Abstract
BackgroundSorghum (Sorghum bicolor L.) is one of the world’s most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency.ResultsWe report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5–2.0 mm in length) isolated 12–15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt-II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs.ConclusionsThis protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.
Highlights
Sorghum (Sorghum bicolor L.) is one of the world’s most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop
Small (1.4–2 mm) immature embryos cultured on CIM with lipoic acid (LA) (1 mg/l) had greater differentiating embryogenic callus (DEC) tissue induction frequency with improved quality of nodular structures compared to CIM without LA
Improved quality refers to the visual appearance of the nodular structures, their colour being soft green to darker green (Fig. 1b) and the relative amount of tissue that had started to develop into globular stage somatic embryos
Summary
Sorghum (Sorghum bicolor L.) is one of the world’s most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Sorghum has been identified as a prospective bioenergy crop and model for C4 grasses [3, 4] It has been recently identified as a potential candidate for making environmentally friendly fuels and chemicals, to engineer leaves and stems of this biomass crop to produce more oil instead of starch that offers alternatives to petroleum-based products. To manipulate these complex pathways using genetic engineering approach, highly efficient transformation methods are prerequisite. An alternative efficient and cost effective method would be desirable for the improvement of sorghum
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