Sorghum is a crop of global importance as a source of grain and forage. It is an important staple cereal in the semi-arid tropics of Africa and Asia, and is widely used as animal feed in industrialised countries. The sorghum plant is well adapted to grow in arid and semi-arid climates, but is susceptible to a broad range of diseases including those caused by Puccinia purpurea (sorghum rust) and Johnsongrass mosaic virus (JGMV). Crop improvement programs increasingly use technologies involving tissue culture and genetic transformation as a means to generate plants with disease resistance.
Until recently, sorghum transformation has relied on the use of microprojectile bombardment to introduce transgenes into embryogenic callus tissue. The use of Agrobacterium tumefaciens to transform sorghum offers several advantages over microprojectile systems. An Agrobacterium method for sorghum was investigated using an established embryogenic callus culture system. Favourable inoculation and ' co-culture conditions for transient and stable transformation were identified using the β-glucuronidase (GUS) visual marker produced by the expression of an intron-containing uidA gene.
Prior to the construction of transformation vectors, designed to confer durable transgenic virus resistance to sorghum, the variation in the coat protein coding region of JGMV isolates was assessed Twenty three isolates from sorghum, maize and johnsongrass were collected from diverse geographical locations over seven years. Differences in the nucleotide sequence of the coat protein coding region ranged between 0.8% to 5.2% and similar variation was found among the deduced amino acid sequences.
Sequence data showed a high degree of similarity (over 94%) between isolates, suggesting that virus variability was low and that a transgenic approach should provide effective stable resistance to JGMV Additionally, two residues were implicated in enabling the JGMV-Kr virus strain to overcome the Krish resistance gene in sorghum. These residues were identified in the core region of the coat protein.
Transformation vectors containing an entire JGMV coat protein sequence or a partial sequence in an inverted repeat orientation were generated Microprojectile bombardment with a particle inflow gun was used to transfer the transgenes into sorghum. Rigorous analysis of the regenerated plants failed to demonstrate that any lines were transgenic.
In addition, four lines of sorghum had previously been transformed with the Rp1-D rust resistance gene .from maize. The progeny of one line had been challenged with the rust pathogen and had shown a putative resistance response. Further genetic analysis and screening of progeny from this line was undertaken. Southern blot hybridisation was used to demonstrate that these progeny did not carry either the rust resistance transgene or selectable marker gene.
The selection of transgenic sorghum plants in tissue culture has relied on the bar gene that confers resistance to the herbicide bialaphos. This system was found to be highly inefficient and resulted in a high frequency of nontransformed cultures being regenerated Field release of transgenic sorghum produced in this way could result in the herbicide resistance gene being transferred to Sorghum halepense, a weedy relative that is already difficult to control. An alternative selection system using mannose as the selective agent was evaluated for use with sorghum. Mannose concentrations above 1.25 gL-1 resulted in poor growth of organogenic sorghum cultures, even in the presence of a regular concentration of sucrose (30 gL-1). An optimal combination of sucrose and mannose to include in selective media was determined to be 20 gL-1 and 2.5 gL-1 respectively.
Transient GUS expression, driven by five promoters derived from Banana bunchy top virus, was assessed in sorghum leaf pieces and embryogenic calli.
Three of these promoters produced equivalent or higher levels of transient expression compared with the maize ubiquitin promoter that is commonly used
A high percentage of sorghum plants failed to grow when transferred from the tissue culture environment to soil. An improved method for this critical stage in the tissue culture system was developed using a sterile 4:1 perlite:vermiculite mix. This system resulted in a 100% survival rate amongst transferred plants.
The genetic diversity of a significant viral pathogen of sorghum in Australia has been characterised and further insight has been provided into the mechanism by which this virus can overcome a host resistance gene. Improvements have been made to the sorghum tissue culture and transformation system to create a more robust and efficient system for the future manipulation of sorghum using a transgenic approach.