Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.

Agrobacterium-mediated sorghum transformation.

A number of parameters related to Agrobacterium-mediated infection were tested to optimize transformation frequencies of sorghum (Sorghum bicolor L.). A plasmid with a selectable marker, phosphomannose isomerase, and an sgfp reporter gene was used. First, storing immature spikes at 4°C before use decreased frequency of GFP-expressing calli, for example, in sorghum variety P898012 from 22.5% at 0 day to 6.4% at 5 days. Next, heating immature embryos (IEs) at various temperatures for 3 min prior to Agrobacterium infection increased frequencies of GFP-expressing calli, of mannose-selected calli and of transformed calli. The optimal 43°C heat treatment increased transformation frequencies from 2.6% with no heat to 7.6%. Using different heating times at 43°C prior to infection showed 3 min was optimal. Centrifuging IEs with no heat or heating at various temperatures decreased frequencies of all tissue responses; however, both heat and centrifugation increased de-differentiation of tissue. If IEs were cooled at 25°C versus on ice after heating and prior to infection, numbers with GFP-expressing cells increased from 34.2 to 49.1%. The most optimal treatment, 43°C for 3 min, cooling at 25°C and no centrifugation, yielded 49.1% GFP-expressing calli and 8.3% stable transformation frequency. Transformation frequencies greater than 7% were routinely observed using similar treatments over 5 months of testing. This reproducible frequency, calculated as numbers of independent IEs producing regenerable transgenic tissues, confirmed by PCR, western and DNA hybridization analysis, divided by total numbers of IEs infected, is several-fold higher than published frequencies.

Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos

This chapter describes a stepwise protocol for Agrobacterium-mediated sorghum genetic transformation. Immature embryos from sorghum plants were used as the target explants. The Agrobacterium strain LBA4404, carrying a "super-binary" vector, was used in this protocol. Agrobacterium co-transformation vectors, one T-DNA containing the selectable marker gene and another T-DNA containing the trait gene(s), were also introduced in sorghum transformation for eliminating the selectable marker gene in the resulting transgenic plants. This chapter provides recommendations for analysis of the transgenic plants to confirm T-DNA integration into the sorghum genome and segregation of the selectable marker gene from the trait gene(s).

Sorghum (Sorghum bicolor L.).

A dual-marker plasmid containing the selectable marker gene, manA, and the reporter gene, sgfp, was used to transform immature sorghum embryos by employing an Agrobacterium-mediated system. Both genes were under the control of the ubi1 promoter in a binary vector pPZP201. The Escherichia coli phosphomannose isomerase (PMI) gene, pmi, was used as the selectable marker gene and mannose was used as the selective agent. The sgfp gene encoding green fluorescence protein (GFP) was the reporter gene and served as a visual screening marker. A total of 167 transgenic plants were obtained from nine different embryogenic callus lines grown on a selection medium containing 1%-2% mannose. Embryoids and shoots regenerated via embryogenesis, that showed strong GFP fluorescence, were selected from two sorghum genotypes: C401, an inbred line, and Pioneer 8505, a commercial hybrid. The GFP accumulation in transgenic plants was observed with a dissecting stereomicroscope. The integration and expression of the manA gene was confirmed by Southern blot and Western blot analyses, and the feasibility of manA selection was demonstrated by the chlorophenol red (CPR) assay. Our results indicated that transgenes segregated in the Mendelian fashion in the T1 generation. The conversion of mannose to a metabolizable fructose carbon source is beneficial to plants. In addition, except in soybean and a few legumes, no endogenous PMI activity has been detected in plant species, indicating that PMI is useful in the transformation of sorghum. In addition, PMI has no sequence homology to known allergens. Optimization of this selection system for sorghum transformation provides an efficient way to produce transgenic plants without using antibiotic or herbicidal agents as selectable markers, and our results showed that the transformation efficiency reached 2.88% for Pioneer 8505 and 3.30% for C401, both values higher than in previously published reports.

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Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system

Morphogenesis and plant regeneration from callus of immature embryos of sorghum

Successful culturing of callus has recently been reported for the following monocotyledonous species: corn (Reference 2, and Linsmaier Bednar, E. M., personal communication), oats (1, 6), rice root (8), asparagus (7), wheat (5), and sorghum (4). For the culture of sorghum callus from roots and tillering nodes, Strogonov et al., (4) used Murashige and Skoog's basal medium plus calcium pantothenate, 5 mg/liter; 2,4-dichlorophenoxyacetic acid, 1 mg/liter; kinetin, 1 mg/liter; casein hydrolysate, 0.5 g/liter; and ascorbic acid, 1 mg/liter. The same basal inorganic medium was also used for the culture of oats (1), asparagus (7), and by Linsmaier Bednar for corn. Corn callus was also cultured on White's medium (2), oats and rice root on Heller's medium (6, 8), and wheat on Hildebrandt's D medium (5). Generally, 2,4-D was used as the auxin, but a-naphthaleneacetic acid as well as 2,4-D was used for corn (2). In other respects the same nutrients and growth conditions were used as those required for the culture of dicotyledonous callus. The ability of monocotyledonous callus tissue to form organs has been demonstrated by the production of embryoids and plantlets from asparagus cells (7), many shoots but few roots from oats (1), roots from wheat callus (5), and roots (2) and both roots and shoots from corn callus (Linsmaier Bednar, E. M., personal communication). This report describes the culture of sorghum callus, the initiation of shoot primordia, and development of plants from callus tissue of this species.

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David J. Holden

Papers

The growth of and organ formation from callus tissue of sorghum.