Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

Abstract

Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3-4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop.

Keywords: Agrobacterium-mediated transformation; Dioscorea rotundata; axillary buds; reporter gene; selection marker gene.

FIGURE 1

Schematic representation of T-DNA of binary plasmids. (A) pCAMBIA1301; (B) pCAMBIA2301; (C) pCAMBIA2300-gfp.

FIGURE 2

Effect of hygromycin and kanamycin concentrations on regeneration of nodal explants of D. rotundata. (A) h0–h15 represent culture conditions with different hygromycin concentrations where h refers to hygromycin and the number after h refers to different concentrations; (B) k0–k200 represent culture conditions with different kanamycin concentrations, where k refers to kanamycin and the number after k refers to different concentrations. The pictures were taken 30 days after culture in shoot elongation medium supplemented with antibiotics.

FIGURE 3

Regeneration and transformation of D. rotundata cv. TDr 2436. (A) Axillary bud induction from nodal explants after 1 week of culture on SBM; (B) shoot induction from nodal explants after 2 weeks culture on SBM; (C) proliferation of shoots within 8 weeks of culture on SBM; (D) rooting of elongated transformed shoot; (E) acclimatized transgenic plant maintained in glasshouse; (F) non-transgenic plant in soil in the glasshouse.

FIGURE 4

Schematic diagram showing various steps of stable genetic transformation of D. rotundata using nodal explants.

FIGURE 5

Expression of reporter genes in tissues of putative transgenic plants of D. rotundata. (A) Transient expression of gusA gene in emerging axillary buds 1 week after Agro-infection; (B) control non-transgenic plantlets; (C) stable expression of the gusA gene in transgenic plantlets; (D) transient expression of gfp gene in emerging axillary buds 3 days after Agro-infection; (E) gene expression in transgenic buds produced ∼1–2 weeks after Agro-infection; (F) leaf of transgenic plantlets viewed under UV light using GFP filter.

FIGURE 6

Molecular analysis of transgenic plants. PCR analysis of genomic DNA of putative transgenic and non-transgenic control plants using primers specific for (A) hpt gene; (B) gusA gene; RT-PCR analysis using primers specific to (C) Actin gene; (D) gusA gene. M- 1 kb plus molecular marker (Fermentas); P- pCAMBIA1301 plasmid DNA; 1–10- transgenic plants; NT- control non-transgenic plant.

FIGURE 7

Analysis of transgenic lines to confirm integration of transgene. (A) Dot blot analysis of transgenic lines. 1–12- transgenic lines in triplicates; P- pCAMBIA1301 plasmid DNA as positive control. (B) Southern blot analysis of genomic DNA of transgenic lines and non-transgenic control plant digested with HindIII. M- DIG-labeled molecular weight marker; 1–3- transgenic lines; NT- non-transgenic plant; and P- pCAMBIA1301 plasmid DNA as positive control.