Abstract
Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1-2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70-80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava.
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Claims 6
Claim · model-written summary
The transformation protocol gave significantly enhanced transformation efficiency over existing cassava protocols.
- Taxon
- cassava
Manihot esculentae0 - Material
- TME14e0
- Outcome
- transformation efficiencye1
- Comparator
- existing cassava transformation protocolse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“recalcitrant cassava cultivar TME14”
e1“This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols”
Claim · model-written summary
About 70-80 independent transgenic lines per ml settled cell volume of friable embryogenic callus were regenerated on selective medium.
- Taxon
- cassavaManihot esculentae0
- Outcome
- independent transgenic linese2
- Value
- 70-80e2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“recalcitrant cassava cultivar TME14”
e2“About 70-80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium”
Claim · model-written summary
GUS assays confirmed gusA expression in transformed calli, somatic embryos and transgenic plants.
- Taxon
- cassavaManihot esculentae0
- Organ / stage
- transformed calli, somatic embryos and transgenic plantse3
- Gene
- gusAe3
- Outcome
- expressione3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“recalcitrant cassava cultivar TME14”
e3“Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants”
Claim · model-written summary
PCR and Southern blot confirmed the presence and integration of the gusA gene.
- Taxon
- cassavaManihot esculentae0
- Gene
- gusAe4
- Outcome
- presence and integratione4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“recalcitrant cassava cultivar TME14”
e4“The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively”
Claim · model-written summary
Use of DKW medium was found to favour production of friable embryogenic callus in cassava.
- Taxon
- cassavaManihot esculentae0
- Treatment
- use of DKW mediume5
- Outcome
- production of friable embryogenic calli (FEC)e5
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“recalcitrant cassava cultivar TME14”
e5“Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium”
Claim · model-written summary
Low Agrobacterium density during co-cultivation enhanced transformation efficiency in cassava.
- Taxon
- cassavaManihot esculentae0
- Treatment
- use of low Agrobacterium density during co-cultivatione6
- Outcome
- transformation efficiencye6
- Direction
- enhancede6
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“recalcitrant cassava cultivar TME14”
e6“transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation”
The same questions in other papers
in vitro · yield · Manihot esculenta (cassava)Results agree
3 papers in the same direction as this paper
- positive
- positive
- positive
Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.