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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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Time course of somatic embryo accumulation after culture initiation for transformation with the binary vector pMDC83 with a MtOLEOSIN4 gene - MtOLEOSIN4 GFP (Ole4-GFP) (a) or with no inserted gene - Null-GFP (b), and total embryos produced per callus (c) (Vertical bars indicate 95% confidence interval).
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pone-0099908-g004: Time course of somatic embryo accumulation after culture initiation for transformation with the binary vector pMDC83 with a MtOLEOSIN4 gene - MtOLEOSIN4 GFP (Ole4-GFP) (a) or with no inserted gene - Null-GFP (b), and total embryos produced per callus (c) (Vertical bars indicate 95% confidence interval).

Mentions: Embryo accumulation after transformation using the standard P4 10∶4 (NAA:BAP in µM) for three weeks before transfer to P4 10∶4∶1 (NAA:BAP:ABA in µM), or P4 10∶4∶1∶5 medium (NAA:BAP:ABA:GA in µM) for two different constructs (see Table 1) is presented in Fig. 4. One construct had no inserted gene for transfer ( construct), while another construct had an MtOLEOSIN4 gene [28] inserted. Hygromycin was in the media as the selection agent. Somatic embryos first appeared in GA+ABA treatments and a rapid increase in numbers occurred about 20 days earlier than the standard protocol (Fig. 4a, b). A large increase in total embryo numbers occurred in the treatments with ABA+GA with both the construct and with the construct containing the MtOLEOSIN4 gene (Fig. 4c). This showed that GA+ABA stimulated somatic embryogenesis under transformation conditions.


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)

Time course of somatic embryo accumulation after culture initiation for transformation with the binary vector pMDC83 with a MtOLEOSIN4 gene - MtOLEOSIN4 GFP (Ole4-GFP) (a) or with no inserted gene - Null-GFP (b), and total embryos produced per callus (c) (Vertical bars indicate 95% confidence interval).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099908-g004: Time course of somatic embryo accumulation after culture initiation for transformation with the binary vector pMDC83 with a MtOLEOSIN4 gene - MtOLEOSIN4 GFP (Ole4-GFP) (a) or with no inserted gene - Null-GFP (b), and total embryos produced per callus (c) (Vertical bars indicate 95% confidence interval).
Mentions: Embryo accumulation after transformation using the standard P4 10∶4 (NAA:BAP in µM) for three weeks before transfer to P4 10∶4∶1 (NAA:BAP:ABA in µM), or P4 10∶4∶1∶5 medium (NAA:BAP:ABA:GA in µM) for two different constructs (see Table 1) is presented in Fig. 4. One construct had no inserted gene for transfer ( construct), while another construct had an MtOLEOSIN4 gene [28] inserted. Hygromycin was in the media as the selection agent. Somatic embryos first appeared in GA+ABA treatments and a rapid increase in numbers occurred about 20 days earlier than the standard protocol (Fig. 4a, b). A large increase in total embryo numbers occurred in the treatments with ABA+GA with both the construct and with the construct containing the MtOLEOSIN4 gene (Fig. 4c). This showed that GA+ABA stimulated somatic embryogenesis under transformation conditions.

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