Robust genetic transformation of sorghum (Sorghum bicolor L.) using differentiating embryogenic callus induced from immature embryos

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.Electronic supplementary materialThe online version of this article (10.1186/s13007-017-0260-9) contains supplementary material, which is available to authorized users.

Up‐regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor

“…Differentiating embryogenic callus was generated from grain sorghum inbred line Tx430 and used as explant for genetic transformation of sorghum and putative transgenic plants were regenerated as described by Belide et al (2017). Negative wildtype controls were taken through tissue culture without antibiotic selection.…”

Comparative Analysis of In Vitro Responses and Regeneration Between Diverse Bioenergy Sorghum Genotypes

“…Plant growth regulators are required for callus induction, and while both LG and WU CIM contained 1 mg/L 2,4-D as the auxin source, WU CIM also contained the cytokinin BA (0.5 mg/L). An analysis of several published sorghum studies has shown that their CIM always includes 2,4-D as the auxin source (Elkonin et al 1995, Carvalho et al 2004, Howe et al 2006, Nguyen et al 2007, Gurel et al 2012, Chen et al 2015, Do et al 2016, Omer et al 2018), sometimes combined with a cytokinin like BA (Belide et al 2017, Espinoza-Sanchez et al 2018) or KIN (Wernicke and Brettell 1980, Kaeppler and Pedersen 1997).…”

“…In addition, a key aspect of the WU regeneration protocol was the use of ABA. A survey of various sorghum somatic embryogenesis studies (Elkonin et al 1995, Kaeppler and Pedersen 1997, Seetharama et al 2000, Gurel et al 2012, Liu and Godwin 2012, Assem et al 2014, Wu et al 2014, Do et al 2016, Visarada et al 2016, Belide et al 2017) indicated that the inclusion of auxin(s) and cytokinin(s) are standard for regeneration, but few use ABA for embryo maturation/regeneration (Assem et al 2014, Wu et al 2014, Belide et al 2017). ABA is known to promote better embryogenic responses, with higher quality embryo formation, and subsequent plantlet conversion (Merkle et al 1995), so the superior performance of explants with the WU protocol may also reflect the inclusion of ABA during regeneration.…”

Genetic modification of sorghum for improved nutritional value: state of the problem and current approaches

“…[23][24][25] However, as a result of intensive research in recent years, significant progress has been made in this area. To date, the methods of introducing transgenes into the genome of sorghum, both by microprojectile transformation 26,27 , and through Agrobacteriummediated genetic transformation [28][29][30][31] were significantly improved.…”

“…29 In this regard, one should mention the report on significant stimulation of callus induction from immature sorghum embryos and plant regeneration by lipoic acid that allowed obtaining high transformation frequency (27.2%) in particle bombardment experiments. 27 Positive effect of lipoic acid might be caused by its anti-oxidative capacity that is important for in vitro cultured sorghum embryos.…”