Division of Plant Industry, CSIRO, Canberra ACT 2601

Abstract

Our aim is to improve the efficiency of Agrobacterium-mediated barley transformation. For Golden Promise, our routine recipient of transgenes, efficiency of transformation is already high at approximately 10% (fertile plants recovered per 100 embryos co-cultivated with Agrobacterium) but other genotypes are significantly lower (approximately 1%). We have compared four barley cultivars for efficiency in two phases of transformation (1) the generation of stably transformed barley callus and (2) the regeneration of plantlets from transformed callus. Using the green fluorescent protein (GFP) gene as a reporter, we have obtained efficient transformation (47-76%) of callus for the four cultivars, Golden Promise, Schooner, Chebec and Sloop. However, regeneration of transformed plantlets (phase 2) is much higher for Golden Promise than for the other three genotypes. A major difference between genotype transformation frequencies, therefore, is in regenerative ability. We have evidence that genes determining regenerability are segregating in these barley cultivars and that favourable gene combinations can be selected to increase regeneration. We are currently screening a number of other genotypes for regenerability with a view to identifying genes that can be combined to improve transformation efficiency.

Introduction

Transformation of barley is now feasible using both biolistic (Wan and Lemaux, 1994) and Agrobacterium based methods (Tingay et al., 1997; Patel et al., 2000) with approximately 6% of bombarded/infected embryos yielding transformed plants. Further optimisation of Agrobacterium-mediated barley transformation in our laboratory has increased our average transformation frequency to 10%, however large amounts of material must still be cultured to generate sufficient numbers of highly expressing stable lines for analysis. This process is time-consuming and involves significant labour so any further improvements in transformation efficiency would be beneficial. In addition, to date, reasonable transformation frequencies (greater than 4%) are only obtained using the cultivar, Golden Promise. The ability to transform other cultivars at reasonable frequencies would be useful as it may reduce the amount of backcrossing needed following the production of transgenic lines. To investigate the process of transformation and better understand the limitations of the procedure we have used green fluorescent protein (GFP) as a reporter gene to follow transformation in four barley cultivars (Golden Promise, Schooner, Chebec and Sloop). Unlike β-glucuronidase (GUS), the other widely used reporter gene, GFP has the significant advantage that it does not require a substrate and its expression can be detected in real time in living cells by light excitation.

Materials and Methods

Barley Transformation and Analysis

Barley plants were grown and transformed as described in Tingay et al. (1997) except immature embryos were isolated and infected with Agrobacterium on the same day (without wounding by bombardment) and excess Agrobacterium was removed from embryos by dragging them across an additional callus induction medium plate. Callus induction medium contained 50 mg/L Hygromycin B (in addition to TimentinTM) as a selective agent, regeneration medium 20 mg/L Hygromycin B and rooting medium 25 mg/L Hygromycin B. Schooner regenerant seed (null segregants from previously transformed Schooner lines) was supplied by Ming-Bo Wang (CSIRO, Plant Industry) and resulted from experiments detailed in Wang et al. (2001). Histochemical assays to monitor GUS activity in plant tissue were carried out according to Jefferson et al. (1987). GFP fluorescence in plant tissues (callus and roots) was visualised using an MZ6 stereomicroscope (Leica) with a fluorescence GFP-Plus filter set.

Reporter Constructs

Plasmids pVec8-GFP and pVec8-GusI were created by subcloning either the sgfpS65T gene (Chiu et al., 1996) or intron-interrupted uidA gene (Ohta et al., 1990) between the maize ubiquitin promoter (containing the first intron) (Christensen et al., 1992) and the Agrobacterium nopaline synthase 3’ region. Each expression cassette was subcloned into the binary vector pWBVec8 (Wang et al., 1998) which contains an intron-interrupted hpt gene driven by a CaMV 35S promoter. Triparental mating was used to introduce binary vectors into Agrobacterium strain AGLO (Lazo et al., 1991).

Results and Discussion

As there have been several reports suggesting GFP can have deleterious effects on suspension cells and plant regeneration, (Wagstaff and Olmstead, 1997; Haseloff and Siemering, 1998) two reporter constructs (Vec8-GFP and Vec8-GusI) were independently used to transform the cultivar Golden Promise. Both constructs use the constitutive maize ubiquitin (Ubi-1) promoter to drive either GFP (Vec8-GFP) or GUS (Vec8-GusI) expression in planta. The results shown in Table 1 indicate plant lines expressing GFP were recovered at a lower frequency (4.4%) than those expressing GUS (9.2%). For each plasmid, no difference in the number of vigorously growing calli was noted six weeks after transformation (results not shown). It was observed that many of the strongly fluorescing GFP calli failed to regenerate suggesting high levels of GFP expression interfered with barley regeneration.

Table 1. Barley Transformation Frequencies using GFP and GUS Reporter Constructs.

For each construct results are combined from at least three independent experiments and scored on the basis of clear reporter gene expression in root tips and leaves.

Despite GFP-expressing lines being recovered at a lower frequency than GUS-expressing lines, use of the Vec8-GFP reporter construct has the advantage that early events in transformation can be followed non-destructively in real time. This construct was therefore used to transform immature embryos from three Australian barley cultivars (Schooner, Chebec and Sloop). Stable callus transformation frequencies six weeks after infection (Table 2) were approximately 50% for Golden Promise, Schooner and Sloop and 76% for Chebec. However, for each Australian cultivar, plant transformation frequencies were very low (0.6%) compared to Golden Promise (4.4%), indicating that in barley, regenerative ability contributes significantly to differences in plant transformation frequencies.

To investigate if genes determining regenerability are segregating in barley, the transformation frequency of immature embryos derived from null segregants of previously transformed Schooner lines (Schooner regenerant) was compared to regular Schooner (Table 2). Similar stable callus transformation frequencies were obtained (46% and 47% respectively), however a significantly higher plant transformation frequency was obtained for Schooner regenerant (3.3%) compared to Schooner (0.6%).

Table 2. Callus and Plant Transformation Frequencies for Barley Cultivars Transformed with Vec8-GFP. For each cultivar, results are combined from at least three independent experiments, *callus fluorescence was scored 6 weeks after infection (number of callus lines expressing GFP per number of embryos infected with Agrobacterium).

Conclusions

Our results indicate that although the GFP gene can be used successfully as a reporter system in the transformation of barley cultivars, caution is also required. When using plasmids pVec8-GFP and pVec8-GusI, we generated transgenic lines expressing GFP at approximately half the frequency as lines expressing GUS. As Ubi-1 is a strong constitutive promoter it is possible that strong GFP expression is interfering with regeneration in barley or that gene silencing has occurred. To date, a similar GFP gene to that used here (driven by Ubi-1 promoter) has been used successfully to transform other monocots such as maize (van der Geese and Petolino, 1998) and sugarcane (Elliott et al., 1999) but there was no mention of deleterious effects.

We have also demonstrated that elite Australian barley lines can be transformed by Agrobacterium infection of immature embryos, although only at low frequencies (0.6%). Analysis of stably transformed callus using the GFP reporter gene indicates this poor transformation frequency is not due to Agrobacterium’s inability to transform barley scutellar tissue but to the inability of the transformed callus to successfully regenerate into green plants. Additional transformation experiments with Schooner regenerant material suggest regeneration in this cultivar is largely controlled by a number of genes segregating within the cultivar. Selection for these genes by using donor material which had already regenerated in tissue culture probably attributed to the increase in transformation frequency from 0.6% in Schooner to 3.3% in Schooner regenerant. These results reiterate the importance of genotype in barley transformation, and suggest favourable gene combinations can be selected to increase transformation frequencies in previously recalcitrant cultivars. We are currently screening a number of other genotypes for regenerability with a view to identifying genes that can be combined to improve transformation efficiency.

Acknowledgements

The support of the Grains Research and Development Corporation (Australia) is gratefully acknowledged.

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