Additive effects of three auxins and copper on sorghum in vitro root induction

A healthy root system is vital for tissue culture plantlet survival and rapid adaptation from the in vitro microenvironment to glasshouse conditions. Optimization of the root induction medium is an effective way to promote root induction and elongation. Levels of three auxins (alpha-naphthaleneacetic acid [NAA], 3-indoleacetic acid [IAA], and 3-indolebutyric acid [IBA]) and copper sulfate (CuSO4) have been investigated in a series of experiments with a sorghum inbred line, Tx430. Significant improvement in root proliferation and shoot growth were observed on Murashige and Skoog (MS) medium supplemented with 1 mu mol/L CuSO4, 1 mg/L NAA, 1 mg/L IAA, and 1 mg/L IBA. On average, one explant (the original in vitro-derived shoot) of Tx430 regenerated 56.7 roots, which was 20-fold higher on the optimal medium than on MS control medium. Another tested genotype SA281 showed similar response patterns as Tx430 across media. In addition, 100% of Tx430 and SA281 plantlets originating from the optimized root induction medium all survived after being transferred to potting soil in the glasshouse. The results demonstrate that a combination of auxins (NAA, IAA, and IBA) and CuSO4 together at optimal concentrations provide additive effects on promoting root proliferation and explant growth of in vitro sorghum in root induction medium, and subsequently resulted in 100% survival rate of plantlets ex tissue culture. Compared with two published and frequently used root induction media, the optimized medium significantly enhanced root induction and plantlet growth

Highly efficient sorghum transformation

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 (T ). 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 T progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T 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

Genetic transformation of sweet sorghum

Sweet sorghum has substantial potential as a biofuel feedstock, with advantages in some environments over alternatives such as sugarcane or maize. Gene technologies are likely to be important to achieve yields sufficient for food, fuel and fibre production from available global croplands, but sorghum has proven difficult to transform. Tissue culture recalcitrance and poor reproducibility of transformation protocols remain major challenges for grain sorghum, and there has been no reported success for sweet sorghum. Here we describe a repeatable transformation system for sweet sorghum, based on (1) optimized tissue culture conditions for embryogenic callus production with >90% regenerability in 12-week-old calli, and (2) an effective selection regimen for hygromycin resistance conferred by a Ubi-hpt transgene following particle bombardment. Using this method, we have produced sixteen independent transgenic lines from multiple batches at an overall efficiency of 0.09% transformants per excised immature embryo. Co-expression frequency of a non-selected luciferase reporter was 62.5%. Transgene integration and expression were confirmed in T(0) and T(1) plants by Southern analysis and luciferase assays. This success using the major international sweet sorghum cultivar Ramada provides a foundation for molecular improvement of sweet sorghum through the use of transgenes. Factors likely to be important for success with other sweet sorghum cultivars are identified

Biolistic DNA delivery and its applications in Sorghum bicolor

Biolistic DNA delivery has been considered a universal tool for genetic manipulation to transfer exotic genes to cells or tissues due to its simplicity, versatility, and high efficiency. It has been a preferred method for investigating plant gene function in most monocot crops. The first transgenic sorghum plants were successfully regenerated through biolistic DNA delivery in 1993, with a relatively low transformation efficiency of 0.3%. Since then, tremendous progress has been made in recent years where the highest transformation efficiency was reported at 46.6%. Overall, the successful biolistic DNA delivery system is credited to three fundamental cornerstones: robust tissue culture system, effective gene expression in sorghum, and optimal parameters of DNA delivery. In this chapter, the history, application, and current development of biolistic DNA delivery in sorghum are reviewed, and the prospect of sorghum genetic engineering is discussed