Highly efficient sorghum transformation

Abstract

A highly efficient microprojectile transformation system for sorghum (Sorghum bicolor L.) has been developed by using immature embryos (IEs) of inbred line Tx430. Co-bombardment was performed with the neomycin phosphotransferase II (nptII) gene and the green fluorescent protein (gfp) gene, both under the control of the maize ubiquitin1 (ubi1) promoter. After optimization of both tissue culture media and parameters of microprojectile transformation, 25 independent transgenic events were obtained from 121 bombarded IEs. The average transformation frequency (the total number of independent transgenic events divided by the total number of bombarded IEs) was 20.7% in three independent experiments. Transgenic events were confirmed by both PCR screening and Southern hybridization of genomic DNA from primary transgenics (T0). More than 90% of transformants were fertile and displayed normal morphology in a containment glasshouse. Co-transformation rate of the nptII and gfp genes was 72% in these experiments. The segregation of nptII and gfp in T1 progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T1 generation. The transformation procedure, from initiating IEs to planting putative transgenic plantlets in the glasshouse, was completed within 11–16 weeks, and was approximately threefold more efficient than the previously reported best sorghum transformation system.

Development of a Simple and Efficient Method for Agrobacterium-Mediated Transformation in Sorghum

“…The sorghum transformation method based on tissue culture has been widely considered challenging since the first research on sorghum transformation was published (Gritz and Davies, 1983;Liu and Godwin, 2012). Recently, the highest efficiency of sorghum transformation was reported at more than 20% using Agrobacterium-mediated and microprojectile transformation (Liu and Godwin, 2012;Wu et al, 2014).…”

“…Recently, the highest efficiency of sorghum transformation was reported at more than 20% using Agrobacterium-mediated and microprojectile transformation (Liu and Godwin, 2012;Wu et al, 2014). However, both of these studies used TX430 immature embryos as explants to induce callus.…”

“…Normally, transformed method based on tissue culture will take six months get transformed seedlings (Liu and Godwin, 2012). Our method eliminates tissue culture process and only requires piercing mature embryos.…”

“…The main problem for sorghum transformation is callus browning in culture tissue and genetic transformation (Gurel et al, 2009). Many studies for sorghum transformation have been done, but the average transformation efficiency has remained below 20% (Zhao et al, 2000;Gao et al, 2005a, b;Gurel et al, 2009;Liu and Godwin, 2012). Additionally, these methods based on tissue culture will take six months to get transformed seedlings.…”

Development of transgenic Sorghum bicolor (L.) Moench resistant to the Chilo partellus (Swinhoe) through Agrobacterium-mediated transformation

“…The first reports of sorghum plants transformed with agronomically important genes were the use of the HT12 gene for higher grain lysine content [29] employing Agrobacterium and the Cry1ACgene for insect resistance [14] employing particle bombardment. Although sorghum tissue culture, including callus induction and plant regeneration, has been successful [13,[30][31][32][33][34][35][36][37][38], genetic transformation of sorghum for commercial use was much less successful because of the lack of effective protocols [36]. Although work on transformation of sorghum began during 1990s, much less success has been achieved than with other crops.…”