 0 KNOo 2 2 KHZP ** Berthelot's Solution: Components per liter of stock 50.0 2.0 .5 005 .05 .20 gm Fe2(SOh)3.nH20 0.10 gm ZnSOh.7H20 MnS .H o .05 CuS ,.5H 0 KI on 2 .lo Becig 2 NiClg.6H2O .05 H3BO C0012.6H20 1.00 ml HZSOh TiCIh TARLE 2. PEECENTAGES 0F CULTURES IN EACH TREATIENT IN LHIOH PROLIFERATIONS APPEARED ON THE ORIGINAL SESNENT. Light Darkness Weeks after 'nith Uithout hith hithout start of Supplement Supplement Supplement Supplement Experiment . Inverted Normal Inverted Normal Inverted Normal Inverted Normal 2 27 36 72 75 15 18 10 22 3 52 h9 85 80 38 33 32 D9 h 58 59 92 93 L9 an 53 60 5 73 83 97 93 79 55 58 6O 6 75 80 100 98 80 78 70 63 8 100 100 100 100 95 89 72 68 10 100 100 100 100 100 100 81 72 TAPLE 3. PEPCENTAOES OF CULTURES IN EACH TREATMENT IN WHICH BULB OR SHOOTS APPEARED ON THE ORIGINAL SEGKENT. Light Darkness Cheeks after Lith hithout gith uithout Start of Supplement Supplement Supplement Supplement Experiment Inverted Normal Inverted Normal Inverted Normal Inverted Normal 5 3O 39 59 59 16 15 68 83 6 37 h8 62 6h 16 13 65 88 8 UB 73 7O 70 1h 13 69 88 10 56 8h 67 7h 17 ll 65 92 TABLE h. -69. EFFECT OF SEVERAL ENVIRCNEENTAL CONDITIONS UPON THE PRODUCTION OF BULS ANL‘SHOCTS PER CULTURE IN EACH TREAIZENT. Polarity Light Darkness Seggint 'Nith Bithout hith hithout Supplement Supplement Supplement Supplement Inverted 1.5 3.1 0.1 0.3 Normal 3.2 h.0 0.1 0.5 TABLE 5. FINAL FRESH'NEISRTS (30.) or Two SETS 0? sacrarTs 111n- TAINED ONE YEAR AFTER TERNINATICU 0F EXPERINENT I. INITIAL NT. NO NO. 11th ‘Nithout Supplement Supplement Cultures 12 1h Kean Final Fresh Wt. (mg) 197L 117h .LaJADJIJE 6 0 FINAL FRESH EEICNT (NO.) OF SECKENTS. TLCH SEGNENT LEISHED INLIVILUALLY. _. —4 “---——-—- Polarity Light Darkness of ‘_4 Segment tith nithout tith Bithout Supplement Supplement Supplement Supplement Inverted A75 + 125 517 + 126 1A1 : 35 96 i 17 Normal Shh‘: 139 523 i 103 125.: 72 97 1 1h .370... TABLE 7. FINAL DR. NEIGHT (r0.) CF SEGRENTS. SEGKENTS LEIGHED COLLECTIVELY FOR EACH TREATNENT. Polarity Light Darkness Segggnt with 'Lithout Eith Lithout Supplement Supplement Supplement Supplement Inverted 65 76 20 11 Normal 70 80 19 12 TABLE 8. PERCEN_ACE DRY LEIGHT OF SECNENTS. Polarity Light Darkness Seggint hith lithout With Mithout Supplement Supplement Supplement Supplement Inverted 13.7 1h.7 1h.2 11.5 Normal 12.9 15.3 15.2 12.6 TABLE 9. PERCENTAGE CF LEAD SEORENTS IN EACH TREATMENT AT END OF HPERILIENT I . Polarity Light Darkness of Segment lith Without Rith Without Supplement Supplement Supplement Supplement Inverted 39 8 5 11 Normal U6 12 8 13 -71... TARLE 10. INITIAL AND FINAL DISPOSITION OF CULTUBES IN EACH TREATMENT OF EXPERILENT 1. Light Darkness With Nithout With Without Disposition Supplement Supplement Supplement Supplement of Cultures Invert Norm Invert Norm Invert Norm Invert Norm Cultures with Shoots & Callus & Living at end of Experiment 36 32 N9 50 38 36 32 35 Cultures Dead at end of Experiment 23 27 h 7 2 3 h 5 Cultures Con- taminated 1 1 7 3 0 1 h 0 Total 60 oo 60 60 to ho ho to Cultures Weighed at end of Experiment 28 2h 35 38 30 30 2h 27 Cultures Saved for Future Experiment 8 8 1h 12 8 6 8 8 Total 36 32 b9 50 38 3o 32 35 -72- TABLE 11. AVERAGE hEIGHT (Le.) OF SESLENTS FOR THE VARIOUS TREATMEITS 0F LALEIC HYDRAZIDE, ACCORDING TO DISTANCE FROM THE APEX. Ealeic hydrazide (ppm) Group Distance 0 l 10 100 1000 from Apex 1 1-5 cm. 55 60 55 62 63 2 6-10 83 95 92 80 87 3 11-15 99 10h 101 100 100 A 16—20 121 132 135 115 129 5 21-25 120 150 160 128 150 6 26-25 165 125 INC 155 130 Average 89 96 9h 88 92 -73... TABLE 12. NUIEER OE ShOOTS (AND-BULB) PRODUCED BY THE CULTURES WITH INCREASING AEOUKTS OE RALEIC hYlfiAZILE. Leeks after start of Experiment Haleic hydrazide 6 9 12 15 18 0 ppm 19 19 25 3b 3b 1 18 27 28 31 31 10 6 6 6 6 6 190 O O O O O 1000 O O O O O TABLE 13. PERCENTAGE OF CULTURES IN ANION THE CALLUS TYPE CE CELLS WAS PRODUCED (IN LIVING CONLITICN AT THE TILE INDICATED). heeks after start of Experiment O“ \O Maleic hydrazide 12 15 18 0 ppm 16 70 92 A9 3 1 ppm 19 AS 83 L3 26 TABLE 1h. DPY MATTER IN CULTURE. -7h- EFFECT OF TEMPERATURE UPON THE PROLUCTION OF FRESH AND Age Init. Final Wt. of Wt. of Final Perc. Incre- Treatment Days 'Wt. of Wet Shoots Callus Dry Dry ment of Segment Wt. Wt. Wt. Growth Part 1 600 Darkness 262 152 251 26 73 83 17.3 99 Room Temp. Light 197 156 572 h6 371 88 15.3 b16 Room Temp. Darkness 197 170 697 120 boa 9h 13.5 527 2890 Darkness 197 161 508 67 280 72 1h.2 3&7 37°C Larkness 262 179 331 O 152 hO 12.2 152 Part 2 Room Temp. Light 137 7h 301 o 227 51 16.8 227 Room Temp. Larkness 137 58 h71 O h13 67 1h.2 h13 2hoc Darkness 137 82 L9h‘ o h12 79 15.9 h12 3000 Darkness 137 7b 373 0 299 h7 12.8 299 37°C Earkness 137 85 156 O 71 18 11.h 71 TABLE 15. EFFECT OF TEKPERATUPE UPON THE PROLUCTION OF SHOOTS, BUIS,ANL ROOTS. Total Treatment No. of Age in Total No. Total No. Length Total No. Cultures Days of Buds of Shoots of Shoots of Roots (cm.) Part 1 6°C Darkness 1b 262 h 6 10 0 Room Temp. Light 13 197 5 15 b7 0 Room Temp. Darkness 9 197 h 8 38 O 2h°C Darkness 19 197 6 12 DB 0 37°C Darkness 12 262 7* O O 0 2.11.3 Room Temp. 5 Light 12 137 O 0 O 1 Room Temp. Larkness 5 137 O O O O 28°C Inrkness 7 137 o o o o 30°C Darkness 7 137 O O O O 37°C Larkness 9 137 o o o o * 7 buds quickly degenerated to callus. TAPLE 16. EFFECT OF REDUCED AERATION UPON THE AVERAGE FRESH AND DRY WEIGHTS (ro.) CF CULTURES. Treatment No. of Initial Final it. of fit. of Final Pct. Incre. Cultures fit. of Wet Shoots Callus Dry Dry of 260 days Segment at. Vt. Wt. Growth Submerged 1h 93 15D 0 61 2h 15.8 61‘: 12 Control 17 92 592 101* 379 105 18.h 880 i 100 * Consisted of two shoots per culture; total length 108 cm. TABLE 17. EFFECT OF INVERTION CF EGMENT IN AGAR MEDIUM UPON THE AVERAGE FRESH AND DRY‘WEIGHTS (NC.) 0F CULTURES. Treatment No. of Initial Final 'Wt. of fit. of Final Pct. Incre. Cultures fit. of let Shoots Callus Dry Dry of 192 days Segment Rt. ht. Et. Growth Inverted 9 62 287 h 181 38 15.5 185 i 53 Normal lb Sh 22L 27 1M3 32 1h.3 170 : hh TACLE 18. RELATIVE PRCDUCTION O? FEDS AND SECCTS AT VALICCS RATIOS OE ALEHIHE SVLEATE AND NAPPTHALEWEACETIC ACID. Treatment Adenine Naphthalene- No. of Total Total Total No. of Shoots Sulfate acetic acid Cultures No. of No. of Length and Buds per mg/l gammas/1 Buds Shoots of Shoots Culture (cm.) 0 130 83 35 22 50 1.3 L0 100 39 1h 7 22 0.5 80 100 h3 1 h 13 0.1 120 100 36 2 11 DB 0.h C so 18 o 5 10 0.3 ho So 19 o 1 3 0.1 80 50 1L 0 o o 0 120 So 10 1 o o 0.1 0 0 22 0 0 0 0 to o 22 o o o o 80 0 23 0 0 0 0 120 0 23 O O O 0 -78— p m.OH p p o Hp mm mm o oNH a o.HH a a 0 Ho 4m mm 0 ca m ~.oa p m o oo am mm o on m o.mH m m 0 mm mm mm o 0 ma m.HH a ma 0 No m: 0H om oNH mm o.NH a mm 0 me am 4H om cm 3 m . NH 3 pm 0 mm 3 ma om 0: op m.afi ma Om OH moa a: ma Om 0 4m m.mH NH mm pm amfl mm pm ooa ONH mm a.HH ma pp p gma mm m: 00H ow 3 92 ON am m Hma 4m mm 03 3 mHH ~.mH mm OOH ma Haa mm ma 00H 0 5.8.5 . £1... . 1...... 99de m poozm . f... p: mam mm H\ m macaw. H\mé mo m5 m5 no mo pm...» we . P...» w 085.30 UH ow 9.8% om. mpwmasm .ppppH .pppm Hanan pnmapg, pgmapa. appaa HpapapH Co .02 -pppapppgapz ppflpppa psoEpmth .mmm: .oav mEmOHM§ E5 979.. mmmdm 20mm QHod UHHmodmzmAGErmmfl/N Q72 Ednfibm MZHzmde r.3 HomaEm .mH mammfi _79- .msaawo op popmHmCCwmm mpsm «* .Uwoa owpmomCCCszpsdwz mo haodsm gmmhm * mph 8.8% 33% and H .QEme Eoom ~.H o+m I I I I I I I I I A0 0v I I I I I I I I I coapmpma 30H .mfima Eoom I I I I I I I I I 8 m4 ”SE 2.0 8 885:8 .QECB Eoom o to o+m 9H 9m .3 9m 2.1.3 .Qame Boom mmmcthm oowm pmpaxppm Doom wmmcxamm oogm I I I I I I I I I I I I a.o o+m I I I I I I mppcepau moo .22 Q I. 03 1.13.2. $.22 .22 1.4.4.2 5.2 as 8 ,1 a? 03 Q1 02 <1 03 CL 03 <32 8388 3238 885889 papaaam spa soap m pupa H pupa ppappppam psasppae maficopa Inmaom Imhma .QECB .Qame oflmamu pgmfiq H» > sH QHHH pHHH HH H pameflpmmxm mo make pom nmpEdz .WMPHQDD mmm mmbm ho mmaZDZ me<2HxQAmm¢ QB mmmmm mmmmon .mAdbo Nm_QMH¢OHQZH mm< onhaflmom mam mmgqomhzoo monz m mazeammB .o+m Homzwm Mae Hm QMB<0HQZH mH mbqqao 93¢ mmzm mo Mozmmmmm .ZOHBUDQcmm QDm QmBHmHmZH wqmmHBzm mo qumHamdm mUHmS mBZMEH .éfiLl- ._25 85 100 115 10 115 1 80 1000 70 ____ ____x .___m ____ ___q, 10 1000 10 1000 2510 0 100 15 00 _1___ 110 100._ 115 10_<_)__ 85 ,_O_ __ 9c 10 0 110 1000 1250 , 80 10 __85 100 1 , 120 10 105 100 95 100 85 ;05 100 1100 110 1000 90 1000 8c , 85 10 125 1 1351 , 85' 1 95 100 _1000_ 125100 _ ‘12510 85 0 100 25 ,100 w 12 ‘0 135 10 80 100 10 1 _1g5 1000 135 100 w 85 85 1000 pp 1 160 0 95 110 00 _10 16510 1&0 1000 85_ N100 109 ________ 200|1___110._____ 10 0 2 10 Averages 90 111 11b 87 77 Averages for First 15 Centimeters 83 103 101 79 71 10 O 1000 100 100 10 1000 100 H1 0 U1 1 i-’ to O F4 F’ C) +4 lcn C) F4 F’ +4 <3 C) C) t' \J'l H \A) U1 I F4 +4 F4 n: c: C) E; <3 [3H 1 J 100 1000 10 1000 10 100 91 DIAGRAMKATIC REPRESENTATION OF THE SHOOTS AND SEGKENTS OF EXPERIEENT II SHOWING INITIAL LEIGHTS (MG.) INSIDE EACH SEOEENT AND TREATKENT OF MALEIC HYDRAZILE (PARTS PER MILLION) AT THE RIGHT OF EACH SEGMENT. Ave 2 Furl N0. 2 ""7;- fifllfi r421 .16.. - ”.5. .--- 5 1 15.0 L22.“ 60 1000 ,___, .2399. - .12 _7_0_1000 .61 1711.2 0 27lQUo087 37-2 1 08 .Lu w? /6 AS 7..po n0 O,nC o/ no 0; 0, no 0. 1L 1) n4 hm . . .11 11 11.11 11 11 . . c u a g . c . . a . O . O . 0. AU AU .AU nu . n0 n0. . AU AU no 01 no AU no AU AU PHI 11 11 11 nu.111 11 my 1f 11.no 11 1.111-11. AU r9 HD.C/ me :2 no w? nu AU v9 «9 :2 nu n6 #3 an 1u.:/ :2 11. 1: no 90 mm go o/ O, o/ mw - , 1 a 1 1 o u a c . AU . no . nu no. no . AU . . 0, AU ms he no my. nSAU .nu AU . menu. . no no n..nu AU 0: nu nv nu. nu nu 00 1O n, no .nu nu no. 0f 11 11 11 11.11-1U1 11 11 11.11 11 11 nu 11- no 11 11 11 111 11 no 11 11 11 .11 n0 11 11 1f mg a) nu no dz :2 a? a) :2 H9 :2 nv.fi/ :2 no 11111111111111111 1111111111111 1 1 1 11‘ 11 11 11 1111 111111 11 1+‘11 111 11 11 11 I C m m 0 . 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ON on 00 Om ~89- 100 _.Percentage of cultures in treatment .‘cz L L I I 1 I -0 3 6 9 12 15 Weeks after start of experiment FIGURE 9. PERCENTAGE OF CULTURES SHOWING ANY TYPE OF GRonTH (CALLUS, " SHOOTS, OR BUIS). 18 -90. .EBQE m5 E mflfimfifim age...“ .SQEH..._,, 82 mi 2.8% mo 5395 mefiéfimoo .2 @3on pcmfifipmcfim mo ppmpm pmpmm mxmm? ma ma ma m o 1 1 q a Sam 0 \\ 0-|u|IIIIIIQ\ thpflso pma mwoozm mo mumpme.p2mo n 5 fimcfl S cm 10 cm 15 cm 1 .--- 2' ..... .9. E o .821 w 0 75 l 85 — ‘1‘ ---_ -_-- 85 0 E0 125 100 0 _1 III— 1501_1___1001 1u0| 0 1100 120 1 1 _.l E. w__+ ,& JEi. ""E P—-' -- --~ 1145‘ 1101 .17_5 fi —I > '— 20 cm ........ ‘ 0. ........... 0 FIGURE 11. POSITION, 0310 PRODUCED PROLIFERATIONS IN CULTURE. -91- h 5 _ ----1_, - ...1 Lab L50 ,__x 35.1 £1 T150 3 1 11.21 ______-__ ‘ 85 0 $22.0 $321 851 ---- Q-__ 0 0 ED ‘— E l.-- .— 1(Dead) l I23 IFSI El 0 U1 L—— H I H 10 EAL WEIGHT, ANIDTREATMENT OF SEGMENTS WHICH OF CONTAMINATION ARE LINED OUT. SEGMENTS LOST BECAUSE 1(Eead) DEAD SEGTENTS ARE INDICATED. l5-QNL 100 l u—c-l-ng O-II-li a.-- 50 ‘1 L___ F— 25.0 O P 29.1 LUEQLEQJ 1 ‘55 ,1 23% W1 _. _. _0_.0 0---, .----¢__, ..... o 11-1 29.0 ZOLqu 25 gm FIGURE 11, (CONTINUED) Burl NO. wl-J O E] O ’1(Dead‘ H 2 El'v [cal [83E °°| 1650 125 1 _93.. Increase in Weight (mg.) 300 . Part 1 200 _ F 100 _ F__ n L I j I 6 20 2h 30 37 Degrees Centigrade boo F '— Part 2 300 . r 200 _ lOO .. n 1 J I 7 6 20 214 30 37 EEgrees Centigrade FIGURE 12. TERMINAL INCREMENTS OF FRESH HEIGHT (MG.) PRODUCED BY CULTURES MAINTAINED IN CONTROLLED TEMPERATURES. -9h- lbO Increase in Yd et G‘s'RJeight (mg) 130 120 100 100 gammas NAA 90- per liter 80. 70. SO gammas NAA per liter 30. w 2Vr lO 0 NAA *0 I 4 n J 0 EC 80 120 Adenine Sulfate (mg/1) FIGURE 13. EFFECT OF ADENINE SULFATE UPON THE TEFIINAL INCREMENTS OF FRESH WEIGHT (MG.) FEOLUCEE BY SEGMENTS CULTURED AT SEVERAL LEVELS OF NAPHTEALENEACETIC ACID (NAA). 100 90 80 50 ho 3O 20 10 FIGURE 1h. EFFECT OF ALENINE SULFATE UPON TE C. -95- Increase in Wet 1.5; e i ght (mg) . lOO gammas NAA per liter r L 50 gammas NRA per liter WI; 0 0 ho 80 120 Adenine Sulfate (mg/l) E TERHINAL INCFEKENTS OF U FEESH'UEIGHT (EG.) 0F CALLUS PE E CED BY SEGEENTS CULTURED AT SEVERAL LEVELS OF NAPHTHALEIEACETIC ACIL (NAA). .BmOHA 2H mmpHdMMmHMH zoom mo mZOHHHQZOo mquEHm 11125 mezflfiHammxm Hammpmm no m-:a£omm HEB wm QMOqumm BmmHmc‘mmmmm 2H mmmdmmOZH .mH mmbon {54> .L. F: ,vrl. New pawEHanxm mo pkmpm pmpma mhma .03 08 02 02 om o n u u c A - H> .00H 0 4.0 E or O . . smegma 83.25 mASmeQEmp Soom 0 HHH . 8N .oom - HH etflhfl 1 03 mhdpmpmmEme 500 335ch LEE W A .mev pzwwq msodCHpcoo psmwm; pm: cfl mmdmpocH 00m _9 7— PLATE 1. REDWOOD BUI’L OF SEQUOLA SEL‘J’ERVIRENS (LA].B.) ENDL. IN SPROUTING CONDITION. x 1/2. 5-“; -~———— v PLATE 2. FPEOHLY CUT SEGMENT FROM SHOOT OF RED-510013 BURL. X 1. . L PLATE 3. TYPICAL GROWTH OF SHOCTS AND CALLUS FROM A SEQA‘ENT AFTER EIGHTY EATS. x 1 1/2. \ \ -lOO— .z’. PLATE h. SHOOT TIPS OF SEQUOIA BURL SHOOTS GROWING IN CULTURE. x 1 1/2. -lOl- PLATE 5. TYPICAL GROWTH OF CALLUS FROM AN EXCISED SEGMENT OF SEQUOIA BURL SHOOT. x 1 1/2. PLATE 6 . -lO 2- DEVELOPL’IENT 0F CALLUS AT BOTH ENDS 0F BURL SHOOT SEGIENT GROWN IN DARKNESS AT 2h0 0. x l. IZOA‘A’I‘ _103_ PLATE 7. METHOD OF GROWING SUBIILERGED CULTURES. X l. §-- -10h- PLATE 8. TYPE OF CALLUS PRODUCED BY SUBNERGED CULTURE S (LIQUID REMOVED). x 1. -105- PLATE 9. PRODUCTION OF CALLUS BY NON-SUBILERGED CULTURE. X 1. ..v'I -~ - .a. C.,-- } :.;_. -106- -— -—~» ~——--— --»——-- » -..._.. -——- -._-- ——-~e— — ‘-——-—‘- -———.‘- PLATE 10. PRODUCTION OF SHOOTS BY NON-SUBMERGED CULTURE. X 1. I-Ay A]... «w Date Due ? LE5“ m USE. BULL, Demco-293