MENU

Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14

Issued on

Routineproductionoflargenumbersoftransgenicplantsisrequiredtofullyexploitadvancesincassavabiotechnologyandsupportdevelopmentofiprovedgermplasmfordeploymenttofarmers.Thisarticledescribesanimproved,high-efficiencytransformationprotocolforrecalcitrantcassavacultivarTME14preferredinAfrica.Factorsthatfavorproductionoffriableembryogeniccalli(FEC)werefoundtobeuseofDKWmedium,crushingoforganizedembryogenicstructures(OES)through1–2mmsizedmetalwiremesh,washingofcrushedOEStissuesandshortexposureoftyrosinetosomaticembryos;andtransformationefficiencywasenhancedbyuseoflowAgrobacteriumdensityduringco-cultivation,co-centrifugationofFECwithAgrobacterium,germinationofparamomycinresistantsomaticembryosonmediumcontainingBAPwithgradualincreaseinconcentrationandvariationsofthefrequenyofsubcultureofcotyledonary-stageembryosonshootelongationmedium.Byapplyingtheoptimizedparameters,FECwereproducedforcassavacultivarTME14andtransformedusingAgrobacteriumstrainLBA4404harboringthebinaryvectorpCAMBIA2301.About70–80independenttransgeniclinespermlsettledcellvolume(SCV)ofFECwereregeneratedonselectivemedium.HistochemicalGUSassaysconfirmedtheexpressionofgusAgeneintransformedcalli,somaticembryosandtransgenicplants.ThepresenceandintegrationofthegusAgenewereconfirmedbyPCRandSouthernblotanalysis,respectively.RT-PCRanalysisoftransgenicplantsconfirmedtheexpressionofgusAgene.Thisprotocoldemonstratessignificantlyenhancedtransformationefficiencyoverexistingcassavatransformationprotocolsandcouldbecomeapwerfultoolforfunctionalgenomicsandtransferringnewtraitsintocassava.

Citation

Nyaboga, E.N., Njiru, J.M., & Tripathi, L. (2015). Factors influencing somatic embryogenesis, regeneration and agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14. Frontiers in Plant Science, 6, 411.

Authors

  • Nyaboga, E.N.
  • Njiru, J.M.
  • Tripathi, L.