Plant Cell Reports (1994) 14:87-93 Plant Cell Reports c~, Springer-Verlag 1994

In vitro regeneration of the tropical multipurpose leguminous tree Sesbania Grandiflora from cotyledon explants

Catherine Detrez, Sai~r Ndiaye, and Bernard Dreyfus

Laboratoire de Microbiologie des sols, [nstitut Franqais de Recherche Scientifique pour le D6veloppement en Coop+ration, ORSTOM, BP 1386, Dakar, S6n~gal

Received 31 May 1993/Revised version received 8 June 1994 - Communicated by G.C. Phillips

S u m m a r y . A system using cotyledon pieces as explants and a BAP/NAA containing medium was developed for in vitro mass propagation of Sesbania grandiflora, a tropical nitrogen-fixing leguminous tree. The age and the lighting conditions of seedlings providing the explants were shown to be critical factors for both bud induction and bud elongation. Optimal choice for an efficient and reproducible bud induction process consisted of dark-grown seedlings, 24/36 h-old-post-imbibition, that yielded up to 96% of explants producing more than 30 buds each, after one week in culture. Bud development occurred throughout a direct organogenesis pathway, from the proximal and adaxial cut surface of the explants as proved by histological studies. Additional sites of regeneration were also obtained after wounding on the epidermal surface of explants, suggesting a large distribution of regenerative cells all along the explants. Bud elongation, i.e. stem differentiation and leaf growth, was improved by bud isolation from cotyledon explants and their further subculture in liquid bud elongation media for one week. Rooting was obtained on an auxin medium after 3 weeks and plants were established in soil with 92% success.

Abbreviations. BAP: 6-BenzyIAminoPurine; NAA: a-Naphthalene Acetic Acid; IBA: Indole-3-Butyrie Acid; 1AA: Indole-3-Acetic Acid; GA3: Gibberellic Acid: MS: Murashige and Skoog (1962) medium

Introduction

Sesbania grandiflora (2n=24) belongs to the subfamily Papilionoideae of the Leguminosae and is placed in the botanical tribe Robinieae (Evans 1990). It is a small tree of the Sesbania family which includes more than 50 species wide-spread in tropical and subtropical areas; 13 species are recognized as woody biennials or perennials. To take advantage of its vigorous early growth, often

Correspondence to: C. Detrez

reaching 8-10 m in height within 2-3 years, S. grandiflora is commonly managed for fodder production purposes as an annual crop. Because of its nitrogen fixing potential due to nodulation by Rhizobium (Harris et al. 1949; Ndoye et al. 1990), it is also planted as green manure or as a component in alley cropping systems to improve or to restore soil fertility in tropical and subtropical farming. Sesbania grandiflora is also used as an ornamental and a source of human food, firewood, gum, tannins and paper (Von Carlowitz 1990). Because of its multiple uses, S. grandiflora is widely recommended in social forestry programs in India (Shanker and Mohan Ram 1990). It has also recently received increased attention for the enhancement of the productivity, diversity and sustainability of ecosystems in Africa.

Despite the ecological and agricultural importance of such a multipurpose tree, there has been little assessment of its genetic variability. The germplasm collections and the genetic improvement of Sesbania perennials have not been well developed (Brewbaker 1990). In this context, micropropagation of tree species could offer a rapid means of producing clones for afforestation, breeding systems and for conservation of elite or rare genotypes. Gene transfer technologies through tissue culture also offers ways of introducing desirable characters in plant genomes from cells which express cellular totipotency (for review see Draper et al. 1988; Gasser and Fraley 1989). Although the majority of tree species have consistently proved to be recalcitrant or difficult to achieve organ formation and plant regeneration in vitro (Bonga 1987; Franclet et aL 1987), some successes have been reported recently with the nitrogen-fixing tree Allocasuarina verticillata (Phelep et al. 1991) and some of the tropical leguminous tree species (Bajaj 1986; Dhawan 1989; Jaiwal and Gulati 1991; Ranga Rat and Prasad 1991; Mathur and Mukunthakumar 1992). In the Sesbania genus, in vitro culture experiments have been focussed

88

on S. rostrata (Vlachova et al. 1987; Pellegrineschi and Tepfer 1993), S. sesban (Khattar and Mohan Ram 1982, 1983; Harris and Moore 1989; Harris and Puddephat 1989; Harris et al. 1989; Dhir and Gupta 1990; Yan-Xiu et al. 1990), S. cannabina (Xu et al. 1984), S. bispinosa (Kapoor and Gupta 1986; Sinha and Mallick 1991), S. formosa and S. exalta (Harris and Puddephat 1989). In these species, micropropagation has been sometimes obtained by enhanced axillary branching from seedling apices or from nodal cuttings of greenhouse plants. However, shoot regeneration via adventitious bud organogenesis is more widely documented and the authors have reported effective and reproducible regeneration from seedling tissues.

In S. grandiflora, Shanker and Mohan Ram (1990) have identified suitable treatments to promote and sustain callus growth, then bud formation, from cotyledon and hypocotyl explants sampled on 10/12-d-old seedlings. In this paper, we show how cotyledon explants are also effective tissue to achieve direct bud organogenesis (without a callus step) and how the lighting conditions and the age of seedling are critical factors to obtain, more quickly, higher percentages of bud inducing explants than those reported by the previous authors. Histological studies were also carried out to confirm the origin and ontogenesis of the multiple shoot formation as a prerequisite for future genetic transformation procedures.

Material and methods

Seed germination and explant isolation. Seeds of Sesbania grandiflora (Inland and Foreign Trading Co, Singapore) were surface sterilized for 1 h in concentrated H2SO 4 and rinsed five times with sterile distilled water. Additionally, seeds were immersed in sterile water for 3 h to promote imbibition. They were finally germinated in 90 x 20 mm Petri dishes containing half-strength MS medium, then incubat~edlfor 24-72 h at 28 + 2~ either under continuous light (99 ktmol.m'Ls of Photosynthetically Active Radiations) or in the dark.

Aseptically dark- or light-grown seedlings, 24- to 72-h-old after seed imbibition, were used as the source of cotyledon explants. Unless specified otherwise, a cotyledon explant consisted of a transversal half (0.4-0.6 cm in length) of a cotyledon, taking care to excise its quiescent axillary bud. After isolation from seedlings, the explants were then transferred to Petri dishes (20 explants per dish) and plated horizontally with the dorsal (abaxial) side down on the bud induction medium.

Media and in vitro growth conditions of the explants. Salts and vitamins of MS were used as a basal medium. Sucrose and the growth regulators (BAP, NAA, IBA, IAA, GA3) were added before autoclaving. All the media were adjusted to pH 5.9 with KOH or HCI, then autoclaved at I10~ for 30 rain. The agar media (0.8% Difco Bacto agar, W/V) were dispensed in Petri dishes (90 x 20 mm) or m culture tubes (20 x 150 mm). The liquid media were dispensed in 250 ml Edenmeyer flasks (40 ml per flask) and agitated at 20 rpm (Schiittelmaschine RO 20, Osi).

The, MS medium, sup.l{lemented with sucrose (30 g.l ' t), NAA (1 mg.l") and BAP (1 mg.l" ) (referred to as MS1 in the text below), was chosen as the bud induction medium because this proved most effective in preliminary attempts (unpublished data). All the explant cultures were incubated at 28 + 2~ under continuous light conditions dispensed by a mixture of Daylight and Gro- fluorescen[ t~bes (Sylvania, QUE) (5 : 1, respectively), providing 99 lamol.m . s of Photosynthetically Active Radiations (PAR) at the Petri dish level.

Data scoring Bud induction - To compare bud induction abilities of different

sources of cotyledon explants (i.e. dark- or light-grown seedlings, 24-, 36-, 48-, 60- or 72-h-old post-imbibition), the percentages of explants giving buds after 3 weeks of culture on MSI were recorded. The assays were statistically analyzed as an unbalanced design with at least 3 separate replications (i.e. 3 Petri dishes) of 20 explants each. Multiple comparisons of data were conducted using the Newman-Keuis test (at the 5% level), after arcsin transformation of the percentages and standard analysis of variance (AOV) (STAT-ITCF, IBM). Bud number per responsive explant was also recorded.

Bud elongation - Elongation of adventitious buds (i. e. formation of shoots including a shoot apex, an elongated stem and expanded leaves with petiole) was first observed on the MS 1 bud induction medium, without any subculture step. The percentage of explants showing bud elongation were scored for each source of explants after 6 weeks of culture on MSI, and statistically analyzed as described above (AOV and Newman-Keuls test at the 5% level). Alternatively, attempts to increase the percentage of bud elongation were performed after isolation of 3-week-old buds from the explants to obtain "propagules". Such propagules generally consisted of a group of 1-3 buds and were subeultured on a fresh bud elongation medium (MS supplemented with NAA, BAP and/or GA3), containing agar or not. The percentages of propagules exhibiting bud elongation, as shoot length, were recorded 3 weeks after subculture, among at least 50 propagules per medium.

Rooting - Rooting of elongated buds was performed on three auxin- containing MS media. The percentages of rooted shoots, as the root number per shoot and root length, were recorded after 4 weeks of culture, from at least 24 shoots per medium.

Cytological investigations. Cotyledon explants were sampled at daily intervals, from the first up to the seventh day of culture on the MS1 bud induction medium. They were fixed in a F.A.A. mixture (formaldehyde/acetic acid/ethanol, 5/5/90, V/V/V) for 24 h, dehydrated in an ethanol series, cleared in xylol and then embedded in paraplast (60~ Sections, 5p.m thick, were stained with paragon or methylene blue (1% aqueous solution) and observed via light

microscopy.

Results and discussion

Bud induction

,0 'q80. * " 2_ [ - 60~

40 = ~

�9 Nll 20"

0 ' 24h 36h 48h 60h 72h Age of light-grown seedlings

% _

100 a_._ iiiililiiiii

80 iiiiiiiiii!!

6( iiiiii!iiiiiii ....-....... ..-......... .....-..... :!:i:i:!:i:i

4( ii!i!iiiiiiiiiiiiiii :i:i:i:i:i:!

2a i~iiiiiiiii ( :::::::::::::

24h Age

ii ii;iiiiiiiili

36h- 48h 601a 72h of dark-grown seedlings

1A: Percentage of explants showing bud induction

% 106

8O"

60

40

2G

0

%

80 t 60

40 -I

20 t "I 0-1

24h 36h 48h 60h 72h 24h 36h 48h 60h 72h Age of light-grown seedlings Age of dark-grown seedlings

1B: Percentage of explants showing bud elongation

Fig. 1. Percentages of half-cotyledon explants cultured on MS1 for 3 weeks in the lighL showing: (1A) bud induction, and/or (1B) bud elongation, for 10 sources of explants: dark- or light- grown Sesbania grandiflora seedlings, 24-, 36-, 48-, 60-, or 72-h-old post-imbibition Percentages recorded from 60 explants per source of explants

89

For each of the 10 sources of cotyledon explants studied q.e. sampled from dark- or light-grown seedlings, 24-, 36-, 48-, 60-, 72-h-old post-imbibition), Figure 1A lists the percentages of explants exhibiting bud induction on the MS 1 medium. These percentages range from 23 to 96%. Analysis of variance of these data, arcsin transformed, shows a very significant effect of both the lighting conditions (referred to as factor 1) and the age (factor 2) of seedlings providing explants, whereas no interaction effect was observed between factors 1 and 2 (Table 1). Concomitantly, the Newman-Keuls test designates the dark-grown, and/or 24/36-h-old seedlings, as being the more efficient sources of bud inducing explants (Table 1); more than 30 buds were moreover routinely obtained from the entire cut surface of these explants. In contrast, adventitious buds on explants from l ight-grown seedlings, 48/60/72-h-old, occurred exclusively at the edge of their adaxial cut surface, and no more than 3 buds were recorded per explant. No budding was observed on explants sampled from 4-d-old or older seedlings (data not shown). Such a decline in the percentage of bud inducing explants with the seedling age (occuring from 48 h post-imbibition, when emergence of cotyledons from their seed coats and their early involvement in photosynthetic functions are observed), is in agreement with data reported by Gulati and Jaiwal (1990) in Vigna. With these authors we suggest that, at such advanced stages of germination, the cotyledon's reserves (already mobilized for cell metabolism of embryo axis growth) have limited availability in terms of prime sources of energy or growth factors to support bud organogenesis. The fact that dark grown cotyledons formed more buds than those developed under light also

rules out the possibility of photosynthates being critical regarding bud induction. The improvement of bud induction by the selection of the youngest explant may also have resulted from the persistance of cells not fully determined, activated by the exogenous and endogenous hormone ratio.

Morphogenesis pathways from cotyledon explants

The localization of regenerative cells and ontogeny of adventitious buds were histologically examined for one culture week, among explants isolated from 36-h-old, dark-grown seedlings. No pre-existing quiescent bud was observed in explants before plating on bud induction medium. By day 2-3 on MS1 medium, cell divisions were observed from the cut surface (Fig. 2A). By day 5-6 cell divisions resulted in the organization of a meris tematic structure (Fig. 2B), which in turn differentiated a bud meristem with overlapping leaf primordia by day 7-8. Buds became macroscopic by day 7-8 and at this stage, vascular bundles connecting the adventitious buds to the vascular tissues of the explants were observed (Fig. 2C, 2D). More than 30 buds shoots were obtained after 2 weeks, but as reported below full shoot differentiation was not frequently observed (Fig. 2E, 2F). The most of buds were arrested in their development and did not express stem differentiation (and a well defined apical dominance), unless subcultured (see also the "Elongation" section).

The pattern of adventitious bud induction and growth, described above, exclusively occurred at the proximal and ventral (adaxial) cut surface of cotyledon explants (Fig. 2E), from dividing cells observed at zero to 400 Jam

Table 1. Statistical analysis of the percentages of explants showing bud induction or bud elongation on the MS1 medium, according to the lighting conditions (factor 1) and the age (factor 2) of seedlings providing the cotyledon explants of Sesbania grandiflora:results

an O) from the analysis of v "ance (F-test) and from the Newman-Keuls test.

Factor studied Statistical values calculated

FACTOR 1 (lighting conditions of seedlings)

FACTOR 2 (age of seedlings)

INTERACTION Factor 1/Factor 2

Morphogenesis step studied Bud induction Bud elongation

Probability value of the F-ratio <0.001 [hs] <0.001 [hs] Percentages of responsive explants (2)

dark 82 b 38 b light 55 a. 50 a

Probability value of the F-ralio <0.001 [Its] <0.001 [hs] Percentages of responsive explants (2)

24hrs 92 a 3 d 36 hrs 93 a 15 c 48 hrs 58 b 68 a 60hrs 42 c 62 ab 72 hrs 58 b 72 a

Probability value of the F-ralio 0.084 [ns] 0.104 [ns]

Coefficiant of variation 13.5 15.4

Biological data recorded from 60 explants per treatment. Explants isolated from dark- or light-grown seedlings, 24-, 36-, 48-, 60-, 72-h-old post-imbibition, then cultured on MS1:MS+NAA1 +BAP1, under continuous lighting conditions for 3 weeks (1): Analysis of variance based on the F-ratio and its probability value calculation (STAT-ITCF, IBM). [hs]: highly significant F-ratio; Ins]: non significant F-ratio (2): In the same column and for each factor studied, the percentages (as mean values) followed by the same letter are not significantly different, after arc sin transformation, according to the Newman-Keuls test, at the 5% level (STAT-ITCF, IBM)

90

posterior to the cut surface, without an intervening unorganized growth phase. At the dorsal (abaxial) cut surface of explants, in contrast, compact calli were frequently produced (Fig. 2F). Densely cytoplasmic cells in the matrix of comparatively vacuolated cells were also sometimes observed in the peripheral regions of the callus tissues. Meristematic nodule formations were subsequently observed, but differentiation of them into buds never occurred.

Increase in the regenerative ability o f cotyledon tissues

Response to addit ional w0undings Additional woundings were performed with a needle on the epidermal surface of cotyledon explants (sampled from dark-grown, 36-h-old seedlings) to study the regenerative ability of cells inside the explant (different from those of the cut s u r f a c e ) . As reported in Table 2, these treatments

Table 2. Bud regeneration from Sesbania grandiflora half-cotyledon explants isolated from dark-grown seedlings, 36-h-old, subsequent to additional wotmdings on their epidermal surface, cultured on MS1 medium.

Type of explants and site of regeneration

% of explants Mean number exhibiting of buds per bud regeneration regenerative site

Control explants (not wounded) from the cut edge surface 98 > 30

W o u n d e d explants from the cut edge surface 96 from the epidermal wounding 100

> 30 3+ 1.3

Data scored from 50 explants per treatment

generated new sites of regeneration per explant: from each wounding site, bud development was noticed (Fig. 2G) and 1-5 buds were recorded, suggesting a large distribution of regenerative cells all along the explant. Effects of the explant size - To study the effects of a decrease in explant size, small transverse sections of cotyledons (i.e. quarters of cotyledon, about 0.2 cm in length, isolated from dark-grown, 36-h-old seedlings) were cultured on the MS 1 medium. In such conditions, polar i ty in the regenerat ive abilities of explants disappeared, and buds were obtained from the proximal as well as from the distal cut surface of the explants. The percentage of bud inducing explants was, however, low (<12%); and high levels of callusing (>85% of bud inducing explants with calli) were frequently obtained around the site of bud formation.

Elongation of adventitious buds

Bud elongation on MS1, without subculture - As reported in Figure 1B, the percentages of explants exhibiting

bud elongation from explants kept on MS1 medium depended on the source of explants (i.e. dark- or light- grown seedlings, 24-, 36-, 48-, 60-, 72-h-old). The highest percentage of explants showing bud elongation was obtained from light-grown, 72-h-old seedlings whereas 24/36-h-old seedlings provided the lowest frequency of elongation. Whatever the sources of explants, only 1-2 well-developed shoots were obtained per explant, the other buds of the explant being arrested in their development (Fig. 2F).

As in the bud induction step, analysis of variance showed that the effects of the lighting conditions and the age of seedlings were highly significant, whereas no interaction between these two factors was observed at the 5% level (Table 1). When comparing the data reported in Fig. 1A and 1B, it is also evident that explants displaying high percentages of bud induction expressed low percentages of elongation and vice versa. As suggested by Gambley and Dodd (1991), the persistance of buds on the explants appeared to be detrimental to further bud elongation, and an apical dominance phenomenon probably occured among the adventitious buds.

Table 3. Bud elongation among Sesbania grandiflora propagules (1) according to the type and the concentration of growth regulators added to the liquid MS medium.

Growth regulators % of Mean length Other morphogenetic and concentrations propagules of shoots (3) pathways commonly (mg.1-1) showing bud observed in the BAP NAA GA3 elongation (2) culture

10 15.0-+5.5

0.1 10 0.5 8

~ .1 - 4 0.1 24 0.5 56

0.1 10 0.1 0.1 26 0.1 0.5 62

12.9_+6.4

24.4~7.8

callusing

0.5 2 vitrification - 0.1 73 30.1+8.7

0.5 64 23.6+7.3

0.1 22 0.1 81 29.7+7.8 0.5 62 19.1+5.6

vitrification

2 axillary branching - 0.1 16 ax.branching;vitrif.

0.5 8 ax. branching

0.1 12 ax. branching; caUi 0.1 46 12.3+4.3 0.5 36

(1): Propagules consisting of a group of 1-3 buds, isolated from cotyledon explants. Explants sampled on dark-grown seedlings, 36-h-old post-imbibition, then cultured on MS1: MS+BAPI+NAA1, for 3 weeks. (2): Bud elongation scored from at least 50 explants per treatment (3): Mean length + standard error

91

Bud elongation after subculture: agar vs liquid media - To improve elongation of buds obtained from the dark-grown, 36-h-old seedling explants, subcultures of buds (sampled as described in the "Material and Methods" section to obtain propagules) were performed on fresh liquid- or agar- media supplemented with hormones (BAP/NAA/GA3, 0.1/0.5/1 mg.l", alone or in combination) (Fig. 2H, 2I). Null or low percentages (less than 15%) of propagules exhibiting bud elongation were obtained on the agar media (Fig. 2I). Axillary branching from adventitious buds was, however, commonly observed in all agar media supplemented with BAP. In contrast, proper bud elongation was easily achieved by subcultures of propagules in liquid MS media, and the highest bud elongation percentages were scored with (in mg.l") BAP0 .5+NAA0.1+GA0.1 or BAP0.5+GA0.1 supplementations (Table 3) (Fig. 2H). The explants were removed from the liquid media after 1 week of culture to avoid vitrification of the newly formed shoots. In such conditions, the percentages of shoot elongation reached 73% and 81% on the two media stated above, respectively.

Rooting and acclimatization in the greenhouse

The rooting of shoots obtained in the BAP0.5+GA0.1 liquid bud elongation medium, was performed on MS media supplemented with sucrose (15 g.l "t) and with NAA (1 mg.ll), or IBA (1 mg.l"), or IAA (0.5 mg.l"). With these auxin-supplementations, 86%, 30% and 21% of rooted shoots were scored, respectively (Table 4).

Table 4. Rooting of Sesbania grandiflora adventitious shoots ( l) on three auxin-containing MS medium.

Type of auxin Concentration (rag.1-1 )

Rooting Meannumber Mean length percentage (2) ofroot of roots

per shoot (3) (mm) (4)

Control (hormone free-MS) 8 1.14_+0.10 3.5+_2.5

NAA 1 86 1.98_+0.43 24~8.4 IBA 1 30 1.33_+0.40 15.0+__5.7 IAA 0.5 21 1.36_~.26 13.5+_4.4

(a): Shoots obtained after i) culture of cotyledon explants (isolated from dark-grown seedlings, 36-h-old) on MSI: MS+NAAI+BAP1, for 3 weeks, then ii) subculture of buds in the liquid bud elongation medium MS+BAP0.5+GA0.1 (2): Rooting percentage scored from at least 24 shoots per medium (3): Mean number + standard error (S.E.) (4): Mean length + S.E.

Leaf shedding was generally observed during the first days following transfer from the elongation to the rooting medium, but did not affect the survival of the elongated buds which rapidly developed non-vitrified leaves within 1 week. The roots appeared within 5-15 d, and plants with a well-branched root system were easily established

after 3 weeks of culture on the NAA-medium (Table 4) (Fig. 2J). The rooted shoots were transferred to the greenhouse in plastic bags filled with a mixture of 1/3 sand, 1/3 polystyrene beads and 1/3 vermiculite (v/v/v). A plastic film was kept over the shoots for 10 d to facilitate gradual adaptation to greenhouse conditions. Four months after transplantation to soil, the survival rote was 92%.

The micropropagated plants thus acclimatizated (157 plants) showed 2 cases of phenotypical variation in their development: early flowering, or fasciation resulting in thick stems with free apices at the tip.

C o n c l u s i o n

This paper provides protocols for rapid adventitious bud and rooted shoot production in the tropical woody legume Sesbaniagrandiflora, using cotyledon explants. In the absence of a callus step during the bud induction process, somaclonal variation should not be promoted (Reisch, 1983) and the low frequency of morphological variants obtained here supports the concept that this technique is suitable for clonal propagation. The use of this clonal propagation process, demanding seed tissues as starting material, will however require evaluation of the clone (i.e. seed) characteristics: no data are available concerning the self- or cross-pollinated status of Sesbania grandiflora and the perennial Sesbania are assumed to be largely outcrossinff the seeds are therefore untested and performance of the genotype unknown.

On the other hand, the regeneration pathway described here may be useful for introducing new genes into the Sesbania grandiflora genome by Agrobacterium tumefaciens- mediated genetic transformatiort Particularly, it displays characteristics which are prerequisites to transformation: i) Totipotent cells are probably distributed all along the explant and are available in large quantities. This is suggested by the ability of different types of transverse sections (distal/proximal half-section or quarter-section) of cotyledons to produce buds; ii) The sites of bud formation are predictible and are associated with a wounding site,, which is required forAgrobacterium- mediated transformation (Bolten et al. 1986; Sheikholeslam and Weeks 1987); iii) Totipotent cells are capable of undergoing differentiation early during culture, and organogenesis seems to be synchronous. Studies are therefore in progress in our laboratory to make shoot regeneration procedures adaptable to gene transfer by Agrobacterium tumefaciens.

Acknowledgements We gratefully acknowledge M. Diouf for his assistance in the cytological studies. Thanks are also due to Y.R. Dommergues and T. Cusack for their comments in the preparation of the English manuscript. This work was supported by a postgraduate grant from AUPELFAJ1;',EF and by EEC contract n ~ TS2-0135B.

93

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Fig. 2 : Ontogeny and pattern of plant organogenesis from Sesbania grandiflora cotyledon explants (transverse half-pieces) [ab: abaxial surface of the explant; ad: adaxial surface of the explant; ca: callus] (A, B, C, D, E): Bud induction on explants isolated from 24-h-old, dark-grown seedlings and cultured on MS1 (MS medium supplemented with BAP (1 mg.1-1) and NAA (1 rag.1 -t) - (A) 3-d-old culture showing cell divisions (arrows), 0-200 jim posterior to the cut surface of a cotyledon explant (Staining: methylene blue) - (B) Organization of meristematic structures (me) along the cut surface in a 6-d-old culture (Staining : paragon) - (C) Longitudinal section along the cotyledon axis of a 10-d-old culture showing leaf primordia (arrows) with a single tunica layer fully emerged from the adaxial tissues; callus formation occurs from the abaxial cut tissues at the cut end surface. Procambial strands (pc) are already differentiated, connecting the buds to the explant. (Staining: methylene blue) - (D) 7-d-old culture with bud primordia (arrows) macroscopicaUy observable at the adaxial cut end surface of the explant. No callus formation occurs at the site of budding - (E) 12-d-old culture showing multiple bud proliferation from the entire cut end adaxial surface of the explant - (F) 14-d-old culture showing the bud elongation and stem differentiation (arrows) of adventitious buds during culture on bud induction medium - (G) Bud formation (arrows) from wounding areas (star) at the epidermal surface of a cotyledon explant (3-week-old culture) (H; I; J): Rooted shoot production from buds grown on explants isolated from 24/36-h-old seedlings - (H) Stem differentiation (arrows) occurring after isolation of buds from the explant and subculture in a liquid medium (3-week-old culture) - (I) Axillary branching and elongation (arrow) of buds subcultured on agar BAP-containing medium (17-d-old culture) - (J) Elongated bud subcultured on MS+NAA (1 mg.1 -~) showing root formation (20-d-old culture).