Plant Cell, Tissue and Organ Culture 68: 225–232, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

225

A specific role for spermidine in the initiation phase of somaticembryogenesis in Panax ginseng CA Meyer

Marta Monteiro, Claire Kevers∗, Jacques Dommes & Thomas GasparPlant Molecular Biology and Hormonology, Institute of Botany B 22, University of Liege, Sart Tilman, B-4000Liege, Belgium (∗requests for offprints; Fax: 32-4-366-3859; E-mail: c.kevers@ulg.ac.be)

Received 18 April 2000; accepted in revised form 30 January 2001

Key words: polyamines, polyamine oxidase, spermidine

Abstract

Somatic embryogenesis of Panax ginseng CA Meyer was initiated from suspension aggregates of an embryogeniccallus, in a liquid medium consisting of half strength Murashige and Skoog (1962) supplemented with the syntheticauxin benzoselenienyl-3 acetic acid. The addition of spermidine to this initiation medium significantly increasedthe production of somatic embryos. In this case, the total polyamine content of the embryogenic mass was higherthan that of cultures without spermidine. At day 6 of the culture, a transient accumulation of free polyamines,mainly spermidine, was observed. After this peak, free and conjugated polyamines levels did not show significantvariation nor did the polyamine oxidase activity. The results clearly demonstrated that spermidine supplied to themedium was oxidised by polyamine oxidase and partially metabolised into putrescine. The role of spermidine andits interaction with auxin in the initiation of the embryogenic process in Panax ginseng are discussed in relation toembryogenic potential.

Abbreviations: BSAA – benzoselenienyl-3 acetic acid; 2,4-D – dichlorophenoxyacetic acid; dcSAM – de-carboxylated S-adenosylmethionine; IAA – indole acetic acid; Kin – kinetin; MS – Murashige and Skoog (1962);PAO – polyamine oxidase; PUT – putrescine; SPD – spermidine; SPM – spermine

Introduction

Panax root has been used in Oriental medicine sinceancient times. The crude root extract is known to havetonic, stimulatory and adaptogenic properties (Hu,1976) due to the presence of a wide range of sapon-ins and sapogenins (Li, 1995). Recently, ginseng hasbecome a popular tonic and health food complement inWestern countries. Therefore, the demand for the planthas increased dramatically worldwide. Ginseng is veryexpensive because of its long-term conventional (5–7years) and troublesome production cycle. As a result,propagation methods of ginseng by plant tissue cultureand particularly by somatic embryogenesis have beeninvestigated. Somatic embryogenesis has been suc-cessfully induced on solid media, directly from root(Chang and Hsing, 1980; Asaka et al., 1992, 1993a,Tirajoh et al., 1998), leaf (Tirajoh et al., 1998) orflower bud derived calli (Shoyama et al., 1997) or dir-

ectly from zygotic embryos (Choi and Soh, 1996) orcotyledons (Choi and Soh, 1994). Culture of ginsengtissues in bioreactors was developed in order to pro-duce fresh material containing ginsenoside saponins(Asaka et al., 1993b). However initiation of somaticembryogenesis in liquid culture has only recently beendescribed (Kevers et al., 2000).

Spermidine (SPD), spermine (SPM) and theirdiamine obligate precursor, putrescine (PUT), aresmall aliphatic polyamines that are ubiquitous in allliving cells. Although the precise mode of action ofpolyamines are yet to be understood (Walden et al.,1997), extensive studies reported their role in a widerange of biological and physiological processes (Gal-ston and Kaur-Sawhney, 1990, Martin-Tanguy et al.,1997) Tiburcio et al., 1997, Walden et al., 1997).Polyamines have been recognised as a new class ofgrowth substances (Bagni and Torrigiani, 1992).

226

Plants possess two parallel pathways to synthesisePUT, one from ornithine mediated by ODC (ornithinedecarboxylase) activity, and the second, from argininemediated by ADC (arginine decarboxylase) activity.dcSAM is the aminopropyl donor to PUT and SPDto produce SPD and SPM, respectively. Polyaminesare catabolised by specific oxidases: PUT by diamineoxidases, SPD and SPM by polyamine oxidases (PAO,Smith 1985).

In recent years, research on polyamines has in-creased tremendously and has provided clues to im-prove plant developmental processes, including so-matic embryogenesis, in a variety of economicallyimportant crops. Several reports have shown the in-volvement of polyamines, particularly in their freeforms, in somatic embryogenesis. High concentra-tions of polyamines were commonly observed in tis-sues undergoing somatic embryogenesis (Santanenand Simola, 1992; Yadav and Rajam, 1997; Minochaet al., 1999; Kevers et al., 2000). Furthermore, thereduction of the endogenous free polyamines by useof inhibitors of polyamine biosynthesis concomitantlyinhibited somatic embryogenesis (Feirer et al., 1985;Hadrami and D’Auzac, 1992; Kevers et al., 2000),indicating the direct role of polyamines in this process.SPD more particularly has been implicated in somaticembryogenesis in tissue cultures of carrot (Feirer etal., 1984, 1985), Vigna (Kaur-Sawhney et al., 1988),Hevea (Hadrami and D’Auzac, 1992) and during theinduction phase in alfalfa explants (Cvikrová et al.,1999).

The aim of the present study was to further ex-tend our understanding of the role of polyaminesparticularly spermidine and its interaction with auxin(BSAA), on the initiation of the embryogenic pro-cess in Panax ginseng callus. For this purpose, thetemporal changes in free and conjugated polyaminesand the PAO activity were monitored during the initi-ation stage of somatic embryogenesis and discussed inrelation to embryogenic potential.

Materials and methods

Plant material and culture media

One-year-old roots of Panax ginseng CA Meyer weresurface sterilised successively in ethanol (70%) for 3min and in sodium hypochlorite (3%) for 20 min, andrinsed three times with sterile distilled water.

Embryogenic calluses were initiated from disinfec-ted root sections of 3 mm length which were cultured

on a solid MS basal medium (Murashige and Skoog,1962) supplemented with 2,4-dichlorophenoxyaceticacid (2,4-D) (0.1 mg l−1 = 0.45 µmol). The cultureswere incubated in darkness at 25 ± 2 ◦C in 9-cmdiameter plastic dishes for 6 weeks. The embryogeniccallus was subcultured onto a solid MS basal medium,the latter diluted two times and supplemented withbenzoselenienyl-3 acetic acid (BSAA, from Acros Or-ganics, Belgium) (1 mg l−1 = 4.18 µmol) and kinetin(from Sigma, USA) (0.3 mg l−1 = 1.40 µmol). Em-bryogenic capacity of the callus was maintained bysubculture at 4-week intervals on the same medium.

Multiplication in liquid medium

Finely minced callus was transferred to liquid mediumin 100 ml Erlenmeyer flasks, at a ratio of 2 g 50ml−1 medium. The cultures were multiplied in half-strength MS (MS/2) medium supplemented with 1 mgl−1 BSAA and 0.3 mg l−1 kinetin for one month andshaken at 80 rpm at 25±2 ◦C in darkness.

Embryogenesis initiation in liquid medium

For embryogenesis initiation, suspension aggregateswere transferred to a MS/2 liquid medium supplemen-ted with BSAA (3 mg l−1 = 12.55 µmol) and with orwithout SPD (10−4mol).

Free and conjugated polyamine levels

The samples collected 0, 3, 6, 9, 12 and 15 days aftertransfer to embryogenesis initiation medium wereground in liquid nitrogen and stored at −20 ◦C. Ex-traction, separation, identification and measurementof polyamine by direct dansylation and HPLC wereperformed as described by Walter and Geuns (1987).TCA-soluble and insoluble conjugated polyamine ex-traction, was done as described by Scaramagli etal. (1999). The separation and measurement wereperformed as described by Walter and Geuns (1987).

[14C]-SPD incorporation in the media

In addition to 3 mg l−1 BSAA and 10−4mol SPD,double labelled [1,4-14C]-SPD 4.14 GBq mmol−1

(Amersham Life Science) was incorporated into themedium at a specific activity of 18.9 MBq mmol−1.Analyses of free polyamine levels were carried outas described above and 0.5 ml HPLC fractions werecollected. Fractions were added to 1 ml scintilla-tion solution (Ecoscint A, National Diagnostics) forradioactivity measurement.

227

Figure 1. Changes in the endogenous levels of polyamine during Panax ginseng embryogenic callus culture: (A) Total free polyamines in thepresence of 3 mg l−1 BSAA with or without 10−4mol SPD; (B) Free PUT, SPD and SPM in the presence of 3 mg l−1 BSAA and 10−4molSPD (n=3, mean ± SE).

PAO activity

Samples were ground in a chilled mortar at a ratio of0.5 g fresh weight ml−1 of 100 mmol Tris HCl (pH7.5) buffer containing 5 mmol EDTA, 10 mmol mer-captoethanol, 1 mmol pyridoxal phosphate, 5 mmoldithiothreitol, 0.5 mol KCl. The extracts were filteredand washed with 500 µl buffer. The filtered fractionswere then dialysed against two litres of 10 mmol Tris-HCl (pH 8) containing 1 mmol EDTA and 10 mmolmercaptoethanol for 8 h.

All the procedures were carried out at 4 ◦C. Thedialysed extracts were used to determine the PAOactivity.

PAO activity was determined by a radiochemicalmethod that measured the [14C] �1-pyrroline forma-tion from [1,4-14C]-SPD. After 15 min preincubationat room temperature, aliquots (50 µl) of extract wereincubated for 1 h at 30 ◦C in a final volume of 0.1ml containing 7.4 KBq of [1,4-14C]-SPD, 0.1 molTris-HCl (pH 8.5), 2.5 mmol SPD and 0.3 mg ml−1

catalase. At the end of incubation, 0.2 ml sodiumcarbonate was added to the mixture and the [14C]-�1-pyrroline immediately extracted in 4 ml toluene byvortexing for 20 sec and centrifuging for 5 min at 2000× g. After this, 1 ml of the toluene phase was addedto scintillation vials containing 4 ml of scintillation

liquid (Ecoscint 0, National Diagnostics) for count-ing in a LS 5000 scintillation counter (Beckman),programmed for tritium determination.

Protein analysis

Soluble protein was determined according to Bradford(1976). Bovine serum albumin was used as standard.

All results are the means of measurements in atleast 3 separate experiments.

Results

Influence of exogenous spermidine

In the previous work by Kevers et al. (2000), the addi-tion of SPD (10−4 mol) to the initiation medium con-taining BSAA was shown to significantly increase theproduction of somatic embryos. Respectively, 20±3and 98±4 somatic embryos were formed from 2.5 gof callus without and with SPD (10−4 mol). Theseresults were reproduced here and formed the basis ofthe present investigation.

228

Figure 2. Changes in the conjugated (TCA-soluble and insoluble) polyamine levels during 15-day cultures of Panax ginseng embryogeniccallus in the presence of 3 mg l−1 BSAA with (A and B) or without (C and D) 10−4mol SPD (n=3, mean ± SE).

Free polyamine levels in embryogenic callus

To assess the role of polyamines in the control ofsomatic embryogenesis, free and conjugated polyam-ine levels were monitored over the 15 days followingtransfer to inductive conditions. First, free polyaminelevels were analysed in embryogenic calluses culturedon medium supplemented with BSAA + SPD and

on medium supplemented with BSAA alone for thecontrol.

The total free polyamine content was stable in thecontrol material, at a low level of about 4 µmol g−1

fresh weight. In embryogenic calluses cultured inBSAA + SPD medium, a transient accumulation offree polyamines was observed at the 6th day of culture(up to 75 µmol g−1 fresh weight). Thereafter, the free

229

polyamine content decreased down to the levels ob-served in control calluses on day 15 (Figure 1A). Thepeak of total free polyamine at the 6th day was mainlydue to an increase in SPD content, the contributions ofPUT and SPM being lower (Figure 1B).

Conjugated polyamine levels

The conjugated polyamine levels in the TCA-solubleand insoluble fractions were determined in the em-bryogenic callus cultured on BSAA and SPD me-dium or on medium with BSAA alone. On the lattermedium, both TCA-soluble and insoluble fractionscontained very low levels of polyamines over the 15days of culture (Figure 2C and D).The highest level(0.2 µmol g−1 fresh weight) was observed for PUTon day 9 in the TCA-soluble fraction. Higher levelsof TCA-soluble polyamines were observed when em-bryogenic calluses were cultured on the medium sup-plemented with BSAA and SPD. A transient accumu-lation of PUT and SPD was shown to occur betweenthe 6th and the 12th day of the culture period (Fig-ure 2A). However these levels were lower than in thecorresponding free fractions (Figure 1).

PUT was the most abundant polyamines in theTCA-soluble fraction, showing two peaks during theculture period. The highest peak was by the 6th day,and a smaller one at the 12th day (Figure 2A). Thesignificant decrease in PUT on the 9th day was con-comitant with an increase in the TCA-insoluble frac-tion (Figure 2B). The level of TCA-soluble SPD wasfar lower than the level of free SPD, but they showedparallel changes. SPM was by far the less abundantof the conjugated polyamine and did not show anysignificant variation during the entire culture period(Figure 2).

PAO activity in embryogenic callus

A dramatic transient increase in the level of freeSPD was monitored in embryogenic callus grown onmedium containing SPD. The reasons for its sharp de-crease after the 6th day were questioned by checkingfor changes in PAO activity.

The PAO activity was assayed in calluses culturedin the presence of the auxin BSAA alone and in thepresence of both BSAA and SPD. In the latter condi-tions, a peak of PAO activity was observed on the 6thday (Figure 3). In the medium without SPD, a sim-ilar peak was observed on the 12th day and a smallerone on the 6th day. SPD degradation by PAO thus oc-curred earlier in medium containing both BSAA and

Figure 3. Time-course of changes in PAO activity in an embryo-genic callus cultured in basal medium in the presence of 3 mg l−1

BSAA with or without 10−4mol SPD (n=3, mean ± SE).

Figure 4. Changes in the exogenous levels of [14C]-SPD during15-day culture of Panax ginseng embryogenic callus on a mediumcontaining 3 mg l−1 BSAA and 10−4 mol SPD, supplemented with456 nmol [14C]-SPD (n=3, mean ± SE).

230

Figure 5. Changes in the endogenous levels of free [14C]-PUT,[14C]-SPD and [14C]-SPM during 15-day culture of Panax ginsengembryogenic callus in a medium containing 3 mg l−1 BSAA and10−4mol SPD supplemented with 456 nmol [14C]-SPD (n=3, mean± SE).

SPD than in medium without SPD. The rapid drop inendogenous SPD after the 6th day of culture on BSAAplus SPD could be correlated with an increase in PAOactivity.

[14C]-SPD incorporation and free polyamine analysis

In order to elucidate the mechanism by which SPDdrastically increased at day 6 and decreased after, thefate of fed radioactive SPD was monitored.

When calluses were cultured in a medium con-taining [14C]-SPD, decrease of radioactivity in themedium was shown to occur after 3 days and to con-tinue throughout the culture period (Figure 4). Anearly accumulation of free [14C]-SPD in callus cellswas observed, with highest levels on day 3 and day6 (Figure 5). Thereafter, the free [14C]-SPD level incallus cells decreased. Labelled SPM was detected ata low level during the first days but showed steadilyincreasing levels throughout the 15 days of culture.The dramatic decrease of free [14C]-SPD after the 6thday was partially due to its metabolisation into PUT,which increased from the day 0 to a maximum on 12thday of culture (Figure 5).

Free polyamine levels in non embryogenic callus

In order to correlate the SPD level with somatic em-bryogenesis initiation, polyamine content was meas-ured in a non-embryogenic callus cultured in the samemedia as the embryogenic callus. Total free polyaminelevels in the former showed almost the same patternof changes as the latter. Peaks were observed in bothmaterials on day 6 and on day 12 (Figure 6A). How-ever on day 6, more polyamines accumulated in theembryogenic callus than in the non-embryogenic one(Figure 6A).

In the non-embryogenic callus, the time course ofchanges in the level of the different free polyamines(Figure 6B) showed that PUT was the main speciesthroughout the culture time. This points to a major dif-ference between embryogenic and non-embryogeniccalluses: the former accumulated mainly SPD on day6 and day 12 (Figure 1B); whereas, the latter accumu-lated mainly PUT but to a lesser extent. SPM level wasmaximum on day 0, then decreased to reach a very lowvalue on day 9. This low level of SPM was observedthroughout the second half of the culture period. SPDlevel showed moderate fluctuations, with a maximumobserved on day 6.

Thereafter, both embryogenic and non-embryogeniccalluses showed transient increases in free polyam-ine levels on day 6 and on day 12 after transferto induction medium (containing BSAA and SPD).However, the relative contribution of the differentpolyamines differed between these materials. The em-bryogenic callus mainly accumulated SPD; whereas,the non-embryogenic callus mainly accumulated PUT.

Discussion

Earlier work with embryogenic callus of Panax gin-seng had shown that exogenous application of polyam-ines in the initiation phase increased the number ofembryos produced (Kevers et al., 2000). It was con-firmed here that among the polyamines, SPD was themost effective (Kevers et al., 2000).

The present study demonstrated a correlationbetween increased endogenous SPD and embryogeniccapacity. A peak of endogenous SPD on the 6thday of culture was observed in the embryogenic cal-lus cultured on the medium with SPD. In the non-embryogenic callus, cultured in the same medium, nosuch SPD peak was seen; instead a transient accumu-lation of PUT occurred, but to a lesser extent.

231

Figure 6. Changes in the endogenous levels of polyamines during 15-day culture in the presence of 3 mg l−1 BSAA and 10−4mol SPD:(A) Total polyamines of Panax ginseng embryogenic callus (EC) and non embryogenic callus (NEC); (B) Free PUT, SPD and SPM of Panaxginseng non-embryogenic callus (n=3, mean ± SE).

Related studies with wild carrot have shown thatSPD alone can restore embryogenesis in culturestreated with polyamine biosynthesis inhibitors, indic-ating a direct role of SPD in somatic embryogenesis(Feirer et al., 1985; Hadrami and D’Auzac, 1992).Such a role of SPD in somatic embryogenesis has alsobeen reported by Tiburcio et al. (1985), Santanen andSimola (1992), Bonneau et al. (1995) Minocha et al.(1999) and Cvikrová et al. (1999). Indeed they ob-served a significant increase in SPD levels associatedwith the formation of somatic embryos in tobacco,Picea abies, Euonymus europeaus L., Pinus radiataand alfalfa explants. SPD was also the most abund-ant polyamine in conditions that allow cell culturesof Papaver somniferum to form embryo-like structures(Nabha et al., 1999).

The increase of endogenous SPD, from day 3 (Fig-ure 1), could be correlated with an incorporation intothe tissues (Figure 5) of this compound present in themedium. Indeed, the level of SPD in the medium de-creased (Figure 4) from day 3. But the amount of thispolyamine in the medium was not sufficient to explainthe endogenous increase. Thus, a synthesis of addi-tional SPD might also occur. The values of the specificactivity (57 Bq mg−1 in the tissues and 130 Bq mg−1

in the medium) also confirmed this pattern. In the sametime, the activity of PAO increased (Figure 3) and was

partially responsible for the decrease of free SPD after6 days while free SPM levels (Figure 1) and conjug-ated polyamine levels (Figure 2) did not significantlyincrease after the SPD peak. Those pathways couldnot be implicated in the decrease of SPD in the tissue.Moreover, our results (Figure 5) clearly demonstratedthat [14C]-SPD supplied to the medium was partiallymetabolised to PUT. In plants this metabolic pathwayis not established. However, studies in tobacco tissuecultured with [3H]-SPD reported that about 14% of thelabel was metabolised to PUT (Apelbaum et al., 1988).In animals this is a well established pathway; a set ofreactions of acetylation and oxidation allow cells toconvert some SPD into PUT (Altman and Levin, 1993;Tiburcio et al., 1997).

The necessity of a powerful auxin as BSAA to al-low the stimulation of organogenic potency by SPDhas never been reported. Exogenous cytokinin applica-tion, is known to increase the endogenous level of IAA(Coenen and Lomax, 1997) and to favour an auxin tocytokinin ratio suitable for embryogenesis (Charrièreet al., 1999). In the absence of cytokinin, the exo-genously applied synthetic auxin BSAA may directlycompensate for an insufficient endogenous IAA level.

232

Acknowledgements

M. Monteiro, from the University of Aveiro (Por-tugal), is greatly indebted to the European Communityfor an Erasmus grant allowing her to work at the Uni-versity of Liège. This research was financed by ORTISLaboratories (Elsenborn, Belgium) and the WallonianMinistry for Technology.

References

Altman A & Levin N (1993) Interactions of polyamines and nitro-gen nutrition in plants. Physiol. Plant. 89: 653–658

Apelbaum A, Canellakis ZN, Applewhite PB, Kaur-Sawhney R &Galston AW (1988) Binding of spermidine to a unique protein inthin-layer tobacco tissue culture. Plant Physiol. 88: 996–998

Asaka I, Ii I, Yoshikawa T, Hirotami M & Furuya T (1992) Em-bryoid formation by high temperature treatment from multipleshoots of Panax ginseng. Planta Med. 59: 345–346

Asaka I, Ii I, Hirotami M, Asada Y, Yoshikawa T & Furuya T(1993a) Mass production of ginseng (Panax ginseng) embryoidson media containing high concentrations of sugar. Plant Med. 60:146–148

Asaka I, Ii I, Hirotami M, Asada Y & Furuya T (1993b) Productionof ginsenoside saponins by culturing ginseng (Panax ginseng)embryogenic tissues in bioreactors. Biotech. Lett. 15: 1259–1264

Bagni N & Torrigiani P (1992) Polyamines: a new class of growthsubstances. In: Karssen CM, van Loon LC & Vreugdenhil D(eds) Progress in Plant Growth Regulation (pp 264–275). KluwerAcademic Publishers, Dordrecht

Bonneau L, Beranger-Novat N, Monin J & Martin-Tanguy J (1995)Stimulation of root and somatic embryo production in Euonymuseuropaeus L. by an inhibitor of polyamine biosynthesis. PlantGrowth Regul. 16: 5–10

Bradford MM (1976) A rapid and sensitive method for the quanti-fication of microgram quantities of protein utilising the principleof protein-dye binding. Anal. Biochem. 72: 248–254

Chang WC & Hsing YI (1980) Plant regeneration through somaticembryogenesis in root-derived callus of ginseng (Panax ginsengC.A. Meyer). Theor. Appl. Genet. 57: 133–135

Charriere F, Sotta B, Miginiac E & Halne G (1999) Induction ofadventitious shoots or somatic embryos on in vitro cultured zy-gotic embryos of Helianthus annuus: Variation of endogenoushormone levels. Plant Physiol. Biochem. 37: 751–757

Choi YE & Soh WY (1994) Origin of somatic embryo induced fromcotyledons of zygotic embryos at various developmental stagesof ginseng. J. Plant Biol. 37: 365–370

Choi YE & Soh WY (1996) Effect of plumule and radicule on so-matic embryogenesis in the cultures of ginseng zygotic embryos.Plant Cell Tissue Org. Cult. 45: 137–143

Cvikrová M, Binarová P, Cenklová V, Eder J, Machácková I (1999)Reinitiation of cell division and polyamine and monoaminelevels in alfalfa explants during somatic embryogenesis. Physiol.Plant. 105: 330–337

Coenen C & Lomax TL (1997) Auxin-cytokinin interactions inhigher plants: old problems and new tools. Trends Plant Sci. 2:351–356

Feirer RP, Mignon G & Litway JD (1984) Arginine decarboxylaseand polyamines required for embryogenesis in the wild carrot.Science 223: 1433–1435

Feirer RP, Wann SR & Einspahr DW (1985) The effects of sper-midine synthesis inhibitors on in vitro plant development. PlantGrowth Regul. 3: 319–327

Galston AW & Kaur-Sawhney R (1990) Polyamines in plantphysiology. Plant Physiol. 94: 406–410

Hadrami I & D’Auzac J (1992) Effects of polyamine biosyntheticinhibitors on somatic embryogenesis and cellular polyamines inHevea brasiliensis. J. Plant Physiol. 140: 33–36

Hu SY (1976) The genus Panax (ginseng) in Chinese medicine.Econ. Bot. 30: 11–28

Kaur-Sawhney R, Shekhawat NS & Galston AW (1988) Polyam-ine levels as related to growth, differentiation and senescencein protoplast-derived cultures of Vigna aconitifolia and Avenasativa. Plant Growth Regul. 3: 329–337

Kevers C, Le Gal N, Monteiro M, Dommes J & Gaspar Th (2000)Somatic embryogenesis of Panax ginseng in liquid cultures: arole for polyamines and their metabolic pathways. Plant GrowthRegul. 31: 209–214

Li TSC (1995) Asian and American ginseng: a review. Hort.Technology 5: 27–34

Martin-Tanguy J, Aribaud M, Carré M & Gaspar T (1997) ODC me-diated biosynthesis and DAO-mediated catabolism of putrescineinvolved in rooting of Chrysanthemum explants in vitro. PlantPhysiol. Biochem. 35: 595–602

Minocha R, Smith DR, Reeves C, Steele KD & Minocha SC (1999)Polyamine levels during the development of zygotic and somaticembryos of Pinus radiata. Physiol. Plant. 105: 155–164

Murashige T & Skoog F (1962) A revised medium for rapid growthand bioassays with tobacco tissue culture. Physiol. Plant. 15:473–497

Nabha S, Lamblin F, Gillet F, Lourain D, Fliniaux M, David A &Jacquin A (1999) Polyamine content and somatic embryogen-esis in Papaver somniferum cells transformed with sam-1 gene.J. Plant Physiol. 154: 729–734

Santanen A & Simola LK (1992) Changes in polyamine metabolismduring somatic embryogenesis in Picea abies. J. Plant Physiol.147: 145–153

Scaramagli S, Biondi S, Capitani F, Gerola P, Altamura MM &Torrigiani P (1999) Polyamine conjugate levels and ethylene bio-synthesis: inverse relationship with vegetative bud formation intobacco thin layers. Physiol. Plant. 105: 367–376

Shoyama Y, Zhu XX, Nakai R, Shiraishi S & Kohda H (1997) Mi-cropropagation of Panax notoginseng by somatic embryogenesisand RAPD analysis of regenerated plantlets. Plant Cell Rep. 16:450–453

Smith TA (1985) Polyamines. Annu. Rev. Plant Physiol. 36: 117–143

Tiburcio AF, Kaur-Sawhney R, Ingersoll RB & Galston AW(1985)Correlation between polyamines and pyrrolidine alkaloids indeveloping tobacco callus. Plant Physiol. 78: 323–326

Tiburcio AF, Altabella T, Borrel A & Masgrau C (1997) Polyaminemetabolism and its regulation. Physiol. Plant. 100: 664–674

Tirajoh A, Kyung TS & Punja ZK (1998) Somatic embryogenesisand plantlet regeneration in american ginseng (Panax quinquefo-lium L.). In Vitro Cell. Dev. Biol. 34: 203–211

Walden R, Cordeiro A & Tiburcio AF (1997) Polyamines: smallmolecules triggering pathways in plant growth and development.Plant Physiol. 113: 1009–1013

Walter H & Geuns J(1987) High speed analysis of polyamines inplant tissues. Plant Physiol. 83: 232–234

Yadav JS & Rajam MV (1997) Spatial distribution of free andconjugated polyamines in leaves of Solanum melongena L. asso-ciated with differential morphogenetic capacity: efficient somaticembryogenesis with putrescine. J. Exp. Bot. 313: 1537–1545