In vitro Bulb Development in Shallot (Allium cepa L. Aggregatum Group): Effects of Anti‐gibberellins, Sucrose and Light

Abstract

Bulbing was studied in shallot plants cultured in vitro. Bulbing occurred under a 16 h photoperiod with fluorescent + incandescent light and 30–50 g l^–1 sucrose in the culture medium. Exogenous gibberellin (10 µm GA₃) inhibited leaf and root growth and bulbing. When added to the medium at a concentration of 10 µm, three inhibitors of gibberellin biosynthesis (ancymidol, flurprimidol and paclobutrazol) promoted bulb formation and the percentage of bulbing. When ancymidol was used in combination with GA₃, it did not reverse the effect of GA₃ applied alone. Under treatments with 30–70 g l^–1 sucrose, bulbing ratios greater than those found in control plants were achieved by addition of ancymidol, and bulb fresh weight was increased in the same way. Ancymidol caused a 66 % decrease in sucrose content in leaf bases but greatly increased the glucose, fructose and fructan contents. The increase in fructan content by ancymidol could result from the three‐fold rise in total [¹⁴C]sucrose uptake per plant from the culture medium associated with a marked increase in leaf base labelling at the expense of root labelling. The possible role of ancymidol is discussed and evidence supports a major regulatory role for gibberellins in bulbing.

INTRODUCTION

Cultivated types of Allium cepa fall into two broad horticultural groups: the Common Onion Group and the Aggregatum Group (Hanelt, 1990). Bulbs of the Common Onion Group are large, normally single, and plants reproduce from seeds. In contrast, bulbs of the Aggregatum Group, which are smaller than those of the common onions, are vegetatively propagated and form clusters of bulbs. Jones and Mann (1963) distinguished two bulb‐forming sub‐groups: multiplier or potato onions, and shallots, the latter being the most important sub‐group of the Aggregatum Group. In comparison with the common onion (Allium cepa L.), few studies have dealt with bulb formation in shallot, though its control seems to be essential for propagation. Jenkins (1954) and Messiaen et al. (1993) showed that long days and relatively high temperatures are necessary for bulb formation in shallots. In an obligate long‐day onion, the spectral quality of light has been shown to influence bulb formation: a low‐spectral ratio of red (R) to far‐red (FR) light was necessary to induce bulbing, indicating the involvement of phytochrome in a high irradiance response (Lercari, 1982a; Mondal et al., 1986; Sobeih and Wright, 1987; Kahane et al., 1992b). Phytochrome and gibberellins (GAs) coordinately regulate multiple aspects of plant development (Kamiya and García‐Martínez, 1999). Onion bulbing is a storage process controlled by daylength and phytohormones, with GAs playing a key role (Kato, 1965; Knypl, 1979; Mita and Shibaoka, 1984).

To assess the possible role of GAs, growth regulators known to inhibit the biosynthesis of GAs have been widely used. Growth retardants with an N‐containing heterocycle, e.g. ancymidol (Ancy), flurprimidol (Flur) and paclobutrazol (PBZ), inhibit GA biosynthesis by blocking the conversion from ent‐kaurene to ent‐kaurenoic acid (Grossmann, 1990; Rademacher, 2000). Ancy and PBZ have been reported to enhance potato tuberization under long and short days at any sucrose concentration (Šimko, 1994; Ochotorena et al., 1999) and to divert assimilates to storage organs (Deng and Prange, 1988; Steinitz et al., 1991).

There is evidence for carbohydrate accumulation in leaf bases during the initiation of bulbing (Butt, 1968; Lercari, 1982a; Sobeih, 1988). The non‐structural carbohydrates in shallots and onions include glucose, fructose and sucrose together with oligosaccharides, the fructans (degree of polymerization up to about 18–20; Darbyshire and Henry, 1978, 1981; Darbyshire and Steer, 1990; Ernst et al., 1998).

This paper deals with the influence of GA biosynthesis inhibitors on in vitro shallot bulb formation at various sucrose concentrations and under two light conditions. The effect of Ancy on sucrose uptake and carbohydrate accumulation in leaves was also investigated.

MATERIALS AND METHODS

Plant material

Experiments were carried out on a cultivar of half‐long Jersey shallot (Mikor), provided by INRA (Ploudaniel, France). Shallots were introduced in vitro as described by Fujieda et al. (1979) and further micropropagated in 150 × 22 mm glass tubes according to Kahane et al. (1992a). After 1 month of culture on this medium, approx. ten plantlets per tube were obtained. These were pooled to conduct proliferation and bulbing experiments.

Culture media and conditions

The proliferation medium contained macro‐ and micro‐elements according to Murashige and Skoog (1962), vitamins according to Morel and Martin (1955), 30 g l^–1 sucrose, 7·5 g l^–1 Microagar (Kalys Biotechnologies, Roubaix, France) and 1 mg l^–1 BAP. The pH was adjusted to 5·8 with 1 m KOH before autoclaving.

The bulbing medium was identical to the proliferation medium with the following exceptions: BAP was omit ted; the sucrose concentration was varied between 30 and 70 g l^–1; and the medium contained 300 mg l^–1 NaH₂PO₄ (Kahane et al., 1992a). GA₃ and inhibitors of GA biosynthesis (Ancy, Flur and PBZ) were sterilized by filtration through a 0·2 µm Dynagard membrane (Poly Labo, Strasbourg, France) and added to the autoclaved bulbing medium to yield a final concentration of 10 µm.

All plantlets were cultured in glass tubes. For proliferation experiments, the cultures were maintained at 24 ± 2 °C under fluorescent light at 45 µmol m^–2 s^–1 with a 13 h photoperiod. Bulbing experiments were performed under a 16 h fluorescent light photoperiod, enriched (F + I) or not (F) by incandescent light, with a total irradiance of 65 µmol m^–2 s^–1.

Measurements

A shallot plant is composed of leaves with a photosynthetic leaf blade and a non‐photosynthetic storage leaf base (scale). During the growth of the plant, the leaf bases (scales) thicken and form the characteristic bulb. The swelling may be expressed as the bulbing ratio (B), i.e. the ratio of the greatest diameter near the base to the diameter at the neck of the plant. A bulbing ratio of > 2 characterizes the onset of bulbing (Clark and Heath, 1962). Bulbing ratios were measured every week or every month.

The fresh weight of roots, bulbs and leaf bases (when the bulbing ratio was < 2) was measured every week or every month. In addition, to determine the dry weight of bulbs, the tissues were heated at 100 °C for 10 min, then at 80 °C until a constant weight was achieved.

The percentages of rooting and bulbing were also determined during the 3 months of culture.

Results reported here are the means of four to six independent experiments carried out on 24 plantlets per treatment.

Sucrose transport

To study the long‐distance transport of [¹⁴C]sucrose, plants grown in vitro for 6 weeks on the bulbing medium, with or without 10 µm Ancy, were transferred to a nutrient solution (Murashige and Skoog, 1962) supplemented with 0·1 mg ml^–1 penicillin, 1 mg ml^–1 gentamycin and 30 g l^–1 sucrose (87·7 mm) and labelled with [¹⁴C]sucrose (53·4 kBq mmol^–1). They were kept at 25 °C under continuous F + I light (65 µmol m^–2 s^–1). After 5 or 24 h, they were harvested and the different organs were separated and rinsed three times (10 min each) with 5 ml of 100 mm mannitol. Washed tissues were transferred to liquid scintillation vials, and the leaf material was decolourized by addition of 0·5 ml 70 % (v/v) HClO₄ and 0·5 ml 30 % H₂O₂ (v/v). The vials were tightly capped and heated in a water bath at 60 °C for 1 h. Ten millilitres of 80 % v/v ethanol was added to all vials and heated to boiling. After evaporation of ethanol the residue was dissolved in 1 ml distilled H₂O.

Radioactivity was measured by liquid scintillation counting (ACS, Amersham, Les Ulis, France and Tricarb 1600 TR counter, Packard Bioscience, Rungis, France). Experiments had three replicates and were repeated twice.

Sugar analysis

After freeze‐drying, tissues were extracted by shaking in water at a ratio of about 100 mg tissue : 5 ml^–1 H₂O (60 °C, 4 h). The resulting solutions were transferred to a volumetric flask for free glucose and free fructose measurements according to Bergmeyer (1974). For oligosaccharides (including sucrose), the procedure was the same except that the mixture of extract and buffer was incubated for 5 h at 25 °C with β‐fructosidase (invertase) (Sigma, Saint‐Quentin Fallavier, France). The oligosaccharide content was calculated from the difference between the amount of fructose and glucose present before and after treatment with β‐fructosidase. Sucrose was separated from other oligosaccharides by thin layer chromatography performed according to Cairns and Pollock (1988). The zone containing sucrose was scraped off the plates and eluted with 500 µl water. The sucrose content was measured after treatment with β‐fructosidase.

Values are the means of two independent extracts analysed in triplicate.

RESULTS

Effects of light quality on bulbing and plant development

One month after transfer to the bulbing medium, noticeable morphological differences between the plantlets cultured under fluorescent light (F) and those grown under a mixture of fluorescent and incandescent light (F + I) were observed. The differences became more marked as the culture duration increased. In both conditions, each plantlet produced only one bulb.

The bulbing ratio (B) of the plantlets grown under fluorescent light never exceeded 2·0 (Fig. 1), which indicates that they failed to bulb. On the other hand, under F + I light, at day 30, 89 % of the plantlets had formed bulbs and 100 % had formed bulbs by day 90. Over the same time‐scale the bulbing ratio gradually increased from 2·45 to 3·2 (Fig. 1). Paralleling bulbing, fresh (Fig. 1) and dry weights (not shown) of bulbs increased concomitantly with a gradual senescence of leaves which was complete by day 90; the senescence of foliage indicated the maturity of bulbs.

Fig. 1.  Effect of adding 10 µm ancymidol, paclobutrazol and flurprimidol to the bulbing medium (30 g l^–1 sucrose) on the fresh weight of roots, leaf bases or bulbs, and on the bulbing ratio, B. The plants were cultured in vitro under fluorescent (F) or fluorescent + incandescent (F + I) light. Data are means ± s.e. of four–six experiments. Each experiment represents 24 plants.

Under the two light conditions, all plantlets developed branched roots; however, in the presence of incandescent light, root growth was stimulated during the first 60 d, then senescence occurred as bulbing progressed.

Effects of sucrose concentration on bulbing

Concentrations of sucrose were varied from 10–70 g l^–1 under a 16 h photoperiod of F and F + I light.

After 3 months under F light, explants cultured on a medium containing 10–30 g l^–1 sucrose did not show bulbing, but their growth was normal (Table 1). With a concentration of 50 g l^–1 sucrose, 10 % of plants formed bulbs, whereas a higher sucrose concentration (70 g l^–1) stopped growth. Within the range 10–50 g l^–1 sucrose, the growth of leaves and roots was maximal at 30 g l^–1 sucrose (Table 1).

Table 1.

Effects of sucrose in the bulbing medium on root, leaf base or bulb fresh weight, on the bulbing ratio, B, and on the bulbing percentage of plants cultured in vitro for 3 months under fluorescent light

Bulb or leaf base

Sucrose concentrations (g l–1)	Root fresh weight (g)	Fresh weight (g)	B	Bulbing percentage
10	0·150 ± 0·013	0·090 ± 0·011	1·20 ± 0·16	0
30	0·234 ± 0·020	0·138 ± 0·015	1·74 ± 0·15	0
50	0·107 ± 0·009	0·220 ± 0·025	1·66 ± 0·16	10
70		No growth of explants	0

Data are means ± s.e. of four independent experiments. Each experiment represents 24 plants.

Vegetative development was markedly stimulated by supplying incandescent light and increasing concentrations of sucrose up to 30 g l^–1. However, a strong inhibition of leaf (not shown) and root formation was noticed at higher sucrose concentrations (Fig. 2A). The bulbing ratio exceeded 2·0 under treatments with 30–70 g l^–1 sucrose and the heaviest bulb was obtained with 30 g l^–1 sucrose (Fig. 2B).

Fig. 2.  Effect of sugar [sucrose (S) or mannitol (M)] and ancymidol (10 µm) in the bulbing medium on the fresh weight of roots, leaf bases or bulbs, and on the bulbing ratio B. Plants were cultured in vitro for 3 months under fluorescent (F) or fluorescent + incandescent (F + I) light. Data are means ± s.e. of five experiments. Each experiment represents 24 plants.

To check whether the increasing external osmotic concentration had the same effect as increases in the sucrose concentration, a non‐permeant osmoticum, mannitol, was combined with sucrose. When 20 g l^–1 of mannitol was added to medium containing 30 g l^–1 sucrose, bulb formation was strongly inhibited compared with treatments with 30 and 50 g l^–1 sucrose alone (Fig. 2A and B).

Effects of plant growth regulators

To assess the effects of plant regulators on bulbing, three growth bioregulators, Ancy, Flur and PBZ, were added to the culture medium containing 30 g l^–1 sucrose under F + I light conditions. Preliminary experiments indicated that 1 or 5 µm Ancy had a significant growth‐promoting effect; maximum growth being obtained at 10–15 µm. After as little as 1 month of culture, the three plant growth regulators, supplied at a concentration of 10 µm, increased bulb formation and percentages of bulbing (Fig. 1), with no effect on the division rate. After 3 months, the three compounds had stimulated bulb fresh weight by 134, 53 and 63 %, respectively (Fig. 1), with an associated decrease in the dry weight percentage (10–11·5 % vs. 13·8 % for the control); Ancy, Flur and PBZ‐treated plants developed bulbs that were approx. 30–60 % larger than controls. Root development (accelerated growth and branching) was also markedly promoted by Ancy and PBZ (+ 288 and 252 %, respectively); in contrast, Flur caused root shortening and thickening, which resulted in a 50 % reduction in root fresh weight (Fig. 1).

Addition of Ancy, while increasing sucrose concentrations up to 50 g l^–1, markedly stimulated leaf (not shown) and root growth (Fig. 2C). At 30 g l^–1 sucrose, B exceeded 2·0 (Fig. 2D). With higher concentrations of sucrose, a reduction in bulb fresh weight was observed although the bulbing ratio still remained greater than 2 (Fig. 2D).

Leaf and root growth and bulbing were inhibited by 10 µm GA₃ (Table 2)_. When Ancy was used in combination with GA_3, it did not greatly reverse the action of GA₃ (Table 2).

Table 2.

Effects of adding GA₃ (10 µm) and ancymidol (10 µm) on root, leaf base or bulb fresh weight, on the bulbing ratio, B, and on the bulbing percentage of plants cultured in vitro for 3 months with 30 g l^–1 sucrose under fluorescent + incandescent light

Bulb or leaf base

Treatment	Root fresh weight (g)	Fresh weight (g)	Bulbing ratio	Bulbing percentage
Control	0·138 ± 0·012	0·908 ± 0·050	3·20 ± 0·30	100
Ancymidol (10 µm)	0·536 ± 0·037	2·859 ± 0·175	4·22 ± 0·35	100
GA3	_	0·089 ± 0·009	1·41 ±0·20	0
GA3 (10µm) + Ancymidol (10 µm)	0·024 ± 0·008	0·149 ± 0·019	1·53 ± 0·16	0

Data are means ± s.e. of four to six independent experiments. Each experiment represents 24 plants.

When plants were cultivated for 3 months under fluorescent light with 10 µm Ancy, no bulbing was induced, but the growth of leaves and roots was markedly increased (by 286 and 257 %, respectively; Fig. 1).

Transport of [¹⁴C]sucrose

In 6‐week‐old control plants, although [¹⁴C]sucrose uptake increased with time, the distribution of radioactivity within the different parts of the plants (expressed as a percentage of total radioactivity) was established after 5 h (Table 3). Labelling was high in the roots, and gradually decreased with increasing distance from the roots.

Table 3.

Effect of ancymidol (10 µm) on the distribution of radioactivity in 6‐week‐old shallots after feeding with [¹⁴C]sucrose (87·7 mm) for 5 or 24 h

	5 h		24 h
	Control	Ancymidol	Control	Ancymidol
Leaf apex	2·8	7·1	5·2	9·8
Leaf base	4·2	10·2	6·4	11·7
Basal plate	8·2	18·8	19·1	24·1
Roots	84·8	63·9	69·3	54·4
Total sucrose uptake (µmol per plant)	1·67	5·46	9·75	25·69

Results expressed as a percentage of the partitioned radioactivity after expressing the radioactivity on a dry weight basis. Total sucrose uptake in µmol per plant. Data are means of two experiments with three replicates.

When plants were grown in the presence of 10 µm Ancy, this compound not only induced a 2·6 to 3·3‐fold rise in total sucrose uptake by plants (Table 3), mainly due to a 2·8‐fold increase in root fresh weight (data not shown), but also induced a marked augmentation in leaf base labelling (10·2–11·7 % vs. 4·2–6·4 %) at the expense of root radioactivity.

Effects of ancymidol on sugar content

Sugar contents (glucose, fructose, sucrose and fructans) were similar in the leaf blades of control and Ancy‐treated plants (Fig. 3). Treatment with Ancy induced a 66 % decrease in sucrose content in leaf bases, but increased the amounts of glucose, fructose and fructan by 188, 274 and 131 %, respectively.

Fig. 3.  Effect of adding 10 µm ancymidol (Ancy) on the carbohydrate content in leaves (base and blade) of plants cultured for 6 weeks in the bulbing medium (30 g l^–1 sucrose) under fluorescent + incandescent light. Results expressed in mg g^–1 d.wt ± s.e. Data are means of two independent extracts analysed in triplicate.

DISCUSSION

Results reported here show that in vitro‐grown shallot plants formed bulbs following culture under fluorescent light enriched in far‐red radiation for 16 h a day; one can conclude that the far‐red effect on the bulbing response is a classical HIR (high irradiance reaction) of phytochrome, as reported for onions by Lercari (1984) and Mondal et al. (1986). The bulbing ratio exceeded 2·0 when the sucrose concentration in the medium was between 30 and 70 g l^–1, and the heaviest bulbs were obtained with 30 g l^–1 sucrose. Nevertheless, the high osmotic pressure developed by 70 g l^–1 sucrose or by the combination of 30 g l^–1 sucrose plus 20 g l^–1 mannitol inhibited root and leaf growth; the basal swelling was probably due to the increase in easily assimilated sucrose in the culture medium. As in other species of bulbous plants, e.g. tulips and onions, increasing sucrose concentration can improve bulb formation (Rice et al., 1983; Kahane et al., 1992b; Keller, 1993). When plants were cultured under fluorescent light in a medium with 50 g l^–1 sucrose, only 10 % of the plants showed signs of bulbing.

Since, under inductive conditions (F + I light), exogenous GA₃ prevented bulb formation, inhibitors of endogenous GA synthesis (Ancy, Flur and PBZ) were added to the bulbing medium. All three compounds favoured bulbing, while simultaneous addition of exogenous GA₃ blocked bulb formation and plant development. However, treatment with Ancy did not cause a basal swelling of the plants under non‐inductive conditions (F light), indicating that the depletion of GAs is not the only cause of swelling.

Application of Ancy strongly stimulated bulbing in high‐sucrose (20–70 g l^–1) medium and greatly promoted rhizogenesis. A stimulatory effect of Ancy on rhizogenesis has been reported previously by Chin (1982) and Burkhart and Meyer (1991), and a better sucrose supply has been suggested as the cause. Indeed, when total [¹⁴C]sucrose (30 g l^–1) uptake per plant was measured, Ancy caused a three‐fold rise in total labelling of the plant, probably due to growth stimulation of the whole plant by Ancy. Furthermore, Ancy changed the partitioning of radioactivity, favouring labelling of the leaf base at the expense of root labelling. This greater mobilizing ability of the leaf base is associated with a higher content of glucose, fructose and fructans; fructans are indeed the major non‐structural carbohydrate reserve of bulbs (Darbyshire and Steer, 1990; Ernst et al., 1998). These results agree with the findings of Sobeih (1988) who showed that long photoperiods at low R : FR ratios led to increased assimilate accumulation in leaf bases. This probably results from the inability of the onion plant to utilize glucose and fructose as rapidly as they are formed and oligosaccharide accumulation could result from an increased rate of oligosaccharide accumulation or from a decrease in oligosaccharide hydrolysis as suggested by Lercari (1982b). Addition of Ancy to the culture medium would reinforce the carbohydrate accumulation induced by the addition of far‐red light to a 16 h photoperiod.

The requirement for light enriched in far‐red radiation indicates that the bulbing response depends on phytochrome action. Phytochrome is known to interact with GAs to regulate certain aspects of plant development either by governing GA biosynthesis or by affecting responsiveness to GAs (García‐Martínez et al., 1987; Martínez‐García and García‐Martínez, 1992; Reed et al., 1996; Jackson et al., 1998; Blázquez and Weigel, 1999; Kamiya and García‐Martínez, 1999). As reported by Mita and Shibaoka (1983, 1984) for onion, GAs stabilized microtubules, and acted conversely to the stimulus of long‐day conditions, which reduced the number of microtubules and disturbed their transverse orientations. A decrease in GA content under long‐day conditions as suggested by these authors would agree with our results obtained with Ancy on bulb development under F + I light, but would fail to explain the lack of effect of Ancy under fluorescent light. Another explanation may be that active phytochrome, via decreased effectiveness of GAs and reorientation of microtubules, would lead to a radial expansion of the cells and to the swelling of the leaf base.

Received: 17 October 2001; Returned for revision: 26 November 201; Accepted 18 December 2001.