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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

Influence of PGR regime on the frequency of shoot regeneration from cotyledonary explants of AZ4270. Each data point represents the mean of 4 replicate plates containing 20 explants per plate.
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Figure 2: Influence of PGR regime on the frequency of shoot regeneration from cotyledonary explants of AZ4270. Each data point represents the mean of 4 replicate plates containing 20 explants per plate.

Mentions: Explants were cultured on plant growth media for four weeks without subculture. Regeneration frequencies (number of explants regenerating per plate) were assessed at this time (Figure 2, Additional file 1: Table S1). A relatively uniform production of plantlets with high levels of regeneration (86-94%) was encountered over the range of PGR combinations and concentrations; however, there were clear visual differences in the fitness and average number of shoots produced per explant. Therefore, we also examined the vigor, hyperhydricity and average number of shoots regenerating per explant in each treatment (data not shown). Treatments resulting in the regeneration of greater than five, morphologically normal, shoots per explant (Figure 1c) were an important parameter in this assessment.


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)

Influence of PGR regime on the frequency of shoot regeneration from cotyledonary explants of AZ4270. Each data point represents the mean of 4 replicate plates containing 20 explants per plate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Influence of PGR regime on the frequency of shoot regeneration from cotyledonary explants of AZ4270. Each data point represents the mean of 4 replicate plates containing 20 explants per plate.
Mentions: Explants were cultured on plant growth media for four weeks without subculture. Regeneration frequencies (number of explants regenerating per plate) were assessed at this time (Figure 2, Additional file 1: Table S1). A relatively uniform production of plantlets with high levels of regeneration (86-94%) was encountered over the range of PGR combinations and concentrations; however, there were clear visual differences in the fitness and average number of shoots produced per explant. Therefore, we also examined the vigor, hyperhydricity and average number of shoots regenerating per explant in each treatment (data not shown). Treatments resulting in the regeneration of greater than five, morphologically normal, shoots per explant (Figure 1c) were an important parameter in this assessment.

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