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

Nolan KE, Song Y, Liao S, Saeed NA, Zhang X, Rose RJ - PLoS ONE (2014)

Bottom Line: It was important to evaluate the GA effect in M. truncatula in order to formulate generic SE mechanisms, given the Arabidopsis information.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.In different species the expression of key genes is probably related to differences in how the hormone networks optimise their expression.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental and Life Sciences, The University of Newcastle, Callaghan, New South Wales, Australia.

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.

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The effect of GA (0, 1, 10 and 100 µM) on somatic embryogenesis induced by P4 10∶4 (NAA:BAP in µM) for three weeks before transfer to P4 10∶4∶1 (NAA:BAP:ABA in µM) shown for two experiments (a) and (b).Five plates of each treatment with six explants per plate. Counts were made 11 weeks from the initiation of culture. Treatments with different letters are significantly different at the 0.05 probability level; vertical bars indicate ± standard error. The numbers on the X-axis represent the GA concentration in µM.
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pone-0099908-g001: The effect of GA (0, 1, 10 and 100 µM) on somatic embryogenesis induced by P4 10∶4 (NAA:BAP in µM) for three weeks before transfer to P4 10∶4∶1 (NAA:BAP:ABA in µM) shown for two experiments (a) and (b).Five plates of each treatment with six explants per plate. Counts were made 11 weeks from the initiation of culture. Treatments with different letters are significantly different at the 0.05 probability level; vertical bars indicate ± standard error. The numbers on the X-axis represent the GA concentration in µM.

Mentions: Our standard protocol for SE is incubation in auxin plus cytokinin for three weeks then subculture into auxin plus cytokinin plus ABA. GA when applied with auxin plus cytokinin from the beginning of leaf explant incubation had little effect on SE at common physiological concentrations of 1 µM and 10 µM (Fig. 1). However, at 100 µM GA was inhibitory. We then 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.


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

Nolan KE, Song Y, Liao S, Saeed NA, Zhang X, Rose RJ - PLoS ONE (2014)

The effect of GA (0, 1, 10 and 100 µM) on somatic embryogenesis induced by P4 10∶4 (NAA:BAP in µM) for three weeks before transfer to P4 10∶4∶1 (NAA:BAP:ABA in µM) shown for two experiments (a) and (b).Five plates of each treatment with six explants per plate. Counts were made 11 weeks from the initiation of culture. Treatments with different letters are significantly different at the 0.05 probability level; vertical bars indicate ± standard error. The numbers on the X-axis represent the GA concentration in µM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061021&req=5

pone-0099908-g001: The effect of GA (0, 1, 10 and 100 µM) on somatic embryogenesis induced by P4 10∶4 (NAA:BAP in µM) for three weeks before transfer to P4 10∶4∶1 (NAA:BAP:ABA in µM) shown for two experiments (a) and (b).Five plates of each treatment with six explants per plate. Counts were made 11 weeks from the initiation of culture. Treatments with different letters are significantly different at the 0.05 probability level; vertical bars indicate ± standard error. The numbers on the X-axis represent the GA concentration in µM.
Mentions: Our standard protocol for SE is incubation in auxin plus cytokinin for three weeks then subculture into auxin plus cytokinin plus ABA. GA when applied with auxin plus cytokinin from the beginning of leaf explant incubation had little effect on SE at common physiological concentrations of 1 µM and 10 µM (Fig. 1). However, at 100 µM GA was inhibitory. We then 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.

Bottom Line: It was important to evaluate the GA effect in M. truncatula in order to formulate generic SE mechanisms, given the Arabidopsis information.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.In different species the expression of key genes is probably related to differences in how the hormone networks optimise their expression.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental and Life Sciences, The University of Newcastle, Callaghan, New South Wales, Australia.

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.

Show MeSH
Related in: MedlinePlus