Plant Growth Regulation30: 133–138, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

133

Promotion by Jasmonic acid of bulb formation in shoot cultures ofNarcissus triandrusL.

Isabel Santos∗ & Roberto SalemaDepartamento de Botânica and Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do CampoAlegre, 823, 4150–180 Porto, Portugal (∗author for correspondence, Instituto de Biologia Molecular e Celular(IBMC) Rua do Campo Alegre, 823; 4150-180 Porto Portugal, e-mail: isantos@ibmc.up.pt)

Received 31 May 1999; accepted in reivsed form 18 August 1999

Key words:bulb formation, Jasmonic acid, micropropagation,Narcissus triandrus, tissue cultures

Abstract

The in vitro bulb formation by shoot clump cultures ofN. triandruson media with jasmonic acid (JA) alone orin association with both 2-isopentenyladenine (2-iP) or naphthalene acetic acid (NAA) and on medium with onlyNAA was studied. The media with JA plus 2-iP or JA plus NAA caused a high multiplication of leaves, significantlyhigher on that with 2-iP. Leaf proliferation was low on medium with JA alone. The media containing JA promotedthe formation of a bulb at the base of the leaves and the bulbs attained different sizes. On the medium with JA plusNAA, the number of bulbs that reached up to 5 mm in diameter was higher than that on the medium with JA plus2-iP, but the highest number was formed on the medium with JA alone. The medium with NAA alone led to theformation of few small bulbs that were elongated instead of roundish as those formed on media with JA. Furtherculture of the bulbs on a growth medium suitable to their enlargement increased their size but the dimensionsattained were dependent upon the medium initially used for bulb formation. Bulbs derived from medium with JAalone attained the largest diameter. Almost all the bulbs grown on the growth medium were rooted. Data describedhere show that JA promotesin vitro bulb formation in shoot cultures ofN. triandrusand suggests that JA mightplay an important role in the formation and enlargement of bulbs inNarcissusplants.

1. Introduction

Natural vegetative propagation ofNarcissusis veryslow, at best 1.6 per annum. Multiplication can beimproved through micropropagation procedures andvarious techniques have been proposed for a morerapid propagation of narcissus [13, 15]. The finalproduct to be transplanted must be the bulbs sincethey survive much better when transferred to soilthan rooted shoots [14]. As result of the increasedcommercial interest in someNarcissuswild species,the naturally growing bulbs of these species havebeen subjected to uncontrolled harvest. Since naturalvegetative propagation does not make up for the num-ber of bulbs collected every season, replanting within vitro produced bulbs seems the way to prevent theeradication of some wild species of narcissus in certainlocales. To obtain a highin vitro yield of bulbs, it is

necessary to determine the culture conditions adequatefor bulb induction and also for their growth. Our workhas been developed with the aim of finding a methodfor the in vitro production of bulbs of threatenedNar-cissuswild species in our country. In previous studies,the in vitro procedure capable of the production ofbulbs with a high transplantation success and withthe capability of flowering during the first season ofgrowth was established forN. bulbocodium[13]. How-ever, the use of the same procedure for bulb productionof bothN. triandrusandN. rupicolawas not success-ful. This failure led us to search an alternate protocolfor bulb production ofN. triandrus.

Jasmonic acid (JA) influences a multiplicity ofphysiological processes in plants by inhibition, pro-motion or induction of various phenomena [2, 6, 9and references therein]. Jasmonates are also potentinducers of vegetative storage protein gene expression.

134

The physiological role of jasmonates in tuber forma-tion of potato and yam plants has been reported [5,18]. Involvement in bulb formation is presumed fromthe fact that JA induces bulb formationin vitro [6].In onion plants it was reported that the bulb forma-tion involves the disruption of cortical microtubulesin the cells of the leaf sheath [8] and the bulb forma-tion is thought to be regulated by hormones. Since JAhas microtubule-disrupting activity in cultured cells oftobacco and potato [1, 7] it was suggested that JA isa bulbing hormone. Nojiri et al. [10] compared thelevel of JA in bulbing and non-bulbing onion plants.They found that the level of JA in bulbing onion plantswas about three times higher than that of non-bulbingonion. However, because JA applied at concentrationof 10−4-10−6 M did not cause the bulbing of onionleaf sheaths, Nojiri and coworkers [10] noted that therelationship between endogenous JA and developmentof onion bulbs remained to be clarified. On the otherhand, the culture of excised roots from onion seedlingson a medium with 10−5 M of JA led to bulb formation[6] and other researchers also found that JA stimulatedbulb formation of garlicin vitro [12]. Bearing in mindall these data, the present study was undertaken toexamine the effect of JA in the formation of bulbs incultures of shoot clumps ofN. triandrus. Since con-tradictory data is available on the effect of NAA onin vitro bulb formation ofNarcissus[3, 17] this auxinwas also used in our study.

2. Material and methods

2.1 Plant material

Previously developed shoot clump cultures ofNar-cissus triandrusL. were used to study the effectof JA on bulb formationin vitro. Shoot cultureswere obtained using twin-scales as primary explantsand the culture procedure followed was similar tothat we previously reported forN. bulbocodium[13],also using an equal modified Murashige and Skoog(MS) medium [4] but supplemented with 10 mgl−1 of BA (benzylaminopurine) plus 1.0 mg l−1 ofNAA (naphthalene acetic acid). The shoot clump cul-tures were grown in a controlled growth chamberat constant 18± 0.1◦C under a 16 h photoperiod(80 µmoles m−2s−1 PAR, supplied by fluorescentlamps Sanyo FL40SS.W/37) and they were transferredto fresh medium every six weeks.

2.2 Culture media, shoot proliferation and bulbformation

MS medium with 3% sucrose was supplemented withgrowth regulators (Sigma, USA), giving the followingfour media:

MS1B with JA (1.0 mg l−1); MS1C with NAA(0.20 mg l−1); MS1 with JA (1.0 mg l−1) +2-iP (1.0 mg l−1) and MS1A with JA (1.0 mgl−1)+NAA (0.12 mg l−1).

The media were solidified with 0.6% agar (Sicomol,Portugal) and autoclaved at 121◦C (0.1 MPa). JA wasfilter sterilized and added to the media after autoclav-ing. The established shoot clump cultures providedthe inocula to be transferred onto these four media.Since the bulb is formed at the base of leaves, theability of the four media to induce leaf prolifera-tion was evaluated. To do this, at the transfer ofshoot clumps to these new fresh media the leaveswere trimmed (leaving a length of about 2 mm) andkept in petri dishes for further counting. The materialfor inoculation had the basal part of the leaves andamorphous achlorophyllous tissue at their base andwas then cut to produce pieces (approximately withsimilar size) used as inocula (3 pieces/flask). The cul-ture flasks, 250 ml, contained about 80 ml of mediumand were sealed with transparent film with a 0.02µmfilter disc (C-6920 Sigma). All cultures were grownin a controlled environment chamber under the sameconditions as were grown the shoot clump cultures.For each medium, 12 flasks were inoculated and allthe experiments were repeated three times. After fiveweeks of culture, the material was removed from theflasks, the leaves were trimmed, counted and the leafmultiplication factor (the number of leaves at the endof the culture divided by the number of leaves at thestart of culture) was calculated. The number of bulbswith diameter≥5 mm and the number of those withdiameter<5 mm formed by the inocula in each flaskwas also calculated for each medium.

2.3 Bulb enlargement

Irrespective of their size, all the bulbs formed onthe four media were transferred onto a fresh mediumadequate to promote their further growth (MS1D– basal media with sucrose increased to 9% plus0.12 mgl−1 NAA). Before the transfer, the larger bulbs(diameter≥5 mm) were separated and roots removedwhenever present. Since the bulbs with smaller dia-meter appeared aggregated, clusters of them were

135

transferred to MS1D as single units. The bulbs werecultured in flasks containing 80 ml of medium for 55days under the same light and temperature conditions.After this period, all bulbs were removed from theflasks and they were graded into two sets; one setwas made with the bulbs with diameter≥5 mm, theother with all the smaller bulbs. Among the group ofbulbs with diameter≥5 mm, some were fairly largeand their percentage was calculated in relation to thepopulation of all bulbs produced on the respectivemedium. The bulbs with a diameter<5 mm weresubjected to a new period of culture on the growthmedium (MS1D) for further enlargement. The bulbswith diameter≥5 mm were prepared forex vitriumculture as described elsewhere [13] and they were thentransferred to a glasshouse.

2.4 Statistical methods

The results of shoot multiplication and of bulb form-ation were compared using the Student’st test, withp <0.05 considered to be significant. The data arepresented as means± SE (SE = standard error of themean attached to 95% confidence limits).

3. Results

3.1 Shoot multiplication and bulb formation

On medium with JA+2-iP and with JA+NAA, severalnewly formed shoots started to grow after two weeksof culture, while on MS1B (JA only) and MS1C (NAAonly) such an occurrence was not detected at this time.At the end of the culture period the differences amongcultures were quite apparent, each one with a charac-teristic growth habit (Figures 1A, 2A, 3A and 4). Theleaf multiplication factors in cultures grown on bothMS1B and MS1C media were 1.2 and 0.7, respectively(Table 1). These results translated the low ability of themedium with JA alone to induce additional leaves andthe negative effect of the medium with only NAA onleaf multiplication; this medium also induced form-ation of some callus tissue (Figure 4). The mediumMS1 and MS1A caused abundant leaf proliferation(Figures 1A and 2A) yet the number of leaves wassignificantly higher on that with JA plus 2-iP (MS1)than on MS1A (Table 1).

Bulb formation was observed on the four media.However, the bulbs obtained on medium with NAAalone were somewhat different from the bulbs pro-duced on the other three media, all with JA. On

the MS1, a few bulbs (about 10%, Table 1), whichappeared at the base of the leaves that began to sen-esce, reached up to 5 mm in diameter while all theothers were smaller, some even appearing as a tinybulb at the base of the youngest shoots (Figures 1Band 1C). On medium MS1A, the formation of a bulb atleaf base was also observed (Figures 2A and 2B); thenumber of the bulbs attaining a diameter≥5 mm washigher (about 30%; Table 1) than that found on MS1medium. Some of the larger bulbs were rooted and allthe others produced bulbs with a smaller diameter. Onmedium with JA alone, the leaves were less developedthan on MS1 and MS1A and at the base of the leaves abulb was formed (Figure 3A). The medium with onlyJA promoted the formation of bulbs larger than theother two media also containing 2-iP or NAA (MS1and MS1A). About 60% of the bulbs formed attaineda diameter≥5 mm (Table 1 and Figures 3B and 3C)and some of them reached 7 mm in diameter; the otherbulbs (about 40%) were smaller. On the medium withNAA alone (MS1C), no additional leaves were pro-duced, only the growth of some of the pre-existingleaves was observed, as assured by the value of themultiplication factor. This medium induced the form-ation of some callus tissue however, the appearance offew bulbs, all with diameter<5 mm (Figure 4) wasalso observed. These bulbs were elongated (Figure 4insert), in contrast with the bulbs formed on the mediacontaining JA that were roundish.

3.2 Bulb enlargement

When the bulbs formed on the four tested mediawere transferred onto MS1D medium to promote theirgrowth, an increase in the size of all bulbs as wellas development of roots was observed. However, thegrowth of the bulbs transferred from the medium withNAA only was very low and so their final size wassmaller than that attained by the bulbs transferred fromthe three media with JA. The bulbs also displayedvery different shapes, deviating from the natural form(compare Figure 5A with Figures 5B, C, and D). Onthe population of bulbs enlarged on the MS1D, dif-ferences in size of the bulbs were found, accordingto the origin of the bulbs. The ones that reachedthe largest diameter were the bulbs that were trans-ferred from the medium with only JA. Consideringthe largest size attained, about 41% (41± 3.0%) ofthe bulbs with diameter of 10–11 mm appeared in thepopulation traceable to MS1B, bulbs with diameter of8–9 mm (36± 2.6%) in the population coming from

136

Figure 1. Shoot cultures on MS1 medium (JA+2-iP). 1A – A view of the culture showing the high leaf proliferation and the formation of a tinybulb at the base of the leaves (arrow); 1B – Bulb formed at the base of a senescent leaf; 1C – The bulbs induced in young shoots are the smallestones.Figure 2. Cultures on the medium MS1A (JA+NAA). 2A – Note the occurrence of bulb formation at the base of the leaves. In general, the bulbsare larger than those formed on MS1, some reaching a diameter>5 mm as shown in 2B.Figure 3. Shoot cultures on the medium MS1B (JA only). 3A – A view of shoot culture showing the formation of a bulb at leaf base; note thatthe bulbs had different diameters; 3B – A bulb reaching 7 mm in diameter; 3C – bulbs with about 5 mm in diameter.Figure 4. Cultures on the medium MS1C (with only NAA) showing the formation of elongated small bulbs and callus tissue (arrow); a highmagnification of an individual bulb is shown in the insert.Figure 5. Bulbs grown for 55 days on MS1D show well developed roots and illustrate that the final size attained reflected their origin. 5A – Acluster of bulbs derived from medium MS1C (with NAA alone) showing the low increase in size; 5B – Bulbs traceable to MS1B (with only JA)when cultured on MS1D increased in size and some of them generated bulblets (arrow); 5C – Bulbs transferred from MS1A; 5D – A clump ofbulbs generated on MS1 medium showing they attained a smaller final size than on other JA containing media.In all figures bar scale represents 5 mm.

137

Table 1. Leaf multiplication and bulb formation by shoot clump cultures ofN.triandruson different media. The data presented are means of the three experiments(n = 36); the means are significantly different at p< 0.05.

Medium

Growth MS1B MS1C MS1 MS1A

Regulators (JA) (NAA) (JA + 2iP) (JA + NAA)

Leaf

Multiplication 1.2± 0.08 0.7± 0.09 1.8± 0.15 1.4± 0.10

N◦ of bulbs with

Diameter<5 mm 5.5± 0.96 8.3± 0.82 18.1± 1.10 11.6± 0.42

N◦ of bulbs with

Diameter≥5 mm 8.7± 0.87 — 2.5± 0.17 5.4± 0.70

Total n◦ of bulbs

per inoculum 14.5± 0.86 8.3± 0.82 20.5± 1.25 17.2± 1.14

MS1A and bulbs with diameter of about 7 mm (28±2.2%) in the population derived from bulbs formedon medium MS1. Besides the large bulbs, smallerones were present in each batch. Since the small bulbswere transferred onto the MS1D in small clusters,aggregates of two, three, four or even five bulbs werefrequently found at the end of the culture (Figure 5D).The large individual bulbs had well developed rootsas well as the clustered bulbs. The appearance of bul-blets attached to some bulbs was frequently observed(Figures 5B and 5C). The rooted bulbs with a diameter≥5mm, after preparation [13], were transplanted to asoil mixture and transferred to a glasshouse to com-plete their growth life cycle. The plants displayed anormal growth and the success of transplantation isunder evaluation.

4. Discussion

Our results show that the inclusion of JA in the cul-ture medium promoted the formation of bulbs in shootcultures. These data are in accordance with resultsfound by others [12] that reported that JA stimulatesshoot and bulb formation of garlicin vitro. For N.triandrus, the JA in the media besides the promotionof bulb formation also had a positive effect on shootproliferation when used alone or in association with2-iP. The synergistic effect of JA and cytokinins waspreviously observed in potato protoplast cultures [11].The number of additional leaves formed on mediumwith only JA was low, possibly due to the concen-tration used in our study. In garlic it was found that

the number of additional shoots was influenced by theconcentration of JA, the highest number obtained with10µM [12]. The medium with NAA alone induced theappearance of some callus tissue and diminution in thenumber of leaves compared to the number at the startof culture. On this medium the formation of bulbs alsooccurred; however, besides the low number of bulbsobtained they were smaller and displayed an elong-ated form. After the period of growth, these bulbsacquired a shape different from the normal morpho-logy. A stimulatory effect [3] as well as an inhibitoryeffect [17] in bulb formation ofNarcissushas beenascribed to NAA. For our material, the NAA behavedas a weak inducer of the formation of bulbs but led toa somewhat abnormal morphology.

The bulbs formed on medium with only JA werelarger than those produced on medium supplementedwith JA + NAA or JA + 2-iP. This result can be dueto the fact that on medium with only JA the formationof the bulbs was initiated earlier than on the other twomedia and so the period for bulb growth was longer.Also, the fact that only JA is present in the medium,a situation precluding possible interactions with theother growth regulator, might well favour the growthof the bulbs. In fact, it has been reported that JAcounteract the auxin-induced elongation [9]. Despitethe difference in the experimental procedure used withgarlic, there was also a delay in the bulb formationwhen the medium contained JA plus 2-iP, comparedto the results on medium with JA only [12]. On thismedium, the garlic bulbs attained a diameter of 3 mmafter six weeks of culture whereas in our material ahigh percentage of bulbs had a diameter≥5 mm after

138

five weeks. Possibly the use of MS medium instead ofGamborg’s B5 medium contributed to this difference.

The results reported here reinforce the suggestionthat JA might play an important role in the bulb form-ation as presumed from the results found for garlic[12] and for onion [6]. The finding that JA promotesthe formation of bulbs in shoot clump cultures ofN. triandrus is an important result. Actually, bulbproduction by shoot clump cultures ofNarcissusisslow, laborious and time consuming in spite of theimprovement recently reported [3, 13]. Furthermore,the methods reported forNarcissussp. bulb formationin vitro [3, 13, 15] were not adequate forN. triandrus.Bulbs formed on media with JA were roundish andall attained a large size on the growth medium. Thismedium promoted the enlargement of the bulbs andthe formation of roots as reported for otherNarcissussp. [3, 13], result indispensable for further transplant-ation of the bulbs. For narcissus it was reported that,before transfer to soil, the bulbs must attain a criticalsize (weight) and develop an abundant root systemto ensure a high percentage of survival [13, 16, 17].These requisites were obtained with bulbs formed onthe three media with JA. These bulbs, after trans-plantation into soil, produced normal plants and thetransplantation success is under evaluation.

The aim of our search was achieved since the cul-ture method adequate forin vitro production of bulbsof N. triandruswas determined. On the other hand,our data showing that JA promotes bulb formation ofN. triandrus, recommends the use of JA as a mean tohasten thein vitro propagation of other species ofNar-cissusand the use of JA as an alternative way for bulbformation inNarcissusspecies that do not respond tothe current micropropagation protocols. At present,JA is being assayed for bulb formation inN. rupic-ola since the use of the micropropagation procedureestablished forN. bulbocodium[13] was unsuccessful.

Acknowledgments

This work was supported by the Parque Naturalda Serra da Estrela (Instituto de Conservação daNatureza-Lisboa, Portugal). The technical assistanceof Mrs. Andrea Costa and Isabel Guimarães is grate-fully acknowledged.

References

1. Abe M, Shibaoka H, Yamane H and Takahashi N (1990)Cell cycle-dependent disruption of microtubules by methyljasmonate in tobacco BY-2 cells. Protoplasma 156: 1–8

2. Creelman RA and Mullet JE (1997) Biosynthesis and action ofjasmonates in plants. Annu Rev Plant Physiol Plant Mol Biol48: 355–381

3. Chow YN, Selby C and Harvey BMR (1992) Stimulation bysucrose ofNarcissusbulbil formationin vitro. J Hort Sci 67(2):289–293

4. Hussey G (1982)In vitro propagation ofNarcissus.1982. AnnBot 49: 707–719

5. Koda Y and Kikuta Y (1991) Possible involvement of jas-monic acid in tuberization in yam plants. Plant Cell Physiol32: 629–633

6. Koda Y (1997) Possible involvement of jasmonates in variousmorphogenic events. Physiol Plant 100: 639–946

7. Matsuki T, Tazaki H, Fujimori T and Hogetsu T (1992) Theinfluences of jasmonic acid methyl ester on microtubules inpotato cells and formation of potato tubers. Biosci BiotechBiochem 56: 1329–1330

8. Mita T and Shibaoka H (1983) Changes in microtubules inonion leaf sheath cells during bulb development. Plant CellPhysiol 24: 109–117

9. Miyamoto K, Oka M and Ueda J (1997) Update on thepossible mode of action of the jasmonates: Focus on the meta-bolism of cell wall polysaccharides in relation to growth anddevelopment. Physiol Plant 100: 631–638

10. Nojiri H, Yamane H, Seto H, Yamaguchi I, Murofushi N,Yoshihara T and Shibaoka H (1992) Qualitative and quanti-tative analysis of endogenous jasmonic acid in bulbing andnon-bulbing onion plants. Plant Cell Physiol 33: 1225–1231

11. Ravnikar M, Vilhar B and Gogala N (1992) Stimulatory effectof jasmonic acid on potato stem node and protoplast culture. JPlant Growth Regul 11: 29–33

12. Ravnikar M, Zel J, Plaper I and Spacapan A (1993) Jasmonicacid stimulates shoot and bulb formation of garlic in vitro. JPlant Growth Regul 12: 73–77

13. Santos J, Santos I and Salema R (1998)In vitro production ofbulbs ofNarcissus bulbocodiumflowering in the first seasonof growth. Scientia Hort 76: 205–217

14. Seabrook JEA (1990) Narcissus (Daffodil). In: Ammirato PV,Evans DA, Sharp WR and Bajaj PS (eds) Handbook of PlantCell Culture, vol. 5, pp 577–597. New York: McGraw-Hill

15. Squires WM and Langton FA (1990) Potential and limitationsof Narcissusmicropropagation: an experimental evaluation.Acta Horticulturae 266: 67–75

16. Squires WM, Langton FA and Fenlon JS (1991) Factorsinfluencing the transplantation success of micropropagatednarcissus bulbils. J Horticult Sci 66(6): 661–671

17. Steinitz B and Yahel H. (1982)In vitro propagation ofNar-cissus tazetta.HortScience 17(3): 333–334

18. Van den Berg JH and Ewing EE (1991) Jasmonates and theirrole in plant growth and development, with special referencesto the control of potato tuberization: a review. Amer Potato J68: 781–794