# Effect of GA3 and paclobutrazol on adventitious shoot regeneration of two Pelargonium sp.

Latifa Hamama, Linda Voisine, Amani Naouar, R. Gala, Denis Cesbron, Gilles Michel, F. Leplat, Fabrice Foucher, Laurence Hibrand-Saint Oyant, Noëlle Dorion

► To cite this version:

Latifa Hamama, Linda Voisine, Amani Naouar, R. Gala, Denis Cesbron, et al.. Effect of GA3 and paclobutrazol on adventitious shoot regeneration of two Pelargonium sp.. 7. ISHS on In Vitro Culture & Horticultural Breeding, Sep 2011, Ghent, Belgium. pp.1. hal-01608780

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<figure><img src="image_1.png" /></figure>
# Effect of GA₃ and Paclobutrazol on adventitious shoot regeneration of two *Pelargonium* sp.

*L. Hamama* ¹,³, L.Voisine¹,³, A. Naouar¹, R. Gala², D. Cesbron¹, G. Michel², F. Leplat¹, F. Foucher², L. Hibrand Saint Oyant² and N. Dorion¹

UMR GenHort A1259, IFR Quasav 149- (1) AGROCAMPUS OUEST Centre d'Angers - INHP, 2 rue Le Nôtre, 49045 Angers cedex 01, France - (2) Centre INRA Angers-Nantes, rue Georges Morel, 49071 Beaucouzé, France - (3) Université d'Angers, UFR Sciences, 2 bd Lavoisier 49045 Angers cedex 01, France. * latifa.hamama@agrocampus-ouest.fr

## OBJECTIVES

In this study, we test the effect of gibberellic acid (GA₃) and Paclobutrazol (PBZ) on callus formation and adventitious shoot regeneration on *Pelargonium* sp., before using this species for functional validation of genes involved in the process of GA regulation via genetic transformation.

## INTRODUCTION

*In vitro* plant regeneration efficient methods are essential in biotechnology. Cell dedifferentiation and differentiation are complex phenomena induced *in vitro* by growth regulators. The effect of auxins and cytokinins are well known whereas the effect of gibberellins (GA) was less studied. However, Hutchinson et al., 1997 showed that GA₃ had an inhibitory effect on development of somatic embryogenesis in *Pelargonium x hortorum*. Conversely, paclobutrazol (PBZ), a GA biosynthetic inhibitor, promoted development of somatic embryogenesis.

## MATERIAL AND METHODS

In a first experiment, adventitious shoot regeneration was performed on *Pelargonium x hortorum* 'Panaché sud' ('P.sud') and *Pelargonium x domesticum* 'Autumn haze' ('P.dom') according to Hassanein and Dorion (2005). First, we evaluated the effect of a range of GA₃ concentrations (1-20µM) and PBZ (0.1-1µM) on callus induction and bud regeneration. In a second experiment GA₃ and PBZ were added to the regeneration medium (SRM) separately or together to check the relationship between PBZ and GA₃ effects.

<figure><img src="image_2.png" /></figure>

## RESULTS AND DISCUSSION

**Shoot regeneration process on SRM.** On leaf discs arising from 4-5 weeks-old mother plant and containing petiole umbilicus both genotypes led to highly efficient formation of adventitious shoots (Table 1). 'P.dom' explants produced limited callus in the cut edges of leaf discs, buds were formed quickly and direct secondary organogenesis took place (Fig.1A). Conversely, 'P.sud' explants were totally dedifferentiated in callus which proliferated extensively before bud formation through several subcultures (Fig.2A).

**Effect of GA₃ on callus formation and shoot regeneration.** In both cases, GA₃ applied alone (Fig.1B, 2B, Table 1), inhibited bud regeneration as soon as 5µM (Fig.3A). Similarly, the rate of callus formation (Fig.3B) decreased drastically when 5 µM GA₃ was applied to 'P.dom' explants while for 'P.sud', callus decreasing is less evident. Same results were observed by Hutchinson et al (1997), It is likely that GA₃ inhibited the dedifferentiation level since callus is also inhibited.

**Effect of PBZ on callus formation and shoot regeneration.** On 'P.dom' explants, callus extended to the whole explant when PBZ was added to SRM (Fig. 1D, 1D'), Regeneration was not significantly promoted but the initiated buds had an early onset and a better appearance provided that PBZ is applied in the early stages of cellular dedifferentiation. Similarly, for 'P.sud', which formed an abundant callus, PBZ did not improve regeneration (Table 1) and led to shoots of poor quality (hyperhydric)(Fig.2C). In our experiment, results on 'P. dom' confirmed more or less those of Hutchinson while 'P.sud' did not.

<figure><img src="image_3.png" /><figcaption>▲Fig. 1. Effect of GA₃ and PBZ on 'P.dom' leaf discs.</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>▲Fig. 2. Effect of GA₃ and PBZ on 'P.sud' leaf discs.</figcaption></figure>

<table><caption>▼Table 1. Effect of GA₃, PBZ or GA₃+PBZ on callus formation and shoot regeneration</caption><thead><tr><th></th><th>Regeneration medium</th><th>SRM control</th><th>SRM GA<sub>3</sub> 10µM</th><th>SRM PBZ 0.3µM</th><th>SRM GA<sub>3</sub> + PBZ</th></tr></thead><tbody><tr><td rowspan="3">'P.dom'</td><td>Explants forming calli</td><td>94%</td><td>48%</td><td>81%</td><td>89%</td></tr><tr><td>Explants forming shoots</td><td>65%</td><td>7%</td><td>44%</td><td>13%</td></tr><tr><td>regulated shoots (mean/explants)</td><td>53 (14.0 ± 7.4) ab</td><td>4 (0.2 ± 0.4) a</td><td>69 (17.3 ± 19.0) b</td><td>9 (2.3 ± 4.5) a</td></tr><tr><td rowspan="3">'P. sud'</td><td>Explants forming calli</td><td>100%</td><td>97%</td><td>100%</td><td>94%</td></tr><tr><td>Explants forming shoots</td><td>83%</td><td>23%</td><td>79%</td><td>13%</td></tr><tr><td>regulated shoots (mean/explant)</td><td>296 (59.2 ± 36.2) b</td><td>28 (5.6 ± 5.7) a</td><td>304 (60.8 ± 30.5) b</td><td>9 (1.8 ± 4.0) a</td></tr></tbody></table>

**Effect of PBZ + GA₃.** PBZ and GA₃ applied at the same time (Table 1) restored totally the ability of callus formation compare to GA₃ alone and therefore but to a lesser extend the regeneration process in 'P.dom'. Conversely, in 'P.sud' explants, no restoration of buds regeneration was observed. This last result was expected since PBZ is an inhibitor of GA's synthesis.

## CONCLUSION

In the experiments we have found complex relationships between GA₃, PBZ and dedifferentiation (callus) versus differentiation (shoot) processes within two cv of *Pelargonium*. The effect of GA's and its inhibitor varied according to the genotype and some of the effects differed from those observed by Hutchinson et al (1997). According to our results and in order to validate by transgenesis the function of gene related to GA's pathway, 'P. dom' which behaved better that 'P.sud' should be used. Moreover attention should be paid to the risk of regeneration inhibition when using overexpression of genes implied in GA₃ synthesis.

Hassanein, A, Dorion, N, (2005). Efficient plant regeneration system from leaf discs of zonal (*Pelargonium x hortorum*) and two scented (*P. capitatum* and *P. graveolens*) Geraniums. Plant Cell, Tissue and Organ Culture. 83: 231-240.
Hutchinson, M.J., KrishnaRaj, S., Saxena, P.K., (1997). Inhibitory effect of GA₃ on the development of thidiazuron-induced somatic embryogenesis in geranium (*Pelargonium x hortorum Bailey*) hypocotyls cultures. Plant Cell Reports.
16: 435-438;
Effect of GA<sub>3</sub> and paclobutrazol on adventitious shoot regeneration of two *Pelargonium* sp.

L. Hamama<sup>1,2</sup>, L. Voisine<sup>1,2</sup>, A. Naouar<sup>1</sup>, R. Gala<sup>3</sup>, D. Cesbron<sup>1</sup>, G. Michel<sup>3</sup>, F. Leplat<sup>1</sup>, F. Foucher<sup>3</sup>, L. Hibrand Saint
Oyant<sup>3</sup> and N. Dorion<sup>1</sup>.

UMR GenHort 1259, IFR149 QUASAV (Agrocampus-Ouest, INRA, Univ. Angers)

1. Agrocampus-Ouest centre d'Angers, 2 Rue Le Nôtre, 49045 Angers, France

2. Université Angers, 2 Bd Lavoisier, 49045 Angers Cedex 01 Angers, France

3. Centre INRA Angers-Nantes, 42 Rue Georges Morel, BP 60057, 49071 Beaucouzé

Latifa.hamama@agrocampus-ouest.fr

Abstract

In vitro plant regeneration methods from differentiated tissues are essential in biotechnology (plant breeding, genetic transformation, functional validation of genes...). Cell dedifferentiation and differentiation are complex phenomena induced in vitro by growth regulators. The effect of auxins and cytokinins has been extensively studied on somatic embryogenesis and organogenesis for several plants species. The effect of gibberellins (GA) is much less studied.

Hutchinson et al., 1997 showed that GA<sub>3</sub> has an inhibitory effect on development of somatic embryogenesis in *Pelargonium x hortorum*. Conversely, paclobutrazol, a GA biosynthetic inhibitor, promotes development of somatic embryogenesis

The objectives of this study was to test the effect of GA₃ and paclobutrazol on adventitious shoot regeneration

Adventitious shoot regeneration was performed on two *Pelargonium* species, *Pelargonium x hortorum* Panaché sud 'P.sud' and *Pelargonium domesticum* Autumn haze 'P.dom' according in the first step to Hassanein and Dorion 2005.

By choosing the explant type and mother plant age, both genotypes led to the highly efficient
formation of adventitious shoots (> 70%) on the same medium. Both genotypes have different
behavior during the shoot regeneration. 'P.dom' explants produces limited callus in the cut edges of
leaf discs, buds are formed quickly and direct secondary organogenesis took place. 'P.sud' explants
are totally dedifferentiated in callus which proliferates extensively before bud formation through
several subcultures.

We evaluated the effect of a concentration range of GA<sub>3</sub> (1-20µM) and paclobutrazol (0.1-3µM) on the callus induction and bud regeneration of the two genotypes. GA<sub>3</sub> and paclobutrazol were added in regeneration medium separately or together to check, in the latter case, if the paclobutrazol can restore GA<sub>3</sub> effect.

In both cases, GA₃ applied alone, completely inhibits the bud regeneration. Moreover, the rate of
callus formation decreased by 50% when 1 µM GA₃ was applied to 'P.dom' explants. The same result
was obtained on 'P.sud' explants using 20µM GA₃. Paclobutrazol (3 µM) applied at the same time that
GA₃ (10µM) can partially restore regeneration process.

In 'P. dom', the use of paclobutrazol alone led to increase callus formation and slightly improved the rate of regeneration. Initiated buds have a better appearance (less hyperhydricity) provided that paclobutrazol is applied only in the early stages of cellular dedifferentiation. For genotype 'P. sud', which forms an abundant callus, paclobutrazol does not improve regeneration and led to hyperhydric shoot formation.

The understanding of gibberellins role on adventitious shoot regeneration in *Pelargonium* sp., is
needed in order to improve the procedure. The modalities of cell differentiation and bud regeneration
through callus or not are probably of major importance.

**Keywords:** Adventitious shoots regeneration; GA<sub>3</sub>; Paclobutrazol; *Pelargonium*.

References

Hassanein, A, Dorion, N, (2005). Efficient plant regeneration system from leaf discs of zonal (*Pelargonium x hortorum*) and two scented (*P. capitatum* and *P. graveolens*) Geraniums. Plant Cell, Tissue and Organ Culture. 83: 231-240.

Hutchinson, M.J., KrishnaRaj, S., Saxena, P.K., (1997). Inhibitory effect of GA3 on the development of thidiazuron-induced somatic embryogenesis in geranium (*Pelargonium x hotorum Bailey*) hypocotyls cultures. Plant Cell Reports. 16: 435-438.