128 001 THS WHESK: 5382 w ‘293 10547 W LIBRAR Y Michigan State litiiRNRHRQy .' ‘W‘ v- «WW-1‘ _ This is to certify that the thesis entitled A Non—Destructive Assay for Gibberellin Production in vitro and its Application to Callus Cultures of Phaseolus and Nicotiana presented by Mrs. Barbara J. Thompson has been accepted towards fulfillment of the requirements for M.S. Horticulture degree in Major professor Date December 22, I980 0-7639 - fl ,/ OVtRDUt #- th3: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records A NON-DESTRUCTIVE ASSAY FOR GIBBERELLIN PRODUCTION IN VITRO AND ITS APPLICATION TO CALLUS CULTURES OF PHASEOLUS AND NICOTIANA By Barbara Lake Thompson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1980 ABSTRACT A NON-DESTRUCTIVE ASSAY FOR GIBBERELLIN PRODUCTION IN VITRO AND ITS APPLICATION TO CALLUS CULTURES OF PHASEOLUS AND NICOTIANA By Barbara Lake Thompson The barley half-seed halo assay was adapted for use as a non—destructive test of gibberellin (GA) content of both callus tissues and culture media. Sterile barley half—seeds were placed in direct contact with callus cultures or media for 24 hours. Gibberellins in the callus or media stimu- lated synthesis of a-amylase in the half-seeds. Seeds were transferred aseptically to starch plates for 72 hours. The plates were flooded with KI/12 solution, whereupon clear halos appeared around the half-seeds where the starch had been digested by the a-amylase. Factors affecting the halo diameters after exposure to GA3 standards were determined. Using these methods a study was made of the gibberellin con- tent of Phaseolus vulgaris and Nicotiana forgetiana tissue cultured in yi££g_to determine the feasibility of using callus as a source of gibberellin production. Callus cul- tures were tested during initiation and subsequent subcul- tures. Both calli produced only small quantities of gibber- ellins in_vitro. ACKNOWLEDGMENTS Special thanks to my husband, Joel, for his love and support, to Dr. Ken Sink and Dr. Peter Carlson for the use of laboratory facilities, and to Dr. Frank Dennis for his guidance during the course of this research. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION LITERATURE I. II. Production of Secondary Metabolites ln’Vitro Gibberellins in Developing Seeds of Phaseolus vulgaris MATERIALS AND METHODS I. II. III. Effect of Stage of Development of Bean Seeds on Content of GA-Like Substances in Methanol Extracts A. Greenhouse Experiment B. Field Experiment Culture of Tissues A. Establishing Cultures of bean, Phaseolus vulgaris L. 'Montcalm' 1. Comparison of media 2. Effect of 2,4-D on callus formation B. Establishing Cultures of Nicotiana forgetiana Hort. ex. Hemsley C. Culture Media Used Assay for Gibberellin-Like Substances A. Barley Half-Seed Halo Assay 1 Direct assay of callus cultures 2. Direct assay of liquid culture medium 3. Assay of extracts in starch/agar plates B. Lettuce Hypocotyl Assay iii Page vi vii 12 14 15 16 16 16 17 17 20 20 20 25 28 29 IV. VI. Gibberellin Content of Established Callus Cultures of Bean and Nicotiana Gibberellin Content of Callus Initiated on Bean Cotyledons and in Subsequent Subculture Gibberellin Content of Nicotiana Callus Following Subculture on Root and Shoot Regeneration Media RESULTS AND CONCLUSIONS I. II. III. IV. VI. Effect of Stage of Development of Bean Seeds on Content of GA-Like Substances in Methanol Extracts A. Greenhouse Experiment B. Field Experiment Establishing Cultures of Bean; Phaseolus vulgaris L. 'Montcalm' A. Comparison ofTMedia B. Effect of 2,4-D on Callus Formation Barley Half-Seed Halo Assay A. Factors Affecting Halo Diameters After Exposure to GA3 Standards in Agar 1. Halo diameters after exposure to known quantities of GA3 2. Incubation time 3. Presoak 4. Light B. Factors Affecting Halo Diameter After Exposure to GA in Aqueous Solution 1. Effect of 2thanol solvent and temperature of medium during addition of GA to liquid standards 2. Incubation timg with starch C. Incubation Time for Assay of Extracts in Starch/Agar Plates Gibberellin Content of Established Callus Cultures of Bean and Nicotiana Gibberellin Content of Callus Initiated on Bean Cotyledons and in Subsequent Subculture Gibberellin Content of Nicotiana Callus Following Subculture on Root and Shoot Regeneration Media iv Page 30 3O 31 32 33 33 33 33 36 36 36 38 44 44 44 44 50 50 57 58 Page DISCUSSION 59 LITERATURE CITED 62 LIST OF TABLES Table 1. Some secondary compounds produced by plant tissue cultures Relative activities of gibberellin-like substances in tissue cultures of various plant species (Nickell, 1958) Components of Murashige and Skoog (1962) basal salts and vitamins medium (MS) and Schenk and Hildebrandt (1972) (SH) Components added to MS basal salts and vitamins medium to prepare test media Treatments used to test effect of adding ethanol and GA3 on halo diameter in barley half-seed halo assay GA—like activity (ug GA3—eq./g f.w.) in methanol extracts of greenhouse grown bean seeds using the lettuce hypocotyl assay Effect of light and medium on gibberellin content (ng/ml) of callus initiated on bean cotyledons Gibberellin content (ng/ml) of first callus subculture Gibberellin content (ng/ml) of Nicotiana callus during culture on MS-O, MS-D3, and UM media vi Page 10 18 19 28 32 57 57 58 LIST OF FIGURES Figure 1. Halos in the barley half-seed halo assay after exposure of half—seeds to GA standard agars for 24 hours and to starch/agar plates for 72 hours GA-like activity in methanol extracts of field grown bean seeds as determined by lettuce hypocotyl assay Effect of 2,4-D concentrations in MS-35 medium on callus formation on bean cotyledon and embryonic axis explants after 19 days of culture Halo diameters in half-seed halo assay after exposure to known concentrations of GA3 in 1 ml aliquots of agar Halo diameters of barley half-seeds incubated for various times on GA3 standard agars and on starch/agar plates Halo diameters of half—seeds after 24 hour exposure to GA standards and 72 hour exposure to stgrch/agar plates. Means for 3 trials Halo diameters in barley half-seed halo assay following 24 hour presoak in water Effect of light during 24 hour incubation on GA standard agars on halo diameters in half-Seed halo assay. A11 half—seeds subse- quently incubated for 72 hours on starch/agar plates in light Effect of method of adding GAi to 2 liquid media on halo diameters in ha f-seed halo assay. Incubation time—~24/72 hours vii Page 24 34 35 37 40 41 43 45 47 Figure Page 10. Effect of incubation time on starch/agar plates following 24 hour exposure to GA3 liquid standards on halo diameters in half-seed halo assay 49 11. Effect of incubation time on halo diameters of barley half-seeds on agar plates contain- ing both GA3 standards and starch 51 12. (A) Gibberellin content and (B) fresh weight of established bean callus during subculture 52 on UM 13. (A) Gibberellin content and (B) fresh weight of established bean callus during subculture on R3B 53 14. (A) Gibberellin content and (B) fresh weight of established Nicotiana callus during subculture on UM 54 15. Gibberellin content per culture of establish- ed callus subcultures 56 viii INTRODUCTION Research on the physiological roles and effects of gibberellins, other than GA3 and GA4+7, is limited by the small quantities available. An adequate supply of these gibberellins would permit extensive studies in areas of plant hormone research thus far untouched. The current interest in tissue cultures that produce pharmaceuticals and other secondary metabolites raises the possibility that gibberellins may be produced in a similar manner. In particular, do they accumulate at some point in culture or would altering environmental conditions induce gibberellins to accumulate in 21339? The major objective of this research was to determine (a) the gibberellin content of callus and (b) effect of subculturing, medium composition, and light exposure on gibberellin concentration. Callus cultures were initiated on explants of immature bean cotyledons, Phaseolus vulgaris L. 'Montcalm'. Beans were chosen for this work because immature bean seeds have high concentrations of gibberellins and thus might retain or attain the capacity to produce them in yitgg. The gibberellin content of callus was determined using a bioassay. Most bioassays for gibberellins involve extraction of the tissue; however, to determine the gibber- ellin content of normally growing callus at various time intervals, the bioassay would have to be non-destructive. The barley half-seed halo assay offered the potential for directly testing callus in_vi££g and was successfully applied to monitor GA—like content of callus cultures in these studies. LITERATURE I. Production of Secondary Metabolites lfl.!i££9 The potential for the industrial production of secondary metabolites through tissue cultures has yet to be fully realized (Staba,et al.,1965; Street, 1973; Butcher, 1977; Alfermann and Reinhard, 1978). The production of these compounds in_vi££g involves developing large scale callus or cell suspension cultures that yield an economical- ly significant quantity of the chemical of interest. These chemicals are generally either secreted into the liquid phase of the medium or compartmentalized within the cell; in either case appropriate extraction methods must be developed. To make extraction profitable the cultures must remain productive over long periods of time. Thus far, plant physiologists have concentrated their efforts on the production of medicinal compounds by tissue culture techniques. To date no published references are known on the possibility of producing plant hormones such as gibberellins (GAS), auxins, or cytokinins on an industri- al scale. Commercially available gibberellins are presently limited to only GA3, GA4, and GA7. These GAs are extracted from cultures of Fusarium moniliforme in its reproductive form, Gibberella fujikuroi. The other known gibberellins 4 are obtained in microgram quantities from plant tissues. Thus, comparisons of physiological activities and commercial use are limited by difficulties in obtaining sufficient quantities. There are many possible approaches to finding an alternative system which yields high quantities of gibberellins. However, a living plant system seems most reasonable since gibberellins are endogenous growth hormones. Because extractions of plant tissues yield low quantities of gibberellins per unit weight, culturing specific tissues which contain high levels of gibber— ellins would appear to be a promising approach. The many advantages of a tissue culture system for the production of secondary compounds have been discussed (Butcher, 1977). Tissue cultures of most plants are easy to establish and maintain either as callus or as cell suspensions. An added advantage in such systems is that environmental conditions are readily controlled. Nutrient and hormone needs can be easily met and precursors of the desired compound can be incorporated into the medium. Recognition of the biosynthetic potential of plant tissue cultures began with the production of rubber from guayule cul— tures (Arreguin and Bonner, 1950). A patent issued in 1956 to Routien and Nickell defined ten cultures with the potential for large scale production of secondary compounds. Since then the possibilities of using tissue cultures for the synthesis of economically valuable compounds have been discussed in several articles (Staba, et al., 1965; Street, 1973; Butcher, 1977; Tabata, 1977; Alfermann and Reinhard, 1978; Dougall, 1979a, 1979b). The use of tissue culture for the production of secondary metabolites is based on the assumption that cells will maintain their chemical as well as morphological totipotency in yigrg. Scientific evidence is available to both support and refute this assumption. With the methods commonly used for callus and cell culture, metabolites normally produced by the intact tissue may or may not be found in the cells cultured, in callus tissue produced by them, or in the culture medium. For example, Digitalis lantana, which produces cardiac glycosides in_vivg, ceases to produce them in_yi£rg. In contrast, Ruta graveolens produces coumarins and alkaloids and Andographis paniculata produces paniculides in vitrg_which are not found in the respective whole plants. When secondary metabolites are produced, the concentrations are often very low. However, some secondary compounds are produced in much greater quantities in_vit£g than ig_vivg. Dougall (1979a, 1979b) gives several examples; cell cultures of Morinda and Galium produce 20 times more anthraquinones than do their intact roots. Numerous secondary metabolites have been identified in plant tissue cultures (Staba, 1963; Carew and Staba, 1965; Staba, et a1., 1965; Puhan and Martin, 1971; Tabata, 1977; Alfermann and Reinhard, 1978; Kemp and Stoltz, 1979; Salem and Reinhart, 1979). Some of these compounds, a few of which are produced at substantially higher levels than in intact plant tissue(s),are listed in Table 1. In some cases a major problem involves the detection of the compound produced. Specific tests for gibberellins, which are color- less, generally require extraction of the tissue, followed by bioassay. Nickell (1958) detected gibberellin-like substances in extracts of tissue cultures using the dwarf pea bioassay (Table 2). Alfermann and Reinhard (1978) suggested a basic strategy for the production of secondary compounds in_viggg. First, cultures of a species that produces the compound of interest are established. Second, cultures of this species are screened for productivity and appropriate strains selected. Third (sometimes included with step two), the factors which influence the growth and productivity of the culture are varied to determine the optimal cultural conditions for production. Fourth, methods for extracting and concentrating the desired chemical are developed. Just how each of these steps is to be carried out must be deter- mined for the specific species and compounds being investi- gated. Analytical techniques for selecting and maintaining productive strains of cells are currently being developed for a wide range of compounds. Freeze-preservation methods permit the storage of viable callus tissues of certain species over long periods of time. Chemostat or turbidostat methods allow the maintenance of cultures in a steady state over extended periods. Radioimmunoassay systems are being ousuHSU deflmaommsmnm oHSDHDo mDHHmouo m H ommn HHmROAz . HHmm azono tam sowunom anomnmu mcmfluoowz : u Baum ownuoomm wnma Ream muma somfiaz mama “maumflaa use nonmm Amv .mm asafiam Rom - onma .Hm um .muman mpou mammmu xoa 9 Am» muma .Hm um .xcoN mHHomwuuHo confluoz RON 0H monocflsvmunucm Hama emanaflmm can nfimahoma< Am.ov msosmm a u whoa eamflnmeesm was eamaxonnm Huaa .Ha um .oanmumaou doma .Hm um .uuoawom Am.0v mammmmoammm xHA u msflcmmoo£uam mocfl> cam mmma .Hw uo .uuoom madman m5£ucmwaom u u cwom caucusawouoxua wRaH mama A m. av wAmH .Hm no .mamn< N H numa .H@ mm .xsom mammou mucuamnwsumo xHA u oaHoHHmEmm mmoaouomom moflommm o>H>.mM .us canoafioo .ocoo ou >Hc m>HumHoH N .ocoo .monauaso osmmwu unwaa mp woodwOHm monsomaoo hhmcfiooom ofiom .H manmfi Ebfluoa «mma amaum Amv mamauahm mason: . >\3 H «momnq mssccm mszucmfl om mmma .Hm um .uuoom cam 2mm mouome . n cwom omeEbm mmoa .Hm um .meEmH munmaw mmflfinuhozaw n q cmmflsuuzomaw mama wnmncmeemm hmv paw camamumm Enownmu mcmfiuooflz u : ocowfiumusaw Amv outflouaop moma .Hm um .Hsmx mouoomowa u m.H nwcowm0flu mmma ovum; wflHmcHonmo can ccmwhoB mnuoaflaoz . n saucesoo mmom cm moma mnmum owxcfio .m>m < i . whom oeunflo mumcwasom moofiuoumfimo : : nHooLuouaEmo Roma ofinwumcoo ADV ouom . i msflmHmuon Au score mmma mxwz cam umoz maocmaaon mmouu< n u ocwmopuw moonmuomom mowooam o>H>.mM .uB wasoafioo .0500 on xuc o>HumHmH N .ocoo A.e.ueoov .H anaae mmma .Hm um .mvoxH muma Hoaawo wmma Hoaaom Amw gowflmu NCMHUOUHZ Am.ov mammou oaumaocfinvflns onma .Hm um .Hmaamn manucmnmcumo xHA - mouncmmumm Amw moma mfiouum EswaoEouum annumm 38b coma wand cam mum: woman mzamhwmomm u i mcflfimaomoom mmma munch Amv mammfiflw xmcmm xH Hm mafiaommm omma Haoxowz can Cofiusom Meson onUM i a peer oflamxo mmma .Hm um .xcoN Amv HoEBHn msoaoo . : uflom oHHmEmon onma .Hm um .mHman mafiumsu mamfiuooflz n Hmm.o ocHuooHG monouowom moflommm o>H>.mm .uB pcsoaaoo .oaoo ou hut o>wumaou N .ofioo A.e.uaooo .H mfinme 10 Table 2. Relative activities of gibberellin-like substances in tissue cultures of various plant species (Nickell, 1958). Relative activity in Species Culture dwarf pea assay Vinca rosea stem, crown gall + Helianthus annuus petiole, crown gall +++ Melilotus officinalis stem, crown gall + " " root, virus tumor + " " stem, virus tumor + " “ root callus + Agave toumeyana leaf callus ++ Ilex aguifolium stem callus + Phaseolus vulgaris cotyledon callus + developed to screen cells for high—yielding strains (Kostenbauder, et a1., 1974; Weiler, 1978). Continuous culture systems may permit cultures to be maintained at a stage of maximum metabolite production. The continuous culture systems make it possible to maintain a constant density, growth rate, chemical composition, and metabolic activity (Butcher and Ingram, 1976). Chemostat cultures maintain the density of the culture by constant addition and removal of nutrient medium in the vessel. In turbidostat systems, the suspension is maintained at a specific optical density automatically by the addition and removal of medium (Wilson, et a1. 1971). Wilson (1978) investigated the use of Chemostat cul- tures for the production of anthraquinones by Galium mollugo. Production was from 7 to 30 times less than that of batch cultures, but was increased to similar levels by 11 manipulation of limiting factors. One of the distinct advantages of the Chemostat system is that the effects of various components of the medium on metabolite produc- tion can be easily verified independently by their addition or removal. Production of secondary compounds on an industrial scale awaits the solution of several major problems. The most prominent of these is the instability of cell strains. While some cells can synthesize the product of interest, other cells do not. Often cells unpredictably lose or regain this ability, especially when subcultured. Thus, loss of productivity can never be totally prevented under present cultural methods (Alfermann and Reinhard, 1978). Cultures must be checked continually for production levels-- an impractical procedure in large scale batch cultures. The production of secondary compounds by tissue culture has received considerable attention in recent years. The emphasis has been on products of pharmaceutical value while the possible production of plant hormones such as gibberel- lins has been neglected. Considering the limited supplies of various gibberellins, a system that would yield useful quantities would be of great value to plant research. Such investigation may provide additional sources of the known gibberellins. 12 II. Gibberellins in Developing Seeds of Phaseolus vulgaris Immature seeds of Phaseolus vulgaris contain high concentrations of gibberellins (Corcoran and Phinney; 1962; Carr and Skene, 1961; Skene and Carr, 1961; Skene, 1970). The free gibberellin content of immature bean seeds is highest between 10 and 15 days after anthesis and declines with maturity. Although the concentration of free gibber- ellins is higher in immature than in mature beans, the concentration of bound gibberellins increases as the seeds mature (Hiraga, et a1., 1974a; Yamane, et a1., 1975). Corcoran and Phinney (1962) measured the gibberellin content in developing seeds of Phaseolus vulgaris 'Black Valentine' using the dwarf corn bioassay. The highest concentration of gibberellin detected was 0.05 ug/g of tissue 10 days after anthesis. Two peaks of gibberellin-like activity were detected in developing seeds of Phaseolus vulgaris 'Hawkesbury Wonder' using the dwarf pea bioassy (Carr and Skene, 1961; Skene and Carr, 1961; Skene, 1970). The higheSt concentrations were observed at 15 days (0.7 ug/g) and 26 days (0.275 pg/g) after anthesis. The average seed weight was 50 mg for the former and 600 mg for the latter. The youngest beans in each case contained the highest gibberellin-like activity. A number of free gibberellins have been identified in immature bean seeds. Durley, et a1. (1971) identified GAl’ GA6, and GA8. Hiraga, et al. (1974b) isolated GAl’ GAS/20’ GA8’ and GA38 and identified GA4, GAS’ GA6, and GA37. l3 Yamane, et al. (1977) added GA17, GAZO’ GA29, and GA44 to the list of free gibberellins known to occur in immature bean seeds. MATERIALS AND METHODS I. Effect of Stage of Development of Bean Seeds on Content of GA-Like Substances in Methanol Extracts Phaseolus vulgaris was chosen for this work since the immature seed contains high concentrations of gibberellins (Corcoran and Phinney, 1962). The gibberellin content of methanol extracts of 'Montcalm' seeds was determined using the lettuce hypocotyl assay (Frankland and Wareing, 1960). A. Greenhouse Experiment Four bean seeds were planted in each of 20 pots (2 gallon volume) in the greenhouse under 12 to 14 hours cool white fluorescent light per day. The temperature was maintained at 200 t 30 C and the plants watered and ferti— lized as needed. Flowers were tagged at anthesis and the pods on all plants were harvested 35 days after the first flowers opened. Seeds were divided into groups according to age, their length was measured, and they were frozen (~10O C) until extracted. For extraction, each group of seeds was mace— rated in a blender with approximately 10 ml cold methanol per gram of tissue. The macerates were filtered under vacuum through Whatman No. 1 filter paper and the filtrate refiltered by gravity. The methanol was evaporated under . . o vacuum and the remaining aqueous extract frozen at -10 C. 14 15 Acidic, neutral, and butanol fractions of the extracts were separated by acid—base fractionation. The pH of the aqueous extract was adjusted to 8.0 with 3 N NH4OH, and the extract partitioned 3 times using 10 ml distilled ethyl acetate. The combined ethyl acetate fraction was washed once with 10 ml distilled water and the water added to the aqueous phase. The pH of the aqueous fraction was adjusted to 3.0 with 3 N formic acid, and the water was partitioned 3 times against ethyl acetate. After washing the combined ethyl acetate fraction (acidic fraction) with 10 ml water and adding the wash to the aqueous fraction, the latter was washed 3 times with n-butanol as described above for ethyl acetate, and the water phase was discarded. The acidic, neutral, and butanol fractions were left overnight at -100 C, then filtered through glass wool in cold Buchner funnels to remove ice particles. The filtrat- es were evaporated and the residues dissolved in 2 to 4 ml of ethanol and stored at -100 C until assayed with the lettuce hypocotyl assay. B. Field Experiment 'Montcalm' bean seeds were planted in the field at the Horticultural Research Center in late June, July, and August. The pods were harvested at varying stages of development and separated by age into 6 groups. A random sample of seeds from each group was dissected and morpholog— ical characteristics of each group were recorded so that similar stages could be identified when selecting samples 16 for culture. Each group of seeds was extracted and the extracts fractionated as previously described. The residue was taken up in ethanol and assayed with the lettuce hypocotyl assay. II. Culture of Tissues A. Establishing Cultures of Bean, Phaseolus vulgaris L. 'Montcalm' Callus cultures were established of immature tissues of bean seeds of Phaseolus vulgaris ‘Montcalm'. Pods were harvested from field grown bean plants when the seeds were 0.6 to 1.0 cm in length and washed to remove soil. Under aseptic conditions, the pods were dipped in ethanol, flamed to sterilized the outer surface, cut open, and the seeds removed. The seed coat was cut away and the cotyledons separated from the embryonic axis. After removal of the proximal tissue, the abaxial surface of the cotyledon was placed in contact with the agar medium in a 2 ounce screw- capped jar. The cultures were grown under cool white lights, 1800 to 2100 uEm-Zs-l, or in the dark. Callus was subcultured about every 4 weeks unless otherwise indicated. 1. Comparison of media Fifteen test media were prepared by adding various hormones to Murashige and Skoog (1962) basal salt and vitamins medium (see Tables 3 and 4). Embryonic axes and cotyledons from greenhouse grown beans were pre- pared as described above, implanted on the media, and 17 cultured in the dark for 5 weeks. Cultures were evaluated weekly for callus texture, color, and size. Root formation and any unusual growth of the explants were also noted. 2. Effect of 2,4-D on callus formation Arnison and B011 (1978) reported that 2,4—D was essential for the growth and maintenance of cell suspension cultures of bush bean cotyledons. Results of the media comparison experiment above suggested that callus growth and friability increased with 2,4-D concentration. No growth occurred on most media which lacked 2,4—D. The medium MS-35 was therefore used with varying concentrations of 2,4-D (0.0, 0.01, 0.5, 1.0, and 3.0 mg/l) added to test the rate of callus initiation and subsequent growth. The pH was adjusted in all cases to 5.8. Embryonic axes and cotyledons were prepared as described above, and the cultures kept in the dark. Four cultures were used for each observation. Fresh weight was recorded after 19 days. B. Establishing Cultures of Nicotiana forgetiana Hort. ex. Hemsley. L. Rapport (personal communication) reported that tobacco callus contained slightly higher concentrations of GA-like substances than did other tissues tested. Nicotiana cultures were obtained from J. Passiatore (personal communication) approximately 3 months after initiation from leaf discs cultured on MS—Zl and transfer to SH (Table 3), followed by subculture 4 weeks later on the same medium. They were then transferred to UM and subcultured once 18 Table 3. Components of Murashige and Skoog (1962) basal salts and vitamins medium (MS) and Schenk and Hildebrandt (1972) (SH). Final concentrations in mg/l unless otherwise specified. Component MS SH NH4N03 1650.0 - NH4H2P04 - 300.0 KNO3 1900.0 2500.0 MgSO4.7H20 370.0 400.0 Mn804.H20 — 10.0 Mn804.4H20 22.3 - Zn804.4H20 8.6 - Zn804.7H20 - 1.0 CuSO4.5H20 0.025 0.2 CaC12.2H20 440.0 200.0 KI 0.83 1.0 CoC12.6H20 0.025 0.1 KHZPO4 170.0 - H3B03 6.2 5.0 Na2M004.2H20 0.25 0.1 FeSO4.7H20 27.85 15.0 NazEDTA 37.25 20.0 Glycine 2.0 - Inositol - 1000.0 Myo-inositol 100.0 — Nicotinic acid 0.5 5.0 Pyridoxine-HCl 0.5 0.5 Thiamine-HCl 0.1 5.0 2,4—D - 0.5 p-CPA - 2.0 Kinetin — 0.1 Sucrose 30.0 g/l 30.0 g/l Agar 8.0 g/l 6.0 g/l pH 5.8 5.8-5.9 19 Table 4. Components added to MS basal salts and vitamins medium to prepare test media. Final concentration in mg/l and pH adjusted to 5.8 unless otherwise specified. Medium Components MS-35‘ ' MS—36 MS-0.01 MS-0.1 MS-l.0 6—BAP 0.5 2.5 - — - 2,4-D 5.0 5.0 0.01 0.1 1.0 MS-Pl R3B MS-21 MS-O 6-BAP 0.5 1.0 0.01 - NAA 2.0 2.0 5.0 - R2A R3 MS-D3 R305 IAA 2.0 5.0 2.0 5.0 2,4-D 2.0 0.5 - - 6-BAP - - 1.0 — NAA - - - 10.0 Kinetin 0.3 0.3 - 0.3 pH 6.0 6.0 5.8 6.0 UM MS-S (Uchimiya and Murashige, 1974) Nicotinic acid 4.4 4.5 Pyridoxine-HCl 9.5 - Thiamine-HCl 9.9 - Folic acid - 0.5 Biotin - 0.05 2,4-D 2.0 3.0 Kinetin 0.25 - Casein hydrolysate 2.0 g/l - Decrease sucrose to 20.0 g/l Increase agar to 9.0 g/l pH 5.8 5.6 20 4 weeks later for these experiments. C. Culture Media Used The components of the media used in these experiments are listed in Tables 3 and 4. III. Assay for Gibberellin-Like Substances A. Barley Half-Seed Halo Assay 1. Direct assay of callus cultures The following procedure based on the half-seed assay by Machi Fukuyama Dilworth (Fukuyama,l97l) was developed to test callus directly in vitrp_for gibberellin content. Sterile barley half-seeds were placed in direct con- tact with callus cultures for 24 hours. The gibberellin in the callus stimulated de_n9vg synthesis of a—amylase in the aleurone layer of the seed. The seed was transferred aseptically to starch plates where the a-amylase diffused into the plates and digested the starch. When flooded with KI/I2 solution, the background turned blue as the starch and iodine reacted. Clear rings or halos appeared around the seeds where the starch had been digested in the agar. Since all procedures were performed under aseptic condi- tions, the cultures tested continued to grow. Preparation of GA3 standard agars. Stock solutions of GA3 were prepared in 95% ethanol and diluted with ethanol (95%) to obtain concentrations of 1.0, 0.1, 0.01, 0.001, 0.0001, and 0.0 pg GAB/10 pl ethanol. These solutions were filter—sterilized through Millipore filters (0.45 um) and 21 stored at 00 C in sterile jars. One ml of 0.8% Difco Bacto agar solution was placed in each of 36 pathology vials, 25 ml volume, the caps were screwed on loosely, and the vials were autoclaved at 15 lbs. pressure for 20 minutes. After autoclaving, the vials were cooled to touch in a 450 C water bath. Ten pl of each GA3 solution was added to each of 6 vials using aanppendorf pipette with sterile tip. The final GA3 concentrations were 1.0, 0.1, 0.01, 0.001, and 0.0001 ug GA3/ml. The control contained 10 p1 of sterile ethanol per m1 of agar. Preparation of barley seeds. Barley seeds, Hordeum vulgare 'Himalaya', diameter 0.3 cm or larger, were cut in half with a razor blade and the embryo half discarded. The remaining half-seeds were stored at 100 C. Before use they were soaked for 8 minutes in a solution of 0.7% Tween 20 and double distilled water and sterilized in a 250 ml flask containing 100 ml of full-strength 'Clorox' (5.25% sodium hypochlorite) and 2 drops of Tween 20. The seeds were sterilized for 1 hour with one change of the 'Clorox' solution and occasional swirling to release air trapped by the seeds. The half-seeds were then rinsed 10 times with 100 ml aliquots of sterile water. Transfer to callus cultures. The callus culture jars were opened aseptically and the cut surface of each sterile half—seed was placed on the callus surface. The same pro- cedure was used to place the half-seeds on the GA3 standard agars. In most cases, 4 half-seeds were placed on each of 22 8 callus cultures for a total of 32 observations per treat- ment. Two half-seeds were placed on each of 6 replicate GA3 controls for a total of 12 observations per concentra— tion. The half-seeds were left on the callus or agar sur- face for 24 hours before transfer to starch/agar plates. Preparation of starch/agar plates. Stock solutions of KHZPO4 (60 mg/ml) and CaC12.2H20 (29.4 mg/ml) were prepared in double distilled water and stored at 100 C until used. The following ingredients were combined while stirring: KHZPO4 stock solution, 30 ml; CaC12.2H20 stock solution, 3 m1; double distilled water, 288 m1; and potato starch, 450 mg. The volume was adjusted to 320 ml with double distilled water and the solution boiled for 1 minute. Following centrifugation at 24 g for 20 minutes, the supernatant was stored at 100 C. Difco Bacto agar (1.5%) was added to the supernatant and the solution was steriliz- ed by autoclaving at 15 lbs. pressure for 20 minutes. After cooling in a 450 C water bath, 25 m1 aliquots were poured into each sterile Petri dish (100 x 15 mm). After solid- ification of the agar, the plates were stored in plastic bags at room temperature for no more than 4 days before use. Transfer of half-seeds to starch/agar plates. After exposure to callus or standard agars, the half-seeds were transferred to agar plates. Four half-seeds were placed on a plate with the cut side contacting the agar firmly but not penetrating the agar surface. The plates were sealed with 'Parafilm' or 'Saran' and incubated for 72 hours at 23 20° c in the light, 400 to 500 uEm‘Zs‘l. Visualization and measurement of response. After incubation, the plates were flooded with KI/I2 solution (300 mg KI plus 30 mg I2 per 100 ml water). After about 3 minutes the solution was poured off and the diameter of the clear halos around the seeds were measured to the near— est mm at the widest diameter of each half-seed (Figure 1). Only circles with clearly defined edges were measured. Circles 0.5 cm (the average diameter of the half-seeds) or less in diameter were tabulated as no response. It was assumed that gibberellins diffused at the same rate through callus as through 0.8% agar. Therefore, 1 ml of 0.8% agar was equivalent to 1 ml of callus and the results were expressed in ug/unit volume. Treatment means and standard deviations were calculated using cultures as replicates. Response to known concentrations of GA3. GA3 standards were prepared at concentrations ranging from 0.0 to 5.0 pg GA3/ml. Half-seeds were incubated on GA3 standard agars for 24 hours and on starch/agar for 72 hours. Incubation time. The effect of incubation time, both on GA3 standards and on starch agar, was investigated to determine the time period which would give the greatest sensitivity and the optimum dose response curve. 'Himalaya' half-seeds were sterilized and exposed to GA3 standard agars and starch/agar plates for varying periods of time in a factorial design using 24, 48, and 72 hours of incubation on GA3 standards and the same periods of exposure to Figure 1. 24 Halos in the barley half—seed halo assay after exposure of half-seeds to GA standard agars for 24 hours and to starch/agar plates for 72 hours. Upper left, 0.0 ug/ml. Upper right, 0.001 pg/ml. Lower left, 0.01 ug/ml. Lower right, 1.0 ug/ml. 25 starch/agar for a total of 3 x 3 - 9 treatments. Two repli— cates of each concentration were used. The entire experiment was repeated. The 24/72 hour treatment was repeated three additional times with 6 replicates of each GA3 concentration x 2 seeds per replicate making 12 observations for each concentration. Presoaking in water. Fukuyama (1971) reported that soak- ing sterile half-seeds for 24 hours prior to use increased the sensitivity of the assay to low concentrations of gibber— ellins. To confirm this observation, barley half-seeds were sterilized, rinsed with sterile distilled water, and soaked in sterile water for 24 hours. The seeds were subsequently transferred to GA3 standard agars for 24 hours and finally transferred to starch/agar plates for 72 hours. Light. Fukuyama (1971) did not investigate the effect of light on the half-seed halo assay. As cultures grown both in the light and in the dark were to be assayed, the effect of light was tested. Two half-seeds were placed on twelve GA3 standard agars of each concentration. Six replicates of each concentration were placed in the dark —ZS-l and 6 under cool white light, 1800 to 2100 “Em , for 24 hours. All seeds were transferred to starch/agar plates_ and held for 72 hours at 200 C and 400 to 500 uEm-zs-l. 2. Direct assay of liquid culture medium To measure gibberellin concentrations in liquid cell cultures, the assay developed by Fukuyama (1971) was modified to enable sampling of continuously growing 26 suspension cultures. The assay involved aseptic opening of suspension cultures 2 to 4 times a week to insert and remove half-seeds. The utmost care in flaming instruments and changing damaged foil caps had to be exercised to avoid contamination. Preparation of GA3 liqpid standards. Since the sus- pension culture assay measured GA in liquid medium, stand- ards were prepared in the same culture medium as the tissue. Media were prepared as described for solidified media except agar was omitted. One ml of the medium.was placed in a culture tube, capped, and autoclaved. Ten p1 of GA3 in ethanol were added to each tube to give concentrations of 1.0, 0.1, 0.01, 0.001, and 0.0001 pg GAB/m1 of medium. Controls included both 10 p1 ethanol per ml of medium and medium alone. Standard solutions were stored at 00 C between assays. Preparation of bagley seeds. Barley half-seeds were prepared as for direct assay of callus cultures previously described. Transfer to liquid cultures. Suspension cultures were opened aseptically and 4 (2 for controls) sterile half-seeds dropped into the medium. The cultures were recapped and returned to thegyrating shaker for 24 hours. Liquid standards were rotated for 24 hours at 1 rpm in a horizontal position on a vertical wheel. The standards were tested in duplicate resulting in 4 observations for each concentration. 27 Preparation of starCh agar plates. Starch/agar plates were prepared as for direct assay of callus cultures pre- viously described. Transfer of halfrseeds to starch/agar plates. Half- seeds were removed from the solutions assayed, blotted on sterile filter paper, and placed out side down in firm contact with the agar surface. The plates were covered and sealed with 'Parafilm' or 'Saran' for 24 hours. Visualization and measurement of response. The direct- assay of liquid cultures was visualized and measured as described above for the direct assay of callus cultures. Effect of ethanol SOlvent and temperature of medium during addition of GA3 to liqpid standards. The method outlined above for preparing liquid GA3 standards was based on the assumption that the ethanol solvent evaporated and thus did not interfere with the assay. To test this premise, GA3 standards were prepared in duplicate as in Table 5, using 10 pl GA3 solution per 1 ml of medium. Two liquid media were tested, UM and R3B. Sterile halfnseeds were incubated in the solutions for 24 hours, then trans- ferred to starch/agar plates for 72 hours. Incubation time with starch. The effect of time of barley half-seed incubation on starch gel was tested. Liquid standards were prepared by adding 10 p1 of ethanolic GA3 solutions to 1 ml aliquots of culture medium. Half- seeds were incubated in these solutions for 24 hours and placed on starch/agar plates for 24, 32, 48, or 72 hours 28 before adding KI/I2 solution. 3. Assay of eXtracts in starch/agar plates The method presented here was adapted from that used by Fukuyama (1971) and involved the addition of an extract of cultured cells or media dissolved in ethanol to the starch/agar plates as they hardened. The halftseeds were placed directly on these plates, allowing both the diffus- ion of GA from the plates into the seed, and of the induced amylase from the seed into the plates. Table 5. Treatments used to test effect of adding ethanol with GA on halo diameter in barley half-seed halo as ay. Solvent Medium 0 for GA3 temperature ( C) Ethanol ca. 00 Ethanol ca. 700 Water ca. 250 Preparation of starch/agar plates with extract. Starch/agar solution was prepared as for the direct assay of callus cultures. Five ml of the autoclaved solution was pipetted into sterile Petri dishes (60 x 15 mm). Before the agar solidified, filter-sterilized extracts of GA3 standards in ethanol were added and the plates swirled to nfix thoroughly. Plates containing extracts and GA3 stand- ards were prepared in duplicate. 29 Preparation of barley seeds. Barley half-seeds were prepared as for direCt assay of callus tissue. After rinsing, the cut surfaces of two half—seeds were placed on the surface of the starch/agar plate. Dishes were sealed with 'Parafilm' or 'Saran' for 60 hours. Visualization and measurement of responSe. Visual- ization and measurement of the halo diameter was performed as for direct assay of callus cultures. Incubation time. Starch/agar plates containing GA3 standards were assayed for 48, 60, and 72 hours to deter- mine optimum time for response. B. Lettuce Hypocotyl Assay The lettuce hypocotyl assay (Frankland and Wareing, 1960) was used to measure the concentration of endogenous GA—like compounds in extracts of fresh bean seeds. High concentrations of 2,4-D in the callus inhibited lettuce hypocotyl elongation making determination of gibberellin content of callus with the lettuce hypocotyl assay impossible without purification of the extract beyond acid-base fractionation. One cm2 filter paper sections (Whatman No. l) were placed in 16 x 65 mm shell vials. Extracts dissolved in ethanol were added to each vial and allowed to dry over- night. An aqueous solution of Tween 80 (0.1%), 0.3 ml, was added. Standards were prepared by adding 0.3 ml of GA3 dissolved in the same solution at concentrations of 30 0.1, 0.03, 0.01, and 0.003 pg GA3/0.3 m1. Extracts and GA3 standards were prepared in duplicate. Ten lettuce seeds cv. Parris Island, were added to each vial and the vials placed on moist paper towel in a clear plastic box on a lab bench for 24 hours. The container was moved to a growth chamber at 23 to 250 C under fluorescent lights -2S-1 (46.5 pEm ) for 72 hours. The length of the five long- est hypoctyls in each vial was recorded. IV. Gibberellin Content of Established Callus Cultures of Bean and Nicotiana Callus cultures of bean and Nicotiana which had been subcultured for the 5th time (Subculture 5) were assayed biweekly for gibberellin content with the half-seed halo assay. Bean cotyledon cultures were grown on R3B and UM and Nicotiana cultures on UM. Cultures were grown in 'zs-l). cool white light (1800 to 2100 uEm Half of the cultures were subcultured after 18 days (Subculture 6). Others were assayed until they turned brown and ceased to grow. The fresh weight of the callus was measured to relate gibberellin concentration to changes in growth rate. V. Gibberellin Content of Callus Initiated on Bean Cotyledons and in Subsequent Subculture If the loss of gibberellin production in established callus was due to subculturing or habituation, freshly derived callus cultures should show a pattern of production similar to that of established cultures. To test this, 31 callus cultures were initiated on bean seed cotyledons 0.6 to 1.0 cm in length. Embryo axes were removed and one cotyledon was placed in each culture jar. Three media were tested: R3B, MS-D3, and UM. Cultures were grown in both light (1800 to 2100 pEm'zs'l) and dark. Gibberellin con- centration was measured with the barley half-seed halo assay once a week from.the time the callus was large enough to support 4 half-seeds until it browned and ceased to grow, approximately 27 days. VI. Gibberellin Content of Nicotiana Callus Following Subculture on Root and Shoot Regeneration Media As gibberellins did not accumulate in meristematic callus, the question arose as to whether or not they might increase in callus as it regenerated plantlets. A system has not yet been devised for regenerating plantlets from bean callus, but has been for NiCotiana. NiCotiana was transferred to MS-D3 for shoot initiation, to MS-O for root initiation, and to UM for maintaining unorganized callus. The gibberellin content was measured by direct assay with the half-seed halo assay. RESULTS AND CONCLUSIONS I. Effect of Stage of Development of Bean Seeds on Content of GA-Like Substances in Methanol Extracts A. Greenhouse Experiment Bean seeds 0.6 to 1.1 cm long (Table 6) had the high— est concentration of active gibberellins when grown in the greenhouse. No GA activity was detected in the neutral fraction except in extracts of the smallest size seeds. Activity in the acidic ethyl acetate fraction was higher than in the neutral ethyl acetate fraction or the butanol fraction. Activity in the acidic ethyl acetate fraction was very low in seeds larger than 1.5 cm. Table 6. GA-like activity (Mg GA3-eq./g f.w.) in methanol extracts of greenhouse grown bean seeds using the lettuce hypocotyl assay. Fraction Days after Length of Neutral Acidic Acidic anthesis bean seed (cm) EtAc EtAc. butanol 10-15 0.6—1.1 0.03 1.50 0.00 15-20 1.1-1.3 0.00 0.20 0.03 25 1.5 0.00 0.30 - 30 1.5 0.00 0.03 - 35 2.0 0.00 0.00 - 32 33 B. Field Experiment The youngest bean seeds tested contained the highest GA-like activity (Figure 2). Activity declined with de- creasing seed size. No activity was detected in the neut- ral EtAc fraction. The activity of the acidic EtAc frac- tion was highest in seeds less than 0.7 mm in length. Seeds 0.3 to 0.5 cm in length were too small to provide viable explants for tissue culture. Seeds from 0.5 to 1.0 cm long were therefore used as a source of explants. II. Establishing Cultures of Bean; Phaseolus vulgaris L. ‘Montcalm' A. Comparison of Media Two types of callus were initiated on the embryonic axes. Clear, yellow, friable callus formed at the base of the primary leaves, while white, firm callus formed just above the tip of the radicle. Callus formed mostly around the edges of the cotyledon where it contacted the surface of the medium. The character of the callus depended upon the medium. Media which produced proliferating callus without roots or unusual growth of the explant for both cotyledons and embryonic axes were UM, MS—35, MS-36, MS-Pl, R33, and R2A (see Materials and Methods page 18-20). Callus which formed on UM was the fastest growing and the most friable. B. Effect of 2,4-D on Callus Formation The initiation and growth rate of callus were direct- ly related to 2,4-D concentration (Figure 3). Media Ii 34 0.20 » .l FRACTION ~——- ACIDIC ETAC x——x ACIDIC BUTANOL o-—«> NEUTRAL ETAc E 0.15 " i E g 0.10 L - 3 a] 'm E 0.05 - A 0.00 ° 4‘3 . . 10 13 15 18 20 25 DAYS AFTER ANTHESIS 0.3- 0.5- 0.5- 1.0- 1.3 1.5 0.5 0.7 0.9 1.4 1.7 LENGTH OF SEED (CM) Figure 2. GA-like activity in methanol extracts of field grown bean seeds as determined by lettuce hypocotyl assay. 35 .onsuaso mo whom ma Houwm mucmaaxo mwxm oafiomunfiw paw coumahuoo soon So Goaumfiuom mDHHmU Go ESHuoE mMnmz cfl mcowumuucoocoo Qu¢.N mo uoommm .m oudwflm 3E5 mlflm o.m m.N o.m m4 0% m5 0.0 . . ed 3 UV wl _.l . 2x< Emmi . m.o m 1: mm . . 04 mm M H . zoomjioo . m4 W . m; o N ( p p h pi p - 36 without 2,4—D did not initiate callus. Embryonic axes required higher concentrations of 2,4-D for growth than did cotyledons. Callus tissue on media containing 2,4-D remained actively dividing and light in color for 4 to 5 weeks. Fresh weight of callus increased with concentration of 2,4—D up to approximately 2.5 mg/l for cotyledons and 2.0 mg/l for embryo axes. Of the 15 media tested previously, all those which induced callus formation contained at least 2.0 mg/l auxin. On media which contained NAA, IAA, or no auxin, callus develop- ed that was brown, slow-growing, and ceased growth after 2 weeks. III. Barley Half-Seed Halo Assay A. Factors Affecting Halo Diameters After Exposure to GA3 Standards in Agar 1. Halo diameters after exposure to known quantities of GA3 Halo diameters of barley half-seeds exposed to increas- ing GA3 concentrations in agar increased at an increasing rate to 10"2 pg/ml GA3 and thereafter increased at a de- creasing rate to 101 pg/ml GA3 (Figure 4). Increases in halo diameter declined sharply above 10-1 pg/ml with only slight additional increases at higher concentrations. The controls (ethanol in agar) consistently resulted in diameters of 0.5 cm. Therefore, the maximum level of GA3 which could be accurately estimated in unknown samples was 10'1 pg/ml. 37 4.0 . 3.5 - 3.0 . $32.5 - Em . 1‘2" s. c215 - O 2:“ 11.0 . 0.5 . 0.0 - 3 -2 -l 0 . 0 10 10 10 10 100 10 GA; 046/ ML) Figure 4. Halo diameters in half-seed halo assay after exposure to known concentrations of GA3 in 1 ml aliquots of agar. 38 2. Incubation time Halo diameter increased with time of exposure of half- seeds to both GA3 and starch/agar (Figure 5). However, the slope of the response curve was maximum with the 24/72 hour treatment. Incubation of half-seeds for 72 hours prior to placement on starch/agar plates essentially negated the response to GA3, since controls produced halos as large as those induced by GA3. These half-seeds were very soft and white exudate remained on the agar surface after they were removed. Almost all of the endosperm had been digested. Similar results were obtained on repeating the experiment (data not shown). On three additional trials, using only the 24/72 hour treatment, the response curve was fairly consistent (Figure 6). 3. Presoak in water The baseline of the dose response curve was increased by soaking barley half-seeds in water for 24 hours prior to incubation on GA3 standards. However, since the slope of the response curve was reduced (Figure 7), the seeds were used without a presoak. 39 Figure 5. Halo diameters of barley half-seeds incubated for various times on GA standard agars and on starch/agar plates. (First figure=hours on GA standards;second figure=hours on starch/agar plgtes.) 4.0 P b -l 3.0" 1 2.0 - . ~ 4 §§1.0 ~ 1 QC :2 t .. 922' < I I e 1 I 230.0 53 _ B- - :‘é l ‘l l. , 72/24 . / ¢ a 2'0 _ \ 4 is . . . . . -4 -3 - -l 0 0 O 10 10 10 2 10 10 Figure 5. 6A3 (pG/ML) 41 HALO DIAMETER (CM) N \N E: E: [—4 O l 0.0 ‘ L l L J L n -4 -3 _ _ 0.0 10 10 10 2 10 l 10 6A3 (”G/ML) Figure 6. Halo diameters of barley half-seeds after 24 hour exposure to GA standards and 72 hour exposure to starch/agar plates. Means for 3 trials. 42 Figure 7. Halo diameters in barley half-seed halo assay following 24 hour presoak in water. Bars repre- sent one standard deviation above and one stand- ard deviation below the mean. 43 Aaa\eao OH OH OH OH .OH o.e o H- N- m- s OH 3| .5 ouswwm OH o.o 1 Al d omxHsvo on on uoabmmm mp3 Home Nw.o mo HE osov .m SH msHHmo mo ustoB x .< .dH .MH .NH moHDme GH unoucoo <0 wchHmHuHsfi he pouwHDono mmB oHDuHso Mom uaouaoo