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University of Johannesburg (2009)

Development of in vitro and transformation methods for Sorghum bicolor (L. Moench)

Mkhonza, Nompumelelo Lucritia

Titre : Development of in vitro and transformation methods for Sorghum bicolor (L. Moench)

Auteur : Mkhonza, Nompumelelo Lucritia

Université de soutenance : University of Johannesburg

Grade : Magister 2009

Résumé
Sorghum [Sorghum bicolor (L.)] is classified as a relatively recalcitrant crop due to its poor amenability to in vitro and genetic manipulation. An efficient and reproducible in vitro plant regeneration method is vital for a successful transformation of any crop. Plant regeneration and transformation of eight selected elite sorghum genotypes was studied. Immature zygotic embryos were used as explants and cultured on two different callus induction media. Three genotypes ICSV1111N, SRN39 and P898012 were found to be highly regenerable producing 5.99 ; 5.1 and 4.74 regenerants per explant respectively on the G2+L-proline callus induction medium. The eight elite sorghum genotypes were co-bombarded with the uidA reporter gene and manA selectable marker gene. Bombarded immature zygotic embryos were selected on G2+L-proline callus induction medium supplemented with mannose as a selective agent. PCR Positive transformants were only obtained from genotype P898012. Furthermore the genotype P898012 was stably transformed with a lower DNA amount of manA minimal transgene. The manA gene presence was confirmed with PCR and southern blot analyses and a transformation efficiency of 0.38% was attained. The fertile transgenic plants displayed simple integration patterns, and the gene was also inherited to the T1 progeny of manA resistant trasnsformants in a Mendelian fashion.

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