Habituation in in Vitro Soybean Cultures

4
Plant Physiol. (1988) 88, 809-812 0032-0889/88/88/0809/04/$0 1.00/0 Communication Habituation in in Vitro Soybean Cultures Received for publication February 1, 1988 and in revised form March 27, 1988 PAUL CHRISTOU Agracetus, 8520 University Green, Middleton, Wisconsin 53562 ABSTRACT The habituation of soybean (Glycine max) callus can be induced rapidly, by exposing the tissue to small amounts (10'molar) of com- pounds including 2.4-dinitrophenol and phenoxyisobutyric acid for brief periods of time. Such compounds reportedly exhibit antiauxin activity. Various soybean callus phenotypes have been isolated which continue to exhibit hormone habituation 14 months following the initiation of the experiment. Protein changes in habituated tissue under selected hormonal regimes were detected indicating changes at the level of gene expression. Habituated tissue exhibits hormonal autonomy in a manner similar to crown gall tissue, suggesting that such studies may help elucidate the mechanism of induction of crown gall disease and genetic transformation by Agrobacterium. in cell phenotype that can result in autonomous growth. The significance of this conclusion lies in the fact that habituation occurs in the absence of a recognizable infectious agent and appears to have an epigenetic basis. This led Braun (3) to speculate that tumor transformation in plants does not necessar- ily involve either the introduction of foreign genetic information or somatic mutation that results in a permanent change in the cell genome. Direct confirmation of this hypothesis, however, depends upon demonstrating that cloned, habituated tissue lines are tumorigenic in grafting tests and that the same lines satisfy the essential criteria for epigenetic changes (17). MATERIALS AND METHODS Plant cells and tissues cultured in vitro sometimes become autonomous of certain growth factors. This phenomenon is termed habituation and was discovered by Gautheret (6) during his work on the in vitro culture of carrot. Habituation relative to auxin has been found to occur in a large number of plants including Scorzonera (7), Vitis vinifera (18), Nicotiana tabaccum (4), Helianthus annuus (10), Lilium longiflorum (20), and Zea mays (9). It has also been encountered for cytokinins, amino acids, and several vitamins (8, 11, 22). Habituation has been observed, in some cases, as a gradual process at the tissue level, while in others it occurs rapidly. By comparing the growth response of tissues on media with and without auxin, it was found that during serial propagation, tissues often increase in degree of habituation (17). During prolonged culture, a few habituated cells arise in the tissue and then, because these cells proliferate more rapidly than nonhabituated cells, the degree of habituation of the tissue increases with time. However, Buiatti and coworkers (4) obtained 100% habituation of tissue from germinated seeds of Nicotiana bigelonii after a very short period of culture on a hormone containing medium. Another example of fast habituation is L. longiflorum which becomes auxin- independent after a short cultivation period (20). Our own work indicates that habituation can be induced rapidly in soybean (Glycine max) and that culturing of callus for a long period is not a prerequisite in this crop. The phenomenon of habituation bears a striking similarity to tumor transformation in crown gall disease where tumor tissue grows independent of exogenous hormones. This similarity may provide an indication as to how the capacity for autonomous growth is maintained in populations of dividing tumor cells. Comparison of the properties of habituated and crown gall cells leads to the conclusion that habituation is a form of neo- plastic transformation involving heritable, progressive changes Plant Material. Soybean seeds (Glycine max cv Corsoy, Wil- liams, No. 209332, and No. 90763) were sterilized for 10 min with commercial bleach diluted 1:5 with water and were germi- nated on filter paper. Callus Induction. Hypocotyl-derived callus was initiated and maintained on Gamborg's B5 medium with IAA (1 mg/L) 2,4- D (mg/L), and K (0.4 mg/L). Callus tissue was subcultured 3 times at 21 d intervals prior to being used in habituation exper- iments. Habituation Experiments. 2,4-DNP' and PiBA were incorpo- rated in Gamborg's B5 media at three concentrations (10-2, 10-6, and 10-9 M). Soybean tissue was cultured on these media with and without hormones as described in other sections. Experi- ments were terminated 30 d following incubation of callus on these media. At that point, fresh weights were recorded, and tissue was frozen in liquid N2 for SDS gel electrophoresis protein analysis. Samples of habituated calli were incubated on hormone- free media for 140 d with a subculturing period of 28 d. Gel Electrophoresis. SDS-PAGE of soybean proteins was car- ried out as follows. Callus tissue (75 mg fresh weight) was frozen and ground to a fine powder in liquid N2. Crude homogenates were extracted (2:1 volume/fresh tissue weight) with buffer con- sisting of 50 mM Tris-HCl (pH 10), 4% SDS, 1 % ,8-mercaptoeth- anol, 0.1% NP-40, 2.5 mM phenylmethylsulfonyl fluoride, 0.6 mg/ml leupeptin, and 0.2 mg/ml each of antipain and pepstatin. The final pH of the crude extract was 7.4. Extracts were boiled for 5 min, sonicated, and spun in a microfuge. The supernatants were collected and subjected to SDS-PAGE as described by Laemmli (14). An estimated 100 gg of protein were loaded per test sample, and the gels were stained with silver. Replicate gels in which half or twice the amount of protein used in the gel shown in Figure 2 gave the same results, confirming the repro- ducibility of the assays. 'Abbreviations: 2,4-DNP, 2,4-dinitrophenol; PiBA, phenoxyisobu- tyric acid; K, kinetin (furfurylaminopurine); NAA, naphthalene acetic acid. 809

Transcript of Habituation in in Vitro Soybean Cultures

Plant Physiol. (1988) 88, 809-8120032-0889/88/88/0809/04/$0 1.00/0

Communication

Habituation in in Vitro Soybean CulturesReceived for publication February 1, 1988 and in revised form March 27, 1988

PAUL CHRISTOUAgracetus, 8520 University Green, Middleton, Wisconsin 53562

ABSTRACT

The habituation of soybean (Glycine max) callus can be inducedrapidly, by exposing the tissue to small amounts (10'molar) of com-pounds including 2.4-dinitrophenol and phenoxyisobutyric acid for briefperiods of time. Such compounds reportedly exhibit antiauxin activity.Various soybean callus phenotypes have been isolated which continue toexhibit hormone habituation 14 months following the initiation of theexperiment. Protein changes in habituated tissue under selected hormonalregimes were detected indicating changes at the level of gene expression.Habituated tissue exhibits hormonal autonomy in a manner similar tocrown gall tissue, suggesting that such studies may help elucidate themechanism of induction of crown gall disease and genetic transformationby Agrobacterium.

in cell phenotype that can result in autonomous growth. Thesignificance of this conclusion lies in the fact that habituationoccurs in the absence of a recognizable infectious agent andappears to have an epigenetic basis. This led Braun (3) tospeculate that tumor transformation in plants does not necessar-ily involve either the introduction of foreign genetic informationor somatic mutation that results in a permanent change in thecell genome. Direct confirmation of this hypothesis, however,depends upon demonstrating that cloned, habituated tissue linesare tumorigenic in grafting tests and that the same lines satisfythe essential criteria for epigenetic changes (17).

MATERIALS AND METHODS

Plant cells and tissues cultured in vitro sometimes becomeautonomous of certain growth factors. This phenomenon istermed habituation and was discovered by Gautheret (6) duringhis work on the in vitro culture of carrot. Habituation relative toauxin has been found to occur in a large number of plantsincluding Scorzonera (7), Vitis vinifera (18), Nicotiana tabaccum(4), Helianthus annuus (10), Lilium longiflorum (20), and Zeamays (9). It has also been encountered for cytokinins, aminoacids, and several vitamins (8, 11, 22). Habituation has beenobserved, in some cases, as a gradual process at the tissue level,while in others it occurs rapidly. By comparing the growthresponse of tissues on media with and without auxin, it wasfound that during serial propagation, tissues often increase indegree of habituation (17). During prolonged culture, a fewhabituated cells arise in the tissue and then, because these cellsproliferate more rapidly than nonhabituated cells, the degree ofhabituation of the tissue increases with time. However, Buiattiand coworkers (4) obtained 100% habituation of tissue fromgerminated seeds of Nicotiana bigelonii after a very short periodof culture on a hormone containing medium. Another exampleof fast habituation is L. longiflorum which becomes auxin-independent after a short cultivation period (20). Our own workindicates that habituation can be induced rapidly in soybean(Glycine max) and that culturing of callus for a long period isnot a prerequisite in this crop. The phenomenon of habituationbears a striking similarity to tumor transformation in crown galldisease where tumor tissue grows independent of exogenoushormones. This similarity may provide an indication as to howthe capacity for autonomous growth is maintained in populationsof dividing tumor cells.Comparison of the properties of habituated and crown gall

cells leads to the conclusion that habituation is a form of neo-plastic transformation involving heritable, progressive changes

Plant Material. Soybean seeds (Glycine max cv Corsoy, Wil-liams, No. 209332, and No. 90763) were sterilized for 10 minwith commercial bleach diluted 1:5 with water and were germi-nated on filter paper.

Callus Induction. Hypocotyl-derived callus was initiated andmaintained on Gamborg's B5 medium with IAA (1 mg/L) 2,4-D (mg/L), and K (0.4 mg/L). Callus tissue was subcultured 3times at 21 d intervals prior to being used in habituation exper-iments.

Habituation Experiments. 2,4-DNP' and PiBA were incorpo-rated in Gamborg's B5 media at three concentrations (10-2, 10-6,and 10-9 M). Soybean tissue was cultured on these media withand without hormones as described in other sections. Experi-ments were terminated 30 d following incubation of callus onthese media. At that point, fresh weights were recorded, andtissue was frozen in liquid N2 for SDS gel electrophoresis proteinanalysis. Samples ofhabituated calli were incubated on hormone-free media for 140 d with a subculturing period of 28 d.Gel Electrophoresis. SDS-PAGE of soybean proteins was car-

ried out as follows. Callus tissue (75 mg fresh weight) was frozenand ground to a fine powder in liquid N2. Crude homogenateswere extracted (2:1 volume/fresh tissue weight) with buffer con-sisting of 50 mM Tris-HCl (pH 10), 4% SDS, 1% ,8-mercaptoeth-anol, 0.1% NP-40, 2.5 mM phenylmethylsulfonyl fluoride, 0.6mg/ml leupeptin, and 0.2 mg/ml each of antipain and pepstatin.The final pH of the crude extract was 7.4. Extracts were boiledfor 5 min, sonicated, and spun in a microfuge. The supernatantswere collected and subjected to SDS-PAGE as described byLaemmli (14). An estimated 100 gg of protein were loaded pertest sample, and the gels were stained with silver. Replicate gelsin which half or twice the amount of protein used in the gelshown in Figure 2 gave the same results, confirming the repro-ducibility of the assays.

'Abbreviations: 2,4-DNP, 2,4-dinitrophenol; PiBA, phenoxyisobu-tyric acid; K, kinetin (furfurylaminopurine); NAA, naphthalene aceticacid.

809

8Plant Physiol. Vol. 88, 1988

RESULTS AND DISCUSSION

We have found that 2,4-DNP and PiBA, which exhibit an-tiauxin activities (1), can induce habituation of soybean tissue invitro. In a preliminary experiment, soybean callus (4 genotypes)was placed on Gamborg's B5 medium (5) enriched with either2,4-DNP or PiBA at three different concentrations (10-2, 10-6,and IO-' M). On days 1, 4, 7, and 14 post-plating, intact piecesof callus were withdrawn from the plates and placed on eitherhormone-free media or media enriched with high cytokinin (K)concentrations which, under normal conditions, are toxic to thetissue. Two controls were included in this preliminary study: (a)soybean callus plated directly onto the 2 media (B50-hormonefree and B5K-high cytokinin) without preexposure to the com-pounds under study, and (b) tissue that remained on the originalmedia enriched with either 2,4-DNP or PiBA. Tissue which wasexposed to the two compounds at concentrations between 10-6and l0-9 M survived and grew normally when transferred tohormone-free (B50) and high cytokinin-(B5K) containing media.Tissue plated directly on B50 and B5K, without prior exposureto 2,4-DNP or PiBA, did not survive upon transfer to the twomedia whereas callus that was left on media containing either ofthe two compounds continued to proliferate 60 d following theinitial plating without any subculturing or transferto fresh media.These results are illustrated in Figure 1. We have isolated anumber of soybean callus phenotypes which sfill exhibit habit-uation 14 months after the initiation of the experiment.

Following the experiments described above, we studied hor-monal involvements during and after habituation induction ingreater detail and also investigated changes in protein patternsof various phenotypes resulting from such an experiment. Soy-bean tissue was plated on 5 induction media as indicated inTable I.At times t = 0, 4, and 24 h as well as 1O and 30 d, tissue was

transferred from each of these induction media onto four mediato study the response of the original tissue to various hormonalregimes. The four new plating media were as follows: B50(hormone-free), B5+IAA (auxin-containing media), B5K (cyto-kinin-containing media), B5+IAA+K (auxin and cytokinin-con-taining media). Five additional subcultures at 28 d intervals wereperformed in order to establish long-term habituated phenotypesfrom the various treatments. At the end of this period, tissue wasanalyzed using SDS gel electrophoresis. Tissue induced on hor-mone-free media (B50) for any period of time failed to growwhen maintained on hormone-free or when transferred to cyto-kinin-ontaining media (B5K). However, the tissue could growwell when it was transferred from B50 to either auxin- (B5+IAA)or auxin plus cytokinin-containing (B5+IAA+K) media up tothe 10 d point. Tissue incubated on hormone-free media forperiods of 30 d could not recover when plated on auxin-contain-ing media. These observations indicate that soybean callus has arequirement for auxin but not for cytokinin. In addition, thetissue can survive in the absence of auxin for at least 10 d,presumably due to utilization of stored auxin. When tissue wasincubated on auxin-containing media (B5+IAA) similar resultswere observed upon subsequent transfer to the plating media,indicating that auxin carryover from the original medium is nota major factor influencing the long-term survival of soybeancallus. It also eliminates the possibility ofattributing the observedeffects on hormonal carryover from the original callus inductionmedium (B5DIK). When either PiBA or 2,4-DNP (data notshown) were incorporated in the induction media, in the absenceof either auxin or cytokinin, the callus could proliferate on allplating media tested as long as the exposure to the compoundswas 24 h or longer. Combining either of the two compoundswith auxin in the induction media resulted in the isolation of acytokinin-requiring phenotype. This is ofinterest because normalsoybean tissue does not have a cytokinin requirement for either

FIG. 1. Induction ofhabituation in Corsoy soybean callus tissue using2,4-DNP and PiBA. Other genotypes tested responded in a similarmanner. Both compounds exhibited severe toxicity at concentrations of0.01 M. In either case, short exposure of callus tissue to the two com-pounds induced habituation. D-O indicates direct plating of callus ontoB50 (hormone-free media) and B5K (high cytokinin-containing media;K at 3 mg/L). Exposure of callus to either of the two compounds for1,4,7,11,14, and 60 d resulted in the induction of habituation in alltissues.

callus induction or proliferation. Contrary to our original obser-vations, this tissue (cytokiin-dependent) could not survive onhormone-free or even auxin-containing media in the absence ofexogenous cytokinin. The above results demonstrate that thephenotype of soybean callus can be manipulated by the use ofcertain compounds, and stable lines can be established which arehabituated to both auxin and cytokinin or are dependent oncytokinin for callus growth. Our results are summarized in TableI. Syono and Furuya (23) demonstrated the induction of auxin-habituated tobacco tissue from wild-type callus at high frequency,and its reversal by short treatments with auxin. They defined 2types of auxin-habituated tissue: type A, derived from treatmentof wild-type tissue with high concentrations of synthetic auxins(NAA and 2,4-D) for short time periods, and type B, derivedfrom wild-type tissue treated with low IAA concentrations forprolonged time periods. They reported that type B tissue couldgrow on high cytokinin-containing media in the absence ofexogenously supplied IAA, whereas type A tissue could not. Thisagrees with our experiments in which short treatments withantiauxins resulted in the recovery of not only auxin-habituatedtissue but also tissue that could grow in the presence of highcytokinin concentrations without any exogenous auxin. Theseinvestigators also reported high quantities of lignin in type Atissue as well as differences in pigment biosynthesis in the twotissue types. The habituated soybean tissue phenotypes we de-scribe here conform to the type B tissue of Syono and Furuya

810 CHRISTOU

HABITUATION IN SOYBEAN CULTURES

Table I. Experimental Design and Resultsfrom Study ofHormonal Implications in Habituation Induction(+) indicates growth of tissue on medium under study; (-) indicates that tissue could not survive on the given medium.

Induction with Platinga

Time B50 B5 + IAA B5K B5 + PiBAb B5 + IAA + PiBAb

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

0 - + - + - + - + - + - + - + - + - + - +24h - + - + - + - + - + - + + + + + - - + +10d - + + + + + + + + + + + +30d - - - - - + - + - - - - + + + + - - + +30 dc + - + + + + + - + - + + + + + NDd ND ND ND

a B50, B5 + IAA, B5K, B5 + IAA + K. b Identical results were obtained when 2,4-DNP was used in place of PiBA. c Indicates tissue plateddirectly on plating media for 30 d. d Not determined.

with reference to lignin content and pigmentation. The habit-uated soybean callus was dark green in color, as opposed to thewhite phenotype of the tobacco callus described by Syomo andFuruya (23). Lignin-like compounds were observed at the initialstages of callus proliferation, but these gradually disappeared onsubsequent subcultures of the habituated tissue.

Figure 2 illustrates some of the protein changes which occurduring induction ofhabituation by PiBA in soybean callus tissue.When the tissue was maintained on hormone-free media andcultured for up to 28 d, no growth could be observed, and theprotein pattern of such tissue is illustrated in Figure 2 (lane 1).However, when the tissue was preincubated on PiBA-containingmedia for 1 d and then transferred to hormone-free medium, analteration in the pattern ofsteady-state proteins became apparent.The most significant change observed was the accumulation ofhigher molecular weight proteins in concert with the disappear-ance of lower molecular weight proteins (Figure 2). Lanes 2 and4 illustrate the effect of cytokinin on the system. In going fromB50 to B5K versus B5+PiBA to B5K we observed a reductionin the expression of the lower molecular weight proteins, but thesynthesis of higher molecular weight proteins, which was seen inthe B5+PiBA to B50 transfer, could not be observed. Habituated

100K

70K

40.

24.5K

1 2 3 4

FIG. 2. SDS gels illustrating protein changes associated with inductionof habituation by PiBA in soybean callus tissue. Lane 1, tissue inducedon hormone-free media; lane 2, tissue transferred from hormone-free tocytokinin-containing media; lane 3, tissue transferred from PiBA-con-taining to hormone-free media; lane 4, tissue transferred from PiBA- tohigh cytokinin-containing media.

cells become capable ofsynthesizing one or more substances thatnormal cells, from which the habituated cells were derived, donot produce. Such activation of biosynthetic systems parallelsthe switching-on of introduced auxin and cytokinin pathways incrown gall cells. In the case of crown gall cells, new genes forauxin and cytokinin biosynthesis are introduced into the plantcell and are stably integrated in the plant's genome. In habituatedcells, it seems that these genes are being turned on. In both cases,cells that have become habituated for one or more essentialfactors have been found to synthesize significant amounts ofthose factors (12, 13). In addition, when cytokinin habituationis attained without a corresponding activation of the auxinsystem, the cells behave in a manner identical to cells trans-formed by certain attenuated strains of Agrobacterium (2, 15).Detailed analysis and comparison of habituation with crown gallformation has shown that habituation is inherited by individualcells and in long-term culture it leads to progressively moreautotrophic cells. Tumor proliferation in crown gall disease canbe accounted for by heritable changes in the pattern of geneexpression (16) in a manner similar to that described earlier forhabituated tissue. Synthesis of a number of proteins is elicited asa result of changes in physiology and/or metabolism in plantsand plant tissues in vitro (21 and references therein), and mostof these proteins appear as an immediate response by the tissueto an altered environment such as temperature, anoxia, osmoticstress, or wounding. Hormonal autonomy in crown gall alsoresults in major metabolic and physiological (morphological)changes in the tissue (19) in a manner similar to habituation,making the latter a useful model for the study of neoplasticphenomena in plants. The neoplastic (tumorous) state in plants,thus appears to be epigenetic in nature and both habituation andcrown gall development depend for their expression on thepersistent, but suppressible, activation of select genes that arepresent in a cell.

Acknowledgments-I would like to thank Barbara Keller for excellent technicalassistance, Joseph Burkholder for SDS gel electrophoresis, and Kenneth Barton fora critical review of the manuscript

LITERATURE ClTlED

1. AMMIRATO PV 1983 Embryogenesis. In Handbook of Plant Cell Culture.Techniques for Propagation and Breeding. MacMillan, New York, pp 1-101

2. BRAUN AC, T LASKARIS 1942 Tumor formation by attenuated crown-gallbacteria in the presence ofgrowth-promoting substances. Proc Natl Acad SciUSA 28: 468

3. BRAUN AC 1978 Plant tumors. Biochim Biophys Acta 516: 167-1914. BUIATI M, A BENNIcI 1970 Callus formation and habituation in Nicotiana

species in relation to the specific ability for dedifferentiation. Rend AccadNaz Lincei 48: 261-269

5. GAMBORG OL, RA MILLER, K OJIMA 1968 Nutritional requirements of sus-pension cultures of soybean root cells. Exp Cell Res 50: 151-158

6. GAUTERET RJ 1942 Hetero-auxines et cultures de tissus v6g6taux. Bull SocChim Biol 24: 13-46

7. GAUTHERET R 1948 Sur la culture de trois types de tissus de Scorsonere; tissues

811

Plant Physiol. Vol. 88, 1988normaux, tissus de crown gall et tissus accountoumes a' 1' hetero-auxine. CR Acad Sci C Paris Ser D 226: 270-271

8. GAUTHERET RJ 1955 The nutrition of plant tissue cultures. Annu Rev PlantPhysiol 6: 433-484

9. HAWES MC, DZ SHARPE, MI PLATA, SG PUEPPKE, PS CHOUREY 1985 Auxin-independent growth of maize tissue culture cells. Plant Sci 40: 197-202

10. HENDERSON JHM 1954 The changing nutritional pattern from normal tohabituated sunflower callus tissue in vitro. Ann Biol 30: 329-348

11. IKEDA M, K OJIMA, K OSHIRA 1979 Habituation in suspension-culturedsoybean cells to thiamine and its precursors. Plant Cell Physiol 20: 733-739

12. KULESCHA Z, R GAUTHERET 1948 Sur 1 'elaboration de substances de croiss-ance par 3 types de cultures de tissus de Scorsonere: cultures normales,cultures de crown gall et cultures accoutoumees a 1'hetero-auxine. C R AcadSci Paris 227: 292-294

13. KULESCHA Z 1952 Recherches sur 1'elaboration de substances de croissancepar les tissus vegetaux. Rev Gen Bot 59: 19-41

14. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

15. LOCKE SB, AJ RICKER, BM DUGGART 1938 Growth substance and the devel-opment of crown gall. J Agric Res 57: 21-39

16. MEINs F JR, A BINNS 1977 Epigenetic variation of cultured somatic cells:Evidence for gradual changes in the requirement for factors promoting celldivision. Proc Natl Acad Sci USA 74: 2928-2932

17. MEINZ F 1982 Habituation of cultured plant cells. In Molecular Biology ofPlant Tumours. Academic Press, New York, pp 3-31

18. MOREL G 1947 Transformations des cultures de tissus de Vigne produites parl'hetero-auxine. C R SOC Biol 141: 280-282

19. MORRIs RO, DE AKIYOSHI, EMS MCDONALD, JW MORRis, DA REGIER, JBZAERR 1982 Cytokinin metabolism in relation to tumor induction by Agro-bacterium tumefaciens. In P Wareing, ed, Plant Growth Substances, Pro-ceedings of the 11th International Conference, Academic Press, London

20. SCHERIDAN WF 1968 Tissue culture of the monocot Lilium . Planta 82: 189-192

21. SINGH NK, AK HANDA, PM HASEYAWA, RA BRESSAN 1985 Proteins associatedwith adaptations of cultured tobacco cells to NaCl. Plant Physiol 79: 126-137

22. STEWARD FC 1969 Growth in organized and unorganized systems-knowledgegained by culture of organs and tissue explants. In FC Steward, ed, PlantPhysiology, Vol Sb: Academic Press, New York, pp 3-224

23. SYoNo K, T FURUYA 1974 Induction of auxin-nonrequiring tobacco callusand its reversal by treatment with auxins. Plant Cell Physiol 15: 7-17

812 CHRISTOU