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Agrobacterium tumefaciens-mediated transformation of Cleome gynandra L., a C(4) dicotyledon that is closely related to Arabidopsis thaliana.

Newell CA, Brown NJ, Liu Z, Pflug A, Gowik U, Westhoff P, Hibberd JM - J. Exp. Bot. (2010)

Bottom Line: Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified.Stable transformants of C. gynandra with detectable amounts of beta-glucuronidase (GUS) were produced at an efficiency of 14%.When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, Downing Street, University of Cambridge, Cambridge CB2 3EA, UK.

ABSTRACT
In leaves of most C(4) plants, the biochemistry of photosynthesis is partitioned between mesophyll and bundle sheath cells. In addition, their cell biology and development also differs from that in C(3) plants. We have a poor understanding of the mechanisms that generate the cell-specific accumulation of proteins used in the C(4) pathway, and there are few genes that have been shown to be important for the cell biology and development of C(4) leaves. To facilitate functional analysis of C(4) photosynthesis, and to enable knowledge from Arabidopsis thaliana to be translated to C(4) species, an Agrobacterium tumefaciens-mediated transformation protocol was developed for the C(4) species Cleome gynandra. A. tumefaciens, harbouring the binary vector SLJ1006, was used to transfer the uidA gene under the control of the CaMV 35S promoter into C. gynandra. Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified. Stable transformants of C. gynandra with detectable amounts of beta-glucuronidase (GUS) were produced at an efficiency of 14%. When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells. This transformation procedure is the first for an NAD-ME type C(4) plant and should significantly accelerate the analysis of mechanisms underlying C(4) photosynthesis.

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Related in: MedlinePlus

Hypocotyl explants of C. gynandra assayed for β-glucuronidase activity 8 d after transfer to regeneration medium A5 with (A–C) or without (D–F) 20 mg l−1 kanamycin. Hypocotyls were initially inoculated with LBA4404 containing the construct SLJ1006 in MSO (A, D), A3 (B, E) or SIM (C, F). Scale bars=2 mm.
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fig1: Hypocotyl explants of C. gynandra assayed for β-glucuronidase activity 8 d after transfer to regeneration medium A5 with (A–C) or without (D–F) 20 mg l−1 kanamycin. Hypocotyls were initially inoculated with LBA4404 containing the construct SLJ1006 in MSO (A, D), A3 (B, E) or SIM (C, F). Scale bars=2 mm.

Mentions: The composition of media used to suspend A. tumefaciens prior to, and during, incubation with plant tissue can alter transformation efficiency (Alt-Mörbe et al., 1989; Godwin et al., 1991; Humara et al., 1999). The influence of three separate liquid suspension media (MSO, A3, and SIM) on the transfer of the uidA gene encoding β-glucuronidase (GUS) into C. gynandra was therefore assessed. Both A. tumefaciens LBA4404 and EHA105 containing the binary plasmids SLJ1006 and pVDH (Jones et al., 1992) with uidA under the control of the CaMV 35S promoter was used for this purpose. β-glucuronidase assays carried out on inoculated explants 8 d or 14 d after transfer to selective or non-selective regeneration medium showed more transformed tissue in those explants treated with LBA4404 compared with EHA105, therefore further experiments were conducted with LBA4404 as it appeared to give the best chance for the regeneration of transformed shoots. LBA4404 continues to be used for Brassica transformation (Chen et al., 2008). A greater number of areas with GUS activity were found when explants were incubated with LBA4404 in SIM compared to either MSO or A3 (Table 2), and transformed areas were larger when SIM was used (Fig. 1). Future experiments were therefore conducted with SIM medium. It was discovered that subsequent explant growth was poor if tissue was left submerged in A. tumefaciens SIM inoculum for too long and so incubation was limited to 30 min.


Agrobacterium tumefaciens-mediated transformation of Cleome gynandra L., a C(4) dicotyledon that is closely related to Arabidopsis thaliana.

Newell CA, Brown NJ, Liu Z, Pflug A, Gowik U, Westhoff P, Hibberd JM - J. Exp. Bot. (2010)

Hypocotyl explants of C. gynandra assayed for β-glucuronidase activity 8 d after transfer to regeneration medium A5 with (A–C) or without (D–F) 20 mg l−1 kanamycin. Hypocotyls were initially inoculated with LBA4404 containing the construct SLJ1006 in MSO (A, D), A3 (B, E) or SIM (C, F). Scale bars=2 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2837259&req=5

fig1: Hypocotyl explants of C. gynandra assayed for β-glucuronidase activity 8 d after transfer to regeneration medium A5 with (A–C) or without (D–F) 20 mg l−1 kanamycin. Hypocotyls were initially inoculated with LBA4404 containing the construct SLJ1006 in MSO (A, D), A3 (B, E) or SIM (C, F). Scale bars=2 mm.
Mentions: The composition of media used to suspend A. tumefaciens prior to, and during, incubation with plant tissue can alter transformation efficiency (Alt-Mörbe et al., 1989; Godwin et al., 1991; Humara et al., 1999). The influence of three separate liquid suspension media (MSO, A3, and SIM) on the transfer of the uidA gene encoding β-glucuronidase (GUS) into C. gynandra was therefore assessed. Both A. tumefaciens LBA4404 and EHA105 containing the binary plasmids SLJ1006 and pVDH (Jones et al., 1992) with uidA under the control of the CaMV 35S promoter was used for this purpose. β-glucuronidase assays carried out on inoculated explants 8 d or 14 d after transfer to selective or non-selective regeneration medium showed more transformed tissue in those explants treated with LBA4404 compared with EHA105, therefore further experiments were conducted with LBA4404 as it appeared to give the best chance for the regeneration of transformed shoots. LBA4404 continues to be used for Brassica transformation (Chen et al., 2008). A greater number of areas with GUS activity were found when explants were incubated with LBA4404 in SIM compared to either MSO or A3 (Table 2), and transformed areas were larger when SIM was used (Fig. 1). Future experiments were therefore conducted with SIM medium. It was discovered that subsequent explant growth was poor if tissue was left submerged in A. tumefaciens SIM inoculum for too long and so incubation was limited to 30 min.

Bottom Line: Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified.Stable transformants of C. gynandra with detectable amounts of beta-glucuronidase (GUS) were produced at an efficiency of 14%.When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, Downing Street, University of Cambridge, Cambridge CB2 3EA, UK.

ABSTRACT
In leaves of most C(4) plants, the biochemistry of photosynthesis is partitioned between mesophyll and bundle sheath cells. In addition, their cell biology and development also differs from that in C(3) plants. We have a poor understanding of the mechanisms that generate the cell-specific accumulation of proteins used in the C(4) pathway, and there are few genes that have been shown to be important for the cell biology and development of C(4) leaves. To facilitate functional analysis of C(4) photosynthesis, and to enable knowledge from Arabidopsis thaliana to be translated to C(4) species, an Agrobacterium tumefaciens-mediated transformation protocol was developed for the C(4) species Cleome gynandra. A. tumefaciens, harbouring the binary vector SLJ1006, was used to transfer the uidA gene under the control of the CaMV 35S promoter into C. gynandra. Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified. Stable transformants of C. gynandra with detectable amounts of beta-glucuronidase (GUS) were produced at an efficiency of 14%. When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells. This transformation procedure is the first for an NAD-ME type C(4) plant and should significantly accelerate the analysis of mechanisms underlying C(4) photosynthesis.

Show MeSH
Related in: MedlinePlus