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.

Resourceful and High Efficiency Agrobacterium Mediated Transformation of Maize (Zea mays L.) Using Coleoptilar Nodal Explants

“…All these conditions of different parameters would help us in developing a congenial high throughput method of Agrobacterium mediated genetic transformation in maize. Similar studies were done in Rice (Ignacimuthu and Raveendar, 2011;Sahoo and Tuteja, 2012) and Yam (Nyaboga et al, 2014). This study will help in maize genetic engineering for agronomically demand traits into recalcitrant maize genotypes.…”

“…Thorough optimizing and tuning those parameters with different genotypes are necessary for getting high frequencies of transformation (Cao et al, 2014), where different genotypes were used by considering the different parameters and achieved 2% transformation frequency in maize using multiple shoots developed from the coleoptilar nodal explant. In contrary to this, in present study transformation studies have been done by using callus cultures developed from the same explant and frequencies were assessed by using hygromycin selection frequency (Nyaboga et al, 2014). In the present study, it was concluded that the parameters viz., one day pre-conditioning of explants, 0.8 optical density of bacterial culture in plain MS liquid infection medium having 5.8 pH along with 200 µM acetosyrengone, 15 minutes infection time for the explants after treating with 0.1% Macerozyme, application of vacuum infiltration for 10 minutes, three days of co-cultivation period, play a vital role in maize transgenic development from coleoptilar nodal explant.…”

Plant regeneration from axillary bud derived callus in white yam (Dioscorea rotundata)

“…The accession was maintained in vitro and multiplied through nodal cuttings on yam basic medium (YBM) containing Murashige and Skoog medium (MS) salts and vitamins, 0.05 mg/l 6-Benzylaminopurine (BAP), 0.02 mg/l Naphthaleneacetic acid (NAA), 25 mg/l Ascorbic acid, 30 g/l sucrose, and 2.4 g/l gelrite, as described by Nyaboga et al (2014) (Fig. 1a).…”

“…The crop has great economic and social significance in sub-Saharan Africa (Ile et al 2006) where more than 95 % of the world's yam is produced (FAO 2013). Of the 600 known Dioscorea species only 10 of the 90 edible species are consistently cultivated for food consumption (Nyaboga et al 2014) and the Dioscorea rotundata species complex is among the most cultivated, especially in West Africa (FAO 2013). Besides its importance as a staple food, yam is also a source of income for people, particularly in the coastal region of West Africa (Asiedu and Sartie 2010).…”

“…Recently the production of transgenic plants of D. rotundata has been reported using nodal segments (Nyaboga et al 2014). Although micropropagation of D. rotundata has been described (Adeniyi et al 2008;Anike et al 2012), there is no report on regeneration through somatic embryogenesis that has many advantages particularly for genetic transformation as embryos are unicellular in origin and avoid chances of chimerism (Mariashibu et al 2013).…”