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Genetic transformation of western clover (Trifolium occidentale D. E. Coombe.) as a model for functional genomics and transgene introgression in clonal pasture legume species.

Richardson KA, Maher DA, Jones CS, Bryan G - Plant Methods (2013)

Bottom Line: Transformation frequencies of up to 7.5% were achieved in 9 of the 17 accessions tested.Development of this protocol provides a valuable contribution toward establishing T. occidentale as a model species for white clover.This presents opportunities for further improvement in white clover through the application of biotechnology.

View Article: PubMed Central - HTML - PubMed

Affiliation: AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand. kim.richardson@agresearch.co.nz.

ABSTRACT

Background: Western clover (Trifolium occidentale) is a perennial herb with characteristics compatible for its development as an attractive model species for genomics studies relating to the forage legume, white clover (Trifolium repens). Its characteristics such as a small diploid genome, self-fertility and ancestral contribution of one of the genomes of T. repens, facilitates its use as a model for genetic analysis of plants transformed with legume or novel genes.

Results: In this study, a reproducible transformation protocol was established following screening of T. occidentale accessions originating from England, Ireland, France, Spain and Portugal. The protocol is based upon infection of cotyledonary explants dissected from mature seed with the Agrobacterium tumefaciens strain GV3101 carrying vectors which contain the bar selection marker gene. Transformation frequencies of up to 7.5% were achieved in 9 of the 17 accessions tested. Transformed plants were verified by PCR and expression of the gusA reporter gene, while integration of the T-DNA was confirmed by Southern blot hybridisation and segregation of progeny in the T1 generation.

Conclusions: Development of this protocol provides a valuable contribution toward establishing T. occidentale as a model species for white clover. This presents opportunities for further improvement in white clover through the application of biotechnology.

No MeSH data available.


Related in: MedlinePlus

Western clover (T. occidentale), shoot regeneration and transformation. (a) white clover (T. repens); compared to (b)T. occidentale(c) prolific regeneration of shoots from an AZ4270 cotyledon following three weeks culture on 1C; (d) shoot regeneration under ammonium glufosinate selection following Agrobacterium-mediated transformation of cotyledon explants; (e) transient GUS expression on cotyledonary explants three days after inoculation with Agrobacterium broth; (f) GUS activity in a trifoliate leaf excised from stably transformed plants approximately eight weeks after transformation.
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Figure 1: Western clover (T. occidentale), shoot regeneration and transformation. (a) white clover (T. repens); compared to (b)T. occidentale(c) prolific regeneration of shoots from an AZ4270 cotyledon following three weeks culture on 1C; (d) shoot regeneration under ammonium glufosinate selection following Agrobacterium-mediated transformation of cotyledon explants; (e) transient GUS expression on cotyledonary explants three days after inoculation with Agrobacterium broth; (f) GUS activity in a trifoliate leaf excised from stably transformed plants approximately eight weeks after transformation.

Mentions: White clover (Trifolium repens L., Figure 1a) is the most extensively used perennial legume in temperate grazed pastoral systems grown for its nutritional value and ability to fix atmospheric nitrogen [1,2]. This stoloniferous, clonal herb [3] originating from the grasslands of Europe, Western Asia and North Africa has an allotetraploid (2n = 4x = 32) genome derived from two diploid ancestors T. pallescens (2n = 2x = 16) and T. occidentale (2n = 2x = 16) [4]. The conventional breeding techniques used for plant improvement have resulted in the release of many commercial cultivars, however, it is now recognised that biotechnology approaches hold the promise of additional genetic gain in this species through the introduction of novel traits [5].


Genetic transformation of western clover (Trifolium occidentale D. E. Coombe.) as a model for functional genomics and transgene introgression in clonal pasture legume species.

Richardson KA, Maher DA, Jones CS, Bryan G - Plant Methods (2013)

Western clover (T. occidentale), shoot regeneration and transformation. (a) white clover (T. repens); compared to (b)T. occidentale(c) prolific regeneration of shoots from an AZ4270 cotyledon following three weeks culture on 1C; (d) shoot regeneration under ammonium glufosinate selection following Agrobacterium-mediated transformation of cotyledon explants; (e) transient GUS expression on cotyledonary explants three days after inoculation with Agrobacterium broth; (f) GUS activity in a trifoliate leaf excised from stably transformed plants approximately eight weeks after transformation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716983&req=5

Figure 1: Western clover (T. occidentale), shoot regeneration and transformation. (a) white clover (T. repens); compared to (b)T. occidentale(c) prolific regeneration of shoots from an AZ4270 cotyledon following three weeks culture on 1C; (d) shoot regeneration under ammonium glufosinate selection following Agrobacterium-mediated transformation of cotyledon explants; (e) transient GUS expression on cotyledonary explants three days after inoculation with Agrobacterium broth; (f) GUS activity in a trifoliate leaf excised from stably transformed plants approximately eight weeks after transformation.
Mentions: White clover (Trifolium repens L., Figure 1a) is the most extensively used perennial legume in temperate grazed pastoral systems grown for its nutritional value and ability to fix atmospheric nitrogen [1,2]. This stoloniferous, clonal herb [3] originating from the grasslands of Europe, Western Asia and North Africa has an allotetraploid (2n = 4x = 32) genome derived from two diploid ancestors T. pallescens (2n = 2x = 16) and T. occidentale (2n = 2x = 16) [4]. The conventional breeding techniques used for plant improvement have resulted in the release of many commercial cultivars, however, it is now recognised that biotechnology approaches hold the promise of additional genetic gain in this species through the introduction of novel traits [5].

Bottom Line: Transformation frequencies of up to 7.5% were achieved in 9 of the 17 accessions tested.Development of this protocol provides a valuable contribution toward establishing T. occidentale as a model species for white clover.This presents opportunities for further improvement in white clover through the application of biotechnology.

View Article: PubMed Central - HTML - PubMed

Affiliation: AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand. kim.richardson@agresearch.co.nz.

ABSTRACT

Background: Western clover (Trifolium occidentale) is a perennial herb with characteristics compatible for its development as an attractive model species for genomics studies relating to the forage legume, white clover (Trifolium repens). Its characteristics such as a small diploid genome, self-fertility and ancestral contribution of one of the genomes of T. repens, facilitates its use as a model for genetic analysis of plants transformed with legume or novel genes.

Results: In this study, a reproducible transformation protocol was established following screening of T. occidentale accessions originating from England, Ireland, France, Spain and Portugal. The protocol is based upon infection of cotyledonary explants dissected from mature seed with the Agrobacterium tumefaciens strain GV3101 carrying vectors which contain the bar selection marker gene. Transformation frequencies of up to 7.5% were achieved in 9 of the 17 accessions tested. Transformed plants were verified by PCR and expression of the gusA reporter gene, while integration of the T-DNA was confirmed by Southern blot hybridisation and segregation of progeny in the T1 generation.

Conclusions: Development of this protocol provides a valuable contribution toward establishing T. occidentale as a model species for white clover. This presents opportunities for further improvement in white clover through the application of biotechnology.

No MeSH data available.


Related in: MedlinePlus