Efficient genetic transformation of Sorghum using a visual screening marker

Abstract

To transform grain sorghum (Sorghum bicolor (L.) Moench) with a visual reporter gene (gfp) and a target gene (tlp), three genotypes (two inbreds, Tx 430 and C401, and a commercial hybrid, Pioneer 8505) were used. We obtained a total of 1011 fertile transgenic plants from 61 independent callus lines, which were produced from 2463 zygotic immature embryos via Agrobacterium-mediated transformation. The reporter gene, gfp, encoding green fluorescent protein (GFP), was used as a visual screening marker, and the target gene, tlp, encoding thaumatin-like protein (TLP), was chosen for enhancing resistance to fungal diseases and drought. Both genes were under the control of the maize ubi 1 promoter in the binary vector pPZP201. A total of 320 plants showing GFP expression, derived from 45 calli, were selected and analyzed by Southern blot analysis. There was a 100% correlation between the GFP expression and the presence of the target gene, tlp, in these plants. Transgenic plants showing strong TLP expression were confirmed by Western blotting with antiserum specific for TLP. The transgene segregated in various ratios among progeny, which was confirmed by examining seedlings showing GFP fluorescence. The progeny also showed different copy numbers of transgenics. This report describes the successful use of GFP screening for efficient production of stably transformed sorghum plants without using antibiotics or herbicides as selection agents.

The impact of over-expression of NPK1 gene on growth and yield of sorghum under drought stress

“…Agrobacterium is a potent transformation tool with the advantage of producing transgenic plants with low transgene copy number. In the present study, the low transgene copy number is consistent with other investigations using Agrobacterium on cereals (Hiei et al, 1994;Ishida et al, 1996;Cheng et al, 1997;Zhao et al, 2000;Gao et al, 2005a;Lu et al, 2009).…”

“…Selection of bialaphos-resistant callus is often conducted at 2 to 3 mg/L bialaphos (Casas et al, 1993;Frame et al, 2002;Grootboom et al, 2010). Bialaphos, as selection agent, has been found to hinder regeneration capacity of sorghum (Casas et al, 1993;Gao et al, 2005a;Lu et al, 2009;Grootboom et al, 2010). In the present investigation, bialaphos imposed a severe selection on sorghum callus.…”

“…To improve sorghum abiotic stress tolerance, HVA1, thaumatin-like protein (TLP), mtld, OsCDPK-7 and TPS1 genes were successfully introduced into sorghum genome (Devi et al, 2004;Gao et al, 2005a;Maheswari et al, 2010;Mall et al, 2011;Yellisetty et al, 2015). In order to make plants more tolerant to stress and restore the cellular balance, transferring a single gene encoding a single specific stress protein, that is, "single-action" genes may not suffice the required tolerance levels.…”

Sorghum Transformation: Overview and Utility

“…In sorghum the visual marker genes green fluorescent protein (gfp) and β-glucuronidase (GUS) are each effective in monitoring for transgenic cells (Jeoung et al 2002). Using the former visual marker Gao et al (2005a) reported a 3.0% transformation efficiency as means to monitor for transgenic differentiating cell lineages from immature embryos of sorghum.…”

Genetic transformation of Sorghum bicolor

“…The PMI structural gene (manA) of Escherichia coli, which is capable of converting mannose to fructose, has been used as a selectable marker gene. The conversion of mannose to a metabolizable fructose carbon source which is beneficial to plants was achieved in sorghum with Agrobacteriummediated system using manA gene (Gao et al, 2005a;Gurel et al, 2009). …”