THE RELATION. 0F GIBBERELLIN TO THE COLD REQUIREMENT 0F LILIUM LONGIFLORUM, THUNB, CV. ACE Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY SANDRA E. KAYS 1969 III' I LIBRARY Michigan Suva University ! .3536'."}l”-‘- 3“ IINDING BY ” I ‘I HMS & SONS' '. " 800K BRIBERY IIIB. ;' ; --' If LIBRARY muons I» -n-nn-n... ”lull. -! ABSTRACT THE RELATION OF GIBBERELLIN TO THE COLD REQUIREMENT OF LILIUM LONGIFLORUM, THUNB., CV. ACE By Sandra E. Kays The cold requirement of Lilium longiflorum, Thunb, is often used in the manipulation of flowering date, but has undesirable effects on plant quality. Hence a chemical substitute for this treatment would be desirable. Previous research has shown that gibberellic acid will supplement or substitute for the cold requirement of certain plants. Therefore, the relation of gibberellic acid to the cold requirement of Lilium longiflorum, Thunb., cv. Ace was studied. The activity of "bound" and "free" fractions of endogenous gibberellins was found to decline during cold storage of precooled and controlled temperature forced bulbs. The activity in controlled temperature forced bul s was always higher than that in precooled bulbs. It was also found that GA3 applied to the bulbs before or after cold storage reduced the bud count but did not affect the number of forcing days to flowering or the number of leaves per plant. Increasing the length 0f 001d Storage, on the other hand, reduced the bud count, the number of days to flowering and the number of leaves per plant. Sandra E. Kays The relation of gibberellic acid to the cold require- nmmt of Lilium longiflorum, Thunb. appears to be only of an indirect or secondary nature and there is no indication of any commercial importance of its application by present standards. THE RELATION OF GIBBERELLIN TO THE COLD REQUIREMENT OF LILIUM LONGIFLORUM, THUNB. , cv. ACE BY Sandra E. Kays A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1969 .4 CI; 0 1r). ACKNOWLEDGMENTS The author wishes to acknowledge the guidance of Dr. W. H. Carlson, Dr. A. A. De Hertogh and Dr. R. L. Anderson in the preparation of the manuscript. Apprecia- tion is also expressed to Dr. Louis Aung for his encourage- ment throughout the course of this study. ii TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . . . . LIST OF TABLES O O O O O O O O O O 0 LIST OF FIGURES . . . . . . . . . . . INTRODUCTION 0 O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . Cold Storage and Flowering of Easter lily Precooling . . . . . . . . . . . Natural cooling . . . . . . . . Controlled temperature forcing Preheating before precooling . Initiation of Flower Primordia Gibberellins and Cold Requirement Gibberellins and Bulbs . . . . Gibberellins and Other Cold Requir PART I INTRODUCTION . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . Source of experimental material . . Cultural conditions . . . . . . . . Isolation and purification of gibberellin-like. substances . . . . . . . . . . . . RESULTS 0 O O O O O O O O O O O O O O in ng EU. 0 O O O O Q l-' (1'. o o o o o o . Quantitative changes in endogenous gibberellin- like substances . . . . . . . . . . Qualitative changes in endogenous gibberellin- like substances . . . . . . . . . . iii Page ii vii La) oooouww 10 12 l4 17 18 l8 18 20 26 26 35 DISCUSSION 0 o O o o o o o o o o v 0 SUMMARY 0 I O o o o o o o o o o o o 0 PART II INTRODUCTION 0 o o o o o o o o o o 0 MATERIALS AND METHODS . . . Source of Experimental Material . . Cultural Conditions . . . . . . . . Treatments . . . . . . . . . . . . Experimental design . . . . . . . . Collection of data . . . . . . . . Analysis of results . . . . . . . . RESULTS 0 O O O O O O O O O O O O O 0 Number of forcing days to flowering Number of flower buds per plant . . Height 0 O O O I O O O O O O O O 0 Number of leaves per plant . . . . DISCUSSION 0 O O O O O O O O O O O O SUMMRY O O O O O O O O O O O O O O 0 LITERATURE CITED iv . . C O O . Page 39 43 44 45 45 47 48 48 49 51 51 51 60 60 74 78 79 LIST OF TABLES Table Page 1. The dates of removal of samples of PC and CTF bulbs of Lilium longiflorum, Thunb., cv. Ace for extraction of gibberellin— like growth substances . . . . . . . . . . . . 21 2. Changes in fresh weight and levels of endo- genous "free" and "bound" gibberellin in PC and CTF Lilium longiflorum, Thunb., cv. Ace at different developmental stages . . . . . . . 35 3. GA3 treatment of controlled temperature forced Lilium longiflorum, Thunb., cv. Ace . . 46 4. Analysis of Variance Table for number of forcing days to flowering of Lilium lon i- florum, Thunb., cv. Ace. Randomized EIocE 3e51gn using all replications . . . . . . . . . 54 5. Analysis of Variance Table for number of forcing days to flowering of Lilium longi- florum, Thunb., cv. Ace. Randomized block deSign using replications l and 4 only . . . . 55 6. Orthogonal comparisons of number of forcing days to flowering of Lilium longiflorum, Thunb., cv. Ace . . . . . . . . . . . . . . . . 56 7. Analysis of Variance Table for number of flower buds per plant of Lilium longiflorum, Thunb., cv. Ace. Randomized block design . . . 61 8. Orthogonal comparisons of number of flower buds per plant of Lilium longiflorum, Thunb., CV. Ace O O O O O O O O O O O O O O O O O O O O 62 9. Analysis of Variance Table for height of Lilium longiflorum, Thunb., cv. Ace at, flowering. Randomized block design . . . . . . 66 Table 10. 11. 12. Page Orthogonal comparisons of height of Lilium longiflorum, Thunb., cv. Ace . . . . . . . . . 67 Analysis of Variance Table for number of leaves per plant of Lilium longiflorum, Thunb., cv. Ace. Randomized block design . . . 71 Orthogonal comparisons of number of leaves per plant of Lilium longiflorum, Thunb., CV 0 Ace O O O O O O O O O O O I O C 72 vi LIST OF FIGURES Figure Page 1. Dates and temperatures for controlled tem- perature forcing and precooling of bulbs of Lilium longiflorum,Thunb., cv. Ace. . . . . . . l9 Histograms showing promotion or inhibition of stem growth of Pisum sativum L. cv. Proqress No. 9 by eluants from chromatogram strips of extracts of "free" gibberellin- like substances from 7 PC bulbs of Lilium longiflorum, Thunb., cv. Ace at different growth stages . . . . . . . . . . . . . . . . . 27 Histograms showing promotion or inhibition of stem growth of Pisum sativum L. cv. Progress No. 9 by eIuants from chromatogram strips of extracts of "bound" gibberellin- like substances from 7 PC bulbs of Lilium longiflorum, Thunb., cv. Ace at different growth stages . . . . . . . . . . . . . . . . . 29 Histograms showing promotion or inhibition of stem growth of Pisum sativum L. cv. - Progress No. 9 by eluants from chromatogram strips of extracts of "free" gibberellin- like substances from 7 CTF bulbs of Lilium longiflorum, Thunb., cv. Ace at different growth stages . . . . . . . . . . . . . . . . . 31 HistOgrams showing promotion or inhibition of stem growth of Pisum sativum L. cv. Progress No. 9 by eluants from chromatogram strips of extracts of "bound" gibberellin- like substances from 7 CTF bulbs of Lilium longiflorum, Thunb., cv. Ace at different growth stages . . . . . . . . . . . . . . . . . 33 Standard curve of growth response of Pisum sativum L. cv. Progress No. 9 to various concentrations of 6A3 . . . . . . . . . . . . . 37 vii Page Figure 7. 10. Effect of 4 concentrations of GA3 applied before and after the cold treatment to planted Easter lily bulbs on the number of forcing days to Opening of the first flower . . . . . . . . . . . . . . . . . . . . 52 Effect of 4 concentrations of GA3 applied before and after the cold treatment to planted Easter lily bulbs on the number of buds produced . . . . . . . . . . . . . . . . . 58 Effect of 4 concentrations of GA3 applied before and after the cold treatment to planted Easter lily bulbs on the height at opening of the first flower . . . . . . . . . . 64 Effect of 4 concentrations of GA3 applied before and after the cold treatment to planted Easter lily bulbs on the number of . . . 69 leaves at anthesis . . . . . . . . . . . viii INTRODUCTION Lilies are a very important flower crop in the United States. Approximately ten million bulbs are pro- duced each year, mainly in Oregon, California and Florida (Gould, 1967). Most of the bulbs produced are forced as pot plants for Easter and many problems are encountered during forcing. It is important that the plants flower in time for Easter and at the same time be of high quality. Normally, the rate of development of the plants is regulated by means of a cold storage treatment (Kiplinger and Langhans, 1967). While this type of treatment can produce a timed lily, it does, however, have an undesirable effect on the number of flowers and leaves and in some cases, the height of the plant (Brierly, 1941). It has been found, with certain plants, that the naturally occurring growth regulating compounds, the gib- berellins, are associated with the low temperature require- ment, either supplementing or substituting for it (Lang, 1965). This may also be true for the Easter lilies. A chemical substitute for the low temperature requirement could lead to the elimination of an expensive, time consuming phase of production and may enhance the accuracy of precision flowering. Hence, the following investigation was carried out to determine the relationship of gibberellic acid (GA3) to the cold requirement of the Easter lily. Changes in the levels of endogenous gibberellin-like substances in the bulb during cold storage were studied. In addition, the effect of exogenously applied GA3 on the subsequent flower— ing and development of the plants was determined. LITERATURE REVIEW Cold Storage and Flowering of Easter Lily Precooling One of the most extensive studies on the Easter lily was carried out by Brierly (1941). He used the method of handling bulbs, now referred to as "precooling," in which unpotted bulbs are subjected to low temperature treatment while still in their original packing cases or similar containers. In a series of experiments, over a period of 3 years, it was shown conclusively that cold storage of lily bulbs had a profound effect on flowering and plant quality. In 1935-36, he tested 3 cultivars (Croft, Creole and Erabu) and found that cold storage of the bulbs in moist peat at 51°F or 36°F for 5 weeks accel— lerated the date of flowering, decreased the height of the lilies and decreased the number of flowers on each plant when compared with bulbs given no cold storage. The mag- nitude of the response varied with the cultivars. The following season Giganteum, Harissii and two types of Creole and Erabu were compared. In addition to the 5 week storage at 50, 40 or 32°F, a 10 week storage period was added. It is interesting to note that, when 3 compared with the shorter period of cold storage, the in- creased duration of the cold treatment reduced the number of days to blooming, the number of flowers produced per plant and in most cultivars, reduced the height. Brierly calculated the number of days to flowering as the time period between the last day of cold storage and the day on which the first flower opened. While the length of this period was reduced by cold storage (5 or 10 weeks) over control, the actual date of flowering was not always advanced. In some cases they flowered at a later date than the untreated controls. In 1937-38, experiments were confined to the cul- tivar Creole, which had not received cold storage in pre- vious handling. In this case, the number of days to flowering was reduced by cold storage of the bulbs (5 or 10 weeks at 32, 40 or 50°F) and an accelleration of the flow— ering date was also noted. Brierly's experiments (1941) established that longer periods of cold storage (15, 20, 30, 45 or 60 weeks at 32°F) resulted in successively fewer days to flowering but correspondingly later flowering dates. The number of flowers per plant was reduced from 8.5 in controls to 5.2, 4.3 and 2.0 in those stored for 5, 15 and 60 weeks, respectively. The number of leaves per plant was also reduced with increasing duration of cold storage. The size of the flower, however, was not significantly altered. Other points that emerge from this study are that cultivan,origin, prehandling of the bulbs, moisture of the packing medium and temperature of the storage compartment all affect the flowering of the Easter lily. Perhaps the most important point indicated by Brierly's work is that the development of lilies could be advanced or retarded through the manipulation of cold storage conditions. Con- sequently, they could be forced accurately for the Easter market or for other times of the year. Trials have been conducted on various sizes, cul- tivars.and origins of lily bulbs with diverse handling, storage and forcing conditions (Stuart, 1943, 1944, 1946, 1947 and 1952; Merritt, 1963; Smith, 1963, and Wilkins, et al., 1967). In each of these trials an increased dura- tion of cold treatment results in approximately the same trends in flowering response as those shown by Brierly (1941). In addition to the lily, other cold requiring plants are known to respond in a similar manner, for exam- ple: Lolium and gg§_(Purvis and Gregory, 1937; Evans, 1960), gyosgyamus (Lang, 1951), Rapnanus (Kojima, et al., 1954; Tashima, 1957), and Daucus (Runger, 1960). Stuart (1952) studied the feasibility of a succes- sion of flowering dates for Easter lilies throughout the winter. Utilizing a wide range of temperatures (36 - 59°F) and storage lengths (2 — 10 weeks) it was found that, for Creole bulbs, each of the temperatures tested reduced the number of forcing days to blooming over unstored bulbs. Neither temperature nor length of storage was "uniformly proportional" to the reduction in flowering date. To achieve a desirable balance between earliness of bloom and number of flowers he recommended storage at 45 - 50°F for no longer than 6 weeks in length. He stated that: Longer storage produces sprouting and rooting of the bulbs before planting and greatly reduces the number of flowers per plant. For a later flowering date he proposed prolonged storage but only at a temperature of 31°F. This can be carried out only if the bulbs are prevented from drying out during the storage period. In an attempt to determine the cause of the accel- leration of flowering by cold storage, Stuart (1952) analyzed stored bulbs for carbohydrate content. Rapid hydrolysis of insoluble carbohydrates occurs with an accumulation of reducing sugars and sucrose in bulbs stored at 32 - 50°F. This hydrolysis takes place at a much slower rate at increasing temperatures (55°, 59° or 70°F). At 32°F there was the greatest release of sugar, however, the intermediate temperature (50°F) resulted in the earliest flowering. Coville (1920), who investigated the breaking of natural dormancy by chilling in trees and shrubs, suggested that the response to cold was closely related to the con- version of stored starch into sugar. An increase in soluble sugars during the cold storage of lily bulbs could account for the accelleration in growth prior to blooming. Whether this shift in metabolism is directly related to the effect of cold treatment on flowering is still not known. Natural Cooling The natural cooling method of handling bulbs is mainly used in the southeastern and western United States. The bulbs are potted immediately after digging and cooled in an outdoor cold frame. Many workers have found that natural cooling of most cultivars of lilies produces better quality plants than the precooling method (Brierly, 1941; Payne, 1963; Box, 1963; and Langhans and Smith, 1965). Natural cooling at 60 and 50°F produces plants with a shorter forcing period, more flowers and shorter height than precooled bulbs (40°F) for both Ace and Georgia lilies (Payne, 1963). Box (1963) reported similar results and stated that when compared to precooled lilies, natural cooling produces rosetted plants with heavier foliage in the earlier stages. This results in more compact, balanced foliage at anthesis and an increased flower number. Langhans and Smith (1965) reported that, with equal lengths of storage, cold framed Ace lilies produced more flowers than precooled bulbs, but that the Croft variety did not give this response. Controlled Temperature Forcing The advantages of natural cooling over precooling of lily bulbs are evident. However, for certain areas of the United States, natural cooling is not feasible since -the prevailing weather conditions are not dependable. The beneficial effects of natural cooling are believed to arise from the growth of the bulbs which takes place during cold framing (Box, 1963; Payne, 1963). Carlson and De Hertogh (1967) and De Hertogh, et a1. (1969) have allowed planted bulbs to develop for a period at 63°F in the greenhouse before cold storage. It was found that better quality plants were produced. For both Ace and Nellie White lilies, there was an increase in bud count and number of leaves without a significant change in the number of days to flower or plant height at flowering. The best combination evaluated was 3 weeks at 63°F followed by 5 weeks at 38°F. Preheating Before Precooling Blaney, et a1. (1963) investigated the effects of preheating Lily bulbs before precooling. Their results showed that preheating of bulbs at 60°, 70° or 80°F pro- duced leafy plants that were slow to force and had a higher bud count than bulbs which were only precooled. A direct correlation was found between number of leaves and the number of days to flowering. They proposed that the 40°F precooling "acts strongly to stOp the initiation of any organ primordia by the growing point once flower bud initia- tion commences." Therefore, any treatment intended to reduce the forcing time must induce flower bud initiation at a low leaf number. Preheating before precooling, how- ever, tends to increase the leafiness and the time required for forcing. This effect has also been investigated by other workers (Brierly and Curtis, 1942; Stuart, 1946, 1967; a'b). Stuart (1946) found that Miller and Kiplinger, 1966 exposing bulbs to 65°F for a period before cooling resulted in production of more flowers while delaying anthesis in Ace, Croft and Estate varieties. This would be of use in delaying flowering for a late Easter in the case of the more rapidly flowering bulbs such as Croft. Later work (Stuart, 1967) on Southern groWn bulbs (Creole) failed to show a beneficial effect of a 65°F treatment before cold storage. Miller and Kiplinger (1966a) subjected Ace bulbs to a range of temperatures (40° to 70°F) before cold storage (40°F for 0 to 7 weeks). In general, bulbs preheated at increasingly higher temperatures required longer periods of cold storage to bring about flowering in the same number 10 of days. Later (1966b) they proposed that a reversal of "vernalization" takes place in the bulbs at high tempera- tures. Ace bulbs subjected to 6 weeks of "vernalization" (40°F) flower in 120 days, while bulbs given an additional 6 weeks at 70°F following the cold treatment require 160 days. Hence, subjecting bulbs to a period of high temp- erature prior to a low temperature, while generally in- creasing vegetative and reproductive growth, also results in a slowing down of the latter so that flowering is con— siderably delayed in most varieties. On the other hand, subjecting planted bulbs of certain varieties to a high temperature before a low temperature has the beneficial effects of increasing leafiness and flower number without any delay in flowering. Initiation of Flower Primordia Very little work has been done on the timing and factors affecting initiation of flower primordia in Easter lily. Pfeiffer (1935) investigated the varieties Eximium and Giganteum and found with the former, that the first indication of floral initiation occurred during cold stor- age. This was observed as the broadening of the apex. Reproductive growth did not proceed beyond this stage until the bulbs were planted. After planting the floral parts differentiated. 11 Emsweller and Pryor (1963) looked at the time of bud differentiation in relation to forcing, rather than the morphology of the floral axis, and found that buds in Creole lilies were not differentiated during cold storage. It is evident that more work is required in this area before accurate conclusions can be reached regarding the time of induction of the flower primordia in relation to the cold treatment. Gibberellins and Cold Requirement As previously mentioned, the manipulation of cold storage conditions can be used to advance or delay the development of the lily to facilitate flowering for various times of the year. However, cold treatment of lily bulbs is not without problems. Cold storage canihave undesirable effects on plant quality with respect to number of flowers, leaves and in some cases height. In addition, cold storage can be eXpensive, time consuming and sometimes unreliable. It has been shown in many species of plants that naturally occurring growth hormones, the gibberellins, are in some way associated with the requirement for low temper- ature in certain plants (Lang, 1965). The gibberellins are believed by some workers to partially, or, in some cases, fully substitute for the cold treatment required to induce a number of diverse physiological responses such as 12 flowering, breaking of seed and bud dormancy and promoting vegetative growth (Lang, 1965). Gibberellins and Bulbs At present no information is available on the rela- tion of gibberellic acid to the cold treatment of Easter lilies. GA3 (0-2,000 ppm) has been Sprayed on the buds of several varieties of lilies (Croft, Ace and Nellie White) but was found to have no effect on time of flowering al- though keeping quality of potted plants was improved (Kelly and Schlamp, 1964). Similarly, no effect on hastening of flowering was observed by Itakura (1958). In both of these experiments, the floral buds had already formed when the gibberellin was applied. In other bulbous crops, some investigations have been conducted on the relationship of GA to the various cold requirements. Rodrigues Pereira (1962) working with excised buds of Wedgewood Iris, found that a medium containing GA3 had a promoting effect on flower induction as well as the rate of subsequent development of the flower primordia. The effect was also observed if the scale leaves were left attached to the excised bud, but not if the primodial leaves were intact. This suggests the presence of some inhibitory material in the leaves. Halevy and Shoub (1964) injected Wedgewood and Prof. Blaauw Iris with GA3 (50 and 500 ppm) both before 13 and after cold storage. These treatments accellerated flowering. The greatest effect was produced by the later application where an increase of 18 days was observed. GA3 was sprayed directly onto the growing plants in another experiment. This also resulted in accelleration of flow— ering and increased foliage. It should be noted that the 2 plants were sprayed with 10— M GA seven times between 3 emergence and anthesis. Changes in levels of growth substances have also been observed on cooling of bulbous plants. Rodrigues Pereira (1964) investigated the Wedgewood Iris, in which flower buds are initiated during the cold period. During cold storage, differences in levels of growth substances were observed in different parts of the bulb. The amounts in the bud were too low to induce flower formation (in excised buds) but the amounts in the scales were found to be high enough. Furthermore, all the growth substances detected during the cold treatment had already been detected in untreated bulbs. Hence, it is believed that the effect of the cold period in Iris may be to mobilize the flower forming substances, to transport them from the scales to the bud, and to enable the apex to respond to them. The accumulation of the different growth substances in the apex at different rates seems to suggest that each may play a different role in flower formation. An attempt to charac— terize the growth substances studied revealed 3 of the fractions to be physiologically related to the gibberellins. f” 1.-.“. .1... 14 Aung and De Hertogh (1967) detected gibberellin- like substances in Tulipa sp. cultivar Elmus. Non-cold treated bulbs showed little or no activity of gibberellins in a variety of bioassays, but far greater activity was found in cold treated bulbs. The same authors (1968) com- pared the levels of "free" (acidic) and "bound" (neutral) forms of gibberellins in cold and non-cold treated bulbs. The levels of the "bound" hormone were higher than those of the "free" hormone in non-cold treated bulbs and those stored for 4, 5 and 8 weeks at 9°C. After 13 weeks at 9°C, however, the level of "free" gibberellin had increased to a higher level than the "bound" form and the total level of the hormone in the bulbs had increased 370 fold over the level in non-cold treated bulbs. Gibberellins and Other Cold Requiring Plants Promotion of flowering by gibberellin was first observed in biennial Hyosgyamus niger (Lang, 1957). Dilute solutions of gibberellin applied repeatedly to the growing point of non—vernalized Hyoscyamus and other biennials, brought about elongation and subsequent flowering under long-day conditions. Similarly in Centaurium minus (Mc Comb, 1967) application of GA to non-vernalized plants also 3 resulted in elongation and flowering under long-day condi— tions. Thus, GA substituted for the vernalization 3 15 treatment. Plants grown under short days elongated but did not flower, indicating that other factors are involved. This general response was also noted by Ketallap and Barbaroo (1966) for Coreopsis grandiflora. Many other examples of gibberellins inducing or promoting flower formation in non-vernalized cold requiring plants have been reported (Lang, 1956E'Wittwer and Bukovac, 1957; Okuda, 1958; Chailakhyan, 1958; Michniewicz and Lang, 1962; Pereira, 1962 and others). Gibberellins are also known to break dormancy in seeds which normally require a period of cold treatment before germination. Frankland (1961), using dormant seeds of Corylus avellana and Fagus sylvatiga found that seeds germinated within 3 weeks on GA but very few seeds ger- 3 minated in distilled water. GA application has also been shown to break dor- 3 mancy in buds of Azalea without the chilling requirement being met (Ballantry, 1966). Likewise, it will substitute for all or part of the chilling requirement necessary for normal Spring growth of rhubarb (Tompkins, 1965; Krause, 1967). As in bulbs, isolation of gibberellins from other cold requiring plants indicates that changes in levels of the endogenous growth substance occur during cold treatment, with substantial differences between cold treated and non cold treated plants (Harada and Nitsch, 1959; Harada, 1960; 16 Lang and Reinhard, 1961; Chailakhyan, et al., 1963; Frank— land and Wareing, 1962). Harada and Nitsch (1959) found quantitative and qualitative changes occurring in the pattern of endogenous growth substances during flower induction and flower devel- opment of Chrysanthemum morifolium after vernalization. Later work by Harada (1960) showed an increase in gibber- ellin-like substances during cold treatment of cold requir- ing Chrysanthemum and Althea. Lang and Rheinhard (1961) obtained evidence that cold induced plants of Hyoscyamus contain slightly more gibberellin than vegetative plants. Chailakhyan, et a1. (1963) also demonstrated that plants derived from vernalized Lolium and Elmyus contained more growth substances than the unvernalized controls. Chailakhyan and Lozhnikova (1962) found an increase of endogenous gibberellins in thermo- induced Lolium and Brassica napus under long day conditions. There is also evidence that cold treatment of dormant Corylus avellana results in higher endogenous levels of gibberellin—like substances (Frankland and Wareing, 1962; Ross and Bradbeer, 1968). In Fagus sylvatica, the same authors found qualitative but not quantitative differences in gibberellins in chilled and unchilled seeds. PART I INTRODUCTION This portion of the dissertation involved an inves- tigation into the changes in levels of endogenous gibber- ellins (GA) in the Lily bulb during low temperature treatments. Two different methods of applying the cold treatment were compared, precooling (PC) and controlled temperature forcing (CTF). At present, precooling is the more popular method of forcing. Controlled temperature forcing is a method currently under investigation at Michigan State University (De Hertogh, Carlson and Kays, 1969). 17 MATERIALS AND METHODS Source of Experimental Material Lilium longiflorum,Thunb., cv. Ace, size 7-8 inch, were provided by the United Bulb Company, Mount Clemens, Michigan. They were planted in Smith River, California, as yearlings in fall 1966, dug on September 22, 1967 and packed immediately. Transport was by truck to East Lansing, Michigan where they arrived on October 26, 1967. Cultural Conditions On arrival, the bulbs were divided into two groups. 80 bulbs for precooling were left in their original packing cases in a medium of moist peat moss and immediately placed at 40°F for 8 weeks. 80 bulbs for the controlled temper- ature forcing were potted immediately, watered and placed in the greenhouse pot to pot for 3 weeks at 63°F. This was followed by 5 weeks in cold storage at 40°F. The dates for the two methods are represented in Figure 1. All bulbs were planted in 6 inch plastic pots. One inch of pea gravel was placed in the bottom of the pot then 1 inch of soil mix and the bulb placed on this. The pot was then filled with a 1:1:1 mixture of steam sterilized 18 19 can Qmumom woe .>o ..nccca mmuoauwmqmw ESHHHA no we mcwouom mucunummfiwu Umaaouuc . Hon 0 use 00 How mmucu m .Hooowum mummfiwu can mwumoll.H wusmam Hm .Uma ezmzedmms halo hoov mw¢mOBw CHOU ma .>OZ mmDom rm 0 M m IZWWMHU mm .900 BfiOQmdeB NN .Bmmm Qm>Hmm¢ QZ< MOdMOBm ODD mace moamosm oqoo Dmumom BZMEBGMMB UZHHOOUmmm HN .UMO 20 sandy loam soil, sphagnum peat moss and a calcine clay, "Turface." Following the cold storage the plants were trans- ferred to the greenhouse and the PC bulbs potted. The greenhouse temperatures were maintained at 65° to 70°F in the day and 63°F at night. Natural photOperiod was utilized and plants were watered as required. On the greenhouse benches, the pots were placed 4 per row with 12 inches from center to center. The two treatments occupied alternate rows on the benches. The plants were fertilized with a mixture of 25-0-25 at a rate of 2—1/2 pounds per 100 gallons of water. This was applied at weekly intervals from January to March. During the last 4 weeks, it was supplemented with a trace element mixture. A solution of magnesium sulphate, 1/2 pound per 100 gallons of water, was applied once in early January. Both treatments received an application of 'Dexon' and 'Terraclor' at 8 and 4 ounces per 100 gallons of water respectively. This was applied as a soil drench shortly after the low temperature treatment (December 26). Isolation and Purification of gibbereIIin-like Substances Sampling.--Samples of the bulbs or plants were removed, on the dates outlined in Table 1, for extraction of the endogenous gibberellins. Each sample consisted of 21 7 plants randomly selected. Sample number one consisted of bulbs flown from Smith River, California directly after digging. Seven of these bulbs were used for the sample. Samples number 4A and 4B consisted of the bulbs plus any plant parts which had developed. Extraction.--Prior to extraction each bulb was washed in running distilled water, separating the leaves, stems, scales and roots and discarding any tissue which was diseased or decayed. These parts were blotted dry and the weight of the combined tissues measured. The tissue was then frozen with liquid nitrogen and homogenized in a Wareing Blender using 3 parts absolute methanol to 1 part of tissue (v/v). The homogenate was then transferred to 1 liter erlenmeyer flasks, an equal volume of methanol added and shaken for 48 hours at room temperature. Table 1.--The dates of removal of samples of PC and CTF bulbs of Lilium longiflorum, Thunb, cv. Ace for extraction of gibberellin:1ike growth substances Sample Date of No. Sampling Method Stage of Development 1 25/Sept./67 - 3 days after digging 2-A 16/Nov./67 PC after 3 weeks at 40°F 2-B CTF after 3 weeks potted at 63°F 3-A 21/Dec./67 PC after 8 weeks at 40°F 3-3 CTF after 5 weeks at 40°F 4-A 30/Jan./68 PC after 6 weeks forcing 4—3 CTF after 6 weeks forcing 22 The methanol extract was filtered under vacuum using a 'Celite' pad and Whatman No. 3 filter paper. The residue was washed and then discarded. The filtrate was evaporated ig_ggggg. The remaining aqueous extract was subsequently lyOphylized. Purification and separation.-— 1. "Free" gibberellin fraction: The dried residue was suSpended in equal volumes of petroleum ether and potassium phosphate buffer, pH 8.0, shaken and allowed to stand overnight. The buffer phase was washed 5 times with petroleum ether and the petroleum ether was washed 5 times with buffer. The buffer phases were combined and the petroleum ether phase discarded. The buffer phase was then washed 3 times with ethyl acetate and the ethyl acetate phase was washed 3 times with buffer. The ethyl acetate phase was discarded. The combined aqueous phases were adjusted to pH 3.0 with concentrated HCl and extracted 5 times with ethyl acetate. The aqueous phase was stored for extraction of the "bound" GA. The ethyl acetate was refrigerated at -18°C for at least 24 hours. The ice was then filtered off under vacuum using a frozen Buchner funnel and Whatman No. 1 filter paper. The water-free ethyl acetate solution of "free" GA was evaporated to dryness in vacuo at 38°C. 23 The residue was then taken up in 20 m1. of absolute methanol and stored at room temperature until the bioassays were carried out. 2. "Bound"_gibberellin fraction: The aqueous phase, withheld after washing with ethyl acetate at pH 3.0 was adjusted to 0.04N HCl and hydrolyzed for 45 minutes at 60°C. After cooling, the aqueous phase was readjusted to pH 3.0 with 5N KOH and extracted 5 times with ethyl acetate. The aqueous phase was discarded and the ethyl acetate phase handled as previously described for the "free" GA fraction. Preparation of sample for bioassay.-- l. Chromatggraphy: 5 ml. of each sample was evap- orated to dryness ig.zaggg. The dried sample was then dissolved in a small volume of absolute methanol to facil- itate preparation of the chromatogram. The sample was then evenly streaked across a strip of chromatography paper (Whatman No. 3 MM) 10 cm. wide by 50 cm. long (from point of application to the solvent front). The solvent consisted of a mixture of isoprOpanol, NH4OH and H20 (10:1:1) and the decending method of separation was utilized. The chromato- gram.was developed for 10-12 hours and then dried. 2. Elution: The chromatogram was divided into 10 equal fractions and each was eluted with 10 ml. of absolute methanol for 24 hours at room temperature. The methanol was decanted off into 10 test tubes and evaporated to 24 dryness on a rotary evaporator. Each fraction was dissolved in 2.0 ml. of a solution of "Tween-80" (0.05% v/v). Dwarf Pea bioassgy.-— l. Assay Plant: Piggm sativum L., var. Progress No. 9 was purchased from Asgro Seed Company. The seeds were initially soaked for 12 hours in running tap water and subsequently drained and treated with "Captan." Uni- formly imbibed seeds were planted in vermiculite in 5x4x2 inch plastic containers. The seeds were planted at 1/2 inch depth with 12 seeds per container in rows 4 by 3. Ten plastic containers were placed in a flat and each con— tainer was partitioned from the others with aluminum foil. The peas were germinated in a growth chamber in the dark at 78°F. After 5 days they were given continuous red light using 40 watt red flourescent lamps at a height of 9 inches above the containers. The plants were watered as needed. 2. Preparation of standards: The standards were prepared from 95% active powdered GA3. Standard solutions 1 -2 -3 of 10' , 10 , 10 and 10’4 ug/ml. GA in 0.05% "Tween-80" 3 were prepared. A control solution of 0.05% "Tween-80" was used. 3. Application: Eight of the most uniform plants were selected from each container and the rest were dis- carded. 0.08 ml. of the test solutions were applied per Plant per day for 3 consecutive days. Applications were normally made on the 7th, 8th and 9th days after planting. 25 The solution was applied to the first trifoliate leaf. A total volume of 2 ml. was added to the plants in each con- tainer for both samples and standards. 4. Collection of data: On the eleventh day after planting, plant height was measured in cm. from the point of application to the growing tip. RESULTS Quantitative Changes_in Endogenous Gibberellin-like Substances As measured by the response of dwarf peas to puri- fied extracts of the bulbs, both methods of cooling (PC and CTF) resulted in a decrease in the activity of endogenous gibberellin-like substances during cold storage (Fig. 2, 3, 4 and 5). In addition, the data offers no evidence that the activity begins to build up again after the end of cold storage and during the first 6 weeks of greenhouse forcing. This general decrease is found to occur in both "free" and "bound" fractions, although there is evidence (Table 2) that the activity of "bound" is always higher than that of "free.“ The extracts from CTF bulbs show a higher activity of gibberellin-like materials than PC bulbs throughout the stages of growth studied here. Some decrease of GA-like activity is observed in CTF bulbs during the period of growth prior to cold storage, but this decrease is not as great as that observed after the 3 weeks cold storage of PC bulbs. After 5 weeks cold storage of CTF bulbs the activity of gibberellin-like substances is still higher than in PC bulbs stored for 3 weeks or 8 weeks (Table 2). 26 27 Figure 2.--Histograms showing promotion or inhibition of stem growth of Pisum sativum L. cv. Progress No. 9 by eluants from chromatogram strips of extracts of "free" gibberellin-like substances from 7 PC bulbs of Lilium longiflorum, Thunb., cv. Ace at different growth stages: A. at dig- ging, B. after 3 weeks cold, C. after 8 weeks cold, and D. after 6 weeks forcing. Histograms have not been corrected for fresh weight. n of ass of ances , 1.5 MM] : d1:- 1 36.15 -| If .graEé CM. GROWTH ABOVE CONI’IOL CM. GROWTH AIOVI CONYIOL 29 Figure 3.-—Histograms showing promotion or inhibition of stem growth of Pisum sativum L. cv. Progress No. 9 by eluants from chromatogram strips of extracts of "bound" gibberellin-like substances from 7 PC bulbs of Lilium longiflorum, Thunb.: cv. Ace at different growth stages: A. at dlg" ging, B. after 3 weeks cold, C. after 8 weeks cold, D. after 6 weeks forcing. Histograms have not been corrected for fresh weight. on of ress s of stances mm, It dig- reeks ms liar: CM. GROWTH AIOVI CON‘I’ROI. 32345670910 H COM. GROW"! ABOVE CONTROL 31 Figure 4.--Histograms showing promotion or inhibition of stem growth of Pisum sativum L. cv. Progress No. 9 by eluants from chromatogram strips of extracts of "free" gibberellin-like substances from 7 CTF bulbs of Lilium longiflorum, Thumb-r cv. Ace at different growth stages: A. at dig- ging, B. after 3 weeks growth, C. after 5 weeks cold, and D. after 6 weeks forcing. Histograms are not corrected for fresh weight. .on of {res s of tans-5 T111131.) at di; 5 week: :ogrars GROWTH AIOVI CONTROL CM. GROWTH AIOVI CONTROL CM. Rf Rf '- . a . Io . c o . . I t o 'n u 'o c . 0. .- I: '- I. '- t. 'u u 0', n. o c- o u. '- u n a o. .- a. II. . v D ” I.O I O 3:}: ,3. .5,- 50.... I... l . . Vl‘io - .s s .o o ..'-O:I:I:: 33 Figure 5.--Histograms showing promotion or inhibition of stem growth of Pisum sativum L. cv. Progress No. 9 by eluants from chromatogram strips of extracts of "bound" gibberellin-like substances from 7 CTF bulbs of Lilium longiflorum, Thunb., cv. Ace at different—Mb stages: A. at dig- ging, B. after 3 weeks growth, C. after 5 weeks cold, and D. after 6 weeks forcing. Histograms are not corrected for fresh weight. .a O I .- 3: U u 3 .2 ( 3 .- 3. I Lon of 9 [ress - )5 Of 50 Sim 12345070910 T's-:9. at ii? m/mL. toga? GROWTH ABOVE CONTROL CM. 35 Table 2.--Shanges in fresh weight and levels of endogenous free" and "bound" gibberellin in PC and CTF Lilium longiflorgmtThumb. cv. Ace at different deveIopmenta stages I Fresh ug GA3 equivalents/kg Weight fresh weight Sample in kg "Free" "Bound" 1 At digging 2.020 .280 .451 Precooled 2-A After 3 weeks cold storage (40°) 0.700 .049 .044 3-A After 8 weeks cold storage (40°) 0.666 .004 .038 4-A After 6 weeks forcing 0.950 Not Detectable .014 CTF 2-B After 3 weeks growth (63°) 0.724 .174 .277 3-B After 5 weeks cold (40°) 0.729 .102 .138 4-B After 6 weeks forcing 1.270 .071 .123 Qualitative Changes in Endogenous Gibberellin-like Substances Little qualitative changes appear to be taking place in the growth-promoting substances detected in lily- bulbs during the time period studied. Peaks are generally observed in the Rf zones 1-2, 5-6 and 10 (Figs. 2, 3, 4 and 5). However, in PC bulbs, substances which inhibited the growth of the dwarf pea plant were detected after 3 weeks 36 cold storage of the bulbs (Fig. 2 and 3). These inhibitory substances were also detected after 8 weeks of cold storage and after the first 6 weeks of greenhouse forcing. 37 Figure 6---Standard curve of growth response of Pisum m L. cv. Progress No. 9 to various con- centrations of GA3. Response was measured as cm. growth of the treated stems above control. CM. GROWTH ABOVE CONTROL 4 5 Loa‘o GA3 CONC. (HO/ML.) DISCUSSION Several workers have noted an increase in gibber- ellin-like materials upon cold treatment of cold requiring plants, suggesting that gibberellins may be in some way involved with the function of the cold treatment (Harada, 1960; Lang and Reinhard, 1961; Frankland and Wareing, 1962; Chailakhyan and Lozhnikova, 1962; Aung and De Hertogh, 1967, 1968). In Easter lily, however, the concentrations of endogenous gibberellins decreased when the bulbs were stored at a low temperature. This was true for both "bound" and “free“ fractions, although the former is generally present at a higher concentration than the latter. From the previous work it would be anticipated that, if gibber- ellins are functioning more or less directly in the cold response of the lily, a similar increase in activity would have been observed. This reversal of the trend may suggest that the concentration of gibberellins in the system is only an indirect effect of the cold treatment. If an indirect relationship is postulated there are certain explanations which might account for this observed decrease in gibberellins during cold storage. 39 40 1. There may be a conversion or a breakdown of gibber- ellins during cold storage to either non-gibberellin— 1ike materials or to other gibberellins that are not detected by this assay, e.g., GA5 (Kende and Lang, 1964). 2. Stuart (1952) stated that hydrolysis of carbohy— drates takes place during cold storage of lily bulbs. It is known that GA3 will induce the syn- thesis of certain hydrolases known to be active in hydrolysis of carbohydrates (Dahlstrom and Sfat, 1961). It is possible that gibberellins are being utilized for this purpose during cold storage of lily bulbs, without the synthesis of gibberellins occurring. 3. Sprouting of the bulbs was observed during cold storage of PC lilies but very little root growth had occurred during this period. GA3 appears to have a very close relationship with stem elongation as seen in its induction of bolting in many plants (Brian et al., 1954, Lang, 1956b). Gibberellins could be utilized in the shoot growth which occurred at this time in the lily bulbs. It has been suggested that the "bound" gibberellins may be the reserve form of the hormone (Hashimoto and Rappaport, 1966). In lily bulbs at the growth stages studied, the "bound" levels of gibberellin-like substances 41 are higher than the "free," especially in CTF bulbs. But the physiological significance of this is not known. CTF lilies are generally of higher quality than PC lilies, with more flowers and leaves per plant (Carlson and De Hertogh, 1967; De Hertogh, et al., in press). It is believed that the beneficial effects of CTF arise from the period of growth before cold storage. During this period there is a decrease in activity of gibberellins but this decrease is not as marked as that in PC bulbs during the synchronous period (i.e., 3 weeks cold storage). In addi- tion, CTF bulbs remain substantially higher in gibberellins throughout the growth stages studied. The level in the CTF bulbs is approximately 1/3 or higher than in PC bulbs after 6 weeks of forcing (the lowest point reached in both). Consequently, this may be considered as a possible contri- buting factor in the beneficial effects of the CTF method. On removal from cold storage to the greenhouse forcing condition, the total level of gibberellins in the plant does not appear to change after the first 6 weeks of forcing. At this stage, substantial rooting occurred and the shoots had emerged well above soil level. While the gibberellin level in the entire plant did not appear to change during this time, the concentrations in various parts of the plant may have shifted. That is, the physio- logical importance of the total level of gibberellins 42 within the system may be of only secondary importance to the concentration or balance within one specific regulatory region or site of action. The same would be true for the inhibitory substances detected. Since the whole plant was extracted, inhibitors present in the extract may be from a specific part of the plant and will consequently mask the activity of any gib- berellins from other parts of the plant. However, it should be pointed out that these sub— stances are inhibitory to the dwarf pea growth and it is not known whether they act as inhibitors in the lily or are caused as artifacts of the extraction procedure. Hence, the physiological significance of the inhibitors is not known. Dwarf peas, when grown in the light, are insensi- tive to certain gibberellins, namely GA5 (Kende and Lang, 1964); consequently, the complete picture may not be rep- resented here. Also, the sensitivity of the dwarf pea to inhibitors may entirely mask a substantial portion of the gibberellin response. While the assay of_gibberellins in this way gives some indication of the activity of the growth promoting substances present in plant extracts, the limits of the system employed here should be kept in mind. SUMMARY 1. As detected by the dwarf pea bioassay activity of gibberellin-like substances decreased during cold storage of PC and CTF Easter lilies. There is a marked increase in the activity of inhibitory substances in PC bulbs during cold storage. 2. The quantity of "bound" gibberellins is generally higher than that of "free" gibberellins at any growth stage studied. 3. In subsequent studies, extraction of gibberellins from individual parts of the plant, rather than the whole plant, would probably be more beneficial in determining the relation of gibberellins to the cold requirement. In addi- tion other bioassays should be employed. 43 PART I I INTRODUCTION This portion of the study was to determine the effect of exogenous applications of gibberellic acid (GA3) on the flowering parameters of the Easter lily. Gibberellin was applied to the bulbs before and after the cold storage period. The controlled temperature forcing method was utilized. 44 MATERIALS AND METHODS Source of Experimental Material Lilium longiflorium,Thunb., cv. Ace, size 7 to 8 inches, were provided by the United Bulb Company, Mount Clemens, Michigan. They were planted in Smith River, Cal- ifornia as yearlings in Fall, 1966, dug on September 22, 1967 and packed immediately. Transport was by truck to East Lansing, Michigan, where they arrived on October 26, 1967. The gibberellic acid was supplied by the Amdal Company of Chicago in the form of a 2% liquid concentrate, "Pro-Gib." Cultural Conditions All bulbs were planted on arrival in 6 inch plastic pots and grown pot to pot in the greenhouse for 3 weeks at 63°F. The bulb was placed on 1 inch of soil mix on 1 inch of pea gravel in the bottom of the pot and the pot was then filled with a 1:1:1 mixture of steam sterilized sandy loam soil, sphagnum peat moss and a calcine clay "Turface." Following 3 weeks in the greenhouse, the bulbs were Placed in cold storage for either 3 or 5 weeks at 40°F. 45 46 Treatments 8, 9, 10, 11 and 12 were stored for 3 weeks and treatments 1, 2, 3, 4, 5, 6 and 7 for 5 weeks (Table 3). Table 3.--GA3 treatment of controlled temperature forced Lilium longiflorum, Thunb., cv. Ace _-f w Treatment number Description 1 weeks potted at 60°F, 5 weeks at 35°F 2 weeks potted at 60°F, GAgslo ppm, 5 weeks at F 3 weeks potted at 60°F, GA3 100 ppm, 5 weeks at 35°F 4 weeks potted at 60°F, GA3 1,000 ppm, 5 weeks at 35°F 5 weeks potted at 60°F, 5 weeks at 35°F, GA3 10 ppm 6 weeks potted at 60°F, 5 weeks at 35°F, GA3 100 ppm 7 weeks potted at 60°F, 5 weeks at 35°F, GA3 1,000 ppm 8 weeks potted at 60°F, 3 weeks at 35°F 9 weeks potted at 60°F, GAgslo ppm, 3 weeks at F 10 weeks potted at 60°F, GA3 léggg ppm, 3 weeks at 11 weeks potted at 60°F, 3 wgegzmat 35°F, GA3 12 weeks potted at 60°F, 3 weeks at 35°F, GA3 1,000 ppm Following cold storage, all bulbs were transferred to the greenhouse where temperatures were maintained at 47 65°F to 70°F in the daytime and 63°F at night. Natural photoperiod was utilized and the plants were watered as required. Four pots were placed per row on the greenhouse benches with 12 inches between centers. The plants were fertilized with a mixture of 25-0—25, at a rate of 2-1/2 lbs. per 100 gallons of water, at weekly intervals from January till March. During the last 4 weeks, this was supplemented with a trace element mixture. A solution of magnesium sulphate, 1/2 pound per 100 gallons of water, was also applied once in early January. After removal from cold storage all treatments received an application of "Dexon" and "Terraclor" at a rate of 4 and 8 ounces per 100 gallons of water, respectively. Treatments The treatments are described in Table 3. The first application of gibberellin was applied either immediately before cold storage (treatments 2, 3, 4, 9 and 10) or imme- diately after cold storage (treatments 5, 6, 7, 11 and 12). A solution of 200 ml was applied to each pot as a soil drench. The concentrations used were 0, 10, 100 and 1,000 ppm on bulbs receiving 5 weeks of cold storage (treatments 1 through 7) and 0, 10 and 1,000 ppm on bulbs receiving 3 weeks of cold storage (treatments 8 through 12). All 48 treatments, with the exception of controls (0 ppm) received a second application of gibberellic acid corresponding to the first application on January 7, 1968. Experimental Design A randomized block design was utilized with 4 rep- lications and 6 observations per treatment. It should be noted here that the temperature in the southeast corner of the greenhouse was 3° lower than that of the rest of the greenhouse. This portion encompassed treatments from replication 2 and replication 3. As a result of this, the date of flowering was considerably delayed in these treatments. Hence, analysis of this parameter was performed on replications l and 4 only. No other parameters were affected by this temperature difference. Collection of data Data were recorded of the date of Opening of the first flower, the date of opening of the last flower, the number of flower buds and leaves produced by each plant and the pedicel height at first flower. From these data the following parameters were calculated: (1) Number of forcing days to flowering (2) Number of flower buds per plant (3) Number of days in flower (4) Number of leaves (5) Height of first flower 49 The number of forcing days to flowering was measured from the day the bulbs were removed from cold storage until the opening of the first flower. The number of days in flower was calculated from the date of opening of the first flower to the date of Opening of the last flower. Height was measured in centimeters from the soil level to the base of the pedicel of the lowest flower. The number Of leaves from soil level to the base of the pedicel of the lowest flower was counted. Analysis of results Analysis of variance was conducted on the means of 6 observations for each treatment and each replication, using STAT series program No. 14 of the Michigan State University Computer Laboratory (analysis of variance with equal frequency in each cell; RAND routine for one—way plus replicate design). The following orthogonal comparisons were then made on the means of 6 observations and 4 replications for each treatment for each parameter: 1. GA3 application versus no GA3 application, overall. 2. 5 weeks versus 3 weeks cold storage when no GA3 is applied. 3. Application before versus application after cold storage between 10 and 1,000 ppm GA3. 4. 5 weeks versus 3 weeks cold storage between 10 and 1,000 ppm GA3. 5. Interaction between 3 and 4 above. 50 6. GA3 1,000 ppm versus 10 ppm. 7. Interaction between 6 and 3 above. 8. Interaction between 6 and 4 above. 9. Interaction between 6 and 3 and 4 above. 10. GA3 1,000 ppm versus no GA3. The last comparison (number 10) is not orthogonal with the previous comparisons. RESULTS Number of forcingdays to flowering GA3 does not appear to have any direct effect on the number of forcing days to flowering (Fig. 7). However, the orthogonal comparisons (Table 6) show a significant difference to occur between 10 and 1,000 ppm GA3. It can be seen from Figure 7 that the lower concentration (10 ppm) when applied after cold storage, causes a reduction in number of days, whereas 1,000 ppm increases the number of days to flowering. The overall effect, as shown by compar- ing 0 versus 1,000 ppm and no GA versus GA, is not significant. Increasing the cold storage period brings about a pronounced reduction in the number Of days to flowering. This parameter was analyzed using replications l and 4 only since the temperature gradient in the greenhouse (mentioned earlier) delayed flowering considerably in cer- tain treatments of replications 2 and 3, thus making them invalid (Tables 4 and 5). Hmeer Of flower buds per plant Increasing concentrations of GA3 resulted in pro- gressively fewer buds per plant, as illustrated in Figure 8 51 58 Figure 8.--Effect of 4 concentrations of GA3 applied before and after the cold treatment to planted Easter lily bulbs on the number Of buds produced. TWO lengths of cold treatment were used. A. GA3 applied before 3 weeks cold storage, B. GA3 applied after 3 weeks cold storage, C. GA3 aP" plied before 5 weeks cold storage, D. GA3 aP" plied after 5 weeks cold storage NUMBER OF 3005 TO I 2 LOGIO GA3 CONCENTRATION (PPM) 52 Figure 7.--Effect of 4 concentrations of GA3 applied before and after the cold treatment to planted Easter lily bulbs on the number of forcing days to opening of the first flower. Two lengths 0f cold treatment were used. A. GA3 applied before 3 weeks cold storage, B. GA3 applied after 3 weeks cold storage, C. GA3 applied before 5 weeks cold storage, D. GA3 applied after 5 weeks cold storage. 60 and supported statistically in Table 8. The strongest effect was from application of 1,000 ppm GA3 after 5 weeks cold storage (Fig. 8). Increasing the length of cold storage also resulted in the production of fewer buds per plant. It should be noted here that 100% of the buds on all plants developed into flowers. Height Application of GA3 to Easter lily bulbs results in an increase in height of the plants as recorded at blooming time. This is illustrated in Figure 9 and supported sta- tistically in Tables 9 and 10. Again the strongest effect is obtained when 1,000 ppm GA3 is applied after 5 weeks cold storage (Fig. 9). Increased duration of cold storage does not appear to have any meaningful effect on height Of the plants. Number of leaves per plant GA application does not appear to affect the 3 number of leaves per plant (Fig. 10 and Table 12). On the other hand, the length of cold storage has a pronounced effect on leaf number. Increasing the length of cold storage tends to decrease the number of leaves per plant. 54 parchmeamem segment. homa.aamv he anuoa mmmm.HH wmom.ehm mm Houum tamooo.ov mvoa.am mvmm.mmm hmsm.mmmm Ha ucoEummHB «amooo.ov oom~.oa mmvm.maa hmeo.mvm m downscaammm mocnowmwcmflm oeumflunum m mumsvm com: mounswm Eoomouh mocnflun> museflxoummd no How mo mmmumwo mo condom mcowunomamms Ham means cmwmmo xopo Umuflfioocmm .mod .>o ..bcssB.EouonflmcoH EOHHMA mo mcflumzoam on what mcfiouom mo Hones: MOM OHQOB mocnwuo> mo mammamcdll.v manna 55 pamoameamem segment. mmm¢.mmNN MN HMUOB momw.m mmmm.mm Ha HOHHm asmooo.ov mooa.hh whmo.mom mmom.NNNN AH ucmfiummua mmo.o moon.v hmmm.NH hoNM.NH H GOHHMOHHmmm OOOOOMMHcmHm oaumflunum m muoowm com: mmumowm Eoommum mocmaum> mumfiflxoummm mo Edm mo mmmnmmo mo condom mcflmo cmfimmo xopo oonwfioocnm .mo¢ Mano e can a mOOMOMOHHme .>o ..oc5£B_EsuoHMHmcoH ESHHMA mo meanmsoam 0» want OOHOHOM mo menace How manna mocnwun> mo mammamc¢||.m manna va.o mm mmmN.H H+ H+ HI HI HI HI H+ H+ m>onn H can m consume coHponHmucH m trees.emm mm eHm.mmeH HI HI HI HI H+ H+ H+ H+ sod ooo.HIOH consume monsoon oHoo mxoms m .> mxmms m e msm.o mm Hom.H HI HI H+ H+ HI HI H+ H+ mac and ooo.H can oH comSumb Hopmn .> OHOMOQ OOHHOOHHmmd m ««me.MMH MN mm.mvm HI H+ OOHHQQO mHmco oc cons monsoon oHoo mxmms m .> mxmms m N ovm.m mN eNm.OH HI HI HI HI m+ HI HI HI HI HI HI m+ HHmHmbo cOHUOOHHmmn mew on .> OOHNMOHHQQO mew H 6 5 UHumHumum mo mumovm U. 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I. 0 8 T. T. to .7 7» cc 0 C._ 0 CH 0 O CH C.. S 0 CH 0 NH HH OH O O O O O O m N H ”when: namsummue OH OH OH OH O OH OH OH OH OH OH O and OH m m m N m N mdw OOHumuucmocoo OOHHOOHHQQO sauna muommm “mums mnommm «o no msHe mmmnoum mxwo3 m mxoms m pHoo mo mxmmz poocHUGOUII.m OHQOB DISCUSSION The effects of increasing lengths of cold storage on flowering of Easter lilies have been investigated by several workers (Brierly, 1941; Stuart, 1946, 1947, 1952; Merritt, 1963; Smith, 1963; Wilkins et al., 1967). The data from this investigation agree with their results and with those of De Hertogh,et a1. (1969) using the controlled temperature forcing method. Increased length of bulb cold storage decreased the forcing time to flowering, the number of flower buds per plant and the number of leaves produced. The height of the plants, measured at flowering, was not affected. On the other hand, gibberellic acid has no effect on the number of days to flowering and the number of leaves produced, but decreased the bud count and increased the height of the plants. Although the decrease in bud count brought about by application of GA3 has little, if any, commercial impor- tance, it is interesting to note that the applied chemical appears to "mimic" the effect of cold storage in this reSpect. Increasing the length of cold storage from 3 to 5 weeks decreases the number of buds from 9.4 to 7.8. 74 75 Application of GA3 (1,000 ppm) reduces the number of buds from 9.4 to 7.0 and from 7.8 to 5.1 in bulbs stored for 3 weeks and 5 weeks, respectively. The decrease in bud count by GA3 is sequential with increasing concentrations of GA3. Reduction in flower number by exogenously applied GA3 has been noted in many plant species, for example, Lycopersicon (Bukovac,et al., 1957); Prunus (Bradley and Crane, 1960); Malus (Guttridge, 1962); Euphorbia (Guttridge, 1963). However, the question of the relation of gibberellic acid to the cold requirement in this experiment must be considered in the light of certain facts. To implicate the function of a natural plant hormone in a physiological process, one must perform the experiment using concentra- tions of the hormone at, or near, the physiological levels. Greater concentrations may merely induce a pharmacological response. In this experiment, since the hormone was ap- plied as a soil drench and the actual amounts entering the plant are not known, the levels of application were quite high; 1,000 ppm (3.5 x 10‘2M), 100 ppm (3.5 x 10‘3M) and 10 ppm (3.5 x 10-4M), to counteract any colloidal binding and leaching. Consequently the possibility of the induc- tion of a pharmacological response cannot be eliminated. The possible relationship between cold and GA is also weakened by the fact that this trend is noted for only one of the several parameters that is normally affected by cold treatment. On the other hand, only GA3 was applied in 76 this experiment, consequently it is possible that one, or several other, gibberellins may be involved. Therefore, the lack of response of other parameters (for example, number of days to flowering) might be explained on the grounds of specificity of the different gibberellins (Brian, et al., 1962; Michniewicz and Lang, 1962; Bentley— Mowat, 1966). It is interesting to note that GA3 (1,000 ppm) ap- plied after 5 weeks cold storage had a very strong effect in reducing the bud count and increasing the height of the lilies. This effect of time of application only occurs with the highest concentration Of the hormone used (1,000 ppm) and only when it is applied to bulbs receiving 5 weeks of cold storage. However there is no valid test for time of application in this experiment since the nature of the study required a second application of GA3 to be made on the same date to all plants that had already received GA3 either before or after cold storage. Hence the enhanced effect of the GA3 applied after 5 weeks cold storage may be due to the closeness of the first and second applications. In all other treatments the time of application had no effect on the number of buds and height of the plants. This suggests either that the time of application is unim- portant or that the second application was more important in bringing about the effect. 77 Gibberellic acid induced an increase in height of the lilies as measured at the time of flowering. This effect has been observed in several other plants (Brian, et al., 1954; Kato, 1955; Lang, 1956b; and Marth, et al., 1956). Height control of lilies is a difficult problem generally solved by forcing practices (Kohl, 1958; Stuart, 1961). For potted lilies a compact plant is the most de— sirable. Hence an increase in height, without an increase in the number of leaves, as Observed here, would not be of commercial advantage. SUMMARY 1. Increasing levels of GA3 (from 0 to 1,000 ppm), applied as a soil drench to Easter lilies resulted in a progressive decrease in the number of flower buds produced per plant. 2. GA3 had the same effect as cold treatment in reduc- ing the number of flower buds per plant, but did not effect the number of forcing days to flowering or the number of leaves per plant. 3. Increasing concentrations of GA3 progressively increased the height of the plants at flowering time. 78 LITERATURE CITED LITERATURE CITED Aung, L. H. and A. A. De Hertogh. 1967. Detection of gibberellin-like substances in tulip bulbs (Tulipa Sp.). Plant and Cell Physiol. 8: 201-205. . 1968. Gibberellin-like substances in non-cold and cold treated tulip bulbs (Tulipa Spp.). 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