An unusual abscisic acid and gibberellic acid synergism increases somatic embryogenesis, facilitates its genetic analysis and improves transformation in Medicago truncatula.

Kim E Nolan,Nasir A Saeed,Ray J Rose,Youhong Song,Xiyi Zhang,Siyang Liao,Josep Bassaganya-Riera

doi:10.1371/journal.pone.0099908

Abstract

Somatic embryogenesis (SE) can be readily induced in leaf explants of the Jemalong 2HA genotype of the model legume Medicago truncatula by auxin and cytokinin, but rarely in wild-type Jemalong. Gibberellic acid (GA), a hormone not included in the medium, appears to act in Arabidopsis as a repressor of the embryonic state such that low ABA (abscisic acid): GA ratios will inhibit SE. It was important to evaluate the GA effect in M. truncatula in order to formulate generic SE mechanisms, given the Arabidopsis information. It was surprising to find that low ABA:GA ratios in M. truncatula acted synergistically to stimulate SE. The unusual synergism between GA and ABA in inducing SE has utility in improving SE for regeneration and transformation in M. truncatula. Expression of genes previously shown to be important in M. truncatula SE was not increased. In investigating genes previously studied in GA investigations of Arabidopsis SE, there was increased expression of GA2ox and decreased expression of PICKLE, a negative regulator of SE in Arabidopsis. We suggest that in M. truncatula there are different ABA:GA ratios required for down-regulating the PICKLE gene, a repressor of the embryonic state. In M. truncatula it is a low ABA:GA ratio while in Arabidopsis it is a high ABA:GA ratio. In different species the expression of key genes is probably related to differences in how the hormone networks optimise their expression.

Highlights

Somatic embryogenesis (SE) in addition to being useful as an in vitro system to study embryogenesis has facilitated the development of clonal propagation, somatic hybridisation and transformation for the study of genes and for transgenic crops
The Effect of Gibberellic acid (GA) on Somatic Embryogenesis Our standard protocol for SE is incubation in auxin plus cytokinin for three weeks subculture into auxin plus cytokinin plus abscisic acid (ABA)
We carried out further experiments to investigate the effect of GA when ABA was given at the beginning of explant incubation to see if this would give similar results

Summary

Introduction

Somatic embryogenesis (SE) in addition to being useful as an in vitro system to study embryogenesis has facilitated the development of clonal propagation, somatic hybridisation and transformation for the study of genes and for transgenic crops. Cytokinin is essential in M. truncatula as with auxin alone roots are initiated [5]. Stress alone is capable of inducing SE in some systems [8]. In this context the stress hormone abscisic acid (ABA) can induce SE in carrot root apices [9]. In M. truncatula, SE is enhanced by ABA when it is added to the auxin plus cytokinin required for SE induction [10]. This is not surprising given what is known about how plant hormone signaling can influence gene expression [11]

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Discussion

Conclusion